Study On Interspecific Somatic Cell Nuclear Transfer Technology Of Sika Deer (Cervus Nippon), Mink (Mustla Vison), Blue Fox (Alopex Lagopus) And Racoon Dog (Nyctereutes Procyonoides Gray)-bovine

In order to explore Somatic Cell Nuclear Transfer (ISCNT) technology using sika deer-bovineInterspecies and to lay a foundation for embryonic engineering of sika deer, we investigatedbiocytoculture and cryopreservation of donor cells, in vitro maturation (IVM) of receptor oocyte,micro-manipulation and in vitro culture of reconstructed embryos, etc. In addition, a technology systemof somatic cell nuclear transfer was validated by experimenting on mink, blue fox and recoon dog.Maincontents were as follow:1. The ear skin fibroblasts of sika deer, mink, blue foxand recoon dog were initially cultured byexplant culture, then purified based on the differences in both enzymatic digestion time and adherencetime (for3~5times). The results showed that the growth of the cells characterized as adherent to thesurface, spindle shaped and with pseudopods. The Cell growth curve of F5generation followed by thepattern of “incubation-logarithmic growth-stagnation”. When DMSO was used as cryoprotectant, thedefrost recovery rate of ESF of sika deer, mink, blue fox and recoon dog was89.64%,92.44%,91.24%and91.04%respectively, which were significantly higher than those when glycerin glycerol was used.Conclusion: the ear skin fibroblasts of sika deer, mink, blue fox and recoon dog possess the typicalgrowth characteristics of skin fibroblast, they can be purified based on the difference inboth enzymaticdigestion time and adherence time.10%DMSO can be used as cryoprotectant during cryopreservation.2. The maturation rate of bovine cumulus oocyte complexes (COCs) after22h IVM was79.1%and75.2%when LH+FSH and PMSG+HCG were used respectively, showed no significant difference(P>0.05), but were significantly higher than the control group (P<0.05). The tinctorial yield of oocyte ofbovine COCs after stained for90min with Brilliant Cresyl Blue (BCB) was68.1%, significantly higherthan the groups of30min and60min (P<0.05) respectively, the identification difficulty classified as“normal”. The tinctorial yield of bovine COCs with BCB of39μM was64.5%, significantly higher thanthat of13μM (P<0.05). The differences between BCB+and control group had no significance, as wellas that between BCB-group and control group (P>0.05). After maturated in vitro for22h, thematuration rate of bovine COCs cultured in medium supplemented with Epidermal Growth Factor (EGF)50ng/mL had been remarkably higher than that with0,10and20ng/mL (P<0.05).Conclusion: the in vitro maturation rate of bovine oocytes can be significantly increased whenhormone such as10IU/mL PMSG,15IU/mL HCG and50ng/mL EGF were added into IVM medium.BCB method is not applicable to identify bovine oocytes of good quality in this study.3. The rate of extrusion of bovine COCs after treatment with0.4μg/mL Demecolcine (DEME) wasnot significantly different after cultured in vitro for20h (61.0%),21h (65.3%), and22h (66.7%)(P>0.05), but was significantly higher after treatment with0.4μg/mL DEME for60min (70.0%) and120min (71.9%) than for30min (P<0.05). The rate of extrusion in the group of0.5μg/mL DEME was78.9%, which was significantly higher than that of0.3and0.4μg/mL groups (P<0.05). The enucleationrate with DEME method (100%) was significantly higher than that with McGrath’s method andextrusion method (P<0.05), but there was no significantly difference in enucleation time (P>0.05).Therate of nuclear injection and the duration of nuclear injection by Perivitelline Microinjection (PM,100%and31.0s) and Whole-Cell Intracytoplasmic Microinjection (WCIM,94.8%and35.1s) were significantly better than by Break-Membrane-Cell Intracytoplasmic Microinjection (BMCIM, P<0.05).There was no significantly difference among the rate of activation, cleavage and blastocyst between theone-step and the two-step methods (P>0.05). The cleavage rate of Sika deer-Bovine embryo treatedwith resveratrol had no significantly difference among the groups of0,0.25,0.50and1.00μM. Theblastocyst rate in group of0.50μM (38.0%) and1.00μM (36.7%) was significantly higher than in thegroup of0μM and0.25μM (P<0.05). The cleavage rate of Sika deer-Bovine embryo in mink ESFco-culture system (55.7%) was significantly higher than the control group (P<0.05). The blastocyst ratein bovine culumus cells co-culture system (59.4%) was significantly higher than that in the two othersystemand control group (P<0.05).Conclusion: this procedures is applicable for the technical sika deer-bovine ISCNT: IVM bovineoocytes for20~22h; process with0.5μg/mL DEME for60min; BMCIM for nuclear transfer;co-culture of activated reconstructed embryo with bovine oocytes.4. When Pizeo was used for cell intracytoplasmic microinjection to construct fur animal-bovineISCNT embryo, there were no significant difference in success rate of nuclear injection, cleavage rateand blastocyst rate, no matter the donor cell was from mink, blue fox or recoon dog (P>0.05).Blastocyst rate of mink-bovine, blue fox-bovine and recoon dog-bovine ISCNT embryo was43.6%,40.1%and42.1%respectively when co-cultured with bovine cumulus cell, all significantly higher thanthose of control group (P<0.05).Conclusion: the procedure of sika deer-bovine ISCNT was also applicable to mink-bovine, bluefox-bovine and recoon dog-bovine ISCNT.