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Aplikované aspekty kryokonzervace rybích spermií
HOROKHOVATSKYI, Yevhen
Fish sperm cryopreservation becomes highly attractive due to increasing number of potential applications and it has been considerably developed over the past decades. Cryopreservation of fish sperm is expected to improve the broodstock management in hatcheries, simplify hybridization and selective breeding, and serve as a useful tool for biodiversity and conservation of endangered species programs. Nevertheless, its status nowadays is highly contentious due to absence of specific techniques and standardization in the developed protocols. Moreover, the heterogeneity of cryoresistance in sperm of some fish individuals and, strong impairment of the sperm quality and survival after cryopreservation, to date, present some challenges inhibiting wide applicability of this approach. Therefore, a better understanding of the mechanisms of spermatozoa cryo-injuries and the development of new and more effective methods for cryopreservation of fish sperm are of primary importance. In the current work, the causes of individual variability in sperm cryoresistance, influence of cryopreservation process on sperm quality parameters regarding application of uncontrolled cooling devices were investigated. One of the study outputs is the founding the possible causes of sperm cryoresistance heterogeneity in common carp (Cyprinus carpio) males. As a result, a negative correlation between changes in the contents of major lipid classes and sperm motility after cryopreservation was found. In particular, the increased level of phospholipid, cholesterol and free fatty acid in samples with low post-thaw motility percentage were observed and related to decrease in sperm cryoresistance in common carp sperm. In the investigation of the consequence of the application of uncontrolled cooling devices on post-thaw sperm parameters of sterlet sperm, it has been shown that sterlet sperm could be cryopreserved by both Styrofoam box and dry shipper devices studied. However, due to inability to precisely control cooling process in these devices, the cooling conditions can be changed by varying straw number during freezing in Styrofoam box or by straw position during freezing in dry shipper. As a consequence, these changes shift the optimal freezing rates needed for successful sturgeon sperm cryopreservation resulting in decrease in post-thaw sperm motility and fertilization ability. These results suggest the way of standardization of fish sperm cryopreservation protocols by selecting appropriate straw number or straws position when uncontrolled cooling devices are used. Finally, to explore the cryopreservation effects on fish spermatozoa, the Percoll density gradient centrifugation technique was elaborated and applied for the first time to sterlet (Acipenser ruthenus) sperm. That technique allows to select and analyse only the sperm population that survived cryopreservation procedure and saved high motility parameters. It was found that this spermatozoa subpopulation possesses minimal changes in protein profile in comparison to native sperm. Moreover, the differences in many proteins which were found in sperm suspension after cryopreservation without separation is related to the presence of non-viable and sperm wreckage subpopulations in the frozen-thawed sperm suspension. Collectively, all of these detected changes in sperm motility parameters, viability, and protein profiles of viable spermatozoa after freeze-thaw process suggest some explanations for the mechanism responsible for cryodamage of sperm and provides the background for further development of an efficient cryopreservation protocols. However, it should be also further studied if the Percoll separation technique could serve as a useful tool for both better understanding of non-lethal sperm cryodamages and improvement of assisted reproduction practice in sturgeon species.
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