332IN VIVO DEVELOPMENTAL CAPACITY OF IN VITRO-PRODUCED EMBRYOS DERIVED FROM SEX-SORTED AND RE-CRYOPRESERVED FROZEN-THAWED RAM SPERM

Abstract

The ability to sort and re-freeze frozen-thawed sperm would significantly increase the potential application of sperm sexing technology to species management. Frozen-thawed, sorted, re-frozen and then thawed ram sperm appear fully functional in vitro with blastocyst production greater than that for frozen-thawed, non-sorted sperm (Hollinshead FK et al. 2003 Theriogenology 59, 209 abst). The aim of this study was to evaluate the in vivo capacity of in vitro-produced embryos derived from frozen-thawed sperm after sorting and a second cryopreservation/thawing step. Frozen semen from 2 rams (n=3 ejaculates per ram) was used throughout. Post-thaw sperm treatments comprised (i) non-sorted (Control); (ii) sorted (Froz-Sort) and (iii) sorted, then re-frozen (Froz-Sort-Froz). X and Y sperm were separated using a high-speed sorter (SX MoFlo®, DakoCytomation, Fort Collins, CO, USA) after incubation with Hoechst 33342 and food dye to eliminate nonviable sperm. Reanalysis revealed high levels (mean±SEM) of purity for X- and Y-enriched samples for all treatments (89±1.2%). At Day 6 post-insemination, 2 embryos (blastocyst stage or greater) were transferred per recipient. Data were analyzed by chi-square and Fisher Exact Test. In vivo embryo survival was similar across sperm treatments (28/64, 43.8% overall) and 20 of 23 (87.0%) sexed lambs were of the predicted sex (Table 1). These results demonstrate high in vivo developmental capacity of in vitro-produced sexed embryos derived from frozen-thawed ram sperm after sorting and a second cryopreservation/thawing step, and increase the potential application of sperm sexing technology. Research support by XY, Inc., Australian Research Council and Zoological Parks Board of NSW. Table 1 In vivo survival of transferred in vitro produced embryos derived from frozen-thawed non-sorted (Control), frozen-thawed and sorted (Froz-Sort) and frozen-thawed, sorted, then frozen-thawed (Froz-Sort-Froz) ram sperm.