Effect of antioxidants on quality and relative expression of fertility related genes of cryopreserved beetal buck semen
Loading...
Date
2023
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
College of Veterinary Science, Assam Agricultural University, Khanapara Campus
Abstract
A total of 120 ejaculates from six Beetal bucks, collected by artificial vagina were used in the study. Immediately after collection each ejaculate was evaluated for volume, mass activity and initial sperm motility and the ejaculates having volume 0.8 ml or more, mass activity (0 to 4+ scale) 3+ or more and initial sperm motility 70 per cent or more were pooled. A total of 48 pooled ejaculates comprising 12 pooled ejaculates for each experiment were evaluated for sperm motility, live sperm, intact acrosome, sperm concentration, HOST-reacted sperm and sperm abnormalities. Each pooled ejaculate was split into two parts and one part was used for assessment of glutathione–S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and malondialdehyde (MDA) level in the seminal plasma. The other part of pooled semen was split into four parts, then centrifuged and the seminal plasma was discarded. The centrifugate of the first three parts was extended separately in Tris extender containing vitamin E @ 1, 2 and 3 mM in experiment I; IGF-1 @ 100, 125 and 150 ng/ml in experiment II; crocin @ 1, 2 and 3 mM in experiment III; and vitamin E @ 2 mM (best of expt. I), IGF-1 @ 125 ng/ml (best of expt. II) or crocin @ 1 mM (best of expt. III) in experiment IV while the fourth part was kept as control in each experiment. Semen was frozen in 0.25 ml French straws using static horizontal vapour freezing. Frozen semen was thawed in warm water at 37ºC for 30 seconds for evaluation. Each semen sample was evaluated after freezing in experiment I, II and III for sperm motility, intact acrosome (Giemsa stain), HOST-reacted sperm and intact DNA (AO stain) and in experiment IV for sperm motility, intact acrosome (FITC-PSA stain), HOST-reacted sperm, viability (CFDA + PI stain), high mitochondrial potential (JC-1 stain) and intact DNA. Semen after freezing in all the experiments was evaluated for GST, SOD, CAT, GR, GPx, ALT, AST, LDH and MDA levels in the extracellular fluid by standard methods. In experiment IV, the relative expression of certain fertility related genes and their correlation with seminal attributes in frozen-thawed Beetal buck spermatozoa as well as fertility rate of frozen semen was also studied. In Beetal bucks all the seminal attributes studied immediately after collection and pooling were within normal ranges. Semen samples extended with Tris extender containing vitamin E @ 1, 2 and 3 mM or no additive (control) and with Tris extender containing IGF-1 @ 100, 125 and 150 ng/ml or no additive differed significantly (P<0.001) in respect of sperm motility, intact acrosome, HOST-reacted sperm, intact DNA, GST, SOD, CAT, GR, GPx, ALT, AST, LDH and MDA after freezing. Semen samples extended with Tris extender containing crocin @ 1, 2 and 3 mM or no additive differed significantly (P<0.05) in respect of sperm motility and intact acrosome after freezing. While HOST-reacted sperm, intact DNA, GST, SOD, CAT, GR, GPx, ALT, AST, LDH and MDA differed significantly (P<0.001) after freezing in Tris extender containing crocin @ 1, 2 and 3 mM or no additive. In semen extended using Tris extender containing best concentration of vitamin E (2 mM), IGF-1 (125 ng/ml) and crocin (1 mM) or no additive differed significantly (P<0.001) in respect of sperm motility, intact acrosome, HOST-reacted sperm, viability, MMP+, intact DNA, GST, SOD, CAT, GR, GPx, ALT, AST, LDH and MDA after freezing.
NFE2L2, GPx4, CAT and SOD2 gene expression was significantly (P<0.05) higher in IGF-1 group compared to that in vitamin E and crocin groups, however, no significant (P>0.05) differences were recorded between vitamin E and crocin groups.
ii
Correlation study revealed that sperm motility showed a significant (P<0.05) positive correlation with all the four target genes, irrespective of the antioxidant treatment. The target genes also showed a positive correlation with all the seminal attributes in different antioxidant groups. Although the kidding rate (doe kidded per inseminated doe) did not differ significantly (P>0.05) between groups, the values were found to be the highest in the IGF-1 @ 125 ng/ml group.
Based on the semen parameters studied it was concluded that IGF-1 @ 125 ng/ml, was found to be superior to other additives studied in maintaining post-thaw semen quality.