Effect of different antioxidant additives in semen diluent on cryopreservability (−196°C) of buffalo semen

Abstract

Aim:The aim of this study was to evaluate the effect of different antioxidant additives in standard tris-fructose-egg yolk-glycerol (TFYG) extender on the cryopreservability of buffalo semen.Materials and Methods:Semen collection using artificial vagina, twice weekly for 5 weeks from three pedigreed health breeding bulls of Mehsani breed, aged between 6 and 8 years. Immediately after initial evaluation all 30 qualifying ejaculates (10/bull) were split into three aliquots and diluted at 34°C keeping the concentration of 100 million spermatozoa/ml with standard TFYG extender as control and TFYG having two antioxidant additives - Cysteine HCl at 1 mg/ml and ascorbic acid at 0.2 mg/ml to study their comparative performance. Semen filled in French Mini straws using IS-4 system and gradually cooled to 4°C and equilibrated for 4 h in cold handing cabinet. After completion of equilibration, straws were cryopreserved in LN2 by Programmable Bio-freezer. Semen was examined at post-dilution, post-equilibration, and post-thaw stages for sperm quality parameters, and at each stage plasma was separated for enzymatic analysis of aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and alkaline phosphatase (AKP).Results:The mean percentage of sperms in TFYG, TFYG + cysteine HCl and TFYG + ascorbic acid diluents at post-thaw stage in terms of progressive motility (52.83±0.52, 57.83±0.52, 57.83±0.52), livability (78.70±0.21, 82.33±0.23, 81.73±0.22), and abnormality (5.43±0.21, 5.03±0.17, 5.23±0.18) varied significantly (p<0.05) between control TFYG and TFYG having antioxidant additives. The mean U/L activities of AST (78.70±0.47, 72.80±0.48, 73.30±0.54), LDH (172.70±0.41, 155.78±0.42, 156.33±0.41), and AKP (103.61±0.34, 90.20±0.34, 91.03±0.34) in semen diluted with TFYG, TFYG + cysteine HCl and TFYG + ascorbic acid diluents at post-thaw stage, respectively, which showed significantly (p<0.05) higher leakage of enzymes in control TFYG than TFYG incorporated with additives.Conclusion:Incorporation of antioxidant additives such as cysteine HCl and ascorbic acid in standard TFYG diluents improves sperm quality parameters, reduces enzyme leakage, and ultimately advances cryopreservability of buffalo semen.

Effect of Different Antioxidant Sources Added to Buffalo Semen Extender During Cryopreservation on Freezability and Fertility of Buffalo Spermatozoa

“…Also, antioxidants properties of NAC can reduce the semen viscosity that important for improving the fertility and pregnancy rate (Verit et al, 2009). According to the fact, GSH production was affected by NAC treatment as a source of L-cysteine (Rushworth and Megson, 2014), it is clear to note that the significant improvement in spermatozoa characteristics in E4 (NAC) compared to control (E1) was in relation with the roles of NAC as ROS scavenger (Patel et al, 2016;Koohestanidehaghi et al, 2020) and cell apoptosis preserver (Dhouib et al, 2016).…”

Post thaw treatment of frozen buffalo semen with antioxidants vitamin C and 2-mercaptoethanol

“…All this amounts to spermatozoon lesions such as oxidative damage or apoptosis, which can lead to negative results during the AI process [7]. Research has shown poor quality of keeping and freezability of buffalo semen [8,9]. Negative changes in buffalo spermatozoa are due to their unique physiology and higher polyunsaturated phospholipid levels in the plasma membrane [1], probably affecting the sperm motility, viability, acrosomal and DNA integrity [10,11].…”

“…The motility and fertilization ability of spermatozoa can improve after addition of various motility enhancing agents or antioxidants [9,12]. The antioxidants check the chemical breakdown of the substrate resulting from oxidation, neutralize the free radicals, and reduce the risk of damage to spermatozoa during cryopreservation [13].…”

“…The major antioxidants naturally present in mammalian semen that regulate ROS are Vitamin C and E. These antioxidants protect the sperm from lipid peroxidation and ensure the integrity of the plasma membrane and mitochondria as well as better kinematics parameters for post-cryopreservation sperm [1,14,15]. Ascorbic acid in the seminal plasma accounts for about 65% of the antioxidant capacity of semen [9]. Tariq et al [16] have shown that the concentration of ascorbic acid in buffalo seminal plasma is 364 lmol/ L, while that in blood plasma is 40 lmol/L.…”

“…The ascorbic acid content of bull semen is significantly higher as compared to buffalo bull semen [17], which could explain the differences in the quality preservability of semen. The addition of antioxidants to the semen extender may lead to mitigation of oxidative damage in buffalo spermatozoa during the post-thawing process [9,18]. In view of these facts, the present investigation aimed to evaluate the effect of antioxidant additives such ascorbic acid (Vit C) and 2-mercaptoethanol (ME) added to buffalo semen samples after thawing for 1 h and 2 h incubation.…”