# Dietaryγ-Aminobutyric Acid (GABA) Promotes Growth and Resistance to Vibrio alginolyticus in Whiteleg Shrimp Litopenaeus vannamei

Authors: Jinho Bae; Ali Hamidoghli; Nathaniel W. Farris; Olumide Samuel Olowe; Wonsuk Choi; Seunghan Lee; Seonghun Won; Mihyang Ohh; Seunghyung Lee; Sungchul C. Bai
Journal: Aquaculture Nutrition
 (2022)

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## Abstract

An 8-week feeding trial was conducted to evaluate the effects of dietaryγ-aminobutyric acid (GABA) on growth performance, immune response, and disease resistance for juvenile whiteleg shrimp, Litopenaeus vannamei. Five isonitrogenous diets were formulated by supplementing 0 (CON), 50 (GABA50), 100 (GABA100), and 300 (GABA300) mg of GABA and 4 g of oxytetracycline (OTC) per kilogram of diet. A total of 225 juvenile whiteleg shrimp with an initial weight of 2.97±0.06 g were randomly distributed and reared in 15 aquaria as triplicates. After 8 weeks of the feeding trial, weight gain, specific growth rate, feed efficiency, and protein efficiency ratio of shrimp fed GABA100 were significantly higher than those of shrimp fed CON, GABA50, and GABA300 diets (P<0.05). However, there were no significant differences among shrimp fed CON, GABA50, GABA300, and OTC diets (P>0.05). After nine days of challenge test with Vibrio alginolyticus, the average cumulative survival rate of shrimp fed GABA50, GABA100, and OTC was significantly higher than that of shrimp fed GABA300 and CON diets (P<0.05). These results may suggest that 100 mg dietary GABA supplementation (including endogenous GABA, 175.6 mg/kg diet) per kilogram of diet could be the optimum dietary level to replace antibiotics and improve growth performance and disease resistance in whiteleg shrimp L. vannamei.


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## Body

## 1. Introduction

Shrimp production is important to global aquaculture growth, and it is the most traded aquaculture product [1]. Whiteleg shrimp, Litopenaeus vannamei, is the most abundantly raised shrimp contributing to 83% of global shrimp aquaculture production, of which the majority (82%) is produced in Asian countries [2]. This shrimp species is also quite resilient to variations in culture salinity and can be raised in both inland and marine water environments. As such, the last ten years has seen an increase of over 51% in L. vannamei production. This rapid growth, in addition to the aforementioned reasons, can largely be attributed to their superior growth rate and survival in high-density cultures, low protein requirements, and ability to successfully spawn in commercial hatcheries [3–6].As with the growth of aquaculture, the intensification ofL. vannamei is characterized by high stocking densities, which have led to increased feed inputs, resulting in oxidative stress to the shrimp as well as local environmental challenges that occur at the farm level [7, 8]. For instance, during feeding events, dissolved oxygen levels can plummet, leading to greater oxidative stress and concomitant increases in such indicators as superoxide dismutase [9, 10]. These crowding and related stressors lower L. vannamei growth rates and impair immune function, thus increasing susceptibility to disease outbreaks and mass mortality events [7, 11, 12]. Furthermore, L. vannamei lacks a true adaptive immune system and is therefore incapable of benefiting from interventions such as vaccination, thereby causing an increased usage of antimicrobial treatments [1, 13]. Though many serious shrimp-related pathogens are viral, the threat posed by bacterial pathogens such as Vibrio spp. is often mitigated by antibiotic usage for either prophylactic or prescriptive purposes [1, 14]. Though the majority of Vibrio spp. are benign, V. parahaemolyticus, V. campbellii, V. harveyi, V. owensii, and V. alginolyticus have wreaked havoc on the industry in recent years [15, 16]. The excessive use of antibiotics likely stems from farmer’s lack of knowledge concerning the hazards related to their abuse such as the natural selection of antimicrobial-resistant strains, inadequate pathogen diagnosis, and ease of availability in some developing countries [17, 18]. This leads to these agents being liberally administered with inadequate information on preparation, dosage, and disease to be treated [18]. The excessive use of antibiotics may pose health threats to both humans and animals alike due to the resulting proliferation of the previously mentioned antimicrobial-resistant pathogens. Sadly, more than 90% of oxytetracycline (OTC) is released into the immediate environment [14]. Because of these serious issues, antibiotics are highly regulated or outright banned in many countries.A potential alternative to the use of antibiotics is feed additives. Feed additives are components of the diet formulation, whether nutritive or otherwise, that are incorporated into the feed in relatively small quantities for purposes other than fulfilling the nutrient requirements of the target species. Such purposes may include but not be limited to enhancement of growth, immunity, pigmentation, feed stability, and final product properties [19, 20]. Numerous studies [21–23] have demonstrated the positive effects of feed additives on the growth rate, survival, immune response, and disease resistance in fish and shrimp.γ-Aminobutyric acid (GABA) is a four-carbon nonessential amino acid and widely known as the principal inhibitory neurotransmitter in the animal central nervous system (CNS). It is also regarded as an interkingdom signaling molecule and serves a myriad of functions across all kingdoms of life ranging from plants and bacteria to invertebrate and vertebrate animals [24]. Recently, it has gained importance as a feed additive [25–27]. Lactic acid bacteria and yeast are the most important producers of GABA due to their commercial availability and scalability [20]. GABA is synthesized from glutamate (Glu) in the neurons via glutamate decarboxylase (GAD) and can play an important role in fish growth enhancement, immune responses, and feed efficiency [28]. Despite the progress made with the evaluation of the effects of other feed additives on various fish species, there is a scarcity of information on the effects of dietary GABA.Though there has been a resurgence of interest in GABA in the context of aquaculture nutrition, it has long been known for its ability to improve reproduction and other health parameters in shellfish. In Pacific oyster (Crassostrea gigas), 10 μmol/L inhibited nitric oxide synthase (NOS) activity and proinflammation cytokines in hemocytes [29]. In the case of red abalone (Haliotis rufescens), GABA induces planktonic larvae to settle and undergo metamorphosis when administered at 1-2 μmol/L [30, 31]. It has also been shown to improve reproduction in rotifer (Brachionus plicatilis) reared in stressful culture conditions at 87.5 mg/L. These trials involved the administration of GABA directly to the water. Though not considered a dietary supplementation, these early trials point to GABA’s usefulness in aquaculture. With regard to dietary supplementation in fish, to date, there have only been a handful of trials evaluating GABA as a feed additive. Wu et al. [32] demonstrated that GABA supplementation in the diet of juvenile grass carp (Ctenopharyngodon idella) significantly improves specific growth rate (SGR) and modulates NPY and ghrelin gene expression. Analysis of their results indicates that 87.5 mg/kg GABA could be the optimal supplemental level. Temu et al. [33] found that supplementation with GABA significantly improved growth and modulated both aspartate aminotransferase (AST) and superoxide dismutase activity (SOD) in juvenile Nile tilapia (Oreochromis niloticus) with a 158 mg/kg determined to be an optimal level in the diet. GABA has also been shown to modulate the immune response and improve survival against bacterial challenge. In a trial by Bae et al. [34], juvenile olive flounder (Paralichthys olivaceus) given a GABA supplementation of 158 mg/kg GABA led to significantly improved growth and enhanced villi length, trypsin activity, and disease resistance against Edwardsiella tarda. In a similar trial, also involving juvenile P. olivaceus, Farris et al. [35] found that in addition to improved growth, supplementation with 100 and 150 mg/kg led to increased lysozyme and amylase activity and disease resistance against Streptococcus iniae. In this trial, an optimal supplementation level of 237 mg/kg was determined based on the total GABA content of the experimental diets (supplemental as well as endogenous to the feed). Furthermore, GABA has been implicated in the modulation of inflammatory pathways as well as modulation of the intestinal microbiome in juvenile turbot (Scophthalmus maximus) [36] at a supplementation level of 160 mg/kg GABA. Feeding trials assessing GABA supplementation in crustaceans have been much more limited than is the case with fish. So far, there have only been a few trials evaluating GABA’s effects. In a recent trial assessing GABA in Chinese mitten crab (Eriocheir sinensis) by Zhang et al. [37], it was found that GABA works to modulate gene expression within orexigenic/anorexigenic pathways which indicates GABA’s ability to affect satiation. As for shrimp, to the best of our knowledge, the only study on the effect of GABA on L. vannamei was carried out by Xie et al. [26] which reported that GABA was effective against ammonia stress and can improve growth performance in low-fishmeal diets when given 150 mg/kg GABA. Therefore, the present study is aimed at evaluating the effects of different dosages of dietary GABA on growth, hematological parameters, nonspecific immune responses, and disease resistance of L. vannamei. Comparisons were also made with a commercial antibiotic (OTC).


## 2. Materials and Methods

### 2.1. Experimental Design and Diets

Presented in Table1 is the formulated experimental diets and proximate composition analysis. To evaluate the dietary γ-aminobutyric acid (GABA) as a feed additive in whiteleg shrimp, five experimental diets were formulated to be isonitrogenous and isocaloric. Diets were supplemented with 0 (CON), 50 (GABA50), 100 (GABA100), and 300 (GABA300) mg of GABA and 4 g of oxytetracycline (OTC) per kilogram of diet. The actual GABA content of each experimental diet was 62.4 (CON), 102 (GABA50), 175 (GABA100), 248 (GABA300), and 57.4 (OTC) mg/kg diet. Ingredients such as fishmeal have some endogenous GABA content, and therefore, diets CON and OTC show some endogenous GABA composition. The composition of the GABA content in each experimental diet was evaluated and analyzed using high-performance liquid chromatography (HPLC, Sykam, Eresing, Germany) following the procedure of Lee et al. [38]. 20 g of each diet was ground and extracted with 100% of MeOH (3×100 mL) for sample preparation. The content of GABA was measured by a reverse-phase column with a gradient elution program (water:acetonitrile=90:10 to 0 : 100 for 40 min). The condition of UV detection was 280 nm. For the preparation of the diets, ingredients were ground to powder using a mechanical grinder and then mixed using an electronic industrial mixer (Hanyoung Food Machinery, Gyeonggi-do, Republic of Korea). The fish oil was added into the mixture and mixed until homogenized. Additionally, 30% of water was added into the mixture. Thereafter, the moistened mixture was passed through a 0.2 cm die pelleting machine (Shinsung, Seoul, Republic of Korea), dried with a dehumidifier, and stored at a temperature of 4°C until the feeding trials.Table 1
Ingredient composition and proximate analysis of the basal diet for juvenile whiteleg shrimp (% dry matter basis).
Ingredients%Sardine fish meal130.0Soybean meal120.0Blood meal13.00Wheat gluten meal15.00Squid liver powder15.00Wheat flour214.0Corn starch18.00Fish oil14.00Calcium phosphate32.50Vitamin premix42.00Mineral premix52.00Cholesterol30.50Lecithin12.00Cellulose12.00Total100Proximate composition (% dry matter basis)Crude protein40.7Crude lipid8.81Crude ash8.28Moisture9.611The Feed Co., Goyang, Rep of Korea. 2Samhwa Flour Mills Co., Seoul, Rep of Korea. 3Sigma-Aldrich Korea, Yongin, Rep of Korea. 4Vitamin premix (as mg/kg premix): A, 1000000 IU; D, 200000 IU; E, 10000; B1, 2000; B6, 1500; B12, 10; C, 10000; calcium pantothenic acid, 5000; nicotinic acid 4500; B-biotin 10; choline chloride, 30000; inositol, 5000. 5Mineral premix (as g/kg premix): ferrous fumarate, 12.50; manganese sulfate, 11.25; dried ferrous sulfate, 20.0; dried cupric sulfate, 1.25; cobaltous sulfate, 0.75; zinc sulfate KVP, 13.75; Calcium iodate, 0.75; magnesium sulfate, 80.20; aluminum hydroxide, 0.75.


### 2.2. Experimental Shrimp and Feeding Trials

The experimental juvenile whiteleg shrimp,Litopenaeus vannamei, was acquired from a commercial farm (Pal-ttak shrimp farm, Go-seong city, Republic of Korea) and acclimated in 250 L rectangular tanks at the FFNRC (Feeds & Foods Nutrition Research Center, Pukyong National University, Busan, Rep. Korea) for two weeks. After the acclimation period, 15 juvenile whiteleg shrimp with an average weight of 2.97±0.06 g (mean±SD) were randomly distributed into 15 aquaria as triplicates. Each aquarium was supplied with filtered seawater in a semirecirculating system during the experiments. The inlet water flow was maintained at 1.3 L/min at a water temperature of 28±1.0°C. Dissolved oxygen was maintained at 6~7 mg/L, and salinity was 33 ppt. The whiteleg shrimp were fed three times per day at 5~7% of wet body weight during the 8 weeks of experiment. Mortality was checked every day, and dead shrimp were removed directly. The feeding ratio was recalculated after the removal of dead shrimp to allow for balance in feeding. Aquarium tanks were maintained by careful cleaning and daily removal of feces by siphoning.Following the 8-week feeding trial, the growth performance parameters such as weight gain (WG), specific growth rate (SGR), feed efficiency (FE), protein efficiency ratio (PER), and survival of shrimp from each tank were measured and calculated according to the following equations [39]:
(1)WG%=finalweightg–initialweightg×100/initialweightg,SGR%/day=100×lnfinalweightg–lninitialweightgdays,FE%=finalweightg–initialweightg×100feedrationg,PER=wetweightgaingproteinintakegSurvival%=100×finalnumberofshrimpinitialnumberofshrimp.


### 2.3. Sample Collection and Analysis

#### 2.3.1. Proximate Composition Analysis

Proximate composition of ingredients, experimental diets, and whole-body shrimp carcass was measured according to the Association of Official Analytical Chemists [40]. Samples for this analysis were grounded and freeze-dried (Advantage 2.0, VirTis, New York, USA) for 72 hours. Total moisture and crude ash contents were measured by drying to constant weight at 105°C for 24 h and combustion at 550°C in a muffle furnace for 3 h, respectively. Nitrogen content (N×6.25) was measured using acid digestion followed by the Kjeldahl method (2300 Autoanalyzer, Foss Tecator AB, Hoganas, Sweden) to analyze crude protein. Crude lipid was measured by using Soxtec system 1046 (Tecator AB, Hoganas, Sweden).


#### 2.3.2. Hemolymph Analysis

At the end of experiment, shrimp were fasted for 1 day and the hemolymph was extracted. Hemolymph was taken from the ventral sinus in the first abdominal segment using a 1 mL syringe (without anticoagulation) and centrifuged at3000×g for 10 minutes to separate the serum. Then, the serum was stored at a temperature of −70°C. Hemolymph biochemical parameters such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glucose (GLU) were measured by a chemical analyzer (Fuji DRI-CHEM 3500i, Fuji Photo Film Ltd., Tokyo, Japan). Antioxidant enzyme such as superoxide dismutase (SOD), nonspecific immune responses such as myeloperoxidase (MPO), and lysozyme activities were also analyzed using the sampled hemolymph. SOD was determined using an assay kit (Enzo ADI-900-157, Enzo Life Sciences, Inc.) following the manufacturer’s instructions. This method relies on inhibition against water-soluble tetrazolium dye and determination of SOD enzyme activity. Samples were incubated for 20 minutes at 37°C using a multiwell spectrophotometer, and absorbance was monitored at 450 nm. Lysozyme activity was determined by the reaction against Micrococcus lysodeikticus and spectrophotometric (Sunrise TECAN, Männedorf, Switzerland) analysis at 530 nm absorbance. Myeloperoxidase (MPO) value was analyzed following the procedure of Quade et al. [41]. Briefly, hemolymph (20 μL); Hanks Balanced Salt Solution (HBSS), 3, 3, 5, 5′tetramethylbenzidinedihydrochloride (TMB, 20 mM; Sigma-Aldrich); H2O2 (5 mM); and 4 M sulphuric acid were diluted in a 96-well plate. The color changes were measured at 450 nm in a microplate reader (Sunrise TECAN, Männedorf, Switzerland).


#### 2.3.3. Challenge Test

After the feeding trial,Vibrio alginolyticus suspension was prepared to conduct a nine-day bacterial challenge test according to Tseng et al. [42]. The concentration of the pathogen after being cultured was adjusted to 2×106 CFU/mL using 0.85% NaCl. Five randomly selected shrimp from each tank were redistributed in 15 tanks (10 L capacity) in a nonrecirculating system to perform the challenge test. The challenge test was conducted in triplicate by injecting 20 μL of the bacterial solution into the ventral sinus of the cephalothorax. The water temperature was maintained at 28±0.5°C (mean±SD) with aeration of the challenge test tanks. Shrimp were not fed during the challenge test. Dead shrimp were removed, and daily mortality was recorded.


#### 2.3.4. Statistical Analysis

After analysis of every parameter, data were checked for normality and homogeneity of variances then analyzed by one-way ANOVA test using SAS program (SAS Institute, Inc. Cary, NC, USA) to analyze the differences of each treatment group. A Fisher Least Significant Difference (LSD) test was used to compare means whenever a significant difference is observed in the ANOVA table. Treatment effects were considered significant at the confidence level ofP<0.05.


## 2.1. Experimental Design and Diets

Presented in Table1 is the formulated experimental diets and proximate composition analysis. To evaluate the dietary γ-aminobutyric acid (GABA) as a feed additive in whiteleg shrimp, five experimental diets were formulated to be isonitrogenous and isocaloric. Diets were supplemented with 0 (CON), 50 (GABA50), 100 (GABA100), and 300 (GABA300) mg of GABA and 4 g of oxytetracycline (OTC) per kilogram of diet. The actual GABA content of each experimental diet was 62.4 (CON), 102 (GABA50), 175 (GABA100), 248 (GABA300), and 57.4 (OTC) mg/kg diet. Ingredients such as fishmeal have some endogenous GABA content, and therefore, diets CON and OTC show some endogenous GABA composition. The composition of the GABA content in each experimental diet was evaluated and analyzed using high-performance liquid chromatography (HPLC, Sykam, Eresing, Germany) following the procedure of Lee et al. [38]. 20 g of each diet was ground and extracted with 100% of MeOH (3×100 mL) for sample preparation. The content of GABA was measured by a reverse-phase column with a gradient elution program (water:acetonitrile=90:10 to 0 : 100 for 40 min). The condition of UV detection was 280 nm. For the preparation of the diets, ingredients were ground to powder using a mechanical grinder and then mixed using an electronic industrial mixer (Hanyoung Food Machinery, Gyeonggi-do, Republic of Korea). The fish oil was added into the mixture and mixed until homogenized. Additionally, 30% of water was added into the mixture. Thereafter, the moistened mixture was passed through a 0.2 cm die pelleting machine (Shinsung, Seoul, Republic of Korea), dried with a dehumidifier, and stored at a temperature of 4°C until the feeding trials.Table 1
Ingredient composition and proximate analysis of the basal diet for juvenile whiteleg shrimp (% dry matter basis).
Ingredients%Sardine fish meal130.0Soybean meal120.0Blood meal13.00Wheat gluten meal15.00Squid liver powder15.00Wheat flour214.0Corn starch18.00Fish oil14.00Calcium phosphate32.50Vitamin premix42.00Mineral premix52.00Cholesterol30.50Lecithin12.00Cellulose12.00Total100Proximate composition (% dry matter basis)Crude protein40.7Crude lipid8.81Crude ash8.28Moisture9.611The Feed Co., Goyang, Rep of Korea. 2Samhwa Flour Mills Co., Seoul, Rep of Korea. 3Sigma-Aldrich Korea, Yongin, Rep of Korea. 4Vitamin premix (as mg/kg premix): A, 1000000 IU; D, 200000 IU; E, 10000; B1, 2000; B6, 1500; B12, 10; C, 10000; calcium pantothenic acid, 5000; nicotinic acid 4500; B-biotin 10; choline chloride, 30000; inositol, 5000. 5Mineral premix (as g/kg premix): ferrous fumarate, 12.50; manganese sulfate, 11.25; dried ferrous sulfate, 20.0; dried cupric sulfate, 1.25; cobaltous sulfate, 0.75; zinc sulfate KVP, 13.75; Calcium iodate, 0.75; magnesium sulfate, 80.20; aluminum hydroxide, 0.75.


## 2.2. Experimental Shrimp and Feeding Trials

The experimental juvenile whiteleg shrimp,Litopenaeus vannamei, was acquired from a commercial farm (Pal-ttak shrimp farm, Go-seong city, Republic of Korea) and acclimated in 250 L rectangular tanks at the FFNRC (Feeds & Foods Nutrition Research Center, Pukyong National University, Busan, Rep. Korea) for two weeks. After the acclimation period, 15 juvenile whiteleg shrimp with an average weight of 2.97±0.06 g (mean±SD) were randomly distributed into 15 aquaria as triplicates. Each aquarium was supplied with filtered seawater in a semirecirculating system during the experiments. The inlet water flow was maintained at 1.3 L/min at a water temperature of 28±1.0°C. Dissolved oxygen was maintained at 6~7 mg/L, and salinity was 33 ppt. The whiteleg shrimp were fed three times per day at 5~7% of wet body weight during the 8 weeks of experiment. Mortality was checked every day, and dead shrimp were removed directly. The feeding ratio was recalculated after the removal of dead shrimp to allow for balance in feeding. Aquarium tanks were maintained by careful cleaning and daily removal of feces by siphoning.Following the 8-week feeding trial, the growth performance parameters such as weight gain (WG), specific growth rate (SGR), feed efficiency (FE), protein efficiency ratio (PER), and survival of shrimp from each tank were measured and calculated according to the following equations [39]:
(1)WG%=finalweightg–initialweightg×100/initialweightg,SGR%/day=100×lnfinalweightg–lninitialweightgdays,FE%=finalweightg–initialweightg×100feedrationg,PER=wetweightgaingproteinintakegSurvival%=100×finalnumberofshrimpinitialnumberofshrimp.


## 2.3. Sample Collection and Analysis

### 2.3.1. Proximate Composition Analysis

Proximate composition of ingredients, experimental diets, and whole-body shrimp carcass was measured according to the Association of Official Analytical Chemists [40]. Samples for this analysis were grounded and freeze-dried (Advantage 2.0, VirTis, New York, USA) for 72 hours. Total moisture and crude ash contents were measured by drying to constant weight at 105°C for 24 h and combustion at 550°C in a muffle furnace for 3 h, respectively. Nitrogen content (N×6.25) was measured using acid digestion followed by the Kjeldahl method (2300 Autoanalyzer, Foss Tecator AB, Hoganas, Sweden) to analyze crude protein. Crude lipid was measured by using Soxtec system 1046 (Tecator AB, Hoganas, Sweden).


### 2.3.2. Hemolymph Analysis

At the end of experiment, shrimp were fasted for 1 day and the hemolymph was extracted. Hemolymph was taken from the ventral sinus in the first abdominal segment using a 1 mL syringe (without anticoagulation) and centrifuged at3000×g for 10 minutes to separate the serum. Then, the serum was stored at a temperature of −70°C. Hemolymph biochemical parameters such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glucose (GLU) were measured by a chemical analyzer (Fuji DRI-CHEM 3500i, Fuji Photo Film Ltd., Tokyo, Japan). Antioxidant enzyme such as superoxide dismutase (SOD), nonspecific immune responses such as myeloperoxidase (MPO), and lysozyme activities were also analyzed using the sampled hemolymph. SOD was determined using an assay kit (Enzo ADI-900-157, Enzo Life Sciences, Inc.) following the manufacturer’s instructions. This method relies on inhibition against water-soluble tetrazolium dye and determination of SOD enzyme activity. Samples were incubated for 20 minutes at 37°C using a multiwell spectrophotometer, and absorbance was monitored at 450 nm. Lysozyme activity was determined by the reaction against Micrococcus lysodeikticus and spectrophotometric (Sunrise TECAN, Männedorf, Switzerland) analysis at 530 nm absorbance. Myeloperoxidase (MPO) value was analyzed following the procedure of Quade et al. [41]. Briefly, hemolymph (20 μL); Hanks Balanced Salt Solution (HBSS), 3, 3, 5, 5′tetramethylbenzidinedihydrochloride (TMB, 20 mM; Sigma-Aldrich); H2O2 (5 mM); and 4 M sulphuric acid were diluted in a 96-well plate. The color changes were measured at 450 nm in a microplate reader (Sunrise TECAN, Männedorf, Switzerland).


### 2.3.3. Challenge Test

After the feeding trial,Vibrio alginolyticus suspension was prepared to conduct a nine-day bacterial challenge test according to Tseng et al. [42]. The concentration of the pathogen after being cultured was adjusted to 2×106 CFU/mL using 0.85% NaCl. Five randomly selected shrimp from each tank were redistributed in 15 tanks (10 L capacity) in a nonrecirculating system to perform the challenge test. The challenge test was conducted in triplicate by injecting 20 μL of the bacterial solution into the ventral sinus of the cephalothorax. The water temperature was maintained at 28±0.5°C (mean±SD) with aeration of the challenge test tanks. Shrimp were not fed during the challenge test. Dead shrimp were removed, and daily mortality was recorded.


### 2.3.4. Statistical Analysis

After analysis of every parameter, data were checked for normality and homogeneity of variances then analyzed by one-way ANOVA test using SAS program (SAS Institute, Inc. Cary, NC, USA) to analyze the differences of each treatment group. A Fisher Least Significant Difference (LSD) test was used to compare means whenever a significant difference is observed in the ANOVA table. Treatment effects were considered significant at the confidence level ofP<0.05.


## 2.3.1. Proximate Composition Analysis

Proximate composition of ingredients, experimental diets, and whole-body shrimp carcass was measured according to the Association of Official Analytical Chemists [40]. Samples for this analysis were grounded and freeze-dried (Advantage 2.0, VirTis, New York, USA) for 72 hours. Total moisture and crude ash contents were measured by drying to constant weight at 105°C for 24 h and combustion at 550°C in a muffle furnace for 3 h, respectively. Nitrogen content (N×6.25) was measured using acid digestion followed by the Kjeldahl method (2300 Autoanalyzer, Foss Tecator AB, Hoganas, Sweden) to analyze crude protein. Crude lipid was measured by using Soxtec system 1046 (Tecator AB, Hoganas, Sweden).


## 2.3.2. Hemolymph Analysis

At the end of experiment, shrimp were fasted for 1 day and the hemolymph was extracted. Hemolymph was taken from the ventral sinus in the first abdominal segment using a 1 mL syringe (without anticoagulation) and centrifuged at3000×g for 10 minutes to separate the serum. Then, the serum was stored at a temperature of −70°C. Hemolymph biochemical parameters such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glucose (GLU) were measured by a chemical analyzer (Fuji DRI-CHEM 3500i, Fuji Photo Film Ltd., Tokyo, Japan). Antioxidant enzyme such as superoxide dismutase (SOD), nonspecific immune responses such as myeloperoxidase (MPO), and lysozyme activities were also analyzed using the sampled hemolymph. SOD was determined using an assay kit (Enzo ADI-900-157, Enzo Life Sciences, Inc.) following the manufacturer’s instructions. This method relies on inhibition against water-soluble tetrazolium dye and determination of SOD enzyme activity. Samples were incubated for 20 minutes at 37°C using a multiwell spectrophotometer, and absorbance was monitored at 450 nm. Lysozyme activity was determined by the reaction against Micrococcus lysodeikticus and spectrophotometric (Sunrise TECAN, Männedorf, Switzerland) analysis at 530 nm absorbance. Myeloperoxidase (MPO) value was analyzed following the procedure of Quade et al. [41]. Briefly, hemolymph (20 μL); Hanks Balanced Salt Solution (HBSS), 3, 3, 5, 5′tetramethylbenzidinedihydrochloride (TMB, 20 mM; Sigma-Aldrich); H2O2 (5 mM); and 4 M sulphuric acid were diluted in a 96-well plate. The color changes were measured at 450 nm in a microplate reader (Sunrise TECAN, Männedorf, Switzerland).


## 2.3.3. Challenge Test

After the feeding trial,Vibrio alginolyticus suspension was prepared to conduct a nine-day bacterial challenge test according to Tseng et al. [42]. The concentration of the pathogen after being cultured was adjusted to 2×106 CFU/mL using 0.85% NaCl. Five randomly selected shrimp from each tank were redistributed in 15 tanks (10 L capacity) in a nonrecirculating system to perform the challenge test. The challenge test was conducted in triplicate by injecting 20 μL of the bacterial solution into the ventral sinus of the cephalothorax. The water temperature was maintained at 28±0.5°C (mean±SD) with aeration of the challenge test tanks. Shrimp were not fed during the challenge test. Dead shrimp were removed, and daily mortality was recorded.


## 2.3.4. Statistical Analysis

After analysis of every parameter, data were checked for normality and homogeneity of variances then analyzed by one-way ANOVA test using SAS program (SAS Institute, Inc. Cary, NC, USA) to analyze the differences of each treatment group. A Fisher Least Significant Difference (LSD) test was used to compare means whenever a significant difference is observed in the ANOVA table. Treatment effects were considered significant at the confidence level ofP<0.05.


## 3. Results

### 3.1. Growth Performance and Body Composition

The growth and survival of juvenile whiteleg shrimp fed different levels of GABA and OTC for eight weeks are shown in Table2. The findings show that shrimp fed GABA100 had significantly higher final body weight, weight gain, feed efficiency, protein efficiency ratio, and specific growth rate in comparison to shrimp fed CON, GABA50, and GABA300 diets (P<0.05). However, there were no significant differences between GABA100 and OTC diets (P>0.05). Furthermore, as shown in Table 3, whole-body composition analysis of shrimp fed the five experimental diets did not show significant differences in crude protein, crude lipid, moisture, and ash contents (P>0.05).Table 2
Growth performance of juvenile whiteleg shrimp fed the five experimental diets for 8 weeks1.
CONGABA50GABA100GABA300OTCIBW22.93±0.072.98±0.042.93±0.072.98±0.102.98±0.04FBW310.1±0.53b10.3±0.41b11.8±0.32a10.5±0.40b11.1±0.71abWG4244±22.6b247±15.6b304±17.4a252±20.7b273±28.6abSGR52.20±0.12b2.22±0.08b2.49±0.08a2.24±0.10b2.35±0.14abFE656.0±5.19b56.6±3.57b69.6±3.98a57.7±4.75b62.5±6.56abPER71.38±0.13b1.39±0.09b1.70±0.10a1.43±0.12b1.54±0.16abSurvival888.9±10.297.8±3.8593.3±0.0088.9±13.991.1±3.851Data are means±SD of triplicate groups of shrimp. Values in each row with different superscripts are significantly different. 2IBW: initial body weight (g). 3FBW: final body weight (g). 4WG: weightgain%=finalwt.−initialwt.×100/initialwt.5SGR:specificgrowthrates%=logefinalwt.−logeinitialwt.×100/days. 6FE: feedefficiencyratios%=wetweightgain/dryfeedintake×100. 7PER: proteinefficiencyratio=wetweightgain/proteinintake. 8Survival%=totalfish–deadfish×100/totalshrimp.Table 3
Whole-body proximate composition (% wet matter) of juvenile whiteleg shrimp fed the five experimental diets for 8 weeks1.
CONGABA50GABA100GABA300OTCCrude protein18.4±0.3018.3±0.6718.2±0.5318.7±0.1518.3±0.76Crude lipid2.64±0.152.84±0.062.77±0.112.85±0.072.82±0.09Crude ash3.55±0.233.65±0.163.66±0.083.75±0.073.72±0.22Moisture75.4±0.6576.2±0.9077.3±0.8575.7±1.3775.3±1.311Data are means±SD of triplicate groups of shrimp.


### 3.2. Hemolymph Biochemical Parameters

As shown in Table4, there were no significant differences among treatment groups in terms of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glucose (P>0.05).Table 4
Hemolymph biochemical analysis of juvenile whiteleg shrimp fed the five experimental diets for 8 weeks1.
CONGABA50GABA100GABA300OTCAST2117±5.03123±7.23119±4.58118±7.64114±3.06ALT31.87±0.452.20±0.462.03±0.512.13±0.292.10±0.30Glucose466.4±2.1265.5±3.1663.9±0.5066.5±2.3868.0±2.831Data are means±SD of triplicate groups of shrimp. 2AST: aspartate aminotransferase (units/liter). 3ALT: alanine aminotransferase (units/liter). 4Glucose, GLU (mg/dL).


### 3.3. Antioxidant Enzyme and Nonspecific Immune Responses

The antioxidant enzyme activity and nonspecific immune responses of shrimp fed diets with different GABA levels and OTC are shown in Table5. At the end of the eight-week feeding trial, there were no significant differences in superoxide dismutase (SOD), lysozyme activity, and myeloperoxidase (MPO) among the different treatments (P>0.05).Table 5
Antioxidant enzyme and nonspecific immune response of juvenile whiteleg shrimp fed the five experimental diets for 8 weeks1.
CONGABA50GABA100GABA300OTCSOD292.4±1.3492.2±0.8792.8±0.7493.7±1.9394.3±2.27Lysozyme30.73±0.070.69±0.040.72±0.020.73±0.090.73±0.03MPO43.53±0.283.73±0.123.81±0.303.66±0.213.78±0.111Data are means±SD of triplicate groups of shrimp. 2SOD: superoxide dismutase (% inhibition). 3Lysozyme activity (U/L). 4MPO: myeloperoxidase (absorbance).


### 3.4. Challenge Test

The cumulative survival rate of juvenile whiteleg shrimp after nine days of challenge test withVibrio alginolyticus is presented in Figure 1. According to these results, shrimp fed GABA50, GABA100, and OTC had significantly higher cumulative survival than those of shrimp fed GABA300 and CON diets (P<0.05). There was no significant difference in cumulative survival of whiteleg shrimp fed GABA50 and GABA100 diets (P>0.05). Also, there was no significant difference between shrimp fed CON and GABA300 diets after nine days of the challenge test (P>0.05).Figure 1
Cumulative survival rate of juvenile whiteleg shrimp fed five different experimental diets for 8 weeks and challenged withVibrio alginolyticus (2×106 CFU/mL) for 9 days. Data points with different letters are significantly different (P<0.05).


## 3.1. Growth Performance and Body Composition

The growth and survival of juvenile whiteleg shrimp fed different levels of GABA and OTC for eight weeks are shown in Table2. The findings show that shrimp fed GABA100 had significantly higher final body weight, weight gain, feed efficiency, protein efficiency ratio, and specific growth rate in comparison to shrimp fed CON, GABA50, and GABA300 diets (P<0.05). However, there were no significant differences between GABA100 and OTC diets (P>0.05). Furthermore, as shown in Table 3, whole-body composition analysis of shrimp fed the five experimental diets did not show significant differences in crude protein, crude lipid, moisture, and ash contents (P>0.05).Table 2
Growth performance of juvenile whiteleg shrimp fed the five experimental diets for 8 weeks1.
CONGABA50GABA100GABA300OTCIBW22.93±0.072.98±0.042.93±0.072.98±0.102.98±0.04FBW310.1±0.53b10.3±0.41b11.8±0.32a10.5±0.40b11.1±0.71abWG4244±22.6b247±15.6b304±17.4a252±20.7b273±28.6abSGR52.20±0.12b2.22±0.08b2.49±0.08a2.24±0.10b2.35±0.14abFE656.0±5.19b56.6±3.57b69.6±3.98a57.7±4.75b62.5±6.56abPER71.38±0.13b1.39±0.09b1.70±0.10a1.43±0.12b1.54±0.16abSurvival888.9±10.297.8±3.8593.3±0.0088.9±13.991.1±3.851Data are means±SD of triplicate groups of shrimp. Values in each row with different superscripts are significantly different. 2IBW: initial body weight (g). 3FBW: final body weight (g). 4WG: weightgain%=finalwt.−initialwt.×100/initialwt.5SGR:specificgrowthrates%=logefinalwt.−logeinitialwt.×100/days. 6FE: feedefficiencyratios%=wetweightgain/dryfeedintake×100. 7PER: proteinefficiencyratio=wetweightgain/proteinintake. 8Survival%=totalfish–deadfish×100/totalshrimp.Table 3
Whole-body proximate composition (% wet matter) of juvenile whiteleg shrimp fed the five experimental diets for 8 weeks1.
CONGABA50GABA100GABA300OTCCrude protein18.4±0.3018.3±0.6718.2±0.5318.7±0.1518.3±0.76Crude lipid2.64±0.152.84±0.062.77±0.112.85±0.072.82±0.09Crude ash3.55±0.233.65±0.163.66±0.083.75±0.073.72±0.22Moisture75.4±0.6576.2±0.9077.3±0.8575.7±1.3775.3±1.311Data are means±SD of triplicate groups of shrimp.


## 3.2. Hemolymph Biochemical Parameters

As shown in Table4, there were no significant differences among treatment groups in terms of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glucose (P>0.05).Table 4
Hemolymph biochemical analysis of juvenile whiteleg shrimp fed the five experimental diets for 8 weeks1.
CONGABA50GABA100GABA300OTCAST2117±5.03123±7.23119±4.58118±7.64114±3.06ALT31.87±0.452.20±0.462.03±0.512.13±0.292.10±0.30Glucose466.4±2.1265.5±3.1663.9±0.5066.5±2.3868.0±2.831Data are means±SD of triplicate groups of shrimp. 2AST: aspartate aminotransferase (units/liter). 3ALT: alanine aminotransferase (units/liter). 4Glucose, GLU (mg/dL).


## 3.3. Antioxidant Enzyme and Nonspecific Immune Responses

The antioxidant enzyme activity and nonspecific immune responses of shrimp fed diets with different GABA levels and OTC are shown in Table5. At the end of the eight-week feeding trial, there were no significant differences in superoxide dismutase (SOD), lysozyme activity, and myeloperoxidase (MPO) among the different treatments (P>0.05).Table 5
Antioxidant enzyme and nonspecific immune response of juvenile whiteleg shrimp fed the five experimental diets for 8 weeks1.
CONGABA50GABA100GABA300OTCSOD292.4±1.3492.2±0.8792.8±0.7493.7±1.9394.3±2.27Lysozyme30.73±0.070.69±0.040.72±0.020.73±0.090.73±0.03MPO43.53±0.283.73±0.123.81±0.303.66±0.213.78±0.111Data are means±SD of triplicate groups of shrimp. 2SOD: superoxide dismutase (% inhibition). 3Lysozyme activity (U/L). 4MPO: myeloperoxidase (absorbance).


## 3.4. Challenge Test

The cumulative survival rate of juvenile whiteleg shrimp after nine days of challenge test withVibrio alginolyticus is presented in Figure 1. According to these results, shrimp fed GABA50, GABA100, and OTC had significantly higher cumulative survival than those of shrimp fed GABA300 and CON diets (P<0.05). There was no significant difference in cumulative survival of whiteleg shrimp fed GABA50 and GABA100 diets (P>0.05). Also, there was no significant difference between shrimp fed CON and GABA300 diets after nine days of the challenge test (P>0.05).Figure 1
Cumulative survival rate of juvenile whiteleg shrimp fed five different experimental diets for 8 weeks and challenged withVibrio alginolyticus (2×106 CFU/mL) for 9 days. Data points with different letters are significantly different (P<0.05).


## 4. Discussion

This study evaluated the effects of different dosages of dietary GABA on growth, survival, whole-body proximate composition, hematology, nonspecific immune responses, and disease resistance of juvenileL. vannamei reared under laboratory conditions. The findings of the present study indicated that the addition of dietary GABA at 100 mg/kg has a positive effect on weight gain, specific growth rate, feed efficiency, and protein efficiency ratio, indicating that GABA could be effective in increasing the growth of whiteleg shrimp. This corroborates with studies on whiteleg shrimp L. vannamei [26], pharaoh cuttlefish Sepia pharaonis [43], Jian carp Cyprinus carpio var. Jian [44], and olive flounder Paralichthys olivaceus [34, 35] on the usage of dietary GABA as a feed additive. GABA is an inhibitory neurotransmitter that has shown to reduce stress indices in animals [45–47]. The ability of GABA to control the neural excitation in animals affects the metabolism, thus, improving the growth performance [48]. The supplementation of diets with GABA has shown to increase feed intake and improve growth performance in cows and protect organs in broilers [49, 50]. Increased fish growth is attributed to effective feeding efficiency which can be improved by dietary glutamine [51]. Based on the previous studies and the results obtained in the present study, it can be inferred that GABA can improve the growth performance of reared animals. However, the effect of GABA on growth is highly dependent on the level of inclusion. The effects of GABA appear to be dosage-dependent. Reference [32] found that the addition of GABA to grass carp diet increased growth rate, superoxide dismutase, and total antioxidant level with an increased GABA level from 50 to 150 mg/kg. This is why at 200 mg/kg, there was a reduction in the mentioned parameters. These authors suggested that a dosage of 85 mg/kg dietary GABA is optimum for juvenile grass carp. A high level of GABA inclusion also decreased the growth rate of the giant freshwater prawn, Macrobrachium rosenbergii [52]. The addition of 150 mg/kg of dietary GABA was suitable for whiteleg shrimp fed with a low-fishmeal diet [26]. These authors asserted that there is a reduction in growth at GABA levels greater than 150 mg/kg, and this validates the findings of our study as the addition of 300 mg/kg of dietary GABA also showed a reduction in growth. This suggests that there is a “dose-effect response” in the usage of dietary GABA in aquafeed [32]. This is likely because GABA has been linked to an increase in feed intake when administered in optimal levels. In a very recent trial by Zhang et al. [37], Chinese mitten crabs that were given an optimal dosage of GABA (84-89 mg/kg) had significantly upregulated orexigenic neural signal-related genes which resulted in an increase in feed intake and molting frequency.In our study, no significant differences were observed in the whole-body composition analysis of whiteleg shrimp fed diets with different levels of GABA. It was previously shown that dietary glutamine and glutamate, which are precursors of GABA, did not influence whole-body proximate composition of Nile tilapia [53]. Also, the body composition of juvenile grass carp was not influenced by the addition of dietary gamma-aminobutyric acid in the diet [32]. However, Pohlenz et al. [54] noted a significant difference in the lipid content composition of channel catfish (Ictalurus punctatus) fed glutamine. A similar result was also reported by Zhao et al. [51] on the addition of supplementary glutamine in the diet of Jian carp. These authors found significant differences in protein and lipid content of fish carcasses compared to the control diet. The inconsistency in the results obtained by previous studies and the present study might have been due to the difference in size and species. Also, shrimp have lower lipid content compared to freshwater and marine fish species; therefore, changes in whole-body lipid content might be negligible [55].Hemolymph biochemical parameters such as AST and ALT are important indicators of hepatopancreas (the equivalent of the liver in vertebrates) health and function. The variations of these enzymes in hemolymph can indicate hepatopancreas damage and are used to diagnose disease in animals [56, 57]. Hemolymph glucose is also an indicator of stress response and is regulated by crustacean hyperglycemic hormone in shrimp [58]. Chang et al. [59] demonstrated that shrimp glucose levels increase after hypothermal stress. In the present study, there were no significant differences in shrimp hemolymph AST, ALT, and glucose levels fed diets with different dietary GABA levels and OTC. This could indicate that our experimental conditions were optimized enough that the effects of GABA seemed negligible on hemolymph biochemical parameters. The same results were observed by Farris et al. [35] when olive flounders were fed different levels of GABA. Further investigations on the effects of GABA on hemolymph biochemical parameters under suboptimal conditions are required.The supplementation of dietary glutamine in farmed fish has been shown to improve not only growth performance but also immune responses [28]. Shrimp are often cultured in highly intensive systems, thereby increasing their susceptibility to high ammonia accumulation from excessive feeding. Oxidative stress can be caused by ammonia or nitrite accumulation through the production of reactive oxygen species [60]. Liang et al. [61] asserted that nonspecific immune response of antioxidant enzymes such as SOD and glutathione peroxidase can eliminate oxidative stress. However, the accumulation of nitrites reduces the levels of these antioxidant enzymes. The addition of glutamine, a precursor of GABA, improved the intestinal antioxidant capacity in grass carp at various levels [62]. The dietary supplementation and the injection of GABA at different dosages significantly increased the total superoxide dismutase and catalase (CAT) activity in L. vannamei [26] and Chinese mitten crab, Eriocheir sinensis [63]. However, in the present study, the nonspecific immune response parameters were not significantly influenced by the dietary GABA levels. This is similar to a previous study on supplementation of dietary GABA in olive flounder, Paralichthys olivaceus [34], and in the inclusion of dietary glutamine in half-smooth tongue sole [45]. Also, Farris et al. [35] tested graded levels of dietary GABA in flounder diet and reported no significant effects on SOD activity. Differences with regard to this parameter in previous studies might be due to variables such as species, developmental stage, feed formulations, or GABA source. The mechanism of action of dietary GABA and its effects on nonspecific immune responses are not well-known. Therefore, further studies are required to understand the effects of this feed additive on immune responses.It has been suggested that the addition of dietary GABA can enhance the immune system by improving the gut microbiota and T-cell pathway [51]. The improvement of gut health of whiteleg shrimp could improve the immune system and resistance against pathogenic bacteria. Zhao et al. [51] suggested that the addition of GABA improves the effectiveness of Enterococcus faecium probiotic. This can help the culture organisms to fight against pathogenic bacteria. Vibrio alginolyticus is a gram-negative, rod-shaped bacterium that has been known to infect Litopenaeus vannamei leading to poor growth performance, anorexia, and death [64]. Disease caused by this bacterium could be treated with antibiotics; however, it has been evidenced that most antibiotics cannot destroy every strain of V. alginolyticus [65]. Increased stress as a result of intensive culture may increase the susceptibility of shrimp to disease and lead to increased dopamine levels. An increase in the dopamine level has been shown to increase the vulnerability of Penaeus monodon to Photobacterium damsela pathogen [66]. In the present study, when challenged with Vibrio alginolyticus, L. vannamei fed diets supplemented with 100 mg of GABA had a significantly higher cumulative survival in comparison to the OTC diet. This is in an agreement with the previous studies regarding the pharmaceutical effects of GABA, which reveal that GABA could inhibit the activity of Vibrio parahaemolyticus. The mechanism of action could be attributed to the ability of GABA to suppress dopamine [67].


## 5. Conclusions

Conclusively, the results from this study indicated that the supplementation of GABA improved the growth performance and disease resistance ofL. vannamei, which may have resulted from increased feeding efficiency and reduced stress. This means GABA can be considered an effective functional feed additive in the prevention of disease infection and also be a potential antibiotic replacer. Our findings suggested that the supplementation of dietary GABA at 100 mg/kg could be the optimum level in whiteleg shrimp diet.



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*Source: 9105068-2022-03-09.xml*