Recovery and evaluation of somatic cells from ovine and bovine semen for use in nuclear transfer




Journal Title

Journal ISSN

Volume Title



Somatic cells in semen are a potential source of nuclei for cloning animals bysomatic cell nuclear transfer. Culture of the cells from frozen semen, if possible, wouldbe extremely valuable for preservation or restoration of endangered, exotic, and extinctanimals when other ways of obtaining somatic cells are unavailable. In the present study,somatic cells isolated from ovine and bovine semen samples were characterized, culturesystems were evaluated for attachment and proliferation of these cells, and usefulness ofthese cells for somatic cell nuclear transfer was determined.Semen samples were collected from eight rams representing three breeds:Dorper, Suffolk, and Hampshire and nine bulls representing three breeds: Charolais,Brahman, and a crossbred Brahman. Somatic cells were isolated immediately postcollection by centrifuging through percoll columns and the epithelial cells wereidentified by immunofluorescence analysis. Culture systems were evaluated for theirability to support attachment and proliferation of the cells. A supplemented mediumcomposed of DMEM/F12, 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 30 g/ml bovine pituitary extract, 5 g/ml insulin, 10 ng/ml cholera toxin, and 50 g/mlgentamicin significantly improved cell proliferation over sheep fetal fibroblastconditionedmedium, 3T3 cell-conditioned medium, and basic medium (p<0.05). Cellproliferation and attachment were further improved when Matrigel-coated culturesurfaces were used (p<0.05). However, the system was not adequate for obtaining cellgrowth from frozen semen.To check the chromosome anomalies, metaphase chromosomal complements ofthe cells cultured from 4 rams were evaluated. The predominant chromosome number ofcells from three of the rams (Dorper 18-month-old; Suffolk 17-month-old; Suffolk 18-month-old) was 2n = 54, which is the normal modal number for sheep. However, thenumbers of chromosomes of cells cultured from the fourth ram (Hampshire, 18-monthold)were near-triploid. These results indicate the need for chromosome analysis of cellsbefore using them for cloning experiments. In our attempts to clone animals, blastocyststage embryos were successfully produced using epithelial cells cultured from semen ofthree different bulls. However, no compact morulae or blastocysts were obtained whensomatic cells isolated from frozen semen but not cultured were used as donor cells.