Effects of Feeding Artemia sp. and Artificial Feed Enriched with Bacillus sp. NP5 to Catfish Pangasianodon hypophthalmus on Growth Performance, Immune Responses, and Resistance to Aeromonas hydrophila Infection

Effects of Feeding Artemia sp. and Artificial Feed Enriched with Bacillus sp. NP5 to Catfish Pangasianodon hypophthalmus on Growth Performance, Immune Responses, and Resistance to Aeromonas hydrophila Infection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of Feeding Artemia sp. and Artificial Feed Enriched with Bacillus sp. NP5 to Catfish Pangasianodon hypophthalmus on Growth Performance, Immune Responses, and Resistance to Aeromonas hydrophila Infection Widanarni Widanarni, Diar Setiawan Aswandi, Rahman Rahman, Waode Munaeni This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1741085/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Motile Aeromonad Septicemia (MAS) is caused by Aeromonas hydrophila and often attacks juvenile catfish. Probiotics could be an alternative to prevent MAS disease. This study aims to determine the dose of probiotics to improve the survival, growth performance, and immune response of juvenile catfish Pangasianodon hypophthalmus to the A. hydrophila infection. This probiotic was contained in Artemia sp. and artificial feed enriched with Bacillus sp. NP5 at concentrations of 10 6 CFU.mL -1 , 10 7 CFU.mL -1 , 10 8 CFU.mL -1 , and the control. Larvae with an average weight of 1.52 ± 0.06 mg and an average length of 0.46 ± 0.007 cm were reared for 28 days in aquariums filled with 10 L of water and a stocking density of 15 individuals.L -1 . After the rearing period, the fish was challenged by the immersion of A. hydrophila with a concentration of 10 7 CFU.mL -1 . This study has revealed that probiotics could increase the fish’s survival, length growth, daily growth rate, and feed conversion ratio. The total bacterial count in larvae and probiotics in juvenile fish is higher than those in the control. After the fish had been challenged with the probiotics at a concentration of 10 8 CFU.mL -1 , they had a better survival rate and immune responses (including leukocyte differential, phagocytosis activity, and respiratory burst activity) than the positive control. Probiotic Bacillus sp. NP5 is effectively administered through bioencapsulated Artemia sp. and commercial feed. Aeromonas hydrophila Bacillus sp. NP5 catfish immune response probiotic Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction One type of fish that has been widely cultivated in various regions in Indonesia is the catfish ( Pangasianodon hypophthalmus ), and its production has increased every year (Marine and Fisheries Ministry of Indonesia 2021 ). Besides Indonesia, several Asian countries, such as China, Thailand, Vietnam, India, and Bangladesh produce P. hypophthalmus in aquaculture (Sathiyanarayanan and Tamilarasan 2019 ). However, the problem of fish farming is a disease, such as P. hypophthalmus which frequently appears during the larval and rearing stages. Meanwhile, P. hypophthalmus fish is frequently attacked by Motile Aeromonad Septicemia (MAS), caused by Aeromonas hydrophila (Hoa et al. 2020 ). Since the disease causes hemorrhage, it is also known as a hemorrhagic disease (Plumb and Hanson 2011 ). Pathogenic bacteria A. hydrophila has caused significant losses in the P. hypophthalmus aquaculture industry (Ruan et al. 2022 ). In addition to causing disease in fish, the bacteria also cause soft tissue wound infections and diarrhea in humans (Singh and Chaudhary 2013 ). Antibiotics are frequently used to treat bacterial infections (Li et al. 2020 ). However, antibiotics can cause resistance to pathogens and disrupt the micro-ecological balance (Liu et al. 2017 ; Tan et al. 2019 ). Probiotics can be used as a safe and environmentally friendly alternative to prevent and control fish diseases (Hoseinifar et al. 2018 ; Zhang et al. 2019 ). Probiotics are live microbes that have beneficial effects on the host by increasing host resistance to pathogens, inhibiting the growth or reproduction of pathogenic bacteria, activating host humoral and cellular immunity, and secreting antagonist substances to inhibit pathogens (Zorriehzahra et al. 2016 ). Bacillus sp. NP5 has been tested to increase the immune response and resistance of tilapia to streptococcosis disease (Agung et al. 2015 ; Tanbiyaskur et al. 2015 ), goldfish to A. hydrophila infection (Djauhari et al. 2016), white shrimp to Infectious Myonecrosis Virus infection (Widanarni et al. 2014 ), catfish to A. hydrophila infection (Tamamdusturi et al. 2016 ), and African catfish to A. hydrophila (Putra et al. 2020 ). Many studies have investigated the application of Bacillus sp. NP5 through artificial or commercial feed on several types of fish and shrimp to conduct an enlargement stage. However, this application has never been tested on catfish P. hypothalamus , especially through the natural feed for larvae and artificial feed for juveniles. In addition, Silva et al. ( 2020 ) assert that the use of different doses of probiotics results in different growth performances in tilapia. The use of probiotics depends on the host species, dose, and duration of administration (Merrifield et al. 2010 ; Cerezuela et al. 2011 ). Thus, it is necessary to gain information related to the optimal dose of probiotic Bacillus sp. NP5 to give juvenile catfish significant growth, immune response, and resistance to A. hydrophila infection. Material And Methods Probiotic Preparation This study employed probiotics Bacillus sp. NP5 from the digestive tract of tilapia (Putra & Widanarni, 2015 ) and made resistance to the antibiotic rifampin as a marker. Bacillus sp. NP5 was cultured on Trypticase Soy Agar (TSA) media and incubated at room temperature (27–30⁰C) for 24 h. Afterward, the bacteria was taken and inoculated on Tryptic Soy Broth (TSB) media. The inoculants were incubated in a water bath shaker at 29⁰C at 140 rpm for 24 h. Preparation of Artemia sp. This study employed 2 g.L − 1 Artemia sp. in the form of a system that was hatched in water with 30 g.L − 1 salinity and was given strong aeration for 24 h. After that, the nauplii of Artemia sp. were harvested by turning off the aeration. The hatching container was covered with dark plastic, and a light source was provided at the bottom. The shell of the hatched system will be on the surface while the unhatched system will settle at the bottom. Nauplii Artemia sp. will approach the light source, which was then siphoned using a hose. Meanwhile, Artemia sp. was given when the fish larvae were 2–5 days old. Preparation of Tubifex sp. (Silkworm) Silkworms or Tubifex sp. were kept in a rearing container and given aeration. Worms had been washed before being placed in different containers according to the treatment used. The finely chopped and washed silkworms were given to fish larvae aged 6–13 days. At this stage, the fish was not given probiotics. Preparation of Artificial Feed The artificial feed was prepared in the form of commercial feed with a protein content of 40%. Feeding was carried out at satiation with a rate of 11% of fish biomass. Artificial feed was given when the seeds were 14–28 days old. Preparation of Maintenance Media Container This study employed 12 aquariums with a size of 25 × 20 × 30 cm 3 . The aquarium was disinfected with 20 ppm potassium permanganate (PK) for 24 h. The aquarium was then cleaned and dried. The cleaned aquarium was placed in a fiber bath measuring 2 × 1 × 0.5 m 3 and filled with 10 L of water. The fiber tub was filled with 200 L of water, and 5 heaters were installed to keep the aquarium’s temperature stable. Each aquarium was added with a hose and an aeration stone to supply oxygen. Test Animal Preparation This study examined catfish larvae from the Center for Development of Catfish Cultivation, Cijengkol, Subang, West Java. Larvae were reared from newly hatched with an average weight and length of 1.52 ± 0.06 mg and 0.46 ± 0.007 cm. The stocking density of fish in each aquarium was 15 fish.L − 1 (150 fish.aquarium − 1 ). Feed Enrichment Process This study employed natural and artificial feed. Natural food was in the form of Artemia sp. and silkworms. Meanwhile, the artificial feed was in the form of commercial feed. The Artemia sp. and commercial feed were enriched with probiotic Bacillus sp. NP5 according to the treatment; this probiotic Bacillus sp. NP5 were 10 6 CFU.mL − 1 , 10 7 CFU.mL − 1 , and 10 8 CFU.mL − 1 . The enrichment process was carried out in each different container. The process of enrichment of Artemia sp. was done by centrifuging probiotics that had been cultured in Tryptic Soy Broth (TSB) and by discarding the liquid supernatant. The precipitated pellet was added with phosphate buffer saline (PBS) as much as the volume of the removed and vortexed supernatants. The ready probiotics were added to the Artemia sp. containing 1 L of water and 30 g.L − 1 salinity and enriched with the treatment for 4 h (Widanarni et al. 2008 ). Afterward, Artemia sp. was harvested using a plankton net and rinsed with water. The harvested Artemia sp. were given directly to the larvae or stored in the refrigerator at 4ºC for later use that day. The commercial feed enrichment process was carried out by centrifuging the probiotics cultured on TSB media and discarding the liquid supernatant. The precipitated pellet was added with PBS as much as the volume of the removed and vortexed supernatants. After that, probiotics were given as much as 2% of the weight of the feed; these probiotics were mixed with a binder in the form of egg whites as much as 2% of the weight of the feed (Wang 2007 ). Next, the feed was sprayed with probiotics according to the treatment and air-dried. The excess feed can be stored in the refrigerator. Maintenance and Challenge Test Juvenile catfish received Bacillus sp. NP5 in feed with different concentrations, namely 10 6 CFU.mL − 1 , 10 7 CFU.mL − 1 , and 10 8 CFU.mL − 1 ; each concentration had three replicates. Meanwhile, the control was without the enrichment of Bacillus sp. NP5, but it consisted of positive control (tested on A. hydrophile ) and negative control (without a test on A. hydrophila ); each control has three replications. Test animals for this study follow (Purwaningsih et al. 2022 ) according to the standard guidelines of the IPB University animal ethics commission (ethical approval number 206–2021). Probiotics were given in two stages. The first stage was giving Artemia sp. to the larvae aged 2–5 days, and the second stage was giving artificial feed to fish aged 14–28 days. Meanwhile, fish aged 6–13 days received silkworms without probiotics. Prior to treatment, catfish larvae had been sampled to determine initial weight and length. 18–24 fish − 1 larvae aged 2–5 days old were fed on the Artemia sp. every 2 h for 4 days of maintenance. Meanwhile, 0.575 g.aquarium − 1 of larvae aged 6–13 days were fed with silkworms without probiotics four times a day: at 8 a.m., 12 p.m., 4 p.m., and 8 a.m. Western Indonesian Time (WIB). Juvenile catfish aged 14–28 days were fed on artificial feed at satiation with a feeding rate (FR) of 11% of fish biomass 3 times a day: at 8 a.m., 12 p.m., and 4 p.m. WIB. The water of the rearing media was changed every other two days, starting from day 6 to 28 of rearing. The temperature (°C) ranged from 29.0-30.3, the dissolved oxygen (DO, mg.L − 1 ) ranged from 5.2–6.3, pH ranged from 6.19–7.12, and the Total Ammonia Nitrogen (TAN; mg.L − 1 ) ranged from 0.016–0.144. After being reared with treated feed for 28 days, the fish were challenged with A. hydrophila . The challenge test process utilized a glass jar with a volume of 3 L for 15 fish. The jar was filled with water (1 L/jar) that had been deposited for 24 h. Juvenile catfish were reared at a density of 15 fish/L and adapted for 2–3 h in the container. The fish were then infected with A. hydrophila at a concentration of 10 7 CFU.mL − 1 . The observation was conducted for seven days post-infection, and dead fish were counted as the fish survival rate data at the end of the challenge test. During the post-challenge fish rearing, the fish were fed artificial feed in the form of commercial feed without probiotic enrichment but with an FR of 11%. This artificial feed was given three times a day. Observation Parameter The survival rate of catfish was calculated at the end of the rearing using the following formulas (Tan et al. 2019 ). Specific growth rate (SGR; %/day) = [ln final weight ln initial weight/days] x 100 Absolute length growth (cm) = Average length of fish at the end of rearing – the average length of fish at the beginning of rearing Total feed consumption = Total weight of feed provided – Total weight of leftover feed Feed conversion ratio (FCR) = (total food intake) × (weight gain) – 1 Survival rate (SR; %) = 100 × (final number of test fish)/(initial number of test fish) Total Bacterial Count (TBC) and abundance of Bacillus sp. NP5 The total bacterial count (TBC) was performed using the plate count method of (Munaeni et al. 2020 ) at the beginning and end of the treatment on larvae (whole-body) and juveniles (intestines). Each treatment used two fish. Meanwhile, the observations were conducted using the Total Bacterial Count (TBC) to measure the larva and the abundance of Bacillus sp. NP5 to measure the juvenile. Meanwhile, the total bacteria employed the Trypticase Soy Agar (TSA) media, while the abundance of Bacillus sp. NP5 employed the TSA + rifampin 50 µg/mL media. The bacteria was calculated using the formula (Madigan et al. 2003 ). Bacterial abundance = number of colonies x 1/diluent factor x 1/mL sample Coefficient of Diversity The coefficient of diversity measured various fish lengths by calculating the coefficient of variance, which was the percentage of the sample standard deviation to the mean value. This measurement used the following formula. Coefficient of variance (%) = root of variance/sample mean x 100 Catfish Immune Response Differential Leukocytes The differential leukocyte was calculated before and after the challenge test. Blood was dripped on the object-glass. Then, another object glass was placed on the end of the first object glass that had already contained blood in a 30° shape. The glass object that formed the corner was pulled to the end. After that, the preparations were air-dried and fixed in methanol solution for five min. The preparations that had been immersed in the methanol solution were air-dried and immersed again in diluted Giemsa solution (1:20) for 15 min. The next steps were rinsing the preparations using distilled water and air-drying them. The finished preparations were observed under a microscope with a magnification of 400 times. Differential leukocytes were calculated according to their types, namely lymphocytes, neutrophils, and monocytes, using the following formula (Amlacher E. 1970 ). % Lymphocytes = (Number of Lymphocytes)/(Lymphocytes + Neutrophils + Monocytes) ×100 % Neutrophils = (Number of Neutrophils)/(Lymphocytes + Neutrophils + Monocytes) ×100 % Monocytes = (Number of Monocytes)/(Lymphocytes + Neutrophils + Monocytes) ×100 Phagocytic Activity The phagocytic activity had been calculated before and after the challenge tests. Fish blood was put into a 50 µL microtube, then 50 µL of Staphylococcus aureus suspension was added to the PBS and homogenized. After that, the blood was incubated for 20 min at 28⁰C. Then, a 5 µL solution was taken to make preparations for review. Then, the solution was fixed with methanol for 5 min and dried. The preparations were soaked for 15 min in Giemsa's solution, washed in running water, and dried. The solution was observed using a microscope with a magnification of 400 times. The phagocytic activity was calculated using the following formula (Dey and Harborne JB 2003). Phagocytic Activity = (Number of phagocytic cells)/(Number of phagocytic cells) ×100 Respiratory Burst (RB) Activity The respiratory burst activity was calculated using the reduction principle of nitroblue tetrazolium (NBT) which produces formazan—the sum of the sizes of superoxide anions. 50 µL of blood was put into the holes of the microplate titer and incubated at 37⁰C for 1 h. The supernatant formed was discarded and rinsed three times with 50 µL of PBS. Then, 50 µL of 0.2% NBT solution was added and incubated at 37⁰C for 1 h. Furthermore, the 0.2% NBT solution was discarded, and the blood was fixed with 50 µL 100% methanol solution for 2 min and rinsed with 30% methanol solution 3 times and dried. The formed formazans blue precipitate was dissolved using 60 L of 2 N KOH solution and 70 µL of dimethyl sulphoxide (DMSO) added to each titer microplate hole. The optical density (OD) of the formazan blue precipitate was then measured using a microplate reader at a wavelength of 630 nm (Divyagnaneswari et al. 2007 ). Data analysis The obtained data were processed using Microsoft Excel 2013 and Minitab 16. Meanwhile, the data were analyzed using the analysis of variance (ANOVA). When the ANOVA results were significantly different, the data were further tested using the Tukey Advanced Test. Results This study has revealed that juvenile catfish in all treatments have significantly different final biomass weight and daily growth rate from the control (Table 1 ). The final biomass weight and the highest daily growth rate of catfish seed were obtained at the treatment with 10 8 CFU.mL − 1 This treatment is not significantly different (P > 0.05) from the treatment of 10 7 CFU.mL − 1 but significantly different (P < 0.05) from the treatment with 10 6 CFU.mL − 1 . On day 14, the treatment with probiotic concentrations of 10 8 CFU.mL − 1 and 10 7 CFU.mL − 1 have resulted in significantly different (P < 0.05) body lengths from the result of the treatment with probiotic concentrations of 10 6 CFU.mL − 1 and the control. Meanwhile, on day 28, the treatment with a probiotic concentration of 10 8 CFU.mL − 1 has resulted in the highest body length which was significantly different (P < 0.05) from the results of other concentrations and the control. Moreover, the treatment with the probiotic concentration of 10 8 CFU.mL − 1 has produced significantly different absolute length growth (P < 0.05) from that of other concentrations and the control. The treatment with probiotic concentrations of 10 8 CFU.mL − 1 and 10 7 CFU.mL − 1 has resulted in significantly different absolute length growth of juvenile catfish from the result of the control. However, the treatment with probiotic concentrations of 10 6 CFU.mL − 1 has produced insignificantly different (P > 0.05) result from that of the control. Table 1 Growth performance of juvenile P. hypophthalmus fed on various concentrations of Bacillus sp. NP5 Parameter Treatment (CFU/mL) Control 10 6 10 7 10 8 Initial biomass weight (g) 1.52 ± 0.06a 1.52 ± 0.06a 1.52 ± 0.06a 1.52 ± 0.06a Final biomass weight (g) 177.67 ± 8.33a 303.33 ± 41.02b 401.67 ± 78.59bc 537.33 ± 35.00c Specific growth rate (SGR; %/day) 18.54 ± 0.33a 20.81 ± 0.68b 22.00 ± 1.04bc 23.32 ± 0.39c Length growth (H0) (cm) 0.46 ± 0.01a 0.46 ± 0.01a 0.46 ± 0.01a 0.46 ± 0.01a Length growth (H14) (cm) 1.86 ± 0.14ab 1.63 ± 0.09a 2.08 ± 0.05bc 2.26 ± 0.04c Length growth (H28) (cm) 2.64 ± 0.10a 2.82 ± 0.15a 3.33 ± 0.11b 4.00 ± 0.04b Absolute length growth (cm) 2.18 ± 0.11a 2.35 ± 0.24a 2.87 ± 0.10b 3.54 ± 0.04c Coefficient of diversity of fish length (H14) (%) 11.14 ± 0.77c 9.46 ± 1.42bc 6.49 ± 1.28ab 4.52 ± 1.16a Coefficient of diversity of fish length (H28) (%) 7.69 ± 1.65ab 9.96 ± 2.9b 4.94 ± 0.70a 4.81 ± 2.00a Total feed consumption 22.21 ± 3.90a 39.86 ± 7.00ab 50.40 ± 11.55b 59.80 ± 10.51b Feed conversion ratio (FCR) 1.90 ± 0,17c 1.71 ± 0.06bc 1.47 ± 0.13b 1.11 ± 0.10a Survival rate (SR; %) 78.44 ± 1.02a 81.33 ± 1.76ab 82.00 ± 1.76b 86.67 ± 0.67c Data are mean ± SD. Different letters in the same line showing significantly different (P < 0.05) On day 14, the treatment with the concentration of probiotics 10 8 CFU.mL − 1 has resulted in the lowest coefficient of the diversity of fish length. This result is not significantly different (P > 0.05) from that of the treatment with the concentration of probiotics 10 7 CFU.mL − 1 but significantly different (P < 0.05) from that of the treatment with the concentration of probiotics 10 6 CFU.mL − 1 and control. On day 28, each treatment has produced diverse lengths, but this result was not significantly different (P > 0.05) from that of the control. The amount of feed consumption, final biomass weight, and SGR at concentrations of 10 7 CFU.mL − 1 and 10 8 CFU.mL − 1 were not significantly different (P > 0.05), but the FCR of the two treatments was significantly different (P < 0.05). The survival rate of the treatment with a concentration of 10 8 CFU.mL − 1 was significantly different (P 0.05) from that of the control. The total bacterial count (TBC) in larvae and the total of Bacillus sp. NP5 in juveniles are higher than those in the control (Table 2 ). The range of total bacteria count at the beginning of the treatment was 1.08–1.80 × 10 4 CFU.larva − 1 while Bacillus sp. NP5 was not found. At the end of the treatment, the larvae has the highest number of bacteria obtained in the treatment of 10 8 CFU/mL and the lowest number of bacteria in the control. Probiotic Bacillus sp. NP5 in juveniles was found in the probiotic treatment, and the highest number of probiotic Bacillus sp. NP5 is found in the treatment of 10 8 CFU.mL − 1 . Table 2 Total bacterial count (TBC) of larvae and total of Bacillus sp. NP5 on juvenile P. hypophthalmus Treatment Total bacterial count (TBC) Total Bacillus NP5 Initial (CFU/larva) Final (CFU/larva) Initial (CFU/ juvenile) Final (CFU/ juvenile) Control 1.08 x 10 4 1.3 x 10 7 0 0 10 6 CFU/mL 1.65 x 10 4 2.1 x 10 7 0 1.2 x 10 5 10 7 CFU/mL 1.41 x 10 5 3.7 x 10 7 0 1.0 x 10 5 10 8 CFU/mL 1.80 x 10 5 4.0 x 10 7 0 2.4 x 10 5 Data are presented as mean ( n = 2). Before the challenge test, each treatment did not have a significantly different total of lymphocytes, neutrophils, and monocytes (P > 0.05). In contrast, after the challenge test, the treatment with the probiotic concentration of 10 8 CFU.mL − 1 (Fig. 1 ) has produced the highest number of monocytes and neutrophils. Furthermore, before the challenge test, the highest percentage of the phagocytic activity was found in the probiotic treatment of 10 8 CFU.mL − 1 , but after the challenge test with A. hydrophila , the highest value of the phagocytic activity is obtained at the treatment of 10 8 CFU.mL − 1 with a significant difference of (P 0.05) from that in each treatment. However, after the challenge test, the respiratory burst activity has increased, and the highest score is obtained at the treatment of 10 8 CFU/ mL. This score is significantly different (P < 0.05) from that at the control (Fig. 3 ). The juvenile catfish has the lowest survival rate after being infected with A. hydrophila in the positive control. This rate is significantly different (P < 0.05) from that in the negative control and the treatment added with the Bacillus sp. NP5. Discussion The final biomass weight and daily growth rate of juvenile catfish in all treatments are significantly different from those of the control (Table 1 ). It is suspected that the provision of probiotics in catfish seed feed can increase nutrient digestibility so that the feed can be easily absorbed by fish. Putra and Widanarni (Putra and Widanarni 2015 ) have revealed that the provision of probiotic Bacillus sp. NP5 from the digestive tract of tilapia can increase nutrient digestibility, digestive enzyme activity, and growth performance of tilapia. Meanwhile, Tamamdusturi et al. (Tamamdusturi et al. 2016 ) state that the Bacillus sp. NP5 can hydrolyze macromolecules in feed to be simpler molecules; thus, the nutrients can be easily absorbed by the intestinal wall of fish and will spread through the circulatory system of the body to serve as an energy source for the growth of catfish. According to Adineh et al. (Adineh and Resources 2013 ), probiotics of Bacillus species can more significantly grow fish larvae. Different doses in this study show different results on the final biomass weight and the highest daily growth rate of catfish parameters; the best treatment is obtained at 10 8 CFU.mL − 1 . Silva et al. (Silva et al. 2020 ) assert that different doses of probiotics affect the growth performance of tilapia. Besides increasing the final biomass weight and daily growth rate, supplementation of probiotics could increase body length. Absolute length growth is obtained at the treatment with a probiotic concentration of 10 8 CFU.mL − 1 . The coefficient of diversity is one of the parameters of seed quality that indicates the level of uniformity of seed size. This study has found that the lowest coefficient of the diversity of fish seed length occurs on day 14. The fish length is classified as uniform if the coefficient of diversity is not more than 15% (Budiardi et al. 2005 ). Postulate that the smaller the coefficient of fish diversity, the better the quality of the produced seeds; consequently, the selling value of the seeds will be higher (Supriyono et al. 2006 ). The value of the coefficient of diversity increases because the fish cannot properly utilize the nutrients on feed for long growth. The concentrations of 10 7 and 10 8 do not produce significantly different (P > 0.05) parameters of feed consumption, final biomass weight, and SGR but produce significantly different (P < 0.05) FCR. These findings show that the use of probiotic doses is necessarily considered in cultivation. This study has also discovered that the treatment of 10 6 CFU.mL − 1 does not produce significantly different (P > 0.05) results from that of the control. However, probiotics have a lower feed conversion value than the control. This indicates that Bacillus sp. NP5 can increase the digestibility of feed and reduce the value of feed conversion. Djauhari et al. (Djauhari et al. 2016) state that Bacillus sp. NP5 can significantly increase the activity of digestive enzymes in carp because it can increase the digestibility of proteins, carbohydrates, and fats. Bacillus sp. NP5 can help the digestive process to produce extracellular enzymes, such as amylase, protease, and lipase. Thus, nutrient absorption and utilization become more efficient. Balcazar et al. (Balcázar et al. 2006 ) explain that digestive enzymes produced by probiotic bacteria, such as amylase, protease, and lipase, can help the digestive process. The lowest feed conversion ratio while using artificial feed is obtained at treatment with the probiotic concentration of 10 8 CFU.mL − 1 . A high growth rate and low FCR are influenced by the probiotic concentration that can be consumed by fish. Such a condition is denoted by the total bacterial count in larvae and the total of probiotics in juvenile fish (Table 2 ). A high concentration of probiotics also gives a higher value. The high total bacteria in larvae and juvenile fish indicates that the probiotic Bacillus sp. NP5 is effectively administered through Artemia sp. and commercial feed. The effectiveness of probiotics depends on the ability of probiotics to survive during the journey to reach the target organs and act on the digestive system. The best fish growth performance is influenced by gut microbes as indicated by an increased total bacterial count and total Bacillus sp. NP5 (Tamamdusturi et al. 2016 ). However, the addition of high doses of probiotics is not always accompanied by an increase in total bacteria in the fish. This condition occurs because not all probiotics added to feed can be taken up by larvae (Widanarni et al. 2010 ). It is suspected that during the bioencapsulation process into Artemia sp. and the coating on the feed, the amount of probiotic does not appropriate for the treatment dose. The interesting finding of this study is that Bacillus sp. NP5 probiotics were no longer found in all treatments after fish larvae had been fed on silkworms without Bacillus sp. NP5 for eight days. This indicates that probiotics should be given continuously during maintenance. Silva et al. (Silva et al. 2020 ) argue that the administration of probiotics gave effects after eight weeks of maintenance. The best survival rate is obtained in the treatment with a concentration of 10 8 CFU.mL − 1 . Other studies have revealed that the addition of probiotic Bacillus sp. NP5 can increase the survival of tilapia (Tanbiyaskur et al. 2015 ) and white shrimp L. vannamei (Widanarni et al. 2014 ). Meanwhile, this study has shown that probiotic supplementation could improve the survival of catfish P. hypophthalmus . Besides improving growth performance, supplementation of Bacillus sp. NP5 could increase the immune response of catfish P. hypophthalmus . The immune system is a coordinating system that aims to protect individuals’ identity and integrity and prevent the invasion of harmful organisms. Parameters or indicators to observe the immune response are leukocyte differential (Fig. 1 ), phagocytic activity (Fig. 2 ), and respiratory burst activity (Fig. 3 ). Differential leukocytes are grouped according to their types, namely lymphocytes, neutrophils, and monocytes. After the challenge test, the highest number of monocytes and neutrophils was found in the treatment of 10 8 CFU.mL − 1 (Fig. 1 ). It is suspected that the phagocytic cells in the fish's body are ready to phagocytize the invading pathogenic bacteria. The differential value of leukocytes before and after the challenge tests has decreased the number of lymphocytes but has increased the number of neutrophils and monocytes. The higher number of monocytes and neutrophils in fish indicates that the fish can produce phagocytic cells, and the blood cells can perform phagocytosis when pathogenic microorganisms attack (Tanbiyaskur et al. 2015 ). Consequently, administering probiotics can increase the body resistance of catfish. Phagocytic activity is the first defense line of the cellular response carried out by monocytes (macrophages) and granulocytes (neutrophils). This process can occur when a foreign object enters the fish body which will be phagocytized by macrophages. These macrophages will destroy antigens by phagocytosis and send signals to the lymphocyte tissue to form specific antibodies. The formed antibodies will reduce the toxicity of the poison and weaken the pathogen from spreading; as a result, phagocytic cells will more easily attack the pathogen (Rodriguez-estrada et al. 2013 ). Unlike the control, the percentage value of phagocytic activity in the probiotic treatment has significantly increased after the challenge test (Fig. 2 ). The highest percentage value of phagocytic activity was found in the treatment of 10 8 CFU.mL − 1 after the challenge tests. This shows that the addition of the Bacillus sp. NP5 can accelerate the phagocyte process carried out by macrophages which is the first line of defense of the immune response. Meanwhile, Djauhari et al. (Djauhari et al. 2016) deliver that the percentage value of phagocytic activity has increased after the challenge test and in the probiotic treatment. Probiotics can interact with mononuclear phagocytic cells (monocytes and macrophages), polynuclear leukocytes (neutrophils), and natural killer cells. Probiotics can act as an effective trigger for phagocytic cells so that they can increase phagocytic activity. The increase in the respiratory burst activity could increase the phagocytic cells of fish when challenged to fight pathogenic bacteria. Tamamdusturi et al. (Tamamdusturi et al. 2016 ) have revealed that an increasing respiratory burst activity occurs after the challenge test which indicates the fish attempts to defend themselves from pathogenic bacterial infection. The probiotic treatment has a higher value of respiratory burst activity than the control. The respiratory burst activity is the basic building block of the antibacterial system found in the fish's body. The increased value of respiratory burst can be associated with an increased value of phagocytic cell activity (Rawling et al. 2012 ). The respiratory explosion can increase the amount of oxygen consumption and form superoxide anions. This process is accelerated by NADPH-oxidase and multi-component enzymes inside the plasma membrane after phagocytic activation (Rieger and Barreda 2011 ). This study has discovered that the supplementation probiotic Bacillus sp. NP5 could increase resistance to pathogenic A. hydrophila . Putra and Widanarni (Putra and Widanarni 2015 ) state that probiotic Bacillus sp. NP5 has the strongest antagonistic activity against pathogenic bacteria, such as Streptococcus sp. Other studies have also discovered that the addition of the Bacillus sp. NP5 could increase the survival of tilapia infected by Streptococcosis (Tanbiyaskur et al. 2015 ) and goldfish injected with A. hydrophila (Djauhari et al. 2016). Probiotics from Bacillus species can increase the fish’s growth, immune response, and resistance to pathogenic infection (Cha et al. 2013 ), such as A. hydrophila in juvenile tilapia fish (Iwashita et al. 2014 ). Conclusions The administration of Bacillus sp. NP5 can increase the total bacterial count in the larvae of P. hypophthalmus . In addition, the administration of Bacillus sp. NP5 can increase survival, growth performance, and immune response (leukocyte differential, phagocytic activity, and respiratory burst activity) of juvenile P. hypophthalmus with the best results found in the dosage of 10 8 CFU.mL − 1 . Abbreviations A. hydrophila : Aeromonas hydrophila ANOVA: analysis of variance CFU : Coloni Forming Units DMSO: dimethyl sulphoxide DO : dissolved oxygen FCR : feed conversion ratio FR : feeding rate MAS : Motile Aeromonad Septicemia NBT: nitroblue tetrazolium OD: optical density P. hypophthalmus : Pangasianodon hypophthalmus PBS: phosphat buffer solutions PK : potassium permanganate RB: respiratory burst SGR : specific growth rate SR : survival rate TAN : total ammonia nitrogen TBC: total bacterial count TSA : Trypticase Soy Agar TSB : Tryptic Soy Broth WIB : Western Indonesian Time Declarations Acknowledgements We thank Mr. Ranta of the Aquatic Organism Health Laboratory at IPB University for the supply of laboratory equipment and technical assistance. We also thank Nurul Novelia Fuandia and other master students in the Department of Aquaculture at IPB University for technical assistance. Funding The authors declare that no funds were received during the preparation of this manuscript. Author information Authors and Affiliations Department of Aquaculture, Faculty of Fisheries and Marine Science, IPB University, Bogor, Indonesia. Widanarni Widanarni (ORCHID ID https://orcid.org/0000-0002-0821-3225) Diar S. Aswandi Rahman Rahman (ORCHID ID https://orcid.org/0000-0002-3682-9529) Department of Aquaculture, Faculty of Fisheries and Marine Science, Khairun University, Ternate, Indonesia. Waode Munaeni (ORCHID ID https://orcid.org/0000-0002-9559-2015) Contributions Widanarni Widanarni: Conceptualization, Methodology, Software, Formal analysis, Writing - original draft. Diar Setiawan Aswandi: Conceptualization, Methodology, Software, Formal analysis, Writing - original draft. Rahman Rahman: Conceptualization, Methodology, Validation, Writing - review & editing. Waode Munaeni: Conceptualization, Methodology, Validation, Writing - review & editing. Corresponding author Correspondence to Waode Munaeni. Data Availability The datasets analyzed during the current study are available from the corresponding author. Ethics Declarations Ethics Approval All the experimental studies considered the welfare of test animals according to the standard guidelines IPB University animal ethics commission (ethical approval number 206-2021). Consent to participate The experimental investigation and design was a collaborative effort amongst all authors. Consent for publication All author approved the publishing of the research. Conflict of Interest The authors declare that there are no competing interests. References Adineh H, Resources N (2013) Effect of Bacillus spp. probiotic on growth and feeding performance of rainbow trout (Oncorhynchus mykiss) larvae. Bulg J Vet Med 16:29–36 Agung LA, Widanarni W, Yuhana M (2015) Application of micro-encapsulated probiotic Bacillus NP5 and prebiotic Mannan oligosaccharide (MOS) to prevent Streptococcosis on tilapia Oreochromis niloticus. Res J Microbiol 10:571–581. https://doi.org/10.3923/jm.2015.571.581 Amlacher E (1970) Textbook of fish disease. TFH Publ. Neptune, New York (USA) Balcázar JL, de Blas I, Ruiz-Zarzuela I et al (2006) The role of probiotics in aquaculture. Vet Microbiol 114:173–186. https://doi.org/10.1016/j.vetmic.2006.01.009 Budiardi T, Salleng R, Utomo NB (2005) Nursery of Penaeus monodon fry in cage culture of intensive pond at different rearing densities. J Akuakultur Indones 4:153–158 Cerezuela R, Meseguer J, Esteban MA (2011) Current knowledge in synbiotic use for fish aquaculture: A review. J Aquac Res Dev 1–7. https://doi.org/10.4172/2155-9546.S1-008 Cha J, Rahimnejad S, Yang S et al (2013) Evaluations of Bacillus spp. as dietary additives on growth performance, innate immunity, and disease resistance of olive fl ounder (Paralichthys olivaceus) against Streptococcus iniae and as water additives. Aquaculture 402–403:50–57. https://doi.org/10.1016/j.aquaculture.2013.03.030 Dey P, Harborne JB (2003) Methods in plant in biochemistry. Academic Press, New York (USA) Divyagnaneswari M, Christybapita D, Michael RD (2007) Enhancement of nonspecific immunity and disease resistance in Oreochromis mossambicus by Solanum trilobatum leaf fractions. Fish Shellfish Immunol 23:249–259. https://doi.org/10.1016/j.fsi.2006.09.015 Djauhari R, Suprayudi MA Jr MZ (2016) Characterization of Bacillus sp. NP5 and its Application as Probiotic for Common Carp (Cyprinus carpio). Res J Microbiol 11:101–111. https://doi.org/10.3923/jm.2016.101.111 Hoa TTT, Annette Simone B, Duyen TTM et al (2020) Nursing stages of striped catfish (Pangasianodon hypophthalmus) in Vietnam: Pathogens, diseases and husbandry practices. Aquaculture 533:736114. https://doi.org/10.1016/j.aquaculture.2020.736114 Hoseinifar SH, Sun Y, Wang A, Zhou Z (2018) Probiotics as Means of Diseases Control in Aquaculture, a Review of Current Knowledge and Future Perspectives. Front Microbiol 9:1–18. https://doi.org/10.3389/fmicb.2018.02429 Iwashita MKP, Nakandakare IB, Terhune JS et al (2014) Dietary supplementation with Bacillus subtilis, Saccharomyces cerevisiae and Aspergillus oryzae enhance immunity and disease resistance against Aeromonas hydrophila and Streptococcus iniae infection in juvenile tilapia Oreoc. Fish Shellfish Immunol xxx 1–7. https://doi.org/10.1016/j.fsi.2014.12.008 Li Q, Liao S, Wei J et al (2020) Isolation of Bacillus subtilis strain SEM-2 from silkworm excrement and characterisation of its antagonistic effect against Fusarium spp. Can J Microbiol 1–38. https://doi.org/doi:10.1139/cjm-2019-0621 Liu G, Zhu S, Liu D et al (2017) Effects of stocking density of the white shrimp Litopenaeus vannamei (Boone) on immunities, antioxidant status, and resistance against Vibrio harveyi in a biofloc system. Fish Shellfish Immunol 67:19–26. https://doi.org/10.1016/j.fsi.2017.05.038 Madigan M, Martinko J, Parker J (2003) Brock, biology of microorganisms, 10th editi. Prentice Hall, New Jersey (US) Marine and Fisheries Ministry of Indonesia (2021) Marine and Fisheries Ministry of Indonesia will Improve Production Facilities of Catfish Cultivation Village in Lebak. https://kkp.go.id/artikel/36716-kkp-akan-tingkatkan-fasilitas-produksi-kampung-budidaya-patin-di-lebak Merrifield DL, Dimitroglou A, Foey A et al (2010) The current status and future focus of probiotic and prebiotic applications for salmonids. Aquaculture 302:1–18. https://doi.org/10.1016/j.aquaculture.2010.02.007 Munaeni W, Widanarni, Yuhana M et al (2020) Impact of dietary supplementation with Eleutherine bulbosa (Mill.) Urb. on intestinal microbiota diversity and growth of white shrimp, Litopenaeus vannamei. https://doi.org/10.1016/j.aquaculture.2020.735466 . Aquaculture Plumb JA, Hanson LA (2011) Health maintenance and principal microbial diseases of cultured fishes, third edn. Wiley-Blackwell, New York (USA) Purwaningsih U, Sukenda S, Lusiastuti AM et al (2022) The phenotypic, genotypic and pathogenicity comparison of Edwardsiella ictaluri Indonesian local isolates causing enteric septicemia of catfish. Aquac Res 1–18. https://doi.org/10.1111/are.15865 Putra AN, Syamsunarno MASB, Ningrum W (2020) Effect of the administration of probiotic Bacillus NP5 in the rearing media on water quality, growth, and disease resistance of African catfish (Clarias gariepinus). Biodiversitas 21:2566–2575. https://doi.org/10.13057/biodiv/d210629 Putra AN, Widanarni W (2015) Screening of amylolytic bacteria as candidates of probiotics in tilapia (Oreochromis sp.). Res J Microbiol 10:1–13 Rawling MD, Merri DL, Snellgrove DL et al (2012) Haemato-immunological and growth response of mirror carp (Cyprinus carpio) fed a tropical earthworm meal in experimental diets. Fish Shellfish Immunol 32:1002–1007. https://doi.org/10.1016/j.fsi.2012.02.020 Rieger AM, Barreda DR (2011) Antimicrobial mechanisms of fish leukocytes. Dev Comp Immunol 35:1238–1245. https://doi.org/10.1016/j.dci.2011.03.009 Rodriguez-estrada U, Satoh S, Haga Y et al (2013) Effects of Inactivated Enterococcus faecalis and Mannan Oligosaccharide and Their Combination on Growth, Immunity, and Disease Protection in Rainbow Trout Effects of Inactivated Enterococcus faecalis and Mannan Oligosaccharide and Their Combination on G. N Am J Aquac 75:416–428. https://doi.org/10.1080/15222055.2013.799620 Ruan Z, Jiang L, Li Y et al (2022) Transcriptomic analysis of the immune response against Aeromonas hydrophila infection in striped catfish Pangasianodon hypophthalmus ☆. Aquaculture 547:737446. https://doi.org/10.1016/j.aquaculture.2021.737446 Sathiyanarayanan A, Tamilarasan N (2019) Development of cell culture systems from the striped catfish Pangasianodon hypophthalmus (Sauvage, 1878). Int J Chem Stud 7:1367–1370 Silva VV, Salomão RAS, Mareco EA et al (2020) Probiotic additive affects muscle growth of Nile tilapia (Oreochromis niloticus). Aquac Res 00:1–9. https://doi.org/10.1111/are.15057 Singh V, Chaudhary DK (2013) Development of diagnostic andvaccine markers through cloning, expression, and regulation of putative virulence-protein-encoding genes of Aeromonas hydrophila. J Microbiol 51:275–282. https://doi.org/10.1007/s12275-013-2437-x Supriyono E, Purwanto E, Utomo NBP (2006) Production of ”Tokolan” white Shrimp Litopenaeus vannamei in the cage with different rearing density. J Akuakultur Indones 5:57–64 Tamamdusturi R, Widanarni W, Munti Y (2016) Administration of microencapsulated probiotic Bacillus sp. NP5 and prebiotic Mannan oligosaccharide for prevention of Aeromonas hydrophila infection on Pangasianodon hypophthalmus. J Fish Aquat Sci 11:67–76. https://doi.org/10.3923/jfas.2016.67.76 Tan CK, Natrah I, Suyub IB et al (2019) Comparative study of gut microbiota in wild and captive Malaysian Mahseer (Tor tambroides). Microbiologyopen 8:1–12. https://doi.org/10.1002/mbo3.734 Tanbiyaskur T, Widanarni W, Lusiastuti AM (2015) Administration of Bacillus NP5 and oligosaccharide to enhance the immune response in Tilapia Oreochromis niloticus towards Streptococcosis. Int J Sci Basic Appl Res 20:304–315 Wang YB (2007) Effect of probiotics on growth performance and digestive enzyme activity of the shrimp Penaeus vannamei. Aquaculture 269:259–264. https://doi.org/10.1016/j.aquaculture.2007.05.035 Widanarni W, Elly E, D T S AS (2008) Administration of Vibrio SKT-b probiotic bacteria on tiger shrimp larvae through Artemia enrichment. J Akuakultur Indones 7:129–137 Widanarni W, Lidaenni MA, Wahjuningrum D (2010) Effects of different doses of skt-b vibrio probiotic bacteria addition on survival and growth rate of tiger shrimp (Penaeus monodon) larva. J Akuakultur Indones 9:21. https://doi.org/10.19027/jai.9.21-29 Widanarni W, Yuhana M, Muhammad A (2014) Bacillus NP5 improves growth performance and resistance against infectious Myonecrosis virus in white shrimp (Litopenaeus vannamei). Ilmu Kelaut -. Indones J Mar Sci 19:211–218 Zhang D, Gao Y, Ke X et al (2019) Bacillus velezensis LF01: in vitro antimicrobial activity against fish pathogens, growth performance enhancement, and disease resistance against streptococcosis in Nile tilapia (Oreochromis niloticus). Appl Microbiol Biotechnol Zorriehzahra MJ, Delshad ST, Adel M et al (2016) Probiotics as beneficial microbes in aquaculture: an update on their multiple modes of action: a review. Vet Q 36:228–241. https://doi.org/10.1080/01652176.2016.1172132 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1741085","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":126831024,"identity":"1967ea35-0c5e-4ed9-86b8-e23a8f58ab3f","order_by":0,"name":"Widanarni Widanarni","email":"","orcid":"","institution":"IPB University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Widanarni","middleName":"","lastName":"Widanarni","suffix":""},{"id":126831025,"identity":"da65bd78-2168-4bed-8344-1cecf49c459b","order_by":1,"name":"Diar Setiawan Aswandi","email":"","orcid":"","institution":"IPB University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Diar","middleName":"Setiawan","lastName":"Aswandi","suffix":""},{"id":126831026,"identity":"809a117e-9777-4796-b5b1-ba859d1426fd","order_by":2,"name":"Rahman Rahman","email":"","orcid":"","institution":"IPB University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rahman","middleName":"","lastName":"Rahman","suffix":""},{"id":126831027,"identity":"704e6495-7d56-488b-9fdc-ad27b73804e3","order_by":3,"name":"Waode Munaeni","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYJACCTgroQLIA3IPgHnMDAZEaDlDshbGNogWGMCqxeD44YM3PjBsS9zOfvjYg4fzDudJzm5+eLiCwU6egZ15A1YtZ9KSLWcw3E7c2ZOWbpC47XCxtMwxg4NnGJING5jZCrBpMTuQYybNA9SyAciQAGpJnCeRYHCwgYE5gYGZB6vDzM6/MZP+A9Jy/v03icQ5IC3pH4Ba6nFruQG0hQGk5UYOm0Riw+HE2RI5IFsO49Rif+NZsmWPwW3jDTeemUkkHEtPnDkjp+Bgg8FxwzYcfpHsTz5440fFbdkN55OfSf6osU6ccSN988eGimp5fv7DWEMMGm7YRNhwqx8Fo2AUjIJRQAAAAM4vZVxFpHruAAAAAElFTkSuQmCC","orcid":"","institution":"Khairun University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Waode","middleName":"","lastName":"Munaeni","suffix":""}],"badges":[],"createdAt":"2022-06-09 08:59:19","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-1741085/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-1741085/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":24920819,"identity":"e8d39957-925c-4c52-a897-c38b7653d1ce","added_by":"auto","created_at":"2022-08-08 13:46:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":91367,"visible":true,"origin":"","legend":"\u003cp\u003ePercentages of lymphocytes (a), neutrophils (b), and monocytes (c) of juvenile catfish \u003cem\u003eP. hypophthalmus\u003c/em\u003e supplemented with various doses of \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 feed. Data are mean ± SD. Different letters in the same patterns showing significantly different results (P \u0026lt; 0.05).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1741085/v2/29affca789fb0f80332a9d4b.jpg"},{"id":24921275,"identity":"8b67d0e9-c785-4d32-90f8-a795ec58f6de","added_by":"auto","created_at":"2022-08-08 13:51:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":190981,"visible":true,"origin":"","legend":"\u003cp\u003ePercentages of the phagocytic activity of juvenile \u003cem\u003eP. hypophthalmus \u003c/em\u003ecatfish supplemented with different doses of \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 on feed. Data are mean ± SD. Different letters in the same patterns showing significantly different results (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1741085/v2/66146f74408694ce8a82fe1b.jpg"},{"id":24920820,"identity":"063ef6da-7347-4be8-9ae5-b7f0fd69903a","added_by":"auto","created_at":"2022-08-08 13:46:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":196997,"visible":true,"origin":"","legend":"\u003cp\u003eRespiratory burst activity of juvenile catfish \u003cem\u003eP. hypophthalmus\u003c/em\u003e supplemented with different doses of \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 on feed. Data are mean ± SD. Different letters in the same patterns showing significantly different results (P \u0026lt; 0.05).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1741085/v2/3605d9c6290323b58aa6ee40.jpg"},{"id":24920821,"identity":"3b4c9d34-bd80-4533-aa93-cc18cebdbdd0","added_by":"auto","created_at":"2022-08-08 13:46:30","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":191714,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival rate of juvenile catfish \u003cem\u003eP. hypophthalmus\u003c/em\u003e supplemented with different doses of \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 on feed before and post challenge test with \u003cem\u003eA. hydrophila. \u003c/em\u003eData are mean ± SD. Different letters in the same patterns showing significantly different results (P \u0026lt; 0.05).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1741085/v2/014c8b3a1d68b41a161e54cb.jpg"},{"id":25515967,"identity":"40c1b540-646d-4e2d-8236-2fe320ddbec7","added_by":"auto","created_at":"2022-08-22 19:44:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":789702,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1741085/v2/f1e07025-1dad-48d8-9c56-14fbc6224355.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of Feeding Artemia sp. and Artificial Feed Enriched with Bacillus sp. NP5 to Catfish Pangasianodon hypophthalmus on Growth Performance, Immune Responses, and Resistance to Aeromonas hydrophila Infection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOne type of fish that has been widely cultivated in various regions in Indonesia is the catfish (\u003cem\u003ePangasianodon hypophthalmus\u003c/em\u003e), and its production has increased every year (Marine and Fisheries Ministry of Indonesia \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Besides Indonesia, several Asian countries, such as China, Thailand, Vietnam, India, and Bangladesh produce \u003cem\u003eP. hypophthalmus\u003c/em\u003e in aquaculture (Sathiyanarayanan and Tamilarasan \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, the problem of fish farming is a disease, such as \u003cem\u003eP. hypophthalmus\u003c/em\u003e which frequently appears during the larval and rearing stages. Meanwhile, \u003cem\u003eP. hypophthalmus\u003c/em\u003e fish is frequently attacked by Motile Aeromonad Septicemia (MAS), caused by \u003cem\u003eAeromonas hydrophila\u003c/em\u003e (Hoa et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Since the disease causes hemorrhage, it is also known as a hemorrhagic disease (Plumb and Hanson \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Pathogenic bacteria \u003cem\u003eA. hydrophila\u003c/em\u003e has caused significant losses in the \u003cem\u003eP. hypophthalmus\u003c/em\u003e aquaculture industry (Ruan et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In addition to causing disease in fish, the bacteria also cause soft tissue wound infections and diarrhea in humans (Singh and Chaudhary \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAntibiotics are frequently used to treat bacterial infections (Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, antibiotics can cause resistance to pathogens and disrupt the micro-ecological balance (Liu et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Tan et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Probiotics can be used as a safe and environmentally friendly alternative to prevent and control fish diseases (Hoseinifar et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Probiotics are live microbes that have beneficial effects on the host by increasing host resistance to pathogens, inhibiting the growth or reproduction of pathogenic bacteria, activating host humoral and cellular immunity, and secreting antagonist substances to inhibit pathogens (Zorriehzahra et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 has been tested to increase the immune response and resistance of tilapia to streptococcosis disease (Agung et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Tanbiyaskur et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), goldfish to \u003cem\u003eA. hydrophila\u003c/em\u003e infection (Djauhari et al. 2016), white shrimp to Infectious Myonecrosis Virus infection (Widanarni et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), catfish to \u003cem\u003eA. hydrophila\u003c/em\u003e infection (Tamamdusturi et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and African catfish to \u003cem\u003eA. hydrophila\u003c/em\u003e (Putra et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Many studies have investigated the application of \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 through artificial or commercial feed on several types of fish and shrimp to conduct an enlargement stage. However, this application has never been tested on catfish \u003cem\u003eP. hypothalamus\u003c/em\u003e, especially through the natural feed for larvae and artificial feed for juveniles. In addition, Silva et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) assert that the use of different doses of probiotics results in different growth performances in tilapia. The use of probiotics depends on the host species, dose, and duration of administration (Merrifield et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Cerezuela et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Thus, it is necessary to gain information related to the optimal dose of probiotic \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 to give juvenile catfish significant growth, immune response, and resistance to \u003cem\u003eA. hydrophila\u003c/em\u003e infection.\u003c/p\u003e"},{"header":"Material And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eProbiotic Preparation\u003c/h2\u003e \u003cp\u003eThis study employed probiotics \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 from the digestive tract of tilapia (Putra \u0026amp; Widanarni, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and made resistance to the antibiotic rifampin as a marker. \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 was cultured on Trypticase Soy Agar (TSA) media and incubated at room temperature (27\u0026ndash;30⁰C) for 24 h. Afterward, the bacteria was taken and inoculated on Tryptic Soy Broth (TSB) media. The inoculants were incubated in a water bath shaker at 29⁰C at 140 rpm for 24 h.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eArtemia\u003c/span\u003e \u003cb\u003esp.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThis study employed 2 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u003cem\u003eArtemia\u003c/em\u003e sp. in the form of a system that was hatched in water with 30 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e salinity and was given strong aeration for 24 h. After that, the nauplii of \u003cem\u003eArtemia\u003c/em\u003e sp. were harvested by turning off the aeration. The hatching container was covered with dark plastic, and a light source was provided at the bottom. The shell of the hatched system will be on the surface while the unhatched system will settle at the bottom. Nauplii \u003cem\u003eArtemia\u003c/em\u003e sp. will approach the light source, which was then siphoned using a hose. Meanwhile, \u003cem\u003eArtemia\u003c/em\u003e sp. was given when the fish larvae were 2\u0026ndash;5 days old.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eTubifex\u003c/span\u003e \u003cb\u003esp. (Silkworm)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSilkworms or \u003cem\u003eTubifex\u003c/em\u003e sp. were kept in a rearing container and given aeration. Worms had been washed before being placed in different containers according to the treatment used. The finely chopped and washed silkworms were given to fish larvae aged 6\u0026ndash;13 days. At this stage, the fish was not given probiotics.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Artificial Feed\u003c/h2\u003e \u003cp\u003eThe artificial feed was prepared in the form of commercial feed with a protein content of 40%. Feeding was carried out at satiation with a rate of 11% of fish biomass. Artificial feed was given when the seeds were 14\u0026ndash;28 days old.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of Maintenance Media Container\u003c/h2\u003e \u003cp\u003eThis study employed 12 aquariums with a size of 25 \u0026times; 20 \u0026times; 30 cm\u003csup\u003e3\u003c/sup\u003e. The aquarium was disinfected with 20 ppm potassium permanganate (PK) for 24 h. The aquarium was then cleaned and dried. The cleaned aquarium was placed in a fiber bath measuring 2 \u0026times; 1 \u0026times; 0.5 m\u003csup\u003e3\u003c/sup\u003e and filled with 10 L of water. The fiber tub was filled with 200 L of water, and 5 heaters were installed to keep the aquarium\u0026rsquo;s temperature stable. Each aquarium was added with a hose and an aeration stone to supply oxygen.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTest Animal Preparation\u003c/h2\u003e \u003cp\u003eThis study examined catfish larvae from the Center for Development of Catfish Cultivation, Cijengkol, Subang, West Java. Larvae were reared from newly hatched with an average weight and length of 1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 mg and 0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007 cm. The stocking density of fish in each aquarium was 15 fish.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (150 fish.aquarium\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFeed Enrichment Process\u003c/h2\u003e \u003cp\u003eThis study employed natural and artificial feed. Natural food was in the form of \u003cem\u003eArtemia\u003c/em\u003e sp. and silkworms. Meanwhile, the artificial feed was in the form of commercial feed. The \u003cem\u003eArtemia\u003c/em\u003e sp. and commercial feed were enriched with probiotic \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 according to the treatment; this probiotic \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 were 10\u003csup\u003e6\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 10\u003csup\u003e7\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The enrichment process was carried out in each different container. The process of enrichment of \u003cem\u003eArtemia\u003c/em\u003e sp. was done by centrifuging probiotics that had been cultured in Tryptic Soy Broth (TSB) and by discarding the liquid supernatant. The precipitated pellet was added with phosphate buffer saline (PBS) as much as the volume of the removed and vortexed supernatants. The ready probiotics were added to the \u003cem\u003eArtemia\u003c/em\u003e sp. containing 1 L of water and 30 g.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e salinity and enriched with the treatment for 4 h (Widanarni et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Afterward, \u003cem\u003eArtemia\u003c/em\u003e sp. was harvested using a plankton net and rinsed with water. The harvested \u003cem\u003eArtemia\u003c/em\u003e sp. were given directly to the larvae or stored in the refrigerator at 4\u0026ordm;C for later use that day.\u003c/p\u003e \u003cp\u003eThe commercial feed enrichment process was carried out by centrifuging the probiotics cultured on TSB media and discarding the liquid supernatant. The precipitated pellet was added with PBS as much as the volume of the removed and vortexed supernatants. After that, probiotics were given as much as 2% of the weight of the feed; these probiotics were mixed with a binder in the form of egg whites as much as 2% of the weight of the feed (Wang \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Next, the feed was sprayed with probiotics according to the treatment and air-dried. The excess feed can be stored in the refrigerator.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMaintenance and Challenge Test\u003c/h2\u003e \u003cp\u003eJuvenile catfish received \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 in feed with different concentrations, namely 10\u003csup\u003e6\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 10\u003csup\u003e7\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; each concentration had three replicates. Meanwhile, the control was without the enrichment of \u003cem\u003eBacillus\u003c/em\u003e sp. NP5, but it consisted of positive control (tested on \u003cem\u003eA. hydrophile\u003c/em\u003e) and negative control (without a test on \u003cem\u003eA. hydrophila\u003c/em\u003e); each control has three replications. Test animals for this study follow (Purwaningsih et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) according to the standard guidelines of the IPB University animal ethics commission (ethical approval number 206\u0026ndash;2021).\u003c/p\u003e \u003cp\u003eProbiotics were given in two stages. The first stage was giving \u003cem\u003eArtemia\u003c/em\u003e sp. to the larvae aged 2\u0026ndash;5 days, and the second stage was giving artificial feed to fish aged 14\u0026ndash;28 days. Meanwhile, fish aged 6\u0026ndash;13 days received silkworms without probiotics. Prior to treatment, catfish larvae had been sampled to determine initial weight and length. 18\u0026ndash;24 fish\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e larvae aged 2\u0026ndash;5 days old were fed on the \u003cem\u003eArtemia\u003c/em\u003e sp. every 2 h for 4 days of maintenance. Meanwhile, 0.575 g.aquarium\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of larvae aged 6\u0026ndash;13 days were fed with silkworms without probiotics four times a day: at 8 a.m., 12 p.m., 4 p.m., and 8 a.m. Western Indonesian Time (WIB). Juvenile catfish aged 14\u0026ndash;28 days were fed on artificial feed at satiation with a feeding rate (FR) of 11% of fish biomass 3 times a day: at 8 a.m., 12 p.m., and 4 p.m. WIB. The water of the rearing media was changed every other two days, starting from day 6 to 28 of rearing. The temperature (\u0026deg;C) ranged from 29.0-30.3, the dissolved oxygen (DO, mg.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) ranged from 5.2\u0026ndash;6.3, pH ranged from 6.19\u0026ndash;7.12, and the Total Ammonia Nitrogen (TAN; mg.L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) ranged from 0.016\u0026ndash;0.144.\u003c/p\u003e \u003cp\u003eAfter being reared with treated feed for 28 days, the fish were challenged with \u003cem\u003eA. hydrophila\u003c/em\u003e. The challenge test process utilized a glass jar with a volume of 3 L for 15 fish. The jar was filled with water (1 L/jar) that had been deposited for 24 h. Juvenile catfish were reared at a density of 15 fish/L and adapted for 2\u0026ndash;3 h in the container. The fish were then infected with \u003cem\u003eA. hydrophila\u003c/em\u003e at a concentration of 10\u003csup\u003e7\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The observation was conducted for seven days post-infection, and dead fish were counted as the fish survival rate data at the end of the challenge test. During the post-challenge fish rearing, the fish were fed artificial feed in the form of commercial feed without probiotic enrichment but with an FR of 11%. This artificial feed was given three times a day.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eObservation Parameter\u003c/h2\u003e \u003cp\u003eThe survival rate of catfish was calculated at the end of the rearing using the following formulas (Tan et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSpecific growth rate (SGR; %/day) = [ln final weight ln initial weight/days] x 100\u003c/p\u003e \u003cp\u003eAbsolute length growth (cm)\u0026thinsp;=\u0026thinsp;Average length of fish at the end of rearing \u0026ndash; the average length of fish at the beginning of rearing\u003c/p\u003e \u003cp\u003eTotal feed consumption\u0026thinsp;=\u0026thinsp;Total weight of feed provided \u0026ndash; Total weight of leftover feed\u003c/p\u003e \u003cp\u003eFeed conversion ratio (FCR) = (total food intake) \u0026times; (weight gain) \u0026ndash; 1\u003c/p\u003e \u003cp\u003eSurvival rate (SR; %)\u0026thinsp;=\u0026thinsp;100 \u0026times; (final number of test fish)/(initial number of test fish)\u003c/p\u003e \u003cp\u003e \u003cb\u003eTotal Bacterial Count (TBC) and abundance of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eBacillus\u003c/span\u003e \u003cb\u003esp. NP5\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe total bacterial count (TBC) was performed using the plate count method of (Munaeni et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) at the beginning and end of the treatment on larvae (whole-body) and juveniles (intestines). Each treatment used two fish. Meanwhile, the observations were conducted using the Total Bacterial Count (TBC) to measure the larva and the abundance of \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 to measure the juvenile. Meanwhile, the total bacteria employed the Trypticase Soy Agar (TSA) media, while the abundance of \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 employed the TSA\u0026thinsp;+\u0026thinsp;rifampin 50 \u0026micro;g/mL media. The bacteria was calculated using the formula (Madigan et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBacterial abundance\u0026thinsp;=\u0026thinsp;number of colonies x 1/diluent factor x 1/mL sample\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCoefficient of Diversity\u003c/h2\u003e \u003cp\u003eThe coefficient of diversity measured various fish lengths by calculating the coefficient of variance, which was the percentage of the sample standard deviation to the mean value. This measurement used the following formula.\u003c/p\u003e \u003cp\u003eCoefficient of variance (%)\u0026thinsp;=\u0026thinsp;root of variance/sample mean x 100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCatfish Immune Response\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003eDifferential Leukocytes\u003c/h2\u003e \u003cp\u003eThe differential leukocyte was calculated before and after the challenge test. Blood was dripped on the object-glass. Then, another object glass was placed on the end of the first object glass that had already contained blood in a 30\u0026deg; shape. The glass object that formed the corner was pulled to the end. After that, the preparations were air-dried and fixed in methanol solution for five min. The preparations that had been immersed in the methanol solution were air-dried and immersed again in diluted Giemsa solution (1:20) for 15 min. The next steps were rinsing the preparations using distilled water and air-drying them. The finished preparations were observed under a microscope with a magnification of 400 times. Differential leukocytes were calculated according to their types, namely lymphocytes, neutrophils, and monocytes, using the following formula (Amlacher E. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1970\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e% Lymphocytes = (Number of Lymphocytes)/(Lymphocytes\u0026thinsp;+\u0026thinsp;Neutrophils\u0026thinsp;+\u0026thinsp;Monocytes) \u0026times;100\u003c/p\u003e \u003cp\u003e% Neutrophils = (Number of Neutrophils)/(Lymphocytes\u0026thinsp;+\u0026thinsp;Neutrophils\u0026thinsp;+\u0026thinsp;Monocytes) \u0026times;100\u003c/p\u003e \u003cp\u003e% Monocytes = (Number of Monocytes)/(Lymphocytes\u0026thinsp;+\u0026thinsp;Neutrophils\u0026thinsp;+\u0026thinsp;Monocytes) \u0026times;100\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePhagocytic Activity\u003c/h2\u003e \u003cp\u003eThe phagocytic activity had been calculated before and after the challenge tests. Fish blood was put into a 50 \u0026micro;L microtube, then 50 \u0026micro;L of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e suspension was added to the PBS and homogenized. After that, the blood was incubated for 20 min at 28⁰C. Then, a 5 \u0026micro;L solution was taken to make preparations for review. Then, the solution was fixed with methanol for 5 min and dried. The preparations were soaked for 15 min in Giemsa's solution, washed in running water, and dried. The solution was observed using a microscope with a magnification of 400 times. The phagocytic activity was calculated using the following formula (Dey and Harborne JB 2003).\u003c/p\u003e \u003cp\u003ePhagocytic Activity = (Number of phagocytic cells)/(Number of phagocytic cells) \u0026times;100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRespiratory Burst (RB) Activity\u003c/h2\u003e \u003cp\u003eThe respiratory burst activity was calculated using the reduction principle of nitroblue tetrazolium (NBT) which produces formazan\u0026mdash;the sum of the sizes of superoxide anions. 50 \u0026micro;L of blood was put into the holes of the microplate titer and incubated at 37⁰C for 1 h. The supernatant formed was discarded and rinsed three times with 50 \u0026micro;L of PBS. Then, 50 \u0026micro;L of 0.2% NBT solution was added and incubated at 37⁰C for 1 h. Furthermore, the 0.2% NBT solution was discarded, and the blood was fixed with 50 \u0026micro;L 100% methanol solution for 2 min and rinsed with 30% methanol solution 3 times and dried. The formed formazans blue precipitate was dissolved using 60 L of 2 N KOH solution and 70 \u0026micro;L of dimethyl sulphoxide (DMSO) added to each titer microplate hole. The optical density (OD) of the formazan blue precipitate was then measured using a microplate reader at a wavelength of 630 nm (Divyagnaneswari et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eThe obtained data were processed using Microsoft Excel 2013 and Minitab 16. Meanwhile, the data were analyzed using the analysis of variance (ANOVA). When the ANOVA results were significantly different, the data were further tested using the Tukey Advanced Test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThis study has revealed that juvenile catfish in all treatments have significantly different final biomass weight and daily growth rate from the control (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The final biomass weight and the highest daily growth rate of catfish seed were obtained at the treatment with 10\u003csup\u003e\u003cem\u003e8\u003c/em\u003e\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e This treatment is not significantly different (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) from the treatment of 10\u003csup\u003e7\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e but significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) from the treatment with 10\u003csup\u003e6\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. On day 14, the treatment with probiotic concentrations of 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 10\u003csup\u003e7\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e have resulted in significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) body lengths from the result of the treatment with probiotic concentrations of 10\u003csup\u003e6\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the control. Meanwhile, on day 28, the treatment with a probiotic concentration of 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e has resulted in the highest body length which was significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) from the results of other concentrations and the control. Moreover, the treatment with the probiotic concentration of 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e has produced significantly different absolute length growth (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) from that of other concentrations and the control. The treatment with probiotic concentrations of 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 10\u003csup\u003e7\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e has resulted in significantly different absolute length growth of juvenile catfish from the result of the control. However, the treatment with probiotic concentrations of 10\u003csup\u003e6\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e has produced insignificantly different (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) result from that of the control.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGrowth performance of juvenile \u003cem\u003eP. hypophthalmus\u003c/em\u003e fed on various concentrations of \u003cem\u003eBacillus\u003c/em\u003e sp. NP5\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eTreatment (CFU/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u003csup\u003e8\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInitial biomass weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFinal biomass weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e177.67\u0026thinsp;\u0026plusmn;\u0026thinsp;8.33a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e303.33\u0026thinsp;\u0026plusmn;\u0026thinsp;41.02b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e401.67\u0026thinsp;\u0026plusmn;\u0026thinsp;78.59bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e537.33\u0026thinsp;\u0026plusmn;\u0026thinsp;35.00c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecific growth rate (SGR; %/day)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLength growth (H0) (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLength growth (H14) (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLength growth (H28) (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbsolute length growth (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCoefficient of diversity of fish length (H14) (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.49\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCoefficient of diversity of fish length (H28) (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7.69\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.96\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.81\u0026thinsp;\u0026plusmn;\u0026thinsp;2.00a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal feed consumption\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.21\u0026thinsp;\u0026plusmn;\u0026thinsp;3.90a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.86\u0026thinsp;\u0026plusmn;\u0026thinsp;7.00ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.40\u0026thinsp;\u0026plusmn;\u0026thinsp;11.55b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e59.80\u0026thinsp;\u0026plusmn;\u0026thinsp;10.51b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeed conversion ratio (FCR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0,17c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06bc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSurvival rate (SR; %)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e78.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e81.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.76b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e86.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eData are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Different letters in the same line showing significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOn day 14, the treatment with the concentration of probiotics 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e has resulted in the lowest coefficient of the diversity of fish length. This result is not significantly different (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) from that of the treatment with the concentration of probiotics 10\u003csup\u003e7\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e but significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) from that of the treatment with the concentration of probiotics 10\u003csup\u003e6\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and control. On day 28, each treatment has produced diverse lengths, but this result was not significantly different (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) from that of the control. The amount of feed consumption, final biomass weight, and SGR at concentrations of 10\u003csup\u003e7\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were not significantly different (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), but the FCR of the two treatments was significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The survival rate of the treatment with a concentration of 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) from that of the other treatments and the control. The treatment with a probiotic concentration of 10\u003csup\u003e6\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was not significantly different (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) from that of the control.\u003c/p\u003e \u003cp\u003eThe total bacterial count (TBC) in larvae and the total of \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 in juveniles are higher than those in the control (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The range of total bacteria count at the beginning of the treatment was 1.08\u0026ndash;1.80 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e CFU.larva\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e while \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 was not found. At the end of the treatment, the larvae has the highest number of bacteria obtained in the treatment of 10\u003csup\u003e8\u003c/sup\u003e CFU/mL and the lowest number of bacteria in the control. Probiotic \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 in juveniles was found in the probiotic treatment, and the highest number of probiotic \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 is found in the treatment of 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTotal bacterial count (TBC) of larvae and total of \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 on juvenile \u003cem\u003eP. hypophthalmus\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eTotal bacterial count (TBC)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eTotal \u003cem\u003eBacillus\u003c/em\u003e NP5\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInitial (CFU/larva)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFinal\u003c/p\u003e \u003cp\u003e(CFU/larva)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInitial (CFU/ juvenile)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFinal\u003c/p\u003e \u003cp\u003e(CFU/ juvenile)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.08 x 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.3 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003csup\u003e6\u003c/sup\u003e CFU/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.65 x 10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.1 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.2 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003csup\u003e7\u003c/sup\u003e CFU/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.41 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.7 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.0 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003csup\u003e8\u003c/sup\u003e CFU/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.80 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.0 x 10\u003csup\u003e7\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.4 x 10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eData are presented as mean (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBefore the challenge test, each treatment did not have a significantly different total of lymphocytes, neutrophils, and monocytes (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In contrast, after the challenge test, the treatment with the probiotic concentration of 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) has produced the highest number of monocytes and neutrophils. Furthermore, before the challenge test, the highest percentage of the phagocytic activity was found in the probiotic treatment of 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, but after the challenge test with \u003cem\u003eA. hydrophila\u003c/em\u003e, the highest value of the phagocytic activity is obtained at the treatment of 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with a significant difference of (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the treatment and control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The value of respiratory burst activity (RB) during the observation before the challenge test was not significantly different (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) from that in each treatment. However, after the challenge test, the respiratory burst activity has increased, and the highest score is obtained at the treatment of 10\u003csup\u003e8\u003c/sup\u003e CFU/ mL. This score is significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) from that at the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The juvenile catfish has the lowest survival rate after being infected with \u003cem\u003eA. hydrophila\u003c/em\u003e in the positive control. This rate is significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) from that in the negative control and the treatment added with the \u003cem\u003eBacillus\u003c/em\u003e sp. NP5.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe final biomass weight and daily growth rate of juvenile catfish in all treatments are significantly different from those of the control (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It is suspected that the provision of probiotics in catfish seed feed can increase nutrient digestibility so that the feed can be easily absorbed by fish. Putra and Widanarni (Putra and Widanarni \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) have revealed that the provision of probiotic \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 from the digestive tract of tilapia can increase nutrient digestibility, digestive enzyme activity, and growth performance of tilapia. Meanwhile, Tamamdusturi et al. (Tamamdusturi et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) state that the \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 can hydrolyze macromolecules in feed to be simpler molecules; thus, the nutrients can be easily absorbed by the intestinal wall of fish and will spread through the circulatory system of the body to serve as an energy source for the growth of catfish. According to Adineh et al. (Adineh and Resources \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), probiotics of \u003cem\u003eBacillus\u003c/em\u003e species can more significantly grow fish larvae. Different doses in this study show different results on the final biomass weight and the highest daily growth rate of catfish parameters; the best treatment is obtained at 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Silva et al. (Silva et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) assert that different doses of probiotics affect the growth performance of tilapia. Besides increasing the final biomass weight and daily growth rate, supplementation of probiotics could increase body length. Absolute length growth is obtained at the treatment with a probiotic concentration of 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe coefficient of diversity is one of the parameters of seed quality that indicates the level of uniformity of seed size. This study has found that the lowest coefficient of the diversity of fish seed length occurs on day 14. The fish length is classified as uniform if the coefficient of diversity is not more than 15% (Budiardi et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Postulate that the smaller the coefficient of fish diversity, the better the quality of the produced seeds; consequently, the selling value of the seeds will be higher (Supriyono et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The value of the coefficient of diversity increases because the fish cannot properly utilize the nutrients on feed for long growth.\u003c/p\u003e \u003cp\u003eThe concentrations of 10\u003csup\u003e7\u003c/sup\u003e and 10\u003csup\u003e8\u003c/sup\u003e do not produce significantly different (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) parameters of feed consumption, final biomass weight, and SGR but produce significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) FCR. These findings show that the use of probiotic doses is necessarily considered in cultivation. This study has also discovered that the treatment of 10\u003csup\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e does not produce significantly different (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) results from that of the control. However, probiotics have a lower feed conversion value than the control. This indicates that \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 can increase the digestibility of feed and reduce the value of feed conversion. Djauhari et al. (Djauhari et al. 2016) state that \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 can significantly increase the activity of digestive enzymes in carp because it can increase the digestibility of proteins, carbohydrates, and fats. \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 can help the digestive process to produce extracellular enzymes, such as amylase, protease, and lipase. Thus, nutrient absorption and utilization become more efficient. Balcazar et al. (Balc\u0026aacute;zar et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) explain that digestive enzymes produced by probiotic bacteria, such as amylase, protease, and lipase, can help the digestive process. The lowest feed conversion ratio while using artificial feed is obtained at treatment with the probiotic concentration of 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA high growth rate and low FCR are influenced by the probiotic concentration that can be consumed by fish. Such a condition is denoted by the total bacterial count in larvae and the total of probiotics in juvenile fish (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A high concentration of probiotics also gives a higher value. The high total bacteria in larvae and juvenile fish indicates that the probiotic \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 is effectively administered through \u003cem\u003eArtemia\u003c/em\u003e sp. and commercial feed. The effectiveness of probiotics depends on the ability of probiotics to survive during the journey to reach the target organs and act on the digestive system. The best fish growth performance is influenced by gut microbes as indicated by an increased total bacterial count and total \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 (Tamamdusturi et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, the addition of high doses of probiotics is not always accompanied by an increase in total bacteria in the fish. This condition occurs because not all probiotics added to feed can be taken up by larvae (Widanarni et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). It is suspected that during the bioencapsulation process into \u003cem\u003eArtemia\u003c/em\u003e sp. and the coating on the feed, the amount of probiotic does not appropriate for the treatment dose. The interesting finding of this study is that \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 probiotics were no longer found in all treatments after fish larvae had been fed on silkworms without \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 for eight days. This indicates that probiotics should be given continuously during maintenance. Silva et al. (Silva et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) argue that the administration of probiotics gave effects after eight weeks of maintenance. The best survival rate is obtained in the treatment with a concentration of 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Other studies have revealed that the addition of probiotic \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 can increase the survival of tilapia (Tanbiyaskur et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and white shrimp \u003cem\u003eL. vannamei\u003c/em\u003e (Widanarni et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Meanwhile, this study has shown that probiotic supplementation could improve the survival of catfish \u003cem\u003eP. hypophthalmus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eBesides improving growth performance, supplementation of \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 could increase the immune response of catfish \u003cem\u003eP. hypophthalmus\u003c/em\u003e. The immune system is a coordinating system that aims to protect individuals\u0026rsquo; identity and integrity and prevent the invasion of harmful organisms. Parameters or indicators to observe the immune response are leukocyte differential (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), phagocytic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and respiratory burst activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Differential leukocytes are grouped according to their types, namely lymphocytes, neutrophils, and monocytes. After the challenge test, the highest number of monocytes and neutrophils was found in the treatment of 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It is suspected that the phagocytic cells in the fish's body are ready to phagocytize the invading pathogenic bacteria. The differential value of leukocytes before and after the challenge tests has decreased the number of lymphocytes but has increased the number of neutrophils and monocytes. The higher number of monocytes and neutrophils in fish indicates that the fish can produce phagocytic cells, and the blood cells can perform phagocytosis when pathogenic microorganisms attack (Tanbiyaskur et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Consequently, administering probiotics can increase the body resistance of catfish.\u003c/p\u003e \u003cp\u003ePhagocytic activity is the first defense line of the cellular response carried out by monocytes (macrophages) and granulocytes (neutrophils). This process can occur when a foreign object enters the fish body which will be phagocytized by macrophages. These macrophages will destroy antigens by phagocytosis and send signals to the lymphocyte tissue to form specific antibodies. The formed antibodies will reduce the toxicity of the poison and weaken the pathogen from spreading; as a result, phagocytic cells will more easily attack the pathogen (Rodriguez-estrada et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Unlike the control, the percentage value of phagocytic activity in the probiotic treatment has significantly increased after the challenge test (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The highest percentage value of phagocytic activity was found in the treatment of 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e after the challenge tests. This shows that the addition of the \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 can accelerate the phagocyte process carried out by macrophages which is the first line of defense of the immune response. Meanwhile, Djauhari et al. (Djauhari et al. 2016) deliver that the percentage value of phagocytic activity has increased after the challenge test and in the probiotic treatment. Probiotics can interact with mononuclear phagocytic cells (monocytes and macrophages), polynuclear leukocytes (neutrophils), and natural killer cells. Probiotics can act as an effective trigger for phagocytic cells so that they can increase phagocytic activity.\u003c/p\u003e \u003cp\u003eThe increase in the respiratory burst activity could increase the phagocytic cells of fish when challenged to fight pathogenic bacteria. Tamamdusturi et al. (Tamamdusturi et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) have revealed that an increasing respiratory burst activity occurs after the challenge test which indicates the fish attempts to defend themselves from pathogenic bacterial infection. The probiotic treatment has a higher value of respiratory burst activity than the control. The respiratory burst activity is the basic building block of the antibacterial system found in the fish's body. The increased value of respiratory burst can be associated with an increased value of phagocytic cell activity (Rawling et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The respiratory explosion can increase the amount of oxygen consumption and form superoxide anions. This process is accelerated by NADPH-oxidase and multi-component enzymes inside the plasma membrane after phagocytic activation (Rieger and Barreda \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study has discovered that the supplementation probiotic \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 could increase resistance to pathogenic \u003cem\u003eA. hydrophila\u003c/em\u003e. Putra and Widanarni (Putra and Widanarni \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) state that probiotic \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 has the strongest antagonistic activity against pathogenic bacteria, such as \u003cem\u003eStreptococcus\u003c/em\u003e sp. Other studies have also discovered that the addition of the \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 could increase the survival of tilapia infected by Streptococcosis (Tanbiyaskur et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and goldfish injected with \u003cem\u003eA. hydrophila\u003c/em\u003e (Djauhari et al. 2016). Probiotics from \u003cem\u003eBacillus\u003c/em\u003e species can increase the fish\u0026rsquo;s growth, immune response, and resistance to pathogenic infection (Cha et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), such as \u003cem\u003eA. hydrophila\u003c/em\u003e in juvenile tilapia fish (Iwashita et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe administration of \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 can increase the total bacterial count in the larvae of \u003cem\u003eP. hypophthalmus\u003c/em\u003e. In addition, the administration of \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 can increase survival, growth performance, and immune response (leukocyte differential, phagocytic activity, and respiratory burst activity) of juvenile \u003cem\u003eP. hypophthalmus\u003c/em\u003e with the best results found in the dosage of 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eA. hydrophila\u003c/em\u003e\u003c/strong\u003e: \u003cem\u003eAeromonas hydrophila\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eANOVA:\u003c/strong\u003e\u0026nbsp; analysis of variance\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCFU\u003c/strong\u003e: Coloni Forming Units\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDMSO:\u003c/strong\u003e dimethyl sulphoxide\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDO\u003c/strong\u003e: dissolved oxygen\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFCR\u003c/strong\u003e: feed conversion ratio\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFR\u003c/strong\u003e: feeding rate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMAS\u003c/strong\u003e:\u0026nbsp;Motile Aeromonad Septicemia\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNBT:\u003c/strong\u003e nitroblue tetrazolium\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOD:\u003c/strong\u003e optical density\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eP. hypophthalmus\u003c/em\u003e\u003c/strong\u003e: Pangasianodon hypophthalmus\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePBS:\u003c/strong\u003e phosphat buffer solutions\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePK\u003c/strong\u003e: potassium permanganate\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRB:\u0026nbsp;\u003c/strong\u003erespiratory burst\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSGR\u003c/strong\u003e: specific growth rate\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSR\u003c/strong\u003e: survival rate\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTAN\u003c/strong\u003e: total ammonia nitrogen\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTBC:\u0026nbsp;\u003c/strong\u003etotal bacterial count\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTSA\u003c/strong\u003e: Trypticase Soy Agar\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTSB\u003c/strong\u003e: Tryptic Soy Broth\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWIB\u003c/strong\u003e: Western Indonesian Time\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Mr. Ranta of the Aquatic Organism Health Laboratory at IPB University for the supply of laboratory equipment and technical assistance. We also thank Nurul Novelia Fuandia and other master students in the Department of Aquaculture at IPB University for technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Aquaculture, Faculty of Fisheries and Marine Science, IPB University, Bogor, Indonesia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWidanarni Widanarni (ORCHID ID https://orcid.org/0000-0002-0821-3225)\u003c/p\u003e\n\u003cp\u003eDiar S. Aswandi\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRahman Rahman (ORCHID ID https://orcid.org/0000-0002-3682-9529)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Aquaculture, Faculty of Fisheries and Marine Science, Khairun University, Ternate, Indonesia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWaode Munaeni (ORCHID ID https://orcid.org/0000-0002-9559-2015)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWidanarni Widanarni: Conceptualization, Methodology, Software, Formal analysis, Writing - original draft.\u003c/p\u003e\n\u003cp\u003eDiar Setiawan Aswandi: Conceptualization, Methodology, Software, Formal analysis, Writing - original draft.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRahman Rahman: Conceptualization, Methodology, Validation, Writing - review \u0026amp; editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWaode Munaeni: Conceptualization, Methodology, Validation, Writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Waode Munaeni.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets analyzed during the current study are available from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the experimental studies considered the welfare of test animals according to the standard guidelines IPB University animal ethics commission (ethical approval number 206-2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental investigation and design was a collaborative effort amongst all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll author approved the publishing of the research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdineh H, Resources N (2013) Effect of Bacillus spp. probiotic on growth and feeding performance of rainbow trout (Oncorhynchus mykiss) larvae. Bulg J Vet Med 16:29\u0026ndash;36\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgung LA, Widanarni W, Yuhana M (2015) Application of micro-encapsulated probiotic Bacillus NP5 and prebiotic Mannan oligosaccharide (MOS) to prevent Streptococcosis on tilapia Oreochromis niloticus. Res J Microbiol 10:571\u0026ndash;581. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3923/jm.2015.571.581\u003c/span\u003e\u003cspan address=\"10.3923/jm.2015.571.581\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmlacher E (1970) Textbook of fish disease. TFH Publ. Neptune, New York (USA)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalc\u0026aacute;zar JL, de Blas I, Ruiz-Zarzuela I et al (2006) The role of probiotics in aquaculture. Vet Microbiol 114:173\u0026ndash;186. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.vetmic.2006.01.009\u003c/span\u003e\u003cspan address=\"10.1016/j.vetmic.2006.01.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBudiardi T, Salleng R, Utomo NB (2005) Nursery of Penaeus monodon fry in cage culture of intensive pond at different rearing densities. J Akuakultur Indones 4:153\u0026ndash;158\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCerezuela R, Meseguer J, Esteban MA (2011) Current knowledge in synbiotic use for fish aquaculture: A review. J Aquac Res Dev 1\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4172/2155-9546.S1-008\u003c/span\u003e\u003cspan address=\"10.4172/2155-9546.S1-008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCha J, Rahimnejad S, Yang S et al (2013) Evaluations of Bacillus spp. as dietary additives on growth performance, innate immunity, and disease resistance of olive fl ounder (Paralichthys olivaceus) against Streptococcus iniae and as water additives. Aquaculture 402\u0026ndash;403:50\u0026ndash;57. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aquaculture.2013.03.030\u003c/span\u003e\u003cspan address=\"10.1016/j.aquaculture.2013.03.030\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDey P, Harborne JB (2003) Methods in plant in biochemistry. Academic Press, New York (USA)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDivyagnaneswari M, Christybapita D, Michael RD (2007) Enhancement of nonspecific immunity and disease resistance in Oreochromis mossambicus by Solanum trilobatum leaf fractions. Fish Shellfish Immunol 23:249\u0026ndash;259. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fsi.2006.09.015\u003c/span\u003e\u003cspan address=\"10.1016/j.fsi.2006.09.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDjauhari R, Suprayudi MA Jr MZ (2016) Characterization of Bacillus sp. NP5 and its Application as Probiotic for Common Carp (Cyprinus carpio). Res J Microbiol 11:101\u0026ndash;111. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3923/jm.2016.101.111\u003c/span\u003e\u003cspan address=\"10.3923/jm.2016.101.111\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoa TTT, Annette Simone B, Duyen TTM et al (2020) Nursing stages of striped catfish (Pangasianodon hypophthalmus) in Vietnam: Pathogens, diseases and husbandry practices. Aquaculture 533:736114. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aquaculture.2020.736114\u003c/span\u003e\u003cspan address=\"10.1016/j.aquaculture.2020.736114\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoseinifar SH, Sun Y, Wang A, Zhou Z (2018) Probiotics as Means of Diseases Control in Aquaculture, a Review of Current Knowledge and Future Perspectives. Front Microbiol 9:1\u0026ndash;18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2018.02429\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2018.02429\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIwashita MKP, Nakandakare IB, Terhune JS et al (2014) Dietary supplementation with Bacillus subtilis, Saccharomyces cerevisiae and Aspergillus oryzae enhance immunity and disease resistance against Aeromonas hydrophila and Streptococcus iniae infection in juvenile tilapia Oreoc. Fish Shellfish Immunol xxx 1\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fsi.2014.12.008\u003c/span\u003e\u003cspan address=\"10.1016/j.fsi.2014.12.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Q, Liao S, Wei J et al (2020) Isolation of Bacillus subtilis strain SEM-2 from silkworm excrement and characterisation of its antagonistic effect against Fusarium spp. Can J Microbiol 1\u0026ndash;38. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/doi:10.1139/cjm-2019-0621\u003c/span\u003e\u003cspan address=\"doi:10.1139/cjm-2019-0621\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu G, Zhu S, Liu D et al (2017) Effects of stocking density of the white shrimp Litopenaeus vannamei (Boone) on immunities, antioxidant status, and resistance against Vibrio harveyi in a biofloc system. Fish Shellfish Immunol 67:19\u0026ndash;26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fsi.2017.05.038\u003c/span\u003e\u003cspan address=\"10.1016/j.fsi.2017.05.038\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMadigan M, Martinko J, Parker J (2003) Brock, biology of microorganisms, 10th editi. Prentice Hall, New Jersey (US)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarine and Fisheries Ministry of Indonesia (2021) Marine and Fisheries Ministry of Indonesia will Improve Production Facilities of Catfish Cultivation Village in Lebak. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://kkp.go.id/artikel/36716-kkp-akan-tingkatkan-fasilitas-produksi-kampung-budidaya-patin-di-lebak\u003c/span\u003e\u003cspan address=\"https://kkp.go.id/artikel/36716-kkp-akan-tingkatkan-fasilitas-produksi-kampung-budidaya-patin-di-lebak\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMerrifield DL, Dimitroglou A, Foey A et al (2010) The current status and future focus of probiotic and prebiotic applications for salmonids. Aquaculture 302:1\u0026ndash;18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aquaculture.2010.02.007\u003c/span\u003e\u003cspan address=\"10.1016/j.aquaculture.2010.02.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMunaeni W, Widanarni, Yuhana M et al (2020) Impact of dietary supplementation with Eleutherine bulbosa (Mill.) Urb. on intestinal microbiota diversity and growth of white shrimp, Litopenaeus vannamei. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aquaculture.2020.735466\u003c/span\u003e\u003cspan address=\"10.1016/j.aquaculture.2020.735466\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Aquaculture\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePlumb JA, Hanson LA (2011) Health maintenance and principal microbial diseases of cultured fishes, third edn. Wiley-Blackwell, New York (USA)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePurwaningsih U, Sukenda S, Lusiastuti AM et al (2022) The phenotypic, genotypic and pathogenicity comparison of Edwardsiella ictaluri Indonesian local isolates causing enteric septicemia of catfish. Aquac Res 1\u0026ndash;18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/are.15865\u003c/span\u003e\u003cspan address=\"10.1111/are.15865\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePutra AN, Syamsunarno MASB, Ningrum W (2020) Effect of the administration of probiotic Bacillus NP5 in the rearing media on water quality, growth, and disease resistance of African catfish (Clarias gariepinus). Biodiversitas 21:2566\u0026ndash;2575. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.13057/biodiv/d210629\u003c/span\u003e\u003cspan address=\"10.13057/biodiv/d210629\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePutra AN, Widanarni W (2015) Screening of amylolytic bacteria as candidates of probiotics in tilapia (Oreochromis sp.). Res J Microbiol 10:1\u0026ndash;13\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRawling MD, Merri DL, Snellgrove DL et al (2012) Haemato-immunological and growth response of mirror carp (Cyprinus carpio) fed a tropical earthworm meal in experimental diets. Fish Shellfish Immunol 32:1002\u0026ndash;1007. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.fsi.2012.02.020\u003c/span\u003e\u003cspan address=\"10.1016/j.fsi.2012.02.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRieger AM, Barreda DR (2011) Antimicrobial mechanisms of fish leukocytes. Dev Comp Immunol 35:1238\u0026ndash;1245. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.dci.2011.03.009\u003c/span\u003e\u003cspan address=\"10.1016/j.dci.2011.03.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodriguez-estrada U, Satoh S, Haga Y et al (2013) Effects of Inactivated Enterococcus faecalis and Mannan Oligosaccharide and Their Combination on Growth, Immunity, and Disease Protection in Rainbow Trout Effects of Inactivated Enterococcus faecalis and Mannan Oligosaccharide and Their Combination on G. N Am J Aquac 75:416\u0026ndash;428. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/15222055.2013.799620\u003c/span\u003e\u003cspan address=\"10.1080/15222055.2013.799620\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuan Z, Jiang L, Li Y et al (2022) Transcriptomic analysis of the immune response against Aeromonas hydrophila infection in striped catfish Pangasianodon hypophthalmus ☆. Aquaculture 547:737446. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aquaculture.2021.737446\u003c/span\u003e\u003cspan address=\"10.1016/j.aquaculture.2021.737446\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSathiyanarayanan A, Tamilarasan N (2019) Development of cell culture systems from the striped catfish Pangasianodon hypophthalmus (Sauvage, 1878). Int J Chem Stud 7:1367\u0026ndash;1370\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSilva VV, Salom\u0026atilde;o RAS, Mareco EA et al (2020) Probiotic additive affects muscle growth of Nile tilapia (Oreochromis niloticus). Aquac Res 00:1\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/are.15057\u003c/span\u003e\u003cspan address=\"10.1111/are.15057\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh V, Chaudhary DK (2013) Development of diagnostic andvaccine markers through cloning, expression, and regulation of putative virulence-protein-encoding genes of Aeromonas hydrophila. J Microbiol 51:275\u0026ndash;282. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12275-013-2437-x\u003c/span\u003e\u003cspan address=\"10.1007/s12275-013-2437-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSupriyono E, Purwanto E, Utomo NBP (2006) Production of \u0026rdquo;Tokolan\u0026rdquo; white Shrimp Litopenaeus vannamei in the cage with different rearing density. J Akuakultur Indones 5:57\u0026ndash;64\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTamamdusturi R, Widanarni W, Munti Y (2016) Administration of microencapsulated probiotic Bacillus sp. NP5 and prebiotic Mannan oligosaccharide for prevention of Aeromonas hydrophila infection on Pangasianodon hypophthalmus. J Fish Aquat Sci 11:67\u0026ndash;76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3923/jfas.2016.67.76\u003c/span\u003e\u003cspan address=\"10.3923/jfas.2016.67.76\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTan CK, Natrah I, Suyub IB et al (2019) Comparative study of gut microbiota in wild and captive Malaysian Mahseer (Tor tambroides). Microbiologyopen 8:1\u0026ndash;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/mbo3.734\u003c/span\u003e\u003cspan address=\"10.1002/mbo3.734\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanbiyaskur T, Widanarni W, Lusiastuti AM (2015) Administration of Bacillus NP5 and oligosaccharide to enhance the immune response in Tilapia Oreochromis niloticus towards Streptococcosis. Int J Sci Basic Appl Res 20:304\u0026ndash;315\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang YB (2007) Effect of probiotics on growth performance and digestive enzyme activity of the shrimp Penaeus vannamei. Aquaculture 269:259\u0026ndash;264. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.aquaculture.2007.05.035\u003c/span\u003e\u003cspan address=\"10.1016/j.aquaculture.2007.05.035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWidanarni W, Elly E, D T S AS (2008) Administration of Vibrio SKT-b probiotic bacteria on tiger shrimp larvae through Artemia enrichment. J Akuakultur Indones 7:129\u0026ndash;137\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWidanarni W, Lidaenni MA, Wahjuningrum D (2010) Effects of different doses of skt-b vibrio probiotic bacteria addition on survival and growth rate of tiger shrimp (Penaeus monodon) larva. J Akuakultur Indones 9:21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.19027/jai.9.21-29\u003c/span\u003e\u003cspan address=\"10.19027/jai.9.21-29\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWidanarni W, Yuhana M, Muhammad A (2014) Bacillus NP5 improves growth performance and resistance against infectious Myonecrosis virus in white shrimp (Litopenaeus vannamei). Ilmu Kelaut -. Indones J Mar Sci 19:211\u0026ndash;218\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang D, Gao Y, Ke X et al (2019) Bacillus velezensis LF01: in vitro antimicrobial activity against fish pathogens, growth performance enhancement, and disease resistance against streptococcosis in Nile tilapia (Oreochromis niloticus). Appl Microbiol Biotechnol\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZorriehzahra MJ, Delshad ST, Adel M et al (2016) Probiotics as beneficial microbes in aquaculture: an update on their multiple modes of action: a review. Vet Q 36:228\u0026ndash;241. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/01652176.2016.1172132\u003c/span\u003e\u003cspan address=\"10.1080/01652176.2016.1172132\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Aeromonas hydrophila, Bacillus sp. NP5, catfish, immune response, probiotic","lastPublishedDoi":"10.21203/rs.3.rs-1741085/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1741085/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMotile Aeromonad Septicemia (MAS) is caused by \u003cem\u003eAeromonas hydrophila\u003c/em\u003e and often attacks juvenile catfish. Probiotics could be an alternative to prevent MAS disease. This study aims to determine the dose of probiotics to improve the survival, growth performance, and immune response of juvenile catfish \u003cem\u003ePangasianodon hypophthalmus\u003c/em\u003e to the \u003cem\u003eA. hydrophila\u003c/em\u003e infection. This probiotic was contained in \u003cem\u003eArtemia\u003c/em\u003e sp. and artificial feed enriched with \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 at concentrations of 10\u003csup\u003e6\u003c/sup\u003e CFU.mL\u003csup\u003e-1\u003c/sup\u003e, 10\u003csup\u003e7\u003c/sup\u003e CFU.mL\u003csup\u003e-1\u003c/sup\u003e, 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e-1\u003c/sup\u003e, and the control. Larvae with an average weight of 1.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 mg and an average length of 0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007 cm were reared for 28 days in aquariums filled with 10 L of water and a stocking density of 15 individuals.L\u003csup\u003e-1\u003c/sup\u003e. After the rearing period, the fish was challenged by the immersion of \u003cem\u003eA. hydrophila\u003c/em\u003e with a concentration of 10\u003csup\u003e7\u003c/sup\u003e CFU.mL\u003csup\u003e-1\u003c/sup\u003e. This study has revealed that probiotics could increase the fish\u0026rsquo;s survival, length growth, daily growth rate, and feed conversion ratio. The total bacterial count in larvae and probiotics in juvenile fish is higher than those in the control. After the fish had been challenged with the probiotics at a concentration of 10\u003csup\u003e8\u003c/sup\u003e CFU.mL\u003csup\u003e-1\u003c/sup\u003e, they had a better survival rate and immune responses (including leukocyte differential, phagocytosis activity, and respiratory burst activity) than the positive control. Probiotic \u003cem\u003eBacillus\u003c/em\u003e sp. NP5 is effectively administered through bioencapsulated \u003cem\u003eArtemia\u003c/em\u003e sp. and commercial feed.\u003c/p\u003e","manuscriptTitle":"Effects of Feeding Artemia sp. and Artificial Feed Enriched with Bacillus sp. NP5 to Catfish Pangasianodon hypophthalmus on Growth Performance, Immune Responses, and Resistance to Aeromonas hydrophila Infection","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2022-08-08 13:46:28","doi":"10.21203/rs.3.rs-1741085/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2022-06-22 16:45:05","doi":"10.21203/rs.3.rs-1741085/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ed458185-c568-48de-8b41-284b08da82fe","owner":[],"postedDate":"August 8th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-08-22T19:44:10+00:00","versionOfRecord":[],"versionCreatedAt":"2022-08-08 13:46:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-1741085","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1741085","identity":"rs-1741085","version":["v2"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}