In-vitro and in-vivo responsiveness of the corpus luteum of the mare to gonadotrophin stimulation

Dispersed horse luteal cells were used to evaluate the ability of horse LH, hCG and PMSG to stimulate progesterone secretion in vitro. Morphological characterization of these cells before gonadotrophin stimulation indicated the presence of two populations of cells based on cell diameters. In luteal cells incubated as suspended cells, horse LH and hCG stimulated (P less than or equal to 0.05) progesterone production at all levels of treatment. Stimulation of progesterone secretion by hCG was greater (P less than or equal to 0.05) than by horse LH over the range of concentrations utilized. When mares (N = 7) received an intramuscular injection of 1000 i.u. hCG on Days 3, 4 and 5 after the end of oestrus, there was an increase (P less than or equal to 0.05), in peripheral progesterone concentrations beginning on Day 7 and continuing until Day 14 compared with controls (N = 7). Peripheral progesterone concentrations continued to be elevated in hCG-treated mares for Days 15-30 after oestrus in those mares that conceived. Although treatment with hCG increased progesterone concentrations, it had no influence on anterior pituitary release of LH as measured by frequency and amplitude of LH discharge. We conclude that the mare corpus luteum is responsive to gonadotrophins in vitro and that exogenous hCG can enhance serum progesterone concentrations throughout the oestrous cycle and early pregnancy.     

Effect of progesterone impregnated vaginal sponges and PMSG administration on estrus synchronization in mares

Estrus synchronization trials with mares were carried out using progesterone impregnated vaginal sponges and pregnant mare serum gonadotropin (PMSG) injections. In Phase 1, 10 non-pregnant, non-lactating mares were administered 1 g progesterone via vaginal sponges (5 x 6 cm) without regard to stage of estrous cycle. Sponges were replaced on day 7 of trial for an additional seven days. On day 12, PMSG (1000 IU, IM) was administered to five mares (Group A); five control mares (Group B) received no injections. There was no difference (P>.05) in estrus synchronization between Group A and Group B. Total sponge retention was 75%. In Phase 2, 11 non-pregnant, non-lactating mares were administered 2 g progesterone via vaginal sponges (10 x 6 cm) without regard to stage of estrous cycle. Sponges were replaced on day 7 of trial for an additional seven days. Estrus behavior was exhibited in 54.5% of mares by day 19. Total sponge retention was 95.4%. There was no difference (P>.05) in estrus synchronization or sponge retention between Phase 1 and Phase 2. The larger Phase 2 sponges showed less (P<.01) posterior movement within the vagina than the smaller Phase 1 sponges.     

Effect of diethylstilboestrol on the relationship between LH, PMSG and progesterone during pregnancy in the mare.

Two studies were conducted to determine the relationship between LH and progesterone and between PMSG and progesterone during pregnancy in mares. In the first, samples of jugular blood were collected daily from 7 mares from the first day of oestrus until Day 28 of pregnancy, and in the second, samples were collected weekly from 14 mares from Day 35 of gestation until parturition. In an attempt to prolong secretion of progesterone from accessory corpora lutea, 7 of these 14 mares were injected with increasing doses (2--10 mg) of diethylstilboestrol (DES) between Days 84 and 142 of gestation. The remaining 7 mares received injections of vehicle. Concentrations of LH, PMSG and progesterone in serum were determined by radioimmunoassay. From the onset of oestrus until Day 4 of gestation, serum concentrations of LH and progesterone were negatively correlated (r = 0.67, P less than 0.01), but from Days 5 to 28 a positive correlation (r = 0.80, P less than 0.01) was noted. Likewise, serum concentrations of PMSG and progesterone were highly correlated between Days 35 and 196 in mares injected with DES (r = 0.72, P less than 0.01) and the vehicle (r = 0.75, P less than 0.01). Injections of DES did not influence serum concentrations of LH, PMSG or progesterone, or affect the length of gestation. It was concluded that DES does not influence the maintenance of pregnancy in the mare.     

Immunochemical demonstration of a new pregnancy protein in the mare

An antiserum against the serum of a pregnant mare was absorbed with stallion serum. This antiserum then gave two precipitates in crossed immunoelectrophoresis with serum from pregnant mares as the antigen. The two precipitates exhibited beta-1 and alpha-2 electrophoretic mobility. Identity was demonstrated between the alpha-2 mobile protein and PMSG. The absorbed antiserum inhibited the biological action of the PMSG preparation when tested in mouse ovarian weight assays. The beta-1 mobile protein was not detected in the serum from non-pregnant mares, stallions or geldings and was detected earlier in pregnancy (Day 30) than was PMSG (Day 42).     

Body weight of mares and foals, estrous cycles and plasma glucose concentration in lactating and non-lactating Lipizzaner mares

This study summarizes weight development, plasma glucose concentrations and reproductive parameters in lactating (n = 46) and non-lactating Lipizzaner mares (n = 11) throughout the breeding season. It was the aim of the study to analyse if an energy deficit with possible effects on reproductive functions occurs at any time during the first 4 months of gestation. Mean gestation length was 334.3 +/- 7.3 days. Gestation of foals born in May/June was shorter (P < 0.01) than for foals born in March/April. Out of the 46 lactating mares, 44 ovulated between Days 8 and 18 postpartum and two mares ovulated on days 30 and 145, respectively. Pregnant mares were significantly (P < 0.001) heavier (600.1 +/- 5.3 kg) than non-pregnant mares (521.8 +/- 10.0 kg) at the beginning of the study. Birth resulted in weight reduction of 64.8 +/- 2.4 kg. During the first 2 weeks postpartum mares lost on average 3.0 +/- 1.8 kg and in the following 2 weeks gained 3.6 +/- 1.4 kg of weight. Thereafter, weight increased slightly but continuously (P < 0.01). At no time after foaling, weight differed significantly between groups. Weight of the foals three days after birth varied between 29 and 67 kg (53.7 +/- 1.1 kg). Average daily weight gain of foals was relatively constant throughout the study period (1.15 +/- 0.17 kg). Although lactation at no time was associated with a major weight loss, it had clear effects on energy metabolism as shown by constantly lower plasma glucose concentrations in lactating mares. Glucose concentrations decreased after foaling and were significantly lower in lactating mares from Weeks 3 to 16 after foaling than at corresponding times in non-lactating mares (P < 0.01). However, glucose concentrations were still within the physiological range. Mares seem to be able to compensate energy losses during lactation mainly by increasing feed intake and not by mobilisation of body fat.     

Induction of ovulation in gilts with cloprostenol

Prepuberal gilts were treated with 750 IU pregnant mare serum gonadotropin (PMSG) followed 72 h later by 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. In this model, ovulation occurred at 42 +/- 2 h post hCG treatment. When 500 mug of cloprostenol was injected at 34 and of 36 h after hCG injection, 78% of the preovulatory follicles ovulated by 38 h compared with 0% in the control gilts. In addition, plasma progesterone concentrations were significantly higher in the cloprostenol-treated group than in the control group (P<0.01) at 38 h, indicating luteinization along with premature ovulation. These results suggest that prostaglandin F(2)alpha (PGF(2)alpha) or an analog can be used to advance, synchronize or induce ovulation in gilts.     

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.     

Endogenous transplacental transmission of Neospora hughesi in naturally infected horses

Over a 2-yr study period, we investigated possible endogenous transplacental transmission of Neospora hughesi in 74 mare and foal pairs following the diagnosis of neuronal neosporosis in a weanling foal. Presuckle and postsuckle serum of each foal, serum and colostrum of each periparturient mare, and serum of each mare and foal pair, collected at 3-mo intervals thereafter, were tested for N. hughesi using an indirect fluorescent antibody test (IFAT). Furthermore, whole blood and colostrum samples and placentae were tested for the presence of N. hughesi by real-time PCR. The mares' seroprevalence at foaling based on IFAT (titer ≥ 160) was 52 and 6% in 2006 and 2007, respectively. Colostral antibodies against N. hughesi were detected in 96 and 11% of the mares in the 2-yr study. With the exception of 3 foals, all remaining foals were born seronegative to N. hughesi. Passive transfer of colostral antibodies to N. hughesi was documented in 15 foals. Three foals born from 2 different mares had presuckle antibodies at a titer ranging from 2,560 to 20,480. All 3 foals were born healthy. Two foals were born to the same dam that also gave birth to the weanling diagnosed with neuronal neosporosis in 2005. The third foal was born to a second mare with no previous foaling history at the farm. Seroconversion was documented in 10 foals and 9 mares over the 2-yr study. All blood and colostrum samples tested PCR negative for N. hughesi. Only 1 placenta collected in 2007 from the mare with the 2 congenitally infected foals tested PCR positive for N. hughesi. In conclusion, N. hughesi persisted in this population via endogenous transplacental infection.     

Efficacy of domperidone gel in an induced model of fescue toxicosis in periparturient mares

The objective was to evaluate the efficacy of domperidone in the prevention of reproductive complications of fescue toxicosis in periparturient mares. Pregnant mares at ≤310 days of gestation were fed ≥200 μg ergovaline per kg diet daily in endophyte-infected fescue hay and seed, starting ≥30 days before their expected foaling date (EFD: 340 days after breeding). Thirty-five mares were randomized to a treatment group to receive either domperidone gel (n = 20, 1.1 mg/kg, PO, once daily) or placebo (n = 15). Treatment was initiated 10 to 15 days before the EFD and continued for 5 days after foaling. “Treatment success” was defined as foaling within 14 days of the EFD, adequate mammary development on the day of foaling, and adequate lactation for 5 days postpartum. Twenty-seven mares were included in the effectiveness analysis. More mares in the domperidone group (12/13, P < 0.0001) were treatment successes than in the control group (1/14). Gestation length was shorter (P = 0.0011), and lactation at foaling (P = 0.0011) was better for the domperidone-group mares. Foals from two control mares were born dead and four others died or were euthanized within a few days after birth, compared with one foal death (an autolyzed twin) from a domperidone-treated mare. Plasma IgG concentrations were evaluated in 24 foals. Failure of passive transfer of immunoglobulins (IgG <800 mg/dL) occurred in 13/16 (81%) foals of domperidone-group mares and 7/8 (88%) foals of control mares. In conclusion, the reproductive complications of fescue toxicosis in periparturient mares induced by a fescue seed/hay model were prevented by treatment with domperidone.     

Patterns of excretion of fecal estradiol and progesterone and urinary chorionic gonadotropin in Grevy’s zebras (Equus grevyi): Ovulatory cycles and pregnancy

The ovulatory cycle and pregnancy of the endangered Grevy’s zebra (Equus grevyi) was characterized by analyzing excretion patterns of fecal estradiol and progesterone. Pregnancy was further described by assay of urinary equine chorionic gonadotropin. Courtship and mating behavior were similar to that seen in the domestic horse, but cycle length was longer, ranging from 28 to 35 days. Steroid hormone profiles during pregnancy were likewise similar to the horse, but gestation length was longer (391 and 406 days) in the two mature mares. A young mare was first seen to mate when 1 year 3 months of age, at which time she conceived but then aborted at 101 days’ gestation. Conception occurred after one and possibly two more abortions. A healthy foal was born after a gestation period of 425 days, when the mare was 3 years 4 months old, the youngest age at first foaling to be reported for this species. Equine chorionic gonadotropin (eCG) was first detected between 35 and 40 days’ gestation, as in the horse, returning to baseline at about 195 days. However, in one young mare, eCG was also detectable around the time of ovulation. The eCG antibody may have cross‐reacted with luteinizing hormone, which would have been reaching peak concentrations at about that time. The general agreement of the endocrine results with observed occurrence of mating behavior and of parturition demonstrates that assay of excreted hormones can be used to document puberty, monitor ovarian cycles, and diagnose and monitor pregnancy in Grevy’s zebra mares. Zoo Biol 20:185–195, 2001. © 2001 Wiley‐Liss, Inc.     

Assessment of luteal function with progesterone enzyme immunoassays in the horse mare

Progesterone (P(4)) concentrations in the horse mare have conventionally been measured by radioimmunoassay (RIA). A commercial quantitative, competitive enzymelinked immunoassay (CELISA) has been recently introduced to the horse breeding industry along with a qualitative CELISA. The objectives of this study were to evaluate these two assays for their ability to detect transitional, cyclic, and pregnancy stages as well as early embryonic death in horse mares. The quantitative CELISA demonstrated a significant difference in P(4) levels during estrus and at Day 24 of pregnancy. There was a significant difference in P(4) concentrations in mares at ovulation and at Days 17 to 18 of pregnancy. However, there was no significant difference between pregnant and nonpregnant P(4) values from Day 1 until Day 16 of pregnancy. There was a significant difference in P(4) concentrations at ovulation and at the peak of the luteal phase in nonpregnant mares. There was also a significant difference between mares in the transitional period and Day 16 pregnant mares. The intraassay coefficient of variation was 3.1%, while the interassay coefficient of variation was 6.2%. The qualitative CELISA provided true positive diagnoses of 76.5% of the time, true negatives 67.3% of the time, false positives 4.1% of the time, and false negatives 3.1% of the time. The test sensitivity was 89.3% and its specificity was 95.7%. The CELISA provided 93% valid positive diagnoses of pregnancy on Day 21, 99% on Day 26, and 100% on Day 30. The use of either of these assay systems will provide a useful adjunct to any breeding program that utilizes one or more of the currently accepted diagnostic techniques, including teasing, palpation, and ultrasound. These assays introduce a new, inexpensive method of confirming the reproductive status in the mare.     

The role of the fetal gonads and placenta in steroid production, maintenance of pregnancy and parturition in the mare.

The effects of fetal gonadectomy on steroid production and the maintenance of pregnancy in the mare were studied. Removal of the fetal gonads resulted in an immediate fall in maternal plasma concentrations of conjugated and unconjugated oestrogens whereas progestagen levels remained unchanged. Hormone profiles in mares carrying sham-operated fetuses remained similar to those in unoperated control mares. Plasma levels of 13,14-dihydro-15-oxo-PGF-2 alpha (PGFM) were much lower, and uterine contractions weaker, during labour in mares carrying gonadectomized foals than in control mares. Pregnancy was maintained until parturition at term in the mares carrying gonadectomized fetuses. However, 3 of the 4 gonadectomized foals were dysmature and died during or soon after birth. The biosynthetic pathways involved in the production of oestrogens by the feto-placental unit and the possible role of oestrogens in fetal development in the pregnant mare are discussed.     

Reproductive function of mares given daily injections of prostaglandin F2alpha beginning at day 42 of pregnancy

Mares at Day 42 of pregnancy received daily intramuscular (i.m.) injection of 5 mg of prostaglandin F2alpha (PGF(2alpha)) until the beginning of the first (Group I, n = 3) or second estrous cycle (Group II, n = 2). All mares aborted 3 to 4 d after the first injection; they displayed estrus 2 to 6 d after this injection. As determined by palpation per rectum and serum progesterone levels, each estrus was accompanied by an ovulation. Endometrial cups did not regress after PGF(2alpha) treatment since serum samples from the mares contained pregnant mare serum gonadotropin (PMSG) for at least 30 d after first injection, as determined by mare immunopregnancy test. After the first estrus, two of three mares in Group I displayed a prolonged diestrus (> 25 d). In contrast, the first estrous cycle was short (8 to 12 d) for mares in Group II. Serum progesterone levels in the first 6 d postovulation were lower (P < 0.05) for Group II than for Group I, indicating that formation of the corpus luteum was impaired by daily injections of PGF(2). Results indicate that 1) daily injections of PGF(2alpha) can induce abortion in mares at Day 42 of pregnancy, 2) abortion is followed by estrus and ovulation, 3) the endometrial cups do not regress as a result of this treatment, and 4) daily injections of PGF(2) can impair early corpus luteum development.     

The relationship between consecutive pregnancies in Thoroughbred mares. Does the location of one pregnancy affect the location of the next, is this affected by mare age and foal heat to conception interval or related to pregnancy success

Delayed uterine involution is a major cause of early reproductive failure in mares. Involution is affected by mare age, and foaling to covering interval. Involution rates vary between the previously non-gravid horn (PNGH), which recovers the quicker, and the previously gravid horn (PGH). Location of a pregnancy and its likely success may, therefore, be affected by its location relative to the previous pregnancy. This study aimed to determine: (i) the location of concepti in consecutive pregnancies; (ii) whether this varies with mare age or foaling to conception interval; (iii) whether location in relation to the previous pregnancy affects success. 1383 Thoroughbred mares were monitored by ultrasonic scanning during oestrus and early pregnancy. Significantly (p < 0.01) more pregnancies were located in the PNGH (79.2%) than the PGH (20.8%). The number of pregnancies in PGH significantly increased with mare age (p < 0.01) and foaling to conception interval (p < 0.05). Significantly (p < 0.001) more pregnancies located in the PGH (16.5%) failed, than those in the PNGH (4.6%). It can be concluded that most pregnancies locate in the PNGH where their chances of success are greatest. The larger number of pregnancies locating in the PGH in older mares and those with shorter foaling to conception intervals may in part account for the higher conceptus mortality rates in such mares. Hence breeding older mares on alternate years and maximising foaling to conception interval may improve reproductive success. Alternatively termination of pregnancies located in the PGH followed by timely recovering may be justifiable as might ET in older mares covered close to foaling.     

On lactation and associated behaviour in a natural herd of horses

Developmental changes in time spent suckling and related mother-foal behaviour are described in an unmanaged herd of Camargue horses. Male foals spent about 40% more time suckling than females during the first 8 weeks. Body weight did not differ between the sexes but time-budgets did: males grazed less and were more active. If pregnant, the typical multiparous mare nursed her foals for 35–40 weeks, males and females alike, and weaned them 15 weeks before the next foaling. Primiparae lactated longer and weaned closer to the next foaling by 5 weeks. The mares played an active role in regulating the time spent suckling in early, and particularly in late lactation.     

Effects of PGF2alpha and PGF2alpha, 1-15 lactone on the corpus luteum and on early pregnancy in the rhesus monkey.

The effects of prostaglandin (PG)F2alpha and PGF2alpha, 1-15 lactone were compared in luteal phase, non-pregnant and in early pregnant rhesus monkeys. Animals treated with either PG after pretreatment with human chorionic gonadotropin (hCG) had peripheral plasma progesterone concentrations that were not statistically different from those in animals treated with hCG and vehicle. However, menstrual cycle lengths in monkeys treated with PGF2alpha, 1-15 lactone were significantly (P less than 0.02) shorter than those in vehicle treated animals. In the absence of hCG pretreatment, plasma progesterone concentrations were significantly (P less than 0.008) lower by the second day after the initial treatment with either PGF2alpha or PGF2alpha, 1-15 lactone than in vehicle treated monkeys. Menstrual cycle lengths in monkeys treated with either PG were significantly (P less than 0.04) shorter than those in animals treated with vehicle. There were no changes in plasma progesterone concentrations in early pregnant monkeys treated with PGF2alpha, and pregnancy was not interrupted. In contrast, plasma progesterone declined and pregnancy was terminated in 5 of 6 early pregnant monkeys treated with PGF2alpha, 1-15 lactone. These data indicate that PGF2alpha, 1-15 lactone decreases menstrual cycle lengths in non-pregnant rhesus monkeys. More importantly, PGF2alpha, 1-15 lactone terminates early pregnancy in the monkey at a dose which is less than an ineffective dose of PGF2alpha.     

Progesterone and estrogens in the pregnant and nonpregnant dolphin, Tursiops truncatus, and the effects of induced ovulation

Baseline serum levels of progesterone and total immunoreactive estrogens were determined for intact and ovariectomized captive female Atlantic bottlenose dolphins (Tursiops truncatus), as well as newly captured wild adult females. Stimulation of ovarian follicular growth and ovulation was attempted by intramuscular injection of pregnant mare's serum gonadotropin (PMSG). High doses of PMSG were required to increase serum estrogen levels. When PMSG was followed by an injection of human chorionic gonadotropin (hCG), ovulation was presumed to have occurred as indicated by subsequent high levels of serum progesterone. From these observations, it appears that 1) females with progesterone levels greater than 3000 pg/ml over an extended period are pregnant, 2) Tursiops truncatus is capable of spontaneous ovulation in captivity without gonadotropin therapy, 3) captive female dolphins, although relatively resistant to PMSG, can be induced to ovulate using a combination of high intramuscular-injected doses of PMSG followed by hCG, and 4) spontaneous ovulation is likely to follow an induced ovulation.     

Plasma prolactin concentrations and cyclic activity in pony mares during parturition and early lactation

Five pony mares were blood sampled during late pregnancy, foaling and early lactation. An homologous assay for horse prolactin was used to measure plasma prolactin concentrations in these samples. Regular estimates of cyclic activity were also made. Plasma prolactin concentrations rose markedly in the last week of pregnancy and remained high although variable in early lactation, before declining to basal levels by 1-2 months post partum. All mares showed a post-partum oestrus 7.0 +/- 0.9 days after parturition. One mare whose foal died shortly after birth showed a rapid decline in plasma prolactin values after death of the foal and an early oestrous period (4 days after parturition). The pattern of prolactin changes reported for the mare are in agreement with those reported for other mammalian species.     

Influence of day of postpartum breeding on pregnancy rate, pregnancy loss rate, and foaling rate in Thoroughbred mares

Records (years 2005-2007) were analyzed from a Thoroughbred stud farm in central Kentucky. Data from all breeding cycles of foaling mares were tabulated (3184 cycles of 2003 foaling mares bred between 7 and 163 days postpartum). A multiple logistic regression model employing Bayesian statistics was used to adjust for factors that significantly affected outcome; odds ratios (ORs) for pregnancy rate, pregnancy loss rate, and foaling rate were determined to examine the influence of day of postpartum breeding on these parameters. Mares bred before Day 22 (Day 0 = day of foaling) postpartum had a decreased OR for becoming pregnant (P < 0.05); the median OR for becoming pregnant (1.00) was not reached until Day 46 postpartum. Mares bred before Day 13 postpartum had an increased OR for pregnancy loss (P < 0.05). The median OR for pregnancy loss did not decline below 1.00 until Day 78 postpartum. Mares bred before Day 20 postpartum had a decreased OR for producing a foal (P < 0.05). The median OR for producing a foal (1.00) was not reached until Day 75 postpartum. We concluded that fertility (in terms of a higher OR for becoming pregnant and a lower OR for pregnancy loss, resulting in a higher OR for producing a foal) continued to improve in Thoroughbred mares for approximately 2.5 mo postpartum. These findings are of importance to management strategies directed at early postpartum breeding, and explain some of the reported drift in subsequent foaling dates of Thoroughbred mares, despite management practices that employ early postpartum breeding.     

Induction of estrus in pubertal miniature gilts

Genetic engineering of miniature pigs has facilitated the development of numerous biomedical applications, such as xenotransplantation and animal models for human diseases. Manipulation of the estrus is one of the essential techniques for the generation of transgenic offspring. The purpose of the present study was to establish a useful method for induction of the estrus in miniature gilts. A total of 38 pubertal miniature gilts derived from 4 different strains were treated with exogenous gonadotropins. Estrus and ovulatory response were examined after treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) as 200 IU PMSG and 100 IU hCG, 300 IU PMSG and 150 IU hCG, or 1,500 IU PMSG only, followed by 100, 150 or 750 IU hCG 72 h later, respectively. The optimal protocol was determined to be the combination treatment of 200 IU PMSG and 100 IU hCG followed by 100 IU hCG. The administration of 200 IU PMSG and 100 IU hCG was effective in inducing estrus regardless of the strain, although there was a strain difference in the ovulatory response. These results indicate that treatment with a low-dose combination of PMSG and hCG provides one of the simplest methods for induction of estrus and ovulation in pubertal miniature pigs.