Effects of Diets Containing Beta-Glucan on Survival, Growth Performance, Hematological, Immunity and Biochemical Parameters of Rainbow Trout (Oncorhynchus Mykiss) Fingerlings

Abstract

This study aimed to investigate the effect of beta-glucan in rainbow trout’s diet on survival, growth performance, hematological, immunity and biochemical parameters. Rainbow trout ( Oncorhynchus mykiss ) with an average weight of 6.35 ± 0.09 were stocked in 30 fiberglass tanks with a volume of 200 liters. Four treatments including: control group (0%), 0.5, 1 and 2% of beta-glucan were considered for 8 weeks. The results showed that the survival and growth performance of rainbow trout in the control group were at the lowest level and showed a significant difference with other treatments. The highest percentage of hematocrit (47.9%), white blood cell count, neutrophil percentage (35.2%) was observed in the treatment with 2% beta-glucan. Also, the highest lysosomal activity (59.7 units’ mL − 1 , IgM (54.7 mg dL − 1 ), C3 and C4 was obtained in 2% beta-glucan treatment. Biochemical parameters showed improvement in treatments fed with 1 and 2% beta-glucan. In general, the present study showed that 2% beta-glucan in the diet of rainbow trout improves growth performance, survival, hematological, immunity and biochemical parameters.

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Effects of Diets Containing Beta-Glucan on Survival,
Growth Performance, Hematological, Immunity and
Biochemical Parameters of Rainbow Trout
(Oncorhynchus Mykiss) Fingerlings
Mohammad Hossein Khanjani (  m.h.khanjani@gmail.com )
University of Jiroft https://orcid.org/0000-0002-3891-8082
Gholamreza Ghaedi
Khorramshar Marine Science and Technology University: Khorramshahr Marine Science and Technology
University
Moslem Sharinia
Iranian Fisheries Research Organization
Research Article
Keywords: Beta-glucan, Immune-stimulation, Rainbow trout, Growth performance, Innate immunity
DOI: https://doi.org/10.21203/rs.3.rs-641367/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
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Abstract
This study aimed to investigate the effect of beta-glucan in rainbow trout’s diet on survival, growth
performance, hematological, immunity and biochemical parameters. Rainbow trout (
Oncorhynchus
mykiss
) with an average weight of 6.35 ± 0.09 were stocked in 30 berglass tanks with a volume of 200
liters. Four treatments including: control group (0%), 0.5, 1 and 2% of beta-glucan were considered for 8
weeks. The results showed that the survival and growth performance of rainbow trout in the control group
were at the lowest level and showed a signicant difference with other treatments. The highest
percentage of hematocrit (47.9%), white blood cell count, neutrophil percentage (35.2%) was observed in
the treatment with 2% beta-glucan. Also, the highest lysosomal activity (59.7 units’ mL− 1, IgM (54.7 mg
dL− 1), C3 and C4 was obtained in 2% beta-glucan treatment. Biochemical parameters showed
improvement in treatments fed with 1 and 2% beta-glucan. In general, the present study showed that 2%
beta-glucan in the diet of rainbow trout improves growth performance, survival, hematological, immunity
and biochemical parameters.
1. Introduction
The aquaculture industry as one of the sources of protein and signicant source of sustainable food is
growing rapidly (Khanjani et al. 2021d; Khanjani and Sharinia 2020,). Success in aquaculture is
achieved by improving genetics, nutrition, immunity and disease control in farmed species. Parameters
such as specic growth rate, feed conversion ratio and survival rate play a key role in evaluating an
aquaculture system (Ghaedi et al. 2015, Khanjani et al. 2020b). The use of immunostimulants is
considered as an effective tool to overcome diseases and strengthen the immune system of farmed
organisms (Meena et al., 2013). In recent decades, much attention has been paid to nding a variety of
immunostimulants with low cost which affect specic and nonspecic immunity and improve the
resistance of sh to a wide range of pathogens (Ghaedi et al. 2015, Dawood et al. 2020a, Mokhbatly et al.
2020, Yang et al. 2021). These stimuli strengthen the immune system of sh by increasing the number of
phagocytes, enhancing the activity of lysozyme, complement and rising the level of immunoglobulin
(Dawood et al. 2020b, Yan et al. 2020).
Recently, more attention has been paid to the use of functional dietary supplements such as probiotics,
prebiotics and immune stimulants in aquaculture. These compounds are useful for improving the
immune system, feed eciency and sh growth performance. Among these immunostimulants used in
aquaculture, beta-glucans are of particular importance for use in the aquaculture industry, as these
compounds reduce stress, disease prevalence, and sh production through biotechnology (Pilarski et al.
2017). β-glucans are homopolysaccharides which composed of glucose molecules linked together by a
glycosidic bonds. This immunostimulant has been widely used to boost innate immunity, and to improve
phagocyte activity, respiratory burst activity, nitric oxide, complement and lysozyme activity; it also
enhances the number of leukocytes (Soltanian et al. 2009, Meena et al. 2013) in some species such as
grass carp (Yang et al. 2021); African catsh (Mokhbatly et al. 2020); pearl gentian grouper (Wei et al.
2020); rainbow trout (Ji et al. 2020) and Nile tilapia (Dawood et al. 2020a). In nature, β-glucans are

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abundant in the cell wall of many plants such as wheat, rye, barley, oats, yeast (
Saccharomyces
) and
members of the Echinaceae family (Tokunaka et al. 2000). β-glucans due to their ability to bind directly to
macrophages and other white blood cells, such as neutrophils and natural killer cells, and activate them,
they provide good resistance to any invader (Herre et al. 2004).
Rainbow trout (
Oncorhynchus mykiss
) is one of the most valuable economic sh and the most important
cold-water species in Iran's aquaculture industry. Efforts to improve the growth indices and boost the
immunity of this sh against various bacterial diseases have increased (Ranjbar et al. 2010). In several
studies, the effects of various immunostimulants such as nettle (
Nigella sativa
), mistletoe (
Viscum
album
), aloe vera (
Aloe barbadensis
), astragalus (
Astragalus gummifer
), Purple Coneower (
Echinacea
purpurea
), Oregano (
Mentha longifolia
), Green tea (
Camellia sinensis
), ginger (
Zingiber ocinale
),
Bakhtiari savory (
Satureja bakhtiarica
) and peppermint (
Mentha piperita
) have been used in rainbow trout
(Haghighi and Sharif Rohani, 2013; Sheikhzadeh et al., 2011). The aim of the present study was to
evaluate the effects of different concentrations of dietary β-glucan on growth performance,
hematological, immunity and biochemical parameters of rainbow trout.
2. Materials And Methods
2.1. Fish and experimental conditions
Altogether, 360 rainbow trout ngerlings were obtained from a private company (Sepidan, Fars, Iran).
Twelve 200-L tanks were prepared and 30 sh were stocked in each tank. water ow rate was 8.2 L min−
1. Water temperature (°C), pH and dissolved oxygen (mg L− 1) were measured weekly and the levels were
determined 12- 13.4°C, 7- 7.4 and 7- 7.6 mg L− 1, respectively. Fish were distributed into four treatments
with three replications for each treatment. The adaptation period was 2 weeks and the feeding was
performed with the commercial diet. Light cycle was 12L:12D, and throughout the experiment, sh were
fed four times daily to apparent satiation at 07:00, 11:00, 15:00 and 19:00 h for 8 weeks.
2.2. Experimental diets
β-glucan (MacroGard®, Biotec-Mackzymal, TromsØ, Norway) was purchased and added to a commercial
diet (Beyza Feed Mill, Fars province, Iran) to obtain diets containing 0.5, 0.1% and 0.2% β-1,3/1,6 yeast
glucan. The diet contained 45% protein, 14% lipid and 15% carbohydrates. The prepared diets were stored
at 4°C in plastic bags until used.
2.3. Growth performance and survival
Feeding was stopped 24 h before weighing. sh were anesthetized with clove powder at a concentration
of 200 mg L− 1.
Weight gain (WG), feed conversion ratio (FCR), specic growth rate (SGR) and survival rate were
calculated using the standard formulas (Khanjani et al. 2021a, Khanjani et al. 2021b):

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Body weight gain (g) = nal weight- initial weight
Body weight index (BWI) (%) = [(nal weight- initial weight)/initial weight] × 100
Growth rate (GR) (mg) = [(nal weight- initial weight)/ days of experiment]
Survival rate (SR) (%) = (number of individuals at end of testing period/initial number of
individuals stocked) × 100.
Specic growth rate (SGR) (%/day) = [(ln nal weight-ln initial weight) ×100]/days of experiment
Feed conversion ratio (FCR) = feed consumed (dry weight)/live weight gain (wet weight)
2.4. Sampling
Sampling was performed 56 days after feeding with beta-glucan. First, the sh were anesthetized with
cloves at a concentration of 200 mg L− 1 and blood was taken from caudal vein (Bohlouli et al., 2015).
Some blood was transferred to tubes containing heparin anticoagulant to measure hematological
parameters and some was transferred to heparin-free tubes to prepare serum and to measure immunity
and biochemical parameters. Heparin-free tubes were centrifuged at 5000 rpm for 5 minutes to separate
serum at 4°C. Serum samples were transferred by sampler to Eppendorf vials and stored in a freezer at
-20°C until biochemical analysis was initiated (Chebanov and Billard 2001, Ghaedi et al. 2015).
2.5. Hematological and biochemical analyses
Blood was diluted and stained with Natt–Herrick’s solution, then red blood cell (RBC) and white blood cell
(WBC) measurement were performed by cell counter method. For differential counts of WBC, blood was
spread on a slide and stained with Gimsa (Hrubec et al. 2001). Hct and Hb were measured by photometric
assay of microhaematocrit and cyanomethemoglobin method, respectively (Houston 1990). Mean
corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin
concentrations (MCHC) were calculated by standard formulas (Ranzani-Paiva et al. 2004). Total protein
was measured using a diagnostic kit (ZiestChem, Diagnostics Co., Iran) according to Vella (1986).
Albumin content was determined following the method of Doumas et al. (1997). Globulin content was
calculated by subtracting albumin from the total protein (Kumar et al., 2005).
2.6. Immunological parameters
The volume of serum complement producing 50% hemolysis (ACH50) was assayed using the method of
Sunyer and Tort (1995). Lysozyme activity was determined according to the lysis of the lysozyme
sensitive Gram-positive bacterium,
Micrococcus lysodeikticus
(Sigma) (Demers and Bayne 1997). Total
immunoglobulin (Ig) level was determined in plasma prior to and after precipitating the Ig molecules
employing a 12% solution of polyethylene glycol (Sigma Chemical) (Puangkaew et al. 2004).
Immunoglobulin M (IgM) was measured using a protein kit (Pars Azmoun Company, Karaj, Iran) and the

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Bradford method (Kruger 2009). Measurement of alternative complements (C3 and C4) activity was
performed based on rabbit red blood cell hemolysis (Amar et al. 2000).
2.7. Statistical analysis
Results were expressed as means ± standard deviation (SD). SPSS software version 21 (SPSS, Chicago,
IL, USA) was used to analyze the data. Kolmogorov–Smirnov and Levene’s tests were used to check
normality and variances for homogeneity, respectively. Then, one-way ANOVA test was applied to
determine differences between hematological and immunological parameters at various levels of b-
glucan. Tukey’s post hoc test was applied to identify treatments with signicant differences (P < 0.05).
3. Results
The results of rainbow trout growth performance are presented in Table1. The results showed that the
lowest rate of body weight gain, specic growth rate and growth rate were obtained in the control group
(P < 0.05), higher values of these factors were observed in the 2% beta-glucan treatment. The highest rate
of feed conversion ratio (1.19) was observed in the control group which showed a signicant difference
with other treatments (P < 0.05).
Table 1
Growth performance and survival rate of rainbow trout fed diets containing different
levels of beta-glucan for 8 weeks
Parameters Control 0.5% 0.1% glucan 0.2% glucan
Initial weight (g) 6.34 ± 0.1 6.35 ± 0.09 6.32 ± 0.08 6.31 ± 0.09
Final weight (g) 15.55 ± 0.59c16.56 ± 0.25b17.38 ± 0.27a17.58 ± 0.61a
WG (g) 9.21 ± 0.59c10.21 ± 0.25b11.06 ± 0.27a11.27 ± 0.61a
SGR (% day− 1)1.6 ± 0.06c1.71 ± 0.03b1.8 ± 0.03a1.82 ± 0.06a
GR (g day− 1)0.164 ± 0.011c0.182 ± 0.01b0.197 ± 0.005a0.2 ± 0.01a
BWI (%) 145.3 ± 9.29c160.8 ± 3.94b175.0 ± 4.25a178.6 ± 9.6a
SR (%) 94.44 ± 1.72b95.57 ± 1.8b97.78 ± 3.44a98.89 ± 1.72a
FCR 1.19 ± 0.07c1.08 ± 0.03b0.99 ± 0.02 a0.98 ± 0.05 a
Means in the same row with different superscripts are signicantly different (P < 0.05).
Blood parameters of rainbow trout fed with different levels of beta-glucan is presented in Table2.
According to the results, the highest white blood cell density and hematocrit percentage (47.9%) were
observed in the treatment of trout fed with 2% beta-glucan (P < 0.05).

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Table 2
Hematological parameters (mean ± SD) of rainbow trout fed with different levels of β-glucan
for 8 weeks.
Parameters Control 0.5% 0.1% glucan 0.2% glucan
RBC (×106 mm− 3)0.9 ± 0.1c1.02 ± 0.13abc 1.04 ± 0.1ab 1.15 ± 0.23a
HB (g dL− 1)6.95 ± 0.18d7.35 ± 0.3c7.95 ± 0.33b8.45 ± 0.22a
HCT (%) 35.35 ± 1.23c40.05 ± 2.13b41.75 ± 4.5b47.9 ± 2.0a
MCV (fL) 392.77 ± 12.2b392.7 ± 14.1b401.44 ± 19.3ab 416.52 ± 17.1a
MCH (pg) 77.22 ± 1.5a72.06 ± 2.7b76.44 ± 3.13ab 73.48 ± 3.2b
MCHC (g dL− 1)19.66 ± 0.5a19.34 ± 0.6a19.05 ± 0.53a19.25 ± 0.63a
WBC (×103 mm− 3)8.75 ± 0.53c8.85 ± 0.43c9.65 ± 0.31b10.45 ± 0.44a
Neutrophils (%) 29.05 ± 2.29b30.15 ± 2b31.25 ± 2.23b35.2 ± 1.8a
Lymphocytes (%) 66.55 ± 3.4a65.8 ± 2.4a64.6 ± 3.1a61.25 ± 2.7b
Monocytes (%) 3.4 ± 0.83a3.35 ± 0.7a3.4 ± 0.9a3.05 ± 0.73a
Eosinophils (%) 1.0 ± 0.61a0.7 ± 0.53a0.75 ± 0.6a0.5 ± 0.55a
Means in the same row with different superscripts are signicantly different (P < 0.05).
The immunological parameters of rainbow trout are shown in Table3. Based on the results, the highest
levels of immunoglobulin M, lysozyme, C3 (47.25 mg mL− 1), C4, ACH50 and total immunoglobulin (28.7
mg mL− 1) were obtained in 2% beta-glucan treatment, which showed a signicant difference with other
treatments (P < 0.05). Improvement of immunity in beta-glucan treatments was observed better than the
control group. The biochemical parameters of rainbow trout in different treatments are presented in
Table4. The results showed that the amounts of albumin (2.81 g dL− 1) and total protein (3.98 g dL− 1) in
1 and 2% beta-glucan treatments were signicantly higher than 0.5% beta-glucan and control group.

Page 7/16
Table 3
Immunological parameters of rainbow trout fed diets containing different levels of β-
glucan for 56 days.
Parameters Control 0.5% 0.1% glucan 0.2% glucan
IgM (mg dL− 1)32.7 ± 2.13d41.9 ± 3.2c47.7 ± 2.14b54.7 ± 3.8a
Lysozyme (units mL− 1)38.14 ± 2.1c44.7 ± 1.8b45.7 ± 2.4b59.7 ± 2.55a
C3 (mg mL− 1)28. 4 ± 3.1c36.24 ± 2.9b39.14 ± 1.8b47.25 ± 2.1a
C4(mg mL− 1)8. 6 ± 1.1c8.8 ± 1.2c10.9 ± 1.8b13.4 ± 1.5a
ACH50 (units mL− 1)59.7 ± 2.1c88.7 ± 3.8b95.7 ± 3.4b109.4 ± 2.85a
Total Ig (mg mL− 1)15.4 ± 1.2d18.1 ± 2.0c23.5 ± 1.4b28.7 ± 2.25a
Means in the same row with different superscripts are signicantly different (P < 0.05).
Table 4
Biochemical parameters of rainbow trout in different treatments
Parameters Control 0.5% 0.1% glucan 0.2% glucan
Globulin (g dL− 1)1.05 ± 0.23a1.14 ± 0.25a1.19 ± 0.22a1.17 ± 0.15a
Albumin (g dL− 1)2.45 ± 0.17b2.4 ± 0.15b2.74 ± 0.2a2.81 ± 0.13a
Total Protein (g dL− 1)3.5 ± 0.18b3.54 ± 0.2b3.93 ± 0.2a3.98 ± 0.14a
Means in the same row with different superscripts are signicantly different (P < 0.05).
4. Discussion
4.1. Growth performance
Immunostimulants such as β-glucans improve aquaculture production, positively affect sh farming,
modify some hematological and immunological parameters (Sánchez-Martínez et al. 2017). In the
current research, growth performance and survival rate in treatments of 1 and 2% beta-glucan were
signicantly higher compared to 0.5 and 0% of beta-glucan treatments. Regarding the effect of beta-
glucan on growth performance, the results of this study are consistent with the results of other
researchers. Misra et al. (2006) showed that oral administration of beta-glucan at 540 mg kg− 1 for 56
days had a positive effect on growth, immunity and survival rate of Indian carp fry. A signicant increase
in growth performance with oral beta-glucan administration in Snapper (Cook et al. 2003), Monodon
shrimp (Chang et al. 2000), Koi carp (Lin et al. 2011) and rainbow trout (Ji et al. 2020) has been reported.

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In the gut, beta-glucans are broken down by beta-glucanase to facilitate the growth of bacteria that utilize
amino acids, which leads to better use of proteins by sh. (López et al. 2003).
Oligosaccharides are a good source of nutrients for the growth and activity of bacteria in the
gastrointestinal tract such as lactic acid bacteria, lactobacilli and bidobacteria and are used as an
energy source by lactic acid bacteria (Staykov et al. 2007). In addition, the most important ultimate
product of metabolism carbohydrate compounds such as beta-glucan are short-chain fatty acids that are
absorbed through the intestinal epithelium and, in addition to providing an energy source for the host,
improve nutrient uptake and increase growth (Waché et al. 2006). Studies have shown that prebiotics like
mannan oligosaccharide and beta-glucan cause the production of hepatic glucose, which provides
energy for metabolism of body tissues and ultimately improves intestinal function by creating the
appropriate conditions for the activity of lactic acid bacteria in the gut (Andrews et al. 2009). Improving
the survival rate of sh fed on beta-glucan, especially in 1% and 2% treatments, may be related to
improving the immune status of sh (Couso et al. 2003, Dalmo and Bøgwald 2008), which is in
consistent with previous research on rainbow trout (Yarahmadi et al. 2016).
In the present study, the highest feed conversion ratio was observed in the control group, which shows
that the presence of beta-glucan in the diet of rainbow trout reduces the feed conversion ratio. As a
supplement in salmon diets, beta-glucan is likely to affect the diversity and abundance of intestinal
microbes, and these microbiota are essential for improving growth function, survival and nutritional
function (Hoseinifar et al., 2015).
4.2. Hematological parameters
Blood parameters are essential tools for assessment of the physiological stress response and general
health conditions of sh during nutritional and environmental changes
The results of the effect of different nutritional strategies with beta-glucan showed that the highest and
lowest red blood cell counts were observed in the diet treated with 2% beta-glucan and the control group,
respectively. Regarding the amounts of hemoglobin and hematocrit, the lowest amount was observed in
the control group. Fish red blood cell and hemoglobin counts change signicantly with seasonal changes,
sexual cycle and other physiological factors (Krajnović-Ozretić et al. 1991). Due to the constant
environmental conditions and sh, the presence of beta-glucan in the diet affected the hemoglobin
concentration. An increase in red blood cell count was observed in 2% beta-glucan treatment. It is
possible that immunostimulants increase metabolism in sh, so that the number and oxygen carrying
capacity of red blood cells enhance (Irianto and Austin 2002). Feeding Oscar sh (
Astronotus ocellatus
)
with yeast signicantly increases the number of red blood cells (Firouzbakhsh et al., 2011), which is
consistent with the results of the present study.
RBC counts in the blood of sh fed on diets supplemented with immunostimulants were higher, indicating
that the sh's immune functions were improved, their defensive mechanisms against pathogens were
activated, and their health was improved (Talpur et al., 2012; Adorian et al., 2018).

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In the control group, a lower hematocrit percentage may indicate they are more susceptible to stress
induced by experimental management or the pathogenic load naturally present in the culture environment
(Tavares-Dias and Moraes, 2004; Mohammadian et al., 2019).
The study also demonstrated this by analyzing other blood variables. It is important to remember that
hemoglobin is a vital component of blood and serves as an oxygen transport system for the body.
However, it should be noted that the levels of hemoglobin were highest at 2% beta-glucan. Its increased
content indicates that sh receive more oxygen, which in turn enhances sh welfare (Talpur et al., 2012).
This indicates that beta-glucan enhances the availability of oxygen in sh blood, leading to benecial
health effects.
In the present study, the presence of beta-glucan in different treatments caused a signicant increase in
white blood cell population, especially blood neutrophils, compared to the control group, but no
signicant difference was observed in the population of monocytes and eosinophils. Jeney et al. (1997)
found that the inclusion of beta-glucan in the diet of rainbow trout signicantly increased the population
of blood neutrophils and decreased lymphocytes. When beta-glucans bind to beta-glucan receptors in
macrophages and neutrophils, they produce oxygen free radicals and increase the antioxidant activity of
enzymes, thereby enhancing immunity, anti-stress activity, and enhancing the invasion of pathogens (Kim
et al. 2009).
Alternative immune responses, in the absence of specic opsonization, could depend on the presence of
mannose receptors and toll-like receptors (TLRs) in microbes, which bind to mannose and glucans,
leading to enhanced phagocytic and bactericidal abilities in phagocytes and neutrophils (Rebl et al.,
2009).
Since WBCs are considered to be the rst line of defense against environmental stress or pathogens, an
increase in the number of them in sh fed with probiotics may reect stimulation of the innate immune
system (Misra et al., 2006). The proportion of leukocytes increased in
Oreochromis niloticus
(Ferguson et
al., 2010) and
O. Mykiss
(Merrield et al., 2011) fed with immunostimulants supplemented diets.
Immunological and biochemical parameters
Herbal medicines are among the immunostimulants that activate the immune cells by affecting the
immune system of sh and lead to increased macrophage cell activity, phagocytic cells (neutrophils and
monocytes), lymphocyte count, serum immunoglobulins and lysozyme activity. The use of these
substances is an effective tool to increase growth indices, immune system capacity and resistance of
sh to common diseases (Hoseinifar et al. 2010). In the current research, a signicant difference was
observed in the values of immune parameters, so that the highest levels of IgM, Lysozyme, C3, C4 and
ACH50 were observed in treatment with 2% beta-glucan, which shows that adding beta-glucan to the diet
improves immunity in trout. Ai et al. (2007) examined the effects of beta-glucan on growth and innate

Page 10/16
immunity indices in
Pseudosciaena crocea
ngerlings. The results of their study showed that 0.09% of
glucan in the diet has the best effect on immunity, but immunity was not signicantly different in the
control group and 0.18%. Moreover, Zhu et al. (2012) reported that channel catsh
Ictalurus punctatus
immune system improved through the phagocytic activity increment owing to feed supplemented (0.3%)
with compounds containing at least of 25% of β-1,3/1,6 glucans.
Studies have indicated that β -1,3-glucan can modulate innate immunity in O. Mykiss (Diao et al., 2013;
Mohammadi et al., 2019) which agrees with what we observed in the present investigation.
The improvement of the immune system in sh fed with a β-1,3-glucan diet could be attributable to lactic
acid-producing bacteria (LAB) fermenting in the large intestine or colon, enhancing their relative
populations, elevated health status and increased colonization of the LAB compared to the control diets.
Based on the results of the present study, a signicant rise in serum proteins (albumin and total) was
observed in the treatments fed with beta-glucan 1 and 2 which may be related to the production of
immunoglobulin. In sh fed higher levels of beta-glucan in the diet, higher levels of albumin and total
protein were observed. Total plasma protein is a dependent parameter for assessing the physiological
status of sh and is a diagnostic factor. Total protein and albumin levels can indicate the nutritional
status and health of sh (Svetina et al. 2002). Increases in protein and albumin levels reect an
improvement in innate immunity; in other words, increases in total protein and albumin concentrations
may be due to stronger nonspecic reactions in sh (Tavares-Dias and Moraes 2007). In the present
study, the increase in total protein in treatments containing 1 and 2% beta-glucan could indicate proper
function of the liver, kidneys and aquatic gastrointestinal tract. These results are in accordance with
reports of previous researchers who reported that immunostimulants increased total serum protein,
albumin and globulin levels in different sh species (Newaj-Fyzul et al., 2007; Sharifuzzaman et al., 2010;
Mohammadian et al., 2019 ).
In conclusion, this study showed that the presence of beta-glucan at the level of 2% in the diet of rainbow
trout improves growth performance, blood and biochemical parameters. Therefore, it is recommended to
use beta-glucan in the diet of rainbow trout to improve production performance.
Likewise, 0.2% of β-glucan supplementation is sucient to stimulate the nonspecic immune system of
rainbow trout and has a positive effect on parameters such as WBC count and neutrophil activity. Further
research is needed on β-1,3 / 1,6-glucan activity and challenge of sh against infectious pathogens.
Declarations
Funding: This research is not nancially supported
Conict of interests /Competing interests: The authors declare that they have no conict of interest.
Ethics approval/declarations:All applicable intuitional guidelines for care and use of sh were followed
by the authors.

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Consent to participate: all authours are agree to participate
Consent for publication:all authours are agree to publication
Availability of data and materials (Data transparency):All the data related to the manuscript are genuine
and available with us.
Code availability:-
Authors’ contribution: All the authors of the manuscript made signicant contribution in the current
study.Mohammad Hossein Khanjani: Conception and design of study, Acquisition of data, Analysis and
interpretation of data, Drafting the manuscript.Gholamreza Ghaedi:Conception and design of study,
Acquisition of data, Analysis and interpretation of data.Moslem Sharinia: Conception and design of
study, Acquisition of data, Analysis and interpretation of data, Revising the manuscript
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