88 Evaluation of embryo transfer results using embryos cryopreserved in ethylene glycol for 8 years or in glycerol for 30 years

Abstract

Various permeating cryoprotectants, such as glycerol and ethylene glycol, have been used in the cryopreservation of embryos to help maintain cellular viability during indefinite and prolonged periods of storage in liquid nitrogen. The objective of this study was to compare the efficiency of glycerol (G) and ethylene glycol (EG) after storage in liquid nitrogen for a considerable period of time before transfer. The work was carried out in Palenque, Chiapas, Mexico. A total of 50 embryos were transferred, 24 Brahman (G) cryopreserved in the 1990s and 26 Brangus (EG) cryopreserved in 2010. Synchronous recipients were selected based on 3 characteristics: body condition (5-7, scale of 1-9), reproductive health, and multiparity. Recipient cows (n=62) were synchronized using a FTET protocol as follows. On Day 0, cows received a progesterone intravaginal device (CIDR) and 2mg of oestradiol benzoate IM. On day 8, the CIDR was removed and all cows received 25mg of dinoprost tromethamine (Lutalyse, Pfizer Animal Health, Montreal, Quebec, Canada), 200IU of eCG, and 0.5mg oestradiol cipionate IM. Day 10 was considered the day of oestrus and embryos were transferred (n=50) to the ipsilateral uterine horn of those recipients with a corpus luteum greater than 1.5cm in diameter on Day 17. The G embryos were produced with 4 bulls whereas the EG embryos were produced with 6 different bulls. The G straws were thawed for 12s in the air plus 12s in 20°C water. Embryos were immersed for 8min in a thawing solution containing 1.0M sucrose (ViGRO One-Step) and then transferred to holding medium (ViGRO Holding) for rehydration before loading into straws for embryo transfer. The EG embryos were thawed by allowing the straws to stand in air for 10s and then immersing them in a 30°C water bath for 10s and were transferred immediately. Pregnancy diagnosis 35 days after the transfer revealed 19 pregnancies of 50 embryos transferred (38%), distributed as 46% embryos in EG (12 pregnant of 26 transferred) and 29% embryos in G (7 pregnant of 24). A Fisher’s exact test was performed showing that no significant difference existed between groups (P>0.05). There was no effect of bull on pregnancy rates, and Brahman breed results by individual bull were 5 pregnancies of 13 (38%), 2 of 6 (33%), 0 of 4 (0%), and 0 of 1 (0%) for bulls I to IV, respectively. Pregnancy rate by Brangus bulls were 6 pregnancies of 7 (86%), 2 of 3 (67%), 2 of 4 (50%), 2 of 4 (50%), 0 of 4 (0%), and 0 of 3 (0%) for bulls 1 to 6, respectively. It is important to remember that the embryos cryopreserved in G remained in the nitrogen tank for more than 30 years, whereas the embryos cryopreserved in EG remained stored in liquid nitrogen for less than 10 years. Although pregnancy rate was numerically lower with Brahman embryos stored in G, pregnancy rates were considered acceptable considering the length of storage. Future research is needed with greater numbers and different breeds to determine whether G or EG will consistently produce higher embryo viability and pregnancies after storage for considerable periods before transfer.