Effect of different levels of bioplex manganese along with probiotics and multi‐enzymes on performance and immune system indices of broilers

Abstract To investigate the effect of different levels of bioplex manganese along with probiotics and multi‐enzymes on the performance and immune system of broilers, 640 one‐day‐old male chicks of the Ross 308 strain were reared and the data analysed in a 4 × 2 × 2 factorial experiment with four levels of bioplex manganese (0, 60, 72 and 84 mg per kg of diet), two levels of Parsilact probiotic (0 and 200 mg per kg of diet) and two levels of Combo multi‐enzyme (0 and 1,000 mg per kg of diet) in a completely randomized design with 16 experimental treatments, 4 replicates and 10 chickens per replicate during a period of 42 days. The results showed that the performance of the broiler chickens in the diets containing 72 and 84 mg bioplex manganese along with probiotics and multi‐enzymes had the greatest difference compared to the control (p < .05). Compared to the control with 0 mg/kg manganese; the bursa of Fabricius weight was greater in chickens fed diets containing additional manganese (p < .05). The concentration of antibodies produced against Newcastle disease virus, as well as the concentrations of IgG, IgM and total immunoglobulins produced against SRBC, were highest in the group fed a diet containing 84 mg manganese along with probiotics and multi‐enzymes (p < .05). The results show combining additional manganese with probiotics and multi‐enzymes in chicken diets leads to better performance as well as a stronger immune system of chickens.


| INTRODUC TI ON
As the highest cost of poultry production is the cost of feed, optimizing feed consumption and reducing feed conversion ratio are very important in the poultry industry (Ahmadi et al., 2018). The efficiency of the digestion and absorption process in poultry is directly related to dietary compounds and microorganisms located in their digestive system. Therefore, several food additives have been developed to improve the diets and gastrointestinal conditions of poultry (Poorghasemi et al., 2013). Such additives often include trace elements (Ahmadi et al., 2019).
Manganese is a key trace element in poultry which plays a very important role in the body's reactions, especially those related to bone and cartilage formation. It is also essential for the prevention of oxidative stress, enzyme activities and metabolism of amino acids, carbohydrates and cholesterol. Suppn has been shown to have a positive effect on the growth performance and immune system of broilers (Ji et al., 2006). Manganese is usually provided in the diet of broilers in the form of inorganic minerals (mostly oxides and sulphates), but organic chelated (biolplex) forms of manganese are also available (Fly et al., 1989). Bioplexes are organic complexes (principally protein-based) of metallic elements. They have a higher rate of absorption in the intestine than inorganic forms (Berta et al., 2004) and their use thus can reduce the required dietary concentration of some minerals, thereby reducing without affecting productivity or product safety (Virden et al., 2003).
Other potential feed additives include probiotics and enzymes.
Probiotics are used to increase the growth of beneficial microbes in the digestive system and to eliminate pathogens in poultry feed.
Probiotics can also have a positive effect on the immune system and production efficiency (Thorat et al., 2015), thereby reducing disease and improving feed conversion ratio without resulting in tissue residues or, unlike antibiotics, causing microbial resistance (Poorghasemi et al., 2017).
Enzymes reduce the quality and quantity of available substrate for microbes, decrease the viscosity of intestinal contents and increase the rate of substance transmission by increasing the rate of digestion and production of simple sugars from diet fibre, thus reducing the harmful microbial population of the intestine. Enzymes also reduce fermentation in the intestine and increase fermentation in the cecum by increasing the digestibility of dietary nutrients (Ghomi et al., 2012;Sun & Kim, 2019). Some substances digested by gastrointestinal enzymes may not be absorbed in the small intestine and be transferred to the cecum, thus increasing bacterial fermentation in the cecum (Ghomi et al., 2012). It has been reported that enzyme supplements can increase the growth performance of poultry by up to 10% (Daghigh Kia et al., 2017). Like probiotics, some of this may be mediated by improvements in immune function (Thorat et al., 2015). Combining enzymes and probiotics may be beneficial; Midilli (2001) stated that enzymes can indirectly prevent the proliferation of harmful bacteria by providing a suitable environment for digestion, and in such conditions, probiotics can lead to more production of useful microbes.
Numerous results from past research have shown that manganese, probiotics and enzymes in broiler diets have positive effects on the performance and immune system of broilers. As these compounds are rarely used in combination in the diets of broilers and on the other hand, the double beneficial effects of the simultaneous use of manganese, probiotics and enzymes can be important in terms of nutritional innovation in broiler breeding, the present experiment was performed to investigate the main effects of manganese, probiotics and multi-enzymes and their interaction on the performance and immune system of broilers.

| MATERIAL S AND ME THODS
This research was conducted under the principles of animal research ethics in the city of Somehsara, Iran (37.2071°N, 50.0034°E).
In this study, 640 one-day-old male chickens of Ross 308 strain were used in a 4 × 2 × 2 factorial experiment with four levels of bioplex manganese (0, 60, 72 and 84 mg per kg of diet), two levels of Parsilact probiotic (0 and 200 mg per kg of diet) and two levels of Combo multi-enzymes (0 and 1,000 mg per kg of diet) in a completely randomized design with 16 experimental treatments, 4 replicates and 10 chickens per replicate during a period of 42 days.
The experimental diets included the starter (1-21 days of age) and finisher (22-42 days of age) diets, which were adjusted, based on the handbook of Ross 308 strain requirements, using UFFDA software (Table 1). During the breeding period, all the groups received the same diets based on maize-soybean. The experimental treatments include: 1-control (without additives), 2-control + probiotics (200 mg per kg of diet), 3-control + multi-enzyme (1,000 mg per kg of diet), 4-control + probiotics (200 mg per kg of diet) + multi-enzyme (1,000 mg per kg of diet), 5-control + manganese (60 mg per kg of diet), 6-control + manganese (60 mg per kg of diet) + probiotics (200 mg per kg of diet),

| PERFORMAN CE
During the study period, breeding conditions were the same for all chickens and feed was available ad-lib. The feeds in each replicate were weighed before consumption and by specifying the weight of the remaining feed at the end of each week, the amount of feed consumed for each treatment for the entire period was calculated.
Chickens were weighed weekly and the weight gain and conversion ratio of the entire period was determined (Poorghasemi et al., 2017).

| Determination of antibody titre produced against Newcastle disease virus
To determine antibody response to Newcastle disease virus vaccination, Newcastle disease vaccine was injected at 22 days of age, and birds blood sampled was pe10 and 20 days later. For blood sampling, two birds were randomly selected from each replicate and 2 ml of blood was taken from the wing vein. Blood samples were transferred to the laboratory and centrifuged for 15 min at 3,000 rpm to extract serum. Antibody titre was determined by agglutination inhibition (HI) assay (Poorghasemi et al., 2015).

| Determination of the titres of the immunoglobulins produced against SRBC
To measure the titre of immunoglobulins produced against sheep red blood cells (SRBC) on 28 and 35 days of age, 4 chickens per experimental unit were injected with 0.2 ml of SRBC suspension (5%) washed in sterile phosphate buffer, in the chest muscle. Seven days after each injection, on days 35 and 42 of age, about 2 ml of blood was drawn from the same birds (marked with colour on the feathers) from the wing vein. Blood samples were kept at laboratory temperature for 6 hr to separate the serum from the blood clot. The obtained serum was then centrifuged at 4,000 rpm for 15 min. The microtitre haemagglutination method was then used to determine IgM and IgG titres as well as the total titres of immunoglobulins produced against SRBC (Poorghasemi et al., 2015).

| Statistical analysis
Data were analysed using SAS statistical software Release 9.4. (SAS Institute, 2004). A general linear model was used:

| RE SULTS
The results on the effect of experimental treatments on the performance of broilers are presented in Table 2. Weight gain during the entire period showed a significant difference between different levels of manganese (p < .05) such that the highest rate of weight gain was related to 84 mg of manganese and the lowest rate was related to the control. With diets containing probiotics and multi-enzymes, the weight gain was significantly higher as compared to the control (p < .05). Weight gain with the combined use of manganese (72 and 84 mg), probiotics and multi-enzymes had the highest significant difference compared to the control (p < .05). Feed intake for the entire period did not show a significant difference between different levels of manganese, probiotics, multi-enzymes and their combined uses (p > .05). The feed conversion ratio was significantly lower than the control when using different levels of manganese as well as probiotics and multi-enzymes (p < .05). In the interacting effects of the combined uses, the conversion ratio of the diets containing 72 and 84 mg manganese with probiotics and multi-enzymes had the lowest significant difference compared to the control (p < .05). but bursa weight significantly increased in all levels of manganese compared to the control (p < .05). Although the use of probiotics and multi-enzymes in broiler diets did not make a significant difference in the weight of lymphoid organs compared to the control, their weights were numerically higher than the control (p > .05). Regarding the combined uses of manganese, probiotics and multi-enzymes on the weight of thymus, spleen and bursa, the results showed that the diets containing different levels of manganese with probiotics and multi-enzymes had the highest difference with the control but this difference was not significant (p > .05).
The results on the levels of antibody titres produced against Newcastle disease virus (NDV) are shown in Table 4. In the second stage, IgM titres in the experimental groups containing manganese and IgG in the experimental groups containing probiotics had a significant increase compared to the control (p < .05). In the combined use, the difference in IgM and total immunoglobulin concentrations produced against SRBC in treatments containing manganese, probiotics and multi-enzymes were significantly increased compared to other treatments (p < .05). The highest significant difference in IgG, IgM concentrations and total immunoglobulin produced against SRBCs were observed for the groups fed by 84mg manganese along with probiotics and multi-enzymes compared to the control (p < .05).

| D ISCUSS I ON
The results of the present experiment showed that increasing the level of manganese significantly increased the weight of broilers in the entire period and improved the feed conversion ratio, which is consistent with the results of Sirri et al., (2016)

TA B L E 2 (Continued)
or probiotics had a significant increase in live weight gain compared to the control and their conversion ratio was significantly reduced.
One reason for the effectiveness of probiotics is their ability to inhibit the growth of harmful bacteria in the digestive system. In addition, bacteria in probiotics increase the activity of digestive enzymes. The release of metabolizable energy in the feed increases due to the effect of digestive enzymes on diet components. Thus, it increases the weight of broilers and improves their conversion ratio (Mountzouris et al., 2015).
Some researchers have reported that the use of multi-enzymes in broiler diets has beneficial effects on weight gain and conversion ratio. According to them, the use of enzymes improves the TA B L E 3 The effects of experimental diets on weight of lymphoid organs  This is especially important in young animals because the digestive system in young animals is not fully developed and the production of internal enzymes is usually not enough for digestion (Hasani et al., 2019).
Regarding the interacting effects of the combined uses, among the different experimental groups, the highest significant difference was observed for weight gain and the lowest significant difference was for conversion ratio in the diets containing 72 and 84 mg of manganese with probiotics and multi-enzymes.
Little is known on the interactive effects of manganese, probiotics and multi-enzymes in diets on broiler performance. In an experiment conducted on broilers by Jin et al., (2002), it was stated that the treatments containing mixed enzymes and probiotics had significant weight gain and improved conversion ratio compared to the control, which is consistent with the results of this experiment.
In the present experiment, the combined use of manganese with probiotics and multi-enzymes in broiler diets increased performance (weight gain and improved conversion ratio) compared to the control and the other groups. Enzyme activity is affected by the pH of
The effect of different levels of manganese, as well as the use of probiotics and enzymes, was not significant on the average feed intake of chickens throughout the entire period. In addition, in the effects of combined uses, the feed intake of the experimental groups did not show a significant difference.
Although probiotics and enzymes used in the diet were able to reduce feed intake, this reduction was not significant, which is consistent with the results of Siadati et al., (2018). They stated that probiotics and enzymes, because of their specific properties, improve the availability of nutrients and their digestion, and may reduce the animal's need to consume more feed for obtaining nutrients (Attia et al., 2017;Siadati et al., 2018).

TA B L E 5
The effects of experimental diets on immune responses of broiler chickens after injection of the first stage of SRBC  The means within the same column with at least one common letter, do not have significant difference (p > .05) a, b, c, d, e, f the means within the same row with different letter, are significantly different (p < 0.05).
Our results show that the difference in the weight of the lymphoid organ of bursa of Fabricius had a significant increase in different levels of manganese compared to the control. Some studies have stated that a sufficient amount of manganese in the diet improves the immune system by increasing the antioxidant activity of superoxide dismutase (SOD) and removing more toxins from cellular metabolism (free radicals). Thus, its deficiency leads to changes and damage to the tissues of immune organs such as the bursa of Fabricius, which are the place where lymphocytes are produced, followed by dysfunction and reduced growth of the organ (Damaceno Faria et al., 2020;Ji et al., 2006).
How manganese, probiotics and enzymes interact is not clear but Majedi et al., (2015) reported that increasing relative weight gain of lymphoid organs is a sign of a strengthened immune system. Probiotics improve the immune system of birds and regu-  Note: ME: Multi-enzyme; P: probiotic; SEM: standard error of the means.
The means within the same column with at least one common letter, do not have significant difference (p > .05) a, b, c, d, e, f the means within the same row with different letter, are significantly different (p < 0.05).
immune system of broilers, as well as by enhancing mature defence cells.
According to Pourakbari et al., (2016) the lack of nutrients in the diet or lack of proper absorption of them in the intestinal tract can reduce the activity of the immune system and that improving digestion and absorption through the use of probiotics and enzymes can cause optimal safety because of access to materials required by the immune system. Wang et al., (2010) also reported that probiotics can strengthen the immune system by improving the utilization of the minerals such as manganese, which is one of the minerals needed to strengthen the immune system.
Consistent with this study on the increased response to antibodies produced against NDV in the diets containing probiotics and multi-enzymes compared to controls, Scott et al., (1997) and Poorghasemi et al., (2017) observed by increasing digestibility the enzymes increase the bird's access to nutrients (proteins and carbohydrates) and probiotics by improving nutrient intake from the small intestinal wall and stimulating the growth of lymphoid follicles in primary and secondary lymphoid organs to produce and proliferate B and T cells, can improve humoral immune response.
According to previous studies, increasing the amount of manganese along with probiotics and multi-enzymes, increased the levels of antibody produced against NDV. It is believed that manganesedependent superoxide dismutase in mitochondria activates the free radicals released inside the cells. The organic form of this mineral enhances the antibody response against the NDV vaccine in broilers. As it is a super oxidation cofactor, the need for manganese also increases during immune responses. Therefore, with sufficient manganese available in the body, the antibody titre will be higher and the immune system will be stronger, which may be the reason for the increased response to antibodies produced against NDV by increasing the level of manganese (Brooks et al., 2012).
In our study, all three levels of manganese were able to increase the concentration of IgM compared to the control. This is consistent with the hypothesis of increasing the antibody titre using manganese chelate and is similar to the works of researchers who stated that the organic form of minerals has a significant positive effect on the immune system. It has been reported that manganese chelate in broilers balanced T-helper lymphocytes (Th1 and Th2), which play a role in activating macrophages and inducing antibody production; it was also shown that they increased the expression of M and G immunoglobulins (Burin Junior et al., 2018).
The probiotic groups showed a significant increase in IgG concentration compared to the control, which is consistent with the results of Khaksefifi and Ghoorchi (2006). These authors report that adding probiotics to poultry diets increases cellular growth and development of cavities or Peyer's patches of the ileum, which is a sign of stimulation of the mucosal immune system which reacts to antigenic stimuli through the secretion of immunoglobulins. They also stated that probiotics activate the immune system to counteract various antigens by increasing the xenophobia of lymphocytes and the production of mediated-cytokines of T-helper cells by lymphoid organ cells, which may increase IgG titres after SRBC injection in probiotic groups.
The level of antibody produced against NDV was increased in the combined uses by increasing the amount of manganese along with probiotics and multi-enzymes. In the results of interacting effects of the combined uses, the groups fed with 84 mg manganese along with probiotics and multi-enzymes showed the highest significant difference in IgG, IgM titres as well as total immunoglobulins produced against SRBC. Little research has been done on the interacting effects of manganese, probiotics and multi-enzymes on the titres of immunoglobulins produced after SRBC injection.
However, probiotics have been shown to stimulate different cells of the immune system to produce cytokines that are involved in inducing and regulating immune responses (Christensen et al., 2002;Hussein et al., 2020). Also, the animal body considers the entry of probiotic bacteria into the digestive system as an external organism and stimulates the immune system against these microorganisms and increases the animal's immunity (Panda et al., 2000). Enzymes reduce the viscosity of digestive substances by breaking large molecules of non-starch soluble polysaccharides into small polymers and improving the immune status of birds by improving the absorption of micronutrients such as manganese (Bedford, 2009).

| CON CLUS IONS
In general, the results of the present experiment show that the simultaneous use of manganese, probiotics and multi-enzymes has additional beneficial effects on the performance and stimulation of the immune system of broilers and it can be produced by supplementing poultry feed with manganese, probiotics and multi-enzymes, birds with high yield and strong immune system against diseases.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

AUTH O R S ' CO NTR I B UTI O N
All authors contributed to and approved the final manuscript. Writing-original draft; Writing-review and editing.

S TATE M E NT O F E TH I C A L CO N S I D E R ATI O N
The experimental protocol was ratified by the Animal Ethic Committee of the Arak Branch, Islamic Azad University, Arak, Iran and the experiment was performed with respect to the International Guidelines for research involving animals (Directive 2010/63/EU).