Equine oocyte competence after in vitro maturation (IVM) was investigated in terms of the diameter of the follicle of origin and the stage of the estrous cycle, with three criteria of maturation: nuclear stage after DNA Hoechst staining, meiotic spindle morphology after tubulin immunocytochemical staining, and cortical granule localization after lectin labeling. Seven successive in vivo ultrasound-guided follicular punctures were performed on 10 cyclic saddle mares, alternatively at the end of the follicular phase (after induction of ovulation with a gonadotropin injection) and in midluteal phase (with or without a gonadotropin injection). Expanded cumulus-oocyte complexes (COCs) were stained at collection, and compact COCs were stained after in vitro culture. They were observed under a confocal microscope. Successive punctures on one mare provided 0.9 preovulatory COCs and 8 immature COCs per 22 days. Among the preovulatory oocytes, 55% had completed nuclear and cytoplasmic maturation, 86% of which displayed a normal meiotic spindle. Of the 262 oocytes cultured in vitro, 37% completed nuclear maturation. The nuclear and cytoplasmic maturation rate significantly increased with follicle diameter. The IVM rate tended to be higher in follicular phase and tended to increase in luteal phase with the gonadotropin injection. The meiotic spindle morphology was not significantly different between the classes of follicular diameters. This study provided the opportunity to increase the number of characterized oocytes collected per cycle and per mare. This is the first report showing the progressive acquisition of meiotic competence in the equine oocyte during antral follicle growth and is the only description of the equine meiotic spindle.

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Follicular development and ovulatory processes in mammals involve local biochemical changes as a result of substantial modifications in cellular metabolism, the most well known of which is steroid variation. In the present study, the intrafollicular variation of several other components was studied using proton nuclear magnetic resonance ((1)H NMR). This approach made it possible to demonstrate that the intrafollicular biochemical content changes during follicular growth and maturation. Follicular fluid was aspirated by ovarian puncture of the dominant follicle at various physiological stages of its development: early dominant, late dominant and preovulatory. Serum samples were collected during each puncture session. (1)H NMR was used to evaluate intrafollicular and circulating glycoconjugates (sugar chains and N-acetyl groups), lipoproteins (CH(3) and CH(2) groups), glucose metabolites (trimethylamines, acetate and lactate), amino acids (glutamine/glutamate and alanine), creatine/creatinine and polyamines. Follicular fluids were assayed by radioimmunoassay for oestradiol and progesterone contents. The intrafollicular contents of alanine and lipoproteins (CH(3) groups) decreased in the dominant follicle during growth, whereas concentrations of progesterone and oestradiol increased significantly. After injection of gonadotrophin to induce ovulation, follicular maturation was characterized by a decrease in glycoconjugates (sugar chains), trimethylamines and acetate, a decrease in oestradiol concentration, and a further increase in CH(3) groups of lipoproteins and progesterone. The results from the present study showed a clear correlation between the intrafollicular content of alanine and that of oestradiol. A correlation between progesterone and glycoconjugates (sugar chains) was also observed. Therefore, (1)H NMR was shown to be effective for studying specific changes in the biochemical composition of the follicular fluid that occur during follicular development. For the first time, the variation of several compounds (glycoconjugates, lipoproteins, glucose metabolites, amino acids and polyamines) in relation to growth and maturation was demonstrated. Some of these changes could be of crucial importance for follicular maturation and ovulation as well as for oocyte maturation and further fertilization.

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BackgroundOviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse.Methods & resultsIn the first experiment, we co-incubated fresh equine spermatozoa treated with calcium ionophore and in vitro matured equine oocytes with or without porcine OEC. We showed that the presence of OEC increases the IVF rates. In the subsequent experiments, we co-incubated equine gametes with OEC and we showed that the IVF rates were not significantly different between 1) gametes co-incubated with equine vs porcine OEC, 2) intact cumulus-oocyte complexes vs denuded oocytes, 3) OEC previously stimulated with human Chorionic Gonadotropin, Luteinizing Hormone and/or oestradiol vs non stimulated OEC, 4) in vivo vs in vitro matured oocytes.In order to identify the proteins responsible for the positive effect of OEC, we first searched for the presence of the genes encoding oviductin, osteopontin and atrial natriuretic peptide A (ANP A) in the equine genome. We showed that the genes coding for osteopontin and ANP A are present. But the one for oviductin either has become a pseudogene during evolution of horse genome or has been not well annotated in horse genome sequence. We then showed that osteopontin and ANP A proteins are present in the equine oviduct using a surface plasmon resonance biosensor, and we analyzed their expression during oestrus cycle by Western blot. Finally, we co-incubated equine gametes with or without purified osteopontin or synthesized ANP A. No significant effect of osteopontin or ANP A was observed, though osteopontin slightly increased the IVF rates.ConclusionOur study shows a beneficial effect of homologous and heterologous oviduct cells on equine IVF rates, though the rates remain low. Furthers studies are necessary to identify the proteins involved. We showed that the surface plasmon resonance technique is efficient and powerful to analyze molecular interactions during fertilization.

Transvaginal ultrasound-guided follicular aspiration was used to study oocyte yield and subsequent ovarian response in mares. In experiment 1, weekly puncture of follicles a 8 mm (an average of 4.3 follicles per mare) produced an average of 0.8 oocytes per mare per week. More follicles were found in saddle mares (5.8) than in pony mares (2.9), and thus saddle mares yielded more oocytes (1.0 vs. 0.6). Compact cumulus surrounded 95% of oocytes recovered from follicles s 20 mm but only 57% of those recovered from follicles > 20 mm. During 11 successive attempts, saddle mares had a linear decrease in the number of follicles. In addition to this general tendency, individual mares showed, from one week to the other, an alternating pattern of either a large follicle (> 20 mm) with several small follicles > 10 mm or no follicle > 20 mm with fewer small follicles. This could be explained by waves of follicular growth. In experiment 2, the puncture in midluteal phase of an average of 5.4 follicles in saddle mares and 3.5 in pony mares produced an average of 1.7 and 1.0 oocytes, respectively. In experiment 3, where successive punctures were performed at 2-5-day intervals, 97% of oocytes obtained at the first puncture had a compact cumulus, but only 74% of those obtained at subsequent punctures did. The percentage of compact cumulus was lower (41% vs. 90%) with a shorter interval (2 days vs. 4-5 days). The suspected cause of this altered morphology was the successive punctures of a follicle, followed by recovery of the oocyte on the second puncture. In experiment 4, the replacement of follicular fluid with PBS, followed 48 h later by oocyte collection, produced oocytes with an expanded cumulus and resumed meiosis, confirming the interpretation of experiment 3.

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In equids, placentation is diffuse and nutrient supply to the fetus is determined by uterine size. This correlates with maternal size and affects intra-uterine development and subsequent post-natal growth, as well as insulin sensitivity in the newborn. Long-term effects remain to be described. In this study, fetal growth was enhanced or restricted through ET using pony (P), saddlebred (S) and draft (D) horses. Control P-P (n = 21) and S-S (n = 28) pregnancies were obtained by AI. Enhanced and restricted pregnancies were obtained by transferring P or S embryos into D mares (P-D, n = 6 and S-D, n = 8) or S embryos into P mares (S-P, n = 6), respectively. Control and experimental foals were raised by their dams and recipient mothers, respectively. Weight gain, growth hormones and glucose homeostasis were investigated in the foals from birth to weaning. Fetal growth was enhanced in P-D and these foals remained consistently heavier, with reduced T3 concentrations until weaning compared to P-P. P-D had lower fasting glucose from days 30 to 200 and higher insulin secretion than P-P after IVGTT on day 3. Euglycemic clamps in the immediate post-weaning period revealed no difference in insulin sensitivity between P-D and P-P. Fetal growth was restricted in S-P and these foals remained consistently lighter until weaning compared to S-D, with elevated T3 concentrations in the newborn compared to S-S. S-P exhibited higher fasting glycemia than S-S and S-D from days 30 to 200. They had higher maximum increment in plasma glucose than S-D after IVGTT on day 3 and clamps on day 200 demonstrated higher insulin sensitivity compared to S-D. Neither the restricted nor the enhanced fetal environment affected IGF-1 concentrations. Thus, enhanced and restricted fetal and post-natal environments had combined effects that persisted until weaning. They induced different adaptive responses in post-natal glucose metabolism: an early insulin-resistance was induced in enhanced P-D, while S-P developed increased insulin sensitivity.