Polyamine synthesis from arginine and proline in tissues of developing chickens.

Polyamines (putrescine, spermidine, and spermine) are synthesized primarily from ornithine via ornithine decarboxylase (ODC) in mammals. Although avian tissues contain ODC activity, little is known about intracellular sources of ornithine for their polyamine synthesis. This study tested the hypothesis that arginase and proline oxidase contribute to polyamine synthesis in chickens. Kidney, jejunum, leg muscle, and liver from 0-, 7-, 14- and 21-day-old broiler chickens were assayed for the activities of arginase, proline oxidase (POX), ornithine aminotransferase (OAT), and ornithine decarboxylase (ODC). Kidney slices were also used to determine 14C-polyamine synthesis from [U-14C]arginine and [U-14C]proline. Furthermore, these tissues and plasma were analyzed for polyamines. Results indicate that all tissues contained OAT (mitochondrial) and ODC (cytosolic) activities, but arginase and POX activities were only detected in the mitochondria of chicken kidneys. Renal POX and arginase activities were greater at 7 days of age compared to newly hatched birds, and declined by Day 14. Renal arginase activity was greater at 21 days compared to 14 days of age, but there was no change in renal POX activity during that same period. Concentrations of polyamines in the kidneys and plasma were greater on Day 7 compared to Day 0 and decreased thereafter on Days 14 and 21. Kidney slices readily converted arginine and proline into polyamines, with peak rates being on Day 7. Concentrations of putrescine, spermidine and spermine in the plasma of chickens were about 20- to 100-fold greater than those in mammals. Our results indicate that polyamines are synthesized from arginine and proline in avian kidneys. Unlike mammals, polyamines released from the kidneys are likely the major source of polyamines in the blood and other extra-renal tissues in chickens.

In vivo emergence of beige-like fat in chickens as physiological adaptation to cold environments.

While it has been hypothesized that brown adipocytes responsible for mammalian thermogenesis are absent in birds, the existence of beige fat has yet to be studied directly. The present study tests the hypothesis that beige fat emerges in birds as a mechanism of physiological adaptation to cold environments. Subcutaneous neck adipose tissue from cold-acclimated or triiodothyronine (T3)-treated chickens exhibited increases in the expression of avian uncoupling protein (avUCP, an ortholog of mammalian UCP2 and UCP3) gene and some known mammalian beige adipocyte-specific markers. Morphological characteristics of white adipose tissues of treated chickens showed increased numbers of both small and larger clusters of multilocular fat cells within the tissues. Increases in protein levels of avUCP and mitochondrial marker protein, voltage-dependent anion channel, and immunohistochemical analysis for subcutaneous neck fat revealed the presence of potentially thermogenic mitochondria-rich cells. This is the first evidence that the capacity for thermogenesis may be acquired by differentiating adipose tissue into beige-like fat for maintaining temperature homeostasis in the subcutaneous fat 'neck warmer' in chickens exposed to a cold environment.

Interorgan Metabolism, Nutritional Impacts, and Safety of Dietary L-Glutamate and L-Glutamine in Poultry.

L-glutamine (Gln) is the most abundant amino acid (AA) in the plasma and skeletal muscle of poultry, and L-glutamate (Glu) is among the most abundant AAs in the whole bodies of all avian tissues. During the first-pass through the small intestine into the portal circulation, dietary Glu is extensively oxidized to CO2, but dietary Gln undergoes limited catabolism in birds. Their extra-intestinal tissues (e.g., skeletal muscle, kidneys, and lymphoid organs) have a high capacity to degrade Gln. To maintain Glu and Gln homeostasis in the body, they are actively synthesized from branched-chain AAs (abundant AAs in both plant and animal proteins) and glucose via interorgan metabolism involving primarily the skeletal muscle, heart, adipose tissue, and brain. In addition, ammonia (produced from the general catabolism of AAs) and α-ketoglutarate (α-KG, derived primarily from glucose) serve as substrates for the synthesis of Glu and Gln in avian tissues, particularly the liver. Over the past 20 years, there has been growing interest in Glu and Gln metabolism in the chicken, which is an agriculturally important species and also a useful model for studying some aspects of human physiology and diseases. Increasing evidence shows that the adequate supply of dietary Glu and Gln is crucial for the optimum growth, anti-oxidative responses, productivity, and health of chickens, ducklings, turkeys, and laying fowl, particularly under stress conditions. Like mammals, poultry have dietary requirements for both Glu and Gln. Based on feed intake, tissue integrity, growth performance, and health status, birds can tolerate up to 12% Glu and 3.5% Gln in diets (on the dry matter basis). Glu and Gln are quantitatively major nutrients for chickens and other avian species to support their maximum growth, production, and feed efficiency, as well as their optimum health and well-being.

Mitochonic Acid 5 Binds Mitochondria and Ameliorates Renal Tubular and Cardiac Myocyte Damage.

Mitochondrial dysfunction causes increased oxidative stress and depletion of ATP, which are involved in the etiology of a variety of renal diseases, such as CKD, AKI, and steroid-resistant nephrotic syndrome. Antioxidant therapies are being investigated, but clinical outcomes have yet to be determined. Recently, we reported that a newly synthesized indole derivative, mitochonic acid 5 (MA-5), increases cellular ATP level and survival of fibroblasts from patients with mitochondrial disease. MA-5 modulates mitochondrial ATP synthesis independently of oxidative phosphorylation and the electron transport chain. Here, we further investigated the mechanism of action for MA-5. Administration of MA-5 to an ischemia-reperfusion injury model and a cisplatin-induced nephropathy model improved renal function. In in vitro bioenergetic studies, MA-5 facilitated ATP production and reduced the level of mitochondrial reactive oxygen species (ROS) without affecting activity of mitochondrial complexes I-IV. Additional assays revealed that MA-5 targets the mitochondrial protein mitofilin at the crista junction of the inner membrane. In Hep3B cells, overexpression of mitofilin increased the basal ATP level, and treatment with MA-5 amplified this effect. In a unique mitochondrial disease model (Mitomice with mitochondrial DNA deletion that mimics typical human mitochondrial disease phenotype), MA-5 improved the reduced cardiac and renal mitochondrial respiration and seemed to prolong survival, although statistical analysis of survival times could not be conducted. These results suggest that MA-5 functions in a manner differing from that of antioxidant therapy and could be a novel therapeutic drug for the treatment of cardiac and renal diseases associated with mitochondrial dysfunction.

Time-course changes in muscle protein degradation in heat-stressed chickens: Possible involvement of corticosterone and mitochondrial reactive oxygen species generation in induction of the ubiquitin-proteasome system.

Heat stress (HS) induces muscle protein degradation as well as production of mitochondrial reactive oxygen species (ROS). In the present study, to improve our understanding of how protein degradation is induced by HS treatment in birds, a time course analysis of changes in the circulating levels of glucocorticoid and N(τ)-methylhistidine, muscle proteolysis-related gene expression, and mitochondrial ROS generation, was conducted. At 25 days of age, chickens were exposed to HS conditions (33 °C) for 0, 0.5, 1 or 3 days. While no alteration in plasma N(τ)-methylhistidine concentration relative to that of the control group was observed in the 0.5 day HS group, the concentration was significantly higher in the 3-d HS treatment group. Plasma corticosterone concentrations increased in response to 0.5-d HS treatment, but subsequently returned to near-normal values. HS treatment for 0.5 days did not change the levels of µ-calpain, cathepsin B, or proteasome C2 subunit mRNA, but increased the levels of mRNA encoding atrogin-1 (P<0.05) and its transcription factor, forkhead box O3 (P=0.09). Under these hyperthermic conditions, mitochondrial superoxide production was significantly increased than that of thermoneutral control. Here, we show that HS-induced muscle protein degradation may be due to the activation of ubiquitination by atrogin-1, and that this process may involve mitochondrial ROS production as well as corticosterone secretion.

Mitochonic Acid 5 (MA-5), a Derivative of the Plant Hormone Indole-3-Acetic Acid, Improves Survival of Fibroblasts from Patients with Mitochondrial Diseases.

Mitochondria are key organelles implicated in a variety of processes related to energy and free radical generation, the regulation of apoptosis, and various signaling pathways. Mitochondrial dysfunction increases cellular oxidative stress and depletes ATP in a variety of inherited mitochondrial diseases and also in many other metabolic and neurodegenerative diseases. Mitochondrial diseases are characterized by the dysfunction of the mitochondrial respiratory chain, caused by mutations in the genes encoded by either nuclear DNA or mitochondrial DNA. We have hypothesized that chemicals that increase the cellular ATP levels may ameliorate the mitochondrial dysfunction seen in mitochondrial diseases. To search for the potential drugs for mitochondrial diseases, we screened an in-house chemical library of indole-3-acetic-acid analogs by measuring the cellular ATP levels in Hep3B human hepatocellular carcinoma cells. We have thus identified mitochonic acid 5 (MA-5), 4-(2,4-difluorophenyl)-2-(1H-indol-3-yl)-4-oxobutanoic acid, as a potential drug for enhancing ATP production. MA-5 is a newly synthesized derivative of the plant hormone, indole-3-acetic acid. Importantly, MA-5 improved the survival of fibroblasts established from patients with mitochondrial diseases under the stress-induced condition, including Leigh syndrome, MELAS (myopathy encephalopathy lactic acidosis and stroke-like episodes), Leber's hereditary optic neuropathy, and Kearns-Sayre syndrome. The improved survival was associated with the increased cellular ATP levels. Moreover, MA-5 increased the survival of mitochondrial disease fibroblasts even under the inhibition of the oxidative phosphorylation or the electron transport chain. These data suggest that MA-5 could be a therapeutic drug for mitochondrial diseases that exerts its effect in a manner different from anti-oxidant therapy.

Effect of heat stress-induced production of mitochondrial reactive oxygen species on NADPH oxidase and heme oxygenase-1 mRNA levels in avian muscle cells.

Heat stress is a major factor inducing oxidative disturbance in cells. In the present study, we investigated the mechanism of overproduction of reactive oxygen species (ROS) in cultured avian muscle cells in response to heat stress, and also focused attention on the interaction of mitochondrial superoxide anions with altered NADPH oxidase (NOX), superoxide dismutase (SOD) and heme oxygenase-1 (HO-1) mRNA levels in heat-stressed cells. Exposure of cells to heat stress conditions (41°C, 6h) resulted in increased mitochondrial superoxide and intracellular ROS levels, and increased carbonyl protein content as compared with that of normal cells (37°C). The mitochondrial uncoupler 2,4-dinitrophenol lowered intracellular ROS levels in heat-stressed cells. Heat stress increased NOX4 mRNA and decreased HO-1 mRNA levels, while SOD1 and SOD2 mRNA levels remained relatively stable in heat-stressed cells. Addition of the superoxide scavenger 4-hydroxy TEMPO to the culture medium of heat-stressed cells restored mitochondrial superoxide and intracellular ROS levels as well as NOX4 and HO-1 mRNA levels to near-normal values. We suggest that mitochondrial superoxide production could play an influential role in augmenting oxidative damage to avian muscle cells, possibly via the up-regulation of NOX4 and down-regulation of HO-1 in heat-stressed avian muscle cells.

Effects of Supplemented Coriander, Ajwain, and Dill Seed Essential Oils on Growth Performance, Carcass Characteristics, Gut Health, Meat Quality, and Immune Status in Broilers.

Numerous studies have assessed the benefits and optimal dosage of supplementation with essential oils (EOs), including extracts from plants of the Apiaceae family, as an alternative to antibiotic growth promoters (AGPs) in broilers. However, little consideration has been given to the actual chemical composition of the evaluated EOs when drawing critical conclusions, even though EO composition can vary with different extraction conditions and plant characteristics. Therefore, the present study was conducted to evaluate the effects of EOs from seeds of plants of the Apiaceae family: coriander (CEO), ajwain (AjEO), and dill (DEO), containing 56.8% linalool, 68.2% thymol, and 41.1% carvone, respectively, on the growth performance, gut health, and immune status of broilers. In total, 660 one-day-old broiler chicks were divided into 11 experimental diet groups and fed for 35 days with either the control diet, basal diet with added AGP (lincomycin, 500 mg/kg), or one of nine EO diets supplemented with CEO, AjEO, or DEO at 200, 400, and 600 mg/kg. Final body weights were improved by supplementation with not only AGP but also any EO except AjEO at 600 mg/kg; within each EO, supplementation of CEO at 400 mg/kg, AjEO at 200 mg/kg, and DEO at 200 mg/kg afforded the best growth performance. EO supplementation had beneficial effects on gut morphology, such as increased villus height in the duodenum, jejunum, and ileum, and against harmful microbiota, such as reduction of Escherichia coli and Salmonella spp. populations. Furthermore, EOs enhanced humoral immunity and improved meat quality by reducing drip loss, likely consequent to their antioxidant properties. Overall, this study presents evidence that CEO, AjEO, and DEO can each play a pivotal role in replacing AGPs, as well as providing information regarding optimal doses for broilers.

Mechanisms underlying the Effects of Heat Stress on Intestinal Integrity, Inflammation, and Microbiota in Chickens.

Poultry meat and egg production benefits from a smaller carbon footprint, as well as feed and water consumption, per unit of product, than other protein sources. Therefore, maintaining a sustainable production of poultry meat is important to meet the increasing global demand for this staple. Heat stress experienced during the summer season or in tropical/subtropical areas negatively affects the productivity and health of chickens. Crucially, its impact is predicted to grow with the acceleration of global warming. Heat stress affects the physiology, metabolism, and immune response of chickens, causing electrolyte imbalance, oxidative stress, endocrine disorders, inflammation, and immunosuppression. These changes do not occur independently, pointing to a systemic mechanism. Recently, intestinal homeostasis has been identified as an important contributor to nutrient absorption and the progression of systemic inflammation. Its mechanism of action is thought to involve neuroendocrine signaling, antioxidant response, the presence of oxidants in the diet, and microbiota composition. The present review focuses on the effect of heat stress on intestinal dysfunction in chickens and the underlying causative factors. Understanding these mechanisms will direct the design of strategies to mitigate the negative effect of heat stress, while benefiting both animal health and sustainable poultry production.

Effect of Dietary 4-Phenylbuthyric Acid Supplementation on Acute Heat-Stress-Induced Hyperthermia in Broiler Chickens.

Hot, humid weather causes heat stress (HS) in broiler chickens, which can lead to high mortality. A recent study found that HS causes endoplasmic reticulum (ER) stress. However, the possible involvement of ER stress in HS-induced physiological alterations in broiler chickens is unclear. This study aimed to evaluate the effect of the dietary supplementation of 4-phenylbutyric acid (4-PBA), an alleviator of ER stress, in acute HS-exposed young broiler chickens. Twenty-eight 14-day-old male broiler chickens (ROSS 308) were divided into two groups and fed either a control diet or a diet containing 4-PBA (5.25 g per kg of diet feed) for 10 days. At 24 days old, each group of chickens was kept in thermoneutral (24 ± 0.5 °C) or acute HS (36 ± 0.5 °C) conditions for 2 h. The results showed that thermoneutral birds supplemented with 4-PBA exhibited no negative effects in terms of broiler body weight gain and tissue weight compared to non-supplemental birds. HS increased body temperature in both the control and 4-PBA groups, but the elevation was significantly lower in the 4-PBA group than in the control group. The plasma non-esterified fatty acid concentration was significantly increased by HS treatment in non-supplemental groups, while the increase was partially attenuated in the 4-PBA group. Moreover, 4-PBA prevented HS-induced gene elevation of the ER stress markers GRP78 and GRP94 in the skeletal muscle. These findings suggest that the 4-PBA effect may be specific to the skeletal muscle in HS-exposed birds and that 4-PBA supplementation attenuated HS-induced muscle ER stress, which could be associated with a supplementation of the body temperature elevation and lipolysis.

Characteristics of Essential Oils of Apiaceae Family: Their Chemical Compositions, in vitro Properties and Effects on Broiler Production.

There has been an upsurge of interest in the phytobiotics coincident with the onset of the potential ban on the use of antibiotic growth promoters (AGPs) in the broiler industry and because many kinds of nutraceuticals play an important role in improving growth performance, feed efficiency, and gut health of broilers. In the previous years, significant biological activities of essential oils (EOs) belonging to phytobiotics were observed, including anti-bacterial, antifungal, antiviral, and antioxidant properties. We found new perspectives on the roles of EOs, particularly extracts from the Apiaceae family, which is one of the largest plant families, in potential replacement of AGPs, and on the chemical composition involved in regulating microorganism activity and oxidative damage. Furthermore, the positive effects of EOs on broiler production and the possible mechanisms inducing the involvement of gut health and growth performance have been studied.

Mitochondrial membrane potential decrease caused by loss of PINK1 is not due to proton leak, but to respiratory chain defects.

Mutations in PTEN-induced putative kinase 1 (PINK1) cause a recessive form of Parkinson's disease (PD). PINK1 is associated with mitochondrial quality control and its partial knock-down induces mitochondrial dysfunction including decreased membrane potential and increased vulnerability against mitochondrial toxins, but the exact function of PINK1 in mitochondria has not been investigated using cells with null expression of PINK1. Here, we show that loss of PINK1 caused mitochondrial dysfunction. In PINK1-deficient (PINK1(-/-)) mouse embryonic fibroblasts (MEFs), mitochondrial membrane potential and cellular ATP levels were decreased compared with those in littermate wild-type MEFs. However, mitochondrial proton leak, which reduces membrane potential in the absence of ATP synthesis, was not altered by loss of PINK1. Instead, activity of the respiratory chain, which produces the membrane potential by oxidizing substrates using oxygen, declined. H(2)O(2) production rate by PINK1(-/-) mitochondria was lower than PINK1(+/+) mitochondria as a consequence of decreased oxygen consumption rate, while the proportion (H(2)O(2) production rate per oxygen consumption rate) was higher. These results suggest that mitochondrial dysfunctions in PD pathogenesis are caused not by proton leak, but by respiratory chain defects.

Meat-type chickens have a higher efficiency of mitochondrial oxidative phosphorylation than laying-type chickens.

Meat-type chickens show high feed efficiency and have a very rapid growth rate compared with laying-type chickens. To clarify whether the type-specific difference in feed conversion efficiency is involved in mitochondrial bioenergetics, modular kinetic analysis was applied to oxidative phosphorylation in skeletal muscle mitochondria of both type chickens. Mitochondria from skeletal muscle of meat-type chickens showed greater substrate oxidation and phosphorylating activities, and less proton leak than those of the laying-type, resulting in a higher efficiency of oxidative phosphorylation. Gene expression and protein content of uncoupling protein (avUCP) but not adenine nucleotide translocase (avANT) gene expression were lower in skeletal muscle mitochondria of meat-type chickens than the laying-type. The current results regarding a higher efficiency of oxidative phosphorylation and UCP content may partially support the high feed efficiency of meat-type chickens.

Possible role of corticosterone in proteolysis, glycolytic, and amino acid metabolism in primary cultured avian myotubes incubated at high-temperature conditions.

Excess glucocorticoid secretion induces oxidative damage and muscle proteolysis and modulates glucose and lipid metabolism. It is known that the high-temperature (HT) treatment enhances corticosterone (CORT) secretion, muscle proteolysis, and mitochondrial reactive oxygen species (mtROS) generation in chickens. The present study investigated the co-effects of CORT on proteolysis and mtROS production, together with glucose, fatty acid, and amino acid metabolism in HT-treated cells. Myoblast cells were isolated from the major pectoralis muscle of five 0- or 1-day-old neonatal chicks and were precultured at 37°C/CO2 conditions for 48 h to reach subconfluent (80%-90%) conditions. Cells were then reseeded onto a 6- or 24-well microplate for the subsequent measurement, followed by the culture under a control temperature (37°C, control) or HT (41°C) conditions for 1 or 6 h. The HT-treated cells were cocultured with physiologically relevant concentrations of CORT (20 ng/mL in dimethyl sulfoxide). The HT treatment decreased cellular protein content (P < 0.05) and increased atrogin-1 mRNA levels and mtROS generation levels compared to the control group (P < 0.05), whereas HT/CORT co-treatment did not induce changes in either parameter. The mRNA level of glucose transporter-1 was decreased in HT-treated cells compared to that in normal cells (P < 0.05), and the decrease was increased in the CORT co-treatment (P < 0.05). While HT treatment did not alter pyruvate dehydrogenase kinase-4 mRNA level, the level was increased in the CORT co-treatment compared to the control and HT-treated cells (P < 0.05). Neither HT nor HT/CORT treatments altered the mRNA levels of fatty acid oxidation-related factors, carnitine palmitoyl transferase-1, and cluster of differentiation 36. The study conducted a metabolic analysis using gas chromatography-mass spectrometry. The results showed that HT/CORT-treated cells had decreased intracellular citrate and α-ketoglutarate levels (P < 0.05) and increased extracellular alanine and amino acid that have gluconeogenic properties, as well as increased aspartate, isoleucine, serine, methionine, and threonine levels (P < 0.05) compared to HT-treated cells. These results suggest that CORT may not affect proteolysis and mtROS production but can suppress pyruvate oxidation and promote alanine production in HT-treated chickens.

Effect of dipeptide on intestinal peptide transporter 1 gene expression: An evaluation using primary cultured chicken intestinal epithelial cells.

Peptide transporter 1 (PepT1) is a transporter responsible for absorbing dipeptide and tripeptide in enterocytes and is upregulated by dipeptide in mammals. It has not been certain whether intestinal PepT1 expression is responsive to dipeptides in chickens because of the lack of in vitro study using the cultured enterocytes. This study established a primary culture model of chicken intestinal epithelial cells (IECs) in two-dimensional monolayer culture using collagen gel by which the response of chicken PepT1 gene expression to dipeptide stimuli was evaluated. The cultured chicken IECs showed the epithelial-like morphology attached in a patch-manner and exhibited positive expression of cytokeratin and epithelial cadherin, specific marker proteins of epithelial cells. Moreover, the chicken IECs exhibited the gene expression of intestinal cell type-specific marker, villin1, mucin 2, and chromogranin A, suggesting that the cultured IECs were composed of enterocytes as well as goblet and enteroendocrine cells. PepT1 gene expression was significantly upregulated by synthetic dipeptide, glycyl-l-glutamine, in the cultured IECs. From the results, we herein suggested that dipeptide is a factor upregulating PepT1 gene expression in chicken IECs.

Effects of plant-derived isoquinoline alkaloids on growth performance and intestinal function of broiler chickens under heat stress.

Broiler chickens reared under heat stress (HS) conditions have decreased growth performance and show metabolic and immunologic alterations. This study aimed to evaluate the effect of supplementation with a standardized blend of plant-derived isoquinoline alkaloids (IQ) on the growth performance, protein catabolism, intestinal barrier function, and inflammatory status of HS-treated chickens. Three hundred sixty 0-day-old Ross 308 male broiler chickens were randomly distributed into 2 treatment groups: control diet (no additives) or diet supplemented with 100 ppm IQ. At day 14, the chicks in each diet group were further divided into 2 groups, each of which was reared under thermoneutral (TN) (22.4°C) or constant HS (33.0°C) conditions until day 42. Each group consisted of 6 replicates with 15 birds per replicate, and chickens were provided ad libitum access to water and feed. During days 15-21, the body weight gain (BWG) and feed intake (FI) were significantly lower in the HS treatment group than in the TN group, and feed conversion ratio was higher (P < 0.05); these factors were not alleviated by IQ supplementation. During days 22-42, the final BW, BWG, and FI of the HS birds were better among those administered IQ than those that were not (P < 0.05). HS treatment increased plasma lipid peroxide, corticosterone, and uric acid concentrations as well as serum fluorescein isothiocyanate-dextran, a marker of intestinal barrier function, and decreased plasma total protein content (P < 0.05). These changes were not observed in the IQ group, suggesting that IQ supplementation improved oxidative damage, protein catabolism, and intestinal barrier function of chickens under HS. Isoquinoline alkaloid supplementation inhibited the expression of intestinal inflammatory factors, IL-6, tumor necrosis factor-like factor 1A, and inducible nitric oxide synthase under HS treatment (P < 0.05). These results suggest that IQ supplementation can improve the growth performance of broiler chickens under HS conditions, which may be associated with amelioration of oxidative damage, protein catabolism, intestinal barrier function, and inflammation.

Growth performance responses to increased tryptophan supplementation in growing barrows fed three different very low crude protein corn and soybean meal-based diets fortified with essential amino acids.

Seventy-five individually fed barrows averaging 35 kg were used in a series of three experiments for 6-week growth assays (25 barrows each) to determine the effects of l-tryptophan (Trp) supplementation on growth performance and to establish the lower limits of dietary crude protein (CP) levels. Corn and soybean meal (SBM)-based diets containing 9% (Experiment 1), 10% (Experiment 2), and 11% CP (Experiment 3) fortified with deficient essential amino acids (AAs) except Trp were used as basal diets for each experiment. The experimental diets were supplemented with 0.00%, 0.02%, 0.04%, or 0.06% Trp. A 16% CP corn-SBM-based diet was set as a positive control in each experiment, and feed and water were provided ad libitum. Average daily gain and gain-to-feed ratio improved quadratically (P < 0.05) as supplemented Trp increased in the 9% and 10% CP group, although these positive effects were not observed in the 11% CP group. Because the maximum performance parameters in 9%, 10%, and 11% CP groups were not different from that of the 16% CP positive control group, the marginally reduced level of dietary CP without growth performance being affected appears to be around 9% at most. A potential reduction of nitrogen intake was clearly indicated.

Oleuropein suppresses mitochondrial reactive oxygen species generation possibly via an activation of transient receptor potential V1 and sirtuin-1 in cultured chicken muscle cells.

This study investigated the intracellular mechanism governing the effects of oleuropein (OLE), a phenolic compound of Olea europaea, on mRNA expression of avian uncoupling protein (avUCP) and mitochondrial biogenesis-related factors, and reactive oxygen species (mitROS) generation in a primary cultured chicken muscle cells. The OLE-treated cells exhibited increases in Avucp and ATP5a1z expression and a decrease in mitROS generation (p < 0.05), while the effects was canceled by sirtuin-1 (SIRT1) or transient receptor potential vanilloid 1 (TRPV1) inhibitors, EX-527 or BCTC, respectively. Intracellular Ca2+ concentration was significantly increased by OLE, while the induction was canceled by BCTC. The study also found that TRPV1 was expressed in the cell membrane and endoplasmic reticulum (ER), and Ca2+ could be released from ER in the OLE-treated cells. The OLE-treated cells exhibited increases in the phosphorylation ratio of AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) protein content. EX-527 and BCTC inhibitors canceled the effects of OLE on p-AMPK ratio and PGC-1α content, while EX-527 SIRT did not change PGC-1α content. The results suggest that the OLE effects may be due to Ca2+ release, possibly from TRPV1 at ER, and increased p-AMPK ratio, followed by SIRT1 activation and PGC-1α protein expression.

Differences in Breast Muscle Mitochondrial Respiratory Capacity, Reactive Oxygen Species Generation, and Complex Characteristics between 7-week-old Meat- and Laying-type Chickens.

The skeletal muscle growth rate is a major feature differentiating meat- and laying-type chickens. A large amount of ATP is required during skeletal muscle synthesis, in which mitochondrial energy production capacities play a significant role. Additionally, mitochondria may participate in muscle protein degradation via reactive oxygen species generation. To investigate the differences in mitochondrial energetic characteristics between chickens exhibiting different growth rates, this study evaluated respiratory capacities in response to different types of respiratory substrate, protein abundances, assembly of individual respiratory complexes (I-V) and supercomplexes, and reactive oxygen species generation rates. These characteristics were compared between mitochondria from the breast muscle (M. pectoralis superficialis) of seven-week-old meat- and laying-type male chickens. Blue native polyacrylamide gel electrophoresis analysis revealed that meat-type chickens exhibited a significantly lower protein abundance of complex III (cytochrome bc 1 complex), complex V (F0F1 ATP synthase), and total amount of supercomplexes than did laying-type chickens. There were no differences between chicken types in the respiration rate of mitochondria incubated with either pyruvate/malate or succinate, each of which drives complex I- and complex II-linked respiration. Carnitine palmitoyltransferase-1-dependent and -independent respiration during ATP synthesis and carnitine palmitoyltransferase-2 enzymatic activity were significantly lower in meat-type chickens than in layingtype chickens. For mitochondria receiving pyruvate/malate plus succinate, the reactive oxygen species generation rate and its ratio to the oxygen consumed (the percentage of free radical leak) were also significantly lower in meat-type chickens than in laying-type chickens. These results suggested that the mitochondrial energetic capacities of the breast muscle of meat-type chickens could be lower than those of laying-type chickens at seven weeks of age. Furthermore, the lower reactive oxygen species generation rate in meat-type chickens might have implications for rapid muscle development, which is possibly related to their lower muscle protein degradation rates.

Heat Stress Directly Affects Intestinal Integrity in Broiler Chickens.

A study using pair-feeding technique was conducted to determine whether heat exposure directly or indirectly (via reduced feed intake) increases intestinal mucosal damage and permeability to endotoxin in broiler chickens. Male broiler chickens (Ross 308), 27-d-old, were subjected to one of the three treatments (n=8): 1) thermo-neutral conditions (24°C) with ad libitum feed intake, 2) heat stress conditions (33°C) with ad libitum feed intake, or 3) pair-feeding under thermo-neutral conditions, with the feed intake identical to that of heat-stressed chickens. Using these groups, two experiments were performed to evaluate temporal changes in the intestinal morphology in response to each treatment. In experiment 1, chickens were sacrificed after 24 h of exposure to the treatment conditions, while in experiment 2, chickens were sacrificed after 12 or 72 h of exposure to the treatment conditions. In experiment 1, exposure to heat stress conditions for 24 h significantly decreased both the villus height to crypt depth ratio and number of proliferating cell nuclear antigen (PCNA)-positive cells in the duodenum and increased the plasma endotoxin concentration. These findings were not observed in pair-fed chickens. In experiment 2, intestinal integrity and function were unaffected by 12 h of heat stress. On the other hand, chickens exposed to heat stress for 72 h exhibited significantly damaged intestinal morphology in the duodenum as well as increased plasma endotoxin concentration; these negative effects were not observed in pair-fed chickens. These findings suggest that the intestinal morphology and permeability changes observed in chickens that are heat-stressed for 24-72 h are due to the heat stress conditions and not due to reduced feed intake.