Vitrification of in vitro-produced bovine embryos by addition of ethylene glycol in one-step.
The objective of this study was to simplify two-step addition of cryoprotectant for vitrification of bovine embryos by developing a one-step procedure. Survival was calculated as a percentage of non-vitrified controls developed from the same batch of oocytes. In experiment 1, bovine blastocysts were vitrified following one- or two-step addition of cryoprotectant. Exposure of embryos to cryoprotectant in one-step resulted in survival rates not significantly lower (p > 0.1) than those obtained by two-step addition (85% vs 98%, respectively). Based on these results, experiments 2-4 were designed to test one-step addition of cryoprotectant more rigorously. Experiment 2 exposed day 7 blastocysts to 6, 7 or 8 M ethylene glycol for 2.5 or 3.5 min. At 24 h post-vitrification, survival of embryos was similar, irrespective of ethylene glycol concentration or exposure time (6 M 38%, 7 M 51%, 8 M 59%; 2.5 min 54%, 3.5 min 45%). In experiment 3, blastocysts were exposed to 7 M ethylene glycol for shorter times (30 or 60 s); 30 s exposure resulted in decreased survival (8% vs 31%, p < 0.05). Experiment 4 concerned one-step addition of cryoprotectant to day 6 bovine morulae, exposed to 7 M ethylene glycol for 1 or 1.5 min. There was no difference in survival between exposure times of 1 or 1.5 min (28% vs 45%, respectively; p > 0.1). It is unclear why many embryos survive vitrification with one-step addition of cryoprotectant, but others do not. Although, one-step addition of cryoprotectant simplifies the vitrification procedure, survival rates were inadequate for routine cryopreservation of in vitro-produced bovine embryos.
Walker
D J
DJ
Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO, USA.
Campos-Chillon
L F
LF
Seidel
G E
GE
D000818:Animals; D001755:Blastocyst / Q000187:drug effects / Q000502:physiology; D002417:Cattle / Q000196:embryology*; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian / Q000187:drug effects* / Q000502:physiology; D019855:Ethylene Glycol / Q000494:pharmacology*; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D009028:Morula / Q000187:drug effects / Q000502:physiology; D013997:Time Factors
D003451:Cryoprotective Agents; D019855:Ethylene Glycol