The objectives of this study were to evaluate the efficiency and reliability of embryo sexing from isolated single blastomeres, and after nuclear transfer to examine the influence of the sex of donor embryos on development in vitro and in vivo up to calving. The sex of the donor embryo was determined by revealing a specific Y DNA sequence by PCR and electrophoresis after isolation of one, two, three, or more than five cells. The efficiency of sex determination was over 90% and reliability was 100% independent of the number of blastomeres used. In a second experiment, sex was determined from a single cell and the other cells were used for nuclear transfer. The effect of sex on in vitro development was studied in 386 male and 314 female reconstructed embryos derived from 19 male and 14 female parent embryos, respectively. Developmental competence in vitro of male and female constructs over 7 days was not statistically different (25.2 and 23.1% blastocysts on day 7, respectively; P > 0.05). After the transfer of predetermined male (n = 30) and female (n = 27) cloned embryos into recipient heifers, no effect of sex was observed on pregnancy rates at day 21, 35 and 90, or on calving rates (P > 0.05). These rates did not differ between single and twin transfer (P > 0.05). The sex of the calves born always corresponded to that determined from a single blastomere. These results show that sex can be determined accurately when using a single blastomere before nuclear transfer and that the sex of the parent embryo does not affect in vitro development or in vivo survival rates of cloned embryos.
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