Development of Probiotics Based Culture System of Macrobrachium rosenbergii Using Different Stocking Densities

The most widely cultured fast growing two giant prawns in the globe including Bangladesh are Penaeus monodon and Macrobrachium rosenbergii. Their culture techniques are traditional, extensive, improved extensive, and mainly based on the stocking density of post larvae and management techniques of the culture ponds. Both monoculture as well as polyculture with fin fish (mullets, carps and tilapia) is the common practice in the South and Southeast Asian countries. Extensive and improved extensive polyculture are widely practiced in the freshwater environment throughout the country [1]. In Bangladesh, prawn farming can play an important role in poverty reduction by increasing job opportunity especially for the rural women that help to enhance their contribution to household income [2]. With increasing demand of environment friendly aquaculture, the use of probiotics in aquaculture is now widely accepted and recently, the use of probiotics to improve and maintain healthy environment for prawn culture has become popular in Bangladesh. Probiotics has been used to supply beneficial bacterial strains in rearing water that will help to increase microbial species composition in the environment and ultimately to improve water quality conditions [3]. This study attempted to make familiar with the fish farmer as well as farm owner’s application of probiotics in the culture system of Macrobrachium rosenbergii and ultimately to minimize or totally stop the antibiotic application in farming system and finally draw the attraction of export market of the aquaculture products.


Introduction
The most widely cultured fast growing two giant prawns in the globe including Bangladesh are Penaeus monodon and Macrobrachium rosenbergii. Their culture techniques are traditional, extensive, improved extensive, and mainly based on the stocking density of post larvae and management techniques of the culture ponds. Both monoculture as well as polyculture with fin fish (mullets, carps and tilapia) is the common practice in the South and Southeast Asian countries. Extensive and improved extensive polyculture are widely practiced in the freshwater environment throughout the country [1]. In Bangladesh, prawn farming can play an important role in poverty reduction by increasing job opportunity especially for the rural women that help to enhance their contribution to household income [2]. With increasing demand of environment friendly aquaculture, the use of probiotics in aquaculture is now widely accepted and recently, the use of probiotics to improve and maintain healthy environment for prawn culture has become popular in Bangladesh. Probiotics has been used to supply beneficial bacterial strains in rearing water that will help to increase microbial species composition in the environment and ultimately to improve water quality conditions [3]. This study attempted to make familiar with the fish farmer as well as farm owner's application of probiotics in the culture system of Macrobrachium rosenbergii and ultimately to minimize or totally stop the antibiotic application in farming system and finally draw the attraction of export market of the aquaculture products.

Materials and Methods
M. rosenbergii can be cultured in earthen ponds, concrete tanks, small reservoirs, cages, pens and paddy fields. In the present experiment, it was conducted in 9 ponds of two rural villages of Chittagong division, South-Eastern part of Bangladesh.

Probiotics selection, mode of action, doses and usage instruction
Methods of selection for probiotics use in Macrobrachium culture were considered the following steps: 1) Collection of background information 2) Acquisition of potential probiotics 3) Estimate the ability of potential probiotics to out-compete pathogenic strains 4) Assessment of pathogenicity of potential probiotics 5) Evaluation of effect of potential probiotics in culture and 6) An economic cost-benefit analysis and to check their availability in the market.

Selection of commercial probiotics
• Sanolife® Pro-1 was used by mixing with feed. Sanolife® Pro-1 contains specific mixture of strains of Bacillus spp. Minimum of 3 × 10 10 cfu/g. Standard Sanolife® Pro-1 application rates instructed 5 g/kg feed. amount of clean water to evenly spread over all the feed particles. Finally, probiotics mixed feed was kept for air drying and then applied to the pond.

Probiotics application in pond water
In this experiment threereplicates of each treatment was practiced with probiotics in pond water. Two commercial probiotics such as Sanolife® Pro-W and Biomin® Pondlife were used. Sanolife Pro-W powder was mixed with clean pond water and then evenly distributed directly to the pond water. For Biomoin® Pond life the calculated amount of powder was kept for germination in a 20-liter clean water and 50 g of sugar mixed solution for 3 hours as per given instruction and then evenly distributed directly to the pond water.

Statistical analysis
t-test was done to find out the value of 'p' by using software-R for the final average body weight, mean growth rate and gross production, where p=0.04018, p=0.03503 and p=0.00036 respectively. For all the three factors p value is smaller than 0.05, which indicates the results are highly significant.
Pearson's product-moment correlation was done for correlation analysis of survival with stocking densities and found, r=-0.98 and p=5.277e-06. r=-0.98 indicates that, there is a high degree of negative association between stocking density and survival rate. That is as the stocking density increases the survival rate decreases. P=5.277e-06 implies that the result is highly significant.

Growth, survival and production performance
The experiment was carried out for 150 days. In the experiment T-1 (2/m 2 ), the final average weight gain was 63.7 g and 55.7 g in probiotics based and non-probiotics based culture systems respectively, in experiment T-2 (3/m 2 ), average weight gain were 55.7 g and 46.7 g and in experiment T-3 (4/m 2 ), average weight gain were 43 g and 37 g in probiotics and non-probiotics based culture system respectively. Mean growth rates were found higher (Table 1) in probiotics then in nonprobiotics culture systems in all replicates.
In three treatments, the mean growth rates were recorded as 0.41 g, 0.36 g and 0.27 g per day in T-1, T-2 and T-3 treatments respectively for Sanolife® Pro-1 culture system. On the other hand, the average growth rates were recorded as 0.35 g, 0.30 g and 0.23 g per day in T-1, T-2 and T-3 treatments respectively with same stocking densities of non-probiotics culture system. Survival rate of cultured M. rosenbergii found different with the same stocking densities among the probiotics and non-probiotics culture system. The highest average survival rates recorded were 69.3% and 68.3% (Table 2) in T-1 (2/m 2 ) for probiotics ponds and nonprobiotics ponds respectively. The lowest average survival rate of 58.3% and 57.7% (Table 2) was found in T-3 (4/m 2 ) for probiotics and nonprobiotics ponds respectively.
Gross production from different culture systems also differs in probiotics and non-probiotics culture system. The estimated average gross production of M. rosenbergii in T-1 at a stocking density of 2/ m 2 was calculated 87.23 g/m 2 and 74.62 g/m 2 ( Table 2) per crop for probiotics and non-probiotics based culture system respectively. On the other hand, the estimated gross productionin T-2 at a stocking density of 3/m 2 was found to be 103 g/m 2 /crop and 87.23 g/m 2 /crop Thiobacillus sp. and Paracoccus sp. Organic carrier product contains a minimum 2 × 10 10 cfu/g. Instructed initial stage dose was 0.175 g/m 2 and final stage dose was 0.3 g/m 2 of pond area.
• Sanolife® Pro-W was the 3 rd type of probiotics used in the study.
The product is a mixture strain of Bacillus spp. of minimum 5 × 10 10 cfu/g. Standard Sanolife® Pro-W application rate was instructed 0.02 g/m 2 .

Site selection and pond construction for freshwater prawn farming
The experiment was conducted in two different rural areas of Chittagong division, South-Eastern part of Bangladesh. Total nine ponds were selected for the experiment from two different villages and size varies from 1200 m 2 to 3600 m 2 . Among the nine selected ponds seven were old and two were newly excavated. Aquatic vegetation and weeds were eradicated from the old ponds. Weed fishes as well as predatory fishes were eradicated by drying the old pond bottom.

Liming and fertilization
Required amount of lime was applied into the ponds to make the desired pH level. Depending upon soil pH, agricultural limestone was applied at 25 g/m 2 to 50 g/m 2 . To increase the fertility and productivity of the ponds both organic and inorganic fertilizers were used. Raw cow dung was applied at 75 g/m 2 to 125 g/m 2 along with 6.25 g/m 2 triple super phosphate and 3.75 g/m 2 Urea. This application was continued for every 7 days interval up to the whole culture period.

Increasing surface area
Like many other crustaceans, M. rosenbergii shows strong territorial defense and hiding behavior. During molting, there is always a considerable risk of predation by larger dominant prawns. Hence, to increase habitat surface area and hiding, substrates are required to reduce predation in grow out ponds. Bushes, palm and coconut-leaves, bamboo, tiers, tree branches, etc. were used indigenously to provide substrates for prawns to cling and to hide in.

Juvenile collection and stocking
Good quality prawn juveniles were collected from a commercial private hatchery of Chittagong where the production of M. rosenbergii was done through probiotics based rearing system. The prawn juveniles were stocked at a size of 2 g to 3 g in the different ponds. In this experiment juveniles were stocked with three different stocking densities. In treatment 1 (T-1) juveniles were stocked at a rate of 2/m 2 , in treatment 2 (T-2) at 3/m 2 and in treatment 3 (T-3) at 4/m 2 .

Feed and feeding
In this experiment stocked juveniles were fed on a commercial pellet feed. Feeds were broadcasted evenly along the marginal areas of the pond. Pellet feeds were given at 3% to 15% of the body weight of prawn daily twice, where 60% to 75% of total feed should be fed at late evening of the day. Initially feeding was started at 15% of total biomass which was gradually reduced to 3% to 4% at the final stage of culture period.

Probiotics application with feed
In the present study, some replicates were practiced with probiotics mixed feed. At first the amount of feed was calculated required for those replicates for the day and then calculated the probiotics (Sanolife® Pro-1) amount at a rate of 5 g/kg feed. Probiotics was mixed with required (Table 2) for probiotics and non-probiotics culture system respectively. In treatment three (T-3) at stocking density of 4/m 2 the final gross production was estimated to be 98.1 g/m 2 /crop for probiotics based culture system and 84.26 g/m 2 /crop ( Table 2) for non-probiotics based culture system.

Influence on water quality
In the present study, the water parameters were recorded weekly basis in all ponds and found the recorded parameters within the range of Macrobrachium culture. Average water temperature range was from 29°C to 31°C in three replicates of all treatments pond which were found optimum for Macrobrachium culture. Average pH ranged from 8 to 8.2 in all ponds. Dissolved oxygen ranged from 4.8 mg/l to 5.5 mg/l, ammonia ranged from 0.1 mg/l to 0.19 mg/l. Nitrite concentration varied from 0.1 mg/l to 0.22 mg/l in all the replicates. Transparency also ranged from 33.5 cm to 35.25 cm. Alkalinity ranged from 77 mg/l to 88 mg/l. The variation of dissolved oxygen, pH and N-ammonia was recorded throughout culture period. Among all the three treatments, only some selected T1R3, T2R3 and T3R3 treatments were practiced with probiotics application in pond water. In those cases, ammonia and nitrite were variable results (Table 3) by the probiotics application. Due to the probiotics action waste feed and other waste matters were degraded more than other treatments those were practiced without probiotics. As a result, the concentration of toxic gases in those ponds was reduced in some extent. In all those three treatments ammonia and nitrite concentration found less (Table 3, highlighted value) compare to other treatments among the three different stocking density groups.

Discussion
Probiotics used in feed and pond water has a significant role in the growth of prawn culture. In the present experiment, it was found that in every replicate of the three different treatments higher growth and higher net gross production were found from the probiotics treated pond. The present may be compared with the following authors. Ziaei-Nejad et al. [4] and Wang [5] reported the benefits of the probiotics supplements improved feed value, enzymatic contribution to digestion, inhibition of pathogenic microorganisms, anti-mutagenic and anticarcinogenic activity, growth promoting factors, and increase immune response. Dalmin et at. [6] reported that use of Bacillus sp. improved water quality, survival, growth rates and health status of the juveniles of P. monodon and reduced the pathogenic vibrios. Keysami et al. [7] mentioned that prawns gained higher weight and better FCR by using B. subtilis in feed.
In the present experiment, different survival rates were recorded in different treatments. Higher survival rate 69.3% was found where the stocking density was maintained at 2/m 2 and in this treatment average growth was also recorded higher in probiotics based treatment. In other two treatments, stocking density (3/m 2 ) and (4/m 2 ) showed lower survival as well as lower growth than the treatment with 2/m 2 stocking density. On the other hand, the average final body weights in three treatments were 55.7 g, 46.7 g and 37 g respectively for T-1, T-2 and T-3. Among three treatments the best result was found at treatments 2 where stocking density was maintained 3/m 2 . Balcazar et al. [8]; Gullian et al. [9] reported that prawn's survival was also found significantly greater in B. subtilis treated groups than in the control group. It is possible that this phenomenon operates by the substitution of opportunist pathogens that reduced growth.
It was found that the final body weight gained 63.7 g and 55.7 g in Sanolife® Pro-1 and non-probiotics based culture systems respectively in T-1 treatment (2/m 2 ). Similarly, the body weight gained 55.7 g and 46.7 g in T-2 (3/m 2 ) and 43 g and 37 g in T-3 (4/m 2 ) in probiotics and non-probiotics treatment respectively. It was found from the above results that body weight was higher in lower stocking densities and lower in higher stocking densities. Siddiqui et al. [10] and Karplus and Sagi [11] reported in their research that, the prawn response to higher  with the introduction of probiotics. Prawn stocking in monoculture has the potentials of increasing total yields as well as farm income with probiotics application. Some ecological advantages of probiotics based prawn culture, such as improved water quality help to increase the sustainability of this system of aquaculture.

Conclusion
The technical potentials of prawn (M. rosenbergii) polyculture using probiotics based system were investigated in this study. This study demonstrated that pond prawn production was significantly increased