Inhibition of sperm motility does not affect live-dead separation of bull sperm by glass beads

Robert H. Foote

Department of Animal Science, Cornell University, Ithaca, NY 14853-4801 , USA

Asian J Androl  2001 Sep; 3: 193-198

Keywords:  sperm motility; glass sphere filters; silicone; fluorides; inhibition

Abstract

Aim: This study was designed to explore factors which influence binding of dead versus live sperm to glass filters. Methods: Multiple semen collections from bulls were used to explore selective filtration of bull sperm as influenced by nonlethal inhibition of sperm motility with fluoride, killing of sperm by quick-freezing, alteration of the glass surface with silicone, and different intervals of sexual rest between semen collections. Results: A comparison of glass spheres100, 200 and 390 m in diameter indicated that 200 m spheres were optimal for selective filtration.  Quantitative separation of live from dead sperm was demonstrated with a correlation between the percentage of motile sperm and retention of sperm by the filter ofr = -0.87 (P < 0.05).  Up to 0.02 mol/L NaFl did not alter the proportion of sperm retained by the filter despite inhibiting sperm motility during filtration, an inhibition which was reversible.  Proportions of live-dead sperm, based upon eosin staining, were unaffected by fluoride.  Coating the glass spheres with silicone greatly reduced selective filtration.  Dead sperm adherence to glass was reduced and resistance to NaFl inhibition was increased by daily ejaculation versus one-week intervals of sexual rest. Conclusion: These studies indicate that the adherence of sperm to glass is primarily due to some form of physico-chemical change accompanying death of the sperm cell independent of active sperm motility.  This attraction between the sperm plasma membrane and glass is modified by the age of the ejaculated sperm.  This information is useful in evaluating different clinical procedures used for sperm separation.

1 Introduction

Dead sperm cells especially have been observed for many years to stick to glass during processing in solutions devoid of organic material.  This can be overcome by adding albumen, egg yolk, milk, or other solutions with macromolecules used for diluting semen.  Bangham and Hancock^[1] and Campbell et al^[2] made use of this observation to separate live from dead bull sperm.  They found that eosinophil stained (dead sperm) primarily adhered to glass spheres in a glass bead filter bed.

Graham and Pace^[3] and Graham et al^[4] used glass fibers to remove dead sperm and other material from bull, boar and turkey sperm.  Paulson and Polakoski^[5] used glass fibers to remove nonmotile sperm and debris from human semen.  Although glass wool filtration of sperm may have a deleterious effect on the ultrastructure of sperm^[6], this filtration procedure has been used extensively as a simple and efficient procedure to increase the proportion of normal motile human sperm^[7-12].

Recently Chandler et al^[13] reported that bull sperm differing in head size could be separated by passing the sperm through a glass sphere bed containing layers of spheres differing in size.  This size difference was presumably associated with sperm containing the X or Y chromosome.

2 Materials and methods

Semen was collected from bulls at the local artificial breeding organization. Generally they were collected twice per week (Experiments 1 to 3), but weekly versus daily semen collections were compared in Experiment 4.  Immediately upon semen collection an aliquot of semen was diluted on a slide, placed on a stage incubator at 37 , and the percentage of progressively motile sperm displayed through a TV monitor connected to the microscope was estimated in several fields.  The sperm concentration was estimated by optical density using a spectrophotometer calibrated with a Coulter Counter.  A portion of each ejaculate was removed and quickly frozen twice in liquid nitrogen to provide dead sperm when it was desired to systematically increase the proportion of dead sperm in the test sample.

The semen was diluted in the various experiments with a buffer for bull sperm used regularly in our laboratory.  It consisted of 8.30 g of NaCl, 0.20 g of KCl; 0.50 g of NaHCO₃, 1.00 g of NaH₂PO₄, and 1.00 g of glucose per 1 L of double distilled water.  When 10 ejaculates of bull sperm were stored at 25 for 8 h they retained 88 %9 % of their initial progressive motility in this buffer compared to 58 %8 % in the buffered-saline used by Bangham and Hancock^[1].  As all experiments were conducted in the laboratory at approximately 25 , the excellent maintenance of progressively motile sperm minimized any effects of time during the short period between semen collection and completion of a test on the ejaculate of semen.

Glass spheres with diameters of 100 m, 200 m and 390 m were obtained from Minnesota Mining and Manufacturing Co. (St. Paul, MN).  They were washed with cleaning solution and rinsed extensively with double distilled water before use.  Details of the glass filter bed are given for each experiment.

Experiment 1.   Optimal characteristics of the filter bed

Experiment 2.  Effect of adding killed sperm on separation

Experiment 3.  Effect of immobilizing sperm with NaFl

This experiment was designed to test the effect of reversible immobilization of live sperm upon the separation of live from dead sperm.  Sodium fluoride was used because of its well-established effects on reversible inhibition of sperm motility (Mann)^[14], it does not alter the plasma membrane and it is readily available.  Semen from three bulls was diluted to 2010⁶ sperm/mL with buffer containing 0.0, 0.002, 0.01, and 0.02 mol/L NaFl.  These treatments were compared with sperm killed by freezing and diluted with buffer containing 0 and 0.02 mol/L NaFl. The filtration conditions were the same as for Experiment 2.  The percentage of progressively motile sperm was determined before and after filtration, and after washing the filtered sperm by centrifugation and resuspending them in the control buffer.  Sperm concentration before and after filtration was measured.  A subset of samples was stained with eosin-nigrosin as a check of membrane integrity.

The experiment was replicated three times.  Based upon these replicates the results were confirmed with eight additional samples of fresh semen suspended in buffer containing 0.0 and 0.01 mol/L NaFl.

In this study bulls were placed on a daily semen collection schedule.  After an equilibration period, 13 ejaculates of semen were collected from bulls with 1 d of sexual rest and compared with 13 ejaculates of semen from bulls on a weekly collection schedule.  The two groups of bulls were selected to be equal based upon their past history of semen quality.  The same filter arrangements and NaFl used previously were included.  Also, the glass spheres were treated with silicone, S-200 (Dow Corning Corporation, Midland, MI).  A 2 % (vol/vol) solution of silicone in methylene chloride was used to immerse the beads.  They were then spread in a very thin uniform layer over a tray in an oven to dry.  Inspection of the treated beads revealed some surface irregularities where the beads touched the surface during drying.  The treated beads were washed with double distilled water and dried before use.

Ten mL of appropriate buffer containing 2010⁶ of fresh sperm or 2010⁶  of killed sperm were filtered through the control and treated beads.  The percentages of motile sperm before and after filtration and the percentages of sperm retained by the filter were determined.

3 Results

The results for Experiment 1 are summarized in Table 1.  The most noticeable effect was the difference in the retention of sperm by the different sizes of glass spheres.  Overall the decrease in sperm in the filtrate from the 100, 200 and 390 m glass spheres was, respectively, 47, 22 and 11 % (P<0.05).  A visual check of the spheres revealed that many motile sperm were retained by the 100 m glass spheres.  The 390 m diameter spheres allowed most of the sperm to pass with little increase in the fraction of motile sperm.  The optimal size sphere, among the sizes tested, was the 200 m diameter spheres with a substantial increase in motile sperm in the filtrate.  These spheres, with a 4 mL filter bed, were used in subsequent experiments.

Table 1.  Effect of bead diameter and volume of the filter bed on sperm filtration in Experiment 1.

In Experiment 2 the mixtures of fresh sperm and dead sperm produced samples with progressively motile sperm that followed closely the expected values.  The fractional removal of the sperm by the filter was inversely proportional to the percentage of motile sperm (Figure 1).  The correlation between these two variables, based upon the individual values for the 10 bulls, was r=-0.87 (P<0.05), in dicating that the glass spheres selectively retained the nonmotile (dead) sperm quantitatively.

In Experiment 3 (Table 2) NaFl was found to inhibit sperm motility in a dose dependent manner, as expected.  The progressive motility of sperm was largely restored following resuspension of the sperm in the control buffer solution.  A live-dead stain check of sperm exposed briefly to NaFl during filtration revealed that a high proportion (82%) of NaFl-exposed sperm remained unstained by eosin versus 83 % for controls.  There was no effect (P>0.05) of NaFl on the efficiency with which sperm were retained by the glass sphere filter, indicating that motility itself was not a factor in sperm retention.  However, killing the sperm resulted in most sperm being retained by the glass spheres.

Table 2.   The effect of NaFl on sperm motility and filtration in Experiment 3.

^a,b,c Onlymeans within a column with different superscripts differ, P<0.05.

In a simplified followup study with eight semen samples suspended in 0.0 and 0.01 mol/L NaFl the fractions of the sperm retained in the filter were 24 and 23 %, respectively (P>0.05), confirming that NaFl inhibition of motility had no effect on retention.

Table 3.   The effect of frequency of semen collection and silicone treatment of glass beads on filtration with and without NaFl in Experiment 4.

^aAll means are based on n=13 ejaculates of semen.
^b,cColumn means of % motile sperm with different superscripts differ, P<0.05.
^A,B,CColumn means of % retained sperm with different superscripts differ, P<0.05.

In the followup check, sperm from bulls ejaculated daily and exposed to 0.0, 0.01 and 0.02 mol/L NaFl for 10 min contained 62%, 52% and 20% motile sperm.  Respective values for sperm collected weekly were 59, 26 and 6% (P<0.05).

4 Discussion

Muller^[15] recently has reviewed many functional tests of semen quality.  Numerous systems for sperm separation, such as glass wool fibers, sedimentation through various types of gradients and swimup have been shown to improve the quality of sperm obtained.  The original studies with glass spheres^[1,2] were modified by using glass fibers^[3,4] for use with bull, boar and turkey sperm.  Paulson and Polakoski^[5] adopted the procedure for separating human sperm.  Numerous papers followed describing the importance of fiber type, tightness of packing and elimination of loose fibers on the efficiency of the system^[7-12].  The simple glass fiber filter generally gave a higher recovery of live sperm separated from the dead sperm retained in the filter than did other procedures.

In the present study the objective was to study factors affecting adhesion of the sperm to glass rather than to optimize a filtration system.  The glass spheres, similar to ones used originally by Bangham and Hancock^[1] were more suitable than glass fibers in treating the glass with silicone and in maintaining filter beds of uniform packing during the several experiments.

The results of Experiment 1 (Table 1) with different sizes of glass spheres indicated that surface area relative to the number of sperm added and compactness of the spheres affected the efficiency of filtration.  The 100 m diameter beads retained many dead sperm, and microscopic examination of beads in the filter bed suggested that there was some clogging.  This prevented some motile sperm from traversing the filter bed.  The 200 m diameter glass spheres were the most effective overall in allowing sperm passage while retaining more dead sperm in the filter.  This sphere was the same size used by Bangham and Hancock^[1].  This size of glass sphere was found in Experiment 2 (Figure 1) to quantitatively selectively remove the nonmotile sperm in the samples of fresh semen as well as the dead sperm added after killing a proportion of sperm from the same ejaculates.

It was of interest to determine if the mechanical motion of motile sperm had any influence on sperm adherence to the glass, or whether adherence was totally due to a surface change in the plasma membrane when the sperm died.  Sodium fluoride was used to reversibly inhibit sperm motility in Experiment 3 (Table 2).  The sperm exposed to NaFl were immobilized during filtration, but motility was largely restored following resuspension of the sperm in fluoride-free buffer.  Also, a check of the permeability of the plasma membrane to eosin following sperm suspension in NaFl solutions revealed no change.  The results from this experiment, and verified in Experiment 4, demonstrated conclusively that the mechanical motility of the sperm cells had no effect on their ability to pass through the filter bed, but rather it appeared to be a function of adherence by the plasma membrane to the glass surface.

To examine further this adherence by dead sperm to glass, an attempt was made to alter the surface of the glass spheres by coating them with silicone (Table 3).  Also, a possible affect of the in vivo aging of sperm on sperm surface properties was tested by obtaining ejaculates on a daily versus weekly basis.  The aging of the sperm did affect retention by control spheres especially of the killed sperm (P<0.05).  Thus, a factor in addition to the live or dead state of the sperm influenced the adherence to the plain spheres.  The greater retention of sperm in semen collected weekly also was associated with the fact that these cells were more susceptible to suppression of motility when exposed to NaFl.  Thus, the sperm obtained daily appeared to be overall more vigorous and resistant to any insult imposed in this experiment.

The silicone treatment allowed more sperm to pass through the filter, as expected. The silicone treatment eliminated the differential effect of frequency of collection of the semen on retention of sperm by the filter bed.

Acknowledgements

Technical help by C. Rude and with manuscript preparation by D. Bevins is appreciated.

References

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[2] Campbell RC, Hancock JL, Shaw IG.  Cytological characteristics and fertilizing capacity of bull spermatozoa.  J Agric Sci 1960; 55:  91-9.
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[4] Graham EF, Schmehl MKL, Evenson BK.  An overview of column separation of spermatozoa.  Proc. 7th Tech. Conf. NAAB, Madison, Wisconsin.  1978: p 69-73.
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[13] Chandler JE, Wilson MP, Canal AM, Steinholt-Chenevert.  Bovine spermatozoal head size variation and evaluation of a separation technique based on this size.  Theriogenology 1999; 52: 1021-36.
[14] Mann T.  Biochemistry of Semen and of the Male Reproductive Tract. New York: John Wiley & Sons, Inc.; 1964.p 383-4.
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Correspondence to: Dr. Robert H. Foote, Department of Animal Science, Cornell University, Ithaca, NY 14853-4801, USA.
Tel: +607-255-2866      Fax: +607-255-9829
E-mail: dgb1@Cornell.edu
Received 2001-07-23    Accepted 2001-08-22