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Full Research Article

Impact of Graded Levels of Dietary Ferrous and Ferric Citrate Supplementation on Expression of Iron Regulatory Genes in Clarias magur

S
Susmita Rani1,2,*
https://orcid.org/0000-0002-9190-0033
P
Prem Prakash Srivastava1,3,*
https://orcid.org/0000-0001-8433-3889
A
Ashutosh D. Deo1
T
Tincy Varghese4
S
Subodh Gupta1
https://orcid.org/0000-0002-9420-480X
A
Abhiman2
P
Pankaj Kumar2
S
Shivendra Kumar3
1Division of Fish Nutrition, Biochemistry and Physiology, ICAR-Central Institute of Fisheries Education, Versova, Mumbai-400 061, Maharashtra, India.
2College of Fisheries, (Bihar Animal Sciences University-Patna) Kishanganj-855 107, Bihar, India.
3College of Fisheries, Dr. Rajendra Prasad Central Agricultural University, Dholi, Pusa, Muzaffarpur-843 121, Bihar, India.
4ICAR-Central Institute of Fisheries Technology, Willingdon Island, Kochi-682 029, Kerala, India.

ABSTRACT

Background: The 60-day experimental study investigated the expression of iron regulatory genes, namely ferritin, transferrin, and hepcidin, in Clarias magur fingerlings fed diets supplemented with two dietary iron sources. The experimental study comprised two groups in which C. magur fingerlings received diets supplemented with ferrous or ferric citrate at doses of 0, 15, 30, and 45 mg Fe/kg above the minimum dietary iron requirement of 30 mg Fe/kg diet, respectively.

Methods: Ferritin, transferrin, hepcidin expression levels in fish intestine, liver, muscle and gill tissues were determined using quantitative-real time PCR (qRT-PCR). Regardless of the dietary iron source, all tissues exhibited expression of the three genes under investigation, however, the degree of expression varied amongst tissues.

Result: Liver showed highest ferritin mRNA expression among all tissues under both iron sources. Maximum expression (6.77-fold) was observed at 45 mg/kg ferrous citrate. Muscle showed modest changes while gill exhibited the lowest ferritin expression. In intestine, under ferrous citrate supplementation, transferrin expression increased with increasing iron level. Maximum expression (4.21±0.14) was recorded at 45 mg/kg iron supplementation. Liver transferrin expression increased with increasing ferrous citrate. Highest expression was observed at T3 (3.31±0.07). In intestine, the highest hepcidin expression occurred at T1 (5.62±0.01). Ferric citrate showed comparatively lower expression in all treatments. Liver showed the highest hepcidin expression among all tissues. Expression increased significantly with increasing iron supplementation. Maximum expression (7.99±0.06) was observed in fish fed 45 mg/kg ferrous citrate (T3).

INTRODUCTION

Iron is an essential micronutrient that plays a crucial role in various physiological processes and immune functions in fish including oxygen transport, drug metabolism, steroid synthesis, DNA synthesis, ATP production, electron transport and cellular respiration (Lim et al., 2001; Ganz and Nemeth, 2015; Sangeetha and Rajan, 2021). In aquaculture, dietary supplementation of various inorganic and organic forms of iron has been reported to significantly enhance growth performance, health status and survival rates of cultured fish species (Yu et al., 2024). However, excessive iron accumulation can promote the generation of reactive oxygen species (ROS), resulting in oxidative stress and cellular damage (Sevcikova et al., 2011; Bresgen and Eckl, 2015).
       
To maintain iron homeostasis, fish possess tightly regulated iron-metabolism proteins that control iron uptake, transport, storage and export (Arosio et al., 2009; Ganz and Nemeth, 2012). These proteins also contribute to host defense by limiting iron availability to invading pathogens, thereby enhancing immune responses during infection. (Shike et al., 2004; Das et al., 2015). Key components of this regulatory network include ferritin, the major intracellular iron-storage protein; transferrin, responsible for iron transport; ferroportin, which mediates cellular iron export; and hepcidin, a central regulator of systemic iron homeostasis that controls iron egress from cells (Torti and Torti, 2002; Nemeth et al., 2004; Bao et al., 2005; Shi and Camus, 2006; Fraenkel et al., 2009; Neves et al., 2009; Gkouvatsos et al., 2012; Yin et al., 2018).
       
The general recommendation for dietary iron content in fish feeds to improve growth and feed utilization ranges from 30-150 mg/kg of dietary feed (NRC, 2011). In channel catfish (Ictalurus punctatus) improved growth and feed efficiency was obtained at 30 g/kg of iron supplementation (Lim et al., 1996). According to Gatlin and Wilson (1986), normal growth and feed efficiency were observed for channel catfish fed 10 mg or more of supplemental iron/kg; however, they observed that 20 mg supplemental iron/kg was required to maintain optimum hematological values. Based on these data, the dietary iron requirement of fingerling channel catfish was determined to be not more than 30 mg Fe/kg diet. Viani et al., (2025) suggested that 60 mg/kg dose of Fe3O4-NPs significantly improves growth, feed conversion and physiological safety in Clarias batrachus.
       
The Indian walking catfish, Clarias magur (Hamilton, 1822), is one of the most important freshwater aquaculture species in India owing to its high market demand and culture potential (Jayasankar et al., 2018; Chakraborty et al., 2020). It is rich in easily digestible high-quality protein, essential omega-3 fatty acids and abundant iron. These properties contribute to cardiovascular health and help prevent iron-deficiency anemia and protein-energy malnutrition (Bolaji et al., 2022). In fish, the minimum dietary iron requirement has been estimated to be approximately 30 mg kg-1 diet (NRC, 2011). However, information regarding the physiological and molecular responses of Clarias magur to supplemental iron sources remains limited. Therefore, present study focuses on evaluating the tissue-specific expression profiles of key iron-regulatory genes (Ferritin, transferrin and hepcidin) in response to graded dietary supplementation of ferric and ferrous citrate in order to have a comprehensive understanding of how organic iron sources influence iron metabolism, storage and regulation at the mechanistic level. Such knowledge can contribute to the development of nutritionally optimized diets that maximize growth performance while maintaining iron homeostasis.

MATERIALS AND METHODS

Experimental fish and laboratory conditions
 
Clarias magur weighing approximately 18.0±0.5 g were obtained from Kolkata, West Bengal, India and acclimatized in tanks containing 500 L freshwater with continuous aeration for 10 days at the wet laboratory of College of Fisheries, Kishanganj, Bihar where the experimental trial was conducted during 2024-2025.
 
Experimental design, diet and feeding schedule
 
In a completely randomized design, fish (n=120) were divided equally among the two experimental groups and fed with basal feed supplemented with Ferrous (Fe2+) and Ferric (Fe3+) citrate, respectively. Each experimental group was further divided into four groups (control and three treatments, T1, T2 and T3) with three replicates consisting of 10 fish each. Control contains 30 mg/kg iron citrate which is the basic dietary iron requirement of fish with no further supplementation. Fish in the three treatment groups received diet containing the minimum dietary iron required by fish (30 mg/kg feed) supplemented with graded dietary concentrations of 15 mg/kg (T1), 30 mg/kg (T2) and 45 mg/kg (T3) of ferric or ferrous citrate, respectively in both the experimental groups. Fish were fed for 60 days and 30-40% water exchange was done on alternate days. During this period, fish were fed with formulated feed consisting of 35% crude protein at the rate of 3% of their body weight (Table 1).

Table 1: Feed composition and proximate composition of the experimental diets.


 
Quantification of mRNA expression of ferritin, transferrin and hepcidin gene
 
Tissue collection
 
At the end of the experiment (60th day), fish (n=2) from the triplicate treatment and control tanks of each dietary group were randomly selected and anesthetized using clove oil (50 μl/L) before sacrifice. The fish were dissected for liver, intestine, gill and muscle tissues. These tissues were collected for gene expression study and preserved immediately in RNAlater™ solution (Qiagen, India) at -80°C until RNA extraction.
 
Total RNA extraction and cDNA synthesis
 
Total RNA was extracted from equal portions of each tissue collected using Trizol TM reagent (Invitrogen, USA) as per manufacturer’s instructions. Briefly, 100 mg of RNA later TM stored tissue was homogenized with 1 ml of Trizol reagent, followed by centrifugation for 5 min at room temperature. To the aqueous phase transferred to a fresh tube, 200 µl of chloroform (0.2 vol/ml Trizol TM reagent) was added, vortexed for 1 min and incubated for 10 min at room temperature. The mixture was centrifuged at 12,000 rpm for 15 min at 4°C and the upper aqueous phase transferred to a fresh 1.5 ml tube. RNA was precipitated out using 500 µl of isopropanol (0.5 vol/ml Trizol TM reagent) followed by centrifugation at 12000 rpm for 15 min at 4°C. The RNA isolated was washed with 70% ethanol, air-dried and re-suspended in 35 µl of nuclease-free water and stored at -80°C. The RNA concentration and purity was estimated based on absorbance at OD260/280 nm using a Nanodrop 2000 (Thermo Fisher Scientific, USA). Total RNA extracted was purified for residual genomic DNA contamination using DNase I (Fermentas International Inc., Canada). Total RNA obtained was reverse transcribed to synthesize cDNA using Revert Aid cDNA synthesis kit (Thermo Scientific) as per the manufacturer’s protocol. cDNA was synthesized using a thermal cycler (Bio-Rad laboratories, Inc) with the following cycling conditions: initial denaturation at 94°C for 4 min followed by 30 cycles of 94°C for 30s, 55°C for 30 s, annealing at 72°C for 30 s and a final extension for 7 min at 72°C. The cDNA was stored at -20°C until use.
 
Primer design for RT-qPCR
 
The primers for RT-qPCR were designed using Gene runner software (version 3.05) based on available gene sequences in NCBI GenBank.  The primer sequences for the gene ferritin, transferrin and hepcidin, their product size and NCBI accession numbers are provided in Table 2. β-actin was used as a reference gene and served as an internal standard gene (Buwono et al., 2015) Primer synthesis was outsourced to Bioserve Biotechnologies, Hyderabad and Bioinnovations, Maharashtra, India.

Table 2: Primers used for expression study in real-time PCR.


 
Quantitative real-time PCR (RT-qPCR) for mRNA expression of ferritin, transferrin and hepcidin gene in different tissues of Clarias magur fingerlings
 
After reverse transcription, target cDNA was amplified and relative quantification of ferritin, transferrin and hepcidin gene in different tissues of Clarias magur fingerlings was done in a quantitative real-time PCR using SYBR green qPCR master mix without ROX (Thermo scientific) in aria MX real-time PCR system (Agilent Technology LDA). The reaction took place in a volume of 10 μl containing 5 μl of 2 × maxima™ SYBR green qPCR master mix (Thermo Scientific), 1 μl of (5 pmol) each gene-specific primer, 1 μl cDNA template and remaining nuclease-free water prepared for quantification. The quantified value of mRNA was expressed in terms of CT (threshold cycle) value. The PCR program included 10-minute activation and initial denaturation step at 95°C for 5 min, followed by 45 cycles for amplification with each denaturation cycle at 95°C for 15s, annealing at 53°C for 30s and extension at 72°C for 30s. At the end, the specificity of reaction was confirmed using melt curve analysis. A comparative CT method was used to estimate the relative expression of mRNA. All PCR reactions were run in duplicate and normalized by the value by 2-ΔΔCT method. The fold change in gene expression was normalized to β-actin and the expression level of the target gene was calculated by the 2-ΔΔCT CT-method (Livak and Schmittgen, 2001).
       
The quantified result was analyzed by the following formulae:
 
Δct = Target gene ct value - reference gene ct value.
 
ΔΔ ct value = Δct value of treatment group - Δct value of control group
 
Statistical analysis
 
For each tissue, the gene transcript values were calculated for triplicate samples and expressed as mean±standard error. The statistical significance of all parameters was determined using one-way ANOVA followed by Duncan’s new multiple range test using the statistical package with SPSS 22.0 for Windows. A significant difference was considered at a p value <0.05.

RESULTS AND DISCUSSION

Table 3, Fig 1-2; Table 4, Fig 3-4; Table 5, Fig 5-6 presents the expression of ferritin, transferrin and hepcidin gene, respectively in different tissues of C. magur fed diets supplemented with varying levels of ferrous or ferric citrate.

Table 3: Relative mRNA expression of ferritin gene in different tissues of Clarias magur fed with experimental diets supplemented with ferrous and ferric citrate.



Table 4: Relative mRNA expression of transferrin gene in different tissues of Clarias magur fed with experimental diets supplemented with ferrous and ferric citrate.



Table 5: Relative mRNA expression of hepcidin gene in different tissues of Clarias magur fed with experimental diets supplemented with ferrous and ferric citrate.



Fig 1: Relative mRNA expression of ferritin gene in different tissues of Clarias magur fed with diets supplemented with ferrous citrate.



Fig 2: Relative mRNA expression of ferritin gene in different tissues of Clarias magur fed with diets supplemented with ferric citrate.



Fig 3: Relative mRNA expression of transferrin gene in different tissues of Clarias magur fed with diets supplemented with ferrous citrate.



Fig 4: Relative mRNA expression of transferrin gene in different tissues of Clarias magur fed with diets supplemented with ferric citrate.



Fig 5: Relative mRNA expression of hepcidin gene in different tissues of Clarias magur fed with diets supplemented with ferrous citrate.



Fig 6: Relative mRNA expression of hepcidin gene in different tissues of Clarias magur fed with diets supplemented with ferric citrate.


 
Ferritin
 
Liver showed the highest ferritin mRNA expression among all tissues under both iron sources. Expression increased significantly with increasing ferrous citrate supplementation. Maximum expression (6.77-fold) was observed in the liver at 45 mg/kg ferrous citrate (T3). Ferric citrate produced considerably lower expression than ferrous citrate.
       
Intestine showed moderate ferritin expression. Under ferrous citrate, expression peaked at T2 (3.44) and then declined. Under ferric citrate, expression increased progressively from T1 to T3, suggesting active regulation of iron absorption and storage.
       
Muscle exhibited relatively low expression. Under Ferrous citrate expression resulted in gradual increase with dose while with ferric citrate expression peaked at T2 and decreased at T3.
       
Gill exhibited the lowest ferritin expression overall. Expression increased significantly at the highest iron supplementation level.
 
Transferrin
 
In intestine, under Ferrous citrate supplementation, transferrin expression increased significantly with increasing iron level. Maximum expression (4.21±0.14) was recorded at T3. Under ferric citrate, expression peaked at T2 (3.98±0.03) and declined at T3. The intestine responds actively to dietary iron supplementation, reflecting its role in iron absorption and transport.
       
Expression remained relatively low in muscle. Ferric citrate produced a sharp increase at T2  followed by a marked decline at T3. Ferrous citrate treatments showed only minor variation. From this it can be concluded that muscle tissue is not a primary site of iron transport regulation and therefore exhibited comparatively lower transferrin expression.
       
Liver transferrin expression increased significantly with increasing ferrous citrate supplementation. Highest expression under ferrous citrate was observed at T3 (3.31±0.07). Ferric citrate resulted in consistently lower expression values. The liver plays a central role in iron metabolism and transferrin synthesis. Higher expression under ferrous citrate suggests better iron utilization.
       
In gill, transferrin expression increased significantly with increasing iron supplementation. Under ferrous citrate, maximum expression occurred at T2 whereas, under ferric citrate, expression increased steadily and reached its highest value at T3. Gill tissue participates in ion and mineral regulation, which may explain the increased transferrin expression with higher iron availability.
 
Hepcidin
 
In intestine, the highest expression occurred at T1 (5.62±0.01), under ferrous citrate supplementation followed by a decline at higher levels. Ferric citrate showed comparatively lower expression in all treatments. Ferrous citrate induced approximately 2-3 times higher hepcidin expression than ferric citrate.  Intestine exhibited a strong response to ferrous iron, indicating activation of iron homeostasis mechanisms.
       
Muscle showed relatively low expression levels of hepcidin compared with other tissues. Under ferrous citrate, expression decreased as iron level increased. Under ferric citrate, expression increased and reached its maximum at T3. Muscle plays a limited role in systemic iron regulation and therefore exhibited lower hepcidin expression.
       
Liver showed the highest hepcidin expression among all tissues. Expression increased significantly with increasing iron supplementation. Maximum expression (7.99±0.06) was observed in fish fed 45 mg/kg ferrous citrate (T3). Ferric citrate also increased expression but remained lower than ferrous citrate. Since the liver is the major site of hepcidin synthesis, elevated expression reflects enhanced regulation of iron metabolism in response to increased dietary iron.
       
In gill under ferrous citrate, hepcidin expression decreased with increasing iron levels. Ferric citrate induced lower expression overall, peaking at T2. Gill showed moderate responsiveness to dietary iron.
       
In this study, Ferrous citrate induced higher ferritin gene expression than ferric citrate in most tissues, indicating better bioavailability and utilization of iron. The liver was the primary site of iron storage, showing the highest ferritin transcription, followed by the intestine. Increasing dietary iron generally increased ferritin expression, particularly in the liver and gill. The highest ferritin expression was observed in the liver at the highest ferrous citrate level (45 mg/kg), indicating enhanced iron storage capacity in response to increased iron availability. The results suggest that ferrous citrate is more effective than ferric citrate in enhancing iron storage mechanisms in magur fingerlings.
       
Transferrin expression levels revealed discrepancies among the tissues tested. Such discrepancies have been previously reported and been attributed to iron sources and concentrations in fish diets (Buyinza et al., 2024). Ferrous citrate generally induced higher transferrin gene expression than ferric citrate, particularly in the intestine, liver and gill. The highest transferrin expression was observed in intestine of fish fed 45 mg/kg ferrous citrate (4.21±0.14), reflecting its crucial role in dietary iron absorption and transport. Elevated transferrin expression in liver and gill further suggests enhanced iron mobilization and distribution within the body. The generally higher transferrin expression observed in fish fed ferrous citrate compared to ferric citrate indicates greater bioavailability of ferrous iron, which may facilitate more efficient iron transport and utilization in magur fingerlings.
       
Hepcidin, another key regulator protein of iron homeostasis is primarily expressed in liver with bacterial challenge and iron overload (Ganz, 2003; Nemeth and Ganz, 2006). It is an iron regulating hormone and can be used as a biomarker in determining the serum iron bioavailability (Rajamanickam et al., 2020, 2021). Here, hepcidin expression exhibited a distinct tissue-specific pattern, with liver showing the highest transcriptional activity among all tissues in both the diets. As the principal regulator of systemic iron homeostasis, hepcidin expression increased markedly with increasing dietary iron levels, particularly in fish fed ferrous citrate. The highest hepatic hepcidin expression observed at 45 mg/kg ferrous citrate suggests activation of regulatory mechanisms to prevent excessive iron accumulation. Increased hepcidin expression under high iron conditions has been widely reported and is considered a feedback response to maintain iron balance by reducing intestinal iron absorption and iron release from storage tissues. This is in accordance to prior studies, where higher transcript of hepcidin was detected in fish liver due to iron overload (Nemeth and Ganz, 2006; Wang et al., 2009; Shen et al., 2019). The genes encoding the hepcidin protein is also regulated by anemia, hypoxia and inflammation thereby limiting iron availability (Shike et al., 2004; Das et al., 2015).
       
Overall, the three gene expression profiles collectively indicate that dietary iron supplementation modulates iron metabolism in Clarias magur through coordinated regulation of iron storage (ferritin), transport (transferrin) and homeostasis (hepcidin). The liver emerged as the principal organ involved in iron regulation, while the intestine played a major role in iron absorption and transport. These findings highlight the effectiveness of ferrous citrate as a dietary iron source and provide molecular evidence for its role in enhancing iron metabolism and homeostasis in Clarias magur fingerlings.

CONCLUSION

Dietary iron supplementation significantly influenced the expression of ferritin, transferrin and hepcidin genes in different tissues of magur fingerlings. The liver showed the highest expression of ferritin and hepcidin, while the intestine exhibited the highest transferrin expression, highlighting their major roles in iron storage, regulation and transport. Ferrous citrate consistently induced greater gene expression than ferric citrate, indicating higher bioavailability and more efficient utilization of ferrous iron. Among the tested levels, 45 mg Fe/kg diet as ferrous citrate produced the strongest transcriptional response, suggesting improved iron metabolism and homeostasis. Overall, ferrous citrate is a more effective dietary iron source for Clarias magur fingerlings.

ACKNOWLEDGEMENT

The authors are thankful to Indian Council of Agricultural Research (ICAR) (Ph.D. student grant 2019-22) for the financial support. Director of ICAR-CIFE (Central Institute of Fisheries Education) for providing necessary infrastructural facilities for completing the experiment. Also, I would like to thank The Dean, College of Fisheries, Kishanganj under Bihar Animal Sciences University, Patna for his guidance and support to complete this research.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

REFERENCES


  1. Arosio, P., Ingrassia, R. and Cavadini. P. (2009). Ferritins: A family of molecules for iron storage, antioxidation and more. Biochimica et Biophysica Acta (BBA). 1790(7): 589- 599.

  2. Bao, B., Peatman, E., Li, P., He, C. and Liu, Z. (2005). Catfish hepcidin gene is expressed in a wide range of tissues and exhibits tissue-specific upregulation after bacterial infection. Developmental and Comparative Immunology. 29(11): 939-950.

  3. Bolaji, O.T., Adeyeye, S.A.O. and Araoye, A. (2022). Effect of smoking and storage conditions on the quality and acceptability of smoked catfish (Clarias gariepinus). Asian Journal of Dairy and Food Research. doi: 10.18805/ajdfr.DR-1933. 

  4. Bresgen, N. and Eckl, P.M. (2015). Oxidative stress and the homeodynamics of iron metabolism. Biomolecules. 5(2): 808-847.

  5. Buyinza, I., Ramena, G., Lochmann, R., Sinha, A. and Jones, M. (2024). Plasma and tissue transferrin and ferritin and gene expression of ferritin, transferrin and transferrin receptors I and II in Ictalurus punctatus fed diets with different concentrations of inorganic or organic iron. Journal of Fish Diseases. 47(8): e13953.

  6. Buwono, I.D., Carsono, N., Mulyani, Y. and Agung, M.U.K. (2015). Isolation Of B-actin promoter derived from walking catfish (Clarias Batrachus). KnE Life Sciences. 1: 201-211.

  7. Chakraborty, A., Das, S., Sarkar, I., Dey, A. and Sinha, M. (2020). Seed production of endangered Indian magur, Clarias magur (Hamilton,1822) in low-cost model with community participation. International Journal of Fauna and Biological Studies. 7(3): 17-20.

  8. Das, A., Mohapatra, A. and Sahoo, P.K. (2015). Cloning and characterization of antimicrobial peptide, hepcidin in medium carp, Puntius sarana. International Journal of Peptide Research and Therapeutics. 21: 139-147.

  9. Fraenkel, P.G., Gibert, Y., Holzheimer, J.L., Lattanzi, V.J., Burnett, S.F., Dooley, K.A., Wingert, R.A. and Zon, L.I. (2009). Transferrin-A modulates hepcidin expression in zebrafish embryos. Blood. 113(12): 2843-2850. 

  10. Ganz, T. (2003). Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation. Blood. 102: 783- 788.

  11. Ganz, T. and Nemeth, E. (2012).  Hepcidin and iron homeostasis. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 1823(9): 1434-1443.

  12. Ganz, T. and Nemeth, E. (2015). Iron homeostasis in host defense and inflammation. Nature Reviews Immunology. 15(8): 500-510.

  13. Gatlin, D.M. and Wilson, R.P. (1986). Characterization of iron deficiency and the dietary iron requirement of fingerling channel catfish. Aquaculture. 52: 191-198.

  14. Gkouvatsos, K., Papanikolaou, G. and Pantopoulos, K. (2012). Regulation of iron transport and the role of transferrin. Biochimica et Biophysica Acta (BBA). 1820(3): 188- 202.

  15. Hamilton, F. (1822). An Account of the Fishes Found in the River Ganges and its Branches (Vol. 1). Archibald Constable.

  16. Jayasankar, P., Mohanta, K.N. and Ferosekhan S. (2018). Freshwater Aquaculture in India. In: Aquaculture in India. [(Edited by): Tripathi, S.D., Lakra, W.S.  and Chadha, N.K.], Narendra Publishing House, Delhi (India). Pages 2349.

  17. Lim, C., Sealey, W.M. and Klesius, P.H. (1996). Iron methionine and iron sulfate as sources of dietary iron for channel catfish Ictalurus punctatus. Journal of World Aquaculture Society. 27: 290-296.

  18. Lim, C., Klesius, P.H. and Shoemaker, C.A. (2001). Dietary Iron and Fish Health. In: Nutrition and Fish Health [Lim, C. and Webster, C. D., (eds.)], Food Products Press, New York. pp 189-199.

  19. Livak, K.J. and Schmittgen, T.D. (2001). Analysis of relative gene expression data using Real-Time quantitative PCR and the 2-ΔΔCT Method. Methods. 25(4): 402-408.  

  20. Nemeth, E. and Ganz, T. (2006). Regulation of iron metabolism by hepcidin. Annual Reviews of Nutrition. 26: 323-342. 

  21. Nemeth, E., Tuttle, M.S., Powelson, J., Vaughn, M.B., Donovan, A., Ward, D.M., Ganz, T. and Kaplan, J. (2004). Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science. 306(5704): 2090-2093. 

  22. Neves, J.V., Wilson, J.M. and Rodrigues, P.N.S. (2009). Transferrin and ferritin response to bacterial infection: The role of the liver and brain in fish. Developmental and Comparative Immunology. 33: 848-857.

  23. NRC, (National Research Council). (2011). Nutrient Requirements of Fish and Shrimp. National Academy Press, Washington D.C.

  24. Rajamanickam, K., Ali, M.S. and Leela, V. (2020). Influence of hepcidin on iron homeostasis during last trimester of gestation in Bos indicus (cattle). Indian Journal of Animal Research. 54(2): 160-162. doi: 10.18805/ijar.B-3743.

  25. Rajamanickam, K., Leela, V., Loganathasamy, K., Latha, B.R. and Balagangatharathilaga, M.  (2021). Serum hepcidin as a clinical prognostic marker to discriminate the outcome of canine babesiosis.  Indian Journal of Animal Research. 55(11): 1323-1329. doi: 10.18805/IJAR.B-4222.

  26. Sangeetha, K. and Rajan, M.R. (2021). Evaluation of different quantity of iron oxide nanoparticles on growth, hematological and biochemical characteristics of Koi carp. Agriculture Science Digest. 41(2): 338-344. doi: 10.18805/ag.D-5236.

  27. Sevcikova, M., Modra, H., Slaninova, A. and Svobodova, Z. (2011). Metals as a cause of oxidative stress in fish: A review. Veterinary Medicine. 56(11): 537-546.

  28. Shen, Y., Zhao, Z., Zhao, J., Chen, X., Cao, M. and Wu, M. (2019). Expression and functional analysis of hepcidin from mandarin fish (Siniperca chuatsi). International Journal of Molecular Sciences. 20(22): 5602.

  29. Shi, J. and Camus, A.C. (2006). Hepcidins in amphibians and fishes: antimicrobial peptides or iron-regulatory hormones? Developmental and Comparative Immunology. 30: 746- 755.

  30. Shike, H., Shimizu, C., Lauth, X. and Burns, J.C. (2004). Organization and expression analysis of the zebrafish hepcidin gene, an antimicrobial peptide gene conserved among vertebrates. Developmental and Comparative Immunology. 28: 747- 754.

  31. Torti, F.M. and Torti, S.V. (2002). Regulation of ferritin genes and protein. Blood. 99(10): 3505-3516.

  32. Viani, V., Santosa, S.J. and Suyanta, (2025). effect of adding nanoparticle magnetite (Fe3O4-NPs) and probiotics in feed on the growth performance of catfish (Clarias batrachus). Aquaculture International. 33(6): 497.

  33. Wang, K.J., Bo, J., Yang, M., Hong, H.S., Wang, X.H., Chen, F.Y. and Yuan, J.J. (2009). Hepcidin gene expression induced in the developmental stages of fish upon exposure to Benzo[a]pyrene (BaP). Marine Environmental Research.  67(3): 159-165.

  34. Yin, X., Mu, L., Bian, X., Wu, L., Li, B., Liu, J., Guo, Z. and Ye, J. (2018). Expression and functional characterization of transferrin in Nile tilapia in response to bacterial infection. Fish and Shellfish Immunology. 74: 530-539.

  35. Yin, X., Mu, L., Bian, X., Wu, L., Li, B., Liu, J., Guo, Z. and Ye, J. (2018). Expression and functional characterization of transferrin in Nile tilapia in response to bacterial infection. Fish and Shellfish Immunology. 74: 530-539. 

  36. Yu, H., Li, L., Ma, C., Zhang, X. and Yu, L. (2024). Effect of dietary iron (Fe) supplementation on growth performance, hematological parameters and anti-oxidant responses of coho salmon Oncorhynchus kisutch (Walbaum, 1792) post-smolts. Aquaculture Reports. 37: 102203.
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    Full Research Article

    Impact of Graded Levels of Dietary Ferrous and Ferric Citrate Supplementation on Expression of Iron Regulatory Genes in Clarias magur

    S
    Susmita Rani1,2,*
    https://orcid.org/0000-0002-9190-0033
    P
    Prem Prakash Srivastava1,3,*
    https://orcid.org/0000-0001-8433-3889
    A
    Ashutosh D. Deo1
    T
    Tincy Varghese4
    S
    Subodh Gupta1
    https://orcid.org/0000-0002-9420-480X
    A
    Abhiman2
    P
    Pankaj Kumar2
    S
    Shivendra Kumar3
    1Division of Fish Nutrition, Biochemistry and Physiology, ICAR-Central Institute of Fisheries Education, Versova, Mumbai-400 061, Maharashtra, India.
    2College of Fisheries, (Bihar Animal Sciences University-Patna) Kishanganj-855 107, Bihar, India.
    3College of Fisheries, Dr. Rajendra Prasad Central Agricultural University, Dholi, Pusa, Muzaffarpur-843 121, Bihar, India.
    4ICAR-Central Institute of Fisheries Technology, Willingdon Island, Kochi-682 029, Kerala, India.

    ABSTRACT

    Background: The 60-day experimental study investigated the expression of iron regulatory genes, namely ferritin, transferrin, and hepcidin, in Clarias magur fingerlings fed diets supplemented with two dietary iron sources. The experimental study comprised two groups in which C. magur fingerlings received diets supplemented with ferrous or ferric citrate at doses of 0, 15, 30, and 45 mg Fe/kg above the minimum dietary iron requirement of 30 mg Fe/kg diet, respectively.

    Methods: Ferritin, transferrin, hepcidin expression levels in fish intestine, liver, muscle and gill tissues were determined using quantitative-real time PCR (qRT-PCR). Regardless of the dietary iron source, all tissues exhibited expression of the three genes under investigation, however, the degree of expression varied amongst tissues.

    Result: Liver showed highest ferritin mRNA expression among all tissues under both iron sources. Maximum expression (6.77-fold) was observed at 45 mg/kg ferrous citrate. Muscle showed modest changes while gill exhibited the lowest ferritin expression. In intestine, under ferrous citrate supplementation, transferrin expression increased with increasing iron level. Maximum expression (4.21±0.14) was recorded at 45 mg/kg iron supplementation. Liver transferrin expression increased with increasing ferrous citrate. Highest expression was observed at T3 (3.31±0.07). In intestine, the highest hepcidin expression occurred at T1 (5.62±0.01). Ferric citrate showed comparatively lower expression in all treatments. Liver showed the highest hepcidin expression among all tissues. Expression increased significantly with increasing iron supplementation. Maximum expression (7.99±0.06) was observed in fish fed 45 mg/kg ferrous citrate (T3).

    INTRODUCTION

    Iron is an essential micronutrient that plays a crucial role in various physiological processes and immune functions in fish including oxygen transport, drug metabolism, steroid synthesis, DNA synthesis, ATP production, electron transport and cellular respiration (Lim et al., 2001; Ganz and Nemeth, 2015; Sangeetha and Rajan, 2021). In aquaculture, dietary supplementation of various inorganic and organic forms of iron has been reported to significantly enhance growth performance, health status and survival rates of cultured fish species (Yu et al., 2024). However, excessive iron accumulation can promote the generation of reactive oxygen species (ROS), resulting in oxidative stress and cellular damage (Sevcikova et al., 2011; Bresgen and Eckl, 2015).
           
    To maintain iron homeostasis, fish possess tightly regulated iron-metabolism proteins that control iron uptake, transport, storage and export (Arosio et al., 2009; Ganz and Nemeth, 2012). These proteins also contribute to host defense by limiting iron availability to invading pathogens, thereby enhancing immune responses during infection. (Shike et al., 2004; Das et al., 2015). Key components of this regulatory network include ferritin, the major intracellular iron-storage protein; transferrin, responsible for iron transport; ferroportin, which mediates cellular iron export; and hepcidin, a central regulator of systemic iron homeostasis that controls iron egress from cells (Torti and Torti, 2002; Nemeth et al., 2004; Bao et al., 2005; Shi and Camus, 2006; Fraenkel et al., 2009; Neves et al., 2009; Gkouvatsos et al., 2012; Yin et al., 2018).
           
    The general recommendation for dietary iron content in fish feeds to improve growth and feed utilization ranges from 30-150 mg/kg of dietary feed (NRC, 2011). In channel catfish (Ictalurus punctatus) improved growth and feed efficiency was obtained at 30 g/kg of iron supplementation (Lim et al., 1996). According to Gatlin and Wilson (1986), normal growth and feed efficiency were observed for channel catfish fed 10 mg or more of supplemental iron/kg; however, they observed that 20 mg supplemental iron/kg was required to maintain optimum hematological values. Based on these data, the dietary iron requirement of fingerling channel catfish was determined to be not more than 30 mg Fe/kg diet. Viani et al., (2025) suggested that 60 mg/kg dose of Fe3O4-NPs significantly improves growth, feed conversion and physiological safety in Clarias batrachus.
           
    The Indian walking catfish, Clarias magur (Hamilton, 1822), is one of the most important freshwater aquaculture species in India owing to its high market demand and culture potential (Jayasankar et al., 2018; Chakraborty et al., 2020). It is rich in easily digestible high-quality protein, essential omega-3 fatty acids and abundant iron. These properties contribute to cardiovascular health and help prevent iron-deficiency anemia and protein-energy malnutrition (Bolaji et al., 2022). In fish, the minimum dietary iron requirement has been estimated to be approximately 30 mg kg-1 diet (NRC, 2011). However, information regarding the physiological and molecular responses of Clarias magur to supplemental iron sources remains limited. Therefore, present study focuses on evaluating the tissue-specific expression profiles of key iron-regulatory genes (Ferritin, transferrin and hepcidin) in response to graded dietary supplementation of ferric and ferrous citrate in order to have a comprehensive understanding of how organic iron sources influence iron metabolism, storage and regulation at the mechanistic level. Such knowledge can contribute to the development of nutritionally optimized diets that maximize growth performance while maintaining iron homeostasis.

    MATERIALS AND METHODS

    Experimental fish and laboratory conditions
     
    Clarias magur weighing approximately 18.0±0.5 g were obtained from Kolkata, West Bengal, India and acclimatized in tanks containing 500 L freshwater with continuous aeration for 10 days at the wet laboratory of College of Fisheries, Kishanganj, Bihar where the experimental trial was conducted during 2024-2025.
     
    Experimental design, diet and feeding schedule
     
    In a completely randomized design, fish (n=120) were divided equally among the two experimental groups and fed with basal feed supplemented with Ferrous (Fe2+) and Ferric (Fe3+) citrate, respectively. Each experimental group was further divided into four groups (control and three treatments, T1, T2 and T3) with three replicates consisting of 10 fish each. Control contains 30 mg/kg iron citrate which is the basic dietary iron requirement of fish with no further supplementation. Fish in the three treatment groups received diet containing the minimum dietary iron required by fish (30 mg/kg feed) supplemented with graded dietary concentrations of 15 mg/kg (T1), 30 mg/kg (T2) and 45 mg/kg (T3) of ferric or ferrous citrate, respectively in both the experimental groups. Fish were fed for 60 days and 30-40% water exchange was done on alternate days. During this period, fish were fed with formulated feed consisting of 35% crude protein at the rate of 3% of their body weight (Table 1).

    Table 1: Feed composition and proximate composition of the experimental diets.


     
    Quantification of mRNA expression of ferritin, transferrin and hepcidin gene
     
    Tissue collection
     
    At the end of the experiment (60th day), fish (n=2) from the triplicate treatment and control tanks of each dietary group were randomly selected and anesthetized using clove oil (50 μl/L) before sacrifice. The fish were dissected for liver, intestine, gill and muscle tissues. These tissues were collected for gene expression study and preserved immediately in RNAlater™ solution (Qiagen, India) at -80°C until RNA extraction.
     
    Total RNA extraction and cDNA synthesis
     
    Total RNA was extracted from equal portions of each tissue collected using Trizol TM reagent (Invitrogen, USA) as per manufacturer’s instructions. Briefly, 100 mg of RNA later TM stored tissue was homogenized with 1 ml of Trizol reagent, followed by centrifugation for 5 min at room temperature. To the aqueous phase transferred to a fresh tube, 200 µl of chloroform (0.2 vol/ml Trizol TM reagent) was added, vortexed for 1 min and incubated for 10 min at room temperature. The mixture was centrifuged at 12,000 rpm for 15 min at 4°C and the upper aqueous phase transferred to a fresh 1.5 ml tube. RNA was precipitated out using 500 µl of isopropanol (0.5 vol/ml Trizol TM reagent) followed by centrifugation at 12000 rpm for 15 min at 4°C. The RNA isolated was washed with 70% ethanol, air-dried and re-suspended in 35 µl of nuclease-free water and stored at -80°C. The RNA concentration and purity was estimated based on absorbance at OD260/280 nm using a Nanodrop 2000 (Thermo Fisher Scientific, USA). Total RNA extracted was purified for residual genomic DNA contamination using DNase I (Fermentas International Inc., Canada). Total RNA obtained was reverse transcribed to synthesize cDNA using Revert Aid cDNA synthesis kit (Thermo Scientific) as per the manufacturer’s protocol. cDNA was synthesized using a thermal cycler (Bio-Rad laboratories, Inc) with the following cycling conditions: initial denaturation at 94°C for 4 min followed by 30 cycles of 94°C for 30s, 55°C for 30 s, annealing at 72°C for 30 s and a final extension for 7 min at 72°C. The cDNA was stored at -20°C until use.
     
    Primer design for RT-qPCR
     
    The primers for RT-qPCR were designed using Gene runner software (version 3.05) based on available gene sequences in NCBI GenBank.  The primer sequences for the gene ferritin, transferrin and hepcidin, their product size and NCBI accession numbers are provided in Table 2. β-actin was used as a reference gene and served as an internal standard gene (Buwono et al., 2015) Primer synthesis was outsourced to Bioserve Biotechnologies, Hyderabad and Bioinnovations, Maharashtra, India.

    Table 2: Primers used for expression study in real-time PCR.


     
    Quantitative real-time PCR (RT-qPCR) for mRNA expression of ferritin, transferrin and hepcidin gene in different tissues of Clarias magur fingerlings
     
    After reverse transcription, target cDNA was amplified and relative quantification of ferritin, transferrin and hepcidin gene in different tissues of Clarias magur fingerlings was done in a quantitative real-time PCR using SYBR green qPCR master mix without ROX (Thermo scientific) in aria MX real-time PCR system (Agilent Technology LDA). The reaction took place in a volume of 10 μl containing 5 μl of 2 × maxima™ SYBR green qPCR master mix (Thermo Scientific), 1 μl of (5 pmol) each gene-specific primer, 1 μl cDNA template and remaining nuclease-free water prepared for quantification. The quantified value of mRNA was expressed in terms of CT (threshold cycle) value. The PCR program included 10-minute activation and initial denaturation step at 95°C for 5 min, followed by 45 cycles for amplification with each denaturation cycle at 95°C for 15s, annealing at 53°C for 30s and extension at 72°C for 30s. At the end, the specificity of reaction was confirmed using melt curve analysis. A comparative CT method was used to estimate the relative expression of mRNA. All PCR reactions were run in duplicate and normalized by the value by 2-ΔΔCT method. The fold change in gene expression was normalized to β-actin and the expression level of the target gene was calculated by the 2-ΔΔCT CT-method (Livak and Schmittgen, 2001).
           
    The quantified result was analyzed by the following formulae:
     
    Δct = Target gene ct value - reference gene ct value.
     
    ΔΔ ct value = Δct value of treatment group - Δct value of control group
     
    Statistical analysis
     
    For each tissue, the gene transcript values were calculated for triplicate samples and expressed as mean±standard error. The statistical significance of all parameters was determined using one-way ANOVA followed by Duncan’s new multiple range test using the statistical package with SPSS 22.0 for Windows. A significant difference was considered at a p value <0.05.

    RESULTS AND DISCUSSION

    Table 3, Fig 1-2; Table 4, Fig 3-4; Table 5, Fig 5-6 presents the expression of ferritin, transferrin and hepcidin gene, respectively in different tissues of C. magur fed diets supplemented with varying levels of ferrous or ferric citrate.

    Table 3: Relative mRNA expression of ferritin gene in different tissues of Clarias magur fed with experimental diets supplemented with ferrous and ferric citrate.



    Table 4: Relative mRNA expression of transferrin gene in different tissues of Clarias magur fed with experimental diets supplemented with ferrous and ferric citrate.



    Table 5: Relative mRNA expression of hepcidin gene in different tissues of Clarias magur fed with experimental diets supplemented with ferrous and ferric citrate.



    Fig 1: Relative mRNA expression of ferritin gene in different tissues of Clarias magur fed with diets supplemented with ferrous citrate.



    Fig 2: Relative mRNA expression of ferritin gene in different tissues of Clarias magur fed with diets supplemented with ferric citrate.



    Fig 3: Relative mRNA expression of transferrin gene in different tissues of Clarias magur fed with diets supplemented with ferrous citrate.



    Fig 4: Relative mRNA expression of transferrin gene in different tissues of Clarias magur fed with diets supplemented with ferric citrate.



    Fig 5: Relative mRNA expression of hepcidin gene in different tissues of Clarias magur fed with diets supplemented with ferrous citrate.



    Fig 6: Relative mRNA expression of hepcidin gene in different tissues of Clarias magur fed with diets supplemented with ferric citrate.


     
    Ferritin
     
    Liver showed the highest ferritin mRNA expression among all tissues under both iron sources. Expression increased significantly with increasing ferrous citrate supplementation. Maximum expression (6.77-fold) was observed in the liver at 45 mg/kg ferrous citrate (T3). Ferric citrate produced considerably lower expression than ferrous citrate.
           
    Intestine showed moderate ferritin expression. Under ferrous citrate, expression peaked at T2 (3.44) and then declined. Under ferric citrate, expression increased progressively from T1 to T3, suggesting active regulation of iron absorption and storage.
           
    Muscle exhibited relatively low expression. Under Ferrous citrate expression resulted in gradual increase with dose while with ferric citrate expression peaked at T2 and decreased at T3.
           
    Gill exhibited the lowest ferritin expression overall. Expression increased significantly at the highest iron supplementation level.
     
    Transferrin
     
    In intestine, under Ferrous citrate supplementation, transferrin expression increased significantly with increasing iron level. Maximum expression (4.21±0.14) was recorded at T3. Under ferric citrate, expression peaked at T2 (3.98±0.03) and declined at T3. The intestine responds actively to dietary iron supplementation, reflecting its role in iron absorption and transport.
           
    Expression remained relatively low in muscle. Ferric citrate produced a sharp increase at T2  followed by a marked decline at T3. Ferrous citrate treatments showed only minor variation. From this it can be concluded that muscle tissue is not a primary site of iron transport regulation and therefore exhibited comparatively lower transferrin expression.
           
    Liver transferrin expression increased significantly with increasing ferrous citrate supplementation. Highest expression under ferrous citrate was observed at T3 (3.31±0.07). Ferric citrate resulted in consistently lower expression values. The liver plays a central role in iron metabolism and transferrin synthesis. Higher expression under ferrous citrate suggests better iron utilization.
           
    In gill, transferrin expression increased significantly with increasing iron supplementation. Under ferrous citrate, maximum expression occurred at T2 whereas, under ferric citrate, expression increased steadily and reached its highest value at T3. Gill tissue participates in ion and mineral regulation, which may explain the increased transferrin expression with higher iron availability.
     
    Hepcidin
     
    In intestine, the highest expression occurred at T1 (5.62±0.01), under ferrous citrate supplementation followed by a decline at higher levels. Ferric citrate showed comparatively lower expression in all treatments. Ferrous citrate induced approximately 2-3 times higher hepcidin expression than ferric citrate.  Intestine exhibited a strong response to ferrous iron, indicating activation of iron homeostasis mechanisms.
           
    Muscle showed relatively low expression levels of hepcidin compared with other tissues. Under ferrous citrate, expression decreased as iron level increased. Under ferric citrate, expression increased and reached its maximum at T3. Muscle plays a limited role in systemic iron regulation and therefore exhibited lower hepcidin expression.
           
    Liver showed the highest hepcidin expression among all tissues. Expression increased significantly with increasing iron supplementation. Maximum expression (7.99±0.06) was observed in fish fed 45 mg/kg ferrous citrate (T3). Ferric citrate also increased expression but remained lower than ferrous citrate. Since the liver is the major site of hepcidin synthesis, elevated expression reflects enhanced regulation of iron metabolism in response to increased dietary iron.
           
    In gill under ferrous citrate, hepcidin expression decreased with increasing iron levels. Ferric citrate induced lower expression overall, peaking at T2. Gill showed moderate responsiveness to dietary iron.
           
    In this study, Ferrous citrate induced higher ferritin gene expression than ferric citrate in most tissues, indicating better bioavailability and utilization of iron. The liver was the primary site of iron storage, showing the highest ferritin transcription, followed by the intestine. Increasing dietary iron generally increased ferritin expression, particularly in the liver and gill. The highest ferritin expression was observed in the liver at the highest ferrous citrate level (45 mg/kg), indicating enhanced iron storage capacity in response to increased iron availability. The results suggest that ferrous citrate is more effective than ferric citrate in enhancing iron storage mechanisms in magur fingerlings.
           
    Transferrin expression levels revealed discrepancies among the tissues tested. Such discrepancies have been previously reported and been attributed to iron sources and concentrations in fish diets (Buyinza et al., 2024). Ferrous citrate generally induced higher transferrin gene expression than ferric citrate, particularly in the intestine, liver and gill. The highest transferrin expression was observed in intestine of fish fed 45 mg/kg ferrous citrate (4.21±0.14), reflecting its crucial role in dietary iron absorption and transport. Elevated transferrin expression in liver and gill further suggests enhanced iron mobilization and distribution within the body. The generally higher transferrin expression observed in fish fed ferrous citrate compared to ferric citrate indicates greater bioavailability of ferrous iron, which may facilitate more efficient iron transport and utilization in magur fingerlings.
           
    Hepcidin, another key regulator protein of iron homeostasis is primarily expressed in liver with bacterial challenge and iron overload (Ganz, 2003; Nemeth and Ganz, 2006). It is an iron regulating hormone and can be used as a biomarker in determining the serum iron bioavailability (Rajamanickam et al., 2020, 2021). Here, hepcidin expression exhibited a distinct tissue-specific pattern, with liver showing the highest transcriptional activity among all tissues in both the diets. As the principal regulator of systemic iron homeostasis, hepcidin expression increased markedly with increasing dietary iron levels, particularly in fish fed ferrous citrate. The highest hepatic hepcidin expression observed at 45 mg/kg ferrous citrate suggests activation of regulatory mechanisms to prevent excessive iron accumulation. Increased hepcidin expression under high iron conditions has been widely reported and is considered a feedback response to maintain iron balance by reducing intestinal iron absorption and iron release from storage tissues. This is in accordance to prior studies, where higher transcript of hepcidin was detected in fish liver due to iron overload (Nemeth and Ganz, 2006; Wang et al., 2009; Shen et al., 2019). The genes encoding the hepcidin protein is also regulated by anemia, hypoxia and inflammation thereby limiting iron availability (Shike et al., 2004; Das et al., 2015).
           
    Overall, the three gene expression profiles collectively indicate that dietary iron supplementation modulates iron metabolism in Clarias magur through coordinated regulation of iron storage (ferritin), transport (transferrin) and homeostasis (hepcidin). The liver emerged as the principal organ involved in iron regulation, while the intestine played a major role in iron absorption and transport. These findings highlight the effectiveness of ferrous citrate as a dietary iron source and provide molecular evidence for its role in enhancing iron metabolism and homeostasis in Clarias magur fingerlings.

    CONCLUSION

    Dietary iron supplementation significantly influenced the expression of ferritin, transferrin and hepcidin genes in different tissues of magur fingerlings. The liver showed the highest expression of ferritin and hepcidin, while the intestine exhibited the highest transferrin expression, highlighting their major roles in iron storage, regulation and transport. Ferrous citrate consistently induced greater gene expression than ferric citrate, indicating higher bioavailability and more efficient utilization of ferrous iron. Among the tested levels, 45 mg Fe/kg diet as ferrous citrate produced the strongest transcriptional response, suggesting improved iron metabolism and homeostasis. Overall, ferrous citrate is a more effective dietary iron source for Clarias magur fingerlings.

    ACKNOWLEDGEMENT

    The authors are thankful to Indian Council of Agricultural Research (ICAR) (Ph.D. student grant 2019-22) for the financial support. Director of ICAR-CIFE (Central Institute of Fisheries Education) for providing necessary infrastructural facilities for completing the experiment. Also, I would like to thank The Dean, College of Fisheries, Kishanganj under Bihar Animal Sciences University, Patna for his guidance and support to complete this research.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

    REFERENCES


    1. Arosio, P., Ingrassia, R. and Cavadini. P. (2009). Ferritins: A family of molecules for iron storage, antioxidation and more. Biochimica et Biophysica Acta (BBA). 1790(7): 589- 599.

    2. Bao, B., Peatman, E., Li, P., He, C. and Liu, Z. (2005). Catfish hepcidin gene is expressed in a wide range of tissues and exhibits tissue-specific upregulation after bacterial infection. Developmental and Comparative Immunology. 29(11): 939-950.

    3. Bolaji, O.T., Adeyeye, S.A.O. and Araoye, A. (2022). Effect of smoking and storage conditions on the quality and acceptability of smoked catfish (Clarias gariepinus). Asian Journal of Dairy and Food Research. doi: 10.18805/ajdfr.DR-1933. 

    4. Bresgen, N. and Eckl, P.M. (2015). Oxidative stress and the homeodynamics of iron metabolism. Biomolecules. 5(2): 808-847.

    5. Buyinza, I., Ramena, G., Lochmann, R., Sinha, A. and Jones, M. (2024). Plasma and tissue transferrin and ferritin and gene expression of ferritin, transferrin and transferrin receptors I and II in Ictalurus punctatus fed diets with different concentrations of inorganic or organic iron. Journal of Fish Diseases. 47(8): e13953.

    6. Buwono, I.D., Carsono, N., Mulyani, Y. and Agung, M.U.K. (2015). Isolation Of B-actin promoter derived from walking catfish (Clarias Batrachus). KnE Life Sciences. 1: 201-211.

    7. Chakraborty, A., Das, S., Sarkar, I., Dey, A. and Sinha, M. (2020). Seed production of endangered Indian magur, Clarias magur (Hamilton,1822) in low-cost model with community participation. International Journal of Fauna and Biological Studies. 7(3): 17-20.

    8. Das, A., Mohapatra, A. and Sahoo, P.K. (2015). Cloning and characterization of antimicrobial peptide, hepcidin in medium carp, Puntius sarana. International Journal of Peptide Research and Therapeutics. 21: 139-147.

    9. Fraenkel, P.G., Gibert, Y., Holzheimer, J.L., Lattanzi, V.J., Burnett, S.F., Dooley, K.A., Wingert, R.A. and Zon, L.I. (2009). Transferrin-A modulates hepcidin expression in zebrafish embryos. Blood. 113(12): 2843-2850. 

    10. Ganz, T. (2003). Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation. Blood. 102: 783- 788.

    11. Ganz, T. and Nemeth, E. (2012).  Hepcidin and iron homeostasis. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research. 1823(9): 1434-1443.

    12. Ganz, T. and Nemeth, E. (2015). Iron homeostasis in host defense and inflammation. Nature Reviews Immunology. 15(8): 500-510.

    13. Gatlin, D.M. and Wilson, R.P. (1986). Characterization of iron deficiency and the dietary iron requirement of fingerling channel catfish. Aquaculture. 52: 191-198.

    14. Gkouvatsos, K., Papanikolaou, G. and Pantopoulos, K. (2012). Regulation of iron transport and the role of transferrin. Biochimica et Biophysica Acta (BBA). 1820(3): 188- 202.

    15. Hamilton, F. (1822). An Account of the Fishes Found in the River Ganges and its Branches (Vol. 1). Archibald Constable.

    16. Jayasankar, P., Mohanta, K.N. and Ferosekhan S. (2018). Freshwater Aquaculture in India. In: Aquaculture in India. [(Edited by): Tripathi, S.D., Lakra, W.S.  and Chadha, N.K.], Narendra Publishing House, Delhi (India). Pages 2349.

    17. Lim, C., Sealey, W.M. and Klesius, P.H. (1996). Iron methionine and iron sulfate as sources of dietary iron for channel catfish Ictalurus punctatus. Journal of World Aquaculture Society. 27: 290-296.

    18. Lim, C., Klesius, P.H. and Shoemaker, C.A. (2001). Dietary Iron and Fish Health. In: Nutrition and Fish Health [Lim, C. and Webster, C. D., (eds.)], Food Products Press, New York. pp 189-199.

    19. Livak, K.J. and Schmittgen, T.D. (2001). Analysis of relative gene expression data using Real-Time quantitative PCR and the 2-ΔΔCT Method. Methods. 25(4): 402-408.  

    20. Nemeth, E. and Ganz, T. (2006). Regulation of iron metabolism by hepcidin. Annual Reviews of Nutrition. 26: 323-342. 

    21. Nemeth, E., Tuttle, M.S., Powelson, J., Vaughn, M.B., Donovan, A., Ward, D.M., Ganz, T. and Kaplan, J. (2004). Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science. 306(5704): 2090-2093. 

    22. Neves, J.V., Wilson, J.M. and Rodrigues, P.N.S. (2009). Transferrin and ferritin response to bacterial infection: The role of the liver and brain in fish. Developmental and Comparative Immunology. 33: 848-857.

    23. NRC, (National Research Council). (2011). Nutrient Requirements of Fish and Shrimp. National Academy Press, Washington D.C.

    24. Rajamanickam, K., Ali, M.S. and Leela, V. (2020). Influence of hepcidin on iron homeostasis during last trimester of gestation in Bos indicus (cattle). Indian Journal of Animal Research. 54(2): 160-162. doi: 10.18805/ijar.B-3743.

    25. Rajamanickam, K., Leela, V., Loganathasamy, K., Latha, B.R. and Balagangatharathilaga, M.  (2021). Serum hepcidin as a clinical prognostic marker to discriminate the outcome of canine babesiosis.  Indian Journal of Animal Research. 55(11): 1323-1329. doi: 10.18805/IJAR.B-4222.

    26. Sangeetha, K. and Rajan, M.R. (2021). Evaluation of different quantity of iron oxide nanoparticles on growth, hematological and biochemical characteristics of Koi carp. Agriculture Science Digest. 41(2): 338-344. doi: 10.18805/ag.D-5236.

    27. Sevcikova, M., Modra, H., Slaninova, A. and Svobodova, Z. (2011). Metals as a cause of oxidative stress in fish: A review. Veterinary Medicine. 56(11): 537-546.

    28. Shen, Y., Zhao, Z., Zhao, J., Chen, X., Cao, M. and Wu, M. (2019). Expression and functional analysis of hepcidin from mandarin fish (Siniperca chuatsi). International Journal of Molecular Sciences. 20(22): 5602.

    29. Shi, J. and Camus, A.C. (2006). Hepcidins in amphibians and fishes: antimicrobial peptides or iron-regulatory hormones? Developmental and Comparative Immunology. 30: 746- 755.

    30. Shike, H., Shimizu, C., Lauth, X. and Burns, J.C. (2004). Organization and expression analysis of the zebrafish hepcidin gene, an antimicrobial peptide gene conserved among vertebrates. Developmental and Comparative Immunology. 28: 747- 754.

    31. Torti, F.M. and Torti, S.V. (2002). Regulation of ferritin genes and protein. Blood. 99(10): 3505-3516.

    32. Viani, V., Santosa, S.J. and Suyanta, (2025). effect of adding nanoparticle magnetite (Fe3O4-NPs) and probiotics in feed on the growth performance of catfish (Clarias batrachus). Aquaculture International. 33(6): 497.

    33. Wang, K.J., Bo, J., Yang, M., Hong, H.S., Wang, X.H., Chen, F.Y. and Yuan, J.J. (2009). Hepcidin gene expression induced in the developmental stages of fish upon exposure to Benzo[a]pyrene (BaP). Marine Environmental Research.  67(3): 159-165.

    34. Yin, X., Mu, L., Bian, X., Wu, L., Li, B., Liu, J., Guo, Z. and Ye, J. (2018). Expression and functional characterization of transferrin in Nile tilapia in response to bacterial infection. Fish and Shellfish Immunology. 74: 530-539.

    35. Yin, X., Mu, L., Bian, X., Wu, L., Li, B., Liu, J., Guo, Z. and Ye, J. (2018). Expression and functional characterization of transferrin in Nile tilapia in response to bacterial infection. Fish and Shellfish Immunology. 74: 530-539. 

    36. Yu, H., Li, L., Ma, C., Zhang, X. and Yu, L. (2024). Effect of dietary iron (Fe) supplementation on growth performance, hematological parameters and anti-oxidant responses of coho salmon Oncorhynchus kisutch (Walbaum, 1792) post-smolts. Aquaculture Reports. 37: 102203.
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