Short-term mode of secretion of equine chorionic gonadotrop in and the effect of GNRH

Five mature Quarterhorse mares were bled every 30 min for 25 h on day 50 of pregnancy to determine the short-term mode of secretion of equine chorionic gonadotropin (eCG). Three other mares with persistent endometrial cups after abortion were administered gonadotropin releasing hormone (GnRH; 1.0 mug/kg of body weight) and were bled immediately prior to and at 15, 30, 45, 60, 90, 120, 180 and 240 min after GnRH. Concentrations of eCG in plasma of pregnant mares were constant over the 24-h period; the variation of each mare's individual values was no greater (P>.05) than the predicted random variation of the radioimmunoassay. Administration of GnRH had no significant effect on eCG concentrations of mares with persistent endometrial cups over a 4-h period. These data are consistent with a model of eCG secretion in the pregnant mare in which there is little short-term regulation of secretion other than the factors which affect the number of healthy endometrial cup cells within the uterus.     

Zebra chorionic gonadotropin: partial purification and characterization

Six samples of pregnant zebra (z) serum from the first and second trimesters of pregnancy were analyzed by RIA and shown to have chorionic gonadotropin levels comparable to that of the mare (0.9-5.3 micrograms/ml); first trimester levels in most cases were higher than second trimester levels. A pool of the sera (10 ml) was fractionated by methods previously employed for the purification of equine (e) and donkey (d) chorionic gonadotropin to achieve a concentration of the zebra chorionic gonadotropin (zCG). A yield of 1.0 mg of glycoprotein was obtained. HPLC analysis of the material indicated the content of zCG to be about 7%. Its molecular size as judged by Ve/Vo values is smaller than eCG, greater than ovine LH, and about the same as equine LH. The zCG was tested in RIAs for LH and eCG, radioreceptor assays (RRA) for LH and FSH, and the rat testis Leydig cell assay for LH. Comparisons were made with equine and donkey chorionic gonadotropin, and equine and zebra LH. The results, preliminary because the preparation is not of high purity, showed that zCG is bioactive as an LH; immunologically similar to eCG, eLH, dCG, and zLH; and competes in RRAs for LH but not FSH receptors. It differs, therefore, from eCG and eLH--which have high levels of intrinsic FSH activity, and is more like dCG, dLH, and zLH--all of which have minimal if any FSH activity.     

Urinary eCG patterns in the mare during pregnancy

Blood and urine samples collected from 12 mares at frequent intervals from 25 to 210 d of pregnancy were analyzed for equine chorionic gonadotropin (eCG). Blood and urine samples were collected daily through two consecutive ovulatory periods from five cyclic mares for comparative purposes. Separate radioimmunoassays (RIA) were developed to detect eCG in the urine and plasma. A simple and quick commercial dipstick enzyme-linked immunospecific assay (ELISA), developed for eCG in the blood, was also utilized in this study to detect eCG in the urine. In the 12 pregnant mares, eCG concentrations in both the plasma and urine as detected by RIA rose significantly on Day 40, peaked by Day 60 and slowly dropped to low levels by Day 200. The dipstick ELISA appeared more reliable for eCG in the plasma than in the urine of the five pregnant mares tested. However, on peak days (50 to 60), both the plasma and urine tested positive in all five mares. Similar eCG profiles were observed when urine samples from seven of the mares were assayed in the dipstick ELISA and RIA. The highest percentage of mares (86%) were positive for eCG by ELISA between Days 65 and 85. The highest concentration of eCG in the urine as detected by RIA was observed between Days 55 and 90. ECG-like immunoactivity was not detected by the ELISA in the urine of cyclic mares, but the RIA showed variable patterns with increases in immunoactivity that could not be correlated with physiological events. In summary, eCG in urine follows a similar profile as the eCG in plasma of mares during their first trimester of pregnancy.     

The use of radioreceptor assay for the detection of pregnancy in the mare

Jugular blood samples were collected between 42-45 days from the last breeding for measurement of pregnant mare serum gonadotropin (PMSG) and progesterone in 46 pregnant mares. A radioreceptor assay (RRA) was developed to measure human chorionic gonadotropin (hCG) and subsequently modified to measure PMSG. Highly purified hCG was iodinated using a lactoperoxidase enzymatic procedure and served as the labeled antigen for both the hCG and PMSG RRA. Standard curves were generated using purified hCG or PMSG. Bovine corpora lutea served as the receptor source. The assay was conducted at 37 degrees C for one hour with a total elapsed time from preparation of the luteal cell homogenate until final results were calculated of 2.5 hours. Twelve of the 46 mares failed to maintain their pregnancy, returned to estrus and reovulated after 45 days post-breeding (non-foaling mares). Thirty-four of the 46 mares delivered foals following a gestation of normal length. Mean concentrations of PMSG in the foaling mares was higher than in non-foaling mares. A concentration of 6.9 I.U. of PMSG/ml was used as the lowest concentration necessary for the confirmation of pregnancy. Five of the mares delivering foals had low concentrations of PMSG and were called non-pregnant. Thus, the incidence of false negatives by RRA was 14.7%. All of the non-foaling mares were correctly diagnosed non-pregnant for an error rate (false positive) of 0.0. Mare Immunological Pregnancy (MIP) tests on the 12 non-foaling mares indicated three false positives - an error rate of 25%. Of the 34 foaling mares, the MIP test indicated 8 inconclusive or false negatives, an error rate of 23.5%. At day 42-45, there was no significant difference in progesterone concentrations (determined by RIA) between foaling and non-foaling mares. RRA is a quick, accurate and quantitative method for measuring PMSG in the mare and can be used to diagnose pregnancy at 42-45 days post-ovulation. Plasma progesterone concentrations at this time were not associated with subsequent pregnancy maintenance as were plasma PMSG concentrations.     

Testosterone secretion during early pregnancy in mares

We have characterized the testosterone secretion pattern during the first 80 d of pregnancy in mares and determined the sources that contribute to circulating testosterone levels during this period. Ten untreated, pregnant mares (Group 1), 10 altrenogest-treated, pregnant mares (Group 2), and 10 altrenogest-treated, pregnant mares in which the CL was eliminated by administration of PGF-2alpha on Day 16 (Group 3) were used in this study. Complete luteolysis occurred following PGF-2alpha administration in all mares in Group 3. Six of the 10 mares in Group 3 did not have an active CL until after Day 60 of pregnancy (Group 3a) and were included in the analysis. The remaining four mares developed a new CL on Days 32, 40, 43 and 49 of pregnancy and were excluded from analysis. Mares without a functional CL (Group 3a) had significantly lower testosterone concentrations than mares with a functional CL (Groups 1 and 2), during the period before equine chorionic gonadotropin (eCG) secretion. At the onset of eCG secretion, testosterone concentrations increased rapidly but the rate of increase decreased with time in mares with a functional CL (Groups 1 and 2). In mares without a functional CL (Group 3a), testosterone concentrations did not increase at the onset of eCG secretion but increased at a gradually increasing rate after Day 50. The lower testosterone concentration in mares without a functional CL before eCG secretion suggests that the CL contributes significantly to the circulating testosterone concentration during the period before eCG secretion. The close time relationship between the onset of eCG secretion and the increase in testosterone secretion in mares with a functional CL and the lack of a testosterone increase in pregnant mares without a functional CL suggest that the increase in testosterone secretion after Day 35 of pregnancy is the result of eCG-stimulated, luteal testosterone synthesis.     

Effect of super-ovulatory doses of pregnant mare serum gonadotropin and human chorionic gonadotropin in blastocyst implantation in golden hamsters

The effect of super-ovulatory dose of pregnant mare serum gonadotropin and human chorionic gonadotropin on ovulation, advancement of ovulation, subsequent embryo development and implantation were studied in the hamster. Groups of hamsters received pregnant mare serum gonadotropin injection on day 1 of the estrous cycle followed by human chorionic gonadotropin injection either at 56 or 76 h later, pregnant mare serum gonadotropin alone on day 1 or human chorionic gonadotropin alone on day 3.The combination therapy (pregnant mare serum gonadotropin and human chorionic gonadotropin) resulted in super-ovulation (an average of 40 mature ova/animal) while human chorionic gonadotropin alone yielded an average of 10 mature ova/animal. Ovulation was advanced by 24 h by giving human chorionic gonadotropin at 56 h instead of 76 h after pregnant mare serum gonadotropin. Subsequent embryo development and implantation occurring under different hormonal regimens were studied. The ova obtained by giving human chorionic gonadotropin injection at 56 h were poorly fertilizablein vivo and hence the pregnancy rate was low (6 %). These ova however, were fertilizablein vitro, suggesting that the low fertilization rate and developmental failure may be due to inhibition of sperm capacitation/transport because of premature human chorionic gonadotropin administration. In the group receiving human chorionic gonadotropin alone on day 3 there was fertilization and cleavage, but no implantation occurred due to failure of functional corpora lutea. However, administration of progesterone and estrone from day 2 of gestation resulted in 80% implantation and sustenance of pregnancy. On the other hand, the pregnant mare serum gonadotropin and human chorionic gonadotropin combination therapy resulted in super-pregnancy. The number of fetuses present at term was higher in the group receiving pregnant mare serum gonadotropin alone than in the group receiving the combination therapy. Embryo resorption however was higher (37%) in the latter group compared with the former (9.5%). However, preimplantation embryos were found to be viable as evidenced by fluorescein diacetate staining.     

Genetic study of gestation length in Andalusian and Arabian mares

The length of gestation in Andalusian, or Spanish Purebred (SPB) and Arabian (AB) mares reared in Spain was analysed, based on 766 spontaneous full-term deliveries appertaining to 141 mares of SPB breed and 72 mares of AB breed in 31 breeding seasons. The data were obtained from the Yeguada Militar de Jerez de la Frontera stud farm in Cádiz, Spain. The mean length of gestation was of 336.8+/-0.48 days in the SPB mares and 340.3+/-0.63 days in AB mares. To assess the accurate prediction of time of birth the potential effect of a number of factors was investigated. The influences of the breed, mare, month and year of mating, age of the mother, number of births and sex of the foal were statistically significant. The factor have the greatest influence over the gestation length was the mare itself, with a correlation among consecutive births of around 0.4. The effect of inbreeding, both of the mare and foal, was negligible. Gestation length shortened as the breeding season progressed: in both breeds, a delay of 1 month in mating corresponded to a decrease of 3 days in the gestation length. According to our results, gestation length decrease as the mare gets older, with the shortest gestation periods when the mare is 10-12 years old, and from this point on, it slowly increases. The gestation period shortens as the 4th or 5th birth approaches, and then gets progressively longer. The range of variation in gestation length due to the number of births to the mare is of 2.9 days for the AB mares, and 2.2 days for SPB mares. The heritability for the gestation length for AB and the SPB breeds was 0.2, with a repeatability of 0.36 and 0.37, for SPB and AB breeds, respectively. With the data from both breeds, and using a classical approach, the response to selection was estimated if mares with extreme gestation lengths were culled, i.e. lengths which are under 310 days, or over 360 days. According to our results, in the case of SPB, a decrease of 14-45% would occur in the number of extreme gestation lengths, while in the AB breed, this value would decrease from 2 to 39%.     

Effects of grazing endophyte-infected tall fescue on eCG and progestogen concentrations from gestation days 21 to 300 in the mare

The influence of grazing endophyte-infected tall fescue on endometrial cup formation and function, progestogen production, and embryonic and fetal development were examined in pregnant mares between Day 21 and Day 300 of gestation. Total immunoreactive progestogens and equine chorionic gonadotropin (eCG) concentrations were compared between untreated controls (endophyte-free, n = 12) and treated mares (endophyte-infected, n = 12). There were no differences in endometrial cup formation or function, as determined by eCG concentrations at Days 45, 60, 75, 90 and 120 (P > 0.05) between mares grazing endophyte-infected and endophyte-free tall fescue. Mares grazing the endophyte-infected tall fescue had lower total progestogen concentrations (P < 0.01) from Days 90 to 120 than the mares grazing endophyte-free tall fescue. Embryonic development based on mean vesicle height was not affected by endophyte exposure. No pregnancies were lost by mares in either treatment group during the trial period. The results of this study indicate that grazing endophyte-infected tall fescue between Day 21 and Day 300 does not alter endometrial cup formation and function, or result in increased pregnancy losses during this period. Lower progestogen concentrations between Days 90 and 120 with exposure to endophyte-infected tall fescue could reflect decreased luteal progesterone production.     

The effect of skin allografting on the equine endometrial cup reaction

This research tested the hypothesis that immunological sensitization of mares by skin allografting, followed by the establishment of pregnancy using semen from the skin-graft donor, would give rise to secondary immune responses to the developing horse conceptus, resulting in an earlier demise of the fetally derived endometrial cups. Maiden mares received skin allografts from a stallion homozygous for Major Histocompatibility Complex (MHC) antigens and/or equivalent autografts and were subsequently mated to the skin-graft donor stallion during the next two breeding seasons. Mares that had been immunologically primed to the foreign MHC class I antigens of the skin-graft donor stallion developed strong secondary antibody responses early in their first pregnancies, whereas autografted mares made weak primary antibody responses in their first pregnancies and strong secondary responses in their second pregnancies. In contrast, histological examination of the endometrial cups after surgical pregnancy termination at Day 60 of gestation revealed no discernible differences between allografted and autografted mares, and there were no significant differences in the concentrations and/or duration of secretion of the endometrial cup-specific hormone, equine chorionic gonadotrophin (eCG), between allografted and autografted mares, nor in either group between first and second pregnancies. The vigorous antibody response observed in the pregnant allografted mares supported the first part of our hypothesis, providing evidence of systemic immunological priming. However, there was a lack of an equivalent heightened cellular response to the endometrial cups. These findings provided strong evidence for an asymmetric immune response to the conceptus, characterized by strong humoral immunity and a dampened cellular response.     

Induction of multiple ovulations during the ovulatory season in mares

The efficacy of an equine pituitary extract for induction of multiple ovulations during the ovulatory season was studied in 112 horse mares in four experiments. Combined for all experiments, 70% of the mares (78 112 ) had multiple ovulations for an average of 3.0 ovulations per mare. The interval between first and last ovulation was decreased (P<0.01) when human chorionic gonadotropin (hCG) was included in the treatment regimen (0.0 +/- 0.0 versus 1.6 +/- 0.4 days). Ovulation rate was lower (P<0.01) when extract treatment was initiated at day 19 (1.3 +/- 0.2) than when initiated at day 15 post-ovulation (2.9 +/- 0.5). Initiation of extract treatment at day 11 was as effective as initiation at day 15, with or without an injection of prostaglandin F(2alpha) on the first day of extract treatment. Increasing the dose of extract from 750 to 1500 or 2250 units per day did not significantly increase the ovulatory response.     

Detection of steroidogenic acute regulatory protein in equine ovaries

A steroidogenic acute regulatory (StAR) protein has been identified in several species as a probable important rate-limiting step in steroidogenesis. This protein is believed to be responsible for transporting cholesterol from the outer to the inner mitochondrial membrane. It is known that equine chorionic gonadotrophin (eCG) stimulates steroidogenesis in the corpora lutea of early pregnant mares and that eCG also upregulates StAR mRNA in bovine ovaries. In the present study, ovarian tissue from cyclic and early pregnant mares was immunostained to detect the distribution of the StAR protein. Western blot analysis was performed, followed by phosphor imaging to establish whether the onset of eCG secretion in pregnancy was associated with increased expression of the StAR protein. Immunostaining for StAR was confined to the theca interna of growing and preovulatory follicles, but 24 h after treatment with hCG, some granulosa cells were positively stained. Positive staining was confined to the large luteal cells of the equine corpus luteum. There was no difference in the distribution of immunostaining before or after onset of eCG secretion in pregnant mares, but increased amounts of StAR were detected in corpora lutea from mares at day 40 or day 41 of pregnancy compared with non-pregnant mares and mares at days 20-30 of pregnancy.     

Expression of 3beta-hydroxysteroid dehydrogenase, cytochrome p450 17alpha-hydroxylase/17,20-lyase and cytochrome p450 aromatase enzymes in corpora lutea of diestrous and early pregnant mares

In the pregnant mare, luteal estrogen production increases at the onset of equine chorionic gonadotropin (eCG) secretion by endometrial cups. In previous studies, we have demonstrated that eCG stimulates luteal androgen and estrogen production in pregnant mares. To further elucidate the regulation of steroidogenesis within the equine corpus luteum (CL) of pregnancy, we examined the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/17,20 lyase (P450(17alpha)) and cytochrome P450 aromatase (P450(arom)) in luteal tissue samples collected during diestrus (Days 7 to 10) and pregnancy before (Days 29 to 35) and after (Days 42 to 45) the onset of eCG secretion. Immunoblot analyses revealed a single protein per enzyme with molecular weights of 48 kDa (3beta-HSD), 58 kDa (P450(17alpha)) and 56 kDa (P450(arom)). Steady-state levels of 3beta-HSD were lower in luteal tissue of diestrus than pregnancy, but expression did not change during pregnancy. Steady-state expression of P450(17alpha) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, P450(17alpha) expression was significantly higher after the onset of eCG secretion. Steady-state expression of P450(arom) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, luteal expression of P450(arom) was significantly lower after the onset of eCG secretion. These data support the hypotheses that eCG has a differential effect on the expression of luteal steroidogenic enzymes, that the eCG-induced increase in luteal estrogen production is the result of an increase in available aromatizable androgen due to an increase in P450(17alpha) expression and activity, and that increased luteal estrogen production is not due to an increase in aromatase expression.     

eFSH in clinical equine practice

Equine follicle stimulating hormone (eFSH) has been used to induce follicular development in transitional mares and problem acyclic mares, as well as superovulate cycling mares. The most efficacious protocol is to administer 12.5 mg eFSH, intramuscularly, twice daily beginning 5 to 7 days after ovulation when the diameter of the largest follicle is 20 to 25 mm. Prostaglandins are to be administered on the second day of eFSH therapy. Treatment with eFSH is continued for 3 to 5 days until follicle(s) are >or=35 mm in diameter. The mare is subsequently allowed to 'coast' for 36 h, after which human chorionic gonadotropin is administered to induce ovulation.     

Management and fertility of mares bred with frozen semen.

Semen quality, mare status and mare management during estrus will have the greatest impact on pregnancy rates when breeding mares with frozen semen. If semen quality is not optimal, mare selection and reproductive management are crucial in determining the outcome. In addition to mare selection, client communication is a key factor in a frozen semen program. Old maiden mares and problem mares should be monitored for normal cyclicity and all, except young maidens, should have at least a uterine culture and cytology performed. Mares with positive bacterial cultures and cytologies should be treated at least three consecutive days when in estrus with the proper antibiotic. With frozen semen, timing the ovulation is highly desirable in order to reduce the interval between breeding and ovulation. The use of ovulation inducing agents such as human chorionic gonadotropin (hCG) or the GnRH analogue, deslorelin, are critical components to accurately time the insemination with frozen semen. Most hCG treated mares ovulate 48h post-treatment (12-72h) while most deslorelin (Ovuplant) treated mares ovulate 36-42h post-treatment. However, mares bred more than once during the breeding cycle appear to have a slight but consistent increase in pregnancy rate compared to mares bred only once pre- or post-ovulation. In addition, the "capacitation-like" changes inflicted on the sperm during the process of freezing and thawing appear to be responsible for the shorter longevity of cryopreserved sperm. Therefore, breeding closer to ovulation should increase the fertility for most stallions with frozen semen. Recent evidence would suggest that breeding close to the uterotubal junction increases the sperm numbers in the oviduct increasing the chances of pregnancy. Post-breeding examinations aid in determining ovulation and uterine fluid accumulations so that post-breeding therapies can be instituted if needed. Average pregnancy rates per cycle of mares bred with frozen semen are between 30 and 40% with a wide range between sires. Stallion and mare status are major factors in determining the success of frozen semen inseminations. Pregnancy rates are lower for barren and old maiden mares as well as those mares treated for uterine infections during the same cycle of the insemination. To maximize fertility with frozen semen, a careful selection of the stallions and mares, with proper client communication is critical. Dedication and commitment of mare owner and inseminator will have the most significant impact on the pregnancy rates.     

Successful transfer of the embryos of Przewalski's horses (Equus przewalskii) and Grant's zebra (E. burchelli) to domestic mares (E. caballus)

Blastocysts were collected non-surgically from 2 Przewalski's horse and 2 Grant's zebra mares and transferred extra-specifically to domestic horse and donkey recipients. Nine Przewalski's horse embryos were transferred surgically, and 2 non-surgically, to domestic Welsh-type pony mares. After surgical transfer, 7 (77.8%) pregnancies were established and 4 foals were born. Twelve Grant's zebra embryos were transferred surgically to 5 pony and 7 domestic donkey recipients respectively and 1 non-surgically to a donkey; 3 (60%) zebra-in-horse pregnancies were established and 2 went to term. Only 2 (28.6%) zebra-in-donkey pregnancies were established but neither went to term, although one zebra foal was aborted alive at Day 292 but failed to survive. No pregnancies resulted from the non-surgical transfers. Measurement of chorionic gonadotrophin concentrations and parental-specific lymphocytotoxic antibodies in the serum of the recipient animals indicated a pronounced maternal immunological response to the extra-specific embryo, but this could not be correlated with success or failure of pregnancy. The results indicate that extra-specific embryo transfer may be a useful aid to breeding exotic equids in captivity.     

Urinary steroid evaluations to monitor ovarian function in exotic ungulates: X. Pregnancy diagnosis in Perissodactyla

Enzymeimmunoassays (EIAs) for estrone conjugates (EC), pregnanediol-3-glucuronide (PDG), and C-19 and C-21 progesterone metabolites (C-19/C-21) were used to analyze urine samples from four nondomestic equid species, four tapir species, and two rhinoceros species in an attempt to identify if these assays could be used for diagnosing and monitoring pregnancy. The same urine samples were also analyzed for the presence of equine chorionic gonadotropin (eCG) activity, using a field dipstick test and a radioimmunoassay (RIA). The EC EIA was validated for three equid species and the Malayan tapir. Neither the PDG nor the C-19/C-21 EIAs were validated in any species evaluated. In equid species, the EC EIA demonstrated a specificity (the percentage of nonpregnant samples identified correctly) of 100% and a sensitivity (the percentage of pregnant samples identified correctly) of ≥ 88%. With the exception of the Grevy's zebra, the C-19/C-21 EIA showed a similar accuracy in identifying pregnant and nonpregnant equids. The PDG EIA was not sufficiently accurate to merit its use in equids or tapirs for pregnancy diagnosis. From the data collected, it appears analysis of a single urine by both the EC EIA and the C-19/C-21 EIA would be the best method of pregnancy detection during the last 2 trimesters of gestation, in equid species. In tapirs, the C-19/C-21 EIA was slightly more accurate for pregnancy diagnosis than the EC EIA. The C-19/C-21 EIA had a specificity of 93%, but a sensitivity of only 73% in tapir species. None of the EIAs evaluated demonstrated a sufficient specificity or sensitivity to be useful, as presently performed, for pregnancy diagnosis from a single sample in the black rhinoceros. The eCG dipstick used in this study did not prove a sufficiently reliable test for routine pregnancy in nondomestic equids. The eCG RIA results in the Przewalski's horses and the Hartman's mountain zebra were positive early in gestation, and indicate that gonadotropin analysis may be useful for pregnancy detection in these species. Only very low amounts of eCG activity was measured by the eCG RIA in the tapir and rhinoceros urine samples. © 1994 Wiley-Liss, Inc.     

Cell proliferation patterns during development of the equine placenta

Placentation involves considerable growth and reorganization of both maternal and fetal tissues. In this investigation, immunohistochemical localization of the proliferation marker Ki-67 antigen was used to monitor cell division during placentation in mares. Endometrial biopsies were obtained from eight mares between day 14 and day 26 of pregnancy and from eight anoestrous mares that had been treated with various combinations of progesterone and oestrogen. Samples of endometrium and fetal membranes were obtained from 19 mares carrying normal horse conceptuses between day 30 and day 250 of gestation and from three failing extraspecific donkey-in-horse pregnancies. Proliferation in the superficial strata of the endometrium was increased by day 18 of gestation and this effect could be mimicked by supplementing with oestradiol benzoate during the last 6 days of a prolonged period (18-36 days) of progesterone administration. Fetal chorionic girdle cells were proliferating vigorously at days 30-32 of gestation, but stopped dividing after they invaded the endometrium, while the trophoblast cells of the allantochorion showed an increase in mitotic activity after day 38. The luminal epithelium of the endometrium started to proliferate only after the primary villi of the true epitheliochorial placenta had been formed, and during days 58-70 this effect was seen only in the pregnant horn in which placentation was further advanced. During the second half of gestation, most of the mitotic activity was confined to the periphery of the microcotyledons which were still growing. In the donkey-in-horse pregnancies, proliferation rates of the maternal and fetal epithelial at day 70 of gestation were markedly reduced in areas of heavy endometrial lymphocyte infiltration and poor placentation. These results provide a basis for further studies on factors that influence invasive and non-invasive placentation.     

An immunochemical demonstration of a pregnancy-specific protein in the horse and its use in the serological detection of early pregnancy

Two-dimensional crossed immunoelectrophoresis of sera from pregnant and non-pregnant horses, using antisera developed against early pregnant mare serum, revealed the presence of two immunologically related proteins one of which appeared to be specific to the pregnant state. This pregnancy-specific protein had beta 2-electrophoretic mobility and was first detectable at Day 6 after successful mating with a stallion. The second protein had gamma 2-electrophoretic mobility and was present in sera from pregnant and non-pregnant horses. The proteins were termed beta 2-horse pregnancy protein and gamma 2-horse protein respectively. The latter appeared to be immunologically related to the former in that the precipitin lines of the 2 proteins showed continuity. Samples from 16 mares mated with a stallion were investigated for the beta 2-protein during the first 3 weeks after mating. Of the 11 successful matings, confirmed by ultrasonic scanning at 90 days or by a successful outcome, 10 mares showed the presence of the protein. In all of 14 non-pregnant sera taken from mares not recently mated, the protein was not detectable. The validity of detection of beta 2-protein as an indication of pregnancy was clinically significant at the 10% level. The presence of the protein in 2 out of the 5 recently mated mares that did not become pregnant may be indicative of a biochemical pregnancy that failed at a later stage of gestation.     

A mare mule--dam or foster mother?

A female, horse-like foal was apparently born to a mare mule from mating to a Shetland stallion. The presence of three pregnant Shetland mares in the same pasture, however, raised some questions about parentage, even though all three mares apparently gave birth to single foals within 6 weeks after the birth of the propositus. The transferrin system indicated that the foal was not born to the mare mule but was the offspring of one of the Shetland mares. Apparently one Shetland mare gave birth to twins 3 to 6 weeks apart, and the mare mule came into lactation spontaneously and adopted the first twin. The offspring was determined to be a horse by appearance, karyotype, blood type, and voice.     

Establishment and maintenance of pregnancy after embryo transfer in ovariectomized mares treated with progesterone

Pregnancy was established and maintained after embryo transfer in 3 ovariectomized mares treated with progesterone only. Four ovariectomized mares were used as recipients, and 7 transfers were performed. Progesterone in oil, 300 mg i.m. daily, was given starting 5 days before transfer of a 7-day embryo. If the mare was pregnant at 20 days, progesterone treatment was continued to 100 days of gestation. The 3 pregnant mares carried to term and delivered live foals with normal parturition, lactation and maternal behaviour. No differences were seen between pregnant and non-pregnant ovariectomized mares in jugular plasma concentrations of oestrogen, LH or FSH from day of transfer (Day 7) to Day 20. Pregnant ovariectomized mares showed a rise in LH, reflecting production of horse CG, starting at Day 36. Oestrogen values remained low until Day 50.