300 BLASTOCYST PRODUCTION BY IN VITRO MATURATION AND DEVELOPMENT OF PORCINE OOCYTES IN DEFINED MEDIA FOLLOWING INTRACYTOPLASMIC SPERM INJECTION

Abstract

Most culture media for in vitro production (IVP) of porcine embryos contain serum or bovine serum albumin (BSA); especially in the case of in vitro maturation (IVM) porcine follicular fluid is added. The present study was carried out to establish porcine-defined IVP. In Experiment 1, we investigated the efficacy of additional 0.6 mM cystine, 100 �M cysteamine (Cys), or cystine + Cys to a defined TCM-199 maturation medium with regard to the developmental competence of in vitro-matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM-199 containing 0.05% (w/v) polyvinyl alcohol (PVA). In Experiment 2, the effect of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development following ICSI. As positive and negative controls, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the basic medium. Five or 10 ng mL-1 of EGF was supplemented in the negative control medium (mPZM-4). All percentage data on cleavage and blastocyst development were subjected to arcsine transformation before statistical analysis using the STATVIEW program (Abacus Concepts, Berkeley, CA). The mean cell numbers per blastocyst (assessed by Giemsa staining method) were analyzed by one-way ANOVA, and the differences among the groups were also analyzed using Tukey-Kramer multiple comparison tests. In Experiment 1, there were no significant differences in the rates of cleavage (31.4 to 64.3%) and blastocyst formation (6.5 to 22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups, without significant differences. In Experiment 2, the rates of cleavage (43.0 to 58.0%) and blastocyst formation (14.0 to 23.6%), and the mean cell numbers per blastocyst (30 to 37) were not significantly different among the groups. However, the addition of 5 ng mL-1 of EGF to the mPZM-4 resulted in a significantly (P d 0.05) higher blastocyst rate (48.6%) to cleaved embryos than the other two defined groups (mPZM-4 and mPZM-4 with 10 ng mL-1 EGF: 23.4% and 23.1%, respectively), but not significantly different with mPZN-3 (40.1%). The present results indicate that TCM-199 with added cysteamine (100 �M) can be used as a defined IVM medium for porcine oocytes, and further addition of cystine into the IVM medium is not necessary. The addition of 5 ng mL-1 of EGF to a defined IVC medium enhanced subsequent development after ICSI. These results show that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI, and IVC). This study was partly supported by a grant-in-aid for Scientific Research (17580242) from the Japan Society for the Promotion of Science.