Effect Of Methionine On Bovine Mammary Epithelial Cells After The Heat Treatment

The present study was performed to investigate the effects of methionine on mammary epithelial cells after the heat treatment. It measured the cell growth, antioxidant activity and apoptosis in response to high temperature and investigated the protective of methionine for the heat stress cows and their breast in the cellular level. This thesis includes two sections.1. Effects of heat stress to bovine mammary epithelial cells in vitro.Put the mammary cells with normal culture (37℃) into thermostat water bath at42℃and then immediately take back into the37℃for recovery. Cell samples were collected at different times (0,3,6,12,24,48h). The cell proliferation of mammary epithelial was assayed by MTT after heat treatment. The greatest recovery time would be selected in which impacted the cell proliferation most. The result showed that:After heat treatment of mammary epithelial cells for42℃,30min, the6h-treated decreased the cell viabilities compared to Oh (P<0.05). Therefore, the recovery time after heat treatment was6h for follow-up test.Cells was cultured in DMEM medium with different methionine concentrations (0,30,60,90,120mg/L) for2d. The37℃groups were placed at37℃water bath for0.5h, and42℃water bath of0.5h for the42℃groups with the recovery time of6h. Cell viability (proliferation) was detected by MTT assay. The content of malondialdehyde (MDA), the activities of superoxide dismutase (SOD) and lactate dehydrogenase (LDH) in the cell culture supernatant were determined by a colorimetric method. Apoptotic cells were detected by Annexin VFITC/PI double labeled flow cytometry. The results showed that: Compared with the37℃groups, LDH, MDA in42℃groups were increased and the cell viability and the levels SOD were decreased. The rate of cell apoptosis was increased. It suggested that heat treatment in42℃0.5h could lead a heat stress. But the stress was rendered insufficient because the heat treatment time was too short.2. Effects of different concentrations of methionine to the bovine mammary epithelial cell after heat treatment in vitro for the proliferation, antioxidant properties and apoptosis.Cells were cultured in DMEM medium with different methionine concentrations (0,30,60,90,120mg/L) for2d. Put the cells into water bath at42℃,0.5h then recovery6h at a temperature of37℃. Cell viability was detected by MTT assay. The content of malondialdehyde (MDA), the activities of superoxide dismutase (SOD) and lactate dehydrogenase (LDH) in the cell culture supernatant were determined by a colorimetric method. Apoptotic cells were detected by Annexin VFITC/PI double labeled flow cytometry. The result showed that:Compared with0mg/L methionine groups, cell viability of60mg/L methionine groups were increased (P<0.05); SOD levels could be increased when added30mg/L,60mg/L methionine in the medium of the heat treatment of mammary epithelial cells (P<0.05);60mg/L methionine groups were the lowest value in the content of MDA (P<0.05). The apoptosis was measured by flow cytometry. Adding30,60mg/L methionine could significantly improve cell viability, reduce early apoptosis rate (P<0.05), and adding60mg/L methionine was the most obvious. It suggested that methionine with concentration of30,60, mg/L could promote proliferation, antioxidant levels and anti-apoptotic of the bovine mammary epithelial cells in the high temperature.