3. Results

3.1. Dietary Supplementation With OLE Improved Growth Performance and Reduced Lipid Accumulation in T. ovatus Fed HFD

After the 8‐week feeding trial, there were significant impacts on final weight, WG, SGR, and SR as dietary OLE levels increased from 0% to 2.0% (Table 2). Final weight, WG, and SGR showed significant quadratic responses (p < 0.05) being significantly increased and then decreased with increasing levels of OLE in the diet, with 1.0% OLE shown to be the optimal concentration. Similarly, SR increased with increasing dietary OLE up to 1.0%, and then decreased as the level of dietary OLE increased further, displaying a significant quadratic response (p < 0.05). Although FCR, CF, HSI, and VSI all showed no significant differences with OLE supplementation, feed efficiency showed an improving trend up to 1.0%, while HSI showed a decreasing trend with dietary OLE supplementation.

Parameters | Dietary OLE level (%) | Pooled SEM | p value
0.0 | 0.5 | 1.0 | 1.5 | 2.0 | ANOVA | Linear | Quadratic
Initial weight (g) | 11.48 | 11.59 | 11.71 | 11.54 | 11.54 | 0.126 | 0.732 | 0.890 | 0.288
Final weight (g) | 60.81bc | 68.20b | 79.57a | 57.74c | 58.65c | 1.844 | <0.001 | 0.030 | <0.001
WG (%) | 429.56bc | 488.50b | 579.35a | 400.48c | 408.79c | 15.807 | <0.001 | 0.027 | <0.001
SGR (% day−1) | 2.98bc | 3.16b | 3.42a | 2.87c | 2.90c | 0.050 | <0.001 | 0.019 | <0.001
FCR | 1.91 | 1.79 | 1.68 | 1.73 | 1.80 | 0.063 | 0.214 | 0.211 | 0.050
SR (%) | 68.33b | 85.00a | 86.67a | 78.33ab | 80.00ab | 3.162 | 0.016 | 0.127 | 0.007
HSI (%) | 2.39 | 2.01 | 2.11 | 2.06 | 2.00 | 0.190 | 0.195 | 0.515 | 0.424
VSI (%) | 8.67 | 9.15 | 8.91 | 8.38 | 8.65 | 0.390 | 0.268 | 0.185 | 0.164
CF (gcm−3) | 3.31 | 3.17 | 3.12 | 3.12 | 3.28 | 0.237 | 0.961 | 0.477 | 0.924

The lipid content of the whole body decreased and then increased with increasing dietary OLE, with 1.0% OLE showing the lowest lipid content, whereas 1.5% OLE did not show any reduction in whole‐body lipid content (Table 3). Levels of TG and TC in serum decreased significantly as dietary OLE supplementation level increased up to 1.0% and then decreased with a further increased level of OLE with obvious linear and quadratic trends (p < 0.05) (Table 4). The levels of HDLC increased with 1.0% dietary OLE (p < 0.05), and then decreased with the further increased OLE addition, while LDLC showed the opposite trend. (Table 4). In addition, total lipid content of liver (% dry weight) also exhibited a significant downward trend as dietary OLE level increased to 1.0% (p < 0.05) (Figure 1A). Moreover, liver contents of total saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs), especially 16:0, 18:0, and 18:1n‐9, showed trends of first increasing and then decreasing, with the critical point being 1.0% OLE (Figure 1B, C). The proportions of long‐chain polyunsaturated fatty acids (LC‐PUFAs), including 20:4n‐6, 20:5n‐3, and 22:6n‐3 showed similar trends (Table S2). However, the proportion of the total PUFA, especially 18:2n‐6, showed the opposite trend (Figure 1B C). ORO staining of liver sections showed that the range of lipid droplet size was the least and lipid droplets were significantly smaller, reduced in number, and distributed more evenly with dietary supplementation with OLE of 1.0% (p < 0.05) (Figure 2).

Figure 1: Lipid content and fatty acid composition of liver of Trachinotus ovatus fed high‐fat diets with different levels of olive leaf extract (OLE) for 8 weeks. (A) Lipid content of liver (% dry weight). (B) Composition (percentage of total fatty acids) of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs), and others (peaks of unknown identity) in liver. (C) The principle individual fatty acids (percentage of total fatty acids) in liver. Bars with different superscript lowercase letters in Figure 1A are significantly different (p < 0.05) as determined by one‐way ANOVA.

Figure 2: Histological examination of liver of Trachinotus ovatus fed high‐fat diets with different levels of olive leaf extract (OLE) for 8 weeks. (A) Sections of liver stained with oil red O (scale bar, 100 μm). (B) Quantitation of the relative area (percentage) of lipid droplets (stained red) in hepatic sections. Values are presented as means ± SEM (n = 3). Bars with different superscript lowercase letters denote significant differences (p < 0.05) as determined by one‐way ANOVA.

Parameters | Dietary OLE level (%) | Pooled SEM | p value
0.0 | 0.5 | 1.0 | 1.5 | 2.0 | ANOVA | Linear | Quadratic
Whole body
Moisture | 69.55 | 67.98 | 70.66 | 67.71 | 70.34 | 1.127 | 0.296 | 0.721 | 0.525
Protein | 15.60 | 16.93 | 15.87 | 17.19 | 15.54 | 0.565 | 0.190 | 0.940 | 0.121
Lipid | 10.72 | 10.68 | 9.13 | 10.80 | 10.17 | 0.378 | 0.052 | 0.433 | 0.179
Ash | 3.30 | 4.09 | 3.33 | 3.57 | 3.20 | 0.238 | 0.135 | 0.359 | 0.174

Parameters | Dietary OLE level (%) | Pooled SEM | p value
0.0 | 0.5 | 1.0 | 1.5 | 2.0 | ANOVA | Linear | Quadratic
TG (mmol L−1) | 2.87a | 1.25c | 1.12c | 2.05b | 2.09b | 0.070 | <0.001 | 0.007 | <0.001
TC (mmol L−1) | 4.56a | 3.70b | 2.31d | 3.29c | 3.14c | 0.055 | <0.001 | <0.001 | <0.001
HDLC (mmol L−1) | 1.20b | 1.47ab | 1.70a | 1.39ab | 1.59ab | 0.102 | 0.047 | 0.055 | 0.102
LDLC (mmol L−1) | 1.14a | 1.01a | 0.47b | 0.66b | 0.65b | 0.046 | <0.001 | <0.001 | <0.001
AKP (U 100 mL−1) | 2.32c | 2.68bc | 3.91a | 3.16b | 2.68bc | 0.151 | <0.001 | 0.030 | <0.001
ALT (U L−1) | 40.19a | 21.95b | 25.36b | 37.69a | 47.60a | 2.329 | <0.001 | 0.002 | <0.001
AST (U L−1) | 30.03a | 23.37b | 19.94b | 22.63b | 32.72a | 1.417 | <0.001 | 0.326 | <0.001
MDA (nmol mL−1) | 5.54a | 2.34c | 2.79bc | 2.34c | 4.10ab | 0.349 | <0.001 | 0.026 | <0.001
SOD (U mL−1) | 12.18b | 15.16ab | 17.08a | 15.76ab | 15.82ab | 1.022 | 0.062 | 0.035 | 0.039
TAOC (U mL‐1) | 23.35b | 28.14ab | 32.93a | 29.87ab | 26.85ab | 1.642 | 0.022 | 0.124 | 0.003

3.2. Dietary OLE Supplementation Reduced Hepatic Lipid Deposition by Inhibiting Lipid Synthesis and Promoting Lipid Catabolism and Transport in T. ovatus Fed HFD

Relative mRNA expression levels of genes related to lipid biosynthesis, including sterol regulatory element binding protein 1 (srebp1), acetyl‐CoA carboxylase (acc) and fatty acid synthase (fas), in liver decreased significantly when 0.5%–1.0% OLE was added to the diet compared to fish fed the control diet (0% OLE) (p < 0.05), but then increased significantly as dietary OLE level exceeded 1.0% (p < 0.05) (Figure 3A–C). The relative mRNA expression of diacylgycerol acyltransferase 1 (dgat1) also showed a similar trend, albeit not statistically significant (Figure 3D). Conversely, mRNA expression levels of genes of lipid catabolism, including peroxisome proliferator‐activated receptor α (pparα), carnitine palmitoyl transferase‐1 (cpt1), and hormone‐sensitive lipase (hsl), and lipid transport, including CD36 molecule (cd36) and fatty acid binding protein 1 (fabp1), increased significantly with dietary supplementation of OLE, particularly 0.5%–1.0%, compared with the control diet without OLE (p < 0.05) (Figure 3E–I).

Figure 3: Relative expression of genes related to lipid metabolism in liver of Trachinotus ovatus fed high‐fat diets with different levels of olive leaf extract (OLE) for 8 weeks. (A–D) mRNA expression levels of genes related to lipid biosynthesis. (E–G) mRNA expression levels of genes related to lipid catabolism. (H, I) mRNA expression levels of genes related to lipid transport. Values are presented as means ± SEM, n = 3. Bars with different superscript lowercase letters are significantly different (p < 0.05) as determined by one‐way ANOVA. acc, acetyl‐CoA carboxylase; cd36, CD36 molecule; cpt1, carnitine palmitoyl transferase‐1; dgat1, diacylgycerol acyltransferase 1; fabp1, fatty acid binding protein 1; fas, fatty acid synthase; hsl, hormone‐sensitive lipase; pparα, peroxisome proliferator‐activated receptor α; srebp1, sterol regulatory element binding protein‐1; β-actin, actin beta.

3.3. Dietary OLE Supplementation Improved Liver Health and Immune and Antioxidant Capacities of T. ovatus Fed HFD

Serum activities of ALT and AST displayed a significant quadratic trend (p < 0.05). Liver health was improved by supplementation of 0.5%–1.0% OLE to the diet, as indicated by significantly lower activities of ALT and AST compared to fish fed the diet without OLE (p < 0.05) (Table 4). However, the activities of ALT and AST tended to rise as the dietary level of OLE increased further (Table 4). On the contrary, serum AKP activity showed a significant quadratic trend (p < 0.05) and increased significantly with dietary supplementation of OLE up to 1.0%, but tended to decrease as dietary OLE increased further. Regarding antioxidant capacity, SOD activity, TAOC, and MDA contents of serum all showed significant quadratic trends. Thus, SOD and TAOC increased as the level of dietary OLE increased to 1.0%, and then decreased as OLE supplementation increased further (p < 0.05), while MDA decreased up to 1.5% OLE before it increased at 2.0% OLE (p < 0.05) (Table 4).

3.4. Dietary OLE Supplementation Alleviated Intestinal Inflammation and Damage by Inhibiting the Expression of Inflammatory Factors in T. ovatus Fed HFD

Histological analysis of the intestine showed that dietary OLE improved the integrity and increased the length of intestinal microvilli of T. ovatus (Figure 4). Moreover, the addition of OLE to the diet reduced the mRNA expression levels of proinflammatory factors in the intestine, including interleukin‐1 (il-1) and il-8 (p < 0.05) (Figure 5A, B). The expression of another proinflammatory factor, tumor necrosis factor α (tnfα), also showed a downward trend in the intestine of T. ovatus fed HFD diet supplemented with OLE (Figure 5C). Conversely, the mRNA expression levels of the intestinal epithelial barrier‐related genes, occludin (ocln) and cadherin 1 (cdh1), were upregulated in the intestine in fish fed diets including 1.0%–1.5% OLE (p < 0.05) (Figure 5D, E), while dietary OLE did not affect the expression of another intestinal epithelial barrier gene, zonula occludens protein 1 (zo-1) (Figure 5F).

Figure 4: Morphology of the anterior intestine of Trachinotus ovatus fed high‐fat diets with different levels of olive leaf extract (OLE) for 8 weeks. (A) The entire section (left; scale bar, 1000 μm) and part section (right; scale bar, 200 μm) of the anterior gut of fish fed diets supplemented with 0.0%, 0.5%, 1.0%, 1.5% and 2.0% OLE, respectively. The location of the black lines, line segments with arrows at both ends and single ended arrows point to wall thickness, villus length and goblet cell, respectively. (B–D) The anterior gut morphology parameters of Trachinotus ovatus. Data shown as means ± SEM of three replicates. Bars in the same subfigure with different superscript lowercase letters are significantly different (p < 0.05) as determined by one‐way ANOVA.

Figure 5: Relative expression of genes related to inflammatory responses and intestinal barrier function in intestine of Trachinotus ovatus fed high‐fat diets with different levels of olive leaf extract (OLE) for 8 weeks. (A–C) mRNA expression levels of genes related to inflammatory responses. (D–F) mRNA expression levels of genes related to intestinal barrier function. Values are presented as means ± SEM, n = 3. Bars with different superscript lowercase letters are significantly different (p < 0.05) as determined by one‐way ANOVA. cdh1, cadherin 1; il-1, interleukin‐1; il-8, interleukin‐8; ocln, occludin; tnfα, tumor necrosis factor alpha; zo-1, zonula occludens‐1; β-actin, actin beta.

3.5. Dietary OLE Supplementation Reduced the Absorption of Lipid by the Intestine and Altered the Composition of Intestinal Microbiota in T. ovatus Fed HFD

As shown in Table 5, the activity of lipase decreased in the intestine of T. ovatus fed HFD with 0.5%–1.5% OLE and then significantly increased with the addition of OLE up to 2.0% (p < 0.05). The addition of 0.5%–1.0% OLE to the HFD diet enhanced the activity of intestinal trypsin, but activity decreased significantly with the addition of more than 1.5% (p < 0.05). Dietary OLE did not affect amylase activity in the intestine of T. ovatus (p > 0.05).

Parameters | Dietary OLE level (%) | Pooled SEM | p value
0.0 | 0.5 | 1.0 | 1.5 | 2.0 | ANOVA | Linear | Quadratic
Lipase | 2.51ab | 1.47bc | 1.49bc | 1.30b | 2.69a | 0.227 | 0.003 | 0.799 | <0.001
Amylase | 0.14 | 0.13 | 0.18 | 0.15 | 0.09 | 0.031 | 0.473 | 0.433 | 0.189
Trypsin | 621.78ab | 649.66ab | 683.08a | 382.70ab | 314.86b | 78.757 | 0.023 | 0.005 | 0.105

Alpha diversity analysis of intestinal flora showed that the coverage of all samples exceeded 98% with no significant differences among the dietary groups (p > 0.05) (Table 6). Orthogonal polynomial contrast analysis revealed significant linear and quadratic trends in Chao1, ACE, Simpson, and Shannon as the level of dietary OLE increased (p < 0.05). Although the Chao1 index was more variable, it was significantly lowest in T. ovatus fed the diet with 2.0% OLE (p < 0.05). Compared to fish fed HFD without the addition of OLE, the ACE index tended to reduce with dietary supplementation with OLE and was lowest with the highest level of supplementation of 2.0% (p < 0.05). On the contrary, the Shannon and Simpson indices both increased significantly in T. ovatus fed OLE‐supplemented diets compared to fish fed the unsupplemented diet, with the highest levels obtained in T. ovatus fed diets with 0.5%–1.0% OLE. Principal coordinate analysis (PCoA) of intestinal microbiota showed that there were clear differences in microbiota clustering between T. ovatus fed diets with and without OLE, but especially between fish fed the diet supplemented with 2.0% OLE and the other dietary groups (Figure 6).

Figure 6: Two‐dimensional principal coordinate analysis (PCoA), using a binary Jaccard distance matrix, of the intestinal bacterial communities of Trachinotus ovatus fed high‐fat diets with different levels of olive leaf extract (OLE) for 8 weeks.

Parameters | Dietary OLE level (%) | Pooled SEM | p value
0.0 | 0.5 | 1.0 | 1.5 | 2.0 | ANOVA | Linear | Quadratic
Feature | 395.00ab | 598.00a | 549.50a | 647.33a | 226.25b | 67.010 | 0.001 | 0.187 | <0.001
ACE | 829.91a | 578.40ab | 517.58bc | 672.65ab | 273.05c | 69.207 | 0.001 | <0.001 | <0.001
Chao1 | 659.26ab | 690.57ab | 580.12ab | 756.05a | 309.31b | 49.456 | <0.001 | 0.001 | 0.017
Simpson | 0.70b | 0.97a | 0.96a | 0.93a | 0.89a | 0.030 | <0.001 | 0.020 | <0.001
Shannon | 3.48c | 7.11a | 6.93a | 6.41ab | 5.48b | 0.350 | <0.001 | 0.006 | <0.001
Coverage | 0.98 | 0.99 | 0.99 | 0.98 | 0.99 | 0.004 | 0.226 | 0.144 | 0.865

At the phylum level, the predominant phyla in overall relative abundance in fish fed HFD without OLE were Proteobacteria, Firmicutes, and Bacteroidetes, with unclassified bacteria also being a major group (Figure 7A). The abundance of Proteobacteria and Actinobacteriota increased progressively with increasing dietary supplementation of dietary OLE, while the abundance of Bacteriodetes and Acidobacteriota showed a trend of first increasing and then decreasing as dietary OLE increased (Figure 7C). At the genus level, the top groups in overall relative abundance in fish fed the control HFD diet without OLE were Lactobacillus, Paucibacter, and Mycoplasma, as well as unclassified bacteria (Figure 7B). Unclassified bacteria and Paucibacter tended to decrease in abundance with dietary supplementation with OLE, while Phascolarctobacterium, Lachnoclostridium, and Bacteroides increased and then decreased in abundance as dietary OLE increased (Figure 7D).

Figure 7: Composition and abundance of bacteria in intestine of Trachinotus ovatus fed high‐fat diets with different levels of olive leaf extract (OLE) at the phylum (A, C) and genus (B, D) levels. The top 10 most abundant (based on relative abundance) bacterial phyla and genera are shown, with all other identified phyla and genera combined and assigned as “Others.” Values are presented as means ± SEM, n = 6. Bars for each phylum or genus in subfigures (C) and (D) with different superscript lowercase letters are significantly different (p < 0.05) as determined by one‐way ANOVA.