Juvenile Pacific white shrimp "Litopenaeus vannamei". Growth performance with lactic acid bacteria "Pediococcus acidilactici"

TABLE OF CONTENTS

Abstract

Acknowledgemen

Table of Contents

List of Figures

List of Tables

List of Appendices

CHAPTER 1. INTRODUCTION
1.1. Background and Rationale
1.2. Significance of the Study
1.3. Objectives of the Study

CHAPTER 2. REVIEW OF RELATED LITERATURE
2.1. Biology of P. vannamei
2.2. Pacific White Shrimp Production
2.3. History and the Definition of Probiotics
2.4. Application of Probiotics in Aquaculture
2.4.1. Promoter of Growth Food utilization and Survival
2.4.2 Improvement of Water Quality
2.5. Selection of Probiotics
2.5.1 Modes of Probiotic Action
2.5.2. Probiotic Organism
2.6. Biology of Pediococcus acidilactici.
2.7. Application of Pediococcus acidilactici as Probiotic
2.7.1. Livestock
2.7.2. Aquaculture
2.7.3. Inclusion in Feeds

CHAPTER 3. METHODOLOGY..
3.1. Experimental Animals and Acclimatization
3.2. Experimental Design
3.3. Sampling and W ater Quality Parameters
3.4. Experimental Feed Preparation
3.5. Feeding Trial
3.6. Proximate Composition of Experimental Diet and Shrimp Carcass
3.6.1. Moisture
3.6.2. Crude Protein
Digestion.
Distillation and Titration
3.6.3. Crude Lipid
3.6.4. Ash
3.7. Statistical analysis

CHAPTER 4. RESULTS
4.1. Growth Performance
4.1.1 Average Body Weight
4.1.2 Percent Weight Gain
4.1.3 Specific Growth Rate
4.1.4 Feed Conversion Ratio
4.1.5 Protein Efficiency Ratio
4.1.6 Protein Productive Value
4.1.7 Survival Rate
4.2. Proximate Analyses of Experimental Diet and Shrimp Carcass

CHAPTER 5. DISCUSSION
5.1. Growth Performance
5.2. Proximate Analyses of Shrimp Carcass

CHAPTER 6. CONCLUSION AND RECOMMENDATION

LITERATURE CITED

APPENDICES

ABSTRACT

Juvenile Pacific white shrimp Litopenaeus vannamei were fed diets incorporated with lactic acid bacteria probiotic Pediococcus acidilactici at 0%, 2%, 4%, 6% and 10% concentrations, to determine its effect on shrimp growth and survival rates for 75 days. The five treatments were triplicated. Diets of about 6-10% of the shrimp's total body weight was divide into four times daily feeding. Fifteen glass aquaria filled with 35 L dechlorinated filtered seawater were each added with 10 pcs of 0.5 g juvenile L. vannamei. Sampling of the five representative shrimp samples per replicate tank were done every 15 days from the start of the feeding trial. Water quality parameters were checked twice a week. Proximate composition of the shrimp carcass was also determined. There is a significant difference in the PPV of shrimps fed with 10% probiotics as compared with the other treatments including the control. Significant differences were also observed in the lipid and protein values of shrimp carcass fed with probiotics-supplemented diets. However, no significant differences were showed in the growth performance parameters and survival rate of shrimps among the different treatments at 75 DOC.

Keywords: Litopenaeus vannamei, Probiotic-supplemented diets, Pediococcus acidilactici, Growth Performance, Survival Rate, Proximate Analyses

LIST OF FIGURES

4.1 Average body weight of shrimps fed with different 17 concentrations of probiotics at 75DOC

4.2 Percent weight gain of shrimps fed with different 18 concentrations of probiotics at 75 DOC

4.3 Specific growth rate of shrimps fed with different 19 concentrations of probiotics at 75 DOC

4.4 Feed conversion ratio of shrimps fed with different 19 concentrations of probiotics at 75 DOC

4.5 Protein efficiency ratio of shrimps fed with different 20 concentrations of probiotics at 75 DOC

4.6 Protein productive value of shrimps fed with different 21 concentrations of probiotics at 75 DOC

4.7 Percent survival of shrimps fed with different 21 concentrations of probiotics at 75 DOC

LIST OF TABLES

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APPENDICES

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CHAPTER 1

INTRODUCTION

Aquaculture is one of the fastest growing industry in the world. World aquaculture has grown tremendously during the last fifty years from a production of less than a million tonne in the early 1950s to 59.4 million tonnes by 2004 (Pandiyan et al., 2013). Recently, it has been suffering great losses due to infectious diseases. To prevent this, the use of antimicrobial drugs, pesticides and disinfectants were developed. However, there was a risk associated with the transmission of resistant bacteria from aquaculture environments to humans, and risk associated with the introduction in the human environment of nonpathogenic bacteria, containing antimicrobial resistant genes, and the subsequent transfer of such genes to human pathogens (Pandiyan et al., 2013). The research on the use of probiotics thus increased in demand for a more environmental-friendly sustainable aquaculture. The use of probiotics in aquaculture was mainly developed to improve the overall gastrointestinal microbiota of aquatic organisms, though studies also showed that microbial intervention can play an important role in aquaculture production, and effective probiotic treatments may provide broad spectrum and greater nonspecific disease protection (Pandiyan et al., 2013).

Probiotics from the Greek word pro and bios, meaning “for life”, the term probiotics is defined as microbial cells that are administered via the diet or rearing water which improves health disease resistance, growth performance, feed utilization, stress response, general vigor, carcass and flesh quality and reduced malformations (Merrified et al., 2010). Probiotics are live microbial food supplements that contain live microorganisms such as Bacillus, Lactobacillus and Pediococcus. Probiotics are known as biocontrol when the treatment is antagonistic to pathogens or bioremediation when water quality is improved (Gatesoupe, 1999). This is why some probiotics are used to treat the rearing medium rather than to supplement the diet. Probiotics in the diets can protect their host from pathogens by producing metabolites that inhibit the colonization or growth of other microorganisms or by competing with them for resources such as nutrients or space (Vine et. al. 2004). In addition, probiotics help in maintaining the balance of intestinal microbiota, produce bacteriocin or inhibitory compounds to combat pathogenic bacteria, complete for adhesion sites and stimulate immune functions which can be beneficial in the production of shrimps (Wongsasak et al., 2015). Probiotics may also improve water quality, nutrient absorption, immune system, and survival and growth rates of hosts (Verchuere et al., 2000). Lactic acid probiotics are beneficial to shrimps by improving the balance of the midgut microbiota, survival, resistance to infection, immunostimulation and digestibility (Andreatta et al., 2016). Prospective probiotics should show antagonism toward the pathogens, an ability to survive and colonize the intestine, and a capacity to increase an immune response in the host.

In this study, the use of lactic acid bacteria Pediococcus acidilactici was tested as probiotics incorporated in the feeds of juvenile white shrimp (Litopenaeus vannamei). Pediococcus acidilactici is a facultative anaerobe bacteria that displays antagonism against pathogens using lactic acid and secretion of bacteria-killing substances. It can also enhance immune responses towards infectious coccidioidal diseases. P. acidilactici was also observed to alleviate vertebral column compression syndrome, elevate blood leucocyte levels, improve intestinal microvilli morphology and increase enterocyte endocytic activity in rainbow trout Oncorhynchus mykiss (Aubin et al. 2005) and improve weight gain, survival, feed conversion ratio and haemolymph total antioxidant status in shrimp (Castex et al., 2008 and 2009).

Studies regarding the improvement of the beneficial effects of probiotics incorporated in shrimp diets are interesting fields to investigate. In fact, an increasing number of reviews on probiotics in aquaculture, particularly in the scope of applications in shrimp larviculture and mechanisms of action enlightens the importance of the development of aquaculture practices (Ninawe and Selvin, 2009). The results of this study will therefore provide information on the efficiency and effectiveness of the different concentrations of lactic acid bacteria, Pediococcus acidilactici, as probiotics incorporated in the diets of juvenile white shrimp (Litopenaeus vannamei). This could be applied in shrimp culture studies and production to improve the weight gain, survival and feed conversion ratio of juvenile L. vannamei.

The study generally aims to evaluate the probiotic effects of the different concentrations of Pediococcus acidilactici incorporated in the feeds of juvenile white shrimp (Litopenaeus vannamei), in terms of growth performance and survival. Specifically, the study is designed to determine which of the probiotic concentrations fed to the shrimps is the optimum concentration, as well as to conduct carcass analyses (i.e., moisture, lipid, protein and ash) to evaluate the nutritive potential of P.acidilactici as probiotics in the diets of juvenile L. vannamei.

CHAPTER 2

REVIEW OF RELATED LITERATURE

2.1. Biology of P. vannamei

Penaeus vannamei are usually located in the Eastern Pacific coast from Sonora, Mexico in the North, through Central and South America as far south as Tumbes in Peru, especially in tropical marine habitat. The adults live and reproduce in the open ocean, while post larvae migrate inshore to spend their juvenile, adolescent and sub­adult stages in coastal estuaries, lagoons or mangrove areas. The males mature from 20 g and females from 28 g onwards at the age of 6-7 months. The hatching happens 16 hrs after spawning and fertilization. The first larval stage (nauplii) swim sporadically, are diurnal and feed on their yolk reserves. The protozoea, mysis and early post larvae stages remain planktonic for a while, feed on phytoplankton and zooplankton, and swim with the tidal currents. About 5 days after molting into post larval stage (PL), they swim inshore and now feed on benthic detritus, worms, bivalves and crustaceans (Boone, 1931).

2.2. Pacific White Shrimp Production

Shrimp farming in the Philippines has developed rapidly due to the tremendous expansions in aquaculture being driven by its potentiality for hig9h profits and demand for high-value seafood products. In addition, the limited supplies from capture fisheries increase the demand for farmed shrimp. In 1992, the Philippines produced the largest shrimp catch of about 120, 000 metric tons making it as one of the leading shrimp producing countries (Vergel, 2017). As of 2013, there are 271 brackish water shrimp farms (3, 617.8 ha) registered at the Bureau of Fisheries and Aquatic Resources of the Philippines and 27% (909.4 ha) of the total shrimp farms are cultured with the Pacific white shrimp (Litopenaeus vannamei). According to the Philippine Statistics Authority in the year 2017, the harvests of white shrimps raised by 2.03 % in the first quarter. The regions of Western Visayas and Negros Island regions were recorded as the contributor to the increase in production of white shrimps.

2.3. History and the Definition of Probiotics

The word ‘probiotic’ comes from Greek language ‘pro-bios' which means ‘for life’. Metchnikoff in 1908 suggested the benefits of probiotic microorganisms on human health and hypothesized that Bulgarians have long, healthy lives because of consuming fermented milk products that have Lactobacillus spp. Therefore, these bacteria were said to have positively affected the gut microflora and decrease microbial toxic activity (Gismondo et al., 1999). In 1965, Lilley and Stillwell used the term ‘probiotic’ for the first time to distinguish microorganisms that promoted the growth of other organisms based on its effect on human health. According to Fuller (1989), probiotic is a live microbial supplement which positively affects the host’s health by improving the intestinal microbial balance. Similarly, Guarino et al. (1998) stated that probiotics can be defined as living microorganisms which upon ingestion in certain numbers exert health benefits beyond inherent basic nutrition. The probiotic perspective is preferred because it is a re-institution of the natural condition, which means fixing an irregularity rather than the addition of extraneous chemicals to the body or as in the case of antibiotics, hasten immunity and compromise subsequent therapy.

2.4. Application of Probiotics in Aquaculture

Due to the increasing demand in environment friendly aquaculture, the use of probiotics became widely accepted. Nowadays, commercially available probiotics has been introduced to fish, shellfish and molluscan farming as feed additives or is incorporated in pond water (Moriarty, 1998). The use of probiotics was considered as safe and effective in supporting the health of aquatic organisms. Initially, the interest in using probiotics focused on their use as growth promoter and in the improvement of animal health. Due to some development, however, new areas such as their effect on reproduction or stress tolerance have been found but scientific development is required.

2.4.1 Promoter of Growth Food utilization and Survival

Different conditions of aquaculture may cause microbial problems and stress regulating resulting in poor growth and mass mortality. Enhancement on growth and survival can be obtained when techniques for rectifying this problem in a safe and biological way are applied on the early stages of development. Probiotics are known to adhere into the intestinal mucosa of the organism to develop and exercise their several benefits. According to Balcazar et al. (2006), probiotic microorganisms are able to colonize the gastrointestinal tract where it can persist for a long time through possessing a multiplication rate that is higher than its expulsion rate.

Probiotics as growth promoters have been reported in many journals. This includes incorporating probiotic Streptococcus strain on the diet of Nile tilapia (Oreochromis niloticus), which resulted in the significant increase of crude protein content and crude lipid of the fish (Lara, 2003). Penaeus monodon fed with probiotic Bacillus supplemented feeds, on the other hand, showed an enhancement on growth and survival. The use of probiotics in shellfish cultures have been successfully tested in increasing the growth rate of small and large abalone by 8% and 34% in about eight months of culture period (Cruz et al., 2012).

2.4.2 Improvement of Water Quality

Due to various anthropogenic activities and indiscriminate uses of different chemicals that cause aquatic pollution, antibiotics are a major obstacle in aqua farming. High concentrations of ammonia nitrite and nitrate in rearing water are considered as harmful for fish larvae. In response to this, the use of probiotics is practiced for reducing ammonia nitrite and nitrate in the rearing water. The application of probiotic strains are more efficient in converting organic matter or large polymers into smaller units which results to the reduction of organic load in the aquatic environment, therefore speeding up the process of water purification. The introduction of probiotics such as Bacillus spp. increased the growth and survival of shrimps since it reduces the chemical oxygen demand and increases shrimp harvest. It also improves the health status of juvenile Penaeus monodon, and reduces the pathogenic vibrio (Cruz et al., 2012). Improved water quality has been associated with the gram positive bacteria, Bacillus sp., a better converter of organic matter back to carbon dioxide. The effectiveness on water quality, population density of bacteria and shrimp production in Penaeus vannamei culture ponds tested with commercially available probiotics resulted to a significantly higher survival rate, feed conversion rate and final production.

2.5. Selection of Probiotics

The selection of probiotic bacteria has been an empirical process based on limited scientific studies. One of the major cause of failure in probiotic research is the selection of inappropriate microorganisms. Probiotic bacteria needs to be adapted for different host species and environment. It is important to first understand and determine the mechanisms of probiotic action and to define selection criteria for potential probiotics. A potential probiotic should have the following characteristics: Capability of exerting beneficial effects on the host animal viz. increased growth or resistance to disease; Non-pathogenic and non-toxic to animals and human; Should be present as viable cells, preferably in large numbers although the minimum effective dose is not fully defined; Ability to withstand processing and storage; High tolerance to bile and gastric acid (low pH); Ability to adhere to epithelium or mucus; Persistency in intestinal tract; Ability to modulate immune response; Ability to produce inhibitory compounds; and Capability of altering microbial activity (Jadhav et al., 2015).

2.5.1 Modes of Probiotics Action

The three general modes of probiotics actions are as follows: suppression of viable count, alteration of microbial metabolism and stimulation of immune system (Jadhav et al., 2015). The suppression of viable count is done by the production of antibacterial compounds (like lactocidin, acidophillin, organic acids and bacteriocins or hydrogen peroxide) and by competitive exclusion. Competition is usually for adhesion sites and nutrients. Nonpathogenic bacteria, like probiotic bacteria, usually have high competitive ability and thus colonize the intestine better than pathogenic bacteria. Probiotics may increase digestive activity through production of enzymes that aid in digestion of various carbohydrates, fat and protein and absorption of nutrients. Probiotics can also decrease bacterial enzyme activity of some pathogenic bacteria and reduce ammonia production. In addition, it can increase antibody levels, elevate microphage activity and improve production of immunoglobulin (Jadhav et al., 2015).

2.5.2 Probiotic organism

Lactic acid bacteria (LAB) have been widely used and researched for human and terrestrial animal purposes, and are also known to be present in the intestine of healthy fish (Pandiyan et al., 2013). Interest in the research of LAB are mainly because they are naturally found in human gastrointestinal tract. This is due to their ability to tolerate the acidic and bile environment of the intestinal tract. LAB are also able to convert lactose into lactic acid, thereby lowering the pH of the gastrointestinal tract and preventing the colonization by many bacteria. The most widely studied and used lactic acid bacteria are the Lactobacilli and Bifidobacteria. Gram-positive obligate or facultative anaerobes are dominant in the gastrointestinal microbiota of man and terrestrial farm animals, while gram-negative facultative anaerobes prevail in the digestive tract of fish and shellfish, though symbiotic anaerobes may be dominant in the posterior intestine of some herbivorous tropical fish (Pandiyan et al., 2013).

2.6. Biology of Pediococcus acidilactici

Pediococcus acidilactici is a gram- positive cocci and a homofermentative bacterium that can grow in a wide range of pH, temperature, pressure, and pH levels. It is a facultative anaerobe because it can survive in conditions where oxygen is in limited quantities or even entirely lacking. Pediococcus acidilactici also prevents the colonization and growth of pathogens like Shigella, Salmonella, Clostridium difficile and Escherichia coli in small animals, and optimize the function of natural micro flora. This is due to the production of lactic acid and secretion of bacteriocins wherein a bacteria-killing compounds known as pediocins are exerted by Pediococc i (Probiotics Database). Furthermore, Pediococcus acidilactici also increases the energy value of the feed and the digestibility of nutrients.

2.7. Application of Pediococcus acidilactici as Probiotic

Incorporating the probiotics in the diet of animals, especially during their stressful periods such as at weaning, at the beginning of the lactation period, and after dietary shift from high forage to readily fermentable carbohydrates shows a significant effect. Pediococcus acidilactici has a wide range of potential benefits of which are still being studied. As a probiotic bacterium, Pediococcus acidilactici presents positive effects on the balance and the role of the intestinal flora which includes strengthening the immune defense and improvement in the production performances and the health of animals.

2.7.1 Livestock

Several studies reported that weanling pigs have increased counts of LAB and deceased Clostridium, E. coli, and Enterobacterium spp. in swine gut (Liao and Nyachoti, 2017). Study shows that diet supplemented with Saccharomyces cerevisiae ssp. boulardii and P. acidilactici, reduced dramatically the levels of E. coli levels in weaned pig after 4 weeks of feeding compared to the pigs fed with non-supplemented diet (Le Bon et al., 2010). Furthermore , Pediococcus acidilactici -based probiotic supplementation have been reported to promote healthy intestines by encouraging an early restoration of the intestinal mucosal thinning that occurs at weaning and would possibly improve local resistance to infection (Di Giancamillo et al., 2008). In monogastric animals, the most common probiotics used are yeasts (Saccharomyces boulardii) and bacteria (Lactobacillus spp., Enterococcus spp., Pediococcus spp., Bacillus spp.) since they target the hindgut where the most diverse microbial population harbors.

Probiotic supplementation to laying hens has been found to improve hen performance which includes feed efficiency, egg production and egg quality, nutrient digestibility, modulation of intestinal microflora, pathogen growth inhibition, and gut mucosal immunity and immunomodulation (Mikulski et al., 2012). According to a study on the effect of dietary probiotic Pediococcus acidilactici (PA) strain MA18/5M on performance, egg traits, egg cholesterol content, and fatty acid composition in laying hens during a 24-wk period (Mikulski, 2012), diets supplemented Pediococcus acidilactici MA 18/5M probiotic show potential for improving egg weight, feed efficiency, yolk color, and eggshell quality during the early laying period. Significant decreases in the number of damaged eggs were recorded as a result of Pediococcus acidilactici supplemented diet. In addition, a decrease in yolk cholesterol levels was noted after 24 weeks of feeding.

2.7.2 Aquaculture

Compared to terrestrial animals, the intricate relationship that an aquatic organism has with its direct environment are taken into account when looking at probiotics (Kesarcodi-Watson et al., 2008). Gram-negative facultative anaerobic bacteria dominates the digestive tract of fish and shellfish. However, the aquatic animals' intestinal microbiota may rapidly vary depending on the intrusion of microbes from water and food. Given the conditions, large numbers of probiotics developed in aquaculture are from bacteria directly originating in the aquatic environment. However, more traditional bacterial or yeast species such as Lactobacillus, Pediococcus, Bacillus, and S. cerevisiae are marketed to use in animal nutrition (Chaucheyras-Durand & Durand, 2010). This species of bacteria targets the fish eggs and larvae, fish juveniles and adults, crustaceans, bivalve molluscs and also live food such as rotifers, artemia, or unicellular algae (Verschuere et al., 2000). Furthermore, a recent study on the distribution of a P. acidilactici-based probiotics under pond conditions shows that it can be an effective treatment in limiting the prevalence and load of Vibrio nigripulchritudo strains in the hemolymph of marine shrimps (Castex et al., 2008).

2.7.3 Inclusion in Feeds

Feed is one of the largest items of expenditure in both agriculture and aquaculture production. The purpose of both industries is to produce maximum profit with minimum input, which is almost impossible given the constant increase in the cost of feed ingredients. Sufficient and effective feeding therefore is the most important requisite in feed production. Probiotics are used as feed additives for better and safe production. According to Wang and Zirong (2006), addition of photosynthetic bacteria (PSB) and lyophilized Bacillus sp. (B) and their mix in common carp basal diets improved growth performances, feed utilization and digestive enzyme activities compare to the control diet. Tilapia fingerlings fed with P. acidilactici -supplemented diet showed increased growth performance and survival rate than the control treatement (Fadl et al., 2013). Similar results found in broiler chicken fed with P. acidilactici (Chafai et al., n.d.). Merrifield et al. (2010) showed improvements in the morphology of the intestinal microvilli of the salmonides when fed probiotics, and Nakandakare et al. (2013) attested that the interactions between the intestinal microflora, morphology of the intestine, immune system and absorption of nutrients may influence on the health and performance of the fishes. In a study of Vieira et al. (2016), there was a decrease in abundance of Vibrio spp. in the intestinal tract of shrimps fed probiotic-supplemented diet due to the increase in the abundance of lactic acid bacteria. The increase in growth performance and feed utilization is attributed to the effect of probiotics on digestive processes by promoting the population of beneficial microorganism, microbial enzyme activity, improve intestinal microbial balance, consequently improving digestibility and absorption of food and feed utilization (EL-Haroun, 2007). Another possible reason for improving growth performance and feed efficiency may be due to probiotics inhibiting the colonization of potential pathogens in the digestive tract by antibiosis or by competition for nutrients and space and alteration of microbial metabolism (EL-Haroun, 2007). In addition, P. acidilactici is a rich source of protein and vitamin B-complex that have the ability to improve the integrity of intestine to absorb nutrients. Probiotics can also increase the area of absorption of small intestine by improving intestinal morphology. Lactic acid bacteria have the ability to hinder pathogenic bacteria to adhere to the intestinal mucosa, and it also secretes antimicrobial substance like plantaricin, hydrogen peroxide and organic acids (Vieira et al., 2016).

CHAPTER 3

METHODOLOGY

3.1. Experimental Animals and Acclimatization

The experiment was conducted at the Institute of Aquaculture Multi­Species Hatchery Building in the University of the Philippines Visayas, Miag-ao campus. The juvenile Litopenaeus vannamei were collected from the hatchery of IA- UPV Hatchery. The shrimps were first acclimatized for 3 days and was fed with control diet before the start of the trial.

3.2. Experimental Design

The experiment was conducted at the University of the Philippines Visayas Hatchery for 75 days, from October, 2017 to January, 2018. In this study, 15 pcs of glass aquaria with a capacity of 35 L were filled with dechlorinated filtered seawater and were used as culture tanks. Five treatments under normal photoperiod of 12h light: 12h dark were triplicated and arranged in random. The water was aerated throughout the culture period. Each tank contains 10 pcs of 0.5 g juvenile L. vannamei.

3.3. Sampling and Water Quality Parameters

The average weight of the five representative shrimp samples per replicate tank were recorded every 15 days from the start of the feeding trial. Each tank was cleaned of shrimp faeces and 20% of the water was renewed daily. The water quality parameters were checked twice a week. The water temperature was held at 24-32°C, dissolved oxygen (DO) level at 3.6-6.5 mg/L and salinity at 5-35 ppt.

3.4. Experimental Feed Preparation

The dietary protein requirements for juvenile L. vannamei under varying conditions ranges from 6-10% of the body weight (Ai et al., 2008). Commercially available lactic acid bacteria Pediococcus acidilactici was used as a probiotic incorporated in the L. vannamei diet. Using the diet formulator software, the chemical composition of the diet is 5.90% Ash, 31.78% Crude Protein, 9.27% Lipid and 1.66% Fiber. Below were the ingredients used in preparing the different diets, based on the studies of De Lima et al. (2016) and Fu et al. (2012).

Table 3.1. Ingredients of the five diet treatments having different probiotic concentrations.

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3.5. Feeding Trial

Feeding was conducted four times as a daily requirement of culture (FAO, 2017). There were five diets containing different probiotic concentrations which were triplicated, namely 0.02, 0.04, 0.08, 0.10 and control. Daily feeding rate was about 6­10% of total body weight (g) and properly regulated according to actual food intake of the white shrimp (Ai et al., 2008). The feeding rate was calculated using the formula, Feeding rate = x feedinq percentage x stock density number of individuals

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3.6. Proximate Composition of Experimental Diet and Shrimp Carcass

3.6.1. Moisture

The ground sample which was weighed to 5-10 g was placed in a drying oven at 105°C for at least 12 h. The sample was then cooled in a desiccator before it was again weighed. Careful not to expose the sample to the atmosphere.

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3.6.2. Crude Protein

Digestion

The 0.5 g of the sample was weighed, homogenized and sieved (0.5 mm) in Whatmann #1 or #42 filter paper. The samples were wrapped in filter paper and was placed inside the digestion tubes, and were labeled. A blank was also prepared for each batch by placing empty filter paper inside the tube. Then 2 g of Selenium catalyst or an alternative catalyst of mixed 1:3 (Copper Sulfate) CuSo4 and (Potassium Sulfate) K2SO4 were also added to the digestion tubes. All six tubes were placed in the stainless rack and 12 mL of concentrated Sulfuric acid (H2SO4) was added. This step was performed inside the hood and with the used of rubber gloves. The stainless rack and the tubes were placed into the digester block. The cover was also placed securely on top of the tubes. The digestion was set to 1 hr at 420°C. After the said time, the stainless rack with the tubes was removed from the digester and was hanged to cool down. The gas was allowed to escape for at least 10 mins before it was opened. The tubes were then transferred from the stainless rack to a tray and then another batch was prepared to be digested.

Distillation and Titration

Water was allowed to flow completely inside the system for 2-3 mins before the distilling unit was turned-on. The system was then waited to heat-up and a beeping sound signaled the equipment was ready to start. The tubings were arranged in the proper container. The suction hose for both the sample chamber and receiving area were rinsed with distilled water. The chamber door was opened and the kjeldahl tubes were placed slowly lowering the level. The suction hose was placed inside the tube containing the digested sample. The receiving flask was prepared with 25 mL of 4% boric solution in 100 mL Erlenmeyer flask and 3-4 drops of mixed indicator were added. The Erlenmeyer flask with the 4% boric solution and mixed indicator were placed in the receiving area. The hose was placed inside the flask. The distillation unit was started and the sample loaded for a few seconds before a 5 min distillation started. The beeping sound signaled the end of distillation. The digestion tube was removed slowly from the sample chamber. The hose was cleaned with distilled water before the next sample tube was placed to be distilled. The same with the receiving area. The Erlenmeyer flask was removed and titrated with 0.1 N HCl. The color changed from blue to faint pinkish color. The pinkish color is the end point.

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3.6.3. Crude Lipids

In the lipid analysis, the modified Bligh and Dyer method was used. The 0.3 g of the sample was weighed (W1) and was homogenized in 1 mL 5% NaCl solution. Then 4 mL MeOH and 2 mL chloroform were added. In a separatory funnel, the mixture was filtered with Whatmann filter paper (GFC). A ratio of 1:1 MeOH:Chloroform were then added. Distilled water was also added to neutralize and create a layer in the filtrate. The clear chloroform layer was collected into a pre­weighted clean dry scintillation vial (W2). The vials were then stored in the drying cabinet to dry for at least a week before weighing again (W3).

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3.6.4. Ash

A sample was weighed to 2 g into a pre-heated crucible. The crucible was then placed into a muffle furnace at 400-600°C for 4hrs or until whitish-grey ash is obtained. Then the crucible was placed in the desiccator and weighed.

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Where:

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3.7. Statistical analysis

Statistical analysis were determined using SPSS 16.0 software. One-way ANOVA was used to detect any significant differences among the analyzed parameters such as the FCR and SGR. The weight gain, survival rate, fed intake, SGR, FCR and PER were also calculated (See Appendix A).

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