Necrotic enteritis (NE) is caused by the ubiquitous bacterium, Clostridium perfringens, together with one or more predisposing factors e.g. coccidiosis and high inclusion of poorly processed fish meal. Necrotic enteritis is an economically important enteric disease prevalent in the fast-growing broilers, particularly in the post-antibiotic era. Among many feed additives, organic acid (OA) blends and plant extracts (PE) are gaining increased attention in alleviating the effects of NE in broilers due to their known antimicrobial properties. However, the data in the literature are not consistent. Moreover, the potentials of these additives are not well-researched under NE challenge, both under subclinical and clinical challenge. The current thesis has examined the potentials of OA blends and PE in improving performance and protecting intestinal health of broilers under NE challenge, both subclinical and clinical.

Chapter 1 presents a brief introduction of the problem, NE control strategies, and the primary objectives to conduct this study.

Chapter 2 presents the summary of the literature related to NE and ongoing strategies to control NE outbreaks with particular emphasis on feed additives, OA blends and PE supplementation.

Chapter 3 examined the potential of blended short-chain fatty acids (SCFA) with medium-chain fatty acids (MCFA) to improve performance, health and welfare of broilers under subclinical NE challenge. The additives used were A) a blend of SCFA, MCFA, and a phenolic compound (SMP); B) a blend of free and buffered SCFA with MCFA (SMF); C) a blend of free and buffered SCFA with a high concentration of MCFA (SHM). Results showed that the birds fed SHM had higher BWG compared to the challenged control group (CHC) (P= 0.001; d 10 to 24). Birds in all 3 additive groups had lower FCR (d 0 to 35), footpad dermatitis and hock burns compared to CHC group (d 35) (P < 0.05). These findings suggest the potential of OA blends to protect birds from NE indicated by improved FCR, digestibility, and bird welfare.

Chapter 4 evaluated the effects of SCFA and MCFA blends and/or phenolic compounds on intestinal integrity, intestinal pH, caecal microbiota, and SCFA profiles of broilers under subclinical NE challenge. Birds fed all 3 additives had decreased fluorescein isothiocyanate dextran (FITC-d) concentration from gut to serum and reduced Bacteroides levels (d 16, P < 0.05) as compared to the CHC group. Birds fed additive SHM had higher concentrations of acetate and butyrate (d 21, P < 0.05) in caecal content than CHC group but were not different from SMP and SMF groups. Birds in all 3 additive groups had similar FITC-d concentrations in serum, acetate, propionate and butyrate concentrations in the caeca, and caecal bacterial loads compared to the BAC group (P > 0.05). However, the SMP group had a higher load ofC. perfringensthan zinc bacitracin (BAC) group (P < 0.05). These findings suggest the potentials of OA blends as alternatives to BAC in protecting birds from NE as indicated by improved intestinal health.

Chapter 5 evaluated the potential of monoglycerides blend (MG), a blend of mono-, di- and tri-glycerides with the main component being 1-monoglycerides, and buffered formic acid (FA) as alternatives to antibiotics on performance and intestinal health in broilers under clinical NE challenge. Results showed that birds fed MG had lower NE-caused mortality than the non-additive control (NC) group (P < 0.05). Birds fed MG had upregulated jejunal tight junction protein1 (TJP1) and immunoglobulin (IgG) gene on d 16, and improved gross energy digestibility on d 24 than NC group (P < 0.05). These findings suggest that supplementation of MG may improve intestinal health and reduce mortality of birds during the clinical NE occurrence.

Chapter 6 examined the potential of PE, a microencapsulated product composed of eugenol and garlic tincture, as an alternative to antimicrobial (AM) agent on performance and intestinal health in broilers under clinical NE challenge. Birds fed PE had improved FCR (P < 0.05) and livability (5.8%) compared to the challenged control (CC) group (d 0 to 35). Inclusion of PE reduced ileal lesion scores in male birds (P < 0.05). Birds fed PE had decreased E. maxima and E. acervulina oocyst counts in caecal content (P < 0.05). Birds fed PE had decreased E. brunetti and total oocyst counts in caecal content, and E. acervulina oocyst counts in ileal content in only female birds (P < 0.05). On d 35, PE supplementation reduced variation of BW in both male and female birds. These findings suggest the potential of PE inclusion to improve the feed efficiency and intestinal health of birds under clinical NE.

Chapter 7 investigated the underlying mode of action of PE in controlling clinical NE by evaluating intestinal integrity, duodenal histomorphology, jejunal gene expressions, ileal and caecal microbiota. Results showed that birds fed PE had reduced FITC-d, increased goblet cell numbers 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 may mitigate the negative effect of NE on intestinal health.

Super dosing copper (Cu) has long been used as an alternative to antibiotic growth-promoters in broiler chickens' diet to improve gut health. This study was designed to compare nutritional and growth-promoting levels of Cu hydroxychloride (CH) with CuSO 4 on gut health bio-markers and liver mineral profile of broiler chickens. Ross 308 chicks (n = 864) were randomly assigned to eight treatments, as basal diet containing no supplemental Cu; the basal diet with 15 or 200 mg/kg Cu as CuSO 4 ; or 15, 50, 100, 150 or 200 mg/kg Cu from CH. The highest liver Cu content was observed in birds fed the diets with 200 mg/kg CuSO 4 (P < 0.01). Serum FITC-d concentration as the leaky gut marker, and liver malondialdehyde concentration were not affected. Copper level or source had no effect on cecal short chain fatty acid and the mRNA expression of five jejunal genes involved in gut integrity. Negative linear responses of Cu were observed on ,Lactobacillus (P = 0.032), Bacteroides (P = 0.033), and Enterobacteriaceae (P = 0.028) counts. The jejunal villus height increased in birds fed CH at 200 and 100 mg/kg (P < 0.05). Increasing Cu levels, linearly and quadratically (P < 0.001), increased Cu excretion.

Published literature indicates that L-arginine (L-Arg), guanidinoacetic acid (GAA) and L-citrulline (L-Cit) can all provide arginine activity in broiler diets (Su and Austic, 1999; Dilger et al., 2013; DeGroot, 2018) and all are commercially available. GAA has been reported to have 77% arginine equivalence for feed conversion (Ringel et al., 2013). This study was conducted to determine the effect of arginine deficiency on performance in reduced protein diets and the efficacy of adding it back as either L-Arg, GAA or L-Cit. Day old male Ross 308 birds (n = 768) were assigned to 8 dietary treatments using a completely randomized design: normal protein (NP, CP levels of 24.9, 23.7 and 21.4% for starter, grower and finisher, respectively), reduced protein deficient in Arg (RP, CP levels 5% lower than those of NP diets for all feeding phases) and RP with 2 levels of L-Arg (238 and 476 g/kg), GAA (309 and 618 g/kg) or L-Cit (238 and 476 g/kg).

Trace minerals, zinc (Zn), copper (Cu) and manganese (Mn) have been well documented as essential components in the diets of poultry since they are involved in various physiological and biological processes within the body in maintaining bird health and performance. Different sources of trace minerals can differently influence growth rate and bird health. Three experiments were conducted to assess the effects of different sources and levels of trace minerals on growth performance and gut health of poultry.

The first experiment (shown in Chapters 3 and 4) evaluated the efficacy of different sources of zinc (Zn) (oxide and sulfate forms - ionic bound forms) versus zinc hydroxychloride (covalent bound form) (ZH) and incremental levels of ZH on growth performance and intestinal health of broilers fed wheat-soybean meal-based diets from d 0 to 35. The dietary treatments consisted of a positive control treatment (50 mg Zn/kg from Zn oxide and 50 mg Zn/kg from Zn sulfate), a negative control treatment (NC) which contained no supplemental Zn; the remaining 5 treatments contained Zn as ZH at 20, 40, 60, 80, 100 mg/kg. The findings obtained in this experiment showed that Zn supplemented from ZH was more efficacious than ionic bound forms in improving growth, feed efficiency, and breast meat yield. Despite the finding that feed intake was not different among treatments, the lower inclusion (40 mg/kg) of Zn as ZH resulted in similar body weight gain (BWG) to birds given a higher level of Zn as the ionic bound forms (100 mg/kg). The results also suggest that lack of Zn supplementation can negatively influence tibia development and gut microbiota composition in broilers. Higher supplemental Zn (80 mg/kg) in the diet alters the cecal microbiota population in favour of Lactobacillus and can decrease the total bacterial load. Supplemental Zn sources and levels in the feed have the potential to manipulate the jejunal gut integrity at a molecular level. The optimal requirement of Zn as ZH for broilers would be 100 mg/kg to meet all metabolic requirements of birds.

The second experiment (shown in Chapters 5 and 6) was designed to determine if nutritional (15 mg/kg) and pharmacological (200 mg/kg) levels of Cu hydroxychloride (CH) could effectively improve the growth performance and intestinal health of broilers, compared with Cu sulfate supplementation. The dietary treatments consisted of a negative control diet (NC) without any supplemental Cu, a basal diet supplemented with either 15 or 200 mg/kg Cu as CuSO4, and 5 diets supplemented with 15, 50, 100, 150, or 200 mg/kg supplemental Cu as CH. The results showed that the supplementation of Cu from CH was more efficient than Cu sulfate in promoting growth performance, both at nutritional and pharmacological levels. Birds fed 200 mg/kg Cu as CH gained more weight (77 g/bird) and had a lower FCR (3.2 points) compared to those fed 200 mg/kg Cu as Cu sulfate. This experiment demonstrated that the higher doses of Cu (up to 200 mg/kg) alter gut microbiota profile without compromising gut structural integrity or promoting tissue oxidation. Also, CH is more effective than Cu sulfate in making intestinal villi longer and reducing Cu excretion. Based on broken-line regression models, the optimal supplemental Cu as CH in the diet for the optimum body weight gain and FCR were estimated to be 109.5 and 72.3 mg/kg, respectively.

The third experiment (shown in Chapter 7) was designed to evaluate the effect of complete replacement of sulfate sources of Cu (15 mg/kg), Zn (80 mg/kg), and Mn (80 mg/kg) - (ITM) with hydroxychloride sources - (HTM) at the same inclusions on productive performance, egg quality, tissue mineralization and feed stability in Hy-Line Brown laying hens during post-peak production and under high ambient temperature. The results showed that HTM supplementation improved egg production, feed efficiency, eggshell quality, and egg mineral profile and produced darker brown eggshell color, although feed intake was not affected. These results are probably attributed to higher bioavailability of HTM compared with ITM in egg formation and pigment deposition into the eggshells, and in reducing the extent of oxidation in the feed, especially during high ambient temperature.

The results of these studies indicate that hydroxychloride trace minerals were more beneficial for broilers and layers over the ionic bound forms (sulfate, oxide) in improving growth performance parameters, intestinal health, productive production parameters, and egg quality of poultry. These effects may relate to the higher bioavailability, less reactivity with other minerals, greater oxidation stability of feed, and modulation of the negative effects of the high ambient temperature by trace minerals from hydroxychloride sources in poultry diets. The mechanism by which trace minerals as hydroxychloride contributed to lipid metabolism of broilers requires further research. An investigation into additional benefits of hydroxychloride trace mineral supplementation in post-peak laying hens could be of interest because commercial hens now lay for longer periods.

1. A study was conducted to assess the effects of supplemental protease (0 or 15,000 units/kg) and diet type (maize- or wheat-based) on apparent jejunal and ileal digestibility and apparent total tract metabolisability of nutrients in Cobb 500 mixed-sex broilers from 6 to 31 d of age.

2. Birds were randomly distributed into 56 metabolism cages (6 birds/cage" 14 replicates/treatment). At 22 d of age, jejunal and ileal digesta contents were collected and pooled from 4 birds/cage to determine apparent digestibility coefficients (DC) and digestible energy (DE). Feed intake was measured, and total excreta were collected from 8 to 11 and 18 to 21 d of age to determine apparent metabolisable energy (AME) and total tract nitrogen and starch metabolisability coefficients.

3. Broilers offered the maize-based diet with protease had greater (P < 0.05) jejunal nitrogen DC, starch DC, and DE (8.2, 6.5, and 14.9%, respectively) and ileal nitrogen DC and DE (5.1 and 6.8%, respectively) than those offered the maize-based diet without protease. Ileal starch DC was increased (P < 0.05) by 1.1% with protease supplementation.

4. Broilers offered maize-based diets had greater (P < 0.05) nitrogen (7.3%) and starch (0.6%) metabolisability coefficients and AME (4.7%) from 8 to 11 d of age, and nitrogen (4.3%) metabolisability coefficients and AME (2.0%) from 18 to 21 d of age compared with those offered wheat-based diets.

5. Thus, protease supplementation and diet type can affect digestive dynamics and nutrient utilisation in broilers.

A study evaluating apparent digestibilities of protein and amino acids and their corresponding digestion rates in four small intestinal sites in broiler chickens was completed to further investigate dietary optimisation via synchronised nutrient digestion and absorption. A total of 288 male Ross 308 broiler chickens were offered semi-purified diets with eight proteinrich feedstuffs, including" blood meal (BM), plasma protein meal (PPM), cold pressed (CCM) and expeller-pressed (ECM) canola meal, high (SBM HCP) and low (SBM LCP) crude protein soybean meals, lupins and peas. Diets were iso-caloric, iso-nitrogenous and the test ingredient was the sole source of dietary nitrogen. Each diet was offered to 6 bioassay cages with 6 birds per cage from day 21 to 28 post hatch. On day 28, all birds were euthanized and digesta samples were collected from the proximal jejunum, distal jejunum, proximal ileum and distal ileum to determine apparent protein and amino acids digestibility coefficients, digestion rates and potential digestible protein and amino acids. Dietary protein source significantly influenced energy utilisation, nitrogen retention, apparent protein (N) digestibilities, digestion rates and potential digestible protein along the small intestine. Diets containing BM and SBM LCP exhibited the highest protein digestion rate and potential digestible protein, respectively. Digestibility coefficients and disappearance rates of the majority of amino acids in four sections of the small intestine were influenced by dietary protein source (P < 0.01) and blood meal had the fastest protein digestion rate. In general, jejunal amino acid and protein digestibilities were more variable in comparison to ileal digestibilities, and the differences in protein and amino acid disappearance rates were more pronounced between types of feedstuffs than sources of similar feedstuffs.

A total of 945 male Ross 308 broiler chicks were used in a growth study to explore the interaction between dietary crude protein concentration and available phosphorus. Nine experimental treatments were constructed factorially by offering low, medium, or standard protein concentrations without or with low, standard, or high available phosphorus. Diets were based on corn, wheat, and soybean meal and all nutrients other than protein/amino acids and available phosphorus were maintained at or above breeder guidelines. Additional synthetic amino acids were used in the diets with low protein concentration in attempt to maintain digestible amino acid supply. Diets were offered to 7 replicate pens of 15 chicks per pen from day 8 to 35. Growth performance was measured during the grower (day 8–24) and finisher (day 25–35) periods. On day 35 carcass composition was determined, blood was drawn for various biochemical measurements and the tibia was excised for mechanical and compositional analyses. Birds that received the low-protein diet had lower terminal body weight and higher feed conversion ratio compared with those that received diets with adequate crude protein content. However, addition of available phosphorus to the low-protein diet resulted in significant reductions in weight-corrected feed conversion that were not evident in the diet with adequate protein content. Bone architecture was only moderately influenced by dietary treatment but birds that ingested the diets containing low and medium protein concentrations had relatively heavier abdominal fat pad weight. Blood biochemistry, especially ammonia, uric acid, and phosphorus, was influenced by both dietary protein and available phosphorus and trends suggested that both axes are involved in protein accretion and catabolism. It can be concluded that performance losses associated with feeding low protein diets to broiler chickens may be partially restored by additional available phosphorus. The implications for use of exogenous enzymes such as protease and phytase and protein nutrition per se warrants further examination.

Controlling enteric diseases of broilers is crucial. Among many additives, organic acids (OA) and their blends are gaining attention to combat diseases in the post-antibiotic era. The current study evaluated the potentials of short-chain fatty acids (SCFA) and medium-chain fatty acids (MCFA) blends and/or phenolic compounds on intestinal integrity, intestinal pH, caecal microbiota, and caecal SCFA profiles of broilers under necrotic enteritis (NE) challenge. The additives used were: (A) a blend of SCFA, MCFA, and a phenolic compound (SMP), (B) a blend of free and buffered SCFA with MCFA (SMF), and (C) a blend of free and buffered SCFA with a high concentration of MCFA (SHM). A total of 1,404 male parental chicks of Ross 308 broilers were randomly allocated to 78 floor pens on hatching day with 6 treatments replicated 13 times with 18 birds per pen. The treatments were: UCC, unchallenged control" CHC, challenged control" BAC, challenged group plus zinc bacitracin" SMP, challenged group plus additive SMP" SMF, challenged group plus additive SMF" SHM, challenged group plus additive SHM. Birds were challenged with field-strain Eimeria spp. on d 9 and Clostridium perfringens on d 14. Birds challenged with NE increased fluorescein isothiocyanate dextran (FITC-d) concentration in serum, reduced acetate and butyrate concentrations, and increased Bacteroides and C. perfringens load in the caeca (P < 0.05). Birds fed additives decreased FITC-d from gut to serum, reduced Bacteroides (d 16, P < 0.05) and numerically reduced C. perfringens load compared to CHC group. Birds fed additive SHM had higher concentrations of acetate and butyrate (d 21, P < 0.05) than CHC group but were not different from SMP and SMF groups. All the additives exhibited similar intestinal protection against NE compared to the BAC group indicated by FITC-d concentration in serum, acetate, propionate and butyrate concentrations in the caeca, and caecal bacterial loads except for the C. perfringens (P > 0.05). The SMP group had a higher load compared to BAC (P < 0.05). These findings suggest the promising effects of OA blends as alternatives to BAC to ameliorate the impact of NE challenge of broilers as indicated by improved intestinal health.