Hobson Sperm Tracker Research Articles

Characterization and biological role of cysteine-rich venom protein belonging to CRISPs from turkey seminal plasma†.

Turkey semen contains cysteine-rich secretory proteins (CRISPs) that belong to the dominant seminal plasma proteins. We aimed to isolate and characterize CRISP from turkey seminal plasma and evaluate its possible involvement in yellow semen syndrome (YSS). YSS, which is well characterized, causes reduced fertility and hatchability. The protein was purified using hydrophobic interaction, gel filtration, and reverse phase chromatography. It then was subjected to identification by mass spectrometry, analysis of physicochemical properties, and specific antibody production. The biological function of the isolated protein was tested and included its effects on sperm motility and migration and sperm-egg interactions. Sperm motility was measured with the CASA system using Hobson Sperm Tracker. The reproductive tract of turkey toms was analyzed for gene expression; immunohistochemistry was used for protein localization in the male reproductive tract, spermatozoa, and inner perivitelline layer. The isolated protein was identified as cysteine-rich venom protein-like isoform X2 (CRVP X2; XP_010706464.1) and contained feature motifs of CRISP family proteins. Turkey CRVP X2 was present in both spermatozoa and seminal plasma. The extensive secretion of CRVP X2 by the epithelial cells of the epididymis and ductus deferens suggests its involvement in post-testicular sperm maturation. The internally localized CRVP X2 in the proximal part of the sperm tail might be responsible for stimulation of sperm motility. CRVP X2 on the sperm head might be involved in several events prior to fusion and may also participate in gamete fusion itself. Although the mechanisms by which CRVP X2 mediates fertilization are still unknown, the involvement of complementary sites cannot be excluded. The disturbance of CRVP X2 expression can serve as an etiologic factor of YSS in the turkey. This study expands the understanding of the detailed mechanism of fertilization in birds by clarifying the specific role of CRVP X2.

Premises for fowl sperm preservation based on applied bioenergetics1

The primary goal of this work was to test whether the sperm mobility assay could be used to derive mathematical relationships from which predictions could be made about sperm cell function. A precondition was random sampling from a pool of sperm. This precondition was met by centrifuging mobile sperm through 12% (wt/vol) Accudenz containing the Ca(2+) chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and then holding washed sperm at 20°C within buffered potassium chloride. These 2 conditions rendered washed sperm immobile at 20°C. Resumption of sperm mobility was independent of time (P > 0.8558) when sperm were reactivated at body temperature with 2 mM Ca(2+) in isotonic sodium chloride at pH 7.4. Reactivated sperm mobility was 93% of the prewash control. Subsequent experiments served to define a dose response, predict optimal conditions for in vitro sperm mobility, and show how sperm can recover from an imposed non-physiological condition. Thus, functions were derived from which predictions were made. Whereas the utility of BAPTA treatment was confirmed in a new context, such utility did not address the question of whole-cell Ca(2+) flux during sperm cell manipulation. This issue is pivotal for the application of bioenergetics to fowl sperm preservation. Therefore, the secondary goal of this research was to investigate sperm cell Ca(2+) flux using a simulation of conditions encountered by sperm during centrifugation through 12% (wt/vol) Accudenz. These conditions included a temperature of 30°C, a Ca(2+) sink, and no exogenous substrate. Sperm motion was measured with a Hobson SpermTracker. Data points conformed to parabolic functions when motile concentration and velocity were plotted as functions of time. In each case, maximums were observed, e.g., 26 min for motile concentration. The upswing was attributed to a redistribution of intracellular Ca(2+) whereas the downswing was attributed to sperm cell Ca(2+) depletion. A pronounced isothermal increase was observed for each variable when the Ca(2+) sink was overcome with exogenous Ca(2+). Experimental outcomes supported four testable premises applicable to fowl sperm preservation research: 1) the importance of sperm mobility phenotype, 2) the relationship between mitochondrial Ca(2+) cycling and sperm mobility, 3) the utility of the sperm mobility assay for predicting experimental outcomes, and 4) understanding mitochondrial Ca(2+) cycling in terms of whole-cell Ca(2+) flux.

Different computer-assisted sperm analysis (CASA) systems highly influence sperm motility parameters

In this study, we examined different computer-assisted sperm analysis (CASA) systems (CRISMAS, Hobson Sperm Tracker, and Image J CASA) on the exact same video recordings to evaluate the differences in sperm motility parameters related to the specific CASA used. To cover a wide range of sperm motility parameters, we chose 12-second video recordings at 25 and 50 Hz frame rates after sperm motility activation using three taxonomically distinct fish species (sterlet: Acipenser ruthenus L.; common carp: Cyprinus carpio L.; and rainbow trout: Oncorhynchus mykiss Walbaum) that are characterized by essential differences in sperm behavior during motility. Systematically higher values of velocity and beat cross frequency (BCF) were observed in video recordings obtained at 50 Hz frame frequency compared with 25 Hz for all three systems. Motility parameters were affected by the CASA and species used for analyses. Image J and CRISMAS calculated higher curvilinear velocity (VCL) values for rainbow trout and common carp at 25 Hz frequency compared with the Hobson Sperm Tracker, whereas at 50 Hz, a significant difference was observed only for rainbow trout sperm recordings. No significant difference was observed between the CASA systems for sterlet sperm motility at 25 and 50 Hz. Additional analysis of 1-second segments taken at three time points (1, 6, and 12 seconds of the recording) revealed a dramatic decrease in common carp and rainbow trout sperm speed. The motility parameters of sterlet spermatozoa did not change significantly during the 12-second motility period and should be considered as a suitable model for longer motility analyses. Our results indicated that the CASA used can affect motility results even when the same motility recordings are used. These results could be critically altered by the recording quality, time of analysis, and frame rate of camera, and could result in erroneous conclusions.

Effects of pH on sperm motility in several Salmoniformes species: Oncorhynchus mykiss, Salvelinus fontinalis, Salmo trutta, Salmo salar and Thymallus thymallus

Summary In this study we investigated the effect of pH (range 5.5–10.5) on sperm motility of rainbow trout (Oncorhynchus mykiss) and other fish species from family Salmonidae: brook trout (Salvelinus fontinalis), brown trout (Salmo trutta), Atlantic salmon (Salmo salar), and grayling (Thymallus thymallus). Video recordings of sperm motility were analyzed using Hobson Sperm Tracker. Since urine contamination of milt was suspected to modulate motility, the effect of pH on rainbow trout spermatozoa was re-evaluated with the use of semen collected with a catheter. Spermatozoa of all the species except for rainbow trout were characterized by a broad pH range for the motility. At a pH of 5.5, motile sperm was observed for both brook trout and brown trout, 31 and 38%, respectively. For rainbow trout spermatozoa collected by stripping, no motility was observed at pH 7.0 and below. On the contrary, when sperm from rainbow trout was collected using a catheter we observed motility at low pH values. Our results demonstrated that salmonid spermatozoa can be motile in a wide range of pH values, including the acidic range. Our results suggest that urine contamination significantly affects pH-dependence of sperm motility activation in fish.

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 < 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.

222 SPECIES SPECIFICITY OF PORCINE SPERM MOTILITY REDUCTION BY A HIGH MOLECULAR WEIGHT FRACTION OF OVIDUCTAL FLUID

The significance of sperm motility with respect to fertilization is widely recognized and used as a criterion to assess the quality of ejaculates. Observations of sperm behavior in the oviductal isthmus of several species have shown that their motility is suppressed in this physiological environment because the spermatozoa bind to the oviductal epithelial cells, forming a sperm reservoir prior to ovulation (Hunter 1981 J. Reprod. Fertil. 63, 109–117; Hunter and Wilmut 1984 Reprod. Nutr. Dev. 24, 597–608). Once the spermatozoa are released from the reservoir, they progress toward the ampullar region to reach the oocyte, and an increase in motility at this point could, potentially, be crucial. It has been demonstrated that a soluble fraction of oviductal epithelial cell apical plasma membrane proteins (sAPM) suppresses sperm motility and enhances sperm survival (Holt et al. 2005 Reprod. Fertil. Dev. 17, 683–692; Satake et al. 2006 J. Exp. Biol. 209, 1560–1572). However, few studies to date have investigated the influence of oviductal fluid (OF), the natural medium into which spermatozoa are released from the reservoir, on sperm motility. Consequently, this study aimed to determine the effects of different soluble fractions of OF on sperm motility and the species specificity of such effects. OF from pigs and cows was collected and selectively filtered to obtain two different fractions with molecular weights higher or lower than 100 kD. Diluted semen samples from 14 different boars were exposed to bicarbonate/CO2 (to stimulate maximum motility) in the presence or absence of OF fractions. Sperm trajectories were measured using a Hobson Sperm Tracker (Hobson Tracker, Ltd., Sheffield, UK) and analyzed by PATN analysis as described previously to identify subpopulations of high and low motility spermatozoa (Abaigar et al. 1999 Biol. Reprod. 60, 32–41; Satake et al. 2006). The results showed that neither of the bovine OF fractions affected the proportions of the fast linear boar sperm subpopulation in the samples, which was similar to that of the control. However, when the high molecular weight fraction of porcine OF was used, a significant suppression of the fast linear sperm subpopulation was observed (P ≤ 0.05). These data support the hypothesis that species-specific, high molecular weight components in OF are involved in the suppression of sperm motility. Further studies are required to confirm the significance of this finding, although it may not be unreasonable to speculate that the OF, in addition to other sperm selection mechanisms, acts to protect oocytes against fertilization by poor quality spermatozoa (Okada et al. 1986 J. Submicrosc. Cytol. 18, 233–247). In fact, similar results demonstrating that oviductal fluid decreases sperm motility have been obtained in cow (Grippo et al. 1995 J. Reprod. Fertil. 105, 57–64) and rabbit (Overstreet and Cooper 1979 J. Reprod. Fertil. 55, 53–59). This work was supported by MEC and FEDER (PR2006-0506 and AGL2006-03495).

227 PHOSPHOINOSITIDE-3-KINASE SIGNALING AND THE CONTROL OF SPERM MOTILITY IN THE PORCINE OVIDUCT

Prior to ovulation, interactions of spermatozoa with the oviductal epithelium lead to the formation of a sperm reservoir; here we investigate the hypothesis that the physiological state of the stored spermatozoa is modulated by the epithelial cells. In the pig, where modulation of sperm motility is sensitive to environmental components, the high bicarbonate content of the oviductal fluid would be expected to stimulate motility to its maximal extent; however, as this is incompatible with reservoir formation, we postulate that a signaling pathway counteracts bicarbonate stimulation and depresses motility (Satake et al. 2006 J. Exp. Biol. 209, 1560–1572). We chose to investigate a signaling mechanism which may suppress sperm motility through phosphoinositide-3-kinase (PI3K). Boar spermatozoa were exposed to solubilized proteins derived from the oviduct epithelial apical plasma membrane (sAPM). We exploited the motility activation effects of bicarbonate on Percoll-washed boar spermatozoa to see whether it was countered by sAPM and reversed by PI3K inhibitors. Washed spermatozoa (n = 9 boars) were incubated (10 min at 38�C) in Tyrode's medium in the presence of sAPM (25 and 50 µg mL–1) or PI3K inhibitors (LY294002 or wortmannin) prior to activation with 15 mm bicarbonate/CO2, and then incubated for a further 35 min. Sperm motility was monitored at 5-min intervals using a Hobson Sperm Tracker (Hobson Tracker, Ltd., Sheffield, UK). Samples were also analyzed with a flow cytometer for mitochondrial activity using JC-1. PATN analysis (Belbin 1993 PATN Pattern Analysis Package, Div. Wildlife & Ecol., CSIRO, Canberra, Australia) was used to distinguish four motility-based sperm subgroups within the data. The proportions of fast and progressive spermatozoa showed significant and rapid (<5 min) bicarbonate-induced increases when there was no sAPM treatment, or when PI3K inhibitors were present. However, there was no significant bicarbonate-induced increase in the presence of sAPM alone, or when spermatozoa were exposed to a mixture of wortmannin and sAPM. PI3K inhibition by LY294002 reversed the sAPM-induced motility suppression (P < 0.001). Mitochondrial membrane potential was also suppressed by sAPM and restored by the presence of LY294002 (P < 0.01). These results support the hypothesis that the sperm–oviduct interaction is modulated by an intracellular signaling pathway involving PI3K and appropriate receptors.

93COMPUTER-ASSISTED ANALYSIS OF BOVINE SPERM MOTILITY BEFORE AND AFTER CRYOPRESERVATION

Sperm cryopreservation causes various types of damage, including membrane injury, oxidative stress, and loss of the acrosome. In cattle, the mortality rate after sperm cryopreservation reaches roughly 50%, and surviving sperm cells have a lower motility and lower fertility than their fresh counterparts. Large variations are also observed between bulls. The aim of this study was to analyse different motility parameters before and after freezing in order to establish correlations. The final objective is to determine, before freezing, parameters that could predict the characteristics of motility after freezing. A computer-assisted sperm analyser (Hobson Sperm Tracker) was used. We analyzed one ejaculate from 30 different bulls before and after freezing (minimum 300 spermatozoa/analysis). Reliable parameters (<10% variation for the same ejaculate) were then selected and included VCL (curvilinear velocity), VAP (average path velocity), MAD (mean angular head displacement), ALH (amplitude of lateral head displacement), STR (straightness of path), and the percentage of motility (%Mot). Linear regressions were established between those parameters before and after freezing. Results are shown in Table 1. The velocity parameters (VAP, VCL, and STR) of the motile sperm were conserved after freezing. Moreover, ejaculates with a high proportion of motile sperm before freezing have, on average, better values for velocity parameters after freezing, while no correlation was found between the percentage of motile sperm before and after freezing. The only parameter of fresh sperm that seems to be correlated with the proportion of motile sperm cells after freezing is MAD (inverse correlation). This could mean that an ejaculate with a high proportion of spermatozoa showing important lateral displacements of the head is more sensitive to cryopreservation. Similarly, a high MAD before freezing was related to a low velocity after thawing. A high MAD could result from a high proportion of capacitated spermatozoa, which is detrimental to their survival and motility. In conclusion, few parameters related to the motility can predict the proportion of motile sperm after freezing. However, by combining several parameters, it seems possible to predict the characteristics of motility of the sperm. Although further investigations are needed, the present evaluation could be of interest to evaluate the freezability of ejaculates, to understand variations between bulls, or to set up new freezing protocols. Table 1 Coefficients of correlation (r2) before and after freezing, calculated from 30 ejaculates from 30 different bulls

Binding of Zona Binding Inhibitory Factor-1 (ZIF-1) from Human Follicular Fluid on Spermatozoa

Previous studies showed that zona binding inhibitory factor-1 (ZIF-1) was the glycoprotein mainly responsible for the spermatozoa zona binding inhibitory activity of human follicular fluid. ZIF-1 has a number of properties similar to glycodelin-A. A binding kinetics experiment in the present study demonstrated the presence of two binding sites of ZIF-1 on human spermatozoa. These binding sites were saturable, reversible, and bound to (125)I-ZIF-1 in a time-, concentration-, and temperature-dependent manner. Glycodelin-A shared one common binding site with ZIF-1 on spermatozoa, and it could displace only 70% of the (125)I-ZIF-1 bound on human spermatozoa. ZIF-1 and glycodelin-A formed complexes with the soluble extract of human spermatozoa. Coincubation of solubilized zona pellucida proteins reduced the binding of ZIF-1 to two complexes of the extract, suggesting that the ZIF-1 binding sites and zona pellucida protein receptors on human spermatozoa were closely related. ZIF-1, but not glycodelin-A, significantly suppressed progesterone-induced acrosome reaction of human spermatozoa. The carbohydrate moieties derived from ZIF-1 reduced the binding of native ZIF-1 on human spermatozoa as well as the zona binding inhibitory activity of the glycoprotein, although the intensity of the effects are lower when compared with the native protein. These effects are not due to the action of the molecules on the motility, viability, and acrosomal status of the treated spermatozoa. Deglycosylated ZIF-1 had no inhibitory effect on both ZIF-1 binding and zona binding capacity of spermatozoa. We concluded that the carbohydrate part of ZIF-1 was critical for the functioning of the glycoprotein.

The human sperm head: a key for successful fertilization.

In order to examine the predictive value of determining the sperm head shape, the acrosomal size, the presence of acrosomal vacuoles, and the challenged acrosome reaction (AR) on the outcome of a standard in vitro fertilization (IVF) program, a prospective study was conducted that included 75 couples undergoing IVF treatment. An assessment of sperm morphology was performed using the Hobson Sperm Tracker (Hobson Tracker Limited, Sheffield, United Kingdom). The assessment of the AR was performed before and after adding pooled undiluted human follicular fluid (FF). The outcome measure was an IVF rate of inseminated oocytes. A positive correlation was found between the fertilization rate (FR%) and the proportion of the sperm with a normal (oval) head shape (P <.001), the sperm exhibiting acrosomal vacuoles (P <.003), the sperm with a normal acrosomal size (40%-70% of total head area, P <.025), and the sperm undergoing AR after adding FF (P <.001). Multiple logistic regression analysis revealed that by incorporating the above 4 parameters, the sensitivity of prediction of IVF FR% values was 79%, and the specificity was 93%, with a positive predictive value of 96%. This study shows that the multiparametric assessment of the sperm head is useful in predicting the FR% values of a standard IVF treatment. The automated analysis used in this study is shown to maintain a level of precision and accuracy acceptable for application in a routine semen analysis situation.

Relationship between semen parameters and mercury concentrations in blood and in seminal fluid from subfertile males in Hong Kong

Relationship between semen parameters and mercury concentrations in blood and in seminal fluid from subfertile males in Hong Kong

Whole blood mercury concentrations in sub-fertile men in Hong Kong.

We aimed to investigate the association between whole blood mercury concentrations and semen quality in sub-fertile men. Fifty-nine male partners of infertile couples attending the Assisted Reproduction Unit of the Chinese University of Hong Kong between 1997 and 1998 were recruited into our study. Blood was taken from each subject for whole blood mercury concentration and hormone profile. Semen samples were obtained for computer assisted semen analysis using the Hobson sperm tracker. The semen parameters and hormone profile were compared between subjects with normal and those with elevated mercury concentrations. Twenty-one subjects (35.6%) had a whole blood mercury concentration higher than the normal range (0-50 nmol/l). All parameters of the semen analysis including the concentration of sperm, percentage of morphologically normal sperm, percentage of motile sperm, curvilinear velocity, straight-line velocity, average path velocity, and amplitude of lateral head displacement, were reduced in those with elevated blood mercury concentrations, although the difference was not statistically significant. We failed to demonstrate a statistically significant effect on the measurement of semen quality, but other adverse effects cannot be excluded. From a public heath perspective, these findings confirm that mercury toxicity is a potentially serious problem affecting the local community.

Sperm mobility: phenotype in roosters (Gallus domesticus) determinedby concentration of motile sperm and straight line velocity.

Previous research demonstrated that sperm mobility, i.e., the net movement of a sperm population, is a quantitative trait of the domestic fowl. However, the cellular basis for this trait was unknown. In the present work, individual motile sperm were evaluated with a Hobson SpermTracker in order to identify one or more properties of motile sperm that could account for variation in sperm mobility observed among males. A method was validated for assessing sperm motion over an erythrocyte monolayer at body temperature. A small-scale experiment with roosters from the tails and center of a normal distribution of sperm mobility phenotypes (n = 33 roosters) demonstrated that straight line velocity (VSL) and motile concentration were critical to expression of phenotype. The importance of these variables was confirmed with a large-scale experiment using a representative subpopulation (n = 100 roosters). VSL of individual sperm at 41 degrees C ranged between 5 and 100 microm/sec. VSL averaged 32, 39, and 40 microm/sec for low, average, and high sperm mobility phenotypes. Sperm were diluted to 1.2 x 10(6)/ml for motion analysis. Mean motile concentrations were 0.52, 0.84, and 0.95 x 10(6)/ml for low, average, and high sperm mobility phenotypes. Motile concentration was correlated with sperm mobility (r = 0.71). VSL appeared to have an additive effect as it was correlated with straightness of sperm cell trajectory (r = 0.79).

Sperm mobility: A primary determinant of fertility in the domestic fowl (Gallus domesticus).

Previous research demonstrated that sperm mobility is a quantitative trait of the domestic fowl. The trait is quantified by measuring the absorbance of an Accudenz solution after overlay with a sperm suspension and brief incubation at body temperature. In the present work, average and high sperm mobility phenotypes (n = 30 males per phenotype) were selected from a base population. Differences were found between sperm oxygen consumption (p < 0.0001), acylcarnitine content (p < 0.05), linear velocity (p < 0.001), and straightness (p < 0.001), a trajectory variable measured with the Hobson SpermTracker. Oxygen consumption and stearoylcarnitine content of sperm from the high-mobility phenotype were twice those observed with sperm from average males, implying a pivotal role for mitochondria. On the basis of these results, a graded relationship was predicted between fertility and sperm mobility. Males (n = 48) were chosen at random from another base population, sperm mobility was measured per male, and each ejaculate was used to inseminate 8-12 hens (8 x 10(7) viable sperm per hen). When fertility was plotted as a function of sperm mobility, data points approximated a skewed logistic function. The hypothesis that vaginal immunoglobulins constitute an immunological barrier to sperm transport was tested and rejected. Therefore, we concluded that sperm mobility is a primary determinant of fertility in the fowl.

Comparative motility of X and Y chromosome-bearing bovine sperm separated on the basis of DNA content by flow sorting.

A combination of flow cytometric sperm sorting of X and Y chromosome-bearing sperm (X and Y sperm) and computer-assisted sperm analysis (CASA) for measuring sperm motility allows assessment of motion parameters in the two populations. Bull sperm were separated into X and Y populations by flow cytometry following staining with the DNA-binding dye Hoechst 33,342. The motion parameters differed depending on sperm concentration. Decreasing sperm concentration resulted in higher velocities and straighter trajectories. The concentrations of control (stained-unsorted and unstained-unsorted) and flow-sorted sperm were therefore adjusted to similar numbers (5 x 10(6) sperm per milliliter). Samples of sorted X and Y sperm and control sperm were transferred to prewarmed slides on a heated stage (37 degrees C) and their motion video recorded for 2 min using a magnification of x 100 and a high-resolution camera. The sperm analysis was carried out on a Hobson Sperm Tracker (HST) using HST 7 software. The following motion parameters were measured: curvilinear, straight-line, and average path velocity; mean angular displacement (MAD); beat cross-frequency; amplitude of lateral head displacement; linearity (LIN); and straightness of path (STR). Sperm movement was unaffected by staining with Hoechst 33,342, excitation by ultraviolet (UV) light, or the physical process of cell sorting. Significant differences were seen between X and Y sperm for MAD, LIN, and STR. No difference was observed for the other parameters. The results indicate that in a simple salts solution, Y bull sperm do not swim faster than X sperm but may be distinguished from X sperm on the basis of LIN and STR.

Semen donor selection by in vitro sperm mobility increases fertility and semen storage in the turkey hen.

Commercial turkey production relies on the artificial insemination (AI) of pooled semen. However, semen quality ultimately depends on the quality of individual ejaculates. The purpose of this study was to evaluate semen from individual toms by means of an objective sperm-mobility assay. Semen was then pooled by mobility phenotype and inseminated into hens, and the percentages of fertile and hatched eggs were determined after egg incubation. To indirectly evaluate hens' sperm storage, we determined the number of sperm holes in the perivitelline layer (PL) of freshly laid eggs. Semen from individual ejaculates (two trials, total of 169 toms) was evaluated by use of the sperm-mobility test (SMT). Semen was diluted to 1 x 10(9) sperm/ml, in prewarmed N-tris-[hydroxymethyl] methyl-2-amino-ethanesulfonic acid (TES)-buffered saline, and was placed over 3 ml of a 2% (w/v) Accudenz solution at 41degrees C. After a 5-minute incubation period, the cuvette was placed in a densimeter, and percentage transmission was recorded after 1 minute. Semen samples from toms ranked, according to sperm mobility, in the highest 10% and the lowest 10%, after three evaluations, were pooled by group and were used to inseminate hens weekly (trial 1: n = 20 hens/group, for 10 weeks, AI dose 150 x 10(6) spermatozoa inseminated fresh and after 24-hour in vitro storage at 5 degrees C; trial 2: n = 60 hens/group, for 16 weeks, AI dose = 75 x 10(6) spermatozoa inseminated fresh). Each week, eggs from day 6 post-AI were evaluated for holes in the PL, vestiges of acrosomal induced hydrolysis. Spermatozoa from toms of different mobility phenotypes were also evaluated individually, for sperm chromatin structure and motility variables, by use of the Hobson Sperm Tracker. Toms characterized by high and low in vitro sperm-mobility phenotype were categorized as "high mobility" and "low mobility," respectively. The percentage of fertile eggs from hens inseminated with semen from the high-mobility toms was higher than the percentage of fertile eggs from the low-mobility group, in each trial (95.8+/-1.3% vs. 90.4+/-2.2%, and 88.7+/-4.0% vs. 82.4+/-0.4%, trials 1 and 2, respectively; P < 0.05). More sperm holes were observed in the PL of eggs fertilized by the high-mobility toms than in the PL of eggs fertilized by the low-mobility toms (P < 0.05). No differences in susceptibility of sperm nuclear DNA to denature in situ, as measured by the flow-cytometric sperm chromatin-structure assay, were detected between toms of differing mobility phenotypes. Sperm-motility variables, straight-line velocity, and average-path velocity were significantly greater for high-mobility toms compared to low-mobility toms (P < 0.05). Sperm-mobility differences between toms (detected by means of the SMT) influenced sperm storage, as indicated by the number of sperm in the PL and by the percentage of fertile eggs produced.

CASA as an aid to selecting sperm suspensions for artificial insemination in Callithrix jacchus

The ability to use sperm motility parameters, obtained by computer-assisted sperm motility analysis (CASA), as an aid to selecting sperm samples for artificial insemination (AI) would have considerable benefits for commercial organizations and for the captive breeding of endangered species. In this study the Hobson sperm tracker (HST) was validated for use with spermatozoa from Callithrix jacchus, the common marmoset, by comparing values for straight line velocity by CASA with those obtained by direct measurement of sperm tracks. Using the settings established during validation, ejaculated and epididymal spermatozoa were analysed with the HST. The range of values for velocity parameters were used to establish expected motility profiles for the two types of spermatozoa as follows: for epididymal spermatozoa (concentration 2.2 - 85.8 x 10(6) spermatozoa/mL) VCL 109.8-155.9, VSL 75.2-141.5, VAP 85.8-142.1, MAD 13.7-40.7, ALH 3.8-7.8, BCF 1.4-4.2, LIN 40.5-91.1% and STR 70.1-97.1%; for ejaculated spermatozoa (concentration 3.2-82.0 x 10(6) spermatozoa/mL) VCL 89.6-136.7, VSL 69.6-110.3, VAP 74.5-121.9, MAD 19.2-29.3, ALH 3.0-9.9, BCF 2.8-5.5., LIN 65.4-85.3% and STR 93.8-97.7%. Epididymal spermatozoa from males which were not sexually active had significantly lower values for VCL, VSL and VAP, while values for MAD were significantly higher than for spermatozoa from sexually active males (p < 0.031). Sperm concentration affected motility parameters significantly. Although motility parameters differed according to the batch of medium used, the differences were not statistically significant. Epididymal sperm samples had significantly higher VCL, VSL and VAP but lower BCF and LIN than ejaculated sperm samples of the same concentration diluted in the same batch of medium, while MAD, ALH and STR were not different. Urine contamination significantly reduced VCL, VSL and VAP (p < 0.008, < 0.016 and < 0.008, respectively, sample size = 7) whereas MAD, ALH, BCF, LIN and STR were not affected. Therefore CASA could be useful in screening ejaculates for use in Al to eliminate samples with unusual motility patterns.

Angiotensin II stimulates sperm motility

The physiological factors which induce and maintain mammalian sperm maturation and motility generally remain unclear, although several agents are known to be involved. We recently described the application of immunocytochemical and immunoblotting methods to identify the angiotensin II type 1 (AT1) receptor in the tails of ejaculated rat and human sperm, and gave evidence to show that angiotensin II may promote sperm motility. These data are extended here by the application of a computerised sperm tracking system (the Hobson Sperm Tracker) to demonstrate that AII has actions on specific motility parameters, including curvilinear velocity, straight line velocity, and amplitude of lateral head movement. Since local tissue renin-angiotensin systems are present in both male and female tracts, the data suggest that angiotensin II has a role in the maintenance of sperm function and fertility.