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Spermatozoan Velocities in Vitro
Spermatozoan Velocities in Vitro A Simple Method of Measurement
F. N. Baker, Ph.D., R. G. Cragle, M.S., G. W. Salisbury, Ph.D., and N. L. VanDemark, Ph.D.
LITTLE IS KNOWN regarding how essential spermatozoan motility is to fertilization. It was formerly believed that the motility of the spermatozoon itself was responsible for its progress through the upper reproductive tract of the inseminated female. However, VanDemark and Moeller have demonstrated in the cow that nonmotile spermatozoa are transported to the oviducts and that motile sperm are transported more rapidly than would be expected from in vitro estimates of spermatozoan velocity. Though apparently not essential for transport of the spermatozoon, motility may be essential for distribution of the sperm cells in the female tract, rendering a meeting of the germ cells more probable and for penetration of the ovum. A simple, rapid method for estimating spermatozoan velocities would greatly facilitate the study of the motility functions of spermatozoa. This paper presents a simple method, requiring a minimum of special equipment, by which spermatozoan velocities can be measured and presents some results from its application to bovine spermatozoa.
METHOD The method is based upon the principle that the number of free-swimming cells passing through a segment of a plane in a given time is dependent upon the dimensions of the segment, the concentration of the cells in suspenFrom the Department of Dairy Science, University of Illinois, Urbana, Ill. 149
150
BAKER ET At.
Fertility & Sterility
sion, and the velocity of the cells. Use of the method is limited to cells in dilute suspension (as are the other methods reviewed below) and, therefore, may not be free from the effects of dilution of extracellular enzymes upon the physiologic functions of the cell. Aliquots of semen samples were diluted to a concentration of 25,000 spermatozoa per cu. mm. in a 1: 9 dilution of egg yolk in aqueous solution of 2.9% sodium citrate dihydrate, and the spermatozoa were dispersed throughout the medium. A clean, dry Petroff-Hausser chamber was filled with the diluted semen, care being taken to avoid overfilling (only the central plateau was filled with liquid). The chamber must be dry and clean or air bubbles will be entrapped in the diluted semen. The Petroff-Hausser chamber was placed on the stage of a microscope and the microscope focused (XlO0-3OO) on the ruled lines on the chamber surface. One side of the smallest square is equal to 0.05 mm. A 0.05-mm. line segment was selected and, upon starting a stopwatch, the cells crossing the chosen line segment were counted. When 100 cells crossed the line, regardless of the direction of travel, the watch was stopped (a hand-tally counter with an audible signal at 100 was used in this laboratory) and the time recorded. The number of nonmotile cells in 80 of the small squares of the PetroffHausser chamber then were counted. This gave the percentage of nonmotile cells, since the concentration was standardized at 25,000 cells per cu. mm. (80 squares contain 0.004 cu. mm. volume or a mean count of 100 cells at the given dilution). Calculation of Velocity The mean gross velocity in millimeters per minute was obtained by dividing 339.4 by the elapsed time in seconds. This gave the mean velocity of all cells in suspension including those of zero velocity (nonmotile). The mean velocity of those cells which were motile was found by dividing the mean gross velocity by the fraction of motile cells present. An explanation of the factor 339.4 used in the calculations is in order. This was a reduction of the separate factors which correct for the number of cells counted, the length of the line segment (one dimension of the plane segment), the depth of the Petroff-Hausser cell (the other dimension of
Vol. 8, No.2, 1957
SPERMATOZOAN VELOCITIES
151
the plane segment), the concentration of cells, the average angle at which a cell crosses the line segment, and the units of time. Thus, Velocity in mm.lmin. = 100
1
x
1 1 X 0.05mm. 0.02 mm.
X
mm. 3 25,000
X
60 1 0.70711 X time (sec.)
339.4 time (sec.)
The factor 1/0.70711 which corrects for angular approach to the line segment needs further explanation. If a spermatozoon starting from any given point approaches the line segment at an angle of 90° - 0 and crosses the line segment in unit time, then the true velocity of the spermatozoon will be equal to the observed velocity divided by the cosine of o. Spermatozoa in dilute suspensions move in random directions, therefore, the average angle of approach to the line segment will be 45°. The cosine of 90° - 45° is 0.70711. This correction is valid for the case in which spermatozoa move in straight lines. It is in error (underestimating true velocity) for the case in which the spermatozoon moves in an arc by the difference between the length of the arc and its chord. In most cases this difference will not be large. The dilution fluid was a modification of the medium routinely used for the extension of bovine semen samples for artificial insemination. The egg yolk was limited in order to increase visibility. In preliminary experiments, spermatozoan velocities declined rapidly in the counting chamber when mineral diluters such as 0.9% NaCI or phosphate buffers were used. The yolk-citrate was superior to other solutions tested, however, other solutions may yet prove to be optimal for spermatozoan velocity. The Petroff-Hausser cell was used because of its internal dimensions, particularly as its depth is only 0.02 mm. and roughly only two and one half times the width of the sperm head. Thus sperm travel was limited in the vertical plane. The ordinary hemacytometer cell permitted wider motility deviations in the vertical plane and was too deep to permit focusing on all cells in the field simultaneously.
RESULTS Preliminary experimentation suggested that the cells crossing the line segment in active semen samples might be counted when the cell concen-
152
BAKER ET
AL.
Fertility & Sterility
tration was 50,000 per cu. mm. An experiment was set up to test the method in which dilution rates of 50,000 and 25,000 cells per cu. mm. were used. Four replications (fillings of the chamber) were made on each semen sample at each dilution. The design permitted a test of the influence of order of replication (a measure of deterioration after dilution). Five bovine semen samples were tested by one author and ten samples by another. The mean velocity of motile spermatozoa obtained by the first observer was 4.46 -+- 0.17* mm. per min. and that obtained by the second was 4.11 -+- 0.16* mm. per min. The over-all average was 4.23 -+- 0.12* mm. per min. There were no significant differences between the two observers, which was not surprising since both observers had obtained semen samples for investigation from the same group of bulls. More interesting were the means obtained under the two dilution rates. At 50,000 cells per cu. mm. the measured rate was 3.95 -+- 0.14* mm. per min., and at 25,000 cells per cu. mm. the rate was 4.50 -+- 0.19* mm. per min. The difference was highly significant and may be explained by the data on nonmotile spermatozoa. A smaller percentage (32.6 -+- 0.05*) of nonmotile spermatozoa was counted in the 50,000 cells per cu. mm. dilution than in the 25,000 cells per cu. mm. dilution (35.7 -+- 0.06*). The difference was highly significant. It is believed that more accurate counts, both of nonmotile spermatozoa and of spermatozoa crossing the line segment, were obtained at the 25,000 cells per cu. mm. dilution. At the lesser concentration the observer could determine more easily the cells crossing a line segment and fewer inactive cells were set in motion as a result of bumping by motile cells or by eddy currents within the observation chamber. The order of making replications did not significantly influence the values obtained at each replication. Highly significant differences were found among semen samples, indicating that the method, as used with four replications, was sufficiently precise to distinguish real differences among the velocities of spermatozoa in different semen samples. The use of this method has highlighted possible errors in visual estimates of per cent motile spermatozoa. Estimates of per cent motility using the method described in this paper with dilutions of 25,000 and 50,000 sperm per cc. as compared to the simple visual estimate are presented in Table 1.
* Standard deviation of the mean.
Vol. 8, No.2, 1957
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SPERMATOZOAN VELOCITIES
TABLE 1. Ten Estimates of the Percentage of Motile Sperm as Obtained by Both the Petroff-Hausser Chamber Method and the Simple Visual-Estimate Method Sample no. 1
2 3 4 .5 6 7 8 9 10
Petroff-Hausser Chamber 25,000 eells/ee.
50,000 eells/ee.
Simple visual estimates
86 85 78 45 55 41 66 57 49 32
85 86 82 60 63 43 70 58 57 39
60 60 60 50 60 40 50 50 40 30
These results, presented in Table 1, indicate that in the highly motile semen samples, the per cent of motile cells may often be underestimated if the simple visual method is used.
DISCUSSION Heretofore three methods, other than simple visual estimates, have been used to assess spermatozoan velocities, all being subject to certain limitations. Phillips and Andrews, and Lamar, Shettles, and Delfs used methods in which spermatozoa were introduced into capillary tubes filled with physiologic liquids or isotonic salt solutions. The change in position of the spermatozoa along the capillary was regarded as a measure of spermatozoan velocity. Such estimates include diffusion effects and may include effects due to turbulence created in the media upon introducing the spermatozoan suspension, as well as the true movements of the spermatozoa. Since they are determined from the first spermatozoa to move through the capillary, they are not indicative of population averages. Moeller and VanDemark timed individual spermatozoa by direct observalion of travel across a microscopic field upon which was superimposed the image of an eyepiece micrometer. This method led to accurate estimates of velocity of those spermatozoa actually timed, but those selected for timing may not have been representative of the population as a whole. Photographic methods have been used by Rothschild and Swann and by Rothschild to estimate sperm speeds. These methods are accurate but have the disadvantages of requiring expensive equipment and being time con-
154
BAKER ET At.
Fertility & Sterility
suming. The Rothschild methods require the taking of photomicrographs at timed intervals, the development of the film, the subsequent projection of the film series, and the measurement of distances traveled by individual sperm on the projected scale or the counting of sperm in individual frames from rapid-sequence photographs. Thus the results of the velocity estimates are not immediately available to the experimenter for judgment of the value of semen samples for further experimentation. The present method is free from diffusion or turbulence effects, is free from sampling bias, is rapid, and, except for the Petroff-Hausser cell, requires no equipment not found in most biological laboratories. One source of bias is inherent in this method as is also inherent in direct timing and in photographic methods. This is the bias due to assumption of travel in a straight line when, in fact, spermatozoa frequently travel in arcs of various magnitudes. This bias is minimized, but not eliminated, in the photographic method by measuring position differences at extremely short time intervals. Correction of this error would require knowledge of the average angular velocity and average radius of arc traveled. No means are known at this time for obtaining this information. Except in the case of semen samples containing many bent- or coiled-tailed spermatozoa, this bias should not be great. Observers with good vision and normal reflexes should have no difficulty keeping count on samples at the 25,000 cells per cu. mm. dilution. If the counting of cells approaching from both sides of the line segment seems too perplexing, one may count the cells approaching from one side only. The latter suggestion has the effect of halving the concentration and this must be taken into account in computing the velocities. If the latter suggestion is adopted, extreme care must be taken that no drifting occurs within the counting chamber. Counting from both sides of the line segments will compensate for drifting if the rate of drift is less than the mean sperm velocity. Actually, little drifting occurs in this type of chamber if it is properly filled with no entrapped air bubbles.
SUMMARY A method for estimating spermatozoan velocities has been described, which is based on the principle that the number of free-swimming cells passing in a given time through a segment of a plane is dependent upon
Vol. 8, No.2, 1957
SPERMATOZOAN VELOCITIES
155
the dimensions of the segment, the concentration of the cells in suspension, and the velocity of the cells. The mean velocity observed in 15 samples of bovine semen was 4.23 mm. per minute.
REFERENCES 1. LAMAR, J. K., SHETTLES, L. B., and DELFS, E. Cyclical penetration of human cervical mucus to spermatozoa in vitro. Am.]. Physiol. 129:234, 1940. 2. MOELLER, A. N., and VANDEMARK, N. L. In vitro speeds of bovine spermatozoa. Fertil. & Steril. 6:506, 1955. 3. PHILLIPS, R. W., and ANDREWS, F. N. Speed of travel of ram spermatozoa. Anat.
Rec. 68:127,1937. A new method of measuring sperm speeds. Nature, London 171:512,1953. 5. ROTHSCIDLD, LORD, and SWANN, M. M. The fertilization reaction in the sea urchin egg: A propagated response to sperm reaction. J. Exper. BioI. 26:164, 1949. 6. VANDEMARK, N. L., and MOELLER, A. N. Speed of spermatozoan transport in reproductive tract of estrous cow. Am.]. Physiol. 165:674, 1951. 4. ROTHSCHILD, LORD.
F. N. Baker, Ph.D., R. G. Cragle, M.S., G. W. Salisbury, Ph.D., and N. L. VanDemark, Ph.D.
LITTLE IS KNOWN regarding how essential spermatozoan motility is to fertilization. It was formerly believed that the motility of the spermatozoon itself was responsible for its progress through the upper reproductive tract of the inseminated female. However, VanDemark and Moeller have demonstrated in the cow that nonmotile spermatozoa are transported to the oviducts and that motile sperm are transported more rapidly than would be expected from in vitro estimates of spermatozoan velocity. Though apparently not essential for transport of the spermatozoon, motility may be essential for distribution of the sperm cells in the female tract, rendering a meeting of the germ cells more probable and for penetration of the ovum. A simple, rapid method for estimating spermatozoan velocities would greatly facilitate the study of the motility functions of spermatozoa. This paper presents a simple method, requiring a minimum of special equipment, by which spermatozoan velocities can be measured and presents some results from its application to bovine spermatozoa.
METHOD The method is based upon the principle that the number of free-swimming cells passing through a segment of a plane in a given time is dependent upon the dimensions of the segment, the concentration of the cells in suspenFrom the Department of Dairy Science, University of Illinois, Urbana, Ill. 149
150
BAKER ET At.
Fertility & Sterility
sion, and the velocity of the cells. Use of the method is limited to cells in dilute suspension (as are the other methods reviewed below) and, therefore, may not be free from the effects of dilution of extracellular enzymes upon the physiologic functions of the cell. Aliquots of semen samples were diluted to a concentration of 25,000 spermatozoa per cu. mm. in a 1: 9 dilution of egg yolk in aqueous solution of 2.9% sodium citrate dihydrate, and the spermatozoa were dispersed throughout the medium. A clean, dry Petroff-Hausser chamber was filled with the diluted semen, care being taken to avoid overfilling (only the central plateau was filled with liquid). The chamber must be dry and clean or air bubbles will be entrapped in the diluted semen. The Petroff-Hausser chamber was placed on the stage of a microscope and the microscope focused (XlO0-3OO) on the ruled lines on the chamber surface. One side of the smallest square is equal to 0.05 mm. A 0.05-mm. line segment was selected and, upon starting a stopwatch, the cells crossing the chosen line segment were counted. When 100 cells crossed the line, regardless of the direction of travel, the watch was stopped (a hand-tally counter with an audible signal at 100 was used in this laboratory) and the time recorded. The number of nonmotile cells in 80 of the small squares of the PetroffHausser chamber then were counted. This gave the percentage of nonmotile cells, since the concentration was standardized at 25,000 cells per cu. mm. (80 squares contain 0.004 cu. mm. volume or a mean count of 100 cells at the given dilution). Calculation of Velocity The mean gross velocity in millimeters per minute was obtained by dividing 339.4 by the elapsed time in seconds. This gave the mean velocity of all cells in suspension including those of zero velocity (nonmotile). The mean velocity of those cells which were motile was found by dividing the mean gross velocity by the fraction of motile cells present. An explanation of the factor 339.4 used in the calculations is in order. This was a reduction of the separate factors which correct for the number of cells counted, the length of the line segment (one dimension of the plane segment), the depth of the Petroff-Hausser cell (the other dimension of
Vol. 8, No.2, 1957
SPERMATOZOAN VELOCITIES
151
the plane segment), the concentration of cells, the average angle at which a cell crosses the line segment, and the units of time. Thus, Velocity in mm.lmin. = 100
1
x
1 1 X 0.05mm. 0.02 mm.
X
mm. 3 25,000
X
60 1 0.70711 X time (sec.)
339.4 time (sec.)
The factor 1/0.70711 which corrects for angular approach to the line segment needs further explanation. If a spermatozoon starting from any given point approaches the line segment at an angle of 90° - 0 and crosses the line segment in unit time, then the true velocity of the spermatozoon will be equal to the observed velocity divided by the cosine of o. Spermatozoa in dilute suspensions move in random directions, therefore, the average angle of approach to the line segment will be 45°. The cosine of 90° - 45° is 0.70711. This correction is valid for the case in which spermatozoa move in straight lines. It is in error (underestimating true velocity) for the case in which the spermatozoon moves in an arc by the difference between the length of the arc and its chord. In most cases this difference will not be large. The dilution fluid was a modification of the medium routinely used for the extension of bovine semen samples for artificial insemination. The egg yolk was limited in order to increase visibility. In preliminary experiments, spermatozoan velocities declined rapidly in the counting chamber when mineral diluters such as 0.9% NaCI or phosphate buffers were used. The yolk-citrate was superior to other solutions tested, however, other solutions may yet prove to be optimal for spermatozoan velocity. The Petroff-Hausser cell was used because of its internal dimensions, particularly as its depth is only 0.02 mm. and roughly only two and one half times the width of the sperm head. Thus sperm travel was limited in the vertical plane. The ordinary hemacytometer cell permitted wider motility deviations in the vertical plane and was too deep to permit focusing on all cells in the field simultaneously.
RESULTS Preliminary experimentation suggested that the cells crossing the line segment in active semen samples might be counted when the cell concen-
152
BAKER ET
AL.
Fertility & Sterility
tration was 50,000 per cu. mm. An experiment was set up to test the method in which dilution rates of 50,000 and 25,000 cells per cu. mm. were used. Four replications (fillings of the chamber) were made on each semen sample at each dilution. The design permitted a test of the influence of order of replication (a measure of deterioration after dilution). Five bovine semen samples were tested by one author and ten samples by another. The mean velocity of motile spermatozoa obtained by the first observer was 4.46 -+- 0.17* mm. per min. and that obtained by the second was 4.11 -+- 0.16* mm. per min. The over-all average was 4.23 -+- 0.12* mm. per min. There were no significant differences between the two observers, which was not surprising since both observers had obtained semen samples for investigation from the same group of bulls. More interesting were the means obtained under the two dilution rates. At 50,000 cells per cu. mm. the measured rate was 3.95 -+- 0.14* mm. per min., and at 25,000 cells per cu. mm. the rate was 4.50 -+- 0.19* mm. per min. The difference was highly significant and may be explained by the data on nonmotile spermatozoa. A smaller percentage (32.6 -+- 0.05*) of nonmotile spermatozoa was counted in the 50,000 cells per cu. mm. dilution than in the 25,000 cells per cu. mm. dilution (35.7 -+- 0.06*). The difference was highly significant. It is believed that more accurate counts, both of nonmotile spermatozoa and of spermatozoa crossing the line segment, were obtained at the 25,000 cells per cu. mm. dilution. At the lesser concentration the observer could determine more easily the cells crossing a line segment and fewer inactive cells were set in motion as a result of bumping by motile cells or by eddy currents within the observation chamber. The order of making replications did not significantly influence the values obtained at each replication. Highly significant differences were found among semen samples, indicating that the method, as used with four replications, was sufficiently precise to distinguish real differences among the velocities of spermatozoa in different semen samples. The use of this method has highlighted possible errors in visual estimates of per cent motile spermatozoa. Estimates of per cent motility using the method described in this paper with dilutions of 25,000 and 50,000 sperm per cc. as compared to the simple visual estimate are presented in Table 1.
* Standard deviation of the mean.
Vol. 8, No.2, 1957
153
SPERMATOZOAN VELOCITIES
TABLE 1. Ten Estimates of the Percentage of Motile Sperm as Obtained by Both the Petroff-Hausser Chamber Method and the Simple Visual-Estimate Method Sample no. 1
2 3 4 .5 6 7 8 9 10
Petroff-Hausser Chamber 25,000 eells/ee.
50,000 eells/ee.
Simple visual estimates
86 85 78 45 55 41 66 57 49 32
85 86 82 60 63 43 70 58 57 39
60 60 60 50 60 40 50 50 40 30
These results, presented in Table 1, indicate that in the highly motile semen samples, the per cent of motile cells may often be underestimated if the simple visual method is used.
DISCUSSION Heretofore three methods, other than simple visual estimates, have been used to assess spermatozoan velocities, all being subject to certain limitations. Phillips and Andrews, and Lamar, Shettles, and Delfs used methods in which spermatozoa were introduced into capillary tubes filled with physiologic liquids or isotonic salt solutions. The change in position of the spermatozoa along the capillary was regarded as a measure of spermatozoan velocity. Such estimates include diffusion effects and may include effects due to turbulence created in the media upon introducing the spermatozoan suspension, as well as the true movements of the spermatozoa. Since they are determined from the first spermatozoa to move through the capillary, they are not indicative of population averages. Moeller and VanDemark timed individual spermatozoa by direct observalion of travel across a microscopic field upon which was superimposed the image of an eyepiece micrometer. This method led to accurate estimates of velocity of those spermatozoa actually timed, but those selected for timing may not have been representative of the population as a whole. Photographic methods have been used by Rothschild and Swann and by Rothschild to estimate sperm speeds. These methods are accurate but have the disadvantages of requiring expensive equipment and being time con-
154
BAKER ET At.
Fertility & Sterility
suming. The Rothschild methods require the taking of photomicrographs at timed intervals, the development of the film, the subsequent projection of the film series, and the measurement of distances traveled by individual sperm on the projected scale or the counting of sperm in individual frames from rapid-sequence photographs. Thus the results of the velocity estimates are not immediately available to the experimenter for judgment of the value of semen samples for further experimentation. The present method is free from diffusion or turbulence effects, is free from sampling bias, is rapid, and, except for the Petroff-Hausser cell, requires no equipment not found in most biological laboratories. One source of bias is inherent in this method as is also inherent in direct timing and in photographic methods. This is the bias due to assumption of travel in a straight line when, in fact, spermatozoa frequently travel in arcs of various magnitudes. This bias is minimized, but not eliminated, in the photographic method by measuring position differences at extremely short time intervals. Correction of this error would require knowledge of the average angular velocity and average radius of arc traveled. No means are known at this time for obtaining this information. Except in the case of semen samples containing many bent- or coiled-tailed spermatozoa, this bias should not be great. Observers with good vision and normal reflexes should have no difficulty keeping count on samples at the 25,000 cells per cu. mm. dilution. If the counting of cells approaching from both sides of the line segment seems too perplexing, one may count the cells approaching from one side only. The latter suggestion has the effect of halving the concentration and this must be taken into account in computing the velocities. If the latter suggestion is adopted, extreme care must be taken that no drifting occurs within the counting chamber. Counting from both sides of the line segments will compensate for drifting if the rate of drift is less than the mean sperm velocity. Actually, little drifting occurs in this type of chamber if it is properly filled with no entrapped air bubbles.
SUMMARY A method for estimating spermatozoan velocities has been described, which is based on the principle that the number of free-swimming cells passing in a given time through a segment of a plane is dependent upon
Vol. 8, No.2, 1957
SPERMATOZOAN VELOCITIES
155
the dimensions of the segment, the concentration of the cells in suspension, and the velocity of the cells. The mean velocity observed in 15 samples of bovine semen was 4.23 mm. per minute.
REFERENCES 1. LAMAR, J. K., SHETTLES, L. B., and DELFS, E. Cyclical penetration of human cervical mucus to spermatozoa in vitro. Am.]. Physiol. 129:234, 1940. 2. MOELLER, A. N., and VANDEMARK, N. L. In vitro speeds of bovine spermatozoa. Fertil. & Steril. 6:506, 1955. 3. PHILLIPS, R. W., and ANDREWS, F. N. Speed of travel of ram spermatozoa. Anat.
Rec. 68:127,1937. A new method of measuring sperm speeds. Nature, London 171:512,1953. 5. ROTHSCIDLD, LORD, and SWANN, M. M. The fertilization reaction in the sea urchin egg: A propagated response to sperm reaction. J. Exper. BioI. 26:164, 1949. 6. VANDEMARK, N. L., and MOELLER, A. N. Speed of spermatozoan transport in reproductive tract of estrous cow. Am.]. Physiol. 165:674, 1951. 4. ROTHSCHILD, LORD.