Age, sexual abstinence duration, sperm morphology, and motility are predictors of sperm DNA fragmentation.

Purpose: Sperm DNA fragmentation (SDF) has recently received attention as a cause of male infertility. However, SDF cannot be fully assessed using conventional semen parameter evaluations alone. Therefore, the authors aimed to elucidate the relationship between SDF and sperm parameters via computer-assisted sperm analysis (CASA) to improve treatment strategies in reproductive medicine. Methods: This retrospective observational study analyzed the relationship between sperm parameters assessed by CASA and SDF values determined by the TUNEL assay in 359 patients who visited the Mie University Hospital for infertility treatment. The methodology involved semen analyses covering concentration, motility, and morphology, followed by SDF quantification using the flow cytometry. Results: Statistical analysis revealed significant correlations between SDF and various factors, including age, sexual abstinence period, and specific CASA-measured parameters. Notably, lower sperm motility rates and abnormal head dimensions were associated with higher SDF values, indicating that these parameters were predictive of SDF. Conclusions: This study highlights the importance of sperm motility and head morphology as indicators of SDF, suggesting their usefulness in assessing male fertility. These findings demonstrate the efficacy of detailed sperm analysis, potentially increasing the success rate of assisted reproductive technologies by improving sperm selection criteria.

Bull Sperm SWATH-MS-Based Proteomics Reveals Link between High Fertility and Energy Production, Motility Structures, and Sperm-Oocyte Interaction.

The prediction of male or semen fertility potential remains a persistent challenge that has yet to be fully resolved. This work analyzed several in vitro parameters and proteome of spermatozoa in bulls cataloged as high- (HF; n = 5) and low-field (LF; n = 5) fertility after more than a thousand artificial inseminations. Sperm motility was evaluated by computer-assisted sperm analysis. Sperm viability, mitochondrial membrane potential (MMP) and reactive oxygen species (mROS) of spermatozoa were assessed by flow cytometry. Proteome was evaluated by the SWATH-MS procedure. Spermatozoa of HF bulls showed significantly higher total motility than the LF group (41.4% vs 29.7%). Rates of healthy sperm (live, high MMP, and low mROS) for HF and LF bull groups were 49% and 43%, respectively (p > 0.05). Spermatozoa of HF bulls showed a higher presence of differentially abundant proteins (DAPs) related to both energy production (COX7C), mainly the OXPHOS pathway, and the development of structures linked with the motility process (TPPP2, SSMEM1, and SPAG16). Furthermore, we observed that equatorin (EQTN), together with other DAPs related to the interaction with the oocyte, was overrepresented in HF bull spermatozoa. The biological processes related to protein processing, catabolism, and protein folding were found to be overrepresented in LF bull sperm in which the HSP90AA1 chaperone was identified as the most DAP. Data are available via ProteomeXchange with identifier PXD042286.

Long-term segmentation-free assessment of head-flagellum movement and intracellular calcium in swimming human sperm.

Human spermatozoa are the archetype of long-term self-organizing transport in nature and are critical for reproductive success. They utilize coordinated head and flagellar movements to swim long distances within the female reproductive tract in order to find and fertilize the egg. However, to date, long-term analysis of the sperm head-flagellar movements, or indeed those of other flagellated microorganisms, remains elusive due to limitations in microscopy and flagellar-tracking techniques. Here, we present a novel methodology based on local orientation and isotropy of bio-images to obtain long-term kinematic and physiological parameters of individual free-swimming spermatozoa without requiring image segmentation (thresholding). This computer-assisted segmentation-free method evaluates, for the first time, characteristics of the head movement and flagellar beating for up to 9.2 min. We demonstrate its powerful use by showing how releasing Ca2+ from internal stores significantly alters long-term sperm behavior. The method allows for straightforward generalization to other bio-imaging applications, such as studies of bull sperm and Trypanosoma, or indeed of other flagellated microorganisms - appealing to communities other than those investigating sperm biology.

A new computational approach, based on images trajectories, to identify the subjacent heterogeneity of sperm to the effects of ketanserin.

The identification of kinematic subpopulations is of paramount importance to understanding the biological nature of the sperm heterogeneity. Nowadays, the data of motility parameters obtained by a computer-assisted sperm analysis (CASA) system has been used as input to distinct algorithms to identify kinematic subpopulations. In contrast, the images of the trajectories were depicted only as examples of the patterns of motility in each subpopulation. Here, python code was written to reconstruct the images of trajectories, from their coordinates, then the images of trajectories were used as input to a machine learning clustering algorithm of classification, and the subpopulations were described statistically by the motility parameters. Finally, the images of trajectories in each subpopulation were displayed in a way we called Pollock plots. Semen samples of boar sperm were treated with distinct concentrations of ketanserin (an antagonist of the 5-HT2 receptor of serotonin) and untreated samples were used as a control. The motility of sperm in each sample was analyzed at 0 and 30 min of incubation. Six subpopulations were found. The subpopulation 2 presented the highest values of velocities at 0 or 30 min. After 30 min of incubation, the ketanserin increased the values of the curvilinear velocity at high concentrations, whereas the linearity and the straight velocity decreased. Our computational model permits better identification of the kinematic subpopulations than the traditional approach and provides insights onto the heterogeneity of the response to ketanserin; thus, it could significantly impact the research on the relationship between sperm heterogeneity-fertility.

Performance evaluation of sperm concentration, motility, and morphological analysis for GSA-810 series of sperm quality analysis system.

BACKGROUND: The performance evaluation of each computer-assisted sperm analysis (CASA) system may provide a basis for the interpretation of clinical results and further improvement of the CASA system. METHODS: The accuracy of the GSA-810 CASA system was evaluated by detecting latex bead quality control products. The precision of sperm concentration, morphology, and percentages of progressively motile sperm (PR) were evaluated by coefficient of variation (CV). Three samples with sperm concentration of about 100 × 106 /mL were diluted to evaluate the linear range. RESULTS: The detection values of latex beads were within the range of target values. The CVs of sperm concentration and PR were significantly and negatively correlated with sperm concentration (r = -0.561, p = 0.001) and PR value (r = -0.621, p < 0.001), respectively. The R2 values of the linear range of sperm concentration were ≥0.99. There was no significant difference in sperm motility and PR within 1-10 min at 36.5°C ± 0.5°C. The coincidence rates of sperm morphology and sperm head morphology for 36 semen samples analyzed by the GSA-810 system and manual method were 99.40% and 99.67%, respectively. The CVs of the percentage of sperm with abnormal morphology and percentage of sperm with abnormal head morphology were less than 5%. CONCLUSION: The GSA-810 system can accurately analyze normal semen samples, but the repeatability of the results is poor for oligozoospermia and asthenozoospermia samples. The future CASA system for analyzing sperm morphology should focus on recognizing the middle and tail segments of a spermatozoon.

Optimization of a non-activating medium for short-term chilled storage of barramundi (Lates calcarifer) testicular spermatozoa.

Reliable short-term chilled sperm storage is a critical prerequisite to using advanced reproductive techniques for captive breeding of barramundi (Asian sea bass; Lates calcarifer). Marine Ringer's solution (MRS) is a common non-activating medium (NAM) and has previously been used to store sperm from wild-caught barramundi. However, MRS-stored spermatozoa from captive-bred barramundi were observed to lyse within 30 min incubation. Therefore, this study aimed to optimize the composition of NAM for short-term chilled storage by characterizing and mimicking the biochemical profile of seminal and blood plasma of captive-bred barramundi. To further understand the effect of each component, osmolality was first examined to determine its effect on sperm viability. Thereafter, the effects of NaHCO3, pH, and Na+ and K+ concentrations on sperm motility were investigated. Optimization of the NAM formula was achieved through iterative adaptions. The increase in NAM osmolality from 260 to 400 mOsm/kg led to a significant improvement in sperm viability. Moreover, using HEPES instead of NaHCO3 as buffering agent significantly enhanced sperm motility and velocity. As a result, sperm samples diluted with optimized NAM (185 mM NaCl, 5.1 mM KCl, 1.6 mM CaCl2·2H2O, 1.1 mM MgSO4·7H2O, 10.0 mM HEPES, 5.6 mM D+ glucose, 400 mOsm/kg, pH 7.4) and stored at 4 °C showed no significant loss in total motility for up to 48 h and retained progressive motility for up to 72 h. The optimized NAM developed in this study significantly extended the functional lifespan of spermatozoa during chilled storage, permitting the ongoing development of advanced reproductive technologies for barramundi.

High concentration of growth differentiation factor-9 (GDF-9) to the ram semen had a negative effect on the sperm positive rheotaxis.

Herein, the microfluidic device technique was used to investigate the effects of GDF-9 concentrations and exposure time on the ram sperm positive rheotaxis (PR). Semen was collected from six rams and utilized for PR, motility and sperm kinetic parameter analysis using a computer-assisted sperm analysis program with controlled flow velocity following 0, 10, 20 or 30 min of incubation at 37°C with GDF-9 (200 , 400 or 600 ng/ml; semen sample without GDF-9 was used as a control). Results revealed that there was not an interaction between effects of GDF-9 concentrations and incubation duration on PR% (p = .457) and TM% (p = .099). A simple main effects analysis showed that GDF-9 concentrations had an effect on PR% (p = .003). However, the incubation duration did not have an effect on PR% (p = .101). GDF-9 concentrations had not an effect on TM% (p = .817). By contrast, the incubation duration affected on TM% (p = .026). A higher PR% was found (p < .05) at 200 ng GDF-9 after 10 min (46.7 ± 10.3) and 20 min (45.5 ± 11.5) of incubation. After 30 min of incubation, the PR% was found lowest (p < .05) at 400 ng of GDF-9 (30.6 ± 14.1) and 600 ng of GDF-9 (32.2 ± 9.6). There was no difference (p > .05) in the sperm kinetic parameters between the four treatment groups. In conclusion, the ram sperms had the best rheotaxis properties after 10 and 20 min of incubation with 200 ng of GDF-9 and were sensitive to high concretions.

In vitro fertilization using sperm activated by ML-2-3 isolated from Morinda lucida Bentham leaves.

Purpose: ML-2-3 is a novel tetracyclic iridoid derived from Morinda lucida Bentham leaves. This compound has anti-trypanosomal and anti-leishmanial effects. In this study, the authors investigated effects of ML-2-3 on in vitro fertilization (IVF) rates, motility, and acrosome reaction of the mouse sperm. Methods: IVF was performed using sperm from BALB/cByJJcl mice treated with ML-2-3. Computer-assisted sperm analysis (CASA) was performed on the sperm of C57BL/6J mice to investigate sperm motility. The effect of ML-2-3 on the acrosome reaction was examined by observing the fluorescence of sperm labeled with the acrosin-EGFP transgene. Results: ML-2-3 improved IVF in BALB/cByJJcl mice with low fertilization rates. The optimum concentration of ML-2-3 in sperm pre-culture medium was 20 µM, and no significant toxicity of ML-2-3 was observed in developing embryos at this concentration. ML-2-3 affected sperm motility but not the acrosome reaction. ML-2-3 increased the IVF rate of mouse sperm that had been refrigerated for 3 days. Conclusions: ML-2-3 can improve the outcome of IVF and motility without inducing the acrosome reaction in mice. These effects of ML-2-3 on sperm behaviors are different from those of the similar drugs.

Influence of sperm cryopreservation on sperm motility and proAKAP4 concentration in mice.

Background: The protein proAKAP4 is crucial for sperm motility and has been suggested as an indicator of male fertility. We determined the relationship between proAKAP4 concentration and sperm motility parameters in mice, and investigated the effects of cryopreservation on these variables. Methods: Computer-assisted sperm analysis and ELISA were applied to determine sperm motility and proAKAP4 concentration in fresh and frozen-thawed epididymal sperm of SWISS, B6D2F1, C57BL/6N, and BALB/c mice. Results: ProAKAP4 levels ranged between 12 and 89 ng/ml and did not differ between fresh and frozen-thawed samples, or between strains. We found a negative relationship between proAKAP4 levels and some sperm motility parameters. Sperm traits differed between strains, and cryopreservation negatively affected sperm velocity but not sperm direction parameters. Conclusion: ProAKAP4 levels in epididymal mouse spermatozoa were unaffected by cryopreservation, highlighting the robustness of this parameter as a potentially time-independent marker for sperm motility and fertility. The high individual variation in proAKAP4 levels supports the potential role of proAKAP4 as a marker for sperm quality, though we found no positive, and even negative relationships between proAKAP4 levels and some sperm motility parameters. Future studies have to investigate the significance of proAKAP4 as an indicator for fertility in mice.

The effects of cryopreservation and cold storage on sperm subpopulation structure of common carp (Cyprinus carpio L.).

The objectives of this study were to identify the presence of different spermatozoa subpopulations (SPs) according to their kinematic characteristics in the sperm of common carp and to test the effects of cryopreservation and prolonged (6-day) storage at room temperature (RT; 23 °C) and 4 °C on spermatozoa motility and subsequently on SP dynamics. Two-step clustering analyses identified three motile SPs based on their kinematic properties: SP1 contained spermatozoa with low velocity and low/moderate STR/LIN values (slow non-linear SP); SP2 was comprised of spermatozoa with high velocities and high STR/LIN values (fast linear SP); SP3 was characterized with high VCL, and moderate LIN/STR (fast non-linear SP); and an additional SP0 was added comprising immotile spermatozoa. Total motility, progressive motility and VCL decreased after cryopreservation to approximately 50% of their value in fresh sperm, while the frequency of SPs characterized by high values of motility parameters declined in favor of those with low motility values and SP0. Motility values of fresh and cryopreserved spermatozoa which were washed with fresh extender after thawing decreased significantly after 24 h of storage at RT and after 72 h of storage at 4 °C, while cryopreserved sperm which remained in the original cryomedium faced a steep decline in motility after only 2 h of storage. As subpopulation frequencies followed this dynamic, this indicates that cryopreserved sperm should be washed with fresh extender in order to obtain favorable sperm kinematic properties after freezing.

Quality assessment of frozen bull semen with the precursor A-kinase anchor protein 4 biomarker.

In this study, the quality of frozen bull semen was evaluated with the proAKAP4 level test. Sixty straws of frozen bull semen from various batches (n = 30) belonging to six bulls were used in the current study. The frozen bull semen samples were analysed in terms of proAKAP4 levels, sperm morphology and sperm movement parameters at hour 0 and hour 3 after thawing. The semen samples were divided into three groups according to the proAKAP4 levels: low concentration (<25 ng/10x106 spermatozoa), moderate concentration (25 to 39 ng/10x106 spermatozoa) and high concentration (≥40 ng/10x106 spermatozoa). A positive correlation was found between the proAKAP4 level and total motility (TM3 ), progressive motility (PM3 ), VSL3 and VCL3 values obtained after the third-hour thermoresistance test (p < .05). There was a negative correlation between the percentage of sperm abnormal tail and the proAKAP4 level (p < .01). In addition, it was observed that the semen samples with proAKAP4 concentrations of 25 ng/106 spermatozoa and higher preserved the TM3 and PM3 motility characteristics. In conclusion, the proAKAP4 has the potential to become a biomarker protein to evaluate in the quality analysis of frozen-thawed semen.

Enhancing liquid-chilled storage and cryopreservation capacities of ram spermatozoa by supplementing the diluent with different additives.

OBJECTIVE: In the present study, we determined efficiency of incorporating caffeine, melatonin or omega-3 polyunsaturated fatty acid in the diluent on mitigating consequences of (a) liquid chilled- and (b) cryo-storage of ram spermatozoa. METHODS: In the first experiment, ejaculates (n = 30) were collected from 5 adult rams and were pooled, diluted (1:10) with Tris-citric acid (base diluent) and were split into 4 aliquots assigned for: control (untreated), caffeine (0.1 mM), melatonin (0.3 mM) or omega-3 fatty acids (0.3 mM) (T0). The diluted specimens were stored at 4°C for 48 h, during which sperm physical and cytological properties were evaluated along with oxidative stress indices (T24, T48). In the second experiment, 15 ejaculates (3 per male) were pooled, diluted with glycerolized base diluent (4% glycerol, v/v) and were split corresponding to the same previous treatment groups before being processed for cryopreservation. Post-thaw physical and kinematic sperm properties were assessed by a computer-assisted sperm analysis system. RESULTS: The results clarified superiority of both melatonin and omega-3 supplementation on maintaining (p<0.05) sperm properties, while reducing (p<0.05) lipid peroxidase reaction and enzymatic activities of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase in preservation medium, compared to caffeine either during liquid-chilled storage or cryopreservation of spermatozoa. CONCLUSION: Melatonin and omega-3 are regarded efficient alternatives to caffeine when processing ram spermatozoa for application of artificial insemination or in vitro fertilization.

[Internal quality control products for computer-assisted sperm analysis: Research and application].

OBJECTIVE: To investigate the application of the self-made semen quality control (QC) product in internal QC of computer-assisted sperm analysis (CASA). METHODS: CASA was calibrated with high- and low-concentration commercially available semen QC product and meanwhile 15 samples of self-made mixed semen QC product were placed in 75 cryotubes containing liquid nitrogen, followed by CASA of the concentration, motility, curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN), wobble (WOB) and straightness (STR) of the sperm using standard procedures and 50 days of continuous monitoring. The Makler counting plate was used to measure the concentration and motility of the self-made sperm. RESULTS: The coefficients of variation (CV) of the commercially available semen QC product at high and low concentrations were 6.18% and 7.85%, respectively. CASA showed that the concentration of the self-made QC product was (25.97 ± 1.41) ×106/ml, with a CV of 5.42%, and the sperm motility, VCL, VSL, VAP, LIN, WOB and STR were (22.15 ± 1.75)% (CV = 7.9%), (59.18 ± 2.05) µm/s (CV = 3.46%), (26.79 ± 1.2) µm/s (CV = 4.48%), (34.98 ± 1.4) µm/s (CV = 4.01%), 46.81 ± 1.55 (CV = 3.3%), 60.52 ± 1.3 (CV = 2.15%) and 76.46 ± 1.98 (CV = 2.59%), respectively. The concentration and motility of the self-made sperm detected with the Makler counting plate were (34.39 ± 2.37) ×106/ml (CV = 6.89%) and (38.04 ± 1.69)% (CV = 4.44%), respectively. Levey-Jennings QC charts were plotted for the indicators using the means and standard deviation. CONCLUSIONS: The self-made internal QC product by liquid nitrogen cryopreservation is feasible and effective for monitoring the accuracy and precision of CASA-derived sperm concentration and motion parameters, and it has a smaller CV than the commercially available QC product in measuring sperm concentration.

Sea urchin reproductive performance in a changing ocean: poor males improve while good males worsen in response to ocean acidification.

Ocean acidification (OA) is predicted to be a major driver of ocean biodiversity change. At projected rates of change, sensitive marine taxa may not have time to adapt. Their persistence may depend on pre-existing inter-individual variability. We investigated individual male reproductive performance under present-day and OA conditions using two representative broadcast spawners, the sea urchins Lytechinus pictus and Heliocidaris erythrogramma. Under the non-competitive individual ejaculate scenario, we examined sperm functional parameters (e.g. swimming speed, motility) and their relationship with fertilization success under current and near-future OA conditions. Significant inter-individual differences in almost every parameter measured were identified. Importantly, we observed strong inverse relationships between individual fertilization success rate under current conditions and change in fertilization success under OA. Individuals with a high fertilization success under current conditions had reduced fertilization under OA, while individuals with a low fertilization success under current conditions improved. Change in fertilization success ranged from -67% to +114% across individuals. Our results demonstrate that while average population fertilization rates remain similar under OA and present-day conditions, the contribution by different males to the population significantly shifts, with implications for how selection will operate in a future ocean.

Interaction of different Chlamydiae species with bovine spermatozoa.

BACKGROUND: Interaction of spermatozoa and Chlamydiae spp. might contribute to reduced fertility in cattle. To proof this hypothesis, bovine semen was incubated with viable or heat inactivated Chlamydia (C.) abortus or psittaci (Multiplicity of infection = 1) and sperm motility was monitored with a computer-assisted sperm analyzer over 24 h. Additionally, the interaction with the spermatozoa was further investigated by means of light and transmission electron microscopy (TEM). RESULTS: Only viable Chlamydiae of both species decreased sperm motility and this only after about 9 h. Taking binding rates into account, the loss of sperm motility after about 9 h could likely be a consequence of Chlamydiae attachment to the spermatozoa. About two thirds of the Chlamydiae elementary bodies were bound to the front third of the sperm, the acrosomal region. No inclusions of Chlamydiae in spermatozoa were observed in TEM after 2 h co-incubation. CONCLUSIONS: As initial motility was not affected following co-incubation of viable Chlamydiae and bovine sperm, it seems likely that sperm could serve as a carrier/vehicle for Chlamydiae facilitating cervical passage of Chlamydiae spp. in cattle. Additionally, our results suggest that spermatozoa carrying Chlamydiae may have no initial disadvantage in reaching the oviduct, but are immotile at the time of ovulation what might have an impact on fertilization capacities of the individual sperm. Consequently, high concentrations of the investigated Chlamydiae in the seminal plasma or female genital tract might play a role in reduced fertility in cattle.

Effects of maternal inhalation of carbon black nanoparticles on reproductive and fertility parameters in a four-generation study of male mice.

BACKGROUND: Previous findings indicate that in utero exposure to nanoparticles may affect the reproductive system in male offspring. Effects such as decreased sperm counts and testicular structural changes in F1 males have been reported following maternal airway exposure to carbon black during gestation. In addition, a previous study in our laboratory suggested that the effects of in utero exposure of nanoparticles may span further than the first generation, as sperm content per gram of testis was significantly lowered in F2 males. In the present study we assessed male fertility parameters following in utero inhalation exposure to carbon black in four generations of mice. RESULTS: Filter measurements demonstrated that the time-mated females were exposed to a mean total suspended particle mass concentration of 4.79 ± 1.86 or 33.87 ± 14.77 mg/m3 for the low and high exposure, respectively. The control exposure was below the detection limit (LOD 0.08 mg/m3). Exposure did not affect gestation and litter parameters in any generation. No significant changes were observed in body and reproductive organ weights, epididymal sperm parameters, daily sperm production, plasma testosterone or fertility. CONCLUSION: In utero exposure to carbon black nanoparticles, at occupationally relevant exposure levels, via maternal whole body inhalation did not affect male-specific reproductive, fertility and litter parameters in four generations of mice.

Gallic and carnosic acids improve quality of frozen-thawed ram spermatozoa.

The objective was to determine effects of gallic acid (GA) and carnosic acid (CA), present in carob pods and rosemary extract respectively, on frozen-thawed ram spermatozoa. Thirty ejaculates were collected from five Merino rams, pooled, diluted in Tris-based extender and divided into five equal portions containing: 0.05 or 2 mM of GA; 0.05 or 0.2 mM of CA; or no additive (control). Extended semen was equilibrated at +4°C, loaded into straws, held 5 cm above liquid nitrogen for 12 min then plunged. Computer-aided sperm analysis was used to assess motility, whereas flow cytometry was used to assess high mitochondrial membrane potential (HMMP) and percentages of spermatozoa with plasma membrane and acrosome integrity (PMAI). Spermatozoa supplemented with 2 mM GA had greater total motility than control spermatozoa (39.9 ± 3.01 vs. 29.2 ± 1.31%, mean ± SEM, p < .05). The PMAI was greatest in 0.2 mM CA (13.3 ± 0.68%), whereas HMMP was highest in 0.05 mM CA but lowest in control (22.9 ± 4.95 and 11.4 ± 3.64% respectively; p < .05). In conclusion, for cryopreservation of ram semen in Tris-based extender, supplementation with 2 mM GA increased post-thaw motility, whereas supplementation with 0.05 mM CA enhanced mitochondrial function.

Automation of human semen analysis using a novel artificial intelligence optical microscopic technology.

Current semen analysis still commonly depends on a manual microscopy method in clinical laboratories worldwide. However, some of the major disadvantages of this technique are that it is labour-intensive, subjective, laboratory-based and time-consuming. Although computer-assisted semen analysers (CASAs) have enabled partial automation of routine semen analysis, they lack wider acceptance due to their complicated operation. Therefore, the development of an accessible, rapid and standardised method for semen analysis is urgently needed. Here, we describe the development and clinical testing of a novel, automated, artificial intelligence optical microscopic (AIOM)-based technology, LensHooke™ X1 PRO (X1 PRO), designed for the quantitative measurement of sperm concentration, motility and seminal pH. We observed high degree of correlation in the results of concentration, progressive motility and progressively motile sperm concentration between the X1 PRO semen analyser and manual method using 135 clinical semen samples. In addition, the seminal pH results obtained by X1 PRO and manual methods were comparable (p = .12). In summary, our results showed that new X1 PRO semen analyser is a reliable diagnostic tool for routine semen analysis providing clinically acceptable results based on World Health Organization (WHO) 5th Edition guidelines.

Method agreement between three different chambers for comparative boar semen computer-assisted sperm analysis.

The computer-assisted sperm analysis (CASA) has become a standard laboratory tool. Although it contributes a lot to the objective sperm motility assessment, its measurements may be affected by many factors. The aim of the study was to evaluate the effect of chamber on boar semen CASA results. Totally, 100 extended (30 × 106  sperm/ml) boar semen samples were analysed by CASA. Each sample was evaluated using Makler, Leja 4 chamber 20 µm and conventional glass slide/coverslip chambers (MC, LC and GSC, respectively). The differences in values between MC and LC and between MC and GSC were significantly positive (higher values for MC compared with LC and GSC) for total motility, progressive, rapid movement, VCL, VSL, VAP, STR and hyperactive, thus indicating a systematic effect. Between LC and GSC, the differences in many parameters (non-progressive, progressive, slow, LIN, STR, hyperactive) were evenly distributed around zero, while in all other parameters the differences were significantly positive (higher values for LC compared with GSC), except for medium movement. Based on the estimated intraclass correlation coefficients, the method agreement between MC and LC and between LC and GSC was overall moderate to good, depending on the parameter; nonetheless, it was poor between MC and GSC. The limits of agreement between methods can vary considerably depending on the parameter and should be considered when comparisons between CASA measurements of different andrology laboratories or studies have to be performed.

Effect of adding different levels of caffeine in the extender on some biochemical constituents, enzymatic activities and physical characteristics of chilled and frozen ram semen.

Two experiments were carried out to determine the efficiency of supplementation of ram semen extender with caffeine on chilled storage and frozen capacity of spermatozoa. In the first experiment, eighty ejaculates were collected by an artificial vagina from five adult Barki rams, aged 2-3 years and weighted 45.0 ± 2.0 kg throughout the experimental period (January to February 2017). The ejaculates were pooled and diluted (1:10) with tris-citric egg yolk extender and were split into five groups. Group 1 served as control, whereas groups 2-5 were supplemented with 0.1, 0.2, 0.3 and 0.4 mM caffeine. All diluted semen specimens were evaluated for physical characteristics immediately after dilution (T0 ) and throughout preservation period of 48 hr at 4°C. Simultaneously, oxidative stress and indices such as total antioxidant capacity (TAC), malondialdehyde concentrations (MDA) and alkaline transaminase (AKP) concentrations and value of resazurin reduction test (RRT) were determined. In the second experiment, the raw pooled ejaculates were diluted (1:10) with glycerolated tris-citric egg yolk extender, receiving the previously mentioned caffeine levels. The post-thaw assessment of cryopreserved spermatozoa, in all groups, was conducted by a computer-assisted sperm analysis (CASA) system. The results revealed that adding caffeine to ram semen extender at low (0.1 mM) or medium (0.2 mM) levels had positive impact on both physical characteristics of ram sperm and the enzymatic activities compared to the other semen groups. Caffeine supplementation also enhanced post-thaw sperm dynamics, which implies its potential as an exogenous antioxidant supplement.