258 THE PRELIMINARY RESULTS OF IN VITRO PRODUCTION OF THE WISENT HYBRID EMBRYOS

Abstract

Wisent (Bison bonasus), also called the European bison, is listed as vulnerable on the International Union for the Conservation of Nature Red List of Threatened Species. In Poland, a program for protection in situ and ex situ is being implemented. One new approach is the use of the in vitro embryo production (IVP) procedures to obtain wisent offspring. In contrast to previous successes with cattle IVP, use of IVP with wisent is limited by the small size of the population (only ~5000 individuals in more than 200 herds in Europe) and seasonal reproduction. The aim of this preliminary study was to obtain hybrid embryos (Bison bonasus × Bos taurus) in vitro. Ovaries were isolated from wisent females outside the reproductive season and eliminated from breeding for reasons other than infertility. Cumulus-oocytes complexes (COC) were isolated from all follicles above 2 mm in diameter. All COC were matured in TCM 199 supplemented with 10% fetal bovine serum, 0.02 IU mL^–1 of porcine FSH, 17 β-oestradiol, 0.2 mM Na pyruvate, and antibiotics. The COC were cultured for 24 h at 38.5°C and 5% CO₂ in humidified air. The matured COC (Bison bonasus) were fertilized in vitro with sperm from Jersey bulls (Bos taurus) in TALP supplemented with 6 mg mL^–1 of fatty acid free BSA (BSA FAF), 0.2 mM Na pyruvate, 20 µM penicillamine,10 µM hypotaurine, 1 µM epinephrine, 2 µg mL^–1 heparin, and antibiotics. Spermatozoa were used at a final concentration of 1 × 10⁵ per oocyte and were co-cultured for 18 h at 38.5°C and 5% CO₂ in humidified air. The hybrid zygotes were cultured in KSOM supplemented with 5 µg mL^–1 of MEM Nonessential Amino Acid Solution (100×), 3 mg mL^–1 of BSA FAF, and antibiotic for 192 h at 38.5°C and 5% CO₂ in humidified air. The medium was partly replaced by fresh medium after 48 and 144 h of culture. Development was evaluated every day. From 25 COC isolated from wisent ovaries, only 18 COC were qualified for in vitro maturation (60%). Of these, 15 COC (83.3%) matured. The percentage of hybrid embryos that cleaved was 80% after 48 h of culture, and the percentage of embryos that developed up to the 8-cell stage was 33% after 96 h of culture. The morula/blastocyst rate was 26.6% after 192 h of culture, as represented by 1 early blastocyst, 2 compact morulae, and 1 morula. The use of the cattle IVP procedure allowed to receive hybrid embryos (Bison bonasus × Bos taurus), but they developed slower than cattle embryos under the same conditions, based on our previous studies. This research will be continued and may make a contribution to the protection of this threatened species.