100 EFFECTS OF USING MICRO-PIPETTE TIPS OF DIFFERENT DIAMETERS AND VOLUME OF VITRIFICATION SOLUTION ON THE VIABILITY OF IVM BOVINE OOCYTES AFTER VITRIFICATION

Abstract

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 2–5 mm follicles of ovaries obtained at a local abattoir. COCs were matured for 19 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 mg mL⁻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 3 min, and then equilibrated for 1 min in 15% EG, 15% DMSO, and 0.5 M 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 0.5 M sucrose; the tips maintained in this medium for 5 min. After washing in HM, oocytes underwent an additional 3 h of maturation. They were then subjected to IVF (Day 0). After IVF, morphologically intact oocytes were cultured. Oocytes matured for 20–21 h 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 0.5 μL 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 (150 μm), 62 (200 μm), and 54 (275 μm). The cleavage rates of morphologically intact oocytes at Day 3 of 150 μm (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 1 μL (60 eggs) of VS in micro-pipette tips with 200-μm ID. The number of morphologically intact oocytes was 40 (0.3 μL), 32 (0.5 μL), and 28 (1 μL). The cleavage rates of morphologically intact oocytes at Day 3 of the 0.3 μL (45.0%), 0.5 μL (37.5%), and 1 μL 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 0.3 μL (15.6%), 0.5 μL (28.1%), and 1 μL 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 0.5 μL of VS.