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Pregnancy diagnosis in the mare by immunoassay of estrone sulfate in serum and milk
PREGNANCY DIAGNOSIS IN THE MARE BY IMMUNOASSAY OF ESTRONE SULFATE IN SERUM AND MILK Mary Dee Sist, DVM; 1 J.F. Williams, PhD, BVSc, MRCVS; 2 Alma M. Geary, BS~
SUMMARY
Concentrations of eslrone sulphate (ES) were determined by radioimmunoassay in serum and milk samples from mares at various stages of gestation and lactation and from non-pregnant cycling and noncycling mares. Serum ES concentrations began to increase within a few weeks postbreeding in pregnant animals, but showed a very sharp rise after 60 days; concentrations generally reached over 30,000 pg/ml at 80 days and were maintained at that level thereafter throughout pregnancy. Differences between pregnant and non-pregnant non-estrous mares were highly significant (P<.001) from 20 days post-breeding onwards. Mean serum ES concentration in non-pregnant mares was approximately 750 pg/ml; however, there were transient peaks of from 2 to 7 times this amount during estrus. By 60 days postbreeding, the majority of pregnant mares have serum ES levels high enough to distinguish them from estrous nonpregnant mares. There was an abrupt fall in serum ES at parturition or after abortion. Concentrations of ES in milk were substantially lower than in serum, but rapidly increased in pregnant mares following a pattern similar to that in serum. By 90 days all pregnant mares had milk values of 2,000 pg/ml or more. In milk from non-pregnant mares the concentration was consistently low (100 pg/ml or Authors' address: 1Probe-TekLaboratories,3010 S. Washington Ave., Lansing,MI 48910; 2Departmentof Microbiologyand Public Health, Collegeof VeterinaryMedicine, Michigan State University, East Lansing, MI 48824. Acknowledgements:Thanks are due to the many central Michigan area horse breeders who cooperatedso willingly in the collection of samplesused in this study and to Michael J. Glade, PhD for assistance with the statistical analysis. 20
less). Determination of ES in serum and milk provides evidence of the persistence of a viable conceptus, and should serve as a valuable adjunct to other means of assessment of the reproductive status of the mare. INTRODUCTION
Determination of circulating estrone sulfate (ES) concentrations in the mare during the course of gestation has been suggested as a reliable means of confirming pregnancy.15 Sustained high ES levels are also a valuable indicator of the integrity and health of the fetoplacental unit, the principal source of conjugated estrogens in the pregnant mare. 16 Recently, workers in Sweden~4 and Australia n have shown that ES levels may increase significantly in some mares as early'as 40 days post-breeding, though a more substantial rise occurs after the first two months of pregnancy. In cows and goats ES also appears in the milk,2,10 and its presence parallels approximately the pattern of plasma concentrations; 6 however, the variables influencing transfer from blood to milk are not wholly understood. 9 Evans e t a l 5 demonstrated that ES was the dominant immunoreactive urinary estrone conjugate in mares sampled up to Day 78 of pregnancy They also proposed that ES estimations would be useful for pregnancy diagnosis in the domestic horse. This was one of few studies conducted in the USA on ES thus far, and no comprehensive account has been developed of the ES profile throughout gestation in any body fluid of the mare. We have established a radioimmunoassay for ES and report herein on data for concentrations of estrone sulfate in serum and milk at various times during gestation and lactation. EQUINE VETERINARY SCIENCE
in immunoreactivity so the majority of estimates were made on batches of stored samples. No changes were detectable when sera were allowed to remain at ambient temperatures for up to four days (approximately 20°C). Serum ES levels in pregnant and non-pregnant mares were significantly different (p<.001) over the course of this study (Figure 1). In pregnant mares, ES concentrations began to rise above those in non-pregnant mares as early as the first week after ovulation. During the first 20 days following ovulation, 8 mares out of the 19 sampled exhibited serum ES concenvations greater than the 99.9% confidence limit of non-estrous mares (1798 pg/ml); only one of these mares was not later confirmed pregnant. Serum ES concentrations were significantly greater (p<.0001) in pregnant mares than in non-pregnant and estrous mares by 4059 days, and increased sharply after 60 days. Some mares showed a slight decline shortly before the steep increase began. Between 120 and 300 days no pregnant mare sample had less than 30,000 pg/ml serum ES, but there was a precipitous decline at parturition (Figure 1), and after abortion in eight mares (data not shown). Mares sample in the first few days post-parturition had serum ES values of around 8000 pg/ml or below. Non-pregnant mares not in estrus showed serum ES concentrations ranging up to 1500 pg/ml though most were generally less than 300 pg/ml. Unbred mares showed fluctuations, declining as samples were taken in the mid-latter parts of the year when cycling activity ceased. Highest estrus values were at foal heat, with lower values recorded at subsequent heat periods (Figure 2). None of 16 pregnant mares sampled in the 40-59 day post-breeding period has a
MATERIALS AND METHODS Animals and sampling: Three hundred and ninety two serum and milk samples were collected from 153 mares; representatives of all major breeds were included (light, draft and mini horses, and donkeys). Care was taken to ensure that animals were included which provided coverage of early post-gestational, estrous, early, mid and late pregnant phases, as well as non-cycling and cycling mares that were not bred. Serum was obtained from 91 pregnant mares, bled 1-6 times at intervals of at least three weeks for a total of 194 samples, and from 62 non-pregnant mares, bled 1-6 times for a total of 120 samples. Milk was obtained from fifteen pregnant and 6 non-pregnant n,ares for a total of 78 samples. In every case, rectal or ultrasound examination data was collected and the success or failure of the pregnancy was recorded. Samples from animals lost from the study because of ownership changes, moves beyond convenient sampling range, etc., were excluded from the data set. Blood was obtained by jugular venepuncture and allowed to clot at ambient temperature before separation of serum. Milk was harvested manually and collected into 10ml plastic scintillation vials. Milk samples were usually mixed portions from each half of the udder, with the first drawn part being discarded. No preservatives were added. Serum and milk were generally stored at -70°C before being used in the assay, though for comparative purposes, values on fresh and stored liquids were determined in preliminary stages of the work. RIA determination of ES: ES-RIA 21 was performed using an antiserum prepared according to the method described by Ke-llie et a113 and 6,7-H3-ES as labeled tracer (50 pg/tube). Cross-reactions were as follows: estriol 0.02%; 17 B estradiol 0.17%; testosterone .005%; progesterone 0.002%; equilin 13%; equilenin 40%; estrone 130%. Recovery of known amounts of ES added to serum was 93.75%. The intra-assay coefficients of variation (CV) were determined from 10.duplicate measurements at 300 and 1500 pg/ml, and the inter-assay CV from 30 separate assays of these two concentrations. Intra-assay CV were 11.96% and 17.37% respectively, and inter-assay CV were 15.01% and 6.83% respectively. Standard curves covering a range of values from 60-6000 pg/ml were plotted for each run with unknown samples. Analysis of Data: The Mann-Whitney U test was used to compare serum ES concentrations in samples collected from pregnant and non-pregnant mares at intervals of 20-39, 40-59, 60-79 and 80-99 days. Confidence limits (99.9%) were computed for non-pregnant, non-estrous mares. The prediction equations for serum ES concentrations in pregnant and non-pregnant mares were compared by regression analysis.
> 50000
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~
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28000
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." I
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o
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o
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200
240
280
•
0~=',
520
-.
,
560
Days following ovulation
RESULTS
Storage of samples at -70°C did not result in any changes Volume 7, Number I
Figure 1. Serum estrone sulfate concentrations in pregnant, non-pregnant and post-partum mares. 21
>8500
>30000
"an"
"~-
Milk
m
SERUM
28000
UJ
.,
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•
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:
8000
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ESTRUS
=
:
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00
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E
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•
o non-pregnant mares
o•
200 80-99DAYS
PREGNANT
C
40
o oo o
o, o
80
120
,
o, o
160
Doys following ovulation
Figure 2. Serum estrone sulfate concentrations in diestrus, estrus and pregnancy.
Figure 3. Milk estrone sulfate concentrations in pregnant and non-pregnant mares.
serum ES value which fell into the range for non-pregnant, non-estrous mares, and from 80-99 days onwards all values recorded were higher than those at estms, excepting foal heat samples. When all values for estrous mares, excluding those for foal heat, were compared with concentrations in the 60-90 day period the difference was still significant (P<.02). ES concentrations in milk were always lower than in serum. Levels in milk taken at foal heat and in the early post-gestational period were up to 1000 pg/ml, but in nonpregnant mares levels quickly dropped. No non-pregnant mare sampled post ovulation gave a value greater than 100 pg/ml for the whole of lactation (Figure 3). In the majority of pregnant mares, on the other hand, ES concentrations began to rise by 40-60 days and by 90 days all had reached >2000 pg/ml, with subsequent values persisting at 8500 pg/ml for the remainder of lactation. In pregnant lactating mares sampled serially there was no evidence of marked fluctuation, and once ES concentrations were higher than those in non-pregnant mares, they continued to be higher.
for identification of pregnant animals. Concentrations of ES in serum early in gestation may be derived fom the endometrium under the influence of an viable embryo, 8 although there is also strong evidence that the ovary contributes significantly to the small peak seen in some mares at 40-50 daysJ 8 By the third month of pregnancy the fetoplacental unit is responsible for ES production, 14 and persistence of a healthy viable conceptus is needed for maintenance of high levels. Measurement of the sharp increase and high concenffations provides a useful test system for pregnancy diagnosis. It also serves as a prognostic aid because adverse effects on the fetoplacental unit, which may result in resorption or abortion will lead to rapidly declining serum ES concenlrations, a2 This is especially important in view of emerging evidence of the frequency of fetal resorption in the mare. Betteridge 1 found that loss rates of echographically confirmed pregnancies were generally in the 9-19% range for normal mature mares, but in young animals this reached 44%. Another recent report found that approximately 10% resorption of fetuses occurred between 2 and 8 weeks post-breeding in over 400 pregnancies detected by echography.2° Additional losses (~0.75%) were recorded after 8 weeks. Estimation of PMSG levels over this period can be misleading because of the persistence of gonadotrophin production by endometrial cup tissue for months after fetal resorption or rejection has taken place.4,7,TM The production of large quantities of estrogens by
DISCUSSION
These results confirm and extend previous observations on the pattern of ES production in pregnant mares.4,sJ 1j4 In addition, they provide evidence for the presence of ES in mare's milk at concentrations which can be readily detected 22
EQUINE VETERINARY SCIENCE
pregnant mares has long been recognized 3,7 and estimation of unconjugated estrogens in serum by RIA has recently been shown to provide a reliable and accurate pregnancy test.19 Conjugated ES determinations offer a further significant advantage in that much higher levels are reached in pregnant mares and distinction from the background in nonpregnant animals is correspondingly easier. Since ES is passed through the glomerulus its presence in urine can also be satisfactorily monitored by RIA for this purpose, 5 although urine collection is not likely to be an attractive means of sample procurement for routine pregnancy testing. In cattle and goats, pregnancy diagnostic services have been established based on ES estimations of milk samples.2,10 The results show an extraordinarily high accuracy in detection rate of true pregnancies, even permitting the unequivocal diagnosis of false pregnancy, a phenomenon increasingly recognized as an important problem in mares, t7 Our data show quite clearly that ES is secreted in mare's milk, and the lower levels detected are entirely comparable to those measurable in cow's and goat's milk, relative to serum ES concentrations. Very low milk ES concentrations in non-pregnant mares permit an appreciable degree o f discrimination between pregnant and non-pregnant animals from 40 days onwards and a very high degree of confidence in the detection o f pregnancy by 90 days. Additional data points collected in the 40-90 day period would be needed to consolidate a position on the earliest reliable sampling time for pregnancy diagnosis using milk samples, with a valuable "practical accuracy;" however since fetal resorptions continue into the third month of gestation, the utility of an ES testing system is especially evident in its capacity to establish the continued presence of a viable fetus. Both serum and milk ES concentrations in our samples show small peaks at first and second estrus post-partum, though for practical purposes this is o f no consequence in a pregnancy test. By 60 days post-breeding, the majority of pregnant mares have serum ES levels high enough to distinguish them from estrous non-pregnant mares. The few pregnant mares that have low 60 days serum ES levels, in the estrous range, have substantially higher ES levels when retested at 75-90 days. Interestingly, in continuing studies (unpublished observations) six mares that eventually aborted showed remarkably low levels of serum ES perhaps indicative of inadequate ovarian function throughout the post-breeding, early gestational phase. This characteristic may also have some prognostic value if ES concentrations were to be included in a monitored hormonal profile for the pregnant mare.
Volume 7, Number I
REFERENCES 1. BeUeridge KJ: Embryonic mortality in horses. Can Vet J 23:280, 1982. 2. Chaplin VM, Holdsworth 1U: Oestrone sulphate in goats' milk. Vet Record 111:224, 1982. 3. Cole HH, Saunders FJ: The concentration of gonad-stimulating hormone in blood serum and of oestrin in the urine throughout pregnancy in the mare. Endocrinology 19:199-208, 1935. 4. Darenius K, Kindahl H, Knudsen O, Madej A, Edqvist LE: PMSG, progesterone and estrone sulphate during normal pregnancy and early fetal death. J Reproduction & Fertility (Supplement) 32:625626,1982. 5. Evans KL, Kasman LH, Hughes JP, Couto M, Lasley BL: Pregnancy diagnosis in the domestic horse through direct urinary estrone conjugate analysis. Theriogenology 22: 615-620, 1984. 6. Gaiani R, Mattioli M, Galeati G, Chiesa F: The relationship between oestrone and oestrone sulphate in the plasma and milk of the cow during pregnancy.Archiv Vet Italiano 33: 86-91, 1982. 7. Ginther O: Reproductive biology of the mare: Basic and applied aspects. Equiservices, Cross Plains Wisconsin, 1979. 8. Heap RB, Hamon M, Allen WR: Studies on oestrogen synthesis in cyclic, pregnant, and postpartum mares. J Reproduction & Fertility (Supplement) 32:343-352, 1982. 9. Heap RB, Hamon M, Fleet JR: Factors affecting oestrone sulfate concentrations in milk. British Vet J 139:79-88, 1983. 10. Holdswolth RJ, Heap RB, Booth JM, Hamon M: A rapid direct radio-immunoassay for the measurement of oestrone sulphate in the milk of dairy cows and its use in pregnancy diagnosis. J of Endocrinol 95:7-12, 1982. I1. Hyland JH, Wright PJ, Manning SJ: An investigation of the use of plasma oestrone sulphate concentrations for the diagnosis of pregnancy in mares. Australian Vet J 61:123, 1984. 12. Hyland JH, Maclean AA, Robertson-Smith GR, Jeffcott LB, Stewart GA: Attempted conversion of twin to singleton pregnancy in two mares with associated changes in plasma oestrone sulphate concentrations. Australian Vet d 62:406-409, 1985. 13. Kellie A.E, Lichman KV, Samarajeewa P: In Steroid Immunoassay, Ed by E.H.D. Cameron, S.G. Hillier, and K. Griffiths, Alpha-Omega Publishing Ltd. Cardiff:33-46, 1975. 14. Kindahl H, Knudsen O, Madej A, Edqvist LE: Progesterone, prostaglandin F2cx, PMSG, and oestrone sulphate during early pregnancy in the mare. J. Reproduction & Fertility (Supplement) 32:353-359, 1982. 15. Parkes RO, Blackmore DJ, Rance TA, Park BK, Dean PDG: Plasma concentrations of equilin and oestrone in the assessment of fetoplacental function in the mare. Vet Record 100:511-512, 1977. 16. ])ashen RC, Allen WR: The role of the fetal gonads and placenta in steroid production, maintenance of pregnancy and parturition in the mare. d Reproduction & Fertility (Supplement) 27:499-509, 1979. 17. Sharp DC: False Pregnancy. Equine Vet Data 3-(23):358-359, 1982. 18. Terqui M, Palmer E: Oestrogen pattern during early pregnancy in the mare. J Reproduction & Fertility (Supplemen0 27:441-446, 1979. 19. Thompson DL, Wright FR: Field trial of an accurate, simple pregnancy test for mares in mid and late gestation. Proc 9th Equine Nutrition and Physiology Symposium:402-406, 1985. 20. Wood, AL, Baker CB, Bilinski J, Hillman RB, Manic EC, Sluijster F: A field study on early pregnancy loss in standardbred and thoroughbred mares. Equine Vet Science 5:264-267, 1985. 21. Wright K, Collins DC, Musey PI. Preedy JRK: A specific radio-immunoassay for estrone sulfate in plasma and urine without hydrolysis. J Clinical Endocrinol & Metabolish 47:1092-1098, 1978.
23
SUMMARY
Concentrations of eslrone sulphate (ES) were determined by radioimmunoassay in serum and milk samples from mares at various stages of gestation and lactation and from non-pregnant cycling and noncycling mares. Serum ES concentrations began to increase within a few weeks postbreeding in pregnant animals, but showed a very sharp rise after 60 days; concentrations generally reached over 30,000 pg/ml at 80 days and were maintained at that level thereafter throughout pregnancy. Differences between pregnant and non-pregnant non-estrous mares were highly significant (P<.001) from 20 days post-breeding onwards. Mean serum ES concentration in non-pregnant mares was approximately 750 pg/ml; however, there were transient peaks of from 2 to 7 times this amount during estrus. By 60 days postbreeding, the majority of pregnant mares have serum ES levels high enough to distinguish them from estrous nonpregnant mares. There was an abrupt fall in serum ES at parturition or after abortion. Concentrations of ES in milk were substantially lower than in serum, but rapidly increased in pregnant mares following a pattern similar to that in serum. By 90 days all pregnant mares had milk values of 2,000 pg/ml or more. In milk from non-pregnant mares the concentration was consistently low (100 pg/ml or Authors' address: 1Probe-TekLaboratories,3010 S. Washington Ave., Lansing,MI 48910; 2Departmentof Microbiologyand Public Health, Collegeof VeterinaryMedicine, Michigan State University, East Lansing, MI 48824. Acknowledgements:Thanks are due to the many central Michigan area horse breeders who cooperatedso willingly in the collection of samplesused in this study and to Michael J. Glade, PhD for assistance with the statistical analysis. 20
less). Determination of ES in serum and milk provides evidence of the persistence of a viable conceptus, and should serve as a valuable adjunct to other means of assessment of the reproductive status of the mare. INTRODUCTION
Determination of circulating estrone sulfate (ES) concentrations in the mare during the course of gestation has been suggested as a reliable means of confirming pregnancy.15 Sustained high ES levels are also a valuable indicator of the integrity and health of the fetoplacental unit, the principal source of conjugated estrogens in the pregnant mare. 16 Recently, workers in Sweden~4 and Australia n have shown that ES levels may increase significantly in some mares as early'as 40 days post-breeding, though a more substantial rise occurs after the first two months of pregnancy. In cows and goats ES also appears in the milk,2,10 and its presence parallels approximately the pattern of plasma concentrations; 6 however, the variables influencing transfer from blood to milk are not wholly understood. 9 Evans e t a l 5 demonstrated that ES was the dominant immunoreactive urinary estrone conjugate in mares sampled up to Day 78 of pregnancy They also proposed that ES estimations would be useful for pregnancy diagnosis in the domestic horse. This was one of few studies conducted in the USA on ES thus far, and no comprehensive account has been developed of the ES profile throughout gestation in any body fluid of the mare. We have established a radioimmunoassay for ES and report herein on data for concentrations of estrone sulfate in serum and milk at various times during gestation and lactation. EQUINE VETERINARY SCIENCE
in immunoreactivity so the majority of estimates were made on batches of stored samples. No changes were detectable when sera were allowed to remain at ambient temperatures for up to four days (approximately 20°C). Serum ES levels in pregnant and non-pregnant mares were significantly different (p<.001) over the course of this study (Figure 1). In pregnant mares, ES concentrations began to rise above those in non-pregnant mares as early as the first week after ovulation. During the first 20 days following ovulation, 8 mares out of the 19 sampled exhibited serum ES concenvations greater than the 99.9% confidence limit of non-estrous mares (1798 pg/ml); only one of these mares was not later confirmed pregnant. Serum ES concentrations were significantly greater (p<.0001) in pregnant mares than in non-pregnant and estrous mares by 4059 days, and increased sharply after 60 days. Some mares showed a slight decline shortly before the steep increase began. Between 120 and 300 days no pregnant mare sample had less than 30,000 pg/ml serum ES, but there was a precipitous decline at parturition (Figure 1), and after abortion in eight mares (data not shown). Mares sample in the first few days post-parturition had serum ES values of around 8000 pg/ml or below. Non-pregnant mares not in estrus showed serum ES concentrations ranging up to 1500 pg/ml though most were generally less than 300 pg/ml. Unbred mares showed fluctuations, declining as samples were taken in the mid-latter parts of the year when cycling activity ceased. Highest estrus values were at foal heat, with lower values recorded at subsequent heat periods (Figure 2). None of 16 pregnant mares sampled in the 40-59 day post-breeding period has a
MATERIALS AND METHODS Animals and sampling: Three hundred and ninety two serum and milk samples were collected from 153 mares; representatives of all major breeds were included (light, draft and mini horses, and donkeys). Care was taken to ensure that animals were included which provided coverage of early post-gestational, estrous, early, mid and late pregnant phases, as well as non-cycling and cycling mares that were not bred. Serum was obtained from 91 pregnant mares, bled 1-6 times at intervals of at least three weeks for a total of 194 samples, and from 62 non-pregnant mares, bled 1-6 times for a total of 120 samples. Milk was obtained from fifteen pregnant and 6 non-pregnant n,ares for a total of 78 samples. In every case, rectal or ultrasound examination data was collected and the success or failure of the pregnancy was recorded. Samples from animals lost from the study because of ownership changes, moves beyond convenient sampling range, etc., were excluded from the data set. Blood was obtained by jugular venepuncture and allowed to clot at ambient temperature before separation of serum. Milk was harvested manually and collected into 10ml plastic scintillation vials. Milk samples were usually mixed portions from each half of the udder, with the first drawn part being discarded. No preservatives were added. Serum and milk were generally stored at -70°C before being used in the assay, though for comparative purposes, values on fresh and stored liquids were determined in preliminary stages of the work. RIA determination of ES: ES-RIA 21 was performed using an antiserum prepared according to the method described by Ke-llie et a113 and 6,7-H3-ES as labeled tracer (50 pg/tube). Cross-reactions were as follows: estriol 0.02%; 17 B estradiol 0.17%; testosterone .005%; progesterone 0.002%; equilin 13%; equilenin 40%; estrone 130%. Recovery of known amounts of ES added to serum was 93.75%. The intra-assay coefficients of variation (CV) were determined from 10.duplicate measurements at 300 and 1500 pg/ml, and the inter-assay CV from 30 separate assays of these two concentrations. Intra-assay CV were 11.96% and 17.37% respectively, and inter-assay CV were 15.01% and 6.83% respectively. Standard curves covering a range of values from 60-6000 pg/ml were plotted for each run with unknown samples. Analysis of Data: The Mann-Whitney U test was used to compare serum ES concentrations in samples collected from pregnant and non-pregnant mares at intervals of 20-39, 40-59, 60-79 and 80-99 days. Confidence limits (99.9%) were computed for non-pregnant, non-estrous mares. The prediction equations for serum ES concentrations in pregnant and non-pregnant mares were compared by regression analysis.
> 50000
• ~
~
e e e ~
m ~
mea=l~e•
~
e
•
28000
24000
Serum |
2000C tO
uJ E °"
;. 16000
~'-
,,j
12000
. non-pregnant m a r e s • pregnant m a r e s • post-partum mares
." I
8o00
#
.....;-
4oooL , .
:-. ~o,~
UO
40
80
120
o
~°o°,s%
160
o
o, °o, 0, * r * * , * , , ~
200
240
280
•
0~=',
520
-.
,
560
Days following ovulation
RESULTS
Storage of samples at -70°C did not result in any changes Volume 7, Number I
Figure 1. Serum estrone sulfate concentrations in pregnant, non-pregnant and post-partum mares. 21
>8500
>30000
"an"
"~-
Milk
m
SERUM
28000
UJ
.,
•
•
6O00
24000
4000 .a 2 0 0 0 0
:D CO W
zo
im
w
16000
e.o
2ooo
&
I,I
I=
,,, 12000
E
:
8000
t 8
4000
0
FOAL SECOND LATE HEAT SEASON
DIESTRUS
ESTRUS
=
:
t 60-79
00
I000
o
E
• pregnant mares
12k 600
•
o non-pregnant mares
o•
200 80-99DAYS
PREGNANT
C
40
o oo o
o, o
80
120
,
o, o
160
Doys following ovulation
Figure 2. Serum estrone sulfate concentrations in diestrus, estrus and pregnancy.
Figure 3. Milk estrone sulfate concentrations in pregnant and non-pregnant mares.
serum ES value which fell into the range for non-pregnant, non-estrous mares, and from 80-99 days onwards all values recorded were higher than those at estms, excepting foal heat samples. When all values for estrous mares, excluding those for foal heat, were compared with concentrations in the 60-90 day period the difference was still significant (P<.02). ES concentrations in milk were always lower than in serum. Levels in milk taken at foal heat and in the early post-gestational period were up to 1000 pg/ml, but in nonpregnant mares levels quickly dropped. No non-pregnant mare sampled post ovulation gave a value greater than 100 pg/ml for the whole of lactation (Figure 3). In the majority of pregnant mares, on the other hand, ES concentrations began to rise by 40-60 days and by 90 days all had reached >2000 pg/ml, with subsequent values persisting at 8500 pg/ml for the remainder of lactation. In pregnant lactating mares sampled serially there was no evidence of marked fluctuation, and once ES concentrations were higher than those in non-pregnant mares, they continued to be higher.
for identification of pregnant animals. Concentrations of ES in serum early in gestation may be derived fom the endometrium under the influence of an viable embryo, 8 although there is also strong evidence that the ovary contributes significantly to the small peak seen in some mares at 40-50 daysJ 8 By the third month of pregnancy the fetoplacental unit is responsible for ES production, 14 and persistence of a healthy viable conceptus is needed for maintenance of high levels. Measurement of the sharp increase and high concenffations provides a useful test system for pregnancy diagnosis. It also serves as a prognostic aid because adverse effects on the fetoplacental unit, which may result in resorption or abortion will lead to rapidly declining serum ES concenlrations, a2 This is especially important in view of emerging evidence of the frequency of fetal resorption in the mare. Betteridge 1 found that loss rates of echographically confirmed pregnancies were generally in the 9-19% range for normal mature mares, but in young animals this reached 44%. Another recent report found that approximately 10% resorption of fetuses occurred between 2 and 8 weeks post-breeding in over 400 pregnancies detected by echography.2° Additional losses (~0.75%) were recorded after 8 weeks. Estimation of PMSG levels over this period can be misleading because of the persistence of gonadotrophin production by endometrial cup tissue for months after fetal resorption or rejection has taken place.4,7,TM The production of large quantities of estrogens by
DISCUSSION
These results confirm and extend previous observations on the pattern of ES production in pregnant mares.4,sJ 1j4 In addition, they provide evidence for the presence of ES in mare's milk at concentrations which can be readily detected 22
EQUINE VETERINARY SCIENCE
pregnant mares has long been recognized 3,7 and estimation of unconjugated estrogens in serum by RIA has recently been shown to provide a reliable and accurate pregnancy test.19 Conjugated ES determinations offer a further significant advantage in that much higher levels are reached in pregnant mares and distinction from the background in nonpregnant animals is correspondingly easier. Since ES is passed through the glomerulus its presence in urine can also be satisfactorily monitored by RIA for this purpose, 5 although urine collection is not likely to be an attractive means of sample procurement for routine pregnancy testing. In cattle and goats, pregnancy diagnostic services have been established based on ES estimations of milk samples.2,10 The results show an extraordinarily high accuracy in detection rate of true pregnancies, even permitting the unequivocal diagnosis of false pregnancy, a phenomenon increasingly recognized as an important problem in mares, t7 Our data show quite clearly that ES is secreted in mare's milk, and the lower levels detected are entirely comparable to those measurable in cow's and goat's milk, relative to serum ES concentrations. Very low milk ES concentrations in non-pregnant mares permit an appreciable degree o f discrimination between pregnant and non-pregnant animals from 40 days onwards and a very high degree of confidence in the detection o f pregnancy by 90 days. Additional data points collected in the 40-90 day period would be needed to consolidate a position on the earliest reliable sampling time for pregnancy diagnosis using milk samples, with a valuable "practical accuracy;" however since fetal resorptions continue into the third month of gestation, the utility of an ES testing system is especially evident in its capacity to establish the continued presence of a viable fetus. Both serum and milk ES concentrations in our samples show small peaks at first and second estrus post-partum, though for practical purposes this is o f no consequence in a pregnancy test. By 60 days post-breeding, the majority of pregnant mares have serum ES levels high enough to distinguish them from estrous non-pregnant mares. The few pregnant mares that have low 60 days serum ES levels, in the estrous range, have substantially higher ES levels when retested at 75-90 days. Interestingly, in continuing studies (unpublished observations) six mares that eventually aborted showed remarkably low levels of serum ES perhaps indicative of inadequate ovarian function throughout the post-breeding, early gestational phase. This characteristic may also have some prognostic value if ES concentrations were to be included in a monitored hormonal profile for the pregnant mare.
Volume 7, Number I
REFERENCES 1. BeUeridge KJ: Embryonic mortality in horses. Can Vet J 23:280, 1982. 2. Chaplin VM, Holdsworth 1U: Oestrone sulphate in goats' milk. Vet Record 111:224, 1982. 3. Cole HH, Saunders FJ: The concentration of gonad-stimulating hormone in blood serum and of oestrin in the urine throughout pregnancy in the mare. Endocrinology 19:199-208, 1935. 4. Darenius K, Kindahl H, Knudsen O, Madej A, Edqvist LE: PMSG, progesterone and estrone sulphate during normal pregnancy and early fetal death. J Reproduction & Fertility (Supplement) 32:625626,1982. 5. Evans KL, Kasman LH, Hughes JP, Couto M, Lasley BL: Pregnancy diagnosis in the domestic horse through direct urinary estrone conjugate analysis. Theriogenology 22: 615-620, 1984. 6. Gaiani R, Mattioli M, Galeati G, Chiesa F: The relationship between oestrone and oestrone sulphate in the plasma and milk of the cow during pregnancy.Archiv Vet Italiano 33: 86-91, 1982. 7. Ginther O: Reproductive biology of the mare: Basic and applied aspects. Equiservices, Cross Plains Wisconsin, 1979. 8. Heap RB, Hamon M, Allen WR: Studies on oestrogen synthesis in cyclic, pregnant, and postpartum mares. J Reproduction & Fertility (Supplement) 32:343-352, 1982. 9. Heap RB, Hamon M, Fleet JR: Factors affecting oestrone sulfate concentrations in milk. British Vet J 139:79-88, 1983. 10. Holdswolth RJ, Heap RB, Booth JM, Hamon M: A rapid direct radio-immunoassay for the measurement of oestrone sulphate in the milk of dairy cows and its use in pregnancy diagnosis. J of Endocrinol 95:7-12, 1982. I1. Hyland JH, Wright PJ, Manning SJ: An investigation of the use of plasma oestrone sulphate concentrations for the diagnosis of pregnancy in mares. Australian Vet J 61:123, 1984. 12. Hyland JH, Maclean AA, Robertson-Smith GR, Jeffcott LB, Stewart GA: Attempted conversion of twin to singleton pregnancy in two mares with associated changes in plasma oestrone sulphate concentrations. Australian Vet d 62:406-409, 1985. 13. Kellie A.E, Lichman KV, Samarajeewa P: In Steroid Immunoassay, Ed by E.H.D. Cameron, S.G. Hillier, and K. Griffiths, Alpha-Omega Publishing Ltd. Cardiff:33-46, 1975. 14. Kindahl H, Knudsen O, Madej A, Edqvist LE: Progesterone, prostaglandin F2cx, PMSG, and oestrone sulphate during early pregnancy in the mare. J. Reproduction & Fertility (Supplement) 32:353-359, 1982. 15. Parkes RO, Blackmore DJ, Rance TA, Park BK, Dean PDG: Plasma concentrations of equilin and oestrone in the assessment of fetoplacental function in the mare. Vet Record 100:511-512, 1977. 16. ])ashen RC, Allen WR: The role of the fetal gonads and placenta in steroid production, maintenance of pregnancy and parturition in the mare. d Reproduction & Fertility (Supplement) 27:499-509, 1979. 17. Sharp DC: False Pregnancy. Equine Vet Data 3-(23):358-359, 1982. 18. Terqui M, Palmer E: Oestrogen pattern during early pregnancy in the mare. J Reproduction & Fertility (Supplemen0 27:441-446, 1979. 19. Thompson DL, Wright FR: Field trial of an accurate, simple pregnancy test for mares in mid and late gestation. Proc 9th Equine Nutrition and Physiology Symposium:402-406, 1985. 20. Wood, AL, Baker CB, Bilinski J, Hillman RB, Manic EC, Sluijster F: A field study on early pregnancy loss in standardbred and thoroughbred mares. Equine Vet Science 5:264-267, 1985. 21. Wright K, Collins DC, Musey PI. Preedy JRK: A specific radio-immunoassay for estrone sulfate in plasma and urine without hydrolysis. J Clinical Endocrinol & Metabolish 47:1092-1098, 1978.
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