Negative consequences of reduced protein diets supplemented with synthetic amino acids for performance, intestinal barrier function, and caecal microbiota composition of broiler chickens.

The consequences of feeding broiler chickens with reduced protein (RP) diets for gut health and barrier function are not well understood. This study was performed to elucidate the effect of reducing dietary protein and source of protein on gut health and performance parameters. Four experimental diets included 2 control diets with standard protein levels either containing meat and bone meal (CMBM) or an all-vegetable diet (CVEG), a medium RP diet (17.5% in growers and 16.5% in finisher), and a severe RP diet (15.6% in grower and 14.6% in finisher). Off-sex Ross 308 birds were assigned to each of the 4 diets and performance measurements were taken from d 7 to 42 post-hatch. Each diet was replicated 8 times (10 birds per replicate). A challenge study was conducted on additional 96 broilers (24 birds per diet) from d 13 to 21. Half of the birds in each dietary treatment were challenged by dexamethasone (DEX) to induce a leaky gut. Feeding birds with RP diets decreased weight gain (P < 0.0001) and increased feed conversion ratio (P < 0.0001) from d 7 to 42 compared with control diets. There was no difference between CVEG and CMBM control diets for any parameter. The diet containing 15.6% protein increased (P < 0.05) intestinal permeability independent of the DEX challenge. Gene expression of claudin-3 was downregulated (P < 0.05) in birds fed 15.6% protein. There was a significant interaction between diet and DEX (P < 0.05) and both RP diets (17.5% and 15.6%) downregulated claudin-2 expression in DEX-challenged birds. The overall composition of the caecal microbiota was affected in birds fed 15.6% protein having a significantly lower richness of microbiota in both sham and DEX-injected birds. Proteobacteria was the main phylum driving the differences in birds fed 15.6% protein. At the family level, Bifidobacteriaceae, Unclassified Bifidobacteriales, Enterococcaceae, Enterobacteriaceae, and Lachnospiraceae were the main taxa in birds fed 15.6% protein. Despite supplementation of synthetic amino acids, severe reduction of dietary protein compromised performance and intestinal health parameters in broilers, evidenced by differential mRNA expression of tight junction proteins, higher permeability, and changes in caecal microbiota composition.

Intestinal permeability, microbiota composition, and expression of genes related to intestinal barrier function of broiler chickens fed different methionine sources supplemented at varying concentrations.

Intestinal health of broiler chickens is influenced by the concentration of dietary amino acids but data are limited on the role of dietary methionine (Met). Two experiments were conducted to investigate the implications of different Met sources for performance, gut barrier function, and intestinal microbiota in broilers. In the first experiment, Ross 308 off-sex birds (n = 900) were assigned to 10 dietary treatments each replicated 9 times in a 35-day study. Three sources of Met included DL-Met, L-Met, or Met hydroxy analog free acid (MHA-FA), each supplemented at suboptimal (SUB) at 80%, adequate (ADE) at 100% and over-requirement (OVR) at 120% of the specifications against a deficient (DEF) diet with no added Met. The second experiment used 96 Ross 308 broilers in a 2 × 4 factorial arrangement. Four diets included 3 sources of Met supplemented at ADE level plus the DEF treatment. On d 17, 19, and 23, half of the birds in each dietary treatment were injected with dexamethasone (DEX) to induce leaky gut. In the first experiment, without an interaction, from d 0 to 35, birds fed DL-Met and L-Met performed similarly for BWG, feed intake, and FCR but birds fed MHA-FA had less feed intake and BWG (P < 0.05). At d 23, mRNA expression of selected tight junction proteins was not affected except for claudin 2. Ileal microbiota of DEF treatment was different from DL-MET or L-MET supplemented birds (P < 0.05). However, microbiota of MHA-FA treatments was only different at OVR from the DEF group. The abundance of Peptostreptococcus increased in DEF treatment whereas Lactobacillus decreased. In the second experiment, DEX independently increased (P < 0.001) intestinal permeability assayed by fluorescein isothiocyanate dextran, but diet had no effect. DL-Met and L-Met fed birds had a higher level of claudin 3 only in DEX-injected birds (P < 0.05). In conclusion, unlike the level of supplementation, DL-Met, L-Met, and MHA-FA were largely similar in their limited impacts on intestinal barrier function and gut microbiota in broilers.

Current experimental models, assessment and dietary modulations of intestinal permeability in broiler chickens.

Maintaining and optimising the intestinal barrier (IB) function in poultry has important implications for the health and performance of the birds. As a key aspect of the IB, intestinal permeability (IP) is mainly controlled by complex junctional proteins called tight junction proteins (TJ) that link enterocytes together. The disruption of TJ is associated with increased gut leakage with possible subsequent implications for bacterial translocation, intestinal inflammation, compromised health and performance of the birds. Despite considerable data being available for other species, research on IP in broiler chickens and in general avian species is still an understudied topic. This paper reviews the available literature with a specific focus on IP in broiler chickens with consideration given to practical factors affecting the IP, current assessment methods, markers and nutritional modulation of IP. Several experimental models to induce gut leakage are discussed including pathogens, rye-based diets, feed deprivation and stress-inducing agents such as exogenous glucocorticoids and heat stress. Although various markers including fluorescein isothiocyanate dextran, expression of TJ and bacterial translocation have been widely utilized to study IP, recent studies have identified a number of excreta biomarkers to evaluate intestinal integrity, in particular non-invasive IP. Although the research on various nutrients and feed additives to potentially modulate IP is still at an early stage, the most promising outcomes are anticipated for probiotics, prebiotics, amino acids and those feed ingredients, nutrients and additives with anti-inflammatory properties. Considerable research gaps are identified for the mechanistic mode of action of various nutrients to influence IP under different experimental models. The modulation of IP through various strategies (i.e. nutritional manipulation of diet) may be regarded as a new frontier for disease prevention and improving the health and performance of poultry particularly in an antibiotic-free production system.

Intestinal Barrier Function and Performance of Broiler Chickens Fed Additional Arginine, Combination of Arginine and Glutamine or an Amino Acid-Based Solution.

Two experiments were conducted to investigate the effect of arginine (Arg); the combination of Arg and glutamine (Gln); as well as an amino acid-based solution (MIX) containing Arg, Gln, threonine (Thr), and grape extract, on performance, intestinal permeability, and expression of selected mechanistic genes. Using 240 male Ross 308 off-sex broiler chickens, four experimental treatments were replicated six times with 10 birds per replicate. The experimental treatments included 5 g/kg Arg, 2.5 g/kg Arg and 2.5 g/kg Gln, and 1 g/kg MIX added to a basal diet as control. In the second study, the four dietary treatments were then given to 24 birds with or without a synthetic glucocorticoid, dexamethasone (DEX), as a gut dysfunction model. Feed conversion ratio was improved by all the supplemented treatments from day 7 to 35 of age (p < 0.001). DEX injections increased (p < 0.001) the intestinal permeability in all treatments, which tended to be reversed by Arg or MIX. Additional Arg, Arg-Gln, and MIX suppressed (p < 0.05) the overexpression of IL-1ß generated by DEX. Feeding birds with MIX treatment increased (p < 0.05) expression of SGLT-1 and glutathione synthetase. In conclusion, tested amino acid supplements were effective in improving feed efficiency and restraining intestinal inflammation caused by DEX through IL-1ß pathway.

A Microencapsulated Mixture of Eugenol and Garlic Tincture Supplementation Mitigates the Effect of Necrotic Enteritis on Intestinal Integrity and Increases Goblet Cells in Broilers.

This study was conducted to examine the effects of a plant extract mixture, a microencapsulated product composed of eugenol and garlic tincture (PE), on intestinal health in broilers under necrotic enteritis (NE) challenge. A total of 960 d-old mixed-sex Cobb 500 chicks were randomly distributed to 48-floor pens housing 20 birds per pen. Six treatments were applied: UC, unchallenged control; CC, challenged control; PE, challenged group plus PE; AM, challenged group plus antimicrobial (AM); FAP, challenged group plus a full dose of AM with PE; HAP, challenged group plus a half dose of AM with PE in starter, grower and finisher phases. Birds in the challenged groups were inoculated with Eimeria spp. on d 9 and Clostridiumperfringens on d 14. On d 16, the CC group had increased serum fluorescein isothiocyanate dextran (FITC-d), reduced villus surface area, goblet cell number, upregulated CLDN1, JAM2 genes and reduced microbial diversity compared to the UC group (p < 0.05). Birds fed PE had reduced FITC-d, increased goblet cell number and Bifidobacterium compared to the CC group (p < 0.05). Birds fed PE had reduced CLDN5 expression in male birds, and Bacteroides spp. in female birds than CC group (p < 0.05). These findings suggest that PE supplementation mitigates the effect of NE by improving the intestinal health of birds.

Balanced nutrient density for broiler chickens using a range of digestible lysine-to-metabolizable energy ratios and nutrient density: Growth performance, nutrient utilisation and apparent metabolizable energy.

Currently, specific nutrient concentration, metabolizable energy (ME) and digestible amino acids are used as feed formulation criteria. A balanced nutrient density (BND) concept based on 2 criteria of nutrient density and balanced amino acids-to-ME ratio may offer more flexibility in optimisation of profit in formulation of diets compared with current formulation based on set values per unit of feed mass. A total of 672 one-d-old off-sex male Ross 308 broiler chickens were used across two 42-d performance trials in a 3 × 2 factorial arrangement of treatments with each diet replicated 8 times (14 birds per replicate). The experimental factors were 2 nutrient density levels (low [LD] and high [HD]) and 3 digestible lysine-to-ME ratios (DLYS:ME; low, medium, and high). Low density diets had ME of 2,876 and 3,023 kcal/kg for starter and finisher, respectively, while values for HD diets were 3,169 and 3,315 kcal/kg with proportionally higher non-nitrogenated nutrients. Separate digestibility and apparent metabolizable energy (AME) assays were conducted at d 21 and 42. Digestibility assays at d 7 were conducted on birds used for performance trials. Regardless of the diet density, birds fed low DLYS:ME had a lower (P < 0.01) feed intake (d 0 to 42) than medium and high DLYS:ME. Without interaction, birds fed low and medium DLYS:ME had a similar body weight gain being the heaviest while birds low DLYS:ME were the lightest. By an interaction (P < 0.05), the highest overall FCR value was observed for birds fed LD × low DLYS:ME and improved linearly when DLYS:ME increased to the highest level reaching a limit for birds fed HD × medium DLYS:ME. Calorie conversion linearly decreased (P < 0.001) with increments in DLYS:ME. Jejunal and ileal starch and protein digestibility were affected on d 21 and 42 but not on d 7 of age. Given the independence of response on BW and feed consumption, the use of BND as a flexible system in diet formulations has the potential to enable more accurate formulation for optimisation of growth performance of broiler chickens.

Expression of selected genes encoding mechanistic pathways, nutrient and amino acid transporters in jejunum and ileum of broiler chickens fed a reduced protein diet supplemented with arginine, glutamine and glycine under stress stimulated by dexamethasone.

Reducing crude protein and supplementation with synthetic amino acids in poultry nutrition is a recent trend to avoid wastage of protein and ammonia in production systems. Stress has been shown to impair intestinal barrier and increase inflammatory response. This study was performed on intestinal tissues of broiler chickens to understand the mechanism of stress induced by a synthetic glucocorticoid, dexamethasone (DEX) and the effect of supplementation of arginine, glutamine and glycine in reduced protein diets. Intestinal tissue samples from a previous study were utilized. Male Ross 308 chickens received a basal diet for the first seven days and then fed with crude protein that was reduced to 194 g/kg in grower experimental diets supplemented with glutamine, glycine and additional arginine at 10, 10 and 5 g/kg respectively. Half of the 96 individual birds were injected with DEX (0.5 mg/kg body weight) or saline on days 14, 16, 18 and 20 of age. mRNA expression for jejunum and ileum for amino acid transporters (y+LAT-1, Bo,+ AT, EAAT-3 and CAT-1), mechanistic genes (SGLT-1, mTOR, IAP and FABP-2) and pro-inflammatory genes (MUC-2, NF-κB, iNOS, IL-8 and IL-1ß) were analysed using real-time PCR. The results showed that DEX decreased y+ LAT1 in jejunum, Bo ,+ AT and EAAT-3 in ileum. Arginine increased CAT-1 in the jejunum and ileum under DEX treatment. Through an interaction, DEX reduced IAP in jejunum of glycine and arginine supplemented group and reduced mTOR in jejunum independently. DEX reduced MUC-2 and iNOS in jejunum and increased iNOS and IL8 in the ileum. Amino acid supplementation did not appear to ameliorate these effects; however, there were some positive effects of glycine on NF-κB and arginine through increased CAT-1. Mechanistic understanding of amino acid supplementation in broiler diets warrants further research particularly when dietary protein is reduced below the level tested in the present study.

Excreta biomarkers in response to different gut barrier dysfunction models and probiotic supplementation in broiler chickens.

Increased intestinal permeability (IP) and inflammation are both linked with functionality of the intestinal barrier and in particular enterocytes. Currently, almost all assessment methods of the intestinal barrier function are invasive. The present study aimed to quantify selected proteins as novel biomarkers in excreta of broiler chickens to facilitate non-invasive assessment of gut barrier function using enzyme-linked immunosorbent assays (ELISA). It was further hypothesised that probiotics as feed additives may counteract gut barrier dysfunction. A 3 × 2 factorial arrangement of treatments was used with the main factors being gut barrier dysfunction models (control, rye-based diet, and dexamethasone-DEX) with and without probiotic supplementation (a three-strain Bacillus) using 72 male Ross 308 day-old chickens. Each of the 6 experimental treatments was replicated 12 times. On d 21 of age, fluorescein isothiocyanate dextran (FITC-d) uptake into serum was examined to test IP. Fresh excreta samples were collected on d 20. The biomarkers included alpha-1 antitrypsin (A1AT), intestinal fatty acid binding protein (I-FABP), lipocalin-2 (LCN2), fibronectin (FN), intestinal alkaline phosphatase (IAP), ovotransferrin (OVT) and superoxide dismutase [Cu-Zn] (SOD1). Only DEX increased (P<0.001) FITC-d passage to the blood on d 21 of age, indicating a greater IP. The excreta concentrations of A1AT, I-FABP and SOD1 were unaltered by the experimental treatments. DEX increased (P<0.05) FN concentration in excreta compared with control birds. Conversely, inclusion of rye in the diet reduced (P<0.05) FN but increased (P<0.001) OVT in excreta. Independently, DEX decreased IAP (P<0.05) in excreta compared with control and rye-fed birds. The excreta concentration of LCN2 tended (P = 0.086) to increase in birds injected by DEX. There was no demonstrable effect of probiotic addition on any of the studied parameters. Among the tested biomarkers, FN, IAP, and LCN2 revealed promise as biomarkers of intestinal barrier function quantified by ELISA kits.

Saltbush (Atriplex nummularia and A. amnicola) as potential plants for free-range layer farms: consequences for layer performance, egg sensory qualities, and excreta moisture.

Highly adapted plant species suited to low rainfall conditions need to be considered for free-range poultry farms, particularly in marginal rainfall areas. A group of suitable plants are the saltbushes, in particular old man saltbush (Atriplex nummularia) can provide shelter, shade, and possibly forage. The first experiment examined whether hens eat old man saltbush while ranging. Free-range Hy-Line Brown layer hens were provided saltbush or "no" saltbush on the outdoor range throughout 11 wk of early production (16 to 27 wk). The quantity of saltbush eaten by the hens was determined by the n-alkane method. Hen interactions with saltbush were video recorded, and hen live weight, feed intake, egg production, and egg quality were measured. Hens ate the saltbush at 5% of their dietary dry matter intake. This level of saltbush intake had no influence on egg production. The video footage revealed hens actively pecked at the saltbush. The second experiment investigated the consequences of hens diluting their diet by eating increased levels of River saltbush (Atriplex amnicola). Air-dried, hammer-milled river saltbush was mixed and pelleted into a standard commercial layer diet at the following levels; 0 (control), 5, 10, 15, and 20%. Hy-Line Brown layer hens were fed the diets for 28 D (32 to 35 wk of age). Seventy-five hens were housed in individual cages, with 15 hen replicates per diet. The saltbush had no significant impact on egg production, hen live weight, or feed intake. Excreta moisture increased significantly (P < 0.001) with increased saltbush (5, 10, 15, and 20%) in the diet. Furthermore, high saltbush eggs (20%) had significantly stronger egg yolk color (P = 0.006) and thicker egg shells (P < 0.001) compared to the control eggs. Consumers were presented with the eggs from the second experiment and they preferred the high saltbush eggs (20%). In conclusion, saltbush would be a valuable plant on free-range poultry farms to provide shade and shelter, especially in those areas with marginal rainfall.