Analysis of production efficiency of cloned transgenic Yucatan miniature pigs according to recipient breeds with embryo transfer conditions.

The purpose of this study was to compare the efficiency of the production of cloned transgenic Yucatan miniature pigs (YMPs) using two recipient breeds, i.e., YMPs and domestic pigs (DPs), under various embryo transfer conditions. We initially assessed the in vitro developmental competence of embryos obtained via somatic cell nuclear transfer (SCNT) from three different transgenic donor cells. No difference was observed among the three groups regarding developmental competence. Furthermore, the cloning efficiency remained consistent among the three groups after the transfer of the SCNT embryos to each surrogate mother. Subsequently, to compare the efficiency of the production of cloned transgenic YMPs between the two recipient breeds using varying parameters, including ovulation status (preovulation and postovulation), duration of in vitro culture (IVC) (incubated within 24 h and 24-48 h), and the number of transferred SCNT embryos (less than and more than 300), we assessed the pregnancy rates, delivery rates, mean offspring counts, and cloning efficiency. Regarding the ovulation status, YMPs exhibited higher pregnancy rates, delivery rates, and cloning efficiency compared with DPs in both statuses. Moreover, the pregnancy rates, delivery rates, and cloning efficiency were affected by the ovulation status in DPs, but not in YMPs. The comparison of IVC duration between groups revealed that YMPs had higher pregnancy rates vs. DPs in both conditions. SCNT embryos cultured for 24-48 h in YMPs yielded higher delivery rates and cloning efficiency compared with those cultured for less than 24 h in DPs. Finally, the analysis based on the number of transferred SCNT embryos showed that both the pregnancy and delivery rates were higher in YMPs vs. DPs. However, the highest average number of offspring was obtained when more than 300 SCNT embryos were transferred into DPs, whereas the cloning efficiency was higher in YMPs vs. DPs. Our results suggest that YMPs are more suitable recipients than are DPs under various conditions for the production of cloned transgenic YMPs.

Overexpression of BRG1 improves early development of porcine somatic cell nuclear transfer embryos.

The epigenetic modification levels of donor cells directly affect the developmental potential of somatic cell nuclear transfer (SCNT) embryos. BRG1, as an epigenetic modifying enzyme, has not yet been studied in donor cells and SCNT embryos. In this study, BRG1 was overexpressed in porcine fetal fibroblasts (PFFs), its effect on chromatin openness and gene transcription was examined, subsequently, the development potential of porcine SCNT embryos was investigated. The results showed that compared with the control group, the percentage of G1 phase cells was significantly increased (32.3 % ± 0.87 vs 25.7 % ± 0.81, P < 0.05) in the experimental group. The qRT-PCR results showed that the expression of H3K9me3-related genes was significantly decreased (P < 0.05), HAT1 was significantly increased (P < 0.05). Assay of Transposase Accessible Chromatin sequencing (ATAC-seq) results revealed that SMARCA4、NANOG、SOX2、MAP2K6 and HIF1A loci had more open chromatin peaks in the experimental group. The RNA-seq results showed that the upregulated genes were mainly enriched in PI3K/AKT and WNT signaling pathways, and the downregulated genes were largely focused on disease development. Interestingly, the developmental rate of porcine SCNT embryos was improved (27.33 % ± 1.40 vs 17.83 % ± 2.02, P < 0.05), the expression of zygotic gene activation-related genes in 4-cell embryos, and embryonic development-related genes in blastocysts was significantly upregulated in the experimental group (P < 0.05). These results suggest that overexpression of BRG1 in donor cells is benefit for the developmental potential of porcine SCNT embryos.

Cloning for the Twenty-First Century and Its Place in Endangered Species Conservation.

Cloning as it relates to the animal kingdom generally refers to the production of genetically identical individuals. Because cloning is increasingly the subject of renewed attention as a tool for rescuing endangered or extinct species, it seems timely to dissect the role of the numerous reproductive techniques encompassed by this term in animal species conservation. Although cloning is typically associated with somatic cell nuclear transfer, the recent advent of additional techniques that allow genome replication without genetic recombination demands that the use of induced pluripotent stem cells to generate gametes or embryos, as well as older methods such as embryo splitting, all be included in this discussion. Additionally, the phenomenon of natural cloning (e.g., a subset of fish, birds, invertebrates, and reptilian species that reproduce via parthenogenesis) must also be pointed out. Beyond the biology of these techniques are practical considerations and the ethics of using cloning and associated procedures in endangered or extinct species. All of these must be examined in concert to determine whether cloning has a place in species conservation. Therefore, we synthesize progress in cloning and associated techniques and dissect the practical and ethical aspects of these methods as they pertain to endangered species conservation.

Effect of additional cytoplasm injection on the cloned bovine embryo organelle distribution and stress mitigation.

Although somatic cell nuclear transfer (SCNT) is a critical component of animal cloning, this approach has several issues. We previously introduced the cytoplasm injection cloning technology (CICT), which significantly improves the quality and quantity of cloned embryos. This study examined the residual status of fused cytoplasmic organelles, such as the endoplasmic reticulum (ER) and lysosomes, in the CICT group during early embryo development. We found that extra-cytoplasmic organelles stained using the ER-Tracker™ Green dye and LysoTracker™ Deep Red probe were fused and dispersed throughout the recipient oocyte and were still visible in day 8 blastocysts. We screened for ER stress, autophagy, and apoptosis-related genes to elucidate the association between the added organelles and improved embryo quality in CICT-cloned embryos. We found that CHOP, ATF4, ATG5, ATG7, and LC3 genes showed non-significantly up- or downregulated expression between CICT- and in vitro fertilization (IVF)-derived embryos but showed significantly (p < 0.05) upregulated expression in SCNT-cloned embryos. Surprisingly, a non-significant difference in the expression of some genes, such as ATF6 and caspase-3, was observed between the CICT- and SCNT-cloned embryos. Our findings imply that compared to conventional SCNT cloning, CICT-derived cloned embryos with additional cytoplasm have much higher organelle activity, lower autophagy, lower rates of apoptosis, and higher embryo development rates.

Astaxanthin enhances the development of bovine cloned embryos by inhibiting apoptosis and improving DNA methylation reprogramming of pluripotency genes.

Low cloning efficiency limits the wide application of somatic cell nuclear transfer technology. Apoptosis and incomplete DNA methylation reprogramming of pluripotency genes are considered as the main causes for low cloning efficiency. Astaxanthin (AST), a powerfully antioxidative and antiapoptotic carotenoid, is recently shown to improve the development of early embryos, however, the potential role of AST during the development of cloned embryos remains unclear. This study displayed that treating cloned embryos with AST significantly increased the blastocyst rate and total blastocyst cell number in a concentration dependent manner, and also alleviated the damage of H2O2 to the development of cloned embryos. In addition, compared with the control group, AST significantly reduced the apoptotic cell number and rate in cloned blastocysts, and the significantly upregulated expression of anti-apoptotic gene Bcl2l1 and antioxidative genes (Sod1 and Gpx4) and downregulated transcription of pro-apoptotic genes (Bax, P53 and Caspase3) were observed in the AST group. Moreover, AST treatment facilitated DNA demethylation of pluripotency genes (Pou5f1, Nanog and Sox2), in accompany with the improved transcription levels of DNA methylation reprogramming genes (Tet1, Tet3, Dnmt1, Dnmt3a and Dnmt3b) in cloned embryos, and then, the significantly upregulated expression levels of embryo development related genes including Pou5f1, Nanog, Sox2 and Cdx2 were observed in comparison with the control group. In conclusion, these results revealed that astaxanthin enhanced the developmental potential of bovine cloned embryos by inhibiting apoptosis and improving DNA methylation reprogramming of pluripotency genes, and provided a promising approach to improve cloning efficiency.

Influence of season and conditions of surrogate sows on efficiency of somatic cell cloning production.

This study was focused on the effects of ovary acquisition season, embryo transfer season, and conditions of surrogate sows on cloning efficiency, with the objective of improving the production of cloned pigs. The statistical analysis documented that cloning efficiency was highest when ovary extraction and embryo transfer occurred in the spring, and lowest when such operations occurred in the autumn. This was evidenced by the higher number of recovered oocytes (3.2 ± 0.47 vs. 2.5 ± 0.51), rate of mature oocytes (57.4 ± 0.07% vs. 48.9 ± 0.06%), rate of developed cloned blastocysts (35.7 ± 0.12% vs. 34.4 ± 0.07%), pregnancy rate of surrogate sows (73.5% vs. 33.3%), delivery rate (67.6% vs. 16.7%), litter size (6.9 ± 2.3 vs. 2.3 ± 2.5), and the number of alive newborns (5.7 ± 2.2 vs. 1.3 ± 1.2). Cloning efficiency was little affected by the ovulatory status of the surrogate sow prior to embryo transfer. The length of pregnancy, the parity, and the length of labor of the surrogate sow significantly affected the efficiency of generating pigs cloned from somatic cells. Specifically, when length of pregnancy ranged from 111 to 117 days, surrogate sows with shorter gestation period had larger litter size (8.9 ± 2.8) and a higher number of stillbirths per litter (2.1 ± 2.0). Moreover, statistical analysis indicated that selecting sows with 2-4 parities as surrogates led to increased litter size (7.7 ± 3.0) and the number of alive newborns (6.4 ± 3.1). In comparison with naturally breeding sows, the surrogate sows spent more time giving birth and suffered higher rates of stillbirth. The data obtained in this study provide valuable insights for improving the production efficiency of somatic cell cloned pigs.

Cloning horses by somatic cell nuclear transfer: Effects of oocyte source on development to foaling.

The cloning of horses is a commercial reality, yet the availability of oocytes for cloned embryo production remains a major limitation. Immature oocytes collected from abattoir-sourced ovaries or from live mares by ovum pick-up (OPU) have both been used to generate cloned foals. However, the reported cloning efficiencies are difficult to compare due to the different somatic cell nuclear transfer (SCNT) techniques and conditions used. The objective of this retrospective study was to compare the in vitro and in vivo development of equine SCNT embryos produced using oocytes recovered from abattoir-sourced ovaries and from live mares by OPU. A total of 1,128 oocytes were obtained, of which 668 were abattoir-derived and 460 were OPU-derived. The methods used for in vitro maturation and SCNT were identical for both oocyte groups, and the embryos were cultured in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 Ham medium supplemented with 10% fetal calf serum. Embryo development in vitro was assessed, and Day 7 blastocysts were transferred to recipient mares. The embryos were transferred fresh when possible, and a cohort of vitrified-thawed OPU-derived blastocysts was also transferred. Pregnancy outcomes were recorded at Days 14, 42 and 90 of gestation and at foaling. The rates of cleavage (68.7 ± 3.9% vs 62.4 ± 4.7%) and development to the blastocyst stage (34.6 ± 3.3% vs 25.6 ± 2.0%) were superior for OPU-derived embryos compared with abattoir-derived embryos (P < 0.05). Following transfer of Day 7 blastocysts to a total of 77 recipient mares, the pregnancy rates at Days 14 and 42 of gestation were 37.7% and 27.3%, respectively. Beyond Day 42, the percentages of recipient mares that still had a viable conceptus at Day 90 (84.6% vs 37.5%) and gave birth to a healthy foal (61.5% vs 12.5%) were greater for the OPU group compared with the abattoir group (P < 0.05). Surprisingly, more favourable pregnancy outcomes were achieved when blastocysts were vitrified for later transfer, probably because the uterine receptivity of the recipient mares was more ideal. A total of 12 cloned foals were born, 9 of which were viable. Given the differences observed between the two oocyte groups, the use of OPU-harvested oocytes for generating cloned foals is clearly advantageous. Continued research is essential to better understand the oocyte deficiencies and increase the efficiency of equine cloning.

Mouse somatic cell nuclear transfer: What has changed and remained unchanged in 25 years.

Somatic cell nuclear transfer (SCNT) is the only reproductive technology used to produce individuals from somatic cells by transferring them to enucleated oocytes. Although more than 25 years have passed since the first mammalian SCNT was reported in sheep, problems such as low birth rates and morphological abnormalities have persisted and limited its practical applications. The mouse is the ideal laboratory animal to unveil these questions due to its established reproductive technologies and extensive knowledge base of its genome and various strains. We investigated the causes of incomplete reprogramming after nuclear transfer of donor somatic cells and found that the loss of imprint in some placenta-specific imprinted genes could induce non-random SCNT abnormalities. By ameliorating aberrantly expressed imprinted genes, we succeeded in increasing the low birth rate and improving morphological abnormalities observed in SCNT fetuses. Furthermore, we sought appropriate mouse strains and cell types as nuclear donors to increase their developmental efficiencies and expand their applications in various fields. Peripheral blood cells are useful as ethical and economical cell species because they can be collected easily, even though SCNT embryos derived from hematopoietic cells show poor developmental abilities after reconstruction. Additionally, it is possible to obtain mice that are reactive to specific antigens of interest by using lymphocytes. Although there are still many limitations to the practical use of SCNT, its utilization is steadily expanding.

First sex modification case in equine cloning.

Somatic cell nuclear transfer (SCNT) is an asexual reproductive technique where cloned offspring contain the same genetic material as the original donor. Although this technique preserves the sex of the original animal, the birth of sex-reversed offspring has been reported in some species. Here, we report for the first time the birth of a female foal generated by SCNT of a male nuclear donor. After a single SCNT procedure, 16 blastocysts were obtained and transferred to eight recipient mares, resulting in the birth of two clones: one male and one female. Both animals had identical genetic profiles, as observed in the analysis of 15-horse microsatellite marker panel, which confirmed they are indeed clones of the same animal. Cytogenetic analysis and fluorescent in situ hybridization using X and Y specific probes revealed a 63,X chromosome set in the female offspring, suggesting a spontaneous Y chromosome loss. The identity of the lost chromosome in the female was further confirmed through PCR by observing the presence of X-linked markers and absence of Y-linked markers. Moreover, cytogenetic and molecular profiles were analyzed in blood and skin samples to detect a possible mosaicism in the female, but results showed identical chromosomal constitutions. Although the cause of the spontaneous chromosome loss remains unknown, the possibility of equine sex reversal by SCNT holds great potential for the preservation of endangered species, development of novel breeding techniques, and sportive purposes.

25 years after Dolly: Update on long-term effects of embryo biotechnologies.

Since the announcement of the birth of Dolly, the world's first mammal produced by cloning, it was demonstrated for the first time that somatic cells could be reprogrammed to produce a whole individual. This represented a considerable change in paradigm in the field of embryo technologies both in humans and animals which led to an intense burst of research on nuclear transfer but also on the establishment of pluripotency and the directed edition of the genome. As such, induced pluripotent cells and gene editing tools, the best known being CRISPR-Cas9, are now available to the scientific community. Nevertheless, cloning was associated with important developmental abnormalities in a variable proportion of pregnancies, raising concern about the long-term effects of embryo technologies at a time when the concept of the developmental origins of health and disease had emerged, together with a better understanding of the underlying epigenetic modifications. The focus of this article is to review current knowledge on long-term effects of artificial reproduction technologies in mammals, leading to globally reassuring information although differences are present and caution remains necessary taking the current increasing number of in vitro-produced ruminant and equine embryos into account and their potential intergenerational consequences.

Live births from urine derived cells.

Here we report urine-derived cell (UDC) culture and subsequent use for cloning which resulted in the successful development of cloned canine pups, which have remained healthy into adulthood. Bovine UDCs were used in vitro to establish comparative differences between cell sources. UDCs were chosen as a readily available and noninvasive source for obtaining cells. We analyzed the viability of cells stored in urine over time and could consistently culture cells which had remained in urine for 48hrs. Cells were shown to be viable and capable of being transfected with plasmids. Although primarily of epithelial origin, cells were found from multiple lineages, indicating that they enter the urine from more than one source. Held in urine, at 4°C, the majority of cells maintained their membrane integrity for several days. When compared to in vitro fertilization (IVF) derived embryos or those from traditional SCNT, UDC derived embryos did not differ in total cell number or in the number of DNA breaks, measured by TUNEL stain. These results indicate that viable cells can be obtained from multiple species' urine, capable of being used to produce live offspring at a comparable rate to other cell sources, evidenced by a 25% pregnancy rate and 2 live births with no losses in the canine UDC cloning trial. This represents a noninvasive means to recover the breeding capacity of genetically important or infertile animals. Obtaining cells in this way may provide source material for human and animal studies where cells are utilized.

Comparison the effects of 5-Aza-2'-deoxycytidine and zebularine on the in vitro development, blastocyst quality, methylation pattern and conception rate on handmade cloned buffalo embryos.

In this study we treated the handmade cloned (HMC) buffalo embryos with the DNA methylation inhibitors; 5-aza-2'-deoxycytidine (AzadC) or Zebularine individually after post-fusion and during in vitro culture till eighth day. The blastocysts production rate significantly improved (p < .01) after treating embryos independently with 5 nM AzadC and 5 nM zebularine compared with 2 and 10 nM AzadC or zebularine groups, respectively. The highest cleavage rates were obtained for 5 nM treatment of AzadC and zebularine compared with other treatments and untreated control group. Quality of blastocysts were evaluated using total cell number (TCN) and the ratio of number of inner cell mass (ICM) cells/total cell number (ICM/TCN). Zebularine treatments (2/5/10 nM) significantly improved both TCN and ICM/TCN ratio compared with AzadC treatments (2/5/10 nM); however, control group TCN and ICM/TCN ratio was found lower. The methylation percentage of pDS4.1 and B. bubalis satellite DNA were comparatively more attenuated with 5 nM zebularine than 5 nM AzadC treatment. The increased in vitro development rates of the treated embryos were correlated with the decreased level of DNA methylation and the improved blastocyst quality. Following transfer of 5 nM zebularine treated embryos to 6 recipients, 4 were found to be pregnant, though the pregnancies were not carried to full term.

Oct-4 activating compound 1 (OAC1) could improve the quality of somatic cell nuclear transfer embryos in the bovine.

Previous studies described aberrant nuclear reprogramming in somatic cell nuclear transfer (SCNT) embryos that is distinctly different from fertilized embryos. This abnormal nuclear reprogramming hampers the proper pre- and/or post-implantation development. It has been demonstrated that SCNT blastocysts aberrantly expressed POU5F1 and POU5F1-related genes. With regard to this, it has been postulated that promoting the expression of POU5F1 in SCNT embryos may enhance reprogramming in SCNT embryos. In this study, we treated either fibroblast donor cells or SCNT embryos with OAC1 as a novel small molecule that has been reported to induce POU5F1 expression. Quantitative results from the MTS assay revealed that lower concentrations of OAC1 (1, 1.5, and 3 µM) are non-toxic after 2, 4, and 6 days, but higher concentrations (6, 8, 10, and 12 µM) are toxic and reduced the proliferation of cells after 6 days. No enhancement in the expression of endogenous POU5F1 was observed when both mouse and bovine fibroblast cells were treated with 1.5 and 3 µM OAC1 for up to 6 consecutive days. Subsequently, we treated either fibroblast as donor cells in the SCNT procedure (BFF-OAC1 group) or SCNT embryos [for 4 days (IVC-OAC1: D4-D7 group) or 7 days (IVC-OAC1: D0-D7 group)] with 1.5 µM OAC1. We observed that neither treatment of fibroblast donor cells nor SCNT embryos improved the cleavage and blastocyst rates. Interestingly, we observed that treatment of SCNT embryos all throughout the in vitro culture (IVC) (IVC-OAC1: D0-D7) with 1.5 µM OAC1 improves the quality of derived blastocyst which was indexed by morphological grading, blastomere allocation, epigenetic marks and mRNA expression of target genes. In conclusion, our results showed that supplementation of IVC medium with 1.5 µM OAC1 (D0-D7) accelerates SCNT reprogramming in bovine species.

Comparative evaluation of production performances of cloned pigs derived from superior Duroc boars.

Since pig was successfully cloned in 2000, somatic cell nuclear transfer (SCNT) became a promising technique in preserving and expanding the genetics of superior boars. Assessing the safety, growth performance, and reproductive performance of cloned pigs and their progeny is critical for their wide application. In this study, three superior Duroc boars were used to construct 61,736 SCNT-cloned embryos. The semen quality and reproductive performance of the cloned Duroc pigs and the growth performances of their progeny were evaluated. Results showed that the cloned pigs derived from superior boars produced semen with normal quality and exhibited similar reproductive performance as the donor boars, whose progenies showed greater growth performance than those derived from non-cloned pigs under the same feed condition. The results shed light on the application of cloning technology in the conservation and expansion of the genetic resources of Duroc pigs.

The many problems of somatic cell nuclear transfer in reproductive cloning of mammals.

In 1996, when Dolly the sheep was born, a new, utopian era was expected to begin. Science fiction and popular culture instantly threatened the public with shortly upcoming human clones, portraying it as a very easy and instant procedure. Practice has proven otherwise; it exposed how little is known about the early development of mammals and epigenetic reprogramming. Unfortunately, somatic cell nuclear transfer success rate in mammals has not changed much since its very beginning. It is not uncommon that hundreds of oocytes need to be reconstructed to obtain a single live birth. In this review we provide a brief summary of the progress and problems of the field; beginning with selection of the donor cells and their susceptibility to different methods of epigenetic reprogramming; methods of the later gene activation, placental abnormalities, and their possible causes; to health issues that such offspring is prone to.

RNAi-mediated knockdown of Xist improves development of the female buffalo (Bubalus bubalis) nuclear transfer embryos.

Xist plays a critical role in the X-chromosome inactivation (XCI), an important epigenetic reprogramming of somatic cell nuclear transfer (SCNT) embryos. Modulation of Xist expression enhanced the developmental ability of mouse cloned embryos. However, the roles of Xist in buffalo SCNT embryos remain unknown. In this study, we investigated the methylation and expression status of Xist in different genders of buffalo donor cells and various stages (two-cell, eight-cell, morula and blastocyst) of in vitro fertilization (IVF) and SCNT embryos. The methylation of Xist in SCNT-♀ and SCNT-♂ embryos was aberrant hypomethylation compared with the buffalo foetal fibroblast (♀-BFF and ♂-BFF), IVF-♀ and IVF-♂ embryos. At the eight-cell stage, Xist expression was significantly higher in SCNT-♀ embryos compared with those in SCNT-♂, IVF-♀ and IVF-♂ embryos (P < 0.05). Meanwhile, no significant difference was found between IVF-♀ and IVF-♂ embryos (P > 0.05). Accordingly, we suppressed Xist expression by RNAi-Xist in SCNT-♀ embryos. Results showed that injection of Xist-shRNA significantly improved the morula and blastocyst rates (P < 0.05). These results indicated that correcting the abnormal expression of the Xist gene contributed to the development of SCNT-♀ embryos.

Evaluation of porcine urine-derived cells as nuclei donor for somatic cell nuclear transfer.

BACKGROUND: Somatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency. OBJECTIVES: This study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs. METHODS: The viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. The in vitro developmental ability of SCNT embryos was evaluated by the blastocyst rate and the expression levels of the core pluripotency factor. Blastocyst cell apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. The in vivo developmental ability of SCNT embryos was evaluated after embryo transfer. RESULTS: Most sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. The in vitro development efficiency between the embryos produced by SCNT using porcine embryonic fibroblasts (PEFs) and urine-derived cells were similar. Moreover, The H3K9me3 in SCNT embryos produced from sow urine-derived cells and PEFs at the four-cell stage showed similar intensity. The levels of Oct4, Nanog, and Sox2 expression in blastocysts were similar in the two groups. Furthermore, there is a similar apoptotic level of cloned embryos produced by the two types of cells. Finally, the full-term development ability of the cloned embryos was evaluated, and the cloned fetuses from the urine-derived cells showed absorption. CONCLUSIONS: Sow urine-derived cells could be used to produce SCNT embryos.

Melatonin promotes the development of sheep transgenic cloned embryos by protecting donor and recipient cells.

The yield efficiency of transgenic animal generation is relatively low[1]. To improve its efficiency has become a priority task for researchers[2]. Melatonin (N-acetyl-5-methoxytryptamine, MT) is a potent-free radical scavenger and antioxidant to protect mitochondria, lipids, protein and DNA from oxidative stress[3]. In this study, we observed that improving the quality of both donor and recipient cells by giving physiological concentration (10-7 M) of MT significantly increase the sheep transgenic embryo development in the in vitro condition. MT promotes the donor cell viability, proliferation, efficiency of monoclonal formation and the electrotransferring efficiency of fetal fibroblast cells (FFCs). The mechanistic exploration indicates that MT has the capacity for the synchronization of cell division cycle, reduction of cellular oxidative stress, apoptosis, and the increase of mitochondrial number and function. All of these render MT's ability to increase the efficiency of animal transgenic processes such as somatic cell nuclear transfer (SCNT) and electroporation. The outcomes are the increased cleavage rate and blastocyst rate of the transgenic sheep embryos after MT treatment. These beneficial effects of MT on transgenic embryo development are worth to be tested in the in vivo condition in the future.

Developmental analysis of reconstructed embryos of second-generation cloned transgenic goats.

To improve the efficiency of the production of transgenic cloned goats by somatic cell nuclear transfer (SCNT), the development of reconstructed embryos of first-generation (G1) and second-generation (G2) cloned transgenic goats was compared and analysed. Primary transgenic foetal fibroblasts were used as donor cells for G1 somatic cell nuclear transfer (SCNT). When the G1 transgenic embryos developed to 35 days in the recipient goats, transgenic foetal fibroblasts were isolated from them and used as donor cells for the G2 clone. In the G1 clones, the average fusion rate of reconstructed embryos was 73.62 ± 2.9%, the average development rate (2-4 cells) was 33.96 ± 2.36%, and the pregnancy rate of transplant recipients was 31.91%. In the G2 clones, the average fusion rate of the reconstructed embryos was 90.29 ± 2.03%, the average development rate was 66.46 ± 3.30%, and the pregnancy rate was 58.14%. These results indicate that in the G2 clones, the fusion rate of eggs, the development rate of reconstructed embryos and the pregnancy rate of transplant recipients were significantly higher than those of G1 clones. We believe these results will lay a solid foundation for the efficient production of transgenic cloned animals in the future.