Effect of short periods of sperm-oocyte coincubation during in vitro fertilization on embryo development in pigs

The present study was conducted to determine if the efficiency of in vitro pig embryo production could be improved by a reduction in the period of time that oocytes are exposed to sperm during in vitro fertilization. A total of 1596 immature cumulus¡oocyte complexes from five replicates were matured in vitro and inseminated with frozen-thawed spermatozoa (2000 spermatozoa/oocyte) for 10, 30, 60 min or 6 h (control group). The oocytes from short coincubation times were washed three times in fertilization medium to remove spermatozoa not bound to the zona and transferred to another droplet of the same medium (containing no sperm) for 6 h. After 6 h, the oocytes from each group were cultured in embryo culture medium for another 6 h to assess fertilization parameters and for 7 days to assess embryo development. After each period of coincubation, some oocytes were stained with Hoechst-33342 to count zona-bound sperm. Although the number of zona-bound sperm increased with the coincubation time (34.1±1.7, 46.8±2.8, 62.8±3.8, and 139.5±6.1 for 10, 30, 60 min and 6 h, respectively, P<0.02), the penetration rate was not significantly different among groups (61.3-68.2%). However, the efficiency of fertilization (number of monospermic oocytes/total number of inseminated oocytes) increased (P<0.04) as the coincubation time was increased (26.6±2.9%, 29.0±4.4%, 39.5±6.2%, and 49.3±3.0% for 10, 30, 60 min and 6 h, respectively). Nevertheless, there were no significant differences among groups in blastocyst formation rates (17.5-25.5%). These results demonstrate that although a sperm¡oocyte coincubation time of as little as 10 min results in fertilization rates similar to a 6-h coincubation, the reduction in the period of time of sperm-oocyte coincubation does not improve the efficiency of in vitro pig embryo production.