Introduction

African catfish, Clarias gariepinus, is a popular freshwater fish in Malaysia, which is highly demanded and affordable (Wei et al. 2024). This species is widely cultivated due to its high disease tolerance, adaptability to various environments, rapid growth, and dietary flexibility. In the economic context, African catfish have a short production cycle (2 to 3 months) reaching marketable size and can be farmed at high stocking density. Therefore, this aquaculture species is the best candidate to boost an aquaculturist’s income. Nevertheless, fish farming at high stocking density causes crowding stress, impairing fish growth and health. Moreover, intensive farming renders fish vulnerable to disease outbreaks and causes major economic losses (Goh et al. 2023a, b). One of the critical diseases in aquaculture is Edwardsiellosis, caused by Edwardsiella tarda. This bacterium has major economic significance as the disease outbreak leads to high mortality and possibly the end of a fish farm operation. E. tarda is found in various hosts and environments, and the infection has been reported worldwide. Furthermore, E. tarda infection occurs in freshwater and marine aquatic animals, such as carp in Japan, India, and China (Pandey et al. 2021; Yamasaki et al. 2013); eels in China and South Korea (Mo et al. 2016; Park et al. 2017); American bullfrog, tilapia, catfish, golden pompano, and Macrobrachium rosenbergii in Malaysia (Lee et al. 2010a, b; Lee et al. 2009; Lee & Wendy 2017; Najiah et al. 2009); Nile tilapia in Egypt (Elgendy et al. 2022); channel catfish and chinook salmon in USA (Loch et al. 2017); and turbot, flounder, sturgeon, catfish, sea horse, farmed crocodile, and soft-shell turtle in China (Du et al. 2017; Liang et al. 2022; Wang et al. 2020; Wu et al. 2022). Several strategies have been applied to mitigate E. tarda infection in aquatic animals, such as vaccination in turbot (Castro et al. 2008). Other studies also revealed the potential of inactivated E. tarda in stimulating immune response in zebrafish, flounder, and tilapia (Kwon et al. 2006; Tang et al. 2017; Xu et al. 2019). However, vaccination is challenging due to the intensive labor, high cost, and species specificity.

Vaccines and antibiotics are used as treatments and preventive measures in aquaculture operations. Nonetheless, vaccination is labor-intensive and expensive (Wei et al. 2022), while antibiotics are banned in numerous countries due to environmental and public health concerns. As a result, feed additives such as probiotics, prebiotics, and phytobiotics are gaining attention in aquaculture as a preventive and treatment strategy (Wee et al. 2024). Phytobiotics, in particular, are inexpensive, abundant, accessible, and effective, offering fish farmers an alternative source of feed additives. Thus, researchers have embarked on a journey to discover the full potential of phytobiotics in aquaculture. This alternative feed additive has shown promising results in preventing and treating infectious diseases (Abdel-Tawwab et al. 2018, 2021; Adeshina et al. 2019) and improving growth and health status of aquatic animals. For instance, dietary Spirulina enhanced the growth and health of stinging catfish, Heteropneustes fossilis (Rahman et al. 2023). Various plant-based polysaccharides also boosted the production of multiple carp species (Goh et al. 2023a Goh et al. 2023b). In addition, fermented water spinach modulated the growth and health of stinging catfish, H. fossilis (Nandi et al. 2023). Dietary papaya leaf extract (Hamid et al. 2022), pineapple waste (Anis Mohamad Sukri et al. 2022, 2023), olive oil by-product (Hazreen-Nita et al. 2022), soybean lecithin (Wee et al. 2023), Peperomia pellucida (Lee et al. 2016), and other phytobiotics (Kari et al. 2023a, b; Kari et al. 2022) positively impacted the growth and health of aquatic animals. Therefore, phytobiotics are a cost-effective, convenient, and effective approach to reducing vaccines and antibiotic usage and boosting aquaculture productivity.

Kaffir lime (KL), Citrus hystrix, is a native lime species in Southeast Asia, including Malaysia. This fruit reportedly contains 78 bioactive compounds, including β-pinene, limonene, sabinene, and citronellal, which benefit human health (Zhao et al. 2023). Phytochemical analysis also demonstrated that KL has terpenoids, phenolic acids, flavonoids, and coumarins as the main constituents (Zhao et al. 2023). Earlier studies revealed that this plant has antimicrobial (Panthong et al. 2013), antioxidant (Panthong et al. 2013), anti-mosquito (Nararak et al. 2017), anti-tumor (Anuchapreeda et al. 2020; Borusiewicz et al. 2017; Sun et al. 2018), anti-inflammatory (Anuchapreeda et al. 2020), and neural-protective properties (Sammi et al. 2016). Furthermore, its fresh, frozen, or dried leaf is widely used in Asian cuisines as a flavoring agent and tea. Nevertheless, limited literature describes the potential of KL leaf as a feed additive in animals, particularly aquatic species. Therefore, this study evaluated the impacts of dietary KL leaf powder on the growth performance, digestive enzyme, hematology, antioxidative response, and disease resistance to E. tarda infection in African catfish, C. gariepinus.

Materials and methods

Kaffir lime leaf powder preparation

Kaffir lime leaves were purchased from a wet market in Tanah Merah, Kelantan. The leaves were oven-dried at 60 °C for 48 h and powdered using a blender (Panasonic, Malaysia). The KL leaf powder was stored in a freezer for further use.

Medicated feed formulation

Five different diets were formulated and prepared, as detailed in Table 1. Four diets were added with 1%, 2%, 3%, and 4% KL leaf powder and labeled KL1, KL2, KL3, and KL4, respectively. All the ingredients were mixed, homogenized, and pelleted using an extruder. All diets were air-dried, placed in sealed, labeled plastic bags, and stored in a freezer for further use. Proximate analyses were carried out to determine carbohydrate, protein, lipid, moisture, and ash content in each diet (Latimer & International 2023). Carbohydrates and protein of the formulated diets ranged from 42.5% to 44.2% and 32.1% to 32.9%, respectively.

Table 1 Feed formulations and nutritional profile of formulated diets
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Feeding trial and experimental design

The experimental fish were purchased from a commercial farm at Tanah Merah, Kelantan, and kept for a week in a holding tank (300 L). The fish were acclimatized for a week and given a control diet (Table 1) once daily in the morning with the feeding rate at 5% of body weight. A complete water exchange was done in the afternoon. At the end of the acclimatization period, only the healthy fish were selected and kept in 50 L aquaria. Each aquarium housed 30 fish and the experiment was conducted in triplicates. The experimental fish received the formulated diets for eight weeks. The experimental fish were given the formulated diets once daily in the morning (feeding rate: 5% of body weight) and 100% water change was carried out in the afternoon. The water quality in the aquaria was maintained as follows: water temperature = 26.8–28.8 °C, ammonia > 0.05 ppm, dissolved oxygen = 6.3–6.8 ppm, and pH 6.1–7.1.

Growth performance determination

At the end of feeding trial, the experimental fish from each treatment were weighed and the growth performance parameters were determined as described in the previous studies: final weight (FW) (final body weight − initial body weight), weight gain (WG) ([total weight gain/initial body weight] × 100), specific growth rate (SGR) (total weight gain × 100/experimental days), feed conversion rate (FCR) (total feed intake/total weight gain), viscerosomatic index (VSI) (total viscera weight/total body weight), and hepatosomatic index (HSI) (total liver weight/total body weight) (Abdul Kari et al. 2021; Kari et al. 2023a, b; Zakaria et al. 2022).

Hematology analysis

Once the feeding trial ended, three fish per replicate from each treatment were anesthetized using clove oil for blood collection. The blood samples were kept in heparinized tubes and later analyzed using a hematology analyzer (Mythic 18 Vet, USA) (Zakaria et al. 2022).

Digestive enzyme activities

The digestive enzyme activity was determined as described in previous studies. The intestines of three experimental fish per replicate from each treatment were harvested and homogenized in phosphate-buffered saline (PBS). The mixture was centrifuged at 7 168 g for 10 min to obtain the supernatant and subjected to digestive enzyme activity assay. The protease and amylase activities were determined through the Folin-Ciocalteu phenol reagent and iodine solution, respectively (Lowry et al. 1951). Meanwhile, the lipase activity was determined, as described by Borlongan (1990).

Antioxidative response determination

The liver of experimental fish (n = 3) per replicate from each treatment was harvested and homogenized in saline. The supernatant of the sample was obtained via centrifugation at 11 200 g for 10 min. Subsequently, the supernatant was used to determine superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities through the colorimetric method via commercial kits (Elabscience, Malaysia). The results were analyzed using a microplate reader (BioRad, USA) at 560 nm (Ashry et al. 2023).

Edwardsiella tarda infection assay

At the end of the feeding trial, 10 healthy fish per replicate from each treatment were challenged with E. tarda infection. The experimental fish was exposed to the bacteria (1 × 108 cfu/mL) via intraperitoneal injection (Lee et al. 2016). The cumulative survival rate of the experimental fish was observed and recorded after four weeks.

Statistical analysis

All data were tested for normality before being analyzed using Statistical Package for Social Sciences (SPSS) version 20.1 (IBM, USA). One-way analysis of variance (ANOVA) was used to determine significant differences among the treatments at p < 0.05, followed by grouping with the Tukey post hoc test. The results were expressed as mean ± standard deviation (SD).

Results

Table 2 demonstrates the growth performance of African catfish after the feeding trial. Dietary KL leaf powder increased the FW, WG, and SGR, with the highest values recorded by the 2% and 3% KL treatment groups (p < 0.05). Furthermore, fish fed with 2% and 3% KL leaf powder exhibited significantly lower FCR values than other groups, while the control group recorded the highest FCR. Meanwhile, the 1% and 4% dietary KL groups demonstrated similar FCR values. Likewise, the HSI and VSI of the 2% and 3% KL groups were the lowest compared to other treatments.

Table 2 Growth performance of experimental fish fed with different percentages of kaffir lime, Citrus hystrix, leaf powder for eight weeks
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The hematological profile of fish fed with 2% and 3% KL was significantly superior (p < 0.05) in WBC, RBC, HGB, and HCT, followed by 1% and 4% (Table 3). Conversely, the control group exhibited significantly lower (p < 0.05) WBC, RBC, HGB, and HCT than other treatments. There were no significant differences in MCH, MCHC, LYM, and MON between the groups in the present study. Dietary KL leaf powder also enhanced digestive enzyme activities significantly (p < 0.05), with the highest activities observed in fish fed with 2% and 3% KL (Figs. 1, 2  and 3), followed by 1% and 4% KL groups. In contrast, the control group exhibited the lowest digestive enzyme activity for amylase, protease, and lipase.

Table 3 Blood parameters of experimental fish fed with different percentages of kaffir lime, Citrus hystrix, leaf powder for 8 weeks
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Fig. 1
figure 1

Amylase enzyme activity of African catfish fed percentage of kaffir lime, Citrus hystrix, leaf powder for 8 weeks. C = control; KL1, 2, 3, and 4 = 1%, 2%, 3%, and 4% of kaffir lime, Citrus hystrix, leaf powder. Values in the same row with different superscript letters indicate significant differences at p < 0.05

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Fig. 2
figure 2

Lipase enzyme activity of African catfish fed percentage of kaffir lime, Citrus hystrix, leaf powder for 8 weeks. C = control; KL1, 2, 3, and 4 = 1%, 2%, 3%, and 4% of kaffir lime, Citrus hystrix, leaf powder. Values in the same row with different superscript letters indicate significant differences at p < 0.05

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Fig. 3
figure 3

Protease enzyme activity of African catfish fed percentage of kaffir lime, Citrus hystrix, leaf powder for 8 weeks. C = control; KL1, 2, 3, and 4 = 1%, 2%, 3%, and 4% of kaffir lime, Citrus hystrix, leaf powder. Values in the same row with different superscript letters indicate significant differences at p < 0.05

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Antioxidant activities (CAT, SOD, and GPx) were significantly higher in the KL-treated groups (p < 0.05) (Fig. 4, 5 and 6). Fish fed with 2% and 3% KL leaf powder demonstrated the highest activities, followed by the 1% and 4% KL treatment groups. The control group recorded the lowest antioxidative responses. In addition, the 2% and 3% KL groups exhibited superior cumulative survival rates post E. tarda infection (Fig. 7) compared to 1% and 4% KL treatments and the control group.

Fig. 4
figure 4

Catalase (CAT) activity of fish fed different percentages of kaffir lime, Citrus hystrix, leaf powder after 8 weeks. C = control; KL1, 2, 3, and 4 = 1%, 2%, 3%, and 4% of kaffir lime, Citrus hystrix, leaf powder

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Fig. 5
figure 5

Superoxide dismutase (SOD) activity of fish fed different percentages of kaffir lime, Citrus hystrix, leaf powder after 8 weeks. C = control; KL1, 2, 3, and 4 = 1%, 2%, 3%, and 4% of kaffir lime, Citrus hystrix, leaf powder

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Fig. 6
figure 6

Glutathione peroxidase (GPx) activity of fish fed different percentages of kaffir lime, Citrus hystrix, leaf powder after 8 weeks. C = control; KL1, 2, 3, and 4 = 1%, 2%, 3%, and 4% of kaffir lime, Citrus hystrix, leaf powder

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Fig. 7
figure 7

Cumulative survival rate of African catfish post-Edwardsiella tarda infection after 4 weeks. C = control; KL1, 2, 3, and 4 = 1%, 2%, 3%, and 4% of kaffir lime, Citrus hystrix, leaf powder

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Discussion

This study evaluated the impacts of KL, Citrus hystrix, leaf powder as a feed additive on the growth performance and health status of African catfish, C. gariepinus. Several assays were conducted in the present study, including a feeding trial (Enis Yonar et al. 2012). Recent research on phytobiotics as a feed additive in aquatic animals revealed promising results in improving the growth and health of aquatic animals. In this study, dietary C. hystrix leaf powder improved the growth and health status of African catfish, C. gariepinus, particularly in FW, WG, and SGR. To the best of our knowledge, this study is the first to report on the effects of KL leaf powder on African catfish. Nonetheless, previous studies have reported the benefits of Citrus products on the growth performance of aquatic animals, particularly Citrus spp. essential oil (EO). For instance, Citrus limon extract (CLE) promoted the growth of Nile tilapia (Oliveira e Silva et al. 2024), limonene and thymol in Nile tilapia (Aanyu et al. 2018), C. sinensis EO in Mozambique tilapia (Acar et al. 2015), bergamot, C. bergamia EO, in European sea bass (Acar et al. 2019), and bitter orange, C. aurantium EO, in juvenile carp (Acar et al. 2021).

The beneficial effects of Citrus spp. on aquatic animals are primarily attributed to the bioactive compounds such as pectin, flavonoids, polysaccharides, and EO (García Beltrán et al. 2017; González-Molina et al. 2010; Macedo et al. 2023). These bioactive compounds are essential in nutrient absorption and promote the protein synthesis of growth factors (Mohamed et al. 2021; Morante et al. 2021; Souza et al. 2023). Moreover, dietary C. hystrix leaf powder significantly enhances fish growth performance and improves digestive enzyme activities, including lipase, amylase, and protease. This positive regulation enhances digestion and amino acid absorption and, subsequently, the fish growth performance (Ashry et al. 2023; Lai et al. 2022). Moreover, C. hystrix leaf powder is a flavoring agent in fish feed to boost their appetite and feed intake. Nevertheless, excess dietary C. hystrix leaf powder may compromise African catfish’s growth performance. C. hystrix leaf powder at 4% reduced the growth performance of African catfish. Similarly, excessive CLE also adversely impacted the growth performance of Nile tilapia (Mohamed et al. 2021).

Dietary C. hystrix leaf powder also decreased HSI and VSI significantly at all inclusion levels, indicating more flesh on the fish’s body. This finding indicated that dietary C. hystrix enhanced lipid metabolism effectively and reduced fat deposition in lipids and viscera (Oliveira e Silva et al. 2024; Weil et al. 2013), possibly due to the presence of flavonoids that are known to exert anti-lipogenic properties (Abdel Rahman et al. 2019). Excessive C. hystrix may trigger an anti-lipogenic effect, resulting in a decline in fish growth performance. Previous studies also reported similar findings (García Beltrán et al. 2017; Macedo et al. 2023; Oliveira e Silva et al. 2024). Therefore, fish farmers should avoid including high doses of C. hystrix leaf powder (4%) in the African catfish diet. Dietary C. hystrix leaf positively regulated the hematological indices of African catfish in this study. The KL treatment groups exhibited significantly higher WBC, HBG, HCT, and RBC. High HBG and HCT indicate that the fish is not anemic with optimal metabolism and nutrient availability in the fish blood (Ashrafizadeh et al. 2020). Furthermore, higher RBC, HCT, and HBG indicate effective hemosynthesis and erythropoiesis activities in the fish body, preventing malnutrition and anemia (Enis Yonar et al. 2012). Meanwhile, there were no significant differences in MCH and MCHC, representing the absence of anemia in the experimental fish (Yonar et al. 2019). Fish fed with C. hystrix leaf have significantly higher WBC, indicating improved health status. However, the WBC levels may be influenced by sex, feeding behavior, season, stress, and pollutants (Ahmed et al. 2020).

Dietary Citrus spp. substantially promoted African catfish’s health, manifested through a higher cumulative survival rate after E. tarda infection. The antioxidative response outcomes in the present study may have contributed to the positive outcomes. Additionally, C. hystrix leaf powder enhanced antioxidative responses, including CAT, SOD, and GPx in KL-treated fish. High antioxidant capacity mitigates stress caused by bacterial infection, resulting in a higher cumulative survival rate in fish supplemented with C. hystrix leaf. Likewise, previous studies reported that Nile tilapia and African catfish that received 1% and 2% lemon peel have higher survival rates after Aeromonas hydrophila infection (Abdel Rahman et al. 2019). In addition, 5% lemon peel EO in the diet of Labeo victorianus can stimulate their disease resistance towards A. hydrophila (Ngugi et al. 2017). Moreover, dietary C. aurantifolia peels at 1.5% and 3% combined with probiotic Bacillus licheniformis protected common carp from A. hydrophila infection (Sadeghi et al. 2021). The dietary tangerine, C. depressa, leaf supplementation in the barramundi diet also promoted their tolerance against A. hydrophila infection (Shiu et al. 2016). On the contrary, dietary C. limon extract did not exert any protective effects in striped catfish against A. hydrophila infection (Macedo et al. 2023).

Conclusion

This study demonstrated that dietary C. hystrix leaf positively impacted African catfish’s growth performance and health status. Their enhanced growth performance can be attributed to the superior digestive enzymes activities, while the disease resistance stimulation is possibly linked to the activation of SOD, CAT, and GPx activities. In summary, 2% or 3% dietary C. hystrix leaf is highly recommended in the African catfish diet to improve their productivity.