Role of Antioxidants in Horse Serum-mediated Vasculitis in Swine: Potential Relevance to Early Treatment in Mitigation of Coronary Arteritis in Kawasaki Disease.

BACKGROUND: Horse serum-induced immune complex coronary vasculitis in swine is the first experimental model to mimic most of the pictures of Kawasaki disease. Immune complex mechanism has been implicated as one of the possible mechanisms in the pathogenesis of vasculitis in Kawasaki disease. Antioxidants have a significant role in the reduction of cardiovascular diseases in both human and animal studies. We tried giving vitamins A, E, and C to treat immune complex vasculitis, in the hope of mitigating coronary vasculitis in Kawasaki disease. METHODS: Our study group consisted of 30 pure bred male piglets of 2-3 months of age, and they were divided into test and control groups. The test (AEC) group (n = 20) received two doses of horse serum, 10 mL (0.65 g protein)/kg body weight at 5-day intervals, and oral vitamins A, E, and C once daily for 14 days. The control group (n = 10) was further divided into the saline group (n = 3) receiving two doses of normal saline and the horse serum group (n = 7) receiving two doses of horse serum at 5-day intervals. Piglets were observed for the rashes and coronary artery dimensions. RESULTS: Both the AEC and the control horse serum group developed rashes after horse serum infusions, but the AEC group developed significantly fewer rashes, and no rashes were seen in the saline group. The control horse serum group (mean ± standard deviation = 2.13 ± 0.72) showed significant coronary artery dilatation, whereas there was no significant dilatation in the AEC group (mean ± standard deviation = 0.81 ± 0.58) or the control saline group (p = 0.002). CONCLUSION: Serum sickness is a prototype of immune complex vasculitis, and the severity can be ameliorated with antioxidants. A trial of therapeutic dosages of vitamins A, E, and C in acute phase of Kawasaki disease, may be effective in mitigation of coronary artery lesion in addition to intravenous immunoglobulin and aspirin.

Histopathological evaluation of horse serum-induced immune complex vasculitis in swine: implication to coronary artery lesions in Kawasaki disease.

BACKGROUND: Immune complex (IC) vasculitis can be experimentally induced in animal models by intravenous injection of horse serum (HS), and the findings of HS-induced IC vasculitis in swine were very similar to that of Kawasaki disease (KD). The IC mechanism may be involved in the pathogenesis of vasculitis in KD. Here, we studied the two-dimensional (2D) echocardiographic and histopathological findings of acute, subacute, and healing phases of vasculitis induced by two different types of HS, and the reproducibility of IC vasculitis in swine. METHODS AND RESULTS: Our study group consisted of 24 pure-bred landrace male piglets of 1.5-3 months of age. They were divided into three HS groups (n = 17), namely, Group A (n = 8) receiving gamma globulin-free HS, and Group B (n = 6) receiving donor herd HS, three doses at 5-day intervals, and Group C (n = 3) that received only one dose of donor herd HS on Day 1, and the saline group (n = 7) that received three doses of intravenous normal saline (NS) at 5-day intervals. The 2D echocardiography was performed every 3-4 days, and all piglets were killed for histopathological studies at different dates from Days 2 to Day 60. All the HS groups developed rashes and demonstrated significant dilation (54-150%) of coronary arteries in Groups A and B; when compared (p < 0.02) with 9-53% dilation in Group C and the saline group. Histopathological changes of test groups were asymmetric coronary vasculitis in various stages, whereas none of the piglets in the control group developed vasculitis. No significant difference in the echocardiographic and histopathological findings was observed among the piglets that received two types of HS. CONCLUSION: HS can induce IC vasculitis in swine. The rashes and 2D echocardiographic and histopathological studies of the acute to healing phases showed close similarities with KD, and it is concluded that swine may serve as a unique experimental model for IC vasculitis and for various therapeutic trials.

Weighted moments estimators of the parameters for the extreme value distribution based on the multiply type II censored sample.

We propose the weighted moments estimators (WMEs) of the location and scale parameters for the extreme value distribution based on the multiply type II censored sample. Simulated mean squared errors (MSEs) of best linear unbiased estimator (BLUE) and exact MSEs of WMEs are compared to study the behavior of different estimation methods. The results show the best estimator among the WMEs and BLUE under different combinations of censoring schemes.

PreZon: prediction by zone and its application to egg productivity in chickens.

Taiwan red-feathered country chickens (TRFCCs) are one of the main meat resources in Taiwan. Due to the lack of any systematic breeding programs to improve egg productivity, the egg production rate of this breed has gradually decreased. The prediction by zone (PreZone) program was developed to select the chickens with low egg productivity so as to improve the egg productivity of TRFCCs before they reach maturity. Three groups (A, B, and C) of chickens were used in this study. Two approaches were used to identify chickens with low egg productivity. The first approach used predictions based on a single dataset, and the second approach used predictions based on the union of two datasets. The levels of four serum proteins, including apolipoprotein A-I, vitellogenin, X protein (an IGF-I-like protein), and apo VLDL-II, were measured in chickens that were 8, 14, 22, or 24 weeks old. Total egg numbers were recorded for each individual bird during the egg production period. PreZone analysis was performed using the four serum protein levels as selection parameters, and the results were compared to those obtained using a first-order multiple linear regression method with the same parameters. The PreZone program provides another prediction method that can be used to validate datasets with a low correlation between response and predictors. It can be used to find low and improve egg productivity in TRFCCs by selecting the best chickens before they reach maturity.

Correlation between radioactivity and chemotherapeutics of the (111)In-VNB-liposome in pharmacokinetics and biodistribution in rats.

BACKGROUND: The combination of a radioisotope with a chemotherapeutic agent in a liposomal carrier (ie, Indium-111-labeled polyethylene glycol pegylated liposomal vinorelbine, [(111)In-VNB-liposome]) has been reported to show better therapeutic efficiency in tumor growth suppression. Nevertheless, the challenge remains as to whether this therapeutic effect is attributable to the combination of a radioisotope with chemotherapeutics. The goal of this study was to investigate the pharmacokinetics, biodistribution, and correlation of Indium-111 radioactivity and vinorelbine concentration in the (111)In-VNB-liposome. METHODS: The VNB-liposome and (111)In-VNB-liposome were administered to rats. Blood, liver, and spleen tissue were collected to determine the distribution profile of the (111)In-VNB-liposome. A liquid chromatography tandem mass spectrometry system and gamma counter were used to analyze the concentration of vinorelbine and radioactivity of Indium-111. RESULTS: High uptake of the (111)In-VNB-liposome in the liver and spleen demonstrated the properties of a nanosized drug delivery system. Linear regression showed a good correlation (r = 0.97) between Indium-111 radioactivity and vinorelbine concentration in the plasma of rats administered the (111)In-VNB-liposome. CONCLUSION: A significant positive correlation between the pharmacokinetics and biodistribution of (111)Indium radioactivity and vinorelbine in blood, spleen, and liver was found following administration of the (111)In-VNB-liposome. The liposome efficiently encapsulated both vinorelbine and Indium-111, and showed a similar concentration-radioactivity time profile, indicating the correlation between chemotherapy and radiotherapy could be identical in the liposomal formulation.

Optimised nano-formulation on the bioavailability of hydrophobic polyphenol, curcumin, in freely-moving rats.

This study has optimised the poly lactic-co-glycolic acid (PLGA) nano-formulation of curcumin to prolong its retention time in the body and improve bioavailability. High-pressure emulsification-solvent-evaporation was designed to obtain curcumin-loaded PLGA nanoparticles (C-NPs) prepared with 2% of PVA containing 20% sucrose as aqueous phase and dichloromethane as oil phase. The size and entrapment efficiency of C-NPs was 158±10nm and 46.6±13.5%, respectively. The stable storage time of C-NPs was one month at 4°C. When curcumin was formulated, a significant increase of curcumin exposure in rat plasma was revealed from the intravenous study (AUC/Dose raised 55%) and the oral study (AUC/Dose increased 21-fold). The oral bioavailability of curcumin at C-NPs was 22-fold higher than conventional curcumin. Excretion results support oral study that absorption of curcumin was significantly increased by nano-formulation. These findings demonstrate that PLGA nano-formulation could potentially be applied to increase bioavailability of hydrophobic polyphenols.

Preparation and characterization of liposomal coenzyme Q10 for in vivo topical application.

Coenzyme Q10 (CoQ10) is an endogenous cellular antioxidant that is used as a nutritional supplement and for medicinal purposes. In recent in vivo investigations, cosmetically applied CoQ10 has demonstrated its ability to reduce photoaging, with a corresponding decrease in wrinkle depth. However, the bioavailability of topical CoQ10 is poor; the development of a practical topical formulation is therefore highly desirable. In this study, a liposomal formulation composed of soybean phosphatidylcholine (SPC) and alpha-tocopherol (Vit E) was utilized to encapsulate CoQ10 for topical application. The liposomes were less than 200 nm in diameter and had a narrow size distribution. Encapsulation of CoQ10 in liposomes composed of SPC and Vit E significantly (p < 0.05) enhanced its accumulation (at least twofold) in rat skin, compared with an unencapsulated suspension. Prolonging the treatment time and increasing the content of CoQ10 in the formulation both raised the amount of CoQ10 in rat skin. However, in skin treated with the highest CoQ10 content formulation, insufficient treatment time limited the amount accumulated. This study demonstrates that liposomal CoQ10 is a promising candidate for the topical application of CoQ10. The treatment duration is the key factor limiting penetration following in vivo topical application.

Herb-drug interaction of Andrographis paniculata extract and andrographolide on the pharmacokinetics of theophylline in rats.

Herb-drug interaction has become a serious problem since herbal medicine is extensively used in the modern world. This study investigates effects of Andrographis paniculata extract (APE) and its major component, andrographolide (AG), on the pharmacokinetics of theophylline, a typical substrate of cytochrome P450 1A2 enzyme, in rats. After APE or AG pretreatment for 3 days, on the fourth day rats were administered theophylline via femoral vein cannula. The blood theophylline levels were monitored by microdialysis sampling combined with HPLC-UV. The results indicated that the clearance of theophylline was significantly increased and the area under concentration-time curve (AUC) was reduced in both AG and APE pretreated groups at low-dose theophylline administration (1mg/kg). The elimination half-life (t(1/2beta)) and mean residence time (MRT) of theophylline were shortened by 14% and 17%, respectively, in the AG pretreated group when high-dose theophylline (5mg/kg) was given. However, theophylline accumulated in rat of the group with APE pretreatment. This phenomenon suggests that some other herbal components contained in APE may interact with theophylline and retard its elimination when theophylline was administered at a high dose. Our results suggest that patients who want to use CYP1A2-metabolized drugs such as caffeine and theophylline should be advised of the potential herb-drug interaction, to reduce therapeutic failure or increased toxicity of conventional drug therapy.

Herb-drug interaction of silymarin or silibinin on the pharmacokinetics of trazodone in rats.

Silymarin, one of the most popular herbal medicines, has been widely used for its hepatoprotective effects. This study investigates the effects of repeated dose of silymarin and its major ingredient, silibinin, on the pharmacokinetics of the antidepressant trazodone. Treatment groups included vehicle control group, concomitant silymarin at 1.0g/kg dose, and four 7-day repeated dose induction groups of 0.5 and 1.0g/kg silymarin and 0.175 and 0.35g/kg silibinin. Microdialysis coupled with high performance liquid chromatography (HPLC) was used to simultaneously monitor blood and bile concentrations of trazodone in the rats. Results indicate that pretreatment with an extremely high dose of 1.0g/kg silymarin significantly decreases trazodone's area under concentration curve (AUC), distribution half-life (t(1/2,alpha)), elimination half-life (t(1/2,beta)), and mean residence time (MRT). In conclusion, the present study finds no marked effects of silymarin and silibinin on the pharmacokinetics of trazodone under normal daily doses and the relative safety of taking the herb with trazodone.

Distribution of blood-muscle for clenbuterol in rat using microdialysis.

Clenbuterol is clinically used as a bronchodilator, but it is also illegally used to increase lean meat in animal husbandry. To investigate the muscle distribution of protein-unbound clenbuterol, a microdialysis technique coupled to liquid chromatography system was applied to simultaneously monitor clenbuterol in rat blood and muscle. Two microdialysis probes were implanted into the jugular vein/right atrium and hind leg muscle of rat for sampling after clenbuterol administration (10 mg/kg) through the femoral vein. Dialysate samples of clenbuterol were separated by a reversed-phase column (250 mm x 4 mm I.D., particle size 5 microm). The results indicate that the maximum concentration of clenbuterol in muscle was found at 30-45 min after clenbuterol administration (10 mg/kg) and the area under concentration curve (AUC) of clenbuterol in blood and in muscle were 942.75+/-101.92 and 174.81+/-13.03 min microg/mL, respectively. The AUC(muscle)/AUC(blood) was 0.20+/-0.03 representing about 20% of the clenbuterol distributing into the muscle. The elimination half-life of clenbuterol in the blood and muscle were about 2 and 6h, respectively. These results suggest that the protein-unbound concentration of clenbuterol sustained a high level and prolonged elimination in the muscle. The accumulation of clenbuterol might result in some clinical effects when clenbuterol-contaminated meat was consumed.

A dual-functional E. coli vector for expressing recombinant protein with high solubility and antigen presentation ability.

A dual-functional Escherichia coli expression vector capable of producing soluble recombinant proteins with high immunogenicity in animals is introduced. This vector expresses polypeptides fused to a PTD-J-domain peptide. The J-domain peptide is derived from murine Hsp40 by using optimized codons for E. coli. The association of the J-domain to the nucleotide binding domain of the DnaK chaperone increases the probability that the fused polypeptide will be folded by the DnaK and hence increases the solubility of the recombinant protein. The PTD-J-domain can also enhance the immunogenicity of the fused chicken IGF-I polypeptide as well as an oligo-peptide derived from haptoglobin in rodents, possibly via the association with either the extracellular or intracellular Hsp70 proteins.

Novel DNA-directed alkylating agents: design, synthesis and potent antitumor effect of phenyl N-mustard-9-anilinoacridine conjugates via a carbamate or carbonate linker.

A series of phenyl N-mustard-9-anilinoacridine conjugates via a carbamate or carbonate linker was synthesized for antitumor evaluation. The carbamate or carbonate linker is able to lower the reactivity of the phenyl N-mustard pharmacophore and thus, these conjugates are rather chemically stable. The in vitro studies revealed that these derivatives possessed significant cytotoxicity with IC(50) in sub-micromolar range in inhibiting human lymphoblastic leukemia (CCRF-CEM), breast carcinoma (MX-1), colon carcinoma (HCT-116) and human non-small cell lung cancer (H1299) cell growth in vitro. Compounds 10a, 10b, 10e, 10i, and 15a were selected for evaluating their antitumor activity in nude mice bearing MX-1 and HCT-116 xenografts. Remarkably, total tumor remission was achieved by these agents with only one cycle of treatment. Interestingly, no tumor relapse was found in mice treated with 10a over 129 days. This agent is capable of inducing DNA interstrand cross-linking in human non-small lung cancer H1299 cells in a dose dependent manner by modified comet assay and has a long half-life in rat plasma.

Pharmacokinetics of gastrodin and its metabolite p-hydroxybenzyl alcohol in rat blood, brain and bile by microdialysis coupled to LC-MS/MS.

Gastrodin is a pharmacologically active substance isolated from Gastrodia elata Blume with sedation, anti-convulsion and anti-epilepsy activities. A rapid and sensitive liquid chromatography technique coupled to tandem mass spectrometry (LC-MS/MS) system was developed to determine gastrodin and its metabolite p-hydroxybenzyl alcohol (HBA) in rat blood, brain and bile collected using microdialysis technique. The analytes were separated using a reversed phase column (4.6 mm x 150 mm, 5 microm). The mobile phase for column separation was 30% methanol with a flow rate of 0.6 mL/min. As a post-column addition, 1% ammonium hydroxide solution (in methanol) was additionally pumped via a T-connection using a chromatographic pump (BAS PM-80, USA) at a flow rate of 0.2 mL/min after the column separation. A LC-MS/MS system equipped with a negative electrospray ionization (ESI) source in multiple reaction monitoring (MRM) mode was used to monitor m/z 285.0-->122.9 and m/z 123.0-->105.0 transitions for gastrodin and HBA, respectively. The lower limit of quantification (LLoQ) for gastrodin and HBA were 0.5 and 2 ng/mL, respectively. The calibration curves were linear over the range of 0.5-5,000 ng/mL and 2-1,000 ng/mL for gastrodin and HBA with a coefficient of determination >0.995, respectively. This selective and sensitive method is useful for the determination of gastrodin and HBA and in the pharmacokinetic studies of these compounds.

Geldanamycin augments nitric oxide production and promotes capacitation in boar spermatozoa.

Sperm capacitation and the acrosome reaction are fundamentally important to fertilization. Nitric oxide (NO) has been shown to have various functions in male reproduction. This work investigates whether boar sperm can generate NO, as well as the effects of NO and geldanamycin (GA), a heat-shock protein 90 (HSP90)-specific inhibitor, on the capacitation of boar spermatozoa. Observations showed that porcine sperm produced low levels of NO under non-capacitating conditions. However, the NO concentration almost doubled under capacitating conditions (P<0.001). Treatment with NG-nitro-L-arginine methyl ester (L-NAME) reduced the production of NO by 30-40% in capacitating sperm (P<0.05). GA treatment increased it by 23-75% in a dose-dependent manner (P<0.05). L-NAME treatment reduced the percentage of sperm undergoing the acrosome reaction, whereas sodium nitroprusside, an NO-releasing compound, and GA treatment increased the percentage of sperm undergoing the acrosome reaction (P<0.05). GA treatment promoted the production of NO and the acrosome reaction. The increase in NO production by GA treatment was similar to that caused by the calcium ionophore, A23187, suggesting that the GA-induced acrosome reaction may be triggered by an increase of the intracellular calcium concentration. The signaling pathway involved in GA-mediated NO production and its biological function in fertilizing boar spermatozoa will be elucidated in further studies.

Preparation and degradation behavior of polyanhydrides nanoparticles.

Polyanhydrides have been used in many drug delivery systems because of their biodegradability and biocompatibility. Their degradation pattern of surface erosion made them suitable for stable drug release applications. However, in nanoparticle systems, this degradation pattern may not hold, and the drug release kinetics will be different also. In this study, copolymers of 1,3-bis(p-carboxyphenoxy)propane (CPP) and sebacic acid (SA) were synthesized to investigate the different degradation patterns of disk and nanoparticle forms of polyanhydride, in addition to the study of the method of preparation of nanoparticles from these copolymers. By using oil-in-water emulsion and poly(vinyl alcohol) (PVA) as emulsifier, nanoparticles of the size 200-500 nm were prepared. The size of the particles can be controlled by varying polymer concentration or PVA concentration, but different SA:CPP ratio did not affect the particle size significantly. Degradation was followed by detecting the amount of monomers released to the medium. It was found that CPP and SA were released at approximately the same rate from nanoparticles; while in disk form, SA was released much faster than CPP. It was found that contrary to general trend in disks, higher CPP content, a more hydrophobic component than SA, in the copolymer actually accelerated the degradation of nanoparticles.

Differentially expressed transcripts in shell glands from low and high egg production strains of chickens using cDNA microarrays.

We have constructed a tissue-specific in-house cDNA microarray to identify differentially expressed transcripts in shell glands from low (B) and high (L2) egg production strains of Taiwanese country chickens during their egg-laying period. The shell gland cDNA library was constructed from the high egg production strain. cDNA clones (7680) were randomly selected and their 5'-end sequences characterized. After excluding overlapping sequences, an in-house cDNA microarray, representing 2743 non-redundant transcripts, was generated for functional genomic studies. Using our microarray, we have successfully identified 85 differentially expressed transcripts from the two different strains of chicken shell glands. In this study, 34 of these transcripts were associated with signal transduction, protein biosynthesis, cell adhesion, cellular metabolism, skeletal development, cell organization and biogenesis. We selected a number of the differentially expressed transcripts for further validation using semi-quantitative RT-PCR. These included elongation factor 2 (EEF2), ovocalyxin-32 (OCX-32) and annexin A2 (ANXA2) which were expressed at high levels in the chicken shell glands of the B strain and, in contrast, the coactosin-like protein (COTL1), transcription factor SOX18 and MX protein were more highly expressed in the L2 strain. Our results suggest that these differentially expressed transcripts may be suitable to use as molecular markers for high rates of egg production, and now need to be investigated further to assess whether they can be applied for use in breeding selection programs in Taiwanese country chickens.

Amphiphilic poly(D,L-lactic acid)/poly(ethylene glycol)/poly(D,L-lactic acid) nanogels for controlled release of hydrophobic drugs.

Photocrosslinked nanogels with a hydrophobic core and hydrophilic shell are successfully fabricated with the goal of obtaining a biocompatible and biodegradable drug carrier for hydrophobic anticancer drugs. These nanogels are composed of amphiphilic triblock copolymers, poly(D,L-lactic acid)/poly(ethylene glycol)/poly(D,L-lactic acid) (PLA-PEG-PLA), with acrylated groups at the end of the PLA segments. The copolymers are synthesized by ring-opening polymerization and possess a low CMC (49.6 mg x L(-1)), which easily helps to form micelles by self-assembly. The acrylated end groups allow the micelles to be photocrosslinked by ultraviolet irradiation, which turn the micelles into nanogels. These nanogels exhibit excellent stability as a suspension in aqueous media at ambient temperature as compared to the micelles. Moreover, the size of the nanogels is easily manipulated in a range of 150 to 250 nm by changing the concentration of crosslinkers, e.g., ethylene glycol dimethacrylate, and ultraviolet light irradiation time. The nanogels achieve a high encapsulation efficiency and offer a steady and long-term release mechanism for the hydrophobic anticancer drug, CPT. It shows that these nanogels are useful for a hydrophobic anticancer drug-carrier system. [pictures: see text] Formation of the PLA-PEG-PLA nanogels.

Analysis of chicken serum proteome and differential protein expression during development in single-comb White Leghorn hens.

Serum is believed to harbor thousands of distinct proteins that are either actively secreted or leak from various blood cells or tissues. Exploring protein composition in serum may accelerate the discovery of novel protein biomarkers for specific economic traits in livestock species. This study analyzed serum protein composition to establish a 2-DE reference map, and monitored protein dynamics of single-comb White Leghorn hens at 8, 19 and 23 weeks after hatching. A total of 119 CBB-stained and 315 silver-stained serum protein spots were analyzed by MALDI-TOF MS. Of these, 98 CBB-stained and 94 silver-stained protein spots were significantly matched to existing chicken proteins. The identified spots represented 30 distinctive proteins in the serum of laying hens. To compare protein expression during development, expression levels of 47 protein spots were quantified by relative spot volume with Melanie 3 software. Ten protein spots increased and 3 protein spots decreased as hen age increased. Previous research has suggested that some of these proteins play critical roles in egg production. The differentially expressed proteins with unknown identities will be valuable candidates for further explorations of their roles in egg production of laying hens.

A reference map and identification of porcine testis proteins using 2-DE and MS.

The development of the testis is essential for maturation of male mammals. A complete understanding of proteins expressed in the testis will provide biological information on many reproductive dysfunctions in males. The purposes of this study were to apply a proteomic approach to investigating protein composition and to establish a 2-D PAGE reference map for porcine testis proteins. MALDI-TOF MS was performed for protein identification. When 1 mg of total proteins was assayed by 2-D PAGE and stained with colloidal CBB, more than 400 proteins with a pI of pH 3-10 and M(r) of 10-200 kDa could be detected. Protein expression varied among individuals, with CV between 4.7 and 131.5%. A total of 447 protein spots were excised for identification, among which 337 spots were identified by searching the mass spectra against the NCBInr database. Identification of the remaining 110 spots was unsuccessful. A 2-D PAGE-based porcine testis protein database has been constructed on the basis of the results and will be published on the WWW. This database should be valuable for investigating the developmental biology and pathology of porcine testis.

Developmental changes of heat-shock proteins in porcine testis by a proteomic analysis.

Heat-shock proteins (HSPs) are important in spermatogenesis. This study investigated developmental changes in the expression of major HSPs in porcine testis. The testis from five immature (mean age 2.9+/-0.1 months) and five mature boars (35.7+/-14.0 months) were examined. Two-dimensional polyacrylamide gel electrophoresis was conducted and proteins were identified by Western blotting and/or matrix-assisted laser desorption/ionization mass spectrometry. Moreover, the 90, 70, and 60 kDa HSPs, 70 kDa heat-shock cognate protein (HSC 70), tubulin, and actin were quantified on two-dimensional gels. Protein spots were quantified by densitometry, combined with a computer-assisted image analysis system. Immunohistochemistry was performed to analyze the expression pattern of major HSPs and beta-tubulin in testis. One isoform of HSP 90 (HSP 90 alpha), two isoforms of HSC 70 (HSC 70a and HSC 70c), one isoform of HSP70 (HSP 70e), and tubulin increased after sexual maturation (P<0.05). A testis-specific HSP70 (P70t) was markedly increased in the testes of sexually mature boars. Meanwhile, levels of actin and some isoforms of HSPs including 60 kDa HSP remained similar in both groups. These observations were further confirmed by immunohistochemistry; therefore, the upregulation of protein expression in the adult testis could be attributed to a higher level of protein expression and the number of cells that were HSPs-positive already resided in the immature testis. The differential expression of major HSPs suggested that they may be important in porcine spermatogenesis.