Integrated transcriptomic and redox–immune responses reveal age-dependent regulation following surgical castration in calves

The objective of this study was to examine the influence of age on redox, immune, and transcriptional responses following surgical castration in calves. Twelve Wagyu calves were allocated to two age groups: 2 months (n = 6) and 4 months (n = 6). Blood was collected before castration and at 6, 24, and 72 hours after castration. Nitric oxide and total antioxidant capacity were measured. Gene expression (IFN-γ, TNF-α, and IL-10) was quantified using quantitative real-time PCR. T-lymphocyte subsets (CD4⁺, CD8⁺, and γδ T cells) were determined by flow cytometry. RNA sequencing of peripheral blood mononuclear cells collected at 6 hours after castration was performed, followed by differential gene expression and pathway enrichment analyses. Nitric oxide concentrations were higher in 4-month-old calves than in 2-month-old calves (p < 0.05), whereas total antioxidant capacity decreased progressively after castration regardless of age (p < 0.05). IFN-γ and TNF-α expression increased significantly at 6 hours after castration, regardless of age. IL-10 expression exhibited an age × time interaction, with 2-month-old calves showing an enhanced anti-inflammatory response at 24-72 hours after castration (p < 0.05). Percentages of CD4⁺, CD8⁺, and γδ T cells changed significantly over time following castration (p < 0.05), and CD8⁺ T-cell percentages were additionally influenced by age (p < 0.05), with 2-month-old calves showing a greater overall magnitude of response. Transcriptomic analysis identified 266 differentially expressed genes between age groups (164 upregulated, 102 downregulated in 4-month calves), with enrichment of immune- and inflammation-related pathways, including NF-κB, MAPK, TNF, IL-17, Toll-like receptor, and NOD-like receptor signaling pathways. Upregulated genes in 4-month calves included CCL14, CSF3, PTX3, ALOX15, and IL17REL, which are associated with inflammatory signaling and leukocyte activation. These findings demonstrate that castration induces coordinated immune and redox responses in calves, with both age and time influencing the magnitude and dynamics of these changes. Older calves exhibited stronger inflammatory and redox-associated responses, whereas younger calves demonstrated distinct regulatory cytokine responses and differences in CD8⁺ T-cell proportions. Integration of physiological, cellular, and transcriptomic analyses suggests age-associated differences in postoperative inflammatory and redox regulation following castration.