Simple SummaryThe cholesterol content in egg yolk is high, and an excessive consumption of egg yolk can have adverse effects on the health of the body. Therefore, it is necessary to systematically study the formation process of cholesterol in egg yolk and reduce the content of cholesterol in egg yolk. After using 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) inhibitors to inhibit the synthesis of cholesterol in the liver of laying hens, this study investigated the changes in the cholesterol content in egg yolk and the surrounding tissues and utilised RNA sequencing (RNA-seq) technology to explore the biological mechanism that is involved when HMGCR inhibitors reduce egg yolk cholesterol. Therefore, this study provides a reference for related research on cholesterol metabolism in laying hens.AbstractThis study aimed to investigate the effect of HMGCR inhibitors on egg yolk cholesterol content and its biological mechanisms. Four groups of 180-day-old laying hens (n= 8 cages/group, 6 laying hens/cage) were fed a corn/soybean-based diet (control) and the control diet supplemented with an HMGCR inhibitor at 60, 150, and 300 mg/kg for 4 weeks. The experimental results showed that adding HMGCR inhibitors of 150 mg/kg or more can significantly reduce the cholesterol content in the liver, yolk, serum, and pectoral muscles of laying hens. The RNA-seq results showed that compared with the control group, the addition of HMGCR inhibitors of 150 mg/kg or more to the diet significantly upregulated genes related to cholesterol synthesis in the liver, and the genes involved in steroid synthesis and metabolism, sterol synthesis and metabolism, and cholesterol synthesis and metabolism were all affected by the HMGCR inhibitors. In summary, adding HMGCR inhibitors of 150 mg/kg or more to the diet of hens can significantly reduce the cholesterol content in egg yolk. After the HMGCR inhibitors inhibited the activity of the liver HMGCR, they also altered the expression of genes related to cholesterol synthesis, bile acid synthesis, and cholesterol transport in the liver, and ultimately reduced cholesterol synthesis and cholesterol transport to the egg yolk.