107 TOXICITY OF ETHYLENE GLYCOL ON FROZEN AND THAWED IVP EMBRYOS IN DIRECT TRANSFER METHOD
S. Matoba A , K. Imai A , Y. Mimaki A , M. Narita A , M. Tagawa A , O. Dochi B and N. Saito AA National Livestock Breeding Center, Fukushima, Japan. email: s0matoba@nlbc.go.jp;
B Department of Dairy Science, Rakuno Gakuen University, Hokkaido, Japan.
Reproduction, Fertility and Development 16(2) 175-176 https://doi.org/10.1071/RDv16n1Ab107
Submitted: 1 August 2003 Accepted: 1 October 2003 Published: 2 January 2004
Abstract
Ethylene glycol (EG) is a cryoprotectant which is highly permeable to mammalian embryos. But the toxicity of this cryoprotectant for embryos after thawing has not been investigated. The aim of this study was to determine the toxicity of EG to embryos frozen and thawed by a direct transfer method. In vitro-produced Day 7 blastocysts (n = 529) of grade 1 quality were used in this study. Embryos were frozen in 1.5 M EG in Dulbecco’s PBS (DPBS) supplemented with 0.1 M sucrose, 4 mg mL−1 BSA and 20% fetal calf serum (FCS). Embryos were transferred into freezing medium, loaded into 0.25-mL straws and kept for more than 15 min for equilibration; then the straws were plunged into a −7°C methanol bath of a programmable freezer for 1 min, seeded at −7°C, held at −7°C for 14 min, cooled to −30°C at the rate of −0.3°C min−1 and then plunged into liquid nitrogen. The straws were thawed by holding in air for 6 sec, and then placed in water at 30°C for 15 s. After thawing, the straws were held for 0, 10, 20, 30 and 60 min (holding time) at either 38.5 or 26.0°C. Ethylene glycol was removed from the embryos by placing them into DPBS supplemented with 20% CS at 38.5°C more than 20 min. The embryos were cultured in TCM-199 supplemented with 20% FCS and 0.1 mM β-mercaptoethanol under a gas phase of 5% CO2 in air at 38.5°C for 72 h. Viability of embryos was evaluated at 0-, 24-, 48- and 72-h incubation by their morphological development. Data were analyzed by ANOVA. There was no significantly difference in the survival rate of thawed embryos held at 38.5°C or 26.0°C for the same holding periods. The survival rate of the thawed embryos held at 38.5°C decreased significantly when the holding period exceeded 30 min compared with no holding period after 24- and 72-h culture (P < 0.05, respectively). On the other hand, the survival rate of the thawed embryos held at 26.0°C decreased significantly when the holding time was 60 min compared with less than 20 min of holding after 24-h culture, and less than 10 min after 72-h culture (P < 0.05, respectively). Therefore, toxicity of EG was observed when thawed embryos were held for 30 and 60 min at 38.5°C and 60 min at 26.0°C. These results suggest the toxicity of EG in direct transfer methods can be avoided by transferring the embryos within 20 min after thawing.
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