Nuclear transfer in cattle: Effect of nuclear donor cells, cytoplast age, co‐culture, and embryo transfer

Abstract

There are many factors affecting the efficiency of nuclear transfer technology. Some are evaluated here using our novel approach by enucleating oocytes at 20-22 hr after in vitro maturation (IVM), culturing the enucleated oocytes (cytoplasts) for 8-10 hr or 18-20 hr to gain activation competence and then conducting nuclear transfer. In the first experiment, we demonstrated that cumulus cell (CC) monolayer can support some cloned embryos to develop into morulae or blastocysts. Co-culture with CC and bovine oviduct epithelial cell (BOEC) monolayers resulted in no differences (P > 0.05) in supporting the development of cloned embryos (Experiment 2). When in vitro matured oocytes were enucleated at 22 hr after IVM followed by nuclear transfer 18-20 hr later, cleavage and morula or blastocyst development of the cloned embryos were similar to those resulting from the enucleated oocytes which had been matured in vivo (Experiment 3). Frozen embryos as nuclear donor cells worked equally well as fresh embryos for cloning in embryo development which was superior to IVF embryos (Experiment 4). However, fresh embryos resulted in a higher proportion (P < 0.05) of blastomere recovery than did frozen of IVF embryos. Finally, embryo transfer of cloned embryos from our procedure produced a viable calf, demonstrating the commercial value of this novel approach of the technology.

Beneficial effect of oocyte activation prior to and during nuclear transfer in cattle using in vitro matured oocytes 24 h of age

“…Our study supports the observation that activation of recipient cytoplasts prior to nuclear transfer improves development of cloned embryos (Barnes et al, 1993;Campbell et al, 1994;Kono et al, 1994;Stice et al, 1994). It is known that the co-culture system was effective in promoting development of bovine embryos in vitro (Eyestone and First, 1989;Hernandez-Ledezma et al, 1993) including cloned embryos (Barnes et al, 1993;Van Stekelenburg-Hamers et al, 1993;Yang et al, 1993a;Kono et al, 1994).…”

“…Indeed, electrical activation of the in vitro matured bovine oocytes was found to be oocyteage-dependent and poor activation results were obtained with oocytes matured in vitro less than 30 h (Ware et al, 1989;Yang et al, 1991 Thus, at one time, most nuclear transfer programs involved using aged oocytes as recipients (Willadsen et al, 1991;Westhusin et al, 1992;Yang et al, 1993a). More recently, improvements on activation procedures have resulted in suc-cessful activation and led to the use of young oocytes for nuclear transfer (First et al, 1992;Barnes et al, 1993;Yang et al, 1993b;Aoyagi ef al, 1994;Heyman et al, 1994;Stice et al, 1994).…”

“…Detailed procedures for nuclear transfer have been described previously (Prather et al, 1987;Stice and Robl, 1988;Yang et al, 1992a;1993a). In this study oocyte enucleation and blastomere insertion were conducted separately 6-7 h apart This effect of the cumulus cell co-culture system reduced embryo cleavage development as compared to that of the BRL cells system (53 vs 66%, p < 0.05).…”

“…Previous nuclear transfer studies involved introducing a donor nucleus into an enucleated mature (metaphase II or Mil) oocyte. Activation was usually conducted at the time of membrane fusion applied by electric stimulation (Willadsen, 1986;Prather et al, 1987Prather et al, , 1989; Stice and Robl, 1988;Smith and Wilmut, 1989;Yang et al, 1992aYang et al, , 1993a; Stice and Keefer, 1993). Because of the high level of maturation promoting factor (MPF) in the Mll oocyte, the transferred nucleus would be quickly forced into metaphase, known as premature chromosome condensation (PCC) (Collas and Robl, 1991;Collas et al, 1992a; Barnes et al, 1993;Campbell et al, 1993).…”

Viability of cloned bovine embryos after one or two cycles of nuclear transfer and in vitro culture

“…Developmental rates to the blastocyst stage similar to those reported using in vivo procedures have been obtained in vitro (3), suggesting that temporary passage in the reproductive tract of nuclear transfer embryos is not necessary. In vitro derived donor embryos have previously been shown to support the development of single generation nuclear transfer embryos with a potential to produce live offspring (3,5,10). Although some authors have reported reduced development from in vitro donor embryos (10), others have shown similar development rates of nuclear transfer embryos derived from in vitro and in vivo donor embryos (2,3).…”