Effect of X-sorted sperm on development grade 1 in vitro-produced embryos derived from bovine ovum pick up oocytes under commercial conditions

Sex-sorted sperm has been used for IVF to produce in vitro sexed bovine embryos. Following IVF, developmental kinetics, specifically the timing of blastocyst formation, has been related to embryo developmental competence (Lonergan et al, Journal of Reproduction and Fertility 117, 159-167, 1999). The objective of this work was to analyze developmental kinetics of OPU-IVP bovine embryos produced with unsorted and X-sorted sperm under commercial conditions. The work was performed at the Biotechnology AURIVA Station located in Denguin, South West, France. Four to fifteen years old Holstein cows (n = 31) and 16-22 months old heifers (n = 18) were used in an OPU-IVP program. Donor animals were stimulated with decreasing pFSH doses (Stimufol; Reprobiol, Liege, Belgium) twice daily during 3 days, (total dose: 350 µg for cows and 250 µg for heifers). Cumulus oocyte complexes (COCs) were collected by OPU 12 to 24 h after the last FSH injection and matured in vitro using a standard IVM protocol. Oocytes were fertilized with frozen–thawed unsorted or X-sorted sperm in modified Tyrode’s bicarbonate buffered medium using different non pre-tested bulls (n = 58). Presumptive zygotes were cultured in SOF medium (Minitub, Tiefenbach, Germany) up to Day 8 at 38.5°C under an atmosphere of 5% CO2, 5% O2 and 90% N2 with maximum humidity. OPU/IVP was repeated one to 16 times (2.3 ± 3.0) for each donor cow or heifer. Grade 1 (G1) expanded blastocysts, according to IETS classification, were recorded on days 6.5, 7.0, 7.5 and 8. Oocyte recovery and embryo production were analyzed by Student T-test and the rate of expanded blastocyst by Chi-Square. Sixty one OPU sessions were performed with unsorted semen and 66 with X-sorted semen, with a mean number of oocytes processed for in vitro maturation 10.9 ± 6.4 and 9.1 ± 4.8 (P > 0.05) and a mean number of G1 produced embryos 7.4 ± 4.5 and 4.7 ± 2.9 (P < 0.05) per session, respectively. The mean embryo developmental rate (G1 embryos/number of oocytes entering maturation process) was 67.8% (unsorted group) and 52.1% (X-sorted group; P < 0.05). On day 6.5, 52.8% of total blastocysts had already expanded after IVF with unsorted sperm, compared with 37.0% with Xsorted sperm (P < 0.05); while 41.3% were developed with unsorted and 53.1% with X-sorted sperm (P < 0.05) on day 7.0. Expansion was observed in 4.6% of embryos produced with unsorted and 9.7% with X-sorted sperm (P < 0.05) on day 7.5. No difference was observed on day 8 in proportion of expanded blastocysts produced with unsorted and X-sorted sperm. Unlike previous studies in which sexed embryos develop on days 8 and 9, in our study, although the number of expanded blastocysts was high with unsorted semen on day 6.5, the majority of expanded blastocysts developed on day 7 with unsorted (94%) and X-sorted sperm (90%).