211 IN VITRO PRODUCTION OF SWAMP BUFFALO EMBRYOS BY INTRACYTOPLASMIC SPERM INJECTION: EFFECT OF CHEMICAL ACTIVATION TREATMENTS

Abstract

Intracytoplasmic spern injection (ICSI) in the buffalo has not yet been well examined. Several factors involved affect the success rates of this technique, particularly the postinjection activation procedure. The objective of this study was to evaluate the effects of chemical activation treatments on in vitro development of oocytes after ICSI. A single spermatozoa was injected into the cytoplasm of an in vitro-matured oocyte using a micromanipulator under an inverted microscope. The ICSI oocytes were assigned to the following chemical activation treatments: (1) exposed to 5 μm ionomycin (Io) in Emcare medium for 5 min and placed in Emcare medium for 3 h, or (2) exposed to 7% ethanol (EtOH) in Emcare medium for 5 min and placed in Emcare medium for 3 h. The treated oocytes that extruded a second polar body were then selected and cultured either in (A) 1.9 mm 6-dimethylaminopurine (6-DMAP) in mSOF medium for 3 h, or (B) 10 μg mL^–1 of cychloheximide (CHX) for 5 h. The treated oocytes were further cultured in mSOF medium supplemented with 3 mg mL^–1 of fatty acid-free BSA at 38.5°C under a humidified atmosphere of 5% O₂, 5% CO₂, and 90% N₂ for 2 d. Thereafter, 8-cell-stage embryos were selected and co-cultured with buffalo cumulus cells in mSOF medium at 38.5°C under a humidified atmosphere of 5% CO₂ in air for another 5 d. The medium was changed daily and the development of embryos was recorded at the same time the medium was changed. The sham-injected oocytes were treated and cultured along with ICSI oocytes. With 8 replications for each activation treatment, 336 oocytes were used for ICSI. With 6 replications for each activation treatment, 211 oocytes were used for sham injection. The cleavage of ICSI oocytes treated with Io + 6-DMAP, EtOH + 6-DMAP, and EtOH + CHX was 76.2, 69.4, and 78.3%, respectively, which was significant higher (P < 0.01) than ICSI oocytes treated with Io + CHX (52.4%) and also significant higher (P < 0.01) than sham-injected oocytes in all treatments. The highest blastocyst rate was observed in ICSI oocytes treated with Io + 6-DMAP (28.6%), which was not significantly different from ICSI oocytes treated with EtOH + CHX (24.4%). The blastocyst rates of ICSI oocytes treated with Io + 6-DMAP and EtOH + CHX were significantly higher than ICSI oocytes treated with Io + CHX (5.9%) and EtOH + 6-DMAP (16.5%) and also were significantly higher than sham-injected oocytes in all treatments. In conclusion, our study demonstrated that activated ICSI of swamp buffalo oocytes with Io + 6-DMAP or EtOH + CHX gave the highest cleavage and blastocyst rates.

This work was supported by the Thailand Research Fund and Suranaree University of Technology.