Altered microRNA composition in the uterine lumen fluid in cattle (Bos taurus) pregnancies initiated by artificial insemination or transfer of an in vitro produced embryo.

Background: MicroRNAs (miRNAs) are presented in the uterine lumen of many mammals, and in vitro experiments have determined that several miRNAs are important for the regulation of endometrial and trophoblast functions. Our aim was to identify and contrast the miRNAs present in extracellular vesicles (EVs) in the uterine lumen fluid (ULF) at the onset of attachment in cattle pregnancies (gestation d 18) initiated by artificial insemination (AI) or by the transfer of an in vitro-produced blastocyst (IVP-ET). A third group had no conceptus after the transfer of an IVP embryo.

Identification of novel cattle (Bos taurus) genes and biological insights of their function in pre-implantation embryo development.

Background: Appropriate regulation of genes expressed in oocytes and embryos is essential for acquisition of developmental competence in mammals. Here, we hypothesized that several genes expressed in oocytes and pre-implantation embryos remain unknown. Our goal was to reconstruct the transcriptome of oocytes (germinal vesicle and metaphase II) and pre-implantation cattle embryos (blastocysts) using short-read and long-read sequences to identify putative new genes.

Non-Alcoholic Fatty Liver Disease Induced by Feeding Medium-Chain Fatty Acids Upregulates Cholesterol and Lipid Homeostatic Genes in Skeletal Muscle of Neonatal Pigs.

Non-alcoholic fatty liver disease (NAFLD) is a range of disorders characterized by lipid accumulation in hepatocytes. Although this spectrum of disorders is associated with adult obesity, recent evidence suggests that this condition could also occur independently of obesity, even in children. Previously, we reported that pigs fed a formula containing medium-chain fatty acids (MCFAs) developed hepatic steatosis and weighed less than those fed an isocaloric formula containing long-chain fatty acids (LCFAs).

Higher abundance of 2-dehydro-d-gluconate in the plasma of sub-fertile or infertile Bos taurus heifers.

Infertility or subfertility impacts approximately 5% and 15% of dairy and beef heifers (Bos taurus), respectively. Heifers that do not produce a calf within an optimum window of time have a significant negative impact on the profitability and sustainability of the cattle industry. Selection of heifers based on their fertility potential remains a challenge yet to be resolved.

Interleukin-6 supplementation improves bovine conceptus elongation and transcriptomic indicators of developmental competence†.

A high incidence of pregnancy failures occurs in cattle during the second week of pregnancy as blastocysts transition into an elongated conceptus. This work explored whether interleukin-6 supplementation during in vitro embryo production would improve subsequent conceptus development. Bovine embryos were treated with 0 or 100 ng/mL recombinant bovine interleukin-6 beginning on day 5 post-fertilization.

Protocol for the electroporation of CRISPR-Cas for DNA and RNA targeting in Bos taurus zygotes.

The use of CRISPR-Cas9 ribonucleoproteins has revolutionized manipulation of genomes. Here, we present a protocol for the electroporation of CRISPR-Cas for DNA and RNA targeting in Bos taurus zygotes. First, we describe steps for production and preparation of presumptive zygotes for electroporation.

Influences of Supplementing Selective Members of the Interleukin-6 Cytokine Family on Bovine Oocyte Competency.

This work explored whether supplementing selective members of the interleukin-6 (IL6) cytokine family during in vitro bovine oocyte maturation affects maturation success, cumulus-oocyte complex (COC) gene expression, fertilization success, and embryo development potential. Human recombinant proteins for IL6, IL11, and leukemia inhibitory factor (LIF) were supplemented to COCs during the maturation period, then fertilization and embryo culture commenced without further cytokine supplementation. The first study determined that none of these cytokines influenced the rate that oocytes achieved arrest at meiosis II.

Ablation of function in cattle embryos by double electroporation of CRISPR-Cas for DNA and RNA targeting (CRISPR-DART).

CRISPR-Cas ribonucleoproteins (RNPs) are important tools for gene editing in preimplantation embryos. However, the inefficient production of biallelic deletions in cattle zygotes has hindered mechanistic studies of gene function. In addition, the presence of maternal RNAs that support embryo development until embryonic genome activation may cause confounding phenotypes.

Extensive rewiring of the gene regulatory interactions between in vitro-produced conceptuses and endometrium during attachment.

Pregnancy loss is a significant problem when embryos produced in vitro are transferred to a synchronized uterus. Currently, mechanisms that underlie losses of in vitro-produced embryos during implantation are largely unknown. We investigated this problem using cattle as a model of conceptus attachment by analyzing transcriptome data of paired extraembryonic membrane and endometrial samples collected on gestation days 18 and 25, which spans the attachment window in cattle.

A multi-omics analysis identifies molecular features associated with fertility in heifers (Bos taurus).

Infertility or subfertility is a critical barrier to sustainable cattle production, including in heifers. The development of heifers that do not produce a calf within an optimum window of time is a critical factor for the profitability and sustainability of the cattle industry. In parallel, heifers are an excellent biomedical model for understanding the underlying etiology of infertility because well-nourished heifers can still be infertile, mostly because of inherent physiological and genetic causes.

Robust identification of regulatory variants (eQTLs) using a differential expression framework developed for RNA-sequencing.

Background: A gap currently exists between genetic variants and the underlying cell and tissue biology of a trait, and expression quantitative trait loci (eQTL) studies provide important information to help close that gap. However, two concerns that arise with eQTL analyses using RNA-sequencing data are normalization of data across samples and the data not following a normal distribution. Multiple pipelines have been suggested to address this.

Sexing of cattle embryos using RNA-sequencing data or polymerase chain reaction based on a complete sequence of cattle chromosome Y.

When necessary, RNA-sequencing data or polymerase chain reaction (PCR) assays can be used to determine the presence of the chromosome Y (ChrY) in samples. This information allows for biological variation due to sexual dimorphism to be studied. A prime example is when researchers conduct RNA-sequencing of single embryos, or conceptuses, prior to the development of gonads.

Tight gene co-expression in BCB positive cattle oocytes and their surrounding cumulus cells.

Background: Cytoplasmic and nuclear maturation of oocytes, as well as interaction with the surrounding cumulus cells, are important features relevant to the acquisition of developmental competence.

Methods: Here, we utilized Brilliant cresyl blue (BCB) to distinguish cattle oocytes with low activity of the enzyme Glucose-6-Phosphate Dehydrogenase, and thus separated fully grown (BCB positive) oocytes from those in the growing phase (BCB negative). We then analyzed the developmental potential of these oocytes, mitochondrial DNA (mtDNA) copy number in single oocytes, and investigated the transcriptome of single oocytes and their surrounding cumulus cells of BCB positive versus BCB negative oocytes.

Delayed processing of blood samples impairs the accuracy of mRNA-based biomarkers.

The transcriptome of peripheral white blood cells (PWBCs) are indicators of an organism's physiological state, thus making them a prime biological sample for mRNA-based biomarker discovery. Here, we designed an experiment to evaluate the impact of delayed processing of whole blood samples on gene transcript abundance in PWBCs. We hypothesized that storing blood samples for 24 h at 4 °C would cause RNA degradation resulting in altered transcriptome profiles.

Isolation of high-quality total RNA and RNA sequencing of single bovine oocytes.

Studying individual mammalian oocytes has been extremely valuable for the understanding of the molecular composition of oocytes including RNA storage. Here, a detailed protocol for isolation of oocytes, extraction of total RNA from single oocytes followed by full-length cDNA amplification, and library preparation is presented. The procedure permits the production of cost-effective and high-quality sequencing libraries.

Rewiring of gene expression in circulating white blood cells is associated with pregnancy outcome in heifers (Bos taurus).

Infertility is a challenging phenomenon in cattle that reduces the sustainability of beef production worldwide. Here, we tested the hypothesis that gene expression profiles of protein-coding genes expressed in peripheral white blood cells (PWBCs), and circulating micro RNAs in plasma, are associated with female fertility, measured by pregnancy outcome. We drew blood samples from 17 heifers on the day of artificial insemination and analyzed transcript abundance for 10,496 genes in PWBCs and 290 circulating micro RNAs.

Beef heifer fertility: importance of management practices and technological advancements.

The development of replacement heifers is at the core of cow-calf beef production systems. In 2020, the USDA, National Agricultural Statistics Service reported 5.771 million beef heifers, 500 pounds and over, are under development for cow replacement.

The blueprint of RNA storages relative to oocyte developmental competence in cattle (Bos taurus).

From the time oocytes leave quiescence, there are constant microenvironmental influences contributing to development, thus acquiring developmental competence is not a simple, linear phenomenon. During folliculogenesis, oocytes experience many morphological and cytological changes that contribute toward the acquisition of developmental competence, a process defined by an oocyte's ability to progress through folliculogenesis, be fertilized, undergo cleavage, and develop into an embryo. Many factors, such as ovarian follicle size, cow age, and the morphology of the cumulus-oocyte complex, have been extensively investigated to understand this process.

Fine-tuned adaptation of embryo-endometrium pairs at implantation revealed by transcriptome analyses in Bos taurus.

Interactions between embryo and endometrium at implantation are critical for the progression of pregnancy. These reciprocal actions involve exchange of paracrine signals that govern implantation and placentation. However, it remains unknown how these interactions between the conceptus and the endometrium are coordinated at the level of an individual pregnancy.

Evaluation of age, weaning weight, body condition score, and reproductive tract score in pre-selected beef heifers relative to reproductive potential.

Background: Artificial insemination is a preferred breeding method for beef heifers as it advances the genetic background, produces a predictive and profitable calving season, and extends the heifer's reproductive life span. As reproductive efficiency in heifers is key for the success of beef cattle production systems, following artificial insemination, heifers are exposed to a bull for the remainder of the breeding season. Altogether, up to 95% of heifers might become pregnant in their first breeding season.

Rainbow-Seq: Combining Cell Lineage Tracing with Single-Cell RNA Sequencing in Preimplantation Embryos.

We developed the Rainbow-seq technology to trace cell division history and reveal single-cell transcriptomes. With distinct fluorescent protein genes as lineage markers, Rainbow-seq enables each single-cell RNA sequencing (RNA-seq) experiment to simultaneously decode the lineage marker genes and read single-cell transcriptomes. We triggered lineage tracking in each blastomere at the 2-cell stage, observed microscopically inequivalent contributions of the progeny to the two embryonic poles at the blastocyst stage, and analyzed every single cell at either 4- or 8-cell stage with deep paired-end sequencing of full-length transcripts.

Plasma metabolomic profiles differ at the time of artificial insemination based on pregnancy outcome, in Bos taurus beef heifers.

Infertility remains the most prevalent reason for cattle being removed from production environments. We utilized metabolomic profiling to identify metabolites in the blood plasma that may be useful in identifying infertile heifers at the time of artificial insemination (AI). Prior to AI, phenotypic parameters including body condition, weight, and reproductive organ measurements were collected.

Transcriptome data of peripheral white blood cells from beef heifers collected at the time of artificial insemination.

The reproductive performance of heifers within their first breeding season influences the success of beef cattle operations. Therefore, a means to identify infertile and late breeding heifers before the start of the breeding season holds great promise for the future of the beef industry. Pubertal beef heifers were subjected to estrous synchronization and fixed time artificial insemination (FTAI).