González-Pérez José Gustavo, Núñez-Ruiz Aleida, Quezada-Casasola Andrés, Rodrigo-García Joaquín, Beristaín-Ruiz Diana Marcela, Luna-Nevárez Pablo, Carrera-Chávez José María
Departamento de Ciencias Veterinarias, Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez, Ciudad Juárez, Chihuahua, 32310, México.
Laboratorio Nacional de Citometría de Flujo, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Coyoacán, Ciudad de México, 04510, México.
Theriogenology. 2025 Dec;248:117615. doi: 10.1016/j.theriogenology.2025.117615. Epub 2025 Jul 31.
Cryopreservation induces negative effects on spermatozoa due to oxidative stress. The aim of this study was to evaluate the effect of the addition of quercetin and gallic acid on sperm characteristics under the cryopreservation process of domestic dog epididymal semen. Samples were collected from sexually mature males by direct epididymal collection technique after orchiectomy and diluted with a commercial extender. Treatments included Control, DMSO (0.005 %), Ethanol (0.05 %), Q25 (quercetin 25 μM), GA45 (gallic acid 45 μM), and a combination of Q25 + GA45. Motility parameters were determined using a Computer-Assisted Sperm Analysis (CASA) system. Viability, mitochondrial membrane potential, acrosomal integrity, DNA fragmentation and reactive oxygen species (ROS) production were analyzed using flow cytometer. Variables were analyzed by ANOVA. Gross and progressive motility in Control and Q25 were similar, and higher than the rest of treatment groups (p < 0.05). Conversely, ethanol showed the lowest mitochondrial membrane potential among all treatments, except for Q25 + GA45, which showed lower than Control and DMSO groups (p < 0.05). For DNA fragmentation all treatments were similar (p > 0.05). Ethanol showed a negative effect on viability and acrosomal integrity compared to Control (p < 0.05). Regarding antioxidant capacity, all treatments showed lower ROS production when compared to Control (p < 0.05). In conclusion, the addition of quercetin and gallic acid reduced ROS production but did not improve sperm parameters during cryopreservation of domestic dog epididymal semen. Furthermore, ethanol used as a solvent for gallic acid adversely affected these parameters, although antioxidants and their combination partially mitigated this negative effect.
由于氧化应激，冷冻保存会对精子产生负面影响。本研究的目的是评估在犬附睾精液冷冻保存过程中添加槲皮素和没食子酸对精子特性的影响。通过睾丸切除术后的直接附睾采集技术从性成熟雄性犬采集样本，并用市售稀释液进行稀释。处理组包括对照组、二甲基亚砜（DMSO，0.005%）、乙醇（0.05%）、Q25（槲皮素25μM）、GA45（没食子酸45μM）以及Q25 + GA45组合。使用计算机辅助精子分析（CASA）系统测定活力参数。使用流式细胞仪分析存活率、线粒体膜电位、顶体完整性、DNA片段化和活性氧（ROS）产生情况。变量采用方差分析。对照组和Q25组的总活力和前向运动能力相似，且高于其他处理组（p < 0.05）。相反，乙醇在所有处理组中显示出最低的线粒体膜电位，除了Q25 + GA45组，该组低于对照组和DMSO组（p < 0.05）。对于DNA片段化，所有处理组相似（p > 0.05）。与对照组相比，乙醇对存活率和顶体完整性有负面影响（p < 0.05）。关于抗氧化能力，与对照组相比，所有处理组的ROS产生均较低（p < 0.05）。总之，在犬附睾精液冷冻保存过程中，添加槲皮素和没食子酸可降低ROS产生，但并未改善精子参数。此外，用作没食子酸溶剂的乙醇对这些参数有不利影响，尽管抗氧化剂及其组合可部分减轻这种负面影响。
