Department of Clinical Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Dierenartsenpraktijk Horst e.o., Horst, The Netherlands.
Cytometry A. 2023 Jun;103(6):479-491. doi: 10.1002/cyto.a.24712. Epub 2022 Dec 22.
Artificial insemination with chilled stallion semen is hampered by a limited period of maximum fertility maintenance (24-48 h). This study used multiparametric flow cytometry to simultaneously measure reactive oxygen species (ROS) production, mitochondrial function or [Ca ] and plasma membrane fluidity in viable, acrosome-intact spermatozoa, with the aim of providing insight into changes in sperm function during storage at 5°C. High proportions of viable and acrosome-intact spermatozoa (71 ± 8%) remained after 96 h of storage demonstrating that the basic integrity of the cells was well preserved (n = 17 stallions). In addition, more than 90% of viable, acrosome-intact spermatozoa had active mitochondria and low intra-cellular or mitochondrial ROS levels. By contrast, the percentage of viable, acrosome-intact sperm with low plasma membrane fluidity and low [Ca ] decreased over time (1 h: 63 ± 16%, 96 h: 29 ± 18%; p < 0.05). The [Ca ] in viable sperm rose 3.1-fold (p < 0.05) over the 4 days, and fewer spermatozoa responded to bicarbonate stimulation (1 h: 46 ± 17%, 96 h: 19 ± 12%) with an increase in plasma membrane fluidity following prolonged storage. Overall, prolonged storage of stallion semen at 5°C resulted in disturbed calcium homeostasis and increased plasma membrane fluidity. The decline in fertility of stallion semen during cooled-storage may therefore relate to aspects of in vitro aging (changes in plasma membrane fluidity and intracellular calcium) which impairs capacitation-associated cell functions.
利用冷藏的种马精液进行人工授精受到最大生育维持期（24-48 小时）的限制。本研究使用多参数流式细胞术同时测量活精子中活性氧（ROS）的产生、线粒体功能或[Ca ]和质膜流动性，目的是深入了解精子在 5°C 储存过程中功能的变化。在 96 小时的储存后，仍有 71±8%的活精子和完整顶体的精子（n=17 匹种马），这表明细胞的基本完整性得到了很好的保存。此外，超过 90%的活的、顶体完整的精子具有活跃的线粒体和低的细胞内或线粒体 ROS 水平。相比之下，活的、顶体完整的精子中具有低质膜流动性和低[Ca ]的比例随着时间的推移而下降（1 小时：63±16%，96 小时：29±18%；p<0.05）。活精子中的[Ca ]在 4 天内增加了 3.1 倍（p<0.05），并且随着储存时间的延长，对碳酸氢盐刺激的反应性（1 小时：46±17%，96 小时：19±12%）减少的精子数量也减少了。总的来说，5°C 下种马精液的长时间储存导致钙稳态紊乱和质膜流动性增加。因此，在冷却储存过程中种马精液生育力的下降可能与体外老化（质膜流动性和细胞内钙离子的变化）有关，这会损害与获能相关的细胞功能。
