GENOMIC EXPRESSION OF CAPACITATION RELATED FERTILITY BIOMARKERS IN BUBALINE SPERMATOZOA
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Date
2021-04
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SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA
Abstract
The objective of the present study was to evaluate the genomic expression of capacitation related fertility biomarkers in Bubaline spermatozoa. Eight adult healthy buffalo bulls between three to six years of age were randomly selected from ABC Semen Station, Veeravalli, Krishna District, Andhra Pradesh. The fresh semen samples were collected from eight buffalo bulls once in a week for four weeks by AV method. The frozen semen straws were also obtained from the same bulls semen. As an preliminary quality check, fresh semen samples (4 samplings from each bull, n=32) were evaluated for semen volume, sperm concentration, mass motility, individual progressive motility, viability, HOST, acrosomal integrity and the mean±SE values were found to be 4.48±0.24 mL, 909.28±82.29 million/mL, 3.79±0.12, 69.92±9.46%, 73.37±0.38%, 74.47±0.37% and 87.14±0.52% respectively. The frozen semen samples (n=32) were also evaluated for concentration, post thaw motility, viability, HOST and acrosomal integrity with mean±SE values of 19.28±0.24 million/straw, 48.13±0.66%, 61.20±0.37%, 52.82±0.68% and 76.37±0.41% respectively. Comparison was made between fresh and frozen samples for parameters like viability, HOST and acrosomal integrity using unpaired t-test. The percent viability, HOST and acrosomal integrity was significantly (p<0.05) higher in fresh compared to frozen semen samples. In order to evaluate the gene expression of capacitation related fertility biomarkers, the fresh and frozen samples were divided in to four groups. Fresh non-capacitated as control (four samples from each bull, n=32), fresh capacitated (n=32), frozen non-capacitated (4 samples from each bull by pooling 4 straws each, n=32) and frozen capacitated (n=32). In vitro capacitation of fresh and frozen semen samples was performed in BO media supplemented containing heparin. Genes such as ADCY10, PRKACA, AKAP4, CatSper2, CRISP2 and HSP90 were selected for the study. The mRNA isolated from respective samples were converted to cDNA and subjected to q-PCR analysis. The target genes were normalized to endogenous control GLUT5 and mRNA expression of fresh capacitated, frozen non-capacitated and frozen capacitated was analyzed relative to fresh non-capacitated as control. Relative fold change was calculated using ΔΔCq method. A significant difference between groups for fold change was analyzed using ANOVA and multiple comparison was performed by Duncan multiple range test. The mRNA expression of ADCY10, PRKACA and AKAP4 was significantly (p<0.05) up-regulated in fresh capacitated, frozen non-capacitated and frozen capacitated compared to fresh non-capacitated control. The mRNA expression of CATSPER2 and CRISP2 was significantly (p<0.05) up-regulated in fresh capacitated and frozen non-capacitated compared to fresh non-capacitated control. The mRNA expression of HSP90 was significantly (p<0.05) higher in frozen non-capacitated compared to fresh non-capacitated control. The mRNA expression of ADCY10, PRKACA, AKAP4, CATSPER2, CRISP2 and HSP90 was significantly lower in frozen capacitated compared to frozen non- capacitated group. From the present study it was concluded that the genes studied in the present study could be used as capacitation related biomarkers. We further state that cryopreservation induces capacitation like changes and these changes have much more impact on expression of capacitation related genes.
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