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Article

Effect of the Inclusion of Natural Pigments on the Performance and Gene Expression of Immune Response and Oxidative Stress of Oreochromis niloticus Cultured in a Biofloc System

by
Andres Elias Castro-Castellon
1,
Maria del Carmen Monroy-Dosta
2,
Aida Hamdan-Partida
3,
Martha Patricia Hernandez-Vergara
4,
Jorge Castro-Mejía
2,
German Castro-Mejia
2,
Arnulfo Misael Martinez-Meingüer
1 and
José Antonio Mata-Sotres
2,*
1
Doctorado en Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana, Unidad Xochimilco, Mexico City 04960, Mexico
2
Departamento El Hombre y su Ambiente, Universidad Autónoma Metropolitana, Unidad Xochimilco, Mexico City 04960, Mexico
3
Departamento de Atención a la Salud, Universidad Autónoma Metropolitana, Unidad Xochimilco, Mexico City 04960, Mexico
4
Tecnológico Nacional de México/Instituto Tecnológico de Boca del Río, Boca del Río 94290, Mexico
*
Author to whom correspondence should be addressed.
Fishes 2025, 10(6), 282; https://doi.org/10.3390/fishes10060282
Submission received: 21 May 2025 / Revised: 5 June 2025 / Accepted: 6 June 2025 / Published: 8 June 2025
(This article belongs to the Special Issue Fish Hematology)
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Abstract

Tilapia (Oreochromis niloticus) is one of the most important species in aquaculture, so the optimization of its culture by using sustainable strategies is fundamental. The aim of this study was to evaluate the effects of incorporating natural pigments (carrot and beetroot meal) as carbon sources in a biofloc system on the growth, immune response, and oxidative stress of O. niloticus. The experiment comprised four treatments: clear water as control (CT), biofloc with molasses (TBM), biofloc with carrot meal (TBC), and biofloc with beetroot meal (TBB). Results showed that biofloc helped to maintain optimal water quality and high survival rates, but specifically, TBC treatment achieved the highest growth rates and feed conversion ratio, along with elevated leukocyte counts, indicating improved health and immunity. Gene expression analysis revealed enhanced antioxidant activity (sod, gpx) and modulated immune responses (tnf-α, il-1β), particularly under thermal stress. Carrot meal emerged as an effective carbon source in biofloc systems, promoting growth, immune resilience, and oxidative stress resistance in tilapia, while beetroot meal improved pigmentation. These findings highlight the potential of natural pigments to enhance aquaculture sustainability and productivity.
Keywords:
carotenes; biofloc; tilapia; gene expression; immunity
Key Contribution: One of the first studies to use carotenoid-rich plant sources as a carbon source for biofloc culture. Natural pigments, such as carrot and beetroot, not only enhance growth performance and feed efficiency but also improve immune resilience and oxidative stress resistance.

1. Introduction

Tilapia is the second most produced organism in aquaculture worldwide and is recognized for its nutritional value and ease of cultivation [1], experiencing exponential growth in recent decades, generating the need to find strategies to optimize its growth and welfare [2,3]. In addition, sustainability in aquaculture production must focus on the design of high-performance production systems that use fewer resources [2] while maintaining economic feasibility [4,5]. In this regard, biofloc technology is one of the most effective aquaculture systems for its profitability and sustainability and allows increasing culture densities with less water use [6,7]. For its definition, biofloc technology is a sustainable fish production, which utilizes recycled nutrients without water exchange, where flocs are composed of the organic particulate matter associated with diverse microorganisms like bacteria, fungi, flagellates, ciliates, algae, nematodes, and mollusks [8,9].
Aquaculture has started using natural phytogenic compounds that can enhance antioxidant activity and innate immunity in various aquatic species [10,11] to neutralize free radicals and mitigate oxidative stress [12]. One of the main stress factors in the farming of tropical fish such as O. niloticus is temperature variations, which have caused large economic losses in aquaculture [13]. Therefore, there is a need to obtain these compounds from natural sources in order to limit the use of synthetic products that represent a high cost for aquaculture and most times their only purpose is the enhancement of color [11,14].
Among the natural phytogenic compounds are carotenoids, which are secondary metabolites that not only help in the coloration of fish but also have important functions in the immune and antioxidant systems by preventing lipid peroxidation, reducing cellular oxidative stress, and reducing inflammatory response in tissues [15,16]. The advantages of using carotenoids include positive effects on the growth, survival, and reproduction of cultivated aquatic organisms [15,17]. There are a variety of plants, fruits, and flowers rich in these compounds that can be used in aquaculture without risk to the cultured organisms and at a low cost. Among these we can find carrots and beets, which have high contents of β-carotenes and betalains, and also have been little used in the aquaculture industry [18].
Although there has been previous research that studied the use of carotenoids as additives in aquaculture, there is a lack of studies specifically investigating agricultural by-products and their integration in biofloc systems, being an important subject since the type of carbon source makes a difference on the water quality, growth performance, body biochemical compounds, immune activity, digestive enzymes, antioxidants, and biofloc compounds produced [8].
Therefore, the aim of this research was to determine the effect of the inclusion of carrot and beetroot meal as a carbon source in a biofloc system on the growth performance, immune response, and oxidative stress in cultured Oreochromis niloticus.

2. Materials and Methods

2.1. Experimental Design

In total, 240 juvenile tilapias, with a mean weight of 1.00 ± 0.01 g, were randomly distributed in 12 water tanks of 200 L capacity, at a density of 20 organisms per tank (1 fish 10 L−1). The experimental treatments were: Control (clear water, CT); biofloc with molasses (TBM); biofloc with carrot meal (TBC); and biofloc with beetroot meal (TBB). All treatments were conducted in triplicate and randomly distributed. The duration of the experiment was 120 days.

2.2. Culture Conditions

The fish were acclimatized for a period of two weeks in an 800 L container with clear water, at a temperature of 27 ± 2 °C, dissolved oxygen of 4 ± 1 mg L−1, pH of 7.5 ± 1, and an ammonium content of 0.1 mg L−1. All biofloc treatments had sufficient aeration to constantly move the entire water column and thus prevent the flocs from settling; previous parameters were taken as baseline values. CT treatment had water changes (30% approximately) every 3 days or when ammonia levels exceeded 1 mg L−1.

2.3. Feeding and Carbon Source Incorporation

The organisms were fed three times a day (9:00, 14:00, and 18:00 h) at a rate of 5% biomass with a commercial tilapia diet containing 32% protein and 5% lipids (Alimentos del Pedregal®, Toluca, Estado de Mexico, Mexico). The ration was adjusted based on the increase in biomass every 15 days.
For the development of the biofloc system, carbon sources (molasses, carrot meal, and beetroot meal) were added to maintain a C:N 15:1 ratio, which allows the production of microbial protein in situ [19]. Carrot and beetroot meal were obtained by drying the vegetables at a temperature of 38 °C, to prevent the degradation of carotenoids. The proximal content of carotenoids in carrot and beet meal was 80 mg 100 g−1 and 50 mg 100 g−1, respectively.

2.4. Monitoring of Physical and Chemical Water Parameters

Water quality parameters were monitored weekly prior to the first feeding. A water sample was taken from each tank and the concentration of nitrite (NO2- mgL−1), nitrate (NO3- mgL−1), ammonium (NH4- mgL−1), and pH were determined using a HANNA® multiparameter photometer (HI-83300).

2.5. Biometry of Organisms

Growth in standard length and weight were obtained every 15 days to determine the performance indexes; for the manipulation of the organisms, low doses of clove oil (5 mg L−1 were used to avoid stress. Survival was recorded daily. The values were calculated as follows:
Weight gain (WG, g) = final weight − initial weight
Length gain (LG, cm): final length − initial length
Specific growth rate (SGR, %) = 100 × [(ln final weight − ln initial weight)/days of feeding]
Feed conversion rate (FCR) = feed intake/weight gain
Survival rate (SR, %) = (total number of fish harvested/total number of stocked fishes) × 100
Condition factor (K) = (weight/length^3) × 100

2.6. Thermal Stress Test

At the end of the study, six fish per treatment were subjected to a cold shock stress test by gradually decreasing the temperature from 27 °C to 15 °C (one degree Celsius every 60 minutes), keeping them at this temperature for 12 h (the 15 °C parameter was used because it is known that physiological effects and even mortalities occur when the temperature drops from 16 °C to 12 °C). Survival was determined, and liver and intestine samples were obtained to evaluate gene expression, using the methodology described in Section 2.8.

2.7. Evaluation of the Immune Response by Leukocyte Count

At the end of the experimental period, the hematological profile of the fish was determined by using the method by Mansour and Esteban [20]. Blood was collected from the caudal vein of three randomly selected fish from each treatment, with a syringe with heparin as anticoagulant. A Neubauer chamber with Shaw’s solution as diluent fluid was used for leukocyte counting [20].

2.8. Evaluation of Oxidative and Immune Response Gene Expression

Six organisms were randomly selected per treatment and sacrificed with 20 mg L−1 clove oil. Once opercular movement ceased, the fish were individually dissected under aseptic conditions on a cold plate at a low temperature. The liver and distal intestine were extracted and placed in 1.5 ml Eppendorf tubes containing RNAlater (Sigma-Aldrich, St. Louis, MO, USA). The tubes were then preserved at −80 °C in a Thermo Scientific (Waltham, MA, USA)™ Revco™ freezer for further processing and analysis.
Total RNA was extracted using the PureLink™ RNA Mini Kit (Invitrogen, Carlsbad CA, USA), following the manufacturer’s instructions. The RNA concentration was determined using a spectrophotometer (Nanodrop® LITE, Thermo Fisher Scientific Inc., Wilmington, NC, USA) at a wavelength of 260 nm. RNA purity was determined by measuring the A260/A280 ratio, which should be between 1.8 and 2.0. Subsequently, cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems; Carlsbad, CA, USA), following the manufacturer’s instructions. The qPCR reactions were performed with 1 ng of cDNA, Fwd and Rev sense primers (200 nM of each), and SYBR® Green Supermix (Bio-Rad, Berkeley, CA, USA). PCR conditions were: an initial denaturation and polymerase activation step for 10 min at 95 °C; 40 cycles of denaturation for 15 s at 95 °C, alignment and extension for 45 s at 60 °C; and finally, a melting curve from 60 °C to 95 °C for 20 min to check for possible dimer artifacts in the primers. Optimization of qRT-PCR conditions was performed at a primer hybridization temperature of 60 °C, at a concentration of 200 nM five 1:10 serial dilutions (10 ng to 100 fg of RNA).
The analyzed genes were gpx (Glutathione peroxidase), sod (Superoxide dismutase), cat (Catalase), hsp70 (Heat shock protein 70), tgfb (Transforming growth factor-beta), tfr (Transferrin receptor), tnfa (Tumor necrosis factor alpha), and il1b (Interleukin 1 beta) (Table 1). β-actin (actb) was used as the internal reference gene. Relative gene quantification was calculated by the ΔΔCT method [21] using an automated threshold and moving baseline to determine the CT values.

2.9. Muscle Carotenoid Content

The method described by Ponce et al. [22] was used to determine changes in muscle coloration. Three fish were randomly taken from each experimental unit and sacrificed with 20 mg/L−1 clove oil, after 1 g of muscle, specifically from the ventral region, was obtained under aseptic conditions. Total carotenoid content was calculated by reading the absorbance at 500 nm in a spectrophotometer (GENESYS 20, Thermo Scientific, Waltham, MA, USA) using the following formula:
Total carotenoid content = 10 × (Absorption at maximum wavelength/0.25 × sample weight (g))
where: 10 is the Dilution factor and 0.25 is the extinction coefficient

2.10. Data Analysis

Prior to statistical analysis, the normality of the data was checked using the Kolmogorov–Smirnov test and Bartlett’s test was applied for homogeneity of variance. Values of water quality parameters, growth and survival rates, hematological values, and expression levels were reported as mean ± standard deviation (SD). Data were analyzed by one-way ANOVA. Significant differences were considered at a level of p < 0.05. When significant differences were observed, Tukey’s multiple mean comparison test was used to identify differences between experimental groups. Statistical programs IBM SPSS Statistics® (V25.0, Armonk, NY, USA) and Microsoft Excel 365® (Redmond, WA, USA) were used to perform the analysis.

3. Results

3.1. Physical and Chemical Water Parameters

Throughout the experimental period, the physicochemical parameters regarding ammonium were maintained at an average of 0.37 ± 0.05 mg L−1 for the three biofloc treatments. Meanwhile, nitrite and nitrate levels were maintained at levels of 2.17 ± 0.31 mg L−1 and 79.16 ± 3.63 mg L−1, respectively (Table 2).

3.2. Growth Performance

Table 3 shows the values obtained for survival, biometric parameters, and FCR after 120 days of experimentation. Fish subjected to TBM, TBC, and TBB treatments had an increase in survival of more than 90%, while CT fish had a survival of 73%, representing a significant improvement in fish viability under biofloc conditions.
Regarding growth in length and weight, the organisms that obtained the highest values in terms of standard length (Figure 1) were from the TBC treatment, followed by the tilapia from the TBB and TBM treatments, with a significant difference between the TBC and CT treatments (p < 0.05). The trend of the results was similar with respect to fish weight (Figure 2), where the organisms that obtained the highest values were from the TBC treatment, followed by TBB and TBM. At the end of the experimentation, the ANOVA test showed significant differences between TBC and CT (p < 0.05).
Regarding FCR, CT showed the highest values, contrasting with a significant reduction in TBC treatment (1.73 ± 0.01 and 1.34 ± 0.02, respectively); on the other hand, in K, TBC presented the highest value with 1.57 ± 0.1, while TC had the lowest value with 1.09 ± 0.2 (Table 3).

3.3. Leukocyte Count

As shown in Table 4, TBC treatment obtained the highest levels of leukocyte count with 6.37 ± 0.45 × 104 μL−1, obtaining significant differences with respect to CT and TBB (p < 0.05).

3.4. Relative Expression of Oxidative Stress Genes at 120 Days of Culture

Regarding sod expression at 120 days of culture (Figure 3), CT and TBC treatments presented the highest expression levels with significant differences (p < 0.056) in comparison with TBM and TBB. In cat expression, the CT and TBM treatments had the highest expression levels with regards to TBC and TBB, also with significant differences (p < 0.05). A similar trend was observed in gpx expression, where the TBC and TBB treatments showed a decrease in expression with respect to CT and TBM (p < 0.05). Finally, biofloc treatments had a significantly lower expression level in the hsp70 gene in comparison to CT (p < 0.05) (Figure 3).

3.5. Relative Expression of Immune Genes at 120 Days of Culture

The expression level of the tnf-α gene showed an increase in TBM and TBC expression levels in comparison with CT and TBB; however, no significant differences were found regarding CT. On the other hand, the relative expression of tgf-β presented an effect of significantly decreased expression in relation to CT (p < 0.05) (Figure 4).
However, in relation to the tfr gene, the TBC and TBB treatments showed significant differences with respect to the CT and TBM treatments (p < 0.05), with a reduction in expression levels with respect to CT. Finally, the il-1β gene showed a reduction in expression levels in biofloc treatments in comparison with CT (p < 0.05) (Figure 4).

3.6. Relative Expression of Oxidative Stress Genes After Temperature Challenge

The relative expression of sod, TBC, and TBB showed a significant increase with respect to CT and TBM (p < 0.05). The opposite was found in cat expression, where the significant reduction in expression levels was observed in TBC and TBB. Only gpx relative expression showed a significant increase in TBB in comparison with the other treatments (p < 0.05). Finally, hsp70’s expression level showed a decreased expression effect in all biofloc treatments, with respect to CT (Figure 5).
Regarding the comparison of expression levels between the end of the culture period and the temperature stress test (both experiments), the sod expression trend was to increase their levels, contrary to CT where there was a reduction in expression. In terms of the expression of cat, a reduction in expression was observed for all treatments with the exception of TBM. On the other hand, gpx expression presented an opposite behavior between experiments. In the case of hsp70 expression, CT showed a significant increase in both experiments in comparison with biofloc treatments.

3.7. Relative Expression of Immune System Genes After Temperature Challenge

No significant differences were found in tnf-α, tgf-β, and tfr in any of the treatments after the thermal stress test. This contrasts with il-1β expression levels, where TBC showed a marked reduction in expression levels (p < 0.05), compared with the rest of the treatments (Figure 6).

3.8. Tissue Carotenoid Concentration

TBB treatment presented the highest value in carotenoid content (9.4 ± 1.05) in comparison with the rest of the treatments, where similar values were obtained between CT, TBM, and TBC (Table 5).

4. Discussion

The obtained results highlight the potential of natural pigments to enhance aquaculture sustainability and productivity. Carrot meal was found to be an effective additive by promoting growth, immune resilience, and oxidative stress resistance in tilapia, while beetroot meal improved pigmentation. Furthermore, both meals proved to be effective carbon sources in biofloc systems.
One of the main sources of stress for cultured organisms is fluctuations in water quality parameters, which can lead to reductions in survival rates [23]. As observed in this experiment, the biofloc system allowed water parameters, mainly nitrogenous compounds such as ammonium, to remain in the optimal range for the growth of O. niloticus. According to the literature, it is mentioned that biofloc technology has been shown to be more effective in removing inorganic nitrogen from water, compared to traditional methods [3,19], due to the action of microbial communities that assimilate them into biomass or transform them into nitrogen gas; this biomass also serves as supplementary feed for the cultured species [3]. The values obtained in this research (0.31–0.41 mg L−1) are similar to those reported by different investigations that performed tilapia culture in biofloc, which had an average TAN of 0.94–1.68 mg L−1 [24], 0.2–0.3 mg L−1 [25], and 0.73–1.17 mg L−1 [26], recalling that the optimum level for tilapia culture is 0.1 mg L−1 and it is up to 7.1 mg L−1 that is highly toxic to cultured fish [27]. The stability of water quality parameters in biofloc positively affects the health and well-being of farmed species [28]. This could be seen in the high survival rates of fish in biofloc treatments compared to other studies using conventional systems with water replacement [29,30]; in addition, floc serves as a food source which contributes to an improvement in tissue development, and the health and nutrition of the organisms [3,31].
The present study shows that carotenoids from carrot meal used as a carbon source increased values in specific growth rates (1.53% cm day−1 and 3.73% g day−1) and FCR (1.34), demonstrating how these bioactive compounds aid in fish metabolism, resulting in increased protein synthesis, digestive efficiency, and the utilization and assimilation of nutrients because it is known that β-carotene stimulates the production of enzymes in fish metabolism as opposed to being stored [8,32,33].
In addition, plant fiber can increase the capacity of the intestinal microbiota to degrade the indigestible components of the food. These results agree with those obtained by Elashry et al. [33], who included β-carotenes from Spirulina platensis, and Judan-Cruz et al. [34], when using carotenes from carrot, who obtained better SGR with 1.91–1.92% g day−1 and weight gains (4.91 g), respectively. These show similar behaviors, but lower than those obtained in the present investigation, where TBC presented values of 3.73 ± 0.24% g day−1 and gain of 31.43 ± 0.13 g. These obtained values by TBC treatment were significantly higher than those of TC, meaning that this is a viable option for farmers to reduce their use of commercial feed and production time, making it more profitable.
Likewise, the results obtained in this study agree with works carried out with other species such as yellow catfish (Pelteobagrus fulvidraco), rainbow trout (Oncorhynchus mykiss), Japanese flounder (Paralichthys olivaceus), sea bass (Micropterus salmoides), and grouper (Argyrosomus regius) [35,36,37,38,39].
Regarding leukocyte count, the results obtained in this work (5.65–6.37 × 104 μL−1) are similar to those of different investigations where β-carotene supplementation increased leukocyte levels, using different sources such as S. platensis [33,40], Curcuma longa [10], and Arthrospira platensis [41], and where they also observed an increase in hematocrit, hemoglobin, and red blood cell values.
This increase in leukocyte levels is due to the fact that β-carotene improves fish health by increasing the immune system’s ability to counter infections and stress by boosting immune parameters such as leukocytes and their derivatives (basophils, monocytes, neutrophils, and lymphocytes), which play an important role in innate immunity during inflammation. Therefore, their counts are considered to be an indicator of fish health status [20,33,41,42], meaning that the increase in the counts of leukocyte in this investigation refers to an improvement in resistance and/or recovery from stress factors.
The balance between ROS generation and the antioxidative system can be affected by different stressors, one of the most important of which is temperature variations. Therefore, it is important to understand the effect of cold stress on antioxidative and immune responses to ensure the future of aquaculture production [13,33]. The antioxidative status of fish can be seen reflected in the levels of sod, cat, and gpx.
The results obtained in the present investigation showed how the inclusion of β-carotenes, mainly from carrot meal, generated an increase in expression in the two periods, unlike beetroot meal which showed this effect only after the stress test. This suggests that the inclusion of carrot and beetroot meal stimulates antioxidant defense and protects against cold-induced oxidative damage. This is in agreement with the results reported by Panase et al. [43], who obtained higher sod and cat activity in O. niloticus fed a diet with Paracoccus carotinifaciens enriched with astaxanthin. The same antioxidant effect was observed in a study where they cultured O. niloticus at high densities (200 fish m−3) fed diets enriched with β-carotene from S. platensis [33].
Similarly, this effect has been observed in different species such as Plectropomus leopardus [44], Lates calcarifer [16], Eriocheir sinensis [45], and Macrobrachium nipponense [46]. It is worth noting that in the present investigation there was a lower expression of the cat gene with the inclusion of carotenoids (TBC and TBB). This can be explained by the double role that carotenoids can play in the antioxidative system: the first is as a precursor to elevate the levels of endogenous enzymes, increasing the antioxidative capacity; while the second is associated with the ability to eliminate ROS directly and maintain the dynamic balance between pro- and anti-inflammatory states [47,48]. When carotenoids eliminate ROS directly, the endogenous enzymes’ activity is reduced; therefore, the cost of production is reduced and can be allocated to other physiological processes, such as immune activation. This enhances the overall antioxidant status [48]. However, the interaction between carotenoids and endogenous antioxidant enzymes may be affected by environmental or pathological conditions, but further studies are required.
Regarding hsp70 expression, in the present investigation a decrease in expression levels was observed in TBC and TBB treatments with respect to CT in the two periods; this effect can be used as an indicator of welfare and improved immunity [48]. HSP70 is responsible for the integrity of fish cells under environmental stress [48,49]. Similarly, in the obtained results, it could be observed how after the stress test, carotenoid treatments increased expression levels compared to the culture period. This supports how carotenoids can directly modulate the mRNA expression levels of essential stress-related proteins and the antioxidant capacity of cells [48]. Taking into account that even in the stress test the levels in TBC and TBB treatments were lower than TC, this proves that the inclusion of carotenes could partially reduce the stress response [50].
In relation to the expression of genes related to the immune system, pro-inflammatory cytokines such as tnf-α and il-1β were influenced by the addition of carotenoid pigments from carrot and beetroot meal, where the levels obtained in tnf-α suggest that β-carotenoids from carrots could be stimulating a controlled inflammatory response, which is essential for defense against pathogens [51,52]. This effect has been seen in several investigations such as that of Li et al. [53], where by including astaxanthin in the culture of Channa argus, they obtained an increase in the expression of tnf-α; and those performed by Al-Deriny et al. [51], and Hassaan et al. [11], with O. niloticus, with similar results when including S. platensis in the diet, which provides β-carotenes and phycocyanins. This may indicate the potential mechanism of carotenoids to modulate the NF-κB signaling pathway, allowing controlled activation [48,54]. Therefore, carrot meals induce an increase in tnf-α expression without reaching harmful proinflammatory levels, reflected in reduced expression of the pro-inflammatory cytokine il-1β, suggesting that natural carotenoid pigments and biofloc help modulate systemic inflammation [54]. In contrast to what was obtained, there are several works where a reduction in the expression of both cytokines was obtained, but with an increase in anti-inflammatory cytokines, like il-10, decreasing the time in the inflammatory processes in fish [13,33,55]. However, knowledge of the mechanisms in the modulation of immune-related gene expression by carotenoids today is incomplete and fragmented [33,48].
Regarding tfr and tgf-β, TBC and TBB treatments obtained the lowest expression values, indicating that natural pigments can decrease oxidative stress and iron chelation demand. Likewise, they optimize the balance between inflammation and repair, avoiding an excessive immune response [16,56].
In the stress test, all treatments showed increased expression of tnf-α and tgf-β in response to cold stress, especially the pigment-added treatments. This reaffirms the importance of carotenoids for the health of aquatic animals, as it increases the efficacy of the immune response and causes the activation of innate immunity [38,45,48]. Similarly decreased levels of il-1β expression support the anti-inflammatory role of carotenoids [16,54].
Regarding the effects on the stress challenge, the obtained results prove the influence of carotenoids in the modulation of stress on an immune-related gene that would help improve the survival and performance of the organisms in case some variation in the temperature occurs, which is an emerging problem due to climate changes [13].
Finally, according to the values obtained regarding the increase in fish muscle coloration, the TBB treatment obtained the greatest increase in carotenoids, followed by TBC, which suggests that pigments from plants can be assimilated to be transported to the muscles of the organisms. This is of great importance since fillet coloration is linked to market value [57] and is associated with the nutritional value of the product, which influences higher demand and consumption [28]. Moreover, this association of the color to the quality of the fillet stimulates consumption behavior due to the preference for bright colored fillet. Losses due to issues such as darkening and loss of luster in meat were reported to be USD 3 billion in the U.S. and USD 14.2 billion globally in recent years [58]. This positive effect from plant-derived carotenoids has been observed in other research where bee pollen has been used in O. mykiss [33], carrot in Oreochromis mossambicus [28], paprika in O. mossambicus [59], marigold in M. nipponense [42], and marigold in Trichogaster trichopterus [60].

5. Conclusions

The present study demonstrated that carrot meal is an alternative as an economical and accessible carbon source for the production of biofloc and inclusion of carotenoid pigments of vegetable origin that allow increasing survival, growth, innate immunity, antioxidative capacity, and coloration in tilapia muscle, improving their health and response to stress in culture systems. The results prove the importance that carrot meal can have in the aquaculture industry by reducing the times of production, improving feed utilization, and providing well-being to the cultured organisms even if periods of stress by temperature variations occur. We suggest that following investigations consider how carotenoids affect mayor signaling like NF-κB and pro- and anti-inflammatory cytokines, as well as different biomarkers like lipid peroxidation and total antioxidant activity.

Author Contributions

Conceptualization, A.E.C.-C.; methodology, A.E.C.-C., A.M.M.-M., A.H.-P. and J.A.M.-S.; data analysis, A.E.C.-C., M.d.C.M.-D. and J.C.-M.; writing—original draft, A.E.C.-C.; writing—review and editing, M.d.C.M.-D., A.H.-P., M.P.H.-V., J.C.-M., G.C.-M., A.M.M.-M. and J.A.M.-S.; supervision, M.d.C.M.-D., A.H.-P. and M.P.H.-V.; funding acquisition, M.d.C.M.-D., J.C.-M., G.C.-M. and J.A.M.-S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the PhD studies in Biological and Health Sciences at Universidad Autónoma Metropolitana—Xochimilco and SECIHTI for the scholarship (CVU: 1010282). This research received no external funding.

Institutional Review Board Statement

The experimental work took place in the facilities of the Live Food Production Laboratory and the Live Food Chemical Analysis Laboratory at the Universidad Autónoma Metropolitana, Unidad Xochimilco. For the use of laboratory animals, the university has the Internal Committee for the Care and Use of Laboratory Animals (CICUAL-UAM-X). The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (CICUAL-UAM-X) of UNIVERSIDAD AUTÓNOMA METROPOLITANA (Sesion 70/20) (Approval Code: UAM-X-CONSEJO DIVISIONAL DE C.B.S 10/20; Approval Date: 10 February 2020).

Informed Consent Statement

Not Applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

We would like to express our sincere gratitude to the PhD program of Biological and Health Sciences at Universidad Autónoma Metropolitana—Xochimilco and SECIHTI for the support to carry out this investigation and for the scholarship (CVU: 1010282); also, to all the members of our laboratory team for their support and collaboration.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design or in any part of the study.

Abbreviations

WGWeight gain
LGLength gain
SGRSpecific growth rate
FCRFeed conversion rate
SRSurvival rate
gpxGlutathione peroxidase
sodSuperoxide dismutase,
catCatalase
hsp70Heat shock protein 70
tgfbTransforming growth factor-beta
tfrTransferrin receptor
tnfaTumor necrosis factor alpha
il1bInterleukin 1 beta
β-act (actb)Beta actin

References

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Figure 1. Standard length (cm) of O. niloticus in the different treatments at 120 days of the experiment.
Figure 1. Standard length (cm) of O. niloticus in the different treatments at 120 days of the experiment.
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Figure 2. Weight gain (g) of O. niloticus in the different treatments at 120 days of the experiment.
Figure 2. Weight gain (g) of O. niloticus in the different treatments at 120 days of the experiment.
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Figure 3. Relative expression of genes related to oxidative stress in O. niloticus after 120 culture days. Different superscript letters represent significantly different values (p < 0.05) (n = 6, mean ± SD).
Figure 3. Relative expression of genes related to oxidative stress in O. niloticus after 120 culture days. Different superscript letters represent significantly different values (p < 0.05) (n = 6, mean ± SD).
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Figure 4. Relative expression of immune-related genes in O. niloticus. Different superscript letters represent significantly different values (p < 0.05) (n = 6, mean ± SD).
Figure 4. Relative expression of immune-related genes in O. niloticus. Different superscript letters represent significantly different values (p < 0.05) (n = 6, mean ± SD).
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Figure 5. Relative expression of oxidative stress-related genes in O. niloticus after the temperature challenge. Different superscript letters represent significantly different values (p < 0.05) (n = 6, mean ± SD).
Figure 5. Relative expression of oxidative stress-related genes in O. niloticus after the temperature challenge. Different superscript letters represent significantly different values (p < 0.05) (n = 6, mean ± SD).
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Figure 6. Relative expression of immune-related genes in O. niloticus after the temperature challenge. Different superscript letters represent significantly different values (p < 0.05) (n = 6, mean ± SD).
Figure 6. Relative expression of immune-related genes in O. niloticus after the temperature challenge. Different superscript letters represent significantly different values (p < 0.05) (n = 6, mean ± SD).
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Table 1. Primer pairs used for q-PCR.
Table 1. Primer pairs used for q-PCR.
Gen (Symbol)Fwd Sequence (5′-3′)Rev Sequence (5′-3′)GenBank
gpxGGAACGACAACCAGGGACTATCCCTGGACGGACATACTTCNM_001279711.1
sodGACGTGACAACACAGGTTGCTACAGCCACCGTAACAGCAGJF801727.1
catGGCCGGGTTTCTAAAAGAAGGCTGTAAACGTGCAAAGTGGXM_019361816.2
hsp70CAAGATCACCATCACCAACGTCTTGTCCTCCTCGCTGATTNM_001279671.2
tgfbCGAGCAGCTGTCCAATATGAAGGTCCATGGCTTAATGTGCNM_001311325.1
tfrGAGCATCGTCCATTCCCTTACTCTGGCATTCAATGGAGGTDQ272465.1
tnfaTCTGGAGTGGAGGAATGGTCTCTGAGTAGCGCCAGATCCTXM_025902124.1
il1bTTTTGGATCCTCAGGACAGGGTAGCAGAACATTGGCAGCAXM_005457887.3
actbGAGCGTGGCTACTCCTTCACGCAGGATTCCATACCAAGGAEF026001.1
Table 2. Physical and chemical water parameters in experimental treatments.
Table 2. Physical and chemical water parameters in experimental treatments.
TreatmentNH4 (mg L−1)NO2 (mg L−1)NO3 (mg L−1)pH
CT0.37 ± 0.052.17 ± 0.5279.16 ± 10.468.2 ± 0.3
TBM0.41 ± 0.171.86± 0.9076.66 ±6.678.3 ± 0.2
TBC0.31 ± 0.072.18 ± 1.9577.49 ± 8.248.2 ± 0.3
TBB0.39 ± 0.172.48 ± 1.3583.33 ± 9.488.1 ± 0.2
Table 3. Growth performance (mean ± SD) of juvenile O. niloticus exposed to different biofloc treatments.
Table 3. Growth performance (mean ± SD) of juvenile O. niloticus exposed to different biofloc treatments.
ParameterCTTBMTBCTBB
WG (g)23.88 ± 0.31 a29.20 ± 0.16 ab31.43 ± 0.13 b29.12 ± 0.25 ab
LG (cm)8.60 ± 0.45 a9.84 ± 0.17 ab11.47 ± 0.06 b10.11 ± 0.25 ab
SGR (%)3.23 ± 0.31 a3.40 ± 0.23 ab3.73 ± 0.24 b3.43 ± 0.33 ab
FCR1.73 ± 0.01 a1.51 ± 0.02 b1.34 ± 0.02 c1.45 ± 0.03 d
SR (%)73.3396.6790.0093.33
K1.09 ± 0.2 a1.39 ± 0.1 ab1.57 ± 0.1 b1.30 ± 0.1 ab
Different letters mean significant differences (p < 0.05).
Table 4. Leukocyte counts of O. niloticus under the different experimental treatments.
Table 4. Leukocyte counts of O. niloticus under the different experimental treatments.
CT TBM TBC TBB
Leukocyte (×104/μL)4.50 ± 0.40 a5.97 ± 0.31 bc6.37 ± 0.45 b5.65 ± 0.45 c
Different letters mean significant differences among treatments (p < 0.05).
Table 5. Average values of optical density and total carotenoid content of O. niloticus tissue with the experimental treatments.
Table 5. Average values of optical density and total carotenoid content of O. niloticus tissue with the experimental treatments.
CTTBMTBCTBB
Optical density (500 nm)0.13 ± 0.010.14 ± 0.010.15 ± 0.020.23 ± 0.02
Carotenoid content (µg)5.45 ± 0.6 b5.71 ± 0.19 b6.33 ± 1.13 b9.4 ± 1.05 a
Different letters mean significant differences (p < 0.05).
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Castro-Castellon, A.E.; Monroy-Dosta, M.d.C.; Hamdan-Partida, A.; Hernandez-Vergara, M.P.; Castro-Mejía, J.; Castro-Mejia, G.; Martinez-Meingüer, A.M.; Mata-Sotres, J.A. Effect of the Inclusion of Natural Pigments on the Performance and Gene Expression of Immune Response and Oxidative Stress of Oreochromis niloticus Cultured in a Biofloc System. Fishes 2025, 10, 282. https://doi.org/10.3390/fishes10060282

AMA Style

Castro-Castellon AE, Monroy-Dosta MdC, Hamdan-Partida A, Hernandez-Vergara MP, Castro-Mejía J, Castro-Mejia G, Martinez-Meingüer AM, Mata-Sotres JA. Effect of the Inclusion of Natural Pigments on the Performance and Gene Expression of Immune Response and Oxidative Stress of Oreochromis niloticus Cultured in a Biofloc System. Fishes. 2025; 10(6):282. https://doi.org/10.3390/fishes10060282

Chicago/Turabian Style

Castro-Castellon, Andres Elias, Maria del Carmen Monroy-Dosta, Aida Hamdan-Partida, Martha Patricia Hernandez-Vergara, Jorge Castro-Mejía, German Castro-Mejia, Arnulfo Misael Martinez-Meingüer, and José Antonio Mata-Sotres. 2025. "Effect of the Inclusion of Natural Pigments on the Performance and Gene Expression of Immune Response and Oxidative Stress of Oreochromis niloticus Cultured in a Biofloc System" Fishes 10, no. 6: 282. https://doi.org/10.3390/fishes10060282

APA Style

Castro-Castellon, A. E., Monroy-Dosta, M. d. C., Hamdan-Partida, A., Hernandez-Vergara, M. P., Castro-Mejía, J., Castro-Mejia, G., Martinez-Meingüer, A. M., & Mata-Sotres, J. A. (2025). Effect of the Inclusion of Natural Pigments on the Performance and Gene Expression of Immune Response and Oxidative Stress of Oreochromis niloticus Cultured in a Biofloc System. Fishes, 10(6), 282. https://doi.org/10.3390/fishes10060282

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