In Vitro Speeds of Bovine Spermatozoa

Transcription

1 In Vitro Speeds of Bovine Spermatozoa A. N. Moeller, M.S., and N. l. VanDemark, Ph.D. THE RATE OF progressive movement of the spermatozoa has been used as one criterion in physiologic studies for evaluation of semen both initially upon ejaculation and after storage or treatment. In most instances the rate of spermatozoan travel is determined by visual estimation with the aid of a microscope, and a rating is then assigned according to some arbitrary scale. Little information has been reported concerning actual individual spermatozoan speeds in vitro. Adolphi reported the average speed of spermatozoa as 4.02 mm. per minute for bull spermatozoa, 3.00 mm. per minute for ram, and 2.58 mm. per minute for dog spermatozoa. Walton used a micrometer eyepiece and a stopwatch in determining the rates of travel by sea-urchin spermatozoa. However, the semen was under the influence of an electrical field and thus the 18"" per seconds reported was not a measure for free-swimming spermatozoa. Grave and Downing reported on some observations of sea-urchin spermatozoa traveling along a capillary tube, but no specific rates of travel were given. A more recent study was made by Rothschild and Schwann,7 who used a time-exposure photographic method to determine that the rate of travel for sea-urchin spermatozoa was approximately 190"" per second. Lamar, Shettles, and Delfs reported a rate of travel of 3 mm. per minute for human spermatozoa in a capillary tube containing cervical mucus. Yamane and Ito also used a capillary-tube method in their study and reported rates of travel for stallion spermatozoa ranging from 87 to 200"" per second. Phillips and Andrews reported an average rate of travel of 4.83 mm. From the Department of Dairy Science, University of Illinois, Urbana, Ill. The authors wish to thank G. W. Salisbury for his encouragement and A. Van Tienhoven for assistance in the experimental determinations. 506

2 Vol. 6, No.6, 1955 BOVINE SPERMATOZOA SPEEDS 507 per minute for ram spermatozoa, with rates up to 15 mm. per minute in the first portion of the capillary tube. Rothschild, 6 in a preliminary report, indicated that the mean speed of bull spermatozoa was 117 p. per second. Using a photographic method and employing the probability-after-eflect principle, the same author reported a rate of travel for bull spermatozoa at 11lp. per second.6 He compared 6 this method to a photograph matching method giving a rate of l23p. per second. The fastest spermatozoon observed traveled at a rate of 250p. per second. Thus the reports on actual sperm speeds to date are based on a very limited number of spermatozoa or ejaculates, and in general the methods reported are too time-consuming for routine use in either the experimental laboratory or the practical field situation. This paper reports the results of an attempt to rapidly assess spermatozoan speeds in vitro by clocking their travel over a measured distance under the microscope. MATERIAL AND METHOD In an attempt to obtain a quick estimate of the rate of travel by an individual spermatozoon and a mean rate for all active spermatozoa in an ejaculate, the time required for each of several spermatozoa to travel a given distance was determined. The time interval was recorded to the nearest %0 of a second with a stopwatch. A lo-power micrometer eyepiece, with a 100-unit scale, was calibrated by means of a hemacytometer field to obtain the distance traveled. The calibration factor was 6.95p. per unit for the 10- power objective and 3.l6p. per unit with the 20-power objective. Preparation of Semen The ejaculates used in this study were obtained from the regular herd bulls maintained at the University of Illinois. All observations were completed within 30 minutes of the time the semen was ejaculated. The semen was prepared for examination by placing a small drop on a warmed microscope slide and dispersing the spermatozoa in a drop of 0.9% saline. A coverslip was placed over the drop and gently pressed down. Thus a thin layer of semen was created which restricted the sperm movement largely to one ~ plane. The slide was held at 38 C. for observation. Speed Determination The time required for each of 10 individual sperm cells to travel the

3 508 MOELLER & VanDEMARK Fertility & Sterility length of the 100-unit scale was recorded. The first 10 sperm cells that could be aligned with the long axis of the scale constituted the group that was timed. Four similar groups of 10 spermatozoa were clocked within consecutive 45-second periods for each of 24 ejaculates. All observations within each group of 10 spermatozoa were made with the same objective power. However, the sequence of powers within each series of 4 groups was changed so that every possible sequence was used in observing the 24 ejaculates. The mean speed of the 10 spermatozoa in each group was used as the estimate of the mean speed of all active spermatozoa in the sample during that 45-second period of observation. Another investigator, using a visual estimation method, assigned to each semen preparation an initial rating for "per cent motile" spermatozoa and rate of travel of the spermatozoa. The percentage ratings were based on the commonly used scale of per cent motile spermatozoa with 10 percentage unit intervals. The speed of travel ratings were based on a scale ranging from 0, indicating no progressive movement, to 4.0, indicating excellent progressive movement, with 0.5 unit intervals for the intermediate estimated speeds of travel. RESULTS The measured speed of the 960 individually observed motile spermatozoa in the 24 ejaculates under the conditions of this study ranged from 10 to 352p. per second, with an average rate of 114p. per second. The mean speed of all 40 spermatozoa in each ejaculate ranged from 32 to 226p. per second. The mean speed of each group of 10 spermatozoa varied from 27 to 254p. per second. The mean speeds in microns per second for each group of 10 spermatozoa observed under the 10- or 20-power microscope objectives and during the consecutive 45-second periods are presented in Table 1. The mean speeds of all spermatozoa observed at the X 100 and the X 200 magnifications were and 111.0p. per second. Differences in Speed An analysis of variance revealed that the differences in the mean speeds of the groups of 10 spermatozoa were significantly greater (0.01 level) for the spermatozoa from different ejaculates than for the spermatozoa from the same ejaculate (Table 2). Also there was as much variation in mean

4 Vol. 6, No.6, 1955 BOVINE SPERMATOZOA SPEEDS 509 speeds between ejaculates from the same bull as there was between ejaculates from different bulls. The differences in mean speeds between those spermatozoa observed under a 10- or 20-power objective were significant TABLE 1. Mean Speed of Sperm Travel (p./sec.) Interval from [;tart of observation (sec.) ~ X ± 43.5a ± ± ± ±45.4 X ± ± ± ± ± 48.4 All ± ± ± ± ± 45.1 a Standard deviation from the mean. (0.01 level). The sperm speeds became significantly lower (0.01 level) with an increase in the length of time from slide preparation to observation. The mean speeds in microns per second were 123.8, 120.3, 113.2, and for the consecutive periods of 0-45, 45-90, , and seconds, re- All TABLE 2. Summary of Analysis of Variance. Degrees at Source freedom Mean square Total 95 Between ejaculatea 23 Between bullsb 9 Within bulls 14 Within ejaculates 72 Powersc 1 Periodsc 3 P 1 VS. P 2 c 1 P 1 + P 2 VS. Pac 1 P1 + P2 + Pa VS. p 4 c 1 Error 68 ~ a Mean square tested by the within ejaculate mean square. b Mean square tested by the within bulls mean square. c Mean squares tested by the error mean square. " Significant at the 0.01 level. 8251" " 2626" a 6485" 118 spectively. Orthogonal comparisons show that there was no significant difference between the first and second period. However, the mean speeds were significantly lower (0.01 level) in periods 3 and 4 than in the preceding periods. Table 2 summarizes the analysis of variance.

5 510 MOELLER & VanDEMARK Fertility & Sterility There was no significant correlation between the mean speed as determined by the clocking speed method and the estimated percentage of motility of the spermatozoa or the original sperm concentration, as the correlation coefficients were 0.06 and 0.04, respectively. Likewise there was no significant correlation between the estimated ratings and the estimated percentage of motility or the original concentration. The correlation coefficients were 0.04 and 0.18, respectively. DISCUSSION AND CONCLUSIONS In comparing the two methods of differentiating between ejaculates on the basis of speed of spermatozoan travel in vitro, the correlation coefficient of 0.05 was not significant. In Table 3 it is seen that the upper half of the TABLE 3. Comparative Rates of Sperm Travel Number of ejaculates Estimated rating Clocked speed in p, per second Meana Rangeb Fastest sperm a Mean speeds of all 40 spennatozoa in each ejaculate. b The range in mean speeds of all 40 spermatozoa in each ejaculate. mean speeds were all grouped in only the top estimated rating assigned. Also there was as great a range in microns per second within the 2.0- through-3.s assigned ratings as between them. There is an indication that the number of spermatozoa present may tend to influence the estimated ratings as the correlation coefficient between estimated rating and original sperm concentration was 0.18 as compared to 0.04 between the clocking speed and original concentration. The data show a wide variation in speeds between spermatozoa within the same ejaculate. Since these great differences exist, it may be of value to determine the individual sperm speeds in physiologic or metabolic studies of semen. Also since the mean speeds for the ejaculates were significantly different, it appears feasible to differentiate between ejaculates or experimental samples of semen on a basis of sperm speed.

6 Vol. 6. No BOVINE SPERMATOZOA SPEEDS 511 Clocking Speed Method The clocking speed method of determining sperm travel has its limitations. The major limitation is the human bias present in the selection of spermatozoa to be measured, reaction times in starting and stopping the watch, and in maneuvering the micrometer eyepiece. Also the spermatozoa may not travel continuously in one plane; thus the speeds might be variable even for an individual spermatozoon. However, in all probability, some of these limitations can be minimized by refinement in technic. On the other hand, the clocking speed method does have some distinct advantageous features. It is simple, quick, and inexpensive. Speeds of individual spermatozoa are easily determinable as well as a mean speed with a standard deviation for an ejaculate. The clocking speed method can be used on a wide range of sample conditions, being limited only to the extent of being able to distinguish individual spermatozoon action. The results obtained are in terms of an objective measure of units of distance during a period of time. The results are not as dependent upon the skill and judgment of an investigator as in estimation methods. In view of these facts the results reported by different workers should prove to be on a much more comparable basis. Thus, the clocking speed method of determining spermatozoon speeds in vitro may be a useful tool especially in semen investigations in the laboratory..., SUMMARY With the aid of a micrometer eyepiece and a stopwatch, a quick measurement of the speeds of spermatozoon travel was made. Groups of 10 spermatozoa were measured under X 100 and X 200 magnification during consecutive 45-second periods for each of 24 ejaculates. The mean speed of the 960 spermatozoa observed was 114ft per second, with a range from 10 to 352ft per second. The mean spermatozoan speeds for ejaculates varied from 32 to 226ft per second. A statistical analysis revealed that the differences between the speeds of spermatozoa from different ejaculates were significantly greater (0.01 level) than the differences between the speeds of sperm cells from the same ejaculate. There was as much variation between ejaculates from the same bull as between ejaculates from different bulls. There was a significant (0.01 level) difference in speeds between those spermatozoa observed at X 100 and

7 .., 512 MOELLER & VanDEMARK Fertility & Sterility X 200 magnification. The mean speed declined significantly (0.01 level) with an increase in the length of time between preparation of a slide and observation. There was no significant correlation between the measured speed and an estimated rate of travel, the estimated "per cent motile" of spermatozoa, or the concentration of spermatozoa in the ejaculate. The clocking speed method to determine either a mean or individual spermatozoan speed may be a more useful tool in semen studies than methods using visual estimation only. REFERENCES 1. AnoLPHI, H. Die Spermatozoen der Saugetiere Schwimmen gegen den Strom. Anat. Anz. 26:549, GRAVE, B. H., and DOWNING, R. C. The longevity and swimming ability of spermatozoa. ]. Exper. Zool. 51 :383, LAMAR, J. K., SHETTLES, L. B., and DELFS, E. Cyclic penetration of human cervical mucus to spermatozoa in Vitro. Am. J. Physiol. 129:234, PHILLIPS, R. W., and ANDREWS, F. N. The speed of travel of the ram spermatozoa. Anat. Rec. 68: 127, ROTHSCHILD, LORD A new method of measuring sperm speeds. Nature, London 171 :512, ROTHSCHILD, LORD A new method of measuring the activity of spermatozoa. ]. Exper. Biol. 30:178, ROTHSCHILD, LORD, and SCHWANN, M. M. The fertilization reaction in the sea urchin egg: A propagated response to sperm reaction. ]. Exper. Biol. 26: 164, WALTON, A. Studies on the physiology of reproduction: 1. The flocculation of sperm suspensions in relation to surface charge. Brit. J. Exper. Biol. 2:12, YAMANE, J., and ITo, T. Uber die Geschwindigkeit der Pferdespermatozoen in Stromenden und Nichtstromenden Flussigkeiten. Cytologia 3: 188,