Temporal and interanimal variation in bloodspot acylcarnitine and amino acid profiles in relation to conventional metabolites and hormones in Holstein dairy cows.

The transition from late gestation to early lactation in dairy cows involves dynamic metabolic adaptations orchestrated by homeorhetic mechanisms, including hepatic fatty acid and AA metabolism. To gain deeper understanding of these mechanisms, we evaluated changes in bloodspot acylcarnitines (AC) and free AA profiles, and conventional blood biomarkers of energy balance (BHB, nonesterified fatty acids [NEFA], glucose, insulin, IGF-1, and fructosamine) along with weekly milk composition and DMI in 2 sequential observational trials. Data were analyzed using correlation and cluster analysis, and linear mixed-effects models with and without repeated measures. Study 1, which involved 28 multiparous Holstein-Friesian cows sampled 7 d before calving and at 3, 6, 9 and 21 d after calving, revealed strong positive correlations between glycine-to-alanine ratio with BHB (r = 0.58) and NEFA (r = 0.59), though these correlations weakened in study 2. Four trajectory patterns in AC, AA, and metabolite ratios were identified. Group 1 (e.g., C5, C16, and C18) showed transient postpartum increase peaking by d 3 or 6, returning to prepartum levels by d 21. Group 2 (e.g., tyrosine, C0:(C16 + C18) exhibited transient postpartum decrease, normalizing by d 21. Group 3 (e.g., C4DC:C3, t[AC]:C0, and valine-to-phenylalanine ratio) displayed variable postpartum responses, whereas group 4 exhibited persistent differences at d 21, with elevated glycine-to-alanine and valine-to-leucine ratios but reduced methionine and ornithine-to-citrulline ratio compared with prepartum levels. Study 2, which examined intercow variations and comprised of 74 cows (83 lactations) sampled at 21 DIM, revealed 2 distinct clusters of clinically healthy cows based on longitudinal time serum BHB profiles: normal and high milk yield-hyperketonemia (HMY-HYK). The HMY-HYK cows had higher milk yield (41.6 ± 1.05 vs. 39.4 ± 0.767 kg/d), average serum BHB and NEFA (0.996 ± 0.086 vs. 0.754 ± 0.062 mmol/L and 0.498 ± 0.051 vs. 0.534 ± 0.071 mmol/L, respectively) and lower insulin (0.343 ± 0.030 vs. 0.368 ± 0.041ng/mL) compared with cows in the normal cluster. The higher milk yield, increased milk urea concentrations, and reduced bloodspot citrulline levels in the HMY-HYK cows suggest enhanced AA catabolism for gluconeogenesis and reduced activity in the ornithine-citrulline cycle. Elevated bloodspot malonylcarnitine, long-chain AC (LCAC) with C16 and C18 carbon chains, acetylcarnitine-to-free carnitine (C2:C0) ratio but lower free carnitine (C0) levels indicate efficient mitochondrial responses, potentially exporting acyl-CoA as C2 and LCAC to mitigate metabolic stress associated with elevated NEFA. In conclusion, bloodspot AC, AA, and metabolite ratios highlight time-dependent and interanimal shifts in adipose and muscle mobilization, as well as adaptive mitochondrial metabolism of NEFA and AA catabolism to support gluconeogenesis and thus, milk synthesis in early lactation.