28 DOUBLING OOCYTE CYTOPLASM VOLUME INCREASES BLASTOCYST QUALITY FOLLOWING INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER IN ARGALI SHEEP (OVIS AMMON)

Abstract

Interspecies somatic cell NT (SCNT) can be used in the conservation of endangered animals but only when there is an abundant source of compatible oocytes and recipients. The objective of this study was to compare the efficiency of intra- and interspecies SCNT in sheep using zona-free embryo reconstruction methods. Skin fibroblasts from either an argali (Ovis ammon) or control (Ovis aries) ram were used as donor cells for SCNT between passages 2 to 5 and following culture in medium containing 0.5% FCS for 4 to 6 days. Single cells were electrically fused to cytoplasts prepared following enucleation of in vitro-matured zona-free metaphase II-arrested oocytes obtained from domestic ewes. In an additional experiment with argali, a double cytoplast (DC-SCNT) procedure was used whereby a second cytoplast was fused to the first reconstruct within 1 h. Reconstructs were artificially activated ~25 h after the start of maturation using ionomycin and 6-DMAP. Zona-free parthenogenote (PG) control oocytes were activated around the same time. In each treatment, 10 to 12 zona-free embryos where cultured in microwells formed in 20-μL drops of modified synthetic oviduct fluid under oil. Half the medium was replaced on Day 3, and developing embryos were transferred to individual 5-μL drops on Day 6. Development on Day 7 was expressed as a percentage of cleaved embryos. Statistical significance was determined using Fisher’s exact test for embryo development and two-tailed t-test for embryo cell numbers. Total embryo development on Day 7 was significantly greater with intraspecies sheep SCNT compared with interspecies argali SCNT (34/157 = 21.7% v. 34/363 = 9.4%, respectively; P < 0.001). Moreover, SCNT embryo development was significantly less than PG controls (122/360 = 33.9%; P < 0.001). Although argali DC-SCNT had no effect on total embryo development compared with SCNT (9/69 = 13.0% v. 7/56 = 12.5%, respectively), doubling cytoplasm volume increased the proportion of grade 1 and 2 embryos on Day 7 (8.7 v. 0%; P < 0.05). Consequently, DC-SCNT blastocysts had greater numbers of nuclei compared with SCNT (108 ± 47 v. 65 ± 9; n = 6, n = 6, respectively; P = 0.054). In comparison, PG blastocysts possessed on average 122 ± 27 nuclei (n = 7). A sample of 14 argali cloned blastocysts were all confirmed to have been derived from the respective ram after genotyping ~6000 ovine single nucleotide polymorphisms on a custom-made chip (Illumina, San Diego, CA, USA). Chromosome spreads of argali embryos revealed a modal number of 4 bi-armed autosomes as opposed to 6 in Ovis aries. In conclusion, in vitro development following interspecies SCNT in argali was about half that compared with domestic sheep. Blastocyst quality was improved by increasing oocyte cytoplasmic volume facilitated by zona-free cloning. Alternative sources of cytoplasm may further improve development. Confirmation that Ovis aries cytoplasm can fully reprogram a differentiated argali nucleus remains to be determined.

This research was supported by AgResearch Core Funding.