VARIATIONS IN LABORATORY SEMEN EVALUATION PROCEDURES AND TESTING

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1 VARIATIONS IN LABORATORY SEMEN EVALUATION PROCEDURES AND TESTING Leonardo F. C. Brito ABS Global DeForest, Wisconsin BRITO Dr. Brito is a research scientist at ABS Global. He received his DVM from the Federal University of Uberlandia in Brazil, M.Sc. from the Sao Paulo State University-Botucatu, Brazil, and M.Vet.Sc. and Ph.D. from the University of Saskatchewan. He has worked as a large animal reproduction clinician at the WCVM at the University of Saskatchewan and the New Bolton Center at the University of Pennsylvania. Dr. Brito is a Diplomate of the American College of Theriogenologists, currently serving on the exam committee of the college. Dr. Brito is also a member of the editorial board of the journal Theriogenology and serves as an ad-hoc reviewer for several international scientific publications. His research interests have included several aspects of animal andrology such as sexual development, testicular thermo-regulation, pathogenesis of sperm defects, semen evaluation, and breeding soundness examinations. Introduction Semen evaluation is a fundamental part of the work of any semen processing center. Although sometimes viewed as trivial, obtaining accurate and precise results requires knowledge of the principles and peculiarities of different tests and equipment, coupled with quality assurance/control measures including technician training, continuing education, and proficiency testing. The objective of this article is to describe some factors that affect the results of sperm motility and concentration evaluations. Sperm Motility There are several sources of variation in subjective sperm motility evaluation and one of the most important is the evaluator. Consistency within and across evaluators can only be obtained with training and continuing proficiency testing, which is difficult due to the lack of standard training materials and the rapid changes in sperm motility in semen samples maintained over time. Although it is virtually impossible to subjectively determine what the real motility is, the precision (variability) of results can be determined, but it is difficult to determine the accuracy of the results. Some obvious technical variations might affect the results of sperm motility evaluation. Sperm cannot be properly visualized with bright-field microscopy. A good microscope with appropriate optics (either phasecontrast or DIC) is essential for proper evaluation of sperm motility; X magnification is commonly used. Uneven distribution of sperm, when wet-mount preparations are used for motility evaluation, might also affect the results. To minimize this problem, several fields of view should be evaluated and areas close to the

2 borders of the coverslip should be avoided. To illustrate how some of other, not-so-obvious, technical variations might also affect the results, two experiments were conducted mimicking procedures commonly used for semen evaluation in the field. There have been reports of the use of a droplet from the very tip of the straw for semen evaluation, but it is unclear how representative this sub-sample is. In the first experiment, frozen semen batches (n = 30) were examined in duplicates in order to determine sperm motility in samples obtained from the tip of the straw. After thawing, the straw was thoroughly dried, shaken to completely move the air bubble towards the sealed end, and the seal was cut. A sample (4.5 μl) was immediately pipetted from the tip of the straws and loaded into a chamber of a 4-chambers Leja slide. The content of the entire straw was then emptied into a glass tube and a sample was obtained and loaded into another chamber. The proportion of progressively motile sperm was determined by evaluating 7 fields per chamber using the Hamilton-Thorne IVOS system. The order of evaluation (i.e. first and second) for tip and straw was alternated among the samples. Sperm motility in samples from the tip of the straw was lower (GLM and Tukey s test; P < ) than in samples from the entire straw (47.9 ± 1.4% vs ± 1.3%, respectively; mean ± SEM). Microscope stage warmers are relatively expensive and evaluation of sperm motility without stage warmers is not uncommon. A second experiment was conducted in order to determine how the temperature of the microscope stage affects sperm motility using frozen semen batches (n = 40). After thawing, the contents of two straws were emptied into a glass tube and a sample (4.5 μl) was loaded into a chamber of a 4-chambers Leja slide; glass tubes and Leja slides were kept at 37 o C using a warming block/plate. The proportion of progresssively motile sperm was determined by evaluating seven fields per chamber using the Minitube Sperm Vision system that included a microscope equipped with a stage warmer. The evaluation was performed with the stage warmer turned on (37 o C) and later repeated (two additional straws) with the stage turned off (room temperature 21 o C). The time between placing the slide on the stage and conclusion of the evaluation was approximately 20 to 30 seconds. Sperm motility was lower (paired t-test; P < ) when the microscope stage warmer was turned off (51.5 ± 1.9%) than when the stage was turned on (55.8 ± 1.6%). The results of these experiments indicate that simple technical variations in the evaluation of sperm motility can have considerable effects on the results. Accordingly, evaluation of sperm motility in samples obtained from the tip of the straw using a microscope without a stage warmer would result in an apparent 10% reduction in sperm motility. Objective evaluation of sperm motility has been made possible by the development of computer-assisted semen analysis (CASA) systems. CASA greatly reduces technician variations in sperm motility results and allows better discrimination of samples than subjective sperm motility evaluation (10,13,32). However, although different CASA systems are based on similar principles, there are several differences in the hardware and in the algorithms used for detection and tracking of sperm among different systems. Temperature, semen extender, sperm concentration, frame acquisition rate, type of chamber, and number of fields (number of sperm) have all been shown to affect CASA sperm motility results. Moreover, motile sperm popula-tions are based on thresholds of average path velocity (VAP), straight-line velocity (VSL), and straightness (VSL/VAP) that are defined by the user (9,22,27,32). Table 1 illustrates the effect of frame acquisition rate on CASA sperm motility, whereas Table 2 illustrates the variability of CASA methods used in studies with bovine semen. Table 1. Effect of video frame acquisition rate on CASA sperm motility results using bovine frozen-thawed semen. Values are the average for results obtained using 30 and 45 frames/field. Modified from (9). 60 Hz 30 Hz Difference Average path velocity (μm/s) Straight-line velocity (μm/s) Straightness (%) Motile sperm (%) Progressively motile sperm (%) Sperm Concentration Determining sperm concentration involves enumerating sperm contained in a defined volume of semen. The process involves preparation and dilution of the semen sample and counting the cells. Gridded-chambers including haemocytometers and Makler chamber, and capillary-filled chambers (Leja, Microcell) used with a microscope eye-piece grid are examples of some manual methods described for counting sperm. Automated sperm counting equipment include CASA, Nucleocounter, and flow-cytometry. As demonstrated in a series of experiments, semen handling and sampling techniques can significantly affect sperm concentration results. In the first experiment, frozen semen batches (n = 40) were used to determine the effect of the material of the tube used for processsing and diluting the semen on sperm concentration. Each sample was prepared by pooling the contents of seven 0.5 ml semen straws into a 12 x 75 mm borosilicate glass tube. The samples were vortexed for 5 sec and 0.5 ml of the semen was transferred into 12 x 75 mm borosilicate glass, polypropylene, or polystyrene tubes

3 Table 2. Methods and settings for determining sperm motility in bull semen using CASA. Semen Extender Concentration (x10 6 /ml) Equipment Chamber No. fields evaluated Frame rate Frames/ field Med VAP (µm/s) Low VAP (µm/s) Threshold Straightness (%) References Fresh Talp 25 Makler (14) (8.1) Fresh, Talp, CUE, 20 Microcell (30) cooled Tris-EY (10) Frozen Tris-EY 40 HT Makler (5) Frozenwashed (10.6) Talp 50 Makler (29) MTM-SM- Minimum Frozen - 2 and 5 Makler (4,16,17) CMA 200 sperm Frozen Tris-EY 50 Leja (7) Eipdidymal HT-Ceros Saline 50 Leja (15) sperm (12.1) Frozen Whole milk - Leja (23) IDENT HT-Ceros Minimum Fresh Saline 30 Leja (19) (12.1) 100 sperm Frozen - 30 Makler (21) (10.9) Fresh and frozen Talp, Bioxcell, Botu-bov 30 (fresh) 100 (frozen) Ultimate Leja, Slide/ coverslip (6) Fresh and Biociphosplus Spermvision Minitube 88 Leja 4 to (19) frozen Fresh and 40 (fresh) Slide/ Bioxcell Custom (25,26) frozen 92 (frozen) coverslip Frozen Tris-EY 120 HT (1) VAP: Average Path Velocity CUE: Cornell University Extender EY: Egg Yolk HT: Hamilton-Thorne

4 in duplicates (80 samples per tube material). Sperm concentration was determined using the Nucleocounter SP-100 (Chemometec, Allerød, Denmark) after dilution (1:11) into the same type of tube that the sample had been dispensed into. The material of the tube into which semen samples were dispensed and diluted into did not affect sperm concentration (28.3 ± 1.4, 28.1 ± 1.4, and 28.5 ± 1.4 x 10 6 sperm/ml for glass, polypropylene, and polystyrene tubes, respectively; mean ± SEM). In the second experiment, frozen semen batches frozen in 0.5 and 0.25 ml straws (n = 50 batches each) were used to determine the effect of semen dispensing method on sperm concentration. After thawing, the tip of the straw was cut, and the cotton polyvinyl alcohol plug end was either pushed along inside the straw using a metal stylet to dispense the semen or the cotton plug was cut to allow draining by gravity. The contents of the straw were collected into a microcentrifuge tube, semen was diluted (1:11), and sperm concentration was determined using the Nucleocounter. Sperm concentration in semen samples dispensed by pushing the straw plug was greater (paired t-test; P < ) than in samples obtained by gravity draining for both 0.5 and 0.25 ml straws (Table 3). On average, sperm concentration in samples obtained by draining was 13.0% lower for 0.5 ml straws and 9.4% for 0.25 ml straws. Table 3. Mean (± SEM) sperm concentration (x 10 6 sperm/ml) in bovine frozen-thawed semen samples obtained by pushing the straw plug along inside the straw or by gravity draining according to straw size. Push plug Draining Difference 95% CI 0.5 ml straws 31.5 ± 3.1 a 28.0 ± 2.9 b ml straws 49.2 ± 2.3 a 44.7 ± 2.3 b a,b Columns with different superscripts differ (P < ). A third experiment was conducted to determine the effects of pipetting method on semen dilution. Four technicians, all with more than 10 years of experience in the laboratory participated in the study. Intra- and intertechnician coefficients of variation (CV) and accuracy for the volume of dispensed water and extender (containing egg yolk and glycerol) were determined using a scale (sensitivity 10-7 g) and an addition-tare method. Each measurement was replicated 20 times by each technician. Z-factor was determined based on room temperature and used to calculate water volume. Extender density was determined based on triplicate weights of 25 ml and used to calculate extender volume. Forward manual pipetting was performed using Pipetman pipettes (Gilson). The pipettes were serviced and calibrated prior to the study and all technicians used the same pipettes. A P-1000 was used to pipette 500 and 1000 μl of water and a P-250 was used to pipette 50, 100, and 200 μl of extender. Automatic pipetting was performed using an auto-diluter (Microlab 500 Series; Hamilton) equipped with 5000 and 100 μl syringes. Water volumes (500 and 1000 μl) were evaluated without aspirating any extender and extender volumes (50 and 100 μl) were determined when diluted with both 500 and 1000 μl of water. The average water weight was subtracted from the total in order to calculate extender volumes; therefore, results on extender volume were actually based on 40 replicates when the auto-diluter was used. The results of this experiment are shown in Table 4. The CV for water manual pipetting were low (<1%) and the results were fairly accurate, mostly 0.5 to 1% lower than the target. However, the CV for extender manual pipetting were slightly greater than that for water (approximately 3%) and the results were less accurate. The volume of manually pipetted extender was approximately 5% lower than the target, in some cases as much as 8% lower. The auto-diluter produced more precise results, as demonstrated by CV that were several orders of magnitude smaller than that observed for manual pipetting for both water and extender. Moreover, the auto-diluter produced more accurate results; the volumes were practically 100% accurate for water and only approximately 0.05% lower than the target for extenders. Another source of variation in sperm concentration results is the counting method. Different manual methods have been described for determining sperm concentration and the improved Neubauer haemocytometer is considered by the World Health Organization the gold-standard method for human semen (33). Makler, Leja, and Microcell chambers have been used for manual sperm count, but the CV observed with these chambers are consistently 1.5 to 2 times greater than those observed with haemocytometers. Moreover, sperm concentrations are overestimated with the Makler chamber and underestimated with the Leja and Microcell chambers when compared to haemocytometers (3, 8, 31). Although haemocytometers are considered the gold standard for evaluation of sperm concentration, variation in results might occur as the result of problems with the haemocytometer (chips and scratches), incorrect use of coverslip (wrong type of coverslip and/or coverslip placement), improper loading (over or under load, presence of air bubbles), deficient counting technique (inconsistent screening pattern, incorrect definition of the area borders, missed or double counted sperm), wrong calculation formulae, and insufficient number of sperm counted. In addition, according to manufacturers the depth of the chamber in haemocytometers is 100 ± 2 μm, but evaluation of empty and loaded chambers from several manufacturers revealed that the actual depth was ± 7 μm (G. Althouse, personal communication). Reported CV s for sperm concentration results using haemocytometers range from 5 to 12% (8, 11, 18).

5 Table 4. Coefficients of variation and accuracy for manual and automatic pipetting of water and semen extender (egg yolk + glycerol). Manual Automatic Water 500 μl intra-technician CV (%) 0.22 to to μl intra-technician CV (%) 0.1 to to μl inter-technician CV (%) μl inter-technician CV (%) μl intra-technician accuracy (%) to to μl intra-technician accuracy (%) to to μl inter-technician accuracy (%) μl inter-technician accuracy (%) Extender 50 μl intra-technician CV (%) 0.73 to to μl intra-technician CV (%) 0.54 to to μl intra-technician CV (%) 0.4 to μl inter-technician CV (%) μl inter-technician CV (%) μl inter-technician CV (%) μl intra-technician accuracy (%) to to μl intra-technician accuracy (%) to to μl intra-technician accuracy (%) to μl inter-technician accuracy (%) μl inter-technician accuracy (%) μl inter-technician accuracy (%) The effect of haemocytometer was evaluated in one experiment using frozen semen batches (n = 40) and two improved Neubauer haemocytometers (Hausser Scientific). Semen was diluted (1:11), both chambers on each side of the haemocytometer were loaded, and sperm were counted in 1 mm 2. The haemocytometers were then washed, dried, and the process was repeated. All procedures were performed by the same technician. There was a small, but significant difference (paired t-test; P < 0.05) in sperm concentration determined with the two haemocytometers (35.4 ± 2.3 vs ± 2.1 x 10 6 sperm/ml; mean ± SEM). There was also a tendency (P < 0.07) of difference for the CV for duplicate counts between the haemocytometers (6.8 vs 7.4%). The overall CV (2 counts per haemocytometer, 4 counts total) was 6.8% (95% CI: 5.9 to 7.7%). Some automatic methods for sperm count include CASA, Nucleocounter, and flow-cytometry. Sperm concentration using CASA has been shown to be either underestimated or overestimated when compared to haemocytometers depending on the species, sperm concentration, and type of chamber used (20, 24, 27, 28). The differences are likely due to erroneous identification of debris as sperm heads, clumping creating objects too large to be accepted as sperm, and collisions with subsequent multiple counts of the same spermatozoon. Although the use of a correction factor to compensate for the uneven distribution of sperm in capillary-filled chambers [the Segre-Silberberg effect (12)] and immobilization of sperm before evaluation might improve the agreement between CASA and haemocytometer results (3), the use of CASA for evaluation of sperm concentration in human semen has not been advised by the WHO (33). The Nucleocounter is an automatic instrument that uses an integrated fluorescent microscope system designed to detect signals from a fluorescent dye (propidium iodide) bound to the nuclei of sperm. Semen samples are diluted with a detergent solution and loaded into a disposable cassette coated with the dye. According to the manufacturer, the depth of the chamber used for the count is determined and the information is embedded in each cassette to be used by the equipment for calculation of the sperm concentration. Results from studies using boar and bull semen have demonstrated that the results obtained with the Nucleocounter agree with those obtained with flow-cytometry (2, 18). Moreover, the reported CV for sperm concentration results using the Nucleocounter are approximately half (2.5 to 4%) of those observed with haemocytometers

6 (8,11,18) and comparable to those observed with flowcytometry. At ABS Global, data obtained from several experiments indicated that the CV for triplicate Nucleocounter results from 186 semen samples with a concentration of ± 41.0 x 10 6 sperm/ml (mean ± SEM) was 3.9% (95% CI 3.5 to 4.2%), whereas the CV for duplicate results from 98 semen samples with a concentration of ± 22.0 x 10 6 sperm/ml was 2.6% (95% CI 2.2 to 3.1%). Moreover, evaluation of two Nucleocounter machines using fresh and frozen (n = 20) semen samples evaluated in duplicate revealed no effect of machine on sperm concentration (214.3 ± 52.3 and ± 51.5 x 10 6 sperm/ml) or CV (2.5 and 2.7%). Practical tips for frozen-thawed bovine semen evaluation The entire contents of one or two semen straws should be pooled for frozen-thawed semen evaluation. A single droplet from the tip of the straw should never be used for evaluation. Frozen-thawed semen straws should be emptied by pushing the cotton plug through the straw and not by gravity draining. Subjective sperm motility evaluation should be performed using phase-contrast or DIC optics under X magnification with a microscope also equipped with a stage warmer set up to 37 o C. Technician training, continuing education, and proficiency testing are fundamental to obtain precise results. CASA sperm motility evaluation should be performed at 37 o C with acquisition frame rate 60 Hz and sperm concentration 40 x 10 6 /ml. Thresholds for defining immotile and motile sperm should be determined empirically under the conditions used in each laboratory (i.e. type of semen sample, extender, equipment, and chamber). The use of auto-diluters and the Nucleocounter is recommended for obtaining accurate and precise sperm concentration results. Acknowledgments I would like to thank Lester Doyle, Paul Golueke, and Kathy Mean for their assistance with experiments; and Eliza Roberts and Jeff Betthauser for reviewing this manuscript. References 1. Amorim, E. A., J. K. Graham, B. Spizziri, M. Meyers, and C. A. Torres Effect of cholesterol or cholesteryl conjugates on the cryosurvival of bull sperm. Cryobiology 58: Anzar, M., T. Kroetsch, and M. M. Buhr Comparison of different methods for assessment of sperm concentration and membrane integrity with bull semen. J Androl 30: Bailey, E., N. Fenning, S. Chamberlain, L. Devlin, J. Hopkisson, and M. Tomlinson Validation of sperm counting methods using limits of agreement. J Androl 28: Ballester, J., A. Johannisson, F. Saravia, M. Haard, H. Gustafsson, D. Bajramovic, and H. Rodriguez-Martinez Post-thaw viability of bull AI-doses with low-sperm numbers. Theriogenology 68: Bilodeau, J. F., S. Blanchette, C. Gagnon, and M. A. Sirard Thiols prevent H2O2-mediated loss of sperm motility in cryopreserved bull semen. Theriogenology 56: Celeghini, E. C., R. P. de Arruda, A. F. de Andrade, J. Nascimento, C. F. Raphael, and P. H. Rodrigues Effects that bovine sperm cryopreservation using two different extenders has on sperm membranes and chromatin. Anim Reprod Sci 104: Chaveiro, A., L. Machado, A. Frijters, B. Engel, and H. Woelders Improvement of parameters of freezing medium and freezing protocol for bull sperm using two osmotic supports. Theriogenology 65: Christensen, P., H. Stryhn, and C. Hansen Discrepancies in the determination of sperm concentration using Burker-Turk, Thoma and Makler counting chambers. Theriogenology 63: Contri, A., C. Valorz, M. Faustini, L. Wegher, and A. Carluccio Effect of semen preparation on CASA motility results in cryopreserved bull spermatozoa. Theriogenology 74: Davis, R. O., and D. F. Katz Standardization and Comparability of CASA instruments. J Androl 13: DeJarnette, J., and K. Lefevre Use of the Nucleocounter in a semen processing laboratory. Proc. 22 nd Tech. Conf. Artif. Insem. and Reprod., NAAB, pp Douglas-Hamilton, D. H., N. G. Smith, C. E. Kuster, J. P. Vermeiden, and G. C. Althouse Capillary-loaded particle fluid dynamics: effect on estimation of sperm concentration. J Androl 26:

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