1. Introduction
The largemouth bass,
Micropterus salmoides, is one of the most important economical freshwater fish in China, and its production in 2019 reached more than 470,000 tons [
1]. Recent research revealed that largemouth bass has a limited ability to utilize starch, the starch content in the feed usually below 10% [
2,
3], so dietary lipids serve as the main provider of energy in the diet.
Besides the energy, dietary lipid provides other important substances such as essential fatty acids, phospholipids and sterols for maintaining cell normal structure and biological function [
4]. Due to the protein-sparing effect of dietary lipids in aquatic feeds, high-fat diet (HFD) is widely used to save costs and reduce nitrogen waste in aquaculture recently [
5,
6,
7]. However, excessive dietary lipid may lead to abnormal lipid accumulation in liver, abdominal adipose tissues and muscle. In many studies, long-term intake of HFD induced reactive oxygen species (ROS) production, oxidative stress and inflammation to fish, and finally negatively affected the growth performance and health of fish [
8,
9,
10]. Previous research indicated that lipid accumulation of largemouth bass increased with the increasing dietary lipid levels [
11], and several studies revealed that high dietary lipid (18–20%) impaired the growth and health of largemouth bass [
12,
13].
Astaxanthin is a potent lipid-soluble marine keto-carotenoid with auspicious effects on human and animal health [
14]. It is found widely in aquatic animals and some other organisms, but its de novo synthesis is limited to several bacteria, protists, fungi, algae and plants, and synthetic astaxanthin accounts for >95% of the world market currently [
15]. Astaxanthin is an important colorant in the crustacean and salmonid feed industry [
16,
17], and is also an additive with auspicious effects on egg quality [
18]. More importantly, astaxanthin acts as a safeguard against oxidative stress through different mechanisms such as scavenging of radicals and neutralizing of singlet oxygen, and astaxanthin also plays a critical role in anti-inflammatory response through modulating the cytokines production, NF-κB signaling pathway and apoptotic pathways [
14,
15,
19,
20]. Dietary supplementation of astaxanthin has been shown to improve the growth performance, anti-oxidative capacity and immune response of shrimp, crab and a variety of fish [
16,
21,
22,
23,
24]. Astaxanthin has been reported to alleviate the oxidative stress of rats induced by HFD [
25,
26], while there is no research evaluating the effect of dietary astaxanthin supplementation on fish fed HFD.
The aim of this study was to evaluate the effects of HFD and astaxanthin supplementation on growth performance, lipid metabolism, anti-oxidative ability and immune response of juvenile largemouth bass.
3. Discussion
HFD has been shown to impair the growth performance of grass carp, giant croaker, tilapia, blunt snout bream and other aquatic animals [
27,
28,
29,
30]. In this study, at the end of the feeding trial, WG, survival and SGR were similar in fish fed diet C and HF, which indicated that HFD (18.08%) did not impair the growth performance of largemouth bass. Our previous research indicated 18.08% of dietary lipid impaired the growth of largemouth bass, Zhou et al. (2020) also reported that largemouth bass fed 20% dietary lipid showed lower WG than those fed 10% dietary lipid [
13]. Meanwhile, in another study, the optimal lipid requirement of largemouth bass was 18.42% [
31]. The different results in lipid requirement of largemouth bass may result from the differences in diet formulation and environmental factors. Present results showed that dietary supplementation of 75 mg kg
−1 astaxanthin significantly improved the growth performance of fish fed the HFD. Previous studies also reported that dietary supplementation with astaxanthin enhanced the growth of kuruma shrimp, golden pompano and red porgy [
22,
23,
32]. Most of those studies evaluated the effects of astaxanthin supplementation in normal aquatic feeds, this is the first study that reveals the beneficial effects of astaxanthin supplementation on growth performance of fish fed HFD.
HFD usually induces the abnormal accumulation of lipid, and finally causes oxidative stress and inflammation. In this study, we investigated the HFD and astaxanthin on lipid accumulation and lipid metabolism firstly. When largemouth bass fed HFD, higher VSI, HSI and IPF were observed, higher lipid contents in muscle, liver and whole body also were observed. These results indicated HFD induced more lipid accumulation in several tissues (viscera and muscle) of largemouth bass, similar results were found in our previous research [
12] and several studies in largemouth bass [
31], giant croaker [
28] and cobia [
10]. The supplementation of astaxanthin in HFD decreased the HSI and IPF of largemouth bass, while having no effects on lipid contents in the whole body, muscle and liver. These results indicate that astaxanthin only reduced the fat deposition of largemouth bass to a certain extent. Similar results were reported in red porgy; dietary supplementation of 100 mg kg
−1 astaxanthin decreased the lipid contents in the whole body and liver [
32]. Meanwhile, 100 mg kg
−1 dietary astaxanthin had no effects on VSI, HSI and whole-body lipid content of golden pompano [
23], and 200–1600 mg kg
−1 astaxanthin did not affect the whole body lipid content of kuruma shrimp [
22]. In the research of mice, Yang et al. (2014) reported that 0.3–3 mg astaxanthin/kg body weight had no effects on body fat of mice [
25], Ikeuchi et al. (2007) reported that 6–30 mg astaxanthin/kg body weight decreased the liver weight [
33]. These previous research studies indicated that dietary astaxanthin supplementation may have larger effects on liver weight than body fat, which revealed that astaxanthin products may have limited effect to control body fat in humans and other animals.
Although astaxanthin has a limited effect on body lipid deposition, it still affected the lipid metabolism of animals and humans in many previous studies. A meta-analysis of randomized controlled trials revealed that astaxanthin supplementation is associated with an increase in HDL [
34]. Similar results were found in the present study; the ratio of HDL/LDL increased when fish fed diet supplemented with 150 mg kg
−1 astaxanthin. HFD also induced high TG and TC in plasma and liver in this study, astaxanthin supplementation reduced the TG in plasma and TC in the liver. In previous studies, astaxanthin had no effects on TG and TC levels when a normal diet was adopted [
34,
35]. When mice were fed an HFD, astaxanthin supplementation also showed similar TG and TC lowering effects as in this study [
25,
26,
33]. Jia et al. (2016) reported that astaxanthin modulates the lipid accumulation of high-fat-fed mice via activation of PPARα and inhibition of PPARγ [
26]. In this study, the
PPARα expression in the liver of largemouth bass showed the opposite tendency as in mice. PPARα is essential in the regulation of genes encoding fatty acid transport, metabolism and fatty acid oxidation in the liver [
36]. The lower expression of
PPARα in the HFA2 group compared to the HF group indicated the lower lipid content in the liver, which was positively correlated with the HSI results.
HMGCR is the rate-limiting enzyme for cholesterol synthesis of largemouth bass [
37], the low
HMGCR expression may associate with the high hepatic TC content. In this study, dietary astaxanthin supplementation enhanced the hepatic mRNA expression of
HMGCR, similar results were found in mice [
33]. In this study, the mRNA expression of lipid metabolism-related gene expressions was consistent with the results of plasma lipids and body fat, which clearly showed that the gene revealed that dietary astaxanthin supplementation may affect the hepatic fatty acid oxidation and cholesterol synthesis of high-fat-fed largemouth bass.
Some previous studies suggested HFD inducing ROS production, inflammatory response, and apoptosis in fish and mammals [
31,
38,
39,
40]. Excessive production of ROS will cause damage to the organism, and antioxidant enzymes such as SOD and GPx are the main participators for eliminating ROS [
41]. As a powerful antioxidant, astaxanthin also has been shown to enhance the anti-oxidative ability of animals under stress or pathological state [
19,
21,
42,
43]. In this study, SOD activity in plasma and
GPx mRNA level in the liver were increased in fish fed HF diet to eliminate the excessive ROS, and dietary supplementation of astaxanthin restored the hepatic redox state of largemouth bass. Excessive production of ROS usually leading to oxidative stress, MDA, a product of lipid peroxidation, is considered a critical marker of oxidative stress [
44]. In this study, MDA contents in plasma and liver were higher in high-fat-fed fish, which revealed that HFD induced oxidative stress in largemouth bass. Dietary supplementation of 150 mg kg
−1 astaxanthin reduced the MDA content in plasma of largemouth bass, which indicated that suitable astaxanthin alleviated the oxidative stress brought by HFD. The gene expression and activity of anti-oxidative enzymes and MDA contents clearly indicated the antioxidant system of largemouth bass was damaged by HFD, and the damage was alleviated by astaxanthin supplementation. Similar results were reported by Yang et al. (2014) [
25], the anti-oxidative ability of mice fed HFD was enhanced by astaxanthin supplementation. Bhuvaneswari et al. (2014) also reported astaxanthin supplementation decreased the ROS induced by HFD [
45].
Besides the anti-oxidative ability, the immune response of fish also was affected by HFD and astaxanthin. IL 15 and TGF-β are two critical inflammatory cytokines [
12], HFD increased the mRNA expression of
IL15 and decreased the mRNA expression of
TGF-β in this study, which indicated hepatic inflammation of largemouth bass may be induced by HFD. Dietary supplementation of astaxanthin reduced the expression of
IL15, but could not enhance the expression of anti-inflammatory cytokines
TGF-β. In previous studies, astaxanthin supplementation reduced the TNFα and IL6 of mice fed HFD [
25,
26], astaxanthin also modulating the immune response of animals through regulating the expression of cytokines. Apoptosis is a necessary condition for the development and homeostasis of cells [
46]. In response to a death signal, activation of the intrinsic apoptotic pathway results in altered expression of the genes including anti-apoptotic (
Bcl-2,
Bcl-xl and
BAG) and pro-apoptotic (
Bax, and
BAD) [
46,
47,
48]. The caspase family is also critical in mediating apoptosis, with Caspase 3 being the key executive molecule, and Caspase 9 being the upstream signaling of Caspase 3 [
49,
50]. Activated caspase triggers compensatory proliferation, referred to as apoptosis-induced proliferation which maintains tissue homeostasis following massive stress-induced cell death, regenerating damaged structures [
51]. In the present study, gene expression of
BAD,
Caspase 3 and
Caspase 9 in the liver of largemouth bass fed HFD were significantly elevated, suggesting HFD may induce apoptosis of largemouth bass. Similar results had been reported in blunt snout bream fed HFD [
30]. The high expression of
Caspase3,
Caspase 9 and
BAD were reduced by the supplementation of 150 mg kg
−1 astaxanthin, which indicating that HFD-induced apoptosis was alleviated by astaxanthin. These results are consistent with research reported in mice that dietary astaxanthin supplementation could inhibit apoptosis induced by HFD [
45]. Although protein expression samples failed to further verify the apoptosis, the present results from gene expression is still robust enough to suggest that astaxanthin supplementation may inhibit apoptosis in largemouth bass by the down-regulation of pro-apoptotic genes.
Overall, our results firstly proved that astaxanthin supplementation decreased the liver weight and alleviated the oxidative stress and inflammation of fish fed HFD, which was similar to many studies in mice. These results revealed the therapeutic potential of astaxanthin to liver inflammation induced by HFD in humans and other animals.