Pregnancy rate and survival in culture of in vitro fertilized bovine embryos frozen in various cryoprotectants and thawed using a one-step system

Abstract

Bovine oocytes surrounded with compact cumulus cells were cultured for 20-22 h (38.5°C, 5 % C02) in modified TCM -199 supplemented with 5 % superovulated cow serum (SCS) and inseminated by in vitro capacitated sperm. Day 7-8 embryos were equilibrated 10 min in 1.3 M methyl cellosolve (MC), 1.1 M diethylene glycol (DEG), 1.8 M ethylene glycol (EG), 1.6 M propylene glycol (PG) and 1.1 M l, 3-butylene glycol(BG) solutions, loaded into 0.25ml straws, placed into an alcohol bath freezer at 0°C, cooled from 0°C to -6°C at -l°C/min, seeded, held for 10 min, cooled at -0.3°C/min or -0.5°C/min to -30°C. Straws were then plunged and stored in liquid nitrogen. After thaw~ng in 30°C water, embryos were rehydrated directly in TCM-199 and then cultured for 48 hours in TCM-199 plus 5% SCS. Embryos were considered viable if they progressed to later developmental stages with good morphology. Some of the embryos frozen in each cryoprotectant were thawed and transferred non-surgically without removing the cryoprotectant. Hatched embryos survived freezing and one step dilution as follows: EG (50.0%), MC (53.6%), DEG (56.9%), PG (58.0%) and BG (11.5%). The survival rate of embryos cooled at 0.3°C/min versus 0.5°C/min was not significantly different (P>0.05). However, blastocysts hatched most often (P< 0.01) in vitro when cooled at a rate of 0.3°C/min [64.6 %(31/48)] compared with 0.5°C/min [22.6% (12 /53)]. Embryo survival in TCM-199 culture relative to cryoprotectant is shown in the table below. Pregnancy rates resulting from embryos frozen in different cryoprotectants were as follows: MC(48%, 10/21), DEG(30%, 3/10), EG(87%, 20 23), and PG(40%, 4/10).