Dietary Alpha-Lipoic Acid Alleviated Hepatic Glycogen Deposition and Improved Inflammation Response of Largemouth Bass (Micropterus salmoides) Fed on High Dietary Carbohydrates

Abstract

In order to mitigate the adverse effects of high carbohydrates on largemouth bass and to investigate the feasibility of LA as a feed additive, the present study observed the effects of added α-lipoic acid (LA) on growth performance, glucose metabolism and immunity in largemouth bass fed on high dietary carbohydrates (10% α-cassava starch inclusion). A total of 315 juvenile largemouth bass (initial body weight, 5.09 ± 0.10 g) were divided into nine tanks (800 L) (upper radius 0.65 m × lower radius 0.5 m × height 1 m), with each holding 35 fish. Three iso-nitrogenous and iso-lipidic diets supplementing with 0 g/kg, 0.5 g/kg and 1 g/kg LA (LA0, LA500, LA1000) were designed to feed juvenile largemouth bass on a satiation diet twice daily for eight weeks with each diet feeding to triplicate groups. The results indicated that the performance in growth was significantly enhanced by the addition of dietary LA (p < 0.05). Meanwhile, hepatic glycogen content was significantly reduced (p < 0.05), and the expression of genes relating to insulin pathway and glycolysis significantly increased with LA inclusion (p < 0.05). The relative expression of insulin receptor a (ira) in the LA500 group was the highest, while the relative expression of glycerol kinase (gk), phosphofructokinase liver type (pfkl) and phosphoenolpyruvate carboxykinase (pepck) was the highest in the LA1000 group (p < 0.05). In addition, LA supplementation significantly increased the activity of lysozyme, which reached its maximum value in the LA500 group (p < 0.05). LA supplementation also promoted the expression of genes relating to anti-inflammatory and inhibited the expression of pro-inflammatory related genes (p < 0.05). Above all, the dietary addition of LA could improve performance in growth, alleviated hepatic glycogen deposition, and improved the immunity function of largemouth bass fed on high dietary carbohydrates. This provides us with ideas to mitigate the adverse effects of high carbohydrates on largemouth bass in actual production and provides a basis for the application of LA in aquatic biology.

1. Introduction

Starch is an economical energy source and a good binder for aquatic feeds [1], which has the advantages of easy accessibility and cost-effectiveness in aquafeed production. However, carnivorous fish could not effectively utilize dietary glucose, and high carbohydrate intake could cause hepatic glycogen deposition, in turn affecting fish growth performance and health status, which has been well demonstrated by studies on largemouth bass (Micropterus salmoides) [2,3], whereas decreased insulin secretion or increased insulin resistance has been suggested to be one of the causes for the limited carbohydrate utilization [4]. Meanwhile, insulin pathway activation is also involved in the improvement of glucose metabolism, which has been proven in largemouth bass [3,5,6]. However, high dietary carbohydrates are not effective in promoting insulin secretion and activating insulin pathways in carnivorous fish [7,8]. Additionally, excessive carbohydrates might also exacerbate inflammatory response in carnivorous fish [9,10], which would negatively affect the health of cultured fish. Consequently, finding an effective additive that could both regulate glucose metabolism and mitigate the adverse effects caused by high carbohydrates is important to produce for carnivorous fish.

Immune function is important for fish health and modern aquaculture exposes fish to acute stresses (e.g., crowding and handling), leading to increased morbidity [11,12]. The improvement of immunity could reduce the incidence of disease in fish [13]. In the immune system, stimulation of specific immunity through vaccines and other means is economically inefficient; therefore, improving non-specific immunity is a key area of focus for aquaculture [14]. Anti-inflammatory cytokines (e.g., IL-10) and pro-inflammatory cytokines (e.g., IL-6) also play important roles in fish immune organs and immune functions [15]. In addition, previous studies have shown that p38-MAPK/I-κB/NF-κB signaling pathway regulates inflammatory cytokines in largemouth bass [16,17,18].

α-Lipoic acid (LA) is a natural compound that can be obtained from microorganisms, plants and animals and has a regulatory role in insulin sensitivity and insulin secretion [19,20]. In mammals, it has been well demonstrated that LA could stimulate the insulin-signaling cascade through its pro-oxide properties [21]. Apart from mammals, in common carp (Cyprinus carpio), LA has been shown to increase the utilization of feed carbohydrates [22], in turn improving glycemic control in fish. Meanwhile, LA also has a role in immunomodulation, which could improve immunity through enhancing the activity of lysozyme, inhibiting the expression genes relating to pro-inflammatory cytokine genes and promoting genes relating to the expression of anti-inflammatory cytokines [16,23]. Therefore, LA shows a potential function in improving carbohydrates utilization and maintaining a state of health for carnivorous fish and has the potential to be used as a feed additive and thus mitigate the adverse effects of high carbohydrates on carnivorous fish. However, studies on LA in aquaculture have mainly focused on its effects on growth performance and non-specific immunity of cultured fish, and there is still a paucity of studies on the regulation of sugar metabolism pathways in fish by LA. It remains to be investigated whether LA can regulate the utilization of carbohydrates in carnivorous fish and mitigate the adverse effects caused by high carbohydrates.

Largemouth bass have been extensively cultured in China. Considerable research has proved the limitation of this fish in utilizing carbohydrates, and 10% starch inclusion could impair its growth and health status [2,3]. Above all, considering the problems identified in the actual culture of largemouth bass, as well as the functions exhibited by LA, this study investigated the effects of dietary LA addition on hepatic glycogen deposition and inflammatory responses in largemouth bass consuming high carbohydrates.

2. Materials and Methods

2.1. Preparation for Diets

Previous studies on LA in a large number of other fish species showed that most of the LA additions in fishes were concentrated at 0.5–1 g/kg [24,25,26,27]. Therefore, three iso-nitrogenous and iso-lipid experimental diets with 0 g/kg (LA0), 500 mg/kg (LA500) and 1000 mg/kg (LA1000) LA additive concentrations were designed (

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

Ingredients	LA0	LA500	LA1000
White fish meal 2	40.00	40.00	40.00
Wheat gluten meal 2	3.00	3.00	3.00
Blood meal 2	3.00	3.00	3.00
Shrimp meal 2	3.00	3.00	3.00
Fermented soybean meal 2	12.00	12.00	12.00
Corn gluten meal 2	12.00	12.00	12.00
Brewer’s yeast meal 2	3.00	3.00	3.00
Squid viscera meal 2	2.00	2.00	2.00
Soybean phospholipids 2	2.00	2.00	2.00
Fish oil 2	2.00	2.00	2.00
Soybean oil 2	2.00	2.00	2.00
α-lipoic acid 1	0.00	0.05	0.10
Microcrystalline cellulose	3.00	2.95	2.90
Vitamin mixture 3	1.00	1.00	1.00
Mineral mixture 4	1.00	1.00	1.00
Ca(H2PO4)2 2	1.00	1.00	1.00
α-cassava Starch 2	10.00	10.00	10.00
Proximate analysis (Mean values, % dry weight)
Crude protein	52.17	51.93	52.03
Crude lipid	11.84	10.54	10.73

¹ Acquired from Sanhua Biotechnology Co. (Zhengzhou, China), purity, ≥98%. ² Provided by Xinxin Tian’en Feed Corporation (Zhejiang, China). ³ Vitamin mixture (mg/kg diet): vitamin B₁, 17.80; vitamin A, 16,000 IU; vitamin B₂, 48; vitamin B₆, 29.52; vitamin B₁₂, 0.24; vitamin C, 800; vitamin D₃, 8000 IU; vitamin E, 160; vitamin K₃, 14.72; choline chloride, 1500; folic acid, 6.40; niacinamide, 79.20; inositol, 320; calcium-pantothenate, 73.60; biotin, 0.64. ⁴ Mineral mixture (mg/kg diet): I [Ca (IO₃)₂], 1.63; Mn (MnSO₄), 6.20; Cu (CuSO₄), 2.00; Fe (FeSO₄), 21.10; Se (Na₂SeO₃), 0.18; Co (CoCl₂), 0.24; Zn (ZnSO₄), 34.4.

). Prior research has demonstrated that 10% dietary starch consistently induces liver glycogen accumulation in largemouth bass, which negatively affects the fish’s health [2,3]. Consequently, 10% of dietary starch addition with 0 mg/kg of LA supplementation in the present study was selected as the control group. After the components were thoroughly mixed, an extrusion swelling machine was used to extrude the experimental diets. The low-lipid components of the experimental meals were pulverized, well combined, and then well blended with the lipid ingredients. Afterwards, water was added to harden the dough, and, after that, the dough was extruded by a pelletizer into two sizes (2.5 mm and 4 mm). The manufactured diets were then placed at −20 °C until the experiment began.

2.2. Experiment Design

This feeding study was carried out in the joint lab of Shanghai Ocean University and Shanghai Nonghao Feed Co., Ltd. (Shanghai, China). The experiment fish were temporarily raised for three weeks with feeding on the commercial feed supplied by Xinxin Tian’en Aquafeed Ltd. (Zhejiang, China), with the same water quality parameters as during the experiment. After the temporary rearing, 315 fish with an average initial body weight of (5.09 ± 0.10 g) were screened and distributed into nine 800 L tanks (upper radius 0.65 m × lower radius 0.5 m × height 1 m), each holding 35 fish. The fish in triplicate tanks were each fed an experiment diet until apparent saturation two times daily (07:30 and 17:30), and intake was recorded for 56 days. Over the process, the water exchange was 5% daily, and the water conditions were as follows: pH 7.1 ± 0.3; temperature 25 ± 0.5 °C; ammonia and nitrate content < 0.1 mg/L.

2.3. Sample Collection

After the trial, fish fasted for 24 h for further sampling, while counting the number of fish in the bucket to calculate survival rate (SR) and measuring the total weight of the fish in each bucket to measure the final mean body weight were conducted. Twelve fish from each tank were taken and anesthetized using eugenol (1:10,000) for body length and weight measurements, and three fish from each tank were randomly taken for the analysis of whole-body composition. After that, liver and viscera were isolated from 8 of the 12 fish, liver and viscera weights were determined and visceral index (VSI) and hepatic index (HSI) were analyzed. The liver samples were separated for the examination of the glycogen content, and the livers and kidneys of the remaining four fish were used for gene expression analysis. Muscle samples used for chemical analysis were obtained from the 12 fish. After being immediately frozen in liquid nitrogen, the samples were maintained at −80 °C for further examination.

2.4. Chemical Analysis

The moisture, crude protein and crude lipid content were analyzed following the method described in AOAC [28]. The whole fish, liver and muscle samples were dried to a consistent weight to assess the moisture content, and the crude lipid content was determined using the Soxhlet extraction method (SX-360, Opsis, Furulund, Sweden). The crude protein content was determined using the Dumas combustion technique (FP828, LECO, St. Joseph, MI, USA). The glycogen content of the liver and muscles was measured with potassium hydroxide/anthrone, using a commercial glycogen assay kit, (operated according to the instructions to accurately organize the weight added to the alkaline solution in a boiling water bath for 20 min). Then, a certain amount of double-distilled water was added to make the blood concentration of the test solution, and a certain amount of detection solution was taken and added as a colorant in the boiling water bath for 5 min. Finally, colorimetry at 620 nm wavelength with 1 cm aperture was conducted (A043-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) [29]. Lysozyme (LZM) activity was determined using a turbidimetric method, using a commercial lysozyme assay kit (the prepared application bacterial solution was put into a 37 °C water bath for more than 10 min, so that the temperature of the bacterial solution reached 37 °C and so that the standard solution and the sample were at the same temperature). And the visible spectrophotometer was set at 530 nm, with 1 cm aperture cuvette and with double-distilled water to adjust the transmittance of 100%. Next, this was placed into the corresponding number of test tubes, and 0.2 mL of the sample was added in order to be tested. Then 2 mL of the application of bacterial solution was quickly rushed into the test tube and was immediately mixed and timed. After that, it was quickly poured it into the cuvette at 530 nm in the visible spectrophotometer, the transmittance value T1 at 15 s was read. The cuvette was not taken out, and the transmittance value T2 at 2 min and 15 s was read, and the difference in the transmittance of the two times was found out and then was calculated according to the instructions (A050-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) [30].

2.5. Relative Gene Expression Analysis

The total liver RNA of cultured fish was extracted using the TransZol Up (Code#ET111-01) (Takara, Shiga, Japan), following the instruction of the manufacturer. Then, a commercial kit (Cat#RR074A) (Takara, Shiga, Japan) was used to reverse the extracted RNA to single strand cDNA for real-time quantification PCR (RT-qPCR). The primers of genes involved in the present study were listed in Table S1. After that, a melting curve analysis was performed after 40 cycles of 95 °C for 10 s, 57 °C for 10 s and 72 °C for 20 s, as well as RT-qPCR using a quantitative thermal cycler (Mastercycler EP Realplex, Eppendorf, Germany). β-actin was selected to be the housekeeping gene, and a 2^−ΔΔCt method was used to analyze relative gene expression [31].

2.6. Calculation and Statistical Analysis

The variance test and the chi-square test were used, respectively, to evaluate the data’s homogeneity and normality. The dose of LA for optimum fish growth was determined using the orthogonal polynomial regression. Using SPSS 24.0 software, one-way ANOVA analysis was performed to analyze all the data, which were represented as mean ± standard error of the mean (SEM). The multiple comparison test, with a significance threshold of 0.05, was Duncan’s multiple range test.

Calculation formula:

$$\mathrm{Survival} \mathrm{rate} (\mathrm{SR}, \%)={\displaystyle \frac{\mathrm{N}\mathrm{f}}{\mathrm{N}0}}\times 100$$

$$\mathrm{Specific} \mathrm{growth} \mathrm{rate} (\mathrm{SGR}, \%/\mathrm{d})={\displaystyle \frac{\left[\ln \left(\mathrm{Wf}\right)-\ln (\mathrm{Wi})\right]}{\mathrm{days}}}\times 100$$

$$\mathrm{Condition} \mathrm{factor} (\mathrm{CF}, \%)={\displaystyle \frac{\mathrm{BW}}{(\mathrm{BL})3}}\times 100$$

$$\mathrm{Feed} \mathrm{intake} \mathrm{rate} (\mathrm{FIR}, \%/\mathrm{d}·\mathrm{individual})={\displaystyle \frac{\mathrm{Wd}}{ [\mathrm{days} \times (\mathrm{Wf}+\mathrm{Wi}) / 2\times \mathrm{Nf}]}}\times 100$$

$$\mathrm{Hepatosomatic} \mathrm{index} (\mathrm{HSI}, \%)={\displaystyle \frac{\mathrm{LW}}{\mathrm{BW}}}\times 100$$

$$\mathrm{Viscerosomatic} \mathrm{index} (\mathrm{VSI}, \%)={\displaystyle \frac{\mathrm{VW}}{\mathrm{BW}}}\times 100$$

N0: number of individuals; Wi: the mean initial body weight; BW: individual body weight; LW: liver weight; VW: visceral weight; BL: fish body length; Wf: the mean of final body weight; Nf: the number of final individuals; Wd: the weight of the meal ingested.

3. Results

3.1. Growth Performance

The growth performance results are given in

Table 2. Effects of adding different doses of LA on growth performance of largemouth bass.

	LA0	LA500	LA1000	p-Value
Initial body weight (g)	5.08 ± 0.01	5.10 ± 0.02	5.09 ± 0.01	0.144, 0.552
Final body weight (g)	40.86 ± 0.84 b	45.53 ± 0.96 a	45.39 ± 1.55 a	0.029, 0.033
Specific growth rate (%/d)	3.72 ± 0.04 b	3.90 ± 0.04 a	3.90 ± 0.06 a	0.034, 0.034
Condition factor (%g/cm3)	2.29 ± 0.02	2.33 ± 0.02	2.33 ± 0.02	0.201, 0.201
Feed intake rate (%/d·individual)	1.91 ± 0.02	1.92 ± 0.03	1.94 ± 0.03	0.796, 0.403
Hepatosomatic index (%)	3.77 ± 0.04 a	3.38 ± 0.16 b	3.52 ± 0.06 ab	0.034, 0.131
Viscerosomatic index (%)	8.32 ± 0.20	8.31 ± 0.20	8.20 ± 0.11	0.990, 0.644

Means standard error of the mean, or SEM, N = 3, indicates that the data inside a row with a comparable superscript letter are not significantly different from the other dietary groups (p > 0.05). The calculation formula is shown below.

. LA supplementation had no significant effect on SR, condition factor (CF) and feed intake rate (FIR) (p > 0.05) but significantly increased specific growth rate (SGR) (p < 0.05). The VSI showed no discernible changes among different groups (p > 0.05). However, the HSI in the LA500 group was substantially lower than that of the control group (p < 0.05). Orthogonal polynomial analysis shows that an LA supplementation of 0.74 g/kg feed resulted in maximum growth in largemouth bass (Figure 1).

3.2. Body Composition and Glycogen Content Analysis

The body composition results are given in

Table 3. Effects of adding different doses of LA (0 mg/kg, 500 mg/kg and 1000 mg/kg) on the body approximate composition of largemouth bass.

	LA0	LA500	LA1000	p-Value
Whole fish
Moisture (%)	73.91 ± 1.16	74.09 ± 0.58	74.06 ± 0.45	0.877, 0.902
Crude lipid (%)	3.91 ± 0.41	3.92 ± 0.38	4.25 ± 0.37	0.986, 0.554
Crude protein (%)	17.26 ± 0.12	17.20 ± 0.14	17.32 ± 0.13	0.756, 0.730
Ash (%)	3.05 ± 0.13	2.97 ± 0.09	3.02 ± 0.02	0.588, 0.849
Liver
Moisture (%)	74.38 ± 0.48	75.38 ± 0.50	73.62 ± 0.41	0.344, 0.528
Crude lipid (%)	2.78 ± 0.04	2.78 ± 0.03	2.74 ± 0.05	0.911, 0.543
Crude protein (%)	9.00 ± 0.04 b	10.47 ± 0.45 a	9.73 ± 0.19 ab	0.030, 0.207
Muscle
Moisture (%)	78.34 ± 0.32	78.00 ± 0.25	78.12 ± 0.17	0.378, 0.555
Crude lipid (%)	1.79 ± 0.14	1.84 ± 0.10	1.48 ± 0.07	0.735, 0.085
Crude protein (%)	19.12 ± 0.36	19.43 ± 0.25	19.39 ± 0.12	0.431, 0.490

Means standard error of the mean, or SEM, N = 3, indicates that the data inside a row with a comparable superscript letter are not significantly different from the other dietary groups (p > 0.05).

, and the glycogen content analysis results are shown in Figure 2. The supplementation of LA had no effect on the proximate composition of the whole fish body (p > 0.05), while liver crude protein content in LA500 was significantly decreased (p < 0.05). Furthermore, the LA addition considerably reduced the hepatic glycogen level and raised the muscle glycogen content (p < 0.05).

3.3. Relative Expression of Genes Related to Insulin Pathway and Glucose Metabolism

The relative expression of genes related to insulin pathway results are shown in Figure 2. The relative expression of ira and irb were significantly enhanced by the addition of dietary LA (p < 0.05), with the LA500 group exhibiting the highest value level among all the groups (Figure 3A,B). The relative expression of akt1 and pi3kr1 in the groups with LA supplementation was significantly higher than the control group (p < 0.05) (Figure 3C,D).

The relative expression of genes related to glucose metabolism are shown in Figure 3. The relative expression of gk and pfkl were considerably increased by dietary LA inclusion, with the LA1000 group exhibiting the highest value among all the groups (p < 0.05) (Figure 4A,B). Nevertheless, there was no discernible change in pk expression (p > 0.05) (Figure 4C). When dietary LA was added, the expression of g6pc and pepck was dramatically increased and the maximum value of the expression of these genes was found in the LA1000 group (p < 0.05) (Figure 4D,E). However, the expression of fbp1 was not affected by dietary LA supplementation (p > 0.05) (Figure 4F).

3.4. Lysozyme Activity and Inflammation Response

The lysozyme activity and inflammation response results are shown in Figure 4. The activity of lysozyme in the LA500 and LA1000 groups was notably enhanced compared to the control group (p < 0.05) (Figure 5A). Additionally, dietary LA inclusion significantly decreased the relative expression of mapk (p < 0.05) (Figure 5B). The relative expression of iκb in LA500 and LA1000 groups was significantly higher than the control group (p < 0.05) (Figure 5C). The relative expression of nfκb in LA500 and LA1000 was significantly lower than the other treatments (p < 0.05) (Figure 5D). The relative expression of interleukin 10 (il-10) was significantly increased with the addition of LA (p < 0.05) (Figure 5E). Among the pro-inflammatory factors, the relative expression of tnf-α was significantly decreased in the LA500 group, whereas the relative expression of il-1β was significantly decreased in both the LA500 and LA1000 groups (Figure 5F,G).

4. Discussion

Due to its variety of applications, LA is a naturally occurring substance that is widely distributed in nature. In recent decades, it has drawn a lot of attention as a frequent feed additive [20,22,24,32,33]. With the addition of LA, the growth performance of largemouth bass increased significantly in this study, but the final body weight in LA1000 decreased slightly compared to LA500. This result suggested that largemouth bass on a high dietary carbohydrate diet would have better growth performance if suitable amounts of LA were added, while excessive addition of LA might inhibit this beneficial effect. This result was consistent with the studies on African catfish [34], juvenile hybrid grouper [26] and northern snakehead [24], which is likely because excess dietary α-LA could increase energy expenditure by modulating hypothalamic AMPK activity and negatively affect growth performance [35].

It has been proved that high carbohydrates could cause excessive glycogen accumulation in the liver of carnivorous fish [7,23]. In recent research, a reduced liver glycogen level indicated the improvement of LA inclusion on the utilization of glucose by largemouth bass. Consistently, in common carp (Cyprinus carpio), LA has been shown to improve glycemic control and carbohydrate metabolism [22]. It has been established that the insulin pathway is essential for preserving glucose homeostasis in animals [36]. The insulin receptor substrate protein (IRS) is phosphorylated as a key node, leading to the activation of downstream pathways of two other key nodes, phosphatidylinositol 3 kinase (PI3K) and serine/threonine kinase (AKT), which control the metabolic actions of insulin [37,38]. Recent studies in largemouth bass suggest that inadequate insulin secretion is responsible for their limited glucose utilization [5]. Therefore, the promotion of insulin secretion or activation of the insulin pathway could improve glucose utilization in carnivorous fish. The study on insulin delivery validates the function of the insulin pathway in controlling the metabolism of glucose in some teleost, including hybrid grouper [39] and largemouth bass [5]. Meanwhile, it has been proposed that the enhanced metabolism of glucose in carnivorous fish on a high carbohydrate diet is caused by the activation of the insulin pathway [3,40], whereas, on the one hand, LA has pro-oxidant properties that stimulate of the insulin-signaling cascade, which in turn increases glucose uptake by muscle and adipocytes, as reflected in muscle glycogen content [21]. Consistent with this, the gene expression results in this study suggested an activation of the insulin-signaling pathway in largemouth bass, which was induced by dietary LA inclusion. Therefore, the activation of the insulin pathway might underline the mechanism for how LA could improve glucose metabolism.

Glycolysis and gluconeogenesis are opposing metabolic pathways in the degradation and synthesis of carbohydrates and play important roles in maintaining glucose homeostasis. Although glycerol kinase (GK), phosphofructokinase (PFK) and pyruvate kinase (PK) catalyze unidirectional reactions and play key roles in glycolysis, most of the enzymes in these two pathways are shared. Phosphoenolpyruvate carboxykinase, fructose-bisphosphatase (FBP), glucose-6-phosphatase catalytic region (G6PC) and phosphoenolpyruvate carboxykinase (PEPCK) are key steps in the control of gluconeogenesis [41,42]. The insulin administration test has shown that teleost’s glycolysis and gluconeogenesis changes when the insulin pathway is activated [5,8]. Recent studies on freshwater prawns confirm that LA promotes glycolysis in the tricarboxylic acid cycle at the enzyme level and positively regulates carbohydrate metabolism [43]. Thus, in recent research, LA proximally promoted glycolysis through the activation of the insulin pathway. However, in recent research, the addition of LA unexpectedly increased the expression of two genes linked to gluconeogenesis, pepck and g6pc of largemouth bass. In line with this, the expression of hepatic genes, pepck and g6pc of zebrafish fed with LA were up-regulated [44]. The presumed reason for this is that LA can influence gluconeogenesis but not through the insulin-signaling pathway.

Excessive carbohydrate intake deteriorates immunity in carnivorous fish such as golden pompano (Trachinotus ovatus) [45], hybrid snakehead (♀ Channa maculate × ♂ Channa argus) [46] and largemouth bass [47]. LA has a potential role in enhancing immunocompetence in fish in addition to regulating the insulin pathway, which has been proven in several fish species, such as northern snakehead [24] and Nile tilapia (Oreochromis niloticus) [48]. In recent research, the addition of LA resulted in a significant increase in lysozyme activity, which confirmed the potential role of LA in enhancing the immunocompetence of the organism. Recent research has proven the effect of the p38-MAPK/I-κB/NF-κB pathway on immune regulation [16,17,18]. Previous research in mammals has shown that LA could inhibit NF-kB by regulating the upstream kinase, MAPK, and by avoiding the degradation of I-κB [49]. Pro-inflammatory factors, such as IL-1β and TNF-α are the primary middlemen of inflammation in fish, and IL-10, as a major anti-inflammatory gene, inhibits the release and synthesis of inflammatory cytokines, thereby modulating the inflammatory response [50,51]. Meanwhile, the modulatory effect of LA upon immunity has also been demonstrated in some fish species, such as grass carp and snakehead. After the addition of LA to their diets, pro-inflammatory factors were down-regulated, and anti-inflammatory factors were up-regulated, which is consistent with our findings [24,52]. In recent research, the addition of LA also activated the expression of iκb, and the expression of mapk and nfκb was inhibited. Meanwhile, LA suppressed the expression of pro-inflammatory factors tnf-α and il-1β and promoted the expression of anti-inflammatory factor il-10. The above results suggest that LA had a modulating effect on immunity.

Hepatic glycogen deposition, growth retardation and reduced immunity are all adverse effects that tend to occur when fed high amounts of carbohydrates, and this study experimentally illustrates the mitigating effects of LA on these three adverse effects, revealing its potential as a high-carbohydrate feed additive. Furthermore, this experiment only discussed the effects of LA on growth, glucose metabolism and the immunity of fish consuming high levels of α-starch, which still has some limitations. The role of LA in the aquaculture process can be further determined by conducting subsequent experiments on the feeding of different carbohydrate sources.

5. Conclusions

Above all, dietary LA improves growth performance of high carbohydrate-fed largemouth bass. In addition, LA could reduce hepatic glycogen accumulation through the activation of the insulin pathway and attenuate the decreased immunity of largemouth bass caused by high carbohydrates. This provides us with ideas to mitigate the adverse effects of high carbohydrates on carnivorous fish in practical production and lays the foundation for the application of LA in aquatic biology.