Iranian Journal of Fisheries Sciences ( ) - DOI: /ijfs. Hematite-biochemical and immune response of Caspian brown trout (Salmo troutta caspius, Kessler, ) juveniles fed different levels of spirulina (Spirulina platensis)

Meshkat Roohani A. ; Fallahi Kapoorchali M. *; Abedian Kenari A. ; Sayyad Borani M. ; Zorriezahra M.J.

Received: July Accepted: August

Abstract The effects of dietary Spirulina platensis on haemato-biochemical and immunity responses of Caspian brown trout (Salmo troutta caspius) juveniles was investigated. For this purpose, diets with five S. platensis inclusion levels (control, FMR ( g kg- spirulina in diet), FMR ( g kg- spirulina in diet), FMR ( g kg- spirulina in diet), and FMR ( g kg- spirulina in diet) were prepared. Six hundred juveniles with an average initial weight of g were assigned to experimental . The experiment lasted for weeks. At the end of the experiment, growth performance, haemato-biochemical parameters including white and red blood cell counts, neutrophils lymphocytes counts, hematocrit, hemoglobin, glucose, albumin, total protein, aspartate amino transferase (AST), alanine amino transferase (ALT), triglyceride, cholesterol, as well as immunity parameters including lysozyme, C , C , Immunoglobulin (IgM), ACH and respiratory burst activity were assessed. The results indicated that fish fed diets supplemented with FMR and FMR had a significantly higher weight gain ( g and g) and specific growth rate ( %bw day- and %bw day- ) compared with control. Furthermore, FMR and FMR treatments had statistically higher protein efficiency ( and ), lipid efficiency ( and ) and statistically lower feed

Downloaded from jifro.ir at 15:41 +0330 on Friday October 1st 2021 conversion ratio ( and ) compared to the other treatments respectively (p< ). The physiological and immunological factors were improved when fish were fed a high level of S. platensis supplement. S. platensis inclusion also increased activity of Lysozyme C , C , IgM and ACH and respiratory burst activity and reduced AST and ALT formation. These results indicate that S. platensis supplement is promising for disease prevention in S. trutta caspius juveniles, at an optimum dietary level of in diet.

Keywords: Spirulina, Salmo trutta caspius, Growth performances, Haemato-biochemial parameters, Immunity.

-Inland Waters Aquaculture Research Center, Iranian Fisheries Science Research Institute, Agriculture Research, Education and Extension Organization (AREEO), Bandar Anzali, Gilan, -Department of Aquaculture, Faculty of Natural Resources and Marine Sciences, Tarbiat Modares University, , Mazandaran, Iran -Iranian Fisheries Science Research Institute, Agriculture Research, Education and Extension Organization (AREEO). Tehran, Iran *Corresponding author's Email: m_fallahi @yahoo.com

Meshkat Roohani et al., Hematite-biochemical and immune response of…

Introduction multi-cell microalgae is gaining more Growth performance and disease attention from medical scientists as a control are the most important priorities nutraceutical and source of potential in current aquaculture. Nowadays, use pharmaceuticals. There are several new of antibiotics and chemotherapeutics peer reviewed scientific studies about has increased significantly, due to spirulina's ability to inhibit viral intensification of culture practices and replication, strengthen and enhancing followed by high prevalence of both the cellular and humoral arms of infectious diseases (Andrews et al., the immune system (Kozlenko et al., ). Accumulation of antibiotic ; Hirahashi et al., ; Andrews residues in fish tissues and environment et al., ). It has been received the causes human and animal health issues most research and public health and led to an urgent need for alternative attention due to its bioactive disease preventive substances and use compounds including vitamins, of immune-stimulants in aquaculture essential amino acids, minerals, (Andrews et al., ; Krishnaveni et essential fatty acids (gamma linolenic al., ). Several types of stimulants acid), and antioxidant pigments such as with different mechanisms and phycocyanine (Belay et al., ; functions such as bacterial products, Regunathan and Wesley, ; Ragap complex carbohydrates, vaccines, et al., ); Ceballos et al., ). immune enhancing drugs, nutritional Additionally, spirulina has high protein factors, animal and plant extracts are content ( – by dry weight) and is

considered, and their abilities to the richest natural source of vitamin B activate the innate immunity have been (Estrada et al., ). This alga studied (Raa, ; Sakai, ; contains a whole spectrum of natural Downloaded from jifro.ir at 15:41 +0330 on Friday October 1st 2021 Shahbazi and Bolhassani, ).One mixed carotene and xanthophylls supplement source, with ease of phytopigments (Belay et al., ; production, processing and distribution Estrada et al., ; Andrews et al., along with a wide range of macro- and ). It has been confirmed that the micronutrients of health benefits is addition of small amounts of algae to spirulina (Ravi et al., ). Research fish feed can exert pronounced effects has shown that this alga contains a wide on growth, lipid metabolism, body variety of compounds such as composition and physiological response phycobiliproteins, carotenoids, to stress and disease (Mustafa et al., phycocyanin, polysaccharides, ; Mustafa and Nakagava, ; unsaturated fatty acids, superoxide Palmegiano et al., ; Abdel- dismutase, different vitamins, and other Tawwab and Ahmad, ; Teimouri et elements which by bring improvement al., ). Spirulina as an immune in body coloration, growth and modulator not only stimulates the immunity of fish (Nakagawa et al., immune system, but also strengthens ). Spirulina (Spirulina platensis), a the body's ability to produce new blood blue-green filamentous, fresh water and cells (Andrews et al., ). Important Iranian Journal of Fisheries Sciences ( ) parts of the immune system, such as endangered species, however, little is bone marrow stem cells, macrophages known about their nutritional cells (number of cells and requirements especially in the field of phagocytosis), T cells (lymphocyte), increasing immunity of species and NK (non-specific cytotoxic), spleen as types of immune-stimulants. Therefore, well as thymus show tangible activities the main objective of this study was to when treated with spirulina extract evaluate the effects of S. platensis meal (Henrikson, ; Watanuki et al., as feed additive on immune system, ; Tongsiri et al., ; Yong-Chin biochemistry and haematology in S. et al., ; Shahbazi and Bolhassani, trutta caspius as a valuable species. ). Recently, spirulina has been speculated to be associated with Materials and methods modulation of the host immune system Experimental diets (Hironobu et al., ). Five artificial diets were formulated Caspian brown trout (Salmo trutta using five levels of microalgae S. caspius, Kessler ), is one of the platensis (Sina microalgae Co., Qeshm, world’s nine subspecies of brown trout Iran) and in three replicates were (Quillet et al., ; Habibi et al., examined in a randomized design. The ) and attains the greatest size and experimental diets were formulated by growth rate of all brown trout partially replacing fishmeal in the basal (Sedgwick, ; Rajabi et al., ). diet ( in control) with spirulina This species is a critically endangered powder at inclusion levels of , , anadromous species distributed in , and , respectively. Diet southern region of the Caspian Sea. In formulations were performed using , this species was declared at risk Lindo software (Lindo copyright, Downloaded from jifro.ir at 15:41 +0330 on Friday October 1st 2021 according to IUCN conditions (Kiabi et release, ), and fishmeal and S. al., ; Coad, ; Kalbassi et al., platensis powder were used as a protein ). Artificial propagation and source. Test diets provided, which were releasing fingerlings to the natural iso-nitrogenous ( - crude protein) waters is an approach to prevent brown and iso-caloric ( MJ kg- ) were trout’s extinction (Rajabi et al., ). analyzed in this regard, only varied in In addition, in the recent years, this terms of fish meal and S. platensis species has attracted interest for content. The proximate composition of aquaculture in cage and raceways in experimental diets was measured intensive culture systems (Kalbassi et according to the standard methods of al., ). S. trutta caspius, like many Association of Official Analytical other species is sensitive to stressors Chemists (AOAC, ) for moisture, and pathogenic agents. Thus, strong protein, fat, and ash determination. defense mechanisms or immune system Briefly, Moisture content was estimated are needed against pathogens to by drying the samples to constant improve the health of the fish. Despite weight at ˚C in a drying oven (GCA, the importance of Caspian salmon as an model EM, Precision Scientific Meshkat Roohani et al., Hematite-biochemical and immune response of… Group, Chicago, IL, USA). N content (kJ g- ) for protein, fat, and was measured using the Labconco carbohydrates, respectively (NRC, Micro Kjeldahl Apparatus (Labconco ). The experimental diet Corporation, Kansas, MO, USA) and formulation and proximate composition crude protein was estimated by are showed in Table , respectively. multiplying N content by . Dietary feed ingredients were ground Lipid content was determined by using a laboratory grinder (Philips acetone extraction using the extraction HR , Finland) and then blended Soxhlet apparatus (Lab-Line into a homogenous doughy matter by Instruments, Inc., Melrose Park, IL, adding water and transferred to a meat USA) for hours, and ash was grinder with a mm-mesh (CGT determined by combusting dry samples Company, mec, Rome, Italy). After in a muffle furnace (Thermolyne drying, diets were broken by hand to fit Corporation, Dubuque, IA, USA) at fish mouths and placed in a grinding °C for hours. Carbohydrate (i.e., machine for breakdown into small nitrogen-free extract plus fiber) was pieces. The resulting diets were then calculated by the difference: dried in food dryer and stored in plastic ( -(protein+fat+ash+moisture)) bags in a refrigerator at - °C until (Aksnes and Opstvedt, ). further use. Gross energy was calculated using the coefficient of , . , and

Table : Dietary formulation and proximate composition of experimental diets. Experimental Diets Ingredient (g kg- dry weight)

Fish meal Spirulina Wheat flour Fish oil

Downloaded from jifro.ir at 15:41 +0330 on Friday October 1st 2021 Soybean oil Lethitin Filler(sand) Mineral mixture Vitamin mixture Anti-oxidant Choline Chloride Mono calcium phosphate Anti fungal Lysine Methionine Vitamin C Chemical analysis (%) protein lipid moisture ash carbohydrate Gross Energy(MJ kg- ) Herring meal, produced by Mirood, Mazandaran province, Iran. Produced in Sina micro algae, Qeshm. Hormozgan province, Iran. Produced by Mirood, Mazandaran province, Iran. , Prepared of Mazandaran aquatic food factory, Sari, Mazandaran province, Iran. Mineral premix consisted of (mg kg- premix): Man= mg, Cu= mg, Fe= mg, Zn= mg, Se= mg, I= mg, Co= mg and carrier up to kg. Vitamin premix consisted of (mg kg- premix) : A= IU, D = IU, E= IU, K = mg, C= mg, B = mg, H = mg, B = mg, B = mg, B = mg, B = mg, B = mg Preparation of the Mazandaran aquatic food factory and its combination including BHT,Etoxyquin ,Synergist Propylgallate. Choline Chloride, , Prepared of livestock and poultry pharmaceutical factory Aras Bazar. , , , Prepared of pharmaceutical factory Aras Taban, Amol, Mazandaran province, Iran. Prepared of pharmaceutical factory Aras Bazar, Amol, Mazandaran province, Iran. Aqutic vitamin C, production of pharmaceutical factory Aras Bazar, Amol, Mazandaran province, Iran.

Iranian Journal of Fisheries Sciences ( )

Fish rearing Specific growth rate (SGR (%))= Fish juveniles (n= ; mean individual (ln W –ln W )/T; where W and W initial weight g) were obtained are the initial and final weight, from Cold Water Breeding and respectively, and T is the number of Restocking Hatchery, Shahid Bahonar days in the feeding period; Center in Kelardasht, Mazandaran, Iran, Feed conversion ratio (FCR) = feed and then were transferred to NIAC consumed (g dry weight)/weight gain multi-purpose co-operative farm fish in (g); Baghbankola village, Amol, Condition Factor (CF) = Weight/Total Mazandaran, Iran. Fish were allowed to length × ; acclimatize for two weeks in indoor Survival rate (%) = (Number of fish at fiberglass tanks prior to the experiment the beginning/Number of fish at the and during this period were fed a end) × . commercial diet twice daily. After the acclimation period, fish were randomly Sampling divided into five equal groups, each At the end of our experiments, to comprising three replicates ( fish per evaluate haemotological parameters, replicate) in separate L fish were fasted for hours fiberglass tanks. The fish were hand-fed immediately prior to blood sampling three times daily ( , and and then six fish from each were h) to apparent satiation by visual sampled randomly. To prevent observation for ten weeks ( Sotoudeh et sampling stress, the fish were al., ) Water temperature, dissolved anesthetized with a stock solution ( oxygen and pH were monitored daily ppm) of clove oil and blood samples (during the experiment, temperature, were collected quickly (approximately Downloaded from jifro.ir at 15:41 +0330 on Friday October 1st 2021 dissolved oxygen and pH were one min/fish) by puncturing the veins in °C, mg L- and the caudal peduncle with a sterile ML respectively). Uneaten feed and fecal syringe (Esmaeili et al., ). The matter were siphoned off every day. collected blood sample was divided into Due to the sensitivity of these juveniles, two portions. One portion was and to prevent mortality, biometry was transferred into Eppendorf tubes performed once at the beginning and containing heparin anti-coagulant ( once at the end of this period. U L- ) for haematological tests and the second portion of blood sample was Growth parameters also transferred into Eppendorf tubes, Growth performance was determined left to clot at ◦C and centrifuged at and feed utilization was calculated as rpm for min at room following (Goytortua-Bores et al., temperature for biochemical and ): Immunological tests. Weight gain (WG %)= ×(final body weight–initial body weight)/initial body weight. Meshkat Roohani et al., Hematite-biochemical and immune response of… Haemotology tests Microhematocrit capillary tubes were Red blood cells (RBC) and white blood centrifuged at rpm for min, and cell (WBC) counts were determined then hematocrit value was calculated using Neubaeur haemocytometer using graded plate. To calculate others following the methods of Blaxhall and haematological indices, MCV (mean Daisley ( ). Blood was diluted to corpuscular volume), MCH (mean with Race solution. Five center corpuscular hemoglobin), MCHC cells of middle square in Neubauer (mean corpuscular hemoglobin chamber were used for counting RBC concentration), the relevant formula and multiplied by (Esmaeili et (Bain et al., ) was used as follows: al., ). For counting WBC, four MCV (fl)=hematocrit/RBC(million per marginal squares in Neubauer chamber mm )× were used after blood dilution ( ) MCH (pg)=hemoglobin/RBC(million with Race solution and the results were per mm ) × multiplied by . Then, blood samples MCHC=hematocrit/hemoglobin× were fixed in methanol and stained using Wright–Giemsa stain for Blood biochemical parameters determination of the differential WBC Total protein (modi-biuret method, (lymphocyte and neutrophil portions of Tietz, ) and Albumin (romocresol total WBC) count (Houston, ). At green binding method, Doumas and least WBCs were counted for Peters, ) assays were performed differential WBC determinations. Thin with Zist Chemistry kits (Zist chemistry slices were used for counting WBC Company, Tehran, Iran).Total protein counting was done according to their content (at wavelength of - nm) shape and reported as percentages. To and Albumin (at wavelength of nm) Downloaded from jifro.ir at 15:41 +0330 on Friday October 1st 2021 measure the Hemoglobin (Hb) levels were determined using an auto analyzer were obtained by the system (Thecnicon, RA , Cyanmethemoglibin method (Houston, NewYork, NY, USA). Globulin levels ). In this method, the RBC are of the samples were calculated from the haemolized and hemoglobin is difference in albumin from total protein converted to Cyanmethemoglobin. (Kumar et al., ). Total cholesterol, Twenty microliters of blood samples triglyceride and glucose measurements with -µl Drabkin’s reagent were were performed using Pars Azmoon mixed and placed in dark place for commercial kits (Pars Azmoon min to form Cyanemethemoglobin. Company, Tehran, Iran) according to Then, absorbance was read at nm in the company’s protocol and using an a UV with spectrophotometer (Jenway auto-analyzer (Thecnicon, RA , , UK) and finally, hemoglobin New York, NY, USA). Briefly, in this value was calculated. Haemotocrit (Ht) method, µl of plasma sample had was measured using the standard micro- mixed with µl of reagent and haemotocrit method (Subhadra et al., incubated for min in room ) and reported as percentages. temperature. Then, the absorbance of Iranian Journal of Fisheries Sciences ( ) the sample against the blank ( µl of system (Tang et al., ). The serum distilled water mixed with µl of C and C reacted with the antibodies reagents) had measured at a wavelength contained in the kit, and then OD had of nm. Similar approach had done measured at a wavelength of nm for other parameters, but with using an auto-analyzer (Thecnicon, RA appropriate sample amount and , New York, NY, USA), compared wavelength according to each to the standard in kits and finally, the parameter’s protocol (Esmaeili et al., C and C amount was calculated ). The Kinetic method was used to based on mg L- . ACH (the activity measure liver enzymes, aspartate of the complement system) was aminotransferase (AST) and alanine determined according to the indirect aminotransferase (ALT), using Pars ELISA method using a commercial kit Azmoon commercial clinical (Wielsa, comple total complement investigation kits (Parsazmon functional screen kit, Sweden). The Company, Tehran, Iran) and were volume of the serum complement analyzed with an auto analyzer producing hemolysis (ACH ) (Technicon RA- ; Technicon was determined, and the number of Instruments, New York, NY, USA). ACH units in ml was calculated for each experimental fish. Immunological parameters Total immunoglobulin levels were The lysozyme activity in serum was determined according to the method determined turbidometrically using the described by Siwicki and Anderson method described by Ellis ( ). In ( ). In this method, analysis of total this method, Micrococcus luteus Ig level in plasma is based on the biuret bacteria was used (Sigma, M ) with colorimetric method. First the Downloaded from jifro.ir at 15:41 +0330 on Friday October 1st 2021 phosphate buffer ( ml, M, pH= ) immunoglobulins were separated from as substrate. For this purpose, activated plasma by precipitation with bacteria were used and cultivated at a polyethylenic glycol (Sigma) concentration in – nm adjusted and remaining supernatant was read. with spectrophotometer (Jenway , That number was subtracted from the UK). Then, µl of substrate and total protein to give total µl of the sample were added to cuvette. immunoglobulin. Protein readings from The decrease of absorption at nm supernatant gave the amount of protein each min for a period of min had taken out by absorption to polyethylene measured through spectrophotometer glycol. To calculate total (Tukmechi et al., ) with chicken immunoglobulin, subtracted these egg white (Sigma, A ) standard. readings from total protein on Turbidometric method and Pars individual samples and compared with Azmoon commercial clinical standards for calculation of protein. investigation kits (Pars Azmoon The production of oxygen radicals by Company, Tehran, Iran) were used to leukocytes was determined by the measure C and C serum complement reduction of Nitro Blue Tetrazolium Meshkat Roohani et al., Hematite-biochemical and immune response of… (NBT, Sigma-Aldrich Chemical, St. obtained at FMR and FMR Louis, MO, USA) according to Rook et treatments, whereas the fish fed control al. ( ). Absorbance was converted diet had the lowest growth to NBT units based on a standard curve performances. According to the results, of NBT diformazan per milliliter of fish fed diet supplemented with blood. FMR and FMR had a significantly higher weight gain ( Statistical analysis g and g) and specific growth rate All the data have been analyzed using ( %bw day- and %bw day- ) the SPSS (version , SPSS, Richmond, compared with those fed the control VA, USA) statistical package as diet ( g and bw day- ). described by Dytham ( ). Mean Furthermore, %FMR and %FMR values of all the parameters were treatments had statistically higher subjected to one-way analysis of protein efficiency ( and ), lipid variance (ANOVA) to study the efficiency ( and ) and treatment effect and comparison of any statistically lower feed conversion ratio mean values had done by Duncan’s ( and ) compared with other Multiple Range Test (DMRT). treatments (p< ). Moreover, there Comparisons were made at the five was no significant difference in the percent probability level (p< ). condition factor among experimental treatments. In addition, fish fed control, Result %FMR and %FMR diets exhibited The growth performance of Caspian equivalent FCR, protein efficiency and brown trout juvenile fed diets lipid efficiency. Survival rate in all containing different levels of spirulina treatments was . Downloaded from jifro.ir at 15:41 +0330 on Friday October 1st 2021 are presented in Table . Overall, the optimum growth performance has been

Table : Mean growth performance of Salmo trutta caspius fed different levels of Spirulina platensis. Experimental Diets Growth performance control % W (g) WG (%) a* ab ab c bc SGR (% / day) a ab ab c bc CF a a a a a FCR b b a a a Survival rate ( ) W : Primary weight WG%= Weight gain FCR: Feed conservation ratio SGR: Specific growth ratio Conditional Factor(CF) *Values are the least square means±standard errors of the means of triplicate samples. P values determined with one- way ANOVA are also provided for the main factors and their interactions. Means marked with the same letter are not significantly different (p> ).Letters a, b, c, d, and e indicate significant differences in treatments, according to Duncan’s test Iranian Journal of Fisheries Sciences ( )

The mean haemotology parameters of increasing levels of dietary S. platensis. S. trutta caspius juveniles fed with diets So, the lowest and the highest levels for containing different levels of S. these parameters (with exception of platensis are shown in Table . Based neutrophils) were observed in the on obtained results, spirulina had a control and treatments, respectively. significant impact on haemotological Other haematological indices such as parameters (RBC, WBC, Hct, Hb, average volume of red blood cells lymphocytes, neutrophils) in S. trutta (MCV), mean hemoglobin in red blood caspius juveniles. RBC increased with cells (MCH), average concentration of increasing levels of dietary S. platensis hemoglobin in a red blood cells to , but in , the rate decreased (MCHC) also have increased with (p< ). WBC, Hct, Hb, differential increasing levels of dietary S. platensis white blood cell percent, lymphocyte (p< ). and neutrophil increased with

Table : Haemotological parameters of Salmo trutta caspius fed with different levels of Spirulina platensis. Experimental Diets Haemotological parameters control RBC (× n mm− ) a* c cd d b WBC (cell ml- ) a b c d e Hct )%( a b c c c Hb (g dl- ) a bc b c d LYM (% of WBC) a b c d d Neut (% of WBC) a a a c c MCV(fl ) a ab b c c MCH(pg ) a a a ab b MCHC )%( a b b b c *Values are least square means±standard errors of the means of triplicate samples. p values determined with two-way ANOVA tests are also provided for the main factors and their interactions. Means marked with the same letter are not significantly different (p> ). Letters a, b, c, d, and e indicate significant differences in treatments, according to Downloaded from jifro.ir at 15:41 +0330 on Friday October 1st 2021 Duncan’s test

The mean blood biochemical protein and globulin decreased and then parameters of S. trutta caspius juveniles increased in treatment (p< ). fed with diets containing different Other blood biochemical parameters levels of S. platensis are summarized in such as glucose, cholesterol and Table & . Based on obtained results, triglycerides have significantly with increasing levels of dietary S. decreased with increasing levels of platensis up to , ALT and AST dietary S. platensis (p< ). enzymes decreased (p< ). Total

Meshkat Roohani et al., Hematite-biochemical and immune response of…

Table : Liver enzymes of Salmo trutta caspius fed different levels of Spirulina platensis. Experimental Diets Liver enzymes control

ALT(U L- ) d* bc b a a

AST(U L- ) d cd bc b a *Values are least square means ± standard errors of the means of triplicate samples. p values determined with two- way ANOVA tests are also provided for the main factors and their interactions. Means marked with the same letter are not significantly different (p> ). Letters a, b, c, d, and e indicate significant differences in treatments, according to Duncan’s test

Table : Blood biochemical parameters of Salmo trutta caspius fed different levels of Spirulina platensis. Experimental Diets Biochemical factors control

Total protein (g dl- ) a* a b c d Albumin (g dl- ) a a a a a Globulin )g dl- ( a a b b c Glucose (mg dl- ( e d b c a Cholesterol (mg dl- ) d c b a b Triglyceride (mg dl- ) e d c b a *Values are least square means ± standard errors of the means of triplicate samples. pvalues determined with two-way ANOVA tests are also provided for the main factors and their interactions. Means marked with the same letter are not significantly different (p> ). Letters a, b, c, d, and e indicate significant differences in treatments according to Duncan’s test

The mean immunity parameters of S. treatment. Complement C , C , trutta caspius juveniles fed with diets ACH , free radical oxygen and containing different levels of S. immunoglobulin were significantly platensis are shown in Table and . affected by dietary S. platensis levels. Based on obtained results, lysozyme The lowest and the highest values for enzyme activity increased with these parameters have been observed in

Downloaded from jifro.ir at 15:41 +0330 on Friday October 1st 2021 increasing dietary S. platensis levels up the control and treatments. to and then decreased in

Table : Lysozyme activity of Salmo trutta caspius fed different levels of Spirulina platensis. . Lysozyme Experimental Diets activity control After sec a* b c d e (µg ml- ) After sec a a b c d )µg ml- ( *Values are least square means ± standard errors of the means of triplicate samples. p values determined with two-way ANOVA tests are also provided for the main factors and their interactions. Means marked with the same letter are not significantly different (p> ).Letters a, b, c, d, and e indicate significant differences in treatments according to Duncan’s test

Iranian Journal of Fisheries Sciences ( )

Table : Immunological parameters of Salmo trutta caspius fed different levels of Spirulina platensis. Immunological Experimental Diets factor control - a* ab b c d C (mg ml ) - a a a b b C (mg ml ) ACH (unit ml- ) a b c d e Igm(mg dl- ) a a b c d O-(RLU s- ) a b c . d e *Values are least square means±standard errors of the means of triplicate samples. p values determined with two- way ANOVA tests are also provided for the main factors and their interactions. Means marked with the same letter are not significantly different (p> ).Letters a, b, c, d, and e indicate significant differences in treatments, according to Duncan’s test

Discussion improvement to dietary spirulina may The present results indicated that be due to the improved feed intake and spirulina significantly improved the nutrient digestibility. Some researchers growth performance of juvenile have been reported that spirulina Caspian brown trout and the fish meal improved intestinal microbial balance, could be replaced with this microalga leading to better growth by improving until without impairments. During food absorption, digestive enzymes the experimental period, no mortality activities and fats transport system and disease in fish fed spirulina were (James et al., ; Teimouri et al., observed compared to control. In ). In addition, the bio-compounds agreement with our research, many in spirulina, delayed absorption of studies have demonstrated a positive dietary nutrients and improved impact of spirulina on growth carbohydrate and protein utilization in performance (Mustafa et al., ; fish. Moreover, there are several James et al., : Palmegiano et al., nutrients especially vitamins, minerals,

Downloaded from jifro.ir at 15:41 +0330 on Friday October 1st 2021 ; Ramakrishnan et al., ; essential amino acids, fatty acids in Tongsiri et al., ; Ungsethaphand et spirulina, that may be beneficial to fish al., ; Promya and Chitmanat, ; growth promotion and might activate Teimouri et al., ; Khanzadeh et al., metabolism and act as growth ; Cao et al., ; Gogoi et al., stimulants (Mustafa et al., ). ). However, contradictory results Generally, in the current study growth have been reported with no beneficial performance in the FMR and effects of dietary spirulina on growth of FMR treatments were higher than common carp Cyprinus carpio the other dietary groups. Differently, (Nandeesha et al., ), Catla Catla similar spirulina content to the FMR catla (Nandeesha et al., ) and Nile ( g kg- ) has been reported to tilapia Oreochromis niloticus (Lu and decrease growth in rainbow trout Takeuchi, ). It is likely that for (Oncorhynchus mykiss) (Teimouri et carnivorous fish, this microalga has had al., ) and Nile tilapia better growth performance and better (Olvera‐Novoa et al., ). There are fish muscle quality and our study is in no reports about anti-nutritional factors agreement with this hypothesis. Growth in spirulina (Vonshak et al., ). Meshkat Roohani et al., Hematite-biochemical and immune response of… However, the slightly lower growth in Phycoerythrin). In fact, these pigments FMR compared to FMR is due to are responsible for the removal of the lower mineral content such as peroxide radicals in the body and phosphorous in spirulina, compared to reduce the rate of haemolysis of RBC fish meal (Olvera‐Novoa et al., ). by oxidants as well as the ability to Perhaps the fish meal content in significantly increase iron absorption FMR ( g kg- in diet) is optimum (Kop and Durmaz, ; Abdel for Caspian brown trout growth and Tawwab and Ahmad, ; Andrews et higher or lower contents is not enough al., ). to reach the highest growth. Reactions caused by WBC are There was a significant difference in mechanisms of cellular nonspecific or haemotology parameters among all early defense in fish that occur in tested treatments. Our results revealed response to various conditions such as that S. platensis has positive impact on bacterial, viral, fungal, protozoan and the health status of S. trutta caspius. parasitic infections, and it is the first Previous studies such as those by Terry indicator to determine the health of et al. ( ) on tilapia, Abdel Tawwab every living being (Andrews et al., and Ahmad ( ) on tilapia, Yong- ). Increasing WBC and their Chin et al. ( ) on vannamei shrimp differentiate percent with increasing (Litopenaeus vannamei), Andrews et al. levels of dietary S. platensis is due to ( ) on Nile tilapia, Promya and stimulating the immune system via Chitmanat ( ) on African catfish, bioactive substances contained in the Ibrahem et al. ( ) on Nile tilapia, microalgae (Tort, ). Furthermore, Ragap et al. ( ) on Nile tilapia, phytocyanins contained in S. platensis Krishnaveni et al. ( ) on catla, activate types of WBC (e.g., Downloaded from jifro.ir at 15:41 +0330 on Friday October 1st 2021 Zamini and Azimi ( ) on Carp koi; macrophages and granulocytes) and are Salehi Farsani et al. ( ) on stellate responsible for repairing damaged sturgeon confirmed that S. platensis has tissues by pathogens (Selmi et al., positive effects on haemotologic ). This bioactive substance with parameters. In addition, increasing induction of cytokine secretion haemotology indices (e.g., RBC, Hb (molecules involved in the immune and Hct) of S. trutta caspius with system by activating macrophages and increasing levels of S. platensis can lymphocytes.) stimulates the production result from a variety of reasons of new WBC marker molecules (Raa, including iron compounds, vitamins ). (B , A and E), and also the Based on these results, levels of liver considerable antioxidant capacity enzymes (AST and ALT) decreased contained in S. platensis. This high with increasing levels of S. platensis. antioxidant capacity is due to the Based on accepted hypothesis, the considerable amount of pigments, increase of these enzymes in animals particularly Phycocyanobilin (e.g., (including fish), indicate damage to the Phycocyanin, Allophycocyanin and liver cells (Jeney and Anderson, ). Iranian Journal of Fisheries Sciences ( ) Numerous studies have confirmed the often referred to as a non-specific effectiveness of S. platensis to reduce marker of stress (Sheikhzadeh et al., cholesterol and liver protection (Khan ). In this study, blood glucose, et al., ; Pieretti and Meineri, ; triglycerides and cholesterol decreased Kim, ; Zeinab et al., ). One of with increasing levels of S. platensis. the most important approaches of Our results are in agreement with data antioxidants is liver cell protection. The reported by other researchers (Abdel- antioxidant properties of S. platensis Tawwab and Ahmad, ; Andrews et have attracted the attention of many al., ; Hernandez, ; Salehi researchers. One of the latest Farsani et al., ). investigations Manoj et al. ( ) Long-chain omega- fatty acids in reported that alcoholic extract of S. the liver by reducing the secretion of platensis accelerates oxidation of fat lipoproteins (VLDL) can be reduced significantly ( ) in comparison to triacylglycerol levels (Harris et al., the antioxidant chemicals such as ). S. platensis is low fat ( fat) alpha-tocopherol ( ), BHA or so, gr. of S. platensis has only gr. Butylated Hydroxy Anisole ( ), and calorie and no cholesterol. S. platensis beta-carotene ( ). particularly in terms of alpha-linolenic In this study, serum total protein, acid ( of the total PUFA) is very albumin and globulin increased with rich. The most essential fatty acids in S. increasing levels of S. platensis. Several platensis that in other food sources are studies have been demonstrated the scarce, is gamma linolenic acid. It has effects of S. platensis on blood been grown under special lighting biochemical parameters (Schaperclaus conditions in the cells, are also able to et al., ; Dunkan and Klesius, ; enrich and enhance it (Khan et al., Downloaded from jifro.ir at 15:41 +0330 on Friday October 1st 2021 Abdel-Tawwab and Ahmad, ; ). Gamma-linolenic acid is a Andrews et al., ; Hernandez, ; precursor of prostaglandins (PGE) and Zeinab et al., ). Blood serum has special effect on the control of protein show changes in health blood cholesterol levels that leads to conditions affected by internal and rapid cell growth. The role of PGE is external factors. Albumin is a blood pressure regulation, cholesterol transporter protein or public transporter synthesis control and cell survival of many organic and inorganic ligands, (Harris et al., ). such as thyroxine, bilirubin, penicillin, In this study, we evaluated the cortisol, estrogen, free fatty acids, immunity status of S. trutta caspius fed calcium, and magnesium during illness, with different levels of S. platensis, malnutrition and stress, blood albumin using lysozyme, complement C and is reduced (Misra et al., ; C , immunoglobulins, oxygen free Alexander et al., ). Glucose, radical and haemolytic activity of the cholesterol and triglyceride levels complement factor. Based on our fluctuate during protein catabolism and results, the activity of lysozyme after glycogenesis. Serum glucose levels are and seconds with increasing levels Meshkat Roohani et al., Hematite-biochemical and immune response of… of S. platensis significantly increased colonization with pathogenic (up to ). Effect of S. platensis on microorganisms, prevent infection and lysozyme by various researchers have pathogenesis. Free radicals of oxygen - confirmed (Watanuki et al., ; (O , H O and O ) together with nitric Abdel Tawwab and Ahmad, ; oxides, lysozyme, cytokines and other Andrews et al., ; Promya and messenger molecules, (prostaglandins, Chitmanat, ; Ragap et al., ; leukotrienes and thromboxanes) Ibrahem et al., ; Krishnaveni et al., increased phagocytic and Pynocytic ; Kim , ; Shimaa, ; Salehi capacity of macrophage cells (Taoka et Farsani et al., ). Lysozyme by al., ). Measuring any of these WBCs (monocytes, macrophages, factors can be effective in determining neutrophils) and using circulating the the activation of macrophages. Several blood in different tissues of the body studies have demonstrated a positive released and leads to non-specific impact of immune-stimulants such as immune organisms (Saurabh and probiotics and micro-algae on amount Sahoo, ). of oxygen free radicals and phagocytic The complement system is a part of activity (Dunkan and Klesius, ; the immune system that enhances the Rengpipat et al., ; Li and Gatlin, ability of antibodies and phagocytic ; Panigrahi et al., ; Taoka et cells to clear microbes and damaged al., ; Watanuki et al., ; Abdel- cells from an organism, promotes Tawwab and Ahmad, ; Andrews et inflammation, and attacks the al., ; Ragap et al., ; Kim, pathogen's plasma membrane (Claire et ; Ibrahem et al., ). Oxidative al., ). Several factors have an stress is the accumulation of ROS impact on immunity, including divalent (Reactive Oxygen Species, ROS). It Downloaded from jifro.ir at 15:41 +0330 on Friday October 1st 2021 ions (especially magnesium and may be an important factor in the calcium), polysaccharides process of developing a variety of immunogenic, season, sexual maturity, diseases. The immune system is prostaglandins, thromboxane and vulnerable to oxidative damage. S. immunoglobulins (Tort et al., ). platensis also contains other pigments The activity of the complement system Phycocyanobilin including and immunoglobulins is increased using Phycocyanin, Allophycocyanin and microalgae (Ragap et al., ; Phycoerythrin that are mainly Krishnaveni et al., ; Shimaa, responsible for antioxidant activity and ). C and C high levels are the removal of peroxide radicals in the related to the health of the fish. High body (Ragap et al., ) serum complement activity in fish fed The results of different impacts S. platensis could be due to the levels of S.platensis on immune and presence of pigment carotenoids haemotology parameters in S.trutta especially beta-carotene. In addition, caspius show that S.platensis may be lysozyme, antibodies and complement beneficial in improve growth, immunity factors due to adhesion and levels in Caspian Sea salmon and create Iranian Journal of Fisheries Sciences ( ) resistance to pathogens. These results AOAC, . Official methods of indicate that S. platensis analysis. th ed. Association of supplementation is promising for Official Analytical Chemists. disease prevention in S. trutta caspius Gaithersburg, MD, USA. P. juveniles and can substitute for up to Bain, B., Bates, I. and Laffan, M., of the fish meal in the diet, although . Dacie and Lewis Practical the optimum performance is obtained Haematology. th Edition, with substitution Published Edinburgh Churchill Livingstone. References Belay, A., Kato, T. and Ota, Y., . Abdel-Tawwab, M. and Ahmad, Spirulina (Arthrospira): Potential M.H., . Live Spirulina application as an animal feed (Arthrospira platensis) as a growth supplement. Journal of Applied and immunity promoter for Nile Phycology, , - . tilapia, Oreochromis niloticus (L.), Blaxhall, P.C. and Daisley, K.W., challenged with pathogenic . Routine haematological Aeromonas hydrophila. Aquaculture methods for use with fish blood. Research, , - . Journal of Fish Biology, ( ), – Aksnes, A. and Opstvedt, J., . . Content of digestible energy in fish Cao, S.P., Zou, T., Zhang, P.Y., Han, feed ingredients determined by the D., Jin, J.Y., Liu, H.K., Yang, ingredient-substitution method. Y.X., Zhu, X.M. and Xie, S.Q., Aquaculture, , – . . 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Downloaded from jifro.ir at 15:41 +0330 on Friday October 1st 2021 nutrition on blood and immunity parameters of koi (cyprinus carpio). Academie Royale DesScience D Outre-Mer Bulletin DesSeances, ( ), - . Zeinab, A.K., Aly, M.S., Faiza, A. and Fatma, E.M., . Effect of Spirulina platensis and Lactobacillus rhamnosus on growth and biochemical performance of Nile Tilapia (Oreochromis niloticus) fingerlings. International Journal of Current Microbiology and Applied Sciences, ( ), -