Embryo transfer as a tool for improving fertility of heat-stressed dairy cattle

The overall objective of the current proposal is to develop procedures to improve the pregnancy rate achieved following transfer of fresh or cryopreserved embryos produced in the laboratory into heat-stress recipients. The overall hypothesis is that pregnancy rate in heat-stressed lactating cows can be improved by use of embryo transfer and that additional gains in pregnancy rate can be achieved through development of procedures to cryopreserve embryos, select embryos most likely to establish and maintain pregnancy after transfer, and to enhance embryo competence for post-transfer survival through manipulation of culture conditions. The original specific objectives were to 1) optimize procedures for cryopreservation (Israel/US), 2) develop procedures for identifying embryos with the greatest potential for development and survival using the remote monitoring system called EmbryoGuard (Israel), 3) perform field trials to test the efficacy of cryopreservation and the EmbryoGuard selection system for improving pregnancy rates in heat-stressed, lactating cows (US/Israel), 4) test whether selection of fresh or frozen-thawed blastocysts based on measurement of group II caspase activity is an effective means of increasing survival after cryopreservation and post-transfer pregnancy rate (US), and 5) identify genes in blastocysts induced by insulin-like growth factor-1 (IGF-1) (US). In addition to these objectives, additional work was carried out to determine additional cellular determinants of embryonic resistance to heat shock. There were several major achievements. Results of one experiment indicated that survival of embryos to freezing could be improved by treating embryos with cytochalasin B to disrupt the cytoskeleton. An additional improvement in the efficacy of embryo transfer for achieving pregnancy in heat-stressed cows follows from the finding that IGF-1 can improve post-transfer survival of in vitro produced embryos in the summer but not winter. Expression of several genes in the blastocyst was regulated by IGF-1 including IGF binding protein-3, desmocollin II, Na/K ATPase, Bax, heat shock protein 70 and IGF-1 receptor. These genes are likely candidates 1) for developing assays for selection of embryos for transfer and 2) as marker genes for improving culture conditions for embryo production. The fact that IGF-1 improved survival of embryos in heat-stressed recipients only is consistent with the hypothesis that IGF-1 confers cellular thermotolerance to bovine embryos. Other experiments confirmed this action of IGF-1. One action of IGF-1, the ability to block heat-shock induced apoptosis, was shown to be mediated through activation of the phosphatidylinositol 3-kinase pathway. Other cellular determinants of resistance of embryos to elevated temperature were identified including redox status of the embryo and the ceramide signaling pathway. Developmental changes in embryonic apoptosis responses in response to heat shock were described and found to include alterations in the capacity of the embryo to undergo caspase-9 and caspase-3 activation as well as events downstream from caspase-3 activation. With the exception of IGF-1, other possible treatments to improve pregnancy rate to embryo transfer were not effective including selection of embryos for caspase activity, treatment of recipients with GnRH.and bilateral transfer of twin embryos. In conclusion, accomplishments achieved during the grant period have resulted in methods for improving post-transfer survival of in vitro produced embryos transferred into heat-stressed cows and have lead to additional avenues for research to increase embryo resistance to elevated temperature and improve survival to cryopreservation. In addition, embryo transfer of vitrified IVF embryos increased significantly the pregnancy rate in repeated breeder cows.