de Andrade André Furugen Cesar, Balogun Kayode, Machaty Zoltan, Knox Robert Victor
Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, Pirassununga, São Paulo, Brazil; Department of Animal Sciences, College of Agricultural, Consumer & Environmental Sciences, University of Illinois at Urbana-Champaign, USA.
Department of Animal Sciences, College of Agriculture, Purdue University, West Lafayette, IN, USA.
Theriogenology. 2023 Apr 1;200:33-42. doi: 10.1016/j.theriogenology.2023.01.025. Epub 2023 Feb 2.
This work aims to evaluate how supplementing a commercial freezing media with butylated hydroxytoluene (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 °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 antioxidants (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.
本研究旨在评估在商业冷冻培养基中添加丁基羟基甲苯（BHT）、还原型谷胱甘肽（GSH）或二者组合对冷冻保存后的公猪精子体外指标的影响。每周轮流从30头高繁殖力公猪采集一次精液。样本在稀释液中按1:1稀释并冷却，然后在17℃下过夜运输至冷冻实验室。到达后，样本在冷冻保存前被分为以下处理组：1）不添加额外抗氧化剂的精液（对照组），2）添加1 mM BHT的精液，3）添加2 mM GSH的精液，4）添加1 mM BHT + 2 mM GSH的精液。解冻后30、120和240分钟对精液的活力动力学进行评估。解冻后60分钟进行流式细胞术评估。在所有评估时间点，用GSH冷冻保存的精液的总活力和前向运动活力均高于对照组（P≤0.05）。在活力、顶体和线粒体膜完整性方面，对照组与其他处理组之间未观察到差异（P>0.05）；然而，与对照组相比，用BHT + GSH处理的精液中获能精子的比例降低了（-21.17%）（P≤0.05）。相反，对照组的超氧阴离子产生量高于BHT + GSH组（P≤0.05，+21.18%）。对于体外受精，与对照组相比，用两种抗氧化剂（BHT + GSH）冷冻保存的精液对受精率有负面影响（P<0.05，-54.11%）。然而，对于囊胚率，与对照组相比，BHT组的囊胚更多（+22.75%）（P≤0.05）。这些结果表明，添加GSH的商业培养基可提高精子活力，但会损害体外受精率。另一方面，添加BHT的培养基可提高冻融精子细胞的体外受精能力。因此，我们建议在本实验所用的商业冷冻培养基配方中添加1 mM BHT。
