Amplitude Of Lateral Head Displacement Research Articles

Oleoylethanolamide Protects Human Sperm Cells from Oxidation Stress: Studies on Cases of Idiopathic Infertility1

N-acylethanolamides are naturally occurring hydrophobic molecules usually present in a very small amount in many mammalian tissues and cells. The presence of N-acylethanolamides has also been demonstrated in human reproductive tracts and fluids, although their biological effects and molecular mechanisms of action are not yet completely elucidated. It is known that some N-acylethanolamides, such as oleoylethanolamide, have antioxidative properties. The aim of this study was to test whether oleoylethanolamide could protect sperm cells from reactive oxygen species-induced oxidative damage in cases of idiopathic infertility, because the excessive generation of these radicals was associated with this pathology. Our results show that 2.5 nM oleoylethanolamide in vitro supplementation significantly reduces DNA strand breaks both in fertile and infertile subjects. Moreover, oleoylethanolamide increases kinematic parameters, such as curvilinear velocity and amplitude of lateral head displacement and hyperactivation, both in the presence and in the absence of oxidative stress. Results of this study support the hypothesis of a possible protective action of oleoylethanolamide against reactive oxygen species, which could explain its beneficial effects on in vitro capacitated spermatozoa.

Kinematic Changes During the Cryopreservation of Boar Spermatozoa

The present study evaluates the effect that various steps of a conventional cycle of cryopreservation have on the patterns of movement exhibited by boar spermatozoa. Sperm-rich ejaculate fractions collected from 24 mature fertile boars (1 ejaculate per boar) were cryopreserved following a standard freeze-thaw procedure with 0.5-mL plastic straws. Overall sperm motility and the individual kinematic parameters of motile spermatozoa (assessed by the computer-aided sperm analysis system Sperm Class Analyzer [SCA]) were recorded in 5 steps of the cryopreservation procedure. These steps were as follows: 1) at the time that the fresh semen was extended, 2) at 17 degrees C, after sperm concentration by centrifugation and re-extension of the pellet with lactose-egg yolk extender; 3) at 5 degrees C, after added freezing extender; 4) at the time that thawed semen was held in a water bath at 37 degrees C for 30 minutes; and 5) at the time that thawed semen was held in a water bath at 37 degrees C for 150 minutes. Data from individual motile spermatozoa, defined by 7 kinematic parameters (curvilinear velocity [VCL], straight-line velocity [VSL], average path velocity [VAP], linearity [LIN], straightness [STR], mean amplitude of lateral head displacement [ALH], and beat cross frequency [BCF]), were analyzed using a pattern analysis technique (PATN) to identify and quantify populations and subpopulations of motile sperm within the semen samples. After the first cluster analysis, 3 motile sperm populations (P) were identified (P1: progressive and/or vigorous cells [90.4%], P2: poorly progressive cells [8.3%], and P3: nonprogressive cells [1.3%]). These populations remained constant (P > .05) throughout the 5-step cryopreservation procedure. A second PATN was carried out within the P1 sperm population, which identified 3 sperm subpopulations (sP) (eg, sP1: cells with progressive and vigorous movement [58.7%], sP2: progressive cells only [24.6%], and sP3: vigorous cells only, hyperactive-like [16.7%]). Although the relative frequency of these 3 subpopulations varied among ejaculates (boars), there was no interaction with any cryopreservation step we examined. Whereas sP1 remained constant (P > .05), sP2 and sP3 varied significantly (P < .05) through the cryopreservation procedure, with the increase in sP3 after centrifugation at 17 degrees C and during cooling at 5 degrees C considered particularly relevant. In conclusion, the present study confirms the heterogeneity of sperm movement patterns in boar semen, patterns that vary through the cryopreservation procedure, especially after removal of the seminal plasma by centrifugation and subsequent extension at 17 degrees C and after the slow cooling at 5 degrees C, when obvious increases in hyperactivated movement appeared. The vast majority of spermatozoa, those exhibiting progressive and vigorous movement, remained constant during the cryopreservation procedure, although the proportion differed among boars.

Sperm Motion Characteristics May Discriminate Fertile From Infertile Men With Normal Parameters

Sperm Motion Characteristics May Discriminate Fertile From Infertile Men With Normal Parameters

Cinétique spermatique d’hyperactivation : influence du milieu de capacitation

Hyperactivation-associated motility is an important marker of capacitation and a prerequisite to fertilization. Computer-Assisted Sperm Analysis (CASA) is a reproducible method that can be used to predict fertilization success, in addition to the migration and survival test. However, published data are contradictory and preparation methods are heterogeneous. Changing one element in the sperm preparation (capacitation medium) may alter its kinetic parameter values. This prospective study with two paired series was performed on sperm considered as normal. The sperm preparation was identical up to the second centrifugation, and there after the centrifugate was divided into two groups and suspended either in the capacitating medium Upgraded B2 INRA Medium(R), or in Ferticult Flushing Medium. Measurements were carried out with a Hamilton Thorne Research Analyzer (IVOS v10.9i). The analysis of the 87 sperm samples included in the study showed differences between the averages of Straight Line Velocity (SLV, p=0.02), Curvilinear Velocity (CLV, p<0.0001), Amplitude of Lateral Head Displacement (ALH, p<0.0001), and algorithmic calculation of the examined hyperactivation (Hyper 5, p<0.0001). The diagnostic validity of hyperactivation motility cannot be dissociated from the preparation conditions. A standardised preparation is a prerequisite for scientific evaluation and validation of this technique. Although higher motility characteristics seem to be desirable in a therapeutic environment, results will have to be validated by clinical tests.

The Stimulatory Role of Estrogen on Sperm Motility in the Male Golden Hamster (Mesocricetus auratus)

To clarify the physiological roles of estrogens in the regulation of sperm motility in the golden hamster, two different approaches were used. In the first experiment, silastic tubes containing either low (low E2 group) or high (high E2 group) amount of estradiol-17beta were implanted (Exp 1). In the second experiment, male golden hamsters were actively immunized against estradiol-17beta (Exp 2). In Exp 1, all sperm motility parameters (including motility, straight velocity, curvilinear velocity, beat/cross frequency, and mean amplitude of lateral head displacement) were significantly increased except linear index in the high E2 group as compared with controls at 20 days after the treatment. In the high E2 group, plasma concentrations of luteinizing hormone (LH) significantly increased, whereas levels of circulating testosterone decreased significantly. Plasma concentrations of follicle-stimulating hormone (FSH) and immunoreactive inhibin were not affected by the treatment with estradiol-17beta. In the Exp 2, titer of circulating antibodies to estradiol-17beta consistently increased after the second immunization until the end of experiment (16 weeks). The sperm motility, straight velocity, and curvilinear velocity were significantly decreased after active immunization to estradiol-17beta. Concentrations of circulating LH and FSH were also decreased significantly by the treatment. In conclusion, the current observations indicate that estradiol-17beta affects sperm motility in adult male golden hamsters.

Capacitation-associated protein tyrosine phosphorylation and membrane fluidity changes are impaired in the spermatozoa of asthenozoospermic patients

Sperm protein tyrosine phosphorylation has been associated with capacitation, motility changes, zona binding, and fertilizing ability. We previously demonstrated that gradient-isolated human sperm subpopulations differ in their plasma membrane composition, their ability to phosphorylate proteins in tyrosine residues, and their capacity to undergo hyperactivation. In this study, we have characterized capacitation-associated changes in protein tyrosine phosphorylation and membrane fluidity in spermatozoa of asthenozoospermic and normozoospermic patients consulting for infertility. Semen samples were studied at baseline and after a capacitating incubation with or without the addition of a permeable cAMP analog and a phosphodiesterase inhibitor. Basic sperm and computer-assisted motion parameters, hyperactivation, protein tyrosine phosphorylation (immunofluorescence and Western blot), and membrane fluidity (fluorescent Laurdan probe) were the main study parameters. In comparison with normozoospermic and proven-fertile donor semen, asthenozoospermic samples showed lower motility, velocity, and amplitude of lateral head displacement, both originally and after a 6-h capacitating incubation. Unlike those in normal samples, asthenozoospermic spermatozoa were unable to increase protein tyrosine phosphorylation during capacitation. Such impairment, however, was overcome when they were incubated with a membrane-permeable cAMP analog and a phosphodiesterase inhibitor, indicating a possible membrane defect. Confirming this hypothesis, plasma membranes of asthenozoospermic sperm showed decreased fluidity (increased Laurdan polarization), even after a capacitating incubation. In conclusion, spermatozoa from functional asthenozoospermic samples may owe their poor motility, and their inability to properly capacitate and develop hyperactivation, to an impairment in the tyrosine phosphorylation of critical proteins caused by decreased membrane fluidity. These findings suggest a molecular pathogenetic mechanism for a common seminal pathology associated with male infertility.

In vitroEvaluation ofin vivoFertilizing Ability of Frozen Rabbit Semen

The present work studied different spermatozoa parameters and the ability of frozen rabbit spermatozoa to fertilize, in vitro, in vivo-matured oocytes, as a test to predict their in vivo fertility and prolificacy. Semen from rabbit bucks was frozen using two freezing protocols [in a freezer at -30 degrees C or in liquid nitrogen vapour (LNV)]. For the in vivo trial, females were inseminated with frozen-thawed spermatozoa. Oocytes used for in vitro testing were recovered 14 h after ovulation induction from donors and co-incubated with 2 x 10(6) frozen-thawed spermatozoa during 4 h at 37 degrees C in Tyrode's medium under an atmosphere of 5% CO(2) in air with maximal humidity. After co-incubation period, presumptive zygotes were cultured in TCM199 supplemented with 20% foetal bovine serum (FBS), under the same conditions described above. Although no statistical differences were observed between freezing protocols in seminal parameters [motility rate: 40 and 35%, VCL: 35 and 46 microm/s, amplitude of lateral head displacement (ALH): 1.7 and 2.4 mum, for semen frozen at -30 degrees C and in LNV, respectively], significant differences were noted in the fertilizing ability in vivo and in vitro. Semen frozen at -30 degrees C showed the highest fertilizing ability in vitro (26.7% vs 6.2 and 8.7% for semen frozen at -30 degrees C, in LNV and fresh semen, respectively) and the lowest fertility rate in vivo (21.7% vs 64.2% and 70.6% for semen frozen at -30 degrees C, in LNV and fresh semen, respectively). Sperm frozen at -30 degrees C seemed to be more capacitated.

Do parameters of seminal quality correlate with the results of on-farm inseminations in rabbits?

This study was conducted to determine if different sperm characteristics correlate with the in vivo fertility of rabbit sperm. A total of 2765 heterospermic inseminations were performed in commercial rabbitries using 50-pooled samples of fresh semen. Sperm motility and morphological evaluations were performed on each of the heterospermic pooled samples to asses the seminal quality, and the percentage of kindling rate (76.2%) and number of kits born alive (9.3) were recorded. Sperm motility parameters, assessed using a computer-assisted sperm analysis (CASA) system (Sperm Class Analyzer, Microptic, Barcelona, Spain), were: average path velocity, curvilinear velocity, straight-line velocity, linearity, amplitude of lateral head displacement, beat cross-frequency, wobble and percentage of total motile spermatozoa. Morphological analyses included the percentage of sperm with a normal apical ridge, the percentage of sperm with cytoplasmatic droplets and the percentage of abnormal sperm. Significant correlations were observed between kindling rate and the percentage of total motile cells ( r = 0.31; P < 0.05), linearity index ( r = −0.32; P < 0.05) and the percentage of abnormal sperm in the sample ( r = −0.32; P < 0.05). Regression models including motility and the morphological parameters explained 45% of the variation in kindling rate. These results indicate that motility parameters, determined by CASA systems, in combination with sperm morphology analyses can provide some information about the fertilizing potential of rabbit sperm.

USEFULNESS OF SPERM QUALITY ANALYZER-V (SQA-V) FOR THE ASSESSMENT OF SPERM QUALITY IN INFERTILE MEN

The aim of this study was to evaluate the correlation of new of Sperm Quality Analyzer (SQA-V) with the computer-assisted sperm analysis (CASA) and manual semen analysis estimates. One hundred five fresh semen samples were analyzed using SQA-V and CASA and manual semen analysis. Significant correlations of sperm concentration (p < 0.0001), sperm motility (p < 0.0001), and normal morphology (p < 0.0001) were observed between SQA-V variables and manual semen analysis estimates. There also were significant correlations of sperm concentration (p < 0.0001), sperm motility (p < 0.0001), and sperm velocity (p = 0.0235) between SQA-V variables and CASA estimates. Meanwhile, it did not correlate with amplitude of lateral head displacement, beat cross frequency, lineality assessed by CASA. The value of the sperm concentration and the sperm motility measured by SQA-V showed high correlations with the value of those measured by CASA and manual semen analysis. In addition, velocity and sperm morphology may also be evaluated to some extent using SQA-V.

Assessment Of Sperm Quality Analyzer Ii B: Comparison With Manual Semen Analysis And Casa

Two hundred and seven patients with male infertility were investigated. Total sperm concentration and percent progressive motility by SQA IIB showed high correlations with those of conventional manual method. Percent of normal morphology showed a significant correlation among these techniques. The sperm motility index (SMI) and total functional sperm concentration (TFSC) demonstrated high correlations with any variables of manual analysis. Only velocity and amplitude of lateral head displacement showed significant correlations with the variables obtained by SQA IIB, especially with SMI and TFSC. It was suggested that SQA IIB could be a useful instrument in the clinical practice of infertility as a screening test for semen quality.

94 ADDITION OF REDUCED GLUTATHIONE TO THAWING MEDIUM IMPROVED THE SPERM MOTILITY AND REDUCED ROS GENERATION IN FROZEN OVINE AND CAPRINE SPERMATOZOA

The processes of cooling and freezing/thawing produce physical and chemical stress on the sperm membrane, and this stress is associated with oxidative stress and reactive oxygen species (ROS) generation that further reduce sperm viability and fertilizing ability. It is known that the process of freezing is associated with a significant reduction of the intracellular reduced glutathione (GSH) content. The aim of these experiments was to investigate the effects of addition of GSH to thawing extenders on motility parameters and ROS generation in frozen-thawed ovine and caprine spermatozoa. Frozen spermatozoa from eight rams (Ovis aries) and eight bucks (Capra hircus) (generously provided by Ovigen, Zamora, Spain) were thawed in a water bath at 37�C for 30 s and resuspended in sperm-TALP medium (Parrish et al. 1986 Theriogenology 25, 591-600) without (control) and with addition of 1 mM or 5 mM GSH. After 30 min of incubation at 37�C, sperm motility was evaluated using a computer-assisted sperm analysis (CASA) system (SCA, Microptic, Barcelona, Spain). The recorded parameters of motility were: % total, % progressive, curvilinear velocity, straight-line velocity, average path velocity, linearity of the curvilinear trajectory, straightness, amplitude of lateral head displacement, wobble of the curvilinear trajectory and beat cross frequency. Another set of sperm samples was incubated in the presence of (0.7 �M) 22,72-dichlorodihydrofluorescein diacetate (Gadea et al. 2005 J. Androl. 26, 396-404) to estimate production of ROS by flow cytometry. Data were analyzed by two-way ANOVA, considering the specific sperm treatment (GSH addition) and the males as the main variables. In ram frozen spermatozoa, all of the motility parameters were significantly improved when the medium was supplemented with GSH (P &lt; 0.01) with even better results when 5 mM GSH was used. As an example, progresive motility increased from 31.16% (control) to 39.17 and 43.97%, respectively, for 1 and 5 mM GSH. Despite of the male effect detected (P &lt; 0.01), all eight rams studied presented a similar pattern (interaction P &gt; 0.05). The generation of ROS was significantly reduced when GSH was added (6.23a for control vs. 5.32b and 3.85c for 1 and 5 mM, respectively; P &lt; 0.01). In buck frozen spermatozoa, % motility and progressive motility were significantly higher in GSH groups than in the control (P &lt; 0.01), with no differences between 1 and 5 mM GSH. However, for the other motility parameters, the differences were not significant, which probably could be related to differences in the pattern shown by different animals (interaction of buck by treatment P &lt; 0.05). ROS generation was significantly reduced when GSH was added (7.50a for control vs. 4.32b and 2.70b for 1 and 5 mM, respectively; P &lt; 0.01). The addition of GSH to the thawing medium had a positive influence on the parameters studied in both species, increasing the motility patterns and reducing the ROS generation. In conclusion, we can assume that the addition of reduced glutathione to the thawing medium exerts a protective effect on spermatozoa functionality. This work was supported by AGL-2003-03144.

Expression and subcellular localization of the μ-opioid receptor in equine spermatozoa: evidence for its functional role

The development of fertilizing ability in sperm cells is associated with changes in the plasma membrane. However, to date the exact nature of sequentially activated primary receptors and channels and the signal transduction pathways derived from these remains elusive. We analyzed the expression and localization of the mu-opioid receptor in equine spermatozoa. A transcript corresponding to the third extracellular loop that selectively binds mu agonists was amplified, sequenced and compared with the known sequences in humans, rats and cattle. The amplification product showed a high degree of nucleotide conservation. By immunofluorescence, mu-opioid receptor labeling was found on the sperm head and on the tail and disappeared in the acrosomal region of acrosome-reacted sperm cells. Immunoblotting revealed two bands of 50 and 65 kDa. Effects of the opioid antagonist naloxone on motility and on viability and capacitation/acrosome reaction were investigated by computer-assisted sperm analysis and Hoechst 33258/chlortetracycline (H258/CTC) staining. Progressive motility was significantly reduced after 3 h incubation in 10(-3) M naloxone (P <0.05), whereas it increased significantly after 5 h in 10(-8) M naloxone (P <0.05). Sperm velocity at 5 h was significantly reduced by the addition of 10(-3) M naloxone (P <0.05), but increased significantly in the presence of 10(-8) M (P <0.001). Curvilinear velocity and amplitude of lateral head displacement in spermatozoa incubated in the presence of naloxone were not indicative of hyperactivation. H258/CTC staining showed that 10(-8) M naloxone significantly stimulated capacitation (P <0.01) after 3 h. However, it had no effect on sperm cell viability and acrosomal status. Overall, this study provides the first evidence that the mu-opioid receptor is expressed in equine spermatozoa and that naloxone significantly affects motility and capacitation.

Use of Powdered Egg Yolk vs Fresh Egg Yolk for the Cryopreservation of Ovine Semen

Egg yolk is a common additive to sperm cryopreservation diluents. Because of its animal origin, however, it also represents a potential risk of microbiological contamination in the diluent. This potential contamination can be avoided by using powdered egg yolk, instead of fresh egg yolk, as it is pasteurized. This study was conducted to determine ram sperm cryosurvival was affected by the type of egg yolk used (powdered egg yolk or fresh egg yolk) and by yolk concentration (10, 15 or 20%) in the diluent. Microbiological analyses were also performed to quantify the microbiological contamination in the diluents containing the two types of egg yolk. Sperm cryosurvival was determined by motility and morphology analyses after thawing. Motility parameters were assessed using a computer-assisted sperm analysis (CASA) system, and the percentage of sperm with a normal apical ridge was evaluated using a differential interference contrast microscope. No significant differences were observed between diluents in the percentage of sperm with normal apical ridge. However, higher percentages of total motile cells were observed for samples containing powdered egg yolk (69%) compared to samples containing fresh egg yolk (60%). However, sperm in diluents containing fresh egg yolk, exhibited higher values for average-path velocity, straight-line velocity and beat cross frequency and lower values for amplitude of lateral head displacement (p <0.05), compared to cells in diluents containing powdered egg yolk. Microbiological contamination was similar (<200 CFU/ml) in both diluents, and no bacterial growth was observed in either, when antibiotics were added. Therefore, powdered egg yolk can be effective used in diluents for the freezing of ram semen. However, the in vivo fertility of sperm frozen in diluents containing powdered egg yolk should be tested, as some motility parameters were different for sperm treated with powdered egg yolk compared to fresh egg yolk.

The effect of an environmental toxin, perfluorooctanoic acid, on cryopreserved-thawed, human sperm

Perfluorooctanoic acid (PFOA) is the final breakdown product of ammonium perfluorooctanoate, which is used in the production of cookware made with Teflon, food wrappers for the fast food industry, clothing, and products that impart stain resistance in carpet, leather, and fabric. The goal of this study was to evaluate the direct effects of PFOA on human live sperm in vitro. A prospective study evaluating the effects of PFOA on human sperm using cryopreserved-thawed semen samples obtained from a population of donor males. Frozen semen samples were thawed in a water bath at 37oC. The samples were washed × 2 in human tubal fluid with HEPES buffer and 5% synthetic albumin (HHTF) and a post-thaw semen analysis was performed. Only samples that were normal by WHO guidelines were used for this study. Perfluorooctanoic acid was dissolved in HHTF and added to individual tubes containing semen for final PFOA concentrations of 0 (control), 0.5, 5, 50, 250, 500 and 1000μM. The concentrations of PFOA used in this study were chosen based on PFOA concentrations that have been measured in human plasma[0.5 to 250μM] and concentrations known to cause Leydig cell toxicity[250–1000μM]. Samples were evaluated for sperm concentration, motility, progression, and vigor by Computer Assisted Semen Analysis (CASA) at baseline, 1, 3, 5, and 7 hours after the initiation of exposure to PFOA. The primary study outcome was sperm motility over time, both between and within groups. Secondary study outcomes included straight-line velocity (VSL), curvilinear velocity (VCL), and amplitude of lateral head displacement (ALH). For statistical analysis, ANOVA and general linear regression were used to determine statistical significance. Tukey's test was used for post-hoc analysis. There was no change in sperm concentration over time. There was a dose dependent decrease in sperm motility in cryopreserved-thawed sperm treated with PFOA as shown in figure 1. There was no significant change in sperm motility in the control group over time. However, all treatment groups demonstrated a significant reduction in sperm motility from baseline including:[0.5μM] at times 3 and 7 hour (p= 0.02);[5μM] from 1 hour onward (p< 0.01); and[50–1000μM] from 1 hour onward (p< 0.001). There was a significant decrease in VSL in the[1000μM] treatment group at all time periods and a significant decrease in VAP in the[1000μM] treatment group at the 1,3 and 7 hour time periods. There was no difference in ALH for any treatment group over time. This is the first study to demonstrate that PFOA treatment of cryopreserved-thawed sperm leads to a dose dependent decrease in sperm motility in vitro. Whether PFOA causes detrimental effects on sperm function in vivo is a subject of ongoing research by our laboratory.

The effects of vaginal lubricants and moisturizers on computer assisted sperm analysis (CASA) parameters associated with cervical mucus penetration

The incidence of vaginal dryness is increased in trying-to-conceive (TTC) couples; however, numerous papers have cited the detrimental effect of common vaginal lubricants and moisturizers on sperm motility. To date, studies have not been done using CASA to evaluate the effects of lubricant products on the motion characteristics of sperm thought to be associated with cervical mucus transport. Specifically, samples of sperm with mean average path velocity (VAP), % straightness (STR) and Amplitude of Lateral Head Displacement (ALH) exceeding a predetermined level have recently been proposed to have a superior likelihood of good cervical mucus penetration in vivo.

Effects of several Chinese herbal aqueous extracts on human sperm motility in vitro.

The effects of six kinds of aqueous extracts of Chinese herbal medicine (Astragalus membranaceus, Acanthopanacis senticosi, Panax genseng and Ophiopogon japonicus, P. genseng and Aconitum carmichaeli, Salviae miltiorrhiae, Polyporus umbellatus polysaccharide) on sperm motility characteristics of 30 infertile male volunteers were studied in vitro with a computer-assisted sperm analysis at 15, 60 and 180 min after incubated with the drugs. The results showed that per cent viability, number of progressive motile spermatozoa, curvilinear velocity, average path velocity and amplitude of lateral head displacement were significantly enhanced by A. membranaceus (P < 0.05 or < 0.01), per cent viability, average path velocity and amplitude of lateral head displacement were significantly enhanced by A. senticosi (P < 0.05), but all the above were not affected by P. genseng and O. japonicus, P. genseng and A. carmichaeli, S. miltiorrhiae and P. umbellatus polysaccharide. It is suggested that A. membranaceus and A. senticosi can enhance the motility of human spermatozoa in vitro.

Visco-elasticity of seminal fluid in relation to the epididymal and accessory sex gland function and its impact on sperm motility.

Seminal viscopathy was shown to be associated with male infertility. However, our knowledge about the regulatory mechanism of this process is still limited. In semen samples from 411 men attending for fertility assessment, traditional semen parameters including visco-elasticity were assessed according to the World Health Organization guidelines. Sperm motility was evaluated by use of computer aided sperm analysis (CASA). Seminal activity of neutral alpha-glucosidase (NAG) and concentrations of prostate-specific antigen (PSA), zinc, and fructose were measured. The activity of NAG, and the concentrations of PSA and zinc were significantly lower in hyper-visco-elastic semen samples (medians: 5 vs. 8 mU/mL; 741 vs. 924 mg/L; 1 vs. 2 mM/L), than in those with normal visco-elasticity (p = 0.004, 0.005 and 0.011, respectively). When comparing the total amounts, only for seminal fructose there was a difference between samples with high visco-elasticity as compared with those of normal visco-elasticity (median: 74 vs. 53 microM/ejaculate, p = 0.007) This seminal marker was the only significant independent parameter in predicting seminal visco-elasticity in a multiple logistic regression analysis (odds ratio for the highest quartile = 4.67). Hyper-visco-elasticity was associated with a lower percentage of motile spermatozoa (43 vs. 50%, p = 0.045). Similar trend was found for the CASA motility characteristics curvilinear velocity (VCL), average path length (VAP), amplitude of lateral head displacement (ALH) (p = 0.008, 0.038 and 0.020, respectively). Our study demonstrated the interplay between the regulatory effect of post-testicular organs on semen visco-elasticity. Hyper-visco-elasticity was associated with asthenozoospermia and lower levels of VCL, VAP and ALH.

330THE EFFECT OF SHEATH FLUID ON THE QUALITY OF SEX-SORTED RAM SPERMATOZOA

The fertility of sexed frozen-thawed ram spermatozoa at low AI doses remains below commercially acceptable levels (Maxwell WMC et al. 2003 Theriogenology 59, 511 abst). The aim of the present study was to determine if the use of an artificial seminal plasma sheath fluid could attenuate the stress of dilution during flow cytometry and thus improve the functional capacity of sexed ram spermatozoa. Semen was collected from 3 Merino rams, processed for sex-sorting (Hollinshead FK et al. 2002 Reprod. Fertil. Dev. 14, 503–508), and then allocated to 2 treatment groups according to the sheath fluid used within the flow cytometer: (i) a tris-citrate-fructose diluent (TRIS), or (ii) an artificial seminal plasma diluent (ASP) (ODonnell JM 1969 J. Reprod. Fert. 19, 207–209). Spermatozoa were sorted using a modified high speed cell sorter (SX MoFlo®, DakoCytomation, Fort Collins, USA) without separation of X and Y bearing gametes, and then frozen and thawed using established protocols (Hollinshead FK et al. 2002). Motility characteristics (HTM-IVOS; Hamilton-Thorne, Beverly, USA) and acrosome integrity (FITC-PNA) were assessed throughout. Statistical analyses were conducted by ANOVA. Results indicate that in all but one instance (VSL pre-freeze), TRIS spermatozoa exhibited higher (P&amp;lt;0.05) total motility (TM), average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) compared to ASP spermatozoa (Table 1). Conversely, ASP treatments displayed enhanced straightness (STR) and linearity (LIN) pre-freezing and immediately post-thaw. In addition, the percentage of hyperactivated (HA) spermatozoa prior to freezing (± SEM) was lower (P&amp;lt;0.05) for ASP (3±1.1) than for TRIS (20±3.4), although post-thaw the differences were NS. Acrosome integrity remained similar (P&amp;gt;0.05) among treatments before and after thawing (range: 92.3–95.9% intact). In conclusion, the ASP sheath fluid resulted in decreased TM, velocity and ALH of sex-sorted frozen-thawed ram spermatozoa, a clear indication of the superiority of TRIS sheath fluid. The partial improvement in STR, LIN and HA that the ASP affords sex-sorted ram spermatozoa does not offset the aforementioned negatives, suggesting ASP is unsatisfactory for use as a sheath fluid during sperm sorting. Research supported by XY, Inc. Table 1 The effect of sheath fluid on motility characteristics (±SEM)

Key parameters of sperm motion in relation to male fertility in rats given alpha-chlorohydrin or nitrobenzene.

This study was undertaken to detect key parameters of rat sperm motion in relation to male fertility by comparing the differences in sperm motion induced by treatment with alpha-chlorohydrin (ACH), known to produce spermatotoxicity, and nitrobenzene (NTB), known to produce testicular toxicity. Male rats received ACH (5 or 20 mg/kg/day) or NTB (60 mg/kg/day) for either 3 days or 18 days. Epididymal sperm was assessed for motility using a Hamilton-Thorne Sperm Analyzer (HTM-IVOS). Numerical data for statistical analysis and graphical renditions of sperm motion using parameters in radar charts and reconstructed sperm tracks were analyzed to evaluate sperm motion. Males were allowed to copulate with untreated females and cesarean sections were conducted in order to examine the effects of drug administration on male fertility. Linearity of sperm track (linearity (LIN) and/or straightness (STR)) decreased and/or beat cross frequency (BCF) increased only in ACH groups (5 or 20 mg/kg/day), although the percentage of motile sperm, sperm velocities (average path velocity (VAP), curvilinear (VCL), and straight line velocity (VSL)) and amplitude of lateral head displacement (ALH) decreased on Day 18 in both ACH and NTB (60 mg/kg/day) groups. Furthermore, from the individual reconstructed sperm tracks, it was clear that ACH-treated spermatozoa were characterized by abnormal motion ("jerking") with low vigor (low velocities) and little or no forward progression. Finally, only ACH treatment led to a reduction in pregnancy rate or infertility. Therefore, our results suggest that linearity (especially VSL, STR and LIN) in sperm motion is a key parameter for assessing a chemical's potential to induce male infertility.

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 (&amp;lt;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