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Impact of commercial probiotics application on growth and production of giant fresh water prawn (Macrobrachium Rosenbergii De Man, 1879)
Aquaculture Reports 4 (2016) 112–117
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Impact of commercial probiotics application on growth and production of giant fresh water prawn (Macrobrachium Rosenbergii De Man, 1879) Alokesh Kumar Ghosh (Assistant professor) a,∗ , Joyanta Bir (Assistant professor) a , Md. Abul Kalam Azad (PhD student) (Senior Upazilla Fisheries Officer) a,b , Abul Farah Md. Hasanuzzaman (Assistant professor) a , Md. Sanaul Islam (Associate professor) c , Khandaker Anisul Huq (Professor) a a b c
Fisheries and Marine Resource Technology Discipline, Khulna University, Khulna-9208, Bangladesh Department of Fisheries (DoF), Balagang, Sylhet, Bangladesh Soil Science Discipline, Khulna University, Bangladesh
a r t i c l e
i n f o
Article history: Received 19 April 2015 Received in revised form 10 August 2016 Accepted 13 August 2016 Keywords: Zymetin Super PS Growth Production Macrobrachium rosenbergii
a b s t r a c t The study was conducted to observe the impact of commercial probiotics application on growth and production performance of fresh water prawn (Macrobrachium rosenbergii) from August 2011 to March 2012. There were four experimental groups viz (a) control or without probiotics treated prawn (T1 ), (b) feed probiotics- Zymetin (T2 ) treated prawn, (c) soil probiotics- Super PS (T3 ) treated prawn and (d) Both Zymetin and Super PS (T4 ) treated prawn. Twelve ponds (each 120 m2 ) were used where stocking density was 2/m2 for all treatments and control and each was triplicated. After pond preparation, prawn PL was reared in the nursery pond for 45 days to become juvenile. At the time of stocking in growout ponds, average body weight of juvenile prawn was 1.04 g. After eight months (240 days) of culture, the mean final weight became 39.5 ± 12.03, 43.4 ± 14.91, 48.0 ± 16.73 and 51.6 ± 15.58 g in T1 , T2 , T3 and T4 respectively. Significance difference was found among all treatments and T4 showed highest growth. The SGR was found to be 1.50 ± 0.13, 1.53 ± 0.13, 1.58 ± 0.13 and 1.61 ± 0.11 (%BW/day) in T1 , T2 , T3 and T4 respectively and the difference was significant. The survival rate did not differ significantly but highest survival rate was found in T4 (90%). The average FCR was significantly lowest in T4 (1.39) and highest in T1 (1.9). The net average production was found to be significantly higher in T4 (914 kg/ha) which was 35% and 21 % higher than the control group (T1 ) and feed probiotics (T2 ) respectively. Water and soil quality parameters were measured and were within the culturable range. The production of probiotics treated ponds was always higher than without probiotics treated ponds but highest growth and production were found in T4 where Zymetin and Super PS were used combinedly. The results of this study can be applied in the farmer’s pond to increase the total production of prawn in the country. © 2016 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Bangladesh is very much blessed with riverine giant prawn (Macrobrachium rosenbergii, locally called as golda) culture because of some suitable conditions like geographic position, vast amount of freshwater and brackishwater, suitable resources, agro climatic environment, water, soil, available culture technique and availability of wild PL (Wahab et al., 2012; Ahmed et al., 2008a). The
∗ Corresponding author. E-mail addresses: alokesh [email protected], [email protected] (A.K. Ghosh).
increase of interest for prawn farming was dramatic with the farmers after 2000 due to the spread of WSSV (White Spot Syndrome Virus) attack in shrimp (Penaeus monodon) farms and because prawn farms were not affected by WSSV; the farmers paid their greater attention for prawn cultivation (Karim, 2000). The production rate of prawn and shrimp in Bangladesh is 713 kg/ha (DoF, 2013), whereas the production in the neighboring counties is, in Thailand 2338 kg/ha (Vicki, 2007), Vietnam 1000–1500 kg/ha (Ridmontri, 2002), China 1500 kg/ha (Weimin and Xianping, 2002) and India 600–1000 kg/ha (Raizeda et al., 2005). The production level of prawn is low in Bangladesh compared to those other countries. With the increasing intensification and commercialization
http://dx.doi.org/10.1016/j.aqrep.2016.08.001 2352-5134/© 2016 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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of aquaculture production, disease problems inevitably emerge. In recent decades, disease prevention and control have led to a substantial increase in the use of antimicrobial drugs, pesticides and disinfectants. The abuse of antimicrobial drugs, pesticides, and disinfectants in aquaculture disease prevention and growth promotion has led to the evolution of resistant strains of bacteria as well as a question of safety (Esiobu et al., 2002). Typically farmers use chemicals and antibiotics in their prawn farming, and very recently they have been introduced to probiotics and employing them in their hatchery and grow-out ponds considering the demand for more disease-free, environment-friendly and sustainable aquaculture practice. There are increasing reports of research works on the application of probiotics in aquaculture (Hai et al., 2009; Zhou et al., 2009; Li et al., 2006) though very few studies related to the usage of probiotics in prawn culture available in Bangladesh. The use of probiotics as a means of reducing disease is widely accepted on the principle of competitive exclusion of potentially pathogenic microorganisms, digestion enhancement by participating in digestion process, supplying fatty acids and vitamins, and providing necessary growth factors, immune response enhancement though modulating non-specific immune responses, water quality improvement by enhancing the domination of heterotrophic bacteria and antiviral effects through the production of antagonistic compounds (Balcázar et al., 2006; Vine et al., 2006). This farming system is mainly done by using various probiotics products which contain beneficial micro-organisms like Bacillus sp., Rhodobacter sp., Rhodococcus sp., Streptococcus sp. etc. Considering the importance of prawn culture and the application of probiotics, the study was developed to examine the effects of commercial probiotics on growth and production of prawn. The scope of using commercially probiotics in the grow-out phase of the prawn culture industry might be developed and expanded. 2. Materials and methods 2.1. Study area and time The experiment was conducted for eight months in twelve experimental ponds (each pond 240 m2 ) in pond complex II at Fisheries and Marine Resource Technology Discipline of Khulna University from August 2011 to March 2012. The experiment was conducted with the giant freshwater prawn (Macrobrachium rosenbergii) together with some commercial probiotic bacteria. 2.2. Experimental design The experiment was conducted with three treatments following a control group. The control group was cultured without any probiotic treatment and was expressed as T1 . The first treatment group of prawn T2 , was cultured using only feed probiotics (Zymetin), the second treatment group of prawn (T3 ) was cultured using only soil probiotics (Super PS) and the third treatment group of prawn was cultured using feed probiotics (Zymetin) and soil probiotics (Super PS) combinedly. Each treatment and control group was triplicated.
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so that undesirable organisms could not enter into the pond from outside. 2.4. PL nursing and stocking The post larvae (PL) of prawn was collected from Shrimp Research Station, Bagerhat and was negative for virus in PCR test. After pond preparation, PL was reared in the nursing pond for 45 days with a stocking density of 25 PL/m2 . After nursing PL, it became juvenile which was stocked in grow-out ponds with a stocking density of 2/m2 while the average individual weight was 1.04 g. 2.5. Feed and feeding management In this experiment, MEGA supplementary feed of APON group was used and was formulated by SPECTRA HEXA FEED LTD., with the following ingredients, fish meal, shrimp head meal, cod liver oil, squid meal, broken rice, soybean meal, wheat flour, cholesterol, phospholipids, vitamins and minerals (Leaflet of MEGA Feed). Before feeding, the feed was analyzed to determine the proximate composition according to the standard procedures given by Association of Official Analytical Chemists (AOAC, 1980). The average percentage of protein, fat and moisture was 31, 7 and 12% respectively in the feed. Feed was given according to the body weight and the age of prawn. At first month, feed was given at 10% of total prawn body weight, then upto five months feed was given at 5% of body weight, and last two months the feed was given at 2% of the total body weight of prawn (the average weight of prawn was multiplied by the total number of prawn to calculate total weight). Feed was spread over the pond surface three times in a day at 30% (6.00 am), 30% (12.00 pm) and 40% (6.00pm) of the total feed allocated for the prawn. 2.6. Composition and application of probiotics Two commercial probiotics were used in the experiment. The trade name of probiotics was Super PS (soil probiotics) and Zymetin (feed probiotics) provided by CP Aquaculture (India) private limited. Before applying the probiotics in the pond, the living condition of the bacteria was tested in standard nutrient agar media. The composition and the mode of application were described on the label (pack) of the probiotics which were given below. 2.6.1. Zymetin Zymetin is made with beneficial bacteria used in feed. It is usually mixed with feed to increase immunity and inhibit the growth of pathogenic bacteria in the gut. It also improves the appetite of the shrimp and increase function of digestion and assimilation for better growth and reduces FCR. It is composed mainly with Streptococcus faecalis, Clostridium butyricum, Bacillus mesentericus, Protease, Lipase and Beer yeast. The total aerobic heterotrophic bacterial load was 1.10 × 106 (CFU/g) (Nimrat and Vuthiphandchai, 2011). The application procedure of this probiotics was as follows; 5 g Zymetin was mixed with 20 g mutagen binder per kg feed for first four days in a week.
2.3. Pond preparation The pond was first dewatered with pump and dried under sun light for one month to allow excavate on the bottom mud and to complete digging of the experimental ponds; thus aquatic weeds and unwanted fauna were removed. The embankment of the pond was repaired and constructed. After testing soil pH, liming was done at a dose of 250 kg/ha (CaCO3 ) that helps to maintain good water quality. The pond was filled with water to a depth of about 1.5 m. The total area of the ponds was fenced with fine meshed nylon net;
2.6.2. Super PS Super PS is a soil probiotics made with beneficial bacteria which is used to improve pond bottom condition, reduce harmful bacteria and keep the environment friendly for aquaculture. It is composed mainly with Rhodobacter sp. and Rhodococcus sp. in 109 CFUml−1 . The application procedure was, spreading 40 liters Super PS/ha before stocking juveniles for the utilization of pond bottom’s organic matter and to increase the beneficial bacteria on the pond bottom. After stocking prawn the dose was 0.5 ppm/week at early
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stage, 1 ppm/week at the middle stage and 1–2 ppm/week at the last stage of the experiment (at sunny day). 2.7. Measurement of water quality parameters Physiochemical parameters of the water quality were measured in the water chemistry laboratory of Fisheries and Marine Resource Technology Discipline, Khulna University at every week interval. Most of the water quality parameters were measured by using HACH kit, produced by HACH, USA (Model FF-2) and also using specific parameters measuring equipment. Salinity was measured by Refractometer (ATAGO CO. LTD, Japan, Master- T 2312, Salinity range 0–100 ppt), and Digital Thermometer (DIGITAL THERMOMETER, made in China, model no WT-2, Temperature range −20–80◦ C) was used to measure temperature. pH was measured by using pH meter (HACH, Sension 3, USA). Alkalinity (titration method with sulphuric acid), hardness (titration method with EDTA solution), ammonia (Nessler Method) and nitrite were measured by HACH kit, produced by HACH, USA (Model FF-2) and dissolved oxygen was measured by DO meter (PDO-519, made in Taiwan, Lutron). 2.8. Measurement of physicochemical parameters of soil Soil pH was determined electrochemically with the help of glass electrode pH meter as suggested by Jackson (1973) and the ratio of soil to water was 1:2.5 (Jackson, 1973). The electrical conductivity of the soil was measured at a soil: water ratio of 1:2.5 with the help of EC meter. Organic carbon of samples was determined by Walkley and Black’s wet oxidation method as outlined by Jackson (1973). Organic matter was calculated by multiplying the percent value of organic carbon with the conventional Van-Bemmelene’s factor of 1.724 (Piper, 1950). The available Ca and Mg were determined from NH4 OAc. (pH-7.0) extract through titrimetric method as described by Jackson (1973). 2.9. Data collection Sampling was done at fifteen days intervals. For length and weight measurements, 50 individuals were collected from each pond and each sampling time by using cast net. Body weight was recorded by electric balance to the nearest 0.001 g accuracy. At the end of the experiment, all the ponds were dewatered by water pump and all the prawn was harvested completely to determine the survival rate, FCR and other production performance parameters. Survival (%) = Nt/N0 ∗ 100% Where Nt is the number of shrimp at the end of the experiment and N0 the initial number of shrimp. Average weight gain (WG) = Wt−W0
2.10. Statistical analysis Recorded data were analyzed using Microsoft excel 2007. Oneway analysis of variance (ANOVA, multiple comparison, Tukey’s test) was used to determine any significant difference of weight of prawn among the treatments using SPSS 16 software and t-test was used to determine any significant differences of survival rate, FCR and production among the treatments and using Microsoft excel 2007. All statistical analyses were considered significant at 5% (P < 0.05). 3. Results 3.1. Growth performance parameters of prawn There were significant differences of final mean prawn body weight among all the treatments (P < 0.05) (Table 1). The average weight of prawn in each sampling is shown in Fig. 1. After 240 days of culture period, the highest mean weight gain (50.6 g) was found in T4 in which the Zymetin and Super PS probiotics were used combinedly. The lowest final weight of prawn (39.6 g) was found in T1 (control). According to Fig. 1 and Table 1, T4 showed the highest final weight (51.6 g) which was 31, 19 and 7% more than T1 , T2 and T3 respectively. The Zymetin treated prawns (T2 ) and Super PS treated prawns (T3 ) showed 10% and 22% higher final weight than the control (T1 ) and it was statistically significant (P < 0.05). At the end of experiment the survival rate of prawn was highest in T4 (90%) and lowest in T1 (88%) but no significant difference was found among the treatments (P>0.05). Highest and lowest SGR was found in T4 (1.61) and T1 (1.5) respectively and other probiotics treated ponds also showed better SGR than the control ponds which was significantly different (P < 0.05) (Table 1). The lowest FCR was found to be 1.39 in T4 and highest FCR was found in T1 (1.9) which was significantly different (Table 1). The FCR for other two treatments were intermediate than T1 and T4 . The average gross production was highest in T4 (933 kg/ha) and gradually reduced 849.65, 772.21 and 693.49 kg/ha in T3 , T2 and T1 respectively; and it was significantly different (P < 0.05) (Table 1). The net production in T4 was highest (Zymetin + Super PS) which was 35% more than T1 (control) and was significantly different. Production in all probiotics treated ponds was higher than the control ponds (Table 1). 3.2. Water quality parameters In the present study, important water quality parameters, like pH, temperature, dissolved oxygen (DO), alkalinity, hardness, ammonia, nitrite, salinity were monitored (Table 2). All the water quality parameters were within the acceptable range for freshwater prawn culture.
Where Wt is the final body weight and W0 the initial body weight of prawn (in grams).
60 50
Where t is the duration of the growth interval
40
Specific growth rate (SGR) = (ln(Wt)−ln(W0 ))/tX100 Where t is the duration of the experiment in days, Wt is the final average individual body weight and W0 the initial average individual body weight (in% BW/day)
Weight (g)
Daily weight gain (DWG) (g/day) = (Wt−W0 )/t
30 20 10 0
Feed conversion ratio (FCR) = Total amount of feed given (g)/Weight gain (g)
Without probiocs (T1) Zymen (T2) Super PS (T3) Zymen+Super PS (T4)
0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240
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Days of culture Fig. 1. Average prawn weight (g) in four treatments from August 2011 to March 2012.
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Table 1 Effect of probiotics on mean final weight and daily weight gain under different probiotics treatments.
Initial weight (g) Final weight (g) Weight gain (g) DWG (g/day) SGR (%BW/day) Survival rate (%) FCR Gross production (kg/ha) Net production (kg/ha)
T1 (control)
T2
T3
T4
1.04 39.56 g ± 12.03a 38.52 ± 12.03a 0.160 ± 0.050a 1.50 ± 0.13a 87.81 ± 1.39a 1.90 ± 0.28a 693.49 ± 99.90a 675.23 ± 100.19a
1.04 43.35 g ± 14.91b 42.31 ± 14.91b 0.176 ± 0.062b 1.53 ± 0.13b 89.10 ± 0.73a 1.69 ± 0.20ab 772.21 ± 86.56ab 753 ± 86.63ab
1.04 48.07 g ± 16.73c 47.03 ± 16.73c 0.196 ± 0.069c 1.58 ± 0.13c 88.50 ± 2.36a 1.54 ± 0.17bc 849.65 ± 93.33bc 831.24 ± 93.56bc
1.04 51.63 g ± 15.58d 50.59 ± 15.58d 0.210 ± 0.064d 1.61 ± 0.11d 90.32 ± 3.47a 1.39 ± 0.08bc 933.02 ± 50.75cd 914.24 ± 50.03cd
Means in the same row with different superscripts are significantly different (Pb0.05).
Table 2 Different water quality parameters (mean ± SD) during the culture period. Water quality parameters
Treatments
Temperature (◦ C) pH Salinity (ppt) DO (mg/L) Alkalinity (CaCO3 mg/L) Hardness (CaCO3 mg/L) Total ammonia (mg/L) Total Nitrite nitrogen (mg/L)
T1 Mean ± SD
T2 Mean ± SD
T3 Mean ± SD
T4 Mean ± SD
27.41 ± 4.51 7.68 ± 0.19 0.87 ± 0.63 5.10 ± 0.68 125.22 ± 16.6 216.3 ± 46.8 1.07 ± 0.11 0.24 ± 0.1
27.22 ± 4.47 7.64 ± 0.27 0.97 ± 0.62 5.29 ± 0.63 138.76 ± 22.33 260.8 ± 44.2 0.34 ± 0.02 0.09 ± 0.07
27.47 ± 4.54 7.76 ± 0.28 1.06 ± 0.69 5.21 ± 0.57 123.57 ± 10.01 250.4 ± 52.8 0.29 ± 0.03 0.07 ± 0.06
27.34 ± 4.36 7.69 ± 0.31 1.01 ± 0.68 5.32 ± 0.53 143 ± 16.75 227 ± 48.45 0.18 ± 0.02 0.06 ± 0.02
3.3. Soil quality parameters In the present study, different soil quality parameters (pH, organic carbon, electrical conductivity, phosphorus, calcium, magnesium) were measured and found within the culturable range (Table 3).
4. Discussion With increasing demand of fish and for environment-friendly aquaculture, the use of probiotics in aquaculture is now considered alternative to antibiotics and chemicals to improve the quality and sustainability of aquaculture production. Since the first use of probiotics in aquaculture, a growing number of studies have demonstrated their ability to control potential pathogens and to increase the growth rates and welfare of farmed aquatic animals (Gatesoupe, 1991; Carnevali et al., 2004; Macey and Coyne, 2005; Wang et al., 2005; Wang and Xu, 2006). The water quality parameters in the experiment were within the culturable range as mentioned by different reports (New, 2002; Firkins and Holdich, 1993; Knowlton and Kirby, 1984; Stone and Thomforde, 2004). The total ammonia and nitrite level was lower in probiotics treated ponds than control and lowest level was found in T4 . This is because the beneficial bacteria used the dissolved organic material as their nutrient and for nitrification rapidly. Suhendra et al. (1997) found that routine use of commercial probiotics in a shrimp farm in West Java resulted in reduced organic matter accumulation, improved water quality and enhanced environmental conditions. Current study data showed the standard concentrations of TP (0.02-
2.67%.), organic carbon (OC) (1.067–1.32%), Ca (0.13–0.17%), Mg (0.08-0.09%) in sediment after the ponds treated with commercial probiotics which is strongly related to the study with Suhendra et al. (1997). During the culture period in the experiment, all the probiotics-supplemented diets resulted in an increase of final weight and weight gain showing that the addition of probiotics increased the growth performance of prawn. This is a very inspiring result for the farmers in prawn culture with probiotics as the size of the prawn is related with the market price and for the earnings of foreign exchange. This study supports the study of Saad et al. (2009) and Hossain et al. (2013) in which better result in growth was found in probiotics treated harvested shrimp and prawn than without probiotics treated shrimp. Gullian et al. (2004) demonstrated a significant growth increase in shrimp inoculated with Bacillus sp. Whilst El-Dakar and Goher (2004) found that enhanced growth was generally obtained in shrimp fed diets with B. subtilis inclusion. The survival rate of prawn in the present study was higher in probiotics treated ponds than control but no significant difference was found. But in some studies the survival rate of post larvae (PL) of prawn was significantly higher in probiotics treated prawns than control (Seenivasan et al., 2011; Hossain et al., 2013). The present study was not similar to those studies because of stocking juveniles in the ponds (before stocking, the PL was first nursed for 45 days). Rinisha et al. (2010) evaluated probiotics effects on the growth and survival of Macrobrachium rosenbergii postlarvae and after 90 days they observed 76% survivability where probiotics was introduced through feed, whereas 64% survivability observed in the control. So, the probiotics effect on survival of prawn was not prominent on juvenile like the PL in the previous studies. The
Table 3 Different soil quality parameters among the treatments. Treatment
pH (ranges)
Organic carbon (%)
Electrical conductivity (ds/m) (ranges)
P (meq/100 g)
Ca (ranges)
Mg (Ranges)
Without probiotics (T1 ) Zymetin (T2 ) Super PS (T3 ) Zymetin + Super PS (T4 )
6.9–7.7 6.7–7.6 6.7–7.6 7–7.6
1.1349 1.06704 1.14738 1.32054
2.3–6.84 5.2–6.15 5.7–6.46 5–6.91
2.67% 2.33% 3% 2.33%
0.15–0.17% 0.12–0.17% 0.15–0.20% 0.11–0.16%
0.08–0.10% 0.08% 0.07–0.10% 0.06–0.09%
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mean FCR value in the present study was lowest in T4 (1.39) which indicates proper utilization of feed due to the presence of beneficial bacteria of probiotics and the highest FCR (1.90) was found in T1 which differed significantly. The present study supported findings of Irianto and Austin (2002) where they revealed probiotics bacteria as a good candidate for improving the digestion of nutrients and growth of aquatic organisms. The probiotic bacteria have been shown to improve the digestive activity by producing digestive enzyme and enhanced digestion and increased absorption of food which ultimately improved feed utilization efficiency and low FCR (Lee and Lee, 1990; Gatesoupe, 1999; El-Dakar and Goher, 2004; Ziaei-Nejad et al., 2006). The gross and net production was higher in probiotics treated ponds than control and T4 showed the highest production. The per hectare total production was 1.35 times higher in T4 than T1 which supports the study of Hossain et al. (2013) in which per hectare total production of P. monodon was 1.72 times better in probiotics treated ponds than control. The highest production in T4 might cause of faster growth rate, higher final weight, higher survival rate, better culture friendly soil and water quality parameters, better digestion and assimilation of feed due to functioning of highest number of beneficial bacteria. In these circumstances, to increase our prawn production in comparing with other developing countries, application of probiotics is essential. A few investment cost for probiotics can give high return in respect of traditional culture. 5. Conclusion It can be generally concluded from the present study that probiotics had significant effect on growth and production of prawns. The combined application of soil and feed probiotics (Zymetin + Super PS) showed best growth performance which was significantly higher. The findings of this study could be applied to the farmer’s level to increase the production of prawn. Competing interest The authors declare that they have no competing interest Authors’ contribution AKG: data analysis using Microsoft Office Excel, drafting and revision of the research article. JB: data collection, in situ measurement, initial set up of the experiment: MAKA: data collection, in situ measurement. AFMH: Field monitoring. MSI: Data collection, soil quality measurement. KAH: Research concept and design, field monitoring, data interpretation. Acknowledgement The research was conducted with the financial support of Bangladesh Academy of Sciences-United States Department of Agriculture Programme in Agricultural and Life Science. The authors would like to acknowledge for their kind support and sincere cooperation during the research period. References AOAC (Association of Official Analytical Chemists), 1980. In: Horwitz, W. (Ed.), Official Methods of Analysis. 13th edition. Association of Official Analytical Chemists, Washington DC, p. 988. Ahmed, N., Demaine, H., Muir, J.F., 2008a. Freshwater prawn farming in Bangladesh: history, present status and future prospects. Aqua. Res. 39 (8), 806–819. Balcázar, J.L., De Blas, I., Ruiz-Zarzuela, I., Cunningham, D., Vendrell, D., Múzquiz, J.L., 2006. The role of probiotics in aquaculture. Vet. Microbiol. 114 (3), 173–186.
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Impact of commercial probiotics application on growth and production of giant fresh water prawn (Macrobrachium Rosenbergii De Man, 1879) Alokesh Kumar Ghosh (Assistant professor) a,∗ , Joyanta Bir (Assistant professor) a , Md. Abul Kalam Azad (PhD student) (Senior Upazilla Fisheries Officer) a,b , Abul Farah Md. Hasanuzzaman (Assistant professor) a , Md. Sanaul Islam (Associate professor) c , Khandaker Anisul Huq (Professor) a a b c
Fisheries and Marine Resource Technology Discipline, Khulna University, Khulna-9208, Bangladesh Department of Fisheries (DoF), Balagang, Sylhet, Bangladesh Soil Science Discipline, Khulna University, Bangladesh
a r t i c l e
i n f o
Article history: Received 19 April 2015 Received in revised form 10 August 2016 Accepted 13 August 2016 Keywords: Zymetin Super PS Growth Production Macrobrachium rosenbergii
a b s t r a c t The study was conducted to observe the impact of commercial probiotics application on growth and production performance of fresh water prawn (Macrobrachium rosenbergii) from August 2011 to March 2012. There were four experimental groups viz (a) control or without probiotics treated prawn (T1 ), (b) feed probiotics- Zymetin (T2 ) treated prawn, (c) soil probiotics- Super PS (T3 ) treated prawn and (d) Both Zymetin and Super PS (T4 ) treated prawn. Twelve ponds (each 120 m2 ) were used where stocking density was 2/m2 for all treatments and control and each was triplicated. After pond preparation, prawn PL was reared in the nursery pond for 45 days to become juvenile. At the time of stocking in growout ponds, average body weight of juvenile prawn was 1.04 g. After eight months (240 days) of culture, the mean final weight became 39.5 ± 12.03, 43.4 ± 14.91, 48.0 ± 16.73 and 51.6 ± 15.58 g in T1 , T2 , T3 and T4 respectively. Significance difference was found among all treatments and T4 showed highest growth. The SGR was found to be 1.50 ± 0.13, 1.53 ± 0.13, 1.58 ± 0.13 and 1.61 ± 0.11 (%BW/day) in T1 , T2 , T3 and T4 respectively and the difference was significant. The survival rate did not differ significantly but highest survival rate was found in T4 (90%). The average FCR was significantly lowest in T4 (1.39) and highest in T1 (1.9). The net average production was found to be significantly higher in T4 (914 kg/ha) which was 35% and 21 % higher than the control group (T1 ) and feed probiotics (T2 ) respectively. Water and soil quality parameters were measured and were within the culturable range. The production of probiotics treated ponds was always higher than without probiotics treated ponds but highest growth and production were found in T4 where Zymetin and Super PS were used combinedly. The results of this study can be applied in the farmer’s pond to increase the total production of prawn in the country. © 2016 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction Bangladesh is very much blessed with riverine giant prawn (Macrobrachium rosenbergii, locally called as golda) culture because of some suitable conditions like geographic position, vast amount of freshwater and brackishwater, suitable resources, agro climatic environment, water, soil, available culture technique and availability of wild PL (Wahab et al., 2012; Ahmed et al., 2008a). The
∗ Corresponding author. E-mail addresses: alokesh [email protected], [email protected] (A.K. Ghosh).
increase of interest for prawn farming was dramatic with the farmers after 2000 due to the spread of WSSV (White Spot Syndrome Virus) attack in shrimp (Penaeus monodon) farms and because prawn farms were not affected by WSSV; the farmers paid their greater attention for prawn cultivation (Karim, 2000). The production rate of prawn and shrimp in Bangladesh is 713 kg/ha (DoF, 2013), whereas the production in the neighboring counties is, in Thailand 2338 kg/ha (Vicki, 2007), Vietnam 1000–1500 kg/ha (Ridmontri, 2002), China 1500 kg/ha (Weimin and Xianping, 2002) and India 600–1000 kg/ha (Raizeda et al., 2005). The production level of prawn is low in Bangladesh compared to those other countries. With the increasing intensification and commercialization
http://dx.doi.org/10.1016/j.aqrep.2016.08.001 2352-5134/© 2016 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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of aquaculture production, disease problems inevitably emerge. In recent decades, disease prevention and control have led to a substantial increase in the use of antimicrobial drugs, pesticides and disinfectants. The abuse of antimicrobial drugs, pesticides, and disinfectants in aquaculture disease prevention and growth promotion has led to the evolution of resistant strains of bacteria as well as a question of safety (Esiobu et al., 2002). Typically farmers use chemicals and antibiotics in their prawn farming, and very recently they have been introduced to probiotics and employing them in their hatchery and grow-out ponds considering the demand for more disease-free, environment-friendly and sustainable aquaculture practice. There are increasing reports of research works on the application of probiotics in aquaculture (Hai et al., 2009; Zhou et al., 2009; Li et al., 2006) though very few studies related to the usage of probiotics in prawn culture available in Bangladesh. The use of probiotics as a means of reducing disease is widely accepted on the principle of competitive exclusion of potentially pathogenic microorganisms, digestion enhancement by participating in digestion process, supplying fatty acids and vitamins, and providing necessary growth factors, immune response enhancement though modulating non-specific immune responses, water quality improvement by enhancing the domination of heterotrophic bacteria and antiviral effects through the production of antagonistic compounds (Balcázar et al., 2006; Vine et al., 2006). This farming system is mainly done by using various probiotics products which contain beneficial micro-organisms like Bacillus sp., Rhodobacter sp., Rhodococcus sp., Streptococcus sp. etc. Considering the importance of prawn culture and the application of probiotics, the study was developed to examine the effects of commercial probiotics on growth and production of prawn. The scope of using commercially probiotics in the grow-out phase of the prawn culture industry might be developed and expanded. 2. Materials and methods 2.1. Study area and time The experiment was conducted for eight months in twelve experimental ponds (each pond 240 m2 ) in pond complex II at Fisheries and Marine Resource Technology Discipline of Khulna University from August 2011 to March 2012. The experiment was conducted with the giant freshwater prawn (Macrobrachium rosenbergii) together with some commercial probiotic bacteria. 2.2. Experimental design The experiment was conducted with three treatments following a control group. The control group was cultured without any probiotic treatment and was expressed as T1 . The first treatment group of prawn T2 , was cultured using only feed probiotics (Zymetin), the second treatment group of prawn (T3 ) was cultured using only soil probiotics (Super PS) and the third treatment group of prawn was cultured using feed probiotics (Zymetin) and soil probiotics (Super PS) combinedly. Each treatment and control group was triplicated.
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so that undesirable organisms could not enter into the pond from outside. 2.4. PL nursing and stocking The post larvae (PL) of prawn was collected from Shrimp Research Station, Bagerhat and was negative for virus in PCR test. After pond preparation, PL was reared in the nursing pond for 45 days with a stocking density of 25 PL/m2 . After nursing PL, it became juvenile which was stocked in grow-out ponds with a stocking density of 2/m2 while the average individual weight was 1.04 g. 2.5. Feed and feeding management In this experiment, MEGA supplementary feed of APON group was used and was formulated by SPECTRA HEXA FEED LTD., with the following ingredients, fish meal, shrimp head meal, cod liver oil, squid meal, broken rice, soybean meal, wheat flour, cholesterol, phospholipids, vitamins and minerals (Leaflet of MEGA Feed). Before feeding, the feed was analyzed to determine the proximate composition according to the standard procedures given by Association of Official Analytical Chemists (AOAC, 1980). The average percentage of protein, fat and moisture was 31, 7 and 12% respectively in the feed. Feed was given according to the body weight and the age of prawn. At first month, feed was given at 10% of total prawn body weight, then upto five months feed was given at 5% of body weight, and last two months the feed was given at 2% of the total body weight of prawn (the average weight of prawn was multiplied by the total number of prawn to calculate total weight). Feed was spread over the pond surface three times in a day at 30% (6.00 am), 30% (12.00 pm) and 40% (6.00pm) of the total feed allocated for the prawn. 2.6. Composition and application of probiotics Two commercial probiotics were used in the experiment. The trade name of probiotics was Super PS (soil probiotics) and Zymetin (feed probiotics) provided by CP Aquaculture (India) private limited. Before applying the probiotics in the pond, the living condition of the bacteria was tested in standard nutrient agar media. The composition and the mode of application were described on the label (pack) of the probiotics which were given below. 2.6.1. Zymetin Zymetin is made with beneficial bacteria used in feed. It is usually mixed with feed to increase immunity and inhibit the growth of pathogenic bacteria in the gut. It also improves the appetite of the shrimp and increase function of digestion and assimilation for better growth and reduces FCR. It is composed mainly with Streptococcus faecalis, Clostridium butyricum, Bacillus mesentericus, Protease, Lipase and Beer yeast. The total aerobic heterotrophic bacterial load was 1.10 × 106 (CFU/g) (Nimrat and Vuthiphandchai, 2011). The application procedure of this probiotics was as follows; 5 g Zymetin was mixed with 20 g mutagen binder per kg feed for first four days in a week.
2.3. Pond preparation The pond was first dewatered with pump and dried under sun light for one month to allow excavate on the bottom mud and to complete digging of the experimental ponds; thus aquatic weeds and unwanted fauna were removed. The embankment of the pond was repaired and constructed. After testing soil pH, liming was done at a dose of 250 kg/ha (CaCO3 ) that helps to maintain good water quality. The pond was filled with water to a depth of about 1.5 m. The total area of the ponds was fenced with fine meshed nylon net;
2.6.2. Super PS Super PS is a soil probiotics made with beneficial bacteria which is used to improve pond bottom condition, reduce harmful bacteria and keep the environment friendly for aquaculture. It is composed mainly with Rhodobacter sp. and Rhodococcus sp. in 109 CFUml−1 . The application procedure was, spreading 40 liters Super PS/ha before stocking juveniles for the utilization of pond bottom’s organic matter and to increase the beneficial bacteria on the pond bottom. After stocking prawn the dose was 0.5 ppm/week at early
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stage, 1 ppm/week at the middle stage and 1–2 ppm/week at the last stage of the experiment (at sunny day). 2.7. Measurement of water quality parameters Physiochemical parameters of the water quality were measured in the water chemistry laboratory of Fisheries and Marine Resource Technology Discipline, Khulna University at every week interval. Most of the water quality parameters were measured by using HACH kit, produced by HACH, USA (Model FF-2) and also using specific parameters measuring equipment. Salinity was measured by Refractometer (ATAGO CO. LTD, Japan, Master- T 2312, Salinity range 0–100 ppt), and Digital Thermometer (DIGITAL THERMOMETER, made in China, model no WT-2, Temperature range −20–80◦ C) was used to measure temperature. pH was measured by using pH meter (HACH, Sension 3, USA). Alkalinity (titration method with sulphuric acid), hardness (titration method with EDTA solution), ammonia (Nessler Method) and nitrite were measured by HACH kit, produced by HACH, USA (Model FF-2) and dissolved oxygen was measured by DO meter (PDO-519, made in Taiwan, Lutron). 2.8. Measurement of physicochemical parameters of soil Soil pH was determined electrochemically with the help of glass electrode pH meter as suggested by Jackson (1973) and the ratio of soil to water was 1:2.5 (Jackson, 1973). The electrical conductivity of the soil was measured at a soil: water ratio of 1:2.5 with the help of EC meter. Organic carbon of samples was determined by Walkley and Black’s wet oxidation method as outlined by Jackson (1973). Organic matter was calculated by multiplying the percent value of organic carbon with the conventional Van-Bemmelene’s factor of 1.724 (Piper, 1950). The available Ca and Mg were determined from NH4 OAc. (pH-7.0) extract through titrimetric method as described by Jackson (1973). 2.9. Data collection Sampling was done at fifteen days intervals. For length and weight measurements, 50 individuals were collected from each pond and each sampling time by using cast net. Body weight was recorded by electric balance to the nearest 0.001 g accuracy. At the end of the experiment, all the ponds were dewatered by water pump and all the prawn was harvested completely to determine the survival rate, FCR and other production performance parameters. Survival (%) = Nt/N0 ∗ 100% Where Nt is the number of shrimp at the end of the experiment and N0 the initial number of shrimp. Average weight gain (WG) = Wt−W0
2.10. Statistical analysis Recorded data were analyzed using Microsoft excel 2007. Oneway analysis of variance (ANOVA, multiple comparison, Tukey’s test) was used to determine any significant difference of weight of prawn among the treatments using SPSS 16 software and t-test was used to determine any significant differences of survival rate, FCR and production among the treatments and using Microsoft excel 2007. All statistical analyses were considered significant at 5% (P < 0.05). 3. Results 3.1. Growth performance parameters of prawn There were significant differences of final mean prawn body weight among all the treatments (P < 0.05) (Table 1). The average weight of prawn in each sampling is shown in Fig. 1. After 240 days of culture period, the highest mean weight gain (50.6 g) was found in T4 in which the Zymetin and Super PS probiotics were used combinedly. The lowest final weight of prawn (39.6 g) was found in T1 (control). According to Fig. 1 and Table 1, T4 showed the highest final weight (51.6 g) which was 31, 19 and 7% more than T1 , T2 and T3 respectively. The Zymetin treated prawns (T2 ) and Super PS treated prawns (T3 ) showed 10% and 22% higher final weight than the control (T1 ) and it was statistically significant (P < 0.05). At the end of experiment the survival rate of prawn was highest in T4 (90%) and lowest in T1 (88%) but no significant difference was found among the treatments (P>0.05). Highest and lowest SGR was found in T4 (1.61) and T1 (1.5) respectively and other probiotics treated ponds also showed better SGR than the control ponds which was significantly different (P < 0.05) (Table 1). The lowest FCR was found to be 1.39 in T4 and highest FCR was found in T1 (1.9) which was significantly different (Table 1). The FCR for other two treatments were intermediate than T1 and T4 . The average gross production was highest in T4 (933 kg/ha) and gradually reduced 849.65, 772.21 and 693.49 kg/ha in T3 , T2 and T1 respectively; and it was significantly different (P < 0.05) (Table 1). The net production in T4 was highest (Zymetin + Super PS) which was 35% more than T1 (control) and was significantly different. Production in all probiotics treated ponds was higher than the control ponds (Table 1). 3.2. Water quality parameters In the present study, important water quality parameters, like pH, temperature, dissolved oxygen (DO), alkalinity, hardness, ammonia, nitrite, salinity were monitored (Table 2). All the water quality parameters were within the acceptable range for freshwater prawn culture.
Where Wt is the final body weight and W0 the initial body weight of prawn (in grams).
60 50
Where t is the duration of the growth interval
40
Specific growth rate (SGR) = (ln(Wt)−ln(W0 ))/tX100 Where t is the duration of the experiment in days, Wt is the final average individual body weight and W0 the initial average individual body weight (in% BW/day)
Weight (g)
Daily weight gain (DWG) (g/day) = (Wt−W0 )/t
30 20 10 0
Feed conversion ratio (FCR) = Total amount of feed given (g)/Weight gain (g)
Without probiocs (T1) Zymen (T2) Super PS (T3) Zymen+Super PS (T4)
0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240
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Days of culture Fig. 1. Average prawn weight (g) in four treatments from August 2011 to March 2012.
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Table 1 Effect of probiotics on mean final weight and daily weight gain under different probiotics treatments.
Initial weight (g) Final weight (g) Weight gain (g) DWG (g/day) SGR (%BW/day) Survival rate (%) FCR Gross production (kg/ha) Net production (kg/ha)
T1 (control)
T2
T3
T4
1.04 39.56 g ± 12.03a 38.52 ± 12.03a 0.160 ± 0.050a 1.50 ± 0.13a 87.81 ± 1.39a 1.90 ± 0.28a 693.49 ± 99.90a 675.23 ± 100.19a
1.04 43.35 g ± 14.91b 42.31 ± 14.91b 0.176 ± 0.062b 1.53 ± 0.13b 89.10 ± 0.73a 1.69 ± 0.20ab 772.21 ± 86.56ab 753 ± 86.63ab
1.04 48.07 g ± 16.73c 47.03 ± 16.73c 0.196 ± 0.069c 1.58 ± 0.13c 88.50 ± 2.36a 1.54 ± 0.17bc 849.65 ± 93.33bc 831.24 ± 93.56bc
1.04 51.63 g ± 15.58d 50.59 ± 15.58d 0.210 ± 0.064d 1.61 ± 0.11d 90.32 ± 3.47a 1.39 ± 0.08bc 933.02 ± 50.75cd 914.24 ± 50.03cd
Means in the same row with different superscripts are significantly different (Pb0.05).
Table 2 Different water quality parameters (mean ± SD) during the culture period. Water quality parameters
Treatments
Temperature (◦ C) pH Salinity (ppt) DO (mg/L) Alkalinity (CaCO3 mg/L) Hardness (CaCO3 mg/L) Total ammonia (mg/L) Total Nitrite nitrogen (mg/L)
T1 Mean ± SD
T2 Mean ± SD
T3 Mean ± SD
T4 Mean ± SD
27.41 ± 4.51 7.68 ± 0.19 0.87 ± 0.63 5.10 ± 0.68 125.22 ± 16.6 216.3 ± 46.8 1.07 ± 0.11 0.24 ± 0.1
27.22 ± 4.47 7.64 ± 0.27 0.97 ± 0.62 5.29 ± 0.63 138.76 ± 22.33 260.8 ± 44.2 0.34 ± 0.02 0.09 ± 0.07
27.47 ± 4.54 7.76 ± 0.28 1.06 ± 0.69 5.21 ± 0.57 123.57 ± 10.01 250.4 ± 52.8 0.29 ± 0.03 0.07 ± 0.06
27.34 ± 4.36 7.69 ± 0.31 1.01 ± 0.68 5.32 ± 0.53 143 ± 16.75 227 ± 48.45 0.18 ± 0.02 0.06 ± 0.02
3.3. Soil quality parameters In the present study, different soil quality parameters (pH, organic carbon, electrical conductivity, phosphorus, calcium, magnesium) were measured and found within the culturable range (Table 3).
4. Discussion With increasing demand of fish and for environment-friendly aquaculture, the use of probiotics in aquaculture is now considered alternative to antibiotics and chemicals to improve the quality and sustainability of aquaculture production. Since the first use of probiotics in aquaculture, a growing number of studies have demonstrated their ability to control potential pathogens and to increase the growth rates and welfare of farmed aquatic animals (Gatesoupe, 1991; Carnevali et al., 2004; Macey and Coyne, 2005; Wang et al., 2005; Wang and Xu, 2006). The water quality parameters in the experiment were within the culturable range as mentioned by different reports (New, 2002; Firkins and Holdich, 1993; Knowlton and Kirby, 1984; Stone and Thomforde, 2004). The total ammonia and nitrite level was lower in probiotics treated ponds than control and lowest level was found in T4 . This is because the beneficial bacteria used the dissolved organic material as their nutrient and for nitrification rapidly. Suhendra et al. (1997) found that routine use of commercial probiotics in a shrimp farm in West Java resulted in reduced organic matter accumulation, improved water quality and enhanced environmental conditions. Current study data showed the standard concentrations of TP (0.02-
2.67%.), organic carbon (OC) (1.067–1.32%), Ca (0.13–0.17%), Mg (0.08-0.09%) in sediment after the ponds treated with commercial probiotics which is strongly related to the study with Suhendra et al. (1997). During the culture period in the experiment, all the probiotics-supplemented diets resulted in an increase of final weight and weight gain showing that the addition of probiotics increased the growth performance of prawn. This is a very inspiring result for the farmers in prawn culture with probiotics as the size of the prawn is related with the market price and for the earnings of foreign exchange. This study supports the study of Saad et al. (2009) and Hossain et al. (2013) in which better result in growth was found in probiotics treated harvested shrimp and prawn than without probiotics treated shrimp. Gullian et al. (2004) demonstrated a significant growth increase in shrimp inoculated with Bacillus sp. Whilst El-Dakar and Goher (2004) found that enhanced growth was generally obtained in shrimp fed diets with B. subtilis inclusion. The survival rate of prawn in the present study was higher in probiotics treated ponds than control but no significant difference was found. But in some studies the survival rate of post larvae (PL) of prawn was significantly higher in probiotics treated prawns than control (Seenivasan et al., 2011; Hossain et al., 2013). The present study was not similar to those studies because of stocking juveniles in the ponds (before stocking, the PL was first nursed for 45 days). Rinisha et al. (2010) evaluated probiotics effects on the growth and survival of Macrobrachium rosenbergii postlarvae and after 90 days they observed 76% survivability where probiotics was introduced through feed, whereas 64% survivability observed in the control. So, the probiotics effect on survival of prawn was not prominent on juvenile like the PL in the previous studies. The
Table 3 Different soil quality parameters among the treatments. Treatment
pH (ranges)
Organic carbon (%)
Electrical conductivity (ds/m) (ranges)
P (meq/100 g)
Ca (ranges)
Mg (Ranges)
Without probiotics (T1 ) Zymetin (T2 ) Super PS (T3 ) Zymetin + Super PS (T4 )
6.9–7.7 6.7–7.6 6.7–7.6 7–7.6
1.1349 1.06704 1.14738 1.32054
2.3–6.84 5.2–6.15 5.7–6.46 5–6.91
2.67% 2.33% 3% 2.33%
0.15–0.17% 0.12–0.17% 0.15–0.20% 0.11–0.16%
0.08–0.10% 0.08% 0.07–0.10% 0.06–0.09%
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mean FCR value in the present study was lowest in T4 (1.39) which indicates proper utilization of feed due to the presence of beneficial bacteria of probiotics and the highest FCR (1.90) was found in T1 which differed significantly. The present study supported findings of Irianto and Austin (2002) where they revealed probiotics bacteria as a good candidate for improving the digestion of nutrients and growth of aquatic organisms. The probiotic bacteria have been shown to improve the digestive activity by producing digestive enzyme and enhanced digestion and increased absorption of food which ultimately improved feed utilization efficiency and low FCR (Lee and Lee, 1990; Gatesoupe, 1999; El-Dakar and Goher, 2004; Ziaei-Nejad et al., 2006). The gross and net production was higher in probiotics treated ponds than control and T4 showed the highest production. The per hectare total production was 1.35 times higher in T4 than T1 which supports the study of Hossain et al. (2013) in which per hectare total production of P. monodon was 1.72 times better in probiotics treated ponds than control. The highest production in T4 might cause of faster growth rate, higher final weight, higher survival rate, better culture friendly soil and water quality parameters, better digestion and assimilation of feed due to functioning of highest number of beneficial bacteria. In these circumstances, to increase our prawn production in comparing with other developing countries, application of probiotics is essential. A few investment cost for probiotics can give high return in respect of traditional culture. 5. Conclusion It can be generally concluded from the present study that probiotics had significant effect on growth and production of prawns. The combined application of soil and feed probiotics (Zymetin + Super PS) showed best growth performance which was significantly higher. The findings of this study could be applied to the farmer’s level to increase the production of prawn. Competing interest The authors declare that they have no competing interest Authors’ contribution AKG: data analysis using Microsoft Office Excel, drafting and revision of the research article. JB: data collection, in situ measurement, initial set up of the experiment: MAKA: data collection, in situ measurement. AFMH: Field monitoring. MSI: Data collection, soil quality measurement. KAH: Research concept and design, field monitoring, data interpretation. Acknowledgement The research was conducted with the financial support of Bangladesh Academy of Sciences-United States Department of Agriculture Programme in Agricultural and Life Science. The authors would like to acknowledge for their kind support and sincere cooperation during the research period. References AOAC (Association of Official Analytical Chemists), 1980. In: Horwitz, W. (Ed.), Official Methods of Analysis. 13th edition. Association of Official Analytical Chemists, Washington DC, p. 988. Ahmed, N., Demaine, H., Muir, J.F., 2008a. Freshwater prawn farming in Bangladesh: history, present status and future prospects. Aqua. Res. 39 (8), 806–819. Balcázar, J.L., De Blas, I., Ruiz-Zarzuela, I., Cunningham, D., Vendrell, D., Múzquiz, J.L., 2006. The role of probiotics in aquaculture. Vet. Microbiol. 114 (3), 173–186.
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