Expression of genes related to ileal barrier function in heritage and modern broiler chickens.

1. An experiment was conducted to determine differences in the expression of genes encoding intestinal barrier proteins between fast, medium and slow-growing chickens. Chicken breeds Athens Canadian Random Bred (ACRB), Longenecker's Heritage (LHR), RedBro, Hubbard H1 (HH1), Cobb500 and Ross708 were raised from hatch for 35 d.2. Ileal samples were collected at embryonic day E19 (-2 days post-hatch), hatch and d 7, 14, 21, 28 and 35 post-hatch to assess the expression of genes encoding tight junction proteins (claudins, CLDN; occludin, OCLN; zonula occludens, ZO; and junctional adhesion molecules, JAM), mucin (Muc2), immunoglobulin A (IgA), polymeric immunoglobulin receptor (pIgR) and fatty acid binding protein (FABP2).3. Expression of CLDN-1 was increased (p < 0.0001) in LHR compared to Cobb500 while CLDN-5 was increased (p < 0.0001) in ACRB, HH1, RedBro and Ross708 compared to LHR as well as in ACRB compared to Cob500. Occludin was upregulated (p = 0.01) in ACRB and LHR compared to Ross708 at d 14 post-hatch. Expression of ZO-1 was upregulated (p = 0.001) in LHR compared to Ross708, HH1 and Cobb500. Tight junction genes, except CLDN-4, JAM-2 and JAM-3 were downregulated (p < 0.0001) at hatch and d 7 post-hatch. Expression of Muc2 was increased (p < 0.0001) in LHR compared to RedBro and from -2 d to d 7 post-hatch.4. Immunoglobulin A was increased (p = 0.001) in LHR compared to Ross708 and HH1 at -2 d post-hatch and in LHR compared to ACRB, Cobb500 and Ross708 at hatch. In addition, IgA expression was increased in all breeds at d 14 post-hatch while pIgR was upregulated (p = 0.02) in Cobb500 and Ross708 compared to ACRB, HH1, LHR and RedBro at hatch.5. The gene expression patterns suggest that selection for growth may have not induced changes in junctional complexes and immune defence genes. However, the results confirmed that the expression of these genes is age dependent.

Expression of genes associated with nutrient uptake in intestines of chickens with different growth potentials show temporal changes but are not correlated with growth.

1. The study was designed to compare the expression of genes that encode proteins located at either the brush border (BB) or basolateral (BL) of the gut epithelium among fast and slow-growing broilers.2. Six broiler breeds with different growth capacities were used: Ross 708, Hubbard H1 (HH1), Cobb 500, Longenecker's Heritage (LHR), Red-Bro, and the Athens Canadian Randombred Control (ACRB). Birds were sampled between embryonic day (ED) 19 and day 35 post-hatch (PH).3. Performance parameters indicated that Ross 708, HH1, and Cobb 500 had the highest body weights (BW) while ACRBs had the lowest.4. Quantitative RT-PCR was performed on 13 genes encoding proteins associated with nutrient processing and uptake. Statistical analysis was carried out (ANOVA) for eight BB genes: Aminopeptidase N (APN), four amino acid transporters, (ATBo,+, BoAT, bo,+AT, EAAT3) a di- and tri-peptide transporter (PepT1), and two sugar transporters (GLUT5 and SGLT1). Analysis of four amino acid transporters (CAT1, CAT2, LAT1, and γ+LAT1), and a single sugar transporter (GLUT2) associated with BL was carried out.5. Four BB associated genes (APN, EAAT3, BoAT, and b0,+AT) in the small intestine were negatively correlated with growth.6. In most cases, genes encoding BB proteins increased in expression over time (P < 0.05) in the small intestine, while, in the caeca, the expression decreased (P < 0.05). The mRNA of BL-associated proteins showed decreased (P < 0.05) expression over time in all gut segments, with exception of GLUT2, which increased in expression in the small intestine.7. The temporal changes in gene expression were consistent among bird lines and BB associated genes tended to increase over time, while BL associated genes tended to decrease over time. Correlation analysis indicated that mRNA expression of nutrient transporter genes may not be a good predictor of growth potential.

The effect of delayed feeding post-hatch on caeca development in broiler chickens.

1. Broiler chicks are frequently deprived of food up to 72 h due to uneven hatching rates, management procedures and transportation to farms. Little is known about the effect of delayed feeding due to extended hatching times on the early neonatal development of the caeca. Therefore, the objective of this study was to investigate the developmental changes and effects of a 48-h delay in feed access immediately post-hatch (PH) on the caeca.2. After hatch, birds (Ross 708) were randomly divided into two treatment groups (n = 6 battery pen/treatment). One group (early fed; EF) received feed and water immediately after hatch, while the second group (late fed; LF) had access to water but had delayed access to feed for 48 h. Contents averaging across all regions of the caeca were collected for mRNA expression as well as for histological analysis at -48, 0, 4 h PH and then at 1, 2, 3, 4, 6, 8, 10, 12 and 14 days PH.3. Expression of MCT-1 (a nutrient transporter), Cox7A2 (related to mitochondrial function) IgA, pIgR, and ChIL-8 (immune function) genes was affected by delayed access to feed that was dependent by the time PH. Expression of immune and gut barrier function-related genes (LEAP2 and MUC2, respectively) was increased in LF group. There was no effect of feed delay on expression of genes related to mitochondrial functions in the caeca, although developmental changes were observed (ATP5F1B, Cox4|1). Caecal mucus and muscle thickness were affected by delayed access to feed during caeca development.4. The data suggested a limited effect of delayed feed access PH on the developmental changes in caecal functions. However, the caeca seemed to be relatively resistant to delayed access to feed early PH, with only a few genes affected.

Effects of limestone particle size and dietary Ca concentration on apparent P and Ca digestibility in the presence or absence of phytase.

The present study evaluated the effects of limestone particle size and Ca concentration on apparent ileal digestibility (AID) of P and Ca in the presence or absence of a 6-phytase derived from Buttiauxella sp., expressed in Trichoderma. Treatment diets were corn-soybean meal (SBM) based with no added inorganic Ca or P. The design consisted of a factorial arrangement of 2 particle sizes of limestone from the same source (mean particle size: pulverized, PUV < 75 µm; particulate, PAR = 402 µm); 3 Ca (0.6, 0.8, and 1.0%) and 2 phytase levels (0 and 1,000 (FTU)/kg) plus the corn-SBM basal diet with no added Ca (0.14% Ca), resulting a total of 13 treatments (Trts, n = 9, 3 birds/n). Starting at 27 d of age, broilers were fed the mash diets for 32 h, then sampled for gizzard pH and distal ileal digestibility. Gizzard pH was 1.94 in birds fed diet without added Ca and was similar to those fed diet with PAR limestone regardless of phytase. Gizzard pH was increased in birds fed PUV limestone diets irrespectively of phytase inclusion compared to birds fed 0.14% Ca (P < 0.05), except in birds fed 0.60% Ca diet. In the absence of phytase, AID P was reduced as a result of increases in limestone inclusion (P < 0.05), with a greater effect seen in PUV as compared to PAR limestone. The AID Ca in birds fed PUV limestone diets was lower than that of those fed PAR limestone diets (22.1% vs. 28.2%; P < 0.05). In the presence of phytase, the AID P was not affected by increasing Ca concentration when PAR limestone was used as the Ca source (average = 64.3%), whereas AID P decreased from 66.9% (0.6% Ca) to 51.0% (1.0% Ca) when PUV limestone was used as the Ca source (P < 0.05). In summary, impact of Ca concentration on AID Ca and P was dependent on limestone particle size and phytase inclusion.

Insulin immuno-neutralization decreases food intake in chickens without altering hypothalamic transcripts involved in food intake and metabolism.

In mammals, insulin regulates blood glucose levels and plays a key regulatory role in appetite via the hypothalamus. In contrast, chickens are characterized by atypical glucose homeostasis, with relatively high blood glucose levels, reduced glucose sensitivity of pancreatic beta cells, and large resistance to exogenous insulin. The aim of the present study was to investigate in chickens the effects of 5 h fasting and 5 h insulin immuno-neutralization on hypothalamic mRNA levels of 23 genes associated with food intake, energy balance, and glucose metabolism. We observed that insulin immune-neutralization by administration of anti-porcine insulin guinea pig serum (AI) significantly decreased food intake and increased plasma glucose levels in chickens, while 5 h fasting produced a limited and non-significant reduction in plasma glucose. In addition, 5 h fasting increased levels of NPY, TAS1R1, DIO2, LEPR, GLUT1, GLUT3, GLUT8, and GCK mRNA. In contrast, AI had no impact on the levels of any selected mRNA. Therefore, our results demonstrate that in chickens, food intake inhibition or satiety mechanisms induced by insulin immuno-neutralization do not rely on hypothalamic abundance of the 23 transcripts analyzed. The hypothalamic transcripts that were increased in the fasted group are likely components of a mechanism of adaptation to fasting in chickens.

Age and adaptation to Ca and P deficiencies: 2. Impacts on amino acid digestibility and phytase efficacy in broilers.

A total of 1,152 straight-run hatchling Heritage 56M×fast feathering Cobb 500F broiler birds were used to determine Ca, age, and adaptation effects on apparent ileal digestibility of crude protein (AID of CP), amino acids (AID of AA) and phytase efficacy. Twelve treatments with 8 replicates, each were fed from 7 to 9 d (6 birds per replicate), 7 to 21 d (6 birds per replicate) and 19 to 21 d (3 birds per replicate) d of age. Diets were prepared with 3 Ca (0.65, 0.80, and 0.95%) and 2 non-phytate P, (0.20 and 0.40%) concentrations. A 6-phytase was added at 500 or 1,000 FTU/kg to the 0.20% nPP diet at each Ca concentration. The age and adaptation effects were determined by comparing the responses between birds fed from 7 to 9 and 19 to 21 d of age, 19 to 21, and 7 to 21 d of age, respectively. An age effect was observed regardless of Ca, nPP, or phytase concentration, with older birds (19 to 21 d) having greater apparent ileal digestibility (AID) of amino acids (AA) and CP than younger birds (7 to 9 d; P<0.05). Response to adaptation varied depending on Ca, nPP, and phytase concentrations. Constant lower AID of CP and AA was seen in adapted birds (7 to 21 d) compared to unadapted bird (19 to 21 d) when 0.20% nPP diets were fed at 0.95% Ca concentrations (P<0.05). At 0.40% nPP, there was no effect of adaptation on AID of CP and AA at any Ca concentration. Phytase efficacy was significantly lower in younger (7 to 9 d) compared to older birds (19 to 21 d; P<0.05), except at 0.65% Ca. Phytase inclusion increased AID of CP and AA regardless of Ca (P<0.05). In conclusion, the AID of CP and AA can be affected by diet, age, and adaptation.

Impact of response criteria (tibia ash weight vs. percent) on phytase relative non phytate phosphorus equivalance.

The current study was conducted to evaluate the impacts of using tibia ash percentage or ash weight as the response criteria on estimated phytase relative equivalence. Straight run broilers were fed treatment (Trt) diets from 7 to 21 d age (6 birds/pen, 8 pens/Trt). The corn-soy based Trt were formulated to contain 0.80% Ca and 4 non-phytate phosphorus (nPP) concentrations (0.20, 0.27, 0.34, and 0.40%). Monocalcium phosphate was the inorganic phosphate source added to achieve 4 different dietary nPP concentrations and against which the nPP relative equivalence of phytase was determined. A 6-phytase (Danisco Animal Nutrition, DuPont Industrial Biosciences, Marlborough, UK) was added at 500 or 1,000 phytase unit ( FTU: )/kg to the 0.20% nPP diet resulting 6 total Trts. Tibia ash was determined at 21 d age. Phytase fed at 500 or 1,000 FTU/kg increased tibia ash weight and ash percentage compared to that of birds fed 0.20% nPP diet without phytase (P<0.05). Graded nPP were log transformed and regressed against tibia ash (weight and percentage) to calculate phytase nPP relative equivalence. The R2 obtained from pen value regressions were 0.81 and 0.84, for tibia ash weight and percentage, respectively. Ash percentage from birds fed 500 and 1,000 FTU phytase/kg fell within the range obtained with the MCP additions. Ash weight (842 mg/tibia) from birds fed 1,000 FTU phytase/kg exceeded (P<0.05) maximum weight (773 mg/tibia) measured in birds fed the greatest nPP Trt (0.40%), thus the nPP relative equivalence was only calculated in birds fed 500 FTU phytase/kg Trt. The nPP relative equivalence in birds fed 500 FTU phytase/kg were 0.117 and 0.168% based on ash percentage and weight, respectively (P<0.05). The nPP relative equivalence in birds fed 1,000 FTU phytase/kg was 0.166% for ash percentage. Results suggested that ash weight better reflects the amount of bone mineralization as compared to ash percentage and using ash percentage may lead to an underestimation of phytase efficacy.

Assessment of postcrumble addition of limestone and calcium-specific appetite in broilers during the starter phase.

A study was done to determine whether broilers can regulate Ca intake when limestone is provided separately or mixed with a crumbled feed of variable Ca and P content, and the influence of this on performance and apparent ileal digestibility (AID) of Ca and P (AIDP). Twelve crumbled diets were fed from 10 to 20 d of age (8 replicates, 8 broilers/replicate). Diets A to D contained 0.28% nonphytate P (nPP) and 0.27, 0.51, 0.77, and 1.02% Ca, respectively. Diets E to H contained 0.48% nPP and 0.41, 0.51, 0.77, and 1.02% Ca, respectively. A large particle size limestone was mixed manually to the crumbled diet on a daily basis to achieve 1.02% total Ca in diets A to H. Diets I to L had the same Ca and nPP as diets A to D, but limestone was provided in a separate feeder to assess spatial importance of limestone supply. Limestone consumption, provided in a separate feeder, decreased as Ca concentration increased in the crumble diet (P < 0.05). Calcium intake increased as Ca concentration in crumbled diets increased (P < 0.05). Increased tibia ash and decreased AIDP were observed as Ca intake increased (P < 0.05). When limestone was added to diets containing 0.28% nPP postcrumble, Ca intake (6.38 g/bird), tibia ash (717 mg/bone), and AIDP (39.78%) were not affected by crumbled diet Ca concentration or consumed Ca. Broilers fed diets containing 0.48% nPP and limestone mixed with the crumble, Ca intake changed (5.96, 6.93, 6.59, and 6.04 g/bird for crumble diet with 0.41, 0.51, 0.77, and 1.02% Ca, respectively). Increasing Ca concentration in the crumble from 0.41 to 1.02% resulted in greater tibia ash (875 mg/bone) but lower AIDP (P < 0.05), although Ca intake was similar. In conclusion, when large particle size limestone was provided ad libitum, the ability of broilers to select for Ca was not sufficient to meet their requirement when crumble Ca was less than 0.77%. The AIDP was highest in birds fed the 0.27% Ca concentration diet.

Expression and activity of the 5'-adenosine monophosphate-activated protein kinase pathway in selected tissues during chicken embryonic development.

The 5'-adenosine monophosphate-activated protein kinase (AMPK) is a highly conserved serine-threonine protein kinase and a key part of a kinase-signaling cascade that senses cellular energy status (adenosine monophosphate:adenosine triphosphate ratio) and acts to maintain energy homeostasis by coordinately regulating energy-consuming and energy-generating metabolic pathways. The objective of this study was to investigate aspects of the AMPK pathway in the liver, brain, breast muscle, and heart from d 12 of incubation through hatch in chickens. We first determined mRNA and protein expression profiles for a major upstream AMPK kinase, LKB1, which is known to activate (phosphorylate) AMPK in response to increases in the adenosine monophosphate:adenosine triphosphate ratio. Expression of LKB1 protein was greatest in the brain, which demonstrated tissue-specific patterns for phosphorylation. Next, AMPK subunit mRNA and protein expression profiles were determined. Significant changes in AMPK subunit mRNA expression occurred in all tissues from d 12 of incubation to hatch. Differences in the levels of active (phosphorylated) AMPK as well as alpha and beta subunit proteins were observed in all 4 tissues during embryonic development. Finally, we determined the protein level and phosphorylation status of an important downstream target for AMPK, acetyl-coenzyme A carboxylase. The expression of acetyl-co-enzyme A carboxylase and phosphorylated acetyl-coenzyme A was greater in the brain than the liver, but was undetectable by Western blotting in the breast muscle and heart throughout the period of study. Together, our results are the first to demonstrate the expression and activity of the AMPK pathway in key tissues during the transition from embryonic to posthatch development in chickens.

Mechanisms regulating feed intake, energy expenditure, and body weight in poultry.

To achieve energy balance and maintain a constant BW, changes in feed intake and energy expenditure must be coordinated and tightly regulated. This may not hold true for some poultry species intensively selected for such economically important traits as growth and meat production. For example, the modern commercial broiler breeder does not adequately control voluntary feed intake to meet its energy requirements and maintain energy balance. As a consequence, feeding must be limited in these birds to avoid overconsumption and excessive fattening during production. It is important to determine a genetic basis to help explain this situation and to offer potential strategies for producing more efficient poultry. This review summarizes what is currently known about the control of feed intake and energy expenditure at the gene level in birds. Highly integrated regulatory systems have been identified that link the control of feeding with the sensing of energy status. How such systems function in poultry is currently being explored. One example recently identified in chickens is the adenosine monophosphate-activated protein kinase pathway that links energy sensing with modulation of metabolic activity to maintain energy homeostasis at the cellular level. In the hypothalamus, this same pathway may also play an important role in regulating feed intake and energy expenditure commensurate with perceived whole body energy needs. Genes encoding key regulatory factors such as hormones, neuropeptides, receptors, enzymes, and transcription factors produce the molecular components that make up intricate and interconnected neural, endocrine, and metabolic pathway networks linking peripheral tissues with the central nervous system. Moreover, coordinate expression of specific gene groups can establish functional pathways that respond to and are regulated by such factors as hormones, nutrients, and metabolites. Thus, with a better understanding of the genetic and molecular basis for regulating feed intake and energy expenditure in birds important progress can be made in developing, evaluating, and managing more efficient commercial poultry lines.

Molecular cloning, genomic organization, and expression of three chicken 5'-AMP-activated protein kinase gamma subunit genes.

The 5'-AMP-activated protein kinase (AMPK) plays a key role in regulating cellular energy homeostasis. The AMPK is a heterotrimeric enzyme complex that consists of 1 catalytic (alpha) and 2 regulatory (beta and gamma) subunits. Mutations of the gamma subunit genes are known to affect AMPK functioning. In this study, we characterized the genomic organization and expression of 3 chicken AMPK gamma subunit genes (cPRKAG). Alternative splicing of the second exon of the cPRKAG1 gene resulted in 2 transcript variants that code for predicted proteins of 298 and 276 amino acids. Use of an alternate promoter and alternative splicing of the cPRKAG2 gene resulted in 4 transcript variants that code for predicted proteins of 567, 452, 328, and 158 amino acids. Alternative splicing of exon 3 of the cPRKAG3 gene resulted in the production of "long" and "short" transcript variants that code for predicted proteins of 382 and 378 amino acids, respectively. We found evidence for differential expression of individual gamma subunit gene transcript variants and, in some cases, tissue-specific expression was observed. The cPRKAG subunit genes displayed similar structural features and high sequence homology compared with corresponding mammalian gamma subunit gene homologues.