Evidence-based ET: What is the best protocol for cryopreserving IVF-derived bovine embryos?

Evidence-based ET: What is the best protocol for cryopreserving IVF-derived bovine embryos?

John F. Hasler

Note: The title of this column was suggested by our AETA board of directors

There has been concern regarding the best way to cryopreserve bovine IVF-derived embryos ever since commercial in vitro embryo production (IVP) started in the early 1990s. Donors in the early days were primarily infertile, problem donors, and annual IVP embryo production in the United States was limited to a few thousand embryos, at most. Production of IVP embryos has increased substantially in recent years, and in 2013, it was reported that 48,112 embryos were produced from OPU collections compared with 301,671 in vivo embryos collected from superovulated cattle. Thus, 13.8% of the total embryos produced were from IVF procedures and 55% of them were reported to have been frozen. It is anticipated that reported IVP production will be substantially higher in 2014. Understandably, the companies providing IVF services are reluctant to share details of their cryopreservation services. However, because fresh IVP embryos are often shipped overnight to ET practitioners/donor owners for transfer on-farm, embryo numbers sometimes exceed the number of available recipients. Consequently, not infrequently, practitioners are faced with cryopreserving leftover IVP embryos. Even today, however, there does not seem to be any widely agreed upon, best protocol for cryopreserving IVP embryos. In the last few years it has been publically reported that IVP embryos have been commercially cryopreserved by vitrification and slow freezing with both ethylene glycol (EG) and glycerol used as the cryoprotectants.

The IVF program at Em Tran, Inc., established in 1992, involved infertile/problem donors exclusively, meaning relatively small numbers of oocytes were usually recovered. Consequently, there was a high priority on producing pregnancies and almost all embryos were transferred fresh. A few IVP embryos were frozen in glycerol and then thawed and transferred at a later date. Day 60 pregnancy rates were 56% for 4,606 fresh and 42% for 67 cryopreserved IVP embryos (Hasler, 1998). The 14 percentage point drop is quite a bit larger than what we usually see when comparing fresh versus frozen in vivo-derived embryos. Most of the Em Tran IVP embryos were grade 1, stage 6–8 blastocysts, and although all were frozen in glycerol and the records are complete and accurate, I have no recollection as to why I chose glycerol versus EG as the cryoprotectant. In those early days, we also performed OPU/IVF on some Holland Genetics-owned donors that failed to superovulate successfully. Holland Genetics requested that we freeze their IVP embryos in glycerol for export, even though EG had largely replaced glycerol as the cryoprotectant of choice for exported in vivo embryos. Holland Genetics did not provide me with data supporting this preference of glycerol versus EG. Note that glycerol is normally removed in 3 steps between thawing and transfer and this necessitates a trained embryologist and a microscope; this is not done when EG is used as the cryoprotectant.

Holland Genetics, now known as CRV, continues today to cryopreserve their IVP embryos in glycerol. In an experiment, on which I collaborated, involving testing a carbon-activated air filtration system (CODA), it was reported that the pregnancy rate for 381 fresh IVP embryos was 46% compared with 41% for 337 cryopreserved IVP embryos (grade 1, stage 5–8; Merton et al., 2007). The 5 percentage point (11%) difference is impressively small, and both pregnancy rates also are noteworthy considering that most of the embryos were transferred into Holstein cows on-farm.

An approach to vitrification of bovine embryos in 0.25 mL straws for direct transfer (DT) was developed at Colorado State University (Campos-Chillòn et al., 2006). Survival following vitrification, in straw-dilution, and culture was moderate for IVP morulae, slightly better for blastocysts, and excellent for in vivo-derived embryos. This system as not been adopted by the commercial ET industry, and there are indications that pregnancy rates for IVP embryos have not been acceptable.

It is very clear that vitrification of bovine IVP embryos that does not involve DT is not a viable approach today owing to the demanding protocol (involving several media and very precise timing) necessary to successfully warm (thaw) them in the field. This leaves us with the question of whether we ought to be using EG (or another rapidly penetrating cryoprotectant) for convenience of DT at thawing, or glycerol because it is demonstrably better, but requires step-wise removal!

In the 1990s a number of studies involving the slow freezing of bovine IVP embryos were published in Japan. Han et al. (1994) reported high survival following thawing and culture of IVP embryos frozen in 10% glycerol and 50% fetal bovine serum (FBS). Grade 1 blastocyst stages 5–8 exhibited similar, high survival rates. Aoki et al. (2004) used 10% glycerol, 20% serum, and 0.25 M sucrose for slow freezing bovine IVP day-7 and 8 (stage 7, grade 1). They reported an overall pregnancy rate of 37.3% following transfer of 3,953 fresh and 3,298 frozen embryos, with no difference in pregnancy rates of fresh versus frozen. The overall pregnancy rate in recipients was 44% for heifers versus 33% for cows. Lastly, Suzuki et al. (1993) tested four rapidly penetrating cryoprotectants for slow freezing of bovine OPU embryos. Survival of OPU embryos frozen in 1.3 M methyl cellosolve, 1.1 M diethylene glycol, 1.8 M EG, 1.6 M propylene glycol were very similar, whereas survival was very poor for 1.1 M butylene glycol. Pregnancy rates following transfer of relatively modest numbers of embryos were highest for 1.8 M EG (74%, 20/27), whereas the other cryprotectants were significantly lower.

It is now been proven with field data that in vivo-derived blastocysts (stages 6 and 7) frozen in EG for DT lead to lower pregnancy rates than stages 4 and 5. It is also a fact that many of the highest quality embryos produced in IVP systems are stage 6 and 7 on day 7. Stage 7 IVP embryos are frequently received in shipments from IVF laboratories by ET practitioners. The sum of the data in the literature do not clearly show that late blastocysts survive slow freezing at a lower rate than stages 4 and 5. We do know that fresh stage 7 IVP blastocysts result in high pregnancy rates compared with stages 4 and 5, but this may very well not be the case with slow frozen stage 7 blastocysts. Barfield (2014) presented a comprehensive review of the literature involving the cryopreservation of expanded blastocysts and the various approaches to reducing the volume of blastocoelic fluid. Barfield found that aspiration of blastocoelic fluid (resulting in collapsing of the blastocyst) greatly improved survival (50.5%) when the embryos were frozen in 1.5 M EG + 0.1 M sucrose compared with control embryos that were not collapsed (26.3%). However, collapsing blastocysts with the aid of a micromanipulator and micro-aspiration syringe is not a practical approach for commercial bovine programs involving a large numbers of embryos. And, it is definitely not practical for practitioners receiving embryos by courier and having to deal with them on farm. However, another approach to reducing the potential deleterious influence of a large volume of blastocoelic fluid is to use an osmotic approach. Iwayama et al. (2011) reported improved survival of human blastocysts that were collapsed with a sucrose treatment compared with non-collapsed controls.

Several practitioners have recently informed me that they have received recommendations to expose stage 6 and 7 IVP blastocysts to 1 M sucrose for 1 minute prior to equilibrating them in 1.5 M EG. Although I have no personal experience with this protocol, it is certainly worth utilizing until a published account demonstrates something better. I think that it would be interesting to see if the addition of robust amounts of FBS (10 to as much as 50%) would improve survival of bovine IVP embryos. At the recent European ET (AETE) meetings in Dresden, Germany, there were presentations on four European bull studs regarding programs involving biopsy for genomic testing of in vivo- and in vitro-derived bovine embryos. One of the studs showed a substantial improvement in survival (based on pregnancy rates) of biopsied in vivo embryos in which serum (amount not specified) was added to the EG freeze medium. This, combined with the two Japanese reports cited above, makes me think that someone should investigate this further. Although serum cannot be used for frozen embryos to be exported, there is nothing to prevent its use for freezing OPU embryos destined for domestic use.


Aoki, S, Murano, S, Miyamura, M, Hamano, S, Terawaki, Y, Dochi, O, and Koyama, H. 2004. Factors affecting of embryo transfer pregnancy rates of in vitro-produced bovine embryos. Reprod. Fertil. Dev. 16:206.


Barfield, JP. 2014. Improving post-thaw viability of bovine embryos by collapsing the blastocoel prior to cryopreservation. Proceedings of the Joint AETA/CETA Conference, Middleton, WI, pp. 46–48.


Campos-Chillòn, LF, Walker, DG, de la Torre-Sanchez, JF, and Seidel, GE Jr. 2006. In vitro assessment of a direct transfer vitrification procedure for bovine embryos. Theriogenology 65:1200–1214.


Han, YM, Yamashina, H, Koyama, N, Lee, KK, and Fukui, Y. 1994. Effects of quality and developmental stage on the survival of IVF-derived bovine blastocysts cultured in vitro after freezing and thawing. Theriogenology 43:645–654.


Hasler, JF. 1998. The current status of oocyte recovery, in vitro embryo production and embryo transfer in domestic animals with an emphasis on the bovine. J. Anim. Sci. 76 (Suppl. 3):52–74.

Iwayama, H, Hochi, S, and Yamashita, M. 2011. J. Assisted Reprod. Gen. 28:355–361.

Merton, JS, Vermeulen, ZL, Otter, T, Mullaart, E, de Ruigh, L, and Hasler, JF. 2007. Carbon-activated gas filtration during in vitro culture increased pregnancy rate following transfer of in vitro-produced bovine embryos. Theriogenology 67:1233–1238.

Suzuki, T, et al. 1993. Pregnancy rate and survival in culture of in vitro fertilized bovine embryos frozen in various cryoprotectants and thawed using a one-step system. Theriogenology 40:651–659.

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