Insulin acutely increases glucose transporter 1 on plasma membranes and glucose uptake in an AKT-dependent manner in chicken adipocytes.

Avian glucose transporters (GLUT) responsible for insulin-responsive glucose uptake into adipocytes remain poorly characterized. We aimed to identify the insulin-responsive GLUT using primary culture of chicken adipocytes. Acute stimulation with 1 µM insulin for 20 min increased 2-deoxyglucose uptake, AKT protein phosphorylation, and GLUT1 protein levels on the plasma membrane of the chicken adipocytes, whereas pretreatment with 10 µM triciribine, an AKT inhibitor, canceled these effects. Furthermore, the insulin stimulation did not affect GLUT12 protein levels on the plasma membrane of the chicken adipocytes. Our results suggest that GLUT1 is an insulin-responsive GLUT in chicken adipocytes.

Dietary Moringa oleifera improves growth performance, oxidative status, and immune related gene expression in broilers under normal and high temperature conditions.

The aim of this study is to evaluate the effects of Moringa oleifera (MO) on the performance, antioxidative status, and immune related gene expression in broilers raised under normal or heat stress conditions. Broiler chickens were distributed into 4 groups and fed diets with dietary MO at 0% or 5% (MO0 or MO5) and raised under ambient temperature 22 ±â€¯1 °C (N) or 35±1 °C (HS). HS conditions negatively affected the weight gain and FCR, while feeding MO exhibited beneficial effects especially under HS conditions. Triglycerides, total cholesterol and low-density lipoprotein cholesterol (LDL) levels were significantly (P < 0.05) higher in chickens raised in HS conditions and fed the basal diet than those in normal condition and fed with or without MO, while MO decreased triglycerides and total cholesterol levels in normal and HS conditions. Blood high-density lipoprotein cholesterol (HDL) was significantly decreased in broilers raised in HS conditions and fed diets without MO, while MO increased HDL level. Blood glutathione peroxidase (GSH-Px) was significantly (P < 0.05) decreased in broilers raised in HS conditions and fed the basal diet without MO. mRNA expression of GSH-Px was significantly (P < 0.05) downregulated in broilers raised in HS conditions and fed diets without MO. Broilers under normal or HS conditions and fed the basal diet exhibited significantly (P < 0.05) downregulated mRNA expressions of superoxide dismutase (SOD) and catalase (CAT) compared to chickens under normal conditions and fed MO. Liver and muscle thiobarbituric acid reactive substance (TBARs) were significantly (P < 0.05) increased in broilers under HS conditions and fed diet without MO. The expressions of interleukins (IL2 and IL6) were significantly (P < 0.05) downregulated in broilers under normal or HS conditions and fed diets without MO. To sum up, HS conditions depressed the performance, antioxidative status, and immune related gene expression in broilers, while MO obviously alleviated these negative effects in broilers.

The ß2-adrenergic receptor is involved in differences in the protein degradation level of the pectoral muscle between fast- and slow-growing chicks during the neonatal period.

The aim of this study was to investigate whether ß2-AR mRNA expression is involved in either atrogin-1/MAFbx mRNA expression or protein degradation in chicken skeletal muscle by comparing fast- and slow-growing chicks during the neonatal period. Based on their body weight gain from 1 to 5 days of age, 5-day-old chicks (Gallus gallus domestics) were divided into a slow-growing and a fast-growing group, the mean weight gains of which were 6.3 ±â€¯1.3 g/day and 11.3 ±â€¯0.9 g/day, respectively. The ratio of pectoral muscle weight to total body weight was higher in the fast-growing group of chicks than in the slow-growing group. In addition, the plasma 3-methylhistidine concentration, an index of protein degradation in skeletal muscle, was significantly lower in the fast-growing than in the slow-growing chicks. The mRNA expression of ß2-AR, which we previously found is involved in decreasing muscle protein degradation by suppression atrogin-1/MAFbx mRNA expression, was significantly higher in the pectoral muscle of the fast-growing group compared with that of the slow-growing group. Concordantly, lower mRNA expression of atrogin-1/MAFbx was observed in the pectoral muscle of the fast-growing chicks. However, in the sartorius muscle, which is a muscle in the thigh, the ratio of the muscle weight to total body weight was not significantly different between the two groups of chicks at 5 days of age. In addition, there was no significant difference in the mRNA expressions of ß2-AR and atrogin-1/MAFbx in the sartorius muscle between these two groups. These results suggest that ß2-AR expression levels might be physiologically significant in the control of protein degradation in the pectoral muscle of neonatal chicks.

ß1- and ß2-adrenergic receptor stimulation differ in their effects on PGC-1α and atrogin-1/MAFbx gene expression in chick skeletal muscle.

Adrenaline changes expression of the genes encoding peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α), which is known as a regulator of muscle size, and atrogin-1/muscle atrophy F-box (MAFbx), which is a muscle-specific ubiquitin ligase. However, the subtype of ß-adrenergic receptor (ß-AR) involved in regulating these genes in skeletal muscle is not yet well defined. In this study, the effects of intraperitoneal injection of adrenaline and three ß1-3-AR selective agonists on chick skeletal muscle metabolism were examined, to evaluate the functions of ß-AR subtypes. Adrenaline decreased atrogin-1/MAFbx mRNA levels accompanied by an increase in PGC-1α mRNA and protein levels. However, among the three selective agonists, only the ß1-AR agonist, dobutamine, increased PGC-1α mRNA and protein levels, while the ß2-AR agonist, clenbuterol, suppressed atrogin-1/MAFbx mRNA levels. In addition, preinjection of the ß1-AR antagonist, acebutolol, and the ß2-AR antagonist, butoxamine, inhibited the adrenaline-induced increase in PGC-1α mRNA levels and the decrease in atrogin-1/MAFbx mRNA levels, respectively. Compared with adrenaline administration, the ß3-AR agonist, BRL37344, decreased PGC-1α mRNA levels and increased atrogin-1/MAFbx mRNA levels. These results suggest that, in chick skeletal muscle, PGC-1α is induced via the ß1-AR, while atrogin-1/MAFbx is suppressed via the ß2-AR.

Effects of first exogenous nutrients on the mRNA levels of atrogin-1/MAFbx and GLUT1 in the skeletal muscles of newly hatched chicks.

The aim of this study was to examine the effects of first exogenous nutrients on the mRNA levels of muscle atrophy F-box (atrogin-1/MAFbx) and glucose transporters (GLUTs) in the skeletal muscles of newly hatched chicks with no feed experience. In experiment 1, newly hatched chicks had free access to feed or were fasted for the first 24h. The chicks having free access to feed for the first 24h increased their body weight and had decreased atrogin-1/MAFbx mRNA levels in their sartorius and pectoralis major muscles compared with the fasted chicks. In experiment 2, newly hatched chicks received a single feed via intubation into the crop. Three hours after intubation, levels of atrogin-1/MAFbx mRNA in the sartorius muscle were decreased whereas the plasma insulin concentration and phosphorylated AKT levels in the sartorius muscle were increased. In addition, the mRNA levels of GLUT1 and GLUT8 were increased in the sartorius muscle after the intubation. However, in the pectoralis major muscle, AKT phosphorylation and levels of atrogin-1/MAFbx, GLUT1 and GLUT8 mRNA were not affected 3h after intubation. The first exogenous nutrients increased the level of phosphorylated AKT in the sartorius muscle of newly hatched chicks, possibly because of the decrease in atrogin-1/MAFbx mRNA levels. Furthermore, the sartorius muscle in newly hatched chicks appeared to be more susceptible to the first feed compared with the pectoralis major muscle.

Gene expression pattern of glucose transporters in the skeletal muscles of newly hatched chicks.

The gene expression pattern of the glucose transporters (GLUT1, GLUT3, GLUT8, and GLUT12) among pectoralis major and minor, biceps femoris, and sartorius muscles from newly hatched chicks was examined. GLUT1 mRNA level was higher in pectoralis major muscle than in the other muscles. Phosphorylated AKT level was also high in the same muscle, suggesting a relationship between AKT and GLUT1 expression.

Clenbuterol changes phosphorylated FOXO1 localization and decreases protein degradation in the sartorius muscle of neonatal chicks.

To investigate the intracellular signaling mechanisms by which clenbuterol reduces muscle protein degradation, we examined the phosphorylation level and intracellular localization of FOXO1 in the sartorius muscle of neonatal chicks. One-day-old chicks were given a single intraperitoneal injection of clenbuterol (0.1 mg/kg body weight). Three hours after injection, AKT protein was phosphorylated in the sartorius muscle by clenbuterol injection. Coincidentally, clenbuterol increased cytosolic level of phosphorylated FOXO1 protein, while it decreased nuclear level of FOXO1 protein in the sartorius muscle. Furthermore, clenbuterol decreased the expression of mRNAs for muscle-specific ubiquitin ligases (atrogin-1/MAFbx and MuRF1) in the sartorius muscle accompanied by decreased plasma 3-methylhistidine concentration, an index of muscle protein degradation, at 3 h after injection. These results suggested that, in the sartorius muscle of the chicks, clenbuterol changed the intracellular localization of phosphorylated FOXO1, and consequently decreased protein degradation via suppressing the expression of genes encoding muscle-specific ubiquitin ligases.

Single injection of the ß2-adrenergic receptor agonist, clenbuterol, into newly hatched chicks alters abdominal fat pad mass in growing birds.

Excessive energy is stored in white adipose tissue as triacylglycerols in birds as well as in mammals. Although ß2-adrenergic receptor agonists reduce adipose tissue mass in birds, the underlying mechanism remains unclear. The aim of the current study was to examine the effects of a single intraperitoneal injection of the ß2-adrenergic receptor agonist, clenbuterol, on the abdominal fat pad tissue development. Thirty-three chicks at 1-day-old were given a single intraperitoneal injection of clenbuterol (0.1mg/kg body weight) or phosphate-buffered saline. At 2 weeks post-dose, the weight of the abdominal fat tissue was decreased in the clenbuterol-injected chicks, and small adipocyte-like cells were observed in the abdominal fat pad tissue of the clenbuterol-injected chicks. Then, the expression of mRNAs encoding genes related to avian adipogenesis was examined in the abdominal fat pat tissue. The expression of mRNAs encoding Krüppel-like zinc finger transcription factor 5 (KLF-5), KLF-15, and zinc finger protein 423 in the abdominal fat pad tissue of the clenbuterol-injected chicks was significantly lower (P<0.05) than that of the control chicks, while the expression of mRNA encoding peroxisome proliferator-activated receptor-gamma was not affected. In addition, both mRNA expression (P<0.05) and enzymatic activity (P<0.05) of fatty acid synthase (FAS) were decreased in the abdominal fat pad tissue of the clenbuterol-injected chicks, while clenbuterol injection did not affect FAS activity in liver. These results suggested that a single injection with clenbuterol into newly hatched chicks reduces their abdominal fat pad mass possibly via disrupting adipocyte development during later growth stages.

Effects of feeding outer bran fraction of rice on lipid accumulation and fecal excretion in rats.

Outer bran fraction of rice (OBFR) contains higher concentrations of crude fiber, γ-oryzanol, and phytic acid compared to whole rice bran (WRB). In this study, we examined the effects of feeding OBFR on lipid accumulation and fecal excretion in rats. Twenty-one male rats at seven-week-old were divided into a control group and two treatment groups. The control group was fed a control diet, and the treatment groups were fed OBFR- or WRB-containing diet for 21 days. There was no significant difference in growth performance. Feeding OBFR diet increased fecal number and weight accompanied by increased fecal lipid content, while it did not affect mRNA expressions encoding lipid metabolism-related protein in liver. In addition, feeding OBFR-diet decreased the abdominal fat tissue weight and improved plasma lipid profiles, while WRB-containing diet did not affect them. These results suggested that feeding OBFR-diet might prevent lipid accumulation via enhancing fecal lipid excretion in rats.

The effects of intraperitoneal clenbuterol injection on protein degradation and myostatin expression differ between the sartorius and pectoral muscles of neonatal chicks.

The purpose of this study was to investigate the effects of injection of the ß2-adrenergic receptor agonist clenbuterol on the skeletal muscles of neonatal chicks (Gallus gallus domesticus). One-day-old chicks were randomly divided into four groups and given a single intraperitoneal injection of clenbuterol (0.01, 0.1, or 1mg/kg) or phosphate-buffered saline. Twenty-four hours after the injection, the sartorius muscles (which consist of both slow- and fast-twitch fibers) of chicks that received 0.01 or 0.1mg/kg clenbuterol were significantly heavier than those of controls, while there were no between-group differences in the weight of the pectoral muscles, which consist of only fast-twitch fibers. Muscle free N(t)-methylhistidine, regarded as an index of myofibrillar proteolysis, was decreased in the sartorius muscle of the clenbuterol-injected chicks, while it was not affected in the pectoral muscles. In the sartorius muscle of the clenbuterol-injected chicks, myostatin and atrogin-1/MAFbx mRNA expressions were decreased, while insulin-like growth factor-I was unaffected. These observations suggested, in 1-day-old chicks, clenbuterol might increase mass of the sartorius muscle by decreasing myostatin gene expression and protein degradation.

Effects of pre-slaughter fasting on antemortem skeletal muscle protein degradation levels and postmortem muscle free amino acid concentrations in broiler chickens.

This study investigated the effects of pre-slaughter fasting time on the relationship between skeletal muscle protein degradation levels at slaughter and chicken meat quality after 48 h of postmortem aging. Twenty-four broiler chicks at 0 d of age were used in this study until 28 d of age. At 27 d of age, the chickens were assigned to 4 treatment groups: 0 h of fasting (0H), 8 h of fasting (8H), 16 h of fasting (16H), or 24 h of fasting (24H). They were slaughtered at 28 d of age. Blood samples were collected before fasting and immediately before slaughter. Plasma Nτ-methylhistidine concentration, an index of skeletal muscle protein degradation level, and muscle free amino acid concentration were analyzed. Antemortem changes in individual plasma Nτ-methylhistidine concentrations were significantly increased in 8H, 16H, and 24H compared to that in 0H (P < 0.05). After 48 h of postmortem storage, the glutamic acid content in the pectoralis major muscles increased with fasting time (P < 0.05), and the umami taste of chicken soup in the fasting groups (8H, 16H, 24H) was higher than that in the 0H group (P < 0.05). The antemortem changes in plasma Nτ-methylhistidine concentrations were correlated with glutamic acid content in the pectoralis major muscles (r = 0.57, P < 0.05) and umami taste (r = 0.66, P < 0.05). These results suggest that skeletal muscle protein degradation levels at slaughter are related to postmortem chicken meat quality, especially glutamic acid content and umami taste.

Effects of Cyclic High Ambient Temperature on Muscle Imidazole Dipeptide Content in Broiler Chickens.

Imidazole dipeptides possess important bioregulatory properties in animals. This study aimed to evaluate the effect of high ambient temperature on muscle imidazole dipeptides (carnosine, anserine, and balenine) in broiler chickens. Sixteen 14-day-old male broiler chickens were divided into two groups, which were reared under thermoneutral (25 ± 1 °C) or cyclic high ambient temperature (35 ± 1 °C for 8 h/day) for 4 weeks. Chickens exposed to cyclic high ambient temperatures displayed lower skeletal muscle anserine and carnosine content than control chickens. Balenine could not be detected in the pectoral muscle of either group. The pectoral muscles of broiler chickens kept under cyclic high-temperature exhibited significantly lower mRNA expression of carnosine synthase 1, which synthesizes carnosine and anserine; but a significantly higher mRNA expression of carnosinase 2, which degrades carnosine and anserine. Our results suggest that heat exposure decreases pectoral imidazole dipeptide content in broiler chickens. This may be attributed to a lower expression of imidazole dipeptide-synthesizing genes, but higher levels of genes involved in their degradation.

Role of prolactin-like protein (PRL-L) in cold-induced increase of muscle mass in chicks.

This study examined the hypothesis that a novel prolactin-like protein gene (PRL-L) is involved in cold-induced growth of skeletal muscle in chicks. Six-day-old chicks (Gallus gallus domesticus) were exposed to cold at 4°C or kept warm at 30°C for 24h. Cold exposure induced significant increases in PRL-L expression that coincided with increases in the weight of the sartorius muscle, which comprises both fast- and slow-twitch fibers. Meanwhile, no induction of PRL-L mRNA was observed in the heart, liver, kidney, brain, or fat. Myoblast cells that expressed PRL-L mRNA grew faster than untransduced cells in media containing 2% serum. These results suggested that PRL-L might be involved in in controlling cold-induced muscle growth of chicks.

Effects of Delaying Post-hatch Feeding on the Plasma Metabolites of Broiler Chickens Revealed by Targeted and Untargeted Metabolomics.

Exogenous nutrients are essential for body and skeletal muscle growth in newly hatched chicks, and delaying post-hatch feeding negatively affects body growth, meat yield, and meat quality. The aim of this study was to investigate the effects of delayed post-hatch feeding on the metabolic profiles of broiler chickens using a combination of targeted and untargeted metabolomics. Newly hatched chicks had either immediate free access to feed (freely fed chicks) or no access to feed from 0 to 2 days of age (delayed-fed chicks); both groups were subsequently provided feed ad libitum until 13 days of age. Untargeted metabolomic analysis was performed using gas chromatography-mass spectrometry, whereas targeted metabolomic analysis of amino acids was performed using high-performance liquid chromatography with ortho-phthalaldehyde derivatization. Delayed feeding increased the plasma levels of sucrose, maltose, serotonin, lactitol, gentiobiose, xylitol, threonic acid, and asparagine, and decreased the plasma levels of creatinine, aspartic acid, and glutamic acid. In addition, the digestibility of the nitrogen-free extract (starch and sugar) and the cecal butyric acid concentration increased in chicks subjected to delayed feeding. In contrast, delayed feeding did not affect muscle protein degradation or digestibility in chicks. Taken together, our results indicate that delaying feeding until 48 h post-hatch alters multiple metabolic pathways, which are accompanied by changes in intestinal carbohydrate digestion and cecal butyric acid content in broiler chickens.

Quantification of Nτ -Methylhistidine and Nπ-Methylhistidine in Chicken Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

The concentration of Nτ-methylhistidine in plasma provides an index of skeletal muscle protein breakdown. This study aimed to establish a quantitative method for measuring the concentrations of Nτ-methylhistidine and its isomer Nπ-methylhistidine in chicken plasma, using liquid chromatography-tandem mass spectrometry with stable isotope dilution analysis. The acceptable linear ranges of detection were 1.56-50.00 µmol/L for Nτ-methylhistidine and 0.78-25.00 µmol/L for Nπ-methylhistidine. The proposed method detected changes in the plasma levels of Nτ-methylhistidine and Nπ-methylhistidine in response to fasting and re-feeding. These results suggest that the method developed in this study can be used for the simultaneous measurement of Nτ-methylhistidine and Nπ-methylhistidine in chicken plasma.

Suppression of FoxO1 mRNA by ß2 -adrenoceptor-cAMP signaling through miR-374b-5p and miR-7a-1-3p in C2C12 myotubes.

ß2 -Adrenoceptor (ß2 -AR) signaling decreases the transcriptional activity of forkhead box O (FoxO), but the underlying mechanisms remain incompletely understood. Here, we investigated how ß2 -AR signaling regulates the protein abundance of FoxO and its transcriptional activity in skeletal muscle. We observed that stimulation of ß2 -AR with its selective agonist, clenbuterol, rapidly decreased FoxO1 mRNA expression, and this was accompanied by a decrease in either FoxO1 protein level or FoxO transcriptional activity. We subsequently observed that miR-374b-5p and miR-7a-1-3p were rapidly upregulated in response to ß2 -AR stimulation. Transfection with mimics of either miRNA successfully decreased FoxO1 mRNA. Moreover, because ß2 -AR stimulation increased cAMP concentration, pretreatment with an adenylyl cyclase inhibitor canceled out these effects of ß2 -AR stimulation. These results suggest that ß2 -AR stimulation results in rapid upregulation of miR-374b-5p and miR-7a-1-3p in myotubes, which in turn results in a decrease in FoxO1 mRNA expression via the ß2 -AR-cAMP signaling pathway.

Effects of Supplementation with Dried Neem Leaf Extract on Lipid Peroxidation and Antioxidant Enzyme mRNA Expression in the Pectoralis Major Muscle of Broiler Chickens.

This study was conducted to evaluate the effects of dietary supplementation of dried neem (Azadirachta indica) leaf extract (DNE) on lipid peroxidation and the expression of genes encoding mRNAs in antioxidant enzymes in the pectoralis major muscle of chickens. A total of 24 male broiler chickens (ROSS308) were divided into three groups (n=8) at 21 days of age. The control group of chickens was fed a basal diet, and the remaining two groups of chickens were fed a basal diet supplemented with DNE at a concentration of 0.5% or 2.0% until 35 days of age. Growth performance (body weight, weight gain, feed intake, and feed conversion ratio) and tissue weights did not differ among the three groups. The 2.0% DNE-supplemented diet decreased the muscle malondialdehyde content, a marker of lipid peroxidation, and drip loss compared to the control chickens. In addition, the expression of genes encoding mRNAs of antioxidant enzymes (i.e., Cu/Zn-superoxide dismutase, Mn-superoxide dismutase, glutathione peroxidase 7, and catalase) were higher in the pectoralis major muscle of chickens fed the 2.0% DNE-supplemented diet than in the control chickens. Therefore, DNE supplementation increased the expression of genes encoding mRNAs in antioxidant enzymes and reduced lipid peroxidation and drip loss in the pectoralis major muscle of broiler chickens.

Analyses of free amino acid and taste sensor traits in egg albumen and yolk revealed potential of value-added eggs in chickens.

To create high-quality eggs by using different breed and feed materials, we investigated free amino acid contents and taste sensor traits using two chicken breeds (Rhode Island Red; RIR and Australorp; AUS) fed two feeds (mixed and fermented feeds). Two-way ANOVA revealed significant breed and feed main and interaction effects on albumen bitterness and a significant interaction effect on yolk bitterness. Albumen from RIR fed mixed feed and AUS fed fermented feed was higher bitterness, whereas yolk from those groups was lower bitterness. Significant breed effects were detected in four albumen amino acid traits (His, Met, Ile, and Lys) and a yolk His, whereas significant feed effects were found in 15 albumen amino acid traits (Asp, Glu, Ser, His, Gly, Thr, Ala, Tyr, Val, Met, Trp, Ile, Leu, Lys, and Pro) and a yolk cystine trait. Compared to albumen amino acids, yolk amino acids had limited effects by breed and feed. The present results suggest that genetic and nutritional factors can alter not only amino acid contents but also sensor values of bitterness, indicating that selecting the combination of breed and feed enable us to make amino acids enriched and taste added designer eggs in future.

Effect of mixed rearing of barrows and gilts on backfat thickness and serum metabolite profiles of the Kagoshima-Kurobuta (Berkshire) pig.

We investigated the effect of mixed rearing of barrows and gilts on the backfat thickness and the serum metabolite profiles of Kagoshima-Kurobuta (Berkshire) pigs. A total of 149 pigs with an average body weight of 35 kg were divided into the following groups: 100%, 90%, 70%, 50%, 30%, 10%, and 0% groups consisting of 10 barrows (1 pen), 9 barrows + 1 gilt (3 pens), 7 barrows + 3 gilts (2 pens), 5 barrows + 5 gilts (3 pens), 3 barrows + 7 gilts (2 pens), 1 barrow + 9 gilts (3 pens), and 9 gilts (1 pen), respectively. All pigs were raised to a shipping weight of 120 kg. Mixed rearing significantly reduced (p < 0.001) backfat thickness, and the optimum mixing ratio of barrows and gilts was 7:3 (the 70% group). Four types of circulating sex steroids were found in both the barrows and gilts in the 50% group but were not detected in barrows from the 100% group. These results indicated that mixed rearing of barrows and gilts was effective for reducing the backfat thickness of barrows, and induced sex steroid hormones may influence the backfat thickness of barrows in mixed-reared groups.

Genetic effect on free amino acid contents of egg yolk and albumen using five different chicken genotypes under floor rearing system.

Chicken eggs play an important role as food resources in the world. Although genetic effects on yolk and albumen contents have been reported, the number of chicken genotypes analyzed so far is still limited. To investigate the effect of genetic background on 10 egg traits, 19 yolk amino acid traits, and 19 albumen amino acid traits, we evaluated a total of 58 eggs from five genotypes: two Japanese indigenous breeds (Ukokkei and Nagoya) and three hybrids (Araucana cross, Kurohisui, and Boris Brown) under a floor rearing system. One-way ANOVA revealed significant effects of genotype on 10 egg traits, 8 yolk amino acids (Asp, Glu, Ser, Gly, Thr, Tyr, Cys, and Leu), and 11 albumen amino acids (Asp, Glu, Asn, Ser, Gln, His, Ala, Tyr, Trp, Phe, and Ile) contents. Moderate to strong positive phenotypic correlations among traits within each trait category (size and weight traits, yolk amino acid traits, and albumen amino acid traits), whereas there were basically no or weak correlations among the trait categories. However, a unique feature was found in the Araucana cross indicating moderate positive correlations of amino acids between yolk and albumen. These results suggest that genetic factors can modify not only the size and weight of the egg and eggshell color but also yolk and albumen free amino acids contents.