Decreased eggshell strength caused by impairment of uterine calcium transport coincide with higher bone minerals and quality in aged laying hens

Deteriorations in eggshell and bone quality are major challenges in aged laying hens. This study compared the differences of eggshell quality, bone parameters and their correlations as well as uterine physiological characteristics and the bone remodeling processes of hens laying eggs of different eggshell breaking strength to explore the mechanism of eggshell and bone quality reduction and their interaction. A total of 240 74-week-old Hy-line Brown laying hens were selected and allocated to a high (HBS, 44.83 ± 1.31 N) or low (LBS, 24.43 ± 0.57 N) eggshell breaking strength group. A decreased thickness, weight and weight ratio of eggshells were observed in the LBS, accompanied with ultrastructural deterioration and total Ca reduction. Bone quality was negatively correlated with eggshell quality, marked with enhanced structures and increased components in the LBS. In the LBS, the mammillary knobs and effective layer grew slowly. At the initiation stage of eggshell calcification, a total of 130 differentially expressed genes (DEGs, 122 upregulated and 8 downregulated) were identified in the uterus of hens in the LBS relative to those in the HBS. These DEGs were relevant to apoptosis due to the cellular Ca overload. Higher values of p62 protein level, caspase-8 activity, Bax protein expression and lower values of Bcl protein expression and Bcl/Bax ratio were seen in the LBS. TUNEL assay and hematoxylin-eosin staining showed a significant increase in TUNEL-positive cells and tissue damages in the uterus of the LBS. Although few DEGs were identified at the growth stage, similar uterine tissue damages were also observed in the LBS. The expressions of runt-related transcription factor 2 and osteocalcin were upregulated in humeri of the LBS. Enlarged diameter and more structural damages of endocortical bones and decreased ash were observed in femurs of the HBS. The lower eggshell breaking strength may be attributed to a declined Ca transport due to uterine tissue damages, which could affect eggshell calcification and lead to a weak ultrastructure. Impaired uterine Ca transport may result in reduced femoral bone resorption and increased humeral bone formation to maintain a higher mineral and bone quality in the LBS.