Effects of in vitro fertilization conditions on preimplantation development and quality of pig embryos

The present study was to investigate the effects of in vitro fertilization conditions on in vitro development and structural integrity of pig embryos. Porcine oocytes matured in vitro were co-incubated with four different spermatozoa concentrations (0.6 × 105, 1.2 × 105, 2.5 × 105 and 5 × 105 cells/ml) for 6 h, and at a spermatozoa concentration (1.2 × 105 cells/ml) for 2, 4 and 6 h, respectively. Spermatozoa penetration and blastocyst formation were observed at 10 and 144 h post insemination, respectively. The allocation of a blastocyst to inner cell mass (ICM) and trophectoderm (TE) cells was determined by using a differential staining method. Polyspermy frequency increased with increasing spermatozoa concentrations. The spermatozoa-oocyte co-incubation period of 2 h provided for decreased in vitro development rate than 4 and 6 h groups (P < 0.05), although no difference was detected in polyspermy frequency between spermatozoa-oocyte co-incubation periods. Interestingly, blastocysts derived from the groups with greater spermatozoa concentrations (2.5 × 105 and 5 × 105 cells/ml) had significantly fewer ICM cell nuclei as compared with those groups with lesser spermatozoa concentrations (0.6 × 105 and 1.2 × 105 cells/ml). There was no difference in the structural integrity of blastocysts among the co-incubation periods. Blastocysts derived from respective experiments were individually classified into three groups (I: <20%; II: 20-40% and III: >40%) based on the ratio of ICM to total cells. Proportion of blastocysts in Group II, with a presumptive normal range of structural integrity, was slightly decreased in the groups with greater spermatozoa concentrations (2.5 × 105 and 5 × 105 cells/ml). The results indicate that the spermatozoa concentration during in vitro fertilization may be important for developmental competence and quality of pig embryos.