| This study was conducted to compare the collection methods of COCs on IVM,artificial activation protocols on parthenogenesis and culture media on scNT embryos’development to establish a platform for embryonic bioengineering research. Resultsmainly included:1. Three methods of aspirating, slicing and integration (aspirating+slicing) wereadopted to collect COCs. The number of useable COCs per ovary collected throughslicing was2.08, which was significantly higher than that collected throughintegration and aspiration(61.4%vs56.4%vs51.5%, P<0.05), The most suitablemethod to collect COCs is slicing.2.0ng/ml EGF,5ng/ml EGF,10ng/ml EGF,20ng/ml EGF was added tomaturation medium respectively to optimize the IVM condition. Result suggested that10ng/ml EGF significantly improved the maturation rate of oocytes, which washigher than those of the0ng/ml and5ng/ml EGF (78.8%vs68.1%,70.2%, P<0.05).There was no significant difference between rates of IVM after10ng/ml and20ng/mlEGF treatment (78.8%vs79.8%, P>0.05), and20ng/ml EGF led to the highest rateof oocyte maturation.3.0.075IU/ml HMG,10ug/ml FSH plus10ug/ml LH were added to maturationmedium respectively. The result indicated that maturation rate from latter wassignificantly higher than that from the former(70.0%vs79.8%,P<0.05).4. Three activating protocols of ionomycin+6-DMAP, ionomycin+6-DMAP+CB and ionomycin+CHX+CB were utilized to artificially atctivate oocytes, and allcould lead to further development of sheep parthenotes effectively. Ionomycin and CBalong with6-DMAP were the best for blastocyst formation, the rate by which wassignificantly higher than that by Ionomycin+6-DMAP group and Ionomycin alongwith CHX and CB group(35.0%vs31.6%vs26.5%, P<0.05).5. In order to optimize culture system for parthenogenetic development, activatedoocytes were cultured under OGS or OEC with SOFaa as the control group. Theresults were as follows: There was no significant difference among three groups’cleavage rates.(69.5%vs68.1%,68.4%,P>0.05). The group with OEC obtained thehighest rate of blastocyst, which was significantly higher than the other two groups.(32.6%vs28.5%vs23.1%,P<0.05).6. Duration of In vitro maturation for ovine COCs were16~18h,19~21h and22~24h. Maturation rate increased with the increase of the maturation time. While theenucleation efficiency showed a trend of decline. Considering maturation rate and enucleation efficiency, the best culture time of sheep oocyte should be19~21h.7. IM was used to reconstruct the scNT embryos with sheep skin fibroblast cellsnucleus as donor. Intervals between injection and activation were designed as0h,1.5h,3h. The development of reconstructed embryos showed a increasing trend wheninterval time increased in the experiment.3h interval resulted in a higher activationrate than those of two others and both the blastocyst rate of24.5%and the cleavagerate of69.8%were also the highest respectively.8. The influence of SOFaa-FBS and G1/G2medium on the development ofreconstructed sheep embryos was studied. The cleavage rate and blastocyst rate werenot significantly different between SOFaa-FBS and G1/G2(P>0.05). In conclusion,the ability of SOFaa-FBS to support the development of reconstructed sheep embryoswas comparable to G1/G2, in which serum was not included, and G1/G2couldsubstitute SOFaa-FBS to support the development of reconstructed embryo in sheep. |
PDF Full Text Request