Br. vet .

7. (1990) . 146, X63

MILK PROGESTERONE DETERMINATION IN BUFFALOES POST-INSEMINATION

MONIKA GUPTA and B . S . PRAKASH*

Division of Cattle Physiology, National Dairy Research Institute, Karnal 132 X)1, India

SUMMARY Milk progesterone levels were studied in 17 buffaloes between post-insemination and 30 days thereafter . Six animals were confirmed pregnant . Seven of 10 animals took > 24 days to return to oestrus and the pattern of change of progesterone levels indicated a steady increase from I ng/ml on day 2 to 20 ng/ ml on day 14 post-insemination, falling subsequently to about 5 ng/ml, 2 days prior to oestrus . In pregnant buffaloes, the milk progesterone levels showed a continuous increase up to 20 days post-insemination but did not decline thereafter . In individual buffaloes returning to oestrus, a cut-off milk progesterone level of > 10 ng/ml was considered for pregnancy diagnosis, 20, 22 and 24 days postinsemination . The test was 60, 75 and 75% accurate for detection of pregnancy on days 20, 22 and 24 respectively and 100% for non-pregnancy diagnosis on all three days . Individual animals showed a highly variable oestrous cycle length, which has been suggested as a contributory factor for false pregnancy diagnosis by milk progesterone test in this species . Milk progesterone assay may also identify silent heats, and clinical disorders .

INTRODUCTION Progesterone determination in whole milk as a tool for monitoring the reproductive status of the cattle herd has been well documented (Hoffmann et al, 1976 ; Bulman & Lamming, 1978 ; Foote et al., 1979 ; Pope & Swinburne, 1980 ; Claus et al., 1983) . The domestic water buffalo-the principal dairy animal in many South and South East Asian countries-has a poor reproductive efficiency, attributed mainly to delayed maturity, poor expression of oestrous symptoms especially during the summer months and long calving intervals . The marked seasonality of buffalo breeding handicaps commercial milk production which alternates between glut and shortage . To offset the problem of poor oestrus expression in buffaloes frequent routine palpation of the ovaries and uterus per rectum becomes essential to monitor reproductive status of the herd . Although this is suitable for checking pregnancy, it is impractical for monitoring oestrous

*Correspondence

to B . S . Prakash .

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cyclicity . The ovaries of the buffaloes are generally smaller than those of cows (see Fahimuddin, 1975) making rectal examination more difficult . The use of the milk progesterone determination for monitoring reproductive status assumes great importance in buffaloes . However, there are very few reports on milk progesterone determination in buffaloes . Batra et al. (1979) and Singh & Puthiyandy (1980) suggested the possibility of confirming pregnancy in buffaloes around days 20-25 post-insemination by milk progesterone determination but no attempt was made in these studies to study the progesterone profiles in individual animals post-Al in order to understand actual causes for false diagnosis of pregnancy . The present study was therefore planned with the following objectives : (1) to determine the progesterone changes in Murrah buffaloes post-insemination, (2) to assess the prospects of using milk progesterone determination for pregnancy, and/or nonpregnancy diagnosis, and (3) to list the possible causes for incorrect detection of pregnancy and/or non-pregnancy diagnosis by milk progesterone estimation .

MATERIALS AND METHODS Experimental animals Seventeen lactating cycling buffaloes were taken from the herd for experimentation . All animals were inseminated at oestrus . Milk sampling Milk samples were collected from all buffaloes on alternate days at 6 .30 p .m . starting from the day of insemination until day 30 post-insemination . The samples were stored at -20° C until tested . Milk analysis for progesterone quantitation Whole milk was analysed by a sensitive, direct radioimmunoassay procedure developed in the laboratory . Milk samples were thawed at 37°C in a water bath and then incubated at 0°C after thorough mixing . A slight precipitation was observed in a few milk samples on thawing but this did not influence the assay . To duplicate 10,ul of milk samples and milk standards (containing progesterone ranging from 0 pg to 500 pg/ 10 µl) were added 300µl of phosphate buffer saline (PBS ; 2 .69 g KH Z PO4 , 8 .3 g Na2HPO 4 , 8 .5 g NaCl, 0 .33 g sodium azide and 1 .0 g gelatin dissolved in 1000 ml of distilled water, pH 7 .2) . Subsequently, 0 .1 ml of progesterone specific antiserum (1 :1500) was added to each tube followed by addition of 0 .1 ml of 3 H-progesterone (101 Ci/mmol ; M/s Radiochemical Centre, Amersham, UK) at 0° C . All tubes were then incubated at room temperature (ranging from 20 to 25 ° C) for 2 h . Free and antisera-bound hormone were separated by the addition of 0 .5 ml of a freshly prepared cold charcoal-dextran suspension (0 .625% activated charcoal plus 0 .0625% dextran in buffer) . The tubes were then stirred, incubated at 0° C in an ice water bath for 10 min, then centrifuged at 2800 gat 4 ° C for 15 min . The supernatant containing the bound progesterone was decanted into scintillation vials and counted in 5 ml of scintillation fluid containing 4 g 2,5-diphenyloxazole and 0 .1 g 1,4-bis[2-(5-phenyloxazolyl) benzene in 1000 ml toluene .



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RESULTS

Assay validation Sensitivity . The sensitivity of the assay calculated on the basis of different standard curves for progesterone in whole milk was 0 .8 ng/ml with 50% binding limit being 12 .5 ng/ml . Precision . Intra-assay coefficient of variation in pooled whole milk samples containing 3 .5 and 7 .6 ng/ml was 8 .5 and 6 .5% respectively while the interassay coefficient of variation was 13 .2 and 11 .6% respectively . Specificity . The cross-reactivity of the antiserum against different steroids is presented in Table I .

Table I Specificity of the progesterone antiserum Steroids

Progesterone Testosterone Cortisol Oestradiol -17ß Oestrone Chloramphenicol Oestrone sulphate PGFM

% Cross-reaction

100 .000 0 .028 0 .004 < 0 .001 10 ng/ml on days 20, 22 and 24 respectively . Of these 10 animals, only six were actually pregnant, giving the accuracy index of pregnancy diagnosis to be 60% on day 20 and 75% on days 22 and 24 post-insemination . However, for non-pregnancy diagnosis, seven, nine and nine animals showing < 10 ng/ml on days 20, 22 and 24 respectively were classified as non-pregnant by milk progesterone test, all of which were later found to be non-pregnant . Thus the milk progesterone test showed 100% accuracy for non-pregnancy detection on all the days . Singh & Puthiyandy (1980) also observed similar results . The present study also attempted to identify causes for the poor accuracy index of progesterone assay for pregnancy diagnosis on day 20 postinsemination since the milk progesterone profiles for individual animals (Figs 1 and 2) were recorded throughout the course of investigation . The present study indicated a possibility of the highly variable oestrous cycle length among buffaloes as a contributory factor since seven of the 10 buffaloes returning to oestrus did so > 24 days post-insemination . Milk progesterone values were greater than 10 ng/ml in four of these animals on



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day 20 post-insemination . However, only two of these four animals showed progesterone levels > 10 ng/ml which contributed to a greater accuracy of the milk progesterone test on days 22 and 24 post-insemination . Finally, the present study has also indicated the possibility of using milk progesterone determinations on a routine basis in order to monitor the reproductive status of the herd . Of the 17 animals studied, one non-pregnant buffalo which did not return to oestrus exhibited cystic ovarian conditions with fluctuating milk progesterone levels (Fig . 3) . One buffalo also did not exhibit behavioural oestrus symptoms (Fig . 1) .

ACKNOWLEDGEMENTS The authors are grateful to Dr H . H . D . Meyer, Institute for Physiology, 8050-FreisingWeihenstephan, West Germany, for the gift of the progesterone antiserum .

REFERENCES BATRA, S . K ., ARORA, R . C ., BACHLAUS, N . K. & PANDEY, R . S . (1979) . Journal of Dairy Science 62, 1390 . BULMAN, D . C . & LAMMING, G . E . (1978) . Journal of Reproduction and Fertility 54, 447 . COCKRILL, W . R . (1974) . In The Husbandry and Health of the Domestic Buffalo, p . 128 . New York : FAO . CLAUS, R ., KARG, H ., ZWIALR, D ., BuFLER, I . V ., PIRCHNER, F. & RATTENBERGER, E . (1983) . British Veterinary Journal 139, 29 . FAII1r 1UDDIN, M . (1975) . In Domestic Water Buffalo. Oxford and IBH . FoorE, R. J., OL IENACL, E . A. B ., KiMMERFELD, H . L ., SmuTH, R. D ., RIEK, P . M . & BRAL N, R . K . (1979) . British Veterinary Journal 135, 550 . HOFFMANN, B ., GLNZLER, O ., HAMBURGER, R . & ScHm im, W . (1976) . British Veterinary journal 132, 469 . MADAN, M. L . (1988) . In Buffalo Production and Health: a Compendium of Latest Research Information Based on Indian Studies, ed . R . R. Lokeshwar, p . 89 . New Delhi, India : ICAR . Pope, G . S . & Swinburne, J . K . (1980) . Journal of Dairy Research 47, 427 . SINGH, M ., CIAUDHARY, K . C . & TAKKAR, O . P . (1988) . In Proceedings of the II World Buffalo Congress, New Delhi, India, p . 271 . A . & PLTHIYANDY, R . (1980) . Journal of Reproduction and Fertility 59, 89 .

SINGH,

(Accepted for publication 5 April 1990)

Milk progesterone determination in buffaloes post-insemination.

Milk progesterone levels were studied in 17 buffaloes between post-insemination and 30 days thereafter. Six animals were confirmed pregnant. Seven of ...
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