Effects of single nucleotide polymorphisms in the 5'-flanking region of heat shock protein 70.2 gene on semen quality in boars.

This study aims to elucidate the effects of single nucleotide polymorphisms (SNPs) in the 5'-flanking region of porcine heat shock protein 70.2 gene (HSP70.2) on semen quality in boars. Genomic DNA isolated from 55 boars (41 Duroc, nine Landrace, and five Yorkshire) was subjected to PCR amplification of the 5'-flanking region of HSP70.2. The nucleotide sequences were determined by automated sequencing. Five SNPs (sites 44, 232, 250, 345, and 393) were detected in this region. Semen quality was evaluated in terms of sperm motility, percentage of normal sperm, percentage of sperm with proximal plasma droplet, percentage of abnormal sperm, sperm concentration, semen volume per ejaculate and total sperm number per ejaculate. The effect of the SNPs on semen quality was evaluated based on breed-corrected data within a season. During the cool season, the sperm motility of boars with AA genotype at the 232 site was significantly higher than that of boars with CC genotype (P<0.05). Meanwhile, boars with AC genotype at the 232 site had higher total sperm number per ejaculate than did those with CC genotype. In the hot season, heterozygotes at both the 232 and 250 sites had significantly higher total sperm number of per ejaculate than AA homozygotes (P<0.05). Semen volume of boars with TT and TC genotypes at the 345 site was significantly larger than that of those with CC genotype (P<0.05). Meanwhile, semen quality for boars with TT genotype at the 345 site was significantly higher than that of boars with TC or CC genotype (P<0.05), that is the semen contained higher percentages of normal sperm and lower percentages of abnormal sperm or sperm with proximal plasma droplets. Results herein suggest that the SNPs in the 5'-flanking region of porcine HSP70.2 are associated with semen quality traits in the hot season.

Effects of birth season, breed, sex, and sire family on cardiac morphology determined in pigs (Sus scrofa domestica) by use of echocardiography.

PURPOSE: Echocardiography played an important role in the screening and diagnosis of hypertrophic cardiomyopathy. In the study reported here, we attempted to evaluate the effects of birth season, breed, sex, and sire family on cardiac morphology determined in pigs by use of echocardiography. METHODS: A total of 411 pigs (mean body weight and age of 105.7 +/- 10.6 kg and 214.4 +/- 25.5 days, respectively) with different genetic backgrounds (Landrace, Yorkshire, and their two-way crossbred) were studied. Cardiac morphologic measurements included thickness of left ventricle and interventricular septum at end-systolic and end-diastolic phases. Meanwhile, the statistical model included the following effects: birth season, breed, sex, interaction between breed and sex, sire family, body weight, and age. RESULTS: Mean cardiac morphologic measurements were as follows: thickness of the interventricular septum at end-systolic and end-diastolic phases was 1.74 and 1.14 cm, respectively; and thickness of the left ventricular free wall at end-systolic and end-diastolic phases was 1.81 and 0.98 cm, respectively. Medium positive correlations existed among the cardiac morphologic measurements r = 0.31 to 0.53; P < 0.001). Pigs born in spring had significantly (P < 0.05) lower cardiac thickness at the end systolic phase than did pigs born in other seasons, and Landrace pigs had higher cardiac morphologic measurements than did Yorkshire and two-way crossbred pigs. Additionally, thickness of interventricular septum at the end-diastolic phase in male pigs was significantly higher than that in female pigs (P < 0.05). Cardiac morphologic measurements for the sire family were significantly (P < 0.05) different, and contributed 77.2 to 87.9% of the total variation, suggesting that genetic variation in cardiac morphology might exist in pigs. CONCLUSIONS: Cardiac morphology of pigs might be influenced by genetic background. The effects of birth season, breed, sire family, and sex should be adjusted when using pigs as an animal model for comparative cardiovascular studies.

Association of heat shock protein 70 with semen quality in boars.

This study attempted to clarify the relationship between the levels of 70kDa heat shock protein (HSP70) and semen quality in boars. Semen samples from 29 (13 Duroc, 9 Landrace, and 7 Yorkshire) boars (mean age=25.2+/-2.2 months) were examined. Three to four ejaculates per boar, collected during cool and hot seasons, were evaluated in terms of the sperm concentration, sperm motility, percentage of normal and abnormal sperm, as well as percentage of sperm with proximal and distal plasma droplets. Significant seasonal and breed differences in semen quality were observed. Experimental results indicate that the semen quality of Landrace boars was better than those of Yorkshire and Duroc boars (P<0.05) and semen quality declined significantly during the hot season (P<0.05). One-dimensional SDS-PAGE analysis of spermatozoa proteins indicated that protein profiles did not significantly differ between seasons and among breeds. Both constitutive and stress-inducible form of HSP70 were detected in boar spermatozoa by Western blot analysis. The level of HSP70, which revealed no difference among breeds within a season, was significantly lower during the hot season in all the three breeds (P<0.05). Although there appeared to be low correlation coefficients between the level of HSP70 and semen quality traits, the semen quality tended to decline significantly in samples with a lower level of HSP70. Results in this study suggest that the levels of HSP70 in boar spermatozoa are significantly lower during the hot season and might be associated with semen quality.

The decline of porcine sperm motility by geldanamycin, a specific inhibitor of heat-shock protein 90 (HSP90).

Sperm motility is an important parameter for fertility. The molecular mechanisms of mammalian sperm motility are still largely undefined. Our previous observations suggested that heat shock protein 90 (HSP90) may be associated with porcine sperm motility. The aim of the present study was to further characterize the plausible novel function of HSP90 on sperm motility. Semen from normal, sexually mature boars with sperm motility higher than 80% was used. An HSP90-specific inhibitor, geldanamycin (GA), was added to diluted semen at 0.5, 1.0, 2.5 or 5.0 microg/mL and the semen was then incubated at 37 degrees C for 15, 30, 45 or 60 min. Sperm motility was determined by using computer-assisted semen analyzer at the end of incubation. The results indicated that GA significantly reduced sperm motility in a dose and time dependent manner. Moreover, incubation of semen with 5.0 microg/mL GA for 15 min completely stopped sperm motility. To test the reversibility of the GA effect on sperm motility, GA was removed after 30 min incubation and was replaced with fresh extender alone or with extender plus 5 mM caffeine, then incubated for another 15, 30, 45 or 60 min. The results showed that simply removing GA did not reverse the inhibitory effect on sperm motility, while adding caffeine partially reversed this inhibitory effect. However, the effect of 2.5 or 5.0 microg/mL GA was not reversed by caffeine. Considering the specificity of GA targeting to HSP90, the above observations suggested that HSP90 may play a crucial role in regulating porcine sperm motility.

Statistical method for characterization of hypertrophic cardiomyopathy by use of morphologic and pathologic measurements in pigs (Sus scrofa domestica).

BACKGROUND AND PURPOSE: Hypertrophic cardiomyopathy (HCM) is characterized by symmetric or asymmetric hypertrophy of the left and/or right ventricle. Morphologic and pathologic indices (MI and PI) of hearts were established for classification of HCM in pigs. METHODS: Fifty on-farm-performance-tested pigs (average body weight, 104.3 kg; age, 224.5 days) were randomly selected. Heart weight, length, width, heart-to-body weight ratio, and thickness of the cranial and middle portions of ventricular septum and left ventricular free wall were measured. Myocyte disorganization and necrosis, myocardial and endocardial fibrosis, and intramural coronary arterial occlusion were scored. Principal component analysis and stepwise regression analysis were used to establish MI and PI. RESULTS: MI was established by using the first principal component as the dependent variable and applying stepwise regression analysis. Hearts were classified as morphologically normal, suspicious, and hypertrophic according to the range of MI. The same statistical method was used to find PI. Hearts were classified as pathologically normal, moderately affected, or seriously affected according to the range of PI. Combining MI and PI, hearts could be classified into five groups: no hypertrophy with minor lesion (normal); hypertrophy but with rare lesion; no hypertrophy but seriously affected; suspicious; and hypertrophy and seriously affected (heart with HCM). Another 119 hearts were collected and classified. The variation of heart measurements was consistent with the original purpose of classification. CONCLUSIONS: Using fewer measurements for identification of HCM objectively in pigs seems to have practical application.

Substantial decrease of heat-shock protein 90 precedes the decline of sperm motility during cooling of boar spermatozoa.

The decline in boar semen quality after cryopreservation may be attributed to changes in intracellular proteins. Thus, the aim of the present study was to evaluate the change of protein profiles in boar spermatozoa during the process of cooling and after cryopreservation. A total of 9 sexually mature boars (mean age = 25.5+/-12.3 mo) was used. Samples for protein analysis were collected before chilling, after cooling to 15 degrees C, after cooling to 5 degrees C, following thawing after freezing to -100 degrees C, and following thawing after 1 wk of cryopreservation at -196 degrees C. Semen characteristics evaluated included progressive motility and the percentage of morphologically normal spermatozoa. Total proteins from 5x10(6) spermatozoa were separated and analyzed by SDS-PAGE. The results revealed that there was a substantial decrease of a 90 kDa protein in the frozen-thawed spermatozoa. Western blot analysis demonstrated that this protein was 90 kDa heat-shock protein (HSP90). Time course study showed that the decrease of HSP90 in spermatozoa initially occurred in the first hour during cooling to 5 degrees C. When compared with the fresh spermatozoa before chilling, there was a 64% decrease of HSP90 in spermatozoa after cooling to 5 degrees C. However, the motility and percentage of normal spermatozoa did not significantly decrease during this period of treatment. Both declined substantially as the semen was thawed after freezing from -100 degrees C. The results indicated that the decrease of HSP90 precedes the decline of semen characteristics. The length of time between a decrease of HSP90 and the decline in sperm motility was estimated to be 2 to 3 h. Taken together, the above results suggested that a substantial decrease of HSP90 might be associated with a decline in sperm motility during cooling of boar spermatozoa.

Heritability estimate of hypertrophic cardiomyopathy in pigs (Sus scrofa domestica).

The purpose of this study was to evaluate the heritability of hypertrophic cardiomyopathy (HCM) in pigs and the relation between HCM and heart measurements, pathologic features, and growth to provide references for HCM line development. A total of 353 on-farm tested gilts (females) and boars (males) from 74 sire families were randomly selected from a single breeding farm where HCM was prevalent. Hearts were collected after animals were slaughtered. Heart length, width, and weight, heart-to-body weight ratio, and thickness of the cranial, middle, and caudal portions of the ventricular septum, left and right ventricles, and apex were measured. Cardiac hypertrophy and myocyte disorganization, myocardial and endocardial fibrosis, and intramural coronary arterial occlusion were used as criteria for HCM. Growth traits were evaluated from average daily body weight gain, ultrasonically determined backfat thickness, loin-eye area, and performance selection index. Heritability of the disease was estimated by treating it as a threshold trait. The prevalence of HCM in three studied breeds was 5.26 in Duroc, 22.98 in Landrace, and 5.56% in Yorkshire pigs. The value in Landrace pigs was significantly (P < 0.001) higher than that in the other pigs. There was no significant difference between sexes. In general the heart of pigs with HCM was heavier, wider, longer, and thicker than that of clinically normal pigs. Backfat was the only growth trait with a difference (P < 0.05) among pig breeds. The HCM pigs were leaner than normal pigs. Leaner pigs may have a higher risk of HCM. Heritability of HCM was > 0.30 for all three breeds, but the standard errors of these estimates were high because of limited sample size, in particular for the Yorkshire and Duroc breeds. The preliminary results of this study indicate that HCM in pigs is moderately heritable; thus development of a high-HCM incidence line by selection is possible.