Enhanced bovine genome annotation through integration of transcriptomics and epi-transcriptomics datasets facilitates genomic biology.

BACKGROUND: The accurate identification of the functional elements in the bovine genome is a fundamental requirement for high-quality analysis of data informing both genome biology and genomic selection. Functional annotation of the bovine genome was performed to identify a more complete catalog of transcript isoforms across bovine tissues. RESULTS: A total of 160,820 unique transcripts (50% protein coding) representing 34,882 unique genes (60% protein coding) were identified across tissues. Among them, 118,563 transcripts (73% of the total) were structurally validated by independent datasets (PacBio isoform sequencing data, Oxford Nanopore Technologies sequencing data, de novo assembled transcripts from RNA sequencing data) and comparison with Ensembl and NCBI gene sets. In addition, all transcripts were supported by extensive data from different technologies such as whole transcriptome termini site sequencing, RNA Annotation and Mapping of Promoters for the Analysis of Gene Expression, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin using sequencing. A large proportion of identified transcripts (69%) were unannotated, of which 86% were produced by annotated genes and 14% by unannotated genes. A median of two 5' untranslated regions were expressed per gene. Around 50% of protein-coding genes in each tissue were bifunctional and transcribed both coding and noncoding isoforms. Furthermore, we identified 3,744 genes that functioned as noncoding genes in fetal tissues but as protein-coding genes in adult tissues. Our new bovine genome annotation extended more than 11,000 annotated gene borders compared to Ensembl or NCBI annotations. The resulting bovine transcriptome was integrated with publicly available quantitative trait loci data to study tissue-tissue interconnection involved in different traits and construct the first bovine trait similarity network. CONCLUSIONS: These validated results show significant improvement over current bovine genome annotations.

Germline ablation achieved via CRISPR/Cas9 targeting of NANOS3 in bovine zygotes.

NANOS3 is expressed in migrating primordial germ cells (PGCs) to protect them from apoptosis, and it is known to be a critical factor for germline development of both sexes in several organisms. However, to date, live NANOS3 knockout (KO) cattle have not been reported, and the specific role of NANOS3 in male cattle, or bulls, remains unexplored. This study generated NANOS3 KO cattle via cytoplasmic microinjection of the CRISPR/Cas9 system in vitro produced bovine zygotes and evaluated the effect of NANOS3 elimination on bovine germline development, from fetal development through reproductive age. The co-injection of two selected guide RNA (gRNA)/Cas9 ribonucleoprotein complexes (i.e., dual gRNA approach) at 6 h post fertilization achieved a high NANOS3 KO rate in developing embryos. Subsequent embryo transfers resulted in a 31% (n = 8/26) pregnancy rate. A 75% (n = 6/8) total KO rate (i.e., 100% of alleles present contained complete loss-of-function mutations) was achieved with the dual gRNA editing approach. In NANOS3 KO fetal testes, PGCs were found to be completely eliminated by 41-day of fetal age. Importantly, despite the absence of germ cells, seminiferous tubule development was not impaired in NANOS3 KO bovine testes during fetal, perinatal, and adult stages. Moreover, a live, NANOS3 KO, germline-ablated bull was produced and at sexual maturity he exhibited normal libido, an anatomically normal reproductive tract, and intact somatic gonadal development and structure. Additionally, a live, NANOS3 KO, germline-ablated heifer was produced. However, it was evident that the absence of germ cells in NANOS3 KO cattle compromised the normalcy of ovarian development to a greater extent than it did testes development. The meat composition of NANOS3 KO cattle was unremarkable. Overall, this study demonstrated that the absence of NANOS3 in cattle leads to the specific deficiency of both male and female germ cells, suggesting the potential of NANOS3 KO cattle to act as hosts for donor-derived exogenous germ cell production in both sexes. These findings contribute to the understanding of NANOS3 function in cattle and have valuable implications for the development of novel breeding technologies using germline complementation in NANOS3 KO germline-ablated hosts.

Gender disparity in survival of early porcine fetuses due to altered androgen receptor or associated U2 spliceosome component.

A single locus on the X chromosome codes for androgen receptor (AR) although this gene is subject to alternative splicing. AR is expressed in multiple tissues in males and females and is essential for reproductive success in the male. Since male and female mice are viable following naturally occurring and engineered loss of function with male mice infertile as anticipated, functional deletion of AR in pigs was hypothesized to provide a genetic containment strategy for males with edited genomes. In addition, deletion of AR might be a method to manage boar taint, hence contributing to a perceived improvement in animal welfare. The CRISPR/Cas9 technology was used to edit either exon 2 or exon 5 of the pig AR gene. Although pregnancies were established following embryo transfer of edited embryos, they were not maintained beyond day 25. Furthermore, normal M:F sex ratios were present in edited blastocysts and 19-day fetuses, but all fetuses recovered on day 21 or later were female. The pig AR gene differs from the mouse in having a U2 spliceosome component encoded in the intronic region. Hence, the absence of fetal survival beyond day 25 may be due to interference with the U2 component rather than AR.

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.

Blastocyst Cell Number and Allocation Affect the Developmental Potential and Transcriptome of Bovine Somatic Cell Nuclear Transfer Embryos.

Cloning cattle using somatic cell nuclear transfer (SCNT) is inefficient. Although the rate of development of SCNT embryos in vitro is similar to that of fertilized embryos, most fail to develop into healthy calves. In this study, we aimed to identify developmentally competent embryos according to blastocyst cell composition and perform transcriptome analysis of single embryos. Transgenic SCNT embryos expressing nuclear-localized HcRed gene at day 7 of development were imaged by confocal microscopy for cell counting and individually transferred to recipient heifers. Pregnancy rates were determined by ultrasonography. Embryos capable of establishing pregnancy by day 35 had an average of 117 ± 6 total cells, whereas embryos with an average of 128 ± 5 cells did not establish pregnancy (P < 0.05). A lesser average number of 41 ± 3 cells in the inner cell mass (ICM) also resulted in pregnancies (<0.05) than a greater number of 48 ± 2 cells in the ICM. Single embryos were then subjected to RNA sequencing for transcriptome analysis. Using weighted gene coexpression network analysis, we identified clusters of genes in which gene expression correlated with the number of total cells or ICM cells. Gene ontology analysis of these clusters revealed enriched biological processes in coenzyme metabolic process, intracellular signaling cascade, and glucose catabolic process, among others. We concluded that SCNT embryos with fewer total and ICM cell numbers resulted in greater pregnancy establishment rates and that these differences are reflected in the transcriptome of such embryos.

Derivation of Bovine Primed Embryonic Stem Cells from Somatic Cell Nuclear Transfer Embryos.

Derivation of bovine embryonic stem cells from somatic cell nuclear transfer embryos enables the derivation of genetically matched pluripotent stem cell lines to valuable and well-characterized animals. In this chapter, we describe a step-by-step procedure for deriving bovine embryonic stem cells from whole blastocysts produced by somatic cell nuclear transfer. This simple method requires minimal manipulation of blastocyst-stage embryos, relies on commercially available reagents, supports trypsin passaging, and allows the generation of stable primed pluripotent stem cell lines in 3-4 weeks.

Histone remodeling reflects conserved mechanisms of bovine and human preimplantation development.

How histone modifications regulate changes in gene expression during preimplantation development in any species remains poorly understood. Using CUT&Tag to overcome limiting amounts of biological material, we profiled two activating (H3K4me3 and H3K27ac) and two repressive (H3K9me3 and H3K27me3) marks in bovine oocytes, 2-, 4-, and 8-cell embryos, morula, blastocysts, inner cell mass, and trophectoderm. In oocytes, broad bivalent domains mark developmental genes, and prior to embryonic genome activation (EGA), H3K9me3 and H3K27me3 co-occupy gene bodies, suggesting a global mechanism for transcription repression. During EGA, chromatin accessibility is established before canonical H3K4me3 and H3K27ac signatures. Embryonic transcription is required for this remodeling, indicating that maternally provided products alone are insufficient for reprogramming. Last, H3K27me3 plays a major role in restriction of cellular potency, as blastocyst lineages are defined by differential polycomb repression and transcription factor activity. Notably, inferred regulators of EGA and blastocyst formation strongly resemble those described in humans, as opposed to mice. These similarities suggest that cattle are a better model than rodents to investigate the molecular basis of human preimplantation development.

Transcriptome annotation of 17 porcine tissues using nanopore sequencing technology.

The annotation of animal genomes plays an important role in elucidating molecular mechanisms behind the genetic control of economically important traits. Here, we employed long-read sequencing technology, Oxford Nanopore Technology, to annotate the pig transcriptome across 17 tissues from two Yorkshire littermate pigs. More than 9.8 million reads were obtained from a single flow cell, and 69 781 unique transcripts at 50 108 loci were identified. Of these transcripts, 16 255 were found to be novel isoforms, and 22 344 were found at loci that were novel and unannotated in the Ensembl (release 102) and NCBI (release 106) annotations. Novel transcripts were mostly expressed in cerebellum, followed by lung, liver, spleen, and hypothalamus. By comparing the unannotated transcripts to existing databases, there were 21 285 (95.3%) transcripts matched to the NT database (v5) and 13 676 (61.2%) matched to the NR database (v5). Moreover, there were 4324 (19.4%) transcripts matched to the SwissProt database (v5), corresponding to 11 356 proteins. Tissue-specific gene expression analyses showed that 9749 transcripts were highly tissue-specific, and cerebellum contained the most tissue-specific transcripts. As the same samples were used for the annotation of cis-regulatory elements in the pig genome, the transcriptome annotation generated by this study provides an additional and complementary annotation resource for the Functional Annotation of Animal Genomes effort to comprehensively annotate the pig genome.

Embryonic microRNAs are essential for bovine preimplantation embryo development.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression after transcription. miRNAs are present in transcriptionally quiescent full-grown oocytes and preimplantation embryos that display a low level of transcription prior to embryonic genome activation. The role of miRNAs, if any, in preimplantation development is not known. The temporal pattern of expression of miRNAs during bovine preimplantation development was determined by small RNA-sequencing using eggs and preimplantation embryos (1-cell, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst). Embryos cultured in the presence of α-amanitin, which permitted the distinguishing of maternal miRNAs from embryonic miRNAs, indicated that embryonic miRNA expression was first detected at the two-cell stage but dramatically increased during the morula and blastocyst stages. Targeting DGCR8 by a small-interfering RNA/morpholino approach revealed a role for miRNAs in the morula-to-blastocyst transition. Knockdown of DGCR8 not only inhibited expression of embryonically expressed miRNAs but also inhibited the morula-to-blastocyst transition. In addition, RNA-sequencing identified an increased relative abundance of messenger RNAs potentially targeted by embryonic miRNAs in DGCR8-knockdown embryos when compared with controls. Results from these experiments implicate an essential role for miRNAs in bovine preimplantation embryo development.

Prediction of transcript isoforms in 19 chicken tissues by Oxford Nanopore long-read sequencing.

To identify and annotate transcript isoforms in the chicken genome, we generated Nanopore long-read sequencing data from 68 samples that encompassed 19 diverse tissues collected from experimental adult male and female White Leghorn chickens. More than 23.8 million reads with mean read length of 790 bases and average quality of 18.2 were generated. The annotation and subsequent filtering resulted in the identification of 55,382 transcripts at 40,547 loci with mean length of 1,700 bases. We predicted 30,967 coding transcripts at 19,461 loci, and 16,495 lncRNA transcripts at 15,512 loci. Compared to existing reference annotations, we found ∼52% of annotated transcripts could be partially or fully matched while ∼47% were novel. Seventy percent of novel transcripts were potentially transcribed from lncRNA loci. Based on our annotation, we quantified transcript expression across tissues and found two brain tissues (i.e., cerebellum and cortex) expressed the highest number of transcripts and loci. Furthermore, ∼22% of the transcripts displayed tissue specificity with the reproductive tissues (i.e., testis and ovary) exhibiting the most tissue-specific transcripts. Despite our wide sampling, ∼20% of Ensembl reference loci were not detected. This suggests that deeper sequencing and additional samples that include different breeds, cell types, developmental stages, and physiological conditions, are needed to fully annotate the chicken genome. The application of Nanopore sequencing in this study demonstrates the usefulness of long-read data in discovering additional novel loci (e.g., lncRNA loci) and resolving complex transcripts (e.g., the longest transcript for the TTN locus).

Acquisition and maintenance of pluripotency are influenced by fibroblast growth factor, leukemia inhibitory factor, and 2i in bovine-induced pluripotent stem cells.

Several opportunities for embryo development, stem cell maintenance, cell fate, and differentiation have emerged using induced pluripotent stem cells (iPSCs). However, the difficulty in comparing bovine iPSCs (biPSCs) with embryonic stem cells (ESCs) was a challenge for many years. Here, we reprogrammed fetal fibroblasts by transient expression of the four transcription factors (Oct4, Sox2, Klf4, and c-Myc, collectively termed "OSKM" factors) and cultured in iPSC medium, supplemented with bFGF, bFGF2i, leukemia inhibitory factor (LIF), or LIF2i, and then compared these biPSC lines with bESC to evaluate the pluripotent state. biPSC lines were generated in all experimental groups. Particularly, reprogrammed cells treated with bFGF were more efficient in promoting the acquisition of pluripotency. However, LIF2i treatment did not promote continuous self-renewal. biPSCs (line 2) labeled with GFP were injected into early embryos (day 4.5) to assess the potential to contribute to chimeric blastocysts. The biPSC lines show a pluripotency state and are differentiated into three embryonic layers. Moreover, biPSCs and bESCs labeled with GFP were able to contribute to chimeric blastocysts. Additionally, biPSCs have shown promising potential for contributing to chimeric blastocysts and for future studies.

Single-Step Genome Editing of Small Ruminant Embryos by Electroporation.

We investigated the possibility of single-step genome editing in small ruminants by CRISPR-Cas9 zygote electroporation. We targeted SOCS2 and PDX1 in sheep embryos and OTX2 in goat embryos, utilizing a dual sgRNA approach. Gene editing efficiency was compared between microinjection and three different electroporation settings performed at four different times of embryo development. Electroporation of sheep zygotes 6 h after fertilization with settings that included short high-voltage (poring) and long low-voltage (transfer) pulses was efficient at producing SOCS2 knock-out blastocysts. The mutation rate after CRISPR/Cas9 electroporation was 95.6% ± 8%, including 95.4% ± 9% biallelic mutations; which compared favorably to 82.3% ± 8% and 25% ± 10%, respectively, when using microinjection. We also successfully disrupted the PDX1 gene in sheep and the OTX2 gene in goat embryos. The biallelic mutation rate was 81 ± 5% for PDX1 and 85% ± 6% for OTX2. In conclusion, using single-step CRISPR-Cas9 zygote electroporation, we successfully introduced biallelic deletions in the genome of small ruminant embryos.

Comparative transcriptome in large-scale human and cattle populations.

BACKGROUND: Cross-species comparison of transcriptomes is important for elucidating evolutionary molecular mechanisms underpinning phenotypic variation between and within species, yet to date it has been essentially limited to model organisms with relatively small sample sizes. RESULTS: Here, we systematically analyze and compare 10,830 and 4866 publicly available RNA-seq samples in humans and cattle, respectively, representing 20 common tissues. Focusing on 17,315 orthologous genes, we demonstrate that mean/median gene expression, inter-individual variation of expression, expression quantitative trait loci, and gene co-expression networks are generally conserved between humans and cattle. By examining large-scale genome-wide association studies for 46 human traits (average n = 327,973) and 45 cattle traits (average n = 24,635), we reveal that the heritability of complex traits in both species is significantly more enriched in transcriptionally conserved than diverged genes across tissues. CONCLUSIONS: In summary, our study provides a comprehensive comparison of transcriptomes between humans and cattle, which might help decipher the genetic and evolutionary basis of complex traits in both species.

Characterization of histone lysine ß-hydroxybutyrylation in bovine tissues, cells, and cumulus-oocyte complexes.

Besides their canonical roles as energy sources, short-chain fatty acids act as metabolic regulators of gene expression through histone posttranslational modifications. Ketone body ß-hydroxybutyrate (BHB) causes a novel epigenetic modification, histone lysine ß-hydroxybutyrylation (Kbhb), which is associated with genes upregulated in starvation-responsive metabolic pathways. Dairy cows increase BHB in early lactation, and the effects of this increase on cellular epigenomes are unknown. We searched for and identified that Kbhb is present in bovine tissues in vivo and confirmed that this epigenetic mark is responsive to BHB in bovine and human fibroblasts cultured in vitro in a dose-dependent manner. Maturation of cumulus-oocyte complexes with high concentrations of BHB did not affect the competence to complete meiotic maturation or to develop until the blastocyst stage. BHB treatment strongly induced H3K9bhb in cumulus cells, but faintly in oocytes. RNA-seq analysis in cumulus cells indicated that BHB treatment altered the expression of 345 genes. The downregulated genes were mainly involved in glycolysis and ribosome assembly pathways, while the upregulated genes were involved in mitochondrial metabolism and oocyte development. The genes and pathways altered by BHB will provide entry points to carry out functional experiments aiming to mitigate metabolic disorders and improve fertility in cattle.

Stem cell-derived porcine macrophages as a new platform for studying host-pathogen interactions.

BACKGROUND: Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. RESULTS: We explored how porcine pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages. Porcine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages and were productively infected by pig pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, porcine PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied either in parental stem cells or directly in the macrophages by lentiviral vector transduction. CONCLUSIONS: We show that porcine PSCdMs exhibit key macrophage characteristics, including infection by a range of commercially relevant pig pathogens. In addition, genetic engineering of PSCs and PSCdMs affords new opportunities for functional analysis of macrophage biology in an important livestock species. PSCs and differentiated derivatives should therefore represent a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in pigs, and also have wider applications in livestock.

Sperm proteins ODF2 and PAWP as markers of fertility in breeding bulls.

Low fertility is the single most important factor limiting livestock reproductive performance, adversely affecting the cattle industry and causing millions of dollars of economic loss. In the livestock industry, male fertility is of crucial importance for the reproductive performance of livestock. However, there is a lack of reliable biomarkers to predict bull fertility in artificial insemination service. The objective of this study was to identify sperm proteins as biomarkers for bull fertility. To discover candidate sperm quality biomarkers, sperm proteome profiling was conducted in extreme high- and extreme low-fertile bulls selected from a pool of 1000 AI sires with varied fertility. Thirty-two differentially expressed proteins were identified. Among them, high levels of sperm outer dense fiber of sperm tails 2 (ODF2) and post-acrosomal assembly of sperm head protein (PAWP/WBP2NL) represented the most extreme differences in quantity between high- and low-fertility bulls. Protein immunodetection and flow cytometry used to validate these putative fertility markers in a combined cohort of 154 AI sires. Both ODF2 and PAWP correlated significantly with fertility. In conclusion, ODF2 and PAWP can be used to assess semen quality and predict sire fertility.

Pig genome functional annotation enhances the biological interpretation of complex traits and human disease.

The functional annotation of livestock genomes is crucial for understanding the molecular mechanisms that underpin complex traits of economic importance, adaptive evolution and comparative genomics. Here, we provide the most comprehensive catalogue to date of regulatory elements in the pig (Sus scrofa) by integrating 223 epigenomic and transcriptomic data sets, representing 14 biologically important tissues. We systematically describe the dynamic epigenetic landscape across tissues by functionally annotating 15 different chromatin states and defining their tissue-specific regulatory activities. We demonstrate that genomic variants associated with complex traits and adaptive evolution in pig are significantly enriched in active promoters and enhancers. Furthermore, we reveal distinct tissue-specific regulatory selection between Asian and European pig domestication processes. Compared with human and mouse epigenomes, we show that porcine regulatory elements are more conserved in DNA sequence, under both rapid and slow evolution, than those under neutral evolution across pig, mouse, and human. Finally, we provide biological insights on tissue-specific regulatory conservation, and by integrating 47 human genome-wide association studies, we demonstrate that, depending on the traits, mouse or pig might be more appropriate biomedical models for different complex traits and diseases.

Regulation of NANOG and SOX2 expression by activin A and a canonical WNT agonist in bovine embryonic stem cells and blastocysts.

Bovine embryonic stem cells (ESC) have features associated with the primed pluripotent state including low expression of one of the core pluripotency transcription factors, NANOG. It has been reported that NANOG expression can be upregulated in porcine ESC by treatment with activin A and the WNT agonist CHIR99021. Accordingly, it was tested whether expression of NANOG and another pluripotency factor SOX2 could be stimulated by activin A and the WNT agonist CHIR99021. Immunoreactive NANOG and SOX2 were analyzed for bovine ESC lines derived under conditions in which activin A and CHIR99021 were added singly or in combination. Activin A enhanced NANOG expression but also reduced SOX2 expression. CHIR99021 depressed expression of both NANOG and SOX2. In a second experiment, activin A enhanced blastocyst development while CHIR99021 treatment impaired blastocyst formation and reduced number of blastomeres. Activin A treatment decreased blastomeres in the blastocyst that were positive for either NANOG or SOX2 but increased those that were CDX2+ and that were GATA6+ outside the inner cell mass. CHIR99021 reduced SOX2+ and NANOG+ blastomeres without affecting the number or percent of blastomeres that were CDX2+ and GATA6+. Results indicate activation of activin A signaling stimulates NANOG expression during self-renewal of bovine ESC but suppresses cells expressing pluripotency markers in the blastocyst and increases cells expressing CDX2. Actions of activin A to promote blastocyst development may involve its role in promoting trophectoderm formation. Furthermore, results demonstrate the negative role of canonical WNT signaling in cattle for pluripotency marker expression in ESC and in formation of the inner cell mass and epiblast during embryonic development. This article has an associated First Person interview with the first author of the paper.

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.

Tissue Resources for the Functional Annotation of Animal Genomes.

In order to generate an atlas of the functional elements driving genome expression in domestic animals, the Functional Annotation of Animal Genome (FAANG) strategy was to sample many tissues from a few animals of different species, sexes, ages, and production stages. This article presents the collection of tissue samples for four species produced by two pilot projects, at INRAE (National Research Institute for Agriculture, Food and Environment) and the University of California, Davis. There were three mammals (cattle, goat, and pig) and one bird (chicken). It describes the metadata characterizing these reference sets (1) for animals with origin and selection history, physiological status, and environmental conditions; (2) for samples with collection site and tissue/cell processing; (3) for quality control; and (4) for storage and further distribution. Three sets are identified: set 1 comprises tissues for which collection can be standardized and for which representative aliquots can be easily distributed (liver, spleen, lung, heart, fat depot, skin, muscle, and peripheral blood mononuclear cells); set 2 comprises tissues requiring special protocols because of their cellular heterogeneity (brain, digestive tract, secretory organs, gonads and gametes, reproductive tract, immune tissues, cartilage); set 3 comprises specific cell preparations (immune cells, tracheal epithelial cells). Dedicated sampling protocols were established and uploaded in https://data.faang.org/protocol/samples. Specificities between mammals and chicken are described when relevant. A total of 73 different tissues or tissue sections were collected, and 21 are common to the four species. Having a common set of tissues will facilitate the transfer of knowledge within and between species and will contribute to decrease animal experimentation. Combining data on the same samples will facilitate data integration. Quality control was performed on some tissues with RNA extraction and RNA quality control. More than 5,000 samples have been stored with unique identifiers, and more than 4,000 were uploaded onto the Biosamples database, provided that standard ontologies were available to describe the sample. Many tissues have already been used to implement FAANG assays, with published results. All samples are available without restriction for further assays. The requesting procedure is described. Members of FAANG are encouraged to apply a range of molecular assays to characterize the functional status of collected samples and share their results, in line with the FAIR (Findable, Accessible, Interoperable, and Reusable) data principles.