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

Abstract

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 degrees C and 32 degrees C. After the 2 experimental groups (A: fresh cortex, B: frozen-thawed cortex) were formed, each group was divided into 2 subgroups (group A1: 4 degrees C, group A2: 32 degrees C [control]; group B1: 4 degrees C, group B2: 32 degrees C). The cortexes were dissected into slices 1-3 mm thick and pieces of 0.5 cm(2). For groups B1 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 degrees C at 2 degrees C/min and held at 7 degrees C for 10 min for manual seeding. The temperature was then lowered by 0.3 degrees C/min to -35 degrees C and thereafter by -10 degrees C/min to -75 degrees C. Vials were plunged into -196 degrees C liquid nitrogen and stored. Cortexes were thawed at 37 degrees 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% CO2, 5% O-2, and 90% N-2 at 38.8 degrees 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 A1: 30.76%, group A2: 38.09%, group B1: 30.65%, and group B2: 33.33%). The rates at which metaphase II (MII) 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 MII stage if transported at 4 degrees C.