A Closer Look

Evidence-based ET

John F. Hasler

Note: I have been using subjects suggested by our board of directors for this column for the past two years.  I would be happy to attempt covering any subjects that the membership might suggest. Let me hear from you!

What is the best synchrony between IVP embryos and recipients?

There is a great deal of evidence that synchrony between the age of in vivo-derived embryos and the day of the estrous cycle in recipients at the time of transfer is affected very little, if at all, when synchrony is within the three day period of 0, plus one or minus one day (zero, meaning day of estrus and age of embryo are the same).  Consequently, the pregnancy rate is not affected when day 7 embryos are transferred into day 6, 7, or 8 recipients.  This holds true for both fresh and frozen in vivo-derived embryos (Hasler, 2001).

Commercial production of bovine embryos through IVF procedures is now being successfully achieved internationally in a number of laboratories and has increased substantially in the USA during the past few years. Today, a routine procedure involves overnight air shipment of in vitro-derived (IVP) embryos maintained in culture medium in a battery-powered incubator.  Upon arrival, the embryos are loaded in straws and transferred into recipients. The choice of day of synchrony, similar to what works for in vivo-derived embryos, i.e., a 3 day span of synchrony works well.  However, the calculation of synchrony with IVP embryos and recipient estrus is skewed one day compared to in vivo-derived embryos, as explained below.

When we started transferring IVP embryos at Em Tran, Inc. in 1992, there were few data available on the possible effect on pregnancy rate of the synchrony between the age of the IVP embryos and the day of estrus in the recipients. Consequently, without regard to their developmental stage or quality, IVP embryos were transferred to zero, plus or minus synchrony recipients (Hasler, 1998). It should be noted that there is a potential for confusion due to the convention of referring to the day of estrus in recipients as Day zero (0) while in IVP systems the day of fertilization is designated as Day 0 (Hasler et al., 1995; Avery et al., 1995).

For example, early blastocysts first start appearing approximately 6 d after fertilization in both in vivo and in vitro. However, due to the difference in the designation of Day 0 in the two systems, this represents Day 6 in vitro and Day 7 in vivo. This is why those of you who receive Day 7 IVP embryos, that were packed and shipped on Day 6, find that many of the embryos have reached stage 7 or the expanded blastocyst stage. These embryos are actually developmentally equivalent to Day 8 in vivo-derived embryos. This means that in determining the degree of synchrony for IVP embryo transfers, zero synchrony involves transferring IVP embryos into recipients whose own embryos would be one day behind in development. This is illustrated in Figure 1.

Figure 1. Comparison of the timing of fertilization (Fert) and embryo development in vivo versus in vitro

      Day of first appearance

Day –  0         1          2          3          4          5          6         7           8          9

In Vivo —      Estrus    Fert                                                                 EB        XB        HB

In Vitro —      Fert                                                                   EB       XB        HB

EB=early blastocyst; XB=expanded blastocyst; HB=hatched blastocyst) (from Hasler, 1998)

Synchrony data resulting from the transfer of 4,598 seven-day old IVP embryos at Em Tran, Inc., are shown in Table 1. The transfer of IVP embryos into recipients that were Day 8 (actually 0 synchrony for Day 7 IVP embryos) resulted in a pregnancy rate of 57.6%, while Day 7 (actually minus 1 synchrony for day 7 IVP embryos) resulted in a 55% pregnancy rate. The 50.3% pregnancy rate that resulted from recipients that were Day 6 (actually minus 2 synchrony for a Day 7 IVP embryo) was significantly lower than both Day 7 and 8 recipients. There were not enough transfers into Day 9 recipients, which would be equivalent to plus 1, to determine a meaningful pregnancy rate.

Also included in Table 1 are pregnancy data from Trans Ova Genetics (unpublished) and Dr. Clay Burnley (unpublished) resulting from the transfer of fresh IVP embryos. In both data sets it is clear that higher pregnancy rates were achieved when IVP embryos were transferred into Day 7 and 8 recipients compared to Day 6. The Trans Ova data were a compilation of transfers at a variety of locations into a combination of heifers and cows of both dairy and beef breeds. The Burnley data were produced in a large Florida dairy.

Lastly, a large data set from Japan is included in Table 1. This study involved transfers into both Holstein cows and heifers and involved roughly equal numbers of both fresh and frozen (10% glycerol + 0.25 M sucrose) IVP embryos.  A higher percentage of heifers (44%) were pregnant compared to cows (33%). Also, the pregnancy rate in Day 8 recipients was significantly higher than either Day 7 or 6 recipients.

The explanation for the outcomes in all four studies is probably due to the fact that minus synchrony of 24 h is actually a 48 h difference between embryo and recipient as depicted in Figure l. The data in Table 1 support the interpretation that the relationship between embryo-recipient synchrony with IVP embryos is different than that observed with in vivo-produced embryos and shown in previous studies involving large numbers of transfers in Holstein heifers (Coleman et al., 1987; Hasler, 2001).

Table 1 – Effect of synchrony between IVP embryo age and recipient estrus on pregnancy rates

^a,b,c < 0.025; ^d,e,f < 0.05

I think that the take home message is that Day 7 IVP embryos can be successfully transferred into recipients showing estrus on days 6, 7 or 8.  However, when a choice is available, Day 7 or 8 and quite possibly Day 9 recipients should be used instead of Day 6.

References

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

Avery, B., H. R. Brandenhoff, T. Greve. 1995. Development of in vitro matured and fertilized bovine embryos, cultured from days 1-5 post insemination in either Menezo-B2 medium or in

HECM-6 medium. Theriogenology 44:935-945.

Coleman, D. A., R. A. Dailey, R. E. Leffel and R.D. Baker. 1987. Estrous synchronization and establishment of pregnancy in bovine embryo transfer recipients. J. Dairy Sci. 70:858-866.

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

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.