Effects of follicular size of cytoplast donor on the efficiency of cloning in cattle

In cattle, oocytes obtained from follicles smaller than 3 mm in diameter can undergo maturation in vitro, progressing to MII and undergoing fertilization, but are developmentally incompetent. Cytoplasts were prepared from in vitro matured oocytes aspirated from small (1-3 mm) or large (6-12 mm) follicles and fused to serum starved mural granulosa cells. Following activation, reconstructed embryos were cultured for 7 days and classified G1 to G4, before being processed for nuclei counting or transferred to synchronized recipients. Oocytes from small follicles had lower rates of polar body extrusion (59.6 vs. 69%; 731/1230 vs. 608/857) and fusion (71.4 vs. 78.8%; 360/497 vs. 364/465; P < 0.06). There were no differences in total rate of blastocysts development (60 vs. 59.8%; small vs. large), or any grade classification. A significant interaction was detected between follicle size and embryo grade with G3 embryos from small follicles having a greater cell number. Developmental competence of G1 and G2 embryos did not differ at day 27 (48 vs. 46%; 16/33 vs. 17/37; small vs. large). Although there were no differences in fetal size between the two groups, differences in allantois length (53 vs. 86 mm; small vs. large; P < 0.002) and allantois width (9.5 vs. 13 mm; small vs. large; P < 0.06) were seen. No differences in survival to term (2/13 in each group) were observed. These results indicate that cytoplasts from follicles of 1-3 and 6-12 mm in diameter are equally developmentally competent when used in a nuclear transfer procedure.