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Hypoosmotic test in equine spermatozoa
?
ELSEVIER
HYPOOSMOTIC TEST IN EQUINE SPERMATOZOA D. Neild, G. Chaves, M. F'lores, N. Mora, M. Beconi and A. Agiiero Area of Theriogenology. Faculty of Veterinary Sciences University of Buenos Aires. Buenos Aires, Argentina Received for publication: February 6, 1997 Accepted: October 7, 1998 ABSTRACT The aim of the study was to evaluate equine sperm membrane integrity using the hypoosmotic swelling (HOS) test and to correlate this test with different sperm parameters in raw and frozen thawed semen. The HOS solutions were made with fructose, sucrose, lactose and sodium citrate each at 300, 150, 100, 50 and 25 mosm. Maximum numbers of swollen spermatozoa were observed in solutions of fructose, sucrose and lactose each at 100, 50 and 25 mosm. Correlations between progressive motility, morphologically normal spermatozoa and the HOS test were r = 0.75 and r = 0.51 in raw semen and r = 0.26 and r = -0.22 in frozen-thawed semen. The correlation between HOS and percentage of intact membranes with the fluorescent stain was r = 0.32 in frozen-thawed semen. The HOS test is a simple and accessible method which could be used as a complement to routine equine semen analysis. It has the added advantages of being less susceptible to the immediate effects of cold shock and of evaluating individual spermatozoa rather than the population as a whole, as does progressive motility. © 1999by ElsevierScience Inc.
Key words: hypoosmotic swelling, membrane functionality, equine spermatozoa INTRODUCTION Standard parameters used to assess male fertility (total number of spermatozoa, progressive motility, morphology) have a limited capacity for predicting the potential of an ejaculate (l). Because the sperm membrane is of fundamental importance in the fertilization process, more attention has been dedicated to this area of study in recent years. Two tests have been available to evaluate membrane integrity: supravital stains and the hypoosmotic swelling (HOS) test. Recently a sensitive fluorescent stain using carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) was developed (4). This method is based on the hydrolysis of CFDA by esterases within the cell producing free carboxyfluorescein, which is retained when the plasma membrane is intact.The PI penetrates membrane-damaged cells and binds to the DNA. The HOS test evaluates whether an intact membrane is functional (5). When exposed to a hypoosmotic solution, functional spermatozoa will undergo swelling to establish osmotic equilibrium, producing the typical swelfing of the tail. Since fertilization will not occur if the sperm membrane is biochemicaily inactive, even if it remains structurally intact, the HOS test is a more useful indicator than the supravital stain (10). Acknowledgments: This work was supported by a grant from the Universidad de Buenos Aires, Secretada de Ciencia y T~cnica. Theriogenology 51:721-727, 1999 © 1999 by Elsevier Science Inc.
O093-691X/99/$-see front matter PII S0093-691 X(99)00021-7
Theriogenology
722
The HOS test has been tried in various species with apparent success (2, 6, 8), and its use has been recommended as an additional fertility indicator. We hypothesized that if. equine spermatozoa have the ability to respond to the HOS test, this would prove to be a useful method for evaluating membrane functionality in this species. Thus, the objectives of the present study were 1) to evaluate the ability of equine spermatozoa to swell in hypoosmotic solutions; 2) to determine the solution and osmolality that produces maximum swelling in equine spermatozoa and the morphological types of swelling that are produced; 3) to establish the correlation between the ability of spermatozoa to swell in the hypoosmotic solution and different sperm parameters in raw and frozen-thawed semen; and 4) to compare the HOS test with the evaluation of progressive motility in ejaculates subjected to cold shock. MATERIALS AND METHODS Materials All chemicals used were purchased from Sigma Chemical Company (St. Louis, MO USA). Raw semen. Semen was collected from 30 stallions of different breeds while under Breeding Soundness Examination, using a Missouri Model artificial vagina and subsequently subjected to different assays. Progressive motifity was estimated visually under light microscopy x 100 magnification at 37°12 on a heated stage. Concentration and total number of spermatozoa were calculated using a hemocytometer. Morphology was evaluated on a wet mount, with spermatozoa fixed in buffered formol saline, using differential interference contrast (DIC) microscopy x 1000 magnification. Sperm membrane functionality was evaluated using the hypoosmotic swelling (HOS) test. The fluorescent stain was not applicable under stud farm conditions so the structural integrity of the plasma membrane was not evaluated by this method in raw semen. Frozen semen. Semen from 15 stallions of different breeds was frozen in 0.5 mL straws (3) and thawed 7 sec at 750C, followed by 5 sec at 37°C. The same parameters were assessed as in raw semen, with the addition of membrane integrity evaluated with a luorescent stain (CFDA/PI). Fluorescent stain (CFDA/PI). Membrane integrity was evaluated with fluorescent microscopy using 6-carboxyfluorescein diacetate (CFDA) and propidium iodide (PI). Samples of semen (100 pL) were incubated at 37° C for 15 min in 1 mL of saline medium as described by Harrison and Vickers (4). The medium contained 20 pL of a stock solution of CFDA (0.5 mg/ml in dimethylsulphoxide), 20 laL of a stock solution of PI (0.5 mg/mL in isotonic saline) and 20 pL solution of formaldehyde (2.5 mg/mL distilled water) in 1 mL of saline medium (4). An average of 100 spermatozoa was assessed in random fields in each of 2 subsamples. Observations were conducted with epifluorescence microscopy x 400 magnification) using a rhodamine and standard fluorescein filter set. Spermatozoa that fluoresced green throughout the entire cell were classified as possessing intact membranes (CFDA positive). Spermatozoa with heads that fluoresced red were considered to have damaged membranes (PI positive). Hvvoosmotic sweilin~ test (HOS). Solutions were made with fructose, sucrose, lactose and sodium citrate each at 300, 150, 100, 50 and 25 mosm (determined by osmometer). Samples of semen (100 BL) were incubated at 37° C for 30 min in 1 mL of hypoosmotic solution.
Theriogenology
723
The types of typical tail abnormalities indicative of swelling were evaluated under light microscopy x 400 magnification and were expressed as percentages. C01d shock. Raw semen samples from three stallions were incubated at 4°12 for 90 min. Every 30 minutes progressive motility, membrane integrity (CFDA/PI) and percentage of swelling in lactose 50 mosm were evaluated. Statistical Analysis Data recorded in percentages were transformed to the arc sine of the square root prior to being analyzed by two-way ANOVA. Pearson's coefficient of correlation was calculated in raw and frozen-thawed semen. Friedman's two- way analysis of variarance by ranks was applied to cold shocked samples. RESULTS Maximum numbers of swollen raw equine spermatozoa were observed with solutions of fructose, sucrose and lactose each at 100, 50 and 25 mosm. All sugars between 300 and 150 mosm as well ~ the solution of sodium citrate in the range of osmolalities used were discarded because the percentage of swelling obtained with them was low (P< 0.05; Table !). Since no differences (P> 0.05) were found between the 3 sugars studied at 100, 50 and 25 mosm, further assays were carded out using a 50 mosm solution of lactose, since this sugar is used in cryopreservation diluents.
Table 1. Percentage of swollen spermatozoa in raw semen using different hypoosmotic solutions (n = 15)
Swollen spermatozoa (%)
Solutions
25 mosm
50 mosm
100 mosm
i 50 mosm
300 mosm
Fructose
43.6 ± 10.5 a
30 ± 12.3 a
48.9 ± 7 a
33.2 ± 16~
26 :t'l 1b
Sucrose
47.7 ± 8.3 a
4 1 . 4 ± 11 a
45.7 ± 4 a
23.3 ± 7 b
7.3_+7 b
lactose
4 1 . 7 ± 12.6 a
49.6±4.6 a
45.6±6.7 a
11.4± 4 b
7.6±7 b
Sodium Citrate
37.7±10.6 b
30.1±13.4 b
29.5±7.4 b
16.4±13 h
6.7± 2b
Experimental conditions were the same as those described in Materials and Methods. Values are mean ± standard deviation. a,b Values with different superscripts are different (P<0.05).
Figures 1 and 2 show the different morphological changes typical of the swelling observed when raw and frozen-thawed equine spermatozoa were subjected to a hypoosmotic solution.
Theriogenology
724
A
A'
B
B'
C
D
E
F
Figure 1. Morphological changes typical of the swelling observed in raw equine semen (samples were incubated in lactose 50 mosm). NS: No swelling. A-F: Types of swelling.
A
A'
B
B'
B"
C
D
E
F
Figure 2. Morphological changes typical of the swelling observed in frozen-thawed equine semen (samples were incubated in lactose 50 mosm). NS: No swelling. A-F: Types of swelling All swelling was observed in the tails of the spermatozoa. When the samples were subjected to cold shock (Figure 3), progressive motility was the first to be affected by the temperature change, and it showed a greater degree of susceptibility to cold shock than did the plasma membrane as observed by both membrane tests. Friedman's test was used to compare the data, finding differences (P<0.05) between the assays used at each point of evaluation.
Theriogenology
725
100 90 8O
m CFOA P O S I I W E n H O S TEST ......
0
~ |
--40
30
80
90
Minutes
Figure 3. Comparative study of progressive motility, fluorescent stain and percentage of swelling in lactose 50 mosm in cold shocked (4°C) raw semen samples from 3 stallions. Values are median.
The HOS test and standard semen parameters were compared in raw (n = 30) and frozenthawed (n = 15) semen, and the mean percentages obtained in each group are shown in Table 2. Correlations between these parameters are presented in Table 3.
Table 2. HOS test and standard semen parameters in raw (n =30) and frozen-thawed (n = 15) equine semen.
Progressive Motility (%) Normal Morphology (%) CFDA positive(%) HOS test (%)
Mean SD Mean SD Mean SD Mean SD
Raw semen 38.4 18.0 42.0 21.9 46.0 19.1
CFDA positive: spermatozoa stained with 6-carboxyfluorescein diacetate. HOS:hypoosmotic swelling test (lactose 50 mosm). Experimental procedures were described in materials and methods.
Frozen-thawed semen 29.7 13.5 54.5 16.8 36.0 10.0 30.2 10.1
Theriogenology
726
Table 3. HOS test and standard semen parameters in raw (n = 30) and frozen-thawed (n =15) equine semen. Parameters HOS vs. PM HOS vs. Norm. Sperm. HOS vs. CFDA positive
Raw semen r = 0.75 * r = 0.51 *
Frozen-thawed semen r = 0.57 * r = - 0.22 r = 0.09
HOS: hypoosmotic swelling test (lactose 50 mosm).PM: progressive motility. Norm. Sperm,: morphologically normal spermatozoa. CFDA positive: spermatozoa stained with 6-carboxyfluorescein diacetate. * significant (P < 0.05) DISCUSSION Differences observed in the percentages of spermatozoa swelling in the sodium citrate solution and the sugar solutions could have resulted from different influence on the availability of water for transport through the plasma membrane. Studies on water permeability of some mammalian sperm membranes (ram and human (5) and bull (13)) show that the osmotic water permeability coefficient is very high and that the associated activation energy is very low, which suggests a porous membrane and the presence of water channel proteins. In ram and human sperm membranes, glucose transporters may have a secondary water channel function (5). In equine semen, high membrane permeability to water can be inferred from the percentages of swelling obtained, which suggests that water transport is produced across a porous membrane. Alterations in the plasma membrane components that contribute to ion and/or metabolite transport produced by freezing and thawing the ejaculate could explain the decrease in swelling that is observed in frozen-thawed semen compared with that of raw semen. The morphological changes observed in equine spermatozoa after being subjected to the HOS test are produced in the tail of those spermatozoa possessing intact membranes. This was confirmed by subjecting the spermatozoa with swelling to the vital fluorescent stain. A high correlation was observed between progressive motility and the HOS test; nevertheless, progressive motility showed a greater susceptibility to cold shock than did either plasma membrane integrity or membrane functionality. This would seem to indicate that sperm motility is more affected by semen handling factors than the plasma membrane. Thus, tests evaluating the plasma membrane would prove to be the more accurate in predicting the fertilizing capacity of a semen sample. The HOS test is a simple and accessible evaluation method which, as in other species, could be a useful complement to routine equine semen analysis. It has the added advantages of being less susceptible to the immediate effects of cold shock and of evaluating individual sperm cells rather than the population as a whole, as does progressive motility. REFERENCES 1. Amann RP. Can the fertility potential of a seminal sample be predicted accurately? J Androl 1989; 10:89-98. 2. Correa JR, Zavos PM. The hypoosmotic swelling test: its employment as an assay to evaluate the functional integrity of the frozen-thawed bovine sperm membrane. Theriogenology 1994;42:351-360.
Theriogenology
727
3. CristaneUi M J, Squires EL, Amann RP, Pickett BW. Fertility of stallion semen processed frozen and thawed by a new procedure. Theriogenology 1984; 22:39-45. 4. Curry MR, Watson PF. Osmotic effects on ram and human sperm membranes in relation to thawing injury. Cryobiology 1994; 31:39-46. 5. Curry MR, Millar JD, Watson PF. The presence of water channel proteins in ram and human sperm membranes. J Reprod Fertil 1995; 104:297-303. 6. Harrison RAP, Vickers SE. Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. J Reprod Fertil 1990; 88:343-352. 7. Jeyendran RS, Van der Ven HH, Perez-Pelaez M, Grabo BG, Zaneveld LJD. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil 1984; 70:219225. 8. Kumi-Diaka J. Subjecting canine semen to the hypoosmotic test. Theriogenology 1993; 39:1279-1289. 9. Schrader SM, Platek SF, Zaneveld JD, Perez-Pelaez M, Jeyendran RS. Sperm viability: a comparison of analytical methods. Andrologia 1986; 18:530-538. 10. Tamuli MK, Watson PF. Effect of temperature of incubation on the development of resistance to cold stress and hypoosmotic stress in boar spermatozoa incubated for up to 24 hours. Proc. 12th. lnt Congr Anim Reprod 1992; 1484-1486. ! 1. Vazquez JM, Martinez EA, Martinez P, Garcia-Artiga C, Roca J. Hypoosmotic swelling of boar spermatozoa compared to other methods for analyzing the sperm membrane. Theftogenology 1997; 47:913-922. 12. Zavos PM. Hypoosmotic swelling test (HOS)/functional integrity of sperm membrane. J Ass Reprod Technol 1990; 2:215-216. i 3. Watson PF, Kunze E, Cramer P, Hammerstedt RH. A comparison of critical osmolarity and hydraulic conductivity and its activation energy in fowl and bull spermatozoa. J Androl 1992; 13: 131-138.
ELSEVIER
HYPOOSMOTIC TEST IN EQUINE SPERMATOZOA D. Neild, G. Chaves, M. F'lores, N. Mora, M. Beconi and A. Agiiero Area of Theriogenology. Faculty of Veterinary Sciences University of Buenos Aires. Buenos Aires, Argentina Received for publication: February 6, 1997 Accepted: October 7, 1998 ABSTRACT The aim of the study was to evaluate equine sperm membrane integrity using the hypoosmotic swelling (HOS) test and to correlate this test with different sperm parameters in raw and frozen thawed semen. The HOS solutions were made with fructose, sucrose, lactose and sodium citrate each at 300, 150, 100, 50 and 25 mosm. Maximum numbers of swollen spermatozoa were observed in solutions of fructose, sucrose and lactose each at 100, 50 and 25 mosm. Correlations between progressive motility, morphologically normal spermatozoa and the HOS test were r = 0.75 and r = 0.51 in raw semen and r = 0.26 and r = -0.22 in frozen-thawed semen. The correlation between HOS and percentage of intact membranes with the fluorescent stain was r = 0.32 in frozen-thawed semen. The HOS test is a simple and accessible method which could be used as a complement to routine equine semen analysis. It has the added advantages of being less susceptible to the immediate effects of cold shock and of evaluating individual spermatozoa rather than the population as a whole, as does progressive motility. © 1999by ElsevierScience Inc.
Key words: hypoosmotic swelling, membrane functionality, equine spermatozoa INTRODUCTION Standard parameters used to assess male fertility (total number of spermatozoa, progressive motility, morphology) have a limited capacity for predicting the potential of an ejaculate (l). Because the sperm membrane is of fundamental importance in the fertilization process, more attention has been dedicated to this area of study in recent years. Two tests have been available to evaluate membrane integrity: supravital stains and the hypoosmotic swelling (HOS) test. Recently a sensitive fluorescent stain using carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) was developed (4). This method is based on the hydrolysis of CFDA by esterases within the cell producing free carboxyfluorescein, which is retained when the plasma membrane is intact.The PI penetrates membrane-damaged cells and binds to the DNA. The HOS test evaluates whether an intact membrane is functional (5). When exposed to a hypoosmotic solution, functional spermatozoa will undergo swelling to establish osmotic equilibrium, producing the typical swelfing of the tail. Since fertilization will not occur if the sperm membrane is biochemicaily inactive, even if it remains structurally intact, the HOS test is a more useful indicator than the supravital stain (10). Acknowledgments: This work was supported by a grant from the Universidad de Buenos Aires, Secretada de Ciencia y T~cnica. Theriogenology 51:721-727, 1999 © 1999 by Elsevier Science Inc.
O093-691X/99/$-see front matter PII S0093-691 X(99)00021-7
Theriogenology
722
The HOS test has been tried in various species with apparent success (2, 6, 8), and its use has been recommended as an additional fertility indicator. We hypothesized that if. equine spermatozoa have the ability to respond to the HOS test, this would prove to be a useful method for evaluating membrane functionality in this species. Thus, the objectives of the present study were 1) to evaluate the ability of equine spermatozoa to swell in hypoosmotic solutions; 2) to determine the solution and osmolality that produces maximum swelling in equine spermatozoa and the morphological types of swelling that are produced; 3) to establish the correlation between the ability of spermatozoa to swell in the hypoosmotic solution and different sperm parameters in raw and frozen-thawed semen; and 4) to compare the HOS test with the evaluation of progressive motility in ejaculates subjected to cold shock. MATERIALS AND METHODS Materials All chemicals used were purchased from Sigma Chemical Company (St. Louis, MO USA). Raw semen. Semen was collected from 30 stallions of different breeds while under Breeding Soundness Examination, using a Missouri Model artificial vagina and subsequently subjected to different assays. Progressive motifity was estimated visually under light microscopy x 100 magnification at 37°12 on a heated stage. Concentration and total number of spermatozoa were calculated using a hemocytometer. Morphology was evaluated on a wet mount, with spermatozoa fixed in buffered formol saline, using differential interference contrast (DIC) microscopy x 1000 magnification. Sperm membrane functionality was evaluated using the hypoosmotic swelling (HOS) test. The fluorescent stain was not applicable under stud farm conditions so the structural integrity of the plasma membrane was not evaluated by this method in raw semen. Frozen semen. Semen from 15 stallions of different breeds was frozen in 0.5 mL straws (3) and thawed 7 sec at 750C, followed by 5 sec at 37°C. The same parameters were assessed as in raw semen, with the addition of membrane integrity evaluated with a luorescent stain (CFDA/PI). Fluorescent stain (CFDA/PI). Membrane integrity was evaluated with fluorescent microscopy using 6-carboxyfluorescein diacetate (CFDA) and propidium iodide (PI). Samples of semen (100 pL) were incubated at 37° C for 15 min in 1 mL of saline medium as described by Harrison and Vickers (4). The medium contained 20 pL of a stock solution of CFDA (0.5 mg/ml in dimethylsulphoxide), 20 laL of a stock solution of PI (0.5 mg/mL in isotonic saline) and 20 pL solution of formaldehyde (2.5 mg/mL distilled water) in 1 mL of saline medium (4). An average of 100 spermatozoa was assessed in random fields in each of 2 subsamples. Observations were conducted with epifluorescence microscopy x 400 magnification) using a rhodamine and standard fluorescein filter set. Spermatozoa that fluoresced green throughout the entire cell were classified as possessing intact membranes (CFDA positive). Spermatozoa with heads that fluoresced red were considered to have damaged membranes (PI positive). Hvvoosmotic sweilin~ test (HOS). Solutions were made with fructose, sucrose, lactose and sodium citrate each at 300, 150, 100, 50 and 25 mosm (determined by osmometer). Samples of semen (100 BL) were incubated at 37° C for 30 min in 1 mL of hypoosmotic solution.
Theriogenology
723
The types of typical tail abnormalities indicative of swelling were evaluated under light microscopy x 400 magnification and were expressed as percentages. C01d shock. Raw semen samples from three stallions were incubated at 4°12 for 90 min. Every 30 minutes progressive motility, membrane integrity (CFDA/PI) and percentage of swelling in lactose 50 mosm were evaluated. Statistical Analysis Data recorded in percentages were transformed to the arc sine of the square root prior to being analyzed by two-way ANOVA. Pearson's coefficient of correlation was calculated in raw and frozen-thawed semen. Friedman's two- way analysis of variarance by ranks was applied to cold shocked samples. RESULTS Maximum numbers of swollen raw equine spermatozoa were observed with solutions of fructose, sucrose and lactose each at 100, 50 and 25 mosm. All sugars between 300 and 150 mosm as well ~ the solution of sodium citrate in the range of osmolalities used were discarded because the percentage of swelling obtained with them was low (P< 0.05; Table !). Since no differences (P> 0.05) were found between the 3 sugars studied at 100, 50 and 25 mosm, further assays were carded out using a 50 mosm solution of lactose, since this sugar is used in cryopreservation diluents.
Table 1. Percentage of swollen spermatozoa in raw semen using different hypoosmotic solutions (n = 15)
Swollen spermatozoa (%)
Solutions
25 mosm
50 mosm
100 mosm
i 50 mosm
300 mosm
Fructose
43.6 ± 10.5 a
30 ± 12.3 a
48.9 ± 7 a
33.2 ± 16~
26 :t'l 1b
Sucrose
47.7 ± 8.3 a
4 1 . 4 ± 11 a
45.7 ± 4 a
23.3 ± 7 b
7.3_+7 b
lactose
4 1 . 7 ± 12.6 a
49.6±4.6 a
45.6±6.7 a
11.4± 4 b
7.6±7 b
Sodium Citrate
37.7±10.6 b
30.1±13.4 b
29.5±7.4 b
16.4±13 h
6.7± 2b
Experimental conditions were the same as those described in Materials and Methods. Values are mean ± standard deviation. a,b Values with different superscripts are different (P<0.05).
Figures 1 and 2 show the different morphological changes typical of the swelling observed when raw and frozen-thawed equine spermatozoa were subjected to a hypoosmotic solution.
Theriogenology
724
A
A'
B
B'
C
D
E
F
Figure 1. Morphological changes typical of the swelling observed in raw equine semen (samples were incubated in lactose 50 mosm). NS: No swelling. A-F: Types of swelling.
A
A'
B
B'
B"
C
D
E
F
Figure 2. Morphological changes typical of the swelling observed in frozen-thawed equine semen (samples were incubated in lactose 50 mosm). NS: No swelling. A-F: Types of swelling All swelling was observed in the tails of the spermatozoa. When the samples were subjected to cold shock (Figure 3), progressive motility was the first to be affected by the temperature change, and it showed a greater degree of susceptibility to cold shock than did the plasma membrane as observed by both membrane tests. Friedman's test was used to compare the data, finding differences (P<0.05) between the assays used at each point of evaluation.
Theriogenology
725
100 90 8O
m CFOA P O S I I W E n H O S TEST ......
0
~ |
--40
30
80
90
Minutes
Figure 3. Comparative study of progressive motility, fluorescent stain and percentage of swelling in lactose 50 mosm in cold shocked (4°C) raw semen samples from 3 stallions. Values are median.
The HOS test and standard semen parameters were compared in raw (n = 30) and frozenthawed (n = 15) semen, and the mean percentages obtained in each group are shown in Table 2. Correlations between these parameters are presented in Table 3.
Table 2. HOS test and standard semen parameters in raw (n =30) and frozen-thawed (n = 15) equine semen.
Progressive Motility (%) Normal Morphology (%) CFDA positive(%) HOS test (%)
Mean SD Mean SD Mean SD Mean SD
Raw semen 38.4 18.0 42.0 21.9 46.0 19.1
CFDA positive: spermatozoa stained with 6-carboxyfluorescein diacetate. HOS:hypoosmotic swelling test (lactose 50 mosm). Experimental procedures were described in materials and methods.
Frozen-thawed semen 29.7 13.5 54.5 16.8 36.0 10.0 30.2 10.1
Theriogenology
726
Table 3. HOS test and standard semen parameters in raw (n = 30) and frozen-thawed (n =15) equine semen. Parameters HOS vs. PM HOS vs. Norm. Sperm. HOS vs. CFDA positive
Raw semen r = 0.75 * r = 0.51 *
Frozen-thawed semen r = 0.57 * r = - 0.22 r = 0.09
HOS: hypoosmotic swelling test (lactose 50 mosm).PM: progressive motility. Norm. Sperm,: morphologically normal spermatozoa. CFDA positive: spermatozoa stained with 6-carboxyfluorescein diacetate. * significant (P < 0.05) DISCUSSION Differences observed in the percentages of spermatozoa swelling in the sodium citrate solution and the sugar solutions could have resulted from different influence on the availability of water for transport through the plasma membrane. Studies on water permeability of some mammalian sperm membranes (ram and human (5) and bull (13)) show that the osmotic water permeability coefficient is very high and that the associated activation energy is very low, which suggests a porous membrane and the presence of water channel proteins. In ram and human sperm membranes, glucose transporters may have a secondary water channel function (5). In equine semen, high membrane permeability to water can be inferred from the percentages of swelling obtained, which suggests that water transport is produced across a porous membrane. Alterations in the plasma membrane components that contribute to ion and/or metabolite transport produced by freezing and thawing the ejaculate could explain the decrease in swelling that is observed in frozen-thawed semen compared with that of raw semen. The morphological changes observed in equine spermatozoa after being subjected to the HOS test are produced in the tail of those spermatozoa possessing intact membranes. This was confirmed by subjecting the spermatozoa with swelling to the vital fluorescent stain. A high correlation was observed between progressive motility and the HOS test; nevertheless, progressive motility showed a greater susceptibility to cold shock than did either plasma membrane integrity or membrane functionality. This would seem to indicate that sperm motility is more affected by semen handling factors than the plasma membrane. Thus, tests evaluating the plasma membrane would prove to be the more accurate in predicting the fertilizing capacity of a semen sample. The HOS test is a simple and accessible evaluation method which, as in other species, could be a useful complement to routine equine semen analysis. It has the added advantages of being less susceptible to the immediate effects of cold shock and of evaluating individual sperm cells rather than the population as a whole, as does progressive motility. REFERENCES 1. Amann RP. Can the fertility potential of a seminal sample be predicted accurately? J Androl 1989; 10:89-98. 2. Correa JR, Zavos PM. The hypoosmotic swelling test: its employment as an assay to evaluate the functional integrity of the frozen-thawed bovine sperm membrane. Theriogenology 1994;42:351-360.
Theriogenology
727
3. CristaneUi M J, Squires EL, Amann RP, Pickett BW. Fertility of stallion semen processed frozen and thawed by a new procedure. Theriogenology 1984; 22:39-45. 4. Curry MR, Watson PF. Osmotic effects on ram and human sperm membranes in relation to thawing injury. Cryobiology 1994; 31:39-46. 5. Curry MR, Millar JD, Watson PF. The presence of water channel proteins in ram and human sperm membranes. J Reprod Fertil 1995; 104:297-303. 6. Harrison RAP, Vickers SE. Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. J Reprod Fertil 1990; 88:343-352. 7. Jeyendran RS, Van der Ven HH, Perez-Pelaez M, Grabo BG, Zaneveld LJD. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil 1984; 70:219225. 8. Kumi-Diaka J. Subjecting canine semen to the hypoosmotic test. Theriogenology 1993; 39:1279-1289. 9. Schrader SM, Platek SF, Zaneveld JD, Perez-Pelaez M, Jeyendran RS. Sperm viability: a comparison of analytical methods. Andrologia 1986; 18:530-538. 10. Tamuli MK, Watson PF. Effect of temperature of incubation on the development of resistance to cold stress and hypoosmotic stress in boar spermatozoa incubated for up to 24 hours. Proc. 12th. lnt Congr Anim Reprod 1992; 1484-1486. ! 1. Vazquez JM, Martinez EA, Martinez P, Garcia-Artiga C, Roca J. Hypoosmotic swelling of boar spermatozoa compared to other methods for analyzing the sperm membrane. Theftogenology 1997; 47:913-922. 12. Zavos PM. Hypoosmotic swelling test (HOS)/functional integrity of sperm membrane. J Ass Reprod Technol 1990; 2:215-216. i 3. Watson PF, Kunze E, Cramer P, Hammerstedt RH. A comparison of critical osmolarity and hydraulic conductivity and its activation energy in fowl and bull spermatozoa. J Androl 1992; 13: 131-138.