Betaine Promotes Milk Protein Synthesis via Alleviating Oxidative Stress Caused by NEFA in Mammary Epithelial Cells of Dairy Cows

ABSTRACT Background Fatty liver is highly prevalent in dairy cows and poses a significant challenge to the dairy industry by reducing both milk yield and quality, thereby inflicting substantial economic losses. Although dietary betaine supplementation has been demonstrated to enhance milk protein content, the underlying molecular mechanisms remain to be fully elucidated. Objectives This study aimed to elucidate the role of betaine in milk protein synthesis and the underlying mechanisms. Methods Blood, liver and mammary gland samples were obtained from healthy and fatty liver‐affected bovines. The serum NEFA levels, oxidation and antioxidant enzyme systems and milk protein synthesis‐related proteins expression were determined. In vitro, the bovine mammary epithelial cells (BMECs) were pretreated with 25 mM betaine/10 mM N‐acetyl‐L‐cysteine (NAC) and then stimulated with NEFA. Results The results indicated an elevation in serum NEFA levels accompanied by enhanced oxidative stress (OS) and reduced protein levels related to milk protein synthesis (phosphorylated (p)‐mechanistic target of rapamycin (mTOR)/mTOR, p‐ribosomal protein S6 kinase 1 (S6K1)/S6K1, p‐Janus kinase 2 (JAK2)/JAK2, p‐signal transducer and activator of transcription 5 (STAT5)/STAT5 and β‐casein) in dairy cows with fatty liver. In vitro, the results revealed betaine decreased the hydrogen peroxide (H 2 O 2 ), malondialdehyde (MDA), and oxygen free radicals (OFR) contents, and lactate dehydrogenase (LDH) activity in the supernatant, but increased the glutathione peroxidase (GPX), catalase (CAT), superoxide dismutase (SOD) and thioredoxin reductase (TrxR) activities; the reduced glutathione‐to‐oxidised glutathione (GSH/GSSG) ratio; and the total antioxidant capacity (T‐AOC); and elevated protein levels related to milk protein synthesis in NEFA‐treated BMECs. Conclusions These findings indicate that betaine reduces NEFA‐induced OS and enhances milk protein synthesis, suggesting its potential as a nutritional intervention for enhancing milk protein content in dairy cows with metabolic disorders.