Genus (United States)

A chromosomal rearrangement such as a reciprocal translocation (RT) in a breeding boar can produce unbalanced gametes during meiosis, leading to a decreased litter size with detrimental economic implications for breeders. FISH and standard karyotyping are currently used to detect RTs, but a fresh sample is required, limiting the shipping conditions. Here, we investigated Hi-C as an alternative technique to diagnose chromosome rearrangements. We show that Hi-C can be used to detect such RTs from either a fresh or a frozen blood sample and therefore this technique represents an alternative to FISH for RT detection.

The objective was to determine the effects of liver abscesses on complete blood counts (CBC) and liver function and to identify blood-based biomarkers for detection of liver abscesses in feedlot cattle. Blood samples were collected on a subset of beef × dairy crossbred heifers with and without liver abscesses (n = 94; n = 113, respectively). From these blood samples, CBC and serum chemistry analytes related to liver health (alkaline phosphatase, gamma glutamyl transferase, bile acids, total bilirubin, and cholesterol) were analyzed to characterize animal health and model for liver abscesses. Abscess presence was predicted using logistic regression models and machine learning algorithms, with individual heifer serving as the experimental unit. In a binary system (abscessed or not), heifers with liver abscesses had lower concentrations of lymphocyte (LYM) and total white blood cell (WBC) concentrations but elevated platelet-to-LYM ratio. Serum chemistry measures were not affected by abscess presence or liver score (severity). In univariate, predictive modeling of CBC measures, only total WBC concentration was accurate (65.85%) at predicting liver abscess presence; however, this was not deemed an acceptable biomarker due to its poor specificity (54.79%). Using total WBC, basophils, red blood cells, and procalcitonin as variables to predict liver abscess condition, the Logit- Boost model produced the greatest accuracy (80.65%) and specificity (85.71%). An accurate blood test that allows for the detection of liver abscesses during the finishing phase may allow for therapeutic management or alternative marketing of cattle; however, the marginal accuracy demonstrated in this study combined with the impracticality and cost of analyzing a blood sample may not be feasible. Regardless, the most explanatory CBC variables in cattle with abscessed livers were suggestive of an immune response. Liver condition influenced the CBC of beef × dairy heifers but not serum chemistry. Multivariate predictive models showed potential for predicting liver abscess condition based on CBC. The results of this study warrant further investigation for biomarkers of the liver abscess condition. An antemortem indicator for liver abscesses could prove useful for making management decisions and in research applications, particularly in evaluating interventions.

Porcine reproductive and respiratory syndrome (PRRS) is one of the world's most persistent viral pig diseases, with a significant economic impact on the pig industry. PRRS affects pigs of all ages, causing late-term abortions and stillbirths in sows, respiratory disease in piglets, and increased susceptibility to secondary bacterial infection with a high mortality rate. PRRS disease is caused by a positive single-stranded RNA PRRS virus (PRRSV), which has a narrow host-cell tropism limited to monocyte-macrophage lineage cells. Several studies demonstrated that the removal of CD163 protein or, as a minimum, its scavenger receptor cysteine-rich domain 5 (SRCR5) precludes the viral genome release, conferring resistance to PRRSV in live animals. Today, very limited information exists about the impact of such edits on animal performance from birth to maturity in pigs. Using CRISPR-Cas9 with dual-guide RNAs and non-homologous end joining (NHEJ), first-generation (E0) pigs were produced with a deletion of exon 7 in the CD163 gene. The selected pigs were bred to produce the next three generations of pigs to establish multiple lines of pigs homozygous for the edited allele, thereby confirming that the CD163 gene with removed exon 7 was stable during multiple breeding cycles. The pigs were evaluated relative to non-edited pigs from birth to maturity, including any potential changes in meat composition and resistance to PRRSV. This study demonstrates that removing the SRCR5 domain from the CD163 protein confers resistance to PRRSV and, relative to unedited pigs, resulted in no detected differences in meat composition and no changes in the growth rate, health, and ability to farrow. Together, these results support the targeted use of gene editing in livestock animals to address significant diseases without adversely impacting the health and well-being of the animals or the food products derived from them.

Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. Our previous work established that gonadotropin-releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope-derived LβT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry (MS) to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterized the temporal dynamics of this modification. Our results show that actin and tubulin are citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. The data show that 10 nM GnRHa induces the citrullination of β-actin, with elevated levels occurring at 10 min. The level of β-actin citrullination is reduced in the presence of the pan-PAD inhibitor biphenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa-induced actin reorganization in dispersed murine gonadotrope cells. GnRHa induces the citrullination of β-tubulin, with elevated levels occurring at 30 min, and this response is attenuated in the presence of PAD inhibition. To examine the functional consequence of β-tubulin citrullination, we utilized fluorescently tagged end binding protein 1 (EB1-GFP) to track the growing plus end of microtubules (MT) in real time in transfected LβT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that the MT average lifetime increases following 30 min of GnRHa treatment, but this increase is attenuated by PAD inhibition. Taken together, our data suggest that GnRHa-induced citrullination alters actin reorganization and MT lifetime in gonadotrope cells.

Profiling bovine blastocyst transcriptome at the single-cell level has enabled us to reveal the first cell lineage segregation, during which the inner cell mass (ICM), trophectoderm (TE), and an undefined population of transitional cells were identified. By comparing the transcriptome of blastocysts derived in vivo (IVV), in vitro from a conventional culture medium (IVC), and in vitro from an optimized reduced nutrient culture medium (IVR), we found a delay of the cell fate commitment to ICM in the IVC and IVR embryos. Developmental potential differences between IVV, IVC, and IVR embryos were mainly contributed by ICM and transitional cells. Pathway analysis of these non-TE cells between groups revealed highly active metabolic and biosynthetic processes, reduced cellular signaling, and reduced transmembrane transport activities in IVC embryos that may lead to reduced developmental potential. IVR embryos had lower activities in metabolic and biosynthetic processes but increased cellular signaling and transmembrane transport, suggesting these cellular mechanisms may contribute to improved blastocyst development compared to IVC embryos. However, the IVR embryos had compromised development compared to IVV embryos with notably over-active transmembrane transport activities that impaired ion homeostasis.

Disease resistance genes in livestock provide health benefits to animals and opportunities for farmers to meet the growing demand for affordable, high-quality protein. Previously, researchers used gene editing to modify the porcine CD163 gene and demonstrated resistance to a harmful virus that causes porcine reproductive and respiratory syndrome (PRRS). To maximize potential benefits, this disease resistance trait needs to be present in commercially relevant breeding populations for multiplication and distribution of pigs. Toward this goal, a first-of-its-kind, scaled gene editing program was established to introduce a single modified CD163 allele into four genetically diverse, elite porcine lines. This effort produced healthy pigs that resisted PRRS virus infection as determined by macrophage and animal challenges. This founder population will be used for additional disease and trait testing, multiplication, and commercial distribution upon regulatory approval. Applying CRISPR-Cas to eliminate a viral disease represents a major step toward improving animal health.

Peroxisome proliferator-activated receptor gamma (PPARG) is a critical regulator of placental function, but earlier roles in preimplantation embryo development and embryonic origins of placental formation have not been established. Results herein demonstrate that PPARG responds to pharmacologic stimulation in the bovine preimplantation embryo and influences blastocyst development, cell lineage specification, and transcripts important for placental function. Peroxisome proliferator-activated receptor gamma (PPARG) is a key regulator of metabolism with conserved roles that are indispensable for placental function, suggesting previously unidentified and important roles in preimplantation embryo development. Herein, we report the functional characterization of bovine PPARG to reveal expression beginning on D6 of development with nuclear and ubiquitous patterns. Day 6 PPARG+ embryos have fewer total cells and a lower proportion of trophectoderm cells compared to PPARG- embryos (P < 0.05). Coculture with a PPARG agonist, rosiglitazone (Ros), or antagonist GW9662 (GW), decreases blastocyst development (P < 0.01). Day 7.5 (D7.5) developmentally delayed embryos exposed to Ros express lower transcript abundance of key genes important for placental development and cell lineage formation (CDX2, RXRB, SP1, TFAP2C, SIRT1, and PTEN). In contrast, Ros does not alter transcript abundance in D7.5 blastocysts, but GW treatment lowers RXRA, RXRB, SP1, and NFKB1 expression. Knockout of embryonic PPARG does not alter blastocyst formation and hatching ability but decreases total cell number in D7.5 blastocysts. The decreased embryo development response and affected pathways following targeted pharmacological perturbation vs embryonic knockout of PPARG suggest roles of both maternal and embryonic origins. These data reveal regulatory contributions of PPARG in preimplantation embryo development, cell lineage formation, and regulation of transcripts associated with placental function.

BackgroundMost genomic prediction applications in animal breeding use genotypes with tens of thousands of single nucleotide polymorphisms (SNPs). However, modern sequencing technologies and imputation algorithms can generate ultra-high-density genotypes (including millions of SNPs) at an affordable cost. Empirical studies have not produced clear evidence that using ultra-high-density genotypes can significantly improve prediction accuracy. However, (whole-genome) prediction accuracy is not very informative about the ability of a model to capture the genetic signals from specific genomic regions. To address this problem, we propose a simple methodology that detects chromosome regions for which a specific model (e.g., single-step genomic best linear unbiased prediction (ssGBLUP)) may fail to fully capture the genetic signal present in such segments—a phenomenon that we refer to as signal leakage. We propose to detect regions with evidence of signal leakage by testing the association of residuals from a pedigree or a genomic model with SNP genotypes. We discuss how this approach can be used to map regions with signals that are poorly captured by a model and to identify strategies to fix those problems (e.g., using a different prior or increasing marker density). Finally, we explored the proposed approach to scan for signal leakage of different models (pedigree-based, ssGBLUP, and various Bayesian models) applied to growth-related phenotypes (average daily gain and backfat thickness) in pigs.ResultsWe report widespread evidence of signal leakage for pedigree-based models. Including a percentage of animals with SNP data in ssGBLUP reduced the extent of signal leakage. However, local peaks of missed signals remained in some regions, even when all animals were genotyped. Using variable selection priors solves leakage points that are caused by excessive shrinkage of marker effects. Nevertheless, these models still miss signals in some regions due to low linkage disequilibrium between the SNPs on the array used and causal variants. Thus, we discuss how such problems could be addressed by adding sequence SNPs from those regions to the prediction model.ConclusionsResidual single-marker regression analysis is a simple approach that can be used to detect regional genomic signals that are poorly captured by a model and to indicate ways to fix such problems.