Effect of Broiler Breeder Age and Glutamine Supplementation on the Development of the Intestinal Mucosa of 7-Day-Old Chicks

The aim of this study was to evaluate the possible effects of glutamine and broiler breeder age on the development of intestinal mucosa in broiler chicks during first week of age. For this purpose, 32 one-day-old broiler chicks were distributed according to a completely randomized experimental design in a 2 x 2 factorial arrangement. Treatments consisted of two broiler breeder ages (30 and 60 weeks) and two dietary glutamine supplementation levels (0% and 1%). The morphological development of intestinal mucosa, particularly villus height and crypt depth, was evaluated. The results showed that the supplementation with 1% of glutamine influenced the development of villus height in the duodenum (p=0.009), jejunum (p = 0.006), and ileum (p = 0.001), as well as crypt depth in the jejunum (p = 0.037) of 7-day-old broilers. These results suggest that the presence of glutamine influenced the development of intestinal mucosa during the first week of age, when these tissues are highly influenced by dietary components, especially by trophic agents. The results show that broiler breeder age (30 or 60 weeks) did not influence the evaluated parameters.


INTRODUCTION
Literature studies have demonstrated the importance of maintaining the health of the intestinal mucosa of poultry to achieve good productivity.This is especially true for broilers, which have a short lifespan.Very good intestinal health status is required because intestinal epithelium cells are responsible for nutrient absorption and for defense against pathogens (Maiorka et al., 2000).
At hatching, despite anatomically complete, the gastrointestinal tract (GIT) is still physiologically immature (Maiorka et al., 2000;Murakami et al., 2007), resulting in inefficient dietary nutrient utilization, and consequently, preventing chicks to achieve their genetic growth potential.
Some compounds are supplemented in pre-starter and starter broiler diets with the aim to stimulate the development of the intestinal mucosa.One of these substances is glutamine, which is a trophic agent.It supplies optimal enterocyte nutrition and it is known for maintaining enterocyte development rate and, in some cases, for stimulating the proliferation of enterocytes after damages of the intestinal mucosa (Rhoads et al., 1997).Well-developed and functional enterocytes are required for proper dietary nutrient absorption.
It was reported that glutamine increases gene transcription by increasing the mitogenic activity of the enzyme protein kinase (Blikslarger & Roberts, 1997), leading to more effective synthesis or turnover of the involved tissues.However, the mechanism by which glutamine stimulates the proliferation of enterocytes is not fully elucidated.Two

Effect of Broiler Breeder Age and Glutamine Supplementation on the Development of the Intestinal Mucosa of 7-Day-Old Chicks
mechanisms have been proposed: glutamine may increase Na + /H + exchange on the cell membrane, as well as enhance the specific activity of the enzyme ornithine-decarboxylase, essential for cell proliferation (Rhoads et al., 1997Moreover, glutamine is a precursor of the synthesis of amino acids, nucleotides, and nucleic acids (Souba, 1993;Murakami et al., 2007).However, endogenous glutamine production is not sufficient to supply the body requirements (Lobley et al., 2001).Therefore, the supply of an exogenous glutamine source may be useful to promote the development of the GIT, particularly of the intestinal mucosa.
There is positive correlation between broiler breeder age and egg size and, consequently, between broiler breeder age and chick size.Chicks from young broiler breeders are lighter when compared with chicks of the same age from older broiler breeders (Dalanezi et al., 2005).This difference may be maintained during the entire broiler life.The study of Wilson (1991), for instance, showed that a difference of one gram in egg weight results in 2-13 gram differences in the body weight of 6-wk-old broilers.Eggs laid by young broiler breeders present lower yolk and albumen contents, and therefore lower nutrient levels available to the developing embryo (Noy & Pinchasov, 1993).
Therefore, the objective of this experiment was to evaluate the effects of the dietary supplementation of glutamine during the first week after hatching on the development of the intestinal mucosa (villus height, crypt depth, number of the villi/segment, surface area of the tip of the enterocytes and length of the small intestine) of broiler chicks derived from breeders of different ages (30 and 60 weeks old).

MATERIAL AND METHODS
A total of 32 one-day-old male Cobb-500 TM chicks (16 hatched from 30-wk-old breeders and 16 hatched from 60-wk-old breeders) were placed immediately after hatching in battery cages located an environmentally controlled chamber at thermoneutral temperature (33 o C).Chicks were distributed according to a completely randomized experimental design in a 2 x 2 factorial arrangement, consisting of two broiler breeder ages (30 and 60 weeks) and two dietary glutamine supplementation levels (0% and 1%), with four treatments of eight birds each.The following treatments were applied: -T1: chicks from 30-wk-old breeders and fed a diet supplemented with 1% glutamine; -T2: chicks from 60-wk-old breeders and fed a diet supplemented with 1% glutamine, -T3: chicks from 30-wk-old breeders and fed a diet not supplemented with glutamine; -T4: chicks from 60-wk-old breeders and fed a diet not supplemented with glutamine.
The diets were based on corn and soybean meal.Diet composition is shown in Table 1.Glutamine was supplemented at 1% of the diet as L-glutamine.Chicks are fed ad libitum during the entire experimental period (7 days).
At the end of the experimental period or seven days after hatching, eight birds per treatment were sacrificed by cervical dislocation.The small intestine was removed for morphological evaluation and its length measured (cm ± 0.01).Tissue samples (approximately 2 cm) were collected from each segment of the small intestine: duodenum -from the pylorus to the distal duodenal loop; jejunum -from the distal duodenal loop to Meckel's diverticulum, and ileum -between Meckel's diverticulum and the opening of the ceca.Samples were evaluated by light microscopy, scanning electron microscopy, and transmission electron microscopy.

Light microscopy
Tissue samples were fixed in Bouin's solution, dehydrated in standard alcohol-toluene series (50, 60, 70, 80, 90, and 100% for 15 minutes each), and embedded in paraffin.Five-micrometer slices were prepared and stained with hematoxylin-eosin.Villus height (mm) and crypt depth (mm) were measured in 60 random microscopic fields in each segment using an image analysis system (Video Plan, Carl Zeiss, Germany).

Sample preparation for electron microscopy
The intestinal content was removed with saline solution buffered with 0.1 M phosphate (pH 7.4), and tissue samples were fixed in 2% glutaraldehyde in phosphate buffer for 24 h at 4 o C. Subsequently, samples were washed in phosphate buffer and postfixed for two hours in 1% osmium tetroxide.The material was washed again with the same buffer solution and dehydrated in increasing ethanol series (50, 60, 70, 80, 90, and 100% for 15 minutes each).

Scanning electron microscopy
Samples were dried in a critical point drier with liquid carbon dioxide.The material was then placed in an appropriate specimen tray, covered with a layer of gold (30 nm), and observed under a scanning electron microscope (model JSM 25SII â , Jeol Ltd, Japan).The average number of villi/segment was obtained by counting the number of villi in six areas measuring 103,269 mm 2 each.

Transmission electron microscopy
Segments of approximately 1 mm 2 were infiltrated with 1:1 ethanol-Epon 812 resin at room temperature for 2 hours and embedded in Epon 812 resin at 60 º C for 72 hours.The samples were then sectioned, contrasted with lead citrate and uranyl acetate and electron-microphotographed by transmission electron microscopy.
Longitudinal sections of the cells were evaluated using an image analyzer (Jeol 1010 ® , Japan) in order to measure enterocyte diameter (tip, mm), microvillus height and width (mm), and microvillus density (microvillus number/1m 2 m).The average of each measurement corresponds to the measurements of 10 samples per bird.
The surface area of the tip of the enterocytes was calculated based on the tip diameter (C d ) of the cell and the extension factor of the microvillus (EFM), according (Ferrer et al., 1995)

Statistical Analysis
Data were analyzed according to a completely randomized experimental design adopted in a 2 x 2 factorial arrangement (broiler breeder age x glutamine supplementation) with eight replicates per treatment, using the General Linear Model procedure of SAS 8.0 software (2000).et al. (1999) reported the influence of breeder age on the intestinal development of their progeny in turkeys.Turkey poults from older breeders presented higher villi than those from younger breeder after hatch, but this difference was not maintained when poults were one week old.Accordingly, in the present study, no differences in the morphology of the intestinal mucosa of 7-d-old broilers derived from broiler breeders of different ages were detected.This suggests that the diet supplied immediately after hatch may minimize the influence of broiler breeder on the initial development of the intestinal mucosa.Applegate et al. (1999) demonstrated that animal metabolism has the ability to shown a rapid response when stimulated.

Applegate
Table 2 shows the results of the supplementation of diets with 1% glutamine on the morphology of the intestinal mucosa of 7-d-old broiler chicks.The chicks fed diets with 1% glutamine presented taller longer villi in the duodenum (p =0.009), jejunum (p =0.006), and ileum (p =0.001) compared with those fed the nonsupplemented diet.In addition, glutamine-fed chicks presented deeper crypts in the jejunum (p =0.037), while no statistical differences were detected in the duodenum and the ileum.Table 2 also shows that broiler breeder age (30 or 60 weeks) did not influence the evaluated parameters.
Therefore, the addition of glutamine to the diet influenced villus height in the duodenum, jejunum and ileum, but not crypt depth.This suggests that the glutamine, despite being a trophic agent, did not affect the number of villi, because these originate from the bottom of the crypt.
Glutamine is known as a trophic agent that stimulates the development and proliferation of developing and regenerating tissues.Because the , using the equation: S=p x C d 2/4 x EFM, where EFM = p x H x d x D + 1 (L = microvillus height; d = microvillus width and D = microvillus number/um 2 ).

Table 1 -
Composition of experimental diets.