Zinc abates sodium benzoate -induced testicular dysfunction via upregulation of Nrf2/ HO-1/ Nf-κB signaling and androgen receptor gene.

BACKGROUND: Sodium Benzoate (SB) is used in daily products such as drinks, juices, sauces, oils, ketchup, toothpaste, mouthwashes, cosmetics, dentifrices, and pharmaceutical products. However, SB has been implicated in gonadotoxicity even at a dosage within the safe limit. Zinc (Zn), on the other hand, has been shown to improve various fertility indices. Hence, this study was designed to explore the possible ameliorative effect of Zn on SB-induced testicular toxicity. METHODS: Animals were randomly divided into control, SB, Zn, and SB+Zn. All treatment lasted for 28 days. RESULTS: SB treatment caused a derangement in reproductive hormone levels, sperm function, and kinematics and a down-regulation of the Androgen receptor (ANDR). Also, a decrease in testicular levels of SOD, CAT, GSH, Nrf2, and HO- 1 activity and an increase in IL-1ß, TNF-α, Nf-κB, and Caspase 3 were observed. These SB-induced distortions were ameliorated in SB-treated rats exposed to Zn. CONCLUSION: Our study suggests that zinc abates SB-induced testicular toxicity by modulating Nrf2/HO-1/ Nf-κB signaling and ANDR upregulation.

Brushing rams before and during electroejaculation improves sperm motility and kinetics with slight changes in stress biomarkers.

The aim of this study was to determine whether brushing rams before and during electroejaculation (EE) reduces their stress response and improves the characteristics of the ejaculate. A single person brushed each ram for 5 min daily, for 15 days, in an individual pen. Semen was collected from five rams brushed before and during EE by the same brusher, while the other five were electroejaculated without being brushed. The treatments were exchanged three days later, so semen was collected from all the rams with both treatments. Brushing increased mass motility (P = 0.05), and curvilinear (P = 0.001), linear (P = 0.02), and average path (P = 0.01) velocities of sperm, as well as the average amplitude of lateral displacement of the sperm head (P = 0.05), and tended to increase sperm concentration (P = 0.09). Brushing tended to reduce the cortisol concentration (P = 0.06) and the duration of head movements when 2 V pulse series V was applied (P = 0.1). Brushing increased creatine kinase concentration (P = 0.04) and tended to increase rectal (P = 0.06) and maximum eye surface temperatures (P = 0.1), total time, and number of electrical pulses administered (P = 0.07 for both variables), as well as the sum of pulses per voltage applied during EE (P = 0.06). In rams accustomed to being brushed by the same person, brushing them before and during EE improved semen quality, with slight changes in the stress responses.

Bioclimatic thermal stress indices and their relationships with andrological characteristics in hair rams.

This study evaluated relationships among reproductive parameters and the bioclimatic indices: temperature and humidity index (THI), equivalent temperature index (ETI), black globe temperature and humidity index (BGTHI), and thermal comfort index (TCI), during the first 45 days of spermatogenesis (SP-45) and during the 15 days of sperm transit through the epididymis (STP-15) that preceded the reproductive assessments (ReA). Such information is useful in determining the optimal breeding season in Northeast Brazil. Santa Inês rams (n = 25) underwent two ReA in three periods of the year (D-P = dry; R-P = rainy and RD-P = rainy/dry transition), and the bioclimatic indices were calculated at the corresponding SP-45 and STP-15 timepoints prior to each ReA. Sperm kinetic parameters in D-P were depressed compared to R-P and RD-P (P < 0.05). The index values had an antagonistic relationship with most parameters and regression analysis demonstrated that the BGTHI and the TCI had a negative association with the progressive motility, curvilinear, straight line, and average path velocities, and a positive association with slow sperm in the ejaculate in SP-45 and STP-15 phases (P < 0.01). Semen quality kinetics is affected throughout the year by the environment and it is apparent that it is impaired in D-P and better in R-P and RD-P seasons. The BGTHI and TCI measured in the sperm production phase classified the environment most coherently and presented better association with the behavior of sperm kinetics.

Can in vitro embryo production be estimated from semen variables in Senepol breed by using artificial intelligence?

Thoroughly analyzing the sperm and exploring the information obtained using artificial intelligence (AI) could be the key to improving fertility estimation. Artificial neural networks have already been applied to calculate zootechnical indices in animals and predict fertility in humans. This method of estimating the results of reproductive biotechnologies, such as in vitro embryo production (IVEP) in cattle, could be valuable for livestock production. This study was developed to model IVEP estimates in Senepol animals based on various sperm attributes, through retrospective data from 290 IVEP routines performed using 38 commercial doses of semen from Senepol bulls. All sperm samples that had undergone the same procedure during sperm selection for in vitro fertilization were evaluated using a computer-assisted sperm analysis (CASA) system to define sperm subpopulations. Sperm morphology was also analyzed in a wet preparation, and the integrity of the plasma and acrosomal membranes, mitochondrial potential, oxidative status, and chromatin resistance were evaluated using flow cytometry. A previous study identified three sperm subpopulations in such samples and the information used in tandem with other sperm quality variables to perform an AI analysis. AI analysis generated models that estimated IVEP based on the season, donor, percentage of viable oocytes, and 18 other sperm predictor variables. The accuracy of the results obtained for the three best AI models for predicting the IVEP was 90.7, 75.3, and 79.6%, respectively. Therefore, applying this AI technique would enable the estimation of high or low embryo production for individual bulls based on the sperm analysis information.

Conditioned medium and secretome from epididymal epithelial cell cultures improve sperm kinetics and capacitation.

Background and Aim: Sperm maturation occurs in the epididymis through interactions with existing molecules inside the lumen. However, the mechanism of epididymis molecular transfer is currently unclear. This study was aimed to determine the necessity of the epididymal epithelial cells (EECs) in the process of sperm maturation in terms of sperm kinetics and tyrosine phosphorylation. Materials and Methods: A true experimental research design was used in this study. The medium tested was a primary culture of mice caput epididymal cells (cells and culture medium), conditioned medium (CM) (supernatant of EECs), and secretome (CM filtered at 0.22 µm). Sperm was cocultured in EEC culture, CM, and secretome for 1, 2, 3, or 4 h. The original culture medium was used as the control. Sperm kinetic analysis was performed after the indicated times using computer-assisted sperm analysis, and tyrosine phosphorylation was detected using the Western blot technique. Results: A primary culture of caput EECs was successfully generated. The results showed increased sperm motility and progressive movement after 3 h of incubation (p < 0.05). There was a significant decrease in the average path velocity (VAP) values after 4 h of incubation (p < 0.05), but there was no significant change in the 1, 2, and 3 h incubation groups. The EEC culture-CM and secretome groups showed a significant increased progressivity and VAP percentage values compared with the control medium (p < 0.05). In terms of percentage motility, the culture and CM groups were significantly different from the control medium, but the secretome group was not. Conclusion: The sperm kinetics of sperm cultured in CM, secretome, and EEC were significantly increased after 3 h of incubation, suggesting that CM and secretome can be used to replace EECs, especially when analyzing molecules secreted by the epididymal epithelium during sperm maturation. The results of this study highlight the potential of CM and secretome as therapy mediums for sperm kinetic abnormalities.

Pogonias courbina sperm characteristcs in its first reproductive season.

Southern black drum (Pogonias courbina) is a species distributed along the western Atlantic Ocean, and it is the largest Sciaenidae observed in the coast of Rio Grande do Sul state, Brazil. However, it is listed as a vulnerable species at The IUCN Red List of Threatened Species™, and their fishing is prohibited. The objective of this study was to determine the sperm characteristics of P. courbina. Sperm samples of five young males (two-year-old fish) were collected through abdominal pressure. The sperm kinetics parameters were sperm motility (MOT) 10.7 ± 5.6%, curvilinear velocity (VCL) 120.07 ± 16.16 mm s ± 1, average path velocity (VAP) 75.64 ± 23.78 mm s ± 1, straight-line velocity (VSL) 62.49 ± 15.83 mm s ± 1, straightness (STR) 83.9 ± 5.3%, wobble (WOB) 61.9 ± 12.7%, beat cross frequency (BCF) 42.981 ± 4.627 Hz and progression (PRG) 1,805.4 ± 564.5 µm. The proportion of normal spermatozoa was 35.6 ± 6.1%. About the abnormalities observed, 22.7% occurred in the tail (short tail = 0.6 ± 0.5%, distally curled tail = 2.4 ± 1.6%, strongly curled tail = 1.9 ± 1.3%, broken tail = 7.9 ± 5.1%, folded tail = 5.5 ± 0.8%, loose tail = 4.4 ± 1.9%); 14.2% occurred in the head (degenerate head = 4.2 ± 1.6%, microcephaly = 1.8 ± 2.5%, loose head = 8.2 ± 2.1%) and 27.5% of the spermatozoa showed cytoplasmatic gouts (proximal gout = 20.0 ± 8.4%, distal gout = 7.5 ± 2.8%). Besides that, a correlation analysis was performed between sperm morphology and kinetics parameters, and the spermatozoa were measured for the morphometric parameters. There was a positive correlation between BCF and normal spermatozoa (r =  0.9269). A negative correlation occurred between BCF and loose head (r =  -0.9047); WOB and strongly curled tail (r =  -0.8911); and PROG and strongly curled tail (r =  -0.9191). The morphometric measures found for the head were length of 2.50 ± 0.21 µm and width of 2.12 ± 0.22 µm, and for the tail it was length of 37.97 ± 2.01 µm. It was possible to verify that the animals have sperm characteristics that indicate reproductive aptitude, but an abnormal behavior on sperm activation and high presence of the cytoplasmic gout abnormality indicates that the animals are not fully mature in their first reproductive season. This work contributes to a better understanding of the P. courbina spermatic parameters, what can be allies to recovery this species population in nature and promote its production in fish farms.

CpG DNA methylation changes during epididymal sperm maturation in bulls.

BACKGROUND: During epididymal transit spermatozoa acquire specific morphological features which enhance their ability to swim in a progressive manner and interact with the oocytes. At the same time, sperm cells undergo specific molecular rearrangements essential for the fertilizing sperm to drive a correct embryo development. To assess epigenetic sperm changes during epididymal maturation, the caput, corpus and cauda epididymis sperm tracts were isolated from eight bulls and characterized for different sperm quality parameters and for CpG DNA methylation using Reduced Representation Bisulfite Sequencing (RRBS) able to identify differentially methylated regions (DMRs) in higher CpG density regions. RESULTS: Caput sperm showed significant variation in motility and sperm kinetics variables, whereas spermatozoa collected from the corpus presented morphology variation and significant alterations in variables related to acrosome integrity. A total of 57,583 methylated regions were identified across the eight bulls, showing a significantly diverse distribution for sperm collected in the three epididymal regions. Differential methylation was observed between caput vs corpus (n = 11,434), corpus vs cauda (n = 12,372) and caput vs cauda (n = 2790). During epididymal transit a high proportion of the epigenome was remodeled, showing several regions in which methylation decreases from caput to corpus and increases from corpus to cauda. CONCLUSIONS: Specific CpG DNA methylation changes in sperm isolated from the caput, corpus, and cauda epididymis tracts are likely to refine the sperm epigenome during sperm maturation, potentially impacting sperm fertilization ability and spatial organization of the genome during early embryo development.

The effect of age and abstinence time on semen quality: a retrospective study.

This study analyzed the effects of male age and abstinence time on semen quality and explored the best abstinence time for Chinese males among different age groups. Semen parameters, including sperm kinetics, morphology, and DNA fragmentation index (DFI), were reviewed from 2952 men. Samples were divided into six age groups (≤25 years, 26-30 years, 31-35 years, 36-40 years, 41-45 years, and >45 years) and were divided into six groups according to different abstinence time (2 days, 3 days, 4 days, 5 days, 6 days, and 7 days). The differences in semen quality between the groups were compared, and the effect of age and abstinence time on semen quality was analyzed. Significant differences were observed in semen volume, progressive motility (PR), and DFI among the age groups (all P < 0.05), and no significant differences were observed in sperm morphological parameters (all P > 0.05). There were significant differences in semen volume, PR, and DFI among different abstinence time groups (all P < 0.05) and no significant differences in sperm morphological parameters (all P > 0.05). Pearson analysis showed that male age and abstinence time were both significantly correlated with sperm kinetics and DFI (both P < 0.05), while no significant correlation was found with sperm morphological parameters (all P > 0.05). The box plots and histograms of men's age, abstinence time, and semen quality show that most semen quality parameters differ significantly between the 2 days and 7 days abstinence groups and other groups at different ages. Except for the sperm morphology parameters, sperm kinetic parameters and sperm DFI are linearly related to male age and abstinence time.

Effect of Extender, Storage Time and Temperature on Kinetic Parameters (CASA) on Bull Semen Samples.

CASA kinetic parameters are often evaluated in a diagnostic centre. How storage conditions affect ejaculates up to evaluation is unclear. We assessed, in 25 commercial bulls electroejaculated in the field, the impact of time until evaluation (0-2 h, 4-6 h, and 24 h post-ejaculation), holding temperature (5 °C vs. room temperature), and extender (AndroMed®, BIOXcell® or INRA96®) on CASA kinetic parameters. Total and progressive motility, VCL, VAP, VCL, ALH, BCF, STR, LIN, and WOB were assessed. CASA kinetic parameters were preserved for up to 4-6 h post-ejaculation, except for AndroMed®. Regardless of extender or temperature, motility decreased from 4-6 h up to 24 h, with the best values obtained with BIOXcell® at 5 °C. Our results suggest that BIOXcell® can preserve sperm motility for up to 6 h, either at 5 °C or room temperature, and also INRA96® at room temperature, with motility assessments and the percentage of the most rapid sperms being the lowest with INRA96® at 5 °C. The kinetic parameters decreased when analyses were performed at 24 h. Therefore, we suggest evaluating seminal quality as soon as possible, before 6 h after collection. These results help to fix adequate protocols for the short-term storage and shipment of bovine semen collected under field conditions.

Potential of watermelon (Citrullus lanatus) to maintain oxidative stability of rooster semen for artificial insemination.

Fruits with antioxidant enrichment can be an economically affordable supplement for mitigating oxidative damage prone spermatozoa membrane pathologies. Computer-assisted sperm analyzer and oxidative status were utilized to evaluate the impact of watermelon (Citrullus lanatus) fortification of dextrose saline as diluent for rooster semen and fertility response of hens inseminated. Watermelon juice and dextrose saline were used to formulate diluent of 7 treatments consisting of unextended semen (positive control), 10%, 20%, 30%, 40%, 50% and only dextrose saline (negative control) designated as Treatments 1-7. Pooled semen was obtained from fertile roosters and equilibrated with diluents at ratio 1:2 in the various treatments and were evaluated using computer software coupled microscope and seminal oxidative status assay. 168 laying hens randomly divided into 7 treatment of 8 replicates and 3 hen per replicate. Hen were everted, and semen (2 × 108 Spermatozoa) deposited intra-vagina and eggs collected over 8 weeks to assess fertility and hatchability of eggs laid. The result obtained revealed that watermelon-dextrose saline rooster semen diluent enhanced progressive motility, sperm kinetics and lowered non-progressive motility in T2-T6 compared to T7 over the 3 hours of evaluation. Watermelon addition to rooster semen diluent enhance the antioxidant capacity of rooster semen and lowered lipid peroxide generation. The percentage fertility was highest in T3 (81.01%) and T4 (81.24%) with lowest value obtained in T7 (73.46%). The hatchability of eggs set of hens inseminated with undiluted semen (71.46%) was lower than values for hens inseminated with watermelon inclusive extended semen (75.71%-80.39%). The optimal inclusion of 30%-40% watermelon in dextrose saline diluent enhance rooster semen kinetics, seminal oxidative stability and egg fertility.

Relationships between Seminal Plasma Metabolites, Semen Characteristics and Sperm Kinetics in Donkey (Equus asinus).

This study aimed to evaluate donkey seminal plasma metabolites and relate this information to the main characteristics of sperm quality. Sperm kinetics from 10 donkey stallions were analyzed with a computerized system at the time of collection (T0) and after 24 h storage at 4 °C (T24). Seminal plasma was frozen at -80 °C for subsequent proton nuclear magnetic resonance (1H NMR) spectroscopy. On three stallions, semen collection was repeated monthly for three times and sperm analysis also included mitochondrial activity and oxidative status. One stallion was azoospermic and a second semen collection was performed after one month. In the seminal plasma, 17 metabolites were identified; their levels showed numerous significant variations between the azoospermic and the normospermic individuals and grouped in well-defined clusters in a multivariate analysis. Comparing individuals with high and low sperm motility, the only discriminating metabolite was phenylalanine, whose levels were lower in the latter, as in the azoospermic individual. Phenylalanine was also the only metabolite highly correlated with all sperm kinematic parameters at T24. In conclusion, the present study has provided relevant information on the chemical characteristics of donkey semen, identified relationships between seminal metabolites, semen parameters, and sperm kinetics, and offered insights for future technological applications.

Aluminum at environmental concentrations affects the sperm quality of the freshwater teleost Astyanax altiparanae: An in vitro study.

Aluminum (Al) is present in rivers and reservoirs in concentrations above that is allowed by regulatory agencies (e.g. 0.5 mg L-1 Al), which can impair fish reproduction. The present study evaluated the in vitro effects on the sperm of Astyanax altiparanae upon Al exposure at different concentrations (0, 0.05, 0.1, 0.3, and 0.5 mg L-1) with various exposure periods (50 s, 10 min, and 30 min). The following biomarkers were evaluated: membrane vitality, DNA fragmentation, morphology, kinetics (10 s and 30 s after sperm activation), and sperm mitochondrial activity. Al damages the membrane vitality of gametes at 0.3 and 0.5 mg L-1 after 50 s of exposure. After 30 min of exposure, there was a decrease in membrane vitality at 0.1 and 0.5 mg L-1, and the membrane vitality decreased with increased exposure time. Within 30 s after sperm activation, Al (0.3 and 0.5 mg L-1) reduced sperm motility by more than 50% at the longest exposure time, while at 0.1 and 0.5 mg L-1, Al exposure reduced motility over time. The average path speed (VAP; 10 s post-sperm activation) was reduced at longer exposure times at 0.05 and 0.5 mg L-1 of Al. Increased exposure time had deleterious effects on mitochondrial activity at the highest concentrations tested. Al did not damage DNA and sperm morphology. In conclusion, Al negatively influences the sperm quality of A. altiparanae with a potential effect of exposure time and increasing concentrations.

Effect of Quercetin Supplementation in Extender on Sperm Kinematics, Extracellular Enzymes Release, and Oxidative Stress of Egyptian Buffalo Bulls Frozen-Thawed Semen.

Buffalo spermatozoa are more sensitive for cryopreservation compared to other species. This study aimed to evaluate the consequences of quercetin against cryodamage of buffalo frozen-thawed spermatozoa characteristics. Semen of Egyptian bulls (n = 4) was extended in OptiXcell extender incorporated with quercetin at 0 (control), 2.5, 5.0, 10.0, 20.0, 40.0, and 80.0 µM before cryopreservation. Frozen-thawed semen was evaluated for sperm motility by computer-assisted sperm analyzer (CASA), viability, morphology, membrane, and acrosome integrities. The kinematics parameters including average path velocity (VAP; µm/s), straight linear velocity (VSL; µm/s), curvilinear velocity (VCL; µm/s), amplitude of lateral head displacement (ALH; µm), beat cross frequency (BCF; Hz), linearity [LIN, (VSL/VCL) × 100], and straightness [STR, (VSL/VAP) × 100] were assessed. The sperm-free extender was evaluated for aspartate aminotransferase (AST), alanine aminotransferase (ALT), and H2O2. Homogenized sperm cells were evaluated for oxidative stress biomarkers [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX)], and lipid peroxidation [malondialdehyde (MDA)]. The highest values of total motility, progressive motility, viability, intact acrosome, and membrane integrity substantially improved with 10 µM of quercetin. STR (%) was substantially low (P < 0.01), and VCL (µm/s) and ALH (µm) were markedly high (P < 0.05) in 10 µM of quercetin. The outflow of ALT enzyme to extracellular fluid was lower with 10 µM of quercetin (P < 0.001) and higher at 2.5 µM of quercetin. The spermatozoa leaked AST was markedly lower at 5.0, 10 (P < 0.001) and 20 µM (P < 0.05) of quercetin. The activity of antioxidant enzymes was eminently low at all quercetin concentrations, and this was accompanied by the decrease in H2O2 in the media. SOD activity at 10-80 µM, CAT at 5.0-40 µM, and GPX at 2.5-80.0 µM of quercetin in spermatozoa were substantially low. MDA level significantly (P < 0.001) decreased at all quercetin concentrations. In conclusion, the incorporation of quercetin at the level of 10 µM is promising in improving buffalo semen characteristics and lower the freezing-thawing oxidative stress.

d-aspartate treatment in vitro improves mouse sperm fertility in young B6N mice.

We previously reported that the administration of d-aspartate (D-Asp) in drinking water over a 2-4-week period to 7-week-old mice resulted in higher sperm quality and increased in vitro fertilisation (IVF) rates associated with a systemic increase of luteinizing hormone and testosterone levels in the serum. The goal of this study was to investigate the effects of in vitro treatment with D-Asp on the IVF rate, embryo transfer, and sperm parameters of cryopreserved-thawed C57BL/6NTacCnrm (B6N) spermatozoa derived from young and adult mice. In this study, cryopreserved-thawed B6N spermatozoa from males aging 9, 11, 13, and 16 weeks were treated for 1 h with 4 mM D-Asp during capacitation. Thereafter, the in vitro fertilisation ability and the embryo transfer efficiency were analysed. Also, the kinetic activity of the treated spermatozoa and the acrosome reaction were measured after 1 h, 2 h, and 5 h of incubation. The capacitation rate of spermatozoa was determined after 1 h of pre-incubation. Spermatozoa from 9- and 11-week-old mice, which were treated with D-Asp, led to significantly increased IVF rates. However, spermatozoa derived from 13- and 16-week-old mice did not lead to a significant improvement in the fertilisation rate. At all ages examined, no differences were observed in the birth rate and sperm kinetic parameters. After 1 h incubation under the same conditions as the IVF was performed, the capacitation rate and the acrosome reaction were significantly higher with the D-Asp-treated spermatozoa from 9-week-old (67.5% vs. 41% and 14.5% vs. 10.5%, respectively) and 11-week-old mice (78.5% vs. 41.1% and 21.0% vs. 3.8%, respectively), corresponding to the improved IVF results. Therefore, the present results demonstrate, for the first time, a direct role of D-Asp in the capacitation process and acrosome reaction.

Is the protective effect of egg yolk against osmotic and cryogenic damage on dog spermatozoa dose-dependent?

Egg yolk (EY) is conventionally used to reduce sperm cryodamage, however, there has not be evaluation of whether there is a dose-dependent effect with inclusion of EY in semen extender. To enhance the knowledge about the protective effect of EY during cryopreservation of dog semen, a specific study was designed to evaluate the dose-dependent protection of the EY against osmotic and cryogenic damage of dog sperm. In the first experiment, sperm stored in an extender that contained graded EY concentrations (0 %, 5 %, 10 %, and 20 %) were diluted with hypo- or hyper-osmotic solutions (final osmolality of 75, 150, 300, 500, 1000 mOsm/kg). Results from sperm kinetic, membrane integrity (MI), mitochondrial activity, and normal morphology evaluations indicated osmotic stress has especially marked effects on the kinetic capacity of spermatozoa, however, there were no direct effects on mitochondrial activity. In both hypo- and hyper-osmotic conditions, EY had a protective effect regardless of concentration. In the second experiment, semen samples were diluted in extenders at increasing EY concentrations (0 %, 5 %, 10 %, and 20 %) and cryopreserved. Effects on sperm kinetics, membrane and acrosome integrity and mitochondrial membrane potential indicated there was improved sperm viability after thawing when the EY concentration was 5 % and 10 %, and lesser viability when it was 20 %. These results indicate, for the first time, that EY reduces osmotic and cryogenic damage when used at 5 % or 10 % concentrations, and that these concentrations can be used to protect dog spermatozoa more effectively than the conventionally used concentration (20 %).

Reduction in Percoll volume increases recovery rate of sex-sorted semen of bulls without affecting sperm quality and early embryonic development.

The aim of the present study was to evaluate the effects of Percoll volume on recovery rate, sperm quality, and embryo development kinetics in in vitro production of cattle embryos. Straws of conventional and sex-sorted semen were allocated to three different volumes of Percoll: 300 µL of each Percoll gradient (90%, 60%, and 30%), Control; 100 µL of each Percoll gradient, P100; and 200 µL of each Percoll gradient, P200. Sperm quality, fertilization rate, and embryo morpho-kinetic development using time lapse cinematography up to 48 h post-insemination were evaluated. For conventionally processed semen, sperm motility, vigor, and recovery rate were greater in the P100 and P200 treatment groups compared to the Control (P < 0.05), whereas reactive oxygen species (ROS) concentrations, lipid peroxidation, and superoxide dismutase enzyme activity were not influenced by treatments. For sex-sorted semen, treatment with P100 increased sperm curvilinear velocity, average path velocity, and amplitude of lateral head displacement (P < 0.05). Recovery rate was greater in the P100 group than Control and P200 groups (P < 0.05), formation of ROS was less in the P100 than Control and P200 groups, and superoxide dismutase enzyme activity was less in the P100 than Control group. Fertilization and cleavage rates, time of first cleavage, and cell number were similar between the P100 and Control groups (P > 0.05). The inclusion of Percoll volumes of 100 µL resulted in an increased sperm recovery rate without damage to sperm quality or affecting early embryonic development.

Physical and kinematic properties of cryopreserved camel sperm after elimination of semen viscosity by different techniques.

This investigation aimed to determine the influence of using different techniques for liquefaction of semen on post-thaw physical and dynamic characteristics of camel spermatozoa. A total of 144 ejaculates were collected from 3 adult camels, Camelus dromedarius, twice-weekly over 3 consecutive breeding seasons. A raw aliquot of each ejaculate was evaluated for physical and morphological properties, whereas the remaining portion was diluted (1:3) with glycerolated Tris lactose egg yolk extender, and was further subjected to one of the following liquefaction treatments: control (untreated), 5µl/ml α-amylase, 0.1mg/ml papain, 5u/ml bromelain, or 40-kHz nominal ultrasound frequency. The post-thaw objective assessment of cryopreserved spermatozoa, in all groups, was performed by a computer-assisted sperm analysis (CASA) system. The results revealed that all liquefaction treatments improved (P<0.05) post-thaw motility, viability and sperm motion criteria. However, an adverse effect (P<0.05) was observed in acrosome integrity, sperm cell membrane integrity and percent of normal sperm in all enzymatically-treated specimens compared to both control and ultrasound-treated semen. These results elucidate the efficiency of utilizing ultrasound technology for viscosity elimination of camel semen. In addition, developing enzymatic semen liquefaction techniques is imperious to benefit from when applying assisted reproductive technologies, particularly AI and IVF, in camels.

Kinetic activity, membrane mitochondrial potential, lipid peroxidation, intracellular pH and calcium of frozen/thawed bovine spermatozoa treated with metabolic enhancers.

Owing to the progressive decline of sperm motility during storage there is a need to find substances capable of enhancing sperm energy metabolism and motility and/or preserving it from oxidative damage. The aim of this study was to evaluate in frozen/thawed bovine spermatozoa the effect of several compounds, such as myo-inositol, pentoxifylline, penicillamine + hypotaurine + epinephrine mixture (PHE), caffeine and coenzyme Q10+ zinc + d-aspartate mixture (CZA), on either kinetic or metabolic parameters. Sperm kinetics was evaluated by Sperm Class Analyser whereas specific fluorochromes were used to evaluated mitochondrial membrane potential (MMP), intracellular pH, intracellular calcium concentration and lipid peroxidation. Lipid peroxidation was also evaluated by TBARS analysis. Treatments significantly affected total and progressive motility with different dynamics in relation to the incubation time. After the first hour of incubation, CZA treatment produced the best performance in total and progressive sperm motility as well as in curvilinear velocity, average path velocity and amplitude of head displacement, whereas pentoxifylline stimulated the highest straight-line velocity. MMP showed higher values (p < 0.01) after treatment with pentoxifylline and PHE. Intracytoplasmic calcium concentration and lipid peroxidation were significantly (p < 0.05) affected by the incubation time rather than the treatments. Intracellular pH varied significantly (p < 0.01) in relation to either the incubation time or treatments. In particular, it showed a progressive increase throughout incubation with values in control group significantly higher than in myo-inositol, PHE, caffeine, pentoxifylline and CZA groups (7.37 ± 0.03 vs. 7.29 ± 0.03, 7.28 ± 0.03, 7.26 ± 0.03, 7.22 ± 0.03 and 7.00 ± 0.03, respectively; p < 0.01).; however, among treatments, CZA displayed the lowest values. Significant correlations were found between sperm kinetic and metabolic parameters. These findings provide new comparative information on the effects of putative metabolic enhancers on kinetics and metabolic activities of bovine spermatozoa. In this study, a rapid methodological approach for evaluating sperm quality is proposed.

Role of calcium on the initiation of sperm motility in the European eel.

Sperm from European eel males treated with hCGrec was washed in a calcium free extender, and sperm motility was activated both in the presence (seawater, SW) and in the absence of calcium (NaCl+EDTA), and treated with calcium inhibitors or modulators. The sperm motility parameters were evaluated by a computer-assisted sperm analysis (CASA) system, and changes in the [Ca(2+)]i fluorescence (and in [Na(+)]i in some cases) were evaluated by flow cytometry. After sperm motility was activated in a medium containing Ca(2+) (seawater, SW) the intracellular fluorescence emitted by Ca(2+) increased 4-6-fold compared to the levels in quiescent sperm. However, while sperm activation in a Ca-free media (NaCl+EDTA) resulted in a percentage of motility similar to seawater, the [Ca(2+)]i levels did not increase at all. This result strongly suggests that increasing [Ca(2+)]i is not a pre-requisite for the induction of sperm motility in European eel sperm. Several sperm velocities (VCL, VSL, VAP) decreased when sperm was activated in the Ca-free activator, thus supporting the theory that Ca(2+) has a modulatory effect on sperm motility. The results indicate that a calcium/sodium exchanger (NCX) which is inhibited by bepridil and a calcium calmodulin kinase (inhibited by W-7), are involved in the sperm motility of the European eel. Our results indicate that the increase in [Ca(2+)]i concentrations during sperm activation is due to an influx from the external medium, but, unlike in most other species, it does not appear to be necessary for the activation of motility in European eel sperm.

Verificación de los valores de referencia del estudio del semen según OMS 2010 en Buenos Aires / Verification of reference values for semen analysis according to WHO 2010 in Buenos Aires / Verificação dos valores de referência da análise do sêmen de acordo com a OMS 2010 em Buenos Aires

La Organización Mundial de la Salud en su edición 2010 ha publicado valores de referencia para los parámetros seminales. Sin embargo, los laboratorios deben verificarlos en su población para transferirlos a su práctica clínica. Para cumplimentar este requerimiento se propuso verificar los intervalos de referencia OMS 2010 en la población de la provincia y Ciudad Autónoma de Buenos Aires. También se determinó el rango de resultados para: número total de espermatozoides móviles progresivos y morfología normal en el eyaculado, parámetros cinéticos y pruebas funcionales. El proceso de verificación fue llevado a cabo de acuerdo a la guía C28-A2 CLSI (Clinical and Laboratory Standards Institute). Mediante difusión se convocó a hombres (n:20) argentinos residentes en Buenos Aires, con fertilidad probada en los últimos 12 meses. Se excluyeron individuos con patología andrológica. Las muestras se procesaron de acuerdo con los criterios OMS 2010. De los resultados se establece que los intervalos de referencia publicados en la última versión han sido verificados, con excepción del volumen seminal. Los rangos de valores obtenidos para los parámetros de movilidad progresiva y morfología en número totales, como también de los parámetros cinéticos y de las pruebas funcionales contribuirán a establecer futuros valores de referencia.

WHO 2010 provided reference values for semen analysis; however clinical laboratories should verify them in healthy fertile males. In order to be able to transfer this reference interval to our unit, it was proposed to examine the available data in the Buenos Aires population. The range of data obtained in this population was also provided for the following: total number of progressively motile and morphologically normal spermatozoa in the ejaculate,nematic parameter values and functional tests, in an attempt to establish standardization in these parameters. The process of reference value verification was carried out according to C28-A2 CLSI (Clinical and Laboratory Standards Institute) guidelines; to that aim, twenty men born in Argentina who reside in Buenos Aires province or city were recruited to participate. They were of proven fertility in the last 12 months. Individuals with pathologies by andrological consultation were excluded. Samples were processed according to 2010 WHO. In Buenos Aires City, WHO reference values were confirmed whereas semen volume was not confirmed. The range of values for total progressive and morphologically normal spermatozoa as well as kinetic parameter values, enrichment by swim up and functional test results were established for the male fertile population studied in an attempt to contribute to the construction of future reference values.

Organização Mundial da Saúde, em sua edição de 2010, tem publicado valores de referência para os parâmetros seminais. No entanto, os laboratórios devem verificá-los em sua população para serem transferidos a sua prática clínica. Para cumprir com esse requisito, decidiu-se verificar os intervalos de referência da OMS 2010, na população da província e da cidade de Buenos Aires. Também foram definidos intervalos de resultados para o número total de espermatozoides móveis progressivos e para a morfologia normal no ejaculado, os parâmetros cinéticos e os testes funcionáis. O processo de verificação foi realizado de acordo com o guia CLSI C28 A2 (Clinical and Laboratory Standards Institute). Por meio de uma convocação, (N 20) homens argentinos residentes em Buenos Aires, com comprovada fertilidade nos últimos 12 meses foram convidados. Indivíduos com patologia andrológica foram excluídos. As amostras foram processadas de acordo com os critérios da OMS de 2010. A partir dos resultados estabeleceu-se que os intervalos de referência publicados na versão mais recente foram verificados, com exceção do volume seminal. Os intervalos de valores obtidos para os parâmetros de motilidade progressiva e morfologia em número total, bem como os parâmetros cinéticos e os dos testes funcionais ajudarão a estabelecer valores de referência futura.