Cornell University, Ithaca, New York

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1 VOLUME OF STRESSED BULL SPERMATOZOA AND PROTO- PLASMIC DROPLETS, AND THE RELATIONSHIP OF CELL SIZE TO MOTILITY AND FERTILITY 1 E STIMATES of bull sperm size have been summarized by Bahr and Zeitler (1964). Considerable variation has been reported, part of which is probably due to artifacts induced during killing of the sperm by fixation and staining while preparing them for microscopic estimation of size. Several studies concerned with sperm size, mass and properties of reflection (Beatty and Sharma, 1960; Leuchtenberger et al., 1956; Lindabl et al., 1952) have indicated differences among males. Electronic sizing (Glover, 1964; Iversen, 1965; Foote and Bredderman, 1969) offers the advantage of sizing directly a large sample of cells while they are exposed to a variety of experimental conditions. However, most studies have involved observations of size distributions without attempting to carefully quantitate the size changes or control the possible factors involved. The extensive swelling observed previously (Drevius and Eriksson, 1966; Foote and Bredderman, 1969) suggested that the capability of spermatozoa to respond to such a test condition as osmotarity of the medium might be used to characterize sperm cells from different bulls. Results of experiments designed to test this idea and relate size changes to other characteristics of semen samples and to fertility are presented in this report. Materials and Methods Two semen samples were obtained from each of 17 bulls in regular use at the Eastern Artificial Insemination Coop., Inc. Twelve of the bulls were ejaculated twice weekly. The other bulls were ejaculated weekly, excepting two ejaculates were obtained after 2 wk. of sexual rest. The percentage of motile spermatozoa and sperm concentration were determined within 10 min. after semen collection. A 0.5-ml. subsample was removed and stored at 20 ~ C, until it could be used for cell sizing. x Supported in part by a grant from Eastern Artificial Insemination Cboperative, Inc. 2 Department of Animal Science. P. J. BREDDERMAN AND R. H. FOOTE v Cornell University, Ithaca, New York 496 The remaining portion of the ejaculate was extended to a final concentration of 10 million motile sperm per milliliter, and used for insemination 1 to 2 days later after storage at 5~ Fertility was measured in terms of 60- to 90-day % nonreturns. A second estimate of each bull's fertility was obtained by including eight additional ejaculates per bull used for insemination just prior to or after the present experiment. To minimize contamination with particulate matter during electronic sizing, all glassware was scrupulously cleaned and rinsed several times with distilled water previously filtered through Millipore HA 0.45 /z diameter membrane filters. Treatment solutions shown in figure 1 were similarly filtered after preparation with distilled water. Otherwise, small contaminating particulate matter could have obscured the protoplasmic droplets. Solutions were stored at 5 o C. and brought to room temperature prior to use. The osmolarity of solutions 1, 2, 3 and 5, determined with an Advanced Instruments Osmometer, were 283, 298, 94 and 712 milliosmols, respectively. A model B Coulter Counter with a 50-~ sizing aperture and attached plotter were used as previously described (Foote and Bredderman, 1969). The aperture apparatus was modified to allow rapid replacement of the solution inside the aperture tube so that it always corresponded to the treatment solution. Polyvinyltoluene monodisperse latex particles with a diameter of ~ were used for calibration, and to determine the proper machine settings to achieve suitable amplification for all treatments. Instrument amplification in the hypotonic saline (treatments 3 and 4) was reduced by a factor of 0.769, so that the extensive cell swelling which occurred with live cells in this solution could be monitored and plotted. Appropriate corrections were applied to data for these treatments prior to the statistical analyses. Semen was added to each treatment solution to achieve a sperm concentration of 150,-

2 BULL SPERMATOZOA I >- u Z o.29% SALINE JKILLED SPERM) 9% TRIS (KILLED SPERM~ 6 SIZE DISTRIBUTION (WINDOW NUMBER) Figure 1. Typical X-Y plot of the six treatments for one of the 34 ejaculates studied. The field of measurement is divided into 25 windows each representing four percent of the field. Background counts in window 1 exceeded the scale used as shown by the flat top. Each successive window (peak) to the right within a graph represents increasing particle size. Graphs 4 and 6 compare distributions of sperm killed by freezing with live sperm in graphs 3 and 5 suspended in the same solutions. 000 cells per milliliter. The suspension was magnetically stirred for approximately 10 seconds and then sized immediately. The time from semen collection to initiation of sizing was 44 (S.D.) minutes. Unfrozen sperm in the various treatments were sized in rapid succession, requiring about 3 rain. each. Approximately 4,000 spermatozoa per treatment were sized. Motility of the undiluted semen was estimated at this time following warming to 35 ~ C. with a stage incubator. Sperm killed by freezing were sized similarly after the undiluted semen had been frozen and allowed to return to room temperature. Precautions were taken to rinse the inside and outside surfaces of the aperture tube and the electrodes between test solutions. On the few occasions when the aperture plugged, the sizing procedure was reinitiated on a freshly prepared cell suspension. Sperm cell volume distribution data from the X-Y plotter were punched on cards for computer processing after correcting data from treatments 3 and 4 for amplification differences. Two statistics of each cell volume distribution were computed: (1) the window representing the mean cell volume for the sperm population, and (2) the percentage of cells that had volumes below the upper border of normal cell volumes in isotonic tris-citric acid (window 14). For brevity only the mean cell volume statistic will be presented in this report, but reference will be made to the latter where distributions were decidedly skewed. This mean window represents the window corresponding to the mean cell volume calculated by summing all cell volumes and dividing by the number of cells. Results Protoplasmic Droplets. Figure 1 shows a typical frequency distribution obtained for each treatment with the X-Y plotter. The first window represents tiny debris and amplified noise in the background, and should be ignored. Treatments 1 and 2 have a distinct bimodal distribution. The mode to the left with a peak most often in window 4, corresponds to protoplasmic droplets shed from the

3 498 BREDDERMAN AND FOOTE sperm tails. This was verified by isolating the droplets centrifugally, examining them microscopically and sizing them. The peak frequency corresponds to a volume of 8.3 /~8, equivalent to the volume of a sphere 2.5~ in diameter. The droplets disappeared microscopically when placed in hypotonic saline (treatment 3) or following freezing and thawing (treatments 4 and 6). In the hypertonic medium of treatment 5, their distribution was markedly shifted to the left, presumably due to shrinkage in this medium. Thus, droplet distributions are distinctly visible only in treatments 1 and 2. Spermatozoa. The major distributions in the graphs shown in figure 1 represent spermatozoa. In treatment 1 sperm in window 12 were most frequent, a value close to the overall mean window of 12.1 for the 34 ejaculates studied. This mean window of 12.1 represents a mean volume of 25.2 /~3. This is based on the calibration with latex particles which showed that each window represented a mean volume increase of 2.08 ~3, with a calibration uncertainty range of 1.93 to 2.25 /z ~. This uncertainty is caused by the 4% width of each of the 25 windows (Iversen, 1965). Therefore, the mean volume of 25.2 ~a for treatment 1 was calculated to be within the range from 23.4 to 27.2 ~a, (12.1X1.93 to 12.1X2.25). The distributions were similar in graphs 1 and 2, where the media were nearly iso-osmotic. In graph 3 a portion of the spermatozoa in hypotonic saline have swollen considerably. After adjusting for amplification some cells appear to have volumes greater than those in window 25 (68/~3). The remainder of the cells in this graph have not changed their volume appreciably, and correspond in size with the smaller sperm cells in physiological saline (treatment 1). Because of the tendency of some sperm cells to swell in a hypo-osmotic medium, leading to a bimodal distribution, the mean window volume statistic does not always reflect accurately the nature of the cell volume changes. A measure of skewness or characterization of two populations would be helpful. In the hypertonic medium of treatment 5 cells appear to have shrunken into a relatively uniformsized population. Killed sperm (treatments 4 and 6) also are uniformly distributed with a reduced volume. Table 1 summarizes the findings for the 34 ejaculates, and it can be seen from the mean window sizes presented that they are similar to the ejaculate illustrated in figure 1. Overall, 86% of the sperm cells in isotonic tris (treatment 2) were below window 14, whereas only 49% of the cells were below this window in hypotonic saline (treatment 3). The large bull effect is present in most of the variables studied. However, the freezing and thawing of sperm without protective agents eliminated bull differences. Standard analysis of variance could not be applied to the sperm volume data because of the heterogeneity of error variances. Arcsin transformation did not eliminate the heterogeneity. However, it was possible to make paired comparisons of treatment means containing orthogonal sets. Mean sperm volume in treatments 1 vs. 2, 3 vs. 4 and 5 vs. 6 all differed (P~.01). The size of frozen sperm in hypo- and hypertonic solutions 4 and 6 no longer differed, although size differences were TABLE 1. SUMMARY OF SEMEN QUALITY, SPERM SIZE CHANGES AND STATISTICAL ANALYSES Bull effects % of total variance Variable Mean S.D.~ Significance due to bulls Sperm conc., 10~/mI P~ % motile after sizing P ~ day % nonreturns 2 ejaculates N.S ejaculates P~ Mean window size: 1. Iso. saline P~ Iso. tris P( Hypo. saline P~ Hypo. saline-frozen sperm N.S Hyper. tris P~ Hyper. tris-frozen sperm N.S. 0 a S.D.:over-all standard deviation.

4 BULL SPERMATOZOA TABLE 2. SIMPLE BULL MEAN CORRELATION COEFFICIENTS day%N.R. Size in different treatmen~ % Variable motile 2 ejac. 10 ejac Sperm concentration % motile after sizing day % N.R., 2 ejac ~~ day % N.R., 10 ejac Size in 1, iso. saline Size in 2, iso. tris Size in 3, hypo. saline Size in 4, hypo. salinefrozen semen Size in 5, hyper, tris Size in 6, hyper, trisfrozen semen P<.05. ~'~ P< ~ ~ 0.57 ~ " ~ " ~ ~ ~ large prior to freezing in these solutions (treatments 3 and 5, respectively). The relationships among the different variables on a bull mean basis are shown by the simple correlations in table 2. Note that sperm volume in the four treatments in which live sperm were used were all significantly correlated with sperm cell motility at the time of sizing. Treatments using killed sperm yielded no statistically significant correlations, as would be expected from the lack of significant bull effects in these two treatments (table 1). Size in iso-osmotic saline was highly correlated with size in iso-osmotic and hyperosmotic tris. Size in iso-osmotic and hyperosmotic tris were also correlated. Various other criteria of semen quality were not correlated, excepting for the two measurements of fertility. Discussion Little attention has been given to the osmotic behavior of protoplasmic droplets. The present studies confirm others reported from this laboratory (Foote and Bredderman, 1969) that there are a large number of protoplasmic droplets in normal ejaculates of bull semen which can be detected by electronic sizing. With their membranous covering, as seen with the electron microscope (Bloom and Nicander, 1961), it is not surprising that the droplets are osmotically reactive (Drevius and Eriksson, 1966). They shrink in hypertonic media and apparently swell and burst in hypotonic media. Debris resembling ghosts was seen microscopically in the present study following hypotonic treatment or freezing. It is likely that a variety of chemical substances would be released upon fragmentation. Thus, in chemical studies of seminal piasma and spermatozoa care should be taken to isolate the droplets. Spermatozoa suspended in iso-osmotic solu- tions, presumed to be approximately isotonic for spermatozoa, resulted in sperm size distributions which are somewhat skewed in the direction of large size. This finding is consistent with fixed preparations in which mass and other parameters do not show a normal or Gaussian distribution (Bahr and Zeitler, 1964; van Duijn, 1960), but show instead a logarithmic-normal distribution. In iso-osmotic media most sperm cells appear to be closer to a fixed minimum volume than to their maximum possible volume. This is substantiated by the considerable increase in size sperm are capable of undergoing in hypotonic solutions compared with the limited decrease in size observed in hypertonic solutions. When hypertonic tris solution was saturated with sucrose, in an experiment not reported, it caused only a slight additional decrease in cell volume. The volume of sperm cells in the unswollen state ranged from about 15 to 32 ~3. This is similar to previous volume measurements obtained for bull spermatozoa by electronic sizing (Glover, 1964; Iversen, 1965; Foote and Bredderman, 1969). Thus, some of the earlier measurements of bull sperm size, which ranged as high as 80 /z a, appear to be gross over-estimates. This is further substantiated by studies of packed cell volume (Foote, 1958) which revealed that an average spermatozoon plus any droplets and intercellular space occupied less than 50 ~3. Suspension of spermatozoa in hypotonic saline (figure 1, treatment 3) generally led to a distinctly bimodal distribution in which some cells had swollen to more than double their normal volume. Maximum recorded cell volumes were about 68 u3, but many cells assumed volumes greater than this. The magnitude of this sweiling is similar to that reported by Drevius and Eriksson (1966) for spermatozoa examined microscopically following suspension in hypotonic solutions and for sper-

5 500 BREDDERMAN AND FOOTE matozoa aged in their own seminal plasma (Foote and Bredderman, 1969). Thus, these results do not support earlier reports (Anderson, 1945; Pursley and Herman, 1950; Rothschild, 1959) which suggested that spermatozoa are incapable of swelling. Motile sperm in hypotonic solution had coiled or hooked tails (Anderson, 1945; Pursley and Herman, 1950; Lindahl and Drevius, 1964; Steinbach and Foote, 1967). The dead spermatozoa had straight tails associated with cell lysis and death. This was accompanied by a decrease in volume and perhaps a loss in intracellular solute (Mann, 1951). These findings are in accord with the observations made by Lindahl and Drevius (1964) and Drevius and Eriksson (1966), utilizing phase and electron microscopy, and with the significant positive correlation of cell size with motility in the present study with a hypotonic solution. Unpublished observations in this laboratory indicate a negative relationship between size and motility of sperm stored in iso-osmotic media. Therefore, the negative correlations between motility and size of sperm in isoosmotic media observed in this study suggest that within 45 rain. of ejaculation changes in the metabolic state already are reflected in changes in cell volume. A high energy level appears to allow the cell to maintain size excepting in the hypotonic solution. In the latter situation the positive correlation coefficient (table 2) is interpreted as representing the ability of highly motile cells to swell without early lysis and subsequent collapse. Spermatozoa killed by freezing and thawing lost their ability to swell. Studies by Saacke and Almquist (1961) suggest that the cytoplasmic membrane would be disrupted by this procedure and therefore are unable to support osmotic swelling. Bulls were a major source of variation in most of the variables excepting when spermatozoa had been killed (table 1). Ejaculates within bulls showed similarities in their size distributions and swelling responses which were characteristic of bulls. However, the mean window size was not significantly correlated with fertility, although the correlation with swelling response in hypotonic saline approached statistical significance (table 2). Further characterization of this size distribution on ejaculates used extensively for breeding is desirable to determine whether or not it might have predictive value for selecting individual ejaculates. Lindahl et al. (1952) suggested that size-related reflecting properties of bull spermatozoa were correlated with fertility. Other studies concerning sperm area or mass and fertility are equivocal (Leuchtenberger, 1956; Beatty and Sharma, 1960). However, the swelling ability of spermatozoa may be of greater value than their initial size as a predictor of fertility. These studies provide additional evidence of the feasibility of sizing spermatozoa electronically and measuring changes under physiological conditions. This technique appears to offer a means of determining the tonicity of a medium as distinguished from its osmotic pressure. It is useful as an additional tool for evaluating the functional integrity of the sperm cell. Studies to evaluate this technique as an indicator of freezing damage (Persidy et al., 1967), are in progress. Summary An electronic sizer (Coulter Counter) was utilized to determine the effects of four solutions of different osmolarities on cell volume of spermatozoa from two ejaculates from each of 17 bulls. Spermatozoa showed a decrease in volume in the hypertonic medium. A portion of the population of cells showed a marked increase in cell volume in the hypotonic medium. This increase was associated with live cells. Sperm cells killed by freezing and thawing did not show osmotic behavior. The absolute mean volumes for spermatozoa at the time of measurement in iso-osmotic saline or Tris, hypo-osmotic saline, hypo-osmotic saline with killed spermatozoa, hyperosmotic Tris and hyperosmotic Tris with killed spermatozoa were estimated to be 25.2, 23.1, 32.4, 20.8, 20.0 and 20.4 poa, respectively. Since dead cells change little, swelling of live cells can be much greater than mean values indicate. These results clearly show the need to control osmolarity in studies of sperm cell size. Protoplasmic droplets were found to be osmotically reactive. Their mean volume in 0.87% saline was estimated to be 8.3 ~a. The droplets appeared to rupture in hypo-osmotic media or upon freezing, leaving only debris visible microscopically. Thus, they may contribute materials to seminal plasma, which could alter its composition, unless the droplets are removed promptly after ejaculation. Bulls contribtued the major source of variation excepting in treatments in which the spermatozoa were frozen before sizing. The significant bull effect in the four sizing media was

6 BULL SPERMATOZOA 501 significantly correlated with the motility at the time of sizing, but not with the bulls' fertility. Literature Cited Anderson, J Semen of Animals and its use for Artificial Insemination. Imperial Bur. An. Breed. and Genetics, Edinburgh, Scotland. Bahr, G. F. and E. Zeitler, Study of bull spermatozoa. J. Cell Biol. 21:175. Beatty, R. A. and K. N. Sharma Genetics of gametes. III. Strain differences in spermatozoa from eight inbred strains of mice. Proc. Roy. Soc. Edinburgh, B, 58:25. Bloom, G. and L. Nicander On the ultrastructure and development of the protoplasmic droplet of spermatozoa. Zeitschrift Zellforschung 55:833. Drevius, L. O. and H. Eriksson Osmotic swelling of mammalian chromosomes. Exp. Cell Res. 42: 136. Foote, R. H Estimation of bull sperm concentration by packed cell volume. J. Dairy Sci. 41: Foote, R. H. and P. J. Bredderman Sizing of aging bull spermatozoa with an electronic counter. J. Dairy Sci. 52:117. Glover, F. A The size distribution of spermatozoa in bull semen. Vth Intern. Congr. An. Reprod., Trento, IV:587. Iversen, S Volume of untreated and ultrasonically :treated bull, boar and human spermatozoa electronically determined. J. Reprod. Fertil. 9:197. Leuchtenberger, C., I. Murmanis, L. Murmanis, S. Ito and D. R. Weir Interferometric dry mass and microspectrophotometric arginine determinations on bull sperm nuclei with normal and abnormal DNA content. Chromosoma 8:73. Lindahl, P. E. and L. O. Drevius Observations on bull spermatozoa in a hypotonic medium related to sperm motility mechanisms. Exp. Cell Res. 36:632. Lindabl, P. E., J. E. Kihlstrom and B. Strom On the relationship between fertility and light reflecting power in bull spermatozoa. J. Agr. Sci. 42: 184. Mann, T Studies on metabolism of semen. 7. Cytochrome in human spermatozoa. Biochem. J. 48:386. Persidy, M. D., V. Richards and J. Leef Volume changes in bone marrow and Ehrlich ascites cells after freezing as an index of preservation efficiency. Cryobiology 3:59. Pursley, G. R. and H. A. Herman Some effects of hypertonic and hypotonic solutions on the livability and morphology of bovine spermatozoa. J. Dairy Sci. 33:220. Rothschild, L Anaerobic heat production of bull spermatozoa. II. The effects of changes in the colligative and other properties of the suspending medium. Proc. Roy. Soc. (London) B; 151:1. Saacke, R. G. and J. O. Almquist Freeze-drying of bovine spermatozoa. Nature 192:995. Steinbach, J. and R. H. Foote Osmotic pressure and ph effects on survival of frozen bovine spermatozoa. J. Dairy Sci. 50:205. van Duijn, C., Jr Mensuration of the heads of bull spermatozoa. Mikroscopie 14:265.