Search

Embryonic morphofunctional competence features regulating post-cryopreservation resumption of development are still poorly understood. In this study, we investigated the correlation between embryonic viability and the speed and ability to resume post-cryopreservation development. Thus, in vitro produced blastocysts were vitrified by the Cryotop method using standard protocols. Subsequently, the embryos were warmed, re-cultured, and classified into groups according to their speed and ability to resume post-cryopreservation development: embryos not re-expanded at 12h (NE12); embryos re-expanded at 12h and hatched at 24h (E12H24); embryos re-expanded at 12h and hatched at 48h (E12H48); embryos re-expanded at 12h and not hatched at 48h (E12NH48). Subsequently, the embryos were subjected to monitoring of total cell number and apoptosis. We identified that the blastocoel's ability to re-expand was negatively affected by the significant higher percentage of apoptotic cells observed in the NE12 group than in the other groups. A greater (P 0.05) number of total cells, found in groups E12H24 and E12H48, seems to have a positive influence on the hatching capacity of blastocysts after cryopreservation. In conclusion, the total number of cells and apoptotic index correlated with the speed and ability to resume post-cryopreservation development. Apoptosis was a determinant for embryonic re-expansion, and the total cell number was crucial for blastocyst hatching.

Made available in DSpace on 2021-06-25T10:17:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The cryosurvival of embryos is a complex process involving dynamic and integrated morphological, functional, and molecular changes. Here, we evaluated the transcriptional profiling of bovine embryos possessing high and low cryotolerance (HC and LC, respectively) by assessing the resumption of development. Embryos were produced in vitro (N = 1137) and cryopreserved (N = 894). Blastocysts samples possessed pronounced group individualization at RNA sequencing. A total of 114 genes were differentially expressed, and 27 and 84 genes were upregulated in HC and LC, respectively. Among the over-represented biological functions, cellular growth and proliferation, cell death and survival, and organismal survival were predicted to be activated, while cellular movement and cell-to-cell signaling were predicted to be inhibited in HC embryos. Enriched canonical pathways and upstream regulators related to cellular proliferation and survival (HC), inflammatory processes, and cell death (LC) were predicted to represent two embryonic molecular profiles present during the resumption of development after cryopreservation. The marked contrast in transcriptional profiles between HC and LC strongly suggests the influence of embryonic competence after cryopreservation on its respective transcriptome and indicated that HC and LC presented two different molecular strategies to overcome cryopreservation-related stress and resume postcryopreservation development. Center for Natural and Human Sciences Federal University of ABC School of Veterinary Medicine Federal University of Pampa In vitro Brazil – IVB ABS Pecplan Department of Pharmacology Laboratory of Phytomedicines Pharmacology and Biotechnology Institute of Biosciences University of São Paulo State (Unesp) Department of Genetics and Evolutionary Biology University of São Paulo Department of Biological Science School of Sciences and Languages University of São Paulo State Department of Genetics and Evolution Federal University of São Carlos Department of Pharmacology Laboratory of Phytomedicines Pharmacology and Biotechnology Institute of Biosciences University of São Paulo State (Unesp)

The expansion of the use of in vitro production techniques has revolutionized the bovine embryo market. In the last decade, we have seen the number of in vitro produced (IVP) embryos surpass the number of in vivo-derived (IVD) embryos obtained worldwide. Concomitantly, other biotechnologies were also improved, following the global trend. Embryo cryopreservation has received special attention, as it is one of the tools capable of disseminating in vitro production. Currently, two protocols are available: slow freezing and vitrification. Both have advantages and disadvantages regarding their application and, many aspects need to be considered before their use. In this review, we discuss in vitro production market trends, cellular and molecular features involved in embryo response to cryopreservation, and addressed cryo-storage period and embryonic developmental stage on cryosurvival. In addition, we also presented an overview of some aspects that impact the pregnancy rate following transfer of fresh and cryopreserved IVP embryos.

Global cattle genetic market is experiencing a change of strategy, large genetic companies, traditionally recognized in the artificial insemination field, have also begun to operate in the embryo market. Consequently, the demand for in vitro produced (IVP) embryos has grown. However, the overall efficiency of the biotechnology process remains low. Additionally, the lack of homogeneity of post-cryopreservation survival results of IVP embryos still impairing a massive dissemination of this biotechnology in the field. A great challenge for in vitro production labs is to increase the amount of embryos produced with exceptional quality after each round of in vitro fertilization. Herein, we discuss the molecular and cellular features associated with the competence and cryosurvival of IVP embryos. First, morphofunctional, cellular and molecular competence of the embryos were addressed and a relationship between embryo developmental ability and quality were established with cryosurvival and pregnancy success. Additionally, determinant factors of embryo competence and cryosurvival were discussed including the following effects: genotype, oocyte quality and follicular microenvironment, in vitro production conditions, and lipids and other determining molecules. Finally, embryo cryopreservation aspects were addressed and an embryo-focused approach to improve cryosurvival was presented.

This study assessed the lipid composition of oocytes from different follicle sizes and compared the expression of lipid-related genes and follicular fluid (FF) molecules between groups. We also investigated the functional consequences of differences on embryo development and blastocyst lipid deposits. Oocytes and FF were recovered from different follicle sizes. Oocytes from small (≤5mm) and large (≥6mm) bovine follicles were used to produce Day 7 expanded blastocysts (Day7Ex) and blastocysts that only became expanded at Day 8 (Day8Ex) after insemination. Oocytes from >8mm follicles had the highest lipid content. Few oocyte phospholipid variations were identified between groups. Very long chain fatty acid elongase 6 (ELOVL6) mRNA abundance was reduced in larger follicle-derived oocytes compared with the ≤2mm group. Increased levels of glucose, reactive oxygen species, glutathione and superoxide dismutase activity were also identified in FF from larger follicles. Large follicle-derived embryo development and lipid content of Day7Ex were greater than those derived from small follicles. Day8Ex had greater lipid deposition than Day7Ex. Oocytes and blastocysts exhibited follicle size-specific lipids. Large-follicle oocytes had increased lipid content and became Day7Ex with greater lipid deposition whereas delayed blastocoel expansion associated with a prolonged period of culture determined the lipid accumulation of Day8Ex. The FF microenvironment of large follicles seems to favour embryo development.

Mammalian preimplantation embryonic development is a complex, conserved, and well-orchestrated process involving dynamic molecular and structural changes. Understanding membrane lipid profile fluctuation during this crucial period is fundamental to address mechanisms governing embryogenesis. Therefore, the aim of the present work was to perform a comprehensive assessment of stage-specific lipid profiles during early bovine embryonic development and associate with the mRNA abundance of lipid metabolism-related genes (ACSL3, ELOVL5, and ELOVL6) and with the amount of cytoplasmic lipid droplets. Immature oocytes were recovered from slaughterhouse-derived ovaries, two-cell embryos, and eight- to 16-cell embryos, morula, and blastocysts that were in vitro produced under different environmental conditions. Lipid droplets content and mRNA transcript levels for ACSL3, ELOVL5, and ELOVL6, monitored by lipid staining and quantitative polymerase chain reaction, respectively, increased at morula followed by a decrease at blastocyst stage. Relative mRNA abundance changes of ACSL3 were closely related to cytoplasmic lipid droplet accumulation. Characteristic dynamic changes of phospholipid profiles were observed during early embryo development and related to unsaturation level, acyl chain length, and class composition. ELOVL5 and ELOVL6 mRNA levels were suggestive of overexpression of membrane phospholipids containing elongated fatty acids with 16, 18, and 20 carbons. In addition, putative biomarkers of key events of embryogenesis, embryo lipid accumulation, and elongation were identified. This study provides a comprehensive description of stage-specific lipidome signatures and proposes a mechanism to explain its potential relationship with the fluctuation of both cytoplasmic lipid droplets content and mRNA levels of lipid metabolism-related genes during early bovine embryo development.

Mammalian pre-implantation embryonic development is a complex, conserved, and well-orchestrated process involving dynamic molecular and structural changes. Understanding the membrane lipid profile fluctuation during this crucial period is fundamental to address cellular and molecular mechanisms governing embryogenesis. A full understanding of stage-specific lipid signatures in early bovine embryo development is, however, still lacking. The aim of the present work was to characterise stage-specific changes in lipid profiles during early bovine embryonic development. Immature oocytes were recovered from slaughterhouse-derived bovine ovaries and assigned among 5 in vitro developmental stages for lipid characterisation: immature oocytes, 2-cell embryos (32–40 h post-insemination), 8 to 16-cell embryos (72 h post-insemination), morulas (120 h post-insemination), and blastocysts (168 h post-insemination). Two different culture media were used for in vitro embryo production, SOFaaci medium supplemented with 2.5% of serum and serum-free SOF-BE1 medium. Cytoplasmic lipid droplets content and membrane phospholipids profiles for each development stage were assessed by lipid staining (Nile red; n = 5–9) and matrix-assisted laser desorption ionization as a mass spectrometry imaging (MALDI-MS; n = 5–9), respectively. For statistical analysis, univariate and multivariate models were used to compare lipid droplets content and membrane phospholipids profiles. Cytoplasmic lipid droplets content increased from minimum in the immature oocyte stage to maximum at the morula stage, followed by a sharp drop at the blastocyst stage (58.4 ± 10.5ac, 62.5 ± 9.4ac, 85.9 ± 8.2a, 148.3 ± 7.4b, 37.4 ± 9.9c of fluorescence intensity per embryo area, respectively, for immature oocyte, 2-cells, 8 to 16-cells, morulas, and blastocysts; abcP

The cryopreservation of in vitro-produced (IVP) bovine embryos is one of the most challenging areas of the assisted reproductive biotechnologies. The aim of the present study was to evaluate the global gene expression pattern of Bos indicus (Nellore) and Bos taurus (Simmental) IVP embryos after vitrification. Follicular aspiration was performed on Nellore (n = 14) and Simmental (n = 14) cows, and oocytes (n = 840 and 450; respectively) were submitted to in vitro maturation and in vitro fertilization. Presumptive zygotes were denuded and cultured in SOFaa with 0.5% BSA and 2.5% FCS during 7 days under standard culture conditions. Blastocysts (grade 1 and 2) were vitrified, warmed, and cultured for an additional 12 h under the same conditions. Nellore (n = 8) and Simmental (n = 8) IVP blastocysts considered viable after vitrification, with re-expanded blastocoel, were submitted to total RNA extraction (PicoPure, Arcturus, Applied Biosystems®, Foster Dity, CA, USA), DNAse I treatment (Qiagen®, Valencia, CA, USA), and amplification (RiboAmp, Applied Biosystems®). Fragmented cRNA were obtained through 3′IVT Express Kit (Affymetrix®, Santa Clara, CA, USA) to perform the hybridization using GeneChip Bovine Genome Array (Affymetrix®). Microarray data analysis was performed using the FlexArray 1.6.1.1 software. Genes with at least a 1.5-fold change and a P-value of less than 0.05 were considered differentially expressed. Of the 1278 genes differentially expressed between Bos taurus and Bos indicus vitrified embryos, 1108 were annotated, with 1193 genes up-regulated and 85 genes down-regulated in Bos taurus compared with Bos indicus IVP vitrified embryos. Differentially expressed genes were associated with the functional networks of cell cycle, cellular movement and DNA replication, recombination and repair; RNA post-transcriptional modifications; gene expression, protein synthesis; RNA damage and repair; cellular function and maintenance; and cell death and survival. The top 6 canonical pathways generated by Ingenuity Pathway Analysis® with the differentially expressed genes were ELF2 signalling, oxidative phosphorylation, tricarboxylic acid cycle, protein ubiquitination pathway, mTOR signalling, and IGF-1 signalling. In conclusion, Bos taurus IVP embryos seem to trigger different cellular response mechanisms to the vitrification stress in comparison with Bos indicus IVP embryos. Differential response is mainly represented by different expression profiles of genes regulating important canonical pathways involved in cellular response to stress that could be related with the higher post-cryopreservation survival capacity observed in Bos taurus embryos.Research was supported by FAPESP, CNPq, FAPERGS, and LNBio – National Laboratory of Biosciences/MCT.