ABSTRACT
Resveratrol (3,5,4'-trihydroxystilbene, RSV) has been widely used in mammalian cells, but whether it can be used during freezing boar semen is still unknown. The effects of RSV treatment during boar semen freezing on its anti-freezing ability, apoptosis, and possible apoptotic pathways were observed in this study. Sperm motility, mitochondrial membrane potential (ΔΨ), adenosine triphosphate (ATP) content, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive apoptotic state, and messenger RNA (mRNA) expression levels of apoptotic genes involved in different apoptotic pathways after freezing with or without RSV treatment were tested. The results showed that: (1) Compared with fresh sperm, the motility, normal acrosome rate, and plasma membrane integrity rate of frozen boar sperm decreased significantly (P0.05), but it did significantly improve the normal acrosome rate (57.65% vs. 47.00%, P<0.05) and plasma membrane integrity rate (46.67% vs. 38.85%, P<0.05). (2) After freezing, most boar sperm showed low mitochondrial ΔΨ. RSV treatment could increase the rate of high mitochondrial ΔΨ of boar sperm. (3) RSV treatment significantly decreased reactive oxygen species (ROS) levels (58.65% vs. 88.41%, P<0.05) and increased the ATP content (0.49 μmol/L vs. 0.25 μmol/L, P<0.05) of boar sperm during freezing. (4) The apoptotic rate of the freezing group (80.41%) with TUNEL detection increased significantly compared to the fresh group (9.70%, P<0.05), and RSV treatment greatly decreased the apoptotic rate (68.32%, P<0.05). (5) Real-time polymerase chain reaction (RT-PCR) showed that not only the genes from the death receptor-mediated apoptotic pathway (tumor necrosis factor-α (TNF-α), Fas ligand (FasL), and Caspase-8), but also the genes from the mitochondria-mediated apoptotic pathway (manganese superoxide dismutase (MnSOD), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-9) were both significantly changed after freezing. RSV treatment during freezing greatly changed their expression levels. Although RSV treatment during boar semen freezing did not significantly increase motility after thawing, it still played an efficient antioxidant role, which could enhance the mitochondrial function and decrease the apoptotic level induced by both the death receptor- and mitochondria-mediated apoptotic pathways.
ABSTRACT
A simple method of trypsin/collagenase I alternative digestion and iterative culture flask adherence to discard fibroblasts for bovine mammary cell culture was established in this study. By immunohistochemistry, flow cytometry, western blot, Electron microscopy analysis, the characteristics of bovine mammary cells were investigated in vitro. Effect of hyperthermia on the cell ultrastructures was also observed. The results showed that the mammary cells were diploid epithelia with intact 30 pairs chromatins, which could secrete alpha-casein into the medium. After exposed to hyperthermia, the cell condensed chromatin like crescent on the nuclei verges, mitochondria occurred expansion and vacuolization, and apoptotic bodies appeared, which suggested that heat stress could induce apoptosis of the mammary epithelia.
Subject(s)
Animals , Cattle , Female , Apoptosis , Blotting, Western , Caseins , Metabolism , Cell Line , Cell Nucleus , Cells, Cultured , Chromatin , Epithelial Cells , Cell Biology , Metabolism , Flow Cytometry , Hot Temperature , Immunohistochemistry , Mammary Glands, Animal , Cell Biology , Metabolism , Microscopy, Electron, Transmission , Mitochondria , Vimentin , MetabolismABSTRACT
Dairy cow mammary tissue was cultured in superfusion system or stationary system, and the influence of these two methods in the activity and ultrastructure of tissue was investigated according to LDH vigor, trypan blue dying, agrose gel electrophoresis, transmission microscope observation. The results showed that the mammary tissue cultured in superfusion system could keep normal tissue activity and ultrastructure within 12-60 h in DMEM plus 10% calf serum, while mammary tissue stationary culture could keep normal tissue activity and ultrastructure within 60-108 h. Both culture systems had some advantages and disadvantages.