Methionine alleviates aflatoxinb1-induced broiler chicks embryotoxicity through inhibition of caspase-dependent apoptosis and enhancement of cellular antioxidant status

Abstract

Practical methods for preventing embryotoxicity in chickens that are caused by aflatoxin-B1 (AFB1) are currently rare. Binding absorbers are commonly used in feeding stuff to reduce laying hens' exposure to off-contaminated diets, thus reducing residue exposure to fertilized eggs. Nonetheless, several adsorbents have been shown to affect the use of nutrients and the absorption of minerals in poultry. Thus, seeking an effective strategy to counter or control embryotoxicity in broiler chicks caused by AFB1 is a problem. A total of 180 embryonated eggs were injected with 36 ng AFB1 with or without 5.90 mg L-methionine (Met) 30 embryonated eggs each, followed by incubation in an incubator until hatching time. The in ovo injection of Met significantly reduced toxicity caused by AFB1 in broiler embryos by enhancing the liver and kidney functions, lipid profiles, and alleviated oxidative stress during the incubation period. Furthermore, the relative gene expressions (SSTR5, TSH-β, Bcl-2, GSH-Px, GST-a, and SOD in the liver) were up-regulated with in ovo injection of AFB1+Met compared to AFB1 alone. Moreover, there was a dowin-regulated trend in Bax, Caspases-3, Caspases-7, Caspases-9, CYP1A1, CYP2H1, and P53 gene expression with in ovo injection of AFB1+Met compared to AFB1 alone. The in ovo injection of Met led to less apoptotic cells in liver tissues. Such results might be necessary for the poultry industry as it is focused on managing the embryotoxicity of AFB1, which affecting poultry production and welfare. Results from this study demonstrated that in ovo Met injection could alleviate AF-induced toxicity in chicken embryos.

Keywords: aflatoxin B1; broiler; embryotoxicity; gene expression; methionine.

Figure 1

Effect of treatments on embryonic mortality. Control = free injected embryos; Saline = 0.75% NaCl-injected; Methanol = injected with 20 µL Methanol 20%; Met = injected with methionine (5.90 mg/L); AFB1 = injected with 36 ng AFB1; AFB1 + Met = injected with 36 ng AFB1+ methionine (5.90 mg/L).

Figure 2

Relative chick weight (A), relative residual yolk sac (B), relative tibia weight (C), tibia length (D), liver weight (E), and liver relative weight (F) in the control and experimental groups. Control = free injected embryos; Saline = injected with 0.75% NaCl; Methanol = injected with 20 µL Methanol 20%; Met = injected with methionine (5.90 mg/L); AFB1 = injected with 36 ng AFB1; AFB1+Met = injected with 36 ng AFB1 + methionine (5.90 mg/L).

Figure 3

SSTR5, and TSHB mRNA expression on the day of hatch detected by quantitative real-time PCR. Data are set to mean ± SD (n = 6). ^{a, d} Values with different letters differ significantly (P < 0.05) in relative expression levels of RNAs. Control = free injected embryos; Saline = injected with 0.75% NaCl; Methanol= injected with 20 µL Methanol 20%; Met = injected with methionine (5.90 mg/L); AFB1= injected with 36 ng AFB1; AFB1+Met=injected with 36 ng AFB1+ methionine (5.90 mg/L).

Figure 4

The relative expression level of GSH-px, GST-a, and SOD at the day of the hatch was detected by quantitative real-time PCR. Data are set to mean ± SD (n = 6). ^a,dValues with different letters differ significantly (P < 0.05) in relative expression levels of mRNAs. Control = free injected embryos; Saline = injected with 0.75% NaCl; Methanol= injected with 20 µL Methanol 20%; Met = injected with methionine (5.90 mg/L); AFB1 = injected with 36 ng AFB1; AFB1+Met = injected with 36 ng AFB1+ methionine (5.90 mg/L).

Figure 5

Effect of treatments on mRNA levels of mitochondrial apoptosis-associated genes (Bcl-2, Bax, CASPASE-3, CASPASE-7, CASPASE-9, P53, CYP1A1, and CYP2H1) liver mRNAs. All data were set as mean ± SD (n = 6). Columns with different letters (a–d) indicate a significant difference at (P < 0.05). Control = free injected embryos; Saline = injected with 0.75% NaCl; Methanol= injected with 20 µL Methanol 20%; Met = injected with methionine (5.90 mg/L); AFB1= injected with 36 ng AFB1; AFB1+Met=injected with 36 ng AFB1+ methionine (5.90 mg/L).

Figure 6

Effect of Met and/or AFB1 in ovo injection on liver histology (1A, B, C, D, E, and F) and apoptotic percentage (2A, B, C, D, E, F, and 3) at the day of hatch. All data were set as mean ± SD (n = 6). Columns with different letters (a–c) indicate a significant difference at (P < 0.05). Control = free injected embryos; Saline = injected with 0.75% NaCl; Methanol = injected with 20 µL Methanol 20%; Met = injected with methionine (5.90 mg/L); AFB1 = injected with 36 ng AFB1; AFB1+Met = injected with 36 ng AFB1+ methionine (5.90 mg/L).