Effects of maternal treatment with β‐hydroxy‐β‐metylbutyrate and 2‐oxoglutaric acid on femur development in offspring of minks of the standard dark brown type

The aim of the study was to evaluate the effect of the diet, mother type and sex of the offspring on the mechanical and geometric parameters of long bones as well as bone tissue density in minks. Primiparous and multiparous dams were supplemented with β‐hydroxy β‐methylbutyrate (a metabolite of leucine, at the daily dosage of 0.02 g/kg of body weight) and/or 2‐oxoglutaric acid (a precursor of glutamine, at the daily dosage of 0.4 g/kg of body weight) during gestation. The diet did not influence bone tissue density and the length of the humerus. An increase in the length of the femur was noted in male offspring delivered by multiparous dams. The diet resulted in an increase in the weight of the humerus in males from multiparous dams and a decrease in offspring from primiparous dams. Heavier femora were noted in male offspring delivered by both types of dams. The maximum elastic strength of the humerus was higher in the offspring delivered by multiparous than primiparous dams, irrespective of the offspring sex. The diet resulted in reduction in the ultimate strength of the femur in the male offspring delivered by primiparous dams. Only females born by multiparous dams, irrespective of the diet, showed a significant increase in the cross‐sectional area of the humerus, while a significant decline was noted in males delivered by multiparous dams and in all the offspring delivered by primiparous dams. An increase in the cross‐sectional area of the femur was noted in the offspring delivered by multiparous dams, while reduction was observed in the offspring delivered by primiparous dams. These results have shown for the first time that the presence of β‐hydroxy‐β‐methylbutyrate or 2‐oxoglutaric acid in the diet of pregnant primiparous or multiparous dams unambiguously affects the geometry and mechanical properties of offspring's long bones.     

β‐carotene attenuates lipopolysaccharide‐induced inflammation via inhibition of the NF‐κB,JAK2/STAT3 and JNK/p38 MAPK signaling pathways in macrophages

β‐carotene is one of the most abundant carotenoids, has potential anti‐inflammatory effect, it has been reported that β‐carotene could suppress LPS‐induced inflammatory responses by inhibiting nuclear factor kappa B (NF‐κB) translocation, but the more detailed molecular mechanisms underlying the anti‐inflammatory action of β‐carotene remain to be fully understood. In this study, we investigated the influence of β‐carotene on the activation of JAK2/STAT3, MAPK, and NF‐κB signaling pathway induced by LPS in RAW264.7 cells and peritoneal macrophages. Cells were treated with different concentrations of β‐carotene for 3 hr after LPS treatment for 24 hr. The mRNA expression and the release of IL‐1β, IL‐6, and TNF‐α were evaluated by RT‐PCR and ELISA, and the level of signaling proteins of JAK2/STAT3, MAPK, and NF‐κB signaling pathway were detected by Western blot. The results showed that β‐carotene significantly suppressed (p < 0.05) LPS‐induced release of IL‐1β, IL‐6, and TNF‐α and their mRNA expression. LPS‐induced JAK2/STAT3, IκB/NF‐κB p65, JNK/p38 MAPK signal activation were significantly attenuated (p < 0.05) by β‐carotene in a dose‐dependent manner. In conclusion, β‐carotene could attenuate LPS‐induced inflammation via inhibition of the NF‐κB, JAK2/STAT3, and JNK/p38 MAPK signaling pathways in macrophages.     

Dietary supplementation of β‐hydroxy‐β‐methylbutyrate in animals – a review

The leucine metabolite β‐hydroxy‐β‐methylbutyrate (HMB) has been studied by many researchers over the last two decades. In particular, the utility of HMB supplementation in animals has been shown in numerous studies, which have demonstrated enhanced body weight gain and carcass yield in slaughter animals; positive immunostimulatory effect; decreased mortality; attenuation of sarcopenia in elderly animals; and potential use in pathological conditions such as glucocorticoid‐induced muscle loss. The aim of this study was to summarize the body of research on HMB supplementation in animals and to examine possible mechanisms of HMB action. Furthermore, while the safety of HMB supplementation in animals is well documented, studies demonstrating efficacy are less clear. The possible reasons for differences in these findings will also be examined.     

β‐hydroxy‐β‐methyl butyrate promotes leucine metabolism and improves muscle fibre composition in growing pigs

The aim of this study was to investigate the effects of excess leucine (Leu) vs. its metabolites α‐ketoisocaproate (KIC) and β‐hydroxy‐β‐methyl butyrate (HMB) on Leu metabolism, muscle fibre composition and muscle growth in growing pigs. Thirty‐two pigs with a similar initial weight (9.55 ± 0.19 kg) were fed 1 of 4 diets for 45 days: basal diet, basal diet + 1.25% L‐Leu, basal diet + 1.25% KIC‐Ca, basal diet + 0.62% HMB‐Ca. Results indicated that relative to the basal diet and HMB groups, Leu and KIC groups exhibited increased Leu concentrations and decreased concentrations of isoleucine, valine and EAAs in selected muscle (p < 0.05) and had lower mRNA levels of MyHC I and higher expression of MyHC IIx/IIb (p < 0.05), and there was no significant difference between the basal and HMB‐supplemented groups. Moreover, the mRNA expression levels of AMPKα and UCP3 were higher but the myostatin mRNA levels were lower in the soleus muscle of the HMB group than those from other groups (p < 0.05). These findings demonstrated that doubling dietary Leu content exerted growth‐depressing effects in growing pigs; dietary KIC supplementation induced muscular branched‐chain amino acid imbalance and promoted muscle toward a more glycolytic phenotype; while dietary HMB supplementation promoted the generation of more oxidative muscle types and increased muscle growth specially in oxidative skeletal muscle, and these effects of HMB might be associated with the AMPKα‐Sirt1‐PGC‐1α axis and mitochondrial biogenesis.     

Effects of β‐carotene‐enriched dry carrots on β‐carotene status and colostral immunoglobulin in β‐carotene‐deficient Japanese Black cows

Data from 18 β‐carotene‐deficient Japanese Black cows were collected to clarify the effects of feeding β‐carotene‐enriched dry carrots on β‐carotene status and colostral immunoglobulin (Ig) in cows. Cows were assigned to control or carrot groups from 3 weeks before the expected calving date to parturition, and supplemental β‐carotene from dry carrots was 138 mg/day in the carrot group. Plasma β‐carotene concentrations in the control and carrot groups at parturition were 95 and 120 μg/dL, and feeding dry carrots slightly improved plasma β‐carotene at parturition. Feeding dry carrots increased colostral IgA concentrations in cows and tended to increase colostral IgG₁, but colostral IgM, IgG₂, β‐carotene and vitamin A were not affected by the treatment. Feeding dry carrots had no effects on plasma IgG₁, IgA and IgM concentrations in cows, but plasma IgG₁ concentrations decreased rapidly from 3 weeks before the expected calving date to parturition. These results indicate that feeding β‐carotene‐enriched dry carrots is effective to enhance colostral IgA and IgG₁ concentrations in β‐carotene‐deficient cows.     

Immunohistochemical expression of p63, Ki67 and β‐catenin in canine transitional cell carcinoma and polypoid cystitis of the urinary bladder

Transitional cell carcinoma (TCC) is a urinary bladder tumour associated with high mortality in dogs. In this study, we investigated the feasibility of using p63, Ki67 or β‐catenin as a clinical marker for predicting biological behaviour and prognosis in canine TCC. Expression levels of these proteins in TCC (n = 25), polypoid cystitis (n = 5) and normal urinary bladder (n = 5) were scored after immunohistochemical staining. The staining scores for p63 (P < 0.01) and β‐catenin (P < 0.05) in TCC were significantly lower than those in normal urinary bladder and polypoid cystitis. In contrast, Ki67 (P < 0.01) staining scores in TCC were significantly higher than those in normal urinary bladder and polypoid cystitis. In TCC, low p63 expression was significantly related to the presence of vessel invasion (P < 0.05) and metastasis (P < 0.01) as well as short survival time (P < 0.05). These findings show that p63 could be a reliable marker for predicting prognosis in canine TCC.     

Expression of VEGFR and PDGFR‐α/‐β in 187 canine nasal carcinomas

Radiotherapy represents the standard of care for intranasal carcinomas. Responses to tyrosine kinase inhibitors (TKIs) have been reported but data on expression of target receptor tyrosine kinases (rTKs) is limited. This study characterizes the expression of vascular endothelial growth factor receptor (VEGFR), platelet‐derived growth factor receptor (PDGFR)‐α and PDGFR‐β in canine intranasal carcinomas. Histological samples from 187 dogs were retrieved. Immunohistochemistry was performed using commercially available antibodies. Expression of rTKs was classified into weak, moderate or intense and additionally recorded as cytoplasmic, membranous, cytoplasmic‐membranous, nuclear or stromal. VEGFR was expressed in 158 dogs with predominantly moderate expression (36.9%) and a cytoplasmic‐membranous expression pattern (70.9%). PDGFR‐α was detected in 133 with predominantly weak expression (57.9%) and cytoplasmic pattern (87.9%). PDGFR‐β was identified in 74 patients with a predominantly moderate expression (17.6%) and cytoplasmic expression pattern (63.5%). Co‐expression of rTKs was common. These results confirm expression of VEGFR, PDGFR‐α and PDGFR‐β in canine intranasal carcinomas and support the utility of TKIs.     

Thiamine deficiency affects glucose transport and β‐oxidation in rats

Thiamine is recognized as a cofactor for many enzymes involved in intermediary metabolism responsible for energy production. Animal model of thiamine deficiency (TD) included direct evaluation of glucose uptake by estimation of ³H‐deoxyglucose transport across red blood cells membranes and β‐oxidation of fatty acids in isolated leucocytes. Feeding of animals with the thiamine‐deficient diet (0.018 mg/kg diet) for 30 days resulted in disturbances in energy production. The thiamine intake was limited not only by vitamin B₁ deficiency in the diet, but also by time‐dependent drop of feed consumption by rats fed this diet. At the end of experiment, diet consumption in this group of rats was 52% lower than in the control group. This was accompanied by low glucose uptake by erythrocytes of rats suffering vitamin B₁ deficiency for longer time. At the end of experimental period, glucose uptake was over 2 times lower in TD erythrocytes than in control RBC. Such drop of energy production was not compensated by delivery of energy from fatty acid degradation. In leucocytes from TD rats, the β‐oxidation was also suppressed. Observed significant decrease of serum insulin from 2.25 ± 0.25 ng/ml (day 0) to 1.94 ± 0.17 ng/ml (day 30) might have significant impact on observed energy production disorders. The results from this study indicate that the thiamine deficiency significantly reduces feed intake and causes modest abnormalities in glucose and fatty acid utilization.     

Effects of supplemental β‐carotene on colostral immunoglobulin and plasma β‐carotene and immunoglobulin in Japanese Black cows

Data from 26 Japanese Black cows were collected to clarify the effects of supplemental β‐carotene on colostral immunoglobulin (Ig) and plasma β‐carotene and Ig in the cows. Cows were assigned to control or β‐carotene groups from 21 days before the expected calving date to 60 days after parturition. Supplemental β‐carotene was provided at 500 mg/day in the β‐carotene group. Supplemental β‐carotene drastically increased plasma β‐carotene concentrations in the cows from parturition to 60 days after parturition, and plasma β‐carotene concentrations in the control and β‐carotene groups at parturition were 202 and 452 μg/dl, respectively. Supplemental β‐carotene had no effects on plasma IgG₁, IgA or IgM concentrations at parturition. Supplemental β‐carotene increased colostral IgG₁ concentrations in the cows, but colostral β‐carotene, IgA and IgM concentrations were not affected by supplemental β‐carotene. These results indicate that supplemental β‐carotene is effective to enhance colostral IgG₁ concentrations and plasma β‐carotene concentrations in Japanese Black cows.     

Effects of supplementation with β‐carotene on the growth performance and intestinal mucosal barriers in layer‐type cockerels

β‐carotene is a robust modulator of mucosal barriers, and it can amplify the immunoglobulin A (IgA) response via the retinoic acid (RA)–mediated pathway. We investigated the influence of β‐carotene on intestinal barriers in layer‐type cockerels. In this study, β‐carotene has a positive influence on growth performance and intestinal morphology. β‐carotene remarkably enhanced serum secretory immunoglobulin A (sIgA) levels, jejunal mucosal sIgA, and IgA concentrations. β‐Carotene significantly enhanced mRNA expression levels of IgA, CC chemokine receptor‐9 (CCR9), polymeric immunoglobulin receptor (pIgR), and retinoic acid receptor α (RARα) in the ileal tissues and pIgR in the jejunal tissues. β‐Carotene improves mRNA expression of intestinal barrier‐related proteins including: mucin‐2 (MUC‐2), zonula occludens‐2 (ZO‐2), occludins (OCLN), and zonula occludens‐1 (ZO‐1) in the ileal tissues. Moreover, β‐carotene decreased the levels of Escherichia coli and elevates the levels of Lactobacillus. The results indicate that β‐carotene can promote growth performance and contribute to the gradual development of intestinal barriers in Hyline Brown chicks. This study enriches our knowledge about the effects of β‐carotene on intestinal barrier and highlights a theoretical basis of β‐carotene application in the poultry industry.     

Integrin heterodimer α9β1 is localized on the surface of porcine spermatogonial stem cells in the undifferentiated state

A previous study found that undifferentiated porcine spermatogonial stem cells (SSCs) did not adhere to tenascin C, indicating that the integrin α₉ and β₁ subunits are inactive on the surface of porcine SSCs. However, that study used recombinant tenascin C without FNIII‐like repeats. Therefore, this study re‐evaluated the existence of integrin α₉β₁ actively functioning on the plasma membrane of porcine SSCs using full‐length native tenascin C with FNIII‐like repeats. The localization and function of the integrin heterodimer were confirmed using immunocytochemistry, attachment and antibody inhibition assays. In undifferentiated porcine SSCs with integrin α₉β₁ on the cell surface, adhesion to native tenascin C was significantly higher compared with cells lacking native tenascin C and functional blocking of integrin α₉β₁ significantly inhibited the attachment to native tenascin C compared with no functional blocking. Accordingly, we confirmed that the integrin α₉ and β₁ subunits function as an active heterodimer on the surface of porcine SSCs in the undifferentiated state.     

Effect of the β‐carotene oxygenase 2 genotype on the content of carotenoids,retinol and α‐tocopherol in the liver,fat and milk of rabbit does,reproduction parameters and kitten growth

Mutations in the β‐carotene oxygenase 2 (BCO2) gene can impair the function of the enzyme that breaks down carotenoids. As a result, gradual accumulation of unoxidized carotenoids in animal tissues gives them a yellow colour. The aim of the study was to determine the content of carotenoids, retinol and α‐tocopherol in the liver, fat and milk of rabbit does with three different genotypes determined by AAT‐deletion mutation at codon 248 of the BCO2 gene and to find out whether differences in the concentrations of the above compounds in the tissues and milk of the does affect reproduction parameters and the rearing rate of kittens. The experimental materials comprised 36 does, 12 of each genotype of the BCO2 gene, with their litters. Females with their litters were placed in individual cages, on deep litter. Between days 7 and 13 of lactation, samples of milk were collected from the does. The kittens stayed with their mothers until 35 days of age. After weaning, the does were sacrificed. Tissue samples of liver and perirenal fat were collected for chemical analyses. Additionally, based on samples taken from one female, RNA expression levels were determined from the mammary gland and liver, adipose tissue and skin. It was found that homozygous does with deletion at codon 248 of the BCO2 gene were characterized by considerably higher concentrations of xanthophylls and beta‐carotene in the liver, adipose tissue and milk than does with the remaining genotypes. However, the differences in the content of the above compounds in milk had no influence on litter weight or the number and rearing rate of kittens. Additionally, RNA expression of the BCO2 gene was found in the mammary tissue of lactating doe and its level was similar to those noted in the liver and adipose tissue.     

Expression analysis of an α‐1, 3‐galactosyltransferase,an enzyme that creates xenotransplantation‐related α–Gal epitope,in pig preimplantation embryos

α‐1,3‐Galactosyltransferase (α‐GalT), an enzyme creating Galα1‐3Gal (α‐Gal) epitope on the cell surface in some mammalian species such as pigs, is known to be a key factor that causes hyperacute rejection upon transplantation from pigs to humans. To establish the RNA interference‐based suppression of endogenous α‐GalT messenger RNA (mRNA) synthesis in porcine preimplantation embryos, we determined the suitable embryonic stage at which stage such approach is possible by using the semi‐quantitative RT‐PCR (qRT‐PCR) and the cytochemical method using a fluorescence‐labeled Bandeiraea simplicifolia Isolectin B₄ (BS‐I‐B₄). Staining with BS‐I‐B₄ demonstrated that α‐Gal epitope expression was first recognized at the 8‐cell stage, and increased up to the hatched blastocyst stage. Single embryo‐based qRT‐PCR also confirmed this pattern. These results indicate that creation of α‐Gal epitope is proceeded by de novo synthesis of α‐GalT mRNA in porcine preimplantation embryos with peaking at the blastocyst stage.     

Effects of transforming growth factor‐β1 treatment on muscle regeneration and adipogenesis in glycerol‐injured muscle

Transforming growth factor (TGF)‐β1 is associated with fibrosis in many organs. Recent studies demonstrated that delivery of TGF‐β1 into chemically injured muscle enhances fibrosis. In this study, we investigated the effects of exogenous TGF‐β1 on muscle regeneration and adipogenesis in glycerol‐injured muscle of normal mice. Tibialis anterior (TA) muscles were injured by glycerol injection. TGF‐β1 was either co‐injected with glycerol, as an ‘early treatment’ group, or injected at day 4 after glycerol, as a ‘late treatment’ group and the TA muscles were collected at day 7 after initial injury. Myotube density was significantly lower in the early treatment group than in the glycerol‐injured group (without TGF‐β1 treatment). Moreover, the Oil red O‐positive area was significantly smaller in the early treatment group than in the late treatment group and glycerol‐injured group. Furthermore, TGF‐β1 treatment increased endomysial fibrosis and induced immunostaining of α‐smooth muscle actin. The greater inhibitory effects of early TGF‐β1 treatment than that of late TGF‐β1 treatment during regeneration in glycerol‐injured muscle suggest a more potent effect of TGF‐β1 on the initial stage of muscle regeneration and adipogenesis. Combination of TGF‐β1 with glycerol might be an alternative to enhance muscle fibrosis for future studies.     

Extended‐Spectrum β‐lactam Resistance in the Enteric Flora of Patients at a Tertiary Care Medical Centre

The dissemination of Enterobacteriaceae expressing resistance to extended‐spectrum cephalosporins, which are therapeutically used in both human and veterinary medicine, is of critical concern. The normal commensal flora of food animals may serve as an important reservoir for the zoonotic food‐borne transmission of Enterobacteriaceae harbouring β‐lactam resistance. We hypothesized that the predominant AmpC and ESBL genes reported in US livestock and fresh retail meat products, bla_CMY‐2 and bla_CTX‐M, would also be predominant in human enteric flora. We recovered enteric flora from a convenience sample of patients included in a large tertiary medical centre's Clostridium difficile surveillance programme to screen for and estimate the frequency of carriage of AmpC and ESBL resistance genes. In‐ and outpatient diarrhoeic submissions (n = 692) received for C. difficile testing at the medical centre's clinical diagnostic laboratory from July to December, 2013, were included. Aliquoted to a transport swab, each submission was inoculated to MacConkey broth with cefotaxime, incubated at 37°C and then inoculated to MacConkey agars supplemented with cefoxitin and cefepime to select for the AmpC and ESBL phenotypes, with bla_CMY and bla_CTX‐M genotypes confirmed by PCR and sequencing. From the 692 diarrhoeic submissions, our selective culture yielded 184 isolates (26.6%) with reduced susceptibility to cefotaxime. Of these, 46 (6.7%) samples harboured commensal isolates carrying the AmpC bla_CMY. Another 21 (3.0%) samples produced isolates harbouring the ESBL bla_CTX‐M: 19 carrying CTX‐M‐15 and 2 with CTX‐M‐27. Our results indicate that β‐lactam resistance genes likely acquired through zoonotic food‐borne transmission are present in the enteric flora of this hospital‐associated population at lower levels than reported in livestock and fresh food products.     

Inactivation of the Wnt/β-catenin signaling contributes to the epithelial barrier dysfunction induced by sodium oxalate in canine renal epithelial cells

High oxalate consumption has been recognized as a risk factor for renal calcium oxalate stones in companion animals (dogs and cats). However, the cellular signaling involved in oxalate-induced dysfunction in renal tubular epithelial cells remains not fully elucidated. In this study, Mardin–Darby canine kidney (MDCK) cells, an epithelial cell line derived from canine kidney tubule, were tested for cell proliferation activity and barrier function after being exposed to sodium oxalate (NaOx). Further, the involvement of Wnt/β-catenin in NaOx-induced renal epithelial barrier dysfunction was evaluated. MDCK cells treated with NaOx exhibited reduction in cell proliferation and migration. Besides, NaOx exposure led to a decrease in transepithelial electrical resistance and an increase in paracellular permeability. The deleterious effects of NaOx on epithelial barrier function were related to the suppressed abundance of tight junction proteins including zonula occludens, occludin, and claudin-1. Of note, protein levels of β-catenin and phosphorylated (p)-β-catenin (Ser552) in MDCK cells were repressed by NaOx, indicating inhibitory effects on Wnt/β-catenin signaling. An inhibition of glycogen synthase kinase-3β (GSK-3β) by SB216763 enhanced the abundance of β-catenin and p-β-catenin (Ser552), and protected against epithelial barrier dysfunction in NaOx-treated MDCK cells. The results revealed a critical role of Wnt/β-catenin signaling in the epithelial barrier function of MDCK cells. Activation of Wnt/β-catenin signaling might be a potential therapeutic target for the treatment of oxalate-linked renal stones.