In vitro and in vivo development of marbled cat and flat-headed cat embryos derived by somatic cell nuclear transfer

EXP. I was conducted to find appropriate in vitro maturation (IVM), fusion and activation protocols for somatic cell nuclear transfer (SCNT) in felid species. The oocytes were obtained from ovaries of ovariohysterectomized domestic cats (DC). The donor cells were obtained from a marbled cat (MC) died in captivity. The developmental rates of MC-DC (donor cell-recipient oocyte) cloned embryos derived from oocytes cultured for 24 h were greater than those cultured for 36 and 42 h (morula stage; 9.2, 4.3 and 0%, respectively, P<0.05). The fusion efficiency of the MC-DC couplets using the 2 pulses of 2.1 and 2.4 kV/cm for 80 µs and 3 pulses of 2.4 kV/cm for 50 µs was 46, 48.5 and 67.7% (P > 0.05). The development of cloned embryos derived from MC and DC fibroblast cells was not different (morula rate; 8 vs. 8.3%, P>0.05). Addition of the cytochalasin B in the activation medium provided a grater blastocyst rate compared to that without the cytochalasin B (8.3 vs. 35%, P<0.05). In conclusion, DC oocyte matured at 24 h is suggested to be used as recipient ooplasm for cloned MC embryos. The MC-DC couplets are fused efficiently by the introducing of 3 pulses of 2.4 kV/cm for 50 [mu]s. The MC fibroblast cells demonstrate the possibility to be reprogrammed in the DC oocytes, resulting in the development to the morula stage. Exposure of mature DC oocytes to cycloheximide and cytochalasin B after electrical stimulation improves the rate of blastocyst formation. EXP. II was conducted to investigate 1) the development of MC and FC cloned embryos reconstructed from DC and rabbit (RB) oocytes and 2) the effect of individual cell line and gender on FC-DC cloned embryo production. Using DC oocytes, the FC-DC couplets yielded a greater percentage of morula (53%) than those of MC- (23%) and DC-DC couplets (11%, P<0.05). However, the number of couplets achieving blastocyst expansion did not differ (MC; 5, FC; 8.3 and DC; 8.5%, P>0.05). Using RB oocytes, The blastocyst rate of FC- (8.5%) and DC-RB embryos (7.7%) was lower than those of RB-RB (14.3%, P<0.05) but not MC-RB embryos (11.5%, P>0.05). The variations of fusion and developmental rate through morula of FC-DC couplets were observed among three cell lines. The development of FC-DC embryos derived from female and male donor cells was not different (P>0.05). These results indicated that the MC and FC embryos could be produced successfully by SCNT of either DC or RB oocytes. Individual cell line but not gender of donor cells influenced the development of cloned FC embryos. EXP. III was conducted to investigate 1) in vitro development of DC IVF embryos and their transfer to recipients and 2) pregnancy establishment of recipients receiving FC-DC cloned embryos. The IVM oocytes were co-cultured with sperm for 18 h. The blastocyst rate and cell number of blastocysts derived from cleaved embryos collected at 18-27 h were greater than those cleaved during >27-42 h (61.4 vs. 18.6% and 106 +- 43 vs. 60 +- 27 cells, P<0.05). Total 150 of cleaved IVF embryos were transferred to 6 recipients (mean 25 +- 9). All recipients became pregnant resulting in five kittens delivered from three recipients. The recipients receiving FC-DC cloned (n=5, 41.4 +- 13 embryos), FC- and DC-DC cloned (n=5, 29.8 +- 20.8 embryos) and FC-DC cloned and DC IVF embryos (n=4, 55 +- 15 embryos) were observed the pregnancy establishment. One of the recipients receiving FC-DC cloned and DC IVF embryos became pregnant and delivered IVF kittens. In conclusion, the pregnancy could be established after DC IVF embryo transfers, and embryos cleaved early at 18-27 h pi would rather be used for transfer than those cleaved later due to the greater development and quality. The FC- and DC-DC cloned offspring were not able to be produced which may be caused by the low development and quality of the embryos.实验一：本研究旨在探索适用于猫科动物体细胞核移植（somatic cell nuclear transfer, SCNT）的体外成熟（in vitro maturation, IVM）、融合与激活方案。实验所用卵母细胞取自经卵巢子宫摘除术的家猫（domestic cats, DC）卵巢，供体细胞则来自1只圈养死亡的纹猫（marbled cat, MC）。以体外培养24小时的卵母细胞构建的MC-DC（供体细胞-受体卵母细胞）克隆胚胎，其桑椹胚发育率显著高于培养36小时与42小时的组别（分别为9.2%、4.3%与0%，P<0.05）。针对MC-DC细胞对的融合效率检测显示，采用2次2.1 kV/cm、80 μs、2次2.4 kV/cm、80 μs及3次2.4 kV/cm、50 μs的电脉冲参数组合，其融合率分别为46%、48.5%与67.7%，组间差异无统计学意义（P>0.05）。纹猫与家猫成纤维细胞来源的克隆胚胎发育能力无显著差异（桑椹胚率分别为8%与8.3%，P>0.05）。激活液中添加细胞松弛素B（cytochalasin B）可显著提升克隆胚胎的囊胚发育率（8.3% vs 35%，P<0.05）。综上，建议采用体外成熟24小时的家猫卵母细胞作为纹猫克隆胚胎的受体胞质；采用3次2.4 kV/cm、50 μs的电脉冲参数可高效实现MC-DC细胞对的融合；纹猫成纤维细胞可在家猫卵母细胞中发生重编程，进而发育至桑椹胚阶段；电刺激后将成熟家猫卵母细胞暴露于环己酰亚胺（cycloheximide）与细胞松弛素B中，可提升克隆胚胎的囊胚形成率。实验二：本研究旨在探究1）以家猫（DC）与家兔（rabbit, RB）卵母细胞构建的纹猫（MC）和渔猫（fishing cat, FC）克隆胚胎的发育能力，以及2）供体细胞系个体差异与性别对渔猫-家猫（FC-DC）克隆胚胎制备的影响。采用家猫卵母细胞时，FC-DC细胞对的桑椹胚发育率（53%）显著高于MC-DC组（23%）与DC-DC组（11%，P<0.05）。但各组的囊胚扩张胚胎占比无显著差异（MC组5%、FC组8.3%、DC组8.5%，P>0.05）。采用家兔卵母细胞时，FC-RB组（8.5%）与DC-RB组（7.7%）的囊胚率显著低于RB-RB对照组（14.3%，P<0.05），但MC-RB组（11.5%）与对照组差异无统计学意义（P>0.05）。3个供体细胞系的FC-DC细胞对的融合率及桑椹胚发育率存在个体差异。来自雌性与雄性供体细胞的FC-DC克隆胚胎发育能力无显著差异（P>0.05）。本研究结果表明，采用家猫或家兔卵母细胞进行体细胞核移植，均可成功制备纹猫与渔猫克隆胚胎；供体细胞系个体差异会影响渔猫克隆胚胎的发育能力，而供体细胞性别无此调控效应。实验三：本研究旨在探究1）家猫体外受精（in vitro fertilization, IVF）胚胎的体外发育能力及其移植后的受体妊娠情况，以及2）移植FC-DC克隆胚胎后的受体妊娠建立情况。将体外成熟的家猫卵母细胞与精子共培养18小时。于受精后18~27小时收集的分裂胚胎，其囊胚发育率与囊胚细胞数均显著高于受精后27~42小时分裂的胚胎（分别为61.4% vs 18.6%，106±43 vs 60±27，P<0.05）。共将150枚分裂期IVF胚胎移植至6只受体母猫（平均每只移植25±9枚），所有受体均成功妊娠，其中3只受体顺利产下幼猫。移植FC-DC克隆胚胎（n=5，每只移植41.4±13枚）、FC-DC与DC-DC克隆胚胎（n=5，每只移植29.8±20.8枚）以及FC-DC克隆胚胎与DC-IVF胚胎（n=4，每只移植55±15枚）的受体均成功建立妊娠。其中1只同时移植FC-DC克隆胚胎与DC-IVF胚胎的受体成功妊娠并产下体外受精幼猫。综上，家猫IVF胚胎移植后可成功建立妊娠，且受精后18~27小时早期分裂的胚胎因发育能力与胚胎质量更优，更适合用于胚胎移植；FC-DC与DC-DC克隆胚胎未能获得成活后代，这可能与克隆胚胎的发育能力与质量较低有关。