Is the exposure time of bovine embryos to ethylene glycol (EG) prior to freezing and/or after thawing critical to survival?
John F. Hasler
Efficient and efficacious cryopreservation of bovine embryos is critical to the commercial ET industry because, as shown by the most recent AETA statistics (2011), 72% of embryos were frozen following collection versus only 28% that were transferred fresh into recipients. Following the published report of Voelkel and Hu in 1992 on cryopreservation with EG, the commercial bovine ET industry rather quickly switched from glycerol to EG as the major cryoprotectant in freezing media. The overall percentage of embryos frozen in EG rose rapidly starting in 1992 and reached 97% in 2008, the last year that the AETA collected data on this specific statistic.
During the past 20 years there has been a continuing debate among ET practitioners regarding the question of whether EG is more toxic than glycerol to bovine embryos. This concern has led some practitioners to limit exposure time of embryos to EG for a maximum of 5 min prior to chilling and seeding. I am not aware of any published reports showing that EG is any more toxic to bovine embryos than is glycerol. Voelkel and Hu (1992) reported that 100% of IVF blastocysts exposed for 20 min to 1.5M EG or 1.5M propylene glycol were viable 48 h after being rehydrated in holding medium and cultured in vitro. The authors’ interpretation was that “neither of the cryoprotectants was overtly toxic to bovine embryos”. Also, in the original US patent filed by Voelkel in 1992, an exposure time of 10 to 20 min to EG prior to freezing, at a temperature of 18° to 25°C, was recommended.
Two studies demonstrated that exposure of in vitro-produced (IVP) embryos to EG for up to 40 min (Hasler et al., 1997) or 60 min (Takagi et al., 1993a) prior to freezing did not decrease survival rate of embryos following thawing and return to in vitro culture. Obviously, there is also embryo exposure to EG after thawing and prior to transfer. In an additional study, Takagi et al. (1993b) reported no differences in the survival of IVP embryos exposed to 1.8M EG for 15 min, frozen in a direct transfer protocol and then thawed and held in the straws for <1, 10 and 30 min at 20-25°C before being cultured in vitro. Matoba, et al. (2004) also examined the effect of EG exposure after thawing of IVP embryos and included temperature as another factor. There were no differences in embryo survival after 0, 10, 20 or 30 min exposure at 26.0°C or of 0, 10 or 20 min exposure at 38.5°C. However, at 38.5°C embryo survival decreased when exposure time exceeded 30 min. Lastly, a recent study in Argentina that involved large numbers of in vivo-derived embryos exposed to EG for different periods of time prior to freezing and then in vitro culture after thawing failed to detect an influence of exposure time (Tribulo, et al., 2012). Specifically, blastocyst re-expansion and hatching rates were similar for embryos exposed to EG for 5, 10, 20 and 30 minutes
Enough on in vitro culture, how about pregnancy results of actual transfers into recipients?
Martinez, et al. (2002) reported that there was no significant difference in the pregnancy rates or calving rates of recipients after transfer of in vivo-derived embryos frozen for DT after 5 min versus 20 min exposure to EG. A study in Japan resulted in very similar pregnancy rates after transfer of embryos exposed to EG for periods ranging from 5 to 45 min compared to embryos frozen in glycerol that were thawed and then diluted in a step-wise protocol (Dochi et al. 1998). Contemporary comparisons of exposure time to EG within one laboratory or ET program provide the most powerful evidence. However, comparisons between ET programs also provide some indication that EG is not toxic when exposure time is more than 5 min. In the data set that I collected in 2012 from 5 large commercial ET units in Canada and the US, there was no evidence that exposure time to EG ranging from 4 minutes to 40 minutes had an influence on conception rates (Hasler, J.F., 2012). One last data set was reported from Canada early in the commercial usage of EG for embryo freezing. McIntosh and Hazeleger (1994) reported a pregnancy rate of 59% for a large number of embryos that were exposed to EG for 10 to 20 min prior to freezing in EG.
The data that I have been able to uncover strongly support the principle that toxicity is not an issue when embryos are exposed to EG for periods up to 30 or more minutes. However, there is some evidence that somewhat shorter maximal exposures of 20 min or so should be used at elevated temperatures.
I do not recommend that anyone currently using minimal exposure times of 5 min or so lengthen the timing of their protocol. If a protocol is working well and does not involve serious inconvenience, there is no reason to change, even if science supports the efficacy of different protocols.
References
Dochi, O., Yamamoto, Y., Saga, H., et al. 1998. Direct transfer of bovine embryos frozen-thawed in the presence of propylene glycol or ethylene glycol under on-farm conditions in an integrated embryo transfer program. Theriogenology 49:1051-1058.
Hasler, J.F. 2012. Effect of embryo stage on pregnancy rate following direct transfer of bovine embryos frozen in ethylene glycol. IETS, January 2012, Phoenix, Arizona. (Reprod. Fertil. Dev. 24:131, 2012).
Hasler, J.F., Hurtgen, P.J., Jin, Z.Q. and Stokes, J.E. 1997. Survival of IVF-derived bovine embryos frozen in glycerol or ethylene glycol. Theriogenology 48:563-579.
McIntosh, A. and N.L. Hazeleger. 1994. The use of ethylene glycol for freezing bovine embryos. Theriogenology 41:253.
Matoba, S., Imai, K., Mimaki, Y., Marita, M., Tagawa, M., Dochi, O. and Saito, N. 2004. Toxicity of ethylene glycol on frozen and thawed IVP embryos in direct transfer method. Reprod. Fert. Dev. 16:175-176.
Martínez, A.G., Brogliatti, G.M., Valcarcel, M.A. de las Heras. 2002. Pregnancy rates after transfer of frozen bovine embryos: a field trial. Theriogenology 58:963-972.
Takagi, M., Boediono, A., Saha, S. and Suzuki, T. 1993a. Survival of frozen-thawed bovine IVF embryos in relation to exposure time using various cryoprotectants. Cryobiology 30:306-312.
Takagi, M., Otoi, T., and Suzuki, T. 1993b. Survival of frozen-thawed bovine IVM/IVF embryos in relation to post-thaw exposure time in two cryoprotectants.
Tribulo, H., Rodriguez, P. , Oviedo, J., Ongarato, F., Cuervo, R., Mapletoft, R., and Bó, G.A. 2013. Survival of in vivo-produced bovine embryos exposed to 1.5M ethylene glycol for different periods of time prior to conventional cryopreservation. Proc. of the 17th ICAR, Reprod. In Dom. Anim. 47:456-456.
Voelkel, S.A. and Hu, Y.X. 1992. Direct transfer of frozen-thawed bovine embryos. Theriogenology 37:23-37.
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