Computer-assisted sperm analysis of canine spermatozoa motility measurements

Computer-assisted sperm analysis (CASA) allows for the determination of specific motion characteristics of sperm cells in vitro. This study was designed to develop a system for the use of CASA to objectively evaluate canine sperm motility, and specifically to determine whether motility characteristics vary between individual dogs. Ejaculates from 10 dogs were collected weekly. Sperm cells were extended in a glucose-free TALP medium, placed on slides and videotaped at 200x. Videotaped samples were then analyzed by the Hamilton-Thorn Motility Analyzer, with 100 cells evaluated per slide. Two slides were made from each ejaculate. Motility characteristics that were evaluated included lateral head displacement, beat cross frequency, path velocity, path linearity, path straightness, percentage of motile cells, and percentage of progressively motile cells. Sperm cell morphology was also evaluated. Canine spermatozoa maintained good overall motility (mean +/- SD, 73 +/- 9%) during the procedure. Mean sperm motility and morphology measurements differed significantly between dogs (P<0.01). There was no difference (P>0.05) between the mean measurements of different ejaculates for an individual dog, or for different slides made from the same ejaculate. Mean motility values for the 10 dogs are reported. There was a significant but not strong correlation (r=0.44) between the percentage of progressively motile sperm cells and the percentage of sperm cells with normal morphology.     

Additional value of computer assisted semen analysis (CASA) compared to conventional motility assessments in pig artificial insemination

In order to obtain a more standardised semen motility evaluation, Varkens KI Nederland has introduced a computer assisted semen analysis (CASA) system in all their pig AI laboratories. The repeatability of CASA was enhanced by standardising for: 1) an optimal sample temperature (39 °C); 2) an optimal dilution factor; 3) optimal mixing of semen and dilution buffer by using mechanical mixing; 4) the slide chamber depth, and together with the previous points; 5) the optimal training of technicians working with the CASA system; and 6) the use of a standard operating procedure (SOP). Once laboratory technicians were trained in using this SOP, they achieved a coefficient of variation of < 5% which was superior to the variation found when the SOP was not strictly used. Microscopic semen motility assessments by eye were subjective and not comparable to the data obtained by standardised CASA. CASA results are preferable as accurate continuous motility dates are generated rather than discrimination motility percentage increments of 10% motility as with motility estimation by laboratory technicians. The higher variability of sperm motility found with CASA and the continuous motility values allow better analysis of the relationship between semen motility characteristics and fertilising capacity. The benefits of standardised CASA for AI is discussed both with respect to estimate the correct dilution factor of the ejaculate for the production of artificial insemination (AI) doses (critical for reducing the number of sperm per AI doses) and thus to get more reliable fertility data from these AI doses in return.     

Exposure to the seminal plasma of different portions of the boar ejaculate modulates the survival of spermatozoa cryopreserved in MiniFlatPacks

Spermatozoa present in the first collectable 10 mL of the sperm-rich fraction (SRF) of the boar ejaculate (portion 1, P1) have higher documented viability during and after cryopreservation than spermatozoa in the rest of the ejaculate (portion 2, P2), probably in relation to different features of the surrounding seminal plasma (SP). In the present study, we investigated whether the SP from these ejaculate portions (SP1 or SP2) was able to differently influence sperm viability and chromatin structure of the P1- or P2-contained spermatozoa from individual boars primarily or secondarily exposed (e.g., following cleansing and re-exposure) to pooled SP1 or SP2 from the same males during 60 min. Spermatozoa were subjected to controlled cooling and thawing in MiniFlatPacks (MFPs) and examined for motility (using computer-assisted sperm analysis, CASA) at selected stages of processing. Moreover, sperm plasma membrane intactness (investigated using SYBR-14/propidium iodide, PI), plasma membrane architecture (examined using Annexin-V-PI staining), and chromatin (deoxyribonucleic acid, DNA) integrity (tested using sperm chromatin structure assay, SCSA) were assessed post-thaw (PT). A higher proportion of P1 spermatozoa than of P2 spermatozoa incubated in their native SP portion were confirmed to be motile from collection to PT. When P1 spermatozoa were cleansed from their original SP and re-exposed to pooled P2-SP, sperm kinematics deteriorated from extension to PT. By contrast, cleansed P2 spermatozoa increased motility to P1 levels, especially PT when re-exposed to pooled P1-SP. Such differential effects on motility were not clearly accompanied by biologically related modifications of sperm membrane or chromatin structure. This influence of the SP on sperm kinematics was not sire-dependent and it was presumably related to different concentrations or either SP proteins or bicarbonate in the different ejaculate portions.     

Season of ejaculate collection influences the freezability of boar spermatozoa

The aim of this retrospective study was to evaluate whether the season of ejaculate collection influences the freezability of porcine sperm. A total of 434 ejaculates were collected from boars of six different breeds over three years (2008–2011) and throughout the four seasons of the year identified in the northern hemisphere (winter, spring, summer and autumn). The ejaculates were cryopreserved using a standard 0.5 mL straw freezing protocol. Sperm quality was assessed before (fresh semen samples kept 24 h at 17 °C) and after freezing and thawing (at 30 and 150 min post-thawing in semen samples kept in a water bath at 37 °C), according to the percentages of total motility, as assessed by the CASA system, and viability, as assessed by flow cytometry after staining with SYBR-14, PI and PE-PNA. The data, in percentages, on sperm motility and viability after freezing and thawing were obtained at each evaluation time (recovered) and were normalized to the values before freezing (normalized). The season of ejaculate collection influenced (P < 0.01) sperm quality before freezing and after thawing (recovered and normalized), irrespective of the breed of boar. Sperm quality was lower in summer, both in terms of motility and viability, and in autumn, in terms of motility, than in winter and spring. Seasonality in the normalized data indicates that the season of ejaculate collection influences sperm freezability, regardless of the season’s influence on sperm quality before freezing. Consequently, the spermatozoa from ejaculates collected during summer and, to a lesser extent, also in autumn, are more sensitive to cryopreservation than those from ejaculates collected during winter and spring.     

Motility and fertility of the subtropical freshwater fish streaked prochilod (Prochilodus lineatus) sperm cryopreserved in powdered coconut water

Streaked prochilod (Prochilodus lineatus) is a freshwater fish inhabiting many South American rivers. The objective was to determine the effectiveness of coconut water (ACP™), combined with methylglycol, as a freezing medium for streaked prochilod sperm. A secondary objective was to compare a computer-assisted sperm analyzer (CASA) system versus subjective microscropic examination as a means of assessing sperm motility. As a control, glucose and methylglycol was used, according to our previous study. Sperm diluted in each medium was loaded into 0.5 mL straws, frozen in liquid nitrogen vapor (in a dry shipper), and stored in liquid nitrogen (-196 °C). Half of the samples were evaluated for sperm motility, both subjectively and with CASA; the remainder were evaluated for fertility. There was no difference (P > 0.05) between subjective or CASA assessment of post-thaw sperm motility. Although sperm motility was higher in sperm cryopreserved in ACP™ (85%) than in glucose (75%), cryopreservation in either extender yielded similar fertilization rates (46-48%) and sperm velocities. There were positive correlations (r = 0.56-0.8) between all sperm velocities and fertilization rate. In conclusion, streaked prochilod sperm cryopreserved in glucose or ACP™ and methylglycol was fertile, and thus could be used for research or commercial settings. Furthermore, although the CASA system provided objective data regarding sperm motility, in the present study, subjective evaluation of sperm motility was practical and a good indication of sperm quality; it could readily be done by well-trained personnel under field or laboratory conditions.     

Microfluidic mixing for sperm activation and motility analysis of pearl Danio zebrafish

Sperm viability in aquatic species is increasingly being evaluated by motility analysis via computer-assisted sperm analysis (CASA) following activation of sperm with manual dilution and mixing by hand. User variation can limit the speed and control over the activation process, preventing consistent motility analysis. This is further complicated by the short interval (i.e., less than 15 s) of burst motility in these species. The objectives of this study were to develop a staggered herringbone microfluidic mixer to: 1) activate small volumes of Danio pearl zebrafish (Danio albolineatus) sperm by rapid mixing with diluent, and 2) position sperm in a viewing chamber for motility evaluation using a standard CASA system. A herringbone micromixer was fabricated in polydimethylsiloxane (PDMS) to yield high quality smooth surfaces. Based on fluorescence microscopy, mixing efficiency exceeding 90% was achieved within 5 s for a range of flow rates (from 50 to 250 μL/h), with a correlation of mixing distances and mixing efficiency. For example, at the nominal flow rate of 100 μL/h, there was a significant difference in mixing efficiency between 3.5 mm (75 ± 4%; mean ± SD) and 7 mm (92 ± 2%; P = 0.002). The PDMS micromixer, integrated with standard volumetric slides, demonstrated activation of fresh zebrafish sperm with reduced user variation, greater control, and without morphologic damage to sperm. Analysis of zebrafish sperm viability by CASA revealed a statistically higher motility rate for activation by micromixing (56 ± 4%) than manual activation (45 ± 7%; n = 5, P = 0.011). This micromixer represented a first step in streamlining methods for consistent, rapid assessment of sperm quality for zebrafish and other aquatic species. The capability to rapidly activate sperm and consistently measure motility with CASA using the PDMS micromixer described herein will improve studies of germplasm physiology and cryopreservation.     

Osmotic effects on feline spermatozoa from normospermic versus teratospermic donors

Effects of osmolality stresses on the sperm of normospermic (>60% normal sperm/ejaculate) versus teratospermic (<40% normal sperm) domestic cats and the normospermic leopard cat and the teratospermic clouded leopard were studied. Spermatozoa were exposed to various anisotonic solutions in a single step or returned to near isotonic conditions in a single step after exposure to anisotonic solutions. The percentage of sperm motility was measured subjectively, and dual fluorescent stains were used to assess membrane integrity by flow cytometry. The percentage of sperm motility declined (P < 0.05) in domestic cat sperm exposed to osmolalities <200 and >450 mOsm. Spermatozoa from all felines underwent marked (P < 0.05) membrane disruption following a hypotonic stress, but sperm from teratospermic donors experienced greater (P < 0.05) membrane disruption in response to decreased osmolality. While feline spermatozoa appeared to be highly resistant to hypertonic (600, 1200, and 2400 mOsm) conditions, with >85% of the cells maintaining intact membranes, severe membrane disruption occurred when cells were returned to isotonicity in a single step. There was no difference (P > 0.05) between a 1- and 5-min exposure to various anisotonic solutions. Similarly, sperm from normospermic and teratospermic domestic cats responded identically after exposure to ionic or nonionic solute. Results demonstrate that: (1) spermatozoa from teratospermic males are more vulnerable to a hypotonic stress than sperm from normospermic counterparts; (2) in response to small deviations in osmolality, feline sperm experience a more rapid decline in motility than membrane integrity; and (3) an abrupt return to isotonicity after a hypertonic stress causes extensive sperm membrane damage regardless of ejaculate quality.     

Evaluation of cryopreserved stallion semen from Tori and Estonian breeds using CASA and flow cytometry

Methods to evaluate the quality of frozen-thawed stallion semen are still needed, particularly those considering the sperm function. The present study evaluated sperm motility, membrane and acrosome integrity and the capacitation status of frozen-thawed spermatozoa from seven Tori and six Estonian breed stallions by way of computer assisted sperm analysis (CASA), a triple fluorophore stain combination and Merocyanine 540, respectively, the latter ones using flow cytometry. Two ejaculates from each stallion were cryopreserved using the Hannover method in 0.5 ml plastic straws. Two straws per ejaculate per stallion were thawed at 37 degrees C for 30s. Motility was analysed with CASA immediately after thawing, while for flow cytometry spermatozoa were cleansed by 70:40% Percoll discontinuous density gradient separation before analysed for sperm viability, acrosome integrity (stained with SNARF, PI and FITC-PSA) and capacitation status (stained with Merocyanine 540/Yo-Pro-1). Results (as least square means) were as follows: the motility of frozen-thawed semen was 43.4% for Tori stallions and 42.3% for Estonian stallions (P>0.05). After Percoll separation 79.3% of the spermatozoa from Tori stallions had intact acrosomes and 1.7% of them showed early signs of capacitation. The same parameters for Estonian stallions were 84.5 and 2.3%, respectively. There were no statistically significant differences between breeds or ejaculates within breed for any evaluated parameter. We conclude that triple staining and flow cytometry are valuable techniques to evaluate frozen-thawed stallion spermatozoa, and that no differences in quality of frozen semen were registered between Tori and Estonian breed stallions, allowing implementation of this technology in the Estonian horse population.     

Sperm motility parameters for Steindachneridion parahybae based on open‐source software

The objective of this work was to evaluate the sperm motility of 13 Steindachneridion parahybae males using open‐source software (ImageJ/CASA plugin). The sperm activation procedure and image capture were initiated after semen collection. Four experimental phases were defined from the videos captured of each male as follows: (i) standardization of a dialogue box generated by the CASA plugin within ImageJ; (ii) frame numbers used to perform the analysis; (iii) post‐activation motility between 10 and 20 s with analysis at each 1 s; and (iv) post‐activation motility between 10 and 50 s with analysis at each 10 s. The settings used in the CASA dialogue box were satisfactory, and the results were consistent. These analyses should be performed using 50 frames immediately after sperm activation because spermatozoa quickly lose their vigor. At 10 s post‐activation, 89.1% motile sperm was observed with 107.2 μm s^?1 curvilinear velocity, 83.6 μm s^?1 average path velocity, 77.1 μm s^?1 straight line velocity; 91.6% were of straightness and 77.1% of wobble. The CASA plugin within ImageJ can be applied in sperm analysis of the study species by using the established settings.     

Environmental factors contributed to circannual rhythm of semen quality

We investigated whether human semen parameters present circannual rhythm or not, and whether environmental factors exert on semen quality. This retrospective study used data of patients mainly from Reproductive Medicine Center and Urology and Andrology Clinic of a general hospital in China. Sperm concentration and motility were measured by computer aided sperm analysis (CASA). Sperm morphology was scored based on the strict criteria (WHO, 2010). The Kruskal–Wallis rank test was used to investigate the relationship between semen parameters and season/month. Partial correlation coefficients were used to analyze the relationship between semen parameters and environmental factors. In this study, we found that sperm concentration and total amount per ejaculate were significantly lower in summer and higher in winter. But, sperm progressive motility and motility were significantly higher in spring and summer (from March to June), lower in autumn and winter (September and October). Unexpectedly, normal sperm morphology and mixed agglutination reaction (MAR) positive rate didn’t vary along with season or month. Furthermore, temperature was negatively related to sperm concentration and total amount per ejaculate. Precipitation was positively associated with progressive motility and normal sperm morphology, but negatively related to sperm head defect percentage. The length of sunlight was positively related to progressive motility. The Air Quality Index (AQI) was positively associated with semen volume and sperm total amount per ejaculate. These suggest seasonal and monthly variation underlying some semen parameters.     

Specificity of the extender used for freezing ram sperm depends of the spermatozoa source (ejaculate, electroejaculate or epididymis)

The objective of this study was to identify possible specificity in the extender formulation for the cryopreservation of ram spermatozoa recovered from three origins (ejaculate, electroejaculate or epididymis), by evaluating post-thawing sperm quality and fertility. Ejaculated, electroejaculated or epididymal spermatozoa samples obtained from identical rams (8) were cryopreserved in four different extenders (TES-Tris-fructose with one of two egg yolk concentrations: 10% Y10 and 20% Y20, and with one of two glycerol rates: 4% G4 and 8% G8). Samples were analyzed before and after cryopreservation by CASA (motility) and flow cytometry (viability with SYBR-14/PI and acrosomal status with PNA/PI). Spermatozoa obtained by electroejaculation were of poorer quality after freezing/thawing, demonstrating that protocols for these samples need to be optimized. Egg yolk at 20% was more appropriate for freezing sperm from any of the sources. In general, 4% glycerol improved the quality of post-thawing samples recovered from ejaculate and electroejaculate, while 8% glycerol was more appropriate for samples recovered from the epididymis. Based on these results, an analysis of fertility was conducted. Fertility rates were similar between ewe groups inseminated with post-thawed sperm obtained from two sources: ejaculate (cryopreserved in Y20+G4), and cauda epididymis (Y20+G8), and this rate was less in the electroejaculated sample (Y20+G4).     

The importance of porcine sperm parameters on fertility in vivo

It would be desirable to use semen parameters to predict the in vivo fertilizing capacity of a particular ejaculate. In animal production, an ejaculate is divided into multiple doses for artificial insemination (AI); therefore, it would be economically beneficial to know the functional quality (i.e., fertility) of the semen before it is inseminated. To identify a predictive assay of the fertilizing capacity of a porcine ejaculate, we performed 4 rapid assays of sperm quality (motility, viability, physiological status as assessed by chlortetracycline fluorescence, and ATP content) on samples from 9 ejaculates, before and after a thermal stress test (42.5 degrees C, 45 min). These parameters were subsequently correlated with in vivo fertility resulting from AI with 2 sperm doses, 3 x 10(9) or 0.3 x 10(9) motile cells in 70 mL (optimal or suboptimal sperm number per insemination, respectively) from these same ejaculates. No parameter was correlated to the fertility rates obtained after inseminating with the optimal semen doses, either before or after the thermal stress test (P > 0.05). However, with respect to the animals inseminated with the suboptimal semen dose, sperm motility (the percentage of motile spermatozoa as assessed visually by microscopy) prior to thermal stress was well-correlated to fertility rates (r = 0.783, P = 0.01). The percentage of spermatozoa displaying the chlortetracycline Pattern AR (acrosome reaction) was also statistically related to fertility (r = 0.05, P = 0.04), but the biological importance of this relationship is questionable given the small variation among ejaculates (range: 0 to 2%). No other sperm parameter was significantly related to fertility rates in this group (P > 0.05). These data, therefore, indicate that sperm motility is a useful indicator of sperm fertilizing capacity in vivo. Moreover, to identify a predictor of semen fertility it is critical that the number of spermatozoa used during insemination is sufficiently low to detect differences in sperm fertilizing efficiency.     

Objective analysis of stallion sperm motility

An image-analysis system utilizing a microcomputer and CellSoft computer-assisted semen analysis software package was evaluated to assess stallion sperm motility characteristics. Analyses were performed at 37°C on a 6 μl drop of diluted semen placed on a glass slide and covered with an 18 mm² coverslip. Four groups of 25 cells each per slide, four slides per ejaculate and four ejaculates from each of three stallions were analyzed in a nested model. The percentage of motile sperm cells, mean velocity (μm/sec), mean linearity, and mean angular head displacement (μm) were measured. Statistical analysis of variance components showed that within ejaculates, more variation was accounted for in the differences among groups of 25 cells than among slides. Predicted standard deviations calculated for combinations of slides and groups of cells showed that a combination of two slides from which a total of 400 cells were analyzed resulted in a mean intra-assay coefficient of variation (CV) of 5.7% for the four measured variables. The following are individual coefficients of variation: percentage of motile cells (7.8%), mean velocity (6.4%), mean linearity (1.9%) and mean angular head displacement (6.6%). When ejaculate differences were included in the model and predicted standard deviations were calculated for a single ejaculate, the mean inter-assay CV was 9.2%. Mean velocity (6.4%) and mean linearity (4.7%) were more repeatable among ejaculates than either the percentage of motile sperm (14.4%) or angular head displacement (11.2%). It was concluded that this system is precise enough to determine differences in motility characteristics of stallion semen samples.     

Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate

Antioxidant supplementation during cooling was assayed to improve the motility of frozen-thawed (FT) boar spermatozoa from two different fractions of the ejaculate, the first component of the sperm-rich fraction (Fraction I) and the rest of the bulk ejaculate (Fraction II). Using a split-sample design, addition of two different concentrations (100 and 200 microMl(-1)) of the water-soluble Vitamin E analogue Trolox (6-hydroxy -2,5,7,8-tetramethylchroman -2-carboxylic acid) was evaluated for an effect on sperm motility (measured both subjectively and by means of a computer assisted motility assessment (CASA)), and on mitochondrial membrane potential using flow cytometry after cell-loading with JC-1. The effect of the Vitamin E analogue was clearly dose-dependent and varied with the fraction of the ejaculate considered. Motility was significantly higher in Trolox-treated spermatozoa (200 microm), from either ejaculate fraction, albeit the effect was more evident in spermatozoa from Fraction II (P<0.05) for any Trolox-concentration. Antioxidant supplementation resulted, also dose-dependent, in a higher number of spermatozoa showing high mitochondrial activity as assessed by the JC-1 staining, in both ejaculate fractions. In the present trial, exogenous Trolox positively affected post-thaw sperm viability (as motility and mitochondrial membrane potential) in both fractions of the ejaculate. The magnitude of the effect appeared, however, to be dependent of the fraction of the ejaculate considered.     

Computer-assisted motion analysis of sperm from the common carp

Computer-assisted semen analysis (CASA) technology was applied to the measurement of sperm motility parameters in the common carp Cyprinus carpio. Activated sperm were videotaped at 200 frames s⁻¹ and analysed with the CellTrak/S CASA research system. The percentage of motile cells and both sperm head curvilinear velocity and straight-line velocity were measured following exposure of carp sperm to three predilution conditions and activation in media of differing ionic strengths and osmotic pressures. The highest percentage of motile sperm was obtained following predilution of sperm in seminal plasma and activation in Na-HEPES buffer pH 8.0. This level of motility was equalled after predilution in 200 m m KCl for 2 h. Straight-line velocities and curvilinear velocities of 130 μm s⁻¹ and 210 μm s⁻¹, respectively, were observed. Duration of motility was higher under seminal plasma predilution conditions (over 50% motile sperm at 55 s post-activation). The application provides a sound basis for the assessment of Sperm Characteristics in fish.     

Effect of feeding a docosahexaenoic acid-enriched nutriceutical on the quality of fresh and frozen-thawed semen in Holstein bulls

The aim of the current study was to investigate the effect of feeding a DHA-enriched nutriceutical on the in vitro quality and sperm motility parameters of fresh and frozen-thawed bull semen assessed by CASA. Samples were obtained from nineteen Holstein bulls used for semen collection at Semen Production Center, Karaj, Iran. Control group (n = 10) were fed a standard concentrate feed while treatment group bulls (n = 9) had this standard feed top dressed with 100 g of a commercially available DHA-enriched nutriceutical. Semen quality was assessed on ejaculates collected at the baseline and after 5, 9, and 12 weeks of supplementation. Classical semen evaluation, assessment of sperm motility (subjective and computer-assisted), viability (eosin-nigrosin), and hypo-osmotic swelling test (HOST) were conducted. Semen volume, sperm concentration, and consequently total sperm output were not affected by dietary treatment (P > 0.05). Feeding the nutriceutical was indeed found to affect sperm motility parameters assessed by CASA after 9 weeks of trial. The treatment has improved total motility (P < 0.01), progressive motility (P < 0.05), average path velocity (P < 0.05), HOST-positive (P < 0.01), and proportion of rapid spermatozoa (P < 0.01) in the fresh semen of bulls. Moreover, the proportion of viable spermatozoa increased (P < 0.05) in the ejaculates collected from nutriceutical-fed bulls compared to the control after 12 weeks of feeding trial. The post-thawed HOST and sperm motility data obtained by CASA did not differ between two groups (P > 0.05). On the other hand, dietary supplementation did not affect body weight, BCS and scrotal circumference. Consequently, it can be concluded that dietary DHA supplementation or its precursors, improve in vitro quality and motility parameters of fresh semen assessed by CASA in Holstein bulls. However, this effect was not pronounced in frozen-thawed semen.     

Effect of feeding a docosahexaenoic acid-enriched nutriceutical on the quality of fresh and frozen-thawed semen in Holstein bulls

The aim of the current study was to investigate the effect of feeding a DHA-enriched nutriceutical on the in vitro quality and sperm motility parameters of fresh and frozen-thawed bull semen assessed by CASA. Samples were obtained from nineteen Holstein bulls used for semen collection at Semen Production Center, Karaj, Iran. Control group (n = 10) were fed a standard concentrate feed while treatment group bulls (n = 9) had this standard feed top dressed with 100 g of a commercially available DHA-enriched nutriceutical. Semen quality was assessed on ejaculates collected at the baseline and after 5, 9, and 12 weeks of supplementation. Classical semen evaluation, assessment of sperm motility (subjective and computer-assisted), viability (eosin-nigrosin), and hypo-osmotic swelling test (HOST) were conducted. Semen volume, sperm concentration, and consequently total sperm output were not affected by dietary treatment (P > 0.05). Feeding the nutriceutical was indeed found to affect sperm motility parameters assessed by CASA after 9 weeks of trial. The treatment has improved total motility (P < 0.01), progressive motility (P < 0.05), average path velocity (P < 0.05), HOST-positive (P < 0.01), and proportion of rapid spermatozoa (P < 0.01) in the fresh semen of bulls. Moreover, the proportion of viable spermatozoa increased (P < 0.05) in the ejaculates collected from nutriceutical-fed bulls compared to the control after 12 weeks of feeding trial. The post-thawed HOST and sperm motility data obtained by CASA did not differ between two groups (P > 0.05). On the other hand, dietary supplementation did not affect body weight, BCS and scrotal circumference. Consequently, it can be concluded that dietary DHA supplementation or its precursors, improve in vitro quality and motility parameters of fresh semen assessed by CASA in Holstein bulls. However, this effect was not pronounced in frozen-thawed semen.     

Evidence of deteriorating semen quality in the United Kingdom: birth cohort study in 577 men in Scotland over 11 years.

OBJECTIVE: To determine whether the quality of semen has changed in a group of over 500 Scottish men born between 1951 and 1973. DESIGN: Retrospective review of data on semen quality collected in a single laboratory over 11 years and according to World Health Organisation guidelines. SETTING: Programme of gamete biology research funded by Medical Research Council. SUBJECTS: 577 volunteer semen donors. Of these, 171 were born before 1959, 120 were born in 1960-4, 171 in 1965-9, and 115 in 1970-4. MAIN OUTCOME MEASURES: Conventional criteria of semen quality including semen volume (ml), sperm concentration (10(6)/ml), overall motility (% motile), total number of sperm in the ejaculate (10(6)), and total number of motile sperm in the ejaculate (10(6)). RESULTS: When the four birth cohort groups were compared a later year of birth was associated with a lower sperm concentration, a lower total number of sperm in the ejaculate, and a lower number of motile sperm in the ejaculate. The median sperm concentration fell from 98x10(6)/ml among donors born before 1959 to 78x10(6)/ml among donors born after 1970 (P=0.002). The total number of sperm in the ejaculate fell from 301x10(6) to 214x10(6) (P=0.0005), and the total number of motile sperm in the ejaculate fell from 169.7x10(6) to 129.0x10(6) (P=0.0065). CONCLUSION: This study provides direct evidence that semen quality is deteriorating, with a later year of birth being significantly associated with a reduced number of sperm in adult life.     

Motility and fertilizing capability of cryopreserved Acipenser ruthenus L. sperm

Motility before and after cryopreservation of Acipenser ruthenus sperm was determined by computer-assisted sperm motion analysis (CASA). In addition, fertility of fresh and frozen/ thawed sperm was tested by in vitro fertilization. Sperm of A. ruthenus was successfully cryopreserved by 1/1 (v/v) dilution with ethylene glycol in final concentrations from 12.5% to 20%. Stepwise cooling using a programmable freezer and fast thawing in a water bath (40°C) for 3 s and 5 s, respectively, resulted in motility rates of 10% to 28% and fry yields of 44% to 94% compared to controls (fresh sperm). With fresh material, different sperm to egg ratios varying from 10³: 1 to 10⁶: 1 gave similar fertilization rates of more than 90%. Using this cryopreservation method the establishment of sperm banks for endangered sturgeon species and the development of a fertility test for seasonally independent testing seem to be possible.