Comparing the stemness and morphobiometry of spermatogonial stem cells from Doom pig on different days of culture
The present study was conducted to compare the stemness and morphobiometry of spermatogonial stem cells (SSCs) from the Doom pig on different days of culture (9th, 30th and 65th day) for the development of long-term culture method. The testes from 7-15-day old piglets were collected and two-step enz...
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| Format: | Article |
| Language: | English |
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Czech Academy of Agricultural Sciences
2020-02-01
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| Series: | Czech Journal of Animal Science |
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| Online Access: | https://cjas.agriculturejournals.cz/artkey/cjs-202002-0004_comparing-the-stemness-and-morphobiometry-of-spermatogonial-stem-cells-from-doom-pig-on-different-days-of-cultu.php |
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| author | Arpana Das Dipak Bhuyan Partha Pratim Das Simanta Koushik Bula Das Arundhati Phookan Suresh Dinkar Kharche Shiva Pratap Singh Manmohan Singh Chauhan |
| author_facet | Arpana Das Dipak Bhuyan Partha Pratim Das Simanta Koushik Bula Das Arundhati Phookan Suresh Dinkar Kharche Shiva Pratap Singh Manmohan Singh Chauhan |
| author_sort | Arpana Das |
| collection | DOAJ |
| description | The present study was conducted to compare the stemness and morphobiometry of spermatogonial stem cells (SSCs) from the Doom pig on different days of culture (9th, 30th and 65th day) for the development of long-term culture method. The testes from 7-15-day old piglets were collected and two-step enzymatic digestion was used to isolate SSCs. Before in vitro culture of SSC-like cells on the Sertoli cell feeder layer, the cells were enriched by differential plating and Percoll density gradient centrifugation. The isolated SSCs were characterised by alkaline phosphatase and immunofluorescence staining and qPCR analysis of SSC specific marker genes. Stemness was compared based on the expression of different SSC specific marker genes. The putative spermatogonial stem cells (PSSCs) from all the days of culture were found to be positive for alkaline phosphatase and immunofluorescence staining. The results from qPCR analysis showed that PSSCs were positive for SSC marker genes, though their expression decreased gradually from day 9 to day 65 of culture. The shape of the cells was found to change from compact round or oval to amorphous shape on day 65 of culture. Colony diameter ranged from 68.92 ± 1.20 µm (day 9) to 213.53 ± 12.52 µm (day 65) and differed significantly from each other. The number of colonies on day 65 of culture was significantly lower than on days 9 and 30. These results suggest that the enriched SSCs from Doom pigs can be maintained up to two months in vitro in the present culture system. |
| first_indexed | 2024-04-10T08:25:17Z |
| format | Article |
| id | doaj.art-a064b9a13fea4ea2b40f7244a824c2f5 |
| institution | Directory Open Access Journal |
| issn | 1212-1819 1805-9309 |
| language | English |
| last_indexed | 2024-04-10T08:25:17Z |
| publishDate | 2020-02-01 |
| publisher | Czech Academy of Agricultural Sciences |
| record_format | Article |
| series | Czech Journal of Animal Science |
| spelling | doaj.art-a064b9a13fea4ea2b40f7244a824c2f52023-02-23T03:33:44ZengCzech Academy of Agricultural SciencesCzech Journal of Animal Science1212-18191805-93092020-02-01652667610.17221/264/2019-CJAScjs-202002-0004Comparing the stemness and morphobiometry of spermatogonial stem cells from Doom pig on different days of cultureArpana Das0Dipak Bhuyan1Partha Pratim Das2Simanta Koushik3Bula Das4Arundhati Phookan5Suresh Dinkar Kharche6Shiva Pratap Singh7Manmohan Singh Chauhan8Department of Animal Genetics and Breeding, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, IndiaDepartment of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, IndiaDepartment of Animal Genetics and Breeding, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, IndiaDepartment of Animal Genetics and Breeding, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, IndiaDepartment of Animal Genetics and Breeding, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, IndiaDepartment of Animal Genetics and Breeding, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, IndiaICAR - Central Institute for Research on Goats, Farah, Mathura, IndiaICAR - Central Institute for Research on Goats, Farah, Mathura, IndiaICAR - Central Institute for Research on Goats, Farah, Mathura, IndiaThe present study was conducted to compare the stemness and morphobiometry of spermatogonial stem cells (SSCs) from the Doom pig on different days of culture (9th, 30th and 65th day) for the development of long-term culture method. The testes from 7-15-day old piglets were collected and two-step enzymatic digestion was used to isolate SSCs. Before in vitro culture of SSC-like cells on the Sertoli cell feeder layer, the cells were enriched by differential plating and Percoll density gradient centrifugation. The isolated SSCs were characterised by alkaline phosphatase and immunofluorescence staining and qPCR analysis of SSC specific marker genes. Stemness was compared based on the expression of different SSC specific marker genes. The putative spermatogonial stem cells (PSSCs) from all the days of culture were found to be positive for alkaline phosphatase and immunofluorescence staining. The results from qPCR analysis showed that PSSCs were positive for SSC marker genes, though their expression decreased gradually from day 9 to day 65 of culture. The shape of the cells was found to change from compact round or oval to amorphous shape on day 65 of culture. Colony diameter ranged from 68.92 ± 1.20 µm (day 9) to 213.53 ± 12.52 µm (day 65) and differed significantly from each other. The number of colonies on day 65 of culture was significantly lower than on days 9 and 30. These results suggest that the enriched SSCs from Doom pigs can be maintained up to two months in vitro in the present culture system.https://cjas.agriculturejournals.cz/artkey/cjs-202002-0004_comparing-the-stemness-and-morphobiometry-of-spermatogonial-stem-cells-from-doom-pig-on-different-days-of-cultu.phpporcine spermatogonial stem celllong-term cultureimmunofluorescence stainingqpcrcolony diametercolony number |
| spellingShingle | Arpana Das Dipak Bhuyan Partha Pratim Das Simanta Koushik Bula Das Arundhati Phookan Suresh Dinkar Kharche Shiva Pratap Singh Manmohan Singh Chauhan Comparing the stemness and morphobiometry of spermatogonial stem cells from Doom pig on different days of culture Czech Journal of Animal Science porcine spermatogonial stem cell long-term culture immunofluorescence staining qpcr colony diameter colony number |
| title | Comparing the stemness and morphobiometry of spermatogonial stem cells from Doom pig on different days of culture |
| title_full | Comparing the stemness and morphobiometry of spermatogonial stem cells from Doom pig on different days of culture |
| title_fullStr | Comparing the stemness and morphobiometry of spermatogonial stem cells from Doom pig on different days of culture |
| title_full_unstemmed | Comparing the stemness and morphobiometry of spermatogonial stem cells from Doom pig on different days of culture |
| title_short | Comparing the stemness and morphobiometry of spermatogonial stem cells from Doom pig on different days of culture |
| title_sort | comparing the stemness and morphobiometry of spermatogonial stem cells from doom pig on different days of culture |
| topic | porcine spermatogonial stem cell long-term culture immunofluorescence staining qpcr colony diameter colony number |
| url | https://cjas.agriculturejournals.cz/artkey/cjs-202002-0004_comparing-the-stemness-and-morphobiometry-of-spermatogonial-stem-cells-from-doom-pig-on-different-days-of-cultu.php |
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