108 the Role of Nitric Oxide Synthase in in Vitro Development of Bovine Oocytes and Pre-Implantation Embryos

Abstract

200 Reproduction, Fertility and DevelopmentDevelopmental Biology for the first experiment, loaded into straws, and assigned to one of 4 treatment groups. Half the straws from each bull were exposed to 90 MPa/30 min, 90 MPa/90 min, 30 MPa/30 min, or 30 MPa/90 min, and then cryopreserved. Controls consisted of straws that were cryopreserved without pressure treatment. Cryopreservation steps were 60 min equilibration at 5 • C, followed by 10 min at −110 • C, and then plunging into liquid nitrogen. Straws were thawed in a 35 • C water-bath for 30 s. Each treatment and control group was replicated 8 times (8 samples per bull). The average post-thaw motility was significantly superior with pressure pre-treatment in each of the pressurized groups compared to the samples frozen without previous pressurization (P < 0.001) (Bull I: 2-3% without pressurization vs. 17-33% with pressurization; Bull II: 0% without pressurization vs. 21-35% with pressure pre-treatment). Among the pressure/time parameters used, 30 MPa/90 min proved significantly superior (33 and 35%; P < 0.05) for each of the bulls. Expt. 2 clearly demonstrates the beneficial effect of a previous pressure treatment on post-thaw motility of bull semen cryopreserved in our experiment. Further investigations are needed, including samples from different bulls, different freezing protocols, and the biological background of the process. Development of improved protocols for cryopreservation of zona pellucida-intact porcine embryos could greatly impact the swine industry. Our aim was to investigate in vitro development following cryopreservation of embryos from Chinese Meishan (M) and occidental white cross (WC) breeds using a modified protocol described previously (Misumi K et al. 2003 Theriogenology 60, 253-260). First-parity M sows (n = 11) and WC gilts (n = 13) were observed for estrus every 12 h and inseminated at 12 and 24 h after estrous onset within breed using semen from 2 different boars. Females were sacrificed between Days 4.5 and 6 after estrus and embryos were collected using Beltsville embryo culture medium (BECM). Compact morula (CM) or blastocyst stage embryos from each female within breed were randomly allocated either directly into the culture system to serve as controls (68 M and 48 WC embryos) or to undergo cryopreservation. A total of 101 M and 78 WC embryos were cryopreserved using the following protocol: (1) 5 min in BECM + 10% ethylene glycol (EG); (2) 5 min in BECM + 10% EG + 0.27 M sucrose + 1% polyethylene glycol (PEG); and (3) 30 to 45 s in BECM + 40% EG + 0.36 M sucrose + 2% PEG. In the last solution, 5 to 10 embryos in a 5-to 10-µL microdrop attached to a fine glass pipette were exposed to the vapor phase of liquid nitrogen (LN 2 ) for 15 s and then plunged into LN 2 . The pipette tip was broken and the tip and associated frozen microdrop were placed inside an LN 2 -submerged 2-mL cryotube containing a hole in the lid for 1 h. Next, embryos were thawed using a 4-step (5 min each) procedure: (1) BECM + 5% EG + 0.57 M sucrose; (2) BECM + 2...