Q022-53,f7 i78/1204·-0425$02.Q0/8 THE r..iODRNAL
Vol. 120, ,:Jttobe:.' Printed in U.S.A.
UROL{}:'.i--~
Copyright © 1978 by Th-e \Villia:c.'ls & \Alilkir_s Co.
EFFECT OF AIVIYLASE
SPERM lVCOTILITY
KATHRYN A. DOUGHERTY, ABRAHAM T. K. COCKETT
AND
VIABILITY RONALD L. URRY
From the Division of Urology, University of Rochester School of Medicine and Dentistry, Rochester, New York
ABSTRACT
The effect of increasing concentrations of amylase on the percentage of active spermatozoa, the quality of their motility and the percentage of viable cells was studied in semen samples in vitro. The amount of amylase needed to liquefy viscous semen samples in vitro also was determined. The percentage of active spermatozoa and viable cells, and the quality of sperm motility were altered in relationship to the amylase levels. Significant decreases in these parameters compared to control values were seen at the higher concentrations of amylase. The lowest level of amylase did not alter these parameters significantly and was sufficient to liquefy 80 per cent of the viscous semen samples. Amylase appears to be effective at low concentrations for use in liquefying viscous semen samples, thus making them easier to analyze during routine semen examination. The level of amylase used and the interval between addition and analysis must be controlled carefully. Technically, highly viscous semen samples are difficult to analyze accurately. Motility estimations and sperm counts in viscous samples may vary greatly, depending upon the particular aliquot taken for analysis, and results from viscous samples must be interpreted with this fact in mind. Several methods have been suggested to reduce semen viscosity in vivo and in vitro, the use of amylase being one such method. Bunge and Sherman first reported that amylase liquefies coagulated semen rapidly when 3 parts of a 5 per cent solution of amylase in Locke's solution is added to 9 parts semen' and no detrimental effect was found on semen quality. Initially, post-coital douching with this solution was found valuable in producing pregnancies in cases in which prolonged semen viscosity was the only apparent fertility abnormality. Subsequently, 50 mg. a amylase vaginal suppositories were reported to be useful in reducing semen viscosity. 2 Although controversial, certain investigators believe that the failure of semen to liquefy may impede sperm motility and, thereby, reduce the fertilizing ability of spermatozoa. Regardless of the influence of semen viscosity on fertility highly viscous samples are difficult to analyze in the laboratory. Therefore, we studied the effect of amylase on sperm motility and viability to determine its effectiveness as a laborator; method to handle viscous samples in vitro. MATERIALS AND METHODS
Experiment 1. Semen samples were obtained from men seen for fertility evaluation. After mixing, 0.5 ml. aliquots of semen (15 minutes post-ejaculation) were placed in test tubes containing 0, l, 5, 10, 20, 40 or 60 mg. amylase powder.* Six samples were analyzed at each dose 15, 30, 60 and 90 minutes after addition of the semen to the amylase. The percentage of active cells at each interval was estimated and the quality of their activity was judged. :i The percentage of viable cells was determined using a supravital staining technique.4 The t statistic was used to characterize the difference between paired samples. The percentage of active cells, motility score and viable cells in each sample at each level of amylase was expressed as a percentage of its control value (0 mg. amylase aliquot). The averages for each dose at each period were calculated. Experiment 2. To determine the minimal dose of amylase needed to liquefy viscous samples 0.5 cc aliquots of viscous semen were added to tubes containing 0, 1, 5 or 10 mg. Accepted for publication February 3, 1978. Supported by the Henry C. Buswell Fund for Urology Research. * Sigma Chemical Company.
amylase powder (10 samples per dose). The samples were mixed and the ability of the sample to form discrete droplets when expelled from a disposable Pasteur pipet was determined. RESULTS
Experiment 1. The percentage of active spermatozoa and viable cells, and the motility score in the semen aliquots appeared to be affected in a dose-response manner at all intervals (see table). However, the 1 mg. level did not affect the measured parameters significantly. Although 5 mg. amylase significantly altered the motility score at the later interval no significant differences were noted between the control and 5 mg. aliquot at the 15 or 30-minute intervals. At each time period significant decreases in most parameters occurred when the dose of amylase was 2::10 mg./0.5 ml. Experiment 2. Low doses of amylase were found to reduce semen viscosity effectively. In 8 samples the semen in tubes containing 1, 5 and 10 mg. amylase liquefied. Each control aliquot (0 mg. amylase) failed to liquefy. In 2 samples only the semen in the 5 and 10 mg. amylase tubes liquefied. DISCUSSION
Since viscous samples are difficult to analyze technically a method is needed to liquefy the sample without altering the semen quality. The results of this report confirm those of other investigators who found that amylase is effective in reducing the viscosity of human semen. 1. 2 In 80 cent of the cases tested a low dose of (l mg./0.5 was sufficient for in vitro liquefaction but semen was not altered significantly. The remaining cases a slightly dose (5 rng./0.5 ml.) but this level of amylase did not alter parameters significantly for at least 30 minutes after its addition. At later time periods, however, significant decreases in sperm activity were noted. Therefore, it appears that while amylase may liquefy viscous semen the amount used and interval between addition of the amylase to the semen and its analysis must be considered so that significant alterations in semen quality are avoided. Since the spermatozoa in all samples may not respond in the same manner to different doses of amylase the possibility of harmful effects with low doses cannot be eliminated. While other investigators have suggested that amylase may be useful in improving the probability of conception in the partner of a man with repeated viscous semen samples our results offer little relative to the safety of its use in vivo or in vitro. The studies do suggest that the dose must be considered
425
426
DOUGHERTY, COCKETT AND URRY
Effects of amylase on the percentage of active cells and viable cells, and the motility scores (values expressed as percentage of control) Mins. After Semen Addition Amylase (mg./0.5 ml.)
1 5 10 20 40 60 *p
15
30
90
60
% Active Spermatozoa
Motility Score
% Viable Spermatozoa
% Active Spermatozoa
Motility Score
% Viable Spermatozoa
% Active Spermatozoa
Motility Score
% Viable Spermatozoa
% Active Spermatozoa
Motility Score
% Viable Spermatozoa
93.7 93.4 85.7 72.5* 28.6* 18.6*
89.8 90.9 67.4* 66.2* 18.2* 9.0*
96.6 94.4 83.2* 77.6* 64.1* 57.8*
101.6 96.6 77.8* 73.4* 25.4* 18.6*
95.1 89.6 57.4* 55.2* 13.1* 9.4*
95.5 105.3 87.6* 92.3 67.0* 70.0*
96.4 88.6* 72.0* 64.0* 17.5* 17.7*
100.0 79.0* 44.6* 48.1* 9.4* 7.6*
100.6 106.3 94.3 84.5* 73.7* 57.9*
102.0 93.2 62.5* 67.3* 20.8* 24.2*
96.4 85.3* 44.1* 42.6* 11.7* 11.4*
102.5 94.3 104.8 80.2* 75.5* 54.4*
= 0.05.
carefully to prevent reduced sperm motility and viability. Further experimental work appears warranted to test the effects of amylase on semen viscosity and fertility. Mrs. Lynn Emilson and Mr. Michael Cunningham provided technical assistance. REFERENCES
1. Bunge, R. G. and Sherman, J. K.: Liquefaction of human semen
by alpha-amylase. Fertil. Steril., 5: 353, 1954. 2. Bunge, R. G.: Alpha amylase suppositories and non-liquefaction of human serum. J. Urol., 99: 350, 1968. 3. Cockett, A. T. K., Netto, I. C. V., Dougherty, K. A. and Urry, R. L.: Semen analysis: a review of samples from 225 men seen at an infertility clinic. J. Urol., 114: 560, 1975. 4. Dougherty, K. A., Emilson, L. B. V., Cockett, A. T. K. and Urry, R. L.: A comparison of subjective measurement of human sperm motility and viability with two live-dead staining techniques. Fertil. Steril., 26: 700, 1975.
Vol. 120, ,:Jttobe:.' Printed in U.S.A.
UROL{}:'.i--~
Copyright © 1978 by Th-e \Villia:c.'ls & \Alilkir_s Co.
EFFECT OF AIVIYLASE
SPERM lVCOTILITY
KATHRYN A. DOUGHERTY, ABRAHAM T. K. COCKETT
AND
VIABILITY RONALD L. URRY
From the Division of Urology, University of Rochester School of Medicine and Dentistry, Rochester, New York
ABSTRACT
The effect of increasing concentrations of amylase on the percentage of active spermatozoa, the quality of their motility and the percentage of viable cells was studied in semen samples in vitro. The amount of amylase needed to liquefy viscous semen samples in vitro also was determined. The percentage of active spermatozoa and viable cells, and the quality of sperm motility were altered in relationship to the amylase levels. Significant decreases in these parameters compared to control values were seen at the higher concentrations of amylase. The lowest level of amylase did not alter these parameters significantly and was sufficient to liquefy 80 per cent of the viscous semen samples. Amylase appears to be effective at low concentrations for use in liquefying viscous semen samples, thus making them easier to analyze during routine semen examination. The level of amylase used and the interval between addition and analysis must be controlled carefully. Technically, highly viscous semen samples are difficult to analyze accurately. Motility estimations and sperm counts in viscous samples may vary greatly, depending upon the particular aliquot taken for analysis, and results from viscous samples must be interpreted with this fact in mind. Several methods have been suggested to reduce semen viscosity in vivo and in vitro, the use of amylase being one such method. Bunge and Sherman first reported that amylase liquefies coagulated semen rapidly when 3 parts of a 5 per cent solution of amylase in Locke's solution is added to 9 parts semen' and no detrimental effect was found on semen quality. Initially, post-coital douching with this solution was found valuable in producing pregnancies in cases in which prolonged semen viscosity was the only apparent fertility abnormality. Subsequently, 50 mg. a amylase vaginal suppositories were reported to be useful in reducing semen viscosity. 2 Although controversial, certain investigators believe that the failure of semen to liquefy may impede sperm motility and, thereby, reduce the fertilizing ability of spermatozoa. Regardless of the influence of semen viscosity on fertility highly viscous samples are difficult to analyze in the laboratory. Therefore, we studied the effect of amylase on sperm motility and viability to determine its effectiveness as a laborator; method to handle viscous samples in vitro. MATERIALS AND METHODS
Experiment 1. Semen samples were obtained from men seen for fertility evaluation. After mixing, 0.5 ml. aliquots of semen (15 minutes post-ejaculation) were placed in test tubes containing 0, l, 5, 10, 20, 40 or 60 mg. amylase powder.* Six samples were analyzed at each dose 15, 30, 60 and 90 minutes after addition of the semen to the amylase. The percentage of active cells at each interval was estimated and the quality of their activity was judged. :i The percentage of viable cells was determined using a supravital staining technique.4 The t statistic was used to characterize the difference between paired samples. The percentage of active cells, motility score and viable cells in each sample at each level of amylase was expressed as a percentage of its control value (0 mg. amylase aliquot). The averages for each dose at each period were calculated. Experiment 2. To determine the minimal dose of amylase needed to liquefy viscous samples 0.5 cc aliquots of viscous semen were added to tubes containing 0, 1, 5 or 10 mg. Accepted for publication February 3, 1978. Supported by the Henry C. Buswell Fund for Urology Research. * Sigma Chemical Company.
amylase powder (10 samples per dose). The samples were mixed and the ability of the sample to form discrete droplets when expelled from a disposable Pasteur pipet was determined. RESULTS
Experiment 1. The percentage of active spermatozoa and viable cells, and the motility score in the semen aliquots appeared to be affected in a dose-response manner at all intervals (see table). However, the 1 mg. level did not affect the measured parameters significantly. Although 5 mg. amylase significantly altered the motility score at the later interval no significant differences were noted between the control and 5 mg. aliquot at the 15 or 30-minute intervals. At each time period significant decreases in most parameters occurred when the dose of amylase was 2::10 mg./0.5 ml. Experiment 2. Low doses of amylase were found to reduce semen viscosity effectively. In 8 samples the semen in tubes containing 1, 5 and 10 mg. amylase liquefied. Each control aliquot (0 mg. amylase) failed to liquefy. In 2 samples only the semen in the 5 and 10 mg. amylase tubes liquefied. DISCUSSION
Since viscous samples are difficult to analyze technically a method is needed to liquefy the sample without altering the semen quality. The results of this report confirm those of other investigators who found that amylase is effective in reducing the viscosity of human semen. 1. 2 In 80 cent of the cases tested a low dose of (l mg./0.5 was sufficient for in vitro liquefaction but semen was not altered significantly. The remaining cases a slightly dose (5 rng./0.5 ml.) but this level of amylase did not alter parameters significantly for at least 30 minutes after its addition. At later time periods, however, significant decreases in sperm activity were noted. Therefore, it appears that while amylase may liquefy viscous semen the amount used and interval between addition of the amylase to the semen and its analysis must be considered so that significant alterations in semen quality are avoided. Since the spermatozoa in all samples may not respond in the same manner to different doses of amylase the possibility of harmful effects with low doses cannot be eliminated. While other investigators have suggested that amylase may be useful in improving the probability of conception in the partner of a man with repeated viscous semen samples our results offer little relative to the safety of its use in vivo or in vitro. The studies do suggest that the dose must be considered
425
426
DOUGHERTY, COCKETT AND URRY
Effects of amylase on the percentage of active cells and viable cells, and the motility scores (values expressed as percentage of control) Mins. After Semen Addition Amylase (mg./0.5 ml.)
1 5 10 20 40 60 *p
15
30
90
60
% Active Spermatozoa
Motility Score
% Viable Spermatozoa
% Active Spermatozoa
Motility Score
% Viable Spermatozoa
% Active Spermatozoa
Motility Score
% Viable Spermatozoa
% Active Spermatozoa
Motility Score
% Viable Spermatozoa
93.7 93.4 85.7 72.5* 28.6* 18.6*
89.8 90.9 67.4* 66.2* 18.2* 9.0*
96.6 94.4 83.2* 77.6* 64.1* 57.8*
101.6 96.6 77.8* 73.4* 25.4* 18.6*
95.1 89.6 57.4* 55.2* 13.1* 9.4*
95.5 105.3 87.6* 92.3 67.0* 70.0*
96.4 88.6* 72.0* 64.0* 17.5* 17.7*
100.0 79.0* 44.6* 48.1* 9.4* 7.6*
100.6 106.3 94.3 84.5* 73.7* 57.9*
102.0 93.2 62.5* 67.3* 20.8* 24.2*
96.4 85.3* 44.1* 42.6* 11.7* 11.4*
102.5 94.3 104.8 80.2* 75.5* 54.4*
= 0.05.
carefully to prevent reduced sperm motility and viability. Further experimental work appears warranted to test the effects of amylase on semen viscosity and fertility. Mrs. Lynn Emilson and Mr. Michael Cunningham provided technical assistance. REFERENCES
1. Bunge, R. G. and Sherman, J. K.: Liquefaction of human semen
by alpha-amylase. Fertil. Steril., 5: 353, 1954. 2. Bunge, R. G.: Alpha amylase suppositories and non-liquefaction of human serum. J. Urol., 99: 350, 1968. 3. Cockett, A. T. K., Netto, I. C. V., Dougherty, K. A. and Urry, R. L.: Semen analysis: a review of samples from 225 men seen at an infertility clinic. J. Urol., 114: 560, 1975. 4. Dougherty, K. A., Emilson, L. B. V., Cockett, A. T. K. and Urry, R. L.: A comparison of subjective measurement of human sperm motility and viability with two live-dead staining techniques. Fertil. Steril., 26: 700, 1975.
