Paratuberculosis is a contagious, chronic and enteric disease in ruminants, which is caused by Mycobacterium avium subspecies paratuberculosis (MAP) infection, resulting in enormous economic losses worldwide. There is currently no effective cure for MAP infection or a vaccine, it is thus important to explore the genetic variants that contribute to host susceptibility to infection by MAP, which may provide a better understanding of the mechanisms of paratuberculosis and benefit animal genetic improvement. Herein we performed a genome-wide association study (GWAS) to identify genomic regions and candidate genes associated with susceptibility to MAP infection in dairy cattle. Using Illumina Bovine 50 K (54,609 SNPs) and GeneSeek HD (138,893 SNPs) chips, two analytical approaches were performed, GRAMMAR-GC and ROADTRIPS in 937 Chinese Holstein cows, among which individuals genotyped by the 50 K chip were imputed to HD SNPs with Beagle software. Consequently, 15 and 11 significant SNPs (P < 5 × 10− 5) were identified with GRAMMAR-GC and ROADTDRIPS, respectively. A total of 10 functional genes were in proximity to (i.e., within 1 Mb) these SNPs, including IL4, IL5, IL13, IRF1, MyD88, PACSIN1, DEF6, TDP2, ZAP70 and CSF2. Functional enrichment analysis showed that these genes were involved in immune related pathways, such as interleukin, T cell receptor signaling pathways and inflammatory bowel disease (IBD), implying their potential associations with susceptibility to MAP infection. In addition, by examining the publicly available cattle QTLdb, a previous QTL for MAP was found to be overlapped with one of regions detected currently at 32.5 Mb on BTA23, where the TDP2 gene was anchored. In conclusion, we identified 26 SNPs located on 15 chromosomes in the Chinese Holstein population using two GWAS strategies with high density SNPs. Integrated analysis of GWAS, biological functions and the reported QTL information helps to detect positional candidate genes and the identification of regions associated with susceptibility to MAP traits in dairy cattle.

Milk fat synthesis in cows mainly includes the synthesis of short- and medium-chain fatty acids, the uptake, transport, and activation of long-chain fatty acids (LCFAs), the synthesis of triglycerides, and the synthesis of the genes, transcription factors, and signaling pathways involved. Although the various stages of milk fat synthesis have been outlined in previous research, only partial processes have been revealed. CLA consists of an aggregation of positional and geometric isomers of linoleic fatty acid, and the accumulated evidence suggests that the two isomers of the active forms of CLA (cis-9, trans-11 conjugated linoleic acid and trans-10, cis-12 conjugated linoleic acid, abbreviated as c9, t11-CLA and t10, c12-CLA) can reduce the fat content in milk by regulating lipogenesis, fatty acid (FA) uptake, oxidation, and fat synthesis. However, the mechanism through which CLA inhibits milk fat synthesis is unique, with most studies focusing only on the effects of CLA on one of the genes, transcription factors, or signaling pathways involved. In this study, we summarized the structure and function of classic genes and pathways (mTOR, SREBP, AMPK, and PPARG) and new genes or pathways (THRSP, METTL3, ELOVL, and LPIN1) involved in each stage of milk fat synthesis and demonstrated the interactions between genes and pathways. We also examined the effects of other substances (melanin, nicotinic acid, SA, etc.). Furthermore, we evaluated the influence of β-sitosterol, sodium butyrate, Met arginine, and Camellia oleifera Abel on milk fat synthesis to improve the mechanism of milk fat synthesis in cows and provide a mechanistic reference for the use of CLA in inhibiting milk fat biosynthesis.

Ketosis is a common nutritional, metabolic disease during the perinatal period in dairy cows characterized by elevated blood β-hydroxybutyrate (BHBA). In this study, RNA sequencing (RNA-seq) was performed to investigate adaptive changes in adipose tissue during the perinatal period of dairy cows. Blood and tailhead subcutaneous white adipose tissue (sWAT) were obtained from ketotic cows (Ket = 8, BHBA ≥ 1.4 mmol/L) and non-ketotic cows (Nket = 6, BHBA < 1.4 mmol/L) 21 d pre-partum and 10 d post-partum. Compared with pre-partum, decreased lipid synthesis due to down-regulation of PCK1 may be in a strong association with clinical ketosis. Simultaneously, PCK2 was downregulated in the Ket postnatally compared to its expression prenatally, and the expression of PCK2 was 2.7~4.2 times higher than that of PCK1, implying a more severe lipid storage impairment in the Ket. Moreover, compared to pre-partum, the upregulated differentially expressed genes post-partum in the Ket were enriched in the inflammatory response biological process. The higher expression of TNC (tenascin C) in the post-partum Ket relative to the Nket suggested that the adipose tissue of ketotic cows might also be accompanied by tissue fibrosis. Notably, pre-partum CD209 was higher in the Ket than in the Nket, which might be used as a candidate marker for the pre-partum prediction of ketosis. Combined with published gene expression traits, these results suggested that inflammation leads to a more widespread downregulation of the lipid synthesis gene network in adipose tissue in ketotic cows. Additionally, sWAT in post-partum cows with ketosis might also be accompanied by tissue fibrosis which could make the treatment of ketosis more difficult.

Targeted sequencing, a pivotal branch of next-generation sequencing (NGS), enables the selective enrichment of specific genomic regions and has demonstrated significant advantages in the detection of animal pathogens. This review systematically explores the underlying principles of targeted sequencing, various enrichment strategies—including PCR amplification, probe hybridization, and CRISPR-Cas systems—and their key applications in veterinary pathogen diagnostics. Due to its high throughput, sensitivity, and cost-effectiveness, targeted sequencing has been successfully applied in the multiplex detection of pathogens in economically significant livestock, such as cattle, as well as in the surveillance of antimicrobial resistance (AMR) genes, pathogen typing, and source tracing. It is particularly effective in identifying mixed infections and low-abundance pathogens. Nonetheless, wide application is restricted by some factors, like incomprehensive reference databases, cost-effectiveness, and limited application in primary-level laboratories. Further development directions are AI-based panel design, multimodal diagnostic platform integration, standard workflow construction, and introduction of a multi-omics method. Such progress focuses on enhancing the targeted sequencing scalability and precision consistent with the “One Health” initiative objective.

Cholesterol in the circulation is partly driven by changes in feed intake, but aspects of cholesterol metabolism during development of fatty liver are not well known. The objective of this study was to investigate mechanisms of cholesterol metabolism in calf hepatocytes challenged with high concentrations of fatty acids (FA). To address mechanistic insights regarding cholesterol metabolism, liver samples were collected from healthy control dairy cows (n = 6; 7-13 d in milk) and cows with fatty liver (n = 6; 7-11 d in milk). In vitro, hepatocytes isolated from 3 healthy female calves (1 d old) were challenged with or without a mix of 1.2 mM FA to induce metabolic stress. In addition, hepatocytes were processed with 10 µmol/L of the cholesterol synthesis inhibitor simvastatin or 6 µmol/L of the cholesterol intracellular transport inhibitor U18666A with or without the 1.2 mM FA mix. To evaluate the role of cholesterol addition, hepatocytes were treated with 0.147 mg/mL methyl-β-cyclodextrin (MβCD + FA) or 0.147 mg/mL MβCD with or without 10 and 100 µmol/L cholesterol before incubation with FA (CHO10 + FA and CHO100 + FA). In vivo data from liver biopsies were analyzed by 2-tailed unpaired Student's t-test. Data from in vitro calf hepatocytes were analyzed by one-way ANOVA. Compared with healthy cows, blood plasma total cholesterol and plasma low-density lipoprotein cholesterol content in cows with fatty liver was markedly lower, whereas the hepatic total cholesterol content did not differ. In contrast, compared with healthy controls, the triacylglycerol content in the liver and the content of FA, β-hydroxybutyrate, and aspartate aminotransferase in the plasma of cows with fatty liver were greater. The results revealed that both fatty liver in vivo and challenge of calf hepatocytes with 1.2 mM FA in vitro led to greater mRNA and protein abundance of sterol regulatory element binding transcription factor 1 (SREBF1) and fatty acid synthase (FASN). In contrast, mRNA and protein abundance of sterol regulatory element binding transcription factor 2 (SREBF2), acyl coenzyme A-cholesterol acyltransferase, and ATP-binding cassette subfamily A member 1 (ABCA1) were lower. Compared with the FA group, the cholesterol synthesis inhibitor simvastatin led to greater protein abundance of microsomal triglyceride transfer protein and mRNA abundance of SREBF2, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), ACAT2, and lower ABCA1 and FASN protein abundance. In contrast, compared with the FA group, the cholesterol intracellular transport inhibitor U18666A + FA led to greater total cholesterol concentration and greater protein and mRNA abundance of FASN. Compared with the MβCD + FA group, the addition of 10 µmol/L cholesterol led to greater concentration of cholesteryl ester and excretion of apolipoprotein B100, and greater protein and mRNA abundance of ABCA1 and microsomal triglyceride transfer protein, and lower concentration of malondialdehyde. Overall, a reduction in cholesterol synthesis promoted FA metabolism in hepatocytes likely to relieve the oxidative stress caused by the high FA load. The data suggest that maintenance of normal cholesterol synthesis promotes very low-density lipoprotein excretion and can reduce lipid accumulation and oxidative stress in dairy cows that experience fatty liver.