Subscribe Now Subscribe Today
Research Article
 

Dietary Betaine Affect Duodenal Histology of Broilers Challenged with a Mixed Coccidial Infection



H. Hamidi, J. Pourreza and H. Rahimi
 
Facebook Twitter Digg Reddit Linkedin StumbleUpon E-mail
ABSTRACT

The purpose of this research was to investigate effect of dietary betaine on intestinal morphology after an experimental coccidiosis. Hence a total of 189 male and female broiler chicks were randomly assigned to 9 floor cages. Chicks were fed a basal diet supplemented with 0, 0.6 or 1.2 g kg-1 betaine. All birds were inoculated orally with Eimeria oocysts on day 28. Duodenal morphology parameters and lesions were scored by microscopic observation on intestine samples which were taken at day 42 of age. Adding 1.2 g kg-1 betaine to diet diminished intestinal lesions (p<0.05). Dietary supplementation with 0.6 or 1.2 g kg-1 betaine significantly (p<0.01) increased intraepithelial lymphocytes as well. Level of additive betaine had no effect on the ratio of villus height/crypt depth or villus surface area. Lamina propria of duodenum became thicker in the intestine of chickens which received more supplemental betaine via their diet. In conclusion, since the number of intraepithelial lymphocytes and thickness of lamina propria represent the condition of gut immune response, it seems that dietary betaine may immunomodulate the gastrointestinal tract of broilers. In addition, betaine effect on villus morphology measured later in life differed from what had been measured already earlier in life of the chicks.

Services
Related Articles in ASCI
Similar Articles in this Journal
Search in Google Scholar
View Citation
Report Citation

 
  How to cite this article:

H. Hamidi, J. Pourreza and H. Rahimi, 2009. Dietary Betaine Affect Duodenal Histology of Broilers Challenged with a Mixed Coccidial Infection. Pakistan Journal of Biological Sciences, 12: 291-295.

DOI: 10.3923/pjbs.2009.291.295

URL: https://scialert.net/abstract/?doi=pjbs.2009.291.295

INTRODUCTION

The intestinal villus and crypt morphology in chickens have been associated with intestine function and chicken growth. Research on villus morphology (Tarachai and Yamauchi, 2000) illustrated that villus morphology is governed by enteral nutrient absorption.

Coccidiosis, caused by the microparasites of the genus Eimeria, is the prevalent pathogens of chickens and an economically important intestinal infection of poultry. The lesions induced by pathogen are visible both macroscopically and microscopically. Furthermore, coccidian infection associated with metabolic and structural changes in the intestinal mucosa of the host animal (Waldenstedt et al., 1999a). Dietary betaine has shown that could stabilize the intestinal epithelial structure in healthy and coccidia-infedted birds (Kettunen et al., 2001). It is possible that the action of betaine is related to its influence on antibody production or phagocytes because these cells are important in protection against coccidia (Yun et al., 2000). On the other hand, the influence of betaine as an osmolyte can assist to improve work and stability of infected intestinal mucous (Allen et al., 1998; Allen and Fetterer, 2002).

Klasing et al. (2002) reported that the number of intraepithelial lymphocyte and thickness of lamina propria were increased by supplemental betine. Kettunen et al. (2001) also observed that the crypt/villus ratio was decreased by the dietary betaine supplementation in healthy and coccidia-infected chicks. Moreover, have been reported inconsistent results of betaine effect on intestinal lesions.

It is probable that some of the morphological parameters of intestine be affected by age, so that rapid growth rate and morphological changes of gastrointestinal track during several early weeks of age (Nitsan et al., 1991) interfere with morphological measurements. Even it has been demonstrated (Iji et al., 2001) that crypt depth and villus height had interaction with age of chickens especially in early weeks. Moreover, practically infection of broilers with coccidiosis predominantly occurs after the 4th (Biffa et al., 2003) or even 6th (Malcolm, 1978) week of age, when the infection has maximum economical and physiological harms, because of short time to redress. In addition Malcolm (1978) described that the appearance of new generation of Eimeria oocystes is relatively late in the life cycle, usually a week after ingestion of sporulated oocystes.

However, almost all of previous studies have examined morphological effect of betaine in coccidiosis condition during 14 to 21 day period. Thus in this research morphological effect of betaine at day 42 was examined on the intestine of chickens which challenged by coccidian-infection at day 28 of age to compare intraepithelial lymphocytes and thickness of lamina propria as indicators of gut immune response as well as intestinal lesions and villus dimension with earlier studies.

MATERIALS AND METHODS

Diet, birds and experimental design: The presented study was performed in the experimental farm of Isfahan University of Technology, Isfahan, Iran. A total of 189 male and female seven day-old broiler chicks (Ross 308) were randomly assigned to 9 floor cages. Chicks were fed a basal diet supplemented with 0, 0.6 or 1.2 g kg-1 betaine. A complete random design was used with 3 replicates during the 7 to 42 day period. The diets were formulated to meet National Research Council (1994) nutrient requirements (Table 1). The chickens were fed starter diet ad libitum until day 21 and grower diet offered from 21 to day 42. Diets contained no coccidiostats.

Experimental infection and tissue sampling: At day 28 of age, all birds were inoculated orally with a mixed culture of Eimeria tenella and Eimeria acervulina to simulate a coccidiosis challenge by 8.5x103 and 7.5x103 sporulated oocysts per chicken respectively. On day 42 the duodenum sections were taken from ~2 cm to the duodenal loop at the apex of the pancreas. Segments of ~1.5 cm in length were flushed with saline and fixed in 100 g L-1 buffered formalin (pH 7.0). The fixed intestinal samples embedded in paraffin wax then were sectioned (5 μm) and stained with hematoxylin-eosin finally were examined by light microscope for the following: lesions score, number of intraepithelial lymphocytes, villus height/crypt depth (V/C) ratio (villus height; from the top of the villus to the top of the lamina propria and crypt depth; from the base upward to the region of transition between the crypt and villus), villus surface area and lamina propria thickness (space between the base of the villus and the top of the muscularis mucosa) (Aptekmann et al., 2001). The tissue morphology and lesions was graded (Zentek et al., 2002) by one person without the knowledge of the origin of the sample. Comparative scores were from 1 to 4 including mediate numbers (1, 1.5, 2, 2.5,…, 4). Data were collected on 10 different villi per chicken on sections of both two chickens per pen.

Table 1: Ingredients and calculated nutrient content of the basal starter and grower diets
A: Vitamin premix provides the following per kilogram: vitamin A 8800 IU; cholecalciferol 3300 IU; vitamin E 16.53 IU; vitamin B 0.023 mg; riboflavin 8 mg; niacin 33 mg; pantothenic acid 35 mg; menadione 1.5 mg; folic acid 0.8 mg; thiamin 3 mg; pyridoxine 2.7 mg; biotin 0.25 mg; ethoxyquin 125 mg; B: Mineral premix provides the following in milligrams per kilogram: Mn, 55; Zn, 50; Fe, 50; Cu, 5; Se, 0.1; I, 1.5, C: Variable amounts of betaine and washed builders sand, D: TSAA: Total sulfur amino acids

Statistical analysis: Data were analyzed using the GLM procedures of SAS Institute (SAS, 1999). Comparisons among the means were made using Dancan’s multiple range test procedure. The means differences were considered significant at a probability p<0.05.

RESULTS AND DISCUSSION

Statistical analysis of data indicated that: there were significantly (p<0.05) less intestinal lesions in chickens which received 1.2 g kg-1 betaine, compared to those unsupplemented with betaine (Table 2). Despite no statistically significant difference of lesion scores between 0 and 0.6 g kg-1 betaine levels, decreasing intestinal lesions numerically was observable. This result is in agreement with findings by Tiihonen et al. (1997) and Hess et al. (1998), who reported that morbidity and macroscopic intestinal lesions resulting from infection with Eimeria diminished by dietary betaine. Also Virtanen et al. (1996) reported a decrease in lesion score of chicks fed betaine in combination with salinomycin, whereas Remus and Virtanen (1996), Zimmermann et al. (1996) and Matthews et al. (1997) didn’t report this effect for betaine added in diet.

The number of intraepithelial lymphocytes in the intestinal section of birds whose diet was supplemented with 0.6 or 1.2 g kg-1 betaine was significantly (p<0.01) greater than those receiving no betaine (Table 2).

Table 2: Effect of betaine levels on histological and morphological parameters
Values are means of comparative scores, V/C: Villus height/Crypt depth, SEM: Standard error of means, a-bValues within variables with no common superscripts differ significantly (p<0.05), NS: Not Significant, *: p<0.05, **: p<0.01, ***: p<0.001

Klasing et al. (2002) reported the same results with adding 0.5 or 1 g kg-1 betaine to the diet. They suggested that increased chemotaxis of monocytes and nitrogen oxide release by macrophages may explain the decreased intestinal pathology but increased leukocyte numbers that were observed when betaine was fed during a coccidia infection. Intestinal lymphocytes are present in two anatomic compartments within the intestinal mucosa, the epithelium and lamina propria (Befus et al., 1980) which these cells and T cells regulate mucosal immune responses. In addition, intraepithelial lymphocytes have been demonstrated to secrete several cytokines, including interleukins, transforming growth factor and interferon-γ (Mayer et al., 1991; Lundqvist et al., 1996; Fan et al., 1998). Furthermore chicken intestinal intraepithelial lymphocytes contain a subpopulation of Natural Killer (NK) cells that serve an important role in controlling coccidiosis, perhaps by cytolysis of infected cells or as a source of interferon (Lillehoj, 1989). Hence these cells are an important part of intestinal immune responses. According to results of this study, dietary supplementation with betaine leads to the improvement of this immune criterion.

As shown in Table 2 the ratio of V/C was unaffected by betaine level. However, Klasing et al. (2002) reported that villi height was decreased by coccidiosis and this was ameliorated by 1.0 g kg-1 dietary betaine. Also Kettunen et al. (2001) observed that the crypt/villus ratio was decreased by the dietary betaine supplementation in healthy and challenged chicks. The sensitivity of the V/C ratio has been demonstrated even after alterations in the lipid composition of the diet (Sagher et al., 1991; Galluser et al., 1993), dietary supplemental calcium (Aptekmann et al., 2001) and Zn deficiency in the diet (Southon et al., 1985). Indeed, in relation to villus height and crypt depth, the important point is that the proliferation in the small intestine increases the crypt proliferative compartment. Instead, the absorptive villus compartment becomes smaller. Consequently, nutrient absorption would be affected.

A more reliable criterion for absorptive area than V/C ratio is villus surface area because this parameter includes villus height, width and amount of folds on villus. Thus in this research, villus surface area was examined to detect more precise effect of betaine (if there be any) on absorptive area. As shown in Table 2 there was no significant difference in duodenal villus surface area between betaine levels. Reduction of villus surface area, in coccidiosis condition, is thought to be responsible for nutrient malabsorption -a typical symptom of coccidiosis (Scott et al., 1982; Girdhar et al., 2006). One result of nutrient malabsorption is the reduction of feed efficiency. According to the presented results, it appears that the improvement of feed efficiency has been observed in some experiments (Waldenstedt et al., 1999a, b) in which coccidia-infected chicks were dietary supplemented with betaine is not due to the effect of betaine on absorptive surface. Probably, stabilizery and osmoregulatory effects of betaine are responsible for it.

Thickness of lamina propria was increased highly significantly by the elevation of dietary supplemental betaine level so that level of 0.6 g kg-1 betaine differed significantly from 0, as well as 1.2 from 0.6 (Table 2). This result supports what has been reported by Klasing et al. (2002). Thickness of lamina propria could be an indicator of gut health, because it contains dendritic cells that survey the contents of the lumen and protect the chicken from infection by stimulating the adaptive immune response, increasing gut motility and modifying mucin production, defensin secretion and immunoglobulin A (IgA) production. On the other hand, only a layer of mucin and a single cell layer of enterocytes separate the deeper tissues of the body from potential opportunistic pathogens found in the lumen of the gastrointestinal tract (Macpherson and Harris, 2004).

CONCLUSION

In conclusion, according to beneficial effect of dietary betaine on intraepithelial lymphocytes and thickness of lamina propria observed in this research and the importance of these tow parameters in immunocompetence of birds it seems that dietary betaine may immunomodulate the gastrointestinal tract of broilers. Furthermore, betaine effect on villus morphology measured later in life differed from what had been measured already earlier in life of the chicks. However, betaine didn’t expand the absorptive surface of the villus.

REFERENCES
Allen, P.C. and R.H. Fetterer, 2002. Recent advances in biology and immunobiology of Eimeria species and in diagnosis and control of infection with these coccidian parasites of poultry. Clin. Microbiol. Rev., 15: 58-65.
CrossRef  |  PubMed  |  Direct Link  |  

Allen, P.C., H.D. Danforth and P.C. Augustine, 1998. Dietary modulation of avian coccidiosis. Int. J. Parasitol., 28: 1131-1140.
CrossRef  |  PubMed  |  Direct Link  |  

Aptekmann, K.P., S.M.B. Arton, M.A. Stefanini and M.A. Orsi, 2001. Morphometric analysis of the intestine of domestic quails (Coturnix coturnix japonica) treated with different levels of dietary calcium. Anatomia Histologia Embryologia, 30: 277-280.
CrossRef  |  PubMed  |  Direct Link  |  

Befus, A.D., N. Johnston, G.A. Leslie and J. Bienenstock, 1980. Gut associated lymphoid tissue in the chicken. I. Morphology, ontogeny and some functional characteristics of Peyer's patches. J. Immunol., 125: 2626-2632.
PubMed  |  Direct Link  |  

Biffa, D., E. Debela and M. Bezabih, 2003. The effects of breed and age of chicken on the amount of faecal excretion of Eimeria oocystes and mortality rate due to coccidiosis. Eth. J. Anim. Prod., 3: 119-130.
Direct Link  |  

Fan, J.Y., C.S. Boyce and C.F. Cuff, 1998. T-helper 1 and T-helper 2 cytokine responses in gut-associated lymphoid tissue following enteric reovirus infection. Cell Immunol., 188: 55-63.
CrossRef  |  PubMed  |  

Galluser, M., B. Czernichow and H. Dreyfus, 1993. Comparison of different lipid substrates on intestinal adaptation in the rat. Gut, 34: 1069-1074.
Direct Link  |  

Girdhar, S.R., J.R. Barta, F.A. Santoyo and T.K. Smith, 2006. Dietary Putrescine (1,4-Diaminobutane) influences recovery of turkey poults challenged with a mixed coccidial infection. J. Nutr., 136: 2319-2324.
Direct Link  |  

Hess, J.B., M.K. Eckman and S.F. Bilgili, 1998. Influence of betaine on broilers challenged with two levels of Eimeria acervulina. Poult. Sci., 77: 43-43.
Direct Link  |  

Iji, P.A., A. Saki and D.R. Tivey, 2001. Body and intestinal growth of broiler chicks on a commercial starter diet. 1. Intestinal weight and mucosal development. Br. Poult. Sci., 42: 505-513.
CrossRef  |  Direct Link  |  

Kettunen, H., K. Tiihonena, S. Peuranena, M.T. Saarinena and J.C. Remusb, 2001. Dietary betaine accumulates in the liver and intestinal tissue and stabilizes the intestinal epithelial structure in healthy and coccidia-infected broiler chicks. Comp. Biochem. Physiol. Part A: Mol. Integr. Physiol., 130: 759-769.
CrossRef  |  PubMed  |  Direct Link  |  

Klasing, K.C., K.L. Adler, J.C. Remus and C.C. Calvert, 2002. Dietary betaine increases intraepithelial lymphocytes in the doudenum of coccidia infected chicks and increases functional properties of phagocytes. J. Nutr., 132: 2274-2282.
Direct Link  |  

Lillehoj, H.S., 1989. Intestinal intraepithelial and splenic natural killer cell responses to Eimerian infections in inbred chickens. Infect. Immunity, 57: 1879-1884.
Direct Link  |  

Lundqvist, C., S. Melgar, M.M. Yeung, S. Hammarstrom and M.L. Hammarstrom, 1996. Intraepithelial lymphocytes in human gut have lytic potential and a cytokine proile that suggest T helper 1 and cytotoxic functions. J. Immunol., 157: 1926-1934.
PubMed  |  Direct Link  |  

Macpherson, A.J. and N.L. Harris, 2004. Interactions between commensal intestinal bacteria and the immune system. Nat. Rev. Immunol., 4: 478-485.
CrossRef  |  PubMed  |  Direct Link  |  

Malcolm, W.R., 1978. Coccidiosis. In: Disease of Poultry, Hofstad, M.S. (Ed.). 7th Edn., Chapter 31, The Iowa State University Press, Iowa, USA.

Matthews, J.O., T.L. Ward and L.L. Southern, 1997. Interactive effects of betaine and monensin in uninfected and Eimeria acervulina-infected chicks. Poult. Sci., 76: 1014-1019.
PubMed  |  

Mayer, L., D. Eisenhardt, P. Salomon, W. Bauer, R. Plous and L. Piccinini, 1991. Expression of class II molecules on intestinal epithelium cells in humans. Differences between normal and inflammatory bowel disease. Gastroenterology, 100: 3-12.
PubMed  |  Direct Link  |  

NRC., 1994. Nutrient Requirements of Poultry. 9th Edn., National Academy Press, Washington, DC., USA., ISBN-13: 9780309048927, Pages: 155.

Remus, J.C. and E. Virtanen, 1996. Use of liquid betaine in low methionine diets for broilers. Poult. Sci., 75: 35-35.

SAS, 1999. SAS statistics User's Guide. Statistical Analytical System. 5th Rev. Edn., SAS Institute Inc., Cary, NC.

Sagher, F.A., J.A. Dodge, C.F. Johnston, C. Shaw, D.K. Buchanan and K.E. Carr, 1991. Rat small intestinal morphology and tissue regulatory peptides: Effects of highdietary fat. Br. J. Nutr., 65: 21-28.
PubMed  |  Direct Link  |  

Scott, M.L., M.C. Nesheim and R.J. Young, 1982. Nutrition of the Chicken. 3rd Edn., M.L. Scott and Associates Ithaca, New York, USA., ISBN-10: 0960272623, Pages: 562.

Southon, S., G. Livesey, J.M. Gee and I.T. Johnson, 1985. Intestinal cellular proliferation and protein synthesis in zinc-deficient rats. Br. J. Nutr., 53: 595-603.
CrossRef  |  PubMed  |  Direct Link  |  

Tarachai, P. and K. Yamauchi, 2000. Effects of luminal nutrient absorption, intraluminal physical stimulation and intravenous parenteral alimentation on the recovery responses of duodenal villus morphology following feed withdrawal in chickens. Poult. Sci., 79: 1578-1585.
CrossRef  |  PubMed  |  Direct Link  |  

Tiihonen, K., H. Kettunen, J. Remus, M. Saarinen and E. Virtanen, 1997. Effects of dietary betaine on broiler chicks with or without mild coccidiosis challenge. Poult. Sci., 76: 18-18.
Direct Link  |  

Virtanen, E., J. Remus, L. Rosi, J. McNaughton and P. Augustine, 1996. The effect of betaine and salinomycin during coccidiosis in broilers. Poult. Sci., 75: 149-149.

Waldensted, L., K. Elwinger, P. Thebo and A. Uggla, 1999. Effect of betaine supplement on broiler performance during an experimental coccidial infection. Poult. Sci., 7: 182-189.
PubMed  |  

Waldenstedt, L., A. Lunden, K. Elwinger, P. Thebo and A. Uggla, 1999. Comparison between a live, attenuated anticoccidial vaccine and an anticoccidial ionophore, on performance of broilers raised with or without a growth promoter, in an initially Eimeria-free environment. Acta Vet. Scand., 40: 11-21.
PubMed  |  

Yun, C.H., H.S. Lillehoj and E.P. Lillehoj, 2000. Intestinal immune responses to coccidiosis. Dev. Comp. Immunol., 24: 303-324.
CrossRef  |  PubMed  |  

Zentek, J., E.J. Hall, A.J. German, K. Haverson and M. Bailey et al., 2002. Morphology and immunopathology of the small and large intestine in dogs with non-specific dietary sensitivity. J. Nutr., 132: 1652-1654.
PubMed  |  Direct Link  |  

Zimmermann, N.G., P. Twining, J. Harter-Dennis and S. Fitz-Coy, 1996. Betaine as a methionine substitute and coccidial deterrent in broilers. Poult. Sci., 75: 154-154.

©  2020 Science Alert. All Rights Reserved