Lipid peroxidation in avian semen.

The main cause of sperm chromatin damage is oxidative stress related to embryo development failure and adult infertility in mammals and also avian. Oxidative stress results in lipid peroxidation (LPO) causing cell damage. Lipid peroxidation is the oxidation of polyunsaturated fatty acids (PUFAs) in biological systems and causes changes in the physical structure and characteristics of the cell membrane. Due to the high amounts of PUFAs in the avian sperm membrane, its sperm seem susceptible to pe-roxidative damage and is a substantial factor in the fertilization capacity of sperm. The most commonly used methods for measuring LPO or its by-products, such as malondialdehyde (MDA) and 4-hydroksy-2-nonenal (4-HNE), in bird semen are based on the colorimetric method TBARS (thiobarbituric acid reactive substances) and on the use of a fluorescence probe (CC 11-BODIPY 581/591) as a marker to evaluate membrane lipid peroxidation. This review aims first to introduce LPO in avian semen and its effects on avian sperm and second to summarize the commonly applied methods of evaluating LPO and its damage in fresh and stored avian semen.

Abnormal Expression of BTLA and CTLA-4 Immune Checkpoint Molecules in Chronic Lymphocytic Leukemia Patients.

Chronic lymphocytic leukemia (CLL) is characterized by the peripheral accumulation of neoplastic B cells and is frequently complicated by the systemic immunosuppression associated with an impairment in B and T lymphocyte activation. We hypothesized that the expression of immune checkpoint suppressors B and T lymphocyte attenuator (BTLA) and cytotoxic T lymphocyte antigen (CTLA-4) is disturbed in both lymphocyte subpopulations in CLL. The expression of CTLA-4 and BTLA mRNA was determined by real-time PCR, while CTLA-4 protein expression (surface or intracellular) was estimated in BTLA+ lymphocytes by flow cytometry. In CLL patients, we observed a higher gene transcript level of BTLA and CTLA-4 than in healthy individuals in both freshly isolated and PMA stimulated B and T cells. Remarkably, lower amounts of both inhibitory proteins were found in peripheral blood (PB) CLL B cells, whereas normal BTLA and elevated CTLA-4 were found in T cells. Consistently, there was a prevalence of CTLA-4+ cells within circulating BTLA+ T cells cells of patients confronting PB healthy cells. After in vitro stimulation, the only change found in CLL patients was a decrease in BTLA expression in B and T lymphocytes. In contrast, healthy lymphocytes responded more vigorously as regards the BTLA and CTLA expression with substantially higher frequency of CD69+ cells under the stimulating condition compared to corresponding cells from the CLL group. Our results indicate that CLL development is associated with the affected expression of BTLA and CTLA-4 checkpoint receptors in PB and its impaired expression might be associated with lowering of the threshold for B cell activation and proliferation, while upregulated CTLA-4 expression in CLL peripheral BTLA+ T cells may contribute to suppressed T cell effector functions. This hypothesis needs to be validated in future studies, which would allow us to explain how the increased or decreased expression of these molecules affects the cell function.

The Viability of Serval (Leptailurus serval) and Pallas Cat (Felis manul) Oocytes after Cryopreservation Using the Rapid-I Method.

BACKGROUND: Vitrification by Rapid-I method could be essential for felid rescue programs to protect wild felid in the future. OBJECTIVE: This study was aimed at adapting the Rapid I method and evaluating the viability of serval and Pallas cat oocytes compared to oocytes of the domestic cat. MATERIALS AND METHODS: Oocytes after collection and in vitro maturation were vitrified using Cryotech medium (Cryotech, Japan) and a Rapid-I device (Vitrolife, Sweden). To evaluate viability, oocytes after warming were stained with fluorescein diacetate and ethidium bromide. RESULTS: Survival rate in the control group (domestic cat) was 75 %. In the experimental group, 70% (serval) and 60% (pallas cat) viable oocytes were found. CONCLUSION: The Rapid-I method can be applied successfully for the vitrification of wild felid oocytes.

Obtaining Wisent early blastocyst in vitro is a basic for protection and creation of biodiversity for this threatened species.

Wisent, or European bison (Bison bonasus), is listed as "vulnerable" on the IUCN Red List of Threatened Species and is therefore protected by international law. For the first time, a Wisent embryo has been obtained in vitro. This procedure creates a new opportunity to protect and increase Wisent reproductive potential and thereby opens new possibilities for the establishment of a controlled and broad reserve of the gene pool.

Effect of different extenders for donkey sperm vitrification in straws.

Aseptic vitrification of semen samples packed in straws has been successfully developed in human but not in donkeys. The aim of this study was to compare the effect of two extenders for donkey sperm vitrification using straws. Ejaculates from four Andalusian donkeys were collected, and samples were extended in INRA-96 (I) or Gent (G) supplemented with sucrose 0.25 M and 1% bovine serum albumin (BSA). Extended samples were cooled for one hour at 5°C. For vitrification, samples were filled in covered 0.25 ml straws and then plunged directly into liquid nitrogen. For warming, straws were immersed in INRA-96 at 43°C. Results showed no significant differences between I and G treatments for TM (34.2% ± 8.7 vs. 30.7% ± 9.6) and PM (26.8% ± 7.3 vs. 24.6% ± 7.9), respectively. In conclusion, donkey sperm could be vitrified in straws either with INRA-96 or with Gent in combination with sucrose and BSA.

Effect of zearalenone on circulating testosterone concentration, testicular and epididymal morphology and epididymal sperm characteristics in wild boars.

This study investigated the effect of exposure to zearalenone (ZEN) and its metabolites on the characteristics of epididymal spermatozoa, testicular and epididymal biometry and histology, and the concentration of testosterone in blood plasma in male wild boars. The study was performed during more than one year on 18 clinically healthy male wild boars with initial and final body weight, of 39 ± 4 kg and 59 ± 3 kg, respectively. The animals were divided into two experimental groups (group I and group II) and one control group (group C) comprising 6 boars per group. Group I animals were administered per os pure zearalenone (ZEN) at 150 µg/kg BW for 7 consecutive days every two months, while group II animals received a dose of 50 µg/kg BW/day via feed that was naturally contaminated with ZEN. These male wild boars were exposed to ZEN over a period of 1 year. Control animals were fed a placebo. Testicles with epididymides of the boars were collected on the last day of the experiment within 3 min after slaughter. Blood samples were collected from each of the male wild boars. Testes and epididymides were measured and sampled for histological examination. Epididymides were dissected and epididymal spermatozoa were harvested. The spermatozoa were diluted with swine-specific BTS extender and stored at 17°C for 144 h. Sperm motility was analyzed with CASA, and other parameters including viability, acrosome integrity, DNA fragmentation index, lipid peroxidation and apoptosis were assessed with flow cytometry. In these wild boars, per os exposure to natural sources of ZEN or a combination of ZEN and its metabolites changed the testicular interstitium and led to modification of some epididymal sperm parameters. The interstitial glands in testes of experimental group I were markedly reduced and hyperemic with evident blood stasis in small capillaries. Also in group I were single degenerating seminiferous tubules. In both groups I and II, immediately after dilution of spermatozoa with BTS remarkable decreases in motility rate as well as in progressive motility and the subpopulation of cells with rapid movement were noted compared with the control group (P < 0.05). But unexpectedly, after 24 h incubation of boar semen in the BTS diluent, these sperm properties improved and were not significantly different from the control group. Thus, exposure to ZEN has no lasting but only a temporary, reversible effect on wild boar sperm motility. There was no influence of exposure to ZEN and its metabolites on the integrity of membranes, intensity of lipid peroxidation and apoptosis or on sperm chromatin structure. This study is the first using these direct measures of sperm motility and integrity to show a redundant adverse effect of ZEN exposure on wild boar sperm characteristics. There were no effects of exposure to ZEN and its metabolites on body weight, testicular and epididymal biometry, gonadosomatic index and the concentration of testosterone in blood plasma in the male wild boars.

Identification and functional analysis of bull (Bos taurus) cauda epididymal fluid proteome.

Despite recent advances in bull epididymal fluid proteome research, significant numbers of proteins secreted to epididymal lumen remain unidentified. The objective of this study was to expand the number of identified cauda epididymal fluid proteins in bulls and to contextualize them in a broader view of their mutual interactions and involvement in biological processes and pathways, to fully elucidate the ways in which epididymal fluid proteins are involved in storage and maturation of spermatozoa in epididymis. We collected postmortem cauda epididymal fluid from 6 mature Holstein Friesian bulls. We performed the identification of proteins using 2-dimensional electrophoresis coupled with MALDI mass spectrometry. Analysis of functionality and pathway involvement of identified proteins was performed using Ingenuity Pathway Analysis software. We identified a total of 189 epididymal fluid proteins, out of which 100 were newly identified in bull epididymal fluid. We have combined our data with 2 previously performed bull epididymal fluid proteome identifications, yielding 280 proteins total, and analyzed it. The main canonical pathways involving epididymal proteins were glycolysis, gluconeogenesis, protein ubiquitination pathway, nuclear factor-erythroid 2-related factor 2-mediated oxidative stress response, and farnesoid X receptor/retinoid X receptor activation. The main biological functions potentially performed by epididymal fluid proteins included carbohydrate metabolism, cellular growth and proliferation, cell death and survival, and small molecule biochemistry. Overall, our results have pointed out multiple novel pathways in bull epididymal fluid that might take part in various aspects of maturation and protection processes of epididymal spermatozoa.

Is the Genetic Background of Co-Stimulatory CD28/CTLA-4 Pathway the Risk Factor for Prostate Cancer?

The impairment of immunological surveillance caused by aberrant T cell activation can lead to an inadequate anti-tumor response. Therefore, deregulation in co-stimulatory pathway might be associated with cancer susceptibility. Here we undertook a prospective study to investigate whether genetic variations in gene encoding molecule CD28 and CTLA-4 playing pivotal role in regulating adoptive immune response can influence susceptibility to prostate cancer. Single nucleotide polymorphisms (SNPs) in CTLA-4 and CD28 genes were genotyped in 301 prostate cancer (PCa) patients and 301 controls. The distributions of the genotypes and haplotypes in the CTLA-4/CD28 SNPs were similar in both studied groups. However, the overrepresentation of carriers of CTLA-4c.49A>G[A] allele and carriers of CTLA-4g.319C>T[T] allele in PCa as compared to controls was observed (p = 0.082 and p = 0.13, respectively). The risk of disease was higher (OR 1.78) for carriers of both susceptibility alleles as compared to carriers of protective genotypes (p = 0.03). The CTLA-4c.49A>G and CTLA-4g.319C>T SNPs might be considered as low risk susceptibility locus for PCa.

Analysis of bull (Bos taurus) seminal vesicle fluid proteome in relation to seminal plasma proteome.

The existing knowledge on the bull seminal vesicle proteome, a major seminal plasma constituent, and its relationship with seminal plasma is limited. This knowledge is prerequisite for a better understanding of seminal plasma variability, which is linked to semen quality. The objective of this study was to characterize the proteomes of seminal vesicle fluid and seminal plasma and to compare them to better understand the origin of seminal plasma proteins. We collected ejaculates and seminal vesicle fluid postmortem from 6 mature Holstein Friesian bulls. We performed the analysis and identification of proteins using 2-dimensional electrophoresis coupled with matrix-assisted laser desorption/ionization mass spectrometry. We identified 105 proteins in bull seminal vesicle fluid and 88 proteins in seminal plasma. For both seminal vesicles and seminal plasma proteins described in our study, top biological functions were cellular movement, cell death and survival, and cellular growth and proliferation. Additionally, seminal vesicle fluid proteins were involved in protein degradation and synthesis. Seminal plasma proteins were also involved in cellular assembly and organization and cell-to-cell signaling and interactions. Proteins of both fluids were involved in the following canonical pathways: glycolysis, gluconeogenesis, liver X receptor/farnesoid X receptor, and farnesoid X receptor/retinoid X receptor activation. Additionally, seminal vesicle fluid proteins appeared to be involved in oxidative stress response mediated by nuclear factor E2-related factor 2. Our results described the bull seminal vesicle fluid proteome for the first time and allowed for significant expansion of the current knowledge on the bull seminal plasma proteome. Moreover, analysis indicated that both bull seminal vesicle fluid and seminal plasma proteomes contained interconnected protein groups related to protective functions, glycolysis, and the morphology and physiology of the spermatozoa. These proteins and their interactions could be targeted in future research.

Evaluation of spermatozoal function-useful tools or just science.

Conventional microscopic semen analysis does not provide precise information on the fertilizing potential of a male. The traditional basis for semen evaluation is that male fertility is dependent on production of a "proper" concentration/number of motile, morphologically normal spermatozoa in excess to achieve conception. Many independent studies, especially in human medicine, have demonstrated that the absolute number of spermatozoa does not accurately determine fertility, but their functional competence. Many functional tests of spermatozoa are developed over the last decades. Computer-assisted sperm analysis (CASA) and flow cytometry have become the gold standard for semen assessment in specialized andrology laboratories. Other functional assays, such as gamete interaction tests, provide additional information regarding the real fertilizing potential of sperm cells. From this point of view, such tests are valuable diagnostic tools in fertility disorders and may be helpful to make a decision which method of treatment to use: pharmacological therapy, intrauterine insemination, introduction of classic IVF, ICSI or exclusion from a breeding programme. The most useful gamete interaction tests include induced acrosome reaction, zona pellucida binding assay, oocyte penetration assay and hyaluronan binding assay. In recent years, andrology has entered into a new era of sophisticated OMICS methods. Genomics, epigenomics, transcriptomics and proteomics brought high hopes for rapid progress in clinical diagnostics.

Mitochondrial DNA Copy Number in Spermatozoa of Fertile Stallions.

Predicting male fertility on non-invasive sperm traits is of big importance to human and animal reproduction strategies. Combining the wide range of parameters monitored by computer-assisted sperm analysis (CASA) with some molecular traits (e.g. mtDNA content) may help to identify markers of the male fertility. The aim of this study was to characterize variation in the mtDNA copy number in equine sperm and to investigate whether mtDNA content is correlated with quality traits of stallion spermatozoa and the age of the male. Ejaculates collected from 53 fertile stallions were divided into four age groups (3-5, 6-10, 11-14 and >15 years) and were subjected to a complex investigation including conventional analysis, CASA, flow cytometry and mtDNA content (real-time PCR). The mean (±SD) number of mtDNA copies equalled 14 ± 9 and varied from 3 to 64. Considering the great number of sperm parameters monitored in this study, only few of them were correlated with the mtDNA content: ejaculate volume (a positive correlation), the amplitude of lateral head displacement (ALH; a negative correlation) and the high mitochondrial activity index (a negative correlation). The stallion age was not correlated with the mtDNA copy number. This study provides the first set of data on mtDNA content in equine sperm and confirms phenomena previously described for humans and dog on associations between sperm mtDNA content and selected motility parameters monitored by the CASA. Basing our study on spermatozoa from fertile stallions could however limit the extent of detected associations.

CTLA-4 and CD28 genes' polymorphisms and renal cell carcinoma susceptibility in the Polish population--a prospective study.

Polymorphisms in co-stimulatory genes are associated with susceptibility to several malignances such as breast cancer, cervical cancer and chronic lymphocytic leukemia, but have been scarcely investigated in renal cell cancer (RCC). A total of 310 RCC patients and 518 controls were genotyped for single-nucleotide polymorphisms (SNPs) in the CTLA-4 and CD28 genes: CTLA-4c.49A>G (rs231775), CTLA-4g.319C>T (rs5742909), CTLA-4g.*6230G>A (CT60; rs3087243), CTLA-4g.*10223G>T (Jo31; rs11571302), CD28c.17+3T>C (rs3116496) and CD28c.-1042G>A (rs3181098). The distribution of the alleles, genotypes and haplotypes in the CTLA-4 and CD28 genes were similar in the RCC patients and in the controls. However, among the patients with a clear cell RCC (CCRCC), the G allele carriers of CT60 and Jo31 SNPs were overrepresented, and the overrepresentation became significant for the carriers of CT60[G] allele in CCRCC patients with necrosis in the primary tumor (P = 0.046). The CTLA-4c.49A>G[A]/CTLA-4g.319C>T[C]/CT60[A]/Jo31[T]/CD28c.17+3T>C[T]/ CD28c.1042G>A[G] haplotype was associated with an approximately threefold increased risk of primary tumor necrosis in CCRCC patients (P corrected = 0.0000007) and with the advanced stage of disease (IV) (P corrected = 0.001). When stratified by gender, CD28c.-1042G>A[GG] genotype was more frequent in the female CCRCC patients compared with healthy women (P = 0.042). Polymorphisms in the CTLA-4 and CD28 genes, in particular considered together as haplotypes, were associated with increased risk of CCRCC, especially with necrosis and with the advanced stage of disease. The CD28c.-1042G>A SNP modulates the risk of CCRCC in women. These findings indicate that the associations of the CTLA-4 and CD28 polymorphisms with the risk of renal cancer are worth further study in a larger group of patients.

Use of fluorescent stainings and flow cytometry for canine semen assessment.

Over the last few decades, appreciable progress has been noted in canine semen assessment techniques. The common use of accurate and sensitive diagnostic methods, such as computer-assisted sperm analysis (CASA), flow cytometry and sperm penetration tests have become routine procedures in specialized andrology laboratories. Many fluorescent probes have been applied to the assessment of specific sperm characteristics in dogs. Flow cytometry enables the observation of cell characteristics such as size, shape and function of the spermatozoon, that can be revealed by a fluorochrome or fluorescent label. The analysis of events detected on dot plots gives accurate and highly reliable information on membrane integrity, acrosomal status, mitochondrial activity, capacitation status, lipid peroxidation, apoptosis and DNA damage. Despite the development of these modern and accurate tools, it is still questionable if the ideal method of semen evaluation, allowing predicting of the fertilizing potential of semen, has been established.

Effects of the inclusion of Equex STM into Tris-based extender on the motility of dog spermatozoa incubated at 5 degrees C.

The aim of this study was to estimate the effects of Equex STM on sperm motion characteristics in chilled dog semen extended in Tris-based diluent. Thirty-two ejaculates were collected from 12 proven German shepherd stud dogs. The sperm-rich fractions were diluted in Tris-based extender with 1% (v/v) Equex STM (sample A) and in Tris-based extender with no addition of detergent (sample B). The extended semen was incubated for 240 h at 5 degrees C and the motility parameters were evaluated by CASA system at 24-h intervals. Addition of Equex STM to Tris-based extender led to an initial activation of motion activity of spermatozoa, followed by a rapid decrease, shortening the lifespan of spermatozoa incubated at 5 degrees C. Computer-assisted sperm analysis clearly showed that Equex STM-induced changes of sperm motion characteristics resemble the hyperactivation (HA) of spermatozoa associated with the capacitation process.

Efficacy of evaluation of rooster sperm morphology using different staining methods.

This work focused on inexpensive methods of evaluation fowl sperm morphology, based on eosin-nigrosin smears, which can determine disorders in spermatogenesis and can be recommended for evaluating the fertilising potency and selecting males in flocks reproduced by artificial insemination. Four fowl breeds (Black Minorca, Italian Partridge, Forwerk and Greenleg Partridge) were used to determine the efficacy of sperm morphology evaluation using four eosin-nigrosin staining methods (according to Blom, Bakst and Cecil, Morisson, Jaskowski) and three examiners of different experience (high, medium, novice). There were significant (P< or = 0.01) differences in sperm morphology between Blom's staining method and those of Bakst and Cecil, Morisson or Jaskowski, irrespective of fowl breed and examiners experience. Blom stain caused sperm head swelling and showed a drastic reduction in the proportion of live spermatozoa with normal morphology. The staining method had a greater influence on sperm morphology evaluation than the experience of the examiners.

[Adenoma oxyphillicum an extremely rare case of tumour of the larynx end cancer lungs which are going together]. / Przypadek gruczolaka oksyfilnego, wyjatkowo rzadkiego guza krtani, wspólistniejacego z rakiem pluca.

There was presented an exstremely rare case of adenoma oxyphillicum of the larynx coexisting with the lung carcinoma. Diagnostical and medical conduct was throughly analyzed. It consisted such matters as detailed graphical examination, spliting of larynx, and radiotherapy and chemiotherapy of lung cancer. The authors want to call attention to limited use fullness of competent lungs X-ray in a-p projection in recognizing the early stadium of lung cancer.

Antidepressant-like activity of CGP 36742 and CGP 51176, selective GABAB receptor antagonists, in rodents.

BACKGROUND AND PURPOSE: A crucial role for the GABAB receptor in depression was proposed several years ago, but there are limited data to support this proposition. Therefore we decided to investigate the antidepressant-like activity of the selective GABAB receptor antagonists CGP 36742 and CGP 51176, and a selective agonist CGP 44532 in models of depression in rats and mice. EXPERIMENTAL APPROACH: Effects of CGP 36742 and CGP 51176 as well as the agonist CGP 44532 were assessed in the forced swim test in mice. Both antagonists were also investigated in an olfactory bulbectomy (OB) model of depression in rats, while CGP 51176 was also investigated in the chronic mild stress (CMS) rat model of depression. The density of GABAB receptors in the mouse hippocampus after chronic administration of CGP 51176 was also investigated. KEY RESULTS: The GABAB receptor antagonists CGP 36742 and CGP 51176 exhibited antidepressant-like activity in the forced swim test in mice. The GABAB receptor agonist CGP 44532 was not effective in this test, however, it counteracted the antidepressant-like effects of CGP 51176. The antagonists CGP 36742 and CGP 51176 were effective in an OB model of depression in rats. CGP 51176 was also effective in the CMS rat model of depression. Administration of CGP 51176 increased the density of GABAB receptors in the mouse hippocampus. CONCLUSIONS AND IMPLICATIONS: These data suggest that selective GABAB receptor antagonists may be useful in treatment of depression, and support an important role for GABA-ergic transmission in this disorder.

[A case of angioma of the spinal cord]. / Przypadek naczyniaka rdzenia kregowego.

A case of angioma situated in the lumbosacral spinal cord is described in a man aged 35 years. During 10 months of conservative treatment and rehabilitation therapy the condition of the patient deteriorated, paralysis of lower extremities developed. The angioma was diagnosed during myelography. The patient was treated surgically, the angioma was removed, and after the operation and rehabilitation therapy an improvement of the neurological state was obtained.