Effects of Soybean Isoflavones on the Growth Performance and Lipid Metabolism of the Juvenile Chinese Mitten Crab Eriocheir sinensis

Abstract

In order to study the effects of soybean isoflavones on the growth performance and lipid metabolism of juvenile Chinese mitten crabs, six experimental diets were formulated by gradient supplementation with 0%, 0.004% and 0.008% soybean isoflavones at different dietary lipid levels (10% and 15%). The groups were named as follows: NF-0 group (10% fat and 0% SIFs), NF-0.004 group (10% fat and 0.004% SIFs), NF-0.008 group (10% fat and 0.008% SIFs), HF-0 group (15% fat and 0% SIFs), HF-0.004 group (15% fat and 0.004% SIFs) and HF-0.008 group (15% fat and 0.008% SIFs). All crabs with an initial weight of 0.4 ± 0.03 g were fed for 8 weeks. The results showed that dietary supplementation with 0.004% or 0.008% SIFs significantly increased the weight gain and specific growth rate of crabs. Diets supplemented with 0.004% or 0.008% SIFs significantly reduced the content of non-esterified free fatty acids and triglycerides in the hepatopancreas of crabs at the 10% dietary lipid level. Dietary SIFs significantly decreased the relative mRNA expressions of elongase of very-long-chain fatty acids 6 (elovl6), triglyceride lipase (tgl), sterol regulatory element-binding protein 1 (srebp-1), carnitine palmitoyltransferase-1a (cpt-1a), fatty acid transporter protein 4 (fatp4), carnitine palmitoyltransferase-2 (cpt-2), Δ9 fatty acyl desaturase (Δ9 fad), carnitine palmitoyltransferase-1b (cpt-1b), fatty acid-binding protein 10 (fabp10) and microsomal triglyceride transfer protein (mttp) in the hepatopancreas of crabs. At the 15% dietary lipid level, 0.008% SIFs significantly increased the relative mRNA expressions of fatty acid-binding protein 3 (fabp3), carnitine acetyltransferase (caat), fatp4, fabp10, tgl, cpt-1a, cpt-1b and cpt-2 and significantly down-regulated the relative mRNA expressions of Δ9 fad and srebp-1. In conclusion, SIFs can improve the growth and utilization of a high-fat diet by inhibiting genes related to lipid synthesis and promoting lipid decomposition in juvenile Chinese mitten crabs.

1. Introduction

Soybean isoflavones (SIFs) are natural phytoestrogens that mainly exists in soybean and other leguminous plants. It has been widely reported that SIFs can enhance non-specific immunity and alleviate human diseases [1,2]. Moreover, they can improve lipid metabolism and associate with the gonadal development of animal. Therefore, SIFs could be a potential feed additive for improving animal growth and health.

Soy isoflavones play an important role in improving immunity and lipid metabolism. It has been reported that diets supplemented with SIFs improved the growth performance of pigs [3] and chickens [4] fed normal-fat diets. The supplementation of high-fat diets with SIFs promoted insulin resistance and growth in rats [5]. However, certain studies have reported contradictory results [6,7].

In mammals, a large number of studies have shown that soy isoflavones are an effective additive for regulating lipid metabolism. Dietary supplementation with SIFs can alleviate lipid accumulation by reducing triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) levels in the liver of obese rats [8,9]. Further studies suggested that SIFs improved lipocatabolic metabolism by up-regulating the relative expression of peroxisome proliferate-activated receptor α (PPARα) [10] and sterol regulatory element-binding protein 1 (srebp-1) [11]. Moreover, some studies reported that SIFs can stimulate liver X receptor α or liver X receptor β phosphorylation and trigger lipid catabolism [12]. In addition to promoting lipolysis, SIFs can also alleviate lipid accumulation through reducing fat synthesis. Some previous studies reported that dietary SIFs inhibited the fat synthesis of obese Zucker rats by down-regulating the expression of peroxisom-proliferator activated receptor γ2 (PPARγ2) and adipose-specific protein 27 (FSP27) [13]. In aquatic animals, diets supplemented with 100 mg/kg and 500 mg/kg genistein significantly down-regulated the genes associated with lipid synthesis of Cyprinus carpio [14]. Some similar results were widely reported in Oncorhynchus mykiss and Paralichthys olivaceus [15,16]. On the contrary, some studies reported that SIFs can increase lipid synthesis. For example, dietary SIFs significantly increased TG and TC content in the serum of Allogynogenetic crucian carp and Paralichthys olivaceus [17,18]. Unfortunately, it is difficult to explain functional variations between species. As such, more studies are needed to understand the function of isoflavones in aquatic animals.

The Chinese mitten crab (Eriocheir sinensis) is an economic crustacean that has been widely farmed. Different from mammals, shrimp and crabs do not have specific adipose tissue, and the lipid metabolism pattern is also different from that of mammals. Whether soy isoflavones can regulate the fat metabolism of crabs is still poorly understood. Therefore, we speculated that soybean isoflavones can improve the utilization of dietary lipid, so soybean isoflavones were used to supplement high-fat diets and normal-fat diets, respectively, to examine the effects of soy isoflavones on the growth performance and lipid metabolism of the Chinese mitten crab.

2. Material and Methods

2.1. Experimental Diets

Six experimental diets were formulated by gradient supplementation with 0%, 0.004% and 0.008% soybean isoflavones at different dietary lipid levels (10% and 15%). The groups were named NF-0, NF-0.004, NF-0.008, HF-0, HF-0.004 and HF-0.008. The formulation and proximate composition of the experimental diets are shown in

Table 1. Formulation and proximate composition of the experimental diets (dry matter, %).

	Normal Fat			High Fat
	0% SIFs	0.004% SIFs	0.008% SIFs	0% SIFs	0.004% SIFs	0.008% SIFs
Ingredients	NF-0	NF-0.004	NF-0.008	HF-0	HF-0.004	HF-0.008
Fish meal	20	20	20	20	20	20
Casein	21	21	21	21	21	21
Gelatin	7	7	7	7	7	7
Corn starch	23	23	23	23	23	23
Soybean lecithin	2	2	2	2	2	2
Cholesterol	0.5	0.5	0.5	0.5	0.5	0.5
Choline chloride a	0.5	0.5	0.5	0.5	0.5	0.5
Fish oil	3	3	3	7	7	7
Soybean oil	3	3	3	7	7	7
Arginine	1.8	1.8	1.8	1.8	1.8	1.8
Methionine	0.5	0.5	0.5	0.5	0.5	0.5
Lysine	0.5	0.5	0.5	0.5	0.5	0.5
Vitamin premix a	1.5	1.5	1.5	1.5	1.5	1.5
Mineral premix b	1.5	1.5	1.5	1.5	1.5	1.5
Sodium carboxymethyl cellulose	2	2	2	2	2	2
Attractant	3	3	3	3	3	3
Butylated hydroxytoluene	0.1	0.1	0.1	0.1	0.1	0.1
SIFs	0	0.004	0.008	0	0.004	0.008
Microcrystalline Cellulose	9.1	9.096	9.092	1.1	1.096	1.092
Proximate analysis (%)
Crude protein	45.18	45.52	44.63	43.68	43.97	45.71
Crude lipid	9.76	9.28	9.57	15.80	16.15	16.55
Moisture	9.13	10.32	10.80	9.63	9.97	10.25
Ash	7.56	7.59	7.59	7.55	7.48	7.49

^a Vitamin premix (per 100 g premix): Ca pantothenate, 0.3 g; para-aminobenzoic acid, 0.1 g; cholecalciferol, 0.0075 g; riboflavin, 0.0625 g; menadione, 0.05 g; ascorbic acid, 0.5 g; biotin, 0.005 g; retinol acetate, 0.043 g; folic acid, 0.025 g; pyridoxine hydrochloride, 0.225 g; thiamin hydrochloride, 0.15 g; niacin, 0.3 g; α-tocopherol acetate, 0.5 g; The remaining part will be used α-cellulose to 100 g. ^b Mineral premix (per 100 g premix): KI, 0.023 g; CuCl₂·2H₂O, 0.015 g; Ca(H₂PO₄)₂, 26.5 g; MnSO₄·6H₂O, 0.143 g; AlCl₃·6H₂O, 0.024 g; KH₂PO₄, 21.5 g; NaH₂PO₄, 10.0 g; CoCl₂·6H₂O, 0.14 g; KCl, 2.8 g; ZnSO₄·7H₂O, 0.476 g; Calcium lactate, 16.50 g; CaCO₃, 10.5 g; MgSO₄·7H₂O, 10.0 g; Fe-citrate, 1 g; The remaining part will be used α-cellulose to 100 g.

.

The ingredients were finely ground and sieved through a 0.25 mm mesh strainer (Pulverizer, 2500Y, Anhui Hualing Xichu Equipment Co., Ltd., Maanshan, China; Strainer, Huafeng Hardware Instrument Co., Ltd., Shaoxing, China). The ingredients were weighed according to the formulation (Table 1) and mixed using an electric mixer (B10K, Foshan Shunhengji Kitchenware Co., Ltd., Foshan, China). The oil and distilled water were subsequently added to make a dough. Finally, the dough was pelleted using a screw-press pelletizer (F-26, South China University of Technology, Guangzhou, China). The pallets were air dried to reach a moisture content of less than 10%. After drying, diet foods were stored at −20 °C.

2.2. Feeding Trial, Sampling and Growth Evaluation

Adult crabs were obtained from a farm in Huzhou, China (East longitude 119 degrees 14′ to 120 degrees 29′, north latitude 30 degrees 22′ to 31 degrees 11′). Crabs were acclimatized to the experimental conditions in 300 L tanks (100 × 80 × 60 cm) before the feeding trial. A total of 1050 crabs (0.4 ± 0.03 g, mean ± SEM) were weighed and put into 30 tanks (100 × 80 × 60 cm), with each tank containing 35 crabs. Four parallel tanks were randomly allotted to one of the experimental diets. Three plastic nets were placed in each tank as shelters to reduce attacking behavior. Diets with a daily ration of 4% body weight were hand-fed to crabs twice daily (6:00 and 18:00). Feces was removed in the morning (09:00), and water 30% of the tank volume was exchanged daily. Dead crabs were immediately removed from the tank, weighed and recorded. The feed intake of each tank was recorded throughout the trial period. During the experimental period, the experimental water temperature in the tanks varied from 18 °C to 24 °C, the dissolved oxygen concentration was >7 mg/L, and the ammonia nitrogen value was <0.05 mg/L.

At the end, crabs were euthanized, after which the hepatopancreases were frozen in liquid nitrogen and kept in an ultra-low temperature freezer (−80 °C) for enzyme activity, gene expression and nutrient composition analyses.

Weight gain, specific growth rate, survival and the hepatopancreas index were calculated using the formulas below:

Weight gain (WG, %) = (final crab weight − initial crab weight)/initial crab weight × 100.

Specific growth rate (SGR, %) = (LN final weight − LN initial weight)/days × 100.

Survival (%) = final number/initial number × 100.

Hepatopancreas index (%) = hepatopancreas weight of crab/whole crab weight × 100.

2.3. Chemical Composition Analysis

The chemical compositions of the experimental diets and crabs were measured according to the standard procedures for proximate composition analysis [19]. Four duplicate samples were measured in each treatment (n = 4). The moisture content was measured after the samples were oven-dried at 105 °C. Crude protein was quantified using the Kjeltec™ 8200 (Foss, Hoganas, Sweden). Crude lipid was extracted using a 1000 mL Soxhlet extraction tube (Fujian minbo toughened glass Co., Ltd., Fuzhou, China). Ash was analyzed using a muffle furnace (SX2-8-10, Shanghai Yiheng Technology Co., Ltd., Shanghai, China) at 550 °C for 6 h. Four duplicate samples were analyzed for each treatment (n = 4).

2.4. Analysis of Biochemical Parameters in the Hepatopancreas

The biochemical parameters in the hepatopancreas were measured using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) in accordance with the instructions of the manufacturer. The source and information of each kit used in this study were as follows: non-esterified fatty acid (NEFA; Cat. No. A042-1-1), lipase (LPS; Cat. No. A054-1-1), total cholesterol (TC; Cat. No. A111-2-1) and triglyceride (TG; Cat. No. A110-2-1). Four duplicate samples were analyzed for each treatment (n = 4).

2.5. Analysis of Gene Expression

Total RNA was extracted from the hepatopancreas using an RNAiso Plus (CAT # 9109, Takara, Kusatsu, Japan). The total RNA concentration and quality were estimated using the Nano Drop 2000 spectrophotometer (Thermo, Waltham, MA, USA). If the ratio of A260/A280 was between 1.8 and 2.0, the sample was used for reverse transcription using a PrimeScript™ RT master mix reagent kit (Perfect Real Time, Takara, Japan). The specific primers for the genes of E. sinensis were designed based on the transcriptome sequencing results and NCBI database using NCBI Primer BLAST (Supplementary Table S1). The RT-PCR amplification reactions were performed in a volume of 10 μL containing 5 μL 2×SYBR Premix Ex TaqTM, 0.25 μL of 10 mM forward primer, 0.25 μL of 10 mM reverse primer and 4.5 μL of diluted cDNA, using a CFX96 Real-Time PCR system (Bio-rad, Richmond, CA, USA). PCR conditions were as follows: 94 °C for 3 min and following 40 cycles at 94 °C for 15 s, 60 °C for 50 s, and 72 °C for 20 s. Samples were run in quintuplicate and normalized with the control gene β-actin and glyceraldehyde-phosphate dehydrogenase (GAPDH). The gene expression levels were calculated by the geometric averaging of multiple internal control genes [20]. Four duplicate samples were analyzed for each treatment (n = 4).

2.6. Statistical Analysis

Statistical analysis was performed using the SPSS 26.0 for Windows (SPSS, Michigan Avenue, Chicago, IL, USA). All data were subject to normality tests and homogeneity of variance by using Shapiro–Wilk and Levene’s equal variance tests, respectively. Data were analyzed by two-way analysis of variance (ANOVA) to determine if there was any interaction between the dietary lipid level and SIFs level. Under the same lipid condition, one-way analysis of variance (ANOVA) was used to analyze the significant differences among crabs fed the diets with different SIFs levels after the normality and homogeneity of variance tests. When the means of each treatment were significantly different, Duncan’s multiple range test was used to compare means among these treatments. At the same SIFs level, an independent-samples T test was used to determine significant differences between crabs cultivated at different lipid levels. Significance was set at p < 0.05. The data were represented as the mean ± standard error of mean (S.E.M.).

3. Results

3.1. Growth Performance

Both with normal-fat diets and high-fat diets, dietary SIFs significantly increased the weight gain (WG), specific growth rate (SGR) and hepatopancreas index (HSI) (p < 0.05, Figure 1a,c,d). At the normal-fat level, the WG, SGR and HSI of crabs fed the diets supplemented with 0.004% and 0.008% SIFs were significantly higher than crabs fed 0% SIFs (p < 0.05, Figure 1a,c,d). At the high-fat level, the WG, SGR and HSI of crabs fed the diets supplemented with 0.008% SIFs were significantly higher than crabs fed 0% SIFs (p < 0.05, Figure 1a,c,d). In the absence of SIFs, the WG and SGR of the crabs in the NF-0 group was significantly higher than that in the HF-0 group (p < 0.01, Figure 1c,d). The dietary lipid level and SIFs did not significantly affect the survival of crabs (p > 0.05, Figure 1b).

3.2. Biochemical Parameters in the Hepatopancreas

At the normal-fat level, diets supplemented with 0.004% and 0.008% SIFs significantly decreased the NEFA and TG contents in the hepatopancreas (p < 0.05,

Table 2. Effects of soybean isoflavones on the biochemical parameters in the hepatopancreas of Chinese mitten crab.

	Parameters
Diets	NEFA (µmol/gprot)	LPS (U/gprot)	TC (mmol/gprot)	TG (mmol/gprot)
NF-0	36.56 ± 16.49 a	1.49 ± 0.74	3.98 ± 0.42 *	34.13 ± 2.26 a
NF-0.004	20.75 ± 7.75 b	1.83 ± 0.38	4.01 ± 0.29	30.29 ± 2.13 b *
NF-0.008	17.47 ± 9.50 b	2.13 ± 0.35	4.01 ± 0.74	26.80 ± 3.53 c *
HF-0	26.42 ± 6.00 A	2.44 ± 0.65	3.15 ± 0.36 *	33.61 ± 4.66
HF-0.004	22.65 ± 3.48 A	1.70 ± 0.59	3.46 ± 0.55	38.03 ± 3.75 *
HF-0.008	21.16 ± 9.12 B	2.73 ± 0.88	3.70 ± 0.54	33.31 ± 2.61 *
Two-way ANOVA (p value)
Lipid level	<0.01	NS	<0.01	<0.01
SIFs	<0.01	NS	NS	<0.01
Lipid level × SIFs	<0.01	NS	NS	<0.01

Note: NEFA, non-esterified fatty acid; LPS, lipase; TC, total cholesterol; TG, triacylglycerol; lipid level; SIFs, soybean isoflavones; NS, no significant differences (p > 0.05). The experimental results are expressed as mean ± SEM. Different superscripts within the first or last three rows of the same column indicate significant differences (p < 0.05). The * marked in the same column represents statistically significant differences (p < 0.05) between the corresponding NF and HF.

). In the absence of SIFs, the NEFA and TG contents in the hepatopancreas of crabs in the NF-0 group was significantly higher than that in the HF-0 group (p < 0.05, Table 2). The dietary lipid level and SIFs did not significantly affect the LPS activities of crabs (p > 0.05, Table 2).

3.3. The mRNA Expressions of Genes Related to Lipid Synthesis in the Hepatopancreas

In the normal-fat level, diets supplemented with 0.004% SIFs significantly down-regulated the mRNA expression of fabp 3 in the hepatopancreas (p < 0.05, Figure 2a). However, at the high-fat level, diets supplemented with 0.004% and 0.008% SIFs significantly up-regulated the mRNA expression of fabp 3 in the hepatopancreas (p < 0.05, Figure 2a). The mRNA expression of fabp 3 of crabs fed the high-fat diets were significantly higher than crabs fed the normal-fat diets (p < 0.01, Figure 2a). At the normal-fat level, dietary SIFs significantly down-regulated the mRNA expression of fabp 4, fabp 10, srebp-1, elovl6 and Δ9 fad in the hepatopancreas (p < 0.05, Figure 2b–f). At the high-fat level, diets supplemented with 0.008% SIFs significantly up-regulated the mRNA expression of fabp 4 and elovl6 in the hepatopancreas (p < 0.05, Figure 2b,e). Diets supplemented with 0.004% and 0.008% SIFs significantly up-regulated the mRNA expression of fabp 10 in the hepatopancreas of crabs under high-fat conditions (p < 0.05, Figure 2c). Diets supplemented with 0.004% SIFs significantly up-regulated the mRNA expression of srebp-1 in the hepatopancreas of crabs under high-fat conditions (p < 0.05, Figure 2d). At the high-fat level, diets supplemented with 0.004% SIFs significantly up-regulated the mRNA expression of srebp-1 in the hepatopancreas (p < 0.05, Figure 2d). On the contrary, diets supplemented with 0.004% and 0.008% SIFs significantly down-regulated the mRNA expression of Δ9 fad in the hepatopancreas of crabs under high-fat conditions (p < 0.05, Figure 2f).

3.4. The mRNA Expressions of Lipolysis-Related Genes in the Hepatopancreas

In the normal-fat level, diets supplemented with 0.004% SIFs significantly down-regulated the mRNA expression of cpt-1a, cpt-2, mttp and tg1 in the hepatopancreas (p < 0.05, Figure 3a,c,d,f). However, at the high-fat level, diets supplemented with 0.004% SIFs significantly up-regulated the mRNA expression of cpt-1a and cpt-1b in the hepatopancreas (p < 0.05, Figure 3a,b). Diets supplemented with 0.004% and 0.008% SIFs significantly up-regulated the mRNA expression of caat and tg1 in the hepatopancreas of crabs under high-fat conditions (p < 0.05, Figure 3e,f). On the contrary, diets supplemented with 0.004% and 0.008% SIFs significantly down-regulated the mRNA expression of mttp in the hepatopancreas of crabs under high-fat conditions (p < 0.05, Figure 3d). Diets supplemented with 0.008% SIFs significantly down-regulated the mRNA expression of caat in the hepatopancreas of crabs under high-fat conditions (p < 0.05, Figure 3e). In the absence of SIFs, the mRNA expression of cpt-1a and cpt-1b in the hepatopancreas of the crabs in the NF-0 group was significantly higher than that in the HF-0 group (p < 0.01, Figure 3a,b). However, dietary SIFs significantly up-regulated the mRNA expression of cpt-1a and cpt-1b of crabs fed the high-fat diets (p < 0.05, Figure 3a,b). Similar results were observed in cpt-2, caat and tg1 (p < 0.05, Figure 3c,f).

4. Discussion

Lipids are important nutrients for aquatic animals, and an optimal dietary lipid level can ensure the growth and development of aquatic animals. In the present study, 15% lipid diets significantly reduced the growth performance of juvenile Chinese mitten crabs, and similar results were reported in Cyprinus carpio [21]. These results showed that an excessive dietary lipid level has a negative impact on aquatic animals. In the present study, the high-fat diet decreased the WG and SGR of juvenile crabs, but dietary SIFs increased the growth performance of juvenile crabs. The possible reason for these results was that dietary SIFs promoted the utilization of the high-fat diet by optimizing the lipid metabolism process, thereby improving the growth performance of juvenile Chinese mitten crabs.

Previous studies have reported that SIFs have lipid catabolism effects in humans and rats [22,23]. Studies have reported that dietary SIFs can reduce blood glucose, serum TG and LDL-C levels in a mouse model of type 2 diabetes induced by a high-fat and high-sugar diet [24]. In the present study, at the 10% lipid level, dietary SIFs significantly reduced the contents of NEFA and TG in the hepatopancreas of the crabs, which indicates that SIFs may be involved in lipolysis and provide energy for the growth of juvenile Chinese mitten crabs. However, the 15% lipid diet decreased the TG and NEFA content in the hepatopancreas when the diet was not supplemented with SIFs, but the dietary supplementation of SIFs increased the TC content, which indicates that SIFs may improve dietary fat utilization in juvenile crabs.

To date, the regulatory mechanisms of SIFs on lipid metabolism in crustaceans are still unknown. Previous studies have shown that SIFs can affect the activities of enzymes and β-oxidation involved in fat metabolism [25,26,27,28]. In the present study, the results showed that SIFs supplementation significantly up-regulated the relative expression of genes related to the fat synthesis and lipolysis of crabs fed the high-fat diets, which indicated that SIFs could improve lipid synthesis and oxidation in the Chinese mitten crabs. A similar result was reported in rainbow trout, which reported that SIFs enhanced the expression of lipid synthesis genes and lipid-binding protein genes [16]. The function of FABPs is the targeted transportation of hepatic fat to catabolic and anabolic sites, which is an important indicator for fat anabolism [29]. In the present study, SIFs supplementation significantly increased the expression of FABPs and elovl6 of crabs fed 15% fat diets. This result indicated that SIFs can improve lipid synthesis in Chinese mitten crabs fed high-fat diets. In contrast, some other studies found that dietary SIFs significantly inhibited the expression of genes related to fat synthesis in rats [30]. The reason for these divergent results may be related to species differences.

Lipid oxidation is one of the key steps in lipid metabolism in mammals, fish and crabs, and β-oxidation is the main pathway of fatty acid oxidation [31,32]. CPT1 and CPT2 form the mitochondrial carnitine palmitoyltransferase system, which plays an important role in the transfer of long-chain fatty acids from cytosolic compartments to the cytoplasm [33]. In the present study, the results showed that SIFs increased the relative expression of genes related to lipolysis (cpt-1a, cpt-1b, cpt-2 and caat) in crabs fed high-fat diets. These results suggested that SIFs supplemented alongside a high-fat diet may accelerate lipid oxidation and improve the utilization of dietary lipids in Chinese mitten crabs.

5. Conclusions

Diets supplemented with 0.004% SIFs or 0.008% SIFs could inhibit the expression of the fatty acid synthesis-related genes srebp-1 and Δ9 fad and promote the expression of the lipolysis-related genes caat, tgl, cpt-1a, cpt-1b and cpt2, thereby improving growth performance and lipid utilization in juvenile Chinese mitten crabs.