Intestinal mucosa morphology in broilers fed a glutamine+glutamic acid+phytogenic additive-supplemented diet

Introduction

Given the restriction in the use of performance-enhancing antibiotics (PEA) in animal nutrition, and the concern of consumers about food quality who demand healthier, drug-free foods, the search has increased for alternative, natural feed additives that, through specific mechanisms, can help to deal with the challenges causing enteric disorders in animals. Research has demonstrated glutamine and glutamic acid (Newsholme, 2003a; 2003b; Yi et al., 2005; Yoo et al., 1997) as well as essential oils and plant extracts (Christaki et al., 2004; Jamroz et al., 2005; Vasconcelos et al., 2010) have the ability of improving the immune response and the intestinal microflora, thus preventing negative effects on the intestinal structure and, therefore, improving nutrient absorption and animal performance at the end. Therefore, these supplements have become important elements for the production of broilers, as alternative additives during challenge periods.

Based on the above, the purpose of this study was to evaluate the effect of feed supplementation with glutamine and glutamic acid, in addition to phytogenic additives, alone or in combination, as an alternative to PEA in the intestinal mucosa morphology in broilers, in the starting phase.

Materials and Methods

Using a completely-at-random experimental design, with 6 treatments (T) and 3 repetitions, 450 one-day-old Cobb male broilers, were used. Chicks were vaccinated in the hatchery for Marek's disease, infectious bursal disease, and fowl pox. The birds were placed in cages arranged in lines, with front feeders and nipple drinkers. Treatments were: a control diet (CD); CD+cocci vaccine; CD+PEA; CD+glutamine+glutamic acid¹ (Gln/Glu); CD+phytogenic additives ² (PAs), and CD+Gln/Glu+PAs.

Nutrient requirements of birds were those recommended by Rostagno et al. (2005). Feed and water were given ad libitum and the lighting program was constant, with 24 hours of light per day throughout the experimental period. Birds in the Cd+vaccine treatment were vaccinated individually by the oral route of administration against coccidiosis (Livacox® - Biopharm).

For the morphological analysis, of the intestinal mucosa at 7 days of age, two birds per repetition were taken at random and killed by cervical dislocation in order to collect segments of the duodenum, jejunum, and ileum. Samples were fixed in 10% formalin for 24 hours. Samples were washed then transferred to 70% alcohol, dehydrated in increasing concentrations of alcohol, clarified in xylol and included in Paraplast. Using a microtome, 5µ-thick sections were obtained and stained with hematoxylin-eosin. Preparations were examined under the optic microscope coupled to an image analyzer and a computer in order to determine villi height and crypt depth, in each segment.

Results were tabulated then analyzed using the GLM system in the SPSS 13.0 for Windows software (2004). The means were compared using Tukey´s test at a 5% probability level.

¹1% AminoGut ®, from 1 to 21 days and 0.5% from 22 to 42 days.

²Imunostart^® (turmeric extract, citric extracts, and grape seed extracts ) at the inclusion level of 700 g/ton from 1 to 10 days of age; 500 g/ton from 11 to 21 days of age) + Enterocox^® (eucalyptus oil, China cinnamon essential oil, Chilean pemus leafs, trigonella seeds) at the inclusion level of 300 g/ton from 1 to 10 days of age, 1,000 g/ton from 11 to 35 days of age, and 500 g/ton from 36 to 42 days of age).

Results and Discussion

Influences of the treatments (P<0.05) were observed only in villi height and crypt depth in the duodenum and ileum (Table 1). The Gln+Glu treatment resulted in increased villi height in the duodenum, as compared to CD, CD+Vaccine and PAs, with no difference from the Gln+Glu+PAs or PEA treatments, which did not differ among themselves or with regard to the other treatments. The increased crypt depth was observed in the Gln+Glu and CD+vaccine treatments, as compared to CD, but no differences were found when compared to the other treatments, that did not differ from CD or among themselves. Increased ileal villi height was seen in the Gln+Glu treatment as compared to the CD, with no statistical difference with all other treatments, that did not differ among themselves or with the CD. Increased ileal crypt depth was seen with the Gln+Glu and Gln+Glu+PAs treatments as compared to all other treatments, which did not differ among themselves.

Table 1. Per-treatment villi height and crypt depth in the duodenum, the jejunum and the ileum of broilers at 7 days of age

Variables	Treatments
CD	CD+Vac.	PEA	Gln+Glu	PAs	Gln+Glu+ PAs	CV (%)
			Duodenum
Height, µm	738.79^b	763.30^b	829.54^ab	974.64^a	788.60^b	803.48^ab	7.95
Cripta, µm	108.84^b	127.98^a	116.86^ab	125.53^a	115.91^ab	124.47^ab	5.05
			Yeyuno
Height, µm	469.50	447.03	485.19	486.77	498.73	475.74	12.14
Crypt µm	110.56	91.50	92.60	110.30	105.85	112.02	17.13
			Ileum
Height µm	300.22^b	324.28^ab	316.55^ab	423.27^a	332.20^ab	397.75^ab	12.57
Crypt µm	73.51^b	74.28^b	70.09^b	115.99^a	73.53^b	106.66^a	13.04

^{a.bMeans followed by different letters in the same line are different as per Tukey´s test (P<0.05).}

Glutamine and glutamic acid, in addition to contributing energy for the metabolic activities of rapidly-proliferating cells, such as those in the intestine (Newsholme, 2003a; 2003b), are also considered precursors for the synthesis of purins and pyrimidines (basic nucleotide constituents, components of the DNA and RNA molecules) and other amino acids, thus supporting the division of crypt cells, leading to increased numbers and heights of villi and aiding the repair of lesions in the intestinal epithelium (Piva et al., 2001). In this study wee observed the actions of these amino acids in the duodenal and ileal segments, with significant increases in villi height and crypt depth. The results of this study are consistent with those obtained by other authors who also found increased villi heights in glutamine-supplemented diets (Bartell and Batal, 2007). The phytogenic additives have been studied as efficacious supplements to reduce bacterial and coccidial (Eimeria spp.) (Jamroz et al., 2005; Vasconcelos et al., 2010). Nevertheless, given that birds were not challenged in this phase of production, we were not able to notice the potential benefits of these additives on the intestinal cell structures. Regarding the increased crypt depth in the duodenum with the CD+Vaccine treatment, this could be due to the cocci vaccine that stimulated the proliferation of crypt cells in an attempt to repair potential villus damages.

Conclusion

Glutamine and glutamic acid associated or not to phytogenic additives can cause improvements in the intestinal structure, and therefore in its function, mainly in regions where the use of performance enhancing antibiotics have been banned.

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