- Email: [email protected]
Evaluation of a modified sperm penetration test in ram semen
EVALUATION OF A MODIFIED SPERM PENETRATION P. Suttiyotin,l C.J. Thwaites,l L.G. Sanchez-Partida
TEST IN RAM SEMEN and B.P. Setchellz
*Department of Animal Science University of New England NSW 235 1 Australia 2Department of Animal Sciences Waite Agricultural Research Institute University of Adelaide SA 5064 Australia Received for publication: ApriZ 14, 1992 Accepted: November 30, 1994 ABSTRACT A modified sperm penetration test, based on the movement of spermatozoa into a capillary tube containing 300 mM Tris-28 mM glucose (TG) solution, was developed for evaluating ram semen. Sperm penetration distance increased from 4.8 mm at 1x 109 sperm/ml to 7.4 mm at 2~x109 sperm/ml (PcO.01) and, with time, from 3.5 at 5 min to 8.1 at 20 min (P
into homologous
mucus has been successfUlly
fertility in human (6) and bull (I I) semell. the tecl!nique
used
involved microscopic
as R test
of
evami ~atl~~nof the
Acknowledgments This work was supported by the Thai-Australian Prince of Songkla University Project and by the University of New England. We thank N. Baillie, G. Chisholm, H. Bozyk and S. Sowerbutts for technical assistance. Theriogenology 44:2940, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0093-691X/95/$1 0.00 SSDI 0093-691 X(95)001 45-X
30
Theriogenology
fertility in human (6) and bull (11) semen, the technique involved microscopic examination of the movement of vanguard spermatozoa into natural mucus, which lack uniformity and is not generally available. A number of alternatives to natural mucus have been investigated, including artificial mucus (22) polyacrylamide (7) and egg white (3) but despite several attempts (4,12) no standardized technique has as yet been generally accepted. Recently (19) we reported the use of Tris-glucose (TG) medium in a capillary tube as the basis of a simple and cheap modified sperm penetration test which was both repeatable and significantly correlated with fertility. The present paper reports experiments designed 1) to standardize conditions of semen concentration and total sperm numbers in test samples; 2) to establish the optimum duration of the technique; and 3) to further validate the test by comparing results with subjective assessment, with known proportions of motile spermatozoa in test samples, and with the results from a computerized motility analyzer, using a range of semen samples typical of those encountered under field conditions. MATERIALS AND METHODS Ejaculates were collected from adult Merino rams by electrical stimulation (Experiments 1 to 3) or artificial vagina (Experiment 4) diluted with 300 mM Tris-28 mM glucose to 2x109 sperm/ml and held at 37°C in a water bath until used. For the modified sperm penetration test, a 100 x l-mm glass capillary tube was filled with TG, one end was sealed with putty, and the tube was inverted so that the open end could be inserted into 0.05 ml of test sample (containing 100x106 spermatozoa) in a round-bottom 75 x 12-mm glass test tube maintained at 37°C. As the spermatozoa swam up into the capillary tube, the turbidity of the TG increased and sperm penetration distance was read by the naked eye, to the nearest 1 mm, against a transparent scale mounted behind the capillary tube. Care was taken to maintain the tubes in a vertical position and to avoid parallax error and any movement of tubes during a test. The sperm penetration distance was routinely read after 15 min. Experiment 1: Effect of Semen Concentration This experiment was designed to investigate possible effects of semen concentration on the results of the modified sperm penetration test when the total sperm number was standardized. Portions of one ejaculate from each of 4 rams were diluted with TG to 1.0, 1.5, 2.0 and 2.5~109 sperm/ml before being subjected, in triplicate, to the modified sperm penetration test. Each test sample contained 100x106 spermatozoa and the volumes were adjusted to 100, 66.7, 50 and 40 ~1, respectively, for the concentrations listed above. The sperm penetration distance was read after 5, 10, 15 and 20 min, the experiment was replicated twice, and repeatabilities were calculated as intra-class correlations. Experiment 2: Effect of Total Sperm Numbers The effect of the total sperm numbers was studied when semen concentration was controlled. Single ejaculates from each of 4 rams were diluted with TG to 2x109 sperm/ml, and test samples of each, containing 50, 100, 150 and 200x 106 spermatozoa (25, 50, 75 and 100 yl, respectively),
31
Theriogenology
were subjected to the modified sperm penetration test. Four replicates were used for each test sample, the sperm penetration distance was noted after 5, 10, 15 and 20 min, and repeatabilities were calculated as intra-class correlations. Experiment 3 : Correlation With Known Proportions of Motile and Immotile Spermatozoa The sperm penetration distance was recorded after 5, 10, 15 and 20 min in duplicate subsamples (0.05 ml) of mixtures of suspensions containing known proportions of motile and immotile spermatozoa. Briefly, to prepare these suspensions (1 S), single ejaculates from at least 5 rams (to obtain sufficient spermatozoa) were pooled, washed and resuspended in TG, frozen-thawed a number of times until microscopic examination revealed that no motile spermatozoa remained. AtIer centrimgation, these sperm cells were washed again and adjusted to 2x 109 cells/ml in TG and used as the immotile sperm suspension. Single ejaculates from each of 4 rams were diluted with TG to 2x109 sperm/ml, the percentages of progressively motile spermatozoa were estimated subjectively, and the resultant motile sperm suspensions were mixed with the previously prepared immotile sperm suspension in the proportions l:O, 3: 1, 1: 1, I:3 and 0: 1. The estimated proportions of motile and immotile spermatozoa in each test sample was then calculated. Experiment 4: Correlation With Subjective Assessment and With Results from a Hamilton Thorn Motility Analyzer To mrther validate the modified sperm penetration test, the results from the technique were correlated with subjective and computer analyzed estimates of sperm motility in a range of semen samples (Table 1) likely to be encountered in the field. A standard modified sperm penetration test procedure was adopted: semen was diluted to 2x109 sperm/ml with TG, and duplicate 0.05-ml subsamples (each containing 100x 106 spermatozoa) were allowed to swim up into a column of TG in 100x 1-mm glass capillary tubes at 37°C for 15 min. For each made by the spermatozoa, on a warming
of the semen samples described in Table 1, concurrent estimates of motility were modified sperm penetration test, subjective evaluation (% progressively motile and motility rating on a scale of 0 to 5, both at x160 in a 60-u thick layer of sample stage at 3 7°C) and by use of a Hamilton Thorn Motility Analyzer (13).
Data were analyzed by analysis of variance, and significant differences between means were tested by Duncan’s new multiple range test. Linear regression analysis was used to relate the sperm penetration distance to subjective motility assessments and the results from the Hamilton Thorn Motility Analyzer. RESULTS Experiment 1 There was a positive effect of semen concentration
on the sperm penetration
distance
2.5x1o9 sperm/ml. the mean value (7.4 mm) was higher than at 2.0, 1.5 and 1.0~10~ sperm/ml
At
Theriogenology
32 Table 1. Details of semen samples evaluated in Experiments 4a to 4f
Experiment
Type of sample
No. of ejaculates
Additional methodology
4a
4
Test samples prepared as in Experiment 3
4b 4c
Motile and immotile sperm suspension Fresh semen Cold-shock semen
21 4
4d
Aged semen
4
4e
Diluted-stored semen
4
4f
Frozen-thawed semen
Evaluated within 10 min of collection 5 aliquots of each cold shocked in ice bath for 0, 0.5, 1, 2 or 5 min. Reading after rewarming to 37°C Reading after incubation at 37°C for 0, 1, 2, 3 or 4 h. Diluted to 2x109 sperm/ml, cooled to 5°C and stored at 5°C for 0, 1, 2, 3, 4 or 7 d. Reading after incubation at 37°C for 0, 1, 2, 3 or 4 h. Semen frozen in pellets (21) and stored in liquid nitrogen. Reading at 0, 1, 2 or 3 h atIer thawing at 37°C in dry test tube
(means of 6.1, 5.7 and 4.8 mm, respectively; WO.01). The sperm penetration distance was also time dependent, mean values increasing from 3.5 mm at 5 min to 5.5, 7.0 and 8.1 mm at 10, 15 and 20 min, respectively (ah P
33
Theriogenology 5 min, y=-48.6+38.3x,
rz0.761
15 min, y=-lU.tl+lO.fix,
rz0.895
r-
10 min. y=-14.1+14.5x, 0
r-0.853 0
d 0 0
cT
20 min, y=-6.2+0.2x, 0
r=O.SOi
0
020
0
0
0
0
8
0
0
k
-
II 0 2 4 6 8 Penetration distance(mm)
10
?rr
i--
0
0 2 4 6 Penetration
I_ 8 IO 12 14 distance(mm)
16
Figure 1 Relationships between known proportions of motile and immotile spermatozoa and the penetration distance (mm) at each observation time in the modified sperm penetration test. homogeneous and that the regression lines at 10 and 15 min were parallel. Mean values for the sperm penetration distance at 5, 10, 15 and 20 min were 2.4kO.1, 4.0+0.4, 5.2kO.6 and 6.1+0.8 mm, respectively. Repeatabilities ranged from 0.88 to 0.97 (P
34
Theriogenology
Experiment 4b In fresh semen, the sperm penetration distance was not significantly correlated with either of the subjective assessments or any of the Hamilton Thorn parameters (Table 2), although the correlations with the percentages of subjective motile cells, rapidly moving and motile approached significance (r=O.398, 0.345 and 0.343 respectively, O.l>P>O.O5). Mean values for the sperm penetration distance, the percentages of subjective motile cells, rapidly moving and motile were 12.9+0.5 mm, 82.9+1.9%, 48.4f3.4% and 61.3+2.8%, respectively. Experiment 4c The sperm penetration distance was significantly correlated with the percentage of subjective motile cells and motility rating (both P
Theriogenology Table 2.
35
Coefficients of correlation between results from a modified sperm penetration test (millimeter penetration distance) and motility assessed subjectively (SMOT: percentage of progressively motile spermatozoa, MR: motility rating) and by a Hamilton Thorn Motility Analyser (PROG%: progressive motility, MOT%: percentage of motile spermatozoa, MPRG: mean progressive velocity in micron/second, MLINo/o: mean linear index, MLDH: mean lateral head displacement, MLIN: mean linearity, MPTH: mean path velocity in micron/second, RAPD%: percentage of rapidly moving spermatozoa, MOD%: percentage of moderately moving spermatozoa, SLOW%: percentage of slowly moving spermatozoa) in Experiments 4b to 4f
Fresh
Sperm treatments Cold-shocked Aged
Diluted-stored
Frozen-thawed
SMOT MR
0.398 0.324
0.918*** 0.875***
0.913*** 0.913***
0.905*** 0.872***
0.897*** 0.873***
PROG% MOT% MPRG MLIN% MLDH MLIN MPTH RAPD% MOD% SLOW%
0.282 0.343 0.183 0.146 0.019 0.241 0.208 0.345 -0.142 -0.291 21
0.741*** 0.846*** 0.553* 0.624** 0.088 0.5 16* 0.512* 0.745*** 0.721*** 0.059 20
0.752*** 0.617** 0.837*** 0.577** 0.553* 0.750*** 0.813*** 0.712*** 0.111 0.105 20
0.909*** 0.885*** 0.838*** 0.571*** 0.303 0.755*** 0.744*** 0.889*** 0.605** -0.376 24
o-913*** 0.918*** 0.825*** 0.341 0.079 0.509* 0.793*** 0.927*** 0.643* 0.080 20
n * PiO.05;
** P
DISCUSSION The results from Experiment 1 clearly indicate that the concentration of semen sample subjected to the modified sperm penetration test has a significant influence on the sperm penetration distance, and that this factor must thus be standardized in the new technique. It is generally agreed that 2x109 sperm/ml represents the lower limit of concentration in normal ram semen (14, 17) and in order to cover as wide a range of semen concentrations as possible with the modified sperm penetration test, this is the value we recommend,for routine use. Previous studies in cervical mucus have indicated that penetration distance increases with concentration of the test sample (IO, 5). The total number of sperm used in the modified sperm penetration test (from 50 to 200~10~) had no effect on the sperm penetration distance (Experiment 2). To use semen sparingly, but at the same time minimize the effects of evaporation during the test, a semen loading of 100~10~ spermatozoa (0.05 ml of the diluted 2x 109 sperm/ml semen) is recommended for routine use.
Theriogenology
36
12
100
If _Ej 10
140 120
I 2 ;:
8
Yj
0
100
c” d
4 2 0
‘;
80
<
60
5
SO.i
d .o ;i 9
70
0
0.6 Cold
1 shock
2
6
00 40
‘0’ ,$
20
20
B
10
Cold
la0
-5. e 10
120 100
4
20
*
I lncubatlon
2 Ume
3 (houre)
4
0
GO
>
40
60
(mln)
au
‘;; ,”
2
80
chock
70
% .Ti S q 2 6 .” 2 4 5
0
so
0
0
140
0
b a
(mln)
14
FA.?DS
% 40 “0
,x
212
dB
a
g .; -s 5,
‘;; 6 2
40 30 20 10 0
0
1 lncuballon
2 Ume
3
4
(hours)
Figure 2. Effects of cold shock (top) and aging of semen samples (bottom) on sperm penetration distance (SPD) and Hamilton Thorn Motility velocity (MPRG: mean progressive velocity, MPTH: mean path velocity) and percentage motility (PROG%: progressive motility, MOT%: percentage of motile spermatozoa, and RAPD%: percentage of rapidly moving spermatozoa) in Experiments 4c and 4d, respectively (* P
Theriogenology
37
Figure 3. Effects of storage of diluted semen on sperm (top) and freezing and thawing of semen (bottom) on penetration distance (SPD) and Hamilton Thorn Motiiity velocity (MPRG: mean progressive velocity, MPTH: mean path velocity) and percentage motility (PROG%: progressive motility, MOT%: percentage of motile spermatozoa, and RAPD%: percentage of rapidly moving spermatozoa) in Experiments 3e and 4f, respectively (* PcO.05; ** P
Penetration of spermatozoa into TG was time dependent in each of Experiments 1 to 3. The homogeneity of the 15 and 20-min regression lines in Experiment 3 indicates no advantage in readings taken beyond 15 mitt, while the higher readings of the 15-min line and its parallelism with the IO-mm one suggests that readings at 15 min are to be preferred. That time is thus adopted for the technique. In validation of the modified sperm penetration test as a predictor of the motility of ram semen, Experiments 1 to 3 confirmed that the repeatability of results from duplicate samples was routinely high, r values of 0.88 to 0.98 being recorded. As a simple, inexpensive technique designed particularly for field use, the modified sperm penetration test could be expected to most commonly be used as an adjustment to, or in place of, traditional subjective procedures (14). In all segments of Experiment 4 apart from 4a (fresh semen), the sperm penetration distance derived from the new technique was highly correlated with the results from both the subjective assessments used (subjective motility and motility rating); correlation coefficients varying from 0.872 to 0.918 (all P
Theriogenology
39
of slowly moving spermatozoa and percentage of moderately moving spermatozoa, the estimated percentages of slowly and moderately moving spermatozoa, and with mean lateral head displacement, since lateral head movement might be expected to be of little significance to the movement of spermatozoa into structureless, low viscosity TG (1) The range of correlations between the sperm penetration distance and the various Hamilton Thorn parameters can thus be seen to have a biological basis, which adds further strength to this validation of the modified sperm penetration test, as does the fact that the effects recorded for cold shock (2, 15) aging (8) storage (8, 16) and freeze-thawing (15, 20, 21) in Figures 2 and 3 are similar to those of previous reports. Taking all aspects of the validations presented into account, we conclude that the modified sperm penetration test is a simple, repeatable, objective technique which can supplement or replace subjective assessments, which is sensitive to progressive changes in the proportions of motile spermatozoa in test samples, and which is correlated in a biologically coherent manner with the range of parameters from the Hamilton Thorn computerized motility analyzer. REFERENCES 1. Aitken RJ, Sutton M, Warner P, Richardson DW. Relationship between the movement characteristics of human spermatozoa and their ability to penetrate cervical mucus and zona-free hamster oocytes. J Reprod Fertil 1985;73:441-449. 2
Choong CH, Wales RG. The effect of cold shock on spermatozoa. Aust J Biol Sci 1962; 15.543-551.
3
Eggert-Kruse W, Gerhard I, Tilgen W, Runnebaum B. The use of hens’ egg white as a substitute for human cervical mucus in assessing human infertility. Int J Androl 1990; 13:258-266.
4
Katz DF, Overstreet JW, Hanson FW. A new quantitative test for sperm penetration into cervical mucus. Fertil Steril 1980;33: 179- 186.
5. Keel BA, Webster BW. Correlation of human sperm motility characteristics with an in vitro cervical mucus penetration test. Fertil Steril 1988;49: 13% 143. 6. Kremer J. A simple sperm penetration test. Int J Fertil 1965; 10:209-215. 7. Lorton SP, Kummerfeld HL, Foote RH. Polyacrylamide as a substitute for cervical mucus in sperm migration tests. Fertil Steril 1981;35:222-225. 8. Martin ICA, Richardson BA. Factors affecting the fertility of diluted ram semen. In: Tomes GJ, Robertson DE, Lightfoot RJ (eds), Sheep Breeding. Perth, Western Australian Institute of Technology, 1976;467-474.
Theriogenology 9. Mortimer D, Pandya IJ, Sawers RS. Relationship between human sperm motility characteristics and sperm penetration into cervical mucus in vitro. J Reprod Fertil 1986; 78:93-102. 10. Murase T, Braun JW. Impact of methodological factors on sperm penetration into cervical mucus. Theriogenology 1990;34:73-80. 11. Murase T, Okuda K, Sato, K. Assessment of bull fertility using mucus penetration test and a human chorionic gonadotrophin stimulation test. Theriogenology 1990;34: 80 l-8 12. 12. Pandya IJ, Mortimer D, Sawers RS. A standardized approach for evaluating the penetration of human spermatozoa into cervical mucus in vitro. Fertil Steril 1986;45:357-365. 13. Quintana Casares P, Maxwell WMC, Wilson HR, Setchell BP. Viability of ram spermatozoa following frozen storage in maxi and mini straws at two concentrations. Aust Sot Reprod Biol 1990;22:67 abstr. 14. Salamon S. Artificial Insemination of Sheep. Chippendale, Australia, Publicity Press, 1976 15. Salamon S, Lightfoot RJ. Effect of cold shock, liquid storage, and pellet-freezing successive ram ejaculates. Aust J Agric Res 1976;18:959-972.
on
16. Salamon S, Maxwell WMC, Firth JII Fertility capacity of ram spermatozoa stored at 5°C. Theriogenology 1977;8:200 abstr. 17. Setchell BP. Male reproductive organs and semen. In: Cupps PT (ed) Reproduction Domestic Animals. New York, Academic Press, 1991;221-249.
in
18. Suttiyotin P, Thwaites CJ. The ability of trypan blue to differentiate live and dead ram spermatozoa. Anim Reprod Sci 1991;25:209-224. 19. Suttiyotin P, Thwaites CJ, Baillie ND Relationships between the results of a modified sperm penetration test and a swim-up technique and the fertility of ram semen. Theriogenology 1992;37:851-857. 20. Visser D. The effect of freezing method on the survival of ram spermatozoa. S Al? J Anim Sci 1974;4:157-163. 21. Visser D, Salamon S. Fertility of ram spermatozoa frozen in a Tris-based diluent. Aust J Biol Sci 1973;26:513-516. 22. Urry RL, Middleton RG, Mayo D. A comparison of the penetration of human sperm into bovine and artificial cervical mucus. Fertil Steril 1986;45: 135-137.
TEST IN RAM SEMEN and B.P. Setchellz
*Department of Animal Science University of New England NSW 235 1 Australia 2Department of Animal Sciences Waite Agricultural Research Institute University of Adelaide SA 5064 Australia Received for publication: ApriZ 14, 1992 Accepted: November 30, 1994 ABSTRACT A modified sperm penetration test, based on the movement of spermatozoa into a capillary tube containing 300 mM Tris-28 mM glucose (TG) solution, was developed for evaluating ram semen. Sperm penetration distance increased from 4.8 mm at 1x 109 sperm/ml to 7.4 mm at 2~x109 sperm/ml (PcO.01) and, with time, from 3.5 at 5 min to 8.1 at 20 min (P
into homologous
mucus has been successfUlly
fertility in human (6) and bull (I I) semell. the tecl!nique
used
involved microscopic
as R test
of
evami ~atl~~nof the
Acknowledgments This work was supported by the Thai-Australian Prince of Songkla University Project and by the University of New England. We thank N. Baillie, G. Chisholm, H. Bozyk and S. Sowerbutts for technical assistance. Theriogenology 44:2940, 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0093-691X/95/$1 0.00 SSDI 0093-691 X(95)001 45-X
30
Theriogenology
fertility in human (6) and bull (11) semen, the technique involved microscopic examination of the movement of vanguard spermatozoa into natural mucus, which lack uniformity and is not generally available. A number of alternatives to natural mucus have been investigated, including artificial mucus (22) polyacrylamide (7) and egg white (3) but despite several attempts (4,12) no standardized technique has as yet been generally accepted. Recently (19) we reported the use of Tris-glucose (TG) medium in a capillary tube as the basis of a simple and cheap modified sperm penetration test which was both repeatable and significantly correlated with fertility. The present paper reports experiments designed 1) to standardize conditions of semen concentration and total sperm numbers in test samples; 2) to establish the optimum duration of the technique; and 3) to further validate the test by comparing results with subjective assessment, with known proportions of motile spermatozoa in test samples, and with the results from a computerized motility analyzer, using a range of semen samples typical of those encountered under field conditions. MATERIALS AND METHODS Ejaculates were collected from adult Merino rams by electrical stimulation (Experiments 1 to 3) or artificial vagina (Experiment 4) diluted with 300 mM Tris-28 mM glucose to 2x109 sperm/ml and held at 37°C in a water bath until used. For the modified sperm penetration test, a 100 x l-mm glass capillary tube was filled with TG, one end was sealed with putty, and the tube was inverted so that the open end could be inserted into 0.05 ml of test sample (containing 100x106 spermatozoa) in a round-bottom 75 x 12-mm glass test tube maintained at 37°C. As the spermatozoa swam up into the capillary tube, the turbidity of the TG increased and sperm penetration distance was read by the naked eye, to the nearest 1 mm, against a transparent scale mounted behind the capillary tube. Care was taken to maintain the tubes in a vertical position and to avoid parallax error and any movement of tubes during a test. The sperm penetration distance was routinely read after 15 min. Experiment 1: Effect of Semen Concentration This experiment was designed to investigate possible effects of semen concentration on the results of the modified sperm penetration test when the total sperm number was standardized. Portions of one ejaculate from each of 4 rams were diluted with TG to 1.0, 1.5, 2.0 and 2.5~109 sperm/ml before being subjected, in triplicate, to the modified sperm penetration test. Each test sample contained 100x106 spermatozoa and the volumes were adjusted to 100, 66.7, 50 and 40 ~1, respectively, for the concentrations listed above. The sperm penetration distance was read after 5, 10, 15 and 20 min, the experiment was replicated twice, and repeatabilities were calculated as intra-class correlations. Experiment 2: Effect of Total Sperm Numbers The effect of the total sperm numbers was studied when semen concentration was controlled. Single ejaculates from each of 4 rams were diluted with TG to 2x109 sperm/ml, and test samples of each, containing 50, 100, 150 and 200x 106 spermatozoa (25, 50, 75 and 100 yl, respectively),
31
Theriogenology
were subjected to the modified sperm penetration test. Four replicates were used for each test sample, the sperm penetration distance was noted after 5, 10, 15 and 20 min, and repeatabilities were calculated as intra-class correlations. Experiment 3 : Correlation With Known Proportions of Motile and Immotile Spermatozoa The sperm penetration distance was recorded after 5, 10, 15 and 20 min in duplicate subsamples (0.05 ml) of mixtures of suspensions containing known proportions of motile and immotile spermatozoa. Briefly, to prepare these suspensions (1 S), single ejaculates from at least 5 rams (to obtain sufficient spermatozoa) were pooled, washed and resuspended in TG, frozen-thawed a number of times until microscopic examination revealed that no motile spermatozoa remained. AtIer centrimgation, these sperm cells were washed again and adjusted to 2x 109 cells/ml in TG and used as the immotile sperm suspension. Single ejaculates from each of 4 rams were diluted with TG to 2x109 sperm/ml, the percentages of progressively motile spermatozoa were estimated subjectively, and the resultant motile sperm suspensions were mixed with the previously prepared immotile sperm suspension in the proportions l:O, 3: 1, 1: 1, I:3 and 0: 1. The estimated proportions of motile and immotile spermatozoa in each test sample was then calculated. Experiment 4: Correlation With Subjective Assessment and With Results from a Hamilton Thorn Motility Analyzer To mrther validate the modified sperm penetration test, the results from the technique were correlated with subjective and computer analyzed estimates of sperm motility in a range of semen samples (Table 1) likely to be encountered in the field. A standard modified sperm penetration test procedure was adopted: semen was diluted to 2x109 sperm/ml with TG, and duplicate 0.05-ml subsamples (each containing 100x 106 spermatozoa) were allowed to swim up into a column of TG in 100x 1-mm glass capillary tubes at 37°C for 15 min. For each made by the spermatozoa, on a warming
of the semen samples described in Table 1, concurrent estimates of motility were modified sperm penetration test, subjective evaluation (% progressively motile and motility rating on a scale of 0 to 5, both at x160 in a 60-u thick layer of sample stage at 3 7°C) and by use of a Hamilton Thorn Motility Analyzer (13).
Data were analyzed by analysis of variance, and significant differences between means were tested by Duncan’s new multiple range test. Linear regression analysis was used to relate the sperm penetration distance to subjective motility assessments and the results from the Hamilton Thorn Motility Analyzer. RESULTS Experiment 1 There was a positive effect of semen concentration
on the sperm penetration
distance
2.5x1o9 sperm/ml. the mean value (7.4 mm) was higher than at 2.0, 1.5 and 1.0~10~ sperm/ml
At
Theriogenology
32 Table 1. Details of semen samples evaluated in Experiments 4a to 4f
Experiment
Type of sample
No. of ejaculates
Additional methodology
4a
4
Test samples prepared as in Experiment 3
4b 4c
Motile and immotile sperm suspension Fresh semen Cold-shock semen
21 4
4d
Aged semen
4
4e
Diluted-stored semen
4
4f
Frozen-thawed semen
Evaluated within 10 min of collection 5 aliquots of each cold shocked in ice bath for 0, 0.5, 1, 2 or 5 min. Reading after rewarming to 37°C Reading after incubation at 37°C for 0, 1, 2, 3 or 4 h. Diluted to 2x109 sperm/ml, cooled to 5°C and stored at 5°C for 0, 1, 2, 3, 4 or 7 d. Reading after incubation at 37°C for 0, 1, 2, 3 or 4 h. Semen frozen in pellets (21) and stored in liquid nitrogen. Reading at 0, 1, 2 or 3 h atIer thawing at 37°C in dry test tube
(means of 6.1, 5.7 and 4.8 mm, respectively; WO.01). The sperm penetration distance was also time dependent, mean values increasing from 3.5 mm at 5 min to 5.5, 7.0 and 8.1 mm at 10, 15 and 20 min, respectively (ah P
33
Theriogenology 5 min, y=-48.6+38.3x,
rz0.761
15 min, y=-lU.tl+lO.fix,
rz0.895
r-
10 min. y=-14.1+14.5x, 0
r-0.853 0
d 0 0
cT
20 min, y=-6.2+0.2x, 0
r=O.SOi
0
020
0
0
0
0
8
0
0
k
-
II 0 2 4 6 8 Penetration distance(mm)
10
?rr
i--
0
0 2 4 6 Penetration
I_ 8 IO 12 14 distance(mm)
16
Figure 1 Relationships between known proportions of motile and immotile spermatozoa and the penetration distance (mm) at each observation time in the modified sperm penetration test. homogeneous and that the regression lines at 10 and 15 min were parallel. Mean values for the sperm penetration distance at 5, 10, 15 and 20 min were 2.4kO.1, 4.0+0.4, 5.2kO.6 and 6.1+0.8 mm, respectively. Repeatabilities ranged from 0.88 to 0.97 (P
34
Theriogenology
Experiment 4b In fresh semen, the sperm penetration distance was not significantly correlated with either of the subjective assessments or any of the Hamilton Thorn parameters (Table 2), although the correlations with the percentages of subjective motile cells, rapidly moving and motile approached significance (r=O.398, 0.345 and 0.343 respectively, O.l>P>O.O5). Mean values for the sperm penetration distance, the percentages of subjective motile cells, rapidly moving and motile were 12.9+0.5 mm, 82.9+1.9%, 48.4f3.4% and 61.3+2.8%, respectively. Experiment 4c The sperm penetration distance was significantly correlated with the percentage of subjective motile cells and motility rating (both P
Theriogenology Table 2.
35
Coefficients of correlation between results from a modified sperm penetration test (millimeter penetration distance) and motility assessed subjectively (SMOT: percentage of progressively motile spermatozoa, MR: motility rating) and by a Hamilton Thorn Motility Analyser (PROG%: progressive motility, MOT%: percentage of motile spermatozoa, MPRG: mean progressive velocity in micron/second, MLINo/o: mean linear index, MLDH: mean lateral head displacement, MLIN: mean linearity, MPTH: mean path velocity in micron/second, RAPD%: percentage of rapidly moving spermatozoa, MOD%: percentage of moderately moving spermatozoa, SLOW%: percentage of slowly moving spermatozoa) in Experiments 4b to 4f
Fresh
Sperm treatments Cold-shocked Aged
Diluted-stored
Frozen-thawed
SMOT MR
0.398 0.324
0.918*** 0.875***
0.913*** 0.913***
0.905*** 0.872***
0.897*** 0.873***
PROG% MOT% MPRG MLIN% MLDH MLIN MPTH RAPD% MOD% SLOW%
0.282 0.343 0.183 0.146 0.019 0.241 0.208 0.345 -0.142 -0.291 21
0.741*** 0.846*** 0.553* 0.624** 0.088 0.5 16* 0.512* 0.745*** 0.721*** 0.059 20
0.752*** 0.617** 0.837*** 0.577** 0.553* 0.750*** 0.813*** 0.712*** 0.111 0.105 20
0.909*** 0.885*** 0.838*** 0.571*** 0.303 0.755*** 0.744*** 0.889*** 0.605** -0.376 24
o-913*** 0.918*** 0.825*** 0.341 0.079 0.509* 0.793*** 0.927*** 0.643* 0.080 20
n * PiO.05;
** P
DISCUSSION The results from Experiment 1 clearly indicate that the concentration of semen sample subjected to the modified sperm penetration test has a significant influence on the sperm penetration distance, and that this factor must thus be standardized in the new technique. It is generally agreed that 2x109 sperm/ml represents the lower limit of concentration in normal ram semen (14, 17) and in order to cover as wide a range of semen concentrations as possible with the modified sperm penetration test, this is the value we recommend,for routine use. Previous studies in cervical mucus have indicated that penetration distance increases with concentration of the test sample (IO, 5). The total number of sperm used in the modified sperm penetration test (from 50 to 200~10~) had no effect on the sperm penetration distance (Experiment 2). To use semen sparingly, but at the same time minimize the effects of evaporation during the test, a semen loading of 100~10~ spermatozoa (0.05 ml of the diluted 2x 109 sperm/ml semen) is recommended for routine use.
Theriogenology
36
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Figure 2. Effects of cold shock (top) and aging of semen samples (bottom) on sperm penetration distance (SPD) and Hamilton Thorn Motility velocity (MPRG: mean progressive velocity, MPTH: mean path velocity) and percentage motility (PROG%: progressive motility, MOT%: percentage of motile spermatozoa, and RAPD%: percentage of rapidly moving spermatozoa) in Experiments 4c and 4d, respectively (* P
Theriogenology
37
Figure 3. Effects of storage of diluted semen on sperm (top) and freezing and thawing of semen (bottom) on penetration distance (SPD) and Hamilton Thorn Motiiity velocity (MPRG: mean progressive velocity, MPTH: mean path velocity) and percentage motility (PROG%: progressive motility, MOT%: percentage of motile spermatozoa, and RAPD%: percentage of rapidly moving spermatozoa) in Experiments 3e and 4f, respectively (* PcO.05; ** P
Penetration of spermatozoa into TG was time dependent in each of Experiments 1 to 3. The homogeneity of the 15 and 20-min regression lines in Experiment 3 indicates no advantage in readings taken beyond 15 mitt, while the higher readings of the 15-min line and its parallelism with the IO-mm one suggests that readings at 15 min are to be preferred. That time is thus adopted for the technique. In validation of the modified sperm penetration test as a predictor of the motility of ram semen, Experiments 1 to 3 confirmed that the repeatability of results from duplicate samples was routinely high, r values of 0.88 to 0.98 being recorded. As a simple, inexpensive technique designed particularly for field use, the modified sperm penetration test could be expected to most commonly be used as an adjustment to, or in place of, traditional subjective procedures (14). In all segments of Experiment 4 apart from 4a (fresh semen), the sperm penetration distance derived from the new technique was highly correlated with the results from both the subjective assessments used (subjective motility and motility rating); correlation coefficients varying from 0.872 to 0.918 (all P
Theriogenology
39
of slowly moving spermatozoa and percentage of moderately moving spermatozoa, the estimated percentages of slowly and moderately moving spermatozoa, and with mean lateral head displacement, since lateral head movement might be expected to be of little significance to the movement of spermatozoa into structureless, low viscosity TG (1) The range of correlations between the sperm penetration distance and the various Hamilton Thorn parameters can thus be seen to have a biological basis, which adds further strength to this validation of the modified sperm penetration test, as does the fact that the effects recorded for cold shock (2, 15) aging (8) storage (8, 16) and freeze-thawing (15, 20, 21) in Figures 2 and 3 are similar to those of previous reports. Taking all aspects of the validations presented into account, we conclude that the modified sperm penetration test is a simple, repeatable, objective technique which can supplement or replace subjective assessments, which is sensitive to progressive changes in the proportions of motile spermatozoa in test samples, and which is correlated in a biologically coherent manner with the range of parameters from the Hamilton Thorn computerized motility analyzer. REFERENCES 1. Aitken RJ, Sutton M, Warner P, Richardson DW. Relationship between the movement characteristics of human spermatozoa and their ability to penetrate cervical mucus and zona-free hamster oocytes. J Reprod Fertil 1985;73:441-449. 2
Choong CH, Wales RG. The effect of cold shock on spermatozoa. Aust J Biol Sci 1962; 15.543-551.
3
Eggert-Kruse W, Gerhard I, Tilgen W, Runnebaum B. The use of hens’ egg white as a substitute for human cervical mucus in assessing human infertility. Int J Androl 1990; 13:258-266.
4
Katz DF, Overstreet JW, Hanson FW. A new quantitative test for sperm penetration into cervical mucus. Fertil Steril 1980;33: 179- 186.
5. Keel BA, Webster BW. Correlation of human sperm motility characteristics with an in vitro cervical mucus penetration test. Fertil Steril 1988;49: 13% 143. 6. Kremer J. A simple sperm penetration test. Int J Fertil 1965; 10:209-215. 7. Lorton SP, Kummerfeld HL, Foote RH. Polyacrylamide as a substitute for cervical mucus in sperm migration tests. Fertil Steril 1981;35:222-225. 8. Martin ICA, Richardson BA. Factors affecting the fertility of diluted ram semen. In: Tomes GJ, Robertson DE, Lightfoot RJ (eds), Sheep Breeding. Perth, Western Australian Institute of Technology, 1976;467-474.
Theriogenology 9. Mortimer D, Pandya IJ, Sawers RS. Relationship between human sperm motility characteristics and sperm penetration into cervical mucus in vitro. J Reprod Fertil 1986; 78:93-102. 10. Murase T, Braun JW. Impact of methodological factors on sperm penetration into cervical mucus. Theriogenology 1990;34:73-80. 11. Murase T, Okuda K, Sato, K. Assessment of bull fertility using mucus penetration test and a human chorionic gonadotrophin stimulation test. Theriogenology 1990;34: 80 l-8 12. 12. Pandya IJ, Mortimer D, Sawers RS. A standardized approach for evaluating the penetration of human spermatozoa into cervical mucus in vitro. Fertil Steril 1986;45:357-365. 13. Quintana Casares P, Maxwell WMC, Wilson HR, Setchell BP. Viability of ram spermatozoa following frozen storage in maxi and mini straws at two concentrations. Aust Sot Reprod Biol 1990;22:67 abstr. 14. Salamon S. Artificial Insemination of Sheep. Chippendale, Australia, Publicity Press, 1976 15. Salamon S, Lightfoot RJ. Effect of cold shock, liquid storage, and pellet-freezing successive ram ejaculates. Aust J Agric Res 1976;18:959-972.
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16. Salamon S, Maxwell WMC, Firth JII Fertility capacity of ram spermatozoa stored at 5°C. Theriogenology 1977;8:200 abstr. 17. Setchell BP. Male reproductive organs and semen. In: Cupps PT (ed) Reproduction Domestic Animals. New York, Academic Press, 1991;221-249.
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18. Suttiyotin P, Thwaites CJ. The ability of trypan blue to differentiate live and dead ram spermatozoa. Anim Reprod Sci 1991;25:209-224. 19. Suttiyotin P, Thwaites CJ, Baillie ND Relationships between the results of a modified sperm penetration test and a swim-up technique and the fertility of ram semen. Theriogenology 1992;37:851-857. 20. Visser D. The effect of freezing method on the survival of ram spermatozoa. S Al? J Anim Sci 1974;4:157-163. 21. Visser D, Salamon S. Fertility of ram spermatozoa frozen in a Tris-based diluent. Aust J Biol Sci 1973;26:513-516. 22. Urry RL, Middleton RG, Mayo D. A comparison of the penetration of human sperm into bovine and artificial cervical mucus. Fertil Steril 1986;45: 135-137.