W. R. Allen

Reproductive efficiency of intensively managed Thoroughbred mares in Newmarket

The findings of a retrospective survey of 1393 Thoroughbred mares visiting 22 studfarms in the Newmarket region of the UK during the 1998 mating season were compared with those of a similar study undertaken in 1983. The effects of mare age and status, stallion, month of mating, application of uterine treatments and other parameters on the rates of singleton and twin conception and subsequent pregnancy losses were analysed. Mare age and status significantly affected the per cycle pregnancy rate and the incidence of pregnancy loss. Overall, the mean number of matings per oestrus was 1.12 and the mean number of times a mare was mated until diagnosed pregnant at 15 days after ovulation was 1.88. An overall mean per cycle pregnancy rate of 59.9% at 15 days after ovulation resulted in 94.8% of the mated mares being pregnant at least once at 15 days after ovulation. This high initial pregnancy rate fell to 89.7% by Day 35 and 87.5% by the time of the October pregnancy test; 82.7% of the mares surveyed gave birth to a live foal at term, which compares favourably with the proportion of mares foaling in 1983 (77%). However, despite improvements in the foaling rates over the last 15 years, the overall rate of pregnancy failure remains high and represents a major loss to the Thoroughbred breeding industry.

Monitoring the fate of autologous and allogeneic mesenchymal progenitor cells injected into the superficial digital flexor tendon of horses: Preliminary study

Autologous mesenchymal progenitor cells (MPCs) purified from bone marrow aspirates are being used in the treatment of superficial digital flexor tendon (SDFT) injuries in the horse with promising results. In this study the fate of autologous and allogeneic MPCs following injection into the SDFT was monitored by stable transfection of MPCs with green fluorescent protein (GFP). Small lesions were created manually in one forelimb SDFT of 2 horses and injected with autologous MPCs, allogeneic MPCs or bone marrow supernatant alone. Post mortem examinations performed after 10 or 34 days revealed GFP labelled cells located mainly within injected lesions, but with a small proportion integrated into the crimp pattern of adjacent healthy areas of tendon. Furthermore, there was no visible cell mediated immune response to allogeneic MPCs in either of the host horses.

Hysteroscopic insemination of small numbers of spermatozoa at the uterotubal junction of preovulatory mares.

Mares were inseminated with motile spermatozoa suspended in 30-150 microliters Tyrodes medium directly onto the uterotubal papilla at the anterior tip of the uterine horn, ipsilateral to the ovary containing a dominant preovulatory follicle of > or = 35 mm in diameter, by means of a fine gamete intrafallopian transfer (GIFT) catheter passed through the working channel of a strobed light videoendoscope. Insemination of 10, 8, 25, 14, 11 and 10 mares with, respectively, 10.0, 5.0, 1.0, 0.5, 0.1 or 0.001 x 10(6) motile spermatozoa resulted in conception rates of, respectively, 60, 75, 64, 29, 22 and 10%. Deposition of 1.0 x 10(6) motile spermatozoa onto the uterotubal papilla began to approach the limit of successful fertilization. These doses are far lower than the 3-15 x 10(9) spermatozoa normally ejaculated by fertile stallions during mating, and the accepted minimum dose of 500 x 10(6) spermatozoa used for conventional uterine body insemination in mares. The simplicity of the technique offers a practical means of exploiting new breeding technologies that require very small numbers of spermatozoa in horse breeding.

Reproductive efficiency of Flatrace and National Hunt Thoroughbred mares and stallions in England

REASONS FOR PERFORMING STUDY Previous surveys of reproductive efficiency in British Thoroughbreds included only mares and stallions standing on studfarms in and around Newmarket. The present study was widened to compare Flatrace (FR) (Group A) and National Hunt (NH) (Group B) mares and stallions on studfarms throughout England. OBJECTIVES To assess the influences of mare type, status and age, and veterinary manipulations on reproductive efficiency parameters. To compare the inherent fertility of stallions, based on singleton and twin pregnancy rates and pregnancy loss rates, in Groups A and B Thoroughbred breeding stock. METHODS Managers of 24 FR and 9 NH public studfarms were asked to complete a questionnaire for each mated oestrous cycle shown by 2321 Group A and 1052 Group B mares throughout the 2002 mating season. Parameters such as per cycle singleton and twin pregnancy rates, and pregnancy loss rates were noted, and the success of hormone treatments to induce oestrus and ovulation assessed. The number of matings per oestrus and per pregnancy were recorded, together with the incidence and effectiveness of uterine and other veterinary treatments. The inherent fertility of 84 Group A and 43 Group B stallions in the study, as measured by the singleton and twin early pregnancy rates and the pregnancy loss rates recorded in the mares they mated, was also estimated. RESULTS Per cycle early pregnancy (Days 13-16) was 63.2% for Group A and 65.3% for Group B mares; and 10.3% and 13.1%, respectively, of those pregnancies were twins or triplets. Early, middle and late pregnancy loss rates were 7.2% vs. 8.0% (Days 15-42), 3.6% vs. 6.1% (Days 42-1st October) and 2.7% vs. 2.1% (October-foaling), respectively. Matings per oestrus and per early pregnancy were significantly higher in Group B vs. Group A mares. For stallions that mated > or = 30 mares, overall early pregnancy rates per cycle in mares mated ranged from 30-89% across the 2 groups. CONCLUSIONS No major differences in reproductive efficiency were identified between FR and NH mares and stallions. Increasing mare age was the single biggest limiting factor to an otherwise high rate of fertility in well-managed English Thoroughbreds. POTENTIAL RELEVANCE This study identified factors that influence reproductive efficiency in the Thoroughbred.

Pregnancy rates in mares after a single fixed time hysteroscopic insemination of low numbers of frozen‐thawed spermatozoa onto the uterotubal junction

REASONS FOR PERFORMING STUDY To compensate for the wide variation in the freezability of stallion spermatozoa, it has become common veterinary practice to carry out repeated ultrasonography of the ovaries of oestrous mares in order to be able to inseminate them within 6-12 h of ovulation with a minimum of 300-500 x 10(6) frozen-thawed spermatozoa. Furthermore, in order to achieve satisfactory fertility, this requirement for relatively high numbers of spermatozoa currently limits our ability to exploit recently available artificial breeding technologies, such as sex-sorted semen, for which only 5-20 x 10(6) spermatozoa are available for insemination. OBJECTIVES This study was designed to evaluate and compare the efficacy of hysteroscopic vs. conventional insemination when low numbers of spermatozoa are used at a single fixed time after administration of an ovulation-inducing agent. METHODS In the present study, pregnancy rates were compared in 86 mares inseminated once only with low numbers of frozen-thawed spermatozoa (3-14 x 10(6)) at 32 h after treatment with human chorionic gonadotrophin (hCG), either conventionally into the body of the uterus or hysteroscopically by depositing a small volume of the inseminate directly onto the uterotubal papilla ipsilateral to the ovary containing the pre-ovulatory follicle. RESULTS Pregnancy rates were similarly high in mares inseminated conventionally or hysteroscopically with 14 x 10(6) motile frozen-thawed spermatozoa (67% vs. 64%). However, when the insemination dose was reduced to 3 x 10(6) spermatozoa, the pregnancy rate was significantly higher in the mares inseminated hysteroscopically onto the uterotubal junction compared to those inseminated into the uterine body (47 vs. 15%, P < 0.05). CONCLUSIONS When inseminating mares with <10 x 10(6) frozen-thawed stallion spermatozoa, hysteroscopic uterotubal junction deposition of the inseminate is the preferred method. POTENTIAL CLINICAL RELEVANCE Satisfactory pregnancy rates are achievable after insemination of mares with frozen-thawed semen from fertile stallions 32 h after administration of human chorionic gonadotrophin (Chorulon). Furthermore, these results were obtained when mares were inseminated with 14 x 10(6) progressively motile frozen-thawed spermatozoa from 2 stallions of proven fertility.

The effects of maternal health and body condition on the endocrine responses of neonatal foals

REASONS FOR PERFORMING THE STUDY Chronic and acute alterations in maternal nutrient intake during pregnancy alter pancreatic and hypothalamo-pituitary-adrenal (HPA) axis function in the offspring, before and after birth. Little is known about these effects. OBJECTIVE To determine whether maternal nutrient restriction caused by natural infection with Streptococcus equi altered endocrine function in neonatal foals born from mares fed a maintenance or high plane of nutrition throughout pregnancy. METHODS Ten primiparous mares received either a diet to maintain moderate body condition score (Moderate, n = 5) or a near ad libitum feeding regime to maintain a high body condition score (High, n = 5) throughout pregnancy. All mares inadvertently became infected with Streptococcus equi in mid gestation and lost approximately 10% body mass. RESULTS Maternal insulin and glucose concentrations decreased (P < 0.05) during, and one month following, the weight loss period. High mares weighed more (P < 0.05) at parturition than Moderate mares; all foals were healthy. Gestational age, foal bodyweights, placental and clinical parameters after birth were no different between the 2 groups. Foal plasma cortisol and glucose responses to exogenous adrenocorticotrophic hormone and insulin, respectively, were similar for both groups. Insulin concentrations during glucose tolerance test were significantly higher (P < 0.05) in foals from Moderate than High mares and compared with foals studied previously from healthy, well-fed mares, suggesting that the beta cell sensitivity to glucose was enhanced in Moderate. CONCLUSION Acute nutrient restriction in mid gestation caused by maternal illness and inappetence, superimposed on a maintenance feed intake throughout pregnancy, enhanced insulin secretion to glucose in foals. Nutritional programming of pancreatic beta cells, but not the HPA axis, appeared to depend on the level of nutrition before and after the weight loss period. POTENTIAL RELEVANCE Disturbances in neonatal pancreatic beta cell function programmed during pregnancy may predispose foals to metabolic problems in later life.

Effects of a Streptococcus equi infection‐mediated nutritional insult during mid‐gestation in primiparous Thoroughbred fillies. Part 1: Placental and fetal development

REASONS FOR PERFORMING STUDY There is a paucity of information on factors that influence placental development in the horse. HYPOTHESIS Changes in nutrition, particularly around the time of proliferative placental growth, can affect development of the placenta and fetal growth. OBJECTIVE To investigate the effects of 2 planes of nutrition and an unforeseen infection-mediated nutritional insult on placental and fetal development in the mare. METHODS Twenty maiden Thoroughbred fillies, age 3 or 4 years, mated to one Thoroughbred stallion, were maintained on either High or Moderate food intake throughout pregnancy. In mid-gestation all the mares unexpectedly became infected with Streptococcus equi and suffered varying degrees of weight loss as a consequence. Gross and stereological measurements of the placenta and measurements of foal birthweight, crown-rump length, ponderal index and plasma IGF-1 concentration were made at term. RESULTS Gains in bodyweight during gestation were significantly higher in the High vs. the Moderate nutrition groups. Placental and fetal growth parameters were not influenced by the plane of nutrition. However, transient weight loss in mid-gestation from acutely reduced food intake resulted in morphological changes to the allantochorion and decreased foal birthweight. CONCLUSIONS Excessive weight gain during gestation in maiden Thoroughbred fillies does not result in nutrient partitioning to the dam at the expense of the fetus. However, sudden weight loss in mid-gestation has detrimental effects on placental development which results in reduced fetal growth. POTENTIAL RELEVANCE Clinicians should appreciate the likely effects of maternal infection and resulting weight loss on pregnancy outcome.

Laparoscopic application of PGE2 to re‐establish oviducal patency and fertility in infertile mares: a preliminary study

REASONS FOR PERFORMING STUDY Mares are occasionally encountered that consistently fail to conceive when inseminated, naturally or artificially, with fertile stallion semen in the absence of any identifiable pathology of either the structure or function of their reproductive tract. HYPOTHESIS Temporary blockage of the oviducts by accumulations of naturally occurring oviducal masses may be preventing oviducal transport of the embryo to the uterus. METHODS Mares, with known reproductive histories, that had exhibited inexplicable failure of conception were treated by laparoscopically guided administration of PGE2-laced triacetin gel directly onto the surface of their oviducts. RESULTS Fifteen mares age 10-21 years that had exhibited inexplicable failure of conception during 1-4 years were treated, of which 14 (93%) conceived within the same or subsequent breeding season. CONCLUSIONS The high success rate of this treatment supports the tentative diagnosis of oviducal obstruction in these mares and indicates that blockage of the mares oviducts may occur in the form of a moveable accumulation of debris rather than from permanent fibrous adhesions resulting from salpingitis. POTENTIAL RELEVANCE This laparoscopic application of PGE2 to the oviducts constitutes a sound and practical method of restoring fertility in mares suffering oviducal obstruction and further studies involving the procedure are warranted.

Maternal dexamethasone treatment in late gestation induces precocious fetal maturation and delivery in healthy Thoroughbred mares

REASONS FOR PERFORMING STUDY The foal requires an active hypothalamo-pituitary-adrenal (HPA) axis for organ maturation and post natal survival. Prenatal administration of synthetic glucocorticoids may provide an effective method for inducing fetal maturation safely in the mare. OBJECTIVES To determine whether dexamethasone administered to late pregnant mares: 1) will induce fetal maturation and precocious delivery; 2) is safe to use and 3) to identify endocrine responses in the mare and foal. METHODS Pregnant Thoroughbred mares received either 100 mg dexamethasone i.m. (treated n = 5) or 50 ml saline i.m. (control n = 5) at 315, 316 and 317 days of gestation. Plasma progestagens, cortisol and prostaglandin F(2α) metabolite (PGFM) concentrations were measured before and after treatment. The foals were weighed, the crown-rump length (CRL) measured and an adrenal stimulation test performed on Day 1. RESULTS Dexamethasone significantly (P<0.01) reduced gestation length in treated mares without apparent adverse effects. Plasma progestagens increased (P<0.05), and cortisol and PGFM (P<0.05) decreased, following dexamethasone treatment compared with control mares. Foals were clinically mature but those from dexamethasone treated mares had reduced (P<0.05) CRL, but not bodyweights, compared with controls. Their cortisol concentrations increased following exogenous adrenocorticotrophic hormone stimulation but 2 foals from dexamethasone treated mares showed evidence of adrenal suppression. CONCLUSIONS Dexamethasone stimulates precocious fetal maturation and delivery in healthy late pregnant mares. However, fetal HPA activity may be suppressed. POTENTIAL RELEVANCE Dexamethasone treatment could be used to improve foal viability in mares at risk of preterm delivery. The endocrine effects of such a therapy must be evaluated before clinical intervention with glucocorticoids can be recommended.

Liposome-mediated uptake of exogenous DNA by equine spermatozoa and applications in sperm-mediated gene transfer.

REASON FOR PERFORMING STUDY Sperm-mediated gene transfer has been reported as a method for production of transgenic animals in a variety of species, and this technique represents a possible method for production of transgenic equids. OBJECTIVES To evaluate the uptake of exogenous DNA (enhanced green fluorescent protein; pEGFP) by equine spermatozoa and to assess the ability of transfected spermatozoa to introduce this transgene into early equine embryos. METHODS To evaluate incorporation of pEGFP into equine spermatozoa, washed spermatozoa were incubated with 32P-pEGFP, with or without lipofection. Spermatozoa were also transfected with fluorescently-labelled DNA (Alexa647-pEGFP) and changes in sperm viability and DNA uptake were assessed. Mares were inseminated with pEGFP-transfected spermatozoa and embryos recovered. Expression of pEGFP was assessed by epifluorescence microscopy of embryos, and the presence of pEGFP DNA and mRNA was assessed by PCR and RT-PCR, respectively. RESULTS Liposome-mediated transfection increased the incorporation of 32P-pEGFP into spermatozoa compared to controls. Flow cytometric evaluation of spermatozoa after transfection with Alexa647-pEGFP revealed a linear increase in the proportion of live, Alexa647+ spermatozoa with increasing DNA concentrations. After insemination with transfected spermatozoa, 8 embryos were recovered. There was no evidence of EGFP expression in the recovered embryos; however, PCR analysis revealed evidence of the pEGFP transgene in 2 of 5 embryos analysed. CONCLUSIONS The incorporation of exogenous DNA by equine spermatozoa was enhanced by liposome-mediated transfection and this did not adversely affect sperm viability, acrosomal integrity or fertility. Although the EGFP transgene was detected in a proportion of Day 7-10 embryos, there was no evidence of expression of EGFP in these embryos. POTENTIAL RELEVANCE Sperm-mediated gene transfer offers a potential technique for the generation of transgenic equids.