Birth of lambs of a pre-determined sex after in vitro production of embryos using frozen–thawed sex-sorted and re-frozen–thawed ram spermatozoa

The characteristics and functional capacity of ram spermatozoa frozen–thawed prior to and after flow cytometric sorting was assessed after incubation (37 °C; 6 h), in vitro fertilisation (IVF), and transfer of fresh and vitrified in vitro produced embryos. Frozen-thawed spermatozoa from two rams were allocated to four treatment groups: (i) non-sorted (Control); (ii) sorted (FS); (iii) sorted then re-frozen (FSF) and (iv) re-frozen control (FCF). Frozen-thawed samples were separated into X- and Y-chromosome bearing spermatozoa using a high-speed sperm sorter after density gradient centrifugation (X: 88 ± 1.5% and Y: 87 ± 1.1% purity). After 6 h incubation (37 °C), the percentage of motile spermatozoa was higher (P < 0.001) for FS (84 ± 2.0%) compared with all other treatments (Control: 36 ± 3.3%, FSF: 28 ± 3.1%, FCF: 20 ± 2.0%). In a sperm migration test greater numbers of FS spermatozoa penetrated 5 mm into the artificial cervical mucus compared with spermatozoa from all other treatments (152 ± 39.4 vs 31 ± 9.2 spermatozoa respectively; P < 0.05). Fertilisation and cleavage rates were higher (P < 0.05) for in vitro matured oocytes inseminated with Control compared with FSF spermatozoa. However, the Day 7 blastocyst development rate was higher for oocytes inseminated with FSF (62.2%) than FS and Control spermatozoa (52.7 and 50.0%; P < 0.05). The number of ewes pregnant (Day 60), lambing and the in vivo embryo survival rate was greater (P < 0.01) after the transfer of fresh embryos rather than vitrified embryos derived from X- and Y-spermatozoa (67.6, 64.7 and 41.2% vs 29.6, 25.9 and 14.8% respectively). Twenty-six of the 30 (86.7%) lambs derived from sex-sorted spermatozoa were of the correct sex. These results demonstrate that frozen–thawed ram spermatozoa can be sex-sorted for immediate or future use after re-cryopreservation and, in conjunction with IVF and embryo transfer, can be used to efficiently produce offspring of pre-determined sex.