Computer-assisted Sperm Analysis System Research Articles

Effects of Bisphenol A on redox balance in red blood and sperm cells and spermatic quality in zebrafish Danio rerio.

Bisphenol-A (BPA) is a potential endocrine disruptor besides being associated with oxidative damage in several vertebrate classes. In the present study we investigated oxidative effects in erythrocytes and sperm cells as well as spermatic quality in Danio rerio exposed to 14 days at BPA concentrations of 2, 10 and 100 μg/L. Organelles structure, reactive species of oxygen (ROS) and lipoperoxidation (LPO) on erythrocytes and sperm cells were measured by flow cytometry and spermatic parameters were analyzed by the computer-assisted sperm analysis (CASA) system. For both cell types, when compared with control BPA treatment induced a significant increase in ROS and LPO production causing the membrane fluidity disorder, loss of membrane integrity and mitochondrial functionality. Furthermore, it was found a significant increase in DNA fragmentation in erythrocytes of zebrafish BPA exposed. Regarding the spermatic quality, results showed lower sperm motility in animals exposed to BPA, and alterations on velocity parameters of spermatozoa. Thus, the present study concludes that BPA affects the oxidative balance of both cell types, and that can directly affects the reproductive success of the adult Danio rerio. The sensitivity of erythrocytes to oxidative damage induced by BPA was similar to sperm cells, indicating a potential use of blood cells as indicators of oxidative damage present in fish sperm.

Ramifications of protease-based liquefaction of camel semen on physical, kinematic and surface glyco-pattern of cryopreserved spermatozoa

The efficiency of incorporating different proteases in the diluent for reducing camel semen viscosity, and subsequent ramifications on morpho-functional and glycan surface properties of cryopreserved spermatozoa were investigated. Ejaculates (n = 48) were collected from three adult camels, Camelus dromedarius, during the breeding season (January - March). A portion of each raw ejaculate was evaluated for sperm physical and morphological traits, whereas the other portion was divided into three aliquots assigned for the following liquefaction treatments: control (untreated), 0.1 mg/mL papain or 5 U/mL bromelain. All samples were diluted with Tris-lactose diluent containing the anti-enzyme E-64 to neutralize both proteases before being processed for cryopreservation. Post-thaw physical and kinematic properties of spermatozoa were analyzed using a computer-assisted sperm analysis (CASA) system. The sperm surface glycocalyx pattern was evaluated with a panel of 14 fluorescent lectins. Although bromelain was more effective in elimination of semen viscosity, there was a negative correlation between bromelain supplementation and values for the variables: normal sperm, intact acrosome and intact sperm cell membrane. Bromelain supplementation, compared to papain-treated and control samples, was positively correlated with secondary sperm abnormalities, increased straight-line velocity (VSL, μm/s) and straightness (%) of spermatozoa. Results from the glycan analysis indicated that both proteases did not affect the N-linked glycan content of the entire sperm surface, whereas the treatment with proteases induced little change in N-acetylgalactosamine and fucose terminating glycans in the tail region of the sperm. Functional studies are needed to evaluate the sperm fertility rates of bromelain- and papain-treated semen for application in camel assisted reproductive technologies.

Alpha‐lipoic acid minimises reactive oxygen species‐induced damages during sperm processing

Shearing forces during sperm preparation for assisted reproduction techniques may lead to excessive production of reactive oxygen species (ROS) which may have an unpleasant effect on embryonic development. In the current study, we assessed the effect of alpha-lipoic acid (ALA) on ROS-induced damages during sperm preparation process. Semen samples were collected from 15 normozoospermic men. Each semen sample was divided into two parts; one part was washed and centrifuged with sperm washing medium plus 0.02mM ALA. Then, sperm pellet was diluted and incubated for 1hr at 37°C in sperm washing media in the absence (ALA-) or presence of 0.02mM ALA (ALA+). The second part was washed and centrifuged with sperm washing media in the absence of ALA, and then, sperm pellet was incubated for 1hr at 37°C in sperm washing media in the absence (ALA-) or presence of 0.02mM ALA (ALA+). Sperm viability, motility, intracellular oxidative stress and DNA fragmentation were assessed by eosin-nigrosin, computer-assisted sperm analysis system, H2 DCFDA staining and acridine orange staining respectively. Our results showed that addition of ALA as a fat- and water-soluble antioxidant to sperm washing media maintains sperm viability and motility by reduction in ROS production and can also protect sperm DNA integrity.

G1-S4 or G2-S16 carbosilane dendrimer in combination with Platycodin D as a promising vaginal microbicide candidate with contraceptive activity.

PurposeHIV-1 and herpes simplex virus type-2 (HSV-2) represent two of the most relevant sexually transmitted diseases (STDs) worldwide. Moreover, each year there are >200 million pregnancies worldwide, and more than half are unintended. Continued high rates of unintended pregnancies and spread of HIV-1 and HSV-2 require new approaches to address these problems. G1-S4 and G2-S16 dendrimers emerge as potential candidates for the development of a topical microbicide due to their safety and effectivity against HIV-1 and HSV-2 infection, both in vitro and in vivo. Our goal is to develop a dual topical microbicide to prevent the transmission of STDs and unintended pregnancies. Platycodin D (PD) was selected for its great spermicidal activity, topical application, and biocompatibility.Materials and methodsToxicology and inhibitory profile of G1-S4/PD and G2-S16/PD were evaluated in vitro and in vivo. Spermicidal activity was assessed by a computer-assisted sperm analysis system (CASA).ResultsG1-S4/PD and G2-S16/PD presented >95% of HIV-1 inhibition in TZM-bl cells and peripheral blood mononuclear cells. CASA assessment determined that 0.25 mM of PD with therapeutic concentrations of G1-S4 or G2-S16 was able to induce 100% immobilization of the sperm in 30 seconds. To evaluate the toxicity in vivo, a vaginal toxicity assay was performed in BALB/c mice. No significant changes or damage to the vaginal epithelium after 7 consecutive days of application were observed.ConclusionOur data indicate that G1-S4/PD and G2-S16/PD combinations are promising candidates to be developed for vaginal microbicides with contraceptive activity.

A Novel Computer Assisted Sperm Analyzer for Assessment of Spermatozoa Motility in Fish; BASA-Sperm Aqua

This study was conducted to determine the working principle and operability of the BASA-Sperm Aqua module software of the newly developed computer-assisted sperm analysis system (BASA) for the evaluation of spermatozoa motility in fish. Sperm samples of trout ( Oncorhynchus mykiss ) species were examined for this purpose. Sperm motility parameters such as VSL (μm/s), VCL (μm/s), VAP (μm/s), LIN (%), BCF (Hz), ALH (μm), ve MAD ( o ) were examined. The investigated parameters were compared with data which analyzed in similar computer systems and published in international manuscripts. Finally, the BASA-Sperm Aqua has been found to be a software that performs its functions very quickly and practical and produces accurate and understandable results in the determining sperm quality parameters of fish.

OpenCASA: A new open-source and scalable tool for sperm quality analysis.

In the field of assisted reproductive techniques (ART), computer-assisted sperm analysis (CASA) systems have proved their utility and potential for assessing sperm quality, improving the prediction of the fertility potential of a seminal dose. Although most laboratories and scientific centers use commercial systems, in the recent years certain free and open-source alternatives have emerged that can reduce the costs that research groups have to face. However, these open-source alternatives cannot analyze sperm kinetic responses to different stimuli, such as chemotaxis, thermotaxis or rheotaxis. In addition, the programs released to date have not usually been designed to encourage the scalability and the continuity of software development. We have developed an open-source CASA software, called OpenCASA, which allows users to study three classical sperm quality parameters: motility, morphometry and membrane integrity (viability) and offers the possibility of analyzing the guided movement response of spermatozoa to different stimuli (useful for chemotaxis, thermotaxis or rheotaxis studies) or different motile cells such as bacteria, using a single software. This software has been released in a Version Control System at Github. This platform will allow researchers not only to download the software but also to be involved in and contribute to further developments. Additionally, a Google group has been created to allow the research community to interact and discuss OpenCASA. For validation of the OpenCASA software, we analysed different simulated sperm populations (for chemotaxis module) and evaluated 36 ejaculates obtained from 12 fertile rams using other sperm analysis systems (for motility, membrane integrity and morphology modules). The results were compared with those obtained by Open-CASA using the Pearson’s correlation and Bland-Altman tests, obtaining a high level of correlation in all parameters and a good agreement between the different used methods and the OpenCASA. With this work, we propose an open-source project oriented to the development of a new software application for sperm quality analysis. This proposed software will use a minimally centralized infrastructure to allow the continued development of its modules by the research community.

Capacitation of mouse sperm is modulated by gamma-aminobutyric acid (GABA) concentration.

In mammals, ejaculated sperm acquire their fertilizing ability during migration through the female reproductive tract, which secretes several factors that contribute to sperm capacitation. Gamma-aminobutyric acid (GABA) is a well-known neurotransmitter in the central nervous system, but additionally enhances the sperm acrosome reaction in the rat and cow. However, the detailed effects of GABA concentration on sperm function remain unclear. In this study, we detected the presence of the GABA type A receptor (GABA A) in mouse epididymal sperm by western blot analysis and in the sperm acrosome by immunocytochemistry. We also investigated the effects of GABA on sperm fertilizing ability. We found that GABA facilitated the tyrosine phosphorylation of sperm proteins, which is an index of sperm capacitation. GABA also promoted the acrosome reaction, which was suppressed by a selective GABA A receptor antagonist. We then found that the effective GABA concentration for the acrosome reaction corresponds to sperm concentration, but we did not detect any marked effect of GABA on sperm motility using a computer-assisted sperm analysis system. Using immunohistochemistry, we also detected GABA expression in the epithelia of the mouse uterus and oviduct. Furthermore, we found that the mRNA levels of glutamate decarboxylase (Gad), which generates GABA from L-glutamate, were higher in the oviduct than in the uterus, and that Gad mRNA levels were higher at estrus than at the diestrus stage. These results indicate that the GABA concentration can act as a modulator of the acrosome reaction and sperm capacitation in the female reproductive tract.

Effect of addition of buserelin acetate to the extender on motility and viability of bovine spermatozoa

The present study was aimed to determine the effect of GnRH analog (buserelin acetate) on the quality of bovine spermatozoa stored at 16° C for 24 h. Semen collected in the summer season from June to September from healthy Polish Holstein–Friesian bulls. Ejaculates were centrifuged, divided and diluted to the final concentration of 240 × 106 spermatozoa/mL using animal protein–free commercial BIOXcell® extender (IMV Technologies, L’aigle, France) (Control) or with BIOXcell® extender supplemented with buserelin acetate and stored 0, 8 and 24 h. Sperm motility parameters analysis was performed using a computer-assisted sperm analysis (CASA) system. The viability of spermatozoa was performed using flow cytometer. The addition of buserelin acetate to BIOXcell® extender did positively affect the total motility (was higher in the observed samples with the addition of 2 µg/mL and 4 µg/mL than in the control group), progressive motile (forward progressing sperm was significantly increased (p < 0.05) over the control group at the 0 h and 8 h of incubation following the supplementation of 2, 4 and 8 μg/mL buserelin acetate) and viability of spermatozoa (the number of live spermatozoa was significantly higher (p < 0.05) in 2 µg/mL and 4 µg/mL samples with buserelin acetate at 8th hour of incubation and in sample with 4 µg/mL at 24th hour of incubation compared to the control group). We recommend adding 4 µg/mL to the extender to improve the quality of bovine semen.

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 2-3years and weighted 45.0±2.0kg 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 0.4mM caffeine. All diluted semen specimens were evaluated for physical characteristics immediately after dilution (T0 ) and throughout preservation period of 48hr 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 (0.1mM) or medium (0.2mM) 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.

Comparison of European eel sperm cryopreservation protocols with standardization as a target

The critical situation of the European eel (Anguilla anguilla) has urged the development of sperm cryopreservation protocols for reproduction in captivity and cryobanking. In the last years, two research groups have developed their own protocols in Spain and Hungary with positive results, but difficult to compare.Here, a series of experiments were conducted to test the quality of thawed sperm after using both protocols, determining which of them produce the best results and aiming for standardization. The quality of thawed sperm was assessed by studying the motility and kinetic values of thawed sperm from both cryopreservation protocols using a computer-assisted sperm analysis (CASA-Mot) system. In addition, a viability analysis was performed using flow cytometry to test if the cryoprotectants or the freezing-thawing process led to a reduction in spermatozoa survival. Furthermore, since during cryopreservation the sperm was treated with methylated cryoprotectants (DMSO or methanol) that may induce epigenetic changes in the sperm DNA (cytosine methylation) and could affect the offspring, we conducted a luminometric methylation assay (LUMA) to study the DNA methylation levels induced by both protocols.In this work, all the above-mentioned parameters were analyzed in fresh and frozen-thawed sperm samples. Our results showed that thawed sperm samples from both protocols presented lower sperm motility and velocity, and lower percentage of live cells than those shown in fresh sperm samples. Furthermore, sperm samples from the methanol based protocol showed significantly higher motility, velocity and percentage of live spermatozoa than the same sperm samples treated with the DMSO based protocol. In addition, the DMSO based protocol induced a hypomethylation of sperm DNA compared to fresh samples whereas the methanol based protocol did not alter sperm DNA methylation level. Our results indicate that the methanol based protocol is a more suitable protocol that preserves better the motility and genetic qualities of the European eel sperm.

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 50µM (Low; L) and 200µM (High; H) H2 O2 , respectively, for 20min 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.

Influence of omega‐3 incorporation in sperm preservation medium on physical and kinematic properties of chilled and cryopreserved ram spermatozoa

Two experiments were carried out to investigate the efficiency of supplementing sperm preservation medium with omega-3 polyunsaturated fatty acids on improving liquid-chilled storage and cryopreservation capacity of ram spermatozoa. Ejaculates (n=100) were collected from five adult rams, Ovis aries, by an artificial vagina twice weekly throughout the period February-April, 2017. After initial evaluation, ejaculates of each collection session from the same males were pooled, diluted (1:10) with Tris-citric acid egg yolk extender, and were further split into five aliquots using a split-sample technique. The first aliquot served as control (omega-free), whereas the other four portions were supplemented with 0.1, 0.2, 0.3 or 0.4mM omega-3, respectively (T0 ). Thereafter, the diluted specimens were stored at 4°C for 48hr, during which sperm physical and morphometric properties were evaluated along with oxidative stress indices (T24 , T48 ). Omega-3 levels that efficiently mitigated the detrimental effects of chilled preservation, and maintained preservation aptitude of spermatozoa were further investigated for sperm cryosurvival against control (untreated). Post-thaw physical and kinematic properties of spermatozoa, in all groups, were objectively evaluated by a computer-assisted sperm analysis (CASA) system. The results showed that, at 48hr of chilled storage, supplementing preservation medium with 0.4mM omega-3 was positively correlated (p<0.01) with each of progressive motility, live sperm, intact acrosome and intact cell membrane (r=0.83, 0.85, 0.85, 0.89, respectively). Furthermore, a positive correlation (p<0.01) was observed between inclusion of omega-3 in cryopreservation medium and each of post-thaw total sperm motility, progressive motility, live sperm, normal sperm, intact acrosome, intact cell membrane, VCL, VSL, VAP, ALH and STR (r=0.76, 0.84, 0.79, 0.90, 0.89, 0.91, 0.61, 0.73, 0.65, 0.78 and 0.60, respectively). These results accentuate efficiency of supplementing the diluent with omega-3 fatty acids on improving chilled and cryopreservation aptitude of ram spermatozoa.

Candidates for reproductive biomarkers: Protein phosphorylation and acetylation positively related to selected parameters of boar spermatozoa quality

Protein post-translational modifications (PTMs) have been reported to be involved in various functions of sperm, yet the exact correlation between PTMs and sperm motility remains unclear. With the goal of contributing to this subject, motility variables were measured by computer-assisted sperm analysis system (CASA), and the amount of PTMs were evaluated using Western blot and immunofluorescence in fresh sperm and liquid stored sperm. Results of the present study indicate that the amount of the phosphorylated substrates of PKA (P-PKAs), protein tyrosine phosphorylation (PTP), global protein acetylation (Pan-Kac) and α-tubulin acetylation (Tub-Kac) was greater in sperm of fresh semen samples with relatively greater motility than in sperm of fresh semen samples with relatively lesser motility. Similarly, the amounts of phosphorylation and acetylation gradually decreased with the reduction in the motility of sperm in liquid stored semen samples. Interestingly, the P-PKAs (r = 0. 634, P < 0. 01) and Pan-Kac (r = 0. 380, P < 0. 05) were positively correlated with sperm motility in fresh semen, whereas only P-PKAs (r = 0.607, P < 0. 01) were positively correlated with sperm motility during liquid storage. Furthermore, it is noteworthy that the amounts of phosphorylation and acetylation were positively correlated with the acrosome integrity and mitochondrial membrane potential of fresh sperm and liquid stored sperm. This study is the first to explore the correlation between PTMs and sperm motility, and it may provide a new reproductive biomarker for evaluating semen quality and predicting sperm capacity for enhancing reproductive performance, which is meaningful for the pig breeding industry.

Liquid Storage of Boar Semen Using Commercial Extenders

The research was focused on the modern evaluation of boar semen parameters, using computer assisted sperm analysis (CASA), before and after liquid storage at 15ºC. Semen was collected from 15 sexually mature boars by manual stimulation. Macroscopical and microscopical evaluation of semen was performed, followed by a detailed CASA analysis of all ejaculates. Subsequently, semen was diluted using 4 different extenders (Semtest, Androstar, MIII and Cronos) and stored at 15ºC for 24 hours. Next, evaluation of progressive motility, total motility and viability was performed, using the same CASA system. All experiments were performed in triplicates and results were statistically analyzed. The average progressive motility after 24 hours was as follows: 69.56 ± 6.38 for MIII, 65.92% ± 2.63 for Semtest, 67.07% ± 5.58 for Androstar Plus and 68.93% ± 3.40 for Cronos. The viability results after 24 hours were: 86.34% ± 1.38 for Semtest extender, 93.55% ± 3.38% for Androstrar Plus, 89.19% ± 3.42 for MIII and 91.35% ± 2.37 for Cronos. The findings of this study suggest that the use of commercial extenders for short-term storage of swine semen is important in order to increase sperm longevity with minimal sperm function deterioration.

Subjective and objective assessment of fish sperm motility: when the technique and technicians matter.

Fish sperm motility is nowadays considered the best sperm quality biomarker in fish, and can be evaluated both by subjective and computerized methods. With the aim to compare the precision and accuracy of both techniques, fish sperm samples were assessed by subjective methods and by a computer-assisted sperm analysis (CASA-Mot) system, and simultaneously by three different technicians with different degrees of expertise on the sperm quality analysis. Statistical dispersion parameters (CV, coefficient of variation; and RG, range) were estimated in order to determine the precision and accuracy of the techniques and the influence of laboratory staff on sperm motion assessments. Concerning precision, there were not much significant differences between the technical support staff (high, medium, and low experimented technician), and statistical dispersion parameters were quite similar between them independent of the technique used and the sperm motility class analyzed. However, concerning accuracy, experimented technician reported subjective motility values very closed to the values provided by the CASA-Mot system, only 10 percentage points away from the data provided by a CASA-Mot system. However, medium and low experimented technicians often overestimate the CASA-Mot values, and amplitudes up to 30 percentage points were detected in several sperm assessments. To sum up, both the technique (subjective or objective) and the technician (degree of expertise) became key factors in order to reach accurate motility estimations, so the use of both qualified staff and novel CASA-Mot systems seems to be a critical requirement for obtaining satisfying results in fish species with similar motility patterns.

Effect of carp pituitary homogenate (CPH) and sGnRHa (Ovaprim) on northern pike (Esox lucius) spermiation stimulation and its effect on quantity and quality of sperm

The effect of carp pituitary homogenate (CPH, n = 7) at a dose of 2.0 mg kg−1 and (D-Arg6, Pro9NET)-sGnRH + domperidone (Ovaprim, n = 7) at a dose of 0.5 ml kg−1 in northern pike (Esox lucius) sperm maturation under controlled conditions was examined. On the control group, 0.9% NaCl at a dose of 1.0 ml kg−1 (n = 7) was used. Sperm was collected 48 h following injection. Sperm quantity (total sperm volume, total sperm production and sperm concentration), sperm motility using the Computer-assisted sperm analysis (CASA) system and sperm and seminal plasma quality (sperm pH, seminal plasma osmotic pressure and seminal plasma pH) were determined for each male separately in each group. The results of the present study demonstrate that hormonal treatment had a positive effect on sperm maturation in northern pike, regardless of the hormonal preparation used. However, even though no differences were found in total sperm volume and total sperm production between fish injected with either CPH or Ovaprim, it should be highlighted that the highest progressive motile sperm (PRG), straight-linear velocity (VSL) and movement linearity (LIN) was noted in fish treated with Ovaprim. It was also found that it is possible to collect sperm from non-hormonally manipulated fish. However, in such a case, only a small sperm volume (around 0.1 ml) characterised by lowered PRG (below 40%) was noted. Considering the fact that only after Ovaprim application sperm motility with progressive movement of sperm above 50% was observed, the hatchery practice of collection of sperm 48 h after its application (at a dose of 0.5 ml kg−1) may be recommended.

Sperm kinematics and subpopulational responses during the cryopreservation process in caprine ejaculates

Sperm cryopreservation in goats has been a challenge for many years due to the detrimental effects of seminal plasma enzymes produced by the bulbo-urethral glands which catalyse the hydrolysis of lecithins in egg yolk to fatty acids and lysolecithins which are deleterious to spermatozoa. This fact implies to carry out additional processing steps during sperm cryopreservation for seminal plasma removal triggering different sperm responses which may affect sperm functionality. The objective of the present study was to determine specific sperm subpopulation responses in different handling steps during the cryopreservation process by using functional sperm kinematic descriptors in caprine ejaculates. Buck ejaculates (n = 40) were analysed for sperm concentration, viability, morphology and acrosome integrity. Moreover, sperm motility was assessed using a computer-assisted sperm analysis (CASA) system after five different handling steps (fresh sperm, 1st washing, 2nd washing, cooling and frozen-thawed sperm) during a standard cryopreservation protocol for goat semen. The results were analysed using Principal Component Analysis (PCA) and multivariate clustering procedures to establish the relationship between the distribution of the subpopulations found and the functional sperm motility in each step. Except for the 1st and 4th steps, four sperm kinematic subpopulations were observed explaining more than 75% of the variance. Based on velocity and linearity parameters and the subpopulations disclosed, the kinematic response varies among processing steps modifying sperm movement trajectories in a subpopulation-specific and handling step-dependent manner (p < 0.001). The predominant motile subpopulation in freshly ejaculated buck sperm had very fast velocity characteristics and a non-linear trajectory (41.1%). Washing buck sperm twice altered the subpopulation structure as well as cooling which resulted in a dramatic reduction in sperm velocities (p < 0.01). Frozen-thawed spermatozoa showed similar characteristics to cooled sperm except there was a further increase in linearity with a large proportion of sperm attributed to new slow, linear cluster (32.5%). In conclusion, this study confirms the variability and heterogeneity of goat sperm kinematic patterns throughout the cryopreservation process and suggests that the predominant motility pattern (assayed in vitro via CASA) of high quality spermatozoa might be typified by high speed and a non-linear trajectory. The relationships among the number and distribution of sperm subpopulations and the different handling steps were particularlly relevant, specially after the cooling and the post-thawing steps, when effects derived from these critical handling steps were evident and altered drastically the sperm motion patterns.

Development of an in vitro toxicological test system based on zebrafish (Danio rerio) sperm analysis.

The effect of seven heavy metals on the motility parameter of zebrafish sperm was tested in order to develop an in vitro toxicological test system as an alternative to live animal testing. In vitro test systems are currently preferred in ecotoxicology due to their practical and ethical advantages and fish sperm can be a suitable model. A number of studies had been carried out previously on this topic, but the described methods had not been standardized in numerous aspects (donor species, measured endpoint, etc.). In this study, heavy metals (mercury, arsenic, chromium, zinc, nickel, copper, cadmium) were used as reference toxicants with known toxicity to develop a standardized fish sperm in vitro assay. The tested concentrations were determined based on preliminary range finding tests. The endpoints were progressive motility (PMOT, %), curvilinear velocity (VCL, μm/s), and linearity (LIN, %) measured by a computer-assisted sperm analysis (CASA) system. According to our results, PMOT was the most sensitive of the three investigated parameters: dose-response curves were observed for each metal at relatively low concentrations. VCL values were less sensitive: higher concentrations were needed to observe changes. Of the three parameters, LIN was the least affected: dose-response relationship was observed only in the case of mercury (e.g., lowest observed effect concentration (LOEC) of Hg at 120min: 1mg/L for PMOT, 2.5mg/L for VCL, 5mg/L for LIN; LOEC of Cu at 120min: 1mg/L for PMOT, 5mg/L for VCL, any for LIN). The order of toxicity as determined by PMOT was as follows: Hg2+ > As3+ > Cd2+ > Cu2+ > Zn2+ > Cr3+ > Ni2+. In conclusion, we found that PMOT of zebrafish sperm was an accurate and fast bioindicator of heavy metal load. Sperm analysis can be adopted to estimate the possible toxic effects of various chemicals in vitro. Future investigations should concentrate on the applicability of this assay to other contaminants (e.g., organic pollutants).

Effects of the supplementation with an high‐polyphenols extra‐virgin olive oil on kinetic sperm features and seminal plasma oxidative status in healthy dogs

The aim of this work was to evaluate the effects of the supplementation of two extra-virgin olive oils (EVOO) having different polyphenols content, on canine spermatozoa kinetic parameters and seminal plasma oxidative status. The study was conducted on 12 clinically healthy dogs of different breeds (2-7years, 5-48kg of body weight) divided into two groups: an experimental group supplemented with EVOO (Coratina cultivar) high in polyphenols (H-P) and a control group fed EVOO (Cima di Bitonto cultivar) low in polyphenols (L-P). The oil was daily administered per os (1ml/3kg BW) before meal. Semen collection was made twice at 15days distance (D01 and D02 ) and then at 30 (D30), 60 (D60) and 90 (D90) days. Semen concentration and kinetic parameters were measured using computer-assisted sperm analysis (CASA) system to evaluate: sperm total count, sperm motile (MOT%), progressive motility (PROGR%) and its fractions, straight-line velocity (VSL, μm/s), curvilinear velocity (VCL, μm/s), average path velocity (VAP, μm/s), amplitude of lateral head displacement (ALH, μm), beat cross frequency (BCF, Hz), straightness (STR%) and linearity (LIN%). On seminal plasma, reactive oxygen species (ROS) and biological antioxidant potential (BAP) were tested. From findings, no differences were found for sperm MOT, VSL, VCL, VAP, ALH, BCF, STR, LIN and BAP. A gradual enhancement of PROGR% was observed in H-P group (p<.01). The ROS levels were higher in dogs H-P compared to the other group (p<.05). In conclusion, our results highlight the positive effects of EVOO polyphenols on sperm PROGR% in healthy dogs.

Effect of Low-Sodium, Choline-Based Semen Diluent on Viability of Bovine Spermatozoa Stored at 4&amp;amp;deg;C

This study evaluated sperm viability over time, after dilution and refrigerator storage of fresh semen extended in either synthetic cauda epididymal plasma (CEP2), or in a low sodium medium (CJ2) supplemented with either AlbuMAX or egg yolk. Semen collected weekly for 4 weeks from 4 bulls and assigned within bulls, across treatments. After extension in either CEP2, or CJ2 containing either egg yolk or AlbuMAX, semen was cooled to 4°C, and evaluated for 7 days. A computer assisted sperm analysis (CASA) system was used for sperm evaluation. Particular emphasis was placed on sperm motility since it is the single most important sperm parameter influencing bull fertility. Total and progressive motility of sperm in CEP2 and CJ2-AlbuMAX were similar (P = 0.85 and P = 0.23, respectively), but both were lower (P < 0.01) when compared to CJ2-yolk. Fewer sperm had rapid motility in CEP2 and CJ2-AlbuMAX compared to CJ2-yolk (P < 0.01). Sperm straightness and linearity were greater in CJ2-AlbuMAX and CJ2-yolk than in CEP2 (P < 0.01). Mean velocity (VAP) and linear velocity (VSL) were greater (P < 0.01) in CJ2-AlbuMAX than either CEP2 or CJ2-yolk. The calculated curvilinear velocity (VCL) of spermatozoa in CEP2 was lower than CJ2-AlbuMAX (P = 0.01), but similar with CJ2-yolk (P = 0.54). Overall, every sperm parameter measured by the CASA system was equal to or higher for sperm stored 7 days in CJ2 medium as compared with CEP2. The CJ2 extender supplemented with egg yolk is a viable alternative for storing fresh bovine semen.