Characterization of β-adrenergic receptors in bovine intramuscular and subcutaneous adipose tissue: comparison of lubabegron fumarate with β-adrenergic receptor agonists and antagonists

Chinese hamster ovary cell constructs expressing either the β ₁-, β ₂- or β ₃-adrenergic receptor (AR) were used to determine whether a novel β-AR modulator, lubabegron fumarate (LUB; Experior, Elanco Animal Health) might exert greater potency for a specific β-AR subtype. EC₅₀ values calculated based on cAMP accumulation in dose response curves indicate that LUB is highly selective for the β ₃-AR subtype, with an EC₅₀ of 6 × 10^–9 M, with no detectible agonistic activity at the β ₂-AR. We hypothesized that the accumulation of lipolytic markers would reflect the agonist activity at each of the β-receptor subtypes of the specific ligand; additionally, there would be differences in receptor subtype expression in subcutaneous (s.c.) and intrmuscular (i.m.) adipose tissues. Total RNA was extracted from adipose tissue samples and relative mRNA levels for β ₁-, β₂-, and β ₃-AR were measured using real-time quantitative polymerase chain reaction. Fresh s.c. and i.m. adipose tissue explants were incubated with isoproterenol hydrochloride (ISO; β-AR pan-agonist), dobutamine hydrochloride (DOB; specific β ₁-AA), salbutamol sulfate (SAL; specific β ₂-AA), ractopamine hydrochloride (RAC), zilpaterol hydrochloride (ZIL), BRL-37344 (specific β ₃-agonist), or LUB for 30 min following preincubation with theophylline (inhibitor of phosphodiesterase). Relative mRNA amounts for β ₁-, β ₂-, and β ₃-AR were greater (P < 0.05) in s.c. than in i.m. adipose tissue. The most abundant β-AR mRNA in both adipose tissues was the β ₂-AR (P < 0.05), with the β ₁- and β ₃-AR subtypes being minimally expressed in i.m. adipose tissue. ISO, RH, and ZH stimulated the release of glycerol and nonesterified fatty acid (NEFA) from s.c. adipose tissue, but these β-AR ligands did not alter concentrations of these lipolytic markers in i.m. adipose tissue. LUB did not affect glycerol or NEFA concentrations in s.c. or i.m. adipose tissue, but attenuated (P < 0.05) the accumulation of cAMP mediated by the β ₁- and β ₂-AR ligands DOB and SAL in s.c. adipose tissue. Collectively, these data indicate that bovine i.m. adipose tissue is less responsive than s.c. adipose tissue to β-adrenergic ligands, especially those that are agonists at the β ₁- and β₃-receptor subtypes. The minimal mRNA expression of the β ₁- and β ₃ subtypes in i.m. adipose tissue likely limits the response potential to agonists for these β-AR subtypes.     

Metformin acts to suppress β-hydroxybutyric acid-mediated inflammatory responses through activation of AMPK signaling in bovine hepatocytes

The occurrence of bovine ketosis involves the accumulation of β-hydroxybutyric acid (BHBA), which contributes to the initiation and acceleration of hepatic metabolic stress and inflammation. Metformin has other beneficial effects apart from its medical intervention for diabetes, such as prevention of laminitis and hyper-triglyceridemic. AMPK maintains energy homeostasis and is the intracellular target of metformin action. This study aims to uncover the role of metformin in modulating BHBA-induced inflammatory responses through the activation of AMPK signaling. The hepatocytes were isolated from the liver tissue of mid-lactation multiparous Holstein cows (~160 d postpartum). Treatments were conducted as follows: treated with PBS for 18 h (control); pretreated with PBS for 12 h followed by treatment of 1.2 mM BHBA for 6 h (BHBA); pretreated with 1.5 mM or 3 mM metformin for 12 h followed by the BHBA treatment (1.2 mM) for 6 h (M(1.5)+B; M(3)+B). The inhibitor of AMPK, Compound C, at a concentration of 10 μM, was applied to substantiate the AMPK-dependent responses. RT-qPCR were applied for the mRNA expression while Western-blots and immunofluorescence were conducted for the target proteins expression. Among dose-dependent assays for BHBA, the concentration of BHBA at 1.2 mM activated NF-κB signaling by upregulating the expression of phosphorylated NF-κB and pro-inflammatory cytokines compared with the control cells (P < 0.05). Along with the upregulation of phosphorylated AMPKα and ACCα, metformin at 1.5 and 3 mM inactivated NF-κB signaling components (p65 and IκBα) and the inflammatory genes (TNFA, IL6, IL1B and COX-2) which were activated by BHBA. Additionally, BHBA inhibited cells staining intensity in EdU assay were increased by pretreatment with metformin. The activation of AMPK resulted in the increased gene and protein expression of SIRT1, along with the deacetylation of H3K9 and H3K14. However, the AMPK inhibitor compound C blocked this effect. Compared with BHBA treated cells, the protein expression of COX-2 and IL-1β were decreased by the pretreatment with metformin, and the inhibitory effect of metformin was released by compound C. The bound of NF-κB onto IL1B promoter displayed higher in BHBA group and this was suppressed by pretreatment with metformin (P < 0.05). Altogether, metformin attenuates the BHBA-induced inflammation through the inactivation of NF-κB as a target for AMPK/SIRT1 signaling in bovine hepatocytes.     

九龙藏黄牛和九龙牦牛β-酪蛋白基因型分析

为比较九龙藏黄牛和九龙牦牛β-酪蛋白（β-CN）的遗传变异体,试验采用酸性尿素聚丙烯酰胺凝胶电泳分析了九龙藏黄牛（n=42）和九龙牦牛（n=17）β-CN的基因型。结果表明,在九龙藏黄牛、九龙牦牛的β-CN中共检测到4 种等位基因,包括A1、A2、B、C,其中在九龙藏黄牛中有7 种基因型：A1A1、A1A2、BB、A1B、A2B、A1C、BC,优势等位基因为A1（频率0.702 4）,优势基因型为A1A1（频率0.547 6）;在九龙牦牛样本中有2 种基因型：A1A2、A2A2,优势等位基因为A2（频率0.764 7 ）。试验表明,九龙藏黄牛与九龙牦牛β-CN均表现出多态性,但优势等位基因明显不同。     

乳制品中A1 β-酪蛋白、A2 β-酪蛋白检测方法研究进展

随着消费者对A2 β-酪蛋白的关注度提升,A2 β-酪蛋白相关产品不断更新换代。A2 β-酪蛋白常用检测方法有反相高效液相色谱法（RP-HPLC）、液相色谱–高分辨串联质谱法、毛细管区带电泳法、试剂盒检测法等。本文对β-酪蛋白遗传变体A1 β-酪蛋白和原始形态A2 β-酪蛋白的功能特点、检测方法、定量分析进行综述,说明检测方法的原理和试剂药品的作用机理,加强对A1 β-酪蛋白、A2 β-酪蛋白检测方法的改进与优化,为A2 β-酪蛋白应用于功能性食品、婴幼儿配方食品提供理论依据。     

A2 β-酪蛋白消化特性与人体健康

β-酪蛋白是牛乳酪蛋白的重要组成成分,具有遗传多态性,特别是A1和A2型与人体健康密切相关。本文综述了A2型β-酪蛋白结构和功能的特性关系、消化特性的人群差异性及不同种类乳β-酪蛋白的消化特性,从消化特性的角度论述了影响人体健康的因素,旨在为不同人群开发具有良好消化特性的乳制品提供参考。     

Successful production of offspring derived from mouse zygotes vitrified with carboxylated ε-poly-L-lysine and polyvinyl alcohol without serum

The vitrification of zygotes is important for their use as donors for generating genome-edited mice. We previously reported the successful vitrification of mouse zygotes using carboxylated ε-poly-L-lysine (COOH-PLL). However, this vitrification solution contains fetal calf serum (FCS), which contains unknown factors and presents risks of pathogenic viral and microbial contamination. In this study, we examined whether polyvinyl alcohol (PVA) can be used as an alternative to FCS in vitrification solutions for mouse zygotes. When COOH-PLL was added to the vitrification solutions, zygotes vitrified with solutions containing 0.01% PVA (PV0.01) and those vitrified in a control solution containing FCS (75.6%) developed into blastocysts (78.4%). In addition, there were no significant differences in the ability to develop to term between the control solution (46.6%) and PV0.01 (44.1%) groups. In conclusion, we clearly demonstrated that PVA can replace FCS in our vitrification solution supplemented with COOH-PLL for mouse zygotes.     

Early detection of urinary bladder carcinogens in rats by immunohistochemistry for γ-H2AX: a review from analyses of 100 chemicals

In safety evaluations of chemicals, there is an urgent need to develop short-term methods to replace long-term carcinogenicity tests. We have reported that immunohistochemistry for γ-H2AX, a well-established biomarker of DNA damage, can detect bladder carcinogens at an early stage using histopathological specimens from 28-day repeated-dose oral toxicity studies in rats. Given the markedly low level of γ-H2AX formation in the bladder urothelium of untreated rats, an increase in γ-H2AX-positive cells following chemical exposure can be relatively easy to identify. Among the 100 compounds examined to date, bladder carcinogens can be detected with high sensitivity (33/39; 84.6%) and specificity (58/61; 95.1%). As expected, γ-H2AX formation levels tended to be high following exposure to genotoxic bladder carcinogens, whereas nongenotoxic bladder carcinogens also increased the number of γ-H2AX-positive cells, probably through secondary DNA damage associated with sustained proliferative stimulation. γ-H2AX formation in the bladder urothelium reflects species differences in susceptibility to bladder carcinogenesis between rats and mice and shows a clear dose-dependency associated with the intensity of tumor development as well as high reproducibility. Some of the bladder carcinogens that showed false-negative results in the evaluation of γ-H2AX alone could be detected by combined evaluation with immunostaining for bladder stem cell markers, including aldehyde dehydrogenase 1A1. This method may be useful for the early detection of bladder carcinogens, as it can be performed by simple addition of conventional immunostaining using formalin-fixed paraffin-embedded tissues from 28-day repeated-dose toxicity studies in rodents, which are commonly used in safety evaluations of chemical substances.     

Different Aβ43 deposition patterns in the brains of aged dogs,sea lions,and cats

Cerebral amyloid β (Aβ) deposition is a pathological hallmark of Alzheimer’s disease (AD). There are several molecular species of Aβ, including Aβ40, Aβ42, and Aβ43, and the pathological roles of Aβ43 have attracted particular attention in recent years. Aβ43 is mainly deposited as senile plaques (SPs) in AD brains, and is known to be more amyloidogenic and neurotoxic than Aβ42 and Aβ40. Aβ40 and Aβ42 deposition have been demonstrated in several animal species, while Aβ43 deposition has not been studied in animals. The brains of sea lions, dogs, and cats exhibit unique age-related Aβ pathologies. In the present study, the deposition patterns of Aβ40, Aβ42, and Aβ43 were examined immunohistochemically in the brains of aged dogs (n=52), sea lions (n=5), and cats (n=17). In dogs, most cerebral amyloid angiopathy (CAA) lesions and primitive SPs were positive for Aβ42, Aβ43, and Aβ40. However, diffuse SPs and capillary CAA lesions were negative for Aβ40. In sea lions, all SPs and most CAA lesions were positive for Aβ42, Aβ43, and Aβ40, while capillary CAA lesions were negative for Aβ40. In cats, Aβ42-immunopositive granular aggregates and arteriole and capillary CAA lesions were positive for Aβ43, but negative for Aβ40. Double-labelling immunohistochemistry revealed the co-localization of Aβ42 and Aβ43. These findings suggest that Aβ43 and Aβ42 are frequently deposited in the brains of Carnivora animals and may play an important role in Aβ pathology.     

5α-Androstenone and Testosterone in Peripheral Plasma of the Boar During and Following Copulation

Copulation was generally followed by increases in peripheral plasma 5α-androstenone and testosterone levels lasting for periods of about 60 to 100 min. The effect of copulation on the plasma levels of these steroids did, however, vary between boars. In seven out of eight boars the maximum levels of 5α-andro-stenone in the period 60 to 100 min. after copulation were from 114 to 218 % (mean 150 %) of the levels in samples collected before copulation. The corresponding figures for testosterone were from 104 to 283 % (mean 190 %). One boar showed decreasing plasma steroid levels after copulation.The coefficient of correlation between the peripheral plasma levels of 5α-androstenone and testosterone was found to be + 0.61 (n = 2.03).     

Modulation of jejunal mucosa-associated microbiota in relation to intestinal health and nutrient digestibility in pigs by supplementation of β-glucanase to corn–soybean meal-based diets with xylanase

This study aimed to evaluate the effects of increasing levels of β-glucanase on the modulation of jejunal mucosa-associated microbiota in relation to nutrient digestibility and intestinal health of pigs fed diets with 30% corn distiller’s dried grains with solubles and xylanase. Forty pigs at 12.4 ± 0.5 kg body weight (BW) were allotted in a randomized complete block design with initial BW and sex as blocks. Dietary treatments consisted of a basal diet with xylanase (1,500 endo-pentosanase units [EPU]/kg) and increasing levels of β-glucanase (0, 200, 400, and 600 U/kg) meeting nutrient requirements and fed to pigs for 21 d. Blood samples were collected on day 19. On day 21, all pigs were euthanized to collect intestinal tissues and digesta. Tumor necrosis factor-alpha, interleukin (IL)-6, and malondialdehyde were measured in the plasma and mid-jejunal mucosa. Viscosity was determined using digesta from the distal jejunum. Ileal and rectal digesta were evaluated to determine apparent ileal digestibility (AID) and apparent total tract digestibility (ATTD) of nutrients. Mucosa samples from the mid-jejunum were utilized for microbiota sequencing. Data were analyzed using the MIXED procedure on SAS 9.4. Overall, increasing dietary β-glucanase tended to increase (linear; P = 0.077) the average daily gain of pigs. Increasing dietary β-glucanase affected (quadratic; P < 0.05) the relative abundance of Bacteroidetes, reduced (linear; P < 0.05) Helicobacter rappini, and increased (linear, P < 0.05) Faecalibacterium prausnitzii. β-Glucanase supplementation (0 vs. others) tended to increase (P = 0.096) the AID of crude protein in the diet, whereas increasing dietary β-glucanase tended to increase (linear; P = 0.097) the ATTD of gross energy in the diet and increased (linear; P < 0.05) the concentration of IL-6 in the plasma of pigs. In conclusion, increasing β-glucanase up to 600 U/kg feed in a diet containing xylanase (1,500 EPU/kg) modulated mucosa-associated microbiota by increasing the relative abundance of beneficial bacteria and reducing potentially harmful bacteria. Furthermore, increasing β-glucanase up to 600 U/kg feed in a diet containing xylanase (1,500 EPU/kg feed) enhanced the status of the intestinal environment and nutrient utilization, as well as reduced systemic inflammation of pigs, collectively resulting in moderate improvement of growth performance. Supplementing β-glucanase at a range of 312 to 410 U/kg with xylanase at 1,500 EPU/kg feed showed the most benefit on jejunal mucosa-associated microbiota and reduced systemic inflammation of pigs.     

Changes in the C-reactive protein and 13,14-dihydro-15-keto-prostaglandin F2α concentrations of uterine lavage samples after the administration of povidone-iodine in cows

Changes in the C-reactive protein (CRP) and 13,14-dihydro-15-keto-prostaglandin F_2α (PGFM) concentrations of uterine lavage fluid were examined in cows given an intrauterine povidone-iodine (PI) infusion. The mean polymorphonuclear leukocyte (PMN) ratios (the ratio of PMN to total cells) and CRP concentration of uterine lavage fluid on the day after the treatment were significantly (P<0.05) greater in the PI infusion group (PMN: 53.0 ± 32.7%, CRP: 50.2 ± 32.3 ng/mL) than in the non-treatment control group (PMN: 7.9 ± 21.9%, CRP: 17.2 ± 5.9 ng/mL), whereas there was no significant difference in the mean PGFM concentration between the two groups. The present findings suggest that the uterine CRP level is a useful biomarker of local uterine inflammation in cows.     

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.     

Glycated Tryptophan PHP-THβC Incorporated into Various Chicken Embryonic Cells Constitutes Cellular Proteins

Glycation is a non-enzymatic reaction, and amino acids are glycated by glucose in vivo. Tryptophan is glycated with glucose to form two types of glycated compounds, tryptophan-Amadori product and (1R, 3S)-1-(D-gluco-1, 2, 3, 4, 5-pentahydroxypentyl)-1, 2, 3, 4-tetrahydro-β-carboline-3-carboxylic acid (PHP-THβC). Although PHP-THβC can be incorporated into various chicken embryonic cells, the mechanism of its incorporation into intracellular fluids has not been clarified. In this study, we examined whether PHP-THβC once incorporated into various chicken embryonic cells can combine with proteins. Embryonic cells from the breast muscle, liver, spleen, kidney, proventriculus, gizzard, and skin were prepared and ³H-PHP-THβC was added to the culture medium at final concentrations of 0, 200, 400, 600, and 800 µM to examine the incorporation of PHP-THβC. After 18 h of incubation, radioactivity was measured in the whole-cell and protein fractions of the chicken embryonic cells. As PHP-THβC concentration increased from 0 to 600 µM, its accumulation in the whole-cell fractions of all types of chicken embryonic cells linearly increased and reached the maximum level. The saturated PHP-THβC accumulation in the whole-cell fractions suggests that PHP-THβC could be incorporated into intracellular fluids across cellular membranes by some transporter proteins. As PHP-THβC concentration increased from 0 to 800 µM, its accumulation in the protein fractions of all types of chicken embryonic cells increased in a linear manner and reached a maximum level in the 800 µM PHPTHβC treatment group. This is the first study to indicate that a part of PHP-THβC incorporated into the whole-cell fraction was detected in the protein fraction of various chicken embryonic cells.     

Short‐term intake of β‐carotene‐supplemented diets enhances ovarian function and progesterone synthesis in goats

The effect of β‐carotene supplementation upon luteal activity, measured as number (CLT) and volume (VLT) of corpus luteum, and P4 synthesis in goats, was evaluated. Goats (n = 22, 34 months) were randomly assigned to one of two experimental groups: (i) β‐carotene [Beta, n = 10; body weight (BW = 44.8 ± 1.45 kg), body condition score (BCS = 3.25 ± 0.07)], and (ii) Control (Control, n = 12; BW = 45.30 ± 1.32 kg, BCS = 3.33 ± 0.06). Upon oestrus synchronization, the Beta group received 50 mg of β‐carotene per day during 35 days pre‐ and 17 days post‐ovulation. The day 4, 8, 12 and 16 post‐ovulation, blood samples were collected for quantification of serum P4 concentrations by radioimmmunoassay, and transrectal ultrasonographic scanning was performed at day 18 for evaluating CLT and VLT. Overall, CLT and VLT mean were 3.10 and 2211.1 mm³ respectively. The Beta‐goats depicted both the largest values for CLT (p = 0.07) and serum P4 levels (p = 0.05), with no differences (p = 0.53) for VLT between treatments. Results suggest a higher efficiency within the cellular‐enzymatic groups defining the steroidogenic pathways in the β‐carotene‐supplemented goats, generating a larger P4 synthesis. The last is essential for ovulation of healthy oocytes, maintenance of uterine quiescence, nourishment and survival of the embryo around implantation; all of them of paramount significance during the maternal recognition of pregnancy process.     

Immunohistochemical analysis of expression of VEGFR2, KIT,PDGFR-β, and CDK4 in canine urothelial carcinoma

Urothelial carcinomas (UCs), also known as transitional cell carcinomas, are the most common canine urinary tract neoplasms. Tyrosine kinases (TKs) are enzymes that tightly regulate cell growth and differentiation through phosphorylation. Receptor TK (RTK) inhibitors are currently used to treat UCs. Toceranib phosphate (Palladia; Pfizer) is an RTK inhibitor that blocks the activity of vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor–alpha and –beta (PDGFR-α, -β), FMS-like tyrosine kinase 3, stem cell factor receptor (KIT, kinase inhibitor targeting), and colony stimulating factor receptor. To better understand UCs and validate treatment targets, we performed immunohistochemical staining for RTKs, as well as a novel target, cyclin-dependent kinase 4 (CDK4, a central regulator of the mammalian cell cycle), on formalin-fixed, paraffin-embedded tissues from bladder biopsies from 17 dogs with UCs, 17 dogs with cystitis (diseased controls), and 8 normal dogs (negative controls). Although immunohistochemical scores could not be extrapolated to prognostic value, response to treatment, and outcome of patients with UC, we demonstrated expression of PDGFR-β and VEGFR2 in UCs; all UC samples staining positively for VEGFR2. Minimal positive staining for KIT was noted in the tumor samples. CDK4 staining intensity was significantly weaker in UCs compared with normal and cystitis bladder samples. The intense staining of VEGFR2 in UC cells suggested that VEGFR2 may be of prognostic and/or therapeutic value in dogs with UC. Overexpression of VEGFR2 in UC cells validates this receptor as a treatment target in UC.     

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.     

Impact of combined α-galactosidase and xylanase enzymes on growth performance,nutrients digestibility,chyme viscosity,and enzymes activity of broilers fed corn–soybean diets

Two experiments were conducted to investigate the effects of a combined α-galactosidase and xylanase preparation on nutrients digestibility and growth performance in broiler chickens. Experiment 1 had 240 broilers allocated to 3 treatments with the dietary supplementation of 0, 300, and 500 g/t of the enzyme combination. Diet and amino acid (AA) digestibility were assessed. Experiment 2 was a 2 × 3 (enzyme × diet) factorial arrangement with 10 replicates of 12 male broilers per replicate. Diets were based on corn–soybean meal (SBM) diet and had 3 nutritional levels (normal, 2% apparent metabolizable energy (AME) and crude protein (CP) reduction, and 4% AME and CP reduction). Each of these diets was fed with or without enzyme supplementation. Growth performance, chyme viscosity, nutrients digestibility, and endogenous enzymes activity were assessed. In experiment 1, enzyme supplementation improved the digestibility of Ca (P = 0.025) and ileal digestibility of total AA, Pro, Alu, Ile, Lys, His, Thr, Glu, Val, Leu, Tyr, and Phe (P < 0.05), and also tended to increase the AME of diets (P < 0.10). In experiment 2, broilers fed the corn–SBM diet with 4% nutrient reduction had better growth performance (P < 0.05), jejunal digesta viscosity at 42 d (P < 0.01), and lower digestibility of gross energy (GE; P < 0.05) when compared with those fed the normal nutrient diet. Enzyme inclusion increased digestibility of CP (P = 0.044), GE (P = 0.009), raffinose (P < 0.001) and stachyose (P < 0.001), improved average daily gain (P = 0.031), and reduced jejunal digesta viscosity at 42 d (P = 0.011). Besides, similar improvements trend in amylase, trypsin, sucrase, and maltase activity with enzyme inclusion were observed as with energy. These data support that the enzyme supplementation increased nutrients and ileal AA digestibility, improved performance and endogenous enzymes activity.     

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.