Chemical activation of in vitro matured dromedary camel (Camelus dromedarius) oocytes: optimization of protocols.
Experiments were conducted to study the efficiency of sequential treatments of ionomycine and ethanol combined with phosphorylation inhibitor (6-dimethylaminopurine) or the specific maturation promoting factor inhibitor (roscovitine) in inducing artificial activation in dromedary M-II oocytes. Cumulus oocyte complexes (COCs), collected from slaughterhouse ovaries were cultured at 38.5 degrees C in an atmosphere of 5% CO2 in air for 24-48 h. In experiment 1, the COCs were either fertilized in vitro or activated with 5 microM ionomycine for 5 min or 7% ethanol for 7 min, both followed by exposure to 6-diethylaminopurine or roscovitine for 4h. After 14-15 h of in vitro culture, the oocytes were fixed and stained with 1% aceto-orcein to evaluate their nuclear status. In experiment 2, the oocytes were activated in the same manner as in experiment 1 but were cultured for 7 days to evaluate their post-parthenogenetic development. In experiment 3, oocytes were exposed to the ionomycine for 2, 3, 4 or 5 min to evaluate the better exposure time while as in experiment 4, the oocytes matured for 28-48 h were activated to see the effect of aging on post-parthenogenetic development. Higher proportion (P<0.01) of oocytes was activated in ionomycine/6-DMAP and ionomycine/roscovitine groups when compared with ethanol/6-DMAP, ethanol/roscovitine and in vitro fertilized groups. However, there was no difference (P>0.05) in the proportion of oocytes activated with ethanol when compared with in vitro fertilized group. No significant difference was seen on the proportion of morula on day 7 of culture, however the development to blastocyst stage was higher (P<0.01) in ionomycine/6-DMAP and ionomycine/roscovitine when compared with ethanol/6-DMAP and ethanol/roscovitine treated oocytes. A higher proportion of oocytes reached blastocyst stage when they were exposed to ionomycine for 3 min but they were not significantly different from the others (P>0.05). The proportion of blastocysts obtained was higher (P<0.05) in oocytes activated after 28 h of maturation when compared with oocytes activated after 32, 36, 40, 44 and 48 h of maturation. In conclusion, a protocol for chemical activation of dromedary camel oocytes with ionomycine/6-DMAP is demonstrated and optimized in the present study for further use in the development of assisted reproductive techniques in this species.
Wani
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Central Veterinary Research Laboratory, Post Box 597, Dubai, United Arab Emirates. nwani@cvrl.ae
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D002162:Camelus / Q000502:physiology*; D002452:Cell Count / Q000662:veterinary; D000431:Ethanol / Q000494:pharmacology; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D015759:Ionomycin / Q000494:pharmacology*; D007476:Ionophores / Q000494:pharmacology*; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D010312:Parthenogenesis / Q000187:drug effects / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011687:Purines / Q000494:pharmacology; D011897:Random Allocation; D000077546:Roscovitine
D007476:Ionophores; D047428:Protein Kinase Inhibitors; D011687:Purines; D000077546:Roscovitine; D000431:Ethanol; D015759:Ionomycin; C001020:N(6),N(6)-dimethyladenine; D000225:Adenine