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.

Morphometric assessment of cryopreserved livestock bull spermatozoa in the tropics.

The identification of different morphometric patterns of spermatozoa serves as a basis for improving our understanding of the diversity in an ejaculate and to relate them to the potential fertility of males. In this study, we aimed to examine the semen subpopulation structure, following dilution in semen of extenders, using a mathematical approach a possible application to fertility analyses. Ten sexually mature Bos taurus bulls were randomly allotted to one of three groups: (1) Tris-citric acid-egg yolk extender (Tris-EY); (2) commercial egg yolk extender OptiXcell® and (3) commercial egg yolk extender Triladyl®. The results showed significant differences (p < .05) between extenders in terms of values for head size and head shape variables of individual sperm, indicating an influence of extender composition. Sperm head width was found to significantly differ (p < .05) according to the extender, decreasing in the following order: OptiXcell® (4.836 ± 0.017 µm), Triladyl® (4.695 ± 0.012 µm) and Tris-EY (4.638 ± 0.010 µm). Principal component analysis allowed us to identify two subpopulations in OptiXcell®, and three subpopulations were each found in Triladyl® and Tris-EY. Overall, we observed significant differences between sperm subpopulations within each extender (p < .05), with differences in sperm head size and shape between bovine species that can be related to functionality and fertility capabilities.

A Bayesian analysis of boar spermatozoa kinematics and head morphometrics and their relationship with litter size fertility variables.

The semen movement and sperm head size patterns of boar ejaculates were analysed using computer-assisted semen analysis (CASA)-Mot and -Morph systems. The aim of the present study was to compare morphometric and kinematics variables from boars and to determine the relationship with sow fertility variables related to litter size. The females were from maternal crossing schemes such as the continuous 3-generation cross between York (Y), Landrace (L), and Pietrain (P) hybrid sows and Pietrain boars. Semen samples were collected from 11 sexually mature boars from two sire lines. Samples were analysed using the ISAS® v1 system to evaluate eight kinematic variables of sperm velocity, progressiveness and undulations. Four morphometric parameters of sperm head size (length, width, area and perimeter) were analysed. Bayesian analysis revealed relevant differences in four kinematic variables (VSL, LIN, STR and WOB) between sire lines, with a probability of relevance (PR ) of 0.79-0.91, and Pietrain boars were associated with higher progressive motility compared with Duroc x Pietrain boars. Moreover, there were relevant differences in all morphometric variables (PR  = 0.82-0.85) between sire lines. The dam line Y-L-50 (½ Y × ½ L) had higher total born per litter and piglets born alive, and YLP-75 (1 /8 Y × 1 /8 L × 3 /4 P) was associated with higher values of litter weight at birth (highest posterior density region at 95% = 9.92, 16.41 kg). There are relevant differences in kinematic variables between the assessed sire lines and the differences in morphometric and litter size variables were also relevant. The York-Landrace hybrid sows had higher total born per litter and piglets born alive, and there were relevant differences when compared with YLP-50 (» York × » Landrace × ½ Pietrain). Differences in kinematic and morphometric variables between sire and dam lines related to fertility need to be further studied.

Combined effect of type and capture area of counting chamber and diluent on Holstein bull sperm kinematics.

The evaluation of sperm motion is crucial for processing of seminal doses for artificial insemination. Here, the combined effect of the type and capture area of three counting chambers, together with the type of diluent employed, on sperm motility was analysed. Ejaculates from thirteen Holstein bulls were used for sperm kinematic analysis with the ISAS® v1 CASA-Mot system, using two capillary-loaded counting chambers (Leja® and Cell-Vu® ) and one drop displacement chamber (Makler® ). Nine fixed positions were analysed per chamber type, considering central and lateral and three longitudinal fields. Independent of the diluent used, differences were found between the three chambers. Independent of the extender, no differences in x-axis were observed with Cell-Vu® , while using Leja® , some parameters showed lower values in the centre than in lateral areas. In both counting chambers, the lowest values were observed in the distal area. Results obtained with the two diluents were highly different with a very low correlation between them. In conclusion, the capture area inside the chambers leads to significant changes in sperm kinematic parameters and different dilution media introduce considerable differences in the motility patterns. It is necessary to optimise sampling methods and specific set-ups to be used with CASA-Mot technology.

Dog sperm swimming parameters analysed by computer-assisted semen analysis of motility reveal major breed differences.

Dogs have undergone an intensive artificial selection process ever since the beginning of their relationship with humans. As a consequence, a wide variety of well-defined breeds exist today. Due to the enormous variation in dog phenotypes and the unlikely chance of gene exchange between them, the question arises as to whether they should still be regarded as a single species or, perhaps, they be considered as different taxa that possess different reproductive traits. The aim of this study was therefore to characterize some male reproductive traits, focusing on kinematic characteristics of dog spermatozoa from several breeds. Thirty-seven dogs from the following breeds were used: Staffordshire Bull Terrier, Labrador Retriever, Spanish Mastiff, Valencian Rat Hunting Dog, British Bulldog and Chihuahua. Semen samples were obtained via manual stimulation and diluted to a final sperm concentration of 50 million/ml, and they were subsequently analysed by the computer assisted semen analysis (CASA-Mot) ISAS® v1 system. Eight kinematic parameters were evaluated automatically. All parameters showed significant different values among breeds and among individuals within each breed. The fastest sperm cells were those of Staffordshire Bull Terriers and the slowest were recorded in Chihuahuas. The intra-male coefficient of variation (CV) was higher than the inter-male CV for all breeds with the Staffordshire Bull Terrier showing the lowest values. When taking into consideration the cells by animal and breed, discriminant analyses showed a high capability to predict the breed. Cluster analyses showed a hierarchical classification very close to that obtained after phylogenetic studies with genome markers. In conclusion, future workers on dog spermatozoa should bear in mind major differences between breeds and realize that results cannot be extrapolated from one to another. Because sperm characteristics are associated with breed diversity, dogs may represent a good model to examine changes in reproductive parameters associated with selection processes.

Effect of frame rate capture frequency on sperm kinematic parameters and subpopulation structure definition in boars, analysed with a CASA-Mot system.

Motility is the most widely used indicator of sperm quality. Computer-Assisted Semen Analysis (CASA) allows the objective evaluation of sperm motility parameters. CASA technology is a common tool to predict semen doses in farm animal reproduction. The kinds of video cameras used until now for image acquisition have presented limited frame rates (FR), which have a negative influence on the quality of the obtained data. The aim of the present work was to define the optimal frame rate for a correct evaluation of boar sperm motility and its subpopulation structure. Eighteen ejaculates from nine mature boars of the Pietrain breed were used. Using the ISAS® v1 CASA-Mot system, with a video camera working up to 200 Hz, six FRs (25, 50, 75, 100, 150 and 200 fps) were compared. ISAS® D4C20 counting chambers, warmed to 37°C, were used. FR affected all the kinematic parameters, with curvilinear velocity (VCL) and BCF the most sensitive ones. All the parameters showed differences among animals. Non-linear correlation showed the asymptotic level for VCL at 212 fps, being the highest FR for all the parameters. For future studies based just on progressive motility, almost 100 fps FR for 0.5 s must be used, while when kinematics must be considered, almost 212 fps for one-second should be analysed. Three principal components were obtained (velocity, progressivity and oscillation), being similar at 50 and 200 fps. Cells were grouped in four subpopulations but with different kinematic and cellular distribution at both FRs.

Comparison of sperm motility subpopulation structure among wild anadromous and farmed male Atlantic salmon (Salmo salar) parr using a CASA system.

Atlantic salmon (Salmo salar) is an endangered freshwater species that needs help to recover its wild stocks. However, the priority in aquaculture is to obtain successful fertilisation and genetic variability to secure the revival of the species. The aims of the present work were to study sperm subpopulation structure and motility patterns in wild anadromous males and farmed male Atlantic salmon parr. Salmon sperm samples were collected from wild anadromous salmon (WS) and two generations of farmed parr males. Sperm samples were collected from sexually mature males and sperm motility was analysed at different times after activation (5 and 35s). Differences among the three groups were analysed using statistical techniques based on Cluster analysis the Bayesian method. Atlantic salmon were found to have three sperm subpopulations, and the spermatozoa in ejaculates of mature farmed parr males had a higher velocity and larger size than those of WS males. This could be an adaptation to high sperm competition because salmonid species are naturally adapted to this process. Motility analysis enables us to identify sperm subpopulations, and it may be useful to correlate these sperm subpopulations with fertilisation ability to test whether faster-swimming spermatozoa have a higher probability of success.

CASA-Mot technology: how results are affected by the frame rate and counting chamber.

For over 30 years, CASA-Mot technology has been used for kinematic analysis of sperm motility in different mammalian species, but insufficient attention has been paid to the technical limitations of commercial computer-aided sperm analysis (CASA) systems. Counting chamber type and frame rate are two of the most important aspects to be taken into account. Counting chambers can be disposable or reusable, with different depths. In human semen analysis, reusable chambers with a depth of 10µm are the most frequently used, whereas for most farm animal species it is more common to use disposable chambers with a depth of 20µm . The frame rate was previously limited by the hardware, although changes in the number of images collected could lead to significant variations in some kinematic parameters, mainly in curvilinear velocity (VCL). A frame rate of 60 frames s-1 is widely considered to be the minimum necessary for satisfactory results. However, the frame rate is species specific and must be defined in each experimental condition. In conclusion, we show that the optimal combination of frame rate and counting chamber type and depth should be defined for each species and experimental condition in order to obtain reliable results.

Effect of counting chamber depth on the accuracy of lensless microscopy for the assessment of boar sperm motility.

Sperm motility is one of the most significant parameters in the prediction of male fertility. Until now, both motility analysis using an optical microscope and computer-aided sperm analysis (CASA-Mot) entailed the use of counting chambers with a depth to 20µm. Chamber depth significantly affects the intrinsic sperm movement, leading to an artificial motility pattern. For the first time, laser microscopy offers the possibility of avoiding this interference with sperm movement. The aims of the present study were to determine the different motility patterns observed in chambers with depths of 10, 20 and 100µm using a new holographic approach and to compare the results obtained in the 20-µm chamber with those of the laser and optical CASA-Mot systems. The ISAS®3D-Track results showed that values for curvilinear velocity (VCL), straight line velocity, wobble and beat cross frequency were higher for the 100-µm chambers than for the 10- and 20-µm chambers. Only VCL showed a positive correlation between chambers. In addition, Bayesian analysis confirmed that the kinematic parameters observed with the 100-µm chamber were significantly different to those obtained using chambers with depths of 10 and 20µm. When an optical analyser CASA-Mot system was used, all kinematic parameters, except VCL, were higher with ISAS®3D-Track, but were not relevant after Bayesian analysis. Finally, almost three different three-dimensional motility patterns were recognised. In conclusion, the use of the ISAS®3D-Track allows for the analysis of the natural three-dimensional pattern of sperm movement.

Sperm kinematic characterization of alpaca (Vicugna pacos L.) during the reproductive season.

Semen analysis is a key factor when determining the fertility ability in males. South American camelids, and in particular the alpaca, have been studied very little when compared with other farm animals. The aim of this work was to perform the kinematic characterization of alpaca spermatozoa collected directly from the deferent duct by using CASA-Mot (Computer Assisted Semen Analysis for Motility) technology. Samples were obtained every three days throughout the reproductive season during two periods and with a break of seven days in the middle. During both periods, the quality of the sample's motility and kinematics increased over the first two days and then subsequently decreased. This pattern was similar in all animals. It was concluded that the introduction of resting times can be useful to improve sperm quality for artificial insemination purposes in natural conditions.

Effect of different oxidative stress degrees generated by hydrogen peroxide on motility and DNA fragmentation of zebrafish (Danio rerio) spermatozoa.

An increase in reactive oxygen species (ROS) or decrease in antioxidant barriers can provoke lipid peroxidation of the membranes or DNA damage of the spermatozoa. The aim of this work is to study the effect of the different degrees of oxidative stress generated by H2 O2 incubation on total motility, kinetics, and DNA fragmentation of zebrafish (Danio rerio) spermatozoa. For this process, experimental groups were incubated in 50 µM (Low; L) and 200 µM (High; H) H2 O2 , respectively, for 20 min at 4ºC. Sperm motility parameters were obtained with a computer-assisted sperm analysis (CASA) system. Sperm DNA fragmentation (SDF) was assessed using the sperm chromatin dispersion test. Both low and high H2 O2 concentration groups showed lower motility than control groups. Progressive motility of spermatozoa incubated in the H group dropped rapidly in comparison with other groups. Regarding SDF, the control and L groups had significantly lower values than the H group (25.0% and 31.6% vs. 48.1% fragmented sperm for C, L, and H groups, respectively; p < 0.05). Sperm motility, mostly progressive motility, decreased as H2 O2 concentration increased, mainly when time after sperm activation increased. SDF increased as the H2 O2 concentration increased. However, measurements of the halo area did not agree with the subjective SDF rate.

A new technique for analysis of human sperm morphology in unstained cells from raw semen.

Sperm morphology analysis is a fundamental component of semen analysis, but its real significance has been clouded by the plethora of techniques used for its evaluation. Most involve different fixation and staining procedures that induce artefacts. Herein we describe Trumorph (Proiser R+D, Paterna, Spain), a new method for sperm morphology analysis based on examination of wet preparations of spermatozoa immobilised, after a short 60°C shock, in narrow chambers and examined by negative phase contrast microscopy. A range of morphological forms was observed, similar to those found using conventional fixed and stained preparations, but other forms were also found, distinguishable only by the optics used. The ease of preparation makes the Trumorph a robust method applicable for the analysis of living unmodified spermatozoa in a range of situations. Subsequent studies on well-characterised samples are required to describe the morphology of spermatozoa with fertilising potential.

Optimization of human semen analysis using CASA-Mot technology.

The purpose of this study is to investigate the optimal framerate (FR) and the use of different counting chambers for improving CASA-Mot technology use in Andrology. Images were captured at 500 fps, then segmented and analyzed in several ranges of FRs (from 25 to 250) to define the asymptotic point that as an optimal FR. This work was replicated using counting chambers based in capillarity (disposable) or drop displacement (reusable) to study their effects on the motility results and kinematic values of the samples under the different experimental conditions. The α value (asymptote corresponding to FRo) of the exponential curve was 150.23 fps, corresponding to a VCL of 130.58 mm/s, far from the value of 98.89 mm/s corresponding to 50 fps (the highest FR used by most current CASA-Mot systems). Our results have shown that, when using reusable counting chambers, type and depth have influence. In addition, different results were obtained depending on the area of image captured inside the different counting chamber types. To have reliable results in human sperm kinematic studies, almost 150 fps should be used for capturing and analyzing and differences between chambers should be considered by sampling from different areas, to obtain a representative value of the whole sample.

Are There Differences between Methods Used for the Objective Estimation of Boar Sperm Concentration and Motility?

Artificial insemination in the swine industry, as in other species, demands adequate semen handling and accurate evaluation for the preparation of seminal doses. Sperm concentration and motility estimates are part of the semen evaluation process and are considered important for maximizing the yield of doses for insemination. In this study, methods were examined for their accuracy in the estimation of boar sperm concentration and motility. Assessments of sperm concentration were carried out using iSperm®, ISAS® v1, Open CASA v2, and the Accuread® photometer. Analyses of sperm motility were performed with iSperm®, ISAS® v1, and Open CASA v2 systems. In this study, boar semen samples were collected from 10 healthy males from two genetic lines. There were no relevant differences between sire lines when sperm concentration was assessed. A Bayesian analysis was applied to the four methods used to assess sperm concentration to examine whether there are relevant differences between them. Results suggested differences in the four methods, with a probability of relevance (PR) of 0.86-1.00. The iSperm® method revealed higher concentration values within the highest posterior density region at 95% confidence interval (HPD95%) = 167.0, 224.2 M/mL, whereas Open CASA v2 showed the lowest values, with HPD95% = 99.3, 155.9 M/mL. The iSperm® demonstrated higher reliability in measuring sperm concentration compared to other methods or devices within the given range of confidence. ANOVAs revealed relevant differences in the three methods of motility estimation. Overall, differences in boar sperm concentration and motility estimates were found using various methods, but further studies are needed for better characterization of these differences.

The Importance of Studying Factors That Affect the In Vitro Evaluation of Semen Quality to Predict Potential Fertility in Males.

The presence of sub-fertile or infertile males in farms or artificial insemination (AI) centres has a great impact on the reproductive and economic performance of the livestock industry [...].

Exploration of semen quality analyzed by casa-mot systems of brahman bulls infected with BLV and BHV-1.

Enzootic bovine leukosis virus (BLV) and bovine herpesvirus 1 (BHV-1) are very important infectious agents for the livestock industry worldwide. The present study aimed to explore the association between natural exposure to BLV and BHV-1 with sperm quality analyzed by Computer-Assisted Semen Analysis (CASA) systems. Ten sexually mature Brahman bulls, with sanitary status BLV+/BHV-1+ (n = 2), BLV-/BHV-1+ (n = 6) and BLV-/BHV-1- (n = 2) were evaluated twice, 30 days apart. Results showed that sanitary status of each bull was not associated with semen quality. It was found that the quality of the semen from the second collection was better due to the interruption of sexual rest. The evidence thus revealed that a bull infected with BLV generated good-quality contaminated semen and, therefore, that it is essential to detect contaminated seminal samples to prevent the spread of BLV. A multivariate analysis showed the presence of four sperm subpopulations in Brahman bulls that differ significantly in their kinematic patterns and with respect to sanitary status (P < 0.05), indicating that infection-free and seronegative bulls present the best kinematic parameters, which improved discrimination of sperm quality according to sanitary status. Overall, the analyses indicate that the seropositive-infected bulls with BLV and BHV-1 should be excluded from beef cattle farms.

Effect of incubation and analysis temperatures on sperm kinematics and morphometrics during human semen analysis.

INTRODUCTION: Human semen analysis must be performed after the liquefaction of the ejaculate. This takes place about 30min after ejaculation and samples must be maintained in the lab during this time. The temperatures for this incubation and the final analysis of motility are crucial but seldom taken into account. This study aims to examine the effect of these temperatures on various sperm parameters both manually (sperm count, motility, morphology, viability, chromatin condensation and maturation and DNA fragmentation) and CASA (kinematics and morphometrics, using an ISAS®v1 CASA-Mot and CASA-Morph systems, respectively) analyzed. METHODS: Seminal samples from thirteen donors were incubated for 10min at 37°C followed by additional 20min at either room temperature (RT, 23°C) or 37°C and then examined following WHO 2010 criteria. RESULTS: The data obtained show that there were no significant differences (P>0.05) in the subjective sperm quality parameters with incubation temperature. On the other hand, the head sperm morphometric parameters were significantly higher after room temperature incubation showing, in addition, lower ellipticity (P<0.05). Furthermore, kinematic parameters were evaluated both at RT and 37°C for the two incubation temperatures. In general, the four temperature combinations showed that kinematic parameters followed this order: RT-RT

Human kinematic and morphometric sperm subpopulation analysis using CASA technology: A new approach to spermatozoa classification.

INTRODUCTION: Semen analysis is a clinical method aimed at determining the fertility of a male individual. The traditional subjective method lacks the reliability that can be achieved by computer-assisted sperm analysis (CASA) technology. Unfortunately, this technology has only been used when taking into consideration individually different sperm characteristics. The aim of this work is to present an integrative mathematical approach that considers different seminal variables to establish human sperm subpopulations. METHODS: Samples were obtained from thirteen volunteers via masturbation and were analyzed by the routine subjective method and two objective systems, CASA Motility (CASA-Mot) and CASA Morphology (CASA-Morph). RESULTS: Seminogram variables were reduced to three principal components (PC) showing two subpopulations. Kinematics and morphometric variables each rendered three PCs for four subpopulations. CONCLUSIONS: These results lay the foundations for future studies including different geographical, social, ethnic and age range conditions with the aim of achieving a definitive view of the human semen picture.

Seminal plasma biochemistry and spermatozoa characteristics of Atlantic cod (Gadus morhua L.) of wild and cultivated origin.

Our objectives were to compare spermatozoa activity, morphology, and seminal plasma (SP) biochemistry between wild and cultivated Atlantic cod (Gadus morhua). Swimming velocities of wild cod spermatozoa were significantly faster than those of cultivated males. Wild males had a significantly larger spermatozoa head area, perimeter, and length, while cultivated males had more circular heads. Total monounsaturated fatty acids and the ratio of n-3/n-6 were significantly higher in sperm from wild males, while total n-6 from cultivated males was significantly higher than the wild males. Significantly higher concentrations of the fatty acids C14:0, C16:1n-7, C18:4n-3, C20:1n-11, C20:1n-9, C20:4n-3, C22:1n-11, and C22:6n-3 were observed in wild males, while significantly higher concentrations of C18:2n-6, C20:2n-6, and C22:5n-3 occurred in cultivated males. Osmolality, protein concentration, lactate dehydrogenase and superoxide dismutase activity of SP of wild males were significantly higher than the cultivated males. Antioxidant capacity of SP was significantly higher in cultivated males, while pH and anti-trypsin did not differ between fish origins. Four bands of anti-trypsin activity and nine protein bands were detected in SP. Performing a discriminant function analysis, on morphology and fatty acid data showed significant discrimination between wild and cultivated fish. Results are relevant to breeding programs and aquaculture development.

New Sperm Morphology Analysis in Equids: Trumorph® Vs Eosin-Nigrosin Stain.

The evaluation of the male fertility potential is based on the analysis of the basic spermatic characteristics of concentration, motility and morphology. Thus, the study of sperm morphology is a fundamental element in the seminal analysis, but its real meaning has been biased by the techniques used for its evaluation. These techniques involve dehydration phases and subsequent staining, which involves the production of artifacts. The aim of the study is to compare two methods for equid semen morphology evaluation, Trumorph® using living sperm vs. eosin-nigrosine stain. A total of 49 ejaculates from stallions and donkeys were used. After semen collection and dilution, an aliquot was placed on the slide and introduced in the Trumorph® device. Then observation was made with a 40x objective and negative phase-contrast microscope. Another aliquot was stained using eosin-nigrosine stain and viewed using 100× magnification. Well-formed sperm were observed, and different abnormalities were identified using Trumorph®. The use of eosin-nigrosin staining method and Trumorph® led to the same results and both techniques can be used for stallion and donkey sperm morphological analysis. However, considering the fact that Trumorph® uses living sperm helps prevent sperm cell alteration during sample preparation. Therefore, Trumorph® can be a good alternative to the conventional staining method, which provides a quick test on live sperm.