Embryo Reconstruction Research Articles

O-130 The spatial conformational features of blastocysts can serve as a new basis for selection

Abstract Study question Can spatial conformational features become a new basis for blastocyst selection? Summary answer The spatial conformational features of blastocysts can be associated with clinical outcomes, which is a new basis for blastocyst selection. What is known already At present, the quality evaluation of blastocysts is two-dimensional, and there are relatively few evaluation indicators. There are now methods for exploring three-dimensional reconstruction of blastocysts, including fluorescence staining to obtain three-dimensional structures or artificial rotation to obtain images and restore three-dimensional conformations. However, one drawback of these methods is that they are far from true clinical applications. There are no reports on the reconstruction techniques that can be used in clinical blastocyst selection techniques, and there are no reports on the spatial conformational features related to them. Study design, size, duration This study included 2,141 frozen thaw single blastocyst transfer cycles at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology from 2020 to 2021. Simultaneously collect multi-focal plane (11) image data for each blastocyst (EmbryoScope+). The inclusion criteria for patients are those under 40 years old, with an endometrial thickness of 7-16mm, and a history of embryo transfer failure of less than or equal to 1 time. Participants/materials, setting, methods This study iteratively interpolated the 11 focal planes using a multi focal plane generation network model. The contour and spatial information of blastocyst TE and ICM were obtained through deep-learning and segmentation networks. Texture information was extracted using depth estimation and image fusion and mapped to a 3D model to obtain a complete blastocyst spatial model. Then calculate a series of new spatial conformational features of the blastocyst and analyze their correlation with clinical outcomes. Main results and the role of chance We validated the model using the fluorescence reconstruction of human blastocysts as the gold standard (key indicator accuracy>90%). The obtained spatial conformational parameters of blastocysts include: Blastocyst: overall volume (BOV), cystic cavity volume (BCV), surface area (BSA); ICM: volume (IV), surface area (ISA), spatial aspect ratio (ISAr), surface sphericity (ISS), relative distance from ICM to TE (IRD), contact area with TE (ICA); TE cells: number (TN), density (TD), distribution difference (TDD), TE cell roundness (TR), circumference (TC), area (TA) and variance, TE cell aspect ratio (TMM) and variance. The BOV, BCV, and BSA of the blastocyst were positively correlated with clinical pregnancy rate (CPR). The quartile results showed significant differences in CPR when the values were >179 wμm3, 165 wμm3, and 25.8 wμm2, respectively. In terms of ICM, there was no significant difference in CPR among the IV, ISA, IRD, and ICA. However, we found ISAr and ISS had higher CPR in the range of 0.715-0.912 and 0.920-0.943, respectively. In terms of TE, TN is positively correlated with CPR. TD, TDD, TR were not significantly associated with CPR. The pleasing finding is that TC, TA, TMM, and their variances are significantly correlated with CPR. Limitations, reasons for caution The overall quality of blastocysts is relatively good, which is related to the priority thawing of the best blastocysts. Some blastocysts may appear partially out of view, leading to data unavailability and potentially affecting 3D reconstruction. Further exploration and discovery are needed for the spatial conformational features of blastocysts. Wider implications of the findings This work provides a 3D reconstruction system for blastocysts that is closer to clinical applications and discovers new spatial features that are related to clinical outcomes. This enables the study of more dimensional biological characteristics of blastocysts, and our research further promotes the development of embryo reconstruction technology. Trial registration number not applicable

Unraveling Renal Arteries Morphogenesis from Tridimensional Human Embryos Reconstruction

During human morphogenesis, the definitive kidneys derive from the metanephros during Carnegie Stage 14 to 23. The pronephros and the mesonephros develop previously and successively to finally lead to the formation of the urinary tract. Renal vascularization, first described in 1912 by Félix using a "ladder theory" model, is highly variable and current available morphogenesis descriptions do not explain all reported anatomical variations. The aim of this work was to study the morphogenesis of the human metanephros and its vascularization by three-dimensional reconstructions of human embryos. Histological sections of 23 human embryos from the Carnegie Collection and 5 human embryos from the French collection (Carnegie stages 14 to 23) were completely digitalized and reconstructed in three dimensions using specific softwares and then analyzed by descriptive method using manual annotation. In all studied embryos, the mesonephric arteries did not reach the metanephros irrespective to the position of the metanephros during its cranial ascent. Before the end of the cranial metanephros migration (15 embryos), at the level of the aorto-iliac bifurcation, a "primitive" vascularization was shown in 9 of them. The renal artery originated from the primitive iliac arteries for 8 embryos and from the inferior mesenteric artery in one embryo. Further, a capillary cluster emerging from the lateral wall of the aorta and extending toward the metanephros was found in 2 embryos (Carnegie stages 21 and 22). This may correspond to a phenomenon of neoangiogenesis responsible of the definitive renal artery. The present study reported the morphogenesis of human renal arteries between Carnegie stages 14 and 23 using an original method of tridimensional computerized reconstructions of historical human embryos. Some original findings, in contradiction with the original Felix's description, may explain the most frequently reported anatomical variations.

Monitoring swine virus transmission in embryos derived from commercial abattoir oocytes.

Pigs are pivotal in agriculture and biomedical research and hold promise for xenotransplantation. Specific-pathogen-free (SPF) herds are essential for commercial swine production and xenotransplantation research facilities. Commercial herds aim to safeguard animal health, welfare, and productivity, and research facilities require SPF status to protect immunocompromised patients. Somatic cell nuclear transfer (SCNT) embryos are the norm for producing cloned and genetically edited animals. Oocytes for embryo reconstruction are most conveniently sourced from commercial abattoirs with unclear disease statuses. However, research on viral clearance from donor oocytes during embryo reconstruction remains limited. SCNT has previously been shown to reduce the transmission of Porcine reproductive and respiratory syndrome virus, Bovine viral diarrhea virus, Porcine Circovirus type 2, and Porcine parvovirus. Still, it is lacking for other pathogens, including endogenous viruses. This project contains two preliminary studies investigating the polymerase chain reaction (PCR) assay detection of common swine viruses through the phases of producing parthenogenic and SCNT embryos. Exogenous pathogens detected in oocyte donor tissue or the oocyte maturation media were not detected in the produced embryos. Porcine endogenous retrovirus type C (PERVC) was not removed by parthenogenic embryo activation and was detected in 1 of the 2 tested SCNT embryos reconstructed using a PERVC-negative cell line. SCNT and parthenogenic embryo construction similarly reduced exogenous virus detection. SCNT embryo construction helped reduce endogenous virus detection. This project demonstrates the importance of screening embryos for endogenous viruses and shows the usefulness of parthenogenic embryos in future exogenous virus clearance studies.

Renal artery morphogenesis rediscovered from 3D reconstructions of human embryos

Renal artery morphogenesis rediscovered from 3D reconstructions of human embryos

P-291 Seeking Arrangements: A New Cleavage-Stage Biomarker from 3D Morphokinetics

Abstract Study question What can we learn from 3D reconstructions of cleavage-stage embryos derived from Hoffman modulation contrast (HMC) time-lapses? Summary answer A simple spatial biomarker extracted from 3D embryo reconstructions at the t4 and t8 stages is associated with blastulation, blastocyst quality, pregnancy and live birth. What is known already Several works have demonstrated significant associations between t4 cell arrangement and blastulation potential. However, no studies have investigated the impacts of cell arrangement beyond the t4 stage in a clinical setting owing to difficulties visualising the 3D structure of embryos in a safe, cost-effective manner. In the previous ESHRE meeting, He et al. presented a deep learning system for the 3D reconstruction of cleavage-stage embryos from HMC focal stacks recorded in standard timelapse incubators. In this work, we use the aforementioned system to understand the spatial networks present in cleavage-stage embryos and investigate their associations with clinical outcomes. Study design, size, duration The study was a retrospective analysis of two imaging datasets from two different clinics. The first dataset (DS1) consisted of 162 t4 embryos with information on blastulation and Gardner grade. The second dataset (DS2) consisted of 202 embryos at t4 and t8 with information on blastocyst grade, pregnancy, live birth and PGT-A. All data was captured at 11 focal planes on Embryoscope incubators between 2018 and 2020. Participants/materials, setting, methods The system proposed by He et al. was used to reconstruct the 3D structure of embryos from their focal stacks. Networks of cell contacts were extracted from the resulting embryo 3D models and each embryo’s mean contacts per cell was computed. Statistical analysis of average cell contacts with respect to outcomes was carried out using unpaired t-tests. Moreover, cell contact networks from different embryos were compared to identify embryos with similar cell arrangements. Main results and the role of chance At t4, a higher average number of contacts per cell was associated with greater rates of blastulation in DS1 (2.59 vs 2.36, blastulated vs non-blastulated, p = 0.029) and blastocyst quality in both DS1 (2.59 vs 2.37, good vs poor, p = 0.010) and DS2 (2.51 vs 2.35, good vs poor, p = 0.017) where a ‘good’ embryo is defined as having an embryologist-provided Gardner grade with EXP>2, ICM>C and TE>C. At t8, a higher average number of contacts was associated with increased blastocyst quality (3.36 vs 3.09, good vs poor, p = 0.017), pregnancy (3.32 vs 2.87, pregnant vs not pregnant, p = 0.003) and live birth (3.40 vs 2.90, live birth vs no live birth, p = 0.0003). No associations were found with miscarriage or aneuploidy. Moreover, average contacts at t4 were not correlated with those at t8 (r = 0.15, 95% CI [-0.043, 0.34]). While 4-cell embryos fell neatly into 9 distinct cell arrangements with the 5 most common (tetrahedral, pseudotetrahedral, planar, closed-Y and linear) accounting for 95% of embryos, 8-cell embryos displayed a great degree of variation with 59 distinct cell arrangements, the largest such group representing only 8 embryos. Limitations, reasons for caution The datasets used in this study were small. Moreover, DS2 was subject to survivorship bias as it only contained embryos with PGT-A results which necessarily entailed successful blastulation. Furthermore, the 3D reconstruction system was not applicable to all cleavage-stage embryos, especially those obscured by the well or having undergone compaction. Wider implications of the findings This work provides evidence for the clinical relevance of cleavage-stage cell arrangement in the human preimplantation embryo beyond the 4-cell stage, which may improve selection techniques for D3 transfers. Moreover, our work provides a strong case for further investigation into spatial biomarkers derived from 3D embryo reconstruction and 3D morphokinetics. Trial registration number N/A

The Effect of Zinc Finger Domain Protein Spalt Like Transcription Factor 4 (SALL4A)-Mediated DNA Demethylation on Cardiac Development and Function

SALL4 is one of the important members of SALL4 gene family and participates in embryo development, including organogenesis, maintenance and reconstruction of pluripotency, such as heart development and function. This study explores the effects of SALL4A-mediated DNA demethylation on heart development and function. The ventricular weight/body weight, ventricular diameter, septal thickness, LVEF% and LVFS% of the SALL4A gene knockout and normal SD mice were compared. ChIP/DIP-Seq and RNA-Seq technology were used to assess the mechanism by how SALL4Adependent DNA demethylation affects heart development and function. We found that compared with control group of SD mice, the ventricular weight/body weight of SD mice in SALL4A knockout group was significantly lower. In addition, SALL4A knockout group showed significantly lower interval thickness, LVEF%, LVFS% and other indicators related to heart development than normal SD mice. In addition, SALL4A-mediated DNA demethylation was closely related to TET. Both TET1 and TET2 were enriched in the SALL4A binding site. SALL4A targeted 5hmC gene in vitro and occupied the enhancer in mouse embryonic stem cells (ESCs) to promote 5hmC oxidation depending on TET enzyme. Therefore, SALL4A promoted oxidation of 5hmC and caused DNA demethylation which finally affected heart development and function. In conclusion, SALL4A, as a gene that can target and bind 5hmC, promotes the oxidation of 5hmC by stabilizing TET enzyme binding, thereby regulating the DNA demethylation process in ESCs to further regulate heart development and function.

O-177 Towards 3D Reconstructions of Human Preimplantation Embryo Development

Abstract Study question Can we use focal stacks collected through Hoffman modulation contrast (HMC) microscopy to generate 3D reconstructions of preimplantation embryos? Summary answer A machine learning system was designed to generate 3D meshes that approximate the structures of embryos captured on HMC microscopes up to the 8-cell stage. What is known already The 3D arrangement of cells in preimplantation human embryos is a topic of clinical interest, with significant associations between the cell arrangement and blastulation potential from as early as the 4-cell stage. In basic research, the use of confocal microscopy for generating 3D reconstructions is commonplace. However, the use of confocal microscopy in the IVF clinic is often infeasible due to cost and concerns for embryos’ wellbeing. The assessment of 3D cell arrangement in clinical settings can thus prove difficult and time-consuming as many embryologists rely on focal stacks captured through the HMC microscopes widely integrated into incubators. Study design, size, duration The study was a retrospective analysis of 581 Embryoscope focal stacks of embryos from 4 clinics collected between 2018 and 2020. The number of planes in each stack ranged from 7-11 and cell outlines were annotated along with the depths at which they were most in-focus. A deep learning system was designed to generate 3D reconstructions of the embryos. Two clinics’ data were used for training (N = 551) and the others’ for evaluation (N = 30). Participants/materials, setting, methods The deep learning system consisted of three stages: a super-resolution module, a cell segmentation module and a depth regression module. The super-resolution stage was used to predict missing planes in focal stacks that did not contain 11 focal planes; the segmentation module identified individual cells; the depth regression module identified the focal plane at which each cell was most “in-focus”. Meshes were then generated under the assumption that blastomeres’ dimensions are similar along each axis. Main results and the role of chance The superresolution module was evaluated by calculating the structural similarity index (SSIM; an image similarity measure ranging from 0-1) between predicted and true planes when tasked with predicting missing frames in focal stacks with up to 4 planes artificially removed (by uniform random sampling). The module achieved an SSIM of 0.80. The predictions were also evaluated by 2 embryologists, a clinician and a developmental biologist on a scale of 1-5 (1=very unrealistic; 3=usable; 5=very realistic), achieving a mean score of 4.11. The segmentation module was evaluated on the proportion of cells it managed to identify (91%) as well as the mean overlap between predicted cell segmentations and the ground truth (intersection-over-union of 0.86). The depth module was evaluated on the mean deviation of predictions from the true most “in-focus” plane (0.73 planes). 3D reconstructions generated by the system were evaluated with reference to the original focal stacks by 2 embryologists on a 1-5 scale similar to before, with a mean score of 3.72. The most common issues with the reconstructions identified by the embryologists were missing cells/fragments, incorrect cell shape due to obstruction by the well’s edge and imprecise depth predictions (with the “true” depth being between focal planes). Limitations, reasons for caution As previously mentioned, some reconstructions had inaccuracies. These would likely be ameliorated through modifications to the system modules and more training data. Moreover, the system was not trained or evaluated on morulae/blastocysts. Finally, each focal stack was analysed independently - future work may examine enforcing temporal consistency within timelapses. Wider implications of the findings This work serves as a first step towards unlocking data captured in IVF clinics for research into cell arrangement in preimplantation embryos. Combined with cell tracking, the system may be useful for research into cell fate. Moreover, the work may find clinical relevance in enabling easier assessment of cell arrangement. Trial registration number N/A

Cell sorting and germ layer formation in reconstructed starfish embryos.

Germ layer formation is driven by embryonic cell sorting during the early developmental stages. Starfish (Patiria pectinifera) embryos have a connected endoderm and ectoderm, albeit with few contact surfaces between the epithelia. To better understand the association between cell sorting and germ layer formation, we reconstructed P.pectinifera embryos and examined their germ layer formation. Initial observations showed that the presumptive endodermal (pEN) and presumptive ectodermal (pEC) portions of the embryonic body at the late-blastula stage were preserved throughout development. Based on this, cells that were dissociated from each dermal fragment were mixed in a reconstruction experiment. Our results showed that the pEN and pEC cells were located inside and outside the reaggregates, respectively, to form an embryonic body containing two epithelial layers, separated by a blastocoel. During this process, the pEN cells were motile and shifted from smaller clumps to form a large clump. In contrast, in reaggregates formed in separate cultures, the pEN cells showed strong adhesion abilities, whereas the pEC cells underwent epithelialization. Unlike that in pEN cells, the reaggregation of pEC cells preceded cadherin expression. Filamentous actin was similarly observed in both reaggregates. These results suggest that during the reconstruction of starfish embryos, germ layer formation occurs via the sorting of pEN and pEC cells, depending on their adhesiveness, motility, and epithelialization. In vivo, these properties might embody the physiological significance of cell adhesion in the germ layers constituting the epithelial monolayer.

High Resolution Episcopic Microscopy for Qualitative and Quantitative Data in Phenotyping Altered Embryos and Adult Mice Using the New "Histo3D" System.

3D imaging in animal models, during development or in adults, facilitates the identification of structural morphological changes that cannot be achieved with traditional 2D histological staining. Through the reconstruction of whole embryos or a region-of-interest, specific changes are better delimited and can be easily quantified. We focused here on high-resolution episcopic microscopy (HREM), and its potential for visualizing and quantifying the organ systems of normal and genetically altered embryos and adult organisms. Although the technique is based on episcopic images, these are of high resolution and are close to histological quality. The images reflect the tissue structure and densities revealed by histology, albeit in a grayscale color map. HREM technology permits researchers to take advantage of serial 2D aligned stacks of images to perform 3D reconstructions. Three-dimensional visualization allows for an appreciation of topology and morphology that is difficult to achieve with classical histological studies. The nature of the data lends itself to novel forms of computational analysis that permit the accurate quantitation and comparison of individual embryos in a manner that is impossible with histology. Here, we have developed a new HREM prototype consisting of the assembly of a Leica Biosystems Nanocut rotary microtome with optics and a camera. We describe some examples of applications in the prenatal and adult lifestage of the mouse to show the added value of HREM for phenotyping experimental cohorts to compare and quantify structure volumes. At prenatal stages, segmentations and 3D reconstructions allowed the quantification of neural tissue and ventricular system volumes of normal brains at E14.5 and E16.5 stages. 3D representations of normal cranial and peripheric nerves at E15.5 and of the normal urogenital system from stages E11.5 to E14.5 were also performed. We also present a methodology to quantify the volume of the atherosclerotic plaques of ApoEtm1Unc/tm1Unc mutant mice and illustrate a 3D reconstruction of knee ligaments in adult mice.

Controlled Cytoplast Arrest and Morula Aggregation Enhance Development, Cryoresilience, and In Vivo Survival of Cloned Sheep Embryos.

Zona-free somatic cell transfer (SCT) and embryo aggregation increase throughput and efficiency of cloned embryo and offspring production, respectively, but both approaches have not been widely adopted. Cloning efficiency is further improved by cell cycle coordination between the interphase donor cell and metaphase-arrested recipient cytoplast. This commonly involves inclusion of caffeine and omission of calcium to maintain high mitotic cyclin-dependent kinase activity and low calcium levels, respectively, in the nonactivated cytoplast. The aim of our study was to integrate these various methodological improvements into a single work stream that increases sheep cloning success. We show that omitting calcium during zona-free SCT improved blastocyst development from 6% to 13%, while caffeine treatment reduced spontaneous oocyte activation from 17% to 8%. In a retrospective analysis, morula aggregation produced high morphological quality blastocysts with better in vivo survival to term than nonaggregated controls (15% vs. 9%), particularly after vitrification (14% vs. 0%). By combining cytoplast cell cycle control with zona-free embryo reconstruction and aggregation, this novel SCT protocol maximizes the benefits of vitrification by producing more cryoresilient blastocysts. The presented cloning methodology is relatively easy to operate and further increases throughput and efficiency of cloned embryo and offspring production. Integration of additional reprogramming steps or alternate donor cells is straightforward, providing a flexible workflow that can be adapted to changing experimental requirements.

RS-1 enhanced the efficiency of somatic cell reprogramming via activation of RAD51 in somatic cell nuclear transfer

Background & Aim Reprogramming efficiency of somatic cell nuclei is very low in both somatic cell nuclear transfer (SCNT) and iPSCs technologies. Recently, it was reported that the genetic instability and DNA damage arise prior to transcriptional activity during reprogramming. Subsequently, in the absence of mitotic nuclear remodeling, DNA replication is delayed and errors are exacerbated (Egli D et al., 2017). Methods, Results & Conclusion Here, in order to find a regulator of DNA damage during reprogramming events, we observed the effect of RAD51 stimulatory compound (RS-1) supplements in DNA repair process of mouse SCNT embryos in the early embryogenesis and compared with in vitro fertilization (IVF)-derived embryos. First, Rad51 mRNA expression in both embryos of SCNT and IVF groups was observed at 1-cell, 2-cell, and 4-cell stages. In IVF group, relative Rad51 mRNA expression was markedly decreased from 1-cell to 2-cell stages and then restored in 4 cell stage. However, in SCNT group, its expression decreased from 1-cell to 2-cell stages at a similar rate with IVF group, but not recovered at 4-cell stage. Based on these results, we have observed DNA damage detected by rH2AX and DNA repair of double strand breaks by Rad51 expressions. The rH2AX expression was highly localized in the nucleus of IVF embryo when compared with SCNT groups until 10 h after reconstruction of embryos. However Rad51 was not expressed in the nucleus until 10 h after SCNT moreover, at 12h after SCNT, rH2AX showed highly dotted in nuclear in both IVF and SCNT+RS-1 group. A large number of embryos were observed Rad51 expression only in IVF and SCNT+RS-1 groups. In this study, we found different expression of some cell cycle-related genes among IVF, SCNT and SCNT+RS-1 groups by RNA-sequencing. The SCNT+RS-1 group significantly enhanced blastocyst formation rate of the cloned embryos when compared to those of the RS-1-untreated group, and reduced the rate of DNA fragmentation. Furthermore SCNT+RS-1 group improved full term clone mice rates and significantly increased the derivation efficiency of pluripotent stem cells from cloned embryos (SCNT-PSCs). Based on these results, we suggest that DNA damage and Rad51-mediated DNA repair during DNA replication could complement each other and its regulation has plays an important role in somatic cell nuclei reprogramming process.

Reinvestigating the early embryogenesis in the flatworm Maritigrella crozieri highlights the unique spiral cleavage program found in polyclad flatworms

BackgroundSpiral cleavage is a conserved, early developmental mode found in several phyla of Lophotrochozoans resulting in highly diverse adult body plans. While the cleavage pattern has clearly been broadly conserved, it has also undergone many modifications in various taxa. The precise mechanisms of how different adaptations have altered the ancestral spiral cleavage pattern are an important ongoing evolutionary question, and adequately answering this question requires obtaining a broad developmental knowledge of different spirally cleaving taxa. In flatworms (Platyhelminthes), the spiral cleavage program has been lost or severely modified in most taxa. Polyclad flatworms, however, have retained the pattern up to the 32-cell stage. Here we study early embryogenesis of the cotylean polyclad flatworm Maritigrella crozieri to investigate how closely this species follows the canonical spiral cleavage pattern and to discover any potential deviations from it.ResultsUsing live imaging recordings and 3D reconstructions of embryos, we give a detailed picture of the events that occur during spiral cleavage in M. crozieri. We suggest, contrary to previous observations, that the four-cell stage is a product of unequal cleavages. We show that that the formation of third and fourth micromere quartets is accompanied by strong blebbing events; blebbing also accompanies the formation of micromere 4d. We find an important deviation from the canonical pattern of cleavages with clear evidence that micromere 4d follows an atypical cleavage pattern, so far exclusively found in polyclad flatworms.ConclusionsOur findings highlight that early development in M. crozieri deviates in several important aspects from the canonical spiral cleavage pattern. We suggest that some of our observations extend to polyclad flatworms in general as they have been described in both suborders of the Polycladida, the Cotylea and Acotylea.

Cloning in livestock agriculture

A tremendous amount is required of the oocyte cytoplasm to reprogramme a differentiated donor nucleus after somatic cell nuclear transfer so that it re-acquires a state of totipotency and can form a cloned individual. These reprogramming events must occur in a relatively short period after embryo reconstruction, quite unlike the situation during gametogenesis. It is remarkable that nuclear transfer can produce physiologically normal animals, but the process is highly prone to epigenetic errors. Aberrant patterns of gene expression are believed to contribute to the cumulative losses and abnormal phenotypes observed throughout development, from embryonic to adult stages. As a consequence, cloning efficiencies with current nuclear transfer technology are low and limit practical applications in farming. These applications depend upon the reliability with which nuclear transfer can reproduce specific genotypes and phenotypes in resulting offspring. Present animal welfare issues are a significant barrier for acceptance of nuclear transfer in agriculture. Any long lasting effects from cloning, as revealed in some mouse studies, need to be comprehensively evaluated in cloned livestock. The safety of food products derived from clones also needs to be thoroughly evaluated. The current indication that the sexually derived offspring of nuclear transfer clones are normal provides confidence for the first applications of the technology in agriculture, namely the generation of cloned, genetically elite animals for subsequent breeding. Future improvements in cloning will come from both technical advances and a greater fundamental understanding of the molecular mechanisms of reprogramming with an aim to control this process better. Increased efficiencies should improve the acceptability and utility of cloning technology.

Nuclear transfer from somatic cells: applications in farm animal species

The reconstruction of mammalian embryos by transfer of a blastomere nucleus to an enucleated oocyte or zygote allows for the production of genetically identical individuals. This has advantages for research (that is, as biological controls) and commercial applications (that is, multiplication of genetically valuable livestock). However, the number of offspring that can be produced from a single embryo is limited both by the number of blastomeres (embryos at the 32-64-cell stage are the most widely used in farm animal species) and the limited efficiency of the nuclear transfer procedure. The ability to produce live offspring by nuclear transfer from cells that can be propagated and maintained in culture offers many advantages, including the production of many identical offspring over an extended period (since cultured cells can be frozen and stored indefinitely) and the ability to modify genetically or to select populations of cells of specific genotypes or phenotypes before embryo reconstruction. This objective has been achieved with the production of lambs using nuclei from cultured cells established from embryonic, fetal and adult material. In addition, lambs transgenic for human factor IX have been produced from fetal fibroblasts transfected and selected in culture.

Reprogramming the genome: role of the cell cycle

In nuclear transfer reconstructed embryos, the co-ordination of donor nuclear and recipient cytoplasmic cell cycle phases is essential to maintain ploidy and prevent DNA damage. However, the stage of the cell cycle at the time of reconstruction and the method of reconstruction may also have a significant impact on the subsequent development of the embryo and fetus through a number of other mechanisms. This paper reviews some of the information currently available and proposes that consideration of the cell cycle may lead to improvement of methods for embryo reconstruction.

Sach-Gaurav:World’s First Cloned Buffalo Born In The Field At An Indian Dairy Farm

Recently, the ICAR-Central Institute for Research on Buffaloes has produced world’s first cloned buffalo in the field, named Sach-Gaurav, on 22 December 2017. In addition to being the world’s first cloned buffalo born in the field, this is also India’s first cloned calf of Assamese buffalo; earlier only Murrah breed of buffaloes was cloned. A small tail tissue of Assamese buffalo was airlifted from the College of Veterinary Science, Guwahati (which is 2000 km away from our cloning laboratory) to establish the cultures of donor cells. This proves that distant and remote animals can be cloned. A singlet method of embryo reconstruction has been used, with one recipient oocyte instead of two (a standard Indian version of animal cloning method). It is a step towards simplification of animal cloning technique, and this approach can reduce the mitochondrial heteroplasmy in cloned calves. Ovaries of Murrah buffalo were used as a source of recipient oocytes, indicating that oocytes from one breed of buffaloes can reprogramme donor cells of other breeds. Also, the Murrah buffalo can serve as a surrogate mother for other buffalo breeds.

High Pregnancy and Calving Rates with a Limited Number of Transferred Handmade Cloned Bovine Embryos.

A major obstacle of widespread commercial application of bovine somatic cell nuclear transfer is the low overall efficiency, that is, healthy calf-late pregnancy per transferred embryo rate. In this study, we report a series of experiments with a limited number of embryos created with handmade cloning (HMC) and transferred without or after open pulled straw vitrification. Embryo reconstruction was performed by using in vitro matured oocytes and adult ear skin fibroblasts. In two experiments, a total of 53 D7 blastocysts were developed from 188 reconstructed embryos. Fresh transfer of seven blastocysts into six recipients has resulted in three early pregnancies, two of them developed over 90 days and eventually resulted in healthy calves (33% offspring/transfer rate). In another two experiments, a total of 11 D7 blastocysts were obtained from 36 reconstructed embryos. Out of these, eight have reexpanded 18 hours after vitrification and warming. Transfer of these blastocysts into eight recipients has resulted in four early pregnancies and two live births; 25% offspring/transfer rate. These results indicate that low overall efficiency may not be an intrinsic feature of cattle cloning, and selection of the right procedures may help to overcome the actual limitations.

Effects of oocyte source, cell origin, and embryo reconstruction procedures on in vitro and in vivo embryo survival after goat cloning

Effects of oocyte source, cell origin, and embryo reconstruction procedures on in vitro and in vivo embryo survival after goat cloning

62 BOVINE OCT4 (POU5F1) KNOCKOUT EMBRYOS FAIL DURING THE SECOND LINEAGE DIFFERENTIATION DUE TO LOSS OF NANOG

We generated a CRISPR/Cas9 mediated knockout (KO) of the OCT4 gene in adult fibroblasts, where biallelic deletion of a single nucleotide leads to a frameshift mutation. Through reconstruction of embryos by somatic cell NT, we were able to study the role of OCT4 during pre-implantation development until Day 7 in vitro. The presence of OCT4 protein was evaluated after immunofluorescent staining by confocal laser scanning microscopy of Day 5 morulae and Day 7 blastocysts; somatic cell NT embryos reconstructed from nontransfected cells of the same source served as control. Whereas control morulae expressed OCT4 in all cells, OCT4 KO morulae showed expression in only 67.8 ± 11.1% (mean ± SD, n = 6) of cells and overall intensity was decreased. By Day 7, no expression of OCT4 was detected in OCT4 KO blastocysts (n = 24), suggesting that maternal stores of the OCT4 protein had decayed. In contrast, control blastocysts (n = 20) showed OCT4 expression ubiquitously in both inner cell mass (ICM) and trophectoderm (TE). Simultaneously to the OCT4 staining, we differentially stained ICM and TE with the TE specific marker CDX2 and counterstained cell nuclei with 4′,6-diamidino-2-phenylindole. No significant differences between OCT4 KO Day 7 blastocysts and controls were detected in total cell numbers (89.6 ± 27.5 v. 96.3 ± 38) and percentage of CDX2 positive cells (50.7 ± 16.8% v. 59.0 ± 20.8%) (P > 0.05, mean ± SD, unpaired, two-tailed t-test). To analyse the role of OCT4 during the second lineage differentiation, we stained Day 5 morulae and Day 7 blastocysts for the epiblast and hypoblast specific markers NANOG and GATA6, respectively. In morulae, both markers were present and co-expressed in OCT4 KO and control embryos. By Day 7, control blastocysts (n = 6) already showed the typical salt and pepper distribution of NANOG and GATA6 positive cells, but expression was not mutually exclusive in all cells and also not restricted to ICM. OCT4 KO embryos lost all NANOG expression at Day 7 blastocyst stage (n = 8) and only stained positive for GATA6 in both TE and ICM. We conclude that OCT4 is not required for the quantitative allocation of cells to either the ICM or the TE during the first lineage differentiation, as total cell number and percentage of CDX2 positive cells was unchanged. Additionally, expression of NANOG seems to be OCT4 dependent and OCT4 KO embryos fail to establish the epiblast lineage—unlike mouse Oct4 KO embryos, where developmental failure was connected to loss of GATA6 expression during second lineage differentiation. This work was funded by the Bavarian Research Foundation (AZ-1031–12).

Tomographic imaging of transparent biological samples using the pyramid phase microscope.

We show how a pyramid phase microscope can be used to obtain tomographic information of the spatial variation of refractive index in biological samples using the Radon transform. A method that uses the information provided by the phase microscope for axial and lateral repositioning of the sample when it rotates is also described. Its application to the reconstruction of mouse embryos in the blastocyst stage is demonstrated.