Distribution and role of microtubules and microfilaments in porcine oocytes during fertilization

Abstract

Use of high sperm concentrations for IVF results in high rates of penetration and polyspermy. To optimize the use of IVM-IVF for comparisons of semen quality, we determined the effect of using decreasing sperm concentrations on penetration, polyspermy and male pronuclear (MPN) formation rates and average number of spermatozoa per oocyte in standardized ejaculate fractions from 3 adult boars of different breeds. Raw semen values of the 3 boars during the period of IVF experiment were recorded for analysis. Standardized aliquots of the first sperm-rich fraction were collected (main plot) on 4 occasions. Each semen sample was serially diluted to 5 × 105, 2.5 × 105, 1.25 × 105, 6.25 × 104 and 3.125 × 104 sperm cell/ml for IVF (sub-plot). Oocyte-cumulus cell complexes collected by aspiration from slaughterhouse ovaries were cultured in vitro using standard procedures. Penetration, polyspermy and MPN formation rates were significantly affected by boars (all P < 0.001), semen dilutions (all P < 0.001) and their interactions (all P < 0.05). Penetration rates were lower (P < 0.05) for the 5 semen dilutions in Boar C (LSM 69.5, 58, 44.3, 25 and 10%) compared with Boar A (LSM 100, 100, 100, 97.5 and 92%) and Boar B (LSM 100, 94.7, 100, 92.8 and 88.3%); these differences were highly correlated to sperm motility assessed at Day 7 after semen collection. Semen dilution in Boar C resulted in a significant decrease in penetration rate but had no effect in Boars A and B over the range of dilutions used. Conversely, although semen dilution in Boars A and B decreased (P < 0.05) rate of polyspermy (LSM 100, 100, 78, 60.8, and 32.2%; and 100, 97.7, 64.3, 37.8, and 34.5%, respectively), and average number of spermatozoa per oocyte (LSM 9.1, 5.8, 3.7, 2.3, and 1.4; and LSM 8, 5.1, 3.1, 1.9 and 1, respectively), there was no effect in Boar C. Because of these interaction effects, the optimal sperm:oocyte ratio for maximal MPN formation rate was different among boars. In conclusion, serial dilution of semen used in the IVM/TVF systems effectively discriminates semen quality. Together with Day 7 motility estimates, this may allow effective prediction of relative boar fertility.