Mucosal and systemic physiological changes underscore the welfare risks of environmental hydrogen sulphide in post-smolt Atlantic salmon (Salmo salar).

Atlantic salmon (Salmo salar) might encounter toxic hydrogen sulphide (H2S) gas during aquaculture production. Exposure to this gas can be acute or chronic, with heightened levels often linked to significant mortality rates. Despite its recognised toxicity, our understanding of the physiological implications of H2S on salmon remains limited. This report details the mucosal and systemic physiological consequences in post-smolt salmon reared in brackish water at 12 ppt after prolonged exposure to elevated H2S levels over 4 weeks. The fish were subjected to two concentrations of H2S: 1 µg/L (low group) and 5 µg/L (high group). An unexposed group at 0 µg/L served as the control. Both groups exposed to H2S exhibited incremental mortality, with cumulative mortality rates of 4.7 % and 16 % for the low and high groups, respectively. Production performance, including weight and condition factors, were reduced in the H2S-exposed groups, particularly in the high group. Mucosal response of the olfactory organ revealed higher tissue damage scores in the H2S-exposed groups, albeit only at week 4. The high group displayed pronounced features such as increased mucus cell density and oedema-like vacuoles. Transcriptome analysis of the olfactory organ unveiled that the effects of H2S were more prominent at week 4, with the high group experiencing a greater magnitude of change than the low group. Genes associated with the extracellular matrix were predominantly downregulated, while the upregulated genes primarily pertained to immune response. H2S-induced alterations in the metabolome were more substantial in plasma than skin mucus. Furthermore, the number of differentially affected circulating metabolites was higher in the low group compared to the high group. Five core pathways were significantly impacted by H2S regardless of concentration, including the phenylalanine, tyrosine, and tryptophan biosynthesis. The plasma levels of phenylalanine and tyrosine were reduced following exposure to H2S. While there was a discernible distinction in the skin mucus metabolomes among the three treatment groups, only one metabolite - 4-hydroxyproline - was significantly impacted by H2S. Furthermore, this metabolite was significantly reduced in the plasma and skin mucus of H2S-exposed fish. This study underscores that prolonged exposure to H2S, even at concentrations previously deemed sub-lethal, has discernible physiological implications that manifest across various organisational levels. Given these findings, prolonged exposure to H2S poses a welfare risk, and thus, its presence must be maintained at low levels (<1 µg/L) in salmon land-based rearing systems.

Intermittent administration of peracetic acid is a mild environmental stressor that elicits mucosal and systemic adaptive responses from Atlantic salmon post-smolts.

BACKGROUND: Fish encounter oxidative stress several times during their lifetime, and it has a pervasive influence on their health and welfare. One of the triggers of oxidative stress in fish farming is the use of oxidative disinfectants to improve rearing conditions, especially in production systems employing recirculation technology. Here we report the physiological and morphological adaptive responses of Atlantic salmon (Salmo salar L.) post-smolts to intermittent exposure to a potent oxidative agent peracetic acid (PAA). Fish reared in semi-commercial scale brackish water recirculating aquaculture system (RAS) were exposed to 1 ppm PAA every 3 days over 6 weeks. Mucosal and systemic responses were profiled before exposure, 22 and 45 days during the intermittent PAA administration. RESULTS: Oxidative stress was likely triggered as plasma antioxidant capacity increased significantly during the exposure period. Adaptive stress response to the periodic oxidant challenge was likewise demonstrated in the changes in plasma glucose and lactate levels. PAA-induced alterations in the transcription of antioxidants, cytokines, heat shock proteins and mucin genes showed a tissue-specific pattern: downregulation was observed in the gills and olfactory rosette, upregulation occurred in the skin, and no substantial changes in the liver. Further, PAA exposure resulted in histological changes in key mucosal organs (i.e. olfactory rosette, skin and gills); pathological alterations were predominant in the gills where cases of epithelial lifting, hypertrophy and clubbing were prevalent. In addition, intermittent PAA administration resulted in an apparent overproduction of mucus in the nasal mucosa. Lastly, PAA did not dramatically alter the ability of salmon to mount a physiological stress response in the presence of a secondary stressor, though some subtle interference was documented in the kinetics and magnitude of plasma cortisol and glucose response post-stress. CONCLUSIONS: The present study collectively demonstrated that intermittent oxidant exposure was a mild environmental stressor that salmon could mount strong adaptive responses at systemic and mucosal levels. The results will be valuable in optimising the rearing conditions of post-smolts in RAS, especially in adopting water treatment strategies that do not considerably interfere with fish health and welfare.

Consequences of continuous ozonation on the health and welfare of Atlantic salmon post-smolts in a brackish water recirculating aquaculture system.

This study investigated the biological consequences of 45-day continuous ozonation on Atlantic salmon (Salmo salar) post-smolts in a brackish water recirculating aquaculture system (RAS). There was no significant difference in survival, operational welfare indicators, and average weight at termination between the ozone-treated and control groups. Plasma biochemical analyses revealed that the creatinine level was significantly higher in the ozone-treated group than in the control at termination. Histological evaluation of skin health showed no significant difference between the two groups. On the other hand, quantitative histopathology disclosed that the ozone group exhibited a better gill health status than did the control group, particularly at the end of the trial. Mucosal transcriptomics revealed a distinct response profile between the gills and skin. At day 45, there were no differentially expressed genes (DEG) identified in the skin, in contrast to 242 ozone-induced DEGs in the gills. Assessment of the transcriptomic profiles over time revealed that temporal effects were of greater impact in skin compared to gills, regardless of the treatment. The treatment did not result in metabolomic dysregulation and the overall profile lent support to the transcriptomics data that temporal effects had a greater influence on the changes observed. Exposure to handling-confinement stress revealed that ozone treatment did not alter the ability of post-smolts to respond to a secondary stressor. In summary, the suite of health and welfare indicators collectively indicated that continuous ozonation resulted in minimal physiological perturbations in salmon post-smolts. The results are expected to contribute to optimising the rearing conditions for post-smolts in RAS.

Striped catfish (Pangasianodon hypophthalmus) use air-breathing and aquatic surface respiration when exposed to severe aquatic hypercarbia.

We investigated the extent to which the facultative air-breathing fish, the striped catfish (Pangasianodon hypophthalmus), uses air-breathing to cope with aquatic hypercarbia, and how air-breathing is influenced by the experimental exposure protocol and level of hypercarbia. We exposed individuals to severe aquatic hypercarbia (up to Pw CO2 = 81 mmHg) using step-wise and progressive exposure protocols while measuring gill ventilation rate, heart rate, mean arterial blood pressure, and air-breathing frequency, as well as arterial blood pH and PCO2 . We confirm that P. hypophthalmus is tolerant of hypercarbia. Under both protocols gill ventilation rate, heart rate, and mean arterial blood pressure were maintained near control levels even at very high CO2 levels. We observed a marked amount of individual variation in the PwCO2 at which air-breathing was elicited, with some individuals not responding at all. The experimental protocol also influenced the onset of air-breathing. Air-breathing began at lower Pw CO2 in the step-wise protocol (23 ± 4.1 mmHg) compared with the progressive protocol (46 ± 7.8 mmHg). Air-breathing was often followed by aquatic surface respiration, at higher PCO2 (71 ± 5.2 mmHg) levels. On average, the blood PCO2 was approximately 43% lower (46 ± 2.5 mmHg) than water Pw CO2 (~81 mmHg) at our highest tested CO2 level. While this suggests that aerial CO2 elimination is an effective, and perhaps critical, respiratory strategy used by P. hypophthalmus to cope with severe hypercarbia, this observation may also be explained by a long lag time required for equilibration.

The Effects of Ozone on Atlantic Salmon Post-Smolt in Brackish Water-Establishing Welfare Indicators and Thresholds.

Ozone is a strong oxidant, and its use in aquaculture has been shown to improve water quality and fish health. At present, it is predominantly used in freshwater systems due to the high risk of toxic residual oxidant exposure in brackish water and seawater. Here, we report the effects of ozone on Atlantic salmon (Salmo salar) post-smolts (~100 g), in a brackish water (12 ppt) flow-through system. Salmon were exposed to oxidation reduction potential concentrations of 250 mV (control), 280 mV (low), 350 mV (medium), 425 mV (high) and 500 mV (very high). The physiological impacts of ozone were characterized by blood biochemical profiling, histopathologic examination and gene expression analysis in skin and gills. Fish exposed to 425 mV and higher showed ≥33% cumulative mortality in less than 10 days. No significant mortalities were recorded in the remaining groups. The skin surface quality and the thickness of the dermal and epidermal layers were not significantly affected by the treatments. On the other hand, gill histopathology showed the adverse effects of increasing ozone doses and the changes were more pronounced in the group exposed to 350 mV and higher. Cases of gill damages such as necrosis, lamellar fusion and hypertrophy were prevalent in the high and very high groups. Expression profiling of key biomarkers for mucosal health supported the histology results, showing that gills were significantly more affected by higher ozone doses compared to the skin. Increasing ozone doses triggered anti-oxidative stress and inflammatory responses in the gills, where transcript levels of glutathione reductase, copper/zinc superoxide dismutase, interleukin 1ß and interleukin were significantly elevated. Heat shock protein 70 was significantly upregulated in the skin of fish exposed to 350 mV and higher. Bcl-2 associated x protein was the only gene marker that was significantly upregulated by increasing ozone doses in both mucosal tissues. In conclusion, the study revealed that short-term exposure to ozone at concentrations higher than 350 mV in salmon in brackish water resulted in significant health and welfare consequences, including mortality and gill damages. The results of the study will be valuable in developing water treatment protocols for salmon farming.

The effects of salinity and photoperiod on aerobic scope, hypoxia tolerance and swimming performance of coho salmon (Oncorhynchus kisutch) reared in recirculating aquaculture systems.

Land-based recirculating aquaculture systems (RAS) have been used to rear salmon from smolt to market-sized adults, but high operating costs have limited their wide spread adoption. One clear advantage of using RAS for salmon aquaculture over open net pens is that fish can be reared under optimal conditions in an attempt to maximize growth and physiological performance and reduce overall production costs. However, few studies have attempted to define the optimal conditions for the long-term rearing of salmon. Thus, the goal of this study is to determine the effects of salinity and photoperiod, two factors that can be easily manipulated in RAS, on the physiological performance of coho salmon (Oncorhynchus kisutch) during long-term rearing. To address this goal, post-smolt coho salmon were reared for 150 days in replicate RAS at 2.5, 5, 10 and 30 ppt under either 12:12 and 24:0 (light:dark) photoperiods. Routine metabolic rate, maximum metabolic rate, aerobic scope and hypoxia tolerance were measured at 60 and 120 days of rearing, while swimming performance was assessed at 60 and 150 days of rearing. There were no effects of salinity or photoperiod on metabolic rate measurements, hypoxia tolerance or swimming performance at any sampling time. There were, however, significant effects of salinity and photoperiod on post-swimming hematology. The results suggest that physiological disturbances continue to manifest due to different environmental conditions, despite acclimation, but do not hinder the animal's ability to cope with physiological stressors. Overall, rearing salinity and photoperiod had very few measurable effects on the physiology and performance of coho salmon except the ionoregulatory disturbances following swimming at salinities of 2.5 and 30 ppt.

Divergent gene expression in the gills of juvenile turbot (Psetta maxima) exposed to chronic severe hypercapnia indicates dose-dependent increase in intracellular oxidative stress and hypoxia.

Elevated concentrations of carbon dioxide are a common stressor for fish and other aquatic animals. In particular, intensive aquaculture can impose prolonged periods of severe environmental hypercapnia, manifold exceeding CO2 concentrations of natural habitats. In order to cope with this stressor, gills are essential and constitute the primary organ in the acclimatization process. Yet, despite a general understanding of changes in ion regulation, not much is known with regard to other cellular mechanisms. In this study, we apply RT-qPCR to investigate changes in the expression of several genes associated with metabolism, stress and immunity within gills of juvenile turbot (Psetta maxima) after an eight-week exposure to different concentrations of CO2 (low = ∼3000 µatm, medium = ∼15,000 µatm and high = ∼25,000 µatm CO2). Histological examination of the gill tissue only found a significant increase of hypertrophied secondary lamella in the highest tested treatment level. gene expression results, on the other hand, implied both, mutual and dose-dependent transcriptional adjustments. Comparable up-regulation of IL-1ß, LMP7 and Grim19 at medium and high hypercapnia indicated an increase of reactive oxygen species (ROS) within gill cells. Simultaneous increase in Akirin and PRDX transcripts at medium CO2 indicated enhanced anti-oxidant activity and regulation of transcription, while reduced mRNA concentrations of COX, EF1α and STAT2 at high CO2 denoted suppressed protein synthesis and reduced metabolic capacity. In addition to upregulated DFAD and ApoE expression, implying compensating repair measures, gills exposed to the highest tested treatment level seemed to operate close to or even beyond their maximum capacity. Thus, fitting the model of capacity limitation, our results provide evidence for accretive intracellular hypoxia and oxidative stress in the gills of turbot, dependent on the level of environmental hypercapnia. Further, genes, such as COX, may be valuable biomarkers when attempting to discriminate between a successful and an overpowered stress response.

The effect of salinity and photoperiod on thermal tolerance of Atlantic and coho salmon reared from smolt to adult in recirculating aquaculture systems.

Land-based, closed containment salmon aquaculture involves rearing salmon from smolt to adult in recirculating aquaculture systems (RAS). Unlike in open-net pen aquaculture, rearing conditions can be specified in RAS in order to optimize growth and physiological stress tolerance. The environmental conditions that yield optimal stress tolerance in salmon are, however, unknown. To address this knowledge gap, we reared Atlantic (Salmo salar) and coho (Oncorhynchus kisutch) salmon in 7 separate RASs for 400 days post-smoltification under 2 photoperiods (24:0 or 12:12, light:dark) and 4 salinities (2.5, 5, 10 or 30 ppt.) and assessed the effects of these conditions on thermal tolerance. We found that over the first 120 days post-smoltification, rearing coho under a 24:0 photoperiod resulted in a ~2 °C lower critical thermal maxima (CTmax) than in coho reared under a 12:12 photoperiod. This photoperiod effect did not persist at 200 and 400 days, which was coincident with an overall decrease in CTmax in coho. Finally, Atlantic salmon had a higher CTmax (~28 °C) compared to coho (~26 °C) at 400 days post-smoltification. Overall, these findings are important for the future implications of RAS and for the aquaculture industry to help identify physiologically sensitive time stages.

The effect of diet, temperature and intermittent low oxygen on the metabolism of rainbow trout.

An automated respirometer system was used to measure VO2, protein catabolism as ammonia quotient and the energy budget to evaluate whether the crude protein content of a standard protein (SP) diet (42·5 %) or a high-protein (HP) diet (49·5 %) influences metabolism in rainbow trout under challenging intermittent, low dissolved oxygen concentrations. In total, three temperature phases (12, 16, 20°C) were tested sequentially, each of which were split into two oxygen periods with 5 d of unmanipulated oxygen levels (50-70 %), followed by a 5d manipulated oxygen period (16.00-08.00 hours) with low oxygen (40-50 %) levels. For both diets, catabolic protein usage was lowest at 16°C and was not altered under challenging oxygen conditions. Low night-time oxygen elevated mean daily VO2 by 3-14 % compared with the unmanipulated oxygen period for both diets at all temperatures. The relative change in VO2 and retained energy during the intermittent low oxygen period was smaller for the HP diet compared with the SP diet. However, in absolute terms, the SP diet was superior to the HP diet as the former demonstrated 30-40 % lower protein fuel use rates, higher retained energy (1-4 % digestible energy) and slightly lowered VO2 (0-8 %) over the range of conditions tested. The decrease in retained energy under low oxygen conditions suggests that there is scope to improve the performance of SP diets under challenging conditions; however, this study suggests that simply increasing the dietary protein content is not a remedy, and other strategies need to be explored.