44 ATTEMPTS TO USE SOMATIC CELLS ISOLATED FROM FROZEN BOVINE SEMEN FOR NUCLEAR TRANSFER

Somatic cells in semen are a potential source of nuclei for nuclear transfer to produce genetically identical animals. This is especially important when an animal has died and the only viable genetic material available is frozen semen. In our previous studies, epithelial cells were cultured from fresh ovine and bovine ejaculates and blastocyst stage embryos were produced using these cells (Liu J et al. 2007 Biol. Reprod. special issue, 177; Liu J et al. 2008 Reprod. Fertil. Dev. 20, 102). However, growing cells from frozen semen can be difficult. We hypothesized that nuclei of the somatic cells in frozen semen can be used for nuclear transfer even though the cell membrane and cytoplasm are damaged during the semen freezing process. Electrical fusion or piezo assisted direct injection was applied to introduce nuclei of somatic cells isolated from frozen bovine semen (not cultured) into enucleated bovine oocytes. With electrical fusion, only 5 of the 64 (7.8%) recombined couplets fused, which is lower than our normal fusion rate of approximately 70%. Of the 5 fused embryos, one cleaved and developed to a 4-cell stage embryo. Staining with propidium iodide indicated that less than 10% of somatic cells isolated from frozen bovine semen were viable. These results suggest that it might not be practical to introduce nuclei of the cells in frozen semen into oocytes by fusion due to the high proportions of dead cells. Membranes of the cells in frozen semen were hard and difficult to break by piezo pulses or drawing in and out of the injection pipette. Therefore, whole cell intracytoplasmic injection was applied. After couplet recombination, activation was induced by applying two 0.3 kV cm–1, 55 μs direct-current pulses delivered by an Eppendorf Multiporator (Eppendorf, North America) in activation medium that was composed of 0.28 m Mannitol (Sigma-Aldrich, St. Louis, MO, USA), 0.1 mm CaCl2 (Sigma-Aldrich), and 0.1 mm MgSO4 (Sigma-Aldrich), followed by incubation in 10 μg mL–1 cycloheximide (Sigma-Aldrich) and 5 μg mL–1 cytochalasin B (Sigma-Aldrich) for 5 h in a humidified 5% CO2, 5% O2, and 90% N2 gas mixture at 38.5°C. Of the 299 recombined embryos, 82 (27.4%) either cleaved or were fragmented. Of the 82 embryos, 48 were examined and 42 (87.5%) were determined to be fragmented (contained intact donor cells). Low cleavage rates were observed in embryos produced by direct injection (4.5%), but no further embryonic development occurred. Cells cultured from fresh bovine semen were used as positive controls for whole cell intracytoplasmic injection. Of the 74 recombined embryos, 73 (98.6%) cleaved and 4 developed into blastocysts. These results highlight the difficulty of obtaining viable embryos by employing nuclear transfer and somatic cells obtained from frozen semen samples. Additional research is warranted given the potential value of this approach for recovering lost genetics.