DISCUSSION

Principal findings

The present study demonstrates the efficacy of GP and, particularly, GO as functional feed additives in fish. Unlike previous investigations in terrestrial animals, this study provides the first evidence in an aquatic species that dietary GO supplementation at 10 g/kg can simultaneously improve pigmentation, carotenoid bioavailability, hematological profile, lipid metabolism, and expression of key immunity-related genes in goldfish. Natural herbs and their extracts have shown substantial benefits in fish nutrition by acting as antioxidants, promoting growth, stimulating appetite, and functioning as immunostimulants [2, 23]. Although several studies have reported the diverse biological functions of gac fruit, including antioxidant, anticancer, anti-inflammatory, antimicrobial, and immunomodulatory properties [23, 37], the specific effects of dietary supplementation with gac aril in fish remain poorly understood. In this study, we evaluated the dietary application of gac aril in powder and oil forms. The results showed that supplementation with GP and GO improved PPV, coloration, total carotenoid deposition, and the expression of immunity-related genes in goldfish. These effects may be attributed to the combined action of the diverse bioactive compounds present in gac arils.

Growth performance and feed utilization

Growth improvement is an important trait in aquaculture because it is closely associated with productivity and profitability [38]. Several studies have reported positive effects of dietary plants or plant extracts on growth parameters in aquatic species [39, 40]. In the present study, increasing dietary levels of GP and GO did not significantly affect final body weight, weight gain, SGR, FCR, PER, somatic indices, or whole-body composition. However, no adverse effects were observed on growth performance, feed utilization, somatic indices, or whole-body composition. Regarding growth and feed utilization, the findings indicated that 30 g/kg GP was associated with the highest growth response and lowest FCR. In addition, dietary supplementation with GP and GO significantly increased PPV compared with the control group. Although data on dietary gac aril supplementation in fish are limited, the improvement in PPV may be attributed to the enhanced nutritional contribution of GP and GO. Similarly, dietary GP supplementation at 0.2–10 g/kg in chickens had no significant effect on growth or feed utilization [26, 41]. Several carotenoid-rich additives, including turmeric at 2 g/kg, red pepper at 2–3 g/kg, and hericium extract at 1 g/kg, have been reported to improve pigmentation and health status in ornamental goldfish [23, 37, 42].

Pigmentation and carotenoid deposition

Natural pigmentation is critical in ornamental fish production because it strongly influences consumer preference and market value [21, 22]. Because fish cannot synthesize carotenoids de novo, they must obtain these pigments from dietary sources to maintain natural skin coloration, which is an essential quality attribute in high-value ornamental species [21, 22]. In the present study, goldfish fed GP- or GO-supplemented diets showed improved coloration, particularly higher L* and a* values in the head, abdominal, and tail regions compared with the control group. Furthermore, dietary GP and GO increased total carotenoid content in several tissues and increased serum carotenoid levels. Both GP and GO are rich sources of β-carotene, lycopene, lutein, and zeaxanthin (Table 8), although carotenoid contents were generally higher in GO than in GP (Table 8). Goldfish require more than 13 carotenoids, including β-cryptoxanthin, lutein, zeaxanthin, diatoxanthin, alloxanthin, and astaxanthin, for coloration [43, 44]. Therefore, the carotenoids present in GP and GO likely contributed to the enhanced coloration (Tables 5 and 6) and higher total carotenoid levels compared with the control group. To date, only one report has evaluated gac aril for coloration in terrestrial animals, showing that dietary supplementation with 10 g/kg GP enhanced egg yolk color in laying hens [26]. The present study introduces GO as a novel and highly effective natural pigmenting agent for ornamental fish, a finding not previously reported.

Items (mg/kg) | GP | GO
β-carotene | 98.34 | 194.64
Lycopene | 65.67 | 401.16
Lutein | 21.37 | 66.32
Zeaxanthin | 3.44 | 8.16
Astaxanthin | N.D. | N.D.
Flavonoids | 281.8 | 702.1

Hematological responses

Medicinal plants can positively influence fish hematological indices, although the magnitude of response varies among plant sources and dietary inclusion levels. Hematological indices are important indicators for evaluating health status, stress response, and disease condition in fish [45]. Previous studies have demonstrated a direct association between RBC and WBC counts and immune response in fish, and elevations in these indices are frequently observed after dietary administration of natural immunostimulants [45, 46]. Gac aril contains several active compounds with antioxidant, antibacterial, and immunomodulatory properties [47, 48], which may partly explain the increased WBC count observed in the present study. RBCs act as carriers of Hb, which facilitates oxygen transport, and tissue oxygen delivery depends on RBC maturity and Hb concentration. In this study, dietary supplementation with GP and GO increased RBC, WBC, and Hb levels in a dose-dependent manner in goldfish (p < 0.05). A similar tendency was observed for Hct, although the difference was not significant (p > 0.05), consistent with other studies evaluating medicinal plant supplementation in fish [49, 50]. The improvement in hematological parameters may be related to increased availability of vitamin E, carotenoids, calcium, and iron in the circulatory system [47, 51]. Humans fed GP showed higher Hb content than the control group [14]. Similarly, the high vitamin A content of gac aril has beneficial effects on iron status in rats. Increasing GP levels, accompanied by higher vitamin A intake, may positively affect erythropoiesis and iron mobilization in other vertebrates [52]. However, these mechanisms require further investigation in fish.

Serum biochemical responses and lipid metabolism

Blood biochemical profiles provide useful information when medicinal plants are incorporated into fish diets. These profiles help assess animal health and monitor metabolic disturbances and diseases caused by biotic and abiotic factors [46]. Albumin, globulin, and the albumin:globulin ratio are components of total serum protein and play important biological roles in fish. In the present study, dietary supplementation with GP and GO did not affect these parameters, indicating that normal biological function was maintained in goldfish. Consistent with these findings, several plant powders or extracts, including Coriandrum sativum [53], _Stachys _lavandulifolia Vahl [54], Rosa canina, and Carthamus tinctorius [55], did not alter serum protein parameters.

Serum AST, ALT, and ALP activities are commonly used as biomarkers in fish because they can indicate disease status and liver injury. In this study, goldfish fed GP and GO diets showed significantly reduced AST levels, whereas ALT and ALP levels did not differ significantly among groups. This result suggests that GP and GO supplementation may have a favorable effect on the health status of goldfish. Previous studies in other vertebrates have shown that gac arils may help alleviate liver disorders [56]. Furthermore, dietary GP and GO supplementation significantly reduced total cholesterol and triglyceride levels, with the lowest values observed in the GO10 group, and increased HDL-c levels in GO-supplemented fish. A previous study demonstrated that dietary gac aril significantly decreased plasma cholesterol, LDL-c, and triglyceride contents in rats [57]. Lin et al. [58] reported that mice fed lyophilized gac aril for 10 weeks showed reduced hepatic triglyceride and cholesterol levels. These findings suggest that GP and GO may exert lipid-lowering effects and indicate potential hypolipidemic activity in goldfish.

Immunity-related gene expression

Inflammatory responses triggered by dietary stimuli can affect tissues and cells. These responses are regulated by cytokines, including pro-inflammatory cytokines, such as TNF-α, _IL-1_β, IL-6, and IL-8, and anti-inflammatory cytokines, such as IL-10 and TGF-β [59, 60]. The pro-inflammatory response is essential for pathogen control, with cytokines regulating innate immunity [60]. As multifunctional regulatory cytokines, IL-10 and _TGF-_β have anti-inflammatory properties that suppress the production of various cytokines and reduce the adverse effects of pro-inflammatory responses, such as excessive reactive oxygen or nitric oxide production during phagocytosis [61]. Therefore, many studies have suggested that evaluating cytokine expression levels may help predict changes in immune responses [60, 61].

In this study, TNF-α and _IL-1_β expression levels were significantly higher in the GP30 and GO groups than in the control group. These findings suggest that GP and GO intake may activate immune responses and improve the ability of fish to respond to pathogen invasion [62, 63]. This effect is likely associated with bioactive compounds present in gac arils, including phenolics, flavonoids, and carotenoids, which may promote the expression of TNF-α, _IL-1_β, and IL-10 mRNA. Previous studies have shown that these bioactive compounds can upregulate the expression of immune-related genes [64–66]. In addition, carotenoids serve as precursors of vitamin A, and carotenoid deficiency negatively affects antioxidant responses and other protective health mechanisms in fish [67]. Dietary supplementation with flavonoids or carotenoids has also been shown to enhance antioxidant and anti-inflammatory capacity in several fish species [68–70].

Lysozyme and innate immunity

Granulocyte-secreted lysozyme is an important immune component that enhances innate immunity by degrading bacterial peptidoglycans and activating the complement system, phagocytic cells, and other immune responses [71]. In the present study, lysozyme mRNA expression was upregulated in fish fed GP- and GO-supplemented diets compared with the control group. This upregulation suggests a positive effect on innate immune responses. Tahir et al. [72] reported that dietary lycopene supplementation significantly increased lysozyme activity in common carp (Cyprinus carpio) compared with the control group. Similarly, Mousavi et al. [73] reported increased lysozyme activity in red fantail male guppy (Poecilia reticulata) after supplementation with carotenoid-rich herbs. In addition, dietary enrichment with β-carotene improved non-specific immune responses in goldfish, as demonstrated by increased total immunoglobulin and lysozyme activity [74]. Recent studies have also shown that flavonoid-rich plants can significantly enhance lysozyme activity and mRNA expression levels in various aquatic species [75–77]. Based on these findings, dietary supplementation with GP and GO may stimulate immune- and cytokine-related gene expression, thereby improving the health status of goldfish. However, pathogen challenge trials are required to confirm the protective efficacy of GO supplementation and substantiate its immunostimulatory effects in ornamental fish. The differential modulation of pro- and anti-inflammatory cytokines and lysozyme gene expression by GP and GO provides new information regarding the immunomodulatory potential of gac-derived products in fish, extending beyond the antioxidant and anti-inflammatory effects previously documented in mammals.

HSP-70 expression and stress response

HSP-70 is a stress-responsive gene associated with stressors such as heat, oxidative stress, starvation, high stocking density, and pollutant exposure [78]. Therefore, under normal rearing conditions, dietary gac supplementation may not markedly affect HSP-70 expression. Gac is rich in antioxidant compounds, including carotenoids, lycopene, and flavonoids, which help maintain cellular homeostasis and reduce oxidative stress. Consequently, the HSP-70-related stress response pathway may not have been activated in the present study. Similar findings have been reported in fish maintained under non-stress conditions or with adequate antioxidant protection [79]. Previous studies have shown that HSP-70 expression increases under stress conditions such as temperature fluctuation, handling, heavy metal exposure, starvation, and high stocking density [78].

Liver histology and safety evaluation

The liver is the largest internal organ in fish and plays a central role in metabolism. It is also a major target organ affected by biological and environmental factors, including feed, pollutants, toxins, parasites, and microorganisms [80]. The microstructural morphology of hepatocytes can directly reflect nutrient absorption and abnormal growth and is therefore useful for assessing fish nutritional status [81]. In this study, dietary supplementation with GP and GO did not alter liver histology (Figures 2A–D). This finding was further supported by unchanged HSI and favorable AST and ALT responses, suggesting that GP and GO did not exert harmful effects on hepatocytes. Studies in terrestrial species have shown that gac aril contains higher lycopene levels than many other fruits and contains antioxidant compounds that may protect tissues, including the liver, colon, and breast, during cancer development [82, 83]. Importantly, this study is the first to confirm the safety of gac aril products on liver morphology in fish, supporting their potential as safe and sustainable feed additives.