Comparative Analysis of Intestinal Morphometry in Mugil cephalus Reared in Biofloc and Water Exchange System

Abstract

This research aimed to evaluate the effect of biofloc technology on the intestinal morphometry, productive performance, and survival of juvenile Mugil cephalus. An 87-day investigation was conducted with two treatments, each with three replicates. Treatment one involved rearing juvenile M. cephalus in a biofloc system with a C/N ratio of 15:1, and treatment two involved rearing juvenile M. cephalus with a water exchange and no carbon addition. Ninety (90) juveniles of Mugil cephalus with an average weight of 117.36 ± 6.48 g were randomly distributed into six (6) circular plastic tanks of 250 L (fifteen fish per tank). At the end of the experiment, 10% of each experimental unit’s population was sacrificed for intestinal morphometry analysis. The productive performance was evaluated every 30 days by randomly sampling fish from each tank for biometric measurements, including the specific growth rate (SGR), feed conversion ratio (FCR), condition factor (K), and survival. No structural changes were observed in the intestinal mucosa. The fish reared in biofloc exhibited a similar gut morphometry (villus length and villus thickness) compared to the fish in the water exchange system. The biofloc system does not compromise the gut health of mullet. No significant differences (p > 0.05) were observed in the final weight, weight gain (WG), daily weight gain (DWG), specific growth rate (SGR), condition factor (K), and survival between the treatments evaluated. M. cephalus can be reared using biofloc technology, demonstrating significant water savings compared to water exchange systems.

1. Introduction

Mugilids represent a fish family with a great potential to promote diversification and sustainable development in aquaculture [1]. Notable species within this family include Mugil cephalus, M. liza, M. platanus, M. capito, Liza aurata, L. ramada, and L. saliens, all of which are considered eurytopic [2,3]. In terms of productive advantages, Mugilidae present low protein requirements, allowing for reduced levels of fishmeal in formulated feeds [4,5]. Due to its commercial relevance, mullet meat is valued for its favorable texture and flavor, making it an important economic resource in the Mediterranean regions, Black Sea, and Asia [6,7].

The flathead mullet (Mugil cephalus) is a migratory coastal marine species with a distributional range of 32 to 700 km. It exhibits a wide habitat plasticity, making it an ecologically successful species [8]. This species spawns in marine waters and migrates to estuarine and brackish environments for breeding [2,9]. Among mugilids, M. cephalus is the most widely distributed species, occurring throughout the Atlantic, Pacific, and Indian Oceans [5,9,10,11]. In the Eastern Pacific, its range extends from Southern California to Chile [12]. Regarding its feeding habits, it has been described as omnivorous [13] and zooplanktivorous. According to Whitfield et al. [2], during the early stages of its life, it is phytophagous, and in adulthood it is considered detritivorous, with a high preference for microalgae. This species constitutes a significant hydrobiological resource for artisanal fisheries in Chile and other parts of the world [5,14,15]. In terms of aquaculture, countries such as Egypt, Senegal, Italy, and other Mediterranean countries practice extensive and semi-intensive culture [16,17,18]. In most cases, wild juveniles are captured and subsequently stocked in lagoons, lakes, or brackish water ponds for grow-out, followed by harvest and commercialization [2].

The flathead mullet has been identified as a suitable candidate for culture in systems characterized by high loads of organic or particulate matter, demonstrating favorable growth performance and survival rates in environments such as fertilized earthen ponds, periphyton-based systems, and biofloc technology (BFT) systems [10,11,12,13,14,15,16,17,18,19,20]. Given its dietary plasticity and detritivorous tendencies, M. cephalus can be reared with cost-effective, nutritionally balanced feeds incorporating alternative ingredients, which may reduce the reliance on conventional fishmeal-based formulations [5,21]. In this context, feeding strategies that integrate biofloc as a natural and supplemental nutrient source have the potential to enhance growth performance across different developmental stages, while also contributing to sustainability in intensive aquaculture systems.

Biofloc technology (BFT) has been extensively studied in species such as white shrimp (Penaeus vannamei) and Nile tilapia (O. niloticus), demonstrating multiple benefits, including an improved water quality management, enhanced growth performance, strengthened immune response, and increased resistance to specific pathogens [22,23,24,25]. Bioflocs are a complex mixture of various microorganisms (bacteria, protozoa, rotifers, nematodes, microalgae), along with other components such as detritus, uneaten feed, and feces from the cultured organisms. They can be applied for multiple purposes, including as an alternative to antibiotics, antifungal agents, probiotics, and prebiotics [26]. These benefits are largely attributed to the microbial community present in the biofloc, which contributes to nitrogen control via heterotrophic assimilation, and provides a continuous source of protein, lipids, and immunostimulants through the microbial biomass [27,28]. According to some studies, bioflocs may help maintain the integrity of the intestinal tract, as indicated by a greater villus length and diameter, and may protect intestinal epithelial cells through their immunostimulatory effect [29]. Despite these advances, the specific effects of BFT on certain biological and productive parameters remain underexplored in emerging aquaculture species such as the flathead mullet (Mugil cephalus) [30]. Therefore, further studies are needed to evaluate the species-specific physiological responses to BFT conditions in M. cephalus, particularly in relation to digestive morphology and nutrient assimilation.

The fish intestine is a vital organ responsible for key physiological functions such as digestion and nutrient absorption [31,32]. These functions can be directly influenced by factors such as diet quality and the inclusion of functional additives, including prebiotics and probiotics. Several studies have reported significant alterations in intestinal morphometric parameters associated with an enhanced structural integrity and absorptive capacity [31,33,34,35,36,37,38,39]. For example, Mirzakhani et al. [23] demonstrated that Nile tilapia fry reared in biofloc systems exhibited significantly larger intestinal villi compared to fish reared in clear water systems. Similarly, Bakhshi et al. [40] found that common carp cultured in biofloc systems supplemented with beet molasses and corn starch as carbon sources showed an increased villus height. Laice et al. [37] evaluated the use of biofloc with and without the addition of prebiotics/probiotics in juvenile Nile tilapia, reporting that the mucosal layer of fish reared in biofloc systems with symbiotic additives was thicker, suggesting an improved nutrient assimilation. Other studies have confirmed that dietary supplementation with probiotics can enhance intestinal morphology, typically reflected by an increased villus length and density [36,41]. Despite this growing body of evidence, there are no published studies evaluating the effects of biofloc systems on the intestinal morphometry in estuarine fish species such as the flathead mullet (M. cephalus).

Considering the increasing application of biofloc technology in sustainable aquaculture and its reported effects on various physiological and productive parameters in different fish species, it is important to investigate how such systems interact with the digestive morphology of emerging cultured species like the flathead mullet. Given the species’ omnivorous–detritivorous feeding habits, broad environmental tolerance, and increasing relevance in aquaculture diversification, characterizing its intestinal structure under different culture conditions may contribute to a better understanding of its digestive physiology and culture potential. In this context, the analysis of intestinal morphometry provides valuable baseline information on gut health and nutrient absorption capacity. Therefore, this study aims to compare the intestinal morphometry of M. cephalus juveniles reared in biofloc and water exchange systems, contributing to the development of informed and sustainable protocols for the intensive culture of this species.

2. Materials and Methods

2.1. Ethics

All experimental operations involving handling (weight monitoring and sampling collection) were approved by the Bioethics and Biosafety Committee of the Pontificia Universidad Católica de Valparaíso (Code: BIOPUCV-BA 465-2021).

Water quality parameters were monitored and controlled in order to ensure animal welfare. During the manipulation of the animals in biometrics, anesthesia was applied to avoid stress and suffering.

2.2. Experimental Design

Ninety (90) juveniles of M. cephalus with an average weight of 117.36 ± 6.48 g were randomly distributed into six (6) circular plastic tanks of 250 L (fifteen fish per tank). Two treatments were evaluated, each with three replicates: biofloc and water exchange (control). In the BFT treatment, the culture was carried out in a biofloc system without water exchange, using unrefined cane sugar (locally named chancaca)—a solidified product from direct sugarcane juice boiling as an organic carbon source [42]. The C:N ratio was maintained at 15:1, and fertilizations were performed according to Ebeling et al. [43]. The control group was kept in tanks with water exchange at a rate of 50% of the total tank volume three times per week, without the addition of organic carbon. The experimental period lasted for 87 days.

2.3. Culture Conditions

Water quality parameters such as temperature, dissolved oxygen (measured with a multiparameter probe (HQ40, HACH company, Iowa City, IA, USA), and salinity were measured daily. pH was monitored every two days using a pH meter (Ohaus STARTER 3100M, Parsippany, NJ, USA). Total ammoniacal nitrogen (TAN), Nitrite, and Nitrate (mg·L⁻¹) were measured weekly using a spectrophotometer (DR3900 HACH company, Iowa City, IA, USA).

The fish were fed with a balanced feed for marine fish (47.5% crude protein, 19% lipids, 19.5 MJ·kg⁻¹) twice a day (morning and afternoon) at a rate of 2% of the total biomass (kg). The feeding protocol followed the guidelines of Barman et al. [44]. The feed ration was adjusted weekly according to the biometric results.

2.4. Histological and Morphometry Analysis of Intestine

At the end of the experiment, nine fish from each treatment group (three fish per experimental unit) were anesthetized and subsequently euthanized with an overdose of tricaine (0.49 g/L Tricaine methanesulfonate 80%, Centrovet Ltd., Santiago, Chile) according to Topic et al. [45]. A longitudinal incision was made from the anus to behind the operculum, below the pectoral fin. The intestine was carefully extracted from the coelomic cavity, measured, and a 2 cm sample was taken from the mid-section.

Once the intestinal portion was extracted, the samples were stored in 10% buffered formalin. Subsequently, the samples were embedded in paraffin for obtaining 4 µm sections using a microtome. The sections were stained using the hematoxylin–eosin (H-E) technique (Figure 1).

Intestinal morphometry was evaluated through microscopic observation (Primo Sta Zeiss company, Minneapolis City, MC, USA). The villus length (µm) and villus thickness (µm) were obtained using electronic images (AxioCam ERc5s, Zeiss company Minneapolis City, MC, USA). These analyses were conducted at the Laboratory of Aquatic Animal Pathology, Federal University of Rio Grande-FURG (Brazil).

2.5. Growth Performance

Biometrics were performed every 30 days to evaluate weight gain (WG), daily weight gain (DWG), specific growth rate (SGR), and survival (%). Weight gain (g) was calculated as WG = Wf − Wi, daily weight gain (g) as DWG = WG/time, and the specific growth rate (SGR %/day) was calculated using the equation SGR (%/day) = [(lnPf − lnPi) × 100]/time (days), where Wf was the final weight and Wi the initial weight (g). Survival was calculated as survival (%) = (final N° of fish harvested/initial N° of fish stocked) × 100 according to [46].

2.6. Statistical Analyses

Normality (Shapiro–Wilk test) and homogeneity of variance (Levene test) assumptions were tested for all variables of interest. To identify differences in performance and water quality parameters between the two treatments (biofloc and water exchange), a t-test was conducted with a significance level of 0.05 (p < 0.05). The non-normal data (length and villus thickness) were analyzed using non-parametric tests (Mann–Whitney U test). All data were analyzed using RStudio (version 4.2.2; 2022)

3. Results

3.1. Culture Conditions

The main water quality parameters (

Table 1. Water quality parameters in the culture of M. cephalus with biofloc and water exchange for 87 days of the study.

Treatment/Parameter	Biofloc	Water Exchange
T (°C)	15.48 ± 0.10	15.91 ± 0.29
O.D. (mg/L)	8.41 ± 0.04	8.37 ± 0.10
pH	7.23 ± 0.18 a	7.86 ± 0.06 b
Alcalinidad (mg CaCO3/L)	369.61 ± 94.15 a	434.25 ± 15.31 b
Sal (ppt)	14.50 ± 0.13	13.88 ± 0.17
TAN (mg/L)	2.07 ± 0.33	2.13 ± 0.44
NO2 (mg/L)	0.91 ± 0.76	0.67 ± 0.27
NO3 (mg/L)	17.34 ± 1.52 a	5.64 ± 0.63 b
TSS (mg/L)	370 ± 129.4 a	150 ± 23.6 b

Data are expressed as mean ± standard deviation. Values with different letters express significant statistical differences (p < 0.05).

) did not present statistically significant differences (p > 0.05) between the treatments, with the exception of pH, alkalinity, TSS, and N-NO₃ (p < 0.05). The three parameters mentioned are directly related to the conditions of the BFT system [43,47].

3.2. Intestinal Morphology

In the present study, the villi in the midgut of fish reared in a biofloc system exhibited a similar length (1570.24 ± 301.84 µm) compared to those in the water exchange (1522.25 ± 409.00 µm) (Figure 2). This indicates that the biofloc treatment did not cause a significant change (p > 0.05) in the height of the intestinal villi. Regarding thickness, it could be expected that thinner villi could favor a greater number per unit area, thereby increasing the absorptive surface. However, when comparing the treatments, villus thickness was similar in both groups: 344.27 ± 92.45 µm in biofloc fish and 364.09 ± 114.71 µm in water exchange fish (Figure 3).

Furthermore, a correlation analysis was performed between the morphometric variables of length (µm) and thickness (µm) of the intestinal villi. The correlation coefficient obtained was close to zero, indicating that the length and thickness are independent of each other. Thus, an increase in thickness does not necessarily imply an increase in length, and vice versa.

3.3. Growth Performance

The productive performance data (including WG, DWG, SGR, Condition Factor K, and survival) are presented in

Table 2. Productive performance parameters of M. cephalus in two culture systems after 87 days of study.

Parameters	Biofloc	Water Exchange
Initial weight (g)	117.62 ± 7.1	117.11 ± 8.7
Final weight (g)	129.70 ± 7.2	124.82 ± 7.8
Weight gain (g)	11.48 ± 2.8	7.72 ± 1.5
Daily weight gain (g)	0.13 ± 0.03	0.09 ± 0.02
Specific growth rate (%/day)	0.11 ± 0.03	0.07 ± 0.02
Initial condition factor (Ki)	1.49 ± 0.01	1.42 ± 0.06
Final condition factor (Kf)	1.54 ± 0.0001	1.51 ± 0.04
Survival (%)	100	100

Data are expressed as mean ± standard deviation. There were no significant differences between the treatments (p> 0.05).

. The fish reared in biofloc showed a similar productive performance compared with the fish reared in the water exchange system. No significant differences (p > 0.05) were observed in the values of final weight, weight gain (WG), daily weight gain (DWG), specific growth rate (SGR), condition factor (K), and survival.

4. Discussion

The intestine plays a key role in mediating interactions between the host and its associated microbiota, whether native or transient, and is highly susceptible to environmental factors, such as culture strategies. The region of the intestine responsible for nutrient absorption contains the highest density of folds, a structural adaptation that increases the effective surface area and enhances the efficiency of the absorption process [48]. Matadamas et al. [49] performed a histological characterization of the intestinal layers of mullet in its natural habitat, identifying the midgut as the primary site for nutrient absorption in this species.

In the present study, no significant differences were observed in the intestinal morphometry of the Mugil cephalus juveniles reared in biofloc systems compared to those maintained under water exchange conditions. Specifically, villus length and thickness remained statistically similar between the treatments, suggesting that the culture system did not affect the structural parameters evaluated. These findings contrast with those of Zaki et al. [50], who reported increases in villus length and width in Liza ramada cultured in biofloc systems. However, our results are consistent with studies such as Laice et al. [37], who found no significant changes in the intestinal morphology of juvenile Nile tilapia reared in biofloc, and Mahadik et al. [51], who reported no histological alterations—including villus number and length—in GIFT strain tilapia cultured using biofloc systems with fermented rice bran as a carbon source.

It is noteworthy that the villus length recorded in this study (1570.24 ± 301.84 µm) was greater than the values reported for Nile tilapia cultured in biofloc systems, regardless of fish size. For instance, Mirzakhani et al. [23] reported values between 893 and 1013 µm in 2.7 g juveniles, while Laice et al. [37] found 212.4 ± 9.7 µm in fish of 30–35 g. Haraz et al. [52] recorded villus heights of 410.22 ± 12.62 µm and 482.32 ± 33.17 µm under different C/N ratios. Although these comparisons are interspecific, they support the idea that M. cephalus may inherently present more developed intestinal structures, possibly related to its detritivorous–omnivorous feeding habits.

On the other hand, it was observed that the morphometric changes in the evaluated variables are independent of each other. In this regard, it is noteworthy that the size of the villi can vary depending on the food provided, adjusting their size to enhance nutrient absorption [32]. Indigestible or toxic foods can cause inflammation, atrophy, or shortening of these structures [53]. In the present study, no signs of degeneration or tissue alteration were found, suggesting that both systems maintained adequate conditions for gut health.

The presence of longer villi in the middle section of the intestines suggests that there may have been a better nutrient absorption in this region without having a direct impact on all growth performance parameters. Silva et al. [54] observed an increase in the length of villi in Nile tilapia but did not observe significant differences in the growth performance. Previous research has linked the presence and activity of microorganisms in bioflocs to changes in villus morphology, demonstrating a substantial increase in their height [23,50,55]. These results suggest the possibility of an expanded absorption area, highlighting the role of biofloc in modulating the anatomical characteristics of the intestine and, consequently, the efficiency of nutrient absorption. A similar trend was observed by Li et al. [38], who indicated that biofloc improves intestinal health, digestive function, and performance in Rhynchocypris lagowskii. Fish farmed using BFT showed a significant increase in intestinal growth rates, as well as positive changes in intestinal morphology, including the length of the villi and the thickness of the muscular layer of the intestine in different segments. In addition, they observed that the fish exhibited a lower intestinal permeability and an increased digestive enzyme activity. Similarly, in previous research, Yu et al. [56] demonstrated that biofloc meal supplied as a dietary supplement reduces oxidative stress, inflammation, intestinal apoptosis, and intestinal barrier dysfunction caused by environmental factors.

In contrast with the findings of the present study, Mirzakhani et al. [23] identified modifications in the morphological characteristics of the intestine, such as increased villus length, in Nile tilapia reared in the biofloc system. Furthermore, they observed an improved productive performance, suggesting that this result may be due to an enhanced nutrient absorption efficiency. Weight gains were up to 319.9% higher than in the control group. Similar findings were reported by Zaki et al. [50]. In the same way, Yu et al. [56] observed improved performance, higher survival rates, and improved intestinal integrity in snakehead fish (Channa argus) cultured with biofloc technology. The findings of this study emphasize that, in addition to enhancing fish growth, biofloc can attenuate inflammation, supported by a decrease in the expression of pro-inflammatory genes. Moreover, they indicate that maintaining a C/N ratio of 15:1 promotes a more efficient antioxidant response. The authors suggest that the improvement in intestinal health may be attributed to the potential probiotic effects of the microorganisms present in the bioflocs.

Debbarma et al. [57] evaluated the culture of butter catfish (Ompok bimaculatus) in BFT using different C/N ratios (0, 10, 15, 20, 25). These authors reported a better performance and survival in the groups with carbon addition, particularly in the treatments with a 15 and 20:1 ratio. They also observed an increase in intestinal morphometric parameters and a higher production of digestive enzymes (amylases, lipases, proteases). Bakhshi et al. [40] observed opposite results when evaluating the culture of common carp fry in BFT using different carbon sources and 75% of the feeding rate. These authors reported a decrease in villus height in the anterior and posterior intestines when beet molasses, sugar, and corn starch were used, compared to a control group. However, the productive performance and antioxidant capacity were not affected. The results of present study and the studies suggest that biofloc integrated into fish culture can contribute to the development of a healthier environment and promote the growth and well-being of fish.

Azim & Little [58] indicate that, in terms of the production performance, cultivation using biofloc technology is superior to a clear water system (high water exchange) because the microorganisms in the biofloc play a significant role in maintaining water quality and excluding pathogens. Also, they are an important source of continuous feed [47]. Biofloc is a complex matrix consisting of bacteria, microalgae, copepods, nematodes, rotifers, protozoa, and ciliates [59,60]. The activity, mainly bacterial, may have a probiotic effect, which could positively impact the intestinal mucosa. Morphological changes in this tissue have been associated with an improved nutrient assimilation [52]. The same authors evaluated biofloc technology with different C/N ratios (0:1, 10:1, and 20:1) and using probiotics (Bacillus subtilis and Lactobacillus acidophilus) as supplements. Tilapias cultured in BFT and BFT with added B. subtilis showed a greater villus height compared to those in the control group (clear water). Furthermore, better production performance parameters were observed in the groups with BFT and BFT + probiotics.

In a previous study, Wei et al. [59] identified that the phylum Proteobacteria is one of the most abundant and common in biofloc. Many bacteria from this phylum and the phylum Actinobacteria have symbiotic effects in aquaculture, improving water quality and functioning as prebiotics and probiotics [61]. Saki et al. [50] observed a higher total bacterial count (TBC) and higher number of Bacillus in the intestine of fish reared in biofloc compared with the intestines of fish in clear water. Some probiotic strains, such as Bacillus megaterium, Exiguobacterium profundum, Pseudomonas balearica, and Pseudomonas stutzeri, isolated from biofloc, have been shown to colonize the intestines of white shrimp, inhibit the proliferation of certain pathogens like Vibrio parahaemolyticus, improve water quality, and enhance the performance of cultured organisms [62].

In this sense, even though no structural differences were observed, the stability of intestinal morphology in fish cultured in biofloc may indicate system safety and physiological compatibility. Future research should assess other aspects of digestive health, such as mucosal integrity, enzyme activity, and gut microbiota composition in M. cephalus under different biofloc regimes and feeding strategies.

5. Conclusions

Based on the findings of this study, it can be concluded that rearing Mugil cephalus juveniles in a biofloc-based culture system does not negatively affect the productive performance or midgut morphometry. The similarity in villus length and thickness between the fish reared in biofloc and water exchange systems indicates that biofloc technology does not compromise intestinal health in this species.

Although biofloc systems have been widely promoted for their benefits in aquaculture, their effects on gut morphology remain understudied in many fish species. The present research contributes to filling this gap by providing baseline information on the intestinal structure of M. cephalus under different culture strategies. Morphometric analysis proved to be a valuable tool for assessing the gut condition and digestive capacity in cultured fish. Future studies are encouraged to evaluate the long-term effects of biofloc systems on intestinal health across different life stages and environmental conditions.