Effect of two activation treatments and age of blastomere karyoplasts on in vitro development of bovine nuclear transfer embryos

The yield and quality of (a) parthenogenetic blastocysts produced by two ac tivation treatments (cycloheximide [CHX] or 6-dimethylaminopurine [DMAP]) a nd (b) nuclear transfer blastocysts generated using these two activation tr eatments and three different ages of karyoplast derived from day 3, 4, or 5 in vitro produced donor embryos, were examined in order to define an optim al nuclear transfer protocol. The two activation protocols comprised calciu m ionophore followed by either CHX or DMAP. Parthenogenetic blastocyst yiel ds were greater (P <0.001) following activation with DMAP than CHX (59.7 +/ - 5.1 vs. 31.4 +/- 4.5 [mean SEMI). In contrast, nuclear transfer blastocys t rates per fused embryo were lower (P <0.0001) using cytoplasts activated with DMAP. The individual rates using day 3, 4, and 5 donors and using CHX and DMAP activation treatments were 31.9 +/- 5.0, 31.7 +/- 6.2, 20.4 +/- 7. 3 and 27.8 +/- 4.7, 20.1 +/- 7.5, 12.7 +/- 8.3, respectively. Blastocyst ra te per fused embryo was negatively correlated (P=0.0091) with the total num ber of blastomeres. per donor embryo. Despite this inverse relationship, th e calculated potential blastocyst yield per donor embryo was positively cor related (P <0.0048) to karyoplast age. The individual potential yields on d ays 3, 4, and 5 and for the two activation protocols (CHX and DMAP) were 4. 7 +/- 0.8, 7.2 +/- 1.2, 10.1 +/- 2.1 and 3.8 +/- 0.8, 5.5 +/- 2.1, 7.3 +/- 4.1, respectively. One possible explanation for the observed inverse relati onship is that differentiation events during early cleavage are able to red uce the ability of the cytoplast to reprogram the transferred karyoplast an d hence reduce blastocyst yields. The mechanism that mediates the different ial effect of the CHX and DMAP on blastocysts yields between parthenogeneti c and nuclear transfer embryos remains to be elucidated. In conclusion, the results indicate that although activation of oocytes with DMAP can produce a higher percentage of blastocysts, CHX activation is superior for use in nuclear transfer. (C) 2001 Wiley-Liss, Inc.