Proportion Of Motile Spermatozoa Research Articles

The effect of hyperprolactinemia on epididymal sperm motility in rats

OBJECTIVE: Hyperprolactinemia (hyperPRL) is a pathological status that might interfere the reproductive function in both male and female mammals. This study is to investigate the effects of pathological levels of prolactin (PRL) on epididymal sperm motility in a hyperPRL rat model induced by pituitary glands allografting. DESIGN: Animal study. MATERIALS AND METHODS: Two anterior pituitary glands were grafted to the subrenal capsule of the recipient rats. Stable hyperPRL status was achieved 6 weeks after the surgery. The control rats were grafted with similar amount of cerebral cortical tissues. Totally 8 rats were assigned for each groups. The blood levels of PRL were measured by radioimmunoassay. Six weeks after grafting, the whole epididymis was removed and transferred to a tube containing 2 ml of Medium 199. The epididymis was cut into small pieces by scissors to release the sperm, and 8 ml of buffer was added to the tube. After mixing, 0.6 ml was transferred to a swim-up chamber and it was layered carefully with 6 ml of medium. The chamber was kept standing still for 2 hours at a 34C environment to allow the sperm to swim up. The samples from the top, middle and bottom part of the chamber were measured for count and motility. RESULTS: The over all sperm count showed relatively similar for both groups. The epididymal sperm motility was 11.9 ± 3.4 % for control groups and 4.0 ± 1.6 % for the hyperPRL rats (P< 0.001). The proportion of motile sperm at the top end of the swim-up chamber was also more abundant in the control group. CONCLUSIONS: We concluded from these results that hyperPRL might influence the motility of epididymal sperm. But we still could not be sure whether the detrimental effects are originated from the testis or the epididymis.

Evaluation of fertilizing capacity of cryopreserved rainbow trout sperm

Experimental insemination was performed using artificially produced low-motility sperm. A mathematical model was applied to the results of the insemination in order to clarify the relationship between sperm motility, the density of sperm and the fertilization rate of eggs. In the model, the probability of fertilization by individual spermatozoa was a function of sperm density in the insemination solution. The results showed that the probability of fertilization clearly decreased with increased sperm density, and the maximum possible fertilizing rate by increasing the sperm density was constrained by the proportion of motile sperm (% motility). The model was also applied to the results of insemination tests of cryopreserved sperm in order to evaluate the fertilizing capacity of cryopreserved sperm. It was proven that cryopreserved sperm needed a higher density to obtain the maximum fertilization rate compared with fresh sperm, and it was anticipated that the ratio of the motile inseminated cryopreserved sperm should be more than 5.0% to achieve an egg fertilization rate greater than 90%.

Constant decline in sperm concentration in infertile males in an urban population: experience over 18 years

To investigate semen quality in infertile men from an urban population over an 18-year period. Retrospective study. Andrology clinic at a city university hospital. A total of 9,327 men were referred to the clinic for infertility investigation. After excluding those with azoospermia, 7,780 samples were evaluated. Semen samples were analyzed within half an hour of production using computer-automated semen analysis and phase microscopy. Sperm concentration, morphology, and motility; semen pH and leukocyte concentration; and patient age. The median patient age over the study period was 31.6 years, with an increase from 30.8 years in 1986 to 34.4 years in 2003. The median sperm concentration was 10.25 million/mL for the entire period, with a decline from 27.75 million/mL in 1986 to 4.60 million/mL in 2003. The median proportion of normally shaped spermatozoa was 15%, and the proportion of motile spermatozoa was 21%. The pH increased from 7.4 in 1986 to 7.9 in 2003, and the median leukocyte concentration was 1.50 million/mL. A constant decline in median sperm concentration was found in the infertile men. The reason for this remains unclear, although the high pH and leukocyte concentration indicates the involvement of infection.

Gametes Alter the Oviductal Secretory Proteome

The mammalian oviduct provides an optimal environment for the maturation of gametes, fertilization, and early embryonic development. Secretory cells lining the lumen of the mammalian oviduct synthesize and secrete proteins that have been shown to interact with and influence the activities of gametes and embryos. We hypothesized that the presence of gametes in the oviduct alters the oviductal secretory proteomic profile. We used a combination of two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry to identify oviductal protein secretions that were altered in response to the presence of gametes in the oviduct. The oviductal response to spermatozoa was different from its response to oocytes as verified by Western blotting. The presence of spermatozoa or oocytes in the oviduct altered the secretion of specific proteins. Most of these proteins are known to have an influence on gamete maturation, viability, and function, and there is evidence to suggest these proteins may prepare the oviductal environment for arrival of the zygote. Our findings suggest the presence of a gamete recognition system within the oviduct capable of distinguishing between spermatozoa and oocytes.

Dietary fish and evening primrose oil with vitamin E effects on semen variables in cockerels

1. Our aim was to determine the effect of n-3 (2%, wt/wt, fish oil rich diet) and n-6 (2%, wt/wt, evening primrose oil rich diet) fatty acid dietary supplementation and their combination with two concentrations of vitamin E (40 vs 200 mg/kg) on semen variables and on fatty acid and vitamin E profiles of spermatozoa in broiler breeders at 32, 42 and 52 weeks of age. 2. The inclusion of fish oil in the cockerel diets increased the docosahexaenoic acid proportion in the sperm phospholipid fraction, which was almost threefold higher compared to the other two groups irrespective of vitamin E supplementation. 3. In contrast, an increase in the proportion of total n-6 polyunsaturates, mainly 22:4n-6, was observed in the evening primrose oil group compared to the control only when the dietary content of vitamin E was increased to 200 mg/kg. 4. Sperm concentration was decreased in the fish and evening primrose oil groups if vitamin E was 40 mg/kg, but such an effect was prevented in the fish, not the evening primrose oil group, by increasing the vitamin E to 200 mg. 5. The proportion of motile spermatozoa was improved by the increased supplementation of vitamin E in all oil treatments.

Exposure of actin on the surface of the human sperm head during in vitro culture relates to sperm morphology, capacitation and zona binding

The aim of this study was to determine the relationship between the proportion of motile sperm with actin exposed on the surface of the head and sperm function. Semen samples were obtained from normozoospermic men and sperm function tests were performed. Motile sperm selected by swim-up were incubated with actin monoclonal antibody (A-mAb, 1:100) for 2 h, then anti-mouse IgG Dynabeads were used to detect sperm-bound A-mAb. Sperm capacitation was increased by phorbol myristate acetate (PMA) and decreased by bicarbonate-free medium. The proportion of sperm with exposed actin increased with time for up to 2 h incubation. Bicarbonate-free medium significantly decreased the proportion of sperm with exposed actin. PMA significantly enhanced this phenomenon. Sperm bound to zona pellucida (ZP) had a significantly higher proportion with exposed actin than did sperm remaining in medium. Of the 79 samples studied, an average of 9.4% (range 1-27%) of motile sperm had exposed actin after 2 h incubation and this was significantly correlated with sperm normal morphology and ZP binding. Exposure of actin on the surface of the sperm head during in vitro culture may be related to membrane modification during sperm capacitation and hence may be a useful marker for this subpopulation of sperm.

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.

Relationship between sperm density, spermatocrit, sperm motility and spawning date in wild and cultured haddock

Semen was collected repeatedly from captive haddock Melanogrammus aeglefinus and the effect of seasonality on various sperm parameters was investigated. No differences in sperm traits were observed for wild and cultured haddock. A highly significant positive relationship existed between spermatocrit and spermatozoa density. A significant increase in mean spermatocrit occurred throughout the spawning season but the amount of variability explained by collection date was low (35·1%) due to variability between males. Each of 10 males sampled repeatedly throughout the spawning season demonstrated an increase in spermatocrit. No relationship existed between spermatocrit and proportion of motile spermatozoa when spermatocrit was ≤70%. Motility was reduced in semen samples with spermatocrits &gt;70%. The proportion of spermatozoa that were motile decreased with time since activation. Some motility was still observed after 60 min in sea water (0·1–15·2%) for sperm collected at all times within the spawning season. Of those spermatozoa that were motile, the proportion that exhibited forward swimming motion decreased and the proportion that had only vibratory movement increased with time post‐activation. The speed of forward swimming spermatozoa showed no significant relationship with spermatocrit at any time between 0 and 60 min after activation. Swimming speed was negatively related to time since activation, decreasing from 174–240 μm s−1 at 0 min to 80–128 μm s−1 at 60 min after activation.

Effect of medium on the kinematics of frozen-thawed ram spermatozoa.

Cervically inseminated cryopreserved ram spermatozoa have reduced fertility due to poor mucus-penetrating ability. This effect is ameliorated by the addition of 20% (v/v) seminal plasma (SP) to the phosphate-buffered saline (PBS) thawing medium. The aims of this study were to determine whether the impaired mucus penetration was due to alterations in the sperm motility and, if so, whether these alterations were due to the SP or its viscosity, or to the medium components. To this end, artificial SP medium (ASP), a medium which supports motility but not capacitation, was compared with PBS and SP. Thawed, pooled semen from seven mature rams was layered under 1 ml each of PBS, SP and ASP and motile spermatozoa allowed to swim up (37 degrees C, 30 min). Upper regions of the overlays were harvested, and the capacitation status of the spermatozoa in each suspension determined by chlortetracycline (CTC) analysis. Sperm movement was videotaped in 300 microm chambers for both computer-aided sperm analysis assessment and manual flagellar curvature analysis. There was no effect of the culture medium on the concentration of spermatozoa recovered by swim up, nor on the proportion of motile spermatozoa. However, the spermatozoa resuspended in PBS did show changes associated with capacitation in both the CTC-binding patterns and in their movement patterns. These changes were significantly greater than those observed in spermatozoa resuspended in SP or ASP. These results indicated that the differences in sperm movement and function observed in SP medium were not due to changes in viscosity, but rather to components of the medium.

Effect of simulated microgravity on testosterone and sperm motility in mice.

We examined changes in the serum testosterone level and in sperm in the testis and epididymis by using tail-suspended mice, which are a simulation model of the body fluid shift in space, to evaluate the possibility of spermatogenesis failure in space environment. We also studied pathological disorders of the testis in the tail-suspended mice. Tail suspension was imposed with a tail harness to a degree at which the hindlegs of mice did not touch the floor of the housing unit. In control mice, the tail was similarly fixed with a tail harness to impose the same stress, except that a hindleg remained on the floor. Body weight was not significantly different between the 2 groups during 7 days, and testicular weight was significantly different. The testosterone level was significantly lower in the tail-suspended group (0.71 +/- 1.24 ng/mL) than in the control group (2.38 +/- 3.50 ng/mL; P <.05). Microscopy with hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) staining showed a small proportion of seminiferous tubules with impairment of spermatogenic function in the tail-suspended group, and multinucleated giant cells were occasionally noted. Terminal deoxynucleotidyl tranferase-mediated nick end-labeling staining revealed positive cells even in animals in which impairment was considered to be mild based on HE and PAS staining. Many cells showed intense p53 immunostaining compared to the control group, with more intense staining of the nucleus in the tail-suspended group. The proportion of motile sperm was slightly but not significantly reduced in the tail-suspended group. However, the mean movement velocity of the motile spermatozoa was significantly decreased.

Regional differences in semen quality in Europe

Recent reports have indicated a decrease in semen quality of men in some countries, and suggested regional differences. A study was undertaken of semen samples from 1082 fertile men from four European cities (Copenhagen, Denmark; Paris, France; Edinburgh, Scotland; and Turku, Finland). Semen analysis was standardized, inter‐ laboratory differences in assessment of sperm concentration were evaluated, and morphology assessment centralized. Lowest sperm concentrations and total counts were detected for Danish men, followed by French and Scottish men. Finnish men had the highest sperm counts. Men from Edinburgh had the highest proportion of motile spermatozoa, followed by men from Turku, Copenhagen and Paris. Only the differences between Paris/Edinburgh and Paris/ Turku were statistically significant (P &lt; 0.003 and P 0.002 respectively). No significant differences in morphology were detected. A general seasonal variation in sperm concentration (summer 70% of winter) and total sperm count (summer 72% of winter) was detected. Semen quality of a ‘standardized’ man (30 years old, fertile, ejaculation abstinence of 96 h) were estimated. Topically, sperm concentrations (x 106/ml) for winter/summer were: Turku 132/93; Edinburgh 119/84; Paris 103/73; and Copenhagen 98/69. These differences in semen quality may indicate different environmental exposures or lifestyle changes in the four populations. However, it remains to be seen whether such changes can account for these differences. These data may also serve as a reference point for future studies on time trends in semen quality in Europe.

Variability in the size of the nucleus in spermatozoa from Houbara bustards, Chlamydotis undulata undulata.

Semen collected from 3-year-old male Houbara bustards contained large proportions (6-40%) of spermatozoa with large nuclei. In these spermatozoa, the length of the nucleus was up to twice the mean length of the nucleus in normal spermatozoa. The lengths of the acrosome, midpiece and flagella were all normally distributed, but the length of the nucleus formed a bimodal distribution. The proportion of spermatozoa with large nuclei varied among males, but not among different semen samples collected from the same male throughout the breeding season. The proportion of motile spermatozoa with large nuclei was half that of normal spermatozoa, but their velocity was significantly greater. After insemination into females, spermatozoa with large nuclei were observed in the outer perivitelline layer of eggs laid, indicating that they were stored and transported within the oviduct and reached the egg at about the time of fertilization. Furthermore, there was no difference in the ability to produce viable progeny in females that were mated with males producing greater proportions of spermatozoa with large nuclei compared with those producing 'normal' spermatozoa. Thus, the abnormal spermatozoa did not appear to impede fertility. There were no signs of triploidy in the males that produced spermatozoa with large nuclei, or in their progeny, as demonstrated by the size of erythrocytes. Therefore, it appears that the spermatozoa with large nuclei were the result of aberrant spermatogenesis.

Variations in sperm motility of the Atlantic herring Clupea harengus

Sperm characteristics and fertilization success were measured in two groups of Manx autumn-spawning herring, Clupea harengus L., captured 1 wk apart. Samples contained motile sperm for up to 45 min after activation (average 7 min). Individual spermatozoa were motile for up to 5 min. The activation of individual spermatozoa is probably phased, although the mechanism for the delay is not clear. This can account for the overall low levels of active sperm at any one time, the periodic fluctuations observed in the proportion of motile sperm, and for successful fertilization rates achieved using samples of apparently immotile sperm. The proportion of motile sperm was not affected by the presence of eggs, but the duration of motility was longer when sperm was activated in the presence of eggs rather than in seawater alone. Individual males differed significantly in the duration of motility in their sperm samples and in the fertilization rates achieved, but not in the proportion of motile sperm in each sample. There were no consistent changes in the characteristics of the spawners or the sperm between the two sample dates. Sperm motility and duration of motility were significantly correlated, but none of the sperm characteristics measured was significantly related to fertilization success.

Sperm motility patterns of Atlantic cod (Gadus morhua) in relation to salinity: effects of ovarian fluid and egg presence

A significantly greater proportion of Atlantic cod (Gadus morhua) sperm were motile in the presence of ovarian fluid and egg compared with seawater. Sperm mean and maximum swimming speeds were also significantly greater in the presence of ovarian fluid and egg compared with seawater. However, there was no significant difference in path linearity among treatments. We also examined the interaction between maternal stimuli (egg and ovarian fluid), time sperm spent in seawater prior to exposure to each stimulus, and salinity on proportion of motile sperm and their swimming behaviours. Proportion of motile sperm decreased significantly with a decrease in salinity in the presence of both maternal stimuli. Proportion of motile sperm also decreased with time spent in seawater prior to exposure to ovarian fluid but not to an egg. Both mean and maximum swimming speeds were maintained in the salinity range of 20-30 ppt and only declined significantly at lower salinities (15 and 17.5 ppt). Mean and maximum swimming speeds decreased with time spent in seawater. This study demonstrates the importance of maternal factors when examining sperm swimming behaviour and highlights the significance of maternal-paternal gametic interactions in understanding fertilization success.

Canine spermatozoa — cryopreservation and evaluation of gamete interaction

Successful cryopreservation of spermatozoa for genome banking or transport for articifial insemination (AI) in domestic and nondomestic Canidae depends on understanding the species-specific conditions required. Experiment 1 was designed to evaluate the post-thaw effects of 5 cooling rates on percentages of motile cells, progressive motility, morphology and acrosomal integrity of domestic dog spermatozoa. Semen samples (n = 18) extended in glycerol-Tris egg yolk buffer were cooled to 0°C, drawn into 0.5-ml straws and subjected to A) cooling at 0.5 °C/min to − 20 °C and at 2 °C/min from −20°C to −70°C, B) cooling at 12 °C/min, C) cooling at 28°C/min, D) cooling at 99 °C/min, and E) cooling at 214 °C/min. Samples were thawed by placing straws into 70 °C water for 6 sec. The contents were poured into microcentrifuge tubes, kept at 0 °C, and evaluated at 0, 2, 4 and 6 h. Overall, the smallest decline in the proportion of motile spermatozoa and in the progressive motility score was with the 12 and 28 °C/min freezing rates, and the greatest with the 214 °C/min rate. There were no differences in morphology of spermatozoa among the protocols, but the proportions of viable spermatozoa and of undamaged acrosomes were highest for the 12 and 28 °C/min rates. In Experiment 2 the capacity of fresh spermatozoa to penetrate fresh, cooled or salt-stored canine oocytes in vitro was evaluated. Canine oocytes harvested from ovaries were either immediately placed into TALP (fresh ova), or into hypertonic salt solution (saltstored ova): for cooled oocytes the ovaries were stored in PBS with BSA at 5 °C overnight before the ova were harvested. Oocytes and spermatozoa were co-incubated for 24 h, stained with Hoechst 33258 and examined under fluorescent light at x 400. Penetration and attachment of spermatozoa to the zona did not differ significantly between fresh and cooled oocytes (73 and 65%; 4.1 and 2.9 sperm/ovum, respectively), but cooled ova tended to show fewer attached spermatozoa. Salt-stored oocytes showed the lowest number of spermatozoa penetrating or attaching to the zona (0.7 sperm/ovum; P < 0.001), and only 36.5% of the oocytes were penetrated (P < 0.02). The results suggest that the optimum cooling rate for freezing canine spermatozoa is ~30 °C/min and that canine spermatozoal function can be evaluated by measuring their penetration of or attachment to homologous oocytes.

The steroid pheromone 4-pregnen-17,20ss-diol-3-one increases fertility and paternity in goldfish.

Previous studies in goldfish (Carassius auratus) showed that the oocyte maturation-inducing steroid 4-pregnen-17,20ss-diol-3-one (17,20ssP) functions after release as a pheromone that increases male serum gonadotropin II (GtH II) concentration, milt (sperm and seminal fluid) volume and sexual activity, effects hypothesized to increase male reproductive success in the sperm competition of multi-male spawnings. The present study tested this hypothesis by determining whether overnight exposure to 17,20ssP increases fertility. In pair spawnings, 17,20ssP-exposed males fertilized a greater percentage of eggs than did control males, apparently because 17,20ssP-exposed males had more releasable sperm at the onset of spawning. Microsatellite DNA paternity analysis showed that 17,20ssP-exposed males also fertilized more eggs in competitive spawnings involving one control male and one 17,20ssP-exposed male. This effect of 17,20ssP on competitive fertility could be due to demonstrated increases in spawning activity, milt volume, duration of sperm motility and proportion of motile sperm. However, it appears that a change in sperm quality is a major component of the pheromonal effect because, in competitive in vitro fertilizations, sperm from 17,20ssP-exposed males fertilized more eggs than did sperm from control males. The results indicate that the response to pheromonal 17,20ssP is a major determinant of reproductive success in male goldfish.

Frequently subnormal semen profiles of normal volunteers recruited over 17 years.

In order to establish semen profiles for men in the general population, we analysed the semen parameters of 187 men attending the Institute of Reproductive Medicine between 1977 and 1993 as volunteers for clinical studies. If several ejaculates were obtained from the volunteer, only the first ejaculate was used for analysis. More than half of the ejaculates of these healthy men showed at least one abnormal parameter, so that only 46% of the volunteers could be classified as being 'normozoospermic' according to WHO guidelines. Aside from a decrease in the proportion of motile spermatozoa, no change in semen parameters could be detected with advancing age of the volunteers. No negative influence of the presence of a varicocele, the grade of varicocele on a history of maldescended testis on semen parameters was detected. The serum levels of FSH, LH and testosterone showed no significant age-related change over the period of investigation. Follow-up analysis of the fertility on the volunteers revealed a proportion of unwanted barrenness similar to or even lower than that in the general population. The analysis demonstrates that the limits of normality for semen parameters may require redefinition.

Evaluation of a modified sperm penetration test in ram semen

A modified sperm penetration test, based on the movement of spermatozoa into a capillary tube containing 300 mM Tris-28 mM glucose (TG) solution, was developed for evaluating ram semen. Sperm penetration distance increased from 4.8 mm at 1 × 10 9 sperm/ml to 7.4 mm at 2.5 × 10 9 sperm/ml (P < 0.01) and, with time, from 3.5 at 5 min to 8.1 at 20 min (P < 0.001). A semen concentration of 2.0 × 10 9 sperm/ml (diluted as necessary with TG) and a penetration time of 15 min (a compromise between high sperm penetration distance and speed of operation) are recommended for routine tests. Sperm penetration distance was validated by the fact that it was highly correlated to both the known proportions of motile spermatozoa in the test samples (r = 0.76 to 0.90; P < 0.001) and, in the aged, cold-shocked, diluted-stored, and frozen-thawed semen samples to most parameters from a computerized Hamilton Thorn Motility Analyzer (r = 0.51 to 0.93; P < 0 05 to < 0.001). The modified sperm penetration test described here is thus a useful addition to the means available for evaluating ram semen. The low cost of the test and the fact that it does not require electric power as well as and the availability and nature of Tris and glucose make the technique particularly useful for field work and in developing countries.

A spectrophotometric method for the assessment of vigour of ram semen

An ideal method of assessment of semen would be simple, rapid and objective with a high correlation with the ability of semen sample to cause conception in the ewes (Ahangari, 1992). Currently the proportion of motile spermatozoa is determined by visual microscopic appraisal. Although, this method of assessment is rapid and easy, it is subjective. Jasko et al (1989) described a spetrophotometric procedure for measuring equine spermatozoan motility objectively. In the present study a modification of the method of Jasko et al (1989) was tested and applied to ram semen.

Andrology: Density adjustment of software settings minimizes bias in automated sperm motility estimation

To minimize overestimation of motility, it is recommended that fresh semen be diluted with seminal plasma prior to automated analysis. However, for glycerolated or cryopreserved semen this is impractical, and alternative methods are needed to minimize automated motility bias. In the present study, the proportion of motile spermatozoa was determined in fresh, diluted and cryopreserved semen (n = 25 ejaculates) using visual and automated methods. The effect of software settings on motility was investigated by assessing samples at a range of modified settings. At standard settings, automated motility was biased in fresh semen (+7.2%) after dilution with cryopreservative (-2.9%) and after cryopreservation (-7.8%) (P < 0.0001 versus visual). Automated motility was inversely related to the minimum number of frames for motility sampling (P < 0.0001), with mean estimates of 41.0, 46.1, 52.0 and 58.2% generated at settings of 8, 4, 2 and 1 frame(s) respectively (n = 15 fresh, diluted and cryopreserved samples). Based on an arbitrary ordinal scale, a method was developed whereby motility sampling was adjusted prior to analysis according to sperm density. Analysis of an independent set of semen samples with density-adjusted software settings reduced bias in automated estimates (n = 30) before and after freezing (P < 0.0001). In addition, bias was no longer related to sperm density. In conclusion, modification of software settings is an effective alternative to dilution to minimize bias in automated motility estimates in fresh, diluted and cryopreserved human semen.