92 INTER-GENERIC MARBLED CAT AND FLAT-HEADED CAT CLONED BLASTOCYSTS GENERATED FROM DOMESTIC CAT AND RABBIT OOCYTES

Abstract

Marbled cats (Pardofelis marmorata; MC) and flat-headed cats (Prionailurus planiceps; FC), the endangered, small wild cats of southeast Asia, are currently at risk of becoming extinct in Thailand. Producing embryos by inter-generic somatic cell nuclear transfer (SCNT) is a way to preserve their genetics, as well as to understand their embryo chronology. The aim of the study was to investigate the development of MC and FC embryos produced from domestic cat (DC) and rabbit (RB) oocytes by SCNT. Intra-species cloned DC served as control. The MC-, FC-, DC-DC, and FC-RB (donor fibroblast cell–recipient oocyte) couplets were prepared as previously described (Thongphakdee et al. 2006 Reprod. Dom. Anim. 41, 219–226), but MC-, FC-, and DC-DC couplets were fused by inducing 3 direct current pulses of 2.4 kV cm^-1, for 50 µs, and subsequently activated in synthetic oviduct fluid (SOF) medium, supplemented with 10% fetal bovine serum (FBS), 10 µg mL^-1 cycloheximide, and 5 µg mL^-1 cytochalasin B, for 4 h. Activated couplets were cultured in SOF medium supplemented with 5% FBS, at 38.5°C, under 5% CO₂ in air, for 5–7 days. Differences in the percentages of fusion and embryo development to a particular stage between species were determined by chi-square analysis. The fusion efficiency of DC-DC couplets (38%; 36/94) was significantly lower than those of MC-DC (74%; 60/81), FC-DC (79%; 60/76), and FC-RB (78%; 47/60) couplets (P < 0.05). No differences in the cleavage rates of MC-DC, FC-DC, FC-RB, and DC-DC fused couplets were found (93%, 97%, 87%, and 89%, respectively). The FC-DC couplets yielded a greater percentage of morulae (53%) than did MC-DC (23%), FC-RB (25.5%), and DC-DC (11%) couplets (P < 0.05). However, the number of couplets that achieved blastocyst expansion did not significantly differ (MC-DC: 5%, FC-DC: 8.3%, FC-RB: 8.5%, and DC-DC: 8.5%). These results demonstrate that MC and FC embryos are successfully produced with reasonable blastocyst formation rates by inter-generic SCNT.

This study was supported by the Zoological Park Organization under the Royal Patronage of H.M. the King, Reproductive Biotechnology Research Unit, Chulalongkorn University, and Royal Golden Jubilee, PhD program of Thailand Research Fund.