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Understanding the biochemical processes associated with ovum fertilization and knowledge about the structure and function of individual substances participating in these processes is crucial for the development of biotechnological methods to improve reproduction of animals and humans. Among many components of seminal plasma, proteins and peptides play a specific role in regulation of the fertilization process, particularly through their ability to bind various types of ligands such as polysaccharides, lipids and ions. Heparin-binding proteins regulate capacitation and acrosome reaction processes. Affinity of plasma proteins to mannans of the fallopian tube epithelium facilitates formation of spermatozoa reservoirs in the female reproductive tract. Ability to bind phosphorylcholine is one of the conditions for the coating of the seminal plasma proteins on the sperm membrane and also determines the formation of oligomeric forms of certain proteins. Zinc binding by seminal plasma proteins regulates sperm chromatin condensation state. It also affects motility of these cells and acrosome reaction. The interspecies analysis indicates significant structural and functional similarities, especially for the proteins with low molecular weight. Fertility associated proteins (FAPs) have been determined in the bull, stallion, boar, ram and dog. The contents of these proteins correlate with the indicators of the fertilizing abilities of sperm. In humans, several seminal plasma proteins were found which serve as diagnostic markers of spermatogenesis, seminiferous epithelium state, and azoospermia. To determine the semen ability for preservation, measurement of some seminal plasma protein content may also be used. Addition of specific plasma proteins to a spermatozoa solution undergoing the process of preservation may be used to retain the features of the cells responsible for efficient fertilization.
The aim of this study was to investigate the effects of PAF addition on selected motility characteristics and plasmalemma integrity of boar spermatozoa following liquid storage in a boar semen extender, Kortowo-3 (K-3), supplemented with lipoprotein fractions extracted from hen egg yolk (LPFh) or lyophilized lipoprotein fractions extracted from ostrich egg yolk (LPFo), at 5°C and 16°C. Sperm motility was evaluated using a computer system (CASA). The determination of AspAT activity in sperm extracts as well as fluorescent analysis, with a fluorochrome, Hoechst 33258, were used to assess the plasmalemma integrity overlying the middle-piece and acrosomal regions of spermatozoa, respectively. It was confirmed that the addition of exogenous PAF to K-3 extender containing LPFh or LPFo had a beneficial effect on the sperm quality parameters during storage at 5°C or 16°C. This phenomenon was manifested by an increase in motility and survivability of spermatozoa. The use of LPFh or LPFo as a component of boar semen extender had a protective effect on the plasmalemma integrity overlying the middle-piece and acrosomal regions of PAF-treated spermatozoa.
Chromatin stability is an important determinant of semen quality, essential for spermatozoa maturation in epididymes and early embryogenesis. A radioisotope method based on the quantitative measurements of tritium-labelled actinomycin D (3H-AMD) incorporation into the spermatozoa nuclei was used to assess chromatin stabilization of boar spermatozoa incubated with physiological (reduced glutathione – GSH, heparin – H and bosine serum albumin – BSA) or non-physiological (dithiothreitol – DTT, disodium ethylenediaminetetra acetate – EDTA, 2-mercaptoethanol – ME and sodium dodecyl sulphate – SDS) decondensing agents.The effect of the composition of seminal plasma and the role of zinc ions in chromatin stability of spermatozoa were also studied. Pre-treatment of spermatozoa with GSH, H, DTT, ME or SDS resulted in an increase in the incorporation of 3H-AMD into the spermatozoa nuclei. In contrast, when sperm samples were treated with BSA or EDTA there was a reduction in the incorporation of 3H-AMD, what was attributed to hyperstabilization of chromatin. A presumed hyperstabilization was also observed when SDS+EDTA+H were used. On the other hand, an exceptionally strong action of decondensation of chromatin was induced by H+BSA. Increased incorporation of 3H-AMD into the spermatozoa nuclei was concomitant with low zinc and protein content in the seminal plasma of boars following depletion test (DT), suggesting disturbances in chromatin stability. The presented radioisotope method based on the application of 3H-AMD is a simple and reliable assay that can be used to monitor the chromatin status of boar spermatozoa.
The aim of this study was to compare the effect of different packaging systems on some parameters of cryopreserved canine spermatozoa. The experimental material consisted of the sperm-rich fractions of ejaculates collected from four Beagle dogs. Semen samples for cryopreservation were stored in 0.25 ml plastic straws and two aluminum tubes with a total volume of 5.0 ml. Semen was frozen in static nitrogen vapor for 10 minutes (0.25 ml straws) or 15 and 20 minutes (aluminum tubes). Post-thaw assessments involved the determination of sperm motility parameters using a computer assisted sperm analyzer (CASA), sperm plasma membrane integrity (SPMI), mitochondrial membrane potential (MMP) and acrosome integrity (normal apical ridge, NAR). Regardless of the packaging system applied, no significant differences in total sperm motility (TMOT) or selected kinematic parameters were observed after freezing-thawing. However, spermatozoa frozen in 0.25 mL straws were characterized by improved functionality, in particular mitochondrial function, after thawing. The results indicate that large quantities of canine semen can be frozen in aluminum tubes. Further studies are required, however, to evaluate different freezing and thawing rates of aluminum tubes.
This study aimed to compare post-thaw quality of boar semen following freezing in an ostrich egg yolk lipoprotein (LPFo) extender supplemented with 0%, 0.25% and 0.50% Orvus Es Paste (OEP). Sperm assessments included total motility (TMOT), mitochondrial function (MF), plasma membrane integrity (PMI) and acrosome integrity (normal apical ridge, NAR). Considerable variations among boars and OEP treatments had a significant effect (P < 0.001) on post-thaw sperm characteristics. It was observed that post-thaw sperm characteristics were significantly compromised in semen samples frozen in the absence of OEP. By contrast, lactose-LPFo-glycerol extender supplemented with either 0.25% OEP or 0.50% OEP markedly enhanced post-thaw sperm characteristics. In all boars, there were no marked differences in post-thaw sperm TMOT between the freezing extenders supplemented with 0.25% and 0.50% OEP. However, a decline in the percentage of post-thaw motile spermatozoa was more pronounced in the extender supplemented with 0.50% OEP following a 120-min incubation period. Furthermore, the proportions of frozen-thawed spermatozoa with MF, PMI and NAR acrosomes varied significantly among the boars in the OEP-supplemented extenders. The findings of this study indicate that different OEP concentrations, in the presence of ostrich egg yolk lipoproteins, could have varying effects on post-thaw sperm survival.
The aim of the present study was to establish the effects of platelet-activating factor (PAF) on selected movement parameters, plasmalemma integrity, capacitation process and acrosome reaction in cryopreserved boar spermatozoa. A positive effect of PAF addition to cryopreserved semen on sperm motility was demonstrated, particularly with the application of phospholipid concentration of 1 x 10-6M-1 x 10-5M. A moderate induction of plasmalemma damage of cryopreserved spermatozoa was observed when PAF was used at a low concentration (1 x 10-8M-1 x 10-7M). The rate at which PAF induced the process of capacitation was inversely proportional to its concentration in the sample (the highest for the concentration of 1 x 10-8M, and the lowest at 1 x 10-5M). In turn, the strongest induction of acrosome reaction of spermatozoa was observed in samples with the addition of PAF at a concentration of 1 x 10-7M. The results obtained suggest that the application of PAF supplement to post-thawed boar semen can be used as a laboratory test of the ability of spermatozoa to induce the acrosome reaction.
The aim of this study was to evaluate the effect of boar semen storage in different short-term extenders (BTS, Kortowo-3, and M III) on the percentage of spermatozoa showing proapoptotic and necrotic changes. For the first time in this study, Annexin V isolated from swine placenta has been used to determine proapoptotic changes in stored boar spermatozoa. Die changes were determined using the IN Cell Analyzer 2000. A gradual decrease in motility was observed on successive days of storage. Spermatozoa incubated in the BTS extender were characterised by the highest average motility, which reached 75% on the 1st d and 39% on day 5. Motility of spermatozoa stored in BTS was significantly higher than those stored in Kortowo-3 and M III extenders after 5 d of storage. Diluted semen contained 1.5% to 2.8% spermatozoa with proapoptotic changes. The discussed process was intensified on the 3rd d of storage when the percentage of apoptotic spermatozoa was determined at 8.3% to 14.6%, and the content of dead spermatozoa exceeded 25%. The analysed extenders differed insignificantly in their ability to protect semen against proapoptotic changes during storage. From the methodological point of view, Hoechst 33258 could be used additionally to stain sperm cells regardless of their status.
 Affinity chromatography on Chelating Sepharose Fast Flow Gel-Zn2+ was used for fractionation of boar seminal plasma proteins. Approximately 30 % of total boar seminal plasma proteins showed affinity for zinc ions (ZnBP fraction). Native electrophoresis (PAGE) of ZnBP revealed six protein fractions which separated into 27 bands under denaturing conditions (SDS/PAGE). Two-dimensional electrophoresis (2D PAGE) showed 148 polypeptides with isoelectric points mostly in the basic and neutral pH range. The zinc-binding proteins comprise mainly 10-20 kDa polypeptides which are probably members of the spermadhesin family. ZnBP present in the incubation mixture of spermatozoa stored for 1 or 24 h at 4 °C allowed preservation of a higher percentage of cells exhibiting linear motility in comparison to a control sample stored in PBS. Presented results indicate that proteins binding Zn2+ ions have a shielding effect on the sperm plasma membrane and acrosome of spermatozoa, protecting these structures against consequences of cold shock.
The aim of this study was to investigate the effect of dog semen dialysis on sperm characteristics and some biochemical components of seminal plasma. Whole ejaculates were dialyzed against Triscitrate-fructose extender for a 5 h period at room temperature (using semi-permeable cellulose tubing of 12-14 kDa molecular weight cut-off). It has been demonstrated that the long-term dialysis of dog semen causes a significant decrease in sperm quality parameters and disrupts the biochemical properties of seminal plasma. This procedure requires further improvement.
Zinc-binding proteins from seminal plasma (ZnBPs) originate in the secretions of different accessory sex glands and are implicated in key events associated with sperm-egg fertilization processes. This study describes the isolation and characterization of the ZnBPs of canine seminal plasma. Ejaculates were collected from three crossbred dogs for a 2-week period. The ZnBPs as well as non zinc-binding proteins (nZnBPs) were isolated by zinc-dependent affinity chromatography. The isolated fractions were subjected to native gel electrophoresis (one-dimensional polyacryamide gel electrophoresis, PAGE) and sodium dodecyl sulphate polyacryamide gel electrophoresis (SDS-PAGE), using denaturing and reducing conditions. Zinc-elution profile using affinity chromatography displayed two protein fractions represented by the nZnBPs and ZnBPs, respectively. Using native gel electrophoresis, it was found that both the nZnBPs and ZnBPs occurred in their native state as aggregates, ranging from 140 to 669 kDa. The nZnBPs were disaggregated into 8 protein bands, with molecular weights ranging from 10.7 to 79.7 kDa, following SDS-PAGE analysis. By contrast, SDS-PAGE analysis of the ZnBPs revealed 13 protein bands, with molecular weights ranging from 11.6 to 152.3 kDa. Densitometric analysis showed that 46-48% of nZnBPs could be accounted by protein fractions with molecular weights of 10.7 and 14.2 kDa. Also, 2 protein fractions with molecular weights of 11.6 and 14.3 kDa, were predominant in ZnBPs, accounting for approximately 28-30% of the total proteins. These results demonstrate the zinc-binding capacity of proteins secreted by the canine prostate. The findings of this study indicate that ZnBPs of canine seminal plasma comprise several protein fractions, which might be implicated in the reproductive processes in the dog.
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