**3. Threonine**

Thr is ranked as the third limiting AA [2, 4] and is very important for the synthesis and maintenance of proteins in the body. About 30–50% of Thr, as well as some other amino acids, is directly used by the small intestine and is not available for extraintestinal tissues. Thr has special importance as an essential nutrient because, compared with other AA, it has the highest metabolism in the portal-drained viscera. One of the primary fates of absorbed Thr is the synthesis of intestinal proteins, which are mainly secreted into the lumen as mucus, whereby protecting the gut from pathogens and antinutritional factors. Mucins are particularly rich in Thr, proline, and serine, with Thr representing as much as 28 to 40% of its total AA profile [43].

The recommended dietary Thr levels for optimum growth performance in broilers varies from 0.80 to 0.68% for starting and growing birds [7] and from 0.85–0.89 and 0.65–0.68 for starting and finishing birds, respectively. Further to this, it has been demonstrated that supplementation of Thr either in ovo or dietary Thr above the recommended level improves the digestive physiology and the cellular and humoral immune responses in nonchallenged birds and those subjected to different immune challenges [43]. IOF of Thr has shown to increase the expression profile of growth factors and immunity-related genes, including higher mucin gene expression on incubation day 18, higher expression of mucin gene on day 14 postinoculation, higher humoral expression of IL-6 and TNF-α, and higher IL-12 cellular gene expression in 26-days-old broilers [22]. 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 several experiments, the IOF of Thr at day 14th of incubation enhanced various immune and digestive responses. IOF of 20 or 30 mg Thr/egg improved the ADWG of broilers from 14 to 28 days of age and enhanced the humoral response to sheep red blood cells; there was a tendency for digestive enzyme activities in proventriculus, jejunum, and pancreas to be higher in Thr-injected chicks at 21 days of age [44]. IOF of 25 mg Thr/egg increased the body weight of broilers at 11, 24, and 42 d and the FI from 1 to 42 days of age; Thr also enhanced the ileum villus height in 11-days-old chicks and the relative weight of the jejunum and ileum and the length of the jejunum in 42-days-old broilers [23]. IOF of 25 mg Thr/egg also increased the ADWG and FI in broilers from 1 to 42 days of age and the antibody titer against sheep red blood cells in broilers at 30 days posthatch [24]. In both studies, hatchability was similar to the control group.

In few experiments, the IOF of Thr in the last days of incubation has been also evaluated. IOF of 10.5, 21.0, 31.5, and 42 mg Thr/egg on day 17.5 of incubation

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

improved the chick hatch weight and growth performance from 1 to 21 days of age; Thr increased the villus height, villus height: crypt depth ratio, and villus area at hatch and 21 days posthatch. At hatch, all Thr levels increased the expression of MUC2 and PepT1 compared to the control group [45]. IOF of 15, 30, and 45 mg Thr/ egg at 18th embryonation d increased the ADWG in broilers up 21 days posthatch, and the FCR was improved at 45 mg Thr; Thr increased the thymus weight (d0), bursa weight (d3), spleen weight (d3 and d7), whereas quadratic effect was observed on weights of bursa, thymus, and spleen at d21. IOF of Thr also increased the weights of gizzard, intestine, and liver at hatch, proventriculus at d7, as well as intestine and liver at d21 [46].

A summary of the results indicates that IOF of Thr at 14 and 17.5–18 days of incubation increased the growth performance of broilers up to 42 days of age, which could be explained by improved immune responses, but especially by increasing the development of the digestive capabilities. The best dosage for IOF of Thr appears to be around 25 mg/egg. In all cases, a high hatchability is maintained.

During the growth out of broilers, there are several studies of Thr supplementation as functional AA in nonchallenged conditions. Ross male broilers fed diets containing 0.8% (NRC [7] requirement), 0.87% (average of NRC and Ross requirement), 0.94% (Ross requirement), and 1.01% (more than Ross requirement) Thr had improved growth responses as dietary Thr increased from 0.8% to 0.87%; similarly, the villi height, crypt depth, and villi surface increased as dietary Thr increased from 0.8% to 0.87% [47]. In broilers from 1 to 21 days fed increasing standardized ileal digestible Thr levels from 0.4 to 1.1%, it was reported that ADWG was higher at 0.84–0.89% Thr, while the villus height in duodenum, jejunum, and ileum were increased linearly up to 1.1% Thr [48]. In broilers from 1 to 21 days of age fed 0.79, 0.87, and 1.07% Thr showed no differences in growth performance due to the supplementation of Thr; opposite to this, Thr supplementation increased the relative weight of spleen and thymus. Thr supplementation linearly increased the intestinal villus height, the ratio of villus height to crypt depth, as well as the goblet cell density and the jejunal immunoglobulin G and M. At the highest Thr supplied, the ileal secretory immunoglobulin A content and mucin-2 mRNA expression were increased, while the mRNA abundances of interferon-γ and interleukin-1β in the ileum were downregulated [49].

In broilers reared in floor pens and fed increasing dietary Thr levels (starter from 0.69–1.21% and grower 0.62–1.12% Thr), which correspond to 85–150% of NRC [7] recommendations, the ADWG and FCR were improved at 100% Thr, whereas the villus height in duodenum and jejunum, crypt depth in duodenum, and villus height/ crypt depth ratio in jejunum were increased a 150% Thr in 21-days-old broilers, and the villus height and villus height/crypt depth ratio in jejunum were increased a 125% Thr in 42-days-old broilers [50]. Floor pen reared broilers fed increasing levels of dietary Thr (starter from 0.94–1.22% and grower from 0.74–0.96% Thr), equivalent to 100–130% of Ross 308 recommendations, had higher growth performance at 110% Thr inclusion, but the antibody titers against NDV and SRBC increased up to 120% Thr supplementation [51]. In two floor pen experiments using slow-growing broilers and a basal feed formula that met the requirements mentioned by Rostagno et al. [8] and added with increasing levels of digestible Thr, it was estimated that the lowest FCR was reached at 0.762 and 0.767 for starter and grower broilers, respectively, while the production of intestinal mucin was highest at 0.697% Thr in the starter phase [52].

In another study, in which broilers from 1 to 21 days of age were fed diets to match the Thr supply to 100% NRC specification, and from 100 to 130% Thr of Vencobb-400 strain specification, the ADWG was highest at 100% Thr of Vencobb-400 strain specification (0.87% Thr); the villus height, crypt depth, villus surface area, goblet cell number/villus, villus width, and goblet cell density were higher at 120% Thr and the weight of bursa and thymus, the total immunoglobulins, titers against Newcastle disease virus, lymphocyte proliferation, and neutrophil phagocytic activity were increased linearly up to 130% Thr [53]. Broilers fed dietary Thr levels that matched 100, 110, and 120% of NRC recommendation and kept in floor pens from 1 to 35 days of age showed enhanced ADWG and FCR at 110% Thr as well as higher villus height, lower crypt depth, greater VCR, greater weight of thymus and bursa, and greater infectious bursal disease titer [54]. Similarly, 1–21 days of age broilers fed dietary Thr level of 100, 120, and 140% of the NRC recommendation had improved performance ADWG and FCR at 120% Thr; anti-SRBC titer were increased at 120% Thr, and the jejunal crypt depth increased and the jejunal and ileal crypt width decreased at 140% Thr [55].

Some experiments were carried out using increasing dietary Thr addition in broilers under bacterial and coccidial challenges. Broilers from 1 to 10 days of age fed two dietary Thr levels (0.857 and 0.956%) and challenged with *Salmonella Enteritidis* at 2 days of age showed no difference in performance, but the intestinal integrity was improved in chicks fed the higher Thr level, including higher villus height, villus:crypt ratio, and goblet cell counts in the jejunum and ileum [56]. In the same way, broilers from 1 to 10 days of age fed two dietary Thr levels (0.81 and 1.00%) and challenged with *Salmonella Enteritidis* at 2 days of age showed no difference in performance, but had increased villus height and villus:crypt ratio in the duodenum [57]. Broilers of 1–21 days age kept in cages and fed two levels of dietary Thr (0.784 and 1.084%), undergoing a challenge using *E. coli* LPS from 17 to 21 days had improved ADWG and FCR at the higher Thr level and reduced serum IL-1β, and TNF-α, IFN-γ in jejunal mucosa and L-1β in ileal mucosa [58]. In three floor-pen experiments, different Thr-to-Lys ratios (from 0.56 to 0.77) were evaluated (as standardized digestibility) in the diets of broilers subjected to a subclinical Clostridium infection at nine d of age; from 9 to 37 days of age, the ADWG in broilers fed the high dietary Thr was increased, but the intestinal damage (incidence and lesion severity) was not affected by Thr supplementation [59].

The results indicate that in eight out of 12 studies, in which nonchallenged and challenged broilers were fed increasing dietary Thr concentrations, the Thr needed to stimulate the immune and digestive system was higher than that needed to improve the growth performance. These results were irrespective of the basis of Thr formulation, the growth rate of the birds, the type of housing (cages or floor pens), and the type and degree of challenge.

### **4. Implications**

According to the literature reviewed, the level of AA required to stimulate the immune and digestive systems in unchallenged and challenged chickens is higher than that required for optimum growth performance; however, from a practical standpoint and the formulation of commercial diets, there is not enough information to confirm any benefits of adding functional AA, especially when issues such as sustainable

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

poultry production, in which the economic return and environmental concerns are key components, come across.

The promotion of concepts such as phase feeding, an ideal AA profile, the addition of AA on a digestible basis, and the use of low-CP diets supplemented with crystalline AA in modern feed formulation aims to maintain high levels of productivity while having a low environmental impact. The recommendations on the required levels of AA in each specific situation have been established by taking into account the stages of development, environmental conditions, management, and degree of immunological challenge due mainly to the presence of infectious agents. All of this is done to ensure that the birds consume the amount and proportion of AA that best suits their maintenance and growth needs while avoiding any excess or deficiency of AA.

The incorporation of functional AA into practical formulation is comlex, owing to the fact that levels of AA above the established requirement for growth must be included. When this occurs, an AA imbalance may exist, affecting digestion, absorption, and metabolism of AA from the same group, as has been demonstrated with dibasic AA such as Arg and Lys. This could result in a deficiency of one or more AAs from the same group, resulting in the deamination of all AA not required in the various metabolic processes, in order to eliminate excess nitrogen in the form of uric acid, resulting in the excretion of excess nitrogen through urine. At the same time, significant amounts of energy associated with uric acid synthesis would be excreted. This problem has not been addressed in the literature.

Experiment models in animals subjected to various challenges attempt to simulate what happens in commercial farms, where animals are exposed to various sources of stress as well as viral, bacterial, and parasitic infectious agents. During the growthout process, the main factors that cause immune challenges (dietary components, management, environment, and infectious agents) can be present simultaneously and sequentially. If experimental and field challenges elicit the same level of immune stimulation and type of immune response, implying that the stimulatory effects of functional AA are similar in both scenarios, it is important to note that in AA nutrition, the ultimate response to AA additions is measured by the productive response. This implies that perhaps, with the information at hand, the use of higher levels of AA beyond the levels necessary to maximize growth and FCR is questioned. In other words, in challenged birds, the use of higher than recommended levels of AA to stimulate a greater immune and digestive response is not worthwhile if this is not reflected in increased growth.

This controversy could probably be explained by drawing on much of the information already known about the metabolic effects of immune challenges to redirect AA to protective functions involving various humoral and cellular mechanisms. To increase the supply of AA, body protein will be broken down and production performance will decrease. This is necessary since the entire immune response process requires amounts and proportions of AA that vary for each type of response. In contrast to this, in most of the reviewed studies, functional AAs have been evaluated individually, or adjusted to a profile to cover the growth recommendations, but in very narrow ranges. This is explained by the difficulty of adjusting the amount and profiles of AA when feeds are balanced with AA concentrations far above the normal requirement, especially in low-CP diets. In addition, the lack of information of a proper AA profile for immune-challenged situations makes this task more difficult.

It is noteworthy that several authors have hypothesized that the addition of synthetic AA would particularly improve the animals' immune response against intracellular pathogens. If this hypothesis is confirmed, the use of functional AA could play a critical role in pathogen reduction and, as a result, in the spread of antimicrobial resistance factors. These advantages should be confirmed in farm animals that are normally subjected to acute and chronic stressors, which may be concurrent and synergistic. It is critical at this point to determine whether episodes of immunosuppression caused by stress can be overcome by functional AA.

It is also unclear whether functional AA should be used continuously or only on a case-by-case basis, particularly when birds are stressed or when there are conditions that increase the risk of disease. If the application is strategic, it should be specified the best moment and the period they should be supplemented.

It is also possible that functional AA should be supplemented during or after an immunological challenge to aid in the recovery of affected individuals and to restore productive parameters to prechallenge levels.
