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 Blood was withdrawn for haematological procedures, whereas plasma and gut tissue were sampled for immune and oxidative stress parameters.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.

Effects of Zinc Source and Enzyme Addition on the Fecal Microbiota of Dogs.

Supplemental zinc from organic sources has been suggested to be more bioavailable than inorganic ones for dog foods. However, the bioavailability of zinc might be affected by dietary constituents such as phytates. The present study aimed to evaluate the effects of two zinc sources (zinc sulfate and zinc proteinate) and the addition of a multi-enzymatic complex from the solid-state fermentation of Aspergillus niger on end-products of fecal fermentation and fecal microbiota of adult Beagles fed a high-phytate diet. The experimental design consisted of three 4 × 4 Latin Squares with a 2 × 2 factorial arrangement of treatments (n = 12 Beagles), with four periods and four diets: zinc sulfate without (IZ) or with (IZ +) enzyme addition, and zinc proteinate without (OZ) or with (OZ +) enzyme addition. Enzyme addition significantly affected Faith's phylogenetic diversity index, whereas zinc source did not affect either beta or alpha diversity measures. Linear discriminant analysis effect size detected nine taxa as markers for organic zinc, 18 for inorganic source, and none for enzyme addition. However, with the use of a negative binomial generalized linear model, further effects were observed. Organic zinc was associated with a significantly higher abundance of Firmicutes and lower Proteobacteria and Bacteroidetes, although at a genus level, the response varied. The DNA abundance of Clostridium cluster I, Clostridium cluster XIV, Campylobacter spp., Ruminococcaceae, Turicibacter, and Blautia was significantly higher in dogs fed IZ and IZ + diets. Higher abundance of genus Lactobacillus was observed in dogs fed enzyme-supplemented diets. End-products of fecal fermentation were not affected by zinc source or enzymes. An increase in some taxa of the phyla Actinobacteria and Firmicutes was observed in feces of dogs fed organic zinc with enzyme addition but not with inorganic zinc. This study fills a gap in knowledge regarding the effect of zinc source and enzyme addition on the fecal microbiota of dogs. An association of zinc bioavailability and bacteria abundance is suggested, but the implications for the host (dog) are not clear. Further studies are required to unveil the effects of the interaction between zinc sources and enzyme addition on the fecal microbial community.

Functional Feeds to Tackle Meagre (Argyrosomus regius) Stress: Physiological Responses under Acute Stressful Handling Conditions.

Marine algae are recognised sources of bioactive compounds that have attracted great interest as nutritional supplements for aquaculture fish. Intensive rearing conditions often expose fish to husbandry-related stressors, rendering fish more susceptible to disease and reducing production yields. The present work evaluated the potential of two marine algae extracts (Fucus vesiculosus and Nannochloropsis gaditana) as nutritional supplements to mitigate stress effects in meagre (Argyrosomus regius) exposed to an acute handling stress (AS). A plant-based diet was used as a control, and three other diets were prepared, which were similar to the control diet but supplemented with 1% of each algal extract or a combination of the two extracts (0.5% each). The effects of supplemented diets on stress biomarkers, antioxidant enzyme activities, and immune response were analysed in fish exposed to AS after 4 weeks of feeding. Supplemented diets did not affect growth performance but the inclusion of F. vesiculosus promoted higher feed efficiency, as compared to the control group. Dietary algal extracts supplementation reduced plasma glucose levels, increased white blood cell counts, and reduced the expression of pro-inflammatory genes when compared with the control. N. gaditana supplementation led to a reduction in hepatic antioxidant enzyme activity and glutathione levels, while F. vesiculosus supplementation increased muscle glutathione reductase activity and reduced lipid peroxidation. These findings support the potential of algal extracts as nutraceuticals in aquafeeds to enhance the ability of fish to cope with husbandry-related stressful conditions and ultimately improve fish health and welfare.

The Use of Defatted Tenebrio molitor Larvae Meal as a Main Protein Source Is Supported in European Sea Bass (Dicentrarchus labrax) by Data on Growth Performance, Lipid Metabolism, and Flesh Quality.

OBJECTIVE: This study aims to determine the maximal inclusion level of defatted (d-) Tenebrio molitor larvae meal (TM) able to replace dietary fishmeal (FM) without compromising growth performance, general metabolism, and flesh quality traits in European sea bass, and to evaluate the major underlying physiological mechanisms. MATERIALS AND METHODS: Fish (55 ± 2 g) were fed with diets containing increasing levels of dTM: 0, 40, 80 and 100% (CTRL, TM40, TM80, and TM100, respectively) to replace FM. After 10 weeks of feeding, the growth performance, nutrient and energy balance, intestinal integrity, plasma metabolites and the expression of genes related to growth and nutrient metabolism, in liver and muscle were determined. The fatty acids (FA) profile, textural properties and color were also evaluated in muscle. RESULTS: Protein and lipids digestibility remained unaltered up to 80% dTM inclusion. Growth performance parameters were similar among dietary treatments. The dTM inclusion increased the hepatosomatic index in fish fed TM100. Muscle eicosapentaenoic acid, docosahexaenoic acid and n-3 long-chain polyunsaturated FA levels were maintained up to 80% dTM inclusion, but total cholesterol and non-esterified FA increased with dietary dTM inclusion. In liver, the expression of elongation of very long-chain FA protein 6 (elovl6) and FA desaturase 2 (fads2) did not change in fish fed TM40 and TM80, but elovl6 decreased whilst fads2 increased in fish fed TM100 when compared to those fed CTRL. The expression of cholesterol 7 alpha-monooxygenase (cyp7a1) decreased with dietary dTM inclusion. In muscle, the expression of myoblast determination protein-2 (myod2) decreased in fish fed TM80 and TM100. CONCLUSION: It is feasible to substitute dietary FM by dTM up to 80% in European sea bass without detrimental effects on nutrient digestibility, growth performance and associated genetic pathways, whilst assuring fillet nutritional value for human consumption.

Supplemental selenium source on gut health: insights on fecal microbiome and fermentation products of growing puppies.

Selenium is an essential trace element that can modulate the gut microbiome with an impact on host health. The present study aimed to evaluate the effects of organic (selenium-enriched yeast) vs inorganic (sodium selenite) selenium source on fecal end-fermentation products and gut microbiome of puppies from 20 to 52 weeks of age. Alpha and beta diversity of the gut bacterial community were affected by age but not by gender or selenium source. The relative abundance of taxa was differently affected by age, and the DNA concentration of all selected bacterial groups increased with age, although total volatile fatty acids (VFA), acetate, propionate, caproate and lactate concentrations decreased. Organic selenium was associated with a higher concentration of total VFA, propionate and butyrate, a higher number of DNA copies of Lactobacillus, and a trend to lower DNA copies of Escherichia coli. Effects on fecal microbiome during growth differed with selenium source. Females had higher fecal end-fermentation products related to protein degradation, whereas males had higher DNA concentration of Bifidobacterium. Organic selenium might be beneficial over inorganic for dog food supplementation due to the positive modulation of the gut microbiome observed in puppies.

Miniaturized Fluorimetric Method for Quantification of Zinc in Dry Dog Food.

Zinc is an essential trace element for animals in several biological processes, particularly in energy production, and it is acquired from food ingestion. In this context, a microplate-based fluorimetric assay was developed for simple, fast, and low-cost determination of zinc in pet food using 2,2'-((4-(2,7-difluoro-3,6-dihydroxy-4aH-xanthen-9-yl)-3-methoxyphenyl)azanediyl)diacetic acid (FluoZin-1) as fluorescent probe. Several aspects were studied, namely, the stability of the fluorescent product over time, the FluoZin-1 concentration, and the pH of reaction media. The developed methodology provided a limit of detection of 1 µg L-1 in sample acid digests, with a working range of 10 to 200 µg L-1, corresponding to 100-2000 mg of Zn per kg of dry dog food samples. Intraday repeatability and interday repeatability were assessed, with relative standard deviation values < 3.4% (100 µg L-1) and <11.7% (10 µg L-1). Sample analysis indicated that the proposed fluorimetric assay provided results consistent with ICP-MS analysis. These results demonstrated that the developed assay can be used for rapid determination of zinc in dry dog food.

Effect of Zinc Source and Exogenous Enzymes Supplementation on Zinc Status in Dogs Fed High Phytate Diets.

Zinc is an essential element, a cofactor of many enzymes, and performs catalytic, structural and regulatory functions. Once in the gastrointestinal tract, zinc can interact with food constituents. Phytic acid, the major phosphorus storage in plants, limits zinc availability from animal feeds due to the formation of insoluble complexes with phytates. This study tested the effect of supplemental zinc source (zinc sulfate and a chelate zinc proteinate) and the addition of exogenous enzymes from a solid-state fermentation product of Aspergillus niger to a high phytate diet. The study was designed according to three Latin Squares 4 × 4 with a 2 × 2 factorial arrangement of treatments, with four periods, four diets, and 12 young adult Beagles. Periods lasted 5 weeks each. Diets were supplemented with 75 mg/kg of zinc sulfate (IZ) or zinc proteinate (OZ), and without or with 200 mg/kg of exogenous enzymes (IZ+, OZ+). Results showed that zinc proteinate increased the bioavailability of phosphorus, yet the zinc biomarkers remained unaffected by the zinc source, with the exception of lymphocyte subsets that benefit from zinc proteinate. The use of exogenous enzymes did not affect zinc availability nor nutrient and energy digestibility.

Flow-Based Dynamic Approach to Assess Bioaccessible Zinc in Dry Dog Food Samples.

This work proposes a simple and easy-to-use flow-through system for the implementation of dynamic extractions, aiming at the evaluation of bioaccessible zinc and the characterization of leaching kinetics in dry dog food samples. The kinetic profile of Zn extraction was determined by flame atomic absorption spectroscopy and the results were fitted in an exponential function (R2 > 0.960) compatible with a two first-order reactions model. Values of fast leachable Zn ranged from 83 ± 1 to 313 ± 5 mg of Zn per kg of sample, with associated rate constants ranging from 0.162 ± 0.004 to 0.290 ± 0.014 min-1. Similar results were observed compared to the static batch extraction. The percentage of bioaccessible Zn ranged from 49.0 to 70.0%, with an average value of 58.2% in relation to total Zn content. Principal component analysis regarding the variables fast leachable Zn, associated rate constant, total Zn, and market segment, has shown that 84.6% of variance is explained by two components, where the second component (24.0%) presented loadings only for the fast leachable Zn and associated rate constant. The proposed method is suitable for the fast evaluation (<1 h) of leaching kinetics and bioaccessibility in dry dog food.

Human exposure to mercury and its hematological effects: a systematic review.

Mercury is a metal found in the environment from natural and anthropogenic sources. It is highly toxic to ecosystems and living beings. Most human exposures come from ingestion of contaminated seafood, outgassing from dental amalgam or occupational exposure (e.g. gold mining), among other cases. Large populations are exposed to mercury, making it a very important issue from the public health perspective. Adverse health effects are commonly seen in the nervous system, but every organ is a potential target, such as the bone marrow. The main goal of this study was to assess the available evidence on human exposure to mercury and its hematological effects. A search strategy was constructed, including key terms (MeSH, text word and equivalents) for querying 2 repositories of master dissertation and PhD thesis (Fiocruz/ARCA and University of São Paulo) and 4 different electronic databases: BVS/LILACS, MEDLINE/PubMed, Scopus and TOXLINE/NIH, for articles published from 1950 to February 2018. There was no language restriction and a tool (EPHPP) was used to assess the quality of included studies. According to pre-established criteria, 80 studies were retrieved, all of them observational (48 case reports, 24 cross-sectional, 6 case series and 2 cohorts), comprising 9,284 people. Despite the fact that most exposed ones (6,012) had normal blood cell count and mercury hematological effects did not seem very usual (1,914 cases: 14 severe and 29 deaths), three studies reported association (ß) for anemia, lymphopenia, neutrophilia and basophilia. We concluded that the gathered information pointed to mercury hematotoxic effects, some of them may be serious and even fatal.

Human exposure to mercury and its hematological effects: a systematic review / Exposição humana ao mercúrio e seus efeitos hematológicos: uma revisão sistemática / Exposición humana al mercurio y sus efectos hematológicos: una revisión sistemática

Mercury is a metal found in the environment from natural and anthropogenic sources. It is highly toxic to ecosystems and living beings. Most human exposures come from ingestion of contaminated seafood, outgassing from dental amalgam or occupational exposure (e.g. gold mining), among other cases. Large populations are exposed to mercury, making it a very important issue from the public health perspective. Adverse health effects are commonly seen in the nervous system, but every organ is a potential target, such as the bone marrow. The main goal of this study was to assess the available evidence on human exposure to mercury and its hematological effects. A search strategy was constructed, including key terms (MeSH, text word and equivalents) for querying 2 repositories of master dissertation and PhD thesis (Fiocruz/ARCA and University of São Paulo) and 4 different electronic databases: BVS/LILACS, MEDLINE/PubMed, Scopus and TOXLINE/NIH, for articles published from 1950 to February 2018. There was no language restriction and a tool (EPHPP) was used to assess the quality of included studies. According to pre-established criteria, 80 studies were retrieved, all of them observational (48 case reports, 24 cross-sectional, 6 case series and 2 cohorts), comprising 9,284 people. Despite the fact that most exposed ones (6,012) had normal blood cell count and mercury hematological effects did not seem very usual (1,914 cases: 14 severe and 29 deaths), three studies reported association (β) for anemia, lymphopenia, neutrophilia and basophilia. We concluded that the gathered information pointed to mercury hematotoxic effects, some of them may be serious and even fatal.

O mercúrio é um metal que pode ser encontrado naturalmente no meio ambiente e através de fontes antropogênicas. É altamente tóxico para ecossistemas e seres vivos. A maior parte da exposição humana provém da ingestão de pescados contaminados, da liberação de gases da amálgama dentária ou da exposição ocupacional (p.ex.: extração de ouro). Vastas populações são expostas ao mercúrio, tornando-se uma questão de saúde pública muito importante. Efeitos adversos à saúde são comumente observados no sistema nervoso, mas todos os órgãos são alvos em potencial, como a medula óssea. O principal objetivo do estudo foi avaliar as evidências disponíveis sobre a exposição humana ao mercúrio e seus efeitos hematológicos. Uma estratégia de busca foi realizada, incluindo termos chave (palavras-chave, palavras do texto e equivalentes), para pesquisar dois repositórios de dissertações de mestrado e teses de doutorado (Fiocruz/ARCA e Universidade de São Paulo) e quatro bases de dados eletrônicas: BVS/LILACS, MEDLINE/PubMed, Scopus e TOXLINE/NIH (artigos publicados de 1950 até fevereiro de 2018). Não houve restrições de linguagem e uma ferramenta (EPHPP) foi utilizada para avaliar a qualidade dos estudos incluídos. De acordo com os critérios pré-estabelecidos, foram encontrados 80 estudos, todos observacionais (48 relatos de caso, 24 estudos transversais, 6 séries de casos e 2 coortes), que compreendiam 9.284 pessoas. Apesar do fato de que as pessoas mais expostas (6.012) tinham contagens de células sanguíneas normais, e os efeitos hematológicos do mercúrio não pareciam muito comuns (1.914 casos, 14 graves e 29 mortes), três estudos relataram a associação de (β) anemia, linfopenia, neutrofilia e basofilia. Concluímos que as informações coletadas indicam efeitos hematotóxicos do mercúrio, alguns dos quais podem ser muito graves e até fatais.

El mercurio es un metal que se puede encontrar de forma natural en el ambiente y mediante fuentes antropogénicas. Es altamente tóxico para los ecosistemas y seres vivos. Entre otras, la mayor parte de la exposición humana, proviene de la ingestión de pescado contaminado, liberación de gases de amalgamas dentales o exposición ocupacional (p.ej. extracción de oro). Vastas poblaciones están expuestas al mercurio, convirtiéndolo en un asunto muy importante desde la perspectiva de la salud pública. Los efectos adversos para la salud se observan comúnmente en el sistema nervioso, pero cada órgano es un objetivo potencial, como la médula ósea. El objetivo principal del estudio fue evaluar las evidencias disponibles sobre la exposición humana al mercurio y sus efectos hematológicos. Se realizó una estrategia de búsqueda, incluyendo términos clave (palabras-clave, palabras del texto y equivalentes), se consultaron 2 registros de trabajos finales de máster y tesis de doctorado (Fiocruz/ARCA y Universidad de São Paulo) y 4 bases de datos electrónicas diferentes: BVS/LILACS, MEDLINE/PubMed, Scopus y TOXLINE/NIH, para artículos publicados desde el año 1950, hasta febrero de 2018. No hubo restricciones de lengua y se usó la herramienta (EPHPP) para evaluar la calidad de los estudios incluidos. De acuerdo con los criterios preestablecidos, se recopilaron 80 estudios, todos observacionales (48 informes de casos, 24 estudios transversales, 6 series de casos, y 2 cohortes), que comprendieron a 9.284 personas. A pesar de que la mayoría de los expuestos (6.012) tenían un recuento normal de células sanguíneas y los efectos hematológicos del mercurio no parecían muy comunes (1.914 casos: 14 severos y 29 muertes), tres estudios informaron de la asociación (β) para anemia, linfopenia, neutrofilia y basofilia. Concluimos que la información recabada indicaba los efectos hematotóxicos del mercurio, algunos de los cuales pueden ser muy serios e incluso fatales.

Mineral Composition of Dry Dog Foods: Impact on Nutrition and Potential Toxicity.

Detailed mineral profile of a selection of commercially available complete dry dog foods was determined using ICP-MS (Se, Cu, Mn, Zn, and nonessential trace elements), flame photometry (Na and K) and atomic and molecular spectrophotometry (Ca, P, Mg, and Fe). The contribution of ingredients to the mineral composition was correlated to the food market segment. Results showed an oversupply of essential elements due to the energy density effect on feed intake. Additives contributed from 40.8 to 55.1% to the total trace elements contents. With the exception of Se, all trace elements were supplied above the nutritional requirements of adult dogs. Legal limits of Cu, Se, and Zn were surpassed. The content of nonessential trace elements included values in the range of nanograms to micrograms per kg, without surpassing safe upper limits. This work brings awareness to the need to find supplementation strategies that ensure nutritional adequacy and avoid waste.

Humoral and mucosal immune responses in meagre (Argyrosomus regius) juveniles fed diets with varying inclusion levels of carob seed germ meal.

Many studies have assessed the effects of incorporation of plant feedstuffs in fish diets on growth performance, whereas few studies have addressed the effects of fish meal replacement by plant protein sources on fish immune parameters. Thus, the aim of this study was to evaluate the effects on immune response of different inclusion levels of carob seed germ meal (CSGM) as partial replacement for fish meal in diets for meagre (Argyrosomus regius) juveniles. Fish were fed four experimental diets with increased CSGM inclusion levels [0% (control), 7.5% (CSGM7.5), 15% (CSGM15) and 22.5% (CSGM22.5)]. After 1, 2, and 8 weeks of feeding fish were sampled to determine haematological profile and several humoral parameters in plasma and intestine. Results showed that dietary inclusion of CSGM did not negatively affect the immune parameters of meagre. In addition, total numbers of red and white blood cells, as well as thrombocytes, lymphocytes, monocytes, and neutrophils counts were not affected by dietary treatments. All parameters evaluated in plasma were unaffected by dietary CSGM inclusion after 1 and 2 weeks of feeding, with only the haemolytic complement activity showing an increase in fish fed diets with CSGM after 1 week and in fish fed CSGM22.5 diet after 2 weeks. Regarding the innate immune parameters analysed in the intestine, it could be highlighted the increase in alkaline phosphatase and antiprotease activities in fish fed the diet with the higher inclusion of CSGM at 8 weeks. Overall, results suggest that high dietary CSGM inclusion do not compromise immune status or induce an inflammatory response in meagre juveniles.

Dietary tools to modulate glycogen storage in gilthead seabream muscle: glycerol supplementation.

The quality and shelf life of fish meat products depend on the skeletal muscle's energetic state at slaughter, as meat decomposition processes can be exacerbated by energy depletion. In this study, we tested dietary glycerol as a way of replenishing muscle glycogen reserves of farmed gilthead seabream. Two diets were tested in duplicate (n = 42/tank). Results show 5% inclusion of crude glycerol in gilthead seabream diets induces increased muscle glycogen, ATP levels and firmness, with no deleterious effects in terms of growth, proximate composition, fatty acid profile, oxidative state, and organoleptic properties (aroma and color). Proteomic analysis showed a low impact of glycerol-supplementation on muscle metabolism, with most changes probably reflecting increased stress coping capacity in glycerol-fed fish. This suggests inclusion of crude glycerol in gilthead seabream diets (particularly in the finishing phase) seems like a viable strategy to increase glycogen deposition in muscle without negatively impacting fish welfare and quality.

Effects of preslaughter stress levels on the post-mortem sarcoplasmic proteomic profile of gilthead seabream muscle.

Fish welfare is an important concern in aquaculture, not only due to the ethical implications but also for productivity and quality-related reasons. The purpose of this study was to track soluble proteome expression in post-mortem gilthead seabream muscle and to observe how preslaughter stress affects these post-mortem processes. For the experiment, two groups of gilthead seabream (n = 5) were subjected to distinct levels of preslaughter stress, with three muscle samples being taken from each fish. Proteins were extracted from the muscle samples, fractionated, and separated by 2DE. Protein identification was performed by MALDI-TOF-TOF MS. Analysis of the results indicates changes on several cellular pathways, with some of these changes being attributable to oxidative and proteolytic activity on sarcoplasmic proteins, together with leaking of myofibrillar proteins. These processes appear to have been hastened by preslaughter stress, confirming that it induces clear post-mortem changes in the muscle proteome of gilthead seabream.