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 Kr ebs-Ringer bicarbonate devised for in vitro fertilization in rats. Ovu lated eggs and epididymal spermatozoa were co-incubated in vitro at 37 degrees 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 ex amined for evidence of fertilization at the pronuclear stage. Although the fertilization rate with spermatozoa preincubated at 1-2 x 10(8) c ells ml(-1) for 2 h was very low (1-13%), it was significantly increas ed (43-51%, P < 0.05) when spermatozoa were preincubated at a lower co ncentration (1-2 x 10(7) cells ml(-1)). Furthermore, the fertilization rate was significantly higher with 1 mmol hypotaurine l(-1) (74.0%) t han without hypotaurine (44.4%, P < 0.05). Fertilization rates of sper matozoa preincubated at 1-2 x 10(7) cells ml(-1) for 0.5 or 2 h were s imilar (69.0% and 73.6%), but a longer preincubation (10 h) resulted i n a significantly lower fertilization rate (56.8%, P < 0.01). Vole spe rmatozoa preincubated for 2 h penetrated the zona pellucida 2 h after insemination, and the sperm heads became decondensed 3 h after insemin ation. 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 bicarbona te supplemented with 1 mmol hypotaurine l(-1).