A Closer Look

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 6^th 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’.

References

Coley, Stanley L. 1987. Improving pregnancy rates from frozen embryos. Proceedings of the 6^th 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 10^th Scientific Meeting of the European Embryo Transfer Association (AETE), Lyon, France. pp.228.