Evidence-based ET: Is the exposure time of bovine embryos to ethylene glycol (EG) prior to freezing and/or after thawing critical to survival?

Categories: Evidence-Based ET
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Published on: April 30, 2013

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.



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.

Evidence-based ET: For what time span can bovine embryos be maintained between recovery and freezing without compromising viability?

Categories: Evidence-Based ET
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Published on: December 21, 2012

John F. Hasler

Introduction – After our annual conference in Winnipeg, our AETA Board of Directors asked me if I would be willing to start a column in A Closer Look that deals with practical ET problems and provides published documentation for the subjects and issues in question. I agreed to do this and the following is my first ‘column’. All of us hold personal opinions on numerous technical aspects of our ET profession. In some cases these opinions are based on well-established principles. However, in some cases we all base opinions primarily on ‘clinical impressions’. I believe that such impressions can sometimes be entirely accurate, but I also know, from personal experience, that unless one accumulates an adequate data base, clinical impressions can sometimes turn out to be wrong. Consequently, this column will present information that has some published documentation and is based on more than just my personal opinion or, in fact, undocumented opinions period. Our board members have submitted a number of suggested topics and have given me the freedom to pick among them. I will attempt to adequately document any subject that is covered in this column. Also, please feel free to suggest future subjects that you feel are of interest to most of our membership.

For what time span can bovine embryos be maintained between recovery and freezing without compromising viability?

For a 10 year period from the mid-1980s to the mid-1990s, Em Tran, Inc. shipped many thousands of frozen embryos from Holstein donors in the US to Holland Genetics, The Netherlands. During one phase of this program, we kept very precise track of the time, down to the minute, between flushing and freezing, out to a maximum of 3 hr or 180 min. Of a total of 2,102 embryos frozen in glycerol during this period, the pregnancy rate for embryos frozen between 0-30 min after flushing was 54.1% and did not vary significantly among 30 minute blocks of time out to the 150-180 min group, which had a pregnancy rate of 55.7%.  These embryos were held at ambient lab temperature in Dulbecco’s modified PBS, which we purchased in the powder form and reconstituted in MilliQ water in our lab, adding antibiotics and 10% FCS. The recipients were primarily Holstein cows.

Another data set generated from 3,570 embryos frozen in the Netherlands at Holland Genetics and transferred within the country showed a slightly different outcome (Otter, 1994). The pregnancy rates were 71%, 66% and 57% for embryos held for 0-2, 2-4 or 4-6 hr respectively. These differences were statistically significant.

I recently uncovered a publication on this subject that I had long forgotten about. At the 6th AETA convention in Orlando FL, in 1987, our former AETA president Stan Coley presented some very interesting frozen embryo data from his ET practice in GA. Stan presented data indicating a very high pregnancy rate (75%) for 300 embryos held in in modified PBS + 0.4% BSA and frozen in glycerol within 4 hr post collection. The pregnancy rate was slightly lower for 76 embryos held at ambient temperatures for 4-8 hr before freezing and lower yet (39%) for a small group of 31 embryos held for over 8 hr. Maintaining a total of 91 embryos at 4°C for over 8 hr did not substantially improve the pregnancy rate (46%) compared to the 8 hr group held at ambient temperature.

Pregnancy rates of frozen-thawed embryos are the best index of embryo viability. However, in vitro culture after thawing also can provide meaningful information. In a study conducted at Virginia Polytech (Jousan, 2003), embryos were recovered from donors, maintained in holding medium (OCM; ECHM-500, Immuno-Chemical Products, Ltd.), at either 5 or 22°C for 2, 6 or 12 hr and then frozen in EG. After thawing the embryos were cultured in a commercial IVC medium and examined after 72 hr of culture. There was no difference in the development rates of embryos held for 2 versus 6 hr prior to freezing (86 vs. 79% respectively), but embryos held for 12 hr had a significantly lower survival rate (54%). Holding embryos at 5 versus 22°C prior to freezing did not improve the survival rates for any of the holding periods.

Unfortunately, none of these studies compared different holding media along with different holding times. Certainly, holding medium quality would enter into this issue as perhaps would temperature if it was highly elevated.

If I was still directly involved with freezing embryos commercially, I would feel most comfortable sticking to a 3-4 hr maximum holding time (in a commercial holding medium) whenever possible. However, a few more hours in holding medium might lower subsequent pregnancy rates but probably would not lead to a ‘disaster’.


Coley, Stanley L. 1987. Improving pregnancy rates from frozen embryos. Proceedings of the 6th Annual Convention of the AETA, Orlando, Florida. pp.40-50

Hasler, John F.  2001. Factors affecting frozen and fresh embryo transfer pregnancy rates in cattle. Theriogenology 56:1401-1415.

Jousan, F.D., Utt, M.D., Whitman, S.S., Hinshaw, R.H. and Beal, W.E. 2003. Effects of varying the holding temperature and interval from collection to freezing on post-thaw development of bovine embryos in vitro. Theriogenology 61:1193-1201.

Otter, T. 1994. Pregnancy rate of fresh and frozen-thawed cattle embryos. Proceedings of the 10th Scientific Meeting of the European Embryo Transfer Association (AETE), Lyon, France. pp.228.

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