Evaluation of culture systems for attachment and proliferation of epithelial cells cultured from ovine semen.

Different culture systems were evaluated for their ability to support attachment and proliferation of the somatic cells obtained from ovine semen. Ejaculates (n=14) were collected from eight rams representing three breeds, Dorper, Suffolk and Hampshire. All samples were processed immediately and somatic cells were obtained from 11 of the 14 ejaculates. These cells had classic epithelial morphology and expressed cytokeratin, indicating they were of epithelial origin. Cells from four rams with the greatest growth rates were used for subsequent studies. Cells were cultured in four different media for 5 days and total numbers of attached cells vs. total numbers of seeded cells were counted and compared each day. Four media were evaluated: (1) a supplemented medium composed of DMEM/F12, 10% fetal bovine serum (FBS), 10 ng/ml epidermal growth factor, 30 microg/ml bovine pituitary extract, 5 microg/ml insulin, 10 ng/ml cholera toxin, and 50 microg/ml gentamycin; (2) sheep fetal fibroblast (SFF)-conditioned medium; (3) swiss 3T3 fibroblast-conditioned medium; and (4) basic medium composed of DMEM/F12, 10% FBS, and 50 microg/ml gentamycin. Cell proliferation was greater in the supplemented medium, SFF-conditioned medium, and 3T3 fibroblast-conditioned medium compared to the basic medium by day 2 of culture (p<0.05, n=24), and greater in supplemented medium compared to the SFF-conditioned medium and 3T3 fibroblast-conditioned medium by day 4 of culture (p<0.05, n=24). Three different surfaces: (1) Matrigel basement membrane matrix-coated plastic; (2) collagen I-coated plastic; and (3) uncoated plastic were evaluated for their ability to support proliferation and attachment of the cells obtained from semen. Cell proliferation was greater when cells were cultured on the Matrigel-coated compared to the collagen I-coated and uncoated plastic by day 2 of culture (p<0.05, n=16). Cell attachment was greater when cells were plated on the Matrigel-coated and collagen I-coated plastic compared to the uncoated plastic (p<0.05, n=16). These studies describe an effective system for the culture and proliferation of epithelial cells obtained from ovine semen samples. The system may increase the likelihood of obtaining cells from frozen semen, which could be used for cloning to recover animals of genetic value in which semen is the only material that is available.