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. 2022 Jun;101(6):101871.
doi: 10.1016/j.psj.2022.101871. Epub 2022 Mar 24.

In ovo injection of betaine promotes adrenal steroidogenesis in pre-hatched chicken fetuses

Halima Abobaker  1 , Nagmeldin A Omer  2 , Yun Hu  2 , Abdulrahman A Idriss  2 , Ruqian Zhao  3
Affiliations

In ovo injection of betaine promotes adrenal steroidogenesis in pre-hatched chicken fetuses

Halima Abobaker et al. Poult Sci. 2022 Jun.

Abstract

Corticosterone is critical for the maturation and survival of chicken fetus around hatching. Betaine is used as a feed additive in poultry industry to promote growth and mitigate stress. However, it remains unknown whether betaine could affect adrenal corticosterone synthesis in pre-hatching chicken fetuses. In this study, betaine (2.5 mg/egg) was injected into developing chicken fetuses at d 11 of incubation (E11) and its impact on adrenal steroidogenesis was investigated at day 19 (E19). Plasma corticosterone concentration was significantly (P < 0.05) elevated in betaine-treated fetuses, together with increased adrenal expression of melanocortin 2 receptor and steroidogenic acute regulatory protein. Accordingly, the corticosterone biosynthetic enzymes, such as cytochrome P450 family 11 subfamily A member 1, 3β-hydroxysteroid dehydrogenase and cytochrome P450 family 21 subfamily A member 2, as well as cholesterol biosynthesis or regulation-related genes, such as sterol regulatory element-binding protein 1, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and low-density lipoprotein receptor, were all significantly (P < 0.05) upregulated in betaine group. Meanwhile, steroidogenic factor-1 and glucocorticoid receptor were significantly (P < 0.05) enhanced, whereas expression of dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene, a nuclear receptor known as a repressor of adrenal steroidogenesis, was significantly (P < 0.05) downregulated. Betaine significantly (P < 0.05) increased adrenal expression of genes involved in one-carbon metabolism and DNA methylation, such as S-adenosyl homocysteine hydrolase, betaine-homocysteine-methyltransferase, methionine adenosyl transferase and DNA methyltransferases, yet the promoter regions of most steroidogenic genes were significantly (P < 0.05) hypomethylated. These results indicate that in ovo injection of betaine promotes adrenal glucocorticoid synthesis in chicken fetuses before hatching, which involves alterations in DNA methylation.

Keywords: adrenal; betaine; chicken; cholesterol; steroidogenesis.

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Figures

Figure 1
Figure 1
Effect of betaine on plasma concentration of corticosterone. (A) Hematoxylin and eosin staining; (B) total plasma cholesterol; (C) plasma corticosterone. Values are means ± SEM, *P < 0.05, compared with control (n = 9). Control = egg injected with 100 μL saline only; Betaine = egg injected with 2.5 mg betaine dissolved in 100 μL saline.
Figure 2
Figure 2
Effect of betaine on adrenal expression of steroidogenic genes. (A) Steroidogenic genes mRNA expression; (B) StAR protein level; (C) CYP11A protein level. Values are means ± SEM, *P < 0.05, compared with control (n = 5 for control group and 4 for the betaine group) for the mRNA and (n = 4) for the protein. Control = egg injected with 100 μL saline only; Betaine = egg injected with 2.5 mg betaine dissolved in 100 μL saline.
Figure 3
Figure 3
Effect of betaine on adrenal expression of cholesterol metabolic genes. (A) Cholesterol biosynthetic genes mRNA expression; (B) SREBP1, HMGCR, and LDLR protein level. Values are means ± SEM, *P < 0.05, compared with control (n = 5 for control group and 4 for the betaine group) for the mRNA and (n = 4) for the protein. Control = egg injected with 100 μL saline only; Betaine = egg injected with 2.5 mg betaine dissolved in 100 μL saline.
Figure 4
Figure 4
Effect of betaine on adrenal expression of transcription factors regulating steroidogenesis. (A) SF-1 mRNA expression; (B) DAX1 mRNA level; (C) GR mRNA expression; (D) GR protein expression; (E) CREB mRNA expression; (F) CREB protein level. Values are means ± SEM, *P < 0.05, compared with control (n = 5 for control group and 4 for the betaine group) for the mRNA and (n = 4) for the protein. Control = egg injected with 100 μL saline only; Betaine = egg injected with 2.5 mg betaine dissolved in 100 μL saline.
Figure 5
Figure 5
Effect of betaine on adrenal expression of methionine metabolic genes. (A) Methionine metabolic gene expression; (B) AHCYL protein expression; (C) BHMT protein expression; (D) GNMT1 protein expression; (E) DNMT1 protein expression. Values are means ± SEM, *P < 0.05, compared with control (n = 5 for control group and 4 for the betaine group) for the mRNA and (n = 4) for the protein. Control = egg injected with 100 μL saline only; Betaine = egg injected with 2.5 mg betaine dissolved in 100 μL saline.
Figure 6
Figure 6
Effect of betaine on DNA methylation at the promoter of steroidogenic gene. (A) Schematic diagram showing the amplified segments (S) on the promoter sequence of VIM. (B) DNA methylation status on the promoter of VIM; (C) schematic diagram showing the amplified segments (S) on the promoter sequence of Star; (D) DNA methylation status on the promoter of StAR; (E) schematic diagram showing the amplified segments (S) on the promoter sequence of CYP11A1 (S); (F) DNA methylation status on the promoter of CYP11A1 Values are means ± SEM, *P < 0.05, compared with control (n = 4). Control = egg injected with 100 μL saline only; Betaine = egg injected with 2.5 mg betaine dissolved in 100 μL saline.
Figure 7
Figure 7
Effect of betaine on DNA methylation at the promoter of transcription factors. (A) Schematic diagram showing the amplified segments (S) on the promoter sequence of SF-1. (B) DNA methylation status on the promoter of SF-1; (C) schematic diagram showing the amplified segments (S) on the promoter sequence of GR; (D) DNA methylation status on the promoter of GR; (E) Schematic diagram showing the amplified segments (S) on the promoter sequence of DAX 1; (F) DNA methylation status on the promoter of DAX1; Values are means ± SEM, *P < 0.05, compared with control (n = 4). Control = egg injected with 100 μL saline only; Betaine = egg injected with 2.5 mg betaine dissolved in 100 μL saline.
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