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).     

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.     

Fertility control using intrauterine devices: an alternative for population control in wild horses

The purpose of this study was to develop a contraceptive method for feral horses. The feral horse population has increased significantly in recent years despite attempts to control numbers. As in most wild animal population control programs, contraceptive methods must be easy to apply, cause minimal disruption to the social structure and be fully reversible. In the present study, we tested the effectiveness of an intrauterine device (IUD) for fertility control in mares. Six mares were fitted with a silastic O-ring-shaped IUD on July 1 of Year 1. The IUD-treated mares were turned out with 12 nontreated mares and a fertile stallion in a large pasture until October 20 (112 d). None of the IUD-treated mares and all the nontreated mares became pregnant. The IUD-treated mares were maintained separately from the stallion during the winter. Following removal of the IUD on April 27 of Year 2, the mares were again introduced to the pasture with the stallion together with 6 nontreated mares. For the 6 mares previously treated with an IUD, the mean interval from introduction to the stallion to conception was 17.5 +/- 5 d or 1.3 cycles per pregnancy, and all mares produced a normal foal at term. Subsequently, 19 recorded mare breeding seasons resulted in 18 foals. Uterine cytology and histopathology indicate that the IUD causes mild chronic endometritis without permanent changes in the endometrium. We conclude that based on our observations, the O-ring-shaped IUD is an effective, safe and practical contraceptive method for mares.     

Reproduction in female Hartmann's zebra, Equus zebra hartmannae

Ovaries, fetuses and plasma were collected from zebra mares shot in the Etosha National Park in Namibia between 15 and 25 August 1983. Ovarian weight was affected by reproductive status and most of the non-pregnant mares were anoestrous. The number of follicles varied between individuals and only pro-oestrous/oestrous mares had follicles larger than 20 mm in diameter. The largest follicle in pregnant mares was only 9 mm in diameter. Corpora lutea and corpora albicantia were found in non-pregnant as well as pregnant mares: 4 pregnant mares had only corpora albicantia. The presence of secondary corpora lutea could not be confirmed in any of the pregnant mares. Implantation was estimated to occur at around 73 days of gestation, and most mares (84%) had conceived between November and April. Peripheral concentrations of plasma progesterone during pregnancy varied from 0.5 to 2.4 ng/ml.     

Concurrent lactation and pregnancy: pregnant domestic horse mares do not increase mother-offspring conflict during intensive lactation

Lactation is the most energy demanding part of parental care in mammals, so parent-offspring conflict arises over milk provided by the mother. In some species females commonly become pregnant shortly after parturition of previous young. This further intensifies mother-offspring conflict due to concurrent pregnancy and lactation. In equids it has been well established that pregnant females wean their foals earlier than non-pregnant ones. Intensified mother-offspring conflict was presumed to associate with pregnancy also during the period of intensive lactation, i.e., before the weaning process starts. We investigated the effect of pregnancy on suckling behaviour characteristics as indicators of mother-offspring conflict in domestic horses. Contrary to expectation, here we provide evidence of a decreased mother-offspring conflict related to pregnancy in lactating females during first two trimesters of pregnancy. Pregnant mares provided longer suckling bouts and did not reject or terminate suckling of their foals more often than non-pregnant mares. Our results suggest that pregnant mares cope with parallel investment into a nursed foal and a foetus through enhancing nursing behaviour in early stages of pregnancy before the initially low requirements of the foetus increase. They compensate their suckling foal with the perspective of its early weaning due to ongoing pregnancy.     

Identification of a murine pregnancy-associated serum protein (alpha 1-PAP) a female-specific acute-phase reactant

A murine pregnancy-associated protein (alpha 1-PAP) with alpha 1-electrophoretic mobility and an estimated molecular weight of 150 000 was present in serum from pregnant C57BL/10 mice but could not be detected in serum of mature non-pregnant females and males. During pregnancy alpha 1-PAP was first detected on Day 7, rose to maximum levels between Days 12 and 14, and thereafter declined during the remainder of pregnancy and was undetectable by Day 8 post partum. The protein was also detected in the serum of females, but not males, subjected to an inflammatory stimulus. Examination of the alpha 1-PAP levels during an acute-phase response in females demonstrated that the protein behaved as a typical classical acute-phase reactant, although the levels were only 10% of those observed during Days 12 to 14 of pregnancy. alpha 1-PAP therefore appears to represent a hitherto undescribed female-specific acute phase reactant in the mouse.     

Sexual behavior following introduction of a stallion into a group of mares

A rested stallion was introduced daily for 30 days into each of three herds of 20 mares. Observations of sexual and mating behavior were made for one hour. The stallion remained with a herd until another stallion was introduced the following day. Other mares were isolated from stallions and were bred by artificial insemination. The diameter or growth rate of the preovulatory follicle for the six days preceding ovulation and the length of the interovulatory interval for mares which did not become pregnant were not affected significantly by the presence of a stallion. The number of breedings per hour of observation (2.4 ±0.2) and the length of the interval from introduction of a stallion into the herd of mares to first breeding (12 ±1 min) were significantly different among stallions, but the length of the interval between breedings (17 ±2 min) was not. The mean number of breedings per stallion per hour was not affected significantly by the number of posturing (estrous) mares. The number of times that the stallion rebred the same mare when more than one mare postured during the observation hour (49%) was greater (P<0.01) than what would be expected to occur by chance (30%). The hypothesis that breeding occurs preferentially in those estrous mares that are closest to ovulation was not supported, except for significantly lower breeding activity in posturing mares on days 8 and 7 before ovulation and on days 0 (day of ovulation) and ?1 (26% bred) than on days 6 to 1 (52%).     

Prostaglandin production by horse embryos and the effect of co-culture of embryos with endometrium from pregnant mares

Embryos, endometrial biopsies, and uterine lavage fluid were collected from pregnant and non-pregnant mares 14 days after ovulation. Embryos were cultured for 20.5 h with and without endometrial tissue from pregnant mares, and endometrial tissue was cultured alone. Endometrial content of PGF tended to be higher (P = 0.06) in non-pregnant than in pregnant mares, but the amount of PGF released from tissue during culture was similar for pregnant and non-pregnant mares. Lavage fluid from non-pregnant mares also tended (P = 0.08) to contain higher concentrations of PGF. Coincubation of embryos with endometrium from pregnant mares significantly (P = 0.01) lowered concentrations of PGF in medium. Tissue concentrations and release of PGE-2 and 6-keto-PGF-1 alpha were similar in endometrial samples from pregnant and non-pregnant mares and prostaglandin production was unaffected by the presence of an embryo during incubation. Horse embryos released all three prostaglandins during a 20.5-h incubation.     

Seasonal changes in blood serum protein fractions and in activity of AspAT and AlAT in Arabian brood mares and their foals

In 34 pure breed Arabian horses divided into four groups (Gr. I--10 pregnant mares, Gr. II--7 barren mares, Gr. III--10 foals born in 1981, Gr. IV--7 foals born in 1982) seasonal changes in total blood serum protein, its electrophoretic fractions and the activity of AspAT and AlAT were studied. Seasonal cyclicity was found in all groups in the amount of total serum proteins, and alpha 2- and beta 1-globulin fractions. Cyclicity was found in the level of albumin and activity of AspAT in three groups, not Gr. II, and in gamma-globulin, not Gr. IV. beta 2-globulin and AlAT cyclicity was found in two groups and alpha 1-globulin cyclicity was found only in Gr. II. Out of nine indices studied, cyclicity was found in eight of them in pregnant mares, Gr. I; in seven in older foals, Gr. III; in six in the young foals, Gr. IV; and in five in barren mares, Gr. II.     

Diagnosis and treatment of four stallions, carriers of the contagious metritis organism--case report

Contagious Equine Metritis (CEM), a venereal disease of horses caused by the bacterium Taylorella equigenitalis, was first diagnosed in 1977 and subsequently spread to many nations [Proc 24th AM Assoc Equine Pract (1979) 287]. The disease was confirmed in the United States in 1978 [Proc Am Assoc Equine Pract (1983) 295]. Specific regulatory procedures for this disease have been established in the United States and 37 other countries. From 1999 through 2001, four of 120 imported European stallions tested positive for CEM at a quarantine facility in Darlington, MD, USA. Two stallions were identified by positive bacterial cultures for T. equigenitalis on arrival. The other two positive stallions were negative on initial bacterial cultures, but were identified as CEM carriers when test mares (that they had mated) were culture-positive for T. equigenitalis. Since T. equigenitalis, is a fastidious slow-growing coccobacillus, additional sets of samples taken over a interval might be required to ensure positive stallions are detected before mating test mares. Likewise, additional sets of samples taken over a long interval after treatment of a stallion for CEM might be required to ensure that positive stallions treated for CEM are detected before mating test mares. Aggressive systemic antibiotic therapy accompanied by routine topical therapy might be required to treat some CEM-positive stallions.     

An investigation of the uterine luminal environment of non-pregnant and pregnant pony mares.

Uterine flushings were collected from 30 non-pregnant Pony mares on Days 8, 12, 14, 16, 18 or 20 after oculation. Mares were allowed a recovery period of one oestrous cycle and were mated at the next oestrus. They were then ovario-hysterectomized on days which corresponded to the day of the oestrous cycle to which they were assigned. Uterine flushings were analysed for total recoverable protein and acid phosphatase activity. Least squares analysis indicated a status X day interaction for total protein (P less than 0.10) and acid phosphatase activity (P less than 0.005) in which the latter was higher in uterine flushings during pregnancy. Peripheral plasma oestrone and oestradiol concentrations were measured by radioimmunoassay and results indicated that plasma oestrone concentrations in pregnant and non-pregnant mares were not different, and oestradiol was lower (P less then 0.005) in the peripheral plasma during pregnancy. conceptus membranes were incubated in vitro for 120 min in a chemically defined medium. Incubation medium was then assayed to assess oestrone and oestradiol production capacilities at Days 8, 12, 14, 16, 18 and 20 of pregnancy. Conceptus membrane production of oestradios (pg/5 ml/h) increased (P less than 0.05) from Day 8 (243 pg/5 ml) to Day 20 (108 763 pg/5 ml). A similar trend, but of lower magnitude, existed for oestrone production.     

Induction of abortion in mice with a monoclonal antibody specific for suppressor T-lymphocyte molecules

A monoclonal antibody, mAb 14-30, which binds T-cell produced suppressor factors (TsF) was used to study the possibility that molecules produced by suppressor T-cells play a role in maintaining pregnancy, presumably by protecting the fetus from the maternal immune system. Female mice were injected with mAb 14-30 at various times after mating. Overall, only 14% of the expected 68% of the mated and treated females were pregnant at term. In addition, Western blots were used to demonstrate the presence of TsF in fetuses, placentae, uteri and spleen of pregnant animals and its presence only in the spleen of non-pregnant animals. These experiments help to confirm results that indicate the importance of immune suppressor factors in maintaining pregnancy and extend these previous observations to include suppressor T-cell molecules.     

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.     

Effect of intrauterine treatment with prostaglandin E2 prior to insemination of mares in the uterine horn or body

Two trials were conducted to investigate the effects of intrauterine infusion of PGE2 and uterine horn insemination on pregnancy rates in mares achieved by breeding with a suboptimal number of normal spermatozoa. Estrus was synchronized and mares were teased daily with a stallion to detect estrus. Mares in estrus were examined by transrectal palpation and ultrasonography to monitor follicular status. On the first day a 35-mm diameter follicle was present, hCG (1500 IU, iv) was administered and the mares were bred the next day. Mares (Trial 1, n = 34; Trial 2, n = 28) were inseminated with 25 million total spermatozoa from either a stallion with good semen quality (Trial 1) or poor semen quality (Trial 2). In each trial, mares were assigned to 1 of 4 treatment groups as follows: Group PGE-HI - infusion of 0.25 mg PGE2 into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the proximal end of the same uterine horn; Group PGE-BI - infusion of 0.25 mg PGE2 into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the uterine body; Group SAL-HI - infusion of 1 mL sterile saline into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the proximal end of the same uterine horn; or Group SAL-BI - infusion of 1 mL sterile saline into the proximal end of the uterine horn ipsilateral to the dominant follicle 2 h prior to insemination in the uterine body. After breeding, mares were examined daily by transrectal ultrasonography to confirm ovulation, and were re-examined 14 to 16 d after ovulation for pregnancy status. Data were analyzed by Chi-square. Overall pregnancy rates were 59% for stallion 1 and 29% for stallion 2. Group pregnancy rates did not differ for mares bred by either stallion (P > 0.10). Pregnancy rates were not altered by horn insemination for either stallion (P > 0.10). Intrauterine infusion of PGE2 improved pregnancy rate in mares bred by the stallion with good quality semen (P < 0.05), but did not alter pregnancy rate in mares bred by the stallion with poor quality semen (P > 0.10). Further research is warranted to determine if intrauterine infusion of PGE2 will enhance spermatozoal colonization of the oviduct and pregnancy rates in mares, and if PGE-treatment will improve pregnancy rates achieved by subfertile stallions.     

Social relationships in a herd of Sorraia horses Part II. Factors affecting affiliative relationships and sexual behaviours

The influence of age, dominance rank, kinship and aggressiveness over affiliative relationships and sexual behaviours were analysed in a herd of Sorraia horses, Equus caballus, kept under extensive management. Subjects were 10 adult mares 5-18 years old that had known each other since birth, and a stallion introduced into the group for breeding for the first time. Kinship coefficient and dominance rank were the most important factors affecting affiliative relationships. Bonds were reciprocal and stronger among mares with higher kinship. Mares spent more time in proximity to close-ranking and lower-ranking females. Mares with stronger affiliative relationships or higher relatedness were not less aggressive towards each other. Affiliative relationships between the stallion and the mares were not reciprocal: lower-ranking mares formed stronger bonds with the stallion but he preferred the less genetically related mares for proximity. However, the stallion was involved in sexual behaviours more frequently with the mares that were more genetically related to him. These results suggest that kinship beyond close relatives may affect affiliative relationships both among familiar and among unfamiliar horses. However, the influence of kinship does not imply that horses possess a kin recognition system and alternative explanations are discussed.     

Depression of Cellular Immunity in Pregnancy due to a Serum Factor

Lymphocytes from pregnant women and non-pregnant individuals were cultured under the stimulus of phytohaemagglutinin in the presence of their own and heterologous (allogeneic) sera. The results indicate that heterologous sera have an inhibitory effect on the lymphocyte transformation rate and suggest that the inhibitory property is more powerful in pregnant and fetal sera. Conversely, the addition of heterologous non-pregnant sera to cultures of pregnant lymphocytes increases their transformation rate. These findings suggest that there is a serum inhibitor in pregnancy and this finding may be relevant to the survival of the fetal allograft.     

The contractile proteins of the human myometrium

The contractile proteins of the human myometrium were quantified and characterized in order to investigate their possible modifications during pregnancy. The myosin concentration was found to be 1 to 5 mg/g of tissue, while actin concentration ranged from 16 to 60 mg/g, leading to an actin/myosin ratio higher (Mean = 14) than in other smooth muscles. Purified myosin submitted to two-dimensional gel electrophoresis exhibited two isoelectric forms for the 17 KD Light Chain, the more acidic being predominant in the pregnant organ, the more basic in the non gravid one. The mobility of myosin of form filaments was studied using electron microscopy. Only the myosin purified from gravid uteri in its phosphorylated form did aggregate in long bipolar filaments. Actin was characterized in crude muscle extracts using two-dimensional gel electrophoresis. It appeared in three forms differing by their isoelectric points. The more basic form (gamma) predominates in the pregnant organ, as soon as 17 weeks of pregnancy, while in the non-pregnant uterus it is the intermediate (beta) form which is predominant.     

Factors influencing conception rates of Arab mares in Tunisia

In order to investigate the factors affecting the reproduction efficiency of Arab breeding mares in Tunisia, breeding data corresponding to 2340 mated cycles, over 5 years (from 2000 to 2004), from 555 mares offspring of 50 sires and bred with 17 stallions were analysed using a multivariate logistic regression. We chose logit link function and binomial distribution and we used log-likelihood-ratio (LL) and Wald tests (X² Wald) to test the mean values. The factors of variation included in the model were the year, the stallion, the age of the mare at mating, the sire and the category of the mare. Logistic regression results showed that age and sire affected both the overall conception rate and the first-cycle conception rate of the mares whilst category and stallion influenced only the overall conception rate. To our knowledge, this is the first study showing that the sire of the mare can affect both the first-cycle conception rate and the overall conception rate of the mares.     

Pregnancy rates and sexual behavior under pasture breeding conditions in mares

Pony mares (n=480) and 16 stallions were assigned to four herds of 60 mares and one stallion (large herds) and to 12 herds of 20 mares and one stallion (small herds). The stallions remained with the herds continuously for all of the large herds and seven of the small herds. In the five remaining small herds the stallion was put into a herd for three hours every two days for 12 observation periods. Pregnancy rates and day of ovulation were estimated by size of embryonal enlargements. Mean pregnancy rates of 51% and 54% were obtained in the small herds and 42% in the large herds during a 48-day period (equivalent to two estrous cycles). Pregnancy rates for herds with the stallion present continuously were higher (P<0.01) for the small herds than for the large herds for days 1-24 (42% versus 19%). There was no effect of herd size on number of mares becoming pregnant per herd on days 1-24, but more mares (P<0.01) became pregnant during days 25-48 in the large herds (13.2 mares per herd versus 1.8). In the herds in which the stallion was present intermittently, the number of times that the stallion rebred the same mare when more than one mare was in estrus was greater (P<0.01) than what would be expected to occur by chance (observed, 21%; expected, 11%). Repeated breeding of the same mare seemed related to the availability or activity of the mare, since such mares more frequently followed and positioned themselves in the vicinity of the stallion. Most of the interferences by a mare which involved keeping the stallion and another mare apart were directed at the mare, whereas most of the interferences during mounting were directed at the stallion (P<0.01). Mares were more likely (P<0.01) to interfere when in estrus than when in nonestrus. When interfering mares were in nonestrus, their hostility was usually directed at the stallion (92%), whereas when in estrus their interference was more frequently directed at a mare (73%, P<0.01).