Supplementary Materials.docx

This study aimed to compare ivermectin residue concentrations in milk following pour-on administration to Holstein cows at different stages of the dry period and to determine the necessary milk withdrawal period. Thirty dry cows were evenly divided into three groups based on estimated calving dates and received a single pour-on treatment of ivermectin at 0.5 mg/kg BW either 10, 20, or 30 days before these dates. Milk samples were collected twice daily for seven consecutive days beginning on the second day after calving. The marker residue, 22,23-dihydroavermectin B1a, was measured using UPLC-MS/MS analysis, with the method showing strong correlation coefficients between 2.5 and 20 ng/mL. The limits of detection (LOD) and quantification (LOQ) were 2 ng/mL and 2.5 ng/mL, respectively. The concentration-time data were analyzed using a non-compartmental model, and the milk withdrawal period was calculated with WTM1.4 software. Results showed that when cows received ivermectin more than 10 days before calving, residue concentrations in milk were undetectable or present only in trace amounts, well below China’s MRL for ivermectin (10 ng/mL). In contrast, cows treated within 10 days before calving had a milk elimination half-life (t_1/2λz) of 3.8 days for 22,23-dihydroavermectin B1a, with a peak concentration in milk of 13.75±8.12 ng/mL at 1.70±1.68 days post-calving. Based on these findings, no milk withdrawal period is required if the interval between administration and calving exceeds 10 days; however, a 4-day milk withdrawal period after calving is recommended if the interval is shorter.