In order to fertilize the egg, sperm cell undergoes several subsequent maturation processes. The final one called acrosome reaction is an exocytosis of acrosome vesicle, which is filled with lytic enzymes. Acrosome reaction is crucial for penetration of the sperm cell through the egg surroundings, especially zona pellucida, as well as for reorganization of a membrane protein composition on its surface. This rearrangement leads to the exposure of proteins essential for fertilization, mainly for gamete recognition, binding and fusion in specific compartments of the sperm head. One of such protein is CD46, which is located in the acrosomal membrane of an intact sperm and after acosomal exocytosis it relocates to the equatorial segment of a sperm head, which is known to be the initial site of interaction of sperm with the egg plasma membrane. The relocation of CD46 is disrupted by inhibition of actin, which reorganization within sperm head is known to play a role in onset of acrosome reaction, however, the precise mechanism of CD46 interaction with actin in sperm is unknown. In this thesis, ezrin - a crosslinker of membrane proteins and actin - has been studied in context of CD46 and its relocation across the sperm head. Analysis of the immunofluorescent detection of ezrin revealed its mutual...

This diploma thesis deals with the quality of ejaculate of shepherd dog breeds. In the first part we will classify shephed dogs within the FCI organization and we will approach the breeds that we assess in this work. We will describe the anatomical and physiological characteristics of the reproductive organ of male dog, its ejaculate, ejaculation, semen and their development. We will also learn about the possibilities of dog ejaculate collection. In the main section we will deal with the evaluation of the ejaculate and we will focus on the macroscopic and microscopic examination of fresh ejaculate. In the course of the work, 100 ejaculates from 28 dogs belonging to I. FCI group will be evaluated, each dog will be taken twice to four times and one shot will be made from each sampling. The dogs will be divided into groups according to age, breed, stabling and feeding, and the examined parameters of the quality of the ejaculate will be discussed and compared. In terms of the monitored indicators, a highly statistically significant effect (P is less than or equal 0.01) of the order of collection on the volume of ejaculate obtained will be demonstrated. A statistically significant difference (P is less than or equal 0.05) between the first and the second sampling will be found in the evaluation of the effect of the order of collection during one day. The second volume will be found to be lower than the first one. It will be also shown that this value is affected by the age of the dog. According to our research, breed type and age of the dog causes a statistically significant difference (P is less than or equal 0,05) for sperm activity. The order of collection and stabling have a lesser influence on this indicator and the feed represents a negligible factor. Concerning the sperm concentration in the ejaculate, we will demonstrate a statistically significant difference (P is less than or equal 0.01) between the age groups of the dogs and the type of their accommodation. We will find a statistically significant difference (P is less than or equal 0.05) in the order of sampling. The factors of breed type and feed will have minimal influence in this case. We will not prove a statistical difference in the influence of the studied factors on color and odor, and we will be able to assert that these factors have negligible influence on these values. As for the qualitative sperm indicators, we will focus on the percentage of altered heads, connecting parts, flagella, degenerate and immature sperm. Concerning the values, we will conclude that the breed type, the age and, in the case of immature sperm, the order of collection have the most important influence on these parameters.

Binding of sperm to the oocyte is mediated by complementary molecules on the surface of both gametes and involves the interaction of sperm protein receptors with the zona pellucida (ZP) saccharide structures. It has been shown that many proteins receptors are involved in the sperm-ZP interaction, and potential primary sperm receptors for ZP glycoproteins have been investigated in various mammals. The majority of proteins with identified sperm-ZP binding activity belong to the plasma membrane proteins. However, the exact methods for isolation of sperm membrane proteins are still to be standardized. This study is focused on investigating how employment of various isolation protocols leads to acquisition of various protein mixtures. Further in the work, two possible approaches towards identification of potential ZP-binding partners are implemented, in order to disclose novel primary ZP-binding receptor candidates. Sperm proteins of ejaculated and in vitro capacitated boar sperms were isolated by: Triton X-100, Triton X-114, acetic acid, sodium dodecyl sulphate (SDS), N-octyl-β-D- glucopyranoside (OBG), rehydration buffer (RHB), and finally by freezing-thawing extraction and they were characterized by 1-D, 2-D protein profiles, glycoprotein staining and substrate zymographic methods. The results have...

Sexual selection plays an important role in the evolution of animals. Today we already know that it takes place not only before copulation (precopulatory sexual selection), but also after copulation. This type of sexual selection is called postcopulatory sexual selection, and occurs mainly in promiscuous species, where females mate with multiple males. In this case, sperm competition occurs in the female reproductive tract. To increase the likelihood of their reproductive success, males began to develop surprisingly diverse sperm adaptations at the morphological, physiological, or behavioural levels. These adaptations often affect sperm velocity (motility), which is a key factor for successful egg fertilization. However, the result of reproductive success can also be influenced by females, who may prefer sperm with a certain phenotype in the process of cryptic female choice, and thus, for example, obtain better genes for offspring. In some species, females even can have the ability to sort and store sperm in specialized organs in which the sperms are nourished for some time, and then used to fertilize the egg. Even in this case, the storage of sperm is often affected by sperm morphology. Due to these mechanisms of postcopulatory sexual selection, sperm are under strong selection pressure, which can...

The ubiquitin-proteasomal system which is the major pathway for intracellular protein degradation is also involved in sperm quality control in the mammalian epididymis. Defective sperm become surface- ubiquitinated during epididymal passage. The level of sperm surface ubiquitination negatively correlates with their quality. Hypothetically it is possible that after fertilization, highly ubiquitinated sperm, naturally present in mammalian ejaculates, would be actively recognized by oocyte (probably via 26S proteasomal complex). Subsequent partial or total sperm degradation should negatively affect the development of the potentially defective embryo. In this study, we examined the effect of sperm ubiquitination on the early embryonic development in pig (Sus scrofa f. domestica) using the method of intracytoplasmic sperm injection (ICSI). In vitro embryonic development to the blastocyst stage after ICSI was comparable with other laboratories. In this study, no significant difference was observed in the formation of pronuclei between oocytes fertilized by lower and highly ubiquitinated sperm cells. On the other hand, significantly better embryonic development to the blastocyst stage was observed in oocytes fertilized by sperm with lower surface ubiquitination (17 %) compared with oocytes fertilized by highly...

The process of sperm capacitation and acrosome reaction (AR) are highly dynamic processes essential for the fertilization, including cytoskeleton proteins in the sperm head. The study of the distribution of actin, spectrin and α-tubulin in the head of representative mammalian sperm indicates that before and after the AR there were changes in the distribution of the cytoskeleton structures in the sperm head of all representatives, mainly in the apical part of the acrosome, but also in the equatorial and postacrosome part of the sperm head. The particularly well-studied actin cytoskeleton plays an important role during the capacitation and before and after the AR. Environmental estrogens can interfere with the function of endogenous hormones in very low concentrations. They can interact with specific receptors affecting several signalling pathways leading to sperm capacitation and AR. The effect of 17β-estradiol, estrone, estriol and synthetic 17α-ethynylestradiol was evaluated by the ratio of tyrosine phosphorylation and the state of the acrosome during in vitro capacitation in the sperm head. This study has provided the evidence that estrogens significantly stimulate capacitation progress in a concentration-dependent manner. Estrogens decrease number of sperm after the induced AR too. The raising...

This thesis provides an improve to existing reproduction technics through investigation of ecology and physiology features of pikeperch broodstock: (I) Spawning behavior and selectivity to different types of spawning substrates as an important part of the biology of pikeperch species. The main output of the study was revealing of strong selectivity to the spawning site with neither water temperature nor availability of spawning places haven't affected substrate preference of pikeperch broodstock. (II) Spawning in captivity is leads to decrease in physiological status of the pikeperch broodstock, especially in females. It was found that one of the reasons of the decrease was male protective behavior, that in limited space of the reproduction unit is forwarded to female. Separation of the sexes significantly reduces number injuries and improved physiological status of the broodstock. (Ill) The study investigated efficiency of different antifungal agents for pikeperch broodstock. The long term exposure to salt bath in concentration of 2.5, 5 and 10 g L-1 and formaldehyde in concentration 0.015 ml L-1 were found to be highly effective and significantly decreased mortality after spawning. In addition, treatment with salt bath are environmentally friendly and recommended for practical use as an effective way to prevent mortality of pond-cultured pikeperch broodstock. (lV) The provision of live feed is crucially important for proper gametes maturation, particularly in pond-cultured fishes not accepting pellets. The study investigated the effect of temperature on daily biomass of prey consumed by pikeperch broodstock. Significantly increased prey consumption was found after temperature reached 4.5 °C. Females exhibited significantly higher prey consumption rate in compare to male. Study revealed the tendency for pikeperch to consume largest available prey after water temperature reached 8.5 °C.

Assessment of semen quality (sperm viability) is an essential for the efficient honeybee semen storage. The motility of spermatozoa is not the ultimate parameter of sperm viability. Instead, the viability of spermatozoa is assessed by testing their structural or functional plasma membrane integrity. These assays are based primarily on the technique of microscopy evaluation of either fluorescent DNA dyes or hypo-osmotic "tail-coiling" reaction of spermatozoa. However, the technique of microscopy evaluation is time-consuming, subjective and often is not accurate. On the other hand, the technique of flow cytometry is rapid, objective and precise. Nowadays, this technique is affordable for many institutions, as the price of "benchtop" flow cytometer is relatively low. To best of our knowledge, there is currently no flow cytometry assay available for evaluation of functional integrity of honeybee spermatozoa. Here we report the establishing of novel flow cytometry assay for simultaneous evaluation of structural and functional plasma membrane integrity. DRAQ7, the new fluorescent DNA dye was used to test the structural integrity of plasma membranes. The hypo-osmotic "tail-coiling" reaction was used for flow cytometric assessment of functional integrity of spermatozoa plasma membranes. The combination of both is a valuable tool for rapid and precise evaluation of sperm viability in honey bees.

The current study attempted to shed light on the regulatory processes and response arrangements of fish spermatozoa during the course of maturation and motility initiation. The first intent of this study was to improve the understanding of the mechanism underlying the acquisition of potential for sperm motility in sturgeon. Up to present work, the physiological process underlying sperm maturation in this species has not been described at all. Our results showed that sperm maturation in sturgeon occurs outside the testes because of dilution of sperm by urine and involves the participation of high molecular weight substances as well as calcium ions present in seminal fluid and/or urine. The second aim of the present study was to investigate the coping mechanisms in fish spermatozoa with osmotic and ionic activating mode, as well as in spermatozoa of euryhaline fishes, to various environmental conditions. We showed that alteration of environmental osmolality might affect the fish sperm in different ways, depending on fish species and modes of spermatozoa motility activation either osmotic or ionic mode. In response to osmotic stress caused by hypotonicity, carp spermatozoa regulated the flow of water across their cell membrane and increased their cytoplasmic volume during their short motility period. In contrast, no indications of sperm volume changes were observed neither in sterlet nor in brook trout spermatozoa, both of which having an ionic mode of motility activation. We also examined the mechanism by which sperm motility triggering in euryhaline fishes can adapt to a broad range of environmental salinity. Our results demonstrated that spermatozoa of euryhaline tilapia, Sarotherodon melanotheron heudelotii, reared in fresh-, sea- or hypersaline water can be activated in hypotonic, isotonic or hypertonic conditions of swimming milieu, provided Ca2+ ions are present at various levels. It was established that the higher the fish rearing salinity or the more hypertonic ambient media at spermatozoa activation, the higher extracellular concentration of Ca2+ ions is required. The results obtained in the present study allow suggesting that osmolality is not the main factor inhibiting sperm motility inside the testis in the S. melanotheron heudelotii. A third aim of this study was investigation of the regulation of motility initiation process and description of flagellar beating initiation in chondrostean spermatozoa. We detected that K+ inhibition of sperm motility in sturgeon can be by-passed due to the pre-exposure of sperm cells to a high osmolality shock prior to its transfer to K+-rich swimming media. Thus, we hypothesized that sturgeon spermatozoa may be activated by use of an unexpected signaling pathway, independent from regular ionic stimulation. The successive activation steps in sturgeon spermatozoa were investigated by high-speed video microscopy, using specific experimental situation, where sperm motility initiation was delayed in time up to several seconds. At motility initiation, the first couple of bends formed at the basal region begins to propagate towards the flagellar tip, but gradually fades when reaching the mid-flagellum. This behavior repeats several times until a stage where the amplitudes of bends gradually reach similar value, what eventually leads to sperm progressive displacement. The total period needed for the flagellum to switch from immobility with rigid shape to full activity with regular propagating bends ranges from 0.4 to 1.2 seconds. In conclusion, the results of the current study bring valuable pieces of information into the general understanding of the processes of maturation of fish spermatozoa, their adaptability to different physical and biochemical circumstances, the extra- and intra-cellular signaling as well as the regulatory mechanisms of motility activation in fish spermatozoa.

Pond and intensive aquaculture of percids relies mainly on juveniles supplied by semi-artificial and artificial reproduction. Fish hatcheries do not always provide optimal conditions for percid reproduction. This project was undertaken to optimize reproduction methods of Eurasian perch (Perca fluviatilis L.) and pikeperch (Sander lucioperca L.). Reproduction parameters were compared in farmed and pond-reared perch broodstock without hormone treatment. Spawning was successful without hormone induction at 92.3% and 76.9% in farmed and pond-reared perch, respectively. Significant differences in morphological characteristics (gonadosomatic, hepatosomatic and viscerosomatic indices), fecundity, hatching, and post-spawning mortality were detected. All results, with the exception of spawning success and post-spawning mortality, were superior in pond reared broodstock. Further studies are required to optimize protocols for using better artificial food in farmed perch and decreased stress and post-spawning mortalities in pond reared perch broodstock. The possibilities of using clove oil, Propiscin, or 2-phenoxyethanol as an alternative to tricaine methane sulphonate (MS222) as anaesthetic, particularly with respect to reducing fish stress, were investigated. The haematological and biochemical profiles of pikeperch anaesthetized with clove oil (33 mg l?1), Propiscin (1.5 ml l?1), 2-phenoxyethanol (0.3 ml l?1), and MS222 (150 mg l?1) were determined. Each tested group was divided into two subgroups; the first subgroup was sampled 10 min after application and the second 24 h post-application. Clove oil was shown to be associated with the lowest effects in pikeperch, and therefore could be recommended as an alternative to MS222, while Propiscin and 2-phenoxyethanol were shown to be unsuitable for use with pikeperch. In Eurasian perch the use of semi-artificial reproduction for mass production of embryos was shown to be simpler and more effective. With mass incubation, 1 134 000 embryos (59% of total production) were obtained with a semi-artificial spawning regime and 796 000 embryos (41% of total production) with artificial spawning. Based on these results, we can recommend the semi-artificial spawning for production of perch embryos. Differing doses of two commonly used hormone preparations: human chorionic gonadotropin (hCG) and mammalian GnRHa (Supergestran) were tested for induction of ovulation in pikeperch. A broad range of effective doses is used in aquaculture of pikeperch. The minimal effective dose based on ovulation success is still missing. Hormone therapy applied to excess can result a detrimental impacts on broodstock fecundity and quality of eggs. Results indicated that a single injection of hCG or Supergestran could be used for successful ovulation, while no spawning was observed in a control group treated by saline solution. Among hormone treated groups, the best results were observed in groups treated with hCG at 500 and 750 IU kg-1 BW and in groups treated with mGnRHa at 25 ?g kg-1 BW. The sperm of pikeperch and Eurasian perch show similar morphology and fine structure, with the exception of differences in arrangement of midpiece and centrioles and the presence of a fin-like structure on the flagellum in pikeperch. Our results suggest use of an activation medium containing Ca2+ for activation of short-term stored sperm. Efficacy of five concentrations of Alcalase enzyme for elimination of egg stickiness compared to a milk/talc suspension was determined. Results indicated that 0.5-2.0 ml l-1 of Alcalase for 2 min may increase pikeperch egg hatching success (80-85%) compared to the milk-talc suspension. The highest hatching rate (85.4%) was observed with 1.5 ml l-1 of Alcalase. The application of Alcalase successfully eliminated pikeperch egg stickiness in 2 min compared to 60 min with traditional milk/clay/talc methods.