Characterization of embryos derived from calf oocytes: kinetics of cleavage, cell allocation to inner cell mass, and trophectoderm and lipid metabolism

Embryos derived from calf oocytes were compared with adult cow oocyte-derived embryos (1) by studying the kinetics of embryo development using time-lapse cinematography (2) by evaluating the ratio between inner cell mass (ICM) and trophectoderm (TE) cells in blastocysts (3) by measuring the triglyceride content of the blastocysts. The rate of calf oocyte-derived embryos reaching the blastocyst stage was reduced (26 vs. 46% for adult derived embryos). Calf oocyte-derived embryos preferably arrested their development before the 9-cell stage. Those that developed into blastocysts had cleaved earlier to reach the 2-cell or 3-cell stages than embryos that arrested before the 9-cell stage. The 9-cell stage tended to appear later in calf oocyte-derived embryo that reached the blastocyst stage than in adult-derived embryos. This difference became significant at the morula stage. Accordingly, the fourth cell cycle duration was longer for calf oocyte-derived embryos. Day 8 blastocysts from both sources had similar total cell numbers (calf: 89 +/- 20; cow: 100 +/- 30) and cell distribution between TE and ICM. The triglyceride content of day 7 blastocysts was similar for both sources (64 +/- 15 vs. 65 +/- 6 ng/embryo, respectively). In conclusion, calf oocyte-derived embryos are characterized by a higher rate of developmental arrest before the 9-cell stage and by a longer lag phase preceding the major onset of embryonic genome expression. These changes might be related to insufficient "capacitation" of the calf oocyte during follicular growth. Despite these differences, modifications in the quality of the resulting blastocysts were not detected.     

Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo

Porcine embryos produced in vitro have a small number of cells and low viability. The present study was conducted to examine the morphological characteristics and the relationship between actin filament organization and morphology of porcine embryos produced in vitro and in vivo. In vitro-derived embryos were produced by in vitro maturation, in vitro fertilization (IVF), and in vitro development. In vivo-derived embryos were collected from inseminated gilts on Days 2-6 after estrus. In experiment 1, in vitro-derived embryos (相似文献   

Development of sheep preimplantation embryos in media supplemented with glucose and acetate

Two experiments were conducted to examine the effect of supplemental glucose (G; 1.5 mM) and/or acetate (A; 0.5 mM) on the development of early sheep embryos to blastocysts when cultured in vitro in glucose-free synthetic oviductal fluid (SOF) + sheep serum or bovine serum albumin (BSA). In Experiment 1, 2- to 4-cell, 8- to 16-cell and >16-cell embryos were cultured in SOF, SOF+G, SOF+A or SOF+G+A. All media were supplemented with 10% sheep serum. In addition, embryos were cultured in either microdrops under polysiloxane oil or in multiwell dishes. Overall, development to the blastocyst stage was 3%, 30% and 68% for 2- to 4-cell, 8- to 16-cell and >16-cell stages, respectively, suggesting that an 8-cell developmental block existed under our culture conditions. Glucose supplementation had little effect on embryo development, and no overall effect was observed from the addition of acetate. In Experiment 2, 8- to 16-cell embryos were cultured in SOF or SOF+G, both supplemented with BSA. Development to the blastocyst stage was 25% and 18%, respectively. The results show that the presence of glucose or acetate did little to enhance embryonic development in our incubation systems. Further work is required to evaluate fully the energy requirements for development of the early sheep embryo.     

Quantification of basigin mRNA in mouse oocytes and preimplantation embryos by competitive RT-PCR

Basigin is a member of the immunoglobulin superfamily and a key molecule related to mouse blastocyst implantation. Whether preimplantation mouse embryos express basigin mRNA is still unknown. The aim of this study was to use a quantitative competitive polymerase chain reaction to assess quantitatively the levels of basigin mRNA in mouse oocyte and preimplantation embryos. Basigin mRNA was detected in the oocyte and all the stages of preimplantation embryos. The levels of basigin mRNA were 0.0606 +/- 0.0282 in the oocyte, 0.0102 +/- 0.0036 in the zygote, 0.0007 +/- 0.0003 in the 2-cell embryo, 0.0031 +/- 0.0017 in the 4-cell embryo, 0.0084 +/- 0.0024 in the 8-cell embryo, 0.0537 +/- 0.0121 in the morula and 0.0392 +/- 0.0161 attomoles in the blastocyst, respectively. The levels of basigin mRNA in the oocyte, morula and blastocyst were significantly higher than those in the zygote and embryos at the 2-cell, 4-cell and 8-cell stages. The high level of basigin expression in the blastocyst may play a role during embryo implantation.     

Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos

After in vitro maturation and fertilization of porcine oocytes, the fertilized embryos were cultured under 5 or 20% oxygen (O2) for 7 days. In embryos cultured under 5% O2 versus 20% O2, development to the blastocyst stage was higher (36.3% versus 22.5%, P < 0.05); the hydrogen peroxide (H2O2) content as a reactive oxygen species was lower (92 pixels versus 111 pixels, P < 0.05); and fragmentation of DNA in 8- to 16-cell stage embryos (estimated by the comet assay) resulted in a shorter (P < 0.05) DNA tail (36 microm versus 141 microm). Antioxidants such as beta-mercaptoethanol (beta-ME) and Vitamin-E (Vit-E) suppressed oxidative damage in the embryos and improved their developmental ability. For embryos cultured under 20% O2, there were the following differences (P < 0.05) between embryos exposed to 0 microM versus 50 microM beta-ME: 28% versus 57% developed to the blastocyst stage; 125 pixels versus 98 pixels per embryo in H2O2 content; and a DNA tail of 209 microm versus 105 microm. In addition, for embryos cultured under 20% O2, there were also differences (P < 0.05) between those exposed to 0 microM versus 50 microM of Vit-E: 28% versus 40% rate of development to the blastocyst stage; 28.9 cells versus 35.9 cells in the expanded blastocyst; 122 pixels versus 95 pixels per embryo (H2O2 content); and 215 microm versus 97 microm length of the DNA tail. Therefore, a low O2 concentration during in vitro culture of porcine embryos decreased the H2O2 content and, as a consequence, reduced DNA fragmentation, and, thereby, improved developmental ability.     

Cleavage pattern and survivin expression in porcine embryos by somatic cell nuclear transfer

Mammalian embryos produced in vitro show a high rate of early developmental failure. Numerous somatic cell nuclear transfer (SCNT) embryos undergo arrest and show abnormal gene expression in the early developmental stages. The purpose of this study was to analyze porcine SCNT embryo development and investigate the cause of porcine SCNT embryo arrest. The temporal cleavage pattern of porcine SCNT embryos was analyzed first, and the blastocyst origin at early developmental stage was identified. To investigate markers of arrest in the cleavage patterns of preimplantation SCNT embryos, the expression of survivin—the smallest member of the inhibitor of apoptosis (IAP) gene family, which suppresses apoptosis and regulates cell division—was compared between embryos showing normal cleavage and arrested embryos.A total of 511 SCNT embryos were used for cleavage pattern analysis. Twenty-four hours post activation (hpa), embryos were classified into five groups based on the cleavage stage as follows; 1-cell, 2-cell, 4-cell, 8-cell and fragmentation (frag). In addition, 48 hpa embryos were more strictly classified into 15 groups based on the cleavage stage of 24 hpa; 1-1 cell (24 hpa-48 hpa), 1-2 cell, 1-4 cell, 1-8 cell, 1 cell-frag, 2-2 cell, 2-4 cell, 2-8 cell, 2 cell-frag, 4-4 cell, 4-8 cell, 4 cell-frag, 8-8 cell, 8 cell-frag, and frag-frag. These groups were cultured until 7 d post activation, and were evaluated for blastocyst formation. At 24 hpa, the proportion of 2-cell stage was significantly higher (44.5%) than those in the other cleavage stages (1-cell: 13.4%; 4-cell: 17.9%; 8-cell: 10.3%; and frag: 13.9%). At 48 hpa, the proportion of embryos in the 2-4 cell stage was significantly higher (32.4%) than those in the other cleavage stages (2-8 cell: 8.2%; 4-8 cell: 12.1%; and frag-frag: 13.9%). Some embryos arrested at 48 hpa (1-1 cell: 5.8%; 2-2 cell: 2.8%; 4-4 cell: 3.8%; 8-8 cell: 6.5%; and total arrested embryos: 18.9%). Blastocyst formation rates were higher in 2-4 cell cleavage group (20.2%) than in other groups. SCNT embryos in 2-4 cell stage showed stable developmental competence. In addition, we investigated survivin expression in porcine SCNT embryos during the early developmental stages. The levels of survivin mRNA in 2-cell, 4-cell stage SCNT embryos were significantly higher than those of arrested embryos. Survivin protein expression showed a similar pattern to that of survivin mRNA. Normally cleaving embryos showed higher survivin protein expression levels than arrested embryos. These observations suggested that 2-4 cell cleaving embryos at 48 hpa have high developmental competence, and that embryonic arrest, which may be influenced by survivin expression in porcine SCNT embryos.     

Pregnancies after in-vitro fertilization of cow follicular oocytes, their incubation in rabbit oviduct and their transfer to the cow uterus

Cow embryos fertilized in vitro (1-8-cell) (n = 113) were transferred surgically to the ligated oviduct of pseudopregnant female rabbits (31 +/- 4 h after 75 i.u. hCG). Rabbits were killed 99 +/- 5 h later and 67 embryos were recovered: 45 (67%) had cleaved at least once, 15 had reached the morula stage and 2 blastocysts were obtained. Transfer of cleaved embryos (2-8-cell) led to a high recovery rate (84%) compared to 39% for one-cell embryos. Of the embryos incubated for more than 99 h in the rabbit oviduct, 41% were at the morula stage. Embryos incubated in vivo (n = 21) (8-cell to blastocyst) were transferred to the uterus of 14 synchronized recipient heifers by a surgical (N = 5) or a non-surgical (N = 9) procedure: 6 pregnancies resulted (4 from the non-surgical procedures). In addition, 27 (2-8-cell) cow embryos developed in vitro were transferred to the oviduct of synchronized heifers by a paralumbar surgical approach on a standing animal, but no pregnancies resulted. It is therefore concluded that (1) the rabbit oviduct can be used to obtain cow eggs at an embryonic stage sufficiently advanced to permit transfer to the uterus of a synchronized recipient; and (2) the pregnancy rate after in-vitro fertilization and incubation in the rabbit oviduct are similar to results with fertilization in vivo.     

Effects of nitrous oxide on preimplantation mouse embryo cleavage and development

Preimplantation mouse embryos were exposed to nitrous oxide for 30 min to determine its effects on subsequent development after short durations of exposure. Two-cell mouse embryos were exposed to 60% nitrous oxide/40% oxygen at 6-7 h, 3-4 h, or 0-1 h prior to the expected onset of their first cleavage in vitro, or at the 4-cell or morula stages. Effects of nitrous oxide were not observed except in 2-cell embryos treated within 4 h of the expected in vitro cleavage. At 3-4 h and 0-1 h prior to the onset of cleavage, exposure to 60% nitrous oxide/40% oxygen resulted in blastocyst development rates of 27.7% and 4.7%, respectively, while control rates ranged from 75% to 77%. The majority of affected embryos were halted at the 2-cell stage before completing cell division. Similar effects were obtained with 80% nitrous oxide/20% oxygen. Thus, we conclude that brief exposure of mouse preimplantation embryos to nitrous oxide may be deleterious to subsequent embryo cleavage, but this effect is highly dependent on the developmental stage at which exposure occurs.     

Requirement for glucose during in vitro culture of sheep preimplantation embryos.

Glucose utilization by sheep embryos was examined in 8-cell (N = 36) and blastocyst (N = 36) stages, by measuring conversion of [5-3H]glucose to 3H2O. Fifty percent glucose utilization occurred at 0.79 +/- 0.69 mM for 8-cell embryos and -0.06 +/- 0.15 mM for blastocysts. Development of 1- and 2-cell sheep embryos (N = 264) was examined under different glucose concentrations (0, 1.5, 3, or 6 mM) and in the presence or absence of 0.33 mM pyruvate and 3.3 mM lactate (PL). Overall, the presence of glucose was detrimental (P less than 0.001) to embryonic development. By contrast, the presence of pyruvate and lactate was beneficial (P less than 0.001) to development. An interaction was observed between the concentration of glucose and presence or absence of PL (P less than 0.05). An optimum level of glucose occurs at 0-3 mM in the presence of PL (P less than 0.1). Development to the blastocyst stage was observed in medium when supplemented with amino acids and albumin alone. Thus, glucose metabolism is not critical for embryonic development, but beneficial at low concentrations. High concentrations can inhibit development, possibly by inhibiting the tricarboxylic acid (TCA) cycle. Sheep embryos may also be using amino acids as an energy source for development.     

The effect of oxygen on the development of preimplantation mouse embryos in vitro

The optimal oxygen tension for development of preimplantation mouse embryos to the blastocyst stage in vitro was found to be between 2.5% and 5%. One- and two-cell embryos had a more sharply defined range of oxygen tension capable of supporting development than 8-cell and morula stages. At all stages of development, more embryos developed to the blastocyst stage under 5% O2 compared to the numbers of developing under higher oxygen tensions (20% and 40% O2). The blastocysts developing under 20% O2 had fewer blastomeres than those which developed under 5% O2. As the time required for development to the blastocyst stage in vitro increased, there were fewer blastomeres present at the blastocyst stage. These results indicate that the cleaving mouse embryo has an optimal oxygen requirement in vitro of about 5%. At higher oxygen tensions, fewer embryos develop to the blastocyst stage and in those which do develop, there are fewer cell divisions. If a gradient of oxygen tension exists across the blastomeres from the outside of the embryo to its centre, the blastomeres might be using this gradient to obtain imformation about their location within the embryo and respond accordingly. Thus blastomeres on the outside at a higher oxygen tension would divide at a slower rate and form trophectoderm whereas those on the inside at a lower oxygen tension would divide more rapidly and contribute to the inner cell mass.     

Ploidy of bovine nuclear transfer blastocysts reconstructed using in vitro produced blastomere donors

The higher rate of embryonic loss in nuclear transfer compared to in vitro produced embryos may be due to chromosome abnormalities that occur during preimplantation in vitro development. Because little is known about ploidy errors in nuclear transfer embryos, this was examined using embryos reconstructed from in vitro produced embryo donors. In vitro matured oocytes were enucleated and then activated using calcium ionophore A23187 followed by 6-dimethylaminopurine (6-DMAP). Subsequently, embryos were reconstructed using blastomeres from day 4-5 in vitro produced donors. The embryos were cultured until day 7 at which time blastocyst nuclei were extracted and chromosome abnormalities were evaluated by fluorescent in situ hybridization using two probes that bind to the subcentromeric regions on chromosomes 6 and 7. In 16 nuclear transfer blastocysts generated from 5 donor embryos, 53.8 +/- 20.2 (mean % +/- SD) nuclei/embryo were examined. Of these 16, 7 embryos (43.8%) were potentially abnormal because in these, 1.1%, 1.4%, 5.3%, 7.5%, 26.3%, 30.4%, and 66.2% % of the nuclei had a chromosome composition deviating from the diploid condition, indicating a wide degree of variation between embryos. These errors comprised mainly triploid (8.2 +/- 10.3 [0-26.3]: % +/- SD [range]) and tetraploid (10.6 +/- 19.9 [0-54.9]) nuclei with other ploidy combinations accounting for only 0.9 +/- 2.1 [0-2.1]% of deviant nuclei. The proportion of completely normal nuclear transfer embryos was no less than those produced by in vitro fertilization but the distribution of chromosome abnormalities was different (p = 0.0002). In conclusion, nuclear transfer embryos reconstructed using blastomere cells can produce over 50% blastocysts with a diploid chromosome complement. However, the contribution of chromosome abnormalities to embryonic loss in the remaining embryos deserves further investigation.     

Effect of embryo developmental stage and culture conditions on number and quality of ovine in vitro produced blastocysts

This study evaluated the final output and quality of in vitro produced blastocysts derived from in vivo recovered sheep embryos cultured at various early developmental stages to blastocyst. A total of 270 embryos were recovered from the oviduct, at different days of the early luteal phase, and were classified into three different developmental stages: 2- to 4-cell (n = 93); 5- to 8-cell (n = 92) and 9- to 12-cell (n = 85). The effect of culture conditions was studied, at the same time, by randomly allocating the embryos to one of four groups: three groups of culture with fresh oviduct monolayers (2, 4 and 5 days old) and a fourth group with 2-day monolayers derived from frozen-thawed oviduct cells. Two control groups were established: first, embryos cultured in semi-defined medium (n = 29) and, second, blastocysts obtained in vivo and cryopreserved (n = 43). Influence on blastocyst yield of embryo developmental stage at the start of culture was statistically significant (p < 0.001). Two- to four-cell embryos showed a significantly lower developmental rate (67.7%) than the 5- to 8-cell (83.6%; p < 0.001) and 9- to 12-cell groups (90.5%; p < 0.0001) and lower quality in terms of blastocyst cryotolerance (56.0 vs. 83.7%; p < 0.005). There were no detected effects relating to the age or handling of the monolayer on the embryo developmental rate, but the day of blastocyst appearance was different between embryos cultured on monolayers derived from fresh or frozen-thawed cells (p < 0.0001); the main influence was on the group of 9- to 12-cell embryos (p < 0.0001). Current results confirm the temporal sensitivities of sheep embryos to in vitro culture, regardless of the culture conditions.     

Effects of antioxidants on the development of bovine IVM/IVF embryos in various concentrations of glucose

This study was undertaken to determine the effects of glucose, antioxidants and different oxygen tensions on the development of bovine embryos cultured in modified synthetic oviduct fluid (m-SOF) medium. In vitro matured (IVM) and fertilized (IVF) oocytes were incubated for 48 h. Embryos reaching at least the 4-cell stage were selected for further culture under various conditions for 6 d. Supplementing the m-SOF media with 4.5 mM glucose resulted in a significantly lower (P < 0.01) embryo developmental rate (21%; Day 8) than was obtained with 1.5 mM glucose (58%; Day 8) or no glucose (53%; Day 8). Antioxidants such as SOD, catalase and mannitol had no positive effect on embryo development in m-SOF medium supplemented with 1.5 mM glucose. However, in m-SOF medium supplemented with 4.5 mM glucose, SOD and mannitol significantly (P < 0.05) improved embryo development: SOD increased the developmental rate from 19 to 35% (Day 8), while mannitol increased it from 13 to 30% (Day 8). Low oxygen concentration improved embryo development significantly (P < 0.05) in m-SOF medium supplemented with 4.5 mM glucose (low O2: 31% vs high O2: 14%; Day 8) but not 0 mM glucose (low O2: 58% vs high O2: 55%; Day 8). Our data suggest that low concentration of glucose during culture of bovine embryos is beneficial, and that generation of free oxygen radicals is partly caused by a high concentration of glucose in the medium.     

慢病毒载体介导的绿色荧光蛋白转基因标记法研究猪嵌合体制作

目的:通过建立慢病毒载体感染猪胚胎体系实现胚胎标记,进而研究不同发育阶段猪孤雌胚胎之间的嵌合能力,为进一步研究猪早期胚胎发育以及细胞分化奠定基础.方法:首先,通过显微注射的方法把2×109I.U./ml、2×108I.U./ml和2×107I.U./ml三个梯度的表达绿色荧光的慢病毒载体分别注射到猪1-细胞胚胎和2-细胞胚胎的透明带下,进行胚胎的GFP转基因标记,在荧光显微镜下观察比较卵裂率、阳性胚胎率、囊胚率、阳性囊胚率和囊胚细胞数.然后,采用凹窝聚合法对同步发育胚胎在不同阶段(2-细胞,4-细胞,8-细胞)进行嵌合,2-细胞胚胎与不同发育阶段(2-细胞、4-细胞、8-细胞)胚胎进行嵌合以及2-细胞胚胎卵裂球互换制作嵌合体胚胎,发育到囊胚时在荧光显微镜下检测胚胎的嵌合状态.结果:2×109I.U./ml的慢病毒感染猪2-细胞胚胎组中,体外受精和孤雌胚胎感染阳性率( 80.00％、76.36％)和阳性囊胚率(90.74％、89.56％)都显著高于其它滴度组(P＜0.05),另外,慢病毒感染的两种胚胎与对照组对卵裂率、囊胚率和囊胚细胞数三个指标没有显著影响(P＞0.05).2-细胞胚胎之间嵌合囊胚率和2-细胞卵裂球互换嵌合囊胚率( 53.85％、62.50％)显著高于2-细胞胚胎与4-细胞胚胎的嵌合率(18.60％,P＜0.05),在同步发育胚胎中8-细胞胚胎之间的嵌合率(75.00％)高于4-细胞胚胎之间和2-细胞胚胎之间的嵌合率( 65.00％、53.80％).结论:2×109I.U./ml的慢病毒感染2-细胞期胚胎效率最高,另外,慢病毒感染对猪胚胎发育没有明显影响.8-细胞间的嵌合率比较高;发育同步胚胎间的嵌合率高于发育非同步胚胎间的嵌合率.     

Relative developmental abilities of hamster 2- and 8-cell embryos cultured in hamster embryo culture medium-1 and -2

The relative developmental abilities of hamster 2- and 8-cell embryos in culture were compared using two versions of hamster embryo culture medium (HECM). These media differed primarily in the number of amino acids present, i.e., 20 amino acids in HECM-1 and four amino acids in HECM-2. When 2-cell embryos were cultured for 24 h, the percentages of greater than or equal to 4-cell embryos obtained in both HECM-1 and HECM-2 were comparable (congruent to 93%); at the end of 48 h, the proportion of greater than or equal to 8-cell embryos obtained in HECM-1 (82.5%) was significantly (P less than or equal to 0.001) more than that obtained in HECM-2 (67.9%). Interchange of media, after 24 h culture, did not enhance the ability of cultured 2-cell embryos to become blastocysts. When 8-cell embryos were cultured for 18 h in HECM-1 and -2, there was no appreciable difference in the proportion of total blastocysts formed (89-91%). However, there were significantly (P less than or equal to 0.001) more late blastocysts in HECM-2 than in HECM-1 (68.2% vs. 38.4%). Embryo development from 2- and 8-cell stages was compared in media that differed by the presence and absence of phenol red and penicillin-G. There was no difference in embryo development when these compounds were present or absent. Similarly, the difference in pyruvate concentration between HECM-1 and -2 (0.5 and 0.2 mM, respectively) did not affect embryo development.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo development of vitrified rat embryos: effects of timing and sites of transfer to recipient females

In cryopreserved rat embryos, survival rates obtained in vitro are not always consistent with the rates obtained in vivo. To determine the optimal conditions for in vivo development to term, rat embryos at the 4-cell, 8-cell, and morula stages were vitrified in EFS40 by a one-step method and transferred into oviducts or uterine horns of recipients at various times during pseudopregnancy. Vitrified and fresh 4-cell embryos only developed after transfer into oviducts of asynchronous recipients on Days -1 to -2 of synchrony (i.e., at a point in pseudopregnancy 1-2 days earlier than the embryos). Approximately half the vitrified embryos transferred into oviducts on Day -1 developed to term, but only a minority of embryos, whether vitrified (10%-34%) or fresh (24%-33%), transferred at later times did so, suggesting that this may not be the most suitable stage for cryopreservation. Very few 8-cell embryos, either vitrified or fresh, developed when transferred into oviducts on Day 0 to -0.5. However, when transferred into uterine horns, high proportions of vitrified 8-cell embryos ( approximately 63%) developed to term in reasonably synchronous recipients (Day 0 to -0.5) but not in more asynchronous ones (6%; Day -1). A majority of vitrified morulae also developed to term (52%-68%) in a wider range of recipients (Days 0 to -1), the greatest success occurring in recipients on Day -0.5. Similar proportions of vitrified and fresh 4-cell embryos, 8-cell embryos, and morulae developed to term when appropriate synchronization existed between embryo and recipient. Thus, vitrification of preimplantation-stage rat embryos does not appear to impair their developmental potential in vivo.     

转人t-PA指形区缺失基因的山羊体细胞核移植及其继代核移植

为了探索转基因体细胞核经连续核移植后的发育潜力,以转人组织型纤溶酶原激活剂(t-PA)指形区缺失基因的山羊胎儿成纤维细胞为核供体,MII期的卵母细胞质为核受体,利用胞质内注射法构建原代核移胚胎(G0),并进行了原代核移植胚胎的继代核移植研究。比较原代和继代核移植胚胎在体外发育能力上的差异;在G1、G2代核移植试验过程中,比较了供体胚胎细胞的发育阶段对核移植胚胎体外发育的影响。结果表明,原代核移植胚胎的卵裂率(76.45%±1.17%)与继代核移植胚胎的卵裂率(72.18%±1.97%,76.05%±2.38%,75.99%±2.84%)无显著性差异(P>0.05)。但原代核移植胚胎的桑葚胚率(47.20%±2.93%)、囊胚率(11.00%±1.42%)显著高于G1、G2、G3代核核移植胚胎的桑葚胚率(34.99%±2.66%,28.23%±2.00%,23.34%±1.99%)、囊胚率(3.87%±0.67%,2.08%±1.66%,0);在G1、G2中,当用16-细胞期核移植胚胎作为核供体时的桑葚胚率(29.57%±1.53%,24.43%±1.87%)、囊胚率(1.96%±1.31%,2.01%±1.34%)低于用32~64-细胞时期的核移植胚胎的桑葚胚率(34.32%±1.31%,29.76%±1.66%)、囊胚率(3.86%±1.03%,3.48%±0.34%),但无显著性差异(P>0.05)。由此得出结论:转基因体细胞核移植胚胎不宜进行多代克隆;胞质内注射法构建核移植胚胎,用32~64-细胞期的胚胎作为核供体构建的核移植胚胎的体外发育率高于用16-细胞期的胚胎作为核供体构建的核移植胚胎的体外发育率。