Serum proteinogram of gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) as a new useful approach for detecting loss of haemostasis.

Proteinograms, a semiquantitative analytical method that separates proteins into multiple bands, have not been explored in teleosts for diagnostic or prognostic purposes. This study aimed to establish reference values for proteinograms in the serum of gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax), two important farmed fish species in the Mediterranean region. Serum proteins were studied using SDS-PAGE, electropherogram, and HPLC-mass spectrometry. SDS-PAGE analysis revealed four major bands of proteins around 11, 25, 70, and 100 kDa in the serum of gilthead seabream and European sea bass. Electropherogram results showed that a protein with a molecular weight of 76.8 kDa was the most abundant protein in the serum of gilthead seabream, while a peak of 75.5 kDa was the most abundant in European sea bass. HPLC-mass spectrometry detected 87 proteins and 119 proteins in the serum of gilthead seabream and European sea bass, respectively, including α1-globulins, α2-globulins, ß-globulins, and γ-globulins. Notably, the albumin sequence was not detected in either of the two species. These results help to characterize the serum protein profile and to establish reference proteinograms for these two fish species. They also provide a basis for the development of novel approaches for the rapid detection of loss of haemostasis due to stress, health disorders or disease in farmed fish.

Comparative analysis of blood protein fractions in two mediterranean farmed fish: Dicentrarchus labrax and Sparus aurata.

Total protein levels in fish are widely used in health and welfare studies, providing a simple and accessible measure. However, the multifaceted role of blood proteins makes it sometimes challenging to link total protein content to specific health issues, while specific protein fractions may offer more precise insights into fish biology and health, particularly in farmed fish species where such data is often lacking. Data were gathered from two experiments involving Dicentrarchus labrax and Sparus aurata, key species in European marine aquaculture. The aim was (1) to assess how different globulin fractions contribute to total protein content in blood and (2) how these contributions vary across different sampling times in healthy animals. In D. labrax, the beta1 globulin fraction emerged as the major contributor (34.16%), followed by albumin and alpha2 globulins (18.24% and 16.41%, respectively). In contrast, pre-albumins and alpha1 fractions had the least contribution (5.49% and 7.71%). S. aurata exhibited albumin as the primary contributor (23.39%), followed by beta1 and alpha2 globulins (19.71% and 19.15%, respectively), with gamma and alpha1 fractions contributing the least (5.34% and 8.63%). Notably, the study revealed relatively stable contributions of globulin fractions to total proteins within both species, albeit with minor variations over time, potentially linked to environmental and individual factors. Furthermore, larger fish displayed higher total protein levels. This research underscores the need for further investigation into the diverse factors influencing globulin contributions to total proteins, ultimately enhancing health and welfare monitoring for farmed fish species.

Short-Term Immune Responses of Gilthead Seabream (Sparus aurata) Juveniles against Photobacterium damselae subsp. piscicida.

Photobacteriosis is a septicaemic bacterial disease affecting several marine species around the globe, resulting in significant economic losses. Although many studies have been performed related to the pathogen virulence and resistance factors, information regarding the host defence mechanisms activated once an infection takes place is still scarce. The present study was designed to understand innate immune responses of farmed juvenile gilthead seabream (Sparus aurata) after Photobacterium damselae subsp. piscicida (Phdp) infection. Therefore, two groups of seabream juveniles were intraperitoneally injected with 100 µL of PBS (placebo) or 100 µL of exponentially growing Phdp (1 × 106 CFU/mL; infected). The blood, plasma, liver, and head kidney of six fish from each treatment were sampled immediately before infection and 3, 6, 9, 24 and 48 h after infection for the broad screening of fish immune and oxidative stress responses. Infected animals presented marked anaemia, neutrophilia and monocytosis, conditions that are correlated with an increased expression of genes related to inflammation and phagocytic activity. Similar studies with different fish species and bacteria can be useful for the definition of health biomarkers that might help fish farmers to prevent the occurrence of such diseases.

Sustained swimming exercise training decreases the individual variation in the metabolic phenotype of gilthead sea bream (Sparus aurata).

Cultured fish can be induced to swim, although the suitability and benefits remain to be tested. Sustained swimming exercise (SSE) training and detraining (DET) were applied in juvenile gilthead sea bream (Sparus aurata) and the metabolic rates were investigated. Fish with a total body mass of 80.5 ± 1.5 g and total length 17.2 ± 0.1 cm were maintained untrained (spontaneously swimming activity, UNT), swim-trained (induced sustained swimming activity, SSE) at 1 BL s-1 for 28 days, or detrained (28 days of swimming followed by 10 days of untraining, DET). Standard metabolic rate (SMR), maximum metabolic rate (MMR), and excess post-exercise oxygen consumption (EPOC) were assessed (n = 10). In addition, the effects of SSE training (51 days) on blood and plasma parameters were investigated before and immediately after applying a high-intensity swimming (HIS) protocol. SMR, MMR, and EPOC values were not different between SSE, UNT, or DET fish (143.2, 465.5 mg O2 kg-1 h-1, and 459.1 mg O2 kg-1, respectively). Spite the lack of differences between treatments, the dispersion in the residuals for SMR, MMR, and absolute aerobic scope (AAS) values followed the order UNT > DET > SSE, indicating that swim training decreases the individual variation of these metabolic parameters. Haematological parameters, plasma glucose, lactate, and cortisol levels were similar between SSE and UNT groups before HIS. Plasma glucose and lactate levels increased in both groups after HIS, being higher in the SSE group. Plasma cortisol levels were similar between both groups after HIS. Results suggest that SSE training improves energy use and reduces individual variation in SMR and MMR, an effect that declines with detraining.

Relation of gilthead seabream (Sparus aurata) seasonal reproductive activity to hematology, serum biochemistry, histopathology, and Brdt gene expression.

This study aimed to find the relation of Sparus aurata (gilthead seabream) reproductive activities to some blood parameters as complete blood count, liver enzymes, some hormones related to reproduction process and microscopic findings of gonads, as well as expression of Bromodomain testis-specific gene. Eighty-eight sexually mature seabream were collected and investigated through the four seasons. Red blood cells were higher in autumn and spring. Hemoglobin was high in summer, MCV highest values ​​were seen in winter and summer, while MCHC was highest in summer. The values ​​of white blood cells increased significantly in spring, summer, and autumn compared with winter. The highest value of lymphocytes was recorded in spring and autumn. Eosinophil was recorded the highest value in the spring. The highest value of segmented neutrophils was recorded in summer. The highest value of band neutrophil was recorded in summer and winter. Alanine aminotransferase and aspartate aminotransferase showed high values in the winter. Luteinizing hormone (LH) was higher in females, males, and hermaphrodites during winter. Follicle-stimulating hormone (FSH) was higher in females during spring. The highest value of estradiol 17-ß and progesterone was recorded in summer. The highest value of total testosterone was recorded in spring. Microscopically, ovaries were immature and inactive during spring and summer but well developed in autumn and winter. During spring and summer, testes were immature and began spermatogenesis process but well developed with the appearance of spermatids and spermatozoa during autumn and winter. The expression of Brdt was higher in testes than ovary. Brdt recorded high expression in autumn and spring than in summer and winter.

Protein changes as robust signatures of fish chronic stress: a proteomics approach to fish welfare research.

BACKGROUND: Aquaculture is a fast-growing industry and therefore welfare and environmental impact have become of utmost importance. Preventing stress associated to common aquaculture practices and optimizing the fish stress response by quantification of the stress level, are important steps towards the improvement of welfare standards. Stress is characterized by a cascade of physiological responses that, in-turn, induce further changes at the whole-animal level. These can either increase fitness or impair welfare. Nevertheless, monitorization of this dynamic process has, up until now, relied on indicators that are only a snapshot of the stress level experienced. Promising technological tools, such as proteomics, allow an unbiased approach for the discovery of potential biomarkers for stress monitoring. Within this scope, using Gilthead seabream (Sparus aurata) as a model, three chronic stress conditions, namely overcrowding, handling and hypoxia, were employed to evaluate the potential of the fish protein-based adaptations as reliable signatures of chronic stress, in contrast with the commonly used hormonal and metabolic indicators. RESULTS: A broad spectrum of biological variation regarding cortisol and glucose levels was observed, the values of which rose higher in net-handled fish. In this sense, a potential pattern of stressor-specificity was clear, as the level of response varied markedly between a persistent (crowding) and a repetitive stressor (handling). Gel-based proteomics analysis of the plasma proteome also revealed that net-handled fish had the highest number of differential proteins, compared to the other trials. Mass spectrometric analysis, followed by gene ontology enrichment and protein-protein interaction analyses, characterized those as humoral components of the innate immune system and key elements of the response to stimulus. CONCLUSIONS: Overall, this study represents the first screening of more reliable signatures of physiological adaptation to chronic stress in fish, allowing the future development of novel biomarker models to monitor fish welfare.

Constant and intermittent hypoxia modulates immunity, oxidative status, and blood components of red seabream and increases its susceptibility to the acute toxicity of red tide dinoflagellate.

Hypoxia is an increasing threat to aquatic ecosystems and its impact on economically and ecologically important marine fish species needs to be studied. Especially, the consequences of hypoxia when occurring along with harmful algal blooms (HABs) are currently not well documented. In this study, we investigated the effect of constant and intermittent (daily and weekly) hypoxia on respiration, immunity, hematological parameters, and oxidative status of red seabream for 2, 4, and 6 weeks. Under constant and daily intermittent hypoxia, respiration rate significantly increased in 2 weeks compared to the control. Constant and daily intermittent hypoxia caused significant decreases in the activity of alternative complement pathway, lysozyme, and the level of total immunoglobulin (Ig), as well as significant increases in the concentrations of cortisol, hemoglobin, red blood cells, and white blood cells. A significantly higher level of malondialdehyde was measured for all hypoxia-exposed groups, indicating lipid peroxidation and oxidative stress. At 4 and 6 week, the level of glutathione and enzymatic activities of glutathione reductase and glutathione peroxidase were significantly decreased after constant and daily intermittent hypoxia challenge. The enzymatic activities of superoxide dismutase and catalase were significantly increased at 2 and 4 weeks, but they were decreased after 6 weeks by constant and daily intermittent hypoxia. Constant and daily intermittent hypoxia with subsequent non-toxin producing dinoflagellate Cochlodinium polykrikoides treatment significantly reduced the respiration rate in 3 and 24 h exposure and survival rate of red seabream. Taken together, the red seabream can be vulnerable to HABs under hypoxia condition through inhibition of immunity and antioxidant defense ability. Our findings are helpful in better understanding of molecular and physiological effects of hypoxia, which can be used in aquaculture and fisheries management.

Inorganic nitrogen compounds reduce immunity and induce oxidative stress in red seabream.

In this study, the effect of ammonia derived from different stocking densities on immunological, hematological, and oxidative stress parameters was analyzed in the blood or liver of red seabream. Density- and time-dependent increases in inorganic nitrogen compounds were measured for 20 days by analyzing the three major inorganic nitrogen compounds, total ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen. Three immunity parameters, alternative complement activity, lysozyme activity, and total immunoglobulin content were significantly decreased in the blood at the highest stocking density (10 kg m-3). The concentrations of hemoglobin and white blood cells were significantly decreased at 10 kg m-3, while there was no significant change in red blood cells. The significant increases in cortisol level and the enzymatic activities of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase at 10 kg m-3 clearly supported inorganic nitrogen compounds-triggered stress. A significant elevation of lipid peroxidation value and depletion of intracellular glutathione were observed at 5 and/or 10 kg m-3 in the liver tissue. The hepatic enzymatic activities of antioxidant defense enzymes, catalase and superoxide dismutase were also significantly increased. When a protein skimmer removes the inorganic nitrogen compounds at the highest density, most parameters showed no significant change. Taken together, these results suggest that accumulated inorganic nitrogen compounds at the highest stocking density inhibit innate immunity and induce oxidative stress in red seabream. This information will be helpful to maintain homeostasis of red seabream by controlling immunity and oxidative status through inorganic nitrogen compounds removal in intensive culture condition.

Water temperature affects osmoregulatory responses in gilthead sea bream (Sparus aurata L.).

Sea bream (Sparus aurata Linneaus) was acclimated to three salinity concentrations, viz. 5 (LSW), 38 (SW) and 55psµ (HSW) and three water temperatures regimes (12, 19 and 26 °C) for five weeks. Osmoregulatory capacity parameters (plasma osmolality, sodium, chloride, cortisol, and branchial and renal Na+,K+-ATPase activities) were also assessed. Salinity and temperature affected all of the parameters tested. Our results indicate that environmental temperature modulates capacity in sea bream, independent of environmental salinity, and set points of plasma osmolality and ion concentrations depend on both ambient salinity and temperature. Acclimation to extreme salinity resulted in stress, indicated by elevated basal plasma cortisol levels. Response to salinity was affected by ambient temperature. A comparison between branchial and renal Na+,K+-ATPase activities appears instrumental in explaining salinity and temperature responses. Sea bream regulate branchial enzyme copy numbers (Vmax) in hyperosmotic media (SW and HSW) to deal with ambient temperature effects on activity; combinations of high temperatures and salinity may exceed the adaptive capacity of sea bream. Salinity compromises the branchial enzyme capacity (compared to basal activity at a set salinity) when temperature is elevated and the scope for temperature adaptation becomes smaller at increasing salinity. Renal Na+,K+-ATPase capacity appears fixed and activity appears to be determined by temperature.

Efficacy of mozuku fucoidan in alternative protein-based diet to improve growth, health performance, and stress resistance of juvenile red sea bream, Pagrus major.

We evaluated the efficacy of mozuku fucoidan supplementation to alternative dietary proteins used in fish meal (FM) replacement to enhance growth, immunity, and stress resistance of Pagrus major. Seven isonitrogenous (45% protein) experimental diets were formulated where diet 1 (D1) was FM-based control diet. Diets 2 to 7 were formulated by replacing 25, 50, and 75% of FM protein with soy protein isolate (SPI) protein, and each replacement level was supplemented without or with fucoidan at 0.4% for diet groups D2 (FM25), D3 (FM25Fu), D4 (FM50), D5 (FM50Fu), D6 (FM75), and D7(FM75Fu), respectively. Each diet was randomly allocated to triplicate groups of fish (4.1 g) for 56 days. Significantly higher weight gain and specific growth rate were observed in fish fed FM50Fu diet group, and it was not differed (P > 0.05) with fish fed FM25Fu diet group. FM-based control diet showed intermediate value, and it was not differed (P > 0.05) with or without fucoidan-supplemented ≤ 50% FM replacement groups and FM75Fu diet group. Significantly lower growth performances were observed in FM75 diet group. At each replacement level, fucoidan-supplemented groups showed nonsignificant improvement of feed utilization performances. Fish fed fucoidan-supplemented diets showed best condition of oxidative and freshwater stress resistance. Lysozyme activity, NBT, and peroxidase activity showed higher (P > 0.05) values in fucoidan-supplemented groups compared with the non-supplemented groups. Catalase activity was significantly lower in FM75Fu diet group. Catalase activity is significantly influenced by the interaction effects of fucoidan and FM replacement level. In conclusion, fucoidan supplementation could increase the efficiency of utilizing SPI (≥ 75%) without any adverse effects on red sea bream performance.

Effects of dietary supplementation of bovine lactoferrin on antioxidant status, immune response and disease resistance of yellowfin sea bream (Acanthopagrus latus) against Vibrio harveyi.

This study investigated the effect of the dietary supplementation of bovine lactoferrin (LF) on growth performance, hematological and immunological parameters, antioxidant enzymes activity and disease resistance against Vibrio harveyi in yellowfin sea bream (Acanthopagrus latus) fingerling. The fish with initial body weight 10 ±â€¯0.3 g were randomly distributed at 10 fish per each 250 L fiberglass tank, and fed with four experimental diets (a control basal diet and three supplemented diets with 400, 800 and 1200 mg LF kg-1 diet) for 8 weeks. The obtained results showed that fish fed with LF supplemented diets had significantly higher final body weight as compared to control diet (P < 0.05). There were no significant differences between LF-treatments and the control group in white blood cell counts, red blood cell counts, hemoglobin and hematocrit. Total protein and complement activity (ACH50) in the serum of yellowfin sea bream were enhanced with increasing the dietary LF supplementation level (P < 0.05). The mucus lysozyme activity in fish fed on 800 and 1200 mg LF kg-1 was significantly higher than those fed on 400 mg LF kg-1 and control fish (P < 0.05). None of the antioxidant enzymes (catalase, glutathione reductase, glutathione S-transferase) was affected by LF supplementation (P > 0.05). Fish fed with dietary LF had a significantly higher survival rate than those fed with the control diet after challenge with Vibrio harveyi (P < 0.05). These results revealed that diet supplementation in A. latus especially with 1200 mg LF kg-1 improve fish growth performance and immune parameters, as well as survival rate against Vibrio harveyi.

The effect of dietary fucoidan on growth, immune functions, blood characteristics and oxidative stress resistance of juvenile red sea bream, Pagrus major.

We determined the supplementation effects of dietary fucoidan on growth, immune responses, blood characteristics, and oxidative stress resistance of juvenile red sea bream. A fishmeal (FM)-based basal diet supplemented with 0% (D1, control), 0.05% (D2), 0.1% (D3), 0.2% (D4), 0.4% (D5), and 0.8% (D6) mozuku fucoidan to formulate six experimental diets. Each diet was randomly allocated to triplicate groups of fish (3.8 g) for 60 days. Results showed that fish-fed diet D5 showed significantly higher (P < 0.05) growth performance compared to the control (D1). Diet groups D2 to D4 also showed intermediate values compared to D1. Feed conversion efficiency and protein efficiency ratio were significantly higher in diet group D5, which was not significantly different with D3. Fucoidan supplementation increased whole-body lipid, which was significantly higher in the D5 group. Condition factor (CF) was significantly higher in fish fed ≥ 0.2% fucoidan-supplemented diet groups. Diet group D5 and D4 showed significantly lower blood urea nitrogen (BUN) and aspartate aminotransferase (AST) level, respectively. Dietary fucoidan reduced the oxidative stress of fish. Among the measured nonspecific immune parameters, only peroxidase activity (PA) and total serum protein (TSP) were significantly influenced by dietary supplementation and it was higher in D4 group. Fucoidan supplementation reduces thiobarbituric acid reactive substance (TBARS) values numerically and it was lowest in fish-fed diet group D5. Under the present experimental condition, finally, we concluded that 0.3-0.4% dietary fucoidan supplementation enhanced the growth and health performance of red sea bream by increasing growth, immune response, blood characteristics, and oxidative stress resistance.

Bacillus subtilis as probiotic candidate for red sea bream: Growth performance, oxidative status, and immune response traits.

The effects of dietary administration of Bacillus subtilis on the growth, digestive enzyme activity, blood chemistry, oxidative status and immune response of red sea bream (Pagrus major) were evaluated in the current study. Fish fed five different levels of B. subtilis at 0 (BS0), 1 × 104 (BS1), 1 × 106 (BS2), 1 × 108 (BS3) and 1 × 1010 (BS4) CFU kg-1 diet for 60 days. The obtained results showed that B. subtilis supplementation significantly improved growth performance (FBW, WG and SGR), feed utilization (FI, FCE, PER and PG) and whole-body protein content when compared to the control group (P < 0.05). Furthermore, the specific activities of amylase, protease and lipase enzymes up regulated significantly upon B. subtilis incorporation in red sea bream diets (P < 0.05). No changes have been reported on blood biochemical variables except for the plasma total protein, which increased significantly in fish fed BS3 diet when compared with the control diet (P < 0.05). Hematocrit, hemoglobin and the nitro blue tetrazolium values also reported the highest values significantly in fish fed B. subtilis, especially in case of BS3 and BS4 diets (P < 0.05). Serum bactericidal activity enhanced significantly in BS2, BS3 and BS4 groups (P < 0.05), while mucus bactericidal activity showed no significant activity among tested groups (P > 0.05). Serum lysozyme activity exhibited higher values in case of BS3 and BS4 groups than BS0 group (P < 0.05), while mucus lysozyme activity increased only in BS3 group. Also, serum peroxidase activity enhanced significantly in fish fed BS2 and BS3 diets (P < 0.05), however, no activities were observed in the collected mucus. All groups reflexed high tolerance ability against oxidative stress except for BS0 and BS1 groups. Additionally, catalase activity increased significantly in all B. subtilis fed groups when compared to BS0 group (P < 0.05). Considering the obtained results, the supplementation of B. subtilis in the diet of red sea bream at 1 × 108 and 1 × 1010 CFU kg-1 diet could improve the growth, feed utilization, health condition and immune response.

Changes in natural haemolytic complement activity induced by stress in gilthead seabream (Sparus aurata L.).

In aquaculture, animals can be continually exposed to environmental stress factors that put their health and even survival at risk. Two experiments were performed to evaluate the impact of different stress conditions (acute crowding and anaesthetic) on the natural haemolytic complement activity in serum and skin mucus of gilthead seabream (Sparus aurata L.). In the first experiment, fish were subjected to 10 kg m-3 (low density, control group) and 50 kg m-3 (high density, crowding group) during 2, 24 and 48 h. In the second experiment, fish were unexposed (control) or exposed to 40 ppm of MS-222 or 5 ppm or 10 ppm of clove oil for 1 h. In fish maintained in acute crowding conditions only an increase of the haemolytic complement activity was observed in the skin mucus after 24 h of exposure. However, a similar statistically significant increase was observed in serum and skin mucus of fish exposed for 1 h to the lowest concentration of clove oil (5 ppm) tested. The results point to a new and alternative way to assess stress in farmed fish by using skin mucus instead of blood serum and confirm that the measurement of natural haemolytic complement activity serves as an indicator of stress in fish.

Influence of dietary inosine and vitamin C supplementation on growth, blood chemistry, oxidative stress, innate and adaptive immune responses of red sea bream, Pagrus major juvenile.

Both inosine (INO) and vitamin C (l-ascorbic acid, AsA) play important roles in growth performance, feed utilization and health status of fish. Therefore, a 56 days feeding trial was conducted to determine the interactive effects of dietary INO and AsA on growth performance, oxidative status, innate and adaptive immune responses of red sea bream. Fish growth performance and fed utilization parameters were significantly affected by dietary INO supplementation but not by AsA. Fish fed diets with INO at 4 g kg-1 diet in combination of high and low levels of AsA (3.1 g kg-1 and 9.3 g kg-1) produced the highest growth and feed utilization performances. In terms of growth and feed utilization performances no significant interaction effects were observed between INO and AsA. Dietary INO significantly influenced hematocrit, glucose and glutamyl oxaloacetic transaminase (GOT) content of red sea bream meanwhile AsA also significantly influenced hematocrit, glucose, total cholesterol, blood urea nitrogen (BUN) and glutamic-pyruvate transaminase (GPT) content of the test fish. No significant interaction effects was also observed between INO and AsA on measured hematological parameters. Reactive oxygen metabolites (d-ROMs) significantly influenced by both INO and AsA. Fish fed diet groups D1, D4 and D6 showed best oxidative stress resistance. Only INO was a significant factor on nitro-blue-tetrazolium activity (NBT) and bactericidal activity (BA). Neither INO nor AsA was a significant factor on serum catalase activity (CAT), total serum protein (TSP), peroxidase activity (PA) and lysozyme activity (LA). No significant interaction effects was observed between INO and AsA on measured innate immune parameters. Agglutination antibody titer was significantly influenced by dietary supplementation, after 15 days of vaccination but not in 21 days. In the day 15th fish and diet group D3 and D5 showed significantly higher values compared to diet groups control and D1. INO was the only significant factor of increasing agglutination antibody titer in 15 t h day. While AsA was not a significant factor on agglutination antibody titer values in day 15 t h, there was an interaction between dietary INO and AsA levels. Finally under the experimental conditions, fish fed high INO and low AsA levels (4 g kg-1 and 0.31 g kg-1 diet, respectively) showed best growth and feed utilization performance. Simultaneously, low level of INO and high level of AsA (2 g kg-1 and 0.93 g kg-1 diet, respectively) improved blood chemistry and immunological parameters. Furthermore, combined use of INO and AsA is possible to improve hemato-immunological responses of red sea bream.

Growth performance, blood health, antioxidant status and immune response in red sea bream (Pagrus major) fed Aspergillus oryzae fermented rapeseed meal (RM-Koji).

This study evaluated the effects of dietary substitution of fishmeal by graded levels of a blend composed of Aspergillus oryzae fermented rapeseed meal [0% (RM0), 25% (RM25), 50% (RM50), 75% (RM75) and 100% (RM100)] on growth performance, haemato-immunological responses and antioxidative status of Pagrus major (average weight 5.5 ±â€¯0.02 g). After 56 days, growth performances were significantly improved in fish fed RM25 diet compared to control (P < 0.05). Meanwhile, up to 50% replacement of fishmeal did not affect growth performance, feed conversion efficiency, protein efficiency ratio, protein apparent digestibility, protease activity, fish somatic indices and survival compared to control. While blood hematocrit and plasma protein were significantly enhanced in groups fed RM0 and RM25 diets, most of the hematological parameters did not change through the trial except glutamic pyruvate transaminase which was significantly increased in RM75 and RM100 groups and blood cholesterol which was gradually decreased with the increasing level of the blend. Interestingly, feeding fish with RM25 and RM50 diets significantly showed enhanced lysozyme, bactericidal and peroxidase activities and fish fed the same diets showed high resistance against oxidative stress (biological antioxidant potential and reactive oxygen metabolites). Additionally, catalase activity and tolerance against low salinity seawater were higher in fish fed RM25 diet. These findings suggested that, at a moderate level (25% and 50%), substitution of fishmeal by the fermented rapeseed meal promoted growth, nutrient utilization, and exerted immune responses and anti-oxidative effects in red sea bream.

Unraveling vasotocinergic, isotocinergic and stress pathways after food deprivation and high stocking density in the gilthead sea bream.

The influence of chronic stress, induced by food deprivation (FD) and/or high stocking density (HSD), was assessed on stress, vasotocinergic and isotocinergic pathways of the gilthead sea bream (Sparus aurata). Fish were randomly assigned to one of the following treatments: (1) fed at low stocking density (LSD-F; 5kg·m-3); (2) fed at high stocking density (HSD-F, 40kg·m-3); (3) food-deprived at LSD (LSD-FD); and (4) food-deprived at HSD (HSD-FD). After 21days, samples from plasma, liver, hypothalamus, pituitary and head-kidney were collected. Both stressors (FD and HSD) induced a chronic stress situation, as indicated by the elevated cortisol levels, the enhancement in corticotrophin releasing hormone (crh) expression and the down-regulation in corticotrophin releasing hormone binding protein (crhbp) expression. Changes in plasma and liver metabolites confirmed a metabolic adjustment to cope with energy demand imposed by stressors. Changes in avt and it gene expression, as well as in their specific receptors (avtrv1a, avtrv2 and itr) at central (hypothalamus and pituitary) and peripheral (liver and head-kidney) levels, showed that vasotocinergic and isotocinergic pathways are involved in physiological changes induced by FD or HSD, suggesting that different stressors are handled through different stress pathways in S. aurata.

Short-term induction of vitellogenesis in the immature male yellowfin seabream ( Acanthopagrus latus) exposed to bisphenol A and 17 ß-estradiol.

Bisphenol A (BPA) is a known environmental endocrine-disrupting chemical that is widely used in plastics manufacturing. BPA enters in the aquatic environment mainly through urban and industrial sewage effluents, thereby posing a potential threat to organisms living in these ecosystems. This study was conducted to investigate the effect of BPA on VTG production with direct (sodium dodecyl sulfate-polyarylamide gel electrophoresis) and indirect (alkali-labile phosphate (ALP), total plasma calcium and protein) methods in immature male yellowfin seabream ( Acanthopagrus latus) as a marine fish model. Fish were randomly distributed into seven groups that were administered 1, 10, 50, and 100 µg g-1 week-1 of BPA and 2 µg g-1week-1 of 17ß-estradiol (E2) over a period of 2 weeks. Solvent controls received olive oil, whereas controls were not injected. The fish were sampled on days 0, 7, and 14, and their blood plasma and liver were obtained. The results showed that the hepatosomatic index of all treated fish was elevated in comparison with controls. Direct and indirect indicators showed that fish VTG protein was induced by BPA and E2 exposure. The protein was found to have two bands with molecular weights around 210 and 190 KDa. ALP, total plasma calcium and protein levels were increased in dose- and time-dependent manners. The results of this study demonstrated that short-term exposure of yellowfin seabream to BPA induced adverse effects in the reproductive system of hermaphrodite fish.

Preliminary studies on haematological and plasmatic parameters in gilthead sea bream (Sparus aurata) held under day/night temperature variations.

This study aimed to evaluate to what extent diel water temperature oscillations over a short period of time (41 h) affected haematologic and plasmatic parameters of gilthead sea bream. Sea bream juveniles (160 fish; 177.2 ± 20.9 g) were divided in two homogenous groups, experimental and control. The experimental group was exposed to water at 22.3 ± 0.6 °C between 5 a.m. and 5 p.m. and to colder water from 5 p.m. to 5 a.m. (temperature variation of 8.6 ± 0.3 °C) during 41h period, whereas for the control group, water temperature was maintained constant (22.4 ± 0.7 °C) for the same period. Fish were fed three times per day (10:30 a.m., 2 p.m., 4 p.m.). Blood was collected from fish caudal veins (n = 6) at different time points (10 a.m., 1 p.m., 5 p.m., 6:30 p.m., 8 p.m. and 10 a.m. of the following day) from each group, for haematologic (haematocrit and haemoglobin) and plasmatic (glucose, cortisol, triglycerides and total protein) determinations. All parameters studied tended to vary throughout the study period, both for experimental and control groups. Significant differences were observed between the two groups for all the studied parameters at different sampling points. These differences were observed near the influence of the cold-water period or during the cooling-water period. Results suggested that colder water temperature cycles depressed fish metabolic activity and affected the levels of the analysed blood-circulating metabolites. Overall, results suggested that stress response was similar for experimental and control groups and that gilthead sea bream were able to cope with diel water temperature variations.

Enhanced dietary formulation to mitigate winter thermal stress in gilthead sea bream (Sparus aurata): a 2D-DIGE plasma proteome study.

Low water temperatures during winter are common in farming of gilthead sea bream in the Mediterranean. This causes metabolic disorders that in extreme cases can lead to a syndrome called "winter disease." An improved immunostimulatory nutritional status might mitigate the effects of this thermal metabolic stress. A trial was set up to assess the effects of two different diets on gilthead sea bream physiology and nutritional state through plasma proteome and metabolites. Four groups of 25 adult gilthead sea bream were reared during winter months, being fed either with a control diet (CTRL) or with a diet called "winter feed" (WF). Proteome results show a slightly higher number of proteins upregulated in plasma of fish fed the WF. These proteins are mostly involved in the immune system and cell protection mechanisms. Lipid metabolism was also affected, as shown both by plasma proteome and by the cholesterol plasma levels. Overall, the winter feed diet tested seems to have positive effects in terms of fish condition and nutritional status, reducing the metabolic effects of thermal stress.