Metabolic and Stress Responses in Senegalese Soles (Solea senegalensis Kaup) Fed Tryptophan Supplements: E ects of Concentration and Feeding Period

The objective of this study was to assess the impact of di erent dietary Trp concentrations on the stress and metabolism response of juvenile Senegalese soles (Solea senegalensis). Fish (38.1 1.9 g) were fed di erent Trp-enriched feeds (0%, 1% and 2% Trp added) for two and eight days, and later exposed to air stress for three min. Samples were taken pre- and 1 h post-stress (condition). Plasma cortisol, lactate, glucose and proteins were significantly a ected by the sampling time, showing higher values at 1 h post-stress. Trp concentration in food also had significant e ects on lactate and glucose levels. However, the feeding period did not a ect these parameters. Post-stress values were higher than in the pre-stress condition for every plasma parameter, except for lactate in two days and 1% Trp treatment. Nevertheless, cortisol, glucose and lactate did not vary significantly between pre- and post-stress samplings in fish fed the 1% Trp-enriched diet for two days. The lack of variability in cortisol response was also due to the high pre-stress value, significantly superior to pre-stress control. The exposure time to Trp feeding did not significantly a ect any enzyme activity; however, Trp added and condition influenced protein-related enzyme activities. In spite of decreasing stress markers, Trp-enriched diets altered the protein metabolism

Vasotocin and isotocin regulate aquaporin 1 function in the sea bream

Aquaporins (AQPs) are specific transmembrane water channels with an important function in water homeostasis. In terrestrial vertebrates, AQP2 function is regulated by vasopressin (AVP) to accomplish key functions in osmoregulation. The endocrine control of aquaporin function in teleosts remains little studied. Therefore, in this study we investigated the regulatory role of vasotocin (AVTR) and isotocin (ITR) receptors in Aqp1 paralog gene function in the teleost gilthead sea bream (Sparus aurata). The complete coding regions of Aqp1a, Aqp1b, AVTR V1a2-type, AVTR V2-type and ITR from sea bream were isolated. A Xenopus oocyte-swelling assay was used to functionally characterize AQP1 function and regulation by AVT and IT through their cognate receptors. Microinjection of oocytes with Aqp1b mRNA revealed regulation of water transport via PKA (IBMX+forskolin sensitive), whereas Aqp1a mRNA injection had the same effect via PKC signaling (PDBU sensitive). In the absence of expressed receptors, AVT and IT (10(-8) mol l(-1)) were unable to modify AQP1 function. AVT regulated AQP1a and AQP1b function only when the AVTR V2-type was co-expressed. IT regulated AQP1a function, but not AQP1b, only when ITR was present. Considering that Aqp1a and Aqp1b gene expression in the sea bream intestine is highly salinity dependent in vivo, our results in ovo demonstrate a regulatory role for AVT and IT in AQP1 function in the sea bream in the processing of intestinal fluid to achieve osmoregulation.Ministry of Education (Spain) [FPU AP2008-01194]; Portuguese National Science Foundation [SFRH/BPD/66808/2009]; Ministry of Science and Education, Spain [AGL2010-14876]; National Science Foundation (FCT, Portugal) [PTDC/MAR-BIO/3811/2012

Evaluation of the Inclusion of the Green Seaweed Ulva ohnoi as an Ingredient in Feeds for Gilthead Sea Bream (Sparus aurata) and European Sea Bass (Dicentrarchus labrax)

Simple Summary The use of seaweeds in aquafeeds is receiving increasing attention due to their potential nutritional and functional benefits. However, several green seaweeds such as Ulva presents nutritional limitations because of the undigestible polysaccharides, although these may exert a positive effect on the immunological status of the fish. The present study developed three different experiments aimed to re-evaluate the presence of protease inhibitors described for Ulva ohnoi, to assess its nutritional value as an ingredient and also to evaluate its potential protective effect on the oxidative metabolism of fish, being experiments developed in two different fish species (European sea bass Dicentrarchus labrax and gilthead sea bream, Sparus aurata). Results indicate the absence of negative effects of U. ohnoi on protein digestion of sea bream but a limited value as a feed ingredient. In contrast, its contents in bioactives seem to be correlated to the observed positive effects on the immune status and oxidative metabolism when fish are challenged by the consumption of highly oxidized dietary oil. This study evaluated the use of Ulva ohnoi as an ingredient in feeds for aquaculture in three different experiments. Experiment 1 was oriented to confirm the negative effect of U. ohnoi on fish digestion. Experiment 2 assessed the effect on growth, feed efficiency, and immune status of juvenile sea bass (Dicentrarchus labrax) fed on diets including U. ohnoi, previously treated or not with carbohydrases used to partially hydrolyze indigestible polysaccharides. Experiment 3 was aimed to evaluate the potential protective effect of U. ohnoi on the oxidative status of sea bream (Sparus aurata) challenged by the consumption of a feed formulated with the oil fraction completely oxidized. Results show a negligible effect of U. ohnoi meal on protein digestion when included in feeds at levels of 10% or less. Moreover, results of growth and feed use evidenced the possibility of using up to 5% inclusion of algal meal in feeds without adverse effects on the zootechnical parameters, while the enzyme pretreatment was ineffective to improve its nutritional use. Finally, the inclusion of U. onhoi in feeds determined both an immunostimulatory effect, evidenced by an increase in skin mucus lysozyme in the two mentioned fish species, and a positive influence on the oxidative metabolism of seabream when fed on a diet including rancid oil.This research was funded by the Excellence Campus of Marine Science (CEIMAR) within the "III Call for Innovative Projects in the field of Blue Economy"

Arginine Vasotocin and Cortisol Co-regulate Vasotocinergic, Isotocinergic, Stress, and Thyroid Pathways in the Gilthead Sea Bream (Sparus aurata)

In teleosts, a complex interaction between several endocrine axes modulates physiological functions related to metabolism, stress, and osmoregulation. Although many studies in fish underline the interconnection between the hypothalamic– pituitary–interrenal (HPI) and hypothalamic–pituitary–thyroid (HPT) endocrine axes, their relationship with the vasotocinergic and isotocinergic systems remains unknown. The aim of the present study is therefore to shed light on the potential cross-regulations between HPT, HPI, and the vasotocinergic and isotocinergic axes in gilthead sea bream (Sparus aurata) at hypothalamic, hypophyseal, and plasma levels. Sea breams were administered with intraperitoneal slow-release implants containing different doses of vasotocin (the active peptide in vasotocinergic system) or cortisol (the last component of HPI axis). Plasma osmolality was higher in active neuropeptides vasotocin (Avt)- treated fish, indicating an osmoregulatory function of this hormone. Low concentrations of Avt increased hypothalamic arginine vasotocin precursor (avt) mRNA levels and increased Avt storage in the pituitary. Avt treatment down-regulated hypothalamic arginine vasotocin receptor v1a-type (avtrv1a), suggesting a negative paracrine coregulation of the HPI axis due to the close location of avtrv1a and adrenocorticotropin hormone (Acth) cells in the anterior pituitary. Furthermore, the up-regulation observed in arginine vasotocin receptor v2-type (avtrv2) suggests their involvement in metabolic and cortisol-related pathways in the hypothalamus. The decrease in isotocin (It) pituitary storage and the up-regulation of it receptor, observed in the Avt-treated group, reinforce the idea of an interconnection between the vasotocinergic and isotocinergic systems. Cortisol and Avt administration each inhibited the HPI axis, down-regulating crh gene expression in the absence of variations in corticotropin releasing hormone binding protein (crhbp). Finally, both hormonal treatments activated the HPT axis via upregulation of trh and down-regulation of thrb. Our results provide evidence for strong interactions among the Avt/It, HPI, and HPT axes of marine teleosts, particularly at the hypothalamic level

Solid-State Hydrolysis (SSH) Improves the Nutritional Value of Plant Ingredients in the Diet of Mugil cephalus

The possibility of improving the nutritional quality of plant byproducts (brewers’ spent grain and rice bran) through an enzyme treatment was tested in a formulated feed for grey mullet (Mugil cephalus). The enzyme treatment was carried out by Solid-State Hydrolysis (SSH) using a commercial preparation including carbohydrases and phytase. A feed prepared without the treatment and a commercial feed for carp were used as controls. In a preliminary short-term trial carried out at laboratory facilities, fish receiving the enzyme-treated feed showed significant improvement in both FCR and SGR when compared to those obtained with the untreated diet, although both experimental diets presented worse values than those obtained with the commercial feed. Different metabolic indicators including higher values of muscle glycogen and plasmatic triglycerides supported the positive effect of the enzyme treatment on the nutritional condition of the fish over those fed on the diet containing non-treated ingredients. Results of growth and feed efficiency that were obtained in a second long-term trial developed for 148 days under real production conditions evidenced the equivalence among the experimental and commercial diets and confirmed that enzyme pretreatment of plant ingredients by SSH may be a useful procedure to improve the nutritive value of high fiber plant byproducts when included in practical diets for this species and others with similar nutritional features

Cloning and molecular ontogeny of digestive enzymes in fed and food-deprived developing gilthead seabream (Sparus aurata) larvae

We have determined the expression pattern of key pancreatic enzymes precursors (trypsinogen, try; chymotrypsinogen, ctrb; phospholipase A2, pla2; bile salt-activated lipase, cel; and α-amylase, amy2a) during the larval stage of gilthead seabream (Sparus aurata) up to 60 days after hatching (dph). Previously, complete sequences of try, cel, and amy2awere cloned and phylogenetically analyzed. One new isoformwas found for cel transcript (cel1b). Expression of all enzyme precursors was detected before the mouth opening. Expression of try and ctrb increased during the first days of development and then maintained high values with some fluctuations during the whole larval stage. The prolipases pla2 and cel1b increased from first-feeding with irregular fluctuation until the end of the experiment. Contrarily, cel1a maintained lowexpression values duringmost of the larval stage increasing at the end of the period. Nevertheless, cel1a expression was negligible as compared with cel1b. The expression of amy2a sharply increased during the first week followed by a gradual decrease. In addition, a fooddeprivation experiment was performed to find the differences in relation to presence/absence of gut content after the opening of the mouth. The food-deprived larvae died at 10 dph. The expression levels of all digestive enzymes increased up to 7 dph, declining sharply afterwards. This expression pattern up to 7 dphwas the same observed in fed larvae, confirming the genetic programming during the early development.Main digestive enzymes in gilthead seabream larvae exhibited the same expression profiles than other marine fish with carnivorous preferences in their juvenile stages

Ontogeny of Expression and Activity of Digestive Enzymes and Establishment of gh/igf1 Axis in the Omnivorous Fish Chelon labrosus

Simple Summary Thick-lipped grey mullet (Chelon labrosus) feeds on the lowest trophic levels during adult stages, for which it is considered a viable candidate for an economically and environmentally sustainable aquaculture. Similar to most of marine fish species, C. labrosus produce a large number of eggs, leading to morphologically and anatomically larvae that are not completely mature and have to pass through substantial differentiation and development in their functional systems to acquire adult features. Therefore, the study of the development of digestive tract and of the growth regulation can provide useful information to adapt the feeding protocols and rearing conditions to the physiological requirements at each stage. This work aimed to evaluate the early ontogeny of key digestive enzymes and somatotropic factors at biochemical and/or transcriptional levels. Our results evidenced that maturation of the digestive system and acquisition of the adult mode of digestion occurs around 60 to 70 days post hatch (dph), when starch or other low-cost carbohydrate-based compounds could be used in formulated diets at increasing levels. Furthermore, our results implied an independent expression of the studied somatotropic genes during the first 40 dph and establishment of a functional growth hormone/insulin-like growth factor 1 axis from 50 dph onward. Abstract Thick-lipped grey mullet (Chelon labrosus) is a candidate for sustainable aquaculture due to its omnivorous/detritivorous feeding habit. This work aimed to evaluate its digestive and growth potentials from larval to early juvenile stages. To attain these objectives the activity of key digestive enzymes was measured from three until 90 days post hatch (dph). Expression of genes involved in digestion of proteins (try2, ctr, pga2, and atp4a), carbohydrates (amy2a), and lipids (cel and pla2g1b), together with two somatotropic factors (gh and igf1) were also quantified. No chymotrypsin or pepsin activities were detected. While specific activity of trypsin and lipase were high during the first 30 dph and declined afterward, amylase activity was low until 57 dph and increased significantly beyond that point. Expression of try2, ctr, amy2a, and cel increased continuously along development, and showed a peak at the end of metamorphosis. Expression of pla2g1b, pga2 and atp4a increased until the middle of metamorphosis and decreased afterwars. Most of these trends contrast the usual patterns in carnivorous species and highlight the transition from larvae, with high protein requirements, to post-larvae/juvenile stages, with omnivorous/detritivorous feeding preferences. Somatotropic genes, gh and igf1, showed approximately inverse expression patterns, suggesting the establishment of the Gh/Igf1 axis from 50 dph

Essential Oils as Stress-Reducing Agents for Fish Aquaculture: A Review

In fish, stressful events initiate a hormone cascade along the hypothalamus-pituitaryinterrenal and hypothalamus-sympathetic-chromaffin (HSC) axis to evoke several physiological reactions in order to orchestrate and maintain homeostasis. Several biotic and abiotic factors, as well as aquaculture procedures (handling, transport, or stocking density), activated stress system inducing negative effects on different physiological processes in fish (growth, reproduction, and immunity). In order to reduce these consequences, the use of essential oils (EOs) derived from plants has been the focus of aquaculture studies due to their diverse properties (e.g., anesthetic, antioxidant, and antimicrobial), which have been shown to reduce biochemical and endocrine alterations and, consequently, to improve the welfare status. Recently, several studies have shown that biogenic compounds isolated from different EOs present excellent biological activities, as well as the nanoencapsulated form of these EOs may potentiate their effects. Overall, EOs presented less side effects than synthetic compounds, but their stress-reducing efficacy is related to their chemical composition, concentration or chemotype used. In addition, their species-specific actions must be clearly established since they can act as stressors by themselves if their concentrations and chemotypes used are not suitable. For this reason, it is necessary to assess the effect of these natural compound mixtures in different fish species, from marine to freshwater, in order to find the ideal concentration range and the way for their administration to obtain the desired biological activity, without any undesired side effects. In this review, the main findings regarding the use of different EOs as stress reducers will be presented to highlight the most important issues related to their use to improve fish welfare in aquaculture

Dietary supplementation of heat-treated Gracilaria and Ulva seaweeds enhanced acute hypoxia tolerance in gilthead sea bream (Sparus aurata)

Intensive aquaculture practices involve rearing fish at high densities. In these conditions, fish may be exposed to suboptimal dissolved O2 levels with an increased formation of reactive O2 species (ROS) in tissues. Seaweeds (SW) contain biologically active substances with efficient antioxidant capacities. This study evaluated the effects of dietary supplementation of heat-treated SW (5% Gracilaria vermiculophylla or 5% Ulva lactuca) on stress bioindicators in sea bream subjected to a hypoxic challenge. 168 fish (104.5 g average weight) were distributed in 24 tanks, in which eight tanks were fed one of three experimental diets for 34 days: (i) a control diet without SW supplementation, (ii) a control diet supplemented with Ulva, or (iii) a control diet with Gracilaria. Thereafter, fish from 12 tanks (n=4 tanks/dietary treatment) were subjected to 24 h hypoxia (1.3 mg O2 l-1) and subsequent recovery normoxia (8.6 mg O2 l-1). Hypoxic fish showed an increase in hematocrit values regardless of dietary treatment. Dietary modulation of the O2-carrying capacity was conspicuous during recovery, as fish fed SW supplemented diets displayed significantly higher haemoglobin concentration than fish fed the control diet. After the challenge, survival rates in both groups of fish fed SW were higher, which was consistent with a decrease in hepatic lipid peroxidation in these groups. Furthermore, the hepatic antioxidant enzyme activities were modulated differently by changes in environmental O2 condition, particularly in sea bream fed the Gracilaria diet. After being subjected to hypoxia, the gene expression of antioxidant enzymes and molecular chaperones in liver and heart were down regulated in sea bream fed SW diets. This study suggests that the antioxidant properties of heat-treated SW may have a protective role against oxidative stress. The nature of these compounds and possible mechanisms implied are currently being investigated.Fil: Magnoni, Leonardo Julián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina. Universidad de Porto; PortugalFil: Martos Sitcha, Juan Antonio. Consejo Superior de Investigaciones Científicas; EspañaFil: Queiroz, Augusto. Universidad de Porto; PortugalFil: Calduch Giner, Josep Alvar. Consejo Superior de Investigaciones Científicas; EspañaFil: Magalhaes Gonçalves, Jose Fernando. Universidad de Porto; PortugalFil: Rocha, Cristina M.R.. Universidad de Porto; PortugalFil: Abreu, Helena T.. ALGAplus; PortugalFil: Schrama, Johan W.. Wageningen University; Países BajosFil: Ozorio, Rodrigo O.A.. Universidad de Porto; PortugalFil: Perez Sanchez, Jaume. Consejo Superior de Investigaciones Científicas; Españ

Targeting the Mild-Hypoxia Driving Force for Metabolic and Muscle Transcriptional Reprogramming of Gilthead Sea Bream (Sparus aurata) Juveniles

Reduced oxygen availability generates a number of adaptive features across all the animal kingdom, and the goal of this study was targeting the mild-hypoxia driving force for metabolic and muscle transcriptional reprogramming of gilthead sea bream juveniles. Attention was focused on blood metabolic and muscle transcriptomic landmarks before and after exhaustive exercise. Our results after mild-hypoxia conditioning highlighted an increased contribution of lipid metabolism to whole energy supply to preserve the aerobic energy production, a better swimming performance regardless of changes in feed intake, as well as reduced protein turnover and improved anaerobic fitness with the restoration of normoxia. On-growing juveniles of gilthead sea bream were acclimated for 45 days to mild-hypoxia (M-HYP, 40-60% O-2 saturation), whereas normoxic fish (85-90% O-2 saturation) constituted two different groups, depending on if they were fed to visual satiety (control fish) or pair-fed to M-HYP fish. Following the hypoxia conditioning period, all fish were maintained in normoxia and continued to be fed until visual satiation for 3 weeks. The time course of hypoxia-induced changes was assessed by changes in blood metabolic landmarks and muscle transcriptomics before and after exhaustive exercise in a swim tunnel respirometer. In M-HYP fish, our results highlighted a higher contribution of aerobic metabolism to whole energy supply, shifting towards a higher anaerobic fitness following normoxia restoration. Despite these changes in substrate preference, M-HYP fish shared a persistent improvement in swimming performance with a higher critical speed at exercise exhaustion. The machinery of muscle contraction and protein synthesis and breakdown was also largely altered by mild-hypoxia conditioning, contributing this metabolic re-adjustment to the positive regulation of locomotion and to the catch-up growth response during the normoxia recovery period. Altogether, these results reinforce the presence of large phenotypic plasticity in gilthead sea bream, and highlights mild-hypoxia as a promising prophylactic measure to prepare these fish for predictable stressful events.This work was financially supported by a grant from the European Commission of the European Union under the Horizon 2020 research infrastructure project AQUAEXCEL2020 (652831) to J.P-S. Additional funding was obtained by a Spanish MICINN project (Bream-AquaINTECH, RTI2018-094128-B-I00). J.A.M.-S. received a Postdoctoral Research Fellowship (Juan de la Cierva-Formacion, Reference FJCI-2014-20,161)