Influence of probiotic Bacillus cereus. Supplemented feed on growth and survival of the ornamental fish, gold fish (Carassius auratus)

Influence of probiotic Bacillus cereus. Supplemented feed on growth and survival of the ornamental fish, gold fish (Carassius auratus) | 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 Article Influence of probiotic Bacillus cereus. Supplemented feed on growth and survival of the ornamental fish, gold fish (Carassius auratus) Mahadevi S, Deivasigamani B, Suthin Raj T, Keerthika P, Padmini N, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4288253/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Marine sediment sample was collected and the total aerobic population was counted and probiotic Bacillus cereus was isolated. Then it was mixed with formulated feed by spraying, while control diet was purchased from a local aquarium. 30 days of feeding experiment was conducted. Carassius auratus fishes were randomly divided into two experimental groups in rectangular tanks. The first group served as a control in which fishes were fed with commercial feed. The second group of fish were fed with probiotic incorporated feed. After 40 days, their survival and growth performance were detected. Bacillus cereus incorporated feed significantly yielded higher survival rate of the fish compared to control. Food conversion ratio (FCR) was significantly lower than the control group. The main aim of this work is to investigate the effect of a marine probiotic bacteria, Bacillus cereus on the survival and growth performance of gold fish Carassius auratus. Biological sciences/Biotechnology Biological sciences/Microbiology Gold fish (Carassius auratus) probiotic bacteria (Bacillus cereus) survival rate growth performance Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Marine sediments or ocean sediments or seafloor sediments are deposits of insoluble particles that have their origins in soil and rocks and have been transported from the land to the sea mainly by rivers but also by dust carried by wind and by the flow of glaciers into the sea. Additional deposits come from marine organisms and chemical precipitation in seawater as well as from underwater volcanoes and meteorite debris. Based on the accumulation, they are classified into Residual sediments, Mechanical sediments, chemical sediments, organic sediments. Continuous sedimentation of organic inorganic substance take place in all aquatic ecosystems, the recently laid sediments of lakes or seas may contain some detritus food and microorganisms Many marine bacteria with antimicrobial activity are foundsome are Pseudomonas putida, Actinomycetes, Bacillus Sp. etc. During the last decades, antibiotics usage was a strategy for fish diseases management and also for improvement of growth and efficiency of feed conversion an alternative approach to manage fish and shrimp health that is fast gaining attention in the aquaculture industry is probiotics,” Major constraint is the cost of feed that is very expensive for fish rearing there is dire among adopted supplementary feeding as substitute technique which is considered promising to enhance production and growth. Now a day’s probiotics have been employed as dietary supplement to enhance fish growth and improve resistance against disease at every stage. Probiotics are often termed as “friendly”,“beneficial”, “good” or “helpful” bacteria, because they help keep the gut healthy. Probiotics are considered as microbes to be administered deliberately to improve gut flora, health of host and to withstand acidity of stomach, bile salts and anti-microbial compounds [ 12 ]; [ 8 ]; [ 14 ]; [ 2 ]. Ornamental fish culture is an important component of aquaculture industry. Ornamental fishes are also called as “living jewels”. Ornamental fishes really are nature’s wonderful creation .it is the second most preferred hobby in the world and the number hobbyists for ornamental fish keeping is rising day by day because it provides a great opportunity for entrepreneurship development and income generation and also one of the most economic and profitable areas of fish farming activities. In India the state west Bengal plays the pioneer role for production of ornamental fishes. The Ornamental fish trade also helps to some extent, in improving the socioeconomic condition of rural people and in upliftment of the condition of rural women in India. But ornamental fishes suffer from different bacterial disease. Among them the most prevalent are infections caused by Aeromonas spp, citrobacter spp, Plesiomonas edwardsiella, and Pseudomonas spp . But there is a broad spectrum or rarely identified bacteria which may be causative agents of disease. The poor culturists now use various chemicals or disinfectants and antibiotics to get rid of the disease outbreaks. Current research related to the search for marine probiotics for application for culturing ornamental fish. Probiotics bacteria proposed as an alternative to antibiotics. Gold fish is used for the present experimental study gold fish is a freshwater fish in the family Cyprinidae of order Cypriniformes. Carassius auratus is considered to be the most popular and attractive pet fish among all ornamental fishes, due to its many variations such as color, fin shape, size and body structure. For experiments Bacillus Sp. is incorporated with traditionally formulated ornamental fish feed are able to significantly reduce the accumulation of organic waste and also increase the feed digestibility, prevent microbial disease, avoid water pollution and also probiotics act as nutrient supplements in artificial feed of gold fish. The increased intensity of Aquaculture has led to a high number of disease outbreaks with an increasing range of pathogens as a result in serious economic losses. With the increase in the intensification and commercialization of aquaculture production come many challenges, such as pathogens diseases especially bacterial infections remain primary constraints to its continued expansion, in addition to feed quality, bacterial infection causes mass mortality Feed quality and feeding methods therefore need to be thoroughly considered in order to improve growth performance and feed efficiency. Therefore, the aim of the present study is to evaluate the effect of probiotics –supplemented feeds on the growth parameters and proximate composition of gold fish. Materials and Methods Sample Collection Sediments were collected from the Annankovil, Parangipettai during December 2022 and transferred aseptically to the laboratory immediately and analyzed for microbial groups within 2–4 hours of sampling. Total aerobic bacterial population study. After collecting marine sediment, prepare serial dilution by taking one gram of the sediment sample was suspended on 10ml sterile distilled water in a test tube and agitated for 10 minutes the suspension was serially diluted by transferring 1ml aliquots to a series of test tubes each containing 9 ml and from respective dilution 0.1ml serially diluted samples was plated using spread plate technique on Zobell Marine Agar (ZMA) for analyzing the total aerobic bacteria. The inoculated agar plates were incubated in an inverted position at 37°C for 24 hours they were examined and the number of colonies were counted, the microbial load in the sample was calculated using the formula given below and it was expressed as Colony Forming Unit (CFU) per gram of the sample. Total microbial load in the given sample (CFU)g -1 ) = Total number of colonies/total volume of the sample × volume of sample plated(0.1ml) × dilution factor Spread plate Technique Enumeration of microbes was done by adopting spread plate technique. In this method, sterile medium was poured into petri dishes aseptically and allowed to solidify. 0.1 ml of the serially diluted sediment sample was pipetted out onto the surface of sterile medium. It was spread on to the surface of sterile medium using a ‘L’ rod. The plates were then incubated in an inverted position at 37 0 c for 24 hours. They were then examined and the numbers of colonies and were counted. Isolation of Probiotic Bacillus spp . using Selective Media To prepare Bacillus culture in nutrient broth, selected the bacterial colonies based on the morphology in Zobell marine agar plates from 10 − 1 to 10 − 5 . The selected bacteria were inoculated with a medium of nutrient broth and placed in an incubator at 37 0 C for 24 hours. The bacterial growth was observed evidenced by obtaining turbidity. The selective media of Bacillus cereus agar was used for the isolation of Bacillus spp. 0.1 ml sample were taken from the bacterial growth in nutrient broth and plated using the spread plate technique on Bacillus cereus agar. Then the media were incubated for 24 hours. The next day, colonies were formed. Isolation of pure culture The selected organisms from the Bacillus cereus agar plates were further streaked in nutrient agar (4.8gm), and they were incubated for 24 hours. Pure cultures were obtained by picking well-isolated colonies and re-streaking on fresh agar plates to obtain the pure isolates. Bacterial Identification All the isolated bacterial strains which were selected based on the morphology were identified biochemical. For the most potent strain in addition to biochemical study.16sRNA partial sequencing was also done (Fig. 4 ). Staining procedure Simple Staining For simple staining, the bacterial smears were treated with crystal violet (60 seconds) and rinsed with distilled water. Then smears were air-dried and observed under a microscope. Gram Staining A thin smear of the isolate was made on a clean glass slide and heat fixed. Then the smear was stained with crystal violet for 1 minute and then washed with water, gram’s iodine was added for 1 minute and decolorized with alcohol. After decolorization, the smear was counter stained with safranin for 1 minute. Finally, the smear was washed with water and air- dried. Then the slide was observed under the microscope Identification of the bacterial isolates All the bacteria isolated were identified by morphological and biochemical tests based on the Bergey’s Manual of Determinative Bacteriology [ 7 ]. Morphological characterization of the bacterial isolates The morphological characteristics such as gram staining [ 4 ], the color of the colony, cell shape, and motility of the organisms were tested following the method of [ 10 ]. Biochemical characterization of the bacterial isolates Biochemical examinations like IMViC (Indole, Methyl red, VogesProskauerand Citrate) test, starch hydrolysis, gelatin hydrolysis, urease test, nitrate reduction, catalase test, oxidase test and carbohydrate fermentation test were followed to identify the different bacterial organisms Then conformation of their identity by RAPD –PCR and sequencing sending the pure culture sample to the ImmugenixBioscience private limited, Perambur, Chennai. Feed preparation Like other fishes, ornamental fishes require food containing protein, lipids, carbohydrates, vitamins, minerals, etc. Protein provides the necessary materials to build up muscle cells and tissue. Most ornamental fish require crude protein level in a range between 30–40%, carbohydrate provides instant energy. Carbohydrate is required between 30–50% lipids provide main energy sources level between 4–8%. Vitamin and minerals help to build up fish health and strengthen bones. Supplementation with probiotics gives resistance for disease, provides nutritional advantage and facilitates improvement of aquarium environment. Ornamental fish feed and feed ingredients are generally selected on the basis of availability, nutrient composition and physical properties. The list of ingredients used for feed preparation were wheat flour, rice bran, maize bran, soybean meal, groundnut cake, fish meal, fish oil, and some other ingredients used for carotenoid are carrot, Hibiscus flower, marigold flower and some spinach leaf as shown in the table (1) Feed prepared based on Pearson square method. Each ingredient was finally powdered in a pulverized, mixed separately and kept in trays. Additionally, some ingredients were prepared for carotenoid content i.e, Hibiscus flower, marigold flower, and spinach leaf, and carrot were dried in sun for 3 days after completely dried the ingredients are powdered in a pulverizes of a mixer separately and then fish oil was prepared for making dough. Fish oil was prepared from the sardine fish ( Sardinella longiceps) were cut into small chunks. Placed the chunks into a container, covered and put into the sun for 2 weeks Then strained the liquid into jars and allowed the oil to separated and float to the top fish oil was scooped out separately. After that, the separated powdered ingredients are mixed thoroughly and added adequate amount of water and fish oil for making dough. Then the dough is cooked for 15 minutes in a pressure cooker for sterilization that improves digestibility. The moist substances are cooled and after that probiotics ( Bacillus cereus ) are added by spraying over them. After cooling the dough is passed through a hand pelletizer to make pellets based on mouth size of fish. Then the pellets are dried at sun for 6 to 7 days to avoid fat oxidation and protein denaturation at high temperature. Finally, the formulated pellets were stored in air tight container to avoid moisture Table 1 Quantity of raw material used in feed preparation Raw materials Quantity of raw materials Wheat flour 50gm Maize bran 50 gm Rice bran 50 gm Soybean meal 20 gm Ground nut oil cake 25 gm Fish meal 50 gm Feed additives – Carrot 5gm Spinach 5gm Hibiscus flower and marigold flower 5gm Fish oil 5ml Fish culture Experimental fish Carassius auratus (gold fish) were collected from local ornamental shop in Cuddalore. The fish initial weight and initial length were measured before introduced into the experimental tank (Fig. 2 ). Experimental setup Forty days of feeding experiment Two rectangular tank length 29.5 cm, height 13.5 cm and width 12.4 cm were taken and fitted by continuous aeration for maintain dissolved oxygen. Two fish were introduced in each tank after proper acclimated to the culture condition. The first group of fishes serves as control in which fishes were fed with commercial feed Fig. 1 . The second group of fishes will be fed with probiotics incorporated with formulated feed Fig. 1 . The commercial food and formulated feed were fed to the fish based on 5% of biomass at the three intervals 7am, 1pm, and 6pm. 75% of water was changed every two days of experimental period. The physiochemical parameters were properly maintained and checked every week during the culture period. The survival and growth performance were determined after completing 40days Fish growth parameters At every 20th day of the sampling period fish were measured Morphometrically for length and weight by using scale and weighing balance. For Morphometric measurement fish were sampled in separate container having water. Fish was taken outside water to weigh and released back to respective aquaria Estimation of fish growth Weight gain (WG) Specific growth rates (SGR), Feed conversion ratio (FCR) Survival rate (SR) Weight Gain = Final weight –initial weight [ 6 ]. Weight gain (%) = final weight - initial weight/initial weight ×100 SGR = 100* In (final weight) – In (initial weight) FCR = Dry weight of feed consumed by fish/Wet weight of fish (g); Survival rate = no of fish at the end of the experiment \ no of fishes at the start of the experiment × 10. RESULTS Identification and characterization of Bacteria Isolation of Bacillus spp from marine sediment was used as probiotics. The preliminary characterization of these Bacillus sp isolates was carried out by the methods recommended by previously published international journals. The isolated bacterial strain which was identified based on the morphology and biochemical test Table (2) Based on the NCBI BLAST analysis, the isolation of bacteria is identified as Bacillus cereus the identification of morphological characteristics of Bacillus cereus is gram positive aerobic rod-shaped bacterium. The present study investigation the application of Bacillus cereus as probiotic feed for the growth of gold fish ( Carassius auratus ) was evaluated. Application of Bacillus cereus in this present study resulted in significant increase in weight gain and length of the ornamental fish gold fish. Table 2 Biochemical tests for B acillus spp . Name of test Observation Gram staining + Morphology Rod Indole test - Methyl red test + Voges Proskauer test + Citrate utilization test + Oxidase test + Nitrate reduction test - Catalase test + Confirmation of bacterial identity by RAPD-PCR and sequencing Randomly amplified polymorphic DNA (RAPD) was first utilized in 1990 by [ 13 ].and it was a PCR- based technique for identifying genetic variation. It involves the use of a single arbitrary primer in a PCR reaction, resulting in the amplification of many discrete DNA products (Fig. 3 ). 16S rRNA Sequences Nature of Sample Pure culture (Sub cultured for isolation). DNA Extraction IGB DNA extraction Kit method (Column Based). 16S rRNA PCR Veriti 96-Well Thermal Cycler (Applied Biosystems, USA) Using broad range pan Eubacterial primers Amplicon Purification Favor Prep PCR Purification Mini Kit (Favorgen, Taiwan). Sequencing ABI 3730XL sequencer (Applied Biosystem, USA) using ABI PRISM® BigDye™ Terminator The 16rDNA sequenced from the strain Bacillus cereus Sequence length is 1170 BP and Resemblance analysis of the 16srDNA sequence was done through the gene bank database using blast method Based on the NCBI BLAST analysis, the isolate is identified as Bacillus cereus with i 100% homology (Fig. 4 ). DNA sequences The sequences produced from this study could be accessed through Gen bank accession number MH762124.1 for Bacillus cereus. 16S Amplicon QC data Sequences producing significant alignments: >MS(C)-1_16SrRNA Growth Parameter study of fishes under experimentation. After 40 days of feeding trails with probiotic and control feed, the growth parameters and length of the fish was analyzed. Measured the growth parameters of gold fish fed the dietary probiotics for 40 days, the fish fed with probiotic feed showed significantly higher growth rate compared to control feed. The growth performance including weight gain (WG), feed conversion ratio (FCR), specific growth rate (SGR) had higher significant difference between diets contained formulated probiotic feed to the control group. Average length and weight of fish FEED GROWTH PARAMETERS INITIAL 0 DAYS AFTER 20 DAYS FINAL 40 DAYS Control (A) Length(cm) 2.4 ± 0.1 3.1 ± 0.2 4.2 ± 0.1 Weight (g) 0.62 ± 0.02 1.01 ± 0.03 1.28 ± 0.12 Experimental (B) Length(cm) 2.5 ± 0.1 3.4 ± 0.1 4.7 ± 0.2 Weight (g) 0.61 ± 0.02 1.26 ± 0.03 1.54 ± 0.03 INITIAL DAY 20 TH DAY 40 TH DAY INITIAL DAY 20 TH DAY 40 TH DAY Growth parameters Parameters Control (c) Experiment(E) Weight gain 0.66 ± 0.02 0.93 ± 0.04 Length (cm) 1.8 ± 0.1 2.2 ± 0.3 Survival rate (%) 50% 100% Food conversion ratio (FCR) 1.878 ± 0.02 1.312 ± 0.03 Specific growth rate (%) 0.431 ± 0.03 0.610 ± 0.04 Water Quality Parameters During the whole experimental period about 4 weeks, the average valve for water quality measurement is TEMPERATURE DISSOLVED OXYGEN PH AMMONIA 29°C 4.72mgL -1 7.9 0.06mgL -1 During the whole experimental period about 40 days water temperature dissolved oxygen, pH and total ammonia are in acceptable level and the experimental diets had no adverse effects on the surrounding water quality of experimental fish. Discussion The effect of probiotics on growth parameters have been studied in a variety of farmed fish and other aquatic species Numerous studies have shown that, the application of probiotics can improve weight gain, feed conversion ratio, specific growth rate [ 1 ]; [ 5 ]; [ 9 ].It is known from this paper that Bacillus cereus act as a significant feed- additive enhancing the growth and survival for gold fish which is also evident from some other articles [ 11 ]; [ 3 ]. Conclusion Based on the obtained result, it is known that bacillus cereus played an important role in enhancing the survival, weight gain, FCR, and SGR of reared Carassius auratus. A better growth performance was obtained in fish fed B. cereus which is highly recommended. Similarly , Bacillus cereus reduced fish mortality. Declarations Author Contribution Mahadevi S1, - Sample collection Keerthika. P1, - Laboratory work Ann Suji. H2, - Microbiology work Padmini.N1, - Laboratory maintenance workDeivasigamani. B*2 - Corresponded of research WORKand Suthin Raj. T2 -overall CORREFCDTION AND TYPING WORK Acknowledgment The authors thank the authorities of the CAS in Marine Biology, Annamalai University for permitting them to carryout their work successfully. Data Availability The sequences produced from this study could be accessed through Gen bank accession number MH762124.1 for Bacillus cereus References Allameh, SayyedKamaleddin&Noaman, Vahid&Nahavandi, Reza. (2017). Effects of Probiotic Bacteria on Fish Performance. Advanced Techniques in Clinical Microbiology. 1. 1–5. Ann Suji, H., T.A.Palavesam, G. Immanuel and T. Suthin Raj. 2014. Production of different enzymes by gut microflora. International journal of science and nature. 5 (1): 24–27. Anuar N. S., Omar N. S., Noordiyana M. N., Sharifah N. E., 2017 Effect of commercial probiotics on the survival and growth performance of goldfish Carassiusauratus. AACL Bioflux 10(6):1663–1670. Coico R. Gram staining. CurrProtocMicrobiol. 2005;Appendix 3:Appendix 3C. doi: 10.1002/9780471729259.mca03cs00 . PMID: 18770544. Gao, Quanxin& Xiao, Changfeng& Min, Minghua& Zhang, Chenjie&Peng, Shiming& Shi, Zhaohong. (2016). Effects of probiotics dietary supplementation on growth performance, innate immunity and digestive enzymes of silver pomfret, Pampusargenteus. Indian Journal of Animal Research. 50. 10.18805/ijar.9640.http://dx.doi.org/10.1093/nar/18.22.6531 . Jafarian S, Gorouhi F, Taghva A, Lotfi J. High-altitude sleep disturbance: results of the Groningen Sleep Quality Questionnaire survey. Sleep Med. 2008;9(4):446–9. doi: 10.1016/j.sleep.2007.06.017 . Epub 2007 Sep 14. PMID: 17869574. Krieg, N.R. and Holt, J.G. (1984) Bergey’s Manual of Systematic Bacteriology. Williams and Wilkins, Baltimore, London. Nikoskelainen S, Salminen S, Bylund G, Ouwehand AC. Characterization of the properties of human- and dairy-derived probiotics for prevention of infectious diseases in fish. Appl Environ Microbiol. 2001;67(6):2430–5. doi: 10.1128/AEM.67.6.2430-2435.2001 . Olmos-Soto and Jorge. (2014). Bacillus subtilis A Potential Probiotic Bacterium to Formulate Functional Feeds for Aquaculture. Journal of Microbial & Biochemical Technology. 6. 361–365. 10.4172/1948-5948.1000169 . Rajan S, Selvichristy R. Experimental procedures in Life Science, Anjana Book House, Old.28, New Delhi. 16, South Madastreet, Koyambedu, Chennai-600 107, 2010. Sinha, Archana Ghosh, Shubhadeep Singh andDharmender (2004) Probiotics as nutrient supplement in artificial feed of gold fish (Carassiusauratus) . Journal of the Indian Fisheries Association, 31. pp. 139–144. Siuta-Cruce, P. &Goulet, J., 2001. Improving probiotic microorganisms in food systems. Food Technology 55, 36–42. Williams, J.G.K., Kubelik, A.R., Licak, J.A., et al. (1990) DNA Polymorphisms Amplified by Arbitrary Primers Are Useful as Genetic Marker. Nucleic Acids Research, 18, 6531–6535. Yanbo, Wang &Zirong, Xu. (2006). Effect of probiotics for common carp ( Cyprinuscarpio ) based on growth performance and digestive enzyme activities. Animal Feed Science and Technology - ANIM FEED SCI TECH. 127. 283–292. 10.1016/j.anifeedsci.2005.09.003 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version 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-4288253","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":299393747,"identity":"3c3aa4c0-2264-42e0-b7be-30413e72acc3","order_by":0,"name":"Mahadevi S","email":"","orcid":"","institution":"Allagappa University","correspondingAuthor":false,"prefix":"","firstName":"Mahadevi","middleName":"","lastName":"S","suffix":""},{"id":299393748,"identity":"d24aa6cc-ce4c-4675-8789-a799b087f13d","order_by":1,"name":"Deivasigamani 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4","display":"","copyAsset":false,"role":"figure","size":76158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSequencing of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. cereus\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"F4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4288253/v1/99f257f3249e615ff98b0523.jpg"},{"id":59383585,"identity":"2942ba40-d273-4eb7-9c53-0f54355483e4","added_by":"auto","created_at":"2024-07-01 06:18:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":833383,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4288253/v1/3137af1d-6fcd-471a-92c5-036aaf20bbc4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Influence of probiotic Bacillus cereus. Supplemented feed on growth and survival of the ornamental fish, gold fish (Carassius auratus)","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMarine sediments or ocean sediments or seafloor sediments are deposits of insoluble particles that have their origins in soil and rocks and have been transported from the land to the sea mainly by rivers but also by dust carried by wind and by the flow of glaciers into the sea. Additional deposits come from marine organisms and chemical precipitation in seawater as well as from underwater volcanoes and meteorite debris. Based on the accumulation, they are classified into Residual sediments, Mechanical sediments, chemical sediments, organic sediments. Continuous sedimentation of organic inorganic substance take place in all aquatic ecosystems, the recently laid sediments of lakes or seas may contain some detritus food and microorganisms Many marine bacteria with antimicrobial activity are foundsome are \u003cem\u003ePseudomonas putida, Actinomycetes, Bacillus\u003c/em\u003e Sp. \u003cem\u003eetc.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eDuring the last decades, antibiotics usage was a strategy for fish diseases management and also for improvement of growth and efficiency of feed conversion an alternative approach to manage fish and shrimp health that is fast gaining attention in the aquaculture industry is probiotics,\u0026rdquo; Major constraint is the cost of feed that is very expensive for fish rearing there is dire among adopted supplementary feeding as substitute technique which is considered promising to enhance production and growth.\u003c/p\u003e \u003cp\u003eNow a day\u0026rsquo;s probiotics have been employed as dietary supplement to enhance fish growth and improve resistance against disease at every stage. Probiotics are often termed as \u0026ldquo;friendly\u0026rdquo;,\u0026ldquo;beneficial\u0026rdquo;, \u0026ldquo;good\u0026rdquo; or \u0026ldquo;helpful\u0026rdquo; bacteria, because they help keep the gut healthy. Probiotics are considered as microbes to be administered deliberately to improve gut flora, health of host and to withstand acidity of stomach, bile salts and anti-microbial compounds [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]; [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]; [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]; [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOrnamental fish culture is an important component of aquaculture industry. Ornamental fishes are also called as \u0026ldquo;living jewels\u0026rdquo;. Ornamental fishes really are nature\u0026rsquo;s wonderful creation .it is the second most preferred hobby in the world and the number hobbyists for ornamental fish keeping is rising day by day because it provides a great opportunity for entrepreneurship development and income generation and also one of the most economic and profitable areas of fish farming activities. In India the state west Bengal plays the pioneer role for production of ornamental fishes. The Ornamental fish trade also helps to some extent, in improving the socioeconomic condition of rural people and in upliftment of the condition of rural women in India.\u003c/p\u003e \u003cp\u003eBut ornamental fishes suffer from different bacterial disease. Among them the most prevalent are infections caused by \u003cem\u003eAeromonas spp, citrobacter spp, Plesiomonas edwardsiella, and Pseudomonas spp\u003c/em\u003e. But there is a broad spectrum or rarely identified bacteria which may be causative agents of disease. The poor culturists now use various chemicals or disinfectants and antibiotics to get rid of the disease outbreaks. Current research related to the search for marine probiotics for application for culturing ornamental fish. Probiotics bacteria proposed as an alternative to antibiotics.\u003c/p\u003e \u003cp\u003eGold fish is used for the present experimental study gold fish is a freshwater fish in the family Cyprinidae of order Cypriniformes. \u003cem\u003eCarassius auratus\u003c/em\u003eis considered to be the most popular and attractive pet fish among all ornamental fishes, due to its many variations such as color, fin shape, size and body structure. For experiments \u003cem\u003eBacillus\u003c/em\u003e Sp. is incorporated with traditionally formulated ornamental fish feed are able to significantly reduce the accumulation of organic waste and also increase the feed digestibility, prevent microbial disease, avoid water pollution and also probiotics act as nutrient supplements in artificial feed of gold fish.\u003c/p\u003e \u003cp\u003eThe increased intensity of Aquaculture has led to a high number of disease outbreaks with an increasing range of pathogens as a result in serious economic losses. With the increase in the intensification and commercialization of aquaculture production come many challenges, such as pathogens diseases especially bacterial infections remain primary constraints to its continued expansion, in addition to feed quality, bacterial infection causes mass mortality Feed quality and feeding methods therefore need to be thoroughly considered in order to improve growth performance and feed efficiency. Therefore, the aim of the present study is to evaluate the effect of probiotics \u0026ndash;supplemented feeds on the growth parameters and proximate composition of gold fish.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample Collection\u003c/h2\u003e \u003cp\u003eSediments were collected from the Annankovil, Parangipettai during December 2022 and transferred aseptically to the laboratory immediately and analyzed for microbial groups within 2\u0026ndash;4 hours of sampling.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTotal aerobic bacterial population study.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter collecting marine sediment, prepare serial dilution by taking one gram of the sediment sample was suspended on 10ml sterile distilled water in a test tube and agitated for 10 minutes the suspension was serially diluted by transferring 1ml aliquots to a series of test tubes each containing 9 ml and from respective dilution 0.1ml serially diluted samples was plated using spread plate technique on Zobell Marine Agar (ZMA) for analyzing the total aerobic bacteria. The inoculated agar plates were incubated in an inverted position at 37\u0026deg;C for 24 hours they were examined and the number of colonies were counted, the microbial load in the sample was calculated using the formula given below and it was expressed as Colony Forming Unit (CFU) per gram of the sample.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTotal microbial load in the given sample (CFU)g\u003c/b\u003e \u003csup\u003e \u003cb\u003e-1\u003c/b\u003e \u003c/sup\u003e \u003cb\u003e)\u0026thinsp;=\u0026thinsp;Total number of colonies/total volume of the sample \u0026times; volume of sample plated(0.1ml) \u0026times; dilution factor\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSpread plate Technique\u003c/h2\u003e \u003cp\u003eEnumeration of microbes was done by adopting spread plate technique. In this method, sterile medium was poured into petri dishes aseptically and allowed to solidify. 0.1 ml of the serially diluted sediment sample was pipetted out onto the surface of sterile medium. It was spread on to the surface of sterile medium using a \u0026lsquo;L\u0026rsquo; rod. The plates were then incubated in an inverted position at 37\u003csup\u003e0\u003c/sup\u003ec for 24 hours. They were then examined and the numbers of colonies and were counted.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIsolation of Probiotic\u003c/b\u003e \u003cb\u003eBacillus spp\u003c/b\u003e. \u003cb\u003eusing Selective Media\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo prepare \u003cem\u003eBacillus\u003c/em\u003e culture in nutrient broth, selected the bacterial colonies based on the morphology in Zobell marine agar plates from 10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e. The selected bacteria were inoculated with a medium of nutrient broth and placed in an incubator at 37\u003csup\u003e0\u003c/sup\u003eC for 24 hours. The bacterial growth was observed evidenced by obtaining turbidity.\u003c/p\u003e \u003cp\u003eThe selective media of \u003cem\u003eBacillus cereus\u003c/em\u003e agar was used for the isolation of \u003cem\u003eBacillus spp.\u003c/em\u003e0.1 ml sample were taken from the bacterial growth in nutrient broth and plated using the spread plate technique on \u003cem\u003eBacillus cereus\u003c/em\u003e agar. Then the media were incubated for 24 hours. The next day, colonies were formed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of pure culture\u003c/h2\u003e \u003cp\u003eThe selected organisms from the \u003cem\u003eBacillus cereus\u003c/em\u003e agar plates were further streaked in nutrient agar (4.8gm), and they were incubated for 24 hours. Pure cultures were obtained by picking well-isolated colonies and re-streaking on fresh agar plates to obtain the pure isolates.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eBacterial Identification\u003c/h2\u003e \u003cp\u003eAll the isolated bacterial strains which were selected based on the morphology were identified biochemical. For the most potent strain in addition to biochemical study.16sRNA partial sequencing was also done (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003eStaining procedure\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eSimple Staining\u003c/strong\u003e \u003cp\u003eFor simple staining, the bacterial smears were treated with crystal violet (60 seconds) and rinsed with distilled water. Then smears were air-dried and observed under a microscope.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eGram Staining\u003c/strong\u003e \u003cp\u003eA thin smear of the isolate was made on a clean glass slide and heat fixed. Then the smear was stained with crystal violet for 1 minute and then washed with water, gram\u0026rsquo;s iodine was added for 1 minute and decolorized with alcohol. After decolorization, the smear was counter stained with safranin for 1 minute. Finally, the smear was washed with water and air- dried. Then the slide was observed under the microscope\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of the bacterial isolates\u003c/h2\u003e \u003cp\u003eAll the bacteria isolated were identified by morphological and biochemical tests based on the Bergey\u0026rsquo;s Manual of Determinative Bacteriology [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eMorphological characterization of the bacterial isolates\u003c/h2\u003e \u003cp\u003eThe morphological characteristics such as gram staining [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], the color of the colony, cell shape, and motility of the organisms were tested following the method of [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical characterization of the bacterial isolates\u003c/h2\u003e \u003cp\u003eBiochemical examinations like IMViC (Indole, Methyl red, VogesProskauerand Citrate) test, starch hydrolysis, gelatin hydrolysis, urease test, nitrate reduction, catalase test, oxidase test and carbohydrate fermentation test were followed to identify the different bacterial organisms\u003c/p\u003e \u003cp\u003eThen conformation of their identity by RAPD \u0026ndash;PCR and sequencing sending the pure culture sample to the ImmugenixBioscience private limited, Perambur, Chennai.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFeed preparation\u003c/h2\u003e \u003cp\u003eLike other fishes, ornamental fishes require food containing protein, lipids, carbohydrates, vitamins, minerals, etc. Protein provides the necessary materials to build up muscle cells and tissue. Most ornamental fish require crude protein level in a range between 30\u0026ndash;40%, carbohydrate provides instant energy. Carbohydrate is required between 30\u0026ndash;50% lipids provide main energy sources level between 4\u0026ndash;8%. Vitamin and minerals help to build up fish health and strengthen bones.\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eSupplementation with probiotics gives resistance for disease, provides nutritional advantage and facilitates improvement of aquarium environment. Ornamental fish feed and feed ingredients are generally selected on the basis of availability, nutrient composition and physical properties. The list of ingredients used for feed preparation were wheat flour, rice bran, maize bran, soybean meal, groundnut cake, fish meal, fish oil, and some other ingredients used for carotenoid are carrot, Hibiscus flower, marigold flower and some spinach leaf as shown in the table (1)\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eFeed prepared based on Pearson square method. Each ingredient was finally powdered in a pulverized, mixed separately and kept in trays. Additionally, some ingredients were prepared for carotenoid content i.e, Hibiscus flower, marigold flower, and spinach leaf, and carrot were dried in sun for 3 days after completely dried the ingredients are powdered in a pulverizes of a mixer separately and then fish oil was prepared for making dough. Fish oil was prepared from the sardine fish (\u003cem\u003eSardinella longiceps)\u003c/em\u003e were cut into small chunks. Placed the chunks into a container, covered and put into the sun for 2 weeks Then strained the liquid into jars and allowed the oil to separated and float to the top fish oil was scooped out separately.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eAfter that, the separated powdered ingredients are mixed thoroughly and added adequate amount of water and fish oil for making dough. Then the dough is cooked for 15 minutes in a pressure cooker for sterilization that improves digestibility. The moist substances are cooled and after that probiotics (\u003cem\u003eBacillus cereus\u003c/em\u003e) are added by spraying over them. After cooling the dough is passed through a hand pelletizer to make pellets based on mouth size of fish. Then the pellets are dried at sun for 6 to 7 days to avoid fat oxidation and protein denaturation at high temperature. Finally, the formulated pellets were stored in air tight container to avoid moisture\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \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\u003eQuantity of raw material used in feed preparation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRaw materials\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQuantity of raw materials\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWheat flour\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50gm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaize bran\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50 gm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRice bran\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50 gm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoybean meal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20 gm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGround nut oil cake\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25 gm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFish meal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50 gm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFeed additives \u0026ndash;\u003c/b\u003e Carrot\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5gm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpinach\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5gm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHibiscus flower and marigold flower\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5gm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFish oil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5ml\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFish culture\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003eExperimental fish\u003c/h2\u003e \u003cp\u003e \u003cem\u003eCarassius auratus\u003c/em\u003e (gold fish) were collected from local ornamental shop in Cuddalore. The fish initial weight and initial length were measured before introduced into the experimental tank (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eExperimental setup\u003c/h2\u003e \u003cp\u003eForty days of feeding experiment Two rectangular tank length 29.5 cm, height 13.5 cm and width 12.4 cm were taken and fitted by continuous aeration for maintain dissolved oxygen. Two fish were introduced in each tank after proper acclimated to the culture condition. The first group of fishes serves as control in which fishes were fed with commercial feed Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The second group of fishes will be fed with probiotics incorporated with formulated feed Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The commercial food and formulated feed were fed to the fish based on 5% of biomass at the three intervals 7am, 1pm, and 6pm. 75% of water was changed every two days of experimental period. The physiochemical parameters were properly maintained and checked every week during the culture period. The survival and growth performance were determined after completing 40days\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFish growth parameters\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAt every 20th day of the sampling period fish were measured Morphometrically for length and weight by using scale and weighing balance. For Morphometric measurement fish were sampled in separate container having water. Fish was taken outside water to weigh and released back to respective aquaria\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of fish growth\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eWeight gain (WG)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eSpecific growth rates (SGR),\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eFeed conversion ratio (FCR)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eSurvival rate (SR)\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eWeight Gain\u0026thinsp;=\u0026thinsp;Final weight \u0026ndash;initial weight [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWeight gain (%)\u0026thinsp;=\u0026thinsp;final weight - initial weight/initial weight \u0026times;100\u003c/p\u003e \u003cp\u003eSGR\u0026thinsp;=\u0026thinsp;100* In (final weight) \u0026ndash; In (initial weight)\u003c/p\u003e \u003cp\u003eFCR\u0026thinsp;=\u0026thinsp;Dry weight of feed consumed by fish/Wet weight of fish (g);\u003c/p\u003e \u003cp\u003eSurvival rate\u0026thinsp;=\u0026thinsp;no of fish at the end of the experiment \\ no of fishes at the start of the experiment \u0026times; 10.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIdentification and characterization of Bacteria\u003c/h2\u003e \u003cp\u003eIsolation of \u003cem\u003eBacillus\u003c/em\u003e spp from marine sediment was used as probiotics. The preliminary characterization of these \u003cem\u003eBacillus\u003c/em\u003e sp isolates was carried out by the methods recommended by previously published international journals. The isolated bacterial strain which was identified based on the morphology and biochemical test Table\u0026nbsp;(2) Based on the NCBI BLAST analysis, the isolation of bacteria is identified as \u003cem\u003eBacillus cereus\u003c/em\u003e the identification of morphological characteristics of \u003cem\u003eBacillus cereus\u003c/em\u003e is gram positive aerobic rod-shaped bacterium. The present study investigation the application of \u003cem\u003eBacillus cereus\u003c/em\u003e as probiotic feed for the growth of gold fish (\u003cem\u003eCarassius auratus\u003c/em\u003e) was evaluated. Application of \u003cem\u003eBacillus cereus\u003c/em\u003e in this present study resulted in significant increase in weight gain and length of the ornamental fish gold fish.\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\u003e\u003cb\u003eBiochemical tests for B\u003c/b\u003e\u003cb\u003eacillus spp\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eName of test\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eObservation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGram staining\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMorphology\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRod\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIndole test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethyl red test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVoges Proskauer test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCitrate utilization test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxidase test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitrate reduction test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCatalase test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eConfirmation of bacterial identity by RAPD-PCR and sequencing\u003c/h2\u003e \u003cp\u003eRandomly amplified polymorphic DNA (RAPD) was first utilized in 1990 by [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].and it was a PCR- based technique for identifying genetic variation. It involves the use of a single arbitrary primer in a PCR reaction, resulting in the amplification of many discrete DNA products (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e16S rRNA Sequences\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eNature of Sample\u003c/strong\u003e \u003cp\u003ePure culture (Sub cultured for isolation).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eDNA Extraction\u003c/strong\u003e \u003cp\u003eIGB DNA extraction Kit method (Column Based).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e\u003cem\u003e16S rRNA\u003c/em\u003ePCR\u003c/strong\u003e \u003cp\u003eVeriti 96-Well Thermal Cycler (Applied Biosystems, USA)\u003c/p\u003e \u003c/p\u003e \u003cp\u003eUsing broad range pan Eubacterial primers\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAmplicon Purification\u003c/strong\u003e \u003cp\u003eFavor Prep PCR Purification Mini Kit (Favorgen, Taiwan).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSequencing\u003c/strong\u003e \u003cp\u003eABI 3730XL sequencer (Applied Biosystem, USA) using ABI PRISM\u0026reg;\u003c/p\u003e \u003c/p\u003e \u003cp\u003eBigDye\u0026trade; Terminator\u003c/p\u003e \u003cp\u003eThe 16rDNA sequenced from the strain \u003cem\u003eBacillus cereus\u003c/em\u003e Sequence length is \u003cb\u003e1170\u003c/b\u003e BP and\u003c/p\u003e \u003cp\u003eResemblance analysis of the 16srDNA sequence was done through the gene bank database using blast method Based on the NCBI BLAST analysis, the isolate is identified as \u003cem\u003eBacillus cereus\u003c/em\u003e with\u003cb\u003ei\u003c/b\u003e100% homology (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eDNA sequences\u003c/h2\u003e \u003cp\u003eThe sequences produced from this study could be accessed through Gen bank accession number MH762124.1 for Bacillus cereus.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e16S Amplicon QC data\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003cp\u003eSequences producing significant alignments:\u003c/p\u003e \u003cdiv id=\"Sec24\" class=\"Section4\"\u003e \u003cp\u003e\u0026gt;MS(C)-1_16SrRNA\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGrowth Parameter study of fishes under experimentation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter 40 days of feeding trails with probiotic and control feed, the growth parameters and length of the fish was analyzed. Measured the growth parameters of gold fish fed the dietary probiotics for 40 days, the fish fed with probiotic feed showed significantly higher growth rate compared to control feed. The growth performance including weight gain (WG), feed conversion ratio (FCR), specific growth rate (SGR) had higher significant difference between diets contained formulated probiotic feed to the control group.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eAverage length and weight of fish\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\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=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFEED\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGROWTH PARAMETERS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eINITIAL\u003c/p\u003e \u003cp\u003e0 DAYS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAFTER\u003c/p\u003e \u003cp\u003e20 DAYS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFINAL\u003c/p\u003e \u003cp\u003e40 DAYS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003cp\u003e(A)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLength(cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWeight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eExperimental\u003c/p\u003e \u003cp\u003e(B)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLength(cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWeight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003cp\u003eINITIAL DAY 20\u003csup\u003eTH\u003c/sup\u003e DAY 40\u003csup\u003eTH\u003c/sup\u003e DAY\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003cp\u003eINITIAL DAY 20\u003csup\u003eTH\u003c/sup\u003e DAY 40\u003csup\u003eTH\u003c/sup\u003e DAY\u003c/h2\u003e \u003cdiv id=\"Sec28\" class=\"Section4\"\u003e \u003ch2\u003eGrowth parameters\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl (c)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExperiment(E)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeight gain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLength (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSurvival rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFood conversion ratio (FCR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.878\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.312\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecific growth rate (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.431\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.610\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eWater Quality Parameters\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDuring the whole experimental period about 4 weeks, the average valve for water quality measurement is\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabc\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTEMPERATURE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDISSOLVED OXYGEN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAMMONIA\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e29\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.72mgL\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.06mgL\u003csup\u003e-1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDuring the whole experimental period about 40 days water temperature dissolved oxygen, pH and total ammonia are in acceptable level and the experimental diets had no adverse effects on the surrounding water quality of experimental fish.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe effect of probiotics on growth parameters have been studied in a variety of farmed fish and other aquatic species Numerous studies have shown that, the application of probiotics can improve weight gain, feed conversion ratio, specific growth rate [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]; [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]; [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].It is known from this paper that \u003cem\u003eBacillus cereus\u003c/em\u003e act as a significant feed- additive enhancing the growth and survival for gold fish which is also evident from some other articles [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]; [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBased on the obtained result, it is known that \u003cem\u003ebacillus cereus\u003c/em\u003e played an important role in enhancing the survival, weight gain, FCR, and SGR of reared \u003cem\u003eCarassius auratus.\u003c/em\u003e A better growth performance was obtained in fish fed \u003cem\u003eB. cereus\u003c/em\u003e which is highly recommended. Similarly ,\u003cem\u003eBacillus cereus\u003c/em\u003e reduced fish mortality.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMahadevi S1, - Sample collection Keerthika. P1, - Laboratory work Ann Suji. H2, - Microbiology work Padmini.N1, - Laboratory maintenance workDeivasigamani. B*2 - Corresponded of research WORKand Suthin Raj. T2 -overall CORREFCDTION AND TYPING WORK\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e \u003cp\u003eThe authors thank the authorities of the CAS in Marine Biology, Annamalai University for permitting them to carryout their work successfully.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe sequences produced from this study could be accessed through Gen bank accession number MH762124.1 for Bacillus cereus\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAllameh, SayyedKamaleddin\u0026amp;Noaman, Vahid\u0026amp;Nahavandi, Reza. (2017). Effects of Probiotic Bacteria on Fish Performance. Advanced Techniques in Clinical Microbiology. 1. 1\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnn Suji, H., T.A.Palavesam, G. Immanuel and T. Suthin Raj. 2014. Production of different enzymes by gut microflora. International journal of science and nature. 5 (1): 24\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnuar N. S., Omar N. S., Noordiyana M. N., Sharifah N. E., 2017 Effect of commercial probiotics on the survival and growth performance of goldfish Carassiusauratus. AACL Bioflux 10(6):1663\u0026ndash;1670.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoico R. Gram staining. CurrProtocMicrobiol. 2005;Appendix 3:Appendix 3C. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/9780471729259.mca03cs00\u003c/span\u003e\u003cspan address=\"10.1002/9780471729259.mca03cs00\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. PMID: 18770544.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao, Quanxin\u0026amp; Xiao, Changfeng\u0026amp; Min, Minghua\u0026amp; Zhang, Chenjie\u0026amp;Peng, Shiming\u0026amp; Shi, Zhaohong. (2016). Effects of probiotics dietary supplementation on growth performance, innate immunity and digestive enzymes of silver pomfret, Pampusargenteus. Indian Journal of Animal Research. 50. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.18805/ijar.9640.http://dx.doi.org/10.1093/nar/18.22.6531\u003c/span\u003e\u003cspan address=\"10.18805/ijar.9640.10.1093/nar/18.22.6531\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJafarian S, Gorouhi F, Taghva A, Lotfi J. High-altitude sleep disturbance: results of the Groningen Sleep Quality Questionnaire survey. Sleep Med. 2008;9(4):446\u0026ndash;9. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.sleep.2007.06.017\u003c/span\u003e\u003cspan address=\"10.1016/j.sleep.2007.06.017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Epub 2007 Sep 14. PMID: 17869574.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrieg, N.R. and Holt, J.G. (1984) Bergey\u0026rsquo;s Manual of Systematic Bacteriology. Williams and Wilkins, Baltimore, London.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNikoskelainen S, Salminen S, Bylund G, Ouwehand AC. Characterization of the properties of human- and dairy-derived probiotics for prevention of infectious diseases in fish. Appl Environ Microbiol. 2001;67(6):2430\u0026ndash;5. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/AEM.67.6.2430-2435.2001\u003c/span\u003e\u003cspan address=\"10.1128/AEM.67.6.2430-2435.2001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOlmos-Soto and Jorge. (2014). \u003cem\u003eBacillus subtilis\u003c/em\u003e A Potential Probiotic Bacterium to Formulate Functional Feeds for Aquaculture. Journal of Microbial \u0026amp; Biochemical Technology. 6. 361\u0026ndash;365. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4172/1948-5948.1000169\u003c/span\u003e\u003cspan address=\"10.4172/1948-5948.1000169\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajan S, Selvichristy R. Experimental procedures in Life Science, Anjana Book House, Old.28, New Delhi. 16, South Madastreet, Koyambedu, Chennai-600 107, 2010.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSinha, Archana Ghosh, Shubhadeep Singh andDharmender (2004) \u003cem\u003eProbiotics as nutrient supplement in artificial feed of gold fish (Carassiusauratus)\u003c/em\u003e. Journal of the Indian Fisheries Association, 31. pp. 139\u0026ndash;144.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiuta-Cruce, P. \u0026amp;Goulet, J., 2001. Improving probiotic microorganisms in food systems. Food Technology 55, 36\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams, J.G.K., Kubelik, A.R., Licak, J.A., et al. (1990) DNA Polymorphisms Amplified by Arbitrary Primers Are Useful as Genetic Marker. Nucleic Acids Research, 18, 6531\u0026ndash;6535.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYanbo, Wang \u0026amp;Zirong, Xu. (2006). Effect of probiotics for common carp (\u003cem\u003eCyprinuscarpio\u003c/em\u003e) based on growth performance and digestive enzyme activities. Animal Feed Science and Technology - ANIM FEED SCI TECH. 127. 283\u0026ndash;292. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.anifeedsci.2005.09.003\u003c/span\u003e\u003cspan address=\"10.1016/j.anifeedsci.2005.09.003\" 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":"Gold fish (Carassius auratus), probiotic bacteria (Bacillus cereus), survival rate, growth performance","lastPublishedDoi":"10.21203/rs.3.rs-4288253/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4288253/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMarine sediment sample was collected and the total aerobic population was counted and probiotic \u003cem\u003eBacillus cereus\u003c/em\u003e was isolated. Then it was mixed with formulated feed by spraying, while control diet was purchased from a local aquarium. 30 days of feeding experiment was conducted. \u003cem\u003eCarassius auratus\u003c/em\u003e fishes were randomly divided into two experimental groups in rectangular tanks. The first group served as a control in which fishes were fed with commercial feed. The second group of fish were fed with probiotic incorporated feed. After 40 days, their survival and growth performance were detected. \u003cem\u003eBacillus cereus\u003c/em\u003e incorporated feed significantly yielded higher survival rate of the fish compared to control. Food conversion ratio (FCR) was significantly lower than the control group. The main aim of this work is to investigate the effect of a marine probiotic bacteria, \u003cem\u003eBacillus cereus\u003c/em\u003e on the survival and growth performance of gold fish \u003cem\u003eCarassius auratus.\u003c/em\u003e\u003c/p\u003e","manuscriptTitle":"Influence of probiotic Bacillus cereus. Supplemented feed on growth and survival of the ornamental fish, gold fish (Carassius auratus)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-07 17:37:29","doi":"10.21203/rs.3.rs-4288253/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":"773f63ad-0579-4ee5-bf40-7ab6298afae3","owner":[],"postedDate":"May 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":31585507,"name":"Biological sciences/Biotechnology"},{"id":31585508,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2024-07-01T06:10:07+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-07 17:37:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4288253","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4288253","identity":"rs-4288253","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}