ABSTRACT In the present experiment, it was assumed that the appropriate dietary ratio of arginine (Arg) to lysine (Lys) can improve the immune status and growth performance of turkeys. The

aim of this study was to evaluate the effects of two inclusion rates of Arg relative to Lys in turkey diets with Lys content consistent with National Research Council (NRC) recommendations

or 10% higher on the immune status of birds and indicators of protein and DNA damage due to oxidation, nitration or epigenetic changes. Another goal was to determine which dietary Arg:Lys

ratio stimulates the immune response of turkeys vaccinated against _Ornithobacterium rhinotracheale_. The experiment was performed on 576 turkeys randomly assigned to four groups with two

it stimulated their immune system and reduced protein nitration as well as protein and DNA oxidation. SIMILAR CONTENT BEING VIEWED BY OTHERS _L. RHAMNOSUS_ IMPROVES THE IMMUNE RESPONSE AND

TRYPTOPHAN CATABOLISM IN LAYING HEN PULLETS Article Open access 01 October 2021 EFFECTIVENESS OF PROBIOTICS AND CLOVE ESSENTIAL OILS IN IMPROVING GROWTH PERFORMANCE, IMMUNO-ANTIOXIDANT

STATUS, ILEUM MORPHOMETRIC, AND MICROBIAL COMMUNITY STRUCTURE FOR HEAT-STRESSED BROILERS Article Open access 01 November 2023 EFFECT OF DIET SUPPLEMENTED WITH FUNCTIONAL AMINO ACIDS AND

POLYPHENOLS ON GUT HEALTH IN BROILERS SUBJECTED TO A CORTICOSTERONE-INDUCED STRESS Article Open access 10 January 2024 INTRODUCTION One of the challenges facing the modern poultry industry

is increased risk of metabolic disorders caused by intensive farming as well as decreased resistance to infections and adverse environmental factors. Turkey diets with a balanced amino acid

profile play a key role in maintaining good health and optimizing the growth potential of birds. Lysine (Lys) and arginine (Arg) are amino acids limiting the biological value of dietary

protein in turkey diets. The optimal inclusion levels and quantitative ratios of Lys to Arg in turkey diets need to be established due to considerable differences between the recommendations

of the NRC1 and breeding companies2, which stir much controversy. According to NRC guidelines1, diets fed to growing turkeys in the first 4 weeks of rearing should contain 1.60% Lys and

1.60% Arg, compared with 1.76% and 1.80%, respectively, recommended by breeding companies2. Similar differences in the amino acid requirements of turkeys can also be observed in later growth

stages. The same applies to the optimal Arg:Lys ratio, since Arg should account for 90–100% of Lys content or 102–105% of Lys content according to the NRC1 and BUT2, respectively. In the

concept of the "ideal protein" for turkeys in the first 4 weeks of life, Boling and Firman3 recommend 105% Arg over Lys, but they do not report Met content but only the sum of

sulfur amino acids. Previous research shows that the dietary Arg:Lys ratio can significantly affect the immune and antioxidant status of birds4,5,6. Arginine is a substrate for the synthesis

of intracellular nitric oxide (NO), and NO levels in the body are determined by the bioavailability of Arg. Nitric oxide plays an important role in the immune response because it

participates in pathogen killing and initiates the signaling pathways that lead to the full expression of the immune response. Lysine and Arg share the same cellular transport systems,

therefore the availability of dietary or extracellular Lys can modulate the entry of Arg into leukocytes and, consequently, NO synthesis by arginase and iNOS7. Closs et al.8 demonstrated

that increasing extracellular Lys concentrations reduced intracellular Arg concentrations and NO synthesis in activated macrophages in a dose-dependent manner. According to many authors, NO

synthesis can be limited by high intracellular Lys concentrations because Lys is capable of inhibiting arginase activity9,10,11. It should be stressed, however, that enhanced NO synthesis in

cells can induce the oxidation of biologically important molecules and compromise immunity in birds. Grisham et al.12 and Hallemeesch et al.13 found that NO exerted immunostimulatory and

antioxidant effects at low concentrations and during short-term exposure. Our previous studies revealed that in turkey diets with Lys content consistent with NRC recommendations1, the

optimal inclusion rate of Arg is 100% Lys, and methionine (Met) level should be higher than that recommended by the NRC1 to improve the growth performance and the immune and antioxidant

status of birds. It was found that in diets with Lys content consistent with NRC recommendations1, the inclusion rate of Arg set at 90% Lys compromised the growth performance and the immune

and antioxidant status of young turkeys during the entire rearing period. Diets with high Arg content (110% Lys) contributed to an increase in the proportion of breast muscles in the final

BW of turkeys and did not induce the oxidation of lipids, proteins or DNA, but promoted undesirable protein nitration and an increase in thyroxine levels14,15,16. In another experiment

conducted by our research team, where turkeys were fed diets with high Lys content (close to the recommendations of breeding companies2), an increase in the inclusion rate of Arg to 110% Lys

increased the protein content of breast muscles, but did not improve the growth performance or the immune status of birds. A decrease in dietary Arg content to 90% Lys did not compromise

performance only when the inclusion level of Met was increased to 45% Lys17. Since our studies of turkeys fed high-Lys diets revealed that low Arg concentration (90% Lys) stimulated the

immune system, the question arose as to whether such an Arg:Lys ratio would be sufficient to effectively mobilize the immune system of birds in response to infection. According to some

authors, a higher dietary Arg:Lys ratio than that recommended by the NRC1 supports the immune system of vaccinated or infected birds18,19,20,21,22,23. Our earlier experiment demonstrated

that in turkeys fed high-Lys diets and infected with _C. perfringens_, Arg content can be decreased to 90% Lys provided that Met content is increased to 45% Lys. The above dietary amino acid

ratios minimized oxidative processes and epigenetic alterations in important molecules in the intestinal wall and blood of turkeys, helped maintain intestinal barrier integrity, and exerted

a beneficial influence on the metabolism and growth performance of birds24. The results of our previous studies also indicate that an increase in the dietary content of Met in excess of the

levels recommended by NRC1 can boost immunity and improve the antioxidant status and growth performance of turkeys, including at the time of vaccination or infection25,26,27,28. In view of

these findings, which clearly show that the inclusion levels of Met in turkey diets should be higher than those recommended by the NRC1, it was assumed that the appropriate dietary ratio of

Arg to Lys can improve the immune status and growth performance of turkeys. The aim of this study was to evaluate the effects of two inclusion rates of Arg relative to Lys (95% and 105%) in

turkey diets with Lys content consistent with NRC recommendations1 or 10% higher on the immune status of birds and indicators of protein and DNA damage due to oxidation, nitration or

epigenetic changes. Another goal was to determine which dietary Arg:Lys ratio stimulates the immune response of turkeys. RESULTS EFFECT OF DIETARY ARG:LYS RATIOS The applied dietary

treatments had no effect on the body weight (BW) of female turkeys or feed conversion ratio (FCR) throughout the experiment. Only in week 8, birds fed diets with the lower Arg level (95%

Lys) had lower BW (_P_ = 0.021) than those receiving diets with the higher Arg content (105% Lys) (Table 1). Diets with the higher Lys level (NRC + 10%) had no effect on the percentages of

CD4+ and CD8 + cells, but contributed to an increase in the percentage of IgM+  B cells (_P_ = 0.008) in the spleen (Table 2), and to a decrease in the levels of CRP (_P_ = 0.034), PC (_P_ 

< 0.001), 8-OHdG (_P_ = 0.002) and Casp 3 (_P_ = 0.008) in the small intestinal wall (Table 3), and a decrease in the plasma levels of 3-NT (_P_ < 0.001) in 16-week-old birds (Table

4). In comparison with the lower Arg concentration (95% Lys), the higher inclusion rate of Arg (105% Lys) led to an increase in the percentage of IgM+ B cells (_P_ = 0.023) in the spleen

(Table 2), and a decrease in the levels of 3-NT in the small intestinal wall (_P_ = 0.030) (Table 5) and blood plasma of turkeys (_P_ = 0.009 and _P_ < 0.001, respectively) (Tables 3 and

4). The levels of 8-OHdG decreased in the blood plasma and small intestinal wall (_P_ = 0.014 and _P_ = 0.015, respectively) of 9-week-old turkeys fed diets with the higher Arg content (105%

Lys) (Tables 3 and 5). The higher inclusion rate of Arg (105% Lys) increased DNA methylation (_P_ = 0.045) in the small intestinal wall (Table 5) but not in the blood plasma of turkeys at 9

and 16 weeks of age. EFFECT OF DIETARY ARG:LYS RATIOS IN VACCINATED AND NON-VACCINATED TURKEYS Diets with the higher Lys level (NRC + 10%) had no effect on IgA gene expression in vaccinated

turkeys, but increased IgA gene expression (_P_ = 0.043) in the liver of non-vaccinated 6-week-old turkeys (Table 6). Vaccine-induced antibody titers against ORT in the blood serum of

vaccinated 16-week-old turkeys were not affected by the higher Lys level (NRC + 10%) (Table 7). Neither low (95% Lys) nor high (105% Lys) Arg levels affected IgA gene expression (Table 6) or

vaccine-induced antibody titers against ORT in the blood serum of vaccinated turkeys (Table 7). DISCUSSION A few experiments have shown that the growth performance of turkeys is affected by

the dietary inclusion levels and ratios of Arg and Lys29,30, and that an Arg:Lys ratio higher than 1:11 improved productivity30. Our previous findings indicate that different dietary

proportions of Arg relative to Lys (90%, 100% and 110% Lys) in diets with high Lys content, close to the recommendations of breeding companies2, have no influence on the growth rate of

turkeys14. In another experiment, different concentrations of Arg (90%, 100% and 110% Lys) in low-Lys diets1 had no effect on the final BW of turkeys, except in the youngest birds (aged 1–8 

weeks) where the lowest Arg level (90% Lys) decreased BW17. In the present study, where turkeys received diets with Lys content consistent with NRC recommendations1 or 10% higher, the

dietary proportions of Arg relative to Lys (95% and 105% Lys) had no influence on the FCR or final BW. However, a decrease in BW was noted in young turkeys fed diets with the lower Arg level

(95% Lys). Lower BWG in growing turkeys administered diets with low Arg content (90–95% Lys) and Lys content consistent with NRC recommendations1 or 10% higher can be attributed to the fact

that young birds are characterized by a faster growth rate and higher Arg requirements than older birds31. Rapid growth in the first weeks of life is accompanied by a high metabolic rate,

and Arg produces creatine which enhances the release of insulin-like growth factor 1 (IGF-1) in the muscles of birds32,33. According to published research, Lys-deficient diets suppress the

synthesis of proteins (including cytokines) and lymphocyte proliferation, decrease cell-mediated immunity and impair the antibody response, thus increasing disease incidence and

mortality34,35,36,37. In the current study, the higher dietary inclusion rate of Lys (NRC + 10%) had no effect on the percentages of CD4+ and CD8+ cells, but induced an increase in the

percentage of IgM+ B cells in the spleen of young turkeys. According to the literature, diets with increased concentrations of Met or Arg can be a major contributor to the synthesis of

immune system proteins, including IgA and IgY21,38. The liver plays a unique role in much immunity and in the physiology of IgA in normal and disease states. In the present experiment, IgA

gene expression in the liver was higher in young turkeys fed diets with the higher Lys level (NRC + 10%) than in those receiving diets with Lys content recommended by the NRC1, but it had no

effect on total IgA and IgY levels in the blood plasma and intestinal wall. C-reactive protein plays an important role in recognizing self and foreign molecules, and leads to the activation

of the adaptive immune system in the early stage of inflammation or infection by interacting with the complement and Fc receptors on phagocytes8. The decrease in CRP levels in the small

intestinal wall of turkeys fed diets with the higher Lys content (NRC + 10%), noted in the present study, suggests that the immune system of turkeys effectively protected them against

pathogens and infection. According to Calder and Yaqoob39, Arg is necessary for efficient proliferation of T and B cells. De Jonge et al.40 reported that Arg deficiency resulting from

arginase overexpression in the small intestine impaired B cell proliferation in the lymphoid organs of transgenic mice. Therefore, a reduced number of B cells leaves the bone marrow and

enters the lymphoid organs, thus considerably reducing the number of B cells in the spleen and lymph nodes and as well as serum IgM levels. In the current experiment, the higher dietary

inclusion rate of Arg (105% Lys) induced an increase in the percentage of IgM+ B cells in the spleen of young turkeys, but it had no influence on IgY and IgA levels or the percentage of T

cells in blood. The appropriate Arg:Lys ratio in turkey diets affects not only the immune system but also redox reactions. High dietary Arg levels can enhance NO synthesis. Endogenous NO has

immunostimulatory properties, but its excess concentrations in cells can stimulate the oxidation and nitration of biologically important molecules, and DNA oxidation41. Excess synthesis of

NO may be suppressed by high concentrations of Lys in cells because Lys can inhibit the activity of arginase responsible for NO synthesis from Arg9,10,11. In the present study, the higher

dietary inclusion levels of Lys (NRC + 10%) and Arg (105% Lys) desirably suppressed protein and DNA oxidation, as manifested by reduced levels of PC and 8-OHdG in the small intestinal wall

and blood plasma of turkeys. The applied dietary treatments had no effect on the levels of DNA repair enzymes, which confirms that increased inclusion rates of Lys and Arg did not induce

cell oxidation. In our previous study, graded dietary Arg levels (90%, 100% and 110%) relative to Lys content consistent with NRC recommendations1 had no influence on the oxidation of

lipids, protein or DNA, but the highest inclusion rate of Arg (110% Lys) induced protein nitration16. However, in the current experiment, the higher dietary inclusion levels of Arg (105%

Lys) and Lys (NRC + 10%) did not promote protein nitration or even inhibited these undesirable reactions. According to Yin et al.42, dietary Lys deficiency impairs amino acid metabolism and

leads to cell apoptosis, thus increasing Casp 3 levels. In one of our previous experiments, neither a decrease in Arg content from 100 to 90% Lys nor its increase to 110% Lys in high-Lys

diets2 affected Casp 3 or Casp 8 levels in turkeys17. In another study of turkeys fed low-Lys diets1, an increase in Arg content to 110% Lys increased Casp 3 levels14. In the present

experiment, the higher Lys concentration (NRC + 10%) decreased Casp 3 levels, which indicates that this amino acid had a positive effect on cell apoptosis and redox reactions. According to

Ulrey et al.43, amino acid metabolism (in particular Met, Lys and Arg) plays an important role in DNA methylation. In the present study, enhanced DNA methylation in the small intestinal wall

of turkeys fed diets with increased Arg content (105% Lys) was undesirable and could be linked to Arg methylation44. Moreover, it could be a consequence of the direct effect of NO-modified

compounds (which affect epigenetic regulations) on DNA. Kubińska et al.26 demonstrated that turkeys that had been re-vaccinated twice against ORT with an inactivated vaccine and received

diets with increased Met content (relative to NRC recommendations1), had considerably higher serum levels of vaccine-induced antibody titres. These studies show that the immune system of

vaccinated turkeys can be additionally stimulated by the increased level of the amino acid. Literature data show that apart from Met, also Arg6,14,20 and Lys14,17 can stimulate the immune

system. The present results do not corroborate the findings of Kubińska et al.26, because the higher dietary inclusion levels of Lys and Arg, relative to NRC recommendations1, had no

influence on vaccine-induced antibody titres in turkeys vaccinated against ORT. According to Tykałowski et al.28, and Guiro and Koncicki45, once-vaccinated turkeys usually do not develop

high levels of serum antibodies, which may explain the present results. In commercial turkeys, veterinarians quite often use a single vaccination against ORT, and then the titre of vaccine

antibodies is lower and lasts less than after re-vaccination. Due to the fact that slaughter turkeys live much shorter than breeding turkeys, usually such protection is sufficient for them

with low pressure of the field ORT. Kubińska et al.26 observed elevated IgA levels in turkeys vaccinated against ORT and fed diets with increased Met content. In the current experiment, a

few days after vaccination against ORT, young turkeys receiving a diet with the higher inclusion rates of Lys (NRC + 10%) and Arg (105% Lys), were characterized by higher IgA gene expression

in the liver than those fed a diet with the higher Lys level and the lower Arg level (95% Lys). Thus, the expression of IgA was influenced by an increased level of Arg in the diet.

l-arginine plays an important role in maintaining immune homeostasis46,47. In studies with chickens, it has been established that increasing Arg in the diet results in an increase in the

secretion of mucosal IgA48. The influence of Arg on the immunity of turkeys is evidenced by the increased expression of IgA in the liver, but no increased level of this immunoglobulin in the

blood was recorded. There was also no effect of the increased Arg level on the other tested parameters of the immune system of turkeys. In our previous study of turkeys administered diets

with Lys content consistent with NRC guidelines1 or higher, as recommended by BUT2, high Arg concentration (110% Lys) did not increase plasma IgA levels13,16. CONCLUSIONS The results of this

study indicate that the Lys content of turkey diets should be 10% higher than that recommended by the NRC1. The increased Lys content should be combined with the higher Arg level (105% of

Lys content). Although the above Arg:Lys ratio did not improve the growth performance of birds, it stimulated their immune system (in particular the immune response following vaccination)

and reduced protein nitration as well as protein and DNA oxidation. MATERIALS AND METHODS EXPERIMENTAL DESIGN AND DIETS The protocol for the study was approved by the Local Ethics Committee

for Animal Experiments in Olsztyn, Poland (decision No. 82/2017), and the animals were cared for under guidelines comparable to those laid down by EU Directive 2010/63/EU. The study was

carried out in compliance with the ARRIVE guidelines. One-day-old female Hybrid Converter turkeys were purchased from the Grelavi commercial hatchery in Kętrzyn. The experiment was performed

on 576 turkeys that were randomly assigned to 32 pens of 18 birds each. The experiment had a completely randomized design with four dietary treatment groups, eight replicate pens per group

and 18 birds per pen. The replicate pens were uniformly distributed in the house. Pens with a floor area of 4 m2 (2.0 m × 2.0 m) were bedded with wood shavings. The stocking density in the

initial stage of rearing was 4.50 birds/m2. Environmental conditions, consistent with Hybrid requirements, were identical for all turkeys in the housing facility. They were controlled

automatically and adjusted to the birds’ age. Throughout the experiment, all birds had free access to feed and water. The feed and drink lines were adjusted to the growth stage of turkeys.

The experimental diets were produced in a local feed mill under the direct supervision of a representative of the Department of Poultry Science and Apiculture, University of Warmia and

Mazury in Olsztyn. According to the experimental procedure, basal diets were prepared for each of the four feeding periods, and their amino acid content was determined analytically (Table

8). Then the diets were mixed with the appropriate amounts of Lys, Met and Arg (Table 8). The actual levels of supplementary Lys, Arg and Met in experimental diets were obtained by adding

supplementary L-Lys HCl, L-Arg HCl and DL-Met on top to the basal feed. Two dietary inclusion levels of Lys were analyzed, low (LL = NRC) and high (LH = NRC + 10%). In diets with the low

level of Lys, l-Lysine HCl was added to the basal diet to obtain 1.60, 1.50, 1.30 and 1.00 g of Lys per 100 g of feed in four successive feeding periods, according to NRC guidelines1.

l-Arginine HCl was added to the basal diet to obtain 95% and 105% Arg relative to the content of dietary Lys (low and high, AL and AH respectively). The effects of four experimental diets,

with two levels of Lys and two levels of Arg (LLAL, LLAH, LHAL, LHAH), were compared in the study. The inclusion rate of Met in experimental diets was identical, and dl-Methionine was added

to obtain 0.62, 0.59, 0.51 and 0.39 g of Met per 100 g of feed in four successive feeding periods. As a result, the Met:Lys ratio was 0.39 in experimental diets with the low Lys level (LL)

and 0.35 in experimental diets with the high Lys level (LH), regardless of the stage of rearing. Throughout the experiment, the birds had free access to feed and water. The diets were

offered as crumbles (days 1–28) and pellets. VACCINE AND VACCINATION At 34 days of age, half of the birds in each replicate were vaccinated against _Ornithobacterium rhinotracheale_ (ORT)

with Ornitin (ABIC Biological Laboratories Ltd., Israel). The vaccine was administered subcutaneously following the manufacturer's instructions. GROWTH TRIAL AND SAMPLE COLLECTION The

body weights (BW) of birds were recorded and calculated on a pen basis. The feed conversion ratio (FCR; kg of feed/kg of body weight gain, BWG) for the experimental period was calculated on

a pen basis from BWG and feed consumption. Mortality rates and causes were recorded daily, and the weights of dead birds were used to adjust the average FCR. Blood samples were collected at

9 and 16 weeks of age from the wing vein. For all analysis besides serological analysis at 9 and 16 weeks blood was collected from 8 birds in each group (one bird per replicate) with BW

similar to the treatment average. While for serological analysis blood samples were collected (into sterile tubes with clot activator) at 16 weeks from 23 birds per treatment that were not

vaccinated and 23 birds per treatment that were vaccinated against ORT. Immediately after collection, blood samples were aliquoted into test tubes containing EDTA K (for flow cytometry

analyses) or heparin (for other analyses) as an anticoagulant. Blood samples for cytometric analyses were directly sent for isolation of mononuclear cells. While the samples for other

analyses were centrifuged for 15 min at 380_g_ and 4 °C, and the resulting plasma was stored at − 20 °C until analysis. At 9 weeks of age, 8 turkeys from each treatment were sacrificed by

cervical dislocation, and the abdominal cavity was opened for the collection of ileal (middle-ileum) tissues and spleen samples (not vaccinated against ORT). Liver samples were collected

from 8 birds per treatment (determined separately for non-vaccinated and vaccinated birds). LABORATORY ANALYSES Peripheral blood mononuclear cells (PBMCs) were isolated according to a

previously described procedure by Koncicki et al.49. The isolation of mononuclear cells from the spleen and the determination of the percentages of CD4+ and CD8+ T cell and IgM+ B cell

subpopulations in PBMCs and spleen were carried out as described by Kubińska et al.50. DNA was isolated from the intestinal wall using QIAGEN kits. Epigenetic changes in the blood and

intestinal wall of turkeys were determined by analyzing global DNA methylation (methylome) with the use of Sigma Aldrich diagnostic kits. The levels of 8_-_hydroxydeoxyguanosine (8-OHdG),

endonuclease 1 (APE-1) and oxoguanine glycosylase (OGG1) in the blood and intestinal wall of turkeys were determined using OxiSelect diagnostic kits (Cell Biolabs, Inc., San Diego, USA).

OxiSelect diagnostic kits (Cell Biolabs, Inc., San Diego, USA) were also used to determine protein carbonyl (PC) and 3-nitrotyrosine (3-NT) derivatives as an indicator of the oxidation of

amino acid residues. The levels of caspase 3 (Casp 3) and caspase 8 (Casp 8) were determined in the blood and intestinal wall of turkeys using an ELISA kit (Cell Biolabs, Inc. San Diego,

USA). The plasma levels of C-reactive protein (CRP) were determined in an ELISA reader using assays from Elabscience Biotechnology Co., Ltd. (Houston, Texas, USA). The levels of

ceruloplasmin (Cp) in the blood and jejunum of turkeys were determined using a Ceruloplasmin ELISA kit (Biomatik, Delaware, USA). The levels of total globulins and immunoglobulins IgA and

IgY, tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6) were determined in an ELISA reader using assays from Elabscience Biotechnology Co., Ltd. (Houston, Texas, USA). Anti-ORT

IgY titres were determined using a commercial immunoenzymatic ELISA kit (IDEXX Laboratories, USA) according to the manufacturer’s recommendations. The ELISA assay was performed using an

epMotion 5075 LH automated pipetting system (Eppendorf), an Elx405 washer, and an Elx800 absorbance microplate reader (BioTek, USA). ANALYSIS OF MRNA TRANSCRIPT LEVELS The relative mRNA

expression levels of genes encoding IgA and IL-6 were quantified in liver samples collected from turkeys 2 days after vaccination against ORT. The protocol of qRT-PCR analysis was described

by Ognik et al.24. The liver samples were immediately frozen in liquid nitrogen. Subsequently, RNA was isolated using the GeneMATRIX Universal RNA Purification Kit (Eurx, Gdańsk, Poland), in

accordance with the manufacturer’s instructions. The concentration of RNA was measured on a NanoDrop spectrophotometer (Nanodrop, NanoDrop Technologies, Wilmington, DE), and RNA integrity

was verified on agarose gel under denaturing conditions. qPCR reactions were performed on a LightCycler 480 II apparatus (Roche Applied Science, CA, USA) using the SG qPCR Master Mix (Eurx,

Gdańsk, Poland). Reaction conditions were as follows: (i) initial denaturation (one cycle at 95 °C for 10 min) and (ii) amplification (35 cycles of denaturation at 95 °C for 10 s, primer

annealing at 58 °C for 10 s and DNA synthesis at 72 °C for 20 s). Endogenous control genes, β-Actin (ACTB) and Vimentins (VIM), were used to normalize gene expression data. The primers for

the target and reference genes used in this study are presented in Table 9. STATISTICAL ANALYSIS The results were analyzed statistically by two-way ANOVA using STATISTICA ver. 13 software

(StatSoft Inc. 2013). The significance of differences between means was determined by Tukey’s test or the non-parametric Kruskal–Wallis test. The variability of data was expressed as

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Article  CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by the National Science Center, Grant No. 2017/27/B/NZ9/01007. AUTHOR INFORMATION AUTHORS

AND AFFILIATIONS * Department of Biochemistry and Toxicology, Faculty of Animal Sciences and Bioeconomy, University of Life Sciences in Lublin, Akademicka 13, 20-950, Lublin, Poland

Katarzyna Ognik, Magdalena Krauze & Anna Stępniowska * Department of Poultry Science and Apiculture, Faculty of Animal Bioengineering, University of Warmia and Mazury in Olsztyn,

Oczapowskiego 5, 10-719, Olsztyn, Poland Dariusz Mikulski, Paweł Konieczka & Jan Jankowski * Department of Poultry Diseases, Faculty of Veterinary Medicine, University of Warmia and

Mazury in Olsztyn, Oczapowskiego 13/14, 10-719, Olsztyn, Poland Bartłomiej Tykałowski * Laboratory of Molecular Diagnostics, Faculty of Biology and Biotechnology, University of Warmia and

Mazury in Olsztyn, Prawocheńskiego 5, 10-720, Olsztyn, Poland Anna Nynca Authors * Katarzyna Ognik View author publications You can also search for this author inPubMed Google Scholar *

Dariusz Mikulski View author publications You can also search for this author inPubMed Google Scholar * Paweł Konieczka View author publications You can also search for this author inPubMed 

Google Scholar * Bartłomiej Tykałowski View author publications You can also search for this author inPubMed Google Scholar * Magdalena Krauze View author publications You can also search

for this author inPubMed Google Scholar * Anna Stępniowska View author publications You can also search for this author inPubMed Google Scholar * Anna Nynca View author publications You can

also search for this author inPubMed Google Scholar * Jan Jankowski View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS K.O. designed the study

concept, interpreted results, performed analyzes and wrote the manuscript. D.M. performed the turkey trial and interpreted statistical data. P.K. performed turkey trial. B.T. performed

vaccination, analyzes and veterinary care. M.K. performed analysis. A.S. performed analysis. A.N. interpreted data regarding genes expression. J.J. designed the study concept, interpreted

results, and edited the manuscript. All authors read and approved the final manuscript. CORRESPONDING AUTHOR Correspondence to Katarzyna Ognik. ETHICS DECLARATIONS COMPETING INTERESTS The

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CITE THIS ARTICLE Ognik, K., Mikulski, D., Konieczka, P. _et al._ The immune status, oxidative and epigenetic changes in tissues of turkeys fed diets with different ratios of arginine and

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