Identification of an optimal protocol for activation of the MII oocytes in a species like camel not only allows us to evaluate the quality of oocytes after their in vitro maturation, but also is required for the success of advanced technologies like cloning. The present study was aimed to determine activation of in vitro-matured dromedary (Camelus dromedarius) oocytes using ionomycin or ethanol followed by sequential culture in phosphorylation inhibitor (6-dimethylaminopurine) or the specific maturation promoting factor inhibitor (roscovitine). Cumulus–oocyte complexes (COCs), collected from slaughterhouse ovaries, were randomly distributed to 4-well culture plates (20–25 COCs/well) containing 500 µL of the maturation medium. The maturation medium consisted of TCM-199 supplemented with 0.15 mg mL–1 L-glutamine, 2.1 mg mL–1 sodium bicarbonate, 0.22 mg mL–1 pyruvate, 20 ng mL–1 epidermal growth factor, 50 µg mL–1 gentamycin, 10 µg mL–1 bFSH, 10 µg mL–1 bLH, 1 µg mL–1 estradiol, and 10% estrous dromedary serum (EDS). The COCs were cultured at 38.5�C in an atmosphere of 5% CO2 in air for 36–40 h. The COCs were either fertilized in vitro (positive control) using epididymal spermatozoa collected from slaughtered males or activated with 5 µm ionomycin for 5 min or 7% ethanol for 7 min, both followed by exposure to 2 mm 6-DMAP or 50 µm roscovitine for 4 h. After being washed thoroughly in embryo culture medium, they were cultured for a period of 7 days at 38.5�C in an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. The embryo culture medium consisted of TCM-199 supplemented with 0.15 mg mL–1 L-glutamine, 2.1 mg mL–1 sodium bicarbonate, 0.22 mg mL–1 pyruvate, 50 µg mL–1 gentamicin, 1% insulin-transferrin-selenium (ITS) media supplement, and 10% EDS. First cleavage was recorded on Day 2 and the number of embryos developing to morulas and blastocysts was recorded on Day 7 of culture. The proportions of oocytes cleaved were 58.6 � 4.4, 55.9 � 4.5, 49.1 � 5.3, 43.2 � 6.05, and 54.1 � 3.3%, while the proportions of cleaved oocytes reaching blastocyst stage were 22.5 � 0.9, 19.1 � 2.8, 9.04 � 3.3, 8.2 � 3.8, and 15.2 � 2.3%, and those at morula stage were 61.1 � 4.9, 54.6 � 6.2, 67.1 � 7.2, 57.8 � 4.6, and 53.6 � 5.6% in the ionomycin/ 6-diethylaminopurine, ionomycin/roscovitine, ethanol/6-diethylaminopurine, ethanol/roscovitine, and IVF groups, respectively. The proportions of blastocysts obtained in the ionomycin/6-diethylaminopurine and ionomycin/roscovitine groups were higher (P < 0.05) when compared with the ethanol/6-diethylaminopurine and ethanol/roscovitine groups. Also, the proportion of blastocysts obtained in the ionomycin/6-diethylaminopurine group was higher than that in the in vitro-fertilized group. In summary, methods for oocyte or cytoplast activation in dromedary camel incorporating ionomycin/6-diethylaminopurine and ionomycin/roscovitine giving better results than those incorporating ethanol/6-diethylaminopurine and ethanol/roscovitine.
Development and pregnancy rates of Camelus dromedarius-cloned embryos derived from in vivo- and in vitro-matured oocytes