Straightness Research Articles

In vitro toxicity of mercuric chloride on rabbit spermatozoa motility and cell membrane integrity

In this in vitro study the effects of mercuric chloride on the motility and structural integrity of rabbit spermatozoa were investigated. The spermatozoa motility was evaluated using CASA method and Annexin analysis was used for detection of structural changes. The concentration of mercury in the medium varied from 5.0 to 83.3 μ g HgCl2/mL. At Time 0 the highest motility was detected in the control group (67.09 ± 8.72%). Motility in groups with mercury administration was lower in comparison with control. Significant differences were detected in groups with 50.0–83.3 μ g HgCl2/mL (P < 0.001) at Time 0. After 60 and 120 minutes of incubation with mercuric chloride the motility significantly decreased almost in all experimental groups. Progressive motility had a decreasing trend in all experimental groups. At time 60 and 120 significant differences were noted in the group receiving 6.25–83.3 μ g HgCl2/mL. Significant differences were detected in all experimental groups, except the group with the lowest mercuric chloride administration. The concentration-dependent decrease of spermatozoa progressive motility up to 50% of control was detected for groups receiving 50.0 – 83.3 μ g HgCl2/mL at Time 0, for groups receiving 12.5–83.3 μ g HgCl2/mL at Time 60 and 120, decreasing from 36.46 ± 18.73% to 1.03 ± 2.50%. Detailed evaluation of spermatozoa distance (DAP, DCL, and DSL) and velocity (VAP, VCL, and VSL) parameters as well as straightness (STR), linearity (LIN), wobble (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) of spermatozoa revealed decrease in groups with the highest mercury concentration in comparison with the control group at all time periods. Detection of spermatozoa with disordered membrane was carried out for groups with higher mercury concentrations and control, using Annexin analysis. Analysis showed higher occurrence of positive spermatozoa in the mercury exposed groups. Some Annexin positive reactions from all spermatozoa were detected in the control group. In mercury-exposed groups positive reaction proved alteration in anterial part of head (acrosome), connection part (connection piece) and in mitochondrial segment. Detected data evidently confirm adverse effects of high mercuric chloride concentrations in rabbit semen on spermatozoa motility parameters.

In vitro gossypol induced spermatozoa motility alterations in rabbits

The objectives of the present study were to: (i) examine the in vitro dose response of rabbit spermatozoa motility to the antifertility agent gossypol (GOS) and (ii) determine whether filtered (FIL) and unfiltered (UNFIL) GOS differ in their magnitude of effect. Rabbit semen belonging to adult males (n = 5; 12–14 months) were cultured with UNFIL GOS and FIL GOS (5% solution) and subsequently diluted (1:1–7) for analysis using a Computer Assisted Semen Analyzer (CASA) system in 5 time periods (0, 60, 120, 180 and 360 minutes). At Time 0, no significant change in rabbit spermatozoa motility (MOT) and progressive motility (PROG) with GOS FIL was noted, while increases were observed with GOS UNFIL. At Time 60, weak changes were noted for MOT and PROG. After 120 minutes of culture with both GOS FIL and GOS UNFIL, MOT and PROG decreased significantly in some experimental groups. However, no differences were recorded for both the parameters at Times 180 and 360, with the exception of PROG in the GOS UNFIL category (groups A, B, E, F and G), where a significant decrease was noticed. Detailed evaluation of the distance and velocity parameters revealed reduction in all these studied markers after 60 and 120 minutes of in vitro culture with both GOS FIL and GOS UNFIL, indirectly confirming the PROG decrease. Straightness (STR), linearity (LIN), wobble (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) mostly remained unaltered at all time periods for GOS FIL, where as some minor alterations were noticed in GOS UNFIL category for STR, LIN, WOB, ALH and BCF parameters at Time 0, 60 and 120. The present study confirms the dose and time dependent alterations of rabbit spermatozoa motility parameters by GOS. The GOS dynamics in our experiment shows that rabbit spermatozoa as a biological material can indicate a GOS inhibition of motility. Obtained data for the first time indicates a higher immobilizing potential of unfiltered GOS in comparison to filtered GOS in its inhibitory action of spermatozoa motility parameters in rabbits.

Assessment of hyperactivation, acrosome reaction and motility characteristics of spermatozoa from semen of men of proven fertility and unexplained infertility.

Semen from men of proven fertility was compared with that of men with unexplained infertility to determine differences in spermatozoal functions such as hyperactivation and acrosome reaction and spermatozoal motility characteristics. The hyperactivated spermatozoa in both groups could be visualised on the monitor of the Computer Assisted Semen Analyser and they exhibited 'circling', 'thrashing', 'starspin' and 'helical' motility patterns and the mean hyperactivation rates were not significantly different. However, 20% of the men with unexplained infertility did not exhibit hyperactivation compared to only 4% in the fertile group. Furthermore, the semen from infertile men when evaluated for hyperactivation could be categorised into two groups with those having lower hyperactivation (< 10% or < 6% after 4 and 6 h of incubation respectively), forming the first group, and those having a higher hyperactivation rate constituting the second group. In the fertile men such distinct groups were not visible and the percentage hyperactivation ranged from 1 to 16%. No significant differences were observed in the rate of acrosome reaction of fertile and unexplained infertile men. The non-hyperactivated spermatozoa from unexplained infertile men showed a significant increase in path velocity (VAP), curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) and a decrease in linearity (LIN) and straightness (STR) compared to spermatozoa from fertile men. Furthermore, the hyperactivated spermatozoa from infertile men also showed an increase in progressive velocity (VSL) (only after 2 h of incubation) and LIN and decrease in ALH and beat cross frequency (BCF) compared to spermatozoa from fertile men. The results are discussed in the light of the importance of the above spermatozoal functions and spermatozoal parameters in fertilization.

Computer-aided sperm analysis (CASA) parameters and its relationship to conventional in-vitro fertilization rates

OBJECTIVE: The CASA is able to measure multiple values for sperm motility. Included within these measurements is VAP (average path velocity), VSL (straight line velocity), VCL (curvilinear velocity), ALH (amplitude of lateral head), BCF (beat cross frequency), STR (straightness), and LIN (linearity). The purpose of this study is to determine if sperm parameters, as calculated by CASA, on sperm used for conventional in-vitro fertilization (cIVF), predict fertilization outcome in cIVF cycles.

Genetic variation in growth, stem straightness and branch thickness in clonal trials of Acacia auriculiformis at three contrasting sites in Vietnam

Acacia auriculiformis has become an important species in Vietnam because it displays adaptability to a wide range of site conditions and produces pulp wood and sawlogs on short rotations. The aim of this study was to estimate genetic parameters and consider implications for development of clonal forestry. Clonal repeatabilities, genotypic correlations, genotype by environment interactions (G × E), genetic selection gains and correlated responses were estimated for total height (HT), diameter at breast height (DBH), stem volume (VOL), straightness (STR), and branch thickness (BRA) measured in three clonal trials of Acacia auriculiformis A. Cunn. ex Benth. in northern, central and southern Vietnam. Assessments were made at age 1, 2 and 3 years on all three trials and also at age 4 years in one of the trials. The estimates of the genetic parameters were based on a total of 130 clones, of which 115 were selected from a progeny test, and 15 were selected from plantations of the Coen River (Queensland, Australia) provenance in Vietnam. Seventy of these clones were planted in all three trials. The clonal repeatabilities for all studied traits increased with age. The repeatabilities at the latest assessment (3 and 4 years old) of the trials ranged from 0.28 to 0.47 for HT, 0.21–0.56 for DBH, 0.21–0.54 for VOL, 0.21–0.32 for STR, and 0.21–0.28 for BRA. Genotypic coefficients of variation ranged from 8.3 to 13.1% for HT and DBH and from 24.6 to 32.6% for VOL. Age–age genotypic correlations were strong for growth traits but ranged more widely from 0.22 to 0.98 for STR and BRA. Straightness and BRA correlated positively with growth traits, with these correlations ranging from 0.13 to 0.76. Low genotypic correlations between the same traits measured in the different trials at age 3 years indicated that G × E was important for this species in Vietnam. Selection of 10% of the clones resulted in gain varying from 14.3 to 23.0% for HT and DBH. Our results indicate that using HT and DBH as selection traits together with STR and BRA would be beneficial in clonal selection of A. auriculiformis for deployment in Vietnam.

Polymorphism of transferrin of carp seminal plasma: Relationship to blood transferrin and sperm motility characteristics

Transferrin (Tf) is a major protein of carp ( Cyprinus carpio) seminal plasma. Its relationship with milt quality is unknown. In this study, we sought to determine if Tf is polymorphic in carp seminal plasma and if this polymorphism is related to sperm motility characteristics. We screened males of purebred common carp line (Polish line R6) for Tf polymorphism in blood plasma. The majority of Tf genotypes represented only DD and DG variants. We then collected milt from preselected DD and DG genotypes and tested their sperm motility characteristics using computer-aided sperm analysis (CASA). Tf polymorphism in seminal plasma was found to be identical with that of blood. However, the relationships between Tf polymorphism and iron metabolic parameters were different for blood and semen. These data suggest different regulation of Tf in liver and testis. We found substantial differences in sperm motility characteristics between both genotypes. Spermatozoa of DG males were characterized by lower curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), higher linearity (LIN) and straightness (STR) of movement as compared to DD males. No differences were found in other sperm characteristics such as sperm concentration and percentage of sperm motility. Our results suggest that sperm motility parameters are related to Tf polymorphism and therefore this polymorphism may be related to sperm competitive ability.

12 COMPUTER-ASSISTED SPERM ANALYSIS OF FRESH EPIDIDYMAL CAT SPERMATOZOA AND THE IMPACT OF COOLED STORAGE (4°C) ON SPERM QUALITY

Feline epididymal sperm is commonly used for in vitro fertilization. It also yields the opportunity to conserve genetic material from valuable males that suddenly die. Epididymal sperm quality parameters vary considerably among laboratories, implicating the need for objective evaluation methods. The aim of the present study was to describe reference values of computer-assisted sperm analysis (CASA) parameters of fresh epididymal cat sperm and to assess the effect of prolonged cooled storage (4�C) on various sample characteristics. Epididymides obtained from tomcats after routine orchiectomy (2–4 pairs/replicate) were sliced to release spermatozoa. The sperm suspension was placed on a 2-layer gradient and, after centrifugation, the sperm pellet was recovered. In Experiment 1 (20 replicates), sperm motility parameters were assessed immediately after retrieval (T0) using the Hamilton Thorne analyzer Ceros 12.1 (HTR; Hamilton Thorne Biosciences, Beverly, MA, USA). In Experiment 2, fresh (T0) sperm samples (4 replicates) were evaluated for motility parameters (HTR), acrosomal status (FITC-Pisum sativum agglutinin staining), morphology (eosin/nigrosin (E/N) staining), and membrane integrity (E/N and SYBR�-14-propidium iodide staining; Molecular Probes, Inc., Eugene, OR, USA). After addition (1:2) of a Tris-glucose-citrate diluent containing 20% egg yolk, samples were cooled and reassessed on Days 1 (T1), 3 (T3), 5 (T5), 7 (T7), and 10 (T10). Results were analyzed in a mixed linear model, with replicate as random factor and time as fixed effect (S-PLUS 7.0; Insightful Corp., Seattle, WA, USA). Results of Experiment 1 were as follows (mean � SD): motility (MOT): 80.8% � 23.5; progressive motility (PMOT): 69.9% � 23.2; velocity average pathway (VAP): 98.7 µm s–1 � 24.2; velocity straight line (VSL): 89.3 µm s–1 � 25.4; velocity curved line (VCL): 134.8 µm s–1 � 31.9; amplitude lateral head (ALH): 4.3 µm � 2.0; beat cross frequency (BCF): 34.6 Hz � 7.0; and straightness (STR): 89.6% � 6.6. In Experiment 2, MOT, PMOT, VAP, VSL, VCL, BCF, and the percentage of normal spermatozoa showed a decrease over time (P &lt; 0.05) compared to fresh samples, starting from T1, T3, T5, T7, T5, T3, and T1, respectively. In contrast, STR, ALH, membrane integrity, and the percentage of acrosome-intact spermatozoa were not affected (P &gt; 0.05) by cooled storage. To summarize, we have presented a set of reference values for CASA-parameters of fresh, epididymal cat spermatozoa. Cooled storage impaired most motility parameters and lowered the percentage of normal spermatozoa, but did not influence membrane integrity or acrosomal status. The effect of cooled storage on DNA fragmentation of sperm and its subsequent influence on in vitro embryo development require further investigation.

162 FREEZING OF SEMEN FROM ENDANGERED ASTURIANA DE LA MONTAÑA BULLS IN ZWITTERONIC LIPIDS-BASED EXTENDERS

This experiment was designed to test the efficacy of 2 different preparation protocols of zwitteronic soyabean-origin lipids for the production of lipidsglycerol liposomes for use in bull semen cryopreservation. Lipids liposomes were prepared at 10% concentration in Tris buffer by 1. highpressure homogenization (Panda 2K, Parma, Italy) and then 8% glycerol were added, extender-1 (E-1); Lipids were homogenized together with glycerol, extender-2 (E-2). Bioxcell extender (E-3) was used as control. Semen was collected 3 times from 3 endangered Asturiana de la Monta�a bulls by the means of an artificial vagina. Ejaculates with at least 70% motility were processed further by a standard freezing protocol used in our AI station. Semen was diluted at 37�C with each of the 4 extenders to a concentration of 92 � 106 spermatozoa per mL, cooled to 4�C over 4 h, aspirated into 0.25-mL plastic straws (IMV Technologies, Aigle, France), frozen in a bio-freezer (IMV Technologies) in 3 steps from 4 to –140�C, and then plunged into liquid N2. Straws were thawed in a water bath at 37�C for 30 s. Sperm motility was analyzed microscopically immediately after collection, after dilution, and after 4, 24, 48, and 72 h of storage at 4�C. Post-thaw semen progressive motility was assessed microscopically, and sperm movement characteristics were analyzed by computer-assisted semen analysis (CASA) (SCA�, Microptic, Barcelona, Spain). Data were compared between extenders and bulls by 2-way ANOVA; percentages were transformed by arcsine transformation before ANOVA. Total and progressive sperm motility at 0 h after dilution ranged from 90 to 70% and was not different between extenders and bulls. There was no difference between bulls in total and progressive motility after 24 h of cold storage; however, both were significantly greater (P &lt; 0.05) for Control (62.4 � 14.7 and 41.4 � 14.9) and E-1 (70.1 � 12 and 33.8 � 7.0) extenders than for the E-2 extender (22.5 � 17 and 1.2 � 1.3). Average post-thaw sperm motility was not different between bulls for either extender, but motility for Bioxcell (Control, 48.1 � 14.6%) and E-1 extenders (43.2 � 13.0%) were significantly greater (P &lt; 0.05) than for E-2 extender (18.7 � 8.8%). There were no differences between bulls for all kinematic semen parameters: curvilinear (VCL), straight line (VSL), average path (VAP) velocities, linearity (LIN) and straightness (STR), evaluated by CASA before and after freezing; however, all were lower (P &lt; 0.05) for the E-2 extender and not different between Control and E-1 extenders. Sperm movements derived from heads (VCL) and linearity of sperm(LIN), both closely related to field fertility, were in the range of 90.9 � 2.1 and 63.0 � 5.5 for E-3 (Control) extender; 99.1 � 3.4 and 49.4 � 3.5 for E-1; and 21.8 � 2.2 and 29.9 � 4.0 for E-2. In summary, zwitteronic soyabean lipid liposomes are an effective egg yolk substitute for the cryopreservation of Asturiana de la Monta�a bull semen; however, the homogenization protocol of the lipids-glycerol mixture must be improved.

Development of a novel CASA system based on open source software for characterization of zebrafish sperm motility parameters

Although computer-assisted sperm analysis (CASA) outperforms manual techniques, many investigators rely on non-automated analysis due to the high cost of commercial options. In this study, we have written and validated a free CASA software primarily for analysis of fish sperm. This software is a plugin for the free National Institutes of Health software ImageJ and is available with documentation at http://rsb.info.nih.gov/ij/plugins/casa.html. That it is open source makes possible external validation, should improve quality control and enhance the comparative value of data obtained among laboratories. In addition, we have improved upon the traditional velocity straight line (VSL) algorithm, eliminating inaccurate characterization of highly curved fish sperm paths. Using this system, the motion of zebrafish ( Danio rerio) sperm was characterized relative to time post-activation and the impact of acquisition conditions upon data analysis determined. There were decreases in velocity and path straightness (STR), but not linearity (LIN), relative to time. From 30 to 300 frames/s, frame rate significantly affected curvilinear velocity (VCL) and STR measurements. Sperm density in the field of view did not affect any measured parameter. There was significant inter-male variation for VCL, VSL, velocity average path (VAP), percent motility, path character (STR, LIN), and duration of motility. Furthermore, relative sperm output (a measure reflecting both semen volume and concentration) was positively correlated to percent motility. For all motion parameters measured (except duration), the average CV was ≤10%, comparable to values obtained using commercial systems.

7 HETEROSPERMIC INSEMINATION AT TWO SPERM CONCENTRATIONS IN TIMED AI: CASA SEMEN PARAMETERS AND PREGNANCY RATES

The latest entry in the field of semen evaluation is computer assisted semen analysis (CASA). Heterospermic insemination has been used to increase pregnancy rates from low fertile bulls. The objective of this study was to evaluate, with the aid of CASA, heterospermic semen characteristics and pregnancy rates using different concentrations of bull semen in a timed artificial insemination protocol. Semen was collected from two bulls of known fertility by artificial vagina and all CASA motility parameters were evaluated individually and combined. Straws were filled using a semi-defined semen extender (Andromed, Minitüb, Tiefenbach, Germany) as follows: single bull A and single bull B (12 × 106 of progressive motile cells after thawing); Mixed bull semen: A + B (12 × 106 of progressive motile cells after thawing) and Supermix bull semen: A + B (28 × 106 of progressive motile cells after thawing). All cows received a P4 intravaginal device (DIB, Syntex, Argentina) and 2 mg of EB i.m. (Syntex) on Day 0, 500 mg cloprostenol (Estroplan, Syntex) at the time of DIB removal (Day 8), and 1 mg EB i.m. on Day 9. Fixed-time insemination (FTAI) was performed at 52 to 56 h after DIB removal. A total of 249 cows were randomly allocated to be inseminated with bulls A and B (n = 76), with Mixed A + B (n = 87), and with Supermixed A+B at a high concentration (n = 86) by a single inseminator. Pregnancy rates were evaluated at 38 days after insemination by transrectal ultrasonography. Means and standard deviations or each characteristic were calculated, compared, and statistically analyzed. The following sperm motility parameters were determined with the Ceros 12.1 sperm analyzer (Hamilton Thorne Biosciences, Inc., Beverly, MA, USA) on at least 1000 spermatozoa: velocity average path (VAP), velocity straight line (VSL), curvilinear velocity (VCL), amplitude lateral head (ALH), beat cross frequency (BCF), straightness (STR), linearity (LIN), and percentage of rapid or static cells. There were no significant differences (P &gt; 0.05) in VAP, VSL, VCL, ALH, STR, or LIN. There was a numerically higher percentage of rapid cells in the Supermix semen. Pregnancy rate from bulls A and B was 61% and from Mixed A + B 60%, while that from Supermixed A + B was 69%. Results from the analysis indicate that semen concentration is an important element to be considered in a timed artificial insemination program. Numerically higher pregnancy rates were obtained with double semen concentration in the straw. More research is required to evaluate the interaction between different breeds within a timed artificial insemination program. This research was supported by Centro Genético Bovino de EOLIA sa and Syntex sa Argentina.

12 CASA PARAMETERS OF FRESH BULL SEMEN COLLECTED BY ARTIFICIAL VAGINA OR ELECTROEJACULATION IN ARGENTINA

The latest entry in the field of semen evaluation is computer assisted semen analysis (CASA). Its greatest advantages are elimination of the subjective nature of routine semen evaluation and the addition of detailed motion analysis unquantifiable by visual examination. The objective of this study was to evaluate CASA motility parameters of fresh bull semen collected by artificial vagina (AV) or electroejaculation (EE) from a total of 56 beef different bulls. Semen samples from a total of 45 beef bulls were collected by AV from winter to the end of spring (740 collections), and from 11 beef bulls by EE (120 collections) in the same period. First and second AV collections were analyzed as individual data. EE collection was performed only one. Means and standard deviations for each characteristic were calculated, compared, and statistically analyzed. A sample of the collection was diluted 1:20 in a semi-defined semen extender (Andromed, Minitüb, Tiefenbach, Germany) and held in a glass tube at 36°C for 5 min before analysis. The sample was loaded into 20-μm chambers, and six microscope fields from each chamber were analyzed. The following sperm motility parameters were determined with the Ceros 12.1 sperm analyzer (Hamilton Thorne Biosciences, Inc., Beverly, MA, USA) on at least 1000 spermatozoa:concentration (CONC), velocity average path (VAP), velocity straight line (VSL), curvilinear velocity (VCL), amplitude lateral head (ALH), beat cross frequency (BCF), straightness (STR), linearity (LIN), and percentage of rapid or statics cells. There were no significant differences (P &gt; 0.05) in VAP, VSL, VCL, ALH, STR, LIN, and the percentage of rapid and static cells of semen collected by AV or EE. The concentration (sperm/mL) of the AV-collected sperm was significantly higher than for the sperm collected by EE. Results from the analysis indicate that semen collected by artificial vagina have motility characteristics similar to those collected by electroejaculation. More research needs to be done to evaluate motility parameters of frozen/thawed semen collected by electroejaculation and by artificial vagina. This research was supported by Centro Genético Bovino de EOLIA sa Argentina.

238 ASSESSMENT OF SPERM MOTILITY, VIABILITY, AND FERTILIZATION RATE OF TWO GROUPS OF BULLS SHOWING A DIFFERENT ABILITY TO PROMOTE EMBRYO DEVELOPMENT IN VITRO

Low developmental rates after IVF with some AI bulls are one of the most frequent problems encountered with in vitro production of bovine embryos. In this study, two groups of high and low in vitro fertility bulls (3 bulls per group) were selected based upon blastocyst rates. We first evaluated sperm motion characteristics using computer-assisted sperm analysis (CASA) before and after Percoll separation. The following parameters were compared between bulls: proportion of motile sperm (% MOT), curvilinear velocity (VCL), and straightness (STR), which allowed us to evaluate the proportion of progressive semen (VCL &gt; 80 μm/s and STR &gt; 60%). Concerning % MOT, differences between bulls were observed before Percoll separation (42 to 59% – ANOVA2: P = 0.017). Following separation, the values of % MOT were globally increased (ANOVA3: P &lt; 0.001) and no further difference was observed between bulls (68 to 76%). The same pattern was obtained for the proportion of progressive spermatozoa (before: 43 to 55% vs. after: 48 to 57% – ANOVA3: P = 0.005). For viability measurements, two DNA-binding fluorochromes, propidium iodide and Hoechst 33342, were used, which respectively stain dead sperm nuclei in red and living sperm nuclei in blue. This viability evaluation was carried out after 0, 2, 6, and 18 h of incubation in TALP medium supplemented with heparin (10 μg/mL), under the same conditions as IVF but without oocytes. In the two groups of bulls, a sharp decrease in the proportion of living sperm was already observed at 2 h (only 50 to 20% of living sperm), while, at 18 h, 17 to 9% of sperm were still intact. Two bulls from the high in vitro fertility group showed a percentage of living sperm significantly more important at 6 and 18 h than the other 4 bulls (chi square – P &lt; 0.05). In a third experiment, we assessed the fertilization (2 pronuclei) and the polyspermy (&gt;2 pronuclei) rates after staining the zygotes with Hoechst at 18 hpi. The high fertility group showed higher fertilization rates than the low fertility one (75 to 80% vs. 59 to 67% – ANOVA2: P &lt; 0.008). No significant difference was noticed between bulls concerning polyspermy. The present results confirm the efficiency of Percoll separation in enhancing some of the motility parameters such as the proportion of motile and progressive sperm. Moreover, this gradient allows us to abolish differences between bulls for those motility parameters. However, the viability and the rate of fertilization still varied between bulls after Percoll separation. In our study, only fertilization rates seemed related to the blastocyst rates. Further investigations are under way to better understand the sperm characteristics of these two groups. This work was supported by the Minestery of Agriculture of the Region wallonne de Belgique.

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.

Boar sperm velocity and motility patterns under capacitating and non-capacitating incubation conditions

This study compares the velocity and motility of boar sperm under capacitating and non-capacitating incubation conditions. Aliquots of pooled, washed boar sperm were incubated in either Tyrode’s complete medium (TCM; a capacitating medium), Ca 2+-free TCM (TCM-Ca 2+), or Ca 2+ and NaHCO 3-free TCM (Tyrode’s basal medium [TBM]; a non-capacitating medium). Motility patterns were determined every hour over a 3 h period of incubation at 38 °C. Capacitation status was assessed by the chlortetracycline assay after 1 and 3 h of incubation. Experiments were repeated five times. Compared to the TBM control, a significant increase was seen in the percentage of capacitated sperm after 1 h of incubation in TCM: the kinematics of these sperm cells were favorably modified. However, the motility patterns of sperm cells incubated in TCM and TCM-Ca 2+ were very similar. Under capacitating conditions (TCM), the coefficients of linearity (LIN) and straightness (STR) significantly increased over time (LIN values were significantly different after 3 h of incubation, while STR values were significantly different after only 2 h). Significant correlations were seen between LIN and the percentage of cells showing the B pattern ( r = 0.334, P < 0.05) and the number of acrosome reacted spermatozoa ( r = 0.301, P < 0.05). This suggests that capacitated boar spermatozoa may have a species-specific motility pattern.

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.

10CASA EVALUATION OF SEXED AND NON-SEXED FROZEN BULL SEMEN

Flow citometry cell sorting has been proven successfully to separate X and Y sperm; however, the technology is still too stressfull for the viability of the sorted semen. The objective of this study was to evaluate nonsexed and sexed frozen sperm motility characteristics using a CASA technology. Ejaculates from 4 different bulls (3 Holstein and 1 Angus) were collected, and processed as split non-sexed and sexed semen samples using Tris egg yolk extenders. X and Y sperm were separated using a high-speed sorter (SX Moflo). Cryopreservation was done at the same time under appropiate conditions using a programmed cryochamber. Thawing procedure was done at 37°C for 30s and a sample of each straw was placed in the evaluation chamber. The experiment was repeated twice and two chambers with 30 observations each were analyzed each time. Mean and standard deviation of each characteristic were calculated, compared and analyzed statistically. The sperm concentration was determined by means of a burker counting chamber. Sperm quality was determined at 0h after thawing, and later at 1h, 2h and 3h after incubation in a glass tube at 30°C. The following sperm motility parameters were determined with the Hamilton Thorne (HTM-ceros 12.1) on at least 1000 spermatozoa: velocity average path (VAP), velocity straight line (VSL), amplitude lateral head (ALH), beat cross frequency (BCF), straightness (STR), linearity (LIN), and percentage of progressively motile spermatozoa (PMS). Linearity of nonsexed spermatozoa was 53±3.5, 47±0.8, 43±7.8 and 42±4.5 for the 0h and the 3 test incubation times and 49.5±3.7, 51.2±3.7, 43.3±7.8 and 44.5±7.6, respectively, for sexed semen. There were no significant differences (P&amp;gt;0.05) in the progressive velocity, track speed and linearity between sexed and nonsexed semen. The percentage of static cells was 33%, 30%, 47% and 50% for the 0h and the 3 test incubation periods; however, the percentage of static cells for the sexed semen was 53%, 71%, 77% and 82%, respectively. Results from the analysis indicate a significant increase (P&amp;lt;0.01) in the number and the percentage of static cells with time. The lateral amplitude of sperm motility for nonsexed semen was 5.9±0.5, 6.8±0.8, 6.0±0.4 and 5.1±0.7, and for sexed semen 6.6±0.7, 6.8±0.4, 6.4±0.4 and 5.5±1.7, respectively. The percentage of progressively motile sperm was significantly different at 0 time 49.7±4.9 and 23.1±4.9 for nonsexed and sexed semen respectively. After 3 hours of incubation the percentage of progressively motile sperm was 38.7±10.2 and 3.7±3.2 for nonsexed and sexed semen, respectively. In conclusion, sexed frozen semen seems to have characteristics similar to those of normal nonsexed semen. However, a significant decrease in the percentage of progressively motile cells could affect pregnancy rates. More research needs to be done to detect differences between bulls and cryoprotectans.Research supported by Centro Genetico Bovino de EOLIA sa Argentina.

Activity of angiotensin-converting enzyme (ACE) in reproductive tissues of the stallion and effects of angiotensin II on sperm motility

A testis-specific isoform of angiotensin-converting enzyme (ACE) has been identified in a number of mammalian species. The purpose of this study was to characterize the activity of ACE in equine spermatozoa, seminal plasma, and testis. Activity of ACE was determined in seminal plasma, ejaculated and epididymal spermatozoa from mature stallions as well as from pre- and postpubertal testis. The effect of addition of angiotensin II on equine sperm motility was also evaluated. The activity of ACE in detergent extracted sperm plasma membrane was approximately 13-fold higher than that detected in seminal plasma (93.7 mU/mg versus 7.0 mU/mg protein, respectively). Activity of ACE in equine testis was significantly higher in postpubertal than in prepubertal males (3.0 mU/mg versus 0.4 mU/mg protein, respectively), and ACE activity was reduced ( P<0.001) in a dose-dependent fashion by the addition of captopril. The effect of angiotensin II on sperm motility was evaluated by computer-assisted semen analysis in sperm incubated with angiotensin II (0, 1, 10, 100 nM) at 38.5 °C. There was no significant effect of angiotensin II on the percent motile sperm; however, there was a significant main effect of angiotensin II ( P<0.01) on the kinematic parameters beat cross frequency (BCF), average path velocity (VAP), and curvilinear velocity (VCL), respectively. In addition, there were significant stallion×concentration interactions for amplitude lateral movement (ALH), BCF, linearity (LIN), straightness (STR), and VCL. This study demonstrates that ACE activity is present in sperm membrane from ejaculated and epididymal spermatozoa and in postpubertal testis. Further studies are required to determine the role of this testis-specific enzyme.

Reactivation of motility of demembranated hamster spermatozoa: role of protein tyrosine kinase and protein phosphatases.

Demembranated cauda epididymidal spermatozoa of hamster, following reactivation with 1 mm ATP, exhibited either a loop or planar type of motility. The spermatozoa with planar motility exhibited increased progressive velocity (VSL), straightness (STR), linearity (LIN) and beat cross frequency (BCF) compared to the spermatozoa with loop type motility. cAMP was observed to have differential effects on the motility parameters of the demembranated spermatozoa depending on the type of motility. For instance, in the loop type, average path velocity (VAP), curvilinear velocity (VCL) and VSL were increased in the presence of cAMP unlike in the planar type. Furthermore, in an attempt to understand the role of protein kinases and protein phosphatases in regulation of sperm motility, the effects of various inhibitors of these enzymes on the motility and phosphorylation of proteins of reactivated demembranated spermatozoa were studied. Inhibitors of PTKase (protein tyrosine kinase) and protein phosphatases inhibited the motility of reactivated demembranated hamster spermatozoa. The activity of the respective enzymes associated with demembranated spermatozoa was concurrently inhibited, thus providing evidence that the effect of the inhibitors on motility was mediated through their inhibitory effects on the activities of the enzymes. The results also demonstrated that two phosphotyrosinylated proteins of molecular weight 65 and 80 kDa showed reduced phosphorylation in the presence of PTKase inhibitors, thus indicating their possible role in reactivation of motility of demembranated hamster spermatozoa.

Overview of studies on rat sperm motion analysis using a Hamilton-Thorne Sperm Analyzer--collaborative working study.

This collaborative study was conducted to determine the utility and sensitivity of nine sperm motion parameters generated by a Hamilton-Thorne Sperm Analyzer (HTM-IVOS) for detecting adverse effects of chemicals on sperm motion in rats. The efficacy of sperm motion parameters was investigated using nine reproductive toxicants: adriamycin, alpha-chlorohydrin (3 different studies were carried out), dinoseb, ethylene glycol monoethyl ether, 2,5-hexanedione, sulfasalazine, trimethyl phosphate, and ornidazole. The percentage of motile sperm (% motile sperm), the only parameter expressing the status of semen containing non-motile sperm, detected adverse effects on sperm motion in 9 out of 10 studies. However, weak effects on sperm motion were not detected by this parameter in 4 out of 7 studies in which sperm motion disorders were noted at medium or low dosages. The percentage of progressively motile sperm (% progressive sperm) and the sperm velocity parameters (average path velocity, straight line velocity, and curvilinear velocity) detected adverse effects on sperm motion in all studies. In 7 studies which noted sperm motion disorders at medium or low dosages, weak effects on sperm motion were detected by the % progressive sperm in 5 studies and by the sperm velocity parameters in 6 studies. In 10 studies, amplitude of lateral head displacement (ALH) did not detect adverse effects on sperm motion in 4 studies, and beat cross frequency (BCF) failed to detect adverse effects on sperm motion in 3 studies. Because ALH and BCF show the swimming pattern of spermatozoa as head movement, the characteristics of these parameters are different from the % progressive sperm and the sperm velocity parameters. Straightness (STR) and linearity (LIN), which are secondary parameters calculated from sperm velocity parameters, could not detect adverse effects on sperm motion when the sperm velocity parameters did not detect adverse effects. On the basis of these results, we concluded that the % progressive sperm and sperm velocity parameters are useful and sensitive indicators for detecting adverse effects on sperm motion. However, in the % progressive sperm, setting up a suitable threshold of VAP and/or STR is important to gain further sensitivity for detecting adverse effects on sperm motion. The % motile sperm is useful for assessment of sperm motion disorder, and ALH and BCF are useful for evaluating the swimming pattern of sperm. STR and LIN are not very useful for detecting adverse effects on sperm motion.