During the experiment, fish were kept in continuously aerated tanks. All the water quality parameters, including temperature (28.70-27.12ºC), pH (7.75-7.73), dissolved oxygen (8.35-7.40 ppm), total alkalinity (156.26-174.64 ppm), total hardness (216.37-251.34 ppm), and NH₃-N (0.055-0.056 ppm) were within the acceptable range as recommended (Boyd, 1998) for fish growth.

Fish feeding was ceased one day prior sampling. At intervals of 0, 30, 60, and 90 days into the experiment, fish from each experimental pool were anesthetized in 2-phenoxyethanol (1 mL L^-1), using 0.45 × 13 mm syringes (Hindustan Syringes & Medical Devices Ltd.). After the fish were weighed and measured for length, they were returned in their designated pools.

Nine fish per treatment or 45 fish were used for blood analysis per sampling session when three rohu fish were randomly selected for blood collection from each FRP pool. Using 1.5-mL syringes washed with a solution containing 25,000 U heparin ammonium salt (Sigma H6279) per 3 mL of 0.9% NaCl, blood samples (100-150 μL) were drawn from the caudal vein.

After centrifugation (4000 g, 3 min, 4°C) to remove the blood serum, the sample was frozen in liquid nitrogen and kept at -80°C until further examination. Subsequently, fish were decapitated and dissected for the brain, liver, and kidney samples and placed in empty sample collection tubes filled with 10% neutral buffered formalin (Sigma-Aldrich, CAS #HT501128). Likewise, flesh samples were obtained for flesh quality analysis and stored at -20°C until analysis.

Growth performance. Monthly records were maintained for fish survival and growth, with measurements taken for total body length and weight. Growth parameters were calculated using standard methodologies as outlined by Halver (1957).

Blood metabolic profile. The blood samples obtained from anaesthetized fish (n=9) were placed in anticoagulant vials. Haematological parameters, including total leucocyte counts (TLC) and total erythrocyte counts (TEC), were measured using a haemocytometer (Naubauer Counting Chamber). The microcapillary method was used to measure the packed cell volume (PCV) or haematocrit, while Sahli’s (1962) approach was used to evaluate hemoglobin (Hb). The blood indices viz., mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were calculated according to Mukherjee (1988). The total protein (Cat. #120231), albumin (A) (Cat. #120223), and glucose (Cat. #120235) were quantified in accordance with standard protocols using ERBA Mannheim Kits.

Liver enzyme profile. The manufacturer’s instructions (ERBA, Lachema s.r.o) for measuring the liver profile parameters, namely aspartate aminotransferase (AST, Cat. #120204) and alanine aminotransferase (ALT, Cat. #120207), were followed while measuring fish serum. A standard technique was followed to quantiy the enzymes using 50 μL serum in a biochemical analyzer (CHEM-7, ERBA, Mannheim). Antioxidant parameters, including superoxide dismutase (SOD) and catalase (CAT), were calculated according to the standard protocol (Nishikimi et al., 1972).

Tissue microarchitecture analysis. After the termination (90 days) of the experiment, fish tissues such as the brain, kidney and liver were removed and preserved in 10% neutral buffered formalin (NBF, Sigma Aldrich, CAS #HT501128). Then, the tissues were processed. Briefly, tissues were dehydrated using different concentrations of methanol (70, 80, 95 and 100%), followed by chloroform (100%) and then embedded in paraffin. After that, tissues were sectioned (4-6 μm) using a Leica RM2125RT rotary microtome. Sections were mounted onto microscope slides (Super Frost, #G10106, Abdos, India) and dried at ~35 °C overnight. The sections were then examined under a microscope using the previously mentioned Harris hematoxylin and eosin staining technique (Shanthanagouda et al., 2014). Then the slides were mounted using DPX and allowed to dry overnight. The images were captured using a digital camera mounted microscope (Nikon 80i).

Statistical analysis of the data was performed using SPSS v16. One-way ANOVA was applied to understand the effect of TM on the growth and blood metabolic profile of fish challenged with AFB1 at p≤0.05 level of significance, followed by Duncan’s multiple comparisons to determine the significant differences among the treatments.

The current study showed percentage (%) average survival of fish as 100.00±0.00, 77.77±2.77, 97.22±2.67, 91.66±0.01 and 80.55±2.77 % in control diet (T1), AF100 (T2), AF25TM (T3), AF50TM (T4) and AF100TM (T5) treatments, respectively. Maximum survival was observed in the negative control diet (100%) followed by AF25TM (97.22%), whereas, in AF100, the lowest survival was observed. The negative control diet group did not experience any mortality during the experiment, and there was a significant (p≤0.05) increase in growth indices as measured by net body weight gain (NBWG) and specific growth rate (SGR) (Table 3). However, the fish in the AF100 group saw a notable decline in growth indices when compared to the negative control group. Conversely, the average NBWG in treatments T3, T4, and T5 was significantly higher (p≤0.05) than that of the positive control (T2) and higher than that of T4 and T5. Similarly, the average final body weight (g) and NBWG (g) were maximum in T1 and minimum in T2 and the differences among different treatments were significant (p≤0.05). Likewise, SGR, condition factor and feed conversion ratio (FCR) demonstrated greater improvements in both the control diet and T3 compared to T2, T4 and T5.

Brain. Fish fed on the negative control (T1) diet displayed no discernible alterations in brain tissue. Across systematically arranged layers from the ependyma to the outer surface, including Stratum periventricular (SPV), Stratum album central (SAC), Stratum griseum central (SGC), Stratum fibrosum et griseum superficial (SFGS), Stratum opticum (SO), and the Stratum marginale (SM), integrity was maintained with no detachment of granular cells or lesions observed in any of the stratified zones in the tectum (Fig. 1A). However, in AF100 group, outer layers such as SO and SM exhibited cell detachments, varied focal necrosis, cell degenerations, and vacuolization in SFGS, SAC, and SPV particularly evident in AF100 (Fig. 1B). On the other hand, different tectum layers were seen strikingly compactly arranged in the AF25TM treatment (Fig. 1C), while in AF100TM treatment (Fig. 1D) less distinction in the separation of layers (LDSL) were observed, accompanied by the presence of pyknotic nuclei (PN).

Kidney. In the negative control group (T1), the kidney exhibited a normal structure, characterized by distal and proximal convoluted tubules and glomeruli (Fig. 2A). However, in the positive control group (AF100), significant histological alterations were observed, including clumping of glomeruli and mononuclear cell infiltration in inter-tubular tissues (Fig. 2B). Conversely, in the AF25TM group (Fig. 2C), dilatation and congestion of blood vessels, multi-clumping of the glomerulus, and deposition of melanomacrophages were observed. Furthermore, the kidney micro-architecture in the AF100TM group (Fig. 2D) showed vacuolization and degenerated glomeruli.

Liver. In the current study, the negative control group, which did not receive AFB1, exhibited normal oval to hexagonal hepatocytes with centrally located nuclei, endothelial cells, and a uniform cytoplasm (Fig. 3A). However, in AF100 (positive control), notable alterations, including vacuolization of hepatocytes, blood cell congestion in the central vein, and depositions were observed (Fig. 3B). In contrast, in AF25TM (Fig. 3C), reduced dilatation of the central vein, vacuolization, and binucleated hepatocytes were recorded. Meanwhile, in AF100TM (Fig. 3D), an obliterated central vein and possible deposition of TM particles were observed.

The high dietary AFB1 levels significantly affected various growth performance and health status parameters in fish. Over a 90-days period of exposure to 100 ppb AFB1 reduced growth indices, including NBWG, SGR, and survival rate compared to diets with 0 and 25 ppb AFB1. These findings indicate that the ingestion of low to moderate doses of AFB1 leads to significant growth depression, reduced weight gain, and decreased feed efficacy in a dose-dependent manner. The amount of toxin and duration of exposure are related to AFB1’s detrimental effect on growth parameters. Similar outcomes have been observed in broiler chicks (Manafi et al., 2014), and in juvenile Nile tilapia (Tuan et al., 2002).

Mahmoud et al. (2014) proposed that the augmented growth response with TM supplementation may stem from enhanced feed consumption and utilization, suggesting increased nutrient digestibility and antioxidant activity of turmeric, thereby promoting protein synthesis through enzymatic systems. Consistent with earlier studies, Ashry et al. (2021) observed significant enhancements in growth rate, SGR, and FCR in Gilthead seabream diet supplemented with TM. Similarly, curcumin has demonstrated improvements in growth performance in L. rohita (Kaur et al., 2020).

The study revealed significant increase (p≤0.05) in haematological parameters (TEC, TLC, Hb, hematocrit value, and MCV) in groups receiving higher doses of AFB1 compared to those receiving lower doses. Similar findings were reported in O. niloticus (Tuan et al., 2002) and L. rohita (Bhatt et al., 2023b). In contrast, diets with low doses of AFB1 and supplemented with TM (5 g kg^-1) exhibited elevated Hb, PCV, TEC, and TLC counts, suggesting potential immune-modulatory effects of TM (Kumar et al., 2013). In line with our findings, green terror (Andinoacara rivulatus) fed TM diets showed an increase in TLC, as observed by Mooraki et al. (2019). Similarly, the elevation in Hb content may be attributed to increased enzyme synthesis or red blood cell size. The decrease in TEC, TLC, Hb, and hematocrit values suggests anemia, possibly due to inhibition of enzyme activities in heme biosynthesis or increased erythrocyte disruption in hematopoietic organs (Jenkins et al., 2003). However, the decline in TLC count might be attributed to epinephrine release during stress, leading to spleen contraction and reduced leukocyte count, thereby compromising the immune system and rendering fish susceptible to infection (Mooraki et al., 2019). Blood indices such as MCV, MCH, and MCHC play a crucial role in assessing erythrocyte relevance and oxygen-carrying capacity, as well as diagnosing anaemic conditions in fish and other animals (Houston, 1997).

In the present study, a high blood glucose level was observed probably due to increased liver glycogenolysis and a decrease in the secretion of insulin as an effect of AFB1, which in turn reduces the process of glycogenesis, resulting in increased blood glucose level. Similar results were obtained by El-Boshy et al. (2008) who reported increased blood glucose levels in AFB1-challenged O. niloticus. The results indicated that the blood glucose level closely correlates to stress level in fish and represents the state of respiratory and nutritional disturbance.

Furthermore, there was a gradual decline in total protein, albumin and globulin in AFB1-exposed fish, indicating decreased protein synthesis as reported in Onchorhynchus mykiss (Ruby et al., 2014), which could be due to the damaging effects of AFB1 on hepatocytes. The current study´s results are consistent with earlier research on O. niloticus (Shehata et al., 2009), showing that fish fed diets containing plant extracts had higher humoral immunity. This suggests that TM supplementation may fortify the immune system and offer long-term resistance (Sahu et al., 2008), exhibiting stronger antioxidant activity. Addition of 0.5% rosemary to AFB1-contaminated food significantly increases total protein and globulin levels, activating the immune system and improving fish health (Naiel et al., 2019).

The liver, being the primary site for AFB1 metabolism, is highly susceptible to damage. Dietary AFB1 significantly increased plasma AST and ALT activity (p≤0.05), indicating potential liver dysfunction or swelling, leading to enzyme release into the bloodstream (Akrami et al., 2015). However, in the current study, fish exposed to 25 ppb AFB1 + 5 g kg^-1 TM supplementation (T3) showed significantly reduced serum AST and ALT activity, indicating ameliorative efficacy. Similar findings were also observed by El-Barbary & Mehrim (2009) in O. niloticus. Moreover, increased SOD and CAT activities in T2 and T5 suggest dietary AFB1-induced oxidative stress, prompting antioxidant enzyme activation as defence mechanisms against AFB1 effects. Furthermore, by converting superoxide anion free radicals, SOD reduces the damage to cellular DNA. Though the fish were challenged with AFB1, supplementation of TM enhanced health by increasing the protein concentration which might be due to the antioxidant and therapeutic properties of TM. This suggests that the application of TM in aquaculture may feed the aquatic species´ general well-being.

Histopathology has been widely used as a suitable biomarker in assessing the health of fish exposed to xenobiotics and or stress factors since they reflect the actual health status of the animals. Amongst several xenobiotics, exposures to aflatoxin have caused serious damage to fish, and the current study has investigated the histopathological responses in key organs and efficiency of TM in amelioration of AFB1 in the most widely cultured tropical fish, rohu.

Brain. In this study, the alterations observed in the fish brain may be caused by AFB1 toxicity, as the brain is the most sensitive tissue to AFB1 toxicity. The severity of alterations observed was more pronounced in the groups which received the highest AFB1 over an extended period in comparison to the group fed less or no contaminated diet. Similar results were also reported in channel catfish (Ictalurus punctatus) (Jantrarotai & Lovell, 1990). Further, the findings advocated that the brain could accumulate AFB1 as per dose and time-dependent manner, and the toxicity could be mainly attributed to free radicals and oxidative stress produced by the aflatoxin (Fig. 4). Typically, it can combine with the thiol group of enzymes working in antioxidant processes and disrupt the normal functioning of the brain (Baldissera et al., 2018) and the intoxicated fish might have suffered from neurotoxicity. The ability of TM to scavenge ROS through interaction with the oxidative cascade to suppress oxidative enzymes and normalize the antioxidant levels, thereby preventing the cell structures of the brain from more damage, has been evidenced (El-Bahr, 2015).

Kidney. In the present study, the acute toxic effects of AFB1 on the tissue micro-architecture of the kidney of L. rohita have been elucidated. The tissue microscopic examinations showed that AFB1 has a great significant effect on the kidney structure that received the highest AFB1 in comparison to the groups fed a less toxin-contaminated diet, as this is consistent with what has been found in previous studies, where fish were exposed to contaminants and pollutants (Hadi & Alwan, 2012). In comparison, fish fed with an aflatoxin-free diet showed normal glomeruli, and proximal and distal convoluted tubules. The damages observed in AFB1-administered groups in the present study could be attributed to the ability of AFB1 to induce free radical generation and increase the activity of catalase in kidney cells (Taheri et al., 2017). This is consistent with several other findings, which report a direct correlation between AFB1-induced oxidant generation and resultant DNA damage (Meki et al., 2001). However, TM seemed to lessen the toxicity by changing the way AFB1 is microsomally activated. It also increased detoxification, protected DNA and stopped the development of future DNA adduct, all of which prevented the toxicity caused by AFB1 from happening.

Liver. The liver is one of the most affected organs due to contaminants, and pollutants. Hepatocytes are the primary target of contaminants. This is evidenced in the present study, where significant alterations observed were more pronounced in the groups which received the highest AFB1 in comparison to the groups fed less or no toxin. Diets contaminated with AFB1 showed dose-dependent changes in histopathological damages similar to those found in the liver of L. rohita (Bhatt et al., 2023c). However, the severity of tissue damage in fish treated with different levels of AFB1 increased with an increase in dosage and period of exposure. The present study highlights that the abnormal changes observed are similar to the previous findings having different stressors. However, in the current study, TM appeared to reduce the AFB1 toxicity for its therapeutic properties and probably mediated through its antioxidant and anti-inflammatory action (Aggarwal et al., 2007). Similarly, in the liver and kidney of mice given AFB1, TM restored the activities of both enzymatic and non-enzymatic antioxidants and restored the levels of lipid peroxidation (Verma & Mathuria, 2008). Fish that were poisoned with aflatoxin may have been able to recover through similar processes.