Protective Effects of Sanyar Prebiotic on Immunity and Tissue Changes of Common Carp ( Cyprinus carpio ) Exposed to CuSO 4 Stress

Prebiotics, as feed additives, can activate the antioxidant and immune systems of fsh and signifcantly improve the survival of aquaculture animals in stressful conditions. Te exposure of fsh to copper sulfate leads to oxidative stress, suppression of the immune system, and destruction of vital body tissues. Tis study aimed to investigate the protective efects of Sanyar prebiotic on the immunity and tissue changes of common carp ( Cyprinus carpio ) exposed to CuSO 4 stress. Two hundred common carp fsh with an average weight of 12.33 ± 0.93 g were stocked in four treatments (triplicates) for 60 days. Te experimental treatments contained Sanyar commercial prebiotics in 100 g of food, which included P 1 (1 ml prebiotic liquid), P 2 (0.1 g prebiotic powder), P 3 (0.5 ml liquid and 0.05 g powder), and treatment C (control treatment without any additives). At the end of the experimental period, the fsh were exposed to acute toxicity of Cu (0.1 m/l) for 96 hours. Gill, kidney, and liver tissue samples were collected and evaluated after 96 hours. According to the results obtained, there were signifcant diferences in specifc growth rate (SGR) and fnal weight ( p < 0.05). Te lowest feed conversion ratio (FCR) was observed in the Sanyar treatments (1.56 ± 0.37), which had a signifcant diference from the control treatment (1.85 ± 0.35) ( p < 0.05). Te results showed that immunity indices such as lysozyme, cortisol, and ACH50 increased in the experimental treatments compared to the control treatment ( p < 0.05). Te highest and lowest lysozyme activities were observed in P 2 (16.35 ± 0.57) and control treatments (13.15 ± 1.00), respectively ( p < 0.05). Generally, no signifcant diference was observed between growth and nutrition indices in Sanyar treatments ( p < 0.05). Symptoms of kidney, liver, and gill tissue damage were reported from mild (hyperemia and infltration of infammatory cells) to severe (hemorrhage and necrosis). Te lowest severity of tissue damage was observed in the treatments fed with Sanyar food supplement. In general, the results of this study showed that the addition of Sanyar prebiotic to the diet of common carp improves the growth indicators and immune response and can also be benefcial in increasing fsh resistance to copper toxicity.


Introduction
Common carp (Cyprinus carpio) is one of the most important fsh cultivated in the world today and belongs to the carp family (Cyprinidae) [1].Tis fsh has an important role in increasing the rate of aquaculture production worldwide as one of the most valuable cultivated species [2,3].Nowadays, strengthening the immune system of aquatic animals against environmental stress is one of the important goals of the aquatic industry, and the use of immune stimulants in the food diet leads to the growth and strengthening of the health of aquatic animals [4].It has been proven that the use of new food supplements such as prebiotics, probiotics, and synbiotics is very efective on growth, food efciency, and production of digestive enzymes [5,6].
A prebiotic refers to a food component that cannot be digested and exerts a positive infuence on the host by specifcally promoting the proliferation and/or functionality of a particular bacterium or a restricted group of bacteria within the colon [7].It has been reported that prebiotics as functional compounds in the aquaculture industry improve growth, feed efciency, intestinal microbiota, digestive enzyme activity, intestinal morphology, immune status, disease resistance, intermediary metabolism, and stress responses [8].
Sanyar prebiotic is derived from Saccharomyces cerevisiae yeast.Sanyar contains cell wall carbohydrates mannan-oligosaccharide and beta-glucan, which mannanoligosaccharide as a prebiotic and beta-glucan as a strong antioxidant are efective in strengthening the immune system of aquatic animals.On the other hand, Sanyar is a very nutritious substance that contains essential amino acids for growth, B vitamins, and growth stimulants including peptides and nucleotides and is also rich in minerals, especially chromium, phosphorus, and selenium.Te types of prebiotics used in the aquaculture industry include beta-glucans, mannan-oligosaccharides, lactose, fructo-oligosaccharides, and inulin, whose efects have been proven in aquatic animals [9][10][11].However, there is not enough scientifc information about the efect of using Sanyar resources on aquatic animals such as common carp.Especially, the protective efects of Sanyar prebiotic against metals stress in fsh have not been investigated.
Copper is an essential trace element that plays a crucial role in the proper development and metabolic processes of living organisms.Nevertheless, exceeding the prescribed limit of copper can pose signifcant risks as it can form persistent metallic compounds that have the potential to accumulate in water and disrupt the delicate balance of the biological system [12].Te rise in environmental pollution can also have adverse efects on aquaculture operations.Te toxicity of copper has been observed to induce harmful efects on fsh species [13].Tis is because fsh are one of the aquatic organisms that are able to accumulate heavy metals in their tissue [12].Based on the importance of the type and quality of food on aquatic production and the role of fsh immunity, the possibility of increasing the resistance of fsh exposed to copper sulfate was investigated after feeding with Sanyar prebiotic diet.Various studies have been carried out related to the resistance of fsh to food supplements including various types of prebiotics [14][15][16][17][18]. Several studies have been conducted on the role of dietary supplements in enhancing resistance against various stresses, such as toxins and diseases [19,20].Farhangi et al. [17] investigated the protective and histopathological efects of garlic extract (Allium sativum) on rainbow trout (Oncorhynchus mykiss) exposed to acute copper toxicity.Komar [21] conducted a study to examine the benefcial and protective efects of a prebiotic, microbial levan, on common carp (Cyprinus carpio) fry during experimental exposure to fpronil.Mokhbatly et al. [22] investigated the protective efects of Spirulina platensis and β-glucan (BG) supplementation in African catfsh (Clarias gariepinus) exposed to chlorpyrifos.Hajirezaee et al. [23] reported the protective efects of vitamin C in common carp (Cyprinus carpio) exposed to titanium nanoparticles.However, this study represents the frst investigation into the efects of Sanyar prebiotic on aquatic animals.Additionally, this study aimed to determine the efectiveness of the Sanyar prebiotic on the growth and resistance of common carp exposed to copper.

Experiment Design.
Two hundred common carp juveniles (12.33 ± 0.93 g) were purchased from the local farm in Golestan province (Voshmgir Dam) and transported to the fsheries laboratory in Gonbad Kavous University.Te fsh were kept in on 1500-L tank for two weeks to acclimatize with the conditions laboratory [24].Ten, fsh with similar weights were selected and distributed in 12 tanks (35-L volume) at a density of 15 fsh per tank (4 treatments with 3 replications).Te tanks were continuously aerated, and 30% of water was exchanged daily to maintain water quality [25].Te fsh were reared for 60 days under a daily feeding rate of 3% of the biomass, divided into three times (7:00, 12:00, and 17:00).Te biomass in the tank was recorded biweekly to adjust the feed amounts.Unconsumed feed and fsh feces were siphoned daily.
Sanyar prebiotic product (liquid and powder) was purchased from Sepehr Javan Kavoshgar Company (registered number 37481, Dezful, Iran) (Table 1) for the resistance of fsh against stress caused by copper sulfate.Sanyar prebiotic in powder and liquid form was used in 100 g of carp fsh diet (SFC code from Faradane company which contained 38% protein, 4% lipid, 3% fber, 7% ash, 5% moisture, and 1% phosphorus) based on the dose suggested by the manufacturer.Generally, four treatments were used in this experiment, which include P1 (1 ml prebiotic liquid), P2 (0.1 g prebiotic powder), P3 (0.5 ml liquid and 0.05 g powder), and treatment C (control treatment without no additives).

Growth Indices.
Bioassay of fsh was carried out in 3-time intervals (initial, middle, and fnal) after anesthesia with 200 mg•l −1 clove powder [26].Half an hour after feeding, the uneaten feed was siphoned, then dried (60 °C for 24 h), and weighed for evaluation of feed efciency.Te weight of the fsh was measured using a digital scale with an accuracy of 0.01 g in each tank.Parameters including weight gain rate (WGR, %), specifc growth rate (SGR), feed conversion ratio (FCR), feed conversion efciency (FCE), protein efciency ratio (PER), and survival percentage (SP) were measured to investigate the performance of experimental diets containing Ajwain supplements on fsh growth based on the following formulas [27] (1)

Pathological Studies under Stress Conditions with Copper
Sulfate.Te fsh were exposed to a concentration of 0.1 mg of copper sulfate for 96 h at the end of the experiment period (60 days period) to determine the level of resistance [28].Te studied concentration is obtained based on the minimum efective concentration (LOEC � lowest observed efect concentration) equal to LC10, 96 h of copper sulfate, and it is a concentration without any losses [29].After the toxicity test and examining the lesions caused by copper toxicity on fsh tissues, 3 fsh samples were randomly caught from each treatment and placed in a 10% formalin solution.Ten, the samples were transferred to the pathobiology laboratory, and tissue sections were prepared.Histological and lesion studies were performed using identifcation keys and light microscopy [30].After determining the volume of water per volumetric unit, copper sulfate was weighed and added to the water to determine the concentration of the poison.Te behavior of fsh exposed to copper poison was recorded during the experimental stages.Te experiment conditions including temperature and pH were constant for all the samples during the test period.Also, feeding was stopped at this stage.

Mucus Immunity Assay.
After 96 h of stress with copper sulfate, the immunity indices of carp fsh fed with Sanyar prebiotic were investigated by direct mucus collection based on the method of Guardiola et al. [31].Seven fsh were randomly caught from each replication, and mucus removal was carried out by massaging the surface of the fsh in less than 2 minutes.Briefy, skin mucus was obtained by gently rubbing the dorsolateral surface of fsh in a front-to-caudal direction, transferred to sterile tubes, and centrifuged for 10 minutes at 3000 rpm at a temperature of 4 °C by a centrifuge machine [32].Ten, the samples were stored in 2 ml microtubes and kept at −80 °C for the mucus immunity test.Mucus protease activity was evaluated based on the azocasein hydrolysis method described by Palaksha et al. [33].Lysozyme enzyme was measured by turbidity method using a spectrophotometer [34].Protease enzyme was measured using the method of Walter [35].Complement 50 (ACH50) was measured using the method of Oriol Sunyer and Trot [36] based on the analysis of rabbit red blood cells, and alkaline phosphatase enzyme activity, glucose, and total protein were measured by a commercial kit of Pars Azmoun using a spectrophotometer [37].Total immunoglobulin (IgM) was

Protective Efects of Prebiotics on Immunity and Mucus
Stress Indices.Te results of the protective efects of Sanyar prebiotic on the mucus immunity of common carp exposed to copper sulfate stress are shown in Table 3. Lysozyme activity showed a signifcant diference between the experimental and control treatments (p < 0.05), with the highest value observed in the P2 treatment (16.35 ± 0.57 U/ml/min) and the lowest in the control treatment (13.15 ± 1.00 U/ml/ min).A signifcant diference in the concentration of ACH50 was found between the diferent Sanyar prebiotic treatments and the control treatment without additives (p < 0.05), with the lowest concentration observed in the control treatment (132.58 ± 3.12).Mucus protein levels also exhibited a signifcant diference between the experimental and control treatments, with the highest amount observed in the P2 treatment.Mucus protease levels were signifcantly diferent in fsh fed with Sanyar prebiotics and exposed to copper sulfate stress, with the highest observed value.Te highest was observed in the P2 treatment and the lowest in the control treatment (p < 0.05) (Table 3).Te results of the protective efects of Sanyar prebiotic on mucus stress indices in common carp exposed to copper sulfate stress are shown in Figure 1.Cortisol concentration in the control and P3 treatments had a signifcant increase compared to the P1 and P2 treatments (p < 0.05).Glucose levels of fsh mucus in the control and P3 treatments were signifcantly increased compared to other treatments (p < 0.05).Alkaline phosphatase had a statistically signifcant increase in the control treatment compared to other experimental treatments (Figure 1).

Te Results of Histological Studies.
After exposing the fsh fed with Sanyar prebiotic to copper sulfate for 96 hours, the highest amount of lesions was observed in the gill tissue and the least amount of lesions was observed in the liver and kidney tissues.However, the severity of lesions observed in the treatments fed with Sanyar was higher than in the control treatment.Complications such as hyperemia and bleeding in the gills and kidneys were more obvious (Table 4).Te main lesions of the gill tissue included hyperemia of the secondary lamellae, edema, detachment of the covering epithelium of the secondary lamellae, hyperplasia at the base of the secondary lamellae, and necrosis of the covering cells (Figure 2).Mild levels of renal duct destruction, hyperemia, bleeding, and the accumulation of melanoma macrophages were observed in the kidney tissue (Figure 3).However, the liver tissue showed minimal damage, indicating that the severity of the lesions was considerably lower in the treatments where Sanyar was administered.Notably, liver lesions such as hyperemia and the accumulation of melanoma macrophages were only observed in the experimental group (Figure 4).It is worth mentioning that no deaths occurred throughout the duration of the experiment (Figure 4).No casualties occurred during the experiment.

Discussion
Resilience and strengthening of aquatic animals against environmental stress is one of the important objectives of the aquaculture industry, which will be implemented by adding immune stimulants to the diet.Tis study was conducted based on the positive results obtained from various probiotic and prebiotic food supplements in the past and investigating the efects of a new prebiotic compound (Sanyar).Based on this, Jafarian et al. [39] reported that adding 0.1 g of Sanyar prebiotic powder to the diet of common carp causes a signifcant increase in the fnal biomass weight, fnal density, and protein yield ratio and a decrease in the feed conversion ratio.In this study, Sanyar prebiotic caused a signifcant increase in growth and nutrition factors (p < 0.05), so the changes in weight gain in the treatments fed with the ration containing Sanyar were more than the control treatment.However, no signifcant diference was observed in the treatments containing Sanyar powder and liquid (p > 0.05).Although 4 Journal of Toxicology Sanyar prebiotic had a good efect on the growth and nutrition of fsh, no diference was observed in the type of combination with food or in addition to water.
Prebiotics are benefcial food compounds that selectively stimulate the growth and activity of a number of bacteria in the digestive system of the host, which are known as growth   stimulants and immune stimulants in aquatic animals [40].
Types of prebiotics used in the aquaculture industry include beta-glucans, mannan-oligosaccharides, lactose, fructo-oligosaccharides, and inulin, which have been proven effective in aquatic animals.Numerous studies have been published on this topic, indicating that the inclusion of   6 Journal of Toxicology Immunogen ® as a dietary supplement, specifcally at a level of 1.5%, can have a benefcial impact on growth parameters and immune responses in shabout (Tor grypus) fsh [41].Te combined efects of thyme essential oil (TEO) and prebiotic (Immunogen ® ) on growth performance and innate im- munity of rainbow salmon, Oncorhynchus mykiss, showed an increase in the performance and well-being of rainbow salmon in the treatment 2% TEO + 0.2% Immunogen [42].Prebiotic compounds improve intestinal microbial fora, facilitate digestion and absorption, and increase the value of food through the production of enzymes and specifc metabolic substances [43,44].In this research, the Sanyar prebiotic facilitates the absorption of nutrients and weight growth in fshes through an indirect efect on the microbial fora of the digestive system.According to the study of Laice et al. [6], adding 0.2 mg/l of synbiotic to water in the biofoc system (Bacillus toyi, Bacillus subtilis, Bifdobacterium bifdum, Enterococcus faecium, Lactobacillus acidophilus, mannan-oligosaccharides, lysine, methionine, choline, vitamin C, vitamin E, and dextrose) caused an increase in the fnal weight, fnal length, weight gain, and specifc growth rate in biofoc + synbiotic treatment compared to the control treatment.In the past years, functional additives such as various prebiotics have been introduced as good candidates to reduce the health efects and disease resistance caused by this replacement by strengthening the fsh immune system [45].
Te results of stress and immunity indices of carp fsh exposed to copper sulfate indicate an increase in fsh resistance due to prebiotic function (Table 3).Cortisol and glucose levels increased in the mucus of the control treatment at the end of the fsh feeding period and exposure to copper sulfate, while the cortisol level in fsh fed with Sanyar prebiotic decreased and had a signifcant diference with the control treatment.Exposure of fsh to copper sulfate as a toxic substance and nervous system stimulant causes stress in fsh.Te results showed that there was less stress in the treatments fed with a ration containing Sanyar, and a signifcant diference was observed compared to the control treatment.Also, this result shows the efect of the Sanyar prebiotic in reducing stress and increasing the level of immunity in common carp fsh.Several studies have shown that the increase of toxic elements in water causes direct efects on fsh organs, severe reduction of oxygen due to stress, and increased oxygen consumption by fsh [46].Te level of alkaline phosphatase as a stress factor in the treatment containing Sanyar prebiotic showed a signifcant decrease compared to the control treatment, and the lowest level was observed in the P2 treatment.Tese diferences show the efect of these food supplements on reducing the stress level in fsh.Alkaline phosphatase activity (ALP) is a polyfunctional enzyme involved in membrane transport activities [47,48].Te increase in the level of these enzymes is probably related to cytolysis and the leakage of enzymes in the immune system, which indicates tissue damage in organs such as the liver and kidney [49].In our study, the use of the Sanyar prebiotic increased the activity of lysozyme and complement 50 (ACH50) in common carp.However, the highest amount was obtained in the P2 treatment (0.1 g per kg of food).Te results of this study are consistent with the fndings of Zarei Nozari et al. [50] and Razavi Amri et al. [51].In this regard, immune factors were signifcantly increased in treatments containing Sanyar commercial prebiotics (0.2 g powder and 1 ml liquid) [39].Te functional results of improving the immune response of common carp in Matala Har are consistent with the fndings of Hosseinifar et al. [11] and Abasubong et al. [52].In research, food supplements with 0.5% of galactomannan and oligosaccharide (GMOS diet) or 0.02% of a combination of essential oils (PHYTO diet) increased fsh serum lysozyme after Vibrio anguillarum infection, so the combined use of both additives can be useful to combat infection in European sea bass [45].Te increase in the activity of lysozyme enzyme and serum complement protein can indicate the strengthening of the fsh immune system, which is probably due to the efectiveness of the active ingredients in Sanyar.Te serum complement protein is a strong nonspecifc defense mechanism to protect fsh against a variety of invasive organisms.Immune stimuli increase the power of xenophagy and increase the production of lysozyme [53].Numerous studies have highlighted the enhanced resistance of aquatic animals when fed with prebiotics, exhibiting increased resilience to various biotic and abiotic stresses.In the context of immunological indicators, a study on rainbow trout (Oncorhynchus mykiss) infected with Aeromonas hydrophila demonstrated the protective efects of a prebiotic on these indicators.Specifcally, the challenge group exhibited higher levels of serum cortisol and glucose.However, fsh that were under challenge and fed a diet supplemented with Immunogen showed lower levels of cortisol and glucose compared to the challenge group [54].
Te study on gilthead sea bream (Sparus aurata) investigated the efects of mannan-oligosaccharide and synbiotic supplementation on growth performance and immune response both before and after thermal stress at 34 °C.Following thermal stress, the experimental diets led to a signifcant increase in monocyte counts, lysozyme activity, and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the fsh.Notably, the fsh fed with a biomin-supplemented diet exhibited the lowest concentration of cortisol.It is important to note that "synbiotic" refers to a combination of prebiotics and probiotics working together synergistically to promote benefcial efects on the host health [55].
Histopathology is a suitable method to observe the toxic efects of chemicals in the aquatic environment.Tis method provides valuable insights into the extent of tissue damage resulting from the intensity and duration of exposure to toxic substances.Additionally, it helps assess the tissue capacity for adaptation and tolerance when exposed to such harmful agents [56].Chemical pollutants stimulate various organs such as gills, kidneys, and liver, and these tissues are suitable evidence to investigate the efects of external pollutants.Te gill tissue has less protection than other tissues and is directly adjacent to water.Terefore, this organ is more sensitive and can be the frst defense barrier of the fsh body against chemical pollutants.In this study, the destruction of gill tissue of fsh exposed to copper sulfate stress included lesions such as hyperemia, bleeding, hyperplasia of the apex in the gill flaments, edema, and expansion of secondary lamellae in all treatments.However, these lesions were diferent and less in fsh fed with Sanyar.Major lesions in liver and kidney tissues included cellular infammation and hyperemia.Several studies have also reported the efects of pollutants and environmental stress on gill tissue [57,58].However, the investigated damages (hyperplasia, hypertrophy, hyperemia, and edema) were more in the control group than in the Sanyar-treated fsh, and fewer damages were observed in the Sanyar-treated fsh.Tis fnding indicates the protective efect of Sanyar on fsh organs exposed to copper toxicity.Similar to our results, several other studies evaluated the positive performance of prebiotics in exposure to toxins.Mokhbatly et al. [22] investigated the protective efects of Spirulina platensis and β-glucan (BG) supplementation in African catfsh (Clarias gariepinus) exposed to chlorpyrifos.Te obtained results showed that the fsh exposed to the poison had lower growth and nutritional factors than the control treatment.Also, the blood factors of the fsh fed with the used supplements had a better condition.Te study conducted by Gupta [59] demonstrates the ameliorative and protective efects of microbial levan, a prebiotic, on common carp (Cyprinus carpio) fry exposed to fpronil experimentally.Te fndings indicate that the inclusion of microbial levan at a concentration of 0.75% efectively mitigates the stress induced by fpronil, owing to its potent nutraceutical properties.One of the methods of removing heavy metals from the environment is the use of microorganisms [60]; therefore, strengthening bacteria with nutrients such as prebiotics can help to increase the population of benefcial bacteria in the digestive system of fsh.According to Zoghi et al. (2014), many microbes have the ability to remove heavy metals, and some of them are lactic acid bacteria (LAB).Lactic acid produced by these bacteria could suppress the growth of pathogenic microbes by making the environment more acidic.Sanyar commercial prebiotic plays a vital role in creating an optimal environment that promotes the growth of benefcial bacteria while suppressing the proliferation of pathogenic bacteria.Tis unique mechanism strengthens the fsh's immune system, empowering it to efectively combat and mitigate the adverse efects of copper sulfate contamination.By enhancing the fsh's natural defense mechanisms, Sanyar prebiotic contributes to a healthier and more resilient aquatic ecosystem.

Conclusion
Te results of this study showed that the use of Sanyar prebiotic can improve the feed and growth of fsh, reduce stress, and increase immunity in fsh exposed to environmental factors.Based on the results, 1 mg/kg Sanyar prebiotic is recommended for common carp feed supplementation.

Figure 1 :
Figure 1: Mean ± SD of stress parameters of common carp fed with diets containing Sanyar after 60 days.Diverse letters show signifcant diferences (p < 0.05) in diferent groups in the Duncan test.Signifcant diferences (p < 0.05) between the means obtained are marked by asterisks.

Figure 2 :
Figure 2: Tissue section prepared from fsh gill exposed to copper sulfate (H&E, bar � 25 μm, ×400).A1: control treatment fed with basal diet and arrowheads: hyperemia of secondary lamellae (a), edema and detachment of covering epithelium in secondary lamellae (b), and basal hyperplasia in secondary lamellae (c).A4: P3 treatment fed with Sanyar supplement and arrowheads: hyperemia of secondary lamellae (a), edema and detachment of covering epithelium in secondary lamellae (b), and basal hyperplasia in secondary lamellae (c).

Table 2
. Te initial weight in all treatments was about 12.33 ± 0.93 g, and no signifcant diference was observed between the treatments (p > 0.05).At the conclusion of the 60-day rearing period, the treatments supplemented with various levels of Sanyar prebiotic demonstrated signifcantly higher fnal weight, weight gain, and weight gain percentage compared to the control group without prebiotic treatment.Te specifc growth rate (SGR) in the treatment groups that received prebiotics exhibited a signifcant increase compared to the control group (1.00 ± 0.17) (p < 0.05).Feed conversion ratio (FCR) was the lowest among the treatments fed with Sanyar compared to the control treatment, so the lowest and highest values were observed in the P2 treatments (1.49 ± 0.31) and the control group (1.85 ± 0.35), respectively.Te feed conversion ratio (FCR) decreased in the treatments fed with Sanyar, feed conversion efciency (FCE), had a signifcant increase (p < 0.05).Protein conversion efciency (PER) of the treatments fed with different levels of Sanyar prebiotic had a signifcant increase compared to the control treatment (p < 0.05).

Table 3 :
Mean ± SD of skin mucus immune parameters of common carp fed with diets containing Sanyar after 60 days.

Table 2 :
Mean ± SD of growth parameters of common carp fed with diets containing Sanyar after 60 days.

Table 4 :
Te common lesions of the fsh exposed to the concentration of copper after 96 h.