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Obsah metabolitů ve spermiích ryb za různých fyziologických podmínek
FEDOROV, Pavlo
Investigation of creatine- and adenylate phosphates involvement in fish spermatozoa metabolism is of high interest for fish spermatology. These compounds are necessary to support normal physiological state and motility of spermatozoa. The simultaneous changes in content of creatine- and adenylate phosphates in fish spermatozoa prior and during their motility are quite unclear. Therefore, studying and development of new methods for the quantification of creatine- and adenylate phosphates in spermatozoa of different fish species under such physiological conditions as maturation and in vitro manipulation are of high importance. One of the study outputs is the developed LC/HRPS (liquid chromatography coupled with high resolution product scan) method for the analysis of creatine- and adenylate phosphates content in fish spermatozoa (Chapter 2). Its main advantage is the possibility to detect and quantify several compounds (creatine, creatine phosphate (CP), AMP, ADP, ATP, and cAMP) simultaneously to obtain maximum information with minimum analytical effort. The method was validated taking into account such key parameters as limit of quantification, selectivity, recovery and repeatability. It represented an excellent performance allowing determination of target compounds in highly diluted fish sperm samples. Consequently, the method was applied for the quantification of aforementioned substances during sterlet (Acipenser ruthenus) spermatozoa maturation and in vitro manipulation with sperm of whitefish (Coregonus lavaretus maraena) and European eel (Anguilla Anguilla). The present study showed that immature sterlet spermatozoa are not able to initiate motility. Significant decrease of CP and stable levels of ATP and ADP content during their maturation were found. The critical importance of ATP regeneration system and oxidative phosphorilation for the maturation process of sterlet sperm as a prerequisite for successful fertilization was assumed (Chapter 3). Further experiments revealed that European eel spermatozoa were not able to initiate motility by activation medium (AM) at the start of the induced spermiation. They acquired the ability to be activated after the dilution with AM at the end of hormonal treatment. This accompanied by the increase of CP and cAMP levels in spermatozoa after activation. That allowed us to assume the involvement of ATP regenerating system and cAMP-dependent regulatory pathways in the process of hormonally induced spermiation (Chapter 4). Current study represents a first successful estimation of cAMP in fish spermatozoa during the motility period using the LC/HRPS. Important issues concerning the short-term storage of European eel sperm were rised. Storage at 4 °C was accompanied by higher marcoergic phosphates content and higher motility in comparison to the storage at 20 °C. It suggests the involvement of macroergic phosphates metabolism in short-term storage. (Chapter 4). Obtained results could contribute to the development of new effective methods for improving of spermiation and short-term sperm storage in European eel aquaculture. Various degrees of energy consumption in response to environment composition were found in whitefish spermatozoa. Energy consumption was significantly higher in motility activating conditions. No effect of osmolality was found on this process. The content of CP and ATP was significantly higher when cells were in motility inhibiting medium comparing to activation medium. No relationship between content of CP, ADP, and ATP and spermatozoa motility parameters in AM of different osmolality was found. Isotonic conditions favor the spermatozoa with longer motility period, higher linearity, and fast velocity without increase in ATP content (Chapter 5). This suggests that whitefish sperm energy management is more efficient after activation in isotonic conditions. Obtained results are of high interest for elaboration of new sperm motility activating media for fisheries practice.
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