Morphology of dromedary camel oocytes and their ability to spontaneous and chemical parthenogenetic activation

The present work was conducted to examine (1) the morphology of dromedary cumulus-oocytes complexes (COCs), (2) to study the incidence of spontaneous development of oocytes in vivo and (3) to assess the ability of in vitro matured dromedary oocytes to chemical parthenogenetic activation compared with in vitro fertilized (IVF) oocytes. COCs were recovered from dromedary ovaries classified according to their morphology into six categories. Oocyte diameter was measured using eye piece micrometer. For chemical activation, COCs with at least three layers of cumulus-cells were in vitro matured (IVM) in TCM 199 + 10 mu g/ml FSH + 10 IU hCG/ml + 10% FCS + 50 mu g/ml gentamycin. COCs were incubated for 40 h at 38.5 degrees C under 5% CO2 in humidified air. After IVM, matured oocytes with first polar body (first Pb) were divided into two groups. Group 1: activated in 7% ethanol (E) for 5 min followed by culture in 2 mM 6-dimethylaminopurin (6-DMAP, E D, subgroup 1) or 10 mu g/ml cycloheximide (CHX, E CHX, subgroup 2) for 3.5 h at 38.5 degrees C under 5% CO2. In group 2, oocytes were activated using 50 mu M Ca A23187 (Ca A) for 5 min followed by culture in 2 mM 6-DMAP (Ca D, subgroup 3) or 10 mu g/ml CHX(Ca CHX, subgroup 4) for 3.5 h at 38.5 degrees C under 5% CO2. For control group, IVM oocytes were fertilized using frozen-thawed camel spermatozoa separated by swim-up method then suspended in Fert-TALP medium supplemented with 6 mg/ml BSA (FAF) + 10 mu g/ml heparin. In all groups, oocytes were in vitro cultured in SOFaa medium + 5% FCS and 5 mu g/ml insulin + 50 mu g/ml gentamycin. Cleavage rate and embryo development were checked on Days 2, 5 and 8. An average of 11.3 mu 0.3 COCs were recovered/dromedary ovary. Categories 1 and 2 represented 33.1% and 34.8%, respectively, and were significantly higher (p < 0.01) than the other categories (19.1, 9.2 and 2.6% for categories 3-5 respectively). Category 6 (embryo-like structures) represented 1.2% of the recovered oocytes, staining of these embryo-like structures with orcien dye indicated the presence of divided cells with condensed nuclei. Dromedary oocytes averaged 166.2 +/- 2.6 mu m in diameter with black cytoplasm. Chemical activation of IVM dromedary oocyte with first Pb in 7% ethanol or 50 mu M Ca A followed by culture in 2 mM 6-DMAP showed significantly higher (p < 0.01) cleavage and developmental rates to the morula stage than oocytes activated using 7% ethanol or 50 mu M Ca A followed by 10 mu g/ml CHX or in vitro fertilized control group. Higher (p < 0.01) proportion of oocytes sequentially cultured in 10 mu g/ml CHX or that in vitro fertilized were arrested at the 2-4-cell stage compared with that cultured in 6-DMAP.