234 S.Y. Huang et al. Animal Reproduction Science 63 2000 231–240
Table 1 Grouping of samples by level of heat shock protein 70
Group 1
2 3
4 5
6 Level of
total HSP70 M+1 S.D. M+1 S.D.,
M+0.5 S.D. M+
0.5 S.D., M
M ,
M−0.5 S.D. M−
0.5 S.D., M−1 S.D.
M− 1 S.D.
2.4. Grouping of samples by level of heat shock protein 70 All the samples were grouped according to the mean M and one-half standard deviation
S.D. of HSP70 level in order to evaluate how HSP70 affects semen quality traits. Six groups were defined as shown in Table 1.
2.5. Statistical analysis The extent to which season and breed affect semen quality traits, level of HSP70 sum of
constitutive and inducible form of HSP70 was analyzed by adopting the GLM procedure of SAS SAS Institute, 1989. The statistical model included season S, breed B, S×B,
and boar S×B. The effect of HSP70 grouping was also analyzed by the GLM procedure. The extent to which the effects differed was determined by the least squares means method.
Correlation coefficients between levels of HSP70 and semen quality traits were Pearson correlation calculated by the CORR procedure of SAS SAS Institute, 1989.
3. Results
3.1. Protein profile and qualitative characterization of heat shock protein 70 in boar spermatozoa
This study analyzed the sperm protein contents of 29 boars using SDS-PAGE. Fig. 1 displays typical protein profiles of boar spermatozoa. Coomassie blue staining of the gels
revealed more than 50 proteins with molecular weights ranging from 30 to 100 kDa Fig. 1a. In addition, different breeds or different seasons did not apparently differ in the overall
protein profile of spermatozoa. Furthermore, an anti-mouse HSP7072 monoclonal antibody was used to detect the level of constitutive and inducible form of HSP70 in boar spermatozoa
Fig. 1b. Both forms of HSP70 could be easily detected with a commercial antibody. However, no HSP70 was detected in the seminal fluid data not shown.
3.2. Seasonal and breed effects on semen quality and level of heat shock protein 70 in boar spermatozoa
The concentration, motility and morphology of the sperm in the semen samples were determined. Table 2 lists these semen quality traits together with the level of HSP70. A
significant seasonal difference was observed in all the traits evaluated P0.05. Semen
S.Y. Huang et al. Animal Reproduction Science 63 2000 231–240 235
Fig. 1. Protein profiles and immunoblotting analysis of boar spermatozoa. Samples from cool Lanes 2, 4 and 6 and hot season Lanes 3, 5 and 7, and different breeds of boar Duroc: lanes 2 and 3; Landrace: lanes 4 and
5; Yorkshire: lanes 6 and 7 were subjected to SDS-PAGE, followed by staining with Coomassie blue a. In a separate experiment, proteins in polyacrylamide gel were transferred to a nitrocellulose membrane and blotted with
monoclonal antibody against HSP70 and b-tubulin b. Each lane contains total proteins from 3×10
6
spermatozoa except lane 1 which contains molecular weight standards. HSC70 and HSI70 are the constitutive and inducible
form of 70 kDa heat shock protein, respectively.
quality in cool season was significantly better than that during the hot season in all three breeds of boars. The semen quality of Landrace boars was better than Duroc and Yorkshire
boars in both seasons P0.05. The level of HSP70 during the hot season was significantly lower than that during the cool season P0.05. Notably, the interactions between season
and breed in all the traits were insignificant data not shown; P0.05.
236 S.Y. Huang et al. Animal Reproduction Science 63 2000 231–240
Table 2 Least squares means of semen quality traits and level of HSP70 in boar spermatozoa
a
Traits Cool season
Hot season Duroc
Landrace Yorkshire
Duroc Landrace
Yorkshire Motility
80.5±1.2 a 82.6±1.5 a
80.5±1.7 a 58.3±1.2 c
71.1±1.5 b 58.9±1.7 c
Normal sperm 55.1±1.9 c
70.7±2.4 a 63.3±2.6 b
37.0±1.8 e 53.3±2.3 c
45.9±2.5 d PPD
b
4.6±0.6 bc 3.2±0.7 c
5.7±0.8 b 7.7±0.6 a
4.6±0.7 b 6.6±0.8 ab
DPD
c
13.0±1.3 c 6.5±1.6 de
2.5±1.8 e 21.6±1.2 a
17.0±1.5 b 8.1±1.7 d
Abnormal sperm 27.3±1.8 c
19.6±2.3 d 28.5±2.6 bc
33.8±1.8 ab 25.1±2.2 cd 39.0±2.5 a Sperm concentration
×10
8
ml 2.38±0.19 b 1.75±0.24 c 2.03±0.27 bc 3.14±0.19 a 2.45±0.23 b 2.28±0.26 c
HSP70
d
1.53±0.05 a 1.40±0.06 a 1.42±0.08 a 1.18±0.05 b 1.00±0.07 b 1.00±0.08 b
a
Means within the same row with different letters differ significantly P0.05.
b
PPD: sperm with proximal plasma droplets.
c
DPD: sperm with distal plasma droplets.
d
HSP70: level of 70 kDa heat shock protein, which was normalized optical density by using b-tubulin as covariate with values of arbitrary unit.
3.3. Correlation between level of heat shock protein 70 and porcine semen quality The correlation coefficients between level of HSP70 and semen quality traits were cal-
culated Table 3, and no significant correlation was found between levels of HSP70 and semen quality traits in either cool season or hot season. Therefore, a different approach
was employed to evaluate the relationship between HSP70 and semen quality traits. The samples were grouped according to their HSP70 level and the semen quality traits were
compared at different HSP70 levels.
The levels of HSP70 for all the samples collected ranged from 0.34 to 3.06 with a mean value of 1.27 and a standard deviation of 0.46. Thus, HSP70 levels of 1.73, 1.5, 1.27, 1.04,
0.81 were used as cutting points for grouping. The HSP70 levels in the six groups were Group 11.73, 1.73Group 21.5, 1.5Group 31.27, 1.27Group 41.04, 1.04Group 50.81,
and 0.81Group 6. Table 4 lists the semen quality traits for the six groups, indicating that the level of HSP70 and semen quality appeared to have a good correlation. The correlation
for boars with a spermatozoa HSP70 level higher than 1.04 Groups 1–4 was not obvious.
Table 3 Correlation coefficients of level of HSP70 and semen quality traits in boars
Traits Cool season
Hot season Sample size
113 112
Motility 0.00
0.14 Normal sperm
0.10 0.15
Sperm with proximal plasma droplets −
0.05 −
0.20
∗
Sperm with distal plasma droplets −
0.11 −
0.08 Abnormal sperm
− 0.03
− 0.04
Sperm concentration −
0.12 0.48
∗∗ ∗
P 0.05.
∗∗
P 0.01.
S.Y. Huang et al. Animal Reproduction Science 63 2000 231–240 237
Table 4 Least squares means of semen quality traits among groups with different levels of HSP70 in boar spermatozoa
a,b
Traits Group 1
Group 2 Group 3
Group 4 Group 5
Group 6 Number of samples
33 29
41 51
38 33
Motility 75.9±2.5 ab
77.9±2.6 a 75.4±2.2 ab
72.5±2.0 ab 70.5±2.3 b
58.5±2.5 c Normal sperm
61.6±3.9 a 59.1±4.2 a
56.5±3.5 ab 54.7±3.2ab
48.0±3.7 bc 39.5±3.9 c
PPD
c
3.4±1.1 b 4.7±1.2 ab
4.6±1.0 ab 5.8±0.9 ab
5.9±1.0 ab 7.2±1.1 a
DPD
d
8.3±2.4 b 8.4±2.6 b
11.7±2.2 ab 14.0±2.0 ab
14.1±2.3 ab 17.9±2.4 a
Abnormal sperm 27.0±2.9 b
27.9±3.1 ab 27.1±2.6 b
25.4±2.4 b 31.7±2.7 ab
35.4±2.9 a Sperm concentration
×10
8
ml 2.60±0.30 a
2.78±0.32 a 2.52±0.27 a
2.60±0.24 a 2.44±0.28 a
1.58±0.30 b HSP70
e
2.07±0.03 a 1.62±0.03 b
1.39±0.02 c 1.15±0.02 d
0.93±0.02 e 0.63±0.03 f
a
Mean and standard deviation of total HSP70 was 1.27 and 0.46. The cut points of HSP70 level for grouping were 1.73, 1.5, 1.27, 1.04, 0.81 as defined in Section 2.
b
Means within the same row with different letters differ significantly P0.05.
c
PPD: sperm with proximal plasma droplets.
d
DPD: sperm with distal plasma droplets.
e
HSP70: level of 70 kDa heat shock protein, which was normalized optical density by using b-tubulin as covariate with values of arbitrary unit.
However, for Groups 5 and 6 with a HSP70 level lower than 1.04, sperm motility, percentage of normal sperm, and sperm concentration significantly decreased and abnormal sperm and
sperm with plasma droplets increased.
4. Discussion
