Comparison of Computer-assisted Sperm Motility Analysis Parameters in Semen from Belgian Blue and Holstein?Friesian Bulls

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1 See discussions, stats, and author profiles for this publication at: Comparison of Computer-assisted Sperm Motility Analysis Parameters in Semen from Belgian Blue and Holstein?Friesian Bulls ARTICLE in REPRODUCTION IN DOMESTIC ANIMALS APRIL 2007 Impact Factor:.52 DOI: 0./j x Source: PubMed CITATIONS 22 READS AUTHORS, INCLUDING: Tom Rijsselaere Ghent University 4 PUBLICATIONS 743 CITATIONS Ann Van Soom Ghent University 43 PUBLICATIONS 5,857 CITATIONS SEE PROFILE SEE PROFILE Dominiek Maes Ghent University 324 PUBLICATIONS 4,247 CITATIONS Aart de Kruif Ghent University 430 PUBLICATIONS 7,642 CITATIONS SEE PROFILE SEE PROFILE All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately. Available from: Tom Rijsselaere Retrieved on: 08 April 206

2 Reprod Dom Anim 42, 53 6 (2007); doi: 0./j x ISSN Comparison of Computer-assisted Sperm Motility Analysis Parameters in Semen from Belgian Blue and Holstein Friesian Bulls G Hoflack, G Opsomer, T Rijsselaere, A Van Soom, D Maes, A de Kruif and L Duchateau 2 Departments of Reproduction, Obstetrics and Herd Health, and 2 Physiology, Biochemistry and Biometrics, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Contents Subjective microscopic sperm motility results have recently been demonstrated to differ between Holstein Friesian (HF) and Belgian Blue (BB) bulls. However, such assessments are rather imprecise. In the present study, sperm motility was assessed objectively by means of the Hamilton Thorne CEROS version 2.2c computer-assisted sperm motility analyser (CASA), and differences between the BB and HF breed could also be demonstrated. Higher percentages of both totally (p < 0.000) and progressively (p < 0.000) motile spermatozoa were encountered in the HF breed compared with the BB breed. Furthermore, a lower kinetic efficiency of the BB spermatozoa, evidenced by a lower beat cross-frequency (p ¼ ) combined with a higher lateral head displacement (p ¼ 0.005), was the basis for the lower velocity of BB sperm cells. Additionally, BB spermatozoa move less straight forward, resulting in a lower straightness (p < 0.000). No sperm motility differences were observed between age groups within the BB breed. The breed differences were observed in the examined bull populations residing at AI centres, in Belgium for the BB bulls and in the Netherlands for the HF bulls. However, these bull populations are selected for fertility. A similar pattern was observed in an unselected bull population of both breeds, although these differences were mostly non-significant for the different CASA parameters. Nevertheless, these data suggest that a genetic component might be responsible for the observed sperm motility breed differences. Introduction Male fertility is an important factor in bovine reproduction as a single bull is generally used to breed numerous cows. To evaluate male fertility potential, semen analysis is the most commonly used procedure (Chong et al. 983; Vantman et al. 988; Comhaire et al. 992; Rijsselaere et al. 2002). Hence, semen evaluation of breeding bulls is of paramount importance (Ott 986; Bruner and Van Camp 992; Chenoweth et al. 994; Barth 997; Garner 997). Although several methods can be used to evaluate the quality of a fresh or frozen thawed semen sample, subjective evaluation using standard optical microscopy is by far the most commonly used procedure (Rijsselaere et al. 2003; Christensen et al. 2005). The conventional semen parameters routinely examined are the concentration, the percentage of motile spermatozoa and their morphology (Vantman et al. 988; Neuwinger et al. 990a). However, subjective evaluation of sperm count, motility and morphology has been shown to be relatively inaccurate and imprecise (Davis and Katz 993; Christensen et al. 999, 2005). Furthermore, the judgement of subjective motility depends largely on the level of training and skills of the investigator (Knuth et al. 989), frequently leading to a lack of agreement between different laboratories examining the same specimens (Davis and Katz 993). Hence, the subjective visual assessment of semen motility of the same sperm samples yielded coefficients of variation of 20% between technicians, and up to 37% between laboratories (Jequier and Ukombe 983; Dunphy et al. 989; Neuwinger et al. 990b). The need for standardization of semen analysis to reduce this high variability has been demonstrated repeatedly (Chong et al. 983; Davis and Katz 993; Verstegen et al. 2002). Computer-assisted semen motility analysis (CASA) procures detailed, accurate and highly repeatable data on different semen motility parameters both in human and animal species (Vantman et al. 989; Gu nzel-apel et al. 993; Farrell et al. 996), largely reducing the subjectivity and overcoming the variability inherent to the routine microscopical semen examination (Verstegen et al. 2002). However, several parameters, such as semen concentration influence CASA outcome (Verstegen et al. 2002; Rijsselaere et al. 2003, 2005). The latter is due to multiple individual collisions of the condensed sperm cells at high concentrations, which interfere with the progressive motility (Vantman et al. 988; Bartoov et al. 99; Rijsselaere et al. 2002, 2003; Hoflack et al. 2005). To avoid this inaccuracy and to obtain more reliable and unbiased kinetic measurements, a concentration between 20 and spermatozoa/ml has been advised in CASA studies (Budworth et al. 988; Vantman et al. 989; Neuwinger et al. 990a; Davis and Katz 993; Farrell et al. 996; Verstegen et al. 2002; Rijsselaere et al. 2003). Although literature data on the relation between sperm motility and fertility are contradictory, with some reports acknowledging this relationship (Wood et al. 986; Kjaestad et al. 993; Correa et al. 997; Zhang et al. 998; Januskauskas et al. 2000) and others not able to demonstrate any relationship (So derquist et al. 99b; Andersson et al. 992; Januskauskas et al. 999), sperm motility is generally considered to be an important semen quality parameter. Most studies on this relationship used frozen thawed semen. In studies using subjectively assessed fresh sperm motility to predict fertility outcome, some found no relation (Fitzpatrick et al. 2002; Holroyd et al. 2002), while others demonstrated that fresh sperm motility is correlated with the fertilizing potential of the inseminate (Christensen et al. 999, 2005). Furthermore, Farrell et al. (998) reported very high correlations (0.99, r 2 ¼ 0.98) between

3 54 G Hoflack, G Opsomer, T Rijsselaere, A Van Soom, D Maes, A de Kruif and L Duchateau combined motility parameters measured by CASA on fresh semen and bull fertility, evidenced by the 59-day non-return rate (NRR) to first service. Other reports on the relation between computerized semen-quality measurements and bull fertility also demonstrated that the combination of several semen-quality parameters correlate better with fertility than single motility parameters (Budworth et al. 987, 988). Hence, CASA has the potential for a more accurate prediction of fertility than the parameters assessed by the routine microscopical semen evaluation (Farrell et al. 998; Christensen et al. 999). In Belgium, the two predominant cattle breeds are the double-muscled Belgian Blue (BB) beef breed, which is famous for its low feed conversion ratio, its high percentage of lean meat and its advantageous carcass classification, and the internationally known Holstein Friesian (HF) dairy breed. In contrast to the HF breed, data on semen quality of BB bulls are scarce. However, recent studies demonstrated a poorer quality of BB sperm in comparison with HF sperm. Concerning motility, both the percentages of totally and progressively motile sperm, as well as sperm velocity were lower in the BB ejaculates (Hoflack et al. 2003, 2006a,b). However, these parameters were subjectively assessed. The aim of the present study was therefore to evaluate and compare several semen motility characteristics of the BB beef breed and the HF dairy breed by means of CASA in order to objectively assess whether the same breed differences occur. Materials and Methods Study population Computer-assisted semen motility analysis was performed on fresh semen of 4 BB and 49 HF ejaculates of 36 BB and 42 HF bulls, respectively, collected at AI centres (in Belgium for the BB bulls and in the Netherlands for the HF bulls, as no HF AI bulls are present in Belgium). All semen samples were collected by means of an artificial vagina and only first ejaculates were used. The examined bulls were all regularly collected (twice a week) in the weeks preceding this study, which was conducted in November 2003 and May 2004 for the BB and HF bulls, respectively. The BB AI bulls were purchased from selection centres as well as from private farms based on their linear classification, which is a scoring system describing several physical characteristics of the bull [concerning height, muscular development, meat type (skeletal conformation), stance and general appearance], and after a quarantine period of month, they were accepted for AI purposes without further selection. However, bulls with poor libido or repeated poor semen motility before (<60% progressive motility) and after (<30% of total or <5% of progressive motility) cryopreservation were discarded keeping only more fertile bulls with increasing age. Furthermore, aggressive or injured bulls were also eliminated. This selection procedure resulted in three groups of BB bulls which could arbitrarily be divided as follows: () the unselected youngest bulls (<2 years), (2) the active breeding bulls between 2 and 4 years old which were intensively selected, and (3) the bulls of proven fertility of over 4 years of age considered to be veterans which had survived culling for different reasons. This was in contrast to the HF bulls, where bull calves were purchased based on their expected genetic value. At approximately months of age, these bulls were transferred to an AI facility, where, after a quarantine period of month semen was collected. Only when these bulls passed a strict semen-quality test (two consecutive ejaculates collected within a 3- or 4-day interval 2ml containing spermatozoa/ml, with 65% motility, 80% normal morphology and 50% intact acrosomes) were they accepted for AI purposes. When an accepted HF bull had produced around 3000 straws, semen collection was stopped for at least 3 years during which the bull s progeny was tested. This resulted in two HF groups comparable with the two youngest groups of BB bulls: () an unselected young group, and (2) a group of selected young bulls that passed an initial semenquality test. Hence, we made three breed comparisons: An overall breed comparison using both unselected and selected bulls of both breeds, stratifying for these respective groups. 2 The unselected young HF and BB bulls <2 years of age, hereafter referred to as the unselected bulls. 3 The fertility selected young HF bulls, and the BB bulls between 2 and 4 years of age, hereafter referred to as the selected bulls. After these three breed comparisons, within-breed comparisons of the three BB groups (unselected selected old BB bulls) were also performed. Computer-assisted sperm motility analysis Immediately after semen collection, a 50-ll semen aliquot was diluted in physiological saline solution at 37 C to a concentration of approximately spermatozoa/ml. This concentration is well within the limits to obtain accurate kinetic measurements (Vantman et al. 989; Neuwinger et al. 990a; Davis and Katz 993; Verstegen et al. 2002; Rijsselaere et al. 2003), which are not biased by multiple individual collisions of condensed sperm cells at higher concentrations falsifying the motility results (Vantman et al. 988; Bartoov et al. 99; Rijsselaere et al. 2002). Motility analysis was directly performed by means of the Hamilton Thorne CEROS version 2.2c computeraided semen analyser (Hamilton-Thorne Research, Beverly, MA, USA) using the green light filter. The software settings of the HTR 2.2c used in the present study were largely based on the recommendations of Davis and Katz (993) and are summarized in Table. Preliminary trials using the playback facility to see whether the sperm cells were correctly identified in physiological saline solution and whether their analysed trajectories were correctly reconstructed, led to these parameter settings, which slightly differ from those advised by the manufacturer. Each ejaculate was assessed twice by loading 7 ll of the diluted semen in a pre-warmed disposable Leja counting chamber (Orange Medical, Brussels, Belgium) on a minitherm stage

4 CASA of Belgian Blue and Holstein Friesian bull sperm 55 Table. Software settings of the Hamilton Thorne Ceros 2.2c used in the present study for the bull sperm motility assessment Parameter Parameter function Value Frame rate (Hz) Image capture 60 Number of frames acquired Image capture 30 Minimum contrast Cell detection 20 Minimum cell size (pixels) Cell detection 0 Non-motile head size (pixels) Cell detection 5 Non-motile head intensity Cell detection 20 Medium VAP cut-off (lm/s, MVV) Progressive cell detection 50 Straightness cut-off (%, STR) Progressive cell detection 70 Low VAP cut-off (lm/s, LVV) Static cell detection 30 Low VSL cut-off (lm/s, LVS) Static cell detection 5 Minimum static intensity limit Cell differentiation 0.5 Maximum static intensity limit Cell differentiation.5 Minimum static size limit Cell differentiation 0.5 Maximum static size limit Cell differentiation 3.0 Minimum static elongation limit Cell differentiation 0 Maximum static elongation limit Cell differentiation 85 warmer (37 C), and by counting at least 000 cells per analysis to avoid poor repeatability, starting in the left upper corner and ending in the right lower corner of the chamber (Verstegen et al. 2002). The average result of these two analyses per ejaculate was used for further data processing. In general, only one ejaculate per bull was assessed by CASA, but in some exceptional cases (five BB and seven HF bulls) two ejaculates per bull, collected on different days, were available. In the latter situation, the CASA results of these two ejaculates were averaged to obtain an as accurate as possible picture of these bull s semen. The assessed parameters were: average pathway velocity (VAP) ¼ the average velocity of the smoothed cell path (lm/s); straight line velocity (VSL) ¼ the average velocity measured in a straight line from the beginning to the end of the track (lm/s); the amplitude of the lateral head displacement (ALH) ¼ the mean width of the head oscillation as the sperm cells swim (lm); the beat cross-frequency (BCF) ¼ frequency of the sperm head crossing the average path in either direction (Hz); the straightness (STR) ¼ average value of the ratio VSL/VAP (%); the percentage of totally motile spermatozoa (MOT; %) and the percentage of progressive spermatozoa (PMOT; %). According to the low (LVV) and medium (MVV) VAP cut-off values, and the low VSL cut-off value (LVS; Table ), the sperm population was additionally divided into four velocity categories: rapid (RAP; % with VAP > MVV), medium (MED; % with MVV > VAP > LVV), slow (SLOW; % with VAP < LVV and VSL < LVS) and static (STATIC; the percentage of spermatozoa which were not moving during the analysis) spermatozoa. To avoid inaccuracy caused by wave motion of the sample, an equilibration time of approximately min after loading the Leja chamber was adopted, and slow and static cells were not considered to determine the percentage of motile spermatozoa. The percentage of progressive spermatozoa (PMOT; %) includes all sperm cells with both VAP > MVV (¼ 50 lm/s) and STR > 70%. Of several motility parameters (VAP, VSL, ALH, BCF, STR), the Hamilton Thorne equipment also calculates the within-ejaculate standard deviations. These withinejaculate standard deviations were averaged over all bulls within each bull group (unselected BB, selected BB, old BB, unselected HF and selected HF bulls) and compared amongst bull groups to assess the homogeneity of (the assessed sperm parameter in) the ejaculates of bulls belonging to a particular bull group. Subjective semen analysis Subjective motility analysis Simultaneously, the percentages of total and progressive motility were subjectively assessed to the nearest 5% by placing 0 ll of diluted semen (0 ll aliquot of pure semen in 790 ll physiological saline solution) on a prewarmed glass slide at 37 C under a coverslip, and by examining five different microscopic fields all in the centre of the coverslip, under a 200 phase-contrast microscope (Hopkins and Spitzer 997). This procedure was repeated twice by the same experienced observer. Additionally, a velocity score ( 4) was attributed to the sample: ¼ very slow semen (e.g. after freeze thawing), 2 ¼ slow semen, 3 ¼ rapid semen, 4 ¼ extremely rapid semen. Subjective morphology analysis Sperm morphology of the examined bulls was also assessed by two experienced observers. This was done by counting 200 spermatozoa on air-dried, eosin nigrosinor eosin aniline blue-stained smears under a 000 light microscope, using immersion oil. Individual spermatozoa were classified according to Barth and Oko (989) into one of five categories: () normal morphology; (2) abnormal head; (3) abnormal midpiece or tail; (4) proximal droplet; or (5) having a distal droplet. When multiple abnormalities were observed in the same sperm cell, only one abnormality was logged. Abnormal heads were given first priority in classification, abnormal midpieces or tails were classified with second priority, proximal droplets with third and distal droplets with least and last priority. Statistical analysis The overall breed comparison for all the motility parameters was based on the fixed-effects model with normally distributed random error term, including bull group (unselected selected) as a stratifying factor. The comparisons between the breeds within the respective groups and the comparisons between the three age categories of the BB breed were also based on the fixedeffects model with normally distributed random error term. The global level of significance for all statistical analyses was 0.05, but for the five pairwise comparisons, the comparisonwise significance level was Bonferroniadjusted to 0.0 (0.05/5 comparisons). The sperm morphology parameters were compared between the two breeds by means of the Mann Whitney test, as these data were not normally distributed. Furthermore, the Spearman s rank correlation coefficient was derived to quantify the relationship between the sperm morphology results on the one hand and the CASA parameters on the other hand.

5 56 G Hoflack, G Opsomer, T Rijsselaere, A Van Soom, D Maes, A de Kruif and L Duchateau Results Computer-assisted sperm motility analysis The CASA results of the overall breed comparison and of the respective groups within both breeds (three BB and two HF groups) for the assessed motility parameters are listed in Table 2. The results for the withinejaculate standard deviations of all the assessed kinetic measurements are given in Table 3. Overall breed differences for the CASA results When the overall results of both breeds were compared, all the assessed parameters (except MED%) differed significantly (Fig., Table 2). The percentages of totally and progressively motile spermatozoa (MOT, PMOT; p < 0.000) and the semen velocity parameters were significantly lower (VAP: p ¼ ; VSL: p ¼ ; STR: p < 0.000) in the BB breed. The ALH was higher (p ¼ 0.005) combined with a lower BCF (p ¼ ) for the BB spermatozoa (Table 2). Fewer rapid (p < 0.000) but more slow (p < 0.000) and static (p < 0.000) spermatozoa were present in the BB breed, while a similar proportion of medium-velocity sperm (p ¼ 0.206) occurred in both breeds (Fig. ). The standard deviations of the parameters of which this was assessed (VAP, VSL, ALH, BCF, STR) were consistently significantly higher in the BB breed (p 0.002; except for VSL which was borderline significantly higher in the BB breed: p ¼ : Table 3). Breed differences within the respective bull groups for the CASA results When comparing the unselected bulls, only the percentage of progressively motile spermatozoa (PMOT; p ¼ 0.009) was significantly lower in the BB breed. The velocity measures were not significantly different, except for the deducted parameter straightness (STR; p ¼ ) which was lower in the BB group (Table 2). However, a higher variability was noticed in the Table 2. Arithmetic mean (±SD) and median (and range) of the motility parameters assessed by CASA for the Holstein Friesian (HF) and Belgian Blue (BB) breed, both for the overall breed results and for the respective groups of both breeds Overall BB Overall HF Unselected BB Selected BB Old BB Unselected HF Selected HF n Parameter MOT (%) 70.4 ± ± ± 6.5 aa 70.5 ± 3.7 aa 58. ± 25.6 a 82. ± 6.2 a 83.8 ± 5.0 b 74.5 ( ) 84.0 ( ) 73.3 ( ) 74.5 ( ) 65.5 ( ) 83.5 ( ) 85.5 ( ) PMOT (%) 53.8 ± ± ± 5.6 aa 53.7 ± 2.6 aa 44.2 ± 20.3 a 67.9 ± 7.2 b 70. ± 5. b 57.5 ( ) 69.8 ( ) 55.3 ( ) 58.0 ( ) 5.0 ( ) 69.0 ( ) 70.0 ( ) VAP (lm/s) 4.0 ± ± ± 5.9 aa 4.5 ±.2 aa 5.9 ± 5.8 a 9.6 ± 2.2 a 22.6 ± 9.9 a 5.9 ( ) 9.7 ( ) 4.6 ( ) 5.9 ( ) 6.0 ( ) 9.3 ( ) 20.2 ( ) VSL (lm/s) 98.8 ± ± ± 5.9 aa 99.0 ± 2.2 aa 99. ± 4.2 a 07.9 ±.0 a.4 ± 9.8 b 00.6 ( ) 08.8 (9. 32.) 98.6 ( ) 00.6 ( ) 02.6 ( ) 07.2 (9. 32.) 0.4 ( ) ALH (lm) 5.0 ± ± ± 0.4 aa 5. ± 0.7 aa 5.6 ±.4 a 4.4 ± 0.7 a 4.5 ± 0.4 b 5. ( ) 4.4 (3.2 6.) 4.7 ( ) 5. ( ) 5.7 ( ) 4.3 (3.2 6.) 4.5 ( ) BCF (Hz) 36.9 ± ± ±.9 aa 36.6 ± 2.2 aa 35.5 ± 2. a 39. ± 2.4 a 39. ± 2.2 b 37. ( ) 39.3 ( ) 37.3 ( ) 37. ( ) 35.3 ( ) 39.5 ( ) 39. ( ) STR (%) 84.9 ± ± ±.6 aa 84.7 ± 3. aa 84.2 ± 2.8 a 88.9 ± 2. b 89.7 ±.7 b 85.5 ( ) 89.5 ( ) 85.8 ( ) 85.0 ( ) 85.5 ( ) 89.5 ( ) 89.5 ( ) The number of assessed bulls within each group is also given (n). A different superscript number (, 2) for the overall results of the HF and BB bulls and a different superscript letter (a, b) for the matched comparisons correspond to a significant breed difference (p < 0.05); a different Greek superscript letter (a,b,c) for the three BB age categories corresponds to a pairwise within-breed significant difference (p < 0.05). Table 3. Arithmetic mean (± SD) and median (and range) of the standard deviations of the motility parameters assessed by CASA for the Holstein Friesian (HF) and Belgian Blue (BB) breed, both for the overall breed results and for the respective groups of both breeds Overall BB Overall HF Unselected BB Selected BB Old BB Unselected HF Selected HF n Parameter VAP SD 46.3 ± ± ± 2.8 aa 45.6 ± 3. aa 46.0 ± 3.7 a 43.9 ± 3.3 a 43.0 ± 3.9 a 46.6 ( ) 43.7 ( ) 47.2 ( ) 46. ( ) 46.5 ( ) 44.3 ( ) 42.5 ( ) VSL SD 47.7 ± ± ± 2.8 aa 47. ± 3.8 aa 46.5 ± 5.4 a 46.3 ± 3.3 a 45.6 ± 3.8 a 47.6 ( ) 46.0 ( ) 48.2 ( ) 47.2 ( ) 48.4 ( ) 46.8 ( ) 45.2 ( ) ALH SD 2.8 ± ± ± 0.2 aa 2.8 ± 0.3 aa 2.9 ± 0.4 a 2.5 ± 0.2 a 2.6 ± 0.3 b 2.8 ( ) 2.5 (2. 3.) 2.7 ( ) 2.8 ( ) 3. (2. 3.2) 2.5 (2. 2.9) 2.6 (2.2 3.) BCF SD 7.5 ± ± ± 0.4 aa 7.4 ± 0.9 aa 7.4 ± 0.4 a 6.9 ± 0.8 a 6.8 ± 0.8 a 7.6 ( ) 6.9 ( ) 7.5 ( ) 7.6 ( ) 7.5 ( ) 6.9 ( ) 7.0 ( ) STR SD 7.3 ± ± ± 0.7 aa 7.4 ±.4 aa 7.2 ±.6 a 5.2 ±.3 b 5.4 ±.3 b 7.5 ( ) 5.5 ( ) 7.3 ( ) 7.5 ( ) 8.0 ( ) 5.5 ( ) 5.5 ( ) The number of assessed bulls in each group is also given (M). A different superscript number (, 2) for the overall results of the HF and BB bulls and a different superscript letter (a, b) for the matched comparisons correspond to a significant breed difference (p < 0.05); a different Greek superscript letter (a,b,c) for the 3 BB age categories corresponds to a pairwise within-breed significant difference (p < 0.05).

6 CASA of Belgian Blue and Holstein Friesian bull sperm Belgian blue Holstein Friesian static sperm compared with both younger BB groups (Tables 2 and 3, Fig. 2). % straightness measures of the BB sperm, evidenced by a significantly higher standard deviation (p ¼ : Table 3) compared with the HF bulls. When the selected bulls were compared, the percentages of totally and progressively motile spermatozoa (MOT, PMOT; p ) and most semen-velocity measures (VSL: p ¼ 0.004; BCF: p ¼ 0.005; STR: p < 0.000) were significantly lower in the BB breed, while ALH was significantly higher (p ¼ ) for BB sperm (Table 2). Fewer rapid (p ¼ ) but more slow (p ¼ ) spermatozoa were present in ejaculates of the BB breed, while a similar proportion of medium (p ¼ ) and static (p ¼ ) sperm were noted in ejaculates of this group of both breeds (Fig. 2). The standard deviations of several sperm parameters (ALH SD: p ¼ ; STR SD: p < 0.000: Table 3) were always significantly higher in the BB bulls compared with their matches in the HF breed. Age differences within the BB breed for the CASA results No differences whatsoever were present between the different age groups within the BB breed, with the sole exception of the oldest bulls having significantly more % Unselected BB Selected BB Old BB Unselected HF 2 Rapid Medium Slow Static Fig.. Sperm subpopulations (rapid, medium, slow and static sperm) for the overall results (unselected and selected bulls) of the Belgian Blue and Holstein Friesian breed. A different superscript number (, 2) corresponds to a significant breed difference (p < 0.05) Selected HF Rapid Medium Slow Static Fig. 2. Sperm subpopulations (rapid, medium, slow and static sperm) for the respective groups of the Belgian Blue and Holstein Friesian breed. A different superscript number (, 2) for the breed comparisons of the matched groups corresponds to a significant breed difference (p < 0.05) and a different superscript letter (a, b, c) for the three BB age categories corresponds to a pairwise within-breed significant difference (p < 0.05) 2 2 a a b 2 Subjective motility analysis The results for the parameters of the subjective motility analysis, both for the overall breed comparison and of the respective groups within both breeds (three BB and two HF groups) are summarized in Table 4. The percentage of totally and progressively motile spermatozoa, as well as the velocity score of these spermatozoa were always significantly (p ) lower in the BB breed, both for the overall breed results and for the matched comparisons. No differences were noted between age groups within the BB breed. Subjective morphology analysis The results on the sperm morphology data of the two breeds are given in Table 5. All the assessed sperm morphology parameters were significantly (p < 0.000) poorer in the BB bulls compared with HF bulls, evidenced by a lower percentage of normal spermatozoa and a higher percentage of abnormal heads and tails, and proximal and distal droplets in the BB breed. Correlations between subjective morphology and CASA outcome The Spearman s rank correlation coefficients between the sperm morphology outcome on the one hand and the CASA parameters on the other hand are presented in Table 6. Significant correlations (p < 0.05) were always present between all the sperm morphology and sperm velocity measures, except for the correlations between all the sperm morphology parameters and the % MEDIUM sperm and between the percentage of proximal droplets and the % STATIC sperm (P > 0.05). The correlation between the percentage of abnormal heads and VAP was borderline significant (p ¼ 0.055), while a tendency (p ¼ 0.097) for correlation was present between the percentage of abnormal tails and VAP. The highest correlation coefficients were obtained between the percentage of normal spermatozoa and several CASA parameters. The percentage of abnormal tails and distal droplets correlated best although negatively with the percentage of motile, progressively motile and rapid spermatozoa and positively with the percentage of static spermatozoa. The percentage of abnormal heads correlated best albeit negatively with the percentage of progressively motile spermatozoa and with the straightness and beat cross-frequency of the sperm cells. Percentage of proximal droplets resulted in the poorest correlation coefficients, of which the correlation with the straightness was the best, but negative. Discussion A significant HF breed advantage for subjectively assessed semen motility parameters has recently been demonstrated when HF bulls were compared with BB bulls (Hoflack et al. 2006a,b), and these results were confirmed in the present study. Similarly, in this study

7 58 G Hoflack, G Opsomer, T Rijsselaere, A Van Soom, D Maes, A de Kruif and L Duchateau Table 4. Arithmetic mean (± SD) and median (and range) of the subjectively assessed motility parameters for the Holstein Friesian (HF) and Belgian Blue (BB) breed, both for the overall breed results and for the respective groups of both breeds Overall BB Overall HF Unselected BB Selected BB Old BB Unselected HF Selected HF n Parameter Motile % 60.5 ± ± ± 8.3 aa 59.6 ± 5. aa 48.6 ± 2.9 a 79.3 ± 8. b 80.6 ± 8.4 b 60.0 ( ) 80.0 ( ) 62.5 ( ) 60.0 ( ) 55.0 ( ) 80.0 ( ) 80.0 ( ) Progressive % 56.5 ± ± ± 8.3 aa 55.5 ± 5.5 aa 46.3 ± 2. a 77.7 ± 8.8 b 80.6 ± 8.4 b 60.0 ( ) 77.5 ( ) 58.8 ( ) 60.0 ( ) 55.0 ( ) 75.0 ( ) 80.0 ( ) Velocity 2.9 ± ± ± 0. aa 2.9 ± 0.3 aa 2.6 ± 0.4 a 3.5 ± 0.2 b 3.5 ± 0.0 b 3.0 ( ) 3.5 ( ) 3.0 ( ) 3.0 ( ) 2.8 ( ) 3.5 ( ) 3.5 ( ) The number of assessed bulls within each group is also given (n). A different superscript number (, 2) for the overall results of the HF and BB bulls and a different superscript letter (a, b) for the matched comparisons correspond to a significant breed difference (p < 0.05); a different Greek superscript letter (a,b,c) for the three BB age categories corresponds to a pairwise within-breed significant difference (p < 0.05). All BB bulls (n ¼ 36) All HF bulls (n ¼ 42) % Normal sperm % Abnormal heads % Abnormal tails % Proximal droplets % Distal droplets 52.9 ± ± ± ± ± ( ) 2.8 ( ) 7.8 ( ) 3.8 ( ).4 ( ) 79.7 ± ± ± ± ± ( ) 7.0 ( ) 8.0 ( ) 2.0 ( ) 0.0 (0.0.0) Table 5. Arithmetic mean (± SD) and median (and range) of the subjectively assessed sperm morphology parameters, for all the Holstein Friesian (HF) and Belgian Blue (BB) bulls All these parameters differed significantly between the two breeds (p < 0.05). The number of assessed bulls is also given (n). Parameters % Normal sperm % Abnormal heads % Abnormal tails % Proximal droplets % Distal droplets VAP )0.28 ns )0.89 ns )0.230 )0.229 VSL )0.33 )0.33 )0.343 )0.333 ALH ) BCF )0.504 )0.332 )0.357 )0.46 STR 0.64 )0.503 )0.486 )0.496 )0.440 % MOT 0.63 )0.485 )0.548 )0.292 )0.533 % PROG )0.537 )0.556 )0.37 )0.546 % RAPID )0.490 )0.532 )0.309 )0.52 % MEDIUM )0.66 ns 0.53 ns ns 0.52 ns ns % SLOW ) % STATIC ) ns Table 6. The Spearman s rank correlation coefficients (r) between the subjectively assessed sperm morphology parameters on the one hand and the CASA parameters on the other hand, for all the bulls of both breeds All the correlation coefficients were significant (p < 0.05), with the exception of those marked by a superscript (ns, not significant). objective sperm motility analysis by means of CASA demonstrated an obvious HF breed advantage when the overall CASA results were assessed: the percentage of totally and progressively motile spermatozoa were higher for HF semen, reflecting the higher proportion of live spermatozoa present in a HF ejaculate (Hoflack et al. 2006a). Additionally, BB spermatozoa apparently move less fluent as evidenced by a lower BCF combined with a higher ALH, compared with HF sperm. This less fluent motility pattern probably results in more resistance because of friction and consequently in a lower velocity. Furthermore, the lower the BCF, the slower the progressive movement will be as a result of less propelling force. Indeed, the BB spermatozoa demonstrated a lower kinetic efficiency as they moved slower and less straight forward compared with HF sperm, as the velocity (VAP, VSL) and direction measures (STR) were significantly lower in the BB ejaculates. This velocity will moreover be influenced by the size of the sperm heads, as small geometrical differences in sperm morphology can result in large differences in sperm hydrodynamics, and subsequently in impaired semen velocity (Dresdner and Katz 98). Size and elongation of the sperm heads are two measures that were also assessed by the Hamilton Thorne analyzer (data not shown), and these data suggest that BB sperm heads are shorter albeit larger compared with HF spermatozoa. This morphometrical difference might in part be responsible for the slower and less fluent velocity of BB spermatozoa (Dresdner and Katz 98). Furthermore, the presence of sperm abnormalities [such as knobbed acrosomes, abnormal acrosomes, nuclear pouches or diadem defects, midpiece defects (segmental aplasia as well as pseudodroplet-like defects), proximal and distal droplets, and accessory and abaxial tails] is generally more common in the BB breed (Hoflack et al. 2006a), and these abnormalities and in particular abnormal midpieces and tails, and (both proximal and distal) droplets might negatively influence semen velocity (Blom 977; Barth and Oko 989; Amann et al. 2000).

8 CASA of Belgian Blue and Holstein Friesian bull sperm 59 In human sperm, a similar influence of morphology on motility has been demonstrated (Johnston et al. 995; Mahmoud et al. 998). Indeed, sperm morphology of the bulls examined in the present study differed significantly between breeds. Moreover, the sperm morphology was significantly correlated to the CASA outcome, suggesting an influence of sperm morphology on motility. Apparently, all the assessed sperm abnormalities negatively influenced sperm motility to some extent. These factors altogether resulted in less rapid and more slow and static (when dead), and subsequently less progressively motile spermatozoa in the BB semen picture. Thus, different subpopulations within an ejaculate exist between breeds. Furthermore, these BB sperm subpopulations were more heterogeneous compared with the HF semen, which was evidenced by the higher standard deviations for most of the assessed CASA parameters in the BB breed. It is noteworthy to remark that even with a rather small number of ejaculates, the overall breed differences were already this obvious and significant. However, these breed differences were less obvious when the matched breed groups were compared. Some differences were still present when the selected bulls were compared, but only PMOT and STR significantly differed when the unselected bulls of both breeds were compared. However, the observed differences between the small group of unselected BB and HF bulls numerically showed the same pattern for any of the CASA parameters as the overall breed comparison. As the quantitative relation is exactly the same for the unselected bulls as for the whole population, it might be possible that there is a genetic component to the overall breed difference. Moreover, the unselected HF bulls were all rather young which might in some cases result in an immature sperm picture with subsequent poor morphology and consequently have a negative influence on semen velocity, minimizing a possibly inherent breed velocity difference (Almquist and Amann 976; Blom 977; Lunstra and Echternkamp 982; Barth and Oko 989; Johnson 997; Amann et al. 2000). However, this suggested genetic component could very well be the mere result of the world-wide long-term selection for highly fertile HF AI bulls (So derquist et al. 99a), a selection that was not performed in the BB breed. In this case, sperm morphological differences would be responsible for the motility differences, as was encountered in the present study. The presented data were however based on a limited number of bulls, and the observed differences can consequently be the result of individual bull factors, as was the case in the oldest BB bulls for the percentage of static spermatozoa. Whether a genetic motility difference is truly present should be confirmed on a larger scale by examining more ejaculates of a high number of bulls of the two breeds by CASA. In this study, the same CASA device with the exact same parameter settings were used to compare the fresh semen motility results of the two breeds. Generally, AI centres have different CASA equipment and moreover use different parameter settings to evaluate the sperm motility, making large-scale comparisons of such motility data irrelevant. Moreover, to elucidate whether the presence of a pure genetic motility difference independent of sperm morphology differences exists, bulls of comparable age with a similar (preferably high) percentage of normal spermatozoa of the two breeds should be compared. This was not possible in the present study. No differences were present between the three different age groups within the BB breed, with the sole exception of the oldest bulls having significantly more static sperm. This exception was due to one bull in the oldest group with an extremely high proportion (84.5%; data not shown) of static spermatozoa, without which this difference no longer occurred. The fact that the sperm motility results did not increase with age, as the BB bulls were selected only keeping the more fertile older bulls, might be explained by the fact that sperm quality in older BB bulls decreases because of testicular degeneration, eventually leading to a quality loss, even in the more fertile bulls, nullifying the selection effect (Kumi-Diaka et al. 98; Hopkins 997; Van Camp 997; Bru ckmann et al. 2000; Brito et al. 2002). In conclusion, we can state that sperm motility differs between the BB and HF breed. The generally higher proportion of live spermatozoa in HF ejaculates compared with BB semen (Hoflack et al. 2006a) apparently results in a higher percentage of both totally and progressively motile spermatozoa. Furthermore, a lower kinetic efficiency of the BB spermatozoa, evidenced by a lower BCF combined with a higher ALH, is the basis for the lower velocity of BB sperm cells compared with HF semen. Additionally, BB spermatozoa move less straight forward, resulting in a lower STR. These differences appear to be related to the sperm morphology and were obvious in the examined bull populations residing at AI centres. In an unselected rather young bull population, the breed differences for the assessed CASA parameters are numerically albeit not significantly in agreement with the above-mentioned results. Hence, we assume that a genetic component could be responsible for the motility differences between the two breeds. Acknowledgements The authors wish to thank the respective AI centres for their hospitality and for the opportunity to examine their bulls. Special thanks are due to CRV Holding for their kind cooperation. 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9 60 G Hoflack, G Opsomer, T Rijsselaere, A Van Soom, D Maes, A de Kruif and L Duchateau Barth AD, Oko RJ, 989: Abnormal Morphology of Bovine Spermatozoa. Iowa State University Press, Ames, IA. Bartoov B, Ben-Barak J, Mayevsky A, Sneider M, Yogev L, Lightman A, 99: Sperm motility index: a new parameter for human sperm evaluation. Fertil Steril 56 (Suppl. ), Blom E, 977: Sperm morphology with reference to bull infertility. First All-India Symposium on Animal Reproduction, Ludhiana, India; pp Brito LFC, Silva AEDF, Rodrigues LH, Vieira FV, Deragon LAG, Kastelic JP, 2002: Effects of environmental factors, age and genotype on sperm production and semen quality in Bos indicus and Bos taurus AI bulls in Brazil. Anim Reprod Sci 70, Bru ckmann A, Yang X, Schallenberger E, 2000: The effects of age on fertility, ejaculate parameters and testosterone, estradiol-7b and luteinizing hormone concentration in breeding bulls. 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10 CASA of Belgian Blue and Holstein Friesian bull sperm 6 sperm quality analyser in predicting the outcome of assisted reproduction. Int J Androl 2, Neuwinger J, Knuth UA, Nieschlag E, 990a: Evaluation of the Hamilton-Thorn 2030 motility analyser for routine semen analysis in an infertility clinic. Int J Androl 3, Neuwinger J, Behre HM, Nieschlag E, 990b: External quality control in the andrology laboratory: an experimental multicenter trial. Fertil Steril 54, Ott RS, 986: Breeding Soundness examination of bulls. In: Morrow DA (ed.), Current Therapy in Theriogenology, Vol. 2. WB Saunders, Philadelphia, PA, pp Rijsselaere T, Van Soom A, Maes D, de Kruif A, 2002: Use of the sperm quality analyzer (SQA II-C) for the assessment of dog sperm quality. Reprod Dom Anim 37, Rijsselaere T, Van Soom A, Maes D, de Kruif A, 2003: Effect of technical settings on canine semen motility parameters measured by the Hamilton-Thorne analyzer. Theriogenology 60, Rijsselaere T, Van Soom A, Tanghe S, Coryn M, Maes D, de Kruif A, 2005: New techniques for the assessment of canine semen quality: a review. Theriogenology 64, So derquist L, Janson L, Larsson K, Einarsson S, 99a: Sperm morphology and fertility in AI bulls. J Vet Med A 38, So derquist L, Rodriguez-Martinez H, Janson L, 99b: Postthaw motility, ATP content and cytochrome C oxidase activity of AI bull spermatozoa in relation to fertility. Zentralbl Veterinarmed A 38, Van Camp SD, 997: Common causes of infertility in the bull. Vet Clin North Am Food Anim Pract 3, Vantman D, Koukoulis G, Dennison L, Zinaman M, Sherins RJ, 988: Computer-assisted semen analysis: evaluation of method and assessment of the influence of sperm concentration on linear velocity determination. Fertil Steril 49, Vantman D, Banks SM, Koukoulis G, Dennison L, Sherins RJ, 989: Assessment of sperm motion characteristics from fertile and infertile men using a fully automated computerassisted semen analyzer. Fertil Steril 5, Verstegen J, Iguer-ouada M, Onclin K, 2002: Computer assisted semen analyzers in andrology research and veterinary practice. Theriogenology 57, Wood PD, Foulkes JA, Shaw RC, Melrose DR, 986: Semen assessment, fertility and the selection of Hereford bulls for use in AI. J Reprod Fertil 76, Zhang BR, Larsson B, Lundeheim N, Rodriguez-Martinez H, 998: Sperm characteristics and zona pellucida binding in relation to field fertility of frozen-thawed semen from dairy AI bulls. Int J Androl 2, Submitted: Author s address (for correspondence): Geert Hoflack, Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 33, 9820 Merelbeke, Belgium. geert.hoflack@ugent.be