Impact of Arginine, Citrulline and Guanidinoacetic Acid Supplementation in Reduced Protein Diets for Chickens

Abstract

<p>Reducing dietary protein has received increasing interest in poultry production due to lower water intake, improved litter quality, and reduced ammonia production resulting in diminished breast blistering and footpad lesions and their sequela. Four experiments were conducted to investigate the effects of supplementing arginine (Arg), guanidinoacetic acid (GAA), and citrulline (Cit) in reduced protein wheat-based basal diets on the performance of broilers and laying hens. Chickens have a high dietary Arg requirement compared to mammalians as they have an incomplete urea cycle and therefore cannot synthesise arginine. Supplementation of arginine becomes crucial in situations where reduced protein diets are used as it has been considered as an essential component of these diets. The work completed in this thesis provides new insights into the effects of Arg, GAA, and Cit supplementation in reduced protein wheat-based diets and may help to develop optimal feeding programs for broilers and laying hens.</p> <p>The first experiment (chapter 3) investigated the effects of Arg source (Arg, GAA or Cit) and level on broiler performance, nutrient digestibility, and carcass quality. The dietary treatments were: normal protein (NP), low protein deficient in Arg (LP) and LP with two levels of either Arg (0.238 and 0.476%), GAA (0.309 and 0.618%), or Cit (0.238 and 0.476%). The LP was 5 percentage points lower in protein level than the NP. Compared to NP, birds fed LP had reduced feed intake (FI), reduced body weight gain (BWG), and increased feed conversion ratio (FCR) from day 0 to 35. Additions of Arg or Cit to the LP at both levels resulted in increased BWG and reduced FCR. Birds fed LP with GAA added had lower FCR but not higher BWG compared to the LP observed from day 0 to 35. Supplementation of Arg, Cit, and the low level of GAA to LP resulted in increased carcass yield but did not increase ileal energy or nitrogen digestibility. The findings indicate that high levels of Arg in low protein pre-starter diets may have a negative effect on performance perhaps because of marginal deficiencies of histidine or phenylalanine. The results suggest that Cit is an efficacious source of Arg in Arg-deficient diets. Supplementation of GAA to broiler diets at the low level (0.309%) was effective in sparing the Arg deficiency but at the higher level (0.618%) was toxic. This information is of importance to the industry.</p> <p>The second experiment (chapter 4) was undertaken to determine the impact of Arg deficiency on laying hen performance, egg quality, and nutrient digestibility in reduced protein diets and the efficacy of adding it to the diet as either Arg, GAA, or Cit. Treatments were: standard protein (17% protein diet; SP), reduced protein diet deficient in Arg (13% protein diet; RP) and RP with added Arg (0.35%, RP-Arg), GAA (0.46% equivalent to 0.35% Arg, RP-GAA) or Cit (0.35%, RP-Cit) to the level of SP. The birds offered RP had reduced egg and albumin weights, lower yolk color score, lower protein intake and excretion than those offered SP. When Arg or Cit was added to RP to make them equivalent in Arg to the SP, FI and egg production were not different than those of RP. The birds offered RP-GAA had decreased FI and egg mass compared to those offered RP. The addition of Arg, Cit, or GAA to the RP did not affect egg quality parameters and protein and energy digestibility. However, birds offered the RP-Cit diet tended to have higher Haugh units and lower shell breaking strength compared to all other treatments while those offered RP-GAA had higher energy digestibility than all other groups but RP. The results suggest that reduction of 4 percentage points crude protein from 17% to 13% is excessive in practical wheat-based diets for laying hens. The lack of effect for Arg, GAA, and Cit supplementation on laying performance in RP diets was possibly due to excessive reduction of dietary CP resulting in the deficiency of some essential and non-essential amino acids and other components of soybean meal in the diets. Citrulline showed more positive effects than the other sources of Arg on internal egg quality in laying hens while GAA was ineffective.</p> <p>The third experiment (chapter 5) explored the effects of Arg or Cit supplementation in broilers fed a reduced protein diet deficient in Arg under cyclic warm temperature (WT). The reductions in protein were not as drastic as the previous two experiments. The treatments were: normal protein (NP) 22.3% and 20.9% crude protein in grower and finisher, respectively, reduced protein (RP) 2.5% lower protein and deficient in Arg, and RP supplemented with 0.28% Arg (RP-Arg) or 0.28% Cit (RP-Cit). A factorial arrangement of treatments was applied during the finisher phase (21 to 35 days). Factors were: diet (four diets above); and temperature, thermo-neutral (NT, 24°C) or cyclic WT (33°C ± 1oC for 6 hours per day) with 6 replicate pens per treatment. During 7 to 35 days and 21 to 35 days, the birds fed the RP diet had lower BWG and higher FCR compared to the NP diet. The addition of Arg or Cit to RP decreased FCR compared to RP. During 21 to 35 days, the birds exposed to cyclic WT had lower FI, lower BWG but similar FCR compared to birds exposed to NT. Diet by temperature interactions were not observed for performance parameters during the period of WT. Birds fed RP had a lower yield of thigh and drumstick and higher fat pad on day 35 but higher nitrogen digestibility on day 21 compared to those offered NP. The results from this study suggest that feeding the NP diets is necessary to maintain growth performance in broilers regardless of the temperature conditions.</p> <p>Finally, the fourth experiment examined the effect of dietary protein level and the replacement of crystalline Arg with Cit in the reduced protein diet on performance, gut health, immunological parameters, and gene expression in the broilers challenged with subclinical necrotic enteritis (NE). The treatments were: standard protein without NE challenge (SP-); with NE challenge (SP+); reduced protein (2 percentage points lower crude protein) without NE challenge (RP-); with NE challenge (RP+); RP+ plus added Arg (103% of RP, RPA+) and RPC+ where supplemental Arg in RPA+ was replaced with Cit. The first 4 treatments were considered as a 2 × 2 factorial arrangement with factors being NE (- or +) and protein level (SP or RP). In addition, all 6 treatments were also analysed by one-way ANOVA. The challenge model consisted of oral inoculation of <i>Eimeria</i> strains on day 9 and <i>Clostridium (C.) perfringens</i> (EHE-NE18 strain) on day 14. The results of this experiment were written in chapters 6, 7, and 8 corresponding to three manuscripts submitted for publication. Chapter 6 investigated the effects of Arg and Cit supplementation to RP diets on growth performance, carcass traits, internal organ weights, serum uric acid, and intestinal lesion score of broilers growing under NE challenge. Feeding RP diets increased FI, increased BWG, and reduced FCR during the grower phase compared to SP diets when birds were challenged with NE. Birds in the RPC+ treatment had lower overall FCR compared to those in the SP+ treatment. Birds in the RPA+ treatment had similar FI, BWG, and FCR compared to those in the RP+ treatment. The results show protective effects of replacing the supplemental Arg with Cit against NE in RP diets as indicated by higher performance during and after challenge. Chapter 7 assessed the effects of Arg and Cit supplementation on intestinal morphology, short-chain fatty acid (SCFA), microbiota count, gut permeability, and pH in broilers fed RP diets and raised under NE challenge. Necrotic enteritis × protein interactions were detected for gut permeability as assessed by serum fluorescein isothiocyanate dextran (FITC-d) level after gavage, <i>C. perfringens</i> count in the ceca, and acetic acid and total SCFA concentrations in the ileum on day 16. Feeding the RP diet reduced serum FITC-d level, reduced the number of <i>C. perfringens</i> in the ceca, and increased acetic acid and total SCFA concentrations in the ileum compared to the SP group only in birds challenged with NE. Birds in the RPC+ treatment had greater jejunal villus height, and lower cecal <i>C. perfringens</i> and Enterobacteriaceae compared to those in the SP+ treatment. The results indicate a benefit to gut health of broilers during NE challenge when replacing crystalline Arg with Cit in RP diets. Chapter 8 investigated the effects of Arg and Cit supplementation on serum immunological parameters, serum mineral composition, and gene expression in broilers fed reduced RP diets during the NE challenge. The NE × protein interactions indicated that serum Na level decreased, and serum K level increased in birds fed the SP diets while serum Ca level decreased in birds fed the RP diets only when challenged with NE. The NE × protein interactions showed that the NE challenge downregulated the mRNA expression of jejunal y+ L amino acid transporter-2, and mucin 2 only in birds fed the RP diets. Feeding the RP decreased serum alpha-1 acid glycoprotein, ovotransferrin, expression of catenin-alpha-1 but increased expression of claudin-5 and tight junction protein genes compared to the SP. Birds in the RPA+ treatment had decreased peptide transporter-2 expression compared to the RP+ treatment (P < 0.01). The results indicate that dietary protein level and infection with NE both have an impact on immune response and expression of genes involved in immunity and nutrient digestibility in broilers.</p> <p>The findings from this study are industry-relevant and may improve performance and general health condition of broilers and laying hens fed moderately lower protein diets. Also, feeding reduced protein diets with the addition of Arg, GAA or Cit will benefit the environment by limiting nitrogenous waste from poultry production resulting from greater nitrogen digestibility. Furthermore, the results reported in this thesis may help reduce feeding of excess protein and improve performance in antibiotic growth promoter-free situations. This will benefit sustainable poultry production.</p>