Sperm Motility Patterns Research Articles

Roles of Na +/K +-dependent ATPase, Na +/H + antiporter and GLUT hexose transporters in the cryosurvival of dog spermatozoa: Effects on viability, acrosome state and motile sperm subpopulation structure

To assess the roles of Na +/K +-dependent ATPase, Na +/H + antiporter and GLUT hexose transporters in the cryosurvival of dog sperm, semen samples were frozen in a standard freezing medium supplemented with the specific inhibitors of these factors ouabain, amiloride or phloretin, respectively. The presence of ouabain did not counteract the effects of freeze-thawing on the percentages of motile sperm and altered acrosomes, although a small recovery effect was observed in motility parameter means. Amiloride had a similar effect, although motility was more intensely recovered. Phloretin significantly (P < 0.05) impaired viability when added at a maximal concentration of 10 −4 M (57.3 ± 5.1% vs 76.5 ± 5.7% in cells frozen without inhibitors), although partial recovery of motility parameters was also observed. These effects were accompanied with specific changes in both motility parameters and the percentages of motile sperm in each of the 4 subpopulations comprising the motile sperm population of the ejaculate. Our findings indicate a role for Na +/K +-dependent ATPase and Na +/H + antiporter in the mechanisms involved in determining specific sperm motility patterns in response to freeze-thawing, although neither pump seems to be important for the resistance of cell membrane structures to freezing-thawing. In addition, a role for GLUTs in regulating water exchange in dog sperm during freeze-thawing seems unlikely. In contrast, the precise structure of dog sperm in terms of its motile subpopulations was found to condition both cryosurvival and sperm cell sensitivity to the inhibitors used.

Effect of platelet activating factor (PAF) supplementation in semen extender on viability and ATP content of cryopreserved canine spermatozoa

The aim of this study was to investigate the effect of platelet activating factor (PAF) on the quality characteristics of cryopreserved canine spermatozoa. Cryopreserved semen of 5 mixed-breed dogs was treated with different concentrations of exogenous PAF (1 x 10(-3) M, 1 x 10(-4) M, 1 x 10(-5) M and 1 x 10(-6) M) and examined at different time intervals (0, 30, 60 and 120 min). Cryopreserved semen treated without PAF was used as the control. Sperm quality was evaluated for motility (computer-assisted semen analysis, CASA), mitochondrial function (JC-1/PI assay) and plasma membrane integrity (SYBR-14/PI assay and Hoechst 33258). Also, ATP content of spermatozoa was determined using a bioluminescence assay. Treatment of cryopreserved semen with 1 x 10(-3) M PAF at 120 min of incubation resulted in significantly higher total sperm motility compared with the control. It was observed that PAF-improved total sperm motility was concurrent with enhanced sperm motility patterns after treatment of cryopreserved semen. Treatment of cryopreserved semen with PAF did not improve either sperm mitochondrial function or plasma membrane integrity, as monitored by different fluorescent membrane markers. Furthermore, ATP content of cryopreserved spermatozoa was significantly higher when PAF was used at a concentration of 1 x 10(-3) M compared with the control and other PAF treatments, regardless of the incubation time. The findings of this study indicated that treatment with 1 x 10(-3) M PAF at 120 min of incubation rendered better quality of cryopreserved canine semen, which was associated with improved sperm motility parameters and ATP content. It can be suggested that exogenous PAF addition is beneficial as a supplement for canine semen extender used for cryopreservation.

A Multiclass Support Vector Machine Model for the Identification of Sperm Motility Patterns in Heterogeneous Populations.

Sperm motility and the capacity to undergo hyperactivation in the female reproductive tract are requirements for normal fertilization in mammals. Quantitative analyses of motility in response to perturbations that modulate sperm metabolism or key signaling events would be facilitated by a method that quickly and accurately distinguishes different types of motility in large populations of sperm. To that end, we generated a multiclass support vector machine (SVM) model that identifies hyperactivated sperm and four other distinct patterns of sperm motility within a heterogeneous population. Our multiclass model is based on 2,434 sperm tracks from twelve CD1 mice that were captured using a Hamilton-Thorne CEROS instrument for computer-assisted sperm analysis (CASA) and visually classified into one of six distinct motility groups: progressive, intermediate, hyperactivated, slow, weakly motile, and bad (noise). The CASA parameters from these classified tracks were used to develop a series of SVM equations using established algorithms, yielding a model that identifies uncharacterized tracks with an overall accuracy of 86%. The model incorporates these equations into a binary decision tree that sequentially sorts tracks into distinct populations. Five standard CASA measurements are factored into each equation, including average path velocity (VAP), straight-line velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). The first equation identifies all CASA tracks as either vigorous or non-vigorous. Vigorous tracks are then classified as progressive, intermediate or hyperactivated, while non-vigorous tracks are classified as slow, weakly motile, or bad. We tested our multiclass model by comparing motility profiles of sperm incubated for 2 h in HTF medium with and without bicarbonate. After capacitation for 90 min in the presence of bicarbonate, 20-30% of sperm tracks were classified as hyperactivated and ~10% as intermediate. Without bicarbonate, >65% of sperm tracks remained progressive throughout the incubation and <2% were classified as hyperactivated or intermediate, as expected. The model also accurately categorized tracks from mice with defects in sperm motility. For example, sperm from mice that lack GAPDHS, an essential sperm glycolytic isozyme, showed no progressive motility and >96% were classified as slow or weakly motile. We also assessed the motility profiles of C57BL6/J, 129S1/SvlmJ, and PWK/PhJ sperm over a 2 h in vitro capacitation period and found significant differences among these strains, both in the proportions of progressive and slow sperm and in the percentage of sperm that undergo hyperactivation. By enabling detailed, quantitative comparisons of motility throughout capacitation, this model will be a valuable tool for assessing potential genetic, biomolecular, and pharmaceutical effects on sperm motility in large, heterogeneous populations. Supported by the Eunice Kennedy Shriver NICHD/NIH through U01 HD60481 and cooperative agreement U54 HD35041 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research. (platform)

Motility and energetic status of Atlantic salmon (Salmo salar L.) sperm after refrigerated storage

Summary Sperm storage in the appropriate diluents brings positive results usually assessed at the level of percent of motile sperm and fertilization rate. Occasionally sperm motility parameters were taken into consideration. The aim of this study was to investigate whether dilution in artificial seminal plasma significantly affects energetic status and motility pattern of Atlantic salmon sperm. The sperm from 11 males was stored separately in two modes on ice (+2–+4°C), for 10 h until examined: one part of milt was stored in thin layer of 5–6 mm under air condition, while the other part was diluted at the ratio 1 : 50 in artificial seminal plasma in a sealed tube. Energetic parameters i.e.: ATP, ADP, AMP, GTP, GDP, GMP, IMP, NAD, NADP, adenosine, guanosine, inosine were determined by HPLC. Motility parameters were measured using CASA. Eight sperm motility parameters were analysed: MOT, VCL, VAP, VSL, LIN, STR, ALH and BCF. After storage the concentrations of ATP, TAN and AEC value of the diluted spermatozoa were significantly (P 0.05). Dilution of salmon milt during short-term storage in artificial seminal plasma is recommended for these species. The technique used in the present study resulted in a significantly better sustenance of the energetic status of spermatozoa and had beneficial effects on the motility parameters at activation such as sperm velocity, linearity and progressivity.

Semen biology of vendace (Coregonus albula L.)

The objective of this study was to describe the morphometry and motility parameters of vendace (Coregonus albula) spermatozoa. Morphometric parameters of vendace sperm head and tail were of values similar to rainbow trout. The effects of pH, sodium, potassium and calcium ion concentrations on computer-assisted sperm analysis (CASA) sperm motility characteristics were tested. Vendace sperm was motile in a wide pH range of 6.0-10.5 with the optimum pH established at 9.0. Increases in potassium and calcium ions caused decreases in the percentage of motile sperm. The CASA parameters and erratic sperm movement pattern of vendace spermatozoa were similar to whitefish (C. lavaretus) sperm motility, suggesting that there is a coregonid-specific sperm motility pattern.

Effect of sugars on characteristics of Boer goat semen after cryopreservation

In order to improve Boer goat semen quality during cryopreservation process, the influence of sugar supplementation on semen characteristics of sperm were investigated. Three experiments were carried out to investigate the effect of (a) addition of two monosaccharides (fructose and glucose) and two disaccharides sugars (trehalose and sucrose) (b) sugar combination (fructose and trehalose, sucrose and trehalose, glucose and trehalose), and control (glucose without trehalose) (c) different concentrations of trehalose on cryopreservation using Tris based extender. The total motility, forward motility, viability, normal spermatozoa, acrosome integrity and membrane integrity were assessed subjectively. Differences were not detected among monosaccharides, but glucose increased ( P < 0.05) sperm forward motility in post-thaw goat semen compared to trehalose or sucrose supplementation. Semen quality did not differ ( P > 0.05) among disaccharide sugar supplementation. Combination of glucose and trehalose significantly improved the characteristics of Boer spermatozoa after cryopreservation ( P < 0.05). Supplementation of trehalose (198.24 mM) into the glucose extender significantly increased total motility, forward motility, live spermatozoa, acrosome integrity and membrane integrity following cryopreservation ( P < 0.05). In conclusion, glucose had the better ability to support Boer sperm motility and movement patterns. Combination of monosaccharide (glucose) and disaccharide (trehalose) improved semen quality following cryopreservation. Trehalose supplementation at the concentration of 198.24 mM to the glucose extender conferred the greater improvement of semen quality for Boer semen cryopreservation.

Rethinking the Relationship Between Hyperactivation and Chemotaxis in Mammalian Sperm1

Hyperactivation, a motility pattern of mammalian sperm in the oviduct, is essential to fertilization. Hyperactivation helps sperm to swim effectively through oviductal mucus, to escape from the sperm reservoir, and to penetrate the cumulus matrix and zona pellucida of the oocyte. There is some evidence that mammalian sperm can undergo chemotaxis; however, the relationship of chemotaxis to hyperactivation is unknown. Ca(2+) signaling is involved in hyperactivation and implicated in chemotaxis as well. In vivo, sperm hyperactivate in the lower oviduct, far from the cumulus-oocyte complex and possibly beyond the influence of chemotactic gradients emanating from the oocyte or cumulus. Thus, sperm are likely to be hyperactivated before sensing chemotactic gradients. Chemotactic signals might modulate hyperactivation to direct sperm toward oocytes as they reach a region of influence. Ca(2+)-directed modulation of hyperactivation is a potential mechanism of this process.

Effect of semen preparation on casa motility results in cryopreserved bull spermatozoa

Computer-assisted sperm analyzers (CASA) have become the standard tool for evaluating sperm motility and kinetic patterns because they provide objective data for thousands of sperm tracks. However, these devices are not ready-to-use and standardization of analytical practices is a fundamental requirement. In this study, we evaluated the effects of some settings, such as frame rate and frames per field, chamber and time of analysis, and samples preparations, including thawing temperature, sperm sample concentration, and media used for dilution, on the kinetic results of bovine frozen-thawed semen using a CASA. In Experiment 1, the frame rate (30–60 frame/s) significantly affected motility parameters, whereas the number of frames per field (30 or 45) did not seem to affect sperm kinetics. In Experiment 2, the thawing protocol affects sperm motility and kinetic parameters. Sperm sample concentration significantly limited the opportunity to perform the analysis and the kinetic results. A concentration of 100 and 50 × 10 6 sperm/mL limited the device's ability to perform the analysis or gave wrong results, whereas 5, 10, 20, and 30 × 10 6 sperm/mL concentrations allowed the analysis to be performed, but with different results (Experiment 3). The medium used for the dilution of the sample, which is fundamental for a correct sperm head detection, affects sperm motility results (Experiment 4). In this study, Makler and Leja chambers were used to perform the semen analysis with CASA devices. The chamber used significantly affected motility results (Experiment 5). The time between chamber loading and analysis affected sperm velocities, regardless of chamber used. Based on results recorded in this study, we propose that the CASA evaluation of motility of bovine frozen-thawed semen using Hamilton-Thorne IVOS 12.3 should be performed using a frame rate of 60 frame/s and 30 frames per field. Semen should be diluted at least at 20 × 10 6 sperm/mL using PBS. Furthermore, it is necessary to consider the type of chamber used and perform the analysis within 1 or 2 min, regardless of the chamber used.

Additional dietary calcium fed to Barred Plymouth Rock roosters reduces blood cholesterol, elevates seminal calcium, and enhances sperm motility, thermo-tolerance and cryosurvivability

Chicken sperm cryosurvivability may be enhanced by manipulating diets fed to roosters because diets modify quality of fresh semen. This study investigated the possibility of added dietary calcium (Ca) improving chicken sperm cryosurvivability due to an elevation in seminal Ca concentration ([Ca 2+]) and a reduction in blood cholesterol (Ch), which may increase fresh sperm quality, thus thermo-tolerance and cryosurvivability. Thirty 21-wk-old Barred Plymouth Rock roosters were fed diets containing 0, 2 or 4% added Ca for 26 wk. Blood [Ca 2+] and Ch in individual males were assessed weekly. Seminal [Ca 2+], Ch and sperm motility at 0, 1, 2 and 3 h after incubation at 41 °C in seminal plasma from males of different groups were evaluated at 28, 29, 30, 31, and 33 wk of age upon pooling the samples per treatment during each collection. Also, sperm motility of fresh pooled semen and of frozen-thawed samples, collected at 34, 35, 36, 37 and 38 wk of age, were analyzed. From 22 wk of age onwards, blood [Ca 2+] was significantly higher and Ch lower in treated males than the control. Seminal [Ca 2+] in treated males was higher than in the control during the sampling period, with 4% group being highest, followed by 2%, and control groups, but seminal Ch was similar among treatments. Regardless of the seminal plasma source used, motility was superior in males fed 2% added Ca, followed by control, and 4% group. Spermatozoa of males fed 2% added Ca had the highest motility in their own seminal plasma or of control males. Also, spermatozoa of all treatment males displayed higher motility in control seminal plasma compared to that of treated males. Frozen-thawed sperm motility pattern was similar to that of fresh semen; with that of 2% group exhibiting superiority to that of control or 4% added Ca-fed males. Indeed, additional dietary Ca elevated seminal [Ca 2+] and lowered blood Ch, which enhanced fresh sperm quality measured as motility, thermo-tolerance and cryosurvivability, especially in 2% group, but not 4% added Ca-fed males.

Effects of permethrin, cypermethrin and 3-phenoxybenzoic acid on rat sperm motility in vitro evaluated with computer-assisted sperm analysis

Pyrethroid pesticides, produced and used worldwide, have been reported to impair male reproductive function by reducing sperm count and sperm motility. They are divided into two types: type I pyrethroids including permethrin, etc. and type II pyrethroids including cypermethrin, fenvalerate, cyfluthrin, etc. Our previous study showed that fenvalerate and cypermethrin could reduce sperm motility in vitro. However, it is not clear whether permethrin and 3-phenoxybenzoic acid (3-PBA, the major metabolite of pyrethroids) affect sperm motility directly or indirectly by affecting spermatogenesis via interaction with androgens and/or their receptors. In this study, rat sperm suspensions were treated respectively with permethrin, cypermethrin and 3-PBA, at various concentrations (0, 1, 4, 16, or 64 mmol/L) for various times (1, 2, or 4 h). The motility parameters of sperm were analyzed with a computer-assisted sperm analysis (CASA) system. The differential effects of permethrin and cypermethrin on sperm motility patterns in vitro were also compared. Our study revealed that permethrin and cypermethrin could reduce sperm motility in vitro in a concentration- and time-dependent manner. Marked differences between the two pyrethroids were not found in this study. Moreover, 3-PBA did not reduce sperm motility directly at all concentrations and treatment periods. These results provide further evidence that permethrin and cypermethrin can directly affect mature rat sperm motility.

Effect of Progesterone and Ionomycin on Domestic Cat Sperm Motility Patterns and Acrosome Reaction

This study was aimed at assessing the changes in sperm motion patterns and the percentage of acrosome reaction (AR) in domestic cat semen after treatment with either ionomycin or progesterone (P(4)). Ten ejaculates were collected from five tomcats using an artificial vagina, and were diluted, centrifuged and resuspended in a capacitation medium. Samples were evaluated and divided into seven equal aliquots and, after 2 h at 25 degrees C, were incubated for 30 min at 38 degrees C in 5% CO(2) and then analyzed. Computer-assisted sperm analysis and a combination of three fluorescent probes were used to assess sperm plasma, acrosomal membrane integrity and mitochondrial transmembrane potential. Thirty minutes after the start of incubation, P(4) was added (10 microg/ml) to the P1 group. Groups P2 and P3 were supplemented with P(4) (10 and 20 microg/ml, respectively) only after 2 h of incubation, and groups I1 and I2 were supplemented with ionomycin (4 and 8 mum, respectively) 2 h after incubation. Group E was supplemented with ethanol (0.6%) at 2 h after incubation and group C received no supplementation. Ionomycin and P(4) treatments led to a hyperactivation-like sperm motion and an increase (p < 0.05) in the percentage of AR. Although a higher (p < 0.05) percentage of AR was obtained in group I2 when compared with all P(4) groups, a decrease (p < 0.05) in total and progressive motility was observed in I2 group. As I1 group was similar to I2 to induce AR without diminishing sperm motility, we can conclude that ionomycin at 4 microm seems to be more suitable to trigger AR in domestic cat sperm.

231 SPERM SUBPOPULATIONS IN THE KOALA (PHASCOLARCTOS CINEREUS) EJACULATE

Koala semen contains a heterogeneous mixture of sperm morphotypes, mainly attributable to extreme degree of shape variability displayed by the hooked sperm head. By analogy with other species, we anticipate that the morphotypes may exhibit correspondingly different sperm-motility behaviors, largely caused by the differences in hydrodynamic interactions with the suspending media. This trend has been shown in human spermatozoa where motility behavior was demonstrably correlated with the sperm head morphology (Overstreet et al. 1981). In this study, we have investigated the heterogeneity of koala sperm motility profiles in semen in an effort to determine whether distinct sperm subpopulations within ejaculates are recognizable by the use of computer-assisted sperm motility analysis. Ejaculates from 5 males were collected by electroejaculation, then diluted and transported in Tris-citrate-glucose (TCG) diluent. Spermatozoa were washed through a 35–60% Percoll gradient to separate seminal plasma and the majority of the prostatic bodies from spermatozoa. Spermatozoa from the washed pellet were then diluted in TCG at 35°C, incubated for 10 min, and video recorded using a negative phase ×10 objective. Sperm motion parameters were then analyzed using the Hobson sperm tracker (Hobson Vision Systems, UK: Holt et al. 1996 J. Androl. 17, 587–596). Multivariate pattern analysis (PATN; CSIRO Australia; Abaigar 1999 Biol. Reprod. 60, 32–41) was used to distinguish 3 sperm subgroups, consistently shown in each ejaculate, within the data (1936 tracks × 6 kinetic parameters; VCL, VAP, MAD, BCF, ALH, LIN). After group allocation by PATN, all parameters showed significant differences between each of the groups (P &lt; 0.0001). Group 1, approximately 25% of the sperm tracks, showed profiles of spermatozoa with fast, non-linear motility (VCL 106.88 ± 28.15; BCF 3.23 ± 3.81; LIN 14.08 ± 10.20). Group 2, approximately 27% of sperm tracks, showed profiles of fast, linear motility (VCL 63.92 ± 13.50; BCF 7.90 ± 3.42; LIN 28.10 ± 12.15). Group 3, 48% of sperm tracks, showed profiles of slow, non-linear or circular patterns of motility (VCL 39.05 ± 11.92; BCF 0.02 ± 0.35; LIN 5.15 ± 4.88). The recognition of 3 clearly identifiable subgroups supports our hypothesis that heterogeneity of sperm motility patterns exists within koala ejaculates. These may be a reflection of the heterogeneity in sperm-head morphotypes in koala semen, but that remains to be investigated in more detail. The clear distinctions between these groups, and the observation that all 3 subpopulations exist in each of the ejaculates, also suggest that the spermatozoa exhibit functional differences, possibly related to biochemical or maturational status. Many thanks to Dr. Michael Pyne and Dr. Vere Nicholson and their teams and animals at Currumbin Wildlife Sanctutary and Dreamwolrd QLD for all their help and support for the collection of samples.

Effect of repeated sequential ejaculation on sperm DNA integrity in subfertile males with asthenozoospermia

The aim of this work was to study the possible beneficial effect of repeated sequential ejaculation on sperm DNA integrity in subfertile males and its possible implementation in assisted reproduction. The study included 20 infertile males with idiopathic asthenozoospermia or oligoasthenozoospermia. They underwent detailed history taking, complete clinical assessment and hormonal assessment. Patients were asked to bring two semen samples (taken within 1-3 h). Two consecutive samples were assessed with regard to semen volume, sperm count, motility grading, and morphology and sperm DNA integrity using the comet assay. There was a significant improvement in the sperm motility pattern and DNA integrity in the second sample in comparison with the first sample. Therefore, it is concluded that due to its positive impact on sperm motility and DNA integrity, repeated sequential ejaculation is recommended in subfertile males with idiopathic asthenozoospermia who pursue assisted reproduction.

Effects of Fenvalerate and Cypermethrin on Rat Sperm Motility Patterns In Vitro as Measured by Computer-Assisted Sperm Analysis

Fenvalerate and cypermethrin were reported to impair male reproductive function, inducing significant reductions in epididymal sperm count. Further, fenvalerate was shown to reduce sperm motility. However, it is not clear whether fenvalerate and cypermethrin might impact sperm motility directly or indirectly by affecting spermatogenesis via interaction with androgens or their receptors. In this study, sperm suspensions were treated with fenvalerate and cypermethrin, respectively, at various concentrations (0, 1, 4, 16, or 64 μmol/L) for various times (1, 2, or 4 h). The motility parameters of sperm treated with these two insecticides were analyzed with a computer-assisted sperm analysis (CASA) system. The differential effects of fenvalerate and cypermethrin on rat sperm motility patterns in vitro were also compared. Our study revealed that fenvalerate and cypermethrin reduced sperm motility in vitro in a concentration- and time-dependent manner. Cypermethrin exerted a greater effect on sperm motility in comparison to fenvalerate. These results provided evidence that fenvalerate and cypermethrin directly influence mature rat sperm motility.

Effects of Different Concentrations of Aflatoxin B on Ram Epididymal and Ejaculatory Sperm Viability and Motility in vitro

The present study was carried to observe the effect of aflatoxin on ovine sperm in vitro. Ram epididymal and ejaculatory sperm cells were added into media containing different concentrations of aflatoxin B1. When ram epididymal sperm were put in different concentrations of aflatoxin, one-hour post incubation in control group, 81.25% of sperm cells were alive of which, 82.88% were motile. Significantly (p < 0.05) higher than medium with 31.25 ppb and higher concentration. The lowest motility (15.93%) was observed in 62.5 ppb aflatoxin. Sperm viability did not significantly changed after 2nd and 3rd h incubation but significantly decreased in 4th and 5th h post incubation. However, motility was not altered in the 2nd, 3rd, 4th and 5th h post incubation. In ejaculatory sperm, one hour post-incubation, in control group, 83.24% of sperm were alive of which 93.98% were motile, significantly (p < 0.05) higher than motility in medium with 7.81 ppb and higher concentrations of aflatoxin but not different with 1.96 ppb aflatoxin. In 62.5 ppb aflatoxin, the lowest motility was observed (18.9%). Sperm motility patterns for both epididymal and ejaculatory sperm cells were different after incubation in different concentrations of aflatoxin. The results of the present experiment showed that aflatoxin could decrease sperm motility obtained from ejaculation or epididymis.

Motion of Spermatozoa by Visual Image Analysis

Spermatozoa are basic male reproductive cells. An analysis of semen characteristics can provide a reasonable basis upon which to develop a strategy for maximizing the fertility. Because it is generally accepted that a high percentage of poorly motile or immotile sperm will adversely affect male fertility, analysis of sperm motility is a central part of the evaluation of male fertility. To study the motility pattern of small particles is difficult. A new development in medical imaging technology can now be applied for this purpose. Here, the author used a new medical imaging technology to predict the motility pattern of sperm. According to this study, it can be seen that the motility of each sperm is horizontal but the motility of all sperms seems to be directionless. In addition, an interesting observation is that there is a whirling type of sperm motility in case of erroneous motion.

Use of computer-assisted sperm analysis (CASA) to evaluate the quality of cryopreserved sperm in red seabream ( Pagrus major)

In the present study, the quality of post-thaw sperm of red seabream Pagrus major frozen with 6–24% DMSO was investigated. The motility, average path velocity and fertilizing capacity of fresh and their corresponding post-thaw sperm were examined for evaluation of the post-thaw sperm motion characteristics and its association with fertilizing capacity. An analysis of sperm motility before and after cryopreservation has been performed using computer-assisted sperm analysis (CASA). For post-thaw sperm frozen with 12–21% DMSO, the percentages of motile sperm were not significantly ( P > 0.05) changed 10 s after activation. Moreover, the main motility pattern and swimming velocity of the motile post-thaw sperm were not significantly ( P > 0.05) changed and the progressive linear motion was still the dominant pattern. However, the total motility of post-thaw sperm (72.3 ± 6.3%) 30 s after activation was ( P < 0.05) lower than the corresponding fresh sperm (82.7 ± 7.2%). Additionally, the fertilizing capacity of post-thaw sperm was investigated with a standardized sperm to egg ratio 500:1. There is a linear regression relationship between the percentage of motile post-thaw sperm and fertilizing capability. These data demonstrate that 12–21% DMSO can provide good protection to the sperm during the freezing–thawing process.

Semen biology and stimulation of milt production in the European smelt ( Osmerus eperlanus L.)

Basic characteristics of the European smelt ( Osmerus eperlanus) sperm are reported here for the first time. Smelt spermatozoa had a bullet-shaped head (1.42 μm length), a short midpiece and a long flagellum (27.72 μm). Two mitochondria were located along the flagella. The volume of smelt sperm was small (30–60 μl) and the duration of sperm motility was short (22 s in distilled water and 41 s in 20 mM sodium bicarbonate solution). Sodium chloride at concentrations ranging from 0–120 mM did not influence the percentage of motile spermatozoa but caused a steady increase in the duration of sperm movement. Potassium ions clearly reduced the percentage of motile sperm at a concentration of 10 mM. Spermatozoa were motile through a broad range of pH with an optimum from 7.5 to 8.5. Testicular spermatozoa had a different motility pattern compared to stripped spermatozoa (the latter exhibiting a reduction of motile spermatozoa by 30%, lower ALH and VCL and higher LIN and VSL). These results indicate that maturation of smelt spermatozoa occurring in sperm ducts is related not only to an increase of the percentage of motile spermatozoa but also to changes in the sperm motility pattern. Maintaining males with females resulted in stimulation of milt production. Our results indicate that European smelt sperm characteristics are similar to those of ayu (Osmeridae).

Effect of 17β-estradiol on gamete quality and maturation in two salmonid species

In the present study the effect of environmentally relevant concentrations of 17β-estradiol on gamete quality and gamete maturation in rainbow trout ( Oncorhynchus mykiss) and grayling ( Thymallus thymallus) was investigated. Male rainbow trout were exposed to 0.5–2.5 ng l −1 17β-estradiol for 35 days during the spawning season. At concentrations of ≥1 ng l −1 the semen volume obtained per male was significantly reduced, and after 50 days also the sperm density and the sperm fertility. When male grayling were exposed to 1.0 ng l −1 17β-estradiol for 50 days during the prespawning season a similar number of males gave semen as in the control. However, the volume of semen produced per male was decreased. The percentage of motile spermatozoa and their sperm swimming velocity were decreased while the percentage of locally motile spermatozoa was increased. In rainbow trout and grayling also the sperm motility pattern was affected by 17β-estradiol exposure. When female rainbow trout were exposed to 0.5–2.5 ng l −1 17β-estradiol and egg portions were stripped in 1 week intervals the egg viability changed in a similar way as in the control indicating that egg overripening processes were not influenced by 17β-estradiol. When female grayling were exposed to 1.0 ng l −1 17β-estradiol during the prespawning time ovulation occurred earlier than in the control group (group exposed to 17β-estradiol: 35 days after the onset of the experiment, control group 35–50 days after the onset of the experiment).

Effect of two insecticides and two herbicides on the porcine sperm motility patterns using computer-assisted semen analysis (CASA) in vitro

The recent decline in sperm concentration observed in men has developed over a short period of time, suggesting that it could be the result of environmental factors. The present study has evaluated the effects of insecticides Malathion and Diazinon, and herbicides Atrazine and Fenoxaprop-Ethyl on porcine sperm viability and motility patterns in vitro using the eosin–nigrosin staining and a computer-assisted semen analyzer (CASA), respectively. Malathion and Fenoxaprop-Ethyl exerted more deleterious effects than Diazinon and Atrazine. Progressive sperm motility was strongly affected whereas the effect on sperm viability was less pronounced. This suggests that a reduction of sperm motility is not necessarily the result of sperm death. Since sperm motility is dependent on energy metabolism the mechanism of action of these pesticides might be mediated at the level of the mitochondrion, producing a delay in motility and eventual cell death.