Effects of Pantothenic Acid on Growth Performance, Oxidative Stress, and Lipid Content in High-Fat-Fed Megalobrama amblycephala

This study was to elucidate the effects of dietary pantothenic acid (PA) on growth, lipid metabolism, and antioxidant status in juvenile blunt snout bream Megalobrama amblycephala. Fish were fed with four experimental diets containing two lipid levels (5% and 11%) and two PA supplementation levels (24mg kg and 240mg kg) for eight weeks. The results showed that diets containing 11% lipid significantly increased condition factor, mesenteric fat index, triacylglycerol (TG), and cholesterol (TCH) in the liver as well as total lipid content and TCH in the muscle (P < 0:05), but significantly decreased specific growth rate and whole-body protein (P < 0:05). As the dietary lipid level increased, hepatic antioxidant enzyme activities were significantly increased (P < 0:05) as well as malondialdehyde content (P < 0:05). Besides, cytosolic cytochrome c concentration was also significantly (P < 0:05) increased, while mitochondrial protein, cytochrome c, and Na-K-ATPase activities were significantly decreased (P < 0:05). There were no significant changes on growth performance and body composition of PA supplementation. Then, significantly negative (P < 0:05) interactions were observed between dietary lipid and PA supplementation on EPA, DHA, and EPA+DHA content of the muscle. Saturated fatty acid (SFA) and polyunsaturated fatty acid (PUFA) contents in the liver were significantly increased (P < 0:05) with dietary lipid level increased, while liver monounsaturated fatty acid (MUFA) was significantly decreased (P < 0:05). Muscle SFA, DHA, and EPA+DHA contents were significantly decreased (P < 0:05) when dietary fat level increased. Meanwhile, EPA, DHA, and EPA+DHA content was significantly decreased (P < 0:05) with dietary high PA supplementation. On the whole, a high PA supplementation level of 240mg kg could alleviate lipid content accumulated and oxidative damage induced by a high-fat diet.


Introduction
The high utility of fat-rich feeds is a current trend in the modern aquaculture industry due to its protein sparing and growth promoting effects [1,2]. However, most freshwater fish cannot tolerate high dietary lipids, and excess lipid may lead to fatty liver and cause dyslipidemia [3,4]. According to our previous studies, the liver of blunt snout bream Megalobrama amblycephala showed a healthy status when fed with a 5% fat diet [4]. When given a 7% fat diet, blunt snout bream showed best weight gain and protein utilization [5]. However, when the dietary lipid level reached 11%, fat accumulation occurred in the liver of blunt snout bream [3]. Under normal physiological conditions, there is a balance between lipid deposition and decomposition to maintain lipid homeostasis in vivo. When blunt snout bream cannot tolerate high dietary lipid, the excess lipid may cause multiple adverse consequences, including retarded growth, fatty liver, fat deposition, and oxidative damage [4]. Thus, it is necessary to find an effective way to alleviate these problems.
Dietary manipulation has been proven to be a useful approach for alleviating the adverse effects. Pantothenic acid (PA) is an essential water-soluble vitamin for fish. PA functions as a component of coenzyme A (CoA) which plays a core role in numerous intermediary metabolic reactions in carbohydrates and lipid metabolism [6]. CoA forms a thioester bond with acyl carbons and thus mediates acyl transfer reaction of fatty acid synthesis, fatty acid oxidation, and cholesterol synthesis in diverse pathways [7]. Besides, PA also donates a 4 ′ -phosphopantetheine moiety to acyl-carrier protein (ACP), which carries the growing acyl chain from one enzyme to another [8]. Previous studies indicated that dietary PA deficiency in grass carp (Ctenopharyngodon idella) and grouper (Epinephelus malabaricus) would result in fish hepatic fat deposition and lipid metabolic dysfunction [9,10]. In a study of blunt snout bream, increased dietary PA levels enhanced the expressions of various genes involved in liver fatty acid synthesis, resulting in the enhanced liver SFA synthesis [11]. However, whether dietary PA has a positive regulatory function on the lipid metabolism in dyslipidemia fish is still unknown. Considering biological functions of PA in lipid metabolism, the investigations of PA on alleviating the adverse effect induced by a high-fat diet are worthwhile.
In order to maintain the body redox equilibrium, the antioxidant systems of the SOD, CAT, and GPX enzymes play important roles, while MDA content represents direct evidence of lipid peroxidation caused by free radicals or disordered lipid metabolism [11]. Besides, mitochondria which play a vital role in cellular homeostasis are considered to be the most critical subcellular organelles for mediating oxidative stress [12,13]. Meanwhile, mitochondria are also recognized as a significant source of reactive oxygen species (ROS) production and endogenous oxidative stress [14]. So far, studies have extensively investigated the effects of nutritional factors on the antioxidant defenses of fish. Recently, PA and its related compounds have been reported to protect the cell membrane against damage caused by lipid peroxidation in an in vitro study [15], indicating the potential correlation between dietary PA levels and body antioxidant capability. Our previous study showed the PA could increase liver CAT activities and decrease liver MDA content [11]. PA supplementation also increased hepatic GSH levels in Jurkat cells that was attributed to increased ATP production as a result of increased mitochondrial CoA levels [16]. Pantothenic supplementation also increased hepatic GSH and liver resistance to irradiation, suggesting that GSH peroxidase and phospholipid-hydroperoxide GSH peroxidase may have detoxified any hydroperoxides formed. Pantothenic acid also prevented the collapse of mitochondrial membrane potential and restored ATP synthesis levels as well as the activity of antioxidant enzymes, such as catalase, GSH peroxidase, GSH reductase, and the NADPH forming malic enzyme in vitro and in vivo [17]. However, little information is given about how dietary PA works on liver antioxidant capacity and how the mitochondrial respiration chain functions in dyslipidemia fish.
Bearing these in mind, the present study was conducted to elucidate the effects of dietary PA supplementation on growth, lipid metabolism, and antioxidant status of juvenile blunt snout bream (Megalobrama amblycephala), an economically important herbivorous freshwater fish widely cultured in China. The data can provide further information about nutritional modulation on antistress mechanisms and dyslipidemia in freshwater fish.

Material and Methods
2.1. Diets. A 2 × 2 factorial design with three replicates was used in the experiment. Four semipurified diets were formulated to contain two lipid levels (5 and 11%) and two PA supplementation levels (24 and 240 mg kg -1 ). Those four experimental diets were named as 5/24, 5/240, 11/24, and 11/240, representing the lipid level with PA supplementation representatively. Formulation and proximate composition of the basal diet were presented in Table 1. The experimental diets were formulated to meet the optimal dietary protein, lipid, and pantothenic acid requirements of blunt snout bream [5,11]. PA supplied in diets was detected to be 26.30 mg kg -1 and 249.70 mg kg -1 via high-performance liquid chromatography. All diets were prepared in the laboratory as detailed by Qian et al. [11].

Fish and Feeding
Trial. Blunt snout bream juveniles were obtained from the Fish Hatchery of Yangzhou (Jiangsu, China). The feeding trial was performed in an indoor recirculating aquaculture system. Fish were reared in several plastic tanks (3 × 0:8 × 0:8 m, L : W : H) for two weeks to acclimate to the experimental conditions before the experiment. During the acclimation period, fish were fed 5/24 experimental diets. Then, fish (25:97 ± 1:01 g) were randomly divided into 12 plastic tanks (3 × 0:8 × 0:8 m, L : W : H) and were fed with experimental diets to apparent satiation thrice daily (8:00, 12:00, and 16:00 h) for eight weeks.

Sample
Collection. At the end of the feeding trial, fish were deprived of feed for 24 h. The fish were then anaesthetized in diluted MS-222 (1 : 10000), counted, and weighed. Six fish per tank were collected and stored at -70°C for body composition analysis. A total of 8 fish per tank were sacrificed for blood, liver, and muscle collection. Then, the blood samples were allowed to clot at 4°C overnight and centrifuged at 5000×g at 4°C for 20 min to collect serum. Samples of the individual liver were quickly collected and stored at -70°C for determination of oxidative stress parameters and mitochondrial status analysis.

Proximate Composition
Analysis. The proximate composition of diets and fish were analyzed following the detailed methods [18]. Moisture was determined by drying to constant weight at 105°C. Crude protein (nitrogen × 6:25) was determined by using the Kjeltec Analyzer Unit (2300, FOSS Tector, Hoganas, Sweden). Crude lipid was determined by ether extraction method using the Soxtec Auto Extraction Unit (2050, Sweden). Ash content was measured by combustion at 550°C for 4 h.

Determination of Oxidative Stress Parameters in the
Liver. For analyzing oxidative stress in liver, we determined the concentration of total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GPX) activities, glutathione (GSH), and malondialdehyde (MDA) following the methods described by Lygren et al. [19]. All the measurements were operated by the commercial kits (Nanjing Jiancheng Bioengineering Institute, China). Aquaculture Nutrition

Isolation of Liver Mitochondrial and Cytoplasmic
Fractions. The isolation of liver mitochondria was followed by the methods of Suarez and Hochachka [20]. Briefly, liver tissue (0.1-0.2 g) was excised and homogenized (w/v = 1 : 10 ), then the homogenate was centrifuged twice at 800×g for 10 min at 4°C. The superficial lipid layer was removed. After that, the remaining supernatant was then centrifuged at 15,000×g for 15 min at 4°C. The supernatant and the residual pellet were considered to be the cytoplasmic fraction and the mitochondrial fraction, respectively. The obtained mitochondrial fraction was washed three times in medium and was then resuspended in the medium with 1 mg ml -1 fatty acid-free bovine serum albumin. Cytochrome c contents in mitochondrial and cytosol fractions were measured according to the method of Sun et al. [21]. For enzymatic analysis, the mitochondrial pellets were suspended in the isolation medium. Succinate dehydrogenase (SDH) was estimated with sodium succinate as substrate according to the method of Philip et al. [22]. The Na + -K + -ATPase (3.6.3.9) activity was measured according to McCormick and Björnsson [23]. Mitochondrial protein concentration was determined by using the method of Lowry et al. [24]. Enzymatic activities were expressed in units (U) per milligram of mitochondrial protein. All the measurements were operated by the commercial kits (Nanjing Jiancheng Bioengineering Institute, China).

Tissue Lipid Concentration and Fatty Acid Composition.
Total lipid content in liver and muscle was determined as described by Folch et al. [25] using chloroform : methanol (2 : 1, v/v) to extract the total lipid. Total triacylglycerol (TG) and total cholesterol (TCH) concentrations were measured with commercial kits (Nanjing Jiancheng Bioengineering Institute, China). According to the method of Xu et al. [26], the relative quantity of each FA of lipid was determined by measuring the area under the chromatograph peak.
2.8. Total RNA Extraction, Reverse Transcription, and Real-Time PCR. According to the method of Xu et al. [26], the relative expression of COX1, COX2, ND1, CYTB, and ATP6 were tested with real-time PCR analysis. Briefly, total RNA in mitochondrial of juvenile blunt snout bream was extracted using Trizol reagent (Invitrogen, USA) and treated with RQ1 RNase-free DNase (Promega, USA) to eliminate genomic DNA contamination. Quantity and purity of RNA was determined by absorbance measures (A260/280), and its integrity was tested by electrophoresis in 1.0% formaldehyde denaturing agarose gels. cDNA was generated from 500 ng DNase-treated RNA using ExScript™ RT-PCR kit (Takara, Japan). The resulting first-strand cDNA from each tissue was then diluted and used as template for PCRs. The set of primers for COX1, COX2, ND1 CYTB, and ATP6 genes were designed by Primer 5 according to the codifying sequences obtained in our laboratory (Table 2). Melting curve analysis of amplification products was performed at the end of each PCR reaction to confirm that the specific products were obtained. Each sample was run in triplicate. PCR reactions without the addition of the template were used as negative controls. At the end of the reaction, the fluorescent data were converted into Ct values. Each gene expression level was normalized to β-actin using the 2 −ΔΔCT method [27].

Statistical Analysis.
According to the method of Xu et al. [26], data were subjected to two-way analysis of variance (ANOVA) using the SPSS statistical package version 20.0 (SPSS Inc., Chicago Avenue, IL, USA) to test the effects of dietary lipid, PA (main effect), and interaction between lipid and pantothenic acid after testing the homogeneity of variances with the Levene test. If an interaction between dietary lipid and pantothenic acid was detected (P < 0:05), Duncan's multiple range test was used to rank the means. A significant (P < 0:05) main effect and no interaction allowed data to be pooled before mean separation (main effect means for dietary lipid or pantothenic acid) by Duncan's multiple range test. All data were presented as means ± S:E:M (standard error of the mean) of three replicates.

Growth Performance.
The growth performance was determined in juvenile blunt snout bream fed with experimental diets containing different lipid level and PA supplementation. The SGR of fish was reduced (P < 0:05) significantly as the dietary lipid levels increased from 5% to 11%, whereas CF and MFI were significantly increased (P < 0:05) (Table 3). VBR, HSI, and DP were not markedly influenced by dietary lipid and PA supplementation (P > 0:05) ( Table 3). There was no significant interaction effect on above mentioned growth performance parameters between PA supplementation and lipid contents (P > 0:05).

Whole-Body Composition.
For analyzing proximate composition of juvenile blunt snout bream, we determined the proportion of moisture, protein, lipid, and ash. As the dietary lipid content increased from 5% to 11%, the lipid content of fish increased markedly (P < 0:05), whereas   Aquaculture Nutrition protein content showed a significant decrease (P < 0:05) ( Table 4). No significant effects were observed on wholebody ash and moisture contents of juvenile blunt snout bream (P > 0:05). There was no significant interaction effect on whole-body composition between PA supplementation and lipid contents (P > 0:05).  Figure 1(a)). The nucleus was ovoid and contained a prominent nucleus (Figure 1(b)). The endoplasmic reticulum was well developed and often adjacent to the nucleus (Figure 1(a)). Hepatocytes displayed dark, slender mitochondria with well-developed cristae and matrix (Figure 1(b)). No significant differences were found between the 5/24 and 5/240 groups. However, some abnormalities were observed in livers of fish fed with diets containing 11% lipid. Hepatocytes exhibited many large, electrondense fat droplets (Figure 1(c)). These extensive intracellular lipid droplets resulted in the displacement of the nucleus to the cell margin and a loss of cytoplasm (Figure 1(d)). The nucleus was atrophic and presented as a polygon ( Figure 1(c)). The endoplasmic reticulum developed poorly and lost most of its ribosomes (Figure 1(d)). Disruption was observed in all of the cellular membranes, including plasma, mitochondria, and nuclear membranes. The mitochondria exhibited a loss of cristae and matrix, as well as great hydropic changes of matrix density (Figure 1(f)).

Lipid Levels in Tissues and Plasma.
To investigate the lipid levels in the liver, muscle, and plasma of blunt snout bream, we detected the total lipid content and the TG and TCH concentration (Table 5). No significant (P > 0:05) interaction effects between dietary lipid and PA were observed. In the liver, we observed significantly higher levels of total lipid, TG, and TCH in fish fed with 11% dietary lipid (P < 0:05). The significantly higher levels of total lipid and TCH were also found in the muscle. Plasma TG was significantly lower (P < 0:05). What is more, dietary PA supplementation showed no significant (P > 0:05) effects on body lipid level.
3.5. Fatty Acid Composition. The fatty acid composition of the liver and muscle was presented in Table 6 and Table 7.
In muscle, SFA and MUFA of C16: 1n-9 and C18: 1n-9 were significantly (P < 0:05) reduced when dietary lipid level increased from 5% to 11%. Meanwhile, PUFA was significantly increased due to C18: 2n-6, C18: 3n-3, and C18: 3n-6 (P < 0:05), rather than DHA (P < 0:05). However, DHA and EPA + DHA content was significantly decreased regardless of increased lipid level or PA supplementation. Significant (P < 0:05) interactions between dietary lipid and PA supplementation were found on muscle EPA, DHA, C22: 5n-3, and EPA + DHA of blunt snout bream, which also showed negative effect with PA supplementation increased.   Aquaculture Nutrition 3.6. Liver Antioxidant Status. Oxidative stress parameters in the liver were remarkably affected by the high-fat diet. As the dietary lipid level increased from 5% to 11%, we found a significant increase in the concentration of MDA, T-SOD, CAT, and GSH-Px, whereas a decrease in the concentration of GSH (P < 0:05) ( Table 8). As dietary PA supplementation increased, GSH-Px activity of the liver was significantly decreased (P < 0:05), whereas the concentration of GSH significantly increased. T-SOD, CAT, and MDA concentrations showed a decreased tendency but without significance (P > 0:05). No significant interaction effects were found on antioxidant status between dietary lipid levels and PA supplementation (P > 0:05).

Discussion
The present study showed that high PA supplementation level of 240 mg kg -1 could improve lipid content accumulated and oxidative damage induced by high dietary fat. Lipids, as essential nutrients including phospholipids and essential fatty acids, play an essential role in the growth and development of various creatures [28]. The proper dietary lipid and protein ratio supports higher growth rates, partly based on protein-sparing in many species, including blunt snout bream [2]. However, the present study showed that dietary 11% lipid significantly suppress the specific growth rate of fish, which is similar to results obtained in blunt snout bream, golden pompano (Trachinotus ovatus), and largemouth bass (Micropterus salmoides) fed with 11%, 18%, and 18% fat diet, respectively [3,26,29,30]. From the perspective of energy utilization, a possible reason may be that excessively high, dietary fat-driven energy intake results in the imbalance of metabolism and uptake of other nonlipid nutrients of fish [31,32]. Higher condition factor, mesenteric fat index, and whole-body crude lipid of fish fed with 11% fat dietary also confirmed the explanation. According to our previous study, the growth performance came to a plateau when the dietary PA supplementation level reached an optimal point of 24 mg kg -1 at 5% fat and 31% protein level diet of blunt snout bream, and no adverse effect was found between high-PA dietary (~64 mg/kg) and growth performance [11]. Though insignificance, the present study still showed a better growth performance of fish fed with high PA supplementation than that of fish fed with low PA supplementation at 11% fat level, which might be due to improved digestive enzyme activities caused by PA supplementation [11]. In addition, grass carp and Bloch (Channa punctatus) fed 38 and 28 mg kg -1 PA diet, respectively, have the highest growth performance, which decreased as dietary PA supplementation increased [9,33]. In this light, despite that the better growth performance of blunt snout bream was observed in the present study (that is, just tiny positive trend), the dose of 240 mg kg -1 PA supplementation is still higher for blunt snout bream fed with 11% fat diet. Considering different sensitivity among species to pantothenic acid, the optimal PA supplementation diet on blunt snout bream fed with an 11% diet needs further study.
As is well known, dietary lipid level is closely associated with fish whole-body lipid content, and an excessive lipid diet is generally accompanied with increased fat deposition in the liver, viscera, and muscle [29,34,35]. The lipid homeostasis of the liver is maintained by the uptake, secretion, and transport of lipid. Previous studies suggested that the blocking of hepatic lipid secretion makes the imbalance between the secretion and uptake of lipid responsible for hepatic lipid accumulation in rats and blunt snout bream, respectively [4,36]. In this study, the liver lipid content was significantly higher in fish fed with a high-fat diet than fish fed with a low-fat diet. These results indicated that the ability to transport lipid out of the liver is attenuated, resulting in higher liver TG and TCH content after the intake of a high-fat diet [4]. As lipids are absorbed from the gut and transported to the liver for further processing, plasma TG and TCH concentrations would be elevated after high-fat feeding [4,37]. Thus, the reduced plasma TG and TCH further indicated that the capacity of endogenous lipid transport out of the liver in fish fed with high-fat diets is 10 Aquaculture Nutrition weakened, resulting in liver fat deposition. Recently, many studies have suggested that PA supplementation and its derivatives can reduce plasma TG and TCH, but the effect showed a dose specificity which is manifested as excess PA supplementation inhibiting the lipid elimination [38][39][40].
In agreement with these data, the lipid content in the present study was increased when dietary 240 mg/kg PA supplementation is at 5% fat level, while lipid content was reduced when dietary 240 mg/kg PA supplementation is at 11% fat level. In addition, increased liver DHA and EPA content may be another factor to explain the attenuated synthesis and secretion of triglycerides [41]. These results indicated that dietary pantothenic acid can regulate lipid metabolism in blunt snout bream fed with high-fat diet. Generally, a high-fat diet is easier to generate more reactive oxygen species (ROS) that cause oxidative stress and oxidative destruction of cellular membranes [29,42]. Fortunately, fish have the enzymatic and nonenzymatic antioxidant system, including SOD, CAT, glutathione peroxidase (GSH-Px), and GSH, which have been confirmed to protect cells and tissues against ROS [43]. However, the imbalance between generation and elimination of ROS would lead to the accumulation of MDA and other lipid peroxides. Therefore, MDA content is considered as an indicator of lipid peroxidation [44]. In the present study, fish fed with a high-fat diet showed higher liver antioxidant enzyme activities and MDA content that indicated oxidant stress does occur and activate the antioxidant system. On the contrary, regardless of dietary lipid level, dietary high-PA supplementation reduces liver oxidative damage and improves liver health, as evidenced by lower MDA content. Consistently, dietary PA supplementation enhanced the antioxidant defense capability of grass carp and white Pekin ducks, thereby reducing liver oxidative damage [9,45]. This beneficial effect of PA, as an antioxidant, is attributed to its ability to protect the cell by increasing glutathione level, promote cellular repair mechanisms through CoA by potentiating synthesis of membrane phospholipids, and directly scavenge ROS generation [11,15,33,45,46]. Moreover, the increased ability of antioxidation and the decreased oxidation damage may be another explanation for the improved growth performance. As the antioxidant system is energy-consuming, liver mitochondria, where most cellular ROS is generated, play a critical role in the process [47]. However, long-term feeding of a high-fat diet caused lipid deposition and nuclear polarization, resulting in damaging the functionality of the liver and mitochondria, which agreed with previous research finding in mice fed with HFD [48]. As the consequence of structural and functional damages of cells and mitochondria, a large amount of cytochrome c is released into the cytoplasm and eventually leads to mitochondrial dysfunction and cell apoptosis which is manifested as ATP deficiency, ROS stress exacerbation, and Na + /K + -ATPase activity inhibition [12,47,[49][50][51]. Dietary PA deficiency might cause oxidative stress of juvenile blunt snout bream. Optimal PA level could alleviate the cellular and/or molecular damages caused by oxidative stress [11,52].
Slyshenkov [53] studied that PA alleviated the ultraviolet-induced decrease of glutathione content, dimin-ished lipid peroxidation, and partly protected the cells against apoptosis produced by ultraviolet irradiation. In the present study, dietary high-PA supplementation decreased lipid droplet accumulation in the liver and alleviated mitochondria structural and functional damages caused by a high-fat diet, which may be explained by higher CoA content synthesized from PA [11]. Hence, sufficient ATP produced by oxidative phosphorylation in mitochondria of the liver cell may support greater antioxidant status, which at least is manifested as higher GSH content, less oxidative damage, higher mitochondrial cytochrome c, and lower cytosolic cytochrome c. Dietary PA may decrease lipid peroxidation caused by high-fat diet to maintain body lipid content.
In conclusion, blunt snout bream fed with high-fat diets showed poor growth performance, severe oxidant stress, and enhanced lipid accumulation in tissues, while dietary PA supplementation increased from 24 mg kg -1 to 240 mg kg -1 could alleviate these negative consequences by reducing lipid deposition and improving energy utilization and antioxidant ability. Though high dose showed a positive effect on alleviating adverse effects caused by a high-fat diet, the optimal dose of PA supplementation in an 11% fat level diet and its mechanism still need further study.

Data Availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.