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Animal reproduction science

In vitro and in vivo developmental competence of dromedary (Camelus dromedarius) oocytes following in vitro fertilization or parthenogenetic activation.


PMID 18789618

Abstract

Parthenogenetic activation of the oocyte represents an important step in the somatic cloning. The aim of the present study was to evaluate the effectiveness (in term of in vitro development) of different methods of parthenogenetic activation of dromedary oocytes. Selected cumulus-oocytes-complexes (n=1264) collected by follicular aspiration from ovaries obtained postmortem were matured in vitro (IVM) for 30 h then divided randomly into seven groups and submitted to artificial activation. Two groups were preactivated with 25 microM of calcium ionophore (CaI) for 20 min then incubated for 4h with either 2mM 6-dimethylaminopurine (6-DMAP) (group 1, n=202) or with 10 microg/mL cycloheximide (CHX) (group 2, n=194). Group 3 (n=172) and group 4 (n=184), oocytes were pretreated with 5 microM ionomycin (Iono) for 5 min then incubated with either 2mM 6-DMAP or 10 microg/mL cycloheximide for 4h, respectively. Group 5 (n=161) and group 6 (n=155) oocytes were preactivated with electrical stimulation (ES) then activated with either 2mM 6-DMAP or 10 microg/mL cycloheximide for 4h, respectively. Group 7 (n=196) oocytes were submitted to in vitro fertilization (IVF) and served as a control. All groups containing oocytes were cultured in vitro following activation or IVF, at 38.5 degrees C under 5% CO(2) in air with >95% humidity. The in vitro development rates of dromedary oocytes exposed to 6-DMAP after CaI (61%), ES (74%) and the IVF group (71%) were similar and significantly greater (P<0.05) than other treatments (10% for group 2, 47% for group 3, 27% for group 4 and 41% for group 6). The blastocyst developmental rate was better (P<0.05) in parthenotes following activation with Iono/6-DMAP (21%) compared to activation with Iono/CHX (12%). However, all were less than that achieved in the IVF group (35%). We conclude that parthenogenetic activation of camel oocytes with 6-DMAP is more effective than activation with CHX for all pre-treatments tested (CaI, Iono or ES). The viability of activated (n=15) or IVF (n=10) hatched-dromedary embryos was examined by transfer to synchronized recipients. An embryonic vesicle was seen by ultrasonography at 15 days post transfer in four females (CaI/6-DMAP: 1/5; 20%, IVF: 3/10; 30%). The only pseudopregnancy obtained with an activated embryo resorbed at 25 days. One of the females receiving the IVF produced embryos aborted at 2 months and the other two females carried to term and gave birth to healthy calves (one female and one male). This study shows that artificial activation of dromedary oocytes with CaI/6-DMAP or ES/6-DMAP is more effective than other treatments in terms of in vitro embryo development. This provides efficient activation conditions which may lead to the development of the somatic cell nuclear transfer procedure in dromedary.