Effects of supplemental antioxidants on in vitro fertility measures for cryopreserved boar spermatozoa

This work aims to evaluate how supplementing a commercial freezing media with butylated hydrox-ytoluene (BHT), or reduced glutathione (GSH), or their combination affected in-vitro measures of boar sperm after cryopreservation. One ejaculate was collected from 30 high-fertility boars in a weekly collection rotation. Samples were diluted 1:1 in an extender and cooled before overnight shipping at 17 degrees C to the freezing lab. On arrival, samples were split into the treatments with the following additions before cryopreservation; 1) semen without additional antioxidants (Control), 2) semen with 1 mM BHT, 3) semen with 2 mM GSH, and 4) semen with 1 mM BHT+2 mM GSH. Semen was evaluated for motility kinetics at 30,120, and 240 min after thawing. Flow cytometry assessments were performed at 60 min after thawing. At all-time points evaluated, total and progressive motility were greater (P < 0.05) in semen cryopreserved with GSH than in Control. No (P > 0.05) differences between Control and other treatment groups were observed in viability, or acrosomal and mitochondrial membrane integrity; however, the proportion of capacitated spermatozoa were reduced (by-21.17%) in semen treated with BHT + GSH compared to Control (P < 0.05). In contrast, there was a higher (P < 0.05, +21.18%) superoxide anion production in the Control than in the BHT + GSH. For IVF, semen cryopreserved with both anti-oxidants (BHT + GSH) had a negative (P < 0.05) impact on fertilization rate (-54.11%) compared to Control. However, for the blastocysts rate, there were more (+22.75%) blastocysts (P < 0.05) for BHT compared to Control. These results indicate that commercial media supplemented with GSH increased motility but impaired in vitro fertilization rate. On the other hand, media supplemented with BHT improved the in vitro fertilizing ability of the frozen-thawed sperm cells. Therefore, we suggest the supplementation with 1 mM of BHT in the formula of commercial freezing media used in the present experiment.(c) 2023 Elsevier Inc. All rights reserved.

Automated summary

This study aimed to evaluate the effect of supplementing a commercial freezing media with BHT, GSH, or their combination on in-vitro measures of boar sperm after cryopreservation. The results showed that adding GSH improved motility but decreased in vitro fertilization rate. Conversely, adding BHT enhanced the in vitro fertilizing ability of the frozen-thawed sperm cells.