key: cord-0925024-8oqrna5b
authors: Adel, Milad; Omidi, Amir Hossein; Dawood, Mahmoud A.O.; Karimi, Behnaz; Shekarabi, Seyed Pezhman Hosseini
title: Dietary Gracilaria persica mediated the growth performance, fillet colouration, and immune response of Persian sturgeon (Acipenser persicus)
date: 2020-09-20
journal: Aquaculture
DOI: 10.1016/j.aquaculture.2020.735950
sha: 771b247982cc8363aff4cc9e9341dc0fb965d504
doc_id: 925024
cord_uid: 8oqrna5b

Algal seaweeds have abundant amounts of active substances and can be used as pharmaceuticals and biomedicals in aquafeeds. In this context, the powder of red macroalgae Gracilaria persica was included in the diets of Persian sturgeon at the rate of 0, 2.5, 5, and 10 g/kg to investigate its role on the growth rate, fillet colouration, haemato-biochemical indices, serum, and skin mucus immunity. The weight gain, SGR, and FCR displayed no significant changes in fish fed varying levels of G. persica (P > 0.05). The level of total carotenoids was significantly higher in the blood and fillet of fish fed 5 and 10 g G. persica/kg diet (P < 0.05). Dietary G. persica significantly altered RBCs, WBCs, and HCT at 5, and 10 g/kg, whereas the Hb was increased in fish fed 5 g/kg (P < 0.05). The blood total protein and albumin were significantly increased in fish fed 5 and 10 g/kg (P < 0.05). No significant alterations were observed on ALT, AST, ALP, and glucose levels of fish fed varying levels of G. persica (P > 0.05). Serum Ig, lysozyme, superoxide dismutase, catalase, and respiratory burst activities were increased in fish fed 5, and 10 g/kg than fish fed 0 and 2.5 g/kg diet (P < 0.05). The level of total protein and lysozyme activity in the skin mucus were significantly higher in the blood and fillet of fish fed 5, and 10 g G. persica/kg diet than fish fed 0 and 2.5 g/kg (P < 0.05). Based on the obtained results, G. persica can be used as a feasible feed additive in the diets of Persian sturgeon at 5–10 g/kg diet.

The new world challenges, including COVID-19, obligates researchers to ensure the production of the healthy and secure food chain. The aquaculture sector is growing to support the increasing population with a nutritious and cheap animal protein source (Magouz et al., 2020) . Persian sturgeon (Acipenser persicus) originally exist in the southern division of the Caspian Sea and has at 3-4% of body weight for eight weeks. The water quality was maintained throughout the experimental period, and partial water exchange was done daily to remove waste feed and fecal materials.

All fish in each tank were deprived of food for 24 h before weighing and sampling, and the following parameters were measured at the end of feeding trial after eight weeks.

Weight gain = W2 (g) -W1 (g) Specific growth rate (SGR) = 100 (ln W2 -ln W1)/T Feed conversion ratio (FCR) = feed intake (g) /weight gain (g) Survival rate (%SR) = (final amount of fish/initial amount of fish) ×100

Where W1 is the initial weight, W2 is the final weight and T is the number of days in the feeding period.

At the end of the feeding trial, 3 fishes were selected from each tank and anesthetized with clove oil (75 mg/l) (Darafsh et al., 2020) . Blood samples (2 ml) were collected from the caudal vein of individual fish and immediately divided into two half parts. One half was transferred to a tube containing an anticoagulant (heparin) for studying the respiratory burst assay and make the hematological analysis, while the other half was assigned to non-heparinized tubes for biochemical and immunological studies. Sera samples were obtained by blood centrifugation (3000 × g, 10 min) and stored at -80 º C until use.

The total red blood cells (RBC: 10 6 /mm 3 ) and white blood cells (WBC: 10 3 /mm 3 ) were enumerated in an improved Neubauer hemocytometer using Hayem and Turck diluting fluids (Blaxhall et al., 1973) . Haematocrit (Ht %) was determined by the standard microhematocrit method and expressed as a percentage. The haemoglobin (Hb, g/dl) level was determined according to cyanomethemoglobin procedure. Furthermore, differential leukocyte cells were measured by preparing Giemsa stained smears. Blood smears were studied by light microscopy to make blood cell counts.

Glucose, albumin, total protein (TP) level, and enzymatic activities of liver including; alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were estimated on fish sera by using commercial kits (Pars Azmoon Company, Tehran, Iran) and a biochemical auto analyzer (Eurolyser, Belgium).

Superoxide dismutase (SOD) activity in serum samples was estimated using the Nitro blue tetrazolium chloride dye reduction test (NBT) (Mohebbi et al., 2012) . Briefly, 100 μL aliquots of serum were added to 2 mL of reaction mixture (containing 0.20 mM xanthine, 0.12 mM NBT, 0.049 IU xanthine oxidase (Sigma Company, St Louis, MO, USA) and 0.1 M phosphate buffer, pH 7.0). The mixture was incubated for 20 min at 37 °C. Then, the absorbance was read at 560 nm. SOD activity was expressed as the percentage inhibition of reduction of NBT.

The serum catalase (CAT) activity was determined according to the method described by Mohebbi et al. (2012) . Briefly, 100 μL of serum was incubated for 1 min with 1 mL reaction mixture consisting of 50 mM potassium phosphate buffer (pH 7.0) and 10.6 mM H 2 O 2 (Merck, Germany) freshly prepared at 37 °C. The reaction was terminated by adding 0.5 mL of 32.4 mM J o u r n a l P r e -p r o o f ammonium molybdate solution (Merck, Germany). A yellow complex of ammonium molybdate and hydrogen peroxide was created. The absorbance of the complex was measured at 405 nm with a spectrophotometer and compared with a blank (distilled water). One unit of CAT activity was defined as the amount of enzyme that catalyses the decomposition of 1 μmol of H 2 O 2 per minute.

The blood carotenoid concentration was measured according to the assay described by Barbosa et al. (1999) . Briefly, 200 μl of serum sample was mixed with 400 μl of ethanol (95 %) and 1 ml of hexane. The mixture was vortexed for 1 min, and hexane was collected following centrifugation at 4500 rpm for 10 min. The carotenoid absorption was read using Tean F-200 multiwell plate reader (Tecan Mannedorf; Zurich, Switzerland) at 450 nm in n-hexane.

Carotenoid concentration was calculated using a specific extinction coefficient (E1 %, 1 cm) 2500 in n-hexane (Weber, 1988) . Approximately 25 g of fish fillet of each Persian sturgeon (n=5) were collected and stored in the freezer (-80 °C). The carotenoid content was extracted according to the method described by Teimouri et al. (2013) . The carotenoid concentration of fillet was determined spectrophotometrically in acetone using extinction coefficients (E1%, 1 cm) 2500 for carotenoids at 450 nm.

Serum immunoglobulin (Ig) level was quantified by microprotein determination method (C-690; Sigma). Precipitation was carried out by 12 % polyethylene glycol solution, and the difference in protein content before and after precipitation was considered as the Ig level (Siwicki et al., J o u r n a l P r e -p r o o f Journal Pre-proof 2000) . The lysozyme activity in serum was determined using the method described by Ellis (1990) with more modifications. Briefly, 50 μl of sera were added to 2 ml of a suspension of Micrococcus lysodeikticus (0.2 mg/ml in a 0.05 M sodium phosphate buffer (pH 6.2) and absorbance was measured at 450 nm after 0.5 min and 3 min by spectrophotometer (Biophotometer, Eppendorf). Respiratory burst activity of blood leukocytes was measured by chemiluminescent assay (LUMI Skan Ascent T392, Finland) employing the strategy of Binaii et al. (2014) . Finally, the alternative complement activity (ACH50) was determined by methods described by Yano (1992) by using 50% hemolysis of the rabbit red blood cells (RaRBC).

The mucus samples were scraped from the anterior to posterior direction on dorsal body surface from 3 fish per tank using a sterile spatula following the method described by Balasubramanian et al. (2013) . The collected mucus was thoroughly mixed with equal quantity of sterilized Trisbuffered saline (TBS, 50 mM Tris HCl, pH 8.0, 150 mM NaCl) and centrifuged at 30,000 × g at 4 o C for 15 minutes (Beckman coulter, Avanti J-26 XPI, Brea, CA, USA). The supernatant was then collected and filtered with Whatman no.1 filter paper. The filtrate was then collected and kept frozen at -80 o C to avoid bacterial growth and degradation until used.

The total protein concentration of mucus was measured according to the method that was described by Lowry et al. (1951) . Total immunoglobulin (Ig) level was determined according to the method described by Siwicki et al. (2000) by using a micro protein determination method (C-J o u r n a l P r e -p r o o f 690; Sigma). The activity of mucus lysozyme was measured similarly to the protocol used for serum lysozyme activity as described above.

The data were subjected to statistical analysis using the SPSS software version no. 20 (SPSS Inc., Chicago, IL, USA). The statistical analysis was done by using one-way analysis of variance (ANOVA) followed by Duncan's multiple range tests. P-value of <0.05 was considered significant. All the tests were performed in triplicate.

The weight gain, SGR, and FCR of Persian sturgeon fed varying levels of G. persica displayed no significant changes among the fish (P>0.05) ( Table 2 ). The survival rate of fish during the trial is 100% without mortality in all groups ( Table 2 ).

The level of total carotenoids was significantly higher in the blood and fillet of fish fed 5, and 10 g G. persica/kg diet than fish fed 0 and 2.5 g/kg (P<0.05) (Table 3) . Additionally, fish fed 10 g/kg had higher total blood carotenoids than fish fed 5 g/kg (P<0.05).

The level of total protein and lysozyme activity in the skin mucus were significantly higher in the blood and fillet of fish fed 5, and 10 g G. persica/kg diet than fish fed 0 and 2.5 g/kg (P<0.05) (Table 7) . Additionally, fish fed 10 g/kg had higher total protein and lysozyme activity than fish fed 5 g/kg (P<0.05).

G. persica appears to be one of the functional additives which have long obtained noticeable responsiveness in fish nutrition (Francavilla et al., 2013) . The supplementation of G. persica can improve the efficacy of aquafeed with friendly and effective feed additives (Mohammadi et al., 2020b ). In the current study, dietary G. persica had no significant impact on Persian sturgeon's growth performance and FCR, which agrees with previous studies that investigated the influence of seaweed belongs to the Gracilaria genus. Longitudinal studies concluded that dietary G. persica powder did not affect the growth performance of zebrafish (Hoseinifar et al., 2018) and

European seabass (Peixoto et al., 2016) . Interestingly, the results also showed that the survival rate was not impaired with dietary G. persica, which explains the safe use of G. persica in the diets of Persian sturgeon. In this context, Xu et al. (2011) reported that dietary Gracilaria lemaneiformis enhanced the growth performance of rabbitfish (Siganus canaliculatu).

Conversely, dietary Gracilaria arcuate reduced the growth performance in Nile tilapia (Younis et al., 2018) and dietary G. lemaneiformis lowered the growth performance in black sea bream (Xuan et al., 2013) . This contradiction refers to the species-specific effect of Gracilaria sp. on the growth performances of aquatic animals.

J o u r n a l P r e -p r o o f

The colouration of the fish fillet is highly requested by consumers and affected by the level of deposited carotenoids in tissue (Baghel et al., 2014) . Fishes are incapable of synthesizing the carotenoids and pigments responsible for colouration and should be included through the diet (Ebeneezar et al., 2020) . It is well documented that fish cannot produce carotenoids entirely, and it must be obtained from external sources such as red and green algae (Araújo et al., 2016; Ramamoorthy et al., 2010) . The results of the present study indicated that the level of carotenoids was increased in the blood and fillet of Persian sturgeon fed G. persica with regards to the control. Similarly, Pezeshk et al. (2019) reported that the skin colouration of yellow cichlid was improved by dietary red macroalga (G. persica). These results confirm the possibility of using G. persica as a natural pigment for skin colouration in fish. The intensity of colouration in Persian sturgeon was probably enhanced through carotenoids ingested and assimilated in blood and fillet (Yeşilayer et al., 2020) .

The evaluation of the haemato-biochemical indices is a reliable tool in the diagnosis of the physiological and health condition of fish (Fazio, 2019; Mohammadi et al., 2020c) . It indicates the impact of different feed additives and feeds formulations on fish performances (Dawood et al., 2017) . The results showed that fish fed G. persica powder at 5-10 g/kg had enhanced RBCs, WBCs, Hb, and Hct values. The effect of G. persica on the RBCs, WBCs, Hb, and Hct indices is probably attributed to the influence of vitamin C, polyphenols, and carotenoids which act as natural immunostimulants that compromised with the lack of anemic and outbreaks in Persian sturgeon under the current trial conditions (Francavilla et al., 2013) . Concurrent with the hematological indices, the blood total protein, albumin, and globulin were increased in fish fed G. persica at 10 g/kg diet for eight weeks. The total protein is also a critical compound involved J o u r n a l P r e -p r o o f in the immune system (Dawood et al., 2020c) . The enhanced total protein, albumins, and globulin are also associated with the improved immune system in Persian sturgeon due to dietary G. persica. Interestingly, G. persica mediated total protein, albumin, and globulin levels, which related to the role of G. persica in regulating the metabolism of the nutrients in fish reared under optimal conditions (Adegbeye et al., 2019). These results also confirm the balanced metabolic rate of proteins and amino acids as well as the sufficient protein content in the experimental diets. Notably, the liver function related enzymes (ALT, AST, and ALP) and glucose level showed no differences among the groups of fish fed varying levels of G. persica, which confirms the safe influence of G. persica on the liver of Persian sturgeon.

The main purpose of including medicinal herbals and their extracts in aquafeed is to maintain the antioxidative capacity (Dawood, 2020; Dawood et al., 2018) . The stressful conditions impair the cell function by deteriorating the redox of reactive oxygen species (ROS) which increase the oxidative stress and harm the cell structure and damage DNA (Al-Deriny et al., 2020; Harsij et al., 2020) . Several enzymes are synthesized to breakdown the high concentration of ROS and keep the antioxidative balance (Dawood et al., 2020a; Lee et al., 2004) . These enzymes include CAT and SOD, which displayed enhanced values in fish, fed G. persica. Similar results were obtained by zebrafish (Danio rerio) fed on Gracilaria gracilis powder (Hoseinifar et al., 2018) .

The enhanced antioxidative condition is probably attributed to the high content of G. persica powder from phenols and polyphenols which has natural antioxidative activity by the degeneration of over ROS (Ahmadifar et al., 2020) .

Skin mucus is the first line of defence that protects the fish body from external and environmental stressors and invaders (Dawood et al., 2017) . Medicinal herbs, seaweeds, and their extracts are potent immunostimulant factors and can enhance the serum and skin mucus immunity (Srichaiyo et al., 2020) . Lysozyme is a lysis enzyme produce by granulocytes during non-specific oxygen-independent pathways that play an essential role in innate immunity (Grinde et al., 1988) . In fish, the lysozyme is a very imperative humoral component in the systemic and mucosal immune system and further acts as a protective factor against pathogenic microorganisms (Saurabh et al., 2008) . Persian sturgeon fed G. persica powder showed enhanced blood total protein compared to the other groups. Serum levels of Ig, ACH50, lysozyme and alternative burst activities were also enhanced by G. persica powder when compared with the control group. Consistent with our findings, Hoseinifar et al. (2018) reported that zebrafish fed on diets supplemented with Gracilaria gracilis powder had enhanced levels of total Ig and lysozyme activity. Additionally, Peixoto et al. (2016) In fish, skin mucus has a physical structure (epithelial lining) and a chemical characters (skin mucus) which build a strong barrier between the fish and the surrounding environment (Mohammadi et al., 2020a) . In our study, Persian sturgeon fed G. persica diets showed enhanced total protein, Ig, and lysozyme activity in the skin mucus when compared with the control group.

Similarly, Hoseinifar et al. (2018) reported that zebrafish fed on diets supplemented with Gracilaria gracilis powder had enhanced levels of skin mucous total protein, total Ig, and lysozyme activity. Seaweeds contain abundant amounts of β-carotenes, polysaccharides, and vitamin C, which induce antioxidative and immunostimulatory influences on the organism (Francavilla et al., 2013) .

The overall results indicate that G. persica can be included in the diets of Persian sturgeon at 5-10 g/kg without adverse features on the growth performances and wellbeing. Additionally, dietary G. persica enhanced the haemato-biochemical indices, serum, antioxidative capacity, and skin mucus immune responses. Attractively, G. persica increased the levels of carotenoids in the blood and fillet, which refers to its role in the colouration of Persian sturgeon. 

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The authors would like to thank the Science and Research Branch, Islamic Azad University, Tehran, Iran, for their financial support of this study.