Pregnancies produced by bovine embryos cloned by nuclear transfer

Abstract

With the aim of obtaining pregnancies from nuclear transfer embryos reconstituted with somatic cells and enucleated oocytes, bovine oocytes from slaughterhouse were matured in vitro and enucleated by micromanipulation. Nuclear donor cells were obtained from the ear of an adult cow, cultured for 9 to 14 days and cryopreserved in liquid nitrogen. Confluent cells were inserted individually in the perivitelline space of enucleated oocytes and treated with 2 electric pulses to induce fusion. The reconstituted zygotes were then cultured for 2 hours and treated with ionomycin and 6-dimethylaminopurine for their activation. The embryos were cultured in synthetic oviduct fluid for 8 to 9 days to obtain the blastocyst stage. These blastocysts were transferred to recipient heifers 7 to 9 days after oestrus. The ultrasound pregnancy diagnosis was done about 30 days after recipient oestrus. Five pregnancies were obtained from 25 transfers (20%). Two of them were lost at 42 days and a third at 120 days. This seems to be the first report on pregnancies obtained by cloned embryos in Chile.