Embryonic quality and survival in the horse: maternal and intrinsic aspects

Early pregnancy loss is a poorly understood but significant cause of economic loss to the equine breeding industry. The majority of equine pregnancy losses occur during the first five weeks of pregnancy, a period characterised by intense fetal-maternal interaction, and known predispositions to reduced embryonic survival include advanced maternal age and in vitro embryo production (IVP). Although a reduction in the quality and/or quantity of oocyte mitochondria has been proposed to contribute to an age-related decline in female fertility in a variety of species, this hypothesis has yet to be proven. In this thesis, we demonstrated that the mitochondria in oocytes from aged mares are more susceptible to damage than those from young mares; using quantitative PCR and transmission electron microscopy we showed that the quantity and quality of mitochondria in oocytes from aged, but not young, mares decrease during maturation in vitro. Moreover, while day 7 blastocysts from young mares contained significantly more mitochondrial DNA copies than pre-ovulatory oocytes, day 7 embryos produced in vitro or recovered from aged mares did not show a similar increase, at least in part because they were developmentally retarded. The observed age and IVP-related decreases in embryo development and quality are likely to result in decreased developmental competence. One potential consequence of oocyte mitochondrial dysfunction is aberrant meiotic spindle function leading to abnormal chromosome disjunction and, as a result, embryonic chromosomal abnormalities. In women, the incidence of embryonic chromosomal abnormalities increases with advancing maternal age and is associated with a marked decline in fertility. Although embryonic chromosomal abnormalities had also been proposed as a significant contributor to early pregnancy loss in the horse, the occurrence of chromosomally abnormal equine embryos had not previously been documented. This thesis includes a study in which fluorescent chromosome specific DNA probes were used to demonstrate that chromosomal abnormalities not only occur in equine embryos, but that their incidence tends to be higher if the embryo is produced in vitro. Correct and properly timed interaction between the developing embryo and the maternal organism is also critical to the establishment and maintenance of pregnancy. Although it has long been clear that maternal circulating progesterone is essential for pregnancy maintenance in the mare, it has been assumed that the effects of progesterone on the developing conceptus were indirect and mediated via the endometrium. In this thesis, we detail gene expression and immunohistochemical studies that demonstrate that early equine embryos express receptors for progesterone and oestrogens, thereby suggesting that these reproductive steroids may also influence embryonic development directly. However, the single most important aspect of fetal-maternal communication during early pregnancy is the production and timely release of the maternal recognition of pregnancy (MRP) signal, i.e. the conceptus factor responsible for prolonging the lifespan of the primary corpus luteum beyond its normal 14-16 days. In this thesis, we examined and rejected the hypothesis that conceptus insulin is the equine MRP signal because chronic administration of insulin to cycling mares during the MRP period neither delayed luteolysis or suppressed the oxytocin-induced luteolytic pathway; moreover, insulin was not present in measurable quantities in the yolk sac fluid of day 10-14 conceptuses

Gastrulation and the establishment of the three germ layers in the early horse conceptus.

Experimental studies and field surveys suggest that embryonic loss during the first 6 weeks of gestation is a common occurrence in the mare. During the first 2 weeks of development, a number of important cell differentiation events must occur to yield a viable embryo proper containing all three major germ layers (ectoderm, mesoderm, and endoderm). Because formation of the mesoderm and primitive streak are critical to the development of the embryo proper, but have not been described extensively in the horse, we examined tissue development and differentiation in early horse conceptuses using a combination of stereomicroscopy, light microscopy, and immunohistochemistry. Ingression of epiblast cells to form the mesoderm was first observed on day 12 after ovulation; by Day 18 the conceptus had completed a series of differentiation events and morphologic changes that yielded an embryo proper with a functional circulation. While mesoderm precursor cells were present from Day 12 after ovulation, vimentin expression was not detectable until Day 14, suggesting that initial differentiation of mesoderm from the epiblast in the horse is independent of this intermediate filament protein, a situation that contrasts with other domestic species. Development of the other major embryonic germ layers was similar to other species. For example, ectodermal cells expressed cytokeratins, and there was a clear demarcation in staining intensity between embryonic ectoderm and trophectoderm. Hypoblast showed clear α1-fetoprotein expression from as early as Day 10 after ovulation, and seemed to be the only source of α1-fetoprotein in the early conceptus

Advancing maternal age predisposes to mitochondrial damage and loss during maturation of equine oocytes invitro

In many mammalian species, reproductive success decreases with maternal age. One proposed contributor to this age-related decrease in fertility is a reduction in the quantity or functionality of mitochondria in oocytes. This study examined whether maternal age or (in vitro maturation). IVM affect the quantity of mitochondria in equine oocytes. Oocytes were collected from the ovaries of slaughtered mares categorized as young (<12years) or aged (≥12years) and either denuded and prepared for analysis immediately (not-IVM) or matured invitro for 30hours before preparation (IVM). The mean oocyte mitochondrial DNA copy number was estimated by quantitative polymerase chain reaction and found to be significantly lower in oocytes from aged mares and that had been subjected to IVM than in any other group. Transmission electron microscopy demonstrated that mitochondria in aged mare oocytes subjected to IVM experienced significantly more swelling and loss of cristae than in other groups. We conclude that maternal aging is associated with a heightened susceptibility to mitochondrial damage and loss in equine oocytes, which manifests during IVM. This predisposition to mitochondrial degeneration probably contributes to reduced fertility in aged mares