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A total of five studies were conducted to determine the nutritional value of co-products of bioethanol production for poultry and pigs. The objective in the first study was to evaluate the relationship between the chemical components of maize- and wheat distillers dried grains with solubles (DDGS) as well as develop prediction equations for indispensable amino acids (IAA), total indispensable amino acid (TIAA) and total amino acid (TAA) contents using nutrient composition data available in literature. The relationship between the chemical constituents of maize- and wheat-DDGS and associated probability values were determined by correlation analysis. Prediction models for determining the IAA, TIAA and TAA contents of maize- and wheat-DDGS from their crude protein (CP) and amino acids (AA) contents were developed using step-wise multiple regression analyses. Maximum improvement in adjusted r2 (adj r2) and reduction in Mallows Cp were the model selection criteria. The chemical composition of maize- and wheat-DDGS varied among sources with coefficient of variation (CV) ranging from 8.5% to 53.5% for total P and Ca respectively in maize-DDGS and 10.5% to 36.1% for CP and acid detergent fibre (ADF) in wheat-DDGS respectively. Of the IAA, Lys, Met and Trp were most variable in maize-DDGS with CV of 13.1%, 12.0%, 10.3%, respectively, whereas Lys, Phe and Met were the most variable IAA in wheat-DDGS with CV of 20.2%, 17.3%, and 16.9%, respectively. For maize-DDGS, there were positive correlations (P < 0.05) between CP and CF, NDF, Ca, ash (r ranged from 0.45 and 0.61). Adjusted r2 ranged from 0.57 to 0.99 in the best models for predicting the IAA in maize- and wheat-DDGS from CP and AA. Except for Trp and Lys, the IAA contents of maize- and wheat-DDGS can be predicted from their CP content alone. The best models for predicting TIAA and TAA in maize-DDGS included Arg, His and Leu (adj r2= 0.98) and His, Leu and Trp (adj r2= 0.90) respectively, the regression equations being TIAA (% DM) = 0.77 + 1.36 (Arg) + 3.87 (His) + 1.99 (Val) and TAA = -3.03 + 14.1 (His) + 3.79 (Leu) + 23.4 (Trp) respectively. For wheat-DDGS, the best three variables for predicting TIAA were Arg, Leu and Val (adj r2=0.99), the regression equation being TIAA (% DM) = -0.07 + 1.11 (Arg) + 0.99 (Leu) + 5.02 (Val). Predicted values were close to actual values in the prediction models for IAA, TIAA and TAA. It was concluded that the IAA, TIAA and TAA contents of both maize- and wheat-DDGS can be predicted from their CP contents with high accuracy. In the second study, the nutritional value of wheat-DDGS without- or with exogenous enzymes for broiler was determined using three experiments. The N-corrected- and apparent metabolisable energy contents (AMEn and AME, respectively) without- or with added admixture of xylanase, amylase and protease (XAP) was determined in experiment 1, true P digestibility without- or with supplemental phytase was determined in experiment 2, whereas the apparent- or standardised ileal digestibility (AID and SID, respectively) of AA without- or with added protease was determined in experiment 3. Birds were fed a nutrient adequate pre-experimental diet from d 1 to 14 post-hatch followed by the dietary treatments from d 14 to 21 in experiment 1 and 2, or from d 25 to 28 in experiment 3, respectively. Each of the 3 experiments was arranged as a randomised complete block design consisting of 7 replicate pens and 3 birds per pen. Six dietary treatments consisting of 3 levels of wheat-DDGS (0, 300 or 600 g/kg of diet) and 2 levels of XAP (0 or 0.25 g/kg) were used in experiment 1. Six diets consisting of 3 levels of wheat-DDGS (200, 400 or 600 g/kg of diet) and 2 levels of phytase (0 or 1000 FTU/kg) were used in experiment 2, whereas four treatments consisting of a nitrogen-free diet (NFD) and an assay diet, both diets without- or with supplemental protease were used in experiment 3. In experiment 1, increasing the level of wheat-DDGS in the basal diet decreased linearly (P < 0.001) dry matter (DM) and energy retention, AME and AMEn. Supplemental XAP tended to improve both the dietary AME (P = 0.059) and AMEn (P = 0.085) values of the diet. The AME value of wheat-DDGS without- or with supplemental XAP was determined to be 15.0 or 15.5 MJ/kg, respectively. Corresponding values for AMEn were 14.0 and 14.5 MJ/kg, respectively. Supplemental XAP did not improve the energy value of wheat-DDGS for broilers. In experiment 2, increasing the level of wheat-DDGS in the diet decreased linearly (P < 0.05) ileal DM digestibility, DM retention and apparent P retention but there was no difference in apparent ileal P digestibility. Except for Fe and Zn at the ileal, and Mn and Zn at the total tract level, increasing the level of wheat-DDGS in the diet increased linearly (P < 0.05) the flow of all other minerals. Flow of minerals at the ileal and total tract level were not different with phytase supplementation. True ileal P digestibility in the wheat-DDGS for broilers was 93.6 or 96% without- or with added phytase, respectively. Corresponding values at the total tract level were 92.4 and 93.5%, respectively. Phytase addition did not improve P utilisation at the ileal or total tract level. In experiment 3, AID ranged from 33% (Asp) to 75% (Pro) without added protease whereas the range was 31% (Asp) to 82% (Pro) with protease supplementation. The AID of Lys was nil regardless of protease supplementation. Supplemental protease improved (P < 0.05) the AID of Arg and Pro and tended to improve (P < 0.10) the AID of Met. Without protease supplementation, SID ranged from 43% (Asp) to 84% (Pro) whereas the range was from 54% (Asp) to 93% (Pro) with added protease. Supplemental protease improved (P < 0.05) the SID of Arg, Leu, Phe, Met, Val and Pro by 21, 14, 13, 26, 13 and 10 percentage points, respectively. It was concluded that wheat-DDGS is a good dietary source of metabolisable energy and P for broilers. The ileal AA digestibility of wheat-DDGS for broilers is quite variable and generally low. Further, the ileal digestibility of some AA in the wheat-DDGS improved with protease supplementation. Using three experiments the third study determined the metabolisable energy content, true P digestibility and retention and AIAAD and SIAAD of wheat-DDGS for turkey. The AMEn and AME content of wheat-DDGS without- or with XAP was determined in experiment 1, the true P digestibility and retention without- or with supplemental phytase was determined in experiment 2, whereas the AIAAD and SIAAD of wheat-DDGS without- or with a protease were determined in experiment 3. Experiment 1 and 2 lasted for 21 days whereas experiment 3 lasted for 28 days. Experimental diets were fed for 7, 5 or 3 d in experiment 1, 2 or 3, respectively. Each of the 3 experiments was arranged as a randomised complete block design consisting of 7 replicate pens and 3 birds per pen. Six dietary treatments consisting of 3 levels of wheat-DDGS (0, 300 or 600 g/kg of diet) and 2 levels of XAP (0 or 0.25 g/kg) were used in experiment 1. Six diets consisting of 3 levels of wheat-DDGS (200, 400 or 600 g/kg of diet) and 2 levels of phytase (0 or 1000 FTU/kg) were used in experiment 2, whereas four diets consisting of a NFD and an assay diet, both diets without- or with supplemental protease were used in experiment 3. In experiment 1, increasing the dietary inclusion of wheat-DDGS from 0 to 600 g/kg decreased linearly (P < 0.05) DM and energy retention. There was wheat-DDGS × XAP interaction (P < 0.05) for dietary AME and AMEn. Dietary AME and AMEn values decreased linearly (P < 0.001) as the level of wheat-DDGS increased in the diets without XAP, whereas there was no effect of increasing wheat-DDGS level on dietary AME or AMEn for the XAP-supplemented diets.

The major reservoirs for most influenza A virus subtypes are wild aquatic birds, especially ducks. However, they are typically resistant to the effects of the infection and usually do not develop clinical disease. In contrast, some influenza viruses cause severe illness or even death in susceptible hosts like chickens and turkeys. Paradoxically, infection of primary duck cells results in rapid cell death, whereas in chicken cells, death occurs less rapidly. Duck cells produce fewer infectious virions in comparison with the longer surviving chicken cells. In order to understand this variation in infectious virus production, chicken and duck embryo fibroblast cells (CEF and DEF) were infected with low pathogenic avian H2N3, and the viruses produced from the two hosts ware characterised. Infectious virus production from chicken cells was significantly greater than that observed from duck cells, from 8–48 hr after infection. Influenza matrix gene and protein expression, analysed by quantitative real time PCR and western blotting of culture supernatants, showed comparable levels between species at 8 and 24 hr post infection. These findings led to investigation of virus budding and morphology following infection of duck and chicken cells with the virus. Differences in morphology of released virions were observed. Budding viruses from duck cells were elongated, while chicken cells produced almost spherical virions. There was a similar clear difference in virus morphology in the duck and chicken culture supernatants. Spherical viruses were observed in chicken supernatants while duck cell supernatants contained pleomorphic virions. No differences between any genes of chicken– and duck–derived viruses were found, suggesting that host cell determinants might be responsible for such variations in virus morphology. DEF cells showed extensive production of filamentous or short filament virions following infection with filamentous (equine H3N8) and non–filamentous (avian H2N3) virus strain, respectively. This was observed even after actin disruption with cytochalasin D (Cyt.D). CEF cells infected with equine H3N8 virus produced extensive filamentous virus, which decreased markedly after disruption of actin with Cyt.D, whereas, following infection with H2N3, spherical virions were observed in the presence or absence of the actin inhibitor. Cells were also transfected with green fluorescent protein – microtubule-associated protein 1A/1B-light chain 3 (GFP–LC3) expression vector and then infected or mock infected with avian H2N3. Short filaments were observed from untransfected and transfected duck cells, while spherical and short filaments were observed from untransfected and transfected chicken cells, respectively. Filamentous virus formation could be enhanced as a result of autophagy which is more marked in duck cells than chicken cells. Further studies such as studying the structure of chicken and duck fibroblast cell membranes, the use of other drugs that inhibit actin in a mechanistically different way, and the role of other cellular proteins in modulating virus morphology should be considered.

T cell immunoglobulin and mucin (Tim) family molecules are cell membrane proteins with four functional Tim family members in mouse, and three in human. They are preferentially expressed on immune cells with Tim1 on Th2 cells, Tim3 on Th1 cells and Tim4 on antigen-presenting cells (APCs). They have several roles, including regulating immune responses and mediating phagocytosis of dead cells. However, little is known about them beyond these two species, and nothing outside mammals. To investigate the Tim family in the chicken, the genes were identified and cDNAs cloned. Differently to mammals, the chicken genome only contains genes for Tim1 and Tim4. Chicken Tim1 (chTim1) has similar mRNA expression patterns to those of mammalian Tim1 in lymphoid tissues and immune cells. Interestingly, chTim4 has at least four splice variants – an extra short isoform (chTimeS) lacking exons 5, 6, 7 and 8, a short isoform (chTim4S) without exons 3, 4 and 5, a long isoform (chTim4L) with all exons and an extra long isoform (chTim4eL), which is similar to chTim4L but with a longer exon 3. The chTim4S is a homologue of mammalian Tim4 with constitutive expression in lymphoid tissues and immune cells; other chTim4 variants showed inducible or cell-specific expression patterns. Like mammalian Tim4, chTim4S is expressed by APCs; but differently to mammals, chTim4S is also expressed by γδ T cells, suggesting a unique role for chTim4 in this population of T cells. The biological activities of the chicken Tim family molecules were also investigated using chTim-Ig fusion proteins. Like mammals, chTim1 and chTim4S fusion proteins can specifically recognise phosphatidylserine (PS), an indicator of apoptotic cells, suggesting they are PS receptors. Pre-incubation with PS blocked binding of the chTim4S fusion protein to PS-exposing apoptotic cells. Physiologically, recognition of PS by the chTim proteins mediates apoptotic cell clearance, which was demonstrated using chTim-transfected fibroblast cells (3T3), which significantly increased their uptake of apoptotic cells compared with untransfected cells. The chTim4-Ig fusion protein also had costimulatory activity on chicken T cells. Monoclonal antibodies against the chTim proteins were generated. They specifically recognise their own antigen tested intensively by different immunological assays. ChTim4L is expressed intracellularly in freshly-isolated splenocytes rather than on the surface, whereas PMA-stimulated splenocytes express chTim4S and chTim4L on the cell surface. Like mammals, chicken splenic macrophages also express chTim4S and chTim4L. Both of them are also expressed by bone marrow-derived macrophages but not bone marrow-derived DCs. The chTim1 protein was detected at high levels in bursal cells and splenocytes by western blot analysis using polyclonal anti-chTim1 serum, which is consistent with its mRNA expression pattern through qRT-PCR analysis, suggesting B and T cells may express chTim1, consistent with its expression in mammals. Mammalian Tim1 is expressed on Th2 cells, its ligand, Tim4, on APCs; the interaction between them drives Th2 cell proliferation. The knowledge from this study will help to further dissect how the chicken’s Th2 responses are regulated through cell surface molecules.

The poultry industry has successfully applied breeding and production programmes to meet growing consumer demands for chicken meat and eggs. Over the last four decades, poultry breeders have selected birds not only for productivity, but also for improved health, welfare, fitness and environmental robustness. Intensive production settings contribute to faster spread of diseases and greater losses in production due to increased morbidity and mortality of the flock. Traditional methods of disease treatment and prevention have played a critical role in control of disease. However, growing resistance of pathogens to therapeutic measures and consumer concerns led to the withdrawal of antibiotics as growth promoting additives in chicken feed. In addition, some vaccines have been overcome by increasing variation and virulence of pathogens and are no longer successful in disease prevention. The emergence of virulent and drug resistant pathogens have emphasised the need to focus on other solutions to disease, particularly natural genetic resistance. Genetic loci or gene expression patterns associated with the differential resistance of lines to specific pathogens have been identified, providing valuable markers for selective breeding. However, to date relatively few of these have been successfully incorporated into commercial lines. An ability to suppress or resist multiple pathogens, by selection for improved innate immune robustness has also been studied but it has not been introduced in commercial production, partly as the phenotype is ill-defined. Previous studies that focused on pro-inflammatory cytokines and their mRNA levels expressed by innate immune effector cells (heterophils and macrophages) identified differences between resistant and susceptible chicken lines, with the former producing stronger responses, supporting efforts to select poultry with an efficient early innate response. Here, small-scale qPCR screening and cellular techniques were evaluated with the conclusion that a more rapid, cheaper and reproducible method needs to be applied. The main objective of this project was therefore to design and validate a diagnostic tool that could be used to phenotype the immune responses of chickens at the level of innate immunity. For this purpose, a panel of 89 genes was selected based on previously published infection studies and on RNA-seq results obtained from stimulation of heterophils, macrophages and dendritic cells with lipopolysaccharide (LPS). Target genes were cloned and sequenced to optimise the design of qPCR reactions and primers. A multiplex qPCR platform, the Fluidigm 96.96 Dynamic Array, was selected as the tool of choice with the capacity to measure transcription of 96 genes of interest in 96 samples simultaneously. The preamplification reaction was optimised and the platform validated using a commercial line of chickens housed in clean or pathogen-challenged environments. Lymphoid tissues, including bursa of Fabricius, spleen, ileum with Peyer’s patches, caecal tonsils, and blood leukocytes were isolated and transcript levels for immune-related genes defined between organs, birds and farms. For qPCR analysis, a panel of reference genes was normalised and TBP, ACTB and GAPDH genes were selected and validated as the most stable. The high-throughput qPCR analysis identified peripheral blood leukocytes as a potentially reliable indicator of immune responses among all the tissues tested with the highest number of genes significantly differentially expressed between birds housed in varying hygienic environments. The research described here could potentially aid the selection of poultry for improved immune robustness. The technical optimisation and validation of a new tool to simultaneously quantify expression of tens of relevant immune-related genes will prime research in many areas of avian biology, especially to define baseline immune gene expression for selection, the basis of differential resistance, and host responses to infection, vaccination or immuno-modulatory substances.

Genetic variation between individuals is of great importance for the development of breeding programmes, to select for animals with the most favourable traits. Many production companies routinely measure the feed intake of their animals, in order to calculate efficiency traits such as feed conversion ratio. The development of electronic feeders which automatically record individual intake on a visit-by-visit basis now allows the short-term feeding behaviour of animals to be monitored and analysed as another source of variation between individuals. Due to differences in the resolutions of these feeders as a measurement tool, a standard unit of feeding event needs to be estimated to allow for comparisons between studies. Different models for estimation of the defining value of a meal, the meal criterion, have been used, with the most recent incorporating the change in satiety with time since last feeding as part of the model. In this study I developed a new methodology, based on these models, for use when a within meal population of intervals cannot be easily modelled. I then used this model for application to data from four lines of broiler chickens to estimate meal criteria and compare feeding behaviour within and between the lines. Significant differences were found between fast and slow growing birds, with the faster growing birds having fewer but larger meals than the slower growing birds. However, the lines showed similar structure and bouting of their feeding behaviour, indicating that the fundamental controls of feeding behaviour, such as hunger and satiety, in these lines had been unaltered despite intensive selection for growth. The models were also applied across poultry species, kept in different experimental conditions. A similar structure to the feeding behaviour was found across all these species, with all showing clear separation of feeding events into bouts. In order to estimate the potential use of these behavioural observations in a breeding programme, the heritabilities and genetic correlations with existing performance traits were calculated for the four broiler lines. Heritabilities of all feeding behaviour traits were found to be moderate to high, and very similar across the lines. Correlations with performance traits, however, were low, meaning that there were no clear links of the traits with the current production goals investigated. This indicates that past selection for production has had limited impact on feeding behaviour and also that potential selection for feeding behaviour will have little effect on production gains. To identify the areas of the genome controlling feeding behaviour, traits were associated with a SNP panel. Many regions were found to have highly significant association with feeding behaviour traits, with the most highly correlated traits showing associations with the same regions, suggesting pleiotropic effects of genes in these regions. Future work in this area should include identification of individual genes controlling feeding behaviour to allow prediction of the effects of selection for favourable feeding behaviour on other traits, and comparison of the genotypes of different lines of broilers, to further understand the control of feeding behaviour.

This thesis is based on the evidence of the use of range by laying hens from two separate studies that were carried out on commercial flocks. The first study involved a direct observation of 6 flocks of laying hens whereas the second study was carried out on a single flock of laying hens using a still digital camera. Mapping of the outdoor range into three zones, namely; apron (0-10metres), enriched (10-50metres) and range (beyond 50metres), was carried out in both studies determine whether it influenced the distribution, behaviour, nearest neighbour distance (NND) and feather condition of the hens. Based on the estimates of the total number of hens recorded outdoors, it was found that 14.5% of the hens used the range in the first study, with 6.0% of the hens seen in the apron, 4.8% in the enriched and the remaining 3.7% in the outer range zone. This ranging pattern was further confirmed by the quadrat head counts, where the hens used the apron most but showed a significant decrease in the use of other outdoor zones as distance increased from the shed (p < 0.001). The results of the two studies showed that range use peaked in the morning and decreased significantly in the afternoon (p < 0.001). Range use was found to increase significantly with age (p < 0.001) in the first study, although there was a significant decrease in the number of hens outside the shed as the flocks got larger (p < 0.001). The age effect reported in the six-flock study was complemented by the significant positive effect of the week of outdoor access on the ability of the hens to use the range (p < 0.001) in the single flock study. Range use increased as outdoor temperature rose in the six-flock study whereas increased outdoor temperature resulted in a decline in range use in the single flock study. NND of the hens was ii measured in the six-flock study and was found to decrease significantly towards the shed (p < 0.001) and as the hens aged (p < 0.001). There was evidence that the use of distant and less crowded outdoor locations by the hens in the first study was associated with improved feather conditions. The evidence presented in the two studies presented in this thesis showed that the hens were unevenly distributed in the range, with reduced hen density (hen/m2) towards the outer range and that an improvement in the use of distant parts of the range may have beneficial effects on the hens.

Necrotic enteritis (NE) is an important enteric disease in poultry production that has re-emerged as a major problem following an EU wide ban on the use of in-feed antimicrobials. Although the primary aetiological agent of disease is Clostridium perfringens type A, a commensal in the gastrointestinal tract (GIT) of chickens, numerous additional influential factors have been reported that can predispose chickens to NE. These precipitating factors mainly include diet, co-infection with other pathogens particularly coccidia, as well as environmental and management factors. Despite being first described almost more than 50 years ago, a reliable, consistently reproducible experimental model for NE induction is still lacking. Here, a series of experiments were conducted to investigate the importance of the various potential predisposing factors, in isolation and in combination, that are believed to play a role in sub-clinical NE development: feed withdrawal, dietary protein sources, co- infection with coccidia, C. perfringens dose and contact with reused litter. In addition, chicken breed sensitivity to NE was assessed, and last, but not least, blood and gut tissue samples were used to identify novel biochemical markers for sub-clinical NE. Chapter 3 reports an experiment showing that feed withdrawal up to 24 hrs in experimentally challenged birds did not result in NE specific lesions. Chapter 4 shows that replacing dietary soyabean meal with potato protein concentrate or canola meal or adding synthetic trypsin inhibitor to the soyabean meal control diet did not induce sub-clinical NE in birds housed on reused litter, a natural source of C. perfringens challenge. Chapter 5 describes that in vitro growth of C. perfringens on in vitro digested grower diets was prolonged following the addition of fishmeal, suggesting that the role of fish meal as a predisposing factor for in vivo sub-clinical NE cannot be excluded. All subsequent diets therefore contained high levels of fish meal. When this was used in combination with high dose of coccidial vaccine, a repeated in-feed challenge for three days at 102 colony forming units (cfu) C. perfringens per g feed did not result in sub-clinical NE, though at 109 cfu/g resulted in 10% of challenged birds (3 out of 30) showing NE-specific lesions (Chapter 6). Further study is needed to determine if the two Ross birds with gross NE lesions compared to the one Hubbard bird (out of 15 birds each) was due to a lower level of NE resistance. 3 The failure to significantly induce sub-clinical NE in the previous experiments suggests that challenging the birds with C. perfringens in the isolated presence of suspected predisposing factors may not provide a suitable experimental model. Indeed, when birds were dosed twice daily with 108 cfu C. perfringens for three days in the presence of high levels of fishmeal, canola meal as main protein source, coccidial and IBD vaccinations, and feed withdrawal prior to challenge, 40.6% of the challenged birds developed lesions of sub-clinical NE without inducing mortality (Chapter 7). This concurred with reduced growth performance relative to the sham-infected control birds, and thus is a successful model for induction of sub-clinical NE. Finally this work has, for the first time provided novel information on potential biomarkers (Chapter 8). Whilst challenge did not impact on the expression of genes previously shown to be differentially expressed upon C. perfringens toxin exposure, the serum ceruloplasmin concentration increased, suggesting that monitoring this acute phase protein may indicate the presence of C. perfringens infection in poultry. However, as such markers generally lack specificity, further research confirming its role in response to sub-clinical NE is needed to provide a fully effective diagnostic and prognostic marker for flock health and welfare, as well as ultimately helping to gain better understanding of the pathophysiology of sub-clinical NE. Improved knowledge of the effect of different dietary components on the growth of C. perfringens may help in the formulation of broiler diets to assist in further reducing the incidence of NE particularly in the absence of antimicrobial growth promoters. It is hoped that host responses in terms of acute phase proteins, and possibley gene expression, will also provide greater insight into the pathogenesis of NE. Provided that the developed experimental sub-clinical NE model is reproducible, this will benefit the understanding of this billion dollar disease and enable further investigation of various chemical and non-chemical interventions to reduce its severity and impact on poultry production.

Four studies consisting of 6 experiments were conducted to investigate the likely mechanisms of actions of feed additives used in the place of antibiotics to improve performance and enteric health in broilers. In the first study, the relationship between digesta pH, body weight and nutrient utilisation in broilers of the same breed but with different propensity for weight gain at different ages were investigated. It was noted that birds in group H (heavy) consumed more feed (P < 0.001) than those in group L (light) during the starter (day 10 to 14) and grower (day 15 to 28) phases. Birds in group H had lower (P < 0.05) caecal pH in the starter and grower phases and lower (P < 0.05) proventriculus pH in grower and finisher (day 29 to 42) phases. In the grower phase, caecal pH was correlated (r = 0.553) with total tract retention of DM and energy utilisation at both the ileal and total tract levels, whereas during the finisher phase, crop pH was correlated with ileal nutrient utilisation, and jejunum pH was correlated with total tract energy utilisation. The data showed that differences in body weight are also reflected in differences in gut pH which is likely indicative of differences in intestinal condition between birds with heavier or lighter body weight. The differences in the gut pH explained about half of the variations in total tract nutrient and energy utilisation. Lower gut pH is advantageous for beneficial bacteria colonization but disadvantageous for pathogenic ones colonization and hence it is likely that birds with the same genetic potential may have differences in growth performance based on the type of bacteria colonizing their gut. In the second study, the response of broiler chickens to the supplementation of benzoic acid (BA) was investigated using growth performance, nutrient and energy utilization, intestinal acidity and histomorphology of the intestine as response criteria, using 945 Ross 308 male broilers in 3 treatments with 7 replicates each for 42 days. In the grower (day 11 to 21) phase, BA supplementation at 0.53 g/kg (BA1) increased (P < 0.001) body weight gain and reduced (P < 0.01) FCR, whereas supplementation at 3.20 g/kg (BA2) reduced (P < 0.005) the feed intake without affecting the body weight gain, resulting in a better FCR. Compared with the control, BA supplementations increased (P < 0.001) the protein and energy efficiency ratios in starter and grower phases and tended (P < 0.10) to increase the energy efficiency ratio in finisher phase (day 22 to 42) or the overall experimental period (day 0 – 42). BA supplementations at both rates reduced the caecal pH. Supplementation of 3.20 g/kg BA stimulated the proliferation of the absorptive cells in the jejunum, as shown in the improvement of the villus and crypt dimensions. The data from this study indicated that dietary supplementation of BA beneficially modified intestinal milieu and improved the growth performance of broiler chicks at 42 d of age. In third study, two experiments were conducted to investigate the benefit of using BA and turmeric meal (TM) individually or in combination using growth performance, nutrient utilization, and intestinal health as response criteria. A total of 300 male one-day old broilers (Ross 308) were assigned in 5 treatments in randomized complete block design with 2x2 + 1 factorial arrangement, with 6 replicate pens and 10 birds each pen. Combination of 1 g/kg BA and 5 g/kg TM improved (P < 0.05) body weight gain relative to the control. Supplementation of 2 g/kg BA reduced the pH in the crop (P < 0.001) and jejunum (P < 0.01), whereas combination of BA and TM at 2 and 10 g/kg respectively reduced digesta pH in the crop (P < 0.001), jejunum (P < 0.01), and caeca (P < 0.05). All of the dietary treatments increased (P < 0.005) villus height, crypt depth and width relative to the control. All dietary treatments increased (P < 0.001) AME and AMEn relative to the control diet, whilst supplementation of 10 g/kg TM only increased energy digestibility (P < 0.05) and ileal digestible energy (P < 0.01). Orthogonal contrasts showed that BA and TM were additive in their effects on the growth performance, digesta pH in the proventriculus, jejunum, and ileum, and energy utilization, but associative on the energy digestibility, as well as the crop and caecal pH. None of the treatment altered the relative weight and length of the digestive tract of 21 days old broiler. These studies pointed out that BA and TM can be used in the diet individually or in combination to improve the enteric health, nutrient and energy utilization, and growth performance of broiler chickens. In the fourth study, two experiments were designed to investigate the efficacy of TM and garlic meal (GM) using growth performance, intestinal pH, and energy and nutrient utilisation as response criteria. Three hundreds male one-day old broilers (Ross 308) were assigned in 5 treatments in randomized complete block design with 2x2 + 1 factorial arrangement, with 6 replicate pens and 10 birds each pen. Results showed that combination of GM and TM at 10 g/kg each increased (P < 0.05) the body weight gain, final body weight, and gain to feed ratio relative to the control and the diet with GM supplementation alone. The crop and caecal pH were reduced (P < 0.05) when the diets were supplemented with TM alone at 10 g/kg. The proventriculus pH also dropped (P < 0.05) following GM and TM mixture supplementation at 10 g/kg each relative to the control. Supplementation of 10 g/kg TM alone or in combination with GM at 5 g/kg each increased (P < 0.05) the apparent ileal energy digestibility and ileal digestible energy. All of the dietary treatments increased (P < 0.001) both AME and AMEn compared with the control. Orthogonal contrasts showed that GM and TM were additive for feed intake, nutrient and energy utilization at both the ileal and total tract levels, but associative in their effects on body weight gain and gain to feed ratio. These studies indicated that GM and TM can be used alone or in combination to support intestinal health, improve energy and nutrient utilization, and stimulate growth performance of broiler chickens. Combination of GM and TM at the rate of 5 g/kg each was optimum for enhancing nutrient and energy utilization and promoting growth performance of broiler chickens. Taken together, these studies showed that benzoic acid and herbal products (garlic and turmeric meal) can be supplemented in the diet alone or in combination to improve the enteric health, nutrient and energy utilization, and growth performance of broiler chickens. Improvements on the growth performance might be attributed to the reduction of the entero-pathogens in the gut, enhancement of intestinal health, alteration of the absorptive cells in the intestinal wall, and improvement in the nutrient and energy utilization.

The overall objective of this thesis was to study the effects of regular and high (super-) doses of phytase in the gut of broilers, with the aim of documenting the mechanism of their action leading to improvements in animal health. Phytase is often supplemented to commercial broiler diets to facilitate the hydrolysis of plant phytate and release of phosphorus for utilisation. Although not the original intention of its addition, phytase supplementation leads to improvements in growth performance parameters and enhanced nutrient utilisation. Further benefits have also been observed following the addition of super-doses of phytase which are not explained by an increase in phosphorus release, and thus have been termed ‘extra-phosphoric effects’. Using diets formulated to be adequate or marginally deficient in available phosphorus (aP) forming the negative control, NC), phytase was supplemented at 1,500 and 3,000 FTU/kg phytase in the first study (both super-doses) and the partitioning of nutrients within the body was investigated. It appeared that there were some metabolic changes between 1,500 and 3,000 FTU/kg, switching between protein and fat accretion, potentially as a consequence of nutrient availability, although these changes were not reflected by changes in growth performance parameters. However, the loss of the NC treatment without phytase on day 12 limits the comparison of the phytase within the NC treatment, but does allow for comparison of each dose at adequate or low dietary aP levels. As expected, a greater degree of phytate hydrolysis was achieved with 3,000 than with 1,500 FTU/kg phytase, but changes in carcass accretion characteristics were greater with 1,500 than 3,000 FTU/kg. Using these findings and the observation that there were no further changes in the parameters measured by increasing phytase from 1,500 to 3,000 FTU/kg (aside from phytate hydrolysis), 1,500 FTU/kg phytase was selected as the super-dose to be used in subsequent studies. The next study considered the influence of regular (500 FTU/kg) and super doses (1,500 FTU/kg) of phytase from within the gut. Overall, it was observed that changes were occurring to the gut environment, which ultimately would influence the absorptive capacity and conditions for further phytate hydrolysis. Dietary treatment influenced gut conditions such as pH, intestinal morphology and bacterial populations which can subsequently influence nutrient utilisation and potential for growth. The subsequent study was designed to investigate the effects within the gut in more detail. The release of nutrients from phytate hydrolysis and their bioavailability within the digesta can influence conditions within intestine, facilitating enhanced absorption. One of the parameters investigated was the expression of genes involved in the transport of nutrients in the intestine. Overall, there were few significant dietary treatment influences on gene expression in the intestine, however there was a dose-dependent response of phytase on the expression of the jejunual divalent mineral transporter. This indicates a change in divalent mineral bioavailability in the intestine, with correlations with inositol phosphate esters (IPs) being identified. This is likely explained by the IPs produced by phytase hydrolysis and accumulating in the digesta, differing between regular and high doses of phytase. It became apparent that interactions between the products of phytate hydrolysis (IP3, IP4) and minerals in the digesta had the potential to influence the gut environment and subsequent nutrient bioavailability and overall phytase action. The final study was designed to increase the content of the IPs, and investigate the influence of phytase under these conditions. As the complete hydrolysis of phytate to myo-inositol has been reported to be beneficial due to its proposed insulin mimetic effects, myo-inositol was also supplemented to one of the diets to see if any further benefits would be observed when supplemented alongside super-doses of phytase. Neither increased concentrations of the higher IP esters (IP6, IP5 and IP4) nor myo-inositol (myo-) had any effect on broiler growth performance, however there were still apparent beneficial influences of phytase supplementation. The results suggest considerable and important interactions between minerals and IP esters within the digesta, which ultimately have the potential to influence gut conditions and thus nutrient utilisation and growth performance. Reduced concentrations of blood glucose in the high IP ester diet with additional phytase supplementation suggest some insulin-like effects of myo- production. Additionally, the lack of effect of myo- supplementation on blood glucose and insulin concentrations suggests a difference between the structure of phytase-produced myo- and supplemented myo-. Although there were no improvements in growth performance by increasing phytase from 500 to 1,500 FTU/kg, there were changes occurring at the level of the gut and expression of genes in the intestine, influencing nutrient utilisation and the partitioning of nutrients within the body. There are many factors to be considered when supplementing phytase, with dietary nutrient content and nutrient release and IP production during phytate hydrolysis having an influence on phytase action, nutrient absorption and conditions within the gut. Super-doses of phytase may be beneficial for maintaining optimal gut conditions, clearing IP esters from the digesta, reducing their potential to form complexes with minerals and other nutrients, ultimately influencing the efficiency of production.