INTRODUCTION:

Aquaculture environments can be stressful, triggering maximum death, particularly in rearing pond^[1]. In ornamental fish culture, probiotics can be mixed into the water or added into the dry or live feed^[2]. Probiotics are risk-free bacteria that assist the well being of the host animal and give, directly or obliquely to defend the host animal next to injurious bacterial pathogens. The short period and long period result of exposure to probiotic was calculated in Arctic charr (Salvelinusalpinus) kept in both tap and brackish water. Fuller^[3]modified the description as ‘Anexist microbial nourish enhancement which helpfully influences the host animal by improving its intestinal microbial balance’. Tannock^[4]proposed the meaning “alive microbial cells administered as nutritional supplements with the mean of improving healthiness”.

A number of positive possessions of probiotics in fish culture have been established, such as higher patience^[5], faster growth^[6], enhanced pressure acceptance^[7], immune structure improvement^[8][9], and general welfare^[10]. These several possessions of probiotics substantiate their flourishing use in fish husbandry areas as well asfood, ecological control and resistance^[11]. Immuno histochemistry is asignificant method that can be used to assess fish resistance even though most studies on probiotics in fish culture center on pathogen test^[12].

Ornamental fish are those tiny sized, live and multicolored fish kept in residence or public aquaria or in backyard pools for relaxation. Fresh water ornamental fish contribute 85% of the total global ornamental fish trade^[11].

Probiotics are well known and regularly used additives in the majorfarm animal’s species. They allege to get better gut strength by steadying gut flora being their effects reflect in anenhancedon the whole physical condition position, welfare and recital of the animals. In aquaculture probiotics are given through feed and/or as a water additive. The supplementation of probiotics through feed is an improved method to make certain the competence of the probiotic bacteria in the gastrointestinal region of the fish. Nevertheless, their use in fish feed making is still meager^[9].

Aquaculture of crustaceans, algal plants, finfish and mollusks is one of the highest emergent food producing sectors, having developed at a yearly rate of approximately 11% from 1984 to 1995 compared with 3% for farm animals’ meat and 1.6% for confine fisheries production^[13]. Disease outbreaks are being progressively more standard as an important restraint on aquaculture production and business, affecting the economic growth of the division in many countries. For example, disease is now measured to be the restraining factor in the shrimp culture subsector. So far, conservative approaches, such as the use of antiseptic and antimicrobial drugs, have had restricted achievement in the avoidance or treat of aquatic disease^[14].

Probiotics offer a treatment of alternating bacterial cultures modifies the microbial load in water and residue decrease or eradicate disease causing microorganism and usually recover the growth and endurance of the targeted species^[15]. Probiotic used in aquaculture to improve the water quality parameters. They aim at promoting the proliferation of beneficial species of bacteria within the aqua system^[16].

Pseudomonas fluorescens anti histocompatibility is caused by the ability of siderophores in the supernatant to efficiently chelate iron, which results in instant iron deprivation of the pathogen Vibrio anguillarum and complete growth arrest ^[17]. In the present study, commercial probiotic is tested for their effect in water quality and growth of the fish in culture tank in lab conditions. This study was designed to evaluate the use of probiotic bacteria such as Bacillus sp, Lactobacillus spas a partial replacement for fishmeal protein in practical diets for giant gourami in terms of growth and survival.

MATERIALS AND METHODS:

The fish Giant gourami, Osphronemusgoramy collected from the farm. The collected fish were transported to the laboratory in a polythene bag containing oxygenated water. The fishes were visually sorted out for their healthy and active conditions and 50 fishes were stocked in each glass tank (50L) for three days for acclimatization to laboratory conditions. The experimental set up and animal was used to treatment in filtered water with aeration. Fish were measured for total length and weight; experiment was conducted for 60 days.

Fish were divided in to four different groups and fed them with feeds. Group I was treated as control, fed with only feed and group II were fed with feed and isolated Bacillus sp; Group III were fed with feed and isolated Lactobacillus sp and group IV were fed with feed consortium a mixture of Bacillus sp and Lactobacillus sp.

The present study, recorded in four tanks fish treated with Bacillus sp, Lactobacillus sp, consortium a mixture of Bacillus sp and Lactobacillus sp and control. The PVC tube was placed in 50L glass tank for the aeration purpose from the two-way aerator. A light stream of bubbles was directed to ensure oxygenation. Each tank was aerated with an air stone. The experimental setup was used for the 60 days’ period. The tanks contained 45L of 0.1 mm filtered freshwater and were maintained at 27^oC. Screens were placed over the tanks to prevent the fish from being jumped out.

The fish feed was made from groundnut oil cake, wheat bran and Soya been which were purchased from the local market and were made into fine granules mechanically. The fine granules were then made into powdered Groundnut oil cake, Soya bean and Wheat bran were mixed in the ratio of 5:3:2and then distilled water was poured and made into a paste. Then the food was sterilized. The paste food was passed through sieve and dried. It was made into pellets. The pellets food was offered to the fish.

Fish were fed 4% of body weight twice a day during the 60 days’ experiment. Weighed feed was given in a feeding tray. One hour later the unconsumed feed was taken and dehydrated in a hot air oven at 90^oC. For every 10 days feed utilization was calculated by subtracting the amount of unconsumed dry feed from the total dry weight of feed given.

Sterilized milk sample was collectedand serially diluted in 0.5% peptone water and it was inoculated by spread plate method on the Nutrient agar (Hi media, India). The inoculated plates were incubated at 37°C for 48 hrs.The isolated organisms were observed based on the colony morphology. These were sub cultured in agar slants and conserved under preservation temperature. Bergey’s Manual of Determinative Bacteriology was referred to identify the bacteria based on the macroscopic and microscopic examination^[18].

The isolated organisms Bacillus sp, Lactobacillus sp, consortium a mixture of Bacillus sp and Lactobacillus sp with the density of 10¹¹ cells per ml were inoculated into the tank with 40L of water and aerated. The inoculation was made twice in aday. In control tanks, no microbial inoculation was made. Water exchange was made for 20% of the culture water every day. Feeding was given at a rate of 5% of the body weight, twice a day. Before feeding water exchange was made, unutilized feed and fecal matter were siphoning out from the culture tank and make up the volume of 40L by water.

The water quality parameters such as Temperature, pH and Dissolved oxygen, Biological Oxygen Demand, Nitrite and Ammonia were monitored. Water samples were transferred carefully to BOD bottles. The modified Winkler’s method described by Strickland and Parsons ^[19]was adopted for the estimation of dissolved oxygen and Biological Oxygen Demand^[20].The tanks were drained twice a week to remove accumulated feces from the bottom and replenished with freshwater.

Once intendays’total length and body weight of the fish were measured for 60 days. Total length was measured with the help of Vernier scale. Fish weight was taken by weighing the animal in live condition in an electronic balance. No considerablevariations in death were found between the four groups various treatments during the 60 days’ post treatment period. Fishes were treated with an intermediate concentration (10⁶CFU/ml) isolated organisms for eight weeks in order to study the long-term effects on survival and growth performance.

RESULTS AND DISCUSSION:

There is no significant difference was noticed between the control and treated tanks. At early stage the temperature was 26^oC but later on it turned into 28^oC (Fig.1). Due to the continuous change of water at the early morning of every day there is no significant difference noticed between the control and experimental tanks. The optimum pH for almost all fish is 6.4 to 7.0 (Fig.2). Due to the continuous supply of oxygen with the help of 2-way aerator there is no huge margin of Dissolved Oxygen between control and treated tanks. Highest dissolved oxygen rate 2.2mg/l was noticed in group IV tank. The lowest dissolved oxygen rate 0.8 mg/l was observed in group IV on 60^th day (Fig.3). Maximum Nitrite level 1.7mg/l was observed in group IV and the minimum 0.9 mg/l was obtained in group I. When the nitrite exceeds 1mg/l, it means the transformation of organic substances is not proceeding regularly. We must clean the filter without leaving sediments on the bottom and we must regularly change small quantities of water. Nitrite levels were significantlyincreased between the control and experimental tanks (Fig.4). When the values of ammonium exceed 0.2mg/l the fish respiration is quickened. For values that exceed 1mg/l we must change the water otherwise the fish risk of dying.Level of ammonia in fish tanks are totally absent (Fig.5).

The growth in terms of length and weight was added in the experimental animals than in control (Fig 6 and 7). The fish maintained in the bacteria inoculated tanks were more active and healthier than those in the control. There was no significant difference between control and treated tanks. However, the linear growth or fish was higher in the probiotic treated than control tank. Growth in terms of wet weight gain was significantly higher in treated than the control tanks. The relative growth rate in fish was greater in treated tanks than in control. The best growth rate was observed in the group IV tanks inoculated with consortium than the group I control.

No mortality occurred in treated tanks during the experiment. A significantly higher growth rate was acquired in fish treated daily with group IV consortium as compared to Group I, II and III. Fish treated regularly with consortium were found to survive at a significantly higher rate (100%) as compared to group I untreated control fish (85%). Thus, a significant improvement of disease resistance was noted in the fish treated with probiotics no deaths were recorded during the experiment. The fish treated with consortium exhibited highest growth rate (Body Weight-4.13+8.24 gm) and survival of 95% at Total Length–4.23+8.47 cm stage. The uppermost growth rate was noted in fish groups treated daily with isolated organisms. This treatment group improved in mean weight from 4.13 to 8.27 with an average growth rate of 0.069% per day. This was significantly better than the control group I which grew from 4.15 to 5.83 g with an average of 0.028 % per day.

Contradictorily, Queiroz and Boyd^[20]experienced a marketable probiotic in catfish (Ictaluruspunctatus), observing anendurance and net fish production drastically higher while the probiotic was added. Though, extremely small differences were important for the determined water quality variables (chemical oxygen demand, dissolved oxygen, nitrate, ammoniaand soluble reactive phosphorus) among the experimental and control ponds. Taoka^[2]calculated effects of marketable probiotics prepared from varied cultures of bacteria and yeast on endurance of Japanese flounder Paralichthysolivaceus and water quality in a clogged recirculating method. The experimental groups showed extensively better survival rate as compared to the control group at the end of rearing experiment (50 days of culture), and water quality parameters were notably lesser in probiotics diet groups. The commercial product made from Bacillus sp., Saccharomyces cerevisiae, Nitrosomonas sp., and Nitrobacter sp., had the ability to increase the beneficial bacterial microbiota of Penaeusvannamei shrimp, further reducing the concentrations of inorganic nitrogen^[21].

In the present study, Probiotics enhances the growth of fish. Probiotic bacteria produces iderophores, enzymes and growth hormones that improve the growth of fish. The choiceof probiotics commonly based on their antagonistic activity towards the pathogens^[22]. Probiotic produces bacteriocins, hydrogen peroxides, proteases, siderophores; that are reduce the pathogenic microbes ^[23]. In aquaculture practice water quality depreciates mostly due to accretion of metabolic leftover. In the current work probiotic bacteria are refining the water quality. Austin^[24]inoculated 21-g on Atlantic salmon, intramuscularly or intraperitoneally with a suspension having the candidate probiotic. The fish were watched for 5 days, after which the survivors were sacrificed Gibson^[25], observed a clear reduction in the level of the pathogen V. tubiashii in a culture of oysters when Aeromonas sp was added, even though the probiotic strain itself could no longer be noticed in the culture after 4 days. Production of enormous quantities of bacterial biomass needs suitable quality control to evadecontagion by other bacteria.

Exact growth rate was improved with the addition of probiotics in fish nourishments. The fish fed diets comprise 10% of B.cereushas significantly (p 0.05) enhanced feeding and growth rate as compared to fish fed the control diet. In the present study the FCR (Feed Conversion Ratio) was higher in control (1.15±0.02) and less in test (0.58±0.13). Different researchers like this, have done and similar result were expressed that adding probiotics to diet of different kinds of fish, increase their growth. In the study by Giri^[26]Lactobacillus plantarum in three concentrations was added to Labeorohita diet, daily weight growth (WG) and feed conversion ratio (FCR) significantly increased. Similar studies also by Sun^[27]on Epinepheluscoioides, on Tilapia niloticus (Oreochromisniloticus)^[6], on gilthead sea bream (Sparusaurata)^[28], obtained same results.

Diseases that may result from pathogenic bacteria are disallowed or condensed by the use of probiotics, which make the fish more challenging^[29][30]. Probiotics are more over occasionally anticipated to have a straight growth endorsing effect on fish also by a direct association in nutrient uptake, or by providing nutrients or vitamins^[31]. It has also been confirmed experimentally that probiotics certainly may improve the growth of fish^[32]
[33].

Various experiments using the similar probiotic may use different dosing schedules; one may be constant and another may use particular or frequent single doses^[34]. Though the minimum and efficient dose of the bacterial mixture was not known, it was found experimentally that no certain negative effects could be detected at a concentration of probiotic was found to have a growth promoting effect on Arctic charr under experimental conditions. Thus, it is suggested that it is feasible to use probiotic for fish farming only at lesser dosage. In addition, they found araise of fish endurance by the probiotic treatment in vivo experiment. These results recommend that a probiotic cause of the consortium could be achieved by supplementing the mixture to the rearing water. This shows that consortium has the ability to act as growth stimulators.

In the current study, group II fed with Bacillus sp showed inhibitory effects revealed in the survival rate and growth. Group IV fed with consortium confirmed better resistance to the stress assessment, telling that fishfed with Bacillus sp and Lactobacillus sp and have better quality than those treated without probiotic.

Olafsen^[35], Picchietti^[12]and Guzmán-Villanueva ^[36]studied the effect of live feed cultured with preferred bacterial strains to progress growth, survivaland immunity. The inoculation of live feed consist probiotics may have been vital in this experiment, because culture tank have apparatus that can obstruct the positive effects of probiotic bacteria added into the water^[37]. In addition, researches must be carried out to assess most favorable probiotic doses for ornamental fish forms.

Fig. 1 Temperature of water in Celsius

Fig.2 Total level of pH

Fig.3 Dissolved oxygenin mg

Fig.4 Total level of Nitrite in mg/l

Fig. 5 Total level of Ammonia in mg/l

Fig. 6 Body Weight of fish in gm

Fig. 7 Total Length of fish in Cm

CONCLUSION:

Probiotics tested in fish under various experimental circumstances. There is good indication that potential probiotic microorganisms are capable to colonize the fish. It should also make substances that are antagonistic to pathogens but harmless to fish, probiotics in fish may restore a good stability of the gut micro flora and thereby contribute to an optimal dietary, growth and health condition of the fish by improving numerous qualities of the water, because they change the bacterial composition of the water and sediments. The usage of probiotics in fish farming shows signs of fetching a success. Conversely, a hugepact of investigation is still required to certify this. Secondly, there is a critical necessary for further studies to decide the best method of administration and dosage level. Enhanced storage time is also vital since the probiotic bacteria will have to be alive.

Quite a few methods of action could report for the favorable effects to fish. Inoculation of probiotics to water containing giant gourami, Osphronemus goramyat concentrations of 10⁶ CFU/ml or lower was found to be safe dose. Probiotics can be recommended in fish farming to improve production since it enhances the growth rate of giant gourami. Probiotics appears to increase the survival of giant gourami. However, this needs further study.

In conclusion the study reveals that the growth of giant gourami, Osphronemusgoramy can be improved and mortality rate was decreased by providing the live feed by Bacillus sp and consortium and also the study reveals the consortium can assist to get better disease resistance in fish. Nevertheless their efficiency may differ between facilities and production units since the influence of many confusing causes. The probiotic property of isolated Bacillus sp, Lactobacillus sp could survive in low pH and can with stand broad range of pH thus indicating that they could be considered as novel probiotic candidates for use in the ornamental fish culture.

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Received on 02.01.2019 Modified on 14.01.2019

Research J. Pharm. and Tech 2019; 12(1): 262-268.

DOI: 10.5958/0974-360X.2019.00050.7

Comparative study of Probiotic Bacteria on ornamental fish giant gourami, Osphronemus goramy for its survival and growth