Capacitation of stallion spermatozoa and fertilisation of equine oocytes in vitro

Summary Two experiments were conducted to: 1) determine the effects of 11 treatments on penetration ability of sperm incubated with zona-free hamster oocytes and frozen-thawed equine oocytes and 2) attempt in vitro fertilisation of in vivo-matured, equine oocytes using sperm treatments that effected greatest penetration in Experiment 1. Treatments evaluated in Experiment 1 included: 1) 1 μM calcium lonophore (A23187), 2) 1 uM A23187 + 5.0 mM caffeine, 3) 10 μM lysophosphatidyl serine (LPS), 4) 10 μM LPS + 5.0 mM caffeine, 5) 10 μM liposomes of phosphatidyl choline (PC 12), 6) 10 iu heparin/ml, 7) 10 iu heparin/ml + 10 μM LPS, 8) 50 μM penicillamine/hypotaurine/epinephrine (PHE), 9) 50 μM PHE + 10 μM LPS, 10) equine pre-ovulatory oviduct fluid diluted 1:1 in glucose-free, modified Tyrodes solution (TALP-g), and 11)TALP-g. Treatments were suspended in TALP-g; A23187 was in glucose-free, BSA-free TALP (TALP-g-BSA). Eight trials were done with five stallions. Oocytes were incubated with sperm immediately after sperm exposure to capacitation treatments 1 to 5. For treatments 6 to 11, oocytes were incubated with sperm after about 5 h of capacitation. For all treatments there were 2×106 sperm/ml and 10 hamster and eight equine oocytes/treatment/trial. Caffeine and LPS were added immediately and 45 mins prior to adding oocytes, respectively. Hamster oocytes were co-incubated 3.5 h and horse oocytes 5 h before examination for penetration. Greatest penetration was achieved using sperm treated with A23187, LPS, and PC12, in hamster assays (36 per cent, 46 per cent, and 33 per cent respectively) and equine assays (8 per cent, 16 per cent and 8 per cent respectively). Number of sperm penetrated per hamster egg was greater for these groups also. Sperm exposed to equine oocytes showed lower penetration rates regardless of treatment; however, similar treatment effects between the two assays suggest a predictive value of the zona-free hamster ova test regarding stallion sperm ability to penetrate homologous zonae. Prolonged exposure of stallion sperm to PC12 may allow capacitation of higher concentrations of sperm by lower levels of PC12 than previously reported. LPS addition to heparin-treated sperm improved penetration rate of hamster vitelli over that of sperm treated with heparin alone. Caffeine addition decreased the ability of ionophore-treated sperm to penetrate hamster vitelli and equine zonae pellucidae. Twenty-six in vivo-matured equine oocytes were recovered via aspiration of pre-ovulatory follicles from hCG-treated mares. After incubation for 6 h in 15 per cent follicular fluid: 85 per cent Ham's F-10 + 10 per cent FCS, oocytes were incubated with sperm treated with either A23187 or LPS as in Experiment 1. Sperm were treated at 2 × 107ml; eggs were exposed to 2 × 106 sperm/ml for 8 h, transferred to Ham's F-10 + 10 per cent FCS and observed for evidence of fertilisation every 8 h for 48 h. Media pH was kept at 7.6 under paraffin oil in 5 per cent CO2 in air at 39°C. Of 26 in vivo-matured equine oocytes incubated in either A23187 or LPS-treated sperm (n=13/treatment), none exhibited cleavage or second polar body extrusion. Structures resembling two pronuclei were noted in one A23187-treated oocyte; a decondensed sperm head and metaphase II chromosomes were found in one LPS-treated ovum after fixation, staining and microscopic examination.