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Intestinal mucosa structure of broiler chickens infected experimentally with Eimeria tenella and treated with essential oil of oregano/Morfometria intestinal de frangos de corte infectados experimentalmente com Eimeria tenella e tratados com oleo essencial de oregano.

INTRODUCTION

Restrictions on the use of animal growth promoter during the last few years have encouraged the use of essential oils because of their metabolic properties. Some properties have been attributed to this plant, such as its useful antimicrobial effect (ELGAYYAR et al., 2001) related to carvacrol and thymol compounds that are primary components of oregano essential oil (KOKKINI et al., 1997). GIANNENAS et al. (2003) observed that after the infection with Eimeria tenella the supplementation with dietary oregano oil resulted in body weight gain and feed conversion ratio not differing from the non-infected group but lower than those of the lasoalocid group. The purpose of these studies was to evaluate possible harmful effect of oregano essential oil on intestinal mucosa structure of broiler chickens after experimental infection with E. tenella.

MATERIAL AND METHODS

Experimental design

Two studies were conducted using in the first trial a total of 250 day-old male Cobb chicks. The broilers were housed indoors on twenty five floor pens and distributed in a completely randomized design divided into five treatment groups with five replicates each (ten birds/box) and given the following diets: a diet with 10mg [kg.sup.-1] avilamycin plus 66mg [kg.sup.-1] salinomycin (positive control); a diet without added avilamycin and salinomycin (negative control); diet with added avilamycin at 10mg/kg, only; a diet supplemented with 0.5g of oregano oil kg [diet.sup.-1]; a diet supplemented with 1.0g of oregano oil kg [diet.sup.-1]. In the second trial a total of 288 day-old male Cobb chicks were used . The broilers were housed indoors on twenty four floor pens and distributed in a completely randomized design divided into four treatment groups with six replicates each (twelve birds/box) and given the following diets: a diet with anticoccidial agent; a diet without anticoccidial agent; a diet supplemented with 0.5g of oregano oil kg [diet.sup.-1]; a diet supplemented with 1.0g of oregano oil kg [diet.sup.-1]. In starter phase (from 0 to 21 days of age), the anticoccidial agent was nicarbazin at 125g tonne-1 and in the grower (from 21 to 40 days of age) salinomycin at 66g tonne-1 was used.

Diets

In all of experiments, the feeding program consisted of a starter diet until 21d and a finisher diet until 42d. The birds were fed with a start diet containing 22% CP and 2900kcal ME [kg.sup.-1], and growing diet with 19% CP and 3000kcal ME [kg.sup.-1]. The basal start diet had 1.5% of soybean oil and the growing diet had 2% of soybean oil. Oregano oil supplementation was obtained by isometrically replacing soybean oil in the basal diet. The oregano essential oil was obtained by hydrodistillation in a modified Clevenger-type apparatus, and their analyses were performed by gaschromatograph with flame ionization detector (GC/FID). Compositions are then expressed as percent of normalized peak areas according to RODRIGUES (2002) and are presented in table 1.

Data collection

Parasitological analysis

In the first trial the number of oocysts per gram litter was determined at 16 and 28 days of age using the procedure described by CONWAY AND MCKENZIE (1991). The litter sample was collected and pooled for the chickens within each pen totalizing five repetitions per treatment. In the second trial each broiler chicken was experimentally infected with Eimeria tenella at 20 days of age by oral inoculation. The oocysts were preserved in 2% potassium dichromate solution to induce sporulation and kept in a refrigerator (2-5 [degrees]C) until use. Each bird was challenged with 5x104 oocysts/chicken of E. tenella. Prior to infection, at 19 days of age, the number of oocysts per gram litter was determined. Excreted oocysts were investigated from 7 and 14 days after infection with E. tenella. Therefore, at 19, 27 and 34 days of age, litter samples were collected from each pen. Pens were used as the experimental units. The litter sample was collected and pooled for the chickens within each pen totalizing six repetitions per treatment.

Intestinal morphometry

In trial 1, jejunum and ceca fragments of four-centimeter length were collected from five individuals/ treatment that were preserved in Bowin solution for morphometric evaluation. The fragments were submitted from the processing inclusion in paraffin, according to the histopathological routine techniques. Paraffin sections of 7[micro]m thick were stained with haematoxylin and eosin. The intestinal villous height was measured from the luminal epithelium to the muscularis mucosa and the crypt depht, from the luminal epithelium of the crypt to the muscularis mucosa in jejunum. In the ceca, the thickness of lamina propria was measured from the basement membrane of the luminal epithelium to the muscularis mucosa (SUN et al., 2005). In the morphometric analysis of the jejunum and ceca a magnification 100x was used. Five villous height and crypt depth in jejunum and cecal lamina propria measurements were taken from each section. The average of the five measurements was treated as an experimental unit. Pictures of villus height, crypt depth, and cecal lamina propria were obtained with a camera with measurements made using the software of SigmaScan Pro 5.

In the second trial, intestinal morphology was determined at 144 hours post-infection with Eimeria tenella and at 42 days of age. Four-centimeter segment of ceca were collected from six individuals/ treatments and were preserved in Bowin solution for morphometric examination. The material collected were submitted from the processing inclusion in paraffin, according to the histopathological routine techniques. Paraffin sections of 7[micro]m thick and was stained with haematoxylin and eosin. Thickness of cecal lamina propria was measured, from the basement membrane of the luminal epithelium to the muscularis mucosa (SUN et al., 2005). The morphometric analysis of the ceca was determined in the same condition of the first trial.

Statistical analysis

The experimental data was submitted to an analysis of variance using the System for Statistical and Genetic Analyses, developed by UFV (1997). Significant differences among averages were determined by SNK test at P<0.05 throughout these studies. Coccidia was exponentially multiplied , not linearly, so pen oocyst counts were log 10 transformed prior to analysis.

RESULTS AND DISCUSSION

The extraction yield of oregano essential oil was determined to be 1.20wt% [+ or -] 0.18wt%, achieved after about 3h of extraction. The chromatogram of oregano essential oil shows that terpinen-4-ol, [gamma]-terpinene and thymol are the major components, followed by [alpha]-terpinene, p-cymene and [alpha]-terpineol, which means a chemical profile very similar to that found by RODRIGUES (2002).

Both villous height and crypt dept are important indicators of broilers digestive health and directly related to the absorptive capacity of mucous membrane (BUDDLE & BOLTON, 1992). From a theoretical point of view, villous height reflects a balance between the mitotic activity of the crypt enteric cells (CERA et al., 1988) and the desquamation produced principally by external aggressors (NABUURS, 1995). In the current study of Trial 1, it was verified significantly treatment effects on crypt dept and villous:crypt ratio in jejunum (P<0.05, Table 2). The nonmedicated group had the highest duodenal crypt depth which differs from chickens fed with growth promoter antibiotic or with 0.5 and 1.0g of oregano oil kg [diet.sup.-1]. Although the antimicrobial and antioxidant properties of plant oils are well known and confirmed in numerous studies (MANZANILLA et al., 2004) there is only slight evidence on morphological and histological investigations referring to active plants oils action in animals fed on diets supplemented with plant extracts. In the present investigation, in which oregano oil was introduced into chicken diets, some morphological changes in gastrointestinal tract walls were registered. GIANNENAS et al. (2003) observed that the oregano oil in broilers diet infected with Eimeria tenella resulted in body weight gains and feed conversion ratios not differed from the non-infected group, but was higher than those of the infected control group and these parameters correspond with the lesion extent score and oocyst numbers and indicates that oregano essential oil exerted an anticoccidial effect against E. tenella.

The villous:crypt ratio is an indicator of the likely digestive capacity of the small intestine. The broilers fed with antibiotic plus anticoccidial agent in the diet had the highest ratio of villous height to crypt depth while that nonmedicated group had the lowest villous:crypt ratio (P<0.05). An increase in this ratio corresponds to an increase in digestion and absorption (MONTAGNE et al., 2003). On the other hand, a decrease in villus:crypt ratio is indicative of a higher rate of enterocyte-cell migration from the crypt to the villous. It has suggested that reduced microbial activity in digesta or microbial activity at the level of the brush border would reduce both damage to enterocytes and the need for cell renewal in the gut (HUGHES, 2003).

In Trial 1 no significant dietary effects were observed on oocyst excretions at 16 days of age (P>0.05) however, at 28 days of age it was observed that the nonmedicated group had the highest oocyst excretion in litter (P<0.05). The opposite was observed for broilers fed with anticoccidial agent or basal diet supplemented with oregano oil (P<0.05, Figure 1).

In Trial 2, after challenge with E. tenella and treated with the oregano essential oil or ionophorous antibiotic, the excreted oocysts of feces were investigated and the results are shown in a figure 2. At 19 days of age, it was observed that broilers fed with basal diet without anticoccidial agent had the highest oocyst excretion in litter (P<0.05). The opposite was observed for broilers fed with basal diet with anticoccidial agent which did not differ from chickens fed with 1.0g of oregano oil/kg diet (P<0.05). At 34 days of age, the broilers fed with basal diet supplemented oregano oil had the lowest oocyst excretion which did not differ from chickens fed with anticoccidial agent in the diet (P<0.05).

Morphological evaluation of cecum wall from broilers orally challenged with Eimeria tenella was observed at 144 hours after infection and broilers fed basal without anticoccidial agent had the highest cecal lamina propria thickeness which differ from chickens fed with anticoccidial agent in diet or supplemented with 1.0 of oregano oil kg [diet.sup.-1] (P<0.05, Table 3). At 42 days of age, the absence of anticoccidial agent in diet resulted in the highest cecal lamina propria thickeness which differ from chickens fed diet supplemented with 1.0 of oregano oil kg [diet.sup.-1] (P<0.05, Table 3). The result of a reduction in cecal lamina propria thickness in birds fed with oregano oil in the diet provides an indirect indicator of reduced pathogen infection in the ceca. It is suspected that addition of antibiotics or essential oils may be efficient at reducing the pathogen load. GREATHEAD & KAMEL (2006) observed that the addition of thymol:carvacrol (1:1) in diet from broilers infected with E. acervulina resulted in improved intestinal integrity probably by reducing the impact of coccidiosis on intestinal integrity.

CONCLUSIONS

The results of these studies indicated that oregano essential oil exerted an anticoccidial effect were similar to the ionophorous antibiotic verified through the intestinal morphometric and excretion of oocysts.

COMMITTEE ON BIOETHICS AND SAFETY

The protocol of animal experimentation is in accordance to CONCEA and was approved by the Ethics Committee in Use of Animals of Espirito Santo Federal Univesity from Protocol No. 01/08.

ACKNOWLEDGMENTS

The authors are grateful to the Banco Nordeste (BNB) e Fundacao de Apoio a Ciencia e a Tecnologia do Espirito Santo (FAPES) for financial support.

REFERENCES

BUDDLE, J.R., BOLTON, J.R. The pathophysiology of diarrhoea in pigs. Pigs News Information, v.13, p.41N-45N, 1992.

CERA, K.R. Effect of age, weaning and postweaning diet on small intestinal growth and jejunal morphology in young swine. Journal Animal Science, v.66, p.574-584, 1988.

CONWAY, D.P., MCKENZIE, M.E. Poultry Coccidiosis diagnostic and testing procedures. 2.ed. New York: Pfizer, 1991. 168p.

ELGAYYAR, M. et al. Antimicrobial activity of essential oils from plants against selected pathogenic and saprophytic microorganisms. Journal of Food Protection, v.64, p.10191024, 2001.

GIANNENAS, I. et al. Effect of dietary supplementation with oregano essential oil on performance of broilers after experimental infection with Eimeria tenella. Archives of Animal Nutrition, v.57, p.99-106, 2003. Disponivel em: < http://www.informaworld.com/smpp/content~db=all?content=10.1080/ 0003942031000107299>. Doi: 10.1080/0003942031000107299.

GREATHEAD, H., KAMEL, C. Encapsulated plant extracts to fight coccidiosis. Feed Mix, v.14, p.18-21, 2006.

HUGHES, R.J. Energy metabolism of chickens physiological limitations. A report for the Rural Industries Research and Development Corporation, RIRDC Publication, n.2, p.151, 2003.

KOKKINI, S. et al. Phytochemistry, v.44, n.5, p.883-886, 1997

MANZANILLA, E.G. et al. Effect of plant extracts and formic acid on the intestinal equilibrium of early-weaned pigs. Journal of Animal Science, v.82, p.3210-3218, 2004.

MONTAGNE, L. et al. A review of interactions between dietary fibre and the intestinal mucosa, and their consequences on digestive health in young non-ruminant animals. Animal Feed Science and Technology, v.108, p.95-117, 2003. Disponivel em: <http://www.sciencedirect.com/ science?_ob=ArticleURL&_udi=B6T42- 48WB6W82&_user=687358&_rdoc=1&_fmt=&_orig=search&_sort=d& view=c&_acct=C000037899&_version=1&_urlVersion=0&_userid=687358& md5=a10356ab60f5c612fca6f5ebf2706c92>. Doi: 10.1016/S0377-8401(03)00163-9.

NABUURS, M.J.A. Microbiological, structural and functional changes of the small intestine of pigs at weaning. Pig News Information, v.16, p.93N-97N, 1995.

RODRIGUES, M.R.A. Estudos dos oleos essenciais presentes em manjerona e oregano. 2002. 148f. Tese (Doutorado em Quimica)--Instituto de Quimica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS.

SUN, X et al. Broiler performance and intestinal alterations when fed drug-free diets. Poultry Science, v.84, p.12941302, 2005.

Universidade Federal de Vicosa--UFV. Manual de utilizacao do programa SAEG (Sistema para Analise Estatistica e Geneticas). Vicosa: UFV, 1997. 150p.

Maria Aparecida da Silva (I) Bruna Mirelly de Sousa Pessotti (II) Surama Freitas Zanini (*I) Geraldo Luiz Colnago (III) Maria Regina Alves Rodrigues (IV) Louisiane de Carvalho Nunes (I) Marcos Santos Zanini (I) Isabella Vilhena Freire Martins (I)

(I) Departamento de Medicina Veterinaria, Centro de Ciencias Agrarias, Universidade Federal do Espirito Santo (UFES), CP 16, 29500-000, Alegre, ES, Brasil. E-mail: surama@cca.ufes.br. * Autor para correspondencia. (II) Programa de Pos-graduacao em Biotecnologia, UFES, Alegre, ES, Brasil. (III) Departamento de Zootecnia, Universidade Federal Fluminense(UFF), Niteroi, RJ, Brasil. (IV) Departamento de Quimica Organica, Universidade Federal de Pelotas (UFPel), Pelotas, RS, Brasil. Received 08.28.08 Approved 04.01.09
Table 1--Components of the Origanum vulgare essential oil obtained
by comparison with terpenes standards and yield (%).

Peak   Retention time   Compounds                    PM

1      10,05            [alpha]-tujeno               136
2      10,35            [alpha]-pineno **            136
3      12,56            Sabineno                     136
4      12,67            [beta]-pineno **             136
5      13,74            mirceno **                   136
6      14,40            [alpha]-felandreno           136
7      15,18            [alpha]-terpineno **         136
8      15,67            p-cimeno **                  134
9      15,93            limoneno **                  136
10     16,06            1,8-cineol **                154
11     16,74            cis/trans [beta]-ocimeno     136
12     17,97            [gamma]-terpineno **         136
13     18,47            trans sabineno hidratado     154
14     19,95            terpinoleno **               136
15     20,56            cis sabineno hidratado       154
16     20,87            linalol **                   154
17     22,16            Trans-p-mentenol             154
18     23,11            Cis-p-mentenol               154
19     25,19            Borneol                      154
20     26,07            4-terpineol **               154
21     27,03            [alpha]-terpineol **         154
22     27,43            trans-piperitol              154
23     29,31            cis-piperitol                154
24     30,26            eter do metil timol          164
25     30,89            eter do metil carvacrol      164
26     31,30            acetato de linalila          196
27     31,88            geraniol/ nerol              154
28     34,41            timol **                     150
29     34,99            carvacrol **                 150
30     40,55            acetato de geranila/nerila   196
31     42,53            [beta]-cariofilleno          204
32     48,43            germacreno                   204
33     52,36            espatulenol                  220
34     52,66            oxido de cariofileno         220

Peak   FM                                 * C (%)

1      [C.sub.10] [H.sub.16]              2,15
2      [C.sub.10] [H.sub.16]              0,51
3      [C.sub.10] [H.sub.16]              5,47
4      [C.sub.10] [H.sub.16]              0,27
5      [C.sub.10] [H.sub.16]              1,71
6      [C.sub.10] [H.sub.16]              2,27
7      [C.sub.10] [H.sub.16]              7,00
8      [C.sub.10] [H.sub.14]              20,38
9      [C.sub.10] [H.sub.16]              6,22
10     [C.sub.10] [H.sub.18] O            0,09
11     [C.sub.10] [H.sub.16]              0,62
12     [C.sub.10] [H.sub.16]              8,31
13     [C.sub.10] [H.sub.18] O            0,97
14     [C.sub.10] [H.sub.16]              3,41
15     [C.sub.10] [H.sub.18] O            2,36
16     [C.sub.10] [H.sub.18] O            1,40
17     [C.sub.10] [H.sub.18] O            0,28
18     [C.sub.10] [H.sub.18] O            0,09
19     [C.sub.10] [H.sub.18] O            0,23
20     [C.sub.10] [H.sub.18] O            11,92
21     [C.sub.10] [H.sub.18] O            2,78
22     [C.sub.10] [H.sub.18] O            0,10
23     [C.sub.10] [H.sub.18] O            0,06
24     [C.sub.11] [H.sub.16] O            1,41
25     [C.sub.11] [H.sub.16] O            2,60
26     [C.sub.12] [H.sub.20] [O.sub.2]    0,21
27     [C.sub.10] [H.sub.18] O            5,30
28     [C.sub.10] [H.sub.14] O            7,88
29     [C.sub.10] [H.sub.14] O            0,50
30     [C.sub.12] [H.sub.20] [O.sub.2]    0,08
31     [C.sub.15] [H.sub.24]              1,12
32     [C.sub.15] [H.sub.24]              0,05
33     [C.sub.15] [H.sub.24] O            1,25
34     [C.sub.15] [H.sub.24] O            0,98

(a) Expressed as percentage of the total peak area of the
chromatograms.

Table 2--Effects on intestinal integrity from feeding oregano oil to
broilers chickens at days of age in Trial 1.

Diet                    Villous height   Crypt depth   Villous:crypt

Positive control        1219.47          215.86 (B)    5.94 (A)
  (with antibiotic
  and anticoccidial)
Negative control        1173.83          343.80 (A)    3.56 (B)
  (without antibiotic
  and anticoccidial)
Only antibiotic         1282.07          245.31 (AB)   5.28 (AB)

0.5g of oregano         1013.72          227.42 (B)    4.52 (AB)
  oil [kg.sup.-1]
1.0g of oregano oil/    994.92           241.92 (B)    4.22 (AB)
  [kg.sup.-1]

(A, B) Averages values within the same column with no common
superscript differ significantly by the SNK test (P<0.05).

Table 3--Effect on segment of ceca from feeding oregano oil to
broilers chickens infected with Eimeria tenella oocysts in Trial 2.

                            Diameter of cecal lamina propria, microns

Diet                        144 hours        42 days of
                            after infection  age

Positive control (with      302.92 (B)       853.25 (AB)
  anticoccidial agent)
Negative control (without   840.18 (A)       967.51 (A)
  anticoccidial agent)
0.5g of oregano oil         516.01 (AB)      486.92 (B)
  [kg.sup.-1]
1.0g of oregano oil         350.14 (B)       469.00 (B)
  [kg.sup.-1]

(A, B) Averages values within the same column with no
common superscript differ.

Figure 1--Oocyst excretions of broilers chickens in Trial 1.

                                 Without
                 Avilamycin      Avilamycin
                 + salinomycin   or salinomycin   Avilamycin

16 days of age   0.70            0.73             0.80
28 days of age   0.75 B          1.54 B           0.67

                 0.5 g of oregano oil   1 g of oregano oil

16 days of age   0.67                             0.65
28 days of age   0.72 B                           0.78 B

Note: Table cree d'un histogramme.

Figure 2--Oocyst excretions of broilers chickens non-infected
or infected experimentally by Eimeria tenella in Trial 2.

                          Without          With anticoccidial
                          Anticoccidial    agent
                          agent

Prior to infection        2.09 A           0.90 C
7 days after infection    2.14             2.01
14 days after infection   1.32 A           1.07 AB

                          0.5 g of         1 g of oregano
                          oregano oil/kg   oil/kg

Prior to infection        1.57 AB          1.31 BC
7 days after infection    1.95             1.71
14 days after infection   0.74 B           0.78 B

Note: Table cree d'un histogramme.
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Title Annotation:Texto en Portuguese
Author:da Silva, Maria Aparecida; Pessotti, Bruna Mirelly de Sousa; Zanini, Surama Freitas; Colnago, Gerald
Publication:Ciencia Rural
Date:Aug 1, 2009
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