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Supravital Staining of Spermatozoa: Relationship of Eosin Concentration to the Percentage of Cells Staining Live
Vol. 118, December Printed in U .SA.
THE JOURNAL OF UROLOGY Copyright © 1977 by The Williams & Wilkins Co.
SUPRAVITAL STAINING OF SPERMATOZOA: RELATIONSHIP OF EOSIN CONCENTRATION TO THE PERCENTAGE OF CELLS STAINING LIVE K. A. DOUGHERTY, R. L. URRY
AND
A. T. K. COCKETT
From the Division of Urology, The University of Rochester School of Medicine and Dentistry, Rochester, New York
ABSTRACT
Supravital staining of human spermatozoa is a useful technique to assess semen quality. We compared 3 concentrations of eosin (1, 2.5 and 5 per cent) for their effectiveness to differentiate viable and non-viable spermatozoa. The percentage of viable cells determined by each concentration was compared as well as the percentage of cells estimated to be active. The results indicate that the percentage of spermatozoa determined to be viable with the supravital stains can be altered by changing the percentage of eosin in the stains. Use of 1 per cent eosin gave values that were significantly higher than the percentage of cells determined to be viable with 5 per cent eosin and the percentage of cells estimated to be active. Better quality slides were produced with 5 per cent eosin, which provided values that correlated favorably with motility estimations. Since inactivity of spermatozoa does not necessarily reflect loss of viability a reliable, quick method to differentiate live and dead spermatozoa has been found useful in assessing semen quality. Various staining procedures to differentiate viable and non-viable spermatozoa have been evaluated. Eosin stains only dead spermatozoa and has served as the basis for these staining techniques. 1 We recently compared the percentage of live spermatozoa determined by eosin-nigrosin and eosin opal blue with the percentage of cells estimated to be active at various intervals after sample collection. 2 At early intervals there were no significant differences between the estimated motility or the percentage of cells stained live with either stain. Only the stain eosin-nigrosin closely compared to the percentage estimated to be active at late intervals. It has been suggested that relatively high concentrations of eosin used in the aforementioned studies (5 per cent) may be capable of penetrating viable cells and, thus, lowering the percentage of cells determined to be viable. 3 We compared 3 concentrations of eosin (1, 2.5 and 5 per cent) for their effectiveness to differentiate viable and nonviable spermatozoa. The percentage of viable cells determined by each concentration was compared as well as the percentage of cells estimated to be active. MATERIALS AND METHODS
Experiment 1. Semen samples were obtained from 50 men attending a fertility evaluation clinic. The samples were evaluated 30 minutes after collection. The percentage of active spermatozoa in the semen samples was estimated by a trained technician, using standard microscopic techniques. Two sets of eosin-nigrosin stained slides were prepared by placing 1 drop of semen on a microscope slide, adding 2 drops of aqueous eosin Y (1 per cent set 1 or 5 per cent set 2) and mixing the solutions. After approximately 15 seconds 3 drops of 10 per cent aqueous nigrosin were added to the solution and mixed. A portion was transferred to a second slide, thin smears were made of both slides and the slides were dried on a warming plate. We counted 200 sperm cells on each slide (deadstained, live-unstained, 400 total sperm cells counted per
sample) and the percentage of live and dead cells was calculated. 2 The means were compared using Student's t test. Experiment 2. Semen samples were evaluated as in experiment 1 but the concentration of eosin was 2.5 per cent (set 1) and 5 per cent (set 2). RESULTS
Experiment 1 . The percentage of cells determined to be viable using 1 per cent eosin was significantly higher than the percentage of viable cells using 5 per cent eosin and the percentage of cells estimated to be active (p less than 0.05). Experiment 2. There were no significant differences between the percentage of viable cells determined with 2.5 or 5 per cent eosin or with these values and the percentage of sperm cells estimated to be active (see table). DISCUSSION
The results of these studies indicate that the percentage of spermatozoa determined to be viable with the supravital stains can be altered by changing the percentage of eosin in the stain. When a lower concentration of eosin is used (1 per cent) a higher percentage of spermatozoa appears to be viable. There does not seem to be a difference in the assessment of sperm viability with the higher concentrations (2.5 and 5 per cent). Also, the percentage of viable cells determined by the higher concentrations of eosin did not differ significantly from the percentage of sperm cells estimated to be active. When the slides prepared with the various concentrations of eosin were assessed it seemed possible that the difference in the percentage staining dead may relate to the percentage eosin Comparison of percentage of viable cells to percentage of active cells using different concentrations of eosin Experiment 1 Active Viable, 1 per cent eosin Viable, 5 per cent eosin
52.2 69.7 56.6
2.40 1. 79 1.76
53.0 46.1 45.3
3.6 3.3 4.5
Experiment 2
Accepted for publication April 22, 1977. Read at annual meeting of American Urological Association, Chicago, Illinois, April 24-28, 1977. Supported by the Henry C. Buswell Fund for Urology Research.
1008
Active Viable, 2.5 per cent eosin Viable, 5 per cent eosin
1009
EOSIN AND SPERM VIABILITY
concentration, since the lower percentage of eosin (1 per cent) may not have imparted a dark enough color to the dead cells to be easily detected" The use of the 5 per cent eosin produced slides of a higher quality that gave values that correlated well with the percentage of cells estimated to be active" The present study, along with previous investigations, indicates that the eosin-nigrosin supravital staining technique is valuable in the assessment of sperm viability and that the 5 cent eosin concentration gives slides of a better quality the use of lower amounts of eosino
Mrso Lynn Emilson and Mro Michael Cunningham technical assistance for this work REFERENCES
L Burgos, Mo Ho and Guillermo, Do Po: Eosin test for the evalnation of sperm vitality" FertiL Sterilo, 2: 542, 195L 20 Dougherty, K Ao, Emilson, L R Vo, Cockett, A To K Urry, R Lo: A comparison of subjective measurements human sperm motility and viability with two live-dead stain° ing techniques" FertiL SteriL, 26: 700, 19750 30 Eliasson, R: Personal communication, 19760
THE JOURNAL OF UROLOGY Copyright © 1977 by The Williams & Wilkins Co.
SUPRAVITAL STAINING OF SPERMATOZOA: RELATIONSHIP OF EOSIN CONCENTRATION TO THE PERCENTAGE OF CELLS STAINING LIVE K. A. DOUGHERTY, R. L. URRY
AND
A. T. K. COCKETT
From the Division of Urology, The University of Rochester School of Medicine and Dentistry, Rochester, New York
ABSTRACT
Supravital staining of human spermatozoa is a useful technique to assess semen quality. We compared 3 concentrations of eosin (1, 2.5 and 5 per cent) for their effectiveness to differentiate viable and non-viable spermatozoa. The percentage of viable cells determined by each concentration was compared as well as the percentage of cells estimated to be active. The results indicate that the percentage of spermatozoa determined to be viable with the supravital stains can be altered by changing the percentage of eosin in the stains. Use of 1 per cent eosin gave values that were significantly higher than the percentage of cells determined to be viable with 5 per cent eosin and the percentage of cells estimated to be active. Better quality slides were produced with 5 per cent eosin, which provided values that correlated favorably with motility estimations. Since inactivity of spermatozoa does not necessarily reflect loss of viability a reliable, quick method to differentiate live and dead spermatozoa has been found useful in assessing semen quality. Various staining procedures to differentiate viable and non-viable spermatozoa have been evaluated. Eosin stains only dead spermatozoa and has served as the basis for these staining techniques. 1 We recently compared the percentage of live spermatozoa determined by eosin-nigrosin and eosin opal blue with the percentage of cells estimated to be active at various intervals after sample collection. 2 At early intervals there were no significant differences between the estimated motility or the percentage of cells stained live with either stain. Only the stain eosin-nigrosin closely compared to the percentage estimated to be active at late intervals. It has been suggested that relatively high concentrations of eosin used in the aforementioned studies (5 per cent) may be capable of penetrating viable cells and, thus, lowering the percentage of cells determined to be viable. 3 We compared 3 concentrations of eosin (1, 2.5 and 5 per cent) for their effectiveness to differentiate viable and nonviable spermatozoa. The percentage of viable cells determined by each concentration was compared as well as the percentage of cells estimated to be active. MATERIALS AND METHODS
Experiment 1. Semen samples were obtained from 50 men attending a fertility evaluation clinic. The samples were evaluated 30 minutes after collection. The percentage of active spermatozoa in the semen samples was estimated by a trained technician, using standard microscopic techniques. Two sets of eosin-nigrosin stained slides were prepared by placing 1 drop of semen on a microscope slide, adding 2 drops of aqueous eosin Y (1 per cent set 1 or 5 per cent set 2) and mixing the solutions. After approximately 15 seconds 3 drops of 10 per cent aqueous nigrosin were added to the solution and mixed. A portion was transferred to a second slide, thin smears were made of both slides and the slides were dried on a warming plate. We counted 200 sperm cells on each slide (deadstained, live-unstained, 400 total sperm cells counted per
sample) and the percentage of live and dead cells was calculated. 2 The means were compared using Student's t test. Experiment 2. Semen samples were evaluated as in experiment 1 but the concentration of eosin was 2.5 per cent (set 1) and 5 per cent (set 2). RESULTS
Experiment 1 . The percentage of cells determined to be viable using 1 per cent eosin was significantly higher than the percentage of viable cells using 5 per cent eosin and the percentage of cells estimated to be active (p less than 0.05). Experiment 2. There were no significant differences between the percentage of viable cells determined with 2.5 or 5 per cent eosin or with these values and the percentage of sperm cells estimated to be active (see table). DISCUSSION
The results of these studies indicate that the percentage of spermatozoa determined to be viable with the supravital stains can be altered by changing the percentage of eosin in the stain. When a lower concentration of eosin is used (1 per cent) a higher percentage of spermatozoa appears to be viable. There does not seem to be a difference in the assessment of sperm viability with the higher concentrations (2.5 and 5 per cent). Also, the percentage of viable cells determined by the higher concentrations of eosin did not differ significantly from the percentage of sperm cells estimated to be active. When the slides prepared with the various concentrations of eosin were assessed it seemed possible that the difference in the percentage staining dead may relate to the percentage eosin Comparison of percentage of viable cells to percentage of active cells using different concentrations of eosin Experiment 1 Active Viable, 1 per cent eosin Viable, 5 per cent eosin
52.2 69.7 56.6
2.40 1. 79 1.76
53.0 46.1 45.3
3.6 3.3 4.5
Experiment 2
Accepted for publication April 22, 1977. Read at annual meeting of American Urological Association, Chicago, Illinois, April 24-28, 1977. Supported by the Henry C. Buswell Fund for Urology Research.
1008
Active Viable, 2.5 per cent eosin Viable, 5 per cent eosin
1009
EOSIN AND SPERM VIABILITY
concentration, since the lower percentage of eosin (1 per cent) may not have imparted a dark enough color to the dead cells to be easily detected" The use of the 5 per cent eosin produced slides of a higher quality that gave values that correlated well with the percentage of cells estimated to be active" The present study, along with previous investigations, indicates that the eosin-nigrosin supravital staining technique is valuable in the assessment of sperm viability and that the 5 cent eosin concentration gives slides of a better quality the use of lower amounts of eosino
Mrso Lynn Emilson and Mro Michael Cunningham technical assistance for this work REFERENCES
L Burgos, Mo Ho and Guillermo, Do Po: Eosin test for the evalnation of sperm vitality" FertiL Sterilo, 2: 542, 195L 20 Dougherty, K Ao, Emilson, L R Vo, Cockett, A To K Urry, R Lo: A comparison of subjective measurements human sperm motility and viability with two live-dead stain° ing techniques" FertiL SteriL, 26: 700, 19750 30 Eliasson, R: Personal communication, 19760