Live microbial feed supplement in aquaculture for improvement of stress tolerance

Two bacterial strains, Lactobacillus fructivorans, isolated from sea bream (Sparus aurata) gut, and Lactobacillus plantarum, isolated from human faeces, were administered simultaneously, during sea bream development, using Brachionus plicatilis and/or Artemia salina as vectors. The probiotic treatment significantly affected gut colonization. To test the probiotic influence on stress responsiveness, sea bream fry, 47 days post-hatching (p.h.), were subjected to pH stress (from 8.6 to 6.3) and cumulative mortality, cortisol levels and HSP70 gene expression were analysed. Cortisol was selected, since under stress conditions its level increases. HSP70 was selected with consideration of its wide involvement in response to a great number of injuries, and because it protects cells probably by binding and refolding damaged proteins. The results obtained indicated that the administration of probiotic to sea bream fry induced higher HSP70 levels, indicating a greater potentiality to respond to the harmful conditions possibly present in fish farms. This hypothesis is supported by the fact that the levels of cortisol found were significantly lower (P < 0.05) in both groups under probiotic treatment. When pH was used as a stressor, it induced a higher cumulative mortality in the control; the mortality was found to be significantly lower in both treated groups. Interestingly, a significant increase (P < 0.05) in HSP70 gene expression was observed in all stressed groups. These results suggest an improvement in tolerance to acute stress of fry fed with probiotics.     

Selection of lactic acid bacteria as candidate probiotics and in vivo test on Artemia nauplii

Lactic acid bacteria (LAB) were isolated from rotifer cultures in a marine hatchery to search for potential probiotics for marine animals. Fifteen strains were first selected among a total of 55, according to antibacterial activity against Vibrio sp. Among eight strains identified as Lactobacillus casei, four were highly adhesive, suggesting some ability for surface colonization. The other strains were identified as Lactobacillus plantarum, Lactobacillus dextrinicus, and Leuconostoc sp. To validate probiotic potential, Artemia were challenged against pathogenic Vibrio alginolyticus, with or without one of six selected LAB strains. The six strains protected Artemia against the pathogen, to some extent on condition that nutrient enrichment was provided. La. casei BR51 and X2 were preferred, as they were efficient even in the absence of nutrient supply. La. casei X2 was finally selected as candidate probiotic, due to the best growth performances of Artemia, with or without the pathogen.     

Preliminary evaluation of growth performance and immune response of Nile tilapia Oreochromis niloticus supplemented with two putative probiotic bacteria

This study evaluated the effect of two bacteria, Bacillus amyloliquefaciens and the dairy yogurt (DY) Lactobacillus sp., on improving the growth performance, feed conversion ratio (FCR) and some immunological and haematological parameters. Nile tilapia (19.1 g) were stocked in 0.42 m³ tanks at 67 fish m^?3 and fed with two probiotic‐incorporated diets and a control diet for 99 days, followed by a normal diet for another 61 days. At the end of the probiotic feeding, the mean weight, FCR and production rate showed no significant difference among the treatments. However, after 61 days of feeding of the normal diet, the B. amyloliquefaciens fish showed significantly superior growth and better FCR than the control. The DY group had the lowest growth and the poorest FCR. Significantly higher serum lysozyme activity, head‐kidney superoxide dismutase, total immunoglobulin and serum bacterial agglutination titres were recorded in the probiotic groups than the control. Haematological parameters showed no difference between treatments. The B. amyloliquefaciens fed fish showed that the gut microflora was dominated by B. amyloliquefaciens even after the withdrawal of the probiotic. On the contrary, the probiotic bacterial species isolated from the dairy yoghurt did not persist longer in the gut. These results indicate the beneficial effect of administering the bacteria isolated from fish gut microflora, B. amyloliquefaciens, in improving growth, FCR and immunological parameters. The high persistence of B. amyloliquefaciens indicates that the probiotic colonization in the gut is essential for the best responses and economics.     

Effects of dietary probiotic Lactobacillus plantarum and whey protein concentrate on the productive parameters,immunity response and susceptibility of Nile tilapia,Oreochromis niloticus (L.), to Aeromonas sobria infection

Immunostimulatory feed supplements have an increasingly interest in aquaculture management. Generally, an individual supplement was used in fish diets but it is expected that the use of multi‐supplements may show synergistic enhancements in fish performance, health, and immunity. Therefore, the present investigation was carried out to evaluate the use of dietary probiotic Lactobacillus plantarum and whey protein concentrate (WPC) in practical diets for Nile tilapia, Oreochromis niloticus. Hence, probiotic L. plantarum, WPC and their mixture were incorporated into a basal fish diet (300 g/kg crude protein) as follows: T1 = a basal control diet, T2 = a basal diet containing L. plantarum, T3 = a basal diet containing 1.0 g WCP/kg diet and T4, T5 or T6 = basal diets containing probiotic L. plantarum + 1.0, 2.0 or 3.0 g WCP/kg diet, respectively. Fish (15.2 ± 0.6 g) were fed on one of the tested diets up to apparent satiation twice a day for 60 days. After that, fish were intraperitoneally injected with pathogenic bacteria Aeromonas sobria and fish mortality was observed for 10 days postchallenge. Fish growth and feed intake were significantly improved by dietary probiotic L. plantarum (T2) and/or WPC (T3) over the control group (T1), and highest fish performance was observed in T5–T6 fish groups. Similarly, highest values of haematocrit, glucose, total proteins, albumin, and globulin were significantly observed in T5–T6 fish groups. Likewise, fish fed dietary probiotic L. plantarum (T2), WPC (T3), and their mixture (T4–T6) showed antioxidants and immune‐stimulating activities better than the control group. Fish fed the control diet were more susceptible to A. sobria infection showing highest fish mortality (75.0%). Meanwhile, dietary probiotic L. plantarum (T2), WPC (T3), and their mixture (T4–T6) enhanced significantly the fish resistance to A. sobria infection resulting in maximum values of relative percent of fish survival (73.3%–80.0%) in T5–T6 groups. The present investigation recommended the use of probiotic L. plantarum with 2.0 g WPC/kg diet to improve the growth, antioxidant, immunity responses and tolerance of Nile tilapia to A. sobria infection.     

Effects of dietary Lactobicillus plantarum on the growth,carcass quality,and immune response of African catfish (Clarias gariepinus) challenged with Salmonella typhi

An experiment was conducted to investigate the effects of Lactobacillus plantarum on the production of African catfish (Clarias gariepinus). Five experimental diets containing 0, 10³, 10⁵, 10⁷, and 10⁹ CFU of Lactobacillus plantarum g^?1 diet (T₁–T₅ treatments respectively) were fed to African catfish (Clarias gariepinus) (9.20 ± 0.1 g initial body mass), for 70 days. Results indicated significantly lower growth performance in the fish fed diet without probiotics and in those fed diet with the least probiotic level. Treatments with probiotics significantly improved the blood profile and carcass protein but significantly reduced the carcass fat (P < 0.05); these treatments also marginally improved the carcass minerals in comparison with the treatment without probiotic (P < 0.05). Challenging the fish fed the experimental diets with Salmonella typhi showed higher immunity of fish fed the probiotic diets than those fed the nonprobiotic diet. Duncan’s multiple range test showed that the best fish performance was observed with 10³ CFU g^?1 L. plantarum for very parameter measured. However, regression analyses showed the optimum level of the bacteria to be 10^4.13–10⁵ CFU g^?1     

Effects of dietary mannan oligosaccharides in early weaning diets on growth,survival, fatty acid composition and gut morphology of gilthead sea bream (Sparus aurata,L.) larvae

Early weaning of marine fish larvae with dry diets delays gut maturation and reduces growth rates. In juvenile and adult forms of several marine fish species, inclusion of dietary mannan oligosaccharides (MOS) improves gut integrity and functionality, but the effects of MOS inclusion in gilthead sea bream (Sparus aurata, L.) larval diets have not been addressed yet. Thus, this study assesses the effects of dietary MOS inclusion on survival, growth performance, gut morphology, feed acceptance and quality of gilthead sea bream larvae. For that purpose, 16 days post‐hatched gilthead sea bream larvae were fed four graded levels of MOS (Biomos^®, Alltech, Nicholasville, KY, USA) in weaning diets as follows: 0 g kg^?1 MOS, 0.5 g kg^?1 MOS, 1.5 g kg^?1 MOS and 2 g kg^?1MOS. Dietary MOS did not affect feed acceptance in gilthead sea bream larvae (P > 0.05). MOS supplementation was correlated in a dose‐dependent way with higher larval survival (P = 0.026). After 15 days of feeding, dietary MOS increased whole larvae (P < 0.01) arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid. Gilthead sea bream larvae fed 2 g kg^?1 MOS presented higher gut occupation with goblet cells after feeding compared with larvae fed the other dietary treatments. Overall, the results suggest that inclusion of MOS in early weaning diets for gilthead sea bream improves essential fatty acid utilization and may promote growth and final survival.     

A drop in ambient temperature results in a transient reduction of interrenal ACTH responsiveness in the gilthead sea bream (Sparus aurata, L.)

This study investigated the effects of a drop in water temperature (18 °C to 9 °C in 24 h) on the pituitary and interrenal hormones of the gilthead sea bream Sparus aurata. The in vitro sensitivity of the interrenal tissue to ACTH, plasma levels of cortisol, ACTH, -MSH, GH, glucose, lactate and ions were determined. In vitro ACTH, stimulated the release of cortisol from isolated interrenal glands from control gilthead sea bream in a concentration dependent fashion. However, the interrenal cells were less sensitive to ACTH as soon as 24 h following the onset of the temperature drop. At this time, plasma cortisol and ACTH levels were raised, and plasma GH concentrations were decreased, whereas no significant changes were found in plasma -MSH. After 96 h plasma ACTH levels had recovered whereas plasma cortisol levels were still higher than controls after 8 days of the beginning of the experiment. Interrenal sensitivity had recovered after 8 days. The results may help to clarify the relationship between the stress response and the aetiology of the winter syndrome in sea bream.     

Modulation of gut microbiota associated with abalone Haliotis gigantea by dietary administration of host-derived Pediococcus sp. Ab1

In the present study, we evaluated how dietary administration of host-derived Pediococcus sp. Ab1 has an effect on the abalone gut microbiota using a culture-dependent method and 16S rRNA gene library analysis. The culturable lactic acid bacteria number in the probiotic sample was 10⁵ higher than that in the non-probiotic sample, and we speculate that this significant increase was due to colonization of Ab1 into abalone gut. The result of a culture-dependent method showed that the proportion of Vibrio halioticoli clade, which is known to be a beneficial resident bacterium to abalone, was much higher in the probiotic sample than in the non-probiotic sample. 16S rRNA gene clone sequences revealed that gut microbiota in the probiotic sample was obviously diverse compared to the non-probiotic sample, probably due to improvement of the gut environment by Ab1 colonization. In addition, some beneficial bacteria–like sequences such as V. halioticoli were only found in the probiotic sample. These results suggest that the dietary administration of Ab1 to abalone gut has a great effect on modulation of not only culturable but also unculturable gut microbiota. Our results are useful for future investigations into understanding the effect of probiotics on gut microbiota.     

Probiotic dietary supplementation in Nile tilapia as prophylaxis against streptococcosis

This study evaluated the effects of the probiotic Lactobacillus plantarum as dietary supplement on growth performance, haemato‐immunological responses, microbiology, histology and transmission electron microscopy of the intestinal epithelium of Nile tilapia challenged with Streptococcus agalactiae. Fish were distributed into two groups: control (unsupplemented) group and the group fed L. plantarum supplemented diet for a period of 58 days. We observed an increase in the concentration of lactic acid bacteria and a reduction in the number of Vibrionaceae in supplemented fish. A significant increase in the final weight, specific growth rate and feed efficiency was also observed in supplemented fish. After challenge, the number of thrombocytes and neutrophils also increased in supplemented animals. Transmission electron microscopy showed damage to the intestinal mucosa and the presence of bacteria similar to S. agalactiae in both infected groups. L. plantarum colonized the intestines of fish, enhanced the growth performance and modulated some haematological parameters.     

Antibacterial effect of Lactococcus lactis subsp. lactis on Artemia franciscana nauplii and Dicentrarchus labrax larvae against the fish pathogen Vibrio anguillarum

The antibacterial effect of Lactococcus lactis subsp. lactis against the fish pathogen Vibrio anguillarum was determined in Artemia franciscana nauplii and in European sea bass (Dicentrarchus labrax) larvae fed with ten pulses of nauplii enriched with L. lactis. The evaluation of the bactericidal activity of the extracellular products of L. lactis in vitro showed inhibition of growth of Vibrio (Listonella) anguillarum and Photobacterium damselae subsp. piscicida. The incorporation of L. lactis in Artemia nauplii did not affect their survival and offered protection in a challenge with V. anguillarum, significantly increasing LD₅₀. The administration of Artemia nauplii enriched with L. lactis for 48 h to sea bass larvae for five consecutive days had no adverse effect on survival of fish. In an in vivo challenge test with V. anguillarum using sea bass larvae, fish treated with nauplii enriched with the probiotic L. lactis showed significantly (P < 0.001) increased survival rates of 81 % compared with the untreated group of challenged fish (24 %). Our results indicate that L. lactis is a probiotic suitable to be used for the prevention of vibriosis in fish larvae and can be safely administered through their live feed Artemia nauplii.     

Using trace elements in otoliths to discriminate between wild and farmed European sea bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> L.) and Gilthead sea bream (<Emphasis Type="Italic">Sparus aurata</Emphasis> L.)

Trace elements in otoliths of sea bass (Dicentrarchus labrax L.) and sea bream (Sparus aurata L.) from fish farms and coastal wild populations in the western Mediterranean Sea were analysed by inductively coupled plasma-mass spectrometry. Results showed that concentrations of Mg, K, and Mn differed significantly between wild and farmed sea bass, while concentrations of Mg, K, Mn, Fe, Zn, Sr, and Ba varied significantly between wild and farmed sea bream. Discriminate analysis and cross-validation classification showed that the trace element profile in otoliths can be used to separate farmed fish from wild stocks with high accuracy on both sea bass (individuals correctly classified: 90.7 %) and sea bream (individuals correctly classified: 96.6 %). Moreover, trace elements in otoliths resulted to be useful to discriminate among wild fish stocks within each species.     

Bacterial host interaction of GFP‐labelled Vibrio anguillarum HI‐610 with gnotobiotic sea bass,Dicentrarchus labrax (L.), larvae

The location and cell damage caused by Vibrio anguillarum, the causative agent of classical vibriosis, within the developing gut of the newly hatched sea bass, Dicentrarchus labrax (L.), is unknown. A gnotobiotic sea bass model was used to investigate the early interactions of V. anguillarum with sea bass larvae. In the present study, germ‐free sea bass larvae were orally exposed to a V. anguillarum HI‐610 pathogen labelled with the green fluorescent protein (GFP‐HI‐610) and sampled at regular intervals. Pathogenic colonization of gut enterocytes was observed 2 h post‐exposure (p.e.) and onwards, whereas bacteria within the swim bladder were visualized 48 h p.e and onwards. Ultrastructural findings demonstrated direct bacterial contact with the host cell in the oesophageal mucosa and putative attachment to microvilli of mid‐ and hindgut enterocytes. The present findings form a starting point for studies assessing the impact of potential candidates (probiotics, prebiotics, antimicrobial peptides) to mitigate bacterial virulence.     

Enhancing growth and resistance to Vibrio alginolyticus disease in catarina scallop (Argopecten ventricosus) with Bacillus and Lactobacillus probiotic strains during early development

In bivalve aquaculture, selecting suitable probiotic treatments can be crucial for improving hatchery‐rearing of larvae and juveniles. We assessed the potential of five bacterial strains, previously selected in vitro, to improve survival, growth and resistance of catarina scallop Argopecten ventricosus during early and late larval and juvenile developmental stages, as well as during exposure to the pathogen Vibrio alginolyticus. Hatchery‐reared larvae and juveniles were treated with eight treatments of single or combined strains of Bacillus and Lactobacillus at 1 × 10⁶ CFU mL^?1 every 48 h for 9 days (larvae) and 21 days (juveniles). Compared with the control, significantly higher survival and growth in size and weight of early veliger larvae occurred with the antibiotic and the RL5 (Lactobacillus graminis) treatments. Significantly enhanced settlement of pediveliger larvae occurred with a different probiotic strain, the mix of Lactobacillus and Bacillus (MIX‐LB), while higher survival and growth of early juveniles occurred with C3 (Lactobacillus plantarum). The mix of Bacillus (MIX‐B) significantly increased survival of juveniles from V. alginolyticus after 120‐h infection, consistent with maximum activity of superoxide dismutase enzyme. In contrast, all untreated and infected scallops died by 96 h. The three Bacillus strains performed poorly when used as single treatments and when given to early developing larvae. Our results indicate that the action mechanism of probiotic strains is stage specific and strain specific, generating different responses by the host, including improved survival and growth (likely from better nutrient assimilation) and higher resistance against pathogens (possibly from strengthening the immune system).     

Effects of Fish Size and Water Temperature on the Acute Toxicity of Copper for Japanese Flounder,Paralichthys olivaceus,and Red Sea Bream,Pagrus major

The acute toxicities of copper were examined for Japanese flounder, Paralichthys olivaceus, and red sea bream, Pagrus major, in terms of fish size and water temperature. Artificial seawater of low pH of 5.4–6.7 was used as testing water to keep dissolved copper concentration at 0.04–41 mg Cu/L. Japanese flounder of 0.3–17 g and red sea bream of 0.5–13 g were exposed to different concentrations of copper for 96 h at 20 C under semistatic condition. Median‐lethal concentration for 96 h of Japanese flounder and red sea bream were 8.7–12.2 and 2.0–5.2 mg Cu/L, respectively. No significant relationships were observed between median‐lethal concentrations for 96 h and fish size for Japanese flounder, while the value decreased significantly with increasing fish size for red sea bream. Effect of water temperature on the acute toxicity was examined for Japanese flounder of 0.3 and 0.4 g at 10, 15, 20, and 25 C and red sea bream of 0.5 and 1.0 g at 12, 15, 20, and 25 C. Ninety‐six‐hour median‐lethal concentrations for Japanese flounder and red sea bream were 5.1–11.2 and 1.0–5.3 mg Cu/L, respectively. No significant relationships were observed between median‐lethal concentrations for 96 h and water temperature for both fish species.     

Reproduction,growth and stress response in adult red sea bream,Pagrus major (Temminck & Schlegel) exposed to different photoperiods at spawning season

Adult red sea bream, Pagrus major (body weight, 1.0–2.0 kg) was exposed to three photoperiods [12 h light:12 h dark (12L:12D), 16 h light:8 h dark (16L:8D) and 24 h light:0 h dark (24L:0D)] from 2 months before spawning till the end of the spawning season to investigate growth, spawning and stress response. During the spawning season, tanks were checked every morning for spawned eggs. The growth performance in fish under 24L:0D was stimulated with significantly higher feed intake than those under other photoperiods (P<0.05). The number of eggs and gonadal histology confirmed that three and five females out of six in each of duplicate tanks of the 16L:8D treatment spawned. In contrast, only two out of six females in one tank of the 24L:0D treatment spawned, and no spawns were observed in the 12L:12D treatment. At the end of the spawning period, both 17β‐estradiol and testosterone levels were significantly higher in fish exposed to 16L:8D followed by 12L:12D and 24L:0D photoperiods (P<0.05). Photoperiod manipulation did not cause significant stress response in fish (P>0.05). The results suggest that stimulating the growth performance of red sea bream at reproductive stage with a 24L:0D photoperiod is possible if the fish are subjected to this photoperiod long before the onset of the spawning season.     

Lactobacillus spp. bacteria as probiotics in gilthead sea bream (Sparus aurata, L.) larvae: Effects on growth performance and digestive enzyme activities

In this study, the influence of commercial probiotic, Lactobacillus spp., supplementation was investigated on growth parameters and digestive enzyme activities in gilthead sea bream, Sparus aurata, during larval development. All experiments were triplicated and designed in three different administrations of probiotic from 3 days after hatching (DAH) concurrently with starting of exogenous feeding. In the first group, probiotic was added to live food (rotifer and Artemia). In the second group, probiotic was supplemented directly to both live food and water. In the third group, probiotic was added directly to water. Also, no probiotic treatment was maintained in control group. Total bacterial counts among probiotic probiotic-supplemented groups were significantly different from total bacterial counts in controls in water and digestive tract of larvae (p < 0.05). The mean of total bacterial counts in control was approximately 4 × 10⁴-fold increased from the experimental groups in the sea water (p < 0.05). Besides, mean digestive enzyme activities of all probiotics treatment groups were significantly different (p < 0.05) with that of the control. Except probiotic water supplementation group, in all treatments, the specific activities of pancreatic and intestinal enzymes were significantly higher (p < 0.05) in larvae to which probiotic had been supplemented by live food and live food with water. Also, S. aurata larvae that had probiotic administered by live food with water demonstrated significant (p < 0.05) increases in both survival (13–105% higher) and specific growth rate (2–9% higher) as compared to controls. As a result, supplementation of probiotic to directly tank water could not significantly increase growth parameters and digestive enzyme activities and therefore, administration of probiotics by this method would not be effective in terms of husbandry parameters and nutritional condition.     

Diet supplemented with probiotic for Nile tilapia in polyculture system with marine shrimp

The aim of this study was to assess the effect of a probiotic (Lactobacillus plantarum) supplemented diet on Nile tilapia (Oreochromis niloticus) in a polyculture system with marine shrimp (Litopenaeus vannamei) as regards culture performance, hematology, and gut bacterial microbiota. Ten 20-m2 pens were arranged in one earthen pond and stocked with 2 fish (41.9 g) m⁻² and 10 shrimp (2.3 g) m⁻², in total of 40 Nile tilapias and 200 shrimp per experimental unit. Tilapia groups in five of the experimental units were fed a commercial diet supplemented with L. plantarum and the other five with an unsupplemented commercial diet (control). After 12 weeks of culture, the tilapia groups fed the probiotic-supplemented diet presented values 13.6, 7.5, and 7.1% higher for feed efficiency, yield, and final weight, respectively. Viable culturable heterotrophic bacteria counts were reduced, and the number of lactic acid bacteria was increased in the gut of fish and shrimp fed the probiotic-supplemented diet. Hematological analyses showed higher number of thrombocytes and leukocytes in tilapia fed the supplemented diet. L. plantarum utilized in this study colonized the gut of tilapia and shrimp and resulted in reduced number of total bacteria and increased tilapia final weight and feed efficiency.     

Effects of diets supplemented with either individual or combined probiotics,Bacillus subtilis E20 and Lactobacillus plantarum 7‐40, on the immune response and disease resistance of the mud crab,Scylla paramamosain (Estampador)

Two trials, including firstly, diets incorporating individual or combined species of probiotics, Bacillus subtilis E20 and Lactobacillus plantarum 7‐40, were fed to the mud crab, Scylla paramamosian, for 28 days to evaluate the growth, immunity and disease resistance, and secondly, the optimal level of B. subtilis E20 in the diet by tracking the growth, immunity and disease resistance after 28 days of feeding were analysed. No significant differences in growth, total haemocyte counts, respiratory bursts, superoxide dismutase, or glutathione peroxidase were found in the two trials. Crabs fed the B. subtilis E20‐containing diet (D20) had significantly increased phenoloxidase (PO) activity, phagocytic activity (PA) and disease resistance compared with crabs fed the control and the mixed probiotics diet (MD). The mortality of crabs fed the D20 was significantly lower than that of crabs fed the L. plantarum 7‐40‐containing diet. L. plantarum 7‐40 had a great inhibitory effect on the growth of B. subtilis E20, which may have led to the decreased probiotic effect of the MD. An analysis of the optimal level of B. subtilis E20 in the diet showed that crabs fed the B. subtilis E20‐containing diet at a level of 10^9–10 cfu kg^?1 had better immune response of PO and PA, and disease resistance in the second trial.