Effect of Plant AntifreezeProteins on Porcine Embryo to Cryopreservaton

ＥｆｆｅｃｔｏｆＰｌａｎｔＡｎｔｉｆｒｅｅｚｅＰｒｏｔｅｉｎｓｏｎＰｏｒｃｉｎｅＥｍｂｒｙｏｔｏＣｒｙｏｐｒｅｓｅｒｖａｔｏｎ￥ＦＥＩＹｕｎ－ｂｉａｏ（费云标）;ＷＥＩＬｉｎｇ－ｂｏ（魏令波）;ＧＡＯｓｕ－ｑｉｎ（高素琴）;ＺＨＡＯＳｈｕ－ｈｕｉ（赵...     

植物体细胞胚发生的分子基础①

ＭｏｌｅｃｕｌａｒＦｏｕｎｄａｔｉｏｎｉｎＰｌａｎｔＳｏｍａｔｉｃＥｍｂｒｙｏｇｅｎｅｓｉｓＸＩＮＧＧｅｎｇ－ＳｈｅｎｇＣＵＩＫａｉ－ＲｏｎｇＳＨＡＮＬｕｎＷＡＮＧＹａ－Ｆｕ（ＴｈｅＳｔａｔｅＫｅｙＬａｂｏｒａｔｏｒｙｏｆＡｒｉｄＡｇｒｏｅｃｏｌｏｇ...     

甘肃柳属一新种

甘肃柳属一新种⒇孙学刚（甘肃农业大学林学院,兰州７３００７０）关键词柳属;舟曲柳;甘肃ＡＮＥＷＳＰＥＣＩＥＳＯＦＳＡＬＩＸＦＲＯＭＧＡＮＳＵＳｕｎＸｕｅ－ｇａｎｇ（ＣｏｌｅｇｅｏｆＦｏｒｅｓｔｒｙ,ＧａｎｓｕＡｇｒｉｃｕｌｔｕｒａｌＵｎｉｖｅｒｓｉｔ...     

固体电极上L－半胱氨酸的电合成

固体电极上Ｌ－半胱氨酸的电合成ＥｌｅｃｔｒｏｓｙｎｔｈｅｓｉｓｏｆＬ－ｃｙｓｔｅｉｎｅａｔｓｏｌｉｄｅｌｅｃｔｒｏｄｅｓ￥／／（英）Ｒａｌｐｈ,ＴＲ．,Ｈｉｔｃｈｍａｎ,ＭＬ,Ｍｉｌｌｉｎｇｔｏｎ,ＪＰ等著张关永摘译陆兆锷校华东理工大学化学系上海２０...     

生物固氮作用机理

生物固氮作用机理李佳格徐继（中国科学院植物研究所,北京１０００９３）ＭＥＣＨＡＮＩＳＭＯＦＢＩＯＬＯＧＩＣＡＬＮＩＴＲＯＧＥＮＦＩＸＡＴＩＯＮＬｉＪｉａ－ｇｅＸｕＪｉ（ＩｎｓｔｉｔｕｔｅｏｆＢｏｔａｎｙ,ＣｈｉｎｅｓｅＡｃａｄｅｍｙｏｆＳｃｉｅｎｃｅ...     

Detection and Analysis of an Estrous－associated Oviductal Glycoprotein DNA Fragment from Primates by PCR

ＤｅｔｅｃｔｉｏｎａｎｄＡｎａｌｙｓｉｓｏｆａｎＥｓｔｒｏｕｓ－ａｓｓｏｃｉａｔｅｄＯｖｉｄｕｃｔａｌＧｌｙｃｏｐｒｏｔｅｉｎＤＮＡＦｒａｇｍｅｎｔｆｒｏｍＰｒｉｍａｔｅｓｂｙＰＣＲ￥ＣＨＥＮＱｉｎｇ－ｘｕａｎ（陈清轩）;ＣｌａｙｔｏｎＥ．Ｗａｌｔｏ...     

河西走廊农户生态系统农田能流和物流数量特征的研究

河西走廊农户生态系统农田能流和物流数量特征的研究李发弟（甘肃农业大学畜牧系,兰州７３００７０）ＱｕａｎｔｉｎｔｉｖｅＣｈａｒａｃｔｅｒｉｓｔｉｃｓｏｆＥｎｅｒｇｙＦｌｏｗａｎｄＮｕｔｒｉｅｎｔＣｙｃｌｉｎｇｏｆＦａｒｍｌａｎｄｉｎａＦａｒｍＥｃｏｓｙｓｔｅｍｉｎＨｅｘｉＣｏｒｒｉｄｏｒ￥ＬｉＦａｄｉ（ＤｅｐａｒｔｍｅｎｔｏｆＡｎｉｍａｌＨｕｓｂａｎｄｒｙ,ＧａｎｓｕＡｇｒｉｃｕｌｔｕｒａｌＵｎｉｔｖｅｒｓｉｔｙ）．ＣｈｉｎｅｓｅＪｏｕｒ－ｎａｌｏｆＥｃｏｌｏｇｙ,１９９３,１２（６）：３７－４０．Ｑｕａｎｔｉｔａｔｉｖｅｃｈａｒａｃｔｅｒｉｓｔｉｃｓｏｆｅｎｅｒｇｙｆｌｏｗａｎｄｎｕｔｒｉｅｎｔｃｙｃｌｉｎｇｏｆｆａｒｍｌａｎｄｓｕｂｓｙｓｔｅｍｉｎａｆａｒｍｅｃｏｓｙｓ－ｔｅｍｉｎＨｅｘｉＣｏｒｒｉｄｏｒｗｅｒｅｓｔｕｄｉｅｄ．Ｔｈｅｒｅｓｕｌｔｓｓｈｏｗｅｄｔｈａｔｔｈｅｄｅｆｌｒｅｅｏｆｏｐｅｎｉｎｇ,ｉｎｐｕｔａｎｄｏｕｔｐｕｔｗｅｒｅｈｉｇｈ,ｔｈｅｒａｔｉｏｏｆｏｕｔｐｕｔｔｏｉｎｐｕｔｏｆｓｕｂｓｉｄｙｅｎｅｒｇｙ,ｎｉｔｒｏｇｅｎａｎｄｐｈｏｓｐｈｏｒｕｓｏｆｔｈｅｆａｒｍｌａｎｄｓｕｂｓｙ     

MPF Induction of Microtubule Assembly at Interphase Kinetochore of CHO Cells

ＭＰＦＩｎｄｕｃｔｉｏｎｏｆＭｉｃｒｏｔｕｂｕｌｅＡｓｓｅｍｂｌｙａｔＩｎｔｅｒｐｈａｓｅＫｉｎｅｔｏｃｈｏｒｅｏｆＣＨＯＣｅｌｌｓＦＥＮＧＭｅｉ;（冯梅）ＺＨＡＮＧＨｕａｎ－ｘｉａｎｇ;（张焕相）ＷＡＮＧＹｏｎｇ－ｃｈａｏ;（王永潮）ＷＡＮＧＹｕｅ...     

G蛋白Rap1活化新机制：GEFs研究进展

小分子Ｇ蛋白Ｒａｓ超家庭成员都是通过与鸟苷酸交换因子（ｇｕａｎｉｎｅ ｎｕｃｌｅｏｔｉｄｅ ｅｘｃｈａｎｇｅ ｆａｃｔｏｒｓ,ＧＥＦｓ）结合而被活化,ＧＥＦｓ能增加这类小分子Ｇ蛋白对ＧＴＰ的摄取,并使之形成有活性的ＧＴＰ结合构象。Ｒａｐ１是Ｒａｓ样的小分子Ｇ蛋白。迄今为止,已发现Ｇ３Ｇ、ＣａｌＤＡＧ－ＧＥＦ１、Ｅｐａｃ、ｃＡＭ－ＧＥＦ１、ｃＡＭＰ－ＧＥＦⅡ、ｎＲａｐＧＥＰ、ＧＦＲ可特异的活化Ｒａｐ     

Existence and Functions of Neurotens in Human Early Placental Villi

ＥｘｉｓｔｅｎｃｅａｎｄＦｕｎｃｔｉｏｎｓｏｆＮｅｕｒｏｔｅｎｓｉｎＨｕｍａｎＥａｒｌｙＰｌａｃｅｎｔａｌＶｉｌｌｉＺＨＡＮＧＣｈｏｎｇ－ｌｉ（张崇理）;ＣＨＥＮＧＬｉ－ｒｅｎ（程丽仁）,ＳＨＥＮＷｅｉ－ｂｉｎ（沈卫斌）;ＹＩＮＨｏｎｇ（殷红）;ＨＵ...     

Clonal lines of transgenic fibroblast cells derived from the same fetus result in different development when used for nuclear transfer in pigs

Different factors are believed to influence the outcome of nuclear transfer (NT) experiments. Besides the cell cycle stage of both recipient cytoplast and donor karyoplast, the origin of the donor cells (embryonic, fetal, and adult) is of interest. We compared in vitro development of NT embryos derived from small serum-starved (G0) or small cycling (G1) porcine fetal fibroblast cells. Serum starvation did not have a positive effect on cleavage rate or the percentage of embryos that developed to the morula and blastocyst stages. Next, we investigated the development of porcine NT embryos derived from different transgenic clonal cell lines that had originated from the same fetus. When different clonal lines of fetal fibroblasts were fused to enucleated metaphase II oocytes, differences in fusion rates as well as in development to the morula and blastocyst stages were observed (P < 0.05). When oocytes derived from sow ovaries were used as recipient cytoplasts, significantly better cleavage (P = 0.03) and blastocyst formation (P < 0.014) was obtained when compared with oocytes derived from gilts. Our data indicate that not only different cell lines, but also different clones derived from one primary cell line, result in different development when used for NT. In addition, the use of sow oocytes as a cytoplast source also improves the efficiency of NT experiments.     

Effects of different states of sheep fetal fibroblasts as donor cells on the early development in vitro of reconstructed sheep embryos

To investigate the effects of different states of donor cells on the development of reconstructed sheep embryos, we designed five treatments of donor cells, including cell passage, cell size, serum starvation,colchicine treatment and gene transfection. Results are as follows: ( Ⅰ ) Compared with 16-18 passage cells, the morula/blastocyst rate of 5-7 passage cells as donor nuclei was significantly higher (17.3%vs. 4.9%, P＜0.05), suggesting the advantage of short-time cultured cells in supporting the development of reconstructed embryos. (Ⅱ) The morula/blastocyst rate of reconstructed embryos derived from medium cells (15-25 μm) as donor nuclei was higher than that from large cells (25-33 μm) and small cells (8-15 μm)( 20.0% vs. 8.0%, 9.7%), indicating that reconstructed embryos from medium cells had a greater potentiality to develop into morula/blastocysts than those from small or large ones. (Ⅲ) The morula/blastocyst rate of reconstructed embryos from donor cells of SS (serum starvation) was lower than that from donor cells of NSS (non-serum starvation), but no significant difference was detected between SS and NSS( 11.8% vs. 18.6%, P＞0.05). (Ⅳ) Fetal fibroblasts treated with 0.05 μmol/L colchicine exhibited a higher morula/blastocyst rate of reconstructed embryos than those treated with 0.10 μmol/L colchicine and untreated ones (27.5% vs. 12.1%, 17.1%), however, no significant difference among the three treatments was detected (P＞0.05). (Ⅴ) The morula/blastocyst rate of reconstructed embryos from fetal fibroblasts transfected with GFP gene only was 3.1%, significantly lower than that from non-transgenic cells (3.1% vs. 20.4%, P＜0.05). In conclusion, our results demonstrated that fetal fibroblasts of fewer passages, medium size could ensure a higher morula/blastocyst rate of reconstructed embryos. Serum starvation of donor cells might be unnecessary to the development of reconstructed embryos. Donor cells treated with 0.05 μmol/L colchicine could facilitate the development of reconstructed embryos. Additionally, as cells transfected with GFP gene were used as donor nuclei, adverse effect on the development of reconstructed embryos was observed. Therefore, the developmental efficiency of reconstructed embryos could be improved if proper treatments to donor cells were used.     

Effects of different states of sheep fetal fibroblasts as donor cells on the early development in vitro of reconstructed sheep embryos

To investigate the effects of different states of donor cells on the development of reconstructed sheep embryos, we designed five treatments of donor cells, including cell passage, cell size, serum starvation, colchicine treatment and gene transfection. Results are as follows: (Ⅰ) Compared with 16-18 passage cells, the morula/blastocyst rate of 5-7 passage cells as donor nuclei was significantly higher (17.3% vs. 4.9%, P<0.05), suggesting the advantage of short-time cultured cells in supporting the development of reconstructed embryos. (Ⅱ) The morula/blastocyst rate of reconstructed embryos derived from medium cells (15-25μm) as donor nuclei was higher than that from large cells (25-33μm) and small cells (8-15μm)( 20.0% vs. 8.0%, 9.7%), indicating that reconstructed embryos from medium cells had a greater potentiality to develop into morula/blastocysts than those from small or large ones. (Ⅲ) The morula/blastocyst rate of reconstructed embryos from donor cells of SS (serum starvation) was lower than that from donor cells of NSS (non-serum starvation), but no significant difference was detected between SS and NSS(11.8% vs. 18.6%, P>0.05). (Ⅳ) Fetal fibroblasts treated with 0.05μmol/L colchicine exhibited a higher morula/blastocyst rate of reconstructed embryos than those treated with 0.10 μmol/L colchicine and untreated ones (27.5% vs. 12.1%, 17.1%), however, no significant difference among the three treatments was detected (P>0.05). (Ⅴ) The morula/blastocyst rate of reconstructed embryos from fetal fibroblasts transfected with GFP gene only was 3.1%, significantly lower than that from non-transgenic cells (3.1% vs. 20.4%, P<0.05). In conclusion, our results demonstrated that fetal fibroblasts of fewer passages, medium size could ensure a higher morula/blastocyst rate of reconstructed embryos. Serum starvation of donor cells might be unnecessary to the development of reconstructed embryos. Donor cells treated with 0.05μmol/L colchicine could facilitate the development of reconstructed embryos. Additionally, as cells transfected with GFP gene were used as donor nuclei, adverse effect on the development of reconstructed embryos was observed. Therefore, the developmental efficiency of reconstructed embryos could be improved if proper treatments to donor cells were used.     

Improvement of the nuclear transfer efficiency by using the same genetic background of recipient oocytes as the somatic donor cells in goats

We have compared the effect of the genetic background of recipient oocytes on the in vitro and in vivo development of nuclear transfer reconstructed embryos in goats. Adult fibroblast cells from Boer goats were used as donor cells, and recipient oocytes were obtained from Boer goats and Boer cross-breeds (Boer♂×Huanghuai♀). Nuclear transfer reconstructed embryos were cultured in vitro, or transferred into recipient goats. The mitochondrial origin of 2 cloned Boer goats was investigated by analysing the D-loop region based on polymorphisms via DNA sequencing. There was no significant difference in the fusion rate and cleavage rate of reconstructed embryos (P>0.05), when using Boer and cross-breeding goat oocytes as recipient cytoplast respectively. However, in vitro morula development of reconstructed embryos from Boer oocytes was significantly higher than that of cross-breeding embryos (34.1% versus 19.1%, P<0.05). There was no significant difference in the rate of pregnancy and foetus loss between the 2 breeds. However, the live-birth rate was significantly higher with Boer goat oocyte recipients than the cross-breeds (3.1% versus 0.8%, P<0.05). Mitochondrial analysis showed that the 2 cloned goats were similar to their respective oocyte donor goats, and significantly different from the nucleus donor. In conclusion, genetic background of recipient oocytes affected in vitro and in vivo development of reconstructed embryos, with the homologous background of cytoplast and nuclear donor benefiting development of reconstructed embryos. The mitochondrial origin of the 2 cloned Boer goats came from recipient oocytes, not donors.     

Duration of in vitro maturation of recipient oocytes affects blastocyst development of cloned porcine embryos

This study investigated the effects of different incubation periods for oocyte maturation and contact inhibition of donor cells as well as different osmolarities for storage of recipient oocytes on fusion rates, cleavage rates, and blastocyst yields of porcine somatic nuclear transfer (SCNT) derived embryos. In addition, the in vivo developmental potential of cloned embryos derived from the most promising SCNT protocol was tested by transfer to recipient gilts. Storage of in vitro-matured oocytes for 7.5 h in calcium-free TL-HEPES medium at 295 or 320 mOsmol prior to activation yielded significantly (p < 0.05) higher parthenogenetic blastocyst rates compared to storage in TL-HEPES with an osmolarity of 270 mOsmol (24.4 +/- 3.0% and 26.2 +/- 4.3% vs. 18.3 +/- 6.4%, respectively, mean +/- SD) and improved the visibility of the polar body. Electrical fusion of fibroblasts to enucleated oocytes matured for 38, 40, or 42 h resulted in similar fusion and cleavage rates (74.8-84.4%). However, nuclear transfer with oocytes matured for 40 h in vitro yielded significantly higher (p < 0.05) development to the blastocyst stage after 7 days of culture (14.7 +/- 1.7%) than with oocytes matured for 38 h (9.5 +/- 2.1%) or 42 h (5.1 +/- 2.1%). Contact inhibition for 24, 48, or 72 h significantly (p < 0.05) increased the proportion of cells at G0/G1 compared with cycling fibroblasts. However, duration of contact inhibition of the donor cells for either 24, 48, or 72 h had no effect on blastocyst rates of SCNT embryos. Four gilts received an average of 150 SCNT embryos (range 138-161) reconstructed with oocytes matured for 40 h; two of these became pregnant; one of them went to term and farrowed four piglets on day 115 of pregnancy. Microsatellite analysis confirmed that the clones were genetically identical with the donor cells. These results show that changes of the in vitro maturation protocol may affect in vitro development of reconstructed porcine embryos, while duration of the contact inhibition period plays a minor role for the success of porcine SCNT. The effects on in vivo development are yet to be determined.     

体细胞的组织来源及培养代数对猪核移植重构胚发育的影响

本研究系统探讨了体细胞的组织来源及培养代数对猪核移植重构胚发育的影响。体外成熟培养40-44h的猪卵母细胞去核后,将经血清饥饿(0．5?s)培养2-9d、0．1mg/L Aphidicolin (APD)培养 0．5?S培养2-9d或一般培养法(10?S)培养的卵丘细胞、颗粒细胞、输卵管上皮细胞和耳皮成纤维细胞,直接注射到去核的卵母细胞质中,或注射到卵周隙中。再经电融合(100V/mm,30μs,电脉冲1次)构建重构胚。重构胚以钙离子载体A23817或电脉冲结合6- DMAP激活处理,体外培养6天。耳皮成纤维细胞和颗粒细胞经0．1mg/L APD 0．5?S培养处理后的重组胚卵裂率,均高于血清饥饿和一般培养处理的同种供体细胞(P＜0．01)。卵丘细胞、颗粒细胞经0．1mg/L APD 0．5?S处理后进行核移植的分裂率和发育率均高于输卵管上皮细胞和耳皮成纤维细胞(P＜0．05)。以猪颗粒细胞为核供体时,电融合法的重构胚分裂率显著高于胞质内注入法(P＜0．05),但囊胚发育率无显著差异(P＞0．05)。培养3代和6代的猪颗粒细胞以及培养6代和10代的耳皮成纤维细胞,其具有正常二倍染色体的细胞比例均无显著差异(P＞0．05);以这2种细胞不同培养代数做供体进行核移植时,各代之间核移胚的体外分裂率、囊胚发育率无显著差异(P＞0．05)。这些结果表明：(1)猪耳皮成纤维细胞和颗粒细胞经培养传代所建立起来的细胞系相对比较稳定;(2)0．1mg/L APD预培养处理供体细胞能提高猪体细胞核移植的效果,血清饥饿培养则无明显效果;(3)猪颗粒细胞和耳皮成纤维细胞等均可做供核细胞．核移植后都能得到体细胞克隆的囊胚,但前者的效果略优于后者,且其核移植效果不受供核细胞培养代数的影响;(4)电融合核移植胚胎的发育率高于胞质内直接注入法,但两者的总体效率相近。     

猪体细胞核移植重构胚的体外发育(英文)

以卵丘细胞为核供体细胞组成重构胚 ,卵裂率达到 5 6.7% ,发育至桑椹胚率达到1 1 .7% ,囊胚率为 6.7% ,显著高于成纤维细胞重构胚 (P <0 .0 5 )。本文还研究了卵母细胞的采集方法、激活程序和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导至G0 G1 期 ,抽吸法 解剖法采集卵母细胞 ,体外培养 3 3～ 44h ,将卵丘细胞放至去核卵母细胞的卵周隙中 ,重构胚以钙离子载体A2 3 81 7或电脉冲结合 6 DMAP激活处理 ,体外培养 6d。研究表明 ,卵母细胞采集方法、激活液中细胞松弛素 (CB)、激活程序并不影响重构胚的发育 (以卵龄 44h的卵母细胞为受体 ) ;而以电脉冲结合 6 DMAP激活处理能提高重构胚发育能力 (以卵龄 3 3h的卵母细胞为受体 ) (P <0 .0 5 )。本研究显示 ,以电脉冲结合 6 DMAP激活卵丘细胞重构胚 ,体外能发育至囊胚     

影响猪体细胞核移植重构胚体外发育的若干因素

以卵丘细胞为核供体细胞组成重构胚,卵裂率达到56．7％,发育至桑椹胚达11．7％、孵化囊胚率为6．7％,显著高于成纤维细胞组成的重构胚（P＜0．05）。我们研究了卵母细胞的采集方法,激活方法和卵龄对卵丘细胞核移植重构胚体外发育的影响。以血清饥饿法将卵丘细胞诱导至G0或G1期,抽吸法／解剖法采集卵母细胞,体外培养33或44h,将卵丘细胞置于去核卵母细胞的卵周隙中,重构胚以钙离子载体A23817或电泳冲结合6－DMAP激活处理,体外培养6天,结果表明,卵 母细胞采集方法、激活液中细胞松弛素（CB）并不影响重构胚的发育（以卵龄44h的卵母细胞为受体）;而以电脉冲结合6－DMAP激活处理能提高重构胚发育能力（以卵龄33h的卵母细胞为受体）（P＜0．05）。本研究显示,以电脉冲结合6－DMAP激活卵丘细胞重构胚,能在体外发育至囊胚。     

Somatic cell nuclear transfer using transported in vitro-matured oocytes in cynomolgus monkey

Somatic cell nuclear transfer (SCNT) is not successful so far in non-human primates. The objective of this study was to investigate the effects of stimulation cycles (first and repeat) on oocyte retrieval and in vitro maturation (IVM) and to evaluate the effects of stimulation cycles and donor cell type (cumulus and fetal skin fibroblasts) on efficiency of SCNT with transported IVM oocytes. In this study, 369 immature oocytes were collected laparoscopically at 24 h following human chorionic gonadotrophin (hCG) treatment from 12 cynomolgus macaque (Macaca fascicularis) in 24 stimulation cycles, and shipped in pre-equilibrated IVM medium for a 5 h journey, placed in a dry portable incubator (37 degrees C) without CO(2) supplement. A total of 70.6% (247/350) of immature oocytes reached metaphase II (MII) stage at 36 h after hCG administration, MII spindle could be seen clearly in 80.6% (104/129) of matured IVM oocytes under polarized microscopy. A total of 50.0% (37/74) of reconstructive SCNT embryos cleaved after activation; after cleavage, 37.8% (14/37) developed to the 8-cell stage and 8.1% (3/37) developed to morula, but unfortunately none developed to the blastocyst stage. Many more oocytes could be retrieved per cycle from monkeys in the first cycle than in repeated cycles (19.1 vs. 11.7, p < 0.05). There were no significant differences in the maturation rate (70.0 vs. 71.4%, p > 0.05) and MII spindle rate under polarized microscopy (76.4 vs. 86.0%, p > 0.05) between the first and repeat cycles. There were also no significant differences in the cleavage rate, and the 4-cell, 8-cell and morula development rate of SCNT embryos between the first and repeat cycles. When fibroblast cells and cumulus cells were used as the donor cells for SCNT, first cleavage rate was not significantly different, but 4-cell (50.0 vs. 88.9%, p < 0.05) and 8-cell (0 vs. 51.9%, p < 0.01) development rate were significantly lower for the former. In conclusion, the number of stimulation cycles has a significant effect on oocyte retrieval, but has no effect on maturation and SCNT embryo development; however, different donor cell types (cumulus and fibroblast) resulted in different developmental potentials of SCNT embryos.     

Nuclear transfer of synchronized african wild cat somatic cells into enucleated domestic cat oocytes

The African wild cat is one of the smallest wild cats and its future is threatened by hybridization with domestic cats. Nuclear transfer, a valuable tool for retaining genetic variability, offers the possibility of species continuation rather than extinction. The aim of this study was to investigate the ability of somatic cell nuclei of the African wild cat (AWC) to dedifferentiate within domestic cat (DSH) cytoplasts and to support early development after nuclear transplantation. In experiment 1, distributions of AWC and DSH fibroblasts in each cell-cycle phase were assessed by flow cytometry using cells cultured to confluency and disaggregated with pronase, trypsin, or mechanical separation. Trypsin (89.0%) and pronase (93.0%) yielded higher proportions of AWC nuclei in the G0/G1 phase than mechanical separation (82.0%). In contrast, mechanical separation yielded higher percentages of DSH nuclei in the G0/G1 phase (86.6%) than pronase (79.7%) or trypsin (74.2%) treatments. In both species, pronase induced less DNA damage than trypsin. In experiment 2, the effects of serum starvation, culture to confluency, and exposure to roscovitine on the distribution of AWC and DSH fibroblasts in various phases of the cell cycle were determined. Flow cytometry analyses revealed that the dynamics of the cell cycle varied as culture conditions were modified. Specifically, a higher percentage of AWC and DSH nuclei were in the G0/G1 phase after cells were serum starved (83% vs. 96%) than were present in cycling cells (50% vs. 64%), after contact inhibition (61% vs. 88%), or after roscovitine (56% vs. 84%) treatment, respectively. In experiment 3, we evaluated the effects of cell synchronization and oocyte maturation (in vivo vs. in vitro) on the reconstruction and development of AWC-DSH- and DSH-DSH-cloned embryos. The method of cell synchronization did not affect the fusion and cleavage rate because only a slightly higher percentage of fused couplets cleaved when donor nuclei were synchronized by serum starvation (83.0%) than after roscovitine (80.0%) or contact-inhibition (80.0%). The fusion efficiency of in vivo and in vitro matured oocytes used as recipient cytoplasts of AWC donor nuclei (86.6% vs. 85.2%) was similar to the rates obtained with DSH donor nuclei, 83.7% vs. 73.0%, respectively. The only significant effect of source of donor nucleus (AWC vs. DSH) was on the rate of blastocyst formation in vitro. A higher percentage of the embryos derived from AWC nuclei developed to the blastocyst stage than did embryos produced from DSH nuclei, 24.2% vs. 3.3%, respectively (P < 0.05). In experiment 4, the effect of calcium in the fusion medium on induction of oocyte activation and development of AWC-DSH-cloned embryos was determined. The presence of calcium in the fusion medium induced a high incidence of cleavage of DSH oocytes (54.3%), while oocyte cleavage frequency was much lower in the absence of calcium (16.6%). The presence or absence of calcium in the fusion medium did not affect the fusion, cleavage, and blastocyst development of AWC-DSH-cloned embryos. In experiment 5, AWC-DSH-cloned embryos were transferred to the uteri of 11 synchronized domestic cat recipients on Day 6 or 7 after oocyte aspiration. Recipients were assessed by ultrasonography on Day 21 postovulation, but no pregnancies were observed. In the present study, after NT, AWC donor nuclei were able to dedifferentiate in DSH cytoplasts and support high rates of blastocyst development in vitro. Incomplete reprogramming of the differentiated nucleus may be a major constraint to the in vivo developmental potential of the embryos.