2.2. Cell Culture and Treatment

MAC-T (a bovine mammary epithelial cell line) cells (a gift from associate researcher G. Xing, Jiangsu Academy of Agricultural Sciences) were incubated in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 medium (Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS) (ScienCell, Carlsbad, CA, USA) and 200 U/mL of penicillin and streptomycin (Hyclone, Logan, UT, USA). The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂.

The cells were divided into five groups through randomization: A control (C) group, heat stress (HS) group, low taurine (HS + LTau) group, moderate taurine (HS + MTau) group, and high taurine (HS + HTau) group. After the cells reached 70% confluence, prior to heat treatment, they were cultured for 24 h under DMEM/F12 with serum-free conditions. Then, the HS group was cultured at 42.5 °C for one hour and recovered at 37 °C for another 12 h; the taurine groups were pretreated with 10 mM, 50 mM, 100 mM of taurine for two hours followed by culturing at 42.5 °C for one hour and recovery at 37 °C for 12 h. The C group was continuously cultured in 37 °C and received no taurine treatment.