Longitudinal characterization of fecal lipids in dairy heifers from birth to first calving

The developmental phases of dairy heifers, from birth to first calving, are characterized by major changes in nutrition, gastrointestinal tract physiology and metabolism. Fecal metabolomics offers a powerful, noninvasive approach to understanding the intricate interplay between host metabolism, gut health, and the evolving gut microbiota. However, the longitudinal dynamics of fecal lipids are poorly characterized across these phases. The aim of this study was to characterize and quantify fecal lipid classes and their temporal dynamics in Holstein heifers at key time points from birth to first calving. For this purpose, 10 Holstein heifers were prospectively enrolled and sampled at 5 critical developmental stages: within 12 h after birth, preweaning (wk 6), at weaning (wk 14), postweaning (mo 8), and at first calving (mean age 26 ± 2.3 mo). Fecal samples were collected rectally at each stage and analyzed by targeted metabolomics (MxP Quant 500 kit) using flow injection analysis-tandem mass spectrometry with normalization to internal standards and strict quality control. Multivariate analyses, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and hierarchical clustering, were used to assess temporal changes in fecal lipid composition. The PCA model explained 69.3% of the total variance (principal components 1, 2, and 3: 39.7%, 18.8%, and 10.8%, respectively). Subsequent permutational multivariate ANOVA (999 permutations) indicated significant stage effects (R² = 0.76). Additionally, PLS-DA, employing 5 components, demonstrated high explanatory power (R² = 0.98, Q² = 0.87), validated through permutation testing. Fecal fatty acids profiling showed a persistent dominance of myristic acid (C14:0) throughout the observation period, suggesting a process independent of direct dietary inputs and possibly related to microbial synthesis or lipid metabolism between host and microbe. Further consideration of the microbial contribution to myristic acid production may have implications of intestinal lipid metabolism processes as well as host-microbe interactions with physiological relevance to gut health. Longitudinal assessments on triglycerides, diglycerides, and monoglycerides revealed stage-dependent changes in lipid metabolism in relation to the process of digestive lipolysis and dietary transition from birth to calving. Additionally, the data indicated shifts toward phospholipids and sphingolipids after the colostrum phase and particularly increased abundances of phosphatidic acid, phosphatidylinositol, and lysophospholipids during the preweaning developmental stage, signaling membrane remodeling via the Lands cycle associated with developmental changes in gut function and immunity. Stage-dependent changes were also evident for fecal cholesteryl esters and acylcarnitines, with early elevations after birth and again during the preweaning stages. This may infer distinct lipid transport and energy metabolism and later fluctuations in signaling physiological changes in response to dietary transitions and rumen development. These findings provide a comprehensive overview of fecal lipid dynamics throughout heifer development, detailing the metabolic signatures related to key physiological changes. Collectively, the findings underscore the potential of fecal lipidomics as a noninvasive approach to monitoring gut maturation, dietary adaptation, and host-microbiota interactions from birth to calving.