Abstract: Background and Objective: The application of probiotics, prebiotics and synbiotics is commonly applied in juvenile and adult shrimp administered through artificial feed, while in larvae, it has still been limited. This study aimed to evaluate growth performance, the total intestinal bacteria, the activity of enzymes and the survival rate of Pacific white shrimp (Litopenaeus vannamei) administered Pseudoalteromonas piscicida 1Ub, mannan-oligosaccharides and synbiotic (the combination of P. piscicida 1Ub and mannan-oligosaccharides) through bio-encapsulation of Artemia sp. Materials and Methods: Bio-encapsulation of Artemia sp. was done by adding P. piscicida 1Ub 106 CFU mL–1, mannan-oligosaccharides 12 mg L–1 and synbiotic (P. piscicida 1Ub 106 CFU mL–1 and mannan-oligosaccharides 12 mg L–1) to the rearing medium of Artemia sp., for 4 h. The administration of the enriched Artemia sp. to the shrimp larvae was done from mysis 3 to Post Larvae (PL) 12. The body length and the body weight of Pacific white shrimp larvae were observed at the beginning and the end of the study, while RNA/DNA ratio, the activity of enzymes, survival rate and total bacteria of shrimp larvae were analyzed at the end of the study. Results: The results showed that daily growth rate, absolute length, RNA/DNA ratio, the activity of enzymes, survival rate and total bacteria of shrimp larvae administered probiotic, prebiotic and synbiotic were higher (p<0.05) than the control. The synbiotic treatment gave the best results in daily growth rate (24.39±0.31%), absolute length (13.00±0.50 mm), RNA/DNA ratio (0.6369±0.0094 μg mL–1), the activity of enzymes (protease 0.033±0.0007, lipase 0.047±0.0010, amylase 0.853±0.008, mannanase 0.148±0.004 U mL–1 min–1), survival rate (92.67±1.26%) and total bacteria (6.7×107 CFU larvae–1). Conclusion: The administration of P. piscicida 1Ub, mannan-oligosaccharides and synbiotic through bio-encapsulation of Artemia sp., effectively improved the growth performance of Pacific white shrimp larvae with the best results demonstrated by the synbiotic treatment.
INTRODUCTION
Pacific white shrimp (Litopenaeus vannamei) is one of major export commodities from Indonesia from the fishery sector. Indonesia is the 3rd largest shrimp exporter in the world after India and Equador, with an export volume of 181,351 t1. The development of Pacific white shrimp production must be supported by the sustainable supply of a high quality shrimp larvae. The high quality larvae would result in a good growth and a high survival rate. The application of probiotics, prebiotics and synbiotics is an alternative that could be used to increase the growth and the survival rate of the shrimp.
A probiotic is a beneficial microbe for the cultivated organism, because it could modify the microbial community, improve the nutritional value, improve the host’s response to disease, improve the environmental quality2 and could improve the immune response3. The results of previous studies have proven the probiotic’s success in improving the shrimp’s growth, survival rate, immune response and resistance4-7. In this study, the probiotic Pseudoalteromonas piscicida 1Ub which was proven to be able to inhibit the growth of the pathogenic bacteria Vibrio harveyi and increase the giant tiger prawn larvae’s survival rate8.
The role of probiotic bacteria could be improved through the application of a prebiotic, a feed material that cannot be digested and is beneficial for the host because it stimulates the growth and the activity of certain bacteria in the intestines and thus improving the host’s health9. A number of studies have demonstrated that prebiotics can improve growth, survival rate, feed digestibility, feed efficiency, the composition of microflora in the intestines, inhibit the growth of pathogens and improve the shrimp’s immune system10-12. The prebiotic used in this study was mannan-oligosaccharides (MOS) which has been demonstrated to improve the growth and the survival rate of Pacific white shrimp juvenile.
If a probiotic and a prebiotic are combined in a single product (synbiotic), the benefits will increase13. The use of synbiotics have been demonstrated to improve growth, survival rate, immune response and resistance in aquatic organisms, including in the European lobster larvae, Homarus gammarus L.14,15 and Pacific white shrimp, L. vannamei 16-18.
In addition, the application of probiotics, prebiotics and synbiotics is commonly applied in the juvenile and the adult shrimp administered through artificial feed10,16-18, while in larvae, it has been limited to the larvae of H. gammarus L.14,15 and L. vannamei larvae with the probiotic Vibrio alginolyticus SKT-b RfR and the prebiotic oligosaccharide derived from sweet potato var., sukuh extract19. The application of probiotics, prebiotics and synbiotics since the larval stadia is important to produce the high quality shrimp larvae with the high growth rate and resistance against certain diseases (specific pathogen resistance), so that when the shrimp larvae are stocked in grow out ponds, they already have the better growth performance and immune response to face attacks from the various pathogens found in the pond. In this study, the application of probiotic, prebiotic and synbiotic in Pacific white shrimp larvae was conducted through the enrichment of Artemia, the main natural feed for shrimp larvae, because it has the appropriate size for larvae, has a high nutritional value and is easy to be digested. This study aimed to evaluate total bacteria, the activity of enzymes and growth performance of Pacific white shrimp larvae administered probiotic P. piscicida 1Ub, prebiotic MOS and synbiotic (a combination between probiotic P. piscicida 1Ub and prebiotic MOS) through bio-encapsulation of Artemia sp.
MATERIALS AND METHODS
Probiotic and prebiotic preparation: The probiotic used was P. piscicida 1Ub, isolated from the nauplii of Pacific white shrimp8. The P. piscicida 1Ub bacterial isolate was marked with the antibiotic rifampicin (1Ub RfR) as a molecular marker. The P. piscicida 1Ub RfR cells were cultured in seawater complete (SWC) slant agar medium (0.5 g bacto peptone, 0.1 g yeast extract, 0.3 mL glycerol, 1.5 g bacto agar, 75 mL seawater and 25 mL distilled water) and were incubated at 29°C for 24 h. Then the bacterial cells were inoculated into SWC broth medium and were incubated in a water bath shaker (29°C, 140 rpm) for 18 h.
The prebiotic used was Bio-MOS (Alltech Inc., KY USA) which contained mannan-oligosaccharides (MOS) derived from the cell walls of Saccharomyces cerevisiae with a composition of 30% crude protein, 1.4% crude fat and 13% crude fiber.
Rearing medium and experimental animal preparation: The rearing medium for the Pacific white shrimp larvae were 12 aquariums (60×30×35 cm3), which were equipped with aeration equipment and heater. The aquariums were filled with 10 L disinfected seawater with a salinity of 30 ppt. The experimental animals were Pacific white shrimp larvae (mysis 3) and were stocked at a density of 200 individuals per aquarium4.
Enrichment of Artemia sp.: Artemia cysts (2 g L–1 seawater) were hatched in strongly aerated seawater (30 ppt), then it were harvested after 24 h. The enrichment of Artemia sp. was conducted on instar 2 stadia of Artemia sp. (approximately 4 h after being harvested) using a plastic container filled with 1 L seawater (30 ppt). The density of Artemia sp. in each container was 100 individuals mL–1 7. The enrichment was conducted by adding the probiotic P. piscicida 1Ub RfR at a concentration of 106 CFU mL–1, 12 mg L–1 prebiotic MOS and a combination between 106 CFU mL–1 probiotic P. piscicida 1Ub RfR and 12 mg L–1 MOS (synbiotic) into each enrichment container of Artemia sp. The enrichment was conducted for 4 h20. And then the Artemia were harvested using a plankton net and were washed with the disinfected seawater, then those were fed to the Pacific white shrimp larvae at a dose of 8-10 individuals per larvae21 and the rest were stored in a refrigerator at 4°C for further use on the same day. During the rearing period, 5-10% of the rearing medium water was siphoned and was replaced every 3 days.
Experimental design: Artemia were fed to the larvae 5 times a day at 06.00 am, 10.00 am, 02.00 pm, 06.00 pm and 10.00 pm. This study was conducted through the completely randomized design with four treatments and three repeats. The treatments were (1) Pacific white shrimp larvae fed Artemia without any probiotic, prebiotic and synbiotic enrichment (control), (2) Pacific white shrimp larvae fed Artemia enriched with probiotic P. piscicida 1Ub RfR at a concentration of 106 CFU mL–1 (probiotic), (3) Pacific white shrimp larvae fed Artemia enriched with prebiotic MOS 12 mg L–1 (prebiotic) and (4) Pacific white shrimp larvae fed Artemia enriched with the combination between the probiotic P. piscicida 1Ub RfR at a concentration of 106 CFU mL–1 and 12 mg L–1 the prebiotic MOS (synbiotic).
Survival rate and growth of Pacific white shrimp larvae: The Pacific white shrimp larvae’s survival rate was calculated at the end of the study using the formula according to Dehaghani et al.22. The growth parameters observed were Daily Growth Rate (DGR) and absolute length (L). The DGR was calculated using the formula according to Nurhayati et al.18 and the absolute length growth (L) was calculated using the formula according to Dehaghani et al.22.
To support the growth parameter data, the calculation of RNA/DNA ratio of the shrimp larvae was done using the gene quant calculator. The extraction of the shrimp larvae RNA and DNA was conducted by placing larvae (n = 3 individuals) from each treatment into 1.5 mL tubes which had been filled with 200 μL isogen on ice, then the sample was ground until it was completely macerated. After all tissues had been macerated, 400 μL isogen was added, then the mixture was stored at a room temperature for 5 min for lysis, then 200 μL chloroform (CHCl3) was added, the sample was homogenized using a vortex for 15 sec and was stored at a room temperature for 2-3 min. The lysis product was centrifuged at 10,000 rpm for 15 min until three layers are formed: The supernatant (clear) on the top layer was chloroform+RNA, the second layer was protein and the pellet was phenol+DNA (blue). The supernatant (chloroform+RNA) and the pellet (phenol+DNA) were collected, then each layer was moved to a new tube which had been filled with 400 μL isopropanol, it was homogenized and stored at a room temperature for 5-10 min, then each layer was centrifuged at 4°C and 10,000 rpm for 15 min. The supernatant was discarded and the pellet remaining at the bottom of the tube was added with 1 mL cold ethanol 70%. Then this was centrifuged again at 4°C and 10,000 rpm for 15 min, the supernatant was discarded and the pellet was air dried. After dried, the pellet was added with 50 μL diethylpyrocarbonate (DEPC), then it was homogenized with a vortex and was stored on ice. The concentrations of the genome’s RNA and DNA were then measured using the gene quant. The concentration of RNA and DNA was the result of the gene quant reading multiplied with dilution factor, while RNA/DNA ratio (μg mL–1) was calculated by dividing the RNA concentration by the genome’s DNA concentration23.
Bacterial population: Total bacterial count and total probiotic P. piscicida 1Ub RfR in the shrimp larvae were enumerated through the spread plate method24. The shrimp larvae from each treatment at a weight of 0.1 g (5-6 shrimps) were macerated and were homogenized in 0.9 mL phosphate buffer saline (0.8% NaCl, 0.15% K2HPO4, 0.02% Na2HPO4 and 0.02% KCl). The serial dilution was performed 10 times, the bacterial suspension from the serial dilution was then spread onto the SWC plate agar medium for the enumeration of total bacteria and onto SWC plate agar+rifampicin (50 μg mL–1) medium for the enumeration of total probiotic P. piscicida 1Ub RfR.
Activity of enzymes: The activity of Pacific white shrimp larvae’s enzymes analyzed, included the activity of protease, lipase, amylase and mannanase. The shrimps used for the analysis were 25-30 shrimps sample (0.5 g sample–1). The procedure for analyzing the activity of protease and amylase followed the method constructed by Bergmeyer et al.25, the procedure for analyzing the activity of lipase followed the method constructed by Borlongan26, while the procedure for analyzing the activity of mannanase followed the method constructed by Hossain et al.27.
Statistical analysis: The data of the daily growth rate, the absolute length, the RNA/DNA ratio, the bacterial population, the activity of enzymes and the survival rate of Pacific white shrimp were analyzed through ANOVA. Differences among treatments were analyzed through the Duncan’s test at a confidence interval 95%. Those statistical analysis were operated by SPSS 16 program.
RESULTS
Survival rate and growth performance: The application of Artemia sp., enriched with probiotic, prebiotic and synbiotic on Pacific white shrimp larvae (mysis 3 to PL 12) had a significant effect (p<0.05) on the larvae’s survival rate (Fig. 1). The highest survival rate (p<0.05) was found in the synbiotic treatment (92.67±1.26%), followed by the probiotic treatment (88.67±1.76%), the prebiotic treatment (87.83±1.76%) and the lowest was found in the control treatment (84.17±1.04%).
The application of probiotic, prebiotic and synbiotic through Artemia sp. in Pacific white shrimp larvae had a significant effect (p<0.05) on the DGR and absolute length, but only the application of probiotic and synbiotic had a significant effect (p<0.05) on the RNA/DNA ratio (Table 1).
| Fig. 1: | Survival rate of Pacific white shrimp larvae given the probiotic Pseudoalteromonas piscicida 1Ub RfR, prebiotic MOS and synbiotic (combination between Pseudoalteromonas piscicida 1Ub RfR and MOS) through the enrichment of Artemia sp., from mysis 3 to PL 12. Different letters within the bars indicate significant different results at p<0.05 |
| Table 1: | Growth performance of Pacific white shrimp larvae given the probiotic, prebiotic and synbiotic through the enrichment of Artemia sp. |
| DGR: Daily growth rate. Different superscript letters in the same column indicate significant different results at p<0.05 | |
The highest DGR value (p<0.05) was found in the synbiotic treatment (24.39±0.31%), followed by the probiotic treatment (21.96±0.24%), prebiotic treatment (21.30±0.39%) and the lowest was in the control treatment (18.54±0.29%). Similar results were found in the absolute length values, the highest value (p<0.05) was found in the synbiotic treatment (13.00±0.50 mm), followed by the probiotic treatment (11.33±0.29 mm) and the prebiotic treatment (10.83±0.58 mm), while the lowest was in the control treatment (8.83±0.76 mm). The RNA/DNA ratio for the Pacific white shrimp larvae during the rearing period ranged between 0.2834-0.6369 μg mL–1. The highest RNA/DNA ratio (p<0.05) was demonstrated by the synbiotic treatment (0.6369±0.0094 μg mL–1), followed by the probiotic treatment (0.4207±0.0459 μg mL–1), while that in the prebiotic treatment (0.3201±0.0349 μg mL–1) was not significantly different (p<0.05) from the control treatment (0.2834±0.0269 μg mL–1).
Bacterial population: The bacterial population values inside the Pacific white shrimp larvae’s bodies in the treatment with the application of the probiotic and synbiotic were higher (p<0.05) than in those treated with the prebiotic and the control. The total bacterial count in the synbiotic treatment was 6.7×107 CFU larvae–1, followed by the probiotic treatment (3.87×107 CFU larvae–1), the prebiotic treatment (3.74×105 CFU larvae–1) and the control treatment (2.42×105 CFU larvae–1). In addition, the probiotic P. piscicida 1Ub RfR was able to survive and colonize in the Pacific white shrimp larvae. The population of the probiotic P. piscicida 1Ub RfR in the bodies of the Pacific white shrimp larvae fed the probiotic treatment was 5.84×105 CFU larvae–1 and that given the synbiotic treatment was 4.75×106 CFU larvae–1 (Table 2).
The results of the sequencing of the dominant bacteria in the bodies of the Pacific white shrimp larvae using the primer 16S rRNA revealed five species of bacteria: Staphylococcus pasteuri, Tenacibaculum mesophilum, P. piscicida, V. alginolyticus and Staphylococcus warneri. The S. pasteuri was predominantly found in the bodies of the shrimp larvae given the probiotic P. piscicida 1Ub RfR and the synbiotic.
| Table 2: | Total bacteria and total Pseudoalteromonas piscicida 1Ub RfR in the bodies of Pacific white shrimp larvae given the probiotic, prebiotic and synbiotic through the enrichment of Artemia sp. |
| Different superscript letters in the same column indicate significant different results at p<0.05 | |
| Table 3: | Activity of enzymes in Pacific white shrimp larvae given the probiotic, prebiotic and synbiotic through the enrichment of Artemia sp. |
| Different superscript letters in the same column indicate significant different results at p<0.05 | |
The T. mesophilum was predominantly found in the bodies of the shrimp larvae given the prebiotic MOS, the probiotic P. piscicida 1Ub RfR and the control. The P. piscicida was predominantly found in the shrimp larvae given the probiotic P. piscicida 1Ub RfR and the synbiotic. The V. alginolyticus and S. warneri were predominantly found in the shrimp larvae given the probiotic P. piscicida 1Ub RfR.
Activity of enzymes: The activity of enzymes in Pacific white shrimp larvae given the probiotic, prebiotic and synbiotic through bio-encapsulation of Artemia sp., demonstrated varied values among treatments (Table 3). The protease activities in the synbiotic and probiotic treatment were higher (p<0.05) than those in the prebiotic treatment and the control. The lipase and amylase activities in the synbiotic treatment were higher (p<0.05) than the other treatments and the control. Similar results were found in the values of mannanase activity; the synbiotic treatment had a higher value (p<0.05) than the control, but the mannanase activity in the synbiotic treatment was not significantly different (p>0.05) from the probiotic and prebiotic treatment.
DISCUSSION
The growth rate and the survival rate of Pacific white shrimp larvae given the probiotic, prebiotic and synbiotic through bio-encapsulation of Artemia sp. were higher than the control. This is estimated to be related to the ability of probiotic P. piscicida 1Ub RfR and prebiotic MOS in modulating the growth and the activity of beneficial microflora in the digestive tract of Pacific white shrimp larvae. They could help increase the feed digestibility, affecting the larval growth and the survival rate. This was demonstrated by the high total bacteria and total P. piscicida 1Ub RfR in the bodies of the Pacific white shrimp larvae given the probiotic, prebiotic and synbiotic through bio-encapsulation of Artemia sp. Endogenous bacteria and the probiotic bacteria P. piscicida 1Ub RfR in the bodies of the Pacific white shrimp larvae contributed in producing the enzymes needed for the digestion, so the feed digestibility, growth performance and survival rate of Pacific white shrimp larvae given the treatments were better than those not given the treatment (control).
Probiotics are live microbial agents which can influence the survival rate, growth and health status of aquatic animals. De Preter et al.28 explained that the survival rate, colonization and beneficial effects of exogenous probiotics can be increased and improved by the simultaneous addition of prebiotics known as synbiotics.
The application of the synbiotic (the combination between P. piscicida 1Ub RfR and MOS) through the bio-encapsulation of Artemia sp. in Pacific white shrimp larvae (mysis 3 to PL 12) resulted in the best DGR, absolute length and RNA/DNA ratio among other treatments and the control. These results were in line with the results shown by the micro-encapsulated synbiotic, which was the combination between Bacillus NP5 RfR and mannan-oligosaccharides in L. vannamei29, the combination between the probiotic V. alginolyticus SKT-b RfR and the oligosaccharide extracted from sweet potato var., sukuh in L. vannamei larvae19, the combination between the probiotic V. alginolyticus SKT-b RfR and the oligosaccharide extracted from sweet potato var., sukuh in L. vannamei18 and the combination between Bacillus spp. and mannan-oligosaccharides in the European lobster larvae15.
The application of the synbiotic (the combination between P. piscicida 1Ub RfR and MOS) through the bio-encapsulation of Artemia sp. in Pacific white shrimp larvae also resulted in the best Survival Rate (SR) among treatments and the control. Some studies have demonstrated that synbiotics could improve the survival rate of aquatic organisms, e.g., the application of the synbiotic (the combination between the probiotic V. alginolyticus SKT-b and oligosaccharide extracted from sweet potato var., sukuh) through the enrichment of Artemia sp., could improve the survival rate of Pacific white shrimp larvae19 and the addition of the synbiotic (the combination between the probiotic Enterococcus faecium and the prebiotic fructooligosaccharide) in feed could improve the survival rate of Carassius auratus gibelio juvenile30. It was caused by an improvement on immunity level of Pacific white shrimp larvae induced by the application of synbiotic. An increase in immunity level will lead to a better protection of the host against a pathogenic infection and environmental stress. Talas and Gulhan31 stated that an increase in immunity level could protect shrimp against the pathogenic infection (white spot disease).
Based on the data of total bacteria and total probiotic P. piscicida 1Ub RfR count in the bodies of the Pacific white shrimp larvae, it could be seen that the application of the synbiotic resulted in a higher total bacterial count and total probiotic P. piscicida 1Ub RfR count than the other treatments and the control. The high total bacterial count and total probiotic P. piscicida 1Ub RfR count in the bodies of the Pacific white shrimp larvae given the synbiotic treatment was possible due to the probiotic P. piscicida 1Ub RfR ability to survive and colonize in the intestinal tract of Pacific white shrimp larvae, because the probiotic P. piscicida 1Ub RfR which was used in this study was bacteria isolated from the nauplii of Pacific white shrimp8 and the ability to utilize mannan-oligosaccharides was caused by the mannanase enzyme produced by the probiotic P. piscicida 1Ub RfR bacteria. Similar results were reported by Widanarni et al.7 who stated that the administration of various doses of the probiotic V. alginolyticus SKT-b through Artemia could increase the total Vibrio count in the bodies of the giant tiger prawn post-larvae and the application of the micro-encapsulated synbiotic (Bacillus NP5 RfR and oligosaccharide) could increase the bacterial population in the intestines of Pacific white shrimp up to log 9 CFU g–1 32.
The results of the sequencing of the dominant bacteria in the bodies of the Pacific white shrimp larvae using the primer 16S rRNA33 revealed that there were five bacterial species: S. pasteuri, T. mesophilum, P. piscicida, V. alginolyticus and S. warneri. These five species of bacteria are commonly found in organisms that live in seawater. The T. mesophilum was isolated from sea sponges and green algae34. The S. pasteuri and S. warneri have potential as probiotics that could influence growth and survival rate in L. vannamei 35, while P. piscicida and V. alginolyticus have been known and applied as probiotics to improve the growth and survival rate of giant tiger prawn and Pacific white shrimp8,18.
The presence of the probiotic bacteria accumulated in Artemia sp., a natural feed could increase the exogenous enzyme production in the bodies of Pacific white shrimp larvae. The probiotic P. piscicida 1Ub RfR can produce a number of exogenous enzymes, including protease, lipase, amylase and mannanase. The probiotic P. piscicida 1Ub RfR ability to produce a number of exogenous enzymes allows the activity of enzymes in Pacific white shrimp larvae that had been given the treatments to be higher than the control. According to Wang et al.36, probiotics are able to produce a number of exogenous enzymes for the digestion, including amylase, protease, lipase and cellulase. Tzuc et al.37 reported that Pseudoalteromonas sp., isolated from the stomach, intestines and hepatopancreas of Pacific white shrimp could produce amylase, lipase and chitinase. Protease is able to hydrolyze protein into peptides and bacteria produce peptidase that breaks down peptides into amino acids that are needed for the metabolism. Amylase can hydrolyze amylum and help the digestion in organisms. Lipase is an enzyme that basically plays a role in the hydrolysis of fats, monoglycerides, diglycerides and triglycerides to produce free fatty acids and glycerol38. The main function of lipase is to digest fats, maintain the function of the gall bladder, maintain the balance of electrolytes in the body, maintain an optimum cell permeability, thus allowing nutrients that are needed to enter the cell to facilitate the metabolism.
The digestion in the stomach and intestines are effective because of the high activity of enzymes. The P. piscicida 1Ub RfR supplemented to Artemia sp. has a function as a supplier of exogenous enzymes and helps the process of the feed breaking down into micro molecules that can be easily absorbed, so that the digestive system of Pacific white shrimp larvae becomes more effective in energy expenditure for the digestive process. The energy that would have been expended is reduced and the energy could be used for the growth instead. The feed materials that have been broken down into simple molecules are then absorbed by the intestines, enter the blood flow are distributed to tissues throughout the body and enter cells39. Inside the cells, glucose will be oxidized to produce energy, while protein and fat are retained inside the body tissues, then those are used for improving the growth performance. Liu et al.40 stated that the increase in the growth of aquatic animals given probiotic could be linked to the increased digestive activities by enzymatic activity and vitamin synthesis which could improve digestibility and weight gain.
Supplementation of the prebiotic mannan-oligosaccharides in Artemia sp., indirectly adds the amount of exogenous enzymes in the bodies of Pacific white shrimp larvae, provides additional energy and nutrition for the natural bacteria in the bodies of the larvae to survive and produce more exogenous enzymes, making the digestion in Pacific white shrimp larvae to be more effective. This can be seen from the high total bacterial count and the activity of enzymes in Pacific white shrimp larvae given the synbiotic treatment. The prebiotic mannan-oligosaccharides used in this study was extracted from the cell walls of the yeast S. cerevisiae with a composition of 30% crude protein, 1.4% crude fat and 13% crude fiber.
Application of the synbiotic (the combination between P. piscicida 1Ub RfR and mannan-oligosaccharides) through the enrichment of Artemia sp., would create a synergy inside the Pacific white shrimp larvae’s body. This can be seen from the total bacterial count and the activity of enzymes (protease, lipase and amylase) of Pacific white shrimp larvae fed the synbiotic which were higher than those given the probiotic or prebiotic in single administration. The prebiotic mannan-oligosaccharides administered could be directly utilized by the probiotic P. piscicida 1Ub RfR or could be utilized by the natural bacteria found in the bodies of Pacific white shrimp larvae. The probiotic P. piscicida 1Ub RfR ability in utilizing mannan-oligosaccharides is closely related to the probiotic P. piscicida 1Ub RfR ability to produce mannanase. This allows the administration of the synbiotic through bio-encapsulation of Artemia sp., resulting in the maximum activity of enzymes and the growth performance in Pacific white shrimp larvae in this study. Similar results were also reported by Zokaeifar et al.6, the administration of the probiotic Bacillus subtilis could increase digestive enzymes in Pacific white shrimp and the administration of the synbiotic (the combination between the probiotic Enterococcus faecium and the prebiotic fructooligosaccharide) could improve the activity of digestive enzymes in the carp juvenile22.
CONCLUSION
The application of the probiotic P. piscicida 1Ub RfR, the prebiotic mannan-oligosaccharides and the combination between the probiotic P. piscicida 1Ub RfR and the prebiotic mannan-oligosaccharides (synbiotic) through bio-encapsulation of Artemia sp., could improve the growth performance, total bacteria, the activity of enzymes and survival rate of Pacific white shrimp larvae with the best results demonstrated by the synbiotic treatment.
ACKNOWLEDGMENTS
Thanks to General Directorate of Higher Education of Indonesia that has provided the doctoral program scholarship for the first author. Thanks also goes to Directorate of Research and Community Service, General Directorate of Strengthening Research and Development, Ministry of Research, Technology and Higher Education of Indonesia that has financially funded this study with a contract number of 079/SP2H/LT/DRPM/II/2016 and to Fauziah Asril Darussamin, DVM for proofreading this manuscript.