Investigation of chia seed (Salvia hispanica L.) extract supplementation to extender on post-thawing ram sperm parameters and genes-related antioxidant capacity

Ram semen cryopreservation is not so widely used compared to bull semen. This study was carried out to explore the potential of methanolic chia seed extract (MCSE) in ram semen extender on kinematics, acrosome, apoptosis, antioxidant capacity, genes-related antioxidants, and caspase-3 in post-thawing ram semen. Semen from Rahmani rams (n=5) was collected, pooled, and diluted with Tris-extender (15 % egg yolk, 1 % soybean lecithin and 5 % glycerol) supplemented with MCSE (0, 125, 250, 375, and 500 µg/mL). MCSE had 67.5 % linolenic acid, 17.99 % linoleic acid, total phenolic, and flavonoid compounds. MCSE had higher antioxidant activity by DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) and FRAP (Ferric-reducing antioxidant power) assays. MCSE (500 µg/mL) significantly improved sperm motility (43.6 vs. 30.0 %), vitality (44.0 vs. 30.7 %), and membrane integrity (43.1 vs. 31.3 %) as well as sperm velocity parameters and acrosome integrity (53.4 vs. 36.2 %) after thawing. MCSE at a level of 500 µg/mL increased viability, total antioxidant capacity level, and decreased apoptotic spermatozoa and malondialdehyde levels in post-thawed semen. MCSE at levels of 250, 375, and 500 µg/mL decreased caspase-3 level to 42.8, 35.6, and 30.0 % compared to control (54.6 %) after thawing, and resulted in a clear up-regulation of antioxidant-related genes of superoxide dismutase 1, catalase, glutathione peroxidase 1, and GA-binding protein transcription factor subunit beta-1 (GAPP1). The highest impact was observed for MCSE on GAPP1 as compared to other profiles as well as for MCSE at 500 µg/mL as compared to with other levels. All MCSE levels decreased Caspase-3 mRNA by about 44, 51, 61, and 61 %, respectively. In conclusion, MCSE had higher anti-oxidative activity. Tris-soya bean lecithin extender supplemented with MCSE at a level of 500 µg/mL, as a natural antioxidant, has a vital role in maintaining the freezing ability of ram spermatozoa after cryopreservation.