Abstract
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.
Highlights
IntroductionConventional sperm selection techniques used in human Assisted reproductive technologies (ARTs) are density gradient centrifugation (DGC), which separates spermatozoa according to their density, and swim up (SU), which separates spermatozoa according to their motility [1,2]
Assisted reproductive technologies (ARTs) consist of all procedures for handling female and male gametes and leading to embryo production aimed to overcome infertility in humans.Conventional sperm selection techniques used in human ARTs are density gradient centrifugation (DGC), which separates spermatozoa according to their density, and swim up (SU), which separates spermatozoa according to their motility [1,2]
VAP was significantly reduced after centrifugation at 300× g for 5 min (p < 0.01), at 300× g for 10 min and 1500× g for 5 and 10 min (p < 0.0001) respectively vs. control; VSL was significantly reduced after centrifugation at 300× g for 5 min (p < 0.001), at 300× g for 10 min and 1500× g for 5 and 10 min (p < 0.0001) respectively vs. control; VCL was significantly reduced after centrifugation at 300× g for 10 min (p < 0.01) and at 1500× g for 5 and 10 min (p < 0.0001) respectively vs. control
Summary
Conventional sperm selection techniques used in human ARTs are density gradient centrifugation (DGC), which separates spermatozoa according to their density, and swim up (SU), which separates spermatozoa according to their motility [1,2]. Such procedures are employed in the animal breeding industry to produce embryos to increase reproductive efficiency, to gain genetic improvement, and to preserve endangered animal species [3]. It was found that SU increased reactive oxygen species (ROS) levels in oligoasthenoteratozoospermic samples [4]
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