R.O.A. Ozório

Dietary probiotic supplementation modulated gut microbiota and improved growth of juvenile rainbow trout (Oncorhynchus mykiss)

In aquaculture, infectious diseases are the major cause of economic losses. Probiotic supplementation may change the microbiota of the digestive tract and modulate the immune defences and nutritional performance. This study was conducted to evaluate the dietary supplementation of multi-species (A: Bacillus sp., Pediococcus sp., Enterococcus sp., Lactobacillus sp.) and single-species probiotics (B: Pediococcus acidilactici) on growth performance and gut microbiota of rainbow trout (Oncorhynchus mykiss). A basal diet was supplemented with probiotic A or B, at two concentrations each (A1, A2, B1 and B2) or not supplemented (control treatment). Diets were distributed to 30 groups of 20 fish, 3 times a day. The gut microbiota was analysed at the end of the feeding trial (96 days) with 16S rDNA denaturing gradient gel electrophoresis (16S-DGGE). Changes in gut microbial community were assessed by Shannon index (H´) and number of operational taxonomic units (OTUs). After 56 days of feeding, weight gain was significantly improved in fish fed diet A1 when compared to the control group. Dietary probiotic supplementation changed the gut microbial composition. Number of OTUs (R) was higher in fish fed A1 (multi-species at lower concentration) than in control group, while H´ was higher in fish fed A1, B1 and B2.

Efeitos benéficos de probióticos no intestino de juvenis de Tilápia-do-Nilo

ABSTRACT.- Mello H., Moraes J.R.E., Niza I.G., Moraes F.R., Ozorio R.O.A., Shimada M.T., En-gracia Filho J.R. & Claudiano G.S. 2013. [Beneficial effects of probiotics on the intesti-ne of juvenile Nile tilapia.] Efeitos beneficos de probioticos no intestino de juvenis de Tilapia-do-Nilo. Pesquisa Veterinaria Brasileira 33(6):724-730. Departamento de Patologia Veterinaria, Faculdade de Ciencia Agraria e Veterinaria, Universidade Estadual Paulista, Via de Acesso Prof. Paulo Donato Castellane s/n, Jaboticabal, SP 14884-900, Brazil. E-mail: julietaengracia@gmail.comThe aim of this study was to evaluate the survival rate, the intestinal microbiota, the mucosal integrity, and the carcass quality of juvenile Nile Tilapia, Oreochromis niloticus , af-ter 80 days being fed on a diet containing probiotic additive ( Bacillus cereus 4.0x10 8 CFUg -1 and Bacillus subtilis 4.0x10 8 CFUg -1 ), at the ratio of 4g/kg of pelleted feed. The completely randomized design with two treatments was used: one control group and one group fed on the mentioned diet. The evaluation of survival rate, the intestinal microbiota analysis by microbiological culture, histomorphometrical analysis of intestinal mucosa and chemical analysis of carcass was performed. The results showed that tilapias from the treated group had higher relative survival rate (P<0.05) than the control group, higher number of colony --forming units (P<0.05) regarding intestinal colonization by

Olfactory sensitivity to amino acids in the blackspot sea bream (Pagellus bogaraveo): a comparison between olfactory receptor recording techniques in seawater

The current study investigated the olfactory sensitivity of the blackspot sea bream to amino acids, odorants associated with food detection in fish, and compared the efficacy of two different experimental methods: multi-unit recording from the olfactory nerve and the electro-olfactogram (EOG). Twenty essential amino acids plus l-DOPA evoked clear, concentration-dependent olfactory responses using both methods, with estimated thresholds of 10−8.5–10−6.2 M (nerve recording) and 10−7.5–10−4.8 M (EOG). The most potent amino acids were l-cysteine, l-methionine (both sulphur-containing), l-alanine, l-leucine (both neutral), l-glutamine (amide-containing) and l-serine (hydroxyl-containing). The least potent were l-proline (secondary α-amino group), the aromatic amino acids and glycine (simplest). Although the rank order of olfactory potency was similar for the two methods used, and the calculated thresholds given by the two methods were positively correlated, the sensitivity of the EOG was consistently lower than multi-unit recording by approximately one order of magnitude, presumably due to the electrical shunting effect of seawater. As in freshwater, the EOG could be a valid method for comparing olfactory potency of different odorants in stenohaline marine fish; however, for absolute ‘biological’ thresholds, a more invasive recording technique, such as multi-unit recording from the olfactory nerve, should be used.

Growth, immune responses and intestinal morphology of rainbow trout (Oncorhynchus mykiss) supplemented with commercial probiotics

The influence of two commercial probiotics on the growth, innate immune parameters and intestinal morphology of rainbow trout (Oncorhynchus mykiss) juveniles (initial weight: 16.4 ± 0.4 g) was evaluated. Two probiotic types: A, multi-species (Bacillus sp., Pedicoccus sp., Enterococcus sp., Lactobacillus sp.) and B, mono-species (Pediococcus acidilactici) were tested at two levels each (A1: 1.5 g kg(-1), 8.6 × 10(5) CFU g(-1); A2: 3 g kg(-1), 1.6 × 10(6) CFU g(-1); B1: 0.1 g kg(-1), 2.6 × 10(4) CFU g(-1); B2: 0.2 g kg(-1), 7.2 × 10(4) CFU g(-1)) versus an unsupplemented diet (C). Diets were distributed to sextuplicate tanks, three times a day to visual satiation for 8 weeks. Growth performance and immune responses (plasma lysozyme, ACH50, peroxidase and head kidney respiratory burst) were determined at 4 and 8 weeks of feeding. Body composition and intestine morphology were determined at the end of the feeding trial. At 8 weeks, the lower dose of multi-species probiotic (A1) improved growth rate, while both probiotic types improved feed conversion rate compared to the control animals, at the lower dose of multi-species (A1) and at the higher dose of mono-species (B2) probiotics. Body composition did not vary between treatments. At 4 weeks, ACH50 activity was significantly higher in fish fed higher dose of B probiotic (B2, 123.7 ± 50.6 vs 44.1 ± 7.7 U.ml(-1) in control). At 8 weeks, lysozyme activity was higher in fish fed A1 (13.1 ± 5.2 μg ml(-1)) diet compared to fish fed control diet (7.8 ± 1 μg ml(-1)). Plasma peroxidase and head-kidney respiratory burst did not differ among the dietary treatments. Villi length and integrity and goblet cell counting of a cross section of the anterior intestine were not significantly different between groups. Results suggest benefits in zootechnical performance and immune humoral responses using both probiotic types, in a dose dependent manner, without apparent alterations in intestinal morphology.

Lethal dose and clinical signs of Aeromonas hydrophila in Arapaima gigas (Arapaimidae), the giant fish from Amazon.

Aeromonas hydrophila is causing substantial economic losses in world aquaculture. This study determined the tolerance limit (LD50-96h) of A. hydrophila in Arapaima gigas, and also investigated the clinical signs after intradermal inoculation. Arapaima gigas fingerlings were inoculated intraperitoneally with 0 (control), 1.0×10(5), 1.0×10(6), 1.0×10(7), 1.0×10(9) and 1.0×10(10)CFU/mL of A. hydrophila for the determination of LD50-96h, which was 1.8×10(8)CFU/mL. In another trial with intradermal inoculation of 1.8×10(8)CFU/mL A. hydrophila, there was a 91.6% of mortality between 8 and 23h, and several clinical signs were found. As follows: depigmentation in the tegument, lesions in the tail and fins, loss of balance, reduction of respiratory movements, hemorrhagic foci, necrotic hemorrhages in the kidney, liver and swim bladder, splenomegaly, ascites in the abdominal cavity and hyperemia, enlargement of the gall bladder, among other clinical signs observed. The results showed that A. gigas has a relative tolerance to A. hydrophila when compared to other Neotropical fish species.

Molecular and functional characterization of a fads2 orthologue in the Amazonian teleost, Arapaima gigas

The Brazilian teleost Arapaima gigas is an iconic species of the Amazon. In recent years a significant effort has been put into the farming of arapaima to mitigate overfishing threats. However, little is known regarding the nutritional requirements of A. gigas in particular those for essential fatty acids including the long-chain polyunsaturated fatty acids (LC-PUFA) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The ability to biosynthesize LC-PUFA is dependent upon the gene repertoire of fatty acyl desaturases (Fads) and elongases (Elovl), as well as their fatty acid specificities. In the present study we characterized both molecularly and functionally an orthologue of the desaturase fatty acid desaturase 2 (fads2) from A. gigas. The isolated sequence displayed the typical desaturase features, a cytochrome b5-domain with the heme-binding motif, two transmembrane domains and three histidine-rich regions. Functional characterization of A. gigas fads2 showed that, similar to other teleosts, the A. gigas fads2 exhibited a predominant Δ6 activity complemented with some capacity for Δ8 desaturation. Given that A. gigas belongs to one of the oldest teleostei lineages, the Osteoglossomorpha, these findings offer a significant insight into the evolution LC-PUFA biosynthesis in teleosts.

Performance of juvenile turbot (Scophthalmus maximus) fed varying dietary L-carnitine levels at different stocking densities

Commercial farming of turbot (Scophthalmus maximus) at high stocking densities may lead to growth depression and increasing production costs. Moreover, the high levels of accumulated waste in an intensive system may cause rapid deterioration of water quality, which may undermine the production. L-carnitine is known as a growth-enhancer which shows promise as mitigator of crowding effects. The effects of stocking densities (4, 8, 11 and 14 kg m2) on growth performance, feed utilization and body composition were evaluated during 75 days on turbot (75.6 ± 2.8 g) fed two dietary L-carnitine levels (40 or 240 mg kg1). At the end of the feeding trial, total ammonia excretion (TAN) was measured postprandially for 24h. Specific growth rate and weight gain decreased with increasing stocking density. Fish held at 4 kg m2 had higher final body weight (94-96 g) than fish held at higher densities (80-87 g). Protein efficiency ratio was higher in fish held at 4 kg m2 (1.33-1.36), in comparison to fish stocked at 8 kg m2 (0.98) or 14 kg m2 (0.45). Voluntary feed intake decreased from 0.70 to 0.56% BW with increasing stocking density. Dietary L-carnitine supplementation did not affect growth performance and body composition, except for body L-carnitine content which increased from 75 to 128 mg kg1 BW with supplementation. Fish fed 240 mg L-carnitine supplements had lower TAN that the ones fed 40 mg L-carnitine (p < 0.05).

Dietary supplementation of heat-treated Gracilaria and Ulva seaweeds enhanced acute hypoxia tolerance in gilthead seabream (Sparus aurata)

ABSTRACT Intensive aquaculture practices involve rearing fish at high densities. In these conditions, fish may be exposed to suboptimal dissolved O2 levels with an increased formation of reactive O2 species (ROS) in tissues. Seaweeds (SW) contain biologically active substances with efficient antioxidant capacities. This study evaluated the effects of dietary supplementation of heat-treated SW (5% Gracilaria vermiculophylla or 5% Ulva lactuca) on stress bioindicators in sea bream subjected to a hypoxic challenge. 168 fish (104.5 g average weight) were distributed in 24 tanks, in which eight tanks were fed one of three experimental diets for 34 days: (i) a control diet without SW supplementation, (ii) a control diet supplemented with Ulva, or (iii) a control diet with Gracilaria. Thereafter, fish from 12 tanks (n=4 tanks/dietary treatment) were subjected to 24 h hypoxia (1.3 mg O2 l−1) and subsequent recovery normoxia (8.6 mg O2 l−1). Hypoxic fish showed an increase in hematocrit values regardless of dietary treatment. Dietary modulation of the O2-carrying capacity was conspicuous during recovery, as fish fed SW supplemented diets displayed significantly higher haemoglobin concentration than fish fed the control diet. After the challenge, survival rates in both groups of fish fed SW were higher, which was consistent with a decrease in hepatic lipid peroxidation in these groups. Furthermore, the hepatic antioxidant enzyme activities were modulated differently by changes in environmental O2 condition, particularly in sea bream fed the Gracilaria diet. After being subjected to hypoxia, the gene expression of antioxidant enzymes and molecular chaperones in liver and heart were down regulated in sea bream fed SW diets. This study suggests that the antioxidant properties of heat-treated SW may have a protective role against oxidative stress. The nature of these compounds and possible mechanisms implied are currently being investigated. Summary: The physiological response and survival rate of gilthead sea bream to hypoxia was highly modulated by dietary supplementation of heat-treated seaweed.

Dietary probiotic supplementation improves growth and the intestinal morphology of Nile tilapia.

Probiotic administration can be a nutritional strategy to improve the immune response and growth performance of fish. The current study aimed to evaluate the effects of a probiotic blend (Bacillus sp., Pediococcus sp., Enterococcus sp., Lactobacillus sp.) as a dietary supplement on growth performance, feed utilization, innate immune and oxidative stress responses and intestinal morphology in juvenile Nile tilapia (Oreochromis niloticus). The probiotic was incorporated into a basal diet at three concentrations: 0 g/kg (A0: control), 3 g/kg (A1: 1.0×106 colony forming unit (CFU)/g) and 6 g/kg (A2: 2.3×106 CFU/g diet). After 8 weeks of probiotic feeding, weight and specific growth rate where significantly higher in fish-fed A1 diet than in fish-fed A0. Alternative complement in plasma was significantly enhanced in fish-fed A2 when compared with A0. The hepatic antioxidant indicators were not affected by probiotic supplementation. Villi height and goblet cell counts increased significantly in the intestine of fish-fed A1 and A2 diets compared with A0. The dietary probiotic supplementation was maintained until 20 weeks of feeding. Then the selected immune parameters, digestive enzymes and apparent digestibility of diets were studied. No effect of probiotic feeding was observed after that longer period supplementation. The dietary supplementation of mixed species probiotic may constitute a valuable nutritional approach towards a sustainable tilapia aquaculture. The improvement of the immune responses and intestinal morphology play an important role in increasing growth performance, nutrient absorption and disease resistance in fish, important outcomes in such a competitive and developing aquaculture sector.

Effects of Dietary Lipid Levels on Growth, Survival, and Molting of Yearling Narrow Clawed Crayfish, Astacus leptodactylus

The influence of dietary lipid level was evaluated on growth, survival, and molting of yearling narrow clawed crayfish, Astacus leptodactylus. Yearling crayfish were fed five isonitrogenous diets (35% protein), formulated to contain 4%, 7%, 10%, 13%, or 16% lipid. Crayfish (10.1 ± 1.6 g) were reared for 56 days in 60-L fiberglass tanks at 23 ± 1.7 °C, with flow-through water and continuous aeration. At the end of the trial, crayfish fed 13% lipid had the highest weight gain and specific growth rate, significantly different from crayfish fed 4% and 7% lipid (p < 0.05). Crayfish fed 13% lipid had a significantly higher survival rate (86.7%) than the animals fed 4% lipid (71%) (p < 0.05). Molting was not affected by lipid level (p > 0.05). We recommend 13% dietary lipid level for optimum growth and survival of Astacus leptodactylus in intensive cultivation.