RELATIONSHIPS BETWEEN SPERM MEMBRANE INTEGRITY AND OTHER SEMEN QUALITY CHARACTERISTICS OF THE SEMEN OF SAANEN GOAT BUCKS

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1 Bull Vet Inst Pulawy 49, , 2005 RELATIONSHIPS BETWEEN SPERM MEMBRANE INTEGRITY AND OTHER SEMEN QUALITY CHARACTERISTICS OF THE SEMEN OF SAANEN GOAT BUCKS ZEKARIYA NUR 1, IBRAHIM DOGAN 1, ULGEN GUNAY 1 AND M. KEMAL SOYLU 1 1 Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, University of Uludag, Gorukle, Bursa, Turkey nurzek@uludag.edu.tr Received for publication December 02, Abstract The aim of the present study was to assess the relationships between sperm membrane integrity, evaluated by the hypo-osmotic swelling (HOS) and water tests, and sperm quality parameters (percentage of dead, motile and abnormal spermatozoa) in goat bucks. For that purpose, a total of 60 fresh ejaculates from 6 Saanen bucks were evaluated. The mean percentage of dead and motile spermatozoa were 12.7±5.7 and 62.5±8.6, respectively. The mean percentages of abnormal spermatozoa with defected acrosome or total morphological defect were 7.2±2.9 and 9.7±3.4, respectively. The mean percentage of swollen tail spermatozoa obtained with the HOS and water tests were 53.7±11.3 and 49.8±9.7, respectively. The mean percentages of swollen tail spermatozoa obtained with the water test were significantly lower than that evaluated from the HOS test (P<0.01). The percentage of swollen tail spermatozoa evaluated with the HOS test was correlated with that obtained with the water test (r=0.657, P<0.001). The percentage of swollen tail spermatozoa evaluated by the water and HOS tests were slightly correlated with the percentage of motile spermatozoa (r=0.554 and r=0.523, P<0.001 and P<0.001). In conclusion, a high correlation between the HOS test and water test was observed in this study. The percentage of swollen tail spermatozoa obtained by the water test was significantly lower than that obtained by the HOS test. Key words: bucks, semen, spermatozoa, morphology, membrane integrity. The traditional evaluation of the quality of ejaculate has been mainly based on routine semen analyses (motility, morphology and acrosomal integrity), but, these have a limited capacity for the prediction of the potential fertility of an ejaculate (8). The process of fertilisation involves complex biochemical and physiological events that are not measured by using the routine semen evaluation. Therefore, different tests have been developed to evaluate the functional and structural integrity of the sperm membrane including supravital staining, hypoosmotic swelling (HOS) test, water test, zona-free hamster ova test, and cervical mucus penetration test (6). Since the plasma membrane functional activity is crucial for the viability and fertilising ability of spermatozoa, it is very important to assess the structural and functional activity of the sperm membrane (6). Supravital stains such as eosin-y and trypan blue make it possible to differentiate spermatozoa that are immotile but alive from those that are dead. However, the supravital staining indicates the structural, but not the functional integrity of the sperm plasma membrane (16). Hypo-osmotic swelling test was developed by Jeyendran et al. (8) to evaluate sperm membrane functional integrity. This test is based on the semipermeability of the intact cell membrane, which allows the sperm to swell under hypo-osmotic conditions. Exposure of spermatozoa with intact plasma membrane to hypo-osmotic solution results in tail swelling and this is a sign that the transport of water across the membranes occurs normally (1). The water test has been developed to evaluate the sperm functional integrity by Lomeo and Giambersio (11), who used distilled water as a hypo-osmotic solution. The last authors suggested that this test was a simpler and quicker method than the HOS test and that its results are also highly correlated with those obtained from routine semen analyses, eosin-y staining and HOS test. There have been a few studies concerning the relationship between HOS test and water test in the human (2) and mouse (17), but to the best of our knowledge, no studies have been documented in fresh goat buck semen. The aim of the current study was, therefore, to assess the relationship between the results of the water and HOS tests of sperm membrane integrity and classical tests based on eosin-nigrosin staining to evaluate the motility and morphology of fresh buck semen.

2 184 Material and Methods Animals. This study was carried out at a farm located in Balikesir, in Western Turkey, during July (time of transition from non-breeding to natural breeding season in the region) under conditions of natural lighting. Six Saanen bucks known to have good fertility, aged 2-4 years, were used as materials. Semen collection and evaluation. Sixty ejaculates (6x10) were collected by the use of an artificial vagina, in the presence of a goat in oestrous, twice a day, at 60 min interval, during 5 d. Semen samples were placed in a water bath at 37 C. Soon after the collection, sperm concentration, ph, percentage of live and dead spermatozoa, and percentage of spermatozoa exhibiting morphologic abnormalities were evaluated according to standard procedures (6). The percentage of spermatozoa with plasma membrane integrity was determined by the HOS and water tests. Sperm motility was assessed by examining a drop of ejaculate, covered with a cover glass, under phase-contrast microscope (x400) equipped with heat plate (37 C) (6). The percentage of live spermatozoa was estimated by supravital staining technique using eosin-nigrosin stain mixture. Two hundred of spermatozoa per slide were evaluated under a light microscope (x1000). Acrosome, head, mid piece, and tail abnormality were evaluated using Giemsa staining procedure (21). A total of 200 spermatozoa were examined under a light microscope (x1000) to determine the percentages of spermatozoa with defective acrosome (DA) and with morphologic defects (TMD). HOS test. Hypo-osmotic solution (8) was made with fructose (8.72 g/l) and sodium citrate (4.74 g/l) at 100 mosm/l and ph The osmolarity of the solution was determined by freezing point depression (Advanced 3D3 Single Sample Osmometer, Advanced Instrument, INC.). A volume of 10.0 µl of semen was added to 1 ml of the solution and incubated at 37 C for 60 min. Immediately after the incubation, 1 drop of the semen was placed on a glass slide covered with cover slide and evaluated under phase-contrast microscope (x400). Microscope fields were selected randomly. At least 200 spermatozoa were evaluated per slide and percentages of swollen tail spermatozoa were calculated. Water test. A volume of 10.0 µl of semen was added to 1 ml of mono distilled water (0 mosm/l, ph 7.02) and incubated at 37 C for 5 min (11). Immediately after the incubation, 1 drop of the semen was placed on a glass slide covered with cover slide and evaluated under phase-contrast microscope (x400). Microscope fields were selected randomly. At least 200 spermatozoa were evaluated per slide and percentages of swollen tail spermatozoa were calculated. Statistical analyses. The mean percentages of swollen spermatozoa obtained with the HOS and water tests were compared using the Wilcoxon rank sum test. Pearson correlation was performed to evaluate relationship among HOS test, water test, and other observed semen parameters (motility, live/dead spermatozoa, defected acrosome, and TMD). SPSS 10.0 was used for all statistical analyses. Results The results of the present study are summarised in Tables 1-2. The mean percentage of HOS test, water test, dead spermatozoa, motility, percentage of defected acrosome and TMD were 53.7±11.3, 49.8±9.7, 12.7±5.7, 62.5±8.6, 7.2±2.9 and 9.7±3.4, respectively (Table 1). The mean semen concentration and ph were 2.2x10 9 /ml and 6.8, respectively. The mean percentages of swollen spermatozoa obtained with the two tests were significantly different (P<0.01). Table 1 Mean percentages of evaluated sperm parameters for the individual bucks (10 ejaculates per buck) No. of buck HOS test Water test Dead sperms () Motility DA TMB ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±2.9 Total 53.7±11.3 a 49.8±9.7 b 12.7± ± ± ±3.4 ± - SD, DA - defective acrosomes; TMD - total morphological defects; a, b - significant differences between HOS test and water test (P<0.01).

3 185 Table 2 Pearson correlation (r) between measurements in sperm analyses (n=60) Characteristics Water test Dead sperms Motility DA TMB HOS test ** ** Water test ** Dead sperms * * Motility DA ** * P<0.05; ** P<0.001; DA - defective acrosomes; TMD - total morphological defects. Buck spermatozoa subjected to the water test showed morphological changes that were similar to HOS test. Correlation among HOS test, water test, dead spermatozoa, motility, percentage of defected acrosome, and total morphological defects are presented in Table 2. There was a correlation between the percentage of spermatozoa that had been swollen under HOS test and water test (r=0.657 P<0.001). The percentage of swollen tail spermatozoa in HOS test and water test were also slightly correlated with motility (r=0.523, P<0.001 and r=0.554, P<0.001, respectively). Relationship between HOS test and water test is given in Fig Water test () r=0.657 P< HOS test () Fig. 1. The distribution of the percentage of swollen spermatozoa obtained by HOS test and water test. Discussion Fresh buck spermatozoa showed morphologic changes, as evidenced by curling-swelling, when exposed to hypo-osmotic milieu. The response of buck semen to hypo-osmotic conditions was similar to that observed in dogs (9), humans (8), bulls (3), boars (19), and stallions (12). The response to the water test was similar to that reported for human (2), dog (7) and mice (17) spermatozoa. In the present study, the mean percentages of swollen spermatozoa obtained by the HOS test were higher than obtained by the water test (P<0.01). In contrast, Bahamondes et al. (2) reported that the mean percentage of swollen spermatozoa obtained in the HOS test and in the water test were not statistically different. On the other hand, Fuse et al. (5) reported that the percentage of g-type swelling tail spermatozoa obtained by the water test was significantly larger than that obtained by the HOS test (P<0.05). These differences may be due to the different sugars, osmolarity and electrolytes (8). Also ph of semen diluents has considerable effect on the activity and metabolism of spermatozoa (18).

4 186 Some previous investigators reported that the percentage of swollen tail spermatozoa obtained by the HOS test correlated well with those obtained by the water test (2, 4, 5, 10). Our finding is in accordance with these earlier data and shows a good correlation between both tests. The relation between the results of these tests is expected since they are all related to the plasma membrane functional status (10). Also this finding indicated that the water test is simple and may be practicable instead of HOS test for the evaluation of tail membrane integrity of buck spermatozoa. In the present study, the percentage of swollen tail spermatozoa obtained with the HOS and water tests showed a correlation with the motility. This finding is not surprising because the motility partly depends on transports of compounds across membrane of spermatozoa (8). Also our finding is in accordance with earlier data of the HOS test performed with fresh or frozen semen of dogs (9), humans (8, 20) bulls (3) and stallions (12), and results of the water test with human fresh semen (5) and epididymial dog semen (7). On the other hand, no significant correlation was reported between the water test results and motility (10, 11). The results obtained in this study indicated that a statistical correlation was found between defective acrosome and TMD (r=0.823, P<0.001) as well as those reported in the post-thaw bull spermatozoa (14). This correlation may be due to the total morphological defect including all observed morphological defects (acrosome, head, mid piece, and tail abnormalities). It has been reported that there is an inverse relationship between the percentage of dead spermatozoa and acrosomal abnormalities, also total morphological defects of dead spermatozoa were higher than those of live spermatozoa (15). In the present study, a low correlation was found between percentage of defected acrosome and dead spermatozoa (r=0.278, P<0.05), and between percentage of dead spermatozoa and TMD (r=0.261, P<0.05), similar to those reported in other experiments (14,15). While the supravital staining depends on the principle that dead cells with damaged membrane take up stain (based on the impermeability for larger dye molecules), the HOS test and water test evaluates the functional integrity of the plasma membrane (based on the osmotic properties of the plasma membrane) (8). As in other studies (2, 10), the percentage of swollen tail spermatozoa, determined with the HOS and water tests in our study, showed insignificant negative correlation with the percentage of dead spermatozoa and percentage of defective acrosome (P>0.05). The findings are, however, in disagreement with the earlier observations of Vigano et al. (20), Lomeo and Giambersio (11), and Sliwa (17); who reported that the water test was significantly correlated with eosin-y staining. There was no correlation between swollen tail spermatozoa obtained by the two membrane integrity tests and TMD (P<0.05). These findings were in agreement with results reported by Jeyendran et al. (8) in human and Correa et al. (3) and Nur et al. (13) in bull spermatozoa for HOS test. In conclusion, a good correlation between the HOS test and water test was observed in this study. The percentage of swollen tail spermatozoa obtained by the water test was significantly lower than that obtained by the HOS test. Also there was a good correlation between motility and percentage of swollen tail spermatozoa obtained by both tests, but no correlation was observed between swollen tail spermatozoa and dead sperms, acrosome defects, and total morphological defects. Acknowledgments: The authors wish to thank vet. surg. Huseyin KARAGOZ for providing the animals. 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