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Effect of methionine supplementation on intestinal morphology in broilers infected with aflatoxicosis B1

Muhsin Al Anas, Lies Mira Yusiati, Cuk Tri Noviandi and Ali Agus

Department of Animal Nutrition and Feed Science, Faculty of Animal Science, Universitas Gadjah Mada, Fauna 03, Bulaksumur, Yogyakarta, Indonesia (55821)
aliagus@ugm.ac.id

Abstract

This study aimed to evaluate methionine supplementation to ameliorate toxicity of aflatoxin B1 (AFB1) on intestinal morphology in broilers. A total of 320 broilers were randomly allocated to 5 treatments with 8 replicates, 8 birds per replicate. The dietary treatments were The dietary treatments (AF = Aflatoxin B1 ) were as follows: CTL: control (basal diet, without AF), AF100 (100 g/kg of AF), AF500 (500 g/kg of AF), AFM100 (100 g/kg AF+ DL-methionine 0.3 g/kg), AFM500 (500 g/kg AF+ DL-methionine 3 g/kg).

The results showed that AFB1 500 g/kg decreased villus height and width(p<0.05). Both 100 and 500 g/kg increased crypt depth (p<0.01), also decreased villus:crypt (V:C) ratio. Supplementation of methionine ameliorated villus height, villus width, and V:C ratio of broilers fed AFB1 contaminated diet. However, it did not affect crypt depth. In conclusion, the addition of methionine reduced the adverse effect of aflatoxicosis B1 on intestinal growth in broilers.

Keywords: amino acid, fungi, mycotoxin, poultry


Introduction

Aflatoxin B1 (AFB1) is a toxic molecule as a secondary metabolite produced by Aspergillus spp. (Ritter et al 2011; Mahato et al 2019). Aflatoxin B1 decreases productivity and increases mortality of broilers, also causes economic loss (Fouad et al 2019). Feeding AFB1 contaminated in the diet inhibited intestinal villus development. The intestinal disorder caused a lowering of nutrient absorption and utilization (Chen et al 2016; Grenier and Applegate 2013).

Aflatoxicosis AFB1 can be reduced through conjugation reaction with glutathione (GSH) in the liver (Murcia 2011; Kim et al 2013). Glutathione is synthesized in the cytosols that are influenced by the availability of cysteine and amino acid sulfur (Lu 2009 2013). Methionine contains sulfur groups as precursors of glutathione formation (Morand et al 1997; Gould and Pazdro 2019). This study aimed to evaluate the effect of methionine supplementation in AFB1-contaminated diet on intestinal morphology in broilers.


Material and methods

Production of AFB1

Crude aflatoxin was produced using maize as substrate. A. flavus was used to inoculate maize and rice in the mash. Sterile distilled water was added until the water content of the maize was 30%, while the rice was soaked in 1 mL of water for every 2 g of rice. After their preparation, the maize and rice were sterilized using an autoclave for 15 minutes at 121C and 105 atm. The substrates were inoculated using 1 mL of the A. flavus mixture dissolved in Tween 2% and then mixed. The substrates were incubated at 30˚C for 5 days. On the second day, 1 mL of distilled water was added to the substrates, which were then mixed. Crude aflatoxin was harvested on day 6. The substrates were sterilized in an autoclave to stop spore growth. Aflatoxin B1 was analyzed using ELISA (enzyme-linked immunosorbent assay) kit (Romer Labs, Singapore).

Table 1. Composition and nutrient content of the experimental diets (g/100 g. as-fed basis)

Composition (%)

CTL

AF100

AF500

AFM100

AFM500

Maize

57.17

57.17

57.17

57.17

57.17

Maize gluten meal

7.00

7.00

7.00

7.00

7.00

Soybean meal

27.53

27.53

27.53

27.53

27.53

Palm oil

3.63

3.63

3.63

3.63

3.63

Limestone (CaCo3)

1.40

1.40

1.40

1.40

1.40

Mono-Calcium Phosphate

1.34

1.34

1.34

1.34

1.34

NaCl

0.22

0.22

0.22

0.22

0.22

L-Lysine HCl

0.37

0.37

0.37

0.37

0.37

DL-Methionine

0.28

0.28

0.28

0.58

0.58

L-Threonine

0.09

0.09

0.09

0.09

0.09

Sodium bicarbonate

0.32

0.32

0.32

0.32

0.32

Trace mineral mix

0.05

0.05

0.05

0.05

0.05

Choline-Cl

0.06

0.06

0.06

0.06

0.06

Vitamin Mix1

0.25

0.25

0.25

0.25

0.25

Sand

0.30

0.00

0.00

0.00

0.00

Proximal analysis

Moisture , %

11.22

11.22

11.22

11.22

11.22

Fat, %

6.30

6.30

6.30

6.30

6.30

Fiber, %

2.33

2.33

2.33

2.33

2.33

Ash,%

5.71

5.71

5.71

5.71

5.71

Protein (%

22.57

22.57

22.57

22.74

22.74

Calcium, %

0.86

0.86

0.86

0.86

0.86

Available phosphorus, %

0.42

0.42

0.42

0.42

0.42

DEB

222.15

222.15

222.15

222.15

222.15

Aflatoxin B1, g/kg

0

100

500

100

500

DEB: Dietary Electrolyte Balance

Experimental design, birds, and diets

One day old chicken unsexed broilers were placed in a brooder. The temperature was kept at 31 C for the first week. On d 8, 320 broilers were divided into 5 treatments with 8 replications, 8 birds each replicate. The experimental diets (Table 1) were formulated based on NRC (1994) for broilers. The dietary treatments (AF = Aflatoxin B1 were as follows:

CTL: control (basal diet, without AF),

AF100 (100 g/kg of AF),

AF500 (500 g/kg of AF),

AFM100 (100 g/kg AF+ DL-methionine 0.3 g/kg),

AFM500 (500 g/kg AF+ DL-methionine 0.3 g/kg).

Birds had ad libitum access to water and feed during the trial. On d 35, one broiler from each replicate treatment was selected randomly and killed by cervical dislocation. The jejunum was removed for morphology analysis.

Intestinal morphology analysis

The jejunal samples (5 birds per treatment) were fixed in 10% formalin. Samples processed by standard paraffin sectioning and stained with hematoxylin-eosin. The jejunal slides were examined under a light microscope fitted with a video camera (Optilab Advance).

Statistics

The data were analyzed by one way ANOVA with a completely randomized design, using IBM SPSS Statistics 19 for Windows.


Results and Discussion

Aflatoxin B1 decreased villus height, villus width, and villus:crypt ratio, also increased crypt depth (Table 2). Methionine supplementation ameliorated the intestinal alteration of broilers fed AFB 1.

Table 2. Intestinal morphology of broilers with or without methionine supplementation in the AFB1 contaminated feed

CTL

AF100

AF500

AFM100

AFM500

SEM

p

Villus height, μm

688b

660ab

645a

691a

666ab

6.52

<0.05

Villus width, μm

110b

100ab

94.5a

106ab

105b

1.99

<0.05

Crypt depth, μm

101a

144b

138b

139b

128b

3.26

<0.01

V:C ratio

6.82c

4.68a

4.73a

5.02b

5.23b

0.14

<0.01

abc. Mean values within rows without common superscript differ at p<0.05

Jahanian et al (2016, 2017) and Wang et al (2018) reported that AFB 1 contaminated in the diet (600-2,000 g/kg) significantly decreased villus height, crypt depth, and V:C ratio. Similar to Aboutalebi (2018), increasing of AFB1 contamination reduced villus height and damaged duodenum tissue.

Aflatoxin B1 can effect on intestinal proliferation (Fleming et al 1994) and lesions, reduced goblet cells that producing mucin (Liu et al 2017; Liu et al 2018; Wang et al 2018), increased pathogen bacteria and depress immune function (Galarza-seeber et al 2016; Jahanian et al 2016, 2017). Aflatoxin B1 disrupts gut microbial metabolism in producing short-chain fatty acids (SCFAs). Moreover, AFB1 had significantly effect to decreased butyric acid that had an important role to increase villus height and cell regeneration (Ahsan et al 2016; Zhou et al 2018; Wu et al 2018).

Methionine has an important role in the villus growth (Shen et al 2015; Seyyedin and Nazem 2017). The addition of methionine showed positive effects in villus growth and maintained the integrity of the small intestine (Yang and Liao 2019). Futhermore, methionine is a precursor to produce glutathione as antioxidants in the intestinal mucin. The glutathione reduced the impact of bacterial pathogens, oxidative stress, and xenobiotic (AFB1) (Yang and Liao 2019).

Gastrointestinal abnormalities caused by AFB1 reduced digestibility and nutrient absorption. Moreover, it will depress the production and quality of broiler products. The addition of methionine in AFB1 contaminated diet plays a role in increasing the growth of the digestive tract.


Conclusions


Acknowledgments

The authors would like to thank the Directorate-General for Science, Technology and Higher Education Resources, Ministry of Research and Technology/National Agency for Research and Innovation of the Republic of Indonesia for financial support of the research through Pendidikan Magister Menuju Doktor untuk Sarjana Unggul (PMDSU) scholarship.


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Received 31 January 2020; Accepted 6 February 2020; Published 2 March 2020

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