Somatic Cell Nuclear Transfer Research Articles

Effects of extracellular vesicles derived from steroids-primed oviductal epithelial cells on porcine in vitro embryonic development

Extracellular vesicles (EVs) play an active role in regulating different physiological events, however, endocrine control of EVs cargo contents remain poorly understood. In this study, we aimed to isolate EVs from the porcine oviductal epithelial cells (POECs) that were primed with steroid hormones including estradiol (E2) and progesterone (P4), mimicking the in vivo conditions of the reproductive cycle and studied their effects on in vitro produced embryonic development. For this purpose, POECs were treated either with 0 concentration (control) or two different combinations of E2 and P4 including 50 pg/mL E2 + 0.5 ng/mL P4 (group H1), and 10 pg/mL E2 + 35 ng/mL P4 (group H2). Embryos were prepared after in vitro maturation either by parthenogenetic activation or somatic cell nuclear transfer (SCNT) technique. Treating parthenogenetic embryo with EVs, led a significantly higher rate of the blastocyst formation in the group supplemented with each EVs, compared to the control group. In addition, TUNEL assay and gene expression level analysis revealed that apoptosis was significantly reduced in the H2 EVs group. Furthermore, EVs from hormone-primed POECs improved the formation rate of porcine SCNT embryos compared to the control group. While in each EVs supplemented group (control EVs, H1 EVs, H2 EVs), the expression of cell reprogramming-related genes in cloned embryos showed a tendency of increase, the effect was stronger in H1 EVs and H2 EVs. In conclusion, EVs derived from POECs cultured in hormonal conditions simulating the in vivo environment had a positive effect on porcine blastocysts formation, which will likely facilitate in the production of cloned embryos.

Development of buffalo ear skin fibroblast cell line for somatic cell nuclear transfer

Fibroblast is the principal active cell of connective tissue. It can be used as a useful tool for reproductive and therapeutic studies for its ability to easily grow in culture, create a preferable environment to growth and can be used as both for primary and permanent cell lines. Somatic cell nuclear transfer (SCNT) is commonly used for complete genetic reprogramming of a fully differentiated cell (e.g., fibroblast). SCNT is influential means for studying genomic imprinting, nuclear-cytoplasmic interaction, totipotency, and the contribution of paternal and maternal genomes to developing embryos. For reproductive cloning, SCNT is a fundamental step. So, the objective of this study was to adapt buffalo fibroblast cell culture protocol in order to develop fibroblast cell line for SCNT. River type buffalo ear skin tissues were used for developing primary cell culture to establish fibroblast cell line. Cells were grown in vitro. The general morphology and growth of cell population and presence of any microbial contaminants were checked regularly under an inverted microscope in phase contrast. Cells were counted by using hemocytometers. Sliced tissue was sown in 15 culture dishes to create primary and subcultures, and cells were cultivated in 6 of those dishes. Cell confluence varied from 70 to 80%. There were performed subcultures. Cell confluence in passages 1 through 10 varied from 73 to 90%. Cell concentration was 2.72 x 105, 2.68 x 105, and 2.65 x 105 per ml in passages 1, 5, and 10, respectively. Viable cells ranged from 84-93% of the total cells in passages 1 through 10. Different passages of cultivated fibroblast cells showed significantly varying viability (p< 0.05). Cultured fibroblast cell lines could now be preserved using cryotechnology.

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.

Luteolin supplementation during porcine oocyte maturation improves the developmental competence of parthenogenetic activation and cloned embryos.

Luteolin (Lut), a polyphenolic compound that belongs to the flavone subclass of flavonoids, possesses anti-inflammatory, cytoprotective, and antioxidant activities. However, little is known regarding its role in mammalian oocyte maturation. This study examined the effect of Lut supplementation during in vitro maturation (IVM) on oocyte maturation and subsequent developmental competence after somatic cell nuclear transfer (SCNT) in pigs. Lut supplementation significantly increased the proportions of complete cumulus cell expansion and metaphase II (MII) oocytes, compared with control oocytes. After parthenogenetic activation or SCNT, the developmental competence of Lut-supplemented MII oocytes was significantly enhanced, as indicated by higher rates of cleavage, blastocyst formation, expanded or hatching blastocysts, and cell survival, as well as increased cell numbers. Lut-supplemented MII oocytes exhibited significantly lower levels of reactive oxygen species and higher levels of glutathione than control MII oocytes. Lut supplementation also activated lipid metabolism, assessed according to the levels of lipid droplets, fatty acids, and ATP. The active mitochondria content and mitochondrial membrane potential were significantly increased, whereas cytochrome c and cleaved caspase-3 levels were significantly decreased, by Lut supplementation. These results suggest that Lut supplementation during IVM improves porcine oocyte maturation through the reduction of oxidative stress and mitochondria-mediated apoptosis.

Blastocyst cell number and allocation affect the developmental potential and transcriptome of bovine Somatic Cell Nuclear Transfer embryos.

Cloning cattle using somatic cell nuclear transfer (SCNT) is inefficient. While the rate of development of SCNT embryos in vitro is similar to that of fertilized embryos, most fail to develop into healthy calves. In this study, we aimed to identify developmentally competent embryos according to blastocyst cell composition and perform transcriptome analysis of single embryos. Transgenic SCNT embryos expressing nuclear-localized HcRed gene at day 7 of development were imaged by confocal microscopy for cell counting and individually transferred to recipient heifers. Pregnancy rates were determined by ultrasonography. Embryos capable of establishing pregnancy by day 35 had an average of 117±6 total cells, whereas embryos with an average of 128±5 cells did not establish pregnancy (p0.05). A lesser average number of 41± 3 cells in the inner cell mass (ICM) also resulted in pregnancies (0.05) than a greater number of 48 ± 2 cells in the ICM. Single embryos were then submitted to RNA sequencing for transcriptome analysis. Using weighted gene co-expression network analysis, we identified clusters of genes in which gene expression correlated with the number of total cells or ICM cells. Gene ontology (GO) analysis of these clusters revealed enriched biological processes in the coenzyme metabolic process, intracellular signaling cascade, and glucose catabolic process, among others. We concluded that SCNT embryos with fewer total- and ICM-cell numbers resulted in greater pregnancy establishment rates and that these differences are reflected in the transcriptome of such embryos.

Epigenetic Iinsights into the Senescence of Porcine Fetal Fibroblasts induced by Passaging

ABSTRACT Epigenetic status of fetal fibroblasts (FFs) is one of the crucial factors accounted for the success of somatic cell nuclear transfer and gene editing, which might inevitably be affected by passaging. But few systematic studies have been performed on the epigenetic status of passaged aging cells. Therefore, FFs from large white pig were in vitro passaged to the 5, 10, and 15 (F5, F10, and F15) passages in the present study to investigate the potential alteration of epigenetic status. Results indicated the senescence of FFs occurs with the passaging, as assessed by the weakened growth rate, increased β-gal expression, and so on. For the epigenetic status of FFs, the higher level both of DNA methylation and H3K4me1, H3K4me2, H3K4me3 was observed at F10, but the lowest level was observed at F15. However, the fluorescence intensity of m6A was significantly higher in F15, but lower (p < 0.05) in F10, and the related mRNA expression in F15 was significantly higher than F5. Further, RNA-Seq indicated a considerable difference in the expression pattern of F5, F10, and F15 FFs. Among differentially expressed genes, not only the genes involved in cell senescence were changed, but also the upregulated expression of Dnmt1, Dnmt3b, Tet1 and dysregulated expression of histone methyltransferases-related genes were detected in F10 FFs. In addition, most genes related to m6A such as METTL3, YTHDF2, and YTHDC1 were significantly different in F5, F10, and F15 FFs. In conclusion, the epigenetic status of FFs was affected by being passaged from F5 to F15.

TGFβ inhibition and mesenchymal to epithelial transition initiation by Xenopus egg extract: first steps towards early reprogramming in fish somatic cell

Xenopus egg extract is a powerful material to modify cultured cells fate and to induce cellular reprogramming in mammals. In this study, the response of goldfish fin cells to in vitro exposure to Xenopus egg extract, and subsequent culture, was studied using a cDNA microarray approach, gene ontology and KEGG pathways analyses, and qPCR validation. We observed that several actors of the TGFβ and Wnt/β-catenin signaling pathways, as well as some mesenchymal markers, were inhibited in treated cells, while several epithelial markers were upregulated. This was associated with morphological changes of the cells in culture, suggesting that egg extract drove cultured fin cells towards a mesenchymal-epithelial transition. This indicates that Xenopus egg extract treatment relieved some barriers of somatic reprogramming in fish cells. However, the lack of re-expression of pou2 and nanog pluripotency markers, the absence of DNA methylation remodeling of their promoter region, and the strong decrease in de novo lipid biosynthesis metabolism, indicate that reprogramming was only partial. The observed changes may render these treated cells more suitable for studies on in vivo reprogramming after somatic cell nuclear transfer.

Dual single guide RNAs-mediating deletion of mature myostatin peptide results in concomitant muscle fibre hyperplasia and adipocyte hypotrophy in pigs

Myostatin (MSTN) is a major gene target for skeletal muscle overgrowth in animals. We hypothesized that deletion of the entire mature peptide encoded by MSTN in pigs would knock out its bioactive form and accordingly stimulate skeletal muscle overgrowth. Thus, we engineered two pairs of single-guide RNAs (sgRNAs) to target exons 1 and 3 of MSTN in primary fetal fibroblasts of Taoyuan black pigs. We found that sgRNAs targeting exon 3, which encodes the mature peptide, had higher biallelic null mutation efficiency than those targeting exon 1. Somatic cell nuclear transfer was conducted using the exon 3 mutation cells as donor cells to generate five cloned MSTN null piglets (MSTN−/−). Growth testing revealed that both the growth rate and average daily weight gain of MST−/− pigs were greater than those of wild-type (MSTN+/+) pigs. Slaughter data demonstrated that the lean ratio of MSTN−/− pigs was 11.3% higher (P < 0.01) while the back-fat thickness was 17.33% lower (P < 0.01) than those of MSTN+/+ pigs. Haematoxylin–eosin staining indicated that the increased leanness of MSTN−/− pigs resulted from muscle fibre hyperplasia rather than hypertrophy.HE staining showed markedly decreased adipocyte size in MSTN−/− pigs. We also critically examined the off-target and random integration by resequencing, which showed that the founder MSTN−/− pigs contained no non-target mutations or exogenous plasmid elements. This study is the first to report the successful knock out of the mature MSTN peptide using dual sgRNA-mediated deletion, leading to the most prominent alteration of meat production traits in pigs published thus far. This new strategy is expected to have a wide impact on genetic improvements in food animals.

Overcoming the H4K20me3 epigenetic barrier improves somatic cell nuclear transfer reprogramming efficiency in mice.

Epigenetic reprogramming during fertilization and somatic cell nuclear transfer (NT) is required for cell plasticity and competent development. Here, we characterize the epigenetic modification pattern of H4K20me3, a repressive histone signature in heterochromatin, during fertilization and NT reprogramming. Importantly, the dynamic H4K20me3 signature identified during preimplantation development in fertilized embryos differed from NT and parthenogenetic activation (PA) embryos. In fertilized embryos, only maternal pronuclei carried the canonical H4K20me3 peripheral nucleolar ring-like signature. H4K20me3 disappeared at the 2-cell stage and reappeared in fertilized embryos at the 8-cell stage and in NT and PA embryos at the 4-cell stage. H4K20me3 intensity in 4-cell, 8-cell, and morula stages of fertilized embryos was significantly lower than in NT and PA embryos, suggesting aberrant regulation of H4K20me3 in PA and NT embryos. Indeed, RNA expression of the H4K20 methyltransferase Suv4-20h2 in 4-cell fertilized embryos was significantly lower than NT embryos. Knockdown of Suv4-20h2 in NT embryos rescued the H4K20me3 pattern similar to fertilized embryos. Compared to control NT embryos, knockdown of Suv4-20h2 in NT embryos improved blastocyst development ratios (11.1% vs. 30.5%) and full-term cloning efficiencies (0.8% vs. 5.9%). Upregulation of reprogramming factors, including Kdm4b, Kdm4d, Kdm6a, and Kdm6b, as well as ZGA-related factors, including Dux, Zscan4, and Hmgpi, was observed with Suv4-20h2 knockdown in NT embryos. Collectively, these are the first findings to demonstrate that H4K20me3 is an epigenetic barrier of NT reprogramming and begin to unravel the epigenetic mechanisms of H4K20 trimethylation in cell plasticity during natural reproduction and NT reprogramming in mice.

Reproduction of Sheep through Nuclear Transfer of Somatic Cells: A Bibliometric Approach.

Somatic cell nuclear transfer (SCNT) is a reproductive biotechnology with great potential in the reproduction of different species of zootechnical interest, including sheep. This study aimed to carry out a bibliometric analysis of scientific papers published on the application of SCNT in sheep reproduction during the period 1997-2023. The search involved the Science Citation Index Expanded and Social Sciences Citation Index databases of the main collection of the Web of Sciences with different descriptors. A total of 124 scientific papers were analyzed for different bibliometric indicators using the VOSviewer software. Since 2001, the number of SCNT-related papers that have been published concerning sheep reproduction has increased and it has fluctuated in ensuing years. The main authors, research groups, institutions, countries, papers, and journals with the highest number of papers related to the application of SCNT in sheep reproduction were identified, as well as the topics that address the research papers according to the terms: somatic cell, embryo, oocyte, gene expression, SCNT, and sheep.

Efficiency of equine oocyte recovery from post mortem ovaries or by ovum pick up

Most oocytes used for cloned embryo production are obtained from abattoir ovaries even though in some countries, slaughtering horses is not allowed. The advantage of scraping post mortem ovaries is the high number of oocytes available in a short period of time to perform somatic cell nuclear transfer. However, mare age, reproductive and health status or ovary manipulation by abattoir staff are not controlled before slaughter and the effect on oocyte quality and early embryo development is uncertain. Ovum pick up (OPU) is an alternative technique to obtain oocytes ex vivo from controlled mares and embryos obtained from those oocytes potentially have better development. Cortez et al. (Journal of Equine Veterinary Science 113 2022; 14) reported higher cleavage and blastocyst rates in cloned embryos from OPU-derived rather than post mortem derived ovaries. The aim of this retrospective study was to determine and compare the oocyte recovery rates from abattoir-sourced ovaries or collected by OPU. Data was collected between September and November 2022 in Argentina. For statistical analysis, generalized linear models were used. A total of 3476 ovaries were collected from the abattoir and 16% (557/3476) were discarded since antral follicles were not visible. Using the scraping technique, 8392 oocytes were recovered in 24 sessions obtaining an averageof 350 oocytes per session. Mean (± SD) oocyte recovered per scraped ovary was 2.9±0.4 and for all collected ovaries per session was 2.4±0.3. On average the scraping time calculated per mare (pair of ovaries) for all collected ovaries per session was 1 min and 36 sec (59 sec-2 min). OPU was performed in 44 mixed breed healthy mares during the southern hemisphere breeding season in which a mean of 16.7 (7-29) follicles were aspirated per mare in an average of 27 min and 7 sec (6 min and 3 sec- 58 min). Oocyte recovery rate per aspirated follicle was 63.3% and a mean of 9.7 oocytes were retrieved per OPU. Mean oocyte retrieval per mare was higher using OPU than scraping (9.7 ± 4.8 vs. 5.8 ± 0.9; p<0.0001). Although oocyte recovery per follicle could not be compared in this study these results could be influenced by mares’ selection for OPU although there was not a minimum number of follicles set as a criteria to perform OPU. The data show that the OPU procedure is a suitable technique to obtain oocytes for equine cloning programs. In some circumstances, the procedures to obtain oocytes by OPU are more expensive than those recovered by scraping, however published data suggest that fewer oocytes recovered by OPU are needed to obtain a cloned embryo.

Forgotten Clones: The Birth of Cloning and the Biological Revolution. By NathanCrowe. Pittsburgh, PA: University of Pittsburgh Press. 2021. pp. 336. $60 (Hardcover).

Forgotten Clones: The Birth of Cloning and the Biological Revolution. By NathanCrowe. Pittsburgh, PA: University of Pittsburgh Press. 2021. pp. 336. $60 (Hardcover).

Deciphering the placental abnormalities associated with somatic cell nuclear transfer at single-nucleus resolution.

Deciphering the placental abnormalities associated with somatic cell nuclear transfer at single-nucleus resolution.

Production of Somatic Cell Nuclear Transfer derived cloned calves in the United Arab Emirates.

Despite the tremendous advancement in somatic cell nuclear transfer (SCNT) in camels, the generation of cloned calf using SCNT in cows is yet to be reported in the United Arab Emirates (UAE). The present study intended to produce cloned calves in the local environment of the UAE. We surgically collected ear tissue from two high-performance Holstein cows (average production of 71 liter/ day) and established the fibroblast cell line to perform SCNT. Fifty- seven (18.48%) blastocysts were derived from 330 fused oocytes for cell donor A, and 51 (19.14%) blastocysts were generated from 289 fused oocytes for cell donor B. We used 102 recipients for embryo transfer; among those, 53 and 49 recipients received embryos reconstructed with cell donor A and B, respectively. A single blastocyst in transfer media was transferred deep into the horn of the uterus of natural cycling recipient cows by trans cervical method on Day 7; where estrus was considered as Day 0. Non-return to estrus (up to 42 days), early pregnancy, and the live birth rate did not differ significantly for the two donor cells. Four (26.67%) and 1 (7.69%) live calves were produced for cell donor A and B, respectively. Microsatellite analysis of 18 bovine-specific loci confirmed the perfect match of cell donors and cloned calves. This is the first reported case of cloned calves using SCNT in the UAE. Our results could benefit the UAE dairy industry by supporting the locally produced high- performance replacement heifers. Keywords: Bovine; Cloning; In vitro maturation; Oocytes; UAE

First gene-edited calf with reduced susceptibility to a major viral pathogen.

Bovine viral diarrhea virus (BVDV) is one of the most important viruses affecting the health and well-being of bovine species throughout the world. Here, we used CRISPR-mediated homology-directed repair and somatic cell nuclear transfer to produce a live calf with a six amino acid substitution in the BVDV binding domain of bovine CD46. The result was a gene-edited calf with dramatically reduced susceptibility to infection as measured by reduced clinical signs and the lack of viral infection in white blood cells. The edited calf has no off-target edits and appears normal and healthy at 20 months of age without obvious adverse effects from the on-target edit. This precision bred, proof-of-concept animal provides the first evidence that intentional genome alterations in the CD46 gene may reduce the burden of BVDV-associated diseases in cattle and is consistent with our stepwise, in vitro and ex vivo experiments with cell lines and matched fetal clones.

Intracellular Strain Evaluation-Based Oocyte Enucleation and Its Application in Robotic Cloning

Since the first cloned sheep was produced in 1996, cloning has attracted considerable attention because of its great potential in animal breeding. Somatic cell nuclear transfer (SCNT) is widely used for creating clones. However, SCNT is very complicated to manipulate and inevitably causes intracellular damage during manipulation. Typically, only less than 1% of reconstructed embryos develop into live cloned animals. This low success rate is considered to be the major limitation in the extensive application of cloning techniques. In this study, we proposed an intracellular strain evaluation-based oocyte enucleation method to reduce potential intracellular damage in SCNT. We first calculated the intracellular strain based on the intracellular velocity field and then used the intracellular strain as a criterion to improve the enucleation operation. We then developed a robotic batch SCNT system to apply this micromanipulation method to animal cloning. Experimental results showed that we increased the blastocyst rate from 10.0% to 20.8%, and we successfully produced 17 cloned piglets by robotic SCNT for the first time. The success rate of cloning was significantly increased compared to that of traditional methods (2.5% vs 0.73% on average). In addition to the cloning technique, the intracellular strain evaluation-based enucleation method is expected to be applicable to other biological operations and for establishing a universal cell manipulation protocol to reduce intracellular damage.

Sperm-borne microRNA-34c regulates maternal mRNA degradation and preimplantation embryonic development in mice

BackgroundStudies have shown that sperm-borne microRNAs (miRNAs) are involved in mammalian preimplantation embryonic development. In humans, spermatozoan miR-34c levels are correlated with in vitro fertilization outcomes, such as embryo quality and the clinical pregnancy and live birth rates. In rabbits and cows, miR-34c improves the developmental competence of embryos generated by somatic cell nuclear transfer. However, the mechanisms underlying the regulation of embryonic development by miR-34c remain unknown.MethodsFemale C57BL/6 mice (6–8 weeks old) were superovulated, and pronucleated zygotes were collected and microinjected with an miR-34c inhibitor or a negative-control RNA. The embryonic development of the microinjected zygotes was evaluated, and the messenger RNA (mRNA) expression profiles of the embryos at the two-cell, four-cell and blastocyst stages (five embryos per group) were determined by RNA sequencing analysis. Gene expression levels were verified by reverse transcription–quantitative polymerase chain reaction. Cluster analysis and heat map visualization were performed to detect differentially expressed mRNAs. Pathway and process enrichment analyses were performed using ontology resources. Differentially expressed mRNAs were systematically analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins database to determine their biological functions.ResultsEmbryonic developmental potential was significantly reduced in zygotes microinjected with the miR-34c inhibitor compared with those microinjected with a negative-control RNA. Two-cell stage embryos microinjected with an miR-34c inhibitor presented altered transcriptomic profiles, with upregulated expression of maternal miR-34c target mRNAs and classical maternal mRNAs. Differentially expressed transcripts were mainly of genes associated with lipid metabolism and cellular membrane function at the two-cell stage, with cell-cycle phase transition and energy metabolism at the four-cell stage; and with vesicle organization, lipid biosynthetic process and endomembrane system organization at the blastocyst stage. We also showed that genes related to preimplantation embryonic development, including Alkbh4, Sp1, Mapk14, Sin3a, Sdc1 and Laptm4b, were significantly downregulated after microinjection of an miR-34c inhibitor.ConclusionsSperm-borne miR-34c may regulate preimplantation embryonic development by affecting multiple biological processes, such as maternal mRNA degradation, cellular metabolism, cell proliferation and blastocyst implantation. Our data demonstrate the importance of sperm-derived miRNAs in the development of preimplantation embryos.

Production of MSTN Gene-Edited Embryos of Buffalo Using the CRISPR/Cas9 System and SCNT.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system and somatic cell nuclear transfer (SCNT) have been used to produce genome-edited farm animal species for improved production and health traits; however, these tools are rarely used in the buffalo and can play a pivotal role in milk and meat production in tropical and subtropical countries. In this study, we aimed to produce myostatin (MSTN) gene-edited embryos of the Murrah buffalo using the CRISPR/Cas9 system and SCNT. For this, fibroblast cells were electroporated with sgRNAs carrying all-in-one CRISPR/Cas9 plasmids targeting the first exon of the MSTN gene. Following puromycin selection, single-cell clonal populations were established and screened using the TA cloning and Sanger sequencing methods. Of eight single-cell clonal populations, one with a monoallelic and another with a biallelic heterozygous gene editing event were identified. These two gene-edited clonal cell populations were successfully used to produce blastocyst-stage embryos using the handmade cloning method. This work establishes the technical foundation for generation of genome-edited cloned embryos in the buffalo.

Methylation Genome-Wide Profiling in Lowly and Highly Efficient Somatic Cell Nuclear Transfer in Pigs

Swine is a common model organism for biomedical research. Epigenetic reprogramming in somatic cell nuclear transfer (SCNT) embryos does not fully recapitulate the natural DNA demethylation events at fertilisation. This study aimed to conduct genome-wide methylation profiling to detect differentially methylated regions (DMRs) responsible for epigenetic differences in stem cells that displayed high and low efficiency of SCNT and to elucidate the low efficiency of cloning rate in pigs. Adipose tissue mesenchymal stem cells (AMSC)s lines were isolated from adipose tissue of adult male pigs (n = 20; high-efficiency cells = 10; and low-efficiency cells = 10). Reduced representation bisulfite sequencing (RRBS) was performed on an Illumina HiSeq1500. Paired-end reads were filtered to remove the adapter contamination, and low-quality reads using TrimGalore! Filtered reads were mapped to the reference genome using Bismark. MethylKit was used to identify differentially methylated regions (DMRs) (bases and tiles), showing statistically significant differential methylation between high and low-efficiency AMSCs. Hierarchical cluster analysis according to methylation patterns clearly defined groups with low and high cloning efficiency. We report 3704 bases with statistically significant differences in methylation and 10062 tiles with statistically significant differences in methylation. Most differentially methylated sites are intergenic 62%, 31% are intronic, 4% are in exons, and 4% in promoters. Moreover, 37% of differentially methylated sites are located in known CpG islands (CGIs), and 4% in CpG island shores (CGSs).

The combination of rolipram and cilostamide improved the developmental competence of cloned porcine embryos

In vitro maturation of porcine oocytes is characterized by asynchronous cytoplasmic and nuclear maturation, leading to less competent oocytes supporting embryo development. The purpose of this study was to evaluate the combined effect of rolipram and cilostamide as cyclic Adenine monophosphate (cAMP) modulators to find the maximum cAMP levels that temporarily arrest meiosis. We determined the optimal time to maintain functional gap junction communication during pre-in vitro maturation to be four hours. Oocyte competence was evaluated by the level of glutathione, reactive oxygen species, meiotic progression, and gene expression. We evaluated embryonic developmental competence after parthenogenetic activation and somatic cell nuclear transfer. The combined treatment group showed significantly higher glutathione and lower reactive oxygen species levels and a higher maturation rate than the control and single treatment groups. Cleavage and blastocyst formation rates in parthenogenetic activation and somatic cell nuclear transfer embryos were higher in two-phase in vitro maturation than in the other groups. The relative levels of BMP15and GDF9 expression were increased in two-phase in vitro maturation. Somatic cell nuclear transfer blastocysts from two-phase in vitro maturation oocytes showed a lower level of expression of apoptotic genes than the control, indicating better pre-implantation developmental competence. The combination of rolipram and cilostamide resulted in optimal synchrony of cytoplasmic and nuclear maturation in porcine in vitro matured oocytes and there by enhanced the developmental competence of pre-implantation embryos.