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Blood analyses provide substantial information about the physiological aspects of animal welfare assessment, including the activation status of the neuroendocrine and immune system, acute and long-term impacts due to adverse husbandry conditions, potential diseases, and genetic predispositions. However, fish blood is still not routinely analyzed in research or aquaculture for the assessment of health and/or welfare. Over the years, the investigative techniques have evolved from antibody-based or PCR-based single-parameter analyses to now include transcriptomic, metabolomic, and proteomic approaches and from hematological observations to fluorescence-activated blood cell sorting in high-throughput modes. The range of testing techniques established for blood is now broader than for any other biogenic test material. Evaluation of the particular characteristics of fish blood, such as its cell composition, the nucleation of distinct blood cells, or the multiple isoforms of certain immune factors, requires adapted protocols and careful attention to the experimental designs and interpretation of the data. Analyses of fish blood can provide an integrated picture of the endocrine, immunological, reproductive, and genetic functions under defined environmental conditions and treatments. Therefore, the scarcity of high-throughput approaches using fish blood as a test material for fish physiology studies is surprising. This review summarizes the wide range of techniques that allow monitoring of informative fish blood parameters that are modulated by different stressors, conditions, and/or treatments. We provide a compact overview of several simple plasma tests and of multiparametric analyses of fish blood, and we discuss their potential use in the assessment of fish welfare and pathologies.

This study evaluated a diverse range of markers of feeding stress to obtain a more precise assessment of the welfare of rainbow trout in relation to inadequate husbandry conditions. A feeding stress model based on dietary soybean meal was employed to identify suitable minimally invasive “classical” stress markers, together with molecular signatures. In a 56-day feeding experiment, rainbow trout were fed diets containing different levels of soybean meal. The impact of these different soybean meal diets on rainbow trout was assessed by water quality analyses, clinical health observations, classic growth and performance parameters, gut histopathology, blood-parameter measurements and multigene-expression profiling in RNA from whole blood. Soybean meal-induced enteritis was manifested phenotypically by an inflammatory reaction in the posterior section of the intestine and by diarrhoea in some trout. These inflammatory changes were associated with decreased supranuclear vacuolation. The haematocrit values and the levels of plasma cortisol and circulating lymphocytes in the blood were increased in trout that had consumed high amounts of SBM. Notably, the increased haematocrit depended significantly on the bodyweight of the individual trout. The transcript levels of certain genes (e.g., MAP3K1, LYG, NOD1, STAT1 and HSP90AB) emerged as potentially useful indicators in the blood of rainbow trout providing valuable information about inadequate nutrition. The expression-profiling findings provide a basis for improved, minimally invasive monitoring of feeding regimens in trout farming and may stimulate the development of practical detection devices for innovative aquaculture operations.

Microalgae are emerging as functional feed ingredients in aquaculture due to their immune-stimulating and stress-modulating properties. We investigated the potential of the microalgae Chlorella vulgaris as a feed supplement to improve the health and modulate microbiota and stress responses of Atlantic salmon. Triplicate groups of Atlantic salmon (~ 126 g) were reared in a recirculating aquaculture system (RAS) at 15 °C and received diets supplemented with 2% (CV2) or 14% (CV14) spray-dried C. vulgaris daily, 14% once weekly (CV14w), or a control diet (CD) for 8 weeks. Subsequently, all groups were exposed to an acute one-hour peracetic acid (CH 3 CO 3 H; PAA) treatment, a commonly used disinfectant in RAS. While CV14 increased feed conversion (FCR) significantly, feeding the diets CV2 and CV14w improved protein retention efficiency. CV14 significantly modulated beta-diversity in the intestinal digesta and mucosa, but this effect was already visible in fish fed CV2. Feeding CV14 and, to a lesser degree, CV2 increased the relative abundances of Paenarthrobacter and Trichococcus in the digesta and mucosa, which are able to metabolize complex carbohydrates. However, the same diets reduced the abundance of the lactic acid bacteria Lactobacillus and Weissella in the digesta and Floricoccus in the mucosa. Peracetic acid exposure induced systemic stress (increase in plasma glucose and cortisol) and a local immune response in the gill, with the most prominent upregulation of several immune- and stress-regulated genes ( clra , cebpb , marco , tnfrsf14 , ikba , c1ql2 , drtp1 ) 18 h after exposure in fish fed the control diet. Fish receiving CV14 once a week showed a reduced transcriptional response to PAA exposure. Catalase protein abundance in the liver increased following exposure to PAA, while superoxide dismutase abundance in the gill and liver was increased in response to C. vulgaris inclusion before stress. Overall, the results highlight that a high (14%) inclusion rate of C. vulgaris in feed for Atlantic salmon impairs feed conversion and shifts the intestinal microbiota composition in digesta and mucosa. Weekly feeding of C. vulgaris proves a viable approach in improving protein retention and improving transcriptional resilience towards oxidative stress in increasingly intensive production systems. Thereby this study may motivate future studies on optimizing temporal feeding schedules for health-promoting aquafeeds.

Via 16S rRNA gene amplicon sequencing, this study explores whether the gut mucus microbiota of rainbow trout is affected by the interaction of a plant-protein-based diet and a daily handling stressor (chasing with a fishing net) across two genetic lines (A, B). Initial body weights of fish from lines A and B were 124.7 g and 147.2 g, respectively. Fish were fed 1.5% of body weight per day for 59 days either of two experimental diets, differing in their fish meal [fishmeal-based diet (F): 35%, plant-based diet (V): 7%] and plant-based protein content (diet F: 47%, diet V: 73%). No diet- or stress-related effect on fish performance was observed at the end of the trial. However, we found significantly increased observed ASVs in the intestinal mucus of fish fed diet F compared to diet V. No significant differences in Shannon diversity could be observed between treatments. The autochthonous microbiota in fish fed with diet V was dominated by representatives of the genera Mycoplasma , Cetobacterium, and Ruminococcaceae, whereas Enterobacteriaceae and Photobacterium were significantly associated with diet F. The mucus bacteria in both genetic lines were significantly separated by diet, but neither by stress nor an interaction, as obtained via PERMANOVA. However, pairwise comparisons revealed that the diet effect was only significant in stressed fish. Therefore, our findings indicate that the mucus-associated microbiota is primarily modulated by the protein source, but this modulation is mediated by the stress status of the fish.

Microalgae are increasingly being investigated as functional feed additives in a variety of fish species, but our knowledge on how microalgae supplementation affects Atlantic salmon remains limited. We hypothesized that microalgae inclusion of 8% in the feed would improve performance, fatty acid and pigment deposition as well as health and immunity of Atlantic salmon reared in recirculating aquaculture systems (RAS). We fed Atlantic salmon smolts with five different microalgae enriched diets containing Tetraselmis chuii (TC), Arthrospira platensis (AP), Schizochytrium limacinum (SL) or Chlorella vulgaris , either intact (CVI) or as broken cell wall derivative (CVB) or a control diet (CD). After eight weeks of feeding in brackish water (13 psu), all groups were transferred to seawater (32 psu) for additional two weeks. Our results indicate that CVB improved feed conversion and protein retention, but reduced condition factor (p < 0.05) compared to fish fed with a control diet. Voluntary feed intake decreased in seawater, but was similar among diet groups. The amount of docosahexaenoic acid was particularly high in SL-fed fish and alpha-linolenic acid was enriched in fish fed CVI, CVB and TC (p < 0.05). Following seawater transfer, fat content and monounsaturated fatty acids decreased in the muscle, while polyunsaturated fatty acids increased. Lutein was present in all muscle samples, but highest concentrations were found in CVB-, CVI- and TC-fed fish. In the anterior intestine, microalgae supplementation induced differentially regulated trout protein 1 ( drtp1 ) expression in CVI- and CVB-fed fish, but reduced the expression of interleukin 1 and 10 receptor ( il1r2 & il10rb ) in CVI-fed fish. In the liver, feeding CVI and SL induced complement C1q like 2 ( c1ql2 ) expression, while reducing serum amyloid A5 ( saa5 ) expression. Superoxide-dismutase protein concentration was induced in the liver of fish fed SL, while myeloperoxidase was reduced in most microalgae-fed groups. In conclusion, we show that commercially relevant microalgae can be used as functional feed additives for Atlantic salmon promoting different health aspects without negatively affecting their growth performance when cultivated in RAS.

Background The aim of the present study was to characterize the effects of handling stress on the microbiota in the intestinal gut contents of rainbow trout ( Oncorhynchus mykiss ) fed a plant-based diet from two different breeding lines (initial body weights: A: 124.69 g, B: 147.24 g). Diets were formulated in accordance with commercial trout diets differing in their respective protein sources: fishmeal (35% in fishmeal-based diet F, 7% in plant protein-based diet V) and plant-based proteins (47% in diet F, 73% in diet V). Experimental diets were provided for 59 days to all female trout in two separate recirculating aquaculture systems (RASs; mean temperature: A: 15.17 °C ± 0.44, B: 15.42 °C ± 0.38). Half of the fish in each RAS were chased with a fishing net twice per day to induce long-term stress (Group 1), while the other half were not exposed to stress (Group 0). Results No differences in performance parameters were found between the treatment groups. By using 16S rRNA amplicon sequencing of the hypervariable region V3/V4, we examined the microbial community in the whole intestinal content of fish at the end of the trial. We discovered no significant differences in alpha diversity induced by diet or stress within either genetic trout line. However, the microbial composition was significantly driven by the interaction of stress and diet in trout line A. Otherwise, in trout line B, the main factor was stress. The communities of both breeding lines were predominantly colonized by bacteria from the phyla Fusobacteriota, Firmicutes , Proteobacteria , Actinobacteriota , and Bacteroidota. The most varying and abundant taxa were Firmicutes and Fusobacteriota , whereas at the genus level, Cetobacterium and Mycoplasma were key components in terms of adaptation. In trout line A, Cetobacterium abundance was affected by factor stress, and in trout line B, it was affected by the factor diet. Conclusion We conclude that microbial gut composition, but neither microbial diversity nor fish performance, is highly influenced by stress handling, which also interacts with dietary protein sources. This influence varies between different genetic trout lines and depends on the fish’s life history.

On 9 June 2008, the UK's largest mass stranding event (MSE) of short-beaked common dolphins (Delphinus delphis) occurred in Falmouth Bay, Cornwall. At least 26 dolphins died, and a similar number was refloated/herded back to sea. On necropsy, all dolphins were in good nutritive status with empty stomachs and no evidence of known infectious disease or acute physical injury. Auditory tissues were grossly normal (26/26) but had microscopic haemorrhages (5/5) and mild otitis media (1/5) in the freshest cases. Five lactating adult dolphins, one immature male, and one immature female tested were free of harmful algal toxins and had low chemical pollutant levels. Pathological evidence of mud/seawater inhalation (11/26), local tide cycle, and the relative lack of renal myoglobinuria (26/26) suggested MSE onset on a rising tide between 06:30 and 08∶21 hrs (9 June). Potential causes excluded or considered highly unlikely included infectious disease, gas/fat embolism, boat strike, by-catch, predator attack, foraging unusually close to shore, chemical or algal toxin exposure, abnormal weather/climatic conditions, and high-intensity acoustic inputs from seismic airgun arrays or natural sources (e.g., earthquakes). International naval exercises did occur in close proximity to the MSE with the most intense part of the exercises (including mid-frequency sonars) occurring four days before the MSE and resuming with helicopter exercises on the morning of the MSE. The MSE may therefore have been a \"two-stage process\" where a group of normally pelagic dolphins entered Falmouth Bay and, after 3-4 days in/around the Bay, a second acoustic/disturbance event occurred causing them to strand en masse. This spatial and temporal association with the MSE, previous associations between naval activities and cetacean MSEs, and an absence of other identifiable factors known to cause cetacean MSEs, indicates naval activity to be the most probable cause of the Falmouth Bay MSE.

BACKGROUND: The harbour porpoise is exposed to increasing pressure caused by anthropogenic activities in its marine environment. Numerous offshore wind farms are planned or under construction in the North and Baltic Seas, which will increase underwater noise during both construction and operation. A better understanding of how anthropogenic impacts affect the behaviour, health, endocrinology, immunology and physiology of the animals is thus needed. The present study compares levels of stress hormones and mRNA expression of cytokines and acute-phase proteins in blood samples of harbour porpoises exposed to different levels of stress during handling, in rehabilitation or permanent human care. Free-ranging harbour porpoises, incidentally caught in pound nets in Denmark, were compared to harbour porpoises in rehabilitation at SOS Dolfijn in Harderwijk, the Netherlands, and individuals permanently kept in human care in the Dolfinarium Harderwijk and Fjord & Belt Kerteminde, Denmark. Blood samples were investigated for catecholamines, adrenaline, noradrenaline and dopamine, as well as for adrenocorticotropic hormone (ACTH), cortisol, metanephrine and normetanephrine. mRNA expression levels of relevant cell mediators (cytokines IL-10 and TNFα, acute-phase proteins haptoglobin and C-reactive protein and the heat shock protein HSP70) were measured using real-time PCR. RESULTS: Biomarker expression levels varied between free-ranging animals and porpoises in human care. Hormone and cytokine ranges showed correlations to each other and to the health status of investigated harbour porpoises. Hormone concentrations were higher in free-ranging harbour porpoises than in animals in human care. Adrenaline can be used as a parameter for the initial reaction to acute stress situations; noradrenaline, dopamine, ACTH and cortisol are more likely indicators for the following minutes of acute stress. There is evidence for different correlations between production of normetanephrine, metanephrine, cortisol and the expression of IL-10, HSP70 and haptoglobin. CONCLUSIONS: The expression patterns of the selected molecular biomarkers of the immune system are promising to reflect the health and immune status of the harbour porpoise under different levels of stress.