Histomorphometric evaluation of melanomacrophage centers (MMCs) and CD3+ T cells of two morphs of brown trout (Salmo trutta) fed diets with immunostimulants.

The brown trout (Salmo trutta) is a salmonid residing in riverine and coastal waters throughout the Northern Hemisphere, whose various populations evolved into distinct ecological morphs, differing in their migratory tendencies and preferred habitats. Unfortunately, due to progressing degradation of natural environment, the conservation of these populations is of growing importance and is undoubtedly a challenging task. Therefore, various means to refine the preparatory protocols for restocking using hatchery-reared fish are being pursued, some of which involve the administration of immunity-boosting substances. The current study assessed the effects of two dietary immunostimulants: Bioimmuno (4% inosine pranobex and 96% ß-glucan) and Focus Plus (commercial preparation by Biomar, Denmark) on two morphs of the brown trout - the river trout (S. trutta morpha fario) and the sea trout (S. trutta morpha trutta). Tissue samples were obtained from ∼75 to 100g fish after 0, 2 and 4 weeks of experimental feeding. Multi-factorial analysis of conducted histological measurements of melanomacrophage centers (MMCs) revealed no changes of their parameters within spleens, but showed a decrease of the occupied tissue area and MMC counts in the livers, progressing with time regardless of the applied diet. Immunohistochemical analysis of CD3+ T cells showed their increased recruitment into mucosal folds of pyloric caeca in the 2-week sampling of trouts fed with the diet with 2% Bioimmuno addition, but this effect was not present in the 4-week sampling. When studying all groups jointly within each morph, there was a significant difference in terms of maintained CD3+ T cells levels, as sea trouts showed significantly higher tissue areas occupied by these cells than river trouts, both in the pyloric caeca and hepatic parenchyma. The study revealed that feeding with a diet enriched with Bioimmuno for 2 weeks may be a favorable enhancement of rearing protocols of brown trout stocks prior to their release, but more studies need to be conducted to test the possibility of an even shorter feeding period.

Effect of Muscle Extract and Graphene Oxide on Muscle Structure of Chicken Embryos.

The effects of CEME and it complex with GO injected in ovo on the growth and development of chicken embryo hindlimb muscle were investigated. First, the preliminary in vitro study on primary muscle precursor cell culture obtained from a nine-day-old chicken embryo was performed to assess toxicity (MTT assay) of CEME, GO (100 ppm) and it complex with different concentrations (1, 2, 5, and 10 wt.%). The effect on cell proliferation was investigated by BrdU assay. CEME at concentrations 1-5% increased cell proliferation, but not the complex with GO. In vitro cytotoxicity was highest in 10% and GO groups. Next, the main experiment with chicken embryos was performed with CEME, GO and it complex injected in ovo on day one of embryogenesis. On day 20 of embryogenesis survival, morphological development, histological structure of the muscle, and biochemical parameters of blood serum of the embryos were measured. No negative effect on mortality, body weight, or biochemistry of blood after use of CEME or GO-CEME complexes was observed. Interestingly, the slight toxicity of GO, observed in in vitro studies, was not observed in vivo. The use of CEME at the levels of 2% and 5% improved the structure of the lower limb muscle by increasing the number of cells, and the administration of 2% CEME increased the number of nuclei visible in the stained cross-section of the muscle. The complex GO-CEME did not further improve the muscle structure. The results indicate that CEME can be applied as an in ovo enhancer of muscle development in broilers.

The Influence of Diet Containing Wheat Gluten Supplemented with Dipeptides or Amino Acids on the Morphology of White Muscle of Yellow Perch (Perca flavescens).

Nutrition affects the metabolism of muscle cells and myogenic progenitor cells which play a crucial role in the growth and development of the muscle tissue. Because of the fact that the development process of yellow perch muscle tissue is not well known, the study aimed to analyze the influence of diets containing wheat gluten and supplemented with Lys and Gly in dipeptides or free form. Fish were allocated into 12 tanks and divided into four groups. Two of the experimental diets were supplemented Lys-Gly in the dipeptide form (DP group) or free amino acids (FAA group). The third was not supplemented with lysine (LF group). The fourth group of fish was fed commercial starter Bio-Oregon (C group). Histological or histomorphometric analyses were conducted: white muscle area, the total number of muscle fibers, the total number of white muscle nuclei, muscle fiber area, number of proliferating myonuclei. Fish fed LF diet showed the lowest number of nuclei and satellite cells proliferation. Results in DP and FAA groups were similar to that observed in fish fed C diet. Summarizing, wheat gluten-based diets supplemented with Lys-Gly dipeptide or free Lys and Gly amino acids exert beneficial effects on the morphology of yellow perch white muscle.

Acute exposure of zebrafish (Danio rerio) larvae to environmental concentrations of selected antidepressants: Bioaccumulation, physiological and histological changes.

Antidepressants have been detected in surface waters worldwide at ng-µg/L concentration. These compounds can exert adverse effects on fish even at low levels. But, all previous analyses have concentrated on adult fish. The aim of the study was to assess the effect of environmental concentrations of sertraline, paroxetine, fluoxetine and mianserin, and their mixtures on such unusual endpoints as physiological and histological changes of zebrafish (Danio rerio) larvae. We also determined the bioconcentration of the pharmaceuticals. Fish Embryo Toxicity test was used to analyze the influence on developmental progression. Histological sections were stained with hematoxylin and eosin. Proliferating cells in liver were determined immunohistochemically by detection of Proliferating Cell Nuclear Antigens. The bioconcentration factor was measured by liquid chromatography coupled to mass spectrometry. Pharmaceuticals were used at low, medium and high concentrations in mixtures and at medium concentration as single compound. Exposure to the analyzed pharmaceuticals increased the rate of abnormal embryo and larvae development, accelerated the hatching time and affected the total hatching rate. Three-times lower proliferation of hepatocytes was observed in larvae exposed to paroxetine, mianserin, sertraline and the mixture of the pharmaceuticals at the highest concentrations. The highest bioaccumulation factor (BCF) was obtained for sertraline. The BCF of the analyzed compounds was higher if the organisms were exposed to the mixtures than to single pharmaceuticals. To conclude, the exposure of zebrafish larvae to selected antidepressants and their mixtures may cause disturbances in the organogenesis of fish even at environmental concentrations.

Cytotoxicity of silver and copper nanoparticles on rainbow trout (Oncorhynchus mykiss) hepatocytes.

Nanoparticles are commonly used in the industry and are present in consumer goods; therefore, evaluation of their potential toxicity is necessary. The aim of the present study was to assess the cytotoxic effects of the nanoparticles of silver (AgNPs) at the concentration of 1.5 mg L-1 and copper (CuNPs) at 0.15 mg L-1 on rainbow trout (Oncorhynchus mykiss) hepatocytes after 28 days of exposure. Histological analysis revealed dilated sinusoids, shrunken hepatocytes, nuclear necrosis, and increased number of Kupffer cells in the liver of fish exposed to nanoparticles. The lowest hepatocyte proliferation index was observed in fish treated with AgNPs. Ultrastructural studies revealed mitochondrial edema and cristolysis, dilated and loosened endoplasmic reticulum, cytoplasm vacuolation, accumulation of lipid droplets, glycogen depletion, and formation of myelin-like bodies. The results also revealed that the liver of fish exposed to copper nanoparticles showed higher regenerative potential indicated by higher proliferation index, more abundant glycogen, and more numerous Kupffer cells compared to the fish treated with silver nanoparticles.

Histopathological effects of silver and copper nanoparticles on the epidermis, gills, and liver of Siberian sturgeon.

The influence of nanoparticles (NPs) on aquatic environments is still poorly documented. The aim of the study was to determine the effects of silver (AgNPs) and copper (CuNPs) nanoparticles on larval Siberian sturgeon (Acipenser baerii) after 21 days of exposure. Acute toxicity of AgNPs on Siberian sturgeon was investigated in a 96-h static renewal study and compared with the toxicity of CuNPs. The AgNPs and CuNPs 96 h mean lethal concentrations (96 h LC50) were 15.03 ± 2.91 and 1.41 ± 0.24 mg L(-1), respectively. Toxicity tests were done in triplicates for each concentration of AgNPs 0.1, 0.5, 1.5 mg L(-1) and CuNPs 0.01, 0.05, 0.15 mg L(-1). The control group was exposed in freshwater. The results indicate that AgNPs and CuNPs exposure negatively influenced survival; body length and mass; and morphology and physiology of the epidermis, gills, and liver of Siberian sturgeon larvae. Fish exposed to AgNPs and CuNPs showed similar pathological changes: irregular structure and pyknotic nuclei of epidermis, aplasia and/or fusion of lamellae, telangiectasis, epithelial necrosis and lifting of the gills, dilation of sinusoidal space, overfilled blood vessels, and pyknotic nuclei of the liver. Fish exposed to CuNPs only demonstrated hyaline degeneration in the gills epithelium and liver. The study shows that CuNPs were more toxic to Siberian sturgeon larvae than AgNPs.

Intersex gonad differentiation in cultured Russian (Acipenser gueldenstaedtii) and Siberian (Acipenser baerii) sturgeon.

Among sturgeons, the occurrence of individuals with gonads containing both testis and ovary components is considered pathological, and such fish are described as intersex individuals or intersexes. Intersexes are observed in both wild and cultured populations of sturgeon, usually at low frequencies. In the present study, intersex Russian (Acipenser gueldenstaedtii) and Siberian (Acipenser baerii) sturgeons constituted 30% of the studied populations. Macroscopically, intersex gonads were recognizable from 500 days posthatching (dph). Initially, gonads with predominantly male characteristics (testis-ova) were observed, but in older fish gonads with predominantly female traits (ova-testis) were more frequent. Using microscopic analysis, intersex gonads were discernible by 130-200 dph. Observations of intersex germinal epithelium development and analysis of sex distribution in the study populations indicated that feminization was occurring. Histological analysis revealed that differentiation of the germinal epithelium in such gonads was accompanied by various morphological alterations (transformations) that were described using quantitative and localization criteria. The most common type of transformations, massive subepithelial transformations, was manifested by the presence of abundant female germinal tissue located under the gonad surface epithelium in the developing testis. These transformations were identified in the early development stage (100-200 dph). In this type of transformation, differentiation of female germinal tissue at the gonad surface and male tissue at the mesorchium/mesovarium resulted in complete formation of both male and female germinal epithelia within the same gonad.

Influence of nanoparticles of platinum on chicken embryo development and brain morphology.

Platinum nanoparticles (NP-Pt) are noble metal nanoparticles with unique physiochemical properties that have recently elicited much interest in medical research. However, we still know little about their toxicity and influence on general health. We investigated effects of NP-Pt on the growth and development of the chicken embryo model with emphasis on brain tissue micro- and ultrastructure. The embryos were administered solutions of NP-Pt injected in ovo at concentrations from 1 to 20 µg/ml. The results demonstrate that NP-Pt did not affect the growth and development of the embryos; however, they induced apoptosis and decreased the number of proliferating cells in the brain tissue. These preliminary results indicate that properties of NP-Pt might be utilized in brain cancer therapy, but potential toxic side effects must be elucidated in extensive follow-up research.

Histopathological, histomorphometrical, and immunohistochemical biomarkers in flounder (Platichthys flesus) from the southern Baltic Sea.

Flounder (Platichthys flesus), collected in late fall of 2009 from four coastal sites in the southern Baltic Sea including the Gulf of Gdansk (GoG), were investigated for a suite of biomarkers of contaminant effects. The biomarkers included liver histopathologies, which were diagnosed and assessed using commonly applied lesion categories, the size and density of melano-macrophage aggregates (MMAs), expression of proliferating cell nuclear antigen (PCNA) and of cytochrome P450 1A (CYP1A) in liver as well as the size and density of MMAs and density of Perls'-positive cells in the spleen. The prevalence of liver lesions differed among the sites. Most frequently occurring were non-specific and early toxicopathic non-neoplastic lesions. Mean MMA size was in the range of 264-519 µm(2) and 717-2137 µm(2) in liver and spleen, respectively, and density was in the range of 6-13 and 15-26 MMAmm(-2), respectively. Mean density of PCNA-positive hepatocytes was in the range of 300-1281 cellsmm(-2). These histomorphometrical biomarkers correlated positively with the muscle Hg, Σ(7)PCB, and ΣDDT residues and negatively with the indices of general liver condition. They showed significant differences between the sites, which were in line with the spatial prevalence of liver lesions and CYP1A induction. Overall, the biomarker responses were more pronounced in the GoG sites in comparison to those outside the Gulf, which confirms some earlier results and broadens the knowledge of contaminant effects in the Polish coastal zone of the Baltic Sea.

The effect of plant protein-based diet supplemented with dipeptide or free amino acids on digestive tract morphology and PepT1 and PepT2 expressions in common carp (Cyprinus carpio L.).

Common carp (Cyprinus carpio) of average body mass 0.07+/-0.02 g were fed three formulated diets: wheat gluten protein-based diet supplemented with Lys-Gly dipeptide (PP), wheat gluten protein-based diet supplemented with free lysine and glycine (AA), and a wheat gluten protein-based control diet without lysine supplementation (CON), frozen zooplankton (Z) (restricted diet), and a commercial starter food Aglo Norse (AN). After 4 weeks of experimental feeding, fish fed AN diet showed the highest body mass and length. Significantly lower mass occurred in groups fed PP, AA, CON, and Z. Fish fed CON diet showed the lowest intestinal folds and the highest number of mucous cells. Fish fed PP diet showed a significantly higher number of gastrin/cholecystokinin (CCK) positive cells. The diameter of lipid vacuoles in hepatocyte cytoplasm of fish fed formulated diets (PP, AA and CON) was significantly higher than in fish fed zooplankton (Z) and the commercial diet (AN). Hepatocytes of fish fed AA and CON showed a higher nucleus proliferation rate than in the other experimental groups. The quantitative analysis of the number of proliferating cell nuclear antigen (PCNA) and caspase-3(rabbit polyclonal antibody CPP-32)-positive cells showed that the highest proliferation rate was accompanied by the high apoptosis in the intestine of fish fed AA and CON. After 4 weeks of experimental feeding the highest relative expression of PepT1 gene was observed in fish fed PP diet, while the lowest expression occurred in fish fed CON. Feeding carp plant protein-based diet supplemented with Lys-Gly dipeptide (PP) had a beneficial influence on fish growth and metabolism in the digestive tract as compared to fish fed control diet without lysine supplementation (CON).

The effect of peptide absorption on PepT1 gene expression and digestive system hormones in rainbow trout (Oncorhynchus mykiss).

The present study evaluates the effect of protein source (dipeptides, free amino acids, and intact protein) on development and growth of Salmonid fish alevin. Specifically, we follow the expression of oligopeptide transporter protein PepT1 in the intestine of rainbow trout (Oncorhynchus mykiss). Fish were fed exogenously one of four diets: three formulated (lysyl-glycine dipeptide supplemented diet - PP, free lysine and glycine supplemented diet - AA, control diet with no lysine - CON) or commercial starter (Aller Futura - AF). Fish increased mean body weight 8 fold with PP- and AA-supplemented diets resulting in significantly higher weight gain than fish fed CON. Statistical analysis revealed a significant increase in relative PepT1 expression of fish fed experimental diets. Immunohistochemical staining with PepT1 antibody showed the presence of the transporter protein in the brush border membrane of the proximal intestinal enterocytes of fish from all experimental groups. Leptin immunoreactivity occurred not only in the gastric glands but also in proximal intestine and pyloric caeca of fish fed PP, AA and AF diets. Leptin immunoreactivity was also observed in hepatocyte cytoplasm and pancreatic acinar cells. Gastrin/CCK immunoreactive cells were present in the proximal intestine and pyloric caeca.

Free amino acids as indicators of nutritional status of silver bream (Vimba vimba), when using commercial and purified diets.

Studies on larval rearing of silver bream (Vimba vimba), a migratory cyprinid fish have addressed on limited scale larval and juvenile rearing using commercial and semipurified diets along with live feeds, such as brine shrimp Artemia nauplii. The objectives of the present study were (1) to determine whether experimental, protein-, peptide-, free amino acid-based diets are adequate for larval silver bream, a stomachless fish, (2) to evaluate whether commercial and purified diets are comparable as the first/exclusive feed for growth and survival of silver bream, and (3) to examine whether free amino acid concentrations in fish body are potential indicators of availability of amino acid sources. We report here the differences in diets acceptance, fish growth and diet utilization in silver bream in comparison to other cyprinid fishes. We specifically address the response in free amino acids in the body to dietary treatments. Experimental diets included: a commercial Aglo Norse feed, casein-gelatin based diet (CG), free amino acid mixture diet (FAA), dipeptide (PP), dipeptide-protein (PP50) based diet, and dipeptide diet without arginine (NoArg). In addition, live Artemia were offered to 3 groups and "fasting" control treatment was included during 3 week long trial. Fish offered Artemia overperformed those offered formulated diets both in terms of mass (80.7+/-26.3 mg) and survival (97.2%). We also indicate that commercial and purified diets are comparable as the first/exclusive feed for growth and survival of silver bream. Our experiment also showed that the whole body free amino acid concentrations of 9 indispensable amino acids (IDAA) out of 10 (His, Thr, Arg, Val, Met, Ile, Leu, Trp, Lys) in the PP50 group was the highest among 7 diet treatments and the totalfree amino acid concentration, total dispensable amino acids (DAA) and total IDAA of the PP50 diet fed fish showed the same trend. This may indicate that diets based on 50% of dipeptides and 50% of protein are adequate for silver bream larvae as they result in similar growth, survival and whole body IDAA concentrations as in fish fed CG (protein-based) diet fed fish.