4.1. Growth performance
Immunostimulants such as β-glucans improve aquaculture production, positively affect fish 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 significantly 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 significant 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. 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 fish. (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 bifidobacteria 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 fish fed on beta-glucan, especially in 1% and 2% treatments, may be related to improving the immune status of fish (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 fish 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 significantly with seasonal changes, sexual cycle and other physiological factors (Krajnović-Ozretić et al. 1991). Due to the constant environmental conditions and fish, 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 fish, so that the number and oxygen carrying capacity of red blood cells enhance (Irianto and Austin 2002). Feeding Oscar fish (Astronotus ocellatus) with yeast significantly 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 fish fed on diets supplemented with immunostimulants were higher, indicating that the fish'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).
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 fish receive more oxygen, which in turn enhances fish welfare (Talpur et al., 2012).
This indicates that beta-glucan enhances the availability of oxygen in fish blood, leading to beneficial health effects.
In the present study, the presence of beta-glucan in different treatments caused a significant increase in white blood cell population, especially blood neutrophils, compared to the control group, but no significant 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 significantly 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 specific 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 first line of defense against environmental stress or pathogens, an increase in the number of them in fish fed with probiotics may reflect 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 (Merrifield 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 fish 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 fish to common diseases (Hoseinifar et al. 2010). In the current research, a significant 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 immunity indices in Pseudosciaena crocea fingerlings. The results of their study showed that 0.09% of glucan in the diet has the best effect on immunity, but immunity was not significantly different in the control group and 0.18%. Moreover, Zhu et al. (2012) reported that channel catfish 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 fish 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 significant 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 fish 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 fish and is a diagnostic factor. Total protein and albumin levels can indicate the nutritional status and health of fish (Svetina et al. 2002). Increases in protein and albumin levels reflect an improvement in innate immunity; in other words, increases in total protein and albumin concentrations may be due to stronger nonspecific reactions in fish (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 fish 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 sufficient to stimulate the nonspecific 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 fish against infectious pathogens.