Effects of Selenium Yeast on Egg Quality, Plasma Antioxidants, Selenium Deposition and Eggshell Formation in Aged Laying Hens

**Background:** Egg quality deterioration in aged laying hens causes significant economic losses. Selenium yeast (SY), an organic selenium source, is known to improve antioxidant status and performance, but its effects on eggshell quality in aged hens and the underlying molecular mechanisms were not fully understood. This study aimed to evaluate the effects of SY on egg quality, plasma antioxidants, selenium deposition, and eggshell formation in aged laying hens.

**Methods:** Five hundred and twenty-five 76-week-old Jing Hong laying hens were fed a selenium-deficient (SD) diet (basal Se 0.056 ± 0.012 mg/kg) for 6 weeks to induce Se depletion. Hens were then randomly allocated to 7 groups (5 replicates of 15 hens each): SD control, SY at 0.15, 0.30, or 0.45 mg/kg, and sodium selenite (SS) at 0.15, 0.30, or 0.45 mg/kg for 12 weeks (83–95 weeks of age). Egg quality was measured at 6-week intervals. Plasma Se content and antioxidant enzymes (T-AOC, T-SOD, GSH-Px) were measured every 2 weeks. After 12 weeks, reproductive organs were collected for Se analysis and histology. Shell gland transcriptomic analysis (RNA-seq) was performed on 4 hens per group, with validation by qRT-PCR.

**Key Results:** During the 6-week Se depletion, plasma Se declined from 0.237 μg/mL to 0.068 μg/mL (p < 0.01), and eggshell strength decreased significantly (from 3.26 to 2.89 kg force, p = 0.04). After 12 weeks of supplementation, the SY0.45 group showed significantly higher eggshell strength (3.48 kg force) compared to SD (2.98 kg force, p < 0.05) and lower shell translucence scores. Plasma Se content in SY0.45 recovered to pre-depletion levels after 8 weeks of supplementation (0.26 μg/mL at week 14), while other groups did not. At week 18, plasma GSH-Px activity was 3545.17 U/L in SY0.45 vs. 2340.10 U/L in SD (p < 0.05), and T-AOC was 5.12 U/mL in SY0.45 vs. 2.09 U/mL in SD (p < 0.01). Organ Se levels increased dose-dependently with SY supplementation. H&E staining showed no pathological lesions in the shell gland. Transcriptomic analysis identified 1308 differentially expressed genes (DEGs) in SY groups vs. SD. Key candidate genes included CEMIP, OVAL, SLC6A17, SLC13A5, POMC, and PENK, enriched in pathways related to eggshell mineralization (calcium signaling, glycosaminoglycan biosynthesis), ion transport, and reproductive hormone response. WGCNA identified modules positively correlated with shell gland Se content and eggshell strength.

**Clinical Implications:** Supplementation with 0.45 mg/kg selenium yeast effectively alleviates the decline in eggshell quality in aged laying hens by enhancing antioxidant capacity, increasing selenium deposition, and modulating expression of genes involved in eggshell mineralization and ion transport. This dietary strategy may help extend the laying cycle, reduce egg breakage, and improve economic outcomes in the poultry industry. The findings also provide molecular targets for further research on selenium's role in eggshell formation.