2008-4buffalo bulletin

Volume 27 No. 4

International Buffalo Information Center

(IBIC)

Aims

IBIC is a specialized information center onwater buffalo. Established in 1981 by KasetsartUniversity (Thailand) with an initial financialsupport from the International DevelopmentResearch Center (IDRC) of Canada. IBIC aims atbeing the buffalo information center of buffaloresearch community through out the world.

Main Objectives

1. To be world source on buffalo information 2. To provide literature search and photocopy services 3. To disseminate information in newsletter 4. To publish occasional publications such as an inventory of ongoing research projects

BUFFALO BULLETINISSN : 0125-6726

Buffalo Bulletin is published quarterly in March,June, September and December. Contributions onany aspect of research or development, progressreports of projects and news on buffalo will beconsidered for publication in the bulletin. Manu-scripts must be written in English and follow theinstruction for authors which describe at inside ofthe back cover.

EditorS. Sophon

Publisher International Buffalo Information Centre, Main Library, Kasetsart University

Online availible:http://ibic.lib.ku.ac.th/e-Bulletin

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

BANGKOK 10903, THAILAND URL : http://ibic.lib.ku.ac.th E-mail : [email protected] Tel : 66-2-9428616 ext. 344 Fax : 66-2-9406688

Buffalo Bulletin (December 2008) Vol.27 No.4

PREVALENCE OF DIFFERENT SPONTANEOUS LIVER LESIONS IN BUFFALOES INMALWA REGION OF MADHYA PRADESH

G.P. Jatav, U.K.Garg and Supriya Shukla

ABSTRACT

The present investigation was carried outto study the various pathological conditions occurringspontaneously in the livers of buffaloes. Examinationof livers from a total number of 510 buffaloesranging from 4 to12 years of age were examinedfor liver affections. The material was collected fromCantonment Board Slaughter House, Mhow (M.P).Among the various pathological conditions of thelivers, toxic hepatitis was found to be the highestfollowed by fatty changes, hydatidosis, amyloidosis,fascioliasis, hypertrophy, cloudy swelling,pigmentation, hyaline degeneration, neoplasm andcirrhosis and suppurative hepatitis.

Keywords: pathology, pathological conditions, liver,buffalo, toxic hepatitis, fatty changes, hydatidosis,amyloidosis, fascioliasis, hypertrophy, cloudy swelling

INTRODUCTION

The buffalo is the predominant domesticanimal for milk and meat production. On average,buffaloes are about four times as productive as anaverage indigenous cow in India. India has world’sbest buffalo dairy breeds and provides superiorbuffalo germplasm to several countries of the world(Kaikini, 1992). Buffaloes are very prone to variousbacterial, viral, fungal and parasitic diseases includingother diseases of diverse etiology. The liver is thefirst organ of the body that undergoes pathologicalchanges when an animal suffers from acuteinfection and the last organ to assume normalcy(Gracey, 1981). The present investigation wascarried out on the livers of buffaloes to investigatethe various pathological conditions of the liver.

MATERIALS AND METHODS

The materials for the present studycomprised livers obtained from buffaloes slaughteredat the Cantonment Board Slaughterhouse, Mhow,(M.P.) which had been brought from different partsof the Malwa region as a source of meat. A totalnumber of 510 buffaloes ranging from 4 to 12 yearsof age were examined for liver lesions. Out of these,10.19% specimen showing gross lesions werecollected and preserved in 10% neutral formalinsolution for hispathological study. After 48- 72 h,these tissue pieces were washed overnight in runningtap water, dehydrated in ascending grades of alcohol,cleared in benzene and embedded in paraffin wax(60-62 0C melting point). Sections of 4-6 micronthickness were cut and stained with haematoxylinand eosin as per the standard procedurerecommended by Lille (1954).

RESULTS AND DISCUSSION

The pathological changes observed duringthe present study, were as shown in (Table 1).Examination of livers of 510 buffaloes revealedvarious types of lesions in 52 animals amounting toan incidence of 10.19%, which was in consonancewith the observations made by Keller (1952), Verma(1963), Kulkarni and Deshpandey (1985), Hussainet al. (1992) and El-Shazley (2002).

In present investigation, toxic hepatitis wasone of the pathological conditions encountered mostoften in the livers, i.e. 2.35%, followed by fattychanges and hydatidosis (1.17%), amyloidosis andfascioliasis (0.98%), hypertrophy, cloudy swelling

Department of Veterinary Pathology, College of Veterinary Science & A.H., Mhow, M.P., India

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and pigmentation (0.78%), hyaline degeneration andneoplasm (0.58%), and cirrhosis and suppurativehepatitis (0.39%), The highest incidence of toxichepatitis is reported by Newsom (1952) andPurushothaman and Rajan (1986)) in livers. Thisfinding was associated with the consumption of toxicfeeds by the animals.

REFERENCES

El-shazley, A.M., S.A. El-wafa, I.M. Haridy,M.Soliman, M.M.A.Rifaat and I.A. Morsy.2002. Fascioliasis among live and slaughteredanimals in nine centers of Dakahliagovernorate. Journal of the Egyptian so-ciety of Parasitology, 32(1), 47-58.

Table 1. Pathological conditions in the livers of slaughtered buffaloes.

Gracey, J.R. 1981. Thronton’s meat Hygiene, 7 th

ed. The English language book society andDailier, Tindall, London. 436p.

Hussain, A., A. Maqbool, S. Hussain, M. Athar, A.Shakoor and M.K. Amin. 1992. Studies onprevalence and organ specificity of hydatidosisin ruminant slaughtered at Karachi andFaisabad abattoir, Pakistan. Indian J. Vet. Sci.,45(9): 454-456.

Kaikini, A.S. 1992. Dimensions of infertility or sterilityin cattle and buffaloes. Indian J. Anim.Reprod., 13: 10.

Kulkarni, V.G. and B.B. Deshpandey. 1985. Anabattoir study of liver diseases in Indianbuffaloes. Livestock Adv., 10(4): 57-61.

Lillie, R.D. 1954. Histologic Technique andPractical Histochemistry. The Blakistan Div.McGraw Hills Book Company, Toronto.

Verma, R.D. 1963. A study on bovine liver lesions.M.V.Sc. Thesis, Jabalpur University.

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S. No.

Pathological Condition

No. of Cases

Percentage of individuals

having a condition among affected animals

Over all percentage out of

total animals examined

1. Atrophy 3 5.76 0.58%

2. Hypertrophy 4 7.69 0.78%

3. Cloudy Swelling 4 7.69 0.78%

4. Fatty Changes 6 11.53 1.17%

5. Amyloidosis 5 9.61 0.98%

6. Hyaline Degeneration 3 5.76 0.58%

7. Cirrhosis 2 3.84 0.39%

8. Suppurative hepatitis 2 3.84 0.39%

9. Toxic hepatitis 12 23.07 2.35%

10. Fascioliasis 5 9.61 0.98%

11. Hydatidosis 6 11.53 1.17%

12. Pigmentation 4 7.69 0.78%

13. Neoplasm 3 5.76 0.58%


PERIPHERAL PLASMA FSH CONCENTRATIONS IN RELATION TO EXPRESSION OFOESTRUS IN MURRAH BUFFALOES (BUBALUS BUBALIS)

ABSTRACT

The present study investigated the changesin peripheral plasma FSH concentrations in relationto expression of estrus in Murrah buffaloes. Out ofa total of seven oestrus, two were accompanied byovert signs whereas the remaining five were silentoestrus. In buffaloes with overt oestrus, plasma FSHconcentrations during perioestrus, early luteal,midluteal and late luteal phase were 2.61±0.64,1.73±0.28, 1.17±0.16 and 1.16±0.22 ng/ml,respectively and the corresponding values inbuffaloes with silent oestrus were 2.42±0.69,0.85±0.12, 0.99±0.18 and 1.62±0.47 ng/ml,respectively. It was concluded that plasma FSHlevels were higher (P<0.01) in buffaloes thatexhibited overt oestrus compared to silent estrus.

Keywords: FSH, oestrus, buffalo

INTRODUCTION

Understanding of the endocrine factors thatregulate oestrus is essential for development ofreproductive strategies to improve the detection ofoestrus behaviour (Allrich, 1994). Poor expressionof oestrus is one of the major factors impedingefficient utilization of tropical Murrah buffaloes.Oestrus is observed by behavioural symptoms, butpractically this is not possible in situations whereherd size is large and animals are stallfed. Oestrusdetection is also very difficult due to lack of expertpersonal, variation of duration of oestrus andreluctance of some teaser bulls to mate. PituitaryFSH is essential for development and maintenance

of ovarian follicles in single and multiple ovulatingspecies (Taya et al., 1991). FSH in peripheral plasmahas also been implicated in expression of oestrusand ovulation (Akbar et al., 1974; Dobson, 1978).There is no information available on peripheralplasma FSH concentrations in relation to expressionof behavioural oestrus symptoms in buffaloes. Thepresent study was, therefore, undertaken to measureperipheral plasma FSH concentrations during theoestrous cycle and to relate them to occurance ofovert or silent oestrus in buffaloes.


Experimental animals and blood samplingFive non-pregnant, non-lactating Murrah

buffaloes (5-6 years old and having body weightbetween 550-600 kg) were selected from theNational Dairy Research Institute animal herd andmaintained under standard feeding and managementschedule as practised in the herd. Blood samples(20 ml) were collected through jugular venipuncturedaily for 32 consecutive days during the wintermonths of January and February. Blood sampleswere centrifuged at 3000 rpm for 30 minutes andplasma was harvested and stored in deep freeze at-20 0C for analysis of progesterone. Estrus wasdetected by parading a vasectomized bull twice dailyand estimating plasma progesterone concentrations.

Hormonal assayPlasma FSH was estimated by double

antibody RIA developed in our laboratory (Palta andMadan, 1995). The sensitivity of the assay was 0.4ng/ml. The intra- and inter-assay coefficients of

S. Mondal1, B.S. Prakash2 and P. Palta2

1National Institute of Animal Nutrition and Physiology, Adugodi Bangalore-560 030, India. e-mail: [email protected] Dairy Research Institute, Karnal, Haryana-132 001, India

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variation were <10% (n = 6). The crossreaction ofFSH antiserum with other hormones were LH 7%;prolactin 0.20%; GH 0.48% and TSH 3.6%. Forstatistical analysis the oestrous cycle was dividedinto four phases, namely, the late luteal phase (day -4 to day -2, day 0 = day of estrus), the periestrusphase (day -1 to day 1), the early luteal phase (day2 to day 5) and the mid luteal phase (day 6 to day14). The changes in peripheral plasma FSH andprogesterone concentrations during different phasesof the cycle in cows exhibiting both overt and silentestrus were analysed by analysis of variance(Snedecor and Cochran, 1967).


Out of a total of seven oestrus, two wereaccompanied by overt signs whereas the other fivewere silent oestrus. The plasma FSH concentrationsin buffaloes that exhibited overt oestrus behaviourand those having silent oestrus during different daysof the cycle are depicted in Table 1. Among differentdays of the oestrous cycle, the mean (±S.E.M.)plasma FSH concentrations ranged from 0.47±0.09to 4.13±0.21 ng/ml and from 0.65±0.15 to 3.42±0.34ng/ml in buffaloes that exhibited overt and silentoestrus, respectively. Plasma FSH concentrationsincreased from 1.67±0.44 ng/ml on day - 4 to reacha peak concentration of 4.13±0.21 ng/ml on day 0(day of oestrus) and decreased thereafter to0.77±0.12 ng/ml on day 8 in buffaloes that exhibitedovert oestrus (Table 1). Plasma FSH concentrationsdecreased from 1.73±0.28 ng/ml during the earlyluteal phase to 1.17±0.16 ng/ml during the mid lutealphase and then decreased to 1.16±0.22 ng/ml duringthe late luteal phase following which they increased(P<0.05) to 2.61±0.64 ng/ml during the perioestrusphase (Figure 1). A similar trend was observed inbuffaloes that had silent oestrus; plasma FSHconcentrations increased from 1.23±0.14 ng/ml onday -4, reached a maximum concentration of3.42±0.34 ng/ml on day 0, and then decreased to0.91±0.04 ng/ml on day 6 (Table 1). Plasma FSHconcentrations increased from 0.85±0.12 ng/mlduring the early luteal phase to 0.99±0.18 ng/mlduring the mid luteal phase and then increased to

1.62±0.47 ng/ml during the late luteal phase followingwhich they increased (P<0.05) to 2.42±0.69 ng/mlduring the perioestrus phase (Figure 1). The patternof plasma FSH levels during the oestrous cycle arein agreement with earlier reports in rats and humans(Gay et al., 1970), monkeys (Boorman et al., 1973),ewes (L’Hermite et al., 1972), cows (Akbar et al.,1974; Barnes et al., 1980; Kaneko et al., 1992),buffaloes (Heranjal et al., 1979; Mondal et al., 2000;Mondal et al., 2001; Razdan et al., 1982) in termsof peak level on the day of oestrus compared toother days of the oestrous cycle. Akbar et al. (1974)reported that serum FSH levels on the day of oestrustended to be higher than follicular or luteal phaselevels in cattle. Plasma FSH concentrationsincreased 9 fold on the day of oestrus compared toother days of oestrous cycle in cattle (Barnes et al.,1980). Kaneko et al. (1992) reported that theconcentrations of plasma FSH were relatively highduring the late luteal phase and then declinedgradually to low levels during the follicular phase incattle. Peak levels of FSH were detected on theday of oestrus in Murrah buffaloes; the levelsdeclined gradually within the next 3-6 days andfluctuated before rising again to the peak levels atthe next oestrus (Heranjal et al., 1979). A suddenrise in LH levels was seen on the day of oestrus incattle, but the levels of both FSH and LH attainedhigh values on this day (Niswender et al., 1974).Following oestrus, decline in FSH is slow whereasthat of LH is sharp. Increasing levels of estradiolduring follicular phase exert a positive feedback onthe hypothalamo-hypophyseal axis resulting inrelease of peak levels of gonadotropins duringoestrus. The increased secretion of FSH and LHmay be due to increased release of GnRH from thethalamus or to increased sensitivity of FSH and LHsecreting cells within the adenohypophysis to GnRH(Niswender et al., 1974).

The growth and development of follicles hasbeen reported to be bimodal in cattle (Ginther et al.,1989; Pierson and Ginther, 1988) and buffalo (Maniket al., 1998). The first and second waves occur atdays 1 and 11, respectively, in cattle and at days0.20 and 9.20, respectively, in buffaloes. In eachwave a dominant follicle emerges along with a cohortof subordinate follicles. Kaneko et al. (1995)reported that plasma levels of FSH were high prior

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to emergence of each follicular wave and low duringthe growing phase of a dominant follicle. Our studysuggests that plasma FSH concentrations weremaximum on the day of oestrus in buffaloes thatexhibited overt and silent oestrus. Following adecrease thereafter, FSH concentration rose againand reached high concentrations on days 7 and 8 inbuffaloes that exhibited overt and silent oestrus,respectively, prior to emergence of the second wave.An inverse relationship has been observed betweenperipheral plasma inhibin and FSH concentrationduring the oestrus cycle in cattle (Taya et al., 1991)

and buffaloes (Mondal et al., 2000). The lowconcentrations of inhibin during the mid luteal phase,an increase through the late luteal and perioestrusphases and a decrease thereafter may play animportant role in regulation of FSH secretion in cattle(Mondal et al.,2001) and buffalo (Mondal et al.,2000).

In conclusion, the results of this studyindicate that plasma FSH levels were higher (P<0.01)in buffaloes that exhibited overt oestrus comparedto silent estrus and might be responsible forexpression of oestrus.

Table 1. Plasma FSH concentrations (ng/ml) during overt and silent oestrus in buffaloes.

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Day of oestrous cycle Overt oestrus Silent oestrus

-4 1.67 ± 0.44 1.23 ± 0.14 -3 1.85 ± 0.66 0.65 ± 0.15 -2 1.48 ± 0.44 1.60 ± 0.50 -1 1.17 ± 0.37 1.39 ± 0.35 0 4.13 ± 0.21 3.42 ± 0.34 1 1.96 ± 0.68 3.01 ± 1.39 2 1.14 ± 0.27 2.58 ± 0.06 3 0.89 ± 0.24 1.26 ± 0.12 4 0.63 ± 0.29 1.98 ± 0.67 5 0.72 ± 0.19 1.09 ± 0.61 6 1.05 ± 0.53 0.91 ± 0.04 7 0.79 ± 0.19 2.07 ± 0.18 8 0.77 ± 0.12 1.09 ± 0.12 9 1.39 ± 0.88 1.49 ± 0.84 10 1.21 ± 0.45 1.03 ± 0.17 11 1.54 ± 0.19 0.88 ± 0.02 12 0.65 ± 0.45 1.01 ± 0.45 13 0.51 ± 0.38 1.22 ± 1.09 14 0.47 ± 0.09 0.84 ± 0.49


Figure 1. Mean peripheral plasma FSH concentrations during different phases of oestrous cycle in buffaloes in relation to overt and silent oestrus (late luteal phase: days -4 to day -2; perioestrus phase: days- 1 to day 1; day 0=day of oestrus; early luteal phase: days 2 to day 5; mid-luteal phase: days 6 to day 14).

ACKNOWLEDGEMENTS

S. Mondal was supported by a Junior NDRIfellowship. The technical assistance rendered by Mr.P. C. Singh and Mrs. A. Ladkhani is acknowledged.

REFERENCES

Akbar, A.M., L.E. Reichert, T.G. Dunn, C.C.Kaltenbach and G.D. Niswender. 1974. Serumlevels of follicle stimulating hormone duringthe bovine estrous cycle. J. Anim. Sci., 39:360-365.

Allrich, R.D. 1994. Endocrine and neural control ofestrus in dairy cows. J. Dairy Sci., 77: 2738-2744.

Barnes, M.A., G.W. Kazmer, S.T. Brierley, M.E.Richardson and J.R. Dicky. 1980. Folliclestimulating hormone and estradiol 17β in dairycows treated with a progesterone releasingintravaginal device. J. Dairy Sci., 63 : 161-165.

Boorman, G.H., G.D. Niswender, V.L. Gay, L.E. JrReichert and A.R. Jr. Midgley. 1973.Radioimmu-noassay for follicle stimulatinghormone in the rhesus monkey using anantihuman FSH and rat FSH131. Endo-crinology, 92: 618-622.

Dobson, H. 1978. Plasma gonadotrophins andoestradiol during oestrus in the cow. J.Reprod. Fertil., 52 : 51-53.

Gay, V.L., A.R. Jr. Midgley and G.D. Niswender.1970. Patterns of gonadotropin secretionassociated with ovulation. FederationProceeding, 29: 1880-1884.

Ginther, O.J., L. Knopf and J.P. Kastelic. 1989.Temporal associations among ovarian eventsin cattle during oestrous cycle with two andthree follicular waves. J. Reprod. Fertil., 87: 223-231.

Heranjal, D.D., A.R. Sheth, R. Desai and S.S. Rao.1979. Serum gonadotropins and prolactinlevels during estrous cycle in Murrahbuffaloes. Indian J. Dairy Sci., 32 : 247-249.

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Kaneko, H., G. Watanabe, K. Taya and S.Sasamoto. 1992. Changes in peripheral levelsof bioactive and immunoreactive inhibin,estradiol-17β, progesterone, luteinizinghormone and follicle stimulating hormoneassociated with follicular development in cowsinduced to superovulate with equine chorionicgonadotropin. Biol. Reprod., 47: 76-82.

Kaneko, H., H. Kishi, G. Watanabe, K. Taya, S.Sasamoto and Y. Hasegawa. 1995. Changesin plasma concentrations of immunoreactiveinhibin, estradiol and FSH associated withfollicular waves during the estrous cycle ofthe cow. J. Reprod. Dev., 41: 311-317.

L’Hermite, M., G.D. Niswender, L.E. Jr. Reichertand A.R. Midgley. 1972. Serum folliclestimulating hormone in sheep as measured byradioimmunoassay. Biol. Reprod., 6 : 325-331.

Manik, R.S., S.K. Singla, P. Palta, and M.L. Madan.1998. Ovarian follicular dynamics monitoredby real-time ultrasonography during oestrouscycle in buffalo (Bubalus bubalis). Asian-australasian J. Anim. Sci., 11: 480-485.

Mondal, S., B.S. Prakash and P. Palta. 2000.Relationship between peripheral inhibin andFSH in cycling Murrah buffaloes (Bubalusbubalis). J. Endocrinol., 167: 67.

Mondal, S., P. Palta and B.S. Prakash. 2001.Peripheral plasma inhibin concentrations in

relation to FSH during estrous cycle in Sahiwalcows and Murrah buffaloes. In AdvanceAbstracts of Physiology Section of 88th

session of Indian Science CongressAssociation, New Delhi, 3 to 7 January, 2001,p. 11-12.

Niswender G.D., T.M. Nett and A.M. Akbar. 1974.The hormones of reproduction. In Hafez,E.S.E. (ed.) Reproduction in Farm Animals.Lea and Fibiger, Philadelphia.

Palta, P. and M.L. Madan. 1995. Gonadotropicresponses to GnRH in Murrah buffaloes(Bubalus bubalis) treated with PMSG.Buffalo J., 11: 205-211.

Pierson, R.A. and O.J. Ginther. 1988. Ultrasonicimaging of the ovaries and uterus in cattle.Theriogenology, 29: 21-37.

Razdan, M.N., M.L Kaker and M.M. Galhotra.1982. Serum FSH levels during estrus and a 4week period following mating in Murrahbuffaloes (Bubalus bubalis). Therio-genology, 17: 175-181.

Snedecor, G.W. and W.G. Cochran. 1967. StatisticalMethods, 6th ed. Oxford and IBH PublicationCo, New Delhi, India.

Taya, K., H. Kaneko, G. Watanabe and S. Sasamoto.1991. Inhibin and secretion of FSH in estrouscycles of cows and pig. J. Reprod. Fertil.,(Suppl) 43: 151-162.

*Continued from page 273

Santos DA- da-S, W.R.R. Vicente, J.C. Canola andE. Lega. 2004. B-mode Ultrasonography incows during lactation to evaluate the teatanatomy using four different techniques.Brazilian Journal of Veterinary Researchand Animal Sciences, 41(5): 349-354.

Saratsis P. and E. Grunert. 1993. Diagnosticultrasound for examination the extension ofteat stenosis or other anomalies of the teat indairy cows. Deut. Tierarztl Wochenschr.,100 (4): 159-163.

Twardon J., M. Dziecio, W. Nizanski and G.J.Dejneka. 2001. Use of ultrasonography indiagnosis of teats disorders. Med. Weter.,57(12): 874-875.

Worstorff H., J.D. Steib, A. Prediger and W.L.Schmidt. 1986. Evaluation of sectional viewsby ultrasonics for measuring teat tissuechanges during milking of cows.Milchwissenschaft, 41: 12-15.

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FETAL ARTHROGRYPOSIS CAUSING DYSTOCIAIN A PLEURIPAROUS BUFFALO

J. Singh1, B. Mideksa1, A.M. Pawde1 and S. Deori2

1Division of Surgery, Indian Veterinary Research Institute Izatnagar , Bareilly, India2Animal Reproduction Division, Indian Veterinary Research Institute Izatnagar , Bareilly, India

ABSTRACT

This communication reports a case ofdystocia in a buffalo due to fetal arthrogryposis alongwith torticollis. The case was successfully handledby caesarian section without any post-operativecomplications.

Keywords: Arthogryposis, dystocia, buffalo

INTRODUCTION

Arthrogryposis is a musculo-skeletaldeformity frequently encountered as a congenitaldisease (Leipold et al., 1996) and is reported in man,farm animals and pets. The present communicationreports a case of fetal arthrogryposis along withtorticollis causing serious dystocia in a pleuriparousbuffalo and its successful management.

HISTORY AND OBSERVATIONS

A pleuriparous buffalo in her third gestationwith the history of full term pregnancy and in labourfor last 12 h was presented. The water bag wasruptured and the animal was suffering from dystocia.The case had been unsuccessfully manipulated bylocal veterinarians. Clinically the animal appearedactive and was straining. The pelvic ligaments wererelaxed and there was let down of milk in the udder.In the per vaginal examination it was found that thecervix was sufficiently dilated and the fetus in thenormal anterior presentation but in dorso-sacralposition. However, the birth canal was dry and therewas lack of lubrication. The fetus appeared

apparently normal and dead. Initial attempts weremade to manually correct the position of the fetusbut it was found that there was contractures of thejoints in the fore limbs. Since the obstetric maneuversfailed, the fetus the removed through lower flanklaprohysterotomy, under local infiltration analgesiawith 2% Lignocaine HCL. The uterine andlaparotomy incisions were closed in a routinemanner. Post-operatively, the animal was given 5liters of 5% dextrose-saline intravenously,Enrofloxacin 20 ml intramuscularly for five days anddiclofenac sodium 15 ml intramuscularly for threedays.


Examination of the fetus revealedcontractures and ankylosis of all the joints withvariable degree of flexion and extension (Figure 1)and hence termed as arthrogryposis. The fetus alsofeatured torticollis. Mahajan et al., (2006) alsodescribed a case of arthrogryposis in a buffalo calfcausing dystocia but it was not characterized bytorticollis as in the present case. The fixation of thejoints may have been due to lack of extensibility ofthe muscles, ligaments or atrophy resulting fromneuropathy (Tyagi and Singh, 1996). Non-hereditarycauses of muscular contractures have also beenreported (Roberts, 1971). In the present case theflexion of joints resulted in the dystocia in the buffalo.Forced extraction is seldom indicated in such casesas it may lead to serious trauma to the uterus andthe birth canal. Hence caeserean section wasapplied to relieve the fetal arthrogryposis and wasfound effective without any post-operativecomplications.

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Figure 1. Arthrogyposis with torticolis in a buffalo calf.

REFERENCES

Leipod, H.W., G. Saperstein and K. Huston. 1996.Large Animal Internal Medicine. 2nd Ed. St.Louis. Mosby, p. 17119-1722.

Mahajan, A., A. Sathya, P. Singh and S. Prabhakar.2006. A case of arthrogryposis in a buffalo

calf causing dystocia. Indian J. Anim.Reprod., 27: 86-87.

Roberts, S.J. 1971. Veterinary Obstetrics andGenital Diseases. 2nd Ed. CBS Publishers,New Delhi, pp. 277-278

Tyagi, R.P.S. and J. Singh. 1996. Ruminant Surgery.CBS Publishers. New Delhi, pp. 200.

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BUFFALO GENETIC RESOURCES IN INDIA AND THEIR CONSERVATION

A.K. Das, Deepak Sharma and Nishant Kumar

Division of Animal Genetics & Breeding, Faculty of Vety. Sci. & Animal Husbandry. SKUAST- J. R.S.Pura.JAMMU. 181102, India

ABSTRACT

The buffalo plays a very important role inIndian economy as it alone contributes about 56%of total milk production in India. The river buffaloes(Bubalus bubalis) of the Indian sub continent aremaintained chiefly for milk production, but all of themare also dual purpose animals, exhibiting good meatcharacteristics, though their potential for meat stillremain unexplored and unexploited. The swampbuffalo is more or less a permanent denizen ofmarshy lands, where it wallows in mud and feed oncoarse marsh grass. The best known breeds areMurrah, Nili-Ravi, Jaffarabadi, Surti, Mehsana,Kundi, Nagpuri and Bhadawari. The germplasm ofsuch well-defined breeds constitute a valuablegenetic resource which needs to be conserved onpriority basis. The situation is further complicatedby the fact that there exists no breed societies orbreed registration/ improvement societies to registeranimals of specific breeds, maintain herd books andensure the purity of the breeds.

Keywords: genetic resources, conservation,domestication, germplasm

INTRODUCTION

The buffalo forms the backbone of India’sdairy industry and is rightly considered as the ‘bearercheque’ of the rural flock considered as India’smilking machine (Balain, 1999). Asian countrieshave been producing over 96 percent of world’s totalbuffalo milk at an annual growth rate of 4.0 percent.India, with 32 million tons, is world’s topmost buffalo

milk producer accounting for 64 percent of theworld’s total of 49 million tons. The world’s buffalopopulation has been estimated to be about 164.9million, and of these more than 56 percent, i.e. 93.8million, are in India (FAO, 2000). These animalsrequire a relatively low level of inputs in thepredominantly mixed farming systems, and are wellknown for their ability to thrive on low-quality cropresidues and green forage (Resali, 2000) under harshclimatic conditions. Furthermore, the contributionsof milk, meat, manure and draft power of the buffaloto the overall national economy have beenoverwhelming (Shrestha and Shrestha, 1998). Thislarge population of buffaloes contributes more than50% of total milk produced by Indian livestock. Soneedless to add, buffalo is one of the most importantlivestock in India and its genetic potential forproduction and reproduction traits has to be improvedto cater to the huge demand of our country.

Conservation is the act or process ofprotection, preservation, management or restorationof wildlife, livestock and natural and culturalresources and management of human use of bio-sphere so that it may yield the greatest sustainablebenefit to present generation.

Breed types, Origin and Domestication The Asiatic and European buffaloes belongto the genus Bubalus, while the African buffaloesto the genus Syncerus. The Asiatic genus, oftendescribed as water or river buffalo, consists of twotypes (river or swamp) distinguishable on the basisof their appearance, behavior, use and habitat. Theriver buffalo (Bubalus bubalis) of the Indian subcontinent, Egypt and Mediterranean basin of Europeand maintained chiefly for milk production (Cockrill,1982) but all of them are also dual purpose animals,

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exhibiting good meat characteristics, though theirpotential for meat still remains unexplored andunexploited. The swamp buffalo is more or less apermanent denizen of marshy lands where itwallows in mud and feed on coarse marsh grass. Itis mainly found in South East Asia and China andhas a very minor or no role in milk production. Inthese areas the principal contribution of the buffalohas been draft power for crop land preparation, ruraltransportation, threshing, water lifting and oilextraction from oil seeds (National Academy ofScience, 1981). The African buffaloes, referred to as thewild buffalo or Cape buffalo, include two sub-species; Syncerus caffer caffer (the African or redbuffalo) and Syncerus caffer nanus (the Congobuffalo). The buffaloes never achieved domesticationand still are wild or semi wild. The origin of thebuffalo and the time of its domestication is lost inantiquity. The name buffalo is derived from the Greekworld ‘boubalos’ used for the cud-chewing ox-likeruminants. The original domesticated stock ofbuffaloes is believed to have been derived from theIndian wild buffalo, Bubalus arnee, while theswamp buffalo was probably first domesticated inChina some 5000 years ago. River buffalo seems tohave originated and domesticated in the Indo-gangticplains some 5000 years ago or even earlier asevidenced by the findings of engraved seals depictingbuffalo bulls along with mangers in belligerent moodand a she buffalo without a manger. This furtherreveals the existence of different feeding treatmentbetween bulls and cows even at that time, the formerperhaps getting a preferential treatment ofconcentrate feeding in a manger.

Buffalo Germplasm of IndiaIndia is a vast country. Major parts of the

country are located in tropical, sub-tropical andtemperate zones. There are agro climatic extremesin respect of soils, rainfall, temperature etc. Thebreeds and types of livestock evolved under suchwidely varying agro-ecological conditions andvarying requirements of man, have acquired adaptivecharacteristics which are reflected in the diversityof animal genetic resources of the country includingbuffaloes. India has been regarded as an extremelyrich gold mine of buffalo germplasm resources as it

harbors all the recognized, high producing breeds ofthis species. Of the estimated 164 million buffaloesin the world and the 159 million in Asia, India aloneaccounts for 94 million (56% the of the world and58% of the Asian populations).

Indian buffaloes may be put into following fivedistinct groups

Group Breeds/TypesMurrah Murrah, Nili-Ravi, KundiGujarat Surti, Mehsana,

JaffarabadiUttar Pradesh Bhadawari, TaraiCentral India Nagpuri, Pandharpuri,

Manda, Kalahandi,Sambalpur

South India Toda, South KanaraThese breed types constitute only about 40%

of the population while the remaining 60% representan admixture of different breeds and are commonlyreferred to as Desi or non-descript types. However,the fact remains that the best known breeds areMurrah, Nili-Ravi, Jaffarabadi, Surti, Mehsana,Kundi, Nagpuri and Bhadawari. The germpasm ofsuch-well defined breeds constitutes a valuablegenetic resource.

Buffalo Genetic Resource in IndiaLittle information is available about the

performance of Indian buffaloes; moreover, itmostly restricted on Murrah, Nili-Ravi and Surti andon the basis of data from different breeds, strains ofinstitutional farms and utility vis-a-vis productionsystem in their home tracts and under farmersmanagement conditions. The situation is furthercomplicated by the fact that there exist no breedsocieties or breed registration/improvement societiesto register animals of specific breeds, maintain herdbooks and ensure the purity of the breeds. There isno controlled breeding in the breeding tract of anyof the breeds. Rather uncontrolled breeding is thecommon order under widely prevalent extensivegrazing situations throughout the country.

In majority of the cases, the true productivepotential of individual breeds in their breeding tractshas not been adequately documented. This hasaffected the detailed description of the breeds andalso their genetic potential. There is thus an urgent

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need for differentiating the real breed differencesby conducting systemic scientific studies. There isan urgent requirement to uniformly describe all theIndian buffalo breeds by utilizing common breeddescriptors, by studying their native environment,management practices, qualitative and quantitativeaspects of morphological, physiological andfunctional traits, blood groups and biochemicalpolymorphisms, cytogenetic parameters, DNAanalyses, utility and demographical and geographicaldistributions. This will lead to the identification ofthe types of genes and gene combinations availablein different breeds and will also assist in formulatingbreeding policies and selection of animals forconservation, propagation and improvementprogrammes.

Conservation of Buffalo Genetic ResourcesThe country’s buffalo genetic resources

need to be used judiciously. The rich biologicaldiversity of this species is progressively being erodeddue to unplanned breeding. Except in few organizedfarms which maintain small herds of pure breed,there is almost unrestricted interbreeding amongdifferent breeds and there is a marked decline inthe availability of unique animals conforming to theattributes of defined breeds, particularly in theirnative breeding tracts. There has been a non-judiciousutilization of buffalo genetic resources in the country.The males are only partially utilized in the form ofbulls and bullocks. There is always a scarcity ofbreeding bulls of superior genetic merit. Above all,the high producing milch buffaloes from the breedingtract, representing the best germplams, are taken tometropolitan cities in large numbers for milkproduction (NDRI, 2006). After completion oflactation, these buffaloes are slaughtered, causing aserious erosion of elite germplasm.

Though the livestock census in India isconducted specie-wise and not breed-wise, it isextremely difficult to determine the exact numberof animals of a particular breed. Preliminary surveysconducted by the National Bureau of AnimalGenetic Resources in the breeding tracts of variousbreeds of buffaloes indicate that the buffalo breedswhich need urgent attention for conservation due totheir vulnerable status are Bhadawari and Toda.Bhadawari buffaloes are famous for the very high

content of fat (10-14%) in their milk. But due totheir low milk yield (600-1000 liters) these buffaloeshave been used for upgrading with Murrah andconsequently the number of pure Bhadawaribuffaloes is restricted to a few thousands only. Ifimmediate attention is not paid, the combination ofgenes imparting high fat percent may be lost.Similarly, the population of Toda buffaloes of Nilgirihills of Tamil Nadu is limited to 10-15 thousand.These animals have been reared traditionally by theToda tribes due to their suitability in the hilly regionsof their home tract and their superior meat productionpotential. The number of Nili-Ravi buffaloes in itsbreeding tract in Punjab is also decreasing at a fasterrate due to the preference for Murrah or Murrahgraded buffaloes. Yet, a large population of this breedis available in neighboring areas of Pakistan.

Approach for ConservationBroadly, there are two means of

conservation i.e. in situ and ex situ. Conserving thelive animals that exist in nature is in situconservation. The animals are maintained in theiroriginal habitats under native conditions with nointerference in their mode of management, feedingand other conditions. The main problem of in situconservation is inbreeding and genetic drift typicalof small populations. The ex situ conservation is tobe used when the endangered population is dismallylow in numbers, as this process has its own innateproblems. It may suffer from spread of disease, orneglect during periods of institutional weakness,besides being costly in long term preservations andlosing the relatedness of current genotype withenvironment when one of these is preserved for longtime (Singh et al., 2004).

Conservation Strategies:ex-situ conservation

Generally sperm, oocytes, embryos, DNAand embryonic stem cell are conserved. It is possiblenow to store a wide variety of living cells for longperiods of time. The techniques can be used for theconservation of endangered breeds as follows:

Sperms and oocytes: Deep freezing ofsemen is suitable for most of the species of domesticanimals.

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Embryos: Cryopreservation of embryos ofcows, buffaloes, sheep, goats and horse hassuccessfully been done to produce offspring. Thisis a better tool for conservation as all the geneticinformation is stored in one diploid zygote.

Storage of DNA: Cryogenic storage ofDNA is another method of preservation of geneticmaterial.

Cloning of somatic cells: Cloning offers theadvantage of producing series of exact replica /copyof the concerned animals.

Embryonic stem cells: Embryonic stem cellsare derived from culture of inner cell mass of a youngblastocyst. These embryonic cells are totipotent andhave potential to develop into viable embryos.

in-situ conservationExplicit efforts to select males from superior

dams under farm conditions and making wider useof the selected best bulls and also preserving theirsemen are necessary. The process has been initiatedfor some of the breeds by NBAGR.

Data Bank Strategy: Maintenance of adatabase containing all relevant breeds, populationcensus and ecological data is essential for designingand implementing conservation strategies. Severalagencies are engaged in generation and disseminationof data/information on Animal Genetic Resources.A useful body of knowledge has already beengenerated/gathered at NBAGR and at otherlocations.

Gene Bank Strategy: Semen fromindigenous breeds has been cryopreserved for usein the future. Ideally sufficient doses should bestoredat at least at two locations remote from eachother. The preserved material should be periodicallyevaluated and put into use.

DNA Bank Strategy: Genetic material canbe preserved in the form of DNA fragments undercryogenic conditions. This has the advantage overstorage of live cells as it is economical, occupiesless space and there is no spread of diseases. Withinand across different countries the storage of DNAhas been made feasible.

Somatic Cell Strategy: With the advent ofDolly sheep, somatic cell technology has received agreat fillip. In future it may be possible to produce alive animal from stored somatic cells. This possibilityis very important since the protocols for collectingsomatic cell samples are less demanding andinexpensive than for collection of spermatozoa andembryos.

REFERENCES

Balain, D.S. 1999. Inflow and outflow of buffalogermplasm resources and their globalcontribution. Invited papers presented inshort course on “characterization andconservation of domesticated livestock andpoultry resources”. 10-19 May, 1999,National Bureau of Animal Genetic Resources(ICAR), Karnal.

Cockrill, N.R. 1982. The water buffalo, A review ,British Veterinary Journal, 137: 8

NDRI. 2006. Compendium of Lectures inAdvanced Animal Breeding Technologiesfor improvement of livestock. 17 March-6April, 2006.

National Academy of Science. 1981. The waterbuffalo. New prospects for an underutilized animal. National Academy of SciencePress, Washington. D.C.

Resali, D.P. 2000. Recent trends in buffaloproduction in Nepal: A review, Buffalo Newsletter. Bulletin of the FAO Inter-Regionalcooperative Research Network on Buffalo,Europe-Near Estate, 14: 6-10.

Shrestha, S.K. and N.P. Shrestha. 1998. Geneticimprovement of buffalo. In Proc. First Nat.workshop on Anim. Genet. ResourcesConserv. Genet. Improvement of Domest.Anim. NARC, Kathmandu, p. 98-102.

Singh, Ram Vir, G.K. Sachdeva, R.C. Garg, S.N.Kaushik and Kripal Singh. 2004. Conservationand Genetic improvement of importantindigenous cattle breed of India. InPreceeding of National Symposium onConservation and Propogation ofindigenous breeds of Cattle Buffaloes. 26-28 Feb, 2004.

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ULTRASONOGRAPHY OF THE UDDER AND TEAT IN BUFFALOES: A COMPARISIONOF FOUR METHODS

K.Rambabu, Makkena Sreenu, R.V.Suresh Kumar and T.S.C.Rao

Department of Veterinary Surgery & Radiology NTR College of Veterinary Science, Gannavaram, SriVenkateswara Veterinary University, Tirupati- 517 502 (A.P). India

ABSTRACT

Ultrasonography of udder and teat inbuffaloes was conducted by direct contact, the gelapplication, water bath and stand off methods. Outof the four-methods, the gel application and waterbath methods gave satisfactory ultrasonographicimages of udder and teat.The merits and demeritsof each method are discussed.

Keywords: ultrasonography,udder and teats,buffaloes

INTRODUCTION

Diseases of the udder and teat are acommon in buffaloes, and any disease conditioninvolving udder or teat ultimately affects the farmer’seconomy. The present study was undertaken tocompare four methods of B Mode ultarasonographyto evaluate the udder and teat anatomy of buffaloessince different methods of diagnosing thepathological conditions of udder and teats areinvogue. Not all the methods may provide completeinformation, and some methods are invasive, thushaving inherent demerits.


The present study was carried on buffaloesrandomly selected from those presented to the clinics

following thorough clinical examination by inspection,palpation, probing, and checking the milk flow fromthe teat. Ultrasonography of udder and teat wasconducted with Logiq 100 V4 model machineand 7.5 MHz linear transducer both in sagital andtransverse planes and the images were recorded onPolaroid paper with a thermal printer. Four differentmethods, namely, direct contact, gel application,water bath, and standoff, were compared for theirmerits and demerits. Before scanning,the udder andteats were cleaned thoroughly with warm potassiumpermanganate solution. In the direct contact method,the surface of linear transducer was placed directlyon the skin surface of the udder and teats. While, inthe gel application method, the transducer was placedlongitudinally/sagitally at the udder or teat afterapplying an acoustic coupling gel (Ultrasound Gel,jaay vee meditech international, Pondicherry,India). In the water bath method, the udder andteats were dipped in a polyethylene bag/condomfilled with water and the transducer was applied invertical/horizontal planes of the outer wall of thepolyethylene bag/ teat dipped in the water filledcondom. A gel filled latex condom was used as astandoff and the transducer was directly applied ina vertical/horizontal planes on the outer wall of thegel filled latex condom and images of the udder andteats were recorded.


Ultrasonographic comparison of differentmethods were carried out and the results weredocumented (Table 1).

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Table 1. Comparative evaluation of different methods for ultrasonography of udder and teats in buffaloes.

++++ : Excellent, +++ : Good, ++ : Moderate, + : Fair, − : Poor

In the direct contact method, the applicationof the transducer on to teat or udder surface waseasy but the shape of the teat could not bemaintained. Visualization of udder parenchyma andgland sinus on ultra sonogram showed good claritywith presence of surface artifacts. The teat canaland sinus showed irregular contour while the threelayers of the teat wall could not be clearlydemarcated. The papillary duct and resette offurstenberg showed overlapped picture.Ultrasonography of udder and teat in cattle wasconducted by direct contact method by Dinc et al.,2000; Santos et al., 2004; Flock et al., 2004 andGungor et al., 2005. Santos et al., 2004 evaluateddirect contact as a useful method in clinical cases.

In the gel application method, the applicationof the transducer on to the teat or udder surfacewas easy but the shape of the teat could not bemaintained in a fixed positon during sonography.Ultrasonographic image of udder parenchyma andgland sinus showed excellent images while the threelayers of teat wall could be clearly demarcated. Thepapillary duct and resette of furstenburg showed anoverlapped pattern with moderate clarity (Figure 1).Bruckmaier and Blum (1992) suggested that thepresence of air between the probe and the tissueexamined must be avoided during ultrasoundexamination; the problem was usually solved by usingcontact gel and applying the probe directly to theanimal’s body. Pressing the ultrasound probe to thetissue leads to teat image deformities. As the

connection between the teat and gland is angular,the application of the probe should simultaneouslyexclude air and prevent teat image deformations asreported by Franz et al., 2001; Twardon et al., 2001;Ayadi et al., 2003; Santos et al., 2004 and Flockand Winter, 2006.

In the water bath method, the applicationof the transducer to the teat or udder surface waseasy. Ultrasonographic image of udder parenchymaand gland sinus showed excellent clarity with minorsurface artifacts. The teat canal and sinus showedregular contour and the three layers of teat wall couldbe clearly demarcated. The papillary duct andresette of furstenberg showed on overlapped patternwith clarity (Figure 2 and 3). Cartee et al. (1986)reported that the use of the water bath in scans ofthe mammary glands was to increase the acousticimpedence difference between the teat wall and thesurrounding medium. The presence of milk in theteat sinus acted similarly as a window of acousticimpedence for imaging the deeper structures andfar wall of the teat. The findings were in accordancewith the observations of Catree et al. (1986);Worstroff (1986); Jenninger (1989); Bruickmaierand Blum (1992); Saratsis and Grunert (1993); Dincet al. (2000); Franz et al. (2001); Hoque et al.(2004); Santos et al. (2004) and Gungor et al.(2005).

In the standoff method, the application ofthe transducer to the teat or udder surface via astand off was easy but the shape of the teat was

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S. No. Parameter Direct

contact Gel

application Water bath

method Standoff method

1 Glandular parenchyma +++ ++++ +++ ++ 2 Gland sinus/cistern +++ ++++ ++++ +++ 3 Teat wall ++ +++ +++ ++

4 Teat sinus/Sinus papillaris/teat cistern ++ +++ +++ ++

5 Papillary duct (teat canal/ductus papillaris) − ++ ++ −

6 Rossete of frusternburg − ++ ++ −


altered and the teat was not in a fixed position duringsonography. The ultrasonographic image of udderparenchyma and gland sinus showed moderateclarity with minor surface artifacts. The teat canaland sinus showed irregular contour while the threelayers of teat wall could be clearly demarcated. Thepapillary duct and resette of furstenberg showedoverlapped pattern as reported Flock et al. (2004).Twardon et al. (2001) and Santos et al. (2004)performed ultrasonography of teats by dipping inliquid and using liquid pressure, respectively. Hoqueet al. (2004) mentioned that the scanning ofsuperficial structures poses a problem by obscuringthe image due to near field reverberations andsuggested the use of a commercially available stand-off pad or home-made fluid filled plastic bag /condomto evaluate superficial structures (less than 3.5 cmdepth). A gel filled condom was used in the presentstudy as a standoff, which provided satisfactoryresults.

In the present study, the glandularparenchyma of the udder appeared as homogenousand hyperechoic with anechoic alveoli which couldbetter be visualized in the water bath and gelapplication methods, whereas the direct and standoff

techniques exhibited some artifacts. Theseobservations were in accordance with Ayadi et al.(2003) in cows and Gungor et al. (2005) in mares.The gland sinus appeared as a homogenous anechoicarea which could be better visualized in all thetechniques without much difference, whereas Carteeet al. (1986) observed the gland sinus as an anechoicarea continuous with the teat sinus. The lining ofthe wall of the gland sinus appeared as mixed hyper-hypoehoic folds. The lactiferous ducts were anechoicareas with in the hypoehoic matrix of the fold.Gungor et al. (2005) mentioned the lactiferous ductas elongated anechoic branches in hyper echoicmammary parenchyma and some of the anechoicareas with in the glandular parenchyma may havebeen blood vessels but others certainly werelactiferous ducts, because they could be seenentering the gland sinus.

Out of four-methods gel application andwater bath methods gave satisfactoryultrasonographic images of the udder and teat. Bothdirect contact and direct contact with standofftechniques were more useful in clinical cases. Thewater bath technique was more ideal for identifyingthe teat anatomy.

Figure 1. Ultrasonographic appearance of teat in a buffalo with the gel technique. Note teat canal and teat cistern.

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Figure 2. Ultrasonographic appearance of teat in a buffalo with the water bath technique. Note the teat as hypoehoic structures with anechoic lumens. The teat wall has three distinct layers the hyperechoic outer layer,hypoechoic thicker middle layer and another more hyperechoic inner layer.The papillary duct(PD) appeared as a thin anechoic area.

Figure 3. Ultrasonographic appearance of teat in a buffaloes with the water bath technique.

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Figure 4. Ultrasonographic appearance of the udder in a buffalo with gel technique. Note the glandular parenchyma (GP) of the udder appears as homogenous and hyperechoic with anechoic alveoli. The gland sinus (GS) appeared as an anechoic area continuous with teat sinus (TS). Note continuation of gland sinus with teat sinus.

REFERENCES

Ayadi M., G. Caja, X. Such and C.H. Knight. 2003.Use of utrasonography to estimate cistern sizeand milk storage at different milking intervalsin the udder of dairy cows. Journal of DairyResearch, 70: l-7.

Bruckmaier R.M. and J.W. Blum. 1992. B-modeutrasonography of mammary glands of cows,goats and sheep during a- and P-adrenergicagonist and oxytocin administration. Journalof Dairy Research, 59(2): 151-159.

Cartee R.E., A.K. Jbrahim and D. McLeary. 1986.B-mode ultrasonography of udder and teat.J. Amer. Vet. Med. Assn., 188: 1284-1287.

Dinc D.A., S. Sendag and I. Aydin. 2000. Diagnosisof teat stenosis in dairy cattle by real- timeultrasonography. Vet. Rec., 147: 270-272.

Flock M. and P. Winter. 2006. Diagnosticultrasonography in cattle with diseases of themammary gland. The Veterinary Journal,171: 314-321.

Flock M., D. Klein and M. Hofman-Parisot. 2004.Diagnostic ultrasonography in cattle with teatdisorders.Wien. Tierarztl. Monatsschr.,91(7): 184-195.

Franz S., M. Hofman-Parisot, W. Baumgartner, G.Windischbauer, A. Suchy and B. Bauder. 2001.Ultrasonography of the teat canal in cows andsheeps. Vet. Rec., 49(4): 109-112.

Gungor O., S.M. Pancarci and A. Kasrabacak. 2005.Examination of equine udder and teat by B-mode ultrasonography. Kafkas UniversitesiFakultesi Dergisi., 11(2): 107-111.

Hoque M., R. Jiwary, S.K. Maiti, G.R. singh, O.P.Gupta and N. Kumar. 2004. Ultrasonographyof bovine udder and teat. In XIth AnnualCongress of Indian association ofadvancement for Veterinary Research(IAAVR), IVRIJzatnagar.

Jennninger S. 1989. Ultrasonography of bovineudder: Physiology and pathologicalfindings.Ultraschalluntersuchungen an derRindes. Physiologische und pathologischeBefunde. p. 99.

*Continued on page 262

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A STUDY ON REPEAT BREEDING OF IRAQI BUFFALO COWS

O. I. Azawi 1, S. N. Omran 2 and J. J. Hadad 3

1 Department of Surgery and Obstetrics, College of Veterinary Medicine, University of Mosul, Mosul, Iraq2 Department of Theriogenology, College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq3 Department of Microbiology, College of Veterinary Medicine, University of Dohuk, Dohuk, Iraq

ABSTRACT

The objectives of the present study were todetermine the relationship between bacteriologicalfindings, clinical signs and histopathological changes,validate diagnostic criteria, and investigate the non-specific aerobic and anaerobic bacterial causes ofendometritis of Iraqi buffalo cows at Ninevehprovince. Data were collected from 60 buffalo cowswith history of repeat breeding in different herds.All buffaloes were subjected to detailed clinicalexamination including external inspection,vaginoscopy and transrectal palpation of the cervix,uterus and ovaries. Swabs for bacteriology andbiopsies for histopathology were collected fromuterine lumen from each buffalo included in thepresent study. Vaginal mucus scored for character,odor, and estimation of polymorphonuclear cells(PMNs). Blood samples were collected from buffalocows for creatine kinase (CK) and aspartateaminotransferase (AST) measurement. Bacteriaidentified using API systems following aerobic andanaerobic cultures, and the bacterial density scoredsemi quantitatively. The most predisposing factor foruterine infection was retained placenta. The mostprevalent bacteria in uterine lumen were E. coli,Archanobacterium pyogenes and Staphylococcusaureus were mostly isolated from buffaloes withrepeat breeding. Vaginal mucus character score wasassociated with the bacterial growth density score.The difference in PMN were highly significant(P<0.01) in animals with repeat breeding and controlgroups. In addition, PMNs was significantly (P<0.01)correlated r = 0.894 with the character of vaginaldischarge. High level of PMNs observed in buffaloes

infected with Archanobacterium pyogenes. Nosignificant difference was found between buffalocows with repeat breeding from normal pluripariousbuffaloes. It could be concluded thatArchanobacterium pyogenes was the only non-specific uterine pathogen directly associated withsevere endometrial lesions. Vaginoscopicexamination combined with palpation of uterusincrease the accuracy of diagnosing endometritis,and cytogenic examination of uterine discharge is amore reliable method of establishing the presenceor absence of uterine inflammation in buffalo cows.

Keywords: buffalo cow, repeat breeding,bacteriology, creatine kinase, aspartate amino-transferase, histopathology

INTRODUCTION

The major problems faced by buffalobreeders and farmers include poor reproductiveefficiency and prolonged intercalving intervals(Jainudeen, 1986; Singh et al., 2000). Karimi et al.(2004) recorded a calving interval of 480-540 daysin Azerbaijan buffaloes. Suboptimal productionpotential and high incidence of fertility diseases wereassociated with buffalo breeding and production(Dobson and Kamonpatana, 1986; Ter-Mulen et al.,1995). Iraqi buffaloes were considered as havinglow reproductive performance and increasedaverage length of calving interval (El-Wisby, 1979).

A repeat breeder is generally defined as anycow that has not conceived after three or moreservices associated with true estrus (Roberts, 1986).Uterine function is often compromised in cattle by

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bacterial contamination of the uterine lumen afterparturition; pathogenic bacteria frequently persist,causing uterine disease, a key cause of infertility(Sheldon and Dobson, 2004). The presence ofpathogenic bacteria in the uterus causesinflammation, histological lesions of the endometrium,delays uterine involution and perturbs embryosurvival (Azawi, 2008). The most common causeof uterine infection is the pathogenic microorganismsaffecting productivity and fertility of cows (Azawiet al., 2007; Azawi et al., 2008). Pathogenicorganisms isolated from an infected uterus are foundgenerally in livestock environments and are capableof infecting other tissues and organs (Azawi, 2008).Thus, uterine infections are classified as non-specificinfections (Bekana et al., 1994; Bonnett and Martin,1995; LeBlanc et al., 2002; Sheldon et al., 2004). Itis called nonspecific infection because the initialcolonizing bacterium is not known and the specificbacteria causing the signs of infection are not known(Lewis, 1997). Even though numerous bacteria in avariety of combinations have been isolated frominfected uterus, Archanobacterium pyogenes andE. coli are usually associated with uterine infectionin cattle (Usmani et al., 2001; Seals et al., 2002).The composition of the uterine flora changessomewhat at each recontamination, and no specificcombination of organisms is associated consistentlywith postpartum infections (Azawi, 2008).Nevertheless, Archanobacterium pyogenes eitheralone or in combination with other bacteria such asthe anaerobic Fusobacterium necrophorum andBacteroides spp. (Bondurant, 1999) is oftenassociated with uterine infections (Lewis, 1997;Sheldon et al., 2004). Intra-uterine oxygen reductasepotential fell in the presence of infection (El-Azabet al., 1988), and mostly with aerobic bacteria,thereby creating an anaerobic environment. Thisdrop in intrauterine oxygen reductase potential maybe associated with either micro-organism metabolismor increased oxygen consumption bypolymorphonuclear inflammatory cells. Of theanaerobic bacteria cultured from cases of uterineinfection, Fusobacterium necrophorum andBacteroides spp. have been identified (Ball et al.,1984). When A. pyogenes was isolated from uterinefluids approximately 21 days postpartum, cowsdeveloped severe endometritis and usually were

infertile at first service (Ball et al., 1984; Dohmenet al., 1995; Noakes et al., 2002). In addition, Griffinet al. (1974) reported that 69% of cows with uterineinfections harbored A. pyogenes often in purecultures. While DelVecchio (1994), reported that64% of the infected cows harbored A. pyogenesusually in combination with E. coli, neither studywas designed to determine the incidence of strictanaerobes. Ruder et al. (1981) determined theincidence of anaerobic and strictly anaerobicorganisms; 27% of infected cows harbored A.pyogenes and this suggested that severe uterineinfections might depend on pathogenic synergismbetween A. pyogenes and anaerobic organisms suchas Fusobacterium necrophorum. The presentstudy was designed to determine the relationshipbetween bacteriological findings, clinical signs, andhistopathological changes in uterine infections,validate diagnostic criteria for endometritis, andinvestigate the non-specific aerobic and anaerobicbacterial causes of repeat breeding.


AnimalsThe present study was conducted on

lactating Iraqi northern buffalo cows in Ninevehprovince. Buffalo cows included in this study wereaged 5 to 15 years. All buffalo cows included in thepresent study were local Iraqi northern breed. Theseprivate buffalo dairy herds consisted ofapproximately 30 to 150 buffaloes. The animals werekept outdoors near the rivers for wallowing andmilked twice daily. A balanced nutritional dietincluding green fodder and concentrate mixture werefed to these animals. Animals with a history ofCaesarean operation, acute mastitis, lameness,abdominal disorders or other undercurrent diseaseswere excluded from the study on the basis of clinicalexaminations to remove any conflicting influence ofnon-uterine bacterial infection during the studyperiod.The following data was recorded for eachbuffalo cow included in the present study: name ofbuffalo, breed, number of parturitions, obstetricalproblems if present, type of last parturition, retained

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placenta, vaginal prolapse, uterine prolapse, abortion,and milk production.

Clinical examinationsThe general health status, appetite, rectal

temperature, pulse rate, respiration rate and otherclinical signs such as arched back, colic, pain,presence of fresh discharge on the vulva, perineum,or tail were recorded. Rectal examination of uterus,uterine tubes, ovaries, and cervix was performed toeach buffalo cow included in this study. When abuffalo cow was examined vaginoscopically,analgesia was preformed by injecting 1 ml of 2%xylasine intravenously. The vulva was wiped cleanwith damp clean towels and then disinfected withiodine-povidine and then washed again with water.A sterile vaginal speculum was lubricated with sterilevaseline and then inserted into the vagina up to thelevel of the external os of the cervix. Inspection ofthe cervix, vagina was performed with illuminationfrom a penlight. The examination lasted for 5 to 30seconds/buffalo cow.

Uterine swab collection and bacteriologySampling for bacteriological examination

was performed immediately after vaginalexamination. After parting the vulval lips an outerprotective sterile plastic soft drink straw sheath wasadvanced into the vagina and fixed in the externalopening of the cervix. A guarded inseminating pipettewas advanced through the outer tube into the uterusby cervical manipulation similar to the technique forartificial insemination. The sterile swab, which wasfixed on the inseminating pipette was pushed out ofits protective sterile plastic drink straw sheath (toprotect from contamination with cervix), and movedabout slightly in the body of the uterus. Afterretraction into its cover, the swab was removed fromthe vagina, with an assistant parting the vulval lips.With the same technique, additional swabs werecollected from the cervix and vagina. Swabs weretransferred into sterile tubes containing thioglycolatebroth as a transport media and transported to thelaboratory at 4 0C and immediately processed forbacteriological examination. Swabs were culturedaerobically on sheep blood agar, MacConkey agarand nutrient agar and anaerobically on sheep bloodagar. After 24 h incubation at 37 0C for aerobic

growth and 7 days for anaerobic bacteria, bacterialgrowth on the cultural plates was scored semiquantitatively, depending on the number of bacterialcolonies detected on the plate; 0: no growth; 1 : <10colonies; 2 : 10-100 colonies; 3 : 100-500 colonies; 4: 500-1000; 5 : > 1000 colonies (Olson et al., 1984).Identification of bacteria was based on thecharacteristic of colony, hemolysis, gram stain,morphology, catalase test, coagulase test, oxidasetest, indole production test, methyl red test, Voges-Proskauer test, citrate production test and sugarutilization, and confirmed by API system (API 20E,API 20A, API Coryn, API Staph, API Strep) suppliedby bioMerieux SA, 69280 Marcy L’Etoile, France.Some isolates were re-confirmed by sending themto the Central Health Laboratory, Ministry of Health,Baghdad.

Biopsy collection and histopathologyBiopsies were taken following the culturing

procedure. Separate tissue samples were obtainedfrom each uterine horn with the biopsy instrument.The endometrial biopsy was immediately placed intoa bottle containing 10% formal saline solution andstored at 4 0C till preparation for sectioning, whichincluded dehydration, clearing, embedding,sectioning, and staining performed as described byLuna (1968).

Estimation of Polymorphonuclear cellsA fluid aliquot was collected from each

buffalo cow included in this study aspirated fromthe uterine lumen using sterile uterine catheter andtransferred into sterile tubes, then transported to thelaboratory at 4 0C for the determination of thepercentage of polymorphonuclear cells. Smears wereprepared from the uterine fluid and fixed withabsolute methyl alcohol and stained by Wright-Giemsa stain. Two stained smears per sample wereused and the average of the two readings used instatistical analysis. Samples were taken before andafter treatment of buffalo cows affected with toxicpuerperal metritis.

Blood sampling and enzyme assaysBlood samples were collected from the

jugular vein. Serum samples were collected aftercentrifugation at 700 g for 10 minutes. Serum

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samples were transferred in plastic tubes and storedat ) -20oC until enzymatic analysis. Aspartateaminotransferase (AST) activity was estimatedusing the Lab kit (Labkit-Josep Tarrad-ellas,8008029, Barcelona, Spain), and a spectrophoto-meter at wavelength of 340 nm. The absorbancewas recorded three times, 1 minutes apart and finallythe mean of the three readings was used as theenzymatic activity. Creatine kinase (CK) wasestimated using the kit CK-NAC (BIOLAB SA, LesHautes Rivers, 02160 Maizy-France). The catalyticactivity of CK was determined by the increase inthe absorbance at 340 nm wavelength of aspectrophotometer by reading the initial absorbanceafter 120 s, followed by three readings, 1 minuteapart and finally calculating the absorbance changeper minute (ΔA D minutes) using the equation CK-activity UA = ΔA D minutes x333.

Statistical analysisEnzymatic activities of CK and AST and

effects of treatment regimens on PMNs were testedby the analysis of variance (ANOVA) and leastsignificance differences (LSD). Student’s two-sample t-test was performed for the differencebetween two means of rectal temperature, pulserate, and respiration rate. For the determination ofcorrelation coefficient between isolates andhistopathological changes of two parameters, thePearson product moment correlation analysis wasused. The differences in percentages of reproductiveproblems were tested by Chi square analysis. Fortesting the differences of bacterial growth densityscore on mucus discharge character score, multiplelinear regressions were used.

RESULTS

Buffalo cows with repeat breeding includedin this study (60 animals) had a history of obstetricalproblems including dystocia (3.3%), retention of fetalmembranes (13.3%), and insertion of foreignmaterials in the vagina for milk let down (11.67).The clinical examination of animals suffering fromrepeat breeding indicated, normal rectal temperature,pulse rate and respiration. On vaginal examination

there was clear mucus (15%), clear mucus withflakes of pus (58.3%), and mucopurulent (26.7%).

The prevalence of bacteria from the genitaltract of buffalo cows with endometritis is listed inTable 1. Between the isolates, E. coli (28.16%),Proteus vulgaris (12.62%) and Pseudomonasaeruginosa (9.7%) were the most prevalentbacteria in the vagina. While in the uterus, the mostprevalent bacteria were E. coli (33.33%),Archanobacterium pyogenes (19.69%) andStaphylococcus aureus (15.15%). In the controlgroups, the most prevalent bacteria in pluripariousbuffaloes and heifers were E. coli. The anaerobicbacteria Lactobacillus acidophilus was only foundin pluriparious, while Lactobacillus fermentusisolated only in heifers.

The most prevalent bacteria isolated inbuffalo cows suffering from severe inflammatorychanges were Archanobacterium pyogenes(38.23%), E. coli (14.71%) and Streptococcuspyogenes (14.71%). However, Archanobacteriumpyogenes and Streptococcus pyogenes were neverisolated from buffalo cows with either moderate ormild forms of histopathological changes, as shownin Table 2. Acute endometritis is characterized byan infiltration predominantly with polymorphonuclearleukocytes in the sub-epithelial zone of the stratumcompactum, and suppuration causing desquamationof endometrium. The exudates are composed mainlyof PMNs with small numbers of lymphocytes andplasma cells. Subacute endometritis characterizedby hypertrophy and thickening of blood vessels, thesub-epithelial infiltration of fibroblasts, andlymphocytes and by slight leukocyte infiltrate aroundthe endometrial glands. Chronic endometritis ischaracterized by predominantly lymphocyticinfiltration, and with the presence of plasma cellsand macrophages and characterized by irreversiblechanges including atrophy of endometrial glands andfibrosis, reduced endometrial gland diameter andsecretary activity and pyknotic nuclei in the glandularepithelial cells. These degenerative changes are duemainly to the presence of perivascular fibrosis. Theseverity of fibrosis is arbitrarily evaluated by thenumber of fibrotic layers: slight (1-3 layers), mild(4-10 layers), and severe (>10 layers). Most of thechronic endometritis cases were of the severe type.Chronic endometrium mainly involves stratum

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spongiosum. In control groups, one pluriparious cowand two heifers had a negative growth in uterinesamples. Among buffaloes with endometritis, sixanimals (10%) had no bacterial growth from theuterine samples and three (5%) had none fromcervical samples. The most prevalent type ofendometritis was chronic type (61.67%) and subacute endometritis (25%), while 13.33% of the cowshad acute endometritis. A correlation was observedbetween the kind of endometritis and character ofdischarge. A high incidence of mucopurulentdischarge was observed in sub-acute and chronicmetritis, 53.33% and 48.65%, respectively. Thebacterial growth density score of the samplescollected from buffalo cows affected withendometritis were significantly different (P<0.05)from the growth score in the cervix, and there wasno significant difference between growth densityscore in vaginal samples, and uterine samples, asshown in Table 3. The vaginal mucus character scorewas associated with the bacterial growth densityscore. Uterine density score appeared to predictmucus score (P<0.05), using the equation:

Mucus score = 0.321+ (0.01 x cervicalgrowth score) + (0.00422 x vaginal growth score) +(0.852 x uterine growth score).

The mean percentage of polymorphonuclearcells was 41.10±11.91, as shown in Table 3. ThePMNs were significantly (P<0.01) correlated r =0.894 with the character of vaginal discharge, aswhen the discharge with fetid smell and purulentdischarge, the PMN were 48.10% and 40.38%,respectively. In addition, an association was foundbetween type of isolates and PMNs. A highpercentage of PMN was observed in samplesinfected with gram-negative anaerobic bacteria(56.35%) and Archanobacterium pyogenes(42.08%).

There were 60 vaginal discharges collectedduring the study (only infected animals wereincluded, and the control not included in this number)and 5.92% had clear or translucent vaginal mucus(score 0), 23.02% had clear mucus with flakes ofpus (score 1), 28.95% had mucopurulent discharge(score 2), 14.47% had purulent discharge (score 3),27.63% had purulent with fetid smell (score 4). Themost prevalent isolates when the discharge was

purulent were Archanobacterium pyogenes, E.coli, and Pseudomonas aeruginosa.

In cows with endometritis, the CK and ASTactivities were above the level of the control group(heifers) and significantly different (P<0.05), but nosignificant difference was observed between buffalocows with endometritis and the pluriparious buffalocow control group, as shown in Table 4.

DISCUSSION

Clinical examination of repeat breedingbuffalo cows revealed normal rectal temperature,respiration rate, pulse rate and normal appetite. Thecharacter of vaginal discharge was mainly clearmucus with flakes of pus (58.3%), mucopurulent(26.7%) and clear mucus (15%). Cervical diameterwas mainly less than 5 cm, with thin uterine walland 63.3% with symmetrical horns. Results ofbacterial examination of repeat breeding buffaloesshowed that E. coli, Archanobacterium pyogenes,Staphylococcus aureus, Streptococcus pyogenes,were the bacterial isolates, 33.33%, 19.69%, 15.15%,7.56% and 7.56%, respectively. Gram-negativeanaerobes were not isolated from endometritis ofrepeat breeder buffaloes. These results confirm theprevious finding by Azawi and Taha (2002), and Tahaand Azawi (2003). The prevalence of gram positiveand gram-negative bacteria seemed to be almostequal, 51.5% and 48.5%, respectively. These resultswere in agreement with Khan and Khan (1989).The high prevalence of Archanobacteriumpyogenes, Staphylococcus aureus, E. coli andStreptococcus pyogenes seems to be related to theseverity of endometritis 38.23%, 20.59%, 14.7% and14.71%, respectively as a percent of prevalence inthe severe type of histopathological changes. Ahighest uterine inflammatory change was associatedwith Archanobacterium pyogenes. This findingwas supported by a study on experimentally inducedendometritis (Chaudhery et al., 1987). Similar resultswere obtained by Lewis (2003) and DelVecchio etal. (1992). Endometrial inflammatory changesassociated with repeat breeder as observed inbuffalo cows included in this study are in agreementwith Dwivedi and Singh (1975). Of buffaloes with

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endometritis, 58.3% had clear mucus with flakes ofpus and 26.7% had mucopurulent discharge.Purulent discharge was associated withArchanobacterium pyogenes, E. coli,Pseudomonas aeruginosa, Staphylococcusaureus, and mucopurulent with E. coli ,Archanobacterium pyogenes, Streptococcuspyogenes and Proteus vulgaris. In clear mucuswith flakes of pus, the most prevalent isolates wereE. coli and Proteus vulgaris. These observationsare in accord with previous reports (Studer andMorrow, 1978; Dohmen et al., 1995; Williams etal., 2005).

It is biologically sensible that the presenceof purulent uterine discharge should be a goodsummary indicator for assessment of postpartumuterine health. The presence of pus discharge onvaginoscopy is highly correlated with active bacterialinfection of the uterus (Dohmen et al., 1995;Williams et al., 2005). Rectal palpation of the uterus,is too subjective and often inaccurate (Miller, 1980).The ability to use rectal palpation of the uterus todiagnose infection accurately is closely related toskill and training, which vary tremendously amongpractitioners (LeBlanc et al., 2002). Thus, rectalpalpation is an error-prone tool for diagnosis.Examination of the vagina with a speculum is astraightforward procedure that permits the evaluationof the characteristics of fluids in the anterior vaginaand external cervical os (Miller, 1980; Williams etal., 2005). Vaginoscopic examination, combined withpalpation of the uterus, should increase the accuracyof diagnosing uterine infection (LeBlanc et al.,2002). Although endometrial biopsy andhistopathology may constitute the ideal method ofdiagnosis of endometritis, the procedure is invasive,expensive and time-consuming (Gilbert et al., 2005).Furthermore, the procedure itself might beassociated with delayed conception (Bonnett et al.,1993). Cytological examination of the reproductivetract often used to evaluate possible reproductivelesions in humans (Glenthoj et al., 1986).Endometrial cytology was used recently as adiagnostic technique for identification of sub clinicalendometritis (Kasimanickam et al., 2004; Gilbert etal., 2005). Cytological examination of buffalo cowswith endometritis revealed a high percentage ofpolymorphonuclear cells. These percentages of

PMNs were significantly higher (P<0.01) than thoseof the control groups. In addition, there wassignificant correlation (P<0.01) between PMNsvalue and bacterial growth score of the uterus.These results are in agreement with the finding byZerbe et al. (1996), and Gilbert et al. (2005). AlsoZargham-Khan et al. (1998) and Ahmadi et al.(2005) claimed that cytogenic examination is a morereliable method of establishing the presence orabsence of uterine inflammation in buffalo cows.

The enzyme activity of creatine kinase(CK) and aspartate aminotransferase (AST) in theserum of buffalo cows measured in this study agreedwith the findings of Sattler and Furll (2004), whoalso observed high elevation of CK and AST in theserum of cows with uterine infection. The highelevations of serum CK and AST in buffaloes withendometritis might be due to the destruction of cellsin the uterine wall mainly in the chronic type ofinflammation, which can induce an increase in serumCK and AST. Buffalo cows with pathological uterinefindings (endometritis) have elevated CK and ASTactivities in serum and differ significantly from theserum activity of the same enzymes in heifers, butno significant difference was observed frompluriparious control group. Other pathologicalconditions such as skeletal muscle damage,hypocalcaemia and liver damage diseases mightinterfere with the enzyme activity. Sattler and Furll(2004) recommended the use of CK as a screeningparameter for the detection of endometritis. Fromthe data obtained in this study, it could be concludedthat CK and AST activities in serum of buffalo cowswith endometritis could be a valuable aid for thedetermination of uterine tissue destruction, but couldnot be used for the determination or diagnosis ofendometritis buffalo cows. No data are availableconcerning enzymatic activity in buffalo cows forcomparing these results with others.

CONCLUSION

It could be concluded that Archano-bacterium pyogenes was the only non-specificuterine pathogen directly associated with severeendometrial lesions. Histopathological studiesindicated a high incidence of chronic metritis. Non-

279


specific bacteria including E. coli, Bacteroidesfragilis, Klebsiella pneumoniae, Pseudomonasaeruginosa, Staphylococcus aureus, Strepto-coccus pyogenes when isolated in high-densitygrowth indicated diseased uteri. Vaginoscopicexamination combined with palpation of uterusincrease the accuracy of diagnosing uterine infection,and cytogenic examination of uterine discharge is amore reliable method of establishing the presenceor absence of uterine inflammation in buffalo cows.CK and AST enzymes in serum of buffalo cowscould not be used for the diagnosis of endometritis.

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Table 2. Bacterial isolates versus histopathological changes of buffalo cows affected with endometritis.

Table 3. Mean ± S.E. of bacterial growth score and polymorphonuclear cells (PMN) of buffalo cows with endometritis.

Table 4. Mean ± S.E. of creatine kinase (CK) and aspartate aminotransferase (AST) of buffalo cows affected with endometritis.

a-b significant at (P< 0.05).

284

Histopathological changes Mild Moderate SevereBacterial isolates

No. % No. % No. %Archanobacterium pyogenes - - - - 13 38.23 Bacillus licheniformis - - - - 2 5.88Escherichia coli 10 90.91 7 33.33 5 14.71 Klebsiella pneumoniae - - 2 9.52 1 2.95Lactobacillus acidophilus 1 9.09 - - - -Proteus vulgaris - - 2 9.52 - -Pseudomonas aeruginosa - - 4 19.05 1 2.95Staphylococcus aureus - - 3 14.28 7 20.59 Streptococcus acidominus - - 2 9.52 - -Streptococcus pyogenes - - - - 5 14.71 Streptococcus ubris - - 1 4.77 - -Total 11 100 21 100 34 100No growth 2 33.33 4 66.67 - -

Bacterial growth score Buffaloes

Vagina Cervix UterusPMN

Tox Endometritis 3.40±0.22a 1.38±0.21d 2.81±0.26 a 41.10±11.91

GroupsNo. of

animals CK (U/L) AST (U/L)

Endometritis Control-heifersControl-pluriparious

1566

321.47±39.06a

162.01±16.41b

208.33±5.84b

133.93±12.43a

97.01±6.86b

156.17±9.65a


SEROPREVALENCE OF LISTERIA MONOCYTOGENESIN BUFFALOES IN GUJARAT, INDIA

Ku. Rathod. P.H., N.M. Shah, H.C. Chauhan, K.A. Vasava, B.S. Chandel,H.N. Kher and A.I. Dadawala

Department of Microbiology, College of Veterinary Science & A.H. SDAU, Sardarkrushinagar -385 5006Dist Banaskantha (Gujarat), India

ABSTRACT

A serological survey of Listeriamonocytogenes was conducted in buffaloes ofGujarat, employing tube agglutination test (TAT), slideagglutination test (SAT), microtitre plate agglutination(MPAT), indirect haemagglutination (IHA), andenzyme linked immunosorbent assay (ELISA). Atotal of 267 serum samples were screened for thepresence of listerial antibodies. Out of these 78(29.21 %), 39 (14.60 %), 67 (25.09%), 72 (26.96%) and 64 (23.97 %) samples were positive. Aftertreatment of serum samples with 2- ME, a dramaticreduction of positive serum samples seen.

Keywords: Listeria monocytogenes, Listeriosis,seroprevalence, buffaloes, Gujarat

INTRODUCTION

Listeriosis is an important bacterial zoonosiscaused by pathogenic strains of Listeriamonocytogenes that occurs world wide and hasbeen reported in variety of animal species and man(Gray and Killinger, 1966). The disease is referredas listeriosis / listerellosis, listerial abortion, and theneural form is also known as circling disease, caprinebacterial encephalitis. Listeria monocytogenes hasgained a great deal of attention due to not onlyincreased reports of clinical disease, manifested bysepticaemia, abortion, stillbirth, meningitis andmeningio-encephalitis in man and animals but alsofor its implication as a food borne pathogen (Lowand Donachie, 1997). Occasionally it also causesmastitis, spinal myelitis or keratoconjunctivitis andopthalmitis (Radostits et al., 1995). L.

monocytogenes is widespread in nature and hasbeen reported from almost all species ofdomesticated animals, as well as many species ofpoultry, fish, wild animals, rodents, ticks and alsofrom man.

The disease can be diagnosed on the basisof clinical signs and symptoms and demonstrationof the organisms in smears by Gram staining, byperoxidase-antiperoxidase method or by FAT, andits confirmation achieved by isolating the pathogenfrom clinical specimens (Malik et al, 2002). Theepidemiological aspects and pathogenicity ofinfection remain poorly understood (Low andDonachie, 1997). Moreover, it can be easily confusedwith nervous form of ketosis, rabies and encephalitisof viral origin (Dedie, 1958). The epidemiology ofthe disease, the extent of food contamination andthe importance of the food-borne route oftransmission remains poorly understood owing to alack of effective detection assays (Kaur and Sood,1996). Therefore, detection of listerial antibodies hasbeen used as the indirect measures of infection invarious domestic animals with the advantage ofscreening large populations in comparatively shortperiods (Teruya et al., 1977).

The present study assessed theseroepidemiology of Listeria monocytogenesinfection in buffaloes in Gujarat by detection oflisterial antibodies.


Test samples A total of 267 serum samples were collectedfrom different regions of Gujarat viz. North Gujarat,Central Gujarat, Saurastra, Navsari and Kuchchh.

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CultureA standard reference culture strain of

Listeria monocytogenes (MTCC-1143) wasobtained from Microbial Type Culture Collection,Institute of Microbial Technology, Chandigarh.

Trypsinized antigens for agglutination testsThe trypsinized antigen for microtitre plate

agglutination (MPAT) and tube agglutination tests(TAT) were prepared by following method ofOsebold et al. (1965) with some modifications.

Reduction of sera samplesEach test serum sample was reduced by

treatment with 2-mercaptoethanol (2-ME) asdescribed by Osebold and Aalund (1968). First 2-ME was diluted to 1:4 in distilled water. The serumsample was diluted to 1:5 in normal tubes. The wholeset was incubated in the a water bath at 50-520Cfor 2 h followed by holding at 4 0C for 24 h. Afterthat, the samples were taken out and left at roomtemperature for 1 h. The observations made byholding the tubes against black background andcomparing them with controls. The highest titreshowing 50 percent or more agglutination was takenas the final agglutination titre.

Slide agglutination test (SAT) The antigen for SAT was prepared as per

the method of Osebold et al. (1965), with somemodifications similar to trypsinized antigen. One drop(0.03 ml) of serum was taken on a glass slide bymicropipette. The antigen bottle was shaken well toensure homogenous suspension and then one drop(0.03ml) of whole cell coloured antigen was added.The antigen and serum were mixed thoroughly witha spreader and then the slide was rotated for 1 to 2minutes. The result was read immediately after 2 to3 minutes. A positive result was indicated by definiteclumping, while in case of negative reaction, themixture remained homogeneous without formationof any clumps (Chand et al., 2001).

Microtitre plate agglutination test (MPAT)The method of microtitre plate agglutination

test as detailed by Vasava et al. (2005) was adopted.The microtitre plate agglutination test was carried

out in Laxbro micro titre plates (MT 96jU). A serialtwofold dilution of both types of serum (whole serumand reduced serum) in NSS @ 200 III was made.After adding equal volume trypsinized antigen, theplates were covered and incubated on a platform ofa serological water bath. The temperature of thewater bath was adjusted to 60 0C so that thetemperature was maintained at 50 0C on the platformat approximately 2.5 cm above the water level. Theplates were then kept at 4 0C for 24 h. After that,they were taken out and left at room temperaturefor 1 h. These plates were examined for agglutination,that is a positive reaction was indicated by matformation, whereas button formation as was takenas negative.

Indirect haemagglutination test (IHA)The preparation of antigen sensitized cells

and testing were performed according Mishra andJaiswal (1992). The whole serum and reduced serumwere tested simultaneously; 0.02 ml of N.S.S. waspipetted in each well. Inactivated serum under testwas added in 0.02 ml quantity in the first well. Serumin first well was mixed and 0.02 ml of the dilutedserum was transferred to the next well. The processwas repeated for making two-fold dilutions in theremaining wells. 0.02 ml-diluted serum from last wellwas discarded. 0.02 ml sensitized cells were addedto each well. The plate was incubated at roomtemperature for two hours and then read. A positivereaction was indicated by mat formation while anegative reaction by button formation. A titre of 1.8and above was considered as positive.

Indirect enzyme linked immunosorbent assay

i-ELISAELI SA for the demonstration of antibodies

in sera of buffaloes was performed according to(Tripathi et al. 2001) as per below.

The trypsinized antigen was used afterdiluting it to 1:50 in coating buffer. (Carbonate/bicarbonate buffer, pH 9.5). Rabbit anti-bovineIgG-HRPO conjugate (Banglore Genei) wasprocured and used in the test. The wells of a flatbottom micro titre plate were coated with 100 pI oftrypsinised antigen. Antigen was diluted to 1:50 incoating buffer (pH 9.6). The antigen-coated plate

286


was incubated at 4 0C for overnight. The plate waswashed thrice with PBS containing 0.05% Tween20 (PBS-Tween) and dried. Then, 100 pI of 1%bovine serum albumin was added to block unreactedsites in the wells and incubated for 1 h at 37 0C. Theplate was washed thrice with PBS-Tween 20. Then100 pI sera (from each positive control, negativecontrol and samples) diluted at 1:50 in PBS wereadded and plate was incubated at 37 0C for two h.The plate was again washed thrice with PBS/Tweenand dried. Then, 100 pI per well of 1:5000 HRPlabeled rabbit anti bovine IgG conjugate diluted inPBS-Tween was added to each well and the platewas incubated at 37 0C for two h. The plate waswashed thrice with PBS-Tween and dried as before.Then, 100 pI of substrate solution was diluted indistilled water and added to each well. The platewas incubated at 37 0C for 45 minutes. The reactionwas stopped by adding 50 μl of 2M H2SO4. Theoptical density (OD) of each well was read at 492nm on an ELISA reader. The OD value, which givesa distinct measurable difference between the controland the test serum, was taken for result inter-pretation.


In the present study a total of 267 buffaloesserum samples were screened to detect listerialantibodies. Of these 78 serum samples were foundpositive by STAT, giving an overall seroprevalenceof 29.21%. Sex wise, female buffaloes showedhigher rate of seroprevalence (70, or 30.43%) thanmale buffaloes (eight, or 21.62%).

Region wise seroprevalence of Listeriamonocytogenes A total of 267 serum samples from differentregions of Gujarat, viz., North Gujarat (Banaskantha,Sabarkantha, Patan and Mehsana), Central Gujarat(Anand, Kheda, Vadodara and Panchmahal),Saurashtra (Junagadh, Jamnagar and Amreli),Navsari and Kuchchh were tested. Of these, thehighest seroprevalence (37.87%) was observed incentral Gujarat whereas the lowest seroprevalence(16.66%) was observed in Navsari. Theseroprevalence observed in north Gujarat was 20.86

percent in, that Saurashtra 35.71%, and that inKuchchh 30.00%.

Seroprevalence of Listeria monocytogenes inwhole serum samples

A total of 267 whole serum samples werescreened by TAT, SAT, MPAT, IHA and 1- ELISAand the rates of seroprevalence recorded were,respectively, 29.21%, 14.60%, 25.09%, 26.96% and23.97%.

Seroprevalence in 2-ME reduced serumsamples

After, treatment of sera samples with 2-ME,seroprevalences of 10.11%, 4.11%, 7.11%, 8.61%were recorded by TAT, SAT, MPAT, and IHA,respectively. There was a dramatic reduction in thenumber of positive samples found.

The interpretation of serological tests forantibodies against L. monocytogenes is madedifficult by the presence of positive reactions of upto 1:200 in clinically normal animals. This is becauseL. monocytogenes is widespread in the environmentand a significant proportion of animals sporadicallyshed the organisms in their feces or milk. (Skovgaardand Morgen, 1988). Osebol and Aalund (1968)studied the antibody response in animals and manand found that all the sera from apparently normalsheep and cattle contained IgM antibody to Listeriamonocytogenes. They also found that normal seradid not contain IgG antibodies but that they appearedregularly following experimental infection with thesebacteria. This was also found in the present studybecause a greater number of sera samples prior tothe treatment with 2-ME were found positive butafter treatment with 2-ME there was reduction inthe number of positive sera samples. It was apparentthat with 2-ME treatment, the agglutinating activityof serum samples was destroyed, suggesting thepresence of IgM antibody in these sera. Theagglutinin activity of rest of the sera samples wasnot destroyed by 2-ME, suggesting the presence ofIgG or IgA.

ACKNOWLEDGEMENTS

We thank Dr. V.P.Vadodaria, Dean, Collegeof Veterinary Science and A.H., for providing the

287


necessary facilities. We also gratefully acknowledgethe help of field veterinarians for collection of serumsamples.

REFERENCES

Chand, P., J. R. Sadana and S. Chand. 2001.Comparison of studies of agglutination test andstandard tube agglutination test for detectionof listeric agglu-tinin in sheep. J. Immunol.immunopathol., 3: 28-33.

Dedie, K. 1958. Listeriosen. Beiheft I, Zentr.Veterinaermed Paul Paray Verlag, Berlin.(Cited by Gray and Killinger, 1966).

Dutta, P.K. and B.S. Malik. 1978. Someepidemiological studies on Listeriosis in manand animals India. Indian J. Public Health.,22: 321-322.

Gray, M.L. and A.H. Killinger. 1966. Listeriamonocytogenes and Listeria infection.Bacteriol. Rev., 30: 309-382.

Kaur, J. and S.K. Sood. 1996. Dealing withLis ter ia monocy togenes in da i ryproducts. Indian Dairyman., 48: 23-30.

Low, J.C. and W. Donachie. 1997. A reviewof Lis ter ia monocy togenes andlisteriosis. Vet. J., 153: 9-29.

Mal ik , S .V.S. , S .B. Barbuddhe and S .P.Chaudhari. 2002. Listeric infection inhumans and animals in the Indian sub

continent: A Review. Trop. Anim. HealthProd., 34: 359-381.

Mishra, S.S. and T.N. Jaiswal. 1992. Transfer ofdelayed hypersensitivity by dialyzablelymphocytes extract from Listeriamonocytogenes sensitized rabbits. Indian Vet.J., 69: 1-4.

Osebold, J.W. and O. Aalund. 1968. Interpretationof serum agglutinating antibodies to Listeriamonocytogenes by immunoglobulindifferentiation. J. Infect. Dis., 118: 139-148.

Radostits, O.M., D.C. Blood and C.C. Gay.1995. Veterinary Medicine, 8th ed. ELBSBailliere Tindall, London.

Skovgaard, N. and C.A. Morgen. 1988. Detectionof Listeria spp. in faeces from animals, in feedsand in raw foods of animal origin. Int. J. FoodMicrobiol., 6: 229-242.

Teruya, J.M., C.A.S. Ross, W. Girogi and R.M.Yauaguita. 1977. Serological studies indomestic animals in Sao Paulo Brazil.International J. Zoonoses., 4: 21-24.

Vasava, K.A., H.N. Kher, H.C. Chauhan, B.S.Chandel and N.M. Shah. 2005. Seroprevalnceof Listeria infection in animals of northGujarat. Indian. Vet. J., 82: 254-256.

Viswanathan, K. R. 1978. Serological studies withListeria monocytogenes in sheep. M.V.Sc.thesis submitted to IVRI.

288


DOCUMENTATION OF CHANGES IN HAEMATOLOGY AND MILK CONSTITUENTSDURING OESTRUS IN MURRAH BUFFALOES

P.K. Pankaj1, A. Mishra2, A.K. Gour3, S.W. Amin3, R. Jain4 and R.P.S. Baghel5

1Livestock Farm, Department of LPM, College of Veterinary Science & A.H., JNKVV, Adhartal, Jabalpur-482004 (M.P.), India. e-mail: [email protected] of Vety.Physiology, CoVSc & A.H., Jabalpur-482001 (M.P.), India3Livestock Farm, CoVSc & A.H., JNKVV, Adhartal, Jabalpur-482004 (M.P.), India4KVK, JNKVV,Rewa (M.P.), India5College of Veterinary Science & A.H., JNKVV, Adhartal, Jabalpur-482001 (M.P.), India

INTRODUCTION

The buffalo has been used in India fromtime immemorial in one form or another. The buffalois also known as an Asian animal due its widedistribution over the Asian continent. The buffalo isof great importance due to its high milk yield withhigher fat percentage and better survival ability underIndian conditions. The native place of the Murrahbuffalo breed is Punjab, but now it is found all overIndia.

Estrus, or heat, is a specific period ofreproductive function when the female becomesreceptive. Estrus in buffaloes is manifested bychanges in the reproductive system and in behavior.Overt signs of oestrus in buffaloes are less obviousthan in cattle. Determination of when a buffalo cowis in estrus is difficult because often the animal showsfew external signs of “heat”. Signs during the normalbreeding season (October to February in Asia andIndia) become still weaker during the hot months ofsummer. This increases the chances of missing acycle. Diurnal patterns of estrus behavior have beenobserved in most buffaloes. Based on variousexperiments, it has been suggested that in buffaloes,majority of estrus occur between 6 PM and 6 AM.Due to the high incidence of silent heat, largenumbers of buffaloes are left unbred. The aim ofthe present study is to see whether there is anychange in the normal blood constituent values andmilk constituents due to estrus from 0 to 36 h. The

duration of oestrus ranges from 19 to 21 h(Jainudeen, 1983).

During estrus mucus is constantly secretedby the columnar epithelial cells of the endocervix,but the amount and consistency vary with the stageof estrus cycle (Kamonpatana, 1982). At estrus, thecervical mucus is profuse, watery and clear. It isoften suddenly discharged from the vulva, especiallyin buffaloes. The cervical milieu at this stage isfavourable for sperm penetration.


Twenty-seven Murrah buffaloes undernormal cyclic condition maintained at CompositeLivestock farm, Adhartal, College of VeterinaryScience and Animal Husbandry, Jabalpur (M.P.)were taken for the present investigation.

MANAGERIAL PRACTICES

The husbandry practices followed during theexperimental period for all the buffaloes were similar.Animals were stall fed and grazing was not allowed.However, the animals were let loose daily in apaddock for about an hour both in the morning andevening. Milking was done manually twice daily at3 A.M. and 3 P.M. All animals were vaccinatedagainst hemorrhagic septicemia, black quarter,anthrax, foot and mouth disease and were free frombrucellosis.

289


* Digestible Crude Protein (DCP) = 17% and Total Digestible Nutrients (TDN) = 70%

Maintenance allowance offered to allanimals was 1.5 kg concentrate per day andproduction ration 0.5 kg concentrate per litre of milk.Green fodder was available to them ad libidum and3 kg of dry-fodder was provided each animal.

COLLECTION OF BLOOD AND MILKSAMPLES

Teaser bulls were used to detect estrus inshe buffaloes. The animals were restrained and heatwas confirmed by rectal palpation and fern patternof cervical mucus. The reproductive organs werepalpated to detect the presence of mature graffianfollicle in the either of the ovaries, turbidity of uterinemusculature and relaxation of the cervical os(Qureshi et al., 1992). Palpation of genital organsusually caused the discharge of the copious, clearand stringy mucus from the cervix through reflexstimulation, swollen vulvar lips, frequent urinationalong with bellowing.

Buffaloes which were detected in heatwere restrained properly and blood was collectedunder aseptic condition with a 16 gauge needle fromthe jugular vein and put in vials having anticoagulantEDTA (Ethylene Diamine Tetra Acetate) 1 mgpowder for 1 ml of blood. A milk sample from therepresentative sample from the buffaloes in estruswas taken carefully in a clean and sterilized testtube. Care was taken to prevent contamination ofthe milk from surrounding environment by pluggingthe mouth of the test tube with a cotton plug andthen both were transported as soon as possible tothe laboratory. Blood and milk samples were collected

at 0, 12, 24 and 36 h of estrus. Changes inhaematology and milk constituents during estruswere studied in the sample collected from buffaloesunder investigation. An automatic blood analyzer andan automatic milk analyzer (Lactoscan) were usedto assess various blood and milk parameters,respectively.Statistical analysis: Mean and standard errorwere evaluated as per Snedecor and Cochran,1989.


Changes in haematology and milkconstituents during estrus were studied in thesamples collected from buffaloes underinvestigation, and the values of different parametersare summarized in Tables 1 and 2.

Fat % was significantly (p<0.05) decreased12 h after oestrus as compared to the average valueand individual 12 hourly intervals values. Milk yieldis usually less at the time of estrus but acorresponding increase in fat % at that time wasnot observed. This decrease in fat % may be due tosudden increase in milk yield after estrous stress.Similar observations were manifested in the case ofSNF %, solids %, freezing point depression (-0C)and Protein %, however, the reverse was in the caseof lactose % which significantly (p<0.05) increased12 h after oestrous. In all cases 36 h after the estrus,the various milk constituents level came to normalstatus. Results were in the similar to those asreported by Qureshi et al., 1989.

The whole episode can better depicted byFigure 1.

290

The composition of the concentrate offered to the animals was as follows:Ingredients Parts

1. Maize 25 2. Mustard Cake 30 3. Arhar Chuni 15 4. Deoiled Rice Polish (DORP) 09 5. Wheat Bran 18 6. Mineral Mixture 1.0 7. Common Salts 2.0


Table 1. Milk constituent variation during peri-estrous period (n=27).

abc; different superscript in a row vary significantly at p<0.05.

Figure 1. Milk constituent variation during peri-estrous period (n=27).

291

Sl. No.

Milk constituents

Avg. value of cyclic animals

Values at Oestrous

Values 12 h after

Oestrous

Values 24 h after

Oestrous

Values 36 h after

Oestrous

1 Fat % 6.63±0.05b 6.04±0.4b 3.52±0.1a 5.06±0.7b 6.52±0.3b

2 SNF % 10.11±0.8b 9.81±0.6b 8.86±0.8a 9.34±0.6ab 9.93±0.8b

3 Protein % 4.08±0.4bc 3.24±0.1ac 2.88±0.1a 3.04±0.2a 4.01±0.4bc

4 Lactose % 4.95±0.4a 5.02±0.4a 6.32±0.6b 5.01±0.5a 4.99±0.3a

5 Density (gm/ml) 33.12±4.1 30.11±1.2 31.97±2.3 31.82±1.2 34.01±3.1

6 Added water 0 0 0 0 0

7 Solids % 0.96±0.06b 0.92±0.03b 0.67±0.9a 0.82±0.02b 0.94±0.01b

8

Freezing point depression (-0C )

0.525±0.03a 0.612±0.05a 0.741±0.02b 0.581±0.03a 0.531±0.04a


Table 2. Hematology during peri-estrous period (n=27).

HEMATOLOGY DURING PERI-ESTROUSPEROID

The haemodynamic values during the peri-estrus period are presented in Table 2. All thehaematological parameters were in the normal range,viz., RBC Time, WBC Time, MCV (Mean cellvolume) 76-96 fl., WBC (white cell count) 4.0-11.0* 10^9/l, RBC (red cell count) 4.5-6.5 * 10^12/l, PLT(platelet count) 150-400 * 10^9/l, HCT (haematocrit),HGB (haemoglobin) 13.5-18.0 g/dl, MCH (mean cellhaemoglobin) 27-32 pg, MCHC (mean cell Hbconcentration) 30-36 g/dl (Lowe, 1999 and Pearson,2001).

ab different superscripts in a row vary significantly at p<0.05.

No significant changes in haematologicalvalues were observed at various intervals of estrus;however, the PLT was significantly (p<0.05)increased 12 h after onset of estrus.

Unlike present observations, reports areavailable regarding increase in the total leucocytecount, particularly neutrophils, and fall in the numberof eosinophils during estrus, increase in neutrophilsduring true estrus with a drop immediately after heat(Sarvaiya and Pathak, 1992). Erythrocytic count andhaemoglobin count decline at estrus while totalnumber of leucocytes increased with a markedneutrophilia and oesinopenia at this stage.Erythrocytic count, haemoglobin, packed cell volume

292

Sl.No. Hematology

Avg. value of cyclic animals

Values at Oestrous

Values 12 h after

Oestrous

Values 24 h after

Oestrous

Values 36 h after

Oestrous

1 RBC Time 13.3±1 13.3±0.07 13.3±0.9 13.2±0.8 13.3±1 2 WBC Time 13.3±0.9 13.3±0.8 13.3±0.5 13.2±0.6 13.3±0.4 3 MCV (fl) 48.9±4 48.4±3 48.7±3.2 48.2±2.5 48.5±5.3 4 WBC (103/ μl) 11.3±1 11.1±0.9 11.6±0.8 12.5±0.7 11.4±0.6 5 RBC (106/ μl) 6.09±0.2 6.05±0.3 6.68±0.1 6.7±0.8 6.1±0.6 6 PLT 20±2a 19±2.1a 27±1.9b 19±1.7a 20±1.6a

7 HCT/ PCV (%) 29.3±3.02 30.2±2.22 32.6±3.11 32.3±2.36 31.22±3.11 8 RDW 20.4±2.1 20.3±1.9 20.3±1.8 19.7±1.6 19.9±1.5 9 LYM (%) 38±3.1 38.1±2.1 37.22±2.2 39.1±3.3 37.9±4.1

10 LYM (103/ μl) 4.7±0.1 4.2±0.3 4.5±0.2 4.3±0.2 5.0±0.4 11 MID % 7.01±0.1 6.7±0.2 7±0.3 6.3±0.4 6.9±0.5 12 MID 0.8±0.01 0.8±0.02 0.9±0.01 0.8±0.01 0.8±0.01 13 RDW % 20.1±0.9 20.3±1.1 20.3±1.7 19.7±1.5 20.1±2.01 14 Hb (gm/dl) 11±1.1 10.5±1.2 11.3±0.94 11.7±0.95 11.1±0.93 15 MCH (pg) 17.2±1.0 17.3±1.2 17±1.3 17.5±2.01 17.1±1.4 16 GRAN (%) 52±4.3 55.3±3.9 55.7±4.4 54±4.8 51±3.2 17 GRAN (103/ μl) 6.2±0.4 6.1±0.3 6.4±0.2 6.7±0.3 6±0.4 18 RDWa 38.8±0.9 38.9±1.2 39.1±1.6 38.1±1.3 38.2±1.4 19 MCHC (%) 36.5±3.1 36.4±3.2 36.1±3.4 37.1±3.2 36.2±3.1


and erythrocyte sedimentation rate are low on theday of estrus (Batra et al., 2004).

Moberg (1952 1953, and 1955) suggestedan increase in the total leucocyte count particularlyneutrophils, and fall in the number of eosinophilsduring estrus, and increase in neutrophils during trueestrus with a drop immediately after heat. Solimenand Selim (1966) suggested that the erythrocyticcount and haemoglobin count decline at estrus whiletotal number of leucocytes increased with a markedneutrophilia and oesinopenia at this stage.Erythrocytic count, haemoglobin, packed cell volumeand erythrocyte sedimentation rate are low on theday of estrus.

CONCLUSION

The present work was designed to studythe changes in haematology and milk constituentsduring estrus in Murrah buffaloes with a view toevaluate the relative merits of various parametersfor diagnosis of the silent estrus problem. This workwas necessitated because of the fact that there weresurprisingly few reports on the changes inhaematology and milk constituents during estrus inMurrah buffaloes, though the basic reproductivephenomena in the females apparently depend uponit, directly or indirectly. From the above experiment,it is observed that after 12 h of estrus there is markedchange in all the constituents of milk.

REFERENCES

Batra, S.K., R.C. Arora, N.K. Bachlaus and R.S.Pandey. 2004. Blood and milk progesteronein pregnant and nonpregnant buffalo. CASlecture manual. Dairy Chemistry Division,National Dairy Research Institute, Karnal(India) 132001.

Jainudeen, M.R., W. Sharifuddin and F.B. Ahmad.1983. Relationship of ovarian contents to

plasma progesterone concentration in swampbuffaloes (Bubalus bubalis). Vet. Rec., 13:369-372.

Kamonpatana, M. 1982. Application of plasmaprogesterone by EIA to estrus confirmationand early pregnancy diagnosis in swampbuffalo, p. 58-67. In Annual Report, TheNational Buffalo Research andDevelopment Center Project, Bangkok,Thailand.

Lowe, G.D. 1999. Rheological influences onthrombosis. Baillieres Best Practice &Research. Clinical Haematology, 12(3): 435-449.

Moberg, R. 1952. Leucocytes during estrual cyclein cow, Proc. II Int.Congr. Phys, and Path.Of An. Repr. And Art. Insem. Copenhagen,vol. I: 129.

Moberg, R. 1953. Leucocytes during various sexualconditions in cattle. In Proc. XV Int. Vet.Congr., Stockholm, Vol.II: 755.

Moberg, R. 1955. The white blood picture insexually mature female cattle with specialreference to sexual conditions.V.M.D.Thesis. Stockholm.

Pearson, T.C. 2001. Evaluation of diagnostic criteriain polycythemia vera. Seminars inHematology, 38(Suppl 2): 21-24.

Qureshi, M.S., M. Ihsan, M. Ashraf, M. Hassanand M.B. Qureshi. 1989. Use of milkprogesterone test in estrus prediction in Nili-Ravi buffaloes. J. Animal Health Prod., 9:80-83.

Sarvaiya, N.P. and M.M. Pathak. 1992. Profile of -Oestradiol, triiodothyronine and bloodbiochemical parameters®progesterone 17 inSurti buffalo heifers. Buffalo J ., 8: 23-30.

Snedecor, G.W. and W.G. Cochran. 1989. Statisticalmethod, 6th ed. The Iowa State UniversityPress, Ames, Iowa, USA.

Solimen, M.K. and R. Selim. 1966. Blood picture ofbuffaloes at the various reproductive phases.Indian J. Dairy Sci., 19: 29.

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ASSOCIATION OF BREED AND POLYMORPHISM OF αS1- AND αS2-CASEIN GENES WITHMILK QUALITY AND DAILY MILK AND CONSTITUENT YIELD TRAITS OF BUFFALOES

(BUBALUS BUBALIS)

S.S. Misra1, Arjava Sharma2, T.K. Bhattacharya3, P. Kumar and S. Saha Roy2

1Division of Animal Genetics and Breeding, Faculty of Veterinary Sciences & Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences & Technology of Kashmir, Shuhama, Alusteng, Srinagar-190006, Jammu & Kashmir, India. e-mail: [email protected] Genetics Division, Indian Veterinary Research Institute, Izatnagar, Bareilly-243 122, Uttar Pradesh,India3Project Directorate on Poultry, Rajendranagar, Andhra Pradesh-50PP0030, India

ABSTRACT

Milk constituents vary widely across breedsand among animals within the same breed. Buffalomilk is highly nutritious and qualitatively rich in higheramounts of different milk constituents compared tocow’s milk. The present investigation aimed atexploring the association of breed and genotypes ofαS1- and αS2-casein genes with different milkqualities (fat, protein, casein, solids not fat and totalsolids percentages), daily milk and constituent yieldtraits in four breeds of buffaloes, namely, Bhadawari,Mehsana, Murrah and Surti. Genotyping of αS1- andαS2-casein genes was done by single strandconformational polymorphism analysis. The milksamples were analysed to estimate different milkquality traits and their yields. The breed hadsignificant (P≤0.05) effect on almost all milk qualitiesand all yield traits. Murrah was the best performingbreed for fat, total protein and casein percentages,while Mehsana was for solids not fat and Bhadawarifor total solids. Mehsana outclassed all other breedsfor all the yield traits, and Bhadawari was found tobe the poorest performer in those traits. Except theeffect of partial αS2-casein exon 4-5 genotypes ontotal solids percentage (p≤0.1), no other genotypesof the two α-casein genes showed any significantassociation with the traits under study.

Keywords: buffalo breed, αS1-casein, αS2-casein,milk quality

INTRODUCTION

India ranks first in the world in terms ofbuffalo population, possessing 97.92 millionbuffaloes, constituting over 57% of the total worldbuffalo population, (Livestock Census Report, 2005;FAOSTAT, 2006) including all the excellent dairybreeds of buffalo. Buffaloes contribute more than55% of the total milk production of India (FAOSTAT,2006). On average, the productivity of the Indianbuffalo is much higher than that of the indigenouscows. Buffalo milk is highly nutritious, and havinghigher amount of fat, protein, vitamins, minerals andlow cholesterol content (BBC, 2005) compared tocow’s milk. In addition, the buffalo has the uniquecapability of metabolizing all dietary carotene intovitamin A, which is passed into and secreted in milk.With too much stress laid down for improvement ofcattle through crossbreeding in the last four decades,the development and conservation of the indigenouswell-adapted species/breeds, especially of buffalo,has been ignored, leading to their gradual geneticdegradation and silent progressive elimination fromthe production system. Thus, despite its hugepotential and superiority over cows in many aspects,the true potentialities of this ‘black gold’ haveremained generally unrealized and been neglectedtill date.

There are significant genetic variationsattributable to sire that exist among different milkconstituents indicating the possibility of altering themto wider limits (Gibson, 1987; Misra and Joshi, 2004).

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Though the milk constituents are almost twice asheritable as compared to the milk production traits(Gonyon et al., 1987), the former have been givenvery little or no attention at all in the geneticimprovement programmes. The excessiveprominence given to boost milk production in Indiahas resulted in dilution of the milk constituents dueto undesirable genetic correlation present amongmilk yield and constituent traits, thus producing milkwith low nutrient quality and reduced acceptance tothe consumers. Of late, the ever-increasing healthconsciousness of consumers has brought in achanging demand for milk and milk products withdesired levels of certain constituent(s). This has ledto the concept of designer milk and establishmentof component-based milk marketing systems in manycountries. Today’s dairy industry is much concernedabout matching the changing preferences of theconsumers. This can be achieved by exploiting theexisting variations of milk constituents attributableto individual and breed differences to permanentlyalter the milk composition through a combinedapproach of molecular and quantitative genetics.

Milk proteins comprise two major groupsof constituents, namely the caseins and wheyproteins. Caseins are the major milk proteins, makingup 80% of the total protein in the milk of ruminants(Dalgleish, 1993). The four types of caseins, i.e. αS1,αS2, β and κ contribute 38%, 10%, 36% and 13% ofthe total casein in milk, respectively (Davies andLaw, 1980). A single autosomal gene codes for eachof these caseins. All the casein genes are located ina 200-300 kb region (Mercier and Vilotte, 1993) ofchromosome number 7 in the buffalo (Iannuzzi etal., 1996).

Milk caseins show breed to breed andanimal to animal variations in their quantities in milkas well as in their polymorphic forms, both at theprotein and DNA levels. Certain genetic variants ofcaseins influence the production, composition,processing and nutritional quality of milk (Ng-Kwai-Hang, 1998; Martin et al., 2002). Among the fourcaseins, αS1- and αS2-caseins are the calcium-sensitive caseins as they are precipitated in thepresence of low concentrations of calcium and stablymaintained in a micelle suspension as a result oftheir interaction with κ-casein (Mackinlay andWake, 1971). They constitute nearly 50% of the total

casein in milk. They show high polymorphism, anddifferent polymorphic forms produce differentquantities of caseins in milk. Like other caseins, theyalso influence the quantity and quality of milk.Several reports relating to studies of the αS1- andαS2-casein genes in cattle, goat and sheep areavailable, but till now reports on buffaloes are veryfew. Hence, the present investigation aimed toexplore the influence of different breeds of buffaloesas well as the polymorphic variants of two α-caseingenes on milk production, quality traits and yields.


Experimental animalsA total of 194 animals of four different

breeds of buffaloes, viz. Bhadawari (n=32), Murrah(n=40), Mehsana (n=65) and Surti (n=57) werestudied. The animals were selected randomly fromfour different organized farms located in differentparts of India. Bhadawari breed is native to Etawahand Agra regions of Uttar Pradesh and Gwalior ofMadhya Pradesh state. Murrah is found in the statesof Haryana and Punjab in Northern India. The othertwo breeds are distributed in Gujarat state of North-west India.

Collection of blood and milk samplesBlood samples were collected from each

animal in sterile polypropylene tubes containingEDTA as an anti-coagulant. Just after milking, about50 ml of fresh and properly mixed milk samples werecollected from those same animals in sterile widemouth plastic bottles containing preservative. Thebottles were tightly capped, thoroughly mixed withthe preservative by gentle shaking and placed in anice box. Both blood and milk samples were thentransported to the laboratory under the coolconditions and analyzed in the quickest possible timefor DNA and different milk constituents,respectively.

Collection of recordsRecords of the target animals were

collected pertaining to different aspects like animalno., sire no., dam no., date of birth, date of calving,

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lactation length and full lactation milk yield from thehistory sheet/daily farm registers maintained at theconcerned farms, according to their availability.

DNA extraction and GenotypingGenomic DNA was extracted from blood

samples following standard protocol (Sambrook andRussell, 2001). The good quality DNA samples wereamplified by polymerase chain reaction (PCR)technique using specific primers for four fragments,corresponding to partial exon 10 to 11 (129 bp) andpartial exon 19 (303 bp) of αS1- and partial exon 4to 5 (141 bp) and partial exon 18 (256 bp) of αS2-casein genes. The amplicons were genotyped bysingle strand conformational polymorphism (SSCP)analysis followed by silver staining (Bassam et al.,1991). The SSCP patterns of individual samples werescored and documented.

Analysis of milk samplesMilk samples of each animal were mixed

properly and analyzed for different milk quality traits,viz. fat, total protein and total casein percentages.The data on solids not fat, total solids percentagesand yields of different milk constituents weregenerated from the estimated data using standardformulae.

Estimation of fat (FP), total protein (TP) andtotal casein (TCN) percentage

The fat percentage of each milk sample wasestimated using the Gerber method (ISI, 1977). The

TP and TCN percentages were estimated by theformal titration method (Pyne, 1932) using thefollowing formulae:TP (%) = Volume of alkali used in secondtitration*1.74TCN (%) = Volume of alkali used in secondtitration*1.38

Estimation of solids not fat (SNF) and totalsolids (TS) percentage

The SNF and TS percentages wereestimated using the following formulae: SNF (%) = CLR/4 + 0.22 F + 0.14where, CLR = corrected lactometer reading afterappropriate adjustment for temperature variation andF = Fat%. TS (%) = Fat % + SNF %

Estimation of daily yields of milk anddifferent milk constituents

The lactation yields of different milkconstituents were generated by multiplying thepercentage data for each trait of an animal with itstotal lactation milk yield (TMY) and then dividing itby 100. As the lactation length was highly variablefrom animal to animal, to adjust the error due tovariability in lactation length (LL), the lactation yieldswere converted into daily basis. Lactation yields ofmilk and each milk constituent was used as followsto estimate their daily yields:

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Daily milk yield (DMY) =Total lactation milk yield (TMY)

LL

LL

LL

LL

LL

LL

Daily fat yield (DFY) =

Daily total protein yield (DTPY) =

Daily total casein yield (DCNY) =

Daily SNF yield (DSNFY) =

Daily TS yield (DTSY) =

Total lactation fat yield (LFY)

Total lactation TP yield (LPY)

Total lactation CN yield (LCNY)

Total lactation SNF yield (LSNFY)

Total lactation TS yield (LTSY)


Statistical AnalysisThe performance of different breeds of

buffaloes as well as genotypes of differentfragments of αS1- and αS2-casein genes with respectto milk quality (fat%, TP%, TCN%, SNF% andTS%), daily milk (DMY) and constituent yield traits(DFY, DTPY, DCNY, DSNFY, DTSY) wereanalysed using the Least Squares MaximumLikelihood (LSML) programme of Harvey (1990).Also, the presence of any statistical association ofbreed and the different SSCP variants with thesetraits was investigated. Pairwise multiplecomparisons of the least squares means were doneby Duncan’s multiple range test as modified byKramer (1957).


GenotypingThe PCR-SSCP analysis revealed three

SSCP genotypes for each of the four fragments ofthe two genes studied. The αS1-casein genotypeswere AB, AC and BC for partial exon 10 to 11 andAB, AC and CC for partial exon 19. The αS2-caseingene showed AB, AC and BC genotypes for partialexon 4 to 5 and AB, AC and BC for partial exon 18region.

Effect of breedMilk quality traits: The effect of breed was

significant (P≤0.05) on FP, SNF and TS, whereas itwas found to be statistically non-significant on TPand TCN percentages (Table 1). Murrah was thebest performing breed for FP, TP and TCN, while itwas Mehsana for SNF and Bhadawari for TS.

Milk and constituent yield traitsThe daily yields of milk and different

constituents showed greater variation acrossbreeds compared to the milk quality traits. Breedhad a significant (P≤0.05) effect on all the yieldtraits (Table 2). Mehsana outclassed all otherbreeds in its performance for all the traits, andBhadawari was found to be the poorest performerin those traits. This extremely poor performanceby the Bhadawari breed may be due to its closed

herd nature with a very small population size.There was no artificial insemination facility andnatural breeding with a very few bulls was theonly way, and this might have resulted in anincreased level of inbreeding. In contrast to thisthe Mehsana herd had a fairly large number ofwell-managed animals, and all the facilities of amodern farm were available.

Effect of αααααS1-casein genotypesMilk quality traits: The αS1-casein gene partialexon 10-11 genotypes had non-significant effect onall the milk quality traits. However, among the threegenotypes, AC showed slightly higher performancefor all the traits as compared to the other genotypes(Table 3). The non-significant effect of thegenotypes may be attributed to the small variabilityamong different genotypes. In some cases, thenumber of animals with a particular genotype wasquite low, and this may have led to disparity incomparison among different classes of animals.

The difference in performance of milkquality traits for three genotypes of αS1-casein genepartial exon 19 was negligible and the effect ofgenotypes was found to be statistically non-significant on all the milk constituent traits. WhileAB genotype performed slightly better for FP, it wasCC which performed marginally higher comparedto the other genotypes for all the other traits (Table4).

Milk and constituent yield traits: As with milkquality traits, there was no significant difference dueto either αS1-casein gene partial exon 10-11 or exon19 genotypes was found in the yield traits. However,the AB genotype excelled in all the milk andconstituent yield traits (Table 5). For exon 19, slightlyhigher performance was noted in AC genotype forDMY, AB for DFY, CC for DTPY and DCNY, andAB for DSNFY and DTSY (Table 6).

Effect of αααααS2-casein genotypesMilk quality traits: Genotypes of partial exon 4-5locus played a significant (p≤0.1) role in the variationof TS percentage with genotype AC showing thebest performance (Table 7). Genotype was notresponsible for the variation present in the othertraits. The partial exon 18 genotypes did not show

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Table 1. Least squares means±SE of milk quality traits (%) of different breeds of buffaloes.

a,b,cMeans within a column under each factor having atleast one superscript in common do not differsignificantly.

significant effect on the milk quality traits. BCgenotype was the best performer for FP, SNF andTS percentages, while AB was for TP and TCN(Table 8).

Milk and constituent yield traits: Though theyield traits showed slight variation among various

genotype groups of partial exon 4-5 and partial exon18, yet the effect was statistically found to be non-significant. However, genotype AC of partial exon4-5 region had the highest performance for all theyield traits (Table 9) and the AB genotype of partialexon 18 performed marginally higher than the othersin all the traits studied (Table 10).

Table 2. Least squares means±SE of daily milk and constituent yield traits (kg) of different breeds of buffaloes.

a,b,c,dMeans within a column under each factor having atleast one superscript in common do not differsignificantly.

Table 3. Least squares means±SE of different genotypes of αS1-casein partial exon 10-11 (129 bp) for different milk quality traits (%).

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Breed FP TP TCN SNF TS Bhadawari 7.43±0.26a 3.92±0.07 3.16±0.06 8.99±0.10ab 17.70±0.28a Mehsana 6.46±0.17b 3.87±0.05 3.07±0.04 9.13±0.06a 15.59±0.18c Murrah 7.53±0.19a 4.03±0.05 3.20±0.04 9.00±0.07a 16.53±0.20b Surti 6.17±0.20b 3.93±0.05 3.11±0.04 8.80±0.07b 14.96±0.21c

Breed DMY DFY DTPY DCNY DSNFY DTSY Bhadawari 2.79±0.23a 0.21±0.019a 0.11±0.010a 0.09±0.008a 0.25±0.022a 0.50±0.040a Mehsana 6.07±0.15b 0.39±0.013b 0.24±0.007b 0.19±0.005b 0.55±0.015b 0.95±0.026b Murrah 4.95±0.16c 0.36±0.014b 0.19±0.007c 0.15±0.006c 0.43±0.016c 0.78±0.028c Surti 3.75±0.17d 0.23±0.014a 0.15±0.008d 0.12±0.006d 0.33±0.017d 0.56±0.030a

Traits FP TP TCN SNF TS Overall 6.96±0.17 3.99±0.04 3.16±0.03 9.04±0.06 16.44±0.18

AB 6.82±0.19 4.00±0.04 3.17±0.04 8.93±0.07 16.09±0.20 AC 7.08±0.44 4.05±0.11 3.19±0.09 9.20±0.16 17.00±0.47 BC 6.98±0.17 3.91±0.04 3.12±0.03 8.98±0.06 16.21±0.17


Table 4. Least squares means±SE of different genotypes of αS1-casein partial exon 19 (303 bp for different milk quality traits (%).

Table 5. Least squares means±SE of different genotypes of αS1-casein partial exon 10-11 (129 bp) for different daily milk and constituent yield traits (kg).

Table 6. Least squares means±SE of different genotypes of αS1-casein partial exon 19 (303 bp) for daily milk and constituent yield traits (kg).

Table 7. Least squares means±SE of different genotypes of αS2-casein partial exon 4-5 (141 bp) for different milk quality traits (%).

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AB 6.87±0.20 3.91±0.05 3.14±0.04 8.96±0.07 16.08±0.22 AC 6.78±0.34 3.91±0.08 3.14±0.07 8.98±0.11 16.02±0.38 CC 6.78±0.51 4.05±0.12 3.25±0.10 9.08±0.17 16.12±0.56

Traits DMY DFY DTPY DCNY DSNFY DTSY Overall 4.26±0.14 0.30±0.01 0.17±0.005 0.13±0.005 0.39±0.01 0.70±0.02

AB 4.60±0.15 0.31±0.01 0.18±0.01 0.14±0.01 0.41±0.01 0.72±0.03 AC 3.88±0.36 0.29±0.03 0.16±0.02 0.13±0.01 0.37±0.04 0.68±0.06 BC 4.29±0.14 0.30±0.01 0.17±0.01 0.13±0.01 0.38±0.01 0.69±0.02

Traits DMY DFY DTPY DCNY DSNFY DTSY overall 4.41±0.18 0.29±0.02 0.17±0.008 0.14±0.006 0.40±0.02 0.69±0.03

AB 4.40±0.16 0.30±0.01 0.17±0.01 0.14±0.01 0.39±0.01 0.70±0.03 AC 4.44±0.27 0.29±0.02 0.17±0.01 0.14±0.01 0.39±0.03 0.69±0.04 CC 4.38±0.40 0.29±0.03 0.17±0.02 0.14±0.01 0.39±0.04 0.69±0.07

a,b,cMeans within a column under each factor having atleast one superscript in common do not differ significantly.


AB 6.75±0.20 3.90±0.71 3.11±0.04 8.95±0.07 16.05±0.21a

AC 7.87±0.57 3.83±2.00 3.15±0.12 9.30±0.21 17.45±0.61b

BC 6.89±0.17 3.99±0.58 3.16±0.03 8.95±0.06 16.14±0.18c


Table 8. Least squares means±SE of different genotypes of αS2-casein partial exon 18 (256 bp) for different milk quality traits (%).

Table 9. Least squares means±SE of different genotypes of αS2-casein partial exon 4-5 (141 bp) for daily milk and constituent yield traits (kg).

Table 10. Least squares means±SE of different genotypes of αS2-casein partial exon 18 (256 bp) for daily milk and constituent yield traits (kg).

CONCLUSIONS

Breed significantly influenced the variationspresent in most of the milk quality traits and all theyield traits. Mehsana was the best performer,whereas Bhadawari found to be the poorestperforming breed. Except the significant (p≤0.1)effect of αS2-casein partial exon 4 to 5 genotypeson total-solids percentage, the effect of genotypesof other loci were statistically non-significant on allother traits. Lack of association of most of the caseingenotypes on the milk quality and production traitsmay be attributable to the small sample size thatwas available for the present study. If screened in afairly large population, these genotypes are expected

to show significant association with milk quality andproduction traits. Any association information willbe useful in genotype assisted selection (GAS) toselect the superior genotypes with bestperformances for certain milk constituents at a veryearly age, thus, improving the quantity and qualityof milk produced.

ACKNOWLEDGEMENT

The first author is grateful to the Director,Indian Veterinary Research Institute for providingthe necessary facilities and financial assistance tocarry out the research work.

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AB 6.82±0.54 4.05±0.13 3.29±0.11 8.91±0.21 16.05±0.63 AC 6.78±0.31 3.86±0.08 3.13±0.07 9.01±0.12 16.17±0.36 BC 6.93±0.32 3.94±0.08 3.21±0.07 9.07±0.12 16.29±0.37


AB 4.18±0.17 0.28±0.01 0.16±0.01 0.13±0.01 0.38±0.02 0.67±0.03 AC 4.85±0.47 0.38±0.04 0.19±0.02 0.15±0.02 0.44±0.04 0.84±0.08 BC 4.57±0.14 0.31±0.01 0.18±0.01 0.14±0.01 0.41±0.01 0.72±0.02


AB 4.89±0.43 0.34±0.04 0.20±0.02 0.16±0.01 0.44±0.04 0.79±0.07 AC 4.63±0.24 0.31±0.02 0.17±0.01 0.14±0.01 0.41±0.02 0.73±0.04 BC 4.58±0.25 0.31±0.02 0.18±0.01 0.14±0.01 0.41±0.02 0.73±0.04


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BBC. 2005. Buffalo milk-the healthy alternative.(http://www.bbc.co.uk/insideout/east/series7/buffalo_milk.shtml)

Dalgleish, D.G. 1993. Bovine milk protein propertiesand the manufacturing quality of milk. Livest.Prod. Sci., 35: 75-93.

Davies, D.T. and A.J.R. Law. 1980. The contentand composition of protein in creamery milksin south-west Scotland. J. Dairy Res., 47: 83-90.

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Gibson, J.P. 1987. The options and prospects forgenetically altering milk composition in dairycattle. Anim. Breed. Abstr., 55: 231-243.

Gonyon, D.S., R.E. Mather, H.C. Hines, G.F.W.Haenlein, C.W. Arave and S.N. Gaunt. 1987.Associations of bovine blood and milkpolymorphisms with lactation traits: Holsteins.J. Dairy Sci., 70: 2585-2598.

Harvey W.R. 1990. User’s Guide for LSMLMWand MIXMDL PC-2 Version. Ohio StateUniversity, Colombus, USA.

Iannuzzi, L., D.S. Gallagher, J.E. Womack, P. DiMeog, C.P. Schelling and M.A.M. Groenen.1996. FISH mapping of the α

S2-casein gene

on river buffalo and cattle chromosomesidentifies a nomenclature discrepancy in thebovine karyotype. Chromosome Res., 4: 159-162.

ISI. 1977. Determination of fat by Gerber method.Part I. Milk (first revision). Indian StandardsInstitution, Manak Bhavan, New Delhi, India.

Kramer, C.Y. 1957. Extension of multiple rangetests to group correlated means. Biometrics,13: 13-18.

Livestock Census Report. 2005. 17th IndianLivestock Census-All India SummaryReport. Ministry of Agriculture, Govt. of India,New Delhi, India.

Mackinlay, A.G. and R.G. Wake. 1971. κ-casein andits attack by rennin (chymosin). In McKenzie,H. A. (ed.), Milk Proteins: Chemistry andMolecular Biology. Academic Press, NewYork, Vol. 2, p. 175-215.

Martin, P., M. Szymanowska, L. Zwierzchowski andC. Leroux. 2002. The impact of geneticpolymorphisms on the protein composition ofruminant milks. Reprod. Nutr. Develop., 42:433-459.

Mercier, J.C. and J.L. Vilotte. 1993. Structure andfunctions of milk protein genes. J. Dairy Sci.,76: 3079-3098.

Misra, S.S. and B.K. Joshi. 2004. Genetic andnon-genetic factors affecting lactationalmilk constituents and yield traits in KaranFries cattle. Ind. J. Dairy Sci., 57(1): 69-72

Ng-Kwai-Hang, K.F. 1998. Genetic polymorphismof milk proteins: Relationships with productiontraits, milk composition and technologicalproperties. Can. J. Anim. Sci.,78 (Supp l.):131-147.

Pyne, G.T. 1932. Determination of milk proteins byformaldehyde titration. Biochem. J., 26: 1006-1013.

Sambrook, J. and Russell, D.W. 2001. MolecularCloning: A Laboratory Manual. 3rd ed. Vol.1. Cold Spring Harbor Laboratory Press, NewYork, USA.


INFLUENCE OF COMMERCIALLY AVAILABLE FOLLICLE STIMULATING HORMONE ONIN VITRO MATURATION OF BUFFALO OOCYTES

S.A. Adlak , K.P. Khillare, C.H. Pawshe and S.W. Mude

Department of Animal Reproduction, Post Graduate Institute of Veterinary and Animal Sciences, Akola 444104,India

ABSTRACT

In the present study, the influence ofcommercially available follicle stimulating hormone(FSH/Folltropin-V) on in vitro maturation of buffalooocytes was evaluated. The highest maturation rate(71.05%) was observed in 10 μg /ml FSH. However,no significant difference was found in the hormonetreated group. The maturation rates at both 1 μg /mlFSH (46.87%) and 20 μg /ml FSH (51.21%) werelow as compared to 10 μg /ml FSH.

Hence we conclude that commerciallyavailable crude FSH preparation has good biologicalactivity and can be used successfully for in vitromaturation of buffalo oocytes.

Keyword: buffalo, oocytes, FSH, hormone

INTRODUCTION

Mammalian oocytes although removed fromthe follicle readily undergo meiotic maturation undergood culture conditions. During maturation, oocytesundergo some spontaneous morphological changeslike nuclear maturation, cumulus expansion andcytoplasmic maturation. The cultural environmentsupplied for in vitro maturation is critical forsubsequent fertilization and embryonic development.

Hormonal conditioning of oocytes at the timeof in vitro maturation is of paramount importancein achieving cytoplasmic maturation, which isnecessary for preparing the female gamete to initiatenormal development through fertilization (Thibaultet al., 1975; Goto et al., 1988). It is well establishedthat gonadotropins play a major role in triggering

the resumption of meiosis and cumulus expansion(Ball et al., 1980). Addition of FSH to culture mediumenhances the degree of cumulus expansion, andhigher rates of penetration and nuclear formationwere observed (Ball et al., 1984; Hensleigh andHunter, 1985).

Nowadays commercially produced crudeFSH and LH are available in the market. Thesecommercially available FSH (Folltropin-V)preparation have both FSH and LH activity, and theratio of FSH and LH varies among commercialproduct batches (Lindsell et al., 1986). Informationregarding the use of commercially available FSH inin vitro maturation of buffalo oocyte is limited.Hence, there is a need to examine the effect of theseproducts on in vitro maturation of buffalo oocytes.


Buffalo ovaries were collected from aslaughterhouse and brought to the laboratory innormal saline at 30-35 0C within 1-2 h of slaughter.Cumulus oocyte complexes (COCs) were isolatedfrom the follicles by the slicing method. Only goodquality oocytes enclosed with compact cumulus cellswere used for maturation.

The collected oocytes were washed thricewith TL-Hepes medium and then two final washingswere given in maturation medium. The oocytes weretransferred to the maturation medium, i.e. Ham’sF-10 supplemented with 10% fetal bovine serum(FBS) in the presence of three differentconcentrations of commercially available FSH(Folltropin-V, Vetrepharm Inc, Ontario, LondonCanada). The in vitro maturation was carried outin six different treatment groups: 1) Ham’s F-10

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alone. 2) Ham’s F-10 + 10% FBS. 3) Ham’s F-10 +10% FBS + 1 µg/ml oestradiol (E2). 4) Ham’s F-10+ 10% FBS + 1 µg/ml oestradiol + 1 µg/ml FSH. 5)Ham’s F-10 + 10% FBS + 1 µg/ml oestradiol + 10µg/ml FSH. 6) Ham’s F-10 + 10% FBS + 1 µg/mloestradiol + 20 µg/ml FSH.

Ten oocytes were placed in 50 µl maturationdrops, covered under sterile mineral oil at 39 0C in5% CO2 in air and 95% relative humidity andincubated for 24 h.

After 24 h of culture, the cumulus cells wereremoved mechanically with the help of a small bore,hand pulled glass pipette. The denuded oocytes werefixed overnight in ethanol: acetic acid (3:1) andstained with 1% aceto orecin stain. They were thenevaluated for germinal vesicle (GV), metaphase I(MI) and metaphase II (MII) and degeneratedoocyte.


In the present study, the effect of differentconcentrations of commercially available FSH(Folltropin-V) on in vitro maturation wasinvestigated. The results are presented in Table 1.

In the present study, the maturation rate ofbuffalo oocytes in Ham’s F-10 alone was 33.33%.The addition of fetal bovine serum improved thematuration rate of buffalo oocyte to 39.47%. Theaddition of hormones like oestradiol and FSH furtherimproved the maturation rate. The maturation ratevaried according to the different concentrations ofFSH. The maximum maturation rate, i.e 71.05%,was found in 10 µg/ml FSH. Whereas, the maturationrates in 1 µg and 20 µg was 46.87% and 51.21%,respectively, but no significant difference wasobserved in any of the hormone treated group.

In the present experiment, we found thatmaturation rate did not increased with increasingFSH concentration.

In the present experiment, three differentconcentrations of commercially available FSH wereused and there effect on in vitro maturation wasevaluated.

In the present study, we found thatmaturation percentage was higher in the hormonetreated group as compared to media containing nohormone. A higher maturation rate (71.05%) wasobserved in 10 µg/ml FSH concentration. The overallmaturation rate in the hormone treated groups wasfound lower in our study as compared to maturation

Table 1. Effect of different concentrations of FSH (Folltropin-V) in the presence of fetal bovine serum and oestradiol on nuclear maturation of buffalo oocytes (r =3).

Note: Means bearing similar superscript shows non-significant difference.GV – Germinal Vesicle stage. MI – Metaphase I stage.MII – Metaphase II stage. Deg –Degenerated.

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Stages of Maturation (%) S.N. Media Sera Hormone

concentrationTotal no. of oocyte GV

(%)MI(%)

MII(%)

Deg(%)

1 Ham's F-10 - - 30 9(30.00)

5(16.66)

10(33.33)

6(20.00)

2 Ham's F-10 FBS - 38 7(18.42)

8(21.05)

15(39.47)

8(21.05)

3 Ham's F-10 FBS E2 (1 μg) 24 4(16.66)

4(16.66)

10(41.66)

6(25.00)

4 Ham's F-10 FBS E2 +FSH (1 μg) 32 4(12.50)

4(12.50)

15(46.87)

9(28.12)

5 Ham's F-10 FBS E2 +FSH (10 μg) 38 3 (7.89) 3 (7.89) 27(71.05)

5(13.15)

6 Ham's F-10 FBS E2 +FSH (20 μg) 41 6(14.63)

9(21.95)

21(51.21)

5(12.19)


rate observed by Pawshe et al. (1993), who observeda maturation rate of 91.86% in presence of 10 μg/ml FSH (Folltropin-V) in goat.

Saeki et al., (1990) used commerciallyavailable FSH; they did not observe any differencein frequencies of maturation, fertilization anddevelopment of embryos amongst the commerciallyavailable FSH and the standard hormone preparationof LH and FSH.

It has been reported that the oocytes do nothave gonadotropin receptors (Lawrence et al.,1980). The receptors are present only in the adjacentfollicle cells. Therefore, signals induced bygonadotropins in follicle cells are transduce into theoocyte via gap junction (Downs et al., 1988). In thepresent study, 10 μg/ml FSH concentration wasfound to be optimum to transduce signals to theoocytes and resulted in a higher maturation rate.The lower concentration of FSH, i.e. 1 μg/ml, didnot seem to be sufficient to induce such signals whilethe 20 μg/ml concentration seemed to cause a definiteinhibitory effect on in vitro maturation of oocytes.

Our results were in agreement with theresults observed by Chauhan and Anand, 1991 whoreported a 69.1% maturation rate in Ham’s F-12supplemented with BSA and FCS in the presenceof FSH.

In conclusion, commercially available FSH(Folltropin-V) preparations can be successfully usedfor in vitro maturation of buffalo oocytes.

ACKNOWLEDGEMENT

Authors are thankful to Dr. D.B. Sarode,Associate Dean, Post Graduate Institute ofVeterinary and Animal Science of Akola, forproviding all necessary facilities.

REFERENCES

Ball, G.D., M.L. Leibfried Rutledge, R.L. Ax, andN.L. First. 1984. Maturation and fertilizationof bovine oocytes in vitro. J. Dairy Sci., 67:2775-2785.

Ball, G.D., R.L. Ax and N.L. First. 1980. Functionalcorrelates of hormone receptors in

reproduction, Elsevier. North Holland, NewYork, p. 561-563.

Chauhan, M.S. and S.R. Anand. 1991. In vitromaturation and fertilization of goat oocytes.Indian J. Exp. Biol., 29: 105-110.

Downs, S.M., S.A.J. Daniel, J.J. Eppig. 1988.Induction of maturation in cumulus cellsenclosed mouse oocytes by follicle stimulatinghormone and epidermal growth factor,evidence for positive stimulus of somatic cellsorigin. J. Exp. Zool., 245: 349-351.

Goto, K., Y. Kajihara, S. Kosaka, M. Koba, Y.Nakanishi and K. Ogawa. 1988. Pregnanciesafter co-culture of cumulus cells with bovineembryos derived from in vitro fertilization ofin vitro matured follicular oocytes. J. Reprod.Fertil., 83: 753-758.

Hensleigh, H.C. and A.G. Hunter. 1985. In vitromaturation of bovine cumulus enclosedprimary oocytes and their subsequent in vitrofertilization and cleavage. J. Dairy. Sci., 68:1456-1562.

Lawrence, T.S., N. Dekel and W.H. Beers. 1980.Binding of human chorionic gonadotropin byrat cumuli oophoron and granulosa cells: Acomparative study. Endocrinology., 106:1114-1118.

Lindsell, C.E., K. Rajkumar, A.W. Manning, S.K.Emery, R.J. Mapletoft and B.D. Murphy.1986. Variability in FSH-LH ratios amongbatches of commercially availablegonadotropins. Theriogenology, 25: 167(abstr).

Pawshe, C.H., S.K. Jain and S.M. Totey. 1993.Effect of commercially available folliclestimulating hormone on in vitro maturation ofgoat oocytes. IJAR, 14 (2): 69.

Snedecor, G.W. and W.G. Cochran. 1994. Statisticalmethod, 8th ed, lowa state University Press,USA, Oxford and IBH Publication Delhi: 591.

Saeki, K., M, Hoshi, M.L. Leibfried-Rutledge andN.L. First. 1990. In vitro fertilization anddevelopment of bovine oocytes matured withcommercially available follicle stimulatinghormone. Theriogenology, 34 (6): 1035-1039.

Thibault, C., M. Gerard and Y. Mennezo. 1975.Acquisition par L’ ovocyte de lapine et de veaudu facterur de deondensation da noyauduspermatosoide fecondant (MPGF). Anim.Biol. Anim. Biochim. Biophys., 15: 705-714.

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BANGKOK 10903, THAILAND URL : http://ibic.lib.ku.ac.th E-mail : [email protected] Tel : 66-2-9428616 ext. 344 Fax : 66-2-9406688

Prevalence of different spontaneous liver lesions in buffaloesin Malwa region of Madhya Pradesh.G.P. Jatav, U.K.Garg and Supriya Shukla………………………………………………...256

Peripheral plasma FSH concentrations in relation to expressionof oestrus in Murrah buffaloes (Bubalus bubalis).S. Mondal, B.S. Prakash and P. Palta……………………………………………………..258

Fetal arthrogryposis causing dystocia in a pleuriparous buffalo.J. Singh, B. Mideksa, A.M. Pawde and S. Deori…………………………………….…...263

Buffalo genetic resources in India and their conservation.A.K. Das, Deepak Sharma and Nishant Kumar…………………………………………...265

Ultrasonography of the udder and teat in buffaloes: A comparison of four methods.K.Rambabu, Makkena Sreenu, R.V.Suresh Kumar and T.S.C.Rao…………………….....269

A study on repeat breeding of Iraqi buffalo cows.O. I. Azawi , S. N. Omran and J. J. Hadad ………………………………...………...…..274

Seroprevalence of Listeria monocytogenes in buffaloes in Gujarat, India.Ku. Rathod. P.H., N.M. Shah, H.C. Chauhan, K.A. Vasava, B.S. Chandel,H.N. Kher and A.I. Dadawala………………………………………………………….…..285

Documentation of changes in haematology and milk constituentsduring oestrus in Murrah buffaloes.P.K. Pankaj, A. Mishra, A.K. Gour, S.W. Amin, R. Jain and R.P.S. Baghel………………289

Association of breed and polymorphism of αS1- and αS2-casein genes withmilk quality and daily milk and constituent yield traits of buffaloes (Bubalus bubalis).S.S. Misra, Arjava Sharma, T.K. Bhattacharya, P. Kumar and S. Saha Roy………….....294

Influence of commercially available follicle stimulating hormoneon in vitro maturation of buffalo oocytes.S.A. Adlak , K.P. Khillare, C.H. Pawshe and S.W. Mude………………………………….302

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