54 CHEMICAL ENUCLEATION OF IN VITRO-MATURED BUFFALO (BUBALUS BUBALIS) OOCYTES INDUCED BY DEMECOLCINE FOR HANDMADE SOMATIC CELL CLONING

Abstract

Cloning by somatic cell nuclear transfer requires enucleation of the recipient oocyte to remove its genetic material. In handmade cloning, a simplified cloning procedure (Vajta et al. 2001 Cloning 3, 89–95), zona-free oocytes are manually bisected, stained, and exposed to UV light for selection of demi-oocytes devoid of chromosomes. Development of procedures for enucleation which avoid exposure to UV light and conserve most of the cytoplasmic volume are necessary for improving the efficiency of handmade cloning. Chemically assisted enucleation protocols involve treating cumulus–oocyte complexes (COCs) during IVM with cytoskeleton-modifying agents, like demecolcine, which induce protrusion cone formation on the surface of the oocytes (Tani et al. 2006 Cloning Stem Cells 8, 61–66). Such a cone-like structure can then be easily excised with a microblade and enucleation of the oocyte achieved without a significant loss of cytoplasm. The aim of the present study was to establish an efficient protocol for demecolcine treatment of buffalo COCs during IVM to obtain the maximal proportion of oocytes where chromosomes are either expelled into the surface protrusion cone or completely enucleated. In Experiment I, COCs (n = 244), obtained from slaughterhouse buffalo ovaries and matured in vitro in TCM-199 (containing 10% FBS, 5 µg mL^–1 pFSH, and 0.81 mm sodium pyruvate) at 38.5°C (in 5% CO₂, 90–95% relative humidity), were treated in two groups with demecolcine (0.5 µg mL^–1) beginning at either 15 h or 19 h from the start of IVM up to the end of IVM at 22 h, and compared to a control group (without demecolcine). Data were analyzed using ANOVA. The proportion of oocytes where a protrusion cone was observed was greatest (P < 0.05) in the 15-h treatment group (84%, n = 72/86) compared to those in either the 19-h or control groups (58%, n = 46/78, and 60%, n = 48/80, respectively). The presence of demecolcine for the last 7 h of IVM appears to have a significant effect on protrusion cone formation in buffalo oocytes. In Experiment II, COCs (n = 276)were divided into four groups, matured in vitro this time for 24 h and treated with demecolcine (0.5 µg mL^–1) from the onset of IVM and up to 18, 21, or 24 h, and compared to a control group without demecolcine. Protrusion cone formation was observed in 72% (52/72), 66% (40/60), 62% (40/64), and 70% (56/80) of oocytes, respectively, in these groups. These percentages did not differ significantly (P < 0.05). However, the 21-h treatment resulted in complete enucleation in 32% (20/64) of oocytes, which was significantly greater (P < 0.05) than that in the other three groups where no such enucleation was observed. It can be hypothesized that demecolcine-free treatment from 21 to 24 h may have assisted in inducing complete enucleation of a significant number of treated oocytes. In conclusion, these results show that demecolcine-assisted and induced enucleation procedures can be used for increasing the efficiency of oocyte enucleation in handmade cloning and other nuclear transfer procedures in buffalo.