Effect of quercetin or butylated hydroxytoluene on cooled or frozen-thawed ram sperm quality

Abstract

Cooling and freezing processes cause physical and chemical damage to sperm by cold shock and oxidative stress. This study aimed to evaluate the effect of two antioxidants on sperm parameters of cooled and frozen-thawed ram semen diluted in an egg yolk-based extender. Semen was collected from 30 rams and processed in two consecutive experiments to test the inclusion of different concentrations of quercetin and butylated hydroxytoluene (BHT) in an egg yolk-based semen extender. Dimethyl sulfoxide (DMSO) was added as a solvent to the semen extender in a ratio of 1 mL DMSO for 90 mg of quercetin and 1 mL DMSO for 880 mg of BHT. After collection, semen was diluted at 200 × 106 motile sperm/mL (control) and split into different groups in each experiment. In experiment 1, semen was diluted with the extender containing quercetin (Q5, 5 μg/mL; Q10, 10 μg/mL; Q15, 15 μg/mL) or DMSO alone (DMSO1, 0.055 μL DMSO per mL; DMSO2, 0.165 μL DMSO per mL). In experiment 2, semen was diluted with the extender with BHT (BHT1, 0.5 μg/mL; BHT2, 1 μg/mL; BHT3, 1.5 μg/mL) or DMSO alone (DMSO3, 0.375 μL DMSO per mL; DMSO4, 1.125 μL DMSO per mL). After dilution, the semen was divided into two aliquots. Treated ram sperm samples were also subjected to different storage methods. The first set of samples was cooled at 5 °C for 24 h, whereas the second set of samples was frozen-thawed. Sperm motility parameters and plasma membrane integrity (PMI) were evaluated immediately after dilution (0h) and 24 h after cooling and in the frozen-thawed samples via computer-assisted sperm analysis and epifluorescence microscopy, respectively. The inclusion of quercetin or BHT did not affect sperm motility parameters or PMI of fresh, cooled, or frozen-thawed sperm in this study (P < 0.05). However, further studies are needed to test the effects of these antioxidants on the fertility of cryopreserved ram semen.

Quercetin: Putative effects on the function of cryopreserved sperms in domestic animals

“…The favourable effects of flavonoids have been connected with different properties comprising the anticipation of oxidative stress (Devi et al, 2021), protection of DNA integrity, and metal chelating ability (Čabarkapa‐Pirković et al, 2021). Hence, supplementation of quercetin to semen extender was found to have beneficial antioxidant activities on post‐thaw properties of sperm cells of rams (Canuto et al, 2022; Jiménez‐Aguilar et al, 2021; Silva et al, 2012); boars (Kim et al, 2014; Ribas‐Maynou et al, 2021), bulls (Avdatek et al, 2018a; El‐Khawagah et al, 2020; Tvrdá et al, 2016), dog (Bang et al, 2022), horses (Seifi‐Jamadi et al, 2016), and bucks (Diniz et al, 2022; Silva et al, 2016). However, reports by Gibb et al (2013) and Silva et al (2012) clarified that the addition of stallion sperm and ram sperm with quercetin before cryopreservation had no significant effect on viability nor acrosome integrity of spermatozoa, indicating that the antioxidant characteristics of quercetin did not affect viability or acrosome integrity parameters.…”

“…Reports from various studies have exemplified the valuable role of quercetin on parameters of fresh and post‐thaw sperm in stallions (Gibb et al, 2013; Seifi‐Jamadi et al, 2017); buffalo bull (El‐Khawagah et al, 2020), ram (Canuto et al, 2022), and rooster (Appiah et al, 2020). Quercetin has been proposed to delay oxidation via interaction with alpha‐tocopherol and the subsequent neutralization and inhibition of free radical generation.…”