Effects of Microencapsulated Organic Acid and Their Salts on Growth Performance, Immunity, and Disease Resistance of Paciﬁc White Shrimp Litopenaeus vannamei

: Use of antibiotics and other chemicals to combat disease outbreaks has been a bottleneck for the sustainable growth of shrimp industry. Among various replacements proposed, organic acid (OA) and their salts (OS) are commonly used by farmers and feed millers. However, in free forms, their requirement is very high (2–3 kg/MT) as they tend to disassociate before reaching the hindgut. The dosage can be reduced by microencapsulation of the ingredients. In this study, a 63-day trial was conducted to assess the effects of OA and OS (COMP) microencapsulated (ENCAP) with fat (HF), fat + alginate (HA), wax esters (WE) and HA + WE (HAWE) on performance, digestive enzymes, immunity and resistance to Vibrio parahaemolyticus . A positive control (PC, 200 g/kg ﬁshmeal-FM) and a negative control (NC, 130 g/kg FM) diet were formulated. Eight other diets were formulated, supplementing an NC diet with microencapsulated OA (OAHF, OAHA, OAWE, OAHAWE) and OS (OSHF, OSHA, OSWE, OSHAWE). Among the ENCAPs, signiﬁcant difference was observed in serum malondialdehyde ( p = 0.026), where HF showed the lowest level (6.4 ± 0.3 mmol/L). Signiﬁcant interactions between COMP and ENCAP were observed in lipid deposition ( p = 0.047), serum alkaline phosphatase, acid phosphatase, hepatopancreatic and serum phenol oxidase ( p < 0.0001). Despite no differences, 96-h mortality during pathogenic Vibrio parahaemolyticus challenge in all treatment diets (45–56%) was lower compared to the NC diets (63%). In conclusion, use of HF microencapsulated OA diets could provide improved performance and disease resistance that could contribute to the reduction of antibiotic use by the shrimp industry.


Introduction
The global farmed shrimp industry is frequently plagued with disease outbreaks starting from yellow head (YHV) and white spot syndrome (WSSV) virus in the 1990s to, more recently, acute hepatopancreatic necrosis disease (AHPND) [1,2]. The frequent outbreaks led to an increased use of antibiotics as a metaphylactic or prophylactic to treat or prevent diseases, respectively, or as antibiotic growth promoters (AGP) [3]. Reducing antibiotic use in farmed animals for disease control and banning GP is a global trend driven mainly by the increasing risk of antibiotic resistant bacteria [4,5].
Various alternatives to AGP, such as phytogenic compounds or plant derived essential oils [6,7], probiotic, prebiotic and synbiotic [8,9], enzymes [10,11], organic acids and their salts [2,[12][13][14][15][16], have been proposed in recent years. Organic acids are "Generally Regarded as Safe" compounds often containing one or more carboxyl groups (-COOH) [17,18]. The most common are those with short chain (C1-C6), such as formic, lactic, propionic, citric acids and their salts. Their probable mode of action includes reducing the digesta pH, stimulating digestive enzyme secretion, promoting intestinal integrity and regulating gut All four products were prepared by spray drying and congealing where active ingredients are dispersed in HF, HA, WE and for the double coated HAWE; the process was conducted first with HA and them repeated with WE using a process slightly modified from Jyothi et al. [48]. Briefly, active ingredients are dispersed in a solution and spraydried where the material solidifies onto the particles of active ingredients such that the microcapsules obtained are of matrix type.
For solubility, 10 g of each test product was mixed with 200 mL of deionized water, then stirred for 6 h at 100 rpm at 19 • C. After 6 h, the supernatant was filtered, and insoluble active ingredients from the filtrate were dried and weighed. A mix of organic acids corresponding to the active ingredients of the micro-encapsulated product was used as a control. The pH of the supernatant was determined after filtration. Each treatment was conducted in triplicate.

Feeding Trial
The feeding trial was conducted for 63 days at the Guangdong Ocean University field experimental station situated at Donghai Island, Zhanjiang of Guagdong province of China. Experimental procedure and animal care were accomplished in accordance with the ethical guidelines for the care and use of laboratory animals provided by the Animal Care Committee of the Guangdong Ocean University.

Experimental Design and Diet Preparation
Ten isoproteic (37.3 ± 0.12% CP) and isoenergetic (16.4 ± 0.02 MJ/kg) diets were prepared: diet 1-positive control with 20% FM (PC); diet 2-negative control with 13% fishmeal and 12% meat and bone meal (NC); diets 3-6 were manufactured by supplementing NC with 0.75 mg/kg of OA microencapsulated with HF, HA, WE and HAWE (OAHF, OAHA, OAWE and OAHAWE, respectively); diets 7-10 were manufactured by supplementing 0.85 mg/kg of OS microencapsulated with HF, HA, WE and HAWE (OSHF, OSHA, OSWE and OSHAWE, respectively) (Tables 1 and 2). It was ensured that microencapsulated products contained the same amount of active ingredients. The microencapsulated test products were supplied by Jefo Nutrition Inc., Quebec, Canada. Diet composition and their proximate chemical composition including amino acid profile are provided in Tables 1 and 2, respectively. All feed ingredients were ground, sieved through 80-mesh screens, mixed with a V-type mixer (Shanghai Tianxiang & Chentai Pharmaceutical Co., Ltd., Shanghai, China), pelleted with a screw pelletizer (South China university of technology, Guangzhou, China) after adding 30% water, air-dried and then stored at −20 • C until used. Pellets of two different sizes, 1.0-and 1.5-mm diameter, were produced for the trial.

Experimental Conditions
Twenty-five thousand PL10 Pacific white shrimp L. vannamei postlarvae were obtained from Allied Pacific Aquaculture Co., Ltd., Zhanjiang, Guangdong, China. The shrimp were acclimatized in two cement pools for 40 days until the average body weight reached 0.3 g. From the cement pools, a total of 1600 white shrimp (0.33 ± 0.02g ABW) were selected and 40 shrimp/tank were randomly distributed into 40 cone-shaped tanks (350-L volume each) with four replicates per treatment.
The shrimp were fed the experimental diets four times daily (7:00, 11:00, 17:00 and 21:00 h) at 8-10% of their body weight. The water was completely exchanged once in every 2-3 days from the first to the fourth week and once daily from fifth to the ninth week.

Sampling
At the end of the experiment, shrimp were fasted for 24 h before the final sampling. For serum and hepatopancreatic analyses, 15 and 10 shrimps were randomly selected from each tank, respectively. Both analyses were not conducted on the same shrimp because of the possibility of interference of one sampling on another. For serum, the blood was drawn using a dispensable 1 mL syringe into 1.5 mL test-tube. The test-tubes were then stored at 4 • C overnight before being centrifuged at 5867× g for 10-min at 4 • C (3K30, Sigma, Hamburg, Germany). The supernatant was then collected into 1.5 mL tubes and stored at −80 • C for subsequent analyses. The hepatopancreas was removed from each shrimp, immediately frozen in liquid nitrogen and then stored at −80 • C for analysis. Another six shrimps from each tank were taken for body chemical composition, ground into slurry, lyophilized and kept at −20 • C until analysis.

Chemical Analyses and Enzymatic Assay
Diets, ingredients and body chemical composition were analyzed following AOAC (1995) protocols. Nitrogen for crude protein (CP, %N × 6.25) was analyzed using a Kjeldahl apparatus (Kjeltec TM 8400, FOSS, Goteborg, Sweden), moisture by drying the samples at 105 • C under atmospheric pressure for 24 h, crude lipid using a Soxhlet apparatus (Soxtec TM 2050, FOSS, Goteborg, Sweden), crude ash by burning the samples at 550 • C using a muffle furnace (Shanghai Boxun industry & Commerce Co., Ltd., Shanghai, China) and gross energy using a bomb calorimeter (Changsha Kaiyuan Instruments, Changsha, China).
The activity of acid (ACP) and alkaline (ALP) phosphatase, total superoxide dismutase (T-SOD), malondialdehyde (MDA), lipase and amylase were determined using diagnostic kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Prophenoloxidase (PO) activity was measured spectrophotometrically by recording formation of dopachrome produced from L-di-hydroxy-phenylalanine (L-DOPA) following a procedure slightly modified from Huang et al. (2010). Briefly, 3 mg/mL L-DOPA solution was prepared by using 1 L of 0.1 M potassium phosphate buffer (0.1 M K 2 HPO 4 ·3H 2 O, 0.1 M KH 2 PO 4 , adjusted to pH 6.6). Shrimp serum (20 µL) was mixed thoroughly with 980 µL L-DOPA solution. A 300 µL sample of the mixture was placed in a 96-well plate and incubated at room temperature. The absorbance was recorded after 6 min (OD sample ) on a Microplate Spectrophotometer (Multilskan spectrum, Thermo Fisher Scientific, Waltham, MA, USA) at 490 nm. At the same time, 300 µl of L-DOPA solution was placed in a 96-well plate and absorbance of the blank control group was recorded (OD blank ). Enzymatic activity for all assays was expressed as the change in absorbance/min.

Resistance to Vibrio Parahaemolyticus
Resistance to the pathogen V. parahaemolyticus was determined from the cumulative mortality of shrimp in 96 h. For this, 10 shrimps for each replicate (3 replicates in each treatment) were used. After injecting each shrimp with 2.4 × 10 7 colony-forming units (CFU) of V. parahaemolyticus, the cumulative mortality in 96 h was recorded.

Calculation
The equations to calculate different parameters are given below: where, SGR is specific growth rate, FBW is final body weight (g) and IBW is initial body weight (g).
where FCR is feed conversion ratio, FI is feed intake (g) and WG is weight gain (g).
where PER is protein efficiency ratio and PI is protein intake (g).
where MDA is malondialdehyde (U/mL), SC is standard concentration (10 nmol/mL) and OD is optical density.
where hepatopancreas protein content is expressed as mg_protin/mL.
where ACP is acid phosphatase (King U/g protein), Std.Conc. is standard concentration (0.1 mg/mL), protein content in hepatopancreas is expressed as g protein/mL.
where ALP is alkaline phosphatase (King U/g protein), Std.Conc. is standard concentration (0.1 mg/mL), protein content in hepatopancreas is expressed as g protein/mL.

Statistical Analysis
All data were expressed as the mean ± SD (standard deviation) and subjected to one-way ANOVA (SPSS 17.0, Chicago, IL, USA). Percentage data were arcsine-square root transformed before statistical analysis. If there was a difference, multiple comparison analyses were performed using Duncan's multiple-range tests. Statistically significant differences were considered when p < 0.05.

Results
During the feeding trial, the water temperature was ranged between 28 • C and 34 • C, and salinity, dissolved oxygen and total ammonia nitrogen content were maintained at 27-28 g/L, >7 mg/L and <0.03 mg/L, respectively. Feed intake was normal, and survival was not affected by the dietary treatments.

Stability of the Microencapsulation Materials
The pH values were similar among the non-protected acids, HF and HA microencapsulation (2.8-2.9) which slightly increased with WE (3.2) and HAWE (3.5) microencapsulation ( Figure 1A). All four microencapsulation materials showed significantly higher recovery than the free acid. Corresponding to the pH values, the recovery was significantly higher for WE (95%) and HAWE (97%) compared to HF (74%) and HA (77%) ( Figure 1B).

Growth Performance and Body Composition
Feed intake and growth were normal, similar to the studies conducted at the laboratory. Effects of the microencapsulated OA and OS on body chemical composition and final body weight, specific growth rate (SGR), feed conversion ratio (FCR) and protein efficiency ratio (PER) are presented in Tables 3 and 4, respectively. The form of organic acids (free or salt) significantly affected the feed intake and FCR where shrimp fed diets with OA showed lower FCR and feed intake compared to those fed the OS diets (p < 0.05). There were no differences (p > 0.05) in body chemical composition among the treatments.

Immune Response and Disease Resistance
No differences in cumulative 96-h mortality when challenged with Vibrio parahaemolyticus ( Figure 2) and serum SOD, hepatopancreatic ALP, ACP and MDA (Table 6) were observed with either the main effects of COMP, ENCAP or their interaction (Table 6). Significant interaction was observed for serum ALP (p < 0.0001), ACP (p < 0.0001) and hepatopancreatic and serum phenol oxidase level (p < 0.0001). A significantly lower serum MDA level (p < 0.026) was observed in HF (6.4) compared to the other ENCAP (HA = 7.7, WE = 6.9 and HAWE = 7.7).

Scoring
Shrimp fed the OA diets showed higher scores in growth performance (58 vs. 38), nutrient utilization (67 vs. 57) and immune response (112 vs. 96) than those fed the OS diets with a combined score of 237 compared to 191 (Table 7). Among the four ENCAP, the overall scores of HF and HA (118 and 117, respectively) were higher than WE (95) and HAWE (98) (p < 0.05) ( Table 7).

Discussion
This study investigated the efficacy of dietary organic acids (free or salt) microencapsulated with hydrogenated fat (HF), hydrogenated fat + alginate (HA), wax esters (WE) and the double coating of HAWE (first coated with HA followed by WE) on the performance of Pacific white shrimp. The organic acid blend contained fumaric acid (pKa = 3.03), sorbic acid (pKa = 4.75) and citric acid (pKa = 2.92-5.21). The organic acid salt blend contained Ca-propionate, Ca-formate and Na-acetate.
Organic acids are low molecular weight aldehyde-containing compounds with one or more carboxyl groups. They are used as a dietary supplement to reduce gastrointestinal tract pH and inhibit the growth of gram-negative bacteria through the disassociation of the acids and production of anions in bacterial cells [49]. As weak acids, the pKa values or the disassociation constant of organic acids are higher than the strong acids, such as HCl or H2SO4 [50]. These acids do not dissociate in the highly acidic stomach pH but tend to dissociate quickly in the proximal intestine as pH increases and the condition becomes alkaline. Shrimp are slow-eating animals taking 1-2 h to hold and chew the pellets. In free-form, organic acid or their salts have considerable risk of leaching in water, preventing them from reaching the hepatopancreas and gut in undissociated form [51]. Coating or encapsulation may significantly reduce leaching and, consequently, can remain effective at a lower dosage [11]. For example, micro-encapsulated organic acid salt blend used by Yao et al. [11] was much lower (835 mg/kg) than in their free form (2000-6000 mg/kg) reported in various studies [52,53]. Micro-encapsulation provides better protection than simple coating that may prevent or reduce the loss of the active ingredient in the case of breakage of the pills, as active ingredients are embedded in the matrix of coating material [54].
Microencapsulation of easily degradable bioactive compounds has become a popular and practical approach for masking unpleasant characteristics of the compounds and delivering them at the intended location of the gastrointestinal tract [24,55]. In this study, despite their lower solubility and recovery, both HF and HA (118 and 117, respectively) had higher total performance scores in vivo compared to WE and HAWE (95 and 98, respectively (Table 7). However, between HF and HA, the growth performance score was higher for HA but lower for immune response than those for HF. No differences in the nutrient utilization scores were observed between the two materials. Both HF and HA were tested in vitro by Omnojio et al. [26], and they observed well-timed release of the active ingredient. Timely release of the active ingredient at the intended location of the digestive tract is utterly important for their efficacy. Hydrogenated fat can be easily digested by intestinal lipase thus guaranteeing the slow release of the active ingredient along the GI tract. In a recent study, the efficacy of HF-based microencapsulated aluminum and iron sulfate in in situ chelation of undigestible phosphorus in the hind gut of rainbow trout were also reported by Ndiyae et al. [56]. The study confirms the release of the active ingredient in the hindgut where it was intended to bind with phosphorus, thus reducing the risk of eutrophication of the surrounding environment. The relatively poor performance of shrimp fed WE diets compared to those fed other treatment diets may be attributed to low solubility and higher retention of active ingredient than hydrogenated fat (Figure 1). Wax-based solid lipid matrix provides better physical stability and more protection against chemical reaction [39]. The positive characteristics, such as slower degradation and mass transfer rate, may not be suitable for shrimp for their short gut-transit time (~2 h) to release the active ingredient.
Blends of organic acids and their salts in free or microencapsulated forms have shown to improve the growth performance of fish [40,57,58] and shrimp [2,11,33,59], as well as antioxidant status [60]. Several studies reported improved growth performance, nutrient utilization and immune response in crustaceans fed a microencapsulated blend of organic acid or acid salts. Safari et al. [61] reported the efficacy of an encapsulated blend of Na-butyrate, Na-lactate and Na-propionate on growth performance and survival of crawfish at 20 g/kg. The OS blend used in the present study contains Ca-propionate, Ca-formate and Na-acetate, and showed higher feed intake compared to those fed the OA diets. Yao et al. [11] also reported improved weight gain and FCR in Pacific white shrimp compared to NC diet with the same OS blend. When compared between the OA and OS treatments of this study, shrimps fed the OA diets showed improved FCR, protein retention and immune response, i.e., higher ALP and PO than the OS blend (Tables 4-6). This is in accordance with the findings of Romano et al. (2015), who reported improved growth performance of Pacific white shrimp with 1-4% microencapsulated OA (blend of formic, lactic, malic and citric acids).
In an in vitro study, Mine and Boopathy [12] demonstrated EC50 values of 0.023%, 0.041%, 0.03% and 0.066% for formic, acetic, propionic and butyric acid, respectively, against Vibrio harveyi. Romano et al. [33] reported similar efficacy in V. harveyi resistance when shrimp were fed OA supplemented diets. Efficacy of organic acid in combination with essential oil against Vibrio sp. Infections was also demonstrated by He et al. [60], where a microencapsulated blend of organic acid (citric acid and sorbic acid) and essential oils (thymol and vanillin) showed significantly higher survival in Pacific white shrimp challenged with V. parahaemolyticus after 48-h compared to those fed the control diets. These are in accordance with the findings of the present study where treatments containing microencapsulated organic acid and organic acid salt blends showed significantly lower cumulative 96-h mortality ranging from 45 to 56% compared to 63% for those fed the NC diets when challenged with pathogenic V. parahaemolyticus (Figure 2).

Conclusions
This is one of the first reports comparing the effects of OA and OS on performance, nutrient utilization, immune response and disease resistance of Pacific white shrimp, as well as comparing different microencapsulation materials and techniques. Finding an effective microencapsulation strategy along with the effective composition of organic acid or their salts is important for sustainable development of the industry.
Based on the findings, it can be concluded that an organic acid blend microencapsulated with hydrogenated fat or hydrogenated fat + alginate may provide better responses in Pacific white shrimp and can be used as an effective strategy to improve immune response and disease resistance. Further studies are recommended to investigate the effects of microencapsulated organic acid compounds on intestinal health, metabolic response and gut microbiome of farmed Pacific white shrimp.  Institutional Review Board Statement: The study was conducted according to the guidelines of the Guangdong Ocean University, China and approved by the Animal Care Committee.