BUFFALO BULLETIN - Kasetsart UniversityBuffalo Bulletin (December 2002) Vol.21 No. 4 77 Table 1. General prevalence rates of tuberculosis, rates by sex and forms of lesions in slaughtered

BUFFALO BULLETIN

Volume 21, No. 4, December 2002 ISSN: 0125-6726

Editor

S. Sophon

International Buffalo Information Center

Buffalo Bulletin (December 2002) Vol.21 No. 4

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SOME EPIDEMIOLOGICAL ASPECTS OF TUBERCULOSIS IN WATER BUFFALOES IN THEAMAZON BASIN OF BRAZIL

Jose de Arimatea Freitas1 and Jose Cezar Panetta2

1School of Veterinary Medicine, Faculty of Agrarian Sciences of Para, Brazil2Veterinar Medicine and Zootecnic Faculty, University of Sao Paulo, Brazil

ABSTRACT

A survey of tuberculosis infection in slaugh-tered buffaloes, coming mainly form Marajo Islandfarms, Para state, Brazil, allowed observations ofcharacteristics and epidemic aspects of the diseasein the Amazon basin. The prevalence rate of thedisease was 7.7%, 72.1% of the lesions werelocalized, 27.9% generalized and more than 50% ofinfected animals were males. The greatest numberof infected animals, which had the highest diseaseinfection, came from Chaves County in the microregion of “Campos de Marajo”. Hot and humidclimate, high rainfall index, pastures flooded with lowsupport capacity and nutritional quality and longanimal permanency in the breeding (higher than 300days breastfeeding) seemed to favor the infection,maintenance and spread of the disease amonganimals.

INTRODUCTION

Bovine tuberculosis is one of the most impor-tant diseases which infect animal herds witheconomic damage to agricultural production (O’Reillyand Daborn, 1995). Direct contact between animalsis one of the most important factors in increasingthe rate of infection and spread of the disease to theanimal herds (Pritchard, 1988).

In the Amazon basin, large bovine and buffaloherds are raised in extensive areas of nativepastures where the climate is hot and humid, withan average annual rainfall 3,000 mm, and with long

rainy and dry seasons. In localized places of theregion, tuberculosis prevalence rates goes up to 8%(Lau, 1990; Freitas et al., 1997).

Despite the potential risk for tuberculosis andthe high incidence of the disease in buffalo herds ofthe region, little is known of the factors that affectinfection in the Amazon basin (Freitas, 1984, 1999).

The objective of this work was to determinesome epidemiological aspects of water buffalotuberculosis in infected animals from regionalbreeding places.

MATERIAL AND METHODS

Tuberculosis-like lesions were observedduring routine slaughtering of 1,735 water buffaloesin the city of Belem, Para, Brazil. The animals wereslaughtered and examined according to officialtechniques and procedures recommended by theFederal Inspection Bureau (Brasi, 1971, 1997). Thediagnosis of the disease was made on the basis oflesion histopathology, isolation of the probableinfection agent and identification of mycobacteriumby recommended biochemical assays (CENTROPANAMERICANO DE ZOONOSIS, 1986).

Data of sex and age of the animals, diseasecharacteristics such as damaged organs, distributionof lesions and spread of the infectious process as

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well as information regarding animal origin (countyand farm) were surveyed during slaughtering and inthe slaughterhouse registration offices. Rain index,rainy and dry seasons, temperature, economicmicro regions, herd management, grass and type ofgrazing were obtained from the local specializedliterature.

RESULTS

Table 1 shows data related to general preva-lence rate of tuberculosis, prevalence rate by sexand forms of lesions in slaughtered animal forconsumption in Belem. The general prevalence ratewas 7.7% distributed between males (4.2%) andfemales (3.5%). The disease lesions were localizedin organs or lymph nodes in 72.1% of the cases andwere generalized in the remaining 27.9% of thecases.

Figure 1 shows the places of origin of infectedanimals. Only two out of the eight counties fromwhich the buffaloes originated were not in the MarajoIsland; one from the county of Marapanim in thestate of Para and the other from the neighboringstate of Amapa.

The prevalence rate by animal origin issummarized in Table 2 and the number of buffaloesand farms in each county of origin of infectedanimal are shown in Table 3. Chaves County hadthe highest number of farms with the disease, eightout of sixteen, and also the highest number ofinfected animals, 55 out of 133, or 41. 4%;Marapanim County had the lowest rate, 0.75%, orjust one female with lesions suggesting tuberculosisinfection.

DISCUSSION

The general prevalence rate of the order of7.7% reported in this survey was inferior to the ratesof 16.4% and 19.55% determined by Letts (1964)

and Moraes (1990) for buffaloes slaughtered inAustralia or submitted to tuberculin ten fold test inBrasil, respectively, and close to the prevalence rateof 8.04% determined in slaughtered animals ofAustralia by Mc Cool and Newton-Tabret (1979).However, it was superior to the rate of 3.99%obtained by Alfinito et al. (1986) in a tuberculin testcampaign in Marajo Island, to the 4.68% rate deter-mined by the Official Sanitary Animal DefenseService (Brasil, 1996), and to the 3.5% prevalencerate reported by Freitas et al. (1997) on the slaugh-tering of animals in the state of Para, Brazil.

Contrary to the statement of Rajaram et al.(1996), the male disease prevalence rates of thissurvey were up to 50% (Table 1). In the meatproducing systems used in the Amazon basin,discard of females is made at the end of 15 years ofproductive life and of reproducer males at eight yearsof age. Animals for the slaughterhouseshould weigh 350 to 370 kg by the age of 4 to 5.5years. (EMBRAPA/EMATER, EMBRAPA/CPATU, 1980).

On the other hand, the 72.1% localized and27.9% generalized forms of lesions determined inthis survey were in accordance with tuberculosisinfection characteristics at slaughtering leveldescribed by Hathaway et al. (1991). The spreadof the infectious process and the lesion characteris-tics allowed the conclusion that buffaloes tubercu-losis is frequently a lung disease with progressiveinfection and that breathing is the primary way ofinfection in slaughtered animals.

In the economic region of “Compos deMarajo”, the origin of the highest number of infectedanimals (Table 2, 3), a hot and humid climate withhigh temperatures prevails throughout the year.Rajaram et al. (1996) reported identical climaticfactors in five Indian farms, including the farm wheremost of the animals showed positive results to thetuberculin test. Edaphoclimatic factors such as highrainfall index, long rainy and dry seasons observedin the “Campos de Marajo” affected the availabilityof native pastures, especially on the swampy areasduring the rainy and flooding periods (EMBRAPA/EMATER-Para, EMBRAPA/CPATU, 1980).


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Table 1. General prevalence rates of tuberculosis, rates by sex and forms of lesions in slaughtered buffaloesfor consumption. Belem, 2002.

Sex Total Total lesions Localized Lesions Generalized LesionsAnimals Number % Number % Number %

Male 1,186 72 4.2 60 45.1 12 9.0

Female 549 61 3.5 36 27.0 25 18.9

Total 1,735 133 7.7 96 72.1 37 27.9

Table 2. Tuberculosis in slaughtered buffaloes for consumption as a function of origin and number of animals by sex with symptoms of the disease. Belem, 2002.

State/County of origin Animals with disease symptomsMale Female Total %

1. Para State 60 54 114 85.71Chaves 33 22 55 41.35Santa Cruz do Arari 6 12 18 13.53Cachoeira do Arari 6 1 7 5.26Ponta de Pedras 2 1 3 2.25Soure 2 8 10 7.52Marapanim 0 1 1 0.75Others1 11 9 20 15.0

2. Amapa State 12 7 19 14.28Total 62 61 133 100.0

1 Origin not defined


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Table 3. Tracing back of animals with tuberculosis infection as a function of county and farm origin. Belem, 2002.

State/County of Number Total of Farms Number of InfectedOrigin Animals Animals

Chaves 55 A 27B 4C 2D 1E 7F 12G 1H 1

Cachoeira do Arari 7 O 3P 4

Santa Cruz do 18 M 1Arari N 1

H 16Soure 10 J 2

K 2L 6

Pontas de Pedra 3 QMarapanim 1 ND 1Others1 20 ND 20Amapa State 19 I 19Total 133 16 133

ND-Not determined


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Figure 1. Geographic location of counties and farms in the economic region of “Campos de Marajo”. Adapted from EMBRATER/EMATER-Para, EMBRAPA/CPATU (1980).


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The low productivity of buffalo herds of“Campos de Marajo” is the result of low nutritionalquality of the pastures planted on soils of low nativefertility without proper management, conditionsclosely associated to climate variability throughoutthe year and heavy rains in the wet season (Teixeiraand Serrao, 1984).

The edaphoclimatic characteristics indicatedand those predominant in the producing systemsrelated to grazing and nutrition seemed to be offundamental importance during flooding of theswampy areas, when the animals stay isolated onhigh elevations of pastures called “tesos”.According to Alfinito et al. (1986), these conditionsand long stays in pastures, with breastfeedingperiods longer than 300 days keep focuses and spreadthe tuberculosis in the regional breeding places.

The conditions described could lead toovercrowding, grazing deficiency and confinement,making easier the spread of the disease amonganimals, a problem also observed in India (Rajaramet al., 1996). Neil et al. (1989, 1991) found thatanimal contact and grazing density were importantfactors for the transmission of tuberculosis underexperimental conditions. Mycobacterium bovisexcretion has been frequently observed in tubercu-losis infection, and in natural transmission conditions,this excretion is observed around 87 days afterinfection.

The tuberculosis in depressed bovines andbuffaloes has been shown to be an infectionprocess with characteristics of endemic disease(personal reports). Alfinito et al. (1986) stated thatthis disease was introduced in Marajo Island fromimported Dutch black and white infected bovinesdiscarded from the Belem milk basin or infectedbuffaloes from farms of southern Brazil. Rajaramet al. (1996) reported that the introduction of bovinetuberculosis in India was in a similar way.Moreover, according to Andrews and Johston (1988),bovine tuberculosis became an endemic disease innorth Australia with the introduction of infectedbovine cattle and as a result of low support capacityof the pastures, high rainfall index in the coastal areaor semiarid conditions in the interior, grazing in open

flat areas and inadequate management of herds,factors which are present on Marajo Island.

The very low support capacity of pastures, oneAU/ha/year, and the low nutritional quality, whichcharacterize the producing system of “campos deMarajo”, are responsible for the nutritional status ofthe animals in the breeding places. Griffin et al.(1993) also indicated the nutritional factor as respon-sible for the outbreak of bovine tuberculosis inIreland.

It is also considered that the high rate of tuber-culosis infection in buffaloes is the result of theirassociative habits (Alfinito et al., 1986). Tullock(1967) cited by Mc Cool and Newton-Tabret (1979),revealed that buffaloes have the common habit ofdefecating and urinating in streams that maycontain large mycobacterium populations and fromwhich they drink water in drought periods. Ac-cording to Wray (1975), cited by Mc Cool andNewton-Tabret (1979), wild pigs frequently sharethese sources of water in which the mycobacteriumpersists for long periods as potential sources for trans-mission of agents of that disease.

CONCLUSIONS

1.Tuberculosis prevalency rates in buffaloes ofMarajo Island were higher in male than in femaleanimals;

2.The proportion of generalized cases of thedisease was high;

3. More than 40% of the infected animalscame from farms (eight out of sixteen) of Chavescounty on Marajo Island;

4. Factors of the physical environment such ashot and humid climate, long rainy and dry periods, aswell as low support capacity and nutritional qualityof the pastures, overcrowding and confinement forlong periods of time in the breeding places duringfloods seem to be of fundamental importance to theinfection and maintenance of the disease in theregion.


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REFERENCES

Alfinito, J., J. William and F.R.I. Olliveira. (1986).Estudo epidemiologico da tuberculose bovinana ilha de Marajo. IN. Congresso Brasileirode Medicina Veterinaria, 20, Cuiaba, 1986.Anais, Cuiaba, : 216-217.

Andrews, L. G. and J.M. Johnston. (1988). Epide-miology of bovine tuberculosis in northernAustralia. Veterinary Acta Scandinavica, 84(Suppl.,) : 139-141.

Brasil. Ministerio da Agricultura. Dipoa. Dicar.(1971). Inspecao de carnes. Padronizacao detecnicas, instalacoes e equipamentos. I.Bovinos. Currais e seus anexos, sala dematanca., Brasilia, 124 p.

Brasil. Ministerio da Agricultura. Secretaria DeDefesa Sanitaria Animal. (1996). Tuberculosebovina. Boletim de Defesa Sanitaria Animal,25 (1-4) : 53-56.

Brasil. Ministerio da Agricultura. Serpa. (1997).Regulamento de inspecao industrial e sani-taria de produtos de origem animal. Brasilia.(s.n.t.)

Centro Panamericano De Zoonosis. (1986). Manualde normas y procedimientos tecnicos parala bacteriologia de la tuberculosis. Martinez,Cepanzo. 43 p.

Embrater/Emater-Para, Embrapa/Cpatu. (1980).Sistema de producao para bubalinos.Microrregiao campos Marajo, Para, Belem, 37p.

Freitas, J. (1984). Tuberculose em um bufalo(Bubalus bubalis var bubalis - Linneus, 1758).Boletim FCAP, 14 : 33-42.

Freitas, J.A., M.J.C.S Barros and J.A.C.Nascimento. (1997). Alteracoes similares atuberculose observadas no abate de bovinos ebubalinos e no nivel de consumo em Belem-Para. FCAP Informe Tecnico, 23 : 22.

Freitas, J.A. (1999). Tuberculose em bubalinos(Bubalus bubalis var bubalis - Linneus, 1758).A doenca em bufalos abatidos paraconsumo: aspectos anatomopatologicos,microbiologicos e consideracoes relativasa saude publica. Sao Paulo: Faculdade deSaude Publica, Universidade de Sao Paulo.(Tese de doutorado).

Gee, R.W. (1986). Bovine tuberculosis eradicationin Australia. Review Scientific and Techno-logic of International Office of Epizooties.5(3) : 789-794.

Griffin, J. M., T. Hahesy, K. Lynch, M.D. Salman,Mc Carthy J., and T. Husley. (1993). Theassociation of cattle husbandry practices,environmental factors and farmer character-istics with the occurrence of chronic bovinetuberculosis in dairy herds in the Republic oflreland. Preventive Veterinary Medicine, 17: 145-160.

Hathaway, S.C., T.J. Ryan, A.C. Johnston, and G.W. De Lisle. (1991). The rolls of slaughter-house inspection in the diagnosis andcontrol of tuberculosis. Publication of MasseyUniversity goes Veterinary Continuing Educa-tion, 132 : 227-234.


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Letts, G. (1964). Feral animals in the northern terri-tory. Australian Veterinary Journal, 40(March) : 84-88.

Lau, H.D. (1990). Common buffalo diseases inAmazonian Brazil. Buffalo Bulletin, 9(4) : 75-77.

Mc Cool, C.J. and D. Newton-Tabrett (1979). Theroute of infection in tuberculosis in reralbuffalo. Australian Veterinary Journal, 55(August) : 401-402.

Moraes, M.J.S. (1990). Rapidas observacoessobre a tuberculose em bubalinos. Macapa:ASTER-AMAPA. p. 16.

Morris, R.S., D.U. Pfeifer, and R. Jackson. (1994).The epidemiology of Mycobacterium infec-tions. Veterinary Microbiology, (10) : 153-177.

Neil, S.D., J. Hanna, J.J. O’brien and R.M. McCracken. (1989). Transmission of tuberculosisfrom experimentally infected-cattle to in-con-tact calves. Veterinary Record, 124, March :269-271, March.

Neil, S.D., J. J. O’brien and J. Hanna. (1991). Themathematical model goes Mycobacteriumbovid excretion from tuberculosis cattle.Veterinary Microbiology, 28 : 103-109.

O’reilly, L.M. and C.J. Daborn. (1995) The epide-miology of Mycobacterium bovide infectionsin animals and man: the review. Tubercle andLung Disease, 76 (Suppl.,) : 1-46.

Rajaram, E.D., V.N.O. Rao, and R. Manickam(1996). Certain epizootiological features ofbovine tuberculosis. Indian Veterinary Jour-nal, 73 (April) : 435-438.

Ropiha, R. (1991). Practical aspects of tubercu-losis detection during beef processing.Massey University Continuing EducationVeterinary Publication, 132 (April) : 235-238.

Teixeira Neto, J.F. and E.A.S. Serrao. (1984).Produtividade estacional, melhoramento emanejo de pastagens na ilha de Marajo.EMBRAPA Comunicado Tecnico, 51 (Janu-ary) : 1-6.


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CLONING OF SWAMP BUFFALO AND CATTLE EMBRYOS USING THEIR EARFIBROBLAST CELLS AS DONOR NUCLEI AND IN VITRO MATURED SWAMP BUFFALO

OOCYTES AS RECIPIENT CYTOPLASTS

Sunpetch Sophon, Kriengsak Tasripoo, and Kitiya Srisakwattana

Research Centre for Bioscience in Animal Production, Faculty of Veterinary Science, Chulalongkorn Univer-sity, Henri Dunant St., Bangkok 10330, Thailand. E-mail : [email protected]

ABSTRACT

The present experiment was conducted toclone swamp buffalo and cattle embryos using invitro matured swamp buffalo oocytes as recipientcytoplasts and swamp buffalo and cattle ear fibro-blast cells (BEFC and CEFC) as donor nuclei andexamine the use of mouse fetal fibroblast cell asco-culture cell during embryos development. Swampbuffalo ovaries were collected from slaughterhouseand cumulus oocyte complexes (COCs) wereharvested by aspiration and then in vitro maturedfor 19-20 h. Matured oocytes were enucleated bymicromanipulation under an inverted microscope.Enucleation was confirmed by staining the removedportion with Hoechst 33342. Individual donor cellsof swamp buffalo or cattle ear fibroblast cells(diameter 14-16 µm) were inserted into perivitellinespace of recipient cytoplasts by micromanipulation.The couplets were fused with two DC pulses of 30V/couplet for 15 µsec each time in fusion medium.After fusion, reconstructed embryos were immedi-ately activated with A23187 followed by culturing inSOFaa-10 which containing cycloheximide andcytochalasin D for 5 h, then cultured in SOFaasupplemented with 1% FCS (SOFaa-1) for 2 days.The embryos at 8-cell stage were harvested andassigned to co-cultured with frozen-thawed buffalooviductal epithelial cell (BOEC) or mouse embry-onic fibroblast cell(MEFC) for another 2 days thenreplaced twice with SOFaa-10 every 2 days. Theresult from the experiment demonstrated that theembryos reconstructed by both BEFC and CEFCcould developed nearly the same rate to 8 cell (53.1

vs 51.0 %), morula(19.8 vs 17.0 %), and blastocyststage(15.6 vs 13.9 %) when co-culture with BOEC.While MEFC could support the reconstructedembryos developed to morula at the same rate (10.8%) and very low rate to blastocyst stage (2.8 vs 1.3%). The cloned cattle embryos could be producedusing swamp buffalo oocytes as recipient cytoplastsand co-culture with MEFC could support thedevelopment but not as good as BOEC. Thedevelopment of the cloned cattle embryos in therecipient cows will be further studied.

INTRODUCTIONThe previous report has demonstrated that

buffalo fetal fibroblast and ear fibroblast could bereprogrammed after transferring into recipientcytoplasts for producing the cloned embryos up toblastocyst stage (Parnpai et al., 1999) and also thereconstructed cattle embryos from ear fibroblast andsubsequent development in vivo (Parnpai et al.,2000), resulted the first cloning calf born alive inMarch 2000 and the second born in April 2001.

Because of the limited of cattle oocyte forcloning the cattle embryo, we would like to try touse buffalo oocytes, which we can obtain easier fromslaughterhouse, produce cattle embryos. Therefore,we have to examine the ability of buffalo oocytecytoplasm to support proliferation of somatic cellnuclei from cattle, fusion efficiency, rate of the firstembryonic cleavage, and rate of blastocystdevelopment of embryos derived by combiningcattle, and buffalo fibroblasts with buffalo recipientcytoplasm.


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Several studies have shown that oocytecytoplasm from bovines, rabbits, and sheep cansupport early development of embryos produced bynuclear transfer of somatic cell nuclei from variousmammalian species (Chen et al., 1999; Dominko etal., 1999; White et al., 1999; Lanza et al., 2000).Recently, the successes of cloning gaur (Lanza etal., 2000), mouflon (Loi et al., 2001), and giantpanda(Chen et al., 1999, 2002) have demonstratedthat the technique of interspecies cloning can bepractically applied.

Fibroblast growth factors (FGFs) make upa large family of polypeptide growth factors that arefound in organisms ranging from nematodes tohumans. In vertebrates, the 22 members of the FGFfamily range in molecular mass from 17 to 34 kDaand share 13-71% amino acid identity. Duringembryonic development, FGFs have diverse roles inregulating cell proliferation, migration anddifferentiation(Ornitz and Itoh, 2001). The use offibroblast cell from mouse embryos as co-culture,to secrete FGFs, may benefit to the embryonicdevelopment.

The present experiment was conducted toclone swamp buffalo and cattle embryos using invitro matured swamp buffalo oocytes as recipientcytoplasts and swamp buffalo and cattle ear fibro-blast cells (BEFC and CEFC) as donor nuclei andexamine the use of mouse fetal fibroblast cell asco-culture cell during embryos development.

MATERIALS AND METHODS Chemicals were obtained from Sigma

Chemical Company (St. Louis, MO) unless other-wise state.

Preparation of donor cell:Ear skin samples were obtained by biopsy

from adult female buffalo and cow, both sampleswere processed identically. Tissue was manually cutinto small pieces and placed on 60 mm cultured dish,top with sterile slide glass and gently pressed thenadd 5 ml αMEM supplemented with 10% FCS and3x Penicillin-Streptomycin-Fungizone, and placed inculture at 37 oC with 5% CO2 in air for 4 days. After

observed the first growth, change the medium andcontinued culture for 10 days (change the mediumevery 3 days). Cells were passaged approximatelyonce a week. Samples from progressively growingcell lines that were established were frozen with 20%DMSO in MEM supplemented with 10% FCS forfuture uses. Three to ten days prior to NT proce-dure, frozen-thawed fibroblasts were cultured inαMEM supplemented with 10% FCS and used asdonor cells for cloning during the 3-8 passages ofculture. A single cell suspension of the donor cellswas freshly prepared by standard trypsinization fewminutes before injection. The cells were pelleted andresuspended in Hepes-buffered TCM 199 supple-mented with 10% FCS (H 199-10) that ready forinjection as donor cells. Ear fibroblast cells,diameter 14-16 µm, were used as donor cells.

Preparation of mouse embryonic fibroblasts :Mouse embryos (13-14 days of gestation)

were decapacitated and it was minced finely in thedish containing Trypsin/EDTA then the tissue wasincubated for 30 minutes. The tissue was trans-ferred into 50 ml cornical tube and the supernatantwas pipetted into a new tube, cells were pelletedand seeded on 25 cm2 culture flask (Nunc) in αMEMsupplemented with 10% FCS. At subconfluence,fibroblasts were trypsinized and cultured up to thethird passage and frozen the same as donor cells.Frozen-thawed fibroblasts were cultured in αMEMsupplemented with 10% FCS and used as co-cul-ture cell by culture in SOFaa-10 4 days before useat concentration 106 cell/ml.

In vitro maturation of oocytes :The maturation procedure was previously

described by Parnpai et al., (1999). Briefly, Swampbuffalo ovaries were collected from slaughterhouseand cumulus oocyte complexes (COCs) wereharvested by aspiration from 3-6 mm follicles. COCswith compact, nonatretic cumulus oophorus-coronaradiata and homogenous ooplasm were selected andwashed 3 times with Dulbeccos phosphate buffersaline (DPBS) plus 0.1% PVA. Group of 10 COCswere then matured in 50 µl droplet of maturationmedium, TCM 199 supplemented with 10% buffalo


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follicular fluid, 1µg/ml E2, 50 IU/ml HCG(Chorulon(R), Intervet) and 0.02 AU/ml FSH. COCswere cultured at 38.5oC in a humidified 5% CO2 inair atmosphere for 19-20 h.

Enucleation, Microinjection and Fusion :Enucleation, Microinjection and Fusion was

performed as previously described (Parnpai et al.,1999). Briefly, cumulus cells were removed fromoocytes at 19-20 h post maturation (hpm) by gentlepipetting in 0.1% hyaluronidase in DPBS and washed4 times with Hepes-buffered TCM 199 supple-mented with 20% FCS (H 199-20). Matured oocyteswere enucleated in H 199-10 containing 7.5 µg/mlcytochalasin B by micromanipulation under invertedmicroscope. Enucleation was confirmed by stainingthe removed portion with 5 µg/ml Hoechst 33342and examined under an epifluorescence microscope.After enucle-ation, the cytoplasts were washed 4times with H 199-20 and kept in this medium untilinjection of the donor cells.Individual donor cells(diameter 14-16 µm) were inserted into perivitellinespace of recipient cytoplasts in H 199-10 by micro-manipulation under an inverted microscope.The couplets were fused at 24-26 hpm by placingindividual couplet between a pair of electrodes toelectrostimulate (BTX 200, Sandiego, CA) with twoDC pulses of 30 V/couplet for 15 µsec each time inZimmermann fusion medium supplemented with0.01% BSA. After electrical stimulus, the recon-structed embryos were washed 4 times with H199-20 and kept in this medium for 30 minutes thenchecked the success of fusion.

Activation :After fusion, reconstructed embryos were

immediately activated with A23187 in SOFaa supple-mented with 10%FCS (SOFaa-10) at roomtemperature (28oC) for 5 minutes then followedby culturing in SOFaa-10 which containing 10 µg/ml cycloheximide and 1.25 µg/ml cytochalasin D at38.5oC in a humidified 5% CO2 in air atmospherefor 5 h.

In vitro Culture of Cloned Embryos :After activation, reconstructed embryos

were cultured in 100 µl droplet of SOFaa supple-mented with 1% FCS (SOFaa-1) at 38.5oC in ahumidified 5% O2, 5% CO2 and 90% N2 for 2 days.At the end of this culture period, embryos at 8-cellstage were harvested and co-culture with BOEC orMEFC. The embryos were observed twice a dayunder an inverted microscope and recorded thedevelopment.

RESULTS AND DISCUSSION The average of maturation rate of

buffalo oocytes cultured in vitro was 35.1%. Byusing normal procedure which co-culture withBOEC, the fusion rate of BEFC and CEFC withbuffalo cytoplast were 80.5% and 93.7%. Cleavagerate of the reconstructed embryos from BEFC andCEFC, developed to 8-cell were 53.1% and 51.0%,Morula 19.8% and 17.0%, and Blastocyst 15.6%and 13.9%. The time from enucleation to blastocyststage of the embryo derived from BEFC was 5 dayswhile CEFC took 6 days (Table 1). The presentresult was lower than the previous worked whichgot 30.9% blastocyst when cloned buffalo embryosderived from fetal fibroblasts(Parnpai et al., 1999)and 49% of reconstructed cattle embryos co-cul-tured with cattle oviductal epithelial cell in SOFaadeveloped to blastocyst (Parnpai et al., 2000).

The cattle embryos in this study develop toblastocyst according to Parnpai et al.,(2000)reported that the first observation of blastocoelformation in reconstructed buffalo embryos was 128h after starting in vitro cultured. The developmentof embryos derived from the same source ofcytoplast but different source of nucleus wasconfirmed again. In vivo and in vitro development,the buffalo embryos develop faster than cattleembryos (Parnpai et al., 1985; Karaivanov et al.,1987; Sophon et al., 1989; Tasripoo andKamonpatana, 1997) which was confirmed again inthis study. In other interspecies cloned embryos,such as sheep-cow, pig-cow, and monkey-cow(Dominko et al., 1999) and mouse-rabbit andcat-rabbit (Chen et al.,2002), the timing of develop-ment was also donor species-specific.


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Table 1. The development of cloned embryo derived from Buffalo ear fibroblast cell (BEFC) and Cattle ear fibroblast cell (CEFC) using in vitro matured buffalo oocytes as cytoplast.

Donor Trials No Timing of No. fused No. of developted embryosnuclei enucle- Develop- oocyte

ated ment 8 cell Morula Blastocystoocyte (day)

BEFC 15 110 5 96(87.3) 51(53.1) 19(19.8) 15(15.6)CEFC 18 218 6 194(89.0) 99(51.0) 33(17.0) 27(13.9)

(number in parenthesis is %)

Development of the cloned embryos to blastocyst were obviously decrease when co-culture withMEFC(Table 2). However, MEFC could support the reconstructed buffalo and cattle embryos developed tomorula at the same rate (10.8 %) but lower rate to blastocyst stage of cattle embryos (2.8 vs 1.3 %).

Table 2. Development of cloned buffalo and cattle derived from Buffalo ear fibroblast cell(BEFC) and Cattle ear fibroblast cell(CEFC) using in vitro matured buffalo oocytes as cytoplast and co-cultured with buffalo oviduct epithelial cell(BOEC) and mouse embryonic fibroblast cell.

Donor Co- Trials No. No. fused No. of developted embryosnuclei culture enucle- oocyte

cell ated 8 cell Morula Blastocystoocyte

BEFC BOEC 15 110 96(87.3) 51(53.1) 19(19.8) 15(15.6)MEFC 15 228 176(77.2) 51(29.0) 19(10.8) 5(2.8)

CEFC BOEC 18 218 194(89.0) 99(51.0) 33(17.0) 27(13.9)MEFC 19 227 223(98.2) 115(51.7) 24(10.8) 3(1.3)

(number in parenthesis is %)

Our results show that the cytoplasm of in vitro matured buffalo oocyte has the ability to support bothBEFC and CEFC nuclear DNA reprogramming and further embryos development. The co-culture with MEFCcould support the development of the embryos but not as good as BOEC. The culture system of MEFC beforeuse as co-culture and the development of cloned buffalo and cattle embryos in the recipient cows should befurther studied.


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Figure 1. Buffalo oviductal epithelial cell(left) and mouse embryonic fibroblast cell(right).

Figure 2. Cloned embryos co-culture with buffalo oviductal epithelial cell at morula stage(left) andblastocyst(right).

Figure 3. Cloned embryos co-culture mouse embryonic fibroblast cell at 16 cell stage(left) and blastocyst(right).


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REFERENCES

Chen, D.Y., Q.Y. Sun, J.L. Liu, G.P. Li, L. Lian, M.K.Wang, Z.M. Han, X.L. Song, J.S. Li, Q. Sun ,Y.C. Chen, Y.P. Zhang and B. Ding (1999).The giant panda (Ailuropoda melanoleuca)somatic nucleus can dedifferentiate in rabbitooplasm and support early development of thereconstructed egg. Sci. China (Series C) 29:324-330.

Chen, D.Y. , D.C. Wen, Y.P. Zhang, Q.Y. Sun, Z.M. Han, Z.H. Liu, P. Shi, J.S. Li, J.G. Xiangyu,L. Lian, Z.H. Kou, Y.Q. Wu, Y.C. Chen, P.Y.Wang and H.M. Zhang (2002). Inter-speciesImplantation and Mitochondria Fate ofPanda-Rabbit Cloned Embryos. Biology ofReproduction 67: 637-642.

Dominko, T., M. Mitalipova, B. Haley, Z. Beyhan,E. Memili, B. Mckusick and N.L. First (1999).Bovine oocyte cytoplasm supports developmentof embryos produced by nuclear transfer ofsomatic cell nuclei from various mammalianspecies. Biol. Reprod., 60: 1496-1502.

Karaivanov, C., K. Vlahov, M. Petrov, D. Kacheva,M. Stojanova, A. Alexiev, O. Polironov, andA. Danev (1987). Studies on preimplantationdevelopment of buffalo embryos.Theriogenology 28: 747-753.

Lanza, R.P., J. Cibelli, F. Diaz, C. Moraes, P.W. Farin,C.E. Farin, C.J. Hammer, M.D. West and P.Damiani (2002). Cloning of an endangeredspecies using interspecies nuclear transfer.Cloning 2: 79-90.

Loi, P., G. Ptak, B. Barboni, J. Fulka, P. Cappaiand M. Clinton (2001). Genetic rescue ofan endangered mammal by cross-speciesnuclear transfer using post-mortemsomatic cells. Nat. Biotechnol. 19: 962-964.

Ornitz, D.M. and N. Itoh (2001). Fibroblast growthfactors. Genome Biology 2(3) : reviews 3005.1 – 3005.12

Parnpai, R., K. Tasripoo and M. Kamonpatana(1999). Development of cloned swampbuffalo embryos derived from fetal fibroblasts: comparison in vitro cultured with or withoutbuffalo and cattle oviductal epithelial cells.Buffalo J. 15(3) : 371-384.

Parnpai, R., K. Tasripoo and M. Kamonpatana(2000). Developmental potential of clonedcattle embryos derived from quiescentand non-quiescent adult ear fibroblastsafter different activation treatments.Theriogenology 53: 239.

Parnpai, R., V. Timsard, M. Kamonpatana, C.Pansin, S. Sophon, T. Jetana, A. Limsakul andC.R. Austin (1985). Recovery of swampbuffalo embryo using the non-surgicaltechnique. Buffalo Journal 1(1) : 77-82.

Sophon, S., R. Parnpai and M. Kamonpatana(1989). Multiovulation leading to embryotransfer in swamp buffaloes: using PMSG andFSH under oestrus regulation by norgestomet.Buffalo Journal 5(1) : 85-98.

Tasripoo, K. and M. Kamonpatana (1997).Assessment of factors contributing to in vitromaturation, fertilization and cleavage rate ofswamp buffalo oocytes. Buffalo Journal 13(3) : 325-336.

White, K.L., T. D. Bunch, S. Mitalipov and W. A.Reed (1999). Establishment of pregnancyafter the transfer of nuclear transfer embryosproduced from the fusion of Argali (Ovisammon) nuclei into domestic sheep (Ovisaries) enucleated oocytes. Cloning 1: 47-54.


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Y. Bingzhuang, L. Zhongquan, Z. Guiwen, Z.Qingkun, Q. Jing. Buffalo Research Institute,Chinese Academy of Agricultural Sciences,Nanning, Guangxi 530001, P.R. China.Present status on cross improvement andutilization of Guangxi buffalo and proposalfor near future. 1st Buffalo Symposium ofAmericas (2002). p. 281-286.

Guangxi has the largest population of buffa-loes in China, with 4.37 million indigenous and about2,000 exotic animals, representing 1/5 of the totalnumber in China. Guangxi Buffalo belongs to swamptype, whose body size is smaller than that of rivertype. It is mainly used for draught, with lowerperformance in terms of milk and meat. In order toimprove the productive performance, it is necessaryto carry out cross improvement. Cross improvementof indigenous buffaloes have been conducted withimported Murrah and Nili-Ravi buffaloes as sire.Significant effects were obtained that the annual milkyield is 1,233 kg for Murrah Cross F1, 1,585 kg forMurrah Cross F2, 2,041 kg for Nili-Ravi Cross F1,2,267 kg for Nili-Ravi Cross F2, and 2294 kg forTriple Cross, respectively. Experiment of slaughterof fattened Triple Cross bulls at 18-24 months showsthat carcass yield and net meat percentage are 53%and 43%, respectively. Productive performance ofcrossbred buffalo is increased 1-2 times more thanGuangxi Buffalo, with broad prospects for develop-ment and utilization.

E.C.A. Neves. Universidade Federal do Para-Departamento de Engenharia Quimica e deAlimentos/CT-Belem-Para-Brazil. Recentprogress concerning buffalo milktechnology in Amazon-Brazil. 1st BuffaloSymposium of Americas (2002). p.312-316.

The North of Brazil presents 50% of thecountry buffalo’s flock, however the milk is still usedin small scale for the obtaining of derived milkproducts, what should be motivated. The interest forthe elaboration of buffalo’s milk derived products isbecoming bigger due its high level of total solids andalso important, its high level of fat, besides thelactose’s and ash, all of them having a great nutri-tional importance, because these elements found inbuffalo milk - in better quality than the cow’smilk- will allow to complement the children’s andadult’s needs for protein, calories and mineralsfilling the required levels for the human diet (4). Therecent researches accomplished at Federal Univer-sity of Para State/CT/DEQAL-Brazil have as theirpurposes the buffalo milk valorization, through thecharacterization of the milk and of some productsalready elaborated in the area, the optimization ofthe technologies already existent and the creationof new products, mainly associated to fruits,increasing the value of the raw material of theAmazon area. There were researched the properprocess for the obtaining of Minas Frescal cheesewithout and with condiments, Ricotta, Mozzarellacheese without and with condiments, Cottage cheesewithout and with condiments or in addition ofcupuacu pulp, Marajoara cheese curd, processedcheese (melted) without and with cupuacu pulpaddition (Theobrama grandiflorum, Shum), yogurtwith regional fruits pulps and mixed pulp (cherry andcarrot), pasty sweet milk added of cupuacu(Theobrama grandiflorum, Shum), passion(Passiflora edulis) and regular or diet pasty sweetmilk added of bacuri (Platonia insignis, Mart) andbuffalo milk powder.

BREEDING AND GENETICS

RESEARCH ABSTRACTS

MANAGEMENT AND PRODUCTION


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B.L. Pander, S.S. Dhaka and S. Singh. Depart-ment of Animal Breeding, CCS HaryanaAgricultural University, Hisar-125 004,India. Genetic improvement of lifetimeperformance and longevity of Indianbuffaloes. Proceedings of the 7th WorldCongress on Genetics Applied to LivestockProduction, Montpellier, France, August,(2002). 7: 0-4.

The heritabilities of lifetime and first lactationtraits and genetic and phenotypic correlations among

J.R.F. Marques, M.S.M. Egito Albuquerque, A.S.Mariante and M.R. Costa. EmbrapaAmazonia Oriental, Trav. Dr. Eneas Pinheiro,s/n, CEP. 66.095-100-Belem PA. Geneticvariability of Brazilian buffaloes: from theintroduction to the molecular markers. 1st

Buffalo Symposium of Americas (2002). p.252-263.

The genetic variability reflects the differencesamong the organisms, which occur due to variationsin the environment and/or alterations in thegenotype of the individuals, in this case, beingtransmitted to future generations. The use ofmolecular markers has contributed a lot to thedevelopment and enhancement of these conceptsand also for the verification of the proposed models.It also permits the determination of the genetic valueof an animal as well, with high precision. Evenbefore the expression of its phenotype. Theproduction of an animal is the result of its geneticvalue (genotype) with the characteristics of theenvironment. The domestic buffaloes in the worldare classified according to many authors aspertaining to the Bubalus bubalis species, with twosubspecies, Bubalus bubalis bubalis -the riverbuffalo and Bubalus bubalis kerabao-the swampbuffalo. In general, the species Bubalus bubalis,known worldwide as water buffalo, is described withthree subspecies: a) Bubalus bubalis, varietybubalis -is the domestic buffaloes, presentingkaryotype 2=50 chromosomes, called river buffalo,including the herd of India, Pakistan, China, Turkeyand many European and American countries, eventhe Italian ones, which also belong to this subspe-cies; in Brazil it is represented by the breedsJafarabadi, Mediterraneo and Murrah, all known byBrazilian Association of Buffaloes Breeders -BABB; b) Bubalus bubalis, variety kerebao - thebuffalo found in Malaysia, Indonesia, Philippines,Ceylon, Thailand and others countries, presentingkaryotype 2=48 chromosomes; in Brazil it is repre-sented by the breed Carabao. It is known in the worldliterature as swamp buffalo; and c) Bubalus bubalis,variety fulvus-the native buffalo of the Northeastregion of India, especially from Assam, living

generally in the wild or semi-domesticated. It is ananimal of smaller size when compared to the pre-vious subspecies. Its tone of color is brownish orreddish, having a resemblance to the Baio type fromBrazil, without having any scientific proof. TheBrazilian buffaloes are four breeds officially knownby the BABB are: Carabao, Jafarabadi,Mediterranian and Murrah. The Baio type can beadded to them, probably belonging to the groupMurrah, with a small number of animals all over thecountry nowadays. At the end of the last centurymany import transactions of buffaloes were madefor our country, although the number of animals weresmall, mainly in the Murrah and Jafarabadi breeds.Molecular markers is a very important tools forelucidate this aspect. But, there aren’t manyreferences with molecular markers in buffaloes, thereare relevant works involving the morphologic,biochemical and molecular markers, comparingdifferent species as in the case of the domesticBuffaloes. This way, the genetic markers can reallyshorten the paths to be followed, especially at thisfirst moment with the similarity information amongthe herd and/or animals to be used as parents in thefuture generations. In order to determine whichcrossbreeding will have better responses and whichanimals inside a herd are genetically superior. Themolecular markers associated with the productivecharacteristics, will also be the most secureindicators to improve the buffaloes breedingprograms.


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FEEDING AND NUTRITION

S. Bartocci, C. Tripaldi and S. Terramoccia. IstitutoSperimentale per la Zootecnia, Via Salaria31, 00016 Monterotondo Sc., Rome, Italy.Characteristics of foodstuffs and diets, andthe quanti-qualitative milk parameters ofMediterranean buffaloes bred in Italyusing the intersive system: an estimateof the nutritional requirements of buffaloherds lactating or dry. Livestock Produc-tion Science (2002). 77(1): 45-58.

The work involved 20 buffalo farms situatedin central Italy; these were divided in three catego-ries: farms with low, intermediate and high milk yield(<8, 8-9, >9 kg/day). Samples of all forages (146)and feeds (134), used by the farms, were taken andanalysed; the net energy was calculated and 258lactating buffaloes were utilized for the experiment(approx equal to 20% were primiparous). Milk yieldwas controlled monthly as also was the number ofsomatic cells. In 2,258 milk samples, protein(Nx6.38), fat, pH and coagulation parameters were

determined. To estimate the requirements of thelactating herd, regression equations were drawn upbetween the daily milk yield (X) and the daily netenergy, crude protein, carbohydrates both structuraland nonstructural (Y) intake. The diets of thelactating buffaloes were administered ad libitum, asignificant difference was evident relative to theenergy level (0.82, 0.77, 0.73 Milk FU/kg DM;P<0.05) and protein values (134.78, 113.96, 101.40g/kg DM;P<0.05) on farms of high, intermediateand low milk yield, respectively. The average dailyintake of dry matter was 16.75 kg/head/day andsignificant differences resulted between farms ofhigh and low milk yield (17.16 vs 16.36 kg/head/day,P<0.05). Considering the average daily yield, dif-ferences were observed between categories (10.46,8.21, 7.27 kg/head/day; P<0.01); the average valueof milk protein was 47.71 g/kg; significantdifferences were found between the intermediate-high yield farms compared to those of low milk yield(47.97, 47.66 vs. 47.02 g/kg; P<0.01); the highestfat values were found in high yield farms, statsticallydifferent from those of intermediate yield but notfrom those of low yield (88.29, 88.26 vs. 84.69 g/kg;P<0.01). The intermediate yield farms showed thehighest somatic cell value (266.38 vs. 182.96, 193.22n x 1000/ml; P<0.01). Milk showing the best clottingability was produced on the high yield farms(A30=49.42 mm, r=14.88 min, K20=3.06 min). Therequirements of dry buffalo herds were reported:dry matter intake of 10.61 kg/day, energy concen-tration of 0.63 Milk Fu/kg DM and protein of 78.96g/kg DM; for a milk yield which oscillates between7 and 12 kg/day (average yield for the shole lacta-tion period), the energy-protein concentrations varyfrom 0.74-0.89 Milk FU/kg DM and from 101.64-150.76 g/kg DM.

lifetime traits and between first lactation andlifetime traits are reviewed. Estimate of heritabilityof lifetime traits were lower than those of firstlactation traits. Genetic correlations among lifetimetraits were generally high. Estimate of geneticcorrelation between first lactation yield (FLY) andlifetime total milk yield (LTMY) was 0.80 andbetween first lactation peak yield (FPY) and LTMYwas 1.0. Indirect selection for LTMY based on FLYplus FPY was found to be more accurate thandirect selection. Some measure of longevity (herdlife/length of productive life/total number of days inlactation) of bull dams and sisters should also beconsidered while selecting young bull for progenytesting.


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HEALTH AND DISEASES

W.L. Pereira, E.C. Cardoso and E. Tury. CaixaPostal 917. CEP. 66077-530. Belem, Pa.Brazil. Hemossiderosis in buffaloes fromMarajo Island, PaRa State, Brazil. 1st

Buffalo Symposium of Americas (2002). p.387-389.

Hipocromic anemia was already evidenced inbuffaloes coming from Marajo Island conditioned athigh concentration of iron (Fe) and low concentra-tion of copper (Cu) in the liver tissue and in the bloodplasma and the present paper is an integrant part ofthis research. The study objectified to quantify theconcentrations of the hemosiderin present in the liverand its relation with the copper and iron concentra-tions. Histological fragments of liver tissue from 90buffaloes coming from several municipal districts ofMarajo Island, Para State, Brazil was analyzedusing Kruskal-Wallis’ test. In order to verify theexistence of relevant differences among copper andiron concentrations in the liver, four groups ofanimals were formed according to thehemossiderosis degree: N (Not observed), + (lowaccumulation), ++ (Moderated) and +++ (highaccumulation). Hemossiderosis became evident in48% of the analyzed cases and did not demonstratea co-relation to the concentration of Cu in the liver.There was a significance co-relation (p<0.01)between hemosiderosis degree and Fe concentra-tion in the liver. The results could demonstratedirectly or indirectly that hemosiderosis possiblebecomes from the Fe concentration in the diet (highFe in soil and in grass) decreases Cu absorptionby intestinal tract and promoted the hipocromicanemia, high concentration of Fe in the liver andhemossiderosis.

H.D. Lau, N.A. Costa, J.B. Lourenco-Junior andR.S.C. Macedo. Research of EmbrapaAmazonia Oriental. P. Box, 48. Belem, ParaState, Brazil. Rotational grazing for hel-minth control of buffaloes in wet tropicalenvironment- Brazil. 1st Buffalo Symposiumof Americas (2002). p.379-381.

This paper examines relationships betweenintensive rotational grazing and buffalogastrointestinal helmintoses. The preliminary resultsindicated that a rotational grazing system consistingof 6 paddocks grazed in sequence for 5 days at atime may permit a control of gastrointestinalparasites in this animal species. The effects ofmanagement on helminth control seen insufficientlyunderstood not only by farmers, but by theirprofessional advisers. Because of the expense ofanthelmintic treatment and anthelmintic-resistancenematodes, rotational grazing deserves furtherinvestigation as a buffalo nematode control in wettropical environment.

D.V. Singh, R.V. Singh and S.P.S. Sodhi. PunjabAgricultural University, Ludhiana, Punjab141 004 India. Effect of hypertonic salinesolution infusion on haemodynamics ofendotoxemic buffalo calves. Indian Jour-nal of Animal Sciences (2002). 72(12): 1083-1086.

Apparently healthy male buffalo calves (10)were subjected to endotoxic shock by i/v infusion ofE. coli endotoxin @ 5 µg/kg BW/h for 3 h. Out ofthese 1 group of 5 calves was left untreated andobserved for next 4 h while the other group of 5calves were given rapid i/v infusion of hypertonic


93

saline solution (7.2% NaCl) @4 ml/kg BW in 6.5min. The endotoxin infusion caused a significantdecline in systolic, diastolic, pulse pressure, meanarterial pressure (MAP) and central venouspressure (CVP) in both groups during endotoxininfusion. In hypertonic saline treated group,following infusion, all these parameters showed vastimprovement while in untreated group, these valueswere significantly lower than normal till end ofobservation period. Mean body temperature variedonly nonsignificantly while respiratory rate wasmarkedly elevated in both groups during and afterinfusion of endotoxin. The various beneficial effectsof hypertonic saline on haemodynamics weretransient in nature and had no effect on bodytemperature and respiration.

E.C. Cardoso, C.F.B. Sa, R. Rodrigues, A.H.H.Minervino, V.S.S.F. Sa and J.B.S. Sousa.FCAP/DZ, P. Box 917, CEP 66.077-530Belem, Para State, Brazil. Effect of thecalcium and copper etilenodinitrilotetracetato (glypodin) on the concentra-tion of copper in the blood plasma ofbuffaloes calves. 1st Buffalo Symposium ofAmericas (2002). p. 390-392.

The study was developed at the “FelisbertoCamargo Experimental Station” of EmpresaAgropecuaria da Amazonia Oriental (EmbrapaEastern Amazon), located in Belem, of Para State,north of Brazil. There was utilized fourteen buffa-loes aging six to eleven months in order to evaluatethe effect of Glypondin, medication on the copper(Cu) concentration in blood plasma of buffaloescalves. The animals were separated in twodifferent treatments (T1 and T2), being T1 overseven animals that received Glypondin applicationat sub-cutaneous doses. The T2 group was used asa control and was constituted of seven animals thatdid not receive the medication. Since the first appli-cation and during 60 consecutive days, samples of

blood were collected fortnightly in order to determi-nate the concentration of Cu in the blood plasma.The experimental delineation was entirely casual,acquiring the bits subdivided model where theperiods were considered as sub-bits inside thetreatments. Glypondin in a sub-cutaneous dose of3ml (0.075 g of calcium and copper etilenodinitrilotetracetato) has increased the concentration of Cuin blood plasma in weaned buffalos calves and it ispossible its utilization as a medication treatment forhipocuprosis cases in these species.

Archana, C.K. Singh and V.S. Senthil. PunjabAgricultural University, Ludhiana, Punjab141 004, India. Detection of rabies anti-gen in experimental buffalo calves byDAS-ELISA. Indian Journal of AnimalSciences (2002). 72(11): 963-964.

Street rabies virus isolated from a sponta-neous case of a dog was experimentally inoculatedinto buffalo calves via oral and intra-thecal routes.DAS-ELISA detected rabies antigen in all braintissues except cerebrum in one group and exceptmedulla oblongata in other group at 30 days post-inoculation (DPI). However, at 60 DPI, all the braintissues were positive for rabies.

Lau, H.D. Research of Embrapa AmazoniaOriental. P. Box. 48. Belem, Para State,Brazil. Management system and healthdisorder relationship in buffalo farming-Brazil. 1st Buffalo Symposium of Americas(2002). p. 376-378.

This prospective observational study wasundertaken to quantify the incidence of health disor-ders of buffalo associated with the farm manage-ment practices. Data were obtained from the 165


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REPRODUCTION

N.A.T. Carvalho, L. Zicarelli, J.A. Visintin, P.S.Baruselli, E.H. Madureira and M.J. D’Occhio.Department of Animal Reproduction,Faculty of Veterinary Medicine, Universityof Sao Paulo, Rua Prof. Orlando Marquesde Paiva, 87, CEP, 05508-000 Sao Paulo,SP, Brazil. Control of ovulation with aGnRH agonist after superstimulation offollicular growth in buffalo: fertilization andembryo recovery. Theriogenology (2002).58(9): 1641-1650.

The potential to use a GnRH agonist bioimplantand injection of exogenous LH to control the time ofovulation in a multiple ovulation and embryotransfer (MOET) protocol was examined in buffalo.Mixed parity buffalo (Bubalus bubalis; 4-15-year-old; 529 ± 13 kg LW) were ranodomly assigned toone of five groups (n=6): Group 1, conventionalMOET protocol; Group 2, conventional MOET with12 h delay in injection of PGF2α ; Group 3, implantedwith GnRH agonist to block the preovulatory surgerelease of LH; Group 4, implanted with GnRHagonist and injected with exogenous LH (Lutropin(R) , 25 mg) 24 h after 4 days of superstimulationwith FSH; Group 5, implanted with GnRH agonistand injected with LH 36 h after superstimulation withFSH. Ovarian follicular growth in all buffaloes wasstimulated by treatment with FSH (Folltropin-V(R),200 mg) administered over 4 days, and was moni-tored by ovarian ultrasonography. At the time ofoestrus, the number of follicles more than or equalto 8 mm was greater (P<0.05) for buffaloes in Group2 (12.8) than for buffaloes in Groups 1 (8.5), 3 (7.3),4 (6.1) and 5 (6.8), which did not differ. All buffa-loes were mated by AI after spontaneous (Groups1-3) or induced (Groups 4 and 5) ovulation. Therespective number of buffalo that ovulated, num-ber of corpora lutea, ovulation rate (%), and

buffalo farms. According the health status, four farmtypes were identified: Type 1- (11.5%) charac-terized by low health disorders incidence. Type 2 -(18.8%) with high incidence for specific reproduc-tive disorders. Type 3 - (38.7%) with high incidenceof calf disorders, and Type 4 - with high incidenceof all health disorders. The variables significantlyassociated with the health disorders in the type 1were: Belem region location, herd with 100 to 200heads, unique pasture cultivated, more than 20 plots,vaccination against all regional diseases, regularlysalt supplementation, strategic helminth control,regularly ectoparasites control and calf healthmanagement. In the others farm types was observedfailures in this practices.

H.F.L. Ribeiro. Section of Animal Reproduction,DPMVP-FCAP, Av. Presidente TancredoNeves, S/N Terra Firme-Po Box 917, CEP66.077-530-Belem-Para-Brazil. Reproduc-tive management of buffalo in Amazonvalley. 1st Buffalo Symposium of Americas(2002). p. 172-785.

Maximizing reproductive efficiency is themajor economic importance to buffalo production.The development of new and improved methods ofincreasing reproductive performance in femalebuffaloes depends on understanding of the physi-ologic and endocrinologic mechanism controlling thereproductive process. The mechanism of puberty andsexual maturity are more accomplished with themanagement and nutrition where the animals wereraised. In Amazon region female buffaloes show apolyestrus continuous pattern, however when it istaken in account the place where the animal is raised,more for the northern or southern regions thespecies show a polyestrus seasonal behaviour. Onthe other hand, factors that modify or completelyinhibit estrus are discussed, and strategies aresuggested for improving the detection of estrusthrough the use of teaser buffalo bulls. Environmentstress play a big role in the reproductive efficiencyand are very close associated with nutrition, housing

and other phenomenom that demand a response fromthe animal to adapt to new environment, mainly inthe tropical humid regios of Brazil. Artificial insemi-nation has allowed rapid genetic improvment inbuffalo herds and has resulted in a marked increasein the buffalo milk and meat production.


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embryos+oocytes recovered were: Group 1 (2, 1.8± 1.6, 18.0 ± 13.6, 0.2 ± 0.2); Group 2 (4, 6.1 ± 2.9,40.5 ± 17.5, 3.7 ± 2.1); Group 3 (0, 0, 0, 0); Group 4(6, 4.3 ± 1.2, 69.3 ± 14.2, 2.0 ± 0.9); and Group 5 (1,2.5 ± 2.5, 15.5 ± 15.5, 2.1 ± 2.1). All buffaloes inGroup 4 ovulated after injection of LH and had arelatively high ovulation rate (69%) and embryorecovery (46%). It has been shown that the GnRHagonist-LH protocol can be used to improve theefficiency of MOET in buffalo.

R.G. Shah, A.J. Dhami, F.S. Kavani, V.B. Kharadiand P.M. Desai. Gujarat AgriculturalUniversity, Anand, Gujarat 388 001, India.Effect of gonadotrophin releasing hormoneon reproductive performance and steroidprofile of postpartum suckled Surti buffa-loes. Indian Journal of Animal Sciences(2002). 72(12): 1076-1082.

The effect of GnRH (250 micro g im) onpostpartum Surti buffaloes with smooth inactiveovaries until day 40 was studied on 6 animals, and 5were kept as control. Blood plasma profiles ofprogesterone (P4) and estradiol-17beta (E2-17beta)

were estimated by standard RIA procedures. Thereproductive performance and ovarian eventsrecorded with the help of rectal palpation findingswere correlated to P4 and E2 profiles. Significantlyhigher P4 levels were observed in the GnRH treatedthan the control buffaloes on 9-10 weeks (2.78 ±0.71 vs. 0.87 ± 0.49 ng/ml) and 11-12 weeks (2.86 ±0.83 vs. 1.04 ± 0.52 ng/ml) postpartum. The plasmaE2-17 beta levels following GnRH treatmentincreased for variable periods with a typical profilein 3 buffaloes followed by oestrus. GnRH treatedbuffaloes resumed ovarian activity on day 45.83 ±0.30 postpartum in contrast to day 64.00 ± 2.27 in 4untreated buffaloes. The occurrence of first heatpostpartum in GnRH treated vs. control group was54.83 ± 2.18 vs. 96.75 ± 14.39 days (P<0.05). Thetreatment to ovulation interval in 5 of 6 treatedbuffaloes was 15.40 ± 2.58 days with an overall meanof 19.00 ± 4.16 days. All GnRH treated buffaloesconceived by 80 days postpartum. Of these, 33%conceived within 60 days and 67% between 61-80days postpartum. In control group, conception ratebefore day 60, 61-80, 81-90, and 121-150 postpar-tum was 0, 20, 20, and 20%, respectively. Theremaining 40% control buffaloes conceived after 370days postpartum. Thus, GnRH treatment has a goodtherapeutic action to initiate early ovarian activityand to enhance reproductive efficiency in Surtibuffaloes.


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BUFFALO BULLETINIBIC, KASETSART UNIVERSITY, P.O. BOX 1084

BANGKOK 10903, THAILANDURL: http//ibic.lib.ku.ac.thE-mail: [email protected]

Tel: 66-2-5795017Fax: 66-2-5611369

CONTENTS

Page

Some epidemiological aspects of tuberculosis in water buffaloes inthe Amazon basin of Brazil.Jose de Arimatea Freitas and Jose Cezar Panetta ........................................ 75

Cloning of swamp buffalo and cattle embryos using their ear fibroblast cells asdonor nuclei and in vitro matured swamp buffalo oocytes as recipient cytoplasts.S. Sophon, K. Tasripoo and K. Srisakwattana ............................................. 83

RESEARCH ABSTRACTS............................................................................ 89

Documents

BUFFALO BULLETIN - Kasetsart UniversityBuffalo Bulletin (December 2002) Vol.21 No. 4 77 Table 1. General prevalence rates of tuberculosis, rates by sex and forms of lesions in slaughtered