S.Y. Huang et al. Animal Reproduction Science 63 2000 231–240 233
2. Materials and methods
2.1. Experimental animals, semen collection and quality evaluation Twenty-nine normal, mature boars 13 Duroc, 9 Landrace, and 7 Yorkshire from an
AI center located in northern Taiwan were used. The average age was 25.2±2.2 months.
Three to four samples per boar were collected during cool average temperature range was 15.0±0.3 to 22.2±0.4
◦
C and hot average temperature range was 23.7±0.3 to 32.0±0.4
◦
C seasons. A phase contrast microscope was used to evaluate semen quality traits including
sperm motility, sperm concentration, percentages of normal sperm, sperm with persistent proximal plasma droplets, sperm with persistent distal plasma droplets, and abnormal sperm
Kuo et al., 1997.
2.2. Gel electrophoresis For protein analysis, the spermatozoa were washed with an extender and centrifuged
at 800×g for 1 min. The pellets were then lysed in Laemmli’s sample buffer Laemmli, 1970, boiled for 5 min and then stored at −20
◦
C until further analysis. In addition, 500 ml of the supernatant were mixed with an equal volume of Laemmli’s buffer and saved for
later analysis. Proteins from 3×10
6
spermatozoa or 10 ml of seminal fluid were separated by 9 SDS-PAGE according to Laemmli 1970. The molecular standards were purchased from
BioRad Hercules, CA, USA. After electrophoresis, the gels were stained with 0.1 Coomassie brilliant blue R-250 for 60 min, followed by destaining with a solution con-
taining methanol and acetic acid until the background was clear.
2.3. Western blot analysis and quantitation of heat shock protein 70 Western blot analysis was performed according to a procedure described elsewhere Lee
et al., 1996; Huang et al., 1999. After electrophoresis, the gels were blotted with monoclonal antibodies against mouse HSP7072 clone N27F3-4, Stressgen, Victoria, Canada; diluted
1:1000 with TTBS containing 1 gelatin and b-tubulin Amersham; diluted 1:500 fol- lowed by biotinylated goat anti-mouse IgG conjugated with alkaline phosphatase Sigma, St.
Louis, MA, USA; diluted 1:5000. The membranes were then developed with a buffer con- taining nitro blue tetrazolium and 5-bromo-4-chloro-3-indoly phosphate BioRad.
The color of immunocomplex was displayed at a proper intensity and within a linear range.
For quantitative analysis of the constitutive HSC70 and the inducible HSI70 forms of HSP70, total proteins from 3×10
6
spermatozoa were Western blotted and the optical densities of HSP70 and b-tubulin bands on nitrocellulose membranes were determined using
a densitometer Molecular Dynamics, Sunnyvale, CA, USA; software was ImageQuant. The levels of HSC70, and HSI70 were normalized using b-tubulin as the covariate, and
their sum was given as the level of HSP70.
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
