286 SPERM–OOCYTE INTERACTION DURING IN VITRO FERTILIZATION IN THE HORSE

Abstract

In vitro fertilization (IVF) has proven to be a surprisingly unsuccessful way of producing horse embryos. The aim of this study was to investigate the interaction between sperm and the cumulus oocyte complex (COC) during IVF. In experiment 1, three IVF conditions were tested: (A) COCs recovered from slaughtered mares were categorized with respect to cumulus morphology (C: compact, n = 86, or E: expanded, n = 55) and matured in TCM199 containing 0.01 IU/mL porcine FSH and equine LH (IVM); after IVM, the oocytes were denuded and those with a visible polar body were incubated with sperm (IVF) in the presence or absence of 150 ng/mL progesterone (P4) to induce the acrosome reaction (AR); (B) IVM oocytes from C-COCS were denuded (n = 52) or not (n = 67) before IVF in the presence of P4;; (C) in vivo-matured oocytes (n = 15) recovered by transvaginal ultrasound-guided aspiration from preovulatory follicles 32 h after the donor mare was treated with hCG, were fertilized in vitro in the presence of P4. In all cases, IVF was performed with frozen-thawed, Percoll-selected sperm from a single stallion, at a final concentration of 1 × 10⁶spermatozoa/ml in fertil-TALP for 20 h (Parrish et al., 1988 Biol. Reprod. 38, 1171–1180). In experiment 2, the possibility that semen cryopreservation or stallion critically influenced IVF was examined by incubating denuded IVM oocytes with fresh or frozen/thawed sperm from the same (fresh;; n = 17 for both C- and E-COCs and frozen-thawed; n = 12 and 21 for C and E-COCs, respectively) or one other stallion (Fresh;; n = 12 and 19 and frozen-thawed; n = 12 and 19 for C and E-COCs, respectively), in the presence of P4 for 20 h. In both experiments, the resulting sperm-oocyte complexes were fixed, permeabilized and labelled with fluorescein-conjugated peanut agglutinin (EY Laboratores, San Mateo, CA, USA) and ethidium homodimer (Molecular Probes, Eugene, OR, USA) to stain the acrosomal membrane and DNA, respectively, so that membrane status and position of the sperm within the oocyte investments could be detected by confocal laser scanning microscopy. The total number of sperm bound per oocyte was compared between treatments using one-way ANOVA with pair-wise multiple comparison (Bonferroni t-test). Despite binding to the zona pellucida (ZP), neither fresh nor frozen/thawed sperm from either stallion acrosome-reacted or penetrated any oocytes, irrespective of cumulus morphology at the onset of IVM, denudation prior to IVF or the presence of P4. However, more sperm bound to the ZP of cumulus-denuded IVM oocytes (65 ± 32 and 62 ± 28 [mean ± sd] for C and E-COCs, respectively), than cumulus-intact IVM (5 ± 4) or in vivo-matured oocytes (23 ± 17: P < 0.001). None of the other factors investigated affected bound sperm numbers. In all cases, ZP-bound sperm failed to AR in the classical fashion, and all oocytes remained arrested at the MII stage. In summary, fertilization failed because sperm did not acrosome-react after binding to the ZP. It is concluded that failure to adequately activate stallion sperm is an important obstacle to successful IVF in horses.