Metabolic Activity of Boar Semen Stored in Different Extenders Supplemented with Ostrich Egg Yolk Lipoproteins.

INTRODUCTION: The aim of this study was to evaluate the effect of lipoprotein fraction isolated from ostrich egg yolk (LPFo) on the metabolic activity of boar spermatozoa following liquid semen storage in different extenders and temperatures. MATERIAL AND METHODS: Boar ejaculates were extended in Androhep, Beltsville thawing solution (BTS), and Martín-Rillo and Alias (MR-A) without (control) or with the addition of LPFo and stored for three days at either 5°C or 16°C. The analysed sperm parameters included total motility (TMOT), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), oxygen consumption, and adenosine triphosphate (ATP) production. RESULTS: The sperm metabolic activity seemed to be higher in the LPFo-based extenders following storage for three days, irrespective of the storage temperature. Compared with the LPFo-free extenders, significantly higher (P < 0.05) sperm PMI and MMP were observed in BTS and MR-A extenders supplemented with LPFo during storage for three days at 5°C. Spermatozoa stored in the BTS-LPFo extender exhibited higher (P < 0.05) TMOT and oxygen consumption, whereas higher (P < 0.05) PMI was observed in spermatozoa stored in Androhep-LPFo and MR-A-LPFo for three days at 16°C. No significant differences (P > 0.05) in ATP content were observed between the LPFo-free and LPFo-based extenders during storage. CONCLUSIONS: Supplementation of LPFo to semen extenders had varying effects on the metabolic activity of boar spermatozoa stored at different temperatures. It can be suggested that the interactions of various components of the extenders and seminal plasma with LPFo exert beneficial effects on the sperm metabolic activity during liquid storage of boar semen.

The activity of N-acetyl-ß-hexosaminidase in boar seminal plasma is linked with semen quality and its suitability for cryopreservation.

The determination of sperm cryotolerance is an important step in the process of developing optimal techniques for the storage of boar semen. The objective of this study was to determine individual proteome variations in boar seminal plasma and spermatozoa and establish their influence on the cryotolerance of ejaculate. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed the presence of protein with estimated molecular weight of 90 kDa in sperm extracts from ejaculates of selected boars. In all cases, dialysis performed at the initial stage of cryopreservation effectively removed the protein from sperm cells. The protein had an affinity for Zn(2+) ions. Mass spectrometry revealed similarities between the discussed protein and the ß subunit of N-acetyl-ß-hexosaminidase (ß-HEX). Seminal plasma ß-HEX was purified 252-fold with approximately 27% recovery and specific activity of 1800 U/mg of protein. Enzyme activity in fresh seminal plasma was correlated with superoxide dismutase activity (r = -0.42, P < 0.05), glutathione peroxidase activity (r = -0.42, P < 0.05), mitochondrial function (r = 0.31, P < 0.05), glutathione content (r = 0.34, P < 0.05), total protein content (r = 0.42, P < 0.05), and total oxidant status of seminal plasma (r = 0.37, P < 0.05). After thawing, ß-HEX activity in seminal plasma was negatively correlated with the total motile sperm count (r = -0.33, P < 0.05), plasma membrane integrity (r = -0.31, P < 0.05), and lipid peroxidation (r = 0.33, P < 0.05). The observed correlations indicate that lower levels of ß-HEX activity in boar seminal plasma are linked with higher quality of sperm after thawing. Based on those observations, the ejaculates were divided into two groups characterized by low (<20,000 U/L) and high (>20,000 U/L) levels of ß-HEX activity in seminal plasma. In plasma with high ß-HEX activity, spermatozoa were characterized by lower plasma membrane integrity (84.7%, P < 0.05). Higher glutathione levels (1250.3 µM), higher total protein content (50 mg/mL), and higher total oxidant status (6.82-µmol H2O2 Equiv/L) were also observed (P < 0.05). After thawing, lower sperm motility (20.4%), lower plasma membrane integrity (41.7%), and higher lipid peroxidation (30.9-nM malondialdehyde/10(8) spermatozoa/h) were reported in ejaculates with high seminal plasma ß-HEX activity. The results of this study indicate that ß-HEX activity in seminal plasma is a useful indicator in preliminary evaluations of boar sperm cryotolerance.

The antigenic character of zinc-binding proteins and their localization in boar reproductive tract--a preliminary study.

In this study immunoelectrophoretic and double immunodiffusion analyses were used to investigate the antigenic character of zinc-binding proteins (ZnBPs), whereas the indirect immunofluorescence technique was used to identify their origin in boar reproductive tract. The mmunoelectrophoretic analysis of ZnBPs of the seminal plasma resulted in the appearance of three antigenic protein complexes, while specific immunoreactivity patterns of the anti-ZnBP serum were detected by double immunodiffusion analysis. Indirect immunofluorescence technique confirmed that ZnBPs were secreted by different reproductive tract tissues, suggesting their contributions to the seminal plasma.

Superoxide dismutase (SOD) in boar spermatozoa: purification, biochemical properties and changes in activity during semen storage (16°C) in different extenders.

The antioxidant system in semen is composed of enzymes, low-molecular weight antioxidants and seminal plasma proteins. Loss of enzymatic activity of superoxide dismutase (SOD) during semen preservation may cause insufficient antioxidant defense of boar spermatozoa. The aim of this study was to isolate and characterize SOD molecular forms from spermatozoa and to describe changes in SOD activity in boar sperm during preservation at 16°C. Sperm extracts were prepared from fresh or diluted semen and used for SOD purification or activity measurement. Ion-exchange chromatography and gel filtration was used to purify SOD molecular forms. BTS, Dilu Cell, M III and Vitasem were used as diluents for 5-day storage of semen at +16°C. The molecular form of SOD released from spermatozoa after cold shock and homogenization had a molecular weight of approximately 67kDa. The activity of the SOD form was the highest at pH 10 within the temperature range between 20 and 45°C. The enzymatic activity of form released after cold shock was inhibited by H2O2 and diethyldithiocarbamate (DDC; by 65 and 40%, respectively). The SOD form released by homogenization was inhibited by H2O2 and DDC (40%). The molecular form released after urea treatment was a 30kDa protein with maximum activity at 20°C and pH 10. Enzymatic activity of this form was inhibited by H2O2 by 35%, DDC by 80% and 2-mercaptoethanol by 15%. The antigenic determinants of SOD isolated from boar seminal plasma and spermatozoa were similar to each other. Susceptibility of spermatozoa to cold shock increased during storage, but the differences between extenders were statistically non-significant.

Zinc-binding proteins from boar seminal plasma -- isolation, biochemical characteristics and influence on spermatozoa stored at 4°C.

Affinity chromatography on Chelating Sepharose Fast Flow Gel-Zn(2+) 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 Zn(2+) ions have a shielding effect on the sperm plasma membrane and acrosome of spermatozoa, protecting these structures against consequences of cold shock.

Seasonal changes in antioxidant defence systems in seminal plasma and fluids of the boar reproductive tract.

This study aimed to analyze seasonal variations in the antioxidant defence systems of the seminal plasma and fluids of the cauda epididymis and vesicular glands of the boar. The analyzed antioxidants included superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total L-glutathione (GSH+GSSG). Seasonal changes in total protein content and total antioxidant status (TAS) of the seminal plasma and reproductive fluids were also analyzed. Compared with the spring-summer period, total protein content in the seminal plasma was significantly higher during the autumn-winter period. Among the antioxidants analyzed, only SOD activity showed marked seasonal variations, being significantly higher during the spring-summer period. Likewise, the fluid of the cauda epididymis exhibited greater SOD and CAT activity during the spring-summer period, whereas TAS levels were markedly higher during the autumn-winter period. Neither GPx activity nor total GSH+GSSG content in the cauda epididymal fluid was significantly affected by the seasonal periods. The vesicular gland fluid exhibited an approximately 4-fold greater level of SOD activity during the autumn-winter period, as compared with the spring-summer period. By contrast, greater CAT and GPx activity, and a higher level of total GSH+GSSG were observed in the vesicular gland fluid during the spring-summer period. In conclusion, the findings of this study indicate that seasonal variations could have varying effects on the antioxidant defence systems in the seminal plasma and fluids of the boar reproductive tract.

Interactions of egg yolk lipoprotein fraction with boar spermatozoa assessed with a fluorescent membrane probe.

The interactions of a fluorescent membrane probe, 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS), with boar spermatozoa were followed through the use of lipoprotein fraction of ostrich egg yolk (LPFo). Semen samples, extended in Kortowo 3 (K3) extender, were supplemented with 2% or 5% LPFo and stored for 3h at 16 degrees C. Additionally, cold shock-treated spermatozoa (1h at 4 degrees C) were stored in K3 extender supplemented with LPFo for 3h at 16 degrees C. In each boar, the fluorescent enhancement of ANS was observed in K3-extended semen supplemented with LPFo, prior to storage. Following storage, there was a significant increase in LPFo-ANS fluorescence, particularly in the sperm membrane overlying the head and midpiece regions. There were significant differences among the boars with respect to the sperm populations defined by the LPFo-ANS fluorescence. Sperm viability was not significantly affected during the storage period. Furthermore, the proportions of spermatozoa defined by the different patterns of LPFo-ANS fluorescence were low and remained unchanged after storage of cold shock-treated spermatozoa with 2% or 5% LPFo, suggesting irreversible damage to the sperm membrane architecture. These findings indicate that the ANS fluorescent probe could be used to shed more light on the nature of the interactions between LPFo and sperm membrane following semen preservation. Such valuable information could contribute to the development of an optimal protocol for cryopreservation of boar semen.

High sequence homology between protein tyrosine acid phosphatase from boar seminal vesicles and human prostatic acid phosphatase.

Boar seminal vesicle protein tyrosine acid phosphatase (PTAP) and human prostatic acid phosphatase (PAP) show high affinity for protein phosphotyrosine residues. The physico-chemical and kinetic properties of the boar and human enzymes are different. The main objective of this study was to establish the nucleotide sequence of cDNA encoding boar PTAP and compare it with that of human PAP cDNA. Also, the amino-acid sequence of boar PTAP was compared with the sequence of human PAP. PTAP was isolated from boar seminal vesicle fluid and sequenced. cDNA to boar seminal vesicle RNA was synthesized, amplified by PCR, cloned in E. coli and sequenced. The obtained N-terminal amino-acid sequence of boar PTAP showed 92% identity with the N-terminal amino-acid sequence of human PAP. The determined sequence of a 354 bp nucleotide fragment (GenBank accession number: GQ184596) showed 90% identity with the corresponding sequence of human PAP. On the basis of this sequence a 118 amino acid fragment of boar PTAP was predicted. This fragment showed 89% identity with the corresponding fragment of human PAP and had a similar hydropathy profile. The compared sequences differ in terms of their isoelectric points and amino-acid composition. This may explain the differences in substrate specificity and inhibitor resistance of boar PTAP and human PAP.

Application of biochemical markers for identification of biological properties of animal semen.

The use of biochemical markers for identification of biological properties of semen will help to develop new criteria that are accurate and objective in predicting and improving male fertility. Understanding and controlling the mechanisms involved in fertility is a key challenge, which is of fundamental importance in successful animal reproductive performance. Moreover, unraveling the unique molecular mechanism associated with sperm function might have considerable diagnostic value in the evaluation of male infertility. This review offered insights into some recent achievements and provided perspectives for possible applications of the biochemical markers of semen.

Aromatization and antioxidant capacity in the testis of seasonally breeding bank voles: Effects of LH, PRL and IGF-I.

Aromatization and antioxidant strategies in the male gonads are important processes, which are involved in control of normal fertility. The objective of this study was to show whether luteinizing hormone (LH), prolactin (PRL), and insulin-like growth factor-I (IGF-I) as well as the length of photoperiod are able to exert an effect on aromatase expression, steroid hormone levels, and antioxidant concentrations in testes of bank voles, seasonally breeding rodents. Mature bank voles that were kept under short light cycles or long light cycles served as the animal model. Testicular sections were used for immunohistochemical visualization of aromatase expression, whereas testicular homogenates were used for radioimmunological measurement of steroids, biochemical determination of superoxide dismutase activity, total antioxidant status (TAS) and protein content. In the testes of hormone-treated voles a stronger immunostaining for aromatase was concurrent with the increase in testosterone and estradiol levels, and total antioxidant status compared with the controls. In contrast, there was a decrease in superoxide dismutase (SOD) activity. The strongest effect on aromatase immunoexpression and steroid hormone levels was detected after combined doses of LH and IGF-I, indicating a stimulatory role of these hormones on estrogen synthesis in the bank vole. An increase in total antioxidant status in testes of hormone-treated bank voles suggests the presence of testicular defense, whereas a decrease in superoxide dismutase activity indicates a protective role of administered hormones against toxic oxygen radicals. The present study also demonstrates a significant, photoperiod-dependent relationship between aromatization and antioxidant capacity in the testis of the bank vole.

Proteomics of boar seminal plasma - current studies and possibility of their application in biotechnology of animal reproduction.

Proteomics is critical to identify the properties and functions of proteins involved in the mechanism regulating the male reproductive tract function. This approach is important in male fertility assessment and clinical diagnosis of the physiological state of individual reproductive organs. Proteomics also provides a tool to understand the interactions of seminal plasma proteins with spermatozoa, which could provide a useful model for studying ligand-cell interaction occurring at the sperm cell surface. This review covers a selection of advances in the realm of functional proteomics of boar seminal plasma proteins and is focused on some fundamental proteomic technologies. Also, this review explores key themes in proteomics and their application in animal reproductive techniques.

Effects of dietary supplementation with polyunsaturated fatty acids and antioxidants on biochemical characteristics of boar semen.

The aim of this study was to investigate the effect of providing a supplement containing polyunsaturated fatty acids and antioxidants (PROSPERM) on the biochemical characteristics of boar semen. Two sexually mature boars were fed a standard diet with PROSPERM (250 g daily) for a 24-week period. Ejaculates collected prior to supplementation were used as the control. Semen quality and biochemical parameters were analyzed. The dietary supplementation enhanced sperm characteristics, including the percentage of spermatozoa with intact plasma membrane and osmotic resistance of the acrosomal membrane. Higher production of malondialdehyde was concurrent with increased activity of superoxide dismutase in the seminal plasma and spermatozoa after 8 weeks of supplementation. These changes were accompanied by a high content of total protein and low-molecular antioxidants of the seminal plasma. It was observed that PROSPERM supplementation enhanced the survivability of boar spermatozoa during storage in a standard semen extender supplemented with lipoprotein fractions, isolated from hen egg yolk or ostrich egg yolk, at 5 degrees C and 16 degrees C. These results indicate that PROSPERM supplementation of boars had a beneficial effect on the biological characteristics of the spermatozoa, which could be useful for semen preservation at different temperatures.

Secretory activity of boar seminal vesicle glands.

This paper reviews the recent research on the physiological role of peptide and protein substances synthesized in boar seminal vesicle glands. Secretions from these glands are the major components of seminal plasma. Unique biochemical properties of seminal vesicle proteins, which determine their numerous functions in the reproductive processes, are discussed. The discovery of phosphotyrosine acid phosphatase in boar seminal vesicle secretions and the first description of the biochemical properties of platelet activating factor acetylhydrolase (PAF-AH) have led to remarkable inroad in molecular andrology. The fundamental role of vesicular low and high molecular antioxidants in the protective function against reactive oxygen species (ROS) is defined. Emphasis is also given to the role of androgens in controlling the secretory activity of boar seminal vesicles.