Progesterone concentration in defatted milk of dairy cows in early pregnancy

Br. vet . J. (1992) . 148, 45

PROGESTERONE CONCENTRATION IN DEFATTED MILK OF DAIRY COWS IN EARLY PREGNANCY

M . B . NOSEIR*, P . GYAWUt and G . S . POPE-1 Department of Endocrinology and Animal Physiology, AFRC Institute of Grassland and Environmental Research, Hurley, Maidenhead, Berks . SL6 5LR

SUMMARY

Progesterone concentrations have been measured in defatted milk of British Friesian cows of four herds during the oestrus cycles (other than short cycles) immediately before artificial insemination (AI) at oestrus and immediately after AI (in non-pregnant cows), and during early pregnancy . Differences in mean progesterone concentrations between herds were significant (P<0 .05) on all days within the day 10-18 period after AI, both in pregnant and in non-pregnant, inseminated cows but were not significant between pregnant and non-pregnant cows within herds until day 17 or 18 . It is concluded that up to this time (that of luteolysis in nonpregnant cows) undefined factors, variable among herds, can have a much greater influence on the rate of progesterone secretion by corpora lutea and consequent progesterone concentration in plasma and milk than does the presence of conceptuses . Maximum mean progesterone concentration reached during early pregnancy in two herds did not differ significantly ; it was reached in the 11-15-day period in one herd but not until 46-50 days in the second . Mean progesterone concentration declined after day 90 .

INTRODUCTION The close correlation found between concentrations of progesterone in bovine milk and systemic plasma (Hoffmann & Hamburger, 1973 ; Pope et al., 1976 ; Bulman et al., 1978 ; Abeyawardene et al., 1984 ; Gao et al., 1988) has led to the widespread use of progesterone concentrations in milk in studies of bovine ovarian function, in dairy husbandry and in veterinary practice (Pope & Swinburne, 1980 ; Ball, 1982 ; Cidoncha & Garcia, 1982 ; Foulkes et al., 1982 ; Heshmat & Taha, 1984 ; *Present address: Faculty of Veterinary Medicine, Alexandria University, Edfina, Behira, Egypt . tPresent address : P .O . Box 446, University Post Office, University of Science and Technology, Kumasi . Ghana. +Correspondence to G .S . Pope, AFRC Institute of Grassland and Environmental Research, Shinfield, Reading, Berks . RG2 9AQ.



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Hoagland & Barnes, 1984 ; Eddy & Clark, 1987 ; Gyawu & Pope, 1990 ; McLeod & Williams, 1991 ; McLeod et al., 1991) . Henricks et al. (1971) reported progesterone concentration in the plasma of pregnant cattle to be higher than in non-pregnant cattle from day 6 after insemination, a finding that was not, however, supported by Baishya (1977) . With the advantage of greater numbers of observations, Lukaszewska & Hansel (1980) and Lamming et al. (1989) have reported mean progesterone concentrations in plasma and milk respectively to be higher in pregnant cattle from as early as day 10 after insemination, thus raising the possibility that interaction between conceptus and corpus luteum (Hansel et al., 1989) results in increased progesterone concentrations in plasma and in milk from this time . The results of further investigation of the changing concentration of progesterone in defatted milk of non-pregnant and pregnant cows and its relevance to corpus luteum function are now reported .

MATERIALS AND METHODS

Cows Study 1 . This study involved 48 autumn-calved and 43 spring-calved cows from British Friesian herds maintained at the Bernard Weitz Centre (National Institute for Research in Dairying) . These herds had been formed to suit husbandry convenience, not by random allocation of heifers . Cows of the two herds were artificially inseminated (AI), with semen (stored frozen) of a British Friesian bull, at the first oestrus on or after 26 November or 26 March respectively, that was 42 days or more after calving . Cows were kept in good body condition ; they were housed during the winter and were at pasture from early spring to late autumn . Study 2 . This study involved autumn-calved cows, 73 at Research Unit (RU) and 36 at Church Farm (CF), both British Friesian herds of the National Institute for Research in Dairying . They were managed as were the autumn-calved cows of Study 1 .

Milk sampling In Study 1 samples were taken from the individual yields, three times weekly from calving until 26 November or 26 March, then daily until 25 days after first AI . In Study 2 samples were taken from individual yields three times weekly from 2 weeks after calving until pregnancy was confirmed (by fetal palpation) and for varying periods thereafter .

Assay of progesterone in defatted milk Defatted milk samples were prepared, stored and their progesterone concentration determined by radioimmunoassay using G465/7 antiserum, as described by Gyawu & Pope (1990), except that in Study 2 samples were not stored at-20 ° C for more than 2 weeks and EDTA was not added .



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Assay of oestradiol-17ß in defatted milk For the autumn-calved cows of Study 1 concentrations of oestradiol-17ß in defatted milk were determined by radioimmunoassay using antiserum G510/6 as described by Gyawu-& Pope (1990) . Statistical analysis Chi-squared tests, analysis of variance and Student's t-tests, employing where appropriate the Satterthwaite method, cited by Snedecor & Cochran (1980), of calculating degrees of freedom, were used . Linear regression was used in Study 2 .

RESULTS Study 1 Oestrus cycles were defined as beginning (on day 0) on the day of detected oestrus or, failing that, on the day of the pre-ovulatory peak in oestradiol-17ß concentration in milk (autumn-calved cows) or on the day of minimum progesterone concentration in milk (spring-calved cows) (Abeyawardene et al., 1984 ; Gyawu & Pope, 1990) . Short cycles were defined as those in which, during the day 6-15 period, there were two (or more) consecutive progesterone concentration measurements below the upper 95% confidence limit of the mean progesterone concentration on day 0 (day of AI) of pregnant cows of the autumn or springcalved group as appropriate . Twenty out of 48 of the autumn and 12 out of 43 of the spring-calved cows became pregnant to first AI ; the difference is not significant (P>0 .05) . Exclusion of cows with short cycles immediately before or after AI left 38 autumn and 25 spring-calved cows in the study, 18 and 12 respectively being pregnant (Fig . 1) . During the 7-18-day period after AI, mean progesterone concentrations in pregnant cows were 32-95% higher in the autumn relative to the spring-calved group (Fig . lb, f) ; the differences were significant (P<0 .05) on all days except day 9 when a very high progesterone concentration (deviation 3 .8xsD) was measured in one autumn-calved cow (Fig . lb) . Also, in non-pregnant, inseminated cows in the day 7-18 period, mean progesterone concentrations were 49-114% higher in the autumn relative to the spring-calved group (Fig . Id, h) . Differences were significant on all days (P<0 .05) . During the 7-16-day period, differences within days between mean progesterone concentrations in pregnant cows and the same cows in the cycle before AI varied from 6% to 63% (median 21%) for the autumn-calved cows (Fig . la, b), and from 4% to 20% (median 10%) for the spring-calved cows (Fig . le, f) ; differences were not significant (P>0 .05) on any day . To have reached significance at this level they would have needed to be 26-84% (median 32%) and 20-56% (median 31%) for the autumn and spring-calved groups respectively. Also in this 7-16-day period, differences within days between mean progesterone concentrations in pregnant cows and non-pregnant, inseminated cows were 0-36% (median 2 .5%) for the autumn calved cows (Fig . lb, d) and 1-27% (median 4%) for the spring-calved cows (Fig . 1f, h) . None of these differences was significant (P>0 .05) ; to have reached significance at this level they would have needed to he



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19-71% (median 25%) and 22-33% (median 26%) for the autumn and springcalved cows respectively . 6

(b)

A(P) n=18 0 2

4 6

8 10 12 14 16 18 20

Fig. 1 . Progesterone concentrations (mean and sri) in defatted milk of cows during oestrus cycles immediately before AI and for 21 days after AI (excluding cows with short cycles immediately before or after AI) . A, autumn-calved ; S, spring-calved ; (P) and P, before and after AI of cows, then pregnant ; (NP) and NP, before and after Al of cows, then not pregnant . Oe, oestrus ; AI, artificial insemination .



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49

Mean progesterone concentrations within days were significantly higher (P< 0 .05) from days 17 or 18 in pregnant cows (autumn and spring-calved) relative to the same cows in the oestrus cycle before AI and also relative to non-pregnant, inseminated cows (Fig . 1) . Study 2

Short oestrus cycles were defined as in Study 1 . Thirty-two cows of 73 and 19 out of 36 became pregnant to first Al in the two herds, RU and CF respectively ; the proportions are not significantly different (P>0 .05) . More cows became pregnant to later AI and 43 and 28 pregnant cows of the RU and CF herds respectively entered the study . Excluding cows with short cycles immediately after AI left 35 and 23 non-pregnant, inseminated cows of the RU and CF herds respectively in the study . In pregnant cows during the periods day 0-5, 6-10 and 11-15 mean progesterone concentrations were respectively 47%, 31% and 75% higher in the CF cows than in those of the RU herd (P<0 .05; Fig . 2a) ; in non-pregnant cows in these three periods, progesterone concentrations were 86%, 45% and 52% higher in the CF relative to the RU cows (P<0 .01 ; Fig . 2b) . During 5-day periods from days 16-20 to 42-45 of pregnancy, mean progesterone concentrations were respectively 28%, 29%, 29%, 19%, 21% and 23% higher in cows of the CF herd relative to those of the RU herd (P<0 .01 ; Fig. 2a) . During later periods of pregnancy, differences in mean progesterone concentrations between herds were not significant (P>0 .05 ; Fig . 2a) . Differences in mean progesterone concentrations between pregnant and nonpregnant cows, within the periods days 0-5, 6-10 and 11-15 were 9%, 6% and 6% respectively for the RU herd and 28%, 4% and 9% respectively for the CF herd (Fig . 2), no difference within any of the three time periods being significant (P>

Progesterone concentrations (mean and SEM) in defatted milk of cows of Research Unit (RU) and Church Farm (CF) herds up to 115 days of pregnancy (a) and of cows not pregnant after AI (b), excluding those with short cycles at this time . •-• RU ; O---O CF . Oe, oestrus; AI, artificial insemination .

Fig . 2 .



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Table I Regression analysis of progesterone concentrations (ng/ml) in milk of pregnant (P) and non-pregnant (NP) cows of two herds, Research Unit (RU) and Church Farm (CF), upon time (days) after AI (on day 0) Period (days)

P or NP

Herd

0-15 0-15 0-15 0-15 16-40 16-40 41-70 41-70 16-70 16-70 71-90 71-90 91-115 91-115

P P NP NP P P P P P P P P P P

RU CF RU CF RU CF RU CF RU CF RU CF RU CF

No. of cows

No. of observations

Regression coefficient

P

43 28 28 16 43 28 39 27 39 27 18 13 8 5

292 169 238 160 452 293 485 342 895 624 150 107 87 52

0 .191 0 .348 0,197 0 .271 0 .042 0 .019 0 .022 0 .003 0 .033 0 .008 0 .070 -0 .019 -0 .057 -0 .036

<0 .001 <0 .001 <0 .001 <0 .001 <0 .005 >0 .2 0 .055 >0 .4 <0 .005 >0 .1 <0 .01 >0 .5 <0 .025 >0 .1

0 .05) for either herd . To have achieved the 95% level of significance, differences would have needed to reach 35%, 11% and 11% for the RU cows and 56%, 19% and 17% for the CF cows . Figure 2 and Table I show that for cows of the RU herd the rise in mean progesterone concentration during days 0-15 (P<0 .05) was followed by further significant rises until day 90 of pregnancy and a fall in the day 91-115 period (P<0 .05), whereas for cows of the CF herd the rise during days 0-15 (P<0 .05) was not followed by further significant rises up to day 90 (P>0 .05) .

DISCUSSION Ginther et al. (1974) found little change in mean progesterone concentrations in whole milk between days 30-90 of pregnancy in Holstein-Friesian cows, a result that resembles that found for cows of the CF herd in this period (Fig. 2a) . Also, the rising mean progesterone concentrations in defatted milk up to day 90 in cows of the RU herd and its subsequent fall (Fig . 2a) bear a strong resemblance to the pattern of mean progesterone concentrations in plasma of British Friesian cows in this period found by Shotton et al. (1978) . The comparisons made between progesterone concentrations in milk of nonpregnant and pregnant cows within herds exclude effects on mean concentrations that would have arisen from premature falls in progesterone concentrations in the non-pregnant cows due to short oestrus cycles . Exclusion from the study of cows having short oestrus cycles before and after AI is justified on the basis that such cycles comprise a population distinct from that of normal length cycles (Odde et A, 1980 ; Gyawu & Pope, 1990) . In cows similar to those of the four herds studied (excluding those with short



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cycles

before or after AI), the results suggest that, within herds, mean progesterone concentrations in defatted milk are unlikely to be greater by a margin of more than approximately 30% in pregnant relative to non-pregnant cows before the time of luteolysis in the latter . This conclusion is not inconsistent with the magnitude of the increased mean progesterone concentration in pregnant cows from day 10 after AI found by Lukaszewska & Hansel (1980) in plasma and by Lamming et al. (1989) in whole milk. The results shown in Figs 1 and 2 suggest that for cows within herds similar to those studied (i .e . excluding those with short cycles before or after AI), the formation and cell organization of corpora lutea leading to maximum rate of progesterone secretion (and also therefore the hormonal control of these events) in the first 15 days of pregnancy differ little from those in the hiteal phase before AI or from those in the luteal phase of cycles immediately after Al in nonpregnant cows. The results suggest that in some pregnant cows there is no further increase in the progesterone secreting activity of corpora lutea from day 16 to 90, whereas in others there is a gradual increase in this activity so that its level is similar in all cows by days 45-70 . Hormonal control in this period either to maintain or to increase the rate of progesterone secretion from corpora lutea could be of conceptus as well as maternal origin . The marked (and statistically significant) differences between herds in mean progesterone concentrations from day 7 to 18 after oestrus, which was found similarly between groups of pregnant cows and between groups of non-pregnant cows after AI, indicates the existence of factors, operating variably among herds, that have a much greater influence than does the presence of conceptuses on the formation and cell organization of corpora lutea and their rate of progesterone secretion in this period . These factors have not been defined ; they could include the chosen interval from calving to AI, other features of herd management or season of the year . The decline in progesterone concentrations in milk from day 90, though significant only in RU herd, is similar to that found in bovine plasma by Shotton et al. (1978) and indicates marked change in the control of progesterone secretion by the corpus luteum at this time .

ACKNOWLEDGEMENTS The authors are grateful for financial support to the British Council (to M .B .N .) and to the University of Science and Technology, Kumasi (to P .G.) . We also thank Dr B . J . A. Furr for gifts of steroid antisera and Mrs M . Surridge and Mr B . Clark for help in preparing the manuscript .

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SHOTTOK, S . M ., Royj H . B . & PoPE, G . S . (1978) . Plasma progesterone concentrations from

before puberty to after parturition in British Friesian heifers reared on high planes of nutrition and inseminated at their first oestrus . Anim. Prod. 27, 89-98 . SNEDECOR, G . W . & COCHRAN, W . G . (1980) . Analysis of variance . In Statistical Methods, 7th edn . pp . 215-37 . Ames, Iowa: Iowa State University Press . (Accepted for publication 15 April 1991)