A nutritional solution based on calcidiol (25-OH-D₃, HyD) and triterpenoid affects muscle protein synthesis pathways in grower-finisher pigs

Abstract To boost profitability in pig production, it is important to achieve increased lean growth rates and maximize efficiency, especially in grower-finisher pigs where feed intakes are high. Molecular research, especially the study of gene expression, is a proven tool for understanding the precise mechanisms behind muscle tissue growth, including a recent focus on pathways such as the mammalian target of rapamycin (mTOR). In the current work, gene expression analyses were used to study the mode of action of the combination of calcidiol (25-OH-D3, HyD) and triterpenoid, which has shown significant growth and efficiency improvements in grower-finisher pigs (McCormack et al., 2024). Three experimental diets were evaluated for 7 wk; Positive control: 0.7% total Ca, 0.36% total P, vit D3 at 1500IU/kg feed (PC), negative control: PC diet + 30% reduced Ca and 10% reduced P (NC), and NC diet with 1,500IU D3 in calcidiol form (25-OH-D3, HyD) and triterpenoid (NC+). At 19 wk of age, 16 animals per treatment were sacrificed and tissue samples were taken from the longissimus muscle and preserved in RNA later solution. The RNA was extracted, quality control checked, and mRNA sequenced using the Illumina platform with 150 base pair paired-end sequencing and 30M read depth. The sequenced data were aligned to the Sus scrofa reference genome (Sscrofa11.1) using Kallisto. Data were normalized and analyzed for differentially expressed genes (DEGs) between treatments using DeSeq2. Genes were assigned to biological pathways using Biofractal’s customisation of the Reactome pathway database. For statistical analysis, RNA integrity number and treatment were included in the model. In the PC compared with the NC, there were 71 DEGs using a false discovery rate (FDR) of < 0.10. Altered pathways included the inhibition of cellular stress pathways and increased cell turnover in the muscle of PC compared with NC pigs. In the NC+ compared with the NC, there were 155 DEGs (FDR < 0.10). The most significantly upregulated gene in the NC+ treatment was adenylate cyclase (FDR < 0.001), an upstream regulator of several pathways related to energy metabolism and muscle protein synthesis. This mechanism was confirmed in the pathway analysis, which found that the NC+ treatment activated PKA-mediated phosphorylation of CREB, a pathway that inhibits muscle atrophy. Furthermore, the NC+ treatment activated several pathways related to muscle growth, including the mammalian target of rapamycin complex 1 (mTORC1). In this study the addition of calcidiol (25-OH-D3, HyD) and triterpenoid to the diet was found to specifically affect molecular pathways in the muscle of grower-finisher pigs involved in both the inhibition of muscle breakdown as well as activating muscle growth.