Marzano Giuseppina, Moscatelli Natalina, Di Giacomo Mariangela, Martino Nicola Antonio, Lacalandra Giovanni Michele, Dell'Aquila Maria Elena, Maruccio Giuseppe, Primiceri Elisabetta, Chiriacò Maria Serena, Zara Vincenzo, Ferramosca Alessandra
Department of Mathematics and Physics E. de Giorgi, University of Salento, Via per Arnesano, 73100 Lecce, Italy.
Institute of Nanotechnology, CNR NANOTEC, Via per Monteroni, 73100 Lecce, Italy.
Biology (Basel). 2020 Jan 27;9(2):22. doi: 10.3390/biology9020022.
Conventional sperm selection techniques used in ARTs rely on centrifugation steps. To date, the different studies reported on the effects of centrifugation on stallion sperm motility provided contrasting results and do not include effects on mitochondrial functionality and different oxidative parameters. The effects of different centrifugation protocols (300 ×g for 5', 300 ×g for 10', 1500 ×g for 5' and 1500 ×g for 10' vs no centrifugation) on motility and oxidative status in cryopreserved stallion sperm, were analyzed. After centrifugation, almost all motility parameters were significantly altered, as observed by computer-assisted sperm analysis. A polarographic assay of oxygen consumption showed a progressive decrease in mitochondria respiration from the gentlest to the strongest protocol. By laser scanning confocal microscopy, significant reduction of mitochondrial membrane potential, at any tested protocol, and time-dependent effects, at the same centrifugal force, were found. Increased DNA fragmentation index at any tested protocol and time-dependent effects at the same centrifugal force were found, whereas increased protein carbonylation was observed only at the strongest centrifugal force. These results provide more comprehensive understandings on centrifugation-induced effects on cryopreserved stallion sperm and suggest that, even at a weak force for a short time, centrifugation impairs different aspects of equine sperm metabolism and functionality.
辅助生殖技术中使用的传统精子筛选技术依赖于离心步骤。迄今为止，关于离心对种马精子活力影响的不同研究得出了相互矛盾的结果，且未涉及对线粒体功能和不同氧化参数的影响。分析了不同离心方案（300×g离心5分钟、300×g离心10分钟、1500×g离心5分钟和1500×g离心10分钟与不离心）对冷冻保存的种马精子活力和氧化状态的影响。离心后，通过计算机辅助精子分析观察到，几乎所有活力参数都发生了显著变化。极谱法测定氧气消耗显示，从最温和到最强的方案，线粒体呼吸逐渐下降。通过激光扫描共聚焦显微镜观察发现，在任何测试方案下，线粒体膜电位均显著降低，且在相同离心力下存在时间依赖性效应。在任何测试方案下均发现DNA碎片化指数增加，且在相同离心力下存在时间依赖性效应，而仅在最强离心力下观察到蛋白质羰基化增加。这些结果为离心对冷冻保存的种马精子的影响提供了更全面的认识，并表明即使在弱力短时间作用下，离心也会损害马精子代谢和功能的不同方面。
