International Buffalo Information Center (IBIC) · International Buffalo Information Center (IBIC) Buffalo Bulletin . ISSN: 0125-6726 (Print), 2539-5696 (Online) Aims . IBIC is a

International Buffalo Information Center (IBIC)

Buffalo Bulletin

ISSN: 0125-6726 (Print), 2539-5696 (Online)

Aims

IBIC is a specialized information center on water buffalo. Established in 1981 by Kasetsart University

(Thailand) with an initial financial support from the International Development Research Center (IDRC) of

Canada. IBIC aims at being the buffalo information center of buffalo research community throughout the world.

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Buffalo Bulletin is published quarterly in January-March, April-June, July-September and October-

December. Contributions on any aspect of research or development, progress reports of projects and news on

buffalo will be considered for publication in the bulletin. Manuscripts must be written in English and follow the

instruction for authors which describe at inside of the back cover.

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Prof. Dr. Charan Chantalakhana Thailand

Prof. Dr. John Lindsay Falvey Faculty of Veterinary and Agricultural Science, University

of Melbourne, Australia

Prof. Dr. Metha Wanapat Department of Animal Science, Faculty of Agriculture,

Khon Kaen University, Thailand

Mr. Antonio Borghese International Buffalo Federation, Italy

Dr. Aree Thunkijjanukij International Buffalo Information Center, Office of the

University Library, Kasetsart University, Thailand

Miss Supanee Hongthong International Buffalo Information Center, Office of the


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Dr. Pakapan Skunmun Thailand

Dr. Kalaya Bunyanuwat Department of Livestock Development, Thailand

Prof. Dr. Federico Infascelli Department of Veterinary Medicine and Animal Science,

University of Naples Federico II, Italy

Dr. Rafat Al Jassim School of Agriculture and Food Sciences, Faculty of Science,

The University of Queensland, Australia

Prof. Dr. Nguyen Van Thu Department of Animal Sciences, Faculty of Agriculture and

Applied Biology, Can Tho University, Vietnam

Prof. K. Sarjan Rao Department of Livestock Production and Management,

College of Veterinary Science, India

Prof. Dr. Masroor Ellahi Babar Virtual University of Pakistan, Pakistan

Asst. Prof. Dr. Asif Nadeem Institute of Biochemistry and Biotechnology, University of

Veterinary and Animal Sciences, Pakistan

Prof. Dr. Raul Franzolin Departamento de Zootecnia, Universidade de São Paulo, Brazil

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Dr. Sunpetch Sophon Thailand

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Mr. Chalermdej Taterian International Buffalo Information Center, Office of the


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Miss Kanchana Anuphan International Buffalo Information Center, Office of the


Miss Jirawadee Wiratto International Buffalo Information Center, Office of the


Buffalo Bulletin

IBIC, Kasetsart University,

P.O. BOX 1084, Bangkok 10903, Thailand

E-mail: [email protected]

Tel: 66-2-9428616 ext. 344

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Buffalo Bulletin (July-September 2016) Vol.35 No.3

CONTENTS Page Case Report

An unusual case of ideopathic fibrinous pericarditis in Nili-ravi buffalo-A case report Muhammad Saqib, Ghazanfar Abbas and Mudassar Niaz Mughal................................................299

Original Article

Effect of lugol’s iodine on estrus induction and fertility response in true anestrus Jaffrabadi buffaloes A.R. Ahlawat, P.U. Gajbhiye, M.D. Odedra, V.B. Dongre and S.N. Ghodasara...........................303

Haemato-biochemical alterations during different stages of lactation in Mehshani buffaloes Hemen Das, A. Lateef, H.H. Panchasara and M. Ayub Ali...............................................................307

Effect of early post-partum GnRH and PGF2 alpha administration on follicular activities in Murrah buffaloes M.V. Ingawale and S.A. Bakshi...............................................................................................................317

Clinical, hemato-biochemical and therapeutic studies on rumen impaction in buffaloes A.K. Tripathi, J.S. Soodan and R.B. Kushwaha....................................................................................325

Diagnosis of rabies in buffaloes: comparison of clinico-pathological, immunohistochemical and immunofluorescent techniques A.B. Beigh, B.S. Sandhu, C.K. Singh, K. Gupta and N.K. Sood.......................................................331

Epidemiology of ixodid ticks in buffaloes (Bubalus bubalis) of Punjab, India N.K. Singh and S.S. Rath..........................................................................................................................347

Epidemiological studies on gastrointestinal parasites of buffaloes in seven agro-climatic zones of Madhya Pradesh, India S. Nath, G. Das, A.K. Dixit, V. Agrawal, S. Kumar, A.K. Singh and R.N. Katuri..........................355

Studies on effect of non-genetic parameters on mortality pattern in Murrah buffaloes Nitin Mohan Gupta, M.L. Mehra and Puneet Malhotra....................................................................365


CONTENTS Page Original Article

Evaluation of breeding values Murrah buffalo bulls under organized farms Vijay Kumar and A.K. Chakravarty.........................................................................................................371

Incidence and fertility status of hydatid cysts in buffaloes A. Sheeba, A. Sangaran, B.R. Latha and A. Raja.................................................................................379

Seroepidemiological study of leptospirosis in buffaloes of south Gujarat, India J.M. Patel, P.D. Vihol, V. S. Dabas, M.C. Prasad, J.H. Patel, C.F. Chaudhari, N.B. Patel and K.M. Patel........................................................................................383

Age related changes in the histomorphology of mandibular gland in prenatal buffalo (Bubalus bubalis) K. Raja, M.S. Lakshmi, G. Purushotham, K.B.P. Raghavende and T.S. Chandrasekhara Rao................................................................................................................389

β(1,4)-Galactosyltransferase-I gene polymorphisms in Pakistani Nili Ravi buffalo Aamir Sohail, Asif Nadeem, Masroor Ellahi Babar, Tanveer Hussain, Akhtar Ali, Wasim Shehzad and Maryam Javed.................................................................................399

Investigation of response to selection for milk traits in dairy buffalo of Iran based on three sale situations Bahareh Taheri Dezfuli and Leonardo De Seno....................................................................................405

Nutritional status and hemato-biochemical profile of anoestrus buffaloes of Malwa region of Madhya Pradesh Nagendra Patil, R.K. Jain and Dharmesh Tewari................................................................................417


CONTENTS Page Original Article

Developmental competence of buffalo (Bubalus bubalis) oocytes: effect of oocytes quality, protein additives, hormonal supplement and type of capacitating agents M.M. Waheed, K.H. El-Shahat and A.M. Hammam............................................................................427

Seminal plasma and sperm membrane proteins of buffalo and cattle bulls: A comparative study Shilpi Dixit, Vijay Pandey, Dilip Kumar Swain, Rajesh Nigam, Ambika Sharma, Deepak Sharma, Atul Saxena and Pawanjit Singh............................................................................437

Incidence of repeat breeding in varying breeds of buffaloes and cattle in different climatic conditions in Khyber Pakhtunkhwa (Pakistan) Amjad Khan, Muhammad Hassan Mushtaq, Mansur ud Din Ahmad, Abid Hussain, Asghar Khan, Ajab Khan and Habibun Nabi.....................................................................................445

Incidence, pain assessment and management of horn affections in buffaloes K. Rama Rao, Makkena Sreenu, K.B.P. Raghavender and P.V.S. Kishore.....................................455

Evaluation of feeding practices and certain minerals status of lactating buffaloes in coastal zone of western India P.L. Sherasia, P.R. Pandya, S. Parnerkar, B.R. Devalia and B.M. Bhanderi.................................467

Gross morphological studies on major salivary glands of prenatal buffalo K. Raja, M. Santhi Lakshmi, G. Purushotham, K.B.P. Raghavender,

T.S. Chandrasekhara Rao and D. Pramod Kumar............................................................................479


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ABSTRACT

An unusual case of idiopathic fibrinous pericarditis in Nili-Ravi buffalo was presented at Veterinary Teaching Hospital, Department of Clinical Medicine and Surgery (CMS), University of Agriculture Faisalabad. The clinical signs and postmortem findings were suggestive of idiopathic fibrinous pericarditis which are discussed in detail.

Keywords: Nili-Ravi buffalo, clinical signs, postmortem findings, idiopathic, fibrinous pericarditis

INTRODUCTION

Nili-Ravi buffalo is also known as Black Gold of Pakistan, because of its adaptability in hot climates and major share in milk and meat production. Inflammation of pericardium along with accumulation of fibrinous material is called as pericarditis (Grunder, 2002). Clinical signs associated with pericarditis are tachycardia, muffled heart sounds, distention of jugular vein, edema of submandibular, brisket and ventral abdominal region. This is usually a disease of developing countries because of poor managemental and feeding practices, affecting ruminants through loss of production and death. This condition is mostly

seen in old, pregnant and recently parturated animals. Ingestion and penetration of any sharp object from gastrointestinal tract to heart usually leads to development of this disease. Incidence of foreign body associated pericarditis higher in buffalo than in cattle (Misk et al., 2001). Among animals, dogs and horses are usually affected by idiopathic fibrinous pericarditis (Jesty et al., 2005). This is infrequently seen condition in buffalo especially Nili-Ravi buffalo (Summet et al., 2012).

CASE PRESENTATION

A 7 year old, Nili-Ravi buffalo was presented in comatosed position at Veterinary Teaching Hospital, Department of Clinical Medicine and Surgery (CMS), University of Agriculture Faisalabad Pakistan. The buffalo was from a dairy farm with 20 other milking animals. She had calved 25 days earlier without any problem. Anamnesis did not reveal any significant abnormality but history of complete anorexia, scanty feces and sudden drop in milk production for the last 5 days. She was treated by referring veterinarian with fluid therapy, antibiotics, analgesics, laxatives and ruminotonics but buffalo did not respond; the condition of the animal had deteriorated despite therapy. On clinical examination, buffalo had severe tachycardia, dyspnoea and mild brisket

AN UNUSUAL CASE OF IDEOPATHIC FIBRINOUS PERICARDITIS IN NILI-RAVI BUFFALO-A CASE REPORT

Department of Clinical Medicine and Surgery, Faculty of Veterinary Sciences, University of Agriculture Faisalabad, Punjab, Pakistan, *E-mail: [email protected]

Case Report

Muhammad Saqib, Ghazanfar Abbas* and Mudassar Niaz Mughal


300

edema. Temperature, respiration and pulse rate were 39.2 oC, 45 breaths/minute, 120 beats/minute, respectively. Before a diagnosis could be arrived, the animal died. Postmortem was conducted with aim of investigation of the cause of death.

NECROPSY FINDINGS

Thoracic and abdominal findings were insignificant except generalized edema most prominent in thoracic region, along with foul smelling straw colored fluid in pericardium having flakes of fibrinous material giving gelatinous and hirsute like appearance to heart especially epicardium and pericardium. Grossly there was no detectable lesion or spots on heart except markedly increased heart size and thickened pericardial wall. Reticulum was full of undigested fodder having four, 5 to 6 centimeter non penetrated metal wires along with six, 3 to 4 centimeter blunt ended nails.

Reticular wall was intact. No other lesions were detected in rest of the body.

DISCUSSION

The clinical signs and postmortem findings were suggestive of fibrinous pericarditis as described by Radostits et al., (2007). Grossly, heart and reticulum were normal, as there was not a single spot or hole on the outer surface. The underlying cause of pericarditis was not of traumatic origin because of absence of fibrous tract from reticulum to pericardium. Generally pericarditis has three forms, effusive, fibrinous and constrictive pericarditis. Accumulation of protein rich fluid in the pericardial sac is called as effusive pericarditis. If there is fibrin deposition along with protein rich fluid then it is termed as fibrinous pericarditis, while maturation of fibrin along with the fibrosis of pericardium is known as constrictive

Figure 1. Accumulation of straw colored fibrinous material in pericardium of affected buffalo.


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pericarditis (Pekins et al., 2004). In ruminants, incidence of fibrinous and septic pericarditis is more in contrast to idiopathic and non septic pericarditis. In ruminants mostly pericarditis is traumatic in origin, where a sharp object such as wire and nail penetrates into pericardium through perforation in reticulum. Postmortem examination of affected animal usually reveals foreign body penetrating into pericardium but sometime the penetrating object may fall back into reticulum after contacting pericardium, so traumatic pericarditis cannot be ignored in this case.Nonetheless, the heart was bereft of any healing or healed trauma. Haematogenous route associated pericarditis has been reported in colibacillosis, salmonellosis and some anaerobic infections. It is less common and is masked by signs of septicemia. Radostits et al. (2007) reported that physical penetration of pericardial sac is not essential for development of pericarditis as in some cases traumatic mediastinitis acts as a vehicle for the development of infection. In humans, presence of large quantity of fibrinous material in the pericardial sac is considered to be a diagnostic parameter for pericarditis (Lewinter and Kabanni, 2005).

Clinical signs, hematological analysis, radiography, pericardiocentesis, ultrasonography and postmortem findings are diagnostic tools for confirmation of pericarditis. Different control measures such as routine administration of magnets to pregnant animals, keeping ruminants away from construction areas, routine analysis of crops for metallic objects, effective feeding and managemental strategies such as screening of fodder and processed feed before serving to animals. This unusual case of idiopathic fibrinous pericarditis has not been reported in Nili-Ravi buffalo in Pakistan. Our case study is based on clinical signs and postmortem findings as described

elsewhere (Radostits et al., 2007; Sumeet et al., 2012). However, this report lacks microbiological and histopathological investigations that need to be investigated to understand the etiopathogensis of this condition.

REFERENCES

Grunder, H.D. 2002. Krankheiten des Herzens und des Herzbeutels, p. 159-181. In Dirksen, G., H.D. Grunder and M. stober (eds.) Innere Medizinund Chirurgie des Rindes, 4th ed. Parey Buchverlag. Berlin, Germany.

Jesty, S.A., R.W. Sweeney, B.A. Dolente and V.B. Reef. 2005. Idiopathic pericarditis and cardiac tamponade in two cows. J. Am. Vet. Med. Assoc., 226: 1555-1558.

LeWinter, M.M and S. Kabbani. 2005. Pericardial diseases, p. 1757-1780. In Zipes, D.P., P. Libby, R.O. Bonow and E. Braunwald (eds.) Braunwald’s Heart diseases. A Textbook of Cardiovascular Medicine, 7th ed. Saunders Elsevier, New York, USA.

Misk, N.A and M.A. Semieka. 2001. The radiographic appearance of reticular diaphragmatic herniation and traumatic pericarditis in buffalo and cattle. Vet. Radiol. Ultrasound., 42: 426-430.

Pekins, S.L., K.G. Maqdesia, W.P. Thomas and S.J. Spier. 2004. Pericarditis and pleuritis caused by corynebacterium pseudotuberculosis in a horse. J. Am. Vet. Med. Assoc., 224: 1133-1138.

Radostits, O.M., C.C. Gay, K.W. Hinchcliff and P.D. Constable. 2007. Veterinary Medicine. A Textbook of the Diseases of Cattle, Horse, Sheep, Pigs and Goats, 10th ed. Saunders Elsevier, PA. p. 189-391.


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Sumeet, S., G.S. Navjot and Varun. 2012. Ideopathic fibrinous pericarditis in a Nili-Ravi buffalo. Buffalo Bull., 31: 173-175.


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ABSTRACT

Anestrus is a functional disorder of reproductive cycle in cattle and buffalo which is characterized by absence of overt signs of estrus and also affecting the livestock enterprise to a great extent. Incidence of anestrus is more in buffalo than the cattle, and problem is severe during summer. The present study was carried out to see the efficacy of lugol’s iodine for the initiation of ovarian cyclicity in postpartum true anestrus buffaloes. Confirmation of true anestrus in 20 buffaloes was done by finding smooth ovaries at rectal examination, out of 20, 10 buffaloes was treated with Lugol’s iodine (1:50) ratio 30 ml I/U once only whereas, the remaining 10 buffaloes were serve as control for treated group and no treatment was given to such animals. The result for induction of estrus was 70% (7/10) and the conception rate was 85.71% (6/7). Lugol’s iodine treatment is cheaper and effective means of management of anestrus but response has been variable.

Keywords: anestrus, conception rate, infertility, ovarian-cyclicity

INTRODUCTION

Anoestrus is the major infertility problem in farm animals. It is important to note that anoestrus is abroad term, which indicates the lack of oestrus expression at an expected time. The meaning depends on age, weight, breed and history. Delay in expression of oestrus is beyond accepted average in anoestrus. It must be understood that a period of sexual quietness in animals is shown by complete absence of oestrus cycles. The incidence and management of the anoestrus have been recognised as age old problems in cattle breeding and there is wealth of documentation on various therapies to induce estrus in cow and buffaloes. The main native tract of Jaffarabadi buffaloes is Saurashtra region of Gujarat. These animals though very good milkers have a very high age at first calving and a long inter- calving period. Delayed resumption of postpartum estrous activity is a most vital factor responsible for poor reproductive efficiency of these animals. Various hormonal and non hormonal therapies have been been used for induction of estrus and fertility in anestrous bovines by various workers. Ovarian massage is known to stimulate the cyclicity in some anoestrous cases. It is proved that the cervical stimulation with lugol’s iodine

EFFECT OF LUGOL’S IODINE ON ESTRUS INDUCTION AND FERTILITY RESPONSE IN TRUE ANESTRUS JAFFRABADI BUFFALOES

A.R. Ahlawat1, P.U. Gajbhiye1, M.D. Odedra1, V.B. Dongre2 and S.N. Ghodasara1

1Cattle Breeding Farm, Junagadh, Agricultural University Junagadh (Gujarat), India, *E-mail: [email protected] of Animal Genetics and Breeding, College of Veterinary Science and Animal Husbandry, Junagadh Agricultural University, Junagadh (Gujarat), India

Original Article

mailto:[email protected]


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at very low concentration as paint or intrauterine infusion gives better response in inducing oestrus.

MATERIALS AND METHODS

The experiment was conducted at cattle breeding farm, Junagadh, Gujarat a total of 20 postpartum pluriparous Jaffrabadi buffalo were selected by rectal palpation. The age and the parity of the buffaloes ranged from 5 to 12 years. The animals in experiment had parities ranging from 2 to 6 The buffaloes which did not any signs of estrus 90 days postpartum were included in the study. The animals were divided in two groups of ten animals each, Group I (treatment group) were given Lugol’s iodine (1:50) 30 ml I/U once only. Whereas the animals of Group II control) animals served as control and no treatment was given. Beginning from the day of injection all the animal were observed for estrus twice a day at 7.00 am and 3.00 pm with a teaser bull. The buffaloes were observed for oestrus activity next day onwards a buffalo was said to be in standing estrus if it allowed the bull to mount. Any buffalo in standing estrus was inseminated with good quality semen

from the buffalo bulls stationed at farm. Artificial insemination was conducted 18 h after sign of estrus was clearly visible. The parameters measured were onset of estrus, duration of estrus, percentage of estrus and conception rate. Buffaloes were rectally palpated to confirm pregnancy 50 days after last AI. All the experimental animals were maintained as a group and were housed in semi open system. Each animal was fed with 30-35 kg of green fodder, 3-5 kg of concentrate (Amul dhan and Cotton seed cake). Management of these animals was nearly similar and they were released extensively during the day for free grazing.

RESULTS AND DISCUSSION

A total of seven animals (70%) out of ten animals injected in group I, responded to the treatment. While out of the control group three animals exhibited heat signs. Out of the seven animals bred six animals conceived with a conception rate of 85.71%. Out of the three animals bred (control group) one animal conceived (conception rate of 33.33%). Various workers have reported variable response ranging from (45%

Table 1. Efficacy of Lugol’s Iodine for induction of estrus in postpartum true anoestrus Jaffrabadi buffaloes.

Sl. No Attribute Group I (Treatment group)

Group II(Control)

1 Total No of animal treated 10 102 Response (animal induced) 7 33 Percentage response (%) 70 304 Animal bred 7/7 3/35 Animal conceived 6/7 1/36 Conception rate 85.71% 33.3%7 Average No of AI / conception 3 2.75


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to 91%) among cattle and buffaloes (Singh and Thakur 1999; Tomar 2004; Gupta et al., 2011). The period of postpartum anoestrus is usually longer in buffalo than the cattle under similar management conditions (Jainudeen and Hafez, 1993), probably due to low LH secretion during early postpartum period (Perera, 2011) It is presumed that painting of Lugol’s iodine on posterior part of the cervix causes local irritation and brings about reflux stimulation at anterior pituitary for secretion of gonadotrophins and consequently cyclicity. Lugol’s iodine is an irritating solution and intrauterine infusion of Lugol’s solution (0.5 to 1.0%) causes hyperemia (enhanced circulation) of uterine mucosa resulting into degree of iodine absorption from uterus. The absorbed iodine probably increases the metabolic rate of body through stimulating the thyroid hormone secretion (Sanchez, 1995). Increased metabolic rate trigger the ovarian functions by enhancing the energy utilization (El–Shahat and Badr, 2011). Injectable Lugol’s iodine has also been used with the same assumption (Sarkar, 2005). Anestrus is a multifactorial problem but its occurrence signals the inadequate nutrition, environmental stress, uterine pathology and improper managemental practices. The return of estrus in the three animals of control group may be explained due to correction in any one of the factors.

CONCLUSION

Anestrus is a multi-causative factors associated problem affecting livestock enterprise to a great extent. Diagnosis of the condition needs to be prompt and at the earliest to prevent its occurrence for effective treatment. Lugol’s iodine treatment is cheaper and effective means of management of

anestrus.The animals given Lugol’s iodine had a better response percentage, faster heat induction and better conception rate.

REFERENCES

El-Shahat, K.H. and A. Badr. 2011. Comparative Study on Efficacy of Different Medicaments on Postpartum Anestrus Dairy Cows. Journal of Applied Biological Sciences, 5(3): 59-63.

Gupta, R., M.S. Thakur and A. Sharma. 2011. Estrus induction and Fertility response in true anestrus buffaloes using lugol’s iodine. Veterinary World, 4(2): 77-78.

Jainudeen, M.R. and E.S.E. Hafez. 1966. Control of estrus and ovulation in cattle with orally active progestin and gonadotrophin. Int. J. Fertil., 11(1): 47.

Perera, B.M. 2011. Reproductive cycles of buffalo. Anim. Reprod. Sci., 124: 194-199.

Sanchez, J.M. 1995. Iodine in bovine nutrition. Nutr. Anim. Tropic., 2: 95-120.

Sarkar, A.K. 2005. Treatment of anestrus cow with diluted logul’s iodine and massage on reproductive oragans-uncontrolled case study. J. Anim. Vet. Adv., 4(8): 734-736.

Singh, S.L. and M.S. Thakur. 1999. Estrus synchronization and fertility with the uterine infusion of prostaglandin F2 alpha and lugol’s iodine in crossbred cows. Indian Journal of Animal Reproduction, 20(1): 12-14.

Tomar, D.S. 2004. Studies on etiology and treatment of subestrus in Murrah buffaloes. M.V.Sc and A.H., Thesis, JNKVV. Jabalpur, India.



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Original Article

ABSTRACT

The present study was carried out to investigate the haemato-biochemical profile of Mehshani buffalo, a native breed of Gujarat, India with the purpose of analyzing the physiological variations under the influence of different lactation stages in terms of determining possible biomarkers to monitor the energetic balance and the metabolic adequacy during lactation. Eighteen clinically healthy lactating buffaloes were categorized into three groups based on the length of their lactation: group I (early stage), group II (mid stage) and group III (late stage). Non significant variations were observed in case of the hematological parameters amongst the three groups of animals. The packed cell volume (PCV), RBC count and haemoglobin (Hb) concentration was lowest in the buffaloes of the early stage of lactation.

Other haematological parameters viz. total leucocyte count (TLC), differential leucocyte count (DLC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and platelet count were recorded to be within the normal limits. Similarly, the values of the blood biochemical analytes varied apparently, but the differences were statistically non significant amongst the groups studied. The glucose level

was recorded to be the lowest in the early stage of lactation; whereas, the protein and creatinine concentrations were slightly higher in this stage. No significant alteration in the concentration of Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and Alkaline phosphatase (ALP) was noticed amongst the three groups of buffaloes under the current study. Data generated during the current study may be useful as reference values for the scientific community as this is the first study of its kind in case of Mehshani buffalo.

Keywords: haemato-biochemical, lactation stage, mehshani buffalo

INTRODUCTION

Blood biochemical parameters vary during different physiological stages of animals (Ahmad et al., 2003). Pregnancy and lactation are two most important stages in the life of dairy animals, which affect metabolism resulting in the alteration of the haemato-biochemical profile (Krajnicakova et al., 2003; Iriadam, 2007). There are numerous reports on the effects of different phases of the reproductive cycle and pregnancy on haemato-biochemical indices in domestic animal species including

HAEMATO-BIOCHEMICAL ALTERATIONS DURING DIFFERENT STAGES OF LACTATION IN MEHSHANI BUFFALOES

Hemen Das1,*, A. Lateef2, H.H. Panchasara2 and M. Ayub Ali1

1Department of Physiology and Biochemistry, College of Veterinary Science and Animal Husbandry, Central Agricultural University, Selesih, Mizoram, India, *E-mail: [email protected] of Veterinary Science and Animal Husbandry, Sardarkrushinagar Dantiwada Agricultural University Sardarkrushinagar, Dantiwada, Gujarat, India


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buffalo (Jain et al., 2009). However, no such study could be traced investigating the blood picture during different stages of lactation in Mehshani buffalo, a unique milch breed of Gujarat, India. It is well established that milk and milk components are directly and indirectly synthesized from blood. The rate at which blood flows to the mammary gland is one of the key-factors in determining milk synthesis.

Approximately, 400 to 500 liters of blood circulate through mammary gland to produce one liter of milk (Fernandez and Hoeffler, 1998). There is a 2 to 6 folds increase in blood flow in the mammary gland starting 2 to 3 days prepartum. During lactation, the mammary gland secretory cells utilize 80% of the blood metabolites for milk synthesis depending on the infiltration of precursors of milk components like amino acids, glucose and fatty acids (Piccione et al., 2009). Hence, blood biochemical parameters including total protein, triglycerides, free fatty acids and urea are important indicators of the metabolic activity in lactating animals (Karapehlivan et al., 2007). Since the milk yield and composition varies across the length of lactation stage, it is, therefore, imperative, to study haematolo-biochemical constituents during different stages i.e. early, mid and late stage of lactation. Accordingly, the present study was undertaken to investigate the variations in haemato-biochemical profile during different stages of lactation in Mehshani buffalo.


Experimental animalsEighteen (18) clinically healthy lactating

Mehshani buffaloes were selected from the herd maintained at Livestock Research Station,

Sardarkrushinagar Dantiwada Agricultural University, Sadarkrushinagar, Gujarat, India. The buffaloes were in various stages of lactation and based on the length of their lactation, the animals were identified as in early (7 to 105 days), mid (106 to 210 days) and late (211 to 315 days) lactational stage. Accordingly, they were categorized into three different groups of six animals each viz. group I (early lactation), group II (mid lactation) and group III (late lactation).

Collection of blood samplesBlood samples were collected aseptically

from each animal of all the three groups by jugular vein puncture into collection tubes containing anticoagulants viz. K

3EDTA and Lithium heparin for hematological and biochemical analysis, respectively.

Haematological analysisCollected blood samples were analyzed

for different hematological parameters including packed cell volume (PCV), haemeoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), total erythrocyte count (TEC), total leucocyte count (TLC), differential leucocyte count (DLC) and platelet count (PLT) using Automated Haematology Analyzer (Cell-Dyn 3700, Abbott Diagnostics, USA).

Biochemical analysisBlood samples were analyzed for different

biochemical analytes viz. Glucose, Total protein, Blood Urea Nitrogen (BUN), Calcium (Ca), Creatinine, Total Bilirubin, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Asparate aminotransferas (AST) by employing Dry Chemistry discs/cartridges in Piccolo Xpress


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Chemistry Analyzer (Abaxis, USA).

Data analysisThe results were statistically analyzed using

one-way ANOVA as per the method of Snedecor and Cochran (1994). P<0.05 was considered to be statistically significant.


The results (mean±SE) of the biochemical and haematological analysis have been presented in Table 1 and Table 2, respectively.

Table 1 reveals no significant difference (P>0.05) in the concentrations of various biochemical constituents amongst the three groups of lactating Mehshani buffaloes. This finding corroborates the report of Hagawane et al. (2012). Present study further indicated that the mean concentration of blood glucose was lowest (40.67±2.04 mgdl-1) in early stage and increased subsequently as the lactation advances. The observed values of blood glucose for mid and late stage of lactation were 42.5±4.57 mgdl-1 and 46.37±4.31 mgdl-1, respectively. Current trend of variations are consistent with earlier report in lactating ewes (Roubies et al., 2006) and in lactating mares (Heidler et al., 2002). In contrast, glucose levels were reported to be the same throughout the three stages of lactation by Peterson and Waldern (1981); whereas, Doornenbal et al. (1988) reported somewhat higher (P<0.05) glucose concentration at parturition that declined during lactation period. The lower level of blood glucose recorded during early stage of lactation may be ascribed to the utilization of large amount of blood glucose by mammary gland for the synthesis of lactose (Schultz, 1968). It is reported

that lactose synthesis and milk yield show a linear positive correlation with glucose uptake and thus the lactose synthesis potential is accompanied by greater glucose uptake by lactating mammary gland (Afshar and Fathi, 2012). The total protein level (8.45 gdl-1) was found to be slightly higher in group I as compared to group II and group III animals. This observation is on the contrary to the finding of Yaylak et al. (2009), who recorded lower protein values in dry and early stages of lactation in case of Holstein cows. Krajnicakova et al. (2003) also observed an increasing trend of total protein level of serum with the progress of lactation in lactating goats and concluded that this is due to the catabolism of protein for milk synthesis. The variation may be attributed to the differences in species, nutrition, husbandry, environment and methods of assay (Beaunoyer, 1992; Osman and Al-Busadah, 2003). However, Hagawane et al. (2012) reported highest protein value in the early stage of lactation, which is comparable to current findings. The possible explanation for this phenomenon may be the haemoconcntration and water losses due to parturition. Further, earlier investigations have clearly shown that the expression of major milk proteins increases dramatically and in a concerted way during the onset of lactation (Bionaz and Loor, 2011).

Similarly, the mean value of blood urea nitrogen (BUN) was also recorded to be higher in initial stage of lactation and decreased as the lactation progresses. This may hold good in relation to observed apparently increased level of total protein. The BUN values observed in the present study at different stages of lactation were higher than those reported in earlier investigation (Hagwane et al., 2012). Reinartz and Hofmann (1989) also found that serum urea concentration was significantly influenced by the lactation stage.


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Table 1. Mean±S.E. values of biochemical analytes at different stages of lactation.

Parameters Early lactation Mid lactation Late lactation Glucose (mg/dl) 40.67±2.04 42.5±4.57 46.37±4.31 Total protein (g/dl) 8.45±0.13 8.12±0.22 8.01±0.29 Blood Urea Nitrogen (mg/dl) 22.67±1.3 20.5±1.58 18.89±2.25Calcium (mg/dl) 7.0±0.93 8.1±0.54 8.19±0.23 Creatinine (mg/dl) 1.33±0.08 1.2±0.07 1.15±0.16 Total Bilirubin (mg/dl) 0.23±0.03 0.22±0.04 0.24±0.04 Alanine aminotransferase (ALT) (U/L) 66.67 ±5.35 72.67±6.37 67.5±5.07 Aspartate aminotransferase (AST) (U/L) 154.0±4.46 148.5±17.97 159.5±7.87 Alkaline phosphatase (ALP) (U/L) 178.67±81.50 168.67±47.03 165.56±45.50

(P<0.05; statistically non significant)

Table 2. Mean±S.E. values of hematological indices at different stages of lactation.

Parameters Early lactation Mid lactation Late lactationWBC (K/µl) 9.63±0.65 8.24±0.79 9.55± 0.98

NEU (%) 30.83±2.19 28.28±1.58 29.12±2.01 LYM (%) 50.17±2.02 48.35±1.69 50.15±2.29

MONO (%) 7.17±0.40 7.18±1.03 8.95±1.96 EOS (%) 5.83±1.49 6.44±0.97 5.36±0.57

BASO (%) 0.841±0.15 0.746±0.16 0.644±0.17 RBC (M/µl) 5.67±0.98 6.38±0.35 7.49±0.41 HGB (g/dl) 12.35±4.48 14.1±4.17 13.73±2.84 PCV (%) 30.87±0.97 32.72±1.87 31.33±1.35 MCV (fL) 48.07±2.021 45.22±2.54 41.93±0.87 MCH (pg) 16.22±0.69 15.48±0.80 15.17±0.58 PLT (K/µl) 326.5±33.63 617.17±104.86 507.17±51.62

(P< 0.05; statistically non significant)


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It is recorded that the efficiency for utilization of metabolisable protein for milk production (0.68) is less than that of maintenance (1.00) (McDonald et al., 1995). So, as the milk production increases, the overall protein utilization efficiency decreases, which consequently leads to more drainage of nitrogen in terms of urea through urine and milk (Roy et al., 2003). An increase in urea value was further observed in the first 8 weeks of lactation (Ndibualonji and Godeau, 1993) and found to be peak at 12 weeks postpartum, which decreased slowly thereafter (Rajcevic et al., 1993). However, other researchers found a different trend of variation in case of BUN. During the first month of lactation lower milk urea (MU) concentration was recorded by Carlsson et al. (1995). Likewise, Whitaker et al. (1995) also reported that cows in early lactation often have much lower MU level. In contrast, no relation was reported between urea concentration in milk and stage of lactation by Erbersdobler et al. (1990) and values were relatively constant between 200 to 300 mg/l. Similarly, Coustumier (1996) also found no correlation between lactation stage and urea levels except just after calving. Similar to our study, Schepers and Meijer (1998) also observed that stage of lactation had no significant influence on BUN and thus on MU concentration. Hence, in the light of varying observations of different researchers, a systemic and critical investigation may be established in this aspect.

In this study, the drop in calcium (Ca) level (7.0±0.93 mgdl-1) was more pronounced during early stage of lactation as compared to mid and late stage. This may be due to excessive drainage of blood calcium pool through colostrum and milk during this stage. As the stage of lactation progresses, the blood calcium level is increased, which is in agreement with the findings of Rowlands et al, (1975) and Nale (2003). It

may be hypothesized that the buffaloes gradually recover from the stress of parturition and excess demand of calcium for initiation of lactation. On the contrary, Ramakrishna (1991) recorded higher values (9.77±0.33 mgdl-1) of calcium in lactating buffaloes. Further, Yokus et al. (2004) also concluded that the levels of Ca decreased slightly in early pregnancy to late pregnancy and then increased during lactation period in sheep.

Present study also indicated that the blood creatinine level was higher in group I as compared to group II and group III buffaloes. The apparent increase in creatinine level at the early stage of lactation may be ascribed to uterine involution and myometrial protein degradation (Bell et al., 2000). Nonetheless, Peterson and Waldern (1981) found no differences in creatinine concentrations amongst the various stages of lactation, but observed that creatinine levels rose in dry cows with increasing days of pregnancy. Kronfeld (1982), working with 21 Holstein herds, reported the highest serum creatinine levels during the peak of lactation. Total bilirubin concentration recorded in this study was found to be consistent throughout the lactation period indicating that its concentration remains unaffected with the stage of lactation. Similarly, no significant alteration in the concentration of Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and Alkaline phosphatase (ALP) was recorded amongst the three groups of buffaloes under the current study. The concentration of AST was found to be highest (72.67±6.37 U/L) in the mid lactation stage. Ling et al. (2003) observed that the blood concentration of AST increases between day 117 and 151 of lactation (mid stage) in Holstein mares, which is in accordance with the present findings. Conversely, Yaylak et al. (2009) reported that the stage of lactation affects AST and ALT activities


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significantly. An increase in ALT, AST and GGT (γ-glutamyl transferase) activity in the blood of ewes during lactation is indicative of increase in hepatic metabolism (Antunovic et al., 2004, 2011). Further, AST and ALP are considered to be effective biomarkers to detect the energetic and mineral imbalance in Saanen dairy goats (Mundim et al., 2007). Changes in activities of these enzymes may also be related to reduced dry matter intake around parturition, which lead to hepatic lipidosis and alter the normal function of the liver (Greenfield et al., 2000). However, no indications were found in the literature to explain the relationship of the recorded trends of variations in the concentrations of these enzymes with different stages of lactation.

Table 2 indicated that although the observed values of the haematological parameters varied apparently, the differences were statistically non significant. Similar types of observations were also recorded by Hagawane et al. (2012). The packed cell volume (PCV), RBC count and haemoglobin (Hb) concentration was found to be lowest in Mehshani buffaloes in early stage of lactation corroborating with those of Esievo and Moore (1979), who concluded that the concentrations of PCV RBC, Hb along with serum iron (SI), iron binding capacity (IBC) and serum albumin decreased in early lactation and rose to pre-lactation levels by mid-lactation. Decline in the number of RBC in the blood of ewes in the early lactation was also reported by Antunovic et al. (2011). Other haematological indices such as the TLC, DLC, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and platelet count were found to be within the limits of normal values laid for the buffaloes. Similar to current investigation, non significant differences in various haematological indices were also reported by Flores et al. (1990) during early and late stage

of lactation.It may be concluded that stage of lactation

does not play significant role in alteration of haemato-biochemical profile in lactating Mehshani buffaloes. The limited sensitivity of these blood parameters to stage of lactation in clinically normal dairy animals is not surprising because, most of these parameters are under the homeostatic control systems (Cozzi et al., 2011). Nonetheless, data generated during the current study may be useful as reference values for the scientific community as this is the first study of its kind in case of Mehshani buffalo. Further, blood profile has traditionally been used to assess the metabolic health status of the animals; hence the present investigation may also be helpful in this regard. In addition, this study may also assist the nutritionists to formulate ration for optimum productivity of the Mehshani buffaloes since blood-biochemical analytes are being widely considered to identify dietary causes of diseases leading to low productivity.

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Original Article

ABSTRACT

The present study was carried out to examine the effect of GnRH and PGF2α administration during early post partum period (PP) on ovarian activities and initiation of PP estrus in Murrah buffaloes. A total of thirty six Murrah buffaloes were selected and divided equally into three groups comprising twelve buffaloes each. Buffaloes in Group I were i.m. injected with GnRH as busreline acetate ( 10 µg) while those in group II were i.m. injected with PGF2α as tiaprost-trometamol (0.750 mg) on day 14 PP.

The buffaloes in Group III were kept as control. The experimental buffaloes were examined with 7.5 transrectal probe on day 14, 21 and 28 PP. On day 14 PP, 14 (38.88%) ovaries showed small follicular development and 4 (11.11%) ones showed multiple follicular activity when observed ultrasonographically. On day 21 post-partum, 4 (11.11%) ovaries showed small follicular development, 11 (30.50%) ovaries showed multiple follicular activity and 18 (50.00%) ones indicated good follicular development when observed ultrsonographically. On day 28 PP, 3 (9.37%) ovaries showed small follicular development and 3 (9.37%) ones showed multiple follicular activities whereas 26 (81.25%) ones indicated good follicular development when observed ultrasonographically. The post partum ovarian activity was initiated with average 3.5±0.150, 3.25±0.130 and 4±0.467

weeks in Group I, II and III, respectively. From the present study it can be concluded that PGF2a administration during early PP hastens the initiation of post-partum estrus in buffaloes and transrectal ultrasonography is good tool for monitoring the slight follicular developments.

Keywords: GnRH, PGF2α, ovarian activity, post-partum estrus

INTRODUCTION

The buffalo has important role in livestock economy of Asia including India. Buffaloes are valued for milk, meat and draught power. Hence the importance of buffaloes to the economy of this country is considerable and cannot be underestimated (Madan, 2010). Low reproductive efficiency in general and buffaloes in particular remains a major economic problem globally, and its incidence is higher (4.66 to 12.66%) in our country (Tomar and Ram, 1993). Failure to resume ovarian activity after calving is the main reason for delay in conception in buffaloes (Parmar et al., 2012). Early post-partum (PP) breeding to reduce the calving interval in buffaloes would increase their reproductive efficiency (Shah et al., 2002). Thus PP period is regarded as an important in the reproductive life of bovines (Fonesca et al., 1983). The low reproductive efficiency of buffaloes, as

EFFECT OF EARLY POST-PARTUM GNRH AND PGF2 ALPHA ADMINISTRATION ON FOLLICULAR ACTIVITIES IN MURRAH BUFFALOES

M.V. Ingawale* and S.A. Bakshi

Department of Animal Reproduction, PGIVAS, Akola (MS) India, *E-mail: [email protected]


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evident from delayed first PP estrus and conception, prolonged service period resulting in extended calving interval (Khasatiya et al., 2006).

Ovarian follicular growth resumes early after calving with the formation of first dominant follicle, detected by ultrasonographcally within 10.12±72 in Bulgarian Murrah buffaloes (Yotov and Atanasov, 2013). Although uterine involution begins and ovarian follicular waves resumes soon after parturition due to rise in FSH concentration (Schallenberger, 1985). However, dominant follicle of these waves fails to ovulate due to failure to undergo final terminal maturation. Failure of PP dominant follicles to undergo final maturation is due to inadequate LH pulse frequency, which result in low foolicular androgen production (Fortune, 1986) and inadequate oestrodiol positive feedback to induce LH surge (Peters et al., 1985), which is perquisite for follicular terminal maturation prior to ovulation. Absence of LH pulses in early post-partum is primarily due to depletion of anterior pituitary LH stores. Following replenishment of LH stores between days 15 and 30 PP absence of LH pulses is due to continued sensitivity of the hypothalamic GnRH pulse generator to the negative feedback effect of estradiol-17b which results in absence of GnRH pulses. The administration of GnRH will therefore overcome the inadequate secretion of pituitary LH in early post-partum period (Shah et al., 1990) and restore ovarian function earlier within PP period.

Prostaglandins plays major role in regulation of reproductive cyclicity (Singh and Madan, 1985). The reproductive cyclicity and its rhythm in terms of its reawakening during early PP period has been linked to temporal changes of prostaglandins in particular (Perera et al., 1981). Lindell et al. (1980) reported that prostaglandin metabolites increased at the time of parturition

and remained high for 8 to 16 days PP. So delay in involution of uterus was due to short period of high prostaglandin F2 alpha metabolite release whereas, long duration of PGF2α release resulted in short period for completion of uterine involution (Lindell, 1981). It has also positive effect on the uterine musculature tone (Lindell and Kindahl, 1983). So PGF2α injection in early PP period (day 14) enhances the uterine involution and reproductive efficiency in normal calved buffaloes (Nazir et al., 1994). This is promoted us to study the follicular dynamics and initiation of PP ovarian activity ultrasonographically and per-rectally following 14 PP injection with GnRH and PGF2α in normally calved buffaloes.


Experimental animalsThe present research work was carried out

using 36 post-partum (PP) Murrah buffaloes at M/S B.G. Chitale Dairy, Research and Development Farm, Bhilawadi in Sangli district, over a period of ten months. The buffaloes were housed in a loose housing barn with four groups of twenty-four buffaloes. The buffaloes were kept indoors and there was no open paddock in the barn. Each lot had twenty-six resting places (1.2x2m) on one side and a manure alley with Delta MasterTM manure scraper (Delaval AB, Sweden) on the other hand side positioned towards the feed rack. Each lot had one automatic concentrate feeding station (AFS) and nine valve-controlled automatic water bowls. The ordinary routine in the barn was adlib feeding of roughages three times a day. The roughages fed during the experiment consisted of fresh, cut and chopped sugercane, alfalfa, napier grass, green maize and jowar straw which were chopped


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and transported to the barn in tractor trolley and dispensed manually into the feed troughs. A pre-calculated quantity of concentrate mixture was fed to each buffalo based on milk yield, body weight and pregnancy status. Concentrate was fed through the automatic concentrate feeding station (AFS) in the barn. If the pre calculated amount was not consumed, the residual was transferred to the next feeding. Residual amounts at the end of a 24 h period were transferred to the next 24 h period. During milking, an in-parlor feeding (IPF) system supplied a fixed amount of concentrates. The buffaloes were provided mineral mixture according to milk production and body weight of the buffaloes.

All the buffaloes were appropriately vaccinated against foot and mouth disease and haemorrhagic septicemia. They were also tested annually to detect possibilities of Brucellosis, Johne’s disease and Tuberculosis and the positive reactors were suitably disposed off. The fecal samples and blood smears were also screened periodically for detection of parasitic infestations and protozoan parasites, respectively. As a routine, all buffaloes were dewormed biannually.

Experimental design Total 36 Murrah buffaloes with second to seventh lactation were selected. These buffaloes were divided into three groups comprising twelve bufaloes and following treatments were given.

Group I Buffaloes were intramuscularly injected with 10 µg Busereline acetate (Intervet, India) on day 14 PP. Group II Buffaloes were intramuscularly injected with 0.750 Tiaprost trometamol (Intervet, India) on day 14 PP. Group III Buffaloes were kept untreated

as control group. All the experimental were observed for

ovarian activity on days 14, 21 and 28 PP per-rectally and ultrasonographically. The ovarian follicular development, corpus luteum development and regression were monitored using real time, B-mode ultrasonograhy machine (Aloka-900) with 7.5 MHz linear array rectal transducer. The follicles appear as black anechoic, roughly circumscribed areas surrounded by hyperechoic ovarian stroma on the ultrasound image. The follicles observed ultrasonographically in the present study were classified according to SFD (slight follicular development): F<0.5cm or follicles as small as 3-4 mm, but few (<5) in number. MSF (Multiple small follicles): F<0.5 cm or follicles as small as 3-4 mm but many (>5) in number. GFD (Good follicular development): Bigger follicles >0.8cm and or oestrual follicles. NFD (No follicular development): Absence of the above picture within ovarian stroma.

The data was analyzed by employing statistical design as recommended by Snedecor and Cochran (1994).


The ultrasound imaging of ovaries in 36 PP buffaloes revealed ovarian activity with respect to follicular growth right from the first day of examination (day 14 PP). The ovaries were characterized by growth and regression of several small (up to 5 mm) and medium sized (>5 and <10 mm in diameter) follicles until the detection of first PP dominant (≥10 mm) and/or ovulatory follicle during the study period.


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Follicular activities A total 424 observations on ovaries were carried out by per-rectally and ultrasonographically. The manual observations were compared with the ultrasonographical observations to study the comparative detectability of ovarian follicles by palpation per-rectum and by ultrasonography. The overall observations during different periods for both the methods are presented in Table (1).

According to Table (1), 6 (16.16%) ovaries indicated small follicular development when observed per-rectally while 14 (38.88%) ovaries showed small follicular development and 4 (11.11%) ovaries showed multiple follicular activity when observed ultrasonographically on day 14 PP. Moreover, two (5.55%) ovaries showed good follicular activity when observed ultrasonographically on day 14 PP.

On day 21 PP, 15 (41.66%) ovaries indicated small follicular development when observed per-rectally while 4 (11.11%) ovaries showed small follicular development and 11 (30.50%)

ovaries showed multiple follicular activity when observed ultrasonographically. Thirteen (36.11%) ovaries indicated good follicular development when observed per-rectally while 18 (50.00%) ovaries indicated good follicular development when observed ultrsonographically. On day 28 PP, 6 (16.16%) ovaries indicated small follicular development observed per-rectally while 3 (9.37%) ovaries showed small follicular development and 3 (9.37%) ovaries showed multiple follicular activity when noticed ultrasonographically. Furthermore, 26 (81.25%) ovaries indicated good follicular development observed per-rectally while 26 (81.25%) ovaries indicated good follicular development when observed ultrasonographically.

The above observations indicate that small follicular development and good follicular development is felt per-rectally, in all the ovaries. Follicular growth (small as well as good) is also noted on ultrasonographic examination in all ovaries. Besides that the follicular growth which was not palpated per-rectally was detected by

Table 1. Ovarian activity monitored by per-rectally and ultrasonographically of Murrah buffaloes following 14 days PP GnRH and PGF2α administration.

GroupsFollicular

Development

Day 14 PP Day 21 PP Day 28 PP

Per- rectally

Ultraono-graphically

Per- rectally

Ultraono-graphically

Per- rectally

Utrasono-graphically

Group-ISFD 3 5 6 1 0 0MSF - 2 - 5 - 0GFD 0 1 5 6 11 11

Group-IISFD 2 5 6 2 0 0MSF - 2 - 4 - 0GFD 0 1 4 6 9 9

Group-IIISFD 1 4 3 1 6 3MSF - 0 - 2 - 3GFD 0 0 4 6 6 6


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ultrasonographic examination. This suggested that rectal examination is a reasonably fair indicator to judge the good follicular development. But ultrasound is better tool for classification of ovarian follicles depending upon its diameter. With the help of ultrasonographical examination it is possible to diagnose fairly good amount of follicular activity which otherwise would have missed on rectal palpation of the ovaries.

It is seen in the present study that higher percentage of follicles have missed or wrongly diagnosed by rectal palpation. The preference of ultrasonography method to compare with rectal palpation in current study is agreed with Pieterse (1990) who compared between trans-vaginal ultrasound and rectal palpation methods for diagnosis the ovaries in bovines. It is observed that ultrasonography permits a better estimation of number and size of the follicles, being similarly with observations in cows Hanzen et al. (2000).

The current observations that by manual diagnosis of smaller follicles was not detected and diagnose of follicles (<5mm) size was more accurate. The ultrasonographic appearance of ovarian structures in the present study was in line with those described by Honparkhe et al. (2003) and Lohan et al. (2004) in buffaloes. Ovarian activity is noted on day 14 post-partum by ultrasonography more effectively than by rectal examination.

It can be concluded that rectal palpation is fairly good technique to diagnose good follicular

activity or prominent follicular growth, but it is possible that slight follicular development or deeply situated follicles could be missed. Thus, rectal palpation is a fairly good indicator of ovarian activity only for routine observations. Ultrasonography should be considered as a tool when daily/frequent monitoring of follicular activity is required. Thus ultrasonographical ovarian scanning should be considered especially in the research techniques and therapeutic purposes.

Initiation of post-partum ovarian activityThe week for initiation of ovarian activity

was noted in the buffaloes from all groups (Table 2).

It was that PP ovarian activity was initiated earliest (P<0.01) in the PGF2a treated buffaloes (Group II) with the average of 3.25 weeks, followed by GnRH treated buffaloes (Group I) and control group (Group III) with averages of 3.50 and 4 weeks, respectively.

Observations regarding the initiation of ovarian activity in the present study was corroborated with those reported by Iqbal et al. (2003) who observed initiation follicular development at days 21.20±5.71 after PGF2a treatment as compared with days 28.20±8.75 in control Nili-Ravi buffaloes. Lohan et al. (2004) observed large follicle>8.5 mm in 75% buffaloes and increases to more than 8.5 mm between day 14-33 PP. Chaudhary et al. (1989) noticed that

Table 2. Average week of initiation of post-partum ovarian activity for all groups in Murrah buffaloes.

ParticularsGroup I(GnRH)

Group II(PGF2a)

Group III(Control)

Average 3.5±0.150 3.25±0.130 4±0.467**

** Significant at P<0.01


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interval from calving to detection of first palpable follicle was averaged 27.43±1.24 days. Sheldon et al. (2000) showed >8 mm between days 14-28 day PP. Concomitant with present study, Bekana et al. (1994) observed the resumption of cyclical ovarian activity within a month in seven animals by rectal palpation as well as on ultrasound. More days required for initiation of follicular activity than present findings were reported by Baruselli (1991), Usmani (1992) and Shah (1999) in buffaloes.

The early return to active follicular development in PP buffaloes, and the fact that some buffaloes ovulated between days 15 and 20 PP, demonstrated the ability of the ovary to resume early PP activity. It suggested that ovarian responsiveness may not be the major reason for the variable duration of the PP anestrus period commonly observed in buffaloes. Usmani et al. (1985) recorded formation of first CL on day 23.8±1.7 after calving as indicated by plasma progesterone level in buffaloes. Arya and Madan, (2001) observed 19.67±3.23 and 19.17±4.53 days for first ovulation in suckled and non-suckled Murrah buffaloes. The small (8.10±5.67) and large number (1.00±0.00) of follicles was detected day six post-partum in buffaloes (Lohan et al., 2004).

The present finding of initiation of PP ovarian activity at comparatively earlier days might be due to careful monitoring of ovarian activity through ultrasound scanning of ovaries, good feeding, health and management practices of the farm as well as due to the PGF2 alpha treatment in early PP period.

Determination of ovulation by rectal palpation during earlier days after calving is tedious and it may be easily missed. Moreover, most of these studies were based on palpation of corpora lutea formed following ovulation. It is accepted that CNS requires prior exposure to progesterone

to elicit behavioral signs of estrus (Noakes et al., 2001). Behavioural signs of estrus did not accompany the first PP ovulation in majority of buffaloes. This finding agreed with the observation reported by Savio et al. (1990) who showed that first PP ovulation occurred without overt estrus behavior in 17 out of 18 dairy cows. Thus, the sign of first estrus is not a true reflection of onset of ovarian activity.

CONCLUSION

It can be concluded that the slight follicular developments is easily detectable through transrectal ultrasonography being a good tool for monitoring of follicular activity. The PGF2a administration during early PP hastens the initiation of estrus in Murrah buffaloes.

REFERENCES

Aboul-Ela, M.B. and A.H. Barkawai. 1988. Pulsatile secretion of LH in cycling buffalo heifers affected by season and stage of the oestrus cycle. In The 11th International Congress on Animal Reproduction and Artificial Insemination in Dublin, Ireland.

Arya, J.S. and M.L. Madan. 2001. Post-partum reproductive cyclicity based on ovarian steroids in suckled and weaned buffaloes. Buffalo J., 3: 361-369.

Baruselli, P.S. 1991. Postpartum ovarian activity and reproductive performance in buffaloes. Anais 9th Congresso Brasileiro de Reproduces Animal Belo Horizonge Brazil, Brazil.

Bekana, M.T. and H. Kindhal. 1994. Ultasonography


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of the bovine post-partum uterus with retained fetal membranes. Zbl. Vet. Med. A., 41: 653-662.

Chaudhary, M.A., M. Ahmed and R.A. Chaudhary. 1989. Postpartum ovarian functions and oestrus interval in Nili-Ravi buffaloes. Pak. Vet. J., 9(4): 155-158.

Fonesca, F.A., J.H. Britt, B.T. Mcdainel, A.H. Rakes and J.C. Wilk. 1983. Reproduction traits in Holland and Jersey. Effect of age, milk yield and clinical abnormalities on involution of cervix and uterus, ovulation, estrus cycle, detection of estrus, CR and days open. J. Dairy Sci., 66: 1128-1147.

Fortune, J.E. 1986. Bovine theca and granulose cells interact to promote androgen production. Biol. Reprod., 35: 292-299.

Hanzen, C., M. Pieterse, O. Scenzi and M. Drost. 2000. Relative accuracy of the identification of ovarian structures in cows by ultrasonography and palpation per-rectum. Vet. J., 159: 161-170.

Honparkhe, M., V.K. Gandotra, A.S. Nanda and S. Prabhkar. 2003. A comparison of rectal palpation and ultrasonography for detection of follicles and corpus luteum in pluriparous buffaloes. Ind. J. Anim. Repro., 24: 149-151.

Iqbal, S., M. Aleem and M.A. Sayeed. 2003. Role of single injection of prostaglandin F2 alpha on breeding efficiency of post-partum buffaloes. Pak. Vet. J., 23(4): 197-201.

Khasatiya, C.T., F.S. Kavani, A.J. Dhami, H.J. Derashri, M.T. Panchal and P.M. Desai. 2006. Studies on puerperal events and reproductive efficiency following hormonal therapy at day 42 post-partum in Surti buffaloes. International Journal Agriculture and Biology, 8(1): 34-41.

Lindell, J.O. 1981. Studies on induced abortion,

parturition and uterine involution in bovine. Veterinary Bull., 051: 064414.

Lindell, J.O. and H. Kindahl. 1983. Exogenous prostaglandin F2 alpha promotes uterine involution in the cows. Acta Veternaia Scandinavica, 24(3): 269-274.

Lindell, J.O., M. Kindahl and L.E. Edquist. 1980. Uterine involution in relation to post partum release of PGF2 alpha in cows. Proceeding of 9th International Congress of Animal Reproduction and AI . III symposia, Medrid, Spain.

Lohan, I.S., R.K. Malik and M.L. Kaker. 2004. Uterine involution and ovarian follicular growth during early postpartum period of Murrah buffaloes (Bubalus bubalis). Asian Austral. J. Anim., 17(3): 313-316.

Madan, M.L. 2010. Concern and conflicts in buffalo production. In Proceedings of International Buffalo Conference. New Delhi, India.

Nazir, F., R.A. Chaudhry, T. Rahil and K.R. Chohan. 1994. Effect of prostaglandin F2 alpha at early postpartum on uterine involution and subsequent reproductive performance in suckled Nili-Ravi buffaloes. Buffalo J., 10(3): 269-271.

Noakes, D.E., T.J. Parkinsons, G.C.W. England and G.H. Arthur. 2001. Arthur’s Veterinary Reproduction and Obstetrics, 1st ed. W.B. Saunders Company, Pennsylvania.

Parmar, K.H., R.G. Shah, P.H. Tank and A.J. Dhami. 2012. Strategies for improving reproductive efficiency of postpartum anestrus Surti buffaloes. Ind. J. Anim. Repro., 33(1): 47-50.

Perera, B.M.A.O., H. Abeygumawardona, A. Thamotharm, H. Kindahl and L.E. Edquist. 1981. Peripheral changes of oestrogen, progesterone and prostaglandin in water


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buffaloes. Theriogenology, 15: 463-467.Peters, A.R., M.G. Pimental and G.E. Lamming.

1985. Hormone response to exogenous GnRH pulses in post-partum dairy cows. J. Reprod. Fertil., 75: 557-565.

Pieterse, M.C., O. Szenci, A.H. Willemse, C.S.A. Bajcsy, S.J. Dieleman and M.A.M. Taverne. 1990. Early pregnancy diagnosis in cattle by means of linear array real-time ultrasound scanning of uterus and a qualitative and quantitative milk progesterone test. Theriogenology, 33: 697-707.

Savio, J.D., M.P. Boland, N. Hynest and J.F. Roche. 1990. Resumption of follicular activity in the early postpartum period of dairy cows. J. Repro. Fertil., 88: 569-579.

Schallenberger, E. 1985. Gonadotropin and ovarian steroids in cattle. III Pulsatile changes of gonadotropin concentration in jugular vein post-partum. Acta Endocrinologica, 109: 37-43.

Shah, R.G. 1999. Hormonal and biochemical profile in fertile and infertile postpartum Surti buffaloes. Ph.D. Thesis, Gujarat Agricultural University, Anand, India.

Shah, S.N.H, A.H. Willemase and D.F.M. Van-de-wiel. 1990. Reproductive performance of Nili-Ravi buffaloes after single injection of GnRH early post-partum. Trop. Anim. Health Pro., 22(4): 239-246.

Shah, R.G., V.B. Kharadi, A.J. Dhami, P.M. Desai and F.S. Kavani. 2002. Effect of gonoadotrophin releasing hormone on reproducive performance and steriod profile of postpartum suckled Surti buffaloes. Indian J. Anim. Sci., 72(12): 1076-1082.

Sheldon, I.M., D.E. Noakes and T.M. Dobson.2000. The influence of ovarian activity and uterine involution determined by ultrasonography

on subsequent reproductive performances of dairy cows. Theriogenology, 54: 409-419.

Singh, M. and M.L. Madan. 1985. Circulating PGF2a during oestrus cycle among buffaloes. Proceddings of the 1st World Buffalo Congress, Cario Egypt. 8: 569-571.

Snedecor, G.W. and U.G. Cochran. 1994. Statistical Methods, 8th ed. Affiliated East West Press. p. 491.

Tomar, S.S. and R.C. Ram. 1993. Factors affecting replacement rule and its comparision in a herd of Murrah buffaloes. Indian Journal of Diary Science, 48: 340-342.

Usmani, R.H. 1992. Effect of past gravid uterine horn on the pattern of resumption of ovarian functions in postpartum Nili-Ravi buffaloes. Buffalo J., 8(3): 265-270.

Usmani, R.H., M. Ahmed, E.K. Inskeep, R.A. Dailey, P.E. Lewis and G.S. Lewis. 1985. The uterine involution and postpartum ovarian activity in Nili-Ravi buffaloes. Theriogenology, 24: 435-448.

Yotov, S.A. and A.S. Atanasov. 2013. Ultrasonographic determination of follicle development and resumption of ovarian activity in post-partum Bulgerian Murrah buffaloes during breeding season. Animal and Veterinary Science, 1(5): 36-41.


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Original Article

ABSTRACT

Ruminal impaction was studied in buffaloes in and around Jammu region the majority of cases were of animals having ingested fibrous feed material, coarse grain, polythene and jute bags and ropes and nonpenetrating metallic objects. Prominent clinical signs noticed were complete cessation of rumination, impaction and atony of rumen, hardening and pelleted mucous coated dung, and inappetance to anorexia. Haematological alterations revealed reversal of neutrophil to lymphocyte ratio. The diseased buffaloes had significantly higher bilirubin, aspartate aminotransferase, glucose, blood urea nitrogen and creatinine, levels and significantly lower calcium, than the control values. The levels of alkaline phosphatase, total protein, albumin, globulin and phosphorus did not differ significantly from the respective control values.

Keywords: buffalo, haematology, biochemical, rumen impaction

INTRODUCTION

Rumen impaction mainly occurs due to feeding of poor quality hay, straw or roughages deficient in protein and readily digestible carbohydrate, overeating of young grasses, ingestion of mouldy or decomposed feed, polythene bags, ropes and other plastic materials, and exposure to hot and dry weather conditions (Radostits et al., 2010). The clinical signs include decreased rumen motility or rumen atony, abdominal distension, anorexia, constipated feces, occasional diarrhoea, normal to increased temperature, increased pulse rate, hard consistency of rumen and solid mass on left side on per rectal examination (Nwity and Chaudhary, 1995). Present study reports the clinical, hematobiochemical alterations and therapeutic studies in various cases of ruminal impaction in buffaloes.


The study was conducted on twenty buffaloes suffering from ruminal impaction, presented at Division of Veterinary Clinic and

CLINICAL, HEMATO-BIOCHEMICAL AND THERAPEUTIC STUDIES ON RUMEN IMPACTION IN BUFFALOES

A.K. Tripathi1, J.S. Soodan2 and R.B. Kushwaha2

1Department of Clinical Medicine, College of Veterinary and Animal Sciences, UP Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan (DUVASU), Mathura, Uttar Pradesh, India, E-mail: [email protected] Division of Veterinary Clinic and Teaching Hospital, Faculty of Veterinary Sciences and Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu (SKUAST-J), R.S. Pura-Jammu, India


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Teaching Hospital Faculty of Veterinary Sciences and Animal Husbandry SKUAST-J, R S Pura-Jammu during the study period from January 2008 to December 2010. The diagnosis of rumen impaction was done on the basis of hard consistency of rumen on per rectal examination (Grymer and Ames, 1981) and exploratory laparorumenotomy. Six apparently healthy buffaloes brought to the clinic for artificial insemination used for studying normal parameters.

Each animal was thoroughly evaluated for its general condition and hydration status. The physical parameters (rectal temperature, heart rate, and respiration rate, colour of mucous membrane, muzzle status, rumen consistency and rumen motility) were recorded at the time of presentation. Quantity and consistency of faeces in rectum, faecal colour, rumen consistency, and rumen size were recorded on rectal examination. Physical and microscopic examination of ruminal fluid was done for color, odour, concistency pH and protozoal motility (Garry, 2002).

Blood samples (2 ml) were collected aseptically from jugular vein in EDTA coated vials. Haemoglobin (Hb, g/dL), packed cell volume (PCV, %), total leukocyte count (TLC, per/µl) and differential leukocyte count (DLC, % and per/µl) were estimated by standard methods (Benjamin, 1985). For glucose estimation blood samples were collected in vials containing sodium fluoride.

Blood samples were also collected in acid free vials without any anticoagulant; serum was separated and transferred to a dry clean vial for storage at-20oC till further evaluation. Following biochemical parameters were estimated using diagnostic kit with help of autoanalyzer viz Total bilirubin, aspartate aminotrasferase (AST), alkaline phosphatase (ALP), glucose, total protein, albumin, blood urea nitrogen (BUN), creatinine, calcium and phosphorus.

The data were subjected to student’s t-test and means and standard errors were calculated for comparison between control and animals with rumen impaction (Snedecor and Cochran, 1994).

Therapeutic management includes exploratiory ruminotomy to evacuate the rumen followed by fluid and electrolyte therapy, broad spectrum antibiotics, mineral oils, antihistaminics, NSAIDS and ruminotorics, ruminal cud transfer was done in some cases as and when required for restoration of normal ruminal flora.


All the animals were in depressed condition and rumination was suspended, muzzle was dry. On visual examination of abdominal contour from rear side, all the animals had unilaterally distended (left side) abdomen, and moderate degree (8-10%) of dehydration. Ruminal motility (1.40±0.25 per 5 minutes) was reduced significantly compared to healthy control (8.50±0.30 per 5 minutes). No significant variation in rectal temperature and respiration rate were noticed in comparison to healthy control. However, heart rate (72.60±4.26 per min) was significantly higher than healthy control (57.80±3.08). No abnormal sounds were herd on auscultation of lung and heart. On per-rectal examination, the consistency of rumen was doughy to moderately hard in all cases, no distension or ballooning of intestinal loops and in majority of animals rectum was found empty and few having constipated dry feces (pellets). These signs are similar to those earlier reported by Nwity and Chaudhary (1995).

Physical and microscopic examination of rumen fluid in impaction cases revealed greenish brown to yellowish brown color, watery


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consistency, and pungent odor, normal pH, poor (+) to nil protozoal motility in all cases, similar findings were reported by Garry, 2002. Exploratory ruminotomy was done in all the cases and it was revealed that impacted materials in majority of cases having ingested fibrous feed material, coarse grain, polythene and jute bags and ropes and nonpenetrating metallic objects.

Hematological studies revealed (Table 1) that mean TLC, and percent neutrophils were significantly higher whereas, the mean lymphocyte was significantly lower. However, no significant variation was observed in the Mean Hb, Mean PCV, in ruminal impaction cases than the healthy control. No variation in the Hb and PCV values in the ruminal impaction cases than healthy control

animals were earlier reported by the Nagarajan and Rajmani, 1973. In the present study leukocytosis and neutrophilia were observed which might have resulted from chronic irritation of the forestomach wall by impacted feed materials, leaving the wall exposed to secondary infection, which resulted in inflammation (Hailat et al., 1996). Decreased lymphocytes could be due to release of corticosteroid as a result of stress (Feldman et al., 2000).

Biochemical studies revealed (Table 1) significantly higher mean Plasma values of total bilirubin, AST, glucose, BUN, creatinine, total protein, globulin and significantly lower mean plasma values of calcium in the ruminal impaction cases than healthy control. However, no

Table 1. hemato-biochemical alterations in buffaloes with rumen impaction.

Parameters Control (Mean±SE), n=6Rumen Impaction (Mean±SE),

n=20Hb (g/dl) 9.80±0.35 8.74±0.76PCV (%) 28.70±1.20 278.40±4.84WBC (x 103/µl ) 5.45±0.56 8.65±0.38*Neutrophil (%) 32.60±3.46 61.60±4.19*Lymphocyte (%) 67.10±1.90 36.40±4.35*N/L ratio 0.49±1.82 1.69±0.96*AST (IU/L) 120.50±4.58 218.80±26.58*ALP (IU/L) 168.40±8.24 178.40±25.20Total bilirubin (mg/dl) 0.42±0.10 3.12±0.68Glucose (mg/dl) 60.32±8.50 120.20±18.80*BUN (mg/dl) 26.40±1.52 81.24±20.20*Creatinine (mg/dl) 0.50±0.05 2.30±0.60*Total protein (g/dl) 7.20±0.20 8.35±0.45Globulin (g/dl) 3.48±0.14 4.40±0.24*Albumin (g/dl) 3.72±0.15 3.94±0.20Calcium (mg/dl) 11.32±0.24 7.65±0.82*Phosphorus (mg/dl) 6.20±0.30 5.95±0.18

Means bearing *differ significantly at P≤0.05


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significant variation in the mean plasma values of ALP, albumin and phosphorus were observed in the ruminal impaction cases than healthy control. The increased bilirubin level in present study may be attributed to absorption of toxic substances from rumen. Impaired uptake and excretion of bilirubin due to deranged liver function, as evident by increased liver enzymes, may have resulted in increased serum bilirubin concentration. The increased bilirubin may also be due to constipation and starvation (Kaneko et al., 2008). The possible cause of higher AST could be necrosis of liver due to toxemia from the damaged rumen mucosa (Garry, 2002). In rumen impaction the putrefied ingesta liberates toxic amines like histamine in rumen which after absorption into circulation increases BUN concentration (Dain et al., 1995). The increased BUN level could also be attributed to decrease in renal blood flow as a part of compensatory mechanism to maintain circulation in hypovolemia associated with dehydration (Kaneko et al., 2008) and same phenomenon may be held responsible for increased creatinine concentration. Moreover, during ruminal disorders there is failure of urea cycling process and urea is not utilized by rumen microbes (Singh et al., 2001). The increase in glucose level may be due to stress of impaction leading to adrenocorticosteroid release, which has glycogenolytic effect, causing hyperglycemia.

Hypocalcaemia may be due to less assimilation of feed materials as a result of long standing anorexia (Sethuraman and Rathore, 1979; Radostits et al., 2010). Daniel (1983) reported that both rumen and abomasal motilities were similarly reduced in hypocalcemia due to general effect of depression of levels of ionised calcium on smooth muscle contractibility. Similarly, reduced calcium level in present study may have contributed to decreased ruminal motility.

All the cases affected with ruminal impaction were treated successfully in a similar principle as suggested by Khose et al., 2010, with slight modification. Initially the left para lumbar fossa was prepared for aseptic surgery. The rumenotomy was performed as per standard technique to evacuate the impacted ruminal content and fresh rumen cud, along with 4 Rumentas (Rumenotoric bolus) were introduced into the rumen before closing it. The Laparotomy incision was closed as per standard technique. Post-operative management included Dextrose Normal Saline 10 liters I/V, Strepto-penicillin 5 gm I/M (7 days), Melonex 15 ml I/M, Anistamina 10 ml I/M and Tribivet 10 ml I/M for 3 days and Floratone 4 boli, Rumentas 2 boli orally bid for 7 days in each cases. Skin sutures were removed on the 10th post-operative day. All The buffaloes affected with ruminal impaction recovered uneventfully.

REFERENCES

Benjamin, M.M. 1985. Outline of Veterinary Clinical Pathology. Kalyani Publisher, New Delhi, India. p. 60-63, 71-75.

Dain, T.A, A.L. Neal and R.W. Dougherty. 1995. The occurrence of histamine and tyramine in ruminal ingesta of experimentally urea fed sheep. J. Anim. Sci., 14: 930-955.

Daniel, R.C.W. 1983. Motility of rumen and abomasum during hypocalcemia. Can. J. Compart. Med., 47: 276-280.

Feldman, B.F., J.G. Zinkl and N.C. Jain. 2000. Indigestion in ruminants. In Garry, F.B. (ed.) Schalm’s Veterinary Haematology, 5th ed. Lee and Febiger, Philadelphia.

Garry, F.B. 2002. Indigestion in ruminants, p. 722-747. In Smith, B.P. (ed.) Large Animal


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Internal Medicine, 2nd ed. Mosby, St. Louis and Baltimore.

Grymer, J. and N.K. Ames. 1981. Bovine abdominal pings. Clinical examination and differential diagnosis. Compendium on Continuing Education of Practicing Veterinarians, 3: 311-318.

Hailat, N., S. Nouth, A. Al-Darraji, S. Lafi, F. Al-Ani and A. Al-Manjali. 1996. Prevalence and pathology of foreign bodies (plastic) in Awassi sheep in Jordan. Small Ruminant Res., 24: 43-48.

Kaneko, J.J., J.W. Harvey and M.L. Bruss. 2008. Clinical Biochemistry of Domestic Animals. 6th ed. Academic Press, London.

Khose, K.A., P.A. Jadhav and V.E. Mahajan. 2010. Ruminal Impaction in a Cow with Indigestible Foreign Bodies and its Surgical Management. Intas Polivet., 11(2): 189-190.

Nagarajan, V. and S. Rajamani. 1973. Alkaline indigestion and rumen putrefaction in cow. Indian Vet. J., 50: 1147-1151.

Nwity, T.N.E. and S.V.R. Chaudhary. 1995. Ruminal impaction due to indigestible materials in the arid zone of Borno state of Nigeria. Pak. Vet. J., 15: 29-33.

Pienkowski, M. 1969. Estimation of the functional state of liver in cows with acid indigestion. Annals universitatis, Marial CurieSkalodowska, Lublin Pulonia, Section, 18: 209-222.

Radostits, O.M., C.C. Gay, K. Hinchcliff and P.D. Constable. 2010. Veterinary Medicine. A Textbook of the Diseases of Cattle, Horses, Sheep, Pigs and Goats, 10th ed. Saunders Elsevier.

Sethuraman, V. and S.S. Rathore. 1979. Clinical, haematological and biochemical studies

on secondary indigestion in bovines due to traumatic reticulitis and diaphragmatic hernia. Indian J. Anim. Sci., 49: 703-706.

Singh, N., R. Kumari and M.A. Akbar. 2001. Ruminal pH as a regulator of blood metabolites in lactating Murrah buffaloes. Indian Vet. Med. J., 25: 253-256.

Snedecor, G.W. and W.G. Cochran. 1994. Statistical Methods, 8th ed. Iowa State University Press. Ames. Iowa, USA.



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Original Article

ABSTRACT

The present study was envisaged to compare the sensitivity of detection of rabies virus antigen by application of Fluorescent Antibody Technique on fresh impression smear (Direct-FAT) and that on formalin fixed nervous tissue (Indirect-FAT), histopathology and immunohistochemistry (IHC) in the species which is highly significant for the economics of the dairy farmer i.e. buffalo. A total of 28 cases of buffaloes suspected for rabies were presented. Out of 28 cases, 18 (64.28%) cases were positive by direct-FAT, indirect-FAT, IHC and 60.71% (17/28) by demonstration of negri bodies and thus, histopathology revealed 94.4% sensitivity in comparison to direct- FAT. While as, indirect-FAT, and IHC revealed 100% sensitivity in comparison to direct-FAT. Percentage of neurons positive for Negri bodies by H and E and IHC were 59.35% and 78.88% and average number of Negri bodies detected per neuron by H & E and IHC were 1.8 and 3.01, respectively. Important clinical signs in rabid animals were anorexia, circling/Head pressing, behavioural change and bellowing. Thus, it is concluded that rabies detection in animals can be accomplished from diagnosis of rabies from fixed brain tissues which offers same sensitivity as detection of rabies in impression smears.

Keywords: buffaloes, FAT, IHC, formalin fixed, rabies

INTRODUCTION

Rabies is a fatal zoonotic disease of worldwide concern caused by a neurotropic negative sense single stranded RNA (ssRNA) virus of the genus Lyssavirus, Order Mononegavirales and of family Rhabdoviridae. Diagnosis of clinical rabies is difficult and is often not made until after death of the animal, so early diagnosis of rabies in animals is necessary for timely administration of post-exposure prophylaxis. At necropsy, rabies is usually diagnosed by subjecting fresh or formalin fixed nervous tissue samples to pathological examination and the routine diagnostic methods used are fluorescent antibody test on brain impression smears and histopathological examination of the brain for Negri bodies. These inclusions are not present in all cases and the use of fresh tissue samples for laboratory examination is hazardous due to possible risk of contamination of the environment with rabies virus. However, in many situations, only formalin-fixed tissue is available for post-mortem diagnosis due to lack of laboratory facilities or presentation of fixed rather than fresh tissues to the laboratory Warner

DIAGNOSIS OF RABIES IN BUFFALOES: COMPARISON OF CLINICO-PATHOLOGICAL, IMMUNOHISTOCHEMICAL AND IMMUNOFLUORESCENT TECHNIQUES

A.B. Beigh1, B.S. Sandhu2, C.K. Singh2, K. Gupta2 and N.K. Sood2

1Department of Veterinary Pathology, Sher-e-Kashmir University of Agricultural Sciences & Technology of Kashmir (SKUAST-K), Srinagar, India, E-mail: [email protected] Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India



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et al. (1997) and Abreu et al. (2012). Hence, there is a need for a better method of diagnosis of rabies using formalin-fixed paraffin-embedded tissues. Immunohistochemistry, indirect FAT and histopathology can be performed on such samples. Immunohistochemistry and indirect FAT technique improves diagnostic accuracy by promoting visualization of the distribution of the infectious disease agent in histological sections Deborah et al. (1991). They provide sufficient amplification of the antibody-antigen interaction to enable detection of antigens immunogenically altered by fixation. So, the present study was envisaged to establish the comparison of sensitivity of routine detection with application of FAT on nervous tissue impression smear with other techniques on formalin fixed nervous tissue.


A total of 28 cases of buffaloes suspected for rabies were presented at Rabies Research-cum-Diagnostic laboratory, Department of Veterinary Pathology, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana from various parts of Punjab. The data regarding age, sex, history of bite, date of bite, source of bite and clinical signs was acquired as per the questionnaire prepared for the purpose from owners of the animals.

Clinical samples for detection of rabiesBrain samples were collected from dogs

suspected for rabies. Three pieces of each tissue sample were stored at 20oC, in 50% glycerol saline solution, and in 10% neutral buffered formalin solution respectively.

Detection of rabies virus antigen in fresh tissue impression smear using fluorescein-labeled antibody (Direct-FAT)

The direct FAT is employed as diagnostic technique because of its sensitivity, accuracy and speed as recommended by World Health Organization (Meslin et al., 1996). A pair of thin impression smears from each of brain tissue were prepared in grease-free labelled glass slides 1 cm in diameter, about 1.5 cm from each end, from either fresh tissue or preserved tissues kept at 20oC in deep freeze.

Control positive slides from known rabies case and control negative from normal uninfected and unvaccinated animal were prepared along with the test smear. Impression smears were air dried for 30 minutes at room temperature. Smears were fixed by immersing in coupling jars containing cold acetone in a deep freeze at 20oC for overnight. Acetone was drained off and impression smear slides were air dried at room temperature for 20 minutes. Lyophilized, anti-rabies nucleocapsid Fluorescein isothiocyanate (FITC) conjugate acquired from Bio-radMarnes-La-Coquette, France was reconstituted with 3 ml of distilled water as recommended by the manufacturer and centrifuged at 1500 rpm for 5 minutes for clarification. The clarified conjugate (0.1 ml) was added on the duplicate impression smears on every slide for each tissue samples and on positive and negative control slides. Then, smears were covered with cover slips and slides incubated at 37oC for 30 minutes by placing in a humidified chamber. Slides were washed twice in 0.01 M phosphate buffered saline (PBS) pH 7.5 for 5 minutes each. Thereafter, slides were air-dried and mounted in 90% buffered glycerol (pH 8.5). Slides were examined using an AHBT3 - RFC reflected light fluorescence attachment (Olympus, Japan).


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Detection of rabies virus antigen in formalin-fixed tissues using fluorescein-labeled antibody (Indirect-FAT) The FAT of formalin-fixed tissues was performed as described by Warner et al., 1997 with the following modifications: The dewaxing and rehydration of tissue sections was carried out by EZ-AR Common solution at 70oC for 10 minutes in microwave oven; Antigen retrieval was done in citrate buffer (0.01 M, pH 6.0-6.2) EZ-RetrieverR System V.2.1 (BioGenex Laboratories Inc., San Ramon, California, USA) at different time and temperature combinations-2 cycles-95oC for 10 minutes and at 98oC for 5 minutes, respectively; Thereafter, slides were washed with PBS washing buffer (pH 7.2 to 7.6) for 3 times, 5 minutes each; and allowed to dry at room temperature. Then Fluorescein isothiocyanate (FITC) conjugate (0.1 ml) was added on every paraffin embedded tissue sections. After this slides were incubated at 37oC for 60 minutes by placing in a humidified chamber. Then slides were washed with PBS washing buffer (pH 7.2 to 7.6) for 2 times 5 minutes each, then twice more in deionized water at room temperature. The sections were air dried and cover glasses were applied using aqueous mounting media FluoromountTM (SIGMA-ALDRICH, Saint Louis, Missouri, USA). The slides were examined using a fluorescent microscope (Nikon, 800i, Japan).

Immunohistochemistry (IHC)Antirabies polyclonal antisera (Rabbit)

available in the Rabies Research-cum-Diagnostic Laboratory of the department of GADVASU; Ludhiana was used as primary antibody for immunohistochemical studies. Different dilutions of 1:50, 1:100, 1:500, 1:1000, and 1:2000 of polyclonal antisera in PBS (pH 7.2 to 7.6) were

used for immunohistochemical staining of brain tissues sections. Maximum dilution of antibody at which these samples revealed positive reaction was 1:1000.

Paraffin embedded tissues were sectioned at 4 to 5µm thickness and mounted on Superfrost/ Plus, positively charged microscopic slides (Fisher Scientific, USA). The slides were then placed in hot air oven to melt the paraffin at 60oC for 30 minutes and stored till further use. Advanced SSTM two step polymer Horseradish Peroxidase (HRPO) Immunohistochemical detection system (BioGenex Laboratories Inc., San Ramon, California, USA) was used for staining of paraffin embedded tissue sections as per recommendation of the manufacturer with some modifications Pedroso et al. (2008). The dewaxing and rehydration of tissues sections were carried out by EZ-AR Common solution at 70oC for 10 minutes in microwave oven.

Antigen retrievalAntigen retrieval was done in EZ-ARTM 3 in

EZ-RetrieverR System V.2.1 (BioGenex Laboratories Inc., San Ramon, California, USA) at different time and temperature combinations-2 cycles-95oC for 10 minutes and at 98oC for 5 minutes, respectively. The slides were cooled and brought to room temperature, washed with PBS buffer (pH 7.2 to 7.6) for 3 times for 3 minutes each. The endogenous peroxidase activity was blocked by incubating slides with a solution of 3% H2O2 in methanol for 25 minutes at room temperature in humidified chamber. Slides were washed with PBS buffer (pH 7.2 to 7.6) for 3 times 3 minutes each and sections were encircled with hydrophobic pen (Pap pen). Non-specific protein binding was blocked using power block solution (BioGenex Laboratories Inc., San Ramon, California, USA) for 15 minutes in moist chamber.

Slides were incubated with primary


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polyclonal rabbit anti-rabies antibody (1:500 and 1:1000 dilution in PBS 1% BSA) for one and half hour in humidified chamber at room temperature. For each staining a negative control was run on sister section in which primary antibody was replaced by PBS. Slides were washed with PBS washing buffer (pH 7.2 to 7.6) for 3 times, 3 minutes each subsequently.

The tissue sections were incubated with secondary antibody ImmPRESSTM UNIVERSAL REAGENT Anti-mouse/Rabbit Ig (Vector Laboratories Inc., Burlingame, U.S.A.) for 30 minutes at room temperature in humidified chamber. Slides were washed with PBS washing buffer (pH 7.2 to 7.6) for 3 times, 3 minutes each. Substrate 3, 3 ‘-diaminobenzidine (DAB) solution, freshly prepared by mixing a drop of ImmPACTTM DAB chromogen with 1 ml of ImmPACTTM DAB buffer (Vector Laboratories Inc., Burlingame, U.S.A) and 5 μl hydrogen peroxide. The antigen-antibody-peroxidase reaction was visualized by adding substrate 3, 3 ‘-diaminobenzidine (DAB) solution on sections for 1 to 2 minutes. Sections were washed in tap water for 5 minutes to stop the antigen-antibody-peroxidase reaction. Slides were counterstained with Gill’s haematoxylin (Merck, Germany) for 30 seconds and washed with running tap water for 5 minutes. Finally the sections were dehydrated in ascending grades of alcohol (70%, 80%, 90%, and absolute alcohol) and cleared in xylene for 2 minutes and mounted with DPX. Slides were examined under microscope (BX 61, Olympus Corporation, Japan).

Histopathology All nervous tissues samples, viz. cerebellum, cerebrum, hippocampus, pons, medulla oblongata from dead animals were collected in 10% neutral buffered formalin solution. These tissues

were routinely processed through ascending grades of alcohol, cleared in benzene and embedded in paraffin wax. The paraffin sections were cut at 4 to 5 μ thickness and stained by haematoxylin and eosin (H and E) method (Luna, 1968). Slides were examined by by BX61 Research Photomicrograph Microscope System of Olympus Corporation, USA.

Sensitivity comparison with direct-FAT Sensitivity of various techniques was calculated in comparison with dFAT as per Perrin and Sureau (1987).

Sensitivity =

True positive

×100(True positive + false negative)


Clinical signs in rabid buffaloesIn case of rabid buffaloes, anorexia was

found in 94.44% (17/18) cases, followed by behavioral change and pressing of head against hard objects in 55.55% (10/18) cases, difficulty in feed intake and hyper-salivation and bellowing in 50% (9/18) cases (Figure1 and 2). Whereas, paralysis and fever in 38.88% (7/18). However, frequent micturition in 27.77% (5/18) cases. Non-recognizing owner 22.22% (4/18) and pica were found in 11.11% (2/18) cases, respectively (Table1). Similar symptoms have been reported by (Salem et al., 1995; Rissi et al., 2008; Pedroso et al., 2009).

Direct FATOut of 28 cases, 17 cases (60.71%) were


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diagnosed positive for the presence of rabies viral antigen (Table2). Characteristic apple green immunofluorescence was observed intra-cytoplasmic in neurons as well as in form of diffused fluorescence in the brain tissue smears (Figure 3). FAT is sensitive, specific, and easy to perform, serves as standard diagnostic procedure and is the preferred test for rabies diagnosis (Smith, 1999; Whitfield et al., 2001).

Indirect FATOut of 28 cases, 18 (64.28%) were

found positive for rabies virus antigen (Table 2) and revealed 100% sensitivity in comparison to direct-FAT on fresh tissue smears (Table 3). The viral antigen in formalin fixed tissue was visible as distinct apple green coloured intracytoplasmic inclusion bodies and finely granular particles along dendritic arborization, axonal tracts and in the stroma (Figure 5 and 6). Similar finding have been reported by Swoveland and Johnson (1979), Johnson et al.(1980), Reid et al.(1983), Umoh et al.(1984), Bourhy and Sureau (1990). Detection of viral antigen was almost same in tissues stored in

formalin for short and long period of time. Johnson et al. (1980) suggested FAT on formalin fixed tissue as complementary to standard diagnostic techniques.

ImmunohistochemistryBrain tissues were positive in 18 out of

28 cases (64.28%) (Table 2), using polyclonal antiserum by immunohistochemistry and it revealed 100% sensitivity in comparison to direct-FAT (Table 4). No positive reaction was observed with monoclonal antibody. The controls were negative and free of endogenous peroxidase (Figure 4). A large amount of distinct, granular rabies viral antigen deposits stained as sharply demarcated brown precipitates of variable sizes were found within the Purkinje cells and in the neurons of the hippocampus, in the axons, in the processes of neurons and in the stroma (Figure 5and 6). These findings were similar as reported by (Gunawardena and Blakemore, 2007; Pedroso et al., 2009).

HistopathologyOut of 28 cases, 17 cases (60.71%) were

Table 1. Clinical signs in rabid buffaloes (Total positive cases=18).

Symptom No. of animals PercentageOff feed 17 94.44Hyper-salivation 9 50Fever 7 38.88Not recognizing owner 4 22.22Circling/Head pressing 10 55.55Difficulty in standing/paralysis 7 38.88Difficult intake of food 9 50Frequent micturition 5 27.77Bellowing 9 50Pica 2 11.11Behavioral change 10 55.55


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Table 2. Comparison of Direct-FAT with other diagnostic techniques for detection of rabies virus antigen.

S. no. Case no. Direct-FAT Indirect-FAT IHC Histopathology

1 RL 25/10 + + + +2 RL 26/10 - - - -3 RL 33/10 + + + +4 RL 36/10 + + + +5 RL 38/10 - - - -6 RL 41/10 + + + +7 RL 02/11 + + + +8 RL 04/11 + + + +9 RL 10/11 - - - -10 RL 17/11 + + + +11 RL 25/11 - - - -12 RL 28/11 + + + +13 RL 31/11 + + + +14 RL 33/11 + + + +15 RL 36/11 - - - -16 RL 37/11 + + + +17 RL 03/12 - - - -18 RL 04/12 - - - -19 RL 10/12 + + + +20 RL 11/12 - - - -21 RL 14/12 + + + +22 RL 16/12 + + + +23 RL 17/12 + + + +24 RL 18/12 - - - -25 RL 19/12 + + + +26 RL 24/12 + + + -27 RL 25/12 + + + +28 RL 27/12 - - - -

% Test positive

64.28% (18/28)

64.28% (18/28)64.28% (18/28)

60.71% (17/28)


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Table 3. Sensitivity of Indirect-FAT in comparison to Direct-FAT on brain tissue smears.

TestDirect-FAT (Positive)

Direct-FAT (Negative) Total

Indirect-FAT (Positive) 18 0 18Indirect-FAT (Negative) 0 10 10Total 18 10 28

Sensitivity of IHC for brain sample =True positive

x 100(True positive + False negative)

= 18/18+ 0 ×100 = 1800/18 = 100%

Table 4. Sensitivity of Immunohistochemistry (IHC) on Brain samples in comparison to FAT on fresh brain tissue smears.



IHC on brain (Positive) 18 0 18IHC on brain (Negative) 0 10 10Total 18 10 28



= 18/18+ 0 ×100 = 1800/18 = 100%

Table 5. Sensitivity of Histopathology on Brain samples in comparison to FAT on fresh brain tissue smears.



Histopathology (positive) 17 0 17Histopathology (negative) 1 10 11Total 18 10 28



= 17/17+ 1 ×100 = 1700/18 = 94.4%


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Table 6. Histopathological and Immunohistochemical evaluation of brain tissues for number of Negri bodies.

Sr. No. Case No.No. of neurons positive for Negri bodies/100 neurons

No. of Negri bodies detected/100 neurons

H &E IHC H & E IHC1 RL 25/10 66 87 172 4082 RL 33/10 46 95 125 3813 RL 36/10 43 88 69 3254 RL 41/10 77 80 114 2305 RL 2/11 59 78 103 1646 RL 4/11 69 83 119 2337 RL 17/11 6 33 7 528 RL 28/11 14 90 18 2179 RL 31/11 76 98 282 580

10 RL 33/11 87 100 152 49511 RL 37/11 39 94 50 19012 RL 10/12 80 96 146 42613 RL 14/12 69 89 174 21014 RL 16/12 41 72 45 14115 RL 17/12 67 91 87 21116 RL 19/12 75 86 150 21917 RL 25/12 95 79 65 178

Total 1009 1341 1878 4044

Table 7. Comparison of histopathology and immunohistochemistry.

Parameter Histopathology (H& E) IHCNeurons having Negri bodies (n=1700) 1009 1341%age of Neurons positive (Negri bodies) 59.35 78.88Total number of Negri bodies detected 1878 4044Average number of Negri bodies per neuron 1.8 3.01


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Figure 1. Rabies suspected buffalo exhibiting head pressing and paralysis.

Figure 2. Rabies suspected buffalo exhibiting hypersalivation.


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Figure 3. Impression smear drawn from hippocampus of a rabid buffalo showing apple green fluorescence in neurons. Direct FAT X 165.

Figure 4. Negative control of IHC-Section of cerebellum showing absence of reaction. IHC-One step polymer HRPO Technique - Original magnification x 400X.


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Figure 5. Section of hippocampus of rabid buffalo showing few Negri bodies with H&E stain- Original magnification x 1000X (A), corresponding IHC stained section - Original magnification x 1000X (B) showing brown coloured Negri bodies (arrow) and corresponding FAT stained section- Original magnification x 400X (C) showing more clearly green coloured Negri bodies (arrow).


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Figure 6. Section of hippocampus of rabid buffalo showing few Negri bodies with H&E stain- Original magnification x 1000X (A), corresponding IHC stained section - Original magnification x 1000X (B) showing brown coloured Negri bodies (arrow) and corresponding FAT stained section- Original magnification x 400X (C) showing more clearly green coloured Negri bodies (arrow).


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found positive for rabies by demonstration of negri bodies (Table 2) and thus, histopathology revealed 94.4% sensitivity in comparison to direct-FAT (Table 5). Negri bodies appeared as single or multiple, eosinophilic intracytoplasmic inclusions within the Purkinje neurons, in the axons and in the neurons of the hippocampus (Figure 5 and 6). Which has been also reported by (Gonzalez and Stephano, 1984; Salem et al., 1995; Lima et al., 2005; Srinivasan et al., 2005; Pedroso et al., 2008 and Rissi et al., 2008).

Comparison of immunohistochemistry and histopathology

FAT on formalin fixed tissue can be used as an alternative to FAT on fresh tissue with the same sensitivity, when only formalin-fixed tissue is available for post-mortem diagnosis. Hundred neurons per case were observed for negri bodies and number of negri bodies in positive neurons (Table 6) and a comparison of IHC and histopathology were done (Table 7). With IHC 78.88% neurons were positive for negri bodies and 59.35% with H and E. It can be concluded that IHC established many more virus infected cells than H and E stained sections which is same as reported by Feiden et al. (1985).

Average number of negri bodies detected per neuron by IHC was 3.01 which were greater than H and E stained brain sections (1.8). The amount of antigen detected with IHC was much more abundant than histopathological findings (Figure 5 and 6) which are reported by several workers (Hamir et al., 1992; Martinez-Burnes, 1997; Jogai et al., 2001 and Suja et al., 2004). Palmer et al. (1985) reported that rabies antigen with IHC was apparent in 62% of the brain area in which inclusion bodies were not found in the corresponding H and E sections. In present study

a comparison of polyclonal and monoclonal antibodies employed in detection of rabies antigen in formalin fixed paraffin embedded tissue sections using IHC revealed polyclonal antibody to be highly efficacious.

As compared to IHC specificity of FAT on formalin fixed tissue was more, this is because of nonspecific binding of polyclonal antibody with nonspecific antigens in case of IHC. There is also enhanced detection of viral antigen due to fluorescence of antigen-antibody complex. Thus, it can be concluded that IHC was more sensitive than histopathology but as sensitive as either of FAT procedures and proved to be a valid method for rabies diagnosis and can replace FAT where fluorescent microscopy is not available or when fresh samples are not available for FAT.

ACKNOWLEDGEMENTS

Authors thank the Director of Research, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana for providing financial support to conduct this study. We also thank Mr. Dan Singh, Mr. Kewal Singh, Mr. Dilchain Singh and staff of rabies diagnostic and histopathology laboratory for their help, cooperation and support extended during the period of study.

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Original Article

ABSTRACT

A total of 3590 buffaloes of all age groups and sex were examined from various districts of Punjab state, India. The association between age, sex of host, season and the prevalence of ixodid ticks on animal level were investigated by multivariate logistic regression. The overall prevalence of ixodid ticks, Rhipicephalus (Boophilus) microplus, Hyalomma anatolicum anatolicum and mixed infestations were 57.68, 31.83, 19.08 and 6.76%, respectively. The results of multivariate analysis showed that the prevalence was associated with season (P<0.001; OR: 3.153; CI 95%: 2.69-3.69), host age (P<0.001; OR: 2.13; CI 95%: 1.67-2.73) and sex (P<0.001; OR: 0.416; CI 95%: 0.24-0.71).

The prevalence of ixodid ticks were highest in monsoon season (74.48%) whereas, maximum prevalence of R. (B.) microplus and H. a. anatolicum were recorded in monsoon (44.73%) and summer (23.32%), respectively and the seasonal variation was significant (P<0.001). Further, higher tick infestation was recorded in calves <6 months of age and also in males. The findings of the current study would provide a basis for evolving effective control strategy for the management of ticks in buffalo population of the region.

Keywords: buffalo, epidemiology, Hyalomma anatolicum anatolicum, Rhipicephalus (Boophilus)

microplus

INTRODUCTION

Ticks and the diseases they transmit are widely distributed throughout the world, particularly in tropical and subtropical regions. Losses attributable to ticks are caused either directly through tick worry, blood loss, damage to hides and udders and the injection of toxins, or indirectly through mortality or debility caused by the diseases transmitted by or associated with the ticks. The global economic loss due to tick infestation has been estimated as US$ 14000 to 18000 million annually and the cost of management of tick and tick borne diseases (TTBDs) in livestock of India is as high as US$ 498.7 million per annum (Minjauw and Mc Leod, 2003). The most common combined effect of TTBDs in Indian dairy system is reduction in milk yield i.e. loss of 14% of the lactation (McLeod and Kristjanson, 1999) and quality of hides for leather industry (Biswas, 2003). Punjab is having a significant percentage of India’s buffalo heads and contributes nearly 9% to the Indian milk production. The state is situated at the North West frontier of India and the climatic condition of the state is highly conducive for growth and development of ticks. In past, several sporadic reports on tick infestation patterns of dairy

EPIDEMIOLOGY OF IXODID TICKS IN BUFFALOES (BUBALUS BUBALIS) OF PUNJAB, INDIA

N.K. Singh* and S.S. Rath

Department of Veterinary Parasitology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India, *E-mail: [email protected]


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animals from Punjab had been published (Gill and Gill, 1977; Singh and Singh, 1999; Ghai et al., 2008; Haque et al., 2011) but a comprehensive epidemiological study covering whole of the state has not been explored so far. Therefore, the present study was undertaken to determine the epidemiological patterns of the ixodid tick in buffaloes of Punjab state, India.


Location, geography and climate of study area The state of Punjab extends from the latitudes 29.30oN to 32.32oN and longitudes 73.55oE to 76.50oE in the northwest region of India. It covers a geographical area of 50,362 km2 and lie between altitudes 180 meters and 300 meters above sea level. The three major seasons in Punjab are summer (April to June; average rainfall 51.6 mm), monsoon (July to September; average rainfall 395.2 mm) and winter (October to March; average rainfall 119.1 mm). The climate of the plains is excessively hot and dry in summers and winters are cool with some frosts. Average rainfall in Punjab is 565.9 mm and ranges from about 915 mm in north to 102 mm in south. (http://punjabonline.in/Profile/Geography/climate.asp).

Collection of ticks Ticks were collected during February, 2010 to August, 2011 from 3590 buffaloes of different villages covered under the eighteen districts. Animals of both sexes and all age groups were examined and each animal examined was considered as one sample. Ticks were searched by passing hands through the animal’s coat and collected manually without damaging their mouthparts. The collected

tick samples were then transferred to plastic tubes and were brought to the laboratory and separately stored in 70% ethanol. Adult ticks were identified under a stereomicroscope, according to general identification keys (Estrada-Pena et al., 2004).

Statistical analysis All data analyses were performed by using statistical software program (SPSS for Windows, Version 19.0, USA). Association between the prevalence of ixodid tick infestation and various factors was carried out by Chi square (χ2-test). Variables with significant association at P<0.05 (two-sided) were subjected to the multivariate logistic regression model. The results were each expressed as P value and odds ratio (OR) with a 95% confidence interval (CI 95%).


Ticks collected from the buffalo population of Punjab state were identified as Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum anatolicum. The prevalence of ixodid ticks, R. (B.) microplus, and H. a. anatolicum were 57.68%, 31.83% and 19.08%, respectively. Most infestations were pure with single species as mixed infestations of both genera occurred less frequently (6.76%). These ticks were present on all over the body but R. (B.) microplus showed preference for the areas with softer skin viz. groin, insides of shoulder and thighs, around the anus and external genetalia, base of tail etc. Whereas, H. a. anatolicum were found to be evenly distributed through out the body and in some animals an atypical circular infestation pattern by the nymphal stages was documented (Figure 1). Results of the current study reveal that R.

http://punjabonline.in/Profile/Geography /climate.asp

http://punjabonline.in/Profile/Geography /climate.asp


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(B.) microplus and H. a. anatolicum are the ixodid ticks infesting buffaloes of Punjab state, India. In contrast, much earlier reports from the region document a large number of ixodid tick species parasitizing buffaloes from the same geographical area (Gill and Gill, 1977; Miranpuri, 1988). The change in the tick population in form of reduced number of species may be related to the adaptation of better animal husbandry practices in the last two to three decades. Newer tick control measures and availability of effective acaricides proved to be detrimental particularly for the survival of ticks other than the two species reported in the study mainly due to their initial low frequencies in the population (Gill and Gill, 1977). Similar to the findings of the current study, recent studies on the tick population of dairy animals from the region also report R. (B.) microplus and H. a. anatolicum as the only tick species infesting dairy animals (Singh and Singh, 1999; Sangwan et al., 2000; Ghai et al., 2008; Haque et al., 2011).

Seasonal dynamics of ixodid ticks The current study indicates that season plays a very important role in population dynamics

of ticks and R. (B.) microplus was recorded as the predominant tick in all seasons of Punjab state. Significant association between prevalence of infestation with ixodid ticks in buffaloes and the season was observed (P<0.001; OR: 3.153; CI 95%: 2.69-3.69). The β value of-0.725 was recorded between the prevalence of ticks and the various seasons (summer followed by monsoon and winter) thus indicating a decrease in tick prevalence with decrease in ambient temperature (Table 1). Several studies are on record with regard to seasonal dynamics of ticks in India (Rajagopalan and Sreenivasan, 1981; Das, 1994; Vatsya et al., 2008). The highest infestation rate was recorded in monsoon season (74.48%), followed by summer (66.64%) and least in winter (38.78%) with a significant variation (P<0.001) (Table 2) in seasonal distribution as the hot and humid environmental conditions in the monsoon is most conducive for the development of various developmental stages of ticks. Whereas, the cold and dry conditions of the winters are unfavourable for its survival and tick passes the winter as engorged females, nymphs, larvae and unfed adults by hiding into

Figure 1. Atypical circular infestation pattern of Hyalomma a. anatolicum in buffalo.


350

the cracks and crevices (Chaudhuri, 1969) thus leading to low infestation levels. The trend was similar for the distribution of R. (B.) microplus with significant statistical variation (P<0.01) among seasons whereas; seasonal prevalence of H. a. anatolicum showed entirely different trend with maximum prevalence in drier months with significant variation (P<0.001) as hot and dry weather is conducive for its development and similar trend has been reported earlier (Bouattour et al., 1996; Estrada Pena, 2008). An earlier study also revealed the gradual overpowering of R. (B.) microplus on H. a. anatolicum in the winter season and is because of its wide distribution and prevalence (Haque et al., 2011).

Prevalence of ixodid ticks in various age groups of buffaloes Animal age significantly affected the prevalence of infestation by ixodid ticks (P<0.001; OR: 2.13; CI 95%: 1.67-2.73) but had a negative correlation (β=-2.61). Thus, among the different age group of buffaloes screened maximum tick infestation was recorded in calves<6 months of age (72.73%), followed by 6 months to 1 year age group (61.30%) and least in >1 year age group (55.53%). Similarly, significant difference (P<0.001) was recorded in the prevalence of R. (B.) microplus among different age groups with highest prevalence in calves<6 months of age, whereas, prevalence of mixed infestations was higher in older animals (Table 2). The age of the host animal plays a significant role on the infestation pattern of tick species (Manan et al., 2007). Younger animals are more prone to tick infestations and can be correlated with the fact that the adult or the productive animals are given utmost care with better animal husbandry practices whereas the younger animals are least attended

with limited use of acaricides leading to higher tick infestations. Also, low tick infestation on adult cattle is probably due to resistance acquired following repeated exposure from early life (Das, 1994). Further, similar infestation pattern of ticks had been reported in past (Nagar et al., 1978; Manan et al., 2007).

Sex wise prevalence of ixodid ticks The effect of sex of host on the prevalence of ixodid tick infestations was significant (P<0.01; OR: 0.416; CI 95%: 0.24-0.71) with positive correlation (β=4.42) and higher prevalence in males. The prevalence of R. (B.) microplus, H. a. anatolicum and mixed infestation in both sexes were significantly variable (P<0.001) (Table 2). Although, Sutherst et al. (1983) reported that the milch animals because of the hormonal stress carry more ticks but in the current study a higher tick infestation was encountered in males and can be attributed to the fact that male animals in this part of the country are neglected and least care is provided with occasional use of acaricides. This is due to the reason that males are now considered useless by the farmers after the popularization of artificial insemination and use to motorized power for farm usage as informed by the owners through the questionnaire. It can hence be concluded that R. (B.) microplus and H. a. anatolicum are the ixodid ticks of buffaloes of Punjab state and the former being the predominant one. Further, as the tick population peaks at the monsoon followed by summer season the control measures should be adopted accordingly to minimize the losses attributed to ticks and thus increase the productivity of the animals.


351

Table 1. Final logistic regression model for factors associated with ixodid ticks infestation in buffaloes on animal levels.

VariablesRegression

coefficient (β)Standard Error

(SE)P value Odds

Confidence Interval (CI 95%)

Season -0.725 0.06 0.000 3.153 2.69-3.69Age -2.61 0.106 0.000 2.135 1.67-2.73Sex 4.42 0.237 0.000 0.416 0.24-0.71

Table 2. Epidemiology of ixodid ticks in buffaloes of Punjab state, India.

Groups Examined Positive+ve for Mixed

infestation+ve for R. (B.)

microplus +ve for H. a. anatolicum

Season

Summer 1256 837 (66.64) 143 (11.38) 401 (31.92) 293 (23.32)Monsoon 921 686 (74.48) 85 (9.22) 412 (44.73) 189 (20.52)Winter 1413 548 (38.78) 15 (1.06) 330 (23.35) 203 (14.36)χ2 value - 358.9* 101.4* 10.40** 27.9*

Age

<6 mon 352 256 (72.73) 16 (4.54) 167 (47.44) 73 (20.73)6 mon – 1 yr 292 179 (61.30) 20 (6.84) 93 (31.84) 66 (22.60)> 1 yr 2946 1636 (55.53) 207 (7.02) 883 (29.97) 546 (18.53)χ2 value - 41.49* 294.1* 999.3* 663.0*

SexFemale 3514 2013 (57.29) 238 (6.77) 1115 (31.73) 660 (18.78)Male 76 58 (76.32) 5 (6.57) 28 (36.84) 25 (32.89)χ2 value - 11.80** 223.4* 1034* 588.6*Total 3590 2071 (57.68) 243 (6.76) 1143 (31.83) 685 (19.08)

Figures in parenthesis are the % prevalence of infested ticks; *P<0.001, **P<0.01


352

ACKNOWLEDGEMENTS

The authors are thankful to The Director of Research, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana for providing facilities to carry out the research work.

REFERENCES

Biswas, S. 2003. Effects of ticks on animal production system. In Proceedings the Natural Seminar Leather Industry in Today’s Perspective. Kolkata, India.

Bouattour, A., M.A. Darghoulh and L. BenMiled. 1996. Cattle infestation by Hyalomma ticks and prevalence of Theileria in H. detritum species in Tunisia. Vet. Parasitol., 65: 233-245.

Chaudhuri, R.P. 1969. Description of the immature stages of Hyalomma kumari and redescription of the adults with notes on its hosts and distribution. Parasitology, 60: 43-53.

Das, S.S. 1994. Prevalence of ixodid tick infestation on farm animals in Pantnagar, tarai of Uttar Pradesh. J. Parasitol. Appl. Anim. Biol., 3: 71-73.

Estrada-Pena, A. 2008. Climate, niche, ticks, and models: what they are and how we should interpret them. Parasitol. Res., 103: 87-95.

Estrada-Pena, A., A. Bouattour, J.L. Camicas and A.R. Walker. 2004. Ticks of Domestic Animals in Mediterranean Region. A Guide to Identification of Species. Bioscience Reports, London, UK. p. 43-131.

Ghai, J.K., M. Singh and A. Singh. 2008. Population dynamics of ixodid ticks infesting cattle in Bathinda and Hoshiarpur districts in the

Punjab State. Ann. Biol., 24: 95-100.Gill, H.S. and B.S. Gill. 1977. Qualitative district-

wise distribution of adult ixodid ticks in the Punjab state. Ixodid Ticks of Domestic Animals in the Punjab State. PAU, Ludhiana.

Haque, M., Jyoti, N.K. Singh, S.S. Rath and S. Ghosh. 2011. Epidemiology and seasonal dynamics of ixodid ticks of dairy animals of Punjab state, India. Indian J. Anim. Sci., 81: 661-664.

Manan, A., Z. Khan, B. Ahmad and Abdullah. 2007. Prevalence and identification of Ixodid tick genera in frontier region Peshawar. J. Agric. Biol. Sci., 2: 21-25.

McLeod, R. and P. Kristjanson. 1999. Tick Cost: Economic Impact of Ticks and TBD to Livestock in Africa, Asia and Australia. International Livestock Research Institute (ILRI), Nairobi, Kenya.

Minjauw, B. and A. McLeod. 2003. Tick-borne diseases and poverty. The impact of ticks and tick-borne diseases on the livelihood of small scale and marginal livestock owners in India and eastern and southern Africa. Research report, DFID Animal Health Programme, Centre for Tropical Veterinary Medicine, University of Edinburgh, UK. p. 59-60.

Miranpuri, G.S. 1988. Ticks parasitising the Indian buffalo (Bubalus bubalis) and their possible role in disease transmission. Vet. Parasitol., 27: 357-362.

Nagar, S.K., R.N. Raizada and V.K. Saxena. 1978. Studies on the rate of infestation of Boophilus microplus on Indian cattle: Its activity and infestation differential. Indian J. Anim. Sci., 48: 173-176.

Rajagopalan, P.K. and M.A. Sreenivasan. 1981. Ixodid ticks on cattle and buffaloes in the


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Kyasanur forest disease area of Karnataka state. Indian Vet. J., 65: 18-22.

Sangwan, A.K., N. Sangwan and M.C. Goel. 2000. Progressive displacement of Hyalomma ticks by Boophilus microplus in Haryana. Journal of Parasitic Diseases, 24: 95-96.

Singh, A.P. and A. Singh. 1999. Seasonal dynamics of ixodid ticks infesting the crossbred cattle of Ludhiana district. Indian Vet. J., 76: 167-168.

Sutherst, R.W., J.D. Kerr and G.F. Maywald. 1983. Effect of season and nutrition on the resistance of cattle to the tick Boophilus microplus. Aust. J. Agric. Res., 34: 329-339.

Vatsya, S., C.L. Yadav, R.R. Kumar and R. Garg. 2008. Prevalence of ixodid ticks on bovines in foothills of Uttarkhand state: a preliminary report. Indian J. Anim. Sci., 78: 40-42.

https://www.cabdirect.org/cabdirect/search/?q=do%3a%22Journal+of+Parasitic+Diseases%22



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ABSTRACT

In the present study 3779 feacal samples of buffaloes were collected from the seven agro-climatic zones of Madhya Pradesh state, India. The study was conducted for a period of one year from April 2011 to March 2012. The prevalence of gastrointestinal (GI) parasitic infection was 55.65% (2143). Amphistomes (28.10%) being the most prevalent GI parasite followed by Strongyle (25.59%), Schistosoma sp. (5.19%), Strongyloides sp. (3.15%), Trichuris sp. (2.59%), Fasciola sp. (2.30%), Toxocara (0.66%) and Monezia sp. (0.42%). Among non helmithic infection coccidian showed prevalence of 19.00%. Out of the seven zones, zone V (Central Narmada valley) had the highest prevalence (61.46%) and the Hills of Jhabua zone XII had the lowest prevalence (50.42%).

Prevalence in calves was more (59.78%) as compared to adult (54.36%). Season wise highest prevalence was observed in monsoon (73.41%) followed by winter (60.47%) and then summer (36.22%). Prevalence of coccidiosis (25.00%) was highest in winter. Monthly prevalence data showed highest prevalence in the month of

August (79.68%) and lowest in the month of April (28.25%). Mean EPG of strongylewas 321.8 and highest intensity of strongyle infection was recorded in the month of July (513.1). Coproculture examination revealed that Haemonchus being the predominant (72.08%) nematode genus, followed by Trichostrongylus (11.42%), Oesophagostomum (10.08%), Bunostomum (3.75%) and Strongyloides (2.67%). The current investigation provide basis to formulate strategic control measures against GI parasitism.

Keywords: buffalo, gastrointestinal parasite, prevalence, strongyle

INTRODUCTION

World population of buffaloes (Bubalus bubalis) is approximately 177.247 million of which 97% (171 million) are found in Asia. India constitutes about 55.7% (98.7 million) of the total world buffalo population (FAO, 2008). Buffaloes are important livestock because of their multifunctional purposes, providing milk,

EPIDEMIOLOGICAL STUDIES ON GASTROINTESTINAL PARASITES OF BUFFALOES IN SEVEN AGRO-CLIMATIC ZONES OF MADHYA PRADESH, INDIA

S. Nath1, G. Das1,*, A.K. Dixit1, V. Agrawal1, S. Kumar1, A.K. Singh2 and R.N. Katuri3

1Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandary, Jabalpur, Madhya Pradesh, India, *E-mail: [email protected] of Veterinary Parasitology, College of Veterinary Science and Animal Husbandary, Mathura, Uttar Pradesh, India3Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandary, Tirupati, Andhra Pradesh, India

Original Article


356

meat and good quality hides. They are considered as “tractors” in Southeast Asia in agriculture farms, means of transportation and their dung act as a good fertilizer and fuel (Liu et al., 2009). Gastrointestinal parasitism is one of the major constraints of livestock industry, severely affecting the animal productivity, retarded growth and increased susceptibility to other diseases. The economic losses may run into millions of rupees (Shah and Chaudhry, 1995).

But, the problem is neglected due to its chronic and insidious nature (Sanyal, 1998). The prevalence of gastrointestinal parasites and its severity depends on many factors such as local environmental conditions and management practices (Regassa et al., 2006). Studies of epidemiological pattern of the parasitic diseases in different agro-climatic zones of the country empower us to develop measures for strategic and tactical control of these diseases. Epidemiology and factors associated with prevalence of gastrointestinal parasites in domesticated animals of the Indian subcontinent is described by Chowdhury and Tada (1994). The incidence of GI parasites in buffaloes has been reported from different states of India (Haque et al., 2011; Wadhwa et al., 2011; Reddy et al., 2012; Singh et al., 2012 and Mir et al., 2013). A few reports are available on the prevalence of GI parasites in buffalo of Madhya Pradesh and adjoining area (Agarwal et al., 2002 and Pal et al., 2001). The present communication records such information pertaining to buffaloes of Madhya Pradesh to formulate control strategies against GI parasites.


The study was carried out in the state of

Madhya Pradesh which extend between latitude 21.20oN-26.87oN and longitude 74.02oE-82.49oE and considered as “Heart of India or Central India” and stretches over an area of 3,08,252 sq km. The average rainfall is about 1,370mm and having subtropical climate. The M.P state has been divided in 11 major agro-climatic zones and 50 districts (Figure 1). Present work has been carried out in 7 zones out of the eleven agro-climatic zones.

The study period was April 2011 to March 2012. A total of 3779 faecal samples were collected per rectally or immediately after defication from the selected villages throughout the year at monthly interval. These samples were collected in sterile labeled polythene bags and brought to the Department of Veterinary Parasitology, College of Veterinary Science& Animal Husbandry, Jabalpur. These samples were subjected to flotation and sedimentation technique (Soulsby, 1982). The positive samples were further checked for their intensity by Mc Master technique as described by Thienpont et al. (1979). The samples positive for nematodes were examined for the generic composition by glass tumbler method. The infective larvae were collected by Bearmann technique (Anon, 1977), cleaned and segregated by repeated centrifugation and decantation. These were identified as per the keys by Van Wyk et al. (2004). Data analysis was done according to Snedecor and Cochron (1980).


Overall prevalence Out of 3779 buffaloes coprologically examined during the period April 2011 to March 2012, 2103 (55.65%) were positive for different gastrointestinal parasitic infections (Table 1).


357

The prevalent parasites constituted strongyles, Strongyloides sp. and Trichuris sp. amongst nematodes and amphistomes, Schistosoma sp., Fasciola sp., Moniezia sp. and coccidian as non nematode GI parasites. Strongyles were the predominant (25.59%) nematodes, followed by Strongyloides sp. (3.15%) and Trichuris sp. (2.59%). Likewise, Kashyap et al. (1997) reported 40.3% prevalence of gastrointestinal helminthiosis in cattle and buffaloes from Madhya Pradesh where strongyle showed highest prevalence. Prevalence of amphistomosis was highest (28.10%) amongst the non-nematode parasites. The prevalence of coccidia, Schistosoma sp. and Fasciola sp. was 19.00%, 5.19% and 2.30% repectively (Table 1). Our study is in accordance with many workers reporting higher prevalence of strongyle in dairy animals (Haque et al., 2011; Singh et al., 2012 and Singh et al., 2008). The higher range of prevalence of amphistomes in the present study is in agreement with Yadav et al., (2004) and Kuchay et al., (2011). It may be due to factors such as wallowing habit, easy dispersion of faeces in water and bulk ingestion of grasses near the water source, increases the risk of amphistomosis due to availability of intermediate host (Radostitis et al., 1994).

Agro-climatic zone-wise prevalence Out of the seven zones, Central Narmada

Valley, zone IV had the highest prevalence (61.46%) and the Hills of Jhabua zone XI had the lowest prevalence (50.42%) (Table 2, Figure 2) however the difference is non-significant. Prevalence of strongyle was highest in zone I (31.11%) minimum in Zone III (19.67%). Zone II (Northen hills zones of Chhattisgarh) showed the highest prevalence of amphistome (36.46) and coccidian infection (23.54%). Zone VIII (Satpura Plateau) and zone I

(chhattisgarh Plains) showed the lowest prevalence of amphistome (18.96%) and coccidia (13.36%) respectively (Table 2).

Age wise prevalence Faecal samples of 2879 adult buffalo and 900 buffalo calf were examined coprologically. Prevalence of GI parasitism in buffalo calves (59.78%) was non-significantly higher compared to the adult (54.36%) (Table 3). The result of present investigation were in accordance with Haque et al. (2011) and Bilal et al. (2009) who reported higher prevalence of GI parasites in calves. In our country, calves are more prone to the parasitic infection due to inadequate attention towards management, treatment and disease control measures (Pfukenyi et al., 2007). Conversly Biswas et al. (2014) reported higher infection in adult. The cause of contradiction may be due to exhausted immune system, different grazing pattern and managemental practices. Adult buffaloes show very low level of prevalence of Toxocara vitulorum (0.07%) there is an inverse relationship between prevalence and age of animal (Halmandge et al., 2005). Coccidiosis was significantly higher in calves (P<0.01) than adult animals as the later exhibited cellular immunity against coccidiosis as a result of the previous exposure to the oocysts (Soulsby, 1982). Moreover the practice of using coccidiostat or coccidiocidal drug was very less in this region. Our finding was in accordance with Haque et al. (2011) and Singh et al. (2012). Moneziosis was not seen in adult buffaloes. Prevalence of amphistomosis was insignificantly higher in adult buffaloes.

Season wise prevalenceThe prevalence of G.I. parasitic infections

in summer, monsoon and winter season was 36.22%, 73.41% and 60.48%, respectively (Table


358

4). The prevalence of gastrointestinal parasites was significantly higher in Monsoon season (P<0.01). The onset and advancement of monsoon rains have profound effect on incidence and seasonality of gastrointestinal infection (Wadhwa et al., 2011). Significant higher infection rate of strongyle and amphistome infection, was seen in rainy season (P<0.01). Same results were seen in the study of Baseshankar and Maske (2000) but results of many scientists vary with the present findings and they have found different seasons for highest prevalence at their working area. Biswas et al. (2013) in Bangladesh, Samanta and Santra (2009) in West Bengal and Mir et al. (2013) in Jammu reported summer to be the prevalent season for GI parasitic incidence. The environmental condition of these regions was hot and humid with normal rainfall during summer providing favourable conditions for development and survival of pre parasitic stage of the parasites where as during rains there was excessive rainfall declining the pastural growth of pre parasitic larvae. In Madhya Pradesh rainfall occur mainly in monsoon providing suitable environment for the survival and propagation of preparasitic stages of parasite. Coccidiosis was significantly higher in winter season (P<0.01) because of the suitable temperature and humidity required for the oocystic sporulation at this time.

Intensity of strongyle infectionThe overall mean EPG (Egg Per Gram)

counts in the seven agro-climatic zones studied were and found to be moderate (321.8) (Figure 2). Wadhawa et al. (2011) reported high overall mean EPG (684.61). It may be due to the climatic and geographical variation. Highest intensity of strongyle infection was recorded in the month of July (513.1). The month wise intensity of strongyle infection is presented in Figure 4. Highest mean

EPG was observed in the rainy season followed by winter and summer. The present finding was supported by the results of Mathur et al. (1996) and Waruiru et al. (2000) reporting the monsoon season to be the highest intensity showing season. The mean intensity of strongyle infection was highest (362.1) in zone II and lowest in zone III (262.7).

Generic composition of nematode larvaeCoprocultural examination of faeces

revealed that Haemonchus was the predominant (72.08%) nematode genus, followed by Trichostrongylus (11.42%), Oesophagostomum (10.08%), Bunostomum (3.75%) and Strongyloides (2.67%). (Figure 3) This finding is consistent with the findings of earlier workers (Yadav et al. 2008) who reported Haemonchus to be the most common and pathogenic genus among various gastrointestinal nematodes causing high mortality and morbidity in India. Jamra et al. (2014) reported Haemonchus to be the most prevalent genera followed by Trichostrongylus and Oesophagostomum. Climatic and geographic condition of this region supports the propagation and growth of Haemonchus larvae.

In order to formulate effective control strategies against parasitic infection in any particular region we have to know the status of infection which can be only possible by the surveys as conducted in the present study. The results give a brief scenario about the prevalent parasites in Madhya Pradesh emphasizing amphistomes and strongyle to be most prevalent GI parasites. Among non-helminth parasites coccidian had the highest prevalence. It is imperative that integrated strategies and measures be taken to control helminth infections in buffaloes in Madhya Pradesh and elsewhere.


359

Tabl

e 1.

Ove

rall

prev

alen

ce (%

) of d

iffer

ent G

I par

asiti

c in

fect

ions

in b

uffa

lo o

f M.P

.

No.

E

xam

ined

Posi

tive

(%)

Posi

tive

for

GI N

emat

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Posi

tive

for

othe

r G

I par

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sSt

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Stro

ngyl

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sTr

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mph

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me

Schi

stos

oma

Mon

iezi

aC

occi

dia

3779

55.6

525

.59

3.15

2.59

0.66

2.30

28.1

05.

190.

4219

.00

Tabl

e 2.

Pre

vale

nce

(%) o

f GI p

aras

itism

in b

uffa

lo in

diff

eren

t Agr

o cl

imat

ic z

ones

of M

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Zon

eN

o.

Exa

min

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sitiv

e (%

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sitiv

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sitiv

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r ot

her

GI p

aras

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Stro

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eSt

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des

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s To

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ra

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I47

960

.13

31.1

13.

344.

180.

841.

4621

.50

6.05

1.04

13.3

6II

480

58.5

427

.50

3.96

3.13

1.04

2.71

36.4

66.

040.

8323

.54

III

900

54.0

019

.67

1.56

0.89

0.00

2.22

33.5

64.

780.

0019

.67

IV48

061

.46

26.4

63.

332.

711.

463.

9633

.75

6.25

0.63

21.8

8V

480

54.3

827

.29

3.33

2.71

0.63

2.08

22.0

85.

210.

8313

.75

VII

I48

052

.08

26.8

82.

712.

501.

251.

0418

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4.38

0.00

16.8

8X

I48

050

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25.4

25.

213.

540.

002.

7125

.63

3.96

0.00

23.3

3


360

Tabl

e 4.

Sea

sona

l pre

vale

nce

(%) o

f GI p

aras

itism

in b

uffa

lo in

M.P

.

Seas

onN

o.

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min

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sitiv

e (%

)Po

sitiv

e fo

r G

I Nem

atod

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sitiv

e fo

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her

GI p

aras

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ngyl

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sTo

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asci

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Am

phis

tom

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hist

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aM

onie

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Coc

cidi

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Sum

mer

1259

36.2

211

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1.03

1.91

0.00

1.43

18.9

82.

860.

2413

.82

Mon

soon

1260

73.4

140

.16

4.84

4.21

1.19

3.02

37.4

67.

620.

7922

.06

Win

ter

1260

60.4

825

.00

3.33

2.14

1.27

1.75

30.4

83.

890.

4825

.00

X2 Va

lue

35*

23*

17*

5.3*

(*)P

<0.0

1

Tabl

e 3.

Age

wis

e pr

eval

ence

(%) o

f diff

eren

t GI p

aras

ites i

n bu

ffalo

of M

.P.

Zon

eN

o E

xam

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361

Figure 1. Different Agro climatic zones of M.P.

All Zone

05

101520253035404550

Apr-11

May-11

Jun-11

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Aug-11

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Figure 2. Intensity of strongyle infection in buffalo of M.P.


362

ACKNOWLEDGEMENTS

The authors are highly thankful to the Dean, College of Veterinary Science and Animal Husbandry, NDVSU, Jabalpur for providing the facilities and “All India Network Programme on Gastrointestinal Parasitism” (ICAR) for fund required for conducting the research work.

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365

ABSTRACT

The theme of investigation was the herd of 1230 Murrah breed buffaloes at the dairy farm of Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana. The study was conducted for the episode of forty years 1971 to 2010, to make out the mortality pattern at this organized herd. The reasons for mortality were the affections of Digestive and Respiratory systems, also circulatory disturbances and the Unclassified reasons. The share of mortality of buffaloes with circulatory disturbances was principal i.e. 44.55%, followed by affections of digestive system 26.61%, unclassified condition were on the tune of 17.95% and the least was accounted at the affections of respiratory system with only 10.89%. The seasonal effect, period and parity have not any concern on the mortality pattern of animal and also it was non-significant too.

Keywords: buffalo, mortality, parity, period, season

INTRODUCTION

The main endeavour of the animal breeder

is to boost the genetic improvement in important economic traits of animals. Disposal of large number of animals from any herd due to various reasons greatly affects its economy. While watching at future genetic gain of the herd the intensive selection to decide an element of disposing off animals remains to be the tough job. Intensive selection remains directly proportional to the quantum of selection differential in large sized herd. The number of born female calves during a year and when they reach at puberty it becomes replacement stock. It is immense necessity to rear the progeny of proven parents to encompass the healthy and high yielding animals and it is the fundamental of any dairy animal improvement programme. Every farm has compulsorily replacements due to the death and culling on performance ground. The calf disposal plays the important role in maintaining the herd strength and farm standards (Reddy and Nagarcenkar, 1989). The present investigation was undertaken in Murrah buffaloes with the objective to know the major cause of mortality and effect of season, period and parity on it.


The statistics pertaining to ancestry,

STUDIES ON EFFECT OF NON-GENETIC PARAMETERS ON MORTALITY PATTERN IN MURRAH BUFFALOES

Nitin Mohan Gupta1,*, M.L. Mehra1 and Puneet Malhotra2

1Department of Animal Genetics and Breeding, Khalsa College of Veterinary and Animal Sciences, Amritsar, Punjab, India, *E-mail: [email protected] of Animal Genetics and Breeding, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India

Original Article



366

production and reproduction for the present exploration were collected from the history-cum-pedigree sheets, growth, production and reproduction records respectively maintained at the dairy farm of GADVASU, Ludhiana. The data with respect to these vital traits were composed over an episode of 40 years, i.e. from 1971 onwards to 2010 and pertained to 1230 animals that were born throughout the period of 1965 to 2010.

Statistical analysisThe statistics on mortality for the present

revision pertained to animals which died during different parity, season and period. The effect of various factors viz. parity, season and period were studied on mortality to appraise relative involvement of each factor. For estimating the effect of these factors, data on mortality of only females were taken, so as to keep away the biasness in results, as males were transferred/ auctioned at early age.

Since the frame of collected data follows discrete allotment, for that reason it was required to get transformed by means of Arcsine transformation and transformed data including sum of squares for various factors incorporated in the statistical model were analyzed by Least-squares analysis as explained by Harvey (1968).

The following statistical model was used to study the variation in mortality (data from 1971 to 2010) due to parity, season and period.

Yijkl = µ + Ri + Sj + Pk + eijkl

Where, Yijkl is the lth observation on mortality of animals

belonging to ith parity, died in jth season and kth period.

µ is the overall average of mortality percentage. Ri is the effect common to all animals belonging

to ith parity (i=1,2,3,4,5,6,7,8,9 and 10 & above parities).

Sj is the effect common to all animals died in jth season (j=1, 2, 3, 4, 5) and summer = 1, rainy = 2, autumn = 3, winter = 4 and spring= 5.

Pk is the effect common to all animals died in kth period (k=1, 2, …………,8).

eijkl is the random error, assumed to be NID (0, σ2e).


Causes of mortalityThe foremost causes for mortality were

acknowledged as disturbances in circulatory system, which accounted for 44.55 percent of entire mortality followed by affections of digestive system (26.93%) and affections of respiratory system (10.89%) while unclassified condition accounted for 17.63% of total mortality. Similar percentage (25.81%) of casualty due to affection of digestive system was reported by Malhotra (2003) in the herd of crossbred cattle.

Unclassified conditions accounted for 17.63 percent of whole mortality which includes NAD (Nothing Abnormality Detected) cases, be deficient in of proper diagnosis and putrefied carcasses ensuing from overdue post mortem of animals.

Season wise incidence of mortalityEach year was alienated into five season’s

viz. summer, rainy, autumn, winter and spring (Table 2). Uppermost mortality was observed in winter (25%) followed by summer (24.03%) and rainy season (23.4%) whereas, highest occurrence of mortality of 38.29% was observed by Patil et al. (1992) during winter in Surti buffaloes. Parallel result was reported by Mourad and Rashwan


367

(2001) and Rana et al. (2010) in winter in Murrah buffaloes. Pooled percentage of these three seasons constitutes round about three forth (72.43%) of total season wise mortality. Towering mortality during summer season may be owing to the less efficient thermo-regulatory mechanism in buffaloes. The black colour of buffalo absorbs supplementary heat during sunny days, as a result it directs to the state of heat stress, which may be compensated by providing better managemental conditions. On the other hand, higher frequency of fatality during rainy season may be owing to increased dampness and underprivileged sanitary environment due to heavy rainfall over tiny period of time. Smallest amount of mortality was evidenced in autumn (11.22%) followed by spring season (16.35%), which is possibly due to temperature which is neither too high nor too low in these seasons. The effect of season on mortality was found to be non significant (P>0.05); this is in agreement with the findings of Khan et al. (2007).

Period wise incidence of mortalityThe mortality accounts were taken from

post-mortem records between 1971 to 2010. These years were alienated into 8 periods of five year each (Table 3). The leader percentage of mortality of 26.28 percent was stuck between the period of 1975 to 1980, which alone constitutes round about one fourth of total period wise occurrence of mortality and the slightest of 6.74 percent of mortality accounted between the periods of 1981 to 1985. The effect of period on mortality was found to be insignificant (P>0.05).

Parity/Lactation wise incidence of mortalityParity wise mortality was calculated in

ten categories viz. first parity to tenth and above parities underneath four disease situation. There

were 312 lactating animals (females) died out of 1230 dead animals which constitutes about 25.36 percent of whole mortality (Table 4). Greatest mortality was observed in first parity or lactation (22.44%), which accounted for one fourth of total mortality, followed by third (16.35%), fourth (14.42) and second parity (13.14%). Least mortality was observed in 10th parity and above, this may be due to the reason that a very few animals can reach up to this parity as most of the animals died due to different diseases prior to reaching up to 10th and above parities. Out of all the diseases, bulk of the animals died owing to affections of circulatory trouble (44.55%) followed next to affection of digestive system (26.9%), unclassified condition constitutes (17.6%) and least mortality was due to affections of respiratory system which contributes (10.89%) of entire mortality. The effect of parity on mortality was established to be non significant (P>0.05).

CONCLUSION

The results of in hand study sketches the attention headed for circulatory disturbances and affections of the digestive system which accounts for more or less three fourth of full amount of mortality. Roughly one fourth of the whole mortality had been occurred all through in initial parity. All these factors designate the call for the improved management and anticipatory measures to be followed for dropping the mortality, so as additional number of animals will be available for future replacements, which will greatly enhance selection intensity, selection differential and moreover genetic gain at the farm.


368

Table 1. Reasons for mortality in buffaloes.

Disease code

Causes of mortalityTotal number of animals died in

40 years

Average number of animal died

per year

Percentage of animals died

A Affections of Digestive system 331 8.27 26.93a

BAffections of Respiratory

system134 3.35 10.89bc

C Circulatory disturbances 548 13.7 44.55a

D Unclassified 217 5.42 17.63ac

Total 1230 30.75 100.00

Table 2. Season wise incidence of mortality in buffaloes.

SeasonTotal number of

animal diedAverage number of

animal died per yearMortality (%)

Summer 75 1.875 24.03a

Rainy 73 1.825 23.40a

Autumn 35 0.875 11.22bc

Winter 78 1.95 25.00a

Spring 51 1.275 16.35bc

Total 312 7.8 100.00

Table 3. Period wise mortality in buffaloes.

PeriodTotal number of

animal diedAverage number of

animal died per yearMortality (%)

1971-1975 29 0.725 9.29ab

1976-1980 82 2.05 26.28a

1981-1985 21 0.525 6.74ac

1986-1990 28 0.70 8.98ac

1991-1995 48 1.20 15.38bc

1996-2000 37 0.925 11.86a

2001-2005 38 0.95 12.18a

2006-2010 29 0.725 9.29ab

Total 312 7.8 100.00


369

Table 4. Parity wise incidence of mortality in Murrah buffaloes.

Parity/ Lactation

Mortality Code No. of animals

died

% of total diedA B C D

1 15 8 35 12 70 22.44a

2 12 3 15 11 41 13.14bc

3 16 3 24 8 51 16.35bc

4 14 5 21 5 45 14.42bc

5 11 7 13 5 36 11.54bc

6 9 3 11 1 24 7.70ac

7 4 3 9 3 19 6.10ac

8 1 0 4 6 11 3.53acd

9 2 2 3 1 8 2.54cd

10 & above 0 0 4 3 7 2.24cd

Total died 84 34 139 55 312 100.00

A-Affections of Digestive system, B- Affections of Respiratory system, C-Circulatory disturbances, D- Unclassified condition Note: The means with at least one common alphabet as superscript do not differ significantly from each other.

Table 5. Analysis of Variance for factors affecting mortality in Murrah buffalo.

SourceDegree of freedom

Mean squares F value

Sire 99 57.51 15.55*Period 7 4.78 1.29NS

Season 4 8.29 2.24NS

Parity 9 6.35 1.71NS

Residual 594 3.69Total 713

* P < 0.05, NS = Non-significant


370

REFERENCES

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Khan, Z.U., S. Khan, N. Ahmad and A. Raziq. 2007. Investigation of mortality incidence and managemental practices in buffalo claves at commercial dairy farms in Peshawar city. Journal of Agricultural and Biological Science, 2(3): 16-21.

Malhotra, P. and O.S. Parmar. 2005. Studies on Culling reasons vis-s-vis Milk production in Cows. Indian Journal of Dairy Science, 58(6): 433-435.

Mourad, M. and S. Rashwan. 2001. Milk production of buffaloes and causes of calf mortality under a semi-intensive production system in Egypt. Revue d’Élevage et de Médecine Vétérinaire des Pays Tropicaux, 54(2): 139-145.

Patil, N.A., S.P. Kumar, S. Mallikarjunappa, K.R. Lakshmaiah and A.R.S. Bhat. 1992. Calf mortality in Surti buffaloes. Indian Vet. J., 69: 1018-1022.

Rana, N., S. Khanna, A.A. Raut, S.R. Bhardwaj, A. Manuja, B. Manuja, A. Saini, S. Kakkar, K.L. Khurana and R.K. Sethi. 2010. Retrospective epidemiological analysis of mortality trends in neonatal and growing Murrah buffalo calves at an organized herd. Indian J. Anim. Sci., 80(10): 976-979.

Reddy, K.M. and R. Nagarcenkar. 1989. Studies on disposal pattern in Sahiwal calves. Indian Journal of Dairy Science, 42: 280-288.

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371

ABSTRACT

In the present study, breeding information spread over a period of 14 years from 1995 to 2008, was collected from the history-cum-pedigree sheets and milk yield registers of Murrah buffaloes maintained in four centres of Network Project on Murrah Buffalo Improvement (National Dairy Research Institute, Karnal; Central Institute for Research on Buffalo, Hisar and Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana and Choudhary Charan Singh Haryana Agricultural University, Hisar). Data on first lactation traits of 832 Murrah buffaloes sired by 95 bulls were used for the study. Farm had significant effect on FL305MY, while season and year of calving did not affect significantly in the present study. Breeding value for first lactation 305 days milk yield was estimated using best linear unbiased prediction (BLUP) method. The breeding value of different bulls varied from 1630.40 kg in fifth set to 2022.61 kg in seventh set.

Keywords: BLUP, breeding value, FL305MY, murrah buffalo

INTRODUCTION

Selection of the superior sires with maximum accuracy is of utmost importance for any breed improvement programme, as sires are easily and rapidly disseminated in various herds under progeny testing programme. Robertson and Randle (1954) opined that as much as 61% of genetic gain in dairy cattle resulted from selection of sires through two paths, i.e. bulls to breed cows and bulls to breed bulls. Hence, accurate selection of bulls used in artificial insemination (AI) programme is of prime importance for long-term genetic progress in the population.

The prediction of breeding values constitutes an integral part of most breeding programmes for genetic improvement of the sire for different economic traits. The accuracy of estimating the breeding value of an animal is the major factor that affects the genetic progress due to selection. The sire evaluation based on milk yield was most widely used criteria. To make rapid genetic progress in performances through selection for traits of economic importance, the animals must be chosen accurately for their superior breeding values. Over the times various methods have been used for sire evaluation, Henderson’s (1973) mixed model or best linear unbiased prediction (BLUP)

EVALUATION OF BREEDING VALUES MURRAH BUFFALO BULLS UNDER ORGANIZED FARMS

Vijay Kumar* and A.K. Chakravarty

Department of Animal Genetics and Breeding, Mathura Veterinary University, UP Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go-Anusandhan Sansthan (DUVASU), Mathura, Uttar Pradesh, India, *E-mail: [email protected]

Original Article


372

procedure has become the method of choice for evaluating the genetic worth of the bulls. BLUP is one of the accurate sire evaluation methods to obtain unbiased estimates of breeding values of sires (Mukherjee et al., 2007). The model of analysis under BLUP takes into account, the fixed effect and relationship among animals. Therefore, the breeding values of animals are estimated with higher accuracy.


The Murrah bulls in 7 sets (11, 12, 15, 14, 15, 16 and 12 bulls) were inducted for progeny testing at Central Institute for Research on Buffalo (CIRB), Hisar, National Dairy Research Institute (NDRI), Karnal Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, Choudhary Charan Singh Haryana Agricultural University (CCSHAU), Hisar. The daughters of first 7 sets have completed their first lactation records. The first lactation 305 days milk yield (FL305MY) records of 832 daughters of 95 bulls calved during 14 years from 1995 to 2008, were used for this study. The period of 14 years was divided into 14 years. Each year of calving was further classified into 2 seasons, viz. most calving season (January to June) and least calving season (July to December) based on calving pattern. All information was classified in four farms viz. NDRI, CIRB, GADVASU and CCSHAU. The breeding value of sires was estimated by best linear unbiased prediction (BLUP) method as given by Henderson (1973). The model of BLUP estimation was considered as follows:

Y= Xb +Za + e where, Y, b, a and e denotes the vector

of observations (FL305MY), fixed effects (farm, season and period effect), random effect (sire effect) and random error and X and Z are incidence matrices pertaining to fixed effects and random effects.


The overall least squares mean of total first lactation milk yield (Table 1) was, however, lower than that reported by Patil (2011) and Geetha (2005) in Murrah buffalo. Higher than this was reported by Katneni (2007). Farm had significant effect on FL305MY in the present study. Centre-wise least-squares means for 305MY for NDRI, CIRB, GADVASU and CCSHAU were found to be 1792.45±31.94, 1684.71±37.88, 1941.02±42.90 and 1969.26±104.80 kg, respectively. Season and year of calving did not affect significantly the FL305MY of Murrah buffaloes in the present study.

The information on bulls along with their breeding values is given in Table 2 to Table7. The breeding value of different bulls varied 1683.44 to 1976.89 kg in first set, 1746.03 to 1952.91 kg in second set, 1720.94 to 1929.56 kg in third set, 1730.12 to 1907.14 kg in fourth set, 1630.40 to 2011.21 kg in fifth set, 1710.75 to 1981.79 kg in sixth set and 1709.76 to 2022.61 kg in seventh set. The highest breeding value was observed for sire 88 (set 7) followed by 66 (set 5) and 69 (set 6). Singh and Singh (1999) observed breeding value of Murrah bulls between 1137.30 to 1329.01 kg using BLUP method. Pandey and Singh (1999) computed breeding value of 52 Murrah bulls for first lactation milk yield by corrected contemporary daughter average index and reported it ranges from 1349.62 to 1934.39 kg.


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Table 1. Least-squares means of first lactation milk yield in Murrah buffaloes.

Factor No. of Observation FL305MYOverall (µ) 832 1846.86±35.94*FarmNDRI 305 1792.45±31.94b

CIRB 314 1684.71±37.88a

GADVASU 188 1941.02±42.90c

CCSHAU 25 1969.26±104.80c

Season of calvingLeast calving season 291 1881.96±43.57 Most calving season 541 1811.75±36.94 Year of calving

1995 5 2070.52±220.35 1996 19 1956.38±117.84 1997 33 1754.71±92.10 1998 9 1665.17±165.24 1999 77 1884.81±62.23 2000 66 1860.91±65.152001 67 1736.42±65.23 2002 72 1804.10±63.26 2003 108 1788.41±53.71 2004 91 1871.62±57.91 2005 81 1903.16±56.12 2006 87 1855.29±57.40 2007 89 1889.94±53.04 2008 28 1814.57±93.51

*Significant P <0.05; Values with different superscript differ significantly: Milk yields are in kg.


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Table 2. Breeding values of Murrah buffalo bulls in set 1.

Sire No. of Daughters Breeding Value Rank1 5 1946.02 112 6 1962.25 63 8 1976.89 44 18 1948.54 105 19 1856.7 446 18 1683.44 947 10 1694.78 938 4 1884.15 329 5 1840.46 5310 2 1804.92 6211 11 1819.11 58

Table 3. Breeding values of Murrah buffalo in set 2.

Sire No. of Daughters Breeding Value Rank12 9 1835.51 5513 8 1889.71 3014 11 1906.64 2315 7 1952.91 716 2 1863.89 4017 8 1882.1 3318 11 1754.49 7719 15 1927.16 1520 10 1841.04 5221 9 1749.77 7922 9 1746.03 8023 13 1771.69 74


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Sire No. of Daughters Breeding Value Rank24 9 1829.47 5625 4 1929.56 1426 8 1905.36 2527 11 1775.59 7328 3 1916.97 1929 6 1920.06 1830 4 1809.36 6031 3 1767.56 7532 2 1892.23 2933 3 1915.45 2034 5 1757.44 7635 21 1781.17 7136 11 1808.65 6137 7 1720.94 8838 9 1875.56 36


Sire No. of Daughters Breeding Value Rank39 18 1860.85 4340 9 1781.36 7041 5 1845.41 4942 9 1793.57 6643 6 1796.92 6544 5 1856.17 4545 6 1730.12 8546 11 1899.66 2747 7 1878.6 3448 9 1743.18 8149 11 1875.07 3750 5 1845.71 4851 11 1907.14 2252 8 1841.94 51


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Sire No. of Daughters Breeding Value Rank53 16 1728.66 8754 3 1848.07 4755 12 1630.4 9556 7 1789.89 6857 6 1800.28 6458 6 1790.09 6759 6 1862.63 4160 8 1842.65 5061 3 1698.94 9262 6 1838.57 5463 11 1888.87 3164 9 1803.98 6365 22 1963.15 566 12 2011.21 267 12 1933.16 13

Table 7. Breeding values of Murrah buffalo bulls in set 6.Sire No. of Daughters Breeding Value Rank68 10 1735.45 8369 20 1981.79 370 7 1788.69 6971 2 1710.75 9072 3 1819.02 5973 4 1905.32 2674 8 1875.61 3575 15 1867.7 3876 14 1925.69 1677 5 1728.86 8678 7 1825.39 5779 10 1949.13 980 10 1906.24 2481 5 1951.98 882 4 1716.71 8983 7 1739.52 82


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REFERENCES

Geetha, E. 2005. Studies on breeding values for persistency in Murrah buffaloes. M.Sc. Thesis, National Dairy Research Institute, Deemed University, Karnal Haryana, India.

Henderson, C.R. 1973. Proceedings of the Animal Breeding and Genetics Symposium in honor of Dr. Jay L. Lush. American society of Animal Science and American Dairy Science Association, Chanpaign, Illinois, USA.

Katneni, V.K. 2007. Studies on breeding values for persistency in Murrah buffaloes. Ph.D. Thesis, National Dairy Research Institute, Deemed University, Karnal, India.

Mukherjee, S., B.K. Joshi and G.K. Gaur. 2007. Comparison of sire evaluation methods In Friewal cattle. Indian J. Anim. Sci., 77: 773-776.

Pandey, A.K. and H. Singh. 1999. Comparison of different methods of sire evaluation in


Sire No. of Daughters Breeding Value Rank84 18 1861.04 4285 8 1749.87 7886 13 1780.19 7287 8 1921.59 1788 6 2022.61 189 4 1735.16 8490 3 1709.76 9191 13 1899.06 2892 6 1909.69 2193 13 1864.92 3994 9 1848.93 4695 17 1934.11 12

Murrah buffaloes. Indian J. Anim. Sci., 69: 1067-1069.

Patil, C.S. 2011. Genetic evaluation of fertility in Murrah buffalo. M.V.Sc. Thesis, National Dairy Research Institute, Deemed University, Karnal, India.

Robertson, A. and J.M. Randel. 1954. The performance of heifers got by artificial insemination. J. Agri. Sci., 44: 184-192.

Singh, P.K. and B.P. Singh. 1999. Efficacy of different methods in genetic evaluation of Murrah sires. Indian J. Anim. Sci., 69: 1044-1047.



379

Original Article

ABSTRACT

Study on the incidence of hydatidosis in food animals meant for human consumption such as buffaloes was done at the time of slaughter by inspecting the carcasses and viscera for the presence of hydatid cysts with particular reference to lungs, liver, spleen etc., Based on the observation, the incidence of hydatid cysts in buffaloes examined was found to be 11.11 percent. With regard to the organ wise involvement, the presence of hydatid cysts was more in lungs, followed by liver and the fertility rate of hydatid cysts was high in lungs.

Keywords: hydatidosis, incidence, fertility, buffaloes

INTRODUCTION

Hydatidosis, a zoonotic parasitic disease of animals and man is caused by the larval stage (metacestode) of the dog tapeworm Echinococcus granulosus, the life cycle involving two mammalian hosts. Definitive hosts are carnivores such as dogs and the intermediate hosts are herbivores and omnivores wherein the development of the cysts occurs in liver, lungs and other organs. Incidence of hydatidosis has been reported earlier by Sundaram and Natarajan (1960) by examination

of animals slaughtered in Chennai. Hydatidosis in animals results in significant economic loss to the meat industry through condemnation of infected organs such as liver, lungs and other organs apart from reduced quality of milk, meat and wool. Hence, a study was done to know the incidence of the hydatid disease in slaughtered buffaloes in Chennai as well as the organ wise involvement and the fertility status of the hydatid cysts.


Buffaloes were observed for the presence of hydatid cysts in lungs, liver and other organs at the time of slaughter in the Corporation slaughter house, Chennai by inspecting the carcasses and viscera of the slaughtered animals. The visceral organs harbouring the hydatid cysts were collected and brought to the laboratory so as to ascertain the fertility or sterile nature of the hydatid cysts based on the presence or absence of protoscolices in the hydatid cyst fluid.


A total of 810 buffalo were screened and observed for the presence of hydatid cysts at slaughter. Out of the 810 buffaloes, 90 buffaloes

Department of Veterinary Parasitology, Madras Veterinary College, Tamilnadu Veterinary and Animal Sciences University, Chennai, India, *E-mail: [email protected]

INCIDENCE AND FERTILITY STATUS OF HYDATID CYSTS IN BUFFALOES

A. Sheeba, A. Sangaran*, B.R. Latha and A. Raja



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showed the presence of hydatid cysts giving an overall incidence of 11.11% in buffaloes. Out of 90 hydatid cysts observed in various organs, 57 hydatid cysts were recovered from lungs, 17 from liver, 15 from lungs and liver and one hydatid cyst from muscle. Forty two cysts were found fertile out of the 90 hydatid cysts observed. With regard to fertility status of the cysts, 27 hydatid cysts from lungs (64.30%), 7 hydatid cysts from liver (16.6%), 7 hydatid cysts from lungs and liver (16.66%) and one hydatid cyst from muscle (2.38%) were found to be fertile.

The prevalence of hydatidosis in cattle has been reported to vary from 7.6% (Deka et al., 1983) to as high as 56.6% (Himonas et al., 1994 and Daryani et al., 2009). The findings on the incidence of hydatid cysts as 11.11% in buffaloes in the present study correlates with the earlier reports.

Variations in the prevalence could be due to the changes in the temperature, environmental conditions, and the management practices adopted in rearing the animals. The present study showed lower prevalence in comparison with the earlier reports. Contrary to past decades, various precautions and changing in behavior pattern such as awareness about the disease, routine deworming of dogs against tapeworms as well as decrease in the number of stray dogs could be the major reasons for the decrease in the incidence of hydatid cysts (Beyhan and Umur, 2011).

With regard to fertility status of hydatid cysts recorded from different viscera, it was observed that 64.30% of hydatid cysts from lungs were found to be fertile, followed by 16.66% from liver. Kosalaraman and Ranganathan (1980) in Madras had reported 35 percent of fertile cysts from lungs, 28% from liver. Koshy (1984) reported 20% of fertile cysts in liver, 28% in lungs and 36% in spleen. In case of food animals like sheep, goats

and buffaloes, maximum number of fertile cysts were recorded in liver (59%) followed by lungs and spleen (Sangaran and Lalitha, 2013). Variation in the cyst fertility might be due to the difference in tissue resistance among the visceral organs. The reason for higher prevalence of fertile cyst in lungs in the present study might be due to softer consistency of lungs and also large capillary fields encountered by the oncospheres (Urquhart et al., 1996). The result of the present study correlates well with the earlier observations of Kosalaraman and Ranganathan (1980) and Koshy (1984) on fertility nature of hydatid cysts.

REFERENCES

Beyhan, Y.E. and S. Umur. 2011. Molecular characterization and prevalence of cystic echinococcosis in slaughtered water buffaloes. Turkish Vet. Parasitol., 181: 174-179.

Daryani, A., M. Sharif, A. Amouei and M. Nasrolahei. 2009. Fertility and viability rates of hydatid cysts in slaughtered animals in the Mazandaram province, northern Iran. Trop. Anim. Health Pro., 20(2): 44-48.

Deka, D.K., G.C. Srivastava and R.C. Chhabra. 1983. Incidence of hydatidosis in ruminants. Indian J. Anim. Sci., 53: 200-202.

Himonas, C., A. Sotiriadou and E. Papadopoulos. 1994. Hydatidosis of food animals in Greece: prevalence of cysts containing viable protoscolices. J. Helminthol., 68: 311-313.

Kosalaraman,V.R. and M. Ranganathan. 1980. A survey of disease conditions of lungs of buffaloes. Cheiron, 9: 281-284.

Koshy, T.J. 1984. Taenid infections in dogs. Ph.D.


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Thesis, Tamil Nadu Agricultural University, Coimbatore, India.

Sangaran, A. and L. John. 2013. Incidence and organ wise involvement of hydatidosis in buffaloes. Buffalo Bull., 32(Special Issue 2): 1009-1010.

Sundaram, R.H. and R. Natarajan. 1960. A study on the incidence of hydatid disease in cattle in the city of Madras. Indian Vet. J., 37: 19-24.

Urquhart, G.M., J. Armour, J.L. Duncan and A.M. Jennings. 1996. Vet. Parasitol., 2nd ed. Blackwell Science Ltd, United Kingdom, p. 307.



383

ABSTRACT

A total of 102 serum samples were collected randomly from buffaloes exposed with different clinical conditions (abortion, repeat breeding, fever, mastitis, anorexia) suspected for leptospirosis and apparently healthy. These serum samples were subjected to seroepidemiogical study using microscopic agglurination test (MAT) having different serovars of Leptospira spp. The seroprevalence of leptospirosis among buffaloes was noted to be 15.69% (16/102). All the three districts of South Gujarat showed the presence of leptospiral antibodies without any significant difference (P≤0.05) with the highest rate in Tapi (50.00%) followed by Navsari (14.89%) and Surat (13.72%). Jafrabadi breed showed 50.00% seropositivity followed by Surati (16.67%), Mehsana (15.00%) and Non-Descript (5.55%). In female buffaloes the seroprevalence positivity was noted in 16.49% cases. However, none of male exhibited seropositivity. In respect of age groups the highest rate of seropositivity (19.23%) was observed in age group of 1 to 4 years followed by above 4 years (15.71%) and below 1 year

(00%) without significant difference (P≤0.05). In buffaloes out of 102 sera screened, 16 were positive with one or more serovars. The highest number of seropositivity was recorded against serovar Kaup (17.39%).

Keywords: buffaloes, Leptospirosis, seroepidemiology, zoonosis, MAT

INTRODUCTION

Leptospirosis is an economically important widespread zoonotic disease caused by pathogenic species of Leptospira interrogans occurs in man and different species of animals like cattle, buffalo, sheep, goat, deer, pig, rodents, camel, horse, sealion, shank, raccoon (Cutler et al., 2005; Cheema et al., 2007). The causative agent is frequently excreted though urine of infected individual and contaminate the environment, there by exposing human being especially farmers, agriculture labour and animal holders.

The state of Gujarat, Maharastra, Tamil Nadu, Kerala and Andaman and Nicobar Islands

SEROEPIDEMIOLOGICAL STUDY OF LEPTOSPIROSIS IN BUFFALOES OF SOUTH GUJARAT, INDIA

J.M. Patel1,*, P.D. Vihol1, V. S. Dabas2, M.C. Prasad1, J.H. Patel3, C.F. Chaudhari4, N.B. Patel5 and K.M. Patel6

1Department of Veterinary Pathology, *E-mail: [email protected], [email protected],2Departmant of Veterinary Surgery and Radiology,3Department of Veterinary Pharmacology and Toxicology, 4Department of Veterinary Gynaecology and Obstetrics, 5Department of Livestock Production and Management, 6Department of Animal Husbandry, Vanbandhu College of Veterinary Sciences and Animal Husbandry, Navasari Agricultural University, Eru cross road, Navsari, Gujarat, India

Original Article

mailto:E-mail:%[email protected]


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are endemic in India and incidences in humans are perennially reported from these states during monsoon season (Maskey et al., 2006). Hence the present seroepidemiological study was conduct to determine the magnitude of occurrence pattern of leptospirosis in buffaloes, reared in rural areas of various district i.e. Navsari, Surat and Tapi of South Gujarat.


Animal seraA total of 102 serum samples were

collected randomly from different age groups and breeds of buffaloes (n=102) of either sex reared in villages of various districts (Navsari, Surat, Tapi) of south Gujarat (Table 1). Whole blood samples were collected from jugular vein directly or during slaughter of buffaloes in sterile 9.0 ml plain vacutainers. To obtain serum, whole blood was kept in slanting position in 9.0 ml plain vacutainers until serum extracted out of the whole blood. Then these 9.0 ml plain vacutainers were centrifuged at 7000 rpm for 10 minutes. The straw coloured serum was collected into 1.5 ml sterile cryo vials and aliquoted and stored at -20oC for carriying out MAT.

MICROSCOPIC AGGUTINATION TEST (MAT)

All the sera were tested for antibodies against live antigens of Leptospira sp. serovars Pyrogenes, Australis, Bankinang, Grippotyphosa, Patoc, Pomona, Icterohaemorrhagiae, Hebdomadis, Canicola, Hardjo, Bellum, Bataviae, Tarassovi, Shermani, Kaup, Hurstbridge and Javanica by

Microscopic agglutination test at Leptospirosis Reference Laboratory, Government Medical College, Surat (Vijayachari et al., 2001) and Project Directorate on Animal Disease Monitoring and Surveillance (PD-ADMAS), Bangalore using standard procedure (WHO-OIE, 2013; Faine, 1982).

STATISTICAL ANALYSIS

Chi-square test was used according to WEB AGRI STAT PACKAGE software developed by Jangam and Wadekar, ICAR research complex, Goa for statistical analysis of data (Jangam and Wadekar, 2012).


In the present study a total 102 sera were screened from three different districts of South Gujarat (Navsari, Tapi, Surat) for leptospiral antibodies. The details of district, breed, sex and age wise seroprevalence results are depicted in Table 1.

The seroprevalence of leptospirosis among buffalo was noted to be 15.69% (16/102) and was comparable to reported prevalence of 14.55% in Gujarat (Savalia, 2001), 14.7% in Uttaranchal (Agrawal et al., 2005) and 15% in Uttaranchal, Tamil Nadu and Uttar Pradesh (Mariya et al., 2007). In contrast to above findings higher seroprevalence was 54.14% in Gujarat (Balakrishnan et al., 2011), 26.66% in Andaman and Nicobar Islands (Varma et al., 2001) and 88.8% (125/111) in Chennai buffaloes (Selvaraj et al., 2010) have been reported.

All the three districts of South Gujarat showed the presence of leptospiral antibodies


385

without any significant difference (P≤0.05) with the highest rate in Tapi (50.00%) followed by Navsari (14.89%) and Surat (13.72%). Comparable findings of leptospirosis seroprevalence were reported by Savalia (2001) from Valsad (17.24%), Navsari (19.35%) and Surat (22.50%) districts.

No significant difference in seroprevalence was observed among samples tested from the different breeds of buffalo. Jafrabadi breed showed 50.00% seropositivity followed by Surati (16.67%), Mehsana (15.00%) and Non-Descript (5.55%). Contrary to this Balakrishnan et al. (2011) reported Murrah breed to be most susceptible (58.25%) followed by Pandharpuri (40.91%) and Jaffrabadi (37.50%). In the present study prevalence rate of leptospirosis in Jafrabadi breed of buffalo was higher (50.00%) than reported earlier by Balakrishnan et al. (2011). There is every likelihood that the breed susceptibility reported by various workers is influenced by the sample size in particular area where specific breed might be prevalent but need further elucidation.

In female buffaloes the seroprevalence positivity was noted in 16.49% cases. However, none of male exhibited seropositivity. Possibly because a wide gap occurred in the number of samples processed (Male-5 and Female-97).

In respect of age groups (above 4 years, 1 to 4 years and below 1 year) the highest rate of seropositivity (19.23%) was observed in age group of 1 to 4 years followed by above 4 years (15.71%) and below 1 year (00%) without significant difference (P≤0.05). It was not in agreement with findings of Balakrishnan et al. (2011) who observed maximum seropositivity (77.05%) among buffaloes between 4 to 7 years (adult group) followed by above 7 years (75.00%, older age group) and below 4 year (26.67%) with significantly different among age group (P≤0.01). Agrawal et al. (2005) studied

the seroprevalence in cattle, buffaloes and goats and reported higher seropositivity in buffalo above 9.0 years of age and concluded that seropositivity increases with advancing age irrespective of animal species involved.

A total of 102 sera screened, 16 were positive with one or more serovars. In the present study highest number of seropositivity was recorded against serovar Kaup (17.39%) followed by Grippotyphosa (13.04%), Pomona (13.04%), Javanica (13.04%), Patoc (8.70%), Canicola (8.70%), Hardjo (8.70%), Bataviae (8.70%), Autumnalis/Bankinang (4.35%), Hurstbridge (4.35%). As against the present observations the most common serovars in Indian buffaloes reported by earlier workers from different states included Hardjo (Agrawal et al., 2005; Balakrishnan et al., 2011), Grippotyphosa (Varma et al., 2001) and Pomona (Selvaraj et al., 2005; Selvaraj et al., 2010). However serovars distribution seen in South Gujarat region in present investigation was comparable with the findings of Savalia (2001) and Balakrishnan et al. (2011) who reported serovars Hardjo, Grippotyphosa, Australis, Hebdomadis, Ballum and Pomona. Other serovars reported from different states of India enlisted Grippotyphosa, Pomona and Australis from Andaman and Nicobar (Varma et al., 2001), Hardjo, Javanica and Australis from Uttaranchal (Agrawal et al., 2005), Pomona, Hebdomadis, Tarassovi, Sejroe, Australis, Pyrogenes, Autumnalis, Grippotyphosa, Ballum, Javanica, Icterohaemorrhagiae, Canicola from Chennai (Selvaraj et al., 2005; Selvaraj et al., 2010).

Major areas of South Gujarat districts are used for paddy and sugarcane cultivation and are rich in natural vegetation with plenty of marshy lands and small ponds/water logging areas with almost neutral pH, suitable humidity and


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Table 1. Seroprevalence of leptospirosis in buffaloes.

Attributes No. of Tested No. of PositivePercent Positive

RegionSouth Gujarat 102 16 15.69

DistrictsNavsari 47 07 14.89Tapi 04 02 50.00Surat 51 07 13.72

Total 102 16 15.69χ2 = 5.99 NS(P<0.05)Breed wise

Surati 60 10 16.67Mehsani 20 03 15.00Jafrabadi 04 02 50.00Nondiscript 18 01 5.55

Total 102 16 15.69χ2 = 7.82 NS(P<0.05)Sex wise

Male 05 00 00Female 97 16 16.49

Total 102 16 15.69χ2 = 3.84 NS(P<0.05)Age wise

<1 year 06 00 001-4 years 26 05 19.23>4 years 70 11 15.71

Total 102 16 15.69χ2 = 5.99 NS(P<0.05)

Note: NS-Non significant at P<0.05


387

temperature which are optimum for growth/survival of leptospires and perpetuation in the environment. Cultivated fields infested with rats, a carrier for leptospires (Collares-Pereira et al., 2000) also play an important role in spreading the infection. Prevalence in buffalo could be attributed to their habit of wallowing in water bodies contaminated with infected urine (Treml et al., 2002) as one of the main sources of transmission of leptospira.

The present study concludes that seroprevalence of leptosprosis among buffaloes in South Gujarat region was ranged from 14.55% (Savalia, 2001) to 54.14% (Balakrishnan et al., 2011) with presently reported seroprevalence was 15.69% without significant difference between age, breed, sex and different district. The continuous presence of Leptospires in this area lead to potential zoonotic risk to slaughter house workers, meat inspectors, animal holder, agriculture labour and farmers. This study also determines the need for continuous monitoring of leptospirosis in animal and humans to combat this zoonotic infection.

REFERENCES

Agrawal, R., M. Kumar, M. Kumar and S.K. Srivastava. 2005. Epidemiological pattern of leptospirosis in livestock of Uttaranchal state. Indian Journal of Comparative Microbiology, Immunology and Infectious Diseases, 26(2): 109-113.

Balakrishnan, G., G.P. Roy, R. Govindarajan, V. Ramaswamy and B.M. Manohar. 2011. Seroepidemiological studies on leptospirosis among bovines in organized farm. IJAVMS., 5(6): 511-519.

Cheema, P.S., S.K. Srivastava, R. Amutha, S. Singh, H. Singh and M. Sandey. 2007. Detection of

pathogenic leptospires in animals by PCR based on lipL21 and lipL 32 genes. Indian J. Exp. Biol., 45: 568-573.

Collares-Pereira, M., M.L. Mathias, M. Santos-Reis, M.G. Ramalhinho and P. Duarte-Rodrigues. 2000. Eur. J. Epidemiol., 16: 1151.

Cutler, S.J., A.M. Whatmore and N.J. Commander. 2005. Brucellosis-new aspects of an old disease. J. Appl. Microbiol., 98: 1270-1281.

Faine, S. 1982. Guidelines for the control of leptospirosis. WHO. Offset Publication 67, Geneva, Italy.

Jangam, A.K. and P. Wadekar. 2012. Web Agri State Package. ICAR research complex for Goa.

Mariya, R., S.K. Srivastava and E. Thangapandian. 2007. Seroprevalence of leptospiral antibodies in Bovine. Indian Vet. J., 84: 547-548.

Maskey, M., J.S. Shastri, K. Saraswathi, R. Surpam and N. Vaidya 2006. Leptospirosis in Mumbai: Post-deluge outbreak 2005. Indian J. Med. Microbi., 24: 337-338.

Savalia, C.V. 2001. Seroprevalence of Leptospirosis in bovines of Gujarat. The Veterianarian, 25(2): 4-5.

Selvaraj, J., B.M. Manohar, R. Govindarajan, V. Jayakumar, T.V. Meenambigai, C. Balachandran and A. Koteeswaran. 2005. Prevalence of leptospiral antibodies in buffaloes (Bos bubalis) at slaughter. Indian J. Comp. Microbiol. Immunol. Infect. Dis., 26(2): 125-127.

Selvaraj, J., B.M. Manohar, R. Govindarajan, V. Jayakumar, T.V. Meenambigai and C. Balachandran. 2010. Seroprevalence of leptospirosis in she-buffaloes (Bos bubalis) at slaughter in chennai, india. Short Communication. Buffalo Bull., 29(2): 95-98.


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Treml, F., M. Pejcoch and Z. Holesovska. 2002. Vet. Med. Czech., 47: 309.

Varma, A., R.B. Rai, P. Balakrishnan, A. Gupta and K.A. Naveen. 2001. Seroprevalence of leptospirosis in animas of Andaman and nicobar islands. Indian Vet. J., 78: 936-937.

Vijayachari, P., A.P. Suganan and S.C. Sehgal. 2001. Role of microscopic agglutination test (MAT) as a diagnostic tool during acute stage of leptospirosis in low and high endemic areas. Indian J. Med. Res., 114: 99-106.

World Organization for Animal Health (Office International des Épizooties-OIE). 2013. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. OIE, Paris; Chapter 2.1.9: 251-264.


389

ABSTRACT

The present work was undertaken on 42 buffalo embryos and fetuses ranging from 40 to 253 days to study the histomorphological features of mandibular salivary glands in the buffalo (Bubalus bubalis) during the prenatal period. The specimens were fixed and processed for serial paraffin sectioning and the sections were subjected to different staining methods. The primordium of mandibular gland appeared first as a solid epithelial bud from the oral epithelium at the base of the tongue at 40 days. Ductal Lumen formation was observed first in the terminal buds and primary cords of mandibular gland at 84 days. The differentiation of terminal buds into terminal tubules was completed at 91 days. The typical compound tubulo - alveolar architecture of the gland was attained first at 125 days. The gland was predominant in mucous type of acini from 140 day onwards. The formation of capsule around the gland was evident at 125 days and it was well developed at 197 days. Differentiation of intercalated, intralobular and interlobular ducts was possible at 125 days.

Keywords: histomorphology, mandibular gland, prenatal buffalo

INTRODUCTION

Major salivary glands of various domestic animals are paired structures, which includes parotid, mandibular and sublingual glands. Salivary glands fulfill important role in the oral biology by producing saliva to provide water for lubrication, as well as supplying electrolytes, mucus, antibacterial compounds and various enzymes to the oral cavity. Loss of salivary glands function can result in the wide spread deterioration of oral health (Hsu et al., 2010). Study of normal development of salivary glands will be helpful for both developmental anatomists and clinicians as they are having important role in several dreadful diseases like rabies, foot and mouth disease and Herpes viral diseases.

AGE RELATED CHANGES IN THE HISTOMORPHOLOGY OF MANDIBULAR GLAND IN PRENATAL BUFFALO (BUBALUS BUBALIS)

K. Raja1,*, M.S. Lakshmi2, G. Purushotham2, K.B.P. Raghavende3 and T.S. Chandrasekhara Rao4

1Department of Veterinary Anatomy, College of Veterinary Science, Korutla, India, *E-mail: [email protected] of Veterinary Anatomy, College of Veterinary Science, Rajendranagar, Hyderabad, Andhra Pradesh, India3Department of Veterinary Surgery and Radiology, College of Veterinary Science, Rajendranagar, Hyderabad, Andhra Pradesh, India4Faculty of Veterinary Science, Sri Venkateswara Veterinary University, Tirupati, India

Original Article


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Total 42 buffalo embryos and fetuses ranging from 40 to 253 day of gestation (2.5 to 79.5 cm) “Curve Crown-Rump Length” (CVRL) were collected from pregnant uteri immediately 1hr after the slaughter from slaughter house of Hyderabad irrespective of the the age and sex. The age of fetuses was determined on the basis of their CVRL by using Soliman’s formula (1975). The whole embryos of earliest possible age (from 40 days i.e. 2.5cm CVRL) and fresh tissue pieces from the mandibular salivary gland of fetuses from 84 day (12.4 cm CVRL) to 253 day (79.5cm CVRL) were collected and fixed in 10% Neutral Buffered Formalin(pH 7). Bouin’s fluids (Singh and Sulochana, 1997) also used for the fixation of the gland because bouins fluid allows crisper and better nuclear staining than 10% neutral-buffered formalin. The tissues were processed in routine for paraffin sectioning of 5 to 8µm thickness and subjected to Mayer’s Haemotoxylin and Eosin method for routine histological developmental study, Van Gieson’s technique for collagen fibres, Masson’s Trichrome for connective tissue fibres (Singh and Sulochana, 1997) staining techniques to study the histomorphological changes in the mandibular salivary gland will be observed by using the Olympus microscope.


The primordium of mandibular salivary gland developed as a solid epithelial bud from the oral epithelium at the base of the tongue in linguo-gingival space at 40 days (Figure 1). Contrary to this the primordium of the gland was reported to be observed at 45 days (Santhi, 2006) and 69 days

(Venkatakrishnan, 1994) in buffalo and during 6th week (Arey, 1965) in human. The migration of epithelial bud into the surrounding mesenchyme as club shaped structure was noticed at 41 days (Figure 2) with surrounding mesenchyme in condensed form at 43 days. The appearance of the anlagen as a solid club shaped structure from the oral cavity in relation with the developing tongue was also reported by Venkatakrishnan (1994), Mc Geady et al., (2006) and Santhi (2006) in buffalo. The glandular mass was composed of undifferentiating basophilic epithelial cells. The epithelial bud was attached to oral epithelium by a single epithelial stalk. The glandular mass began to branch into several terminal epithelial buds at 45 days in dichomotous pattern surrounded with large amount of dense mesenchyme. The terminal buds were formed by multilayered polyhedral cells with basophilic cytoplasm and spherical nuclei. The gland reached the space between the tympanic bulla and the angle of the mandible at 54 days.

At 84 days the gland was formed by groups of luminized terminal buds (terminal tubules) and primary cords (Intercalated ducts) (Figure 3) with dense mesenchyme and rich vasculature. However Venkatakrishnan (1994) reported the similar finding at 105 days in buffalo. The emergence of duct system was reported to be observed at 21 day in utero in the submandibular gland of rat (Ogawa et al., 2000). Most of the terminal tubules were lined with 2 to 3 layers of cells with central lumen from 91 days. The terminal tubules attained the structure of acini at 115 days in which the lining epithelium was changed to single layer (Figure 4), which was reported to be established at 5 weeks postnatally in rat (Ogawa, 2000).

The cytoplasm of the acinar cells was lightly basophilic with spherical basal nucleus. Typical compound tubulo - alveolar nature of the


391

gland was attained first at 125 days of foetal life (Figure 5). From 140 day onwards the gland was predominantly mucous type along with serous demilunes. The cells of the mucous acini were pyramidal in shape with distinct cell borders and basement membrane. The cytoplasm was lightly basophilic with flattened basal nuclei. The cells lining the serous acini were pyramidal with spherical and darkly stained nuclei. Myoepithelial cells were observed first around the acini and intercalated ducts at 140 days between the basement membrane and acinar cells. The developing mandibular gland was compound tubulo-alveolar type with predominantly mucous alveoli during the late foetal stage, which was reported to occur in all foetal age groups of animals by Venkatakrishnan (1994) in buffalo.

The differentiation of embryonic mesenchymal tissue into stroma was observed first in foetal mandibular gland at 84 days (Figure 6). The gland showed primitive lobules separated by stroma with fine collagen fibers at 91 days. The lobulation of the gland was distinct at 101 days (Figure 7). The lobules of the gland were reported to be formed during 9th to 10th week (Merida-Velasco et al., 1993) in human beings and 14 to 26 days of prenatal development (Knopse and Bohme, 1995) in cat. The gland was highly vascular between 84 and 101 days. Dense lobulation of the gland with steep increase in the number of lobules and capsule formation was evident at 125 days (Figure 5). The connective tissue septa were formed at 131 days and were traversed by several blood vessels, nerves and ducts. Capsule showed large amount of collagen fibres and few elastic fibres at 197 days. The parenchyma of the gland was predominant in mucous acini from 125 days and 253 days (Figure 9). A large quantity of connective tissue was observed around the groups

of interlobular ducts between 140 and 188 days of foetal life.

The developmental changes in the parenchyma and stroma of the mandibular salivary gland of fetuses were gradual in buffalo, which is in agreement with the findings of Venkatakrishnan (1994) in buffalo, Arey (1965) and El- Mohandes et al. (1987) in man. The stromal content was reduced and replaced by the parenchyma as the age of the foetus advanced. The parenchyma and the duct system of the foetal mandibular gland nearing the full term of pregnancy had achieved its identity to that of glands in adult buffalo.Differentiation of intercalated and intralobular ducts was observed at 84 days of foetal age. The intercalated ducts and striated ducts were lined by a double layered epithelium from 91 to 130 days with inner low columnar or cuboidal cells and outer flattened cells. All types of ducts i.e., intercalated (acinar ducts), intralobular (striated ducts) and interlobular ducts were observed at 125 days (Figure 6) in the developing mandibular gland. However the differentiation of striated ducts, intercalated ducts and terminal buds was reported to occur at the time of the birth (Cutler and Chaudhry, 1973) and at 16 weeks (El-Mohandes et al., 1987) in human being. The intercalated ducts were lined with single layered cuboidal epithelium and were closer nearest to the secretory end pieces from 140 days. The number of intercalated ducts increased as the foetal age increased.

The intralobular ducts appeared first at 84 days in the form of solid epithelial cell cords. Double layered intralobular ducts composed of inner and outer cuboidal cells were evident at 140 days of foetal life (Figure 8). The mature striated ducts were lined by a layer of tall inner columnar cells with flattened myoepithelial cells in the basal area. The inner cells lining the striated ducts


392

Figure 1. Photomicrograph of cross section of foetal head showing the primordium of mandibular (M) salivary gland at 40 days.

SL- Sublingual salivary gland , LN-Lingual nerve, T-Tongue H and E X 20.

Figure 2. Photomicrograph of cross section of 41 day buffalo foetal head showing the migration of epithelial bud of mandibular (M) gland into the mesenchyme.

SL-Primordium of sublingual gland, O-Oral cavity, MG- Mandibular ganglion H and E X 20.


393

Figure 3. Photomicrograph of cross section of 84 day foetal mandibular gland showing groups of terminal buds (TB), terminal tubules (TT) with dichomotus intercalated (ICD) and intralobular (LD) ducts.

ILD-Interlobular ducts, EM-Embryonal mesenchyme, BV- Blood vessels Van Gieson’s method X 10.

Figure 4. Photomicrograph of cross section of 115 day foetal mandibular gland showing the earliest appearance of the mucous acini (A) lined with single layer epithelium (E).

ICD-Intercalated duct, ILD-Interlobular duct, BV-Blood vessel H and E X 20.


394

Figure 5. Photomicrograph of cross section of 125 day foetal mandibular gland showing compound tubulo-alveolar architecture predominant in mucous acini (A) and developing capsule (C) around the gland.

LD-Intralobular duct, ILD-Interlobular duct, E- Excretory duct, L-LobuleVan-Gieson’s method X 10.

Figure 6. Photomicrograph of cross section of 84 day foetal mandibular gland showing the differentiation of stroma (ST), capsule (C) and septa (SE) from embryonic mesenchyme.

TT-Terminal tubule Van-Gieson’s method X 10


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Figure 7. Photomicrograph of cross section foetal mandibular gland showing distinct lobulation (L) at 101 days.

TT- Terminal tubule, ICD-Intercalated duct, LD-Intralobular duct, M-MesenchymeH and E X 10.

Figure 8. Photomicrograph of cross section of 140 day foetal mandibular (M) gland showing Two layered intralobular ductal structures composed of inner and outer cuboidal cells.

MA-Mucous acini, SA-Serous acini, SD-Serous demilune ---Goblet cells H and E X 20.


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Figure 9. Photomicrograph of cross section of 253 day foetal mandibular gland showing parenchyma predominant in mucous acini (MA).

ICD-Intercalated duct, SD-Serous demilune, SA- Serous acini H and E X 10.

showed intensely eosinophilic cytoplasm and darkly stained nuclei. These ducts showed a well developed adventitia with abundant vasculature.

Two layered immature interlobular ducts were evident at 125 days. The tunica adventitia was prominent around the interlobular ducts with well developed collagen fibres, elastic fibres, blood vessels and nerves at 131 days. Groups of mature interlobular ducts were also prominent in the interlobular space from 140 days and lined with multilayered cuboidal or columnar epithelium with abundant goblet cells (Figure 8).

The excretory ducts were lined by a double layered columnar epithelium at 54 days. The caruncula sublingualis was distinct at 56 days as reported by Santhi (2006) in prenatal buffalo. The excretory ducts were larger than the other ducts in all age groups studied and were lined by stratified cuboidal or columnar epithelium in advanced age groups. Vacuolated cells were seen occasionally. The ducts had a well developed adventitial layer

with collagen fibres, elastic fibres, blood vessels and nerves. Findings of this work gave a valuable informations and tremendous scope for the clinicians and Developmental anatomists.

REFERENCES

Arey, L.B. 1965. Developmental Anatomy, 7th ed. W.B. Saunders Company, Philadelphia. p. 411-419.

Cutler, L.S. and A.P. Chaudhry, 1973. Differentiation of myoepithelial cells in the rat submandibular gland in vivo and in vitro-An ultrastructural study. J. Morphol., 140: 343-354.

El-Mohandes, E.A., K.G. Botros and A.A. Bondok. 1987. Prenatal development of the human submandibular gland. Acta Anatomica, 130(3): 213-218.

Hsu, J.C.F., M. Kenneth and Yamada. 2010.


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Salivary gland branching morphogenesis, recent progress and future opportunities. International Journal of Oral Science, 2(3): 117-126.

Knospe, C. and G. Bohme. 1995. Zur Prsanatalen Entwicklung Derglandula Manibularis und Glandula parotis der katze. Anatomia, Histologia and Embryologia, 24: 1-6.

Mc Geady, T.A., P.J. Quinn, E.S. Fitzpatrick and M.T. Ryan. 2006. Veterinary Embryology.Wiley-Blackwell Publishers, UK. p. 184-204.

Merida-Velasco, J.A., I. Sanchez-Montesinos, J.F. Espin, J.D. Garcia-Garcia, S. Garcia-Gomez and V. Roldan-Schilling. 1993. Development of the human submandibular salivary gland. Journal of Dental Research, 72(8): 1227-1232.

Ogawa, Y., S. Toyosawa, T. Ishida and N. Ijuhin. 2000. Keratin 14 Immunoreactive cells in Pleomorphic Adenomas and Adenoid Cystic carcinomas of Salivary glands. Virchows Arch., 437(1): 58-68.

Santhi, L., 2006. Prenatal development of the skull in the buffalo (Bubalus bubalis). Ph.D. Thesis, Sri Venkateswara Veterinary University.

Singh, U.B. and S. Sulochana. 1997. A Practical Manual of Histopathological and Histochemical Techniques. Kothari Publication, Bombay. p. 1-41.

Soliman, M.K. 1975. Studies on the physiological chemistry of the allantoic and amniotic fluids of buffaloes at the various periods of pregnancy. Indian Vet. J., 52: 106-112.

Venkatakrishnan, 1994. Morphogenesis, histomorphology and histochemistry of the mandibular gland of the buffalo (Bubalus bubalis). Thesis, TANUVAS, Chennai,

India.



399

ABSTRACT

β4GalT-I interacts with α-lactalbumin to form the lactose synthase in mammary gland. Considering the biological function of the lactose synthase complex, β4GalT-I gene can be considered as a candidate gene for milk production in dairy animals. The bovine β4GalT-I gene, 3,283 base pairs in length, is mapped on chromosome 8. The present research work was planned to identify the polymorphism in β4GalT-I gene in Nili Ravi buffalo. Blood Samples were collected, DNA was extracted and primers were designed for PCR. After amplification, PCR products were sequenced for the identification of allelic variation. Thirteen polymorphic, six in coding and seven in non-coding region of gene, were identified. This is a first report toward genetic screening of β4GalT-I gene at molecular level in Nili Ravi buffalo. The present study will provide a better selection to develop association of identified polymorphisms with production traits in buffalo population.

Keywords: polymorphisms, β4GalT-I Gene, Nili Ravi, buffalo, Pakistan

INTRODUCTION

The β 1, 4-galactosyltransferase-I (β4GalT-I) belongs to a family of enzymes called galactosyl transferases. In the mammalian mammary gland, lactose is synthesized from blood glucose and galactose by a lactose synthase enzyme (Strucken et al., 2015). The βeta 1, 4-galactosyltransferase-I gene (β4GalT-I) interacts with α-lactalbumin, the calcium binding non-catalytic protein, in the golgi complex of mammary secretory cells to form the lactose synthase complex (Ramakrishnan et al., 2002; Shahbazkia et al., 2010; Strucken et al., 2015). This enzyme is a membrane-bound glycoprotein which is widely distributed among in mammals, non-mammalian vertebrates and also in some plants (Powell and Brew, 1974). Considering the biological function of the lactose synthase complex, β4GalT-I gene can be a potential candidate gene for milk production in dairy animals. It is considered a housekeeping gene in the biosynthesis of glycans which occurs in essentially all cell types (Shaper et al., 1998). β4GalT-I act as a cell surface component, adhesion and recognition molecule, signal transducer, tumor marker (Berger and Rohrer, 2003) and also involve in angiogenesis, wound healing and collagen deposition in the skin (Shen et al., 2008). However, in spite of its involvement in various physiological

β(1,4)-GALACTOSYLTRANSFERASE-I GENE POLYMORPHISMS IN PAKISTANI NILI RAVI BUFFALO

Aamir Sohail1, Asif Nadeem1,*, Masroor Ellahi Babar2, Tanveer Hussain2, Akhtar Ali2, Wasim Shehzad1 and Maryam Javed1

1Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences Lahore, Pakistan, *E-mail: [email protected] University of Pakistan, Lahore, Pakistan

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and biochemical reactions, little is known regarding the presence of polymorphisms in dairy animals and any consequent effects on production traits. It is a membrane-bound glycoprotein widely distributed in the mammals, non-mammalian vertebrates and also in some plants. Two distinct isoforms of the β4GalT-I gene product have been reported in the human, mouse and in cattle.

The bovine β4GalT-I gene is 53,283 base pairs in length, comprises of six exons and five introns and is located on chromosome 8. The bovine β4GalT-I protein consists of four domains- the cytoplasmic domain (residues 8-24), the transmembrane domain (residues 25-44), the stem region (residues 45-145) and the catalytic domain (residues 146-402) (Qasba et al., 2008). Thus, it was accepted that the polymorphisms which are located in the catalytic domain of the protein may affect some of the catalytic properties of the enzyme. This study was aimed to identify the probable polymorphism in the β4GalT-I gene of Nili Ravi buffalo breed of Pakistan.


Blood samples (n=50) of Nili Ravi buffaloes were collected from Livestock Production Research Institute (LPRI) Bahadarnagar, Okara and preserved in EDTA (0.5 M) coated falcon tubes. Genomic DNA was extracted from blood samples using inorganic method (Sambrook and Russel 2001). DNA quantification was carried out using 0.8% Agarose gel. 50 ng/uL of genomic DNA was used for the amplification of coding regions of the β4GalT-I gene. PCR primers were designed from GenBank accession no. AC_000165.1 by web based software “Primer3” (http://bioinfo.ut.ee/primer3-0.4.0/) (Untergasser et al., 2012).

All primers were amplified by touchdown PCR protocol with annealing temperature (62-52°C) on Bio-Rad and peQ Lab thermocycler. After the precipitation with 70% ethanol in dark, PCR products were sequenced through ABI prism 3100 genetic analyzer (Applied Biosystems Inc., Foster City, CA). Sequencing results were analyzed with BioEdit software (http://www.mbio.ncsu.edu/bioedit/bioedit.html). Pair wise alignment of sequence was done with the help of blast2 sequence. Allelic and genotypic frequencies for the identified polymorphisms were calculated using bioinformatics software POPGENE (Yeh et al., 1999).

RESULT

The overall sequence variation across the bovine β4GalT-I locus is high. Thirteen polymorphic sites were identified by using BLAST in local Nili Ravi buffalo breed. Aligned sequence represent that six identified substitutions were in coding region of the gene while seven were positioned in intronic (near to exonic) region of the gene. All these identified polymorphism can be used as strong association markers with economic traits in Nili Ravi buffalo population. The reported exonic polymorphisms do not bring amino acid change and appeared as synonymous substitution. However, these sites may be related to detect causative mutation or adjacent QTL. The distribution pattern of alleles and genomic frequencies against each identified polymorphisms are presented in Table 1. However the identified polymorphic sites were considered breed specific and might be correlated to milk production and other economic traits.

http://bioinfo.ut.ee/primer3-0.4.0/

http://bioinfo.ut.ee/primer3-0.4.0/

http://www.mbio.ncsu.edu/bioedit/bioedit.html

http://www.mbio.ncsu.edu/bioedit/bioedit.html


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DISCUSSION

The β4GalT-I encodes the catalytic part of lactose synthase enzyme which is responsible for lactose synthesis in the mammary gland. The whole coding region of the gene was screened for the presence of allelic variation among a sample of

fifty buffaloes, using PCR technique followed by Sanger sequencing. Altogethersixteen polymorphic sites were identified across the whole bovine β4GalT-I locus.

Two distinct isoforms of the β4GalT-I gene product have been reported in the mouse (Shaper et al., 1998) and similar variants have been

Table 1. Change in nucleotide, genotypic and allelic frequency of all identified polymorphisms in bovine β4GalT-I gene.

SNP IDChromosomal

PositionChange in Nucleotide

Genotype Frequency Allele Frequency

GALT1 76203115 G>AGG AG AA G A

0.5806 0.0646 0.3548 0.5968 0.4032

GALT2 76183169 C>TCC CT TT C T

0.5484 0.3187 0.1329 0.6613 0.3387


0.4516 0.2113 0.3371 0.4861 0.5139

GALT4 76183243 T>GTT TG GG T G

0.5881 0.2791 0.1328 0.8226 0.1774

GALT5 76183252 T>CTT TC CC T C

0.5137 0.3319 0.1544 0.4677 0.5323

GALT6 76183296 T>ATT TA AA T A

0.5741 0.2897 0.1362 0.3548 0.6452

GALT7 76183328 T>CTT TC CC T C

0.6134 0.1844 0.2022 0.4918 0.5082

GALT8 76183346 A>GAA AG GG A G

0.3147 0.2741 0.4112 0.3953 0.6047

GALT9 76183447 T>CTT CT CC T C

0.4918 0.3147 0.1935 0.5173 0.4827

GALT10 76180158 C>GCC CG GG C G

0.5178 0.1928 0.2894 0.5712 0.4288

GALT11 76180262 G>AGG AG AA G A

0.4617 0.2349 0.3034 0.4911 0.5089


0.4143 0.2316 0.3541 0.6197 0.3803


0.5349 0.2199 0.2452 0.5763 0.437


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identified in the bovine (Russo et al., 1990) and human (Mengle-Gaw et al., 1991) homologues. Both of these isoforms, ‘‘short’’ and ‘‘long’’ are differ only by an N-terminal sequence of 13 residues (Shahbazkia et al., 2012). The amount of β4GalT-I enzyme in the lactating mammary gland increases during the lactation period to meet the demand for lactose synthesis.

The most important mechanism by which this increase is ensured is the switch from the long to the short variant, which has the effect of raising β4GalT-I transcript level (Shaper et al., 1998). Thus, allelic variation which affects the transcription start codon probablydisturbs this mechanismand prevents the synthesis of higher levels of lactose.

Shahbazkia et al., (2012) reported the exon 1 T→A (14Lys) transversion alters the second transcription start site of the codon and may have impact on the gene expression. However, no such type of transversion was seen in this study. Other polymorphisms (174 Thr and 220 His) in exon 2 were identified in the catalytic domain that may alter some of the catalytic properties of the enzyme (Shahbazkia et al., 2012). In another study, Qasba et al., 2008 reported that the Phe280 residue, together with Tyr286, Gln288, Tyr289, Phe360 and Ile363, were concerned in the relations between β4GalT-I and α-lactalbumin and may alter this interaction and the properties of the lactose synthase complex.

In our study all the identified substitutions GTG> GTA (236Val), AAT> AAC (296Asp), GGA> GGG (302Gly), ATC> ATT (326 Ile), GTG> GTA (369Val), and ACG> ACA (387Thr) were synonymous in nature and located in the catalytic domain of enzyme. However, this Phe280 Tyr substitution was not identified in Nili Ravi buffaloes.

To conclude, it was the first screening of

β4GalT-I gene single nucleotide polymorphisms in Nili Ravi buffalo breed of Pakistan. The above discussion just explains the potential and molecular basis of identified polymorphisms. These SNPs may serve as a potent genetic reserve for the development of molecular markers to assist selection in dairy breeding.

REFERENCES

Berger, E.G. and J. Rohrer. 2003. Galactosyltransferase-still up and running. Biochimie, 85: 261-274.

Mengle-Gaw, L., M.F. McCoy-Haman and D.C. Tiemeier. 1991. Genomic structure and expression of human beta-1, 4-galactosyltransferase. Biochem. Bioph. Res. Co., 176: 1269.

Powell, J.T. and K. Brew. 1974. Glycosyltransferases in the Golgi membranes of onion stem. Biochem. J., 142: 203.

Qasba, P.K., B. Ramakrishnan and E. Boeggeman. 2008. Structure and function of -1, 4-galactosyltransferase. Curr. Drug Targest., 9: 292.

Ramakrishnan, B., E. Boeggeman and P.K. Qasba. 2002. Beta-1, 4-galactosyltransferase and lactose synthase: molecular mechanical devices. Biochem. Bioph. Res. Co., 291(5): 1113-1118.

Russo, R.N., N.L. Shaper and J.H. Shaper. 1990. Bovine beta1-4-galactosyltransferase: two sets of mRNA transcripts encode two forms of the protein with different amino-terminal domains. In vitro translation experiments demonstrate that both the short and the long forms of the enzyme are type II membrane-bound glycoproteins. J. Biolog. Chem., 265:


403

3324.Sambrook, J. and D.W. Russell. 2001. Molecular

Cloning: A Laboratory Manual, 3rd ed. Cold spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA.

Shahbazkia, H.R., M. Aminlari and A. Cravador. 2012.Association of polymorphism of the β (1, 4)-galactosyltransferase-Igene with milk production traits in Holsteins. Mol. Biol. Rep., 39: 6715-6721.

Shahbazkia, H.R., M. Aminlari, A. Tavasoli, A.R. Mohamadnia and A. Cravador. 2010. Polymorphisms of the β-1, 4 galactosyltransferase-I gene in Holsteins. Livest. Sci., 131: 297-300.

Shaper, N.L., M. Charron, N.W. Lo and J.H. Shaper. 1998. 1, 4-Galactosyltransferase and lactose biosynthesis: recruitment of a housekeeping gene from the nonmammalian vertebrate gene pool for a mammary gland specific function. J. Mammary Gland Biol., 3: 315-324.

Shen, A., J. Qian, L. Liu, H. Liu, J. Chen, S. Niu, M. Yan, X. Chen, C. Shen and J. Gu. 2008. The role of [beta]-1, 4-galactosyltransferase-I in the skin wound healing process. Am. J. Dermatopath., 30: 10.

Strucken, E.M., Y.C.S.M. Laurenson and G.A. Brockmann. 2015. Go with the flow-biology and genetics of the lactation cycle. Front. Genet., 6: 118.

Untergasser, A., I. Cutcutache, T. Koressaar, J. Ye, B.C. Faircloth, M. Remm and S.G. Rozen. 2012. Primer3 - new capabilities and interfaces. Nucleic Acids Res., 40(15): 115.

Yeh, F., C. Boyle, T. Rongcai, Z.Y. Ye and J.M. Xian. 1999. POPGENE, Version 1.31. A Microsoft window based free ware for population genetic analysis. University

of Alberta, Edmonton. http://www.mbio.ncsu.edu/bioedit/bioedit.html. Biological sequence alignment editor.



405

ABSTRACT

The aim of the present study was to estimate the index and individual responses to selection for milk, fat percentage and protein percentage for different breeding goals in three buffalo milk sale situations in Khuzestan State of Iran characterized by: 1) selling just milk, 2) selling milk and Sarshir cream and 3) selling milk and Mozzarella. The current payment policy is based exclusively on milk volume. Index responses to selection were calculated for three different breeding goals (BG): 1) milk yield (MY) exclusively; 2) milk yield and milk fat percentage (MY+FP), 3) milk yield and milk fat and protein percentage (MY+FP+PP), 4) fat percentage (FP) exclusively and 5) fat and protein percentage (FP+PP). Index responses for the milk sale situation were US$102.85 (BG1), US$103.30 (BG2), US$103.52 (BG3), US$5.26 (BG4) and US$5.94(BG5). For the Sarshir (local cream of the region) sale situation, index responses were US$143.99 (BG1), US$143.97 (BG2), US$144.12 (BG3), US$2.78 (BG4) and US$2.80(BG5) and for Mozzarella sale situation, index responses were US$185.13 (BG1), US$185.08 (BG2), US$ 184.59 (BG3) US$4.54 (BG4) and US$9.22(BG5). The results suggest that for the present circumstances, selection for milk components is not advantageous

when milk is produced for sale only milk. However, when Sarshir cream or Mozzarella making is added to the production, the selection for components and milk volume is economically beneficial.

Keywords: buffalo, Iran, selection index, economic weight, Sarshir cream

INTRODUCTION

According to statistical data of Food and Agriculture Organization (FAO) in 2010, there is 650 thousand head of buffaloes in Iran that is ranked as 16th country among the 43 countries which breed buffaloes (FAO, 2010). There are buffalo in three different regions of Iran: 1) cold highlands, 2) Mediterranean humid temperate and 3) warm lowland. Khuzestan province in warm lowland region of Iran is one of the provinces that have buffalo. The suitable climatic conditions of Khuzestan for rearing buffalo (existence of important large rivers and ponds and also having a special weather conditions) causes to employment of more than 5 thousand rural families to buffalo rearing (Anonymous, 2008). Relative importance of buffalo products was the same in different areas of the province and in all regions buffalo are

INVESTIGATION OF RESPONSE TO SELECTION FOR MILK TRAITS IN DAIRY BUFFALO OF IRAN BASED ON THREE SALE SITUATIONS

Bahareh Taheri Dezfuli1 and Leonardo De Seno2

1Animal Science Research Department, Khuzestan Agricultural and Natural Recourses Research and Education Center, Agriculture Research, Education and Extension Organization (AREEO), Ahwaz, Iran, *E-mail: [email protected] Sciences Faculty, Grande Dourados Federal University, Dourados, MS, Brazil

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bred primarily for milk production, so that about 40 percent of Khuzestan dairy products produced from buffalo (Anonymous, 2008). There is not government payment policy for buffalo milk and all of its milk is sold to the private sector with free pricing system based on its volume. However, in some cities, most buffalo owners separate fat from milk with completely traditional method and sell it as Sarshir (a local cream) along with remaining milk (skim) to market. Buffalo milk has special features because of high fat percentage (6 to 7%), and its products are of high economic value. Therefore, increasing production capacity of buffalo and full use of its genetic potential can be effective in improving buffalo production efficiency and economic buffalo breeding, in addition to providing a significant portion of needed protein.

Generally, to achieve maximum genetic progress, it is necessary to define the target traits for selecting animals (Groen, 1988; Ponzoni, 1988; Bekman and Van Arendonk, 1993; Charfeddine, 2000) and selecting these traits should be such that maximize economic profit of the production system. In dairy animals, milk production and its components have higher economic importance than other traits that with changes in these traits, the greatest changes in profitability is obtained. Komlosi et al. (2010) in a study of 15 traits of milk production, growth and carcass characteristics of Hungarian Holstein-Friesian cows, reported milk production as the most economically important trait. Seno et al. (2006) also to provide a good selection program for buffaloes of Sao Paulo state in Brazil, studied the response of single trait and multi trait breeding purposes determined based on milk yield, fat and protein yield. The authors found that the high fat and protein percentages in buffalo milk provide high economic returns to Brazilian farmers that produce their own Mozzarella cheese.

The aim of this study was to estimate the total and component selection response for milk production traits (milk yield, fat and protein percentage) with different selection purposes in current milk sale situations in Khuzestan province of Iran.


In this study, the current status of the sale of milk and fat (Sarshir) was used to calculate the economic values of milk yield (MY) and fat percentage (FP). Also, the proposed sale status of Mozzarella cheese was used to calculate economic value of protein percentage (PP) beside milk yield and fat percentage. Production, demographic, economic and management data of 30 buffalo herds with average of 30 head of buffalo cows were used to estimate the parameters required for the calculation of cost and revenue equations. Herds were selected from the main areas of buffalo breeding, so that covered different weather conditions and the production and sale of milk and dairy products, and also different managements. Management system of the herds in the study was traditional and in all herds, milking is performed twice a day by hand and in the presence of calves in order to expedite the removal of milk. Also, cows after the morning milking and feeding are taken to wetlands to bath for 2 to 3 hours.The aim of the present study was to estimate the index and individual responses to selection for milk, fat percentage and protein percentage for different breeding goals for three buffalo milk sale situations in Khuzestan state of Iran characterized by: 1) selling just milk, 2) selling milk and Sarshir and 3) selling milk and Mozzarella. The current payment policy is based exclusively on milk volume. Index


407

responses to selection were calculated for three different breeding goals (BG): 1) milk yield (MY) exclusively; 2) milk yield and milk fat percentage (MY+FP), 3) milk yield and milk fat and protein percentage (MY+FP+PP), 4) fat percentage (FP) exclusively and 5) fat and protein percentage (FP+PP).

Revenues and costs for both present sales situations were considered for all herds: 1) sale of only milk and 2) separation of milk fat to produce Sarshir, and sale it and remaining milk along with selling buffalo milk and 3) production and sale of Mozzarella cheese substitute of milk and Sarshir. Sarshir or oily part of milk is produced from milk fat particles with different sizes which are spread on the surface of the milk by heating the milk. We produced Sarshir by 1 kilogram milk with different amount of fat percent. Based on this experiment the correlation between fat percentage of milk and the amount of Sarshir was obtained 0.72 and the amount of Sarshir increased 11 gram with increasing one percent of milk fat percentage. To calculate the amount of Mozzarella cheese produced (PKM) from one kg milk in the proposed sale of the study, the equation or Mozzarella index proposed by Altiero et al. (1989) was used:

Where, MY is the milk yield and %F and %P are the milk fat and protein percentage, respectively.

The significant sources of income for considered buffalo herds were sale of milk, Sarshir and remaining low-fat milk (skim), sale of animals (calves, heifers, culled cows and sires) and fertilizer. The annual costs for herds were: the cost of producing one kilogram of milk (food and non-food costs), the feed cost to produce one percent of

additional fat, the feed cost to produce one percent of additional protein, variable costs include of the average cost of transporting, construction and maintenance, electricity, water, labor, health, and the average cost of feed for one buffalo herd, and also, the fixed cost of herd with an average size of 30 buffalo cows. During collecting data from the herds, measure the amount of feed used by each buffalo was not possible, feed intake and daily food requirements of maintenance, growth, reproduction and milk production of buffaloes were extracted using Borghese (2005).

Economic weights of milk traits were calculated in maximized profit breeding perspective, using following equation:

Where, TP is total annual herd profit per head of buffalo cow ($), TR is total annual herd revenues per head of buffalo cow ($) and TC total annual herd costs per head of buffalo cow ($).

The current payment policy is based on exclusively on milk volume. Milk and Sarshir prices were 0.73 US$/kg and 8.16 US$/kg, respectively. Mozzarella cheeses price were considered equal to Sarshir because it was proposed to substitute Sarshir. Exchange rate at the time of calculations for this paper: 1 US$ = 12260 Rials (March 2013).

Economic values (Table 1) were calculated for milk yield, fat percentage and protein percentage, taking into accounts the differences of the three sale situations. For the calculation of economic values, the prices of production components and product were obtained from questionary forms with going to each buffalo herd (Taheri Dezfuli et al., 2011).

Expected responses were calculated for five different breeding goals (BG) in each sale situation:1) Milk Yield (MY) exclusively

TP = TR-TC

PKM - MY x 3.5 (%P)+1.23 (%F)-0.88 100

( ),


408

2) Milk Yield (MY)+Fat Percentage (FP) 3) Milk Yield (MY)+Fat Percentage (FP)+Protein Percentage (PP) 4) Fat Percentage (FP) 5) Fat Percentage (FP)+Protein Percentage (PP)

Index responses to selection were calculated based on the selection index theory (Hazel, 1943). Solutions for weighting factors (b) were calculated as b = var (X)-1 cov (X,A). X is a vector with the information sources, var (X) is a matrix with (co)variances between these sources and cov (X,A) is a vector of covariances between each information source and the true genotype. The index equations are then Pb = Gv or b = P-1Gv. P is used to describe the matrix with variances and covariances between the information sources in X. It is a variance-covariance matrix between the means of phenotypic observations. G is the matrix with covariances between X and A. Finally, v is a vector with economic values of traits in the

breeding goal. After calculating the b values, the index (σ2

I ) and breeding goal (σ2H ) the standard

deviation were obtained from: σI = sqrt(b’Pb) and σH = sqrt(v’Cv), where C = matrix of (co)variances for traits in the breeding goal. The correlation between the index and the breeding goal (rIH) was given by σI/σH.

Index responses were then obtained from R = i × σI = i × rIH × σH, where i is the selection intensity, σI is the standard deviation of the index, rIH is the correlation between the index and the breeding goal and σH is the standard deviation of the breeding goal.

Individual responses to selection were also calculated for the studied traits. The average of genetic and phenotypic parameters of traits used in response calculations are presented in Table 2 (Taheri et al., 2012; Da-you et al., 2008; Rosati and Van Vleck, 2002; Thevamanoharan et al., 2000; Seno et al., 2010; Castillo et al., 2001; Tonhati et al., 2000; Tonhati et al., 2004; Aspilcueta-Borquis

Table 1. Economic values (EV), expressed in US$ for milk (MY), fat (FP) and protein percentage (PP) for Milk, Milk and Sarshir, and Milk and Mozzarella sale situations.

Sale Situation EVMY EVFP EVPP

Milk 0.40 -15.92 -14.91Milk and Sarshir 0.56 8.43 -12.60Milk and Mozzarella 0.72 13.73 61.01

Table 2. Standard deviations (σp), heritabilities (bold), genetic (above diagonal), and phenotypic correlation coefficients (below diagonal) for milk yield (MY), fat (FP) and protein (PP) percentages.

Sale Situation σp MY FP PPMY 635.4 0.22 -0.08 -0.12FP 0.93 -0.19 0.18 0.31PP 0.32 -0.20 0.48 0.18


409

et al., 2010). Progeny test was considered to select best buffalo bulls with an average progeny size of 50 daughters per sire. Selection intensity was assumed equal to one.

Relative selection efficiencies (RSE) were expressed as a percentage of the expected responses to selection obtained for BG1 with each selection index, as follows:

RSE2= (BG2/BG1) × 100, and RSE3= (BG3/BG1) × 100. RSE4= (BG4/BG1) × 100. RSE5= (BG5/BG1) × 100.


The weighting factors for milk yield, fat percentage and protein percentage, standard deviations of indexes and of breeding goals, correlations between selection indexes and breeding goals, according to the breeding goals, are presented in Table 3, for the three sale situations.

In general, the weighting factors of traits showed magnitude and signals that were in agreement with the breeding goals and economic values of traits in the breeding goals. In the research of Seno et al., (2005), taking into account the differences of the two production systems (milk and Mozzarella systems), economic values were calculated for MY, FY and PY as 0.26, -0.27 and -0.30 for milk production system and 1.12, 6.90 and 20.14 in Mozzarella production system, respectively. Also, Pieters et al., (1997) calculated economic values of milk traits for three different Italian payment systems. The first system is based on a payment system for regions, where milk is produced for direct consumption or fresh dairy products, second system is based on using milk for

the production of Parmesan cheese, where protein is of high value and payment system suggested by Badino dealing for the 1985 price and production circumstances for an optimal cheese production (Rozzi, 1989). The results showed that Parmesan payment system resulted in the highest marginal value of milk with average composition in situations without herd output limitation.

The selection indexes, calculated for the three studied sale situations and different breeding goals are presented in Table 4. XM, XF and XP refer to the average performance of sires’ daughters for milk yield and milk fat and protein percentage, expressed as deviations from the average performance of all females. XF and XP were standardized for the b value obtained for milk yield.

The index and individual responses to selection, calculated for each breeding goal in the three sale situations (Milk, Milk and Sarshir, Milk and Mozzarella), are presented in Table 5.

In the case of milk sale situation, when breeding goal was only milk, the individual response for milk yield was 257.12 kg. For breeding goal 2 and 3, when we select with two and three-trait indexes, the individual response for milk yield approximately decreased (256.96 and 256.91 kg, respectively), but decreasing amount is negligible. About breeding goal BG2 and BG3, individual responses for fat and protein percentage were negative. In the fourth and fifth breeding goals, as the results show due to the negative economic weights for both fat and protein percent, selection based on the attributes of the characters and for these traits, reduced fat and protein percentage (-0.3 and -0.06 %).

Regarding the milk sale situation, the greatest economic response to the index was observed for BG3 (US$103.52), when three studied


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traits were in the breeding goal, while the lowest index response was observed for BG1 (US$102.85) when selection was only on the milk yield. This was expected, taking into account the current milk payment policy with no differential payment for milk components in this region and negative correlations between fat and protein percentage and milk yield. However, the expected index response for breeding goal BG3 (US$103.52) was the same as BG2 (US$103.30) and both of them were close to BG1 (US$102.85) and the differences were low.

The relative efficiencies of selection were 100.43 and 100.63% for BG2 and BG3, respectively, indicating that the selection exclusively on each selection index (BG1, BG2, and BG3) would be efficient. But this parameter was calculated as 5.11 and 5.77% for BG4 and BG5, respectively, indicating that selection exclusively on fat percent of fat and protein percent would not be efficient in this case. Totally, these results suggest that under the current payment policy, it is not desirable to select on fat and protein percentage, given that the revenues are based exclusively on milk sale. So, selection for milk is the easiest index and more suitable for milk sale situation. In the case of milk and Sarshir sale situation, when breeding goal was only milk, the individual response for milk was approximately the same for three first breeding goals (257.10, 257.20, 257.30 kg). The economic responses were also the same for these three breeding goals. For BG2, with increasing the number of traits in the index, the individual result for milk did not change basically, but with adding fat and protein percentage in this index, the individual response for fat percentage was improved (from -0.03 to -0.01%). In breeding goal 3, the three-trait indexes (257.30 kg) were superior to selection on milk and fat percentage for milk response. But there were not observed any

advantage between two and three-trait indexes for fat percentage response (-0.01 and -0.01%). The expected index responses, in the milk and Sarshir sale situation, were US$143.99 (BG1), US$143.97 (BG2) and US$144.12 (BG3). Despite the positive economic weigh for fat percentage, the individual responses to selection for FP were negative and similar for BG2 and BG3. It seems that negative correlation between fat percentage and milk yield and also greater response for milk yield cause to decrease fat percent. In the fourth and fifth breeding goals, as the results show, selection based on and for fat percent, increased fat percentage as 0.3%, but the response for protein percentage is negative. It was expected because in this case the protein percentage is not important. The relative efficiency of selection for BG2 and BG3 was 100% (99.98 and 100.09%, respectively). This parameter was calculated as 1.93 and 1.94% for BG4 and BG5, respectively, indicating that selection exclusively on fat percent of fat and protein percent would not be efficient. So, comparing these results suggest that with selling of Sarshir besides the milk, selection on milk and fat percentage can be desirable as easiest selection index.

In the case of milk and Mozzarella sale situation the individual response for milk was also approximately the same for all three breeding goals with increasing the number of traits in index (257.12, 257.30, 257.00 kg) with insignificant differences. The economic responses were also the same for indexes of MY and MY & FP, in this sale situation. For BG2 with two-trait index, with adding fat and protein percentage in the index, the individual response for fat percentage was improved (from -0.03 to -0.01%). The economic response also had slight increasing for this breeding goal. In breeding goal 3, selection on milk and fat percentage or milk, fat and protein percentage


411

(-0.004 and -0.005%) were superior to selection index on only milk. For protein percentage also there were not observed any advantage between two and three traits (-0.01 and -0.01%). In this case, the economic response increased for BG3 with adding traits in selection index.

In the milk and Mozzarella sale situations, the expected index responses were US$185.13 (BG1), US$185.08 (BG2) and US$184.59 (BG3). The individual responses to selection for FP and PP were negative but less than those in milk and Sarshir sale situation. In this case, the relative efficiency of selection for BG2 and BG3 was 100%, too (99.97 and 99.70%, respectively). This parameter was calculated as 2.45 and 4.98% for BG4 and BG5, respectively. The greatest selection response in these two breeding goals was obtained in milk and Mozzarella sale situation.

In spite of the economic importance of milk components (fat and protein percentage) in the production of Mozzarella cheese, in this situation the results showed that the greatest response was observed when the breeding goal included just the milk yield. But, in the production of Sarshir, the results showed that the responses were approximately the same for three breeding goals. Comparing the individual responses to selection for FP and PP obtained for the milk and Mozzarella and milk and Sarshir sale situations, in milk sale situation if selection were based on these two traits (BG2 and BG3), negative responses were obtained because of negative economic values. In two other situations, these individual responses also were obtained negative because of their negative correlation with milk yield but its magnitude has decreased from milk sale situation.

The differences in both index and individual responses for milk production traits between the two sale situation (milk and, milk and

Sarshir) show the importance of implementing appropriate selection indexes for buffalo dairy herds taking into account the local production and market circumstances, with special reference to regions where the Sarshir represents the main product of the buffalo dairy activity. Also, the incorporation of the Mozzarella-making process on the farm level (as proposed sale situation) resulted in positive economic values for FP and PP, and consequently improved individual responses for these traits. Also, at the individual responses obtained for BG3, it is observed that the individual responses for FP and PP were around 40 and 50%, respectively, superior to those of BG2. The results also suggest that the additional payment for fat and protein percentage could benefit not only milk producers, but also the industry.

Seno et al., (2006) in study of the index and individual responses to selection for milk (MY), fat (FY) and protein (PY) yields for different breeding goals for two commercial buffalo milk production systems in São Paulo State (1. all milk produced is sold to the industry and 2. all milk produced is used in the Mozzarella cheese-making process at the farm), suggest that when milk is produced for sale to the industry, selection for milk components is not advantageous and when Mozzarella making is added to the system, the selection for components and milk volume is the most economically beneficial.

CONCLUSION

The results obtained in the present study suggest that for the milk sale situation, selection for milk components is not advantageous. However, when the sale situation shifted to milk and Sarshir or the manufacturing of Mozzarella cheese is


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Table 3. Weighting factors (b) for milk Yield (MY), fat (FP) and protein (PP) percentage, standard deviations of indexes (σI) and of breeding goals (σH), correlations between selection indexes and breeding goals (rIH), according to the breeding goal (BG), for the three sale situations.

Sale Situation BGB

σI σH rIHMY FP PP

Milk

1 0.5954 - - 102.85 119.22 0.862702 0.5968 -7.306 - 103.30 119.89 0.861643 0.5979 -9.939 3.905 103.52 120.18 0.861384 - -22.35 - 5.26 6.28 0.837875 - -22.41 -17.43 5.94 7.17 0.82773

Milk & Sarshir

1 0.8336 - - 143.99 166.91 0.862702 0.8373 33.05 - 143.97 166.67 0.863773 0.8385 30 10.06 144.12 166.88 0.863664 - 11.84 - 2.78 3.33 0.837875 - 12.47 -20.19 2.80 3.23 0.86399

Milk & Mozzarella

123

1.072 - - 185.13 214.59 0.862701.077 46.56 - 185.08 214.23 0.863921.079 40.5 123.3 184.59 213.46 0.86475

4 - 19.28 - 4.54 5.42 0.837875 - 17.9 84.14 9.22 11.21 0.82258

Table 4. Selection indexes standardized for the b value obtained for milk yield (MY), for the three sale situations (Milk, Milk and Sarshir cream and Milk & Mozzarella) and different breeding goals.

Sale Situation BG Index b value(MY)

Milk

123

1 (Xmy) 0.59441 (Xmy) - 12.24 (Xfp) 0.59681 (Xmy) - 16.62 (Xfp) + 6.53 (Xpp) 0.5979

4 - 22.35 (Xfp) -5 - 22.41 (Xfp) - 17.43 (Xpp) -

Milk and Sarshir

123

1 (Xmy) 0.83301 (Xmy) + 39.47 (Xfp) 0.83731 (Xmy) + 35.78 (Xfp) + 12 (Xpp) 0.8385

4 11.48 (Xfp) -5 12.47 (Xfp) – 20.19 (Xpp) -

Milk and Mozzarella

123

1 (Xmy) 1.0721 (Xmy) + 43.23 (Xfp) 1.0771 (Xmy) + 37.53 (Xfp) + 114.27 (Xpp) 1.079

4 19.28 (Xfp) -5 17.9 (Xfp) + 84.14 (Xpp) -


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Table 5. Individual* and expected index responses to selection for Milk, Milk and Sarshir, and Milk and Mozzarella sale situations.

Sale Situation BGIndividual Response Index

Response ($)MY (kg) FP (%) PP (%)

Milk

1 257.10 - - 102.852 256.90 -0.03 - 103.303 256.90 -0.03 -0.01 103.524 - -0.3 - 5.265 - -0.3 -0.06 5.94

Milk and Sarshir

1 257.10 - - 143.992 257.20 -0.01 - 143.973 257.30 -0.01 -0.01 144.124 - 0.3 - 2.785 - 0.3 -0.03 2.80

Milk and Mozzarella

1 257.12 - - 185.132 257.30 -0.01 - 185.083 257.00 -0.005 -0.01 184.594 - 0.3 - 4.545 - 0.23 0.10 9.22

*For milk yield (MY), fat percentage (FP) and protein percentage (PP).

adopted, selection for components and milk volume is the most beneficial from decreasing fat and protein percentage. The differences in index responses for milk production traits between the three sale situations (Milk, Milk and Sarshir, Milk and Mozzarella) suggest that it is necessary to take into account the local production and market circumstances, when designing breeding programs for buffalo milk production situations in Iran.

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Anynoumus. 2008. Buffalo breeding in Khuzestan, p. 23-20. Publications committee of the agriculture promotion and exploitation of the Khuzestan province.

Aspilcueta-Borquis, R.R., R.C. Sesana, M.H.M. Berrocal, L.D.O. Seno, A.B. Bignardi, L.El Faro, L.G. de Albuquerque, G.M.F. de Camargo and H. Tonhati. 2010. Genetic parameters for milk, fat and protein yields in Murrah buffaloes (Bubalus bubalis Artiodactyla, Bovidae). Genet. Mol. Biol., 33(1): 71-77.

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Da-you, F.A.N., X.U. Shang-zhong, L.I. Jun-ya, R.E.N. Hong-yan and Y.A.N.G. Xue-li. 2008. Genetic and statistical analysis between production traits and secondary traits in Chinese Semintal. Acta Vet. et Zoote. Sin., 39(8): 1025-1032.

Dezfuli, B.T., A.N. Javaremi, M.A. Abbasi, J. Fayazi and M. Chamani. 2011. Economic Weights of Milk Production Traits for Buffalo Herds in the Southwest of Iran Using Profit Equation. World Applied Sciences Journal, 15(11): 1604-1613.

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Pieters, T., F. Canavesi, M. Cassandro, E. Dadati and

J.A.M. Van Arendonk. 1997. Consequences of differences in pricing systems between regions on economic values and revenues of a national dairy cattle breeding scheme in Italy. Livest. Prod. Sci., 49: 23-32.

Ponzoni, R.W. 1988. The derivation of economic values combining income and expense in different ways: an example with Australian Merino sheep. J. Anim. Breed. Genet., 105: 143-153.

Rosati, A. and L.D. Van Vleck. 2002. Estimation of genetic parameters for milk, fat, protein and Mozzarella cheese production in Italian river buffalo population. Livest. Prod. Sci., 74: 185-190.

Rozzi, P. 1989. Indici economici adottati dall’Anafi nella se lezione; da1 IX Congresso ANAFI. Bianco Nero giugno 1989, 6: 23-27.

Seno, L.O. 2005. Valores econômicos para as características de produção de leite de búfalos (Bubalus bubalis) no Estado de São Paulo. Dissertação (Mestrado em Genética e Melhoramento Animal), Faculdade Estadual Paulista, Jaboticabal.

Seno, L.O., V.L. Cardoso and H. Tonhati. 2006. Responses to selection for milk traits in dairy buffaloes. Genet. Mol. Res., 5(4): 790-796.

Seno, L.O., V.L. Cardoso, L. El Faro, R.C. Sesana, R.R. Aspilcueta-Borquis, G.M.F. de Camargo and H. Tonhati. 2010. Genetic parameters for milk yield, age at first calving and interval between first and second calving in milk Murrah buffaloes. Livestock Research for Rural Development, 22(2).

Taheri, D.B., A.N. Javaremi, M.A. Abbasi, J. Fayazi and M. Chamani. 2012. Performance study and estimating genetic parameters of production and reproduction traits of

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Khuzestani buffaloes. Iran J. Vet. Res., 8(3): 45-53.

Thevamanoharan, K., W. Vandepitte, G. Mohiuddin and M. Shafique. 2000. Genetic, phenotypic and residual correlation between various performance traits of Nili-Ravi buffaloes. Buffalo Bull., 19: 80-86.

Tonhati, H., M.F.C. Muñoz, J.Á. Oliveira, J.M.C. Duarte, T.P. Furtado and S.P. Tseimazides. 2000. Parâmetros genéticos para a produção de leite. Gordura e proteína em bubalinos. R. Bras. Zootec, 29: 2051-2056.

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ABSTRACT

In Malwa region of Madhya Pradesh reproductive failure (anoestrus) is a major problem in buffaloes under field conditions due to under feeding and non availability of balanced ration. To find out the nutritional causes behind anoestrus, thirty anoestrus buffaloes {10 heifers (average body wt. 262.80±22.51 Kg)+20 buffaloes (average body wt. 461±10.83 Kg, milk yield 7.62±0.48 litre/h/d)} were selected randomly that have normal genitalia from ten villages of Indore district. Average daily feed intake of each animal was recorded and proximate principles, major elements like Calcium (Ca) and Phosphorus (P) and trace elements like iron (Fe), zinc (Zn), manganese (Mn), copper (Cu) and cobalt (Co) in available feedstuffs were determined to find out nutrient availability. Deficiency of various nutrients was calculated by comparing with the standard requirements of the animals. Blood samples were also collected from the animals and analyzed for different haemato-biochemical constituents. The average values for different blood parameters in heifers and lactating buffaloes were hemoglobin (Hb) 10.32±0.19 and 11.15±0.32 g/dl; blood glucose, 50.62±1.80 and 59.29±1.05 mg/dl; plasma protein 5.06±0.19 and 6.33±0.18 g/dl, respectively. Major mineral profile in heifers and lactating buffaloes were Ca,

8.13±0.38 and 9.59±0.25 mg/dl; Inorganic-P (iP) 5.06±0.14 and 5.07±0.17 mg/dl; Total-P 24±2.21 and 17.50±1.42 mg/dl, respectively. Trace mineral levels in heifers and lactating buffaloes were Fe 22.05±0.58 and 17.68±0.84ppm; Zn 1.04±0.05 and 0.87±0.05 ppm; Cu 0.90±0.05 and 0.77±0.04 ppm; Mn 0.84±0.05 and 1.14±0.10 ppm; Co 1.92±0.41 and 1.13±0.09 ppm, respectively.

Results indicated that values of Hb, blood glucose, total protein, Ca, iP, Zn were marginally low but Cu was deficient in heifers, while levels of iP, Cu and Zn were marginally low in lactating buffaloes. Fe levels were found high in both heifers and lactating buffaloes. It may be concluded that dietary deficiency reflected the hemato-biochemical profile of anoestrus buffaloes and a strategic supplementation is needed to these animals for exploitation of their genetic potential for optimum production and reproduction.

Keywords: nutritional status, blood profile, anoestrus, buffalo, Malwa, Madhya Pradesh

INTRODUCTION

Reproductive failure of dairy animals is the major area of concern now days in all over the country, which causes a huge economical loss to the

NUTRITIONAL STATUS AND HEMATO-BIOCHEMICAL PROFILE OF ANOESTRUS BUFFALOES OF MALWA REGION OF MADHYA PRADESH

Nagendra Patil, R.K. Jain and Dharmesh Tewari*

Department of Animal Nutrition, College of Veterinary Science and Animal Husbandry, Narendra Deva University of Agriculture Technology, Kumarganj, Faizabad, Uttar Pradesh, India, *E-mail: [email protected]

Original Article


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dairy owners. Among the various factors affecting it, nutrition is one of the most important factors which receive less attention than what actually it should be. For normal development and activity of reproductive organs, feeding of balanced ration is of utmost importance, because most field cases of reduced fertility or sterility are of nutritional origin. The interaction between nutrition and reproduction needs particular attention in our country, to overcome nutritional inadequacies either in terms of quantitative or qualitative nutrient deficiencies/ imbalances.

The nutritional deficiency causes several infertility conditions in buffaloes and the highest (50.26%) prevalence was observed for anoestrus, while cases of repeat breeder, metritis, pyometra and prolapse were only about (25.69%) among the common field cases Malwa region of Madhya Pradesh (Shukla et al., 2007). Thus anoestrus remains a major condition which constitutes about half of the reproductive problems occurring in buffaloes.

In Malwa region of Madhya Pradesh most of the farmers rear crossbred cattle and buffaloes as the dairy animal and anoestrus is the major reproductive problem among dairy animals. Farmers are following traditional feeding practices, usually cotton seed cake is the only concentrate source fed to lactating cattle along with straw (wheat/ masoor/ gram/ soybean) and mineral supplementation is a rare inhabitant among the farmers (Mudgal et al., 2003). Other factors associated with anestrous are energy deficiency (Ling et al., 2007) and minerals also play important role in the regulation of hormones and enzymes for initiation of estrus (Dhoble and Gupta, 1986). This nutritional deficiency also reflects the blood profile. So that blood profile of anoestrus buffaloes of Malwa region was evaluated in this experiment.


Thirty buffaloes {10 heifers (average body wt. 262.80±22.51 Kg)+20 buffaloes (average body wt. 461±10.83 Kg, milk yield 7.62±0.48 litre/h/d)} with normal genitalia (by per rectal examination) and without any clinical infection, showing anoestrus were selected randomly from 10 villages (Borkhedi, Harsola, Kevti, Piplihamalhar, Umaria, Panda, Rau, Rangwasa, Sonvay and Bhaslai) around Veterinary College, Mhow of Indore District (from Malwa region of Madhya Pradesh). Body weights (Kg) of the animals were determined by recording the length (inch) and girth (inch) of each animal and then putting them in Shaeffer’s formula (Sastry et al., 1982). Feed offered and residues left of each animal were weighed with the help of spring balance at both times (morning and evening) for three consecutive days. Then average feed intake of each animal was calculated. The representative samples of each feed were subjected to proximate analysis (AOAC, 1995), Ca and P (Talpatra et al., 1940) content and trace mineral estimations by Atomic Absorption Spectrophotometer.

Milk yield (litre) of each lactating animal was measured during milking (morning and evening) for three consecutive days. After that average milk yield was calculated. Availability of DM, DCP, TDN, major (Ca and P) and trace elements (Fe, Cu, Mn, Zn and Co) for each animal was calculated on the basis of chemical composition of feedstuffs and their intake. Selenium, carotene and vitamin E intake were worked out using values given in the literature. Finally, the nutrient availability of individual animal was compared with the standard nutrient requirements calculated out for specific body weight and productivity of individual animal with the help of feeding standards (Kearl, 1982) and thus the deficiencies/ excess to specific nutrient


419

was worked out.Blood samples of around 15 to 20 ml were

collected from all 30 anoestrus buffaloes (heifer and lactating) from jugular vein in a sterilized plastic tubes containing anti-coagulant heparin solution (0.2 mg/ml of blood).

The tubes containing blood samples were kept in ice and brought to the laboratory. In laboratory blood was analyzed for hemoglobin (Oser, 1979), glucose (Folin and Wu, 1920) and total phosphorus (AOAC, 1995) and remaining blood was centrifuged for separating the plasma. The plasma sample then stored in glass vials for analysis of protein (Teitz, 1986), Ca (Ichaylova and Iikova, 1971), inorganic P (Fiske and Subbarow, 1925) and trace minerals (Fe, Zn, Cu, Mn and Co) using atomic absorption spectrophotometer [Perkin Elmer Aanalyst 100, USA] after wet digestion.


The chemical composition of specific feed ingredients being consumed by the animals has been presented in Table 1. The feeds being offered to the heifers were mainly the agricultural by products including wheat straw, gram straw, masoor straw and soybean straw, while wheat bran, cotton seed cake or at some places concentrate mixtures were also being used additionally in the ration of lactating buffaloes.

In Table 2, the availability of different nutrients to the animals was worked out and compared with the standard requirement and hence the excess or deficiency of specific nutrient has been presented. As compared to the standard requirements (Kearl, 1982), availability of DM was about 4-6% less, which may be associated with the deficiency of TDN too. Similar findings was also

been reported by Mudgal et al. (2003) and Tiwary et al., (2007). Between the groups deficiency of major nutrients was observed in heifers for digestible crude protein which was only about 48% to that of the requirements, while limited deficiency (11%) was seen in lactating buffaloes and which may be associated with the supply of concentrate to the lactating buffaloes. Similar observations were also recorded by earlier workers (Sinha, 1982; Sohal et al., 1982; Mudgal et al., 2003 and Tiwary et al., 2007). The lower levels of energy and /or protein may be associated with the ovarian inactivity and anoestrus (Wiltbank et al., 1965) as negative energy balance depresses the ovarian activity by inhibiting pulsatile LH release (Butler and Smith, 1989).

When the major minerals were compared it showed that Calcium was the element supplied in excess (33 to 283%) to the requirement and which may be due to supply of higher amounts of leguminous straws in their ration. This reduces the availability of phosphorus on one hand and over supply the calcium on other hand as leguminous straws having a wider Ca: P ratio and having a fair deficiency of element phosphorus. Phosphorus is necessary for normal energy and phospholipids metabolism as well as normal skeletal development and its severe deficiency may delay the onset of puberty and postpartum anoestrus and increased incidence of cystic follicles because of inactive ovaries, whereas moderate and low conception rates (Pugh et al., 1985). Many other workers also have found lower levels of inorganic phosphorus in serum of anoestrus heifers /buffaloes than cyclic animals (Naidu and Rao, 1982; Kumar et al., 1992 and Dutta et al., 2001).

Among trace elements the supply of iron and cobalt remained on plus side, while of zinc and copper remained deficient, while Mn was deficient only in buffaloes but not in heifers. The presence


420

Tabl

e 1.

Mac

ro a

nd m

icro

nut

rient

con

tent

s of f

eeds

tuffs

(DM

bas

is).

Feed

stuf

fsW

heat

stra

wG

ram

stra

wM

asoo

r st

raw

Soyb

ean

Stra

wW

heat

bra

nC

otto

nSe

ed c

ake

Con

cent

rate

m

ixtu

reC

P (%

)3.

95±0

.22

6.24

±0.2

46.

52±0

.32

6.14

±0.3

413

.99±

0.52

22.6

0±0.

4216

.16±

2.70

EE (%

)0.

99±0

.06

0.63

±0.5

01.

50±0

.11

0.80

±0.0

63.

45±0

.11

10.2

2±0.

643.

50±0

.18

CF

(%)

33.0

8±0.

6939

.16±

0.74

36.9

1±0.

8241

.77±

1.48

9.99

±0.9

227

.15±

1.72

15.1

9±1.

36N

FE (%

)50

.31±

0.66

45.9

5±1.

2046

.21±

0.87

45.5

2±1.

7468

.71±

1.63

35.7

5±1.

1549

.29±

2.51

TA (%

)11

.64±

0.64

7.98

±0.3

48.

84±0

.30

6.59

±0.3

84.

51±0

.74

4.25

±0.2

213

.98±

1.79

AIA

(%)

5.84

±0.1

62.

44±0

.15

4.17

±0.2

30.

69±0

.06

0.48

±0.1

00.

18±0

.02

6.08

±1.4

8C

a (%

)0.

23±0

.02

1.54

±0.0

81.

46±0

.07

0.94

±0.0

40.

21±0

.01

0.22

±0.0

10.

32±0

.05

P (%

)0.

06±0

.01

0.04

±0.0

00.

05±0

.00

0.24

±0.0

150.

61±0

.05

0.51

±0.0

20.

17±0

.02

Fe (p

pm)

414.

76±6

.47

364.

14±1

2.8

605.

28±8

.05

461.

99±6

2.70

298.

51±1

5.23

275.

24±1

6.96

258.

45±5

.94

Zn(p

pm)

13.4

8±1.

128.

41±0

.28

23.2

4±1.

9226

.43±

1.37

63.4

0±8.

1941

.64±

3.03

28.8

3±2.

49M

n (p

pm)

39.8

1±1.

1815

.68±

0.85

87.0

7±7.

3567

.63±

2.38

71.3

7±3.

2215

.80±

0.56

18.8

2±1.

37C

u (p

pm)

7.91

±0.3

64.

36±0

.29

4.86

±0.4

110

.01±

0.70

11.6

8±1.

1610

.12±

0.80

2.66

±0.3

2C

o (p

pm)

0.16

±0.0

30.

72±0

.06

0.71

±0.0

60.

18±0

.02

0.69

±0.1

70.

55±0

.05

0.86

±0.0

5


421

Tabl

e 2.

Dai

ly re

quire

men

ts a

nd a

vaila

bilit

y of

nut

rient

s in

anoe

stru

s buf

falo

es.

Para

met

ers

Req

uire

men

ts

for

300

kg

Bod

y w

t.

Avai

labi

lity

for

262.

80±

22.5

1 B

ody

wt.

Defi

cien

cy

/ Exc

ess

(%)

Req

uire

men

ts fo

r 50

0 kg

B

ody

Wt a

nd 8

lits

/day

pr

oduc

tion

(7%

fat)

Avai

labi

lity

For

461±

10.8

3 kg

B

ody

wei

ght

Defi

cien

cy

/Exc

ess

(%)

Hei

fers

Buf

falo

esD

MI (

Kg)

5.99

5.75

±0.4

34

(-)

1211

.33±

0.47

6 (-

)D

CP

(g)

374

179.

32±2

1.80

52 (-

)77

268

4.69

±39.

9711

(-)

TDN

Kg)

3.55

2.83

±0.2

120

(-)

7.28

6.06

±0.2

617

(-)

Ca

(g)

1557

.38±

8.92

283

(+)

46.4

61.9

3±6.

3933

(+)

P (g

)12

8.16

±1.2

832

(-)

35.8

24.4

1±2.

1531

(-)

Fe (m

g)29

9.5

2779

.56±

252.

6482

8 (+

)60

038

01.7

3±23

9.87

534

(+)

Zn (m

g)17

911

8.24

±13.

7434

(-)

480

294.

24±2

1.01

39 (-

)M

n (m

g)23

9.6

353.

96±3

8.74

48 (+

)48

039

6.24

±24.

8317

(-)

Cu

(mg)

59.9

44.6

1±3.

2226

(-)

120

91.4

0±4.

0924

(-)

Co

(mg)

0.59

2.45

±0.5

131

5 (+

)1.

204.

68±0

.38

290

(+)

Se (m

g)0.

6-1.

80.

78±0

.06

Ade

quat

e1.

2-3.

62.

4A

dequ

ate

Vit

A (I

U)

1200

057

17.8

1±45

52 (-

)21

000

1141

8.95

±0.2

046

(-)

Vit

E (I

U)

159.

49±1

.54

38 (-

)15

57.9

6±4.

9828

6 (+

)


422

of zinc is highly essential for certain enzymatic activities related to reproduction and indirectly it may act through the pituitary to influence the release of gonadotropic hormones or directly through complexing with specific legend in gonads (Miller, 1979). Deficiency of copper may also be reflected on reproductive behavior as well as performance of animals. Inactive ovaries, delayed oestrus and early embryonic death have been reported to occur due to deficiency of copper (Hidiroglou, 1979 and Singh and Vadnere, 1987).

Due to practice of least greens supply in farmers vitamin A remained the most deficient among the animals and vitamin A is very important for maintaining the health status of epithelial tissue of the reproductive tract. The deficiency of vitamin E was only observed in heifers and which indicates the lacking of concentrate in their ration, but not in lactating buffaloes. The negative impact of insufficient vitamin E was also been observed on ovulation rates (Harrison et al., 1984) and postpartum activities (Arechiga et al., 1994) of the

animals.The average values of blood Hemoglobin,

glucose and total phosphorus and plasma levels of Protein and different major and micro elements of the anoestrus buffalo heifers and lactating buffaloes are shown in Table 3. It was observed that Hb contents in anoestrus heifers (10.32±0.19 g/dl) and buffaloes (11.15±0.32 g/dl) were lower than the normal value (13.14±0.06 g/dl) as reported by Das et al. (2003). It may be due to deficient levels of copper. Similar lower values (8.79-10.24 g/dl) were also observed by other workers (Sharma et al., 1983 and Perumal et al., 2007). The average values of blood glucose and total protein were also found lower in anoestrus lactating buffaloes and in heifers compared to the values of healthy animals (Mandal et al., 2002 and Nayyar et al., 2003). Similar findings were reported by different workers (Sharma et al., 1983; Tandle et al., 1998; Jani et al., 2001; Sharma et al., 2004; Singh and Singh, 2005; Singh and Singh, 2006, Perumal et al., 2007). In contrast to above findings Giri and

Table 3. Hemato-biochemical profile* of anoestrus buffaloes.

Parameters Heifers BuffaloesHemoglobin (g/dl) 10.32±0.19 11.15±0.32Glucose (mg/dl) 50.62±1.80 59.29±1.05Total Phosphorus (mg/dl) 24±2.21 17.50±1.42Total Protein (g/dl) 5.06±0.19 6.33±0.18Calcium (mg/dl) 8.13±0.38 9.59±0.25Inorganic Phosphorus (mg/dl) 5.06±0.14 5.07±0.17Iron (ppm) 22.05±0.58 17.68±0.84Zinc (ppm) 1.04±0.05 0.87±0.05Copper (ppm) 0.90±0.05 0.77±0.04Manganese (ppm) 0.84±0.05 1.14±0.10Cobalt (ppm) 1.92±0.41 1.13±0.09

* The values of hemoglobin, Glucose and Total Phosphorus were reported in blood, while others in plasma.


423

Yadav (2001) and Jagathesan et al. (2006) reported that Hemoglobin, glucose and total protein values were in normal range in anoestrus animals and which may be associated with change in feeding practices of those animals.

The plasma concentration of total P, Fe, Mn and co were within range in heifers (McDowell et al., 1984) but Ca (8.13±0.38 mg/dl), inorganic phosphorus (5.06±0.14 mg/dl) and Zn (1.04±0.05 ppm) and Cu (0.90±0.05 ppm) were below the normal values (Underwood, 1977; Prasad and Rao, 1997; Das et al., 2002; Mandal et al., 2002; Das et al., 2003 and Sharma et al., 2004). In lactating buffaloes the values of Ca (9.59±0.25 mg/dl), total P (17.5±1.42 mg/dl), Fe, Mn and Co were in range but iP, Zn, and Cu were marginally low (Underwood, 1977; McDowell et al., 1984; Mandal et al., 1996; Paul et al., 2000 and Yadav et al., 2002). The concentration of Fe in plasma of heifers (22.05±0.58 ppm) and lactating buffaloes (17.68±0.84 ppm) observed many times higher than the reported normal values (Underwood 1977; Prasad and Rao, 1997; Das et al., 2002; Das et al., 2003 and Sharma et al., 2004) indicates the higher levels of it in the feed ingredients used.

It may be concluded that dietary deficiency reflected the hemato-biochemical profile of anoestrus animals and a strategic supplementation is needed to these animals for exploitation of their genetic potential for optimum production and reproduction.

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Arechiga, C., F.O. Ortiz and P.J. Hansen. 1994.

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Butler, W.B. and R.D. Smith. 1989. Interrelationship between energy balance and poetpartum reproductive function in dairy cattle. J. Dairy Sci., 72: 767.

Das, A., T.K. Ghosh and S. Haldar. 2003. Mineral distribution in soil, feeds and grazing cattle of different physiological stages in the red laterite and new alluvial agro climatic zones of West Bengal. Indian J. Anim. Sci., 73(4): 448-454.

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Dhoble, D. and S.K. Gupta. 1986. Serum calcium and inorganic phosphorus levels during postpartum anoestrus in buffaloes. Indian J. Anim. Health, 25: 123-126.

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Fiske, C.H. and Y.S. Subbarao. 1925. Determination of inorganic phosphorus in blood. J. Biol. Chem., 66: 375.

Folin, O. and H. Wu. 1920. A system of blood analysis. Supplement IA simplified and


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Jagatheesan, P.N.R., N. Selvaraju, V.R.S. Kumar and C. Chandrahasan. 2006. Blood biochemical profile and estrus induction to arguments fertility in anoestrus Murrah buffaloes. Tamil Nadu Journal of Veterinary and Animal Science, 2(1): 10-12.

Jani, R.G., B.R. Prajapati, P.R. Patel and M.R. Dave. 2001. Certain haematological and biochemical changes in normal fertile and infertile cross bred cows. Indian Journal of Dairy and Biosciences, 12: 22-25.

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Ling, K., A. Waldmann, J. Samarutel, H. Jaakson, T. Kart and A. Leesmae. 2007. Field trial on the relation ship of blood metabolites and body condition score with recurrence of luteal activity in Holstein cows. J. Vet. Med. A., 54(7): 337-341.

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Mudgal, V., M.K. Mehta, A.S. Rane and S. Nananavati. 2003. A survey on feeding practices and nutritional status of dairy animals in Madhya Pradesh. Indian J. Anim. Nutr., 20(2): 217-220.

Naidu, K.V. and A.R. Rao. 1982. Short course on role of minerals and vitamins in livestock health and production. In Kumar, H. 2004. Reproductive disorders due to deficiency of minerals and vitamins in livestock. IVRI, Izatnagar, U.P., Indian Vet. J., 59: 781.

Oser, B.L. 1979. Blood analysis. Hawk’s Physiological Chemistry, 14th ed. Tata Mcgraw Hill Publishing Co. Ltd., New Delhi. p. 1054-1093.

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Prasad, K.S.N. and S.V.N. Rao. 1997. Blood mineral profile of anestrous and repeat breeder cross bred cows-A field study. Indian J. Anim. Nutr., 14(2): 135-137.

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426

beef female through 2nd calving. Agriculture Research. Service, U.S.D.A., Washington, D.C., USA. Tech. Bull., 1314.


427

Original Article

ABSTRACT

Four experiments were conducted to study the effect of media supplements on in-vitro maturation, cleavage and embryo development of buffalo oocytes. In experiment 1, oocytes were cultured in TCM-199+10% fetal calf serum (FCS) and kept at 39oC under 5% CO2 for in-vitro embryo development. In experiment 2, excellent quality oocytes were subjected to TCM-199 enriched with either 10% FCS or estrous buffalo serum (EBS; 20 to 40 pg/ml) and then fertilized using frozen semen in TALP medium containing heparin (0.02 mg/ml) and caffeine (3.89 mg/ml). In experiment 3, oocytes were cultured in-vitro maturation (IVM) medium supplemented or not with 20 IU/ml equine chorionic gonadotropins (eCG). Experiment 4 was carried out to examine the suitable capacitating agent, either heparin or caffeine or both. Excellent and good quality oocytes produced higher (P<0.05) maturation and morula development rates. In-vitro maturation and cleavage rates were significantly higher (P<0.05) in IVM medium plus EBS or eCG. Heparin and caffeine produced significantly (P<0.05) higher embryo developmental rates. In conclusion, excellent quality oocytes cultured with either EBS or eCG and fertilized with buffalo spermatozoa capacitated with heparin and caffeine

progressively enhanced developmental competence of buffalo oocytes.

Keywords: buffalo, oocytes, eCG, heparin, caffeine

INTRODUCTION

The application of superovulation and embryo transfer in buffaloes has been slow (Mehmood et al., 2011; Kandil et al., 2012). In-vitro production of buffalo embryos has been gaining attention for its research and commercial applications (Mehmood et al., 2011). In-vitro embryo production (IVEP) would be an effective technique to improve the efficacy of transferable embryos (Di Francesco, 2010). Aspiration and slicing methods were mostly used for the recovery of buffalo oocytes from abattoir ovaries (Di Francesco, 2010). The efficacy of this method was compared on the basis of cumulus oocyte complexes per ovary (COCs/ovary; Cremonesi et al., 2010). However, these techniques are severely hampered by poor recovery of total oocytes and in-vitro maturation and fertilization (IVMF; Masudul Hoque et al., 2011). Conditions during in- vitro maturation, fertilization and embryo culture

DEVELOPMENTAL COMPETENCE OF BUFFALO (BUBALUS BUBALIS) OOCYTES: EFFECT OF OOCYTES QUALITY, PROTEIN ADDITIVES, HORMONAL SUPPLEMENT

AND TYPE OF CAPACITATING AGENTS

M.M. Waheed1, K.H. El-Shahat2 and A.M. Hammam3

¹Department of Clinical Studies, College of Veterinary Medicine and Animal Resources, King Faisal University, Kingdom of Saudi Arabia, E-mail: [email protected] of Theriogenology, Faculty of Veterinary Medicine, Cairo University, Egypt3Department of Animal Reproduction and Artificial Insemination, National Research Center, Egypt


428

(IVMFC) are believed to play a pivotal role in the acquisition of development competence of embryos (Alvarez et al., 2013). Therefore, the present study aimed to increase the developmental competence of buffalo’s oocytes by studying the effect of oocytes quality, protein additives, hormonal supplement and type of capacitating agents.


All chemicals used in the study were reagent grade (Sigma-Aldrich, American Samoa, USA) or as otherwise indicated.

Collection and culture of oocytes Ovaries were collected at a local abattoir within 20 to 30 minutes after slaughter of buffaloes and transported in a warm saline solution (0.9% NaCl) to a well-equipped laboratory within 2 h. At the laboratory, ovaries were washed 3 times in normal saline containing 100 IU/mL penicillin and 100 ug/mL streptomycin. Non atretic antral follicles (2 to 6 mm diameter) were aspirated with an 18- gauge needle connected to a 10 mL disposable syringe. The aspiration medium consisted of modified phosphate buffer (M-PBS) enriched with sodium pyruvate (0.036 g/mL), 10% fetal calf serum (FCS) and the above mentioned antibiotics. Follicular oocytes were recovered and counted under stereomicroscope. The recovered oocytes were washed 3 times in IVM medium. According to the number of cumulus cell layers and ooplasm morphology, oocytes were divided into three groups as adapted after Dadashpour Davachi et al. (2012): (1) Excellent COCs; (2) Good POCs (partial oocytes complexes); (3) Fair DO (denuded oocytes).

Experimental design Experimental 1: Effect of oocytes quality on IVMFC of buffalo oocytes. Oocytes (n=320) were cultured in TCM-199 plus 10% FCS and 50 ug/mL gentamicin and covered with mineral oil in CO2 incubator containing 5% CO2 and 95% relative humidity at 39oC. Maturation rate was assessed either by the degree of cumulus mass expansion (Srinivasa Prasad et al., 2013) or by staining with 1% aceto-orcein stain (1% orcein in 45% glacial acetic acid) for observation of the 1st polar body (Prentice-Biensch et al., 2012).

Sperm capacitation and IVF One 0.5 mL straw of frozen buffalo semen was thawed in a water bath at 37oC for 30 seconds. Spermatozoa were washed twice by centrifugation in TALP medium supplemented with 3.89 mg/mL sodium caffeine benzoate and 0.02 mg/mL heparin. Sperm pellet was suspended in 2mL TALP medium enriched with 20 mg/mL bovine serum albumin (BSA) plus the above mentioned additives. The sperm cell concentration was adjusted to 2×106 /mL sperm cells (Di Francesco, 2010). A 100 ul aliquot of the sperm cell suspension was placed into a four well cultured dish and covered with warm mineral oil. After maturation, oocytes were washed in the same sperm suspension medium and then 15 to 20 oocytes were transferred into the sperm suspension droplet and cultured under the previous conditions in CO2 incubator for 5 h. After fertilization, oocytes were washed 3 times in IVM medium and then cultured for 6 to 7 days in CO2 incubator. The cleavage rate and the frequency of morula and blastocyst were recorded. Experimental 2: Effect of protein additives (EBS) on cleavage and embryo developmental rates of buffalo oocytes. Only excellent quality oocytes (n=237) were washed 3 times in IVM medium then


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cultured for maturation in either TCM-199+10% FCS that considered as a control group or in TCM-199+10% estrous buffalo serum (EBS; Jamil et al., 2007). IVM medium was supplemented with 50 ug/mL gentamicin. In both groups, fertilization was performed using frozen thawed buffalo semen capacitated in TALP medium containing heparin (0.02mg/ml) and sodium caffeine benzoate (3.89mg/ml). Maturation, cleavage and embryo developmental rates were carried out as in experiment1. Experimental 3: Effect of gonadotropin (eCG) added to the maturation medium on cleavage and embryo developmental rates of buffalo oocytes. oocytes (n=290) were classified into two groups; Group 1 washed 3 times in TCM-199 enriched with 10% FCS without gonadotropins and served as a control and Group 2 in which IVM medium was supplemented with 20 IU/ml equine chorionic gonadotropins (eCG; Intergonan®, Intervet, Holland). Fertilization, maturation, cleavage and embryo developmental rates were performed as in experiment 1. Experimental 4: Selection of suitable capacitating agent added to TALP medium. This experiment (n=210 fertilized oocytes) was carried out to examine the suitable capacitating agent added to TALP medium, either heparin (0.02 mg/ml) or caffeine (3.89 mg/ml) or both. IVM/IVFC

was accomplished as previously mentioned in experiment 1.

STATISTICAL ANALYSIS

Experiments were repeated five times. Data were pooled and analyzed by Chi-square test using SPSS 22.0 statistical software (2013).

RESULTS

Experiment 1: The effect of oocytes quality on maturation, cleavage and developmental rates of IVF buffalo oocytes is shown in Table 1. Maturation rate and cleavage rates in COCs and POCs groups was significantly (P<0.05) higher than with Do type (Figures 1 and 2). In addition, the proportion of embryo that developed to the morula and blastocyst stage was significantly (P<0.05) higher in COCs and POCs groups than in DO type.

Experiment 2: Table 2 denotes that addition of EBS to the culture medium (TCM-199) produces a significantly (P<0.05) higher maturation and cleavage rates than those cultured in the same medium supplemented with FCS. The proportion of embryos in term of morula and blastocyst doesn’t

Table 1. Effect of oocytes quality on the maturation, cleavage and Embryo development rates (%).

Oocytes quality

No. of oocytes cultured

Maturation rate (%)

Cleavage rate (%)

Embryo development rates (%)

Morula BlastocystCOCs 100 70 (70.00)a 35 (50.00)a 15 (42.85)a 10 (28.57)a

POCs 100 65 (65.00)a 30 (46.15)a 10 (33.34)a 5 (16.67)b

DO 120 40 (33.34)b 10 (25.00)b 1 (10.00)b 0 (00.00)c

Percentages with dissimilar superscripts in the same column are significantly different at P<0.05


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Table 2. Effect of estrous buffalo serum (EBS) added to the culture medium (TCM-199) on the maturation, cleavage and embryo developmental rates (%).

Culture condition


Maturation rate (%)

Cleavage rate (%)


Morula BlastocystTCM-199+10% FCS

100 45 (45.00)a 15 (33.34)a 4 (26.67)a 3 (20.00)a

TCM-199+10% EBS

137 100 (72.99)b 50 (50.00)b 15 (30.00)a 10 (20.00)a


Table 3. Influence of commercially available source of gonadotropins (eCG) on in vitro maturation, cleavage and embryo developmental rates (%).

Culture condition


Maturation rate (%)

Cleavage rate (%)


Morula BlastocystTCM-199+10% FCS

143 84 (58.74)a 25 (29.76)a2

(8.00)a

1(4.00)a

TCM-199+10% FCS+20 IU

eCG147 110 (74.82)b 50 (45.46)b 10 (20.00)b 8 (16.00)b


Table 4. Influence of heparin and/or sodium caffeine benzoate addition on the cleavage and embryo developmental rates (%) of buffalo oocytes.

TreatmentNo. of fertilized

oocytesCleavage rate

(%)Embryo development rates (%)

Morula BlastocystHeparin (H) 60 20 (33.34)a 2 (10.00)a 2 (10.00)a

Caffeine (C) 70 25 (35.72)a 3 (12.00)a 2 (8.00)a

H+C 80 45 (56.25)b 15 (33.34)b 10 (22.23)b

Percentages with dissimilar superscripts in the same column are significantly different at P<0.05.


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Figure 1. Buffalo embryo developed in-vitro to 4-cell stage.

Figure 2. Buffalo embryo developed in-vitro to morula stage.


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significantly vary between EBS and FCS groups.

Experiment 3: As shown in Table 3, maturation, cleavage and embryo developmental rates up to blastocyst are significantly (P<0.05) higher when eCG added to IVM medium compared with hormonal free medium.

Experiment 4: Table 4 shows that the addition of heparin together with sodium caffeine benzoate to TALP medium resulted in significantly (P<0.05) higher cleavage and embryo developmental rates compared with the use of heparin or caffeine alone.

DISCUSSION

In the present study, data showed that good quality buffalo oocytes surrounded by multilayer of compact investment with a homogenous ooplasm had a significantly higher maturation cleavage, and developmental rates up to blastocyst compared with oocytes of poor quality. This finding identifies the essential role of cumulus cells in promoting normal cytoplasmic maturation of oocytes necessary for fertilization and embryo development of buffalo oocytes. Our results are similar to those previously reported for buffalo oocytes (Gasparrini, 2013). The presence of cumulus cells surrounding the oocyte is essential to facilitate the transport of nutrients and signals into and out of oocytes (Kharche and Birade, 2013). The cumulus cells improve fertilization rate first by providing a capacitating- inducing mechanism and secondly by facilitating the interaction between capacitated spermatozoa and the zona pellucida surface (de Souza et al., 2013). However, Zhang et al. (2012) found that cumulus cells had no influence on fertilization.

The result of this experiment indicated that, addition of EBS to the IVM medium progressively enhanced the developmental competence of buffalo oocytes as compared to FCS additives to the same medium. This is in agreement with the result of Jamil et al. (2007) in buffalo. A possible explanation for the beneficial role of ESB might be a result of its relatively high LH and estradiol levels (Terzano et al., 2012). LH hormone may affect the cytoplasmic maturation of oocytes by increasing the calcium distribution within the ooplasm and promote glycolysis, combined with an increased mitochondrial glucose oxidation metabolism within the oocytes (Silverstre et al., 2007). The beneficial effects of EBS for oocyte maturation may also act via cumulus cells or directly on the oocytes. EBS contains a number of known growth factors that have an important role in the regulation of oocyte maturation, and it also prevents the hardening of the zona pellucida (Jamil et al., 2007). Moreover, Alm et al. (2002) stated that the higher maturation rates of equine oocytes could be due to the increased concentration of insulin like growth factor-1 in estrous mare serum (more than twice that found in FCS). In the present study, IVM of buffalo oocytes in TCM-199 medium supplemented with eCG increased maturation cleavage, and developmental rates up to blastocyst as compared to control medium. These findings run parallel to those previously reported for buffalo (Hegab et al., 2009). eCG as a source of gonadotropin (more FSH and less LH) stimulation lead to the generation of positive factors that acted on the oocytes to override the inhibitory influence and induced germinal vesicle breakdown (Wang et al., 2009). Therefore, cAMP dependent protein kinase regulated by cumulus cells following FSH- stimulation play a role in the complex mechanism of chromatin


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condensation leading to meiotic resumption in bovine oocytes (Tatemoto and Terada, 1998). Moreover, FSH enrichment to the culture medium enhances early embryonic development (Anderiesz et al., 2000). In this study, the addition of heparin and caffeine to TALP medium resulted in a significantly higher cleavage and embryo developmental rates as compared to the addition of heparin or caffeine alone to the same medium. This is in agreement with the results reported in buffalo by Scholkamy (2002). The role of caffeine may be through increasing the concentration of cAMP which accelerate the rate of capacitation (Breininger et al., 2010); whereas, heparin appeared to be necessary for capacitation and acrosome reaction (Cormier and Bailey, 2003). Heparin stimulates the conversion of proacrosin to acrosin and also it was reported to be responsible for change of calmodulin and calmodulin binding protein at capacitation (Leclerec et al., 1990). It seems therefore that, there is a synergistic action for both heparin and caffeine in penetration of oocytes in-vitro, which depends on their compensatory action to induce capacitation and/or to increase penetration of oocytes (Tajik and Niwa, 1998).

CONCLUSIONS

Excellent quality oocytes cultured in IVM medium supplemented with either protein additives (EBS) or hormonal supplement (eCG) and fertilized with capacitated buffalo spermatozoa in TALP medium enriched with heparin and caffeine progressively enhanced developmental competence of buffalo oocyte.

ACKNOWLEDGEMENT

The study was conducted in the Veterinary Research Division, National Research Centre, Dokky, Egypt.

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Álvarez, C., C. García-Garrido, R. Taronger and G. González de Merlo. 2013. In vitro maturation, fertilization, embryo development and clinical outcome of human metaphase-I oocytes retrieved from stimulated intracytoplasmic sperm injection cycles. Indian J. Med. Res., 137(2): 331-338.

Anderiesz, C., A.P. Ferraretti, C. Magli, A. Fiorentino, D. Fortini, L. Gianaroli, G.M. Jones and A.O. Trounson. 2000. Effect of recombinant human gonadotrophins on human, bovine and murine oocyte meiosis, fertilization and embryonic development in vitro. Hum. Reprod., 15(5): 1140-1148.

Breininger, E., P.D. Cetica and M.T. Beconi. 2010. Capacitation inducers act through diverse intracellular mechanisms in cryopreserved bovine sperm. Theriogenology, 74(6): 1036-1049.

Cormier, N. and J.L. Bailey. 2003. A differential mechanism is involved during heparin-and cryopreservation-induced capacitation of bovine spermatozoa. Biol. Reprod., 69(1):


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177-185.Cremonesi, F., K. Anderson and A. Lange-

Consiglio. 2010. Efficacy of tuohy needle in oocytes collection from excised mare ovaries. Vet. Med. Int., 2010. http://dx.doi.org/10.4061/2010/102591.

Dadashpour, D.N., H. Kohram and S. Zainoaldini. 2012. Cumulus cell layers as a critical factor in meiotic competence and cumulus expansion of ovine oocytes. Small Rumin. Res., 102: 37-42.

Di Francesco, S. 2010. Effect of season on reproductive performances in buffalo species (Bubalus bubalis). Ph.D. Thesis, Napoli, Italy. p. 166.

Gasparrini, B., 2013. In vitro embryo production in buffalo: yesterday, today and tomorrow. Buffalo Bull., 32(1): 188-195.

Hegab, A.O., A.E. Montasser, A.M. Hammam, E.M.A. Abu El-Naga and S.M. Zaabel. 2009. Improving in vitro maturation and cleavage rates of buffalo oocytes. Anim. Reprod., 6(2): 416-421.

Jamil, H., H.A. Samad, N.U. Rehman, Z.I. Qureshi and L.A. Lodhi. 2007. In vitro Maturation and Fertilization of Riverine Buffalo Follicular Oocytes in Media Supplemented with Oestrus Buffalo Serum and Hormones. Acta Vet. Brno., 76: 399-404.

Kandil, O.M., A.S.S. Abdoon, D. Kacheva, C.H. Karaivanov, M.F. Fadel, N.A. Hemeida, B. Georgiev, T.I. Maslev, W.M. Ahmed and H.R. Badr. 2012. Successful embryo transfer in Egyptian buffaloes. Glob Vet., 8(4): 320-327.

Kharche, S.D. and H.S. Birade. 2013. Parthenogenesis and activation of mammalian oocytes for in vitro embryo production: A review. Advances Biosci

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R.D. Lambert. 1990. Decreased binding of calmodulin to bull sperm proteins during heparin induced capacitation. Biol Reprod., 42: 483-489.

Masudul Hoque, S.A., S.K. Kabiraj, M.A.M. Yahia Khandoker, A. Mondal and K.M.A. Tareq. 2011. Effect of collection techniques on cumulus oocyte complexes (COCs) recovery, in vitro maturation and fertilization of goat oocytes. Afr. J. Biotechnol., 10(45): 9177-9181.

Mehmood, A., M. Anwar, S.M.H. Andrab and M. Afzal. 2011. In vitro maturation and fertilization of buffalo oocytes: the effect of recovery and maturation methods. Turkish J. Vet. Anim. Sci., 35(6): 381-386.

Prentice-Biensch, J.R., J. Singh, B. Alfoteisy and M. Anzar. 2012. A simple and high-throughput method to assess maturation status of bovine oocytes: comparison of anti-lamin A/C-DAPI with an aceto-orcein staining technique. Theriogenology, 78(7): 1633-1638.

Scholkamy, T.H. 2002. Biochemical aspects in media and their supplements used for oocytes in vitro maturation, cleavage and sperm capacitation in Egyptian buffalo. Ph.D. Thesis, Cairo University, Egypt.

Silvestre, M.A., I. Alfonso, E. García-Mengual, I. Salvador, C.C. Duque and I. Molina. 2007. Effect of recombinant human follicle-stimulating hormone and luteinizing hormone on in vitro maturation of porcine oocytes evaluated by the subsequent in vitro development of embryos obtained by in vitro fertilization, intracytoplasmic sperm injection, or parthenogenetic activation. J.


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Souza de, J.M.G., N. Duffard, M.J. Bertoldo, Y. Locatelli, E. Corbin, A. Fatet and V.J.F. Freitas. 2013. Influence of heparin or the presence of cumulus cells during fertilization on the in vitro production of goat embryos. Anim. Reprod. Sci., 138(1-2): 82-89.

Srinivasa, P.C.H., A. Palanisamy, V.S. Gomathy, S. Satheshkumar, A. Thangavel and G. Dhinakar Raj. 2013. Effect of TCM-199 and synthetic oviductal fluid (SOF) medium and cysteamine supplementation to in vitro maturation media on maturation, cleavage rate and subsequent embryonic development of buffalo oocytes. Buffalo Bull., 32(3): 182-188.

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Tatemoto, H. and T. Terada. 1998. Involvement of cumulus cells stimulated by FSH in chromatin condensation and activation of maturation promoting factor in bovine oocytes. Theriogenology, 49: 1007-1020.

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ABSTRACT

The seminal plasma proteins and sperm membrane proteins of six each Bhadawari buffalo and Hariyana cattle were isolated by using the protein isolation kits and protein profiling were carried out by 1-D SDS-PAGE.

Keywords: buffalo, cattle, SDS-PAGE, seminal plasma protein, sperm membrane protein

INTRODUCTION

Seminal plasma is complex fluid containing a wide variety of both organic and inorganic components, among which proteins are an important part of the high-molecular-weight substances. The protein composition of mammalian seminal plasma varies in species and has important effect on sperm functions such as sperm motility (Henricks et al., 1998), viability (Brandon et al., 1999) and freezability (Asadpour et al., 2007), sperm capacitation and fertilization (Rodriguez et al.,1998) and also serve to protect sperm from damage or to maintain their longevity which were evident from the correlation observed among

semen characteristic and seminal plasma proteins reported by Sharma et al. (2015).

Besides proteins of seminal plasma, sperm surface proteins reported to have important role in recognition of zona proteins for binding to zona and plasma lemma of ovum and also in acrosome reaction for successful fertilization (Jagadish et al., 2005). It has been reported that, the loss of integrity of the sperm plasma membrane is frequently associated with infertility in male, despite normal semen parameters (Rajeev and Reddy, 2004).

Looking to the importance of buffalo in India, it becomes necessary to understand the structural and functional attributes of the buffalo spermatozoa and its comparison with that of cattle which will help in better understanding of molecular features that make the buffalo sperm less fertile than cattle. In the scenario of climate change, indigenous animals are of choice for better dissemination of gemplasm and it urges the science to have a picture of the protein profiles of the semen as a whole to make it more suitable for artificial insemination. Keeping in this mind the present study was designed for comparative evaluation of protein profiles of seminal plasma and sperm membrane in buffalo and cattle bull semen.

Original Article

SEMINAL PLASMA AND SPERM MEMBRANE PROTEINS OF BUFFALO AND CATTLE BULLS: A COMPARATIVE STUDY

Shilpi Dixit1, Vijay Pandey1,*, Dilip Kumar Swain1, Rajesh Nigam1, Ambika Sharma1, Deepak Sharma1, Atul Saxena2 and Pawanjit Singh2

1College of Biotechnology, *E-mail: [email protected],2College of Veterinary Science and Animal Husbandry, Deen Dayal Upadhyaya Veterinary and Animal Science University (DUVASU), Mathura, India



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Experimental animalsSemen samples were collected by an

artificial vagina from six each sexually mature Haryana cattle and Bhadawari buffalo bulls (2 to 4 years old) from the District Dairy Demonstration farm of College of Veterinary Science and Animal Husbandry, Mathura. The animals were maintained in nearly identical nutritional and management conditions throughout the period of study. The animals utilized were clinically healthy and regularly dewormed and vaccinated against common ailments.

Semen evaluationEjaculates were collected twice a week

from each bull in morning hours (8:00 to 9:00 AM) and total six ejaculates were collected from each bull during the study period. Immediately after collection, semen volume was determined with a graduated plastic tubes and concentration of spermatozoa (million/ml) was determined by automatic sperm counter.

Preparation of seminal plasma The seminal plasma was prepared by

centrifugation and protein extraction was carried out by using the Triprep extraction kit (Fisher Scientific). Fresh semen was centrifuged at 5000 rpm for 10 minutes. The total protein was estimated by spectrophotometric method at 280 nm (protein’s absorbance at 280 nm). After the estimation of total protein, the protein extraction was carried out as per the protocol of the kit. Proteins were recovered by centrifugation at 10,000 rpm for 10 minutes, re-suspended in phosphate buffered saline (PBS) and stored at -20oC until further analysis of seminal plasma proteins. The SDS-PAGE analysis was

done after one day of sample processing.

Preparation of sperm membrane extractThe sperm membrane proteins were

extracted by method described by Cheema and Babbar (2008) with some modification. The sperm pellets were washed three times in PBS (pH 7.4) and resuspended in lysis buffer containing 1.5% (w/v) Tris [hydroxylmethyl] aminomethane buffer (pH 6.8), 20% (w/v) sucrose, 10% (w/v) SDS, 5% (v/v) β-mercaptoethanol and 0.05% (w/v) bromophenol blue (3’3”5’5” tetrabromophenol-sulfonephthalein). The mixture was kept in boiling water bath for 5 minutes and centrifuged at 5000 rpm for 10 minutes and protein extraction was carried out by using the Triprep extraction kit (Fisher Scientific). Proteins were recovered by centrifugation at 10,000 rpm for 10 minutes, re-suspended in phosphate buffered saline (PBS) and stored at -20oC until further analysis by SDS-PAGE.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE was performed for separation and determination of molecular weight of seminal plasma and sperm membrane proteins. Proteins samples were subjected to SDS-PAGE as per method of Laemmli (1970), using a 12% polyacrylamide gel. The relative molecular weights were determined by using the (broad range molecular weight markers of Merck, Germany), Gel documentation and analysis system (Gel-doc. Model- Alpha imager TM1220, Alpha Innotech Corporation, USA).

Image analysis and statistical analysis Gel images were analysed to determine molecular weight and relative protein content


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using the Gel doc system. Data were analyzed using SPSS software program (SPSS version 16.0 for Windows). The results were presented as mean ± standard error of mean (S.E.M) and analysis of significance was attributed at P<0.05.


The results of semen characteristics and protein concentration of seminal plasma and sperm membrane extract of buffalo and cattle bulls are depicted in Table 1 and 2, respectively. The statistical analysis of the result did not show significant difference in ejaculate volume and sperm concentration in buffalo and cattle bull semen while protein concentration of seminal plasma as well as sperm membrane extract showed significant difference (P<0.01) in buffalo and cattle bull semen. The lower ejaculate volume and sperm concentration was observed in buffalo compared to cattle bull in present study corroborate the reports of Khalek et al. (2008) in Nilliravi buffalo and Holestein cattle bulls. In spite of the insignificance of differences between the two species in semen characteristics, there was tendency of lower values of ejaculate volume and sperm concentration in buffalo than cattle bulls (Table 1).Variation in semen volume and sperm concentration might be due to differences in frequency of collection, season, nutrition, management, genetics, reproductive health status and age of bulls (Soderquist et al., 1992). Variations can also be due to skill of semen collector/attendant and temperature of AV.

The seminal plasma protein concentration in present investigation showed a significant difference (P<0.01) between buffalo and cattle bull semen. Comparable seminal plasma protein concentration was observed by Arangasamy et al. (2005) and Singh et al. (1995) in cattle and Nendre

(2007) in Surti and Dhanju et al. (2001) in Murrah buffaloes. The concentration of sperm membrane extract protein showed significant difference (P<0.05) in buffalo and cattle bull spermatozoa. The concentration of membrane protein extracted from sperms of buffalo and cattle in present study was observed lower than the reports of Cheema and Babbar (2008) in crossbred cattle and Dhanju et al. (2001) in buffalo bulls. The variation in the concentration of extracted proteins may be due to genetic variability in the animals or may be due to variations in extraction methods used by different authors.

Table 2 shows the protein profile of seminal plasma and sperm membrane protein of buffalo and cattle bulls. The electrophoretogram of buffalo semen revealed 24 protein bands ranging between 6.0 to 200 kDa in seminal plasma and 14 protein bands ranging from 16.0 kDa to 205 kDa in sperm membrane extract proteins. Nine protein bands of molecular weight 20, 26.5, 36.5, 38, 44, 66, 70, 72 and 84 kDa were observed common in seminal plasma and sperm membrane of buffalo which indicated that these proteins are structural as well as secretory in nature. Asadpour et al. (2007) and Sharma et al. (2014) revealed 25 protein bands on SDS - PAGE analysis of seminal plasma in buffalo bulls, and most of the bands were observed to be comparable with results of present study. Selvaraju et al. (2010) and Dhanju et al. (2001) also reported protein bands of comparable molecular weight in seminal plasma and sperm membrane of buffalo spermatozoa, respectively. The reports of these authors simulate the findings of present study.

SDS-PAGE of cattle seminal plasma revealed 13 protein bands ranging from 6.5 kDa to 204 kDa. The proteins of comparable molecular weight were reported by Jobim et al. (2004) and Bellin et al. (2012) in seminal plasma of different


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Table 1. Mean semen characteristics of Haryana cattle and Bhadawari buffalo bulls.

Semen attributes Cattle BuffaloSemen volume (ml) 4.038±0.22a 2.958±0.18a

Sperm concentration (106/ml) 1736.944±60.46a 1678.05±86.68a

Seminal plasma protein (gm/dl) 7.86±0.34a 4.63±0.16b

Sperm membrane protein (mg/109sperms) 2.81±0.25a 4.42±0.63b

Means bearing at least one common superscript alphabet in one parameter did not differ significantly (P≥0.05), otherwise significant at 5% level (P<0.05).

Table 2. SDS-PAGE protein profile of seminal plasma and sperm membrane of cattle and buffalo bull semen (kDa).

S. No.Cattle Buffalo

Seminal Plasma Sperm Membrane Seminal Plasma Sperm Membrane1 6.5 6.5 6.5 162 8.5 8.5 12.5 203 18.5 12 18.5 22.54 26.5 22.5 20 26.55 43 25 24 36.56 66 26.5 26.5 387 70 32.5 28 428 75 34.5 32 449 84 38 35 6610 88 43 36.5 7011 96 48 38 7212 160 58 40.5 8413 204 66 44 17414 69 46 20515 70 4816 84 6017 174 6618 7019 7220 8421 8622 9623 18424 200


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breeds of cattle. Bellin et al. (1996) reported proteins of 75, 84, 66 kDa molecular weight in seminal fluid of cattle bulls. Fernandez et al. (2009) reported eight bands ranging from 15 to 63kDa. Out of these eight bands, two similar proteins (22 and 25kDa) reported by Fernandez et al. (2009) in Bos taurus taurus bulls simulates the findings of present study. The cattle sperm membrane proteins revealed 17 protein bands ranging between 6.5 to 174 kDa. Five proteins of comparable molecular weight (84, 66, 48, 24 and 12 kDa) were reported by Bellin et al. (1996) in sperm membrane of vasectomised bulls. Cheema et al. (2011) reported proteins of comparable molecular weight (10, 25, 40, 65, and 70 kDa) in sperm membrane of buck. The reports of these authors simulate the findings of present study. Proteins observed other than these proteins in present study may be specific to cattle bulls. Seven protein bands of molecular weight 6.5, 8.5, 26.5, 43, 66, 70, and 84 kDa were observed common in seminal plasma and sperm membrane of cattle which indicated that these proteins are structural as well as secretory in nature.

The protein bands of molecular weight 6.5, 10 and 12 kDa in cattle and 26.5, 36.5 and 174 kDa in buffalo sperm membrane were observed to be most abundant proteins. Seven protein bands (6.5, 18.5, 26.5, 66, 70, 84 and 96 kDa) in seminal plasma and seven protein bands (22.5, 26.5, 38, 66, 70, 84, and 174) in sperm membrane were detected similar in both the species in present investigation. Proteins detected other than these proteins in present study may be said to be species specific proteins.

In conclusion, not all the seminal plasma and sperm membrane proteins are similar in buffalo and cattle only seven proteins are observed to be similar in seminal plasma and sperm membrane of buffalo and cattle semen. The difference in protein

profile indicates species specific variations may account for their variable fertility and freezability of buffalo and cattle semen. Further studies may be carried out by employing the modern tools to characterize these proteins and their putative role in the regulation of variable fertility and freezability in buffalo and cattle bull semen. This will help in the formulation of extenders during semen processing so as to increase the post thaw quality of the buffalo semen.

ACKNOWLEDGEMENTS

Authors are thankful to Vice Chancellor, UP Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishvavidyalya evam Gau Anusandhan Sansthan, (DUVASU), Mathura, for providing necessary facilities to carry out this work. The authors are also thankful to Dean, College of Biotechnology and College of Veterinary Science, Mathura for his cooperation and guidance in carrying out the research work.

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ABSTRACT

Change in climatic factors poses formidable challenge to the livestock sector development in Pakistan. Repeat breeding (RB), defined as adult buffalo and cow’s failure to conceive from more than 3 times regularly spaced AI or natural services in the absence of any detectable reproductive abnormalities, is a costly problem for the dairy sector. An active surveillance was conducted aimed to address the impact of climate change on incidence of RB in different cattle and buffalo breeds in Khyber Pakhtunkhwa (KPK), Pakistan. Through multistage cluster sampling 3 different climatic and geographic clusters were selected. Total of 1167 animals were included in the study. Out of total 586 were cows and 581 were buffaloes. The sampled population was stratified on parity basis into primiparous and multiparous cow’s and buffalo sub-groups. The overall incidence of RB was calculated 27.33%.

RB incidence was significantly (P<0.05) higher in buffaloes (33.04%) than in cattle (21.67%).

Whereas RB in multiparous (29.28%) were significantly (P<0.05) higher than primiparous

(23.71%) cattle and buffaloes. Significant variations in incidence of RB with season were observed. The results also elicit the significant impact of monthly mean temperature, humidity, average annual rainfall, altitude and breed on the incidence of RB. The culling percentage was significantly (P<0.05) higher in repeat breeder buffaloes (77%) than in cattle (23%). In conclusion the result shows that exotic and cross bred cattle breeds were more efficient reproductively than buffaloes and non descriptive breeds of cattle in varying environmental conditions. It was also concluded that RB is a multi-factorial problem that involves a number of intrinsic and extrinsic factors fixed to the animal.

Keywords: repeat breeding, geographic, primiparous, multiparous, incidence

INTRODUCTION

Repeat breeding (RB) has been considered from many decades one of the most important reproductive disorder in cattle and buffaloes. Incidences of RB in lactating large dairy

INCIDENCE OF REPEAT BREEDING IN VARYING BREEDS OF BUFFALOES AND CATTLE IN DIFFERENT CLIMATIC CONDITIONS IN

KHYBER PAKHTUNKHWA (PAKISTAN)

Amjad Khan1,*, Muhammad Hassan Mushtaq1, Mansur ud Din Ahmad1, Abid Hussain2, Asghar Khan3, Ajab Khan4 and Habibun Nabi5

1Department of Epidemiology and Public Health, University of Veterinary and Animal Sciences, Lahore Pakistan, *E-mail: [email protected] of Poonch Rawlakot Azad, Jammu and Kashmir, India3Department of Clinical Medicine and Surgery, 4Department of Pathology, University of Veterinary and Animal Sciences, Lahore, Pakistan 5Veterinary Research Institute (VRI), Peshawar, Khyber Pakhtunkhwa (KPK), Pakistan

Original Article



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animals varied among regions, management and environments. Internationally, declined have been reported in calving rates to 1st service from 60% to 40% over the past twenty five years (Bulman et al., 1978). Environment all over the world is the major factor in these days affecting reproductive and productive efficacy respectively. Breeding efficiency in buffaloes is affected greatly by season. Previous studies have shown a certain tendency of having better performance in cool months, July to February (70 to 80% conception rate) in buffaloes. It is also known that buffaloes are activated sexually by decrease in day length and temperature (Agrawal et al., 2003). As they have poor thermal regulation system. That’s why it is important to protect them in summer from extreme heat allowing them wallowing and also in winter from extreme cold, that may give chance to many other diseases to attack the animal (Ramesh et al., 2002).

A lower number of services per conception are needed during the July to February. RB syndrome is a major cause of economic losses and deprived reproductive performance in the dairy sector (Bartlett et al., 1986; Bage et al., 2002). It contributes to lower the dairy profit by insemination costs and wasting semen, increasing culling, increasing interval to conception and replacement costs and also reduces fertility (Gustafsson et al., 2002). The incidence of RB ranges from 10.1 to 24% mostly in exotic cattle breeds (Bartlett et al., 1986; Bage et al., 2002; Gustafsson et al., 2002). Though, specific causes of RB are not clear but it has found to be a multi-factorial problem involving a number of intrinsic as well as extrinsic one (Gracia et al., 2007). Ever since numerous factors affect incidence of RB in dairy animals, therefore it is intricate to make generalizations about major causes (Silvia, 1994).

Acclimation is a phenotypic reaction by the animals developed within the environment to an individual source of stress (Fregley, 1996). To cope with the thermal challenges that leads to reduced feed intake and many physiological functions alterations i.e. productive and reproductive efficiency (Beede and Collier, 1986; Wolfenson et al., 2000) reported that more that 50% of bovine population is being located in the tropical region and estimated economic losses in about 60% of the dairy farms due to heat stress around the world. It compromises oocyte growth in dairy animals by altering progesterone level, follicle-stimulating hormone, secretion of luteinizing hormone (LH) and dynamics throughout the estrus cycle (Ronchi et al., 2001).

According to the Intergovernmental Panel on Climate Change (IPCC) evaluation reports, about 0.6oC increase has occurred in average global temperature since the industrialization and in future increase of 2 to 4.5oC is almost expected by the end of 21th century (IPCC, 2007). Although climate changes at the global level, its positive impacts as well as negative, will be experienced at the local level.

The average annual temperature in South Asia by the end of 21th century could go up from 3.5 to 5.8oC because this region occurs in the arid and semi-arid zone (IPCC, 2007). Therefore the South Asian region will be more affected by the consequential climate change effects. In Pakistan these impacts are already visible particularly since 1990 Pakistan meteorological department (PMD records). For having great topographic contrasts and as a result the climate of the Pakistan has large temporal and spatial variations. In Pakistan no such research has conducted before addressing the impact of changing climatic factors at different altitudes on incidence of RB in different breeds of


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cattle and buffaloes. This research will also provide clue for future research to specify most significant risk factors responsible for incidence of RB.


Study design and site selectionAn active surveillance based study was

conducted in account to pile the incidence and impact of climate change on Repeat breeding (RB) in different breeds of cattle and buffaloes in Khyber Pakhtunkhwa (KPK) Pakistan. The target population in the study area was about 808068 lactating, 184229 dry and 97664 not yet calved buffaloes and 6059041 lactating cows, 743852 dry and 419547 cows that were not yet calved (Livestock census, 2006). Administratively the northern province of Pakistan KPK is divided into three agricultural zones i.e. Semi-Arid, Sub-Humid and Humid region (Fig 1). Eight districts were selected randomly, at least two from each region and one from Federally Administered Tribal Areas (FATA) (Figure 1). Geographically KPK could be separated into two zones: the northern and the southern one. The northern zone is winters with heavy rainfall and moderate summers. While southern zone is arid with hot summers and cold winters with scanty rainfall. The climate of KPK varies immensely as compared to its size, encompassing the majority of many types of climates found in the country.

Repeat breeder’s definition and exclusion criteria

A cow or buffalo was considered as a repeat breeder when it did not conceived after three inseminations or natural services (in case of buffaloes mostly), apart from having no detectable

clinical reproductive disorders. The animals were excluded on the following conditions.

• Having any detectable reproductive disorder.

• If not available for three consecutive AI or natural services.

• If the owner is not willing.• No farm animal was included in

the study.

Sampling technique and Sample size calculationThrough multistage cluster sampling the

study frames were selected. Then the end sample was drawn through simple random method for higher accuracy of results. The number of cow’s and buffaloes to be sampled for estimate prevalence with a confidence interval of 95% was estimated using the formula by Thrusfield, (1995).

N = 1.962 ×Pexp (1− Pexp)/d2

Where n = required sample size, Pexp = expected prevalence, and d = desired absolute precision.

Since the prevalence estimates of RB in the area were not available, therefore a 50% random estimate was chosen. The minimum number of cattle and buffaloes needed to be included from the study site calculated was 384. More animals were selected to take care of excluded animals during study.

Data collectionData regarding cattle and buffaloes from

smallholder dairy owners of the rural areas of KPK were obtained on a predesigned questionnaire. Only house hold animals were observed purposely to see the climatic impact under the local management


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practices in the study area. Various surveys were encountered from December 2012 to mid December 2013 to observe the status of RB in the included sampled animals at different seasons. Data about each cow and buffalo: breed, parity, season of heat and services, any reproductive disorder (pyometra, fetal membranes retention, endometritis, ovarian cysts and urovagina) was recorded.

Statistical analysesAnalysis was performed by using the

statistical package (SPSS 16.0). Chi square test was used to calculate the association between the categorical variations studied in the study i.e. repeat breeding with season, climatic region, parity, animal species, breed of the animal, average mean temperature and culling status of the repeat breeders.

RESULTS

A total of 1216 animals were included in the present study, in which 49 animals were lost in follow up not falling in the inclusion criteria. Thus 586 cows and 581 buffalo’s were included in the study. The study population was also stratified on parity basis to see the impact of climate change in relation to age of the animal. Total of 409 primiparous, 758 multiparous cows and buffaloes were selected (Table 1). Statistical analysis showed (27.33%) cumulative incidence of repeat breeding (RB); in cows (21.67%) and in buffalo (33.04%) significantly (P<0.05) higher than in cattle. The result elicits the insignificant impact (P>0.05) of climatic region on the incidence of RB (Table 1).

Parity of the animal was evaluated for the incidence of RB in different climatic conditions. It was found significantly (P<0.05) higher in

the multiparous cattle and buffaloes; 23.71% in primiparous and 29.28% in multiparous. Average rainfall, altitude and monthly average mean temperature showed a significant impact on the incidence of RB especially in the cows breed. Different breeds were included in the study both from the cattle and buffalo population prevailing in the study area. Most of the breeds are used to bring from the Punjab province of Pakistan into this area for milk purpose. The results illustrated higher cumulative incidence rate of (35.87%) in the non descriptive breed of cattle. While the most efficiently reproductive and highly conceptive breed were exotic breeds of cattle (Table 1). The local breed of cattle (Achai) was also found to be highly conceptive as compared to the non descriptive ones. Same was the case with buffalo population; in which the local Aza kheli breed was found with the lesser incidence rate of RB than Nili ravi and non descriptive buffalo breeds.

Seasonal variation was observed; significantly higher (P<0.05) RB incidence rate of (34%) in the late summer (July to September). While a higher conception rate of almost 83% from the month of January to April (Table 1). The most important factor that was found in the present study was the culling practice of the farmers after an animal gone to be a RB. Total of 200 (62.69) animals were culled out of 319 RB; 154 (77%) buffaloes and 46 (23%) cows. Amongst the culled animals 146 (73%) were multiparous RB while 54 (27%) were primiparous ones. A significant (P<0.05) difference of culling for species and parity was observed in the present study (Figure 2). Seasonal variation with culling was also observed.


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DISCUSSION

The present study was conducted to understand the impact of extent of change in the climatic factors i.e. monthly mean temperature, seasonal rainfall, humidity variation at different altitudes and geographical conditions. In this study, all the cows and buffaloes considered as RB had normal estrous cycles and returning to sexual cycle within 18 to 24 days, but did not got conceived, accordingly to the findings of (Allen et al., 1996).The reproductive performance of various breeds of cattle and buffalo’s was also evaluated in different environmental conditions. The overall cumulative incidence recorded in the present study was 27.33%. This was much higher than the previous cumulative incidence recorded by Rabbani et al. (2010) in Faisalabad district (Pakistan). The difference may be due the time span or environmental conditions as well different management practices in the two study areas. Also the later study was conducted in one of the hotter regions of the country where buffalo are considered to be more adoptive to the hot environmental conditions as compared to the climate of KPK. The cumulative incidence in the buffalo (33.04%) population was significantly (P<0.05) higher than cow’s (21.67%). This difference can be attributed to the difference in nature of two different species in terms of reproductive physiology. As previously discussed by Vale et al., (1988) and Danell (1987) that higher RB incidence in buffalo population might be due to low level of steroidal hormones and high progesterone level. The silent heat in buffalo is also the most reasonable cause of higher RB incidence. The incidence of RB in cows was in agreement with the reported incidence by Yusuf et al. (2010). The results of our study were also similar to that of (Perez et al., 2007; Kendall et al.,

2009).Table 1 depicts cumulative incidence

of RB in different climatic regions showing in significant (P>0.05) variation. This is because of the overall incidence calculated for both the species studied. There was significant variation observed in the incidence of RB in between the species and also amongst the different breeds based on parity. It was confirmed; RB incidence in primiparous (23.71%) lesser than multiparous (29.28%). This substantial variation could be due to the stress of high production and change in environmental conditions on the aged animals as compared to young ones (primiparous). These estimates were in coincidence with that of (Rabbani et al., 2010) reporting (28.35%) of RB incidence in 2nd lactating cows. Though Yusuf et al. (2010) reported higher conception rate in 1st parity cows and higher RB incidence totally opposite to the results of the present study. In this study we concluded higher conception rate in primiparous cows and buffaloes that multiparous ones (Robert et al., 2011) reported (26.61%) incidence of RB in primiparous cows almost in acceptance with the results of our study. To our knowledge, in Pakistan no accessible data exists on about the incidence of RB in different cattle and buffalo breeds. Breed variation was recorded in the present study in both the species that varied 15 to 35% in cattle and 26 to 34% in buffaloes; that falls in the range reported by Robert et al., (2011) of 0.00% to 42.42% on basis of breed variations.

Seasonal variation was significantly recorded among all the breeds of both the species (Table 1). Such environmental stresses on reproduction have also been explained earlier by Dobson et al. (2000) and Gwazdauskas et al. (1981) that heat stress shortens the duration and intensity of estrous expression leading to silent


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Table 1. Definitions and descriptive statistics of repeat breeding in different breeds of cattle and buffaloes, in KPK, Pakistan (2012-2013).

Variable code Description Values/level*RB

Incidence[%(n/N)]P-value

Host specie Cattle/Buffaloes Cattle 21.67 (127/586) <0.05Buffaloes 33.04 (192/586)

Geography Climate based Semi-Arid 28.23(83/294) 0.071Sub-Humid 22.15(76/343)Humid Region 29.94 (159/531)

Breed Cattle breeds Zebu cattle (Achai) 20.68 (6/29) <0.05Cross bred 18.06 (58/321)Exotic Breeds 15.23 (16/105)Non descriptive 35.87 (47/131)

Buffalo breeds Nili Ravi 34.59 (137/396) <0.05Aza-kheli 26.60 (29/109)Non descriptive 34.21 (26/76)

Season Winter (Dec-March) 16.61(53/319) <0.05Summer (Apr-June) 23.00(73/319)Monsoon (July-Sep) 34.00(107/319)

Post Monsoon (Oct-Mid Dec) 26.29 (86/319) Age Cow’s Parity Primiparous 16.45 (38/231) <0.05

Multiparous 25.07 (89/355)Buffalo’s Parity Primiparous 33.14 (59/178) 0.500

Multiparous 33.00 (133/403)

*n=number of repeat breeders, N= total animals observed.


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Figure 2. Showing the culling status of repeat breeders in the cattle and buffalo population in the studied animals.

Figure 1. Geographic representation of the study Area KPK, Pakistan.


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ovulation. These variations are also similar to the findings of (Agrawal et al., 2003). The astonishing major factor that is the economic loss for the smallholder dairy farmers in this region of the study observed was the culling of RB, s. Where 62.70% repeat breeders were culled in one year period that in much higher than (39.17%) reported by Rabbani et al. (2010) from Pakistan five years back. As can be judged in Figure 2 higher culling percentage is for multiparous buffalo repeat breeders as compare to cows. It is because of the higher RB incidence rate for that group and low conception rate after calving. Also the RB season played an important role in mass culling because of feed scarcity at that season. The longer the animal will take to conceive the greater chance it has to be culled. These involuntary culling of RB reduces the rural farmer’s profitability significantly because it is never correlated with dairy production.

CONCLUSION

To increase the reproductive performance of cattle and buffaloes in future in warming environmental challenges strategies shall be adopted, making selection of breeds for adequate production purpose. Accordingly the genetics must match the environment in future.

ACKNOWLEDGEMENT

The authors are highly thankful to the farmers of rural areas for their full cooperation during study period.

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ABSTARCT

Pre and post operative pain in animals suffering with horn affection should be attended to relieve stress on the animal as it may affect its production. The incidence and, pain evaluation was studied. Out of 126 cases 76 (60.3%), 26 (20.16%), 14 (11.11%), 9 (7.14) and 1 (0.79%) were fractures, avulsion, septic horn, overgrown horns and horn cancer respectively. The incidence of horn affections were 62 (49.2%) 55 (43.7%) and 9 (7.1%) right, left and both horn affections. The common pain symptoms like restlessness, twitching of ears, shaking of head, bruxism, rubbing against fixed objects, pawing at the affected site and evading the affected site were observed in horn affections. Conservative and surgical Management of various affections of horn in buffaloes was recorded and discussed.

Keywords: incidence, pain symptoms, horn affections, buffaloes

INTRODUCTION

Horns of buffaloes are massive, angular and well developed with a wider base as compared to cattle. The thickness of the horn shell increases towards apex until it becomes solid. The corium is traversed by numerous blood vessels. The horn is prone to various affections like avulsion, fracture, overgrowth, sepsis, fissures and cancer. Pre and post operative pain in animals suffering with horn affection should be attended to relieve stress on the animal as it may affect its production. Most of these affections do not respond to the routine medical management and demand amputation of the horn (Sreenu and Kumar, 2006). This Paper reports about the incidence, pain symptoms and management of various horn affections in buffaloes.


The overall incidence of horn affections in buffaloes was recorded in terms of number of cases presented to the Department of Veterinary

INCIDENCE, PAIN ASSESSMENT AND MANAGEMENT OF HORN AFFECTIONS IN BUFFALOES

K. Rama Rao1, Makkena Sreenu2, K.B.P. Raghavender3 and P.V.S. Kishore4

1Veterinary Dispensory, Gollapudi Krishna (Dt), Andhra Pradesh, India2Department of Veterinary Surgery and Radiology, NTR College of Veterinary Science, Sri Venkateswara Veterinary University Gannavaram, Krishna (Dt), Andhra Pradesh, India, E-mail: [email protected] of Veterinary Surgery and Radiology, College of Veterinary Science, Rajendra Nagar, Hyderabad, Andhra Pradesh, India4Department of Veterinary Anatomy and Histology, NTR College of Veterinary Sciecne, Gannavaram, Krishna (Dt), Andhra Pradesh, India

Original Article


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Surgery and Radiology, NTR College of Veterinary Science, Gannavaram, Andhra Pradesh, India over a period of one and a half years i.e. January 2010 to August 2011 with available and relevant technical records. The various ailments, particular to horn affections were tabulated and incidence of the each affection was noticed to arrive in terms of percentages. The clinical signs of the various horn affections, side of horn affected and parity were recorded. The various affections associated with horn in buffaloes were recorded and the pain exhibited by the animals with a particular affection was evaluated using pain score system modified as per the procedure (appendix) of Holton et al. (2001). The horn affections recorded were treated with appropriate method and discussed.


Incidence Out of 126 cases of horn affections 76 (60.3%), 26 (20.16%), 14 (11.11%), 9 (7.14) and 1 (0.79%) were fractures, avulsion, septic horn, overgrown horns and horn cancer respectively. The site of fracture was at tip, middle, lower third and base in 8 (10.5%), 11 (14.5%), 22 (30%) and 35 (46%) respectively out of 76 cases of fracture.

Out of 126 cases 62 (49.2%) 55 (43.7%) and 9 (7.1%) were right, left and both horn affections. In avulsion cases 13 (50%) and 13 (50%) were on right and left side. Among the fracture cases 40 (52.6%) and 36 (47.4%) were right and left affections. Out of 14 cases of septic horn 6 (42.9%) and 8 (57.1%) were right and left horn affections. The over grown horns reported in the present study were bilateral in 100 percent of the cases. In one buffalo with horn cancer it was observed to be a left horn affection.

In the present study higher incidence of fractures was recorded followed by avulsion, septic horn, overgrown horns and a case of cancer. The higher incidence of fractures was in accordance with the observations of Shivaprakash et al. (2007), Salgar (2008) and Mistry (2009) while Sreenu and Kumar (2006) and Mahida et al. (2009) reported higher incidence of Avulsion. The higher incidence of fractures among horn affections in the present study might be due to vigorous and infighting nature of buffaloes. The site of fracture was at tip, middle, lower third and base. The higher incidence of fractures at the base was more due to curly horns in buffaloes of this region which are mostly graded murrah and its crosses, which might be locked during fighting. The incidence of right side horns affection was more compared to the left in the present study. Similar observations were also made by Sreenu and Kumar (2006). On the contrary, Deshpande (1983) recorded involvement of left horn more than that of the right.Mahida et al. (2009) observed highest overgrown horns/ misshapen horns followed by avulsion of the horn.The avulsion of the horn was noticed to be the major affection of the horn in Surti buffaloes whereas the Mehsana buffaloes had more incidence of the overgrown horns misshapen horns. Naik et al., 1969; Kaul and Kalra, 1973 and Somvanshi, 1991 stated that incidence of horn cancer is a rare condition in buffaloes. Only one case was observed in the present study and it is in accordance with Kumar and Tilagar (2000) who recorded a bilateral sqamous cell carcinoma of the horn, while Salgar (2008) reported that four Mehsani buffaloes suffered from horn cancer. Damodaran and parthasarathy (1979) reported a case of neoplastic growth in a murrah buffalo bull. The observations suggestive of a very distinct animal husbandry practices followed in the area where in the livestock owners are not interested in


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rearing the male as all the cases studied during the present study were only the female buffaloes.

Pain symptomsThe pain symptoms exhibited by the

buffaloes with horn affections were given in Table 1. Avulsion of the horn: The prominent pain symptom observed in the buffaloes suffering with Avulsion of the horn included restlessness (87.5%) followed by twitching of ears (75%), decreased appetite and water intake (75%), rubbing the affected part against fixed objects (37.5%), and piloerection (37.5%) while all the animals tried to evade aside on palpation of affected area. The symptoms like dilatation of pupil, abnormal posture and bruxism were not observed with any animal. Fracture of the horn: The symptom observed in the buffaloes suffering with fracture of the of the horn included pilo erection , dilatation of pupil, restlessness, twitching of ears/shaking of head, bruxism , rubbing of the affected part against fixed objects, tries to evade aside on palpation of affected area and decreased appetite and water intake.

The pain reflexes varied with the site of the fracture. The symptoms like animal trying to evade the side on palpation of affected area in 100 percent and twitching of ears/shaking of head in 50 percent of the buffaloes were seen when the fracture was at the tip.

The symptoms like animal trying to evade the side on palpation of affected area (100%), rubbing of the affected part against fixed objects (66.66%), twitching of ears/shaking of head (66.66%) and restlessness (33.33%) were noticed in buffaloes suffering with the fracture of the horn at its middle third.

The buffaloes affected with fracture of

the horn at its lower third showed pain symptoms like animal trying to evade aside on palpation of affected area (100%), rubbing of the affected part against fixed objects (71.42%), twitching of ears/shaking of head (71.42%) , restlessness (42.85%) bruxism (28.57%) and decreased appetite and water intake (28.57%).

The symptoms like animal trying to evade the side on palpation of affected area (84.61%), rubbing of the affected part against fixed objects (76.92%), twitching of ears/shaking of head (76.92%) and restlessness (61.53%) decreased appetite and water intake (30.76%) and bruxism (23.07%) were observed in buffaloes suffering with the fracture of the horn at its base. The symptom like pilo erection was noticed in only one buffalo (7.69%) when the fracture was at the base of the horn.

Septic horn: The symptoms like twitching of ears/shaking of head (100%) rubbing of the affected part against fixed objects (100%), animal tries to evade the side on palpation of affected area (75%), restlessness (50%) decreased appetite and water intake (50%) and bruxism (25%) were observed in buffaloes suffering with septic horn.

Over grown horn: The prominent pain symptom observed with over grown horn in all buffaloes was animal trying to evade the side on palpation of affected area.

Horn Cancer: The symptoms like twitching of ears/shaking of head (100%) rubbing of the affected part against fixed objects (100%), animal tries to evade the side on palpation of affected area (100%) and restlessness (100%) were observed in buffaloes suffering with horn cancer. Pain score index and grading In avulsion cases the average pain score index was 12.00±0.46 and the pain was graded as severe. The average pain score index was 3.50±0.50, 6.67±0.88,


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7.14±0.34 and 6.54±0.31 in the fracture at the tip, middle third, lower third and base of the horn respectively. The pain was graded as moderate in all the fractures while the average pain score index in septic horn was 9.40±1.03 and the pain was graded as moderate. The average pain score index in over grown horn cases was 2.50±0.50 and the pain was graded as low. In the horn cancer case the average pain score index was 11.00±0.00 and the pain was graded as severe.

In the present study the pain symptoms observed in the buffaloes suffering from horn affections includes restlessness, rubbing the affected part against fixed objects, twitching of ears/shaking of head, decreased appetite and water intake, trying to evade the side on palpation of affected area, dilatation of pupil, bruxism and pilo erection. The findings are in partial agreement with the report of George (2003). The pain symptoms are much in avulsion and cancer which might be due to the exposed corium considered to be the sensitive part of the horn. The pain reflexes varied with the site of the fracture. The symptoms observed with other conditions are specific to the problem. Assessment of individual animal behavioral changes in response to pain is very subjective and can be influenced by differences in individual perception and interpretation.

According to the pain score index recorded, severe pain scores were noticed in avulsion followed by horn cancer, septic horn and fractures with moderate pain which might be due to sudden exposure of the core in avulsions and induration of the animal for slow growing cancer. The average pain score index in over grown horn cases was graded as low as this is not a serious pathological problem. The behavioral changes observed due to pain were more in the cancer followed by avulsion which indicates more distress to animal in these

conditions and is also supported through the changes in biochemical parameters. Sandford et al. (1986) also stated that behavior was commonly used to recognize and assess the pain and distress in animals. Anil et al. (2002) mentioned that behavior was a more sensitive indicator of pain than other physiological measures. According to Broom (2000) pain is an aversive feeling or sensation associated with actual or potential tissue damage and resulting in physiologic, neuroendocrine, and behavioral changes that are indicative of a stress response.

MANAGEMENT OF HORN AFFECTIONS

The animals brought for treatment were evaluated to adopt treatment options. The animals with avulsion showed loss of outer shell (Figure 1) with severe excitement or resistance on touching, fracture at tip (Figure 2), middle (Figure 3), lower third (Figure 4) and base (Figure 5) and few cases were fractures with discharging pus (Figure 6). A case of horn cancer (Figure 7) and few cases were overgrown horns causing pressure necrosis at the back of the poll region (Figure 8) where the horn touches the skin was observed in the present study.

Avulsion of the horn All the buffaloes with avulsions were

treated as outpatient cases by applying tincture benzoin seal over the core of the horn after thorough irrigation with 1: 5,000 potassium permanganate under surface analgesia with 4% Lignocaine Hydrochloride.

Fracture of the hornThe fracture cases at the tip were treated


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by applying Zinc oxide paste and bandage as conservative therapy. In cases which had complete fracture at middle or last third or the base of the horn exposing the communication of frontal sinus amputation was carried out. Two case of the chronic fractures showed deep fissure and were effectively treated with packing off the cavitary area with Zinc oxide and wax (Figure 9). Due to anatomical peculiarity of the buffaloes the horns with incomplete fractures were also amputated to avoid unnecessary complications (Figure 10).Two cases with both fracture and avulsions were also amputated (Figure 11 and Figure 12).

Septic horn Animals with septic horn (Figure 13) were

given prophylactic antibiotic therapy with 5g of streptopenicillin for a period of 5 days along with antiseptic bandage using tincture benzoin so as to maintain antibiotic levels at the time of surgery and also to curtail the production of pus at the operative site. None of these four cases showed any postoperative sepsis or wound dehiscence which suggested that the adopted therapy yielded good results.

Over grown horn The overgrown horns were noted to cause varying degrees of damage ranging from simple discomfort to severe degree punctured wounds on the frontal, occipital or cervical regions based on the shape of the horn and angle of curvature and duration of contact (Figure 14). In some cases suppuration was also recorded as complications of soft tissue damage due to the overgrown horns. Trimming of overgrown horns was done at a point just above the junction of upper and middle third using a hack saw without reaching the core. This was followed by treatment of the damaged soft

tissues with antiseptic dressing using povidone iodine.

Horn cancerA case of horn cancer was recorded in

a buffalo during the period of study which had a history of fracture at the base, one and half a month earlier. It was treated by horn amputation. In this case there were discharges from the nostrils varying from frank blood in the initial days to mucoid and purulent discharges in later stages.The management of horn affections vary depending on the affection. The conditions reported under present study were managed with appropriate measures. All cases of avulsion were successfully treated on outpatient basis, by spraying 4% lignocaine spray as surface analgesic and applying tincture benzoin seal over the stump of the horn after thorough irrigation with mild antiseptics like (1: 5,000) potassium permanganate as reported by Sreenu and Kumar (2006). Verma and Kumar (1999) treated avulsion of horn by covering it with an antiseptic dressing with pine tar and carbolic acid in oil soaked bandage and the authors opined that the avulsion of horn was not a serious condition except that there was profuse bleeding. The response was good in the present study as the tincture benzoin has the properties of adhesiveness, antiseptic and styptic. In the present study treatment was given by spraying 4% lignocaine spray as surface analgesic effectively abolished the pain as evidenced by the absence of pain symptoms. Verma and Kumar (1999) opined that cornual nerve block should be performed before treating avulsion of horn to avoid pain.

The fracture cases at the tip were treated by packing off the cavity area with Zinc oxide as means of conservative therapy. In cases that deserved surgery, amputation by flap/modified flap


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Figure 1. Avulsion of horn in a buffalo. Note the exposed core.

Figure 2. Fracture at the tip of the horn in a buffalo.

Figure 3. Fracture at the middle third of horn in a buffalo.

Figure 4. Facture at the lower third of horn in a buffalo.

Figure 5. Fracture at the base of the horn in a buffalo.

Figure 6. Septic horn discharging thick pus in a buffalo.


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Figure 7. Cancerous growth at the base of the horn in a buffalo.

Figure 8. Overgrown horns causing pressure necrosis at the occipital region in a buffalo.

Figure 9. Deep fissure as a sequelae to fracture of the horn in a buffalo.

Figure 10. incomplete fracture in a buffalo.

Figure 11. An old fracture of the horn at its lower third along with avulsed horn in a buffalo.

Figure 12. A recent fracture of the horn at its lower third along with avulsed horn in a buffalo.


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method was carried out. Balappanavar (2005) used bamboo sticks as external splints for stabilization of the fractured horn at the base with intact skin. The normal horn was used to stabilize the fractured horn in a criss cross pattern using splints. Sreenu and Kumar (2006) treated horn fractures effectively by flap method of amputation of horn whereas Patil et al. (2007) treated a case of horn fracture using external splints using iron bars, rings, nuts and bolts. The treatment options followed in cattle to treat fractures such as application of plaster of paris bandage with or without splints, usage of aluminium wire were not practcable due to anatomical variations of the horn in buffaloes as suggested by Sreenu and Kumar (2006). Animals with septic horn were given prophylactic antibiotic therapy with 5g of streptopenicillin for a period of 5 to 7 days in addition to antibiotic bandage so as to maintain antibiotic levels at the time of surgery and also to curtail suppuration at the operative site if they warrant amputation. This procedure was also followed by Sreenu and Kumar (2006) and Mahida et al. (2009). Mistry (2009) observed leucocytosis in septic horn of buffaloes which could be attributed to the trauma and inflammation.

The overgrown horns were noted to cause varying degrees of damage ranging from simple

discomfort to severe degree punctured wounds on the frontal, occipital or cervical regions based on the shape of the horn and angle of curvature and duration of contact. In some cases suppuration was also recorded as complications of soft tissue damage due to the overgrown horns. In the present study, trimming of overgrown horns was done at a point just above the junction of upper and middle third using a hack saw without exposing the core. This was followed by treatment of the damaged soft tissues with routine antiseptic dressing using povidone iodine. Oheme and Prier (1974) opined that trimming of horn in bovine practice was essential in case of its excess growth otherwise it causes pressure sore of the head as well as obstruction in vision. Sreenu and Kumar (2006) suggested sawing the curved portion of the horn without touching the corium.

A case of horn cancer, recorded in the present study was treated by horn amputation by flap method as reported by Angelo and Das (1970). Kumar and Thilagar (2000) who reported a case of bilateral horn cancer in a buffalo mentioned that the incidence of horn cancer was rare in buffaloes. Various authors worked to treat horn cancer in bovine with use of 5% liquor formaldehyde after amputation in cattle (Pandya,1932), flap method of

Figure 13. Septic horn showing pus discharge from the base of the horn in a buffalo.

Figure 14. Punctured wounds at occipital region due to over grown horns in a buffalo.


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APPENDIXMultidimensional pain scale for assessing pain in horn affections in buffaloes

Details of animal:Case No: Species: Breed: Sex: Calvings:Case History:Clinical examination:Pain scale:

ScoreObjective assessment

A) Physiological Dilatation of pupil Yes:1 No:0Piloerection Yes:1 No:0Respiratory rate Within reference range : 0

Above the reference range : 0Heart rate Within reference range : 0

Above the reference range : 0Temperature Within reference range : 0

Above the reference range : 0B) Behavioral Posture Normal:0

Rigid:1Arched back:2Any abnormal:3

Behavior patterns Comfortable: 0Restlessness:1Twitching of ears/Shaking of head: 2Bruxism: 3Rubbing against fixed objects:4Pawing at the site: 5

Vocalization Absent:0Bellow/grunt:1

Mental state Change in mental state: yes:1 No:0Evoked behavior No reaction on palpation of area:0

Trying to evade aside:1Activity Appetite

Normal: 0Reduced: 1Absent: 2

Water intakeNormal: 0Reduced: 1Absent: 2

Total pain index 1-5 Low pain6-10 Moderate pain>10 severe

C) Biochemical Plasma cortisol C-reactive protein


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amputation (Angelo and Das, 1970), radiotherapy after horn amputation (Joslin, 1972), flap method of horn amputation following triphining of frontal sinus (Mohanty et al., 1972), administration of autogenous vaccine (Pachauri and Singh, 1978), flap method of horn amputation using wire saw (Kumar and Thilagar, 2000), flap method (Yadav et al., 2002) amputation of horn by rising ventral flap after resection of horn (Sreenu and Kumar 2006) and modified flap method horn amputation (Mahida et al., 2010).

REFERENCES

Angelo, S.J. and S.C. Das. 1970. Amaurosis in dog-a case report. Indian Vet. J. 47(3): 268-270.

Anil, S.S., L. Anil and J. Deen. 2002. Challenges of pain assessment in domestic animals. J. Am. Vet. Med. Assoc., 220: 313-319.

Balappanavar, B.R. 2005. External splints for the treatment of horn fracture of cattle. The Indian Cow: The Scientific and Economic Journal, 2(6): 434-435.

Broom, D.M. 2000. The evolution of pain. Vlaama Diergeneeskundig Tijdschrift, 69: 385-411.

Damodaran, S. and K.R. Parthasarathy. 1979. Indian Vet. J., 49: 649.

Deshpande. 1983. Studies on horn cancer in cattle. M.Sc. Thesis, Marathwada Agricultural University, Parbhani, India.

George, L.W. 2003. Pain control in food animals. In Steffey, E.P. (ed.) Recent Advances in Anesthetic Management of Large Domestic Animals. International Veterinary Information Service.

Holton, L.L., J. Reid, E.M. Scott and A.M. Nolan. 2001. Development of a behaviour-based

scale to measure acute pain in dogs. Vet. Rec., 148(17): 525-531.

Ithaca, NY. and C.A. Joslin. 1972. After loading methods in radiotherapy. In Halnan, K.E. (ed.) Recent Advances in Cancer and Radiotherapeutics. Churchill, Livingstone, London. p. 353.

Kaul, P.L. and D.S. Kalra. 1973. Incidence of horn cancer in Haryana state. Haryana Agric. Univ. J. Res., 3: 161-165.

Kumar, R. and S. Thilagar. 2000. An unusual case of bilateral horn cancer in a buffalo. Indian Vet. J., 77: 48-49.

Mahida, H.K., P. Tank, M.A. Dhami, D.O. Joshi, A.S. Karle and H.S. Vedapathak. 2009. Epidemiological status of surgical affections of the buffalo horn at hospital population and ambulatory villages. Indian Journal of Veterinary Surgery, 30(2): 192-193.

Mistry, J.N. 2009. Surgical affections of horn in Mehsani buffaloes and their management. Indian Journal of Veterinary Surgery, 30(2): 112-113.

Naik, S.N., C.R. Balakrishna and H.P. Randella. 1969. Epidemiology of horn cancer in Indian Zebu cattle: Breed incidence. Brit. Vet. J., 125: 222-230.

Oheme, F.W. and J.E. Prier. 1974. Text Book of Large Animal Surgery, Williams and Wilkins Co., Philadelphia, p. 1208.

Pachauri, S.P. and N.P. Singh. 1978. Clinicopathological studies on horn cancer in bovines. Phillipp. J. V. Med., 17: 181-184.

Pandya, B.S. 1932. Liquor formaldehyde in cases of horn cancer. Indian Vet. J., 9: 147.

Patil, A.S., S.L. Patil, P. Suvarna and C.M. Sajjanavar. 2007. Malignant melanoma over nasal ridge in a bullock and repair of


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horn fracture using external splints in two bullocks. Indian Journal of Veterinary Surgery, 28(1): 44.

Salgar, B.S. 2008. Surgical affections of horn in Mehsani buffaloes and their management. MSc. Thesis abstract. Indian Journal of Veterinary Surgery, 30(2): 128.

Sandford, J., R. Ewbuck, V. Motony, W.D. Tavernor and Uvarovo. 1986. Guidelines for the recognition and assessment of pain in animals. Vet. Rec., 118: 334-338.

Shivaprakash, B.V., D. Kumar and S.M. Usturge. 2007. Dehornig with primary closure, a preferred technique over traditional amputation and open healing: Long term study on 500 cases. Indian Journal of Veterinary Surgery, 28(1): 24-25.

Sreenu, M. and Kumar, N.R. 2006. Affections of horn in buffaloes Indian Vet. J., 83: 1206-1207.

Somavanshi, R. 1991. Horn cancer in Indian cattle. Veterinary Bulletin, 61: 901-911.

Verma, R. and N. Kumar. 1999. Affections of horn and their management. Intas Polivet, 2(2): 134-139.

Yadav, G.U., P.T. Jadhao, S.D. Moregaonker, A.V. Bhikane and R.R. Mugale. 2002. An unusual horn cancer in a cow. A case report. Indian Vet. J., 79: 515-516.



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ABSTRACT

To evaluate the nutritional status of buffaloes (n=31), samples of feeds, fodder and blood serum were analyzed for proximate and certain mineral contents collected from the selected villages of coastal zone (Porbandar district) of western (Gujarat state) India. Average intakes of protein and metabolizable energy were 101 and 111% of requirement, respectively. Calcium (Ca) content ranged from 0.22 to 1.74% in roughages, as compared to 0.02 to 0.19% in concentrates. Average phosphorus (P) content in concentrates (0.42%) was almost three times higher than that of roughages (0.14%). Copper (Cu) level was recorded low in most of the feed resources.

Straws of jowar (Sorghum bicolor) (8.11 ppm), wheat (Triticum aestivum) (5.71 ppm) and bajra (Pennisetum glaucum) (9.82 ppm) were found low in zinc (Zn). Manganese (Mn) content in feeds and fodder ranged from 9.65 to 73.0 ppm. Average blood serum levels of Cu, Zn and Mn in buffaloes were 0.63, 0.79 and 0.05 ppm, respectively. As compared to critical level of Cu (0.65 ppm) and Zn (0.80 ppm) in blood serum, more than 60% of the animals screened showed low Cu and Zn status. Based on the calculated intakes of protein, energy, Ca, P, Cu, Zn and Mn from various feed resources, suggestions for correcting supply of protein and energy, and extent of supplementation

required through area specific mineral mixture, for obviating deficiency in the ration of buffaloes were given to the farmers of Porbandar district.

Keywords: feedstuffs, nutritional status, buffalo, porbandar, India

INTRODUCTION

India possesses the world’s largest livestock population having 57% of the world’s buffalo (Bubalus bubalis) population. Currently, India is the largest milk producing country in the world and buffalo is the main milk producing animal, contributing more than 50% of the total milk production in India with an average lactation yield of ~1300 kg. This low yield is mainly due to feeding of poor quality feed resources, particularly crop residues and agro industrial by-products fed to animals in rural households. Moreover, buffalo milk has a much higher fat content at 6 to 7% in comparison to 3.1 to 4.5% in cow milk.

Accurate assessment of nutritional status of dairy buffaloes is invaluable in modern livestock production. For efficient production, reproduction and maintenance of normal health in dairy animals, it is essential to provide protein, energy and minerals according to their requirement. Limited information was available about the extent of

EVALUATION OF FEEDING PRACTICES AND CERTAIN MINERALS STATUS OF LACTATING BUFFALOES IN COASTAL ZONE OF WESTERN INDIA

P.L. Sherasia*, P.R. Pandya, S. Parnerkar, B.R. Devalia and B.M. Bhanderi

Animal Nutrition Research Department, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, India, *Email: [email protected]

Original Article



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nutrients availability from different feeds and fodder fed to lactating buffaloes in western zone of India. Therefore, present study was undertaken to evaluate the existing feeding practices to know the status of protein, energy and certain minerals in lactating buffaloes of Porbandar district of Gujarat state and to recommend necessary modifications.


Sampling procedure The survey was conducted in Porbandar

and Ranavav tehsils of Porbandar district in Western India. Two villages were selected at random from each tehsil, for collection of samples of feeds, fodder and blood serum (Figure 1). In each village, about 15 dairy farmers were selected at random. The selected farmers were interviewed and the desired information was collected in the pro-

forma developed for the purpose. While selecting farmers, due care was taken to ensure that selected farmers were evenly distributed in the village and truly represent animal management practices of the village. The recorded parameters from each farmer included number of livestock, land area, irrigated facilities, fodder and other crops being grown etc. In addition, information regarding the amount and types of feeds and fodder offered to their milch animals, rate of actual daily feed intake, milk yield and fat percent, physiological status of animal etc were collected with the fair degree of precision on a questionnaire, using standard sampling procedure.

Analytical methods Composite samples of green and dry fodder, individual concentrate ingredients and homemade concentrate mixtures were collected from the surveyed area. Samples were dried in oven, ground (1 mm) and stored in airtight bags

Figure 1. Map of Porbandar district.


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until analysis. The amount of dry matter (DM), crude protein (CP) and Metabolizable energy (ME) available to buffaloes were calculated from the records of intake of feeds and fodder, using digestibility coefficients/nutritive values given by ICAR (1998). Average body weight of buffaloes in the district was considered at 500 kg and requirements for protein (Kearl, 1982), energy (Mandal et al., 2003), calcium (Ca), phosphorus (P), copper (Cu), zinc (Zn) and manganese (Mn) (NRC, 2001) were worked out. Feed and fodder samples were analyzed for proximate constituents (AOAC, 2007). For mineral analysis, samples were prepared and digested using 5 ml concentrated HNO3 plus 1 ml concentrated HCl, by microwave digestion method and total volume of mineral extract was made to 25 ml with de-ionized water. Blood from jugular vein was collected from the individual buffalo and centrifuged at 2000 rpm for 10 minutes, to harvest the serum. Serum samples were preserved in deep freeze till further analysis. All the samples were analyzed for Ca, P, Cu, Zn and Mn, by Inductively Coupled Plasma-Optical Emission Spectrometer; Optima 3300 RL, Perkin Elmer, Waltham, MA, USA.Statistical analysis Data were statistically analyzed using SAS 9.3 software package (2012), SAS Institute, USA as per Snedecor and Cochran (1994). Overall differences between treatment means were considered significant when P<0.05. The data have been presented as mean±S.E.


Current feeding practices Survey revealed that most of the dairy

animal owners/farmers reared their animals on grazing and supplementation of wheat straw (Triticum aestivum), maize straw (Zea mays), groundnut gotars (by-products of leguminous crops) or local grasses (a mixture of leguminous and non-leguminous species in varying proportions) collected from the wasteland, as the basal roughages. Tur gotar (by product of Cajanus cajan) was also fed, but availability was seasonal. Some of the farmers had employed the practice of feeding with cultivated fodder like lucerne (Medicago sativa) or jowar (Sorghum bicolar), bajra (Pennisetum typhoides) and maize (Zea mays). Green fodder availability was only for limited period due to water scarcity and frequent drought in the area. Those farmers, who did not have irrigation facilities, were feeding local green grasses and cotton balls with leaves available at the time of cotton crop harvesting. Whole carrot (Daucus carota) plants, as subsidiary green fodder was available for feeding to milch buffaloes. The practice of feeding compounded cattle feed was rare in the area. Concentrates were mostly offered twice a day, at the time of milking. Interestingly, it was observed that none of the farmers supplemented the ration of animals with mineral mixture, except for therapeutic purpose or on recommendation of the veterinary practitioners. However, some of the farmers were supplementing common salt to their animals. These observations are consistence with the reports from Gujarat state (Garg et al., 2002; Chavda, 2003; Ghogale, 2003; Bhanderi, 2007). Some rich progressive farmers/cattle owners were also feeding concentrates to pregnant (for steaming up) and growing animals. Crushed wheat (Triticum aestivum), bajra (Pennisetum typhoides), cottonseed cake (Gossypium spp.) and edible oil (approximately 100 g) were commonly used for steaming up.


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Proximate composition Chemical composition revealed that whole cottonseed and cottonseed cake were good source of protein and energy (Table 1). Amongst green fodders, lucerne (18.4%) had highest CP, followed by whole carrot plant (11.3%), bajra (10.1%) and jowar (8.6%). Straws were low in CP content, except gotars of groundnut and tur. Silica content was highest in jowar straw (5.8%), followed by maize straw (4.3%). Data are in agreement with the reports of Anonymous (2006) and Bhanderi (2007).

Nutritional status Average DM intake (DMI) of buffaloes in the district was 15.1 kg/d. The DMI of buffaloes in different tehsils, as well as in different villages within tehsils did not differ significantly (Table 2). Lal et al. (1998) reported similar values of DMI (15.7 kg/d) by lactating buffaloes yielding 10 to 14 kg milk/d. Similarly, Singh et al. (2001) reported 15.3 kg DMI/d by Murrah buffaloes yielding 7 to 18 kg milk/d in their native breeding tract. Average protein intake in villages of Porbandar and Ranavav tehsils was 0.88 and 0.90 kg/d, respectively, which did not differ significantly. However, the same differed (P<0.05) between villages within tehsils. Protein intake as percent of requirement in the district was 101. Lal et al. (1998) reported 1.28 kg protein intake as against requirement of 1.20 kg/d by lactating buffaloes yielding 10 to 14 kg milk/d. Lal et al. (1999) reported protein intake of 1.25 kg/d in Murrah buffaloes yielding 16 to 18 kg milk/d. Average protein intake in the district (0.89 kg/d) was lower as recorded by these workers. The ME intake in Porbandar and Ranavav tehsils was 36.21 and 38.53 Mcal/d, respectively, which did not differ significantly. However, the same

differed (P<0.05) between villages of Porbandar tehsil. Average energy intake as % of requirement in the district (111) was slightly higher than that of in Porbandar tehsil, whereas, lower than that of in Ranavav tehsil. Lal et al. (1998) reported 43.23 Mcal/d ME intake against the requirement of 46.44 Mcal/d by lactating buffaloes yielding 10 to 14 kg milk/day. The average ME intake (37.40 Mcal/d) by buffaloes in the district was in agreement with these authors. To fulfill the nutrient requirements of buffaloes in villages Rajpar and Vadwala, about 250 g protein meal may be included in the ration. The buffaloes in the district were fed adequately in terms of protein, whereas energy intake was slightly higher than the requirements.

Minerals profile of feeds and fodder The average Ca content ranged from 0.22

to 1.74% in roughages as compared to 0.02 to 0.19% in concentrates (Table 3). Average P level in concentrates (0.42%) was almost three times higher than that of roughages (0.14%). Similar finding were also reported by Ramana et al. (2001), Udar et al. (2003) and Garg et al. (2008). About 100, 28.6 and 50.0% samples of concentrates, green fodder and dry fodder, respectively, were found to be below the critical level of Ca. All samples of green and dry fodders were also found to be deficient in P (Table 4).

Lucerne (26.7 ppm) and carrot leaves (24.2 ppm) were good source of Cu as compared to bajra, maize and jowar green. Tur gotar had highest Cu content (12.5 ppm) amongst the dry fodder, however, most of the concentrate feed ingredients were low in Cu. Zinc content was found to be very low in wheat straw (5.71 ppm). These values are in agreement with values reported by Yadav et al. (2002) and Mandal et al. (2004). Wheat bran (51.6 ppm) was a good source of Zn. Mn levels in the


471

Table 1. Chemical composition of feeds and fodder (% on DM basis; mean±S.E.).

Feedstuffs CP EE CF NFE Ash Silica

Grains/seedsBajra (7) 10.3 ± 0.71 3.3 ± 0.25 1.5 ± 0.06 82.3 ± 0.45 1.5 ± 0.05 0.7 ± 0.04Wheat (6) 8.9 ± 0.48 1.4 ± 0.02 1.4 ± 0.07 86.7 ± 0.51 1.5 ± 0.01 0.5 ± 0.04Maize (6) 8.8 ± 0.40 2.8 ± 0.27 2.5 ± 1.13 83.9 ± 3.14 2.0 ± 0.74 0.5 ± 0.41Whole cottonseed (10)

22.3 ± 0.21 18.2 ± 0.68 18.6 ± 0.74 37.1 ± 0.33 3.7 ± 0.17 0.6 ± 0.15

Brans and cakesWheat bran (4) 16.3 ± 0.32 3.3 ± 0.06 5.0 ± 0.08 71.3 ± 0.25 4.1 ± 0.14 0.8 ± 0.04Cotton seed cake (16)

27.0 ± 1.08 5.7 ± 0.53 21.2 ± 0.27 42.1 ± 0.21 4.0 ± 0.05 0.5 ± 0.06

Green fodder/grassesJowar green (4) 8.6 ± 0.31 1.8 ± 0.05 23.4 ± 0.46 60.6 ± 0.28 5.5 ± 0.26 2.6 ± 0.32Maize green (5) 6.5 ± 0.60 1.3 ± 0.03 27.5 ± 0.96 58.2 ± 0.36 6.5 ± 0.15 2.3 ± 0.77Whole carrot plant (4)

11.3 ± 0.35 3.5 ± 0.18 12.3 ± 0.32 60.4 ± 0.27 12.5 ± 0.22 2.7 ± 0.05

Lucerne green (7) 18.4 ± 0.95 2.2 ± 0.05 28.1 ± 1.49 41.7 ± 3.74 9.6 ± 0.54 0.5 ± 0.07Cotton ball with leaves (3)

5.5 ± 0.71 1.1 ± 0.06 26.0 ± 0.37 61.2 ± 0.40 6.3 ± 0.26 3.8 ± 0.20

Bajra green (4) 10.1 ± 0.84 2.5 ± 0.05 28.3 ± 0.70 45.8 ± 0.28 13.3 ± 0.36 1.4 ± 0.08Local grass (3) 5.9 ± 0.80 3.1 ± 0.20 35.4 ± 3.45 40.1 ± 0.55 15.4 ± 2.84 6.8 ± 0.11Dry fodderGroundnut gotar (7)

9.0 ± 0.80 1.4 ± 0.08 24.8 ± 1.24 56.2 ± 0.43 8.6 ± 0.38 3.2 ± 0.35

Jowar straw (8) 3.9 ± 0.28 1.2 ± 0.14 26.8 ± 0.36 60.9 ± 0.39 7.2 ± 0.57 5.8 ± 0.46Tur gotar (3) 8.8 ± 0.22 3.0 ± 0.10 27.6 ± 0.28 51.5 ± 0.35 8.7 ± 0.36 0.2 ± 0.03Maize straw (10) 4.9 ± 0.18 0.6 ± 0.04 31.6 ± 2.36 55.0 ± 0.46 7.9 ± 0.67 4.3 ± 0.36Wheat straw (8) 3.2 ± 0.28 0.8 ± 0.06 39.4 ± 3.36 46.6 ± 0.44 9.9 ± 1.67 2.3 ± 0.26Bajra straw (6) 3.1 ± 0.88 0.9 ± 0.14 26.6 ± 3.36 59.5 ± 0.31 9.9 ± 1.67 3.3 ± 0.30

Figures in the parentheses indicate number of samples analyzed.


472

Tabl

e 2.

Nut

ritio

nal s

tatu

s of l

acta

ting

buffa

loes

in P

orba

ndar

dis

trict

.

Part

icul

arM

Y(k

g/d)

DM

I(k

g/d)

Inta

keR

equi

rem

ent

Inta

ke a

s % o

f req

uire

men

tPr

otei

n(k

g/d)

ME

(Mca

l/d)

Prot

ein

(lg/d

)M

E(M

cal/d

)Pr

otei

nM

E

Raj

par

9.7

± 0.

3215

.2 ±

0.8

40.

85a ±

0.0

834

.19a ±

0.4

60.

87 ±

0.0

67.

74 ±

0.6

497

.7 ±

5.3

510

0a ± 6

.23

Deg

am9.

4 ±

1.22

14.2

± 0

.66

0.91

b ± 0

.11

38.2

4b ± 0

.75

0.90

± 0

.10

7.79

± 0

.82

100

± 4.

7311

1b ± 3

.24

Tehs

il av

erag

e9.

5 ±

0.71

14.7

± 0

.73

0.88

± 0

.10

36.2

1 ±

0.67

0.89

± 0

.07

7.77

± 0

.79

98.9

± 4

.38

106a ±

4.3

4

Vadw

ala

8.6

± 1.

6514

.6 ±

0.4

30.

83a ±

0.1

238

.95

± 0.

440.

85 ±

0.1

27.

63 ±

0.6

297

.6a ±

2.4

511

6 ±

3.98

Bile

shw

ar9.

6 ±

0.88

16

.3 ±

0.4

90.

96b ±

0.0

638

.11

± 0.

750.

87 ±

0.0

97.

50 ±

0.4

511

0b ± 4

.25

115

± 4.

34Te

hsil

aver

age

9.1

± 0.

9415

.5 ±

0.4

60.

90 ±

0.0

738

.53

± 0.

360.

86 ±

0.1

27.

57 ±

0.5

110

4 ±

3.50

116b ±

3.4

2

Dis

trict

av

erag

e9.

3 ±

0.80

15.1

± 0

.61

0.89

± 0

.08

37.3

7 ±

0.42

0.87

± 0

.12

7.67

± 0

.64

101

± 2.

1011

1 ±

3.95

a

,b v

alue

s diff

er si

gnifi

cant

ly in

a c

olum

n (P

<0.0

5)


473

Table 3. Minerals profile of feeds and fodder (DM basis; mean±S.E.).

Feedstuffs/critical level

Ca (%)< 0.30

P (%)< 0.25

Cu (ppm)< 8.0

Zn (ppm)< 30.0

Mn (ppm)< 40.0

Grains/seedsBajra (7) 0.04 ± 0.01 0.29 ± 0.01 5.58 ± 0.09 32.4 ± 0.47 13.2 ± 2.07Wheat (6) 0.04 ± 0.00 0.29 ± 0.01 4.68 ± 0.18 23.0 ± 0.72 32.5 ± 1.08Maize (6) 0.02 ± 0.00 0.21 ± 0.01 3.07 ± 0.20 17.6 ± 0.54 9.93 ± 0.32Whole cottonseed (10)

0.15 ± 0.01 0.40 ± 0.01 7.77 ± 0.29 27.1 ± 0.49 9.65 ± 0.21

Brans and cakesWheat bran (4) 0.11 ± 0.00 0.84 ± 0.01 12.7 ± 0.43 51.6 ± 1.44 48.9 ± 1.69Cotton seed cake (16)

0.19 ± 0.02 0.48 ± 0.01 8.74 ± 0.43 31.6 ± 1.44 19.0 ± 1.69

Green fodder/grassesJowar green (4) 0.41 ± 0.01 0.15 ± 0.01 8.60 ± 0.25 23.8 ± 0.18 50.9 ± 2.27Maize green (5) 0.22 ± 0.01 0.12 ± 0.00 12.9 ± 0.24 26.5 ± 0.89 44.7 ± 1.96Whole carrot plant (4)

1.03 ± 0.05 0.23 ± 0.02 24.2 ± 0.26 20.5 ± 0.57 40.8 ± 0.89

Lucerne green (7) 1.22 ± 0.06 0.22 ± 0.01 26.7 ± 0.61 23.2 ± 1.36 40.2 ± 2.21Cotton ball with leaves (3)

0.36 ± 0.03 0.17 ± 0.01 8.41 ± 0.57 28.8 ± 0.40 30.7 ± 0.84

Bajra green (4) 0.26 ± 0.02 0.15 ± 0.01 14.3 ± 0.57 32.4 ± 0.40 30.2 ± 0.84Local grass (3) 1.58 ± 0.11 0.13 ± 0.01 15.1 ± 0.54 24.4 ± 1.48 73.0 ± 2.10Dry fodderGroundnut gotar (7) 1.74 ± 0.09 0.15 ± 0.02 9.82 ± 0.45 16.6 ± 1.27 54.1 ± 5.38Jowar straw (8) 0.52 ± 0.05 0.11 ± 0.02 6.04 ± 0.25 8.11 ± 0.76 15.9 ± 0.65Tur gotar (3) 1.07 ± 0.14 0.18 ± 0.03 12.5 ± 0.72 16.1 ± 1.44 23.0 ± 2.80Maize straw (10) 0.28 ± 0.03 0.10 ± 0.02 5.64 ± 0.25 13.4 ± 0.16 24.3 ± 0.65Wheat straw (8) 0.28 ± 0.04 0.08 ± 0.02 6.81 ± 0.85 5.71 ± 1.28 28.9 ± 7.65Bajra straw (6) 0.24 ± 0.00 0.09 ± 0.01 6.34 ± 0.12 9.82 ± 0.08 34.3 ± 0.83


474

Table 4. Feedstuffs having below critical levels of minerals.

Feed stuff/critical level

Ca(< 0.30%)

P(< 0.25%)

Cu(< 8.0 ppm)

Zn(< 30.0 ppm)

Mn(< 40.0 ppm)

Concentrate All (100%)Maize

(16.7%)

All, except cottonseed cake,

wheat bran (66.7%)

Wheat, maize, cottonseed

(50%)

All, except wheat bran

(83.3%)

Green fodderMaize green, Bajra green

(28.6%)All (100%)

Jowar green,Maize green,Whole carrot plant (42.5%)

All, except bajra green

(85.7%)

Cotton ball, bajra green

(28.6%)

Dry fodder

Maize straw, Wheat straw, Bajra straw

(50%)

All (100%)

All, except groundnut gotar

and tur gotar (66.7%)

All (100%)All, except groundnut

gotar (83.3%)

Table 5. Minerals content in blood serum of buffaloes.

ParticularCa (mg/dl)

< 8.0P (mg/dl)

< 4.50Cu (ppm)

< 0.65Zn (ppm)

< 0.80Mn (ppm)

< 0.02

Buffaloes 8.75±0.23

(6.33-12.19)5.31±0.23(4.65-6.89)

0.63±0.8(0.54-1.43)

0.79±0.03(0.53-0.97)

0.05±0.00 (0.04-0.12)

% of buffaloes showing deficiency

47.0 56.0 57.6 66.7 0.0

Figure in the parenthesis indicate range.


475

district ranged from 15.9 to 73.0 ppm in roughages and from 9.65 to 49.0 ppm in concentrate feeds.

Mineral profiles of blood The average blood serum Ca and P were 8.75

and 5.31 mg/dl, respectively (Table 5). Buffaloes screened in the district showed 47 and 56% lower serum Ca and P, respectively. These findings are similar to those of Ramana et al. (2001) and Mandal et al. (2004). Average serum Cu and Zn content were 0.63 and 0.79 ppm, respectively. As compared to critical level of Cu (0.65 ppm) and Zn (0.80 ppm) in blood serum (Cuesta et al., 1993) more than 50% of the buffaloes screened showed low Cu and Zn values. The Mn content of blood serum was within the normal range. The lower concentration of these minerals in feeds and fodder might have resulted in lower level in blood serum (Bhattacharya et al., 2004). However, blood serum mineral levels are not always true indicators of mineral deficiency as minerals may be mobilized from the target tissue, during low dietary intake and complex interrelationships (McDowell et al., 1993). Hence, regular supplementation of mineral mixture in the ration of buffaloes is necessary.

CONCLUSION

Study revealed that majority of lactating buffaloes in Porbandar district was deficient in essential minerals like Ca, P, Cu, Zn and Mn. It is necessary to supplement these deficient minerals, through supplementation of area specific mineral mixture along with protein meal in the ration of buffaloes. Deficient trace minerals may be supplemented in the form of chelates, for better minerals bio-availability.

ACKNOWLEDGEMENTS

The authors are grateful to the Department of Animal Nutrition, College of Veterinary Science and Animal Husbandry, Anand Agricultural University and the Management of National Dairy Development Board, for providing necessary facilities and financial support to carry out this research work.

REFERENCES

Anonymous, 2006. Progress Report Presented in the Meeting of Research Sub-Committee on Animal Production. Published by Animal Nutrition Research Department, Faculty of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand Campus, Anand, India.

AOAC. 2007. Official Methods of Analysis, 18th ed. Association of Official Analytical Chemists, Washington, D.C., USA.

Bhanderi, B.M. 2007. Assessment of feeding practices and status of certain minerals in dairy animals of Jamnagar district of Gujarat state. M.V.Sc. Thesis, Anand Agricultural University, Anand, Gujarat, India.

Bhattacharya, B.N., B.C. Sarmah, A. Baruah, K.K. Baruah, K.C., Goswami, R N and D.J. Kalita. 2004. Mineral profile of lactating cattle of two agro-climatic zones of Assam under field conditions. Indian J. Anim. Sci., 74: 1206-1207.

Chavda, M.R. 2003. Assessment of feeding practices and status of certain minerals in dairy animals of Patan district of north Gujarat. M.V.Sc. Thesis, Gujarat


476

Agricultural University, Sardarkrushinagar, Gujarat, India.

Cuesta, P.A., L.R. McDowell, W.E. Kunkle, F. Bullock, A. Drew, N.S. Wilkinson and F.G. Martin. 1993. Seasonal variation of soil and forage mineral concentrations in North Florida. Communication in Soil Science and Plant Analysis, 24(3-4): 335-347.

Garg, M.R., B.M. Bhanderi and S.K. Gupta. 2008. Effect of supplementing certain chelated minerals and vitamins to overcome infertility in field animals. Indian J. Dairy Sci., 61(1): 181-184.

Garg, M.R., B.M. Bhanderi and P.L. Sherasia. 2002. Trace minerals status of feeds and fodders in Junagadh district of Gujarat. Indian J. Dairy Sci., 55: 154-158.

Ghogale, P.P. 2003. Assessment of feeding practices and status of certain minerals in dairy animals of Sabarkantha district of North Gujarat. M.V.Sc. Thesis, Gujarat Agricultural University, Sardarkrushinagar, Gujarat, India.

ICAR. 1998. Nutrient Requirements of Livestock and Poultry. Indian Council of Agricultural Research, New Delhi, India.

Kearl, L.C. 1982. Nutrient Requirements of Ruminants in Developing Countries. International Feedstuffs Institute, Utah State University, UMC 46, Logan, Utah 84322 USA.

Lal, D., V.B. Dixit, U. Arora and T.R. Chauhan. 1999. Nutritional status of high yielding buffaloes – A case study. Indian J. Anim. Prod. Manage., 15(1): 39-41.

Lal, D., V.B. Dixit, T.R. Chauhan and V.S. Solanki. 1998. A critical analysis of feeding system of lactating buffaloes in Hisar. Indian J. Anim. Prod. Manage., 14(3): 164-166.

Mandal, A.B., S.S. Paul and N.N. Pathak. 2003. Nutrient requirements and feeding of buffaloes and cattle, 1st ed. International Book Distributing Co., Lucknow, U.P., India.

Mandal, A.B., P.S. Yadav and V. Kapoor. 2004. Mineral status of buffaloes under farm feeding condition of Faridabad district of Haryana state. Indian J. Anim. Nutr., 21: 104-110.

McDowell, L.R., J.H. Conrad and F.G. Hembry. 1993. Minerals for grazing ruminants in tropical regions. Animal Science Department, Centre for Tropical Agriculture, University of Florida. The U.S. Agency for International Development and Caribbean Basin Advisory Group (CBAG).

NRC. 2001. Nutrient Requirements of Dairy Cattle, 6th ed. National Research Council, National Academy of Science, Washington, DC.

Ramana, J., C.S. Prasad, N.K.S. Gowda and K.S. Ramachandra. 2001. Levels of micro-nutrients in soil, feed, fodder and animals of North East transition and dry zones of Karnataka. Indian J. Anim. Nutr., 18: 235-242.

SAS. 2012. Base SAS 9.3 Procedures Guide: Statistical Procedures, 2nd ed. SAS Institute Inc. Cary, NC, USA.

Singh, J., K.K. Yadav and A.B. Mandal. 2001. Feeding plane of milch Murrah buffaloes in its native breeding tract. Buffalo J., 17(1): 1-12.

Snedecor, G.W. and W.G. Cochran. 1994. Statistical Methods, 6th ed. Oxford and IBH Publication Company, New Delhi, India.

Udar, S.A., S. Chopde and R.N. Dhore. 2003. Mineral profile of soil, feeds and fodder and buffaloes in Western Agro-climatic Zone of


477

Vidarbha. Anim. Nutr. Feed Techn., 3: 165-172.

Yadav, P.S. A.B. Mandal and D.V. Dahiya. 2002. Feeding pattern and mineral status of buffaloes in Panipat district of Haryana state. Anim. Nutr. Feed Techn., 2: 127-138.



479

ABSTRACT

Gross morphological study was conducted on the major salivary glands of 42 buffalo foetuses ranging from 73 to 253 days. The mandibular gland was the largest salivary gland during the prenatal life. Dense compact lobulation of the parotid and mandibular glands was observed first at 155 days of foetal age. The mandibular gland was covered by the confluence of linguo-facial, occipital and maxillary veins laterally and developing thymus caudally throughout the prenatal period. Differentiation of dorsal and ventral parts of the sublingual gland was observed at 108 days of foetal age.

Major salivary glands of various domestic animals are paired structures, which includes parotid, mandibular and sublingual glands. Salivary glands fulfill important role in the oral biology by producing saliva for lubrication, as well as supplying electrolytes, mucus, antibacterial compounds and various enzymes to the oral cavity. Loss of salivary glands function can result in the wide spread deterioration of oral health (Hsu et al., 2010). Study of normal development of salivary glands will be helpful for both anatomists and clinicians as they are having important role in several dreadful diseases like rabies, foot and mouth disease and other viral diseases.

Keywords: buffaloes, Bubalus bubalis, Gross

morphological, salivary glands, mandibular duct


The prenatal specimens of unknown age, irrespective of the sex, nutritional status of the mother were collected from slaughter houses the CVRL (Curved Crown Rump Length) of specimens was measured to estimate the age of by Soliman’s (1975) formula. Gross morphological changes were studied in feasible foetal age from of 73 days to 253 days regarding shape, size, location and relation of the parotid, mandibular and sublingual salivary glands.


The parotid gland was situated along the caudal border of the masseter muscle extending from the region of the external auditory canal to the level above the angle of mandible. It was in the form of a pyramid with loosely arranged multiple lobules at 73 day foetal age. However the shape of the gland was found to be evident as triangular with a broad base and apex during 84 days to 253 days. Gradual change in the shape may be due to growth of the gland during the prenatal life. The base of the gland was superior with a notch placed

GROSS MORPHOLOGICAL STUDIES ON MAJOR SALIVARY GLANDS OF PRENATAL BUFFALO

K. Raja, M. Santhi Lakshmi*, G. Purushotham, K.B.P. Raghavender,

T.S. Chandrasekhara Rao and D. Pramod Kumar

Department of Veterinary Anatomy, College of Veterinary Science, Rajendranagar, Andhra Pradesh, India, *E-mail: [email protected]

Original Article


480

around the external auditory canal, while the apex was situated little above the angle of the mandible (Figure 1). Subsequently the gland reached the space between the base of the ear, vertical ramus of the mandible and sterno-mandibularis muscle in mid and late foetal stages (Figure 2).

The colour of the parotid gland varied from light yellow to light brown during the prenatal period, variation may be attributed to increased vasculature to the gland. The weight of the parotid gland ranged from 2.0 to 7.1 g during 84 to 253 days. The length and width of the parotid gland ranged between 0.8 to 2.1cm and 0.5 to 2.4 cm respectively from 84 to 253 days. Gradual increase in length, width and weight of the gland was due to increased proliferation of ducts, increased lobulation and connective tissue formation during the foetal stage. Compact lobulation and adult characteristic features of the gland were attained at 155 days (Figure 3). The lateral surface was covered by parotid fascia, developing parotido - auricularis muscle and a muscle extending from the zygomatic arch to cervical fascia. The gland was reported to be attached superiorly to the zygomatic arch. The middle part of the gland was penetrated by the maxillary vein from lateral to medial surface throughout the prenatal life (Figure 1). The medial surface was uneven and related to great cornu of hyoid, digastricus, occipito-hyoideus and sterno-mastoideus muscles, external carotid artery, external jugular vein and its tributaries, facial nerve and its branches. The anterior border was in contact with the parotid lymph node above and masseter muscle below throughout the prenatal period (Figure 1). The medial surface of the parotid gland showed impressions for the mandibular salivary gland, parotid lymph node and masseter muscle as reported in human embryos and

foetuses of 36 to 53 days (Guizetti and Radlanski, 1996). The duct of the parotid gland was opened into the mouth cavity at the level of upper 2nd erupting cheek tooth till 145 days, the same was evident at the level of upper erupting 3rd cheek tooth from 155 days to 253 days of foetal life. The mandibular gland was the largest among the three major salivary glands in prenatal buffalo, while the parotid gland was reported to be the largest major salivary gland in human beings during prenatal life (Attie and Sciubba, 1981). It was long, narrow and curved throughout the prenatal period (Figure 1).

The mandibular gland was light yellow in colour during the prenatal period. It was long, narrow and curved and extended from the region of tympanic bulla to the level above the angle of the mandible behind the parotid salivary gland in early age groups (Figure 1). However the position of the gland gradually changed to that of adult at 123 days, but it was located caudo-medial to the parotid salivary gland as reported in day old kid by Rauf et al. (2004) which continued throughout the mid and late foetal age groups. The average length, width and weight of the mandibular gland ranged from 1.23 to 2.84 cm, 0.45 cm to 1.6 cm and 2.1 to 8.00 g respectively during the prenatal period from 84 to 253 days.

The mandibular gland was covered laterally by fascia and the confluence of the external jugular vein with maxillary, linguo-facial and occipital veins (Figure 1), which agree partly with the findings of Rauf et al. (2004) in one day old kid. The gland was related to the larynx, division of the common carotid artery, external carotid artery, 9th ,10th and 11th cranial nerves, stylo-hyoideus muscle and great cornu of hyoid medially and developing thymus caudally. The anterior extremity was narrow and placed at the


481

level of the angle of the mandible above the sterno-mandibularis muscle and the posterior extremity at the level of fossa atlantis as reported in domestic animals (Budras and Habel et al., 2003). However the anterior extremity was carried to the level of root of the tongue in mid and late age groups. The mandibular gland showed loosely arranged lobules at 73 days. Dense compact lobulation of the gland was observed between 155 to 253 days of prenatal life (Figure 3). Mandibular lymph node was placed above the anterior extremity of the gland, a little cranial to or at the angle of the mandible. At 253 days the mandibular gland attained the characteristic shape, colour and position to that of adult. The mandibular duct was well developed at 84 days but not amenable for dissection. It was found to be leaving the gland at lower third of the inferior border between 84 to 123 days and middle of the concave border between 145 to 253 days. The duct running on the floor of the mouth cavity in

close association with the sublingual duct opened closely along the side of monostomatic sublingual duct at the caruncula sublingualis.

The sublingual gland was the smallest among the major salivary glands (Figure 3). It was composed of two parts, the dorsal (polystomatic) and ventral (monostomatic) parts. The dorsal and ventral parts of the gland were distinguishable at 108 days of foetal age. The dorsal part of the gland was arranged in a chain of lobules from the palato-glossal arch to the incisive part of the mandible, while the ventral part was lying beneath the mucosa of the floor of the mouth above the mylohyoid muscle between the mandible laterally and the muscles of tongue medially. The dorsal and ventral parts of the gland were distinguished at 108 days. The colour of the dorsal part varied from light yellow to light brown during prenatal development, while the ventral part appeared as light yellow between 189 to 253 days of prenatal life.

Figure 1. Photograph of 84 day buffalo foetal head showing parotid (P) and mandibular (M) salivary glands and their relation to adjacent structures. PL- Parotid lymph node, EJ- External jugular vein, SM-Sterno-mandibularis, D-Dorsal buccal nerve, MA-Masseter muscle,E-External ear, T- Thymus.


482

Figure 2. Photograph of 145 day buffalo foetus showing compact lobulation of parotid gland (P) and loosely arranged lobules of mandibular (M) salivary glands. E- External auditory canal, EJ- Ext jugular vein, T -Thymus,SM- Sterno mandibularis muscle, MA- Masseter muscle

Figure 3. Photograph showing dense compact lobulation in foetal parotid (P), mandibular (M), and sublingual (L) salivary glands at 155 days in buffalo.


483

The ventral part of the gland was elongated and situated beneath the mucosa of the floor of the mouth above the mylohyoideus muscle between the mandible laterally and the muscles of tongue medially. The ventral part of the sublingual gland was drained by only one duct, which opened along the side of mandibular duct at caruncula sublingualis as reported by Latshaw (1987) in domestic animals. But the ducts of the dorsal part of sublingual gland were not visible to the naked eye during the prenatal period.

The weight, length and width of the ventral part of sublingual gland ranged from 1.45 g to 2.65 g, from 1.7 to 4.2 cm and 0.25 to 1.1 cm from 84 days to 253 days of embryonic life. Increase in the size and weight of the gland was due to increased proliferation of ducts, increased lobulation and connective tissue formation.

REFERENCES

Attie, J.N. and J.J. Sciubba. 1981. Tumors of Major and Minor Salivary Glands, Clinical and Pathologic Features in Current Problems in Surgery. Medical Book Publishers Inc, Chicago, p. 80-81.

Budras and Habel. 2003. Colour Atlas of Bovine Anatomy, 1st ed. Schlutersche Verlagsgesellschaft mb H and Co. KG., Hans - Bochler - Alle 7, 30173 Hannover, p. 38-39.

Guizetti, B. and R.J. Radlanski. 1996. Development of the submandibular gland and its closer neighbouring structures in Human embryos and fetuses of 19-67 mm CRL. Ann. Anat., 178: 509-514.

Hsu, J.C.F., M. Kenneth and Yamada. 2010. Salivary gland branching morphogenesis,

Recent Progress and Future Opportunities. Int. J. Oral. Sci., 2(3): 117-126.

Latshaw, W.K. 1987. The Veterinary Developmental Anatomy. B.C. Decker Co., INC, Toronto. p. 128-129.

Rauf, S.M.A., M.R. Islam and M.K. Anan. 2004. Macroscopic and Microscopic study of the mandibular salivary gland of black bengal goats. Bang. J. Vet. Med., 2(2): 137-142.

Soliman, M.K. 1975. Studies on the physiological chemistry of the allantoic and amniotic fluids of buffaloes at the various periods of pregnancy. Indian Vet. J., 52: 106-112.

http://www.google.com/search?tbo=p&tbm=bks&q=inauthor:%22Klaus-Dieter+Budras%22

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