INTRODUCTION
During the past decade there have been substantial efforts to optimize conditions for in vitro maturation (IVM) and fertilization (IVF) of bovine oocytes and culture (IVC) of resulting embryos. Gonadotropins and estradiol are commonly employed for IVM, and insulin has enhanced in vitro development of bovine oocytes inseminated with unsorted sperm. This experiment was to determine effects of various hormones added to IVM medium and insulin added to IVC medium on the outcome of bovine IVF with flow cytometrically-sorted sperm.
MATERIALS AND METHODS
A total of 484 oocytes in 3 replicates were matured in the following 3 media for 22 to 24 h at 39°C in 5% CO2 in air: 1) M199 + 10% estrous cow serum (ECS) and 15 ng FSH, 1 µg LH, and 1 µg E2/ml (FLE); 2) M199 + 10% ECS + 10 µg LH/ml (LH) and 3) M199 + 10% ECS + 2 x 106 granulosa cells/ml (GC). Sperm for IVF were flow cytometrically-sorted without regard for DNA content (bulk-sorted) and frozen. The basic procedures for IVF and IVC were as described elsewhere (1). Gametes were co-incubated in a chemically defined medium + 0.5 mM Na citrate (CDM-1(2)) for 18 to 20 h for fertilization, and presumptive zygotes were then cultured in fresh CDM-1. Cleaved embryos from each of above 3 IVM treatments were allocated to 2 sub-groups of CDM-2 (CDM-1 + essential amino acids, and no EDTA), with or without 0.12 IU/ml insulin 48 h after culture in CDM-1. Blastocysts per oocyte were determined on D7-8 after insemination. Data were analyzed by 2.
RESULTS AND DISCUSSIONS
Cleavage rate with GC treatment (74.7%) was higher (P<0.1) than that with FLE treatment (65.8%) but not different from the GC and LH (72.7%) group. There was no significant difference in blastocyst rate on D7 either among the IVM treatment groups (16.5, 19.5 and 21.8% for FLE, LH and GC treatment, respectively) or between IVC treatments with (22.8%) and without (16.8%) insulin. However, percentages of grade 1 to 2 embryos and expanded blastocysts on D7 and hatched blastocysts on D8 with insulin treatment (62.4, 50.9, 35.6) was significantly higher (P<0.05) than that without (45.1, 14.2, 14.2). From this study, we conclude that 1) there is no benefit of adding hormones to IVM medium if GC are included; and 2) insulin added to IVC medium improves embryo quality and accelerates development of bovine oocytes inseminated with flow cytometrically-sorted sperm.
REFERENCES
1. Lu, K.H., Cran, D.G. & Seidel, G.E., Jr. (1999) Theriogenology 52:1393-1405.
2. Olson, S.E. & Seidel, G.E., Jr. (2000) J. Anim. Sci. 78:in press.