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The effects of citral on the intestinal health and growth performance of American bullfrogs (Aquarana catesbeiana)

BMC Veterinary Research volume 21, Article number: 49 (2025) Cite this article

Abstract

Bullfrogs (Aquarana catesbeiana) are increasingly cultivated for their high nutritional value and adaptability to intensive aquaculture systems. However, ensuring optimal intestinal health and growth performance remains a challenge due to poor water quality and high stocking densities. This study evaluated the effects of varying dietary concentrations of citral, a natural compound from lemongrass essential oil, on the intestinal health, microbiota, antioxidant capacity, and growth performance of juvenile bullfrogs. A total of 200 juvenile bullfrogs (initial weight 6.85 ± 0.71 g) were randomly assigned into six groups, each receiving diets supplemented with citral at 0, 1, 2, 4, 8, and 16 mL/kg feed for 8 weeks. Citral supplementation significantly improved intestinal morphology, with goblet cell numbers, mucosal thickness, and villus-to-crypt ratios peaking at 2–4 mL/kg (P < 0.05). Optimal doses of 2–4 mL/kg also enhanced digestive enzyme activities, with α-amylase, lipase, and pepsin activities showing significant increases compared to the control group (P < 0.05). Antioxidant markers, including total antioxidant capacity (T-AOC) and glutathione (GSH), were highest at 2 mL/kg, while higher citral concentrations reduced superoxide dismutase (SOD) and catalase (CAT) activities, indicating potential oxidative stress at 8–16 mL/kg (P < 0.05). Citral also modulated the intestinal microbiota, increasing the relative abundance of beneficial bacteria such as Cetobacterium at 1–2 mL/kg (P < 0.05). However, microbial diversity decreased significantly at concentrations above 4 mL/kg. Growth performance analysis revealed that 4 mL/kg citral supplementation significantly improved weight gain rate (WGR), specific growth rate (SGR), carcass weight (CW), and feed efficiency (FE), while survival rates declined at 16 mL/kg (P < 0.05). A linear regression model determined the optimal dietary citral concentration to be 3.216–3.942 mL/kg. This study concludes that dietary citral at 2–4 mL/kg optimally enhances growth performance, intestinal health, and antioxidant capacity in juvenile bullfrogs, while higher concentrations may disrupt gut health and oxidative balance. These findings provide valuable insights into the use of natural compounds like citral for sustainable aquaculture practices.

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Introduction

Aquaculture has rapidly emerged as a pivotal sector in global food production, driven by the escalating demand for sustainable and high-quality protein sources [1]. Among the species cultivated, the bullfrog (Aquarana catesbeiana) has garnered significant attention due to its rapid growth, high nutritional value, and adaptability to intensive farming conditions [2, 3]. These attributes make bullfrogs a promising candidate for aquaculture, particularly in regions where traditional protein sources are limited or unsustainable [4]. In China, bullfrog production reached 1 million tons in 2023, with an economic value of 100 billion CNY (about 14.23 billion USD) [5]. However, despite its potential, optimizing the health and growth of juvenile bullfrogs remains challenging, with intestinal health—an essential factor for nutrient absorption, disease resistance, and overall development—posing a significant hurdle.

The gastrointestinal tract (GIT) of aquatic organisms, including bullfrogs, is a complex system that performs dual roles: enabling nutrient digestion and absorption, while acting as a primary barrier against pathogens and environmental toxins [6]. Maintaining intestinal health is thus essential for ensuring the overall well-being and growth efficiency of farmed species. The integrity of the gastrointestinal tract (GIT) is upheld by three principal barriers: the physical barrier, constituted by intestinal epithelial cells and tight junctions that effectively obstruct harmful substances from infiltrating the gut lining; the chemical barrier, which includes digestive and antioxidant enzymes that play key roles in breaking down food and neutralizing harmful substances, such as free radicals, to protect gut tissues from oxidative damage; and the microbial barrier, established by gut microbiota that outcompete pathogens and modulate immune responses [7, 8]. However, in intensive aquaculture, bullfrogs are frequently subjected to stressors such as suboptimal water quality, high stocking densities, and inconsistent feed quality, which may compromise gut integrity, impair nutrient absorption, and increase susceptibility to infections [9]. Addressing these challenges is essential for enhancing the sustainability and productivity of bullfrog aquaculture.

Current strategies to improve intestinal health and growth performance in aquaculture increasingly focus on dietary supplements, particularly those derived from natural sources [10, 11]. One such compound that has garnered considerable attention is citral, a monoterpenoid found in the essential oils of plants like lemongrass and lemon myrtle [12]. Citral is known for its potent antimicrobial, anti-inflammatory, and antioxidant properties, which have been shown to benefit a range of aquatic species by improving gut health, enhancing immune responses, and reducing oxidative stress [13,14,15]. In previous studies, dietary citral supplementation has been associated with improved growth, feed conversion ratios, and disease resistance in species such as silver catfish (Rhamdia quelen) [16], tilapia (Oreochromis niloticus) [17], and brine shrimp (Artemia franciscana) [12].

Despite promising findings in other species, the specific effects of citral on the growth and intestinal health of juvenile bullfrogs remain underexplored. The existing literature lacks comprehensive data on how citral supplementation specifically affects intestinal morphology, gut microbiota composition, and overall health in bullfrogs under intensive farming conditions. This gap in research is particularly significant given the unique physiological and environmental challenges inherent in bullfrog aquaculture. Without a clear understanding of how citral influences these factors in bullfrogs, it is difficult to develop optimized feeding strategies that fully leverage the potential benefits of this natural compound.

The present study seeks to address this gap by investigating the effects of different dietary concentrations of citral on the growth performance, intestinal morphology, digestive and antioxidative enzyme activities, and gut microbiota of juvenile bullfrogs. This research provides valuable insights into how natural compounds like citral can be leveraged to enhance the sustainability and productivity of aquaculture systems.

Materials and methods

Preliminary preparation

Six different citral concentrations were formulated as follows: 0 mL/kg (control), 1 mL/kg, 2 mL/kg, 4 mL/kg, 8 mL/kg, and 16 mL/kg. The citral (95% purity) was sourced from SIGMA, USA. Citral solutions were diluted to these concentrations and evenly sprayed onto the surface of the feed. The control feed was sprayed with an equivalent amount of distilled water. After spraying, the feed was sealed and dried in the shade before being administered to the animals. The feed was prepared fresh and used immediately.

Animal and feeding

This experiment was conducted at the Zhuhai Experimental Base of the South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences. The bullfrog A. catesbeiana specimens were purchased from Wubo Animal Husbandry and Aquaculture Co., Ltd. (Huizhou, China). The juvenile American bullfrogs (6.85 ± 0.71 g) were acclimated for one week under laboratory condition in a 250 × 148 × 102 cm canvas pool filled with continuously aerated fresh water (20 cm depth, temperature 24.5 ± 0.5 °C; pH 7.1 ± 0.3; 11 L:13D light/dark cycle). During the acclimatization period, the bullfrogs were fed with commercial feed, and excrement and food residues were regularly removed.

After the acclimatization, the bullfrogs were randomly divided into six treatment groups, 0 mL/kg (control), 1 mL/kg, 2 mL/kg, 4 mL/kg, 8 mL/kg, 16 mL/kg with each group consisting of three replicates and 30 bullfrogs per replicate. The experiments were conducted in an indoor environment, with feedings administered twice daily at 8:30 and 18:30. The bullfrogs were fed to apparent satiation during the 8-week trial. Feed intake was monitored by providing an excess amount of feed, and any uneaten feed was collected, weighed, and dried 40 min after feeding to calculate the actual intake. Feed amounts were adjusted daily according to the observed feed intake, with considerations for mortality rate to ensure consistent feeding conditions across all groups. The environmental conditions during the experiment were consistent with those during the acclimatization period, with water changes every two days (100% change). This study utilized commercial feed from SKRETTING biotechnology Co., Ltd (Zhuhai, China), containing crude protein ≥ 42.0%, moisture ≤ 10.0, crude fat ≥ 4.0%, crude fiber ≤ 4.0%, effective lysine ≥ 2.20, crude ash ≤ 15.0, total phosphorus ≥ 1.20, calcium ≤ 4.50. The experimental workflow, including the sampling and analysis procedures, is summarized in Fig. 1.

Fig. 1
figure 1

Schematic representation of the experimental workflow. This figure summarizes the key steps of the study, including animal sampling, sample preparation, biochemical analysis, histological examination, and gut microbiota analysis

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Biochemical analyses

At the end of the experiment, the bullfrogs were euthanized using pithing prior to sampling [18]. This method, which involves destroying the central nervous system, is widely accepted for amphibians as it quickly and effectively induces loss of consciousness without the need for additional anesthesia. Intestinal and blood samples were collected in centrifuge tubes and immediately stored at -80 °C for future analysis. During analysis, the frozen intestinal samples were thawed, cut into small pieces using scissors, and grounded with a cryogenic homogenizer. The homogenate was centrifuged at 3,500 revolutions per minute (rpm) for 10 min at 4 °C, and the supernatant was collected and stored at -80 °C for the determination of biochemical indices.

Bullfrog liver tissues were also collected to measure total antioxidant capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA) and reduced glutathione (GSH) using diagnostic kits from the Jiancheng Bioengineering Engineering Research Institute (Nanjing, China).

Histological examination

Histological examinations were performed on intestinal tissues of three bullfrogs per replicate group. Tissues were collected and fixed in 10% formaldehyde for 24 h, preserved in 75% ethanol, embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (HE). The slides were analyzed using a Nikon Eclipse E100 Micronal microscope (Japan) equipped with a Nikon DS-U3 digital imaging system. Morphological alterations in the intestinal tissue were examined under 400× magnification.

Gut microbial population analyses

The intestinal contents were collected in sterile tubes and stored at -80 °C until analysis. Total microbial DNA was extracted using a DNA extraction kit, and the concentration and purity of DNA were assessed by 1% agarose gel electrophoresis. DNA was amplified using primers specific for the V3 to V4 region of the 16 S rRNA gene, and the purified PCR products were sequenced by Illumina NovaSeq 6000. Sequencing services were performed by Wekemo Tech Group Co., Ltd (Wekemo Bioincloud, Shenzhen, China). The reads obtained from sequencing were filtered to remove low-quality reads and processed to remove noise and correct sequencing errors, resulting in the final valid data. The taxonomic annotation of the feature sequences was performed using SILVA as a reference database, and the diversity and composition of the microbial communities were further analyzed based on the sequencing results using QIIME and R language.

Calculating and statistical analyzes

The formulas for calculating growth performance are presented in Table 1. The statistical comparisons were performed using One-way analysis of variance (ANOVA) with Duncan’s multiple-range test to identify significant differences between the treatments, using IBM SPSS 26.0 statistical software [5]. Statistical significance was considered when P < 0.05. The results are presented as the mean ± standard deviation (SD) of three replicates. Graphs and plots were generated using GraphPad Prism 10 and Origin 2021.

Table 1 Index formula for growth performance and survival rate of American bullfrogs (Aquarana catesbeiana)
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Results

Effects of citral on the intestinal morphology in bullfrogs

The intestinal morphology and related parameters are presented in Fig. 2 and summarized in Table 2. Histopathological analysis revealed significant changes in the intestinal structure across treatment groups. As shown in Table 2, the number of goblet cells in the 1, 2, and 4 mL/kg treatment groups was significantly higher than in the control group (P < 0.05). Goblet cells play a crucial role in maintaining intestinal mucosal integrity by secreting mucus, which acts as a protective barrier. The observed increase suggests an enhanced protective response in these treatment groups, followed by a gradual decline at higher citral concentrations. The inclusion of citral significantly enhanced the thickness of the muscular layer and mucosal thickness (P < 0.05), peaking at 2.0 mL/kg before subsequently decreasing. The thickened muscular and mucosal layers may indicate an adaptive mechanism to support increased nutrient absorption and mechanical digestion.

Villus length was significantly increased in the citral-treated groups compared to the control group, with the maximum observed at 8 mL/kg (P < 0.05). Longer villi are associated with increased surface area for nutrient absorption, indicating improved intestinal functionality in the treatment groups. Similarly, the villus-to-crypt (V/C) ratio was significantly higher in the 1, 2, 4, and 8 mL/kg groups (P < 0.05). A higher V/C ratio reflects better epithelial cell turnover and intestinal health, suggesting a positive impact of citral at these concentrations. However, in the 16 mL/kg treatment group, there were no significant differences in the V/C ratio compared to the control group (P > 0.05), indicating potential detrimental effects at higher citral concentrations.

Histopathological examination also showed signs of epithelial disorganization and mild inflammation at 16 mL/kg, which could account for the diminished benefits at this concentration. These findings collectively suggest that while citral positively affects intestinal morphology at optimal concentrations, excessive levels may induce stress or adverse structural changes.

Fig. 2
figure 2

Photomicrography of the intestinal tissue of A. catesbeiana. All images were captured at a magnification of 10x, accompanied by a scale bar measuring 240 μm. Notes the structure of intestinal tissue, such as (A) 1 mL/kg, (B) 2 mL/kg, (C) 4 mL/kg, (D) 8 mL/kg, F) 16 mL/kg, muscle thickness (MT), villus length (VL), villus width (VW), crypt depth (CD), goblet cell (GC)

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Table 2 Effect of dietary citral on the intestinal morphology indices of A. catesbeiana
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Influences of citral on the digestive enzyme activities in the intestinal

The intestinal digestive enzyme activities showed that the addition of citral increased the activity of α-amylase (AMS), with the highest activity observed in the 4 mL/kg treatment group (P < 0.05). The AMS activity decreased in the 8 mL/kg and 16 mL/kg groups, showing no significant difference compared to the control group (P > 0.05) (Fig. 3B). Also, citral significantly enhanced pepsin (PP) activity in the treatment groups, with PP activity rising as citral concentration rose (P < 0.05) (Fig. 3A). As shown in Fig. 3C, lipase (LPS) activity was significantly higher in the 1 mL/kg, 2 mL/kg, and 4 mL/kg groups compared to the control group, peaking in 4 mL/kg (P < 0.05). Conversely, LPS activity in the 8 mL/kg and 16 mL/kg groups did not differ significantly from the control.

Fig. 3
figure 3

Effects of citral on the levels of digestive enzymes in A. catesbeiana. (A) α-amylase (AMS). (B) pepsin (PP). (C) lipase (LPS). Distinct lowercase letters indicate significant differences (P < 0.05)

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Influences of citral on antioxidant and oxidative indicators in the intestine

As displayed in Fig. 4A, the abundance of the antioxidant SOD in the intestines significantly decreased in the 2 mL/kg citral group compared to the control group (P < 0.05). This decrease became even more pronounced at higher dietary citral levels (8.0 mL/kg and 16.0 mL/kg). Similarly, CAT activity also significantly decreased at 8.0 mL/kg and 16.0 mL/kg citral levels (P < 0.05). Compared to the control group, the production of MDA was significantly reduced (P < 0.05) in the intestines of the 2.0 mL/kg group. However, the MDA levels increased in the 4 mL/kg group, though this increase was not statistically significant compared to the control group. MDA levels then significantly decreased in the 8 mL/kg and 16 mL/kg groups (Fig. 4C). The GSH activity in the intestines was significantly higher in the treatment groups than in the control group (P < 0.05), with the highest activity observed in the 2 mL/kg group, followed by a decrease and then another increase. Figure 4E showed that the total antioxidant capacity (T-AOC) was significantly higher in the citral-treated groups compared to the control group, with the 2 mL/kg group showing the highest T-AOC. The overall trend exhibited an initial increase, followed by a decrease, and then a subsequent increase (P < 0.05).

Fig. 4
figure 4

Effects of citral on biomarkers of oxidative stress in the A. catesbeiana intestine. (A) Superoxide dismutase (SOD). (B) catalase (CAT). (C) malondialdehyde (MDA). (D) reduced glutathione (GSH). (E) total antioxidant capacity (T-AOC). Distinct lowercase letters indicate significant differences (P < 0.05)

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Effect of citral with different concentrations on the intestinal microbial diversity of bullfrogs

Alpha and beta diversity

Figure 5A revealed that all experimental groups shared 107 OTUs. Compared to the control group, the number of OTUs increased in the 1 mL/kg treatment group but decreased in the 2 mL/kg, 4 mL/kg, and 8 mL/kg groups. There were no significant differences in the Chao1, Shannon, and Simpson indices among all the groups (P > 0.05). However, the Faith’s Phylogenetic Diversity index indicated that the microbial diversity was significantly lower in the 4 mL/kg, 8 mL/kg, and 16 mL/kg treatment groups compared to the control group (P < 0.05) (Fig. 5B, C, D, E). The microbiota analysis for the OTUs, conducted using Partial-Least Squares Discriminant Analysis (PLS-DA), is presented in Fig. 5E. The analysis demonstrates a clear separation between the control group and the citral treatment groups, with less pronounced differences between the 2 mL/kg, 4 mL/kg, 8 mL/kg, and 16 mL/kg groups and the 1 mL/kg group (P < 0.05).

Fig. 5
figure 5

Intestinal microbial diversity analysis of A. catesbeiana fed with citral-supplemented diets. (A) Petal diagram showing shared and unique operational taxonomic units (OTUs) among groups. (B) Chao1 index. (C) Shannon index. (D) Faith’s Phylogenetic Diversity index. (E) Simpson index. (F) The x-axis and y-axis represent the two selected principal components (X-variate1 and X-variate2), with their corresponding percentages indicating the proportion of sample variation explained by each component. Different colors represent samples from various treatment groups. The proximity of sample groups reflects the similarity in their microbial compositions; closer groups have more similar compositions, while distant groups are more dissimilar. * indicates P < 0.05, ** indicates P < 0.01, and NS (not significant) indicates P > 0.05

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The impact of varying citral concentrations on bullfrog intestinal microbial composition

At the phylum level, the dominant phyla were Firmicutes and Proteobacteria, which together accounted for more than 80% of the total bacterial community. As shown in Fig. 6A, the relative abundance of Firmicutes in the control, 1 mL/kg, 2 mL/kg, 4 mL/kg, 8 mL/kg and 16 mL/kg groups was 82.98%, 48.09%, 70.61%, 83.81%, 88.80%, and 80.94%, respectively. The abundance of Firmicutes was significantly reduced when the citral concentration was 1 mL/kg (P < 0.05). The relative abundance of Proteobacteria in the control, 1 mL/kg, 2 mL/kg, 4 mL/kg, 8 mL/kg and 16 mL/kg groups was 5.14%, 29.34%, 10.78%, 9.02%, 6.81% and 12.30%, respectively. The abundance of Proteobacteria in the 1 mL/kg group significantly increased (P < 0.05), while the 2 mL/kg, 4 mL/kg, 8 mL/kg, and 16 mL/kg groups showed higher levels in relation to the control group, though these increases were not statistically significant (P > 0.05). The next most abundant phyla were Bacteroidota and Fusobacteriota. There were no significant differences in the abundance of Bacteroidota among all experimental groups (P > 0.05). The relative abundance of Fusobacteriota was 2.83% in the control group, and 8.55%, 9.30%, 0.43% and 0.81% in the 1 mL/kg, 2 mL/kg, 4 mL/kg, 8 mL/kg and 16 mL/kg groups, respectively. The abundance of Fusobacteriota in the 1 mL/kg and 2 mL/kg groups increased compared to the control group, but these changes were not statistically significant (P > 0.05) (Fig. 6C).

The relative abundance of intestinal bacteria at the genus level is shown in Fig. 6D. Listeria was the dominant genus across all groups, with the highest relative abundance observed in the 8 mL/kg group (75.76%) and the lowest in the 1 mL/kg group (15.96%), though the differences were not significant. Citral significantly increased the relative abundance of Cetobacterium in the intestinal microbiota of bullfrogs in the 1 mL/kg group (P < 0.05). The relative abundance of Malacoplasma in the 1 mL/kg group was higher compared to the control group, but this increase was not statistically significant (Fig. 6D).

Fig. 6
figure 6

Effects of citral with different levels of A. catesbeiana on Intestinal microbial composition. (A) phylum level. (B) genus level. (C)-(D) Results for the phylum and genus level are represented as means ± SEM (n = 6) and analyzed using ANOVA followed by Duncan’s test. Distinct lowercase letters indicate significant differences (P < 0.05)

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Effects of citral on the growth performance of bullfrogs

Table 3 showed that, the survival rate of the treatment group receiving 16 mL/kg of citral in the bullfrogs feed was significantly lower than that of the control group (P < 0.05). No significant differences were observed in survival rates among the other groups (P > 0.05).

As shown in Table 3, after 8 weeks of the experiment, the group treated with 4 mL/kg of citral showed a significant increase in WGR, SGR, carcass weight (CW), and feed efficiency (FE) compared to the control group, with all values being the highest among the experimental groups (P < 0.05).

The hind leg index (HLI) increased with higher concentrations of citral, with the highest HLI observed in the group treated with 8 mL/kg (P < 0.05). At week 8, the groups treated with 4 and 16 mL/kg also exhibited significantly higher FE compared to the control group, with the highest FE observed in 16 mL/kg (P < 0.05).

Based on the analysis of SGR, WGR, and FE using a linear regression model, the optimal concentration of citral in bullfrog feed was determined to be 3.216–3.942 mL/kg (Fig. 7).

Table 3 Growth performance of A. catesbeiana fed with diets containing different doses of citral for 8 weeks
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Fig. 7
figure 7

(A) Broken-line model between the specific growth rate and dietary acidifier levels. (B) Broken-line model between the weight gain rate and dietary acidifier levels. (C) Broken-line model between the feed efficiency and dietary acidifier levels. The letter “a” represents the optimal supplementation level of dietary citral

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Discussion

The intestinal barrier comprises mechanical, chemical, microbial, and immune barriers, which collectively form the first line of defense in maintaining intestinal health [19]. Therefore, maintaining gut health depends not only on the physical integrity of the intestinal structure but also on the balance of gut microbiota, which plays a pivotal role in nutrient absorption, immune regulation, and metabolic processes [20,21,22]. Citral, a compound predominantly derived from lemongrass oil and other essential oils, has been demonstrated to modulate gut microbiota and enhance gut health [17, 23]. Additionally, antioxidant enzymes are essential formaintaining redox balance and protecting the intestine from oxidative damage [24]. This study investigates the effects of citral on bullfrog gut health by examining its regulatory influence on intestinal enzymes and gut microbiota across various concentrations, thereby elucidating the interplay between digestive enzyme activity, microbiota composition, and antioxidant defense in sustaining optimal gut health.

The intestinal physical barrier is crucial for digestion and absorption, acting as a defense against harmful substances and playing an essential role in immune defense, which impacts growth, development, and overall health [25, 26]. The physical barrier primarily consists of tight junctions between epithelial cells and the mucous layer covering the surface of intestinal mucosal epithelial cells [27]. Among these, intestinal villi are finger-like projections of the intestinal epithelial cell layer, serving to significantly enhance the surface area of the intestine and facilitate nutrient absorption. Larger villus length and width indicate a greater absorptive surface area and enhanced absorption capacity [28]. The intestinal muscularis also plays a role in the transport and absorption of food within the intestine and can serve as an indicator of intestinal function [29]. Crypt depth is associated with the maturity and secretory function of intestinal cells; shallower crypts indicate better cellular maturity and secretory function. The ratio of villus height to crypt depth (V/C) assesses the overall intestinal function. A longer villus length and shallower crypt depth result in a higher V/C ratio, reflecting stronger intestinal absorption capability, and vice versa [30]. In the present study, compared to the control group, the thickness of the muscularis, villus length, and villus width increased with higher concentrations of citral. Conversely, crypt depth decreased with increasing citral concentration. Except for the highest concentration group (16 mL/kg), the V/C ratio in other experimental groups was significantly higher than that in the control group (P < 0.05).

Goblet cells are situated between epithelial cells and play a crucial role in secreting mucus that coats the intestinal lining, thereby forming a protective mucus layer [31]. This mucus layer functions by trapping pathogens and preventing their direct contact with epithelial cells. Consequently, the number and secretory capacity of goblet cells are vital to maintaining the integrity of the physical barrier. Insufficient goblet cells secretion or impaired function can lead to a thinner intestinal mucus layer, reducing barrier efficacy and increasing susceptibility to infections or inflammation [32]. Conversely, an increased number of goblet cells correlates with enhanced mucus secretion, thereby providing a more effective physical barrier [33]. In this experiment, bullfrogs treated with citral concentrations of 1, 2, and 4 mL/kg was significantly more goblet cells compared to the control group. This finding suggests that citral enhances the integrity of the intestinal physical barrier in bullfrogs. The results of this experiment suggest that citral may enhance the gut health of bullfrogs, thereby serving as an effective feed additive to improve growth efficiency and economic returns in aquaculture.

The gut’s chemical barrier consists of a diverse array of chemicals and enzymes that regulate the intestinal environment’s chemical balance, protecting it from pathogens and harmful substances [34]. Digestive enzyme activity, influenced by factors such as feed composition, rearing conditions, and feeding strategies, is commonly used as an indicator for assessing physiological function and nutritional status [35, 36]. Protease, amylase, and lipase responsible for the digestion and absorption of proteins, carbohydrates, and fats, respectively [37]. Increased activity of these digestive enzymes can enhance nutrient digestion and absorption, thereby improving growth performance [36].

In this study, citral supplementation demonstrated a dose-dependent effect on digestive enzyme activity in bullfrogs, significantly enhancing pepsin (PP), α-amylase (AMS), and lipase (LPS) activities at specific concentrations. The PP activity notably increased with higher citral concentrations (P < 0.05), suggesting that citral may promote protein digestion by enhancing pepsin secretion or activation [38]. Similarly, AMS activity was significantly elevated in the 4 mL/kg group (P < 0.05), indicating enhanced carbohydrate digestion, though no further increase was observed at 8 mL/kgor16 mL/kg, possibly due to citral’s adverse effects on the intestinal environment at higher doses [39]. For LPS, significant improvements were observed in the 1 mL/kg, 2 mL/kg, and 4 mL/kg groups, with the greatest effect in the 4 mL/kg group (P < 0.05). However, at higher concentrations (8 mL/kg and 16 mL/kg), LPS activity showed no significant difference from the control group, mirroring the trends in growth performance. Overall, citral exerted significant benefits on digestive enzyme activity at optimal doses, while excessive concentrations may reduce its efficacy.

While citral has been shown to enhance the activities of digestive enzymes, which are crucial for nutrient absorption and overall metabolic function, it is imperative to consider the potential oxidative stress that may arise from increased metabolic activity. The heightened breakdown of nutrients, particularly at elevated doses of citral, could result in the production of reactive oxygen species (ROS) as byproducts of metabolism, thereby affecting the antioxidant defense mechanisms within the intestine. Figure 4 shows a significant reduction in the activities of superoxide dismutase (SOD) and catalase (CAT) in the intestine when citral was added at concentrations of 8.0 mL/kg and 16.0 mL/kg (P < 0.05). SOD and CAT are crucial antioxidant enzymes responsible for eliminating excess reactive oxygen species (ROS) and maintaining cellular redox balance [40]. Experiments conducted by Kang et al. [41] and Ju et al. [42] indicate that citral can induce an elevation in ROS levels within cellular environments. Furthermore, the study by Lukić et al. [43] demonstrated that oxidative stress exposure in green frogs (Pelophylax esculenta) resulted in a significant reduction of CAT and SOD activity. Therefore, high doses of citral may induce oxidative stress in the intestine, leading to elevated ROS levels, which could influence the activities of SOD and CAT. These findings align with Tawfeek et al. [44], who reported the modulation of antioxidant enzymes under different oxidative stress conditions in aquatic animals, highlighting the dual role of environmental factors and dietary interventions. However, it is also possible that citral may reduce ROS production, leading to a decrease in the need for antioxidant enzyme activity [45, 46]. This dual effect suggests that citral may trigger oxidative stress, it might also regulate ROS levels, potentially reducing the necessity for high antioxidant enzyme activity.

Malondialdehyde (MDA), a marker of lipid peroxidation, reflects oxidative damage to cell membranes [47]. In the 2.0 mL/kg citral group, MDA levels were significantly reduced (P < 0.05), indicating that this concentration provides antioxidant protection by reducing lipid peroxidation. However, at higher doses (4–16 mL/kg), MDA levels initially rose before declining, suggesting that higher citral concentrations might induce oxidative stress, which the body counteracts by activating other antioxidant mechanisms like increasing non-enzymatic antioxidants such as glutathione (GSH).

GSH, a key non-enzymatic antioxidant [48], was significantly elevated in the 2 mL/kg group (P < 0.05), indicating enhanced antioxidant capacity. However, as citral doses increased, GSH activity initially decreased due to oxidative stress but later rose, likely as a compensatory response, aligning with findings in Salmo salar [49].

Total antioxidant capacity (T-AOC), an overall measure of antioxidant ability [50], followed a similar pattern, peaking at 2 mL/kg and fluctuating at higher doses (P < 0.05). This indicates that moderate citral supplementation (1–2 mL/kg) significantly enhances antioxidant capacity, reducing oxidative damage, while higher doses may temporarily induce oxidative stress.

A relatively balanced microecosystem is established between intestinal microbes and their hosts, serving as a microbial barrier against pathogen infection [51]. Intestinal microbiota plays a crucial role in maintaining barrier functions, enhancing nutrient absorption, and boosting immunity, making it closely related to overall health [52]. Research indicates that citral, a natural plant extract with antimicrobial and antioxidant properties, can enhance immune response and disease resistance by promoting beneficial bacteria growth and reducing pathogenic bacteria numbers [23, 53, 54].

In this study, the petal plot and alpha diversity analysis showed that citral concentrations above 4 mL/kg significantly reduced richness of intestinal microbiota in bullfrogs. PLS-DA results indicated a clear separation between the CON group and the all citral-treated groups, demonstrating that citral supplementation significantly altered the intestinal microbiota composition. As citral concentration increased, the intestinal microbiota structure in the experimental groups became more similar. This may be due to increased selective pressure, which favors the survival of more tolerant strains, thereby reducing microbial diversity and leading to a convergent microbiota structure.

At the phylum level, Firmicutes and Proteobacteria were dominant in all experimental groups, comprising for over 80% of the total bacterial community, consistent with findings from Wang et al. [54] and Wang et al. [55]. When citral was added at 1 mL/kg, the abundance of Firmicutes significantly decreased, while Proteobacteria abundance significantly increased (P < 0.05). With higher concentrations, there were no significant differences in the abundances of Firmicutes and Proteobacteria compared to the control group. Firmicutes are typically associated with energy absorption and fat deposition [56]. The decrease in Firmicutes abundance might indicate an inhibitory effect of low citral concentrations on these bacteria. The increase in Proteobacteria abundance could be related to the decrease in Firmicutes, as the reduction in Firmicutes might create ecological space that reduces competition with beneficial bacteria, thereby facilitating Proteobacteria growth [57].

At the genus level, the relative abundance of Bacteroides significantly increased in the 1 mL/kg group (P < 0.05). Cetobacterium, which is associated with amino acid metabolism in fish and amphibians, increased significantly, which might contribute to improved nitrogen balance and protein metabolism in bullfrogs [58,59,60]. This suggests that low doses of citral could have beneficial effects on the gut health of bullfrogs by promoting the growth of this beneficial bacterium.

Citral supplementation significantly alters the composition and diversity of the bullfrog intestinal microbiota. Low doses of citral may optimize the gut microbiota by inhibiting some bacteria and promoting beneficial species, thereby enhancing gut health. Similar to findings by Sherif et al., dietary modifications were shown to influence the diversity of intestinal microbiota, suggesting that selective pressures induced by citral may create an ecological niche favoring beneficial species [61]. However, high doses of citral may induce substantial shifts in microbial structure, potentially impairing the digestive function and general health of the bullfrogs.

Intestinal health is essential for the growth and productivity of animals, as it directly affects nutrient absorption, metabolic efficiency, and immune function [62]. A well-functioning digestive system ensures that animals can efficiently convert feed into energy and biomass, leading to better growth performance and feed utilization [63]. In contrast, poor gut health can impair digestion, reduce nutrient absorption, and increase susceptibility to disease, all of which negatively impact growth rates and feed conversion [64]. Therefore, maintaining gut integrity and functionality is critical in aquaculture settings. In this study, after 8 weeks of cultivation, the experimental group with 4 mL/kg of citral in their feed showed a significant increase in weight gain rate (WGR), specific growth rate (SGR), and feed efficiency (FE) compared to the control group. Brum et al. [65] demonstrated that 5% Zingiber officinale essential oil (containing 41.1% citral) can increase the WGR and SGR in Nile tilapia (Oreochromis niloticus). Similarly, Hadi Pratama et al. [66] showed that dietary citral could increase the SGR and FE of shrimp at concentrations of 75 mg/kg and 100 mg/kg. These findings are consistent with the results of this experiment. Slaughter performance is a crucial index for evaluating the growth performance of meat animals [67]. Sherif et al. demonstrated that vitamin-based dietary interventions significantly enhanced growth and immune performance in Nile tilapia, paralleling the growth-promoting effects observed with citral supplementation in bullfrogs [68]. Also, the results of this experiment indicate that after 8 weeks of citral supplementation in the feed, there was an improvement in the carcass weight (CW) and hind leg index (HLI) of bullfrogs. The increase in CW and HLI suggests that citral can enhance the slaughter performance of bullfrogs, which is consistent with the findings of Rahman et al. [69] and Safwat et al. [70].

Conclusion

This study demonstrates that citral supplementation has a significant impact on bullfrog gut health by modulating gut microbiota composition, digestive enzyme activity, and antioxidant enzyme levels. At low concentrations (1–4 mL/kg), citral significantly enhanced the growth performance of bullfrogs, digestive enzyme activity, and the abundance of beneficial bacteria, such as Cetobacterium, which are associated with improved nutrient absorption and gut health. However, higher citral concentrations (above 4 mL/kg) led to reduce in microbial diversity and a decrease in antioxidant enzyme activities, including superoxide dismutase (SOD) and catalase (CAT), indicating the onset of oxidative stress. This suggests that while citral offers considerable benefits at certain concentrations, excessive amounts may compromise gut health and oxidative balance. Therefore, optimal citral dosages (2–4 mL/kg) should be carefully considered to maximize its positive effects while mitigating potential adverse impacts on bullfrog gut health.

Recommendation

To promote the sustainability and efficiency of bullfrog farming, it is advisable to incorporate citral as a feed additive in commercial formulations. Future research should focus on validating the field applicability of citral supplementation and evaluating its cost-effectiveness under large-scale farming conditions. Moreover, examining the long-term effects of citral on immune function and stress resistance, as well as exploring its potential utility in other aquaculture species, could significantly broaden its industrial applications.

Data availability

Data will be made available on request.

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Acknowledgements

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Funding

This work was supported by Key-Area Research and Development Program of Guangdong Province (2021B0202030001), Guangdong Province Modern Agricultural Industrial Technology System Innovation Team building project (2022KJ150), Guangzhou Rural Science and Technology Commissioners project (2023E04J1299), Central Public-interest Scientific Institution Basal Research Fund, CAFS (NO.2023TD97, 2021SD19), Guangdong Agricultural Technology Service Light Cavalry Major Agricultural Technology Rural Promotion Project (NJTG20240250), Guangdong Province Science and Technology Commissioner Supporting Village Project (KTP20240452).

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Authors and Affiliations

  1. Key Laboratory of South China Sea Fishery Resources Exploitation & Utilization, Ministry of Agriculture and Rural Affairs, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, 510300, China

    Xiaoting Zheng, Qiuyu Chen, Xueying Liang, Jingyi Xie, Hongbiao Dong & Jiasong Zhang

  2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, 201306, China

    Xiaoting Zheng & Jinlong Yang

  3. Faculty of Maritime Engineering and Marine Sciences (FIMCM), Escuela Superior Politecnica del Litoral (ESPOL), Guayaquil, 09015863, Ecuador

    Alfredo Loor

  4. Sanya Tropical Fisheries Research Institute, Sanya, 572018, China

    Hongbiao Dong & Jiasong Zhang

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  1. Xiaoting Zheng
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Contributions

X. Zheng contributed to the study by assisting in the experimental design, providing critical revisions, and editing the manuscript for clarity and coherence. Q. Chen was responsible for the conceptualization of the study, overseeing validation processes, managing data curation, conducting investigations, creating visualizations, and drafting the original manuscript. X. Liang performed key experimental analyses that contributed to the study’s findings. J. Xie participated in data curation, ensuring accurate collection and organization of the data used in the study. A. L. contributed by conducting formal analysis of the data and providing important revisions to improve the manuscript. H. Dong assisted in the design of the experiments, contributing valuable insights to the study’s methodological framework. J. Yang was involved in data curation and contributed to the study’s methodology by ensuring the proper application of experimental techniques. J. Zhang secured the funding for the research, contributed to the experimental design, performed formal analysis, and provided critical revisions to the manuscript. And all authors agreed to be accountable for all aspects of the work.

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Correspondence to Jiasong Zhang.

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This study was conducted using bullfrogs, with animal care strictly adhering to the Management Rule of Laboratory Animals (Chinese Order No. 676 of the State Council, revised 1 March 2017). The protocols for animal collection and handling were strictly followed in accordance with the guidelines of the Institutional Animal Care and Use Committee on Laboratory Animal Welfare and Ethics at the South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences (SCSFRI–CAFS; Institutional Review Board approval No. nhdf2024–18).

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Zheng, X., Chen, Q., Liang, X. et al. The effects of citral on the intestinal health and growth performance of American bullfrogs (Aquarana catesbeiana). BMC Vet Res 21, 49 (2025). https://doi.org/10.1186/s12917-025-04498-1

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BMC Veterinary Research

ISSN: 1746-6148

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