Assessment Of Sperm Motility Research Articles

Evaluation of human spermatozoa in cervical mucus: comparison of different microscopic and extraction techniques

This study was designed to describe an accurate and consistent microscopic technique for the assessment of sperm number and motility in sperm–cervical mucus samples, such as those of postcoital tests (PCTs), and to identify a suitable method to extract functional spermatozoa from cervical mucus (CM). Sperm–CM preparations containing various sperm concentrations were counted using three different microscopic illuminations. The dark field-Makler technique was compared with the more classical bright field-slide technique currently used by our clinicians. Several sperm extraction techniques were applied first to bovine (BCM) and then to human (HCM) cervical mucus. Dark field microscopic illumination provided accurate, fast, and easy sperm identification. Counting variability was significantly greater with bright field-slide than with dard field-Makler, while sperm motility was always higher with this latter methodology. A high degree of agreement (intraclass correlation coefficient = 0.965) among three raters, i.e., low interobserver variability, was obtained only with dark field-Makler. Extraction procedures based on “swim-out,” Percoll, trypsin, an enzyme cocktail, and mercaptoethanol resulted in small sperm yields in BCM. Mercaptoethanol and trypsin also showed poor sperm recovery in HCM. Among the protocols with the largest yields, the mechanical technique had the largest amount of residual CM, and bromelain reduced sperm motility. The extraction with dithiothreitol (DTT) showed the best results with a mean sperm recovery of 76% and enhanced sperm motility. Sperm viability as well as spontaneous and induced acrosome reaction were conserved in all techniques. In conclusion, use of the dark field-Makler counting technique in combination with DTT extraction of spermatozoa from CM samples, such as those of PCTs, would allow accurate and functional assessment of spermatozoa for preliminary contraceptive efficacy or infertility evaluation.

A high molecular weight glycoprotein in seminal plasma is a sperm immobilizing factor in the teleost Nile tilapia, Oreochromis niloticus.

Sperm that have acquired potential for motility are kept immotile in seminal plasma in the teleost, Nile tilapia. In order to investigate the mechanism of immobilization, several experiments were performed using a previously characterized monoclonal antibody (TAT-30) against a molecular weight (Mr) = 120,000 protein that is secreted by Sertoli cells and epithelial cells of the sperm duct, and is also bound to the head of the spermatozoon. First, we assessed sperm motility in the seminal plasma protein fraction (SPP), and demonstrated that the sperm motility is inhibited by SPP in a concentration-dependent manner. Furthermore, sperm motility was recovered if SPP was pretreated with TAT-30, suggesting that the TAT-30 antigen is one of the components of the sperm immobilizing factor. Calibration by gel filtration followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting with TAT-30 demonstrated that the sperm immobilizing factor was more than Mr = 1,000,000 in seminal plasma, suggesting that it is a homopolymer of the Mr = 120,000-TAT-30 positive protein. Additionally, lectin blot analysis showed that the TAT-30 antigen was reactive with Lens culinarin agglutinin (LCA) and Conavalia ensiformis agglutinin (ConA), indicating that it is a glycoprotein. Immunohistochemical studies showed that the TAT-30 antigen was localized specifically on the heads of spermatozoa and on the apical surface, lysosomes and rough endoplasmic reticulum of Sertoli cells.

Effect of cooling rates on post-thaw sperm motility, membrane integrity, capacitation status and fertility of dairy bull semen used for artificial insemination in sweden

We studied the effects of 2 different cooling rates during equilibration of semen from room temperature to 4 °C, at 4.2 °C/min (control split sample) or at 0.1 °C/min (treatment split sample) on in vitro sperm viability post thawing and fertility after AI. Forty batches of split-frozen semen from 14 dairy bulls (Swedish Red and White breed) aged 14 to 16 mo or 66 to 79 mo were evaluated post-thawing for sperm motility (visual and computer-assisted sperm analysis [CASA], membrane integrity (fluorescent microscopy and flow cytometry post-loading with the combined fluorophores Calcein AM/EthD-1 and SYBR-14/PI); acrosomal status (with Pisum sativum agglutinm [PSA] staining); and capacitation status (CTC-assay). Fertility values (56-d nonreturn rate) of the slow cooling batches (treatment) were 0.4% units higher than for faster cooled (control) batches, but the difference was not statistically significant. Fertility values for the older bulls were 1.6 % units higher than for the group of younger sires. No statistically significant correlations were found between semen viability parameters assessed in vitro and 56-d nonreturn rate. Visually assessed sperm motility, membrane integrity, capacitation and acrosomal status post-thawing did not differ significantly between cooling procedures, however the percentage of motile spermatozoa and the kinetic characteristics of spermatozoa — average path velocity (VAP), straight path velocity (VSL) and curvilinear velocity (VCL) — assessed by CASA differed significantly between cooling procedures. The results indicate that most of the in vitro sperm viability parameters post-thawing and the fertility results for bulls after AI did not differ significantly between the 2 semen cooling procedures tested.

QUALITY OF CRYOPRESERVED TESTICULAR SPERM IN PATIENTS WITH OBSTRUCTIVE AND NONOBSTRUCTIVE AZOOSPERMIA

Sperm retrieved by testicular sperm extraction is routinely used to attempt pregnancy by in vitro fertilization-intracytoplasmic sperm injection. We evaluated the efficacy of cryopreserving testicular sperm collected by testicular sperm extraction at diagnostic biopsy. A total of 73 men with obstructive and 42 with nonobstructive azoospermia underwent testicular sperm extraction at diagnostic biopsy. Sperm was retrieved and cryopreserved in all cases of obstruction and in 15 of nonobstructive azoospermia cases. Before freezing we determined sperm count, motility, morphology and viability, and after thawing we assessed sperm motility and viability. In 17 couples a total of 20 cycles of in vitro fertilization-intracytoplasmic sperm injection were performed and fertilization, cleavage and pregnancy rates were determined in cases of obstruction and nonobstruction. Sperm count and morphology were lower in the testicular biopsies of men with nonobstructive versus obstructive azoospermia. Motility was low or absent in all testicular sperm extraction specimens. Importantly, pre-freeze (63%) and post-thaw (31%) viability was the same in both patient groups. After in vitro fertilization-intracytoplasmic sperm injection using frozen and thawed testicular sperm the fertilization, cleavage, implantation and clinical pregnancy rates were 60, 86, 16 and 50%, respectively. Using cryopreserved sperm we observed no differences in outcome of any in vitro fertilization-intracytoplasmic sperm injection procedure in patients with obstructive versus nonobstructive azoospermia. Cryopreservation of testicular sperm provides enough good quality sperm after thawing to result in excellent in vitro fertilization-intracytoplasmic sperm injection outcomes. Cryopreservation does not adversely affect intracytoplasmic sperm injection outcomes, including pregnancy rate. Therefore, we recommend routine testicular sperm extraction and cryopreservation of sperm at testicular biopsy.

In vitro maturation and fertilization techniques for assessment of semen quality and boar fertility.

The reliability of using different in vitro-derived measures of sperm quality to predict boar fertility was examined. On three occasions during a 20-wk period of breeding, special collections of the first sperm-rich fraction of the ejaculate from six boars were carried out. After in vitro capacitation procedures, three dilutions (5 x 10(5), 1.25 x 10(5), and 3.125 x 10(4) sperm/mL) of these semen samples were used in a standardized in vitro fertilization (IVF) test with oocytes recovered from prepubertal slaughterhouse ovaries and matured in vitro. Routine assessments of sperm motility, concentration, and morphology were also carried out for all collections used for AI during the 20-wk period. Semen from the same ejaculate, processed according to normal commercial practice using the AndroHEP extender, was used to inseminate equal numbers of recently weaned sows with either 3 x 10(9) or 2 x 10(9) total sperm, three times during the estrous period. Data from a total of 444 sows were used to determine boar fertility; between 12 and 54 sows were bred with each semen dose across the six boars. All measures of sperm fertilizing ability in vitro were different among boars (all P < .05) and use of different semen dilutions for IVF allowed further discrimination of apparent sperm quality among boars. The laboratory evaluation of semen collected during the period of breeding indicated effects of boar on ejaculate volume, total number of sperm per ejaculate, motility, and the percentage of sperm with normal morphology (all P < .01). Sperm dose used in AI had no effect on farrowing rate (80.7 vs 81.5%), but the lower AI dose resulted in a reduction (P < .05) in total numbers born (10.8 vs 10.0). For all three semen dilutions, estimated potential embryo production rate accounted for up to 70% of the variation in litter size obtained with 3 x 10(9) sperm per AI dose, and the number of sperm attached per oocyte was a major factor accounting for variation in litter size obtained with 2 x 10(9) sperm per AI dose. These IVF variables may, therefore, be effective indicators of boar sperm quality for use in AI. With 2 x 10(9) sperm per AI dose, the percentage of sperm with normal morphology also explained a large part of the variance in litter size born (R2 = .59), indicating that morphological characteristics are a useful measure of semen quality.

Fluorometric assessments of mitochondrial function and viability in cryopreserved bovine spermatozoa.

Mitochondrial function and sperm viability were quantified in samples of cryopreserved bovine spermatozoa from 12 bulls using fluorometric techniques. The active mitochondria of the spermatozoa were fluorescently stained using three different fluorophores: rhodamine 123 (R123), 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyan ine iodide (JC-1) or MitoTracker Green FM (MITO). The stained spermatozoa, and companion aliquots that had been stained with SYBR-14 (a living-cell nucleic acid stain) and propidium iodide to assess viability, were quantified using flow cytometry. The resulting fluorescent measurements of mitochondrial function were compared with microscopic assessments of progressive sperm motility immediately after thawing, with motility after 3-h incubation at 37 degrees C, and with the fluorescent assessment of sperm viability. Staining with either R123 or MITO resulted in a single green population. In contrast, the JC-1 staining of mitochondria produced both green and red-orange populations of spermatozoa and sometimes a progressive gradient between the two populations. The ability of JC-1 to discriminate between mitochondria exhibiting high membrane potential from those having low to medium membrane potential provided a more rigorous estimate of metabolic function than the other two fluorescent stains. Overall, the three fluorometric measurements of mitochondrial function were highly correlated with each other, with the SYBR-14 assessment of viability, and with the microscopic estimates of motility.

A technique for standardization and quality control of subjective sperm motility assessments in semen analysis

Objective: To establish a quality control method to monitor and eventually to standardize the subjective assessment of sperm motility in conventional semen analysis. Design: Quality control study running over 2 years. Setting: University infertility clinic and andrology laboratory. Patient(s): Randomly chosen patients attending the clinic. Main Outcome Measure(s): Conventional semen analysis with sperm motility assessed by grading according to the World Health Organization (WHO) criteria and analysis of individual sperm tracks with a computer-aided sperm analysis (CASA) system. Formulas were established to identify, from the track data, the threshold velocity values for distinguishing between motility grades a and b and motility grades b and c for each technician. Result(s): The subjective thresholds above which technicians categorized sperm as WHO grades a and b were determined by CASA to be 61 ± 1 and 11 ± 1 μm/s (straight-line velocity), respectively. Agreement among three to five technicians over 2 years was reasonable (coefficient of variation < 20%), but threshold values were variable. Conclusion(s): Agreement within and between laboratories in the assessment of sperm motility grades could be achieved by agreeing on designated values for threshold velocities for grade a and b sperm. On the basis of such values, CASA analysis could be used to provide the expected percentages of grades a, b, and c forms for quality control samples recorded and distributed on videotapes, against which technicians could adjust their subjective assessments.

Påverkan av spermιeantalet per strån på uptιnιngs vιabιhtet och fertιlιtet hos SRB-semιn tjurar

Semen from 5 Swedish Red and White bulls, approved in the fertility and progeny testing programme of a bull center, was split-frozen to produce straws with 15 or 10 x 10(6) spermatozoa each (control and treatment dose, respectively). Post-thaw sperm viability was evaluated by visual assessment of sperm motility (MOT), measurement of ATP (Adeinosin Tri Phosphate) contents by luminometry, assessment of membrane integrity with combined fluorophore probes [Calcein AM (CAM)/Ethidium homodimer (EthD-1)] and by using a hyposmotic swelling test (ORT). The straws were used for a total of 16,651 artificial inseminations (A.I.). No statistically significant difference was recorded between the 2 treatments for any of the post-thaw sperm viability parameters. In addition, a significant bull effect was evident for most post-thaw sperm traits assayed. Significant variation in overall fertility (56-days NRR) was recorded among the bulls used. A.I. with a reduced number of spermatozoa (10 x 10(6)/straw) resulted in a 2%-units decrease (n.s.) compared with controls (67.8% +/- 4.8%, means +/- SD) in overall fertility. In the control split-sample (15 x 10(6) spermatozoa/straw), MOT did not show any statistically significant correlation with fertility (r = 0.41, p = 0.07). However, MOT was correlated with the percentage of spermatozoa depicting progressive motility (category A1, r = 0.45, p < 0.05) as assessed with CAM/EthD-1. The latter was correlated with ATP contents (r = 0.57, p < 0.01), expressed as the percentage of viable spermatozoa. Both CAM/EthD-1 and ATP contents showed a statistically significant correlation with ORT (r = 0.45, p < 0.05 and r = 0.61, p < 0.05, respectively). In the straws with the reduced sperm number (10 x 10(6) spermatozoa), post-thaw motility was significantly correlated with fertility (r = 0.50, p < 0.05) and ATP-total contents (r = 0.48, p < 0.05). CAM/EthD-1 and ATP contents (as million viable spermatozoa) were significantly correlated (r = 0.47, p < 0.05), CAM/EthD-1 was significantly correlated with ORT (r = 0.48, p < 0.05). In conclusion, the results indicated that the freezing-thawing of straws with a reduced sperm concentration (10 x 10(6) spermatozoa) did not alter the post-thaw viability or overall fertility of the bull semen used. However, in view of the significant bull effect found in the limited population studied, we recommend that such a reduction in sperm number/straw be based on the fertility of the bull in question.

An in vivo and in vitro investigation into the effects of α-chlorohydrin on sperm motility and correlation with fertility in the han wistar rat

Following a recommendation from the International Conference on Harmonisation, pharmaceutical companies are now monitoring possible drug effects on sperm motility in the rat during preclinical safety studies by assessing sperm motility (velocity). However, it is not known precisely how changes in sperm motility relate to fertility. Therefore, the effects of α-chlorohydrin on sperm motility were investigated and related to fertility both in vivo and in vitro. α-Chlorohydrin was given orally to male rats using a range of doses: 0, 5, 10, and 20 mg/kg for at least 5 consecutive days. Sperm were then assessed for motility using a standard scoring system (operators' observation of sperm) that graded degree of motility (i.e., 0 = i mmotile to 4 = very motile). The results showed a dose-related decrease in sperm motility. The sperm also appeared to move with a “jerky” action. Surprisingly, when this was correlated to fertility, none of the females mated with treated males became pregnant. A dose-related decrease in pregnancy would perhaps have been expected. There was no effect on sperm morphology, and testicular and epididymal pathology were only seen after doses of 20 mg/kg. When sperm from untreated rats were incubated with α-chlorohydrin in vitro at concentrations of 0, 0.5, 1.0, and 1.5 mM, sperm motility and motion were similarly affected as observed in vivo. However, the fertilization capacity (in vitro fertilization) of the treated sperm showed a concentration-related reduction in percentage fertilization, and there was also evidence of abnormal embryo development. These findings suggest that the present standard scoring system used in preclinical safety studies is not a comprehensive indicator of sperm function and/or fertility. A better understanding of sperm movement, therefore, is desirable so effects on sperm motility can be related to fertility.

Methods for assessing sperm motility, morphology, and counts in the rat, rabbit, and dog: A consensus report

Reproductive toxicity studies are increasingly including assessments of sperm parameters including motility, morphology, and counts. While these assessments can provide valuable information for the determination of potential reproductive toxicity, the methods for conducting the assessments have not been well developed in all laboratories and are continually evolving. The use of different methods in different laboratories makes comparison of data among laboratories difficult. To address the differences in methods, a working group was convened to discuss methods currently in use, share data, and try to reach consensus about optimal methods for assessing sperm parameters in rats, rabbits, and dogs. This article presents the consensus report, as well as future research needs, with the hope that optimized common methods will aid in the detection of reproductive effects and enhance interlaboratory comparisons.

Estimation of sperm viability in frozen-thawed semen from Swedish A.I. bulls.

From a total of 40 Swedish diary A.I. bulls, the number of viable spermatozoa in frozen-thawed semen was estimated using a commercial luminometer (SVT) to determine ATP contents and by fluorescence microscopic examination of samples stained with the combined fluorophore probes Calcein AM and Ethidium homodimer (CAM/EthD-1). The bulls were of two breeds (24 Swedish Red and White and 16 Holstein) and two age classes (1-2 and 6-10 years old, prior to and after progeny testing, respectively). The SVT-instrument was able to estimate a frequency of viable spermatozoa (47.0 +/- 14.4%; means +/- SD) close to that recorded by the fluorophore probes (spermatozoa with intact plasmalemma and progressively motile, 53.6 +/- 11.5%) and by subjective visual assessment of sperm motility (53.4 +/- 6.0%). The percentage of motile spermatozoa assessed under phase-contrast was positively (r = 0.33, P < 0.01) correlated with both the total ATP content, the percentage of viable spermatozoa recalculated from the SVT reading (r = 0.26, P < 0.05) as well as the frequency of spermatozoa having intact membranes as assessed by CAM/EthD-1 (r = 0.45, P < 0.001). The latter was also significantly correlated (r = 0.59, P < 0.001) with the total ATP contents. No significant variation in ATP contents was present between bulls, breeds or age. A significant difference, however, was seen between bulls for sperm membrane integrity. The results indicate that the SVT instrument seems to provide a quick and easy estimation of the number of viable spermatozoa in frozen-thawed bull semen. The assessment of membrane integrity with fluorophore probes allows the estimation of sperm subpopulations within the sample and seems therefore to be more useful and objective to determine sperm viability than visual assessment of sperm motility.

Micromachining: A new direction for clinical analyzers

Abstract

Assessment of sperm function and clinical aspects of impaired sperm function.

Fertility is dependent on a complex set of events, involving both male and female components. Normal sperm function involves many steps, including motility, capacitation, acrosome reactivity and, ultimately, fertilization of the oocyte. While male fertility is most often assessed by means of gross semen parameters, infertility may also be caused by abnormal sperm function, and only by performing specific tests of this function, may the reasons for infertility become evident. Specific tests which may be helpful include semen analysis, detailed sperm motility assessment, motility longevity, hypo-osmotic swelling test, mucus penetration assay, acrosome reactivity, antisperm antibody tests, sperm penetration assay and in vitro fertilization. Relatively well-defined syndromes of abnormal sperm function include immunologic infertility, immotile-cilia syndrome, anejaculation and nifedpine-associated infertility.

Simple Methods for Objective Assessment of Sperm Viability and Motility with MTT Assay and Sperm Quality Analyzer (SQA) in Rats

AbstractTo confirm the applicability of MTT assay and sperm quality analyzer (SQA) to the examination of sperm viability and motility in rats, epididymal spermatozoa derived from male rats (Slc:SD) treated with nitrobenzene at a dose of 60 mg/kg/day for 16 days were examined by these methods. Epididymal fluid was suspended in Dulbecco's modified Eagle's medium containing 25 mM fructose in ratio of 1:125. Sperm concentrations in the epididymal fluid were 2.48 ± 0.55 (× 106/μl, mean ± SD, n = 8) in the control and 2.20 ± 0.54 in the nitrobenzene group, and did not significantly differ between the groups. Both absorbance unit of the MTT assay (1.497 ± 0.406 vs. 1.084 ± 0.350) and SMI (sperm motility index) value of the SQA (204.0 ± 15.3 vs. 182.5 ± 21.6) were significantly decreased in nitrobenzene group. The results suggest that the examinations of sperm viability and motility with MTT assay and SQA are available for the reproductive toxicology in rats.

Assessment of Sperm Viability by Measurement of ATP, Membrane Integrity and Motility in Frozen/Thawed Bull Semen

Control of sperm quality after commercial freezing/thawing of bull semen is still restricted to the subjective assessment of sperm motility, despite its low correlation to fertility (Soderquist et al. 1991, Kjaestad et al. 1993). Although no single in vitro method has yet been designed to predict the fertilizing ability of a given semen sample, the quantitation of viable spermatozoa (with intact plasma and acrosome membranes, and metabolically active) seems to be most promising (Woelders et al. 1991). The present report describes the use of a bioluminiscence technique to determine ATP-levels and a novel supravital stain (using fluorescent dyes) to assess the amount of viable spermatozoa in frozen/thawed semen from 3 A.I. dairy bulls with significantly different motility after thawing.

Synchronous assessment of sperm motility and fertilizing ability in the hamster following treatment with alpha-chlorohydrin.

To investigate the relationship between sperm motion parameters and fertilizing ability, a model was developed to assess both of these endpoints synchronously using a toxicant that inhibits sperm motion. alpha-Chlorohydrin (ACH) was administered daily for 4 days to male hamsters at 0, 33, 49, 66, and 83 mg/kg body weight. These males were then allowed a 45-minute breeding period with untreated estrus females on the morning of day 5. One hour after breeding, sperm samples were surgically recovered from the uteri of the females for motility analysis. Six hours later, eggs were flushed from the oviducts and evaluated for fertilization. Cauda epididymal sperm were also collected from the males shortly after breeding. Proportions of motile and progressively motile sperm were manually quantified, and overall sperm velocity and the velocity of representative vigorously swimming sperm in both the uterine and epididymal samples were measured by computer-aided sperm analysis. Significant decreases in in vivo fertilization rates and epididymal sperm motion parameters were observed at 66 and 83 mg/kg ACH, whereas uterine sperm motion was adversely affected at all ACH dosages used. All sperm motion parameters except the percentage of motile sperm in the epididymis were significantly correlated with fertilization rates by both linear and logistic regression. Overall, uterine and epididymal sperm endpoints predicted fertilizing ability comparably well. Stepwise multiple linear regression gave a model containing epididymal sperm velocity (EVCL) and uterine sperm percent motility (UMOT) with an R2 value of 0.649. Stepwise multiple logistic regression gave models containing EVCL alone and EVCL and UMOT in binary (fertile/infertile) and quantal models, respectively.

Computer assisted semen analysis systems: sperm motility assessment.

Computer assisted semen analysis systems: sperm motility assessment.

Superimposed Image Analysis System (SIAS) software: a new approach to sperm motility assessment

To apply Superimposed Image Analysis System (SIAS) software (Delta Sistemi, Rome, Italy) to the objective analysis of sperm motility. To test a new procedure for sperm motility assessment based on image superimposition. The system can superimpose six sequential frames onto a monitor producing a final image with a motion effect similar to the multiple exposure photography method, but bypassing the film processing stage. Viewing the final image of a complete series of six superimposed frames allows the evaluation of the percentage of motile spermatozoa and their kinetic characteristics. Compared with multiple exposure photography, SIAS has the following main advantages: [1] the procedure is quick; [2] projecting the first frame alone allows the easy evaluation of sperm concentration; and [3] the system allows quick and easy analysis and measurement of each sperm trajectory frame by frame, thus avoiding misleading interpretations because of crossing sperm trajectories, collisions, and overlapping of motile spermatoza with round cells or immotile spermatozoa. Superimposed image analysis is a new approach to sperm motility assessment. It seems to be useful particularly in selected situations, such as oligozoospermia or in cases of increased round cell concentration. In these situations, SIAS can even serve as a support or an alternative to the computer-aided sperm analysis system widely used for objective sperm analysis.

Improved accuracy of sperm motility assessment using a modified Micro-Cell sperm counting chamber

To assess the effect of modifications made in the Micro-Cell sperm counting chamber on the motility of washed human spermatozoa. In a 2 x 2 experimental design, human sperm samples were washed with or without protein supplementation, loaded into modified or unmodified Micro-Cell chambers, and assessed by automated semen analysis for changes associated with sperm adhesion to the glass chamber surfaces. Twenty-one men who were undergoing semen analysis and presented with > or = 50% motile sperm, or normal donors. Reduced motility and elevated lateral head displacement associated with sperm adhesion to glass surfaces were compared using a doubly repeated measures analysis of variance. Addition of protein to the sperm washing solution partially reversed the adhesion of spermatozoa to the glass of unmodified Micro-Cell chambers. Chambers manufactured to reduce cell adhesion to glass surfaces yielded the highest motility and lowest lateral head displacement, whether the sperm were washed in a solution supplemented with or without protein. These findings indicate that, as in raw semen, kinematics of washed sperm can be measured reliably in the modified Micro-Cell chamber.

The routine assessment of sperm motility at room temperature and 37 degrees C.

The World Health Organization (WHO) laboratory manual (1992) states that assessment of sperm motility can be performed at either 37 degrees C or room temperature (20-24 degrees C). The motility of spermatozoa in 44 semen samples (22 fresh samples and 22 frozen-thawed samples) was assessed at both of these temperatures and a significant difference in the motility profiles was noted, specifically an increase at 37 degrees C in the percentage (expressed here as median and ranges) of spermatozoa with excellent progressive motility and an overall increase in the percentage with total progressive motility. With fresh samples the excellent progressive motility increased from 41 (19-53) to 54 (30-66) and the overall motility from 58.5 (39-74) to 65.0 (40-79). With the frozen-thawed samples the excellent motility increased from 14 (1-33) to 25 (6-45) and the overall motility from 30.5 (14-51) to 33.0 (16-52). As the WHO laboratory manual was published 'In response to a growing need for the standardisation of procedures for the examination of human spermatozoa' it is proposed that only one temperature for routine analysis should be used, namely 37 degrees C, which may have more physiological relevance and eliminate effects of fluctuations in ambient laboratory temperature.