Effect of L-glutamine levels in piglets diets challenged with Escherichia coli lipopolysacharides

Objective. To evaluate the effect of different levels of L-glutamine on weaned and immunologically challenged piglets with Escherichia coli lipopolysaccharides (LPS) on performance parameters, serum cortisol and defense cells. Materials and methods. Four levels of L –glutamine were evaluated (0, 1.0, 1.5, 2.0%) as well as the addition, or no addition, of LPS (0.3μg). 96 piglets were used (48 castrated males and 48 females) of Agroceres x PenArlan lineage, with an initial age of 21 days and 6.06±0.852 kg live weight. An experimental design was used on randomized blocks in a factorial setting 4 x 2 (levels of Lglutamine with or without challenge). Results. Cubic effect was shown for daily weight gain of unchallenged animals, and was better with the addition of 0.41% Lglutamine. Feed conversion improved with increased levels of L -glutamine for challenged animals. In the evaluation of defense cells, there was interaction of leukocytes with the levels of Lglutamine and the immune challenge. Eosinophils and lymphocytes showed a quadratic effect for the levels of L –glutamine, with a maximum value of 1.30% and 0.5%, respectively. Conclusions. L -glutamine supplementation of up to 2% in the diet improves feed conversion and favors the immune serum of weaned piglets challenged with LPS of E. coli.


INTRODUCTION
Weaning in the swine industry is done between 21 and 28 days of age, at this stage psychological factors (maternal and litter segregation), social (establishment of the hierarchy in the new group) and nutritional (physical change of diet and type of food) significantly affect the development of the animals (1,2).
From the dietary or nutritional point of view, interruption of breastfeeding and substitution of this source of energy/protein with plant products requires morphological, enzymatic and metabolic adaptation (3).This leads to alterations in intestinal flora, causing a reduction in the population of beneficial bacteria as a result of pathogens, which produce toxic metabolic, causing inflammation and exfoliation of the piglet's intestinal mucous membrane, generating diarrhea and underdevelopment, when death does not occur (4).
The set of alterations causes a typical picture of stress, increasing levels of corticotropin releasing factor (CRF) and serum cortisol, influencing the behavior of the animal and corresponding to an increase in the production of immune cells, which may interfere with metabolism and animal performance (5).
Various remedies have been used to minimize problems resulting from weaning.The inclusion of glutamine in the piglets' diet is one of the recent tools that favor high replication of gastrointestinal tract and immune system cells (6).Glutamine is the most abundant free amino acid in physiological fluids (plasma, cytoplasm, milk and fetal fluids), and is also present in animal and plant protein (7,8).It is considered the main energy substrate for rapidly proliferating cells such as activated enterocytes and lymphocytes (9); moreover, it is essential in the synthesis of mucin and for maintaining barriers against bacterial attacks (10).
Pardo -Effect of L-glutamine levels in piglests diets el mantenimiento de las barreras contra ataques de bacterias (10).
Las dietas experimentales tuvieron diferentes inclusiones (0, 1.0, 1.5 y 2.0%) de L-glutamina (Ajinomoto Brasil) y asociados o no a un desafío Due to the many important metabolic processes in which glutamine participates, such as nitrogen transport and donation, control, acid-base balance and in the integrity of tissues (11), research has intensified with the aim of establishing glutamine as an amino acid conditionally essential for health (12).
Administration of lipopolysaccharide (LPS) of E. coli causes a series of direct morphological alterations in the gastrointestinal tract, characterized by the decrease in height of intestinal villi and the increase of intestinal crypts (13), increased levels of pro inflammatory cytokines TNF-α and IL-6 (14), and reduced consumption.
The aim of this study was to evaluate different levels of L-glutamine in the diets of weaned piglets challenged with Escherichia coli lipopolysaccharides, on performance parameters, serum cortisol and defense cells.

MATERIALS AND METHODS
Study site and animals.The experiment was performed in the Swine Sector of the Farm School at Londrina State University (Paraná-Brazil) located at an altitude of 532 meters above sea level with coordinates between 23°20'23.45"South latitude and 51°12'32.28"West longitude; with a pre mountain humid tropical climate; with an average temperature of 20°C, ranging from 16 to 27ºC with annual rainfall of 1588 mm.96 piglets were used with Agroceres x PenArlan lineage (48 castrated males and 48 females) weaned at 21 days of age, with an average weight of 6.06 ± 0.852 kg, housed in 48 cages (24 metal cages with raised floors and 24 on concrete floors) with one male and one female per cage.The animals received food and water "ad libitum" during the entire experimental period.
Treatments.The experimental diets were formulated to meet recommendations described by Rostagno (15) for pigs in the pre-initial phase I (1-15 days) and initial phase II (15-30 days).The nutritional and centesimal composition is found in table 1.

RESULTADOS
El consumo diário de alimento y la conversión alimenticia no fueron influenciados por la inclusión mL of skim milk and administered orally between 7:30 and 8:00 h.The other animals also received 5 mL of skim milk free of LPS orally.
Sampling.Weekly growth performance, feed intake, weight gain and feed conversion of the animals were estimated.For the cortisol and leukogram analysis, blood samples were collected from all animals by puncturing the cranial cava vein.The sample was collected in tubes containing 0.1 mL anticoagulant EDTA 10%.The first sample was taken on the 43rd day of life between 7:30 and 8:00 h, before LPS inoculation and after an 8-hour fast.Other samples were taken at the same time, day 45°, 47° and 49° of life.Leukogram was determined by the method of impedance in hematology BC-280Vet (Mindray) in an authorized clinical veterinary laboratory.
For determination of serum cortisol, samples were collected in tubes without anticoagulant, then were centrifuged at 2000 rpm for 10 minutes.The serum obtained was then transferred to 1.5 ml micro tubes, identified and stored at -20°C for later analysis.Cortisol was determined by the ELISA technique (Enzyme Linked Immunosorbent Assay) competitive in the immunology IV laboratory at the State University of Londrina.Pardo -Effect of L-glutamine levels in piglests diets de L-glutamina ni por el desafio inmunológico de LPS de E. coli.Entre tanto, el desafio inmunológico influyó en la ganancia de peso (Tabla 2).
El conteo de leucocitos y monocitos no fue influenciado por los niveles de L-glutamina, entre tanto, se presentó un efecto cuadrático en los eosinófilos y linfocitos influenciados por los diferentes niveles de L-glutamina; siendo el valor máximo de 1.30% y Experimental design.An experimental design was used on randomized complete blocks with a factorial arrangement 4x2 (four levels of L-glutamine and immune challenge with or without LPS of E. coli).The data obtained was subjected to analyses of normality, additivity and homoscedasticity and then subjected to an analysis of variance and regression using the statistical package ExpDes from the R statistical program (16).For feed intake, feed conversion and weight gain, the cage was considered as an experimental unit, with two animals per unit.For all other variables the animal was considered the experimental unit.

RESULTS
Daily feed intake and feed: gain rate conversion were not influenced by the inclusion of L-glutamine or by the E. coli LPS immunological challenge.Meanwhile, the immunological challenge influenced weight gain (Table 2).0.59% de inclusión, respectivamente.Los neutrófilos presentaron un efecto lineal creciente con cada nivel de inclusión de la L-glutamina (Tabla 6).
Interaction between levels of glutamine and the LPS challenge were observed for weight gain and feed conversion (Table 3).Unchallenged animals had a cubic effect of glutamine levels on weight gain, the maximum point of inclusion of L-glutamine being 0.41%.
In addition, there were no observed differences in daily weight gain between challenged or unchallenged animals supplemented with 1.0 and 2.0% glutamine (Table 3).
For animals that were not supplemented with L-glutamine and were not challenged, increased daily weight gain was observed.However, challenged animals supplemented with 1.5% glutamine obtained a greater weight gain compared to non-challenged (Table 3).
Feed conversion of the challenged animals showed a decreasing linear effect with increasing inclusion of L-glutamine, indicating a reduction of 0.254 kg for each inclusion level (Table 3).The animals that were not supplemented with L-glutamine showed a (p<0.05)difference in feed conversion.
Serum cortisol concentration was not influenced by the immunological challenge or by the inclusion of L-glutamine (Table 4).
Meanwhile, there was interaction between levels of glutamine and the LPS challenge for cortisol values (Table 5).Unchallenged animals showed a quadratic effect, obtaining a maximum value of cortisol with the inclusion of 1.45% of L-glutamine.
The challenged animals showed a cubic effect reaching a maximum cortisol value with a level of 1.68% of L-glutamine inclusion, and a minimum value with 0.51% of L-glutamine inclusion.
There was also a difference between glutamine levels 0, 1.0 and 1.5 for immunologically challenged animals or unchallenged animals.Las diferencias obtenidas en la conversión alimenticia para los animales que no recibieron inclusión de L-glutamina (que fueron o no desafiados con LPS de E. coli), pueden ser debidas a que durante los procesos de estrés metabólico ocurre una reducción en el transporte luminal de la glutamina y de la actividad de la glutaminasa de la mucosa (19), existiendo la posibilidad de que el intestino supla las necesidades de otros órganos como parte del proceso para priorizar la síntesis proteica y de esa manera no sea comprometido el aprovechamiento de los nutrientes.
Meanwhile, cortisol levels in challenged and unchallenged animals supplemented with 2% L-glutamine remained similar throughout the experimental period (Table 5).
The counting of leukocytes and monocytes was not influenced by levels of L-glutamine, meanwhile, there was a quadratic effect on eosinophils and lymphocytes influenced by different levels of L-glutamine; with a maximum value of 1.30% and 0.59% of inclusion, respectively.Neutrophils showed an increasing linear effect with each level of L-glutamine inclusion (Table 6).
The immunological challenge influenced the leukocyte count; with higher values for the animals that were challenged with E. coli LPS (Table 6).
For leukocytes, there was interaction between L-glutamine levels and the immunological challenge (Table 7).
There was a difference between the animals supplemented with 0, 1.0 and 1.5% of L-glutamine and the immunologically challenged, meanwhile, challenged animals supplemented with L-glutamine (1.0, 1.5 and 2.0%) had increased lymphocyte counts compared with unchallenged; however, this difference was reversed by supplementing piglets with 1.5% of L-glutamine (Table 7).

DISCUSSION
Food consumption is one of the factors that have great importance on the development of the digestive tract of recently weaned piglets.Enzyme production is proportional to the amount of substrate in the intestinal tract, therefore, the observed effect on weight gain with the inclusion of 1.5% of L-glutamine is largely due to the increase in activity of amylase and pancreatic trypsin in the piglets, improving digestibility (17).
The positive effect on feed conversion can be explained by glutamine action on the metabolism, intestinal structure and function, which inhibits the decrease of growth and atrophy of the mucosal villi in animals challenged with LPS, increases absorption of dietary nutrients and improves feed efficiency (18).
The differences obtained in feed conversion for animals that didn't receive inclusion of L-glutamine (which were not challenged with E. coli LPS) may be due to metabolic stress processes during which there is a reduction in luminal transport of glutamine and in the activity  El estrés es un proceso físico-químico o emocional que promueve la liberación de citocinas proinflamatorias, hormona liberadora de corticotropina y cortisol.Cuando los estímulos estresantes se vuelven crónicos hacen que los niveles de cortisol se mantengan elevados, lo que induce a un desequilibrio de las interacciones neuroendocrinoinmunológicas (20).La exposición a bajas dosis de LPS de E. coli causa la activación del eje hipotálamico-hipofisiario-adrenal, aumentando los niveles de cortisol en la sangre, e indica el nivel de estrés a que el animal está siendo sometido (19,21).
Cuando los animales son desafiados con LPS de E. coli y son suplementados con L-glutamina, se of the mucosal glutaminase (19), with the possibility of the intestine meeting the needs of other organs as part of the process for prioritizing protein synthesis and thus not compromising the use of nutrients.
Stress is a physical-chemical or emotional process which promotes the release of pro-inflammatory cytokines, corticotropin-releasing hormone and cortisol.When stressors become chronic they cause cortisol levels to remain elevated, which leads to an imbalance of neuro-endocrine immunological interactions (20) (20).Glutamine may reduce the synthesis of cortisol in the adrenal cortex due to reduction in key enzyme activities or in the availability of NADPH.
In normal situations, cortisol values may vary, it is therefore necessary to consider normal periodic changes and physiological responses to stress (21).
The observed increase in the number of lymphocytes in challenged animals is attributed to the role of glutamine as a source of ATP for lymphocytes and macrophages, enhancing the immune response of the performance of the intestinal barrier (22).In addition, a simple stress stimulus causes changes in the number of leukocytes in the blood, which can affect the immune system's availability in responding to these changes.
The difference observed in leukocytes for challenged or unchallenged animals indicates that LPS stimulates increased production of leukocytes and this increase is dependent on extracellular glutamine present at the time of challenge; however, these concentrations may also vary depending on genetics, environment, the health of the animals and serum cortisol values (23).
The evident effect for neutrophils was possibly caused by the inclusion of L-glutamine on the immune response, as the amino acid enhances proliferation of phagocytic activity and the rate of production of superoxide (free radical necessary for bacterial death), decreasing the number of E. coli present after a challenge (24).

Agradecimientos
A la Fundación de Amparo a la Investigación de Mato Grosso-Brasil (FAPEMAT) por la beca de investigación, así como a Ajinomoto Brasil/ Animal Nutrition por la donación de la L-glutamina utilizada y a CAPES por la beca de estudio del programa PEC-PG.
When animals are challenged with E. coli LPS and are supplemented with L-glutamine, one can observe the beneficial effect of the amino acid, attenuating the decrease in growth and inefficient use of nutrients.In this case, L-glutamine serves as a metabolic regulator to increase protein synthesis and reduce protein catabolism in infectious and inflammatory processes, maintaining a rate of protein deposition in the skeletal muscle (25).
Depending on the severity of the challenge, it is difficult to correctly estimate the beneficial effect of L-glutamine inclusion, since inclusion levels determine different responses in the evaluated parameters.Furthermore, it is known that immunological challenges cause inflammatory alterations and a variety of physiological responses (26).When challenged and not supplemented with L-glutamine, the release of cytokines by macrophages occurs, and the activation of the hypothalamic-pituitary axis, which causes protein degradation in skeletal muscle, reducing intracellular concentration of glutamine, altering the intermediary metabolism and absorption of nutrients (27).
In conclusion L-glutamine inclusion of up to 2% in the diet improves the feed conversion of animals subjected to the challenge with E. coli LPS.Therefore, positive responses regarding cellular immunity serum in weaned piglets are due to the inclusion of L-glutamine, and its use is recommended at this stage.

Table 1 .
Calculated percentage composition of the basal diet.

Table 2 .
Production parameters of weaned piglets supplemented with different levels of glutamine and immunologically challenged with E. coli LPS.

Table 3 .
Interaction between glutamine and LPS levels for variables daily weight gain (DWG) and feed conversion (FGT).
1Values followed by different letters in the row differ statistically by the F test (p<0.05).

Table 4 .
Serum cortisol values (ng/mL) of weaned piglets immunologically challenged with E. coli LPS and supplemented with L-glutamine.

Table 5 .
Interaction between levels of glutamine and LPS for the serum cortisol.
1 Levels of L-glutamine inclusion.Values followed by different letters in the row differ statistically by the F test (p<0.05).

Table 7 .
Interaction between levels of glutamine and LPS for the Leukocyte variable.
1Levels of L-glutamine inclusion.Values followed by different letters in the line differ statistically by the F test (p<0.05).

Table 6 .
Defense cell values for weaned pigs fed different levels of L-glutamine and immunologically challenged with E. coli LPS.
. Exposure to low doses of E. coli LPS causes activation of the hypothalamic-pituitaryadrenal axis, increasing cortisol levels in the blood, and indicates the level of stress to which the animal is being subjected (19, 21).Using 0.3 µg of LPS/pig/day did not follow the physiological pattern of moderate or transient cortisol during collections, as the values were expected to decrease in comparison to the first challenge.Increased cortisol values in challenged animals can be explained by the effects of endotoxemia induced by E. coli LPS and the effect of stress