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Dietary supplementation with succinic acid improves growth performance and flesh quality of adult Nile tilapia (Oreochromis niloticus) fed a high-carbohydrate diet
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Manxia Caoa, b, Ningning Xiea, Jianmin Zhanga, Ming Jianga, Feng Huangb, Lixue Donga, Xing Lua, Hua Wena, *, Juan Tiana, b, *
Animal Nutrition | 2024, 18(1) : 390 - 407
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Animal Nutrition | 2024, 18(1): 390-407
• Original Research Article •
Dietary supplementation with succinic acid improves growth performance and flesh quality of adult Nile tilapia (Oreochromis niloticus) fed a high-carbohydrate diet
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Manxia Caoa, b, Ningning Xiea, Jianmin Zhanga, Ming Jianga, Feng Huangb, Lixue Donga, Xing Lua, Hua Wena, *, Juan Tiana, b, *
Affiliations
  • aKey Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China
  • bKey Laboratory for Animal Nutrition and Feed Science of Hubei Province, Wuhan Polytechnic University, Wuhan 430023, China
Published: 2024-09-10 doi: 10.1016/j.aninu.2024.04.016
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To evaluate the effects of dietary supplementation with succinic acid on growth performance, flesh quality, glucose, and lipid metabolism of Nile tilapia (Oreochromis niloticus) fed a high-carbohydrate diet (HCD), five iso-nitrogenous and iso-lipidic diets were prepared as follows: HCD (control group) consisting of 55% corn starch and HCD supplemented with 0.5%, 1.0%, 2.0%, and 4.0% succinic acid, respectively. Tilapia with an initial body weight of 204.90 ± 1.23 g randomly assigned to 15 tanks with 3 replicates per group and 10 fish per tank fed for 8 weeks. Increasing dietary succinic acid supplementation resulted in significant second-order polynomial relationship in the weight gain rate (WGR), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency rate (PER), viscerosomatic index, condition factor, and contents of muscular crude lipid and glycogen (P < 0.05). The hepatosomatic index, mesenteric fat index, liver glycogen content and crude lipid contents of the whole-body and liver demonstrated significantly linear and second-order polynomial relationship (P < 0.05). Quadratic curve model analysis based on WGR, SGR, PER, and FCR demonstrated that optimal supplementation with succinic acid in the HCD of Nile tilapia ranged from 1.83% to 2.43%. Fish fed with 1.0% succinic acid had higher muscular hardness, increased the contents of alkali-soluble hydroxyproline in collagen, docosahexaenoic acid (DHA) and n-3 polyunsaturated fatty acid (n-3PUFA) in muscle, and lower total fatty acid content in muscle (P < 0.05) compared with the control group. Compared to the control group, dietary supplementation with 1.0% succinic acid significantly increased the contents of total bounding amino acid (arginine, histidine, isoleucine, lysine, methionine, alanine, proline), total flavor amino acid (free aspartic acid), the catalase (CAT) activity and total antioxidant capacity, and the mRNA relative expression levels of CAT, superoxide dismutase (SOD), and nuclearfactor erythroidderived 2-like 2 (Nrf2) in muscle (P < 0.05). Furthermore, succinic acid supplementation significantly up-regulated mRNA relative expression levels of glycolysis genes (hexokinase 2 [HK2], phosphofructokinase, muscle-A [PFKMA], and phosphofructokinase, muscle-B [PFKMB]), a key glycogen synthesis gene (glycogen synthase [GYS]), and lipid catabolism genes (carnitine palmitoyltransferase-1B [CPT1B], hormone sensitive lipase [HSL], and lipoprotein lipase [LPL]), while down-regulating the mRNA relative expression level of fatty acid synthase (FASN) in muscle (P < 0.05). In conclusion, dietary supplementation with 1.83% to 2.43% succinic acid improved muscle quality by increasing muscle antioxidant capacity and hardness, changing muscle amino acid and fatty acid composition, and regulating muscle glucose and lipid metabolism.

Oreochromis niloticus  /  High-carbohydrate diet  /  Succinic acid  /  Antioxidant capacity  /  Muscular nutritional value  /  Muscle growth
Manxia Cao, Ningning Xie, Jianmin Zhang, Ming Jiang, Feng Huang, Lixue Dong, Xing Lu, Hua Wen, Juan Tian. Dietary supplementation with succinic acid improves growth performance and flesh quality of adult Nile tilapia (Oreochromis niloticus) fed a high-carbohydrate diet[J]. Animal Nutrition, 2024 , 18 (1) : 390 -407 . DOI: 10.1016/j.aninu.2024.04.016
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1.　Introduction
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With the scarcity and sudden rise in the price of fish meal, enhancing the utilization of non-protein nutrients in aquatic animals is of vital importance (Aragão et al., 2022). Carbohydrates are abundantly available and affordable, and the optimum dietary carbohydrate level might enhance growth and feed efficiency (Kamalam et al., 2017). Therefore, high-carbohydrate diets (HCD) have been extensively used in both omnivorous (Boonanuntanasarn et al., 2018b) and herbivorous (Tan et al., 2009) fish cultures to minimize protein and lipid catabolism in fish (Krogdahl et al., 2005). Nonetheless, the ability of fish to utilize carbohydrates is limited, and they are considered “glucose intolerant” (Polakof et al., 2012). Excessive dietary carbohydrate ingestion causes metabolic disorders, reduced growth performance (Li et al., 2016), prolonged hyperglycemia (Kostyniuk et al., 2019), and extreme lipid deposition (Prisingkorn et al., 2017; Viegas et al., 2016). Moreover, excessive carbohydrate intake decreased the content of polyunsaturated fatty acids (PUFA) and affected the amount of essential amino acids (EAA) in muscle (Wang et al., 2017; Wu et al., 2021; Li et al., 2014), resulting in poor fish flesh quality. Hence, the pursuit of effective methods to improve the utilization of carbohydrates and reduce metabolic disorders and muscle quality degradation caused by HCD has attracted increasing attention.
Long-term consumption of HCD has been found to lead to changes in concentrations of intermediate metabolites from the tricarboxylic acid cycle (TCA), such as cis-aconitate and malic acid (Zakim et al., 1967; Zhang et al., 2021). Succinic acid, situated at the core position of various metabolic pathways, can be produced by glycolysis and fatty acid β-oxidation pathways, as well as by intestinal microbial fermentation of dietary fiber (Cummings et al., 1987; De Vadder et al., 2016). Additionally, succinic acid is also a downstream product of the α-ketoglutarate dehydrogenase complex and is involved in the formation and elimination of endogenous reactive oxygen species (ROS) (Liang, 2018). The above results raise speculation that supplementation with exogenous succinic acid may be an effective way to enhance the utilization of HCD. Studies have indicated that dietary supplementation with 0.5% succinic acid promotes the growth, feed utilization, and immunity of white shrimp (Litopenaeus vannamei) (Duan et al., 2018). Dietary supplementation with 0.15% succinate also promoted growth, feed intake, and protein deposition in juvenile zebrafish (Danio rerio) (Ding et al., 2022). Succinic acid has also been reported to induce the conversion of fast-twitch muscle fibers to slow-twitch muscle fibers in the skeletal muscle of mice, change the proportion of muscle fibers, and significantly improve muscle quality (Wang et al., 2019). However, the role of succinic acid in improving the muscle quality of farmed fish remains unexplored.
Nile tilapia (Oreochromis niloticus) is a crucial economic fish farmed globally, playing a significant role in addressing the issue of fish scarcity in developing nations due to its ease of cultivation, substantial flesh, and high protein content (Deng et al., 2010; He et al., 2021). Tilapia have been found to tolerate 330 g/kg of carbohydrate (Boonanuntanasarn et al., 2018a), and a diet with a carbohydrate/lipid ratio of 2:1 to 6.5:1 was most conducive to growth (Kabir et al., 2020). Diets are classified as HCD when the dietary carbohydrate content exceeds 380 g/kg (Ning et al., 2023). Prior research has indicated that diets with carbohydrate levels of 420 and 449 g/kg decrease muscle quality (Wu et al., 2021), systemic lipid accumulation, and glucose metabolism disorders (Liu et al., 2018). Therefore, the aim of the current study was to investigate the effects of succinic acid supplementation in a HCD containing 55% corn starch on the growth performance, flesh quality, glucose, and lipid metabolism of Nile tilapia and the underlying mechanisms. Our study might provide a new method for alleviating HCD stress and improving the quality of fish flesh.
2.　Materials and methods
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2.1.　 Animal ethics statement
In China, tilapia is widely farmed and is neither an endangered nor a protected species. The Yangtze River Fisheries Research Institute's Institutional Animal Care and Use Committee gave its approval for this work (Permit Number: YFI 2018-40).
2.2.　 Experimental diets
Five iso-nitrogenous and iso-lipidic diets were designed for this experiment. Succinic acid (99% purity) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). In the current study, casein and gelatin were utilized as protein sources, fish and soybean oils were utilized as lipid sources, and corn starch was utilized as a carbohydrate source. The experimental diets were divided into a HCD (CON group, containing 550 g/kg corn starch and 60 g/kg lipid) and four supplementary diets supplemented with 0.5% (S0.5), 1.0% (S1.0), 2.0% (S2.0), and 4.0% (S4.0) succinic acid (Table 1). All components were precisely weighed, ground through a 60-mesh screen, and mixed evenly. Oil and water (about 30% of the dry ingredients) were added and mixed thoroughly. The well-mixed ingredients were then extruded (2.0 mm in diameter) using a laboratory pellet mill (TY-432, Shanghai Taiyi Machinery, China). The resultant noodle-like diets were dried in a convection oven at 60℃ for 3 h. For later usage, the feeds were crushed into tiny pellets and kept at −20℃.
2.3.　 Experimental fish and feeding trial
The feeding study was carried out at the Yangtze River Fisheries Research Institute (Wuhan, China) with an indoor recirculating aquaculture system. Tilapia were purchased from the Guangxi Tilapia National Breeding Station (Nanning, China) and cultured in an indoor recirculating aquaculture system with commercial tilapia feed (supplied by Shenzhen Alpha Feed Co., Ltd.) for 2 weeks to acclimate to the environment. Prior to the feeding trial, the experimental fish were fasted for 24 h and anesthetized with 50 mg/L of MS-222 (tricaine methane sulfonate). About 150 healthy fish with initial body weights (204.90 ± 1.23 g) were chosen, weighed, and arranged in 15 tanks with a total water volume of 400 L and 10 fish each. Three tanks were selected at random for each diet. The fish were hand-fed 3 times a day, at 08:30, 12:30, and 17:30, until their behavior demonstrated signs of satiety. The water temperature, feeding behavior, and mortality of tilapia were recorded daily. Approximately 30% of the water in each tank was exchanged every 2 days. The parameters of the water throughout the experiment were as follows: pH 6.8 to 7.2; dissolved oxygen content > 5 mg/L; ammonia nitrogen mass content < 0.05 mg/L. The temperature of the water was between 28 and 33℃. The feeding trial lasted for 8 weeks.
2.4.　 Sample collection
At the end of the feeding trial, fish were fasted for 24 h. All fish were anesthetized with 80 mg/L of MS-222 before sampling. To calculate the specific growth rate (SGR), weight gain rate (WGR), survival rate (SR), and feed conversion ratio (FCR), the number and total weight of fish in each tank were recorded. Two fish were randomly selected from each tank for whole-body composition determination. The other six fish from each tank were randomly chosen to measure the body weight and length to calculate the condition factor (CF). Then, blood samples were drawn from the caudal vein of these 6 fish using a 2-mL syringe. Blood samples were centrifuged (960 × g, 4℃) to obtain serum samples and stored at −80℃ prior to analysis. The above six fish were disinfected with 75% alcohol before dissection. The viscera, liver, and mesenteric fat were weighed to calculate the viscerosomatic index (VSI), hepatosomatic index (HSI), and mesenteric fat index (MFI). Then, the dorsal muscle was separated, immediately frozen in liquid nitrogen, and stored at −80℃ for RNA isolation. To examine the texture, the muscles (10 mm × 10 mm × 5 mm) at the third to fifth dorsal fins above the lateral line of two fish from each tank were cut and stored at 4℃. The muscle (5 mm × 5 mm × 5 mm) of the other two fish per tank was collected and then fixed in 4% phosphate buffer saline (PBS) for histology determination. The muscle and liver of the remaining fish were isolated and stored at −20℃ for further nutritional value analysis.
2.5.　 Growth indices and biochemical analyses
2.5.1.　 Growth indices
The following formulae were used to calculate the experimental fish's SR, WGR, SGR, feeding rate (FR), feeding intake (FI), FCR, HSI, VSI, CF, MFI, and protein efficiency ratio (PER) based on the measured data.
where Wt is final body weight (g); W0 is initial body weight (g); L is body length (cm); Nt is final fish number; N0 is initial individual fish number; t is experimental days (d); Wf is dry feed consumed (g); Wd is total death weight (g); Wh is hepatopancreas weight (g); Wv is viscerosomatic weight (g); Wp is feed crude protein content (%); Wm is mesenteric fat weight (g).
2.5.2.　 Composition analysis
The standard methodology was used to determine the proximate composition of diets, liver, muscle, and whole fish (AOAC, 2005). Diets were dried at 105℃ to a consistent weight according to AOAC method 2001.12 in order to measure their moisture content. The whole-body, muscle, and liver were freeze-dried for 72 h in a vacuum freeze dryer (Christ Beta 2-4 LD plus LT, Marin Christ Corporation, Osterode, Germany) to determine their moisture content. The crude protein content was measured with an auto Kjeldahl system (Kjelflex K-360; BUCHI Labortechnik AG, Flawil, Switzerland) according to the AOAC method 2001.11. Crude lipid content was determined by petroleum ether Soxhlet extraction (Sox606, Hanon Advanced Technology Group Co., Ltd., China) according to AOAC method 920.39. The ash content was determined by calcination in a muffle furnace (SX-4-10, Nanbei Instrument Limited, China) for 10 h at 550℃ according to AOAC method 942.05. An adiabatic bomb calorimeter (SDC311; Hunan Sundy Science and Technology Development Co., Ltd., China) was used to measure the gross energy of the meals by direct combustion.
2.5.3.　 Serum biochemical analysis
The contents of total protein (TP) (Sysmex, 290618), total cholesterol (TCHO) (Sysmex, 290723, 290724), triglyceride (TG) (Sysmex, 80945, 80946), glucose (GLU) (Sysmex, 290713, 290714), and albumin (ALB) (Sysmex, 290615) in serum were determined by the methods of biuret, cholesterol oxidase-peroxidase aminoantipyrine (CHOD-PAP), glycerol kinase-glycerol phosphate oxidase-peroxidase (GK-GPO-POD), hexokinase, and bromocresol green (BCG), respectively. The activities of aspartate aminotransferase (AST) (Sysmex, 290705, 290706), alanine aminotransferase (ALT) (Sysmex, 290703, 290704), and alkaline phosphatase (ALP) (Sysmex, 290701, 290702) in serum were determined by the methods of lactate dehydrogenase-ultraviolet (LDH-UV), malate dehydrogenase-ultraviolet (MDH-UV), and nitrophenyl-disodium phosphate -amino methy propanol (NPP-AMP), respectively. The kits were acquired from Sysmex Corporation in order to use an automated biochemical analyzer (BX-3010, Sysmex Corporation, Tokyo, Japan) to identify serum indicators.
2.5.4.　 Activities of liver and intestinal digestive enzymes
The fresh liver and intestine samples (about 0.6 g per sample) were accurately weighed and put into 10 mL centrifuge tubes. The samples were homogenized with ice-cold physiological saline solution (1:9, w/v) on ice using a portable homogenizer and then centrifuged at 960 × g for 30 min at 4℃. The resulting supernatants were collected and kept at −80℃ to analyze the enzyme activity. The lipase (LPS, A054-2-1) activity, amylase (AMS, C016-1-1) activity, and total protein (TP, A045-4) content of the liver and intestine were determined by commercial kits (Nanjing Jiancheng Bioengineering Institute, China). The protease activity was determined by the Folin-phenol method as follows: 0.25 mL of tissue homogenate supernatant was added with 0.5 mL of 0.5% casein preheated in advance and reacted in a water bath for 15 min. Then 0.75 mL of 10% trichloroacetic acid was added and centrifuged (960 × g, 10 min) to remove precipitation. Then 1.25 mL of sodium carbonate (0.55 mol/L) and 0.25 mL of Folin reagent were added to 0.25 mL of the resultant supernatant, respectively. The color was developed in a water bath for 15 min. The corresponding optical density (OD) values were recorded at 680 nm wave length for subsequent calculations.
2.5.5.　 Determination of glycogen content of liver and muscle
The fresh liver and muscle tissue were weighted (about 0.1 g), and 3 times the volume of alkali solution was added and hydrolyzed in a boiling water bath for 20 min. The resultant hydrolysate was collected, and the glycogen contents of the liver and muscle were determined by commercial kits (A043-1-1, Nanjing Jiancheng Bioengineering Institute, China).
2.5.6.　 Muscle texture properties analysis (TPA)
Complete fresh back muscle blocks were selected and cut into 1 cm × 1 cm × 1 cm squares. Texture profile analysis was used to measure muscle hardness, springiness, gumminess, chewiness, and resilience with the TVT-300XP texture instrument (Perten Instruments (Beijing) Co., Ltd., Beijing, China). The P-cy5s cylindrical probe was used for two tests; the test speed was 1 mm/s, the interval was 5 s, the deformation was 60%, and the data acquisition rate was 200 pps. The test speed was 2 mm/s before and after the test. Each sample was pressed twice.
2.5.7.　 Determination of collagen content
The hydroxyproline (Hyp) content was used to determine the collagen concentration. The muscle alkaline-soluble Hyp and alkaline-insoluble Hyp were separated by the method provided by Li et al. (2005). In brief, following the homogenization of muscle samples, sodium hydroxide (0.2 mol/L) and 8 mol/L hydrochloric acid (HCl) were used to extract the alkaline-soluble and alkaline-insoluble Hyp in an ice bath, respectively. The Hyp was determined by the method provided by Zhang et al. (2013). The Hyp content was calculated using a standard curve.
2.5.8.　 Determination of muscle antioxidant capacity
The fresh muscle samples (about 0.6 g per sample) were accurately weighed and put into 10 mL centrifuge tubes. The samples were homogenized with ice-cold physiological saline solution (1:9, w/v) on ice using a portable homogenizer and then centrifuged at 960 × g for 30 min at 4℃. The resulting supernatants were collected and kept at −80℃ to analyze the enzyme activity. Using assay kits acquired from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China), the total protein (TP, A045-4) content, total antioxidant capacity (T-AOC, A015-2-1), catalase (CAT, A007-1-1) activity, superoxide dismutase (SOD, A001-3) activity, and malondialdehyde (MDA, A003-1) content of muscle were determined.
2.5.9.　 Histological properties analysis
Following ethanol dehydration and xylene cleaning, muscle samples were embedded in paraffin. Hematoxylin and eosin were used to stain the 5 μm tissue sections, followed by observation and photography using the light microscope DM2500 (Leica DM2500, Leica, Solms, Germany). The myofiber characteristics, including the number of muscle fiber and density, were measured using Image-J Launcher software. The muscle fiber density (N/mm2) was expressed as the rate of all the fibers in the entire region.
2.5.10.　 Analysis of amino acid and fatty acid contents
Bound amino acids were determined using an automated amino acid analyzer (HITACHI L-8900, Tokyo, Japan). Samples of muscle were prepared using the following method: freeze-dried muscle samples weighing about 0.1 g were put into glass tubes that were sealed, and 12 mL of HCl (6 mol/L) was added to hydrolyze the samples for 24 h at 110℃. The hydrolysate was then filtered and diluted with distilled water to a volume of 100 mL. A vacuum drier was used for 24 h to remove the HCl from the filtrate (2 mL), which was then evaporated to dryness at 60℃. A second 24 h evaporation of the 2 mL of distilled water was added. Then, 8 mL of 0.1 mol/L HCl was used to deliquesce the sedimentation and filter the mixture through a 0.22 μm millipore membrane, leaving 1 mL of the supernatant for analysis of bound amino acids.
The free amino acids were determined using an automated amino acid analyzer (HITACHI L-8900, Tokyo, Japan). Samples of muscle were prepared using the following method: fresh muscle tissues weighing approximately 1.0 g were homogenized with 3 mL of 10% sulfosalicylic acid and then centrifuged (18,000 × g, 4℃, 15 min). Then 1 mL of supernatant was filtered through a 0.22 μm millipore membrane and used for the measurement of free amino acids.
The fatty acids were isolated from the muscle in accordance with the Chinese standard (GB 5009.168-2016). Briefly, approximately 0.4 g of muscle tissue was put into a 5 mL centrifuge tube, then 4 mL of isooctane was added, vortexed, and mixed for 30 s, and then shaken in a shaker at 25℃ overnight for extraction. Then, 8 mL of 2% sodium hydroxide in methanol solution was added to the resultant extraction solution and gently boiled for 40 min. Following this, 20 min of gentle boiling was spent adding 7 mL of 15% boron trifluoride to the methanol solution. Twenty milliliters of n-heptane were added and boiled gently for 1 min. The mixture was allowed to stratify after adding the saturated sodium chloride solution. Five mL of the upper n-heptane extraction solution was taken. Then 5 g of anhydrous sodium sulfate was added and stirred for 1 min and stood for 5 min. The resulting supernatants were put in a 2 mL centrifuge tube and kept at −20℃ until analysis. Gas chromatography (GC, Agilent 7890A, Agilent Technologies, Santa Clara, USA) was used to determine individual fatty acids. A polydicyanopropyl siloxane capillary column with a strong polar stationary phase separated the lipids under the following GC conditions: the injection volume was 1 μL, and the temperatures of the injection port and the detection port were 270 and 280℃, respectively. The temperature schedule was set up as follows: after 13 min at 100℃, the temperature was increased to 180℃ at a rate of 10℃ per min and maintained for 6 min. After that, the temperature increased to 200℃ at a rate of 1℃ per min and stayed there for 20 min. After reaching 230℃ at a rate of 4℃ per min, the temperature was maintained for 10.5 min. The carrier was nitrogen gas, and the split ratio was 100:1. The ratio of the peak areas of various fatty acids to the internal standard's peak areas (C11:0) was used to compute the fatty acid content.
2.5.11.　 cDNA synthesis and quantitative reverse transcription polymerase chain reaction (RT-qPCR)
TRIzol reagent (Life Technologies, Carlsbad, CA, USA) was used to extract total RNA from muscle, and PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara Biotech, Dalian, China) instructions were followed to generate cDNA. TB Green Premix Ex Taq II (Tli RNaseH Plus) (TaKaRa Biotech, Dalian, China) was utilized to quantify the mRNA expression of genes. The RT-qPCR was carried out according to published papers (Xie et al., 2021). The primers are listed in Table 2 (synthesized by Sangon Biotech). β-actin was selected as the housekeeping gene. Using the 2−ΔΔCt method, relative quantification of transcript expression was computed, with the relative mRNA expressions of the CON group serving as the reference.
2.6.　 Statistical analysis
The experiment data were analyzed with SPSS 26.0 software (SPSS Inc., Chicago, IL, USA). All results were presented as the means ± standard deviation (SD). Normality and homoscedasticity were confirmed initially. Then, a one-way analysis of variance was performed on all the data, and Tukey's multiple range tests were performed. Furthermore, orthogonal polynomial contrasts were used to evaluate all the data in order to determine if the tendency (or pattern) was linear or second-order polynomial. Statistical differences were evaluated at P < 0.05. GraphPad Prism 8 was used to create the column charts (GraphPad Software, Inc., La Jolla, CA, USA). The quadratic regression graphs were made using OriginLab 2019 (OriginLab Inc., Massachusetts, USA).
3.　Results
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3.1.　 Growth performance and feed utilization
The SR of tilapia was 100%. With the increase in succinic acid supplementation in the CON diet, the WGR, SGR, PER, FCR, VSI and CF of tilapia demonstrated a significantly second-order polynomial relationship (P < 0.05), and the HSI and MFI revealed a significantly linear and second-order polynomial relationship (P < 0.05). The minimum of VSI and HSI and the maximum of CF were recorded in the S1.0 group. The maximum of WGR, SGR, and PER and the minimum of FCR were recorded in the S2.0 group (Table 3). Dietary supplementation with succinic acid had no considerable effect on the FR and FI (P > 0.05). Quadratic curve model analysis based on WGR, SGR, PER, and FCR demonstrated that the optimal supplementation with succinic acid in HCD ranged from 1.83% to 2.43% for tilapia (Fig. 1).
3.2.　 Proximate composition and muscle collagen content
With the increase in succinic acid supplementation in the CON diet, the contents of whole fish moisture and muscle crude protein exhibited a significantly linear relationship (P < 0.05), and the contents of whole fish crude lipid, muscle crude lipid, muscle glycogen, liver moisture, and collagen alkali-soluble hydroxyproline demonstrated a significantly second-order polynomial relationship (P < 0.05). The contents of liver crude lipid, collagen alkali-insoluble hydroxyproline, and total hydroxyproline revealed a significantly linear and second-order polynomial relationship (P < 0.05). The maximum contents of muscle ash, muscle crude protein, and total hydroxyproline appeared in the S0.5 group; the minimum crude lipid contents of the whole fish, muscle, and liver were recorded in the S1.0 group; and the maximum contents of muscle glycogen and collagen alkali-soluble hydroxyproline were presented in the S2.0 group. Dietary supplementation with succinic acid had no considerable effect on the contents of whole fish ash, muscle moisture, and liver crude protein (P > 0.05, Table 4).
3.3.　 Serum biochemical indices
With the increase in succinic acid supplementation in the CON diet, the activities of AST, ALT, and TG content in serum exhibited a significantly second-order polynomial relationship (P < 0.05), and the contents of TP, ALB, TCHO, and GLU demonstrated a significantly linear and second-order polynomial relationship (P < 0.05). The maximum contents of TP and ALB in serum were recorded in the S1.0 group, and the minimum amounts of AST and ALT activities and the contents of ALP and TG appeared in the S1.0 group. The minimum amount of serum TCHO content was presented to S4.0 group (Table 5).
3.4.　 Activities of liver and intestinal digestive enzymes
With the increase in succinic acid supplementation in the CON diet, the activities of AMS and LPS in the liver demonstrated a significantly second-order polynomial relationship (P < 0.05), and the activity of LPS in the intestine revealed a significantly linear (P < 0.001) and second-order polynomial relationship (P = 0.001). The highest AMS activity in the intestine was recorded in the S0.5 group, the maximum AMS activity in the liver was recorded in the S1.0 group, the maximum LPS activity in the liver appeared in the S2.0 group, and the maximum LPS activity in the intestine was presented in the S4.0 group. Dietary supplementation with succinic acid had no considerable effect on the activity of protease in the liver and intestine (P > 0.05, Table 6).
3.5.　 Muscle texture analysis
With the increase in succinic acid supplementation, muscle springiness and chewiness displayed a significantly second-order polynomial relationship (P < 0.05), and muscle hardness and resilience demonstrated a significantly linear and second-order polynomial relationship (P < 0.05). The maximum muscle hardness, gumminess, and chewiness were recorded in the S1.0 group, and the maximum muscle springiness and resilience were present in the S2.0 group (Table 7).
3.6.　 Histological properties analysis
Compared with the CON group, the density of muscle fibers in the S1.0 group was higher, and the gap between muscle fibers was smaller (Fig. 2). In comparison to the CON group, the muscle fiber density of the S1.0 and S2.0 groups was significantly greater (P = 0.001). In contrast to the other groups, the number of muscle fibers with a diameter of 70 to 110 μm was significantly increased in the S1.0 group (P < 0.001), and the number of muscle fibers with a diameter of less than 70 μm was significantly decreased in the S1.0 group (P < 0.001) (Table 8).
3.7.　 Muscle antioxidant capacity
With the increase in succinic acid supplementation, the activity of CAT and T-AOC in muscle displayed a significantly second-order polynomial relationship (P < 0.05), while the MDA content demonstrated a significantly linear (P = 0.005) and second-order polynomial relationship (P = 0.003). The maximum muscular SOD activity and the minimum MDA content appeared in the S0.5 group. The maximum muscular CAT activity was recorded in the S1.0 group, and the maximum T-AOC was recorded in the S2.0 group (Table 9). The mRNA expression levels of muscular CAT, SOD, and Nrf2 in the succinic acid supplementation group were significantly up-regulated (P < 0.05) (Fig. 3).
3.8.　 Fatty acid profiles of the muscle
There were 22 different types of fatty acids found in muscle tissue: 7 types of saturated fatty acids (SFA), 7 types of monounsaturated fatty acids (MUFA), and 8 types of PUFA (Table 10). With the increase in succinic acid supplementation, the contents of C18:2n-6c and ΣMUFA exhibited a significantly linear relationship (P < 0.05), and the contents of C18:3n-3 (LA) and Σn-3PUFA demonstrated a significantly second-order polynomial relationship (P < 0.05). The contents of C16:0, C18:1n-9c, C20:3n-6, C20:4n-6 (ARA), C20:5n-3 (EPA), C24:1n-9, and ΣSFA demonstrated a significantly linear and second-order polynomial relationship (P < 0.05). The maximum contents of ΣPUFA and Σn-6PUFA were present in the S0.5 group; the minimum content of ΣMUFA was recorded in the S1.0 group; the maximum contents of Σn-3PUFA and DHA appeared in the S1.0 group; and the maximum content of ΣSFA was recorded in the S2.0 group. The maximum contents of ARA and EPA were present in the S4.0 group.
3.9.　 Free and bound amino acid profiles in muscle
A total of 17 kinds of bound amino acids were detected in this study, including 9 kinds of EAA (except Try) and 8 kinds of non-essential amino acids (NEAA) (Table 11). With the increase in succinic acid supplementation, Arg content demonstrated a significantly second-order polynomial relationship (P = 0.017), and His, Ile, Lys, and Met contents displayed a significantly linear and second-order polynomial relationship (P < 0.05). The maximum content of Thr was recorded in the S0.5 group, the maximum content of Arg and the minimum content of Leu were recorded in the S1.0 group, the maximum content of Met was recorded in the S2.0 group, and the maximum contents of His, Ile, and Lys were recorded in the S4.0 group. Among the NEAA, the content of Tyr demonstrated a significantly linear relationship (P = 0.048), the content of Ala demonstrated a significantly second-order polynomial relationship (P = 0.003), and the contents of Asp and Cys demonstrated a significantly linear and second-order polynomial relationship (P < 0.05). The maximum contents of Pro and ΣNEAA were recorded in the S1.0 group; the maximum contents of Ala and Cys were recorded in the S2.0 group; and the maximum contents of Asp, ΣEAA and total amino acids (ΣTAA) were recorded in the S4.0 group. Dietary supplementation with succinic acid had no discernible effect on the content of Phe, Val, Gly, Glu, and Ser (P > 0.05).
A total of 14 kinds of free amino acids were detected in the muscle tissue, including 8 kinds of EAA and 6 kinds of NEAA (Table 12). With the increase in dietary succinic acid supplementation, the contents of Leu and Phe demonstrated a significantly linear relationship (P < 0.05), and the contents of Ile, Met, Asp, Gly, Pro, and total bitter amino acid (ΣBAA) followed a significantly second-order polynomial relationship (P < 0.05). The content of Arg followed a significantly linear (P = 0.001) and second-order polynomial relationship (P < 0.001). The minimum contents of Ile and Leu were found in the S0.5 group; the maximum contents of Ala, ΣEAA, ΣNEAA, and TAA were recorded in the S0.5 group; the maximum contents of His, Lys, Pro, and ΣFAA were recorded in the S1.0 group; and the minimum contents of Met, Val, Gly, and Tyr were recorded in the S1.0 group. The maximum content of Asp and the minimum content of ΣBAA appeared in the S2.0 group, and the minimum content of Arg was recorded in the S4.0 group. The level of dietary supplementation with succinic acid had no discernible effect on the content of Glu (P = 0.077).
3.10.　 Muscle regulatory factor (MRF) gene expression in the muscle
The mRNA expression levels of MyoD, MyoG, Myf5, and MRF4 were significantly up-regulated in the succinic acid supplementation group (P < 0.05). The S2.0 group had a significantly greater MyoD mRNA expression level compared to the other groups (P < 0.001). The S0.5 group had the highest MyoG mRNA expression level. With the increase in succinic acid supplementation, the mRNA expression levels of Myf5 and MRF4 demonstrated a significantly second-order polynomial relationship (P < 0.05), and the maximum was recorded in the S1.0 group (Fig. 4).
3.11.　 Glucose lipid metabolism in the muscle
The mRNA expression level of FASN was significantly down-regulated in the succinic acid supplementation group (P = 0.004). With the increase in succinic acid supplementation, the mRNA expression levels of CTP1B and HSL demonstrated a significantly second-order polynomial relationship (P < 0.05), and the maximum was reflected in the S1.0 group and S2.0 group, respectively. The mRNA expression level of LPL demonstrated a significantly linear and second-order polynomial relationship (P < 0.05), and the minimum was recorded in the S4.0 group (Fig. 5).
The mRNA expression levels of genes related to glycolysis (PFKMA and PFKMB), glycogen synthesis (GYS), and gluconeogenesis (PCK) were significantly increased in the succinic acid supplementation group (P < 0.05). The mRNA expression levels of HK1A, HK1B, HK2, and GLUT4 demonstrated a significantly linear and second-order polynomial relationship (P < 0.05). The maximum mRNA expression levels of HK1B and HK2 were present in the S0.5 group, and the maximum mRNA expression level of HK1A was recorded in the S1.0 group. The maximum mRNA expression level of GLUT4 was recorded in the S2.0 group. The GLUT1 mRNA expression level revealed a significantly second-order polynomial relationship (P = 0.004), and the maximum appeared in the S1.0 group (Fig. 6).
4.　Discussion
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4.1.　 Succinic acid supplementation in HCD improves the growth and feed utilization of tilapia
Carbohydrates are used in fish diets as the cheapest energy source to enhance growth performance and promote protein-sparing action (Li et al., 2020). The majority of research has revealed that the optimal dietary carbohydrate level improves growth, but excessive dietary carbohydrate negatively affects fish health (Han et al., 2021). In recent years, glucose metabolism intermediates or derivatives have been used as feed additives to improve growth performance and feed utilization in aquatic animals (Duan et al., 2018). For example, dietary supplementation with 0.2% malic acid and 0.2% citric acid significantly improved the growth performance of Gibel carp (Carassius auratus gibelio) (Zhang et al., 2020), and dietary supplementation with 0.15% succinate promoted the growth and whole-body protein deposition of zebrafish (Ding et al., 2022). Our study demonstrated that supplementation with 2.0% succinic acid in HCD significantly improved the WGR, SGR, and PER of tilapia and reduced the FCR, indicating that succinic acid ameliorated the adverse effects of HCD on tilapia. Previous studies in zebrafish have reported that the growth promotion effect of succinate was related to the increase in FI (Ding et al., 2022). Nevertheless, succinic acid had no impact on the FI of tilapia, which might be related to different feeding strategies, fish size, and cultivation density. Quadratic curve model analysis based on WGR, SGR, PER, and FCR demonstrated that the optimal supplementation level with succinic acid in HCD ranged from 1.83% to 2.43% for tilapia, which was higher than that of zebrafish (0.15%) (Ding et al., 2022) and white shrimp (0.5%) (Duan et al., 2018), which might be due to fish needing more succinic acid to increase body metabolism in HCD and alleviate HCD stress.
Generally, herbivorous and omnivorous fish have a stronger capacity to utilize dietary carbohydrates, which may be related to enhanced AMS activity in vivo (Bautista et al., 1988). Organic acids have the capacity to slightly reduce the pH of the intestinal environment, which further facilitates the improvement of digestive enzyme activity and boosts the digestion and absorption of nutrients (Li et al., 2006; Luckstadt, 2012). Prior research revealed that the addition of organic acids enhanced the digestive enzyme activity in red drum (Sciaenops ocellatus) (Castillo et al., 2014). Furthermore, dietary supplementation with 0.5% succinic acid promoted both digestive enzyme activity and feed utilization in white shrimp (Duan et al., 2018). In the current study, dietary supplementation with 0.5% succinic acid enhanced the activities of AMS and LPS, indicating that succinic acid might enhance the ability to utilize carbohydrates and promote the digestion and absorption of nutrients by improving digestive enzyme activity to adapt to a HCD, which was conducive to the growth of tilapia.
4.2.　 Succinic acid supplementation in HCD improves the muscle antioxidant capacity of tilapia
Excessive dietary carbohydrate induces excessive production of free radicals, especially ROS, which leads to the oxidation of lipids and proteins in muscle, resulting in a decline in muscle quality (Cai et al., 2022; Wang et al., 2015). In vitro studies on the muscle cells of olive flounder (Paralichthys olivaceus) found that HCD induced oxidative stress and led to the apoptosis of muscle cells (Liu et al., 2022). In addition, oxidative stress reduced the physicochemical and textural properties of common carp (Cyprinus carpio) muscle (Morachis-Valdez et al., 2015). Organisms have an antioxidant defense system, which includes enzymatic and non-enzymatic antioxidants like SOD and CAT, to avoid oxidative damage (Cai et al., 2021). Dietary supplementation with organic acids enhances the antioxidant capacity of aquatic animals (Duan et al., 2017; Wang et al., 2015). Dietary supplementation with 0.5% succinic acid increased the antioxidant capacity of the hepatopancreas of white shrimp (Duan et al., 2018). In this study, dietary supplementation with 0.5% to 1.0% succinic acid significantly enhanced the antioxidant capacity of tilapia by improving the activity of endogenous antioxidant enzymes, such as CAT, SOD, and T-AOC. The exogenous addition of succinic acid stimulates the activity of succinate dehydrogenase (SDH), a common antioxidant, which simultaneously links the TCA with oxidative phosphorylation and imports electrons from the TCA into the electron transport chain (Adisa et al., 2019). Dietary succinic acid might accelerate the flow of paired electrons in the electron transport chain and accelerate the generation of electrochemical transmembrane potential to prevent the leakage of unpaired electrons, causing ROS production and the formation of lipid peroxides (Grishina et al., 2015). Therefore, succinic acid improves muscle quality after slaughter and during storage by reducing oxidative damage in muscle.
4.3.　 Succinic acid supplementation in HCD improves the muscle hardness of tilapia
Muscle texture is one of the most important parameters for reflecting muscle quality. The assessment of muscle texture is commonly based on its textural properties (Liu et al., 2020). Muscle hardness is the main indicator for consumers to evaluate aquatic products, and is positively correlated with flesh quality (Cai et al., 2022). Long-term intake of HCD significantly reduces muscle hardness, gumminess, and chewiness (Bao et al., 2022; Wu et al., 2021). In the current study, dietary supplementation with 1.0% succinic acid significantly increased muscle hardness, gumminess, chewiness, and resilience, indicating that succinic acid improves muscle texture, thereby improving taste. Our results are consistent with studies on mammals such as cattle (Gao et al., 2014) and fattening pigs (Liang, 2018).
The textural properties of fish flesh are significantly influenced by the diameter and density of muscle fibers (Koganti et al., 2020). Studies have shown that the muscle fiber diameter of Atlantic salmon (Salmo salar) (Johnston et al., 2000), sea bass (Dicentrarchus labrax) (Periago et al., 2005), and gilthead sea bream (Sparus aurata) (Valente et al., 2011) were negatively correlated with muscle hardness, while muscle fiber density was positively correlated with muscle hardness, chewiness, and collagen content (Bao et al., 2022). Dietary supplementation with succinic acid promoted the transformation of fast-twitch muscle fibers into slow-twitch muscle fibers in mice by activating the Ca2+ signaling pathway mediated by the succinate receptor 1 (SUCNR1) gene (Wang et al., 2019). In the present study, dietary supplementation with 1.0% succinic acid increased the muscle fiber density and the number of muscle fibers with a diameter ranging from 70 to 110 μm, and decreased the number of muscle fibers greater than 110 μm, which might promote the transformation of muscle fiber type, change the proportion of muscle fibers and thus significantly improve muscle quality.
The development of muscle fibers is regulated by MRF genes; MyoD and Myf5 regulate myoblast proliferation, while MyoG and MRF4 regulate myoblast differentiation (Koganti et al., 2020). Prior research has confirmed that excessive carbohydrate intake reduces the mRNA expression level of MRF gene and inhibits muscle fiber hyperplasia (Bao et al., 2022; Chapalamadugu et al., 2009). In the current study, dietary supplementation with 0.5% to 2.0% succinic acid significantly up-regulated the mRNA expression levels of MyoD, MyoG, Myf5, and MRF4, indicating that succinic acid promotes the development of muscle fibers. Myogenic regulatory factors are regulated by the mammalian target of rapamycin (mTOR) signaling pathway, and the anabolic process of mTOR signaling requires adequate energy from the TCA (Meng et al., 2023; Periago et al., 2005). Therefore, succinic acid might promote the mTOR signaling pathway by accelerating the oxidative phosphorylation process, thereby up-regulating the expression level of MRF gene, stimulating muscle cell development, and thereby increasing muscle fiber density. The aforementioned statement provides additional clarification regarding the potential mechanism underlying succinate-induced alterations in muscle fiber diameter and density.
Collagen content, which is directly proportional to muscle hardness, is a crucial determinant of fish flesh quality (Dong et al., 2022). The concentration of hydroxyproline is used to measure collagen (Hofman et al., 2011). Hydroxyproline is a metabolite of Pro catalyzed by proline hydroxylase (PHD). Proline hydroxylase is a type of dioxygenase dependent on oxygen and α-ketoglutarate, which is also the basis for collagen to maintain its triple helix structure (Yang et al., 2021). In addition, collagen is mainly synthesized by fibroblasts using Gly and Pro (Li and Wu, 2018). In this study, supplementation with 0.5% succinic acid significantly increased the contents of Pro, Gly, and hydroxyproline in muscle, suggesting that succinic acid might increase the content of muscle collagen by increasing the raw materials for collagen synthesis. On the other hand, succinic acid might activate PDH to promote collagen synthesis by increasing the concentration of α-ketoglutarate. Other research revealed that the content of hydroxyproline in muscle was positively correlated with the muscle texture of Atlantic salmon (Li et al., 2005) and Atlantic halibut (Hippoglossus hippoglossus) (Hagen et al., 2007), which improves muscle hardness. In the current study, fish fed with 0.5% to 1.0% succinic acid had higher muscle fiber density and total hydroxyproline content, which might be one of the reasons for the increase in the muscle hardness of tilapia.
4.4.　 Succinic acid supplementation in HCD improves the muscular nutritional value and flavor of tilapia
The levels of protein and amino acids in muscle determine its nutritional value and flavor (Jiang et al., 2016). In the present research, dietary supplementation with 1.5% succinic acid significantly increased muscle crude protein content, which might be related to the activation of Akt/mTOR/S6 and other signaling pathways related to protein synthesis (Liang, 2018). Protein is composed of amino acids, and EAA content is an important indicator of nutritional value in muscle. Studies have shown that TCA cycle intermediate metabolites are precursors for the synthesis of some amino acids. For example, oxaloacetate is a precursor for the synthesis of aspartate (Ling et al., 2023). Exogenous addition of α-ketoglutaric acid significantly increased the contents of amino acids such as Arg, Ala, Gly, and Glu in cells (Mühling et al., 2010). In the current study, dietary supplementation with 0.5% to 4.0% succinic acid significantly increased the contents of muscle ΣEAA and TAA, suggesting that succinic acid might promote the synthesis of bound amino acids and increase protein content by increasing the concentration of TCA cycle intermediate metabolites, thereby improving the nutritional value of tilapia muscle.
Muscle fatty acid contents and composition are important for human health and are important indicators of the nutritional value of muscle (Wood et al., 2008). According to the World Health Organization (WHO) report, reducing the intake of SFA and increasing the intake of unsaturated fatty acids (especially n-3PUFA and n-6PUFA) in the human diet effectively prevents chronic disease (Diet nutrition and the prevention of chronic diseases, 2003). The human body cannot synthesize EPA and DHA, so fish is the preferred source of high-quality PUFA for consumers. Studies have shown that dietary supplementation with 1.2% citrate significantly increases PUFA content in muscle (Xu et al., 2020). In addition, unsaturated fatty acids (especially n-3PUFA) are prone to lipid peroxidation, which reduces the content of PUFA in the body (Kamal-Eldin and Yanishlieva, 2002). In the current study, fish fed with 1.0% succinic acid had higher n-3PUFA content and lower total fatty acid content, suggesting that succinic acid changes the composition of fatty acids and increases the nutritional value of tilapia muscle by reducing lipid peroxidation, which was also confirmed by the significant reduction of MDA content in muscle.
Free amino acids are the main basis of muscle flavor in aquatic animals, including umami and bitterness (Herranz et al., 2006). Among the 14 free amino acids detected in this study, Asp and Glu were related to umami, Ala and Gly were related to sweetness, and Val, Met, Ile, Leu, His, and Arg were associated with bitterness. The delicious taste of fish depends on the composition and contents of free amino acids in muscle, especially the contents of free Asp, Glu, Gly, and Ala (Cheng, 2021). Studies have shown that dietary supplementation with 1.5% α-ketoglutaric acid significantly increased the contents of Thr, Ala, and Pro, as well as TAA and EAA, in the muscle of mirror carp (Wang et al., 2017). Furthermore, supplementation with 1.0% α-ketoglutaric acid significantly increased the contents of Glu, Lys, Ala, and Pro in the skeletal muscle of growing pigs (Chen et al., 2018). Succinic acid is a natural flavor enhancer that increases muscle umami (Subbaraj et al., 2016). In the present research, dietary supplementation with 1.0% succinic acid increased the contents of ΣFAA (Asp and Glu) and sweet amino acids (Ala and Gly) and decreased the ΣBAA (Val, Met, Ile, Leu, His, and Arg). Therefore, succinic acid might increase the flavor of muscle by increasing the content of flavor amino acids.
4.5.　 Succinic acid supplementation in HCD affects the glucose and lipid metabolism of tilapia muscle
Gluconeogenesis and glycolysis are two crucial metabolic processes for carbohydrate metabolism. Hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK) are three key enzymes in the glycolytic pathway, and phosphoenolpyruvate carboxykinase (PCK), fructose-1, 6-diphosphatase (FBPase) and glucose-6-phosphatase (G-6-pase) are the rate-limiting enzymes of gluconeogenesis (Enes et al., 2009; Polakof et al., 2011). Studies have shown that succinate significantly reduced liver G6Pase activity by regulating intestinal gluconeogenesis in mice, thereby reducing liver glucose production (De Vadder et al., 2016). In the current study, dietary supplementation with 1.0% succinic acid up-regulated the mRNA expression levels of glycolytic genes (HK2, HK1A, HK1B, PFKMA and PFKMB), indicating that succinic acid might promote glycolysis to accommodate HCD. In addition, excessive carbohydrates were converted into glycogen, which was stored in the muscles and liver to maintain blood glucose balance (Enes et al., 2009). In the current study, dietary supplementation with 1.0% succinic acid increased the contents of liver and muscle glycogen and significantly up-regulated the expression levels of glycogen synthesis (GYS) and glucose transporters (GLUT1 and GLUT4) genes, which was consistent with the results of previous studies in zebrafish (Ding et al., 2022). Therefore, succinic acid might promote glycolysis and glycogen deposition to maintain glucose homeostasis.
Long-term feeding of HCD might result in metabolic problems that cause fat storage and impair the growth performance of fish (Prisingkorn et al., 2017; Viegas et al., 2016). Thus, lipid anabolism plays a crucial role in maintaining glucose homeostasis. Fatty acid synthase is a key enzyme involved in lipid synthesis. Carnitine palmitoyltransferase-1 is considered to be the rate-limiting enzyme in fatty acid β-oxidation (Morash et al., 2008). Hormone sensitive lipase is an intracellular neutral lipase that hydrolyzes lipids such as triglycerides and cholesterol esters (Lampidonis et al., 2011). In this study, dietary supplementation with 0.5% succinic acid reduced the crude lipid content of the whole fish and liver, MFI, and muscle total fatty acid content, down-regulated the expression of the lipid synthesis gene (FASN), and up-regulated the expression of lipid catabolic genes (CTP1B, HSL, LPL), indicating that succinic acid might induce a change in muscle fatty acid content by promoting fatty acid decomposition and β-oxidation and inhibiting fatty acid synthesis. Furthermore, dietary supplementation with 0.5% to 2.0% succinic acid significantly reduced the HSI and VSI of tilapia, confirming that succinic acid reduces abnormal fat deposition, thereby increasing the proportion of edible parts (muscle). In addition, the contents of serum TG and TCHO reflected the status of lipid metabolism (Zhang et al., 2021). In this study, fish fed 1.0% to 4.0% succinic acid had lower contents of serum TG and TCHO. These results further confirm the promoting effect of succinic acid on lipid catabolism. Therefore, succinic acid might reduce muscle total fatty acid content by promoting fatty acid β-oxidation and inhibiting the fatty acid synthesis process.
5.　Conclusion
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Dietary supplementation with 1.83% to 2.43% succinic acid promoted the growth performance, digestion, absorption, and antioxidant capacity of Nile tilapia. Additionally, succinic acid changed the proportion of muscle fibers by up-regulating muscle regulatory factor genes to increase muscle hardness and altering muscle amino acid and fatty acid composition to improve muscle flavor and nutritional value (Fig. 7). Succinic acid also promoted glycolysis and fatty acid β-oxidation, inhibited lipogenesis, decreased the accumulation of fat in the whole-body, and maintained glucose homeostasis.
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doi: 10.1016/j.aninu.2024.04.016
  • Receive Date：2023-12-07
  • Online Date：2026-01-28
  • Published：2024-09-10
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  • Received：2023-12-07
  • Revised：2024-02-20
  • Accepted：2024-04-29
Affiliations
    aKey Laboratory of Freshwater Biodiversity Conservation, Ministry of Agriculture, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China
    bKey Laboratory for Animal Nutrition and Feed Science of Hubei Province, Wuhan Polytechnic University, Wuhan 430023, China

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(J. Tian).
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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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