Effect of catalase, superoxide dismutase and reduced glutathione in LDL extender on ovine cryopreserved sperm viability

Abstract

The aim of this study was to evaluate the motility, kinetics and membrane integrity of ovine sperm cryopreserved in extenders containing 8% LDL with enzymatic antioxidants at different concentrations. Four Santa Inês rams were used to form four pools of semen (each pool containing ejaculates from four ram, totaling four ejaculates per animal). Each seminal pool was divided into eight aliquots for the following treatments: 1) Tris-glucose-glycerol (TGG) + (16%) egg yolk (control 1); 2) TGG + 8% (w/v) LDL (control 2); 3) TGG + 8% LDL + catalase 100 U/mL; 4) TGG + 8% LDL + catalase 200 U/mL; 5) TGG + 8% LDL + superoxide dismutase 100 U/mL; 6) TGG + 8% LDL + superoxide dismutase 200 U/mL; 7) TGG + 8% LDL + reduced glutathione 5 mM; and 8) TGG + 8% LDL + reduced glutathione 10 mM. The samples were packed into 0.25 mL straws, cooled (-0.25 °C/ min), maintained at 5 °C for 2 h and then frozen (-25 °C/ min) using a TK4000 ® . Immediately after thawing (38 °C/ 30 s), sperm motility and movement characteristics were assessed by computer sperm analysis (CASA). The structural integrity of the plasma and acrosomal membranes was analyzed using fluorescent dyes. The functional integrity of membranes was assessed using a hypoosmotic swelling test. As assessed by ANOVA, significant differences (P<0.05) among treatments were only observed for VCL, VSL and VAP. For the VCL variable, the 2, 3, 4, 5, 6 and 7 extenders were similar and higher than 1 and 8 extenders, the latter being similar to each other. For the VSL variable, the 3, 4, 5, 6 and 7 extenders were similar and higher than 1, 2 and 8 extenders, the latter being similar to each other. For the VAP variable, the 3, 4 and 6 extenders were similar and higher than 1, 2, 5, 7 and 8 extenders, the latter being similar to each other. In conclusion, enzymatic antioxidants as catalase and superoxide dismutase improve the protective activity of extenders containing LDL on frozen ovine sperm. concentrações. Quatro carneiros da raça Santa Inês foram utilizados para formar quatro pools de sêmen (cada pool contendo ejaculados provenientes dos quatro carneiros, totalizando quatro ejaculados por animal). Cada pool de sêmen foi dividido em oito alíquotas para os seguintes tratamentos: 1) Trisglicose-glicerol (TGG) + (16%) gema de ovo (controle 1); 2) TGG + 8% (g/L) LDL (controle 2); 3) TGG + 8% LDL + catalase 100 U/mL; 4) TGG + 8% LDL + catalase 200 U/mL; 5) TGG + 8% LDL + superóxido dismutase 100 U/mL; 6) TGG + 8% LDL + superóxido dismutase 200 U/mL; 7) TGG + 8% LDL + glutationa reduzida 5 mM; and 8) TGG + 8% LDL + glutationa reduzida 10 mM. As amostras foram envasadas em palhetas de 0,25 mL, resfriadas (-0,25 °C/min), mantidas a 5 °C por duas horas e em seguida congeladas (-25 °C/ min) usando uma máquina de congelar TK4000 ® . Imediatamente depois da descongelação (38 °C/30 s), as amostras foram submetidas à análise computadorizada (CASA) para avaliação da motilidade e cinética. A integridade estrutural das membranas plasmática e acrossomal foi analisada utilizando corantes fluor...

Post-thaw quality of ram sperm frozen with different concentrations of low-density lipoproteins associated with non-enzymatic antioxidants

“…Although ROS generation is primarily required for physiological signaling pathways, a precise balance between its production and scavenging is crucial for sperm viability ( Bast et al, 1991 ; Gibb et al, 2020 ). Therefore, the addition of antioxidants to the extender during semen cryopreservation has been tested in several species to improve semen quality, including in sheep ( Cirit et al, 2013 ; Snoeck et al, 2015 ; Amidi et al, 2016 ; Fang and Zhong, 2020 ; Galarza et al, 2020 ).…”