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.     

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.     

Transrectal Doppler sonography of uterine blood flow during early pregnancy in mares

Transrectal color Doppler sonography was used for the noninvasive investigation of uterine blood flow in five mares. Both the left and right uterine arteries were scanned to obtain blood flow velocity waveforms during two consecutive estrous cycles and two early pregnancies in each mare. Blood flow was expressed as the time-averaged maximum velocity (TAMV) and the resistance index (RI). In all pregnancies the embryonic vesicle could be detected for the first time on Day 11 (day of ovulation: Day 0). No differences in mean TAMV and RI values of both uterine arteries were observed in comparison to the corresponding days of the estrous cycle until Day 11 of pregnancy (P>0.05). From Day 11 onwards, mean TAMV values were higher and mean RI values lower in pregnant mares than in cyclic mares (P<0.05). During the estrous cycle TAMV and RI values did not differ between the right and left uterine arteries (P>0.05). From Days 15 to 29 of pregnancy, TAMV values were consistently higher and RI values lower in the uterine artery ipsilateral to the conceptus and they had a more distinct rise and decline, respectively, compared to the contralateral uterine artery (P<0.05). The variance component estimates for the effect of mare on TAMV and RI values during pregnancy were 60 and 53%, respectively, and for the effect of day of pregnancy, they were 29 and 34%, respectively (P<0.0001). Within mares there were no significant differences between the two pregnancies with regard to blood flow (P>0.05). The results show that uterine blood supply increases in mares during the second week of pregnancy compared to cyclic mares. Furthermore there are individual variations in blood flow between mares.     

Urinary and plasma estrogen conjugates, estradiol and estrone concentrations in nonpregnant and early pregnant mares

A direct radioimmunoassay for estrogen conjugates (EC) was applied to paired blood and urine samples collected from 20 mares and compared against estrone (E(1)) and estradiol-17beta (E(2)) to monitor changes in estrogen production during ovulatory cycles and early pregnancy. Blood samples were taken daily from five mares through two consecutive ovulations and from six mares at 6-h intervals starting 48 hours prior to ovulation and continuing after ovulation had occurred. Blood samples were also collected daily or three times per week from conception until Day 60 of pregnancy in nine pregnant mares. The mean urinary EC, plasma EC and plasma E(2) dynamics were parallel in nonpregnant mares, with a 3-fold increase in mean urinary EC concentrations from baseline to the ovulatory peak, a 1.8-fold increase in mean plasma EC concentrations and a 1.4-fold increase in mean plasma E(2) concentrations. In early pregnancy, a two-fold increase in mean plasma E(1) and EC concentrations occurred in concert with a five-fold rise in mean urinary EC concentrations, whereas plasma E(2) did not change. Following hydrolysis and chromatographic separation, E(1) and E(2) were identified as the hydrolytic products in the urine of nonpregnant and pregnant mares; however, an unidentified estrogen was the major hydrolytic product in nonpregnant mares and pregnant mares prior to Day 38 of pregnancy. The increased resolution of the EC profiles compared with the profiles of other estrogen components indicates that the determination of EC in urine or plasma provides a useful alternative method for monitoring reproductive events in mares.     

Effects of different artificial insemination techniques and sperm doses on fertility of normal mares and mares with abnormal reproductive history

The effects of different artificial insemination (AI) techniques and sperm doses on pregnancy rates of normal Hanoverian breed mares and mares with a history of barrenness or pregnancy failure using fresh or frozen-thawed sperm were investigated. The material included 187 normal mares (148 foaling and 39 young maiden mares) and 85 problem mares with abnormal reproductive history. Mares were randomly allotted into groups with respect to AI technique (routine AI into the uterine body, transrectally controlled deep intracornual AI ipsilateral to the preovulatory follicle, or hysteroscopic AI onto the uterotubal junction ipsilateral to the preovulatory follicle), storage method of semen (fresh, frozen-thawed), AI volume (0.5, 2, 12 ml), and sperm dose (50 x 10(6) or 300 x 10(6) progressively motile sperm (pms) for fresh semen and 100 or 800 x 10(6) frozen-thawed sperm with >35% post-thaw motility). The mares were inseminated once per cycle, 24 h after hCG administration when fresh semen was used, or 30 h for frozen-thawed semen. Differences in pregnancy rates between treatment groups were analyzed by Chi-squared test, and for most relevant factors (insemination technique, mare, semen, and stallion) expectation values and confidence intervals were calculated using multivariate logistic models. Neither insemination technique, volume, sperm dose, nor mare or stallion had significant effects (P > 0.05) on fertility. Type of semen, breeding mares during foal heat, and an interaction between insemination technique, semen parameters, and mares did have significant effects (P < 0.05). In problem mares, frozen semen AI yielded significantly lower pregnancy rates than fresh semen AI (16/43, 37.2% versus 25/42, 59.5%), but this was not the case in normal mares. In normal mares, hysteroscopic AI with fresh semen gave significantly (P < 0.05) better pregnancy rates than uterine body AI (27/38, 71% versus 18/38, 47.3%), whereas in problem mares this resulted in significantly lower pregnancy rates than uterine body AI (5/15, 33.3% versus 16/19, 84.2%). Our results demonstrate that for problem mares, conventional insemination into the uterine body appears to be superior to hysteroscopic insemination and in normal mares, the highest pregnancy rates can be expected by hysteroscopic insemination.     

Seasonal variation and opioidergic regulation of growth hormone release in cyclic, ovariectomized, and pregnant pony mares

Modulation of reproductive functions is one of the multiple effects of growth hormone (GH). To investigate effects of reproductive functions on GH release in the horse, plasma GH concentrations in ovary-intact (n = 7) and ovariectomized (n = 8) mares during the anovulatory and breeding seasons and in pregnant mares (n = 6) at various stages of gestation were determined. To analyze an opioidergic regulation of GH release, repeated blood samples were taken over 3 h, and mares were injected with the opioid antagonist naloxone (0.5 mg/kg i.v.) or saline. GH was determined by RIA with an antiserum raised against porcine GH and equine GH as standard. In ovariectomized and ovary-intact, cyclic mares, GH concentrations were low and not different between the two groups in November and December. GH concentrations increased significantly (P < 0.05) in cyclic mares during May and June but were not affected by stage of the cycle and were low in ovariectomized mares. In pregnant mares, plasma GH concentrations remained high throughout pregnancy and did not decrease during winter but increased significantly (P < 0.05) postpartum. Naloxone induced a significant GH release in ovary-intact mares; this response was most pronounced (P < 0.05) during the breeding season. Naloxone did not affect GH in ovariectomized mares. During pregnancy, naloxone induced a significant release of GH around Day 280 (P < 0.05) but not at other times of pregnancy. In conclusion, GH release is influenced by season. The seasonal changes depend on ovarian factors, are absent in ovariectomized mares, and can be modulated by pregnancy. GH release is regulated at least in part by opioidergic pathways.     

Monoclonal antibodies against the beta subunit of human chorionic gonadotropin

The aim of investigation was creation of hybridomas, which produce monoclonal antibodies to the beta-subunit of human chorionic gonadotropin (beta-hCG), characterization of monoclonal antibodies, which necessary for hCG immunoassay in biological fluids, as an immunological methods of detection of early stage of pregnancy and choriocarcinoma. 4 hybridomas, producing monoclonal antibodies to-hCG of IgG1 isotype, were created. On the base of monoclonal antibodies, which produced by D2 hybridoma cell line, test-systems for RIA of hCG in blood serum and urine were elaborated. These test-systems can be used in medical practice for diagnosis of early stages of pregnancy and choriocarcinoma.     

Analysis of several factors of variation of gestation loss in breeding mares

The files for ultrasound diagnosis of gestating mares belonging to the French equine herd recorded for 3 consecutive years were joined with the files for foal birth of these same mares, allowing the statistical analysis of factors of pregnancy loss. For 28 872 positive diagnoses of gestation, 2898 losses were recorded, that is, a global rate of gestation interruption of 9.12%. The etiology of these interruptions is mainly extrinsic: the year and month of insemination, as well as region for climatic reasons. The intrinsic causes that are implicated are breed of the father (heavy breeds except the hypermetric ones lose fewer pregnancies than warm-blooded breeds), age of the mother (losses are lower in mares of 7 to 10 years of age) and status (mares with foals have fewer pregnancy losses than mares not having foaled the previous year), as well as fetuses with consanguinity (when this increases, the pregnancy losses increase as well). However, the additive genetic effect is extremely low; it corresponds to heritability below 5% and few effects of the environment, common to the offspring of the same mare, were identified. This therefore gives little hope of being able to select against the ‘gestation loss’ trait.     

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.     

Suppression of lymphocyte reactivity by culture supernatant from horse embryos and endometrium

The mechanisms that permit maternal tolerance of the conceptus allograft during early pregnancy in the mare have not been investigated. Embryos and endometria were collected from mares 14 days after ovulation and cultured for 20.5 h. The effect of addition of culture supernatant on incorporation of [3H]thymidine by equine peripheral blood lymphocytes was studied. Culture supernatant from endometrium of nonpregnant mares did not affect lymphocyte blastogenesis, but supernatant from both embryos and endometrium of pregnant mares reduced concanavalin A (Con A)- and phytohemagglutinin-induced blastogenesis. Five of six cultures performed in the present of indomethacin did not contain immunosuppressive factors. The suppressive effect on Con A-induced blastogenesis was eliminated by charcoal treatment of the supernatants and reduced by treatment with trypsin or heat. Blastogenesis of bovine lymphocytes was inhibited by culture supernatant of endometrium from pregnant mares, but not by embryo supernatant. Preincubation of blood lymphocytes with supernatants from endometrium of pregnant mares enhanced subsequent incorporation of [3H]thymidine by lymphocytes. A 24-h delay in addition of embryo culture supernatants significantly reduced the degree of immunosuppression. These results suggest that probably more than one substance interacts with the lymphocyte cultures and the observed blastogenesis reflects the end result of the interaction between suppressive and stimulating factors. The lymphocyte inhibitory effect evident in supernatants from embryos and endometrium from pregnant mares may be important in local immunosuppression and maternal acceptance of pregnancy.     

Relationship between ultrasonic characteristics of the corpus luteum, plasma progesterone concentration and early pregnancy diagnosis in Friesian mares

The purpose of the present study was to evaluate the change in cross-sectional area of the early corpus luteum (CL) and progesterone production in relation to subsequent pregnancy diagnosis. The cross-sectional area of the CL of 75 Friesian brood mares was measured by ultrasonography on Day 1 or 2 and Day 8 or 9 after ovulation. The change in cross-sectional area was expressed in a volume ratio. Plasma progesterone concentrations were measured on Days 8 to 9, and ultrasonography to determine pregnancy status was carried out on Day 17. The data obtained were analyzed by using a multiple logistic regression model. There were significant differences in the age, volume ratio and progesterone concentration between pregnant and nonpregnant mares. Pregnancy on Day 17 was related to the change in size of the CL up to Days 8 to 9 and progesterone concentration on Days 8 to 9. These differences between pregnant and nonpregnant mares might reflect the first luteal response to pregnancy.     

Effect of Timing of Frozen Semen Insemination on Pregnancy Rate in Mares

Thirty-four mares were inseminated with frozen semen from one stallion during 2 oestrous cycles, every 48 h until ovulation took place and within 12 h after ovulation. Semen was frozen using the Colorado method. The insemination dose was from 200 to 400×106 progressively motile spermatozoa. Ovaries were examined every 12 h to determine time of ovulation. Examination for pregnancy was carried out using ultrasonography, 15 days after ovulation. Thirty-five per cent of mares inseminated < 24 h and 23% of mares inseminated between 24 - 48 h before ovulation were pregnant (p = 0.388). The pregnancy rate in all mares inseminated before ovulation was 30%. In the mares inseminated within 12 h of ovulation, it was 18% (p = 0.253). Younger mares (aged 4-10 yr) had a higher pregnancy rate (59%) than older mares (aged 11-15 yr) (23%), but the difference was not statistically significant (p = 0.057).     

Perceptions of obesity in a UK leisure-based population of horse owners

Pregnancy rates with cooled equine semen can be unsatisfactory and show great variation. Information about first cycle pregnancy rates and pregnancy rates per cycle are often lacking from publicly available records. This retrospective cohort study was performed to evaluate the fertility of the Norwegian Coldblooded trotter. The aim of the study was to compare the breeding results after insemination with fresh, extended with those of cooled, shipped semen among Norwegian Coldblooded trotter mares. First cycle pregnancy rate was the main parameter used to measure fertility. Stud-books were collected from four studs from the years 2006–2010. Statistical analyses were done in Stata using Chi square test and multivariable analyses where different models were compared based on Akaike’s information criterion. First cycle pregnancy rate, seasonal pregnancy rate and foaling rate all showed significant differences (P < 0.0001) when comparing mares inseminated at stud with mares inseminated with cooled, shipped semen, favoring artificial insemination (AI) at stud. First cycle pregnancy rate was 55.1 % for mares inseminated at stud with fresh extended semen and 42.2 % for mares inseminated with cooled shipped semen. The overall pregnancy rate per cycle was 84.4 % for AI at stud and 66.9 % for cooled, shipped semen. The parameters stud, mare age, number of inseminations within an estrus cycle and individual stallion were also investigated for influence on fertility. Few retrospective studies include the parameter of first cycle pregnancy rates. Our study does not differ dramatically when comparing seasonal pregnancy rates and foaling rates with similar studies. Fertility parameters for the Norwegian Coldblooded trotter do not differ significantly from most other studies of Coldblooded mares and other mare breeds around the world. But the difference in fertility parameters between AI at stud to AI with cooled semen between our study and others, indicates that higher pregnancy rates in Norwegian Coldblooded trotter may be possible.     

Relationships between sexual behaviour, dominant follicle area, uterus ultrasonic image and pregnancy rate in mares of two breeds differing in reproductive efficiency

Weak or equivocal expression of oestrous behaviour, related to different level of mares' reactivity, may cause problems in oestrus detection and thus influence the reproductive efficiency. The aim of the study was to test whether a breed characterised by low pregnancy rate and high emotional reactivity (Thoroughbreds) differs in oestrous behaviour from a primitive breed with higher reproductive efficiency (Koniks). Additionally, the follicle size was examined to determine how it influences the intensity of oestrus and uterine ultrasonic image and whether the pregnancy rate differs in relation to the intensity of oestrous behaviour and the size of the dominant follicle(s) area on day preceding ovulation in both breeds. During four reproductive seasons the behaviour of 20 Konik polski (K) and 37 Thoroughbred (T) was observed during daily teasing. Simultaneously, the ultrasonic examinations of the reproductive system were carried out. The behaviour of mares was quantified by scoring on an 8-point scale (behavioural score, BS), according to increasing sexual receptivity. Cross-sectional follicular area (FA) was taken as a product of the two largest perpendicular follicular diameters and mean values for each breed were estimated on 1693 and 1982 mm2 for K and T mares, respectively (P < 0.05). Mares were classified according to the pooled area of dominant follicle (FA) during the preovulatory period: group A (FA < or = breed mean) and group B (FA > breed mean). Uterus image (UI) was scored (1-5) according to the increasing uterine echogenicity. The BS was higher (P < 0.01) in K mares (BS = 5.19) than in T mares (BS = 4.04). The BS was significantly related to increasing follicular area (FA). There was no breed difference in uterus image (UI) score. However, significant regression of UI on FA was found in K mares. The intensity of oestrus was positively related with UI (r = 0.29; P < 0.01) only in K mares, no such relationship was found in T mares. The pregnancy rate was significantly higher for K mares (88.5%) than for T mares (46.0%) and lower for T mares with less intense oestrous behaviour (29.4%) as compared to T mares with more intense oestrus (60.0%). No differences in pregnancy rate was found in mares belonging to A or B group of follicular area. The ascertained weaker oestrous behaviour in Thoroughbred was related to lower pregnancy rate. It is hypothesised that oestrus intensity may be the result of breed differences in the response of the neural system to follicular secretions, or may be an effect of higher incidence of multiple non-synchronic ovulation and/or higher sensitivity to stress in Thoroughbred mares.     

Biological and clinical significance of anti-Müllerian hormone determination in blood serum of the mare

Anti-Müllerian hormone (AMH), a member of the transforming growth factor β superfamily of growth and differentiation factors, is expressed in granulosa cells of preantral and small antral ovarian follicles. In humans, AMH appeared to regulate recruitment and growth of small ovarian follicles. Furthermore, circulating AMH concentrations were elevated in women with granulosa-cell tumors (GCT). In the horse, GCTs are the most common tumor of the ovary, and a variety of endocrine assays have been used to diagnose presumptive GCTs. The objectives of the present study were to validate a heterologous enzyme immunoassay for determination of serum AMH in the horse, and to determine concentrations of AMH in the blood of mares during the estrous cycle, pregnancy, and in mares with granulosa-cell tumors. Mares with normal estrous cycles (n = 6) and pregnant mares (n = 6) had blood samples collected throughout one interovulatory period and monthly throughout gestation, respectively. Mares diagnosed with GCT had blood samples taken before (n = 11) and after ovariectomy (n = 5). Tumors were sectioned and fixed for immunohistochemistry and snap frozen for immunoblot analyses and RT-qPCR. In normal cyclic mares and in pregnant mares, there was no effect of cycle stage or month of gestation on serum AMH concentrations. In GCT mares, serum concentrations of AMH (1901.4 ± 1144.6 ng/mL) were higher than those in cyclic (0.96 ± 0.08 ng/mL) or pregnant (0.72 ± 0.05 ng/mL) mares and decreased after tumor removal. Both AMH and AMH receptor (AMHR2) immunolabeling and expression were detected by immunohistochemistry in the tumor and cyst fluid obtained from mares with GCTs. Therefore, we concluded that AMH was a useful biomarker for detection of granulosa-cell tumors in mares.     

Pregnancy determination in uncaptured feral horses by means of fecal steroid conjugates

This study was carried out to develop an accurate, rapid and inexpensive method for diagnosing pregnancy in uncaptured feral horses by analysis of fecal steroid metabolites and to compare the accuracy of this method with diagnosis by urinary estrone conjugates (E(1)C). Paired urine and fecal samples were collected from 40 sexually mature feral mares during August and October. Urine samples were extracted directly from the soil and analyzed by enzymeimmunoassay (EIA) for E(1)C. Water extracts of fecal samples were assayed by EIA for E(1)C and nonspecific progesterone metabolites (iPdG). Urinary E(1)C, fecal E(1)C and fecal iPdG concentrations for seven mares which produced foals were 3.9 +/- 1.3 (SEM) mug/mg creatinine, 4.2 +/- 0.8 ng/g feces and 1.411 +/- 569.6 ng/g feces, respectively. Urinary E(1)C and fecal E(1)C and iPdG concentrations for the 33 mares which did not produce foals were 0.1 +/- 0.0 mug/mg creatinine and 0.5 +/- 0.1 and 32.8 +/- 4.5 ng/g feces, respectively. These differed (P < 0.01) from values in mares which produced foals.     

The equine frozen semen industry.

Recent acceptance of frozen semen as a method to produce registered foals by two of the worlds largest breed associations, the American Quarter Horse and American Paint Horse, has stimulated new interest in frozen semen technology. This review will: (a) attempt to identify the major impediments to the development of the frozen semen industry, (b) suggest alternative methods for marketing and application of frozen semen, and (c) present the results of a recent study in our laboratory. The objective of which was to compare pregnancy rates of insemination with cooled and frozen semen. Major impediments to the development of the frozen semen industry include 1. Lower fertility with frozen semen as compared to cooled semen for many stallions. 2. Increased costs associated with management of mares for AI with frozen semen using current insemination protocols. 3. Unfavorable marketing practices for frozen semen. Reports of fertility with cooled transported semen in commercial breeding programs indicate seasonal pregnancy rates ranging from 60 to 90%. We compiled data from three commercial transported cooled semen programs in which semen from 16 stallions was used for insemination of 850 mares throughout North America by local veterinarians. During the 1999 and 2000 breeding seasons, first cycle and seasonal pregnancy rates of 59.4 and 74.7% were obtained. During that same period, first cycle and seasonal pregnancy rates of 51.3 and 75.6% were obtained following insemination of 876 mares with frozen semen from 106 different stallions processed by our laboratory and distributed through our commercial distribution program. First cycle and seasonal pregnancy rates were higher for mares bred outside of North America than for mares bred within North America (53.5 and 81.9 versus 49.4 and 65.6%, respectively). Seasonal pregnancy rates were higher presumably because of the better mare management employed for mares bred with exported semen and the fact that some of the domestic mares were switched to cooled semen insemination after a failed first cycle attempt with frozen semen. These data support the position that comparable seasonal pregnancy rates may be obtained using frozen and liquid cooled semen in a commercial setting.     

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.     

Administration of oxytocin immediately after insemination does not improve pregnancy rates in mares bred by fertile or subfertile stallions

It is probable that reduced pregnancy rates in mares bred to subfertile stallions is attributable, in part, to the reduced number of normal spermatozoa that colonize the oviduct. Administration of oxytocin stimulates both uterine and oviductal contractility. The hypothesis that oxytocin may enhance sperm transport to/into the oviducts, and thereby increase pregnancy rates, was tested in 2 trials. For both trials, fertile estrous mares with follicles > or = 35 mm in diameter were inseminated once at 24 h after administration of 1500 to 2000 U hCG. The inseminate dose was limited to 100 million spermatozoa in order to lower pregnancy rates and thus increase the chance of detecting a treatment effect. Pregnancy status was determined by transrectal ultrasound examination 14 to 16 d after insemination. In Trial 1, 49 mares were inseminated with 4 mL extended semen from 1 of 3 stallions (1 fertile and 2 subfertile males). Immediately after insemination, the mares were administered either 20 U oxytocin or 1 mL saline intravenously. In Trial 2, 51 mares were inseminated with 4 mL extended semen from 1 of 4 stallions (1 fertile and 1 subfertile male used in Trial 1, and 2 additional fertile males). Immediately after insemination, and again 30 min later, mares were administered either 5 U oxytocin or 0.25 mL saline intramuscularly. To test for effects of treatment with oxytocin and for the interaction between semen quality and treatment, a generalized linear mixed regression model was used that accounted for the split-plot design (treatment within stallions), the random effect of stallion, the fixed effect of semen quality, the binary outcome of a single breeding trial, and the varying number of trials per stallion/treatment groups. Three treatment protocols or regimens were used: placebo, 5 U oxytocin injected twice intramuscularly, and 20 units oxytocin injected twice intravenously. Semen was classified as high (fertile stallions) or low (subfertile stallions) quality. No interaction between semen quality and treatment was detected (P > 0.10). The pregnancy rate of mares treated with oxytocin immediately after insemination was 30% (15/50) compared with 50% (25/50) for mares treated with saline immediately after breeding. Administration of oxytocin did not affect pregnancy rates (P > 0.10).     

Luteolysis and cycle synchronization with a new prostaglandin analog for artificial insemination in the mare

Over a period of three years, 165 cyclic or anestrous Hanoverian mares received 177 treatments with 2, 3 or 4 mg of a novel luteolytic prostaglandin analog, K 11941. Heat and ovulations, indicating luteolysis, were observed after an average of 3.98 and 7.62 days, respectively, in 88.04% of 142 treated cyclic, postpartum and anestrous mares, and mares after an early loss of the conceptus. All doses tested were effective in inducing luteolysis as confirmed by determination of progesterone blood levels in samples collected daily, in 70 of 80 mares studied (87.5%). Attempts to achieve control of the cycle by various methods revealed that with K 11941 given once or twice, alone or in combination with hCG and/or an GnRH analog (Hoe 766), one can effectively concentrate estrus periods and follicular growth patterns, but can neither synchronize nor concentrate ovulations. Since most of the mares treated were confirmed problem mares, the pregnancy rate of 40.0% from first insemination at drug induced estrus, was regarded as satisfactory when compared to a pregnancy rate of 43.8% obtained with natural breeding in the same population. No drug related clinical signs of side-effects were observed.