Freshwater Recirculating Aquaculture Systems Research Articles

Smoltification, seawater performance, and maturation in Atlantic salmon (Salmo salar) fed different fat levels

IntroductionThe use of recirculating aquaculture systems (RAS) to produce Atlantic salmon smolts has resulted in exceptionally high fish growth rates. However, there are potential negative trade-offs between fast growth and key physiological processes, such as inadequate smoltification and early sexual maturation, which can both be linked to body energy reserves.MethodsThis study determined the effect of i) dietary fat levels on Atlantic salmon whole-body fat and fatty acids composition, growth performance and smoltification and ii) a previous dietary regime on seawater growth performance and male early sexual maturation. In freshwater RAS, salmon parr (~19g) were fed 3 fat levels (20, 24, and 28%) over a 14-week period at 12°C. Subsequently, in seawater flow-through systems, smolt (~96g) were fed a control diet (26%) for 12 weeks at 2 temperatures (12 and 16°C).ResultsDietary fat levels resulted in differences in k-factor, fish whole-body fat and fatty acids composition; the low fat diet resulted in fish with a 23% lower whole-body fat when compared to the other two dietary groups at the end of the freshwater phase. These differences in whole-body fat faded at the end of the seawater phase. During the freshwater phase, all three feeds resulted in a comparable growth performance and smoltification indicator values, including k-factor, gill Na+, K+-ATPase, blood serum chloride and smolt index score. In contrast, water temperature resulted in fish body weight differences, where groups reared at 16°C were larger than those reared at 12°C. Nevertheless, both temperature regimes supported a similar thermal growth coefficient. Smolts grown at 16°C showed a higher level of the sex steroids androstenedione compared to those at 12°C. However, 11-ketotestosterone and testosterone levels did not differ despite a trend for higher levels at 16°C. Furthermore, testis histology at the final sampling indicated that some individuals showed initial signs of maturation (stage 3).DiscussionIn conclusion, varying dietary fat levels (20 - 28%) during the freshwater phase did not influence smoltification or male early sexual maturation during the subsequent grow-out phase. However, a temperature increase from 12 to 16°C resulted in larger fish and appeared to stimulate early male maturation in some fish individuals.

Performance, processing yields, and fillet composition of specific United States diploid and triploid rainbow trout (Oncorhynchus mykiss) lines reared in a semi-commercial scale freshwater recirculating aquaculture system

Large rainbow trout marketed as steelhead are gaining considerable attention for production in recirculating aquaculture systems (RAS). Diploid and triploid trout with varying genetics are available from several commercial suppliers; however, the performance and processing metrics of these lines have not been compared when raised in freshwater RAS. Current data on these outcomes could support more accurate cost and profitability estimates for potential RAS projects and optimize operation for existing farms. Accordingly, the objective of this study was to quantify performance metrics, processing yield, and fillet composition of six all-female rainbow trout lines (three diploid and three triploid) exposed to an artificial winter (12:12 light: dark at 40 g (Artificial Winter Photoperiod; AWP)) or continuous, 24-h light (No Winter Photoperiod; NWP) grown in a semi-commercial scale freshwater RAS to 3.5 kg. Overall, growth was impacted by line, ploidy, and artificial winter photoperiod. Diploid and AWP trout were smaller than triploids and NWP trout at stocking and 500 g. Mean diploid and triploid weight was similar at larger sizes (>1 kg); however, triploid performance compared to diploids varied by commercial supplier. Mortality was lower in lines from commercial supplier 1 than lines from commercial supplier 2 and the United States Department of Agriculture (14.4 ± 0.8 vs 27.5 ± 2.0 and 29.8 ± 4.3%, respectively). Head-on gutted and skin-on fillet yields were related with larger fish weight. Mature prevalence in diploids may have led to differences observed between diploids and triploids in fillet lipid (10.9 ± 0.8 vs 13.3 ± 0.6%) and moisture (67.0 ± 0.6 vs 65.1 ± 0.4%). The results provide guidance for rainbow trout RAS producers to make informed decisions on optimal harvest size targets and tailor their rainbow trout line or ploidy selection to those harvest sizes.

Influence of dietary phosphorus on orthophosphate accumulation in recirculating aquaculture systems with rainbow trout (Oncorhynchus mykiss)

Dietary phosphorus (P) levels affect the excretion of orthophosphate (PO4-P) from fish. Since most recirculating aquaculture systems (RAS) do not specifically remove PO4-P within the recirculating loop, dietary P levels may influence PO4-P concentrations in RAS water. A feeding trial was performed in freshwater RAS, aiming to determine the relationship between dietary P levels and PO4-P accumulation in RAS water, and to examine potential effects of dietary P levels on microbial activity and abundance in RAS water given that P is an essential mineral for all microorganisms. Three diets with 1.49% (HiP), 1.20% (MeP), and 0.89% (LoP) total P, respectively, were each fed to triplicate pilot-scale RAS with juvenile rainbow trout (Oncorhynchus mykiss) for five weeks, applying a daily feed loading of 1.67 kg feed/m3 make-up water. Digestible dietary P levels correlated significantly with the PO4-P concentrations in RAS water at the end of the trial, while no similar correlations with microbial abundance or activity were observed. Nitrate-N concentrations measured by the end of the trial matched predicted equilibrium concentrations. In contrast, measured PO4-P concentrations were approximately 40 – 50% lower than predicted regardless of diets, suggesting that PO4-P was likely utilized by RAS microbes. We therefore speculate whether lowering dietary P levels could become an effective tool for microbial management in RAS.

A novel method to estimate biofilm activity based on enzymatic oxygen release from hydrogen peroxide decomposition

Biofilm is central for biological water treatment processes in recirculating aquaculture systems (RAS). A lack of suitable methods for quantifying biofilm activity, however, makes it difficult to assess and compare the microbial status of biofilm. This type of information of the biofilm will be useful to assess the colonization status of nitrifying biocarriers or to evaluate the effect of disinfectants on the biofilm activity.Here we introduce a novel assay for rapid assessment of microbial activities in the biofilm attached on bioelements from a RAS biofilter. The assay consisted of an intermittent respirometer platform where biofilter elements were exposed to 10 mg/L hydrogen peroxide (H2O2) for 1 h, following concurrent measurements of oxygen release from the decomposition of H2O2 caused by biofilm-associated enzymes. A different number of colonized, mature bioelements from a moving bed biofilter in a freshwater RAS were tested with repeated H2O2 exposure, and compared against their autoclaved forms. A substantial increase in dissolved oxygen (DO) concentration (0.92–2.31 mg O2/L) occurred with mature bioelements during 1 h of H2O2 exposure, compared to small amounts of DO release (≤0.27 mg O2/L) with autoclaved bioelements. This substantiates that H2O2 decomposition by biofilm is mainly governed by microbial enzymatic activities. A monomolecular model fitted well with the observed oxygen release profiles of tested mature bioelements after H2O2 exposure (R2 > 0.98). The kinetic rate constant of net oxygen release (kor, h−1) was proportional (R2 for linear fit = 0.99) to the number of mature bioelements tested. Repeated exposure of H2O2 to the same bioelements did not change kor, which indicates that 10 mg/L H2O2 with an exposure time of 1 h does not suppress enzymatic activity in biofilm. Our study provides a new rapid method that allows simple quantification of microbial activity in biofilm samples from aquaculture systems, which could potentially be also applied to study biofilm from wastewater treatment plants and other industries.

Comparision of nitrogen removal characteristic and microbial community in freshwater and marine recirculating aquaculture systems

Recirculating aquaculture system (RAS) has a good prospect in aquaculture, but its nitrogen removal characteristics and microbial community changes in freshwater and marine water remain unclear. In this study, six RAS were designed and divided into freshwater group and marine water group with salinity of 0‰ and 32‰, respectively, and operated for 54 days to test changes in nitrogen (NH4+-N, NO2--N, NO3--N), extracellular polymeric substances and microbial communities. The results showed that ammonia nitrogen was rapidly reduced and almost converted to nitrate nitrogen in the freshwater RAS but to nitrite nitrogen in marine RAS. Compared with freshwater RAS, marine RAS had lower tightly bound extracellular polymeric substances and worse stability and settleability condition. 16S rRNA amplicon sequencing reflected significantly lower bacterial diversity and richness in marine RAS. Microbial community structure at phylum level showed lower relative abundance of Proteobacteria, Actinobacteria, Firmicutes, Nitrospirae, but higher abundance of Bacteroidetes under a salinity of 32‰. High salinity decreased the abundance of funtional genera (Nitrosospira, Nitrospira, Pseudomonas, Rhodococcus, Comamonas, Acidovorax, f_Comamonadaceae), which may account for nitrite accumulation and low nitrogen removal capacity in marine RAS. These findings could provide theoretical and practical basis for improving the start-up speed of high-salinity nitrification biofilm.

Simultaneous biomethane production and nutrient remineralization from aquaculture solids

The rapid expansion of the aquaculture industry has brought about a heightened focus on the waste produced by high intensity fish farming. In closed-containment, recirculating aquaculture systems (RAS), fish solids are mechanically separated and/or coagulated before being disposed as waste. Subsequent revalorization is typically limited to the direct dispersal of aquaculture solids onto agricultural fields. Here, we developed a novel, continuous flow, low-cost solids waste treatment system for freshwater and saline RAS. Rotating drum filter backwash was collected as the primary feedstock for anaerobic digestion. A laboratory scale set up was used to monitor the conversion of the solids into a methane-rich (60–80% purity) biogas stream. Iron supplementation (ferric iron at 100 mg/L and 1000 mg/L) improved salt tolerance of the methanogenic community, leading to higher methane yields in a supplemented (FeCl3 at 1000 mg/L) saline treatment than the saline control. The application of iron additionally improves pH stability and volatile fatty acid utilization. The methane yield ranged from 0.1 to 0.4 NL CH4/ g VS across the three freshwater treatments and the iron-supplemented saline treatment, however, it was significantly lower for the saltwater control: ranging between 0.08 and 0.25 NL CH4/ g VS. These values correspond to a percentage yield of 57–86% of the total biomethane potential. Overall, implementing anaerobic digestion for RAS waste valorization may generate significant amounts of biomethane to be used in electricity and heating for large-scale aquaculture facilities, while even for smaller facilities it may off-set costs and mitigate environmental impacts of the waste streams.

Closing the gap – producing black soldier fly larvae on aquaculture side streams

The aim of the study was to investigate if black soldier fly (Hermetia illucens) larvae can be grown on sludge from a salmonid fresh-water recirculating aquaculture system facility in a small pilot-scale trial. The growth and transfer of both nutrients and unwanted components from the sludge to the insect larvae was analysed. A commercial chicken feed was used as a control growth substrate and up to 100% of the chicken feed was replaced with aquaculture sludge. Replacing 40% of the chicken feed with aquaculture sludge did not influence the growth of the larvae. A gradual decrease in growth was, however seen at a sludge inclusion of higher than 40%. Proximate (crude protein and fat, dry matter) and amino acid composition of the larvae stayed stable across treatments, while ash content increased with higher sludge inclusion. An accumulation of elements such as Zn, Fe and Se was observed in the larvae with increasing amount of sludge in the growth substrate. The larvae also accumulated unwanted elements regulated by EU feed legislation such as As, Hg and Cd. Both As and Hg in the larvae exceeded the legal EU limits for feed ingredients, but only in the two highest levels of sludge inclusion (80 and 100% sludge). Fatty acid composition reflected the substrate, with the sludge-fed larvae being enriched in marine n-3 poly unsaturated fatty acids. No salmon pathogens were detected in the larvae. The trial showed that it is possible to grow black soldier fly larvae on aquaculture sludge but points at a few feed safety risk factors which need to be further elucidated before a use of sludge as growth material for insects can be considered.

Metagenomics and metabarcoding experimental choices and their impact on microbial community characterization in freshwater recirculating aquaculture systems

BackgroundMicrobial communities in recirculating aquaculture systems (RAS) play a role in system success, nutrient cycling, and water quality. Considering the increasing socio-economic role of fish farming, e.g., regarding food security, an in-depth understanding of aquaculture microbial communities is also relevant from a management perspective, especially regarding the growth, development, and welfare of the farmed animal. However, the current data on the composition of microbial communities within RAS is patchy, which is partly attributable to diverging method choices that render comparative analyses challenging. Therefore, there is a need for accurate, standardized, and user-friendly methods to study microbial communities in aquaculture systems.ResultsWe compared sequencing approach performances (3 types of 16S short amplicon sequencing, PacBio long-read amplicon sequencing, and amplification-free shotgun metagenomics) in the characterization of microbial communities in two commercial RAS fish farms. Results showed that 16S primer choice and amplicon length affect some values (e.g., diversity measures, number of assigned taxa or distinguishing ASVs) but have no impact on spatio-temporal patterns between sample types, farms and time points. This implies that 16S rRNA approaches are adequate for community studies. The long-read amplicons underperformed regarding the quantitative resolution of spatio-temporal patterns but were suited to identify functional services, e.g., nitrification cycling and the detection of pathogens. Finally, shotgun metagenomics extended the picture to fungi, viruses, and bacteriophages, opening avenues for exploring inter-domain interactions. All sequencing datasets agreed on major prokaryotic players, such as Actinobacteriota, Bacteroidota, Nitrospirota, and Proteobacteria.ConclusionThe different sequencing approaches yielded overlapping and highly complementary results, with each contributing unique data not obtainable with the other approaches. We conclude that a tiered approach constitutes a strategy for obtaining the maximum amount of information on aquaculture microbial communities and can inform basic research on community evolution dynamics. For specific and/or applied questions, single-method approaches are more practical and cost-effective and could lead to better farm management practices.

Relationship between gut microbiota and Chinook salmon (Oncorhynchus tshawytscha) health and growth performance in freshwater recirculating aquaculture systems.

Gut microbiota play important roles in fish health and growth performance and the microbiome in fish has been shown to be a biomarker for stress. In this study, we surveyed the change of Chinook salmon (Oncorhynchus tshawytscha) gut and water microbiota in freshwater recirculating aquaculture systems (RAS) for 7 months and evaluated how gut microbial communities were influenced by fish health and growth performance. The gut microbial diversity significantly increased in parallel with the growth of the fish. The dominant gut microbiota shifted from a predominance of Firmicutes to Proteobacteria, while Proteobacteria constantly dominated the water microbiota. Photobacterium sp. was persistently the major gut microbial community member during the whole experiment and was identified as the core gut microbiota for freshwater farmed Chinook salmon. No significant variation in gut microbial diversity and composition was observed among fish with different growth performance. At the end of the trial, 36 out of 78 fish had fluid in their swim bladders. These fish had gut microbiomes containing elevated proportions of Enterococcus, Stenotrophomonas, Aeromonas, and Raoultella. Our study supports the growing body of knowledge about the beneficial microbiota associated with modern salmon aquaculture systems and provides additional information on possible links between dysbiosis and gut microbiota for Chinook salmon.

The effects of Fennel (Foeniculum vulg0are) Essential Oils on Growth Performance and Digestive Physiological Traits in Black Sea Salmon (Salmo labrax PALLAS 1814) Juveniles

The present study was carried out in the freshwater recirculating aquaculture system to determine the effects of the fennel (Foeniculum vulgare) essential oil on Black Sea salmon (Salmo labrax). Fish were distributed randomly to 50 L experimental tanks, and 45 fish were in each tank. The experiment was triplicate in each group, and the results were averaged. Five diets of equal isonitrogenous and isocaloric content with 50, 100, 200, and 400 mg kg-1 of fennel essential oil were prepared. Fish were fed with diets at the rate of 3% of live body weight four times daily by hand for 90 days. The results revealed that dietary supplementation with fennel essential oil did not have any significant effect on the growth performance. Supplementation with 200 mg fennel kg-1 increased lipase activity in contrast with control group. Supplementation with 400 mg kg-1 fennel showed similar results with the control group in terms of pepsin activity. The intestinal villi length of fish fed 200 mg kg-1 fennel was higher than the control group. The thickness of muscularis in group fed with 50, 100 and 200 mg kg-1 fennel was similar to each other and higher than the control group. Lactic acid bacteria were reduced by fennel essential oil supplementation. Results showed that fennel essential oil can be used in diets of Black Sea salmon without the growth performance.

Effect of biocarrier material on nitrification performance during start-up in freshwater RAS

Biofilters are key components in recirculating aquaculture systems (RAS). For ensuring cost-efficient function of biofilters, characteristics of biocarriers such as shape, material, surface area, density, longevity, and price needs to be evaluated. In this study, we compared otherwise identical biocarriers made of polyethylene (PE) against ones made of polypropylene (PP) and PP biocarriers with three different levels of pigment (carbon black) addition (without added pigment, semi-pigmented, fully-pigmented). The four types of biocarriers were tested in 12 parallel moving bed bioreactors under identical conditions receiving water from a semi-commercial freshwater RAS with rainbow trout. Measurements of total ammonium nitrogen (TAN) and nitrite-nitrogen (NO2--N) removal rates and nitrification kinetics were carried out by spiking events with NH4Cl or NaNO2 two, four, and six weeks after start-up. The results showed all biofilters removed TAN only after two weeks of operation, and that biofilters with PP biocarriers had a shorter maturation time, reaching their maximum TAN removal rate at week 4, while PE biofilters reached equal maximum at week 6. Biofilters with PE had generally lower NO2--N removal rates and first-order reaction constants than PP biofilters. Pigmentation level had a minor effect on TAN and NO2--N removal rates and kinetics, as fully pigmented biocarriers initially were found to have lower removal rates and first order constants for TAN and NO2--N than semi-pigmented or biocarriers without added pigment. Overall, the results suggest that PE is inferior biocarrier material than PP, due to the longer start-up period and nitrite accumulation, which might be related to the differences in the surface characteristics between these two materials. Furthermore, the results indicate that pigment addition does not provide any extra benefit for nitrification.

Water quality, waste production, and off‐flavor characterization in a depuration system stocked with market‐size Atlantic salmon Salmo salar

AbstractLand‐based Atlantic salmon, Salmo salar, grow‐out facilities utilize depuration to remediate off‐flavor. Water used in this process is either discharged or repurposed as supply water in recirculating aquaculture systems (RAS). Both approaches require an understanding of water quality and waste production for water treatment decisions and compliance with pollution discharge standards; however, these data were lacking. Therefore, a study was carried out to characterize these parameters. To begin, 311 salmon (5–6 kg) originally cultured in freshwater RAS were stocked at 100 kg/m3 in an 18 m3 depuration tank. Feed was withheld 1 day before transfer and throughout the 7‐day study period. Hours after stocking, total suspended solids (TSS), total phosphorus (TP), and total ammonia nitrogen (TAN) levels spiked, and concentrations declined thereafter. Delta TSS and TP were negligible by the end of the trial; however, TAN plateaued, indicating that salmon began to catabolize somatic tissue in the absence of feeding. Geosmin and 2‐methylisoboreol levels in water and fish were low throughout the study. This research indicates that residual waste production occurs while depurating Atlantic salmon. Procedural refinements and recommendations were gleaned including locality for introducing depuration system water within RAS and extension of the feed withholding period before depuration.

Estimation of nitrifying and heterotrophic bacterial activity in biofilm formed on RAS biofilter carriers by respirometry

Biofilm, the aggregates of microbial layers that are attached on the surface of biofilter carriers, plays a central role in the removal and conversion of nutrients in recirculating aquaculture systems (RAS). The bacterial activity in biofilm involves autotrophic and heterotrophic processes. These biological processes are crucial for the water quality in RAS but are difficult to monitor. In this study, we demonstrate a new method based on intermittent respirometry to selectively estimate bacterial activity in biofilm following a spike in substrate concentration. The method was tested with three different biofilter carriers (extruded polypropylene, injection molded polypropylene and polymeric foam) from moving bed biofilm reactors in a common freshwater RAS. Oxygen consumption rates of biofilm-associated bacteria were measured in closed metabolic chambers under standard conditions. The protocol included sequential flushes of either pure tap water, or tap water spiked with nitrite, ammonium or acetate. The results showed consistent carrier-specific metabolic activities of endogenous respiration, nitrite-oxidizing bacteria (NOB), ammonia-oxidizing bacteria (AOB) and heterotrophic bacteria (HB) in biofilm. The highest activities normalized to volumetric oxygen consumption rates, were found in polymeric foam with values of 677 ± 125, 764 ± 156 and 166 ± 36 g O2·m−3·d−1 for NOB, AOB and HB, respectively. The biofilter performance evaluation based on respirometric data showed satisfactory accordance with the values from substrate degradation batch kinetic tests for all three tested carriers, confirming the feasibility and robustness of the method as a means to assessing biofilter performance. Our study provides an on-site tool in biofilter performance tests and leads to a better understanding of dynamic and relationship between biofiltration and water quality.

Exposing Atlantic Salmon Post-Smolts to Fluctuating Sublethal Nitrite Concentrations in a Commercial Recirculating Aquaculture System (RAS) May Have Negative Consequences

Salmon farmers are interested in extending the time post-smolts are reared in recirculating aquaculture systems (RAS). However, there is a lack of knowledge regarding optimal water quality for post-smolts in RAS, and regarding potential consequences of long term exposure to different toxic compounds, such as nitrite, in the RAS water. To address this issue, we conducted a case study at a Faroese Atlantic salmon farm, that rears large post-smolts in fresh water RAS for 22 months before sea transfer, with no additional chloride salt, a known treatment for nitrite toxicity. The aim was to document the potential effects of long-term exposure of fluctuating sub-lethal nitrite concentrations in fresh water RAS on blood physiology of large post-smolts. The study was conducted over six weeks, at the end of the RAS production cycle. Our case study shows that after ~22 months in RAS with no additional chloride, the fish had accumulated a plasma nitrite concentration 8 to 16 times higher than the ambient water. Our results indicate that the accumulation may have resulted in extracellular hyperkaliemia, since there was a positive correlation between plasma nitrite and potassium levels (p=0.00095), with potassium levels almost twice as high than previously reported for Atlantic salmon. This could indicate that Atlantic salmon health is challenged due to prolonged sub-lethal nitrite exposure in fresh water RAS. Further research related to long-term nitrite exposure in RAS is needed to asses the potential negative impact, in order to optimize welfare and growth performance during production of Atlantic salmon post-smolts.

Effects of water temperature on the gut microbiome and physiology of Chinook salmon (Oncorhynchus tshawytscha) reared in a freshwater recirculating system

Climate change and global warming pose particular concerns to the aquaculture industry as water temperature is one of the most important influences of health and growth performance in poikilotherms. Despite increased recognition of the importance of the gut microbiome in fish health, knowledge of temperature effects on the holobiont and implications for aquaculture performance are still limited. We assessed the effect of water temperature in a freshwater recirculating aquaculture system on the gut microbiome and the physiology, health and growth of New Zealand's most important farmed fish, the Chinook Salmon (Oncorhynchus tshawytscha). Fish were reared at four temperatures (8, 12, 16, 20 °C) for 41 to 49 days. Health, growth and feed intake were assessed at the beginning and end of the experiment. Haematology, plasma biochemistry, histology and tissue composition (e.g. fatty acids, amino acids, chemical composition) and gut microbiota (faeces and hindgut wall mucosa) were characterized. We found that increased temperature resulted in an increase in feed intake and growth (plateauing as it approached 20 °C) but did not impact feed conversion efficiency. Gut microbiome community composition assessed from both sample types changed with increasing temperature, with the dominance of Vibrionaceae (Photobacterium phosphoreum and/or pisciola) being gradually replaced by Fusobacteriaceae (Cetobacterium somerae) and Brevenemataceae (Brevinema). A significant effect of temperature was also observed on fish histology, fatty acids, chemical composition, haematology, plasma biochemistry and several internal biometrics, with significant correlation of most of these data layers with the gut microbiota. Notably, glucose, lactate and magnesium concentration in plasma, whole-body palmitic and the sum of the saturated fatty acids, and Cetobacterium were positively associated with increased temperature. This study provides further knowledge on how the Chinook salmon holobiont is influenced by water temperature and how the changes observed relate to fish growth performance and health. These results will help make more informed decisions for monitoring fish gut microbiota and tailoring dietary requirements to rearing water temperature.

Monitoring of Enzymatic Activity in the Gastrointestinal Tract of Black Sea Salmon (Salmo labrax Pallas, 1814)

In this study, it was aimed to determine the activity of digestive enzymes at different time intervals after-feeding throughout the gastrointestinal tract of Black Sea salmon (Salmo labrax). The study was conducted at freshwater recirculating aquaculture systems (RAS). The fish were fed by hand up to satiation for 60 days with diets containing 46.25% protein and 14.90% lipid. The fish gastrointestinal tract including stomach, anterior (with pyloric caeca), middle and posterior sections were taken together at 45th minute, 3rd, 6th, 12th, 24th, 48th, 72nd, and 96th hours post-feeding. The tissues were were stored at -80°C until analyzed. In terms of digestive enzyme activity, at the end of the study, anterior and middle intestine sections had the highest level at 3rd-hour post-feeding. Similarly, pepsin, trypsin, and lipase in the stomach had the highest activity at 3rd-hour post-feeding. However, amylase in the stomach was the highest level at 45th minute and 3rd-hour post-feeding. At 3rd-hours post-feeding, the pepsin in the stomach, the trypsin, and lipase in the anterior section, and the amylase in the middle intestine had the highest levels. Moreover, the correlations between these enzymes were strong in the positive direction. Also, the difference in pepsin, trypsin, amylase, and lipase levels in the samples dissected at different time intervals after feeding was statistically significant. Our results revealed that stomach, anterior, middle, and posterior sections in the gastrointestinal tract of Black Sea salmon had enzyme activity in the different levels, and enzyme activity of these sections changed depending on time post-feeding.

Mode of Application of Peracetic Acid-Based Disinfectants has a Minimal Influence on the Antioxidant Defences and Mucosal Structures of Atlantic Salmon (Salmo salar) Parr.

Peracetic acid (PAA) is an oxidative disinfectant with a broad spectrum of antimicrobial activity and low environmental impact. In this study, we investigated the physiological impacts of PAA application in Atlantic salmon (Salmo salar) parr reared in freshwater recirculating aquaculture systems over a 4-week period. PAA at a target concentration of 1 mg/L was administered either in pulse (every 3 days) or continuous. The group that did not receive PAA served as a control. Fish tissue samples were collected for histology, gene expression, and biochemical analyses at day 0 and after 2 and 4 weeks of exposure. The expression of genes encoding for antioxidant defence in the olfactory organs, skin, and gills changed during the trial, but the temporal effects were more pronounced than inter-treatment impacts. The glutathione group of antioxidant genes was more responsive to PAA. In most cases, an upregulation was observed. Significantly lower levels of reactive oxygen species were identified in the plasma and skin mucus of the two PAA-exposed groups at week 4; nonetheless, significantly increased levels of total antioxidant capacity were only observed in the skin mucus of fish from the continuous treatment group. Additional markers of oxidative stress (i.e., 8-oxo-2′-deoxyguanosine and o,o'-dityrosine) were analysed in the skin, gills, liver, and dorsal fins. These markers were unaffected by the two PAA treatments. Sporadic reversible structural alterations were observed in the three mucosal organs; the changes were time-dependent, and the effects of PAA treatment were minimal. The number of mucous cells varied over time but not within treatments except in the skin of the pulse group at week 4 where a reduction was observed. The ratio of acidic and neutral mucous cells in the skin and gills were affected by PAA treatments especially in the pulse group. Overall, this study revealed that Atlantic salmon parr mobilised mucosal and systemic antioxidant defences against the oxidative disinfectant PAA, but it was evident that the mode of application did not impose a strong influence. The minimal effects of PAA application on the indicators of health and welfare underscore the potential use of PAA as a routine disinfectant in recirculating aquaculture systems.

RNA-Seq Analysis of the Growth Hormone Transgenic Female Triploid Atlantic Salmon (Salmo salar) Hepatic Transcriptome Reveals Broad Temperature-Mediated Effects on Metabolism and Other Biological Processes.

This study examined the impact of rearing temperature (10.5, 13.5 or 16.5°C) on the hepatic transcriptome of AquAdvantage Salmon (growth hormone transgenic female triploid Atlantic salmon) at an average weight of 800 g. Six stranded PE libraries were Illumina-sequenced from each temperature group, resulting in an average of over 100 M raw reads per individual fish. RNA-sequencing (RNA-seq) results showed the greatest difference in the number of differentially expressed transcripts (1750 DETs), as revealed by both DESeq2 and edgeR (q < 0.05; fold-change > |1.5|), was between the 10.5 and 16.5°C temperature groups. In contrast, 172 and 52 DETs were found in the 10.5 vs. 13.5°C and the 13.5 vs. 16.5°C comparisons, respectively. Considering the DETs between the 10.5 and 16.5°C groups, 282 enriched gene ontology (GO) terms were identified (q < 0.05), including “response to stress”, “immune system process”, “lipid metabolic process”, “oxidation-reduction process”, and “cholesterol metabolic process”, suggesting elevated temperature elicited broad effects on multiple biological systems. Pathway analysis using ClueGO showed additional impacts on amino acid and lipid metabolism. There was a significant positive correlation between RNA-seq and real-time quantitative polymerase chain reaction (RT-qPCR) results for 8 of 9 metabolic-related transcripts tested. RT-qPCR results also correlated to changes in fillet tissue composition previously reported in these salmon (e.g., methionine and lysine concentrations positively correlated with hsp90ab1 transcript expression), suggesting that rearing temperature played a significant role in mediating metabolic/biosynthetic pathways of AquAdvantage Salmon. Many transcripts related to lipid/fatty acid metabolism (e.g., elovl2, fabpi, hacd2, mgll, s27a2, thrsp) were downregulated at 16.5°C compared to both other temperature groups. Additionally, enrichment of stress-, apoptosis- and catabolism-relevant GO terms at 16.5°C suggests that this temperature may not be ideal for commercial production when using freshwater recirculating aquaculture systems (RAS). This study relates phenotypic responses to transcript-specific findings and therefore aids in the determination of an optimal rearing temperature for AquAdvantage Salmon. With approval to grow and sell AquAdvantage Salmon in the United States and Canada, the novel insights provided by this research can help industry expansion by promoting optimal physiological performance and health.

Temporal changes in skin and gill microbiomes of Atlantic salmon in a recirculating aquaculture system – Why do they matter?

Mucosal surfaces are key components of teleost health, providing defence against opportunistic pathogens and other insults. Maintaining the integrity of mucosal surfaces and their associated microbial communities, especially the gill and skin that have large surface areas exposed to the environment is essential. Production of Atlantic salmon in land-based recirculating aquaculture systems (RAS) has increased significantly in recent years as it allows greater control over stability of the environment in which fish are reared, reduces water demand and minimises environmental impacts. However, little is known about the impact of the RAS environment upon the temporal dynamics of skin and gill mucosal microbiomes. In this study we examined microbial communities in gill mucus, skin mucus and rearing water throughout freshwater (FW) RAS production, and at 1-week and 4-weeks following transfer to seawater (SW) in open cage production using 16S rRNA sequencing. Microbial diversity and richness in skin and gill mucus of fish reared in a RAS system were temporally dynamic. Dynamics in richness and diversity were similar in the two mucosal tissues, and to some extent also mirrored that of the surrounding water. Dysbiosis indicated by an abrupt decline in diversity during FW production coincided with an increase in the relative abundance of two taxa belonging to the RAS-biofilter-associated nitrogen-cycling genus Hydrogenophaga in RAS tank water and this was also observed in gill and skin mucus. Extensive overlap in core taxa was observed between gill and skin mucus, but host-specific cores were non-existent during the dysbiotic event with all cores present in the rearing water. Diversity remained stable during the transition from FW to SW, but distinct community composition and core taxa were observed in the two environments. Although RAS are closely controlled, significant temporal variation could be observed in temperature as well as levels of CO2 and nitrogen compounds, reflecting the increasing biological load within the system over time. The results presented here suggest that, in terms of microbiomes, dysbiosis may occur in both the RAS environment and fish mucosal surfaces over time, but microbial communities have the capability to recover.

Effects of ozonation and foam fractionation on rainbow trout condition and physiology in a small-scale freshwater recirculation aquaculture system

The combined use of ozonation and foam fractionation significantly improves the water quality in freshwater recirculating aquaculture systems (RAS). In a recent study, numbers and total volume of suspended particles were reduced by 89% and 76%, respectively, water turbidity was improved by 79% and bacterial activity was reduced by 90% compared to control RAS. In the current study, we assessed the effects of those improvements in water quality on the welfare status of the rainbow trout juveniles present in the RAS. Ozone and foam fractionation were applied separately or in combination in a two-by-two factorial design with triplicated individual RAS. The trial was 8-week long and the welfare of the fish was assessed on week 4 (mid-term) and on week 8 (final). Fish welfare was assessed by using a combination of variables related to physiological stress, oxidative stress, growth performance, energy metabolism and external damage. Feed amounts were kept constant during the study to maintain homogeneous water quality conditions through the study, resulting in a decreasing specific feeding ration during the experiment. Restrictive feeding affected a number of variables related to the metabolic condition of the fish, but those effects were similar for all treatments. Effects of ozonation, foam fractionation and their combination on fish welfare were modest. Both ozone and foam fractionation led to improved skin condition of the fish, and seemed to act complementarily when applied together. Furthermore, foam fractionation had a small significant effect in improving the fish condition factor with respect to the fish in the RAS without foam fractionation. While the size of the described effects was small, they were the result of short-term exposure to the improved water quality. Further studies should focus in investigating the long-term effects of ozonation and foam fractionation on fish welfare in RAS-based aquaculture.