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CHAIRMANDr. M. R. SaseendranathDirector (Academics & Research)

MEMBERSDr. S. RamkumarDirector of Entrepreneurship

Dr. P.C. SaseendranDean, College of Veterinary & Animal Sciences, Mannuthy

Dr. Leo JosephDean, College of Veterinary & Animal Sciences, Pookode

Dr. R. RajendrakumarDean, College of Dairy Science & Technology, Mannuthy

Dr. Jose John ChungathProfessor, Veterinary Anatomy and Histology,College of Veterinary & Animal Sciences, Mannuthy

Dr. P.I. GeevargheseProfessor, Dairy Microbiology, College of Dairy Science &Technology, Mannuthy

Dr. K.N. Aravinda GhoshProfessor, Animal Reproduction, Gynaecology & Obstetrics,College of Veterinary & Animal Sciences, Mannuthy

Dr. A. D. MercyProfessor, Animal Nutrition,College of Veterinary & Animal Sciences, Mannuthy

ANIMAL SCIENCESE D I T O R I A L B O A R D

EDITORDr. K. DevadaProfessor and Head

Veterinary Parasitology

ASSOCIATE EDITORSDr. Shibu Simon

Assistant Professor, Animal Reproduction,Gynaecology & Obstetrics

Dr. Indu. V. Raj Assistant Professor

Veterinary Anatomy and Histology

Dr. H. Shameem Assistant Professor

Veterinary Parasitology

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Journal of Veterinary and Animal Sciences is a half yearly publication of theKerala Veterinary and Animal Sciences University (KVASU) devoted to the publication of originalresearch papers on various aspects of Veterinary and Animal Sciences and clinical articleswhich are of interest to research workers and practitioners engaged in livestock and poultryproduction. Research papers on wild life, laboratory animals and environmental problemsaffecting livestock production; short communications of importance in Veterinary and AnimalSciences are also accepted. From 2013 onwards, the editorial board has decided to publisharticles in respect of Dairy Science / Technology and other related Science. The editorialboard look forward to continual support and co operation from all well wishers in future for apromising and prospective venture.

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This journal was first published in 1970 as The Kerala Journal of Veterinary Scienceby the Director of Animal Husbandry, Kerala on behalf of the Kerala Veterinary ResearchCouncil. The journal was renamed in 1990 as the Journal of Veterinary and Animal Sciences.From 1972 till 2007, it was published by Kerala Agricultural University and thereafter it isbeing published by Kerala Veterinary and Animal Sciences University.

CONTENTS

REVIEW ARTICLE

1. Ultrasonographic diagnosis and monitoring of pregnancy in the bitch - a review...............1P. Sridevi

RESEARCH ARTICLES

1. Gene expression profile in prepubertal bovine mammary parenchyma and epithelial cells inresponse to ovariectomy ................................................................................................8B. T. Velayudhan, M. L. McGilliard, H. Jiang, S. E. Ellis and R. M. Akers

2. Effect of fish oil and coconut oil on lipid profile and oxidative stress in liver and heartof rats .........................................................................................................................15K. P. Sreeji and Sisilamma George

3. Anatomical studies on the bones of the pelvic limb in Indian Muntjac(Muntiacus muntjak)....................................................................................................21C. V. Rajani, Leena Chandrasekhar, George Chandy and J. J. Chungath

4. Screening of cattle blood samples from Kasargod district for presence of HCH and DDTresidues.......................................................................................................................26V. Dineshkumar, A. R. Nisha, P. T. A. Usha, C.B. Vineetha, A. P. Usha and Siju Joseph

5. Job satisfaction and stress among the veterinarians of Kerala state in India....................31

Soumya Sankar, P. Reeja George and P.J. Raj Kamal

6. Comparing ELISA using serum antibodies and coproantigens for detecting earlyamphistomosis.............................................................................................................35A. Kandasamy and K. Devada

7. Effect of genetic and non-genetic factors on seroprevalence of Mycoplasma mycoidessubsp. capri in goat......................................................................................................38C. N. Dinesh , A. Sonawane, A. Kumar, R. Rana, A. Chauhan and D. Sharma

8. Ovulation synchronisation for improving fertility in postpartum dairy cows...................42P. S. Shameem Abubaker, M. O. Kurien, K. N. Aravinda Ghosh, Shibu Simon, K. S. Aniland B. Bibin Becha

9. Training needs of dairy farm instructors in fodder production and management ............46N. Vimal Raj Kumar

, R. S. Jiji

and P. J. Rajkamal

10. Detection of anthelmintic resistance in small scale goat rearing units in Thrissur...........51Asha Rajagopal, R. Radhika, H. Shameem and K. Devada

11. Gross anatomical studies on the lungs of wild sow (Sus scrofa cristatus).......................54A. R. Sreeranjini, C. V. Rajani, V. R. Indu and N. Ashok

12. Assessment of oxidative stress during peripartum period in crossbred Malabari goats....57J. Rejitha and K. Karthiayini

13. In vitro studies on the effect of Allium sativum (garlic) on Damalinia caprae................61Bindu Lakshmanan, R. Radhika, R. Sreekrishnan and H. Subramanian

14. Gross observations on the cervical vertebrae of leopard (Panthera pardus)....................63V. R. Indu, A. R. Sreeranjini, C. V. Rajani and N. Ashok

JOURNAL OF VETERINARY AND ANIMAL SCIENCESVolume 44 2013 Issues 1 & 2

JOURNAL OF VETERINARY AND ANIMAL SCIENCESVolume 44 2013 Issues 1 & 2

15. Diagnosis of canine trypanosomosis by polymerase chain reaction.................................66P. V. Tresamol, N. P. Usha, T. V. Aravindakshan, Reji Varghese and M. R. Saseendranath

16. In vitro efficacy of Butea Monosperma against amphistomosis in cattle.........................70N. P. Usha, V. R . Dhanya and Subin Jose

17. Levels of calcium, sodium and potassium in plasma as influenced by anticoagulants....72K. V. Pramina, P. T. Mincy, P. Anu Joseph, V. Lisha, K. A. Mercy and V. Ramnath

18. Aflatoxin levels in feeds and feed ingredients of livestock and poultry in Kerala............76B. Bibin Becha and S. S. Devi

SHORT COMMUNICATIONS

1. Surgical management of gynecomastia in a Sirohi buck by mastectomy..............................79K. Vinod Kumar

2. Prevalence of helminth parasites of camels (Camelus dromedaries) in the Anseba regionof Eritrea in North East Africa..............................................................................81Basharat Ahmed Pandit, Michael Kahsay,, Sanjay Devarajan and Paulo Luis Valerino Cambra

3. Myoglobinuric acute renal failure in a dog....................................................................83P. P. Kanaran, N. P. Usha, V. R. Ambily and P.C. Alex

4. Cerebral babesiosis due to Babesia gibsoni in a dog - a case report...............................85P.V. Tresamol, Usha Narayana Pillai, J. Anumol, K. Devada and M. R. Saseendranath

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Received - 20.08.13Accepted - 29.09.13

ULTRASONOGRAPHIC DIAGNOSIS ANDMONITORING OF PREGNANCY IN THEBITCH - A REVIEW

The use of ultrasound as a tool in smallanimal reproduction has expanded from its initialrole in the evaluation of pregnancy in the femaleto its current use in monitoring fetal development,timing gestation and predicting parturition,diagnosis and management of reproductive tractdisease and in supplementing breedingsoundness examinations. In fact, B-modeultrasonography has become an indispensabletool in veterinary practice. When ultrasonographicdiagnosis of pregnancy is being performed it isimportant to remember that all events in caninepregnancy are related to day of the pre-ovulatoryLH surge (day 0) or to the time of ovulation (whichoccurs 2 days after the LH surge) and not to themating dates. Parturition typically occurs 64 to66 days after the LH surge. This article aims toreview the use of ultrasonography in monitoringpregnancy in the bitch.

A 5-7.5 MHz transducer is adequatefor most dogs while for large and giant breedsof dogs a 3.5 MHz transducer is ideal. Fordiagnosis of mid to late-term pregnancy a 5.0-MHz transducer may be sufficient but may notprovide adequate resolution for study of moresubtle pathologic changes of the reproductivetract (Yeager and Concannon, 1990, 1995,1996 and Yeager et al., 1992). Ultrasonographyis usually performed with the dog in dorsal orlateral recumbency. Preparation of theabdomen includes clipping the hair andapplying acoustic coupling gel on the skin. Theability to ultrasonographically detect ordiscriminate a developmental feature at one dayversus another during gestation is highlydependent on the equipment used (England

et al., 2003). Forced respiration or panting canseriously affect the stillness of the image andthus make its interpretation much more difficult.Temporarily closing either the mouth or thenostrils of the dog can reduce the disturbingeffect of respiratory movements, or momentarilyremove them (Wolfgang Kahn, 2004).

DIAGNOSIS OF PREGNANCY

Real time ultrasonography has provento be a valuable tool for diagnosing caninepregnancy and assessing fetal viability (Inabaet al., 1984). The ultrasonographic appearanceof a gravid uterus in Beagle bitches at knowntime of gestation was studied in detail byYeager and Concannon (1990) and Yeageret al. (1992). They detected cardiac activityand fetal movements as early as 25 and 34days respectively after LH surge. Mattoon andNyland (1995) reported detection of gestationalsac at 20 days post breeding as the first signof confirming pregnancy using ultrasonography but preferred to wait until day 30 asgestational sac with viable embryo could beidentified with high level of confidence at thattime. Yeager et al. (1992) reported that thegestational sacs or embryonic yolk sacs canbe first imaged approximately 18 to 19 daysafter the LH surge and appear as sphericalanechoic structures 1 to 2 mm in diameterwithin the lumen of the uterus. The embryonicheartbeat has been detected as a brightechogenic flicker as early as 23 days after theLH surge. The embryo has a bipolar shape byday 28 of pregnancy. The head region isidentifiable as containing an anechoic area by

P. Sridevi*Department of Clinics,Madras Veterinary College, Chennai.e-mail: [email protected]

* Professor,

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Ultrasonographic diagnosis and monitoring of pregnancy in the bitch - a review

day 30; limb buds are usually identifiable fromday 32 to 34 onwards. The fetal skeleton isevident by day 34 of pregnancy. The bones ofthe head appear first, followed by those of thelower body. At this stage the hyperechoic heartvalves can be imaged and are seen to bemoving and the great vessels can be tracedcranially and caudally.

The zonary, circumferential placentawraps around the central portion of theconceptus like a waistband, and is observedultrasonographically between the fetus and theuterine walls in all planes. When imaged inthe longitudinal plane, the placenta appearsas two thick bands one on either side of thefetus, between the fetus and the uterine wall(Concannon et al., 2003).

Fig. 2. Fetal head and neck. Sonogram of aday 47 pregnant bitch showing a fetal head andneck in frontal section. The hyperechoic bones(bright white) of the calvarium outline the skull.The bones of the vertebrae are likewisehyperechoic. The bright echogenic area belowthe head is an acoustical shadow caused bythe density of the head bones. The scale onthe left is marked in 1.0 cm increments.

Fig.1. Sonogram of two transverse sections ofthe uterine horn in a 6 year old cross bred bitchshowing marked enlargement of the uterus withanechoic material The uterine wall is mildly thick.

After day 36 - 38 after the LH surge,it is possible to identify the fluid-filled fetalstomach caudal to the liver in more than 90%of fetuses. A day or so later the fetal bladderis identifiable in the caudal abdomen and withcareful examination the urachus may beimaged. These changes are obvious by day40 - 45 after the LH surge. In late gestationthe skeleton becomes more obvious in latepregnancy and the skull, spinal column andribs are easily identifiable Figures 1-5 showthe ultrasonographic appearance of variousfeatures at different days of gestation.

Fig.4. Note the zonary placenta between thefetus and abdominal wall

Fig.3. Day 37 yolk sac and placenta. Sonogramshowing an uterine gestational sac inlongitudinal plane at day 37 of gestation. Thelong, echogenic remnant of the tubular yolk sacextends the full length of the chorionic sac,being attached at each end to the chorionicpoles. The chorionic sac containing the dark,anechoic chorionic fluid extends at each endbeyond the margins of the placental girdle.

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Fig. 5. Day 55 M-mode examination of fetal heart rate. Sonogram of a day 55 canine fetus(Left) and M-mode display of the heartbeat (right). The sonogram is centered on the fetal heart,the chambers and great vessels of which appear as anechoic (dark) areas within the fetalthorax. The thorax is delineated by the two lines of hyperechoic (bright white) ribs, above andbelow the heart in this view.

PREDICTION OF GESTATIONAL AGEShille and Gontarek (1985) in a study

involving 23 Grey hound bitches have reporteddiameter of the gestational vesicle determinedby ultrasound technique at seven differentstages of gestation. Pregnancy was timed fromthe calculated date of ovulation to the day whenfirst pup was born. The results revealed thatthe gestational sac diameter increased asgestation days progressed. On days 27 to 34,35 to 44 and 47 to 56 after ovulation the averagevesicle diameter ranged between 23 and 30mm,25 and 49mm and 46 and 89 mm respectivelywith a mean diameter of 26.5mm, 36.1mm and68.3mm respectively. Yeager et al. (1992) usedultrasonography to estimate the gestational agein 8 pregnant Beagle bitches. The gestationalage was based from day of preovulatory LHsurge which was denoted as “0”day of gestation.Serial ultrasonographic examinations of eachpregnant bitch began on day 28 to 37 after theLH surge. It was reported that measurementsof chorionic cavity diameter (CD) was the mostaccurate predictor of gestational age. It had theleast variation compared with all othermeasurements on the fetus. From days 38 to60, the fetal head diameter was the moreaccurate predictor of gestational age.

Fetal measurements and estimation offoetal age (Yeager et al., 1992)

· For pregnancy < 40 days, GestationalAge (GA) is: GA = (6 x GSD or ICC ) + 20where GSD is Gestational sac diameterand ICC is Inner chorionic cavity diameter

· For pregnancy > 40 days Gestational

Age (GA) is: GA = (15 x BPD or HD) + 20where HD is Head diameter and BPD isBiparietal diameter.

Days Before Parturition (DBP) = 65-GA

The formulae currently used by Luvoni (2013)are as follows:

·ICC in small size bitches: days beforeparturition = (mm – 68.68)/1.53

·ICC in medium size bitches: days beforeparturition = (mm - 82.13)/1.8

·BP in small size bitches: days beforeparturition= (mm - 25.11)/0.61

·BP in medium size bitches: days beforeparturition=(mm - 29.18)/0.7

A study was undertaken by Luvoni andGrioni (2000) to estimate the gestational agein medium size dogs by ultrasonographicexaminations. Formulae were derived toestimate the expected date of delivery bymeasuring the anatomical fetal structures andthe gestational sac was found to be 90.9 percent accurate and that of biparietal diameter70.8 per cent accurate for predicting the dateof parturition with ±1 day in medium size dogs.The accuracy of prediction of date of parturitionfor small size dogs was 90.9 per cent usinggestational sac diameter and 68.2 per centusing biparietal diameter.

When measuring the gestational sacs,two transverse plane measurements shouldbe taken at 90° angles to each other and thevalues averaged before using the above

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formulas. Head and body diameters aremeasured to the transverse planes. Whentaking measurements of fetal or extra-fetalstructures at least two distinct fetuses orgestational sac should be measured wheneverpossible and the measurements averagedbefore applying them to formulas. Thisbecomes difficult if it is a singleton fetus andmeasurement of multiple features such asGSD, HD, CRL or body diameter may becarried out to increase the accuracy.

Lenard et al. (2007) assessed theaccuracy of estimating the gestational age andlitter size in 76 bitches using one or twotechniques. The first method used thedifferential features of fetal organ developmentthat occur in early and mid-pregnancy, basedon published tables for Beagles. The secondmethod used biparietal head and trunkdiameters to predict gestational age based ontables published for late gestational period forLabrador Retrievers. The accuracy of the twomethods was compared to evaluate the effectof maternal body weight and litter size. Littersize and maternal body weight did not affectthe accuracy of gestational age prediction.Using a combination of both the methods, theoverall accuracy of predicting parturition dateto within 65 ± 1 day and ± 2 days was 70.8per cent and 86.1 per cent, respectively. Thecorrect litter size was predicted in 65 per centof cases and in 89.5 per cent of cases for ± 1pup. It was concluded that the optimum timefor sonographic estimation of fetal age andlitter size was early and mid-pregnancy.

PREDICTION OF PARTURITION

Accurate prediction of the date ofparturition in the bitch is clinically useful toprevent or minimize reproductive losses bytimely intervention. For example, an accuratemethod of predicting parturition date isnecessary to plan an elective caesareansection. Intervening when the pregnancy is fullterm can reduce losses of offspring frombitches having obstructions of the pelvis orvagina, histories of primary or secondaryuterine inertia, or prolonged parturition withresultant puppy mortality. For bitches withhistories of pyometra, abortion, embryonicreabsorption, or insufficient luteal phase,accurate assessment of gestational age canassist in therapeutic decision making. Finally,progress in assisted reproductive techniquesin this species, such as estrous synchroni-zation and embryo transfer, requires accurate

prediction of ovulation, gestational age, andparturition date (Kim et al., 2007).

The duration of canine gestation, astimed from the preovulatory serum LH peak,is 65 ±1d. However full-term gestation,calculated from insemination, is reported torange from 57 to 72 d (Concannon et al.,1993). The difference between thesemeasurements was attributed to the potential6-day viability of sperm in the femalereproductive tract and the long period ofreceptivity in the bitch. Therefore the key totiming the duration of canine gestation wasthe preovulatory LH surge rather than mating/ insemination date or estrus onset (Meyers-Wallen, 1995). Ultrasound provides anaccurate estimate of parturition date and themost accurate prediction was obtained whenthe ultrasound examination was conducted atDay 30 (Kutzler et al., 2003). However,prediction was inaccurate when made fromfetal measurements in late gestation (>Day39). Prediction accuracy was also significantlyaffected by non-pregnant body weight of thedam. Fetal growth was linear from Days 17 to30 and subsequently became exponential(England, 1998). Kutzler et al. (2003) reportedthat after day 30, fetuses of small bitches (<9kg) grew slower, and fetuses of giant bitches(>40 kg) grew faster, than those of medium orlarge bitches. When corrected for body weightof the dam, the overall accuracy for parturitiondate prediction by the ultrasound method was75% for the Day 65 ± 1 prediction, 87% forthe Day 65 ± 2 prediction, and 100% for theDay 65 ± 3 prediction.

MONITORING FETAL WELL BEINGFetal Death

Recognition of fetal death at or nearparturition is of extreme importance to theveterinarian and breeder. As the date ofparturition approaches, the following areindications for fetal monitoring withtransabdominal ultrasonography:

· Failure to initiate parturition asexpected.

· Unusual vaginal discharge

· Vague signs of illness

· Delay in parturition after delivery ofpart of a litter

Fetal heart rate has beenrecommended as a very useful parameter toestimate the survival possibilities of the

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foetuses (England, 1998). Likewise, theevidence of fetal motion can be consideredvery important to determine fetal survival inthe ultrasonographic point of view. Fetal stressis diagnosed by reduced fetal heart rate thatis due to hypoxia. Normal fetal heart rate is220 to 240 bpm while rates of < 180 bpm areindicative of fetal distress (Zone and Wanke,2001). One should take care to remember thatintermittent uterine contractions over a fetusmay cause a temporary, substantial reductionin heart rate which would return to normalwithin 1-2 minutes and would remain withinthe normal range if there is no fetal distress(Lopate, 2008). Increase in echodensity of fetalfluids is suggestive of passage of meconium(Zone and Wanke, 2001) or haemorrhage(Lopate, 2008) due to premature separationof placenta. Abdominal: biparietal diametersof < 2 from days 48 to term are indicative ofgrowth retardation. Puppies with lowabdominal: biparietal diameter ratios tend toweigh < 20 per cent of the average birth weightfor the breed and are at risk for early neonatalloss (Zone and Wanke, 2001). Edema andthickening of the placenta indicateabnormalities or alterations in blood flow,reduced ability of placenta to drain fluids orplacentitis. At any stage of pregnancy,regardless of the breed the normal canineplacenta should not exceed 1.2 cm at itscentremost point (Lopate,2008).

Fetal death is recognized by a loss ofcardiac activity. Poorly defined organogenesishas been also reported to be an importantultrasonographic feature for the diagnosis offetal death (Poffenbarger and Feeney, 1986).On assessment of near-term or term fetuses,cardiac activity should immediately berecognized. Fetal movements, such asswallowing, hiccoughs, and body and limbmovements, should also be seen. Sonographicrecognition of fetal structures rapidlydiminishes after death. After a day or two, onlymineralized skeletal structures may berecognized by characteristic hyperechogenicityand acoustic shadows. Intrauterine or intrafetal gas may also be identified (Matton andNyland, 1995).

Should embryonic death occur before35 days after ovulation, there is usuallycomplete resorption of the conceptus. This canoccur without vaginal discharge as late as Day30. The sonographic aspects of a resorption

are generally a reduction in the volume of theconceptus, an increased echogenicity of theembryonic fluid (sometimes particles may beidentified free-floating within the allantoic fluid),an absence of the embryonic heartbeat,disintegration of the embryonic mass andultimately collapse of the conceptus withinward bulging of the uterine wall. The uterusoften remains slightly enlarged in this region,and there may be a small volume of freeluminal fluid; the uterine wall often appearsmoderately hyperechoic (Concannon et al.,2003). Konde (1988) has reported poorlydefined fetal anatomy with amorphousechodensity, distortion of the gestational sac,and presence of hyperechoic material withinthe uterus as ultrasonographic signs of fetaldeath. Gas within the stomach of the fetusobserved on ultrasound has been also reportedas a sign of fetal death (England, 1998).

Death of fetuses occurring after Day35 of pregnancy is usually followed by abortionand vaginal discharge and is associated withexpulsion of fetal material and fluid. The earlyfeatures of fetal abortion are an increase inechogenicity of the allantoic and amniotic fluidoften with echogenic particles, followed by anabsence of the fetal heartbeat and sometimesa thickening of the uterine wall (Fig.6). Afterexpulsion, the uterus assumes an appearancethat is similar to that observed in thepostpartum bitch (Concannon et al., 2003).

Fetal AbnormalitiesIt is uncommon to detect fetal

abnormalities in the bitch, since there areusually multiple fetuses and it is difficult to fullyexamine each. However, a number of strikingabnormalities have been detected, some of

Fig.6. Fetal death and resorption at lategestation. Note the presence of fetal remnants.

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which have necessitated delivery of the litter bycaesarean operation. Such abnormalities includehydrocephalus, fetal anasarca (Fig.7), herniationof the ventral abdominal wall and fetal monsters(Poffenbarger and Feeney, 1986).

APPLICATION OF DOPPLERULTRASONOGRAPHY IN CANINEPREGNANCY

Miranda and Domingues (2010)evaluated blood flow in the uterine (UA) andumbilical (Uma) arteries in the pregnant bitch,by measuring the resistive index (RI) andpulsatility index (PI); and performed conceptusecobiometry for fetal growth assessmentduring pregnancy.Triplex Doppler and B-modeultrasonography were used to assess bloodflow and conceptus ecobiometry. Allpregnancies ended with a normal whelping andbirth of live puppies. Prior to whelping, allconceptus dimensions increased significantly,whereas RI and PI of both the Uma and UAdecreased significantly. For the UA, RI and PIwere (mean +/- SEM) 0.95 +/- 0.02 and 2.75+/- 0.41, respectively, on Day -44, and were0.60 +/- 0.01 and 0.99 +/- 0.03 on Day -4. Forthe Uma, RI and PI were 0.99 +/- 0.01 and2.42 +/- 0.03 on Day -31, and were 0.62 +/-0.01 and 1.15 +/- 0.02 on Day -4. The completedisappearance of the early diastolic notch inthe UA, and the appearance of diastolic flowin the Uma occurred on Days -16 +/- 5 and -21 +/- 1. The authors concluded that UA andUma perfusion were important end points toassess fetal vitality in bitches. Furthermore,the current reference values provided abaseline for monitoring normal and abnormalpregnancies in bitches

Blanco et al. (2011) described thechanges of uterine artery, umbilical artery andfetal abdominal aorta, renal and internalcarotid arteries blood flow in abnormal caninepregnancy. Color and pulsed-wave Dopplerexaminations of uterine artery were conductedevery 10 days from Day 20 to 50 fromestimated luteinizing hormone peak. Dopplerultrasonography was also conducted in thefetuses to assess umbilical artery, abdominalaorta, renal and internal carotid arteries fromDay 40 to 60 of gestation. Throughout thestudy, resistance index (RI) of uterine,umbilical and fetal renal arteries decreased upto -15% compared to -36% (P<0.01), -11%compared to -23% (P<0.05) and 2% comparedto -13% (P<0.05), respectively in the abnormaland normal bitches. Fetal abdominal aorta andinternal carotid did not differ between groups(P>0.05). They concluded that in dogs, uterineartery, umbilical artery and fetal renal arteryRI differ between normal and abnormalgestation and therefore were useful for theprediction of adverse obstetric outcome.

Thus, to conclude ultrasonographicexamination is an indispensable tool forpracticing veterinarians for monitoring fetalgrowth, for assessing gestational age and forprediction of parturition which is particularlyvaluable for providing clinical assistance duringwhelping or elective caesarean sections.

ReferencesBlanco, P. G., Rodríguez, R., Rube, A., Arias,

D.O., Tórtora, M., Díaz, J. D.andGobello. C. 2011. Dopplerultrasonographic assessment ofmaternal and fetal blood flow inabnormal canine pregnancy. Anim.Reprod. Sci. 126 (1-2) :130-135.

Concannon, P. W., England, G., Verstegen J.and Linde-Forsberg C. 2003.Ultrasound imaging of reproductivetract of the bitch. InternationalVeterinary Information Service(www.ivis.org), Ithaca, New York, USA.

Concannon, P. W., Whaley, S., Lein, D.,Wissler. R. 1993. Canine gestationlength: variation related to time ofmating and fertile life of sperm. Am.J. Vet. Res. 44 : 1819–1821.

England, G. 1998. Ultrasonographicassessment of abnormal pregnancy.Vet. Clin. North Am. Small Anim.Pract., 28: 1233-1256

Fig.7. Ultrasonographic image of fetal anas-arca. Note the increased subcutaneousoedema (arrow) and serous effusions (astrix).

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England, G., Yeager, A. and Concannon, P. W.2003. Ultrasound Imaging of theReproductive Tract of the Bitch. In: Recentadvances in small animal reproduction.IVIS, Ithaca. New York, USA.

Inaba, T., Matsu, N., Shimizu, R. and Imori, T.1984. The use of echography inbitches for detection of ovulation andpregnancy. Vet. Rec. 115 : 267-277.

Kim, B. S. and Son. C. H. 2007. Time of initialdetection of fetal and extra-fetalstructures by ultrasonographicexamination in Miniature Schnauzerbitches. J. Vet. Sci. 8 : 289-93

Kim Se Ra, Kang Hyun Gu, Oh KiSeok, Park InChul, Park SangGuk, Kim Sung Ho, SonChang Ho. 2000. Establishment ofprediction table of parturition day byultrasonography in Korean Jindo bitches.Korean J. Vet. Res. 40 : 373-381.

Konde, L.1988. Diagnostic ultrasound incanine pregnancy and uterinedisease. Proc. Ann. Meet Soc. forTheriogenology, Orlando, Florida,September 16–17, 247-249.

Kutzler, M. A., Yeager, A. E., Mohammed H.O. and Meyers. W. V. N. 2003.Accuracy of canine parturition dateprediction using fetal measurementsobtained by ultrasonography.Theriogenology. 60:1309-1317.

Lenard, Z., Hopper, B., Lester, N., RichardsonJ. and Robertson. I. 2007. Accuracyof prediction of canine litter size andgestational age with ultrasound. Aust.Vet. J. 85:222-225.

Lopate, C. 2008. Estimation of gestational ageand assessment of canine fetalmeasurements using radiology andultrasonography.: A review.Theriogenology, 70 (3): 397-402.

Luvoni, G.C. 2013. Ultrasonographic study ofgestation in dogs and cats. Rev. Bras.Reprod. Anim., Belo Horizonte, 37:172-173.

Luvoni, G. C. and Grioni . A. 2000.Determination of gestational age inmedium and small size bitches usingultrasonographic fetal measurements.J. Small. Anim. Pract. 41:294-296.

Mattoon, J. S. and Nyland T. G. 1995.Ultrasonography of the genital system:In Veterinary Diagnostic Ultrasound.

Eds. Nyland, T.G. and Mattoon, J.S.,W.B. Saunders, USA. pp146-148.

Meyers-Wallen, V. N.1995. The electivecesarean section. In: Bonagura J.D.,Kirk R.W., (Eds.) Current veterinarytherapy XII. Philadelphia, WBSaunders Co., pp. 1085–1089.

Miranda, S. A. and Domingues, S. F. S. 2010.Conceptus ecobiometry and triplexDoppler ultrasonography of uterineand umbilical arteries for assessmentof fetal viability in dogs. Theriogenology. 74: 608–617.

Poffenbarger, E. and Feeney, D. 1986. Use ofgray-scale ultrasonography in thediagnosis of reproductive disease inthe bitch: 18 cases (1981-1984). J.Am. Vet. Med. Assoc. 189 : 90-95.

Shille, V. M. and J. Gontarek.1985. The useof ultrasonography for pregnancydiagnosis in the bitch. J.Am.Vet.Med.Assoc. 187: 1021-1025.

Yeager, A. E. and Concannon, P. W. 1990.Association between the preovulatoryLH surge and the early ultrasonographic detection of pregnancy andfetal heartbeats in beagle dogs.Theriogenology. 34 : 655-665.

Yeager, A. E. and P. W. Concannon .1995.Ultrasonography of the reproductive tractof the female dog and cat. In: BonaguraJ. D., Kirk R.W., (Eds.) Kirk’s CurrentVeterinary Therapy XII. Philadelphia:W.B. Saunders Co.,1040-1052.

Yeager, A.E. and Concannon, P. W. 1996.Uterus. In: Green R.W., (Ed.) SmallAnimal Ultrasound. Philadelphia:Lippincott-Raven, 265-292.

Yeager, A. E., Mohammed, H. O., Meyers-Wallen,V., Vannerson L. and Concannon, P. W.1992. Ultrasono graphic appearance ofthe uterus, placenta, fetus, and fetalmembranes throughout accurately timedpregnancy in beagles. Am. J. Vet. Res.53:342-351.

Wolfgang Kahn. 2004. Veterinary Reproductive Ultrasonography. SchlûterscheVerlagsgesellschaft mbH & Co. KG,Hans·BOc:klcr·Allee 7, 30173 Hannover, pp.233-245.

Zone, M. A. and Wanke M. M. 2001. Diagnosisof canine fetal health by ultrasonography. J. Reprod. Fertil. 57: 215-219.

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8 Gene Expression Profile...

GENE EXPRESSION PROFILE INPREPUBERTAL BOVINE MAMMARYPARENCHYMA AND EPITHELIAL CELLSIN RESPONSE TO OVARIECTOMY

B. T. Velayudhan1, M. L. McGilliard2, H.Jiang3, S. E. Ellis4, and R. M. Akers5*

Department of Dairy Science,Virginia Polytechnic Institute and State University,Blacksburg, VA 24061.

Received - 18. 06.13Accepted - 20. 08.13

Abstract

This study was carried out to comparethe expression profile of steroid receptors andselected genes associated with cell signalingand proliferation in Mammary parenchyma(PAR) homogenates compared with mammaryepithelial cells (MEC) in response toovariectomy. Prepubertal dairy heifers wererandomly assigned to one of two treatments,ovariectomized (n = 7) or sham operated (n =12) and tissues were harvested 30 d aftersurgery. Parenchymal samples were snapfrozen in liquid nitrogen for total RNA isolation.Samples of MEC were prepared fromcryosections of parenchymal tissues usinglaser assisted microdissection and capture.Coupling two precise technologies like lasercapture microdissection and quantitative real-time PCR enabled measurement of transcriptabundance in MEC even at a very low level.Data were analyzed using Mixed procedure ofSAS and significance was declared at P d”0.05. The data strongly indicate a disparity inthe response to ovariectomy between MECand PAR tissue and suggest that ovariectomyvery strongly impacted the non-epithelial cellsin the PAR rather than the MEC themselves.

Keywords: mammary parenchyma,mammary epithelial cells, laser capturemicrodissection.

Mammary parenchyma (PAR) is a

1. Visiting Assistant Professor, Department of Biology, James Madison University, Harrisonburg, VA 228072. Professor, Department of Dairy Science3. Professor, Department of Animal and Poultry Sciences4. Program Director, Physiological and Structural Systems Integrative Organismal Systems,BIO Directorate, National Science Foundation, Boulevard, Arlington VA 222305. Department Head & Horace E. & Elizabeth F. Alphin Professor of Dairy Science Corresponding author: R. Michael Akers [email protected]

complex tissue consisting of mammaryepithelial cells (MEC) surrounded by intralobular stroma composed of fibroblasts,adipocytes and blood vessels. It is well knownthat the ovary is an integral regulator ofprepubertal mammary development and thatmany of the effects of estrogen are mediatedthrough stromal cells (Haslam, 1988; Hoveyet al., 1998). This is supported by the findingthat isolated mammary epithelial cells inculture did not respond to estrogen (Woodwardet al., 1994). We have previously reported thatovariectomy impacted transcript abundance ofproliferation markers as well as selectedestrogen responsive genes (Velayudhan et al.,2012). However, the magnitude of geneexpression responses to estrogen and ovarianstatus were shown to be greater in mammaryfat pad compared with parenchyma (Meyer etal., 2006). Moreover, the area occupied byepithelium per unit area of parenchyma wasonly 13-15 %, indicating that tissuehomogenates prepared from parenchymalsamples contain a mixture of cells other thanepithelial cells. Therefore, we hypothesizedthat the response to ovariectomy in thetranscript abundance in mammaryparenchyma (PAR) is mainly from the intralobular stromal cells, rather than the mammaryepithelial cells (MEC). Advent of laser assistedmicrodissection (LMD) of histologicalpreparations in 1990s has enabled precise

*

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separation of even single cells from tissuesconsisting of complex cell types. A combinationof LMD along with quantitative real timepolymerase chain reaction (qRT-PCR) iswidely used to determine transcript abundancein single cell types. The objective of the studywas to compare the gene expression profilebetween the PAR homogenate and MEC inresponse to ovariectomy employing thetechniques of LMD and qRT-PCR.

Materials and Methods

Animals and Treatments

All animal care and use protocolsused in the study were approved by theClemson University Institutional Animal Careand Use Committee. Briefly, 24 Holsteinheifers were fed with commercial milkreplacers and calf starter diets according tothe manufacturer ’s instructions prior toweaning and fed grains and hay thereafter.After a week’s adaptation period at the facility,heifers were randomly assigned to eitherovariectomy (OVX; n = 12) or sham operation(INT; n = 12) either at 2, 3 or 4 months of age.Animals of different age groups were acquiredin batches and hence surgery was performedat different time points. In OVX heifers ovarieswere removed surgically while in INT heiferssurgery was performed, but ovaries were keptintact. Mammary tissues were harvested 30 dafter surgery by humanely sacrificing theheifers using captive bolt pistol stunning andexsanguination. Only 19 animals were usedin this study (INT, n = 12 and OVX, n = 7) forcomparative gene expression analysisbecause of inadequate amount of tissueavailable from some animals.

Sample Collection and Analyses

Mammary parenchymal sampleswere collected consistently from the innerparenchymal region of the left hind quarter forPAR homogenate preparation and from the leftfront quarter for MEC samples. For preparingtissue homogenate, PAR samples wereimmediately frozen by dipping tissue piecesinto liquid nitrogen. For preparing MECsamples, parenchymal tissue pieces were firstplaced in plastic moulds containing a tissueembedding compound that ensures optimalcutting temperature (OCT) (Sakura FinetekU.S.A. Inc.; Torrence, CA) and then the tissueswere also frozen in liquid nitrogen. Bothsamples were stored at -80oC until further

processing. Isolation of total RNA from PARtissue homogenate was performed asdescribed (Velayudhan, et al., 2012).

Cryo-preserved samples in OCTblocks were prepared for LMD followingprotocols of (Becker et al., 1997; Saal et al.,2003). Briefly, seven micrometer thickcryosections were made from OCT blocks andplaced on polyethylene naphthalate membranecoated glass slides (Molecular Devices,Sunnyvale, CA). Slides were previouslyirradiated in a UV cross linker (FB UVXL-1000,Fisher Scientific, Pittsburgh, PA) for 30 min atmaximum power to ensure cross linking ofRNA onto the membrane. Slides were kept ondry ice while sectioning and stored at -80oCuntil LMD. Cryosections were thawed for lessthan 30 s and subjected to a quick stainingprocedure. Tissue sections were firstrehydrated in 70 % ethanol followed by a rinse

Figure 1. Representative figures of mammaryparenchymal tissues before and aftermicrodissection and catapulting. MammaryPAR tissue sections stained with toluidine bluebefore (A) and after (B) laser assistedmicrodissection and capture by catapulting.Thick arrows show the epithelial structures thatwere dissected and collected for RNA isolationfor MEC samples and thin double headedarrows show the intralobular stroma.

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in RNAse-free water for 30 s each. Sectionswere then treated with 1 % Toluidine blue stain(Fisher Scientific) containing 0.1 U/ ìL ofRNAse-inhibitor (Qiagen Inc., Valencia, CA )for 30 s followed by washing in RNAse-freewater and dehydration in 80, 90 and100 %ethanol solutions, 30 s each. Tissue sectionswere made completely moisture free by airdrying. Mammary epithelial cells wereidentified and the selected areas were preciselyexcised (Fig 1) using a UV-laser which wascoupled through the illumination path of themicroscope (PALM micro beam system, CarlZeiss Micro Imaging, Inc.; Thornwood, NY).The excised cell zones were then catapultedagainst gravity into collecting tube capscontaining 40 ìL of lysis buffer (RNeasy microkit; Qiagen Inc.). The cells were then lysedimmediately by vortexing in 350 ìL lysis buffer.On an average 85 to 90 epithelial zonesdepending on size were collected per heifer. TotalRNA from MEC was isolated and purified usingRNeasy Micro Kit (Qiagen Inc.). Quantity and

quality of RNA were determined in a 2100 seriesBioanalyzer from Agilent (Quantum Analytics,Inc, Foster City, CA) using pico-chips. Total yieldof RNA from microdissected samples rangedfrom 0.84 – 4.04 ng and were not differentbetween INT and OVX (P = 0.549; Fig 2).

qRT-PCR

From each sample a total of 0.4 ngRNA was used to make single stranded cDNAin a 20 ìL reaction volume using the HighCapacity cDNA Archive Kit (AppliedBiosystems, Foster City, CA). A reaction tubecontaining no reverse transcriptase enzymewas used for each sample and this was laterused in qRT-PCR as controls. A test run ofqRT- PCR for different genes was performedwith different concentrations of cDNA (stockand diluted) and 10 fold dilution of cDNA stockwas used for qRT-PCR. A total of 4 pg cDNAwas used in each reaction, along with 12.5 mlof SYBR Green dye (Applied Biosystems), 9.5ml of sterile distilled water, 0.5 ml of 10 mMeach of forward and reverse primers. The PCRconditions were: 95 °C for 10 min, 95 °C for15 s, and 60 °C for 1 min. The reaction wasset for 40 cycles in 7300 Series Real-TimeSystem (Applied Biosystems).

Relative mRNA abundances ofgrowth hormone receptor (GHR), insulin-likegrowth factor-1 ( IGF-1), IGF-I receptor (IGF-1R), IGF binding protein (BP)-3, proliferatingcell nuclear antigen (PCNA), signaltransducers and activators of transcription(Stat)-5b, and suppressors of cytokinesignaling (Socs)-2 were determined in bothPAR and MEC for OVX relative to INT heifersby comparative Ct (2-ÄÄCt) method afternormalizing the Ct values to the geometricmean of three endogenous control genes(Piantoni et al., 2008). Primer sequences oftarget and endogenous reference genes aregiven in Table 1.

Statistical Analysis

Relative abundance of mRNA in PARhomogenates was not different betweendifferent age groups and there was nointeraction between treatment and age (P >0.05). Additionally, the number of samplesavailable for the 2 mo surgery group was verylimited for MEC samples due to limited tissueresources. Therefore, for statistical analysisof comparative gene expression between thetwo types of sample preparations (PAR and

Figure 2. Quantity and quality of total RNAisolated from microdissected mammaryepithelial cells. (A) Total yield of RNA isolatedfrom microdissected mammary epithelial cellsfrom sham operated (INT; n = 12) andovariectomized (OVX; n = 7) Holstein heifers.Total yield of RNA was not different betweenINT and OVX (P = 0.549). Data are presentedas LSM and bars represent SEM. (B) Gel imageshowing the quality of RNA isolated from OVXand INT heifers. Lane 1 represents the ladderand lanes 2-20 represent RNA samples fromthe isolated mammary epithelial cells.

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MEC) data were pooled within treatments andonly treatment effect was tested. All data wereanalyzed using Mixed procedure of SAS (SAS9.2; Cary, NC). Random effect error was heiferwithin treatment. Data distribution wasanalyzed using the “Proc Mixed Boxplot”statement (Appendix A.6). Significance wasdeclared at P d” 0.05 for all analyses.

The model used in all analyses was

Yij = ì + Ti + e(i)j where

Yij = variable being tested

ì = overall mean

Ti = fixed effect of treatment (INT orOVX) (i = 1, 2)

e(i)j = residual error

Gene expression data for PAR andMEC were analyzed separately. The ÄCt datawere used for statistical analyses andsignificance was determined based on the Pvalues for ÄCt data. However, data waspresented as fold change in gene expressionfor OVX relative to INT control using thecomparative Ct (2-ÄÄCt) method.

Results and Discussion

There was a moderate degree ofdisintegration in the RNA isolated from MECsamples for both OVX and INT samples.However, we succeeded in obtaining a sufficientamplification for all the mRNA speciesevaluated and no-RT template controls hadundetectable amounts in all the samples tested.Dissociation curves for each target gene hadthe same single peak in PAR and MEC samples

indicating amplification of the same singleproduct in both sample types. Additionally, therewas no difference in the yield of total RNAbetween treatments (Fig 2). Therefore, we madethe assumption that the low integrity of RNAdid not hamper our ability to evaluate therelative gene expression between treatmentsusing the highly sensitive qRT-PCRmethodology. Quantitative determination oftranscript abundance even in tissue extractscontaining partially fragmented RNA is possibleby qRT-PCR because this technique enablesamplification of targets of very small size(Gibson et al., 1996; Heid et al., 1996) indicatingthat even though RNA undergo degradation, theresulting fragments are still large enough to bedetected by qRT-PCR.

Relative abundance of individualmRNA species present in MEC samples werevery low compared with that of PARhomogenate as indicated by greater Ct valuesfor MEC samples in the qRT-PCR reactions(Fig 3). This could be due to the difference inthe amount of total RNA used in the reversetranscription reactions. Because of the limitedamount of MEC available for RNA extractionwe could only use 0.4 ng total RNA to makecDNA whereas, 4 ug RNA was reversetranscribed for each PAR sample. Wemeasured mRNA expression of IGF-1, IGF-IR, IGFBP-3, PCNA, GHR, Stat5b and Socs2in PAR as well as MEC from OVX and INTheifers. Relative mRNA abundance of IGF-1and PCNA was down regulated and IGF-1RmRNA was up regulated in PAR homogenatedue to ovariectomy (P < 0.05; Fig 5). We and

Table 1. Primer pair sequences used in real-time PCR assays

1Whether the gene is treated as a target gene or endogenous control gene

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others (Akers et al., 2000; Berry et al., 2003)observed a decrease of IGF-1 mRNA in PARdue to ovariectomy. Transcript abundance ofthe proliferation marker gene PCNA wasreduced in PAR (P < 0.05) but there was nodifference in PCNA mRNA in MEC betweenOVX and INT. In support we did not see adifference in epithelial cell proliferationbetween intact and ovariectomized heifers(Velayudhan, et al., 2012). Therefore, it seemslikely that the reduction in PCNA mRNA in PARas a response to ovariectomy was from thenon-epithelial components of PAR.

Insulin-like growth factor-1 mRNAwas not expressed in isolated primary MEC

(Berry, et al., 2003). Contrary to this report wemeasured IGF-1 mRNA in MEC. However, IGF-1 mRNA expression in MEC was not affectedby ovariectomy. Although there was numericaldecreases in IGF-1 (46 %) mRNA in MEC byovariectomy, the decrease was not significant(P = 0.295) which could be due to the lownumber of samples used in the current study.Similarly, transcript abundance of IGF-1R inMEC was also increased numerically (38 %),but was not statistically significant (P = 0.207).It could be due to the high animal to animalvariation and comparatively less number ofsamples used in the assay. Bovine mammaryepithelial cells express IGFBP-3 both in vivoand in cell culture systems (Cohick and Turner,1998; Weber et al., 2000). Like previous reportswe did not find difference in IGFBP-3 mRNAabundance between OVX and INT in PAR orMEC (Berry et al., 2001). Even though GHR isexpressed in stromal and epithelial cells (Jianget al., 1999; Plath-Gabler et al., 2001) ligandbinding assays failed to demonstrate directbinding of GH in mammary parenchyma (Akersand Keys, 1984). However, GH increasestranscription of milk protein genes in MAC-Tcells expressing GHR, mediated through Stat5(Zhou et al., 2008). Therefore, the activity ofGHR in MEC may differ under in vivo and invitro conditions as well as under differentendocrine states. In the current study there wasno detectable expression of Stat5b mRNA inMEC. However, Socs2 mRNA was detected inMEC although transcript abundance was notaffected by ovariectomy. There was nodetectable expression of Stat5b in mammaryepithelial cells. Transcript abundance inmammary epithelial cells was not affected byovariectomy.

In summary, coupling the techniqueof laser assisted microdissection and pressurecatapulting with the precise and sensitivemethod of qRT-PCR enabled measurement oftranscript abundance in MEC and PARdiscretely from prepubertal mammary glandin ovariectomized and intact heifers. Ourresults strongly indicate a disparity in theresponse to ovariectomy between MEC andPAR tissue homogenate and suggest thatresponse to ovariectomy in the geneexpression profile in PAR is markedly impactedby the presence of the stromal cells adjacentto epithelium.

AcknowledgementsThe authors extend their gratitude to

Figure 3. Effect of ovariectomy on transcriptabundance in mammary parenchyma. RelativemRNA expression (2–ÄÄCt) in PAR tissuehomogenate samples collected from shamoperated (INT; n = 12) and ovariectomized(OVX; n = 7) heifers. There was a reduction intranscript abundance for IGF-1 and PCNA whileIGF-1R mRNA increased in OVX relative to INT.

Figure 4. Effect of ovariectomy on transcriptabundance in mammary epithelial cells.Relative mRNA expression (2–ÄÄCt) inmicrodissected MEC collected from shamoperated (INT; n = 12) and ovariectomized(OVX; n = 7) heifers. There was no detectableexpression of Stat5b in mammary epithelialcells. Transcript abundance in mammaryepithelial cells was not affected byovariectomy.

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Dr. Anthony Capuco (Bovine FunctionalGenomics, USDA, Beltsville, MD) for technicalas well as intellectual support. The authorsalso thank Cathy Parsons (Virginia Tech) fortechnical support. In addition, we would liketo acknowledge the facility center at FralinLifescience Institute, Virginia Tech, Blacksburgfor letting us use their laser capturemicrodissection microscope. This project wassupported by National Research InitiativeCompetitive Grant no. 2006-35206-16699,‘Ovarian Regulation of Stem Cells andIGF-I Axis Molecules in Prepubertal HeiferMammary Gland’ to R. M. Akers and S. E. Ellisfrom the USDA National Institute of Food andAgriculture.

References

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Akers, R. M., McFadden, T. B., Purup, S.,Vestergaard, M., Sejrsen, K. andCapuco, A. V. 2000. Local igf-i axis inperipubertal ruminant mammarydevelopment. J. Mammary Gland Biol.Neoplasia. 5:43-51.

Becker, I., Becker, K. F., Rohrl, M. H. andHofler, H. 1997. Laser-assistedpreparation of single cells fromstained histological slides for geneanalysis. Histochem. Cell Biol. 108:447-51.

Berry, S. D., McFadden, T. B., Pearson, R. E.and Akers, R. M. 2001. A localincrease in the mammary igf-1: Igfbp-3 ratio mediates the mammogeniceffects of estrogen and growthhormone. Domest. Anim. Endocrinol.21:39-53.

Berry, S. D., R. D. Howard, P. M. Jobst, H.Jiang and Akers., R. M. 2003.Interactions between the ovary andthe local igf-i axis modulate mammarydevelopment in prepubertal heifers. J.Endocrinol.177:295-304.

Cohick, W. S. and Turner, J. D. 1998.Regulation of Tgf binding proteinsynthesis by a bovine mammaryepithelial cell line. J. Endocrinol.157:327-336.

Gibson, U. E., C. A. Heid and Williams, P. M.1996. A novel method for real-timequantitative pcr. Genome Res. 6:995-1001.

Haslam, S. Z. 1988. Local versus systemicallymediated effects of estrogen onnormal mammary epithelial celldeoxyribonucleic acid synthesis.Endocrinology.122:860-867.

Heid, C. A., J. Stevens, K. J. Livak and Williams,P. M. 1996. Real time quantitative pcr.Genome Res.6:986-994.

Hovey, R. C., Davey, H. W., Mackenzie, D. D.and McFadden, T. B. 1998. Ontogenyand epithelial-stromal interactionsregulate igf expression in the ovinemammary gland. Mol. Cell.Endocrinol.136:139-44.

Jiang, H., Okamura, C. S. and Lucy, M. C.1999. Isolation and characterizationof a novel promoter for the bovinegrowth hormone receptor gene.J. Biol. Chem. 274:7893-900.

Meyer, M. J., A. V. Capuco, Y. R. Boisclair andAmburgh., M. E. V. 2006. Estrogen-dependent responses of themammary fat pad in prepubertal dairyheifers. J. Endocrinol.190:819-27.

Piantoni, P., Bionaz, M., Graugnard, D. E.,Daniels, K. M., Akers, R. M. and Loor,J. J. 2008. Gene expression ratiostability evaluation in prepubertalbovine mammary tissue from calvesfed different milk replacers revealsnovel internal controls for quantitativepolymerase chain reaction.J Nutr.138:1158-64.

Plath-Gabler, A., C. Gabler, Berisha, B. andSchams., D. 2001. The expression ofthe igf family and gh receptor in thebonvine mammary gland.J. Endocrinol.168:39-48.

Saal, I., Gustin, A., Rombaut, K., Pelaez, P.,Rorive, S., Remmelink, M. andSalmon, I. 2003. Laser-assistedmicrodissection applied to frozensurgical pathologic specimens -methodological aspects on rt-pcr.J. Exp. Ther. Oncol. 3:325-35.

Velayudhan, B. T., Huderson, B. P., McGilliard,M. L., Jiang, H., Ellis, S. E. and Akers,

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�

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Woodward, T. L., J. D. Turner and Akers, R.M. 1994. Lack of mitogenic responseto egf. Pituitary and ovarian hormonesin bovine mammary epithelial cells.Endocrine.2:529-535.

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K. P. Sreeji and Sisilamma George

Received - 10.10.12Accepted - 18.0713

EFFECT OF FISH OIL AND COCONUT OIL ONLIPID PROFILE AND OXIDATIVE STRESS INLIVER AND HEART OF RATS*

K. P. Sreeji1 and Sisilamma George2

Department of Veterinary BiochemistryCollege of Veterinary and Animal SciencesMannuthy- 680 651, Thrissur, Kerala

Abstract

Effect of fish oil and variouspreparations of coconut oil viz., copra oil/Refined, bleached and deodorized (RBD)coconut oil, seasoned coconut oil and virgincoconut oil, was evaluated in liver and heartof rats. The oils were administered @16.4 g/kg body weight. Administration of copra oilsignificantly (P<0.05) increased the levels oftotal cholesterol (TC), triacylglycerol (TAG),lipid peroxides (LP) and reduced glutathione(GSH) in both the tissues. Rats fed withseasoned coconut oil showed asignificant(P<0.05) increase in the level of TCin both the organs and GSH in heart. Levelsof TAG and LP did not show any significantvariation. Virgin coconut oil significantly(P<0.05) decreased the levels of TC and GSHin both the organs whereas, TAG levelincreased only in heart and LP level did notshow any significant variation. There was nosignificant variation in the level of TAG in boththe organs of fish oil fed rats, while TCdecreased significantly (P<0.05) in liver withno significant variation in heart. However, LPincreased (P<0.05) in both the organs withincrease (P<0.05) in the content of GSH onlyin heart. Significant (P<0.05) increase wasobserved in the weight of both the organs incopra oil and fish oil fed rats whereas, virgincoconut oil did not cause any significantchange. Rats fed with seasoned coconut oilshowed a reduction (P<0.05) in weight of liverwith no variation in weight of heart. The studysuggested that virgin coconut oil consumptioncaused least adverse effects on both the

organs followed by seasoned coconut oil. Highlevel of oxidative stress was observed in fishoil while copra oil undesirably affected allparameters except for the level of GSH.

Key words: Fish oil, GSH, Lipid peroxides,RBD coconut oil, Seasoned coconut oil, Virgincoconut oil

Coronary heart disease (CHD) hasbecome a major public health problem in manydeveloping countries. It has been predictedthat by 2020, the most common cause ofdeath, globally, would be CHD. The annualglobal mortality due to CHD is estimated tobe 14.3 million and of these, two thirds areoccurring in developing countries (Mandal etal. 2009). CHD is high among people in theIndian subcontinent and a greater risk appearsto be at younger age (Ahmed and Bhopal,2005). Sedentary life style and increasedconsumption of calories and saturated fatresult in abdominal obesity, insulin resistanceand atherogenic dyslipidaemia (Singh andSen, 2003). High incidence of CHD in Keralais believed to be due to consumption ofcoconut kernal and oil that contain highamount of saturated fats (Kumar, 1997). Thisgeneral belief made the people of Kerala toshift to alternate cooking oils such as,sunflower oil, which is rich in linoleic acid, anessential, ù-6 fatty acid (Sabitha et al. 2009).Various preparations of coconut oil areavailable commercially. Refined, bleached anddeodorized (RBD) coconut oil or Copra oil andVirgin coconut oil (VCO) are important amongthese. Seasoned coconut oil is the one, which

*Part of M.V.Sc thesis submitted by the first author to the Kerala Agricultural University, Thrissur1. M.V.Sc scholar2. Professor and Head, Department of Veterinary Biochemistry, CVAS, Pookode

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16 Effect Of Fish Oil And Coconut Oil...

is used by the people in the western coastalregion of India, where heated RBD coconutoil is treated with mustard seeds, onion, curryleaves and turmeric powder which is added incurried dishes to increase its taste and flavour.

It has become a practice to consumefish oil to increase ù-3: ù-6 fatty acid ratio ofthe diet (Luotola and Luotola, 1985).Althoughfish oils are good in preventing the incidenceof atherosclerosis, it may increase the level ofincorporation of long-chain ù-3 fatty acids inmembrane phospholipids thereby, potentiateperoxidation of cellular membranes (Iritani andFuiikawas, 1982). Increased production ofhydroperoxides may be deleterious tomembrane integrity and can result in theaccumulation of degradative products ofperoxidized lipids (Hammer and Wills, 1978).

Present study, compares the effect ofvarious preparations of coconut oil and fishoil on lipid profile and oxidative stress in liverand heart of rat.


Commercial coconut oil (Copra oil/RBD coconut oil) was procured from KeralaAgricultural University. Commercial virgincoconut oil (RUBCO Nutri-ko) and commercialshark liver oil were procured from local market.Seasoned coconut oil was prepared in thelaboratory as described in our earlier studies(Sreeji and George, 2011). Thiobarbituric acid(TBA), 1, 1, 3, 3 Tetra methoxy propane (TMP),disodium hydrogen phosphate, monosodiumdihydrogen phosphate and 5, 5’ Dithio bis-2-nitrobenzoic acid (DTNB) were purchased fromHimedia Laboratories Pvt Ltd, Mumbai.Sodium dodecyl sulphate (SDS) andTrichloroacetic acid (TCA) were procured fromSigma–Aldrich India, Bangalore andQualigens Fine Chemicals, Glaxo Smith KlinePharmaceuticals Ltd, Mumbai, respectively.All other chemicals were procured from MerckIndia Ltd, Mumbai.

Male Wistar rats weighing 180-220 gwere housed in appropriate cages in a well-ventilated experimental animal room under 12:12 hr LD cycle at 22 to 28°C and 45 to 55%relative humidity with free access to standardrat pellet diet and drinking water. Experimentswere conducted with the approval of InstitutionalAnimal Ethics Committee. Rats were randomlydivided into 5 groups, G1 to G5, eachcomprising 6 animals. Except G1, rats underall other groups were administered with various

oils for a period of 90 days as follows:

G1 – Normal control (NC)

G2 – Copra oil/ RBD coconut oil (CO)

G3 – Seasoned coconut oil (SCO)

G4 – Virgin coconut oil (VCO)

G5 – Fish oil (FO)

Dose was fixed based on per capitaworld average oil consumption level (17.8 kg/head/year), consumption level of developedwestern world (44 to 48 kg/head/year) and thetotal coconut oil consumption in Kerala (freeoil + oil derived from kernel), which comesaround 14 kg/head/year (Rajamohan, 2004).A dose of 30 kg/head/year was fixed, whichcomes to a rat dose of 16.4 g/kg body weight.The dose fixed was an average value of percapita world average consumption andconsumption of developed western world.Moreover, it was nearly double the per capitacoconut oil consumption in Kerala. Oils wereadministered every day orally using anorogastric tube in two divided doses, atmorning and evening.

On completion of experiment (on day91) the rats were euthanized and liver and heartwere collected. The organs were washed in icecold saline, dried on a filter paper and weighed.Samples from liver and heart were collectedfor the preparation of tissue homogenates.

Total cholesterol and Triacylglycerolwere estimated by the method of Haug andHostmark (1987). Level of lipid peroxides inliver and heart tissue homogenates wasdetermined by the method of Ohkawaet al.(1979) and that of reduced glutathione (GSH)by the method of Moron et al. (1979).

Data obtained were compared byanalysis of variance (ANOVA) (Snedecor andCochran, 1989) followed by Duncan multiplerange test to determine the level of significance(Steel and Torrie, 1980).


Total cholesterol level increasedsignificantly (P<0.05) in both the organs of G2and G3 (Table 1). Very high level of TAG wasobserved in both the tissues of G2 rats whileit was maintained to that of control rats in G3.It has been reported thatlarge amounts oflauric (C: 12: O) and myristic(C: 14: 0) acidsin coconut oilare capable of increasing totalcholesterol (Dauqan et al., 2011) and

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increased HMG CoA reductase activity, canlead to the increased level of tissue cholesterol(Zulet et al. 1999). Asai and Miyazawa (2001)showed that supplementation of curcuminoidsin the diet significantly increased hepatic acyl-CoA oxidase activity and lowered lipid level inrat liver and the lipid lowering effect ofcurcuminoids might be due to alterations infatty acid metabolism.

Significantly (P<0.05) decreased levelof cholesterol in both liver and heart wasobserved in G4 rats. However, TAG level wassignificantly increased in heart while it wasmaintained to that of control rats in liver (Table1) as against the findings of Nevin andRajamohan (2004). They observed reducedlevels of cholesterol and TAG in liver, heartand kidney of rats fed with VCO for 45 daysand suggested that this could be due to thereduced activity of HMG CoA reductase andthe relative rate of synthesis and oxidation offatty acids in liver. The reason for the increasedlevel of TAG observed in heart in the presentstudy needs to be explored further.

Rats in group G5 showed decreased(P<0.05) level of liver cholesterol while, thatof heart and TAG in both the tissues weresimilar to that of control rats (Table 1). Similarobservations have also been reported byearlier workers (Nalbone et al. 1988). Ahmedet al. (2006) showed significantly lower levelof liver cholesterol in rats fed with FO dietcompared to soybean and palm oil diets andsuggested that this decrease could be mainlydue to an increased rate of excretion ofcholesterol and bile acids in faeces.

Significant (P<0.05) increase wasobserved in the level of LP in both the tissuesand GSH in heart of G2 rats. Although, therewas an increase in the level of GSH in liver, itwas not significant statistically. Rats in G3 didnot showany significant variation in the levelof LP in both the tissues and GSH in liverwhereas, the GSH content increasedsignificantly (P<0.05) in heart (Table 2). Thisis in agreement with the findings of Nevin andRajamohan (2006), who suggested that risein the level of GSH could be a compensatorymechanism to scavenge the free radicalsgenerated. Flavonoids and other polyphenolsin SCO have been reported to exert a varietyof biological actions such as free radicalscavenging, chelation of metal ions,modulation of enzyme activity, signaltransduction, activation of transcription factorsand gene expression (Rice-evans et. al., 1995;Arulselvan and Subramanian, 2007; Bensonand Devi, 2009).

Rats in G4 exhibited numericallydecreased level of LP in both liver and heartfrom that of G1, but not significant statistically(Table 2). However, a significant (P<0.05)decrease was observed in the level of GSH inboth the organs. Similar observations on thelevel of LP and related enzymes were reportedby earlier workers (Nevin and Rajamohan,2006 and 2008). They suggested that highcontent of unsaponifiable components suchas, vitamin E and polyphenols might havecontributed to the beneficial effect of VCO andthe reduction in GSH content of both thetissues might be attributed to the reduced

Table 1. Effect of various preparations of coconut oil and fish oil on total cholesterol andtriacylglycerol in liver and heart (mg/g wet tissue) of rats.

(Mean ± SE, n = 6)

Level of significance was determined column wise between groups. Values not bearing a commonsuperscript letter (a, b, c and d) in a column differ significantly (P<0.05).

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oxidative stress in the organs.

Rats in G5 showed significant(P<0.05) increase in LP level in both the organsand GSH in heart whereas, liver GSH did notvary significantly from that of control rats(Table 2) indicating a higher oxidative damagein liver compared to heart. Glutathionefunctions as a direct free radical scavengerand stabilizes membrane structure through theremoval of products of lipid peroxidation(Khajuria et al., 1997). As an adaptiveresponse to increased oxidation, GSH contentwould have been increased in both the tissueswhile an insignificant increase in the level ofGSH and a significant rise in the level of lipidperoxides in liver, suggest that there mighthave been a rapid utilization of the newly

synthesized/transported GSH for theelimination of reactive oxygen species (ROS)and quenching the products of lipidperoxidation. Gonzalez et al.(1992) reportedincreased level of tissue lipid peroxides inheart, skeletal muscles and mammary glandof mice fed with fish oil diet containing addedsynthetic antioxidant while, Sen et al.(1997)showed that addition of the antioxidant, vitaminE in FO supplemented diets decreased thelevels of lipid peroxides in tissues of rats.Increased lipid peroxidation observed in ratson FO diet could be due to either reduced levelof á-tocopherol or its rate of absorption fromthe intestine (Meydani et al., 1987).

Significant (P<0.05) increase wasobserved in the weight of liver and heart of G2

Table 2. Effect of various preparations of coconut oil and fish oil on lipid peroxides (nM/g) andreduced glutathione (GSH) in liver and heart (µg/g wet tissue) of rats.

(Mean ± SE, n = 6)

Level of significance was determined column wise between groups. Values not bearing acommon superscriptletter (a, b, c and d) in a column differ significantly (P<0.05).

Table 3. Effect of various preparations of coconut oil and fish oilonweight of liver and heart(g) of rats

(Mean ± SE, n = 6)

Level of significance was determined column wise between groups. Values not bearing acommonsuperscript letter (a, b, and c) in a column differ significantly (P<0.05).

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rats while in G3, SCO administration decreasedliver weight and maintained the weight of heartsimilar to that of control rats (Table 3).

The observations could be correlatedto the oxidative stress and TAG content of boththe organs. Increased weight of both the organsin rats on CO rich diet could be attributed tooxidative damage and resultant inflammation(Onyema et al. 2006), which is evident from thelevel of lipid peroxides. Very high content of TAGobserved in both the tissues also contribute tothe rise in weight. Zulet et al. (1999) reportedsimilar findings. Rats in group, G4 did not showany significant variation in the weight of boththe organs (Table 3), which is supported by thebiochemical parameters. Significant (P<0.05)increase in weight of both the vital organs (Table3) of G5 rats could be attributed to oxidativedamage and resultant inflammation of tissues,which is evident from the increased level of lipidperoxidation, which is in accordance with thefindings of Parrish et al. (1991). They showedthat adipose tissue (epididymal and perirenal)was the only tissue whose mass decreased afterFO supplementation, whereas mass of liver andspleen increased.

It is evident from the present study thatthe level of tissue lipids and oxidative stressincreased in CO consumption. Though, therewas an increase in tissue TC, the TAG leveland oxidative status was similar to that ofcontrol rats in SCO fed group. VCOadministration showed a better lipid profile (lowlevel of TC in both the tissues and liver TAGsimilar to that of control animals)except for TAGlevel in heart and a reduced oxidative stresswhereas, fish oil exhibited a better lipid profilein both the tissues but with a higher oxidativestress. The study revealed least adverse effectsin both the organs on consumption of virgincoconut oil followed by seasoned coconut oil.High level of oxidative stress was observed infish oil while copra oil undesirably affected allparameters except for the level of GSH.

Acknowledgement

This study has been carried out as part ofMasters Research programme and thefinancial support provided by KeralaAgricultural University is acknowledged.

References

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Received - 17. 09.12Accepted - 22. 10.12

Abstract

Anatomical studies on the bones of the pelviclimb of three Indian muntjac were carried out.The long axis of ilium and ischium were in thesame line and the pelvic floor had a caudalslope. The elliptical pelvic inlet was obliquelyplaced. The gluteal line was a rounded crest.The iliopubic eminence was a small ridge. Theischiatic spine was tall and sharp. The tuberischii was large and trituberculate. The rim ofacetabulum presented a narrow deep notch.The shaft of femur was curved cranially in thedistal third. The trochanter major projectedabove the level of the head of femur. Themedial trochlear ridge was more prominentthan the lateral. The supracondyloid fossa waswell-developed and the patella was long,narrow and triangular with pointed apex. Thepopliteal line and the muscular ridges on tibiawere less prominent. The medial intercondylartubercle of tibia was taller. Lateral malleoluswas a small quadrilateral bone. The tarsuscomprised of tibial tarsal, fibular tarsal, centraland fourth fused tarsal, first tarsal and secondand third fused tarsal. The shaft of the largemetatarsal was distinctly four-sided. The smallmetatarsal was a quadrilateral sesamoid bone.Two chief digits each with three phalanges andthree sesamoids were present. The proximaland middle phalanges were more slender thanthat of the pectoral limb. The middle phalanxwas one-third shorter than the proximal oneand the distal phalanx had the shape of a hoof.

Key words: Morphology, pelvic limb,Indian muntjac

The Indian muntjac or barking deer isthe most numerous one among the variousmuntjac deer species. The male Indianmuntjac has small, unbranched antlers andtusk-like upper canine teeth. Information aboutthe anatomical peculiarities of the pelvic limbin Indian muntjac is scanty. Hence this studywas undertaken to elucidate the anatomicalfeatures of the pelvic limb in Indian muntjac.

Materials and MethodsPelvic limb bones were collected from

three Indian muntjac died of natural causesand brought for post-mortem examination tothe Department of Pathology, College ofVeterinary and Animal Sciences, Pookode.Bones were processed (Young, 1980) forstudying the anatomical features.


The pelvic girdle comprised of two oscoxae each had three bones: ilium, pubis andischium (Fig. 1). The bone was 16.1 cm long.The pelvic floor formed by pubis and ischiumsloped a little caudally as in small ruminants(Getty, 1975). The long axis of ilium andischium were placed in the same line. Theelliptical pelvic inlet was obliquely positioned.The middle transverse diameter of the pelvicinlet and transverse diameter of outlet were3.8 cm and 2.4 cm respectively. The ilia ofboth sides were almost parallel and comprisedof a broad wing and an elongated shaft as ingoat (Nickel et al., 1986). The sacropelvicsurface consisted of an extensive rougharticular area and a narrow elongated smooth

ANATOMICAL STUDIES ON THE BONES OFTHE PELVIC LIMB IN INDIAN MUNTJAC(Muntiacus muntjak)

C. V. Rajani1, Leena Chandrasekhar2,George Chandy3 and J. J. Chungath4

Department of Veterinary Anatomy and HistologyCollege of Veterinary and Animal Sciences,Pookode, Lakkidi- 673 576, Wayanad, Kerala

1,2Assistant Professors,. 3Assistant Professor, Department of Veterinary Surgery and Radiology, 4Professor & Head, Dept. of Veterinary Anatomy & Histology, CVAS, Mannuthy

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22 Anatomical Studies On The Bones....

iliac surface. The gluteal line was a roundedcrest parallel to the lateral border of wing ofilium. This concurred with the reports of Nickelet al. (1986) in small ruminants where thegluteal line was a rounded crest but disagreedwith the observations in cattle where it wasindistinct. The arcuate line and the psoastubercle on the shaft were indistinct. The crestof ilium was thin, concave and sharp. The coxaltuber was thick while the sacral tuber was thinwith a wide gap separating it from the oppositeside. The greater ischiatic notch was deep.These features agreed with observations madeby Dyce et al. (1996) in large animals.

The pubis consisted of a body andtwo rami- a cranial ramus and a caudal ramus.The cranial border of pubis showed laterallyiliopubic eminence as a small ridge similar tothat in sheep (Getty, 1975). But in cattle, itwas a large roughened tubercle. Thetransverse groove on the cranial border wasvery faint that faded before reaching theacetabular notch. The ischium made up of abody, a ramus and a tabula. The ventralsurface of ischium had a rough ridge for themuscles. The ischiatic spine located caudalto the greater ischiatic notch was tall and sharpwhich showed vertical rough lines laterally. Thelarge tuber ischii presented three tubercles of

which the lateral tubercle was more prominentas recorded in goat (Fig. 1). However itdisagreed from sheep where the lateraltubercle of the tuber ischii was much longer(Nickel et al., 1986) and the ischiatic spine waslow and everted (Getty, 1975). The lesserischiatic notch was very shallow. The depthand articular circumference of the rim ofacetabulam were 1 cm and 5.3 cmrespectively. Caudomedially it had a narrowdeep notch as seen in small ruminants (Nickelet al. 1986). Nevertheless the additional cranialnotch observed in cattle was absent. Theischiatic arch was narrow and deep with anangle of about 520. The pelvic floor presentedlarge elliptical obturator foramen of 3.8 cmlong and 1.8 cm wide.

The shaft of femur was slender,cylindrical and was curved cranially in thedistal third as reported in Sambar deer (Rajaniet al., 2012). But the shaft was almost straightin cattle. A nutrient foramen was located inthe proximal third of cranial surface asrecorded by Peters (1988) in domestic cattle.The caudal surface was narrow, longitudinallyconcave and showed in its middle third thefacies aspera bounded by the prominent lateraland faint medial femoral lips. The distalextremity showed a well-developedsupracondyloid fossa. These featurescorroborated with Rajani et al. (2012) inSambar deer. On the other hand, Nickel et al.(1986) recorded a shallow supracondyloidfossa in large ruminants and supracondyloidtuberosity in small ruminants.

The proximal extremity of femurcomprised of a head, neck and two trochanters(Fig. 2). The stout convex head projectedmore medially. It presented a shallow foveacapitis in the middle. Medially, the neck wasdistinct as in smaller species (Dyce et al.,1996). The undivided trochanter major wasmassive (Hyman, 2004) whose summit

Fig.1. Ventrolateral view of pelvic girdle of IndianmuntjacC- Coxal tuber of Ilium, G- Gluteal line, AR- Articular surface,P- Pubis, IL- Iliopubic eminence, SP- Pubic symphysis,S- Ischium, A- Acetabulum, N- Notch of acetabulum, T- Tuberischii, O- obturator foramen

Table Mean morphometrical parameters of the pelvic limb bones of Indian muntjac

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C. V. Rajani et al.

projected 0.7 cm above the level of the head.This finding concurs with the observationsmade by Nickel et al. (1986) in large ruminantsbut disagreed with that made by Rajani et al.(2012) in Sambar deer where the trochantermajor was in level with the head. A prominenttrochanter minor was located at thecaudomedial aspect of proximal third of shaft.The extensive trochanteric fossa was 1.2 cmdeep and the intertrochanteric crest was welldeveloped. The distal extremity consisted ofsagittally oblique, caudodistally projected twolarge condyles and a cranial trochlea (Fig. 2).The trochlear groove had a length of 3.1 cmand breadth of 1.5 cm. It was bounded bymedial and the lateral trochlear ridges, ofwhich the medial one was more prominent.This feature was similar to that of domesticcattle (Peters, 1988) and Sambar deer (Rajaniet al., 2012) where the medial ridge was moreprominent, but disagreed with the appearancein small ruminants where both the ridges wereequal (Nickel et al., 1986). The lateral condyleand epicondyle were bigger than the medialone. The inter-condyloid fossa was rough,

oblique and wide.The patella was long and simulated a

narrow triangle with pointed apex (Fig. 2) asrecorded by Getty (1975) in cattle. It had alength and breadth of 2.7 cm and 1.7 cmrespectively. Conversely, Rajani et al. (2012)reported it as ovoid, long and narrow inSambar deer. The base was broader inSpotted deer, whereas in the cattle it was inthe form of a thick transverse ridge (Nickel etal., 1986). The rough cranial surface wasconvex while the caudal articular surface wassmooth. The caudal articular surface showeda blunt sagittal ridge which divided it into amedial smaller and a lateral larger area as inlarge ruminants (Nickel et al., 1986) andSambar deer (Rajani et al., 2012). But thecaudal surface of patella was concavetransversely in small ruminants as opined byNickel et al.(1986).

The tibia was moderately curved asin cattle. The proximal third of the shaft wastriangular in cross section with the distal endoval (Fig. 3). Medial and lateral surfaces werelongitudinally convex and concaverespectively. The caudal surface was flat; butthe popliteal line and the muscular ridges wereless prominent. But Getty (1975) describedmore prominent ridges in this area in cattle.The cranial border was blunt and laterallycurved as in cattle. The proximal extremityconsisted of two condyles separated caudallyby the popliteal notch. The medialintercondylar tubercle was taller than thelateral one as recorded in cattle but, in smallruminants both the tubercles were equal(Nickel et al., 1986). The lateral condyleshowed cranially an extensor sulcus for thepassage of tendons.

The distal extremity had two sagittaloblique grooves and an intermediate ridge. Themedial malleolus had a pointed end. Laterally,distal extremity articulated with the lateralmalleolus. The fibula comprised of only twoextremities (Fig. 3). The head was fused withthe tibia in the form of a small prominence onthe lateral condyle of the tibia. Lateralmalleolus was a small, quadrilateral and sideto side compressed bone of about 1.1 cm long.

The tarsus comprised of five bonesarranged in three rows: tibial and fibular tarsalsin the proximal row, central and fourth fusedtarsal in the middle row and the first and thesecond and third fused tarsals in the distal row(Fig. 4). The tuber calcis of fibular tarsal was

Fig.2. Caudal and cranial view of femur andpatella of Indian muntjacH- Head, TM- Trochanter Major, T- Trochanteric fossa,I- Inter trochanteric crest, M- Trochanter minor,S-Shaft,M-Medialcondyle, R- Trochlea, P- Patella, ML- Medial condyle

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marked by a wide shallow groove. Central andfourth fused tarsal showed a medioplantarhook like projection directed proximally. Thelateral plantar tuberosity of central and fourthfused tarsal was more prominent than themedial one. This differed from cattle (Getty,1975) wherein the medial tuberosity was moreprominent. The first tarsal was a smallquadrilateral bone while second and third fusedtarsal was broad and flat.

The metatarsals were two in number:a large metatarsal and a small medialmetatarsal. The shaft of large metatarsal wasfour-sided in the proximal 2/3rd, while distallyit was almost oval. It was longer than the largemeatacarpal. The dorsal longitudinal sulcuswas wide, deep and distinct (Fig. 3). Theplantar longitudinal sulcus was distinct in theproximal 2/3rd. The proximal perforatingforamen of the plantar surface opened on theplantar aspect of the proximal extremity. Thesmall metatarsal bone was a smallquadrilateral sesamoid bone.

Two chief digits (III) and (IV), eachwith three phalanges and three sesamoids

were observed (Fig. 5). Second and fifth digitswere dewclaws. The proximal and middlephalanges were more slender than that ofpectoral limb. The flexor tubercles of proximalphalanx were positioned more proximally as

Fig.4. Dorsal and lateral views of tarsus ofIndian muntjacT- Tibial tarsal, F- fibular tarsal, S-Sustentaculum tali,C- fused Central and fourth tarsal, S- sustentaculum tali

Fig.3. Medial view of tibia; dorsal view ofmetatarsus of Indian muntjacL- Lateral malleolus, T- Tibial tuberosity, LC- Lateralcondyle, F- Fibula, C- Crest of tibia, S- Shaft,D- Dorsal longitudinal groove of large metatarsus, C-condyle of large metatarsus

Fig.5. Dorsal and palmar views of chief digitsof Indian muntjacP-Proximal phalanx, M- middle phalanx, D- Distal phalanx

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recorded by Nickel et al. (1986) in smallruminants. The three sided middle phalanxwas one-third shorter than the proximalphalanx as reported by Getty (1975) in cattle.The distal phalanx resembled the shape of ahoof. The dorsal border was narrow and sharp.This was similar to the observations in smallruminants where the dorsal border was narrowbut differed from the features in cattle whereit was broader (Nickel et al., 1986). Theextensor process was rough and prominent.

ReferencesDyce, K. M., Sack, W. O. and Wensing, C. J.

G. 1996. Textbook of VeterinaryAnatomy, 2nded. W B SaundersCompany, Philadelphia. 43p

Getty, R. 1975. Sisson and Grossman’s TheAnatomy of the Domestic AnimalsVol.II. 5thed. W B SaundersCompany, Philadelphia. 755p

Hyman, L. H. 2004. Anatomy of ComparativeVertebrates. Satish Serial PublishingHouse, Delhi. 138p.

Nickel, R., Schummer, A. and Seiferle.E.1986. The Locomotor System ofDomestic Mammals.Verlag PaulParey, Berlin, Hamburg. 75p

Peters, J. 1988. Osteomorphological featuresof the appendicular skeleton of Africanbuffalo, Syncerus caffer (Sparrman,1779) and of domestic cattle, Bospriigenius f. taurus Bojanus, 1827. Z.Säugetierkunde 53: 108-123

Rajani, C. V., Shijina Raj, Leena Chandrasekhar, Maya, S., Pradeep, M. andSajitha, I. S. 2012. Morphological studies on the femur and patella of SambarDeer (Cervus unicolor). Tamilnadu J.Vet. Anim. Sci. 8:19-21

Young, T. H. 1980. Preparation of skeletalspecimen. Equine Practice. 2: 276

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26 Screening Of Cattle Blood Samples...


Abstract

A study was undertaken to investigatepresence of HCH and DDT residues in bovineblood samples of Enmakaje panchayat ofKasargod district. Retrospective analysis ofcase sheets for last three years at the localveterinary dispensary was also carried out.Whole blood samples were collected from 20randomly selected cows for pesticide residueand haematological analysis. Collectedsamples were processed by various clean upprocedures and analysed using GasChromatography for HCH and DDT residues.Residues of DDE and DDT were belowdetection limit (BDL) in all the analysed serumsamples. The haematological values of cattlefrom study area remained within the normalrange except differential leucocyte count whichexhibited marked lymphocytopenia. In thepresent study, the level of pesticide residuesin the serum samples in the study area wasnot enough to produce any visible healthhazards in cattle.

Key words: HCH, DDT residues, Cattle blood,Gas Chromatography

Organochlorine pesticides (OCPs)such as hexachlorocyclohexane (HCH) anddichlorodiphenyltrichloroethane (DDT) arepersistent, broad-spectrum toxicants. They resistbiodegradation and therefore can concentratethrough food chains to produce a significant

SCREENING OF CATTLE BLOOD SAMPLESFROM KASARGOD DISTRICT FORPRESENCE OF HCH AND DDT RESIDUES

V.Dineshkumar1, A.R. Nisha 2,P.T.A.Usha 3, C.B. Vineetha 4,A.P. Usha 5 and Siju Joseph6

Dept. of Veterinary Pharmacology and Toxicology,College of Veterinary and Animal Sciences,Mannuthy- 680 651, Thrissur, Kerala

* Part of M.V.Sc.thesis submitted by the first author to the Kerala Veterinary and Animal Sciences University, Pookode, Wayanad1Research associate2Assistant Professor (on study leave)3Associate Professor and Head4Project Fellow5Professor (CPPR)6Assistant Professor, Department of Veterinary Microbiology

magnification of the original concentration in thefood web with high risks to the ecosystem andhuman health (Leena et al., 2011).

Enmakaje panchayath of Kasargoddistrict has long been in the media due to itsburden of illness generated by intensive andbroad usage of Endosulfan by PlantationCorporation of Kerala. Research of past yearshas identified potential entry routes ofpesticides into the environment such as spraydrift, volatilisation and precipitation, surfacerunoff/overland flow, leaching, drainflow andbase flow seepage (Carter, 2000). There hasbeen reports of birth of calves with deformedlimbs in the animal population of this area.Therefore this study was structured toinvestigate for the presence of HCH and DDTresidues in cattle blood and also to study thehaematological pattern of the same animals.


Padre, Perla, Swarga and Vani nagarvillages of Enmakaje Panchayath of Kasargoddistrict were selected as study area.

A preliminary survey was conductedamong 60 farmers belonging to thePanchayath for identifying the disease patternin cattle. For this a proforma was designed toassess the disease conditions of cattle, rearingsystem, the clinical course of illness, treatmentadopted and disease outcome and then

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distributed among the farmers. The responsesindicated that conditions as infertility, cancer,congenital anomalies, repeated abortions werefrequent in animals as in humans. Thesampling locations were identified and fixedafter consulting veterinary surgeon,panchayath officials and health officials of thisarea. Further, a retrospective analysis of casespresented at the Badiadka Veterinarydispensary in the past three years was doneto ascertain the disease pattern and trendsprevailing in the area.

Blood samples were collected fromrandomly selected 20 adult cattle of the areawhich were maintained for a period of threeyears or more. Further, blood samples werecollected from 20 healthy cattle in theUniversity Livestock Farm (ULF), Mannuthyand these were used as control forhaematological analysis. Samples collectedwere immediately placed in cool boxmaintained at 4-8p C and transported in similarcondition to the laboratory and stored at -20pC until analyzed.

Pesticide residue extraction by Pitarchet al. (2005) was followed. The serumseparated from blood was stored in clean dryglass vials with stopper. From this, 2 ml serumwas taken in a 20 ml separating funnel. It was

diluted with 3 ml deionised water. To this added5 ml dichloromethane and shaken well for fiveminutes. Repeated this procedure and extractwas centrifuged to remove the emulsionobtained. The organic layer formed afterextraction was then separated and dried bypassing through a column packed with 3 cmlayers of anhydrous sodium sulphate. Theextract was evaporated to dryness under agentle stream of air and residue was dissolvedin 1ml of n-hexane for injection into the GasChromatography (GC).

Hexachlorocyclohexane anddichlorodiphenyltrichloroethane content inserum samples were quantified by GC.Analysis was done in SHIMADZU-2010 GCwith electron capture detector (ECD) having63Ni as radioactive source.

Haematological parameters werestudied using blood samples with potassiumEDTA added as anticoagulant. Totalerythrocyte count (TEC), haemoglobin (Hb),volume of packed red cells (VPRC), totalleucocyte count (TLC), differential leucocytecount (DLC) and Erythrocyte indices like Meancorpuscular volume (MCV), Mean Corpuscularhaemoglobin (MCH), Mean corpuscularhaemoglobin concentration (MCHC) were alsocalculated using techniques given byBenjamin, 1985.

Statistical comparison of results fromexposed and non-exposed animals wasperformed using the student t-test by usingStatistical Package for the Social Sciences(SPSS) 17 version.


A total of 8843 cases had beenrecorded at the Badiadka Veterinarydispensary in the following pattern ofdecreasing occurrence: digestive disorders(53.4%), parasitic diseases (13.2%),reproductive diseases (10.48%), respiratorydisease (6.5%), skin disorders (6.22%),metabolic disorders (3.7%), others (3.5%),fever (2.8%) and poisoning (0.2%).

Analytical method for estimation ofHCH and DDT

The chromatogram of mixedstandards depicted each isomer of HCH(alpha, beta, gamma and delta) and DDT (p’p-DDE, p’p-DDD and p’p-DDT) as a separateand well defined peak (Fig.1). Under theidentical operating condition of GC, the

Fig.1. Chromatogram of HCH and DDTStandard and their retention time

Fig.2. Chromatogram of Blood sample

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retention time (RT) for each component was8.981, 9.812, 10.466, 11.017, 26.691, 30.256and 34.339 minutes respectively. Recoverystudies revealed the percentage of recoveryof HCH and DDT to be about 70-80 per centwhich is satisfactory to carry out sampleanalysis. HCH and DDT residues were belowdetection limit in all of the blood samplescollected from study area. The chromatogramof a blood samples collected from study areais shown in Fig.2. The human and animalspesticide residue in serum is a biological indexof pesticide exposure and studies on blood canbe used for assessing the total body burdenof pesticides in the general population (Saxenaet al., 1987). Mean value of total HCH andDDT in serum samples of cattle from the studyarea were below the detection level. Kaphaliaand Seth (1984) detected total HCH level inbuffalo and goat serum samples were belowthe generally accepted safe level of 50ppb.Blood sampling is considered as a valid methodfor measuring body burden of persistentorganochlorine contaminants (Dhananjayanand Muralidharan, 2010). Present studyrevealed that, HCH and DDT were not presentat a detectable level in serum samples.

Haematological Parameters

Total erythrocyte count (TEC),haemoglobin (Hb), volume of packed red cells(VPRC), total leucocyte count (TLC), differentialleucocyte count (DLC), mean corpuscularhaemoglobin concentration (MCHC), meancorpuscular volume (MCV) and meancorpuscular haemoglobin (MCH) were estimatedand the values are presented in Table.1.

Mean RBC count (millions/µl) of thesamples from Kasargod and samples fromUniversity Livestock Farm (ULF), Mannuthywere 5.721±0.29835 and 5.2740±0.20974respectively. The values were in accordancewith the normal erythrocyte count in cattle of5 to 10 millions/ µl (Kaneko et al., 1997).

The mean WBC count (number/ µl) ofthe cattle from study area was 9918±919.443 andthat of the ULF, Mannuthy was 8528±407.439.Thus, the TLC in the cattle of study area wasmore than that of control group. But, the valueslie within the normal range of 4000 to 12,000numbers/ µl (Kaneko et al., 1997).

Volume of packed red cells (%) ofblood samples from Kasargod and ULF,Mannuthy area were 26.06±2.08967 and

24.83±1.01686 respectively. No significantstatistical difference was observed between thetwo groups. The samples from study areaexhibited VPRC values in the normal range of24 to 48 per cent (Benjamin, 1985).

Average Hb concentration (g %) ofsamples from Kasargod and samples from ULF,Mannuthy were 9.67±0.41313 and 8.38±0.32857respectively. The values from cattle of both areasfound to be within the normal range of 8 to 15gper cent (Benjamin, 1985).

The mean neutrophil percentage inblood samples from Kasargod area and ULFMannuthy samples were 25.3±2.70 and18.60±2.77 per cent respectively. Nosignificant difference existed between the twogroups (Table 1). The mean lymphocytepercentage in blood samples from study area(59±4.03 per cent) was significantly (P<0.05)lower than the samples of ULF, Mannuthy(76.8±3.60 per cent). Mean eosinophilpercentage of blood samples from study areaand Mannuthy area were 14.80±3.54 per centand 12.70±7.64206 per cent respectively. Nosignificant statistical difference was observedbetween the two groups. The mean monocytecount in blood samples of study area and ULF,Mannuthy samples were 3.55±0.50 per cent and2.2±0.554 per cent respectively. No significantdifference existed between the two groups.

Mean corpuscular volume (MCV) ofcattle from Kasargod and ULF, Mannuthy areawere 49.41±1.67 and 47.37±1.78 (fl)respectively. There was no significantdifference between the two groups (Table 1).The mean corpuscular haemoglobin (MCH)values of cattle from study area and controlsamples were 17.00±0.41 and 15.97±0.47 (pg)respectively. No significant difference wasobserved between the values (Table 1). Meancorpuscular haemoglobin (MCHC) value ofcattle from Kasargod and ULF, Mannuthy were34.53±0.38 and 33.79±0.35 (g%) respectively.Statistical analysis showed that there was nosignificant difference observed between thetwo groups (Table).

The present study revealed that theHCH and DDT residues in the blood of cattlefrom Enmakaje panchayath of Kasargoddistrict are below the detectable level and thehaematological picture showed that theseanimals are healthy.

Acknowledgement

Authors are grateful to Kerala State

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Council for Science, Technology andEnvironment (KSCSTE) for sponsoring thisproject. The facilities provided by the Dean,College of Veterinary and Animal Sciences,Mannuthy is also appreciated.

References

Benjamin, M. M. 1985. Outline of VeterinaryClinical Pathology. 3rd ed. KalyaniPublishers, New Delhi, 310p.

Carter. A, 2000. How pesticides get into water– and proposed reduction measures.Pesticide Outlook. 149-156

Dhananjayan, V. and Muralidharan, S. 2010.Levels of organochlorine pesticideresidues in blood plasma of various

species of birds from India. Bull.Environ. Contam. Toxicol. 85: 129-136

Kaneko, J. J., Harvey, J. W. and Bruss, M. L.1997. Appendizes: ClinicalBiochemistry of Domestic Animals. 5th

ed.Harcourt Brace and Company AsiaPTE. Ltd. pp. 885-905.

Kaphalia, B. S. and Seth, T. D. 1984. Screeningof blood serum of food animals, chickenand human beings for organochlorinepesticides and electrolytes. Indian J.Anim. Hlth. 23: 23-28*

Leena, S., Choudhary, S. K. and Singh, P. K.2011. Organochlorine and organophosphorous pesticides residues in waterof river Ganga at Bhagalpur, Bihar,

Table. Haematological parameters of cattle in Kasargod and ULF, Mannuthy

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India. Int. J. Res. Chem. Environ. 1:1(77-84)

Pitarch, E., Lopez, F. J., Serrano, R. andHernandez, F. 2001. Multiresiduedetermination of organophosphorusand organochlorine pesticides inhuman biological fluids by capillarygas chromatography. Fresenius J.Anal. Chem. 369: 501-509

Sandhu, H. S. and Brar, R. S. 2000. Textbookof Veterinary and Toxicology. KalyaniPublishers, Delhi, India, 255p.

Saxena, S. P., Khare, C., Farooq, A.,Murugesan, K. and Chandra, J. 1987.DDT residues in blood of residents ofareas surrounding a DDT manufacturing factory in Delhi. Bull. Environ.Toxicol. 38 :392-395

WHO-IPCS (World Health Organization-International Programme on Chemi calSafety),1991a. Lindane. Environ mentHealth Criteria 124. World HealthOrganisation, Geneva, Switzerland.

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Abstract

A study was undertaken to know theimportant aspects of job situation in terms ofstress factors which influence job satisfaction.The two top ranking stress factors found wereprescribing costly medicines to poor livestockowner and large animal practices especiallyattending dystocia. However majority of theveterinarians perceived medium level of stress.

Key words: Job stress, Job dissatisfaction,Job situation, Veterinarians

The salience of job stress as an areaof research has been due in part to themagnitude of its effects. In addition to beingassociated with a variety of physical diseasesincluding hypertension, (O’Connor et al.,2000), high levels of job stress can have anegative effect on emotional well being(Paterniti et al., 2002). At the organizationallevel, high levels of job stress have been linkedto low levels of productivity (Gandham, 2002).In general, job stress has been viewed as anantecedent of job satisfaction and the twoconstructs have been treated as related yetdistinct (Stanton et al.,2002). An inverserelationship between job stress and jobsatisfaction among various populations hasbeen reported constantly in literature (Cottonet al., 2002 ; Heslop et al., 2002). Theveterinary profession has been identified by anumber of studies as a stressful occupation(Gardner and Hini, 2006;Heath,2002).

JOB SATISFACTION AND STRESS AMONGTHE VETERINARIANS OF KERALA STATEIN INDIA*

Soumya Sankar1, P. Reeja George2,and P.J. Raj Kamal3

Department of Veterinary and Animal Husbandry ExtensionCollege of Veterinary and Animal Sciences,Mannuthy-680 651,Thrissur, Kerala

A better understanding of thestressors in veterinary practice may allow foridentification of strategies to improve theworking conditions of veterinarians withresulting benefits for the quality of veterinaryhealth care. This paper specifically exploresthe perception of veterinarians regarding jobdissatisfaction as a potential cause of stress.


A simple random sample of 460veterinary surgeons and 135 senior veterinarysurgeons were selected from a list of 920veterinary surgeons and 270 senior veterinarysurgeons who perform both clinical andextension functions in the Department ofAnimal Husbandry of Kerala. A structuredschedule prepared by consulting veterinariansthemselves was sent to the selectedrespondents by mail or distributed at monthlymeetings. A total of 170 veterinary surgeonsand 47 senior veterinary surgeons returned thefilled up questionnaires. The study samplehence consisted of 155 veterinary surgeonsand 45 senior veterinary surgeons. Dataprovided by this sample was collated andanalysed.

Job satisfaction referred to the degreeof satisfaction that the veterinarian derived outof the various aspects of his/her job such asprofessional activities, salary, job security andrecognition. Possible areas of stress due tojob dissatisfaction were identified through

* Part of the MVSc thesis submitted by the first author to the Kerala Veterinary and Animal SciencesUniversity,Pookode,Wayanad1Teaching Assistant2.Assistant Professor(Corresponding Author)3Professor and Head

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extensive review of literature and expertopinion. The perception of veterinarians aboutthe most important aspects of their worksituation that influenced job satisfaction wasarrived at through extensive personalinterviews with 20 veterinarians. A scale wasthen constructed to assess the stressexperienced by veterinarians due to jobdissatisfaction on the work place.

The scale consisted of eight stressors.The respondents were asked to indicate theirfeeling regarding the importance of thestressors on the work place on a five pointcontinuum viz: - almost always true, mostlytrue, sometimes true, rarely true and not at alltrue with scores of 4,3,2,1 and 0 respectively.The minimum and maximum scores that arespondent could obtain were 0 and 32. Therespondents were then classified into threecategories viz., high, medium and low applyingthe Dalenius- Hodges cumulative root f method.


In response to items that indicatepotential causes of stress, it is evident from Table1 that majority of the respondents (50.5%)experienced medium level of stress due to jobdissatisfaction. Thirty-one per cent of therespondents were highly dissatisfied with theirjobs while 18.5 per cent respondents experiencedlow level of stress due to dissatisfaction with theirjobs. Lehal (2007) reported that there was asignificant relationship between job stress andjob satisfaction. Low job satisfaction has beenreported to be associated with high level of stress(Hollingworth, et al.1988; AbudulHalim, 1981;Leigh et al.1988). Landsbergis (1988) alsoreported that high level of work stress wereassociated with low level of job satisfaction.

Data in table 2 indicate thatprescribing costly medicines to poor livestockowners was the most stressful factor underjob satisfaction.Salleh et al. (2008) observedthat among the major factors of stress

identified, viz:- job integrity conflict, security,adaptability and support, the factor jobintegrity conflict had the highest beta value andhence was the most important factor that wasassociated with stress. This is in consonancewith the findings of the present study that fromthe eight items on job satisfaction, the itemwhich reflected the job integrity conflict of theveterinarian, the difficulty of prescribing acostly medicine to a livestock owner who wouldfind difficulty in buying it was the mostimportant cause of job dissatisfaction andstress for the veterinarian. The second itemthat was a source of stress amongveterinarians was large animal practice,generally and especially cases of dystocia thatare difficult to handle due to physical problemslike back pain. Hansez et al. (2008) alsoobserved that veterinary surgeons faced anumber of professional risks associated withanimal care which included musculoskeletaldisorders due to uncomfortable workingpositions especially in rural practice settings.

These views were endorsed byJeyaretnam et al. (2000) and Reijula et al.(2003). In many instances, the success of theclinical cases would require physical exertionand veterinarians have to perform themdespite physical discomfort since a failure todo so would entail loss of face. Similar findingswere made among veterinarians in NewZealand where physical demands of the jobwere more of a stressor in large animalpractice (Gardner, 2009). Reijula et al. (2003)showed that inadequate facilities andassistance were major risk factors for injuriesand musculoskeletal disorders inveterinarians. Manual handling/lifting ormoving animals, rectal palpation, feet-trimming,obstetric procedures and surgical procedureslasting less than one hour were veterinaryprocedures associatedwith musculoskeletalproblems in veterinarians according to Cattell2000, O’Sullivan and Curran 2008 andScuffhamet al. 2010. Scuffham et al.(2010)

Table 1. Distribution of respondents based on level of stress due to job dissatisfaction N =200

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suggested that multi-modal interventions, suchas the use of training in combination with theuse of mechanical lifting aids, would beeffectiveas long-term preventive strategiesagainst musculoskeletal problems. (Silversteinand Clark, 2004).

Table 2 also reveals the difficulty inworking efficiently because of the inadequacyof medicines which was perceived as the fourthimportant stressor contributing to jobdissatisfaction among veterinarians in Kerala.So also, not being able to keep abreast ofnew developments in this field was perceivedto be a cause of stress among the veterinarians.This finding is endorsed by Sallehet al. (2008)who observed that the need to keep up withtechnological changes was a major cause ofstress in the broad area of career development.The arduous nature of the veterinary professionreflected in other studies is also seen in thefindings of the present study.

Past research among veterinarianshas suggested that proactive measures witha view to creating awareness about jobresources such as external support in planning,extra help in administration and in clinical workare important in improving work in theveterinary profession. Several countries suchas UK, USA, Canada and Norway have begunconcerted attempts to adopt stressmanagement interventions specifically for

veterinarians. Similar measures in our countrycould also be instrumental in improving wellbeing in the veterinary profession.

Acknowledgement

The authors are thankful to the Dean,College of Veterinary and Animal Sciences,Mannuthy for the facilities provided.

References

Abudul-Halim, A.1981.Social support andManagerial affective adaptation. J. ofOccupational Behaviour. 6: 117-230.

Ailsby R.L.1996. Occupational arm, shoulder, andneck syndrome affecting large animalpractitioners.Can. Vet. J. 37: 411.

Cattell, M. B. 2000. Rectal palpation associatedcumulative trauma disorders andacutetraumatic injury affecting bovinepractitioners. Bovine Practitioner. 34: 1–5.

Cotton, S. J., Dollard, M. F., & de Jonge, J.2002. Stress and student job design:Satisfaction, well-being, andperformance in university students. Int.J. of Stress Management. 9: 147-162.

Gandham, S. R. 2000. Occupational stress:Time for a policy. The Health& SafetyPractitioner. 18: 20-21.

Gardner, D. 2009.Vets and stress, New

Table 2. Item-wise mean scores of dimension- Job satisfaction

Analysis of various items of Job satisfaction

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Zealand Veterinary Association.

Gardner, D.H. and Hini, D. 2006. Work-relatedstress in the veterinary profession inNew Zealand. N Z Vet J. 54:119-124.

Hansez, I., Schins, F. and Rollin, F.2008.Occupational stress, work-homeinterference and burnout amongBelgian veterinary practitioners. IrishVet. J. 61 : 233-241.

Heath T.J. 2002. Longitudinal study ofveterinarians from entry tothe veterinarycourse to 10 years after graduation:attitudesto work, career andprofession.Aust. Vet J 80:474-478.

Heslop, P., Smith, G. D., Metcalfe, C., Macleod,J., & Hart, C. 2002. Change in jobsatisfaction, and its association withself-reported stress, cardiovascularrisk factors and mortality. SocialScience and Medicine. 54: 1589-1599.

Hollingworth, C.,Mathews, G. and Hartnett,O.1988. Job satisfaction and mood : Anexploratory Study.Work and Stress.2:225-232.

Jeyaretnam, J., Jones, H. and Philips, M.2000. Disease and injury amongveterinarians. Australian Vet. J. 78:625-629.

Landsbergis, P.A. 1988. Occupational stressamong health care workers: A test ofthe job demands- control model. J. ofOrganizational Behavior. 9: 217-239.

Lehal, R. 2007. A Study of Organisational RoleStress and Job Satisfaction amongExecutives in Punjab. Indian manage.studies J. 11: 67-80.

Leigh, J.H., George, H. and Woodman,R.W.1988. Effect of perceivedorganizational and factors on rolestress job attitude relationship. J.of

Manage. 14:16-25.

O’Connor, D. B., O’Connor, R. C., White, B.L., & Bundred, P. E. 2000. Job strainandambulatory blood pressure inBritish general practitioners:A preliminary study. Psychology,Health and Medicine. 5 : 241-250.

O’Sullivan, K. and Curran, N. 2008. It shouldn’thappen to a vet ... Occupational injuriesin veterinary practitioners working inIreland. Irish Vet. J. 61: 584–587.

Paterniti, S., Niedhammer, I., Lang, T.,&Consoli, S. M. 2002. Psychosocialfactors at work, personality traits anddepressive symptoms: Longitudinalresults from the GAZEL study. BritishJ. of Psychiatry. 181: 111-117.

Reijula, K, Rasanen K; Hamalainen M;Juntunen K; Lindbohm M.L, TaskinenH; Bergbom, B; Rinta-Jouppi M. 2003.Work environment and occupationalhealth of Finnish veterinarians.American J. of Industrial Medicine.44: 46–57.

Salleh, A., Bakar, A., and Keong, W. 2008.How Detrimental is Job Stress? : ACase Study of Executives in theMalaysian Furniture Industry. Int.Rev. of Business Res. 4: 64-73.

Scuffham, A. M., Legg, J. S., Firth, E. C.andStevenson, M. A.2010. Prevalenceand risk factors associated withmusculoskeletal discomfort in NewZealand veterinarians. AppliedErgonomics. 41: 444–453.

Stanton, J. M., Bachiochi, P. D. Robie,C., Perez, L. M., & Smith, P. C. 2002.Revising the JDIWork Satisfa ctionsubscale: Insights into stress andcontrol.Educational and PsychologicalMeasurement. 62: 877-895.

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A.Kandasamy and K. Devada


Abstract

Hundred cattle known to be infectedwith Gastrothylax crumenifer, the mostprevalent amphistome species were selectedat random for the present study. An indirectELISA was performed to detect serumantibodies using somatic antigens of the fluke.Fifty one samples were found to beseropositive. Similarly, ELISA was employedto detect coproantigens in faecal supernatantsof the same cattle using rabbit hyper immuneserum against the somatic fluke antigens.Seventy four samples were found to containdetectable amounts of coproantigens indicatinga sensitivity of 74 per cent for the test. Theresults indicate that coproantigen detectionwhich revealed a higher sensitivity than thatof serum antibodies by ELISA is reasonableand practical for the detection of amphistome

infections in bovines.

Key words: Coproantigen, Indirect ELISA,Amphistomes

Conventional methods of diagnosis ofamphistomosis caused by Gastrothylaxcrumenifer in bovines like observation ofclinical signs, aided by coprologicalexamination and serological methods havemany limitations. (Hafeez, 1981; Johnson etal., 1996; Dumenigo et al., 1996). As a resultemphasis has been given on the detection ofparasitic antigens in biological samplesespecially if it does not involve collection ofblood samples (Johnson et al.,1996).Kandasamy and Devada (2011) ensured thatdiagnosis of amphistome infection in cattle

COMPARING ELISA USING SERUMANTIBODIES AND COPROANTIGENS FORDETECTING EARLY AMPHISTOMOSIS*

A. Kandasamy1 and K. Devada2

Department of Veterinary ParasitologyCollege of Veterinary and Animal ScinecesMannuthy 680 651, Thrissur, Kerala

was feasible using coproantigens in an ELISA.The present study was undertaken to comparethe sensitivity of ELISA using serum antibodiesand coproantigens in the diagnosis ofamphistomosis in cattle.


Live flukes from the rumen of infectedcattle slaughtered at the Municipal slaughterhouse, Thrissur, were collected and broughtto the laboratory in chilled saline. The mostprevalent amphistome species, Gastrothylaxcrumenifer were separated from the rest in thelaboratory based on morphology. Somaticantigens were prepared from the above flukesas per Jithendran et al. (1996) and its proteincontent was determined by Biuret methodusing photometer 5010. Hyper immune sera(HIS) against somatic antigens were raised inrabbits (Johnson et al., 1996) and the serawere tested for antibodies by AGPT (Kaganand Norman, 1976).

Serum samples from 100 cattle withknown Gastrothylax infection were collectedand stored in sterile vials at -20 º C for antibodydetection in an ELISA. Faecal samples of thesame 100 cattle infected with Gastrothylaxspecies, from which the sera were obtainedwere collected just before slaughter in smallplastic vials for coproantigen detection.

An indirect ELISA was performed as perthe method describes by Craig et al. (1995) andAnderson et al.(1999) to detect serum antibodies.The optimum concentration of somatic flukeantigen, test serum samples and rabiit anti-bovineIgG-HRP conjugate (Genei, Pvt, Ltd.) were

*Part of M.V.Sc. thesis submitted by the first author to the Kerala Agricultural University, Thrissur1-Veterinary Officer, Venky’s India, Palladam, Tamil Nadu2- Professor & Head

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standardized by checker board titration.

An indirect ELISA using coproantigens was performed as described by Ahmadand Nizami (1998) in 96 well flat bottomedmicrotitre plates. The optimum concentrationof coproantigens, anti somatic HIS raised inrabbits and goat anti-rabbit IgG-HRPconjugate ( Genei, Pvt, Ltd.) were standardizedby checker board titration.

In both the tests, sera from cattle withknown Gastrothylax infection formed thepositive control and those from cattleabsolutely free from any amphistome infectionformed the negative control. Optimumdilutions of the reagents which gave thehighest titre with the positive sample and thelowest titre with the negative sample wereselected as the working dilution for the presentstudy. The plates were read at 450 nm in aMultiscan MS ELISA reader. Samples that hadan OD value above the sum of the mean ofnegative controls and three times the standarddeviation (cut-off point) were taken as positive.


The protein content of somatic antigensprepared for raising HIS in rabbits was found tobe 4 mg/ml and that of faecal supernatantcontaining coproantigens was found to be 1 mg/ml. The optimum concentrations of somatic flukeantigen, test serum and rabbit anti bovine IgG-HRP conjugate for serum ELISA were found tobe 1:200, 1:100 and 1: 1000 respectively andthose of coproantigens, anti somatic HIS andgoat anti rabbit IgG –HRP used for coproantigenELISA were 1:64, 1:100 and 1:100 respectively.

The cut off point for the samples inthe serum antibody ELISA was 0.300 and 51serum samples were found to have an ODvalue above this point and marked as seropositive. The sensitivity of the test was 51 percent. The low sensitivity obtained in the presentwork, might be attributed to crude somaticantigen used in the present study. According

to Craig et al. (1995) and Anderson et al.(1999) formation of circulating immunecomplexes, parasite induced immunesuppression, nutritional status and antigenicvariation could contribute to the lowering ofantibody levels.

In the indirect ELISA for detection ofcoproantigens the cut-off point was 0.225.Using this criterion, 74 faecal samples werefound to be positive for coproantigens and thesensitivity of copro antigen ELISA was 74 percent. Here too, the sensitivity was lower, dueto the utilisation of somatic antigens whileAhmad and Nizami (1998) and Rahman et al.(1999) were able to obtain a higher sensitivityof 100 percent utilizing excretory /secretoryantigens of the parasites.

The results of ELISA using serumantibodies and coproantigens are comparedand presented in the table.

Fifty one animals were positive forserum antibodies and 74 for coproantigens outof 100, with known G. crumenifer infection.Forty three animals revealed both serumantibodies and coproantigens while 18 animalsdid not show either of them. Thirty one animalsthat were negative for serum antibodies werefound positive for coproantigens. But eightanimals that were negative for coproantigenswere found seropositive. The above resultsindicate the possibility of patent infectionssince 43 cattle had both the antigens andantibodies. It also signifies the advantages ofcoproantigens that they are devoid of immunecomplex formation and immune suppressionwhich naturally interfere with the detection ofantibodies. The observation that 8 animalsfound negative for coproantigens butseropositive suggests the possibility ofprevious exposure as remarked by Craig etal. (1995). The reason for 18 cattle foundnegative in both the tests reflects thelimitations of the assays as discussed above.

Table: Comparison of results of Serum antibody ELISA and Coproantigen ELISA

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References

Ahmad, G. and Nizami,W. A. 1998.Coproantigens: Early detection andsuitability of an immunodiagnosticmethod for echinococcosis in dogs.Vet.Parasitol., 77: 237-244

Anderson, N., Luong, T.T., Vo, N.G., Bui, K.L.,Smooker, P.M. and Spithill, T.W 1999.The sensitivity and specificity of twomethods for detecting Fasciolainfections in cattle. Vet. Parasitol.,83:15-24.

Craig. P. S., Gasser, B., Parada, l., Cabrera,P., Parietti, S., Borgues, C., Acuttis,A., Snowden, K. and Paolillo, E. 1999.Diagnosis of canine echinococcosis:comparison of coproantigen andserum antibody tests with arecolinepurgation in Uruguay. Vet. Parasitol.,56: 293-301

Dumenigo, B.E., Espino, A.M. and Finlay,C.M.1996. Detection of Fasciola hepaticaantigen in cattle faeces by a mono clonalantibody based sandwich immunoassay.Res. Vet. Sci. 60: 278-279

Hafeez, M.D. 1981. Studies on certainimmunological aspects of amphistomiasis of sheep and goats with special

�

reference to the effect of ionizing radiation.Ph.D thesis, Andhra Pradesh AgriculturalUniversity, Hyderabad. 300 p.

Jithendran, K. P., Vaid, J. and Krishna, L. 1996.Comparative evaluation of AGPT,CIEP and PHA tests for the diagnosisof Dicrocoelium dentriticum infectionin sheep and goats. Vet. Parasitol., 61:151-156

Johnson, M. J., Behnke, J. M. and Coles, G.C. 1996. Detection of gastrointestinalnematodes by a coproantigen captureELISA. Res.Vet. Sci., 60: 7-12

Kagan, I.G. and Norman, L. 1976.Serodiagnosis of parasitic diseases.In: Manual of Clinical Immunology.American Society for Microbiology.1976. pp. 382-409

Kandasamy, A. and Devada, K. 2011.Feasibility of coproantigen detectionin amphistomosis. J.Vet Anim.Sci.,42: In press

Rahman, S. M. A., Reilly, K. L. O. and Malone,J. E. 1999. Biochemical characterization and localisation of Fasciolahepatica 26-28 kDA diagnosticcoproantigens. Parasite Immunol., 21:279-286.

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Abstract

A cross sectional study was done tounderstand the effect of genotypes, farmmanagement and agro-climatic conditions inKerala and Uttar Pradesh on seroprevalenceof Mycoplasma mycoides Subsp. capri (Mmc)in goats. Goat serum was screened by SlideAgglutination test (SAT) for Mmc antibodies. Itwas found that there was no statisticallysignificant difference (P>0.05) in mycoplasmaseroprevalence between the two states andamong the genotypes-Malabari, Attapadi Blackand non-descript goats. However, the chi-square probability distribution revealed thatthere was significant difference (P>0.05) inseroprevalence in goats reared in governmentfarms than farmer herds and the likelihood ofgetting mycoplasmosis from farmer herds was3.40 than government farms.

Key words: Goat, Mycoplasma mycoidesSubsp.capri,seroprevalence,Slide Agglutination Test.

Mycoplasmas are responsible for avariety of diseases in domestic animals thatare mainly associated with ailments of thelung, genitourinary tract, joints, mammarygland and other tissues (Simecka et al. 1992).CCPP is a very severe disease condition ofgoats which is highly contagious and often fatalwith morbidity and mortality rates can reachup to 100% and 80%, respectively, in naïve

EFFECT OF GENETIC AND NON-GENETICFACTORS ON SEROPREVALENCE OFMYCOPLASMA MYCOIDES SUBSP. CAPRIIN GOAT

C. N. Dinesh1, A. Sonawane2, A.Kumar3, R. Rana4, A. Chauhan5 andD. Sharma6

Animal Genetics Division, Indian VeterinaryResearch Institute (IVRI),Izatnagar, Bareilly, Uttar Pradesh, India-243 122

herds. Food and Agricultural Organization(FAO) recognizes CCPP as a major trans-boundary livestock disease of goat and classifyit under OIE List B (Singh et al. 2007).

In India, many workers haveconducted seroprevalence studies onMycoplasma mycoides Subsp. capri (Mmc) insmall ruminants. A seroprevalence of 5.02 %in goats and 4.44 % in sheep was reported inHimachal Pradesh (Ramdeva et al. 2008). InNagpur district of Vidarbha region, 33.67%goats were found to be seropositive againstmycoplasma (Ingle et al. 2008). Outbreaks ofmycoplasmosis in sheep were also reportedin Andhra Pradesh (Kumari et al. 2011), ingoats in Gujarat (Kumar et al. 2011) and WestBengal (Mondal et al. 2004). These findingssuggest that mycoplasmosis is a major threatto the small ruminant production in India.

In this scenario, it was exploredwhether breed differences and agro-climaticand farm management conditions have anyrole on mycoplasma seroprevalence in goats.


Blood samples were collected fromAttapadi Black goats reared in the GovernmentGoat Farm, Agali, (Palakkad district) andMalabari goats maintained in the GovernmentGoat Farm, Kommeri (Kannur district), theGovernment Goat Farm, Agali (Palakkad

1. Assistant Professor, Department of Animal Genetics and Breeding, CVAS,Pookode2. Scientist3. Scientist4. Scientist5. Principal Scientist Bacteriology and Mycology Division, IVRI, Izatnagar, Bareilly, Uttar Pradesh, India-243 1226. Principal Scientist & Head

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district) and two farmer herds (located atCalicut and Wayanad districts) of Kerala andserum was separated and brought to IndianVeterinary Research Institute (IVRI), Izatnagar,Bareilly maintaining cold chain and stored at -20o till further processing.

From Uttar Pradesh, serumseparated from blood samples collected fromgoats reared in the Goat Farm of IVRI andfrom goats owned by farmers of nearbyvillages was used for the study. These bloodsamples were collected during a span of sevenmonths from July, 2012 to January, 2013. Allthe 247 serum samples were screened formycoplasma antibodies by Slide Agglutinationtest (SAT) with Mmc coloured antigen procuredfrom the National Referral Laboratory onMycoplasma, IVRI, Izatnagar. To describe thescreening test, in brief, 25µl each of sampleserum and coloured antigen were mixed on aclean glass plate using a glass rod followedby rotating the plate in clock wise and anticlockwise direction. Alongside, as positive control,25µl each of standard positive serum andcoloured antigen were also mixed. Then theresults were read against a source of light byobserving for the presence of agglutination inthe serum-antigen mix. Samples where flackswere formed within two minutes wereconsidered as positive for mycoplasmaantibodies; agglutination after two minutes wastaken as negative. Statistical analysis wasperformed using PROC FREQ procedure ofSAS 9.3 and ODDs ratios were calculated.

Results and DiscussionThe results of serum screening

are given in Table 1. The prevalence of Mmc

antibodies were 23.13 % and 31.03% amonggoat population from Kerala and UP,respectively and the difference in percentseroprevalence between the states was notsignificant (P>0.05). This shows that differentagro-climatic conditions prevailing in Keralaand UP have no effect on mycoplasmaseroprevalence. A comparable mycoplasmaseroprevalence of 28.92% was reported in thegoat population of Wayanad district in Kerala(Priya et al. 2008). However, a relatively lowmycoplasma seroprevalence of 9.64% wasreported in the goat population of UttarPradesh (Kashoo et al. 2011).

To elucidate the effect ofmanagement conditions on mycoplasmaprevalence, the data from government farmsand farmer herds in Kerala as well as in UttarPradesh were compared. In Kerala, aseroprevalence of 15.97% in goats reared ingovernment farms was significantly (Pd”0.05)lower than prevalence in farmer herds whichstood at 43.90%. Similarly, in Uttar Pradesh,a seroprevalence of 20% in government farmswas significantly (Pd”0.05) lower thanprevalence of 38.46% in farmer herds. It wasestimated that the ODDs of gettingseropositive cases for mycoplasmosis fromfarmer herds was 3.40 (1.88 to 6.14; 95% CI)as against government farms.

A seroprevalence study on CCPP inthe migratory goat flocks in India found that97 per cent of the goats were positive withLatex agglutination (LA) test and 78% werepositive with enzyme linked immunosorbentassay (ELISA) (Rana et al. 2009). A highseroprevalence of 62.5% was recorded in a

Table 1. Results of serum screening by SAT for Mmc antibodies

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40 Effect of genetic and non-genetic factors....

private farm in Kannur district of Kerala.(Ravishankar et al. 2011). The probablereason for a high level of prevalence in farmerherds could be due to the extensive methodof rearing that is popular among goat farmersand the consequent high chance for contactinginfection from infected and /or carrier animals.In addition, stress factors due to exposure tocold and wet conditions, malnutrition andmovement over long distances couldpredispose the animal to disease (Vihan 2010).As against this, a probable consistentminimum adherence to veterinary, hygienicand husbandry measures in the governmentfarms under the supervision of veterinariansreduce the possibility of contacting and/orspreading infection in the government farmsand hence the prevalence was low.

To evaluate the effect of genotypeon seroprevalence of mycoplasma, goatsreared under similar management conditionsi.e. intensive rearing under government farmsand extensive system at farmer’s door werecompared separately. Seroprevalence ofmycoplasma was 15.38%, 16.67% and 20%respectively, in Malabari, Attapadi Black andnon-descript goats reared in the governmentfarms. The chi-square probability distributionshows that there was no significant (P>0.05)genotype difference in the prevalence ofmycoplasma. There are several reports onbreed differences for mycoplasma infection ingoat as well as other livestock species.Kwantesav and Harbyb (1995) reported breeddifference in susceptibility to mycoplasmalarthritis among Batinah, Dhofari and JebelAkthar goats, while Abegunde et al. (1981)reported differences in vulnerability toMycoplasma putrefaciens infection in goatbreeds of California. Similarly, significantdifferences in susceptibility to Mycoplasmahyorhinis arthritis have been reported in PineyWoods Miniature swine and Yorkshire swineby Barden et al. (1973). Moreover, brownleghorns were found to be more susceptiblethan white varieties to amyloid arthropathyassociated with Mycoplasma synoviae(Landman and Bronneberg, 2003). However,our results of non-significant differences inMmc seroprevalence between differentgenotypes were contrary to these reports.Similarly, in the goats reared in the farmer’shouseholds, the seroprevalence ofmycoplasma was 43.9% in Malabari and38.46% in non-descript and this difference was

also non-significant statistically (P>0.05).

It was found out that the likelihood ofgetting mycoplasmosis in farmer herds was 3.40than government farms. This might be due tothe high chance for exposure to infected and /orcarrier animals and stress factors in the extensivemethod of rearing. Therefore, it could beconcluded that the managemental environmentas a whole played an important role in thedevelopment of mycoplasmosis in goat.

Acknowledgements

The authors gratefully acknowledgethe Director, IVRI, Izatnagar, Director, AnimalHusbandry Department, Kerala, Professor &Head, Livestock Production ManagementDivision, Professor & Head, Bacteriology andMycology Division and In-charge, NationalReferral Laboratory on Mycoplasma, IVRI,Izatnagar for the facilities provided for theconduct of this study.

References

Abegunde, T. O., Adler, H. E., Farver, T. B.,DaMassa A. J. 1981. A serologicsurvey of Mycoplasma putrefaciensinfection in goats. Am. J. Vet. Res.,42:1798-801.

Barden, J. A., Decker, J. L., Dalgard, D. W. andAptekar, R. G.1973. Mycoplasmahyorhinis Swine Arthritis III. ModifiedDisease in Piney Woods Swine. Infect.Immun., 8: 887-890.

Ingle, V. C., Sivakumar P., Kalorey, D. R., Pote,D. E., Dhamanna Patil, P. S. Chavhan,S. K., Nagdive, A. A. and Hatkar, D. N.2008. Seroprevalence of ContagiousCaprine Pleuropneumonia in goats inNagpur district of Vidarbha region. Vet.World., 1: 270-271.

Kashoo, Z. A., Singh, V. P., Rana, R., Sankar,M., Gazalli, H. and Cheema, P.S.2011. Evaluation of different serological tests for diagnosis of contagiousagalactia in goats. Indian J. Anim. Sci.,81: 1201-1203.

Kumar, P., Roy, A., Bhandari, B. B. and Pal, B.C. 2011. Isolation, identification andmolecular characterization of Mycoplasma isolates from goats of GujaratState, India. Vet. Arhiv. 81: 443-458.

Kumari, R., L., Muqueeth, M. A., Mrunalini, N.and Sudersan Rao, M. 2011.

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Mycoplasma Infections in SmallRuminants. Andhra Pradesh Veterinarian., 14:32-33.

Kwantesav, L. J. and Harbyb, H. A. M. 1995.Caprine mycoplasmal arthritis in theSultanate of Oman. Small RuminantRes., 16: 287-289

Landman, W. J. and Bronneberg, R. G. 2003.Mycoplasma synoviae-associatedamyloid arthropathy in WhiteLeghorns: case report. TijdschrDiergeneeskd., 128:36-40.

Mondal, D., Pramanik, A. K. and Basak, D. K.2004. Clinico-haematology andpathology of caprine mycoplasmalpneumonia in rain fed tropics of WestBengal. Small Ruminant Res., 51:285–295.

Priya, P. M., Baburaj, N. K., Kiran, N., Rajan,S., Ravishankar, C. and Jayesh, V.2008. A cross sectional investigationon contagious caprine pleuropneumonia among goats of Wayanaddistrict of Kerala, India. Biosci.Biotech. Res. Comm., 1:140-142.

Ramdeva, J., Sharma, Katoch, V. C., Dhar, P.and Chahota, R. 2008. Seroprevalance of mycoplasmosis in sheepand goats of Himachal Pradesh. IndianJ. Small Ruminants., 14: 276-278.

Rana, R., Mehra, S., Kumar, V., Kumar, A. andVihan,V.S. 2009. Seropreval ence ofcontagious caprine pleuropneumoniain goats using native M. mycoidessubsp. capri isolate. Indian Vet. J., 86:115-117.

Ravishankar, C., Priya, P. M., Ravindran, R.and Ajithkumar, S. 2011. Outbreak ofmycoplasmosis in an organized goatfarm in Kannur district of Kerala. J.Vet. Anim. Sci., 42: 66-67.

Simecka, J. W., Davis, J. K., Davidson, M. K.,Ross, S. E., Stadtlander, C. T. K. H.and Cassell, G.H. 1992. Mycoplasmadiseases of animals. In: Maniloff (Ed).Mycoplasmas-Biology and Pathogenesis. American Society for Microbiology, Washington.D.C : 391-396.

Singh, N., Singh, V. P. and Rana, R. 2007.Mycoplasma infections in goat andsheep in Indian perspective. In: V.S.Vihan, A. Kumar, V.K. Gupta, and N.P.Singh, (Eds) Small Ruminant-Emerging Diseases, Containmentunder WTO Regime. Sathish SerialPublishing House, Delhi: 241-247.

Vihan, V. S. 2010. Mycoplasmal and rickettsialDiseases. In: Diseases of SmallRuminant. Sathish Serial PublishingHouse, Delhi. 197-211.

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42 Ovulation synchronisation for improving fertility...


Abstract

The aim of the study was to evaluatethe different ovulation synchronisationprotocols for improving fertility in postpartumdairy cows. The work was conducted in 28lactating crossbred cows at UniversityLivestock Farm, Mannuthy starting on day 40postpartum (day 0) in three experimental andone control group comprising seven cowseach.The cows in Group I (Ovsynch group)were administered GnRH on day 0 followedby PGF2á on day 7 and a second dose of GnRHon day 9. Cows in Group II (Doublesynchgroup) were administered PGF2á on day 0,GnRH on day 2, a second PGF2á on day 9 anda second GnRH on day 11. Cows in Group III(CIDR group) were inserted with CIDR followedby administration of GnRH on day 0. The CIDRinsert was removed and PGF2á administeredon day 7 and a second dose of GnRH on day9. Timed artificial insemination (TAI) wasperformed 18 h after the second GnRHinjection in all experimental groups. Cows inGroup IV (Control group) that showed naturalpostpartum oestrus after day 0 wereinseminated at detected oestrus. Pregnancydiagnosis was conducted 45 days post AI bytransrectal palpation. The first serviceconception rate in animals of groups I to IVwere 42.86, 28.57, 42.86, 28.57 per cent andthe overall conception rates were 71.43, 71.43,85.71, 57.14 per cent, respectively. The resultsdemonstrated that the CIDR progesteroneinsert protocol with highest overall conception

OVULATION SYNCHRONISATION FORIMPROVING FERTILITY IN POSTPARTUMDAIRY COWS*

P. S. Shameem Abubaker1, M. O. Kurien2,K. N. Aravinda Ghosh3, Shibu Simon4,K. S.Anil5, B. Bibin Becha6

Department of Animal Reproduction,Gynaecology and Obstetrics,College of Veterinary and Animal Sciences,Mannuthy-680 651, Thrissur, Kerala

rate was superior to Ovsynch andDoublesynch protocols. However, Ovsynchand Doublesynch protocol had better overallconception rates than control. Hence it isrecommended that ovulation synchronisationprotocols viz., Ovsynch, Doublesynch andCIDR can be effectively employed forimproving the fertility in post partum dairy cows.

Key words: Ovulation synchronisation, postpartum dairy cows.

Synchronisations of ovulationprotocols to improve the fertility of the dairycows involve programmed folliculardevelopment, regression of corpus luteum andsequentially timed artificial insemination.Initially, the Ovsynch protocol was developedas a breeding strategy to eliminate the needfor oestrus detection. Later, the controlledinternal drug release (CIDR) protocol byinclusion of an exogenous progestogen wasdeveloped. Recently, a double synchronisation(Doublesynch) protocol by administering anadditional prostaglandin two days before theOvsynch protocol was developed. All theseprotocols produced varied success rates. Theprecise control of the oestrous cycle andovulation in cattle were achieved only whenboth, the life span of the corpus luteum andthe follicular wave status at the end of thetreatment were controlled.

This scenario clearly shows the needfor an in depth study of different ovulation

*Part of MVSc Thesis submitted by first author to the Kerala Veterinary and Animal Sciences University, Pookode, Wayanad 1MVSc Scholar 2Associate Professor, CVAS, Pookode 3Professor and Head 4Assistant Professor, University Veterinary Hospital, Kokkalai.5Associate Professor and Head, University Livestock Farm, Mannuthy. 6Assistant Professor.

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synchronisation strategies for increasing thereproductive efficiency in dairy cattle.With thisobjective the present research work usingdifferent ovulation synchronisation protocolsviz., Ovsynch, Doublesynch, CIDR progesterone insert was conducted.


The study was conducted in 28lactating crossbred cows at University LivestockFarm, Mannuthy starting day 40 postpartum(day 0) in three experimental and one controlgroup comprising seven cows each.

Experimental animals of Group I(Ovsynch group) were administered Buserelinacetate, a Gonadotrophin Releasing Hormone(GnRH) analogue, 20 µg intra muscular (i.m)on day 0 (Receptal®, Intervet, 10 ml vial)followed by Cloprostenol, a PGF2á analogue,500 µg i.m on day 7 (Cyclix®, Intervet, 2 mlvial) and a second dose of Buserelin acetate20 µg i.m on day 9.

Animals of Group II (Doublesynchgroup) were administered PGF2á analogueinjection on Day 0, GnRH analogue on Day 2,a second PGF2á analogue on Day 9 and asecond GnRH analogue on Day 11 at the samedose schedule and route mentioned in group I.

Animals of Group III (CIDR group)were inserted with CIDR intra vaginal device(1.38 g progesterone in elastic rubber moldedover a nylon spine, EAZI BREED®, Pfizer IndiaLtd) followed by administration of GnRHanalogue on Day 0. CIDR insert was removedand PGF2á analogue administered on Day 7and a second dose of GnRH analogue on Day9 at the same dose schedule and routementioned in group I.

TAI was performed 18 h after thesecond GnRH injection in all threeexperimental groups. Animals of Group IV(Control group) that showed natural oestrusafter 40 days postpartum were inseminatedat detected oestrus.

Pregnancy was confirmed in all thegroups 45 days post AI by rectal examination.Conception rate with respect of first serviceand overall conception rate were determined.The overall conception rate was calculated aspercentage of animals pregnant in threeconsecutive AI out of the number of animalstreated in each group.


The first service conception rate

observed in animals of experimental groups Ito III were 42.86, 28.57 and 42.86, respectivelyand the corresponding values in control groupIV was 28.57 per cent. The overall conceptionrate observed in this study in animals ofexperimental groups I to III were 71.43, 71.43and 85.71, respectively and the correspondingvalues in control group IV was 57.14 per cent.(Table). Statistical analysis revealed that nosignificant difference in the first service andoverall conception rates among groups.

The first service conception rate wassimilar in Groups I and III and higher whencompared with Group IV whereas the firstservice conception rate was similar in Group IIand Group IV. The overall conception rate washigher in group I, II and III and highest being ingroup III when compared with Group IV.

The first service conception rate of42.86 per cent obtained in the Ovsynch groupin present study concurred with the findingsVijayarajan et al. (2009) who reportedconception rate of 50 per cent in postpartumdairy cows. However, relatively higherconception rates of 55 to 90 per cent werereported in cows by Ansari et al. (2008) andSathiamoorthy and Kathirchelvan (2010). Onthe contrary, lower conception rates of 30 to 40per cent in cows were reported by Dagli et al.(2008) and Ghallab et al. (2009). The possiblereason for the variation could be thereproductive status or stage of oestrous cycleat the beginning of the protocol in addition tothe variations due to nutrition, management,lactation, drug, season, age, breed and species.

The first service conception rates of28.57 per cent obtained in Doublesynch groupconcurred with the findings of Cinar et al.(2012). However, higher conception rate of 71per cent was reported by Ozturk et al. (2010).

The conception rate of 42.86 per centobtained in the CIDR group in present studyconcurred with the findings of Sathiamoorthyand Kathirchelvan (2010) and Cevik et al.(2010) who reported 42.74 and 53.3 per centconception rate, respectively. Contrary to this,higher conception rates of 69 to 80 per centwere observed in dairy cows by Tauck et al.(2007) and Aali et al. (2008). However,Chenault et al. (2003) reported a relativelylower conception rate of 32.7 per cent wasrecorded in dairy cows.

The highest overall conception rateobtained in CIDR group concurred with the

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findings of Ammu et al. (2012) who reported83.33 per cent overall conception rates. El-Zarkouny et al. (2004); Stevenson et al. (2006)and Bollwein and Lüttgenau (2013) reportedimproved conception rates when CIDR wasincluded in the Ovsynch protocol. On thecontrary, Lima et al. (2009) reported nodifference in conception rates when CIDR wasincluded in the Ovsynch protocol. However,Galvao et al. (2004) reported reducedconception rate when CIDR was included inOvsynch protocol.

The overall conception rate of 71.43per cent obtained in Ovsynch group concurredwith the findings of Ammu et al. (2012) whoreported 66.66 per cent overall conceptionrate. On the contrary, lower overall conceptionrate of 35 to 50 per cent were reported inOvsynch group by Aali et al. (2008) and higheroverall conception rate of 90 percent wasrecorded by Muneer et al. (2009) in dairy cows.The overall conception rate of 71.43 per centobtained in Doublesynch group concurred withthe findings Ozturk et al. (2010) who reported72.8 per cent overall conception rate.

Thus, the results demonstrated thatthe CIDR progesterone insert protocol withhighest overall conception rate was superiorto Ovsynch and Doublesynch protocols whichindicated that inclusion of progesterone duringthe period between GnRH and PGF2á

prevented premature ovulation afterspontaneous luteolysis during the treatmentperiod in cows whose dominant follicles failedto respond to GnRH. However, Ovsynch andDoublesynch protocol had better overallconception rates than control.

Hence it is recommended thatovulation synchronisation protocols viz.,Ovsynch, Doublesynch CIDR can beeffectively employed for improving the fertilityin post partum dairy cows.

References

Aali, M., Pretheeban, T., Giritharan, G. andRajamahendran, R. 2008. Pregnancyrates and peripheral progesteronelevels following Ovsynch or CIDRovulation synchronisation/timedartificial insemination protocols inpostpartum dairy cows. Can. J. Anim.Sci. 88: 457-461.

Ammu, R., Dhami, A. J., Naikoo, M., Parmar,B.C. and Divekar, B. S. 2012. Oestrusinduction and fertility response inpostpartum anoestrus gir cows. IndianJ. Anim. Reprod. 33: 37-42.

Ansari, S. M. A., Rao, K. S. and Raju, K. G. S.2008. Studies on post-partumanoestrus with special emphasis oninduction of oestrus in crossbredcows. Proc. of 24th Annu. Conventionof ISSAR and Nati. Symp., 11th to 13th

December, 2008, Bangalore.KVAFSU. p.23.

Bollwein, H and Lüttgenau, J. 2013. Lowprogesterone levels—A cause for lowfertility in dairy cattle. Reprod. Biol.139 (Supp.2): 2

Çevik, M., Selcuk, M. and Dogan, S. 2010.Comparison of Pregnancy Rates afterTimed Artificial Insemination inOvsynch, Heatsynch and CIDR-Based Synchronisation Protocol inDairy Cows. Kafkas Univ.Vet. Fak.Derg. 16: 85-89.

Chenault, J.R., Boucher, J.F., Dame, K.J.,Meyer, J.A and Wood-Follis, S.L.2003. Intra vaginal progesteroneinsert to synchronize return to estrusof previously inseminated dairycows. J. Dairy Sci. 86: 2039–2049.

Çinar, M., Aydýn Güzeloðlu and HüseyinErdem. 2012. Effect of presence ofcorpus luteum at the beginning of

Table. First service conception rates (%) and Overall conception rates (%) in postpartumcrossbred dairy cows treated with different ovulation synchronisation protocols

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Ovsynch protocol on pregnancy ratesin lactating dairy cows. Kafkas Univ.Vet. Fak. Derg. 18: 513-516.

Dagli, N.R., Gulawane, S.V. and Bakshi, S. A.2008. Use of Ovsynch an effectivereproductive managemental tool fora dairy farm. Proc. of 24th AnnuConvention of ISSAR and Natl. Symp,11th to 13th December, 2008,Bangalore. KVAFSU. p.62.

El-Zarkouny, S. Z., Cartmill, J. A., Hensely,B. A. and Stevenson, J.S. 2004. Pregnancyin dairy cows after synchronized ovulationregimens with or without presynchronisation and progesterone. J. Dairy Sci. 87:1024-1037.

Galvão, K. N., Santos, J. E. P., Juchem, S.O., Cerri, R. L. A., Coscion, A. C. andVillaseñor, M. 2004. Effect of additionof a progesterone intravaginal insertto a timed insemination protocol usingestradiol cypionate on ovulation rate,pregnancy rate, and late embryonicloss in lactating dairy cows. J. Anim.Sci. 82: 3508-3517

Ghallab, A. M., Moghney, A. F. A. and Osman,R. H. 2009. Efficacy of Ovsynchprotocol on pregnancy in lactatingdairy cattle during summer season.Vet. Med. J. 57: 193-202.

Lima, J. R., Rivera, F. A., Narciso, C. D., Oliveira,R., Chebel, R.C. and Santos, J. E. P.2009. Effect of increasing amounts ofsupplemental progester one in a timedAI protocol on fertility of lactating dairycows. J. Dairy Sci. 92: 5436-46.

Muneer, S., Rao, K. S. and Raju, K. G. S. 2009.Efficacy of GnRH- PGF2á -GnRH,PMSG and PMSG + hCG inpostpartum anestrous crossbred cows.Indian J. Anim Reprod. 30: 7-9.

Ozturk, O. A., Cirit, U., Baran, A. and Ak. K.2010. Is Doublesynch protocol a newalternative for timed artificialinsemination in anestrous dairy cows.Theriogenology. 73: 568-576

Sathiamoorthy, T. and Kathirchelvan, M. 2010.Efficacy of PGF2á, CIDR and Ovsynchtreatment on oestrus response andfertility rate in crossbred cows. IndianJ. Anim. Reprod. 31: 43-45.

Stevenson, J. S., Pursley, J. R., Garverick,H. A., Fricke, P. M., Kesler, D. J.,Ottobre, J. S. and Wiltbank, M. C.2006. Treatment of cycling andnoncycling lactating dairy cows withprogesterone during Ovsynch. J.Dairy Sci. 89: 2567-2578.

Tauck, S.A., Wilkinson, J. R. C., Olsen, J. R.and Berardinelli, J.G. 2007. Comparison of using 7-d or 14-d CIDRtreatments in an estrous synchronisation protocol that included PGF2á,TAI and GnRH in primiparous, suckledbeef cows. Proc. Western Section,Am. Soc. Anim. Sci. 58: 271-273.

Vijayarajan, A., Chandrahasan, C. and Napolean,R.E. 2009. Synchroni sation of ovulationin repeat breeding crossbred cows.Indian J. Field Vet.,5: 57-58.

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46 Training needs of dairy farm instructors in fodder...


Abstract

The study was carried out on thetraining needs of Dairy Farm Instructors (DFIs)of the Dairy Development Department ofKerala. The data were collected from a sampleof 75 DFIs by means of structuredquestionnaires. Knowledge and skill needswere assessed in the subject matter area offodder production and management.Knowledge (78.99) and skill (78.99) needs offodder production ranked first followed byknowledge (77.55) and skill (76.44) needs offodder preservation. Under fodder production,knowledge (82.66) and skill (80.88) needs offodder disease management and fertilizerapplication ranked first. The most preferredtype of training was institutional learning(58.67%). Practice in demonstration (76.00%)was the most preferred method of training.Experts from outside the parent organizationwere the most preferred trainers (58.64%).The most preferred durations for short termand long term training programmes were oneto seven days (76%) and fifteen days to onemonth (48%) respectively.

Key words: Training needs, Dairy FarmInstructors, Dairy Development Department,Dairy extension, Training strategy.

One of the major constraints to dairyproduction in Kerala is the shortage of qualityfodder. The marginal and small farmers who

TRAINING NEEDS OF DAIRY FARMINSTRUCTORS IN FODDER PRODUCTIONAND MANAGEMENT *

N. Vimal Raj Kumar1, R.S.Jiji2

andP.J.Rajkamal3

Department of Veterinary &Animal Husbandry Extension,College of Veterinary and Animal Sciences,Mannuthy-680 651, Thrissur, Kerala, India

are the predominant cattle owners of the statehave either limited or no land at all for fodderproduction. The lack of fodder and high cost ofcattle feed result in increased cost of milkproduction. Realizing this precarious situation,the State Dairy Development Department (DDD)gives thrust to the implementation of fodderpromotion schemes. The Dairy Farm Instructors(DFIs) of the department being the educatorsand change agents at the block level need to beupdated with advances in fodder production andpreservation technologies through regular in-service training programmes.

The present study was thereforedesigned to identify the perceived trainingneeds of the Dairy Farm Instructors in thesubject matter area of fodder production andmanagement and also to explore the trainingstrategy preferred by the respondents.


At the time of data collection, 120DFIs were actually in position with the DDDand structured questionnaires were either sentto them by post or distributed in person duringthe district level monthly meetings. Out ofthem, 75 DFIs returned the filled inquestionnaires within the stipulated period ofone month. Hence the sample of the studycomprised of 75 DFIs.

Determination of training need

In the present study, training need

* Part of M. V. Sc. thesis submitted by the first author to the Kerala Agricultural University, Thrissur1. Assistant Professor, Department of Veterinary and Animal Husbandry Extension, Madras Veterinary College, Veppery,Chennai, Tamilnadu – 6000072. Associate Professor3. Professor and Head

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was operationalised as the perceived trainingneeds of the DFIs, obtained in a checklist ofitems in the subject matter area of fodderproduction and management under whichselected items to assess the knowledge needsof the respondents and those to assess theskill needs were given separately. Therespondents were asked to rate both theknowledge and skill need items separately ona three point continuum viz., required,somewhat required and not required withscores of three, two and one respectively.

The Training Need Index (TNI) for each itemwas calculated using the formula, Sum ofscores obtained for an

TNI of an item = item by all the respondents———————— X 100Maximum possible score for the item

The items were ranked based on thetraining need indices.

Training strategy preferredThe respondents were asked to

mention the type, method, trainers, durationand venue of training they preferred the mostfrom among the given categories, for thesubject matter area of fodder production andmanagement and preference ranking wasdone accordingly.


1. Perceived Training needs

Table 1 illustrates the perceivedtraining needs of the DFIs with regard toknowledge and skills in the subject matter areasof fodder production and fodder preservation.

For both knowledge and skill needs, fodderproduction stood first followed by fodderpreservation. Under the domain of fodderproduction, fodder disease management andfertilizer application received top priority for bothknowledge and skill needs. With regard to thedomain of fodder preservation, hay makingreceived precedence.

The fodder production programmesimplemented by the Department of DairyDevelopment envisage training to farmers infodder production and management as apriority area. Lack of scientific knowledge offodder cultivation was viewed as the majorconstraint by 40 per cent of rural respondentsas per a study carried out among the livestockfarmers of Belgaum district of Karnataka byPushpa (2006). In a study conducted in theMahabubnagar and Anantapur districts ofAndhra Pradesh and Tumkur district ofKarnataka, Misra et al (2007) found thatthrough exposure visits and farmer-to-farmerinteraction, many farmers realized thatintegration of livestock and fodder productionwithin their limited land and water resourcesprovided a better livelihood option in dry lands.

As the training sessions for farmers infodder production are dealt by the Dairy FarmInstructors, they need to be equipped withadvanced knowledge and skills in this realm.Training need analysis among the DFIs in termsof both knowledge and skill requirements revealhigher preference for fodder production thanpreservation. Further, under fodder production,disease management and fertilizer applicationwas assigned the topmost priority as against the

Table.1. Training needs of Dairy Farm Instructors in the domain of fodder production andmanagement

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basic cultivation practices that received the leastpriority. Probably, the respondents were confidentof their knowledge and skills pertaining to thebasic cultivation practices. Higher preference fordisease management and fertilizer applicationtraining might be attributed to the devastatingimpact of fodder diseases, pest incidences andenvironmentally harmful pesticides and fertilizerson smallholder dairy production. The trainingneed in the subject matter area of fodderproduction and management for extensionpersonnel is in accordance with that of Saini andSandhu (1993).

2. Preferred training strategy

a) Type of training, method andtrainers

It is obvious from Table 2 thatinstitutional learning was the most preferredtype of training followed by integrated learning.The most preferred training method waspractice in demonstration followed by studytour, workshop, lecture and seminar. Expertsfrom outside the parent organization but withinthe state were preferred the most as trainers.

The preference of the respondents forinstitutional training might be attributed to theopportunities for interactive and face to facelearning. This finding is in agreement with thatof Sakthivel (2001) but in contrast with that ofPatil and Kokate (2011).

It could be noted that practice indemonstration was the most preferred methodof training by the respondents. This might bedue to their appreciation of the demonstrationtechnique as an effective tool in skill teaching.This finding is in accordance with that of

Sudeepkumar and Subramanian (1993). Themethod of study tour ranked second. Therespondents might have perceived theopportunities to visit professional institutes,research stations, farms and dairy plantselsewhere that would widen their practicallearning experience. The preference for studytours by trainees was also reported bySudeepkumar and Subramanian (1993) andMathiyalagan and Subramanian (1998).Workshop as a method of training wasassigned the third rank. Perhaps, workshopmight have been perceived as a better meansto exchange ideas, experiences and skills thatwould in turn help the participants to produce aproduct, prepare a document, report orprogramme for future action. In a descriptivestudy probing into the in-service training needsof extension agents in West Iran, Alibaygi andZarafshani (2008) observed that cooperativelearning techniques were the most preferredtraining methods (50%), followed by workshops(25.55%), group discussions (15.45%), andlectures (9%). It was interesting to note thatrole play was the least preferred method oftraining. This might be because the respondentsmight have perceived role play as embarrassingsince they were required to act roles.

Regarding preference for trainers,most of the respondents liked to invite trainersfrom outside the parent organization but withinthe state. The respondents might haveprobably felt that interacting with experiencedpersons from outside the parent organizationwould be beneficial. This finding is inagreement with that of Naik (1982) andSakthivel (2001).

Table. 2. Type of training, method and trainers preferred in the domain of fodder production andmanagement

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b) Duration of Training

Data in Table 3 shows that more thanthree fourth of the respondents were in favourof short term residential training programmesof 1-7 days duration for which the respondentwould probably have to stay away from his/her home. Almost half of the respondents werein favour of long term residential trainingprogramme of 15 days to one month durationfor which they would have to stay away fromhome sometimes.

Duration is an important criterion forthe success of any training programme. It isessential that the duration of training isadequate to deliver the content of training. Itshould be convenient to the trainees as well.The finding that most of the respondentspreferred a duration of one to seven days fora short-term training programme and fifteendays to one month for a long-term trainingprogramme deserves consideration whiledeciding upon the duration of training. Thisfinding is in accordance with those of Bhagat(1989), Sudeepkumar and Subramanian(1993) and Khan et al (2011).

c) Venue of Training

Table 4 shows that the most preferredvenue for training in fodder production andmanagement was Kerala AgriculturalUniversity followed by identified centres ofDairy Development Department (DDD), KeralaCooperative Milk Marketing Federation(MILMA), Kerala Livestock DevelopmentBoard (KLDB) and premier institutes outsideKerala such as Tamil Nadu AgriculturalUniversity (TNAU) and Indian AgriculturalResearch Institute (IARI). None preferred anyinstitute other than these.

The physical facilities andenvironment of training institutes haveconsiderable influence on the learningexperience of trainees. The selection of avenue with all the required facilities such assuitable physical environment, teaching aidsand resource persons is essential for thesuccess of the training programme. The findingsreveal that most of the respondents preferredinstitutes within Kerala. Attending trainingprogrammes outside their home state may not

Table 3. Preferred training duration

Table. 4. Preferred venue of training

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be convenient in view of domestic obligations.This finding is in agreement with those of Naik(1982), Bhaghat (1989) and Mani (1996).

Acknowledgements

The financial support in the form ofJunior Research Fellowship extended by theICAR for the conduct of this research work isprofoundly acknowledged herewith. Also, theauthors are thankful to the Dean, College ofVeterinary and Animal Sciences, Mannuthy,Thrissur, Kerala for the facilities provided.

References

Adhikari, S. 2003. Training needs of frontlineextension workers of mid-westerndevelopment region of Nepal. M. Sc.Thesis, Department of AgricultureExtension and Rural Sociology, IAAS,Rampur, Chitwan, Nepal. 80p.

Alibaygi, A. and Zarafshani, K. 2008. Trainingneeds of Iranian extension agentsabout sustainability: The use ofBorich’s need assessment model.AJAR., 3 : 681-687.

Bhagat, G. R. 1989. A study on training needsof Horticulture Inspectors in PunjabState.M.Sc (Ag.) thesis, PunjabAgricultural University, Ludhiana.125p.

Halim, A. and Islam, A. F. M. S. 1973. Attitudeof the Front Line Extension Workerstowards in-service training programme. Indian J. Extn. Edn., 9: 108-114.

Khan, M. Z., Haq, Z. U., Khan, N. U., Pervez,U. and Khan, M. A. 2011. Trainingneeds of Agricultural extension agentsin Khyber Pakhtunkhwa. Sarhad J.Agric., 27: 133-137.

Mani, S. 1996. A study on training needs ofAOs under Tamil Nadu AgriculturalDevelopment Project (TNADP). M.Sc.(Ag.) thesis, Tamil Nadu AgriculturalUniversity, Coimbatore. 115p.

Mathiyalagan, P. and Subramanian, R. 1998.Perception of training strategy bypoultry farmers and extensionpersonnel. Cheiron., 27: 55-58.

Misra, A K., Rama Rao, C. A., Subrahmanyam,K.V., Vijay Sankar Babu, M.,Shivarudrappa, B. and Ramakrishna,

Y. S. 2007. Strategies for livestockdevelopment in rain fed agro-ecosystem of India. Livest Res forRural Dev.,19: Article #83 retrievedfrom http://www.lrrd.org/lrrd19/6/misr19083.htm.

Naik, S. 1982. A critical analysis of in-servicetraining needs of the AssistantAgricultural Officers of Karnataka.M.Sc. (Ag.) thesis, University ofAgricultural Science, Dharwad.116P.

Patil, S. S. and Kokate, K. D. Training NeedAssessment of Subject MatterSpecialists of Krishi Vigyan Kendras.Indian Res. J. Ext. Edu., 11: 18-22.

Pushpa, P. 2006. A study on livestockproduction systems of rural andperiurban livestock owners. M.Sc.(Ag.) thesis, University of AgriculturalScience, Dharwad.104p.

Reddy, A. A. and Reddy, K. J. 1966. Criteriafor the training needs of AgriculturalExtension Officers. Indian J. Extn.Edn., 2: 59-65.

Sakthivel, K. M. 2001. Analysis of the trainingneeds of Veterinary Surgeons ofKerala for continuing veterinaryeducation. M.V.Sc. thesis, KeralaAgricultural University, Thrissur.140P.

Sandhu, A. S. and Bilang, K. S. 1977. Trainingneeds of Agricultural Extension Officers.Indian J. Extn. Edn., 13: 71-73.

Sharma, D. and Shukla, A. N. 1986. Trainingneeds of Agricultural ExtensionOfficers of Punjab. Indian J. Extn.Edn., 22: 80.

Singh, B. N. and Singh, A. P. 1966. In-servicetraining needs of AgriculturalExtension Officers. Indian J. Extn.Edn., 2: 17-21.

Sudeepkumar, N. K. and Subramanian, R.1993. An analysis on the trainingneeds preference of dairy trainees.J. Extn. Edn., 4: 726-729.

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Abstract

Fecal Egg Count Reduction Test(FECRT) was employed for the in vivodetection of anthelmintic resistance toalbendazole and ivermectin in small scale goatrearing units in VallachiraPanchayath ofThrissur District, Kerala. Resistance wasdetected against albendazole with per cent eggcount reduction of 71 per cent and lower 95per cent confidence interval less than 90.Ivermectin was found effective with per centegg count reduction of 97 per cent. Resistanceto albendazole can be attributed to theprolonged and intensive use of the drug. Theuse of ivermectin orally for deworming hasbeen introduced recently and the drug is stillefficacious. Detection of resistance in smallholder farmers’ flocks warrants judicious useof these drugs to prevent further selection forresistance. Haemonchus contortus was thepredominant species among gastrointestinalnematodes identified by coproculture and thisfactor also contributed to the rapid selectionfor resistance.

Key words:- Anthelmintic resistance, FECRT,goats, Haemonchus contortus

Anthelmintic resistance has emergedas a major problem hampering the successfulcontrol of gastrointestinal nematodes in smallruminants. There have been several reportsof anthelmintic resistance from different statesin India (Dhanalakshmi et al. 2003;Jeyathilakan et al. 2003; Deepa and Devada,2007; Easwaran et al. 2009 and Buttar et al.2012), mainly from organized sheep or goatfarms. Reports of anthelmintic resistance from

DETECTION OF ANTHELMINTICRESISTANCE IN SMALL SCALE GOATREARING UNITS IN THRISSUR

Asha Rajagopal1, R. Radhika2,H. Shameem3 and K. Devada4

Department of Veterinary Parasitology,College of Veterinary and Animal Sciences,Mannuthy-680651,Thrissur, Kerala.

1,2&3Assistant Professor4Professor & Head

small holder farmers’ flocks are rare. Effectivemonitoring of resistance is vital in order tomaintain the efficacy of the currently availableanthelmintics and to prevent further selectionfor resistance. The present study reports in vivodetection of anthelmintic resistance in smallgoat rearing units in Vallachira Panchayath ofThrissur District in Kerala.


The study was conducted in adjacent,small holder farmers’ flocks with flock size of15 to 30 animals, maintained under identicalmanagemental and feeding practices inVallachira Panchayath. The farmersadministered anthelmintics without properveterinary advice and did not follow any regulardeworming schedule.

Fecal egg count reduction test(FECRT)was performed for detection ofresistance to the two commonly usedanthelmintics, albendazole and ivermectin as perthe guidelines of WAAVP (Coles et al.2006).Thirty kids, aged three to six months which havenot been dewormed eight weeks prior to thestudy, were randomly allocated into three groupsof ten animals each. Rectal samples werecollected from all the animals and they weregiven recommended dose of anthelmintics.Group I was given albendazole (Valbazen,Pfizer)@ 5 mg/kg orally; group II – ivermectin(Neomec, Intas)@ 0.2 mg/kg orally and groupIIIwas left untreated as the control group.

Rectal samples were again collectedon day 14 post treatment. Egg counts weremade on the pretreatment and post treatment

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52 Detection of anthelmintic resistance in small scale goat ...

samples by Modified Mc master technique.The data wereanalysed statistically for findingout the per cent reduction in egg counts usinga programme, RESO. Reduction in egg countsof less than 95 per cent with lower 95 per centconfidence limit less than 90 was consideredas indicative of resistance against the drug(Coles et al. 1992).

Copro culture was done on pooled pretreatment samples and post treatmentsamples for identifying the species of infectingnematodes. Mature third stage larvae wereidentified based on morphological characters(Van Wyk et al.2004).


Results of FECRT are presented in thetable. Resistance was detected againstalbendazole while ivermectin was found effective.

The results indicate the developmentof resistance against albendazole in smallholder farmers’ flocks in Kerala. Resistanceto albendazole could be attributed to theprolonged and intensive use of the drug overthe years. The drug is being widely used bythe farmers for deworming their livestock evenwithout proper veterinary advice, often leadingto underdosing.

In this study, ivermectin was foundeffective with a per cent egg count reductionof 97 per cent. This can be attributed to thefact that the use of oral ivermectin fordeworming has been introduced only recentlyand its use is not widespread. However thereare increasing reports of development ofresistance to the drug in farms in the recentyears (Deepa and Devada, 2007; Buttar et al.2012). This warrants the judicious use of thedrug to maintain its efficacy.

Previous reports of anthelminticresistance are mainly from organized farms

with intensive anthelmintic treatmentschedules. Existence of drug resistant GInematodes in breeding animals in farmsincreases the risk of dissemination of resistantstrains to small holder farmers’ flocks as farmbred animals are distributed to farmers(Easwaran et al.2009). Reports of anthelminticresistance from small holder farmers’ flocksare rare or uncommon, but if the present useof anthelmintics is continued, the situation canbecome unmanageable (Harikrishnan, 2012).Thus the detection of anthelmintic resistancein small holder farmers’ flocks is significantand warrants implementation of properanthelmintic treatment strategies to checkfurther development of resistance.

GInematode species identified bycoproculture was predominantly Haemonchuscontortus(85%), followed by Trichostrongylussp. (8%) and Strongyloides sp. (7%). Highbiotic potential of GI nematodes especiallyH.contortus contributes to rapid selection forresistance as large number of generations ofworms are produced within a short time(Deepa and Devada, 2007). Since H. contortuswas the predominant GI nematode speciesobserved in this study, this factor also mighthave contributed to the selection for resistance.

In conclusion, the detection ofanthelmintic resistance in GI nematodes ofgoats necessitates implementation of urgentmeasures to slow down the development ofresistance. Farmer perception regarding thejudicious use of anthelmintics should beincreased through extension activities.Periodical screening of flocks for resistanceby FECRT is also recommended.

Acknowledgements

The authors acknowledge the AnimalHusbandry Department, Kerala for funding the

Table: Results of FECRT

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study through the project on ‘Detection ofanthelmintic resistance in GI nematodes of goats’.

References

Buttar, B. S., Hari H. S. , Singh,N. K., Jyoti, M. Haque and Rath, S.S. 2012. Emergence of anthelminticresistance in an organized sheep farmin Punjab. J.Vet. Parasitol. 26: 69-71.

Coles, G. C., Bauer, C., Borgasteede, F. H. M.,Geerts S., Klei, T. R. Taylor, M. A. andWaller, P.J. 1992. World Associationfor the Advancement of VeterinaryParasiotology (WAAVP) methods fordetection of anthelmintic resistance innematodes of veterinary importance.Vet. Parasitol. 44:35-44.

Coles, G. C., Jackson F., Pomroy W. E.,Prichard R. K., Himmel Stjerna,Silvestre, A, Taylor, M. A., andVercruysse, J. 2006. The detection ofanthelmintic resistance in nematodesof veterinary importance. Vet.Parasitol. 136.167-185.

Deepa,C.K and Devada, K. 2007. Anthelminticresistance in GI nematodes of goats.J.Vet. Anim. Sci.38: 52-54.

Dhanalakshmi, H., Jagannath, M. S. andD’Souza, Placid E. 2003. Multipleanthelmintic resistance in GI nematodesof sheep. J.Vet. Parasitol. 17(2): 89-91.

Easwaran,C., Harikrishnan, T. J., Raman, M.2009. Multiple anthelmintic resistancein GI nematodes in South India. VetArhiv. 79:611-620.

Harikrishnan, T. J. 2012. Management ofanthelmintic resistance in GIhelminthes of sheep and goats inTamil Nadu- the way forward. Proc.,International conference on HolisticApproaches for combating anthelmintic resistance–An update inParasito logy.46-53.

Jeyathilakan, N., Radha G., Gomathinayagam, S. and John, L. 2003.Emergence of anthelmintic resistancein nematodes of sheep in Tamil nadu.J.Vet.Parasitol. 17:159-160.

Van Wyk, J. A., Cabaret J. and Michael, L.M.2004. Morphological identifi cation ofnematode larvae of small ruminantsand cattle simplified. Vet Parasitol. 119:277-306.

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54 Gross anatomical studies on the lungs of wild sow


Abstract

A study was conducted on the lungsof an adult wild sow collected soon after itsdeath. The lungs were bluish pink in colour withdistinct lobulations. Each lung was covered bypulmonary pleura and presented an apex,base, two surfaces and three borders. Theright lung was larger than the left lung. Itcontained four lobes namely- cranial, middle,caudal and accessory lobes. The left lungpresented a divided cranial lobe and a caudallobe. A small tracheal bronchus originated fromthe trachea and entered the undivided craniallobe of the right lung. Apart from the apicalbronchus, the right lung presented middle,accessory and caudal lobar bronchi, ventilatingcorresponding lobes. The cranial and caudallobar bronchi entered the cranial and caudallobes of the left lung respectively. It wasconcluded that the gross anatomical featuresof the lungs of wild sow were similar to thoseof domestic pig.

Key words:- Gross anatomy, lungs, wild sow

Wild pigs are considered as theancestors of domestic pigs. The number of thewild pigs is fast diminishing as a result ofpoaching and the loss of habitat. The lungsare principal structures of the respiratorysystem and contain special phagocytic cellsthat scavenge the foreign materials enteringthe organ. The comparable volumes of porcineand human lungs are thought to beadvantageous for the study of respiratoryfunction (Jones et al., 1975). According toRendas et al. (1978), the vascular circulation

GROSS ANATOMICAL STUDIESON THE LUNGS OF WILD SOW(Sus scrofa cristatus)

A. R. Sreeranjini1, C.V. Rajani2,V. R. Indu3 and N. Ashok4

Department of Veterinary Anatomy and Histology,College of Veterinary and Animal Sciences,Pookode - 673 576, Wayanad, Kerala.

1&3. Assistant Professors, CVAS, Mannuthy, Thrissur2. Assistant Professor4. Professor, CVAS, Mannuthy, Thrissur

of both humans and pigs has a commonpattern of organization and development.Riquet et al. (2000) reported that the lymphaticdrainage of lungs in domestic pigs is similarto that of human beings.

Reports pertaining to the grossanatomical features of lungs in wild pigs arescanty and hence the present study wasundertaken with an attempt to bridge the gap.


The study was conducted on the lungscollected from an adult wild sow brought forpost mortem examination to the Departmentof Pathology, College of Veterinary and AnimalSciences, Pookode, Wayanad. Both lungswere collected, cleaned and preserved in 10%neutral buffered formalin for studying themorphological features.


The lungs of the wild sow were bluishpink in colour with distinct lobulations (Fig.1).Mc Laughlin et al. (1961) observed that porcineand human lungs were characterized by welldeveloped lobularity with each lobuledemarcated by relatively thick, collagenousinterlobular septa. Each lung was covered bythe pulmonary pleura and presented an apex,base, two surfaces and three borders. Apexwas directed cranially and in right lung it waslarger than that of the left as in domestic pig(Getty, 1975). The caudally placed base wasconcave with the concavity directed towardsdiaphragm. The lateral or costal surface wasconvex and was more extensive than the

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medial surface. The dorsal border was thickand rounded while the ventral border was thinand irregular, with deep fissures. The basalborder was thick and rounded dorsally and thinand sharp ventrally.

The right lung was larger than theleft lung similar to the findings of Dyce et al.(1996) in domestic pig. It was divided into fourlobes, viz. cranial, middle, caudal and

accessory lobes as reported by Cabral et al.(2001) in wild boar. The cranial lobe was fairlylarge and undivided (Fig.2). The middle lobewas pyramid shaped and was separated fromcranial and caudal lobes by moderately deepfissures. The caudal lobe was the largest andwas related cranially to cranial and middlelobes. The accessory lobe was attached to themedial surface of the caudal lobe posterior tothe hilus of the lung. This lobe was irregularlytriangular with a blunt anterior apex and adeeply concave posterior base unlike thefindings of Getty (1975) in domestic pig wherethe accessory lobe was pyramidal in shapewith slightly concave base. Through the hilus,bronchi, blood vessels, lymph vessels andnerves entered the lungs as observed in otherdomestic animals (Nickel et al.,1979).

The left lung presented a dividedcranial lobe and a caudal lobe. Its cranial lobewas L- shaped as seen in domestic pig (Getty,1975). The larger caudal lobe was placedposterior to the cranial lobe. The cranial andcaudal bronchi entered the cranial and caudallobes respectively. Contrary to this, Nakakuki(1994) observed that the left lung of domesticpig was having a bilobed middle lobe and acaudal lobe.

A small tracheal bronchus originatedfrom the trachea and entered the undividedcranial lobe of the right lung (Fig.3). Two inchesposterior to the tracheal bronchus, the tracheadivided into right and left principal bronchi.Close to the tracheal bifurcation, a middlebronchus originated to enter the middle lobe.Just caudal to middle bronchus, an accessorybronchus also originated to ventilate theaccessory lobe. The right caudal bronchus wasposterior most and entered the right caudal

C-Cranial lobe, M-Middle lobe,Ca-Caudal lobe

Fig.1. Lungs of wild sow- Parietal surface

C - Cranial lobe, M - Middle lobe,A - Accessory lobe, Ca - Caudal lobe.

Fig.2. Lungs of wild sow-Visceral surface

T-Tracheal bronchus, M-Middle lobar bronchus,A- Accessory lobar bronchus, C-Caudal lobar bronchus,F- Frill like mucosal folds, B - Tracheal bifurcation.

Fig.3. Lungs of wild sow showing division of bronchi

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lobe. The mucosa of trachea posterior to theorigin of apical bronchus and that of principalbronchi showed few longitudinal frills like folds.

References

Cabral, V. P., Oliveira, F. S., Machado, M.R.,Ribeiro, A. A. and Orsi, A. M. 2001.Study of lobation and vascularisationof the lungs of wild boar (Sus scrofa).Anat. Histol. Embryol. 30 : 205- 209.

Dyce, K. M., Sack, W. O. and Wensing, C. J.G. 1996. Textbook of VeterinaryAnatomy, 2nd ed., W. B. SaundersCompany, Philadelphia. pp. 783-784.

Getty, R. 1975. Sisson and Grossman’s TheAnatomy of the Domestic Animals-Vol. II.

5th ed. W. B. Saunders Company, Philadelphia.pp. 1292 - 1296.

Jones, R., Baskerville, A. and Reid, L. 1975.Histochemical identification ofglycoproteins in pig bronchialepithelium: (a) normal and (b) hyper

trophied from enzootic pneumonia.J. Pathol. 116: 1–11.

Mc Laughlin, R. F., Tyler, W. S. and Canada,R. O.1961. A study of the subgrosspulmonary anatomy of variousmammals. Am. J. Anat.108: 149–165.

Nakakuki, S. 1994. Bronchial tree, lobulardivision and blood vessels of the piglung. J. Vet. Med. Sci. 56: 685-89.

Nickel, R., Schummer, A. and Seiferle, E.1979. The Viscera of the DomesticMammals, 2nd ed. Verlag Paul Parey,Berlin, Hamburg. pp. 241- 246.

Rendas, A., Branthwaite, M. and Reid, L. 1978.Growth of pulmonary circulation innormal pig– structural analysis andcardiopulmonary function. J. Appl.Physiol. 45: 806–817.

Riquet, M., Souilamas, R., Hubsch, J. P., Briere,J., Colomer, S. and Hidden, G. 2000.Lymphatic drainage of heart and lungs,comparison between pig and man.Surg. Radiol. Anat. 22: 47 - 50.

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J. Rejitha and K. Karthiayini


Abstract

A study was conducted to assess theoxidative stress status of cross bred Malabarigoats during peripartum period. Serum levelsof malondialdehyde (MDA), a degradationproduct of lipid peroxidation, reducedglutathione (GSH), serum ascorbic acid andoxidative stress factor (OSF) were used toassess the oxidative stress status of animalsin advanced pregnancy, day of kidding and oneweek after kidding. Changes in haematology(PCV and Hb) associated with pregnancy andkidding were also analyzed. The results of thestudy showed that MDA and OSF weresignificantly (P£0.01) increased in animals onday of kidding and one week after kidding whencompared to non pregnant animals. Serumascorbic acid concentration of animals wasreduced on day of kidding and one week afterkidding. Lowest ascorbic acid concentrationwas noticed at one week after kidding.However, serum GSH level was notsignificantly affected either by pregnancy orby kidding. Haemoglobin level of pregnantanimals was significantly higher (P£0.05) whencompared to that of non- pregnant animals.

Key words:- Oxidative stress, peripartumperiod, goats.

Oxidative stress (OS) is a conditioncaused by excess production of free radicalsand or depletion of antioxidant reserve withinthe body. During normal metabolic reactions,reactive oxygen species (ROS) like superoxideanion radicals, hydrogen peroxide andhydroxyl radical are formed continuously inliving cells which cause oxidative damage to

ASSESSMENT OF OXIDATIVE STRESSDURING PERIPARTUM PERIOD INCROSSBRED MALABARI GOATS*

J. Rejitha 1 and K. Karthiayini 2

Department of Veterinary Physiology,College of Veterinary and Animal Sciences,Mannuthy-680651, Thrissur, Kerala.

the cells. However, under physiologicalconditions, there is a balance betweenendogenous oxidants and various antioxidantdefenses in the body. Normally, body hassufficient antioxidant reserves to manage theproduction of free radicals (Miller et al., 1993;Castillo et al., 2005), but the ROS productionmay increase in pathological and other stressconditions (Roth, 1997) so that theendogenous antioxidant reserves may not besufficient to remove the ROS leading to OS.During peripartum period, increased metabolicactivity and negative energy balance mightalso lead the animal to a state of OS. Body iscapable to resist OS, but depletion ofantioxidant reserve, sudden hormonal changesand physiological adaptation to increasedmetabolic need for lactogenesis andgalactopoesis might be the reasons for OSduring peripartum period. Hence the presentstudy was undertaken.


The experiment was conducted usingeight pregnant and eight non pregnantMalabari crossbred does, aged one to six yearsmaintained at the University Goat and SheepFarm, College of Veterinary and AnimalSciences, Mannuthy. The blood samples werecollected from pregnant animals at 128 to 138th

day of pregnancy, within 12 h of kidding andone week after kidding. Blood samples werecollected from animals by jugular venipunctureusing sterile scalp vein. Samples werecollected with and without anticoagulant(Sodium EDTA @ 1.5mg/ml of blood; Heparin20 IU/ml of blood). Whole blood and separatedserum from each animal were used for

* Part of MVSc thesis submitted by the first author to the Kerala Veterinary and Animal Sciences University, Pookode

1. MVSc Scholar2. Associate Professor, CVAS, Pookode, Wayanad

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58 Assessment of oxidative stress during peripartum...

haematological parameters and analysis ofantioxidant status. Haemoglobin (Hb)concentration and volume packed red cell(VPRC) were estimated as per the standardtechnique described by Schalm (1986). Levelof lipid peroxides in serum was determined byestimating malondialdehyde (MDA) level (Yagi,1984). Reduced glutathione level in blood wasdetermined by the method of Moron et al.(1979). Ascorbic acid level was determined bymethod of Sonnenwirth and Jarett (1980).Oxidative stress factor was calculated usingthe formula of Bisla et al. (2003).

OSF=

Statistical Analysis

Independent ‘t’- test was used tocompare the pregnant and non pregnantanimals. Paired ‘t’ test was used to comparebetween the different peripartum periods (Latepregnancy, day of kidding and one week afterkidding) by using computerized softwareprogram SPSS Ver. 17.0.


The Hb concentration, VPRC valueand oxidative stress status of pregnant animalsduring peripartum period and non pregnantanimals are given in Table 1, Table 2, Table 3and Table 4. The Hb concentration of pregnantanimals was higher than that of non pregnantanimals. There was no significant variation inthe VPRC value of pregnant and non pregnantanimals. These hematological parameterswere also similar in animals in advanced stageof pregnancy, 12 h of kidding and one weekafter kidding. On day of kidding the level oflipid peroxidation was significantly high whencompared to that of animals in advanced stage

of pregnancy. However, it reduced one weekafter kidding. The serum GSH level was similarin both pregnant and non pregnant group ofdoes. The GSH level of animals during theperipartum period was also similar. This mightbe due to the increased production of GSHwithin the body according to the needespecially during the physiological states likepregnancy and parturition. Georgeson et al.(2002) reported that when pregnant animalwas exposed to some OS, antioxidant activitiesincreased in placenta which limited the effectof free oxygen radicals in the embryonic tissue.There was no significant variation in the valueof serum ascorbic acid between pregnant andnon pregnant animals. But on day of kiddingand one week after kidding the serum ascorbicacid concentration of does were less whencompared to non pregnant animals. Theconcentration of ascorbic acid was lowest(0.68±0.07 mg/dl) in animals at one week afterkidding which was significantly lower than thevalue observed in animals of advanced stageof pregnancy. These observations showed thatthere might be increased production ofantioxidants within the body during the periodof pregnancy to meet the increased demandsof the body. However, it was observed that thelevel of production of antioxidants is quiteinsufficient to meet the demand during the dayof kidding and immediate post partum periodwhich indicate the need to supplement it toalleviate the OS during these periods.

The study revealed that duringperipartum period the animals were under OSindicated by high levels of MDA, a product oflipid peroxidation and reduced serum levels ofantioxidants like GSH and abscorbic acid. ThisOS might disturb the body homeostasis leadingto reduced performance of the dam and kid.

Table 1. Oxidative stress status and haematological status of non pregnant Vs pregnant animals(Mean±SE, n=8).

* - P<0.05; difference significant at 5% level** - P<0.01; difference significant at 1% level

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Table 2. Oxidative stress status and haematological status of non pregnant Vs animals on dayof kidding (Mean±SE, n=8).

* - P<0.05; difference significant at 5% level ** - P<0.01; difference significant at 1% level

Table 3. Oxidative stress status and haematological status of non pregnant Vs animals at oneweek after kidding (Mean±SE, n=8).

* - P<0.05; difference significant at 5% level ** - P<0.01; difference significant at 1% level

Table 4. Oxidative stress status and haematological status of animals on advanced pregnancy,on day of kidding and at one week after kidding (Mean±SE, n=8).

a, b Means within a row with no common superscripts are significantly different at 5% level

References

Bisla, R.S., Singh, J., Singh, K. andKrishnamurthy, D. 2003. Assessment ofvitamin E as an antioxidant in pre andpost-operative treatment schedule ofbuffaloes subjected to trans-abdominaldiaphragmatic herniorrhaphy. IndianJ. Vet. Surg., 24: 11-15

Castillo, C., Hernandez, J., Bravo, A., Lopez-Alonso, M., Pereira, V. and Benedito,

J.L. 2005. Oxidative status during latepregnancy and early lactation in dairycows. Vet. J., 169: 286-292

Georgeson, G. D., Szony, B. J., Streitman, K.,Varga, I. Z., Kovacs, A., Kovacs, L.,Laszlo, A. 2002. Antioxidant enzymeactivities are decreased in preterminfants and in neonates born viacaesarean section. Eur. J. Obstet.Gynecol. Reprod. Biol., 103:136-139

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Miller, J.K., Brzezinska-Slebodzinska, E. andMadsen, F.C. 1993. Oxidative stress,antioxidants and animal function. J.Dairy. Sci., 76: 2812-2823

Moron, M.A., De Pierre, J.W., Mannervick, B.1979. Levels of glutathione,glutathione reductase, glutathione- S-transferase activities in rat liver.Biochem. Biophys. Acta., 582: 67-68

Roth, E. 1997. Oxygen free radicals and theirclinical implications. Acta Chir Hung.,36: 302-305

Schlam, O.M., Jain, N.C. and Carroll, E.J. 1986.Veterinary Haematology. 4th ed. Leaand Febiger, Philadelphia. pp. 45-48

Sonnenwirth, A.C. and Jarett, L. 1980.Gradwohl’s Clinical LaboratoryMethods and Diagnosis. 8th ed. The C.V. Mosby Company, Torrento, 2339p.

Yagi, K. 1984. Assay for blood plasma or serum.Methods Enzymol., 105: 328-331

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Bindu Lakshmanan et al.


Abstract

Use of phytopesticides has manyadvantages over conventional chemicalinsecticides and is receiving worldwide attentionas an alternative means of pest control.Preliminary studies were conducted to assessthe lousicidal activity of alcoholic extracts ofAllium sativum bulbs. In vitro study was carriedout on the goat louse, Damalinia caprae usingfilter paper method. It was found that thesecrude extracts caused cent percent mortalityof adult lice at a concentration of 100 mg/ml at32 h post- exposure whereas at 50 mg/ml, thesame mortality was observed at 48 h post-exposure. It is opined that further purificationstudies and in vivo trials are needed to ascertainthe active ingredients responsible for theinsecticidal properties of garlic juice extracts.

Key words:- Allium sativum, Damalinia caprae

Lice are permanent ectoparasites thatconstitute a very large group of more than theehundred species. The temperature and relativehumidity prevailing in Kerala, coupled with ashort life cycle enable lice to proliferate quickly,thus leading to high degree of infestation withina short span of time. Damalinia caprae is abiting louse belonging to the familyTrichodectidae, infesting goats. Though thespecific deleterious effect of D. caprae in goatsin Kerala has not been studied in detail, it isthought to contribute to loss in production.

Topical insecticides remain themainstay of control of lousiness in animals andthis has led to several problems including thedevelopment of resistance, toxic manife

IN VITRO STUDIES ON THE EFFECTOF ALLIUM SATIVUM (GARLIC) ONDAMALINIA CAPRAE

Bindu Lakshmanan1, R. Radhika2,R. Sreekrishnan3 and H.Subramanian4

Department of Veterinary Parasitology,College of Veterinary and Animal Sciences,Mannuthy-680651, Thissur, Kerala

1 &2. Assistant Professors 3. Associate Professor, RGCOVAS, Pondicherry 4. Finance Officer, KVASU

stations in animals, and concerns of residuesin milk, meat as well as environment. The useof eco -friendly herbal plants individually or incombination may provide an alternative toconventional insecticides (Houghton, 1995).Ponnudurai et al. (2007) recorded 100 per centreduction of bird louse treated with herbalformulation containing Azadirecta indica,Cadrus deodara, Brassica compestris andOcimum sanctum. Allium sativum (garlic), amember of family Alliaceae has been shownto possess arthopocidal properties (Jarial,2001 and Stjernberg and Berglund, 2000).Miticidal activity of garlic is also wellestablished (Birrenkott et al.2000; Dwivedi andSharma, 1986 and Magi et al., 2006).In viewof this, a preliminary study was designed toexplore the in vitro lousicidal activity of A.sativum bulbs against Damalinia caprae.


Bulbs of A. sativum (garlic) wereprocured from the local market and allowedto dry in the shade. The dried products weregrated in an electric grinder. Ethanolic extractswere prepared from 200g of the grated materialusing the extraction technique described byAzhahianambi et al. (2004) with somemodifications. Briefly, 200g of the gratingswere macerated in one litre of rectified spiritin a closed container for 24 h with intermittentvigorous mixing. The supernatant wascollected in large petridishes and the alcoholwas allowed to evaporate. The residuesobtained after evaporation were used for invitro assays at different concentrations (5 percent and 10 per cent) made with one per cent

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62 In vitro studies on the effect of Allium sativum ...

Tween 80(v/v) in aqueous medium. Filter papermethod was adopted for testing the efficacyof A.sativum extracts as described byNalamwar et al. (2009) for extract of Acoruscalamus rhizomes. Different concentrations ofcrude A. sativum extract (50 mg/ml (AS 1) and100 mg/ml (AS 2)) and 1 per cent Tween 80(negative control) were poured on WhatmanNo.1 filter paper and allowed to dry. Driedfilter papers were placed in differentpetridishes. Adult Damalinia caprae werecollected from naturally infested goats. Twelvelice each were randomly selected andtransferred to these petridishes, which werethen maintained in a humidity chamber at atemperature of 280 C and relative humidity of85 per cent. Observations related to themortality of lice were taken at 2 h, 4h, 6h, 12h,24 h, 32h and 48 h intervals.


The result of in vitro treatment of D.caprae with ethanolic extracts of A. sativumbulbs showed that the lice survived AS1 andAS2 treatments up to 24 h. It was observedthat AS1 treatment caused 50 per cent and100 per cent mortality of lice after 32 h and 48h of treatment respectively. AS2 treatmentresulted in 100 per cent mortality at 32 h itself.All the lice in negative control were found tobe alive even after 48 h of treatment. Thisseems to indicate that extracts from A. sativumbulbs possess certain lousicidal propertieswhich were directly proportionate to theconcentration. The results also concur with thatof Kumar et al. (2011) who reported thatmortality percentage of adult ticks increasedas the concentration of herbal extractsincreased and as the time interval progressed.Nchu et al. (2005), evaluating the toxic effectsof extracts of A. sativum bulbs on adults ofHyalomma and Rhipicephalus sp. of ticksconcluded that ethanolic extracts causedmortality of adult ticks after 24 h of exposurewhile dichloromethane extracts causedmortality in less than an hour. Nalamwar etal. (2009) inferred that A.calamus rhizomescontain certain constituents responsible formortality of D. caprae. Preliminary investigations on lousicidal activity of A. sativumextracts are encouraging so that one moreeffective herbal lousicide can be used tocontrol D. caprae infestation. However, furtherstudies are needed to identify the activecomponents in the garlic extract responsiblefor this activity.

AcknowledgementThe authors acknowledge the

financial support of Animal HusbandryDepartment, Government of Kerala.

References

Azhahianambi, P., Hariharan, P., Punniamurthy,N. and Gomathinayagam, S. 2004.Insecticidal property of leaves andseeds of sitaphal (Annona squamosa).J. Vet. Parasitol. 18:55-57.

Birrenkott, G. P., Brockenfelt, J. A., Greer, J. A.and Owens, M. D. 2000. Topicalapplication of garlic reduces Northernfowl mite infestation in laying hens.http://www.poultryscience.org:1575-1577.

Dwivedi, S. K and Sharma, M. C. 1986.Studieson a herbal preparation againstscabies in indigenous pigs. Indian J.Vet. Med.6:51-53.

Houghton, P. J. 1995. Plants in traditional medicine,their potential as source of a new drug.J. Alter. Complimentary Med. 1:102.

Jarial, M. S. 2001. Toxic effect of garlic extracts onthe eggs of Aedes aegypti (Diptera:Culicidae): A scanning electron microscopestudy.J. Med. Entomol. 38:446-450.

Kumar, A., Singh, S., Mahour, K. and Vihan,V.S. 2011. In vitro and in vivoacaricidal activity of some indigenousplants under organised and farmerflock. Pharmacologyonline.3:361-369.

Nalamwar, V. P., Khadabadi, S. S., Aswar,P. B., Kosalge, S.B. and Rajurkar,R.M. 2009. In vitro licicidal activity ofdifferent extracts of Acorus calamusLinn.(Araceae) rhizome. Int. J. Pharm.Tech. Res.1:96-100.

Nchu, F., Magano, S. R. and Eloff, J. N. 2005.In vitro investigation of the toxic effectsof extracts of Allium sativum bulbs onadults of Hyalomma marginatumrufipes and Rhipicephalus pulchellus.J. S. Afr. Vet.Assoc.76:99-103.

Ponnudurai, G., Harikrishnan, T. J. andAnna, T. 2007. Evaluation of herbalformulation against ectoparasites oflivestock and poultry. IndianJ.Anim.Sci.77:234-235.

Stjernberg, L. and Berglund, J. 2000. Garlicas an insect repellent. J. Am. Med.Assoc. 284:1-3.

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V. R. Indu et al.


Abstract

A study was conducted on the cervicalvertebrae of two leopards. There were sevencervical vertebrae and among them the first twowere atypical and differed greatly from all others.The third, fourth and fifth vertebrae had onlyminor differences. The sixth and seventhvertebrae presented distinct features to maketheir identification possible. There were somemajor structural variations observed from thatof other small carnivores. In the atlas,transverse foramen was seen on its caudalborder. The posterior extremity of spinousprocess of axis presented a distinct pointedtubercle. The ventral crest was indistinct in theaxis to sixth cervical vertebrae. The cranialventral tubercle of the transverse process ofthe third to sixth cervical vertebrae were verybroad and plate like. These features might beinterpreted as adaptations for the thick andstrong extensor muscles on the nape of theneck for the predatory behavior seen in leopard.

Keywords: Cervical vertebrae, leopard,osteology

The cervical vertebrae give flexibilityto the neck and are built to enhance the speedof running rather than to support the bodyweight. Several adaptations are seen in theskeleton and muscles of the neck region inFelidae, Hyaenidae and Canidae to providenecessary force for downward stabbing, tear-ing and swift walking by carrying large prey(Spoor and Badoux, 1986). Among the sevencervical vertebrae present, the first two areatypical and highly specialized for the move-

GROSS OBSERVATIONS ON THE CERVICALVERTEBRAE OF LEOPARD(Panthera pardus)

V. R. INDU1, A. R. SREERANJINI2,C. V. RAJANI3 and N. ASHOK4

Department of Veterinary Anatomy & Histology,College of Veterinary & Animal Sciences,Pookode- 673 576, Wayanad, Kerala

1&2 Assistant Professors, Dept. of Veterinary Anatomy & Histology, CVAS, Mannuthy, Thrissur.3 Assistant Professor4 Professor, Dept. of Veterinary Anatomy & Histology, CVAS, Mannuthy, Thrissur.

ment of head on the neck (Anderson andAnderson, 1994). The remaining five are typi-cal vertebrae. Review of literature revealedscanty information on the cervical vertebraeof leopard. Hence the present study was un-dertaken.


Cervical vertebrae were collected fromtwo adult leopards that died of natural causesand were brought to the Department of Pathologyfor postmortem examination. Osteologicalstudies were conducted on the bones processedby the method of Young (1980).


The atlas formed a bony ringcomprising two large lateral masses whichwere joined by dorsal and a ventral arches.The former was wider than the latter asreported in dogs by Miller (1965). The dorsalarch presented a distinct bifid dorsal tuberclemedially on its cranial border. The ventral archpresented a small ventral tubercle in the caudalborder (Fig.1). Contrary to this, in canines bothdorsal tubercle and conical ventral tuberclewere present in the caudal border (Miller,1965).The shelf-like transverse processes or thewings extended laterally on both sides fromthe point where the two arches met each other.The fossae atlantis were depressions ventralto the wings for the passage of vertebral arteryand vein. The root of each wing was piercedby a canal like transverse foramen on itscaudal border that lead into the atlantal fossa.However, in dogs the transverse foramenperforated the dorsal surface of the base of

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64 Gross observations on the cervical vertebrae...

wing at the junction of the wing with the lateralmasses (Smith, 1999). This might be due tothe different relative positions of the traversingvertebral artery, vein and nerves between thesetwo species. At the origin of transverse processon its cranial and caudal aspects cranial /alarand caudal notches were present. Medial tothe alar notch there was a groove leading tothe lateral vertebral foramen. The foramen waspresent on the craniodorsal part of the dorsalarch. The cranial articular surface consistedof two cotyloid cavities that articulated withthe occipital condyles of skull to permit freeextension and flexion of the head. The caudalarticular surface consisted of two shallowglenoid cavities which formed a freely movablearticulation with the second cervical vertebra.The caudal part of the dorsal surface of ventralarch presented the concave fovea dentis.Similar observations were made by Nickel etal. (1986) and Smith (1999) in carnivores.

The body of axis was dorsoventrallyflattened, laterally compressed and extremelylong characterized by the presence of odontoidprocess that reached almost to the occipitalbone as reported in dogs (Nickel et al., 1986;Dyce et al., 1996) (Fig.2). The odontoidprocess was ‘dens- like’ as in tiger (Gaikwadet al., 2010). The ventral crest on the bodywas indistinct cranially whereas, it was distinctcaudally. Contrary to this, Nickel et al. (1986)reported that in dogs the ventral crest wasprominent both cranially and caudally anddivided the ventral surface of the body of axisinto two fossae. The dorsal spinous processwas ridge like and thin cranially and thickcaudally. Its anterior and posterior extremitiesextended towards the atlas and third cervicalvertebrae, respectively. The posterior extremityof spinous process presented a distinct pointedtubercle and had lateral ridges runningdownward to meet the caudal articularprocess. Below the caudal extremity of thespinous process a deep irregular fossa wasnoticed. Similar observations were made byArora (2009) in tiger. However, in dogs thetubercle and deep fossa in the posteriorextremity was indistinct (Miller,1965). Thetransverse processes were narrow and blade-like with only a caudal process and projectedcaudolaterally from the body. The root of thetransverse process was perforated by arelatively large transverse foramen. Lateral tothe dens, the body presented two distinct andtriangular convex cranial articular surfaces.The caudal articular process was fused to the

Fig. 2. Axis of leopard- left view1.Odontoid process, 2.Dorsal spinous process3.Tubercle on caudal extremity of spinous process4. Lateral ridges 5. Caudal articular process6. Transverse process 7.Transverse foramen8. Cranial articular surface 9.Cranial vertebral notch10. Caudal vertebral notch

Fig.1. Atlas of leopard- caudal view1.Dorsal tubercle in dorsal arch, 2.Ventral tubercle inventral arch 3.Fovea dentis, 4.Transverse process,5.Transverse foramen 6.Caudal articular surface

Fig.3. Third to seventh cervical vertebrae ofleopard- left viewIII, IV, V, VI VII – Third to seventh cervical vertebrae1.Dorsal spinous process2.Transverse processa.cranial ventral tubercleb.caudal dorsal tubercle3.Tubercles on caudal articular surface4.Cranial articular process5.Caudal articular process

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ventral surface of the spinous process. Thecranial and caudal vertebral notches concurredon either side with those of the atlas and thirdcervical vertebra respectively to form the largeintervertebral foramina, as in canines(Miller,1965; Nickel et al., 1986).

The length of the body of vertebraedecreased progressively from third to seventh(Fig.3.). Their cranial extremities weremoderately convex and caudal were slightlyconcave. The seventh vertebra had costalfacets on the caudal extremity. Theseobservations were in accordance with that ofNickel et al. (1986) in carnivores. The ventralcrest was indistinct in third, fourth, fifth andsixth cervical vertebrae unlike in dogs wherein it was distinct and became more pronouncedcaudally (Smith, 1999). On the dorsal surfaceof the bodies of third to seventh cervicalvertebrae spinous processes were flattenedfrom side to side, inclined cranially andincreased in length from before backwards.The dorsal spinous process of third vertebrawas ridge like with a small cranial tubercle butin the seventh vertebra it was almost in verticaldirection with a rough blunt tip. Theseobservations tally with the findings of Arora(2009) in tiger. The transverse processes ofthe third, fourth and fifth cervical vertebraewere divided into a cranial ventral tubercle andcaudal dorsal tubercle that was three sided.The cranial ventral tubercle was broad andplate like bending slightly backwards anddownwards and became distinct caudally. Thetransverse process of the fifth cervicalvertebrae was shortest, however its ventraltubercle was broader. The unique transverseprocess of the sixth vertebra allowed it to bedistinguished from the others. Its ventraltubercle was the broadest and formed asagittal plate that had cranial and caudal partsseparated by a notch. However, Nickel et al.(1986) observed that the ventral tubercle wasa slim branch in the third, fourth and fifthvertebrae while in the sixth, the ventral laminawas a broad sagittal plate and the notch wasabsent in dogs. These differences might beattributed to the well developed scalenusmuscles that are inserted on to the transverseprocess to depress the neck with unusual forcefor the predatory behavior seen in leopards.In the seventh cervical vertebra, the transverseprocess was single and only caudal tuberclewas present in the form of a distincttransversely directed rod. These observations

concur with the reports of Arora (2009) inIndian tiger. The transverse foramina of thethird to sixth vertebrae were distinct and wereabsent in the seventh, as reported in dogs(Nickel et al., 1986). The cranial and caudalvertebral notches were deep and formed wideintervertebral foramina that were overlappedlaterally by the cranial articular processes.Dorsally the caudal articular processespresented tubercles for the multifidus muscle.The size of the tubercles decreased from thirdto seventh cervical segment. The interarcuatespaces were very narrow.

References

Arora, B. M. 2009. Osteological profile ofIndian tiger (Panthera tigris tigrisLinneaus 1821). Indian Wild Life YearBook 8 & 9: 44-78

Anderson,W. D. and Anderson B. G. 1994.Atlas of Canine Anatomy. Lea andFebiger, Philadelphia. pp: 341-345

Dyce, K. M., Sack, W. O. and Wensing, C. J.G.1996. Textbook of VeterinaryAnatomy. 2nd ed., W. B. SaundersCompany, Philadelphia. pp: 395-397

Gaikwad, S. A., Dhande P. L., Goel, A.,Lambate, S. B. and Ghule, P. M. 2010.Comparative quantitative analysis ofosseous anatomy of the cranio-vertebral junction of tiger, horses, deerand human. Proceedings of the XXVannual convention of IAVA andnational symposium. pp:42

Miller, M. E. 1965. Anatomy of the Dog. W. B.Saunders Company, Philadelphia. pp:51-52.

Nickel, R., Schummer, A. and Seiferle, E.1986. The Locomotor System ofDomestic Mammals. Verlag PaulParey, Berlin, Hamburg. pp: 38-39

Smith, J. B.1999. Canine Anatomy. LippincottWilliams and Wilkins, Philadelphia.pp: 215-219.

Spoor, C. F. and Badoux, B. M. 1986.Descriptive and functional myology ofthe neck and forelimb of the stripedHyaena. Anat. Anz. 161: 375-387.

Young, J. H. 1980. Preparation of skeletalspecimen. Equine Practice.,2:29-32.

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66 Diagnosis of canine trypanosomosis...


Abstract

A clinical case of trypanosomosis in aGerman Shepherd dog and its diagnosis bypolymerase chain reaction(PCR) wasdescribed.The dog was presented with clinicalsigns such as pyrexia, bilateral corneal opacity,pale mucous membranes, emaciation andlymphadenopathy. Wet film and stained smearexamination of peripheral blood revealedTrypanosoma evansi. Blood sample was alsosubjected to PCR for detection of parasitic DNAusing the forward and reverse Kin primers, Kin-1: 5’-GCG TTC AAA GAT TGG GCA ATG-3’and KIN-2: 5’-CGC CCG AAA GTT CAC C-3’,which bind to an internal transcribed spacerregion (ITS1). Polymerase chain reactionshowed positive result and the amplicon sizefor T.evansi was 540bp. The case wassuccessfully treated with a single dose ofdiminazine aceturate @10 mg/kg BW.

Keywords: Trypanosoma evansi, PCR, KinPrimers, Diminazine aceturate.

In India Trypanosoma evansi causesacute and chronic form of caninetrypanosomosis. Biting flies are primarilyassociated with the spread of the disease. Indogs the disease is characterised byintermittent fever, progressive emaciation,oedema of head, pharyngeal region and otherdependant parts, corneal opacity, anaemia anddeath (Gill,1991). Diagnosis of trypanosomosis can be done by conventional wet filmand blood smear examination and by usingdifferent serological tests. Polymerase chain

DIAGNOSIS OF CANINE TRYPANOSOMOSISBY POLYMERASE CHAIN REACTION

P.V.Tresamol1, N.P. Usha2, T.V.Aravindakshan,3 Reji Varghese4 andM.R. Saseendranath5

Dept. of Veterinary Epidemiology &Preventive MedicineCollege of Veterinary and Animal Sciences,Mannuthy-680651, Thrissur, Kerala

1. Associate Professor and Head2. Associate Professor, Dept. of Clinical Veterinary Medicine3. Professor, Centre for Advanced studies in Animal Breeding and Genetics4. Veterinary Surgeon, Animal Husbandry Department5. Director (Acad. & Research), KVASU

reaction (PCR) was reported to be moresensitive than the conventional methods andis useful for detection of low levels ofparasitaemia. A case of trypanosomosis in aGerman Shepherd dog and its confirmationusing PCR is reported.


A female German Shepherd dog ofthree years of age was presented at theUniversity Veterinary Hospital, Mannuthy witha complaint of clouding of eyes since a week.The dog was reported to be weak and off feed.Detailed clinical examination of the dog wasperformed and clinical data were recorded. Theperipheral blood was collected from the eartip and subjected to wet film and blood smearexamination. The whole blood samples werecollected and analyzed for various haemato-biochemical parameters. Blood sample wasalso subjected to PCR for detection of parasiticDNA (Desquesnes and Davila, 2002).

Blood sample was collected usingEDTA (1mg/ml of blood) as anticoagulant andstored at 40C till processed. Extraction ofgenomic DNA was done using phenol-chloroform method (Sambrooke et al., 1989)Polymerase chain reaction was carried outusing the forward and reverse Kinprimers, Kin-1:5’-GCG TTC AAA GAT TGG GCA ATG-3’ andKIN-2:5’-CGC CCG AAA GTT CAC C-3’(McLaughlin et al., 1996). These primers bindto an internal transcribed spacer region (ITS1)situated between 18S and 5.8S ribosomalsubunit genes on nuclear DNA. The primers

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were custom synthesized and reconstituted insterile water for assay. A 540 bp fragment ofthe ITS1 gene was amplified using PCR.

The PCR reaction was carried out ina total reaction volume of 25 µl, using 200 µlthin walled reaction tubes. The reagentconcentrations were : 20pM of each primer, 1unit of DyNAzmeII DNA polymerase(finnzymesoy), 1X PCR buffer (10mMtris-HCl(pH 9.0), 1.5mMMgCl2, 50mMKCl and 0.01 percent gelatin), 200 µM of each dNTPs,approximately 50ng of genomic DNA.

The PCR products were loaded on 2%agarose gel and electrophoresis wasperformed at 120v for one hour and visualizedon a gel documentation system.

Results and DiscussionClinical examination of the dog

revealed pyrexia (104.4OF), bilateral cornealopacity, pale mucous membranes, emaciationand lymphadenopathy (Fig.1). Wet film andstained smear examination of peripheral bloodrevealed Trypanosoma evansi (Fig.2). Theresults of haemato-biochemical estimationsare given in the table. There was severeanaemia with reduced haematocrit values andthrombocytopaenia. Blood glucose level wasgreatly reduced indicating hypoglycaemiawhich is consistent with the condition.Biochemical estimations revealed hypoproteinaemia with hypoalbuminaenia. Clinical signsand haemato-biochemical findings observedin this case were in concordant with thefindings of many workers (Balakrishnan et al.,1994., Arora and Pathak, 1995 and Varshneyet al., 2003).

Polymerase chain reaction showedpositive result and the amplicon size forT.evansi was 540bp (Fig.3). Several diagnosticassays based on detection of trypanosomalDNA by PCR have been developed and isreported to be more sensitive thanconventional diagnostic techniques in differenthosts and has the advantage that it can identifyparasites at the species level (Wuyts et al.,1995., Desquesnes and Davila, 2002.,Ravindran et al., 2008). The Kin primer basedPCR was reported as an effective tool fordetecting trypanosomes in naturally infectedcattle ( Solano et al., 1999).

The dog was treated with single doseof Berenil (diminazine aceturate) (10 mg/KgBW) by deep intramuscular route. Supportivetherapy was also given with 50 ml of 25%dextrose solution intravenously and one ml

Table showing the touch down PCRthermocycling protocol used.

Table. Haemato-biochemical parameters

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68 Diagnosis of canine trypanosomosis...

each of iron dextran (Inferon) and B complexvitamins intramuscularly. On day two, the bodytemperature came down to 102.40F and therewas slight improvement in the appetite andgeneral activity. Wet film and blood smearswere negative for trypanosomes. Blood samplewas also subjected to PCR which showed anegative result (Fig.3). Corneal opacity wasreduced by 5th day (Fig. 4) and the dogresumed its normal appetite and generalactivity by one week post treatment. Treatmentwith diminazine aceturate was very effectivein eliminating trypanosomes in dogs asreported by Varshney et al. (2003).

References

Arora, J. K. and Pathak, K. M. L. 1995. Clinico-haematological and biochemicalchanges associated with T. evansiinfection in dogs. Indian J. Anim. Hlth.,34: 33-38.

Balakrishnan, V. S., Alex, P. C., Jayakumar,K. M., Baby, P.G. and Saseendranath,M.R.1994. Canine Trypanosomiasis.Cherion., 23:2-5.

Desquesnes, M. and Davila, A. M. R. 2002.Application of PCR-based tools fordetection and identification of animalTrypanosomes-a review and perspective. Vet.Parasitol., 109:213-231

Gill, B. S. 1991. Trypanosomes andTrypanosomiasis of Indian livestock.1st revised ed. Publication andinformation division, Indian Council ofAgricultural Division, Pusa, NewDelhi. pp 22-37.

McLaughlin, G. L., Ssenyonga, S. S., Nantez,E., Rubaire-Akiki, Wafukla, O.,Hansen, R.D., Vodkin, M.H.,Novak,R.J . ,Gordon,V.R. ,Montenegro-james,S.,.James,M. , Aviles, H.,Armijos.R., Santrich, C., Weigle, K.,Saravia, N., Wozniak, E., Gaye, O.,Mdachi, R., Shapiro, S. Z., Chang, K.P. and Kakoma, I 1996. PCR baseddetection and typing of parasites.In:Azcel, M.A., Alkan, M.Z. (Eds)Parasitology for the 21st century.CABInternational, Wollingford,Oxon, U.K.Chapter 25, pp. 261-287.

Ravindran, R., Rao, J. R., Mishra, A. K.,Pathak, K. M. L., Babu, N., Satheesh,C. C. and Rahul, S. 2008. Trypanosoma evansi in camels, donkeys, and

Fig.1: Bilateral Corneal Opacity

Fig.2: T. evansi in blood smear

Fig.3: PCR 1,3,5 positive bands at 540bp

Fig.4: After Treatment

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dogs in India. Comparison of PCRand light microscopy for detection.Vet.archiv., 78: 89-94.

Sambrook, J., Fritsch, E. F. and Maniatis, T.1989. Molecular cloning: a laboratorymanual, 2nd ed. Cold Spring HarborPress,New York.

Solano, P., Michel, J. F., Lefrancois, T.,deLaRocque, S., Sidibe, I., Zoung rana,A. and Cuisance, D. 1999. PCR as adiagnosis tool for detecting trypanosomeson naturally infected cattle in BurkinaFaso. Vet. Parasitol., 86:95-103.

Varshney, J. P., Bandopathyay, S., Ranjan, R.and Saghar, S. S. 2003. Diagnosisand treatment of T. evansi associatedcorneal opacity in dogs. J.Vet.Parasitol. 78: 89-94

Wuyts, N., Chokesajjawatee, N., Sarataphan,N. and Panyim, S. 1995. PCRamplification of crude blood onmicroscopic slides in the diagnosis ofTrypanosoma evansi infection in dairycattle. Ann.Soc.Belge.Med.Trop., 75:229-237.

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70 In vitro efficacy of Butea Monosperma ...


Abstract

Butea monosperma is a moderatesized tree widely distributed throughout India.Hot aqueous extract of the Butea monospermaseeds were prepared. Ethyl acetate, ethanoland methanolic fractions were performed. Invitro analysis of Methanolic fraction of Buteamonosperma revealed the active ingredientagainst amphistomosis in cattle.

Keywords:- Butea monosperma, Amphistomosis, Methanolic extract

Helminthosis is one of the mostimportant animal diseases worldwide that cancause heavy production losses in grazinganimals’ especially in developing countries andis always associated with poor managementpractices and inadequate and inappropriatecontrol strategies. Various problems haveemerged with the use of anthelmintics such asresistance, chemical residues and toxicityproblems. A number of medicinal plants havebeen used to treat parasitic infections in manand animals Butea monosperma is a moderatesized deciduous tree which is widelydistributed throughout India, known as ‘dhak’or palas’, commonly known as ‘Flame of forest’.Its seeds have anthelmintic property especiallyfor roundworms and tapeworms (Kumar andSamantha 2012).The in vitro efficacy of Buteamonosperma against amphistomosis wasstudied in the present study.


Preparation of hot aqueous extract

About 250 g of shade dried powderedseeds were gathered in a clean muslin cloth

IN VITRO EFFICACY OF BUTEAMONOSPERMA AGAINST AMPHISTOMOSISIN CATTLE

N.P. Usha1, V.R . Dhanya 2 andSubin Jose3

Department of Veterinary Clinical Medicine,College of Veterinary and Animal Sciences,Mannuthy-680651, Thrissur, Kerala.

1Associate Professor2&3 Research Associates

bag and tied properly. This was kept immersedin a large bowl containing 4 litres of water tofacilitate extraction. When the volume of waterwas about half, two more litres of water wasadded and repeated till 12 litres of water wasadded totally. Finally the muslin bag was takenout and the extract was placed on a boilingwater bath till complete evaporation of water.The residue was taken out and weighed toassess the yield 8%.

Phytochemical analysis

Phytochemical analysis was done asper the procedure of Harbourne (1991).

In vitro trials - Effect of hot aqueous extractof the seeds against amphistomes was studiedinvitro. Amphistomes were maintained inTyrode’s saline containing the hot aqueousextract at three concentrations, viz., one, two,and five percent. This was compared withanother triplicate of untreated control.Observations were taken for a period of 24 hand results were recorded.

Fractionation studies - Soxhlet extraction ofhot aqueous extract of seeds was carried outusing ethyl acetate, ethanol and methanol andthe yield was two, twenty and twenty fourpercent respectively. The resulting fractionswere further analysed for the in vitro activity.

In vitro analysis of fractions –In vitro trialswere conducted on each fraction. Ten percentof ethyl acetate, ethanolic and methanolicfractions was prepared in Tyrode’s saline.Amphistomes were maintained as statedearlier and six amphistomes were transferredto each tube containing the fractions. One tube

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was maintained as control containing only 5ml of Tyrode’s saline. The tubes wereincubated for 24 h at 37

o

C.


Phytochemical analysis -Phytochemicalanalysis of the seeds revealed the presenceof alkaloids, tannins, terpenes and saponins.Glycosides and flavanoids were not presentin the sample.

In vitro trials of hot aqueous extract

The hot aqueous extract of seeds wasfound to be effective in killing amphistomesover a period of 24 h in vitro. Five percentsolution could kill all the worms by 6 h. Also,activity of the flukes reduced drastically within3 to 4 hours in all the test groups (Table-1).

In vitro analysis of fractions

The methanolic fraction was found tobe effective in killing all amphistomes within2 h. Ethyl acetate and ethanolic fractions did

not show any activity. No mortality occurred incontrol sample (Table 2).

It was concluded that the methanolicfraction contained the active ingredient againstamphistomosis and further studies arenecessary to separate the active componentfrom the seeds of Butea monosperma.

AcknowledgementThe authors are thankful to the ICAR,

New Delhi,for providing financial support.

References

Harbourne, J.B.1991. Phytochemical methods- Guide to modern techniques of plantanalysis.2nded. Chapman andHall,India. 653p

Kumar,A. and Samantha,K. 2012. AMultipurpose Medicinal Tree- ButeaMonosperma.International J DrugDiscovery and Herbal Res. 2: 436-441.

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Table 2. In vitro analysis of fractions of seeds of Butea monosperma

Table 1. Invitro trials of hot aqueous extract of seeds of Butea monosperma

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72 Levels of calcium,sodium and potassium...


Abstract

Influence of anticoagulants in thelevels of major electrolytes like calcium, sodiumand potassium in plasma was studied andcompared with their levels in serum. Forty twoblood samples were collected from sevenspecies namely human, elephant, cattle,sheep, goat, dog and rabbit. Heparin andPotassium Ethylene Diamine Tetra Acetate(EDTA) were used as anticoagulants. It wasfound that the levels of these minerals in theblood plasma got altered significantly by theaddition of heparin and EDTA. However, lessinterference was rendered by heparin thanEDTA, on comparison with the serum values.

Keywords:- EDTA, Heparin, Serumelectrolytes, Plasma electrolytes.

Anticoagulants are added in requiredquantity to inhibit the clotting of blood, therebyensuring that the concentration of substanceto be measured is changed as little as possiblebefore analysis (Guder, 2001). Heparin andEthylene Diamine Tetra Acetic acid (EDTA)exert their actions as anticoagulants byinhibiting thrombin in blood and chelating thecalcium ions. Even though serum fromcoagulated blood is the most preferredspecimen for clinical biochemistry, plasmaobtained with an appropriate anticoagulant canalso be served equally good. The mostpreferred anticoagulant meant for plasmaclinical biochemistry is heparin, while forhematological examination is either sodium orpotassium salt of EDTA. Since the harvest ofserum requires a minimum of 30 min wait forthe completion of coagulation before

LEVELS OF CALCIUM, SODIUM ANDPOTASSIUM IN PLASMA AS INFLUENCEDBY ANTICOAGULANTS

K.V. Pramina1, P. T. Mincy2, P. Anu Joseph3,V. Lisha4, K. A. Mercy5 and V. Ramnath 6*

Department of Veterinary Physiology,College of Veterinary and Animal Sciences,Mannuthy-680 651, Trichur, Kerala.

centrifugation, use of plasma is more specifiedin emergency situations. Furthermore, plasmayield from the given volume of whole blood isalways greater than the yield of serum (Youngand Bermes,1999).

The effects of various types ofanticoagulants on plasma biochemistry havebeen studied in man and various animals. Nocomprehensive information is available withrespect to levels of blood minerals like calcium,sodium and potassium in the serum andplasma collected using different anticoagulantsof farm animals. Thus, the study wasundertaken to compare the variations in theserum and plasma levels of calcium, sodiumand potassium in various farm animal speciesand human and to determine and recommendthe most suitable anticoagulant and its dosefor separation of plasma from blood.


About three ml of blood withoutanticoagulant was collected from clinicallyhealthy subjects belonging to each species,viz. human, elephant, cattle, sheep, goat, dogand rabbit and serum was separated as perstandard procedure. Anticoagulated bloodsamples (3ml) were collected simultaneouslyfrom each species with anticoagulant,dipotassium EDTA at concentrations of one,two, three and five mg per three ml blood aswell as with lithium heparin, 60 units per threeml blood. All samples were chilled immediatelyafter collection for 30 min, centrifuged at 1800g for 10 min. The levels of calcium in the serumand anticoagulant added plasma wereestimated spectrophotometrically using

1 to 4: Veterinary Graduates 5. Professor & Head, Dept.of Statistics;6. Associate professor and *corresponding author: 94446579057; [email protected]

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commercial kits following Arsenazo III method(Bauer, 1981). Sodium and potassium levelswere estimated spectrophotometrically usingcommercial kits by the modified methoddescribed by Maruna and Trinder (1958). Theresults were analysed statistically using one-way ANOVA method.


The serum and plasma levels ofcalcium, sodium and potassium of human andsix domesticated species of animals viz.elephant, cattle, sheep, dog, goat and rabbitwere estimated and compared with in group.It was found that the concentration of calciumin serum significantly (p<0.05) differed fromthat of plasma in all the species tested. Thelevel of calcium was found to be significantlymore in heparinised plasma compared toserum and other plasma samples (heparin andEDTA at varying concentrations). It was foundthat in cattle blood, addition of EDTA at therate of 1 mg/ 3 ml completely chelated calciumin the blood. However in other species, thisdose was not sufficient. Blood samples ofhuman and sheep showed zero concentrationof calcium when EDTA was added at the rateof 2mg/3ml. The blood from elephant andrabbit when collected with 5 mg EDTA/3ml,were left with trace amounts of free calcium in

the plasma (Table 1).

Addition of heparin as anticoagulantfor plasma separation resulted in significantly(p<0.05) high levels of sodium in the plasmasamples when compared to serum in allspecies. Addition of EDTA to blood resultedsignificantly (p<0.05) higher levels of sodiumin all the concentrations tested.. However, thevariation seen in sodium level was not muchrelevant with different EDTA concentrationsutilized (Table 2).

Heparinised plasma showed lesserdegree of variations in potassium levels whencompared to serum in all the species tested.However, significantly (p<0.05) higherincrease in plasma potassium level wasnoticed when EDTA at different concentrationswas used as anticoagulant (Table 3).

In the present study it was found thatthe levels of minerals like calcium, sodium andpotassium in the blood was alteredsignificantly by addition of naturalanticoagulant heparin and EDTA. Changes inelectrolyte contents induced by EDTA additionwere described earlier in man ,dog, cattle,horse and sheep (Morris et al., 2002; Mohri etal., 2007 a, b; Ceron et al., 2004). They havereported an increase in sodium level in

Table 1. Levels of calcium (mg/dl) in the serum and plasma.

Means with the same superscripts in a row did not differ significantly (P<0.05)

Table 2. Levels of sodium (mmol/l) in the serum and plasma.


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74 Levels of calcium,sodium and potassium...

heparinised plasma, while a decrease inpotassium level as compared to the serumvalues. In the present study sodium as wellpotassium levels were on the higher side inthe plasma samples collected with EDTA.Serum and heparinised plasma yielded similarresults for sodium and potassium levels, whilethe calcium level in EDTA chelated bloodplasma was significantly low. The reason forincreased potassium level in heparinised andEDTA chelated blood plasma could beattributed to the activation of platelets.

EDTA acts as a chelating agentforming complexes with calcium which isessential for coagulation, is recommended forroutine haematology, since it provides a verygood staining quality of blood cells. It can beused as disodium, dipotassium or tripotassiumsalts. The latter two being preferred as theyare more soluble.

The use of serum or plasma in clinicalpathology remains debatable. Serum ispreferred by many laboratories for clinicalbiochemical tests, since it avoids the additionof anticoagulants which can interfere withsome analytical methods or change theconcentration of parameters being measured.Using EDTA as anticoagulant caused asignificant difference for concentrations ofurea, creatinine, total protein, calcium, andmagnesium and the activity of aspartateaminotransferase and alkaline phosphatase,in plasma comparing with serum (Mohri andRezapoor, 2008).

In the serum stored at lowtemperature, the chance of formation of fibrinstrands is lower when compared to plasmaand thus there is less risk of occlusion ofautomated analyzers. However the use ofplasma is preferred in some centres as it savestime because there is no necessity to wait until

coagulation is completed and also 15 to 20per cent more plasma can be obtained fromthe same volume of blood.

With the results of the study, it isrecommended that the relative amounts ofanticoagulants that can be used for separatingplasma using heparin is 20U/ml of blood forall species, while that of dipotassium EDTAfor cattle blood is 1mg/3ml, for sheep andhuman blood is 2mg/3ml, for goat and dogblood is 5mg/3ml and for rabbit and elephantblood is a minimum of 5mg/3ml or slightlyabove. Excess addition of EDTA and otheranticoagulants would result in changes in thelevels of other biochemical parameters.Results of the present study is also suggestiveof the less interference rendered by heparin inthe plasma electrolytes level.

References

Bauer, P. J.1981. Affinity and stoichiometry ofcalcium binding by Arsenazo III.Anal.Biochem. 110: 61

Ceron, J. J., Subiela, M. S., Hennemann, C.and Tecles, F. 2004. The effects ofdifferent anticoagulants on routinecanine plasma biochemistry. The Vet.J. 167: 294–301.

Guder, W. G. 2001. The quality of diagnosticsamples. Blood Gas News. 10: 18–24.

Maruna, R. F. L. and Trinder.P. 1958. Clin.Chem. Acta. 2: 581.

Mohri, M. and Rezapoor, H. 2009. Effects ofheparin, citrate and EDTA on plasmabiochemistry of sheep: Comparisonwith serum. Res.Vet. Sci.86: 111–114.

Mohri, M., Shakeri, H. and Zadeh, L. S. 2007.Effects of common anticoagulants(heparin,citrate and EDTA) on routine

Table 3. Levels of potassium (mmol/L) in the serum and plasma.


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plasma biochemistry of cattle.Comp.Clin. Pathol. 16: 207-209

Mohri, M., Sharifi, K. and Eidi, S.2007.Hematology and serum biochemistryof Holstein dairy calves: Age relatedchanges and comparison with bloodcomposition in adults. Res.Vet. Sci.83:30–39.

Morris, D. J., Fernandez, J. M., Chapa, A. M.,Gentry, L. R., Thorn, K. E. and Weick,

T. M. 2002. Effects of sample handling, processing, storage, and hemolysis on measurements of key energymetabolites in ovine blood. SmallRuminant Res. 43: 157-166.

Young, D. S. and Bermes, E. W. 1999. Specimencollection and processing: sources ofbiological variation. Tietz Textbook ofClinical Chemistry. 3rd ed. W.B.Saunders, Philadelphia. pp. 42-72

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76 Aflatoxin levels in feeds and feed ingredients...


Abstract

Total aflatoxin and aflatoxin B1 levels infeeds and feed ingredients of livestock andpoultry in Kerala were analysed in 709 samplesby fluorometry after extraction using specificmonoclonal antibody based immune-affinitycolumns. The levels of total aflatoxin varied widelyfrom 1 to 400 ppb in feeds and levels were foundto be higher in poultry, duck and quail feeds whencompared to their accepted dietary limits. Eventhough the levels of total aflatoxin varied from1 to 680 ppb among different feed ingredients,the mean levels were found to be very high inmaize (122 ± 53.36 ppb) and ground nut cake(139.75 ± 31.1 ppb). Aflatoxin B1 contributed to66 to 82% of total aflatoxins in the feed. Totalaflatoxin levels were found to be very high duringthe months of October and November (North-east monsoon season).

Keywords:- Aflatoxin, aflatoxin B1, livestockfeed, feed ingredients

Aflatoxins are a closely related het-erocyclic group of toxic secondary metabolitesof certain strains of fungi, chiefly Aspergillusflavus and A. parasiticus. Since warm and hu-mid climatic conditions favour aflatoxin produc-tion, contamination of feed grains and oil cakeswith aflatoxin are more common in tropicalcountries. Aflatoxins produce most severe andchronic problems in agriculture, livestock,poultry and human beings. They are associ-ated with low productivity, immunosuppres-sion, liver and renal damages, carcinogenicand teratogenic effects. Derivatives of aflatox-ins are found in milk, egg, meat and their prod-ucts which point to its public health impor-tance. This study was undertaken to estimate

AFLATOXIN LEVELS IN FEEDS AND FEEDINGREDIENTS OF LIVESTOCK ANDPOULTRY IN KERALA

B. Bibin Becha1 and S.S. Devi2

Avian Disease Diagnostic Laboratory,Manjadi, P.O., Thiruvalla, Kerala – 689 105

1. Assistant Professor, Dept. of Animal Reproduction, Gynaecology and Obstretics, CVAS, Mannuthy2. Veterinary Surgeon, Regional Animal Husbandry Centre, Thiruvananthapuram

the level of aflatoxin contamination in livestockand poultry feeds and feed ingredients inKerala during various seasons.


A total of 709 samples of feed andfeed ingredients were collected from differentlivestock and poultry farms of Kerala.Aflatoxins were extracted by blending 50 g offeed sample with salt and 100 ml solution ofmethanol and distilled water (80:20). Thefiltered extract is diluted (1:4) using distilledwater and filtered again through glass fibrefilter paper. From 10 ml of the filtrate, aflatoxinswere adsorbed onto a monoclonal antibodybased immunoaffinity column by passing itthrough the column at the rate of two drops/sec. Aflatoxins were eluted out into a cuvetteusing one millilitre of HPLC grade methanol.This elute was derivatized with one millilitre ofdeveloper solution and the levels of aflatoxinswere estimated quantitatively using acalibrated fluorometer (Cigic and Prosen,2009). Levels of aflatoxin B1 was alsoestimated in 17 samples collected at randomby the same procedure using immunospecificcolumns for Aflatoxin B1. The mean levels oftotal aflatoxin, aflatoxin B1 and proportion ofaflatoxin B1 in different feeds and feedingredients were also analysed. Seasonalvariations influencing level of total aflatoxin infeeds were also analysed.


The levels of aflatoxin in differentfeeds and feed ingredients are summarised inTable 1 and 2. The levels of aflatoxin variedwidely in all types of feeds from 1 to 400 ppb.The mean level of aflatoxin was found to behigher in poultry, duck and quail feeds

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B. Bibin Becha and S. S. Devi

compared to their acceptable dietary limits asper the recommendations of package ofpractices. Acceptable limit of aflatoxin in feedof poultry is 20 ppb and that of duck is 10ppb. The levels of aflatoxin varied widely in alltypes of feed ingredients from 1 to 680 ppb.The mean level of aflatoxin was found to bevery high in maize (122 ± 53.36 ppb) andground nut cake (139.75 ± 31.1 ppb). However,these levels are lower than the levels of totalaflatoxin recorded in poultry feeds and feedingredients by Khan (1994).

The levels total aflatoxin and aflatoxinB1 in different feeds are summarised in Table3. Aflatoxin B1 contributed 66 to 82 per centof total aflatoxins present in the feed.

Levels of total aflatoxin in feeds andfeed ingredients during different season of theyear were analysed and the results aresummarised in Table.4. Among the 709samples tested during the different season ofthe year, total aflatoxin levels were found tobe very high during the north-east monsoonseason (October and November). Storage ofcereals in warm and humid conditions in-creases the aflatoxin contamination(Saleemullah et al., 2006). Giridhar andKrishnamurthy (1977) found that 25.7 per centof commercial ground nut oil contained morethan 100 ppb aflatoxin and higher toxin con-centration was found in oil extracted fromkharif (rainy) season crop.

It is practically difficult to producefeeds and feed ingredients free from aflatoxin.But the very high levels of aflatoxin in maizeand ground nut cake warrants to limit their usein feed to minimum required levels and onlyafter proper testing for its levels. Reddy et al.,

(2009) reported that only two per cent of therice samples collected from different states ofIndia showed aflatoxin B1 contamination abovethe permissible limits. Jowar and broken ricecould be used to replace maize as energysupplements in feed, since these ingredientswere found to contain only permissible levelsof toxin. Aflatoxin B1 contributes to the majorportion of total aflatoxin in feed. Bhat et al.,(1996 and 1997) showed that aflatoxin B1

levels exceeded the permissible Indianregulatory limit of 30 ppb in 21 per cent ofground nut samples and 26 per cent of maizesamples collected from 11 different states.Since aflatoxin B1 is the most toxic aflatoxin,its level has to be considered along with totalaflatoxin before accepting or choosing a feedor feed ingredient for dietary use in animals.

References

Bhat, R.V., Vasanthi, S., Sashidhar Rao, B.,Nageswara Rao, R., Sudershan Rao,V., Nagaraja, K.V., Girija Bai, R.,Krishna Prasad, C.A., Vanchinathan,S., Roy, R., Saha, S., Mukherjee, A.,Ghosh, P.K., Toteja, G.S., Saxena,B.N. 1996. Aflatoxin B1 contaminationin groundnut samples collected fromdifferent geographical regions ofIndia: a multicentre study. Food Addit.Contam. 13: 325–331

Bhat, R.V., Vasanthi, S., Sashidhar Rao, B.,Nageswara Rao, R., Sudershan Rao,V., Nagaraja, K.V., Girija Bai, R.,Krishna Prasad, C.A., Vanchinathan,S., Roy, R., Saha, S., Mukherjee, A.,Ghosh, P.K., Toteja, G.S., Saxena,B.N. 1997. Aflatoxin B1 contaminationin maize samples collected from

Table 1. Levels of aflatoxin in different feeds

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different geographical regions of India– a multicentre study. Food Addit.Contam. 14: 151–156.

Cigic, I. K. and Prosen, H. 2009. An overviewof conventional and emerginganalytical methods for determinationof mycotoxins. Int. J. Mol. Sci.,10: 62 – 115.

Giridhar, N., Krishnamurthy, G.V. 1977.Studies on aflatoxin content ofgroundnut oil in Andhra Pradesh withreference to climatic conditions andseasonal variations. J. Food Sci.Technol. 14: 84–85

Khan, B. A. 1994. Aflatoxin contamination ofPoultry feed and resulting disordersin chicken. Thesis . University ofKarachi, Pakistan.

Reddy, K.R.N., Reddy, C.S. and Muralidharan,K. 2009. Detection of Aspergillus spp.and aflatoxin B1 in rice in India. FoodMicrobiol. 26: 27–31.

Saleemullah, Iqbal, A., Khalil, I.A and Shah,H. 2006. Aflatoxin content of storedand artificially inoculated cereals andnuts. Food Chem. 98: 699 – 703.

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Table 4. Levels of total aflatoxin in feeds during different season

Table 2. Levels of aflatoxin in different feed ingredients

Table 3. Levels of total aflatoxin and aflatoxin B1 in different feeds

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SURGICAL MANAGEMENT OFGYNECOMASTIA IN A SIROHI BUCKBY MASTECTOMY

Gynecomastia in bucks is a glandulartissue proliferation leading to unilateral orbilateral enlargement of mammary glands withoccasional secretory activity (Oskam etal.,2005). The condition has been reportedamong Nubian (Wooldridge et al., 1999) andPolish White (Jaszczak et al., 2010) goats. Theage of occurrence varies from seven monthsto several years and milk yield from 20 ml to1500 ml/day (Marx et al., 1975). The cytological,histological, hormonal and semen quality of thebucks are usually unaffected (Jaszczak et al.,2010). In this article the occurrence of bilateralgynecomastia in a two year old Sirohi buck andits surgical correction is reported.

A Sirohi buck, aged two years, was

presented to the Veterinary Polyclinic,Kunnamkulam with a history of abnormal growthnear the testes. The buck was purchased fourmonths before and was being utilised as a stud.It had showed normal male phenotype from birthto puberty and had sired 11 kids before purchaseby the present owner. Enlargement of mammaryglands became apparent two months afterpurchase (Fig 1). At the time of examination bothglands were well developed (Fig.2) with secretionof approximate yield of 25 ml of milk from eachAppetite, activities and libido of the animal wereunaffected. Mastectomy of the animal wasundertaken at the request of the owner of theanimal.

Xylazine @ 0.1mg/kg body weightgiven intramuscularly, followed by localinfiltration of 7ml of xylocaine 2% solutionprovided sedation and analgesia. Ellipticalincisions were placed below the base of eachhalf with retention of enough skin for completeapposition after removal. Blunt dissection wasused to separate the gland from the skin.External pudental artery and external pudentalvein were ligated. Both glands were removedseparately (Fig 4). Scrotal/mammary lymphnodes were not resected. Subcutaneoussutures with plain silk (No.2) were placedinorder to obliterate dead space. Continuoussutures with braided silk (No.2) were used for

Fig.1. Early appearance of enlargedglandular tissue.

Fig. 2 Pendulous glands after one month

Fig. 3. Individual surgical removal and closure

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closure (Fig 5). Post operative care includedTetanus Toxoid one ml and Meloxicam @ 0.3mg/kg body weight followed by Strepto-penicillin injection @ 10,000 IU /kg bodyweight for five days. Recovery was uneventful.

Gynaecomastia is more frequentamong bucks compared to rams and bulls.Familial predisposition, endocrine imbalancesand manipulation of rudimentary teats eitherby the animal or other animals have beenimplicated as possible predisposing causes.But the condition has not yet been conclusivelyinterpreted based on cytological, hormonal orseminal aberrations. In the present case, theanimal also had normal values for the clinicalparameters. Mastectomy was performed sincethe condition is presumed to predispose theanimal to mammary tumours or eventualreduction in fertility.

Summary

A case of gynaecomastia and itssurgical correction in a Sirohi buck is reportedand discussed.

References

Jaszczak, K., Sysa,P ., Romanowicz, K.,Boryczko, Z., Parada, R., Sacharczuk,M., Witkowski, M., Kawka, M. andHorbanczuk, K. 2010. Cytogenetic,histological, hormonal and semenstudies in male goats with developed

1.Assistant Professor

udders. Animal Science Papers andReports. 28: 71-80

Marx, D., Klempp, J., Loeffler, K. and Bohm,S.1975.Gynecomastia in a male goat.I. Sexual activity and sperm and milkquality. Zuchthygiene. 10: 125-34

Smith, MC and Sherman, D. M.1994. GoatMedicine. Lea and Febiger.,Philadelphia. 446-63

Wooldridge, A A., Gill, M S., Lemarchand, T.,Eits, B., Taylor, H W and Otterson, T(1999). Gynecomastia and mammarygland carcinoma in a Nubian buck.Can Vet. J. 40: 663-65

Oskam, I .C.,Lyche, J L., Krogenaes,A.,Thomassen, R., Skaare,J.U.,Wiger,R., Dahl, E., Sweeney, T., Stien,A and Ropstad, E. 2005. Effect of longterm maternal exposure to low dosesof PCB 126 and PCB 153 on therepeoductive system and relatedhormones of young male goats.Reproduction. 130: 731-32.

K. Vinod Kumar1

Department of Veterinary Epidemiologyand Preventive Medicine,College of Veterinary and Animal Sciences,Mannuthy-680651, Thrissur, Kerala

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Basharat Ahmed Pandit et al.

Received - 10. 10. 2010Accepted - 10. 12. 2010

PREVALENCE OF HELMINTH PARASITESOF CAMELS (Camelus dromedaries)IN THE ANSEBA REGION OF ERITREAIN NORTH EAST AFRICA

Despite the susceptibility of camelsto parasitic infections, they may not usuallydie or manifest obvious clinical signs.However, the slow, constant drain in theirhealth often means a substantial loss inincome to the farmers, camels being animportant animal in Eritrea located in NorthEast Africa. Hence the present study wasundertaken to monitor the prevalence ofgastrointestinal parasites of camel in andaround Zoba Anseba region of Eritrea. Camelsof different villages of Zoba Anseba werescreened for the study. A total of 230 faecalsamples were collected and examined forobserving the prevalence of gastrointestinalparasitic fauna in the area during 2009 & 2010.The samples were collected from both maleand female camels of different age groups.The faecal samples were collected in polythenebags and brought to laboratory for examinationand examined by both sedimentation andfloatation techniques. All the parasitic eggswere identified on the basis of morphologicalfeatures given by Soulsby, 1968 and 1982. Thedata was subjected to statistical analysis usingstandard‘t’test (Snedecor and Cochran, 1967).

Out of the 230 faecal samplescollected, 106 (46.08%) were found positivefor different types of helminth eggs. Most ofthe camels were showing mixed type ofinfection with two or three species of helminthparasites. The parasites identified on the basisof morphology of ova were Strongyle sp 98(92.45% ), Trichuris sp 21 (19.81%) Monieziasp 9 (8.49%) and Fasciola sp 2 (1.88%). Sincemixed infection was observed with two to threespecies of helminths, the percent positivity wascalculated from the total positive cases.

Out of 106 camels positive for differenttype of parasites, 60(56.60%) were males and46(43.39%) females. The males were foundmore susceptible due to the fact that they areused for draught purpose and hence moreexposed to parasitic infections(although thedata were not significant statistically ).Theprevalence rate of different helminths are givenin Table.

According to Gebrehiwet (1998), theprevalence of diseases in camels was highestin the rainy season(47.7%) and lowest in thedry season (19.2%). Parsani et al (2008) had

Table. Prevalence rate of different helminthes

Null Hypothesis: There was no significant relation between the mean effects of males and females due to different Parasiticinfections, t = 0.728, Probability = 0.48 and Probability > 0.05 hence not significant at 5% level of significance. Agewise , outof 106 positive camels, 35 were young ones (<3 years) (33.01%) and 71 were adults(>3 years) (66.98%)

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recorded the common gastro intestinalnematodes of camels as Haemonchus,Nematodirus, Nametodirella, Trichostrongylus,Strongyloides, Ostertagia, Marshallagia,Cooperia, Trichuris and Camelostrongylus fromthe camels of Rajasthan, India. They have alsoobserved maximum prevalence with theseparasites during rainy season. Mostly the areaunder Zoba Anseba remains dry and getsmaximum rainfall during July and Augustwhich frames the baseline for the infection inanimals. The present study is also inagreement with the findings of Parsani et al(2008). Further, Manefield and Timson(1997)reported that camel was the least likely of alldomestic livestock to suffer from heavyburdens of helminthes although low grades ofnematode infection were common, during highrainfall. In the neighboring Eastern Sudan,Haemonchus spp and Trichostrongylus sppwere among the commonest helminthsspecies found (Fadle et al ,1992). In thepresent study the Trichuris was found as highas 19.81% and is in agreement with that ofManefield and Timson(1997).

In the present study only one camelwas found positive for Fasciola which mightbe due to the fact that the camel migratedfrom another Zoba, as Zoba Anseba has noor meagre availability of surface water.Gebrehiwet (1998) also observed that camelsmove in Eritrea during early morning hours andlate evening. During this time, the infective thirdstage larvae actively move up on the leavesand camels acquire infection while browsing.

SummaryThe prevalence of helminth parasites

in the camels of Zoba Anseba was studied. Forthe study a total of 230 fecal samples werecollected and investigated. The investigationshowed an infection rate of 46.08%. Thehelminthe ova identified were Strongyle 98(92.45% ), Trichuris sp 21(19.81%) Monieziasp 9(8.49%) and Fasciola sp 2 (1.88%). Mixedinfection with two to three species of helminthswas recorded. The infection rate due to varioushelminths in males and females as well as youngones and adults were non significant (P > 0.05).

AcknowledgmentThe authors are thankful to Mr. Semere

Amelsom, Dean, Hamelmalo AgriculturalCollege for enabling us to conduct the studyand to Dr. G. S. Rao Assistant Professor,Statistics, for his assistance in analysis of thedata.

References

Bissrat Ghebru and Thomas Kholer. 1999. Soiland Water Conservation andManagement in Eritrea. Proceeding ofthe AEAS/University of Berne.Collaborative Workshop, Asmara, 17-20 February, 1998.

Fadle, M., Magzoub, M., Burger, H.J.1992.Prevalence of Gastro – intestinal nematode infection in the dromedaries(Camelus dromedaries) in Butana plains,Sudan., Revue d Elevase et de MedicineVeterinaraire des Pays Tropicaux., 44(3 – 4): 291 – 293.

Gebrehiwet. T.1998.Camels in Eritrea – An allpurpose animal., World AnimalReview 91: 34 – 42p.

Manefield, G. W. and Tinson, A. H. 1997.Camels – A Compendium SydneyPost Graduate Foundation VadeMecum Series C No. 22.

Parsani, H. R., Veer Singh and Momin, R. R.2008 Vet. World Vol.1, No.10 www.veterinaryworld.org.

Snedecor, G. W and Cochean, W.C.1967.Statistical methods 6th ed. Oxford andIndian Book House, Calcutta.

Soulsby.,E.J.L.1982. Helminths, Arthropodsand Protozoa of DomesticatedAnimals, 7th ed.ELBS and BailliereTindall Publication, London.

Basharat Ahmed Pandit 1, MichaelKahsay2, Sanjay Devarajan3 and PauloLuis Valerino Cambra4

Hamelmalo Agricultural College,P. O. Box No. 397, Keren, Eritrea,North East Africa

1 Professor Veterinary Parasitology,2 Veterinary Pathologist & Head of Department,3 & 4- Veterinary Epidemiologists.

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MYOGLOBINURIC ACUTE RENALFAILURE IN A DOG

Rhabdomyolysis is an importantcause of acute renal failure. Early diagnosismay provide the opportunity to reverse thedecline in renal function and obviate thedevelopment of established acute renal failure.This case report highlights the importance ofconsidering rhabdomyolysis in all animalpatients with unexplained acute renal failure.

A three year old Weimarnnerweighing about 50 kg was referred to theCollege Veterinary Hospital, Mannuthy witha history of discoloured urine, melena andhaematemisis of two day duration. The animalwas vaccinated against rabies about two daysback in a nearby hospital. At the time ofpresentation of local veterinary hospital therectal temperature was 106°F and prescribedNimesulide on that day. Upon presentation atthe College hospital the rectal temperature was102°F, heart rate was 160 bpm and respirationrate was 58/min. Echymotic and petechiaewere seen on the ventral abdomen, thigh andpenile mucosa. Ulceration was observed onoral mucosa and tongue. Animal was oliguricand recumbent. The urine was dark brown andturbid. Specific gravity of urine was 1.005.Urine sample was positive for Ammoniumsulphate precipitation test indicative ofmyoglobinuria. Ultrasonographic examinationof abdomen revealed hypoechoic andhomogenous kidney but no otherabnormalities could be detected on otherorgans. Initial treatment indicated fluids (NS),Dexamethasone sodium phosphate andAmoxicillin Cloxacillin @ 10 mg/kg b.wt i/v.Blood smear was negative for haemoparasite.Blood sample was collected forhaematobiochemical analysis. Serum had anunusual greenish brown colour.Haematological examination revealedleukocytosis (38.6 x 103/ml) with neutrophiliaand lymphopenia. The values of haemoglobin,RBC and PCV were 16.8 g/dl, 6.87 x 106/cc

and 56.8% respectively. The most strikinghaematological abnormality observed wassevere thrombocytopenia (26,000/cc). Thecharacteristic biochemical abnormalities onthe day of admission was increased serumlevels of creatine kinase (50,000 IU/L), ALT(1300 IU/L) and AST (2300 IU/L).Otherbiochemical values were protein (5.6 g/dl),albumin 2.7 g/dl, A:G ratio 0.9, BUN 123 mg/dl and creatinine 5.6 mg/dl. The very next daythe creatinine value was elevated to 13 mg/dl.The same treatment were continued for threemore days. In spite of therapy the conditionof the dog deteriorated and it died on the 4th

day. Based on the history of strenuousphysical activity during transportation and thevalues of serum biochemical and enzymaticstudies obtained a diagnosis of ARF due torhabdomyolysis was made.

Summary

Rhabdomyolysis is an uncommondisease process with profound sequelae if itis not identified and treated expediently.Clinical presentation varies, ranging from anearby asymptomatic illness to fulminate andlife threatening disease process withmultiorgan system failure. Rhabdomyolysisresults in the release of myoglobin, a haempigment, which through multiple effects canresult in nephrotoxicity and ARF (Zagar, 1989and Better and Steain, 1990). The mainmechanism is likely to be intra-luminalobstruction by haem pigment casts andischemia ( Luscher, 1991) and a direct or iron–mediated proximal tubular injury. Treatmentshould be directed at reducing the nephro-toxicpotential of the released pigments which canbe achieved by rehydration, correction ofacidosis, maintaining good urine output andestablishing a urinary pH of 7.0 by forcedalkaline diuresis ( Prabhakar et al., 2000)

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References

Better, O. and Steain, J. 1990. Earlymanagement of shock and prophylaxis of acute renal failure in traumaticrhabdomyolysis. N. Engl. J. Med.,322: 825-827.

Luscher, T. 1991. Endothelium derived relaxing and contracting factors : Perspective in nephrology. Kidney Int.,39: 575-577

Prabhakar, K.S., Pall, A.A. and Woo, K.T. 2000.Rhabdomyolysis and acute renal failurecomplicating detergent ingestion.Singapore Med. J., 41: 182-183.

P. P. Kanaran1, N. P. Usha2, V. R.Ambily3 & P.C. Alex4

Department of Clinical MedicineCollege of Veterinary and Animal SciencesMannuthy-680651 Thrissur, Kerala

1.Veterinary Surgeon, AHD, Kerala2.Associate Professor3.Veterinary Surgeon, AHD, Kerala4.Professor and Head

Zagar, R. 1989 . Studied of mechanisms andprotective maneuvers in myoglobinuric acute renal injury. Lab. Invest.,60: 619-621.

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Babesiosis is an importanthaemoprotozoan disease causing highmorbidity and mortality in dogs. It is caused byBabesia canis and B.gibsoni and is transmittedby brown dog tick Rhipicephalus sanguineus.Cerebral babesiosis is characterized by nervoussymptoms such as limb weakness, muscularpain, paresis and sudden death. There areseveral reports of cerebral babesiosis due toBabesia canis in India (Bharti, 2003; Sriram andGomathi Nayakam, 2004). Reports of cerebralbabesiosis due to Babesia gibsoni is scarce(Chaudhary et al., 2009). The present paperdescribes a case of cerebral babesiosis in adog and its successful treatment withdiminazene aceturate.

A four year old male boxer waspresented to the University Veterinary Hospital,Mannuthy with a history of depression,weakness, ataxia, occasional seizures andanorexia. On clinical examination, the animalwas anaemic with pale mucous membranesand was dehydrated. Clinical data were withinnormal range except for a rise in bodytemperature (104ºF). Wet film examination ofblood did not reveal any motile parasites.Peripheral blood smear examination revealedcharacteristic ring shaped B.gibsoni organismsin the erythrocytes. Haematologicalexamination revealed low haematocrit valueswith neutropaenia and eosinophilia.

The animal was treated with a singleinjection of diminazene aceturate @7.5mg/Kgbody weight, deep intra muscular. Alsosupportive therapy with chloramphenicol @ 25mg/Kg body weight i/v for five days, dextrose10% solution i/v and neurobion forte, one tabdaily orally. The animal showed markedimprovement and remission of clinical signsafter five days. Blood smears collected on thefifth day were negative for B.gibsoni.

Cerebral babesiosis occurs with the

CEREBRAL BABESIOSIS DUE TO BABESIAGIBSONI IN A DOG - A CASE REPORT

sludging of erythrocytes within central nervoussystem capillaries leading to tissue hypoxia,weakness, ataxia, seizures and vestibular orcerebellar signs (Suresh et al., 2010). Epilepticfits, ataxia and/ or paresis have been reportedin dogs with cerebral babesiosis caused byB.canis (Jacobson, 1994). Moore and Williams(1979) also suggested that sludging ofparasitized erythrocytes in the smaller vesselsor capillaries of the brain, owing todisseminated intra vascular coagulation maybe responsible for the CNS symptoms.Jacobson and Lobetti (1996) also observedmuscular pain and tremors in dogs withbabesiosis and opined that it could be due torhabdomyolysis owing to release ofinflammatory cytokines and nitric oxide. In thepresent case there was clinical recovery usingdiminazene aceturate at a dose rate of 7.5mg/kg BW as suggested by Taboada (1998). Eventhough the incidence of cerebral babesiosis islimited, it should be included in the differentialdiagnosis of neurological disorders in dogs.

Summary

A case of cerebral babesiosis due toB.gibsoni in a dog charecterised by depression,weakness, ataxia, seizures and anorexia wasdescribed. It was successfully treated with asingle dose of diminazene aceturate @7.5 mg/kg BW. The report stresses the need forincluding cerebral babesiosis in the differentialdiagnosis of neurological disorders in dogs.

ReferencesBharti, C.R. 2003. Cerebral babesiosis in a dog-

a case report. Intas Polivet. 4: 93-94.

Chaudhary, S., Changkiga, B. and Varshney, J.P.2009. Cerebral babesiosis in dogs-areport. Indian. J. Vet. Med. 29: 60-61.

Jacobson, L.S. 1994. Cerebellar ataxia as apossible complication of babesiosis intwo dogs. J. South African Vet. Assoc.

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65:130-131.

Jacobson, L.S. and Lobetti, R.G. 1996.Rhabdomyolysis as a complication ofcanine babesiosis. J.Small Anim.Pract. 37:286-291.

Moore, J. and Williams, M.C. 1979.Disseminated intravascular coagulation: a complication of B.canisinfection in the dog. J. South AfricanVet. Assoc. 50:265-275.

Suresh, R.V., Prasad, A.A., Prathaban, S., Simon,M.S. and Balachandran, C. 2010.Cerebral Babesiosis in a dog-a casereport. Indian. J. Field Vet. 5:31-32.

Sriram, P. and Gomathinayakam, S.2004.Cerebral babesiosis in a spitz

Tresamol P.V.1, Usha Narayana Pillai2,Anumol J.3, Devada K.4 andSaseendranath M.R. 5

Dept. of Veterinary Epidemiology &Preventive MedicineCollege of Veterinary and Animal Sciences,Mannuthy - 680651, Thrissur, Kerala.

1. Associate Professor and Head2. Associate Professor, Department of Veterinary Clinical Medicine3. M.V.Sc. Scholar4. Professor and Head, Department of Veterinary Parasitology5. Director of Academics and Research, KVASU

dog. Indian Vet. Med. J. 28:295-297

Taboada,J.1998. Babesiosis. In: Greene,C.E.(Ed). Infectious diseases of thedog and cat.WB Saunders,Philadelphia .pp. 473-481

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