71 THE DEVELOPMENT OF INTRA- AND INTER-SPECIES CLONED EMBRYOS DERIVED FROM RABBIT OOCYTES: THE EFFECT OF DONOR CELL SOURCES

Abstract

Generating cloned embryos using rabbit oocytes as the recipient cytoplasm is useful for studying the interaction between a foreign donor nucleus and the recipient cytoplasm. The aims of the study were to (1) investigate the development of inter-species cloned embryos derived from rabbit oocytes, using cow (no. 1 and no. 2), swamp buffalo, pig, and elephant ear fibroblasts as donor cells, and (2) observe the developmental capacity of cloned rabbit embryos derived from adult and fetal fibroblasts. Mature oocytes were collected from superovulated rabbit does by oviductal flushing. A single fibroblast (passages 3–5) in the starvation stage (1–3 days prior to nuclear transfer) was transferred into an enucleated oocyte, which was confirmed by Hoechst staining. Reconstructed oocytes were later fused by electrical stimulation using 3 DC pulses, 3.2 kV.cm^-1, for 20 µs. Fused couplets were activated by inducing the same electrical stimulation, and subsequently incubated in activation medium comprised of 5 µg mL^-1 cycloheximide, 2 mM mL^-1 6-DMAP and 10% fetal bovine serum (FBS), in synthetic oviduct fluid (SOF), for 1 h. They were then cultured in SOF supplemented with 10% FBS for 5 days. Differences in the percentages of fusion and embryo development to a particular stage between source of donor cells were determined by chi-square analysis. The fusion rates of cow no. 1 (62%; 82/133), cow no. 2 (54%; 41/76), buffalo (63%; 53/84), pig (69%; 60/87), elephant (59%; 44/74), adult rabbit (55%; 41/74), and fetal rabbit couplets (70%; 56/80) did not differ significantly. The cleavage rates of cows no. 1 and 2, buffalo, pig, and elephant embryos were 33%, 83%, 57%, 67%, and 100%, respectively. The rabbit fibroblasts derived from an adult gave a significantly lower cleavage rate than those derived from a fetus (54% vs. 95%) (P < 0.05). The developmental rates at morula and blastocyst of cloned embryos reconstructed from fetal rabbit fibroblasts were significantly greater than those reconstructed from adult rabbit, buffalo, pig, and elephant (morula: 39%, 12%, 4%, 7%, and 14%; blastocyst: 23%, 7%, 0%, 3%, and 9%, respectively; P < 0.05). While cow no. 2 donor cells provided a significant embryo development success rate in terms of blastocyst formation, cow no. 1 cloned embryos could not develop beyond the cleavage stage (morula: 10% vs. 0%; and blastocyst: 7% vs. 0%). In conclusion, cow, swamp buffalo, pig, and elephant fibroblasts can be produced by using enucleated rabbit oocytes as recipient cytoplasm. The developmental capacity of cloned embryos is affected by the sources and the individual ability of donor cell used, and varies according to the species.

This study was supported by Ratchadaphiseksomphot Endowment Fund, Reproductive Biotechnology Research Unit, Chulalongkorn University, and Royal Golden Jubilee, PhD program of Thailand Research Fund.