**2. Arginine**

Arg is known as an essential amino acid (AA) for birds due to its inability to synthesize Arg, so it must be supplemented in their diet [13]. Arg plays an important function in serving as the building block of proteins and polypeptides and fulfills several physiological roles through the regulation of key processes such as maintenance, growth, reproduction, and immunity. The recommended dietary Arg levels for optimum growth performance in broilers varies from 1.25 to 1.10% for starting and growing birds [7] and from 1.37–1.43 and 1.0–1.1 for starting and finishing birds, respectively [8]. There are also dietary Arg recommendations for different commercial strains of broilers. In addition to this, it has been documented that the addition of

#### *Advances in the Nutrition of Functional Amino Acids in Healthy and Immunologically… DOI: http://dx.doi.org/10.5772/intechopen.101895*

Arg either in ovo or dietary Arg above the recommended level improves the digestive physiology and the cellular and humoral immune responses in nonchallenged and challenged birds; hence Arg has gained the distinction of belonging to the group of functional nutrients. The readers are referred to several comprehensive reviews about this topic [14–18]. In the next section, the recent findings on Arg feeding from in ovo to unchallenged and challenged broilers will be presented with emphasis on simultaneous effects on immunity/digestive physiology and productivity.

In ovo feeding (IOF) of Arg. IOF of Arg has been shown to boost glucose synthesis in the liver, which correlates with enhanced glucose 6-phosphatase activity at hatch [19, 20]. In this regard, it has been shown that IOF of Arg improved posthatch growth performance in chicks, and the effects of Arg have been linked to glucose synthesis and hormone production. Furthermore, supplementing with Arg has been proven to improve gut morphology (a sign of gut health), implying that it may affect the metabolism of this oxygen-demanding tissue [21]. IOF of Arg also stimulates the intestine mucin gene expression at 18th d of incubation and 14th d posthatch, as well as the IL-6 and IFN-γ humoral gene expression in 26-days-old birds [22]. In this section, the available literature was examined to find the best dosage of Arg that improves the immune response and gut morphology and that leads to enhanced growth performance in posthatch broilers.

In some experiments, the Arg IOF at day 14th of incubation has been evaluated. IOF of 35 mg Arg/egg depressed hatchability but increased the body weight of alive broilers at 11, 24, and 42 days of age; Arg inclusion increased the length of jejunum and ileum at 42 d of age and led to the greatest villus height and crypt depth in jejunum at 11 days of age [23]. In a similar experiment, Arg reduced hatchability again but increased the body weight in 42 days old broilers, and the relative weight of spleen and bursa of Fabricius at 11 days posthatch and antibody titer against SRBC at 30 days posthatch [24].

In several recent experiments, lower levels of IOF (0.6 mg Arg/egg) at 17.5 d of incubation have been tested. A summary of the results indicates that Arg IOF did not negatively affect the hatchability and improved the body weight at 7 and 21 d posthatch, and the ADWG from 1 to 21 d of age; Arg also increased the weights of digestive organs, the activities of digestive enzymes, alkaline phosphatase, maltase, and sucrase in the jejunum, the mRNA expressions of jejunal sensing receptors of taste and nutrient transporters of solute carriers [21]. Arg also increased the absolute weights of lymphoid organs, the activity and the mRNA expression and protein abundance of iNOS, the contents of IL-2, IL-4, and sIgA, the mRNA expressions of TLR-2 and TLR-4 in intestinal mucosa and serum; conversely, Arg decreased the iNOS promoter methylation percentage in jejunal mucosa [25].

In other reports, it was found that IOF of 0.6 mg Arg increased the weight of embryos at 19 days of incubation and the ADWG in chicks from 1 to 7 days posthatch [26], and the ADWG of broilers from 1 to 21 days and from 1 to 42 days of age [27]. Arg also increased the duodenum activities of alkaline phosphatase, maltase, sucrase, and inducible nitric oxide synthase of 7-days-old posthatch broilers, and the villus height and the ratio of villus height to crypt depth in duodenum of broiler embryos and posthatch birds and increased the density of goblet cells [26, 27]. The hatchability was high and similar to the control group in Arg supplemented eggs. Other benefits of Arg were the increased percentage of proliferating cell nuclear antigen positive cells of villus, and the mRNA expressions of mucin-2, claudin-1, and zonula occludens-1 and -2 in jejunal mucosa of 21-day-old broilers [27]. Furthermore, IOF of 0.6 mg Arg/egg increased the relative weight of breast muscle

at hatch and 7, 14, and 21 days posthatch, and increased the concentration of some essential AA in the breast muscle such as Thr, valine (Val), phenylalanine (Phe), lysine (Lys), and Arg at hatch and 21 days posthatch [20].

Some studies have also been published using higher levels of IOF Arg with positive results. The IOF of 2.5 mg Arg/egg at 18 days of incubation resulted in similar hatchability to the control group, and in higher chick weight at hatch and lower transit weight lost from the hatchery to the farm; Arg also improved the body weight and ADWG in broilers up to 21 days of age [28]. When using even higher levels of IOF Arg (11 and 22 mg Arg/egg) at 18 days of incubation, the hatchability was similar to the control group, and greater body weight in chicks at 7 days posthatch was reported with 11 mg Arg; improved development of duodenal villi in 7-days-old chicks and enhanced cell-mediated immune response after 24 and 48 h in 28-days-old broilers was observed with 11 and 22 mg Arg [29].

A summary of the results indicates that IOF of 0.6 mg Arg/egg at 17.5 days of incubation increased the growth performance of broilers up to 42 days of age, which could be explained by the enhanced immune humoral and cellular response and the early maturation of the digestive capabilities.

In several studies, the stimulatory properties of increasing levels of dietary Arg on the immune and digestive systems have been documented in broilers kept in nonchallenged and under-challenged conditions [30–33]. From this, a number of studies have been published that have evaluated the productive performance along with the immune and digestive responses to high levels of Arg supplementation. These studies, while few, may provide insight into whether improvements in the immune and digestive responses can be associated with increased productivity at the same level of Arg supplementation.

In nonchallenged, 1–28 days of age broilers kept in cages and fed 1.48% (considered a normal level in corn/soybean meal diets) and 1.58 dietary Arg (keeping an Arg:Lys ratio of 1.20), no differences in growth performance were found, whereas the addition of 1.58% Arg increased the percentage of mucosa T helper (CD4+TCRvβ1+) and T cytotoxic (CD8+CD28+) [34]. In nonchallenged chicks housed in floor pens from 1 to 21 days of age and fed increasing levels of Arg [1.00, 1.125, 1.250, 1.375, and 1.50% of NRC [7] recommendations for Arg requirements], the performance was improved at 1.25% Arg, while the relative weight of thymus increased in a nearly linear manner, and the cell-mediated immune response to phytohemagglutinin P and antibody titer against NDV increased linearly up to 1.375% Arg [35]. Similarly, using increased Arg levels (0.86, 1.31, 1.76, 2.21, and 2.66%, based on the recommended Arg requirement by NRC [7]), the ADWG and FCR were improved at 1.31% Arg, whereas serum total immunoglobulins and IgA increased up to 1.76 and 2.21% dietary Arg, respectively [36]. Furthermore, low growth potential chicks fed increasing total Arg levels (0.85, 0.97, 1.09, 1.21, and 1.33%, based on the nutritional requirements for Qingyuan partridge chickens) for 30 days showed maximum ADWG and FCR at 0.97% Arg, while mucosal jejunum IgG and ileum sIgA increased linearly up to 1.21% [37].

In challenged broilers vaccinated against *Salmonella enteritidis* at nine days of age and kept in cages from 1 to 28 days of age, similar performance was observed with 1.48 and 1.58% dietary Arg (keeping an Arg:Lys ratio of 1.20); however, increased suppressors monocytes (Kul+MHCII−) were found in birds supplemented with 1.58% Arg [34]. In broilers fed diets: deficient, normal, and excessive in Arg (1.05, 1.42, and 1.90% according to NRC [7]), kept in cages and challenged with an *Escherichia* (*E.*) *coli* lipopolysaccharide (LPS), showed higher ADWG and FCR at 1.42 and 1.90% Arg, respectively, after the challenge; depressed TLR4 and NF-kB in cecal tonsils relative

#### *Advances in the Nutrition of Functional Amino Acids in Healthy and Immunologically… DOI: http://dx.doi.org/10.5772/intechopen.101895*

mRNA expression at 1.42% Arg and PPAR-γ in spleen and IL-1b in cecal tonsils relative mRNA expression at 1.90 Arg were observed after the challenge [38].

In several studies, the coccidiosis challenge has been used as a mean to demonstrate the functional benefits of Arg. A summary of some studies is given below:

Broilers from 1 to 26 days of age allocated in metabolic cages fed increasing levels of dietary Arg (1.04, 1.14. 1.24, 1.34, and 1.44%) and challenge with Eimeria sporulated oocysts at 12 days of age, showed better ADWG and FCR at 14 days postchallenge at 1.14% Arg. Higher levels of Arg (1.34%) improved the intestinal permeability at five d postchallenge and the tight junction proteins zonula occludens-1 and zonula occludens-2 at six d postvaccination, while the addition of 1.44% Arg increased the zonula occludens-1 and zonula occludens-2 at 14 days postvaccination [16]. In chicks also housed in cages from 1 to 21 days, added with 1.11, 1.33, and 2.01% dietary Arg and challenged with a coccidiosis vaccine at 14 d of age, showed similar ADWG regardless of the dietary Arg level and lower FCR at 1.33% Arg. Increased sucrase, sIgA, and relative IL-1RI mRNA expression and reduced abundance of TLR4 and MyD88 in jejunum at 7 days postchallenge were observed at 1.33% Arg, and increased mucosal density in the jejunum was observed at 2.01% Arg at 7v postchallenge [38].

In floor-pens reared broiler given 100, 105, and 110% of the standard recommended values of dietary Arg for Ross broilers, and challenged with a mixture of Eimeria species from 16 to 20 days of age, addition of 105 and 110% Arg, prevented depressed ADG in coccidia-infected broiler chickens during the finisher period. The FCR was improved at 110% Arg supplementation during the grower and finisher periods. Increased villi height to crypt depth ratio at 105% Arg and increased villi surface area at 110% Arg were found, as well as a linear decrease in fecal oocyst count [39]. Broiler chicks reared in pens fed 100, 125, and 150% Arg levels, according to Ross recommendations, and infected with Eimeria on day 21, showed better ADWG, FI, and FCR from 22 to 42 days of age at 125% dietary Arg (starter 1.71%, grower 1.54%, and finisher 1.375%); furthermore, at 125 and 150% dietary Arg, increased levels of serum NO and proinflammatory cytokine concentrations (IL-1β IL-2 IL-6 TNF-α IFN-γ) and reduced fecal oocysts were found [40].

In two additional studies with broilers subjected to viral challenges, it was observed that productivity and immune responses were improved with higher levels of dietary Arg than recommended. Broiler chickens fed diets exceeding by 2.5 times, the recommended NRC levels (starter 1.34 vs. 3.35, grower 1.13 vs. 2.8, and finisher 1.1 vs. 2.58), and challenged with an intermediate plus strain of IBD virus (10-fold greater than normal vaccination doses) at 28 days of age, showed enhanced body weight, ADWG, and FCR, as well as higher serum level of IFNα, IFNγ, immunoglobulin G, and lower lesion scores in the bursa and spleen compared to the control birds [41]. In the same way, broiler chicks fed 2% supplementary dietary Arg and vaccinated and challenged against hydropericardium syndrome virus showed higher body weight, lymphoproliferation, and cutaneous basophil hypersensitivity reactions, lymphoid organ weights, and highest survival rate compared to unvaccinated non-Arg supplemented chicks [42].

Results in four available studies, in which nonchallenged broilers were fed increasing dietary Arg concentration, indicate that the Arg needed to stimulate the immune system was higher than that needed to improve the growth performance. These results were irrespective of the basis of Arg formulation, the growth rate of the birds, and the type of housing (cages or floor pens).

The information also denotes that in four out of eight studies available, in which challenged broilers were fed increasing dietary Arg concentration, the Arg needed to stimulate the immune system was also higher than that needed to improve the growth performance. These results were irrespective of the basis of Arg formulation, the growth rate of the birds, the type of housing (cages or floor pens), and the type and degree of challenge.

It was found that in three out of four studies, in which the growth performance and immune and digestive responses were enhanced at the same Arg levels, the Arg levels were higher than those recommended for optimum growth; it is noteworthy that in one of these studies, ADWG and FCR were improved in 1–49-days-old broilers with Arg levels 2.5 times higher than recommended.
