Ten fertile feral mares and 6 domestic horses (4 fertile mares, 1 infertile mare, 1 gelding) were immunized with heat-solubilized pig zonae pellucidae by 4 injections equivalent to 2000 or 5000 zonae each at 2-4-week intervals and a booster injection of 20,000 zonae 6-10 months after the last of the initial inoculations. The immune response was reflected by high antibody levels as measured by an enzyme-linked immunosorbent assay (ELISA) using immobilized pig zona antigen. In-vivo inhibition of fertility occurred in 12 (86%) of the 14 fertile mares studied and persisted for a minimum of 7 months. Repeated mating of the fertile domestic mares resulted in conception when anti-pig zona antibody concentrations had decreased from initial peak absorbance ratios (greater than 1.0) to relatively lower levels (0.64 or less with one exception). An indirect immunofluorescence assay, revealed a considerably lower cross-reactive antibody titre with horse oocytes as compared to pig oocytes. Clinical, endocrinological and histological analyses of the ovaries and their function following regained fertility after immunization revealed no abnormalities. One mare remained infertile.

Summary On Day 7 of pregnancy, when the inner cell mass coalesces to one side of the blastocyst, a discontinuous layer of cells is present underlying the inner cell mass and isolated cells were found around the mural trophoblast. As early as Day 8, these cells that were scattered around the blastocyst cavity unite to form a continuous endodermal layer, resulting in a bilaminar blastocyst. The endodermal cells have slightly bulging apical surfaces towards the cavity of the blastocyst, but in many areas have flanges and projections that form complex basolateral compartments within the endoderm and especially between the endoderm and trophoblast. These cells also develop an extensive series of unusual apical ridges. With the development of the primitive streak and the trilaminar blastocyst, angiogenesis proceeds from the forming embryo into the yolk sac. At first the anastomotic vessels form a simple mesh, but by Day 20, erythroblasts are present in the newly formed sinus terminalis. The loss of the capsule (Day 21) and the development of the embryonic circulation establish the relationships of the definitive yolk sac. There is a bilaminar region with endodermal cells showing basolateral compartmentalisation and a specialised sinus terminalis region bordering the choriovitelline placenta. The largest endodermal cells are associated with the choriovitelline placenta, but a substantial cuboidal lining also covers the splanchnopleuric portion of the vascular yolk sac. The allantochorion rapidly assumes predominance in respiratory exchanges, and the choriovitelline placenta not only decreases in relative area but increases in thickness. However, the 3 major regions of the yolk sac persist for several weeks after their formation, and both the vasculature and especially the endodermal cells remain robust despite the decrease in size of the yolk sac. It is concluded that the pleomorphic endodermal cells of the equine yolk sac differentiate in response to local signals. By Day 25 the allantochorion is an increasingly significant exchange unit, and the continued robustness of the vascular portions of the yolk sac and especially its endodermal layer suggests that synthetic and metabolic activities of the endodermal cells will be the dominant function of the yolk sac subsequently.

Domestic ewes (Ovis aries) were immunised with porcine zonae pellucidae (pZP) or pZP conjugated to keyhole limpet haemocyanin (KLH) in adjuvant(s) to examine the feasibility of the species to serve as a model for further development of pZP-based vaccines in ungulates. Two immunisation groups were employed, with a third group receiving only adjuvant (n = 5 per group). Early in the study, oestrous activity was monitored by the use of a vasectomised ram fitted with a marking harness. Eventually, ewes were exposed to an intact ram for breeding. In addition, weekly serum and every-other-day faecal samples were collected to measure pZP antibodies and progesterone metabolite concentrations respectively. At the conclusion of the study, fecundity was established, and ovarian tissue was examined. Ewes immunised against pZP : KLH with adjuvant produced minimal antibody absorbance levels, displayed normal oestrous cycles, became pregnant upon introduction of the intact ram and exhibited normal ovarian histopathology. Ewes immunised against pZP with adjuvant produced high antibody absorbance levels, were acyclic following primary immunisation and were infertile. Examination of the ovarian tissue revealed atrophic changes that included: (1) the absence of growing follicles; (2) significant reduction in the number of primordial follicles; and (3) the presence of abnormal granulosa cell clusters lacking oocytes. Antisera displayed immunoreactivity to the major components of pZP, and immunohistochemical labelling of ovarian tissue showed specificity to the ZP. These data are the first generated in an ungulate species showing deleterious effects of pZP immunisation on folliculogenesis and oestrous cyclicity.

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The structure of the equine chorionic girdle between days 28 and 42 of gestation was examined. Of particular interest were differentiation of trophoblastic cells within the girdle, adhesion between girdle and endometrium, invasion and displacement of the uterine epithelium, and the nature of the endometrium when girdle cells migrate into it to form endometrial cup cells. The chorionic girdle, identified initially as a band of tall columnar cells, becomes a stratified columnar epithelium indented by clefts and pits. Adhesion to and penetration through the endometrial luminal epithelium are rapid and occur initially in very limited areas. Stromal invasion occurs as strands of contiguous trophoblast cells invade through the basal lamina. Only girdle cells that are adjacent to the basal lamina or have entered the endometrial stroma undergo hypertrophy and differentiate into cup cells. At the initiation of trophoblastic invasion, the luminal epithelium contains numerous, large, intraepithelial, granular lymphocytes; small lymphocytes then accumulate in the stroma, but by day 42 lymphocytes are largely confined to the periphery of the cup. Although adhesion of trophoblast to the endometrial surface is initiated by small groups of girdle cells on restricted areas of the endometrial folds, the area is then increased by new areas of adhesion and by expansion of the initial invasion. Areas of girdle cells that do not attach undergo necrosis, as do superficial portions of areas of invasion. Consequently the girdle cells that form cups may be a minority of the original population. It is suggested that the differentiation of girdle cells is closely programmed and that cells that do not reach the stroma become necrotic at the same time that endometrial cup cells are differentiating.