Heat Shock-Induced Apoptosis in Preimplantation Bovine Embryos: MATERIALS AND METHODS(6)

MATERIALS AND METHODS(6)

Heat-induced apoptosis in bovine embryos at the 2- or 4-cell stage. Embryos at the 2- or 4-cell stage were collected at 28-30 and 37-39 h after insemination, respectively. Embryos were transferred to a new drop of modified KSOM and cultured at 38.5°C for 24 h or were heat shocked at 41°C for 9 h followed by 38.5°C for 15 h. Embryos were fixed in 4% paraformaldehyde, transferred to a poly-L-lysine coated slide and saved at 4°C until analysis by TUNEL. The experiment was replicated seven times using 491 embryos (94-146 embryos/group).

Heat-induced apoptosis in bovine embryos at the 8- to 16-cell stage on Day 3 after insemination. Embryos at the 8- to 16-cell stage were collected on Day 3 after insemination and transferred to a new drop of modified KSOM. Embryos were then cultured at 38.5°C for 24 h or were heat shocked at 40 or 41°C for 9 h followed by 38.5°C for 15 h. At the end of culture, embryos were fixed and subjected to TUNEL analysis. The experiment was replicated three times using 188 embryos (52-64 embryos/group).

Induced thermotolerance in bovine embryos at the 8- to 16-cell stage on Day 4 after insemination. Embryos at the 8- to 16-cell stage were collected on Day 4 after insemination and transferred to a new drop of KSOM (15-30 embryos per drop). Embryos were then subjected to four treatments: control (38.5°C for 24 h), mild heat shock (40°C for 80 min followed by 38.5°C), severe heat shock (41°C for 9 h followed by 38.5°C for 15 h), or thermotolerance (40°C for 80 min followed by 2 h at 38.5°C and 9 h at 41°C). At 24 h after initiation of temperature treatment, embryos were fixed in 4% paraformaldehyde, transferred to a poly-L-lysine coated slide and saved at 4°C until analysis by TUNEL. The experiment was replicated four times using 473 embryos (111-120 embryos/group).

Caspase activity in bovine embryos. Embryos at the 2-cell stage or a 16-cell stage were collected 28-30 h after insemination or on Day 5 after insemination, respectively. Embryos were transferred to a new drop of modified KSOM and cultured at 38.5°C for 24 h or heat shocked at 41°C for 9 h followed by 38.5°C for 15 h. After heat shock, embryos were washed three times in a 50-|xl drop of prewarmed Hepes-TALP and analyzed for caspase activity as described above. The experiment was replicated five times using 350 embryos. Caspase activity was quantified using 135 embryos produced in two replicates (22-42 embryos/group).

Statistical Analysis

Data were analyzed by least-squares analysis of variance using the General Linear Models procedure of SAS. Percentage data were transformed using the arcsin transformation before analysis. Dependent variables were total cell number, percentage of apoptotic cells, and the arcsin of the percentage of cells displaying apoptosis. Independent variables varied according to the experimental design and included treatment, day (i.e., replicate), and stage of embryonic development. The mathematical model included main effects and all interactions. All main effects were considered fixed. Orthogonal contrasts and a means separation procedure of SAS called pdiff were performed when appropriate to determine differences between levels of individual treatments.

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