Effects of feeding yeasts on blood physiology and gut microbiota of rainbow trout

Yeast represents a sustainable protein alternative to fishmeal in diets for farmed fish, although more than 40% replacement has been shown to reduce fish growth and welfare. This thesis investigated the effects of feeding high inclusions of inactivated and live yeasts to fish in order to replace fishmeal without negative health consequences. The specific focus was on red blood cell characteristics, plasma amino acid uptake, gut microbial communities and stress/immune responses of rainbow trout (Oncorhynchus mykiss). Post-prandial blood samples were collected from dorsal aorta-cannulated rainbow trout fed diets in which 60% fishmeal protein was replaced with two yeast species, Saccharomyces cerevisiae and Wickerhamomyces anomalus, inactivated by heat extrusion. Blood analysis showed that feeding both yeasts resulted in higher blood pH and haemoglobin levels, which were associated with lower buffering capacity of yeast and possible haemolytic anaemia from metabolism of high levels of nucleic acid. Plasma analysis revealed that amino acid uptake was similar in fish fed both yeasts and fishmeal, except for higher methionine in fish fed yeasts attributed to higher supplementation. In a later study, fish were fed live S. cerevisiae and reared at 11 and 18°C. No adverse effects on blood physiology were found, although most cells survived digestion and were not metabolised. These results indicate that reduced growth in fish fed yeast may not be due to lower amino acid content, but rather to metabolism of high levels of nucleic acid leading to impaired red blood cell function. In separate studies, fish were fed inactivated yeasts that replaced 20, 40 and 60% of fishmeal protein and fish kept at 11 and 18°C were fed 40% replacement with live yeast. High-throughput sequencing of the distal gut revealed that inactivated W. anomalus affected bacterial diversity and abundance, while both inactivated and live S. cerevisiae had minor effects. Increased temperature reduced the abundance of lactic acid bacteria and reduced bacterial diversity. In both studies, Debaryomyces hansenii, S. cerevisiae and Rhodotorula spp. were naturally present in the fish gut and feeding live yeast, but not inactivated, increased the gut yeast load. Fish at 18°C had higher plasma cortisol levels and suppressed expression of inflammatory cytokines, which were further suppressed when fed live yeast. This suggests that increased temperature subjected fish to chronic stress and that feeding live yeast may impair the innate immune response. In conclusion, this thesis suggest that impaired red blood cell and immune function are key factors reducing growth and welfare of rainbow trout fed yeast and managing these factors may enable sustainable replacement of fishmeal

Evaluation of membrane filtration and UV irradiation to control bacterial loads in recirculation aquaculture systems

Ultraviolet (UV) irradiation is commonly used to control pathogen loads in recirculation aquaculture systems (RAS), although these micro-organisms can be shielded by particles in the water, and some species tolerate very high UV doses. The objective of this study was to evaluate membrane filtration (MF) as an alternative, or complimentary, treatment to UV irradiation for pathogen control in RAS, as well as examine the operation and cost of each treatment. In a pilot-scale RAS, both MF and UV were used to treat wastewater for 30 days and water samples were collected biweekly and analysed for culturable bacteria, suspended solids, UV transmittance and other parameters. Bacterial control efficiencies were similar between both MF and UV treatments, which removed 99% of total bacteria and 98% of heterotrophic bacteria, respectively. Surface fouling was negligible for the UV while MF required biweekly cleaning to maintain operation. However, MF had the additional benefit of removing 96% of suspended solids, which resulted in increased UV transmittance. Capital and operating costs of MF were similar to UV, but only when MF treated a fraction of the wastewater compared with UV. We conclude that MF represents a potential complimentary technology to enhance UV irradiation, especially to minimise pathogens in RAS that are shielded by particles or tolerate UV

High-throughput sequencing of gut microbiota in rainbow trout (Oncorhynchus mykiss) fed larval and pre-pupae stages of black soldier fly (Hermetia illucens)

Black soldier fly (Hermetia illucens) meal is a potential alternative to fishmeal and plant proteins in diets for farmed fish since it can be produced on organic waste substrates, requires little energy and water inputs and contains high levels of essential amino acids. Recent studies have partially replaced fishmeal with black soldier fly meal, however, research on their impact on gut microbiota of fish is limited. In a five week experiment, juvenile rainbow trout (Oncorhynchus mykiss) were fed either a reference diet based on fishmeal or three diets with 30% inclusion of black soldier fly meals in the form of pre-pupae, larvae or defatted-larvae. The combined luminal content and mucosa were collected from the distal intestine of three fish per tank with four tanks per diet (n = 12) and 16S rRNA gene amplicons were sequenced using the Illumina MiSeq platform. Feeding the insect-based diets increased the alpha-diversity of bacteria and abundance of lactic acid bacteria, which may be due to the addition of dietary chitin. Compared with fishmeal, feeding insects resulted in higher abundance of phyla Firmicutes and Actinobacteria with lower abundance of Proteobacteria. Fish fed the full-fat meals had higher abundance of Corynebacterium that was attributed to its ability to produce lipase and the high content of dietary lipids as a substrate. Bacillaceae was increased in fish fed both larvae diets and unchanged in the pre-pupae diet, which indicated that life-cycle stage of the insect influenced the gut microbiota. Based on these results, we found that feeding black soldier flies increased diversity and altered the composition of gut bacteria of rainbow trout, which were further influenced by life-cycle stage and lipid content of the insect meal

Haematological and intestinal health parameters of rainbow trout are influenced by dietary live yeast and increased water temperature

Live yeast may be a sustainable protein source in salmonid diets while exhibiting a probiotic effect to counteract environmental stressors, such as increased water temperature that is being exacerbated by climate change. The objective of this study was to evaluate the effects of feeding a high dietary inclusion of live yeast and increased water temperature on growth, haematological and intestinal physiology of rainbow trout. For six weeks, 129 g fish in 16 tanks (n = 4) were fed either a diet based on fishmeal or based on live yeast (214 g kg−1 of diet or 7.6 log CFU g−1 of Saccharomyces cerevisiae) that replaced 40% of fishmeal protein while fish were reared in water temperatures of either 11 °C (cold) or 18 °C (warm). Fish weights, caudal blood and proximal and distal intestines were collected and analysed. Fish fed live yeast resulted in reduced growth (SGR and WG) and higher FCR, while growth in cold and warm water was similar despite differences in TGC. However, increased mortality, plasma cortisol, and intestinal oedema and villous damage indicated fish reared in warm water were subjected to chronic stress. Temperature had a significant effect on haematocrit and red blood cell counts that resulted in significantly higher haemoglobin levels in fish kept in warm water attributed to an elevated oxygen demand. In the proximal intestine, increased temperature resulted in reduced expression of pro-inflammatory cytokines, e.g. TNFα and IL8, that were further reduced in fish fed live yeast. In addition, feeding live yeast reduced gene expression of CLD6 involved in gut barrier function, which suggests that the level of yeast was too high and masked any beneficial effects on fish health. In conclusion, feeding a high inclusion of live yeast reduced fish growth and expression of intestinal genes, while increasing the temperature from 11 to 18 °C subjected fish to chronic stress that restricted growth, suppressed innate immunity and induced intestinal damage. Replacing 40% of fishmeal protein with live yeast did not counteract negative effects caused by increased temperature, thus alternative strategies need to be explored and implemented to protect the growth and health of rainbow trout from seasonal and long-term rises in water temperature

The application of single-cell ingredients in aquaculture feeds-a review

Single-cell ingredients (SCI) are a relatively broad class of materials that encompasses bacterial, fungal (yeast), microalgal-derived products or the combination of all three microbial groups into microbial bioflocs and aggregates. In this review we focus on those dried and processed single-cell organisms used as potential ingredients for aqua-feeds where the microorganisms are considered non-viable and are used primarily to provide protein, lipids or specific nutritional components. Among the SCI, there is a generalised dichotomy in terms of their use as either single-cell protein (SCP) resources or single-cell oil (SCO) resources, with SCO products being those oleaginous products containing 200 g/kg or more of lipids, whereas those products considered as SCP resources tend to contain more than 300 g/kg of protein (on a dry basis). Both SCP and SCO are now widely being used as protein/amino acid sources, omega-3 sources and sources of bioactive molecules in the diets of several species, with the current range of both these ingredient groups being considerable and growing. However, the different array of products becoming available in the market, how they are produced and processed has also resulted in different nutritional qualities in those products. In assessing this variation among the products and the application of the various types of SCI, we have taken the approach of evaluating their use against a set of standardised evaluation criteria based around key nutritional response parameters and how these criteria have been applied against salmonids, shrimp, tilapia and marine fish species

Screening of intact yeasts and cell extracts to reduce Scrapie prions during biotransformation of food waste

Yeasts can be used to convert organic food wastes to protein-rich animal feed in order to recapture nutrients. However, the reuse of animal-derived waste poses a risk for the transmission of infectious prions that can cause neurodegeneration and fatality in humans and animals. The aim of this study was to investigate the ability of yeasts to reduce prion activity during the biotransformation of waste substrates—thereby becoming a biosafety hurdle in such a circular food system. During pre-screening, 30 yeast isolates were spiked with Classical Scrapie prions and incubated for 72 h in casein substrate, as a waste substitute. Based on reduced Scrapie seeding activity, waste biotransformation and protease activities, intact cells and cell extracts of 10 yeasts were further tested. Prion analysis showed that five yeast species reduced Scrapie seeding activity by approximately 1 log10 or 90%. Cryptococcus laurentii showed the most potential to reduce prion activity since both intact and extracted cells reduced Scrapie by 1 log10 and achieved the highest protease activity. These results show that select forms of yeast can act as a prion hurdle during the biotransformation of waste. However, the limited ability of yeasts to reduce prion activity warrants caution as a sole barrier to transmission as higher log reductions are needed before using waste-cultured yeast in circular food systems

Effects of dietary yeast inclusion and acute stress on post-prandial whole blood profiles of dorsal aorta-cannulated rainbow trout

Yeast is a potential alternative to fish meal in diets for farmed fish, yet replacing more than 50 % of fish meal results in reduced fish growth. In a 4-week experiment, 15 rainbow trout (Oncorhynchus mykiss) were cannulated and fed three diets each week: 30 % fish meal as a control (FM); 60 % replacement of fish meal protein, on a digestible basis, with Saccharomyces cerevisiae (SC); and 60 % replacement with Wickerhamomyces anomalus and S. cerevisiae mix (WA). Blood was collected at 0, 3, 6, 12 and 24 h after feeding. In the final week, fish were exposed to a 1-min netting stressor to evaluate possible diet-stress interactions. Significant increases in pH, TCO2, HCO3 and base excess were found after fish were fed the SC and WA diets compared with FM, which elevated blood alkaline tides. Yeast ingredients had lower buffering capacity and ash content than fish meal, which explained the increase in alkaline tides. In addition, fish fed the WA diet had significantly reduced erythrocyte area and fish fed SC and WA diets had increased mean corpuscular haemoglobin levels, indicating haemolytic anaemia. Higher levels of nucleic acid in yeast-based diets and potentially higher production of reactive oxygen species were suspected of damaging haemoglobin, which require replacement by smaller immature erythrocytes. Acute stress caused the expected rise in cortisol and glucose levels, but no interaction with diet was found. These results show that replacing 60 % of fish meal protein with yeasts can induce haemolytic anaemia in rainbow trout, which may limit yeast inclusion in diets for farmed fish

Effect of dietary oil from Camelina sativa on the growth performance, fillet fatty acid profile and gut microbiome of gilthead Sea bream (Sparus aurata)

Background In the last two decades, research has focused on testing cheaper and sustainable alternatives to fish oil (FO), such as vegetable oils (VO), in aquafeeds. However, FO cannot be entirely replaced by VOs due to their lack of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA), particularly eicosapentaenoic (EPA; 20:5n-3) and docosahexaenoic (DHA; 22:6n-3) acids. The oilseed plant, Camelina sativa, may have a higher potential to replace FO since it can contains up to 40% of the omega-3 precursors α-linolenic acid (ALA; 18:3n-3) and linoleic acid (LA; 18:2n-6). Methods A 90-day feeding trial was conducted with 600 gilthead sea bream (Sparus aurata) of 32.92 ± 0.31 g mean initial weight fed three diets that replaced 20%, 40% and 60% of FO with CO and a control diet of FO. Fish were distributed into triplicate tanks per diet and with 50 fish each in a flow-through open marine system. Growth performance and fatty acid profiles of the fillet were analysed. The Illumina MiSeq platform for sequencing of 16S rRNA gene and Mothur pipeline were used to identify bacteria in the faeces, gut mucosa and diets in addition to metagenomic analysis by PICRUSt. Results and Conclusions The feed conversion rate and specific growth rate were not affected by diet, although final weight was significantly lower for fish fed the 60% CO diet. Reduced final weight was attributed to lower levels of EPA and DHA in the CO ingredient. The lipid profile of fillets were similar between the dietary groups in regards to total saturated, monounsaturated, PUFA (n-3 and n-6), and the ratio of n-3/n-6. Levels of EPA and DHA in the fillet reflected the progressive replacement of FO by CO in the diet and the EPA was significantly lower in fish fed the 60% CO diet, while ALA was increased. Alpha and beta-diversities of gut bacteria in both the faeces and mucosa were not affected by any dietary treatment, although a few indicator bacteria, such as Corynebacterium and Rhodospirillales, were associated with the 60% CO diet. However, lower abundance of lactic acid bacteria, specifically Lactobacillus, in the gut of fish fed the 60% CO diet may indicate a potential negative effect on gut microbiota. PICRUSt analysis revealed similar predictive functions of bacteria in the faeces and mucosa, although a higher abundance of Corynebacterium in the mucosa of fish fed 60% CO diet increased the KEGG pathway of fatty acid synthesis and may act to compensate for the lack of fatty acids in the diet. In summary, this study demonstrated that up to 40% of FO can be replaced with CO without negative effects on growth performance, fillet composition and gut microbiota of gilthead sea bream