This body of research explores the dietary application of a bioactive, solid-state fermentation (SSF) product in contemporary and lupin-containing diets of Nile tilapia (Oreochromis niltoticus) and rainbow trout (Oncorhynchus mykiss). Consequently, the work provides holistic assessment of the influences of SSF products on animal health and performance; depending upon rearing temperature, nutritional physiologies, feed formulations and extrusion conditions; alongside information on the performance of lupins in aquafeeds. The SSF product (at 0.1 % inclusion) improved growth performance of Nile tilapia fed diets containing lupins. Phosphorous retention appeared higher when the SSF product was included in a yellow lupin (Lupinus luteus) diet whilst Mg retention was significantly higher in fish fed narrow-leaf lupin (Lupinus angustifolius). The experimental ingredients did not appear to show any clear effects upon midgut macrostructure. At an ultrastructural level, the fish fed yellow lupin alone, displayed poorest brush border characteristics but those fed yellow lupin and the SSF product showed signs of amelioration since they did not differ significantly from those fed narrow-leaf lupin. Focus was then turned towards a yellow lupin-based diet in rainbow trout, with two inclusion levels of the SSF product. The SSF product significantly improved growth performance and feed efficiency at 0.5 %, with values closer to a fishmeal-based diet than the lupin control. The SSF product increased the digestibility of protein and energy and bioavailability of numerous elements. However, the digestibility and bioavailability of certain nutrients, e.g. fibre and Zn, were only increased with a 0.5 % inclusion. The SSF product influenced vertebral Ca:P ratio but no effect on vertebral morphology was identified. Fish fed yellow lupin kernel meal displayed high Mn concentrations throughout a number of tissues. The intestinal environment was explored in depth, revealing large differences dependent upon SSF product inclusion rate. Fish fed a 0.1 % inclusion exhibited deteriorated brush border characteristics and high diversity of microbes, including increased proportions of key salmonid pathogens. Those fed a 0.5 % inclusion displayed signs of increased surface area at an ultrastructural level, reduced goblet cell numbers and a low microbial diversity; with domination of one particular family, Enterobacteriaceae. Activities of alkaline phosphatase and leucine aminopeptidase within the anterior intestine also appeared to be influenced by SSF product inclusion. Variations in haemato-immunological parameters were also observed between the treatments. In the final experiment, the SSF product was applied, pre-extrusion, to a contemporary rainbow trout formulation, at 0, 0.5, 1.0 and 1.5 %. No significant improvements to performance were identified following SSF product inclusion. However, crude protein digestibility from SSF-supplemented diets was significantly higher than the control and tendencies towards elevated retention of Ca, P, and Mg were apparent. In vitro analysis of free-phosphate release was conducted upon pre- and post-extruded diets, at varying temperatures. This indicated that neither extrusion conditions (105 °C) nor an ambient temperature of 10 °C were sufficient to cease P-liberating capabilities by the SSF product; suggesting that the two conditions combined limited the product’s efficacy in practice. This research evidenced that SSF products can be an effective means of improving the available nutrient profiles of compound diets for both omnivorous, warmwater and carnivorous, temperate finfish. Lupins are a promising alternative protein source but their nutritional value can be substantially improved by SSF product application. Exploration of the effects of SSF products on the intestinal environment revealed that both negative and positive effects on intestinal health can occur, which is highly dependent upon product inclusion rate. The holistic approaches adopted within this series of studies have seldom been performed on monogastric animals and thus provide valuable, transferable information for advancing knowledge in the application of SSF products, exogenous enzymes and lupins to farm animal feeds, in general.

Research has been actively looking for alternative feed ingredients to reduce the reliance of the aquafeed industry on marine ingredients, namely fish meal (FM) and fish oil (FO). In this context, insects, in particular housefly (Musca domestica) and black soldier fly (BSF, Hermetia illucens) larvae, have been identified as promising candidates. Although a global insect farming industry is emerging, it is for now constrained by regulatory and technical bottlenecks that raise the question ‘where and how insect-based products could be integrated into aquaculture’. The literature indicated a high interspecies variability of the results when replacing FM with insect meals in fish diets and previous work failed to consider the existing challenges related to the insect production to demonstrate commercial relevance and applicability. In this thesis, maggot meals (MM) and frass (insect digestate) were assessed as strategic feed ingredients for two commercially important farmed species: Atlantic salmon, (Salmo salar) and Nile tilapia (Oreochromis niloticus), in their relevant contexts. Case studies showed that both housefly and BSF MM are high quality feed ingredients and suitable alternative to FM. Specifically, dietary inclusions of up to 200 g/kg of crude or defatted housefly larvae meal did not compromised the feed digestibility and utilisation and the growth performance and body composition of salmon parr (freshwater stage), compared to a FM-based control diet. Hormone (17α-methyltestosterone) treated diets containing between 250 and 1000 g/kg BSF or housefly meal were found as effective as a commonly used pure hormone-treated FM in sex-reversal process leading to 99.8 to 100% males, high survival and evenness of the fish produced. In a commercial diet for advanced nursing of Nile tilapia fingerlings, up to 80 g/kg BSF meal was included without impairing the fish performance and body composition; dietary inclusion was limited by the lipid content of the crude MM. Finally, BSF frass derived from brewery spent grains or processed food wastes were found more effective when used as soil bio-fertilisers with minimum application rate of 10.0 tonnes/ha or 5.0 tonnes/ha, respectively (for a spring onion culture), rather than supplemental feeds for tilapia farmed in semi-intensive conditions (fertilised pond). The study also indicated that site-specific conditions should be accounted to support appropriate and sustainable use of insect-based products but in any case, juvenile fish should be strategically targeted given their requirements. It is expected that this approach, could support the sustainable intensification of aquaculture and contribute more broadly to food security whilst contributing to the development of a circular economy.

Aquaponics, the integrated production of fish and hydroponic crops in a recirculating system, is an intensive cultivation method in which metabolic fish wastes fertilize plants. This study compares the effects of two aquafeeds on Red amaranth (Amaranthus tricolor) productivity and on water quality under cultivation of Blue tilapia (Oreochromis aureus), with three aquaponic units (n=3) per treatment over a 60-day trial. The fishmeal-based control feed contains higher crude protein (40%) and phosphorus (1.12%) than the plant-based alternative feed (32% and 0.40%). The alternative feed resulted in a significantly higher amaranth crop yield (p

Intensive and semi-intensive aquaculture systems are dependent on nutrient input either in the form of supplemental or complete feeds. Most complete diets still include high fish meal (FM) levels (≥10%). However, as the industry attempts to reduce its reliance on FM, feeds must now be formulated with much lower levels especially for omnivorous species such as tilapia. By 2015, mean FM inclusion in tilapia diets was projected to fall below 3% and be further reduced to 1% by 2020. In the global context of competition for crops, finding suitable plant-based replacers for FM and meeting the increasing demand for seafood, lower-cost and under-utilised plant feedstuffs are now receiving greater attention. The study was divided into three distinct components – field survey, growth experiments, and life cycle assessment. Field surveys were used to contextualise the growth experiments and assess commercialisation opportunities for multi-enzyme inclusion in tilapia feeds. Two sets of digestibility and growth experiments were designed to evaluate the feasibility of using high inclusions of plant-based ingredients sourced from locally available feedstuffs in Thailand to substitute FM at low inclusion levels (0 – 5%). The research evaluated the hypothesis regarding the potential of exogenous enzymes (protease, xylanase and phytase) to minimize anti-nutritional effects on nutrient digestibility of proteins, polysaccharides and phosphorus in tilapia. The research also assessed the secondary effects of enzyme supplementation on economic efficiency and life cycle environmental impacts. Tilapia is the second most cultured finfish globally and Thailand is the sixth largest producer. Based on the findings of the field survey, feeding practices of Thai tilapia farmers were confirmed to be diverse. Feed inputs included, but were not limited to, agro-industrial by-products (e.g. rice bran, corn bran etc.) and commercial diets. Commercial diets contained 15 – 30% crude protein and lower protein livestock diets (i.e. pig ration) were often used for supplemental feeding or “fattening”. The experimental low FM diets were therefore formulated as grow-out or “fattening” diets for semi-intensive green-water systems, a prominent feature (>60%) of Thai tilapia farming. In Phase 1, the digestibility experiment assessed the digestibility and growth in tilapia fed 0%, 3% and 5% FM diets with and without xylanase (0.385 g kg-1) and phytase (0.075 g kg-1). Performance decreased significantly with declining FM levels. No differences in feed intake, feed conversion ratio (FCR), specific growth rate (SGR) and weight gain were observed between the enzyme and control diets. Nevertheless, tilapia fed the enzyme supplemented 3% FM and control 5% FM performed similarly (P < 0.05). No enzyme-related effects were noticed for protein digestibility but phosphorus (P) digestibility improved by 9%, except at 0% FM level (P > 0.05). The enzymes had no apparent influence on nitrogen (N) retention contrary to previous studies, however, higher retention for P was observed. Villus length decreased with declining FM levels yet no improvements were seen in tilapia fed enzyme diets. In a simultaneous grow-out experiment, the six experimental diets were compared to an industry 10% FM standard. Conversion ratio was the lowest (1.66) in adult tilapia fed 10% FM diet however the enzyme supplemented 0% FM fed fish had a comparatively low FCR of 1.67. There were no significant enzyme-related effects on weight gain¸ SGR and protein efficiency. Proximal villi results were inconsistent. The cost of feed decreased with declining FM levels but increased with enzyme inclusion. Nevertheless, the economic returns per kg of whole fish produced were better using enzyme supplemented diets compared to the controls. Though the size of the effects on growth and nutrient utilisation were modest, the findings suggested that xylanase and phytase had some level of synergistic action on the targeted anti-nutrients. However, further research was required. In Phase 2, two control diets (2% FM, negative control (NC) and 10% FM, positive control (PC)) were compared with three enzyme supplemented 2% FM diets (NO-PRO, 0.385 g kg-1 xylanase and 0.075 g kg-1 phytase only; LO-PRO, xylanase + phytase + 0.2 g kg-1 protease and HI-PRO, xylanase + phytase + 0.4 g kg-1 PRO). Growth performances improved with enzyme supplementation compared to the NC (P < 0.05). Of the enzyme supplemented diets, the LO-PRO diet showed the highest improvements in weight gain (26%) and feed intake (19%), the latter comparing statistically to the 10% FM PC diet. The HI-PRO diet had the best FCR (1.88), again comparable to the PC (1.73). The NO-PRO diet had the highest protein, P, lipid and energy digestibility, suggesting no additive effect of protease on these coefficients. In terms of gut histomorphology, the LO-PRO and PC diets had the highest measurements and were statistically similar which may have explained similarities in feed intake. Compared to the NC, the HI-PRO diet produced the highest level of change in net profit due to gains in feeding efficiency however, the LO-PRO showed better improvements in terms of growth. Based on these findings, the ternary combination of protease with xylanase and phytase (LOPRO) has potential in limiting FM use for tilapia grow-out feeds, however, the economic efficiencies were still below that of a 10% FM diet. Future considerations for research should target the indigestible dietary components in order to optimise enzyme dosages and maximise the benefits of each enzymes. In conclusion, a comparative life cycle assessment (LCA) was used to evaluate the environmental impacts of low FM diets and commercial feeds associated with tilapia production in Thailand. The study showed that the low FM enzyme supplemented diets had lower impact potentials and were environmental superior to the average (10% FM) commercial standard. LCA modules are recommended for least-cost formulation programmes as an option going forward. Additionally, LCA can be used as a predictive tool to guide farmers, especially small-scale producers, on the potential impacts of feed input choices and feeding practices. This will ensure higher product quality but also demonstrate environmental responsibility on the part of aquafeed and fish producers to final seafood consumers.

The most abundant organic compound produced by plants is cellulose, however it has long been accepted that animals do not secrete the hydrolytic enzymes required for its degradation, but rely instead on cellulases produced by symbiotic microbes. The recent discovery of an endogenous cDNA transcript encoding a putative GHF9 endoglucanase in the parastacid crayfish Cherax quadricarinatus (Byrne et al., 1999) suggests that similar cellulase genes may have been inherited by a range of crustacean taxa. In this study, the evolutionary history of the C. quadricarinatus endoglucanase gene and the presence of additional GHF9 genes in other decapod species were investigated. The activity of endoglucanase and endoxylanase enzymes within several cultured decapod species were also compared. The evolutionary history of the C. quadricarinatus endoglucanase gene was assessed by comparing intron/exon structure with that of other invertebrate and plant GHF9 genes. The coding region of the gene was found to be interrupted by eleven introns ranging in size from 102-902 bp, the position of which was largely conserved in both termite and abalone GHF9 genes. These structural similarities suggest GHF9 genes in crustaceans and other invertebrate taxa share a common ancestry. In addition, two introns were observed to share similar positions in plant GHF9 genes, which indicates this enzyme class may have been present in ancient eukaryote organisms. The presence of GHF9 genes in C. quadricarinatus and various other decapod species was then explored via degenerate primer PCR. Two distinct GHF9 gene fragments were determined for C. quadricarinatus and several other Cherax and Euastacus parastacid freshwater crayfish species, and a single GHF9 gene fragment was also determined for the palaemonid freshwater prawn Macrobrachium lar. Phylogenetic analyses of these fragments confirmed the presence of two endoglucanase genes within the Parastacidae, termed EG-1 and EG-2. The duplication event that produced these two genes appears to have occurred prior to the evolution of freshwater crayfish. In addition, EG-2 genes appear to have duplicated more recently within the Cherax lineage. The presence of multiple GHF9 endoglucanase enzymes within the digestive tract of some decapod species may enable more efficient processing of cellulose substrates present in dietary plant material. Endoglucanase and endoxylanase enzyme activities were compared in several parastacid crayfish and penaeid prawn species using dye-linked substrates. Endoglucanase activity levels were higher in crayfish compared with prawn species, which corresponds with the known dietary preferences of these taxa. Endoglucanase temperature and pH profiles were found to be very similar for all species examined, with optimum activity occurring at 60°C and pH 5.0. These results suggest endoglucanase activity in penaeid prawns may also be derived from endogenous sources. Additional in vitro studies further demonstrated crayfish and prawn species liberate comparable amounts of glucose from carboxymethyl-cellulose, which indicates both taxa may utilise cellulose substrates as a source of energy. Endoxylanase temperature and pH profiles were also similar for all crayfish species examined, with optimal activity occurring at 50°C and pH 5.0. These results suggest xylanase activity in crayfish may originate from endogenous enzymes, although it is unclear whether this activity is derived from GHF9 enzymes or a different xylanase enzyme class. In contrast, no endoxylanase activity was detected in the three prawn species examined. Together, these findings suggest a wide range of decapod crustacean species may possess endogenous GHF9 endoglucanase genes and enzymes. Endoglucanases may be secreted by various decapod species in order to digest soluble or amorphous cellulose substrates present in consumed plant material. Further biochemical studies may confirm the presence and functional attributes of additional endoglucanase genes and enzymes in decapods, which may ultimately assist in the design of optimal plant based crustacean aquaculture feeds.