Culture of Embryos Fertilized In Vitro and Assessment of Early Embryonic Development
Some of the denuded oocytes were washed three times with mR1ECM and observed for evidence of sperm penetration by means of an inverted phase-contrast microscope (Nikon Diaphot; Nikon Corp., Tokyo, Japan). Only penetrated zygotes were washed again three times with mR1ECM, transferred (10-20 zygotes) into 400 ^l of the same medium, and cultured in an atmosphere of 5% CO2 in air at 37°C. In vitro development of the zygotes was determined after 24, 72, 96, and 120 h after insemination by using an inverted phase-contrast microscope (Nikon Diaphot). Embryos showing compaction and blastocyst cavity formation were classified as morulae and blastocysts, respectively. ampicillin antibiotic
Transfer of Morulae and Blastocysts to Recipients
Embryos that had developed to the morula and blastocyst stages 99-101 h after insemination were transferred into the uteri of pseudopregnant female Wistar rats as described by Miyoshi et al.. To induce pseudopregnancy, female rats were stimulated by insertion of a glass rod connected to an electric vibrator into the vagina between 1930 h and 2000 h on the day of proestrus (Day 0). Between 1000 h and 1200 h on Day 4, the females were anesthetized with an i.p. injection of tribromoethanol (Avertin, 0.012 ml/ g body mass; Aldrich Chemical Co., Inc., Milwaukee, WI), and the uterus was exposed through a dorsal incision.