61 ISOLATION AND CULTURE OF SOMATIC CELLS OBTAINED FROM SEMEN AND MILK OF GULF COAST NATIVE SHEEP

In addition to the sperm found in semen, somatic cells (SC) are frequently present. Furthermore, SC are also present in fresh milk. These SC could also be a potential source of biomaterial for the preservation of endangered species. In the present study, the possibility of isolating SC from fresh, cooled and frozen semen, as well as from fresh milk, was evaluated in the endangered Gulf Coast Native sheep. Semen from 7 mature rams was collected by electro-ejaculation and a 0.5-mL sample from each ejaculate was then diluted in PBS (22°C). The remainder of the ejaculate was cooled (4°C), extended in a TRIS-yolk-glycerol extender, loaded into 0.5-mL plastic straws and frozen. Semen from the same collection (2–5 straws) was also diluted in PBS and allotted to the cooled semen treatment group (4°C). Sheep milk samples (n = 26) from 5 Gulf Coast Native (GCN) ewes and 11 crossbred ewes (GCN × Suffolk) were collected and stored at 4°C. Samples of fresh (0.5 mL), cooled (2–5 straws) and frozen–thawed (2–5 straws) semen and refrigerated milk (15–50 mL) were washed with PBS, centrifuged at 500g for 10 min and the re-suspended pellet cultured in tissue culture dishes containing 2 mL of DMEM with 15% fetal bovine serum (FBS) plus 100 µM nonessential amino acids, supplemented with antibiotics and incubated at 38.5°C in 5% CO2 in air. After 24 h, the semen sample culture dishes were rinsed twice with fresh culture medium to remove remaining sperm cells and then returned to the the incubator. After 3 to 7 d of incubation, groups of epithelial-like cell colonies were detected in 1 sample from the fresh semen group (1 of 7 rams, 14.3%) and 4 semen samples from the cooled group (4 of the 7 of rams, 57%). These cells subsequently proliferated until a monolayer was observed; the cells were then harvested and plated (in a single culture dish) for further passage. One cell line survived from the cooled semen group, which was frozen in DMEM medium with 15% FBS and 10% DMSO and then stored in LN2. No cells were derived from the frozen-thawed semen group. Colonies of fibroblast-like or epithelial-like cells were detected after culture of the milk samples. Of the 26 colonies, 23 (88%) initially plated cells, 11 (41%) proliferated to the 1st passage, 5 (19%) resulted in cell lines that were frozen and 2 (8%) of these subsequently proliferated during post-thaw incubation. The genetic status of the cell lines that proliferated until cryopreservation was assessed by PCR amplification of microsatellite markers. Results confirmed that the cell populations derived from both the semen and milk samples originated from the appropriate donor animals. Also, immuno-histochemical analyses for vimentin and cytokeratin were performed on these cell populations to determine the morphological cell type. Cells derived from semen samples were identified as epithelial cells by the presence of cytokeratin, whereas the milk samples produced epithelial and fibroblast cells. In the present study, we demonstrated that it is possible to establish a somatic cell line from cooled semen, as well as from fresh milk, in an endangered sheep. This approach could provide cell lines from valuable animals when other means of obtaining cells are not possible. This project was partially funded by the J. Bennett Johnston Science Foundation.

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