Effects of a Short Co-Incubation Time for Spermatozoa-Oocytes Using Commercial Liquid Stored Boar Semen on Porcine in vitro Fertilization Efficiency

This study was conducted to evaluate if the efficiency of porcine In Vitro Fertilization (IVF) could be improved by a short co-incubation tun. e for spermatozoa-oocytes using commercial Liquid Stored (LS) boar semen For this study, LS semen was supplied weekly from the Veterinary Service Laboratory (Department of Livestock Research, Yong-in, Gyeonggi-do, Republic of Korea) and kept at 17 degrees C for 5 days. A total of 808 immature cumulus-oocyte complexes from six replicates were matured in vitro and inseminated with LS semen (5x10(5) mL(-1) sperm concentration) for 5, 20, 90 or 360 min (control group). To evaluate the developmental competence of each group, all IVF embryos were examined for the following: fertilization rate, cleavage rate, blastocyst formation rate and cell numbers. The oocytes from the 5, 20 and 90 min co-incubation periods were washed in IVF medium (mTBM) to remove spermatozoa, transferred to another droplet of the same medium (containing no spermatozoa) and incubated until 6 h post-insemination. Thereafter, all IVF embryos were cultured in porcine zygote medium-3 for 10 h after insemination to assess fertilization parameters. A co-incubation period of 5 min resulted in a significantly (p < 0.05) lower spermatozoa penetration rate (66.8%) as compared with 20, 90 and 360 min (91.8, 100 and 100%, respectively) but there was no difference in Male Pronucleus(MPN) formation rate. Co-incubation for 5 and 20 min resulted in a significantly higher monospermy rate (39.7 and 38.4%, respectively) as compared with the 90 min incubation (4.2%) while no monospermic oocytes were observed in the 360 mm group. The polyspermy rate was significantly higher with co-incubation periods of 90 and 360 min (95.8 and 100%, respectively) as compared with 5 and 20 min. (60.3 and 57.8%, respectively). The mean number of penetrating spermatozoa per oocyte was significantly lower in the 5 and 20 min groups (1.7 and 2.3, respectively) as compared with the 90 and 360 mm groups (9.2 and 17.6, respectively). The efficiency of fertilization (number of monospermic oocytes/total number of inseminated oocytes) was significantly higher in the 20 min (35.8%) co-incubation period as compared with the other groups (25.1 and 4.2% for 5 and 90 min, respectively). The cleavage rate for the 5 and 20 min (525 and 65.6%, respectively) groups was significantly higher than the 90 and 360 min groups (32.1 and 10.5%, respectively). The 20 min (18.8 and 113.2%) co-incubation period resulted in a significantly higher blastocyst formation rate and total blastocyst cell number compared to the 90 min. group (3.6 and 68.0%) but there was no significant difference between the 5 min (10.7 and 90.3%) and 20 min groups. The embryos in the 360 min co-incubation period did not develop into blastocysts. Concerning cell numbers, blastocysts that developed with the 20 min (24.6) co-incubation period had a greater number of cells in the inner cell mass than the 5 min (12.4) group however, there was no difference in the number of trophectoderm cells and total cell number. Therefore, researchers recommend a two-step IVF procedure for porcine oocytes with a co-incubation period with spermatozoa for 20 min followed by an additional culture until 6 h without spermatozoa when using commercial LS semen for porcine IVF experiments.