Dynamics of transcription is affected by oxygen tension and developmental speed during in vitro production of bovine embryos.

This study examines the impact of oxygen tension and embryo kinetics on gene transcription dynamics in pathways crucial for embryonic preimplantation development, including lipid metabolism, carbohydrate transport and metabolism, mitochondrial function, stress response, apoptosis and transcription regulation. Bovine embryos were generated in vitro and allocated into two groups based on oxygen tension (20% or 5%) at 18 h post insemination (hpi). At 40 hpi, embryos were categorized into Fast (≥4 cells) or Slow (2 cells) groups, resulting in four experimental groups: FCL20, FCL5, SCL20 and SCL5. Embryo collection also occurred at 72 hpi (16-cell stage; groups FMO20, FMO5, SMO20 and SMO5) and at 168 hpi (expanded blastocyst (BL) stage; groups FBL20, FBL5, SBL20 and SBL5). Pools of three embryos per group were analysed in four replicates using inventoried TaqMan assays specific for Bos taurus, targeting 93 genes. Gene expression patterns were analysed using the K-means algorithm, revealing three main clusters: genes with low relative abundance at the cleavage (CL) and 16-cell morula (MO) stages but increased at the BL stage (cluster 1); genes with higher abundances at CL but decreasing at MO and BL (cluster 2); and genes with low levels at CL, higher levels at MO and decreased levels at BL (cluster 3). Within each cluster, genes related to epigenetic mechanisms, cell differentiation events and glucose metabolism were particularly influenced by differences in developmental kinetics and oxygen tension. Fast-developing embryos, particularly those cultured under low oxygen tension, exhibited transcript dynamics more closely resembling that reported in vivo-produced embryos.

The central role of pyruvate metabolism on the epigenetic maturation and transcriptional profile of bovine oocytes.

In brief: Pyruvate metabolism is one of the main metabolic pathways during oocyte maturation. This study demonstrates that pyruvate metabolism also regulates the epigenetic and molecular maturation in bovine oocytes. Abstract: Pyruvate, the final product of glycolysis, undergoes conversion into acetyl-CoA within the mitochondria of oocytes, serving as a primary fuel source for the tricarboxylic acid (TCA) cycle. The citrate generated in the TCA cycle can be transported to the cytoplasm and converted back into acetyl-CoA. This acetyl-CoA can either fuel lipid synthesis or act as a substrate for histone acetylation. This study aimed to investigate how pyruvate metabolism influences lysine 9 histone 3 acetylation (H3K9ac) dynamics and RNA transcription in bovine oocytes during in vitro maturation (IVM). Bovine cumulus-oocyte complexes were cultured in vitro for 24 h, considering three experimental groups: Control (IVM medium only), DCA (IVM supplemented with sodium dichloroacetate, a stimulant of pyruvate oxidation into acetyl-CoA), or IA (IVM supplemented with sodium iodoacetate, a glycolysis inhibitor). The results revealed significant alterations in oocyte metabolism in both treatments, promoting the utilization of lipids as an energy source. These changes during IVM affected the dynamics of H3K9ac, subsequently influencing the oocyte's transcriptional activity. In the DCA and IA groups, a total of 148 and 356 differentially expressed genes were identified, respectively, compared to the control group. These findings suggest that modifications in pyruvate metabolism trigger the activation of metabolic pathways, particularly lipid metabolism, changing acetyl-CoA availability and H3K9ac levels, ultimately impacting the mRNA content of in vitro matured bovine oocytes.

Adaptative response to changes in pyruvate metabolism on the epigenetic landscapes and transcriptomics of bovine embryos.

The epigenetic reprogramming that occurs during the earliest stages of embryonic development has been described as crucial for the initial events of cell specification and differentiation. Recently, the metabolic status of the embryo has gained attention as one of the main factors coordinating epigenetic events. In this work, we investigate the link between pyruvate metabolism and epigenetic regulation by culturing bovine embryos from day 5 in the presence of dichloroacetate (DCA), a pyruvate analog that increases the pyruvate to acetyl-CoA conversion, and iodoacetate (IA), which inhibits the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), leading to glycolysis inhibition. After 8 h of incubation, both DCA and IA-derived embryos presented higher mitochondrial membrane potential. Nevertheless, in both cases, lower levels of acetyl-CoA, ATP-citrate lyase and mitochondrial membrane potential were found in blastocysts, suggesting an adaptative metabolic response, especially in the DCA group. The metabolic alteration found in blastocysts led to changes in the global pattern of H3K9 and H3K27 acetylation and H3K27 trimethylation. Transcriptome analysis revealed that such alterations resulted in molecular differences mainly associated to metabolic processes, establishment of epigenetic marks, control of gene expression and cell cycle. The latter was further confirmed by the alteration of total cell number and cell differentiation in both groups when compared to the control. These results corroborate previous evidence of the relationship between the energy metabolism and the epigenetic reprogramming in preimplantation bovine embryos, reinforcing that the culture system is decisive for precise epigenetic reprogramming, with consequences for the molecular control and differentiation of cells.

Insulin-like growth factor-1 (IGF-1) selectively modulates the metabolic and lipid profile of bovine embryos according to their kinetics of development.

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.

Lipid availability during in vitro maturation alters oocyte lipid content and blastocyst development and metabolism.

Lipids play a crucial role in various biological functions, including membrane composition, energy storage, cell signalling, and metabolic and epigenetic processes. Abnormal lipid accumulation and metabolism during in vitro maturation (IVM) of oocytes have been linked to the use of fetal bovine serum (FBS), even though it provides several beneficial molecules, contributing to the oocyte competence. Delipidating agents have been used to mitigate these deleterious effects, but they can have adverse effects on embryonic development. In this study, we explored how lipids present in fetal bovine serum (FBS) can impact the composition of oocytes and their resulting blastocysts in vitro. For that, we used organic solvents to separate the polar and nonpolar (lipid enriched) phase of FBS. Oocytes were in vitro matured in the presence of 10% whole FBS (control), 10% FBS plus 10% nonpolar lipids (lipid enriched - OL) or 10% polar lipids only (partially delipidated - ODL). After 24 h, part of the matured oocytes was collected and those remaining in each group underwent in vitro fertilization (IVF) and culture (IVC) under the same conditions and expanded blastocysts were collected at day 7 (control, BL and BDL). Oocytes and embryos were analysed by Multiple Reaction Monitoring mass spectrometry (MRM-MS) to determine their lipid composition. Interestingly, principal component analysis (PCA) revealed a clear distinction in the lipid profile of oocytes and blastocysts from both treatments compared to the control group. Control oocytes and blastocysts had higher triacylglycerol and cholesterol ester enrichment while the OL, ODL, BL and BDL groups had higher amounts of free fatty acids (FFAs). The structural and signalling phospholipids also differed among groups. Our findings suggest that the lipid-enriched fraction of FBS can be manipulated for IVM to ensure proper maturation, resulting in oocytes and blastocysts with less accumulated intracellular lipids and an improved metabolic status.

Metabolism-epigenetic interactions on in vitro produced embryos.

Metabolism and epigenetics, which reciprocally regulate each other in different cell types, are fundamental aspects of cellular adaptation to the environment. Evidence in cancer and stem cells has shown that the metabolic status modifies the epigenome while epigenetic mechanisms regulate the expression of genes involved in metabolic processes, thereby altering the metabolome. This crosstalk occurs as many metabolites serve as substrates or cofactors of chromatin-modifying enzymes. If we consider the intense metabolic dynamic and the epigenetic remodelling of the embryo, the comprehension of these regulatory networks will be important not only for understanding early embryonic development, but also to determine in vitro culture conditions that support embryo development and may insert positive regulatory marks that may persist until adult life. In this review, we focus on how metabolism may affect epigenetic reprogramming of the early stages of development, in particular acetylation and methylation of histone and DNA. We also present other metabolic modifications in bovine embryos, such as lactylation, highlighting the promising epigenetic and metabolic targets to improve conditions for in vitro embryo development.

Silencing mark H3K27me3 is differently reprogrammed in bovine embryos with distinct kinetics of development.

The kinetics of the first cleavages is a predictor of blastocyst development and implantation. For bovine embryos, this attribute was previously related to distinct metabolic, molecular and epigenetic profiles, including DNA and histone modifications. In the present work, we described the dynamics of trimethylation of lysine 27 on histone H3 (H3K27me3) in fast and slow developing embryos and verified if this epigenetic mark was also influenced by the speed of the first cleavages. In vitro-produced bovine embryos were classified as fast (4 or more cells) or slow (2 cells) at 40 hr post fertilization (hpf) and either collected or cultured until 96 hpf or 186 hpf. Immunofluorescence analysis was performed in these three time points and showed that although both groups presented the same levels of H3K27me3 at 40 hpf, slow embryos presented a pronounced increase in this mark at 186 hpf when compared to fast embryos, resulting in blastocysts with remarkable differences in H3K27me3 levels. In conclusion, the increased levels of this repressive histone post-translation modification (PTM) might be an attempt of slow embryos to promote gene expression control and chromatin integrity, since it was already reported that these embryos present reduced levels of other epigenetic repressive marks as DNA methylation and trimethylation of lysine 9 on histone H3 (H3K9me3).

Metabolism-driven post-translational modifications of H3K9 in early bovine embryos.

Metabolic and molecular profiles were reported as different for bovine embryos with distinct kinetics during the first cleavages. In this study, we used this same developmental model (fast vs slow) to determine if the relationship between metabolism and developmental kinetics affects the levels of acetylation or tri-methylation at histone H3 lysine 9 (H3K9ac and H3K9me3, respectively). Fast and slow developing embryos presented different levels of H3K9ac and H3K9me3 from the earliest stages of development (40 and 96 hpi) and up to the blastocyst stage. For H3K9me3, both groups of embryos presented a wave of demethylation and de novo methylation, although it was more pronounced in fast than slow embryos, resulting in blastocysts with higher levels of this mark. The H3K9ac reprogramming profile was distinct between kinetics groups. While slow embryos presented a wave of deacetylation, followed by an increase in this mark at the blastocyst stage, fast embryos reduced this mark throughout all the developmental stages studied. H3K9me3 differences corresponded to writer and eraser transcript levels, while H3K9ac patterns were explained by metabolism-related gene expression. To verify if metabolic differences could alter levels of H3K9ac, embryos were cultured with sodium-iodoacetate (IA) or dichloroacetate (DCA) to disrupt the glycolytic pathway or increase acetyl-CoA production, respectively. IA reduced H3K9ac while DCA increased H3K9ac in blastocysts. Concluding, H3K9me3 and H3K9ac patterns differ between embryos with different kinetics, the second one explained by metabolic pathways involved in acetyl-CoA production. So far, this is the first study demonstrating a relationship between metabolic differences and histone post-translational modifications in bovine embryos.

Less is more: Reduced nutrient concentration during in vitro culture improves embryo production rates and morphophysiology of bovine embryos.

Reproducing the environment to which the embryo is naturally exposed may be an alternative to improve viability of embryos produced in vitro. In the first part of this work, we describe a novel culture media, namely Embryonic Culture Supplementation (ECS100). The composition of this media was based on the contents of carbohydrates and amino acids found in oviductal and uterine fluids. Because it was a new formulation, we investigated the performance of ECS100 in comparison with conventionally used SOFaa, and possible benefits to embryo development. Embryo production rates (cleavage, morula and blastocyst conversion, blastocyst and hatching rates) and morphophysiological parameters (total cell number, cell allocation, Mitochondrial membrane potential (MMP), Reactive Oxygen Species (ROS), NADH, FAD+ and ATP content) were similar between ECS100 and SOFaa. Next, we tested if a reduction of ECS100 concentration could positively contribute to embryo viability by resembling the more dynamic availability of nutrients that reach the embryos in vivo. Therefore, embryos were cultured in ECS100 or in its serial dilution (ECS75, 50 and 25). Despite the fact that the lowest concentration (ECS25) still supported blastocyst formation, halving the concentration of metabolites (ECS50) actually improved embryo production rates. Thus, embryos produced in ECS100 or ECS50 were submitted to further analyses on Days 4 and 7. Embryos cultured in ECS50 presented better developmental rates and morphophysiological profile than embryos cultured in ECS100. Additionally, physiological traits (MMP, ROS and NADH levels) of embryos cultured in ECS50 presented the expected pattern for embryos produced in vivo. In conclusion, we presented a novel, more personalized and effective culture media for bovine IVP embryos. And although the ECS media formulation was based on the contents of female reproductive fluids, it is worth mentioning that adaptations must be specifically directed for in vitro conditions rather than reproduced exactly from in vivo state.

Erasing gametes to write blastocysts: metabolism as the new player in epigenetic reprogramming.

Understanding preimplantation embryonic development is crucial for the improvement of assisted reproductive technologies and animal production. To achieve this goal, it is important to consider that gametes and embryos are highly susceptible to environmental changes. Beyond the metabolic adaptation, the dynamic status imposed during follicular growth and early embryogenesis may create marks that will guide the molecular regulation during prenatal development, and consequently impact the offspring phenotype. In this context, metaboloepigenetics has gained attention, as it investigates the crosstalk between metabolism and molecular control, i.e., how substrates generated by metabolic pathways may also act as players of epigenetic modifications. In this review, we present the main metabolic and epigenetic events of pre-implantation development, and how these systems connect to open possibilities for targeted manipulation of reproductive technologies and animal production systems.

The dynamics between in vitro culture and metabolism: embryonic adaptation to environmental changes.

Previous studies have discussed the importance of an optimal range of metabolic activity during preimplantation development. To avoid factors than can trigger an undesirable trajectory, it is important to learn how nutrients and metabolites interact to help launching the correct developmental program of the embryo, and how much the in vitro culture system can impair this process. Here, using the bovine model, we describe a factorial experimental design used to investigate the biochemical and molecular signature of embryos in response to different combinations of morphological features-i.e. speed of development-and external stimuli during in vitro culture-i.e. different oxygen tensions and glucose supplementation. Our analyses demonstrate that the embryos present heterogeneous metabolic responses depending on early morphological phenotypes and the composition of their surroundings. However, despite the contribution of each single stimulus for the embryo phenotype, oxygen tension is determinant for such differences. The lower oxygen environment boosts the metabolism of embryos with faster kinetics, in particular those cultured in lower glucose concentrations.

Tricarboxylic Acid Cycle Metabolites as Mediators of DNA Methylation Reprogramming in Bovine Preimplantation Embryos.

In many cell types, epigenetic changes are partially regulated by the availability of metabolites involved in the activity of chromatin-modifying enzymes. Even so, the association between metabolism and the typical epigenetic reprogramming that occurs during preimplantation embryo development remains poorly understood. In this work, we explore the link between energy metabolism, more specifically the tricarboxylic acid cycle (TCA), and epigenetic regulation in bovine preimplantation embryos. Using a morphokinetics model of embryonic development (fast- and slow-developing embryos), we show that DNA methylation (5mC) and hydroxymethylation (5hmC) are dynamically regulated and altered by the speed of the first cleavages. More specifically, slow-developing embryos fail to perform the typical reprogramming that is necessary to ensure the generation of blastocysts with higher ability to establish specific cell lineages. Transcriptome analysis revealed that such differences were mainly associated with enzymes involved in the TCA cycle rather than specific writers/erasers of DNA methylation marks. This relationship was later confirmed by disturbing the embryonic metabolism through changes in α-ketoglutarate or succinate availability in culture media. This was sufficient to interfere with the DNA methylation dynamics despite the fact that blastocyst rates and total cell number were not quite affected. These results provide the first evidence of a relationship between epigenetic reprogramming and energy metabolism in bovine embryos. Likewise, levels of metabolites in culture media may be crucial for precise epigenetic reprogramming, with possible further consequences in the molecular control and differentiation of cells.

Follicular environment as a predictive tool for embryo development and kinetics in cattle.

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.

Corrigendum to "Oxidative Stress Alters the Profile of Transcription Factors Related to Early Development on In Vitro Produced Embryos".

[This corrects the article DOI: 10.1155/2017/1502489.].

Genome-wide screening of DNA methylation in bovine blastocysts with different kinetics of development.

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.

Oxidative Stress Alters the Profile of Transcription Factors Related to Early Development on In Vitro Produced Embryos.

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.

Role of insulin-like growth factor 1 on cross-bred Bos indicus cattle germinal vesicle oocytes exposed to heat shock.

Germinal vesicle (GV) oocytes are susceptible to heat stress. However, neither the cellular mechanisms triggered by elevated temperature nor the thermoprotective effects of insulin-like growth factor (IGF) on GV oocytes are completely understood. Therefore, a series of experiments was conducted to determine the direct effects of IGF1 (0, 12.5, 25, 50 and 100ng mL-1) on heat-treated GV oocytes. Butyrolactone-arrested GV oocytes were cultured at 38.5°C (control) or 41°C (heat shock; HS) for 14h in the presence of different concentrations of IGF1. Exposure of GV oocytes to 41°C increased (P<0.05) the number of terminal deoxyribonucleotidyl transferase-mediated fluorescein-dUTP nick end-labelling (TUNEL)-positive oocytes. At concentrations of 12.5 and 25ng mL-1, IGF1 tended to minimise these negative effect of HS (P=0.07). However, neither HS nor IGF1 had any effect on caspase activity. HS also decreased (P<0.05) GV oocyte mitochondrial activity and developmental competence to the blastocyst stage. These deleterious effects of HS were alleviated (P<0.05) by 12.5ng mL-1 IGF1. This concentration of IGF1 did not affect cleavage rate, the percentage of TUNEL-positive blastomeres and total blastocyst cell number regardless of temperature. In conclusion, exposure of GV oocytes to HS triggered the apoptotic cascade and compromised oocyte developmental competence. Physiological concentrations of IGF1 had a beneficial effect on heat-shocked GV oocytes.

Thermoprotective effect of insulin-like growth factor 1 on in vitro matured bovine oocyte exposed to heat shock.

The role of insulin-like growth factor 1 (IGF1) on cellular function and developmental capacity of heat-shocked oocytes has not been completely understood. Therefore, the objective of this study was to determine the effect of IGF1 on apoptosis, mitochondrial activity, cytoskeletal changes, nuclear maturation, and developmental competence of bovine oocytes exposed to heat shock. Cumulus-oocyte complexes were submitted to control (38.5 °C for 22 hours) and heat shock (41 °C for 14 hours followed by 38.5 °C for 8 hours) in the presence of 0 or 100 ng/mL IGF1 during IVM. Heat shock increased the percentage of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)-positive oocyte and reduced oocyte mitochondrial activity. However, addition of 100 ng/mL IGF1 minimized these deleterious effects of temperature. Caspase activity was affected neither by heat shock nor IGF1. Exposure of bovine oocytes to 41 °C during the first 14-hour IVM affected cortical actin localization and microtubule organization at the meiotic spindle and reduced the percentage oocytes that reached the metaphase II stage. However, in the presence of IGF1, cortical actin and percentage of metaphase II oocytes were not different between control and heat-shocked oocytes, suggesting a partial beneficial effect of IGF1. There was no effect of IGF1 on microtubule organization. Heat shock also reduced the percentage of oocytes that reached the blastocyst stage, blastocyst cell number, and increased the percentage of TUNEL-positive blastomeres. However, there was no effect of 100 ng/mL IGF1 on oocyte development to the blastocyst stage and blastocyst quality. Therefore, 100 ng/mL IGF1 prevented some heat shock-induced cellular damage in bovine oocytes but had no effect on oocyte developmental competence. In contrast, a low IGF1 concentration (25 ng/mL) had a thermoprotective effect on oocyte developmental competence to the blastocyst stage. In conclusion, IGF1 prevented part of the damage induced by heat shock on oocyte function. This effect was modulated by IGF1 concentration.

Early cleavages influence the molecular and the metabolic pattern of individually cultured bovine blastocysts.

Embryo 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.