Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain; Department of Animal Science, Universidade Federal de Viçosa, Viçosa, 36570-900, Brazil.
Theriogenology. 2024 Nov;229:127-137. doi: 10.1016/j.theriogenology.2024.08.021. Epub 2024 Aug 22.
Conservation of equine semen in the liquid state is a central procedure in horse breeding and constitutes the basis of associated reproductive technologies. The intense mitochondrial activity of the stallion spermatozoa increases oxidative stress along the storage period, leading to sperm demise within 24-48 h of storage, particularly when maintained at room temperature. Recently, the relationship between metabolism and oxidative stress has been revealed. The study aimed to extend the period of conservation of equine semen, at room temperature through modification of the metabolites present in the media.
Processed ejaculates (n = 9) by single-layer colloid centrifugation were split in different aliquots and extended in Tyrode's basal media, or modified Tyrode's consisting of 1 mM glucose, 1 mM glucose 10 mM pyruvate, 40 mM glucose, 40 mM Glucose 10 mM pyruvate, 67 mM glucose and 67 mM glucose 10 mM pyruvate. At time 0h, and after 24 and 96 h of storage, motility was evaluated by CASA, while mitochondrial production of Reactive oxygen species (ROS), and intracellular Ca+ concentrations were determined via flow cytometry using Mitosox Red and Fluo-4 respectively. ROS and Ca were estimated as Relative Fluorescence Units (RFU) in compensated, arcsin-transformed data in the live sperm population.
After 48 h of incubation, motility was greater in all the 10 mM pyruvate-based media, with the poorest result in the 40 mM glucose (41 ± 1.1 %) while the highest motility was yielded in the 40 mM glucose 10 mM pyruvate aliquot (60.3 ± 3.5 %; P < 0.001); after 96 h of storage highest motility values were observed in the 40 mM glucose 10 mM pyruvate media (23.0 ± 6.2 %) while the lowest was observed in the 1 mM glucose media was 9.2 ± 2.0 % (P < 0.05). Mitochondrial ROS was lower in the 40 mM glucose 10 mM pyruvate group compared to the 40 mM glucose (P < 0.01). Over time Ca increased in all treatment groups compared to time 0h.
Viable spermatozoa may experience oxidative stress and alterations in Ca2+ homeostasis during prolonged storage, however, these effects can be reduced by regulating metabolism. The 40 mM glucose- 10 mM pyruvate group yielded the highest sperm quality parameters.
在马的繁殖中，精液的液体保存是一个核心程序，它构成了相关生殖技术的基础。公马精子的线粒体活性很强，这会导致在储存期间产生氧化应激，从而使精子在 24-48 小时内死亡，尤其是在室温下储存时。最近，代谢与氧化应激之间的关系已经被揭示。本研究旨在通过改变培养基中的代谢物来延长马精液在室温下的保存时间。
通过单层胶体离心处理的精液（n=9）被分为不同的等分试样，并在 Tyrode 基础培养基中或由 1mM 葡萄糖、1mM 葡萄糖+10mM 丙酮酸、40mM 葡萄糖、40mM 葡萄糖+10mM 丙酮酸、67mM 葡萄糖和 67mM 葡萄糖+10mM 丙酮酸组成的改良 Tyrode 培养基中延长保存时间。在 0h 时以及储存 24 和 96 小时后，通过 CASA 评估运动性，同时通过使用 Mitosox Red 和 Fluo-4 通过流式细胞术测定线粒体产生的活性氧物质 (ROS) 和细胞内 Ca+浓度。在活精子群体中，用补偿后的、反正弦转换的数据来估计 ROS 和 Ca 的相对荧光单位 (RFU)。
孵育 48 小时后，所有基于 10mM 丙酮酸的培养基中的运动性都较大，而在 40mM 葡萄糖中的结果最差（41±1.1%），而在 40mM 葡萄糖+10mM 丙酮酸等分试样中运动性最高（60.3±3.5%；P<0.001）；储存 96 小时后，在 40mM 葡萄糖+10mM 丙酮酸培养基中观察到最高的运动性值（23.0±6.2%），而在 1mM 葡萄糖培养基中观察到最低的运动性值为 9.2±2.0%（P<0.05）。与 40mM 葡萄糖相比，40mM 葡萄糖+10mM 丙酮酸组中的线粒体 ROS 较低（P<0.01）。随着时间的推移，与 0h 相比，所有处理组中的 Ca 都增加了。
在长时间储存过程中，存活的精子可能会经历氧化应激和 Ca2+稳态的改变，然而，通过调节代谢可以降低这些影响。40mM 葡萄糖-10mM 丙酮酸组产生了最高的精子质量参数。
