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Aquaculture has been steadily increasing global production; however, there remains a progressively greater demand for sustainable sources of protein, whilst the prevalence of disease represents a major constraint to industry growth. Feed additives are gaining traction as useful tools to help combat disease and promote health, and previous research has documented benefits to the mucosal surfaces and microbial communities within a range of teleost species. Three experimental in vivo feeding trials were undertaken using a range of in-depth analyses, such as growth performance, haematology, immunology, intestinal histology, intestinal gene expression and intestinal bacterial 16S rRNA sequence libraries. Studies were conducted on three important aquaculture species: rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and Nile tilapia (Oreochromis niloticus). All three experiments revealed that dietary B-GOS® applications modulated the intestinal bacterial communities and intestinal gene expression. Some of these modulations may have potential benefits at the localised level for the hosts. However, no significant improvements of growth performance metrics were observed at the whole organism level, where FCR, SGR, survival, weight gain, condition factor and body composition remained unaffected by treatment. Despite the lack of statistical significance, numerical improvements indicated that GOS at the inclusion rate of 4g kg¯¹ was the optimal concentration. Results from the experimental in vivo trials conducted during this research have provided evidence that a novel GOS tested in three commercially important teleost species had scope to improve localised intestinal health but offered little benefit to growth performance of fish reared with highly nutritious diets and excellent rearing conditions. Further research should prioritise the 4g kg¯¹ B-GOS® inclusion level for further investigation in these fish species whilst under pathogenic or environmental challenge, as this concentration presented the most potential to improve fish health and growth performance.

The mucosal surfaces of fish are considered highly active immunological sites that work to maintain homeostasis and play a key role in the defence against pathogens (Yu et al., 2020). While disease represents a major constraint to the aquaculture industry, the potential influence of dietary additives upon the mucosal barriers and defences of fish is the subject of increasing amounts of research. RT-q-PCR gene expression analysis and histological examinations of experiment one jointly indicate the potential of a dietary prebiotic mixture, a parabiotic and their synbiotic to positively influence the mucosal barrier defences of the Gill-(GIALT), Skin-(SALT) and Gut- associated lymphoid tissues (GALT) of rainbow trout. Significant increases in goblet cell area fraction (CAF) were observed in the SALT, in addition to significantly elevated expressions of tight junction-, mucosal- and immunity-associated genes, within the GIALT, SALT and GALT. Similar results were observed for Atlantic salmon fed prebiotic- and probiotic-supplemented diets in experiment two: differences in goblet cell abundance and CAF were observed between treatments, while RT-q-PCR analysis revealed transcriptional regulation of mucosal- and immune-related genes. Additionally, following pathogen challenge, significantly fewer sea-lice (Lepeophtheirus salmonis) were observed on fish fed the additive supplemented diets. Investigations of experiment three were conducted on Atlantic salmon housed in open sea pens, reflective of commercial aquaculture practices. Significant reductions in sea-lice prevalence and changes to epidermal mucous secretions were observed between treatments. Furthermore, Microfluidic q-PCR analysis allowed for the evaluation of 59 gene targets within the GIALT, SALT, and GALT at two different time-points, providing a holistic view of the expression alterations occurring in response to the prebiotic and probiotic additives investigated, and the latent copepod infection. Through the implementation of in vivo feeding trials, results of the experiments conducted during this research collectively support the proposed interconnectivity of the MALT tissues in both rainbow trout and Atlantic salmon, and serve to highlight the potential of dietary additives to enhance the mucosal barrier defences and reduce sea-lice prevalence.

Fungal contamination of agricultural commodities, particularly by mycotoxigenic fungi, represents a huge concern for global food security in terms of feeding the world's growing population with sufficient and safe food. Not only do they reduce crop yield and quality, but also produce vast number of mycotoxins, which pose serious adverse health effects in human and animals. The overall aim of this thesis was to determine exposure of farm animals to multiple mycotoxins and to assess wide range of products, for their potential to bind mycotoxins. A longitudinal feeding trial (between May 2017 and December 2019) was used to investigate the occurrence and performance-related effect of mycotoxins on broiler chickens. Feed samples given to birds were collected at the beginning of each trial and analysed for multi-mycotoxins using a validated LC-MS/MS method. Furthermore, parameters including feed intake, body weight and feed efficiency were recorded on a weekly basis. Results showed that deoxynivalenol (DON), zearalenone (ZEN), fumonisins (FBs), apicidin, enniatins, emodin, diacetoxyscirpenol (DAS) and beauvericin (BEV), were the most prevalent mycotoxins. Moreover, multiple linear regression analysis also revealed an interaction amongst mixtures of DON, ZEN, FBs, BEV, and DAS, which resulted in a significant impact on broiler chickens feed efficiency. The study clearly showed that prolonged exposure of farm animals to low doses of multiple mycotoxins, below European Union guidance limits can negatively influence performance. Furthermore, three novel natural products with potential to bind or inactivate aflatoxins, DON and ZEN have been identified and characterised. These products have the potential as future technological feed additives, to reduce farm animal exposure to mycotoxins.

Given the rapid expansion of aquaculture globally and with the Litopenaeus vannamei sector making a significant contribution to seafood production, there is an urgent need to address diet and feed formulation focused on sustainability and novel ingredients, without using chemotherapeutics. This thesis explores three bioactive natural agents used in shrimp diets to mitigate the threat of disease and confer a more resilient shrimp: (i) β-glucans and yeast-derived components as functional feed additives; (ii) a blend of phytobiotic herbal extractives and yeast (YAH); and (iii) Tuna Protein Hydrolysate (TLH) as growth-promoting and immunostimulants. All studies were conducted on juvenile shrimp in cages within an open system environment under high density with fluctuating, oxygen, temperature, salinity and photoperiod. Zootechnical parameters, and indices relating to haemocyte levels for immune competence and histological assessment of the intestine, with emphasis on gut morphology and integrity, were analysed. Also, the aim was to ascertain the effect of the functional feed additives on the gut microbiota associated with their potential modulatory capacity under commercial conditions. The findings confirmed that β-glucans and yeast combinations enhanced growth and survival of juvenile shrimp. For YAH, a 1% inclusion gave excellent improvements in growth and survival. For TLH, a 2% inclusion rate gave the best overall performance. These trials revealed a marked shift in the microbial enteric community with effects on phyla but more pronounced at the genera taxonomic level. The thesis discusses these aspects for securing a more efficient shrimp production industry with a cost-benefit assessment for economic appraisal of using function feed additives in this important aquaculture sector. The results are expected to be a comparative basis for other studies, and relevant for the aquaculture industry. The findings are expected to contribute to the advance of animal nutrition and health and for the increasing of shrimp farming industry.

Experiment one revealed fishmeal (FM) replacement with soy protein concentrate (SPC) alone, and in combination with pea protein concentrate (PPC) and saponins (S) modulated the intestinal bacterial communities of D. labrax, increasing the presence of lactic acid bacteria. Intestinal histology revealed significantly reduced goblet cell’s (GC’s) in fish fed the SPC+S, epithelial microvilli densities (MD) in fish fed the SPC+PPC, SPC+PPC+S and SPC+S after two weeks feeding. Significant reductions in GC’s and intraepithelial leukocytes (IEL’s) in fish fed the SPC+S, and MD’s in fish fed the SPC+S and SPC+PPC+S after four weeks feeding, relative to fish fed the FM control. Furthermore, fish fed all plant based diets appeared to exhibit a loss of membrane integrity at the microvilli tips, most pronounced in fish fed the SPC+S diet. These results suggest a sub-acute enteritis response in the posterior intestine of D. labrax, which was deemed to be most pronounced in fish fed the SPC+S diets. Experiment two utilised the SPC+S diet as a sub-optimal basal diet to assess the potential of the probiotic Bacillus subtilis and the prebiotic Previda®, individually and in combination, in alleviating the enteritis-like effects induced by this diet, observed in the first experiment. Microbiological analyses revealed B. subtilis modulated the allochthonous bacterial communities. Fish fed the combination of B. subtilis and Previda® (synbiotic) diet exhibited a significantly increased intestinal perimeter ratio, compared to fish fed the basal. Significantly elevated GC’s in fish fed the probiotic and synbiotic treatments, and significantly elevated epithelial MD’s, and intestinal absorptive surface index in fish fed the probiotic diet was observed, relative to fed fish the basal. The loss of membrane integrity induced by the basal diet, was reduced in fish fed the probiotic, prebiotic and synbiotic diets. The intestinal gene expression of the pro-inflammatory cytokines IL-1β and TNFα was significantly up-regulated in fish fed all experimental diets, relative to fish fed the basal. The intestinal gene expression of HSP70, CASP3 and PCNA was significantly down-regulated in fish fed the probiotic, prebiotic and synbiotic relative to fish fed the basal. At the end of the experiment intestinal samples were exposed to one of four treatments [1. PBS (control), 2. B. subtilis, 3. Vibrio anguillarum and 4. B. subtilis + V. anguillarum], ex vivo, to determine if the feed additives could mitigate enteric pathogen damage. All feed additives revealed the potential to reduce the morphological damage caused by the pathogen. Experiment three assessed B. subtilis and the phytobiotic Next Enhance 150® on the growth and health of D. labrax. B. subtilis modulated the allochthonous bacterial communities and reduced the presence of some potential pathogens. The intestinal gene expression of HSP70, CASP3, PCNA and CAL was significantly down-regulation in fish fed the probiotic diet relative to fish fed the control. Significantly elevated IEL’s were observed in fish fed the probiotic and Next Enhance 150® diets relative to fish fed the control. Growth performance was remained unaffected. The present research demonstrates that dietary B. subtilis modulates the allochthonous bacterial communities, as well as, improving the intestinal morphology and localised immunity in European sea bass. Dietary Previda® and Next Enhance 150® were also observed to confer beneficial effects on the gut health of this species. No detrimental effects were observed as a consequence of any of the feed additives used in the present research.

Urgency to reduce anthropogenic sources of greenhouse gases (GHG) has substantially increased in recent years. Livestock, particularly cattle, represent a large majority of methane (CH4) generated in agriculture due to their digestive anatomy as a ruminant and their widespread presence supporting human populations worldwide. Careful manipulation of rumen fermentation and other related pathways to synthesize less CH4 could be achieved through the supplementation of feed additives with unique non-nutritional profiles. The first chapter of this dissertation outlines feed additives that either inhibit methanogenesis directly or have rumen modifying capacity to potentially produce less CH4 with a focus on in vivo models. The subsequent three chapters investigate implementing distinct additives into diets of beef or dairy cattle and their ability to suppress CH4 production along with potential impacts on performance. Various plant species are of interest as anti-methanogenic feed additives because of their phytochemical profile and ubiquity in nature. The second chapter of this dissertation evaluated lemongrass, a plant often characterized as rich in phytochemical substances, as a potential anti-methanogenic feed additive in growing beef steers. A random complete block design was organized with 20 beef steers half of which were assigned to a control diet with 0% lemongrass (CON) and the remaining 10 receiving a diet with lemongrass included at a rate of 2% of DMI (LGT). Gas production (CH4, CO2, and H2) was measured over 15 weeks. Analyses revealed that steers in the LGT versus CON group produced 9.9% more CH4 (P < 0.05) overall likely due to the displacement of high energy feed with a highly fibrous additive. Both treatment groups were similar regarding DMI, ADG, and total gain over the experiment (P > 0.05). I conclude that low concentrations of condensed tannins were responsible and no improvements in performance. The third chapter of this dissertation compared effectiveness of monensin, an ionophore, and RumenProof, a peroxidase antimicrobial, on animal performance including CH4 emissions, digestibility characteristics, blood metabolites, milk yield (MY), energy corrected milk (ECM), milk composition, and feed conversion (FCE) efficiency using a 3 x 3 Latin Square Design. Eighteen Holstein lactating dairy cattle in early to mid-lactation were blocked by milk production and parity. Dairy cows were similar across treatments for CH4 production, yield (CH4 g/kg DMI), and intensity (CH4 g/kg ECM) (P > 0.05). Milk production of cows was not affected by treatments, but milk urea nitrogen and solids non-fat tended to decrease in both monensin and RumenProof groups compared to control (P < 0.10). Digestibility of protein, starch, and NDF were unaffected by either treatment (P > 0.05). The lack of reduction in emissions with supplementation of monensin aligns with inconsistent results in the literature. Antimicrobials have previously demonstrated effects on ruminant digestibility; however, dietary conditions, length of adaptation, and dose levels are subject to cause variability in results. The fourth and final chapter of this dissertation explored the effectiveness of Amplio, a Bacillus subtilis direct fed microbial, on CH4 production and performance in lactating dairy cattle. Eighteen multiparous Holstein cows early to mid-lactation were blocked by DIM and balanced by MY and randomly assigned to a treatment of either 0 colony forming units (CO; CFU), 10 billion CFU (LD), or 30 billion CFU (HD) of B. subtilis in a 3 x 3 Latin Square Design. Animal performance, including DMI, MY, ECM and FCE were assessed, and no differences (P > 0.05) were detected due to treatment. Production of CH4 was 443, 426, and 425 (g/d) in the CO, LD, and HD group, respectively (P > 0.05). Other gases including CO2 and H2 remained unaffected by treatment inclusion of either dose of Amplio in addition to CH4 yield (g/kg DMI) and CH4 intensity (g/kg ECM). This experiment demonstrated that at these two dosages of Amplio versus CO neither animal performance nor enteric CH4 emissions were different. Lack of effects due to inclusion of B. subtilis is likely due to either lacking the ability to impact methanogenesis, carryover effects of the study design, or be a combination of a suboptimal dose and the once per day offering. Further experiments with greater levels of the probiotic and consistent treatment consumption by the animal may show better effectiveness in reducing CH4 emissions.

To secure animal welfare and maximum production, feed additives are often added to nursery pig diets to increase nutrition digestibility, enhance intestinal health, and prevent or mitigate disease, with the ultimate goal of improving growth performance. This thesis performed a longitudinal analysis using next-generation sequencing to investigate the dynamic changes of gut microbiota in weaned pigs fed commercial feed additives. Chapter II was used to evaluate the effects of organic acid mixture on growth performance and gut microbiota of weaning pigs. This study demonstrated that the inclusion of 0.035% (SBA0.035) or 0.070% (SBA0.070) sodium butyrate in a diet containing 0.5% benzoic acid improved the growth performance of weaning pigs. The SBA0.035 diet revealed the greatest gut microbial diversity and the enrichment of several potentially beneficial bacterial taxa such as Oscillospira, Blautia, and Turicibacter. The results showed that the gut community benefitted more in pigs fed this diet compared to the other diets used in this study. Chapter III evaluated the impact of peptides along with probiotic and ZnO on the growth performance and fecal microbiome of weaning pigs. Results indicated that the combination diet of peptides and ZnO can offer equivalent growth performance but at a lower cost as compared to the diet containing SDPP and ZnO. Gut microbiota analysis clearly showed that diet containing peptides plus ZnO had similar impacts on specific bacteria as dietary SDPP in combination with ZnO, which may have contributed to increased growth performance. In Chapter IV, we evaluated the growth performance and gut microbiota of weaning pigs subjected to different levels of peptides with or without the pharmaceutical level of ZnO supplementation in a nutrient‐deficient diet. Results revealed that the pharmaceutical level of ZnO conditioned the gut community to a point where the peptides could effectively restore growth performance in nursery pigs fed nutrient-deficient diets.

Research has demonstrated that supplementing exogenous enzymes to ruminants has potential to improve feed digestion and animal performance. Enzyme products with fibrolytic, proteolytic and amylolytic activities and diets with diverse composition have been used to test enzyme efficacy. Responses to these conditions have been variable. A series of in vitro experiments were designed to 1) screen enzyme products in ruminal batch culture to determine effects on digestibility and gas production and 2) further examine effective enzymes in dual-flow continuous culture to determine their effect on microbial fermentation. In Exp. 1, seven treatments, including a multi-enzyme blend (MEB), ferulic acid esterase (FAE), protease (PRO), α-amylase (AAM), β-glucanase (BGL), xylanase (XYL) or control (CON), were added to each of 3 diets with forage:concentrate ratios of 50:50 (Diet 1), 30:70 (Diet 2) or 10:90 (Diet 3), at 6 dosage rates (0, 125, 250, 500, 1000 and 10,000 mg of enzyme/kg of diet DM) in batch culture. Dose was removed from analysis and all doses were combined and examined together. In vitro total dry matter digestibility (IVTDMD) was greater (P < 0.05) for BGL compared with all other treatments in Diet 1 and Diet 2. In Diet 3, BGL was greater (P < 0.05) than AAM and CON. Control and XYL had greater (P < 0.05) total gas production than MEB, FAE, and AAM. Rate of gas production (mL/h/g of DM) was also affected (P < 0.05) by diet and enzyme for the first 24 h of fermentation with CON having the fastest rate at 3 h, generally. Overall, BGL increased digestibility without generating as much gas as CON. In Exp. 2, BGL and PRO from Exp. 1, a cellulase (CEL) preparation and control (CON) were examined in eight dual-flow continuous culture over 3 periods. The diet was a 40:60 (forage:concentrate) and enzyme was supplied at 1000 mg/kg of diet DM. Digestibility of apparent DM and OM tended to be greater (P < 0.10) for CON compared with CEL. Volatile fatty acid (VFA) concentration and nitrogen metabolism were not affected (P > 0.05) by enzyme treatment. In summary, the increase in DM and OM digestibility found with BGL and other effects of enzymes on fermentation in batch culture were not observed in dual-flow continuous culture.

Three monensin levels during a stocker phase (0, 800, 1600 g/ton in a free choice mineral) and two levels during finishing (0 [U] or 37.5 mg/kg diet DM [M]) were used to determine the effects of monensin supplementation during a stocker and subsequent finishing phase on beef calves. Steers (n = 605, BW = 278 ± 27 kg) were fed pearl millet hay with soybean hull and corn gluten feed supplement (0.5% BW daily [AF basis] Block 1) or grazed fall wheat (Block 2), spring wheat (Block 3), bermudagrass (Blocks 4 & 5) or wheat with mixed-grass baleage (Block 6). A subset of calves were transported 1,068 km to Canyon, TX (blocks 1, 2, and 4) or 636 km to Stillwater, OK (block 6) for finishing. Rumen fluid was collected on a subset of cattle (n = 30) throughout the experiment to characterize rumen microbiota by next generation sequencing. There were no treatment × diet interactions (P ≥ 0.96) during the stocker phase or location × treatment interactions (P ≥ 0.19) during finishing, therefore data were pooled for statistical analysis. During the stocker phase, cattle consuming monensin had greater (P = 0.01) final BW compared with the 0 treatment. Steers on 800 and 1600 had greater ADG (P = 0.02) and total gain (P = 0.02) than 0. Mineral intake decreased (P < 0.01) as monensin level increased. Stocker × feedlot treatment interactions were not observed for feedlot performance (P ≥ 0.50). Feedlot cattle consuming monensin had decreased (P < 0.01) DMI and increased (P < 0.01) G:F than those that did not. Cattle consuming monensin during finishing had increased (P = 0.03) fat thickness. During the stocker phase, monensin treatments had decreased alpha community (P ≤ 0.04) compared to the 0 treatment. Cattle previously on the 0 and 1600 treatments during the stocker phase and were fed monensin at the feedlot had decreased alpha diversity (P = 0.04) on feedlot d 14 compared to those that did not.

Weaning is a stressful event of newborn animals which can lead to dysbiosis in the GIT causing invasion of pathogens, retarded growth, high incidence of diarrhea, and increased neonatal mortality. Since antibiotics use in livestock production have been regulated, various feed additives have been designed as antibiotic alternatives to use in newborn animal during weaning. This thesis investigated the dynamics of bacterial composition of GIT in weaned animals fed commercial feed additives by analysis of high throughput sequencing data generated from PCR-amplified DNA targeting V1-V3 region of 16S rRNA gene. Experiment 1 investigated the rumen environment of neonatal calves fed concentrate pellet and milk replacer supplemented with a commercial blend of EO. This study demonstrated higher propionate concentrations and higher relative abundance phyla Bacteroidetes in samples from EO fed calves than the control. Two bacterial OTUs were significantly more abundant in EO fed calves; SD_Bt-00966 was found to be a close relative of Prevotella ruminicola (97%), while SD_Bt-00978 likely corresponded to an uncharacterized species of Gammaproteobacteria. Experiment 2 evaluated the impact of low inclusion of peptide-based commercial product Peptiva on the performance and fecal microbiome of weaning pigs that were assigned phase diets. Results demonstrated no significant difference in body weight (BW), daily gain, and feed efficiency between control and treatment animals. OTUs analysis revealed that Lactobacilli, represented by four main OTUs (Ssd-00002, Ssd-00019, Ssd-00025, and Ssd-00053), were more abundant at the end of Phase II (P < 0.05), while Streptococci, mostly represented by OTUs Ssd-00039 and Ssd-00048, were in higher abundance at the end of Phase III (P < 0.05). This experiment provided insight that Peptiva can modulate the composition of swine fecal microbiome during a specific window of the nursery stage, potentially by accelerating its maturation. Experiment 3 was aimed to investigate the effects of peptide based commercial product Peptiva along with mannose oligosaccharides (MOS) and protease on growth performance and fecal microbiome composition of weaned piglets on standard phase feeding program. Results revealed no significant difference on body weight on all phases, while pigs fed Peptiva added with MOS and protease at phase II showed higher daily gain and pigs fed Peptiva added with MOS had higher feed efficiency compared to control. At the OTUs level, Lactobacillus, represented by two OTUs, Ssd-00001 and Ssd-00123 were most abundant (P < 0.05) in phase III, while Ruminococcus, represented by one OTU was highly abundant (P < 0.05) in phase II. Together, these results showed Peptiva along with MOS and protease can modulate the swine gut microbiome during nursery period.

The following study was conducted to investigate the antibacterial and feed additive potential of medicinal plants. Ethanol extracts of different medicinal plants including Curcuma longa (Turmeric), Zingiber officinale (Ginger), Piper nigrum (Black Pepper), Cinnamomum cassia (Cinnamon), Thymus vulgaris (Thyme), Laurus nobilis (Bay leaf), and Syzgium aromaticum (Clove) were tested using the disc diffusion method for their antimicrobial activity against the common poultry pathogens E. coli, S. typhimurium, E. faecium, and E. faecalis. Cinnamon extract (CE), at 130 mg/disk, exhibited antibacterial activity against E. coli, S. typhimurium, and E. faecalis. Thyme extract (TE), at 30 mg/disk, exhibited antibacterial activity against E. coli, E. faecium, and E. faecalis while the remaining medicinal plants extracts showed no activity. The minimum inhibitory concentration (MIC) of the cinnamon and thyme ranged from 31.25 to 250 mg/ml by the dilution method. From this in vitro antibacterial study, cinnamon and thyme were selected for a 21-d feeding trial in broilers to study their influence on feed consumption, body weight gain, and feed conversion. There were 6 dietary treatments groups: 1) negative control (NC) containing no plant extracts or antibiotic, 2) positive control (PC) containing BMD (bacitracin) at 50g/ton of feed, 3) Diet 1 plus low level of cinnamon extract (LCE) at 290 gm/100 kg of feed, 4) Diet 1 plus high level of cinnamon extract (HCE) at 580 gm/ 100 kg of feed, 5) Diet 1 plus low level of thyme extract (LTE) at 290 gm/100kg of feed, and 6) Diet 6 plus high level of thyme extract (HTE) at 580 gm/100 kg of feed. No significant changes in body weight gain were observed with the cinnamon extracts compared to the NC or PC at 7, 14, or 21 d. The HTE reduced body weight gain compare to the NC and PC at 7, 14, and 21 d (P < 0.02). No difference in feed efficiency was observed with any of the treatments except LCE which reduced feed efficiency compared to other treatments. No difference in feed consumption was found among any of the treatments. These results suggest that cinnamon and thyme have antibacterial activity in vitro, and thyme has an activity that reduces body weight. Since cinnamon caused no significant change in body weight gain compared to positive or negative controls, it warrants further study as a substitute for antibiotics in the diet.