109. AGGREGATING CLONED WITH IN VITRO FERTILISED EMBRYOS RESULTS IN CHIMAERAS AND IMPROVED FETAL SUVIVAL IN CATTLE

Abstract

Cloning cattle by somatic cell nuclear transfer (NT) results in low survival and high frequencies of abnormal placentation and fetal development. We postulate that such anomalies may be overcome by complementing NT embryos with in vitro fertilised (IVF) embryos to form chimaeras. The gender and germline composition of chimaeras can be experimentally manipulated. Using embryological methods, we aim to produce chimaeric fetuses that are functionally male and produce sperm derived from the somatic NT embryo. Provided sufficient contribution from the IVF embryo, such chimeras should develop more normally than clones. At the 12- to 16-cell stage, individual male NT embryos were aggregated with female IVF embryos derived from X-sorted sperm. Following aggregation, there were no significant difference in blastocyst development between NT/IVF aggregates and disaggregated and re-aggregated IVF and NT controls (86/183 = 47% v. 77/233 = 33% v. 47/109 = 43%, respectively). Suitable quality embryos were transferred individually into synchronised recipient animals. Pregnancy establishment at Day (D) 35 was not significantly different between aggregate, IVF and NT groups (18/57 = 32% v. 11/45 = 24% v. 6/31 = 19%, respectively). Whilst there was no difference in survival between aggregates and IVF controls to ~D100, aggregates survived significantly better than NT controls (16% v. 18% v. 0%; respectively; P < 0.05). In the aggregate group, 7/8 fetuses recovered were phenotypically male. Using RT-PCR, expression of the female-specific mRNA for Xist was detected in 4/5 liver samples, indicating chimaerism. Despite improved survival to ~D100 compared to NT, 3/7 fetuses in the aggregate group still displayed evidence of abnormalities, such as fetal overgrowth. Further studies will explore alternative aggregation strategies and germline transmission of the NT-derived genome in chimaeras.