Integrated analysis of genomics and transcriptomics revealed the genetic basis for goaty flavor formation in goat milk.

Goat milk exhibits a robust and distinctive "goaty" flavor. However, the underlying genetic basis of goaty flavor remains elusive and requires further elucidation at the genomic level. Through comparative genomics analysis, we identified divergent signatures of certain proteins in goat, sheep, and cow. MMUT has undergone a goat-specific mutation in the B12 binding domain. We observed the goat FASN exhibits nonsynonymous mutations in the acyltransferase domain. Structural variations in these key proteins may enhance the capacity for synthesizing goaty flavor compounds in goat. Integrated omics analysis revealed the catabolism of branched-chain amino acids contributed to the goat milk flavor. Furthermore, we uncovered a regulatory mechanism in which the transcription factor ZNF281 suppresses the expression of the ECHDC1 gene may play a pivotal role in the accumulation of flavor substances in goat milk. These findings provide insights into the genetic basis underlying the formation of goaty flavor in goat milk. STATEMENT OF SIGNIFICANCE: Branched-chain fatty acids (BCFAs) play a crucial role in generating the distinctive "goaty" flavor of goat milk. Whether there is an underlying genetic basis associated with goaty flavor is unknown. To begin deciphering mechanisms of goat milk flavor development, we collected transcriptomic data from mammary tissue of goat, sheep, cow, and buffalo at peak lactation for cross-species transcriptome analysis and downloaded nine publicly available genomes for comparative genomic analysis. Our data indicate that the catabolic pathway of branched-chain amino acids (BCAAs) is under positive selection in the goat genome, and most genes involved in this pathway exhibit significantly higher expression levels in goat mammary tissue compared to other species, which contributes to the development of flavor in goat milk. Furthermore, we have elucidated the regulatory mechanism by which the transcription factor ZNF281 suppresses ECHDC1 gene expression, thereby exerting an important influence on the accumulation of flavor compounds in goat milk. These findings provide insights into the genetic mechanisms underlying flavor formation in goat milk and suggest further research to manipulate the flavor of animal products.

Effect of strontium on transcription factors identified by transcriptome analyses of bovine ruminal epithelial cells.

BACKGROUND: Strontium (Sr) has similar physicochemical properties as calcium (Ca) and is often used to evaluate the absorption of this mineral. Because the major route of Ca absorption in the bovine occurs in the rumen, it is essential to understand whether Sr impacts the ruminal epithelial cells and to what extent. RESULTS: In the present study, RNA sequencing and assembled transcriptome assembly were used to identify transcription factors (TFs), screening and bioinformatics analysis in bovine ruminal epithelial cells treated with Sr. A total of 1405 TFs were identified and classified into 64 families based on an alignment of conserved domains. A total of 174 differently expressed TFs (DE-TFs) were increased and 52 DE-TFs were decreased; the biological process-epithelial cell differentiation was inhibited according to the GSEA-GO analysis of TFs; The GO analysis of DE-TFs was enriched in the DNA binding. Protein-protein interaction network (PPI) found 12 hubs, including SMAD4, SMAD2, SMAD3, SP1, GATA2, NR3C1, PPARG, FOXO1, MEF2A, NCOA2, LEF1, and ETS1, which verified genes expression levels by real-time PCR. CONCLUSIONS: In this study, SMAD2, PPARG, LEF1, ETS1, GATA2, MEF2A, and NCOA2 are potential candidates that could be targeted by Sr to mediate cell proliferation and differentiation, as well as lipid metabolism. Hence, these results enhance the comprehension of Sr in the regulation of transcription factors and provide new insight into the study of Sr biological function in ruminant animals.

Feeding a Saccharomyces cerevisiae fermentation product during a gut barrier challenge in lactating Holstein cows impacts the ruminal microbiota and metabolome.

Through its influence on the gut microbiota, the feeding of Saccharomyces cerevisiae fermentation products (SCFP) has been a successful strategy to enhance the health of dairy cows during periods of physiological stresses. Although production and metabolic outcomes from feeding SCFP are well-known, its combined impacts on the ruminal microbiota and metabolome during gut barrier challenges remain unclear. To address this gap in knowledge, multiparous Holstein cows (97.1 ± 7.6 DIM [SD]; n = 8/group) fed a control diet (CON) or CON plus 19 g/d SCFP for 9 wk were subjected to a feed restriction (FR) challenge for 5 d, during which they were fed 40% of their ad libitum intake from the 7 d before FR. The DNA extracted from ruminal fluid was subjected to PacBio full-length 16S rRNA gene sequencing, real-time PCR of 12 major ruminal bacteria, and metabolomics analysis of up to 189 metabolites via GC/MS. High-quality amplicon sequence analyses were performed with the TADA (Targeted Amplicon Diversity Analysis), MicrobiomeAnalyst, PICRUSt2, and STAMP software packages, and metabolomics data were analyzed via MetaboAnalyst 5.0. Ruminal fluid metabolites from the SCFP group exhibited a greater α-diversity Chao 1 (P = 0.03) and Shannon indices (P = 0.05), and the partial least squares discriminant analysis clearly discriminated metabolite profiles between dietary groups. The abundance of CPla_4_termite_group, Candidatus Saccharimonas, Oribacterium, and Pirellula genus in cows fed SCFP was greater. In the SCFP group, concentrations of ethanolamine, 2-amino-4,6-dihydroxypyrimidine, glyoxylic acid, serine, threonine, cytosine, stearic acid, and pyrrole-2-carboxylic acid were greater in ruminal fluid. Both Fretibacterium and Succinivibrio abundances were positively correlated with metabolites across various biological processes: gamma-aminobutyric acid, galactose, butane-2,3-diol, fructose, 5-amino pentanoic acid, ß-aminoisobutyric acid, ornithine, malonic acid, 3-hydroxy-3-methylbutyric acid, hexanoic acid, heptanoic acid, cadaverine, glycolic acid, ß-alanine, 2-hydroxybutyric acid, methyl alanine, and alanine. In the SCFP group, compared with CON, the mean proportion of 14 predicted pathways based on metabolomics data was greater, whereas 10 predicted pathways were lower. Integrating metabolites and upregulated predicted enzymes (NADP+-dependent glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, serine: glyoxylate aminotransferase, and d-glycerate 3-kinase) indicated that the pentose phosphate pathway and photorespiration pathway were most upregulated by SCFP. Overall, SCFP during FR led to alterations in ruminal microbiota composition and key metabolic pathways. Among those, we identified a shift from the tricarboxylic acid cycle to the glyoxylate cycle, and nitrogenous base production was enhanced.

Role of diacylglycerol O-acyltransferase 1 (DGAT1) in lipolysis and autophagy of adipose tissue from ketotic dairy cows.

High-yielding dairy cows in early lactation often encounter difficulties in meeting the energy requirements essential for maintaining milk production. This is primarily attributed to insufficient dry matter intake, which consequently leads to sustained lipolysis of adipose tissue. Fatty acids released by lipolysis can disrupt metabolic homeostasis. Autophagy, an adaptive response to intracellular environmental changes, is considered a crucial mechanism for regulating lipid metabolism and maintaining a proper cellular energy status. Despite its close relationship with aberrant lipid metabolism and cytolipotoxicity in animal models of metabolic disorders, the precise function of diacylglycerol o-acyltransferase 1 (DGAT1) in bovine adipose tissue during periods of negative energy balance is not fully understood, particularly regarding its involvement in lipolysis and autophagy. The objective of the present study was to assess the effect of DGAT1 on both lipolysis and autophagy in bovine adipose tissue and isolated adipocytes. Adipose tissue and blood samples were collected from cows diagnosed as clinically ketotic (n = 15) or healthy (n = 15) following a veterinary evaluation based on clinical symptoms and serum concentrations of BHB, which were 3.19 mM (interquartile range = 0.20) and 0.50 mM (interquartile range = 0.06), respectively. Protein abundance of DGAT1 and phosphorylation levels of unc-51-like kinase 1 (ULK1), were greater in adipose tissue from cows with ketosis, whereas phosphorylation levels of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) were lower. Furthermore, when adipocytes isolated from the harvested adipose tissue of 15 healthy cows were transfected with DGAT1 overexpression adenovirus or DGAT1 small interfering RNA followed by exposure to epinephrine (EPI), it led to greater ratios and protein abundance of phosphorylated hormone-sensitive triglyceride lipase (LIPE) to total LIPE and adipose triglyceride lipase (ATGL), while inhibiting the protein phosphorylation levels of ULK1, PI3K, AKT, and mTOR. Overexpression of DGAT1 in EPI-treated adipocytes reduced lipolysis and autophagy, whereas silencing DGAT1 further exacerbated EPI-induced lipolysis and autophagy. Taken together, these findings indicate that upregulation of DGAT1 may function as an adaptive response to suppress adipocytes lipolysis, highlighting the significance of maintaining metabolic homeostasis in dairy cows during periods of negative energy balance.

Interferon-tau protects bovine endometrial epithelial cells against inflammatory injury by regulating the PI3K/AKT/ß-catenin/FoxO1 signaling axis.

Endometritis is one of the most common causes of infertility in dairy cows, and is histopathologically characterized by inflammation and damage of endometrial epithelium. Interferon-tau (IFN-τ) is a novel type I interferon secreted by ruminant trophoblast cells with low cytotoxicity even at high doses. Previous studies suggested that IFN-τ plays an important role in inflammation. However, the mechanisms whereby IFN-τ may modulate the inflammatory responses in the bovine endometrium are unknown. In the present study, primary bovine endometrial epithelial cells (BEEC) isolated from fresh and healthy uterine horns were used for in vitro studies. The integrity of BEEC was assessed by immunofluorescence staining for cytokeratin 18 (CK-18, a known epithelial marker). For the experiments, BEEC were stimulated with different concentrations of lipopolysaccharide (LPS; 0-20 µg/mL) for different times (0-24 h). Cell viability and apoptosis were assessed via CCK-8 and flow cytometry. In a preliminary study, we observed that compared with the control group without LPS, 10 µg/mL of LPS stimulation for 24 h induced apoptosis. In a subsequent study, 20 or 40 ng/mL of IFN-τ alleviated LPS-induced apoptosis. Relative to the LPS group, western blotting further revealed that IFN-τ inhibited the protein abundance of TLR4 and phosphorylated (p-) p65 (p-p65) and Bax/Bcl-2 ratio, suggesting that IFN-τ can protect BEEC against inflammatory injury. Furthermore, the protein abundance of p-phosphoinositide 3-kinase (p-PI3K), p-protein kinase B (p-AKT), p-glycogen synthase kinase-3ß (p-GSK3ß), ß-catenin, and p-forkhead box O1 (p-FoxO1) was lower in the LPS group, whereas IFN-τ upregulated their abundance. The use of LY294002, a specific inhibitor of PI3K/AKT, attenuated the upregulation of p-PI3K, p-AKT p-GSK3ß, ß-catenin, and p-FoxO1 induced by IFN-τ, and also blocked the downregulation of TLR4, p-p65, and Bax/Bcl-2 ratio. This suggested that the inhibition of TLR4 signaling by IFN-τ was mediated by the PI3K/AKT pathway. Furthermore, compared with the LPS group, the ß-catenin agonist SB216763 led to greater p-FoxO1 and lower p-p65 and cell apoptosis. In contrast, knockdown of ß-catenin using small interfering RNA had the opposite effects. To explore the role of FoxO1 on the inhibition of TLR4 by IFN-τ, we employed LY294002 to inhibit the PI3K/AKT while FoxO1 was knocked down. Results revealed that the knockdown of FoxO1 blocked the upregulation of TLR4 and p-p65 induced by LY294002, and enhanced the inhibition of IFN-τ on TLR4, p-p65, and cell apoptosis. Overall, these findings confirmed that IFN-τ can protect endometrial epithelial cells against inflammatory injury via suppressing TLR4 activation through the regulation of the PI3K/AKT/ß-catenin/FoxO1 axis. These represent new insights into the molecular mechanisms underlying the anti-inflammatory function of IFN-τ in BEEC, and also provide a theoretical basis for further studies on the in vivo application of IFN-τ to help prevent negative effects of endometritis.

Residual feed intake is related to metabolic and inflammatory response during the preweaning period in Italian Simmental calves.

Residual Feed Intake (RFI) is defined as the difference between measured and predicted intake. Understanding its biological regulators could benefit farm profit margins. The most-efficient animals (M-Eff) have observed intake smaller than predicted resulting in negative RFI, whereas the least-efficient (L-Eff) animals have positive RFI. Hence, this observational study aimed at retrospectively comparing the blood immunometabolic profile in calves with divergent RFI during the preweaning period. Twenty-two Italian Simmental calves were monitored from birth through 60 d of age. Calves received 3 L of colostrum from their respective dams. From 2 to 53 d of age, calves were fed a milk replacer twice daily, whereas from 54 to 60 d (i.e., weaning) calves were stepped down to only one meal in the morning. Calves had ad libitum access to concentrate and intakes were recorded daily. The measurement of BW and blood samples were performed at 0, 1, 7, 14, 21, 28, 35, 45, 54, and 60 d of age. Calves were ranked and categorized as M-Eff or L-Eff according to the median RFI value. Median RFI was -0.06 and 0.04 kg of DMI/d for M-Eff and L-Eff, respectively. No evidence for group differences was noted for colostrum and plasma IgG concentrations. Although growth rate was not different, as expected, (0.67 kg/d [95% CI = 0.57-0.76] for both L-Eff and M-Eff) throughout the entire preweaning period (0-60 d), starter intake was greater in L-Eff compared with M-Eff calves (+36%). Overall, M-Eff calves had a greater gain-to-feed ratio compared with L-Eff calves (+16%). Plasma ceruloplasmin, myeloperoxidase, and reactive oxygen metabolites concentrations were greater in L-Eff compared with M-Eff calves. Compared with L-Eff, M-Eff calves had an overall greater plasma concentration of globulin, and γ-glutamyl transferase (indicating a better colostrum uptake) and Zn at 1 d. Retinol and urea were overall greater in L-Eff. The improved efficiency in nutrient utilization observed in M-Eff was paired with a lower grade of oxidative stress and systemic inflammation. L-Eff may have had greater energy expenditure to support the activation of the immune system.

Diacylglycerol O-acyltransferase (DGAT) isoforms play a role in peridroplet mitochondrial fatty acid metabolism in bovine liver.

Hepatocellular lipid accumulation characterizes fatty liver in dairy cows. Lipid droplets (LD), specialized organelles that store lipids and maintain cellular lipid homeostasis, are responsible for the ectopic storage of lipids associated with several metabolic disorders. In recent years, non-ruminant studies have reported that LD-mitochondria interactions play an important role in lipid metabolism. Due to the role of diacylglycerol acyltransferase isoforms (DGAT1 and DGAT2) in LD synthesis, we explored mechanisms of mitochondrial fatty acid transport in ketotic cows using liver biopsies and isolated primary hepatocytes. Compared with healthy cows, cows with fatty liver had massive accumulation of LD and high protein expression of the triglyceride (TAG) synthesis-related enzymes DGAT1 and DGAT2, LD synthesis-related proteins perilipin 2 (PLIN2) and perilipin 5 (PLIN5), and the mitochondrial fragmentation-related proteins dynamin-related protein 1 (DRP1) and fission 1 (FIS1). In contrast, factors associated with fatty acid oxidation, mitochondrial fusion and mitochondrial electron transport chain complex were lower compared with those in the healthy cows. In addition, transmission electron microscopy revealed significant contacts between LD-mitochondria in liver tissue from cows with fatty liver. Compared with isolated cytoplasmic mitochondria, expression of carnitine palmitoyl transferase 1A (CPT1A) and DRP1 was lower, but mitofusin 2 (MFN2) and mitochondrial electron transport chain complex was greater in isolated peridroplet mitochondria from hepatic tissue of cows with fatty liver. In vitro data indicated that exogenous free fatty acids (FFA) induced hepatocyte LD synthesis and mitochondrial dynamics consistent with in vivo results. Furthermore, DGAT2 inhibitor treatment attenuated the FFA-induced upregulation of PLIN2 and PLIN5 and rescued the impairment of mitochondrial dynamics. Inhibition of DGAT2 also restored mitochondrial membrane potential and reduced hepatocyte reactive oxygen species production. The present in vivo and in vitro results indicated there are functional differences among different types of mitochondria in the liver tissue of dairy cows with ketosis. Activity of DGAT2 may play a key role in maintaining liver mitochondrial function and lipid homeostasis in dairy cows during the transition period.

Fatty acids promote M1 polarization of monocyte-derived macrophages in healthy or ketotic dairy cows and a bovine macrophage cell line via impairing mTOR-mediated autophagy.

Excessive concentrations of free fatty acids (FFA) are the main factors causing immune dysfunction and inflammation in dairy cows with ketosis. Polarization of macrophages (the process of macrophages freely switching from one phenotype to another) into M1 or M2 phenotypes is an important event during inflammation induced by environmental stimuli. In non-ruminants, mammalian target of rapamycin (mTOR)-mediated autophagy (a major waste degradation process) regulates macrophage polarization. Thus, the objective was to unravel the role of mTOR-mediated autophagy on macrophage polarization in ketotic dairy cows. Four experiments were performed as follows: (1) In vitro differentiated monocyte-derived macrophages from healthy dairy cows or dairy cows with clinical ketosis (CK) were treated with 100 ng/mL lipopolysaccharide (LPS) and 100 ng/mL interferon-γ (IFN-γ) or 10 ng/mL interleukin-4 (IL4) and 10 ng/mL interleukin-10 (IL10) for 24 h; (2) Immortalized bovine macrophages were treated with 0, 0.3, 0.6, 1.2 mM FFA and LPS and IFN-γ or IL4 and IL10 for 24 h; (3) Macrophages were pretreated with 2 µM 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine (MHY1485) for 30 min before treatment with LPS and IFN-γ or IL4 and IL10; (4) Macrophages were pretreated with 100 nM rapamycin (RAPA) for 2 h before treatment with LPS and IFN-γ or IL4 and IL10. Compared with healthy cows, cows with CK had a greater mean fluorescence intensity (MFI) of CD86+, but lower MFI of CD206+ and lower number of autophagosomes and autolysosomes in macrophages. Exogenous FFA treatment upregulated protein abundance of inducible nitric oxide synthase (iNOS) and mean fluorescence intensity of CD86, whereas it downregulated the protein abundance of arginase 1 (ARG1) and mean fluorescence intensity of CD206. In addition, FFA increased the p-p65/p65 protein abundance and tumor necrosis factor α (TNFA), interleukin-1B (IL1B), and interleukin-6 (IL6) mRNA abundance, but decreased LC3-phosphatidylethanolamine conjugate (LC3-II) protein abundance and autophagosomes and autolysosomes number. Pretreatment with MHY1485 promoted macrophage M1 polarization and inhibited macrophage M2 polarization via decreased mTOR-mediated autophagy. Activation of mTOR-mediated autophagy by pretreatment with RAPA attenuated the upregulation of inflammation in M1 macrophages that was induced by FFA. These data revealed that high concentrations of FFA promote macrophage M1 polarization in ketotic dairy cows via impairing mTOR-mediated autophagy.

Plasma and milk metabolomics profiles in dairy cows with subclinical and clinical ketosis.

Ketosis, a commonly observed energy metabolism disorder in dairy cows during the peripartal period, is distinguished by increased concentrations of ß-hydroxybutyrate (BHB) in blood. This condition has a negative impact on milk production and quality, causing financial losses. An untargeted metabolomics approach was performed on plasma samples from cows between 5 and 7 DIM diagnosed as controls (CON, BHB <1.2 mM, n = 30), subclinically ketotic (SCK, 1.2 < BHB <3.0 mM, n = 30), or clinically ketotic (CK, BHB >3.0 mM, n = 30). Cows were selected from a commercial farm of 214 Holstein cows (average 305-d yield in the previous lactation of 35.42 ± 7.23 kg/d; parity, 2.41 ± 1.12; body condition score, 3.1 ± 0.45). All plasma and milk samples (n = 90) were subjected to Liquid Chromatography-Mass Spectrometry (LC-MS)-based metabolomic analysis. Statistical analyses was performed using the Graph Pad Prism 8.0, MetaboAnalyst 4.0 and R packages (version 4.1.3). Compared with the CON group, both SCK and CK groups had greater milk fat, freezing point, and fat-to-protein ratio and lower milk protein, lactose, solids-nonfat, and milk density. Within 21 d after calving, compared with CON, the SCK group experienced a reduction of 2.65 kg/d in milk yield, while the CK group experienced a decrease of 7.7 kg/d. Untargeted metabolomics analysis facilitated the annotation of a total of 5,259 and 8,423 metabolites in plasma and milk. Differentially affected metabolites were screened in CON vs. SCK, CON vs. CK, and SCK vs. CK (unpaired t-test, False discovery rate <0.05; and absolute value of log(2)-fold change >1.5). A total of 1,544 and 1,888 differentially affected metabolites were detected in plasma and milk. In plasma, glycerophospholipid metabolism, pyrimidine metabolism, tryptophan metabolism, sphingolipid metabolism, amino sugar and nucleotide sugar metabolism, phenylalanine metabolism, steroid hormone biosynthesis were identified as significant pathways. Weighted gene co-expression network analysis (WGCNA) indicated that tryptophan metabolism is a key pathway associated with the occurrence and development of ketosis. Increases in 5-Hydroxytryptophan and decreases in kynurenine and 3-indoleacetic acid in SCK and CK were suggestive of an impact at the gut level. The decrease of most glycerophospholipids indicated that ketosis is associated with disordered lipid metabolism. For milk, pyrimidine metabolism, purine metabolism, pantothenate and CoA biosynthesis, amino sugar and nucleotide sugar metabolism, nicotinate and nicotinamide metabolism, sphingolipid metabolism, fatty acid degradation were identified as significant pathways. The WGCNA indicated that purine and pyrimidine metabolism in plasma was highly correlated with milk yield during the peripartal period. Alterations in purine and pyrimidine metabolism characterized ketosis, with lower levels of these metabolites in both milk and blood underscoring reduced efficiency in nitrogen metabolism. Our results may help to establish a foundation for future research investigating mechanisms responsible for the occurrence and development of ketosis in peripartal cows.

Nuciferine protects bovine hepatocytes against free fatty acid-induced oxidative damage by activating the transcription factor EB/peroxisome proliferator-activated receptor γ coactivator 1 alpha pathway.

Excessive free fatty acid (FFA) oxidation and related metabolism are the major cause of oxidative stress and liver injury in dairy cows during the early postpartum period. In nonruminants, activation of transcription factor EB (TFEB) can improve cell damage and reduce the overproduction of mitochondrial reactive oxygen species. As a downstream target of TFEB, peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α, gene name PPARGC1A) is a critical regulator of oxidative metabolism. Nuciferine (Nuc), a major bioactive compound isolated from the lotus leaf, has been reported to possess hepatoprotective activity. Therefore, the objective of this study was to investigate whether Nuc could protect bovine hepatocytes from FFA-induced lipotoxicity and the underlying mechanisms. A mixture of FFA was diluted in RPMI-1640 basic medium containing 2% low fatty acid bovine serum albumin to treat hepatocytes. Bovine hepatocytes were isolated from newborn calves and treated with various concentrations of FFA mixture (0, 0.3, 0.6, or 1.2 mM) or Nuc (0, 25, 50, or 100 µM), as well as co-treated with 1.2 mM FFA and different concentrations of Nuc. For the experiments of gene silencing, bovine hepatocytes were transfected with small interfering RNA targeted against TFEB or PPARGC1A for 36 h followed by treatment with 1.2 mM FFA for 12 h in presence or absence of 100 µΜ Nuc. The results revealed that FFA treatment decreased protein abundance of nuclear TFEB, cytosolic TFEB, total (t)-TFEB, lysosome-associated membrane protein 1 (LAMP1) and PGC-1α and mRNA abundance of LAMP1, but increased phosphorylated (p)-TFEB. In addition, FFA treatment increased the content of malondialdehyde (MDA) and hydrogen peroxide (H2O2) and decreased the activities of catalase (CAT) and glutathione peroxidase (GSH-Px) in bovine hepatocytes. Moreover, FFA administration enhanced the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactose dehydrogenase (LDH) in the medium of FFA-treated hepatocytes, but reduced the content of urea. In FFA-treated bovine hepatocytes, Nuc administration increased TFEB nuclear localization and the protein abundance of t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, decreased the contents of MDA and H2O2 and the protein abundance of p-TFEB, and enhanced the activities of CAT and GSH-Px in a dose-dependent manner. Consistently, Nuc administration reduced the activities of ALT, AST, and LDH and increased the content of urea in the medium of FFA-treated hepatocytes. Importantly, knockdown of TFEB reduced the protein abundance of p-TFEB, t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, and impeded the beneficial effects of Nuc on FFA-induced oxidative damage in bovine hepatocytes. In addition, PPARGC1A silencing did not alter Nuc-induced nuclear translocation of TFEB, increase of the protein abundance of t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, or decrease of the protein abundance of p-TFEB, whereas it partially reduced the beneficial effects of Nuc on FFA-caused oxidative injury. Taken together, Nuc exerts protective effects against FFA-induced oxidative damage in bovine hepatocytes through activation of the TFEB/PGC-1α signaling pathway.

Homeostatic crosstalk among gut microbiome, hypothalamic and hepatic circadian clock oscillations, immunity and metabolism in response to different light-dark cycles: A multiomics study.

The accelerated pace of life at present time has resulted in tremendous alterations in living patterns. Changes in diet and eating patterns, in particular, coupled with irregular light-dark (LD) cycles will further induce circadian misalignment and lead to disease. Emerging data has highlighted the regulatory effects of diet and eating patterns on the host-microbe interactions with the circadian clock (CC), immunity, and metabolism. Herein, we studied how LD cycles regulate the homeostatic crosstalk among the gut microbiome (GM), hypothalamic and hepatic CC oscillations, and immunity and metabolism using multiomics approaches. Our data demonstrated that central CC oscillations lost rhythmicity under irregular LD cycles, but LD cycles had minimal effects on diurnal expression of peripheral CC genes in the liver including Bmal1. We further demonstrated that the GM could regulate hepatic circadian rhythms under irregular LD cycles, the candidate bacteria including Limosilactobacillus, Actinomyces, Veillonella, Prevotella, Campylobacter, Faecalibacterium, Kingella, and Clostridia vadinBB60 et al. A comparative transcriptomic study of innate immune genes indicated that different LD cycles had varying effects on immune functions, while irregular LD cycles had greater impacts on hepatic innate immune functions than those in the hypothalamus. Extreme LD cycle alterations (LD0/24 and LD24/0) had worse impacts than slight alterations (LD8/16 and LD16/8), and led to gut dysbiosis in mice receiving antibiotics. Metabolome data also demonstrated that hepatic tryptophan metabolism mediated the homeostatic crosstalk among GM-liver-brain axis in response to different LD cycles. These research findings highlighted that GM could regulate immune and metabolic disorders induced by circadian dysregulation. Further, the data provided potential targets for developing probiotics for individuals with circadian disruption such as shift workers.

Molecular mechanisms in the miR-33a/LPPR4 pathway regulating unsaturated fatty acid synthesis in bovine mammary epithelial cells.

The regulatory mechanisms governing metabolism of fatty acids in cow mammary gland are crucial for establishing relationships between milk quality and fatty acid content. Both, microRNAs (miRNAs) and protein-coding genes are important factors involved in the regulation of milk fat synthesis. In this study, high-throughput sequencing of miRNAs and mRNAs in bovine mammary gland tissue was performed during peak lactation (3 samples) and late lactation (3 samples) periods to characterize expression profiles. Differential expression (DE) analyses of miRNA and mRNA and miRNA-mRNA regulatory pathway screening were performed. Two-hundred eighty regulatory miRNA-mRNA pairs were identified, including the miR-33a-lipid phosphate phosphatase-related protein type 4 (LPPR4) pathway. Bioinformatics prediction, dual-luciferase reporter system detection, qRT-PCR, and Western blotting revealed that miR-33a can directly target LPPR4 and inhibit its expression. Experiments also revealed that miR-33a promotes the synthesis of triglycerides and increases the content of unsaturated fatty acids (UFAs) in bovine mammary epithelial cells (BMECs). These results indicate that miR-33a via LPPR4 plays an important role in the regulation of milk fat synthesis and UFA levels.

Regulation of cholesterol metabolism during high fatty acid-induced lipid deposition in calf hepatocytes.

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-ß-cyclodextrin (MßCD + FA) or 0.147 mg/mL MßCD 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, ß-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 MßCD + 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.

Sirtuin 3 relieves inflammatory responses elicited by lipopolysaccharide via the PGC1α-NFκB pathway in bovine mammary epithelial cells.

Excessive inflammation in bovine mammary endothelial cells (BMEC) due to mastitis leads to disease progression and eventual culling of cattle. Sirtuin 3 (SIRT3), a mitochondrial deacetylase, downregulates pro-inflammatory cytokines in BMEC exposed to high concentrations of nonesterified fatty acids by blunting nuclear factor-κB (NFκB) signaling. In nonruminants, SIRT3 is under the control of PGC1α, a transcriptional cofactor. Specific aims were to study (1) the effect of SIRT3 on inflammatory responses of lipopolysaccharide (LPS)-challenged bovine mammary epithelial cells (bovine mammary alveolar cells-T, MAC-T) models, and (2) the role of PGC1α in the attenuation of NFκB signaling via SIRT3. To address these objectives, first, MAC-T cells were incubated in triplicate with 0, 50, 100, 150, or 200 µg/mL LPS (derived from Escherichia coli O55:B5) for 12 h with or without a 2-h incubation of the NFκB inhibitor ammonium pyrrolidine dithiocarbamate (APDC, 10 µM). Second, SIRT3 was overexpressed using adenoviral expression (Ad-SIRT3) at different multiplicity of infection (MOI) for 6 h followed by a 12 h incubation with 150 µg/mL LPS. Third, cells were treated with the PGC1α agonist ZLN005 (10 µg/mL) for 24 h and then challenged with 150 µg/mL LPS for 12 h. Fourth, cells were initially treated with the PGC1α inhibitor SR-18292 (100 µM) for 6 h followed by a 6-h culture with or without 50 MOI Ad-SIRT3 and a challenge with 150 µg/mL LPS for 12 h. Data were analyzed using one-way ANOVA with subsequent Bonferroni correction. Linear and quadratic contrasts were used to determine dose-responses to LPS. There were linear and quadratic effects of LPS dosage on cell viability. Incubation with 150 and 200 µg/mL LPS for 12 h decreased cell viability to 78.6 and 34.9%, respectively. Compared with controls, expression of IL1B, IL6, and TNFA was upregulated by 5.2-, 5.9-, and 2.7-fold with 150 µg/mL LPS; concentrations of IL-1ß, IL-6, and tumor necrosis factor-α (TNF-α) in cell medium also increased. Compared with the LPS group, LPS+APDC increased cell viability and reversed the upregulation of IL1B, IL6, and TNFA expression. However, mRNA and protein abundance of SIRT3 decreased linearly with increasing LPS dose. Ad-SIRT3 infection (50 MOI) reduced IL1B, IL6, and TNFA expression and also their concentrations in cell medium, and decreased pNFκB P65/NFκB P65 ratio and nuclear abundance of NFκB P65. The PGC1α agonist increased SIRT3 expression, whereas it decreased cytokine expression, pNFκB P65/NFκB P65 ratio, and prevented NFκB P65 nuclear translocation. Contrary to the agonist, the PGC1α inhibitor had opposite effects, and elevated the concentrations of IL-1ß, IL-6, and TNF-α in cell medium. Overall, data suggested that SIRT3 activity could attenuate LPS-induced inflammatory responses in mammary cells via alterations in the PGC1α-NFκB pathway. As such, there may be potential benefits for targeting SIRT3 in vivo to help prevent or alleviate negative effects of mastitis.

Effects of choline deficiency and supplementation on lipid droplet accumulation in bovine primary liver cells in vitro.

Rumen-protected choline (RPC) supplementation in the periparturient period has in some instances prevented and alleviated fatty liver disease in dairy cows. Mechanistically, however, it is unclear how choline prevents the accumulation of lipid droplets (LD) in liver cells. In this study, primary liver cells isolated from liver tissue obtained via puncture biopsy from 3 nonpregnant mid-lactation multiparous Holstein cows (∼160 d postpartum) were used. Analyses of LD via oil red O staining, protein abundance via Western blotting, and phospholipid content and composition measured by thin-layer chromatography and HPLC/mass spectrometry were performed in liver cells cultured in choline-deficient medium containing 150 µmol/L linoleic acid for 24 h. In a subsequent experiment, lipophagy was assessed in liver cells cultured with 30, 60, or 90 µmol/L choline-chloride. All data were analyzed statistically using SPSS 20.0 via t-tests or one-way ANOVA. Compared with liver cells cultured in Dulbecco's Modified Eagle Medium alone, choline deficiency increased the average diameter of LD (1.59 vs. 2.10 µm), decreased the proportion of small LD (<2 µm) from 75.3% to 56.6%, and increased the proportion of large LD (>4 µm) from 5.6% to 15.0%. In addition, the speed of LD fusion was enhanced by the absence of choline. Among phospholipid species, the phosphatidylcholine (PC) content of liver cells decreased by 34.5%. Seventeen species of PC (PC [18:2_22:6], PC [15:0_16:1], PC [14:0_20:4], and so on) and 6 species of lysophosphatidylcholine (LPC; LPC [15:0/0:0]), PC (22:2/0:0), LPC (20:2/0:0), and so on] were decreased, while PC (14:1_16:1) and LPC (0:0/20:1) were increased. Choline deficiency increased the triglyceride (TAG) content (0.57 vs. 0.39 µmol/mg) in liver cells and increased the protein abundance of sterol regulatory element binding protein 1, sterol regulatory element binding protein cleavage activation protein, and fatty acid synthase by 23.5%, 17%, and 36.1%, respectively. Upon re-supplementation with choline, the phenotype of LD (TAG content, size, proportion, and phospholipid profile) was reversed, and the ratio of autophagy marker LC3II/LC3I protein was significantly upregulated in a dose-dependent manner. Overall, at least in vitro in mid-lactation cows, these data demonstrated that PC synthesis is necessary for normal LD formation, and both rely on choline availability. According to the limitation of the source of liver cells used, further work should be conducted to ascertain that these effects are applicable to liver cells from postpartum cows, the physiological stage where the use of RPC has been implemented for the prevention and treatment of fatty liver.

Alterations in skeletal muscle abundance of protein turnover, stress, and antioxidant proteins during the periparturient period in dairy cows fed ethyl-cellulose rumen-protected methionine.

Skeletal muscle turnover helps support the physiological needs of dairy cows during the transition into lactation. We evaluated effects of feeding ethyl-cellulose rumen-protected methionine (RPM) during the periparturient period on abundance of proteins associated with transport AA and glucose, protein turnover, metabolism, and antioxidant pathways in skeletal muscle. Sixty multiparous Holstein cows were used in a block design and assigned to a control or RPM diet from -28 to 60 d in milk. The RPM was fed at a rate of 0.09% or 0.10% of dry matter intake (DMI) during the prepartal and postpartal periods to achieve a target Lys:Met ratio in the metabolizable protein of ∼2.8:1. Muscle biopsies from the hind leg of 10 clinically healthy cows per diet collected at -21, 1, and 21 d relative to calving were used for western blotting of 38 target proteins. Statistical analysis was performed using the PROC MIXED statement of SAS version 9.4 (SAS Institute Inc.) with cow as random effect, whereas diet, time, and diet × time were the fixed effects. Diet × time tended to affect prepartum DMI, with RPM cows consuming 15.2 kg/d and controls 14.6 kg/d. However, diet had no effect on postpartum DMI (17.2 and 17.1 ± 0.4 kg/d for control and RPM, respectively). Milk yield during the first 30 d in milk was also not affected by diet (38.1 and 37.5 ± 1.9 kg/d for control and RPM, respectively). Diet or time did not affect the abundance of several AA transporters or the insulin-induced glucose transporter (SLC2A4). Among evaluated proteins, feeding RPM led to lower overall abundance of proteins associated with protein synthesis (phosphorylated EEF2, phosphorylated RPS6KB1), mTOR activation (RRAGA), proteasome degradation (UBA1), cellular stress responses (HSP70, phosphorylated MAPK3, phosphorylated EIF2A, ERK1/2), antioxidant response (GPX3), and de novo synthesis of phospholipids (PEMT). Regardless of diet, there was an increase in the abundance of the active form of the master regulator of protein synthesis phosphorylated MTOR and the growth-factor-induced serine/threonine kinase phosphorylated AKT1 and PIK3C3, whereas the abundance of a negative regulator of translation (phosphorylated EEF2K) decreased over time. Compared with d 1 after calving and regardless of diet, the abundance of proteins associated with endoplasmic reticulum stress (XBP1 spliced), cell growth and survival (phosphorylated MAPK3), inflammation (transcription factor p65), antioxidant responses (KEAP1), and circadian regulation (CLOCK, PER2) of oxidative metabolism was upregulated at d 21 relative to parturition. These responses coupled with the upregulation of transporters for Lys, Arg, and His (SLC7A1) and glutamate/aspartate (SLC1A3) over time were suggestive of dynamic adaptations in cellular functions. Overall, management approaches that could take advantage of this physiological plasticity may help cows make a smoother transition into lactation.

AMPK-endoplasmic reticulum stress axis contributes to lipopolysaccharide-caused mitochondrial dysfunction by regulating mitochondria-associated membrane function in bovine hepatocytes.

Mitochondrial homeostasis is closely associated with cellular homeostasis process, whereas mitochondrial dysfunction contributes to apoptosis and mitophagy. Hence, analyzing the mechanism of lipopolysaccharide (LPS)-caused mitochondrial damage is necessary to understand how cellular homeostasis is maintained in bovine hepatocytes. Mitochondria-associated membranes (MAM), a connection between endoplasmic reticulum (ER) and mitochondria, is important to control mitochondrial function. To investigate the underlying mechanisms of the LPS-caused mitochondrial dysfunction, hepatocytes isolated from dairy cows at ∼160 d in milk (DIM) were pretreated with the specific inhibitors of adenosine 5'-monophosphate-activated protein kinase (AMPK), ER stress, RNA-activated protein kinase-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α), c-Jun N-terminal kinase, and autophagy followed by a 12 I1/4g/mL LPS treatment. The results showed that inhibiting ER stress with 4-phenylbutyric acid decreased the levels of autophagy and mitochondrial damage with AMPK inactivation in LPS-treated hepatocytes. The AMPK inhibitor compound C pretreatment alleviated LPS-induced ER stress, autophagy and mitochondrial dysfunction by regulating the expression of MAM-related genes, such as mitofusin 2 (MFN2), PERK, and IRE1α. Moreover, inhibiting PERK and IRE1α mitigated autophagy and mitochondrial dynamic disruption by regulating the MAM function. Additionally, blocking c-Jun N-terminal kinase, the downstream sensor of IRE1α, could reduce the levels of autophagy and apoptosis and restore the balance of mitochondrial fusion and fission by modulating the B cell leukemia 2 (BCL-2)/BCL-2 interacting protein 1 (BECLIN1) complex in the LPS-treated bovine hepatocytes. Furthermore, autophagy blockage with chloroquine could intervene in LPS-caused apoptosis to restore mitochondrial function. Collectively, these findings suggest that the AMPK-ER stress axis is involved in the LPS-caused mitochondrial dysfunction by mediating the MAM activity in bovine hepatocytes.

Integrated meta-omics analyses reveal a role of ruminal microorganisms in ketone body accumulation and ketosis in lactating dairy cows.

The extent to which a nutrition-related disorder such as ketosis alters the ruminal microbiota or whether microbiota composition is related to ketosis and potential associations with host metabolism is unknown. We aimed to evaluate variations occurring in the ruminal microbiota of ketotic and nonketotic cows in the early postpartum period, and how those changes may affect the risk of developing the disease. Data on milk yield, dry matter intake (DMI), body condition score, and blood ß-hydroxybutyrate (BHB) concentrations at 21 d postpartum were used to select 27 cows, which were assigned (n = 9 per group) to a clinical ketotic (CK, 4.10 ± 0.72 mmol BHB/L, DMI 11.61 ± 0.49 kg/d, ruminal pH 7.55 ± 0.07), subclinical ketotic (SK, 1.36 ± 0.12 mmol BHB/L, DMI 15.24 ± 0.34 kg/d, ruminal pH 7.58 ± 0.08), or control (NK, 0.88 ± 0.14 mmol BHB/L, DMI 16.74 ± 0.67/d, ruminal pH 7.61 ± 0.03) group. Cows averaged 3.6 ± 0.5 lactations and a body condition score of 3.11 ± 0.34 at the time of sampling. After blood serum collection for metabolomics analysis (1H nuclear magnetic resonance spectra), 150 mL of ruminal digesta was collected from each cow using an esophageal tube, paired-end (2 × 300 bp) sequencing of isolated DNA from ruminal digesta was performed via Illumina MiSeq, and sequencing data were analyzed using QIIME2 (v 2020.6) to measure the ruminal microbiota composition and relative abundance. Spearman correlation coefficients were used to evaluate relationships between relative abundance of bacterial genera and concentrations of serum metabolites. There were more than 200 genera, with approximately 30 being significant between NK and CK cows. Succinivibrionaceae UCG 1 taxa decreased in CK compared with NK cows. Christensenellaceae (Spearman correlation coefficient = 0.6), Ruminococcaceae (Spearman correlation coefficient = 0.6), Lachnospiraceae (Spearman correlation coefficient = 0.5), and Prevotellaceae (Spearman correlation coefficient = 0.6) genera were more abundant in the CK group and were highly positively correlated with plasma BHB. Metagenomic analysis indicated a high abundance of predicted functions related to metabolism (37.7%), genetic information processing (33.4%), and Brite hierarchies (16.3%) in the CK group. The 2 most important metabolic pathways for butyrate and propionate production were enriched in CK cows, suggesting increased production of acetyl coenzyme A and butyrate and decreased production of propionate. Overall, the combined data suggested that microbial populations may be related to ketosis by affecting short-chain fatty acid metabolism and BHB accumulation even in cows with adequate feed intake in the early postpartum period.

Store-operated Ca2+ entry-sensitive glycolysis regulates neutrophil adhesion and phagocytosis in dairy cows with subclinical hypocalcemia.

Hypocalcemia in dairy cows is associated with a decrease of neutrophil adhesion and phagocytosis, an effect driven partly by changes in the expression of store-operated Ca2+ entry (SOCE)-related molecules. It is well established in nonruminants that neutrophils obtain the energy required for immune function through glycolysis. Whether glycolysis plays a role in the acquisition of energy by neutrophils during hypocalcemia in dairy cows is unknown. To address this relationship, we performed a cohort study and then a clinical trial. Neutrophils were isolated at 2 d postcalving from lactating Holstein dairy cows (average 2.83 ± 0.42 lactations, n = 6) diagnosed as clinically healthy (CON) or with plasma concentrations of Ca2+ <2.0 mmol/L as a criterion for diagnosing subclinical hypocalcemia (HYP, average 2.83 ± 0.42 lactations, n = 6). In the first experiment, neutrophils were isolated from blood of CON and HYP cows and used to analyze aspects of adhesion and phagocytosis function through quantitative reverse-transcription PCR along with confocal laser scanning microscopy, mRNA expression of the glycolysis-related gene hexokinase 2 (HKII), and components of the SOCE moiety ORAI calcium release-activated calcium modulator 1 (ORAI1, ORAI2, ORAI3, stromal interaction molecule 1 [STIM1], and STIM2). Results showed that adhesion and phagocytosis function were reduced in HYP cows. The mRNA expression of adhesion-related syndecan-4 (SDC4), integrin ß9 (ITGA9), and integrin ß3 (ITGB3) and phagocytosis-related molecules complement component 1 R subcomponent (C1R), CD36, tubulinß1 (TUBB1) were significantly decreased in the HYP group. In the second experiment, to address how glycolysis affects neutrophil adhesion and phagocytosis, neutrophils isolated from CON and HYP cows were treated with 2 µM HKII inhibitor benserazide-d3 or 1 µM fructose-bisphosphatase 1 (FBP1) inhibitor MB05032 for 1 h. Results revealed that the HKII inhibitor benserazide-d3 reduced phagocytosis and the mRNA abundance of ITGA9, and CD36 in the HYP group. The FBP1 inhibitor MB05032 increased adhesion and phagocytosis and increased mRNA abundance of HKII, ITGA9, and CD36 in the HYP group. Finally, to investigate the mechanism whereby SOCE-sensitive glycolysis affects neutrophil adhesion and phagocytosis, isolated neutrophils were treated with 1 µM SOCE activator thapsigargin or 50 µM inhibitor 2-APB for 1 h. Results showed that thapsigargin increased mRNA abundance of HKII, ITGA9, and CD36, and increased adhesion and phagocytosis in the HYP group. In contrast, 2-APB decreased mRNA abundance of HKII and both adhesion and phagocytosis of neutrophils in the CON group. Overall, the data indicated that SOCE-sensitive intracellular Ca2+ levels affect glycolysis and help regulate adhesion and phagocytosis of neutrophils during hypocalcemia in dairy cows.

Liver fibrosis is a common pathological change in the liver of dairy cows with fatty liver.

Fatty liver (i.e., hepatic lipidosis) is a prevalent metabolic disorder in dairy cows during the transition period, characterized by excess hepatic accumulation of triglyceride (TG), tissue dysfunction, and cell death. Detailed pathological changes, particularly hepatic fibrosis, during fatty liver remain to be determined. Liver fibrosis occurs as a consequence of liver damage, resulting from the excessive accumulation of extracellular matrix, which distorts the architecture of the normal liver, compromising its normal synthetic and metabolic functions. Thus, we aimed to investigate liver fibrosis status and its potential causal factors including oxidative stress, hepatocyte apoptosis, and production of inflammatory cytokines in the liver of cows with fatty liver. Forty-five dairy cows (parity, 3-5) were selected, and liver biopsy and blood were collected on the second week postpartum (days in milk, 10-14 d). On the basis of the degree of lipid accumulation in liver, selected cows were categorized into normal (n = 25; TG <1% wet wt), mild fatty liver (n = 15; 1% ≤ TG <5% wet wt), and moderate fatty liver (n = 5; 5% ≤ TG <10% wet wt). Compared with normal cows, blood concentrations of nonesterified fatty acids and ß-hydroxybutyrate, along with alanine aminotransferase and aspartate aminotransferase activities, were greater in the cows with fatty liver (mild and moderate). Hepatic extracellular matrix deposition, as indicated by Picrosirius red staining, was greater in cows with fatty liver than those with normal ones. In addition, we observed an increased proportion of collagen type I fiber in extracellular matrix with increased lipid accumulation in the liver. Compared with normal cows, the area of α-smooth muscle actin (α-SMA)-positive staining along with the mRNA abundance of collagen type I α 1 (COL1A1), ACTA2 (gene encoding α-SMA), and transforming growth factor-ß (TGFB) were greater in cows with fatty liver. Compared with normal cows, hepatic contents of malondialdehyde, glutathione disulfide, and 8-isoprostane were greater, whereas total antioxidant capacity, the hepatic content of glutathione, and activities of antioxidant indicators, including superoxide dismutase, glutathione peroxidase, and catalase, were lower in cows with fatty liver. The number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells and abundance of apoptosis-related molecules BAX, CASP3, CASP8, and CASP9 were greater in cows with fatty liver. However, mRNA abundance of the anti-apoptotic gene BCL2 did not differ. The mRNA abundance of pro-inflammatory cytokines including tumor necrosis factor-α (TNFA), interleukin-1ß (IL1B), and interleukin-6 (IL6) was greater in the liver of cows with fatty liver. Overall, the present study indicated that fibrosis is a common pathological response to liver damage and is associated with oxidative stress, hepatocyte death, and inflammation.