Intact tissue preservation methods towards construction of the slaughterhouse-derived bovine somatic cell transfer donor cell bank for a conservation of bovine genetic resource

Abstract

It will become strategically important to preserve a variety of bovine somatic cells, including somatic cells of economically superior cattle, in order to conserve bovine genetic resourse, due to the advent of cloning technology using somatic cell transfer for the mammal production. It is efficient to collect the cells from the slaughterhouse, for this strategy. First, we investigated the conditions of both temporary PBS soakage-based and semi-permanent vitrification-based methods suitable for preservation of intact tissue, by using the fresh aorta tissue and the masseter muscle tissue collected immediately after slaughter. The quality of the tissue preservation was evaluated by the degree of the proliferation of cells dispersed from the preserved tissue. As to the temporary methods, addition of 10% calf serum and 0.01% calcium chloride to PBS enhanced the quality of the tissue preservation, and maintained its preservation until 8 days, while cutting of the tissue into small pieces decreased its quality. Further, we developed the semi-permanent methods using the vitrification liquid (PBS containing 25% ethylen glycol and 25% glycerol) and a 0.5ml bovine semen preservation straw, which maintains a higher quality of the tissue preservation. Next, we demonstrated that the established semi-permanent method can also applied to the kidney tissue collected 4 days after slaughter. Finally, we showed that either the established preservation methods are successful in the production of somatic cell transfer-derived cloned embryos, through using the somatic cell obtained from the preserved tissues. We are now constructing slaughterhouse-derived bovine somatic cell transfer loner cell bank, by using the established intact tissue preservation methods for genetic resource concervation of economically superior Japanese Black cattle.