Effects of Thymol Supplementation in Health Care Sand on Growth Performance, Serum Biochemical Parameters, Fecal Microbial Diversity, and Fecal Metabolites in Squabs

This study aimed to investigate the effects of thymol supplementation in health care sand on growth performance, serum biochemical parameters, fecal microbial diversity, and fecal metabolites in Grey King squabs. A total of 160 healthy 28-day-old Grey King squabs were selected and randomly assigned to four treatment groups: the CON, 2%Thy, 3%Thy, and 4%Thy groups. Each group consisted of 10 replicates with four squabs per replicate, resulting in a total of 40 squabs per group. Under identical feeding management conditions and dietary nutrient levels, squabs in the CON group received the basal diet plus 5 g of health care sand; those in the 2%Thy group received the basal diet plus 5 g of health care sand containing 2% thymol; those in the 3%Thy group received the basal diet plus 5 g of health care sand containing 3% thymol; and those in the 4%Thy group received the basal diet plus 5 g of health care sand containing 4% thymol. The experiment lasted for 30 days. During the experimental period, the pigeons were housed in cages measuring 45 cm × 50 cm × 60 cm. Each pigeon was provided with 40 g of diet and 5 g of health care sand daily and had free access to water. After Benjamini–Hochberg false discovery rate correction, no significant differences were observed in growth performance, body measurements, apparent nutrient retention, or energy metabolizability among treatments (q > 0.05). However, thymol-supplemented groups exhibited significantly increased serum total antioxidant capacity and superoxide dismutase, glutathione peroxidase, and catalase activities, together with decreased malondialdehyde levels (q < 0.05). Fecal microbiome and metabolomic analyses were restricted to the CON and 3%Thy groups, with the 3%Thy group selected after evaluation of the phenotypic and physiological responses rather than being prespecified before the experiment. These omics comparisons were therefore considered exploratory. No statistically significant difference in overall fecal microbial community composition was detected between the two groups, whereas 17 candidate differential fecal metabolite features were observed. These features were mainly putatively annotated as lipids and lipid-like molecules, organic acids and derivatives, organoheterocyclic compounds, and benzenoids. Among the supplementation levels evaluated, the 3%Thy group showed comparatively favorable numerical responses in several measured parameters and may be considered a candidate level for further investigation. However, the present findings do not establish an optimal thymol supplementation level, and the biological significance of the exploratory fecal omics results requires further validation.