5. RESULTS
5.1 Experiment 1. Improvement of assessment of stallion sperm quality by Chicago sky blue
staining method
Subjective evaluation
In a preliminary study, we evaluated different fixation (2, 3, 4, 6 min) and Giemsa staining times (1-4 h, and overnight) following TB and CSB staining. Three different concentrations of CSB (0.26%, 0.16%, 0.13%) also were tested. Among these, the 0.16% CSB solution proved best, resulting in similar sperm heads, but better tail live/dead differentiation compared to 0.27% TB (Figs. 13-14).
Figure 13. Frozen stallion spermatozoa stained with 0.16% CSB/Giemsa a) Spermatozoon with intact head, tail and acrosome membrane b) Spermatozoon with damaged head, tail and acrosome membrane
c) Spermatozoon with intact head and damaged tail membrane Bar = 10 µm
Figure 14. Frozen stallion spermatozoa stained with 0.27% TB/Giemsa a) Spermatozoon with intact head, tail and acrosome membrane b) Spermatozoa with damaged head, tail and acrosome membrane d) Spermatozoon with damaged head and intact tail membrane
e) Spermatozoon with damaged head and tail membrane with no acrosome Bar = 10 µm
Fixation for 4 min resulted in darker “dead” staining with acceptable background.
Giemsa exposure for 2-4 h was sufficient for acrosome staining. The differences we found between CSB and TB viability staining are shown in Table 10.
Acrosome staining was the same for both procedures: intact acrosomes were purple or pink, loose and damaged acrosomes were lavender, and the anterior part of the sperm head with no acrosome was pale gray or pale grayish pink.
Table 10. Differences in CSB and TB staining
Chicago sky blue Trypan blue
Live heads white, pale grayish blue pale blue, pale grayish blue Live tails light pink grayish pink
Dead heads dark grayish-blue stronger dark blue Dead tails black, stronger dark blue dark blue
Repeatability and method-agreement analyses
The average difference between the repeated counts by microscopy of the intact cells on smears stained by TB was 0.28% (SD = 2.73%); the BSI repeatability coefficient (2 SD) was 5.47% (Fig. 15). The average difference between the repeated counts by microscopy of the intact cells in smears stained by CSB was -0.77% (SD = 2.83%); 2 SD was 5.67% indicating good repeatability (Fig. 16). The mean of the differences between the intact cells counted on the smears stained by the two viability stains was 0.03% (SD = 2.18%); (P = 0.78).
Figure 15. Repeatability of TB/Giemsa staining for counting intact cells on the smears.
The differences between the repeated measurements are plotted against their average.
The mean of the differences (d) and the British Standards Institution repeatability coefficient (2 SD) are presented (n =30)
Fig. 16. Repeatability of CSB/Giemsa staining for counting intact cells on smears. The differences between the repeated measurements are plotted against their average. The mean of the differences (d) and the British Standards Institution repeatability coefficient (2 SD) are presented (n = 30)
The corrected SD = was 3.08% and the corrected repeatability coefficient (2 corrSD) was 6.18%. The 95% limits of agreement (d ± 2 corrSD) were -6.13 and 6.19% (Fig. 17). This interval was small and close to the d ± 2 SD of the repeated measurements of both methods (TB: -5.19, 5.75%; CSB: -6.44, 4.90%).
The average difference between the repeated counts by microscopy of cells with a damaged tail with TB/Giemsa staining was -0.19% (SD = 3.06%) and the BSI repeatability coefficient (2 SD) was 6.11%. The average difference between repeated measurements of the cells with a damaged tail with CSB/Giemsa staining was 0.64%
(SD = 2.96%). The BSI repeatability coefficient (2SD) was 5.91%. The mean of the differences between the cells with a damaged tail counted on the smears stained by the two viability stains was -0.09% (SD = 2.40%). The corrected SD was 3.39% and the corrected repeatability coefficient (2 corrSD) was 6.79%. The limits of agreement (-6.88 and 6.70%) were small enough for us to be confident that the new method can be used instead of the old one (Fig. 18). counting intact cells on smears. The differences between the paired measurements are plotted against their average. The mean of the differences (d) and the limits of agreement (d ± 2corrSD) are presented (n =30)
-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10
0 10 20 30 40 50 60 70 80 90 100
Ave rage of paire d measurements (%) Differences of paired measurements (%)
d + 2 corr SD = 6.70%
d
d - 2 corr SD = -6.88%
d = -0.09% 2 corr SD = 6.79%
Figure 18. Agreement between the TB/Giemsa and CSB/Giemsa staining methods for counting cells with damaged tail on smears. The differences between the paired measurements are plotted against their average. The mean of the differences (d) and the limits of agreement (d ± 2corrSD) are presented (n =30)
Densitometry (image analysis)
Measuring the composite RGB value was the most objective and relevant method for comparing differences between the color intensity produced by the two stains (Figs.
11-12). The histograms showed the highest RGB values for intact tails unstained by CSB (171.2 ± 6.9) and the lowest RGB values for the stained tails by CSB (96.7 ± 10.6; Fig. 19). TB staining resulted in a small difference (~ 30 RGB) between the live and dead tails on half of the smears (Fig. 20).
60 70 80 90 100 110 120 130 140 150 160 170 180 190
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Individual samples (n =20)
RGB values intact tail CSB
damaged tail CSB intact tail TB damaged tail TB
Figure 19. Means of RGB values of stained (damaged) and unstained (intact) spermatozoon tails in the individual samples
0 10 20 30 40 50 60 70 80 90 100 110
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Individual samples (n = 20)
RGB values
CSB TB
Figure 20. Comparison of RGB value differences between stained (damaged) and unstained (intact) spermatozoon tails
using the two viability stains
The 20-30 RGB difference between live and dead sperm tails after TB staining was visible on the screen with higher magnification, but it was more difficult to distinguish the viability status by subjective evaluation by microscopy. We found an average of 80% higher measured values for differences in the brightness levels between the live and dead tails after CSB than after TB staining (mean ± S.D: 74.5 ± 12.7 vs. 40.9 ± 12.3; P < 0.0001; n = 20; Figs. 19-20). The difference between the two stains was also clearly seen by conventional subjective visual evaluation (Figs. 13-14). The measured values of the differences between live and dead heads obtained by TB staining were 14% higher than those obtained by CSB staining (mean ± S.D: 67.6 ± 16.4 vs. 59.3 ± 11; P = 0.028; n = 20). This difference was very small (average of 8.3 RGB) and not definitely visible by microscopy. There was no problem distinguishing live and dead heads in smears stained by either TB or CSB (Figs. 13, 14, 21).
0 10 20 30 40 50 60 70 80 90 100 110 120
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Individual samples (n=20)
RGB values
CSB TB
Figure 21. Comparison of RGB value differences between stained (damaged) and unstained (intact) spermatozoon heads
using the two viability stains.