90 Guinea pig sperm is capable of fertilising bovine zona-intact oocytes in vitro

Heterologous (He) IVF may be an effective aproach to evaluate the fertilising capacity of guinea pig sperm as it is often difficult to obtain adequate numbers of naturally corresponding oocytes in this species outside of South America. We aimed to evaluate the fertilising capacity of guinea pig sperm in He IVF with zona-intact bovine oocytes in two different media. Sperm were collected from the vas deferens of two guinea pigs individually by gently mincing with fine scissors in 1 mL of normal saline. Motile sperm for He and homologous (Ho) IVF were selected by BoviPure® density gradient centrifugation (Nidacon International AB). In vitro-matured zona-intact bovine cumulus-oocyte complexes (COCs; 30/drop) were subjected to He IVF with guinea pig motile selected sperm (mL⁻¹) in 60-µL drops of (A) Tyrode’s medium with 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate, 6 mg mL⁻¹ fatty acid-free BSA and 10 mg mL⁻¹ heparin (FERT) (HeA; n = 499), and (B) minimal culture medium with 0.25 mM Na-pyruvate, 20.0 mM Na-lactate, 5.56 mM glucose, 2.7 mM KCl, 0.3% BSA, and 10 mg mL⁻¹ heparin (HeB; n = 491). In parallel, Ho IVF (n = 243) and parthenogenesis (non-fertilised oocytes; n = 75) were performed in 60-µL drops of FERT medium. Sperm and COCs were co-incubated for 18 h, then presumptive zygotes or (parthenogenetic) oocytes were cultured in synthetic oviductal fluid (SOF) supplemented with 5% fetal calf serum at 38.5°C and 5% CO₂ to complete a total of 48 h of incubation. A subset of co-incubated oocytes/presumptive zygotes were vortexed for 3 min, fixed in 4% paraformaldehyde, and stained with Hoechst 33342 at 2.5 h post-insemination (hpi) (n ∼ 13/group/replicate) to assess sperm-oocyte interaction by evaluating the number of bound sperm to the zona pellucida; at 18, 20, 22, 24 and 26 hpi for He and at 18 hpi for Ho IVF (n ∼ 25/group/replicate) to evaluate polyspermy and pronuclear formation (PrF); and at 48 hpi to evaluate cleavage rate in all groups (n ∼ 30/He-Ho/replicate and n ∼ 18/parthenogenetic/replicate) using a fluorescence microscope (Zeiss ApoTome 2). Data obtained from four replicates was analysed by one-way ANOVA. The number of bound sperm was similar between groups (Ho = 0.5 ± 0.1, HeA = 0.4 ± 0.1, and HeB = 0.4 ± 0.1). The PrF for HeA group was significantly higher (P ≤ 0.05) than for the HeB group at every time point evaluated (18 hpi: 58.8 ± 1.9 vs. 45.3 ± 2.5%; 20 hpi: 58.2 ± 1.5 vs. 46.2 ± 1.1%; 22 hpi: 61.5 ± 4.2 vs. 45.4 ± 4.2%; 24 hpi: 64.4 ± 4.9 vs. 47.2 ± 4.2%; 26 hpi: 64.9 ± 2.1 vs. 45.3 ± 5.7%, respectively). Homologous IVF was associated with higher (P ≤ 0.05) percentages of PrF at 18 hpi (77.6 ± 2.0%) compared to HeA and HeB IVF and no polyspermy was detected in any group. As expected, the cleavage rate at 48 hpi was higher (P ≤ 0.05) in Ho (83.5 ± 0.7%) than HeA (69.8 ± 1.7%) and HeB (49.1 ± 1.1%) IVF. Spontaneous parthenogenetic activation in mature unfertilised bovine oocytes at 48 hpi was observed (4.2 ± 2.5%). In conclusion, guinea pig sperm can penetrate zona-intact bovine oocytes and lead to hybrid embryo formation, indicating that heterologous IVF is a promising method to analyse the fertilising capacity of guinea pig sperm obtained from the epididymis.