Ultrasound-guided transvaginal follicular aspiration to provide a source of bovine oocytes for gene microinjection

Abstract

Three experiments were conducted to determine an efficient method of obtaining oocytes from cows via ultrasound-guided transvaginal follicular aspiration. Transvaginal oocyte recovery combined with in vitro maturation, fertilization, and culture (IVMlIVFIIVC) can produce pre-implantation stage bovine embryos and also supply a source of ova for gene microinjection. In Experiment 1, once- (IX) vs. twice-weekly (2X) oocyte recovery frequencies were compared. No differences in recovery per session were observed (IX = 6.8 vs. 2X = 6.3 oocytes/session; p>O.1 0). However, the 2X group generated more oocytes on a weekly basis (IX = 6.8 vs. 2X = 12.6 oocytes/week; p<O.OS). In Experiment 2, in vitro embryo production was compared among the groups aspirated once-weekly (1 X), twice-weekly (2X), and twice-weekly after receiving I5 mg FSH (2XF). No differences existed among the aspiration groups in the proportion of blastocysts produced following IVMIIVFIIVC (IX = 23.10/0, 2X = 26.1%, 2XF = 18.0%, % viable). However, the 2X group generated more oocytes and embryos throughout the experiment (2X = 83/318, 2XF = 38/211, IX = 58/2SI viable blastocyst/total oocytes; p<O.O5) than the other groups. A higher proportion of 2X or 2XF generated embryos were of excellent quality (2X = 60.2%, 2XF = 60.5%) compared to the IX group (37.9%) In Experiment 3, in vitro embryo development rates were compared among oocytes from follicular aspiration (TVFA) and oocytes derived from slaughterhouse ovaries (SHD). Oocytes (65%) from both sources were subjected to pronuclear-microinjection of foreign DNA while the remainder served as non-injected controls. Only control oocytes differed, with TVF A-derived oocytes developing to blastocyst more successfully than SHD oocytes (40.8% vs. 30.0olo~ p<0.05). Microinjected embryo development was similar between the groups (TVFA 15.9%, vs. SHD = 12.8% viable blastocyst/total;) with the TVFA oocytes holding a slight but non-significant numerical advantage (p>O.IO) In these experiments, twice-weekly follicular aspiration without exogenous FSH, was the best scheme of oocyte recovery. This program provided a source of consistent, high quality oocytes that responded favorable to the IVMIIVFIIVC system and microinjection.