Interspecies Somatic Cell Nuclear Transfer Research Articles

Current status of interspecies somatic cell nuclear transfer and meta‐analysis of the effects of phylogenetic distance on embryonic and fetal development

Abstract Interspecies somatic cell nuclear transfer (iSCNT) has the capacity to rescue endangered species, ethically produce human embryonic stem cells, and allow for more specific analysis of pre‐implantation embryos. However, several obstacles negatively affect iSCNT efficiency. Over 100 iSCNT studies have been published between 1999 and the spring of 2023. This review details the possible benefits of iSCNT, describes the hypothesized issues that prevented the development of some embryos and/or live offspring from developing; and analyzes the cleavage, blastocyst, pregnancy, live birth, and iSCNT efficiency rates based on published data. Embryos produced by iSCNT may help with the rescue of endangered species and the production of human embryonic stem cells with the use of oocytes from domestic species, as well as in‐depth analyses of embryonic genome activation (EGA) and mRNA transcripts. But the combination of the cells of two species in a single embryo presents several challenges: lack of EGA synchronisation, presence of mitochondrial DNA (mtDNA) from both species in the embryo, requirement of cell and embryo culture optimization for each species combination, lack of embryo transfer data, and an extremely low overall efficiency in terms of producing live offspring. Additional research on the aforementioned topics is warranted to increase iSCNT efficiency.

18 Development of interspecies somatic cell nuclear transfer (iSCNT) caprine-bovine embryos injected with demethylase mRNA and mitochondrial extract

18 Development of interspecies somatic cell nuclear transfer (iSCNT) caprine-bovine embryos injected with demethylase mRNA and mitochondrial extract

Bovid Interspecies Somatic Cell Nuclear Transfer with Ooplasm Transfer.

Interspecies somatic cell nuclear transfer (iSCNT) contributes to the preservation of endangered species, albeit nuclear-mitochondrial incompatibilities constrain its application. iSCNT, coupled with ooplasm transfer (iSCNT-OT), has the potential to overcome the challenges associated with species- and genus-specific differences in nuclear-mitochondrial communication. Our iSCNT-OT protocol combines the transfer of both bison (Bison bison bison) somatic cell and oocyte ooplasm by a two-step electrofusion into bovine (Bos taurus) enucleated oocytes. The procedures described herein could be used in further studies to determine the effects of crosstalk between nuclear and ooplasmic components in embryos carrying genomes from different species.

Interspecific Nuclear Transfer Blastocysts Reconstructed from Arabian Oryx Somatic Cells and Domestic Cow Ooplasm.

Cloning, commonly referred to as somatic cell nuclear transfer (SCNT), is the technique of enucleating an oocyte and injecting a somatic cell into it. This study was carried out with interspecific SCNT technology to clone the Arabian Oryx utilizing the oryx's fibroblast cells and transfer it to the enucleated oocytes of a domestic cow. The recipient oocytes were extracted from the cows that had been butchered. Oryx somatic nuclei were introduced into cow oocytes to produce embryonic cells. The study was conducted on three groups, Oryx interspecific somatic cell nuclear transfer into enucleated oocytes of domestic cows, cow SCNT "the same bovine family species", used as a control group, and in vitro fertilized (IVF) cows to verify all media used in this work. The rates of different embryo developmental stages varied slightly (from 1- cell to morula stage). Additionally, the oryx interspecies Somatic cell nuclear transfer blastocyst developmental rate (9.23%) was comparable to that of cow SCNT (8.33%). While the blastula stage rate of the (IVF) cow embryos exhibited a higher cleavage rate (42%) in the embryo development stage. The results of this study enhanced domestic cow oocytes' ability to support interspecific SCNT cloned oryx, and generate a viable embryo that can advance to the blastula stage.

Effect of Mechanical Micro-Vibrations on The Efficiency of Leopard Inter-Species Somatic Cell Nuclear Transfer.

Scarcity of oocytes for assisted reproduction in endangered species can be bypassed by interspecies somatic cell nuclear transfer (iSCNT). In Felids, domestic cat (Felis catus) oocytes can serve as recipients for the nucleus of the endangered Persian leopard (Panthera pardus saxicolor). However, in vitro oocyte maturation is still suboptimal in cats, whereas it has been reported to benefit from micro-vibration in non-felid species. Therefore, the present study is aimed to determine whether micro-vibration, applied during in vitro maturation (IVM), improves the embryogenic potential of cat oocytes transplanted with fibroblast nuclei of the Persian leopard. In the experimental study, cat cumulus-oocyte complexes (COCs) were randomly assigned to the treatment group (micro-vibration) or control group (static culture). Resultant metaphase II (MII) oocytes were enucleated and reconstructed with nucleus transplants from leopard fibroblasts, followed by artificial oocyte activation and embryo culture under the same condition (static) for 7 days. While cumulus cell expansion and oocyte maturation profited from micro-vibration (P<0.05), the quantity and quality of blastocysts were significantly lower in micro-vibration than in the control group (P<0.05). The total number of blastocyst cells tended to be lower in the micro-vibration than in the control group (P=0.075). Nevertheless, the proportion of ICM and TE cells did not differ between the micro-vibration and control groups (P>0.05). The present study indicated that micro-vibration at a frequency of 44 Hz for 5 secs per hour enhanced nuclear maturation and cumulus cell expansion of cat oocytes. However, exposure to micro-vibration during IVM impaired the survival rate of reconstructed oocytes during the iSCNT process and their developmental competence toward the blastocyst stage.

Low Expression of Mitofusin 1 Gene Leads to Mitochondrial Dysfunction and Embryonic Genome Activation Failure in Ovine-Bovine Inter-Species Cloned Embryos.

Inter-species somatic cell nuclear transfer (iSCNT) is significant in the study of biological problems such as embryonic genome activation and the mitochondrial function of embryos. Here, we used iSCNT as a model to determine whether abnormal embryo genome activation was caused by mitochondrial dysfunction. First, we found the ovine-bovine iSCNT embryos were developmentally blocked at the 8-cell stage. The reactive oxygen species level, mitochondrial membrane potential, and ATP level in ovine-bovine cloned embryos were significantly different from both bovine-bovine and IVF 8-cell stage embryos. RNA sequencing and q-PCR analysis revealed that mitochondrial transport, mitochondrial translational initiation, mitochondrial large ribosomal subunit, and mitochondrial outer membrane genes were abnormally expressed in the ovine-bovine embryos, and the mitochondrial outer membrane and mitochondrial ribosome large subunit genes, mitochondrial fusion gene 1, and ATPase Na+/K+ transporting subunit beta 3 gene were expressed at lower levels in the ovine-bovine cloned embryos. Furthermore, we found that overexpression and knockdown of Mfn1 significantly affected mitochondrial fusion and subsequent biological functions such as production of ATP, mitochondrial membrane potential, reactive oxygen species and gene expressions in cloned embryos. These findings enhance our understanding of the mechanism by which the Mfn1 gene regulates embryonic development and embryonic genome activation events.

Use of Bisection to Reduce Mitochondrial DNA in the Bovine Oocyte.

Interspecies somatic cell nuclear transfer (iSCNT) may be used to rescue endangered species, but two distinct populations of mitochondrial DNA (mtDNA) exist within the reconstructed embryo: one within the recipient ooplasm and one within the donor somatic cell. This mitochondrial heteroplasmy can lead to developmental issues in the embryo and the fetus. Handmade cloning protocols include oocyte bisection, which can be used to decrease the mtDNA copy number, reducing the degree of mitochondrial heteroplasmy in a reconstructed embryo. Centrifugation of denuded, mature bovine oocytes produced a visible mitochondria-dense fraction at one pole of the oocyte. Oocytes' zonae pellucidae were removed by exposure to a pronase solution. Bisection was performed using a microblade to remove the visible mitochondria fraction. qPCR was used to quantify the mtDNA present in DNA samples extracted from whole oocytes and bisected ooplasts, providing a comparison of mtDNA copy numbers before and after bisection. Copy numbers were calculated using cycle threshold values, a standard curve's regression line formula, and a ratio that included the respective sizes of mtDNA PCR products and genomic PCR products. One bovine oocyte had an average mtDNA copy number (± standard deviation) of 137,904 ± 94,768 (n = 38). One mitochondria-depleted ooplast had an average mtDNA copy number of 8,442 ± 13,806 (n = 33). Average mtDNA copies present in a mitochondria-rich ooplast were 79,390 ± 58,526 mtDNA copies (n = 28). The differences between these calculated averages indicate that the centrifugation and subsequent bisection can significantly decrease the mtDNA copy numbers present in the mitochondria-depleted ooplast when compared to the original oocyte (P < 0.0001, determined by one-way ANOVA). The reduction in mtDNA should decrease the degree of mitochondrial heteroplasmy in a reconstructed embryo, possibly fostering standard embryonic and fetal development. Supplementation with mitochondrial extract from the somatic donor cell may also be essential to achieve successful embryonic development.

In vitro and in vivo development of interspecies Asian elephant embryos reconstructed with pig enucleated oocytes

Interspecies somatic cell nuclear transfer (iSCNT) has an immense potential to rescue endangered animals and extinct species like mammoths. In this study, we successfully established an Asian elephant’s fibroblast cell lines from ear tissues, performed iSCNT with porcine oocytes and evaluated the in vitro and in vivo development of reconstructed embryos. A total of 7780 elephant–pig iSCNT embryos were successfully reconstructed and showed in vitro development with cleavage rate, 4-cell, 8-cell and blastocyst rate of 73.01, 30.48, 5.64, and 4.73%, respectively. The total number of elephant–pig blastocyte cells and diameter of hatched blastocyte was 38.67 and 252.75 μm, respectively. Next, we designed species-specific markers targeting EDNRB, AGRP and TYR genes to verify the genome of reconstructed embryos with donor nucleus/species. The results indicated that 53.2, 60.8, and 60.8% of reconstructed embryos (n = 235) contained elephant genome at 1-cell, 2-cell and 4-cell stages, respectively. However, the percentages decreased to 32.3 and 32.7% at 8-cell and blastocyst stages, respectively. Furthermore, we also evaluated the in vivo development of elephant–pig iSCNT cloned embryos and transferred 2260 reconstructed embryos into two surrogate gilts that successfully became pregnant and a total of 11 (1 and 10) fetuses were surgically recovered after 17 and 19 days of gestation, respectively. The crown-rump length and width of elephant–pig cloned fetuses were smaller than the control group. Unfortunately, none of these fetuses contained elephant genomes, which suggested that elephant embryos failed to develop in vivo. In conclusion, we successfully obtained elephant–pig reconstructed embryos for the first time and these embryos are able to develop to blastocyst, but the in vivo developmental failure needs further investigated.

C23 gene regulates the nucleolin structure and biosynthesis of ribosomes in bovine intraspecific and interspecific somatic cell nuclear transfer embryos.

Somatic cell nuclear transfer (SCNT) can reprogram differentiated somatic cells to produce individual animals, thus having advantages in animal breeding and chromatin reprogramming. Interspecies SCNT (iSCNT) provides extreme cases of reprogramming failure that can be used to understand the basic biological mechanism of genome reprogramming. It is important to understand the possible mechanisms for the failure of zygotic genome activation (ZGA) in iSCNT embryos in order to improve the efficiency of SCNT embryos. In the present study, we compared the development of bovine-bovine (B-B), ovine-ovine (O-O) SCNT, and ovine-bovine (O-B) iSCNT embryos and found that a developmental block existed in the 8-cell stage in O-B iSCNT embryos. RNA sequencing and q-PCR analysis revealed that the large ribosomal subunit genes (RPL) or the small ribosomal subunit genes (RPS) were expressed at lower levels in the O-B iSCNT embryos. The nucleolin (C23) gene that regulates the ribosomal subunit generation was transcribed at a lower level during embryonic development in O-B iSCNT embryos. In addition, the nucleolin exhibited a clear circular-ring structure in B-B 8-cell stage embryos, whereas this was shell-like or dot-like in the O-B embryos. Furthermore, overexpression of C23 could increase the blastocyst rate of both SCNT and iSCNT embryos and partly rectify the ring-like nucleolin structure and the expression of ribosomal subunit related genes were upregulation, while knockdown of C23 increased the shell-like nucleolin-structure in B-B cloned embryos and downregulated the expression of ribosomal subunit related genes. These results implied that abnormal C23 and ribosome subunit gene expression would lead to the developmental block of iSCNT embryos and ZGA failure. Overexpression of the C23 gene could partly improve the blastocyst development and facilitate the nucleolin structure in bovine preimplantation SCNT embryos.

Comparison of Blastocyst Development between Cat-Cow and Cat-Pig Interspecies Somatic Cell Nuclear Transfer Embryos Treated with Trichostatin A

We previously demonstrated that the histone deacetylase inhibitor, trichostatin A (TSA), improves the development of cat-cow interspecies somatic cell nuclear transfer (iSCNT) embryos. In this study, we investigated if 50 nM TSA treatment for 24 h would improve the rate of embryo development to the blastocyst stage in cat-pig iSCNT embryos, compared with cat-cow iSCNT embryos under same treatment. Despite the SCNT technique (Piezo technique) differing from our previous study (conventional technique), we confirmed that TSA supplementation at 50 nM improved the development of cat-cow iSCNT blastocysts. Porcine oocytes showed the capability to support cat iSCNT embryo development up to the blastocyst stage without TSA treatment although bovine oocytes did not. The blastocyst developmental rate of TSA treated cat-pig iSCNT embryos was significantly lower than that of the non-treated groups (0.68 and 7.95%, respectively, p < 0.05). We used in vitro-fertilized (IVF) porcine embryos to compare the effects of acetylation levels on H3K9 between cat-pig iSCNT embryos and naturally fertilized embryos. We found no differences in acetylation levels of H3K9 between the TSA treated and non-treated iSCNT groups, and the levels were lower than that of IVF embryos. In conclusion, the effects of TSA treatment on iSCNT embryo production were found to be species-specific and dependent on individual characteristics.

Mitochondrial metabolism assessment of lycaon-dog fetuses in interspecies somatic cell nuclear transfer

Many studies have reported that interspecies somatic cell nuclear transfer (iSCNT) is considered the prominent method in preserving endangered animals. However, the development rate of iSCNT embryos is low, and there are limited studies on the molecular mechanism of the iSCNT process. This study evaluated the developmental potential of interspecies lycaon (Lycaon pictus)-dog embryos and assessed the mitochondrial content and metabolism of the produced cloned lycaon-dog fetus. Of 678 collected oocytes, 516 were subjected to nuclear transfer, and 419 reconstructed embryos with male lycaon fibroblasts were transferred into 27 surrogates. Of 720 oocytes, 568 were subjected to nuclear transfer and 469 reconstructed embryos with female lycaon fibroblasts were transferred into 31 surrogates. Two recipients who received female reconstructed embryos were identified as pregnant at 30 days. However, fetal retardation with no cardiac activity was observed at 46 days. Microsatellite analysis confirmed that the cloned lycaon-dog fetus was genetically identical to the lycaon donor cell, whereas mitochondrial sequencing analysis revealed that oocyte donor dogs transmitted their mtDNA. We assessed the oxygen consumption rate and mitochondrial content of the aborted lycaon-dog fetus to shed some light on the aborted fetus’s cellular metabolism. The oxygen consumption rates in the lycaon-dog fetal fibroblasts were lower than those in adult dog, lycaon and cloned dog fetal fibroblasts. Furthermore, lycaon-dog fetal fibroblasts showed decreased proportions of live and active mitochondria compared with other groups. Overall, we hypothesized that nuclear-mitochondrial incompatibility affects pyruvate metabolism and that these processes cause intrauterine fetal death.

The perspective of the incompatible of nucleus and mitochondria in interspecies somatic cell nuclear transfer for endangered species.

Taking into account the latest Red List of the International Union for Conservation of Nature in which 25% of all mammals are threatened with extinction, somatic cell nuclear transfer (SCNT) could be a beneficial tool and holds a lot of potential for aiding the conservation of endangered, exotic or even extinct animal species if somatic cells of such animals are available. In the case of shortage and sparse amount of wild animal oocytes, interspecies somatic cell nuclear transfer (iSCNT), where the recipient ooplasm and donor nucleus are derived from different species, is the alternative SCNT technique. The successful application of iSCNT, resulting in the production of live offspring, was confirmed in several combination of closely related species. When nucleus donor cells and recipient oocytes have been used in many other combinations, very often with a very distant taxonomical relation iSCNT resulted only in the very early stages of cloned embryo development. Problems encountered during iSCNT related to mitochondrial DNA (mtDNA)/genomic DNA incompatibility, mtDNA heteroplasmy, embryonic genome activation of the donor nucleus by the recipient oocyte and availability of suitable foster mothers for iSCNT embryos. Implementing assisted reproductive technologies, including iSCNT, to conservation programmes also raises concerns that the production of genetically identical populations might cause problems with inbreeding. The article aims at presenting achievements, limitations and perspectives of iSCNT in maintaining animal biodiversity.

Developmental competence of interspecies cloned embryos produced using cells from large Japanese field mice (Apodemus speciosus) and oocytes from laboratory mice (Mus musculus domesticus).

The large Japanese field mouse (Apodemus speciosus) is endemic to Japan and may be used as an animal model for studies related to environmental pollution, medical science, and basic biology. However, the large Japanese field mouse has low reproductive ability due to the small number of oocytes ovulated per female. To produce experimental models, we investigated the in vitro developmental potential of interspecies somatic cell nuclear transfer (iSCNT) embryos produced by fusing tail tip cells from the large Japanese field mouse with enucleated oocytes from laboratory mice (Mus musculus domesticus). Only a small number of iSCNT embryos developed to the 4-cell (0–4%) and blastocysts (0–1%) stages under sequential treatment using trichostatin A (TSA) and vitamin C (VC) supplemented with deionized bovine serum albumin (d-BSA). This sequential treatment led to the reduction in H3K9 trimethylation and did not affect H3K4 trimethylation in at least the 2-cell stage of the iSCNT embryos. Moreover, iSCNT embryos that received tail tip cells with exposure treatment to ooplasm from cell fusion to oocyte activation or VC treatment prior to cell fusion did not exhibit significant in vitro development improvement compared to that of each control group. This suggests that large Japanese field mice/laboratory mice iSCNT embryos that received sequential treatment using TSA and VC with d-BSA may have slightly better developmental potential beyond the 4-cell stage. Our results provide insights into the reprogramming barriers impeding the wider implementation of iSCNT technology.

Establishment of immortalized primary cell from the critically endangered Bonin flying fox (Pteropus pselaphon).

The Bonin flying fox (Pteropus pselaphon) is one of the most critically endangered species of animals. The number of this species is estimated to be around 150; being classified at the top rank in the list by International Union of Animal Conservation. Our group previously showed that expression of CDK4, CYCLIN D1, and telomerase reverse transcriptase (TERT) efficiently induce immortalization of human, bovine, swine, monkey, and buffalo-derived cells. In this manuscript, we successfully established the primary cells from Bonin flying fox. We introduced CDK4, CYCLIN D1, and TERT into the primary cells. The established cells showed efficient expression of introduced genes at the protein level. Furthermore, the established cells were free from senescence, indicating it reached to immortalization. Moreover, we showed that interspecies somatic cell nuclear transfer of Bonin flying fox derived cell into bovine embryo allowed the development of the embryo to 8 cell stages. Our established cell has the potential to contribute to species conservation.

17 Effect of the zona-free aggregation on the developmental competence of kodkod (Leopardus guigna) embryos generated by interspecies somatic cell nuclear transfer

The kodkod is considered a vulnerable species by the International Union for Conservation of Nature. Phylogenetically, the kodkod is classified in the Leopardus genus, which has only 36 chromosome pairs compared with the domestic cat, which has 38. The proposed hypothesis was that domestic cat oocytes are capable of reprogramming somatic cells from kodkod after interspecies somatic cell NT (SCNT), allowing the in vitro embryo development up to blastocyst stage. Five experimental groups were made based on the technology and culture system: (1) cat embryos generated by IVF (IVF), (2) cat embryos generated by SCNT (Ca1x), (3) aggregated cat embryos generated by SCNT (Ca2x), (4) kodkod embryos generated by interspecies SCNT (K1x), and (5) aggregated kodkod embryos generated by interspecies SCNT (K2x). Interspecies SCNT was performed using a zona-free method. Reconstructed embryos were activated by 2 electrical pulses of 140 kV cm−1 for 40 µs and then incubated for 5h in 10μg mL−1 of cycloheximide and 5μg mL−1 of cytochalasin B. Embryos were cultured in SOF media using the well of the well system in a 5% O2, 5% CO2, and 90% N2 atmosphere at 38.5°C for 8 days. The morulae and blastocysts rates were estimated, and diameter of cloned blastocysts was measured. The relative expression of OCT4, SOX2, and NANOG was evaluated in blastocysts by RT-qPCR using the standard curve method; SDHA was used for normalization. The Kruskal-Wallis test was used to evaluate the developmental parameters and gene expression. The t-test was used to evaluate blastocyst diameter. Statistical differences were considered at P&amp;lt;0.05. The number of replicates was IVF=10, Ca1x=8, Ca2x=6, K1x=3, and K2x=8. The morulae rate was lower when clone embryos were cultured individually (IVF=97/153, 63.4%; Ca2x=28/51, 54.9%; K2x=63/110, 57.3%; Ca1x=48/126, 38.1%; K1x=22/87, 25.3%; P&amp;lt;0.05). In the domestic cat, blastocysts rate was higher in IVF (58/153, 37.9%) and Ca2x (28/51, 29.4%) groups than in the Ca1x group (21/126, 16.7%; P&amp;lt;0.05). No blastocysts were generated in the K1x group (0/87), whereas 5.5% of blastocysts were obtained from the K2x (6/110; 5.5%); this was not statistically different compared with the K1x group (P&amp;gt;0.05). No differences were found in blastocyst diameter between the Ca1x (220.4µm) and Ca2x (251.2µm) groups (P&amp;gt;0.05). However, the diameter of the blastocysts from the K2x group (172.8µm) tended to be lower than that of the blastocysts from the Ca2x group (P=0.05). Regarding gene expression, only 1 of the 6 kodkod blastocysts expressed OCT4, and none expressed SOX2 and NANOG. On the other hand, the relative expression of OCT4 tended to decrease in blastocysts from the Ca1x and Ca2x groups compared with the IVF group (P=0.09), but no differences were found in the expression of SOX2 and NANOG among groups (P&amp;gt;0.05). In conclusion, after interspecies SCNT, domestic cat oocytes support the development of kodkod embryos until the morula stage. However, the embryo aggregation did not significantly improve the blastocyst rate and gene expression.

Epigenotoxic Effect of Dimethyl Sulfoxide on Buffalo Somatic Cells and Buffalo-Bovine Interspecies Somatic Cell Nuclear Transfer Embryos.

Objective In the present study, we investigated the possible epigenotoxic effect of dimethyl sulfoxide (DMSO) on buffalo fibroblast cells and on reconstructed oocytes during buffalo-bovine interspecies somatic cell nuclear transfer (iSCNT) procedure and its effect on rate and quality of blastocyst which derived from these reconstructed oocytes. Materials and Methods In this experimental study, cell viability of buffalo fibroblasts was assessed after exposure to various concentration (0.5, 1, 2 and 4%) of DMSO using MTS assay. The epigenetic effect of DMSO was also assessed in terms of DNA methylation in treated cells by flowcytometry. Reconstructed oocytes of buffalo-bovine iSCNT exposed for 16 hours after activation to non-toxic concentration of DMSO (0.5%) to investigate the respective level of 5-methylcytosine, cleavage and blastocyst rates and gene expression (pluripotent genes: OCT4, NANOG, SOX2, and trophectodermal genes: CDX2 and TEAD4) of produced blastocysts. Results Supplementation of culture medium with 4% DMSO had substantial adverse effect on the cell viability after 24 hours. DMSO, at 2% concentration, affected cell viability after 48 hours and increased DNA methylation and mRNA expression of DNMT3A in fibroblast cells. Exposure of reconstructed oocytes to 0.5% DMSO for 16 hours post activation did not have significant effect on DNA methylation, nor on the developmental competency of reconstructed oocyte, however, it decreased the mRNA expression of NANOG in iSCNT blastocysts. ConclusionDepending on the dose, DMSO might have epigenotoxic effect on buffalo fibroblast cells and reconstructed oocytes and perturb the mRNA expression of NANOG in iSCNT blastocysts.

Effect of DNA and Histone Methyl Transferase Inhibitors on Outcomes of Buffalo-Bovine Interspecies Somatic Cell Nuclear Transfer.

Somatic cell nuclear transfer (SCNT) derived embryos suffer from abnormal epigenetic reprogramming, which handicaps pre- and postimplantation development. It was hypothesized that epigenetic modifiers, including zebularine (DNA methyltransferase inhibitors) and BIX-01294 (histone methyltransferase inhibitors), could decrease the respective levels of 5-methylcytosine and H3K9me2 in reconstructed oocytes (RO). Accordingly, we investigated whether treating RO with zebularine and BIX-01294 for 16 hours after activation could improve developmental competence and quality of buffalo-bovine interspecies SCNT (iSCNT) embryos. Treatment of RO with zebularine but not BIX-01294 significantly increased two-cell formation at 16 hours postactivation. Conversely, early cleaved embryos had significantly lower rate of blastocyst formation in zebularine treated RO compared to their counterparts in control and BIX-01294 groups. Treatment of RO with zebularine and BIX-01294 did not improve blastocyst rate of buffalo-bovine iSCNT embryos compared to their control counterparts. However, these two epigenetic drugs might have some beneficial effects on buffalo-bovine iSCNT compared to bovine SCNT embryos. The quality of iSCNT blastocysts was improved due to significant expression of OCT4 and CDX2 in BIX-01294 and CDX2 in zebularine treated RO. Furthermore, treatment of RO with zebularine and BIX-01294 did not affect DNA fragmentation in derived blastocysts against control group. In conclusion, treatment with zebularine and BIX-01294 did not enhance developmental competence of iSCNT embryos, but may have some beneficial effects on epigenetic makeup and quality of derived blastocysts.

35 Interspecies Somatic Cell Nuclear Transfer Embryos that Form Nucleoli do not Always Activate Mitochondrial Functional Differentiation at the Time of Embryonic Genome Activation

Embryonic genome activation (EGA) is a complex process that needs a good orchestration of all biochemical processes at the time of maternal-to-embryonic transition. Mitochondria are strictly dependent on the nucleus for their correct activity as ~1500 mitochondrial genes have nuclear localisation. The finding of transcriptional activation and accumulation of mRNAs related to mitochondrial biogenesis (Mtango et al. 2008 Reprod. Fertil. Dev. 20, 846-859) around the time of EGA confirmed the role of nucleus in this process. Studying mitochondria behaviour in interspecies somatic cell nuclear transfer (iSCNT) embryos (Lagutina et al. 2010 Reproduction 140, 273-285), we have found that at the time of EGA, mitochondria activation could be demonstrated by JC-1 accumulation. We suggested that comparison of the mean green fluorescence intensity (FI) that corresponds to the fluorescence of the monomeric form of the dye and correlates to relative mitochondrial mass (Mancini et al. 1997) in iSCNT and control nuclear transfer (NT) could serve as a test to assess EGA in iSCNT embryos. The aim of this study was to estimate nuclear-cytoplasmic interaction in iSCNT embryos that formed nucleoli at the time of EGA (Lagutina et al. 2011 Reproduction 141, 453-465) such as embryos derived from bovine oocytes and bovine (control), buffalo or ovine donor nuclei, and from porcine oocytes and porcine (control), horse, or rabbit nuclei. Embryos 72 h after activation were stained with 2 μM JC-1 in SOF-HEPES with 10% FCS at 37°C for 1 h. Images were collected using a fluorescein isothiocyanate (FITC) filter and analysed with Adobe Photoshop Elements 2 (Adobe Systems, San Jose, CA, USA). The data are presented as mean FI of the embryo. To demonstrate the effect of EGA inhibition on mitochondria, bovine and porcine NT embryos were cultured in medium supplemented with 25 μg/mL α-amanitin (AA) from 48 to 72 h after activation. The analyses of mean FI of the embryos showed that ovine and buffalo nuclei were able to support mitochondrial mass accumulation in iSCNT embryos with bovine oocytes equal to control bovine NT embryos (35 ± 11.2; 41.9 ± 14.8; 36.2 ± 7.6, respectively) that was significantly higher than in bovine embryos treated with AA (15.4 ± 4.9; P &lt; 0.05). In the iSCNT embryos composed of porcine oocytes and equine or rabbit nuclei, mean FI values (20 ± 13.4; 18.3 ± 5.5, respectively) were comparable with those in porcine NT embryos treated with AA (16.2 ± 6.2), and were significantly lower than in porcine control (91 ± 47.7; P &lt; 0.05) NT embryos, demonstrating the inability of equine and rabbit nuclei to properly govern the porcine mitochondria mass growth at the time of EGA. In conclusion, nucleolus formation and activation of nuclear encoded mitochondrial genes at the time of EGA cannot serve, per se, as a marker of correct embryonic genome activation in iSCNT embryos, because, in our conditions, no iSCNT embryos developed to blastocyst (Lagutina et al. 2010 Reproduction 140, 273-285). This knowledge about behaviour of different embryo compartments at the time of EGA could extend our understanding of the whole process. This work was funded by Translink (EU FP7 no. 603049) and Xenoislet projects (EU FP7 no. 601827).

34 Recent Advances in Cloning by Somatic Cell Nuclear Transfer in Camelids

In addition to its application for production of elite males, racing champions, animals with the highest potential for milk production, and the prized beauty camels, somatic cell nuclear transfer (SCNT) can be utilised for the conservation of endangered wild Bactrian camels and vicunas by using the technique of interspecies SCNT (iSCNT). In the present study, embryos were reconstructed by using skin fibroblast cells from a Bactrian camel (Camelus bactrianus), a llama (Llama glama), and a dromedary camel (Camelus dromedarius) as donor karyoplasts and dromedary oocytes as recipient cytoplasts to evaluate in vitro and in vivo developmental potential of these embryos. Mature oocytes were collected from super-stimulated dromedary camels by ultrasound guided transvaginal ovum pick-up. Serum-starved skin fibroblast cells were injected into the perivitelline space of enucleated oocytes. The fibroblast cell and recipient cytoplasm were fused by 2 DC pulses of 100V for 15 µs each. Reconstructs were activated 1 to 1.5 h post-fusion with 5 µM ionomycin, followed by exposure to 6-DMAP for 4 h. The activated oocytes were then cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2. The proportion of oocytes that cleaved was recorded on Day 3, and those that reached morula and blastocyst stages were recorded on Day 7 of culture. A lower blastocyst production rate was observed when llama fibroblast cells were used as karyoplast (13.3 ± 3.3) compared with those obtained from the embryos reconstructed with Bactrian (34.4 ± 3.9) and dromedary (32.2 ± 6.2) fibroblast cells; however, no difference was observed in their cell numbers. Out of 26 and 20 blastocysts from reconstructs with Bactrian and dromedary fibroblast cells, transferred to 23 and 12 synchronized dromedary recipients, 3 and 2 pregnancies were achieved at Day 60, respectively. One pregnancy from each group reached term and both (a Bactrian and a dromedary) calves were delivered normally after completing the gestation period. We demonstrated birth of a Bactrian calf conceived from the reconstructed embryo by iSCNT using the somatic cell from a Bactrian camel and enucleated dromedary oocyte. The present study also demonstrated that dromedary camel can be used as a surrogate to carry these pregnancies to term (Wani et al. 2017 PLoS One 12, e0177800; https://doi.org/10.1371/journal.pone.0177800). This study has opened doors for enhanced multiplication and preservation of the wild Bactrian camels, which are threatened with extinction, being the eighth most endangered large mammal on the planet.

Suberoylanilide hydroxamic acid during in vitro culture improves development of dog-pig interspecies cloned embryos but not dog cloned embryos.

This study was conducted to investigate whether the treatment of dog to pig interspecies somatic cell nuclear transfer (iSCNT) embryos with a histone deacetylase inhibitor, to improve nuclear reprogramming, can be applied to dog SCNT embryos. The dog to pig iSCNT embryos were cultured in fresh porcine zygote medium-5 (PZM-5) with 0, 1, or 10 µM suberoylanilide hydroxamic acid (SAHA) for 6 h, then transferred to PZM-5 without SAHA. Although there were no significant differences in cleavage rates, the rates of 5-8-cell stage embryo development were significantly higher in the 10 µM group (19.5 ± 0.8%) compared to the 0 µM groups (13.4 ± 0.8%). Acetylation of H3K9 was also significantly higher in embryos beyond the 4-cell stage in the 10 µM group compared to the 0 or 1 µM groups. Treatment with 10 µM SAHA for 6 h was chosen for application to dog SCNT. Dog cloned embryos with 0 or 10 µM SAHA were transferred to recipients. However, there were no significant differences in pregnancy and delivery rates between the two groups. Therefore, it can be concluded that although porcine oocytes support nuclear reprogramming of dog fibroblasts, treatment with a histone deacetylase inhibitor that supports nuclear reprogramming in dog to pig iSCNT embryos was not sufficient for reprogramming in dog SCNT embryos.