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ThesisItem Open Access GENOMIC EXPRESSION OF ATP SYNTHESIS ASSOCIATED FERTILITY BIOMARKERS IN BUBALINE SPERMATOZOA(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2021-03) SAI KIRAN, B.V.S; SRINIVASA PRASAD, CH (MAJOR); RAMBABU NAIK, B; ASWANI KUMAR, KThe present study was designed to study the genomic expression of ATP synthesis associated fertility biomarkers in Bubaline spermatozoa. The present research was conducted on both fresh and frozen semen, collected from eight Murrah buffalo bulls of age (4-7 years) maintained at ABC semen bank, Veravalli, A.P. Fresh semen samples (n=32) were collected using a sterilized artificial vagina (AV) over a male dummy bull once in a week for four weeks from eight bulls. The samples were transferred to the laboratory for physio-morphological evaluation i.e. volume, sperm concentration (millions/ml), mass-motility, individual motility %, live and dead %, HOST reactive % and acrosome integrity % and the Mean±SE were found to be 4.46±0.23 mL, 921.63±77.65 million/mL, 3.8±0.14, 71.48±0.7%, 73.73±0.70%, 74.53±0.85 and 87.05±0.75 % respectively. In the case of frozen semen (n=32), straws from same selected eight bulls were obtained and assessed for sperm concentration, post thaw motility %, live and dead %, HOST reactive % and acrosome integrity % and the Mean±SE were found to be 19.15±0.23 million/straw,51.17±0.81 %, 62.15±0.45 %, 53.78±0.85 % and 75.31±0.83 % . Comparison between fresh and frozen samples was performed for viability, HOST and acrosome integrity and found that the percent was significantly (p<0.05) higher in fresh compared to frozen semen samples. For gene expression study the fresh and frozen semen samples were grouped into four groups, fresh non capacitated (control, four samples from each bull, n=32), fresh in vitro capacitated (32), frozen non capacitated (four samples from each bull by pooling four straws each, n=32) and frozen in vitro capacitated (n=32). qPCR was performed to evaluate the relative gene expression of GAPDHS, PGK2, ENO4, MDH2, ATP5F1B, AK1 and HSP 70 and normalized to GLUT5 in all the four groups. The mRNA expression of GAPDHS, ENO4, PGK2, MDH2 and ATP5F1B were significantly (p<0.05) up-regulated in frozen non-capacitated compared to fresh non capacitated group (control). The mRNA expression of ENO4 was significantly (p<0.05) up-regulated in fresh capacitated and frozen non capacitated groups compared to the frozen capacitated group. The mRNA expression of PGK2 was significantly (p<0.05) up-regulated in frozen non-capacitated compared to rest of the three groups. Whereas significant (p<0.05) upregulation in the mRNA expression levels was also found in frozen capacitated compared to the fresh non capacitated group (control). The mRNA expression of MDH2 was significantly (p<0.05) up-regulated in frozen non-capacitated compared to rest of the three groups. Whereas significant (p<0.05) upregulation in the mRNA expression levels was also found in fresh capacitated and frozen capacitated groups compared to the fresh non capacitated group (control), however there was no significant variation seen between fresh capacitated and frozen capacitated groups. The mRNA expression of ATP5F1B was significantly (p<0.05) up-regulated in fresh capacitated and frozen non-capacitated compared to rest of the other two groups. On the contrary, the mRNA expression of AK1 was significantly (p<0.05) up-regulated in fresh capacitated compared to rest of the three groups. The mRNA expression of HSP 70 was significantly (p<0.05) down-regulated in frozen non capacitated and frozen capacitated compared to fresh non capacitated (control), group. From the present study, it was concluded that evaluation of critical metabolic enzymes such as Adenylate Kinase 1 which was down regulated during cryopreservation would help us to understand the handling efficiency of semen post collection to ensure a proper fertility after it is thawed/ when it is actually intended to AI or in vitro fertilization.
