314. 5-Hydroxytryptophan Attenuates Toxic Tall Fescue-Induced Oxidative Stress on Fetal Satellite Cells.

Abstract Satellite cells (SAT) are muscle precursor cells that support muscle growth and regeneration. Nutrient supplementation plays a significant role in satellite cell activity and may alter the abundance or transcriptional state of a specific cell population. This study aims to identify and compare single-cell populations across treatments to determine how 5-hydroxytryptophan (5-HTP) supplementation and toxic fescue (E+) exposure affect single-cell gene expression patterns in fetal lamb semimembranosus (SM) muscle. Twenty-seven ewes were confirmed pregnant with twins and allocated to one of the three treatments: 1) negative control (CON), 2) 5-HTP (2.5 mg/kg BW) and E+ seed containing 1.77 mg ewe-1 d-1 ergovaline (E+/5-HTP), or 3) positive control, fed E+ seed containing 1.77 mg ewe-1 d-1 ergovaline (E+) from gestational day (gd) 86 to d 111. Terminal necropsy was performed on gd 111, and the fetal semimembranosus muscle (SM) was obtained. SAT were isolated from SM for each treatment (n = 2 for CON and E+/5-HTP; n = 1 for E+) and then activated. Cells were quantified using Acridine Orange/Propidium Iodide (AO/PI), and only those with 80% live cells were sequenced. Illumina Single Cell 3’ RNA Prep, T10 protocol, was used for cell suspension and single cell (SC) library preparation. The FASTQ file was generated after sequencing, and a sensitivity analysis was performed using Pipseeker. Sensitivity analysis showed a total read ranging from 292,679,871 to 481,938,226, while mean reads per cell ranged from 41,771 to 75,080. Also, gene expression levels in cells ranged from 24,418 to 25,524. Seurat results showed 20 cell clusters across treatments. Three contrasts, E+/5-HTP vs. CON, E+ vs. CON, and E+/5-HTP vs. E+, were tested. Across all 20 clusters, E+/5-HTP upregulated 21 genes and downregulated 98 genes relative to E + (adj p < 0.05). The downregulated genes play roles in tissue remodeling, oxidative stress and inflammatory signaling, suggesting that 5-HTP may be mitigating the toxic effects of fescue on satellite cells. Compared with CON, E+/5-HTP treatment produced a predominant downregulation (adj p < 0.05) of differentially expressed (DE) genes across clusters, including MXRA8, SPP1, PDPN, OVAR-DRB1, SLITRK5, SLITRK2, and COL6A1, which are related to neural-associated signaling and stress response activity. For E+ vs CON, 401 upregulated and 305 downregulated genes (adj p < 0.05) were observed across clusters. The upregulated genes suggest that E+ induces a strong stress response, an inflammatory regulatory program, and reactivates the remodeling program, while reducing normal tissue maintenance pathways and extracellular matrix support genes such as DCN, COL3A1, FGF7, and LUM. In conclusion, 5-HTP supplementation altered single-cell transcriptional profiles in satellite cells from fetal lamb SM. 5-HTP showed evidence of attenuating E+ toxic fescue-associated gene expression changes; whereas E+ exposure was associated with increased expression of stress, inflammatory, and tissue-remodeling.