EFFECTS OF LOW-TEMPERATURES ON IN-VITRO PRODUCED BOVINE ZYGOTES

The objective of this research was to investigate the effects of cooli ng on the development of bovine zygotes. One-cell bovine embryos were maintained at 39 degrees C (control), 20 degrees C, 10 degrees C, or 0 degrees C for 5, 10, or 20 minutes, then cultured in vitro for 7 days and the proportion of embryos developing to the compact morula or bla stocyst stage compared between different treatments. Duration of expos ure time had no effect on development. Development rates to the compac t morula or blastocyst stage were 3.9%, 11.4%, 17.4%, and 24.4% for zy gotes maintained at 0 degrees C, 10 degrees C, 20 degrees C, and 39 de grees C, respectively, with differences in embryo yield between every treatment (P < 0.05). In a second experiment, bovine pronuclei (karyop lasts) and cytoplasts were cooled at 0 degrees C or maintained at 39 d egrees C for 5 minutes. Pronuclear transplantation was then utilized t o create 4 types of reconstructed embryos, those with: 1) non-cooled p ronuclei and non-cooled cytoplasm, 2) non-cooled pronuclei and cooled cytoplasm, 3) cooled pronuclei and non-cooled cytoplasm, and 4) cooled pronuclei and cooled cytoplasm. The proportion of embryos developing to the blastocyst stage was highest when non-cooled pronuclei were tra nsferred into non-cooled cytoplasm (18.9%), and similar to that of non -cooled, non-manipulated control zygotes (13.2%, P > 0.05). No embryos developed to the blastocyst stage when pronuclei (cooled or non-coole d) were transferred into cooled cytoplasm. However, zygotes with coole d pronuclei transferred into non-cooled cytoplasm yielded 4.5% blastoc ysts (P < 0.05). More embryos developed to the compact morula or blast ocyst stage when non-cooled vs. cooled cytoplasm was utilized, regardl ess of whether the pronuclei were cooled (P < 0.05). These data demons trate that pronuclei are more tolerant to low temperature exposure tha n is ovum cytoplasm. (C) 1997 Wiley-Liss, Inc.