Biplot analysis and correlations between changes in terpenes in calf milk and immune variables, performance and rumen variables

This study aimed to investigate the effects of selected terpenes derived from thyme, sage, and lavender essential oils on growth performance, rumen fermentation parameters, and immune responses in pre-weaned Holstein calves. A total of 32 calves were randomly assigned to four groups: a control group receiving whole milk and starter feed, and three treatment groups supplemented with thyme (150 µL/day), sage (150 µL/day), or lavender oil (1500 µL/day) in milk. The chemical composition of the aromatic oils was determined by gas chromatography/mass spectrometry (GC/MS), and major terpenes included thujone, cineole, camphor, cis-ocimene, α-caryophyllene, terpinen-4-ol, phellandrene, linalool, and linalyl acetate. Biplot and Pearson correlation analyses revealed distinct associations between specific terpenes and physiological responses. Cineole (r = 0.61), thujone (r = 0.53) camphor (r = 0.58), camphor (r = 0.53), cis-ocimene (r = 0.68), α-caryophyllene (r = 0.62) and terpinene_4_ol (r = 0.48) were positively correlated with feed intake (FI) P < 0.01). Thujone, cis-ocimene and α_caryophyllene were acetic (AA), butyric (BA), propionic (PA), and iso-valeric (IVA) acids (P < 0.05) in calf rumen fluid. The ruminal ammonia-nitrogen (NH₃-N) level was negatively correlated with α-caryophyllene (r= -0.39) and thujone (r= -0.44), but positively correlated with terpinene_4_ol, phellandrene and linalyl acetate (P < 0.05). The tumor necrosis factor-α (TNF-α) showed inverse correlations with oxygenated monoterpenes, particularly cineole (r = - 0.51), camphor (r = - 0.50), terpinene_4_ol (r= -0.44), linalyl_acetate (r= -0.39) and linalool (r= -0.41) (P < 0.05). These findings suggest that certain terpenes may have dual functionality, improving both digestive fermentation and immune modulation during early calf development. In conclusion, the study demonstrates that targeted terpene supplementation via aromatic oils can beneficially influence rumen fermentation and immune responses in neonatal calves. These results highlight the potential of phytogenic compounds as functional feed additives in antibiotic-free calf-rearing systems.