Abstract
The aim of the present study was to investigate the effect of different technical settings and semen processing on sperm motility parameters measured by the Sperm Class Analyzer (SCA). Semen was collected from 3 dogs, pooled, and diluted in phosphate buffered saline and subsequently assessed by the SCA for the different sperm motility characteristics. The data were statistically analyzed by ANOVA and the repeatability was assessed by coefficient of variation (CV). After a principal component analysis, the reliability was determined with intra-class correlation coefficient (ICC). In experiment 1, the CV's were below 10% for all evaluated parameters. Significant differences (P < 0.05) were found between the different sperm concentrations (25, 50, and 75 x 106 spermatozoa/ml) in all of the motion parameters assessed, yielding the highest ICC (0.81) at 25 x 106 spermatozoa/ml. No significant differences (P > 0.05) in SCA read-outs were found between the number of microscopic fields captured (1, 2, 3, 4, or 5 fields), yielding the highest ICC (0.83) when 3 fields were captured. No significant differences (P > 0.05) in motility parameters were found between the number of cells analyzed in each field (20, 50, and 100 spermatozoa) with the exception of beat cross frequency. Reliability of the SCA was good (ICC = 0.71 to 0.90) for all motility measurements when 20 (ICC = 0.89) or 50 (ICC = 0.77) cells were captured in each field, but only just acceptable (ICC = 0.51 to 0.70) when 100 cells were counted (ICC = 0.67). The frame settings significantly (P < 0.05) influenced most of the measured motility characteristics. Scanning 60 frames at a frame rate of 30 Hz improved the reliability of the results (ICC = 0.92). In conclusion, we suggest that the measurements with the SCA are ideally performed at a sperm concentration of 25 x 106 spermatozoa/ml, counting at least 100 cells in three microscopic fields. We also propose that the SCA should analyze 60 frames at 30 Hz to yield consistent results of a set of measurements or a measuring instrument thus obtaining reliable motility results.
Introduction
The evaluation of sperm motility is one of the most important parameters for the assessment of a sperm sample. It has been previously implicated as an important parameter for fertility in dog sperm analysis [Iguer-Ouada and Verstegen Citation2001b]. Until recently, canine semen analysis was routinely assessed using standard optical microscopy [Rijsselaere et al. Citation2005]. However, microscopic measurements of motility have been shown to be inaccurate and imprecise and subject to high subjectivity [Davis and Katz Citation1993; Verstegen et al. Citation2002; Rijsselaere et al. Citation2005]. Moreover, this parameter has often shown a poor correlation with fertility potential, making the interpretation of the data very difficult [Rijsselaere et al. Citation2005].
In order to overcome these limitations, automated systems have been developed. Computer-assisted semen analysis (CASA) systems were shown to produce accurate and highly repeatable data on different semen motion parameters in many mammalian species such as dogs [Rijsselaere et al. Citation2002], cats [Filliers et al. Citation2008], bulls [Leite et al. Citation2010], goats [Dorado et al. Citation2007], horses [Hoogewijs et al. Citation2010], and pigs [Didion Citation2008]. However, semen handling procedures and parameter settings still differ between laboratories, making it difficult to compare results [Davis and Katz Citation1993; Verstegen et al. Citation2002; Rijsselaere et al. Citation2003]. During the last decade, many systems have been tested and validated for the objective analysis of canine spermatozoa [Günzel-Apel et al. Citation1993; Iguer-Ouada and Verstegen, Citation2001a; Smith and England Citation2001; Rijsselaere et al. Citation2003; Schäfer-Somi and Aurich Citation2007]. However, to our knowledge, the Sperm Class Analyzer (SCA) has not yet been validated for dog semen analysis or compared with other CASA systems. The aim of the present study was therefore to assess the effect of different technical settings and semen handling procedures on the results obtained by the SCA for dog semen analysis.
Results
The experimental strategy is outlined in . The mean (± SD) volume of the second sperm-rich fraction of the ejaculate was 1.18 ± 0.45 ml. The sperm concentration as measured using a Spermacue (Minitüb, Tiefenbach, Germany) was 262.29 ± 98.88 x 106 spermatozoa/ml, and the pH was 6.14 ± 0.20. Principal component analysis (PCA) identified two principal components that accounted for 84.35% of the variance. Sperm motility (mean, minimum and maximum values) and the CV of each CASA-derived motility parameter are summarized in using adjusted parameters outlined in . For all parameters analyzed, measurements were repeatable, as reflected by a low CV (below 10%).
Table 1. Description of the handling characteristics proposed for each experiment: 1) Precision of measurements, 2) Effect of concentration on sperm motility parameters, 3) Effect of the number of scanned microscopic fields on sperm motility parameters, 4) Effect of the number of scanned microscopic fields on sperm motility parameters, and 5) Effect of frame setting on sperm motility parameters.
Table 2. Mean, minimum and maximum values, and the coefficient of variation (CV) for each sperm motility parameter obtained with the SCA system.
Table 3. Technical settings of the Sperm Class Analyzer (SCA) as used in the present study.
Effect of concentration on sperm motility parameters
Significant differences (P < 0.05) were found between the different sperm concentrations for all of the evaluated sperm motion parameters (). Mean values of total motile spermatozoa (MOT) and progressive motile spermatozoa (PMOT) were significantly higher (P < 0.05) at sperm concentrations of 50 and 75 x 106 spermatozoa/ml. A significant reduction (P < 0.001) in sperm velocity: curvilinear velocity (CLV), straight line velocity (SLV), and average path velocity (APV), in motion quality: linearity (LIN), straightness (STR), and wobble (WOB), and in beat cross frequency (BCF) was observed for sperm concentration of 75 x 106 spermatozoa/ml, whereas the opposite was found for lateral head displacement (LHD). As shown in reliability based on the ICC was good (ICC = 0.71 to 0.90) in samples assessed at 25 x 106 spermatozoa/ml (ICC = 0.81), acceptable (ICC = 0.51 to 0.70) in those scanned at 50 x 106 spermatozoa/ml (ICC = 0.69), and poor (ICC = 0.31 to 0.50) at sperm concentration of 75 x 106 spermatozoa/ml (ICC = 0.40).
Table 4. Effect of different sperm concentrations (25, 50, and 75 x 106/ml) on SCA measurements (mean ± SD). Each value is the average of the SCA read-outs of pooled dog semen repeated in four replicates (n = 20).
Effect of the number of scanned microscopic fields and counted cells in each field on sperm motility parameters
No significant differences (P > 0.05) in SCA read-outs were observed between the number of microscopic fields captured (1, 2, 3, 4, or 5 fields; ). In all cases, the SCA was reliable (ICC ≥ 0.80), yielding the highest ICC (0.83) when three fields were captured. No significant differences (P > 0.05) in motility parameters were found between the number of cells analyzed in each field (20, 50, or 100 cells) with the exception of BCF (). Mean values of BCF were significantly higher (P < 0.05) when 20 cells were counted in each field. The reliability of the SCA was good for all motility measurements when 20 (ICC = 0.89) or 50 (ICC = 0.77) cells were captured in each field, and just acceptable when 100 cells were counted (ICC = 0.67, ).
Table 5. Effect of the number of scanned microscopic fields (1, 2, 3, 4, and 5 fields) on SCA measurements (mean ± SD). Each value is the average of the SCA read-outs of pooled dog semen repeated in four replicates (n = 20).
Table 6. Effect of the number of counted cells in each field (20, 50, and 100 spermatozoa) on SCA measurements (mean ± SD). Each value is the average of the SCA read-outs of pooled dog semen repeated in four replicates (n = 20).
Effect of frame acquisition rate and of the number of analyzed frames on sperm motility parameters
Significant differences (P < 0.05) in SCA read-outs were found between different frame settings () with the exception of LHD. The motility (MOT and PMOT) and velocity parameters (CLV, SLV, and APV) increased significantly (P < 0.05) when 25 subsequent images were analyzed at 25 Hz. A decrease was observed for BCF when 30 frames were analyzed at 15 Hz. The analysis of 30 frames at 15 Hz provided significantly higher measures for motion quality as assessed for LIN, STR, WOB, and LHD. Reliability based on the ICC was excellent (ICC > 0.90) when 60 frames were captured at 30 Hz (ICC = 0.92), good (ICC = 0.83) in samples assessed at sampling intervals of 1 sec (25 data points at 25 Hz), and just acceptable when 30 frames were captured at 15 Hz (ICC = 0.55, ).
Table 7. Effect of frame setting (25 frames at 25 Hz, 30 frames at 15 Hz, and 60 frames at 30 Hz) on SCA measurements (mean ± SD). Each value is the average of the SCA read-outs of pooled dog semen repeated in four replicates (n = 20).
Discussion
In this study, the SCA system was evaluated for analyzing dog semen for the first time. High repeatability of measurements was observed for all parameters assessed, since the CV's of the results remained below 10%. These findings are in agreement with findings in previous studies in dogs [Iguer-Ouada and Verstegen Citation2001b; Rijsselaere et al. Citation2003; Schäfer-Somi and Aurich Citation2007] and bulls [Hoflack et al. Citation2005] using different types of CASA systems. It is, however, important to emphasize that variations of 30 – 60% were obtained when the motility of the same ejaculate was assessed using subjective microscopy by different observers within the same laboratory [Verstegen et al. Citation2002].
As raw dog semen is highly concentrated it cannot be analyzed by CASA. A proper dilution ratio of the semen sample is required to accurately reconstruct the sperm trajectories [Mortimer Citation2000]. However, previous studies reported that the concentration of a specimen could affect motility results recorded by CASA [Davis and Katz Citation1993]. We observed a clear influence of sperm concentration on motility measures and the reliability of measurement. The majority of the sperm motility parameters measured with SCA (CLV, SLV, APV, LIN, STR, WOB, and BCF) was statistically lower at a sperm concentration of 75 x 106 spermatozoa/ml, probably because sperm cells are so concentrated that they are unable to move freely within the Makler counting chamber, resulting in multiple individual collisions [Bartoov et al. Citation1991; Rijsselaere et al., Citation2002], and consequently their motion parameters are slightly altered [Mortimer Citation2000]. In contrast, total motility increases significantly with higher concentrations. This finding could be due to rapid death of spermatozoa after extensive dilution in PBS, known as the ‘dilution effect’ [Contri et al. Citation2010]. Alternatively, this finding could be due to the amount of spermatozoa detected in 50 and 75 x 106 spermatozoa/ml samples. When an excessive concentration is used there is the risk that the tracks may be incorrectly reconstructed [Mortimer Citation2000] due to erroneous head detection in following frames, collision, and cross-tracks, and consequently this could affect the percentage of motile spermatozoa. Rijsselaere et al. [2003] showed a similar trend in the effect of sperm concentration on CASA results using the CEROS sperm analyzer. This likely affected the ability of the device to perform the analysis. Therefore, a different sperm concentration was proposed for CASA evaluation of dog semen (50 x 106 spermatozoa/ml). Other studies in human and several domestic species recommended that sperm concentration be adjusted to 20 – 50 x 106 spermatozoa/ml to obtain reliable kinematic measurements [Mortimer et al. Citation1988; Farrell et al. Citation1996; Contri et al. Citation2010]. The differences in the concentration suggested for specimen analysis in the different studies likely reflects different CASA devices. In our study, the reliability of the SCA system was highest (ICC = 0.81) when the sperm motility was assessed at 25 x 106 spermatozoa/ml. We suggest that measurements are most accurately performed at a concentration of 25 x 106 spermatozoa/ml when using the SCA for canine semen; using the playback option, this concentration permitted continuous tracking of each spermatozoa whereas a higher concentration (50 and 75 x 106 spermatozoa/ml) was frequently too dense to individually verify the correct trajectory of each spermatozoa.
There are several factors which can influence the reliability, accuracy, and precision of CASA analyses, particularly the analysis duration, the number of spermatozoa analyzed, the number of fields to analyze, the user's training and experience, and the type of counting chamber used [Davis and Katz Citation1993; Verstegen at al. 2002]. In order to reduce the influence of the observer on CASA read-outs all motility analyses were performed under controlled experimental conditions as the same technician read all the pooled semen samples. Regarding the effect of examined field, no influence of the number of microscopic fields captured per analysis on motility values could be elucidated. However, most of the motion parameters decreased when the number of microscopic fields analyzed increased, probably because a large number of fields captured increases the analysis duration. Contri et al. [2010] reported that the time between the sample loading and the analysis affects velocity parameters, regardless of chamber used.
The depth of the chamber is another factor that may influence sperm motility either by restricting the displacement or by some interaction with the chamber walls. Douglas-Hamilton et al. [2005a;b] recently showed that the distribution of spermatozoa in suspension is regulated by the Segre-Silberberg effect. This effect produces a transverse lifting force on suspended particles, which are driven toward two stable planes situated a short, calculable distance from the walls. Consequently, a relatively lower concentration of cells is counted when evaluating the center of the microscopic field. This underestimates sperm concentration as determined by CASA [Maes et al. Citation2010]. The Segre-Silberberg effect can also modify total and progressive motility through a different and apparently selective distribution of motile and non-motile spermatozoa, as hypothesized by Douglas-Hamilton et al. [2005a;b]. Fluid dynamics and chamber depth only partially explain why the analysis of the same sample can result in such an increase in non-motile spermatozoa [Contri et al. Citation2010]. Further studies should be performed to assess the effect of fluid dynamics of sperm suspension in different chambers. This would permit standardization of counting chambers and procedures for all laboratories, with the ultimate goal of improving accuracy and reliability of CASA results.
The number of spermatozoa analyzed influence the accuracy and precision of all measures [Davis and Katz Citation1993]. It has been widely known that if there are too many spermatozoa in the field of view, it may not be possible for the instrument to accurately reconstruct their trajectories [Mortimer Citation2000]. It is also accepted that the minimum number of cells counted for sperm motility analysis should be at least 100 [Verstegen et al. Citation2002] for a reliable kinematic estimation using CASA. In this study, no significant differences were found for the majority of the assessed sperm parameters between the number of cells counted in each field. However, SCA reliability was good for all motility measurements when 20 or 50 cells were captured in each field. This suggested that the analysis of 100 spermatozoa is sufficient for the motility assessment of canine semen. Nevertheless, the assessment of three microscopic fields at a concentration of 25 x 106 spermatozoa/ml permitted measurement of at least 100 spermatozoa per sample. Hence, we are inclined to suggest the assessment of three fields for the analysis of dog semen diluted to a concentration of 25 x 106 spermatozoa/ml, using the Makler chamber.
Significantly different measurements were obtained for all of the motility parameters assessed, analyzing the same sperm populations at different frame settings. Motility (MOT and PMOT) and velocity (CLV, SLV, and APV) parameters increased at a scanning time of 1 sec (25 subsequent images at 25 Hz). It has been widely demonstrated that both the acquisition frequency of images and the number of scanned frames influence the accuracy and precision of all measures [Davis and Katz Citation1993]. Moreover, the shape of the reconstructed trajectory is influenced by the frame rate [Mortimer Citation2000]. These findings suggest that the reduction in the acquisition rate can result in significant errors for various CASA parameters, as previously reported [Owen and Katz Citation1993; Rijsselaere et al. Citation2003]. However, the number of frames analyzed seems to affect CASA results. The reconstruction of the trajectory using 60 points, besides more accurate measures, resulted in different values from the reconstruction using either 25 or 30 points. The clear increase in BCF observed in our study suggests that high frequency analysis might be necessary to obtain an accurate measurement of this parameter. The analysis of 60 frames at 30 Hz yielded the highest ICC (0.92), which means that this acquisition frequency of images yields the most repeatable results of the evaluated frame settings. These findings demonstrate how the number of points sampled per flagellar beat and the sampling interval are critical in determining the precision of CASA analysis. Our results suggest that analyzing 25 data points at 25 Hz (acquisitions of 1 sec) appeared sufficient to reconstruct the sperm trajectory. However, increasing both the number of frames scanned (60 frames) and the sampling time (30 Hz) improved reliability for dog sperm motion evaluation, as previously reported in other species [Owen and Katz Citation1993; Mortimer and Swan Citation1995; Kraemer at al. 1998]. It is important to emphasize that the choice of the optimal frame acquisition rate is still under discussion in many species, as it is equipment-dependent and depends on species and experimental conditions [Davis and Katz Citation1993; Morris et al. Citation1996; Verstegen et al. Citation2002]. Moreover, it has been established that the calibration of the system and the use of different systems in various animal species may influence the variability of a measurement [Maes et al. Citation2010]. For these reasons, further studies should be performed to improve the accuracy and precision of SCA for the assessment of dog sperm motility parameters, providing an objective value for the comparison of results between laboratories.
In conclusion, this study clearly shows that SCA is dependent on several technical settings and semen handling procedures. The precision of the SCA is acceptable for dog semen analysis based on our findings, and we suggest that measurements using the SCA are best performed at 25 x 106 spermatozoa/ml, analyzing three microscopic fields and counting at least 100 cells per sample. Finally, we also propose that the SCA instrument used should analyze 60 frames at 30 Hz to obtain reliable kinematic measurements.
Materials and Methods
Animals
Three clinically healthy experimental dogs of unknown fertility were used: two sexually mature Spanish Greyhounds (2 and 3 years old) and one Beagle (9 years old). All the experimental dogs were obtained from the kennel of the Clinical Veterinary Hospital of the University of Cordoba, Spain. They were housed individually in indoor-outdoor runs with natural lighting, and were fed twice a day with commercial dog food, and given water ad libitum. This study was performed in accordance with the Spanish Animal Protection Policy RD1201/2005, which conforms to the European Union Directive 86/609 regarding the protection of animals used in scientific experiments.
Semen collection and evaluation
Semen was collected from all 3 experimental dogs twice weekly by digital manipulation as described by Linde-Forsberg [1991] and if necessary in the presence of a teaser bitch. The second sperm-rich fraction and the third fraction (prostatic fluid) were collected into two separate pre-warmed (38°C) plastic tubes (BDFalconTMTubes, BD Biosciences, Erembodegem, Belgium), avoiding the first fraction (pre-spermatic fluid).
After semen collection, the undiluted sperm-rich fraction from the three dogs was evaluated for pH with a quantitative system (Basic pH meter, Denver Instrument Co, Colorado, USA) and for sperm concentration (x 106 spermatozoa/ml) with a photometer (Spermacue, Minitüb, Tiefenbach, Germany) as described by Schäfer-Somi and Aurich [2007]. Prior to motility analysis, the sperm rich fractions were pooled, diluted with phosphate buffered saline (PBS D5773, Sigma-Aldrich Chemie GmbH, Steinheim, Germany), and stored at 38°C for 5-10 min. All analyses were performed within 5-10 min after semen collection.
Use of the Sperm Class Analyzer
The Sperm Class Analyzer computer-aided semen analyzer, version v.3.2.0 (SCA®2005, Microptic SL, Barcelona, Spain), was used to simultaneously evaluate different sperm motility parameters. This cell motion analyzer includes a trinocular negative phase-contrast microscope (Eclipse 50i, Nikon, Tokyo, Japan), a minitherm stage warmer (OK 51-512, Osaka, Digifred SL, Barcelona, Spain), a high-speed digital camera (A312fc, BaslerTM AG, Ahrensburg, Germany) that recognizes 53 frames/sec, and a computer (Intel inside®, Pentium® 4, Intel Labs, Barcelona, Spain) to save and analyze the acquired data.
In the different experiments, the aliquots of the sperm rich fraction were diluted with PBS, as described by Keenan [1998]. For each analysis, 5 µl of extended semen was placed on a pre-warmed (38°C) Makler cell chamber (Sefi Medical Instruments Ltd., Haifa, Israel) and was allowed to settle for 1 min on the minitherm stage warmer (38°C) before the analysis [Smith and England Citation2001]. Five different randomly selected microscopic fields were scanned in five different drops (five times with one drop per time), obtaining 25 scans for every semen sample. The mean of the five scans for each microscopic field was used for the statistical analysis.
The following ten parameters were measured by the SCA (as described by the manufacturer): curvilinear velocity (CLV, μm/sec): the average velocity measured over the actual point to point track followed by the cell, straight line velocity (SLV, μm/sec): the average velocity measured in a straight line from the beginning to the end of the track, average path velocity (APV, μm/sec): the average velocity of the smoothed cell path, the linearity (LIN, %): average value of the ratio SLV/CLV, the straightness (STR, %): average value of the ratio SLV/APV, the wobble (WOB, %): average value of the ratio APV/CLV, the mean amplitude of the lateral head displacement (LHD, μm): the mean width of the head oscillation as the spermatozoa swim, the beat cross frequency (BCF, Hz): frequency of the sperm head crossing the average path in either direction, the overall percentage of motile spermatozoa (MOT, %), and the percentage of spermatozoa with a progressive motility (PMOT, %) i.e., cells with an average path velocity > 50 µm/sec and straightness > 75%.
The software settings recommended by the manufacturer were adjusted in order to obtain a clear identification of the individual spermatozoa (). The playback option of the device, used after every scan, showed the video sequence of the last scan, providing an additional control to validate whether all spermatozoa in the microscopic field were identified and whether their trajectory could be reconstructed correctly.
Experimental design
The handling characteristics proposed for each experiment in the present study are presented in .
Precision of measurements
To test the precision of measurements, pooled semen samples were diluted to 25 x 106 spermatozoa/ml. Five randomly allocated fields were scanned in five different drops, including a minimum of 500 spermatozoa. This experiment was repeated four times at a two-day-interval. For each parameter, the coefficient of variation (CV) per sample was calculated and reported as mean CV over all samples [Arunvipas et al. Citation2003].
Effect of concentration on sperm motility parameters
To detect possible influences of a different sperm concentration on the measurements obtained by the SCA, pooled semen samples were diluted to 25, 50, and 75 x 106 spermatozoa/ml and analyzed as described before. This experiment was repeated four times at a two-day-interval.
Effect of the number of scanned microscopic fields on sperm motility parameters
In order to assess the possible effects of the number of scanned fields for each analysis, semen was collected four times at a two-day-interval. Ejaculates obtained on the same day were pooled and analyzed as described before. Thereafter, motility data were compared among different microscopic fields scanned (1, 2, 3, 4, or 5 fields).
Effect of counted cells in each field on sperm motility parameters
To determine the effect of counted cells in each field, the same semen samples were used as in experiment 3. A total of 100 spermatozoa were analyzed per field (500 per sample). Subsets of 20, 50, and 100 spermatozoa were randomly selected from the initial reference group of 100 and compared.
Effect of frame acquisition rate and of the number of analyzed frames on sperm motility parameters
To detect possible effects caused by both frame rate or number of analyzed frames, pooled semen samples were analyzed at three different frame settings: 25 frames at 25 Hz (as recommended by the manufacturer), 30 frames at 15 Hz, and 60 frames at 30 Hz. For each frame setting, five randomly allocated fields were analyzed in five different drops. This experiment was repeated four times at a two-day-interval.
Statistical analysis
Throughout the study, results were expressed as mean values and variation was expressed as standard deviation (SD). Five randomly allocated fields were scanned in 5 different drops (five times with one drop per time), obtaining 25 scans for every semen sample. Each experiment was repeated four times at a two-day-interval. Therefore, as the mean of the five scans for each microscopic field was used for the statistical analysis, 20 data points were compared between sampling factors. To exclude the effect of the replica on motility results, we represent mean values of 4 replicates as a single pooled semen sample. The CV was used as a parameter for repeatability when assessing precision. In all other cases the different sampling factors were compared using a general linear model (ANOVA). Dependent variables expressed as percentages were arcsine-transformed before the analysis. Differences between mean values were analyzed by the Duncan method. Thereafter, the PRINCOMP procedure was applied to perform principal component analysis (PCA) of the motility data. The purpose of PCA is to derive a small number of linear combinations (principal components) from a set of variables that retain as much of the information in the original variables as possible. Principal components were then analyzed using intra-class correlation coefficient (ICC) as reliability statistics [Doros and Lew Citation2010]. Reliability (degree to which a set of measurements or a measuring instrument gives consistent results) is expressed as a decimal value between 0.00 and 1.00, with higher values indicating greater reliability. The statistical analysis was carried out with SAS statistic package v9.0 (SAS Institute Inc., Cary, NC, USA). Values were considered to be statistically significant when P < 0.05.
Declaration of Interest: The authors report no conflict of interest.
Abbreviations
| SCA: | = | Sperm Class Analyzer |
| CV: | = | coefficient of variation |
| ICC: | = | intra-class correlation coefficient |
| MOT: | = | total motile spermatozoa |
| PMOT: | = | progressive motile spermatozoa |
| CLV: | = | curvilinear velocity |
| SLV: | = | straight line velocity |
| APV: | = | average path velocity |
| LIN: | = | linearity |
| STR: | = | straightness |
| WOB: | = | wobble |
| LHD: | = | lateral head displacement |
| BCF: | = | beat cross frequency. |
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