RESULTS

Growth performance, feed utilization, and survival

To our knowledge, this is the first report demonstrating that GO outperforms GP in improving carotenoid deposition and immunity-related gene expression in fish. All experimental groups showed survival rates exceeding 96.67% (Supplementary Figure 1C), with no significant differences among dietary treatments (p > 0.05; Table 3). Growth performance, including final body weight, weight gain, and SGR, and feed utilization, including FCR and PER, were not significantly affected by dietary supplementation with GP or GO (Table 3). Nevertheless, the highest weight gain and SGR and the lowest FCR were observed in the GP30 group (p > 0.05; Supplementary Figures 1A and B). Interestingly, dietary supplementation with GP10, GP30, and GO5 significantly affected PPV compared with the control group (p = 0.001). The highest PPV value was recorded in the GP30 group, followed by the GO5 and GP10 groups. No adverse effects, abnormal behavior, external lesions, or treatment-related mortality were observed. Fish in all groups remained healthy and exhibited normal feeding and swimming behavior throughout the experiment.

Parameters | Control | GP10 | GP30 | GO5 | GO10 | p-value
Initial body weight (g/fish) | 9.02 ± 0.00 | 9.02 ± 0.01 | 9.03 ± 0.00 | 9.02 ± 0.01 | 9.02 ± 0.01 | 0.315
Final body weight (g/fish) | 16.65 ± 0.58 | 17.89 ± 1.57 | 18.81 ± 0.82 | 18.65 ± 0.73 | 17.65 ± 0.93 | 0.130
Weight gain (%) | 84.17 ± 6.46 | 98.28 ± 17.47 | 108.38 ± 8.96 | 106.77 ± 8.15 | 95.65 ± 10.32 | 0.133
Specific growth rate (%/day) | 0.88 ± 0.05 | 0.97 ± 0.13 | 1.05 ± 0.06 | 1.04 ± 0.06 | 0.96 ± 0.08 | 0.127
Feed conversion ratio | 3.42 ± 0.21 | 3.30 ± 0.62 | 2.96 ± 0.15 | 3.15 ± 0.12 | 3.29 ± 0.26 | 0.512
Protein efficiency ratio | 0.63 ± 0.02 | 0.73 ± 0.12 | 0.79 ± 0.04 | 0.71 ± 0.03 | 0.69 ± 0.03 | 0.160
Protein productive value (%) | 9.29 ± 0.20ᵇ | 14.47 ± 1.23ᵃ | 15.01 ± 1.45ᵃ | 14.67 ± 2.34ᵃ | 8.67 ± 2.29ᵇ | 0.001
Survival (%) | 96.67 ± 2.89 | 100.00 ± 0.00 | 100.00 ± 0.00 | 98.33 ± 2.83 | 96.67 ± 2.89 | 0.233

Somatic indices and whole-body composition

Dietary supplementation with GP and GO did not significantly affect condition factor, HSI, or VSI in goldfish (p > 0.05; Table 4). Similarly, dietary GP and GO supplementation did not alter dry matter, crude protein, crude fat, or ash content compared with the control group (p > 0.05).

Body coloration

The coloration of goldfish fed different levels of GP and GO is shown in Table 5. Dietary GP and GO supplementation significantly affected the L* value of the head region, with higher values observed in the GO5 and GO10 groups than in the control group (p = 0.015). However, the L* value was not significantly affected by GP10 or GP30 supplementation (p > 0.05). Similarly, the L* value of the abdominal region was significantly increased in the GO10 group, whereas the L* value of the tail region was significantly increased in the GO5 and GO10 groups. The a* value was significantly increased in the head, abdominal, and tail regions of goldfish. The highest a* values were observed in the GO5, GO10, and GO10 groups for the head, abdominal, and tail regions, respectively (p < 0.05). The b* value of the head region differed significantly in goldfish fed GP10, GP30, and GO10 compared with the control group. Similarly, the GP10 group showed a significantly higher b* value in the abdominal region than the control group, whereas the b* value in the tail region was not affected by dietary GP or GO supplementation (p > 0.05).

Items | Control | GP10 | GP30 | GO5 | GO10 | p-value
Somatic indices |  |  |  |  |  | 
Condition factor (g/cm³) | 3.95 ± 0.61 | 4.00 ± 0.88 | 4.21 ± 0.53 | 4.60 ± 1.09 | 4.24 ± 1.14 | 0.677
Hepatosomatic index (%) | 1.26 ± 0.84 | 1.94 ± 0.71 | 1.24 ± 0.56 | 1.24 ± 0.44 | 1.43 ± 0.23 | 0.080
Viscerosomatic index (%) | 11.02 ± 2.04 | 9.76 ± 1.30 | 9.65 ± 0.84 | 10.91 ± 1.41 | 10.94 ± 1.21 | 0.342
Whole-body composition (%) |  |  |  |  |  | 
Dry matter | 29.43 ± 1.69 | 35.07 ± 1.68 | 32.34 ± 1.77 | 33.71 ± 1.21 | 34.34 ± 0.46 | 0.063
Crude protein | 12.23 ± 0.91 | 12.53 ± 0.70 | 13.39 ± 1.01 | 13.18 ± 0.13 | 11.34 ± 0.48 | 0.106
Crude fat | 19.06 ± 0.84 | 19.18 ± 0.27 | 19.35 ± 0.57 | 20.29 ± 0.58 | 19.26 ± 0.40 | 0.728
Ash | 1.88 ± 0.67 | 2.36 ± 0.13 | 2.40 ± 0.20 | 2.14 ± 0.14 | 2.22 ± 0.04 | 0.373

Parameters | Control | GP10 | GP30 | GO5 | GO10 | p-value
Head |  |  |  |  |  | 
Luminosity (L*) | 32.25 ± 2.07ᵇ | 33.44 ± 3.87ᵇ | 37.66 ± 2.08ᵃᵇ | 39.90 ± 1.70ᵃ | 40.12 ± 0.51ᵃ | 0.015
Redness (a*) | 8.75 ± 2.14ᵇ | 17.48 ± 1.52ᵃ | 19.51 ± 1.83ᵃ | 21.31 ± 2.04ᵃ | 18.94 ± 0.50ᵃ | <0.001
Yellowness (b*) | 29.44 ± 1.59ᵇ | 39.35 ± 2.31ᵃ | 35.69 ± 6.42ᵃ | 31.93 ± 1.59ᵃᵇ | 41.38 ± 3.03ᵃ | 0.001
Abdominal region |  |  |  |  |  | 
Luminosity (L*) | 61.73 ± 5.11ᵃᵇ | 55.62 ± 3.36ᵇ | 55.64 ± 1.57ᵇ | 55.38 ± 1.62ᵇ | 64.27 ± 1.97ᵃ | <0.001
Redness (a*) | 8.08 ± 0.88ᶜ | 18.17 ± 0.83ᵃᵇ | 18.79 ± 1.29ᵃᵇ | 16.93 ± 2.31ᵇ | 21.04 ± 0.80ᵃ | <0.001
Yellowness (b*) | 35.49 ± 3.93ᵇᶜ | 47.37 ± 0.87ᵃ | 43.43 ± 2.98ᵃᵇ | 41.41 ± 5.62ᵃᵇᶜ | 31.90 ± 4.05ᶜ | 0.004
Tail |  |  |  |  |  | 
Luminosity (L*) | 37.68 ± 2.70ᵃᵇ | 33.91 ± 0.56ᵇ | 36.26 ± 2.84ᵃᵇ | 43.05 ± 3.92ᵃ | 41.07 ± 1.12ᵃ | 0.009
Redness (a*) | 16.28 ± 1.96ᵇ | 23.64 ± 1.07ᵃ | 22.34 ± 1.13ᵃᵇ | 23.05 ± 1.88ᵃ | 26.03 ± 4.27ᵃ | 0.006
Yellowness (b*) | 32.07 ± 0.97 | 30.96 ± 0.76 | 35.77 ± 4.75 | 33.44 ± 3.49 | 32.25 ± 2.73 | 0.390

Total carotenoid content

The total carotenoid content of goldfish fed different levels of GP and GO is shown in Table 6. Total carotenoid contents in various tissues were affected by dietary GP and GO supplementation. Dietary supplementation with GP and GO increased the total carotenoid content in fins, with the highest level observed in fish fed GP30, followed by the GO10 group (p < 0.05). Fish fed GO-supplemented diets showed significantly higher total carotenoid contents in the muscle, liver, and serum than fish fed GP-supplemented diets and the control diet (p < 0.05).

Items | Control | GP10 | GP30 | GO5 | GO10 | p-value
Fin (µg/g) | 168.70 ± 6.75ᶜ | 157.15 ± 5.51ᶜ | 206.39 ± 10.66ᵃ | 173.25 ± 10.01ᵇᶜ | 195.82 ± 14.11ᵃᵇ | 0.001
Muscle (µg/g) | 53.63 ± 1.49ᶜ | 51.97 ± 1.81ᶜ | 53.92 ± 0.88ᶜ | 61.95 ± 1.27ᵇ | 72.88 ± 1.79ᵃ | <0.001
Skin (µg/g) | 152.25 ± 14.20ᵇ | 152.25 ± 10.96ᵇ | 163.45 ± 13.03ᵇ | 254.10 ± 30.26ᵃ | 280.00 ± 16.07ᵃ | 0.026
Liver (µg/g) | 20.62 ± 1.99ᵈ | 21.49 ± 1.19ᵈ | 31.99 ± 2.24ᶜ | 37.45 ± 2.26ᵇ | 52.43 ± 4.12ᵃ | <0.001
Serum (µg/mL) | 1.35 ± 0.63ᵇ | 1.55 ± 0.24ᵃᵇ | 1.72 ± 0.23ᵃᵇ | 1.75 ± 0.24ᵃᵇ | 1.95 ± 0.20ᵃ | 0.019

Hematological and biochemical parameters

RBC and Hb levels increased significantly with increasing dietary GP and GO levels. Dietary supplementation with both GP and GO resulted in significantly higher RBC and Hb levels (Table 7). Dietary inclusion of GO also significantly increased WBC levels (p < 0.05); however, Hct did not differ significantly among dietary treatments (p > 0.05). Total protein, albumin, globulin, albumin:globulin ratio, ALT, ALP, and LDL-c levels did not differ significantly in response to dietary supplementation with GP or GO (p > 0.05). AST levels were affected by dietary GP and GO supplementation; specifically, AST levels were markedly lower in GO-supplemented groups than in the GP and control groups (p < 0.05). The results also showed reduced total cholesterol and triglyceride levels, with the lowest values observed in the GO10 group (p < 0.05). In addition, HDL-c levels were significantly increased in fish fed GO5 and GO10 diets (p < 0.05).

Blood parameters | Control | GP10 | GP30 | GO5 | GO10 | p-value
Hematological parameters |  |  |  |  |  | 
Red blood cells (×10⁶ cells/µL) | 9.99 ± 0.84ᶜ | 14.03 ± 0.67ᵇ | 14.81 ± 1.48ᵇ | 18.25 ± 0.73ᵃ | 20.03 ± 1.76ᵃ | <0.001
White blood cells (×10³ cells/µL) | 13.73 ± 1.27ᵇ | 15.09 ± 2.87ᵃᵇ | 20.88 ± 5.22ᵃᵇ | 21.85 ± 2.32ᵃ | 22.96 ± 1.49ᵃ | 0.011
Hematocrit (%) | 31.67 ± 2.98 | 37.22 ± 4.86 | 37.66 ± 5.75 | 39.00 ± 6.40 | 38.55 ± 8.80 | 0.306
Hemoglobin (g/dL) | 12.98 ± 1.37ᶜ | 14.61 ± 0.73ᵇᶜ | 15.72 ± 1.19ᵃᵇ | 17.26 ± 1.06ᵃ | 16.15 ± 0.89ᵃᵇ | <0.001
Biochemical parameters |  |  |  |  |  | 
Total protein (g/dL) | 2.97 ± 0.23 | 2.91 ± 0.00 | 2.88 ± 0.23 | 3.16 ± 0.36 | 3.16 ± 0.37 | 0.744
Albumin (g/dL) | 1.16 ± 0.08 | 1.17 ± 0.06 | 1.23 ± 0.06 | 1.36 ± 0.16 | 1.28 ± 0.08 | 0.335
Globulin (g/dL) | 1.81 ± 0.16 | 1.74 ± 0.06 | 1.64 ± 0.17 | 1.80 ± 0.19 | 1.88 ± 0.28 | 0.784
Albumin:globulin ratio | 0.64 ± 0.01 | 0.66 ± 0.06 | 0.75 ± 0.04 | 0.75 ± 0.00 | 0.68 ± 0.05 | 0.151
Aspartate transaminase (U/L) | 60.61 ± 9.05ᵃ | 33.26 ± 4.45ᵇ | 33.69 ± 5.40ᵇ | 27.55 ± 1.34ᵇ | 27.50 ± 3.33ᵇ | 0.007
Alanine transaminase (U/L) | 16.56 ± 6.45 | 18.71 ± 4.90 | 18.66 ± 2.04 | 13.64 ± 2.12 | 17.63 ± 0.63 | 0.687
Alkaline phosphatase (U/L) | 29.62 ± 1.95 | 32.27 ± 2.91 | 31.17 ± 2.76 | 23.74 ± 2.36 | 30.61 ± 2.14 | 0.092
Cholesterol (mg/dL) | 326.11 ± 5.75ᵃ | 300.60 ± 6.36ᵇ | 310.12 ± 4.21ᵃᵇ | 301.07 ± 8.58ᵇ | 264.96 ± 4.07ᶜ | 0.013
Triglycerides (mg/dL) | 198.10 ± 14.01ᵃ | 189.67 ± 6.41ᵃ | 164.67 ± 23.43ᵃᵇ | 174.45 ± 22.58ᵃᵇ | 122.33 ± 7.28ᵇ | 0.033
High-density lipoprotein cholesterol (mg/dL) | 57.04 ± 3.73ᵈ | 67.30 ± 5.79ᶜ | 75.28 ± 4.43ᵇ | 85.24 ± 8.49ᵃ | 88.31 ± 1.63ᵃ | 0.043
Low-density lipoprotein cholesterol (mg/dL) | 204.59 ± 6.48 | 195.81 ± 0.57 | 202.18 ± 4.29 | 206.14 ± 15.54 | 193.02 ± 7.17 | 0.552

Immunity-related gene expression

Compared with the control group, all GP- and GO-supplemented groups exhibited significantly higher expression levels of _IL-1_β, IL-10, and lysozyme (p < 0.05; Figures 1B, C, and E). The highest expression levels of IL-10 and lysozyme transcripts were observed in the GO10 group, showing a 24.5-fold increase compared with the control group. The GO5 and GO10 groups showed higher _IL-1_β expression than all other groups (p < 0.05). However, HSP-70 expression was not altered by dietary supplementation with GP or GO (p > 0.05; Figure 1D). The expression of TNF-α mRNA was upregulated in the GP30, GO5, and GO10 groups (p < 0.05), with 6-, 7-, and 18-fold increases, respectively, compared with the control group; however, no change was observed in the GP10 group (p > 0.05; Figure 1A).

Figure 1: (A) Relative expression of tumor necrosis factor-alpha. (B) Relative expression of interleukin-1β. (C) Relative expression of interleukin-10. (D) Relative expression of heat shock protein-70. (E) Relative expression of lysozyme in the liver of goldfish fed diets supplemented with gac aril powder and gac aril oil. Data are presented as mean ± SD (n = 3), representing three biological replicates per treatment. Different lowercase letters indicate significant differences among treatments (p < 0.05).

Liver histology

The liver of fish fed the control diet showed normal architecture, with polyhedral mononucleated hepatocytes containing centrally located nuclei and heterochromatin distributed centrally and peripherally (Figure 2A). A semi-quantitative score of 0, indicating normal liver histology, was assigned to the control group. Similarly, the GP and GO groups showed no significant histological differences compared with the control group. No inflammatory, degenerative, or enlarged vacuolar changes were observed in the examined liver sections (Figures 2B–D). The GP and GO groups also showed a semi-quantitative score of 0, indicating normal liver histology.

Figure 2: Representative liver histology of goldfish fed diets supplemented with different levels of gac aril powder (GP) and gac aril oil (GO) for 10 weeks (hematoxylin and eosin staining; magnification ×40; scale bar = 50 µm). (A) Control diet without GP or GO supplementation. (B) Diet supplemented with 10 g/kg GP. (C) Diet supplemented with 30 g/kg GP. (D) Diet supplemented with 5 g/kg GO. (E) Diet supplemented with 10 g/kg GO. Black arrowheads indicate hepatocytes with centrally located nuclei. No inflammatory infiltration, hepatocellular degeneration, or vacuolar enlargement was observed in any treatment group.