[Application of the blastomere count variations "skip value" in the embryo AI assessment].

Objective: To explore the correlation between blastomere count variations "skip value" which extracted from by time-lapse technology (TLT) combined with artificial intelligence (AI) and morphological features of in vitro fertilization (IVF) embryo, and to test its feasibility in clinical applications. Methods: This study was a diagnostic experiment (AI reassessment of embryo transferred patients), a total of 6 545 embryos from 1 226 patients who underwent IVF at the Women and Children's Hospital of Chongqing Medical University from December 2020 to December 2021 were retrospectively analyzed, of which 2 869 embryos were attempted to cultured to blastocyst stage by TLT. The embryo dynamic map (EDM) was drawn by Embryo Viewer, a TLT recording software, based on embryo developmental kinetics. The self-developed AI embryo evaluation software identified and recorded the number of cleavages in real time during embryonic development, and compared with the EDM, the correlation between the skip value formed by the change of cleavage sphere counts and the outcomes of the embryos was analyzed. The correlation among skip value, morphological score of embryo, implantation rate and live birth rate were performed by Spearman and step-up logistic regression. The receiver operating characteristic (ROC) curve was selected for reporting there relationship of skip value and morphology. Finally, predicting power of skip value for implantation and live birth rate were performed by ROC analysis. Results: The total skip values extracted from the blastomere count of embryos (72 hours post-fertilization) were negatively correlated with abnormal cleavage, blastocyst formation rate, day 3 (D3)-cell score, uneven size and fragmentation (the ß values were -0.268, -0.116, -0.213, -0.159 and -0.222, respectively; all P<0.001); positively correlated with D3-cell number (ß=0.034; P<0.001); negatively correlated with blastocyst formation rate and implantation rate (OR=0.97, 95%CI: 0.93-0.99, P=0.034; OR=0.96, 95%CI: 0.93-0.98, P=0.044). The power of predicting implantation were similar between the order selection of skip values and traditional morphology criteria [area under curve (AUC): 0.679 vs 0.620]. Live birth rate were negatively correlated with female age (OR=0.91, 95%CI: 0.88-0.93; P<0.001), D3 general score (OR=0.77, 95%CI: 0.59-0.99; P=0.045) and order selection of skip values (OR=0.98, 95%CI: 0.96-0.99; P=0.038), while positively correlated with retrieved oocyte number and endometrial thickness in embryo transferred (OR=1.08, 95%CI:1.05-1.11, P<0.001; OR=1.09, 95%CI:1.06-0.12, P<0.001, respectively) from multivariate regression analysis, and the power of predicting live birth was 0.666 for AUC. Conclusions: The skip value and its order form is a systematic quantification of embryo development, correlated with embryo developmental quality and clinical outcome. It could be an addition parameter for embryo culture and selection.

In vitro developmental competence of bovine demi-embryos generated by blastomere separation and blastocyst bisection.

The efficiency of bovine in vitro embryo production can be significantly improved by splitting embryos at different stages. However, the blastocyst quality of in vitro-produced demi-embryos remains unexplored. The objective of this research was to compare embryo developmental rates and quality of bovine demi-embryos produced by two different strategies: (a) embryo bisection (BSEC) and (b) 2-cell blastomere separation (BSEP). To determine demi-embryos quality, we evaluated total blastocyst cell number and proportion of SOX2+ cells. Additionally, the expression of SOX2, NANOG, OCT4, CDX2, IFNT, BAX and BCL genes and let-7a and miRNA-30c Micro RNAs was analysed. BSEP resulted in improved blastocyst development, higher ICM cells and a significantly higher expression of IFNΤ than demi-embryos produced by BSEC. Let-7a, which is associated with low pregnancy establishment was detected in BSEC, while miRNA-30c expression was observed in all treatments. In conclusion, BSEP of 2-cell embryos is more efficient to improve in vitro bovine embryo development and to produce good quality demi-embryos based on ICM cell number and the expression pattern of the genes explored compared to BSEC.

Efficiency of embryo complementation and pluripotency maintenance following multiple passaging of in vitro-derived bovine embryos.

Context Current methods to obtain bovine embryos of high genetic merit include approaches that require skilled techniques for low-efficiency cloning strategies. Aims The overall goal herein was to identify the efficacy of alternative methods for producing multiple embryos through blastomere complementation while determining maintenance of cell pluripotency. Methods Bovine oocytes were fertilised in vitro to produce 4-cell embryos from which blastomeres were isolated and cultured as 2-cell aggregates using a well-of-the-well system. Aggregates were returned to incubation up to 7days (Passage 1). A second passage of complement embryos was achieved by splitting 4-cell Passage 1 embryos. Passaged embryos reaching the blastocyst stage were characterised for cell number and cell lineage specification in replicate with non-reconstructed zona-intact embryos. Key results Passage 1 and 2 embryo complements yielded 29% and 25% blastocyst development, respectively. Passage 1 embryos formed blastocysts, but with a reduction in expression of SOX2 and decreased size compared to non-reconstructed zona-intact embryos. Passage 2 embryos had a complete lack of SOX2 expression and a reduction in transcript abundance of SOX2 and SOX17, suggesting loss of pluripotency markers that primarily affected inner cell mass (ICM) and hypoblast formation. Conclusions In vitro fertilised bovine embryos can be reconstructed with multiple passaging to generate genetically identical embryos. Increased passaging drives trophectoderm cell lineage specification while compromising ICM formation. Implications These results may provide an alternative strategy for producing genetically identical bovine embryos through blastomere complementation with applications towards the development of trophoblast and placental models of early development.

BF170 hydrochloride enhances the emergence of hematopoietic stem and progenitor cells.

Generation of hematopoietic stem and progenitor cells (HSPCs) ex vivo and in vivo, especially the generation of safe therapeutic HSPCs, still remains inefficient. In this study, we have identified compound BF170 hydrochloride as a previously unreported pro-hematopoiesis molecule, using the differentiation assays of primary zebrafish blastomere cell culture and mouse embryoid bodies (EBs), and we demonstrate that BF170 hydrochloride promoted definitive hematopoiesis in vivo. During zebrafish definitive hematopoiesis, BF170 hydrochloride increases blood flow, expands hemogenic endothelium (HE) cells and promotes HSPC emergence. Mechanistically, the primary cilia-Ca2+-Notch/NO signaling pathway, which is downstream of the blood flow, mediated the effects of BF170 hydrochloride on HSPC induction in vivo. Our findings, for the first time, reveal that BF170 hydrochloride is a compound that enhances HSPC induction and may be applied to the ex vivo expansion of HSPCs.

Capturing totipotency in human cells through spliceosomal repression.

The cleavage of zygotes generates totipotent blastomeres. In human 8-cell blastomeres, zygotic genome activation (ZGA) occurs to initiate the ontogenesis program. However, capturing and maintaining totipotency in human cells pose significant challenges. Here, we realize culturing human totipotent blastomere-like cells (hTBLCs). We find that splicing inhibition can transiently reprogram human pluripotent stem cells into ZGA-like cells (ZLCs), which subsequently transition into stable hTBLCs after long-term passaging. Distinct from reported 8-cell-like cells (8CLCs), both ZLCs and hTBLCs widely silence pluripotent genes. Interestingly, ZLCs activate a particular group of ZGA-specific genes, and hTBLCs are enriched with pre-ZGA-specific genes. During spontaneous differentiation, hTBLCs re-enter the intermediate ZLC stage and further generate epiblast (EPI)-, primitive endoderm (PrE)-, and trophectoderm (TE)-like lineages, effectively recapitulating human pre-implantation development. Possessing both embryonic and extraembryonic developmental potency, hTBLCs can autonomously generate blastocyst-like structures in vitro without external cell signaling. In summary, our study provides key criteria and insights into human cell totipotency.

The first two blastomeres contribute unequally to the human embryo.

Retrospective lineage reconstruction of humans predicts that dramatic clonal imbalances in the body can be traced to the 2-cell stage embryo. However, whether and how such clonal asymmetries arise in the embryo is unclear. Here, we performed prospective lineage tracing of human embryos using live imaging, non-invasive cell labeling, and computational predictions to determine the contribution of each 2-cell stage blastomere to the epiblast (body), hypoblast (yolk sac), and trophectoderm (placenta). We show that the majority of epiblast cells originate from only one blastomere of the 2-cell stage embryo. We observe that only one to three cells become internalized at the 8-to-16-cell stage transition. Moreover, these internalized cells are more frequently derived from the first cell to divide at the 2-cell stage. We propose that cell division dynamics and a cell internalization bottleneck in the early embryo establish asymmetry in the clonal composition of the future human body.

Should we should consider day 3 blastomere number during single vitrified-warmed blastocyst transfer cycle? A retrospective study.

The present study investigated whether day 3 blastomere number has an effect on the clinical outcomes during single vitrified-warmed blastocyst transfer cycles. A total of 3294 vitrified-warmed single day 5 blastocyst transferred cycles were analyzed in this retrospective study from January 2018 to December 2021. The cycles were divided into ≥ 7 and < 7 blastomere groups depending on the day 3 embryo blastomere number. The clinical outcomes were compared between the two groups, moreover multivariate logistic regression analysis was conducted to investigate the correlation between the number of day 3 blastomeres and clinical outcomes. The chi-square test demonstrated that the rates of clinical pregnancy and live birth were significantly higher in the ≥ 7 blastomere group compared to the < 7 blastomere group with respect to single high-quality blastocyst transfer cycles. Conversely, these rates were similar in the two groups with respect to single low-quality blastocyst transfer cycles. These results were confirmed by multivariate logistic regression analysis. However, the miscarriage rate was higher in the < 7 blastomere group than in ≥ 7 group during low-quality blastocyst transfer cycles. These results suggested that day 3 blastomere number should be considered during single vitrified-warmed blastocyst transfer cycles. Thus, blastocsyts derived from ≥ 7 blastomere embryos are preferred when choosing the same quality blastocysts.

A clinical consensus-compliant deep learning approach to quantitatively evaluate human in vitro fertilization early embryonic development with optical microscope images.

The selection of embryos is a key for the success of in vitro fertilization (IVF). However, automatic quality assessment on human IVF embryos with optical microscope images is still challenging. In this study, we developed a clinical consensus-compliant deep learning approach, named Esava (Embryo Segmentation and Viability Assessment), to quantitatively evaluate the development of IVF embryos using optical microscope images. In total 551 optical microscope images of human IVF embryos of day-2 to day-3 were collected, preprocessed, and annotated. Using the Faster R-CNN model as baseline, our Esava model was constructed, refined, trained, and validated for precise and robust blastomere detection. A novel algorithm Crowd-NMS was proposed and employed in Esava to enhance the object detection and to precisely quantify the embryonic cells and their size uniformity. Additionally, an innovative GrabCut-based unsupervised module was integrated for the segmentation of blastomeres and embryos. Independently tested on 94 embryo images for blastomere detection, Esava obtained the high rates of 0.9940, 0.9121, and 0.9531 for precision, recall, and mAP respectively, and gained significant advances compared with previous computational methods. Intraclass correlation coefficients indicated the consistency between Esava and three experienced embryologists. Another test on 51 extra images demonstrated that Esava surpassed other tools significantly, achieving the highest average precision 0.9025. Moreover, it also accurately identified the borders of blastomeres with mIoU over 0.88 on the independent testing dataset. Esava is compliant with the Istanbul clinical consensus and compatible to senior embryologists. Taken together, Esava improves the accuracy and efficiency of embryonic development assessment with optical microscope images.

External bending of cryodevice during vitrification leads to cryoprotectant cracks and damage to embryo blastomeres.

RESEARCH QUESTION: Embryo blastomeres and the zona pellucida are occasionally damaged during vitrification; is this a result of crack-induced mechanical damage in the glass state, caused by external bending of the device? DESIGN: A stereomicroscope was used to observe external bending-induced cracks in a cryoprotectant. Thereafter, 309 human cleavage-stage embryos derived from abnormally fertilized eggs were used to assess embryo damage under two external bending conditions: forward bending and backward bending, with three bending degrees applied. Three distinct embryo positions were used to examine the correlation between bending and embryo damage. Damage was assessed by looking at blastomere lysis rates, and overall rates of damaged and surviving embryos. RESULTS: A series of parallel cracks were identified in the cryoprotectant used for external bending, which led to damage to the embryo blastomeres. Compared with forward bending and control, the embryos were found to be more easily damaged by backward bending, indicated by significantly higher blastomere lysis and embryo damage rates, and lower embryo survival rate of backward bending than forward bending (P < 0.001). The degree of embryo damage also increased as the degree of external forces increased. Embryo position correlated with degree of embryo damage. CONCLUSIONS: Cryoprotectant crack-induced damage was identified as the cause of embryo damage. Mechanical damage to the glass state occurs because of improper external bending of the cryodevice strip in liquid nitrogen during vitrification. To prevent damage, bending of the strip should be avoided and the embryos should be placed near the tip of the strip.

Manipulation of Embryonic Cleavage Geometry Using Magnetic Tweezers.

The geometry of reductive divisions that mark the development of early embryos instructs cell fates, sizes, and positions, by mechanisms that remain unclear. In that context, new methods to mechanically manipulate these divisions are starting to emerge in different model systems. These are key to develop future innovative approaches and understand developmental mechanisms controlled by cleavage geometry. In particular, how cell cycle pace is regulated in rapidly reducing blastomeres and how fate diversity can arise from blastomere size and position within embryos are fundamental questions that remain at the heart of ongoing research. In this chapter, we provide a detailed protocol to assemble and use magnetic tweezers in the sea urchin model and generate spatially controlled asymmetric and oriented divisions during early embryonic development.

Nr5a2 ensures inner cell mass formation in mouse blastocyst.

Recent studies have elucidated Nr5a2's role in activating zygotic genes during early mouse embryonic development. Subsequent research, however, reveals that Nr5a2 is not critical for zygotic genome activation but is vital for the gene program between the 4- and 8-cell stages. A significant gap exists in experimental evidence regarding its function during the first lineage differentiation's pivotal period. In this study, we observed that approximately 20% of embryos developed to the blastocyst stage following Nr5a2 ablation. However, these blastocysts lacked inner cell mass (ICM), highlighting Nr5a2's importance in first lineage differentiation. Mechanistically, using RNA sequencing and CUT&Tag, we found that Nr5a2 transcriptionally regulates ICM-specific genes, such as Oct4, to establish the pluripotent network. Interference with or overexpression of Nr5a2 in single blastomeres of 2-cell embryos can alter the fate of daughter cells. Our results indicate that Nr5a2 works as a doorkeeper to ensure ICM formation in mouse blastocyst.

Generation of allogenic chimera carrying mutations in PDX1 and TP53 genes via phytohemagglutinin-mediated blastomere aggregation in pigs.

The generation of genetically engineered pig models that develop pancreas-specific tumors has the potential to advance studies and our understanding of pancreatic cancer in humans. TP53 mutation causes organ-nonspecific cancers, and PDX1-knockout results in the loss of pancreas development. The aim of the present study was to generate a PDX1-knockout pig chimera carrying pancreas complemented by TP53 mutant cells via phytohemagglutinin (PHA)-mediated blastomere aggregation using PDX1 and TP53 mutant blastomeres, as a pig model for developing tumors in the pancreas with high frequency. First, the concentration and exposure time to PHA to achieve efficient blastomere aggregation were optimized. The results showed that using 300 µg/mL PHA for 10 min yielded the highest rates of chimeric blastocyst formation. Genotyping analysis of chimeric blastocysts derived from aggregated embryos using PDX1- and TP53-edited blastomere indicated that approximately 28.6% carried mutations in both target regions, while 14.3-21.4% carried mutations in one target. After the transfer of the chimeric blastocysts into one recipient, the recipient became pregnant with three fetuses. Deep sequencing analysis of the PDX1 and TP53 regions using ear and pancreas samples showed that one fetus carried mutations in both target genes, suggesting that the fetus was a chimera derived from embryo-aggregated PDX1 and TP53 mutant blastomeres. Two out of three fetuses carried only the PDX1 mutation, indicating that the fetuses developed from embryos not carrying TP53-edited blastomeres. The results of the present study could facilitate the further improvement and design of high-frequency developing pancreatic tumor models in pigs.

Heterogeneity of nucleolar morphology in four-cell mouse embryos after IVF: association with developmental potential.

In mammals, around fertilization, the nucleolus of embryos transforms into the nucleolus precursor bodies (NPBs), which continue to mature until the blastocyst stage, leading to distinct morphological changes. In our study, we observed two types of nucleolar morphology in mouse in vitro fertilized embryos at the four-cell stage, which we refer to single nucleolus (SN) and multiple nucleoli (MN). To visualize nucleolar morphology, four-cell embryos were immunostained with anti-NOPP140 antibody. These embryos were categorized into five types based on the number of blastomeres carrying SN: SN4/MN0, SN3/MN1, SN2/MN2, SN1/MN3, and SN0/MN4, with percentages of 13, 27, 21, 23 and 9, respectively. Next, using a light microscope, we divided the four-cell in vitro fertilized embryos without fixation into two groups: those with at least two blastomeres displaying SN (SN embryos) and those without (MN embryos). Notably, significantly more SN embryos developed into blastocysts and offspring at 18.5 dpc compared with MN embryos. Furthermore, SN embryos displayed a higher NANOG-positive cell number at the blastocyst stage, significantly lower body and placental weights, resulting in a higher fetal/placental ratio. These findings suggest a close association between nucleolar state at the four-cell stage and subsequent developmental potential.

Induced formation of primordial germ cells from zebrafish blastomeres by germplasm factors.

The combination of genome editing and primordial germ cell (PGC) transplantation has enormous significance in the study of developmental biology and genetic breeding, despite its low efficiency due to limited number of donor PGCs. Here, we employ a combination of germplasm factors to convert blastoderm cells into induced PGCs (iPGCs) in zebrafish and obtain functional gametes either through iPGC transplantation or via the single blastomere overexpression of germplasm factors. Zebrafish-derived germplasm factors convert blastula cells of Gobiocypris rarus into iPGCs, and Gobiocypris rarus spermatozoa can be produced by iPGC-transplanted zebrafish. Moreover, the combination of genome knock-in and iPGC transplantation perfectly resolves the contradiction between high knock-in efficiency and early lethality during embryonic stages and greatly improves the efficiency of genome knock-in. Together, we present an efficient method for generating PGCs in a teleost, a technique that will have a strong impact in basic research and aquaculture.

Biópsia embrionária: qual a melhor escolha? / Embryo biopsy: what is the best choice?

Introdução: a biópsia embrionária tem como objetivo selecionar embriões geneticamente normais. Essa seleção ocorre por meio de testes genéticos pré-implantacionais. Espera-se, com isso, uma diminuição dos riscos de doenças genéticas e um aumento das taxas de implantação em fertilização in vitro. Objetivo: verificar, por meio de revisão bibliográfica, qual técnica de biópsia embrionária é considerada mais apropriada para feitura de testes genéticos pré-implantacionais. Método: pesquisa bibliográfica, na forma de revisão de publicações científicas, por meio das redes US National Library of Medicine (Pubmed), Literatura Latino-Americana e do Caribe em Ciências da Saúde (Lilacs), Google Acadêmico e Biblioteca Virtual em Saúde (BVS). Resultados e conclusão: existem três maneiras de efetuar a biópsia para reprodução humana assistida. A primeira consiste em retirar o primeiro e/ou o segundo corpúsculo polar estruído pelo oócito. Também se pode fazer a biópsia a partir de um blastômero do embrião em estágio de clivagem ou usar cinco a dez células do trofoectoderma de blastocisto. Normalmente as técnicas usadas para o diagnóstico são PCR, Fish, CGH array e SNP array, entre outras. Acredita-se que a biópsia de blastocistos é a melhor técnica para manter o potencial de implantação embrionária. Essa tendência se justifica por causa da maior quantidade de material genético disponível em fase avançada de desenvolvimento embrionário. Admite-se que nessa fase a incidência de mosaicismo seja menor em relação à biópsia de blastômeros, com consequente aumento na eficácia dos testes genéticos. Outra questão importante é que na biópsia de blastocistos as células são retiradas do trofoectoderma, enquanto que na biópsia em estágio de clivagem a remoção de um blastômero pode prejudicar o desenvolvimento embrionário.

Introduction: the embryo biopsy aims to select genetically normal embryos. This selection occurs through pre- implantation genetic testing. It is expected the reduction of risk ofgenetic disorders and increase implantation rates in IVF.Objective: to verify, through bibliographical revision, which embryo biopsy technique is considered more suitable for pre-implantation genetic diagnosis. Method: bibliographical research, in the form of literary review of scientific publications via networks, US National Library of Medicine (Pubmed), Latin-American Literature and Caribbean Health Sciences (Lilacs), Google Scholar and Virtual Health Library. Results and conclusion: there are three ways to perform the biopsy on assisted human reproduction.The first one consists in removing the 1st and/or 2nd polar body (if there wasfertilization). You can also perform the biopsy from the one blastomere of embryo cleavage stage or use 5-10 trophoectoderm cells blastocyst. Usually the techniques used for diagnosticpurpose are PCR, Fish, CGH array, SNP array and others. Nowadays it is believed that blastocyst biopsy is the best technique in order to maintain the embryonic implantation. This tendency is justified by the larger amount of genetic material available in an advancedstage of embryonic development. It is assumed that in this stage the incidence of mosaicism is reduced with the consequent increase in the effectiveness of genetic testing. Another important question is that the blastocyst biopsy cells are removed from the trophoectoderm while inbiopsy incleavage stage, the removal of one blastomere can impair embryonicdevelopment.

Perspectivas de uso da hibridizacão genômica comparativa como rastreamento pré-implantacional em biópsias de embrião humano no estágio de blastocisto / Prospects of using comparative genomic hybridization in blastocyst stage human embryo biopsy as pre-implantation genetic screening

O rastreamento genético pré-implantacional parece melhorar as taxas de gravidez em certos grupos de pacientes com mau prognóstico nos tratamentos de fertilização in vitro. Mais recentemente tem-se sugerido seu uso para a seleção visando à transferência de um embrião único. O presente artigo tem como objetivo discorrer sobre a técnica de hibridização genômica comparativa, a biópsia do embrião na fase de blastocisto e as perspectivas de uso desses como rastreamento genético pré-implantacional. Foi feita revisão bibliográfica em artigos científicos, livros e periódicos. O método de hibridização genômica comparativa é capaz de analisar todos os pares cromossômicos e mostra-se promissor na identificação das aneuploidias. A biópsia do embrião na fase de blastocisto parece ser menos agressiva e fornece mais material genético do que aquela feita na fase de clivagem. A transferência de um embrião geneticamente normal aumenta as chances de gravidez nos procedimentos de fertilização in vitro. Entretanto, ainda não temos estudos randomizados controlados suficientes para assegurar que a hibridização genômica comparativa em blastocisto tenha impacto prático suficiente para aumentar as chances de gravidez.

Pre-implantation genetic screening could improve pregnancy rates in certain groups of patients with poor prognosis undergoing in vitro fertilization procedures. Recently its use as screening has been suggested aiming single embryo transfers. This paper evaluates comparative genomic hybridization technique, blastocyst stage embryo biopsy and analyzes the prospects of using both as pre-implantation genetic screening. A bibliographical review in scientific articles, books, journals and websites was done. Comparative genomic hybridization is able to analyze all chromosomes being promising as pre-implantation genetic screening. Blastocyst biopsy seems to be less aggressive to the embryo and provides additional genetic material compared to cleavage stage biopsies. The transfer of a genetically normal embryo increases pregnancy rates in in vitro fertilization procedures. However there are insufficient randomized controlled trials to ensure that comparative genomic hybridization in blastocysts cells has a practical impact in pregnancy rates.

Desarrollo embrionario del capaz pimelodus grosskopfii (Steindachner, 1879) / Embryonic development of capaz pimelodus grosskopfii (Steindachner, 1879)

El conocimiento del desarrollo embrionario en los peces es especialmente importante en especies nativas con potencial para la piscicultura, en virtud que permite identificar eventos morfológicos y cronológicos, necesarios para establecer prácticas de manejo durante las fases de incubación y larvicultura. El capaz (Pimelodus grosskopfii) es una especie con potencial para cultivo comercial, por sus hábitos alimenticios omnívoros y aceptación de su carne en el mercado. Para estudiar el desarrollo embrionario de la especie, ejemplares adultos sexualmente maduros fueron inducidos a la reproducción con extracto de hipófisis de carpa (5,75 y 4,0 mg Kg-1, hembras y machos, respectivamente). Los óvulos seminados fueron incubados en un sistema de flujo ascendente de 30 L a 27 +/- 1 °C. Las muestras (n=30) fueron colectadas al momento de la extrusión, durante la fertilización y cada 15 minutos a partir de las 0 horas postfertilización (HPF) hasta las 2 horas y cada 30 minutos desde las 2 HPF hasta 5 HPF; finalmente, entre las 5 HPF y la eclosión, cada 60 minutos. Los óvulos fertilizados presentaron forma esférica, sin adherencias y con amplio espacio perivitelino. El desarrollo embrionario finalizó a las 12 HPF. La diferenciación del polo animal y vegetal ocurrió a las 0,2 HPF, el primer clivaje a las 0,3 HPF, el blastodisco alto y estratificado a las 1,8 HPF, el blastodisco achatado a las 3,3 HPF, la epibolia < a 50 por ciento se observó a las 4 HPF, el cierre del blastoporo a las 5,7 HPF, la diferenciación cráneo caudal e inicio de la neurolación a las 7 HPF, la diferenciación de las vesículas ópticas, óticas y vesícula de Kupffer a las 8,5 HPF, la liberación de la cola del vitelo a las 10 HPF, los primeros movimientos se observaron a las 10,5 HPF y finalmente la eclosión ocurrió a las 12 HPF. Las larvas al eclosionar presentaron una longitud total de 2987+/-67 um, sin pigmentación, tracto digestivo rudimentario, sin abertura bucal ni anal y presencia de cromatóforos...

The knowledge of embryonic development in fish is important in native species with potential for fish farming, by virtue of which it makes possible to identify morphological and chronological events to establish management practices during incubation periods and larviculture. The capaz (Pimelodus grosskopfii) is a species with potential for commercial crop, due to their omnivorous eating habits and acceptance of its meat in the market. To study the embryonic development of the species, sexually mature adult specimens were induced to reproduce with carp pituitary extract (5.75 and 4.0 mgKg-1, females and males, respectively). The inseminated oocytes were incubated in an upward flow system 30 a 27 +/- 1 ° C. The samples (n = 30) were collected at the same time of the extrusion, during fertilization, and every 15 minutes starting from 0 to 2 hours post fertilization (HPF) and every 30 minutes from 0 to 2 HPF, and every 30 minutes from 2 to 5 HPF; finally, between 5 HPF and hatching every 60 minutes. The fertilized oocytes had a spherical shape without adhesions and large perivitelline space. Embryonic development took 12 HPF. The differentiation in animal and vegetal pole occurred at 0.2 HPF, the first cleavage at 0.3 HPF, stratified and high blastodisc at 1.8 HPF, flattened blastodisc at 3.3 HPF, the epiboly <50 percent was observed at 4 HPF, the closure of the blastopore at 5.7 HPF, cranial-caudal differentiation and starting the neurolation at 7 HPF, the differentiation of the optic vesicles, otic and Kupffer's vesicle at 8.5 HPF, tail of the vitelum was released at 10 HPF, first movements were observed at 10.5 and finally hatching occurred at 12 HPF. When the larvae hatched, they showed a total length of 2987+/-67 µm, without depigmentation, rudimentary digestive system without oral and anal opening and the presence of chromatophores on the yolk sac.

Developmental rates of bovine nuclear transfer embryos derived from different fetal non transfected and transfected cells

Since the first successful somatic cell nuclear transfer (SCNT) experiments were carried out, a number of domestic and agriculture species have been cloned using donor cells derived from different sources and origin. However, differences in nuclear transfer efficiency both in vitro and in vivo have been generally observed. These differences may be accentuated when transgenic cell lines are used as nuclear donors in an attempt to generate transgenic cloned offspring. The present study examined the suitability of cell lines derived from 3 different fetal sources and the effects of genetic manipulation of donor fetal fibroblasts with a red fluorescent plasmid, on the in vitro developmental potential and quality of nuclear transfer derived bovine embryos. We observed no differences in the cleavage rate of nuclear transfer embryos generated with any of the cell lines evaluated. However, the blastocyst rate was significantly affected when cell lines were derived from the 3 different fetal sources (21, 18 and 11 percent, respectively) or from 2 transgenic clonal cell lines that had originated from the same primary fetal cell (18 and 10 percent, respectively). Despite this difference, quality of embryos as measured by the total number of cells and by assessing some morphology aspects of their appearance was not different. Together these results indicate that fetal fibroblast cell lines derived from different fetal sources and transgenic clonal cell lines that had originated from the same fetus results in different in vitro developmental potential when used as donors for nuclear transfer experiments. Further studies, including evaluation of pregnancy rates, development to term, and epigenetic modifications of these cell lines will be necessary to better understand the differences observed in nuclear transfer efficiency.

Survival of vitrifi ed mouse blastocysts loaded intoglass micro-capillaries / Sobrevivencia embrionaria de blastocistos murinos vitrificados en microcapilares de vidrio / Sobrevivência embrionária de blastocistos murinos vitrificados em micro capilares de vidro

The purpose of this study was to determine the in vitro expansion and hatching rates of vitrified mouseblastocysts loaded into glass micro-capillaries (Brand® - 5 μL). Early morning on day 4 of pregnancy,blastocysts were collected from donors, morphologically evaluated, and then allocated in three groups:Group 1 (Control): embryos were transferred into 100 μL of KSOM medium drops and in vitro culturedduring 72 h; Groups 2 and 3: embryos initially exposed to the equilibration solution (PBSm + 10% EG+ 10% PROH and 0.5% PVA) for 1 min, and then to vitrification solution (PBSm + 20% EG + 20%PROH + 0.5% PVA) for 30 sec. After that, blastocysts were loaded into glass micropipettes (GMP) or glassmicrocapillaries (GMC) and plunged into super-cooled liquid nitrogen (-200 °C). Embryo warming andcryoprotectant dilution were carried out into 500 μL droplets of PBSm supplemented with 0.25 M sucrosemaintained at 37 °C. After 5 min embryos were transferred to 100 mL droplets of KSOM medium andcultured in vitro for 72 h. Blastocyst expansion rates after in vitro culture were 77% (138/177) and 74%(131/175), for blastocysts vitrified in GMP and GMC, respectively. Blastocyst hatching rate (control group)was 91% (134/146), which was higher than for embryos loaded in GMP 61% (108/177) and GMC 53%(93/175). ICM number in control group embryos contained 25.7 ± 2.5 cells and did not differ from themean cell number observed in vitrified embryos loaded in GMP (24.2±2.3) or GMC (22.5±2.59). Regardingthe trophoectoderm cell number, Group 1 embryos displayed 63.1±3.0 cells, and also not differ from the cellnumbers of the embryos loaded into GMP (58.0±1.8) or GMC (58.0±.3.7). In conclusion, manufacturedGMC (Brand®) tested in this study showed same efficiency as GMP for vitrification of mouse blastocysts.

El objetivo de este estudio fue determinar las tazas de expansión y eclosión in vitro de los blastocistosmurinos vitrificados en micro-capilares de vidrio (Brand® - 5 μL). En el día 4 de preñez, los blastocistoseran colectados de las donantes, evaluados morfológicamente y localizados en tres diferentes grupos:Grupo 1 (Control): compuesto por los embriones que eran transferidos a gotas de 100 μL de medio KSOMy cultivados in vitro por un periodo de 72 h; Grupos 2 y 3: compuesto por los embriones que eran expuestosinicialmente a la solución de equilibrio (PBSm + 10% EG + 10% PROH and 0.5% PVA) por 1 min,y posteriormente a la solución de vitrificación (PBSm + 20% EG + 20% PROH + 0.5% PVA) por unperiodo de 30 seg. Posteriormente, los blastocistos, eran almacenados dentro de micro-pipetas de vidrio(GMP) o micro-capilares de vidrio (GMC) y sumergidos en nitrógeno líquido (-200 °C). La dilución delos crioprotectores y desvitrificación de los embriones fue realizada al colocarlos en gotas de 500 μLde PBSm suplementado con 0.25 M de sacarosa a una temperatura de 37 °C. Después de 5 minutos losembriones fueron transferidos a gotas de 100 μL de medio KSOM y cultivados in vitro por 72 h. Las tazasde expansión de los blastocistos, posteriores al cultivo fueron de 77% (138/177) y 74% (131/175), para losblastocistos vitrificados en GMP y GMC, respectivamente. Las tazas de eclosión fueron de 91% (134/146)para el grupo control, siendo mayores que para los embriones vitrificados en GMP 61% (108/177) y GMC53% (93/175). El número del índice de masa celular interna (ICM) para los embriones del grupo controlfue de 25.7 ± 2.5 células, no teniendo diferencia significativa con el número de células observado en losembriones vitrificados en GMP (24.2±2.3) ó GMC (22.5±2.59).

O objectivo de este estudo foi determinar as taxas de expansão e eclosão in vitro dos blastocitos murinosvitrificados em micro capilares de vidro (Brand® - 5 μL). No quarto dia de prenhes, os blastocitos foramcolectados das doadoras, avaliados morfologicamente e localizados em três diferentes grupos: Grupo 1(Controle): composto por os embriões que foram transferidos a gotas de 100 μL de KSOM e cultivados invitro por um período de 72 h; Grupos 2 e 3: composto por os embriões que foram expostos inicialmente ásolução de equilíbrio (PBSm + 10% EG + 10% PROH e 0.5% PVA) por 1 min, e posteriormente á soluçãode vitrificação (PBSm + 20% EG + 20% PROH + 0.5% PVA) por um período de 30 seg. Posteriormente,os blastocistos, foram armazenados dentro de micro pipetas de vidro (GMP) ou micro capilares de vidro(GMC) e submergidos em nitrogénio líquido super-resfriado (-200°C). A diluição dos crioprotetores edesvitrificação dos embriões foi realizada ao colocar-lhos em gotas de 500 μL de PBSm suplementadocom 0.25 M de sacarose a uma temperatura de 37 °C. Depois de 5 minutos, os embriões foram transferidosa gotas de 100 μL de KSOM e cultivados in vitro por 72 h. As taxas de expansão dos blastocistos, posterioresao cultivo foram de 77% (138/177) e 74% (131/175), para os blastocistos vitrificados em GMP e GMC,respectivamente. As taxas de eclosão foram de 91% (134/146) para o grupo controle, e foram maioresos embriões vitrificados em GMP 61% (108/177) e GMC 53% (93/175). O número do índice de massacelular interna (ICM) para os embriões do grupo controle foi de 25.7 ± 2.5 células, não havendo diferenciasignificativa com o número de células observado em embriões vitrificados em GMP (24.2±2.3) ou GMC(22.5±2.59). Alem do mais, as células do trofoectodermo, no grupo controle apresentaram 63.1±3.0células, no sendo diferente às células dos embriões vitrificados em GMP (58.0±1.8) ou GMC (58.0±.3.7).