INTRODUCTION

Alongside pangasius, shrimp is one of the top two aquaculture products in Vietnam, with the majority of production concentrated in the Mekong Delta region. According to the General Statistics Office of Vietnam (2025), shrimp production was 1,000,080 tons in 2023, accounting for nearly 85% of the country’s total shrimp output. Super-intensive whiteleg shrimp farming has rapidly expanded in many provinces in the Mekong Delta region because of its high expected profits; however, frequent and intensive extreme weather events are a major factor negatively affecting shrimp growth and health. To minimize antibiotic usage in aquaculture farming, synbiotic products are increasingly being developed and used as growth boosters and health-promoting agents for aquatic animals (Khanjani et al., 2024). The synergistic supplementation of probiotics and prebiotics (indigestible carbohydrates) like fructo-oligosaccharides (FOS), galacto-oligosaccharides, and mannan oligosaccharides has demonstrated beneficial effects on the host (Knipe et al., 2021). Recently, several studies have shown the advantageous effects of synbiotics on aquatic animals, particularly shrimp species. For example, Nababan et al. (2022) demonstrated Pseudoalteromonas piscicida combined with FOS enhanced the resistance of the P. vannamei to co-infection with White Spot Syndrome Virus (WSSV) and Vibrio harveyi, thereby improving shrimp development. In addition, Hong et al. (2022) reported that the administration of Pediococcus pentosaceus combined with FOS has demonstrated synergistic effects in enhancing growth, immunity, and resistance to Vibrio parahaemolyticus infection in whiteleg shrimp. According to Chen et al. (2020), the combination of mannan oligosaccharides and Bacillus licheniformis positively influences the overall growth, nutrient utilization, and immunological health of P. vannamei. Similar positive effects have been reported in other crustacean species supplemented with synbiotics in their diets, including prawn (Macrobrachium rosenbergii) (Li et al., 2024; Halim et al., 2018), brown shrimp (P. aztecus) (Kaya, 2025), and tiger shrimp (P. monodon) (Chin et al., 2025). Moreover, Ismail et al. (2019) reported that synbiotics can be used as replacement for antibiotics for disease control in aquatic organisms, including Nile tilapia (Oreochromis niloticus). Recently, the utilization of natural plant extracts as prebiotics in combination with probiotics has emerged as a sustainable strategy for eco-friendly aquaculture farm management.

Plants, and their by-products, which are abundant in many biologically active phytochemicals, vitamins, minerals, and fibers, are used as a prebiotic that is extracted with different solvents and combined with animal feed, promoting health or stimulating growth (Mabrouki et al., 2017; Citarasu, 2010). Moreover, plant extracts have antimicrobial activity against various bacteria that cause diseases in aquatic animals (Citarasu, 2010; Bondad-Reantaso et al., 2023). The noni tree (Morinda citrifolia) is used in traditional medicine and is widely cultivated in tropical regions, especially in Southeast Asia (Chan-Blanco et al., 2006). In Vietnam, this tree naturally grows in humid areas, particularly alongside canals and rivers. Mubarokah et al. (2023) reported that noni leaf extracts exhibit a diverse range of phytochemical constituents such as flavonoids, coumarins, terpenoids, and carotenoids. Moreover, noni leaf extract is rich in dietary fiber (24.53%) and polysaccharides (37.82%) (Nwakanma et al., 2022) that serve as a prebiotic, potentially enhancing the benefits of probiotics through synergistic effects, thereby improving nutrient digestibility and promoting growth in animals. In previous studies of diets supplemented with noni, noni levels in shrimp diets differ markedly depending on the plant parts used, including fruit, seed, and leaf, ranging from 1.25 to 10 g kg^-1 of diet (Abidin et al., 2022; Phan et al., 2023). The probiotic Lactobacillus plantarum CMT1 at 10⁸ CFU kg^-1 diet in shrimp feed is the optimal dose to promote weight gain and digestion and absorption, which can modulate intestinal microorganisms and could consequently enhance production efficiency of whiteleg shrimp (Phan et al., 2024). To develop synbiotics from natural plants or their by-products as prebiotic components in aquaculture species, optimization of prebiotic and probiotic concentration is a key prerequisite for their practical and effective application. Limited research has addressed the potential of probiotics and noni leaf extracts to promote aquatic animal growth and health, especially in strengthening resistance to pathogenic bacteria. Previous studies have not consistently demonstrated the synergistic effects of noni leaf extract and L. plantarum in the whiteleg shrimp. Therefore, this study evaluated the effect of prebiotics, noni leaf extract plus L. plantarum CMT1 probiotics, on weight gain, digestive enzymes, intestinal bacteria population, and resistance against pathogenic bacteria in whiteleg shrimp. The present study also underscores the prospective effects of synbiotics and determines the optimal dosage for their applications in shrimp farming.

MATERIALS AND METHODS

Experimental diets: Noni leaf extract was prepared using the procedure described by Phan et al. (2023). Briefly, dried noni leaf powder was extracted with 70% methanol for 48 h. The filtered liquid was then concentrated and stored at 4^oC until further use in experiments. Lactobacillus plantarum CMT1 was provided by the Laboratory of Probiotics, Can Tho University, Can Tho city, Vietnam. Bacteria were cultured in glass tubes without shaking in MRS (De Man, Rogosa and Sharpe) medium (Himedia, India) at 37^oC. The bacteria were adjusted to a concentration of 10⁹ CFU mL^-1 using sterile sodium chloride (0.9%). The noni leaf extract and diluted bacterial suspension were thoroughly incorporated into commercial feed to prepare the shrimp diets for different experimental groups. After preparation, all diets, including the control diet, were uniformly coated with 0.5% binder (Aqua Vina, Vietnam) to ensure consistent pellet stability, then dried and stored at 4°C until use.

Experiment design: The experiment was conducted from February to April, 2025 at the wet laboratory, College of Aquaculture and Fisheries, Can Tho University, Vietnam. Post-larval shrimp were sourced from a commercial hatchery and acclimatized for two weeks. The experiment was designed as a completely randomized design (CRD) with three replicates per treatment. A total of 1500 shrimp juveniles (average weight: 0.6 ± 0.18 g) were divided into 5 experimental groups, with three replicates of 100 shrimp each (300 shrimp per group). The different diet groups included (1) control; (2) diet 1 (0.5% noni); (3) diet 2 (1% noni); (4) diet 3 (0.5% noni + 10⁸ CFU kg^-1L. plantarum CMT1); (5) diet 4 (1% noni + 10⁸ CFU kg^-1L. plantarum CMT1). The commercial feed used in this study was sourced from GrowMax group (Vietnam) and contained 40 - 43% protein and 6% lipid. Shrimp were stocked into 500-L composite tanks equipped with a recirculation system and continuous aeration. Different diets were provided four times per day during the 60-day experimental period. Feed intake was recorded for each replicate by collecting uneaten feed, drying it to constant weight, and subtracting it form the total feed provided. . Throughout the culture period, minerals were added weekly to all treatments. Daily measurements and recordings were made using a Handheld Multiparameter (YIS model, USA), and the culture conditions were 26.8 - 27.6^oC for temperature, 7.6 - 7.9 for pH, and DO 5.1 - 5.7 mg L^-1. Alkalinity, ammonium nitrogen, and nitrite were analyzed weekly (APHA, 2017), and the values were in the range of 102.7 - 150.9 mgCaCO₃ L^-1, 0.106 - 0.304 mg L^-1, and 0.101 - 0.337 mg L^-1, respectively.

Challenge analysis: After the 60-day feeding period, shrimp of all groups were injected with pathogenic V. harveyi. Bacteria were adjusted to 10⁸ CFU mL^-1 and V. harveyi was cultured according to a standard procedure provided by Phan et al. (2025).The challenge dose was based on the median lethal dose (LD₅₀), which was approximately 5.0 × 10⁷ CFU mL⁻¹ at 96 h post-challenge, as determined in a preliminary test using the Reed and Muench method, and was selected to induce approximately 50% total mortality (data not shown). Each shrimp was injected with 20 μL of 5.0 × 10⁷ CFU mL^-1bacterial suspension into the second abdominal segment. A total of sixty shrimp were randomly selected from each diet group (20 shrimp per replicate tank). Shrimp in the control were also injected with an equivalent level of NaCl (0.85%) as the negative control. During 96 hrs, dead shrimp of each group were monitored and documented, and nine shrimp in the diet group (3 shrimp per replicate tank) were collected to measure total hemocyte count (THC), phenoloxidase (PO), and hemocyanin.

Growth performance: After a 60-day culture period, shrimp growth performance and feed conversion ratio (FCR) were evaluated. Individual body weight was measured using an electronic analytical balance (± 0.01 g accuracy, model: XY600-2C, XingYun, China). The individual survival in each replicate was counted manually, and FCR, defined as the total feed consumed divided by the weight gain, was calculated. Feed intake was determined by subtracting the uneaten feed from the amount of feed offered. Growth parameters were determined as follows:

Weight gain (WG, g) = final body weight – initial body weight

Specific growth rate (SGR) (% day^-1) = ([Ln final weight - Ln initial weight]/ days) × 100

Daily weight gain (DWG) = (Final weight – Initial weight)/ days

Survival rate (%) = (number at harvest ∕ number at stocking) × 100

FCR = feed intake/WG

Biomass (kg m^-3) = (mean final weight × final number of shrimp)/ volume of water

Digestive enzyme and intestinal bacteria analysis: For digestive enzyme and intestinal bacterial analyses, five shrimp per replicate were randomly sampled. Following the previously reported method by Phan et al. (2024), protease and amylase were measured according to the methods of Lowry et al. (1951) and Bernfeld (1955), respectively. For leu-aminopeptidase determination, the enzyme parameters were analyzed following the procedure outlined in Ezquerra et al. (1999). Enzyme activity was expressed as U mg protein⁻¹, where one unit (U) is defined as the amount of enzyme required to catalyze the conversion of 1 µmol of substrate per minute under the assay conditions. Each measurement included three replicates.

To minimize the number of animals used, the same individual shrimp was used for total bacteria, Vibrio spp., and Lactobacillus spp. counts, and immune parameters. The intestines were collected, weighed, and homogenized in 0.85% NaCl. Then, 100 µL of the intestinal homogenate was plated on TSA, MRS, and TCBS agar petri dishes to measure the total bacteria, Lactobacillus spp., and Vibrio spp. counts, respectively. The plates were stored at 30◦C for TSA and TCBS, and at 37◦C for MRS for 24 - 48 h, and density was expressed as CFU g^-1.

Analysis of immune parameters: THC was assessed based on the procedure outlined in Le Moullac et al. (1997), with minor change. Shrimp hemolymph was drawn and immediately added to an anticoagulant solution (pH 7.55). Hemocytes were counted under a microscope at 40× magnification. PO was measured using 3 mg mL^-1 L-dihydrophenylalanine (L-DOPA) as the substrate (Giang et al., 2011). Hemocyanin in hemolymph was detected following the procedure reported by Pascual et al. (2003) and Hagerman (1983).

Data analysis: All results are reported as means ± standard errors (SE). The data met the assumptions of normality and homogeneity of variances, as confirmed by Shapiro-Wilk and Levene’s tests, respectively. Data were analyzed using one-way ANOVA. When significant differences were detected, means were separated using Tukey’s honestly significant difference (HSD) test as a post hoc analysis. All statistical analyses were performed using SPSS (version 22.0).Differences were considered significant at p ≤ 0.05.

RESULTS

Growth performance and survival: At the end of the 60-day feeding trail, growth parameters were significantly higher in shrimp fed diets 1, 2, 3, and 4 (four diets) compared to the control group (Table 1). Specifically, the final weight, weight gain, and DWG values were greatest for diet 3 group and reported significant differences compared with other diets (p ≤ 0.05). Shrimp fed diet 3 exhibited the highest SGR, which was significantly higher than that of shrimp fed diet 1, diet 2, and the control (p ≤ 0.05). Shrimp survival rate increased, and FCR values decreased in four diet groups compared with those in the control diet (p ≤ 0.05). No statistical significances in survival rates were observed among diets 1-4.

Table 1. Growth performance of the whiteleg shrimp fed different diets for 60 days

Data are expressed as mean ± SE.; Different letters (a, b, c) within the row indicate significant differences (p ≤ 0.05).

Digestive enzyme activity and bacterial count: The levels of digestive enzyme activity in all diet groups are shown in Fig. 1. Amylase level was significantly greater in the diet 1 and 3 groups compared with the other groups (p ≤ 0.05) (Fig. 1a). All the experimental diets, except diet 2, resulted in higher protease activity compared to the control diet (p ≤ 0.05); however, no significant variation was detected among diets 1, 3, and 4 (Fig. 1b). The diet 3 group exhibited the highest leu-aminopeptidase activity, while no significant differences were observed among diets 1, 2, and 4 (Fig. 1c).

Fig. 1. Digestive enzyme activity of Amylase (a), Protease (b), and Leu-aminopeptidase (c) (U mg protein^-1) in the shrimp’s intestine.

^abcdDifferent letters indicate significant differences among treatments (p ≤ 0.05).

Intestinal bacteria population data are shown in Fig. 2. Supplementation with four different diets had no significant effect on total bacteria density (p > 0.05), except for the diet 3. Shrimp in the experimental groups exhibited significantly reduced Vibrio spp. densities in the intestine than the control diet (p ≤ 0.05). The opposite result was found Lactobacillus spp. density, in which the densities in the diets 1, 2, 3, and 4 showed greatly higher population than the control group (p ≤ 0.05).

Fig. 2. Density of total bacteria (a), Vibrio spp. (b), and Lactobacillus spp. (c) counts in the intestine of whiteleg shrimp after 60 days of feeding experimental diets.

^abcdDifferent letters above the bar indicate significant differences between treatments (p ≤ 0.05).

Capacity for resistance to Vibrio harveyi: The result of the one-way ANOVA showed that the survival rates in the diet 1, 2, 3, and 4 groups were markedly greater compared with the positive control (p ≤ 0.05) (Fig. 3). However, insignificant differences were found between diets 1, 2, 3, and 4 groups (p > 0.05). The negative control group exhibited no mortality, indicating the test conditions did not adversely affect shrimp survival.

Fig. 3. Survival rate of the whiteleg shrimp at 96 h post-challenge with Vibrio harveyi.

Different letters (a, b, c) indicate significant differences between treatments (p ≤ 0.05).

Immune indicators of whiteleg shrimp after challenge with V. harveyi are presented in Fig. 4. The diet 3 group exhibited the highest THC level, which was significantly higher than that of the control group and the other three diet groups (p ≤ 0.05). Similarly, the diet 3 group also showed the greatest values for PO and haemocyanin (Fig. 4b, c), which were greater than in the remaining groups (p ≤ 0.05). Overall, the immune response was markedly higher in the group fed diet 3, including 0.5% noni plus L. plantarum CMT1, compared with the groups fed diets supplemented with noni alone (p ≤ 0.05).

Fig. 4. Immune parameters including Total haemocyte count, THC (a), Hemocyanin (b), and Phenoloxidase, PO (c) in the whiteleg shrimp.

^abcdDifferent letters indicate significant differences among treatments (p ≤ 0.05).

DISCUSSION

Several studies have shown that synbiotics can serve as alternatives to antibiotics, resulting in higher yields and improved sustainability in shrimp aquaculture. In recent years, synbiotics developed from prebiotics - derived from plants and fibers - have been widely recognized in shrimp farming. Notably, plant extracts might serve as prebiotics, supplying substrates for probiotic growth (Kaur et al., 2021; Zhang et al., 2024), which consequently increases growth performance of aquatic animals.Moreover, these extracts metabolized by probiotics are typically absorbed and utilized more efficiently by the host.This study clearly showed that diet administration of either noni leaf alone or combined with probiotic L. plantarum led to improved growth performance in whiteleg shrimp. In addition, the synbiotic treatment containing 0.5% noni leaf extract and L. plantarum produced higher growth performance than diets supplemented with noni leaf extract alone, suggesting a synergistic interaction between the plant extract and the probiotic. This result agrees with the report of Abidin et al. (2022), who demonstrated that the combiningMoringa oleifera leaf extract with L. acidophilus in the diet resulted in greater weight gain (595.2 ± 11.6%) in whiteleg shrimp than either supplementation alone (459.0 ± 23.4%). Previous studies have shown that increased digestive enzyme activity and higher total intestinal bacterial counts can enhance feed utilization efficiency, thereby supporting growth (Abdel-Ghany et al., 2020; Khanjani et al., 2024). A 60-day feeding trial with noni leaf extract either alone or in combination with Lactobacillus plantarum CMT1 increased the survivability in L. vannamei, likely due to the regulation of intestinal microbial population by the prebiotic and synbiotic treatments.

Shrimp intestinal bacteria are essential for maintaining shrimp health and overall well-being by improving digestion and nutrient absorption, protecting against pathogens, and regulating immunity (Holt et al., 2021). As indicated by the findings of this study, the addition of noni leaf extract or its combination with L. plantarum CMT1 significantly decreased Vibrio density and increased Lactobacillus spp. population in shrimp intestine. These findings suggest that synbiotic supplementation effectively modulates the microorganism flora in the shrimp intestines. Similarly, the report of Obeng-Boateng et al. (2024) indicated that bioactive compounds of noni leaf are natural antibiotics and can suppress pathogenic bacteria. In addition, noni leaves are rich in carbohydrates (Singhet al., 2008), which can serve as a substrate to promote the proliferation of probiotics. These probiotics produce organic acids during fermentation, thereby reducing the intestinal pH (Hassan et al., 2020) and creating unfavorable conditions for pathogenic organisms, such as Vibrio spp. Another explanation is that prebiotics could promote the proliferation of beneficial bacteria while suppressing harmful species by competing with glycoconjugates on the intestinal epithelial surface. This interaction enhances mucus secretion and increases the synthesis of short-chain fatty acids and cytokines, contributing to enhanced gut health and immune responses (Hoseinifar et al., 2017). The probiotic component, L. plantarum in synbiotics produces antimicrobial compounds, such as bacteriocins, which inhibit growth of pathogens on the intestinal wall and help reinforce the mucosal barrier (Echegaray et al., 2023). Prabawati et al. (2022) demonstrated that synbiotic-supplemented diets for whiteleg shrimp significantly increased Lactobacillus sp. with a concurrent decrease in Vibrio genus bacteria in the digestive tract. Moreover, Chin et al. (2025) revealed that administration of synbiotics derived from L. plantarum L20 and Sargassum polycystum significantly increased the abundance of Lactobacillaceae in the black tiger shrimp intestines. This finding indicated that supplementation of 0.5-1% noni leaf extract combined with L. plantarum CMT1 through the diet markedly increased the total Lactobacillus population in the gastrointestinal tract of L. vannamei.

The findings also showed that enzyme activity in shrimp fed with a synbiotic-supplemented diet plus 0.5% noni leaf extract was greater than that in shrimp fed the remaining diets. These findings suggest a synergistic interaction between noni leaf extract and L. plantarum CMT1 that enhances digestive function and supports nutrient absorption and growth performance. This may be explained by phytochemicals in noni leaves that enhance the probiotic activity of L. plantarum by acting as selective growth-promoting substrates and quorum-sensing modulators. They may also exert antimicrobial effects that suppress competing pathogens, thereby improving gut microbial balance. However, higher inclusion levels of plant extracts may introduce excessive bioactive compounds, such as phenolics, tannins, or other secondary metabolites, which could negatively affect feed palatability, digestive processes, or nutrient utilization. Therefore, a moderate level (0.5%) of noni leaf extract may provide an optimal balance of beneficial compounds, while higher levels (1%) may reduce its positive effects. Notably, dietary administration of synbiotic also showed the highest levels of proteases and leu-aminopeptidase, which are vital for protein digestion and assimilation. Similarly, Pardede et al. (2024) showed that synbiotic supplementation enhanced amylase and protease levels, resulting in increased digestive system and whiteleg shrimp growth. Abidin et al. (2022) showed significant enhancement in intestinal enzyme activities in L. vannamei fed a mixture of L. acidophilus and Moringa oleifera leaf. Similarly, Hasyimi et al. (2020) observed that a combination of 0.5% honey and Bacillus sp. greatly influenced protease, amylase, and lipase activities in the gut of the whiteleg shrimp. These improvements in digestive enzyme production are likely associated with the increased abundance of beneficial bacteria in the intestine, which could produce exogenous enzymes and potentially stimulate endogenous enzyme activity (Maas et al., 2021; Rahayu et al., 2024).

Dietary supplementation of noni leaf extract individually or mixed with L. plantarum CMT1 improved the defensive responses against infection with V. harveyi (10⁸ CFU mL^-1) in the whiteleg shrimp. Several studies have demonstrated reductions in THC and PO activity in whiteleg shrimp after pathogen injection (Ekasari et al., 2014; Wang and Chen, 2005). In this study, although THC, PO, and hemocyanin decreased after the challenge test in all groups, shrimp fed noni leaf extract alone or in combination with L. plantarum CMT1 showed higher levels compared to the control group. This finding is partially consistent with the observation of Oktaviana et al. (2014), who demonstrated the administration of synbiotics in shrimp diet markedly increased THC and PO after co-injection of IMNV and V. harveyi. The enhancement in survival from the experimental groups was associated with an increase in PO and THC values compared with the control group. Previous studies have reported that increased immune responses to various infections are due to enhanced THC and PO level, which promote protection against pathogens, thereby reducing mortality (Oktaviana et al., 2014; Zubaidah et al., 2015). The highest levels of THC, hemocyanin, and PO were recorded in whiteleg shrimp fed 0.5% noni leaf extract combined with L. plantarum CMT1 at 10⁸ CFU kg^-1, which indicates a dose-dependent effect of the diet on the host’s immune response.

The current findings indicate that the supplementation of noni leaf extract, either alone or in combination with L. plantarum CMT1, enhances digestive enzyme activities in whiteleg shrimps, leading to better growth performance, survival, and feed conversion ratios. Supplementation with a synbiotic formulation containing 0.5% noni leaf extract and L. plantarum CMT1 at 10⁸ CFU kg^-1is recommended to promote overall growth rate, feed utilization, and disease resistance in shrimp culture. As noni leaves are a readily available plant resource in Vietnam, their use as a low-cost, locally sourced prebiotic offers practical potential for aquaculture applications.Overall,these results provide a theoretical basis for utilizing plant products in the development of cost-effective prebiotics and synbiotics, thereby supporting sustainable aquaculture and advancing the circular economy. Future studies are needed to optimize dosage and evaluate efficacy under field conditions.

Acknowledgments: This work was supported by the Vingroup Innovation Foundation (VINIF) through the Postdoctoral Scholarship Programme, code VINIF.2024.STS.33, awarded to Phan Thi Cam Tu.

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