146 Improvement in hyperactivation, capacitation, and heterologous zona pellucida binding of cryopreserved equine spermatozoa after brief exposure to ionophore A23187

Spermatozoa must undergo three events: capacitation, hyperactivation, and acrosome reaction to fertilise an oocyte. In different species, like murine and bovine, the calcium ionophore A23187 has been used to study the in vitro acquisition of fertilising ability in spermatozoa. A23187 increases events related to capacitation and hyperactive motility without inducing PKA-activation (pPKA) or protein tyrosine phosphorylation (pTyr). Therefore, it has been used to increase the embryo production rate when combined with routine IVF protocols in these species. Given the difficulties in equine embryo production by the IVF method because conditions under which the spermatozoa acquire their fertilising ability in vitro are still unknown, this work aimed to study hyperactivation and capacitation induction in cryopreserved equine sperm after a brief exposure to A23187, as well as its ability to bind to the zona pellucida of bovine oocytes. Using cryopreserved semen samples from six different stallions, we first studied total motility by computer-assisted sperm analysis (CASA), where results showed that the spermatozoa were first immobilised by A23187 (1 μM, Treated) in comparison with sperm incubated in non-capacitating conditions (nontreated) (motile sperm: Treated = 27.6% ± 2.4%**, nontreated = 46.1% ± 6.1%; P < 0.01, n = 5) without affecting viability (live sperm evaluated by HOS test: Treated = 27.3% ± 4.2%, nontreated = 23.4% ± 2.6%; P > 0,05, n = 5) or acrosome status (reacted sperm evaluated by PSA-FITC and IF: Treated = 79.3% ± 2.7%, nontreated = 84.3% ± 2.8%; P > 0.05, n = 5), but they initiated hyperactive motility after ionophore wash by centrifugation and BSA addition. (Motility parameters studied by CASA: VCL: Treated = 153.7 µm/s ± 10.4 µm/s*, nontreated = 100.4 µm/s ± 10.9 µm/s; LIN: Treated = 45.2% ± 5.4%*, nontreated = 64.5% ± 3.1%; ALH: Treated = 7.5 µm ± 0.5 µm*, nontreated = 4.3 µm ± 0.5 µm; CASA; P < 0.05, n = 5.) Also, our results suggest that A23187 (1 μM) induces events associated with capacitation, such as an increase in acrosomal reaction induced by progesterone (reacted live sperm evaluated by PSA-FITC and IF: Treated = 10.2% ± 1.5%*, nontreated = 2.1% ± 2.4%; P < 0.05, n = 3) through a signalling pathway that would omit pPKA and pTyr activation (evaluated by IF: Treated = 85.2% ± 3.0%, nontreated = 80.0% ± 3.0%; P > 0.05; n = 3). Finally, the pre-incubation of spermatozoa with A23187 increased their ability to bind to the zona pellucida of mature bovine oocytes (studied by IF after heterologous IVF with sperm pre-incubated with Hoechst: Treated = 81.4 spz/oocyte ± 9.9 spz/oocyte***; nontreated = 25.2 spz/oocyte ± 2.8 spz/oocyte; P < 0.001, n = 7), suggesting a possible improvement in their fertilising potential. Taking into account our results, a brief exposure of cryopreserved equine spermatozoa to A23187 should be further studied as a possible method to increase acquisition of their in vitro fertilising ability.

We thank GeneTec by Ativet for providing semen and the funding support (FONCYT PICT 2020-1872).