Effect of weaning duration (3 vs. 6 weeks) on plasma acylcarnitine and free fatty acid profiles in ad libitum–fed dairy calves

Early-life milk allowance is a major determinant of growth and metabolic programming in dairy calves, yet biochemical adaptation to weaning under elevated milk feeding remains insufficiently characterized. Plasma acylcarnitines provide an integrated readout of mitochondrial acyl-group transport and β-oxidation and can therefore capture systemic substrate partitioning during the transition from liquid to solid feeding. This study evaluated the effects of weaning duration (3 vs. 6 wk) on plasma acylcarnitine and free fatty acid profiles in dairy calves fed milk replacer (MR) ad libitum. Twenty Holstein calves were enrolled at approximately 1 wk of age, group housed and fed ad libitum MR (peak allowance up to 25 L/d) until wk 7. Calves also had access to concentrate and hay, and from wk 7 onward to ad libitum TMR, with identical solid-feed management across treatments. At wk 8, MR allowance was standardized to 12 L/d, and calves were stratified by BW and sex, then randomly assigned (n = 10/group) to a 3-wk or 6-wk linear step-down weaning protocol, with identical solid-feed management across groups. Plasma samples collected at wk 10, 13, and 16 were stored at -80°C and analyzed using the Biocrates MxP Quant 500 kit. As intended, calves assigned to the 6-wk protocol maintained higher MR intake for longer, whereas calves in the 3-wk group consumed more concentrate during the shared weaning period, consistent with partial compensation through solid feed intake. However, weaning duration had no overall main effect on average daily gain or BW, although both traits changed over time and showed sex-related differences in growth trajectories. Plasma acylcarnitines underwent pronounced temporal remodeling across the weaning transition, with sampling week explaining most multivariate variance. Free carnitine (C0) was affected by both age and weaning strategy and was higher in 6-wk calves during early transition stages. Short-chain acylcarnitines (including C2, C3, C3-DC/C4-OH, and C4) were primarily driven by developmental stage, consistent with maturation of intermediary metabolism, whereas weaning-duration effects were selective: C5, C5-DC, and C5:1 were greater in 6-wk calves, whereas C6 and C6:1 were higher in 3-wk calves, and C8 increased transiently after rapid milk reduction, suggesting acute changes in lipid flux and acyl-group handling. Medium-chain species were comparatively stable, although C10:2 remained higher in 6-wk calves later in transition, and most long-chain acylcarnitines were driven by age; nevertheless, C14:2, C16, and C16:1 displayed treatment-dependent trajectories, with higher concentrations in gradually weaned calves during transition. Plasma fatty acids showed parallel temporal restructuring, including declines in several C18-C20 and polyunsaturated fatty acids by wk 16, while treatment effects were selective and week dependent, and correlation network analysis demonstrated substantial rewiring of fatty acid-acylcarnitine associations from wk 10 to wk 16. Collectively, these findings indicate that ontogeny is the dominant driver of systemic metabolic remodeling during weaning under ad libitum MR feeding, whereas gradual 6-wk weaning selectively modulates mitochondrial lipid-handling pathways and may support a more stable oxidative adaptation during the shift from milk lipid dependence to a postweaning, rumen-driven metabolic phenotype.