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In vitro fertilization and embryo development of Japanese field voles (Microtus montebelli)

Optimal conditions for in vitro fertilization of Japanese field voles (Microtus montebelli) were analysed. The medium used was a modified Krebs–Ringer bicarbonate devised for in vitro fertilization in rats. Ovulated eggs and epididymal spermatozoa were co-incubated in vitro at 37°C under 5% CO₂ in air for 6 h, and the eggs were fixed with 2.5% (w/v) glutaraldehyde, stained with 0.25% (v/v) acetolacmoid and examined for evidence of fertilization at the pronuclear stage. Although the fertilization rate with spermatozoa preincubated at 1–2 x 10⁸ cells ml^–1 for 2 h was very low (1–13%), it was significantly increased (43–51%, P < 0.05) when spermatozoa were preincubated at a lower concentration (1–2 x 10⁷ cells ml^–1). Furthermore, the fertilization rate was significantly higher with 1 mmol hypotaurine l^–1 (74.0%) than without hypotaurine (44.4%, P < 0.05). Fertilization rates of spermatozoa preincubated at 1–2 x 10⁷ cells ml^–1 for 0.5 or 2 h were similar (69.0% and 73.6%), but a longer preincubation (10 h) resulted in a significantly lower fertilization rate (56.8%, P < 0.01). Vole spermatozoa preincubated for 2 h penetrated the zona pellucida 2 h after insemination, and the sperm heads became decondensed 3 h after insemination. At 6 h after insemination, male and female pronuclei were found in most penetrated eggs. When the eggs were left in the fertilization medium without washing and cultured for 96 h after insemination, they developed to two-cell (82.6%), four-cell (60.9%), eight-cell (23.2%) and morula/blastocyst (8.7%) stages in modified Krebs–Ringer bicarbonate supplemented with 1 mmol hypotaurine l^–1.