Isolation and culture of epithelial cells from stored buffalo semen and their use for the production of cloned embryos
Monika Saini A * , Naresh L. Selokar
A B * , Rasika Rajendran A , Dharmendra Kumar A , Pradeep Kumar A and Prem Singh Yadav A B
A ICAR-Central Institute for Research on Buffaloes, Sirsa Road, Hisar-125001, Haryana, India.
B Corresponding author. Email: selokarnareshlalaji@gmail.com; psycirb@gmail.com
Reproduction, Fertility and Development 31(10) 1581-1588 https://doi.org/10.1071/RD18356
Submitted: 16 October 2017 Accepted: 30 April 2019 Published: 13 June 2019
Abstract
The aim of the present study was to isolate somatic cells from semen, a non-invasive source of donor somatic cells, for somatic cell nuclear transfer (SCNT) experiments. The study had two parts: (1) isolation and culture of somatic cells from semen, which was stored at 4°C; and (2) investigating the SCNT competence of semen-derived somatic cells. We successfully cultured somatic cells from freshly ejaculated semen, which was stored for different times (0, 4, 12, 24, 72 and 144 h after semen collection) at 4°C, using a Percoll gradient method. Up to 24 h storage, 100% cell attachment rates were observed; cell attachment rates of 66% were observed for the 72 and 144 h storage groups. The attached cells observed in all groups examined were proliferated (100%). Cultured cells exhibited epithelial cell morphology and culture characteristics, which was further confirmed by positive expression of cytokeratin 18, an epithelial cell-type marker. We compared the SCNT competence of semen-derived epithelial cells and skin-derived fibroblasts. The cleavage rate, blastocyst production rate, total number of cells in blastocysts and the apoptotic index of blastocysts were similar for embryos produced from semen-derived epithelial cells and skin-derived fibroblasts, indicating that semen-derived epithelial cells can serve as donors for SCNT experiments. In conclusion, we demonstrate a method to culture epithelial cells from stored semen, which can be used to produce cloned embryos of breeding bulls, including remote bulls.
Additional keywords: blastocyst, breeding bulls, SCNT, semen-derived epithelial cells.
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