The initial development of mammalian embryos represents a highly intricate and meticulously orchestrated process. This journey encompasses distinct cellular fate determinations and complex regulatory mechanisms. In the scope of our study, we conducted a comprehensive examination of the lipid and metabolic profiles characterizing these distinct cell populations within bovine embryos. In vitro-produced embryos, were submitted to microsurgery and/or immunosurgery techniques to recover trophectoderm (TE) and inner cell mass (ICM) cells. After different cell type obtaining ICM and TE samples underwent lipid and metabolic profiling utilizing multiple reaction monitoring (MRM) profiling mass spectrometry. We unveiled clear and distinct patterns of metabolites expression, including lipids, with TE cells demonstrating a heightened abundance of a variety classes of lipids, whereas ICM cells exhibited specific abundance increase of amino acids. The substantial presence of amino acids in ICM cells aligns with their pivotal role in orchestrating the development of diverse tissues and organs within the emerging organism. Conversely, TE cells are primarily dedicated to preparations for placentation, maternal recognition, and providing essential support to the ICM throughout the developmental process. In summary, our study furnishes valuable insights into the intricate metabolic dynamics that underlie early embryonic development in bovine embryos. It underscores the unique lipidomic and metabolic signatures of ICM and TE cells, reflecting their respective roles in shaping the developmental trajectory. These findings significantly contribute to our deeper understanding of the molecular mechanisms governing embryonic development, offering potential implications for bovine reproduction and broader insights into mammalian embryology.

In vitro-produced embryos are constantly exposed to stressful conditions that can lead to the activation of the apoptotic pathway. The nuclear Kappa B factor (NF-κB) is an inflammatory mediator that induces the expression of tumor necrosis factor (TNF-α), a pro-inflammatory cytokine, while interleukin-10 (IL-10), an anti-inflammatory cytokine, inhibits NF-κB activity. This study aimed to investigate the effects of IL-10 and TNF-α on the competence and cryosurvival of in vitro-produced bovine embryos. Embryos were produced in vitro using standard protocols, and Grade I blastocysts were vitrified using the Cryotop method. Non-vitrified and vitrified blastocysts were subjected to the TUNEL assay. In Experiment I, on day 6.5 (156 h post-insemination), the embryos were treated with PBS (control), 50 ng/mL of IL-10, or a combination of 25 ng/mL of TNF-α and 50 ng/mL of IL-10. Embryonic development and apoptotic rates were monitored. In Experiment II, the same groups were set up, with the addition of a group treated with 25 ng/mL of TNF-α alone. Grade I blastocysts were vitrified 5 h after treatment, and cryosurvival was monitored at until 48 h post-warming. The apoptosis rate and total cell number were investigated in the vitrified-hatched blastocysts. IL-10 alone did not affect developmental competence or cryosurvival (P > 0.05). The IL-10-treated embryos, when exposed in combination with TNF-α, presented a detrimental effect (P < 0.05) in the embryonic development of non-vitrified embryos. However, vitrified blastocysts had no negative effect (P > 0.05). The TNF-α treatment reduced (P < 0.05) the re-expansion rate at 6 h post-warming and increased (P < 0.05) the apoptosis rate in vitrified hatched blastocysts, whereas no effect (P > 0.05) of the treatments was detected in the hatching rate and total cell number post-warming. In conclusion, TNF-α has a detrimental effect on embryonic developmental competence and cryosurvival by compromising the development of non-vitrified embryos and apoptotic-related events of vitrified blastocysts, whereas IL-10, when in combination with TNF-α, appears to attenuate the detrimental effects of TNF-α.

A wide-ranging review study regarding the molecular characterization of the first cell lineages of the developmental embryo is lacking, especially for the primary events during earliest differentiation which leads to the determination of cellular fate. Here, a systematic review and meta-analysis were conducted according to PRISMA guidelines. MEDLINE-PubMed was searched based on an established search strategy through April 2021. Thirty-six studies fulfilling the inclusion criteria were subjected to qualitative and quantitative analysis. Among the studies, 50 % (18/36) used mice as an animal model, 22.2 % (8/36) pigs, 16.7 % (6/36) cattle, 5.5 % (2/36) humans, and 2.8 % (1/36) goats as well as 2.8 % (1/36) equine. Our results demonstrated that each of the first cell lineages of embryos requires a certain pattern of expression to establish the cellular determination of fate. Moreover, these patterns are shared by many species, particularly for those molecules that have already been identified in the literature as biomarkers. In conclusion, the present study integrated carefully chosen studies regarding embryonic development and first cellular decisions in mammalian species and summarized the information about the differential characterization of the first cell lineages and their possible relationship with specific gene expression.

Supplementation of culture media with IGF-1 during in vitro culture of embryos has had controversial results over the years. In the present study, we show that differences previously observed in response to IGF addition might be related to intrinsic heterogeneity of the embryos. In other words, the effects exerted by IGF-1 are dependent on the characteristics of the embryos and their ability to modulate metabolism and overcome stressful conditions, such as the ones found in a non-optimized in vitro culture system. To test this hypothesis, in vitro produced bovine embryos with distinct morphokinetics (fast- and slow-cleavage) were submitted to treatment with IGF-1 and then evaluated for embryo production rates, total cell number, gene expression and lipid profile. Our results show that remarkable differences were found when fast and slow embryos treated with IGF-1 were compared. Fast embryos respond by upregulating genes related to mitochondrial function, stress response, and lipid metabolism, whereas slow embryos presented lower mitochondrial efficiency and lipid accumulation. We conclude that indeed the treatment with IGF-1 selectively affects embryonic metabolism according to early morphokinetics phenotypes, and this information is relevant for decision-making in the design of more appropriate in vitro culture systems.

Each living organism is unique because of the lipid identity of its organelles. The diverse distribution of these molecules also contributes to the role of each organelle in cellular activity. The lipid profiles of whole embryos are well documented in the literature. However, this approach can often lead to the loss of relevant information at the subcellular and consequently, metabolic levels, hindering a deeper understanding of key physiological processes during preimplantation development. Therefore, we aimed to characterize four organelles in vitro-produced bovine embryos: lipid droplets (LD), endoplasmic reticulum (ER), mitochondria (MIT), and nuclear membrane (NUC), and evaluate the contribution of the lipid species to each organelle evaluated. Expanded blastocysts were subjected to cell organelle isolation. Thereafter, lipid extraction from cell organelles and lipid analysis using the Multiple Reaction Monitoring (MRM) profiling method were performed. The LD and ER displayed a greater number of lipids (Phosphatidylcholine - PC, Ceramide - Cer, and Sphingomielin - SM) with high signal-to-noise intensities. This result is due to the high rate of biosynthesis, lipid distribution, and ability to store and recycle lipid species of these organelles. The NUC had a more distinct lipid profile than the other three organelles, with high relative intensities of PC, SM, and triacylglycerols (TG), which is consistent with its high nuclear activity. MIT had an intermediate profile that was close to that of LD and ER, which aligns with its autonomous metabolism for some classes of phospholipids (PL). Our study revealed the lipid composition of each organelle studied, and the roles of these lipids could be associated with the characteristic organellar activity. Our findings highlight the lipid species and classes that are relevant for the homeostasis and function of each associated organelle and provide tentative biomarkers for the determination of in vitro embryonic development and quality.

Although well-established and adopted by commercial laboratories, the in vitro embryo production system still requires refinements to achieve its highest efficiency. Early embryonic development is a dynamic event, demanding suitable conditions to provide a high number of embryos with quality and competence. The first step to obtaining an optimized in vitro environment is to know the embryonic metabolism and energy request throughout the different stages of development. Oxygen plays a crucial role in several key biological processes necessary to sustain and complete embryonic development. Nonetheless, there is still controversy regarding the optimal in vitro atmospheric concentrations during culture. Herein, we discuss the impact of oxygen tension on the viability of in vitro-produced embryos during early development. The importance of oxygen tension is addressed as its roles regarding essential embryonic traits, including embryo production rates, embryonic cell viability, gene expression profile, epigenetic regulation, and post-cryopreservation survival. Finally, we highlight the damage caused by in vitro unbalanced oxygen tensions and strategies to mitigate the harmful effects.

Intermediates of the energy metabolism (as acetyl Co-A) can donate their acetyl group to introduce acetylation in histones, establishing a relationship between metabolism and epigenetic control in somatic and embryonic stem cells. Embryos with different kinetics during the first cleavages also have alterations in epigenetic profile as well as in metabolism and energy substrate consumption during invitro culture. The aim of this work was to verify if and how this relation between metabolism and epigenetic parameters was also presented in invitro-produced bovine embryos. For that, we first characterised the pattern of H3K9ac, and the molecular pattern of enzymes involved with histone acetylation and acetyl-CoA production in female blastocysts derived from fast and slow cleavage embryos. To validate the results, we also produced bovine embryos cultured with an inhibitor of the pyruvate production, and consequently the acetyl Co-A generation to check if this could interfere in the H3K9ac pattern. For this, embryos were invitro produced following standard protocol and classified at 40 hours post-insemination as fast (4 or more cells) or slow (2 cells) and collected at the blastocyst stage. Blastocysts were immunostained to H3K9ac and the fluorescence intensity of each nucleus was quantified using ImageJ and analysed by Student's t-test. For transcript quantitation, RNAseq data were accessed from a previous report using the same kinetics classification model (Milazzotto et al. 2016Mol. Rep. Dev. 83, 324-336; https://doi.org/10.1002/mrd.22619) and analysed using limma-voom on Galaxy 3.38.3. To validate the results, bovine embryos were produced and cultured until Day 4 and then incubated until the blastocyst stage with different doses of iodoacetate (IA; 2 and 5mM) to reduce the intracellular levels of acetyl CoA. These blastocysts were also assessed by H3K9 acetylation. Slow blastocysts presented higher fluorescence intensity for H3K9ac than fast blastocysts (fast 13.33±0.37 AU vs. slow 38.14±1.17 AU; P<0.0001). Despite the fact that there were no differences in transcripts related to this acetylation (ELP3 and HAT2), slow blastocysts presented higher levels of transcripts for PDHB1 and PDHA1, responsible for acetyl-CoA production (PDHB1: fast 11.6±0.2CPM vs. slow 13.1±0.2 counts per million; P<0.01 PDHA1: fast 12.6±0.2 CPM vs. slow 13.2±0.3 CPM; P<0.01). The reduction of acetyl-CoA in blastocysts induced by IA led to lower levels of H3K9ac in 1 and 2mM doses when compared with the control (control: 43.8±0.7 AU; 1 mM: 34.7±0.5 AU; 2 mM: 30.1±0.6 AU; P<0.0001). Interestingly, H3K9ac levels were similar for 5mM IA and control group (5 mM: 41.2±1.4; P>0.05), suggesting a compensatory mechanism in extreme cases to maintain the histone acetylation. As far as we know, this is the first work that describes a relation between metabolism and epigenetics in bovine embryos. Although the pattern of genes related to acetylation seems to be unaltered, changes in acetyl Co-A production pathway exert an influence on H3K9ac status. Grant support was provided by grant 2017/18384-0 and 2018/11668-6 from FAPESP and CAPES.

Human embryo studies have proposed the use of additional morphological evaluations related to the moment of the first cell divisions as relevant to embryo viability. Nevertheless, there are still not enough data available related to morphokinetic analysis and its relationship with lipid composition in embryos. Therefore, the aim of this study was to address the lipid profile of bovine embryos with different developmental kinetics: fast (four or more cells) and slow (two or three cells) at 40 h post-insemination (hpi), at three time points of in vitro culture (40, 112 and 186 hpi) and compare these to profiles of in vivo embryos. The lipid profiles of embryos were analyzed by matrix-assisted laser desorption ionization mass spectrometry, which mainly detected pools of membrane lipids such as phosphatidylcholine and sphingomyelin. In addition to their structural function, these lipid classes have an important role in cell signalling, particularly regarding events such as stress and pregnancy. Different patterns of lipids in the fast and slow groups were revealed in all the analyzed stages. Also, differences between in vitro embryos were more pronounced at 112 hpi, a critical moment due to embryonic genome activation. At the blastocyst stage, in vitro-produced embryos, despite the kinetics, had a closer lipid profile when compared with in vivo blastocysts. In conclusion, the kinetics of development had a greater effect on the membrane lipid profiles throughout the embryo culture, especially at the 8-16-cell stage. The in vitro environment affects lipid composition and may compromise cell signalling and function in blastocysts.

Pyruvate is a key energy substrate for the oocyte during maturation and acquisition of developmental competence. Mitochondrial activity is also essential for oocyte competence. Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase that indirectly stimulates pyruvate dehydrogenase (PDH), increasing pyruvate oxidation. PDH converts pyruvate into acetyl coenzyme A (acetyl-CoA) and thereby modulates the entry of glucose-derived carbons into the tricarboxylic acid (TCA) cycle, the main ATP production pathway within the oocyte. It was reported that DCA addition to embryo culture media improves embryo development in aged mice, by enhancing mitochondrial membrane potential (MMP) and decreasing oxidative stress (McPherson et al. 2014 Fertil. Steril. 101, 1458-1466). We hypothesised that increased pyruvate metabolism through the oxidative pathway, by stimulating PDH activity with DCA, could influence in vitro oocyte maturation. The aim of this work was to evaluate the effect of different concentrations of DCA during in vitro maturation (IVM) of bovine oocytes on maturation rate and mitochondrial activity, by assessing MMP and levels of flavin adenine dinucleotide (FADH2), nicotinamide adenine dinucleotide hydride (NADH), and reactive oxygen species (ROS). Abattoir-derived bovine cumulus-oocytes complexes (COC; n=360, over 4 replicates) were in vitro-matured with 0 (Control; n=120), 0.5mM (n=120) and 5mM (n=120) of DCA. After maturation, all matured COC were denuded by mechanical pipetting and meiotic progression was assessed by Hoechst 33342 staining and MMP by MitoTracker Red CMXRos test (Thermo Fisher Scientific, Waltham, MA, USA). Moreover, FADH2 and NADH levels were evaluated by autofluorescence (Dumollard et al. Development 134, 455-465) and ROS levels by CellRox® Green test (Thermo Fisher Scientific). Data were analysed by ANOVA, and the Tukey post hoc test was used to evaluate the difference among groups. The α-level was set at 0.05. Treatment with both concentrations of DCA decreased maturation rate (86.1, 67.8, and 67.6% in 0, 0.5, and 5mM groups, respectively; P<0.05). The MMP increased in oocytes matured with the highest concentration of DCA (3.42±0.28, 4.44±0.51, and 6.32±0.89 pixel/mm2, with 0, 0.5, and 5mM DCA, respectively; P<0.05). In line with this, higher levels of FADH2 (3.16±0.15, 3.96±0.24, and 3.83±0.20 pixel/mm2, with 0, 0.5, and 5mM DCA, respectively; P<0.05) and NADH (3.86±0.14, 4.80±0.16, and 4.95±0.17 pixel/mm2, with 0, 0.5, and 5mM DCA, respectively; P<0.05) were found in both DCA-treated groups compared with the control. Unexpectedly, ROS levels increased in the presence of DCA (0.9±0.07, 1.30±0.12, and 1.54±0.16 pixel/mm2, with 0, 0.5, and 5mM DCA, respectively; P<0.05) compared with the control. These results suggest that DCA was effective in stimulating mitochondrial activity of bovine oocytes, but also resulting in increased oxidative stress that likely accounts for the decreased maturation rate. Therefore, alternative strategies should be identified for the manipulation of the oocyte metabolic profile to improve oocyte developmental competence.

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.

Buffaloes are tendentially short-day breeders, and seasonality is one of the main factors affecting the feasibility of ovum pickup and in vitro embryo production technology in this species. An improvement of oocyte developmental competence during decreasing daylight months was previously reported in Italian Mediterranean buffalo (Di Francesco et al. 2011 Anim. Reprod. Sci. 123, 48-53). The aim of this work was to evaluate whether season also affects embryo quality and cryotolerance. Abattoir-derived buffalo cumulus-oocyte complexes were collected during the breeding season, characterised by decreasing daylight length (n=349 over 6 replicates), and the non-breeding season, characterised by increasing daylight length (n=770 over 12 replicates). Buffalo cumulus-oocyte complexes were in vitro matured, fertilized, and cultured according to standard procedures (Di Francesco et al. 2011 Anim. Reprod. Sci. 123, 48-53). The embryos obtained by the end of culture (i.e. on Day 7 post-IVF) were scored for quality and developmental stage, and the percentages of total transferable embryos (tight morulae and blastocysts) were recorded. Embryos (n=107 and 110 in the breeding and non-breeding seasons, respectively) were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide, and 0.5M sucrose (Boccia et al. 2013 Ital. J. Anim. Sci. 12, 492-496). Warming was carried out by plunging the cryotop strip into a 0.25M sucrose solution and transferring the embryos into 0.15M sucrose for 5min. Embryos were then washed and cultured in SOF for 24h to evaluate post-culture viability. The resistance to cryopreservation was evaluated by assessing the survival rate, on the basis of morphological criteria, and development rate (i.e. the percentage of embryos that resumed their development and reached a more advanced developmental stage) after 24h post-warming culture. Data were analysed by Student’s t-test. Both cleavage (82.8±4.3v. 73.1±1.7 in the breeding and non-breeding seasons, respectively; P<0.05) and blastocyst (32.9±3.5v. 18.3±1.7 in the breeding and non-breeding seasons, respectively; P<0.01) rates increased during the breeding season, confirming previous observations. Due to the different efficiency, a higher number of replicates was required during the non-breeding season to obtain an equal number of embryos. In addition, a seasonal effect was recorded on embryo quality, indicated by poorer cryotolerance of in vitro-produced buffalo embryos during the non-breeding season. Indeed, both survival (94.6±2.7% and 74.0±5.5% in the breeding and non-breeding seasons, respectively; P<0.01) and development (67.3±7.6% and 40.0±7.2% in the breeding and non-breeding seasons, respectively; P<0.01) rates of vitrified blastocysts decreased after 24h post-warming culture in the non-breeding season. These findings suggest that the reduced developmental competence of buffalo oocytes during the non-breeding season may also lead to lower blastocyst quality. This is in contrast to the evidence in cattle that embryo quality is mainly determined by culture conditions, whereas blastocyst production depends on oocyte quality.

During in vitro production (IVP), blastocysts can be differentiated based on the kinetics of early cleavage. These groups present distinct patterns of global DNA methylation, an epigenetic characteristic generally responsible for suppression (presence of methylation) or activation (absence of methylation) of genes from different biological pathways. This work investigated the DNA methylation and mRNA levels of genes related to embryo development and viability. For this purpose, bovine embryos underwent IVP using conventional protocols. After 40 h of insemination, embryos were classified as FBL (fast cleavage: 4 cells or more) or SBL (slow cleavage: 2 or 3 cells), remaining in culture until blastocyst stage. Sexed semen was used to prevent differences due to sex, even without statistical differences in male:female ratio already reported. Blastocysts (40 per group) were analysed by EmbryoGENE Methylation DNA Array (Ispada et al. 2016 Proc. 49th SSR: 181) and later analysed through BioMark™HD (Fluidigm Corp., South San Francisco, CA, USA) for the transcripts profile. The PPIA gene was used as endogenous control for ΔCt calculation and submitted to Student’s t-test. Genome-wide DNA methylation analysis identified 47,713 methylated regions (7976 hypermethylated in FBL and 3608 hypermethylated in SBL). Fast embryos presented more hypermethylations distributed throughout the genome, such as introns, exons, promoter and repeat elements, whereas hypermethylation were more present in CpG islands in slow embryos. Differentially methylated regions were clustered by means of biological processes and the most affected pathways were related to lipid metabolism and cell differentiation and survival. Regarding the gene expression analysis, all results are presented in FBL in relation to SBL. Of genes involved in lipid metabolism, ACSL3, ELOVL6, PPARA, and FADS, previously identified as hypermethylated genes, were down-regulated, whereas PPARG and PTGS2 showed no statistical difference; SCD and FASN, although hypomethylated, were also down-regulated, and ACSL6, which did not differ in DNA methylation status, was down-regulated. Of genes involved in survival/death, BAX, HSPA1A, BID, NFE2L2, and GPX1 were hypermethylated; however, the first 2 were up-regulated, BID was down-regulated, and the last 2 were not statistically different. Although CASP9, TXNRD1, and FOXO3 were all hypomethylated, only CASP9 was up-regulated. Also, DDIT3 was down-regulated and NOS2 was up-regulated, although they did not differ in DNA methylation between groups. Of genes involved in cell differentiation, POU5F1 and SALL4 were both hypermethylated, but only the POU5F1 was down-regulated; NANOG, which did not differ in DNA methylation status between groups, was also down-regulated. In conclusion, although we did not find correlation in DNA methylation and RNA levels for all genes evaluated, the chosen pathways were indeed different between groups, which could lead to their potential suppression/activation and affect embryo viability. Also, this lower correlation may be a result of the influence of other epigenetic mechanisms differently activated between groups.

Follicular fluid composition and the transcription pattern of granulosa cells were analysed to better comprehend associations between embryo development and morphokinetics. Bovine follicles were punctured and their respective follicular fluid and granulosa cells were collected. Cumulus-oocyte complexes derived from these follicles were matured and fertilised invitro. Embryo morphology and kinetics were evaluated at 40h after insemination, when embryos were classified as fast (FCL, four or more cells), slow (SCL, 2-3 cells) or non-cleaved (NCL). Their development was followed until the blastocyst stage. Glucose, pyruvate, cholesterol and oestradiol were quantified in the follicular fluid and the transcription pattern of 96 target genes was evaluated in granulosa cells by large-scale quantitative reverse transcription polymerase chain reaction. Follicular fluid from the blastocyst group had increased levels of glucose, total cholesterol and pyruvate compared to the non-blastocyst group, whereas higher levels of oestradiol were observed in the follicular fluid of embryos and blastocysts with fast cleavage. The transcriptional pattern revealed altered metabolic pathways between groups, such as lipid metabolism, cellular stress and cell signalling. In conclusion, both follicular fluid and granulosa cells are associated with the possibility of identifying follicles that may generate embryos with high potential to properly develop to the blastocyst stage.

Background: The timing of the first cell divisions may predict the developmental potential of an embryo, including its ability to establish pregnancy. Besides differences related to metabolism, stress, and survival, embryos with different speeds of development present distinct patterns of gene expression, mainly related to energy and lipid metabolism. As gene expression is regulated by epigenetic factors, and that includes DNA methylation patterns, in this study we compared the global DNA methylation profile of embryos with different kinetics of development in order to identify general pathways and regions that are most influenced by this phenotype. For this purpose, bovine embryos were in vitro produced using sexed semen (female), classified as fast (four or more cells) or slow (two cells) at 40 hpi and cultured until blastocyst stage, when they were analyzed. Results: Genome-wide DNA methylation analysis identified 11,584 differently methylated regions (DMRs) (7976 hypermethylated regions in fast and 3608 hypermethylated regions in slow embryos). Fast embryos presented more regions classified as hypermethylated distributed throughout the genome, as in introns, exons, promoters, and repeat elements while in slow embryos, hypermethylated regions were more present in CpG islands. DMRs were clustered by means of biological processes, and the most affected pathways were related to cell survival/differentiation and energy/lipid metabolism. Transcripts profiles from DM genes connected with these pathways were also assessed, and the most part disclosed changes in relative quantitation. Conclusion: The kinetics of the first cleavages influences the DNA methylation and expression profiles of genes related to metabolism and differentiation pathways and may affect embryo viability.

High oxygen levels during in vitro culture (IVC) can induce oxidative stress through accumulation of reactive oxygen species (ROS), negatively affecting embryo development. This study evaluated the effect of different O2 tensions during IVC on bovine blastocyst development and transcriptional status, considering transcription factors that play an essential role during early embryo development. For this purpose, embryos were produced in vitro by conventional protocols and cultured in two different oxygen tensions, physiological (5%) and atmospheric (20%). Expanded blastocysts were subjected to transcript quantitation analysis by RT-qPCR with Biomark™ HD System (Fluidigm, US), using 67 TaqMan assays specific for Bos taurus. Differences were observed in genes related to oxidation-reduction processes, DNA-dependent transcription factors, and factors related to important functional pathways for embryo development. Blastocyst rate was higher in the 5% O2 group and the number of cells was assessed, with the 5% O2 group having a higher number of cells. ROS concentration was evaluated, with a higher ROS presence in the 20% O2 group. Taken together, these results allow us to conclude that IVC of embryos at atmospheric O2 tension affects the expression of important transcription factors involved in multiple cell biology pathways that can affect embryo development, quality, and viability.

Estabeleceu-se um protocolo novo e eficaz de cultivo individual de embriões bovinos sem o uso de cocultivo e sem compartilhamento de meio visando à análise do metabolismo individual do embrião. Para isso, embriões foram produzidos in vitro por protocolos convencionais em três diferentes tipos de meio: KSOM, SOFaa e KSOM seguido por SOFaa no dia 2. Os zigotos presumíveis foram divididos em seis grupos: controles (cultivo em grupo – C-KSOM, C-SOFaa e C-KS) e sistema de poços individuais (W-KSOM, W-SOFaa e W-KS). As taxas de clivagem foram avaliadas nos dias 2 e 7, respectivamente. Além disso, a quantificação relativa de transcritos relacionados a importantes processos metabólicos (GLUT1, GLUT3, GSK3, SOD1, HSPD1 e G6PD) foi avaliada nos blastocistos dos grupos C-KS e W-KS. Os resultados mostram que as taxas de clivagem foram maiores apenas no grupo W-KSOM quando comparado ao grupo C-KSOM, enquanto a taxa de blastocistos diferiu apenas entre os grupos C e W-SOF. Além disso, a análise da expressão gênica mostrou que blastocistos cultivados em grupo ou em sistema de poços individuais são semelhantes quanto à expressão gênica. Assim, a conclusão obtida foi que o sistema individual proposto pode ser utilizado como um protocolo alternativo eficiente para o cultivo individual de embriões de bovino, uma vez que suas características permanecem semelhantes àquelas do sistema convencional de produção de embriões.

The timing of the first embryonic cell divisions may predict the ability of an embryo to establish pregnancy. Similarly, metabolic profiles may be markers of embryonic viability. However, in bovine, data about the metabolomics profile of these embryos are still not available. In the present work, we describe Raman-based metabolomic profiles of culture media of bovine embryos with different developmental kinetics (fast x slow) throughout the in vitro culture. The principal component analysis enabled us to classify embryos with different developmental kinetics since they presented specific spectroscopic profiles for each evaluated time point. We noticed that bands at 1076 cm(-1) (lipids), 1300 cm(-1) (Amide III), and 2719 cm(-1) (DNA nitrogen bases) gave the most relevant spectral features, enabling the separation between fast and slow groups. Bands at 1001 cm(-1) (phenylalanine) and 2892 cm(-1) (methylene group of the polymethylene chain) presented specific patterns related to embryonic stage and can be considered as biomarkers of embryonic development by Raman spectroscopy. The culture media analysis by Raman spectroscopy proved to be a simple and sensitive technique that can be applied with high efficiency to characterize the profiles of in vitro produced bovine embryos with different development kinetics and different stages of development. (C) 2016 Society of Photo-Optical Instrumentation Engineers (SPIE)

Secreted molecules could be correlated with the potential of embryonic development. The development of new technologies, such as mass spectrometry (MS), has enabled analyzes in culture medium to favor the determination of embryos viability in order to improve embryo selection.

DNA methylation is an epigenetic control mechanism essential during embryonic development, directing the differentiation of cell lineages and preventing regression to an undifferentiated state. Based on the fact that embryos with different development kinetics present differences in gene expression, this study aimed to characterize the differences between fast and slow embryos regarding the methylome status. For that, COCs from slaughterhouse ovaries were subjected to IVM for 22-24 h, IVF using sexed semen (female) for 18 h and IVC for 7 days. After 40 hours of insemination, embryos were classified as fast (four or more cells) or slow (2 or 3 cells) remaining in culture until the blastocyst stage (40 blastocysts per group in 4 replicates). These embryos were analyzed by EmbryoGENE Methylation DNA Array (EDMA). Briefly, the DNA extracted from 10 embryos was fragmented with MseI restriction enzyme and the fragments were extracted using methylation-sensitive digestion and ligation-mediated amplification PCR- (LMA-PCR). Hybridization was performed according to the manufactureru0027s instructions (Agilent Technologies). It was considered as differentially methylated regions (DMRs) fragments with methylation differences with P 1.5 between groups. From probes used for microarray, 9082 were expressed above background only in fast blastocysts (FBL), 20670 in slow blastocysts (SBL) and 47713 were observed in both groups. FBL presented 7976 DMRs and SBL presented 3608 DMRs. While fast embryos presented more hypermethylations in DMRs distributed throughout the genome, such as introns, exons, promoter and repeat elements, slow embryos presented more methylation of various densities (High - FBL: 17; SBL: 35; Intermediate - FBL: 81; SBL: 143 and Low - FBL: 31; SBL: 68) focused on CpG islands independent of length (Long - FBL: 14; SBL: 41; Intermediate - FBL: 79; SBL: 152 and Small - FBL: 36; SBL: 53). With Ingenuity Pathway Analysis 25 pathways from FBL and SBL were identified with differences in DNA methylation. These pathways include DNA damage, repair and replication, development disorder, cell morphology, gene expression, post-transcriptional modifications, metabolic diseases, lipid metabolism, post-translational modifications and embryonic development (33-35 genes observed in each pathway). Hypermethylation differences were also observed in genes related to pluripotency (NANOG, OCT4), epigenetic control (DNMT3A, HDAC8, MECP2) and embryonic development (EGFR, FGF8, IGFR1). In conclusion, the DNA methylation profile is different between embryos with fast or slow kinetics of development, thus influencing the phenotype and response to the environment.

SUMMARYEmbryo morphokinetics suggests that the timing of the first embryonic cell divisions may predict the developmental potential of an embryo; however, correlations between embryonic morphokinetics and physiology are not clear. Here, we used RNA sequencing to determine the gene expression profile of in vitro‐produced early‐ and late‐dividing bovine embryos and their respective blastocysts, and compared these profiles to in vivo‐produced blastocysts to identify differentially expressed genes (DEGs). Principal component analysis revealed that fast‐ and slow‐dividing embryos possess similar transcript abundance over the first cleavages. By the blastocyst stage, however, more DEGs were observed between the fast‐ and slow‐dividing embryo groups, whereas blastocysts from the slow‐dividing group were more similar to in vivo‐produced blastocysts. Gene ontology enrichment analysis showed that the slow‐dividing and in vivo‐produced blastocysts shared biological processes related to groups of up‐ or down‐regulated genes when compared to the fast‐dividing blastocysts. Based on these DEG results, we characterized the relationship between developmental kinetics and energy metabolism of in vitro‐produced bovine embryos. Embryos from fast‐ and slow‐dividing groups exhibited different pyruvate and lactate metabolism at 22 hr post‐in vitro culture (hpc), glucose consumption at 96 hpc, and glutamate metabolism at 168 hpc. Glycogen storage was similar between cleavage‐stage and morulae groups, but was higher in the blastocysts of the slow‐dividing group. On the other hand, blastocysts of the fast‐dividing group had a higher concentration of lipids. Taken together, these data identify transcriptomic and metabolic differences between embryos with different morphokinetics, suggesting that sorting embryos based on cleavage speed may select for different metabolic patterns. Mol. Reprod. Dev. 83: 324–336, 2016. © 2016 Wiley Periodicals, Inc.