Zugl.: Leipzig, Universiẗat, Diss., 2005.

Tierärztliche Hochsch., Diss., 2005--Hannover.

An egg yolk is a common component of diluents used as protectants of sperm cells during the process of cryopreservation. Its substitution by low density lipoproteins (LDL) has shown that it is the LDL that provides the egg yolk with its cryoprotective characteristics: it protects sperms against cold shock and other changes, thus helping to preserve their fertilization ability even after the freezing-thawing process. However, the sperm quality is affected by many other internal and external factors. Therefore, the aim of this study was to evaluate if the effect of the addition of LDL to the diluent of the ejaculate is significantly influenced by the following selected factors - the type of used diluent, the bull´s breed and its individuality and the date of the sampling. Experimental insemination doses were repetitively (four times) obtained from a group of six bulls (three Holstein bulls and three Czech Fleckvieh bulls) at the Natural Hradišťko insemination service s.r.o. The samples of semen were diluted with two types of non-egg diluents containing soybean lecithin extracts (AndoMed and BioXCell). To each of these diluents, LDL at 4, 6, and 8% concentration was added; a non-LDL diluent served as a check. All the insemination doses were frozen by a standard procedure and then stored in liquid nitrogen. The CASA system (Computer Assisted Sperm Analysis) was selected for the evaluation of the sperm motility. Immediately after thawing and then after two hours of incubation in water bath (37 °C), the values of kinematic parameters were obtained from the samples - the total percentage of motile sperm and the percentage of progressively motile sperm. These data were then statistically processed; based on the outputs, VAP, VCL, ALH and the percentage of progressively motile sperm (PMOT) were chosen as representative kinematic parameters. The values of the parameters were higher in almost all evaluated samples diluted with BioXCell, compared to those diluted with AndroMed. Even though we demonstrated the existence of a high variability of results depending on the time of incubation, bull breed, the individuality of the bull and the date of the sampling, it could be said that the best concentration of added LDL is 6 % for BioXCell and 8 % for AndroMed. It would be useful to perform more experiments evaluating the effect of adding LDL to non-egg diluents on the quality of the thawed sperm. In these experiments, more sperm quality parameters should be examined and factors influencing the variability of results demonstrated in this work should be taken into consideration.

The aim of this study was to evaluate the preservation ability of five commercial boar semen extenders and compare CASA (Computer Assisted Sperm Analysis) method and classic subjective method of the sperm motility evaluation. The second objective of this study was to examine the boar seminal plasma selenium and zinc concentration and their relation to semen quality and quantity. The third objective of this study was to analyse the effect of twelve natural substances characterized by antibacterial activity on boar semen. Furthermore, the last objective of this study was to assess the effect of herbal extract supplement prepared from Eurycoma longifolia, Tribulus terrestris and Leuzea carthamoides on libido, semen quality and quantity in boars. The sperm progressive motility decreased among all tested extenders, depending on the storage time and increasing dilution ratio. The highest sperm progressive motility was achieved in Androhep Plus extender, followed by extenders VIP 5 and VIP 3. The lowest progressive sperm motility was typical for both BTS type extenders. The sperm progressive motility was influenced by the method and by the method and interactions with a variety of other factors including dilution ratio, sample storage time and sperm movement quality. There was no significant relation between the boar seminal plasma zinc concentration and sperm quality or quantity parameters. The boar seminal plasma selenium concentration positively correlated with sperm progressive motility, sperm concentration, total number of spermatozoa per ejaculate and negatively with distal protoplasmic droplet occurrence. Natural substances characterized by antibacterial activity had a significant negative effect on the sperm progressive motility. The positive effect of the herbal extract supplement containing natural stimulants on the libido of boars has been demonstrated in this study. The quality or quantity of boar sperm was not highly affected by this herbal extract supplement.

Sperm damage during cryopreservation is considered a major obstacle to the expansion of sperm storage technology in fish. In-depth knowledge of cryoinjury and cryoprotectants with respect to the quality of fish sperm can enhance future use of cryopreservation. We used antifreeze proteins as cryoprotective agents to improve the quality of frozen/thawed spermatozoa, along with optimization of cryopreservation protocols. Reviews vitrification, a promising cryopreservation technique for fish sperm storage. Vitrification requires rapid cooling/warming, small volume containers, and use of permeable cryoprotectants at high concentrations to solidify both intra- and extra-cellular materials. While high concentration of cryoprotectant show toxicity to cells. The quantity of permeable cryoprotectant can be reduced or eliminated by use of apparatus or techniques that dramatically increase freezing and warming rates by treating a much smaller quantity of sperm. Thus, vitrification may be more suitable for fish producing small quantities of highly concentrated sperm, but not sturgeon producing high quantities of sperm with low concentration. As second aim of the present study, proteomic methods were applied to characterize the protein profiles of sterlet spermatozoa and seminal plasma and assess their effect on spermatozoa function in conventional cryopreservation. The motility variables of cryopreserved sterlet sperm were also investigated. The motility rate of sterlet sperm significantly decreased after cryopreservation, while no difference in mean curvilinear velocity of fresh and cryopreserved sperm was detected. Six proteins were altered in seminal plasma and thirteen in spermatozoa following cryopreservation. Among them, eight proteins were positively identified: a) two (mitochondrial ATP synthase subunit alpha and heat shock protein 70) were from seminal plasma, associated with metabolism and response to stress; b) four (triosephosphate isomerase, mitochondrial ATP synthase subunit ?, glycerol-3-phosphate dehydrogenase [NAD(+)], enolase B) in spermatozoa are involved in metabolic pathways such as gluconeogenesis and glycolysis to provide efficient energy for spermatozoon movement; c) the other two (tubulin ? chain and tubulin ? chain, testis-specific) in spermatozoa are major constituents of sperm microtubules, playing important roles in the organization of the microtubule cytoskeleton. These results broaden the understanding of protein-related cryoinjury in sperm, which may help to determine the function of altered proteins and provide new insights into improving sperm cryopreservation. Since cryopreservation is known to cause lethal and sublethal damage to sperm, different concentrations of antifreeze proteins (AFPI or AFPIII) were employed as cryoprotectants. The flow cytometry analysis revealed that supplementation with antifreeze proteins was associated with significantly higher membrane integrity in cryopreserved sterlet sperm, except with the use of 0.1 ?g/ml of AFPI. However, motility rate, curvilinear velocity, straight-line velocity, and fertilization rate of frozen-thawed sperm did not differ from that without addition of antifreeze proteins. It was concluded that addition of antifreeze proteins to cryopreservation medium was the source of the protective effects on sperm plasma membrane integrity.

Zugl.: Heidelberg, Univ., Diss., 2007

Tierärztl. Hochsch., Diss., 2004--Hannover.

Tierärztl. Hochsch., Diss., 2004--Hannover.

The reproduction of fish either in nature or controlled condition in aquaculture is a biological event that can be strongly related to the reproductive success. In addition, study of sperm physiology and control of sperm quality can be taken into account as an issue to obtain successful production in aquaculture systems. It is often admitted that sperm quality as one of the limiting factors of the fertilization success is affected by different factors, so some researches have paid attention to sperm quality. Motility is regarded as one of the most reliable parameters for predicting sperm quality, and to evaluate the sperm fertilizing potential, observation of decrease in sperm motility performance under in vivo or in vitro circumstances has been commonly used. These studies were conducted based on the hypothesis that aims to assess sperm motility characteristics and some physiological changes of spermatozoa (such as antioxidant enzymes activity and lipid composition) in out range of spawning temperature of each species. The investigation of the fish spermatozoa motility function and enzymatic response in relation to different in vitro temperatures was carried out using activation of spermatozoa from taxonomically distant fish species (common carp, rainbow trout and sterlet) at 4, 14, and 24° C. Special attention was taken to the sperm motility parameters (motility rate, duration and velocity), activity of catalase (CAT) and superoxide dismutase (SOD), and thiobarbituric acid-reactive substance (TBARS) content as marker of lipid peroxidation. As results of this study, duration of motility in three aforementioned species were shown to be longest at 4° C compared to 14 and 24° C. In rainbow trout and carp, velocity was significantly increased at 14 °C after 10 and 30 s of spermatozoa activation respectively. While, at 60 s post-activation, the velocity of spermatozoa in sterlet was highest at 24° C. Activity of CAT was in the highest level at 4° C in rainbow trout and sterlet, while no difference was observed in carp spermatozoa. As second aim of the present study, lipid composition was analyzed in motile or immotile carp spermatozoa at different in vitro temperatures (4, 14 and 24° C). The lower proportions of some fatty acids, 18:3 (n-3) and 22:6 (n-3) in motile spermatozoa were detected at 24° C. With respect to importance of phospholipids as source of energy in fish spermatozoa movement, the total phospholipid content was lesser in motile than in immotile spermatozoa at 24° C. At 24° C also, phosphatidylcholine and phosphatidylserine proportions were lesser in motile than immotile spermatozoa. On the basis of these data, lipid composition of carp motile spermatozoa is affected by temperature, with decrease in proportion of some lipid compositions at higher temperature, elevation of sperm curvilinear velocity and decreased duration of the period over which motility is sustained. The third study was designed to investigate role of different factors that might control the activation of spermatozoa motility, using physiological functions of osmolality, calcium (Ca2+) ion and particularly temperature in burbot. Spermatozoa motility was tested at temperatures of 4 and 30° C in following media: seminal fluid (SF), isotonic media (plus Ca2+ and without it) and hypotonic media (plus Ca2+ and without it). With regard to activation of burbot spermatozoa motility in seminal fluid at high temperature (30° C), it seems that no risk of semen handling will occur during hatchery routine practice. The results suggest that initiation of spermatozoa motility in burbot is mediated by the simultaneous involvement of Ca2+ and temperature. Thus, it might be concluded that burbot spermatozoa is more sensitive to high temperature for initiation of motility relative to other freshwater fish species.

No description available.