Introduction

The increasing global population presents a major challenge in maintaining a stable and nutritious food supply. Aquaculture has emerged as a sustainable solution, providing a controlled environment for the production of nutritious food sources1,2,3,4,5. As one of the fastest-growing sectors in food production, aquaculture plays an essential role in enhancing global food security6,7. In 2020, aquaculture accounted for approximately 178 million metric tonnes (MMT) of global fish supply8. India’s total fish production in 2021-22 reached 16.24 MMT9. Fish serves as a vital food source while also contributing significantly to income generation, employment, and recreational activities4. Fish serves as an important source of nutrients for addressing global health challenges10. Its affordability and rich protein content make it a vital source of nutrition10. This has increased dietary acceptance of fish in developed and developing countries11. India relies mainly on freshwater fish farming to tackle rising fish demand12. Choosing high-demand species is crucial for achieving a greater return on investment for fish farmers13. Catla catla, a significant cultivable fish species in the Indian subcontinent, contributes 5.6% to global aquaculture production and ranks 6th among the most cultivated aquaculture species14. Native to freshwater and primarily farmed in Asia, especially the Indian subcontinent, this crucial species plays a vital role in the region’s food security15. As a surface feeder, C. catla generally consumes plankton, including phytoplankton and zooplankton, thriving in the riverine systems of northern and central India16. Its growth potential and high consumer preference have made it the most crucial freshwater candidate species for culture in India17.

Catla and other Indian major carps (IMC) are the powerhouse behind India’s freshwater aquaculture, contributing around 87% of its production16.Catla fingerlings primarily feed on zooplankton, planktonic algae, and vegetable waste, with adults showing a preference for zooplankton18,19. Live food also has limitations that will likely hinder its effective utilization20. However, the intensification of aquaculture has led to environmental concerns, particularly the accumulation of cyanobacteria biomass, resulting in harmful algal blooms (HABs)21. These blooms, characterized by bright green, yellow-brown, and red colours, pose severe water quality problems. Freshwater HABs, typically comprising cyanobacteria, are unsightly and produce foul odours22,23. Cyanobacteria release toxic secondary metabolites that adversely affect aquatic animals24. HABs erupt when algae and their kin, like dinoflagellates, diatoms, and cyanobacteria, experience explosive growth25. The intensification of aquaculture has increased the use of inputs like fertilizers and feed per unit of land, leading to higher waste generation within production systems26. This waste contributes to nutrient loading in culture systems and local water bodies, raising sustainability concerns27,28. The feed used in aquaculture systems is a significant waste source28,29. Decomposed or uneaten feed releases nitrogen (N) and phosphorus (P), which trigger algal bloom production and eutrophication30. Blue-green algae (cyanobacteria) are most prevalent due to their hardy nature and adaptability to diverse climatic conditions31. Cyanobacterial toxins significantly disrupt ecosystems, persisting and accumulating as they move through the food web32. Dating back nearly 3 billion years, cyanobacteria are ancient photosynthetic bacteria (not plants) that played a key role in shaping Earth’s atmosphere33,34,35. Cyanotoxins can cause cytotoxic and genotoxic effects in teleost fishes by generating reactive oxygen species (ROS) and down-regulating antioxidant biomarkers, leading to oxidative stress, genotoxicity, cytotoxicity, and apoptosis32. Microcystin-LR, a toxin produced by cyanobacteria, generates excessive reactive oxygen species to exert its toxic effects36. Anatoxin-a (ANTX-a), a toxin found in freshwater algae blooms worldwide, poses a growing threat to consumers37. Multiple types of cyanobacteria, including Anabaena and Aphanizomenon, churn out this neurotoxin38,39,40,41,42,43,44. This neurotoxin bioaccumulates in aquatic life, harming them and the ecosystem25,45.???

The present study aims to evaluate the effects of increasing dietary levels of the cyanobacterium Anabaena on the growth performance and physio-metabolic responses of the economically important freshwater species Catla catla. This research provides insights into the impacts of cyanobacterial blooms, often a consequence of eutrophication, and explores the potential use of eutrophic algal biomass as a sustainable dietary supplement in aquaculture. Such practices are critical for enhancing production efficiency in response to rising protein demand and pressures on aquatic ecosystems.

Table 1 Physico-chemical parameters of water in different experimental tanks during the experimental period of 60 days.
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Results

Physico-chemical parameters of water

The average range of physicochemical parameters of the water studied, including temperature (°C), dissolved oxygen (mg/L), pH, nitrite (mg/L), nitrate (mg/L), free CO2 (mg/L), and ammonia (mg/L), is presented in Table 1.

Table 2 Proximate composition of Anabaena powder (% dry matter basis).
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3

Proximate composition of the Anabaena powder

The proximate composition of the Anabaena powder is given in Table 2. The Anabaena powder was from outdoor mass culture, not pure culture.

Table 3 The composition and proximate analysis of experimental diets.
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Proximate analysis of the experimental diets (% dry matter basis)

The proximate composition of the experimental diets is summarized in Table 3. The dry matter (DM) content ranged from 91.90 to 92.00%, crude protein (CP) from 30.20 to 30.98%, ether extract (EE) from 6.41 to 6.54%, crude fibre (CF) from 8.41 to 8.65%, nitrogen-free extract (NFE) from 47.79 to 48.58%, and total ash content (TA) from 6.24 to 6.40%. The diets’ estimated digestible energy (DE) values were between 377.86 and 379.17 kcal/100 g of feed.

Whole body composition of C. catla fingerlings (% wet weight basis)

Table 4 details the biochemical composition of C. catla fingerlings, encompassing moisture, CP, EE, TC, and TA. The analysis indicated that the whole-body composition parameters remained consistent across different dietary treatments, showing no significant differences (P > 0.05).

Table 4 Whole body chemical composition of C. catla fingerlings of different experimental groups (% wet weight basis).
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Growth performance and nutrient utilization indices

Table 5 illustrates growth performance and nutrient utilization parameters subjected to various experimental diets. Percentage weight gain (PWG) did not differ significantly (P > 0.05) among the treatment groups. Similarly, the specific growth rate (SGR) did not exhibit significant variation (P > 0.05) across the experimental groups. Additionally, the feed conversion ratio (FCR), feed efficiency ratio (FER), and protein efficiency ratio (PER) analysis also showed no significant differences (P > 0.05) across the dietary treatments. These findings suggest that the different diets did not significantly impact the growth performance or nutrient utilization of C. catla fingerlings.

Table 5 Growth performances of C. catla fed different experimental diets for 60 days.
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Apparent digestibility coefficient of the experimental diets

Table 6 shows the apparent dry matter digestibility coefficient (ADMDC) values for C. catla fingerlings among all treatment groups (A0, A3, A6, A9, A12, and A15). The ADMDC values did not differ significantly (P > 0.05) from the control group (A0).

Table 6 Apparent digestibility coefficient (ADC) dry matter and protein of experimental diets.
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Digestive enzyme activities

Table 7 shows the recorded activities of digestive enzymes, including protease, amylase, and lipase, in the intestines of C. catla fingerlings across various experimental groups. The specific activities of intestinal protease in C. catla did not exhibit significant differences (P > 0.05) among the different dietary treatment groups. In contrast, the intestinal amylase activities varied significantly (P < 0.05) among the groups, with the highest levels observed in the control (A0) and A3-fed groups. Similarly, the intestinal lipase activities showed significant variation (P < 0.05) across all dietary treatment groups.

Table 7 Digestive enzyme activities in the intestine of C. catla fingerlings fed with different experimental diets.
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Metabolic enzymes

Protein metabolic response

Table 8 presents the aspartate aminotransferase (AST) activities in the liver and muscle of C. catla fingerlings subjected to various experimental diets. Notably, there were no significant differences (P > 0.05) in AST activity between the control (A0) and other experimental groups in both muscle and liver tissues. Similarly, alanine aminotransferase (ALT) activities in the liver and muscle of C. catla fingerlings across different dietary treatments showed no significant (P > 0.05) variation among the dietary treatment groups.

Table 8 Activities of protein metabolic enzymes in C. catla fed with different experimental diets.
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Carbohydrate metabolic response

The lactate dehydrogenase (LDH) activities in the liver and muscle of C. catla across various experimental groups are presented in Table 9. Notably, there was no significant (P > 0.05) difference in liver LDH activity between the control group and the dietary treatments in muscle. However, a significant increase (P < 0.05) was observed in fish fed an Anabaena-based diet in the liver. The malate dehydrogenase (MDH) activities in the liver and muscle of C. catla fingerlings showed enhanced MDH activities observed in the muscle and liver fed with various dietary inclusions of Anabaena powder.

Table 9 Activities of carbohydrate metabolic enzymes in C. catla fingerlings fed with different experimental diets for 60 days.
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Oxidative stress response

Figure 1 illustrates the superoxide dismutase (SOD) activities in the gills and liver of C. catla fingerlings subjected to various experimental diets. The results indicate a significant increase (P < 0.05) in SOD activities in both tissues for the groups supplemented with Anabaena. Similarly, Fig. 2 depicts the catalase (CAT) activity in the gills and liver, and notably, the Anabaena supplemented groups exhibited significantly higher (P < 0.05) CAT activities in both the liver and gills.

Fig. 1
figure 1

Illustrate the oxidative stress activity in C. catla fingerlings subjected to various experimental diets.

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Fig. 2
figure 2

Illustrate the oxidative stress activity in C. catla fingerlings subjected to various experimental diets.

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Serum parameters

Table 10 summarizes the serum concentrations of glucose, total protein, albumin, globulin, and the albumin-to-globulin (A/G) ratio, along with the results of the Nitroblue Tetrazolium (NBT) assay for the experimental groups. Serum glucose levels differed significantly (P < 0.05), showing an increase with the inclusion of dietary Anabaena; the A15 group exhibited the highest levels (140.41 ± 3.34), while the control group (A0) exhibited the lowest (38.65 ± 0.64). Furthermore, serum albumin levels also varied significantly (P < 0.05), with the control group (A0) having higher levels compared to the other groups. There were no significant differences (P > 0.05) in total protein concentrations, globulin levels, or the A/G ratio between the control and treatment groups. NBT activity in C. catla fingerlings showed no significant (P > 0.05) variation across the different diets.

Table 10 Serum total protein, albumin (A), globulin (G), a: G ratio, and glucose content of the different experimental groups.
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Haematological parameters

Table 11 details the haematological parameters of C. catla fingerlings subjected to various dietary treatments. Significant differences (P < 0.05) were observed in haemoglobin concentration (Hb), total erythrocyte count (RBC), and packed cell volume (PCV) among the different diet groups, with the control group exhibiting higher Hb and PCV levels. In contrast, total leucocyte count (WBC) did not differ significantly (P > 0.05) across the groups.

Table 11 Haematological parameters of C. catla fingerlings fed different experimental diets.
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Discussion

The present study explores the multifaceted effects of incorporating Anabaena, a cyanobacterial genus, into the diet of Catla catlafingerlings, focusing on key physiological and biochemical parameters. The investigation aims to evaluate the impact of this dietary modification on nutrient utilization, growth performance, and overall health status of the fish. Fish are sensitive to changes in their aquatic environment, so maintaining optimal water quality in aquaculture systems is essential. Ensuring stable water parameters is crucial for maximizing their survival, growth, and overall health47. In this study, water quality parameters were meticulously maintained within optimal ranges suitable for freshwater fish culture. Given that fish are poikilothermic organisms, their metabolic rates are significantly influenced by temperature48. Optimal metabolic activity and maximum yield for C. catlaare achieved within a specific temperature range19. In this study, observed temperatures aligned with the optimal range. Moreover, pH levels across all experimental groups were within the recommended limits49. The experimental tanks’ DO levels were maintained within the desired concentrations, aligning with the recommended values for optimal fish health. In addition, ammonia concentrations in the water were controlled within the safe range19. By maintaining these physico-chemical characteristics, the study establishes a stable environment that supports the health and growth of C. catla, highlighting the importance of water quality management in aquaculture.

The experimental diets were formulated to be iso-nitrogenous (30% protein) and iso-lipidic (6% lipid), based on the optimal levels recommended for Indian major carps (IMC) fingerlings50. The test diets provided usable energy within a range consistent with previous findings suggesting a digestible energy requirement of 350–400 kcal/100 g for Catla fingerlings51.

Dietary supplementation with Anabaena had no significant effect on the proximate composition of the fish, including moisture, crude protein (CP), ether extract (EE), total carbohydrates (TC), and ash content. This outcome is consistent with previous studies on Nile tilapia fingerlings, where Anabaena supplementation similarly showed no substantial influence on body composition52. Conversely, Goldfish fed with lyophilized blue-green algae (BGA) powder exhibited significant changes in body composition after 16 weeks, showing significant reductions in CP, EE, and total ash levels53. The protein content of IMCs typically ranges from 16 to 19%54. The fish-fed diets with or without Anabaena for 60 days exhibited no significant difference in body protein content, indicating that the inclusion of Anabaena had minimal impact on protein levels.

Growth parameters, including weight gain (WG), percent weight gain (%WG), specific growth rate (SGR), feed efficiency ratio (FER), and protein efficiency ratio (PER), were assessed, and results indicated that Anabaena supplementation did not significantly influence any of these growth metrics. This finding is consistent with a study that showed no significant changes in growth among female Nile tilapia-fed diets containing different levels of dried cyanobacterial biomass and Arthrospira(cyanobacteria) over a 28-day period55. Adding up to 15% Anabaena to the fish diet had no significant effect on their growth compared to the control group.

A crucial factor in selecting ingredients for fish feed is their digestibility by the targeted fish species56. In this study, the apparent crude protein digestibility coefficient (ACPDC), apparent lipid digestibility (ALDC), and apparent dry matter digestibility coefficient (ADMDC) of Anabaena-containing diets did not significantly differ from the control diet. Our results support that dietary Anabaena had no significant impact on liver enzymes in Nile tilapia52.

The breakdown of complex nutrients into smaller, absorbable components in an animal’s digestive system is highly dependent on the presence and accessibility of specific enzymes20. This study found that C. catlafingerlings fed different diets had varying levels of amylase activity in their intestines. Despite variations in dietary regimens, no significant differences were observed in lipase and protease activities. Certain cyanobacteria exhibit a multifaceted impact on the gastrointestinal tract. These microorganisms can disrupt the intestinal barrier function, alter the composition of gut microbiota, and interfere with the production of digestive enzymes and inflammatory signaling molecules57. Consequently, these effects may influence the activation of immune genes within the intestinal mucosa58. Silver and bighead carp can digest Microcystis, albeit with poor digestibility59. A study found that silver carp weren’t very good at digesting or using M. aeruginosaas a food source60. Microcystis, due to its low digestibility, passes through the digestive system of the roach (Rutilus rutilus) undigested and undergoes exponential growth following excretion61.

Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are essential enzymes moving nitrogen groups between different amino acids within the body62. AST and ALT catalyze transamination reactions, transferring an amino group between amino acids and α-keto acids63. AST exchanges the amino group of aspartates with the keto group of α-ketoglutarate, yielding oxaloacetate and glutamate. ALT performs a similar reaction, replacing aspartate with alanine, producing glutamate and pyruvate64. These pyruvate and oxaloacetate molecules are essential for synthesizing non-essential amino acids such as alanine, asparagine, and glutamine, which are important for animal protein synthesis and growth. Additionally, amino acids can undergo deamination to generate intermediates for the tricarboxylic acid (TCA) cycle, thereby contributing to ATP production and energy metabolism65. The present study assessed ALT and AST activities in the muscle and liver, revealing no significant differences among the dietary treatments with Anabaena supplementation. While microcystins have been reported to impact fish liver function66,67,68, our findings did not indicate significant alterations in ALT and AST activities due to Anabaena supplementation. Interestingly, some previous studies showed that the levels of glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT) in the blood plasma of common carp (Cyprinus carpio L.) increased after intraperitoneal injections of MC-LR, indicating that cyanotoxins may adversely affect liver function67,69.

Lactate dehydrogenase (LDH) acts as the final enzyme in the glycolytic pathway, catalyzing the conversion of lactate to pyruvate in the presence of the coenzyme NADH, which is simultaneously oxidized to NAD +70. This reaction is crucial for sustaining glycolysis by regenerating NAD+. In aerobic conditions, pyruvate proceeds into the Krebs cycle; however, under anaerobic conditions, it is reduced back to lactate71. In the present study, elevating the inclusion level of Anabaena significantly enhanced LDH activities in both the liver and muscle. This increase is likely due to elevated lactate production, which serves as fish’s preferred substrate for gluconeogenesis during anaerobic metabolism72. Generally, LDH activity increases under stress conditions such as temperature stress73, starvation stress74, and confinement stress75. Exposure to antimetabolites stimulates liver cells to neutralize and eliminate them76. This mechanism entails heightened aryl hydrocarbon hydroxylase (AHH) activity, an enzymatic system crucial for detoxifying hazardous hydrocarbons that rely on molecular oxygen for their processing77. As a result, there is a competition for oxygen between detoxification processes and the electron transport chain (ETC.), which can lead to increased activity of LDH, an enzyme in the liver78. Malate dehydrogenase (MDH), a key enzyme in the energy-generating citric acid cycle, can switch malate into oxaloacetate and back again79. The activity of malate dehydrogenase (MDH), an enzyme derived from amino acids, is expected to increase during gluconeogenesis. Our study observed a significant elevation in muscle MDH activity as the proportion of Anabaena in the diet increased.

Organisms utilize diverse defence mechanisms against reactive oxygen species (ROS), such as antioxidant enzymes like superoxide dismutase (SOD) and catalase (CAT)80. Oxidative stress arises when pro-oxidant factors overpower antioxidant defences, resulting in insufficient removal of ROS81. SOD facilitates the dismutation of superoxide ions into oxygen and hydrogen peroxide, whereas CAT decomposes hydrogen peroxide into water and oxygen82. These enzymes are pivotal in serving as antioxidant defences in organisms subjected to oxygen exposure. In instances of heightened oxidative stress, the levels of SOD and CAT generally increase. Loach fish fed with Microcystismixed with commercial eel food powder exhibited significantly increased activities of antioxidant enzymes (SOD, CAT)83. A simultaneous induction response in SOD and CAT activities was noted after exposure to contaminants84, while enhanced SOD and CAT activities were observed in MC-LR-induced common carp hepatocytes84,85. Similarly, increased activity of antioxidant enzymes (SOD, CAT, GPx, GR) was observed in Tilapia fish subjected to cyanobacterial cells86. Our findings corroborate previous studies, demonstrating that Anabaena supplementation significantly increased superoxide dismutase (SOD) activity in fish compared to the control. Furthermore, higher levels of Anabaena inclusion (12% and 15%) were correlated with elevated catalase (CAT) activity, suggesting increased oxidative stress at these inclusion rates. This suggests that antioxidant enzymes are triggered as a defensive mechanism against Microcystistoxins in Tilapia86.

Glucose is a crucial animal energy source and a stress indicator influenced by physical factors87,88,89. During stress, catecholamine secretion increases, leading to glycogen breakdown and elevated blood glucose levels90. Blood glucose levels are controlled by the absorption of glucose in the intestines, the production of glucose in the liver, and the uptake of glucose by tissues via processes such as hepatic glycogenolysis, glycolysis, and gluconeogenesis91. The present study found that dietary supplementation with 15% Anabaena increased blood glucose levels, suggesting physiological stress in the fish. Furthermore, serum protein levels were evaluated, as they serve as important biomarkers of non-specific immunity and overall fish health20,92. Immune stimulants can boost protein synthesis, producing immune-related molecules such as immunoglobulin, complement, lysozyme, and anti-proteases, which play roles in immunity93. The addition of Anabaena in this study did not result in any changes to serum protein levels. Serum albumins and globulins are vital components that contribute significantly to immune responses94. The liver plays a crucial role in synthesizing albumins, which help maintain vascular fluid volume, and globulins, especially gamma globulins, are vital for immune function95. Anabaena supplementation significantly lowered serum albumin levels. While serum globulin levels and the A: G ratio remained relatively unchanged, haematological parameters were adversely affected, with decreases in RBC count, Hb, and PCV, and an increase in WBC count. These findings suggest a detrimental effect of Anabaena inclusion on overall protein status and haematopoiesis. Nile Tilapia-fed diets containing Anabaena exhibited comparable results to those reported in previous studies, with a negative impact observed at higher inclusion levels96. The Nitroblue tetrazolium (NBT) assay revealed the lowest respiratory burst activity in the A15 group and the highest in the control group. Plant ingredients might reduce RBC and Hb concentrations97. Reduced protein metabolism could decrease RBC synthesis, lowering Hb content and NBT-induced immune response97. The elevated presence of toxic metabolites in Anabaena-based diets was likely a contributing factor to the observed changes in haematological and immune parameters in the fish.

Materials and methods

Experimental site

The 60-day feeding trial was conducted at the ICAR-Central Institute of Fisheries Education, Kolkata Centre. Subsequently, laboratory analyses were performed in the Fish Nutrition, Biochemistry, and Physiology Laboratory at the same institute.

Procurement and acclimatization of experimental animals

Specimens of C. catla were sourced from a fish seed farm located in Naihati, West Bengal, India, and transported to the ICAR-CIFE Kolkata Centre in oxygenated plastic bags to ensure their viability. Upon arrival, the fish were promptly placed into three 1000 L FRP tanks and allowed to rest overnight to recover from transportation stress. To reduce handling-induced stress, they were treated with a mild solution of salt and vitamin C in aerated tanks before undergoing a 15-day acclimatization period on a standard diet. Following acclimatization, the experiment commenced with their transfer to the designated rearing tanks.

Experimental units

For the feeding trial, 18 rectangular FRP tanks (800 L capacity each) were used. The tanks were covered with mosquito netting to prevent fish escape. Before the experiment, the tanks were disinfected with a 4-ppm potassium permanganate (KMnO₄) solution overnight. After disinfection, they were thoroughly washed and sun-dried for 12 h. The tanks were then filled with 550 L of dechlorinated bore-well water, and continuous aeration was provided using air stones.

Experimental design and feeding

Two hundred seventy C. catla fingerlings (initial average weight 9.45 ± 0.15 g) were divided into six experimental groups (in triplicate). Six diets containing varying levels of Anabaena blue-green algae (0%, 3%, 6%, 9%, 12%, and 15%) were prepared, with a stocking density of 15 fish/ tank in a completely randomized design (CRD). The fish were fed to apparent satiation twice daily (7.30 h, 17.00 h). Daily tank maintenance included cleaning and replacing some portion of water using a siphon.

Physico-chemical parameters of water

The water temperature was checked twice daily using a Master test kit water thermometer (TSi15, UK). The MERCK dissolved oxygen (DO) meter (Germany) was used to measure DO. Daily, the pH of the water was assessed using a Master test kit pH solution (XL, UK). Total ammonia nitrogen concentrations were measured using an ammonia-nitrite test kit (ICAR-CIFE, Mumbai, India), and the results were expressed in mg/L. Nitrite-N concentrations were measured every three days using the same test kit, and the values were reported in mg/L. Nitrate-N concentrations were reported in mg/L using the same kit. Dissolved free carbon dioxide (CO2) levels were determined by a titrimetric method98.

Formulation and preparation of the experimental diets

Six experimental diets were prepared (Table 1), with varying levels of Anabaena blue-green algae at 0%, 3%, 6%, 9%, 12%, and 15% (designated as A0, A3, A6, A9, A12, and A15). These diets were standardized to maintain equal nitrogen and energy content, incorporating fishmeal, soybean meal, mustard oil cake, and Anabaena blue-green algae powder as primary protein sources. Lipids were derived from fish and soybean oil, while carbohydrates were sourced from rice bran, maize, and wheat flour. Choline chloride was used as an attractant, supplemented by a comprehensive vitamin-mineral mix to meet the nutritional needs of the fish. Additional nutrients, including vitamin C, were included as per the dietary requirements. All ingredients were precisely measured and combined in a large plastic container. The mixture was homogenized to form a dough with the desired water content. The dough was steamed in a pressure cooker for 20 min, then cooled. Oils and a vitamin-mineral premix were added and mixed thoroughly. The dough was pelletized (SB, Panchal, Mumbai, India) into uniformly sized (2 mm) pellets. The pellets were air-dried on paper sheets and packaged in airtight polythene bags, each labeled with its respective treatment.

Proximate analysis of diets and carcass

The proximate composition (moisture, crude protein (CP), ether extract (EE), crude fiber (CF), and total ash of both the experimental diets and whole fish bodies (% dry matter basis) were analyzed13,99.The nitrogen-free extract (NFE) for diets and total carbohydrates (TC) in whole fish bodies were calculated using the subtraction method13.

The proximate analysis was calculated using the following formulas:

$$\:\text{Moisture}\:\left({\%}\right)=\frac{\text{W}\text{t}.\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\:\text{b}\text{e}\text{f}\text{o}\text{r}\text{e}\:\text{d}\text{r}\text{y}\text{i}\text{n}\text{g}\:\left(\text{g}\right)\:-\:\text{W}\text{t}.\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\:\text{a}\text{f}\text{t}\text{e}\text{r}\:\text{d}\text{r}\text{y}\text{i}\text{n}\text{g}\:\left(\text{g}\right)}{\text{W}\text{t}.\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\:\text{b}\text{e}\text{f}\text{o}\text{r}\text{e}\:\text{d}\text{r}\text{y}\text{i}\text{n}\text{g}\:\left(\text{g}\right)}\text{x}\:\:100$$
$$\:\text{Dry}\:\text{m}\text{a}\text{t}\text{t}\text{e}\text{r}\:\left({\%}\right)\:=\:100\:\:\text{M}\text{o}\text{i}\text{s}\text{t}\text{u}\text{r}\text{e}\:\left({\%}\right)$$
$$\:\text{C}\text{P}\:\left({\%}\right)\:=\:\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{n}\text{i}\text{t}\text{r}\text{o}\text{g}\text{e}\text{n}\:\text{c}\text{o}\text{n}\text{t}\text{e}\text{n}\text{t}\:\left({\%}\right)\:\text{X}\:6.25$$
$$\:\text{E}\text{E}\:\left({\%}\right)=\frac{\text{W}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\text{f}\text{l}\text{a}\text{s}\text{k}\:\text{p}\text{l}\text{u}\text{s}\:\text{e}\text{x}\text{t}\text{r}\text{a}\text{c}\text{t}\text{e}\text{d}\:\text{l}\text{i}\text{p}\text{i}\text{d}\:\left(\text{g}\right)\:\text{W}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\:\text{f}\text{l}\text{a}\text{s}\text{k}\:\left(\text{g}\right)}{\text{W}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{o}\text{f}\:\text{t}\text{h}\text{e}\:\text{d}\text{r}\text{i}\text{e}\text{d}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}\:\left(\text{g}\right)}$$
$$\:\text{C}\text{F}\:\left({\%}\right)=\frac{\text{W}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{o}\text{f}\:\text{c}\text{r}\text{u}\text{c}\text{i}\text{b}\text{l}\text{e}\:\text{w}\text{i}\text{t}\text{h}\:\text{d}\text{r}\text{y}\:\text{r}\text{e}\text{s}\text{i}\text{d}\text{u}\text{e}-\text{W}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{o}\text{f}\:\text{c}\text{r}\text{u}\text{c}\text{i}\text{b}\text{l}\text{e}\:\text{w}\text{i}\text{t}\text{h}\:\text{a}\text{s}\text{h}}{\text{W}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{o}\text{f}\:\text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}\times\:100$$
$$\:\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{a}\text{s}\text{h}\:\left({\%}\right)=\frac{\begin{array}{c}Weight\:of\:the\:ash\:\left(g\right)\end{array}}{\begin{array}{c}Weight\:of\:the\:dry\:sample\end{array}\:\text{t}\text{a}\text{k}\text{e}\text{n}\:\left(\text{g}\right)}\text{x}\:100$$
$$\:\text{N}\text{F}\text{E}\:\left({\%}\right)\:=\:100-\:(\text{C}\text{P}{\%}\:+\:\text{E}\text{E}{\%}\:+\:\text{C}\text{F}\:+\:\text{T}\text{A}{\%})$$
$$\:\text{T}\text{C}\:\left({\%}\right)\:=\:100\:-\:(\text{C}\text{P}{\%}\:+\:\text{E}\text{E}{\%}\:+\:\text{T}\text{A}{\%})$$

Growth and nutrient utilization

Fish weight was measured at the beginning and end of the experiment using an electronic weighing balance (UniTech, Kolkata, India) to assess growth parameters. Before weighing, the fish were fasted 24 h. Feed and protein intake values were employed to calculate feed and nutrient utilization. The growth and nutrient utilization were calculated using the following formula100,101.

$$\:\text{W}\text{G}\left(\text{g}\right)=\text{F}\text{i}\text{n}\text{a}\text{l}\:\text{w}\text{e}\text{t}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\:\left(\text{g}\right)-\text{I}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l}\:\text{w}\text{e}\text{t}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\left(\text{g}\right)$$
$$\:\text{W}\text{G}{\%}=\frac{\text{F}\text{i}\text{n}\text{a}\text{l}\:\text{w}\text{e}\text{t}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\:\left(\text{g}\right)-\text{I}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l}\:\text{w}\text{e}\text{t}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\left(\text{g}\right)}{\text{I}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l}\:\text{w}\text{e}\text{t}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\left(\text{g}\right))}\text{x}\:\:10$$
$$\:\text{S}\text{G}\text{R}{\%}=\frac{\text{ln}\text{o}\text{f}\text{f}\text{i}\text{n}\text{a}\text{l}\:\text{w}\text{e}\text{t}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\:\left(\text{g}\right)-\text{ln}\text{o}\text{f}\text{i}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l}\:\text{w}\text{e}\text{t}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\left(\text{g}\right)\:}{\text{E}\text{x}\text{p}\text{e}\text{r}\text{i}\text{m}\text{e}\text{n}\text{t}\text{a}\text{l}\:\text{p}\text{e}\text{r}\text{i}\text{o}\text{d}\:\left(\text{d}\text{a}\text{y}\text{s}\right)}\text{x}\:\:100$$
$$\:\text{F}\text{C}\text{R}=\frac{\text{F}\text{e}\text{e}\text{d}\:\text{i}\text{n}\text{t}\text{a}\text{k}\text{e}\:\left(\text{d}\text{r}\text{y}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{i}\text{n}\:\text{g}\right)}{\text{B}\text{o}\text{d}\text{y}\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{g}\text{a}\text{i}\text{n}\:\left(\text{w}\text{e}\text{t}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{i}\text{n}\:\text{g}\right)}\text{x}\:\:100$$
$$\:\text{P}\text{E}\text{R}=\frac{\text{B}\text{o}\text{d}\text{y}\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{g}\text{a}\text{i}\text{n}\:\left(\text{w}\text{e}\text{t}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{i}\text{n}\:\text{g}\right)}{\text{P}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n}\:\text{i}\text{n}\text{t}\text{a}\text{k}\text{e}\:\left(\text{d}\text{r}\text{y}\:\text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\:\text{i}\text{n}\:\text{g}\right)}\text{x}\:\:100$$

Digestibility study

A 30-day digestibility study, following a feeding trial, using chromic oxide (Cr₂O₃) as an inert marker. Fish were acclimated to test diets containing 0.5% Cr₂O₃ for a ten-day period prior to the trial. Daily feedings were administered at 10:00 AM, with subsequent fecal collection at 8:00 AM using a modified method. Collected feces were freeze-dried and stored at -20 °C for subsequent analysis. The samples’ crude protein, lipid, and ash content were determined99. The chromic oxide content in feed and fecal matter was measured102. Apparent digestibility coefficients (ADCs) for dry matter, protein, lipid, and carbohydrate were calculated103.

$$\:\text{A}\text{D}\text{C}\:\text{o}\text{f}\:\text{d}\text{r}\text{y}\:\text{m}\text{a}\text{t}\text{t}\text{e}\text{r}\:{\%}\:=\:100\:\times\:\left(1-\frac{{\%}\text{C}\text{h}\text{r}\text{o}\text{m}\text{i}\text{c}\:\text{o}\text{x}\text{i}\text{d}\text{e}\:\text{i}\text{n}\:\text{d}\text{i}\text{e}\text{t}\:\:}{{\%}\text{C}\text{h}\text{r}\text{o}\text{m}\text{i}\text{c}\:\text{o}\text{x}\text{i}\text{d}\text{e}\:\text{i}\text{n}\:\text{f}\text{e}\text{c}\text{e}\text{s}}\right)$$
$$\:\text{A}\text{D}\text{C}\:\text{o}\text{f}\:\text{n}\text{u}\text{t}\text{r}\text{i}\text{e}\text{n}\text{t}\text{s}\:{\%}\:=\:100\:\times\:\left(1-\frac{{\%}\text{C}\text{h}\text{r}\text{o}\text{m}\text{i}\text{c}\:\text{o}\text{x}\text{i}\text{d}\text{e}\:\text{i}\text{n}\:\text{d}\text{i}\text{e}\text{t}\:\text{x}\:{\%}\:\text{n}\text{u}\text{t}\text{r}\text{i}\text{e}\text{n}\text{t}\:\text{i}\text{n}\:\text{f}\text{e}\text{c}\text{e}\text{s}\:}{{\%}\text{C}\text{h}\text{r}\text{o}\text{m}\text{i}\text{c}\:\text{o}\text{x}\text{i}\text{d}\text{e}\:\text{i}\text{n}\:\text{f}\text{e}\text{c}\text{e}\text{s}\:\text{x}\:{\%}\:\text{n}\text{u}\text{t}\text{r}\text{i}\text{e}\text{n}\text{t}\:\text{i}\text{n}\:\text{t}\text{h}\text{e}\:\text{d}\text{i}\text{e}\text{t}}\right)$$

Enzyme assays

Sample collection and tissue homogenate preparation

After the experiment, nine fish per group were anesthetized with clove oil (50 µl/L) and dissected to collect liver, intestine, and muscle tissues. Under cold conditions, these tissues were homogenized (5% in chilled 0.25 M sucrose) using a Teflon-coated mechanical homogenizer (HM30, REMI, India). The homogenate was then centrifuged (5000 rpm, 10 min, 4 °C) to separate the liquid portion (supernatant), which was stored frozen (-20 °C) for enzyme analysis.

Estimation of tissue protein

Lowry’s method measured Protein content in tissues104. Tissue samples (0.1 ml) were treated with an acid solution (TCA) and centrifuged to isolate the protein fraction. The remaining protein was then dissolved in a sodium hydroxide solution. This solution was mixed with specific chemicals and incubated in stages. The resulting solution’s colour intensity (optical density) was measured at 660 nm to determine protein concentration. A standard protein (BSA) helped calculate the actual protein amount in the fish tissues.

Digestive enzyme activities

Protease

In 1974, a method was described for casein digestion to assess protein activity in intestine tissue homogenates105.

Amylase

The amylase activity of intestine tissue homogenate was determined using the DNS (3,5-dinitro salicylic acid) method106.

Lipase

Intestinal lipase activity was assessed using the titrimetric method107.

Metabolic enzyme activities

Aspartate aminotransferase (AST) activities in the liver and muscle of C. catlawere assayed108. The procedure for estimating alanine aminotransferase (ALT) activity mirrored that of AST activity, with the distinction that the substrate used was 0.2 M DL-alanine instead of L-aspartic acid. ALT activity was quantified as nanomoles of pyruvate produced per milligram of protein per minute at 37 °C. Lactate dehydrogenase (LDH) and Malate dehydrogenase (MDH) activity in various tissues was assessed109,110.

Oxidative stress response

Superoxide dismutase (SOD) activity in liver tissue homogenates was quantified111, which is based on the enzyme’s inhibition of epinephrine oxidation to adrenochrome. Catalase (CAT) activity in liver tissue homogenates was determined112.

Haematological and serum parameters

Blood and serum collection

Fish were anesthetized with clove oil (50 µL/L) prior to blood collection. Blood was drawn from the caudal vein and immediately transferred to EDTA coated vials to prevent clotting. Aliquots were used for hemoglobin (Hb), red blood cell (RBC), white blood cell (WBC), and nitroblue tetrazolium (NBT) assays. A separate blood sample was collected without anticoagulant, allowed to clot, and centrifuged. The resulting serum was stored at -20 °C for further biochemical and immunological analyses.

Serum glucose

Serum glucose levels were quantified using the Erba diagnostic kit (Transasia Bio-medicals Pvt. Ltd., India). The glucose oxidase oxidizes glucose to generate hydrogen peroxide (H2O2). The H2O2 subsequently reacts with 3,5-dichloro-2-hydroxybenzene sulfonic acid and 4-aminoantipyrine, forming a pink dye, and absorbance was measured at 514 nm.

Serum total protein

Blood serum protein was measured using a Biuret kit (Erba, India). The assay is based on the reaction of proteins with copper ions, resulting in a blue-violet complex whose intensity increases with protein concentration. The protein levels were quantified by measuring the absorbance of the solution at 546 nm.

Serum albumin

Serum albumin levels were quantified using the Erba diagnostic kit (Transasia Bio-medicals Pvt. Ltd., India). This method is based on the interaction between albumin and Bromocresol Green (BCG) (pH 4.2), which leads to a shift in the absorbance of the BCG dye. The resultant blue-green hue, directly proportional to the albumin concentration, was assessed within the 580–630 nm wavelength range.

Serum globulin

Serum globulin levels were determined by subtracting albumin levels from total protein content.

Albumin-globulin (A/G) ratio

The albumin to globulin ratio (A/G ratio) was calculated by dividing albumin levels by globulin levels.

Nitro blue tetrazolium (NBT) assay

Blood samples were drawn from the caudal vein into a test tube containing 2.7% EDTA. 50 µl of blood were then aliquoted into U-bottom microplate wells and incubated at 37 °C for 1 h to facilitate cell adhesion. Following incubation, the wells were rinsed with PBS and treated with 50 µl of 0.2% NBT for 1 h. Subsequently, the cells were fixed using 100% methanol for 2–3 min, rinsed with 30% methanol, and air-dried. A solution comprising 60 µl of 2 N KOH and 70 µl of dimethyl sulfoxide was added to each well to solubilize the formazan blue precipitate. The optical density (OD) was then measured at 450 nm using an ELISA reader113.

Haematological parameters

Blood parameters such as erythrocyte count (RBCs), hematocrit (PCV), hemoglobin (Hb), and leukocyte count (WBCs) were assessed using an Olympus BH-2 light microscope at 100× magnification114.

Statistical analysis

The data were statistically analyzed using SPSS version 22.0. Normality was assessed via the Shapiro-Wilk test, and homoscedasticity was evaluated using Levene’s test. A one-way ANOVA was performed, followed by Duncan’s multiple-range test to determine significant differences among the means, with a significance level set at 5%.

Conclusion

In summary, incorporating Anabaena blue-green algae (ABGA) into the diet of Catla catla fingerlings at levels up to 15% does not negatively impact growth performance. However, higher inclusion rates may induce adverse physiological and metabolic effects. Specifically, increased ABGA levels were associated with significant reductions in amylase activity, alterations in hematological parameters, elevated oxidative stress markers, and increased serum glucose levels. Thus, while up to 15% ABGA can be safely included in the diet without impairing growth, the physiological stress at higher concentrations requires further investigation to establish a safe and optimal inclusion level.

This study adhered to the guidelines set forth by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), under the Ministry of Environment and Forests (Animal Welfare Division), Government of India, for the ethical care and use of animals in scientific research. All procedures involving fish handling and treatment received approval from the Ethics and Animal Care Committee of ICAR-CIFE, Mumbai, in compliance with institutional and national standards.