The Effects of Dietary Gracilaria Corticata (Marine Macroalgae, Rhodophyta) Extraction on Growth Performance, Antioxidant Defence, Plasma and Mucosal Immune Components and Immune-related Gene Expressions in Goldsh, Carassius Auratus

The present study examined the effects of the hydro-alcoholic extraction of the red seaweed Gracilaria corticata (GCE), as food additive on growth, antioxidant defence and immunity in the goldsh, Carassius auratus. Four experimental treatments in three replications were established and fed the experimental diets for 60 days. The groups were: a control (sh fed only a basal diet), GCE1: sh supplemented with 0.5 % GCE/kg diet, GCE2: sh supplemented with 1 % GCE/kg diet, GCE3: sh supplemented with 1.5 % GCE/kg diet. After feeding period, the antioxidant [superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT)] and immune responses were evaluated. Dietary GCE had no effect on growth performance (P>0.01). The plasma immune responses including alternative complement (ACH 50 ) and lysozyme activities elevated in sh supplemented with 1 % and 1.5 % GCE compared to those fed 0.5 % GCE and basal diet (P<0.01). The plasma and mucosal immunoglobulin (Ig) signicantly elevated in all GCE supplemented sh (P<0.01). The plasma peroxidase activity signicantly increased only in the sh receiving GCE at dietary level of 1.5 % GCE compared to control and those supplemented with 0.5 % and 1 % GCE (P<0.01). The mucosal lysozyme, protease activity and alkaline phosphatase signicantly increased in sh supplemented with 1 % GCE compared to other experimental diets (P<0.01). The activity of antioxidant enzymes (SOD, CAT, GPx) showed signicant increases in sh supplemented with 1 % and 1.5 % GCE (P<0.01). Furthermore, the expression of the immune-related genes, complement and lysozyme signicantly elevated in the treatments of 1 % and 1.5 % GCE compared to other experimental diets (P<0.01). The results of this study revealed that


Introduction
Seaweeds (SWs) contain valuable bioactive compounds, making them a good choice for food and pharmaceutical usages for human and husbandry animals (Kolanjinathan et al., 2014;Kasimala et al., 2015;Khalil et al., 2017). During last decade, use of plant-based materials has considerably increased in aquaculture to enhance sh and shell sh immune system (Van Hai, 2015; Awad and Awaad, 2017). However, using SWs as food additive is a relatively new approach in sh nutrition and needs more attentions. In recent years, the potentials of SWs in sh nutrition have been studied by many studies. It was recognized that SWs improve growth (  , where sh fed diets containing 9-13.5% Clarias gariepinus and 5% Gracilaria bursa-pastoris, respectively. These studies clearly indicated that the effects of dietary SWs could be different depending on species and their dietary levels. Therefore, it is necessary to optimize the dietary levels of SWs, when used as food additive in the diet of sh.
The red alga, Gracilaria corticata, belonging to the family Gracilariacea and order Gracilariales and is one of the most predominant seaweeds of the world including Oman sea and Persian Gulf coasts (

Fish and experimental design
A total number of 240 gold sh (initial weight: 31.1 ± 0.6 g; initial length: 12.5 ± 0.5 cm) were distributed into 12 tanks (300 l) (20 sh/tank) containing aerated and disinfected water. After 10-day's acclimation period, sh were fed diets containing different levels of Gracilaria corticata extract (GCE), as three experimental groups and one basal diet supplemented group as control in three replicates. The experimental groups were: control: non-GCE-supplemented sh, GCE1: 0.5% GCE/kg diet, GCE2: 1% GCE/kg diet, GCE3: 1.5% GCE/kg diet. A commercially basal diet (STARTER for Common carp: crude protein: 41.5%; lipid: 6%, Fiber: 4.5%; Ash: 9%, Faradaneh CO., Shahrekord, Iran) was used to make the experimental diets. To prepare the diets, the basal diet was mill-powdered, the dried GCE added and then pasted by adding 100 ml of distilled water. After that, the paste was passed through a sieve to form granule particles (mean diameter: in 3 ± 0.1 mm; mean length: 2.6 ± 0.3 mm). The foods were incubated at 35°C to dry and then stored at 4°C for further usages. Feeding was carried out daily at a rate of 5% of total sh weight. Feeding size was adjusted every ve days by weighing 5 sh from each tank. Throughout the experiment, the water quality parameters were checked daily, which were at normal ranges for temperature: 21.1 ± 0.15°C, ammonia: 0.03 ± 0.004 mg/L (colorimetrically at 670 nm) and dissolved oxygen: 7.1 ± 0.2 mg/L (OxyGouard) and pH: 7.1 ± 0.1 (APX15/C-WTW-330i).

Growth parameters
The growth parameters were measure after feeding experiment according to following formula: Weight gain (WG) = Wf (g) -Wi (g).
Where Wi is the initial weight, Wf is the nal weight and T is the number of days in the feeding period.
2.4. Mucus, kidney, liver and blood sampling After 60 days feeding, sh (n= 20 sh/tank) were starved for 24 h and placed in a nylon bag and the mucus samples were collected according to Subramanian et al. (2007). The liver and kidney tissues were taken by dissecting out the sh after sedation with 30 mg/l clove oil. The blood samples were taken by heparinized syringe from the caudal vein and then centrifuged at 13000 g for 5 min to separate the plasma. The plasma and liver samples were kept at liquid nitrogen (-196°C) for biochemical analysis. In this study, the samplings and manipulation of the sh were carried out according the ethical standards of the University of Tehran, Iran.

Liver biochemical assays
The activity of antioxidant enzymes in liver were assayed using commercial assay kits (Sigma-Aldrich CO, USA) based on manufacturer's instructions.
The activity of SOD was measured colorimetrically at 440 nm through oxidation of xanthine to superoxide radicals (Marklund and Marklund 1974). The activity of GPx levels were spectrophotometrically assayed at 340 nm through enzymatic generation of oxidized glutathione from glutathione under GPx action. Catalase (CAT) activity was spectrophotometrically assayed at 240 nm through catalyzing hydrogen peroxide (H 2 O 2 ) and production of water and oxygen (Claiborne, 1985). The lipid peroxidation was measured by thiobarbituric acid reaction method at 532 nm (Utley et al., 1967).

Plasma and mucosal immunological assays
The activity of plasma alternative complement activity (ACH 50 ) was assayed by haemolysis of rabbit red blood cells according to Karimi et al. (2020). The volume of plasma causing 50% haemolysis was considered to estimate the ACH 50 activity. The total immunoglobulin (Ig) of plasma and mucus was measured according to Siwicki and Anderson (1993). To this end, the total protein was assayed and then the Ig molecules were precipitated by adding 12% polyethylene glycol solution. The difference in protein content before and after precipitation was estimated as plasma Ig concentration. The lysozyme activity in plasma and mucus was estimated using the turbidity assay according to Parry et al. (1965). Brie y, 50 µl of the plasma was added to 2 ml of the bacterial suspension [Chicken egg lysozyme (as the standard) + 0.2 mg/ml lyophilised Micrococcus lysodeikticus in 0.04 M sodium phosphate buffer (pH 5.8)] and the reduction in the absorbance at 540 nm was determined in two times of 0.5 and 4.5 min incubation at 22°C. A rate of 0.001 min −1 reduction in absorbance was considered as one unit of lysozyme activity.
The plasma peroxidase activity was measured by a colorimetric method at 450 nm according to Quade and Roth (1997)  and an iQ5 optical system software version 2.1 (Bio-Rad) was used for data analysis. The expression of the β-actine gene was used as reference gene.

Statistical analysis
Data analysis was carried out by SPSS software. The data normality was evaluated by Kolmogorov-Smirnov test. After that, the one-way analysis of variance (ANOVA), followed by Tukey's test were applied to nd the statistical differences and to compare the means between the experimental groups, respectively. All data are presented as mean ± SD.

Growth and survival rate
Use of GCE in the diet of the sh had no effects on growth parameters i.e. nal weight (g), weight gain (g) (Fig. 1) and SGR (Fig. 2, P>0.01). Similarly, the survival rate (%) showed no changes between the experimental groups over the course of the experiment (Fig. 3, P>0.01). unde ned

Malondialdehyde
Supplementation with GCE had no signi cant effect on MDA levels in liver (Fig. 11, P>0.01).

Gene expressions
The expression of immune-related genes, complement and lysozyme signi cantly increased in the sh fed 1% and 1.5% GCE compared to other experimental diets (Fig. 12, P<0.01).  (Thépot et al., 2020). However, providing information about algae species, especially endemic species could be valuable. In the present study, the immunostimulatory and antioxidant effects of an endemic macroalgae, Gracilaria corticata extracts was examined in the gold sh by adding it in the diet. Based on our results, the growth performance the sh were not affected by dietary GCE. In general, the use of dietary Gracilaria sp. has resulted various results related to growth performance of sh. In Persian sturgeon, Acipenser persicus, dietary Gracilaria sp. (2020) reported a decrease in GPx activity in the gilthead seabream fed 2.5% and 5% Gracilaria sp. In the present study, GCE at dietary levels of 1 % increased the activity of mucosal lysozyme, alkaline phosphatase and protease. Mucosal proteases play an essential role in the breakdown of proteins involved in in ammatory, coagulation, apoptotic and tissue regeneration processes (Ahmad et al., 2021). Skin mucus alkaline phosphatase is known as part of sh mucosal immune system, though its actual mode of action is still unknown. However, some antibacterial properties have been reported for skin mucus alkaline against water pathogens (Esteban et al., 2015). . In this study, the MDA levels did not show signi cant changes between the experimental groups, which could indicate that the GCE did not induce oxidative stress. However, GCE at high concentrations (1-1.5 %) enhanced the activity of liver antioxidant enzymes (CAT, SOD, GPx), which clearly shows the enhancing effects of GCE on antioxidant defense system of the sh.

Conclusion
In conclusion, the ndings of the current study demonstrated the antioxidant and immunostimulatory effects of dietary GCE in the gold sh, without negative impacts on the sh growth. The datasets used during the current study are available from the corresponding author on reasonable request.

Compliance with ethical standards
The authors declare that they have no competing interests.

Funding
There is no government or organizational fund for this work.   Effects of different dietary levels of Gracilaria corticata extracts (GCE) on survival rate of the gold sh, Carassius auratus. Different superscripted letters indicate signi cant differences between the dietary groups (P<0.01). Data are presented as mean ± SD.

Figure 4
Effects of different dietary levels of Gracilaria corticata extracts (GCE) on plasma immunoglobulin (Ig) concentrations and ACH 50 activity in the gold sh, Carassius auratus. Different superscripted letters indicate signi cant differences between the dietary groups (P<0.01). Data are presented as mean ± SD.

Figure 5
Page 17/20 Effects of different dietary levels of Gracilaria corticata extracts (GCE) on plasma lysozyme and peroxidase activity in the gold sh, Carassius auratus. Different superscripted letters indicate signi cant differences between the dietary groups (P<0.01). Data are presented as mean ± SD.

Figure 6
Effects of different dietary levels of Gracilaria corticata extracts (GCE) on mucosal immunoglobulin in the gold sh, Carassius auratus. Different superscripted letters indicate signi cant differences between the dietary groups (P<0.01). Data are presented as mean ± SD.

Figure 7
Page 18/20 Effects of different dietary levels of Gracilaria corticata extracts (GCE) on skin mucus lysozyme and protease activity in the gold sh, Carassius auratus. Different superscripted letters indicate signi cant differences between the dietary groups (P<0.01). Data are presented as mean ± SD.

Figure 8
Effects of different dietary levels of Gracilaria corticata extracts (GCE) on skin mucus alkaline phosphatase in the gold sh, Carassius auratus. Different superscripted letters indicate signi cant differences between the dietary groups (P<0.01). Data are presented as mean ± SD.

Figure 9
Effects of different dietary levels of Gracilaria corticata extracts (GCE) on liver antioxidant enzyme, catalase in the gold sh, Carassius auratus. Different superscripted letters indicate signi cant differences between the dietary groups (P<0.01). Data are presented as mean ± SD.

Figure 10
Effects of different dietary levels of Gracilaria corticata extracts (GCE) on liver antioxidant enzymes, GPx and SODin the gold sh, Carassius auratus. Different superscripted letters indicate signi cant differences between the dietary groups (P<0.01). Data are presented as mean ± SD.

Figure 11
Effects of different dietary levels of Gracilaria corticata extracts (GCE) on liver malondialdehyde (MDA) levels in the gold sh, Carassius auratus. Different superscripted letters indicate signi cant differences between the dietary groups (P<0.01). Data are presented as mean ± SD.

Figure 12
Effects of different dietary levels of Gracilaria corticata extracts (GCE) on immune-related gene expressions in the gold sh, Carassius auratus. Different superscripted letters indicate signi cant differences between the dietary groups (P<0.01). Data are presented as mean ± SD.