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Milk progesterone measurement in dairy cows: Correlation with estrus and pregnancy determination
THERIOGENOLOGY MILK PROGESTERONE MEASUREMENT IN DAIRY COWS: CORRELATION WITH ESTRUSAND PREGNANCY DETERMINATION M. Shemesha, N. Ayalonb,
E. ShalevC,
A. Neryac,
H. Schindlerd,
F. Milguira Received
for
Publication:
January 9, 1978
ABSTRACT Progesterone levels in fore milk, determined by a highly specific radioinmunoassay, were compared for the assessment of estrus by a veteran herdsman and an experienced inseminator, in cows presented for insemination. In addition, an examination was made of the relative accurac:y of using milk progesterone levels for the determination of pregnancy at 24, 40 and 44 days after insemination, as compared with rectal palpation at 45-50 days post-breeding. Fat-free fore milk progesterone levels were similar to jugular plasma levels at 24 days post-insemination and reached roughly 60% of the level of unextracted fore milk at this time. Accuracy of estrus diagnosis by herdsman, inseminator and milk nroeesterone level was 840, 93% and 96%. respectively. For pregnancy diagnosis, milk progesterone determination in 85 cows showed 78% accuracy in predicting pregnancy,and 100% _acc_uracy in predicting non-pregnancy. At 40 days post-insemination false positives dropped to 10% and at 44 days only 7% of the cows were incorrectly diagnosed as pregnant. The false positives in this study were largely due to embryonic mortality as reflected by abnormal intervals of return to estrus. Two milk progesterone determinations, at 24 and either 40 or 44 days post-insemination ensure maximum reliability for early pregnancy diagnosis. INTRODUCTION Accurate determination of estrus is essential for good results in artificial insemination, and early information on pregnancy status is important in intensive dairy farming where the aim under present conditions is to achieve an interval between calvings of about one year. These considerations are of special significance in countries such as Israel, where dairy cattle are kept in large units, with a relatively small staff of dairy workers.
:Dept. of Dept. of iKibbutz Division
APRIL
Hormone Research, Kimron Veterinary Institute, Beit Dagan, Israel Reproduction, Kimron Veterinary Institute, Beit Dagan, Israel Givat Haim Hamuhad, Israel of Animal Reproduction, Volcani Center, Beit Dagan, Israel
1978 VOL. 9 NO. 4
343
THERIOGENOLOGY
Natural service has been shown to give better conception rates than artificial insemination (1,2), and at least part of the explanation for this difference may be ascribed to inaccurate diagnosis of estrus as reflected by progesterone concentrations in blood or milk of cows presented for insemination (3,4). The minimal values of plasma and milk progesterone present around the time of estrus have been well documented (S-10). A striking difference in peripheral plasma progesterone levels between pregnant and nun-pregnant cows was observed 19 days after insemi nation (ll), and it was suggested that this difference might furnish the This has since been verified by basis for a very eariy pregnancy test. a number of studies (12,13,14). The discovery that the levels of progesterone in milk parallel the concentrations of this hormone in blood (15) has spurred a number of reports concerning the use of milk progesterone levels as a diagnostic and for the determination of estrus and pregnancy in dairy cows (15-20). The following report deals with a pilot field trial in which milk progesterone levels were compared with the assessment of estrus by a veteran herdsman and an experienced inseminator in cows presented for insemination. In addition, an examination was made of the relative accuracy of using milk progesterone levels for the early determination of pregnancy or non-pregnancy. MATERIALSAND METHODS The cows investigated were part of a large, well-managed high producing (average production: 8,200 kg) herd of Israeli-Friesian cattle, maintained under conditions of loose housing. Estrus checks were carried out twice daily at 6 AM and 6 PM. Cows were presented for insemination based upon the criteria of standing passively to mounting by other cows, or else repeated mounting of other cows. The inseminator decided if the cow was in estrus on the basis of uterine tone, the presence of mucus typical of estrus and, in cases of doubt, examination of the cervix with the aid of a vaginal speculum. Milk samples (10 ml with potassium dichromate as a preservative) were collected from the foremilk of the afternoon milking, Milk samples were collected for progesterone evaluation on the day of presentation for insemination to assess the accuracy of estrus determination. Milk samples were also collected from inseminated cows 24, 40 and 44 days after breeding to diagnose pregnancy. Pregnancy was confirmed by rectal palpation 45-50 days post-insemination. A total of 55 cows were presented with data on estrus assessment and milk progesterone evaluation, whereas pregnancy determination was carried out on 85, 35 and 29 cows at the intervals: 24, 40 and 44 days, respectively, after insemination.
344
APRIL
1978 VOL. 9 NO. 4
THERIOGENOLOGY
Progesterone Assay - Progesterone content in fore milk samples was determined by radio-immunoassay (RIA) using an antibody raised in our Laboratory against progesterone-11-hemisuccinate-BSA. This antibody showed less than 0.01 per cent cross reaction wit!: cortisol, corticosterone, cholesterol, 17 hydroxy progesterone, estrone, androstendione, prostaglandin E and F. The estradiol , testosterone, RIA was carried out with 0.05 ml samples of unextracted milk and The RIA technique was essentially as described by 0.1 ml of plasma. Shemesh and Hansel (21). Separation of free, from antibody-bound For progesterone, was accomplished with dextran-coated charcoal. purpose:s of comparison, blood plasma levels of progesterone were determi:ned by the same RIA technique in 33 samples collected 24 days post -insemination. comparisons were made at 24 days In addition, post-breeding between paired samples of unextracted fore milk and fat-free fore milk obtained by centrifuging at 1,SOOO g for twenty minutes at room temperature. equivalent to Assay Evaluation - Knohn amounts of progesterone 2, 3,T and 8 ng/ml were added to plasma containing 0.3 ng/ml and to milk containing 0.5 ng/ml. The recovered progesterone was 92f3% (n=20) and 55?4% (n=GO) respectively. The lower percentage of recovery found with the unextracted milk suggests the presence of progesterone-binding protein, like transcortin, which binds progesterone and results in lower recovery rates. We found that this interference could be overcome by heating the milk or else by adding the same amount of milk, which contained 0.5 ng/ml of progesterone, to the tubes containing the standard amounts of progesterone. The addition of milk, in order to overcome the interference encountered in the progesterone assay of milk, has already been reported (22). The intra-assay coefficient of variation between duplicate pairs was 7.9% and the interassay coefficient of variation was 10.2%. The results found were corrected by multiplying the values determined by lOO/SS. The calibration curve was fitted by computer according to the method of Rodbard and Lewald (22)) and the sample concentration calculated as the mean of duplicates and expressed as ng/ml.
APRIL
1978 VOL. 9 NO. 4
345
THERIOGENOLOGY
RESULTS IWD DISCUSSION
Progesterone
levels
after
insemination
Table I presents the findings regarding of unextracted and fat-free fore milk and of intervals of collection after insemination.
Table
Days after insemination
I.
Progesterone concentration (ng/ml?S.E.) foremilk and jugular plasma in pregnant non-pregnant cows
Foremilk (unextracted)
Foremilk (Fat-free)
Pregnant
Not Pregnant
Pregnant
24
4.73f2.2 (n=44)
0.1-0.6 (n=41)
2.63tl.8 (n=Zl)
40
3.921.9 (n=30)
~0.1-0.6 (n=S)
44
4.83k2.1 (n=27)
346
the progesterone content jugular plasma at
in and
Jugular
Not Pregnant
co.1 (n=29)
APRIL
Pregnant
2.9f0.9 (n=Zl)
Plasma Not Pregnant
1978 VOL. 9 NO. 4
THERIOGENOLOGY
The presence of progesterone in cow’s milk has been well proven. However there has been considerable disagreement on the concentration .__ of progesterone in the milk. Progesterone concentration has been reported to be higher in cream than in skim milk (23) and it has been noted that concentrations vary depending on when the sample was collected with regard to time of day and whether samples were from foremilk, strippings, or composite milk collections. Laing and Heap (15) determined the progesterone levels in samples taken from composite milk samples collected in the afternoon. The average progesterone concentration was 5.1 ng/ml with a range of 1.3 to 15.9 ng/ml in lactating, non-pregnant Friesian cows, whereas in pregnant cows average progesterone concentration was 19.7 ng/ml, with a range of 7.1 to 35.6 ng/ml. However, Heap et al (24) reported a serious interference in the radioimmunoassay technique used to qualify the progesterone. They observed that milk. samples analyzed by a highly specific antiserum gave values consistently lower than those measured with a less specific. antiserum. They suggested that another and, as yet,unidentified compound(s) was present in milk which cross react(s) with the antisera. The considerable difference between the level of values found in this study as compared with other reports could be explained by the fact that in this investigation a highly specific antisera was used which resulted in minimal cross-reaction with other metabolites. Accuracy
of
estrus
diagnosis
Table II presents the accuracy of diagnosis of estrus herdsman, inseminator and on the basis of milk progesterone Table
Accuracy
II.
No.
of cows
of Estrus
In Estrus No. %
Diagnosis
Not in Estrus % No.
Herdsman
55
g
=
Inseminator
45
334
=93
5
=
Milk Progesterone
55
g
=
;
=
APRIL
1978 VOL. 9 NO. 4
a4
96
by the determination.
Accuracy %
Overall No. 46 s
=
84
100
$$
=
93
100
g
=
96
-
347
THERIOGENOLOGY Sixteen per cent of the cows presented for insemination by the herdsman were not in estrus as judged by milk progesterone levels. In two other studies,between 10 and 22% (4) and between 14 and 26% (17) were not in estrus when inseminated as judged by plasma or milk progesterone levels respectively. The two errors in estrus diagnosis on the basis of milk progesterone in the present study were in cases where the cows were not inseminated due to the judgment of the inseminator, and were later shown to have conceived from a previous insemination. The high degree of accuracy of the inseminator is noteworthy and it indicates that it would be worthwhile to invest time and effort in training inseminators to achieve a high degree of skill in the diagnosis of estrus. Accuracy of pregnancy diagnosis Table III presents the accuracy of pregnancy diagnosis by milk progesterone determination at three periods, as compared with return to estrus and/or rectal palpation at 45-50 days post-breeding. Table III.
Days after breeding
Accuracy of pregnancy diagnosis progesterone determination
No. Diagnosed of correct COWS NO. %
by milk
Pregnant Diagnosed error not pregnant NO. % correct
24
Overall accuracy No. %
100
75 E
88
40
35
g
90
3 E
10
5
100
32 E
g1
44
29
g
93
E2
7
T 2
100
E 27
g3
The improved results
of pregnancy testing
at the two later
periods
are more apparent than real because at least some of the cows which were falsely diagnosed as pregnant at 24 days had returned to estrus before testing at 40 and 44 days and the potential for error was therefore less.
348
APRIL
1978 VOL. 9 NO. 4
THERIOGENOLOGY
The accuracy of diagnosis at 24 days, when compared with rectal was quite similar to that of other palpation and/or return to estrus, reports for testing about this time (17,18,19). Thus, the 100% accuracy for negative diagnosis found in this investigation was matched b:y the 98-100% accuracy recorded in the other three studies. Likewise, the figure of 78% accuracy for positive determination in the present study, was paralleled by 78% accuracy for positive tests at 20 days in Germany (17), 77% accuracy at 23 days in the USA (18) and 80% correct answers for cows diagnosed as pregnant at 24 days in England (19). It is worthwhile considering the nature of the relatively high incidence of false positives. An analysis of the time of return to estrus of these cases indicates that the source of error is not in the technique of the assay itself, but rather primarily because of inseminating cows which were not in estrus and because of embryonic mortality. In the present study, the influence of cows which were inseminated when they were not in estrus on the basis of milk progesterone levels, was not of significance. When positive pregnancy results were separately worked up for cows clearly in estrus on the basis of standing to mounting by other cows, positive inseminator assessment, and.minimal milk progesterone levels - accuracy for positive pre,gnancy diagnosis rose only slightly to 81%. In contrast, the 10 cows ltiith false positive determinations all returned to estrus between 29 and 55 days, mostly at times suggestive of embryonic mortality. The distribution of time intervals between insemination and return to estrus for these cows was as follows: 29-35 days - 4 cows, 36-43 days 4 cows, 52 days - 1 cow and 55 days - 1 cow. Similar observations regarding the importance of embryonic mortality between 21 and about 50 days post-insemination, have been recorded (19,20,25). In conclusion, the results of this study support the opinion of those investigators who contend that the principle value of milk progesterone testing for pregnancy is to detect those cows which are not pregnant:. The results of positive pregnancy assays from several countries with varying conditions of management, nutrition, and level of production indicate that with present techniques positive assays cannot be expected to give an accuracy of more than 80%. In practical terms this means that under present conditions what is needed are more efficient methods for estrus detection and milk progesterone assay will have its chief application for early detection of non-pregnant cows as well as an aid to clinical diagnosis of such conditions as cystic ovaries, subestrus, anestrus, and for monitoring changes in progesterone levels before and after gynecological treatment.
APRIL
1978 VOL. 9 NO. 4
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THERIOGENOLOGY
ACKNOWLEDGMENTS Thanks are due to Mrs. Sarah Levi and to Mrs. Ella Katz for excellent technical assistance in performing the progesterone assays. We are also grateful to Mr. Yehuda Vilmosh for examining the cows presented for insemination. REFERENCES 1.
Milk Marketing Board (England and Wales). A survey of bovine fertility in the north of England. Annual Report of Production Division No. 7: 105 (1956/7) .
2.
Clinical observations Mylrea, P.J. Aust. Vet. J. 38:253 (1962).
3.
Gunzler, O., Hamburger, R. and Hoffman, B. Observations from field trials, using milk progesterone determination as a tool for the surveyance of fertility. Zuchthyg. 8:154 (1973) .
4.
Appleward, W.T. and Cook, cattle _ Vet. Rec. 99:X3
5.
Shemesh, M., Ayalon, N. and Lindner, H.R. Plasma progesterone concentrations in dairy cows during the oestrous cycle. Refuah Vet. 25: 270 (1968) .
6.
Stabenfeldt, G.H., Ewing, L.L. and McDonald, L.E. Peripheral plasma progesterone levels during the bovine oestrus cycle. J. Reprod. Fert. 19:433 (1969).
7.
Donaldson, L .E . , Bassett, J.M. and Thorburn, G.D. Peripheral plasma progesterone concentration of cows during puberty, oestrous cycles, pregnancy and lactation, and the effects of under nutrition or exogenous oxytocin on progesterone concentration. J. Endocr. 48:599 (1970).
S.
Shemesh, M., Lindner, H.R. and Ayalon, N. Competitive proteinbinding assay of progesterone in bovine jugular venous plasma during the oestrou; cycle. J. Reprod. Fert. 26:167 (1971).
9.
Robertson, H .A. Sequential changes in plasma progesterone in the during the oestrous cycle, pregnancy, at parturition and post -partum * Can. J. Anim. Sci. 52-645 (1972).
on reproduction
B. The detection (1976) .
in dairy cows.
of oestrus
in dairy
COW
350
APRIL
1978 VOL. 9 NO. 4
THERIOGENOLOGY
10.
Glencross, R.G., Munro, I.B., Senior, B.C. and Pope, G.S. Co,ncentrations of oestradiol-17B, oesteone, and progesterone in jugular venous plasma of cows during the oestrus cycle, and in early pregnancy. Acta Endocrinologica 73:374 (1973).
11.
Shemesh, M., Ayalon, N. and Lindner, H.R. Early effect ot conceptus on plasma progesterone level in the cow. J. Reprod. Fert. 15:161 (1968).
12.
Shemesh, M., Ayalon, N. and Lindner, H.R. Early pregnancy diagnosis based upon plasma progesterone levels in the cow and ewe. J. Anim. Sci. 36:726 (1973).
13.
Robertson, H.A. and Sarda, I.R. A very early pregnancy test for mammals; its application to the cow, ewe and sow. J. Endocr. 49:407 (1971).
14.
Ginther, O.J., Nuti, L., Wentworth, B.C. and Tyler, W.J. Progesterone concentration in milk and blood during pregnancy in.cows. Proc. Sot. Exp. Biol. Med. 146:354 (1974).
15.
La.ing,J.A. and Heap, R.B. The concentration of progesterone in the milk of cows during the reproductive cycle. Brit. Vet. J. 127:X1X (1971).
16.
Wishart, D.F., Head, V.A. and Horth,'C.E. Early pregnancy diagnosis in cattle. Vet. Rec. 96:34 (1975).
17. Hoffman, B., Gunzler, O., Hamburger, R. and Schmidt, W. Milk progesterone as a parameter for fertility control in cattle; methodological approaches and present status of application in Germany. Brit. Vet. J. 132:469 (1976). 18.
Pennington, J.A., Spahr, S.L. and Lodge, J.R. Factor affecting progesterone in milk for pregnancy diagnosis in dairy cattle. Brit. Vet. J. 132:487 (1976).
19. Heap, R.B., Holdsworth, R.J., Gadsby, J.E., Laing, J.A. and Welters, E.E. Pregnancy diagnosis in the cow from milk progesterone concentration. Brit. Vet. J. 132:445 (1976). 20.
Pope, G.S., Majzlik, I., Ball, P.J. and Leaver, J.D. Information on reproduction in female cattle obtainable from concentrations of progesterone in plasma and milk. Proc. VIII Int'l. Cong. Anim. Reprod. Krakow. Vol. 111:172.
APRIL
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THERIOGENOLOGY
21 .
Shemesh, M. and Hansel , W. Levels of prostaglandin F (PGF) in bovine endometriwn, uterine venous, ovarian arterial and jugular plasma during the estrous cycle. Qroc. Sot. Exp. Biol. Med. 148:123 (1975).
22.
Ball, Q.J.11. and Pope, G.S. Measurement of concentrations progesterone in fat-free cows’ milk: its potential value studies of reproduction. J. Endocr. 69:4DQ (1976).
of in
23.
Rodbard, D. and Lewald, J.E. Computer analysis of radioligand In: Karolinska symposia on assay and radioimmunoassay data. Research Methods in Reproductive Endocrinology. 2nd Symposium. Steroid assay by protein binding. pp. 79:103. Editor, A. Diczfalusy Karolinska Institute, Stockholm (1970).
24.
Darling, steroids
25.
Heap, R. B., Gwyn, M., Laing, J.A. and Walters, D.E. Pregnancy diagnosis in cows, changes in milk progesterone concentration during the oestrous cycle and pregnancy measured by a rapid radioimmunoassay.
26.
Boyd, II. Oestrous cycles in Ayrshire cows before insemination. Vet. Rec. 92:427 (1973).
352
J.A.B., Laing and Harkness, R.A. A survey of the in cow’s milk. J. Endocrinol. 62:291 (1974).
and after
APRIL 1978 VOL. 9 NO. 4
E. ShalevC,
A. Neryac,
H. Schindlerd,
F. Milguira Received
for
Publication:
January 9, 1978
ABSTRACT Progesterone levels in fore milk, determined by a highly specific radioinmunoassay, were compared for the assessment of estrus by a veteran herdsman and an experienced inseminator, in cows presented for insemination. In addition, an examination was made of the relative accurac:y of using milk progesterone levels for the determination of pregnancy at 24, 40 and 44 days after insemination, as compared with rectal palpation at 45-50 days post-breeding. Fat-free fore milk progesterone levels were similar to jugular plasma levels at 24 days post-insemination and reached roughly 60% of the level of unextracted fore milk at this time. Accuracy of estrus diagnosis by herdsman, inseminator and milk nroeesterone level was 840, 93% and 96%. respectively. For pregnancy diagnosis, milk progesterone determination in 85 cows showed 78% accuracy in predicting pregnancy,and 100% _acc_uracy in predicting non-pregnancy. At 40 days post-insemination false positives dropped to 10% and at 44 days only 7% of the cows were incorrectly diagnosed as pregnant. The false positives in this study were largely due to embryonic mortality as reflected by abnormal intervals of return to estrus. Two milk progesterone determinations, at 24 and either 40 or 44 days post-insemination ensure maximum reliability for early pregnancy diagnosis. INTRODUCTION Accurate determination of estrus is essential for good results in artificial insemination, and early information on pregnancy status is important in intensive dairy farming where the aim under present conditions is to achieve an interval between calvings of about one year. These considerations are of special significance in countries such as Israel, where dairy cattle are kept in large units, with a relatively small staff of dairy workers.
:Dept. of Dept. of iKibbutz Division
APRIL
Hormone Research, Kimron Veterinary Institute, Beit Dagan, Israel Reproduction, Kimron Veterinary Institute, Beit Dagan, Israel Givat Haim Hamuhad, Israel of Animal Reproduction, Volcani Center, Beit Dagan, Israel
1978 VOL. 9 NO. 4
343
THERIOGENOLOGY
Natural service has been shown to give better conception rates than artificial insemination (1,2), and at least part of the explanation for this difference may be ascribed to inaccurate diagnosis of estrus as reflected by progesterone concentrations in blood or milk of cows presented for insemination (3,4). The minimal values of plasma and milk progesterone present around the time of estrus have been well documented (S-10). A striking difference in peripheral plasma progesterone levels between pregnant and nun-pregnant cows was observed 19 days after insemi nation (ll), and it was suggested that this difference might furnish the This has since been verified by basis for a very eariy pregnancy test. a number of studies (12,13,14). The discovery that the levels of progesterone in milk parallel the concentrations of this hormone in blood (15) has spurred a number of reports concerning the use of milk progesterone levels as a diagnostic and for the determination of estrus and pregnancy in dairy cows (15-20). The following report deals with a pilot field trial in which milk progesterone levels were compared with the assessment of estrus by a veteran herdsman and an experienced inseminator in cows presented for insemination. In addition, an examination was made of the relative accuracy of using milk progesterone levels for the early determination of pregnancy or non-pregnancy. MATERIALSAND METHODS The cows investigated were part of a large, well-managed high producing (average production: 8,200 kg) herd of Israeli-Friesian cattle, maintained under conditions of loose housing. Estrus checks were carried out twice daily at 6 AM and 6 PM. Cows were presented for insemination based upon the criteria of standing passively to mounting by other cows, or else repeated mounting of other cows. The inseminator decided if the cow was in estrus on the basis of uterine tone, the presence of mucus typical of estrus and, in cases of doubt, examination of the cervix with the aid of a vaginal speculum. Milk samples (10 ml with potassium dichromate as a preservative) were collected from the foremilk of the afternoon milking, Milk samples were collected for progesterone evaluation on the day of presentation for insemination to assess the accuracy of estrus determination. Milk samples were also collected from inseminated cows 24, 40 and 44 days after breeding to diagnose pregnancy. Pregnancy was confirmed by rectal palpation 45-50 days post-insemination. A total of 55 cows were presented with data on estrus assessment and milk progesterone evaluation, whereas pregnancy determination was carried out on 85, 35 and 29 cows at the intervals: 24, 40 and 44 days, respectively, after insemination.
344
APRIL
1978 VOL. 9 NO. 4
THERIOGENOLOGY
Progesterone Assay - Progesterone content in fore milk samples was determined by radio-immunoassay (RIA) using an antibody raised in our Laboratory against progesterone-11-hemisuccinate-BSA. This antibody showed less than 0.01 per cent cross reaction wit!: cortisol, corticosterone, cholesterol, 17 hydroxy progesterone, estrone, androstendione, prostaglandin E and F. The estradiol , testosterone, RIA was carried out with 0.05 ml samples of unextracted milk and The RIA technique was essentially as described by 0.1 ml of plasma. Shemesh and Hansel (21). Separation of free, from antibody-bound For progesterone, was accomplished with dextran-coated charcoal. purpose:s of comparison, blood plasma levels of progesterone were determi:ned by the same RIA technique in 33 samples collected 24 days post -insemination. comparisons were made at 24 days In addition, post-breeding between paired samples of unextracted fore milk and fat-free fore milk obtained by centrifuging at 1,SOOO g for twenty minutes at room temperature. equivalent to Assay Evaluation - Knohn amounts of progesterone 2, 3,T and 8 ng/ml were added to plasma containing 0.3 ng/ml and to milk containing 0.5 ng/ml. The recovered progesterone was 92f3% (n=20) and 55?4% (n=GO) respectively. The lower percentage of recovery found with the unextracted milk suggests the presence of progesterone-binding protein, like transcortin, which binds progesterone and results in lower recovery rates. We found that this interference could be overcome by heating the milk or else by adding the same amount of milk, which contained 0.5 ng/ml of progesterone, to the tubes containing the standard amounts of progesterone. The addition of milk, in order to overcome the interference encountered in the progesterone assay of milk, has already been reported (22). The intra-assay coefficient of variation between duplicate pairs was 7.9% and the interassay coefficient of variation was 10.2%. The results found were corrected by multiplying the values determined by lOO/SS. The calibration curve was fitted by computer according to the method of Rodbard and Lewald (22)) and the sample concentration calculated as the mean of duplicates and expressed as ng/ml.
APRIL
1978 VOL. 9 NO. 4
345
THERIOGENOLOGY
RESULTS IWD DISCUSSION
Progesterone
levels
after
insemination
Table I presents the findings regarding of unextracted and fat-free fore milk and of intervals of collection after insemination.
Table
Days after insemination
I.
Progesterone concentration (ng/ml?S.E.) foremilk and jugular plasma in pregnant non-pregnant cows
Foremilk (unextracted)
Foremilk (Fat-free)
Pregnant
Not Pregnant
Pregnant
24
4.73f2.2 (n=44)
0.1-0.6 (n=41)
2.63tl.8 (n=Zl)
40
3.921.9 (n=30)
~0.1-0.6 (n=S)
44
4.83k2.1 (n=27)
346
the progesterone content jugular plasma at
in and
Jugular
Not Pregnant
co.1 (n=29)
APRIL
Pregnant
2.9f0.9 (n=Zl)
Plasma Not Pregnant
1978 VOL. 9 NO. 4
THERIOGENOLOGY
The presence of progesterone in cow’s milk has been well proven. However there has been considerable disagreement on the concentration .__ of progesterone in the milk. Progesterone concentration has been reported to be higher in cream than in skim milk (23) and it has been noted that concentrations vary depending on when the sample was collected with regard to time of day and whether samples were from foremilk, strippings, or composite milk collections. Laing and Heap (15) determined the progesterone levels in samples taken from composite milk samples collected in the afternoon. The average progesterone concentration was 5.1 ng/ml with a range of 1.3 to 15.9 ng/ml in lactating, non-pregnant Friesian cows, whereas in pregnant cows average progesterone concentration was 19.7 ng/ml, with a range of 7.1 to 35.6 ng/ml. However, Heap et al (24) reported a serious interference in the radioimmunoassay technique used to qualify the progesterone. They observed that milk. samples analyzed by a highly specific antiserum gave values consistently lower than those measured with a less specific. antiserum. They suggested that another and, as yet,unidentified compound(s) was present in milk which cross react(s) with the antisera. The considerable difference between the level of values found in this study as compared with other reports could be explained by the fact that in this investigation a highly specific antisera was used which resulted in minimal cross-reaction with other metabolites. Accuracy
of
estrus
diagnosis
Table II presents the accuracy of diagnosis of estrus herdsman, inseminator and on the basis of milk progesterone Table
Accuracy
II.
No.
of cows
of Estrus
In Estrus No. %
Diagnosis
Not in Estrus % No.
Herdsman
55
g
=
Inseminator
45
334
=93
5
=
Milk Progesterone
55
g
=
;
=
APRIL
1978 VOL. 9 NO. 4
a4
96
by the determination.
Accuracy %
Overall No. 46 s
=
84
100
$$
=
93
100
g
=
96
-
347
THERIOGENOLOGY Sixteen per cent of the cows presented for insemination by the herdsman were not in estrus as judged by milk progesterone levels. In two other studies,between 10 and 22% (4) and between 14 and 26% (17) were not in estrus when inseminated as judged by plasma or milk progesterone levels respectively. The two errors in estrus diagnosis on the basis of milk progesterone in the present study were in cases where the cows were not inseminated due to the judgment of the inseminator, and were later shown to have conceived from a previous insemination. The high degree of accuracy of the inseminator is noteworthy and it indicates that it would be worthwhile to invest time and effort in training inseminators to achieve a high degree of skill in the diagnosis of estrus. Accuracy of pregnancy diagnosis Table III presents the accuracy of pregnancy diagnosis by milk progesterone determination at three periods, as compared with return to estrus and/or rectal palpation at 45-50 days post-breeding. Table III.
Days after breeding
Accuracy of pregnancy diagnosis progesterone determination
No. Diagnosed of correct COWS NO. %
by milk
Pregnant Diagnosed error not pregnant NO. % correct
24
Overall accuracy No. %
100
75 E
88
40
35
g
90
3 E
10
5
100
32 E
g1
44
29
g
93
E2
7
T 2
100
E 27
g3
The improved results
of pregnancy testing
at the two later
periods
are more apparent than real because at least some of the cows which were falsely diagnosed as pregnant at 24 days had returned to estrus before testing at 40 and 44 days and the potential for error was therefore less.
348
APRIL
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The accuracy of diagnosis at 24 days, when compared with rectal was quite similar to that of other palpation and/or return to estrus, reports for testing about this time (17,18,19). Thus, the 100% accuracy for negative diagnosis found in this investigation was matched b:y the 98-100% accuracy recorded in the other three studies. Likewise, the figure of 78% accuracy for positive determination in the present study, was paralleled by 78% accuracy for positive tests at 20 days in Germany (17), 77% accuracy at 23 days in the USA (18) and 80% correct answers for cows diagnosed as pregnant at 24 days in England (19). It is worthwhile considering the nature of the relatively high incidence of false positives. An analysis of the time of return to estrus of these cases indicates that the source of error is not in the technique of the assay itself, but rather primarily because of inseminating cows which were not in estrus and because of embryonic mortality. In the present study, the influence of cows which were inseminated when they were not in estrus on the basis of milk progesterone levels, was not of significance. When positive pregnancy results were separately worked up for cows clearly in estrus on the basis of standing to mounting by other cows, positive inseminator assessment, and.minimal milk progesterone levels - accuracy for positive pre,gnancy diagnosis rose only slightly to 81%. In contrast, the 10 cows ltiith false positive determinations all returned to estrus between 29 and 55 days, mostly at times suggestive of embryonic mortality. The distribution of time intervals between insemination and return to estrus for these cows was as follows: 29-35 days - 4 cows, 36-43 days 4 cows, 52 days - 1 cow and 55 days - 1 cow. Similar observations regarding the importance of embryonic mortality between 21 and about 50 days post-insemination, have been recorded (19,20,25). In conclusion, the results of this study support the opinion of those investigators who contend that the principle value of milk progesterone testing for pregnancy is to detect those cows which are not pregnant:. The results of positive pregnancy assays from several countries with varying conditions of management, nutrition, and level of production indicate that with present techniques positive assays cannot be expected to give an accuracy of more than 80%. In practical terms this means that under present conditions what is needed are more efficient methods for estrus detection and milk progesterone assay will have its chief application for early detection of non-pregnant cows as well as an aid to clinical diagnosis of such conditions as cystic ovaries, subestrus, anestrus, and for monitoring changes in progesterone levels before and after gynecological treatment.
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ACKNOWLEDGMENTS Thanks are due to Mrs. Sarah Levi and to Mrs. Ella Katz for excellent technical assistance in performing the progesterone assays. We are also grateful to Mr. Yehuda Vilmosh for examining the cows presented for insemination. REFERENCES 1.
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