PRODUCTION OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS USING MALE AND FEMALE LLAMA (LAMA GLAMA) CELL LINES

M. SANSINENA, S.A. TAYLOR, P.A. TAYLOR, R.S. DENNISTON, R.A. GODKE

Auckland, Nueva Zelanda

Congreso; International embryo transfer society (IETS) meetings; 2003

IETS

Interspecies nuclear transfer can be used as a tool to produce embryos in endangered species and also in species where oocyte availability is limited. In this laboratory, we have previously used the interspecies model to study the effect of different horse cell lines on in vitro embryonic development (Sansinena et al., 2001, in press). Due to the limited availability of llama oocytes, we used the interspecies approach to produce nuclear transfer llama embryos, and to compare female and male adult llama fibroblast cell lines by monitoring in vitro embryo development. Two male and female (treatment A & B) adult llama fibroblast cell lines were evaluated. All cell lines used were between 2rd and 3th passage, serum starved for at least 3 consecutive days. Cell line identity was blinded until the culmination of the experiment, to avoid bias. Results are summarized in table 1. Of a total of 585 bovine metaphase-II stage oocytes, 130 were randomly assigned to activation only control group (Control D). Of the remaining 455 oocytes, 438 (96%) survived enucleation. Of these, 124 were assigned to an enucleation/activation control group (Control C). The remaining enucleated oocytes were assigned to the following cell treatments: 164 were reconstructed with female llama fibroblasts (Treatment A), 150 were reconstructed with male llama fibroblasts (Treatment B), and all reconstructed couplets were fused with two pulses of direct current (1.9 to 2.3 kV/cm, 30 msec each). Fusion was confirmed by microscopic examination, and activation was immediately performed by 5-minute exposure to 5mM ionomycin followed by 3-hour incubation in cycloheximide (10mg/ml) in CR1aa medium. All embryos were cultured in 35 ml drops of CR1aa under mineral oil at 38.5ºC in 90% N2, 5% O2, and 5% CO2 for 7 days. There was no significant difference in fusion or cleavage rates between treatments. However, there was a significant difference (P < 0.05) in the number of lysed couplets post activation between treatments and controls. From fused units, the proportions of those completing the first mitotic cycle were 53%, 51% 62%, and 67% for Treatments A, B, Controls C and D, respectively. Embryos from Treatments A and B progressed to the 8 to 16 cell stage, but did not develop further in vitro. Presence of nuclei was confirmed using Hoechst 33258. Our results indicate that the cow oocyte is capable of activation of mRNA after transfer of a llama donor karyoplast, and supporting the first mitotic cycles in vitro. Although embryos developed beyond the first mitotic cycles, and in vitro block was observed between 8-16 cell stage. Further research is needed to determine the reason for this in vitro block in interspecies nuclear transfer embryos.