Dietary Salicornia ramosissima improves the European seabass (Dicentrarchus labrax) inflammatory response against Photobacterium damselae piscicida.

Introduction: Modern fish farming faces challenges in sourcing feed ingredients, most related with their prices, 21 availability, and specifically for plant protein sources, competition for the limited cultivation space for 22 vegetable crops. In that sense, halophytes have the added value of being rich in valuable bioactive compounds and salt tolerant. This study assessed the inclusion of non-food fractions of S. ramosissima in European seabass diets. Methods: Different levels (2.5%, 5%, and 10%) were incorporated into seabass diets, replacing wheat meal (diets ST2.5, ST5, and ST10) or without inclusion (CTRL). Experimental diets were administered to seabass juveniles (8.62 ± 0.63 g) for 34 and 62 days and subsequent inflammatory responses to a heat-inactivated Photobacterium damselae subsp. piscicida (Phdp) were evaluated in a time-course manner (4, 24, 48, and 72 h after the challenge). At each sampling point, seabass haematological profile, plasma immune parameters, and head-kidney immune-related gene expression were evaluated. Results: After both feeding periods, most parameters remained unaltered by S. ramosissima inclusion; nonetheless, seabass fed ST10 showed an upregulation of macrophage colony-stimulating factor 1 receptor 1 (mcsf1r1) and cluster of differentiation 8 (cd8ß) compared with those fed CTRL after 62 days of feeding. Regarding the inflammatory response, seabass fed ST10 showed lower plasma lysozyme levels than their counterparts fed ST2.5 and ST5 at 24 h following injection, while 4 h after the inflammatory stimulus, seabass fed ST10 presented higher numbers of peritoneal leucocytes than fish fed CTRL. Moreover, at 4 h, fish fed ST2.5, ST5, and ST10 showed a higher expression of interleukin 1ß (il1ß), while fish fed ST5 showed higher levels of ornithine decarboxylase (odc) than those fed CTRL. An upregulation of macrophage colony-stimulating factor 1 receptor 1 (mcsf1r1) and glutathione peroxidase (gpx) was also observed at 72 h in fish fed ST10 or ST5 and ST10 compared with CTRL, respectively. Discussion: In conclusion, incorporating up to 10% of the non-food fraction S. ramosissima in feed did not compromise seabass growth or immune status after 62 days, aligning with circular economy principles. However, S. ramosissima inclusion improved the leucocyte response and upregulated key immune-related genes in seabass challenged with an inactivated pathogen.

Exploring the Effects of Acute Stress Exposure on Lumpfish Plasma and Liver Biomarkers.

This study aimed to expand knowledge on lumpfish stress physiology by investigating the effects of acute stress on primary (i.e., cortisol) and secondary (e.g., metabolites) stress responses, as well as oxidative stress biomarkers, from stress exposure to a recovery phase. The results showed that the lumpfish physiological response to 1 min air exposure is mild, in line with recent studies, and comparable to that described for white sturgeons. Cortisol seems to be the most reliable acute stress biomarker in lumpfish, with a significant increase in plasma 30 min after stress exposure, returning to resting levels 2 h after exposure. In contrast, glucose and lactate were not significantly altered by short-term air exposure. Effects on hepatic energy mobilisation were also detected following the acute stress. This study showed that acute 1 min air exposure seems tolerable, allowing a swift recovery. However, more studies on the impacts of air exposure and repeated acute stressors on lumpfish stress and immune responses are required to develop industry standards for lumpfish health and welfare monitoring.

Chlorella vulgaris Extracts as Modulators of the Health Status and the Inflammatory Response of Gilthead Seabream Juveniles (Sparus aurata).

This study aimed to evaluate the effects of short-term supplementation, with 2% Chlorella vulgaris (C. vulgaris) biomass and two 0.1% C. vulgaris extracts, on the health status (experiment one) and on the inflammatory response (experiment two) of gilthead seabream (Sparus aurata). The trial comprised four isoproteic (50% crude protein) and isolipidic (17% crude fat) diets. A fishmeal-based (FM), practical diet was used as a control (CTR), whereas three experimental diets based on CTR were further supplemented with a 2% inclusion of C. vulgaris biomass (Diet D1); 0.1% inclusion of C. vulgaris peptide-enriched extract (Diet D2) and finally a 0.1% inclusion of C. vulgaris insoluble fraction (Diet D3). Diets were randomly assigned to quadruplicate groups of 97 fish/tank (IBW: 33.4 ± 4.1 g), fed to satiation three times a day in a recirculation seawater system. In experiment one, seabream juveniles were fed for 2 weeks and sampled for tissues at 1 week and at the end of the feeding period. Afterwards, randomly selected fish from each group were subjected to an inflammatory insult (experiment two) by intraperitoneal injection of inactivated gram-negative bacteria, following 24 and 48 h fish were sampled for tissues. Blood was withdrawn for haematological procedures, whereas plasma and gut tissue were sampled for immune and oxidative stress parameters. The anterior gut was also collected for gene expression measurements. After 1 and 2 weeks of feeding, fish fed D2 showed higher circulating neutrophils than seabream fed CTR. In contrast, dietary treatments induced mild effects on the innate immune and antioxidant functions of gilthead seabream juveniles fed for 2 weeks. In the inflammatory response following the inflammatory insult, mild effects could be attributed to C. vulgaris supplementation either in biomass form or extract. However, the C. vulgaris soluble peptide-enriched extract seems to confer a protective, anti-stress effect in the gut at the molecular level, which should be further explored in future studies.

Dietary supplementation with Gracilaria gracilis by-products modulates the immune status and oxidative stress response of gilthead seabream (Sparus aurata) stimulated with Photobacterium damselae subsp. piscicida.

This study evaluated the effects of agar waste (AW) dietary supplementation, obtained from the seaweed Gracilaria gracilis cultivated under two different spectral lights, neutral (NT) and blue (BL), on haematological parameters, inflammatory response, and antioxidant biomarkers of gilthead seabream (Sparus aurata). Three diets were prepared: i) a basal diet (CTR), ii) a diet supplemented with 2.5% NT, and iii) a diet supplemented with 2.5% BL. After 15 days of feeding, fish were injected with PBS (placebo) or inactivated Photobacterium damselae subsp. piscicida (stimulated) and sampled at 4 h and 24 h post-stimulus. Results indicated that fish fed NT and BL supplemented diets had lower Ht value and mean corpuscular volume (MCV) than fish fed the CTR diet, regardless of the stimulus and the sampling time. No differences in mean corpuscular haemoglobin (MCH) were found between fish fed the different diets, while the mean corpuscular haemoglobin concentration (MCHC) increased in fish fed AW supplemented diets compared to fish fed the CTR diet, regardless of the stimulus and the sampling time. In response to inflammation, fish fed the NT diet displayed higher neutrophils count in blood when compared to the CTR group, regardless of the stimulus and sampling time. Thrombocyte count was higher in fish fed NT and BL diets than in the CTR group, especially in the stimulated fish (Diet*injection (D*I), P = 0.004). An increase in plasma protease activity was detected in fish fed NT or BL diets in both placebo and stimulated fish regardless of the sampling time. Hepatic catalase activity was higher in fish fed the NT and BL than in the CTR group, particularly in the stimulated fish (D*I, P < 0.001). In addition, both stimulated and placebo fish that received the BL diet showed an increase in hepatic GR activity compared to the CTR group, regardless of the sampling time. Dietary supplementation with AW by-products obtained from G. gracilis cultured under NT and BL conditions showed to improve the inflammatory and antioxidant mechanisms in gilthead seabream in response to a UV-killed bacterial stimulus, having valuable applications for the sustainable use of seaweed toward improving the health and welfare of cultured fish.

Dietary Histidine, Threonine, or Taurine Supplementation Affects Gilthead Seabream (Sparus aurata) Immune Status.

AAs have become interesting feed ingredients to be used in functional fish feeds as not only are they protein building blocks, but they also participate in several other key metabolic processes. In the present study, a comprehensive analysis of transcriptomics, hematology, and humoral immune parameters (plasma and skin mucus) were measured twice over the course of the feeding trial (four weeks). Plasma antiprotease activity increased in fish fed Thr compared to those fed the CTRL and Tau treatments, regardless of sampling time. The bactericidal activity in skin mucus decreased in fish fed Tau and His treatments compared to those fed the CTRL diet after two weeks. The membrane IgT (mIgT) was upregulated in fish fed Tau after four weeks, while C-type lectin domain family domain 10 member (clec10a) was downregulated in fish fed Thr after two weeks of feeding. By comparing the molecular signatures of head-kidney by means of a PLS-DA, it is possible to visualize that the main difference is between the two sampling points, regardless of diet. Altogether, these results suggest that dietary supplementation with these AAs at the tested levels causes mild immune-modulation effects in gilthead seabream, which should be further studied under disease challenge conditions.

Short-Term Supplementation of Dietary Arginine and Citrulline Modulates Gilthead Seabream (Sparus aurata) Immune Status.

Several amino acids (AA) are known to regulate key metabolic pathways that are crucial for immune responses. In particular, arginine (ARG) appears to have important roles regarding immune modulation since it is required for macrophage responses and lymphocyte development. Moreover, citrulline (CIT) is a precursor of arginine, and it was reported as an alternative to ARG for improving macrophage function in mammals. The present study aimed to explore the effects of dietary ARG and CIT supplementation on the gilthead seabream (Sparus aurata) immune status. Triplicate groups of fish (23.1 ± 0.4 g) were either fed a control diet (CTRL) with a balanced AA profile, or the CTRL diet supplemented with graded levels of ARG or CIT (i.e., 0.5 and 1% of feed; ARG1, CIT1, ARG2, and CIT2, respectively). After 2 and 4 weeks of feeding, fish were euthanized and blood was collected for blood smears, plasma for humoral immune parameters and shotgun proteomics, and head-kidney tissue for the measurement of health-related transcripts. A total of 94 proteins were identified in the plasma of all treatments. Among them, components of the complement system, apolipoproteins, as well as some glycoproteins were found to be highly abundant. After performing a PLS of the expressed proteins, differences between the two sampling points were observed. In this regard, component 1 (61%) was correlated with the effect of sampling time, whereas component 2 (18%) seemed associated to individual variability within diet. Gilthead seabream fed ARG2 and CIT2 at 4 weeks were more distant than fish fed all dietary treatments at 2 weeks and fish fed the CTRL diet at 4 weeks. Therefore, data suggest that the modulatory effects of AA supplementation at the proteome level were more effective after 4 weeks of feeding and at the higher inclusion level (i.e., 1% of feed). The bactericidal activity increased in fish fed the highest supplementation level of both AAs after 4 weeks. Peripheral monocyte numbers correlated positively with nitric oxide, which showed an increasing trend in a dose-dependent manner. The colony-stimulating factor 1 receptor tended to be up-regulated at the final sampling point regardless of dietary treatments. Data from this study point to an immunostimulatory effect of dietary ARG or CIT supplementation after 4 weeks of feeding in the gilthead seabream, particularly when supplemented at a 1% inclusion level.

Dietary tryptophan supplementation induces a transient immune enhancement of gilthead seabream (Sparus aurata) juveniles fed fishmeal-free diets.

European aquaculture is an industry with a high sustainability profile contributing to the supply of safe seafood. However, several diseases can affect farmed fish and it is imperative to find alternatives for chemotherapeutic treatments when disease outbreaks occur. Maintenance of health through nutrition is well-establish in modern animal farming, and amino acids (AA) are promising candidates as functional additives to improve fish health. Therefore, the goal of this research is to provide a better understanding of the influence of tryptophan supplementation on nutritional condition and immune mechanisms in fish. Triplicate groups of fish (13.3 ±â€¯0.3g) previously fed with a fishmeal-based diet were either fed a control diet with an extreme formulation (0% fishmeal) but meeting the AA requirements (CTRL), or the SUP diet, formulated as the CTRL with an increase in tryptophan (TRP) content. After 2 and 13 weeks of feeding, head-kidney (HK), liver (L) and white skeletal muscle (WSM) were collected for gene expression, whereas plasma was suited for humoral immune parameters. A holistic approach using transcriptomic, humoral and zootechnical parameters was undertaken. The expression of 29-31 genes for WSM, L or HK confirms an effect due to the treatment across time. A two-way ANOVA analysis revealed that 15-24 genes varied significantly depending on the tissue, and the multivariate analysis by means of PLS-DA explained (R2) and predicted (Q2) with four components up to 93% and 78% of total variance, respectively. Component 1 (R2 = 50.06%) represented the time effects, whereas components 2 (24.36%) and 3 (13.89%) grouped fish on the basis of dietary treatment, at early sampling. The HK results in particular suggest that fish fed SUP diet displayed an immunostimulated state at 2 weeks. No major differences were observed in plasma humoral parameters, despite an increase in antiprotease and peroxidase activities after 13 weeks regardless of dietary treatment. These results suggest that tryptophan supplementation may improve the seabream immune status after 2 weeks. Hence, the use of functional feeds is especially relevant during a short-term feeding period before a predictable stressful event or disease outbreak, considering that these putative advantageous effects seem to disappear after a 13 weeks feeding period.

Dietary Creatine Supplementation in Gilthead Seabream (Sparus aurata) Increases Dorsal Muscle Area and the Expression of myod1 and capn1 Genes.

Creatine (Cr) is an amino acid derivative with an important role in the cell as energy buffer that has been largely used as dietary supplement to increase muscle strength and lean body mass in healthy individuals and athletes. However, studies in fish are scarce. The aim of this work is to determine whether dietary Cr supplementation affects muscle growth in gilthead seabream (Sparus aurata) juveniles. Fish were fed ad libitum for 69 days with diets containing three increasing levels of creatine monohydrate (2, 5, and 8%) that were compared with a non-supplemented control (CTRL) diet. At the end of the trial, the fast-twist skeletal muscle growth dynamics (muscle cellularity) and the expression of muscle-related genes were evaluated. There was a general trend for Cr-fed fish to be larger and longer than those fed the CTRL, but no significant differences in daily growth index (DGI) were registered among dietary treatments. The dorsal cross-sectional muscle area (DMA) of fish fed Cr 5 and Cr 8% was significantly larger than that of fish fed CTRL. The groups supplemented with Cr systematically had a higher relative number of both small-sized (≤20 µm) and large-sized fibers (≥120 µm). Dorsal total fibers number was highest in fish fed 5% Cr. In fish supplemented with 5% Cr, the relative expression of myogenic differentiation 1 (myod1) increased almost four times compared to those fed the CTRL diet. The relative expression of calpain 3 (capn3) was highest in fish fed diets with 2% Cr supplementation, but did not differ significantly from those fed the CTRL or Cr 5%. The myod1 gene expression had a positive and significant correlation with that of capn1, capns1a, and capn3 expression. These results suggest that the observed modulation of gene expression was not enough to produce a significant alteration in muscle phenotype under the tested conditions, as a non-significant increase in muscle fiber diameter and higher total number of fiber was observed, but still resulted in increased DMA. Additional studies may be required in order to better clarify the effect of dietary Cr supplementation in fish, possibly in conjunction with induced resistance training.