Transgenic production from in vivo-derived embryos: Effect on calf birth weight and sex ratio

We examined transgenic-cattle production by DNA microinjection into 1-, 2-, and 4-cell embryos, analyzing the impact on calf size and subsequent viabi lity. Embryos were either collected at an abattoir by flushing oviducts fro m superovulated and artificially inseminated cows (in vivo-derived) or obta ined by in vitro maturation and in vitro fertilization of oocytes aspirated from excised ovaries (in vitro-derived). A human serum albumin (hSA) milk- expression DNA construct was microinjected, either in one of the visible pr onuclei of in vitro- and in vivo-derived 1-cell embryos or in the nuclei of two blastomeres of 2- and 4-cell in vivo-derived embryos. Microinjection-i nduced mortality (lysis and developmental block) was equivalent (similar to 40%) for all microinjected embryos. Embryos were co-cultured with BRL cell s in B-2 medium containing 10% fetal calf serum (FSC). Overall, embryo deve lopment to morulae/blastocysts was significantly greater for in vivo-derive d ova (15.5%) than for in vitro-derived oocytes (9.3%). All morulae and bla stocysts were transferred to synchronized recipient females on Days 6-8 pos t-fertilization. A total of 189 calves were delivered. Birth weights were s ignificantly greater for calves generated from in vitro-derived oocytes com pared with those generated from in vivo-derived oocytes. One transgenic bul l calf was obtained from the microinjection of a 2-cell embryo. Fluorescenc e in situ hybridization (FISH) analysis of lymphocytes detected one transge nic integration site in all cells. Transmission frequency of the hSA transg ene in embryos obtained through IVM/IVF/IVC utilizing the semen of the tran sgenic calf confirmed that it was not mosaic.