After coculture of cumulus-oocyte complexes with spermatozoa in the above-mentioned modified mR1ECM-BSA for 10 h, the incidence of sperm penetration was compared to that in mKRB. Further, to examine the effect on sperm penetration of supplementation to adjust osmolarity, the osmolarity of mR1ECM-BSA was increased to the same level (310 mOsm) in mKRB by increasing NaCl concentration (106.7 mM) or by adding 60 mM sorbitol. After coculture of cu-mulus-oocyte complexes with spermatozoa in the modified mR1ECM-BSA for 10 h, the incidence of sperm penetration was compared to that in mR1ECM-BSA. Cheap Diskus Advair
To determine the optimal concentration of NaCl in mR1ECM-BSA for sperm penetration, cumulus-oocyte complexes were cocultured with spermatozoa in mR1ECM-BSA containing various concentrations (90-140 mM) of NaCl for 10 h.
To compare the developmental competence of embryos fertilized in mR1ECM-BSA containing a high NaCl concentration with that of embryos fertilized in mKRB, zygotes fertilized in mKRB or mR1ECM-BSA containing various NaCl concentrations (100-130 mM) were cultured in mR1ECM. The incidence of embryos that had developed to the 2-cell, 4-cell, morula, and blastocyst stages was determined at 24, 72, 96, and 120 h after insemination, respectively. Furthermore, some of the embryos fertilized in mR1ECM-BSA containing 110 mM NaCl that had developed in mRIECM to the morula and early blastocyst stages were transferred into the uterine horn of 7 recipients (9-13 embryos per recipient) 86-88.5 h after induction of pseudopregnancy as described previously. The developmental competence of the embryos to term was also examined.
Data from four replicate trials were expressed as means ± SEM. Statistical analyses for treatment comparisons were carried out by ANOVA and Duncan’s multiple range test using the STATVIEW program (Abacus Concepts, Inc., Berkeley, CA). All percentage data were subjected to arc sine transformation before statistical analysis. When ANO-VA revealed a significant treatment effect, the treatments were compared by Duncan’s multiple range test. A probability of p < 0.05 was considered to be statistically significant.