Myostatin down‐regulates the IGF‐2 expression via ALK‐Smad signaling during myogenesis in cattle

Myostatin (MSTN) is a negative regulator during muscle differentiation, whereas insulin‐like growth factors (IGFs) are essential for muscle development. MSTN and IGFs act oppositely during myogenesis, but there is little information on the mutual relationship of MSTN and IGFs. The present study was conducted to examine whether MSTN affects IGF expression during early myogenesis in cattle. IGF‐1 mRNA was similarly expressed in M. longissimus thoracis of double‐muscled (DM) and normal (NM) Japanese shorthorn cattle. IGF‐2 mRNA expression was consistently higher in the normal and regenerating muscle of DM cattle than those of NM cattle. When myoblasts were isolated from regenerating M. longissimus thoracis, IGF‐2 mRNA expression showed a significant increase in differentiating DM derived myoblasts (DM‐myoblasts) as compared with differentiating NM derived myoblasts (NM‐myoblasts). An addition of recombinant mouse myostatin (rMSTN) to myoblast cultures attenuated IGF‐2 mRNA expression and decreased myotube formation, but did not effect IGF‐1 mRNA expression. An activin‐like kinase (ALK) inhibitor, SB431542, mediates MSTN action, suppressed the translocation of Smad2/3 into the nucleus in DM‐myoblasts, and restored the attenuated IGF‐2 mRNA expression and the decreased myotube formation induced by rMSTN in myoblast cultures. The findings indicate that MSTN may negatively regulate myoblast differentiation by suppressing IGF‐2 expression via ALK‐Smad signaling.     

Quantitative real‐time PCR monitoring of Escherichia coli and Clostridium perfringens with oral administration of Lactobacillus plantarum strain Lq80 to weaning piglets

Levels of fecal or intestinal lactobacilli, Escherichia coli and Clostridium perfringens, and the prevalence of clostridial alpha toxin gene and heat‐stable toxin (ST) gene of enterotoxigenic E. coli (ETEC) were monitored in weaned piglets before (day 0) and during (days 7, 14, and 21) the administration of Lactobacillus plantarum strain Lq80. Lactobacilli were enumerated in a culture‐dependent method. The remainders were determined by quantitative real‐time PCR. In this quantitative real‐time PCR method, the detection limit was proved to be as low as 10³ cells/g feces or intestinal contents. Number of lactobacilli increased from day 0 to day 7 (P < 0.05), to day 14 (P < 0.05), and to day 21 (P = 0.07) in the Lq80‐administered group. L. plantarum contributed to as low as 10% of the lactobacillal population in the Lq80‐administered group. The number of E. coli and C. perfringens, and the prevalence of alpha toxin gene in feces or intestinal contents of the Lq80‐administered group decreased, at least in the first week of the postweaning period. Oral administration of L. plantarum strain Lq80 can stimulate the growth of indigenous lactobacilli and decrease ST‐producing ETEC and C. perfringens in the intestine of postweaning piglets.     

Oncolytic reovirus synergizes with chemotherapeutic agents to promote cell death in canine mammary gland tumor

The oncolytic effects of reovirus in various cancers have been proven in many clinical trials in human medicine. Oncolytic virotherapy using reovirus for canine cancers is being developed in our laboratory. The objective of this study was to examine the synergistic anti-cancer effects of a combination of reovirus and low doses of various chemotherapeutic agents on mammary gland tumors (MGTs) in dogs. The first part of this study demonstrated the efficacy of reovirus in canine MGTs in vitro and in vivo. Reovirus alone exerted significant cell death by means of caspase-dependent apoptosis in canine MGT cell lines. A single injection of reovirus impeded growth of canine MGT tumors in xenografted mice, but was insufficient to induce complete tumor regression. The second part of this study highlighted the anti-tumor effects of reovirus in combination with low doses of paclitaxel, carboplatin, gemcitabine, or toceranib. Enhanced synergistic activity was observed in the MGT cell line treated concomitantly with reovirus and in all the chemotherapeutic agents except toceranib. In addition, combining reovirus with paclitaxel or gemcitabine at half dosage of half maximal inhibitory concentration (IC₅₀) enhanced cytotoxicity by activating caspase 3. Our data suggest that the combination of reovirus and low dose chemotherapeutic agents provides an attractive option in canine cancer therapy.     

Implantation of permanent transvenous endocardial pacemaker in a dog with atrioventricular block

A 10-year old male mongrel dog was presented to the University Veterinary Teaching Hospital with a two-month history of episodic syncope. Twenty-four hr Holter electrocardiographic (ECG) recording revealed frequent episodes of advanced atrioventriculer block with long periods of ventricular asystole. The cause of syncope was determined to be Adams-Stokes syndrome exhibited bradyarrhythmia. After the animal failed to respond to medical therapy, permanent transvenous pacemaker implantation was performed. Postoperative Holter ECG showed 100 beat per min programmed pacemaker rhythm, which indicated successful capture of the artificial pacing. The dog recovered smoothly from the operation and syncopal episodes completely disappeared. Six months after the surgery, no complications were observed and the dog's quality of life has dramatically improved.     

Lesional expression of thymus and activation-regulated chemokine in canine atopic dermatitis

Glucocorticoids are reported to bias cytokines to a Th2 phenotype. However, this dogma has been advanced largely from studies utilizing potent glucocorticoid analogs. The current study was conducted to revisit the issue of glucocorticoid modulation of Th1/Th2 cytokine production and evaluate migration inhibitory factor (MIF) mRNA expression in cultured pig splenocytes treated with physiologically relevant concentrations of cortisol (CORT). Dexamethasone (DEX) was included for comparison. In Experiment 1, DEX, at 150 and 300 nM, suppressed concanavalin (ConA)-stimulated IFNgamma at both 12 and 24 h in culture, and IL-10 at 24h (P<0.05). Both 150 and 300 nM CORT suppressed IL-10 at 24 h (P<0.05), but neither concentration affected IFNgamma at 24 h. In Experiment 2, cells were cultured with a broader range of CORT for 48 h following ConA. Parallel cultures with identical treatments also were conducted in separate plates for evaluation of glucocorticoid regulation of MIF mRNA. IFNgamma was reduced by 300 nM DEX at 12, 24, and 48 h (P<0.05), whereas 150 and 300 nM CORT blunted IFNgamma at 24 h (P<0.05), but not 48 h. ConA increased IL-2 (P<0.01), but none of the steroid treatments affected IL-2. At both 12 and 24 h, IL-10 was reduced by 300 nM DEX and by 150 and 300 nM CORT (P<0.05). ConA increased relative abundance of MIF mRNA (P<0.001), but no steroid treatment affected MIF mRNA. In Experiment 3, steroid additions were delayed by 24 h after ConA, and cytokine concentrations evaluated 48 h later. Again, separate cultures were used for determination of effect of treatments on MIF mRNA. None of the steroid treatments affected IFNgamma, but 300 nM DEX reduced IL-10 (P<0.05). All of the CORT treatments (75-300 nM) reduced MIF mRNA (P<0.05), whereas DEX did not affect MIF mRNA in this experiment. The current experiments suggest that both DEX and high physiological concentrations of CORT can suppress both type 1 and type 2-like cytokines in cultured pig splenocytes. But, IL-10 was generally more sensitive to CORT suppression with increased time in culture than was IFNgamma. In addition, MIF mRNA could be suppressed by delayed addition of CORT to porcine splenocytes. Taken together, the data do not support the hypothesis that CORT directs the cytokine milieu toward a type 2 bias in cultured pig splenocytes.     

Effects of protein phosphatase inhibitor calyculin a on the postacrosomal protein serine/threonine phosphorylation state and acrosome reaction in boar spermatozoa incubated with a cAMP analog

In order to reveal the involvement of the sperm postacrosomal region in the acrosome reaction, we examined the effects of the protein phosphatase inhibitor calyculin A on the postacrosomal protein serine/threonine phosphorylation state and acrosome morphology in boar spermatozoa incubated with a cAMP analog. Proteins were highly phosphorylated on the serine/threonine residues only in the postacrosomal region before incubation. After 90-min incubation without calyculin A, the protein phosphorylation state declined in the postacrosomal region irrespective of the capacitation state while it remained under the detectable level in the other regions of the sperm head. However, addition of calyculin A effectively suppressed the decline in protein phosphorylation state and increased an inactive form of protein phosphatase 1 in the postacrosomal region. On the other hand, this inhibitor had no influence on the protein phosphorylation state in the acrosome and equatorial segment. After incubation without calyculin A for 180 or 360 min, many spermatozoa exhibited acrosomal changes and loss that indicated occurrence of the acrosome reaction. However, addition of calyculin A significantly blocked these events. These results are consistent with our suggestion that postacrosomal serine/threonine-phosphorylated proteins are involved in suppression of the acrosome reaction in boar spermatozoa in vitro.     

氧化鱼油与棕榈油对花鲈肝脏抗氧化酶及组织结构的影响

在21个200 L圆形流水水槽中各放养花鲈(Lateolabrax maculatus)幼鱼,初始体质量为(1.73±0.01)g.在相同的环境条件下投喂以棕榈油(P)与新鲜鱼油(F)或氧化鱼油(OF)不同比例混合的7组饲料(10P,10F,6F4P,4F6P,100F,60F4P和40F6P),每个实验组设3个重复.60d饲养实验结束后,通过测定花鲈肝脏部分抗氧化酶(超氧化物歧化酶SOD,过氧化氢酶CAT和谷胱甘肽过氧化物酶GSH-Px)活力和总抗氧化能力(T-AOC),并对肝脏,肠以及肌肉进行组织学观察,研究在配合饲料中添加棕榈油替代氧化鱼油对花鲈肝脏抗氧化酶活力及其对组织结构的影响.结果表明,在含有氧化鱼油的实验组中,添加40%的棕榈油对花鲈肝脏的CAT活力以及GSH-Px活力有显著提高作用(P<0.05),对SOD活力有一定的提高效果(P>0.05),各组间总抗氧化能力差异不显著(P>0.05).新鲜鱼油组(10F,6F4P,4F6P)间抗氧化酶活力差异不显著(P>0.05).非氧化组花鲈肝脏和肌肉组织结构完整,100F组花鲈肝脏和肌肉组织呈现氧化脂肪中毒症状,添加棕榈油后(60F4P和40F6P)症状减轻,各组花鲈肠结构无明显差异.由此认为,棕榈油在花鲈饲料中部分替代鱼油是可行的,另外,在贮存不当的饲料中,后喷涂一定比例的棕榈油能对其毒性产生缓解作用.     

Dietary Soy Peptide Enhances Thermotolerance and Survival of Juvenile Japanese Flounder,Paralichthys olivaceus

Soy peptide (SP), a soy protein enzymatic hydrolysate, contains bioactive substances that could be utilized as an immune‐stimulating feed ingredient. The experiment evaluated the efficacy of dietary SP on promoting growth, and enhancing tolerance and survival to heat stress in juvenile Japanese flounder, Paralichthys olivaceus. Four diets were incorporated with different levels of SP (0, 2, 5, and 10%) and a 6‐wk feeding trial ensued. Following the feeding trial, the experimental groups were subjected to heat stress to measure survival rate and heat shock protein 70s (HSP70s) in gill, liver, and skin. Fish fed diets with SP inclusion showed considerable decrease in percent weight gain. Significantly higher lethal time values to 50% mortality (LT₅₀) value were recorded for fish fed 10% SP. Moreover, LT₅₀ values of fish fed 2 and 5% SP were significantly higher compared with fish fed control diet. HSP70s produced in all the tissues were significantly highest in fish fed 10% SP. HSP70s values were significantly higher in fish fed 2 and 5% SP compared with fish fed control diet. A significant reduction in HSP70s among all groups during recovery period was also observed. These results suggest that SP can be used to enhance the immune response and survival of P. olivaceus under heat stress.