Effects of Barranca yajiagengensis Powder in the Diet of Trachinotus ovatus on the Growth Performance, Antioxidant Capacity, Immunity and Morphology of the Liver and Intestine

Barranca yajiagengensis, a novel filamentous microalga, can accumulate lutein under high-light and low-nitrogen conditions. It is well known that lutein has antioxidant, anti-inflammatory and immune-modulating properties. The purpose of this study is to evaluate the effects of including lutein-rich B. yajiagengensis powder in the diet of Trachinotus ovatus on the growth performance, antioxidant capacity, immunity, liver, and intestinal morphology. For this aim, three experimental diets containing 0% (BY0), 1% (BY1), and 5% (BY5) B. yajiagengensis powder were formulated for six-week feeding trials. The results indicated that growth performance, feed utilization, and intestinal morphology were not affected by different diet treatments. Fish fed with the BY5 diet promoted antioxidant ability by activating the Nrf2-ARE signal pathway and enhancing antioxidant enzymes activities. Furthermore, the BY5 diet improved non-specific immunity and antibacterial ability by activating lysozymes and the complement system and increasing the nitric oxide (NO) content and total nitric oxide synthase activity. Dietary B. yajiagengensis supplementation improved the liver morphology and exerted hepatoprotective effects. Therefore, as a natural source of lutein, B. yajiagengensis has the potential as a safe and non-toxic immunostimulant for T. ovatus. A diet supplemented with 5% B. yajiagengensis is recommended to improve the growth, antioxidant capacity, immune response, and liver health of T. ovatus.


Introduction
The golden pompano (Trachinotus ovatus) is a marine fish widely distributed in China, Japan, Australia, and Southeast Asia. It has become one of the most economically important mariculture fish species in southern China because of its fast growth rate, high-quality flesh, wide salinity tolerance, increasing market demand, and considerable economic efficiency. In China, the annual production of T. ovatus in 2020 was 101,693 metric tons [1]. At present, inshore net pens and intensive breeding mode are widely used in the culture of T. ovatus in China and Southeast Asian. However, owing to the polluted inshore water and extreme weather (e.g., typhoon, high temperature, rainstorm), T. ovatus cultured in intensive modes are more susceptible to infectious diseases and experimental stresses. Antibiotics are often used against such diseases, which not only does not result in satisfactory effects, but also threats food safety. Therefore, dietary supplementation with immunostimulants to enhance were collected by mesh filtration, and then freeze-dried by freezing dryer to obtain algae powder. The B. yajiagengensis powder contained 0.33% lutein and 4.4% linole ( Figure 1). Three isonitrogenous and isolipidic experimental diets were formulated to c 0% (BY0), 1% (BY1), and 5% (BY5) B. yajiagengensis powders. The formula and nu levels of experimental diets were presented in Table 1. All ingredients were thoro ground using a grinder and then sifted through a 100 μm sieve to obtain a uniform ple. Based on the feed formula, all required ingredients were weighed and thoro mixed. Then, distilled water (300 g kg −1 dry matter) was added to the mixture. Th pared mixtures were transferred into a puffing apparatus (Institute of Chemical Eng ing, South China University of Technology, Guangdong, China), and the final 2. diameter puffed pellets were produced. The diets were dried in an air-conditioned until the moisture level was below 10%, and then stored at −20 °C.  Three isonitrogenous and isolipidic experimental diets were formulated to contain 0% (BY0), 1% (BY1), and 5% (BY5) B. yajiagengensis powders. The formula and nutrient levels of experimental diets were presented in Table 1. All ingredients were thoroughly ground using a grinder and then sifted through a 100 µm sieve to obtain a uniform sample. Based on the feed formula, all required ingredients were weighed and thoroughly mixed. Then, distilled water (300 g kg −1 dry matter) was added to the mixture. The prepared mixtures were transferred into a puffing apparatus (Institute of Chemical Engineering, South China University of Technology, Guangdong, China), and the final 2.5-mm-diameter puffed pellets were produced. The diets were dried in an air-conditioned room until the moisture level was below 10%, and then stored at −20 • C.

Fish and Feeding Trial
The nutritional feeding trial was conducted in Lingshui bay, Hainan, China. T. ovatus juveniles were obtained from a local commercial company and acclimatized in a floating sea cage (3.0 m × 3.0 m × 5.0 m) for 14 days. During the acclimation, fish were fed two times daily with the control diet. At the beginning of the trial, all fish were fasted for 24 h, and then 180 fish of similar size (7.06 ± 0.12 g) were selected and randomly assigned into 9 sea cages (1.0 m × 1.0 m × 1.5 m) at a density of 20 fish per cage. Each experimental diet was randomly applied to three cages. Fish were fed slowly by hand to apparent satiation twice daily (08:00 and 17:00) for 42 days. Feed consumption and the number and weight of dead fish were recorded every day.

Sampling Collection
The animal use protocol listed below has been reviewed and approved by the Institution Animal Care and Use Committee, Sun Yat-Sen University (approval code: SYSU-IACUC-2022-B0159). At the end of the trial, the feeding process stopped for 24 h and then fish were anesthetized with tricaine methanesulfonate (Sigma, St. Louis, MO, USA). All fish from each replication were counted and weighed to determine growth performance. Blood samples were collected from six fish per cage by a 1 mL syringe from the caudal vein, then stored overnight at 4 • C, and finally centrifuged (4 • C, 4000 r/min, 10 min) to collect the serum for analysis of hematological parameters and antioxidant enzyme activities. The liver and midgut of the aforementioned six fish were removed and washed with double distilled water (4 • C) on ice, and then rapidly frozen in liquid nitrogen for analysis of digestive enzymes, antioxidant enzymes, and gene expression. Finally, the liver and midgut samples from three fish per cage were removed and fixed in 4% paraformaldehyde for histological analysis.

Biochemical Analysis of B. yajiagengensis and Experimental Diets
The total lipids in the B. yajiagengensis powder were determined with the gravimetric method following the procedures described by Gao et al. (2016) [16]. The fatty acid profiles of B. yajiagengensis were analyzed by gas chromatograph (6890 N GC, Agilent Technologies, Palo Alto, CA, USA) according to the procedures designed by Zhang et al. (2018) [17]. Total carbohydrate content of B. yajiagengensis powder was measured by the phenolsulfuric acid method (Wang et al. 2019) [18]. Total protein of B. yajiagengensis powder was quantified by commercial assay kits (Sangon Biotech, Shanghai, China) based on the Lowry method. The lutein content in the B. yajiagengensis powder was measured according to Gao et al. (2022) [13].
Crude protein, crude lipid, moisture, and ash contents in experimental diets were measured following the method described by the Association of Official Analytical Chemists (AOAC, 1995) [19].

Enzyme Activity Assays
The midgut and liver samples were weighed and homogenized in ice-cold normal saline at a volume ratio of 1:9, and then centrifuged at 3000 r/min (4 • C) for 10 min to obtain the supernatant. The supernatant of liver and midgut tissues as well as serum were used for the determination of enzyme activity.
Antioxidant parameters from the lipid soluble antioxidant system, including superoxide dismutase (SOD, EC1.15.1.1), catalase (CAT, EC1.11.1.6), and glutathione peroxidase (GSH-PX, EC1.11.1.9), plus total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content, were measured with kits NJBI (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, the supernatant of the liver sample was diluted 30 times and used for the determination of SOD activity at 37 • C, and the absorbance was measured at 530 nm. The supernatant of the liver sample was diluted eight times and used for the determination of CAT activity at 37 • C, and the absorbance was measured at 405 nm. The supernatant of the liver sample was used for the determination of GSH-PX activity at 37 • C, and the absorbance was measured at 412 nm. The serum SOD activity was measured at 37 • C, and the absorbance was measured at 530 nm.
The activities of digestive enzymes in the midgut, including amylase (AMS, EC3.2.1.1), pepsin (PEP, EC3.4.23.1), and lipase (LPS, EC3.1.1.3), were measured spectrophotometrically by kits from NJBI, China. Briefly, the supernatant of the midgut sample was diluted 20 times and used for the determination of AMS activity at 37 • C, and the absorbance was measured at 660 nm. The supernatant of the midgut sample was used for the determination of PEP activity at 37 • C, and the absorbance was measured at 660 nm. The supernatant of the midgut sample was used for the determination of LPS activity at 37 • C, and the absorbance was measured at 580 nm.

RNA Extraction and Gene Expression Analysis
The total RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were performed according to Zhao et al. (2020) [14]. Briefly, total RNA from the liver in each cage was isolated using a reagent kit (TaKaRa, Dalian, China) following the manufacturer's protocol. RNA sample quality was assessed by 1% agarose gel electrophoresis and quantified by spectrophotometric analysis (OD260/280) using the Nanodrop Lite (Thermo Scientific, Waltham, MA, USA). Then, the total RNA samples were diluted to the same concentration with diethylpyrocarbonate treated water for normalization. Subsequently, cDNA was synthesized using a PrimeScript RT Reagent kit with gDNA Eraser (TaKaRa, Dalian, China) according to the provided instructions. qRT-PCR analysis was performed on a LightCycler 480 Real-Time System (Roche Applied Science, Basel, Switzerland) with SYBR ® Premix ExTaq™ II (TaKaRa, Dalian, China).
All data were presented as the means ± standard error (SE) and analyzed using SPSS 20.0 statistical software (SPSS, Chicago, IL, USA). All data were checked for normality with the Kolmogorov-Smirnov test and homogeneity with the Levene's test. The differences in data were assessed by using one-way analysis of variance (ANOVA) followed by Tukey test. Differences with p < 0.05 were considered to be statistically significant.

Biological Performance
The results of biological performance of fish fed with experimental diets were summarized in Figure 2. The results showed that final body weight, SGR, WGR, FCR and SR were not affected by different diet treatments (p > 0.05).

Serum Biochemical Parameters
The results obtained from the determination of serum biochemical parameters of fish fed with experimental diets were presented in Table 2. The results showed that the contents of TG, GLU, LDL-C, HDL-C, and HDL-C/LDL-C ratio in different diet treatments did not show a significant difference (p > 0.05). Values are presented as mean ± SE (n = 18). TG, triglyceride (mmol L −1 ); GLU, glucose (mmol L −1 ); LDL-C, low-density lipoprotein cholesterol (mmol L −1 ); HDL-C, high-density lipoprotein cholesterol (mmol L −1 ).

Antioxidant Parameters
As shown in Figure 3, the T-AOC and activities of CAT, GSH-PX, and SOD in the liver of the BY5 diet treatment increased significantly compared to the BY0 and BY1 diet treatments, while MDA content in the liver decreased significantly in the BY5 diet treatment (p < 0.05). Serum SOD activity and T-AOC in the BY5 diet treatment was significantly higher than that of the BY0 and BY1 diet treatments (p < 0.05). However, serum MDA contents were not affected by different diet treatments (p > 0.05).

Immune Biochemical Parameters
As shown in Figure 4, the NO content and TNOS activity in the liver as well as lysozyme activity and C4 content in the serum of the BY5 diet treatment increased significantly compared to the BY0 and BY1 diet treatments (p < 0.05).

Immune Biochemical Parameters
As shown in Figure 4, the NO content and TNOS activity in the liver as well as lysozyme activity and C4 content in the serum of the BY5 diet treatment increased significantly compared to the BY0 and BY1 diet treatments (p < 0.05).

Morphology of Liver and Midgut and Activities of Digestive Enzymes in Midgut
As shown in Figure 5, no obvious histological alterations were observed in the midgut among all diet treatments. In addition, the activities of digestive enzymes in the midgut, including LPS, AMS, and PEP, were not affected by different diet treatments (p < 0.05).   As shown in Figure 6A-C, the liver of fish fed with diet supplemented with B. yajiagengensis showed a healthy morphology, while mild inflammatory cell infiltration was observed in the liver from the BY0 diet.
As shown in Figure 6A-C, the liver of fish fed with diet supplemented with B. yajiagengensis showed a healthy morphology, while mild inflammatory cell infiltration was observed in the liver from the BY0 diet.

Expression Analysis of Antioxidant-Related and Immune-Related Genes in Liver
Fish fed with the BY5 diet showed significantly higher mRNA levels of TGF-β1 (p < 0.05), while the mRNA levels of IL-1β, IL-8, and IL-10 were not affected by different diet treatments (p > 0.05) ( Figure 6D).
Compared with the BY0 and BY1 diet treatments, the mRNA levels of GR, Nrf2, Mn-SOD, HO-1, C-Lyz, and C4 were significantly enhanced in the BY5 diet treatment (p < 0.05), while the mRNA level of Keap1 decreased significantly in the BY5 diet treatment (p < 0.05) (Figure 7).

Expression Analysis of Antioxidant-Related and Immune-Related Genes in Liver
Fish fed with the BY5 diet showed significantly higher mRNA levels of TGF-β1 (p < 0.05), while the mRNA levels of IL-1β, IL-8, and IL-10 were not affected by different diet treatments (p > 0.05) ( Figure 6D).
Compared with the BY0 and BY1 diet treatments, the mRNA levels of GR, Nrf2, Mn-SOD, HO-1, C-Lyz, and C4 were significantly enhanced in the BY5 diet treatment (p < 0.05), while the mRNA level of Keap1 decreased significantly in the BY5 diet treatment (p < 0.05) (Figure 7).

Discussion
The findings obtained in the current study showed that dietary B. yajiagengensis powder supplementation had no significant effects on growth performance and feed utilization of T. ovatus. The results were consistent with similar studies on the effect of lutein on the growth and feed utilization of fish, such as Amphiprion ocellaris [21], Carassius auratus [11], and Larimichthys croceus [22]. Digestive enzyme activity is closely related to the digestion and absorption of nutrients and the growth of aquatic animals [14,23]. In this study, the activities of digestive enzymes (LPS, AMS, and PEP) in the midgut were not affected by different diet treatments, which was consistent with growth performance. The results demonstrated that dietary B. yajiagengensis powder supplementation did not accelerate the digestion process to improve the feed utilization and growth of T. ovatus. However, Ettefaghdoost and Haghighi (2021) [10] indicated that adding 50-200 mg/kg of lutein to the diet enhanced growth, feed utilization, and digestive enzyme activities (LPS, AMS, protease) of Macrobrachium nipponense. Lutein acts as a mediator in metabolism, which accelerates the digestion process and ultimately promotes growth performance [10]. Comparison of results from previous studies with those of the current study revealed that there are species differences in the effects of lutein on growth performance and feed utilization of aquatic animals.
The observation of tissue morphology can intuitively show the health status of fish. In the current study, liver morphological examination revealed mild inflammatory cell infiltration in fish fed with BY0 diet, which was largely due to the influence of environmental stress. Fish cultured in offshore cages are more vulnerable to various environmental stressors (e.g., typhoons, rainstorm, hypoxia, temperature, pollutants), which may result in peroxidation and inflammatory response in liver. The liver of fish fed with diets supplemented with B. yajiagengensis showed a healthy morphology, which demonstrated that B. yajiagengensis exerted hepatoprotective effects. In fish, inflammatory response is

Discussion
The findings obtained in the current study showed that dietary B. yajiagengensis powder supplementation had no significant effects on growth performance and feed utilization of T. ovatus. The results were consistent with similar studies on the effect of lutein on the growth and feed utilization of fish, such as Amphiprion ocellaris [21], Carassius auratus [11], and Larimichthys croceus [22]. Digestive enzyme activity is closely related to the digestion and absorption of nutrients and the growth of aquatic animals [14,23]. In this study, the activities of digestive enzymes (LPS, AMS, and PEP) in the midgut were not affected by different diet treatments, which was consistent with growth performance. The results demonstrated that dietary B. yajiagengensis powder supplementation did not accelerate the digestion process to improve the feed utilization and growth of T. ovatus. However, Ettefaghdoost and Haghighi (2021) [10] indicated that adding 50-200 mg/kg of lutein to the diet enhanced growth, feed utilization, and digestive enzyme activities (LPS, AMS, protease) of Macrobrachium nipponense. Lutein acts as a mediator in metabolism, which accelerates the digestion process and ultimately promotes growth performance [10]. Comparison of results from previous studies with those of the current study revealed that there are species differences in the effects of lutein on growth performance and feed utilization of aquatic animals.
The observation of tissue morphology can intuitively show the health status of fish. In the current study, liver morphological examination revealed mild inflammatory cell infiltration in fish fed with BY0 diet, which was largely due to the influence of environmental stress. Fish cultured in offshore cages are more vulnerable to various environmental stressors (e.g., typhoons, rainstorm, hypoxia, temperature, pollutants), which may result in peroxidation and inflammatory response in liver. The liver of fish fed with diets supplemented with B. yajiagengensis showed a healthy morphology, which demonstrated that B. yajiagengensis exerted hepatoprotective effects. In fish, inflammatory response is regulated by pro-inflammatory cytokines and anti-inflammatory cytokines. Pro-inflammatory cytokines, such as IL-1β and IL-8, are initially activated during inflammatory responses; however, subsequent release of antiinflammatory cytokines, such as IL-10 and TGF-β1, can robustly inhibit this effect, thereby counteracting the hyperactivity of immune responses triggered by pro-inflammatory cytokines [24,25]. Therefore, to further clarify the hepatoprotective mechanism of B. yajiagengensis, the current study determined the effects of B. yajiagengensis on the mRNA levels of inflammation-related genes. The findings obtained in the current study showed that fish fed with the BY5 diet showed significantly higher mRNA level of TGF-β1. The current results indicated that B. yajiagengensis has anti-inflammatory properties and potently suppresses inflammatory responses in the liver, which may be mainly attributed to the lutein contained in B. yajiagengensis. Lutein exhibits anti-inflammatory activity by decreasing inflammatory proteins and pro-inflammatory cytokines, and increasing anti-inflammatory cytokines [26][27][28]. In addition, midgut morphological examination showed no obvious pathological alterations among all diet treatments. The results of morphological observation confirmed that B. yajiagengensis is a safe and nontoxic feed additive and exerted hepatoprotective effects by upregulating the expression of anti-inflammatory cytokines.
The Nrf2-ARE pathway plays a significant role in protecting cells from oxidative stress by removing reactive oxidants [29]. Nrf2 and Keap1 are two key transcription factors in the Nrf2-ARE pathway. Keap1, as a repressor of Nrf2, combines with Nrf2 to form an Nrf2-Keap1 complex under normal homeostatic conditions, which prevents the translocation of Nrf2 from cytoplasm to nucleus and promotes Nrf2 degradation [30]. Once stimulated by oxidative stress, Nrf2 dissociates from Keap1 and subsequently binds to antioxidantresponsive elements in the nucleus to induce transcription of antioxidant enzyme genes, such as GR, HO-1, and Mn-SOD [29]. Therefore, to evaluate the antioxidant properties of microalgae, the current study determined the effects of B. yajiagengensis on the mRNA levels of genes related to the Nrf2-ARE pathway. The results showed that fish fed with the BY5 diet upregulated Nrf2 mRNA levels and downregulated Keap1 mRNA levels in the liver. Further analysis showed that the mRNA levels of Mn-SOD and HO-1 were also significantly upregulated in the BY5 diet treatment. Similarly, a previous study reported that lutein exerts protection against oxidative stress by upregulating Nrf2 and HO-1 expression [27]. The findings obtained in the current study indicated that 5% B. yajiagengensis powder promoted the antioxidant ability of T. ovatus by activating the Nrf2-ARE signal pathway.
It is well known that lipid-soluble antioxidant systems, such as SOD, CAT, and GSH-PX, protect the cell against free radical-induced oxidation. As the first line of antioxidant defense in cells to resist the toxicity caused by free radicals, SOD and CAT can prevent the formation of free radicals and protect cell membrane lipids from peroxidation [10]. SOD can convert superoxide ( • O 2 − ) to H 2 O 2 via the dismutation of superoxide, which helps to maintain the redox balance in cells and protect cells from peroxidative damage. CAT is an oxidoreductase enzyme that converts H 2 O 2 into water and molecular oxygen. GSH-PX is a peroxidase with strong reducibility, which catalyzes the reduction of H 2 O 2 and lipid hydroperoxides, protecting the liver from oxidative damage. The increased enzyme activity is caused by the increased synthesis of enzyme protein, which largely relies on its gene transcription and translation [31]. Therefore, to further confirm whether the elevated Nrf2 mRNA level contributes to increasing the activities of its downstream antioxidant enzymes, the current study measured the effects of B. yajiagengensis on lipid-soluble antioxidant system activities that protect polyunsaturated fatty acids (PUFAs) in biomembranes. In the current study, fish fed with the BY5 diet boosted the lipid-soluble antioxidant systems. Similar results have also been reported in which dietary lutein improved the antioxidant potential of mice by increasing the activities of SOD, CAT, and GSH-PX in the liver [32]. In addition, T-AOC and MDA are indicators for evaluating the balance between oxidants and antioxidant factors. T-AOC directly reflects the antioxidant capacity of fish, while the MDA level indirectly reflects the damage degree of cells attacked by free radicals [33]. According to the study results, T-AOC increased significantly in the liver and serum of fish fed with the BY5 diet, while MDA decreased significantly in the liver. The aforementioned results suggested that 5% B. yajiagengensis boosted the lipid-soluble antioxidant systems, protecting PUFA in dietary oil and protecting PUFAs in biomembranes. Based on the results of antioxidant parameters determined in this study, the dietary supplement of 5% B. yajiagengensis powder improved the antioxidant capacity of T. ovatus by activating the Nrf2-ARE pathway and enhancing lipid-soluble antioxidant systems including SOD, CAT, and GSH-PX and protected lipids from peroxidation.
Compared with pond-and factory-farming models, offshore cage culture is often affected by environmental stressors and increases the risk of pathogen infection in fish. Therefore, antibacterial ability is an important indicator to evaluate the effect of immunostimulants on fish immunity. NO, as a gaseous signaling molecule, is involved in the regulation of neuronal transmission and anti-inflammatory, anti-tumor, and antibacterial activities [34]. NO can react with free radical superoxide to generate active substances including nitrogen dioxide, dinitrogen trioxide, and peroxynitrite, which cause severe nitrosation and oxidative stress to bacteria, eventually destroying cell membranes and causing cell dysfunction of bacteria [35]. Therefore, NO is considered to be an effective bactericidal agent that kills broad-spectrum bacteria, especially drug-resistant ones [36]. NO is produced by NOS-catalyzed reaction of L -arginine with molecular oxygen [37]. Therefore, there was a positive correlation between NO content and NOS activity. NO and NOS are considered to be antibacterial molecules against pathogen infection in aquatic animals [38,39]. In this study, TNOS activity and NO content increased significantly in liver of fish fed with the BY5 diet. The findings obtained in the current study indicated that 5% B. yajiagengensis powder promoted the defense ability of T. ovatus against pathogenic infection.
The non-specific immune system of teleost fish plays a more important role in resisting pathogen invasion and secondary damage than that of other vertebrates [40]. In fish, the complement system plays a central role in the immune response of fish against pathogen infection, and is responsible for the clearance of cellular debris, apoptotic cells, and foreign invaders [41]. In addition, it can bind to specific sites on the surface of phagocytes to promote phagocytosis [42]. Lysozyme, as an antibacterial molecule, can cleave bacteria by hydrolyzing the β-1,4 glycosidic bond of peptidoglycan layer of the bacterial cell wall [43,44]. Previous studies have shown that Lyz and the complement system can act synergistically in bactericidal processes [45,46]. In this study, fish fed with the BY5 diet upregulated the mRNA levels of C-Lyz and C4 in the liver and increased the C4 content and Lyz activity in the serum. Similarly, dietary astaxanthin supplementation improved the immunity of Pseudosciaena crocea by increasing lysozyme activity and complement content in the serum [47]. Zhao et al. (2021) [48] also demonstrated that T. ovatus fed with astaxanthin-rich Haematococcus pluvialis upregulated the mRNA expression of C-Lyz and C4 in the liver. The current results suggested that 5% B. yajiagengensis could improve the non-specific immune response and antibacterial ability of T. ovatus by activating Lyz and the complement system.

Conclusions
This study demonstrated that Barranca yajiagengensis exerted beneficial effects in T. ovatus. The diet supplementation of 5% B. yajiagengensis powder promoted antioxidant responses of T. ovatus by activating the Nrf2-ARE signal pathway and enhanced the lipid-soluble antioxidant system, improving non-specific immunity and antibacterial ability by activating the complement system and Lyz, and increasing the NO content and TNOS activity. In addition, dietary B. yajiagengensis supplementation improved the liver morphology and exerted hepatoprotective effects. Therefore, as a natural source of lutein, B. yajiagengensis has potential as a safe and non-toxic immunostimulant for T. ovatus. The application of B. yajiagengensis can enrich the source of lutein in aquafeed.