Iron supplementation in the diets of hybrid catfish (Ictalurus punctatus × I. furcatus) juveniles affected haematocrit levels and potentially decreased disease resistance to Edwardsiella ictaluri.

To prevent catfish idiopathic anaemia, diets fortified with iron have been adopted as a regular practice on commercial catfish farms to promote erythropoiesis. However, the effects of prolonged exposure of excess dietary iron on production performance and disease resistance for hybrid catfish (Ictalurus punctatus × I. furcatus) remains unknown. Four experimental diets were supplemented with ferrous monosulphate to provide 0, 500, 1000, and 1500 mg of iron per kg of diet. Groups of 16 hybrid catfish juveniles (~22.4 g) were stocked in each of 20, 110-L aquaria (n = 5), and experimental diets were offered to the fish to apparent satiation for 12 weeks. At the end of the study, production performance, survival, condition indices, as well as protein and iron retention were unaffected by the dietary treatments. Blood haematocrit and the iron concentration in the whole-body presented a linear increase with the increasing the dietary iron. The remaining fish from the feeding trial was challenged with Edwardsiella ictaluri. Mortality was mainly observed for the dietary groups treated with iron supplemented diets. The results for this study suggest that iron supplementation beyond the required levels does affect the blood production, and it may increase their susceptibility to E. ictaluri infection.

Influence of probiotic and prebiotic supplementation on intestinal microbiota and resistance to Edwardsiella ictaluri infection in channel catfish (Ictalurus punctatus) following florfenicol administration.

Enteric septicemia of catfish (ESC), caused by the gram-negative enteric bacteria Edwardsiella ictaluri, is a significant threat to catfish aquaculture in the southeastern United States. Antibiotic intervention can reduce mortality; however, antibiotic use results in an imbalance, or dysbiosis, of the gut microbiota, which may increase susceptibility of otherwise healthy fish to enteric infections. Herein, recovery of the intestinal microbiota and survivability of channel catfish in response to ESC challenge was evaluated following a 10-day course of florfenicol and subsequent probiotic or prebiotic supplementation. Following completion of florfenicol therapy, fish were transitioned to a basal diet or diets supplemented with a probiotic or prebiotic for the remainder of the study. Digesta was collected on Days 0, 4, 8 and 12, beginning on the first day after cessation of antibiotic treatment, and gut microbiota was characterized by Illumina sequencing of the 16S rRNA gene (V4 region). Remaining fish were challenged with E. ictaluri and monitored for 32 days post-challenge. Florfenicol administration resulted in dysbiosis characterized by inflated microbial diversity, which began to recover in terms of diversity and composition 4 days after cessation of florfenicol administration. Fish fed the probiotic diet had higher survival in response to ESC challenge than the prebiotic (p = .019) and negative control (p = .029) groups.

Dietary Supplementation of a Commercial Prebiotic, Probiotic and Their Combination Affected Growth Performance and Transient Intestinal Microbiota of Red Drum (Sciaenops ocellatus L.).

In the present study, the potential synergism between beneficial lactic acid bacteria (Pediococcus acidilactici) contained in a probiotic and a mixture of fermentable complex carbohydrates and autolyzed brewer's yeast (or prebiotic) were explored in red drum. Four experimental diets were formulated from practical ingredients, and the basal diet was supplemented with either probiotic, prebiotic, or both supplements. Red drum juveniles (~5.5 g) were offered the four experimental diets for 56 days, and at the end of the feeding trial fish fed diets supplemented with probiotic had significantly better weight gain than those fed the non-supplemented diets, and higher protein content in their whole-body composition. Transient intestinal microbiome alpha and beta diversity were significantly affected by the dietary treatments. Interestingly, a higher relative abundance of the lactic acid genus Pediococcus was observed for fish fed diets supplemented with the prebiotic. A higher relative abundance was also observed for the predicted functions of the microbial metagenome, and many of these pathways involved the biosynthesis of essential amino acids, vitamins, and nucleotides. Even though no potential synergistic effect was observed, the individual inclusion of these prebiotic and probiotic supplements positively affected the intestinal health and growth performance of red drum, respectively.

Use of deuterium oxide (2H2O) to assess muscle protein synthesis in juvenile red drum (Sciaenops ocellatus) fed complete, and valine-deficient diets.

The use of 2H2O in tank water to assess protein synthesis rates in fish is a relatively novel methodology that could allow for a better understanding of the effects of particular nutritional and environmental variables on rates of protein accretion. As such, this study involved an assessment and comparison of protein synthesis rates in the muscle of juvenile red drum fed a control diet (nutritionally complete) versus a valine (Val)-deficient diet. Six groups of 12 juvenile red drum, initially weighing ~ 4.5 g/fish, were stocked in six separate 38-L aquaria operating as a recirculating system. Fish were acclimatized to experimental conditions for 2 weeks while being fed the control diet. Just prior to initiating the protein synthesis assay, one aquarium of fish was fed the control diet while a second aquarium of fish was fed the Val-deficient diet. Immediately after consuming the experimental diets, each group of fish was moved to an independent aquarium containing 2H2O, and the fractional synthetic rate (FSR) of protein synthesis was obtained at 12, 24, 36 and 48 h after feeding by collecting two fish per treatment at each time point. This protein synthesis assay procedure was performed in three separate sessions, and considered as replicates over time (n = 3) for fish fed the control or Val-deficient diets immediately before initiating the session. Results indicated that a one-time feeding of a diet deficient in Val significantly reduced protein synthesis rates in the muscle of red drum. In addition, a significant effect of time after feeding was found, where observed FSR values peaked at 12 h after feeding and decreased as time progressed. In conclusion, deuterium methodologies were applicable to red drum, and this approach had the sensitivity to assess differences in protein synthesis rates when dietary perturbations were introduced.

Delivery of selenium using chitosan nanoparticles: Synthesis, characterization, and antioxidant and growth effects in Nile tilapia (Orechromis niloticus).

This study aimed to elucidate the effects of selenium-loaded chitosan nanoparticles used as a dietary supplement on Nile tilapia (Oreochromis niloticus) antioxidant and growth responses. First, chitosan-based nanoparticles containing selenium (Se) were synthesized using the ionotropic gelation method and their physicochemical characteristics, controlled release profile, and antioxidant activity properties were investigated. Thereafter, the effects on glutathione peroxidase and antioxidant activities (by radical scavenging activity), growth, and whole-body composition of Nile tilapia were evaluated when they were fed with Se-loaded chitosan nanoparticles and compared with other selenium dietary supplements. Se-loaded chitosan nanoparticles showed high entrapment efficiency (87%), spherical shape, smooth surface, and broad size distribution. The controlled release of Se consisted of an initial burst followed by a gradual release over 48 h. Se-loaded nanoparticles presented significantly higher antioxidant activity compared to free Se. A 60-day feeding trial was conducted to compare the effects of supplementing different dietary Se sources, including selenomethionine (as organic source), sodium selenite (as inorganic source), and Se-loaded chitosan nanoparticles (Se-Nano and Se-Nano x1.5) on antioxidant and growth responses of Nile tilapia. A basal diet without Se supplementation was used as the control. The dietary supplementations with different Se sources (free and encapsulated selenium) lead to significant improvements in final weight and feed efficiency of Nile tilapia fingerlings. However, dietary treatments did not affect whole-body protein and lipid content. Diets containing Se-Nano and Se-Nano x1.5 were more effective than sodium selenite and selenomethionine in preventing oxidative stress and improving antioxidant activity in Nile tilapia. Overall, Se-loaded nanoparticles presented a great potential as an efficient source for delivering dietary Se to Nile tilapia, directly affecting the growth performance, feed efficiency, oxidative stress, and antioxidant activity of this species.

Efficacy of purified nucleotide supplements on the growth performance and immunity of hybrid striped bass Morone chrysops x Morone saxatilis.

Fishmeal is being increasingly replaced in aquatic animal diets with alternative plant protein feedstuffs such as soybean meal which have lower concentrations of nucleotides; therefore, supplemental sources of exogenous nucleotides in diets could become increasingly important. A 9-week feeding trial was conducted with triplicate groups of juvenile hybrid striped bass (average initial body weight ± standard deviation, 5.6 ± 0.1 g) to determine the effects of supplementing single purified nucleotides on the growth performance and immune parameters. The basal diet, which utilized menhaden fishmeal (25%) and soybean meal (75%) as protein sources, contained 44% protein, 10% lipid and an estimated digestible energy level of 3.5 kcal g-1. Single additions of 5'- adenosine monophosphate (AMP), 5'- uridine monophosphate (UMP), 5'- cytidine monophosphate (CMP), 5'- guanosine monophosphate (GMP), and 5'- inosine monophosphate (IMP) disodium salts (Chem-Impex International, Wood Dale, Illinois, USA) were evaluated with each nucleotide added to the basal diet at 0.5% of dry weight at the expense of cellulose. A positive control diet in this trial was a diet containing 5'- AMP from Sigma-Aldrich also supplemented at 0.5% by weight. Results showed significantly (P < 0.05) improved weight gain between fish fed AMP-supplemented diets and the basal diet. No statistical significance (P > 0.05) was detected in whole-body proximate composition and protein retention of fish fed any of the dietary treatments. The respiratory burst of whole blood phagocytes also was significantly (P < 0.05) higher in fish fed the AMP Sigma diet compared to the other dietary treatments. Dietary IMP and AMP both significantly (P < 0.05) enhanced the capacity of isolated phagocytes to generate extracellular superoxide anion compared to all other dietary treatments. No significant differences were seen in other innate immune parameters such as plasma lysozyme, total plasma protein, and total immunoglobulin. The ability of isolated B lymphocytes to proliferate prompted by the presence of lipopolysaccharides was significantly (P < 0.05) different among dietary treatments with the highest simulation index observed in fish fed the diets containing AMP Sigma and UMP; however, it was not significantly different from that of fish fed the basal diet. Based on all the measured responses, it is concluded that AMP at 0.5% of diet had the most positive influence on growth performance and innate immunostimulation of hybrid striped bass.

The effect of ß-1,3-glucan derived from Euglena gracilis (Algamune™ ) on the innate immunological responses of Nile tilapia (Oreochromis niloticus L.).

Algamune™ is a commercial additive produced from Euglena gracilis, providing a rich source of the ß-1,3-glucan paramylon. Isolated kidney phagocytes of Nile tilapia were incubated with graded doses (0, 8, 16, 32, 64 and 128 µg/ml) of Algamune™ and purified paramylon to gauge their ability to elicit the production of reactive oxygen species. A linear response was observed for extracellular superoxide anion for both sources but only Algamune™ for intracellular superoxide anion. After corroborating the immunostimulant properties ex vivo, a feeding trial was conducted to evaluate the dietary supplementation of Algamune™ (0, 100, 200, 400 and 800 mg/kg of diet) for Nile tilapia. Fish were fed for 3 weeks, after which, fish were sampled for blood and head kidney phagocytes. The remaining fish were challenged with Streptococcus iniae. Macrophage extracellular superoxide anion production was significantly elevated in fish fed diets with 200 mg of Algamune™ kg-1 when compared to fish fed the basal diet. Even though the disease challenge did not show statistical differences, it is worth mentioning that fish fed intermediate doses of Algamune™ had lowest numerical mortality values. Therefore, Algamune™ was demonstrated to enhance some immunological responses of tilapia both in ex vivo and in vivo.

l-glutamine in vitro supplementation enhances Nile tilapia Oreochromis niloticus (Linnaeus, 1758) leukocyte function.

Under appropriate conditions, glutamine (Gln) is an essential nutrient for immunological responses, acting as a metabolic substrate for proliferation of enterocytes and lymphocytes, and having positive effects on the function of stimulated immune cells. Thus, specific components of both innate and adaptive immune systems of Nile tilapia were evaluated after supplementing Gln to cell culture media. Primary cell cultures of kidney leukocytes were used for respiratory burst and phagocytic activity assessment. The ability of macrophages to kill Streptococcus iniae also was evaluated. Additionally, a proliferation assay was conducted with peripheral blood lymphocytes (PBL) exposed to non-specific mitogens. Results showed that macrophage phagocytosis, anion superoxide production, and bactericidal capacity were significantly (P < 0.05) enhanced by Gln supplementation to the culture media. The proliferation of lymphocytes upon mitogenic exposure also was significantly (P < 0.05) enhanced by Gln supplementation to the media. Our results suggest that in vitro, different levels of Gln were necessary for optimal immunological responses of leukocytes and lymphocytes. As such, Gln supplementation was able to enhance and modulate both innate and adaptive responses of Nile tilapia leukocytes, highlighting its potential application as an immunonutrient.

ß-1,3 glucan derived from Euglena gracilis and Algamune™ enhances innate immune responses of red drum (Sciaenops ocellatus L.).

To reduce susceptibility to stressors and diseases, immune-modulators such as ß-glucans have been proven effective tools to enhance the innate immune responses of fish. Consequently, commercial sources of this polysaccharide are becoming increasingly more available. Algamune™ is a commercial additive produced from Euglena gracilis, as a source of linear ß-1,3-glucan. In order to evaluate the immunomodulatory effects of this ß-glucan product, the present study assessed the innate immune parameters of red drum (Sciaenops ocellatus) exposed to Algamune™ ex vivo and in vivo. Isolated kidney phagocytes were incubated with graded concentrations (0, 0.2, 0.4, 0.8, 1.6 and 3.2 mg L-1) of dried Euglena gracilis (Algamune™) as well as purified Paramylon (linear ß-1,3 glucan). Increased bactericidal activity against Streptococcus iniae, and production of intracellular O2- anion superoxide were stimulated by both ß-glucan sources. A reduced activity of extracellular anion superoxide was observed by the phagocytes incubated with Algamune ™. After corroborating the effectiveness of the glucan source ex vivo, a feeding trial was conducted using red drum juveniles (∼26.6 g initial weight). Fish were fed diets with graded levels of Algamune™ (0, 100, 200, 400 and 800 mg kg-1) twice daily for 21 days. No significant differences were detected regarding production performance parameters. At the end of the feeding trial, blood, intestinal content, and kidney were sampled. Intestinal microbiota from fecal material was analyzed through denaturing gradient gel electrophoresis (DGGE) and found to be similar among all treatments. No significant differences were detected for oxidative radical production from whole blood, and isolated phagocytes, and plasma lysozyme activity. However, the total hemolytic activity of red drum plasma was increased in fish fed 100 and 200 mg kg-1 of dietary Algamune™ when compared to fish fed the basal diet. Based on results from both ex vivo and in vivo trials, ß-glucan from Algamune™ was demonstrated to have a moderate immunostimulatory effects on red drum.