Administration of Bacillus subtilis strains in the rearing water enhances the water quality, growth performance, immune response, and resistance against Vibrio harveyi infection in juvenile white shrimp, Litopenaeus vannamei

In this study, vegetative cell suspensions of two Bacillus subtilis strains, L10 and G1 in equal proportions, was administered at two different doses 105 (BM5) and 108 (BM8) CFU ml−1 in the rearing water of shrimp (Litopenaeus vannamei) for eight weeks. Both probiotic groups showed a significant reduction of ammonia, nitrite and nitrate ions under in vitro and in vivo conditions. In comparison to untreated control group, final weight, weight gain, specific growth rate (SGR), food conversion ratio (FCR) and digestive enzymatic activity were significantly greater in the BM5 and BM8 groups. Significant differences for survival were recorded in the BM8 group as compared to the control. Eight weeks after the start of experiment, shrimp were challenged with Vibrio harveyi. Statistical analysis revealed significant differences in shrimp survival between probiotic and control groups. Cumulative mortality of the control group was 80%, whereas cumulative mortality of the shrimp that had been given probiotics was 36.7% with MB8 and 50% with MB5. Subsequently, real-time RT-PCR was employed to determine the mRNA levels of prophenoloxidase (proPO), peroxinectin (PE), lipopolysaccharide- and β-1,3-glucan- binding protein (LGBP) and serine protein (SP). The expression of all immune-related genes studied was only significantly up-regulated in the BM5 group compared to the BM8 and control groups. These results suggest that administration of B. subtilis strains in the rearing water confers beneficial effects for shrimp aquaculture, considering water quality, growth performance, digestive enzymatic activity, immune response and disease resistance

Effect of dietary linolenic acid (18:3n–3)/linoleic acid (18:2n–6) ratio on growth performance, tissue fatty acid profile and histological alterations in the liver of juvenile Tor tambroides

This study was conducted to determine optimal ratios of dietary linolenic acid (18:3n-3, LnA) to linoleic acid (18:2n-6, LA) for Tor tambroides. Juveniles were fed three trial diets with different ratios of LnA/LA (0.0, 0.5 and 1.0) for 10 weeks. Another diet contained 100% palm oil, which was similar tothe diet with 0.0 LnA/LA ratio but different in total amounts of C18polyunsaturated fatty acid, was also used as a control. At the end of the experiment, no significant difference in growth performance was observed among treatments. The overall fatty acid composition in muscle of T. tambroides fed experimental diets was similar in terms of saturated fatty acid and monounsaturated fatty acid. The muscle of fish fed diet with LnA/LA ratio of 0.0 contained significantly lower (p<.05) amount of overall n-3 PUFA than those fed the other diets. Intense accumulation of lipid in the liver parenchyma of all fish except for those fed control diet led to severe degeneration of hepatocytes indicating fatty liver. However, most of the hepatocytes of fish fed control diet were also swollen with nuclei migrated. T. tambroides fed diet with LnA/LA ratio of 0.0 showed degenerated enterocytes with an epithelium with disrupted edges. In conclusion, using vegetable oils contained high level of either LA or LnA in T. tambroides diet seemed to have no advantage over using palm oil contained high level of saturated fatty acid

Probiotic administration of Litopenaeus vannamei: Is there any negative effect on the fatty acid profile of meat?

It has been found that appropriate probiotic applications increased growth performance and disease resistance in shrimp. Bacillus subtilis has been suggested as a potent probiotic in improving growth performance and enhancing immune response in white shrimp, Litopenaeus vannamei. The aim of this work was to evaluate the possible effect of B. subtilis administration on the meat fatty acid profile of white shrimp, L. vannamei. Two groups of shrimps received B. subtilis strains L10 and G1 from the B. subtilis-supplemented feed (10^5 and 10^8 CFU g^-1) while two other groups received it from the rearing water (10^5 and 10^8 CFU ml^-1). One group received no B. subtilis and served as control. According to the results, there was no significant difference between the muscle fatty acid profiles of shrimps administrated by probiotic and control group. This study showed that B. subtilis administration, in either diets or water, did not have any negative effect on fatty acid profiles of L. vannamei meat

Detection and identification of antibiotic biosynthesis genes in Bacillus subtilis strains

Two Bacillus subtilis strains L10 and G1, previously isolated from fermented pickles, were tested for antibacterial activity against Vibrio harveyi using a well-diffusion agar assay. Antibiotic biosynthesis genes were then detected in both bacterial strains and included the following genes: bacA, bacB and bacD genes for bacilysin production, ppsE/fenB gene for plipastatin/fengycin production, albF and albA genes for subtilosin production, and srfAB and sfP genes for surfactin production. Based on these results, two B. subtilis strains could be considered as potential biological control agents in aquaculture

Potential probiotics for white shrimp (Litopenaeus vannamei, Boone 1931) derived from pickle homogenate

The present study focused on the use of candidate probiotic bacteria derived from a vegetable pickle to improve the growth performance, survival rate, culture condition and reduce the infectious disease problems in shrimp aquaculture. This study was conducted in four experiments to investigate probiotic abilities of isolated bacteria from pickle in juvenile white shrimp L. vannamei. Two potential probiotic strains were isolated from vegetable pickle based on the antagonistic activity against two shrimp pathogens Vibrio harveyi and Vibrio parahaemolyticus. These probiotics were named as strain L10 (the isolate number 10 from vinegar) and strain G1 (the isolate number 1 from garlic). Both bacteria were identified by biochemical test, followed by 16s ribosomal RNA gene sequence analysis as Bacillus subtilis, and characterized by PCR amplification repetitive bacterial DNA elements (rep-PCR). Subsequently, B. subtilis, L10 and G1 strains were tested for antibacterial activity under different physical conditions, including culture medium, salinity, pH, and temperature using the agar well diffusion assay. Among the different culture media, LB broth was the most suitable medium for antibacterial production. Both strains showed the highest level of antibacterial activity against the two pathogens at 30 ºC and 1% NaCl. Under the pH conditions strain G1 showed the greatest activity against V. harveyi at pH 7.3-8.0 and against V. parahaemolyticus at pH 6.0-8.0, whereas strain L10 showed the greatest activity against the two pathogens at pH 7.3. In addition wide ranges of tolerance to NaCl, pH and temperature were also recorded for both strains. The potential probiotic strains were subjected for characterization of antibacterial substances and detection of antibiotic biosynthesis genes. Two strains of B. subtilis, L10 and G1, were thus examined against Vibrio harveyi for minimum inhibitory activity and temperature stability of the cell-free supernatants using well-diffusion agar assay. For minimum inhibitory activity a serial dilution of the cell-free supernatant was made at 0, 25, 50, 75% using sterile phosphate buffered saline and those supernatants were then tested for antibacterial activity. The result showed the reduction of antibacterial activity with the higher dilution factors, however, a reasonable activity (7 mm) was observed for both strains even at the highest dilution factors (75%). Temperature stability of the cellfree supernatant was tested at -20 ºC after 7 months preservation at 60, 80, 100, and 120 ºC for 20 min. Both strains showed great antibacterial activity of the cell-free supernatant preserved at -20 ºC, but not those treated at 60, 80, 100, and 120 ºC. Additionally, in order to characterize the antibacterial substances the cell-free supernatant of both strains were treated with four different enzymes. Results showed considerable reduction of antibacterial activity for both strains, indicating the proteinaceous nature of the antibacterial substances. Extra-cellular enzyme production revealed the ability of both strains in secretion of protease and amylase in PG and SA agar. On the other hand no production of cellulase enzyme was recorded for both strains in CMC agar. Subsequently, the antibiotic biosynthesis genes were detected and identified using molecular techniques. Both strains showed similar pattern of antibiotic biosynthesis genes including bacD, bacA and bacB genes for production of bacilysin substances, ppsE/FenB gene for production of plipastatin/fengycin substances, albF and albA genes for secretion of subtilosin, and srfAB and sfP genes for secretion of surfactin. On the basis of the great antibacterial activity, extracellular enzyme production and possessing of antibiotic biosynthesis genes, both strains were considered for further in vivo studies via different application methods (feed and water) for white shrimp, L.vannamei. The first in vivo study was carried out to evaluate the safety and effects of both probiotic candidates, Bacillus subtilis strains L10 and G1 through dietary administration, on the growth performance, digestive enzyme activity, immune gene expression and disease resistance of juvenile white shrimp L. vannamei. The safety of candidate probiotics was determined by injection a dose of 10 10 CFU ml-1 into third abodominal segment of shrimp L. vannamei. Both strains were harmless to shrimp as no mortality was observed after the injection for 15 days. A mixture of two probiotic strains, L10 and G1 in equal proportions, was administered at two different doses 105 (BM5) and 108 (BM8) CFU g-1 feed to shrimp for eight weeks. In comparison to untreated control group, final weight,weight gain and digestive enzyme activity were significantly greater in shrimp fed BM5 and BM8 diets. Significant differences for specific growth rate (SGR) and survival were recorded in shrimp fed BM8 diet as compared with the control; however, no significant differences were recorded for food conversion ratio (FCR) among all the experimental groups. Eight weeks after the start of the feeding period, shrimp were challenged with Vibrio harveyi. Statistical analysis revealed significant differences in shrimp survival between probiotic and control groups. Cumulative mortality of the control group was 63.3%, whereas cumulative mortality of the shrimp that had been given probiotics was 20.0% with BM8 and 33.3% with BM5. Subsequently, real-time PCR was employed to determine the mRNA levels of prophenoloxidase (proPO), peroxinectin (PE), lipopolysaccharide- and B-1,3-glucan- binding protein (LGBP) and serine protein (SP). The expression of all immune-related genes studied was significantly up-regulated (P@ 0.05) in the shrimp fed BM5 and BM8 diets compared to the control group. These findings demonstrate that administration of B. subtilis strains, L10 and G1, can improve growth performance and disease resistance through an enhanced immune response in shrimp. The second in vivo experiment was carried out to investigate the effect of B. subtilis strains L10 and G1 on the water quality, ion reduction, growth performance, digestive enzyme activity, immune response, and disease resistance of juvenile white shrimp L.vannamei. A mixture of two probiotic strains, L10 and G1 in equal proportion, was administered at two different doses 105 (BM5) and 108 (BM8) CFU ml-1 rearing water to shrimp for eight weeks. Both probiotic groups showed promising effects of the improvement of water quality and ion reduction. In comparison to untreated control group, final weight, weight gain, specific growth rate (SGR), and digestive enzyme activity were significantly greater in BM5 and BM8 groups. Significant differences for survival were recorded in the BM8 group as compared with the control; however, no significant differences were recorded for food conversion ratio (FCR) among all the experimental groups. Eight weeks after the start of experiment, shrimp were challenged with Vibrio harveyi. Statistical analysis revealed significant differences in shrimp survival between probiotic and control groups. Cumulative mortality of the control group was 80%, whereas cumulative mortality of the shrimp that had been given probiotics was 36.7% with BM8 and 50% with BM5. Subsequently, real-time PCR was employed to determine the mRNA levels of prophenoloxidase (proPO), peroxinectin (PE), lipopolysaccharide- and B-1,3-glucan- binding protein (LGBP) and serine protein (SP). The expression of all immune-related genes studied was significantly up-regulated in the BM5 and BM8 groups compared to the control group. These results suggest that administration of B. subtilis strains, L10 and G1, can improve growth performance,immune response and disease resistance of shrimp. In conclusion, both strains, B. subtilis L10 and G1, have the potential to be probiotic candidates for the improvement of shrimp culture

Probiotic administration of Litopenaeus vannamei: is there any negative effect on the fatty acid profile of the meat?

It has been found that appropriate probiotic applications increased growth performance and disease resistance in shrimp. Bacillus subtilis has been suggested as a potent probiotic in improving growth performance and enhancing immune response in white shrimp, Litopenaeus vannamei. The aim of this work was to evaluate the possible effect of B. subtilis administration on the meat fatty acid profile of white shrimp, L. vannamei. Two groups of shrimps received B. subtilis strains L10 and G1 from the B. subtilis-supplemented feed (105 and 108 CFU g-1) while two other groups received it from the rearing water (105 and 108 CFU ml-1). One group received no B. subtilis and served as control. According to the results, there was no significant difference between the muscle fatty acid profiles of shrimps administrated by probiotic and control group. This study showed that B. subtilis administration, in either diets or water, did not have any negative effect on fatty acid profiles of L. vannamei meat

Effect of Bacillus subtilis on the growth and survival rate of shrimp (Litopenaeus vannamei)

The effect of Bacillus subtilis, isolated from digestive tract of Macrobrachium rosenbergii was investigated on growth and survival rate of Litopenaeus vannameiduring 60 days of culture. Sixteen aquaria with four replicates were used for treatments and controls. Treatment groups were consisted of і) shrimp fed diet withB. subtilis (T1), and ii) shrimp fed diet mixed with B. subtilis and commercial probiotic (T2). Control groups were consisted of і) shrimp fed diet with commercial probiotic as positive control, and ii) shrimp fed unaltered diet as negative control. Results showed that B. subtilis was proliferated in digestive tract of treated shrimps, and the number of Vibrio spp. was reduced in digestive tract during the cultural period. Survival rate, 75.5± 4.62 %, and yields of shrimps, 190.00 ± 13.13 g, treated with B. subtilis were significantly greater (P<0.05) than the other treated and control groups. Also population density of total viable bacteria and B. subtiliscounted in digestive tract of shrimps treated with B. subtilis were significantly higher (P<0.05) than the other treated groups. Results of this study indicate that the addition of B. subtilis can improve shrimp (L. vannamei) survival rate and yield

Effects of Bacillus subtilis on the growth performance, digestive enzymes, immune gene expression and disease resistance of white shrimp, Litopenaeus vannamei

AbstractWe studied the effect of two probiotic Bacillus subtilis strains on the growth performance, digestive enzyme activity, immune gene expression and disease resistance of juvenile white shrimp (Litopenaeus vannamei). A mixture of two probiotic strains, L10 and G1 in equal proportions, was administered at two different doses 105 (BM5) and 108 (BM8) CFU g−1 feed to shrimp for eight weeks. In comparison to untreated control group, final weight, weight gain and digestive enzyme activity were significantly greater in shrimp fed BM5 and BM8 diets. Significant differences for specific growth rate (SGR) and survival were recorded in shrimp fed BM8 diet as compared with the control; however, no significant differences were recorded for food conversion ratio (FCR) among all the experimental groups. Eight weeks after the start of the feeding period, shrimp were challenged with Vibrio harveyi. Statistical analysis revealed significant differences in shrimp survival between probiotic and control groups. Cumulative mortality of the control group was 63.3%, whereas cumulative mortality of the shrimp that had been given probiotics was 20.0% with BM8 and 33.3% with BM5. Subsequently, real-time PCR was employed to determine the mRNA levels of prophenoloxidase (proPO), peroxinectin (PE), lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP) and serine protein (SP). The expression of all immune-related genes studied was significantly up-regulated (P < 0.05) in the shrimp fed BM5 and BM8 diets compared to the control group. These findings demonstrate that administration of B. subtilis strains, L10 and G1, can improve growth performance and disease resistance through an enhanced immune response in shrimp