Effect of Siberian Ginseng Water Extract as a Dietary Additive on Growth Performance, Blood Biochemical Indexes, Lipid Metabolism, and Expression of PPARs Pathway-Related Genes in Genetically Improved Farmed Tilapia (Oreochromis niloticus)

Abstract

Overnutrition in high-density aquaculture can negatively affect the health of farmed fish. The Chinese herbal medicine Siberian ginseng (Acanthopanax senticosus, AS) can promote animal growth and immunity, and regulate lipid metabolism. Therefore, we conducted an 8-week experiment, in which Oreochromis niloticus was fed with a diet supplemented with different concentrations of AS water extract (ASW) (0‰, 0.1‰, 0.2‰, 0.4‰, 0.8‰, and 1.6‰). The ASW improved the growth performance and increased the specific growth rate (SGR). Linear regression analysis based on the SGR estimated that the optimal ASW amount was 0.74‰. Dietary supplementation with 0.4–0.8‰ ASW reduced the triglyceride and total cholesterol levels in the serum and liver, and regulated lipid transport by increasing the high-density lipoprotein cholesterol concentration and lowering the low-density lipoprotein cholesterol concentration. Dietary supplementation with ASW increased the activities of superoxide dismutase and catalase in the liver, thereby improving the antioxidant capacity. Moreover, ASW modulated the transcription of genes in the peroxisome proliferator-activated receptor signaling pathway in the liver (upregulation of PPARα, APOA1b, and FABP10a and downregulation of PPARγ), thereby regulating fatty acid synthesis and metabolism and slowing fat deposition. These results showed that 0.4–0.8‰ ASW can slow fat deposition and protected the liver from cell damage and abnormal lipid metabolism.

1. Introduction

As essential nutrients, lipids play an important physiological role in maintaining the normal function of the body, promoting fish growth, and improving the survival rate [1,2]. In addition, they comprise the main structure of biofilms. Moreover, they serve as carriers of fat-soluble vitamins and as cofactors of hormones and enzymes [3,4]. However, in recent years, with the development of intensive farming models, excessive energy intake [5] and environmental pollution [6] at high densities can result in excessive fat deposition. This can eventually cause fatty liver disease, which damages liver cells and affects liver metabolism, leading to slow growth and weakened immunity of farmed fish [7,8,9]. Therefore, the excessive deposition of fish fat is one of the urgent problems to be solved in aquaculture.

Tilapia is one of the main freshwater farmed fish in China, with a production of 1,655,410 tons in 2020 [10]. Genetically improved farmed tilapia (Oreochromis niloticus, GIFT) is bred from selected tilapia populations from African and Asian strains [11]. Owing to its excellent growth performance and disease resistance, it has extremely high market value and broad market potential [12,13], and currently accounts for nearly 75% of the total farmed tilapia in China. Therefore, from both economic and biological perspectives, it is particularly important to better understand the potential mechanism of fatty liver in GIFT to develop prevention or treatment strategies against excessive fatty deposits.

Lipid metabolism research can no longer be simply based on experimental observations and descriptions; however, it should involve genomic data in-depth analysis. The peroxisome proliferator-activated receptor (PPAR) signal transduction pathway is one of the main pathways of lipid metabolism. It has a wide range of biological effects in animals, and regulates important physiological processes, such as lipid metabolism, energy homeostasis, and cell division and differentiation [14]. At present, studies on PPARα and PPARγ in fish are mainly focused on lipid metabolism. PPARα regulates lipid metabolism by regulating target genes encoding enzymes involved in peroxisome and mitochondrial fatty acid beta-oxidation [15,16]. PPARγ is a superfamily of nuclear receptors that regulate lipid metabolism in adipose tissue and control the differentiation, proliferation, and adipogenesis of adipocytes [17,18]. The regulatory effects of PPARα and PPARγ on lipid metabolism have been verified in zebrafish (Danio rerio) [19] and large yellow croaker (Pseudosciaena crocea R.) [20]. Apolipoprotein A1 (ApoA1) is the main protein component of high-density lipoprotein (HDL). In an animal model study, it was found that the body fat content of ApoA1-knockout mice was significantly higher than normal mice [21]. Fatty acid-binding protein (FABP) belongs to the family of intracellular lipid-binding proteins (ILBPs) and is mainly involved in the transport of fatty acids and in fat metabolism in cells [22]. FABP can specifically bind to intracellular fatty acids and reduce the cell membrane damage caused by unsaturated fatty acids.

Traditional Chinese herbal medicines have considerable potential as aquafeed additives since they are natural products, innocuous, easy to prepare, inexpensive, and have few side effects for the fish or the environment [23]. Acanthopanax senticosus (AS) is a typical oriental folk medicinal herb, and several studies have shown that it is rich in the nutrients and trace elements that are required by animals, as well as a variety of active ingredients, such as polysaccharides, saponins, and flavonoids [24,25]. This herb has been demonstrated to have a positive effect on animal growth [26], improve antioxidant capacity [27], enhance immunity [28], treat inflammation [29,30], and regulate lipid metabolism [31]. The physiological role of AS in lipid metabolism has been shown by many experimental studies. For example, dietary supplementation of AS saponins alleviated fatty liver changes caused by a high-fat diet in mice [32]. Ethanol extracts from the stem bark of AS enhanced the insulin effect in the liver, reduced the synthesis of triglycerides, and improved liver steatosis in mice on a high-fat diet [31]. A metabolomics analysis of mice with Parkinson’s disease showed that the therapeutic effect of AS extract may involve regulating metabolic pathways, such as mitochondrial β-oxidation of long chain saturated fatty acids and fatty acid metabolism [33]. Therefore, in this study, GIFT was fed with diets containing different concentrations of AS water extract (ASW), and then its effects were determined on biochemical and physiological parameters and on the transcript levels of lipid metabolism-related genes in the PPAR pathway (PPARα, PPARγ, APOA1b, and FABP10a).

To explore the effects of ASW on fat deposition, serum biochemical parameters and liver enzyme activities were measured under different levels of ASW supplementation. In addition, differences in lipid metabolism-related gene transcript levels among the different ASW supplementation groups were detected using quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. We aimed to explore the method in which ASW regulates lipid metabolism in GIFT at the physiological and molecular levels, and to provide a theoretical basis for the application of ASW as a green feed additive.

2. Materials and Methods

2.1. Ethics Statement

The study protocols were approved by the Ethics Committee at the Freshwater Fisheries Research Centre of the Chinese Academy of Fishery Sciences (FFRC, Wuxi, China). All experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals in China.

2.2. Experimental Diets

In Saito’s research [34], 0.5‰ and 1.0‰ AS fruit supplementation improved obesity-associated insulin resistance and hepatic lipid accumulation in high-fat diet-fed mice. Therefore, in this study, six groups supplemented with 0‰, 0.1‰, 0.2‰, 0.4‰, 0.8‰, and 1.6‰ ASW were prepared (

Table 1. Feed formulae of experimental diets (containing 0‰, 0.1‰, 0.2‰, 0.4‰, 0.8‰, and 1.6‰ ASW).

	Control	0.1‰	0.2‰	0.4‰	0.8‰	1.6‰
Ingredients
Fish meal	10.00	10.00	10.00	10.00	10.00	10.00
Wheat middling	10.60	10.60	10.60	10.60	10.60	10.60
Corn starch	16.80	16.80	16.80	16.80	16.80	16.80
Soybean oil	5.00	5.00	5.00	5.00	5.00	5.00
Soybean meal	16.00	16.00	16.00	16.00	16.00	16.00
Cottonseed meal	16.00	16.00	16.00	16.00	16.00	16.00
Rapeseed meal	16.00	16.00	16.00	16.00	16.00	16.00
Vitamin premix	0.50	0.50	0.50	0.50	0.50	0.50
Mineral premix	0.50	0.50	0.50	0.50	0.50	0.50
Choline chloride	0.50	0.50	0.50	0.50	0.50	0.50
Sodium vitamin C phosphate	0.20	0.20	0.20	0.20	0.20	0.20
Ca(H2PO4)2	1.50	1.50	1.50	1.50	1.50	1.50
Microcrystalline cellulose	6.40	6.39	6.38	6.36	6.32	6.24
AS water extract	0.00	0.01	0.02	0.04	0.08	0.16
total	100	100	100	100	100	100
Nutrient composition (%, DM)
Dry matter	92.61	92.60	92.94	92.88	92.83	93.02
Crude protein	28.15	28.54	28.88	28.72	28.52	28.77
Crude Lipid	6.79	6.72	6.7	6.7	6.71	6.76
Ash	5.32	5.15	5.45	5.28	5.38	5.41

). We used commercially available ASW, which was provided by the Beijing Yujing Biotechnology Co., Ltd. (Beijing, China). The feed preparation method was in accordance with Tao, Qiang [35]. The nutrient composition of the experimental diets was determined using previously published methods [36,37]. The moisture content was determined by weighing the samples, drying them at 105 °C for 24 h, and then reweighing them. The crude protein content was determined using the Kjeldahl method after acid digestion. In addition, the crude lipid content was determined by the ether extraction method. Moreover, the ash content was determined by combusting the dry samples in a muffle furnace (Thermolyne Corporation, Dubuque, IA, USA) at 550 °C for 6 h, and then weighing the residue.

2.3. Experimental Facility and Fish Rearing

The experimental GIFT juveniles were selected from the Yixing experimental base of the Freshwater Fisheries Research Center. Juveniles were acclimated in an aerated flow-through system and fed with commercial feed for 1 week. The breeding experiment was carried out in a circulating water system. A total of 540 healthy GIFT juveniles of similar size (2.00 ± 0.02 g) were selected and randomly divided into six groups, each with three replicate tanks (30 fish/tank). The tanks were filled with 600 L of dechlorinated freshwater. The fish were fed with experimental feed twice per day (08:00 and 17:00) to apparent satiation for 56 days. During the entire experimental period, the water temperature was maintained at 28 ± 0.5 °C, the pH was 7.4, and dissolved oxygen was ≥5 mg/L. A third of the water was exchanged every 3 days.

2.4. Sample Collection

Following 56 days of feeding, the fish were starved for 24 h. Then, they were quickly and deeply anesthetized with 100 mg/L MS-222 (Argent Chemical Laboratories, Redmond, WA, USA) and the number and total weight of all fish in each tank were recorded. Three fish were randomly captured from each tank and blood samples were taken from the tail vein and divided into two parts, one for blood cell determination and the other, which was for serum analysis, was immediately centrifuged as described by Ma, Qiang [38]. The serum was stored at −80 °C for further biochemical analyses. Three fish per tank were weighed and dissected, and then their liver tissues were weighed. In addition, nine fish were picked from six groups, and the liver tissues were collected, quickly frozen in liquid nitrogen, and stored at −80 °C until analysis. The liver samples were separated into three portions, one was used to determine physiological indexes, one was used for Oil Red O staining for section examination, and one for RNA extraction.

2.5. Fish Growth Performance

Differences in the growth performance of GIFT among the six groups were determined by comparing the initial body weight (IBW), final body weight (FBW), specific growth rate (SGR), and hepatosomatic index (HSI). During feeding, the feed consumption was calculated daily and the number of dead fish was recorded. The feed conversion ratio (FCR) was calculated as an indicator of feed use. These parameters were calculated as described elsewhere [39].

2.6. Blood Biochemical Analysis

Red blood cells (RBC, 10¹²/L), white blood cells (WBC, 10⁹/L), hemoglobin concentration (Hb, g/L), and hematocrit levels (Ht, %) were determined using an automatic blood cell analyzer (bc-5300, MINDRAY, Shenzhen, China), in accordance with the instructions of each test kit. A fully automatic biochemical analyzer (bs-400, MINDRAY, Shenzhen, China) was used to measure serum total protein (TP), triglyceride (TG), total cholesterol (TC), glucose, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) contents, as well as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities. All test kits were purchased from MINDRAY.

2.7. Hepatic Biochemical Analysis

Liver samples (about 1.0 g) were homogenized in ice-cold phosphate-buffered saline (PBS, 50 mmol/L, pH 7.3), and then centrifuged for 15 min at 5000× g at 4 °C [39]. Liver TG, TC, malondialdehyde (MDA), free fatty acid (FFA) concentrations, and superoxide dismutase (SOD) and catalase (CAT) activities were measured using enzyme-linked immunosorbent assay (ELISA) kits. All kits were purchased from Shanghai Lengton Bioscience Co., Ltd. (Shanghai, China).

2.8. Liver Tissue Sections

The preparation and analysis of liver sections was conducted as described previously [35]. Liver samples collected from fish were washed with physiological saline. The samples were flash-frozen in liquid nitrogen, and then cut into 10-mm frozen sections using a freezing microtome (Model 3050S, Leica, Wetzlar, Germany). The sections were stained with Oil Red O solution for 15 min, rinsed with distilled water for 30 s, counterstained with Mayer’s hematoxylin for 3 min, and then viewed and photographed under an optical microscope. Images were analyzed using Image-Pro plus (v6.0.0.260, Media Cybernetics Corporation, Silver Spring, MD, USA), and at least six different microscope fields per group were analyzed to calculate the area of lipid droplets. The reagents used for histological staining were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

2.9. Quantitative Reverse Transcription PCR Analyses of Lipid Metabolism-Related Genes

We selected four genes related to lipid metabolism in the PPAR signaling pathway for qRT-PCR analysis: PPARα, PPARγ, APOA1b, and FABP10a. All primers used to amplify the genes (

Table 2. Sequences of primers used for qRT-PCR.

Name	Primer Sequence (5′-3′)
PPARα *	F: 5′-TCCAAAAGAAGAACCGAAACA-3′ R: 5′-TTCCACCTCTTTCTCAACCAT-3′
PPARγ	F: 5′-TTTACCCATCAAACTGACCAC-3′ R: 5′-GAGGAAATGGAGGCGTAGT-3′
APOA1b	F: 5′-TGGCTCTAGCTCTCACCCTT-3′ R: 5′-AGAGCCTTTGACAAGCGAAGA-3′
FABP10a	F: 5′-GTGGCCAAAGACATCAAGCC-3′ R: 5′-ACCTTGATCTTCCTTCCGTCC-3′
β-actin	F: 5′-CCACACAGTGCCCATCTACGA-3′ R: 5′-CCACGCTCTGTCAGGATCTTCA-3′

* PPARα: Peroxisome proliferator-activated receptor α; PPARγ: Peroxisome proliferator-activated receptor γ; APOA1b: Apolipoprotein A-1b; FABP10a: Fatty acid binding protein 10a.

) were synthesized by Suzhou GeneWiz Biotechnology Co., Ltd. (Suzhou, China). Total RNA was extracted from liver samples using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and was reverse transcribed into cDNA using Prime Script™ RT Master Mix (Takara, Dalian, China). The qRT-PCR analyses were performed on a CFX96™ Real-time PCR System (Bio-Rad, Hercules, CA, USA) in accordance with the instructions of the SYBR^® Premix Ex Taq kit (Takara). The internal reference gene was β-actin. Each PCR mixture (25 μL) contained 8 μL of RNase-free water, 12.5 μL of SYBR Premix Ex Taq II (2×), 0.5 μL of ROX Dye (50×), 2 μL of forward and reverse primers (10 μM), and 2 μL of cDNA working solution. The amplification conditions were as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s [40]. Each reaction was repeated three times. The relative gene transcript levels in the different treatments were calculated using the 2^−ΔΔCT method [41].

2.10. Data Analysis

The results are reported as mean ± standard error of the mean (SEM). The Shapiro-Wilk test and the Levene test were used to analyze the data results for normal distribution and homogeneity of variance. One-way analysis of variance (ANOVA) with post-hoc Duncan’s multiple range test was used to determine the significance level among the groups. p < 0.05 was considered significant. Statistical analyses were conducted using SPSS v22.0 (IBM Corp., Armonk, NY, USA). The relationship between SGR and dietary ASW levels was analyzed using a two-slope broken-line model [42].

3. Results

3.1. Growth Performance, FCR, and Survival

As shown in

Table 3. Effect of ASW on growth performance of GIFT (mean ± SEM).

ASW (‰)	0	0.1	0.2	0.4	0.8	1.6
IBW (g)	2.003 ± 0.003	2.000 ± 0.015	2.007 ± 0.009	2.00 ± 0.012	2.013 ± 0.007	1.993 ± 0.007
FBW (g)	74.28 ± 0.73 a,*	76.07 ± 1.10 a	77.87 ± 2.08 ab	83.87 ± 3.57 bc	86.48 ± 2.33 c	79.58 ± 0.72 ab
SGR (%/d)	6.23 ± 0.02 a	6.27 ± 0.03 a	6.31 ± 0.05 ab	6.44 ± 0.06 bc	6.48 ± 0.05 c	6.36 ± 0.02 abc
HSI (%)	1.39 ± 0.08 c	1.26 ± 0.09 bc	1.17 ± 0.07 abc	1.15 ± 0.08 ab	1.01 ± 0.08 a	1.16 ± 0.06 abc
FCR	1.307 ± 0.023	1.290 ± 0.012	1.270 ± 0.012	1.267 ± 0.012	1.253 ± 0.014	1.250 ± 0.006
SR (%)	95.56 ± 1.11	97.78 ± 1.11	96.67 ± 0.00	97.78 ± 2.22	97.78 ± 1.11	98.89 ± 1.11

* Data with different superscript lowercase letters in the same row indicate a significant difference (p < 0.05).

and Figure 1, the FBW and SGR of the 0.4–0.8‰ ASW treatment groups were significantly higher than those of the control group, with the 0.8‰ ASW group showing the highest performance (p < 0.05). Compared with the control, the 0.4–0.8‰ ASW treatment groups showed significantly lower HSI levels (p < 0.05). However, ASW supplementation did not significantly affect the FCR or survival rate of GIFT (p > 0.05).

3.2. Hematological Indexes

The WBC count differed significantly between the control group and the GIFT fed with 0.4–0.8‰ ASW supplementation, with the highest performance observed in the 0.8‰ diet group (p < 0.05;

Table 4. Effect of ASW on blood parameters of GIFT (mean ± SEM).

ASW (‰)	0	0.1	0.2	0.4	0.8	1.6
WBC (109/L)	215.74 ± 4.76 a,*	222.99 ± 6.39 a	230.69 ± 3.16 ab	243.59 ± 2.89 bc	253.62 ± 7.03 c	229.82 ± 6.83 ab
RBC (1012/L)	2.65 ± 0.12	2.71 ± 0.11	2.75 ± 0.11	2.80 ± 0.07	2.80 ± 0.08	2.84 ± 0.10
Hb (g/L)	92.67 ± 3.62	94.08 ± 4.90	94.08 ± 3.30	95.83 ± 3.47	96.00 ± 5.61	100.75 ± 3.96
Ht (‰)	31.93 ± 1.42	31.69 ± 2.25	33.72 ± 0.72	34.54 ± 1.38	34.48 ± 1.68	32.78 ± 1.78

* Data with different superscript lowercase letters in the same row indicate a significant difference (p < 0.05).

). However, we found that the RBC, Hb, and Ht were not significantly different between the treatment groups and the control group (p > 0.05).

3.3. Serum Biochemical Parameters

Dietary supplementation with ASW did not significantly affect the TP and Glu contents in the serum of GIFT (p > 0.05) (

Table 5. Effect of ASW on serum biochemical parameters of GIFT (mean ± SEM).

ASW (‰)	0	0.1	0.2	0.4	0.8	1.6
ALT (U/L)	1.84 ± 0.09 c,*	1.70 ± 0.11 bc	1.59 ± 0.12 abc	1.36 ± 0.08 a	1.46 ± 0.11 ab	1.55 ± 0.08 abc
AST (U/L)	1.88 ± 0.22 c	1.67 ± 0.17 bc	1.66 ± 0.17 bc	1.29 ± 0.16 ab	0.95 ± 0.12 a	1.51 ± 0.20 bc
TP (g/L)	7.19 ± 0.50	7.61 ± 0.43	7.76 ± 0.21	8.40 ± 0.41	8.17 ± 0.47	7.64 ± 0.38
TG (mmol/L)	1.31 ± 0.11 c	1.16 ± 0.09 bc	1.06 ± 0.10 abc	0.88 ± 0.07 a	0.93 ± 0.06 ab	1.09 ± 0.10 abc
TC (mmol/L)	1.61 ± 0.10 c	1.49 ± 0.12 bc	1.33 ± 0.09 bc	1.04 ± 0.07 a	1.22 ± 0.07 ab	1.38 ± 0.11 bc
Glu (mmol/L)	1.20 ± 0.11	1.13 ± 0.08	1.07 ± 0.08	1.00 ± 0.08	0.90 ± 0.07	1.07 ± 0.06
HDL-C (mmol/L)	1.05 ± 0.04 a	1.17 ± 0.06 a	1.29 ± 0.09 bc	1.41 ± 0.05 c	1.38 ± 0.07 bc	1.31 ± 0.12 bc
LDL-C (mmol/L)	2.34 ± 0.09 c	2.07 ± 0.13 b	1.98 ± 0.10 a	1.78 ± 0.12 ab	1.88 ± 0.13 a	1.99 ± 0.14 ab

* Data with different superscript lowercase letters in the same row indicate a significant difference (p < 0.05).

). However, the TG and TC levels significantly decreased with the increasing dietary ASW in a dose-dependent manner. The TG and TC levels were significantly lower in the 0.4–0.8‰ treatment groups than the control group (p < 0.05). Similarly, ALT and AST activities were significantly lower in the 0.4–0.8‰ treatment groups (p < 0.05) than the control. The HDL-C and LDL-C contents showed opposite trends. To be specific, compared with the control group, the groups supplemented with 0.2–1.6‰ ASW showed significantly increased HDL-C contents and significantly decreased LDL-C contents (p < 0.05).

3.4. Hepatic Biochemical Parameters

There were significant differences in liver TG and TC levels between the control group and the 0.4–0.8‰ ASW treatments groups, with the lowest TG and TC levels in the 0.8‰ treatment group (p < 0.05) (

Table 6. Effect of ASW on liver lipids and antioxidant enzymes in GIFT (mean ± SEM).

ASW (‰)	0	0.1	0.2	0.4	0.8	1.6
TC (mmol/L)	16.01 ± 0.82 c,*	15.39 ± 0.81 bc	13.97 ± 0.56 abc	13.65 ± 0.66 ab	13.00 ± 0.54 a	14.06 ± 0.77 abc
TG (mmol/L)	23.56 ± 2.48 b	23.35 ± 2.94 b	17.37 ± 1.63 ab	16.35 ± 2.00 a	13.71 ± 1.21 a	18.07 ± 2.30 ab
FFA (mmol/L)	232.69 ± 24.98 c	210.56 ± 20.85 bc	182.23 ± 21.67 abc	160.05 ± 23.07 ab	130.46 ± 16.69 a	198.83 ± 24.55 bc
MDA (mmol/L)	60.35 ± 5.99 b	51.68 ± 4.36 ab	45.85 ± 5.99 ab	36.79 ± 4.97 a	39.66 ± 5.59 a	47.59 ± 4.80 ab
SOD (ng/mg prot)	3.49 ± 0.23 a	3.74 ± 0.20 ab	4.14 ± 0.26 abc	4.51 ± 0.22 c	4.41 ± 0.22 bc	3.91 ± 0.24 abc
CAT (ng/mg prot)	1.40 ± 0.08 a	1.60 ± 0.13 ab	1.62 ± 0.11 ab	1.90 ± 0.10 b	1.86 ± 0.10 b	1.68 ± 0.12 ab

* Data with different superscript lowercase letters in the same row indicate a significant difference (p < 0.05).

). In addition, compared with the control group and the 0.1‰, 0.2‰, and 1.6‰ ASW treatment groups, the 0.4–0.8‰ ASW treatment groups showed significantly decreased FFA levels in the liver (p < 0.05). The activities of CAT and SOD in the GIFT liver were significantly higher in the 0.4–0.8‰ treatment groups than the other groups and the control (p < 0.05). The liver MDA content was significantly lower in the 0.4–0.8‰ ASW treatment groups than the control group (p < 0.05).

3.5. Histological Structure of Liver

The lipid droplets area stained with Oil Red O occupied almost the entire area of the liver section of the GIFT in the control group (Figure 2A), whereas it did not in the fish supplemented with ASW in the diet (Figure 2B,C), with the 0.4‰ supplementation group showing the least lipid droplets (

Table 7. Effect of ASW on the area of hepatic lipid droplets in GIFT (mean ± SEM).

ASW (‰)	0	0.4	1.6
Lipid droplet (Object/Total, %)	11.85 ± 0.69 c,*	3.34 ± 0.26 a	7.84 ± 0.42 b

* Different superscript lowercase letters in the same row indicate a significant difference (p < 0.05).

).

3.6. Expression Level of Lipid Metabolism Genes in the PPAR Signaling Pathway

As shown in Figure 3, ASW at a low concentration (0–0.2 and 1.6‰) did not have a significant effect on lipid metabolism-related genes in the PPAR signaling pathway (p > 0.05); however, the expression level of PPARα, PPARγ, APOA1b, and FABP10a mRNA under 0.4–0.8‰ ASW supplementation was significantly changed (p < 0.05). Compared with the control group, the expression levels of PPARα, APOA1b, and FABP10a mRNA in the GIFT liver with 0.4–0.8‰ ASW added to the diet were significantly increased (p < 0.05). Among these genes, the expression of PPARα, APOA1b, and FABP10a was the highest in the 0.4‰ ASW group. In addition, compared with the control, in the 0.4–0.8‰ ASW diet groups, the PPARγ mRNA expression level was significantly downregulated (p < 0.05), with the lowest expression observed in the 0.4‰ supplementation group.

4. Discussion

Chinese herbal medicines are rich in a variety of nutritional elements and biologically active substances, which can promote the synthesis of proteins and enzymes, accelerate the absorption and use of nutrients, stimulate the metabolism of animals, and thus be beneficial to animal growth [43,44]. In recent years, AS has been proven to play an active role in the growth of animals, such as yellow catfish (Pelteobagrus fulvidraco) [45], swamp eels (Monopterus albus) [46], weaned piglets [26], and broiler chickens [47]. In this study, we found that adding 0.4–0.8‰ ASW to the diet significantly promoted the growth of GIFT, and that 0.74‰ of ASW was the optimum concentration.

The fish blood composition is affected by metabolism, nutritional status, and immune response. The levels of various parameters, such as RBC, WBC, Hb, and Ht are considered to be effective physiological indicators, and thus are commonly used to evaluate fish nutrition, health, and the stress response to the environment [48,49]. We found that supplementation of 0.4‰ and 0.8‰ ASW significantly increased the WBC level in GIFT, which includes granulocytes, monocytes, and lymphocytes that are closely related to the fish immunity [50]. Therefore, we considered that the fish in these two groups may have better non-specific immunity.

As an important metabolic transport system, the blood participates in the regulation of fish lipid metabolism. The levels of TG and TC in the serum reflect the fat metabolism [51]. Fu, Fu [52], Li, Qiang [53], and Nishida, Kondo [54] have reported that AS reduced the concentration of TC and TG in animals, and improved lipid metabolism in the blood. This is consistent with our results that the serum TG and TC concentrations were significantly reduced in the 0.4–0.8‰ ASW treatment groups. In addition, we observed that the 0.4‰ ASW group had the highest HDL-C concentration, which was significantly higher than the control group, but the LDL-C concentration was the lowest in 0.8‰ ASW group. Relevant studies have shown that HDL-C transports excess blood lipids to the liver, which are excreted after processing and decomposing [55]. When fish consume excessive energy, high concentrations of fatty acids will lead to a decrease in HDL-C content [56]. LDL-C transports lipids from the liver to other tissues; when the LDL-C content is too high, the lipids accumulate on the arterial wall, which can easily cause arteriosclerosis and vascular damage [57]. Therefore, we speculated that dietary supplementation with 0.4–0.8‰ ASW can regulate the lipid transport in fish by affecting the concentration of lipoproteins, thereby improving lipid metabolism in the blood. Previous studies have shown that AS extracts have a hypoglycemic effect [58,59]. In our study, the serum Glu content did not differ significantly between the ASW treatment groups and the control group, but there was a decreasing trend with the lowest Glu content in the 0.8‰ ASW group.

The liver is an important organ for lipid synthesis and storage [60]. In this study, the liver FFAs concentration in GIFT in the control group was the highest and significantly higher than the 0.4–0.8‰ ASW treatment groups. This finding is consistent with the results of the increase in the serum HDL-C content and the decrease in the serum LDL-C content in the ASW supplementation groups. Furthermore, studies have shown that a high concentration of FFAs in the liver may cause sever cytotoxicity and lead to liver cell damage and the induction of abnormal lipid metabolism [61]. We found that adding 0.4–0.8‰ ASW to the diet significantly reduced the HSI in GIFT. In this case, high levels of HSI represent liver fat deposition and liver cell damage. The liver TG and TC concentrations were similar to those in the serum, and significantly decreased under 0.4–0.8‰ ASW supplementation. These results indicate that ASW reduced lipid synthesis, increased catabolism by regulating lipid transport, and reduced fat deposition in the body, and thereby improved liver steatosis. In addition, histopathological analysis further confirmed the lipid-lowering effect of ASW in GIFT. The group of fish that did not receive ASW clearly showed fat deposition in the liver, and the Oil Red O staining showed that almost all of the cytoplasm in the control group was filled with red lipid droplets, whereas the liver fat deposition was significantly improved under 0.4‰ ASW supplementation, which is similar to the results reported in mice [54].

During the formation of a fatty liver, the massive production of active oxygen destroys the antioxidant defense system and oxidative stress occurs [62]. Studies have shown that several oxygen free radicals, such as superoxide and hydroxyl radicals, as well as hydrogen peroxide (H₂O₂), caused oxidative stress in the body, which may lead to structural alterations in the cellular and intracellular organelle membranes [63]. SOD and CAT are the first line of enzymatic antioxidant defense. In this study, the activities of liver SOD and CAT enzymes were significantly increased by 0.4–0.8‰ ASW addition. Therefore, we believe that ASW can significantly increase the activity of antioxidant enzymes in GIFT liver to maintain the intracellular redox balance, and thus protect the body from oxidative stress. Consistently, the MDA content, which is the end product of lipid peroxidation and an indirect indicator of the degree of cell damage [64], was lower in the 0.4–0.8‰ ASW treatment groups than the control group.

PPARs are members of the nuclear hormone receptor superfamily, which can be activated by various xenobiotics and natural fatty acids [65]. This pathway regulates fatty acid synthesis and metabolism, adipocyte proliferation and differentiation, and inflammation through specific target genes [66]. PPARs play an important role in lipid metabolism and homeostasis. Therefore, to further examine the effect of ASW on the regulation of GIFT lipid metabolism, we selected four genes related to lipid metabolism in the PPARs signal transduction pathways for qRT-PCR analysis. PPARα is highly expressed in liver cells, integrates carbohydrate and lipid signals in liver cells, and stimulates the transcription of genes essential for fatty acid oxidation [67]. The lipid-lowering effect of PPARα activation has been confirmed in carp (Cyprinus carpio) [68], yellow catfish [69], and Nile tilapia (Oreochromis niloticus) [70]. PPARγ is a ligand-activated transcription factor that plays a key role in controlling the expression of genes related to various physiological processes. Adipocytes are the core cells that control the energy balance and systemic lipid homeostasis. When preadipocytes differentiate into adipocytes, PPARγ is induced and highly expressed in white adipose tissue [71], resulting in the accumulation of TG droplets [72]. Studies have shown that PPARγ mutants with dominant-negative activity inhibit adipogenesis in cultured preadipocytes [73]. In our study, the liver PPARα mRNA expression in GIFT was significantly upregulated by 0.4–0.8‰ ASW supplementation, which stimulated the oxidative metabolism of lipids in the liver. In contrast, the PPARγ gene was significantly downregulated, indicating that ASW effectively reduced adipogenesis and lipid droplet accumulation.

Growth and energy transfer depend on the efficient transport of lipid molecules between tissues and cells. FABPs are a class of small molecule proteins, whose physiological functions include the uptake and use of fatty acids, the metabolism of fatty acids, and protection of cell structures from the effects of fatty acids [74,75]. FABP10 is specifically expressed in the liver [76], and the ablation of this gene greatly induces the accumulation of liver TC and TG, as well as weight gain [77]. We found that the expression level of FABP10a mRNA in GIFT that did not receive an ASW diet was significantly lower than the 0.4–0.8‰ ASW groups. The control fish showed severe fat deposition, which was consistent with the above biochemical indicators and liver histology results. Apolipoprotein is an important part of plasma lipoproteins, which are involved in the regulation of enzyme activity, fat transport, absorption, and energy storage [78]. ApoA1 is considered as the main protein in HDL, and plays an important role in reversing TC transport by extracting TC and phospholipids from various cell types, and then transferring them to the liver [79]. In our study, compared with the control group, the 0.4–0.8‰ ASW treatment groups had higher transcript levels of APOA1b, indicating that ASW can promote the transcription of APOA1b to regulate fat transport.

5. Conclusions

The Chinese herbal medicine Siberian ginseng (Acanthopanax senticosus, AS) contains a variety of nutrients and active ingredients. In this study, we found that when feeding the genetically improved farmed tilapia (Oreochromis niloticus, GIFT) with different AS water extract (ASW) concentrations, 0.4–0.8‰ of ASW effectively promoted the growth of GIFT, reduced the levels of triglyceride (TG) and total cholesterol (TC) in the blood and liver, adjusted lipid transport by changing the lipoproteins’ concentrations, as well as modulated the transcription of specific target genes in the PPAR signaling pathways to regulate fatty acid synthesis and metabolism and reduce the size of the liver. In addition, certain ASW concentrations reduced the concentration of malondialdehyde (MDA), and enhanced the activity of antioxidant enzymes in the liver, thereby improving antioxidant capacity and reducing stress hazards. Therefore, based on the current research, aiming at the high-lipid load of overnutrition under intensive aquaculture, the addition of 0.4–0.8‰ ASW to the diet of GIFT may effectively slow down fat deposition and protect the liver from cell damage and abnormal lipid metabolism (Figure 4).