Studies On The Cryopreservation Conservation And Chimeras Production Of HeiFeng BCs

As a potential of embryonic stem cells, chicken stage X blastodermal cells (BCs) had important application value in avian embryo development research, breeding and breed conservation, improving production performance and disease resistance, etc.This paper focused on the HeiFeng black silkies chicken BCs, which were explored the effects of the blastoderm discrete degree, density of cells, centrifugal force when BCs were cultured. Then built a cryodamage evaluating method via adherent ratio of cells cultivated after 24 h(CAR), combined with the cell viability (CV) witch calculated by fluorescein diacetate (FDA), we studied on the effect factors of different DMSO concentration, freezing and thawing rate and different antioxidant on cryopreservation of BCs. Finally, we developed an efficient cryopreservation and cryodamage valuation technology of BCs that provided the technical supportstoring for the preserve of female poultry genetic resources. At the same time, this paper also optimized the chimeras production technology which contrast the influence of eggs hatchability of different sealing method, returned air chamber, different turning angle and relatively humidity in hatching, added order of sealing material, different setting method and different injection. The results were as follows:1.Established the indirect damage evaluation method for BCsCAR method, the chicken Blastodermal cells (BCs) were isolated from stage X embryos, and cultured using the high glucose DMEM medium (10% FBS, Penicillin and streptomycin) without feeder layers cells. The best BCs culture procedures was separating chicken embryos with filter paper ring and beating the embryos in the medium for 20 times, centrifuging the cell suspension by 5590 X g and cultured the cells at the density of 5×104 cells/mL in the 96-well cell culture plates. Also the adherent ratio of cells cultivated after 24 h would be efficient for BCs activity detection.CV method, droped FDA into cell suspension, the FDA's final concentration was 1 ug/mL, dyeing 3?5 min at 37? in darkroom after mixing, then count the number by fluorescence microscopy, be sure to complete within 10 min. The living cells were green, but the dead were not and had obvious contrast.2. Established the efficient freezing method of BCsThe BCs was frozen using dimethyl sulfoxide (DMSO) and straw, cooling at -1 ?·min-1 to -7? for 10 min, continuing cooling to -35? at the same ratio and plunging into liquid nitrogen finally, then thawed cells by 37? water bath for 1?2 min, could get not only good CV, but also CAR.3. Studied on the effect of antioxidant for cryopreservation of BCsThe addition of NAC, CAT or LP into the cryoprotectant for freezing BCs, could not only improve the CV (had not significant difference), but also the CAR (had extremely significant difference).The best for CV was CAT(200 U/mL), but for CAR was LP(0.05 mg/mL). We also found it had no significant difference between using after 2 months refrigerated in -20? and using it right after it was ready. But the protection effect decreased significantly in 5? cold storage after 2 months.4. A preliminary study on chimeras production technology via BCsThe results prove that the best method of sealing was dropping instant glue first and sprinkled the Straw Powder next, then put eggs narrow end down immediately after sealed what get the highest hatchability 68.8%. We also found there were highly positive correlation between air chamber rate and hatchability, the higher air chamber rate for eggs, the higher hatchability happened. Another experiment showed the best hatching conditions was 90° turning angle and 70%RH while incubated. Also the result indicated drilling window or injection could both extremely significant influence the hatchability (P<0.01). The hatchability would dramatic decline when the injection dose increased and injected luL DMEM could get the highest hatchability(48.4%) which had extremely significant difference with 3uL,5uL andlOuL(P<0.01).Therefore, this paper had set up an efficient system of method from several key aspects such as the BCs in vitro culture, viability detection, cryopreservation and the window technology, which obviously affect survival rate of BCs chimeras. Also it had laid the foundation not only for cryopreservation poultry genetic resources via BCs but also the related biotechnology research.