4. Discussion

4.1. OLE Improved Growth Performance and Reduced Lipid Accumulation in T. ovatus Fed HFD

Plant extracts have a range of potential beneficial applications, including growth promotion and appetite stimulation in cultured fish [28] and could represent an acceptable and environmentally friendly strategy as feed additives in fish culture in the era of the green economy [29]. In the current study, OLE displayed similar beneficial effects, with WG, SGR, and SR all increased in T. ovatus fed HFD supplemented with 0.5% and 1.0% OLE. However, it is important to note that the addition of excessive levels of additives can have negative impacts on the growth and health performance of fish [30]. This was apparent in the current study, where concentrations of OLE above 1.0% had negative effects on the growth of T. ovatus, which suggested that dietary supplementation of 1.0% OLE was the optimum level for promoting the growth of T. ovatus fed HFD. Similarly, it was reported previously that 200 mg/kg of OLE had a positive effect on the growth of common carp (Cyprinus carpio), while higher concentrations showed no beneficial effects [15]. However, other studies reported no significant differences in the growth of rainbow trout (Oncorhynchus mykiss) [31] and common carp [32] fed diets with various levels of OLE. The reasons for the differential impacts of OLE on fish growth in different studies may be due to differences in species and biology (age and developmental stage) of the fish studied, the OLE product used (geographical origin, extraction process, and contents/composition of active substances), diet formulations (OLE concentration, lipid content [HFD or not], and other ingredients), or the experimental conditions of the trials.

Dietary lipid level usually impacts whole‐body lipid contents of fish, and excessively high lipid contents in feed are accompanied generally by increased lipid accumulation in tissues including liver, muscle, and abdominal viscera [33]. In the current study, we utilized a lipid level of ~16.0% in the feed to establish an HFD model in T. ovatus and found that OLE supplementation of that diet reduced the lipid content of the liver (with significant linear and/or quadratic trends) and whole body. This suggested that dietary OLE can reduce lipid accumulation in tissues of HFD‐fed T. ovatus, which was supported by the reduction in both lipid droplet content and size in the liver. These data were consistent with a previous mammalian study that reported that oral administration of 16 mg/kg of oleuropein‐ and hydroxytrosol‐rich OLE improved dyslipidemia induced by HFD in rats [34]. Furthermore, TG and TC concentrations in serum were decreased in T. ovatus fed HFD supplemented with OLE, further confirming the impacts of OLE on the regulation of lipid metabolism. The reduced serum TC in T. ovatus fed HFD supplemented with 1.0% OLE was associated with decreased serum LDLC but increased HDLC concentrations, reflecting transport of excess cholesterol from peripheral tissues to the liver for metabolism or excretion, accompanied by reduced transport of cholesterol from the liver to peripheral tissues [35].

In general, the accumulation of excess lipid in fish tissues can increase the rate of lipid oxidation that, in turn, can induce the production of reactive oxygen species (ROS) and impact antioxidant capacity [36]. Previous studies showed that oleuropein can increase antioxidant enzyme activities in rats fed HFD [37] and protect human erythrocytes and HeLa cells against oxidative damage [38, 39]. In the current study, supplementation of HFD with OLE increased SOD activity and TAOC and decreased the concentration of MDA in serum, with the optimum effects obtained with 1.0% OLE. This indicated that the antioxidant defense system was increased effectively and oxidative damage reduced by dietary OLE in T. ovatus fed HFD. In addition, dietary inclusion of 0.5%–1.0% of OLE decreased activities of AST and ALT in the serum of fish fed HFD, suggesting that an appropriate level of dietary OLE could have an important function in preventing or alleviating liver damage induced by long‐term HFD.

To further explore the impacts of OLE on the liver lipid metabolism, mRNA expression levels of transcription factors and genes related to lipid metabolism were determined. Srebp1 is a transcription factor playing a crucial role in lipogenesis through modulating the expression of adipogenic genes such as fas, acc, and dgat1 [40]. In the current study, expression levels of srebp1, acc, fas, and dgat1 were all reduced in the liver of fish fed HFD supplemented with OLE, particularly at inclusion levels of 0.5% and 1.0%, suggesting that OLE could reduce lipid deposition in the liver by decreasing the rate of lipid synthesis. This was consistent with earlier studies that demonstrated that hydroxytyrosol (a significant component of OLE) mitigated the HFD‐induced liver expression of lipogenesis‐related genes (SREBP1, ACC, FAS, and SCD1) in rat [41]. In addition, olive polyphenols were found to have positive effects on lipid metabolism as an antilipogenic agent in medaka (Oryzias latipes) [42]. Another important mechanism to reduce lipid deposition is increased lipolysis and fatty acid oxidation. Hsl is a key enzyme in adipocytes, catalyzing TG hydrolysis and providing free fatty acids and glycerol in a hormone‐controlled lipolytic process [43]. In the current study, hsl expression in the liver was increased by OLE supplementation, which suggested that OLE may also increase lipolysis in fish fed HFD. Furthermore, pparα is an important transcription factor for mitochondrial β‐oxidation of fatty acids, in which Cpt1 is a pivotal enzyme in long‐chain fatty acid transport into the matrix of mitochondria for oxidation [44]. The transcription of fatty acid transporter genes, cd36 and fabp1, is also regulated by positive feedback of pparα, resulting in increased cellular uptake of fatty acids, which are then transported to mitochondria for β‐oxidation [45]. The current study showed up‐regulated levels of expression of pparα, cpt1, hsl, cd36, and fabp1 in T. ovatus fed HFD supplemented with OLE, further suggesting that OLE could increase lipid catabolism in fish liver. The main component of the OLE used in the present study was oleuropein (40%), which has been shown to be an agonist of pparα in rats, playing a role in regulating blood lipids [46, 47]. Thus, it appears that pparα is likely a key factor in the OLE in the regulation of lipid metabolism, but this requires further research. The analysis of liver fatty acid composition revealed that the addition of 0.5%–1.5% OLE to HFD may enhance the ability of T. ovatus to utilize C18 PUFA, particularly 18:2n–6, whose major fate was indicated to be β‐oxidation for energy production [48], thereby saving LC‐PUFA. Therefore, the impacts of OLE on lipid metabolism in the liver of T. ovatus fed HFD may involve the following metabolic mechanisms: (1) reducing lipid synthesis by downregulating the expression of lipogenesis‐related genes and (2) reducing lipid deposition by upregulating the expression of genes related to lipolysis and lipid transport and enhancing β‐oxidation of C18 PUFA. However, the detailed mechanisms underpinning the effects that dietary OLE has on the lipid metabolism of fish require further study.

4.2. OLE Improved Intestinal Damage and Altered Intestinal Digestive Capacity and Microbiota Composition in T. ovatus Fed HFD

As the primary organ for digestion and absorption of nutrients, it is crucial that intestinal functions and morphological integrity are maintained [49]. Well‐developed intestinal folds are conducive to the absorption of nutrients [50] and, in the current study, the height of intestinal folds was increased when 0.5% and 1.0% OLE was supplemented to the diet, which indicated that OLE contributed to a well‐developed intestinal villi area. Supplementation of HFD with 1.0% OLE also increased intestinal wall thickness, which suggested that appropriate dietary OLE did not damage the intestinal mucosa and, rather, was beneficial in maintaining the integrity of the intestinal tissue. In the intestine, goblet cells not only produce mucus that can protect various site‐specific functions but are also involved in immune responses [51]. Previously, we showed that, compared to a standard lipid diet, HFD (18% lipid) triggered intestinal inflammatory responses as evidenced by increased expression of proinflammatory factors il-8 and tnfα in the intestine of T. ovatus fed HFD [20]. The current study showed that dietary supplementation with OLE reduced expression levels of proinflammatory cytokines il-1, il-8, and tnfα, and increased goblet cell number per fold in T. ovatus fed HFD, which together suggested that OLE can possibly ameliorate HFD‐induced inflammatory responses and support normal protection and lubrication of intestinal mucosa. Moreover, dietary OLE also enhanced intestinal barrier function, as evidenced by upregulation of intestinal barrier function‐related genes cdh1 and ocln in the intestine of T. ovatus fed HFD supplemented with 1.0%–1.5% of OLE. This was consistent with the reported effects of OLE on improving intestinal permeability and intestinal barrier function [52]. Furthermore, it was interesting that the addition of OLE at 0.5%–1.5% reduced the activity of intestinal lipase in T. ovatus fed HFD. A previous study reported that HFD stimulated the secretion of lipase and induced chronic intestinal inflammation in Nile tilapia [53] and, thus, inhibiting intestinal lipase activity could contribute to the mitigation of inflammation and increased fat deposition in the liver caused by HFD. Therefore, we speculate that one mechanism of action of OLE may be to reduce excess fat deposition in T. ovatus fed HFD by lowering the secretion of intestinal lipase and decreasing the digestion and absorption of lipid, thereby breaking the high‐fat intake → excessive fat absorption and deposition → fatty liver pathway. However, further research is required on the mechanism whereby OLE affects the secretion of intestinal lipase.

Intestinal bacteria (microbiota) play important roles in not only the gut barrier but also intestinal function and health [54]. Increased Simpson and Shannon indices, which indicate that intestinal microbiota diversity had increased, were observed with dietary supplementation of OLE in T. ovatus fed HFD in the present study. Furthermore, the abundance of bacterial taxa such as Bacteroides was increased, while harmful bacterial taxa like Mycoplasma were decreased in T. ovatus fed diets supplemented with OLE. Studies in rats showed previously that OLE can prevent the reduction of Bacteroides caused by HFD [52]. Bacteroides are considered to be beneficial bacteria that can reduce lipid accumulation, decrease hyperlipidemia, and prevent hepatic steatohepatitis and liver injury in rats [55]. Other studies reported that Lactobacillus can promote the in vivo conversion of oleuropein to hydroxytyrosol [56]. It is noteworthy that ~20% of the bacteria were unclassified in the intestinal microbiota of T. ovatus fed the control HFD, and this was reduced as dietary supplementation with OLE increased. The reduction of uncategorized bacteria in fish fed diets containing OLE may be a further reason for the alleviation of intestinal inflammation and damage caused by feeding HFD in the present study. Further identification of the unclassified bacterial species will be required in the future to further reveal possible mechanisms by which OLE alleviated intestinal damage caused by HFD. Overall, the results suggested that OLE supplementation favorably altering the composition of the intestinal microbiota in T. ovatus fed HFD, which can possibly supplement the roles of oleuropein in decreasing blood lipids, supporting antioxidation, and improving immunity.