238 ASSESSMENT OF SPERM MOTILITY, VIABILITY, AND FERTILIZATION RATE OF TWO GROUPS OF BULLS SHOWING A DIFFERENT ABILITY TO PROMOTE EMBRYO DEVELOPMENT IN VITRO

Abstract

Low developmental rates after IVF with some AI bulls are one of the most frequent problems encountered with in vitro production of bovine embryos. In this study, two groups of high and low in vitro fertility bulls (3 bulls per group) were selected based upon blastocyst rates. We first evaluated sperm motion characteristics using computer-assisted sperm analysis (CASA) before and after Percoll separation. The following parameters were compared between bulls: proportion of motile sperm (% MOT), curvilinear velocity (VCL), and straightness (STR), which allowed us to evaluate the proportion of progressive semen (VCL > 80 μm/s and STR > 60%). Concerning % MOT, differences between bulls were observed before Percoll separation (42 to 59% – ANOVA2: P = 0.017). Following separation, the values of % MOT were globally increased (ANOVA3: P < 0.001) and no further difference was observed between bulls (68 to 76%). The same pattern was obtained for the proportion of progressive spermatozoa (before: 43 to 55% vs. after: 48 to 57% – ANOVA3: P = 0.005). For viability measurements, two DNA-binding fluorochromes, propidium iodide and Hoechst 33342, were used, which respectively stain dead sperm nuclei in red and living sperm nuclei in blue. This viability evaluation was carried out after 0, 2, 6, and 18 h of incubation in TALP medium supplemented with heparin (10 μg/mL), under the same conditions as IVF but without oocytes. In the two groups of bulls, a sharp decrease in the proportion of living sperm was already observed at 2 h (only 50 to 20% of living sperm), while, at 18 h, 17 to 9% of sperm were still intact. Two bulls from the high in vitro fertility group showed a percentage of living sperm significantly more important at 6 and 18 h than the other 4 bulls (chi square – P < 0.05). In a third experiment, we assessed the fertilization (2 pronuclei) and the polyspermy (>2 pronuclei) rates after staining the zygotes with Hoechst at 18 hpi. The high fertility group showed higher fertilization rates than the low fertility one (75 to 80% vs. 59 to 67% – ANOVA2: P < 0.008). No significant difference was noticed between bulls concerning polyspermy. The present results confirm the efficiency of Percoll separation in enhancing some of the motility parameters such as the proportion of motile and progressive sperm. Moreover, this gradient allows us to abolish differences between bulls for those motility parameters. However, the viability and the rate of fertilization still varied between bulls after Percoll separation. In our study, only fertilization rates seemed related to the blastocyst rates. Further investigations are under way to better understand the sperm characteristics of these two groups. This work was supported by the Minestery of Agriculture of the Region wallonne de Belgique.