Assessment of fertilizing ability of Iberian ibex (Capra pyrenaica) vitrified and frozen epididymal sperm by in vitro heterologous fertilization of bovine oocytes

The aim of this study was to evaluate the fertilizing ability of vitrified and frozen Iberian ibex sperm by assessing heterologous IVF using bovine oocytes. Testes were obtained from mature ibexes that were legally hunted in the Tejeda and Almijara Game Reserve, in southern Spain. Epididymal spermatozoa were collected by the retrograde flushing method. Sperm from right epididymis was vitrified with TCG-6% egg yolk plus 100 mM sucrose while sperm from left epididymis was conventionally frozen with TCG-6% egg yolk and 5% glycerol. In vitro matured zona-intact bovine oocytes were subjected to heterologous IVF with vitrified-warmed (n=495) or frozen-thawed ibex sperm (n= 565) and homologous IVF (n=299). A non-fertilized group was included as control for parthenogenesis (n=81). For heterologous fertilization, sperm pool of three males was used for each treatment. Sperm-oocyte interactions were evaluated at 2.5 hours post-insemination (hpi) by the number of attached and bound spermatozoa whereas penetration and polyspermy were evaluated after 12 hpi. Presumptive zygotes were fixed and stained with Hoechst 33342 at 18, 20, 22, 24 and 26 hpi to assess pronuclear formation using a phase contrast and confocal microscopy. Besides, cleavage rate was evaluated in all groups at 24 hpi. Data obtained was analyzed using one way ANOVA (Sigma Stat, Jandel Scientific, San Rafael, CA) Results showed a higher number of bound and attached spermatozoa in both heterologous groups compared to homologous group (P<0.001). The homologous IVF group as expected, showed the highest percentage of pronuclear formation at 18 hpi (67.7±9.8%), significantly different to both heterologous groups (Frozen: 21.3±13.9%; Vitrified: 28.8±15.5%, P<0.05). Indeed, pronuclear formation was delayed in both heterologous groups with the highest percentage at 24 hpi (30.3 ± 15.1%) for frozen sperm and at 20 hpi (31.7 ± 21.5%) for vitrified sperm. In addition, cleavage rate was higher in homologous group compared with heterologous frozen and vitrified groups (76.1±15.9% vs. 31.3±27.2% and 45.1±24.4%, respectively, P<0.05). No differences were observed between heterologous vitrified and frozen sperm in all parameters evaluated. In conclusion, Iberian ibex epididymal sperm can be vitrified successfully, maintaining its fertilization ability in the same extend as frozen sperm. To our knowledge, this is the first report of successful epididymal sperm vitrification in a mammal species being capable of fertilization as a standard tool for genome conservation in threatened species.