The present study explored a suitable parthenogenetic activation (PA) procedure for rabbit oocytes and investigated the developmental potential of somatic cell nuclear transfer (SCNT) embryos using rabbit foetal fibroblasts (RFFs). The electrical activation had the optimal rate of blastocyst (14.06%) when oocytes were activated by three direct current (DC) pulses (40V/mm, 20s each) followed by 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) treatment; the blastocyst rate of ionomycin (ION)+6-DMAP+CHX (12.07%) activation was higher than that of ION+6-DMAP (8.6%) activation or ION+CHX (1.24%) activation; there was no significant difference in blastocyst rate between ION+6-DMAP+CHX and DC+6-DMAP+CHX groups. The blastocyst rate of ION+6-DMAP+CHX-activated oocytes in the basic rabbit culture medium (M-199)+10% foetal bovine serum (FBS; 14.28%) was higher than that in buffalo conditioned medium (5.75%) or G1/G2 medium (0), and the blastocyst rate was increased when M-199+10% FBS was supplemented with amino acids. Refreshing culture medium every day or every other day significantly increased the blastocyst rate. Treatment of donor cells with 0.5% FBS for 3-5days increased blastocyst rate of SCNT embryos (33.33%) than no serum starvation (22.47%) or 0.5% FBS treatment for 6-9days (23.61%); the blastocyst rate of SCNT embryos derived from nontransgenic RFFs was higher than that derived from transgenic RFFs by electroporation. The blastocyst development ability of SCNT embryos derived from RFFs by electroporation (32.22%) was higher than that of liposome (19.11%) or calcium phosphate (20.00%) transfection, and only the embryos from electroporation group have the EGFP expression (24.44%). In conclusion, this study for the first time systematically optimized the conditions for yield of rabbit embryo by SCNT.

【Objective】The influencing factors of rabbit embryo transplantation were explored to provide references for improving rabbit embryo transplantation efficiency and producing transgenosis rabbits. 【Method】The embryo were collected after mating and cultured in vitro, and then transplanted into recipients according to the number of embryo, recipients type(young hare multiparous hare) and synchronic oestrus methods. The pregnancy rate was observed after transplantation. 【Result】The results showed that 10-20 or 20-30 embryos were respectively transferred into each recipient. There was no significant difference in the pregnancy rate between the two groups(23.1% vs 20.0%, P0.05). When the multiparous rabbits were used as recipient, the pregnancy rate was significantly higher than that of nulliparity rabbits(27.8% vs 13.3%,P0.05). Embryos were respectively transferred into natural estrus recipients and induced estrus recipients. The pregnancy rate of natural estrus recipients was extremely higher than that of induced estrus recipients(30.7% vs 10.0%, P0.05).【Conclusion】These results suggested that the natural estrus of multiparity rabbits were better to be used for recipients than nulliparity rabbits for ET, and the 10-30 transfer embryos were the best.

This study was conducted to examine the effects of different serum on the in vitro development of buffalo embryos.Early buffalo embryos derived from parthenogenetic activation(PA) or in vitro fertilization(IVF) were cultured in the medium with 5%BSA,fetal bovine serum(FBS),oestrus serum of buffalo(OBS),oestrus serum of cattle (OCS) Or mixed serum(OCS:FBS= 1:1),respectively.And then,the developmental potential of buffalo embryos was evaluated after cultured in 30μL drop of granulosea cell monolayers for 7-9 days.The result showed that the cleavage(73.52%,68.07%vs.38.37%,44.97%,52.02%;P0.05) and blastocyst rate(30.59%,27.73%vs 6.40%,10.05%and 17.04%) of PA embryos cultured in with OCS or mixed serum groups were significantly higher than those of embryos treated with BSA,OBS,FBS groups.Also,the highest cleavage rate and blastocyte development rate of IVF embryos were the embryos culture in OCS group(62.50%,22.62%),which significantly higher than that of embryos cultured in BSA(30.50%,6.38%),OBS(35.48%,7.10%;P0.05) and FBS(40.11%, 11.76%) groups,but there was no significant difference in either cleavage or blastocyte development rate compared with mixed serum group(60.42%,21.35%,P0.05).The results indicated that addition 5%OCS or mixed serum in the culture medium could improve the development of buffalo embryos effectively.

The effect factors of the superovulation in rabbit were investigated in this study.The results showed: ①The average number of oocytes recovered from FSH-treated group(30.05±8.97) was significantly higher than PMSG-treated group(22.46±5.18)(P<0.05).② When the superovulation was conducted in seasons of Spring,Summer,Autumn and Winter,the number of recovered oocytes were 32.34±5.58,15.96±4.11,24.18±4.95 and 22.61±3.75,respectively,the number of recovered oocytes of the treatment group in Spring was higher than that of the other three groups(P<0.05),and there were no difference between Autumn and Winter groups(P>0.05),but both of them were higher than Summer group(P<0.05).③ The average number of oocytes recovered from superovulation in pluriparous rabbit group(30.56±6.79) was higher than young rabbit group(21.60±3.66)(P<0.05).These results indicated that the effect of FSH on superovulation in rabbit was more effective than PMSG,and the season of spring and pluriparous rabbit were comparatively suitable for superovulation.

To study the effects of in vitro maturation(IVM) of rabbit oocytes,rabbit ovaries were collected under different seasons,temperature and storage time.The experiment adopted the rabbit ovaries to be placed in normal saline at various temperature of 25-30 ℃,31-35 ℃,36-40 ℃ respectively.The ovaries would be transported to a laboratory as quickly as possible.And then the rabbit oocytes collected were cultured in maturation system for 22-24 hours.The rabbit oocytes in vitro maturation were studied during different storage time at <3 h,≥3-≤4 h,>4 h affected.The different seasons were compared to reveal the effect on rabbit oocytes in vitro maturation with the same ovaries storage temperature and transport time.The results showed that the proportion of maturation in temperature 25-30 ℃ group was 61.09%,significantly higher than 31-35 ℃ and 36-40 ℃ groups(26.30% 15.79%,P<0.05).The maturation rate of >4 h group of transported time was 50.23%,which was significantly lower than the <3 h and ≥3-≤4 h groups(64.24%,59.64%,P<0.05).The summer group had obtained a percentage rate of matured oocytes 32.65%,significantly lower than that of spring,autumn and winter groups(64.26%,58.37%,64.86%,P<0.05),but there were no significant differences among the three groups.The study demonstration that the effect of collecting and culturing rabbit oocytes in spring,autumn and winter would be better,and the ovaries were stored at a 25-30 ℃ physiological saline and ovaries transported to the lab not exceed four hours would get a pretty maturation rate.

Methods for activating rabbit oocytes were investigated in this study to establish a favorable foundation for rabbit somatic cell ntlclear transfer. Rabbit oocytes were matured in vitro for 20-22 h, then carried on experiments as follows. Exploring to the optimal condition of direct current （DC）field intensity and pulse width for rabbit oocytes electro-activation; comparing to the effect of the ooctyes activated by different ways, Ⅰ：Ion5min＋6-DMAP3h,Ⅱ：Ion5min＋CHX3h,Ⅲ：Ion5min＋6-DMAP and CHX 3 h,Ⅳ ：Electro-activation （1 time） ＋6-DMAP and CHX 3 h;aptimizing the joint action time of 6-DMAP and CHX. The result showed that when using 40 V/mm,20 μs electrical pulse to rabbit oocytes,the cleavage and blastocyst rate of the 3 treatments can reach 80.64% and 14.51%. In experiment 4, the development ability of embryos treated with IV group （80.64%,13.71% ） were significantly higher than that of oocytes treated with Ⅰ group （66.67% ,8.6%） or Ⅱ group （43.24% ,1.35% ,P〈0.05） ,but no significant difference comparing with Ⅲ group （77.59 %, 12.07 %, P〉 0.05）. These results indicated that the comparatively suitable electro-activation condition for rabbit oocyte was that of 40 V/mm, 20 μs and 3 times of direct current pulses; and the methods of Ion 5 min or electric pulse （1 time）,then 6-DMAP＋CHX 1-3 h could activate the rabbit oocytes more effectively.