Introduction
Vitamin C (L-ascorbic acid) is a crucial water-soluble micronutrient that plays a vital role in supporting antioxidant defense, immunity, stress resistance, and collagen synthesis. These functions are essential for improving physiological processes, maintaining tissue integrity, and enhancing overall health, survival, and growth performance in aquatic animals.1–4 Most teleost fish, including eel species, lack the enzyme L-gulonolactone oxidase required for endogenous vitamin C synthesis and therefore depend entirely on dietary sources.5,6 Additionally, ascorbic acid is unstable when exposed to temperature and light, resulting in a significant loss of its activity during processing and storage. Therefore, supplementing vitamin C in artificial feeds used in aquaculture is essential.2,7,8 Although fish can tolerate moderate excesses of vitamin C, excessive supplementation may disrupt redox balance and impair other physiological processes.9 Inadequate dietary vitamin C supplementation may result in detrimental effects in farmed animals, including growth retardation, skeletal deformities, gill filament distortion, fin erosion, hemorrhages, anorexia, and elevated mortality.10,11 Previous research has shown that the dietary vitamin C requirements for freshwater aquatic animal species may range from 150 to 1,200 mg/kg, while for marine and brackish species, it is approximately 156–259.09 mg/kg, depending on the specific species, life stages, farmed conditions, and the forms of vitamin C used (pure or derivative).4,12,13
The swamp eel (M. albus) is a freshwater species that breathes air and can breathe through its skin and mouth. It can live in many different places, such as rice fields, canals, ponds, swamps, and other shallow, muddy freshwater habitats, even when the oxygen levels are low and the salinity levels change across East and Southeast Asia.14–16 M. albus meat is characterized by high protein content, low lipid levels, and the presence of bioactive compounds with nutritional and medicinal value.17,18 The rapid increase in consumer demand, together with advances in artificial breeding and larval rearing technologies, has driven the expansion of swamp eel aquaculture in Vietnam.19–21 However, production efficiency is still limited because this scaleless teleost species has slow growth, high size variations in populations, poor use of artificial feed, and high death rates in the early stages of life.22–24 Moreover, its natural populations have declined markedly due to overexploitation and habitat degradation.24,25 These challenges show the need to implement optimized nutritional strategies to effectively enhance swamp eel production for both conservation and commercial purposes.
Hematological indices are crucial for evaluating the physiological and immunological status of M. albus, which often inhabits areas with poor, highly variable water quality.26–28 Numerous studies have shown that dietary vitamin C has positive effects on hematological and immune parameters in aquacultured species. For example, it has been shown to significantly increase hematocrit, hemoglobin, and red blood cell counts, thereby improving oxygen transport and overall stress resistance.29–32 The available literature on dietary vitamin C in M. albus, however, is limited. Swamp eel fry at the onset of the exogenous feeding stage (≈0.028 g) were fed diets supplemented with pure L-ascorbic acid (80–140 mg/kg). The best growth performance, survival rate, and coefficient of variation were exhibited at 140 mg/kg after 60 days of rearing.33 A 10-week feeding trial on M. albus (≈32.6 g) showed dietary vitamin C in the L-ascorbyl-2-monophosphate (35% ascorbic acid equivalent, Roche) form at 0.1–278.5 mg kg⁻¹ significantly improved growth, antioxidant capacity, immunity, and disease resistance, with quadratic analysis indicating optimal growth at approximately 80.66 mg/kg.23 In migratory eel species, Anguilla japonica juveniles (≈11.0 g), fed diets supplemented with calcium L-ascorbate at 3–3,135 mg/kg, exhibited an estimated optimal ascorbic acid requirement for growth exceeding 27 mg/kg, while higher inclusion levels (≥645 mg/kg) enhanced hematological indices and tissue vitamin C concentrations34; meanwhile, the juvenile (≈75.5 g) showed a significant improvement in liver vitamin C levels, blood biochemical indices, and non-specific immune responses when fed a diet supplemented with ascorbyl-2-monophosphate (762 mg/kg).35 This study aimed to evaluate the effect of dietary vitamin C (L-ascorbic acid) dosage on the rearing performance and hematological indices of M. albus juveniles (≈ 5.6 g). These findings are expected to provide stage-specific data on vitamin C supplementation and contribute to the development of nutritionally balanced feeds for swamp eel aquaculture.
Materials and Methods
Experimental animals and rearing conditions
M. albus juveniles, averaging 5.61 g in weight and 18.69 cm in length, were sourced from a local hatchery and transferred to the Wet Lab at Tra Vinh University. They were transported in slightly oxygenated Styrofoam boxes filled with hatchery freshwater. The travel time was approximately 30 minutes. The juveniles were acclimated to the experimental conditions for one week prior to the start of the investigation. Freshwater for the experiment was sourced from tap water, purified using a reverse osmosis (RO) system, and vigorously aerated for 24 hours to remove chlorine before use. Rectangular Styrofoam basins, each with a volume of 16.4 liters (31 cm long x 22 cm wide x 24 cm high), were used as raising units. Each basin was filled with 7 cm of water and equipped with nylon bundles that covered approximately one-fifth of the bottom surface, creating artificial substrates for the eels. A commercial shrimp pellet feed from Grobest Company (Vietnam), with a particle size ranging from 1.2 to 1.7 mm and a crude protein content of 42%, was supplemented with the powder vitamin C (L-ascorbic acid) at dosages of 0, 80, 140, 200, and 260 mg/kg. The vitamin C was dissolved in water and thoroughly mixed with the pellets approximately 15 minutes before feeding the eels. Proximate nutritional components of basal feed used in experiments are presented in Table 1.
Experimental procedures and data collection
This study was conducted from July to September 2025 at the Aquaculture and Agriculture Wet Lab, Tra Vinh University, Vietnam. A completely randomized design with three replicates was used over a 60-day period. The main objective of the study was to assess the rearing performance and hematological indices of M. albus juveniles under different dosages of dietary vitamin C supplementation. Detailed descriptions of the experiment are provided below:
A total of 1,500 juvenile M. albus eels were randomly stocked in 15 rearing basins, with 100 individuals assigned to each basin. Five dietary vitamin C levels (0, 80, 140, 200, and 260 mg/kg) were tested, with each level replicated in three basins. The juveniles were fed the trial diets twice daily at 07:00 and 17:00 until they appeared satiated, approximately 3–4% of their body weight. Two hours after feeding, uneaten feed and waste were removed, and the water in the basins was completely renewed. Key water quality parameters were monitored daily and remained within the optimal range for M. albus.15,36–38 Water temperature varied from 26.34°C in the morning to 30.68°C in the afternoon, while pH levels ranged from 8.10 to 8.25, as measured with a HANNA portable meter (Model HI98103, Romania). Total ammonia nitrogen (TAN) and nitrite (NO₂⁻) levels in the rearing basins were consistently low and stable, remaining below 0.01 mg/L.
Every 15 days during the rearing period, five eels were randomly sampled from each basin (15 eels per dietary vitamin C level) to assess growth performance. Individual body weights were measured using an electric balance with a precision of 0.01 g, while total lengths were measured with a scaled ruler (in mm). At the conclusion of the experiment, the survival rate and feed conversion ratio (FCR) were recorded and calculated. The following formulas were utilized to determine the monitored performance parameters:
Average weight (AW, g) = total weight of 15 eels / 15
Average length (AL, cm) = total length of 15 eels / 15
Weight gain (WG, g) = final weight - initial weight
Length gain (LG, cm) = final length - initial length
Daily weight gain (DWG, g/day) = (final weight - initial weight) / number of rearing days
Daily length gain (DLG, cm/day) = (final length - initial length) / number of rearing days
Specific growth rate in weight (SGRw, %/day) = ((ln (final weight) - ln (initial weight)) / number of rearing days) × 100
Specific growth rate in length (SGRL, %/day) = ((ln (final length) - ln (initial length)) / number of rearing days) × 100
Survival rate (%) = (final eel number / initial eel number) × 100
Feed conversion ratio (FCR) = total dry feed fed (g)/total wet weight gain (g)
At the end of the experiment, three eels were randomly collected from each basin (nine eels for each dietary vitamin C level) for hematological examination. The eels were anesthetized using ice. Approximately 0.4 mL of blood was drawn from each eel’s caudal vein using a syringe and transferred to EDTA containers (BD Microtainer, UK) to act as an anticoagulant. All hematological determinations were performed according to established fish hematology protocols.39,40 Blood samples were examined for hematological indices, including white blood cells (WBC, 10⁹ cells/L), lymphocytes (LYM, 10⁹ cells/L), mid-sized cells (MID; monocytes, eosinophils, and basophils; 10⁹ cells/L), granulocytes (GRAN, 10⁹ cells/L), red blood cells (RBC, 10¹² cells/L), hemoglobin (HGB, g/L), hematocrit (HCT, %), mean corpuscular hemoglobin (MCH, pg), and mean corpuscular hemoglobin concentration (MCHC, g/L). Analyses were performed using a Urit 3000 Vet Plus hematology analyzer (Urit Medical, China).
Data analysis
All data were analyzed using SPSS v20.0 (Windows). One-way ANOVA followed by Tukey’s post hoc test was used to assess differences among treatments at a significance level of P≤0.05. Levene’s test evaluated variance homogeneity, and percentage data were arcsine-transformed prior to analysis.
Results
From day 15 onward, average weight (AW₁₅–₆₀), WG, DWG, and SGRw increased progressively with dietary vitamin C levels ranging from 80 to 260 mg/kg, with significant improvements at levels ≥ 200 mg/kg, except for MW30 (P ≤ 0.05, Table 2); however, no significant differences were found in all weight growth parameters between the 200 and 260 mg/kg levels (P ≥ 0.05, Table 2).
The effects of dietary vitamin C levels between 0 and 200 mg/kg on the length growth performance of experimental eels were unclear, as most length growth parameters showed no significant differences within this range (P ≥ 0.05, Table 3). However, ML15, ML30, ML60, DLG, and SGRL were significantly higher at 260 mg/kg than at 0 mg/kg (P ≤ 0.05; Table 3).
Survival remained 100%, and FCR was similar across all tested dietary vitamin C levels (P ≥ 0.05; Table 4).
Most hematological indices, including WBC, LYM, RBC, HGB, HCT, and MCHC, showed no significant differences among the dietary vitamin C levels tested (P ≥ 0.05, Table 5). However, MCH values decreased with increasing vitamin C levels, with significant reductions observed at levels ≥ 200 mg/kg (P ≤ 0.05, Table 5). In contrast, MID and GRAN values showed no consistent trend across the tested vitamin C levels (Table 5).
Discussion
Rearing performance
The present study demonstrated that dietary vitamin C (L-ascorbic acid) supplementation markedly improved the weight growth performance of M. albus juveniles. This improvement was indicated by increased values of MW₁₅–₆₀, WG, DWG, and SGRw at dietary vitamin C levels ranging from 80 to 260 mg/kg. These results align with previous findings in fry (Anguilla bicolor) eel reared in cage containers,41 juvenile tilapia (Oreochromis karongae),42 and juvenile Waigieu seaperch (Psammoperca waigiensis)43 reared in aquaria, indicating that dietary vitamin C supplementation significantly increased weight gain. Vitamin C has been documented to promote fish health and growth through multiple physiological and biochemical mechanisms. From a biological perspective, swamp eels have a carnivorous feeding habit and a high capacity for protein accretion, which may increase their demand for micronutrients involved in protein synthesis and tissue repair.18,44 It has been shown that vitamin C is vital for collagen synthesis, tissue repair, and bone formation, which helps maintain structural integrity and promote muscle growth.1,8,45 Additionally, in intensive aquaculture systems, vitamin C helps mitigate oxidative damage induced by high stocking density, handling stress, and fluctuating environmental conditions.2,7,46,47 It also enhances immune function by stimulating leukocyte proliferation, increasing phagocytic activity, promoting lysozyme production, and improving resistance to bacterial pathogens.3,48
Remarkably, the weight growth response stabilized between 200 and 260 mg/kg. Similar growth plateaus at this level have been reported in Anguilla bicolor eels41 and Labeo rohita fish,49 wherein growth parameters also improved with increasing dietary vitamin C levels up to approximately 200 mg/kg, while no further enhancements were noted at higher levels. The finding suggests that 200 mg/kg may be the optimal dietary level for maximizing weight growth efficiency in M. albus juveniles in the current study.
Another observation in this study indicated that dietary vitamin C had a limited effect on length growth in M. albus juveniles, as most length-related indices showed no significant differences up to 200 mg/kg, with clear improvements observed only at 260 mg/kg compared to the control group. The discrepancy between weight and length responses is consistent with previous studies in M. albus23 and other elongated or eel-like species, such as Anguilla japonica.48 Swamp eels exhibit significant morphological plasticity, with growth frequently characterized by increases in body mass rather than linear extension, especially in captivity.24,50 Consequently, vitamin C supplementation may preferentially support muscle accretion and connective tissue development rather than skeletal elongation.
The survival rate of M. albus juveniles was 100%, and no significant differences in FCR were observed among treatments after the 60-day feeding period. Furthermore, throughout the rearing period, no typical signs of vitamin C deficiency, such as lordosis or scoliosis, were detected in fish fed the control diet,10,11,51 indicating that the basal diet probably provided sufficient vitamin C to support survival and normal development of the juveniles, although not optimal growth. Similar results have been reported for juvenile Psammoperca waigiensis, in which dietary vitamin C supplementation significantly enhanced growth performance, and survival remained at 100% across supplementation levels of 0–400 mg/kg.43 Likewise, Anguilla bicolor fry showed no significant differences in survival between vitamin C-supplemented diets and the control.41 The FCR exhibited no significant variation among the treatments, suggesting that the tested dietary levels of vitamin C did not adversely affect feed utilization in the experimental M. albus juveniles.
Hematological responses
In this study, most blood parameters, such as WBC, LYM, RBC, HGB, HCT, and MCHC, did not differ among treatments. This suggests that dietary vitamin C from 0 to 260 mg/kg did not noticeably affect the blood profile of M. albus juveniles under experimental conditions. Similar results were reported in Brycon amazonicus52 and Oncorhynchus mykiss,53 in which higher vitamin C levels did not significantly change hematological values. These findings indicate that once basic vitamin C needs are met, extra supplementation may not further influence baseline blood parameters of swamp eel juveniles. The MCH, however, decreased significantly at dietary vitamin C levels ≥ 200 mg/kg. Similar changes in erythrocyte indices have been reported in matrinxã and rainbow trout fed high vitamin C diets.52,54 The reduced MCH at higher vitamin C supplementation levels likely reflects movement in erythrocyte dynamics without impaired oxygen transport, as HGB and HCT levels remained stable. Thus, it may be an adaptive hematopoietic response rather than a pathological effect.53,55,56 MID and GRAN counts showed some variability among treatments but lacked a clear dose-dependent pattern with dietary vitamin C and were not accompanied by changes in total WBC or lymphocyte counts. The observed variations are likely within normal physiological limits and do not indicate immune activation.1,3,52 Overall, these findings suggest that dietary vitamin C, within the tested range, supports hematological stability in M. albus juveniles.
In summary, this study demonstrated that dietary vitamin C (L-ascorbic acid) supplementation significantly increased weight gain in M. albus juveniles at levels ≥ 200 mg/kg; however, there was no difference between 200 and 260 mg/kg. In contrast, length growth exhibited a relatively limited response to the vitamin C dosages used. Additionally, while the MCH decreased at higher dietary vitamin C levels, the MID and GRAN values showed no consistent trends, and survival rates remained at 100%. FCR, as well as most hematological indices, remained stable across treatments. These findings suggest that the tested dietary vitamin C range had a minimal impact on hematological indices in trial eels. Overall, a dietary vitamin C supplement at 200 mg/kg is recommended as optimal for rearing M. albus juveniles under the study conditions. This study was limited by controlled experimental conditions, a relatively short 60-day duration, and the absence of dose–response modeling to estimate the dietary vitamin C requirement of the trial eels. Additionally, there was a lack of tissue vitamin C quantification and stress or immune challenge tests, which are needed in future research to better support practical aquaculture applications for M. albus.
Acknowledgments
We acknowledge the support of time and facilities from Tra Vinh University (TVU) and Ton Duc Thang University for this study.
Authors’ Contribution
Conceptualization: Huong K. Huong (Equal), Diep X. Doan (Equal). Methodology: Huong K. Huong (Equal), Nam K. Ho (Equal), Diep X. Doan (Equal). Investigation: Huong K. Huong (Equal), Nam K. Ho (Equal), Diep X. Doan (Equal). Writing – original draft: Huong K. Huong (Equal), Diep X. Doan (Equal). Supervision: Huong K. Huong (Lead), Diep X. Doan (Equal). Formal Analysis: Nam K. Ho (Lead). Writing – review & editing: Diep X. Doan (Lead).
Competing of Interest – COPE
No competing interests were disclosed
Ethical Conduct Approval – IACUC
The animal experiments adhered to relevant national and international guidelines. Only the M. albus juveniles underwent weighing and measuring and were anesthetized using ice for blood sampling, ensuring no harm was caused. After the experiments, the eels were returned to the storage tanks for further research.
Informed Consent Statement
All authors and institutions have confirmed this manuscript for publication.
Data Availability Statement
All are available upon reasonable request.