100EFFECTS OF USING MICRO-PIPETTE TIPS OF DIFFERENT DIAMETERS AND
VOLUME OF VITRIFICATION SOLUTION ON THE VIABILITY OF IVM BOVINE OOCYTES AFTER
VITRIFICATION
The objective of this study was to investigate the effects of the diameters of micro-pipette tips and the volume of vitrification solution (VS) on viability of IVM bovine oocytes after vitrification. COCs were aspirated from 2–5mm follicles of ovaries obtained at a local abattoir. COCs were matured for 19h in TCM-199 supplemented with 5% calf serum (CS) and 0.02mgmL−1 FSH at 38.5°C in an atmosphere of 5% CO2 in air. The matured oocytes were then vitrified on the basis of Kuwayama and Kato (2000 J. Assist. Reprod. Genet. 17, 477 abst). Matured oocytes were first exposed to 7.5% ethylene glycol (EG) and 7.5% DMSO in holding medium (HM; Dulbecco’s PBS supplemented with 20% CS) for 3min, and then equilibrated for 1min in 15% EG, 15% DMSO, and 0.5M sucrose in HM. Ten oocytes were loaded into each micro-pipette tip (MidAtlantic Diagnostics, Inc., Marlton, NJ, USA), and directly plunged into liquid nitrogen. Warming was performed by placing the narrow end of the micro-pipette tips directly into HM containing 0.5M sucrose; the tips maintained in this medium for 5min. After washing in HM, oocytes underwent an additional 3h of maturation. They were then subjected to IVF (Day 0). After IVF, morphologically intact oocytes were cultured. Oocytes matured for 20–21h were used as a control. The cleavage rate at Day 3 and blastocyst rate at Day 7 to 9 were based on the number of cultured oocytes, and analyzed using the chi-square method. In experiment 1, the oocytes were vitrified with 0.5μL of VS in micro-pipette tips with 150-, 200-, or 275-μm inner diameters (ID) (100 eggs per tip size). The number of morphologically intact oocytes was 64 (150μm), 62 (200μm), and 54 (275μm). The cleavage rates of morphologically intact oocytes at Day 3 of 150μm (45.3%) and 200-μm tips (45.2%) were significantly lower than that of 275-μm tips (53.7%) and the control (63.6%) (P<0.05). The blastocyst rate of morphologically intact oocytes at Day 7 to 9 of 150-μm (9.4%) and 275-μm tips (14.8%) were significantly lower than that of the control (33.0%) (P<0.05), and that of 200-μm tips (19.4%) also showed a tendency of being lower than that of the control (P<0.1). In experiment 2, the oocytes were vitrified with 0.3 (70 eggs), 0.5 (60 eggs), or 1μL (60 eggs) of VS in micro-pipette tips with 200-μm ID. The number of morphologically intact oocytes was 40 (0.3μL), 32 (0.5μL), and 28 (1μL). The cleavage rates of morphologically intact oocytes at Day 3 of the 0.3μL (45.0%), 0.5μL (37.5%), and 1μL solutions (35.7%) were significantly lower than that of the control (67.6%) (P<0.05). However, there were no differences in the blastocyst rate of morphologically intact oocytes at Day 7 to 9 among 0.3μL (15.6%), 0.5μL (28.1%), and 1μL solutions (17.9%), and control (23.9%). These results suggest that the viability of IVM bovine oocytes after vitrification may be improved by using micro-pipette tips with 200-μm ID and containing 0.5μL of VS.