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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.

Fedorov P., Grabic R., Fedorova G., Cosson J., Boryshpolets S., Dzyuba B. (2017): Development and application of LC/HRPS for quantification of adenine nucleotides, creatine phosphate, and creatine in sturgeon spermatozoa. Czech J. Anim. Sci., 62, 67-74.The objective of this study was to investigate the applicability of liquid chromatography coupled with high resolution product scan (LC/HRPS) mass spectrometry for simultaneous quantification of adenine nucleotides, creatine phosphate, and creatine levels after fixation of sturgeon sperm by perchloric acid. This approach has been used for the determination of adenine nucleotides, creatine, and creatine phosphate in biological tissues, but no applications to sperm cells have been reported to date. The results of method validation showed that specific correction factors for the calculation of macroergic phosphate content in sperm cells extracted by perchloric acid should be used to get accurate concentration values. The proposed LC/HRPS method is beneficial for the analysis of adenine nucleotides because it allows simultaneous quantification of all target analytes at low concentrations in a single run. This is an advantage compared to conventional methods based on colorimetric or chemiluminescent assays and even compared to sophisticated methods based on high resolution nuclear magnetic resonance.

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