Abstract
In many fish species, sperm cryopreservation has deleterious effects and leads to a significant decrease in spermatozoa viability. However, the effect of cryopreservation on sperm cells that survive this process and are still viable is not fully understood. The objective of this study was to compare the viability and proteomes of fresh and cryopreserved sterlet (Acipenser ruthenus) sperm samples before and after live-dead cell separation using Percoll density gradient centrifugation. Both fresh and cryopreserved sperm samples were divided into two groups (with or without application of Percoll separation). At each step of the experiment, sperm quality was evaluated by video microscopy combined with integrated computer-assisted sperm analysis software and flow cytometry for live-dead sperm viability analysis. Sperm motility and the percentage of live cells were reduced in the cryopreserved group compared to the fresh group from 89% to 33% for percentage of motility and from 96% to 70% for live cells. Straight line velocity and linearity of track were significantly lower in cryopreserved samples than in those separated by Percoll before and after cryopreservation. However, the percentages of motile and live spermatozoa were higher than 90% in samples subjected to Percoll separation. Proteomic analysis of spermatozoa by two-dimensional differences in-gel electrophoresis coupled with matrix-assisted laser-desorption/ionization time-of-flight/time-of-flight mass spectrometry revealed that 20 protein spot abundances underwent significant changes in cryopreserved samples compared to fresh ones. However, only one protein spot was significantly altered when samples before and after cryopreservation followed by Percoll separation were compared. Thus, the results of this study show that cryopreservation leads to minimal proteomic changes in the spermatozoa population, retaining high motility and viability parameters. The results also suggest that global differences in protein profiles between unselected fresh and cryopreserved samples are mainly due to protein loss or changes in the lethal and sublethal damaged cell subpopulations.
Highlights
Worldwide demand for sturgeon boneless meat and caviar has resulted in an increased requirement for sturgeon aquaculture [1]
Fish sperm cryopreservation is considered to be a powerful tool in aquaculture as it affords an opportunity for gamete availability synchronization of sperm and eggs, the rational use of sperm, a decrease in the number of males needed in a hatchery, simplification of broodstock maintenance and gamete transport between fish farms [2]
Spermatozoa are exposed to the influence of numerous physical and chemical factors caused by a high solute concentration during slow freezing or intracellular ice formation during rapid freezing [7]
Summary
Worldwide demand for sturgeon boneless meat and caviar has resulted in an increased requirement for sturgeon aquaculture [1]. Despite all of these advantages, cryopreservation presents challenges, as it may strongly impair sperm function and survival and decrease reproductive capacity. To be successfully cryopreserved and used for artificial reproduction in aquaculture, fish sperm must be of the highest quality before freezing as well as after thawing. In many fish species, the cryopreservation procedure has deleterious effects and is associated with a significant decrease in sperm viability [4,5,6]. Spermatozoa are exposed to the influence of numerous physical and chemical factors caused by a high solute concentration during slow freezing or intracellular ice formation during rapid freezing [7]. Water can recrystallize into larger lethal-sized ice crystals during warming as a result of differences in surface free energy and damage the cell [8]
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