280LAMBS BORN AFTER IN VITRO EMBRYO PRODUCTION FROM PREPUBERTAL LAMB OOCYTES AND FROZEN-THAWED UNSORTED AND SEX-SORTED SPERMATOZOA

Abstract

Developments in sperm sexing technology have resulted in the birth of a number of offspring after IVF of oocytes from adult animals (Johnson LA, 2000 An. Reprod. Sci. 60–61, 93–107). The aim of this study was to combine sperm sexing technology with juvenile breeding. Merino lambs, 2–3 weeks (n=43) were hormone stimulated (Morton KM et al., 2003 Proc. Soc. Reprod. Fert., P18), and COCs were matured in TCM-199 (Sigma) with 10μgmL−1 p-FSH (Folltropin-V; Bioniche Animal Health Australasia), 10μgmL−1 pLH (Bioniche), and 20% sheep serum (v/v) in a humidified 6% CO2, 5% O2, 89% N2 atmosphere for 22h. Semen collected from Merino rams was diluted and frozen as pellets (Unsorted), or stained with H33342, separated into X and Y sperm using a SX MoFlo (Cytomation Inc., Fort Collins, CO, USA), and frozen as pellets (Sorted). Sperm were prepared for IVF by swim-up under 0.5mL of SOF with 2% sheep serum (v/v; SOF+) for 45min (Unsorted), or diluted in 0.5mL of Sydney IVF Sperm Buffer (Cook IVF, Brisbane, Australia) and centrifuged at 650g for 3min (Sorted). After IVM, oocytes were transferred to SOF+, and cultured with 0.5×106mL−1 (Unsorted) or 1.0×106mL−1 (Sorted) motile sperm for 18h. Presumptive zygotes were transferred to Sydney IVF cleavage and blastocyst medium (Cook IVF) for 3 and 5 days, respectively. Oocyte maturation and fertilization were assessed by orcein staining 18h post-insemination (hpi). Two Day-7 blastocysts were transferred to each recipient ewe (n=9; 3 per group) and pregnancies diagnosed by ultrasound on Day 57 of gestation. Data were analyzed by chi-square test. Oocyte maturation was 83.9% (73/87), and monospermic fertilization did not differ for Unsorted (22/32; 68.7%), X- (6/14; 42.8%), and Y-sperm groups (15/27; 55.6%). Polyspermic fertilization was 9.4% (3/32) and 7.4% (2/27) for the Unsorted and Y groups. Cleavage was reduced with X- and Y-sperm compared with Unsorted, but blastocyst formation (from cleaved oocytes) did not differ (Table 1). There were three (100%), zero (0%), and one (33.3%) pregnancies from Unsorted, X- and Y-embryos, respectively, all of which survived to birth, demonstrating that juvenile breeding can be successfully combined with sperm sexing. Table 1 Cleavage and blastocyst formation after IVF with Unsorted, X- or Y-sperm. Values in parenthesis are percentages