. the total distance traveled by a sperm during the acquisition divided by the time taken ,
Ž lateral head displacement LHD, mm; deviation of the sperm head about the axis of
. Ž
. Ž
average path , linearity LIN s VSLrVCL = 100 , and straightness STR s VSLrVAP .
Ž =
100 . Each observation was based upon the average of three different fields Anzar et .
al., 1991 . 2.4.3. Hypo-osmotic swelling HOS .
Ž .
Plasma membrane integrity PMI of spermatozoa was assessed using the HOS-assay as Ž
. described earlier Jeyendran et al., 1984 . HOS solution was prepared by dissolving
Ž 0.735 g sodium citrate and 1.351 g fructose in 100 ml distilled H O osmotic pressure
2 y1
. Ž
. ;
190 mOsm kg , according to World Health Organization 1992 manual. The HOS
assay was performed by mixing 50 ml of the semen samples to 500 ml of the prewarmed Ž
. 378C HOS solution and incubated at 378C for 30–45 min. After incubation, a drop of
Ž .
semen sample was examined under phase-contrast microscope =400 . Two hundred spermatozoa were counted for their swelling, characterized by coiled tail, indicating
intact plasma membrane.
2.4.4. Normal acrosomes. Ž
. Ž
. Semen sample 500 ml was fixed by 50 ml of 1 formal citrate Hancock, 1957 which
Ž .
was prepared by adding 1 ml of 37 commercial formaldehyde Merck in 99 ml of Ž
. 2.9 wrv sodium citrate. Acrosomal integrity characterized by normal apical ridge
Ž .
Ž .
NAR was assessed under oil immersion =1000 using phase-contrast microscope Ž
. Leica, Leitz Wetzlar, Germany . Two hundred spermatozoa were counted.
2.5. Statistical analysis Results in text are presented as mean SEM. Data for each variable were analysed
Ž .
Ž .
by the analysis of variance ANOVA . When the F-ratio was significant P - 0.05 ,
Tukey’s Honestly Significant Difference Test was used, to compare the treatment means Ž
. SYSTAT, 1996 .
3. Results
3.1. Effect of buffers on post-thaw motility of buffalo spermatozoa Data pertaining to the effect of buffers on motility after thawing in buffalo spermato-
Ž .
zoa is presented in Table 1. The buffers tended to be different P s 0.07 , amongst each other in visual motility of spermatozoa. The average percent visual motility of spermato-
Ž .
Ž .
zoa was highest in TEST and HEPEST 60.0 1.0 , intermediate in TCA 53.0 4.6 , Ž
. and lowest in CITRATE 48.0 2.5 . Percentage of motility assessed using CASA, did
not differ due to the buffering systems and the overall average was 54.5 4.4. Percentage of linear motility of spermatozoa showed the tendency to be different
Ž .
Ž .
P s 0.09 due to buffers. It was highest in CITRATE and TCA 75.0 3.2 , intermedi- Ž
. Ž
. ate in HEPEST 63.2 7.4 , and lower in TEST 52.0 6.9 . There was a significant
Table 1 Ž .
Ž .
Effect of buffers on post-thaw motility of buffalo spermatozoa meanSEM; ns 5 VMOT sVisual motility, CMOT s computer-assisted motility, LMOT s linear motility, and CIRMOT s
circular motility. Variable
CITRATE TCA
TEST HEPEST
P VMOT
48.02.5 53.04.6
59.03.7 61.02.9
0.07 CMOT
48.05.7 48.66.5
67.06.4 54.46.5
0.16 LMOT
71.88.3 78.25.5
52.06.9 63.27.4
0.09
a,b b
a a,b
CIRMOT 13.05.1
11.62.8 29.85.6
19.04.6 0.05
a,b
Ž .
Values in the same row with different superscripts differ significantly P - 0.05 .
Ž .
P - 0.05 variation due to the buffers on the percentage of circular motility of
Ž .
spermatozoa. This was lowest in TCA 11.6 2.8 , intermediate in CITRATE and Ž
. Ž
. HEPEST 16.0 3.0 , and highest in spermatozoa extended with TEST 29.8 5.6 .
3.2. Effect of buffers on post-thaw Õelocities, lateral head displacement, linearity, and straightness of buffalo spermatozoa
Data on the effect of buffers on velocities, lateral head displacement, linearity and straightness after thawing in buffalo spermatozoa are presented in Table 2. The buffers
did not vary the straight-line and average path velocities of spermatozoa, and the overall Ž
y1
. Ž
y1
. averages were 48.6 1.3 mm s
and 55.8 1.6 mm s , respectively. There was a
Ž .
significant effect P - 0.02 of buffers on curvilinear velocity of spermatozoa. This was minimal in TCA, maximal in TEST and intermediate in CITRATE and HEPEST.
Ž .
Similarly, lateral head displacement mm of spermatozoa was recorded as the lowest Ž
. Ž
. Ž
. P - 0.03
in TCA 1.7 0.2 , highest in TEST
3.7 0.6 , and intermediate in Ž
. CITRATE and HEPEST 2.5 0.3 . The percentage of linearity and straightness of
spermatozoa did not differ due to treatments and the overall averages were 62.0 3.4 and 87.2 2.3, respectively.
Table 2 Effect of buffers on post-thaw velocities, lateral head displacement, linearity, and straightness of buffalo
Ž .
spermatozoa meanSEM; ns 5 VSL sStraight-line velocity, VAP saverage path velocity, VCL s curvilinear velocity, LHDs lateral head
displacement, LINs linearity, and STR sstraightness. Variable
CITRATE TCA
TEST HEPEST
P
y1
Ž .
VSL mm s 51.42.2
47.03.3 46.03.7
50.02.2 0.54
y1
Ž .
VAP mm s 58.42.9
51.02.7 56.42.5
57.21.3 0.18
y1 a,b
b a
a,b
Ž .
VCL mm s 79.05.8
69.42.0 87.21.6
82.63.0 0.02
a,b b
a a,b
Ž .
LHD mm 2.20.5
1.70.2 3.70.6
2.80.3 0.03
Ž . LIN
66.66.1 67.63.5
52.94.8 61.03.9
0.15 Ž .
STR 88.43.4
91.92.4 81.03.3
87.32.6 0.11
a,b
Ž .
Values in the same row with different superscripts differ significantly P - 0.05 .
Table 3 Ž .
Ž .
Effect of buffers on post-thaw membrane integrity of buffalo spermatozoa mean SEM; ns 5 PMI s Plasma membrane integrity, and NAR s normal apical ridge.
Variable CITRATE
TCA TEST
HEPEST P
PMI 45.21.2
32.63.8 41.45.2
40.83.9 0.16
NAR 69.20.8
61.82.4 53.28.3
65.84.6 0.16
3.3. Effect of buffers on post-thaw membrane integrity of buffalo spermatozoa Average plasma membrane integrity and the normal acrosomes of buffalo spermato-
Ž .
Ž . zoa did not vary due to buffering systems Table 3 . Their overall values were
averaged 40.0 2.7 and 61.4 4.6, respectively.
4. Discussion
