Estrogen and progesterone concentrations in bovine milk during the estrous cycle

THERIOGENOLOGY

ESTROGEN AND PROGESTERONE CONCENTRATIONS BOVINE MILK DURING THE ESTROUS CYCLE1 R. Narendran,

R. R. Hacker,

V. G. Smith'

IN

& A. Lun,

Department of Animal & Poultry Sciences, University of Guelph, Guelph, Ontario, Canada, Received

for Publication:

April

NlG

2Wl

16, 1979

ABSTRACT Estrogen and progesterone concentrations,in milk during the estrous cycle were estimated in 18 normally cycling Holstein dairy The estrogen and progesterone concentrations in milk during cows. the estrous cycle followed the pattern described for them in blood in the corresponding period. During most of the estrous cycle, estrogen concentration remained at approximately 200 pg/ml and reached a proestrous peak of 360 + 127 pg/ml on day 19. The progesterone concentration in milk during the estrous cycle increased to a peak on day 13 (45.5 * 6.6 ng/ml) and thereafter declined towards estrus. Estrus detection/prediction based on milk progesterone concentrations appears feasible in view of the significant differences in milk progesterone concentrations between the early luteal (postluteal and rapid follicular growth periods of the estrous ovulatory), cycle. INTRODUCTION The characteristic changes in blood progesterone, estrogen or luteinizing hormone concentrations permit estrus to be anticipated accurately (6). Except in those cows with follicularcysts showing nymphomania, basal progesterone concentrations in blood coincided with estrus recorded from behavioural signs observed on videotapes But the difficulty of obtaining blood samples or by herdsman (14). routinely in the field limits practical application of this technique. Milk provides an ideal alternative to blood for endocrinological studies of the reproductive cycle in the dairy cow and is amenable to long term and frequent sampling. The possibility of estrous detection (3) in the cow based upon milk progesterone concentrations has been previously studied. Cur aim was to measure the concentrations of both estrogens progesterone in milk of dairy cows during the estrous cycle. MATERIALS

AND METHODS

Milk samples from 18 Holstein cows, and preceding the day of estrus at which -1 Research National 2

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for the 21 days including they were successfully bred,

supported by the Ontario Ministry Research Council of Canada, Grant

Present address: 49001, U.S.A.

1979

Upjohn

Company,

VOL. 12 NO. 1

and

Unit

of Agriculture No. A-6247.

9602-25-5,

Kalamazoo,

& Food

and

Michigan,

19

THERIOGENOLOGY

were obtained daily from the weigh jar at the end of the morning milkings and stored within an hour of collection at -18C. The feeding and husbandry practices were uniform for all animals. Estrus was confirmed by visual signs and pregnancy diagnosis at 40 days postinseminaMilk samples from alternate days during tion was by rectal palpation. the estrous cycle were allowed to thaw overnight at 4C and were homogenized for 3 min using an ultrasonic device (Biosonik III, Bronwell New York) at maximum intensity, prior to being Scientific, Rochester, analysed for estrogen, progesterone and fat. Samples for fat analysis (Mercuric Chloride + Potassium dichromate; were preserved with "Lactabs" prior to being dispatched to the Thompson and Capper Ltd., Liverpool) Guelph, Ontario for the analysis. Central milk testing laboratories, Percent fat was determined using an infra-red milk analyser. Milk samples were assayed for total estrogens and progesterone by radioimmunoassay and competitive protein binding assays respectively, using material and procedures reported previously (16). The milk estrogen and progesterone values measured in the assays were corrected No corrections were made to account for the for procedural losses. Student's blank estimates in the estrogen and progesterone assays. t-test (22) was used to test the significance of the differences in hormone concentrations between different periods within the estrous Correlation coefficients between fat percentages and estrogen cycle. and progesterone concentrations in milk were also determined. RESULTS

AND DISCUSSION

Mean concentrations of total estrogens and progesterone in milk during the 21 days culminating in estrus (day 21) are presented in of 360 + 127 pg/ml figure 1. The peak estrogen concentration (Mean ?r S.E.) on day 19 of the cycle declined to 235.5 i 21 pg/ml on Estrogen concentrations of between 200 to 209 pg/ the day of estrus. ml were seen during the first 12 days, but started to increase commencing on day 13 to reach a proestrus peak on day 19. The estrogen concentrations increased when the progesterone concentrations were on Total free estrogen concentrations the decline commencing on day 13. in milk were probably under estimated in this study, because estradiol178 standard was used for quantification and the rate of displacement of 3H-estradiol-176 by standard estradiol-17a or estrone was .5 that of standard estradiol-178 at 50% displacement of 3H-estradiol-17@ Monk et al. (15) reported total milk estrogen (estradiol + estrone) values of 142 * 5 pg/ml and 137 + 5 pg/ml of day of estrus and the day The mean estrogen concentrations refollowing estrus respectively. ported during the remaining days of the cycle ranged between 55 and 85 pg/ml and gradually increased up to estrus and declined thereafter. Although the estrogen concentrations reported herein are higher than those reported by Monk et al. (15), the overall pattern of variation in estrogen concentration in milk over the estrous cycle is in conformity with those reported in milk (15) and blood (2,9). There were wide variations in milk estrogen concentration between cows on day 19 of the estrous cycle, which is reflected in the large standard error. Existence of such variation was also recognized by Monk et al. (15).

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1979 VOL. 12 NO. 1

THERIOGENOLOGY Estrogen concentrations in milk during the estrous cycle do not hold any promise as aids to estrous detection, since the proestrous peak (figure 1) is transitory and during the rest of the cycle the estrogen concentrations are not subject to much fluctuation. Milk estrogen concentrations and percent fat were not correlated during the estrous cycle and no such relationship have been previously reported. Progesterone concentrations in milk increased from 17 2 3.2 ng/ml on day 1 to 45.5 + 6.6 ng/ml on day 13 and thereafter declined to reach lower levels at estrus (24 + 5.2 ng/ml). Milk progesterone concentrations at estrus ranging from 1 ng/ml (12.20) to 3 ng/ml (3) and at midcycle ranging from 9 ng/ml (20) to 34 ng/ml (13) have been previously Heckman et al. (13) have reported that progesterone concenreported. trations at time of insemination for 13 COWS not conceiving was 12.5 2 4.7 ng/ml at estrus, whereas it was 1.2 _+ 2 ng/ml for 14 cows which conceived. Dobson et al. (3) also r eported very high concentrations of milk progesterone on the day of estrus in one cow and referred The high to similar observations by Heap (personal communication). standard errors observed for milk progesterone concentration in the Even the cows present study reflect the variation between cows sampled. exhibiting high concentrations of milk progesterone at estrus had been successfully bred at this estrus, in contrast to what was observed by Heckman et al. (13). The overall pattern of variation in progesterone concentrations in milk over different days of the estrous cycle is in conformity with those reported in milk (3,13) and blood (9,lO). Estrous detection/prediction using milk progesterone concentrations appears feasible in view of the milk progesterone profile seen during the estrous cycle (Figure 1). Significant(W.05) differences were noted in milk progesterone concentrations between the days 1 to 5, 7 to 17 and 19 to 21 corresponding to the early luteal (postovulatory), lutcal and rapid follicular growth phases, respectively, of the estrous cycle. The correlation between milk progesterone concentration and percent fat was Ginther et al. (8) not significant during the estrous cycle (r = .l). reported that fat and progesterone concentrations in milk during the estrous cycle and pregnancy were correlated (r= .98), whereas Oltenacu and Foote (17) reported a correlation of 0 between percent fat and milk progesterone for cycling cows. The high concentrations of progesterone and estrogen in milk appear to be at least in part due to an active uptake and metabolic role by the functioning mammary gland (7,11,15,18,19,21). Evidence has been presented for the possible conversion of estradiol to estrone (4) and estrone and estradiol-17B to biologically inactive estradiol-17cr (5) by the cow mammary gland. Conversion of estrone to estradiol-176 in udders of goats has been reported (l), but evidence for a similar conversion has not been reported for the cow. An active role by the bouptake and metabolism during early vine mammary gland in progesterone The lactation as in the goat (11,21) is also a distinct possibility. differences in times of milking (S), methods of sample collection (12) storage and handling prior to analysis (17), have been shown to affect the progesterone concentrations measured in milk and the correlation between milk fat and progesterone. Heap et al. (12) have discussed the

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1979 VOL. 12 NO. 1

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THERIOGENOLOGY possibility that with respect to milk progesterone estimations, the competitive protein binding assay and radioimmunoassays using less specific antisera, may be measuring an yet unidentified compound(s) present in milk. Such a possibility cannot be precluded from the present study. The possibility of a time lapse before changes in blood concentrations of estrogen and progesterone become reflected in milk and the importance of the time of sampling milk relative to any such time lapse also have to be borne in mind when comparing results from different studies. The differences observed between the present and other studies for milk estrogen and progesterone concentrations during the estrous cycle may be partly ascribed to the above factors and partly to those arising from differences in procedures employed to measure and adjust for nonspecific binding. In summary, the results of this study suggest milk as a suitable and convenient alternative to blood in studies on bovine reproductive endocrinology involving measurement of estrogens and progesterone. The estrogen and progesterone concentrations in milk conform to the pattern seen in blood during the estrous cycle. Estrous detection and/or prediction using progesterone concentrations in milk appears feasible. Futher field studies are required prior to routine application of this technique, on account of the existence of wide variation between cows in the basal concentrations of progesterone in milk at the time of estrus and the consequent difficulty of laying down standards to distinguish between luteal and basal levels of progesterone in milk during the estrous cycle. REFERENCES 1.

Challis, J. R. G. and Linzell, J. L. Oestrone metabolism in pregnant and lactating goats. J. Endocrinol. 57:457. (1973).

2.

Chenault, J. R., Thatcher, W. W., Kalra, P. S., Morans, R. M. and Wilcox, C. J. Transitory change in plasma progestins, estradiol and luteinizing hormone approaching ovulation in the bovine. J. Dairy Sci. 57:709 (1975).

3.

Dobson, H., Midmer, S. E. and Fitzpatrick, R. J. Relationship between progesterone concentrations in milk and plasma during the bovine oestrous cycle. Vet. Rec. 96:233. (1975).

4.

Erb, R. E., Chew, B. P. and Keller, H. F. Relative concentrations of estrogen and progesterone in milk and blood and secretion of estrogen in urine, J. Anim. Sci. 46:617. (1977).

5.

Erb, R. E., Chew, B. P., Keller, H. F. and Malven, P. V. Effect of hormonal treatments prior to lactation on hormones in blood plasma, milk and urine during early lactation. J. Dairy Sci. 60:557. (1977).

6.

Foote, F. H. Estrus detection and estrus detection aids. Dairy Sci. 58:248. (1975).

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7.

Ginther, 0. J., Nuti, L., Wentworth, B. C. and Tyler, W. J. Progesterone concentrations in milk and blood during pregnancy in cows. Proc. Sot. Exp. Biol. and Med. 146:354. (1974).

8.

Ginther, 0. J., Nuti, L. C. Garcia, M. C., Wentworth, B. C. and Tyler, W. J. Factors affecting progesterone concentrations in cows milk and dairy products. J. Anim. Sci. 42:155. (1976).

9.

Glencross, R. G., Munro, I. B., Scrinor, B. E. and Pope, G. S. Concentrations of oestradiol-178, oestrone and progesterone in jugular venous plasma of cows during the oestrous cycle and in early pregnancy. Acta Endocrinol. 73:374. (1973).

10.

Hansel, W., Concannon, P. W. and Lukaszawaka, J. H. Corpora lutea of the large domestic animals. Biol. Reprod. 8:222. (1973).

11.

Heap, R. B. and Linzell, J. L. Arterial concentration, ovarian secretion and mammary uptake of progesterone in goats during the reproductive cycle. J. Endocrinol. 36:389. (1966).

12.

Heap, R. B., Gwyn, M., Laing, J. A. and Walters, D. E. Pregnancy diagnosis in cows. Change in milk progesterone concentration during oestrous cycle and pregnancy measured by rapid radioimmunoassay. J. Agric. Sci. 81:151. (1973).

13.

Heckman, G. S., Katz, L. S., Foote, R. H., Oltenacu, E. A. B., Scott, N. R. and Marshall, R. A. Estrous cycle patterns in cattle monitored by electrical resistance and milk progesterone. J. Dairy Sci. 62:64. (1979).

14.

King, G. J., Hurnik, J. F. and Robertson, H. A. Ovarian function and estrus in dairy cows during early lactation. J. Anim. Sci. 42:688. (1976).

15.

Monk, E. L., Erb, R. E. and Mollett, T. A. Relationship between immunoreactive estrone and estradiol in milk, blood and urine of dairy cows. J. Dairy Sci. 58:34. (1975).

16.

Narendran, R., Hacker, R. R., Smith, V. G. and Lun, A. Hormonal induction of lactation in the bovine: Estrogen and progesterone levels in milk. J. Dairy Sci. (in print). (1979).

17.

Oltenacu, E. A. B. and Foote, R. H. The relationship between milk progesterone level and milk fat percentage, and the effect of storage on measurement of milk progesterone for pregnant and mid-cycle cows. Program. 71st Ann. Meeting, Amer. Dairy Sci. Assoc., p. 102 (Abstr.) (1976).

18.

Pearlman, W. S., DeHertogh, R., Lauman, D. R., Brueggemann, J. A. and Pearlman, M. R. J. Metabolism and tissue uptakes of steroid hormones in patients with advanced carcinoma of the breast and normal rats. Steroid Dynamics, Cr. Pincus et al. ed. Academic Press, New York. (1966).

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19.

Sar. Madhabananda and Stumpf, W. E. Autoradiography of mammary glands and uteri of mice and rats after the injection of (3H)-Estradiol. J. Steroid Biochem. 7:391. (1976).

20.

Schiavo, 3. .J.,Matusczak, R. L., Oltenacu, E. B. and Foote, R. H. Milk progesterone in postpartum and pregnant cows as a monitor of reproductive status. J. Dairy Sci. 58:1713. (1975).

21.

Slotin, C., Heap, R. B., Christiansen, J. M. and Linzell, J. L. Synthesis or progesterone by the mammary gland of goat. Nature London, 22:385. (1970).

22.

Steel, R. G. D. and Torrie, J. H. Principles and procedures of statistics. McGraw-Hill Book Co., Inc. New York/Toronto/ London: 57. (1960).

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THERIOGENOLOGY

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Oestrogen and progesterone during

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1979

the estrous

VOL. 12 NO. 1

cycle

concentrations (n=lS).

in bovine

milk

25