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20221
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Subject: Animal Genetics and Breeding × Author: Lalmasawma, Timothy × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    Isolation, characterization and morpho-biometric evaluation of pre-pubertal porcine spermatogonial stem cells in different culture media
    (College of Veterinary Science, Assam Agricultural University, Khanapara Campus, 2022-03) Lalmasawma, Timothy; Das, Arpana
    Testes samples were collected from 7-15 days old pre-pubertal male crossbred piglets (Local × Hampshire) for isolation, enrichment and in vitro culture of porcine spermatogonial stem cells (SSCs). Isolation of spermatogonial stem cells like cells was performed by double enzymatic digestion using four enzymes viz., collagenase, DNase I, hyaluronidase type II and trypsin-EDTA. Isolated cells were further enriched by differential plating and percoll density gradient centrifugation method. Enriched cells were cultured on Sertoli cell feeder layer in three different culture media. All the three media consisted of same concentration of DMEM, NEEA, L-glutamine, FBS, EGF and FGF, however in addition to these, LIF was added to media I, GDNF was added to media II and both LIF and GDNF were added to media III. Characterization of SSCs was done by alkaline phosphatase and immunoflourescence staining. Expression of SSC specific pluripotent marker genes by putative SSCs was also studied by RT-PCR study. Porcine SSCs were observed as dome shaped round or oval bodies on 5th -6th day of culture in all the three media. Clustering of cell was observed from 4th -5th day of culture and single, paired or multiple colonies were observed from 8-10th day of culture. The SSCs colonies appeared as mulberry, grape or rosette shaped with irregular distinct boundary from feeder layer on 15th - 19th day of culture in all the three media. However, the shape of the SSCs was found to be distorted with increase in the days of culture. The morphology of the SSC colonies was maintained best up to 30th day of culture in media III. The SSC colony number was recorded as 82.14 ± 2.91, 60.07 ± 2. 78 and 48.43 ± 1.96 on 5th, 15th and 30th day of culture respectively in media I. The corresponding numbers were 91.71 ± 2.62, 67.00 ± 2.05 and 57.29 ± 2.17 in media II and 105.93 ± 2.82, 80.21 ± 2.45 and 62.50 ± 2.09 in media III respectively. The SSC colony diameter was found to be 64.26 ± 0.85, 125.30 ± 1.88 and 123.01 ± 5.49μm on 5th, 15th and 30th day of culture respectively in media I. The corresponding values were 69.67 ± 1.12, 139.58 ± 3.93 and 142.08 ± 5.72μm in media II and 76.49 ± 1.61, 152.55 ± 4.07 and 172. 08 ± 4.96μm in media III respectively. The day of culture and culture media had significant effect (P≥0.01) on SSC colony number and significantly higher number of SSC colony was observed on day 5 and lower was on day 30 of culture in all the three media. The SSC colony number was significantly higher in media III containing both GDNF and LIF. The diameter of SSC colony differed significantly (P≥0.05) due to day of culture and culture media. The interaction between day of culture and culture media was also significant (P≥0.01). The colony diameter recorded on day 30 of culture was significantly higher, whereas lower number was recorded on day 5 of culture in all the culture media. Diameter of SSC colony obtained in media III was found to significantly higher and the lower diameter was obtained in media I on all the day of culture. It was observed that the SSC colony number decreased and colony diameter increased with the day of culture from day 5th to 30th day of culture in all the media.The putative SSCs in all the three media showed positive result for alkaline phosphatase and immunofluorescence staining. The putative SSCs in all the three media were also found to express SSC specific pluripotent marker genes viz., OCT4, SOX2, NANOG and maximum expression was observed in media III, however, no expression was recorded for c-KIT and PPARγ which were known to be the markers for differentiated SSCs. BAX4, an apoptopic marker gene was also expressed by putative SSCs in all the three media. Based on the findings of the present study, it may be concluded that a pure population of porcine spermatogonial stem cells (SSCs) could be obtained and successfully maintained in vitro up to 30th day of culture. Media III containing DMEM, FBS, NEAA, L-glutamin, FGF, EGF, LIF and GDNF was found to be the best for in vitro culture of porcine SSCs.