Effect of different transport temperatures on in vitro maturation of oocytes collected from frozen-thawed sheep ovaries

摘要

The aim of this study was to determine the effects of 2 different transport temperatures on the in vitro maturation of oocytes collected from frozen-thawed sheep ovaries. Sheep ovaries were transferred into saline at temperatures of 4 °C and 32 °C.After the 2 experimental groups (A: fresh cortex, B: frozen-thawed cortex) were formed, each group was divided into 2 subgroups (group Al: 4 °C, group A2: 32 °C [control]; group Bl: 4 °C, group B2: 32 °C). The cortexes were dissected into slices 1-3mm thick and pieces of 0.5 cm~2. For groups Bl and B2, 1-2 cortex pieces were placed in cryogenic vials containing 1 mL of freezing medium modified with Earle's salts (TCM-199) and supplemented with 10% fetal calf serum (FCS) (FCS + 2.5 M ethylene glycol+ 0.1 M sucrose). The vials were then cooled to -7 °C at 2 °C/min and held at -7 °C for 10 min for manual seeding. The temperature was then lowered by -0.3 °C/min to -35 °C and thereafter by -10 °C/min to -75 °C. Vials were plunged into -196 °Cliquid nitrogen and stored. Cortexes were thawed at 37 °C. Collected oocytes were matured in their own groups in 700 mu L of TCM-199 (supplemented with luteinizing hormone, follicle-stimulating hormone, pyruvate, and FCS) for 23 h in a gas mixture of 5%CO_2, 5% O_2, and 90% N_2 at 38.8 °C. After maturation, oocytes were fixed in acetic acid and ethyl alcohol (1:3) for 48 h. Oocytes were stained with aceto-orcein and then examined. At the end of the study, maturation rates for reaching metaphase I (MI) were similar in all groups (group Al: 30.76%, group A2: 38.09%, group Bl: 30.65%, and group B2: 33.33%). The rates at which metaphase II (Mil) was reached were 18.58%, 34.69%, 7.25%, and 6.48%, respectively. The best development was seen in group A2 (P< 0.001). Sheep oocytes obtained from fresh and frozen-thawed cortexes reached the Mil stage if transported at 4 °C.