91 Fertilising capacity of guinea pig spermatozoa by heterologous fertilisation with zona-intact murine oocytes

The guinea pig is an important meat production species in South America and a valuable animal model for the study of reproduction in humans and mammals. In vitro fertilisation (IVF) in this species is poorly developed mainly because of the limited accessibility to homologous (Ho) oocytes outside of South America. Thus, heterologous (He) IVF represents an alternative to improve the procedure. We aimed to evaluate the fertilising capacity of guinea pig sperm using two capacitation protocols in He IVF with murine oocytes. Spermatozoa were collected from the vas deferens of three guinea pigs and processed separately using two protocols: (A) spermatozoa were isolated by flushing the lumen of the vas deferens with 2 mL of 0.15 m NaCl and the sperm suspension was washed twice by centrifugation at 600 × g for 3 min and then incubated in minimal culture medium with 0.25 mM Na-pyruvate, 20.0 mM Na-lactate, and 5.56 mM glucose (MCM-PLG), during 2 h at 5% CO₂ and 37°C; (B) the vas deferens was gently minced with fine scissors and spermatozoa suspended in 500 μL of HTF medium supplemented with 1% bopvine serum albumin (BSA), and incubated for 1 h at 5% CO₂ and 37°C. Zona-intact murine oocytes collected from superovulated female mice were used for He IVF with spermatozoa capacitated through protocol A (HeA, n = 285) or B (HeB, n = 296) into drops of 500 µL of MCM-PLG with 2.7 mM KCl and 0.3% BSA or HTF medium, respectively. In parallel, Ho IVF (n = 243) and parthenogenesis (non-fertilised oocytes, n = 75) was performed in HTF medium. In vitro-matured oocytes were co-incubated for 5 h with 1 × 10⁶ spermatozoa mL⁻¹ and then presumptive zygotes or (parthenogenetic) oocytes were cultured in KSOM medium at 37°C and 5% CO₂ to complete a total of 48 h of incubation. Sperm-oocyte interaction was assessed at 2.5 h post-insemination (hpi) by evaluating the number of bound spermatozoa. Presumptive zygotes were fixed and stained with Hoechst at 6, 18, and 22 hpi to assess polyspermy and pronuclear formation (PrF) by widefield fluorescence microscope with structured illumination. Cleavage rate was evaluated at 24 and 48 hpi. Data obtained from three replicates were analysed using one-way ANOVA. The number of bound sperm was similar between groups (Ho = 0.9 ± 0.1; HeA = 0.8 ± 0.1; HeB = 0.8 ± 0.1). No differences were seen in PrF at any time point (ranged from 24.4 ± 0.6 to 25.1 ± 0.9%) or cleavage rate between HeA or HeB IVF at 24 hpi (25.6 ± 0.9 and 25.7 ± 2.9%, respectively) or 48 hpi (24.2 ± 1.7 and 25.0 ± 1.3%). Homologous IVF was associated with higher percentages of PrF at 6 hpi (79.8 ± 2.1%) compared to HeA (24.4 ± 0.6%) and HeB (25.1 ± 0.9) IVF, and no polyspermy was detected. As expected, the cleavage rate at 24 or 48 hpi was higher (P < 0.05) in Ho (80.8 ± 0.9 and 81.4 ± 1.0, respectively) than He IVF. In addition, spontaneous parthenogenetic activation in mature unfertilised oocytes from mice at 24 and 48 hpi was observed (1.9 ± 1.9 and 2.6 ± 2.6%, respectively). In conclusion, He IVF is a promising method to assess the fertilising ability of guinea pig spermatozoa obtained from epididymis, revealing their ability to penetrate zona-intact murine oocytes, leading to hybrid embryo formation.