81 THE EFFECT OF FOLLICULAR AND OVIDUCT OOCYTES ON THE DEVELOPMENT OF RABBIT NUCLEAR TRANSFERRED EMBRYOS IN VITRO

Abstract

This study was designed to examine the effect of rabbit oocytes collected from oviducts v. follicles on the developmental potential of nuclear transplant (NT) embryos. Rabbit oocytes were flushed from the oviducts (oviduct oocytes) or collected from the ovarian Graafian follicles(follicular oocytes) of superovulated does at 12 h post-hCG injection (hpi). Cumulus cells were then removed from the oocytes by incubation in 0.5% hyaluronidase and pipetting. Oocyte enucleation was conducted in TCM-199 +10% fetal bovine serum (FBS) and confirmed under fluorescent microscopy. Skin fibroblasts from an adult rabbit were prepared and cultured to passage 8 to 10 before use as nuclear donors. A donor cell with a diameter of approximately 15 to 19 μm was transferred into the perivitelline space of an enucleated oocyte and subsequently fused with the recipient oocyte by applying 3 direct current pulses at 3.2 kV cm-1 for 20 μs per pulse. Fused oocytes were activated by the same electrical stimulation described above, and then cultured in TCM-199 + 10% FBS containing 2.0 mM 6-DMAP and 5 μg mL-1 cycloheximide for 1 h. Cloned embryos were cultured in 2.5% FBS B2 medium in 5% CO2 and 95% humidified air at 38.5°C for 3 d. Embryo development to cleavage (2- to 4-cell), 8-cell, and morula/blastocyst (Mor/BL) stages was evaluated. The data were analyzed by the General Linear Model procedure (SPSS 11.0, SPSS Inc., Chicago, IL, USA).The total number of oocytes collected per animal was 27.6 ± 1.3, with 47.8% from oviducts, and 52.2% from follicles. The percentage of oviduct oocytes that showed the first polar body was 98.3% (n = 150) at the time of collection, whereas follicular oocytes only had 54.8% at collection (n = 93), but it reached 92.4% when immature follicular oocytes were cultured for 3 h in vitro. The enucleation rates were similar between the follicular (82.7%) and the oviduct (79.1%) groups. Table 1 shows that a significantly higher fusion rate was found in follicular oocytes compared with that in the oviduct group (90.8 v. 63.4%; P < 0.05). There was no difference in the cleavage rate and Mor/BL development between the 2 groups, although the 8-cell(78.4 v. 63.9%; P = 0.11) and the overall efficiencies (30.6% v. 17.9%; P = 0.14) appeared higher in the follicular group. These results demonstrated that rabbit follicular oocytes at 12 hpi have potential equivalent or maybe better (fusion) than that with oviduct oocytes for promoting the preimplantational development of NT embryos. Table 1.The effect of follicular and oviduct oocytes on the development of rabbit NT embryos Supported by NIH1R43 RR023774-01A1 and 5R44HL091605-03.