Effects of dietary Curcuma longa extract supplementation on growth performance, hematological responses, antioxidant status, and intestinal protease activity in grass carp (Ctenopharyngodon idella) fingerlings

Introduction

Aquaculture is one of the fastest-growing food production sectors and plays a vital role in meeting the increasing global demand for animal protein (FAO, 2024) [12] . However, intensive aquaculture practices often increase stress, disease susceptibility, and oxidative damage, negatively affecting fish health and productivity (Dawood et al., 2018) [11] . Although antibiotics and synthetic growth promoters have traditionally been used to improve fish performance, their extensive use has raised concerns regarding antimicrobial resistance, environmental contamination, and food safety (Cabello, 2006) [9] . Consequently, natural feed additives have gained considerable attention as sustainable alternatives in aquaculture. Grass carp (Ctenopharyngodon idella) is an economically important herbivorous freshwater fish species widely cultured in Asia because of its rapid growth, efficient feed utilization, and high consumer acceptance (FAO, 2024; Wang et al., 2015) [12,26] . Therefore, improving the growth performance and health status of grass carp fingerlings is essential for sustainable aquaculture production. Phytogenic feed additives derived from medicinal plants have emerged as promising alternatives to synthetic growth promoters due to their growth-promoting, antioxidant, antimicrobial, immunostimulatory, and digestive-enhancing properties (Reverter et al., 2014; Dawood et al., 2018) [22,11] . Among these, turmeric (Curcuma longa L.) has attracted considerable interest because of its diverse biological activities, primarily attributed to curcuminoids, particularly curcumin (Ammon & Wahl, 1991; Aggarwal et al., 2007) [4,3] . Curcumin is known to enhance antioxidant defense by stimulating enzymes such as superoxide dismutase, catalase, and glutathione peroxidase, while also improving digestive enzyme secretion, nutrient utilization, and intestinal health (Menon & Sudheer, 2007; Sivaram et al., 2004; Abdel-Tawwab et al., 2010) [18,24,1] . Previous studies have demonstrated that dietary turmeric supplementation improves growth performance, hematological indices, antioxidant status, and immune responses in several fish species, including rohu, Nile tilapia, common carp, gilthead seabream, and orange-spotted grouper (Sahu et [23,14,21,5] . Moreover, curcumin has been reported to activate antioxidant signaling pathways and reduce oxidative stress in fish (Ming et al., 2020) [19] . However, information regarding the effects of graded dietary levels of C. longa extract on growth, hematology, antioxidant status, and digestive physiology in grass carp fingerlings remains limited. Therefore, the present study aimed to evaluate the effects of dietary supplementation with graded levels of Curcuma longa extract (0.2%, 0.4%, and 0.6%) on growth performance, survival, hematological parameters, antioxidant enzyme activities, and intestinal protease activity in grass carp fingerlings during a 30-day feeding trial.

Materials and Methods

Experimental fish and acclimatization

Healthy fingerlings of grass carp (Ctenopharyngodon idella) with an average initial body weight of 11 ± 4 g were procured from a local fish farm and acclimatized to laboratory conditions for one week prior to the commencement of the feeding trial. During the acclimatization period, fish were fed a basal diet and maintained under optimal water quality conditions with continuous aeration and regular water exchange.

Experimental design and preparation of diets

A 30-day feeding trial was conducted following a completely randomized design consisting of four dietary treatments with three replicates per treatment. Each replicate contained 10 fish fingerlings, resulting in a total of 30 fish per treatment group. Fish were fed their respective experimental diets at a feeding rate of 5% of body weight per day throughout the experimental period. The daily feed ration was divided into two equal portions and administered twice daily, in the morning and evening.

Effects of Curcuma longa extract supplementation on growth performance

Growth performance was evaluated at 15-day intervals throughout the experimental period (days 15 and 30). Individual body weight ( ± 1 g) of all fish from each replicate was recorded during each sampling period. The collected data were used to calculate weight gain (WG), percentage weight gain (%WG), specific growth rate (SGR), and survival rate (SR) according to the standard formulae described by Weatherley and Gill (1987) [27] .

Effects of Curcuma longa extract supplementation on hematological parameters

At the end of the feeding trial (day 30), blood samples were collected aseptically from the caudal vein using heparinized syringes. Hematological parameters including hemoglobin concentration (Hb), red blood cell count (RBC), white blood cell count (WBC), and packed cell volume (PCV) were determined according to the methods described by Blaxhall and Daisley (1973) [8] .

Effects of Curcuma longa extract supplementation on antioxidant enzyme activities

At the termination of the experiment, liver tissues were excised and homogenized in ice-cold phosphate buffer followed by centrifugation to obtain the supernatant for enzymatic assays. Antioxidant status was evaluated by measuring the activities of superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) using standard spectrophotometric methods described by Misra and Fridovich (1972) [20] , Aebi (1984) [2] , and Beutler et al.

(1963) [7] , respectively.

Effects of Curcuma longa extract supplementation on intestinal protease activity

At the end of the feeding trial, intestinal tissues were collected and homogenized in chilled phosphate buffer. The homogenate was centrifuged and the resulting supernatant was used for enzyme analysis. Intestinal protease activity was determined according to the method described by Walter (1984) [25] , while total protein concentration in the enzyme extract was estimated following the method of Lowry et al. (1951) [16] .

Statistical analysis

All experimental data are presented as mean ± standard deviation (SD) in tables. For graphical presentation, mean values with standard error (SE) as error bars were used to improve visualization of treatment differences. Statistical analyses were performed using IBM SPSS Statistics software. Growth parameters measured at different sampling intervals (days 15 and 30) were analyzed using two-way ANOVA, whereas hematological parameters, antioxidant enzyme activities, and intestinal protease activity measured at the end of the experiment were analyzed using one-way ANOVA, followed by Tukey's multiple comparison test. Differences among treatment means were considered statistically significant at p < 0.05.

Results and Discussion

Effects of Curcuma longa extract supplementation on growth performance and survival of Ctenopharyngodon idella fingerlings

The growth performance and survival of grass carp fingerlings were significantly influenced by dietary supplementation with Curcuma longa extract during the 30day feeding trial (p < 0.05), as presented in Table 2. No significant differences were observed in the initial body weight among the treatment groups (11.30-11.64 g), indicating homogeneity of the experimental fish at the beginning of the study (Table 2). At the end of the feeding trial, fish fed turmeric-supplemented diets exhibited improved growth performance compared with the control group. The highest final body weight was recorded in the T 3 group (16.92 ± 1.63 g), followed by T 2 (15.96 ± 0.79 g), T 1 (15.60 ± 1.28 g), and the control group (14.69 ± 0.28 g) (Table 2). Similarly, the highest weight gain (2.93 ± 0.43 g), percentage weight gain (20.97 ± 2.93%), and specific growth rate (1.27 ± 0.16% day⁻¹) were observed in fish receiving 0.6% C. longa extract supplementation. The T 2 and T 3 treatments resulted in significantly higher percentage weight gain and specific growth rate compared with the control treatment (p < 0.05) (Table 2). Furthermore, dietary turmeric supplementation positively affected fish survival, with T 2 and T 3 groups exhibiting 100% survival, whereas the control group recorded the lowest survival rate (80.33%) (Table 2). The improvement in growth performance observed in turmeric-supplemented groups may be attributed to the presence of bioactive compounds, particularly curcumin, which enhances feed utilization efficiency, nutrient absorption, and digestive processes. Similar improvements in growth performance following dietary turmeric supplementation have been reported by Sivaram, V. et al. (2004) [24] , who observed enhanced growth and disease resistance in juvenile Orange-spotted grouper fed turmeric-enriched diets.

The present findings are also in agreement with the observations of Abdel-Tawwab, Mohamed et al. (2018) [1] , who reported that phytogenic feed additives improved growth performance and feed utilization in cultured fish species through enhanced digestive activity and improved intestinal health. Moreover, the growth-promoting effects of turmeric may be associated with its antioxidant and immunomodulatory properties, which reduce oxidative stress and allow more metabolic energy to be directed towards growth rather than maintenance and cellular repair. Similar conclusions were reported by Reverter, Mireia et al.

(2014) [22] , who demonstrated that phytogenic feed additives can improve growth performance and health status in aquaculture species by enhancing antioxidant defense and immune responses. The improved survival observed in the turmeric-fed groups may also be associated with the antimicrobial and immunostimulatory properties of curcumin, which contribute to enhanced stress tolerance and disease resistance in fish. Comparable findings were reported by Citarasu, Thavasimuthu (2010) [10] , who highlighted the beneficial role of herbal feed additives in improving fish health and survivability under aquaculture conditions. Overall, the present study demonstrates that dietary supplementation with Curcuma longa extract, particularly at inclusion levels of 0.4% and 0.6%, effectively enhances growth performance and survival of grass carp fingerlings and may serve as a promising natural feed additive in sustainable aquaculture practices.

Effects of dietary Curcuma longa extract on haematological parameters

Dietary supplementation of Curcuma longa extract significantly affected the haematological profile of Ctenopharyngodon idella fingerlings (Table 3), with highly significant effects on Hb (p < 0.001) and significant effects on RBC (p = 0.007) and WBC (p = 0.008) counts, while PCV remained statistically unaffected (p = 0.062). RBC and Hb increased in a clear dose-dependent manner, from 1.38 ± 0.05 × 10⁶/µL and 5.81 ± 0.13 g/dL in the control to peak values of 1.64 ± 0.08 × 10⁶/µL and 6.84 ± 0.17 g/dL at T 2 -T 3 , respectively. Both indices plateaued beyond 0.4% inclusion, with no significant difference between T 2 and T 3 . This trend is consistent with Ashry et al. (2021) [5] , who reported dose-dependent increases in RBC and Hb in gilthead seabream (Sparus aurata) fed curcumin at 2-3%, and with Sahu et al. (2008) [23] , who observed elevated RBC and Hb in rohu (Labeo rohita) supplemented with graded C. longa doses. The improvement is generally attributed to the antioxidant activity of curcuminoids, which protects erythrocyte membranes from oxidative damage and supports haemoglobin synthesis via improved hepatic function (Manju et al., 2013) [17] . PCV rose only marginally (23.24% to 24.81%) without reaching significance, mirroring Manju et al. (2013) [17] , who similarly found that Hb and RBC responded significantly to dietary curcumin in Anabas testudineus while related volumetric indices did not reflecting the lower sensitivity of PCV, which depends on both cell number and corpuscular volume rather than erythrocyte count alone. WBC count increased significantly at T 2 (68.26 × 10³/µL) and T 3 (67.84 × 10³/µL) relative to control, indicating an immunostimulatory effect of C. longa, consistent with Pereira et al. (2020) [21] , who reported elevated leukocyte counts in Nile tilapia fed 2.5% Curcuma longa hydrolate, with the effect plateauing at higher doses a pattern also seen here between T 2 and T 3 . Notably, this leukocyte response is not universal: Manju et al. (2013) [17] found WBC unaffected by curcumin in A. testudineus, suggesting the response is species-and dose-dependent. [5,17,21] . The plateau beyond 0.4% suggests T 2 (0.4%) as the near-optimal inclusion level, consistent with dose-saturation trends reported for curcumin in rainbow trout (Bakhtiari Aqmasjed et al., 2023) [6] and Nile tilapia (Pereira et al., 2020) [21] .

Effects of dietary Curcuma longa extract on antioxidant parameters

Dietary supplementation with Curcuma longa extract resulted in a significant and dose-dependent enhancement of antioxidant enzyme activities in Ctenopharyngodon idella fingerlings (Figure 1). Superoxide dismutase (SOD) activity increased progressively from 45.3 ± 0.39 U/mg protein in the control group to 48.5 ± 0.42, 55.43 ± 0.56, and 58.47 ± 0.77 U/mg protein in T 1 , T 2 , and T 3 , respectively (Figure 1A), representing an approximately 29% increase over the control at the highest supplementation level. Similarly, catalase (CAT) activity increased significantly from 12.43 ± 0.20 U/mg protein in the control group to 19.27 ± 0.33 U/mg protein in the T 3 group (Figure 1B). Reduced glutathione (GSH) levels also exhibited a progressive increase from the control to the highest supplementation level, indicating enhanced non-enzymatic antioxidant defense in turmeric-fed fish (Figure 1C). The observed increase in SOD, CAT, and GSH suggests that dietary C. longa supplementation enhanced both enzymatic and nonenzymatic antioxidant defense mechanisms in grass carp fingerlings. Similar findings were reported by Ming et al.

(2020) [19] , who observed elevated antioxidant enzyme activities and glutathione content in grass carp supplemented with dietary curcumin. Likewise, Giri et al.

(2019) [14] reported improved antioxidant status in common carp following dietary turmeric supplementation. The antioxidant effects of turmeric are primarily attributed to curcumin, which functions both as a direct scavenger of reactive oxygen species (ROS) and as an activator of the Nrf2/Keap1 signaling pathway, thereby stimulating the expression of antioxidant defense genes (Ming et al., 2020) [19] . Overall, the progressive increase in antioxidant parameters observed in the present study (Figure 1) indicates that dietary C. longa extract effectively improved the antioxidant capacity and physiological resilience of C. idella fingerlings. Protease activity increased progressively with increasing dietary inclusion levels of turmeric extract, with all treatment groups differing significantly from one another (p < 0.05). The highest protease activity was recorded in the T 3 group (14.60 ± 0.29), followed by T 2 (13.47 ± 0.23), T 1 (10.83 ± 0.19), and the control group (9.17 ± 0.16). As illustrated in Figure 5, the improvement in protease activity suggests enhanced digestive efficiency and protein utilization in turmeric-supplemented fish. This effect may be attributed to curcumin and other bioactive constituents of C. longa, which stimulate digestive secretions and enzyme synthesis, thereby improving nutrient digestion and absorption. Similar improvements in digestive enzyme activities following dietary turmeric supplementation have been reported by Sivaram, V. et al. (2004) [24] and Abdel-Tawwab, Mohamed et al. (2010) [1] . Furthermore, Reverter, Mireia et al. (2014) [22] suggested that phytogenic feed additives enhance digestive physiology and nutrient utilization, ultimately contributing to improved growth performance in cultured fish species. Overall, the results indicate that dietary inclusion of C. longa extract, particularly at 0.4% and 0.6%, effectively improves proteolytic activity and digestive capacity in grass carp fingerlings.

Conclusion

The present study demonstrated that dietary supplementation with Curcuma longa extract positively influenced the growth performance, hematological profile, antioxidant status, and digestive physiology of grass carp (Ctenopharyngodon idella) fingerlings during the 30-day feeding trial. Fish fed turmeric-supplemented diets exhibited higher weight gain, percentage weight gain, specific growth rate, and survival compared to the control group. Dietary inclusion of C. longa extract also significantly improved hematological parameters, particularly hemoglobin concentration, red blood cell count, and white blood cell count, indicating enhanced physiological condition and immune competence of the fish. Furthermore, supplementation with turmeric extract resulted in a dosedependent increase in antioxidant enzyme activities, suggesting improved protection against oxidative stress and enhanced cellular defense mechanisms. The observed increase in intestinal protease activity further indicates improved digestive efficiency and nutrient utilization in turmeric-fed fish, which may have contributed to the enhanced growth performance observed in the supplemented groups. Overall, the findings of the present study suggest that Curcuma longa extract is an effective natural phytogenic feed additive for improving the health and productivity of grass carp fingerlings. Among the tested inclusion levels, dietary supplementation with 0.4% and 0.6% C. longa extract produced the most favorable responses, with 0.4% appearing to be the optimal level considering the comparable physiological benefits achieved relative to the higher inclusion level. Therefore, dietary turmeric supplementation may represent a sustainable and environmentally friendly strategy for improving fish performance and health in freshwater aquaculture.

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