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Use of triple stain technique for simultaneous assessment of vitality and acrosomal status in boar spermatozoa
Theriogenology 38:843-%52, 1992
USE OF TRIPLE STAIN TECHNIQUE FOR SIMULTANEOUS ASSESSMENT OF VITALITY AND ACROSOMAL STATUS IN BOAR SPERMATOZOA J.M. Vkquez’,
E. Martinez’,
P. Co+ and S. Ruid
J. Rod,
Departments of ‘Animal Pathology and 2Animal Biology Facultad de Veterinaria, Universidad de Murcia 30071 Murcia, Spain Received for publication: kbrch Accepted: JUZY
3, 5,
I 992 1x2
ABSTRACT
The object of this study was to adapt the triple stain technique to diluted and incubated boar spermatozoa. Freshly ejaculated semen was resuspended in MR-A diluent to contain 3x10’ cells/ml (diluted spermatozoa) and was subsequently capacitated (incubated spermatozoa). Experiments were conducted to show the conditions required for optimal staining quality and validation of triple stain technique. The most satisfactory staining solutions for diluted spermatozoa were 2% Trypan blue at 37°C for 15 minutes, 0.8% Bismarck brown in 30% ethyl alcohol (PH 2.8) at 40°C for 10 minutes and 0.8% rose Bengal in 0.1 M of Tris (pH 4.3) at 21°C for 20 minutes. Satisfactory results were obtained for incubated spermatozoa with rose Bengal when the staining time was 10 minutes. Triple stain technique seemed to be a useful method for the simultaneous assessment of sperm vitality and acrosomal status; consequently, it should be valuable tool, for use in porcine in vitro fertilization systems, Key words: sperm, acrosome, capacitation, triple stain technique, boar
INTRODUCTION The mammalian spermatozoon must undergo morphological changes, termed the acrosome reaction, before it is capable of fertilizing the ovum (1). This event, occurring only in live spermatozoa, is referred to as the true acrosome reaction. In contrast, false acrosome reaction occurs only in dead spermatozoa as a postmortem change (2).
Acknowledgments This research was supported in part by CICYT nQAGF92/0521.
Copyright 0 1992 Butterworth-Heinemann
844
Theriogenology
The analysis of acrosomal structure and function is currently receiving much attention (3-7). Although the ideal assay to assess acrosomal status (simultaneous assessment of motility and acrosomal status on the same living cell) are not yet available, existing techniques, if appropriately employed, may prove to be adequate. A triple stain technique has been developed for human spermatozoa to categorize true or false acrosome reaction using the bright-field microscope (3). This technique has also been adapted for use with mouse (8), bull (4), horse (9) and goat (6) spermatozoa. The triple stain technique employs Trypan blue to distinguish live spermatozoa, which exclude the dye; Bismarck brown is used to stain the postacrosomal region of all spermatozoa; while rose Bengal is used to stain the acrosomal region of acrosome-intact spermatozoa. The aim of this study was to adapt and validate the triple stain technique with diluted and incubated boar sperm.
MATERIALS
for use
AND METHODS
Sperm Treatment The sperm-rich fractions were collected from mature Landrace boars using the gloved hand method. Semen from 2 boars was pooled and used for each assay. The freshly ejaculated semen was resuspended in MR-A diluent (10) to contain 3 x 10’ cells/ml (diluted spermatozoa). These were subsequently capacitated (incubated spermatozoa) by using a basic method described by Cheng et al. (11). Briefly, the diluted spermatozoa were incubated for about 24 hours at 16°C and were then centrifuged at 50 g for 3 minutes. The supernatant was washed by centrifugation (3 x 3 minutes) at 1,200 g in 0.9% saline containing 1 mg/ml of bovine serum albumin (BSA: Fraction V, Sigma Chemical Co., St Louis, MO, USA), with a pH 7.2. The spermatozoa were subsequently diluted to 2 x 10’ cells/ml in modified TCM-199 (Boehringer, Matmheim, Germany; TCM-199m) as has been described earlier (12), and the pH was adjusted to 7.8. Then, the spermatozoa were preincubated for 40 minutes at 39°C in a tightly capped test tube. Then, the spermatozoa were diluted to 10’ cells/ml in TCM-199m enriched with 2 mM caffeine and 5.8 mM calcium lactate, at a pH of 7.4. The incubations were carried out at 39°C in an atmosphere of 5% CO, in air. The spermatozoa were removed at 2,4 and 6 hours and processed for triple staining technique. Staining Procedures
- Diluted
Spermatozoa
The basic triple stain technique used in the experiment was developed by Talbot and Chacon (3) and modified by Kusunoki et al. (6). We further adapted the triple stain technique for use with boar spermatozoa. I&pan bm Assays with Trypan blue were carried out using a 4 x 6 factorial design and were used to determine both 1) the adequate concentration of
845
Theriogenology
Trypan blue and 2) the exposure time of diluted spermatozoa to the staining solutions to achieve good staining quality. Spermatozoa suspended in MR-A were diluted with an equal volume of 0.4, 1, 2 or 3% Trypan blue in TCM-199 and were scored for quality of staining at 1, 5, 15, 25, 35 and 55 minutes. Quality of the staining was determined in 5 replicates and is reported as either understained or good. Osmolarity of Trypan blue solutions, determined by microosmometer (Advanced Mod. 3MO), ranged from 300 to 340 mOsm/kg. To prove that Trypan blue stains only the dead spermatozoa, semen samples were plunged into liquid nitrogen and thawed (3 times), and were then washed in MR-A (2 times). Samples were prepared with ratios of live to killed spermatozoa of 10&O, 75:25,50:50,25:75 and 0:lOO. The spermatozoa were incubated with 2% Trypan blue in TCM-199 at 37°C for 15 minutes. Spermatozoa were placed on slides and were air-dried. The slides were examined under a bright-field microscope at 1000x magnification, and the spermatozoa were scored for the percentage of blue (dead) spermatozoa. The slides were counted for a total of 400 spermatozoa per sample for each of 5 replicates. rck brown w This experiment (2 x 2 x 2 factorial) was conducted to examine the effects of the concentration (0.5 or 0.8%), diluent (100% purified water and 30% ethyl alcohol in water solution), and pH (1.8 or 2.8) of Bismarck brown solutions on staining quality. Bismarck brown solutions were buffered to pH 1.8 or 2.8 with 0.5 M of TrisHCl. Sperm suspensions were diluted in an equal volume of the TCM-199 containing 2% Trypan blue; they were then incubated at 37°C for 15 minutes and smeared on prewarmed glass slides and air-dried. The smears on slides were fixed in 3% glutaraldehyde solution in 0.1 M of cacodylate buffer at 20°C for 30 to 45 minutes, rinsed in water and air-dried. Finally, the smears were stained with the different Bismarck brown solutions described above at 40°C for 10 minutes, rinsed briefly in water and air-dried. The slides were microscopically examined (1000x) for the quality of staining as for the Trypan blue assay. Rose Bw This experiment was conducted to find the optimal pH of rose Bengal solutions. The spermatozoa were stained as described for the Bismarck brown assay. The spermatozoa were stained in 0.8% rose Bengal solutions in 0.1 M of TrisHCl at pH 4.3,5.3 or 6.3 for 20 minutes at 20°C. The spermatozoa were then rinsed well in water and were air-dried. To evaluate the staining quality, classed as understained, good or overstained, the slides were microscopically examined (1000x) for each of 5 replicates. Staining Procedures
- Incubated
Spermatozoa
Modification of the triple stain technique for diluted spermatozoa was necessary for incubated spermatozoa. First the spermatozoa were triple stained as described above except that the staining periods for Bismarck brown (5 or 10 minutes) and in rose Bengal (10, 15 or 20 minutes) solutions were modified (2 x 3 factorial). Staining quality was evaluated as described above for the rose Bengal assay.
846 Validation
Theriogenolog y of Triple Stain Technique
To validate the triple stain technique as a method to determine sperm vitality, the Trypan blue staining method was used. The spermatozoa were incubated with 2% Trypan blue in TCM-199 at 37°C for 15 minutes. Spermatozoa were placed on slides and were air-dried. The percentage of spermatozoa with damaged acrosomes assessed by triple stain technique was compared with that of damaged acrosomes determined under phase contrast microscopy of samples fixed in 2% glutaraldehyde according to the method of Purse1 et al. (13); 400 cells were examined for each of 5 replicates. Statistical Analysis The statistical significance square analysis (14).
of treatment
comparisons
was determined
by Chi-
RESULTS Diluted Spermatozoa The staining quality of the different Trypan blue concentrations and staining periods are shown in Table 1. When spermatozoa were incubated with 0.4, 1, 2 or 3% Trypan blue at 1 or 5 minutes, the quality of staining was classed as understained in all the trials. When 1% Trypan blue was used, a minimum of 25 minutes was required for good staining. The minimum period of incubation for optimal quality staining was 15 minutes when a concentration of 2% of Trypan blue was used. Data from samples with different ratios of live to killed spermatozoa (Table 2) indicate that there were no significant differences in percentages of live spermatozoa between observed and estimated spermatozoa. Results for the Bismarck brown assay are presented in Table 3. Good quality staining was obtained only in 0.8% Bismarck brown in 30% ethyl alcohol at a pH of 2.8. The pH of rose Bengal solutions affected the ability of the dye to produce good staining of the acrosome in the triple stain procedure. In all instances the spermatozoa were overstained by rose Bengal at a pH of 6.3. At a pH of 5.3, good staining was achieved in 3 replicates but overstaining occurred in 2 trials. At a pH of 4.3, the spermatozoa had a good quality. This triple stain protocol distinguishes four categories of spermatozoa: 1) unstained or light brown postacrosomal regions with pink acrosomes (live spermatozoa with normal acrosomes); 2) unstained or light brown postacrosomal regions with unstained or poorly pink stained acrosomal regions (live spermatozoa without normal acrosomes); 3) dark brown postacrosomal regions with dark pink acrosomes (dead spermatozoa with normal acrosomes); and 4) dark brown postacrosomal regions with unstained or poorly pink stained acrosomal regions (dead spermatozoa without normal acrosomes).
847
Theriogenology
Table 1. Influence of concentration of Trypan blue solutions and periods of sperm exposure on the quality of staining a*b Period of staining (minutes) Trypan blue (%)
1
15
5
0.4
uuuuuu
1
UUUGGG
2
UUGGGG
3
UUGGGG
25
35
L Evaluations of the quality of staining of spermatozoa Staining quality: understained (U) and good (G).
Table 2. Validation of Trypan killed spermatozoaa
55
were made on 5 replicates.
blue stain with different proportions
of live and
Live spermatozoa (%) Ratiob Observed’
Estimated
100/o
85.1526.31
85.15k6.31
7512
66.3 1” 5.69
64.17k5.40
so/so
43.7Ok3.54
42.50k3.24
25175
21.5022.19
21.272 1.56
o/100
0
0
a Spermatozoa were incubated 15 minutes in 2% Trypan blue in TCM-199 before scoring. b Ratio of fresh spermatozoa to those killed by plunging semen into liquid nitrogen then thawing (3 times). ’ Values represent means f SD of 5 replicates; 400 spermatozoa were counted per replicate.
Theriogenolog y
848 Table 3. Quality of staining for the different conditions sohrtiorPb Bismarck brown (%)
of Bismarck brown
Water
30% Ethyl alcohol pH 1.8
pH 2.8
pH 1.8
pH 2.8
0.5%
U
U
U
U
0.8%
U
G
U
U
a Evaluations of the quality of staining of spermatozoa b Staining quality: understained (U) and good (G).
were made on 5 replicates.
Table 4. Effect of length of staining time of Bismarck brown and rose Bengal dyes in diluted and incubated spermatozoaaPb Duration of incubation (hours) Timec
0
2
4
6
5/10
U
U
U
U
5/15
U
U
U
U
5120
U
U
U
U
lO/lO
U
G
G
G
lO/ 15
U
.
.
.
10120
G
.
.
.
a Evaluations of the quality of staining of spermatozoa were made on 5 replicates. b Staining quality: understained (U), good (G) and overstained (m). ’ The times in Bismarck brown/time in rose Bengal is given in minutes.
849
Theriogenology Incubated
Spermatozoa
The effect of length of staining time in Bismarck brown and rose Bengal stains is shown in Table 4. When the staining procedure for diluted spermatozoa was used on incubated spermatozoa, the latter, were overstained. Satisfactory results were obtained when the staining period for rose Bengal was 10 minutes. Validation
of Triple Stain Technique
Shown in Table 5 are the percentages of unstained (live) cells when sperm vitality was assessed in diluted and incubated spermatozoa using either triple stain technique or Trypan blue method. The data indicate that there were no significant differences between the parameters tested. Similar results were found for the percentages of normal acrosome spermatozoa assessed by triple stain technique and by 2% glutaraldehyde method.
Table 5. Validation of the triple stain technique (TST) procedure for the simultaneous assessment of vitality and acrosomal status of boar spermatozoa* Trypanb Blue
TST
Glutaraldehyde’
% Live
% Acrosome damage
% Live
Diluted
80.6k2.3
19.8-t 1.9
81.3+3.1
19.0+ 4.4
Incubatedd
23.2? 5.0
82.0* 6.7
23.8 + 4.9
80.4 t 6.8
Spermatozoa
% Acrosome damage
a Values represent means+SD of 5 replicates; 400 spermatozoa were counted per replicate. b Spermatozoa were incubated 15 minutes in 2% Trypan blue in TCM-199 before scoring. ’ Spermatozoa were fixed in 2% glutaraldehyde and were examined by phase contrast microscopy. d A 6 hours of incubation was used in the trial.
DISCUSSION It is very important to distinguish degenerative acrosomal modifications in dead spermatozoa from the physiological acrosome reactions that occur in live motile spermatozoa. Spermatozoa with large acrosomes (e.g., guinea pig) can be inspected satisfactorily without fixing by phase-contrast microscopy. Since spermatozoa of many mammals (including the pig) have either thin or small acrosomes, and their
850
Theriogenology
acrosome reactions are difficult to detect in a live state, several techniques have been developed to aid in the assessment of acrosome reaction. The triple stain technique is perhaps the most widely used, having been applied to several species, but to date, it has not been used on boar spermatozoa. Although the methods and reagents in the triple stain technique of human and bull spermatozoa have been described (3,4), modifications were necessary for application to boar spermatozoa. Thus our results indicate that boar spermatozoa need to be incubated in 2% Trypan blue for 15 minutes or in 1% Trypan blue for 25 minutes to assure that dead spermatozoa are well stained. Since Trypan blue solutions are made up readily, we recommend that these solutions be prepared immediately before use. This produces uniform staining quality. Bamba (5), by contrast, using eosin-nigrosin staining, found difficulty in classifying spermatozoa as live or dead, because some spermatozoa (lo%-15% in smears from certain ejaculates) become stained only in the postacrosomal region (half-stained). In our study, half-stained spermatozoa were present at a low rate (< 2%) except when inadequate manipulation of spermatozoa during the staining procedure occurred (unpublished observations). The efficiency of Trypan blue staining is extremely dependent upon the conditions under with it is used. We attributed the half staining of spermatozoa to changes occurring in the membrane, and like Bamba (5), we classified these cells as dead spermatozoa. Mortimer et al. (15) suggest that Trypan blue staining leads to overestimates in the proportion of dead cells in human spermatozoa. However, in our study, no significant differences were observed in the percentage of live spermatozoa observed and that estimated between the ratios of live to dead cells. Our results indicate that Trypan blue does stain all the dead spermatozoa in boar semen. The chemical mechanisms of triple stain technique are poorly understood (16). Although it is well known that Bismarck brown stains the postacrosomal region light brown, and prevents nonspecific staining of these region by rose Bengal, which stains the acrosome pink (17), some modifications of Bismarck brown solutions are necessary for use on boar spermatozoa, unlike that of other species such as the human, bovine and caprine, to obtain good specific-site staining. Likewise, the pH of rose Bengal solutions may affect staining quality. In human spermatozoa optimal staining using rose Bengal occurs at a pH of 5.3 (17). This pH has also been used on bovine and caprine spermatozoa (4,6). In our study, when the pH of rose Bengal solutions were adjusted to 4.3, excellent results were obtained, perhaps due to the specific characteristic of the boar sperm membrane. This same pH produced understained cells when used on human spermatozoa.
We also detected variation in the optimal time for rose Bengal staining when incubated spermatozoa were used. Presumably this occurred because the plasma membrane was directly exposed to the capacitating medium.
851
Theriogenology
The assays we used validated that triple stain technique is an efficacious method for the simultaneous assessment of sperm vitality and acrosome status. This technique might also be useful for quantifying the number of spermatozoa with true acrosome reactions. As such it would be a valuable instrument for use in porcine in vitro fertilization systems. However, the efficiency of triple stain technique is extremely dependent upon the staining conditions under with it is used for optimal differentiation of 4 classes of spermatozoa: live unreacted, live reacted, dead unreacted and dead reacted.
REFERENCES 1. Austin, C.R. and Bishop, M.W.H. Role of the rodent and acrosome and perforatium in fertilization. Proc. Roy. Sot. London Ser. B. I&?: 241-248 (1958). 2. Bedford, J.M. Sperm capacitation 2 (Suppl.): 128-158 (1970).
and fertilization
in mammals.
Biol. Reprod.
3. Talbot, P. and Chacon, R.S. A triple stain technique for evaluating normal acrosome reactions of human sperm. J. Exp. Zool., 215: 201-208 (1981). 4. Didion, B.A. and Graves, C.N. In vivo capacitation and acrosome reaction of bovine sperm in estrous and diestrous cows. J. Anim. Sci., a: 1029-1033 (1986). 5. Bamba, K. Evaluation of acrosomal integrity of boar spermatozoa by bright field microscopy using an eosin-nigrosin stain. Theriogenology Be: 1245-1251 (1988). 6. Kusunoki, H., Sakaue, M., Kato, S, and Kanda, S. Induction of the acrosome reaction in ejaculated goat spermatozoa by preincubation in chemically defined medium. J. Exp. Zool. m: 322-328 (1989). 7. Didion, B.A., Dowrinsky, J.R., Giles, J.R. and Graves, C.N. Staining procedure to detect viability and the true acrosome reaction in spermatozoa of various species. Gamete Res. 2: 51-57 (1990). 8. Talbot, P. and Chacon, R.S. Observation on the acrosome sperm in vitro. Am. J. Primatol. 1: 211-219 (1981).
reaction of human
9. Varner, D.D., Ward, CR., Storey, B.T. and Kenney, R.M. Induction and characterization of acrosome reaction in equine sperm. Am. J. Vet. Res. as: 1383-1389 (1987). 10. Martin, S., Perez, C. and Alias, E. Fertility results by using boar semen diluted 1:lO after 5 days storage. 34 Reuni6n Anual de F.E.Z. (Madrid) 2: 755 abstr. (1983).
852
Theriogenology
11. Cheng, W.T.K.. Moor, R.M. and Polge, C. In vitro fertilization of pig and sheep oocytes matured in viva and in vitro. Theriogenology a: 146 abstr. (1986). 12. Mattioli, M., Galeati, G. and Seren, E. Effect of follicle somatic cells during pig oocyte maturation on egg penetrability and male pronucleus formation. Gamete Res. 21): 177-184 (1988). 13. Pursel, V.G., Johnson, L.A. and Rampacek, G.B. Acrosome morphology of boar spermatozoa incubated before cold shock. J. Anim. Sci. 3: 278-283 (1972). 14. Steel, R.G.D. and Torrie, J.H. Principles and procedures of statistics: Biometrical Approach. McGraw-Hill, New York, 1980, pp. 469-475.
A
15. Mortimer, D., Curtis, E.F. and Camenzind, A.R. Combined use of fluorescent peanut agglutinin lectin and Hoechst 33258 to monitor the acrosomal status and vitality of human spermatozoa. Human Reprod. 5: 99-103 (1990). 16. Cross, N.L. and Meizel, S. Methods for evaluating the acrosomal mammalian sperm. Biol. Reprod. U: 635-641 (1989). 17. Talbot, P. and Dudenhausen, E. Factors affecting triple staining sperm. Stain Technol. 56: 307-309. (1981).
status of
of human
USE OF TRIPLE STAIN TECHNIQUE FOR SIMULTANEOUS ASSESSMENT OF VITALITY AND ACROSOMAL STATUS IN BOAR SPERMATOZOA J.M. Vkquez’,
E. Martinez’,
P. Co+ and S. Ruid
J. Rod,
Departments of ‘Animal Pathology and 2Animal Biology Facultad de Veterinaria, Universidad de Murcia 30071 Murcia, Spain Received for publication: kbrch Accepted: JUZY
3, 5,
I 992 1x2
ABSTRACT
The object of this study was to adapt the triple stain technique to diluted and incubated boar spermatozoa. Freshly ejaculated semen was resuspended in MR-A diluent to contain 3x10’ cells/ml (diluted spermatozoa) and was subsequently capacitated (incubated spermatozoa). Experiments were conducted to show the conditions required for optimal staining quality and validation of triple stain technique. The most satisfactory staining solutions for diluted spermatozoa were 2% Trypan blue at 37°C for 15 minutes, 0.8% Bismarck brown in 30% ethyl alcohol (PH 2.8) at 40°C for 10 minutes and 0.8% rose Bengal in 0.1 M of Tris (pH 4.3) at 21°C for 20 minutes. Satisfactory results were obtained for incubated spermatozoa with rose Bengal when the staining time was 10 minutes. Triple stain technique seemed to be a useful method for the simultaneous assessment of sperm vitality and acrosomal status; consequently, it should be valuable tool, for use in porcine in vitro fertilization systems, Key words: sperm, acrosome, capacitation, triple stain technique, boar
INTRODUCTION The mammalian spermatozoon must undergo morphological changes, termed the acrosome reaction, before it is capable of fertilizing the ovum (1). This event, occurring only in live spermatozoa, is referred to as the true acrosome reaction. In contrast, false acrosome reaction occurs only in dead spermatozoa as a postmortem change (2).
Acknowledgments This research was supported in part by CICYT nQAGF92/0521.
Copyright 0 1992 Butterworth-Heinemann
844
Theriogenology
The analysis of acrosomal structure and function is currently receiving much attention (3-7). Although the ideal assay to assess acrosomal status (simultaneous assessment of motility and acrosomal status on the same living cell) are not yet available, existing techniques, if appropriately employed, may prove to be adequate. A triple stain technique has been developed for human spermatozoa to categorize true or false acrosome reaction using the bright-field microscope (3). This technique has also been adapted for use with mouse (8), bull (4), horse (9) and goat (6) spermatozoa. The triple stain technique employs Trypan blue to distinguish live spermatozoa, which exclude the dye; Bismarck brown is used to stain the postacrosomal region of all spermatozoa; while rose Bengal is used to stain the acrosomal region of acrosome-intact spermatozoa. The aim of this study was to adapt and validate the triple stain technique with diluted and incubated boar sperm.
MATERIALS
for use
AND METHODS
Sperm Treatment The sperm-rich fractions were collected from mature Landrace boars using the gloved hand method. Semen from 2 boars was pooled and used for each assay. The freshly ejaculated semen was resuspended in MR-A diluent (10) to contain 3 x 10’ cells/ml (diluted spermatozoa). These were subsequently capacitated (incubated spermatozoa) by using a basic method described by Cheng et al. (11). Briefly, the diluted spermatozoa were incubated for about 24 hours at 16°C and were then centrifuged at 50 g for 3 minutes. The supernatant was washed by centrifugation (3 x 3 minutes) at 1,200 g in 0.9% saline containing 1 mg/ml of bovine serum albumin (BSA: Fraction V, Sigma Chemical Co., St Louis, MO, USA), with a pH 7.2. The spermatozoa were subsequently diluted to 2 x 10’ cells/ml in modified TCM-199 (Boehringer, Matmheim, Germany; TCM-199m) as has been described earlier (12), and the pH was adjusted to 7.8. Then, the spermatozoa were preincubated for 40 minutes at 39°C in a tightly capped test tube. Then, the spermatozoa were diluted to 10’ cells/ml in TCM-199m enriched with 2 mM caffeine and 5.8 mM calcium lactate, at a pH of 7.4. The incubations were carried out at 39°C in an atmosphere of 5% CO, in air. The spermatozoa were removed at 2,4 and 6 hours and processed for triple staining technique. Staining Procedures
- Diluted
Spermatozoa
The basic triple stain technique used in the experiment was developed by Talbot and Chacon (3) and modified by Kusunoki et al. (6). We further adapted the triple stain technique for use with boar spermatozoa. I&pan bm Assays with Trypan blue were carried out using a 4 x 6 factorial design and were used to determine both 1) the adequate concentration of
845
Theriogenology
Trypan blue and 2) the exposure time of diluted spermatozoa to the staining solutions to achieve good staining quality. Spermatozoa suspended in MR-A were diluted with an equal volume of 0.4, 1, 2 or 3% Trypan blue in TCM-199 and were scored for quality of staining at 1, 5, 15, 25, 35 and 55 minutes. Quality of the staining was determined in 5 replicates and is reported as either understained or good. Osmolarity of Trypan blue solutions, determined by microosmometer (Advanced Mod. 3MO), ranged from 300 to 340 mOsm/kg. To prove that Trypan blue stains only the dead spermatozoa, semen samples were plunged into liquid nitrogen and thawed (3 times), and were then washed in MR-A (2 times). Samples were prepared with ratios of live to killed spermatozoa of 10&O, 75:25,50:50,25:75 and 0:lOO. The spermatozoa were incubated with 2% Trypan blue in TCM-199 at 37°C for 15 minutes. Spermatozoa were placed on slides and were air-dried. The slides were examined under a bright-field microscope at 1000x magnification, and the spermatozoa were scored for the percentage of blue (dead) spermatozoa. The slides were counted for a total of 400 spermatozoa per sample for each of 5 replicates. rck brown w This experiment (2 x 2 x 2 factorial) was conducted to examine the effects of the concentration (0.5 or 0.8%), diluent (100% purified water and 30% ethyl alcohol in water solution), and pH (1.8 or 2.8) of Bismarck brown solutions on staining quality. Bismarck brown solutions were buffered to pH 1.8 or 2.8 with 0.5 M of TrisHCl. Sperm suspensions were diluted in an equal volume of the TCM-199 containing 2% Trypan blue; they were then incubated at 37°C for 15 minutes and smeared on prewarmed glass slides and air-dried. The smears on slides were fixed in 3% glutaraldehyde solution in 0.1 M of cacodylate buffer at 20°C for 30 to 45 minutes, rinsed in water and air-dried. Finally, the smears were stained with the different Bismarck brown solutions described above at 40°C for 10 minutes, rinsed briefly in water and air-dried. The slides were microscopically examined (1000x) for the quality of staining as for the Trypan blue assay. Rose Bw This experiment was conducted to find the optimal pH of rose Bengal solutions. The spermatozoa were stained as described for the Bismarck brown assay. The spermatozoa were stained in 0.8% rose Bengal solutions in 0.1 M of TrisHCl at pH 4.3,5.3 or 6.3 for 20 minutes at 20°C. The spermatozoa were then rinsed well in water and were air-dried. To evaluate the staining quality, classed as understained, good or overstained, the slides were microscopically examined (1000x) for each of 5 replicates. Staining Procedures
- Incubated
Spermatozoa
Modification of the triple stain technique for diluted spermatozoa was necessary for incubated spermatozoa. First the spermatozoa were triple stained as described above except that the staining periods for Bismarck brown (5 or 10 minutes) and in rose Bengal (10, 15 or 20 minutes) solutions were modified (2 x 3 factorial). Staining quality was evaluated as described above for the rose Bengal assay.
846 Validation
Theriogenolog y of Triple Stain Technique
To validate the triple stain technique as a method to determine sperm vitality, the Trypan blue staining method was used. The spermatozoa were incubated with 2% Trypan blue in TCM-199 at 37°C for 15 minutes. Spermatozoa were placed on slides and were air-dried. The percentage of spermatozoa with damaged acrosomes assessed by triple stain technique was compared with that of damaged acrosomes determined under phase contrast microscopy of samples fixed in 2% glutaraldehyde according to the method of Purse1 et al. (13); 400 cells were examined for each of 5 replicates. Statistical Analysis The statistical significance square analysis (14).
of treatment
comparisons
was determined
by Chi-
RESULTS Diluted Spermatozoa The staining quality of the different Trypan blue concentrations and staining periods are shown in Table 1. When spermatozoa were incubated with 0.4, 1, 2 or 3% Trypan blue at 1 or 5 minutes, the quality of staining was classed as understained in all the trials. When 1% Trypan blue was used, a minimum of 25 minutes was required for good staining. The minimum period of incubation for optimal quality staining was 15 minutes when a concentration of 2% of Trypan blue was used. Data from samples with different ratios of live to killed spermatozoa (Table 2) indicate that there were no significant differences in percentages of live spermatozoa between observed and estimated spermatozoa. Results for the Bismarck brown assay are presented in Table 3. Good quality staining was obtained only in 0.8% Bismarck brown in 30% ethyl alcohol at a pH of 2.8. The pH of rose Bengal solutions affected the ability of the dye to produce good staining of the acrosome in the triple stain procedure. In all instances the spermatozoa were overstained by rose Bengal at a pH of 6.3. At a pH of 5.3, good staining was achieved in 3 replicates but overstaining occurred in 2 trials. At a pH of 4.3, the spermatozoa had a good quality. This triple stain protocol distinguishes four categories of spermatozoa: 1) unstained or light brown postacrosomal regions with pink acrosomes (live spermatozoa with normal acrosomes); 2) unstained or light brown postacrosomal regions with unstained or poorly pink stained acrosomal regions (live spermatozoa without normal acrosomes); 3) dark brown postacrosomal regions with dark pink acrosomes (dead spermatozoa with normal acrosomes); and 4) dark brown postacrosomal regions with unstained or poorly pink stained acrosomal regions (dead spermatozoa without normal acrosomes).
847
Theriogenology
Table 1. Influence of concentration of Trypan blue solutions and periods of sperm exposure on the quality of staining a*b Period of staining (minutes) Trypan blue (%)
1
15
5
0.4
uuuuuu
1
UUUGGG
2
UUGGGG
3
UUGGGG
25
35
L Evaluations of the quality of staining of spermatozoa Staining quality: understained (U) and good (G).
Table 2. Validation of Trypan killed spermatozoaa
55
were made on 5 replicates.
blue stain with different proportions
of live and
Live spermatozoa (%) Ratiob Observed’
Estimated
100/o
85.1526.31
85.15k6.31
7512
66.3 1” 5.69
64.17k5.40
so/so
43.7Ok3.54
42.50k3.24
25175
21.5022.19
21.272 1.56
o/100
0
0
a Spermatozoa were incubated 15 minutes in 2% Trypan blue in TCM-199 before scoring. b Ratio of fresh spermatozoa to those killed by plunging semen into liquid nitrogen then thawing (3 times). ’ Values represent means f SD of 5 replicates; 400 spermatozoa were counted per replicate.
Theriogenolog y
848 Table 3. Quality of staining for the different conditions sohrtiorPb Bismarck brown (%)
of Bismarck brown
Water
30% Ethyl alcohol pH 1.8
pH 2.8
pH 1.8
pH 2.8
0.5%
U
U
U
U
0.8%
U
G
U
U
a Evaluations of the quality of staining of spermatozoa b Staining quality: understained (U) and good (G).
were made on 5 replicates.
Table 4. Effect of length of staining time of Bismarck brown and rose Bengal dyes in diluted and incubated spermatozoaaPb Duration of incubation (hours) Timec
0
2
4
6
5/10
U
U
U
U
5/15
U
U
U
U
5120
U
U
U
U
lO/lO
U
G
G
G
lO/ 15
U
.
.
.
10120
G
.
.
.
a Evaluations of the quality of staining of spermatozoa were made on 5 replicates. b Staining quality: understained (U), good (G) and overstained (m). ’ The times in Bismarck brown/time in rose Bengal is given in minutes.
849
Theriogenology Incubated
Spermatozoa
The effect of length of staining time in Bismarck brown and rose Bengal stains is shown in Table 4. When the staining procedure for diluted spermatozoa was used on incubated spermatozoa, the latter, were overstained. Satisfactory results were obtained when the staining period for rose Bengal was 10 minutes. Validation
of Triple Stain Technique
Shown in Table 5 are the percentages of unstained (live) cells when sperm vitality was assessed in diluted and incubated spermatozoa using either triple stain technique or Trypan blue method. The data indicate that there were no significant differences between the parameters tested. Similar results were found for the percentages of normal acrosome spermatozoa assessed by triple stain technique and by 2% glutaraldehyde method.
Table 5. Validation of the triple stain technique (TST) procedure for the simultaneous assessment of vitality and acrosomal status of boar spermatozoa* Trypanb Blue
TST
Glutaraldehyde’
% Live
% Acrosome damage
% Live
Diluted
80.6k2.3
19.8-t 1.9
81.3+3.1
19.0+ 4.4
Incubatedd
23.2? 5.0
82.0* 6.7
23.8 + 4.9
80.4 t 6.8
Spermatozoa
% Acrosome damage
a Values represent means+SD of 5 replicates; 400 spermatozoa were counted per replicate. b Spermatozoa were incubated 15 minutes in 2% Trypan blue in TCM-199 before scoring. ’ Spermatozoa were fixed in 2% glutaraldehyde and were examined by phase contrast microscopy. d A 6 hours of incubation was used in the trial.
DISCUSSION It is very important to distinguish degenerative acrosomal modifications in dead spermatozoa from the physiological acrosome reactions that occur in live motile spermatozoa. Spermatozoa with large acrosomes (e.g., guinea pig) can be inspected satisfactorily without fixing by phase-contrast microscopy. Since spermatozoa of many mammals (including the pig) have either thin or small acrosomes, and their
850
Theriogenology
acrosome reactions are difficult to detect in a live state, several techniques have been developed to aid in the assessment of acrosome reaction. The triple stain technique is perhaps the most widely used, having been applied to several species, but to date, it has not been used on boar spermatozoa. Although the methods and reagents in the triple stain technique of human and bull spermatozoa have been described (3,4), modifications were necessary for application to boar spermatozoa. Thus our results indicate that boar spermatozoa need to be incubated in 2% Trypan blue for 15 minutes or in 1% Trypan blue for 25 minutes to assure that dead spermatozoa are well stained. Since Trypan blue solutions are made up readily, we recommend that these solutions be prepared immediately before use. This produces uniform staining quality. Bamba (5), by contrast, using eosin-nigrosin staining, found difficulty in classifying spermatozoa as live or dead, because some spermatozoa (lo%-15% in smears from certain ejaculates) become stained only in the postacrosomal region (half-stained). In our study, half-stained spermatozoa were present at a low rate (< 2%) except when inadequate manipulation of spermatozoa during the staining procedure occurred (unpublished observations). The efficiency of Trypan blue staining is extremely dependent upon the conditions under with it is used. We attributed the half staining of spermatozoa to changes occurring in the membrane, and like Bamba (5), we classified these cells as dead spermatozoa. Mortimer et al. (15) suggest that Trypan blue staining leads to overestimates in the proportion of dead cells in human spermatozoa. However, in our study, no significant differences were observed in the percentage of live spermatozoa observed and that estimated between the ratios of live to dead cells. Our results indicate that Trypan blue does stain all the dead spermatozoa in boar semen. The chemical mechanisms of triple stain technique are poorly understood (16). Although it is well known that Bismarck brown stains the postacrosomal region light brown, and prevents nonspecific staining of these region by rose Bengal, which stains the acrosome pink (17), some modifications of Bismarck brown solutions are necessary for use on boar spermatozoa, unlike that of other species such as the human, bovine and caprine, to obtain good specific-site staining. Likewise, the pH of rose Bengal solutions may affect staining quality. In human spermatozoa optimal staining using rose Bengal occurs at a pH of 5.3 (17). This pH has also been used on bovine and caprine spermatozoa (4,6). In our study, when the pH of rose Bengal solutions were adjusted to 4.3, excellent results were obtained, perhaps due to the specific characteristic of the boar sperm membrane. This same pH produced understained cells when used on human spermatozoa.
We also detected variation in the optimal time for rose Bengal staining when incubated spermatozoa were used. Presumably this occurred because the plasma membrane was directly exposed to the capacitating medium.
851
Theriogenology
The assays we used validated that triple stain technique is an efficacious method for the simultaneous assessment of sperm vitality and acrosome status. This technique might also be useful for quantifying the number of spermatozoa with true acrosome reactions. As such it would be a valuable instrument for use in porcine in vitro fertilization systems. However, the efficiency of triple stain technique is extremely dependent upon the staining conditions under with it is used for optimal differentiation of 4 classes of spermatozoa: live unreacted, live reacted, dead unreacted and dead reacted.
REFERENCES 1. Austin, C.R. and Bishop, M.W.H. Role of the rodent and acrosome and perforatium in fertilization. Proc. Roy. Sot. London Ser. B. I&?: 241-248 (1958). 2. Bedford, J.M. Sperm capacitation 2 (Suppl.): 128-158 (1970).
and fertilization
in mammals.
Biol. Reprod.
3. Talbot, P. and Chacon, R.S. A triple stain technique for evaluating normal acrosome reactions of human sperm. J. Exp. Zool., 215: 201-208 (1981). 4. Didion, B.A. and Graves, C.N. In vivo capacitation and acrosome reaction of bovine sperm in estrous and diestrous cows. J. Anim. Sci., a: 1029-1033 (1986). 5. Bamba, K. Evaluation of acrosomal integrity of boar spermatozoa by bright field microscopy using an eosin-nigrosin stain. Theriogenology Be: 1245-1251 (1988). 6. Kusunoki, H., Sakaue, M., Kato, S, and Kanda, S. Induction of the acrosome reaction in ejaculated goat spermatozoa by preincubation in chemically defined medium. J. Exp. Zool. m: 322-328 (1989). 7. Didion, B.A., Dowrinsky, J.R., Giles, J.R. and Graves, C.N. Staining procedure to detect viability and the true acrosome reaction in spermatozoa of various species. Gamete Res. 2: 51-57 (1990). 8. Talbot, P. and Chacon, R.S. Observation on the acrosome sperm in vitro. Am. J. Primatol. 1: 211-219 (1981).
reaction of human
9. Varner, D.D., Ward, CR., Storey, B.T. and Kenney, R.M. Induction and characterization of acrosome reaction in equine sperm. Am. J. Vet. Res. as: 1383-1389 (1987). 10. Martin, S., Perez, C. and Alias, E. Fertility results by using boar semen diluted 1:lO after 5 days storage. 34 Reuni6n Anual de F.E.Z. (Madrid) 2: 755 abstr. (1983).
852
Theriogenology
11. Cheng, W.T.K.. Moor, R.M. and Polge, C. In vitro fertilization of pig and sheep oocytes matured in viva and in vitro. Theriogenology a: 146 abstr. (1986). 12. Mattioli, M., Galeati, G. and Seren, E. Effect of follicle somatic cells during pig oocyte maturation on egg penetrability and male pronucleus formation. Gamete Res. 21): 177-184 (1988). 13. Pursel, V.G., Johnson, L.A. and Rampacek, G.B. Acrosome morphology of boar spermatozoa incubated before cold shock. J. Anim. Sci. 3: 278-283 (1972). 14. Steel, R.G.D. and Torrie, J.H. Principles and procedures of statistics: Biometrical Approach. McGraw-Hill, New York, 1980, pp. 469-475.
A
15. Mortimer, D., Curtis, E.F. and Camenzind, A.R. Combined use of fluorescent peanut agglutinin lectin and Hoechst 33258 to monitor the acrosomal status and vitality of human spermatozoa. Human Reprod. 5: 99-103 (1990). 16. Cross, N.L. and Meizel, S. Methods for evaluating the acrosomal mammalian sperm. Biol. Reprod. U: 635-641 (1989). 17. Talbot, P. and Dudenhausen, E. Factors affecting triple staining sperm. Stain Technol. 56: 307-309. (1981).
status of
of human