Effect of semen preparation on casa motility results in cryopreserved bull spermatozoa

Computer-assisted sperm analyzers (CASA) have become the standard tool for evaluating sperm motility and kinetic patterns because they provide objective data for thousands of sperm tracks. However, these devices are not ready-to-use and standardization of analytical practices is a fundamental requirement. In this study, we evaluated the effects of some settings, such as frame rate and frames per field, chamber and time of analysis, and samples preparations, including thawing temperature, sperm sample concentration, and media used for dilution, on the kinetic results of bovine frozen-thawed semen using a CASA. In Experiment 1, the frame rate (30-60 frame/s) significantly affected motility parameters, whereas the number of frames per field (30 or 45) did not seem to affect sperm kinetics. In Experiment 2, the thawing protocol affects sperm motility and kinetic parameters. Sperm sample concentration significantly limited the opportunity to perform the analysis and the kinetic results. A concentration of 100 and 50 × 10⁶ sperm/mL limited the device's ability to perform the analysis or gave wrong results, whereas 5, 10, 20, and 30 × 10⁶ sperm/mL concentrations allowed the analysis to be performed, but with different results (Experiment 3). The medium used for the dilution of the sample, which is fundamental for a correct sperm head detection, affects sperm motility results (Experiment 4). In this study, Makler and Leja chambers were used to perform the semen analysis with CASA devices. The chamber used significantly affected motility results (Experiment 5). The time between chamber loading and analysis affected sperm velocities, regardless of chamber used. Based on results recorded in this study, we propose that the CASA evaluation of motility of bovine frozen-thawed semen using Hamilton-Thorne IVOS 12.3 should be performed using a frame rate of 60 frame/s and 30 frames per field. Semen should be diluted at least at 20 × 10⁶ sperm/mL using PBS. Furthermore, it is necessary to consider the type of chamber used and perform the analysis within 1 or 2 min, regardless of the chamber used.     

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

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

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.     

Rappels technologiques,biais méthodologiques possibles et conditions de mesure

Computer-aided sperm analysis (CASA) was developed in the last decade to overcome the problems related to the inherent subjectivity of manual semen analysis. A typical CASA system includes a video camera, a microscope equiped with phase optics and a warming stage, a microcomputer with hardware and software dedicated to motion analysis, a monitor and a printer. The image of the sperm cells under the microscope is transformed into discrete pixels producing a voltage proportional to the intensity of the light as light strikes the camera’s CCD array. In standard video technology, the first field (set of rows; odd lines) is scanned in one sixtieth of a second and remains illuminated, then the alternate set of rows (even lines) is scanned in the next one sixtieth of a second to display the complete video frame which requires one thirthieth of a second. The first step called digitization is the encoding of video pixels as numbers following a gray level scale between white and black. From this process the detection of the sperm heads in the field is performed and repeated on a field by field basis. The centroïd coordinates of the sperm heads in the field are calculated and sequentially transmitted to the hard disk. A path finder algorithm connects the centroïds through time allowing the reconstruction of trajectories and the calculation of various parameters. Many factors modulating CASA measurements can invalidate the results of analysis. Consequently, a standardization of CASA is necessary. Various guidelines for accurate analysis are proposed. In summary, basic knowledge on sperm physiology, CASA technology and rigorous standardized conditions of analysis are required for providing reliable and accurate results because CASA systems are not ready-to-use “robots”. CASA is useful for routine semen analysis and essential for the measurement of sperm kinematics for clinical as well as for research purposes.     

Which fields under a coverslip should one assess to estimate sperm motility?

Under field conditions the motility of bull semen often has to be estimated under a coverslip on a microscope slide. This study was aimed at determining which combination of fields under coverslips provides measurements of sperm motility that best represent the motility in semen specimens as measured in a specially designed chamber for use in a computer-assisted sperm analyzer (CASA). We measured the motility (percentages motile, progressively motile, and aberrantly motile spermatozoa) in each of four straws of frozen-thawed semen from each of 10 bulls five times, ranging from 5 to 120 minutes after thawing with each bull by straw by time combination yielding one semen specimen. Motility was measured in duplicate in a Hamilton, Thorne IVOS CASA; once in each of 12 fields equally spaced along the equatorial radius of a coverslip (Field 0 at the edge and Field 11 at the center) and once in each of eight equally spaced fields along the equator of a Leja 4 chamber designed for use in a CASA. We used the weighted average motility of all fields in a chamber as gold standard and compared it to the average motility of each the following combinations of fields under the coverslip: all 12 fields, Fields 2 to 4, Fields 2 and four, Field 3 and the center three fields. The concordance correlation coefficient (CCC) was determined between the motility in each combination of fields under coverslips and the chambers as a reproducibility index, which evaluates the agreement between the readings under the coverslips and the gold standard readings in the chambers (n = 187 for each CCC). We performed pairwise comparisons of the CCCs (P < 0.005 for each comparison) and established that the average motility under all 12 fields better reproduced the motility in the chamber than the center three fields or Field 3. The averages of Fields 2 to 4 and Fields 2 and 4 reproduced chamber motility as well as the average of all 12 fields, except for the percentage motile sperm, where the average of all 12 fields was better. Using the average motility of Fields 2 and 4, 50% of estimates fell within 6%, 4% and 3% above or below the percentages motile, progressively motile and aberrantly motile spermatozoa in the Leja 4 chamber, 80% of estimates fell within 12%, 8% and 7% thereof and 95% fell within 23%, 13% and 12% thereof. In conclusion, for the method of spreading semen under a coverslip and the range in motility values used, this study shows that the average of the motility over the 12 fields along the equatorial radius under a coverslip provides the best estimate of the motility of a semen specimen, while the average of Fields 2 and 4 is also suitable for the subjective estimation of motility under field conditions, although the estimated motility is expected to fall within 6% above or below the motility of the specimen in only 50% of semen specimens.     

Computer-assisted semen analysis and its utility for profiling boar semen samples

Achieving and maintaining a successful swine AI program depends on a number of factors, including accurate semen evaluation, typically sperm motility, morphology and concentration. Computer-Assisted Semen Analysis or CASA (i.e., image analysis with a phase-contrast microscope and computer measurements of motion parameters) objectively evaluates sperm motion characteristics, morphology and concentration. A total of 3077 semen collections were evaluated with CASA (on the day of collection), and a semen dose subset was used for single-sire AI of 6266 females over 6 months. Fertility data from these inseminations were fitted with models including farm/stud, line, boar, parity, mating week, semen age at mating and boar age at mating. The residuals from these models showed no correlation for any CASA semen unique motion parameter, which could be due to the level of sperm concentration, the number of inseminations per estrus, and the low number of females mated per boar. Future studies to expand CASA/fertility analysis need to address these constraints and may include analysis of extended boar semen after storage for 1 week.     

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.     

The comparison of assessment of pigeon semen motility and sperm concentration by conventional methods and the CASA system (HTM IVOS)

The aim of these experiments was to compare conventional, microscopic methods of evaluating pigeon sperm motility and concentration to those measured by computer-assisted sperm analysis (CASA system). Semen was collected twice a week from two groups of pigeons, each of 40 males (group I: meat-type breed; group II: fancy pigeon) using the lumbo-sacral and cloacal region massage method. Ejaculates collected in each group were diluted 1:100 in BPSE solution and divided into two equal samples. One sample was examined subjectively by microscope and the second one was analysed using CASA system. The sperm concentration was measured by CASA using the anti-collision (AC) system and fluorescent staining (IDENT). There were not any significant differences between the methods of evaluation of sperm concentration. High positive correlations in both groups were observed between the sperm concentration estimated by Thom counting chamber and AC (r=0.87 and r=0.91, respectively), and between the sperm concentration evaluated by Thom counting chamber and IDENT (r=0.85 and r=0.90, respectively). The mean values for CASA measurement of proportion of motile spermatozoa (MOT) and progressive movement (PMOT) were significantly lower than the values estimated subjectively in both groups of pigeons (p< or =0.05 and p< or =0.01, respectively). Positive correlations in MOT and PMOT were noted between both methods of evaluation. The CASA system is very rapid, objective and sensitive method in detecting subtle motility characteristics as well as sperm concentration and is recommended for future research into pigeon semen.     

The effect of adjusting settings within a Computer-Assisted Sperm Analysis (CASA) system on bovine sperm motility and morphology results

Semen motility is the most widely recognized semen quality parameter used by Artificial Insemination (AI) centers. With the increasing worldwide export of semen between AI centers there is an increasing need for standardized motility assessment methods. Computer-Assisted Sperm Analysis (CASA) technology is thought to provide an objective motility evaluation; however, results can still vary between laboratories. The aim of present study was to verify the impact of different setting values of the CASA IVOS II on motility, concentration, and morphology of bovine semen samples frozen in an extender with or without egg yolk and then decide on optimal settings for a further validation step across AI centers. Semen straws from 30 different bulls were analyzed using IVOS II with twelve modified settings. No significant changes were observed in semen concentration, percentage of motile sperm or kinetic results for either extender type. However, increasing settings for both STR and VAP progressive (%) from Low, Medium, and High cut-off values significantly (p<0.05) reduced the percentage of detected progressive spermatozoa, in egg yolk extender from 49.5±15.2, 37.2±11.9 to 11.9±5.3%, and in clear extender from 51.9±9.1, 35.8±7.3 to 10.0±2.4%, respectively. In clear extender only, the modification of droplet proximal head length significantly affected the detection of normal sperm percentages (88.0± 4.7 to 95.0±0.6 and 96.0±0.6%) and of the percentage of detected proximal droplets (12.2±4.7, 2.5±2.7 to 0.6±0.2%) for Low, Medium and High values respectively (p<0.05). The identification of sensitivity within the CASA system to changes in set parameters then led to the determination of an optimal IVOS II setting. The existing variability among centers for these phenotypes was reduced when the standardized settings were applied across different CASA units. The results clearly show the importance of applied settings for the final CASA results and emphasize the need for standardized settings to obtain comparable data.     

Sperm concentration determination between hemacytometric and CASA systems: why they can be different

Determination of sperm concentration is a critical component of semen analysis. Traditionally, the hemacytometer has been the standard for calibrating other technologies used to estimate sperm concentration, including photometry, Coulter counters, flow cytometry, and computer-automated semen analysis (CASA). Disposable capillary-loaded slides are commonly used in conjunction with most CASA systems currently in use. Questions have been raised regarding differences in sperm concentration measurements between CASA systems (using 20 microm disposable slides) and hemacytometry. This review explains that these differences are largely due to the Segre-Silberberg (SS) effect, which occurs during Poiseuille flow in thin, capillary-loaded slides. The SS effect can lead to errors in estimation of particle concentration, as demonstrated with latex beads and suspensions of human or porcine spermatozoa. The SS effect does not appear to have time to develop in the hemacytometer, which at 100 microm is considerably deeper than most disposable slides. Thus, hemacytometry, when properly performed, remains the gold standard for estimation of sperm concentration. When using thin (20 microm) slides with CASA systems, recognition of the appropriate compensation factor to adjust for the SS effect is critical for accuracy.     

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.     

Dimethylformamide is no better than glycerol for cryopreservation of canine semen

The objective of this study was to compare the effects of dimethylformamide (DMF) and glycerol in canine (Canis lupus familiaris) semen cryopreservation based on postthaw motility and velocity evaluated by computer-assisted sperm analysis (CASA) and the effects on subjective progressive motility, percentage of live sperm, and plasma membrane functional integrity. The semen was diluted in two steps with an egg-yolk Tris extender containing 6% glycerol or DMF, frozen in 0.25-mL straws, and stored in liquid nitrogen. Immediately after thawing, samples were accessed for subjective sperm motility, sperm membrane functional integrity, percentage of live sperm, and evaluation by CASA. There were differences (P < 0.05) between glycerol and DMF with regard to subjective progressive motility (43.1% vs. 21.5%), objective progressive motility (11.8% vs. 6.2%), velocity average pathway (31.1 vs. 23.1 μm/sec), and amplitude of lateral head (3.3 vs. 3.9 μm), which confirmed the efficiency of glycerol. In conclusion, objective analysis performed by CASA confirmed that no benefits were derived by using DMF to replace glycerol for cryopreservation of canine 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.     

Validation and usefulness of the Sperm Quality Analyzer V equine for equine semen analysis

Routine semen analysis includes evaluation of concentration combined with seminal volume, morphology and motility. Subjective analysis of these parameters is known to be inaccurate, imprecise and subject to variability. Automated semen analysis could lead to an increased standardization in and between laboratories but for that to happen automated devices need to be validated. A new device, the sperm quality analyzer V equine (SQA-Ve) version 1.00.43, was evaluated for its repeatability and agreement with light microscopy (LM), for raw and extended equine semen. Results were compared with computer assisted sperm analysis (CASA), which was also tested for its repeatability and agreement with LM. The SQA-Ve showed a good repeatability and fine agreement for assessing sperm concentration of raw semen based on scatter and Bland-Altman plots. This was in contrast with the motility parameters, which had a low repeatability. Morphology assessment with SQA-Ve was poorly repeatable as well as in poor agreement with LM. For extended semen, the findings were comparable. The SQA-Ve did well for concentration, whereas for the motility parameters repeatability was only just acceptable, with no agreement with LM. This sharply contrasted the CASA findings that were highly repeatable and almost in perfect agreement with LM. Based on these findings, the tested version of the SQA-Ve is insufficiently accurate to be used for analyzing raw or extended equine semen.     

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.     

Quantification of bull sperm characteristics measured by computer-assisted sperm analysis (CASA) and the relationship to fertility

Two experiments were conducted to evaluate semen quality of bulls housed under controlled conditions at a large AI facility and relate results to fertility. In Experiment 1 semen was collected from six 6-yr-old bulls twice daily at 3- to 4-d intervals for 3 d. In Experiment 2 eleven 6- to 11-yr-old bulls were used. Extensive breeding information was available and semen was collected as in Experiment 1 but replicated 4 times. Standard semen analysis and computer-assisted sperm analysis (CASA) with the Hamilton Thorne IVOS, model 10 unit, were performed on 36 first and second ejaculates in Experiment 1 and on 44 first ejaculates in Experiment 2. Sixteen fields (2 chambers with 8 fields per chamber) were examined per sample. In Experiment 1 the correlation between estimated sperm concentration by spectrophotometry and CASA was 0.91 (P < 0.01). Among bulls the range in the percentage of motile spermatozoa was 52 to 82 for CASA versus 62 to 69 for subjective measurements made by highly experienced technicians. Thus, CASA, with high repeatability, provided a more discriminating estimate of the percentage of motile sperm cells than did the subjective procedure. Bull effect was much greater than any other variable in the experiments. Chamber differences were small and so the results for the 2 chambers with 8 fields each were combined. One to five CASA values were correlated with bull fertility, defined as 59-day nonreturn rates corrected for cow and herd effects. The percentage of motile spermatozoa accounted for a small fraction of the total variation in fertility (r2 = 0.34). However higher r2 values (0.68 to 0.98) were obtained for 2 to 5 variables used in the multiple regression equations. The results are promising, and further testing will determine more precisely which of these CASA variables are most useful in estimating bull fertility potential.     

Standardization of computer-assisted semen analysis using an e-learning application

Computer-assisted semen analysis (CASA) is primarily used to obtain accurate and objective kinetic sperm measurements. Additionally, AI centers use computer-assessed sperm concentration in the sample as a basis for calculating the number of insemination doses available from a given ejaculate. The reliability of data is often limited and results can vary even when the same CASA systems with identical settings are used. The objective of the present study was to develop a computer-based training module for standardized measurements with a CASA system and to evaluate its training effect on the quality of the assessment of sperm motility and concentration. A digital versatile disc (DVD) has been produced showing the standardization of sample preparation and analysis with the CASA system SpermVision™ version 3.0 (Minitube, Verona, WI, USA) in words, pictures, and videos, as well as the most probable sources of error. Eight test persons educated in spermatology, but with different levels of experience with the CASA system, prepared and assessed 10 aliquots from one prediluted bull ejaculate using the same CASA system and laboratory equipment before and after electronic learning (e-learning). After using the e-learning application, the coefficient of variation was reduced on average for the sperm concentration from 26.1% to 11.3% (P ≤ 0.01), and for motility from 5.8% to 3.1% (P ≤ 0.05). For five test persons, the difference in the coefficient of variation before and after use of the e-learning application was significant (P ≤ 0.05). Individual deviations of means from the group mean before e-learning were reduced compared with individual deviations from the group mean after e-learning. According to a survey, the e-learning application was highly accepted by users. In conclusion, e-learning presents an effective, efficient, and accepted tool for improvement of the precision of CASA measurements. This study provides a model for the standardization of other laboratory procedures using e-learning.     

The breeding management affects fresh and cryopreserved semen characteristics in Melopsittacus undulatus

Melopsittacus undulatus is a companion parrot worldwide diffused. Many parrots are considered endangered or vulnerable. The preservation of semen is crucial in endangered species, thus, M. undulatus could be a good model to study sperm characteristics and semen cryopreservation in these other endangered parrots. In this study the effect of the breeding management (males bred in promiscuous aviary or in couple) on sperm characteristics (motility, membrane integrity and morphometry) of fresh and cryopreserved semen was evaluated. The computer-assisted sperm analysis (CASA) revealed a significant effect of the husbandry method on semen characteristics in budgerigars: male housed in couple with the female in individual cages allowed the higher results in term of both semen quantity and sperm quality. Total and progressive motility were significantly higher in males bred in couple (68.7 ± 8.9% and 54 ± 15.9%, respectively) than in promiscuous aviary (48.3 ± 15.1% and 24.4 ± 12.4%, respectively), such as sperm velocity (average path velocity, straight line velocity, and curvilinear velocity). The type of sperm movement (amplitude of lateral head displacement, beat cross frequency, straightness, and linearity), sperm membrane integrity and morphometry parameters seemed not affected by the husbandry method. The standardization of a CASA procedure for the semen analysis in M. undulatus allow further studies on parrot semen manipulation and cryopreservation, but the method used for the breeding of the male could have a significant effect on the semen quality.     

Place de l’analyse du mouvement dans un bilan de fertilité: Valeurs prédictives des différents paramètres mesurés

Routine evaluation of semen characteristics-spermiogram-includes estimation of the percentage of motile sperm; however it does not provide quantitative informations about sperm movement characteristics, except under the form of qualitative appreciations (slow, sluggish, yawing, non progressive, etc.). Flagellar function is indeed directly involved in the migration of spermatozoa through the female genital tract, and in the fertilization process by itself (migration through the zona pellucida requires special motility state, generally called “hyperactivation”). Sperm flagellar movements can now be indirectly investigated by analysing movements of sperm head, which are more easily detectable under phase contrast illumination: video signals are digitalized then sperm tracks are reconstructed by the computer from coordinates of sperm centroids (these systems are called “computer-assisted semen analysis” or “CASA”). CASA systems are now so performing and rapid that sperm movement analysis (SMA) can be proposed in the same time of routine semen analysis. However SMA, together with other functional tests, offer more interest in some particular situations as unexplained infertility, or unexpected failure of IVF. Numerous studies have tried to identify the most discriminant parameters, generally by means of multiple regression analysis. The interpretation of litterature data is difficult because of several differences in the protocol design, concerning either the measurements conditions (before or after sperm washing and selection) or the nature of the functional test: migration into cervical mucus, zona-free hamster egg penetration, IVF, etc. Moreover, the most discriminant factors are generally represented by classical parameters as % of normal forms or of motile sperm. In the present study we showed that only the factor “% of motility” allowed a significant discrimination according to different classes of fertilization rate (FR) in an IVF system. FR increased with hyperactivation rate (HA), but the statistical test was not significant. However, the more numerous cases of IVF failure were found in the group corresponding to very low HA rate (0–5%). We conclude that one major interest of SMA is to reveal some flagellar dyskinesia (i.e. corresponding to low values of amplitude lateral displacement of the head, or straight line velocity). These cases could then benefit of assisted reproductive techniques well adapted to this motion dysfunction, as subzonal insemination (SUZI) or intra-cytoplasmic sperm injection (ICSI).