Antioxidants protect proteins' anchorage to the bilayer by improving plasma membrane integrity of ram spermatozoa during liquid preservation in a soya lecithin‐based diluent

Abstract

Antioxidants are known to prevent the reactive oxygen species (ROS)-mediated peroxidative damage to the membrane lipids during hypothermic storage of mammalian spermatozoa. We hypothesized here that ROS also affect the lipid-protein interactions, thereby diminishing the membrane's integrity and proteins' anchorage to the bilayer. Antioxidants prevent these damages by scavenging the ROS. Ejaculates from Patanwadi rams were pooled after subjective evaluation and centrifuged using Percoll . Sperm pellet was resuspended in soya lecithin-Tris-fructose diluent (400 × 10 cells/ml) containing either antioxidants (100 IU/ml catalase + 10 mM reduced glutathione) or no antioxidant. Aliquots were chilled to 5°C in a cabinet and stored in a refrigerator at 3-5°C for 72 hr. Sperm motility, viability, lipid peroxidation (LPO) and hypo-osmotic swelling test (HOST) were performed at 0, 24, 48 and 72 hr. Sperm proteins extracted with 0.5% Triton X-100 were resolved by SDS-PAGE and quantified using Quantity One software (Bio-Rad, USA). The rapid motility, linearity and straight-line velocity (VSL) were found significantly (p < .05) higher in the antioxidant-treated group compared to the control at 48 hr of storage. Sperm viability was found comparable between the groups. Higher HOST response and lower LPO were found in the antioxidant-treated sample compared to the control both at 48 and at 72 hr. Overall, the proteins P1 (106.09 kDa), P2 (87.00 kDa) and P4 (51.14 kDa) were lower (p < .05) in the sperm extract of antioxidant-treated group compared to the control. The content of P4 (51.14 kDa) in sperm extract was found to increase (p < .05) earlier (48 vs. 72 hr) in the control group compared to the antioxidant-treated group. Altogether, the results suggested that antioxidants reduced LPO in spermatozoa, resulting in higher sperm motility, plasma membrane integrity and protection of proteins' anchorage to the plasma membrane at 48 and 72 hr of storage.

Effects of soy isoflavones on intake, body weight, sex hormones, antioxidant performance, and semen quality in Xinong Saanen goats

“…Similar results were also obtained in our study. We concluded that increase of plasma membrane integrity of sperm had a high correlation with antioxidant performance induced by SI (Paul and Smk 2017). The HOST was descried as a classical assay to evaluate function of sperm and integrity of physiological function of sperm plasma membrane (Lechniak et al 2003).…”

The enriched Y-bearing sperm combined with delayed fixed-time artificial insemination for obtaining male Simmental crossbred offspring

“…Seminal plasma contains sodium, potassium, protein, ascorbic acid, and many nutrients. Ascorbic acid, an antioxidant, keeps the plasma membrane from oxidative phosphorylation [23]. Meanwhile, sodium and potassium ions in seminal plasma play a key role in sperm metabolism through the plasma membrane [24].…”

Recent approaches in the use of antioxidants and proteomic modifications in ram semen preservation

“…However, other studies have shown the beneficial effects of catalase in semen cryopreservation. For instance, Paul et al (2017) found that the addition of catalase (100 IU/mL) and glutathione (10 mM), in soya lecithin-Tris-fructose extenders at 3-5°C for 48 h, improved various parameters of ram semen, such as motility and sperm velocity, improved the plasma membrane integrity, and reduced significantly the levels of MDA. Furthermore, Alomar et al (2016) demonstrated that adding catalase to ram semen extenders significantly reduced the formation of H 2 O 2 by live and dead sperm.…”