Effect of cold stress on cell morphology and cell viability. Cells were allowed to grow in the incubator at 28°C in 5% CO2 and then treated with low temperature (13 and 18°C). Ctr, the control group; Dg, the domesticated group; Ng, the non-domesticated group. (A) Morphological pictures of ZF4 cells under normal or low temperature conditions. Cell morphology was examined under an inverted microscope at a 20 × magnification. Scale bar = 100 μm. (B,C) The CKK-8 assay was used to measure the viability of groups of short-term low temperature stress and long-term low temperature acclimation cells. The experiments were repeated three times. The results are presented as the mean ± SD (n = 6); *P < 0.05, **P < 0.01.

Low temperature acclimation helps to maintain the ZF4 cell cytoskeleton. F-actin was labeled with rhodamin-phalloidin following exposure to low temperatures at different time points in ZF4 cells. The cell cytoskeletons were stained with phalloidin (red) and the staining of nuclei was performed with DAPI (blue). Laser scanning confocal microscopy was used to visualize the cytoskeleton of the cell. Scale bar = 25 μm. Ctr, the control group; Dg, the domesticated group; Ng, the non-domesticated group.

Effects of cold stress on the antioxidant capacity of ZF4 cells. The effects of short-term low temperature stress and long-term cold acclimation on the antioxidant activities of SOD (A,B), CAT (C,D), and GSH-Px (E,F) in the ZF4 cells were measured using the corresponding detection kits. Ctr, the control group; Dg, the domesticated group; Ng, the non-domesticated group. The experiments were repeated three times. The results are presented as the mean ± SD (n = 3); *P < 0.05, **P < 0.01.

Cold acclimation suppressed cold-induced oxidant stress in ZF4 cells. Ctr, the control group; Dg, the domesticated group; Ng, the non-domesticated group. (A,B) Cellular ROS production under cold stress. ROS was detected by flow cytometry and analyzed using FlowJo software. The results are presented as the mean ± SD (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001.

The effect of cold stress on mitochondrial membrane potential. Ctr, the control group; Dg, the domesticated group; Ng, the non-domesticated group. (A) Representative images were scanned by a confocal microscope after staining with JC-1. The red fluorescence indicates high mitochondrial membrane potential, while the green indicates low mitochondrial membrane potential. Scale bar = 50 μm. (B,C) Quantitative results of mitochondrial membrane potential by flow cytometry. Each experiment was performed in triplicate. The results are presented as the mean ± SD (n = 3); *P < 0.05, **P < 0.01.

Cold acclimation alleviated cold-induced cell apoptosis in ZF4 cells. Ctr, the control group; Dg, the domesticated group; Ng, the non-domesticated group. (A) Analysis of cell apoptosis by flow cytometry. Apoptosis was evaluated with AnnexinV-FITC/PI staining. (B,C) Apoptotic cells were calculated and graphed according to the flow cytometry results in GraphPad Prism 8 software. The results are presented as the mean ± SD (n = 3); **P < 0.01.