Our previous studies demonstrated prompt elevation of proteinase activity in mammary secretion of drying-off cows and goats. The current study examined the progressive changes in composition of cow mammary secretion following drying-off and, in parallel, characterized the mode of peptide neogenesis using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography-electrospray-ionization (LC-ESI) MS/MS. The results show that the percentage of casein of total milk protein at time of drying-off was 76%, which dropped to 41%, 24%, and 16%, respectively, 1, 2, and 3 weeks after drying-off. Levels of β-lactoglobulin and α-lactoalbumin in mammary secretions of drying-off cows decreased prominently while levels of lactoferrin, BSA, and casein derived-proteins increased concomitantly compared with regular milk. A fractionation procedure was applied to remove molecules larger than 10 kDa before MALDI-TOF MS and LC-ESI MS/MS and the results show that the MALDI-TOF MS peptide profile of mammary secretion ranging from m/z 600 to 4000 was apparently modified after drying-off for 1 week, whereas species 1590 m/z and 2460 m/z were most obviously enriched compared with regular milk. LC-ESI MS/MS results were used to map peptide sequence with Mascot search server and under no post translational modification to reduce database size and 202 novel β-casein-derived peptides were successfully identified in mammary secretion after drying-off for 1 week in contrast to regular milk. Accordingly at least 48 additional cleavage positions were assigned on β-casein for mammary secretion. Among the 202 novel peptides, 5 are homologous with confirmed opioid agonists, angiotensin 1-converting enzyme inhibitors, or immuno-modulators. In conclusion, peptides are released in situ from milk proteins within short intervals following drying-off in cows. They might play roles in the transition of mammary glands from lactating to non-lactating. With specified post-translational modifications and focused functional screening, novel peptides are yet to be discovered in dry cow mammary secretion.

Interactions between inflammatory cytokines and plasminogen (Pg) activation system on immune cells are yet to be established. In previous studies we reported a somatic cell-associated elevation of proteolytic activity in mammary secretion of drying-off goats and cows. The purposes of the present study were to examine the role of TNF-α in polymorphonuclear neutrophil (PMN)-associated Pg activation, and the significance of this activation pathway for overall plasmin (Pm) activity in mammary secretion of drying-off cows. Results of experiments in vitro showed that the spontaneous Pg activation observed on fresh preparations of bovine blood PMN was completely blocked by anti bovine TNF-α antibody, and was further up-regulated by exogenous bovine TNF-α. Monitoring the parameters of mammary secretion of drying-off cows revealed that both somatic cell counts and differential PMN ratio was significantly elevated at weeks 1, 2 and 3 of milk stasis. Nevertheless, specific activity of soluble Pm in mammary secretion increased and the level of 17-kDa TNF-α decreased immediately following milk stasis. Iimmunoblotting revealed that although both 26-kDa pro-TNF-α and 17-kDa TNF-α were consistently present in somatic cells of mammary secretion collected at weeks 0, 1, 2 and 3 of milk stasis, only 26-kDa pro-TNF-α was present in somatic cells of milk during lactation. In-vitro assay indicated that cell-free mammary secretion of drying-off cows exerted no Pg activation bioactivity towards bovine blood PMN. Altogether, the current study suggests the existence of an active TNF-α-Pg-Pm autocrine/paracrine loop on the massively infiltrated PMN inside udders of drying-off cows, which involves extensive binding and internalization of 17-kDa TNF-α on PMN and consequently activation of Pg, resulting in high Pm activity and low 17-kDa TNF-α level in mammary secretion. These coordinated mechanisms may play a role in the defence of drying-off mammary gland.