244 EFFECT OF β-MERCAPTOETHANOL IN A TRANSIENT-IVF SYSTEM ON SPERM PENETRATION INTO PORCINE IVM OOCYTES

Abstract

This study was carried out to determine the effect of β-mercaptoethanol (bME; at 50 μM) during a brief co-culture and the additional culture of inseminated oocytes on sperm penetration of porcine IVM oocytes. Cumulus-oocyte complexes (COCs) collected from 3–∼6-mm follicles were cultured in modified NCSU-37 supplemented with bME, eCG, hCG, and dibutyryl cAMP for 20 h and then in the same medium without eCG, hCG, and dibutyryl cAMP for additional 24 h. After IVM, the oocytes were denuded with 0.1% hyarulonidase, randomly divided into three groups (as described below) and fertilized in vitro. Treatment 1 (T1, control): Oocytes were co-cultured with fresh spermatozoa for 10 min in modified M199 medium (mM199) supplemented with 5 mM caffeine (+caff) in the absence of bME (bME-), then washed gently with caffeine-free mM199, and cultured for further 9 h in mM199; T2: co-culture for 10 min in mM199+caff bME− and additional culture in mM199 in the presence of bME (bME+); and T3: co-culture for 10 min in mM199+caff bME+ and additonal culture in mM199 bME+. At 9 h after insemination, oocytes were fixed and stained for the evaluation of sperm penetration. Results from four replicates were analyzed by ANOVA and Duncan's multiple range test. Sperm penetration rate was lower in T3 (79.8% in 92 oocytes) compared with T1 (95.3% in 84 oocytes) and T2 (93.3% in 93 oocytes). Percentage of eggs with male and female pronuclei in penetrated oocytes was higher in T1 (89.6%) than in T2 (56.4%) and T3 (57.2%). Monospermy rate was lower in T1 (48.9%) than in T2 (59.5%) and T3 (64.3%). These results demonstrated that, although the presence of β-mercaptoethanol during co-culture and the additional culture reduced the fertilization rate, the presence during the additional culture period affected the process of fertilization and increased the incidence of monospermy at 9 h after insemination. In conclusion, the progression of sperm penetration may be controlled by utilization of β-mercaptoethanol in porcine IVF system. This work was supported by JSPS-KAKEN (C16580230).