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, 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, 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; 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. DNA extracted from ruminal fluid was subjected to PacBio Full-Length 16S rRNA gene sequencing, RT-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 Targeted Amplicon Diversity Analysis (TADA), MicrobiomeAnalyst, PICRUSt2, and STAMP software, while 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 PLS-DA 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 abundance 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, while 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, there was a shift from the tricarboxylic acid (TCA) cycle to the glyoxylate cycle and nitrogenous base production was enhanced.

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.

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.

Effects of LED Light Colors on the Growth Performance, Intestinal Morphology, Cecal Short-Chain Fatty Acid Concentrations and Microbiota in Broilers.

This study aimed to explore the effects of light-emitting diode (LED) light colors on growth, intestinal morphology, and cecal microbiota in broilers. A total of 360 healthy male Arbor Acres (AA) broilers with similar weights were selected and divided into four groups with six replicates in each group and 15 broilers in each replicate: LED white light (W), LED green light (G), LED blue light (B), and LED blue-green composite light (BG). The experimental period was 42 d, the light cycle of each treatment group was 23L:1D (23 h of light, one hour of darkness) from 1 to 3 d, and the light cycle from 4 to 42 d was 16L:8D; light intensity was 20 Lux. The results showed that the average daily feed intake and final weight of broilers receiving the B group were the highest in 21 d and 42 d compared with other groups. The average daily feed intake of the BG group was lower than that of the B group. In the same light color, small intestine villus height grows with age. On days 21 and 42, compared with other groups, the ileal villus height was higher, the crypt depth was lower, and the V/C ratio (villus to crypt ratio) was higher in the BG group. The combination of blue-green composite light was beneficial to increase the content of propionate, isobutyrate, butyrate, isovalerate, and valerate in the cecum of 21-day-old broilers and the content of isobutyrate in the cecum of 42-day-old broilers, and a decrease in cecal short-chain fatty acid concentrations with age. The B group and the BG group had higher abundances of Bacteroidetes at day 21 of age and lower abundances of Phascolarctobacterium at day 42. However, no cecal microbiota differences were detected by the Bonferroni-corrected test. In general, our research results showed that light color could promote the growth of broilers by affecting intestinal morphology, microbiota abundance (needs to be validated by further experiments), and cecal short-chain fatty acid concentrations. And blue and blue-green composite lights are more suitable for broiler growth.

Impact of a Saccharomyces cerevisiae fermentation product during an intestinal barrier challenge in lactating Holstein cows on ileal microbiota and markers of tissue structure and immunity.

Feeding a Saccharomyces cerevisiae fermentation product (SCFP; NutriTek, Diamond V, Cedar Rapids, IA) during periods of metabolic stress is beneficial to the health of dairy cows partially through its effect on the gut microbiota. Whether SCFP alters the ileal microbiota in lactating cows during intestinal challenges induced by feed restriction (FR) is not known. We used 16S rRNA sequencing to assess if feeding SCFP during FR to induce gut barrier dysfunction alters microbiota profiles in the ileum. The mRNA abundance of key genes associated with tissue structures and immunity was also detected. Multiparous cows (97.1 ±â€…7.6 days in milk (DIM); n = 7 per treatment) fed a control diet or the control plus 19 g/d NutriTek for 9 wk were subjected to an FR challenge for 5 d, during which they were fed 40% of their ad libitum intake from the 7 d before FR. All cows were slaughtered at the end of FR. DNA extracted from ileal digesta was subjected to PacBio Full-Length 16S rRNA gene sequencing. High-quality amplicon sequence analyses were performed with Targeted Amplicon Diversity Analysis and MicrobiomeAnalyst. Functional analysis was performed and analyzed using PICRUSt and STAMP. Feeding SCFP did not (P > 0.05) alter dry matter intake, milk yield, or milk components during FR. In addition, SCFP supplementation tended (P = 0.07) to increase the relative abundance of Proteobacteria and Bifidobacterium animalis. Compared with controls, feeding SCFP increased the relative abundance of Lactobacillales (P = 0.03). Gluconokinase, oligosaccharide reducing-end xylanase, and 3-hydroxy acid dehydrogenase were among the enzymes overrepresented (P < 0.05) in response to feeding SCFP. Cows fed SCFP had a lower representation of adenosylcobalamin biosynthesis I (early cobalt insertion) and pyrimidine deoxyribonucleotides de novo biosynthesis III (P < 0.05). Subsets of the Firmicutes genus, Bacteroidota phylum, and Treponema genus were correlated with the mRNA abundance of genes associated with ileal integrity (GCNT3, GALNT5, B3GNT3, FN1, ITGA2, LAMB2) and inflammation (AOX1, GPX8, CXCL12, CXCL14, CCL4, SAA3). Our data indicated that the moderate FR induced dysfunction of the ileal microbiome, but feeding SCFP increased the abundance of some beneficial gut probiotic bacteria and other species related to tissue structures and immunity.

Stressors, including limited access to feed, heat stress, transportation, and disease are factors that reduce integrity of the gut epithelial barrier in livestock. Feeding Saccharomyces cerevisiae fermentation products (SCFP) mitigated immunological, aflatoxin, and subclinical mastitis challenges, heat stress, and grain-based subacute ruminal acidosis indicating it also could alleviate gut damage. Microbiota profiling of ileal epithelium using 16S rRNA sequencing and bioinformatics revealed that Lactobacillales and Animalis abundance was greater in cows fed SCFP versus controls during a 5-d feed restriction to induce intestinal dysfunction. Some genera of Firmicutes, Bacteroidota phylum, and Treponema genus were correlated with mRNA abundance of genes associated with integrity and inflammation of ileal epithelium. Thus, feeding SCFP can increase the abundance of beneficial bacteria during a gut challenge.

Alterations in ileal transcriptomics during an intestinal barrier challenge in lactating Holstein cows fed a Saccharomyces cerevisiae fermentation product identify potential regulatory processes.

Stressors such as lack of access to feed, hot temperatures, transportation, and pen changes can cause impairment of ruminal and intestinal barrier function, also known as "leaky gut". Despite the known benefits of some nutritional approaches during periods of stress, little is understood regarding the underlying mechanisms, especially in dairy cows. We evaluated the effect of feeding a Saccharomyces cerevisiae fermentation product (SCFP; NutriTek, Diamond V, Cedar Rapids, IA) on the ileal transcriptome in response to feed restriction (FR), an established model to induce intestinal barrier dysfunction. Multiparous cows [97.1 ±â€…7.6 days in milk (DIM); n = 5/group] fed a control diet or control plus 19 g/d SCFP for 9 wk were subjected to an FR challenge for 5 d during which they were fed 40% of their ad libitum intake from the 7 d before FR. All cows were slaughtered at the end of FR, and ileal scrapping RNA was used for RNAseq (NovaSeq 6000, 100 bp read length). Statistical analysis was performed in R and bioinformatics using the KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO databases. One thousand six hundred and ninety-six differentially expressed genes (DEG; FDR-adjusted P ≤ 0.10) were detected in SCFP vs. control, with 451 upregulated and 1,245 downregulated. "Mucin type O-glycan biosynthesis" was the top downregulated KEGG pathway due to downregulation of genes catalyzing glycosylation of mucins (GCNT3, GALNT5, B3GNT3, GALNT18, and GALNT14). An overall downregulation of cell and tissue structure genes (e.g., extracellular matrix proteins) associated with collagen (COL6A1, COL1A1, COL4A1, COL1A2, and COL6A2), laminin (LAMB2), and integrins (ITGA8, ITGA2, and ITGA5) also were detected with SCFP. A subset of DEG enriched in the GO term "extracellular exosome" and "extracellular space". Chemokines within "Cytokine-cytokine receptor interaction pathways" such as CCL16, CCL21, CCL14, CXCL12, and CXCL14 were downregulated by SCFP. The "Glutathione metabolism" pathway was upregulated by SCFP, including GSTA1 and RRM2B among the top upregulated genes, and GSTM1 and GPX8 as top downregulated genes. There were 9 homeobox transcription factors among the top 50 predicted transcription factors using the RNAseq DEG dataset, underscoring the importance of cell differentiation as a potential target of dietary SCFP. Taken together, SCFP downregulated immune-, ECM-, and mucin synthesis-related genes during FR. Homeobox transcription factors appear important for the transcriptional response of SCFP.

Stressors such as lack of access to feed, hot temperatures, transportation, and disease contribute to diminished gut epithelial barrier integrity in livestock. RNA-sequencing technology and bioinformatics were used to evaluate genome-wide mRNA abundance profiles in ileal tissue from dairy cows fed Saccharomyces cerevisiae fermentation product (SCFP) or an unsupplemented control diet during an intestinal challenge induced by feed restriction. Molecular responses were characterized according to metabolic pathways and other biological categories. Genes associated with "Mucin type O-glycan biosynthesis" and "Extracellular matrix-receptor interaction" were downregulated due to SCFP relative to controls. Alterations in cytokine and chemokine mRNA profiles induced by SCFP underscored differences in tissue immune response. Overall, SCFP altered the transcriptome of ileal tissue damaged by feed restriction.

Potential of phage EF-N13 as an alternative treatment strategy for mastitis infections caused by multidrug-resistant Enterococcus faecalis.

Bovine mastitis is the most common and costly disease affecting dairy cattle throughout the world. Enterococcus faecalis is one of the environmental origin mastitis-causing pathogens. The treatment of bovine mastitis is primarily based on antibiotics. Due to the negative impact of developing antibiotic resistance and adverse effects on soil and water environments, the trend toward use of nonantibiotic treatments is increasing. Phages may represent a promising alternative treatment strategy. However, it is unknown whether phages have therapeutic effects on E. faecalis-induced mastitis. Thus, the objective of this study was to investigate the degree of protection conferred by a phage during murine mastitis caused by multidrug-resistant E. faecalis. Enterococcus faecalis was isolated from the milk of dairy cows with mastitis, and a phage was isolated using the E. faecalis isolates as hosts. The bactericidal ability of the phage against E. faecalis and the ability to prevent biofilm formation were determined in vitro. The therapeutic potential of the phage on murine mastitis was evaluated in vivo. We isolated 14 strains of E. faecalis from the milk of cows with mastitis, all of which exhibited multidrug resistance, and most (10/14) could form strong biofilms. Subsequently, a new phage (EF-N13) was isolated using the multidrug-resistant E. faecalis N13 (isolated from mastitic milk) as the host. The phage EF-N13 belongs to the family Myoviridae, which has short latent periods (5 min) and high bursts (284 pfu/cell). The genome of EF-N13 lacked bacterial virulence-, antibiotic resistance-, and lysogenesis-related genes. Furthermore, bacterial loading in the raw milk medium was significantly reduced by EF-N13 and was unaffected by potential IgG antibodies. In fact, EF-N13 could effectively prevent the formation of biofilm by multidrug-resistant E. faecalis. All of these characteristics suggest that EF-N13 has potential as mastitis therapy. In vivo, 1 × 105 cfu/gland of multidrug-resistant E. faecalis N13 resulted in mastitis development within 24 h. A single dose of phage EF-N13 (1 × 104, 1 × 105, or 1 × 106 pfu/gland) could significantly decrease bacterial counts in the mammary gland at 24 h postinfection. Histopathological observations demonstrated that treatment with phage EF-N13 effectively alleviated mammary gland inflammation and damage. This effect was confirmed by the lower levels of proinflammatory cytokines IL-6, IL-1ß, and tumor necrosis factor-α in the mammary gland treated with phage EF-N13 compared with those treated with phosphate-buffered saline. Overall, the data underscored the potential of phage EF-N13 as an alternative therapy for bovine mastitis caused by multidrug-resistant E. faecalis.

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.

Low abundance of insulin-induced gene 1 contributes to SREBP-1c processing and hepatic steatosis in dairy cows with severe fatty liver.

Fatty liver is a major metabolic disorder of high-producing dairy cows during the transition period. In nonruminants, it is well established that insulin-induced gene 1 (INSIG1) plays a crucial role in regulating hepatic lipogenesis by controlling the anchoring of sterol regulatory element-binding protein 1 (SREBP-1) on the endoplasmic reticulum along with SREBP cleavage-activating protein (SCAP). Whether the INSIG1-SCAP-SREBP-1c transport axis is affected in cows experiencing fatty liver is unknown. Thus, the aim of this study was to investigate the potential role of INSIG1-SCAP-SREBP-1c axis in the progression of fatty liver in dairy cows. For in vivo experiments, 24 dairy cows at the start of their fourth lactation (median; range 3-5) and 8 d in milk (median; range 4-12 d) were selected into a healthy group [n = 12; triglyceride (TG) content <1%] and a severe fatty liver group (n = 12; TG content >10%) according to their hepatic TG content. Blood samples were collected for detecting serum concentrations of free fatty acids, ß-hydroxybutyrate, and glucose. Compared with healthy cows, cows with severe fatty liver had higher serum concentrations of ß-hydroxybutyrate and free fatty acids and lower concentration of glucose. Liver biopsies were used to detect the status of INSIG1-SCAP-SREBP-1c axis, and the mRNA expression of SREBP-1c-target lipogenic genes acetyl-CoA carboxylase α (ACACA), fatty acid synthase (FASN), and diacylglycerol acyltransferase 1 (DGAT1). Cows with severe fatty liver had lower protein expression of INSIG1 in the hepatocyte endoplasmic reticulum fraction, greater protein expression of SCAP and precursor SREBP-1c in the hepatocyte Golgi fraction, and greater protein expression of mature SREBP-1c in the hepatocyte nuclear fraction. In addition, the mRNA expression of SREBP-1c-target lipogenic genes ACACA, FASN, and DGAT1 was greater in the liver of dairy cows with severe fatty liver. In vitro experiments were conducted on hepatocytes isolated from 5 healthy 1-d-old female Holstein calves, and hepatocytes from each calf were run independently. First, hepatocytes were treated with 0, 200, or 400 µM palmitic acid (PA) for 12 h. Exogenous PA treatment decreased INSIG1 protein abundance, enhanced the endoplasmic reticulum to Golgi export of SCAP-precursor SREBP-1c complex and the nuclear translocation of mature SREBP-1c, all of which was associated with increased transcriptional activation of lipogenic genes and TG synthesis. Second, hepatocytes were transfected with INSIG1-overexpressing adenovirus for 48 h and treated with 400 µM PA 12 h before the end of transfection. Overexpressing INSIG1 inhibited PA-induced SREBP-1c processing, upregulation of lipogenic genes, and TG synthesis in hepatocytes. Overall, the present in vivo and in vitro results indicated that the low abundance of INSIG1 contributed to SREBP-1c processing and hepatic steatosis in dairy cows. Thus, the INSIG1-SCAP-SREBP-1c axis may be a novel target for treatment of fatty liver in dairy cows.

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.

AdipoRon alleviates fatty acid-induced lipid accumulation and mitochondrial dysfunction in bovine hepatocytes by promoting autophagy.

During the transition period in dairy cows, high circulating concentrations of nonesterified fatty acids (NEFA) increase hepatic lipid deposits and are considered a major pathological factor for liver damage. We investigated whether AdipoRon, a synthetic small-molecule agonist of adiponectin receptors 1 and 2 shown to prevent liver lipid accumulation in nonruminants, could alleviate NEFA-induced lipid accumulation and mitochondrial dysfunction. Bovine hepatocytes were isolated from 5 healthy Holstein female newborn calves (1 d of age, 30-40 kg, fasting), and independently isolated hepatocytes from at least 3 different calves were used for each subsequent experiment. The composition and concentration of NEFA used in this study were selected according to hematological criteria of dairy cows with fatty liver or ketosis. First, hepatocytes were cultured with various concentrations of NEFA (0, 0.6, 1.2, or 2.4 mM) for 12 h. In a second experiment, hepatocytes were treated with AdipoRon at different concentrations (0, 5, 25, or 50 µM for 12 h) and times (25 µM for 0, 6, 12, or 24 h) with or without NEFA (1.2 mM) treatment. In the last experiment, hepatocytes were treated with AdipoRon (25 µM), NEFA (1.2 mM), or both for 12 h after treatment with or without the autophagy inhibitor chloroquine. Hepatocytes treated with NEFA had increased protein abundance of sterol regulatory element-binding protein 1c (SREBP-1c) and mRNA abundance of acetyl-CoA carboxylase 1 (ACACA), and decreased protein abundance of peroxisome proliferator-activated receptor α (PPARA), proliferator-activated receptor gamma coactivator-1 α (PGC-1α), mitofusin 2 (MFN2), cytochrome c oxidase subunit IV (COX IV), and mRNA abundance of carnitine palmitoyltransferase 1A (CPT1A), along with lower ATP concentrations. AdipoRon treatment reversed these effects, suggesting this compound had a positive effect on lipid metabolism and mitochondrial dysfunction during the NEFA challenge. In addition, upregulated expression of microtubule-associated protein 1 light chain 3-II (LC3-II, encoded by MAP1LC3) and downregulated expression of sequestosome-1 (SQSTM1, also called p62) indicated that AdipoRon enhanced autophagic activity in hepatocytes. The fact that chloroquine impeded the beneficial effects of AdipoRon on lipid accumulation and mitochondrial dysfunction suggested a direct role for autophagy during NEFA challenge. Our results suggest that autophagy is an important cellular mechanism to prevent NEFA-induced lipid accumulation and mitochondrial dysfunction in bovine hepatocytes, which is consistent with other studies. Overall, AdipoRon may represent a promising therapeutic agent to maintain hepatic lipid homeostasis and mitochondrial function in dairy cows during the transition period.

Mitochondrial Calcium Uniporter Regulator 1 (MCUR1) Relieves Mitochondrial Damage Induced by Lipopolysaccharide by Mediating Mitochondrial Ca2+ Homeostasis in Bovine Mammary Epithelial Cells.

The metabolic stress triggered by negative energy balance after calving induces mitochondrial damage of bovine mammary epithelial cells. Mitochondrial calcium uniporter regulator 1 (MCUR1) is a key protein-coding gene that mediates mitochondrial calcium ion (Ca2+) uptake and plays an important role in mediating homeostasis of mitochondria. The aim of the present study was to elucidate the effects of MCUR1-mediated Ca2+ homeostasis on mitochondria of bovine mammary epithelial cells in response to an inflammatory challenge with lipopolysaccharide (LPS). Exogenous LPS resulted in upregulation of the MCUR1 mRNA and protein abundance, mitochondrial Ca2+ content, and mitochondrial reactive oxygen species (Mito-ROS) content while decreasing mitochondrial membrane potential, causing mitochondrial damage, and increasing the rate of apoptosis. Ryanodine pretreatment attenuated the upregulation of the mitochondrial Ca2+ content and Mito-ROS content induced by LPS. Overexpression of MCUR1 increased the mitochondrial Ca2+ content and Mito-ROS content, while it decreased mitochondrial membrane potential, damaged mitochondria, and induced cell apoptosis. In addition, knockdown of MCUR1 by small interfering RNA attenuated LPS-induced mitochondrial dysfunction by inhibiting mitochondrial Ca2+ uptake. Our results revealed that exogenous LPS induces MCUR1-mediated mitochondrial Ca2+ overload in bovine mammary epithelial cells, which leads to mitochondrial injury. Thus, MCUR1-mediated Ca2+ homeostasis may be a potential therapeutic target against mitochondrial damage induced by metabolic challenges in bovine mammary epithelial cells.

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.

Abundance of Amino Acid Transporters and mTOR Pathway Components in the Gastrointestinal Tract of Lactating Holstein Cows.

Data from non-ruminants indicate that amino acid (AA) transport into cells can regulate mTOR pathway activity and protein synthesis. Whether mTOR is expressed in the ruminant gastrointestinal tract (GIT) and how it may be related to AA transporters and the AA concentrations in the tissue is unknown. Ruminal papillae and the epithelia of the duodenum, jejunum, and ileum collected at slaughter from eight clinically healthy Holstein in mid-lactation were used. Metabolites and RNA were extracted from tissue for liquid chromatography-mass spectrometry and RT-qPCR analysis. The glycine and asparagine concentrations in the rumen were greater than those in the intestine (p < 0.05), but the concentrations of other AAs were greater in the small intestine than those in the rumen. Among the 20 AAs identified, the concentrations of glutamate, alanine, and glycine were the greatest. The mRNA abundances of AKT1 and MTOR were greater in the small intestine than those in the rumen (p < 0.05). Similarly, the SLC1A1, SLC6A6, SLC7A8, SLC38A1, SLC38A7, and SLC43A2 mRNA abundances were greater (p < 0.05) in the small intestine than those in the rumen. The mRNA abundances of SLC1A5, SLC3A2, and SLC7A5 were greater in the rumen than those in the small intestine (p < 0.05). Overall, the present study provides fundamental data on the relationship between mTOR pathway components and the transport of AAs in different sections of the gastrointestinal tract.

One-carbon metabolism and related pathways in ruminal and small intestinal epithelium of lactating dairy cows.

Physiological and environmental stresses such as the transition into lactation and heat load contribute to gastrointestinal tract (GIT) dysfunction. The nonruminant gastrointestinal tract has mechanisms to cope with pro-oxidant and pro-inflammatory stressors arising from the gut lumen or within intestinal cells. One-carbon metabolism (OCM) contributes to anti-oxidant capacity via the production of glutathione (GSH) and taurine, and the synthesis of phospholipid, creatine, and the osmolyte glycinebetaine among others. A multipronged approach was used to assess the biological relevance of OCM and closely-related pathways on GIT function in dairy cows. Ruminal papillae (Rum) and scrapings from duodenum (Duo), jejunum (Jej), and ileum (Ile) were collected at slaughter from eight multiparous Holstein cows averaging 128 ± 12 d in milk and producing 39 ± 5 kg/d. A MIXED model ANOVA with preplanned orthogonal contrasts was used for statistical analysis. Methionine adenosyl transferase 1 activity (MAT) was ~10-fold greater (P < 0.01) and cystathionine ß-synthase activity doubled in Rum vs. small intestine. Total glutathione peroxidase (GPX) activity was greatest (P = 0.03) in Ile, but similar to Rum. Activity and mRNA abundance of betaine-homocysteine S-methyltransferase were undetectable. There was a 2.5-fold greater protein abundance of GPX1 (P < 0.01) and a ~2-fold greater abundance of GPX3 (P < 0.01) in Rum vs. small intestine. Among the various amino acids (AA) with roles in OCM or closely-related pathways (e.g. creatine synthesis), concentrations of arginine, aspartate, glutamine, methionine, and serine were lower (P < 0.01) in Rum vs. small intestine. Unlike AA, concentrations of OCM-related intermediates S-5'-adenosyl-homocysteine (SAH), glycinebetaine, carnitine, creatine (CRE), and cysteinesulfinic acid were greater (P < 0.01) while taurine was lower in Rum vs. small intestine. Intermediates of the folate cycle were undetectable. The fact that S-adenosylmethionine (SAM) was undetectable while MAT activity and SAH were greater in Rum suggested that availability of SAM (a methyl donor) is a key determinant of flux through the folate and methionine cycles in the GIT. Except for adenosine, concentrations of glutamate, glycine, α-ketoglutarate, hypotaurine, and GSH were lowest in Ile. Together, the data underscored unique differences in activity of one-carbon metabolism and related pathways across sections of the GIT.

The gastrointestinal tract serves a number of essential functions in the animal and exposure to physiological and environmental stressors can lead to disruption of its barrier function and compromise nutrient absorption. In nonruminants, mechanisms to cope with pro-oxidant and pro-inflammatory stressors are essential for maintaining gut function. One-carbon metabolism contributes to anti-oxidant capacity via the production of glutathione and taurine, synthesis of phospholipids, energy-producing compounds, and the osmolyte glycinebetaine among others. A multipronged approach was used to assess the biological relevance of one-carbon metabolism and closely-related pathways in the rumen and small intestine of lactating dairy cows. Enzyme activities, mRNA and protein abundance, and metabolite profiling revealed unique patterns in the rumen versus small intestine. Methyl donor synthesis, transsulfuration, glutathione synthesis, and glutathione peroxidase activity are active mechanisms in ruminal tissue. Research targeting the alteration of these pathways through specific nutrients during stressful periods such as the transition into lactation, weaning, and heat load is warranted.

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.

Feeding a Saccharomyces cerevisiae fermentation product before and during a feed restriction challenge on milk production, plasma biomarkers, and immune function in Holstein cows.

Periods of decreased feed intake may disrupt function of the intestinal barrier. Feeding NutriTek® (NTK; Diamond V, Cedar Rapids, IA), a postbiotic from S. cerevisiae fermentation (SCFP), improved health and supported anti-inflammatory functions. We investigated the effects of feeding NTK to cows before and during a period of feed restriction (FR) designed to model periods of intestinal barrier dysfunction. In total, 16 multiparous cows (97.1 ± 7.6 DIM; n = 8/group) were fed a control diet (CON) or CON plus 19 g/d NTK for 9 wk (Phase 1; P1) and then were subjected to an FR challenge for 5 d, during which they were fed 40% of their ad libitum intake from the 7 d prior to FR. Milk yield (MY) and DMI were collected daily. During FR, milk was collected daily for composition, blood daily to measure plasma biomarkers and to measure monocyte and neutrophil phagocytosis and oxidative burst on d 1, 3, and 5. Data were analyzed using a mixed model in SAS 9.4. All data were subjected to repeated measures ANOVA. Dietary treatment (TRT), Day, and their interaction (TRT × Day) were considered as fixed effects and cow as the random effect. For analysis of P1, data collected during a 7-d adaptation phase were used as a covariate. During P1, NTK cows tended to have greater DMI and had greater fat, ECM and FCM yields, and feed efficiency (ECM/DMI and FCM/DMI). Protein yield tended to be greater in NTK compared with CON cows. A tendency for greater monocyte phagocytosis was detected with NTK. However, during FR, feeding NTK led to lower MY and lactose yield and tended to lower solids percentage. While NTK cows tended to have reduced neutrophil oxidative burst than CON cows during FR (NTK: 26.20%, CON: 36.93%), there was no difference in phagocytosis (NTK: 7.92%, CON: 6.31%). Plasma biomarkers of energy metabolism, liver function, inflammation, and oxidative stress during the FR period did not differ. Overall, results suggested that feeding NTK increased the yield of FCM, ECM, feed efficiency and milk components prior to FR.

Postbiotic fermentation products have the potential to improve health and support anti-inflammatory functions when fed to lactating dairy cows. Since dairy cows experience disruptions of the intestinal barrier function at various stages of their life, for example, the transition into lactation, we sought to investigate potential beneficial effects of feeding a Saccharomyces cerevisiae fermentation (NTK) before and during a period of feed restriction to challenge gut function. Although feeding NTK increased yield of energy-corrected milk and feed efficiency prior to feed restriction (FR), it had no effect on production or plasma indices of metabolism, inflammation, and liver function during a period of abrupt FR to 40% of baseline feed intake.

Taraxasterol alleviates fatty acid-induced lipid deposition in calf hepatocytes by decreasing ROS production and endoplasmic reticulum stress.

Increased concentrations of free fatty acids (FFAs) induce reactive oxygen species (ROSs) generation and endoplasmic reticulum (ER) stress, thus, increasing the risk of fatty liver in dairy cows during the periparturient period. In non-ruminants, Taraxasterol (Tara; a pentacyclic triterpenoid found in medicinal plants) plays an important role in anti-inflammatory and antioxidant reactions. Whether Tara can alleviate or prevent fatty liver in ruminants is unknown. We addressed whether Tara supply could dampen lipid accumulation, ROSs production, and ER stress caused by FFAs in calf hepatocytes. Primary calf hepatocytes were isolated from five healthy calves (1 d old, female, 30-40 kg, fasting, rectal temperature 38.7-39.7 °C). In the first experiment, hepatocytes were incubated with various concentrations of Tara (2.5, 5, and 10 µg/mL) for 12 h prior to the 1.2-mM FFAs challenge. Results indicated that the level of ROSs was lowest with 5 µg/mL Tara. Thus, to further characterize the molecular mechanisms whereby Tara protects from FFAs-induced lipid deposition in calf hepatocytes, we performed incubations with 5 µg/mL Tara for 12 h prior to a 1.2-mM FFAs challenge for an additional 12 h. Results indicated that 1.2-mM FFAs challenge increased mitochondrial membrane potential (MMP), enhanced expression of proteins and mRNA associated with ER stress (PERK, IRE1, GRP78, ATF6, and CHOP) and fatty acid synthesis (FASN, ACC1, and SREBP-1c), and ultimately led to increased lipid droplet synthesis. In contrast, Tara treatment alleviated these negative effects after 1.2-mM FFAs challenge. To determine whether Tara protects against FFAs-induced lipid droplet synthesis by alleviating oxidative stress, hepatocytes were treated with 5 µg/mL Tara for 22 h prior to H2O2 (440 µM) challenge for 2 h. Compared with H2O2 treatment alone, results revealed a marked decrease in ROSs, MMP, and protein abundance of ER stress (GRP78, ATF6, and CHOP) and lipid droplet synthesis in response to Tara prior to H2O2 challenge. Data suggested that the increase in mitochondrial ROSs production contributes to lipid accumulation in calf hepatocytes. Collectively, our in vitro data indicate that Tara alleviates fatty acid-induced lipid deposition. Further research is warranted to ascertain that Tara can be helpful in the therapeutic management of early lactating cows to control or alleviate excessive hepatic lipid deposition.

Fatty liver is a common occurrence in the early postpartum period, partly due to the large influx of fatty acids into the liver during adipose tissue lipolysis. Because there is a linkage between fatty acid metabolism, oxidative stress, and lipid deposition in hepatocytes of nonruminant animals, we evaluated the potential therapeutic roles of Taraxasterol on reactive oxygen species and endoplasmic reticulum (ER) stress in vitro. This compound found in medicinal plants alleviated oxidative and ER stress and reduced lipid accumulation. Thus, it may represent a novel therapeutic tool for the management of dairy cows around parturition.

Activation of PINK1-mediated mitophagy protects bovine mammary epithelial cells against lipopolysaccharide-induced mitochondrial and inflammatory damage in vitro.

Increased metabolic stress during early lactation results in damage of mitochondria and inflammatory responses in bovine mammary epithelial cells, both of which could be aggravated by inhibition of mitophagy. PTEN-induced putative kinase 1 (PINK1)-mediated mitophagy is essential in the removal of damaged mitochondria and the regulation of inflammatory responses. The aim of the present study was to elucidate the role of PINK1-mediated mitophagy on mitochondrial damage and inflammatory responses in bovine mammary epithelial cells challenged with lipopolysaccharide (LPS). Exogenous LPS activated mitophagy and led to lower protein abundance of oxidative phosphorylation (OXPHOS) complexes (COI-V) and lower oxygen consumption rate (OCR) along with increased mitochondrial reactive oxygen species (Mito-ROS) content. These effects were also associated with increased protein abundance of Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) in a time-dependent manner. Pretreatment with 3-Methyladenine (3-MA) or knockdown of PINK1 aggravated the downregulation of COI-V protein abundance, the increase in Mito-ROS content, and the protein abundance of NLRP3, Cleaved-Caspase-1 and IL-1ß induced by LPS. Overexpression of PINK1 activated mitophagy and alleviated LPS-induced NLRP3 inflammasome activation by reducing Mito-ROS production. Overall, the data suggested that PINK1-mediated mitophagy is a crucial anti-inflammatory mechanism that removes damaged mitochondria in bovine mammary epithelial cells experiencing an increased inflammatory load.

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.