Genetic Background of Blood ß-Hydroxybutyrate Acid Concentrations in Early-Lactating Holstein Dairy Cows Based on Genome-Wide Association Analyses.

Ketosis is a common metabolic disorder in the early lactation of dairy cows. It is typically diagnosed by measuring the concentration of ß-hydroxybutyrate (BHB) in the blood. This study aimed to estimate the genetic parameters of blood BHB and conducted a genome-wide association study (GWAS) based on the estimated breeding value. Phenotypic data were collected from December 2019 to August 2023, comprising blood BHB concentrations in 45,617 Holstein cows during the three weeks post-calving across seven dairy farms. Genotypic data were obtained using the Neogen Geneseek Genomic Profiler (GGP) Bovine 100 K SNP Chip and GGP Bovine SNP50 v3 (Illumina Inc., San Diego, CA, USA) for genotyping. The estimated heritability and repeatability values for blood BHB levels were 0.167 and 0.175, respectively. The GWAS result detected a total of ten genome-wide significant associations with blood BHB. Significant SNPs were distributed in Bos taurus autosomes (BTA) 2, 6, 9, 11, 13, and 23, with 48 annotated candidate genes. These potential genes included those associated with insulin regulation, such as INSIG2, and those linked to fatty acid metabolism, such as HADHB, HADHA, and PANK2. Enrichment analysis of the candidate genes for blood BHB revealed the molecular functions and biological processes involved in fatty acid and lipid metabolism in dairy cattle. The identification of novel genomic regions in this study contributes to the characterization of key genes and pathways that elucidate susceptibility to ketosis in dairy cattle.

Population Structure and Selection Signal Analysis of Nanyang Cattle Based on Whole-Genome Sequencing Data.

With a rich breeding history, Nanyang cattle (NY cattle) have undergone extensive natural and artificial selection, resulting in distinctive traits such as high fertility, excellent meat quality, and disease resistance. This makes them an ideal model for studying the mechanisms of environmental adaptability. To assess the population structure and genetic diversity of NY cattle, we performed whole-genome resequencing on 30 individuals. These data were then compared with published whole-genome resequencing data from 432 cattle globally. The results indicate that the genetic structure of NY cattle is significantly different from European commercial breeds and is more similar to North-Central Chinese breeds. Furthermore, among all breeds, NY cattle exhibit the highest genetic diversity and the lowest population inbreeding levels. A genome-wide selection signal analysis of NY cattle and European commercial breeds using Fst, θπ-ratio, and θπ methods revealed significant selection signals in genes associated with reproductive performance and immunity. Our functional annotation analysis suggests that these genes may be responsible for reproduction (MAP2K2, PGR, and GSE1), immune response (NCOA2, HSF1, and PAX5), and olfaction (TAS1R3). We provide a comprehensive overview of sequence variations in the NY cattle genome, revealing insights into the population structure and genetic diversity of NY cattle. Additionally, we identify candidate genes associated with important economic traits, offering valuable references for future conservation and breeding efforts of NY cattle.

Integrative analyses of the mRNA expression profile reveal the involvement of STC1 in chicken folliculogenesis.

Efficient ovarian follicle development, maturation, and ovulation are critical for egg production performance. Previous research has underscored the importance of messenger RNAs (mRNAs) in regulating development and folliculogenesis in chicken ovarians. However, the molecular mechanism is not fully understood, especially in the late period of the laying cycle. In the present study, ovarian tissues from 80-week-old Hy-Line Brown layers (three with high and three with low rates of egg laying) were collected for transcriptome sequencing. A total of 306 differentially expressed genes (DEGs) were identified in this study, at a false discovery rate (FDR)-corrected P-value < 0.05 and a log2|fold change| (log2|FC|) ≥1.5. Among these DEGs, stanniocalcin 1 (STC1) was mainly related to cellular processes, single-organism processes, biological regulation, metabolic processes, developmental processes, and reproductive processes. Then, we further investigated the regulation of STC1 during chicken follicle development and found that STC1 inhibited the proliferation and stimulated the apoptosis of follicular granulosa cells (GCs), and decreased the expression of progesterone (P4) and estradiol (E2). Collectively, these results suggest that STC1 plays an important role in chicken follicle development by decreasing GC proliferation and steroidogenesis and stimulating GC apoptosis. This study contributes to the understanding of the reproductive biology of laying hens in the late period of the laying cycle and further lays a foundation for the improvement of egg production in poultry breeding.

The egg production performance of chickens is an essential economic trait that differs significantly between high- and low-egg-laying breeds. In addition to external factors such as feeding, light, and environment, the periodic recruitment of pre-hierarchical follicles and the normal development of hierarchical follicles affect this difference. Thus, we used high-throughput sequencing technology to perform transcriptome analysis of ovarian tissues from 80-wk-old Hy-Line Brown layers with high- and low-egg-laying rates (HH and HL), and an association with the laying performance gene stanniocalcin 1 (STC1) was found. The proliferation and apoptosis of granulosa cells (GCs), as the basic functional cells of ovarian follicles, are highly correlated with the normal development and regression of follicles. Therefore, this study used ovarian follicular GCs cultured in vitro to study the effects of the STC1 gene on the proliferation, apoptosis, and secretion function of GCs and to explore its mechanism of action, laying a foundation for the study of the regulation of the STC1 gene on follicular development.

Occurrence, Distribution, and Risk Assessment of Antibiotics in the Aquatic Environment of the Karst Plateau Wetland of Yangtze River Basin, Southwestern China.

In this study, the occurrence, distribution, and ecological risk of 40 commonly used antibiotics, including 15 sulfonamides (SAs), 9 fluoroquinolones (FQs), 7 macrolides (MCs), 3 tetracyclines (TCs), 2 chloramphenicols (CAPs), and 4 other categories, in the aquatic environment of the karst plateau wetland Caohai of the Yangtze River basin in southwestern China are reported. In total, 27 antibiotics were detected, with the detection rate ranging from 5% to 100%. The total concentration at each site ranged from 21.8 ng/L to 954 ng/L, with the average concentration being 189 ng/L. FQs and MCs were the most predominant categories, contributing 29.3% and 25.0% of the total antibiotic burden. The five most commonly detected antibiotics were ciprofloxacin (CIP), oxytetracycline (OTC), acetyl sulfamethoxazole (ASMZ), norfloxacin (NOR), and florfenicol (FF). The spatial distribution of the total concentration at each site demonstrated a decreasing trend from the southeastern area upstream adjoining the main counties to the northwestern area downstream, indicating that human activities have a great impact. Meanwhile, the natural attenuation rates of different types of antibiotics in the direction of flow ranged from 17.6% to 100%, which implied the natural purification potential of the wetland for antibiotics. The cluster analysis results indicated that domestic sewage and wastewater from agriculture and animal husbandry were the main sources of contamination in the surrounding wetland. Risk quotients (RQs) assessment showed that most of the individuals were at low to medium risk and that the adverse risks posed by mixtures of antibiotics were higher than those posed by the individual antibiotics.

Genome-wide association study identifies SNPs for growth performance and serum indicators in Valgus-varus deformity broilers (Gallus gallus) using ddGBS sequencing.

BACKGROUND: Valgus-varus deformity (VVD) is a lateral or middle deviation of the tibiotarsus or tarsometatarsus, which is associated with compromised growth, worse bone quality and abnormal changes in serum indicators in broilers. To investigate the genetic basis of VVD, a genome wide association study (GWAS) was performed to identify candidate genes and pathways that are responsible for VVD leg disease, serum indicators and growth performance in broilers. RESULTS: In total, VVD phenotype, seven serum indicators and three growth traits were measured for 126 VVD broilers (case group) and 122 sound broilers (control group) based on a high throughput genome wide genotyping-by-sequencing (GBS) method. After quality control 233 samples (113 sound broilers and 120 VVD birds) and 256,599 single nucleotide polymorphisms (SNPs) markers were used for further analysis. As a result, a total of 5 SNPs were detected suggestively significantly associated with VVD and 70 candidate genes were identified that included or adjacent to these significant SNPs. In addition, 43 SNPs located on Chr24 (0.22 Mb - 1.79 Mb) were genome-wide significantly associated with serum alkaline phosphatase (ALP) and 38 candidate genes were identified. Functional enrichment analysis showed that these genes are involved in two Gene Ontology (GO) terms related to bone development (cartilage development and cartilage condensation) and two pathways related to skeletal development (Toll-like receptor signaling pathway and p53 signaling pathway). BARX2 (BARX homeobox 2) and Panx3 (Pannexin 3) related to skeleton diseases and bone quality were obtained according to functional analysis. According to the integration of GWAS with transcriptome analysis, HYLS1 (HYLS1 centriolar and ciliogenesis associated) was an important susceptibility gene. CONCLUSIONS: The results provide some reference for understanding the relationship between metabolic mechanism of ALP and pathogenesis of VVD, which will provide a theoretical basis for disease-resistant breeding of chicken leg soundness.

Nontarget and high-throughput screening of pesticides and metabolites residues in tea using ultra-high-performance liquid chromatography and quadrupole-orbitrap high-resolution mass spectrometry.

A Sin-QuEChERS, coupled to UHPLC Q-Exactive Orbitrap MS, was used for nontargeted high-throughput rapid screening and quantitative analysis of residual pesticides and metabolites in green teas. The sample was extracted with 0.1% formic acid in acetonitrile with shaking, salted out and centrifuged, and purified with Sin-QuEChERS Nano solid phase extraction column; with Full MS/ddMS2 as the data collection mode, the database containing 384 pesticides combined with Trace Finder 3.0 software, In the absence of standard products, rapid screening and confirmation of potential pesticide residues in tea samples with accurate mass, isotope abundance ratio, secondary fragment ions, etc. 20 pesticides were used as quality controls to verify the screening method, and the linearity of these pesticides was between 1 and 200 µg/L, and the correlation coefficients were all greater than 0.9922. Moreover, the LOQ was between 0.002 and 0.01 mg/kg. The average recoveries of spiked tea samples were 74%-111%. Efficiency and reliability of this method were investigated by the analysis of 38 Chinese green tea samples. 18 potential residual pesticides were detected by non-targeted screening. The researchers then conducted a quantitative analysis of the 18 potential residual pesticides.

The Chicken Pan-Genome Reveals Gene Content Variation and a Promoter Region Deletion in IGF2BP1 Affecting Body Size.

Domestication and breeding have reshaped the genomic architecture of chicken, but the retention and loss of genomic elements during these evolutionary processes remain unclear. We present the first chicken pan-genome constructed using 664 individuals, which identified an additional approximately 66.5-Mb sequences that are absent from the reference genome (GRCg6a). The constructed pan-genome encoded 20,491 predicated protein-coding genes, of which higher expression levels are observed in conserved genes relative to dispensable genes. Presence/absence variation (PAV) analyses demonstrated that gene PAV in chicken was shaped by selection, genetic drift, and hybridization. PAV-based genome-wide association studies identified numerous candidate mutations related to growth, carcass composition, meat quality, or physiological traits. Among them, a deletion in the promoter region of IGF2BP1 affecting chicken body size is reported, which is supported by functional studies and extra samples. This is the first time to report the causal variant of chicken body size quantitative trait locus located at chromosome 27 which was repeatedly reported. Therefore, the chicken pan-genome is a useful resource for biological discovery and breeding. It improves our understanding of chicken genome diversity and provides materials to unveil the evolution history of chicken domestication.

Differentially Expressed lncRNAs Related to the Development of Abdominal Fat in Gushi Chickens and Their Interaction Regulatory Network.

Chickens are one of the most important sources of meat worldwide, and the growth status of abdominal fat is closely related to production efficiency. Long noncoding RNAs (lncRNAs) play an important role in lipid metabolism and deposition regulation. However, research on the expression profile of lncRNAs related to the development of abdominal fat in chickens after hatching and their interaction regulatory networks is still lacking. To characterize the lncRNA expression profile during the development of chicken abdominal fat, abdominal adipose tissues from 6-, 14-, 22-, and 30-week-old Chinese Gushi chickens were herein used to construct 12 cDNA libraries, and a total of 3,827 new lncRNAs and 5,466 previously annotated lncRNAs were revealed. At the same time, based on the comparative analysis of five combinations, 276 differentially expressed lncRNAs (DE-lncRNAs) were screened. Functional enrichment analysis showed that the predicted target genes of these DE-lncRNAs were significantly enriched in pathways related to the posttranscriptional regulation of gene expression, negative regulation of cell proliferation, cell adhesion and other biological processes, glycosphingolipid biosynthesis, PPAR signaling, fatty acid degradation, fatty acid synthesis and others. In addition, association analysis of the lncRNA transcriptome profile was performed, and DE-lncRNA-related lncRNA-mRNA, lncRNA-miRNA and lncRNA-miRNA-mRNA interaction regulatory networks were constructed. The results showed that DE-lncRNA formed a complex network with PPAR pathway components, including PPARD, ACOX1, ADIPOQ, CPT1A, FABP5, ASBG2, LPL, PLIN2 and related miRNAs, including mir-200b-3p, mir-130b-3p, mir-215-5p, mir-122-5p, mir-223 and mir-125b-5p, and played an important regulatory role in biological processes such as lipid metabolism, adipocyte proliferation and differentiation. This study described the dynamic expression profile of lncRNAs in the abdominal fat of Gushi chickens for the first time and constructed the DE-lncRNA interaction regulatory network. The results expand the number of known lncRNAs in chicken abdominal fat and provide valuable resources for further elucidating the posttranscriptional regulatory mechanism of chicken abdominal fat development or deposition.

Integrating RNA-Seq with GWAS reveals novel insights into the molecular mechanism underpinning ketosis in cattle.

BACKGROUND: Ketosis is a common metabolic disease during the transition period in dairy cattle, resulting in long-term economic loss to the dairy industry worldwide. While genetic selection of resistance to ketosis has been adopted by many countries, the genetic and biological basis underlying ketosis is poorly understood. RESULTS: We collected a total of 24 blood samples from 12 Holstein cows, including 4 healthy and 8 ketosis-diagnosed ones, before (2 weeks) and after (5 days) calving, respectively. We then generated RNA-Sequencing (RNA-Seq) data and seven blood biochemical indicators (bio-indicators) from leukocytes and plasma in each of these samples, respectively. By employing a weighted gene co-expression network analysis (WGCNA), we detected that 4 out of 16 gene-modules, which were significantly engaged in lipid metabolism and immune responses, were transcriptionally (FDR < 0.05) correlated with postpartum ketosis and several bio-indicators (e.g., high-density lipoprotein and low-density lipoprotein). By conducting genome-wide association signal (GWAS) enrichment analysis among six common health traits (ketosis, mastitis, displaced abomasum, metritis, hypocalcemia and livability), we found that 4 out of 16 modules were genetically (FDR < 0.05) associated with ketosis, among which three were correlated with postpartum ketosis based on WGCNA. We further identified five candidate genes for ketosis, including GRINA, MAF1, MAFA, C14H8orf82 and RECQL4. Our phenome-wide association analysis (Phe-WAS) demonstrated that human orthologues of these candidate genes were also significantly associated with many metabolic, endocrine, and immune traits in humans. For instance, MAFA, which is involved in insulin secretion, glucose response, and transcriptional regulation, showed a significantly higher association with metabolic and endocrine traits compared to other types of traits in humans. CONCLUSIONS: In summary, our study provides novel insights into the molecular mechanism underlying ketosis in cattle, and highlights that an integrative analysis of omics data and cross-species mapping are promising for illustrating the genetic architecture underpinning complex traits.

Technical note: Development and application of KASP assays for rapid screening of 8 genetic defects in Holstein cattle.

Specific DNA mutations underlying several genetic defects associated with embryo loss or reduced calf survivability have been identified in dairy cattle, and a convenient and cost-effective platform is required for their routine screening. We developed Kompetitive allele-specific PCR (KASP) assays for discrimination of the wild-type alleles from the associated defective alleles at each of 8 common genetic defects in Holstein cattle, involving 5 SNP [HH1, HH3, HH4, bovine leukocyte adhesion deficiency (BLAD), and complex vertebral malformation (CVM)] and 3 insertion or deletion mutations [HH5, haplotype for cholesterol deficiency (HCD), and brachyspina (BS)]. A total of 390 cows from a Chinese Holstein herd were genotyped and the carriers identified at 7 of these 8 loci (except HH4), with the highest carrier frequencies found for CVM (10.5%) and HH1 (10.0%), followed by HH3 (2.6%), BS (2.1%), HCD (1.3%), HH5 (0.8%), and BLAD (0.5%). Surprisingly, 102 cows (26.2%) carried at least 1 of the 7 defective alleles. Our results demonstrate that these KASP assays are simple, rapid, and reliable for the detection of multiple genetic defects. The high carrier frequency of these genetic defects indicates an urgent need for routine molecular testing to eliminate the deleterious alleles from Chinese Holstein cattle.

Genome-wide association study identifies QTLs for displacement of abomasum in Chinese Holstein cattle1.

Displacement of abomasum (DA) is one of the most common and important disorders in dairy cattle. The objective of the present study was to detect the quantitative trait loci (QTL) for DA in Chinese Holstein using single-step genomic BLUP methodology. A total of 60,556 producer-recorded DA event records from 32,190 cows, together with 2,336 genotyped animals with 40,054 SNP markers, were used for the analysis. Genomic data were incorporated into a threshold model for variance component estimation, and the estimated heritability of DA was 0.108 (SE = 0.086). Results of genome-wide association studies were reported as the proportion of genetic variance explained 20-SNP windows. Eight QTLs covering 129 genes on Bos taurus autosomes 2, 4, 7, 10, 14, 17, 20 showed associations with DA. Ten genes, namely BMP4, SOCS4, GCH1, DDHD1, ATG14, ACBP/DBI, SMO, AHCYL2, CYP7A1, and CACNA1A, involved in insulin metabolism and lipid metabolism pathways may be considered as candidate genes of DA in dairy. The identified QTLs, biological pathways, and associated genes underlying DA identified from the present study will contribute to the understanding of the genetic architecture of this complex disease.

Estimation of genomic inbreeding coefficients based on high-density SNP markers in Chinese Holstein cattle.

In livestock, inbreeding coefficient based on pedigree information is usually used to evaluate the level of inbreeding. Recently, with cost reduction of high-density SNP genotyping, it's possible to analyze real genomic inbreeding degree using genomic information. In this study, utilizing high-density SNP chip data, we analyzed the frequency and distribution of runs of homozygosity (ROH) in 2107 Chinese Holstein cattle in Beijing area, and calculated 2 genomic inbreeding coefficients, i.e., 1) the proportion of ROH length in the total length of autosomal genome (Froh), and 2) the percentage of homozygous SNPs (Fhom). Then we analyzed the correlation between 2 genomic inbreeding coefficients and the correlation between genomic and pedigree inbreeding coefficients. We totally detected 44 676 ROHs that mainly ranged from 1 to 10 Mb. Various lengths of ROHs existed in the genome. There were more short ROHs than long ROHs. ROHs aren't evenly distributed in chromosomes. The area with most ROHs is in the middle part of chromosome 10. Strong correlation (r > 0.90) existed between 2 kinds of genomic inbreeding coefficients, but the correlation between pedigree and genomic inbreeding coefficients were much lower (r < 0.50). Our finding suggests that pedigree completeness influences the correlation between genomic and pedigree inbreeding. Genomic inbreeding measures may reflect individuals' real inbreeding, which could be a useful tool to evaluate population inbreeding.

Screening for JH1 genetic defect carriers in Jersey cattle by a polymerase chain reaction and restriction fragment length polymorphism assay.

An autosomal recessive genetic defect termed JH1 has been associated with early embryonic loss in the Jersey cattle breed. The genetic basis has been identified as a cytosine to thymine mutation in the CWC15 gene that changes an amino acid from arginine to a stop code. To screen for JH1 carriers in an imported Jersey population in China, a method based on a polymerase chain reaction amplification followed by a restriction fragment length polymorphism assay (PCR-RFLP) was developed for the accurate diagnosis of the JH1 allele. A total of 449 randomly chosen cows were examined with the PCR-RFLP assay, and 31 were identified as JH1 carriers, corresponding to a carrier frequency of 6.9%. The PCR-RFLP method was validated by DNA sequencing of 8 positive and 13 negative samples, with all 21 samples giving the expected DNA sequence. In addition, 3 negative and 3 positive samples were confirmed by a commercial microarray-based single nucleotide polymorphism assay. Finally, samples from 9 bulls in the United States of known status were correctly identified as carriers (5 bulls) or noncarriers (4 bulls). As the JH1 defect has most likely spread worldwide, implementing routine screening is necessary to avoid the risk of carrier-to-carrier matings and to gradually eradicate the deleterious gene.