Fructooligosaccharide and Bacillus subtilis synbiotic combination promoted disease resistance, but not growth performance, is additive in fish.

Species diversification from major to minor carps for their sturdiness and initial higher growth, and also a quest for antibiotic-free aqua farming in the subcontinent, mandates search for and evaluation of alternatives. An experiment was performed to investigate the potential of fructooligosaccharide (FOS) and Bacillus subtilis (BS) (alone or as synbiotics) in promoting growth and immunity against infections in Labeo fimbriatus fingerlings. Six iso-nitrogenous and iso-lipidic diets containing combinations of two levels of FOS (0% and 0.5%) and three levels of BS (0, 104, 106 CFU/g feed) were fed to fish for 60 days. At the end of the feeding trial, twenty-four fish from each group were injected intra-peritoneally with pathogenic strain of Aeromonas hydrophila O:18 to test the immunoprotective efficacy of the supplements against bacterial infection. BS, but not FOS, significantly improved (P < 0.05) growth and feed utilisation attributes like percentage weight gain (PWG), specific growth rate (SGR) and feed conversion ratio (FCR). There were interactive effects of FOS and BS on PWG, SGR and FCR; however, the effects were not additive in nature. These beneficial effects of BS, alone or in combination with FOS, were corroborated by increased protease activity, microvilli density and diameter and number of goblet cells. Overall beneficial effects of FOS and BS included improved erythrocyte (RBC), hemoglobin (Hb), total protein and globulin levels. Total leucocyte (WBC) count and immunological parameters like respiratory burst activity of leucocytes (NBT reduction), lysozyme activity, albumin: globulin ratio and post-challenge survival were significantly improved by both FOS and BS, and their dietary combination yielded the highest improvement in these parameters. Synergistic effects of FOS and BS as dietary supplements indicate that a combination of 106 CFU/g BS and 0.5% FOS is optimal to improve growth, feed utilisation, immune functions, and disease resistance in L. fimbriatus fingerlings.

Mitigating multiple stresses in Pangasianodon hypophthalmus with a novel dietary mixture of selenium nanoparticles and Omega-3-fatty acid.

Effects of a novel dietary mixture of selenium nanoparticles (Se-NPs) and omega-3-fatty acids i.e., Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on mitigating arsenic pollution, high-temperature stress and bacterial infection were investigated in Pangasianodon hypophthalmus. To aim this, four isocaloric and iso-nitrogenous diets were prepared: control feed (no supplementation), Se-NPs at 0.2 mg kg-1 diet with EPA + DHA at 0.2, 0.4 and 0.6% as supplemented diets. Fish were reared under normal condition or concurrent exposure to arsenic (2.65 mg L-1), and temperature (34 °C) (As + T) stress for 105 days. The experiment was conducted with eight treatments in triplicates. Response to various stresses i.e., primary (cortisol), secondary (oxidative stress, immunity, and stress biomarkers) and tertiary stress response (growth performance, bioaccumulation and mortality due to bacterial infection) were determined. Supplementation of dietary Se-NPs at 0.2 mg kg-1 diet and EPA + DHA at 0.2 and 0.4% reduced the primary stress level. Exposure to arsenic and temperature (As + T) and fed with control diet and EPA + DHA at 0.6% aggravated the cortisol level. Anti-oxidative enzymes (Catalase, superoxide dismutase, glutathione peroxidase and glutathione-s-transferase) and immunity (Nitroblue tetrazolium, total protein, albumin, globulin, A:G ratio, total immunoglobulin and myeloperoxidase) of the fish were augmented by supplementation of Se-NPs and EPA + DHA at 0.2 and 0.4%. Neurotransmitter enzyme, HSP 70, Vitamin C were significantly enhanced (p < 0.01) with supplementation of Se-NPs at 0.2 mg kg-1 and EPA + DHA at 0.2 and 0.4%. Whereas total lipid, cholesterol, phospholipid, triglyceride and very low-density lipoprotein (VLDL) were reduced (p < 0.01) with the supplementation of Se-NPs at 0.2 mg kg-1 diet and EPA + DHA at 0.2 and 0.4%. Tertiary stress response viz. growth performance was also significantly enhanced with supplementation of Se-NPs at 0.2 mg kg-1 and EPA + DHA at 0.2 and 0.4% reared under As + T. Whereas arsenic bioaccumulation in fish tissues was significantly reduced with dietary supplementation of Se-NPs and EPA + DHA. Cumulative mortality and relative percentage survival were reduced with Se-NPs at 0.2 mg kg-1 and EPA + DHA at 0.2 and 0.4%. The investigation revealed that a novel combination of Se-NPs at 0.2 mg kg-1 and EPA + DHA at 0.4% followed by 0.2% has the potential to alleviate temperature stress, bacterial infection and arsenic pollution. Whereas diet containing Se-NPs at 0.2 mg kg-1 diet and EPA + DHA at 0.6% was noticeably enhanced the stress in P. hypophthalmus.

Dicamba elevates concentrations of S-adenosyl methionine but does not induce oxidative stress or alter DNA methylation in rainbow trout (Oncorhynchus mykiss) hepatocytes.

Dicamba is a benzoic acid herbicide used to target woody and broadleaf weeds in industrial, domestic, and municipal spheres. Because of its widespread use, dicamba is frequently detected in surface waters near sites of application. However, little is known regarding the effects of dicamba on freshwater fishes. In the present study, primary cultures of hepatocytes from rainbow trout (Oncorhynchus mykiss) were exposed to either an environmentally relevant (0.22 or 2.2 µg L-1) or supra-environmental (22 µg L-1) concentration of dicamba for 48 h to investigate if oxidative stress is a mechanism of toxicity. mRNA abundances of genes involved in the response to oxidative stress, levels of lipid peroxidation, and concentrations of glutathione and s-adenosyl methionine (SAM) were quantified. Results indicate that dicamba does not induce oxidative stress. However, exposure to 2.2 µg L-1 of dicamba did cause a 5.24-fold increase in concentrations of SAM. To investigate the mechanisms of increased SAM, effects of dicamba on global and genome-wide DNA methylation were quantified. Dicamba did not cause changes to DNA methylation. Overall, dicamba was not acutely toxic to hepatocytes and did not cause oxidative stress or changes in DNA methylation at environmentally relevant concentrations.

The effects of dietary selenomethionine on tissue-specific accumulation and toxicity of dietary arsenite in rainbow trout (Oncorhynchus mykiss) during chronic exposure.

The interactive effects of different doses of dietary selenium [as selenomethionine; 1.8 µg g-1 (control), 10 µg g-1 and 40 µg g-1 diet] on the toxicity of dietary arsenic [as arsenite (As3+); 80 µg As per g diet] were investigated in rainbow trout over an exposure period of 30 days. Fish fed with As3+ alone showed an increased hepatic lipid peroxidation (LPO) and a concomitant decline in cellular redox potential (determined as GSH:GSSG) in the liver tissue relative to the control fish. Interestingly, fish fed with low (10 µg g-1) or high (40 µg g-1) concentration of dietary selenomethionine in combination with As3+ showed an even higher degree of hepatic LPO and a further decrease in GSH : GSSG molar ratio relative to the fish treated with As3+ alone. Our study also revealed that exposure to dietary selenomethionine (both at low and high levels) resulted in significantly higher levels of arsenic in target tissues (liver, kidney, and muscle) relative to fish treated with As3+ alone. Similarly, the synchrotron-based X-ray fluorescence imaging analysis also suggested a dose-dependent increase in the co-localization of arsenic and selenium in the brain of fish co-treated with dietary As3+ and selenomethionine. These observations suggested that selenomethionine facilitated arsenic deposition in the brain and likely in other tissues, possibly via bio-complexation. Overall, our findings indicated that elevated dietary selenomethionine can increase the tissue-specific accumulation and toxicity of As3+ in fish during chronic dietary exposure.

A FTIRM study of the interactive effects of metals (zinc, copper and cadmium) in binary mixtures on the biochemical constituents of the gills in rainbow trout (Oncorhynchus mykiss).

We employed Fourier Transform Infrared Microspectroscopy to examine, in situ, the effects of waterborne Cu, Cd and Zn, alone and in binary mixtures, during acute exposure on the integrity of major lipid and protein constituents of the gill of a model teleost species, rainbow trout (Oncorhynchus mykiss). Our findings demonstrated that acute exposure to metals, both individually and in binary mixture, resulted in the degradations of various components of proteins and lipids in the gill tissue. Generally, when comparing the effects of individual metals, Cu was found to induce the maximum adverse effects followed by Cd and Zn, respectively. Among the binary metal-mixture combinations, Cu and Cd produced additive effects on the degradation of major proteins and lipid moieties, whereas the co-exposure of Zn with Cd or Cu elicited ameliorative effects, indicating antagonistic (less than additive) interactions between Zn and Cd or Cu in the rainbow trout gill. Overall, the present study demonstrates that FTIRM can be a useful tool to gain novel mechanistic insights into the biochemical changes induced by metals in the fish gill, which could influence the overall toxicity of metals to fish.

Distribution and speciation of zinc in the gills of rainbow trout (Oncorhynchus mykiss) during acute waterborne zinc exposure: Interactions with cadmium or copper.

We utilized micro X-ray fluorescence imaging (µ-XFI) and micro X-ray absorption near-edge spectroscopy (µ-XANES), which are both synchrotron-based techniques to investigate Zn distribution profile, its co-localization patterns with Ca, S, and Fe and speciation in the gills of rainbow trout (RBT). Fish (~100 g) were exposed to acutely toxic levels of waterborne Zn alone and in combination with waterborne Cd or Cu for 24 h (each at 1 × 96 h LC50). Gill sections were prepared and analyzed at the VESPERS beamline of the Canadian Light Source. The primary lamellae of the fish gill were found to be the primary area of Zn accumulation. These regions also correspond to the zones of mitochondria rich cells localization in fish gills, supporting the putative roles of these cells in metal uptake. Zn was also found to predominantly co-localize with Ca and S, but not with Fe, indicating the roles of Ca and S in intracellular Zn handling. Zn distribution in the gill was markedly reduced during co-exposure to Cd, but not to Cu, suggesting a competitive interaction between Zn and Cd for uptake. The speciation of Zn in the gill was dominated by Zn-phosphate, Zn-histidine and Zn-cysteine species; however, the interactions of Zn with Cd or Cu resulted in the loss of Zn-cysteine. Overall, our findings provide important novel insights into the interactions of Zn, Cd and Cu in the fish gill, which may ultimately help to explain the mechanisms underlying the acute toxicity of these metals in binary mixture to fish.

Interactive effects of chronic dietary selenomethionine and cadmium exposure in rainbow trout (Oncorhynchus mykiss): A preliminary study.

The present study investigated the interactive effects of dietary cadmium (Cd) and selenium (Se) on the tissue-specific (liver, kidney, and muscle) accumulation of these two elements, hepatic oxidative stress response, and morphometrics in rainbow trout (Oncorhynchus mykiss) during chronic exposure. Fish were exposed to elevated dietary Cd (45 µg g-1 dry wt.), and medium (10 µg g-1 dry wt.) or high (45 µg g-1 dry wt.) dietary selenium (added as selenomethionine), both alone and in combination, for 30 days. Exposure to dietary Cd alone caused oxidative stress in fish as reflected by reduced thiol redox (GSH:GSSG), increased lipid peroxidation, and induction of anti-oxidative enzymes (catalase, superoxide dismutase, and glutathione peroxidase) in the liver. Also, an increase in tissue-specific Cd burden and impaired morphometrics (hepato-somatic index and condition factor) were also recorded in fish following exposure to dietary Cd. In contrast, the dietary co-exposure to Cd and Se (at both medium and high doses) resulted in a decrease in Cd burden in the liver and kidney of fish. However, co-exposure to medium, but not high, dose of dietary Se completely alleviated Cd-induced oxidative stress and impaired morphometrics in fish, indicating that the reduced Cd tissue burden might not have been the primary factor behind the amelioration of Cd toxicity by Se. Overall, our study demonstrated that the protective effect of Se against the chronic Cd toxicity in fish is mainly mediated by the anti-oxidative properties of Se, but this protective effect is dose-specific and occurs only at a moderate exposure dose.

Chronic Dietary Selenomethionine Exposure Induces Oxidative Stress, Dopaminergic Dysfunction, and Cognitive Impairment in Adult Zebrafish (Danio rerio).

The present study was designed to investigate the effects of chronic dietary exposure to selenium (Se) on zebrafish cognition and also to elucidate possible mechanism(s) by which Se exerts its neurotoxicity. To this end, adult zebrafish were exposed to different concentrations of dietary l-selenomethionine (control, 2.3, 9.7, 32.5, or 57.7 µg Se/g dry weight) for 30 days. Cognitive performance of fish was tested using a latent learning paradigm in a complex maze. In addition, we also evaluated oxidative stress biomarkers and the expression of genes involved in dopaminergic neurotransmission in the zebrafish brain. Fish treated with higher dietary Se doses (32.5 and 57.5 µg Se/g) exhibited impaired performance in the latent learning task. The impaired learning was associated with the induction of oxidative stress and altered mRNA expression of dopamine receptors, tyrosine hydroxylase, and dopamine transporter genes in the zebrafish brain. Collectively, our results illustrate that cognitive impairment in zebrafish could be associated with Se-induced oxidative stress and altered dopaminergic neurotransmission in the brain.

Effects of chronic exposure to waterborne copper and nickel in binary mixture on tissue-specific metal accumulation and reproduction in fathead minnow (Pimephales promelas).

The current study evaluated the interactive effects of chronic waterborne copper (Cu) and nickel (Ni) exposure on tissue-specific metal accumulation and reproductive performance in fathead minnow (Pimephales promelas). Fish trios (1 male: 2 female; n = 5-6) were exposed for 21 days to: (i) control (no added Cu or Ni), (ii) waterborne Cu (45 µg/L), (iii) waterborne Ni (270 µg/L), and (iv) binary mixture of waterborne Cu and Ni (45 and 270 µg/L, respectively). Fish fecundity (cumulative egg production) was found to be the most sensitive reproductive endpoint, and the interaction of Cu and Ni elicited an additive effect on egg production. Tissue-specific accumulation of both metals was not influenced by the interaction of Cu and Ni, except an increased Cu and Ni burden in the carcass and ovary, respectively, were recorded. The expressions of hepatic estrogen receptor genes (ER-α and ER-ß) and the circulating estradiol level in females were also not affected by the metal-mixture treatment. However, co-exposure to waterborne Cu and Ni resulted in a significant downregulation of the hepatic vitellogenin gene in females, which was associated with the maximum upregulation of the hepatic metallothionein gene. In addition, a significant alteration of ovarian histopathology (decreased abundance of post-vitellogenic follicles, and increased follicular atresia) was also observed only in females exposed to Cu and Ni in mixture. Collectively, these observations suggest that chronic waterborne exposure to Cu and Ni in binary mixture may impair fish reproductive capacity by inducing histopathological damage in ovarian tissue, and disrupting of energy homeostasis in fish.

Dose and chemical species-specific effects of selenium against arsenite toxicity in cultured hepatocytes of rainbow trout (Oncorhynchus mykiss).

The present study evaluated the mechanistic underpinnings of the interactive effects of selenium (Se), both inorganic (selenite) and organic (selenomethionine (SeMet)), against arsenite (As-III) cytotoxicity using rainbow trout (Oncorhynchus mykiss) hepatocytes in primary culture. Arsenite is known to induce cytotoxic effects by disrupting cellular redox homeostasis. In contrast, Se is essential for the maintenance of cellular anti-oxidative machinery, but when present above a threshold concentration, can also induce reactive oxygen species (ROS) generation and cause oxidative damage. In this study, hepatocytes were exposed to 100 µM arsenite independently or in combination with selenite or SeMet (5-40 µM) for 24 h. Exposure to arsenite alone reduced cell viability by inducing intracellular ROS generation, which also corresponded with a concomitant decrease in cellular thiol (GSH : GSSG) ratio and the activities of enzymatic antioxidants (GPx and SOD). Both selenite and SeMet were found to ameliorate the arsenite-induced loss of cell viability and thiol balance significantly, but only at low-intermediate exposure levels (5-20 µM), with selenite being more effective than SeMet. Further analyses of cellular antioxidative pathways, using specific pharmacological treatments, revealed that selenite and SeMet mediate their protective effects against arsenite toxicity via different mechanisms. Selenite ameliorates arsenite-induced oxidative stress primarily by augmenting enzymatic antioxidants (especially SOD), whereas SeMet elicits its protective response essentially by upregulating the non-enzymatic antioxidative pathway that involves GSH. Overall, our study demonstrated that the antagonistic interactions of arsenite and Se at the cellular level are influenced by the exposure dose as well as the chemical speciation of Se.

Effects of chronic waterborne cadmium and zinc interactions on tissue-specific metal accumulation and reproduction in fathead minnow (Pimephales promelas).

The present study was designed to evaluate the interactive effects of chronic waterborne cadmium (Cd) and zinc (Zn) on tissue-specific metal accumulation and reproduction in fathead minnow (Pimephales promelas). Trios (1 male: 2 female; n=6-7) of fish were exposed for 21 days to: (i) control (no added Cd or Zn), (ii) waterborne Cd (7µg/L), (iii) waterborne Zn (170µg/L), and (iv) Cd and Zn in mixture (7 and 170µg/L, respectively). Exposure to Cd or Zn alone did not elicit any significant effect on reproductive output (cumulative egg production) relative to the control, however exposure to Cd and Zn in mixture resulted in a ~50% decrease in fish fecundity. Plasma estradiol in females was reduced by Cd and Zn exposures, both individually and in mixture, with the maximum reduction in the metal mixture exposure. The expression of hepatic estrogen receptor genes (ER-α and ER-ß) in females was affected by exposure to Zn, alone and in mixture with Cd, but not to Cd alone, whereas hepatic vitellogenin gene expression was downregulated across all treatments. Increased follicular atresia in the ovary was also recorded, but only in fish exposed to Cd and Zn in mixture. The interactions of Cd and Zn in mixture decreased Cd accumulation in tissues (gill and liver), however no reciprocal reduction in tissue Zn accumulation was observed. In addition, the expression of the hepatic metallothionein gene was upregulated following exposure to Zn, alone and in combination with Cd, with no additive effects in the latter treatment. Overall, our findings suggest that chronic exposure to waterborne Cd and Zn in mixture may induce additive reproductive toxicity, essentially by disrupting estrogen-mediated functions in fish.

Dopamine receptors participate in acquisition and consolidation of latent learning of spatial information in zebrafish (Danio rerio).

There is growing appreciation that various aspects of learning and memory are strongly influenced by dopamine neurotransmission, and that zebrafish hold particular promise in the study of neurotransmitter systems. In this study, we sought to investigate the effect of dopamine receptors on acquisition and consolidation of memory in zebrafish using a latent learning paradigm. To this end, fish were subjected to a 30 min training trial each day for 16 days during which fish were allowed to freely explore a complex maze with the left or right path blocked and without the presence of a reward. During 16 days fish were treated with dopaminergic agonists (apomorphine, SKF-38393, and quinpirole) and antagonists (SCH-23390 and eticlopride) before or after training trials. To assess cognitive performance of fish, a subsequent probe trial was performed on day 17 while all paths leading to a reward chamber were open and the maze now contained stimulus fish as a reward. Pre- and post-training exposure to apomorphine, SKF-38393, and quinpirole significantly impaired learning and memory in fish. In contrast, fish exposed to eticlopride before and after training exhibited improved performance in a latent learning task. Administration of SCH-23390 before training did not affect zebrafish learning ability, but produced significant memory enhancement when given after training trials. Taken together, these findings are the first indications that D1 and D2 receptors are critically involved in acquisition and consolidation of latent learning in zebrafish, with a more prominent role for D2 receptors. The current study opens the door to future studies to investigate the involvement of dopamine receptors in various aspects of cognitive processes.

Modulatory effects of dopamine receptors on associative learning performance in zebrafish (Danio rerio).

The zebrafish (Danio rerio) has been shown to be an insatiable rival for mammalian model organisms, in many research areas including behavioral neuroscience. Despite a growing body of evidence on successful performance of zebrafish in learning paradigms, little progress has been made toward elucidating the role of neuromodulatory systems in regulation of cognitive functions in this species. Here, we investigated the modulatory effect of dopamine, one of the major neurotransmitters of importance in the brain, on cognitive performance of zebrafish. To this end, a plus maze associative learning paradigm was employed where fish trained to associate a conditioned visual stimulus with the sight of conspecifics as the rewarding unconditioned stimulus. Experimental fish were exposed to dopaminergic agonists (SKF-38393 and quinpirole) and antagonists (SCH-23390 and eticlopride) immediately before training, after training, and just before probe. Pre- and post-training administration of SKF-38393 and SCH-23390 enhanced learning and memory performance of zebrafish in the maze but not when given immediately before the probe trial. Quinpirole also enhanced probe trial performance when administered immediately before training and before the probe but not when given after training. Furthermore, fish that received eticlopride before training, after training or before the probe showed impairment in associative learning performance. Taken together, our results shed first light on modulatory role of dopamine receptors in different aspects of learning and memory in zebrafish.

An in vitro examination of selenium-cadmium antagonism using primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes.

The present study evaluated the ameliorative properties of selenium (Se) against cadmium (Cd)-induced oxidative stress, using isolated rainbow trout (Oncorhynchus mykiss) hepatocytes in primary culture as the model experimental system. Cadmium (Cd) is known to induce cytotoxic effects by disrupting cellular oxidative homeostasis. On the other hand, selenium (Se) is an essential component of biological antioxidative machinery, and thus may provide protection against the toxic insults of Cd by augmenting the cellular antioxidant response. However, Se, when present above the threshold concentration, can also induce reactive oxygen species (ROS) generation and cause oxidative damage. In this experiment, trout hepatocytes in primary culture were exposed to 100 µM Cd, alone or in combination with different concentrations (25-500 µM) of selenite (SeO3(2-)) or selenomethionine (SeMet) for 48 h. Our findings indicated that both chemical forms of Se, at the lowest concentration used (25 µM), significantly reduced Cd-induced cytotoxicity (measured as cell viability). In contrast, Se at higher concentrations (≥ 50 µM) did not offer any protection against a Cd induced decrease in cell viability. The reduced cytotoxicity of Cd in the presence of 25 µM selenite or SeMet was associated with reduced intracellular ROS production, recovery of the cellular thiol status (ratio of reduced and oxidized glutathione), and amelioration in the activities of major enzymatic antioxidants (superoxide dismutase, catalase, and glutathione peroxidase). Co-treatment of hepatocytes with Cd and pharmacological antioxidants (TEMPO and NAC) also reduced Cd-induced oxidative stress in trout hepatocytes. This provided further evidence that Se likely ameliorates Cd toxicity via different antioxidative mechanisms.