Combined chitosan and baicalein promote mammary involution during early dry period in dairy cows

The timely onset of mammary involution during early dry period is important for subsequent udder health and productive performance. However, mammary involution is also accompanied by oxidative stress and inflammation, and existing strategies rarely boost involution while reducing related damage. Chitosan (CS) has the potential to promote involution, while Baicalein (BAI) can counter oxidative stress, yet their combined effects during early dry period in dairy cows remain unclear. Two independent cow experiments assessed a combined chitosan/baicalein formulation (C/B) for non-antibiotic intramammary use. In a preliminary trial, 15 Holstein cows (n = 3 per group) received Vehicle (solvent control without C or B), Ceftiofur Hydrochloride dry-cow therapy (CEF), CS, BAI or C/B via intramammary infusion immediately after the final milking at dry-off. Compared with Vehicle, C/B increased somatic cell count, bovine serum albumin, and lactoferrin concentrations in mammary secretions, indicating enhanced involution-associated changes. C/B also improved antioxidant and inflammatory profiles, as shown by increased superoxide dismutase activity and reduced malondialdehyde, interleukin-6, and tumor necrosis factor-α levels. In a validation experiment, 54 cows were assigned to C/B, CEF, or untreated abrupt dry-off control groups (n = 18 cows/group). Compared with control groups, C/B reduced post-dry-off milk leakage and clinical signs suggestive of new intramammary infection. It also promoted the resolution of udder distension and recovery of eating time, with overall responses comparable to CEF and no evident biochemical safety abnormalities. To investigate the underlying mechanisms, mammary epithelial cells were treated with C/B in vitro. C/B (30 μg/mL) reduced the expression of tight junction-associated proteins, impaired cell migratory capability, induced mitochondrial dysfunction, and triggered transcriptomic changes associated with epithelial remodeling and apoptosis. These findings suggest that C/B may facilitate physiological mammary involution while mitigating oxidative stress and inflammation, thereby supporting dry-off management and offering a potential non-antibiotic approach in dairy production.