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Full Research Article

Impact of Plant-based Protein Substitutes on Growth Performance, Histology and Gene Expression in Striped Snakehead, Channa striata (Bloch, 1793) Fingerlings

S
S. Ajidhaslin1,*
C
Cheryl Antony1
A
Arul Victor Suresh2
B
B. Ahilan1
A
A. Uma1
K
K. Ravaneswaran1
C
C Grace Angel2
1Dr. M.G.R Fisheries College and Research Institute, Dr. J. Jayalalithaa Fisheries University, Ponneri-601 204, Tamil Nadu, India.
2Growel Feeds and Private Limited, Chevuru-521 329, Andhra Pradesh, India.

ABSTRACT

Background: This research assessed the effects of incorporating various plant-based protein sources, including corn gluten meal (CGM), cottonseed meal (CSM), groundnut meal (GNM) and mustard seed meal (MSM), alongside soybean meal (SBM) in the diet of juvenile snakehead (Channa striata). Building on our previous findings that established an optimal SBM inclusion level of 25%, this subsequent trial explored the addition of these plant protein ingredients at a 10% inclusion rate, supplemented by SBM.

Methods: The experimental diets were designed to be isonitrogenous (42% crude protein), isolipidic (9% crude fat) and isoenergetic (16.1-16.2 MJ/kg). The trial was conducted over eight weeks with 400 healthy murrel fingerlings (average weight 27.0±0.06 g), sourced from Growel Feeds Pvt Ltd, a commercial fish hatchery in Andhra Pradesh. The fingerlings were reared in fiberglass reinforced plastic (FRP) tanks (150 L capacity, with 20 fish per tank).

Result: Evaluations of growth performance, haematological parameters, histological analyses and gene expression showed no significant differences among the various experimental groups. These results suggest that all the plant-based protein ingredients tested provided comparable growth performance and can be effectively used as alternative protein sources in conjunction with SBM for Channa striata feed formulations. This study highlights the potential for integrating cost-effective, plant-based protein sources into the feed for striped murrel fingerlings, promoting sustainability and economic viability.

INTRODUCTION

The striped murrel (Channa striata) is a rapidly growing and versatile carnivorous fish found in Southeast and Southern Asia, prized for its strong market demand (Gustiano et al., 2021; Kumar et al., 2022). Its commercial cultivation has played a significant role in advancing global aquaculture (FAO, 2020). Being a carnivorous species, C. striata necessitates high-protein diets, which are conventionally supplied by fishmeal (FM). However, this reliance raises sustainability concerns due to limited availability and fluctuating prices (Vo et al., 2015; Glencross et al., 2024). Consequently, there is a growing interest in alternative protein sources, such as plant proteins, that can replace or supplement fishmeal while ensuring adequate growth performance (Maksimenko et al., 2024).
       
Plant proteins, including soybean meal (SBM), are economically advantageous and contribute to the expansion of aquaculture; yet, their use can be restricted by anti-nutritional factors and lower digestibility in carnivorous fish (NRC, 2011; Kumari et al., 2013). Addressing these issues requires the development of optimized feed formulations (Glencross et al., 2024). Techniques such as fermentation and enzymatic treatment may enhance the quality of plant proteins (Maksimenko et al., 2024).
       
This study aims to assess the supplementation of SBM with CGM, CSM, GNM and MSM in C. striata diets to foster sustainable and cost-effective aquaculture practices. Degossypolized cottonseed protein (DCP) is produced by the solvent extraction of water-soluble carbohydrates and free gossypol from cottonseed meal, which finally contains high-quality protein and a low concentration of free gossypol (Wang et al., 2019). Detection of aflatoxins in corn gluten meal and groundnut meal is carried out by high-performance thin-layer Chromatography based on their fluorescence under UV light with an acceptable level below 20 μg kg-1 (Jaiswar et al., 2022).

MATERIALS AND METHODS

Ethical statement
 
The experiment was conducted following the procedures of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals), Ministry of Environment and Forests (Animal Welfare Division), Govt. of India on care and use of animals in scientific research. This study was approved by the ethical committee of Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Nagapattinam, Tamil Nadu, India. The experiment was conducted for 60 days at Growel Fish Hatchery, Perikegudem, Andhra Pradesh, India.
 
Experimental diets
 
Five distinct diets were formulated to evaluate the effects of alternative plant protein sources, including Corn Gluten Meal (CGM), Cottonseed Meal (CSM), Groundnut Meal (GNM) and Mustard Seed Meal (MSM), alongside soybean meal (SBM) for Channa striata. The diets comprised a control group with only SBM and four additional groups featuring CGM (T2), CSM (T3), GNM (T4) and MSM (T5). All diets were designed to be isonitrogenous, isolipidic and isoenergetic, containing 42% crude protein, 9% crude fat and gross energy levels of 16.1-16.2 MJ/kg. The feed was processed into floating pellets of 1.2 mm in diameter using a twin-screw extruder. A detailed composition can be found in Table 1.

Table 1: Feed formulation and proximate composition of experimental diets (g.kg-1 diet).


 
Fish husbandry
 
Juvenile Channa striata were acquired from Growel Feeds Hatchery located in Andhra Pradesh and were acclimatized for a duration of 20 days on a commercial diet (42% CP, 9% CL). The feeding trial was executed in a Recirculating Aquaculture System (RAS) utilizing 20 fiberglass-reinforced plastic (FRP) tanks, each with a capacity of 150 L and supplied with sand-filtered water, aeration and covered with fiber mats. The FRP tanks were linked to biofiltration units, with water recirculated four times per day.
       
A total of 400 juveniles (average weight 27.0±0.06 g) were randomly allocated into the 20 tanks (20 fish per tank). Fishes were hand fed their respective diets till apparent satiety three times a day (8:00, 12:00 and 17:00 h) at 10% body weight until satiation for 8 weeks. Cannibalism cannot be completely eliminated in snakehead culture, but maintaining size uniformity and ensuring satiation feeding effectively minimized cannibalistic interactions in the experimental setup. Initial body composition analyses were conducted by sampling three fish per tank, while final body composition was assessed at the conclusion of the study. Water quality parameters such as temperature (26.1± 0.5oC), dissolved oxygen (5.5±0.1 ppm), pH (7.5±0.1) and ammonia levels (0.02±0.006 ppm) were monitored twice daily, adhering to APHA (2005) standards.
 
Sampling methods and calculations
 
Following the eight-week feeding period, fish were deprived of food for 24 hours, weighed collectively. The fishes were anesthetized with clove oil (50 μl/L) and three fish per tank were selected for measuring length, body weight, visceral weight and liver weight to compute the condition factor (K), viscero-somatic index (VSI) and hepato-somatic index (HSI). For blood analysis, blood samples were collected from the caudal vein into EDTA-coated tubes. Plasma was obtained via centrifugation (3000 × g for 5 minutes) and subsequently stored at -20oC. Muscle samples and foregut sections were also collected for gene expression and histological assessments. Bio-growth parameters were calculated using standard formulas.
 
Analytical chemistry
 
Proximate analysis for crude lipid, crude protein, moisture, dry matter, ash and energy content in both diets and fish samples was performed following the AOAC (2000) guidelines. Moisture content was determined by drying samples at 105oC until a constant weight was reached. Crude protein levels were ascertained via nitrogen content using the Kjeltec system, while crude lipid was assessed through petroleum ether extraction using the Soxtec system. Crude fiber was analyzed using acid-alkali digestion with the Fibretech system and ash content was determined by incineration at 550oC for six hours.
 
Antioxidant enzyme analysis
 
Plasma concentrations of superoxide dismutase (SOD) and catalase (CAT) were measured. SOD activity was evaluated by tracking the increase in absorbance of Epinephrine at 480 nm, expressed as U/mg protein (Marklund and Marklund, 1974). Catalase activity was assessed by monitoring the decrease in H2O2 absorbance at 240 nm, expressed as ìmol/mg protein/min (Aebi, 1984). Protein concentration in serum was determined using AOAC’s method (1951).
 
Haematological and biochemical parameters
 
Three fish per tank were anesthetized with clove oil (50 μLL) for blood sampling from the caudal vein using EDTA-treated syringes. Haematological parameters (RBC, WBC, haematocrit, MCV, MCH, MCHC) were analyzed using an automatic analyzer (Cell Tech 380, India). Biochemical markers (glucose, serum cholesterol, triglycerides) were evaluated using a semi-automatic biochemical analyzer.
 
Histological analysis
 
Foregut samples were fixed in Davidson’s fixative, then dehydrated in ethanol and embedded in paraffin. Sections (7 μm) were stained with haematoxylin and eosin. Measurements of villus height, thickness and muscular thickness were conducted using image analysis software. This work was performed at Madras Veterinary College, Vepery, Tamil Nadu.
 
Quantitative Real-time PCR (qRT-PCR)
 
Muscle samples from three fish per tank were collected for gene expression analysis of MyoD and MyoG. RNA extraction was performed using RNA iso-plus (Takara Bio) and cDNA synthesis was conducted with a first-strand synthesis kit (Thermo Scientific). The list of primers given in Table 2. Gene expression was quantified through qRT-PCR on a CFX96 system (Bio-Rad) under specific cycling conditions. Beta-actin was used as the housekeeping gene and relative expression was calculated using the 2-ΔΔCt method. The study was carried out at the State Referral Laboratory for Aquatic Animal Health, Dr. M.G.R. Fisheries College and Research Institute, Madhavaram, Tamil Nadu.

Table 2: Primers used for qRT-PCR analysis of selected genes for Channa striata fed experimental diets.


 
Statistical analysis
 
Data were expressed as mean±standard deviation (SD) across four replicates. Statistical significance was assessed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (p<0.05). All statistical analyses were conducted using SPSS software version 20.0 (IBM), with variations analyzed at p<0.05 levels. The quantitative results were presented as mean±SD.

RESULTS AND DISCUSSION

Growth performance, feed utilization and somatic parameters
 
There were no significant differences in final weight (FW), weight gain (WG), specific growth rate (SGR), or feed conversion ratio (FCR) among the various treatment groups, which included both the SBM control and the diets supplemented with plant proteins. The protein efficiency ratio (PER) and SGR remained consistent across all dietary treatments. Furthermore, the addition of plant proteins (CGM, CSM, GNM, MSM) did not significantly influence the viscerosomatic index (VSI), hepatosomatic index (HSI), survival rates, or condition factor (p>0.05) given in Table 3.
 

Table 3: Growth performance and survival of C. striata fingerlings fed different experimental diets after 8 weeks of feeding trial.



Whole body composition
 
Proximate analysis of whole body composition indicated no significant differences across the different diet treatments (p>0.05) given in Table 4. All evaluated parameters were stable, suggesting that variations in diet did not affect overall body composition.

Table 4: Chemical composition of the whole body of C. striata fingerlings fed different experimental diets before and after feeding trial (% wet weight basis).


 
Intestinal morphology
 
The inclusion of alternative plant proteins did not result in significant changes in villi length. However, a notable reduction in villi width was observed in these treatment groups when compared to the control group, provided in Table 5. Despite this decrease in villi width, the structural integrity of the intestine, as measured by muscular thickness, remained consistent across all treatment groups.

Table 5: Histological parameters of fore-gut cross sections of juvenile Channa striata at the end of the 8 week feeding trial.


 
Haematological and biochemical indices
 
No significant alterations were noted in haematological parameters (RBC, WBC, haemoglobin, MCV, MCH) or biochemical indicators (albumin, globulin, total protein) among the treatment groups. The addition of plant proteins (CGM, CSM, GNM, MSM) did not adversely affect these health indicators, thus ensuring the well-being of the fish presented in Table 6.

Table 6: Changes of haematological and biochemical indices in C. striata fingerlings fed different experimental diets after feeding trial.


 
Antioxidant enzymes
 
The Nitroblue Tetrazolium (NBT) assay revealed no significant differences in phagocyte activity. Additionally, the activities of catalase (CAT) and superoxide dismutase (SOD) showed no significant changes in serum and liver samples, tabulated in Table 7. There were also no significant variations in albumin, globulin, or total protein levels, indicating stable protein quality and metabolic health.

Table 7: Effects of different inclusion of alternative plant protein ingredients along with SBM on antioxidant enzymes activity in serum of juvenile Channa striata.


 
Relative mRNA expression
 
No significant increase in the expression of MyoD and MyoG mRNA was detected in fish that were fed diets containing alternative plant proteins (CGM, CSM, GNM, MSM). This finding suggests that these plant protein sources did not influence the transcription levels of myogenic regulatory factors when compared to the SBM control diet.
       
The current study focused on evaluating the incorporation of various plant protein sources, including CGM, CSM, GNM and MSM, in the feed formulations for snakehead (Channa spp.). The findings revealed no significant differences in growth performance, feed conversion ratio (FCR), or weight gain, suggesting that these ingredients can effectively substitute fishmeal, consistent with previous research (Liu et al., 2023; Fei et al., 2024) (Fig 1). There was no statistically significant difference found in the length-weight relationship of sea bream fed diets with varying protein levels (Korkut et al., 2018). The plant proteins demonstrated their potential as sustainable alternatives, showing no detrimental impact on growth or feed efficiency, in alignment with studies conducted on other fish species (Zhang et al., 2014; Watanabe and Pongmaneerat, 1993; Fan et al., 2023). In the study by Priyatharshni et al., 2023 replacement of soybean meal study has also been evaluated with sesame meal in Tilapia fishes.

Fig 1: Weight gain (%) of C. striata fingerlings fed different experimental diets after 8 weeks of feeding trial (mean±SD; n = 3).


       
Proximate body composition analysis indicated no significant variations between the fish fed SBM alone and those supplemented with plant proteins, confirming that these alternative proteins did not compromise the nutritional quality of the fish (Love, 1980; Saliu et al., 2017; Zhang et al., 2014). Histological evaluations showed healthy intestinal tissue with no notable differences across diets, contrasting with earlier studies that reported negative impacts on intestinal morphology at high SBM levels (Zhang et al., 2018; Wang et al., 2017) (Fig 2).

Fig 2: Histological comparison of intestinal differences of snakehead fish in the control (A), SBM+CGM (B), SBM+CSM (C), SBM+GNM (D) and SBM+MSM (E). The magnification was 10x.


       
Using a Plant Protein blend of Wheat Meal, Wheat Gluten and Soybean Meal in rainbow trout diet was found faesible without apparent decrement in growth or intestinal health status (Calabria et al., 2021). The investigation of Madhubabu et al., 2021 demonstrated that a combination of plant protein sources could substitute 50% of fishmeal in the diet of Asian seabass without having any adverse effect on growth performance.
       
The antioxidant enzymes, SOD and CAT, are vital for the immune function of fish. Although some studies have suggested that plant protein inclusion may reduce SOD activity (Huang et al., 2023), this study found no significant differences regarding antioxidant activity or respiratory burst activity (RBA) (Fig 3). In the treatment with GNM, cholesterol levels were noted comparatively high, that implies with the study of Xie et al., 2025 where SBM diet with cholesterol supplementation could increase the activities of intestinal enzymes (e.g., amylase) and restore the structural integrity of the intestinal lining, including villus height and goblet cell count. Additionally, no notable changes in serum biochemical indices (total protein, albumin, glucose) were observed, affirming that the inclusion of plant proteins did not adversely affect immune or metabolic health, in line with findings in rainbow trout and snakehead (Nazir et al., 2020; Suratip et al., 2023). Das et al., 2015 has evaluated cost effective floating feeds by replacement of soybean meal with alternative feed ingredients. Also net profit with respect to total biomass harvested and feed cost reduction was also recorded in diets using alternative plant protein ingredients like CGM, CSM, GNM and MSM.

Fig 3: SOD and CAT activity of C. striata fingerlings fed different experimental diets after 8 weeks of feeding trial (mean±SD; n =3).


               
Regarding muscle growth, the lack of significant upregulation of MyoD and MyoG gene expression suggests that plant protein supplementation does not negatively impact muscle development, supporting the feasibility of these ingredients for aquafeed. Overall, these results highlight the viability of using alternative plant proteins in snakehead diets without compromising growth, immunity, or tissue health.

CONCLUSION

This study showed that plant protein ingredients such as Corn gluten meal (CGM), cottonseed meal (CSM), Groundnut meal (GNM) and Mustard meal (MSM), when combined with soybean meal (SBM), supported comparable growth performance in Channa striata at a 10% inclusion level. The lack of significant differences suggests these alternatives can partially replace traditional protein sources without compromising fish performance. Future research should explore higher inclusion levels of these plant proteins to identify optimal formulations that support sustainability and cost-effectiveness in aquafeed development. This may provide insights into incorporating higher levels without negatively affecting growth or feed conversion efficiency. Histological and gene expression analyses showed no adverse effects, highlighting the potential for integrating these plant proteins into future snakehead feed formulations.

ACKNOWLEDGEMENT

This research was supported by Dr. M.G.R. Fisheries College and Research Institute, Ponneri, Tamil Nadu Dr. J. Jayalalithaa Fisheries University and Growel feeds Pvt., Ltd. andhra Pradesh. The authors are grateful to the institutions for their assistance in the research work.

CONFLICT OF INTEREST

All authors declare that they have no conflict of interest.

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    Full Research Article

    Impact of Plant-based Protein Substitutes on Growth Performance, Histology and Gene Expression in Striped Snakehead, Channa striata (Bloch, 1793) Fingerlings

    S
    S. Ajidhaslin1,*
    C
    Cheryl Antony1
    A
    Arul Victor Suresh2
    B
    B. Ahilan1
    A
    A. Uma1
    K
    K. Ravaneswaran1
    C
    C Grace Angel2
    1Dr. M.G.R Fisheries College and Research Institute, Dr. J. Jayalalithaa Fisheries University, Ponneri-601 204, Tamil Nadu, India.
    2Growel Feeds and Private Limited, Chevuru-521 329, Andhra Pradesh, India.

    ABSTRACT

    Background: This research assessed the effects of incorporating various plant-based protein sources, including corn gluten meal (CGM), cottonseed meal (CSM), groundnut meal (GNM) and mustard seed meal (MSM), alongside soybean meal (SBM) in the diet of juvenile snakehead (Channa striata). Building on our previous findings that established an optimal SBM inclusion level of 25%, this subsequent trial explored the addition of these plant protein ingredients at a 10% inclusion rate, supplemented by SBM.

    Methods: The experimental diets were designed to be isonitrogenous (42% crude protein), isolipidic (9% crude fat) and isoenergetic (16.1-16.2 MJ/kg). The trial was conducted over eight weeks with 400 healthy murrel fingerlings (average weight 27.0±0.06 g), sourced from Growel Feeds Pvt Ltd, a commercial fish hatchery in Andhra Pradesh. The fingerlings were reared in fiberglass reinforced plastic (FRP) tanks (150 L capacity, with 20 fish per tank).

    Result: Evaluations of growth performance, haematological parameters, histological analyses and gene expression showed no significant differences among the various experimental groups. These results suggest that all the plant-based protein ingredients tested provided comparable growth performance and can be effectively used as alternative protein sources in conjunction with SBM for Channa striata feed formulations. This study highlights the potential for integrating cost-effective, plant-based protein sources into the feed for striped murrel fingerlings, promoting sustainability and economic viability.

    INTRODUCTION

    The striped murrel (Channa striata) is a rapidly growing and versatile carnivorous fish found in Southeast and Southern Asia, prized for its strong market demand (Gustiano et al., 2021; Kumar et al., 2022). Its commercial cultivation has played a significant role in advancing global aquaculture (FAO, 2020). Being a carnivorous species, C. striata necessitates high-protein diets, which are conventionally supplied by fishmeal (FM). However, this reliance raises sustainability concerns due to limited availability and fluctuating prices (Vo et al., 2015; Glencross et al., 2024). Consequently, there is a growing interest in alternative protein sources, such as plant proteins, that can replace or supplement fishmeal while ensuring adequate growth performance (Maksimenko et al., 2024).
           
    Plant proteins, including soybean meal (SBM), are economically advantageous and contribute to the expansion of aquaculture; yet, their use can be restricted by anti-nutritional factors and lower digestibility in carnivorous fish (NRC, 2011; Kumari et al., 2013). Addressing these issues requires the development of optimized feed formulations (Glencross et al., 2024). Techniques such as fermentation and enzymatic treatment may enhance the quality of plant proteins (Maksimenko et al., 2024).
           
    This study aims to assess the supplementation of SBM with CGM, CSM, GNM and MSM in C. striata diets to foster sustainable and cost-effective aquaculture practices. Degossypolized cottonseed protein (DCP) is produced by the solvent extraction of water-soluble carbohydrates and free gossypol from cottonseed meal, which finally contains high-quality protein and a low concentration of free gossypol (Wang et al., 2019). Detection of aflatoxins in corn gluten meal and groundnut meal is carried out by high-performance thin-layer Chromatography based on their fluorescence under UV light with an acceptable level below 20 μg kg-1 (Jaiswar et al., 2022).

    MATERIALS AND METHODS

    Ethical statement
     
    The experiment was conducted following the procedures of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals), Ministry of Environment and Forests (Animal Welfare Division), Govt. of India on care and use of animals in scientific research. This study was approved by the ethical committee of Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Nagapattinam, Tamil Nadu, India. The experiment was conducted for 60 days at Growel Fish Hatchery, Perikegudem, Andhra Pradesh, India.
     
    Experimental diets
     
    Five distinct diets were formulated to evaluate the effects of alternative plant protein sources, including Corn Gluten Meal (CGM), Cottonseed Meal (CSM), Groundnut Meal (GNM) and Mustard Seed Meal (MSM), alongside soybean meal (SBM) for Channa striata. The diets comprised a control group with only SBM and four additional groups featuring CGM (T2), CSM (T3), GNM (T4) and MSM (T5). All diets were designed to be isonitrogenous, isolipidic and isoenergetic, containing 42% crude protein, 9% crude fat and gross energy levels of 16.1-16.2 MJ/kg. The feed was processed into floating pellets of 1.2 mm in diameter using a twin-screw extruder. A detailed composition can be found in Table 1.

    Table 1: Feed formulation and proximate composition of experimental diets (g.kg-1 diet).


     
    Fish husbandry
     
    Juvenile Channa striata were acquired from Growel Feeds Hatchery located in Andhra Pradesh and were acclimatized for a duration of 20 days on a commercial diet (42% CP, 9% CL). The feeding trial was executed in a Recirculating Aquaculture System (RAS) utilizing 20 fiberglass-reinforced plastic (FRP) tanks, each with a capacity of 150 L and supplied with sand-filtered water, aeration and covered with fiber mats. The FRP tanks were linked to biofiltration units, with water recirculated four times per day.
           
    A total of 400 juveniles (average weight 27.0±0.06 g) were randomly allocated into the 20 tanks (20 fish per tank). Fishes were hand fed their respective diets till apparent satiety three times a day (8:00, 12:00 and 17:00 h) at 10% body weight until satiation for 8 weeks. Cannibalism cannot be completely eliminated in snakehead culture, but maintaining size uniformity and ensuring satiation feeding effectively minimized cannibalistic interactions in the experimental setup. Initial body composition analyses were conducted by sampling three fish per tank, while final body composition was assessed at the conclusion of the study. Water quality parameters such as temperature (26.1± 0.5oC), dissolved oxygen (5.5±0.1 ppm), pH (7.5±0.1) and ammonia levels (0.02±0.006 ppm) were monitored twice daily, adhering to APHA (2005) standards.
     
    Sampling methods and calculations
     
    Following the eight-week feeding period, fish were deprived of food for 24 hours, weighed collectively. The fishes were anesthetized with clove oil (50 μl/L) and three fish per tank were selected for measuring length, body weight, visceral weight and liver weight to compute the condition factor (K), viscero-somatic index (VSI) and hepato-somatic index (HSI). For blood analysis, blood samples were collected from the caudal vein into EDTA-coated tubes. Plasma was obtained via centrifugation (3000 × g for 5 minutes) and subsequently stored at -20oC. Muscle samples and foregut sections were also collected for gene expression and histological assessments. Bio-growth parameters were calculated using standard formulas.
     
    Analytical chemistry
     
    Proximate analysis for crude lipid, crude protein, moisture, dry matter, ash and energy content in both diets and fish samples was performed following the AOAC (2000) guidelines. Moisture content was determined by drying samples at 105oC until a constant weight was reached. Crude protein levels were ascertained via nitrogen content using the Kjeltec system, while crude lipid was assessed through petroleum ether extraction using the Soxtec system. Crude fiber was analyzed using acid-alkali digestion with the Fibretech system and ash content was determined by incineration at 550oC for six hours.
     
    Antioxidant enzyme analysis
     
    Plasma concentrations of superoxide dismutase (SOD) and catalase (CAT) were measured. SOD activity was evaluated by tracking the increase in absorbance of Epinephrine at 480 nm, expressed as U/mg protein (Marklund and Marklund, 1974). Catalase activity was assessed by monitoring the decrease in H2O2 absorbance at 240 nm, expressed as ìmol/mg protein/min (Aebi, 1984). Protein concentration in serum was determined using AOAC’s method (1951).
     
    Haematological and biochemical parameters
     
    Three fish per tank were anesthetized with clove oil (50 μLL) for blood sampling from the caudal vein using EDTA-treated syringes. Haematological parameters (RBC, WBC, haematocrit, MCV, MCH, MCHC) were analyzed using an automatic analyzer (Cell Tech 380, India). Biochemical markers (glucose, serum cholesterol, triglycerides) were evaluated using a semi-automatic biochemical analyzer.
     
    Histological analysis
     
    Foregut samples were fixed in Davidson’s fixative, then dehydrated in ethanol and embedded in paraffin. Sections (7 μm) were stained with haematoxylin and eosin. Measurements of villus height, thickness and muscular thickness were conducted using image analysis software. This work was performed at Madras Veterinary College, Vepery, Tamil Nadu.
     
    Quantitative Real-time PCR (qRT-PCR)
     
    Muscle samples from three fish per tank were collected for gene expression analysis of MyoD and MyoG. RNA extraction was performed using RNA iso-plus (Takara Bio) and cDNA synthesis was conducted with a first-strand synthesis kit (Thermo Scientific). The list of primers given in Table 2. Gene expression was quantified through qRT-PCR on a CFX96 system (Bio-Rad) under specific cycling conditions. Beta-actin was used as the housekeeping gene and relative expression was calculated using the 2-ΔΔCt method. The study was carried out at the State Referral Laboratory for Aquatic Animal Health, Dr. M.G.R. Fisheries College and Research Institute, Madhavaram, Tamil Nadu.

    Table 2: Primers used for qRT-PCR analysis of selected genes for Channa striata fed experimental diets.


     
    Statistical analysis
     
    Data were expressed as mean±standard deviation (SD) across four replicates. Statistical significance was assessed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (p<0.05). All statistical analyses were conducted using SPSS software version 20.0 (IBM), with variations analyzed at p<0.05 levels. The quantitative results were presented as mean±SD.

    RESULTS AND DISCUSSION

    Growth performance, feed utilization and somatic parameters
     
    There were no significant differences in final weight (FW), weight gain (WG), specific growth rate (SGR), or feed conversion ratio (FCR) among the various treatment groups, which included both the SBM control and the diets supplemented with plant proteins. The protein efficiency ratio (PER) and SGR remained consistent across all dietary treatments. Furthermore, the addition of plant proteins (CGM, CSM, GNM, MSM) did not significantly influence the viscerosomatic index (VSI), hepatosomatic index (HSI), survival rates, or condition factor (p>0.05) given in Table 3.
     

    Table 3: Growth performance and survival of C. striata fingerlings fed different experimental diets after 8 weeks of feeding trial.



    Whole body composition
     
    Proximate analysis of whole body composition indicated no significant differences across the different diet treatments (p>0.05) given in Table 4. All evaluated parameters were stable, suggesting that variations in diet did not affect overall body composition.

    Table 4: Chemical composition of the whole body of C. striata fingerlings fed different experimental diets before and after feeding trial (% wet weight basis).


     
    Intestinal morphology
     
    The inclusion of alternative plant proteins did not result in significant changes in villi length. However, a notable reduction in villi width was observed in these treatment groups when compared to the control group, provided in Table 5. Despite this decrease in villi width, the structural integrity of the intestine, as measured by muscular thickness, remained consistent across all treatment groups.

    Table 5: Histological parameters of fore-gut cross sections of juvenile Channa striata at the end of the 8 week feeding trial.


     
    Haematological and biochemical indices
     
    No significant alterations were noted in haematological parameters (RBC, WBC, haemoglobin, MCV, MCH) or biochemical indicators (albumin, globulin, total protein) among the treatment groups. The addition of plant proteins (CGM, CSM, GNM, MSM) did not adversely affect these health indicators, thus ensuring the well-being of the fish presented in Table 6.

    Table 6: Changes of haematological and biochemical indices in C. striata fingerlings fed different experimental diets after feeding trial.


     
    Antioxidant enzymes
     
    The Nitroblue Tetrazolium (NBT) assay revealed no significant differences in phagocyte activity. Additionally, the activities of catalase (CAT) and superoxide dismutase (SOD) showed no significant changes in serum and liver samples, tabulated in Table 7. There were also no significant variations in albumin, globulin, or total protein levels, indicating stable protein quality and metabolic health.

    Table 7: Effects of different inclusion of alternative plant protein ingredients along with SBM on antioxidant enzymes activity in serum of juvenile Channa striata.


     
    Relative mRNA expression
     
    No significant increase in the expression of MyoD and MyoG mRNA was detected in fish that were fed diets containing alternative plant proteins (CGM, CSM, GNM, MSM). This finding suggests that these plant protein sources did not influence the transcription levels of myogenic regulatory factors when compared to the SBM control diet.
           
    The current study focused on evaluating the incorporation of various plant protein sources, including CGM, CSM, GNM and MSM, in the feed formulations for snakehead (Channa spp.). The findings revealed no significant differences in growth performance, feed conversion ratio (FCR), or weight gain, suggesting that these ingredients can effectively substitute fishmeal, consistent with previous research (Liu et al., 2023; Fei et al., 2024) (Fig 1). There was no statistically significant difference found in the length-weight relationship of sea bream fed diets with varying protein levels (Korkut et al., 2018). The plant proteins demonstrated their potential as sustainable alternatives, showing no detrimental impact on growth or feed efficiency, in alignment with studies conducted on other fish species (Zhang et al., 2014; Watanabe and Pongmaneerat, 1993; Fan et al., 2023). In the study by Priyatharshni et al., 2023 replacement of soybean meal study has also been evaluated with sesame meal in Tilapia fishes.

    Fig 1: Weight gain (%) of C. striata fingerlings fed different experimental diets after 8 weeks of feeding trial (mean±SD; n = 3).


           
    Proximate body composition analysis indicated no significant variations between the fish fed SBM alone and those supplemented with plant proteins, confirming that these alternative proteins did not compromise the nutritional quality of the fish (Love, 1980; Saliu et al., 2017; Zhang et al., 2014). Histological evaluations showed healthy intestinal tissue with no notable differences across diets, contrasting with earlier studies that reported negative impacts on intestinal morphology at high SBM levels (Zhang et al., 2018; Wang et al., 2017) (Fig 2).

    Fig 2: Histological comparison of intestinal differences of snakehead fish in the control (A), SBM+CGM (B), SBM+CSM (C), SBM+GNM (D) and SBM+MSM (E). The magnification was 10x.


           
    Using a Plant Protein blend of Wheat Meal, Wheat Gluten and Soybean Meal in rainbow trout diet was found faesible without apparent decrement in growth or intestinal health status (Calabria et al., 2021). The investigation of Madhubabu et al., 2021 demonstrated that a combination of plant protein sources could substitute 50% of fishmeal in the diet of Asian seabass without having any adverse effect on growth performance.
           
    The antioxidant enzymes, SOD and CAT, are vital for the immune function of fish. Although some studies have suggested that plant protein inclusion may reduce SOD activity (Huang et al., 2023), this study found no significant differences regarding antioxidant activity or respiratory burst activity (RBA) (Fig 3). In the treatment with GNM, cholesterol levels were noted comparatively high, that implies with the study of Xie et al., 2025 where SBM diet with cholesterol supplementation could increase the activities of intestinal enzymes (e.g., amylase) and restore the structural integrity of the intestinal lining, including villus height and goblet cell count. Additionally, no notable changes in serum biochemical indices (total protein, albumin, glucose) were observed, affirming that the inclusion of plant proteins did not adversely affect immune or metabolic health, in line with findings in rainbow trout and snakehead (Nazir et al., 2020; Suratip et al., 2023). Das et al., 2015 has evaluated cost effective floating feeds by replacement of soybean meal with alternative feed ingredients. Also net profit with respect to total biomass harvested and feed cost reduction was also recorded in diets using alternative plant protein ingredients like CGM, CSM, GNM and MSM.

    Fig 3: SOD and CAT activity of C. striata fingerlings fed different experimental diets after 8 weeks of feeding trial (mean±SD; n =3).


                   
    Regarding muscle growth, the lack of significant upregulation of MyoD and MyoG gene expression suggests that plant protein supplementation does not negatively impact muscle development, supporting the feasibility of these ingredients for aquafeed. Overall, these results highlight the viability of using alternative plant proteins in snakehead diets without compromising growth, immunity, or tissue health.

    CONCLUSION

    This study showed that plant protein ingredients such as Corn gluten meal (CGM), cottonseed meal (CSM), Groundnut meal (GNM) and Mustard meal (MSM), when combined with soybean meal (SBM), supported comparable growth performance in Channa striata at a 10% inclusion level. The lack of significant differences suggests these alternatives can partially replace traditional protein sources without compromising fish performance. Future research should explore higher inclusion levels of these plant proteins to identify optimal formulations that support sustainability and cost-effectiveness in aquafeed development. This may provide insights into incorporating higher levels without negatively affecting growth or feed conversion efficiency. Histological and gene expression analyses showed no adverse effects, highlighting the potential for integrating these plant proteins into future snakehead feed formulations.

    ACKNOWLEDGEMENT

    This research was supported by Dr. M.G.R. Fisheries College and Research Institute, Ponneri, Tamil Nadu Dr. J. Jayalalithaa Fisheries University and Growel feeds Pvt., Ltd. andhra Pradesh. The authors are grateful to the institutions for their assistance in the research work.

    CONFLICT OF INTEREST

    All authors declare that they have no conflict of interest.

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