Identification of Melatonin-Regulated Genes in the Ovine Pituitary Pars Tuberalis, a Target Site for Seasonal Hormone Control

The pars tuberalis (PT) of the pituitary gland expresses a high density of melatonin (MEL) receptors and is believed to regulate seasonal physiology by decoding changes in nocturnal melatonin secretion. Circadian clock genes are known to be expressed in the PT in response to the decline (Per1) and onset (Cry1) of MEL secretion, but to date little is known of other molecular changes in this key MEL target site. To identify transcriptional pathways that may be involved in the diurnal and photoperiod-transduction mechanism, we performed a whole genome transcriptome analysis using PT RNA isolated from sheep culled at three time points over the 24-h cycle under either long or short photoperiods. Our results reveal 153 transcripts where expression differs between photoperiods at the light-dark transition and 54 transcripts where expression level was more globally altered by photoperiod (all time points combined). Cry1 induction at night was associated with up-regulation of genes coding for NeuroD1 (neurogenic differentiation factor 1), Pbef / Nampt (nicotinamide phosphoribosyltransferase) , Hif1α (hypoxia-inducible factor-1α), and Kcnq5 (K channel) and down-regulation of Rorβ, a key clock gene regulator. Using in situ hybridization, we confirmed day-night differences in expression for Pbef / Nampt, NeuroD1, and Rorβ in the PT. Treatment of sheep with MEL increased PT expression for Cry1, Pbef / Nampt, NeuroD1, and Hif1α, but not Kcnq5. Our data thus reveal a cluster of Cry1-associated genes that are acutely responsive to MEL and novel transcriptional pathways involved in MEL action in the PT

Expression profiles of genes regulating dairy cow fertility: recent findings, ongoing activities and future possibilities

Subfertility has negative effects for dairy farm profitability, animal welfare and sustainability of animal production. Increasing herd sizes and economic pressures restrict the amount of time that farmers can spend on counteractive management Genetic improvement will become increasingly important to restore reproductive performance. Complementary to traditional breeding value estimation procedures, genomic selection based on genome-wide information will become more widely applied. Functional genomics, including transcriptomics (gene expression profiling), produces the information to understand the consequences of selection as it helps to unravel physiological mechanisms underlying female fertility traits. Insight into the latter is needed to develop new effective management strategies to combat subfertility. Here, the importance of functional genomics for dairy cow reproduction so far and in the near future is evaluated. Recent gene profiling studies in the field of dairy cow fertility are reviewed and new data are presented on genes that are expressed in the brains of dairy cows and that are involved in dairy cow oestrus (behaviour). Fast-developing new research areas in the field of functional genomics, such as epigenetics, RNA interference, variable copy numbers and nutrigenomics are discussed including their promising future value for dairy cow fertility

Identification of molecular genetic causes of bull infertility

This is a cumulative thesis that includes three different topics around male infertility in cattle and canine diseases. Whole genome association analysis (GWAS) and whole genome sequencing (WGS), as well as additional experimental approaches, were used to identify the candidate genes for these disorders. One of the current challenges facing the dairy industry is to predict and improve the fertility of bulls. We have detected a Holstein bull, who had been approved for artificial insemination based on his semen characteristics, but did not produce offspring after 412 first inseminations, resulting in a non-return rate (NR-Abw) of -29. To identify causative mutations underlying this idiopathic infertility, GWAS and WGS were performed on different non-return rate bulls followed by genotyping of candidate variants in a large population. Finally, a nonsense mutation (AC_000170.1: g.54429815G>A, rs468948776) in α/β-Hydrolase D16B (ABHD16B) on chromosome 13 was identified as a potential candidate variant. Protein analysis revealed expression of ABHD16B in the testes and epididymis of control bulls, and lipidomic analysis showed significant differences in lipid content of the plasma membrane between carriers and controls. We conclude that ABHD16B may play a role in lipid changes during spermatogenesis or sperm maturation and that the altered lipid content may explain the reduced fertilization capacity of mutant sperm. Hemophilia B is a monogenic, X-chromosome recessive bleeding disorder caused by genetic variants in the coagulation factor IX gene (F9). Here we identify a Hovawart family with hemophilia B Leyden caused by a deletion (NC_006621.3:g.109501492delC) in the F9 promoter within the conserved binding region of hepatocyte nuclear factor 4α (HNF-4α) and androgen receptor (AR). Using EMSA we found that the deletion only eliminated the binding of HNF-4α to the promoter, but did not affect the binding of AR. In vitro approach utilizing luciferase reporter gene assay revealed a significant expression reduction of the mutated promoter compared to wild-type. Our results suggest that dogs provide a natural occurrence model for understanding hemophilia B. Congenital deafness is prevalent in many modern dog breeds, but few causative genes have been identified to date. Here, we attempt to find the genetic cause of congenital deafness around three deaf Australian Stumpy Tail Cattle Dogs (ASCD) using GWAS and WGS. A GWAS was performed on 3 bilateral deaf ASCDs, 43 herding dogs, and one unaffected ASCD, resulting in the identification of 13 significantly associated loci on 6 chromosomes (CFA3, 8, 17, 23, 28, and 37). More than half of the significantly associated signals were localized on CFA37, containing the most significantly associated variant. Further WGS was conducted on same three deaf ASCDs and one control, whereupon a missense variant (NC_006619.3:g.15562684G>A; XP_022270984.1:p.Leu173Phe) of kruppel-like factor 7 (KLF7) on CFA 37 was considered as a candidate variant. Genotyping of the KLF7 variant in an additional cohort of 55 ASCD samples (28 deaf and 27 normal hearing dogs) indicated that the variant was still associated with deafness (p = 0.014), with a calculated penetrance of 0.75. In conclusion, KLF7 is a promising candidate gene for the etiology of deafness in ASCD.2021-11-2

Functionally Annotating Regulatory Elements in the Equine Genome Using Histone Mark ChIP-Seq.

One of the primary aims of the Functional Annotation of ANimal Genomes (FAANG) initiative is to characterize tissue-specific regulation within animal genomes. To this end, we used chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to map four histone modifications (H3K4me1, H3K4me3, H3K27ac, and H3K27me3) in eight prioritized tissues collected as part of the FAANG equine biobank from two thoroughbred mares. Data were generated according to optimized experimental parameters developed during quality control testing. To ensure that we obtained sufficient ChIP and successful peak-calling, data and peak-calls were assessed using six quality metrics, replicate comparisons, and site-specific evaluations. Tissue specificity was explored by identifying binding motifs within unique active regions, and motifs were further characterized by gene ontology (GO) and protein-protein interaction analyses. The histone marks identified in this study represent some of the first resources for tissue-specific regulation within the equine genome. As such, these publicly available annotation data can be used to advance equine studies investigating health, performance, reproduction, and other traits of economic interest in the horse

Polymorphism discovery and allele frequency estimation using high-throughput DNA sequencing of target-enriched pooled DNA samples

BACKGROUND: The central role of the somatotrophic axis in animal post-natal growth, development and fertility is well established. Therefore, the identification of genetic variants affecting quantitative traits within this axis is an attractive goal. However, large sample numbers are a pre-requisite for the identification of genetic variants underlying complex traits and although technologies are improving rapidly, high-throughput sequencing of large numbers of complete individual genomes remains prohibitively expensive. Therefore using a pooled DNA approach coupled with target enrichment and high-throughput sequencing, the aim of this study was to identify polymorphisms and estimate allele frequency differences across 83 candidate genes of the somatotrophic axis, in 150 Holstein-Friesian dairy bulls divided into two groups divergent for genetic merit for fertility. RESULTS: In total, 4,135 SNPs and 893 indels were identified during the resequencing of the 83 candidate genes. Nineteen percent (n = 952) of variants were located within 5' and 3' UTRs. Seventy-two percent (n = 3,612) were intronic and 9% (n = 464) were exonic, including 65 indels and 236 SNPs resulting in non-synonymous substitutions (NSS). Significant (P < 0.01) mean allele frequency differentials between the low and high fertility groups were observed for 720 SNPs (58 NSS). Allele frequencies for 43 of the SNPs were also determined by genotyping the 150 individual animals (Sequenom(® )MassARRAY). No significant differences (P > 0.1) were observed between the two methods for any of the 43 SNPs across both pools (i.e., 86 tests in total). CONCLUSIONS: The results of the current study support previous findings of the use of DNA sample pooling and high-throughput sequencing as a viable strategy for polymorphism discovery and allele frequency estimation. Using this approach we have characterised the genetic variation within genes of the somatotrophic axis and related pathways, central to mammalian post-natal growth and development and subsequent lactogenesis and fertility. We have identified a large number of variants segregating at significantly different frequencies between cattle groups divergent for calving interval plausibly harbouring causative variants contributing to heritable variation. To our knowledge, this is the first report describing sequencing of targeted genomic regions in any livestock species using groups with divergent phenotypes for an economically important trait

Prioritizing single-nucleotide polymorphisms and variants associated with clinical mastitis

Next-generation sequencing technology has provided resources to easily explore and identify candidate single-nucleotide polymorphisms (SNPs) and variants. However, there remains a challenge in identifying and inferring the causal SNPs from sequence data. A problem with different methods that predict the effect of mutations is that they produce false positives. In this hypothesis, we provide an overview of methods known for identifying causal variants and discuss the challenges, fallacies, and prospects in discerning candidate SNPs. We then propose a three-point classification strategy, which could be an additional annotation method in identifying causalities

The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing

Kinetoplastid parasites—trypanosomes and leishmanias—infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5'-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and accumulation of peri-nuclear granules. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, the results indicate that mRNA processing is a primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing, and the EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Molecular modeling results suggested that inhibition of CPSF3 by AN7973 is feasible. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition and resistance after CPSF3 expression did not, however, always correlate and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. It is therefore possible that the modes of action of oxaboroles that target trypanosome mRNA processing might extend beyond CPSF3 inhibition

Comparative Analysis of V-Akt Murine Thymoma Viral Oncogene Homolog 3 (AKT3) Gene between Cow and Buffalo Reveals Substantial Differences for Mastitis

AKT3 gene is a constituent of the serine/threonine protein kinase family and plays a crucial role in synthesis of milk fats and cholesterol by regulating activity of the sterol regulatory element binding protein (SREBP). AKT3 is highly conserved in mammals and its expression levels during the lactation periods of cattle are markedly increased. AKT3 is highly expressed in the intestine followed by mammary gland and it is also expressed in immune cells. It is involved in the TLR pathways as effectively as proinflammatory cytokines. The aims of this study were to investigate the sequences differences between buffalo and cow. Our results showed that there were substantial differences between buffalo and cow in some exons and noteworthy differences of the gene size in different regions. We also identified the important consensus sequence motifs, variation in 2000 upstream of ATG, substantial difference in the “3′UTR” region, and miRNA association in the buffalo sequences compared with the cow. In addition, genetic analyses, such as gene structure, phylogenetic tree, position of different motifs, and functional domains, were performed to establish their correlation with other species. This may indicate that a buffalo breed has potential resistance to disease, environment changes, and airborne microorganisms and some good production and reproductive traits