198 Comparison of in vitro production of bison and cattle embryos and effect of L-carnitine during maturation of bison oocytes

Abstract

Bison are an important species in North America, both economically and culturally. Although assisted reproductive technologies have been applied to preserve the genetic diversity of bison, development of these technologies remains limited for this species. The objective of the present study was to compare success rates of oocyte maturation, fertilization, and embryo development in vitro in bison versus cattle (experiment 1). Cumulus-oocyte complexes obtained from abattoir-derived cattle and bison ovaries were matured, fertilized with frozen semen, and cultured in vitro using standard procedures (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 585-596). At least three replicates were repeated for each experimental group. Oocyte recovery rate was lower in bison than in cattle (4.3; 2797/699 vs. 6.7; 4138/677, oocyte/ovary; P < 0.01, t-test). Nuclear maturation (oocytes at MII, 23 h post-IVM) and fertilization rates (oocytes with 2 pronuclei 18 h post-insemination; p.i.) evaluated by Hoechst stain were lower (P < 0.01, chi-square) for bison (65.1%; 56/86 and 32.7%; 18/55, respectively) than for cattle (88.3%; 83/94 and 70.9%; 39/55, respectively). Polyspermy tended to be higher in bison than in cattle (12.7% vs. 3.6%, P = 0.08). The percentages of 2-cell embryos tended to be lower in bison than in cattle (13.5% vs. 25.0%, P > 0.05) at 24 h p.i. but by 30 h p.i., this difference increased (33.7% vs. 67.0%, P < 0.01, chi-square). Cleavage (Day 3) and blastocyst (Day 7) rates were lower (P < 0.01, chi-square) for bison (58.2%; 280/481 and 14.6%; 70/481, respectively) than for cattle (90.8%; 405/446 and 22.9%; 102/446, respectively). Total cell number (74.9 ± 4.8 vs.114.2 ± 5.8), trophectoderm cell numbers (57.9 ± 4.6 vs. 89.2 ± 4.8) and inner cell mass cell numbers (16.9 ± 2.3 vs. 25 ± 1.9) as determined by Hoechst and propidium iodide were all lower (P < 0.01, t-test) in bison than in cattle blastocysts. To improve oocyte competence in bison, we evaluated effects of L-carnitine (LC) supplementation during IVM on developmental potential of bison oocytes (experiment 2). Cumulus-oocyte complexes were matured in IVM medium supplemented with 0, 0.15, 0.3, 0.6, or 1.2 mg mL⁻¹ LC. No differences were observed in cleavage rates of control (0 mg mL⁻¹ LC) and LC-treated groups (values ranged from 60.0 to 66.4%). Interestingly, a dose-dependent increase in blastocyst development was found with the lowest value recorded in control group (10.4%; 14/134) and the highest value in the 1.2 mg mL⁻¹ LC supplemented group (22.2%; 23/105; P < 0.01, chi-square, n = 4). Adding 1.2 mg mL⁻¹ LC to the IVM medium improved the percentage of hatching blastocysts compared with the control. In conclusion, bison oocytes exhibited lower in vitro maturation, fertilization, and developmental rates compared with cattle oocytes using our system, and bison embryos were delayed in the timing of first cleavage. L-Carnitine supplementation during IVM of bison oocytes improved the preimplantation development and quality of in vitro-produced blastocysts.