Effects of Supplementation of Ferulic Acid (FA) on Growth Performance, Activities of Digestive Enzymes, Antioxidant Capacity and Lipid Metabolism of Large Yellow Croaker (Larimichthys crocea) Larvae


 A 30-day feeding trial was conducted to investigate effects of supplementation of ferulic acid (FA) on growth performance, activities of digestive enzyme, antioxidant responses and lipid metabolism of the large yellow croaker (Larimichthys crocea) larvae. Four isonitrogenous and isolipidic micro-diets were formulated with graded levels of FA (0, 20, 40, and 80 mg/kg). Results showed that larvae fed the diet with supplementation of 40 mg/kg FA had significantly higher survival rate, while the specific growth rate was significantly higher in larvae fed diets with 40 and 80 mg/kg FA than the control group (P < 0.05). Activities of trypsin in pancreatic segments (PS) and intestinal segments, lipase in PS and Alkaline phosphatase in brush border membrane were significantly increased by supplementation of FA compared to the control group (P < 0.05). Supplementation of FA significantly increased activities of total superoxide dismutase and catalase, and reduced the malondialdehyde content compared to the control group. Meanwhile, activities of lysozyme, total nitric oxide synthase and nitric oxide content were significantly improved by supplementation of FA in diets. Furthermore, results revealed that supplementation of FA reduced the lipid accumulation in visceral mass of larvae fed the diet with 40 mg/kg FA probably through inhibiting gens expression of lipogenesis-related genes (scd1, fas and dgat2) and promoting expression of lipid catabolism-related genes (aco, cpt1, and hl). In conclusion, appropriate supplementation of 40 mg/kg FA could improve the survival and growth performance of large yellow croaker larvae through increasing digestive, antioxidant capacity and promoting lipid metabolism.


Introduction
Ferulic acid (FA) is a hydroxycinnamic acid extracted from natural products, especially the Chinese herb Ferula sinkiangensis K. M. Shen ( (Zhou et al. 2020) and anti-cancer (Chaudhary et al. 2018;Middleton et al. 2000). In recent years, researches in the application of FA were carried out in aquatic animals. Previous studies have shown that supplementation of FA can improve the growth performance, antioxidant capacity and immune response of Nile tilapia (Oreochromis niloticus), and promote the development of digestive system by improving intestinal morphology and microbiome composition (Dawood et al. 2020;Yu et al. 2020a, b). Moreover, the supplementation of FA in diets enhanced the integument color and inhibited oxidative stress in Red Sea Bream (Pagrus major) (Maoka et al. 2008).
Large yellow croaker (Larimichthys crocea) is a carnivorous marine sh species which was widely cultured in southeast China due to delicious taste and commercial value (Feng et al. 2017). Compared with juvenile and adult sh, larval phase is critical to ful ll the development of organ systems of the physiological functions (Huang et al. 2020; Liu et al. 2020). However, the production of larvae is still hampered by high mortality during the metamorphosis and weaning (Yao et al. 2020). Nutritional studies on sh larvae had focused on feeding habits, digestive physiology and nutritional requirements (Ai et  Nevertheless, there was no report even concerned about activities of digestive enzyme and the lipid metabolism of supplementation of FA in marine sh larvae. Thus, the present study intended to evaluate the potential effects of supplementation of FA on growth performance, activities of digestive enzyme, antioxidant capacity and lipid metabolism of large yellow croaker larvae.

Sampling and dissection
At the beginning of the experiment, initial body length (IBL) and initial body weight (IBW) were measured by fty larvae of 15 DAH collected randomly from each tank. At the termination, the survival rate (SR) was determined by counting the remaining individuals in each tank. All 45 DAH larvae were fasting for 24 h at the end of the experiment to empty digestive tract, and then were sampled. Fifty individuals were randomly selected from each tank to monitor the nal body length (FBL) and nal body weight (FBW). Twenty larvae in each tank were dissected on ice to obtain visceral masses containing a crude mixture of pancreas, liver, heart, spleen, and intestine. The visceral masses were quickly put into 2.0 mL RNase-free cryogenic vials, then immediately frozen in liquid nitrogen for gene expression analysis. Fifty larvae were separated under a dissecting microscope on a glass plate maintained at 0 ℃ to obtain pancreatic segments (PS) and intestinal segments (IS) for the determination of activities of digestive enzyme.

Body composition
After sampling, remaining larvae were taken out from each tank to measure total crude protein, crude lipid, and moisture. The moisture content was determined by placing the sample in a ventilated drying oven at 105 ℃ until the larval weight was constant. The crude protein and the crude lipid of the sample were determined following the method of Association with O cial Analytical Chemists (AOAC 2003).
Digestive enzyme activities assay Samples of 0.2-0.3 g PS and IS were homogenized in 2 mL 0 ℃ phosphate-buffered saline (pH = 7.4) respectively and centrifuged at 3300 g for 10 mins. The supernatant was collected for further determination.
Puri ed brush border membranes (BBM) from homogenate of the intestinal segment were obtained according to a method described by Crane et al. (1979). The activity of trypsin was assayed in accordance with Holm et al. (1988). The activity of leucine-aminopeptidase (LAP) was assayed according to Ji et al.
(2013) and Maroux et al. (1973). Several assay kits including total protein quantitative assay kit, α-Amylase assay kit, lipase assay kit, and alkaline phosphatase (AKP) assay kit were purchased from Nanjing Jiancheng Institute of Biological Engineering, China. All experiments were carried out in strict accordance with the instructions.

Antioxidant and innate immune enzyme activities assay
The visceral mass of sh larvae was weighed and homogenized in 0 ℃ phosphate-buffered saline (pH = 7.4). The proportion of tissue (g) and saline (mL) was 1:9. The homogenate of visceral mass was then centrifuged at 3300 g for 10 mins, and the supernatant was used for the assay of activities of antioxidant and innate immune enzyme. Activities of total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC) catalase (CAT), and Malondialdehyde (MDA) content as well as activities of lysozyme (LZ), total nitric oxide synthase (T-NOS), inducible nitric oxide synthase (iNOS) and nitric oxide (NO) content in visceral mass were determined by commercial reagents and kits (Nanjing Jiancheng Bio-Engineering Institute, China).

RNA extraction and real-time quantitative PCR
The sample was pulverized in liquid nitrogen and added with Trizol reagent (Takara, Japan). The total RNA was extracted according to the manufacturer's protocol. The integrity of RNA was evaluated by electrophoresis, and the total RNA concentration was measured using a Nano Drop® 2000 spectrophotometer (Thermo Fisher Scienti c, USA). RNase-Free DNase (Takara, Japan) was using to remove the DNA contaminant in RNA. The RNA was reversely transcribed to cDNA by Prime Script-RT reagent Kit (Takara, Japan). The real-time quantitative polymerase chain reaction was carried out in a quantitative thermal cycler (CFX96TM Real-Time System, BIO-RAD, USA). The primers sequence of stearoylcoenzyme A desaturase 1 (scd1), fatty acid synthase (fas), diacylgycerol acyltransferase (dgat2), sterolregulatory element binding protein 1 (srebp1), acyl-CoA oxidase (aco), carnitine palmitoyl transferase 1 (cpt1), peroxisome proliferators-activated receptor (pparα), hepatic lipase (hl), lipoprteinlipase (lpl), fatty acid binding protein 3 (fabp3), fatty acid binding protein 10 (fabp10), fatty acid binding protein 11 (fabp11), microsomal TAG transfer protein (mtp), apolipoprotein b100 (apob100) and β-actin, were synthesized based on the published sequences from Cai et al.  Table 2). Real-time quantitative PCR temperature pro le was 95 ℃ for 2 mins, followed by 39 cycles of 95 ℃ for 10 s, 59 ℃ for 10 s, and 72 ℃ for 20 s. The uorescence data acquired during the extension phase were normalized to β-actin via 2 −ΔΔCT methods as described by Livak and Schmittgen (2001). The parameters were calculated as follows: Survival rate (SR, %) = N t × 100 / N 0 Speci c growth rate (SGR, %/day) = (Ln W t -Ln W 0 ) × 100 / d where N t is the nal number of larvae in each tank, and N 0 is the initial number of larvae in each tank; W t is the nal wet body weight (g), and W 0 is the initial wet body weight; d is the experimental period in days.
All data were subjected to perform statistical analysis by using SPSS Statistics 25.0 software (SPSS Inc., USA). The data were rstly analyzed by using one-way analysis of variance (ANOVA), and then determined by Tukey's multiple range test. The level of signi cance was chosen at P < 0.05. Results were expressed as mean ± S.E.M. (Standard error of means).

Survival, growth performance and body composition
With the supplementation of FA increasing from 0 to 40 mg/kg, the SR of large yellow croaker larvae signi cantly increased from 15.50 to 21.42% (P < 0.05) ( Table 3). Meanwhile, larvae fed the diet with 40 and 80 mg/kg FA showed signi cantly higher SGR than the control group (P < 0.05) ( Table 3). The crude protein of larvae showed an increasing trend, while the total lipid was decreasing from 20.45 to 19.32% with FA supplementation from 0 to 20 mg/kg, but no signi cant differences were observed among dietary treatments (P > 0.05) ( Table 4).  No signi cant difference in the activity of amylase in larval PS and IS were observed among all dietary treatments (P > 0.05) ( Table 5). The activity of trypsin in PS of larvae fed the diet with 20 mg/kg FA and trypsin in IS in larvae fed diets with 20 and 40 mg/kg FA were signi cantly higher than the control group (P < 0.05) (Table 5). Meanwhile, the activity of lipase in larval PS were signi cantly higher in larvae fed diets with 20 and 40 mg/kg FA than the control group (P < 0.05) ( Table 5). The activity of AKP in BBM of larvae fed diets with 20 and 40 mg/kg FA had signi cantly higher activities compared to the control group (P < 0.05), while no difference was observed among dietary treatments in LAP of larvae BBM (P > 0.05) ( Table 5).

Antioxidant and innate immunity capacity
The activity of T-SOD was signi cantly higher in larvae fed the diet with 40 mg/kg FA compared to the control group (P < 0.05) (Fig. 1A). However, the activity of T-AOC was not signi cantly different among dietary treatments (P > 0.05) (Fig. 1B). The activity of CAT in larvae fed diets with 20 and 40 mg/kg FA was signi cantly higher than the control group (P < 0.05) (Fig. 1C). Meanwhile, the MDA content in larvae fed diets with 20 and 40 mg/kg FA were signi cantly lower than the control group (P < 0.05) (Fig. 1D). The activity of LZ in visceral mass was signi cantly higher in larvae fed diets with 20 and 40 mg/kg FA than the control group (P < 0.05) ( Fig. 2A),while larvae fed the diet with 40 mg/kg FA had the signi cantly higher activity of T-NOS compared to the control group (P < 0.05) (Fig. 2B). No signi cant difference was observed in the activity of iNOS among dietary treatments (P < 0.05) (Fig. 2C).The NO content in larvae fed diets with 40 and 80 mg/kg FA were signi cantly higher than the control group (P < 0.05) (Fig. 2D).
Triglyceride (TG) content and mRNA expression of lipid metabolism-related genes in visceral mass TG content of visceral mass in larvae fed the diet with 40 mg/kg FA was signi cantly lower than the other groups (P < 0.05) (Fig. 3A). In terms of the lipogenesis-related mRNA expression, mRNA expression of scd1 and fas were signi cantly lower in larvae fed the diet with 80 mg/kg FA than the control group (P < 0.05), while supplementation of 40 mg/kg FA signi cantly reduced the mRNA expression of dgat2 (P < 0.05) (Fig. 3B). For the fatty acid catabolism-related genes expression, compared to the control group, larvae fed the diet with 80 mg/kg FA had signi cantly higher mRNA expression of aco, and larvae fed diets with 40 and 80 mg/kg FA had signi cantly higher mRNA expression of cpt1 and hl (P < 0.05) (Fig. 3C). No signi cant difference was found in the mRNA expression of pparα and lpl among all treatments (P > 0.05) (Fig. 3C). For the lipid transport-related genes, the mRNA expression of fabp3 was signi cantly lower in larvae fed diets with 20 and 80 mg/kg FA (P < 0.05) and the mRNA expression of fabp11 was signi cantly reduced by supplementation of 20 and 40 mg/kg FA (P < 0.05) compared to the control group (Fig. 3D).
Larvae fed the diet with 40 mg/kg FA had signi cantly higher fatp1 expression than the control group (P < 0.05) (Fig. 3D). No signi cant differences was found in the mRNA expression of mtp, fabp10 and apob100 among all dietary treatments (P > 0.05) (Fig. 3D).

Discussion
Results of the present study demonstrated that the survival and growth performance of large yellow croaker larvae were signi cantly enhanced by supplementation of 40 mg/kg FA, which was consistent with previous In the present study, FA in diets not only improved the activity of trypsin (PS and IS) and lipase (PS), but also signi cantly improved activities of AKP (BBM), indicating the improvement of digest ability of large yellow croaker larvae. These results indicated that FA could improve activities of digestive enzymes of large yellow croaker larvae, which were consistent with the results in Nile tilapia (Yu et al. 2020b).
The antioxidant function of sh larvae is not mature, which made the larvae susceptible to the in uence of external environment and resulted in slower growth (Birnie-Gauvin et al. 2017). Various kinds of stresses may trigger the production of reactive oxygen species (ROS), then cause oxidative injuries such as lipid peroxidation and DNA damage (Martínez-Álvarez et al. 2005). In order to cope with the stresses, antioxidant defenses system has been developed, containing T-SOD, CAT, and T-AOC (Martínez-Álvarez and Morales 2005). In the present study, activities of SOD and CAT in larvae fed diets with FA were signi cantly higher than the control group. The result was similar to the study in Nile tilapia (Yu et al. 2016(Yu et al. , 2020a, which found that the supplementation of dietary FA could decrease the MDA content and increase activities of SOD and CAT. MDA is the biomarker of oxidative damage (Del et al. 2005). In the present study, MDA content in larvae fed diets with 20 and 40 mg/kg FA were signi cantly decreased compared to the control group, suggesting that FA reduced the oxidative damage of large yellow croaker larvae. Also, Maoka et al. . Compared with the control group, supplementation of FA activated aco, cpt1 and hl genes thereby activating fatty acid oxidation and accelerating the lipid consumption process. The result was similar in mice with Ma et al. (2019). The mRNA expression of fabp3 and fabp11 in larvae fed diets with FA were markedly decreased compared to the control group. Results above showed that FA could reduce the lipogenesis and accumulation of large yellow croaker larvae, which was achieved by reducing the expression of lipogenesis-related genes, up-regulating the expression of lipid catabolismrelated genes and down-regulating the relative expression of transport-related genes. Combined with the growth of large yellow croaker larvae in this study, we have reason to suspect that supplementation of FA in diets can promote the SR and SGR of large yellow croaker larvae through improving the lipid metabolism of larvae.

Conclusions
In conclusion, results of the present study demonstrated that appropriate supplementation of FA (40 mg/kg) could promote growth performance of large yellow croaker larvae, which was probably due to its improvement in activities of digestive enzymes, antioxidant capacity and lipid metabolism.