Epigallocatechin-3-gallate protects bovine ruminal epithelial cells against lipopolysaccharide-induced inflammatory damage by activating autophagy.

BACKGROUND: Subacute ruminal acidosis (SARA) causes an increase in endotoxin, which can induce immune and inflammatory responses in the ruminal epithelium of dairy cows. In non-ruminants, epigallocatechin-3-gallate (EGCG), a major bioactive ingredient of green tea, is well-known to alleviate inflammation. Whether EGCG confers protection against SARA-induced inflammation and the underlying mechanisms are unknown. RESULTS: In vivo, eight ruminally cannulated Holstein cows in mid-lactation were randomly assigned to either a low-concentrate (40%) diet (CON) or a high-concentrate (60%) diet (HC) for 3 weeks to induce SARA (n = 4). Cows with SARA had greater serum concentrations of tumor necrosis factor (TNF)-α and interleukin-6, and epithelium had histological signs of damage. In vitro, immortalized bovine ruminal epithelial cells (BREC) were treated with lipopolysaccharide (LPS) to imitate the inflammatory damage caused by SARA. Our data revealed that BREC treated with 10 µg/mL LPS for 6 h successfully induce a robust inflammatory response as indicated by increased phosphorylation of IκBα and nuclear factor kappa-B (NF-κB) p65. Pre-treatment of BREC with 50 µmol/L EGCG for 6 h before LPS challenge promoted the degradation of NLR family pyrin domain containing 3 (NLRP3) inflammasome through activation of autophagy, which further repressed activation of NF-κB pathway targeting Toll-like receptor 4 (TLR4). Analyses also revealed that the ECGG upregulated tight junction (TJ) protein expression upon incubation with LPS. CONCLUSIONS: Subacute ruminal acidosis causes ruminal epithelium injury and systemic inflammation in dairy cows. However, the anti-inflammatory effects of EGCG help preserve the integrity of the epithelial barrier through activating autophagy when BREC are exposed to LPS. Thus, EGCG could potentially serve as an effective therapeutic agent for SARA-associated inflammation.

Research Progress on the Role of M6A in Regulating Economic Traits in Livestock.

Reversible regulation of N6-methyladenosine (m6A) methylation of eukaryotic RNA via methyltransferases is an important epigenetic event affecting RNA metabolism. As such, m6A methylation plays crucial roles in regulating animal growth, development, reproduction, and disease progression. Herein, we review the latest research advancements in m6A methylation modifications and discuss regulatory aspects in the context of growth, development, and reproductive traits of livestock. New insights are highlighted and perspectives for the study of m6A methylation modifications in shaping economically important traits are discussed.

Varying the ratio of Lys: Met through enhancing methionine supplementation improved milk secretion ability through regulating the mRNA expression in bovine mammary epithelial cells under heat stress.

Introduction: The ratio of lysine (Lys) to methionine (Met) with 3.0: 1 is confirmed as the "ideal" profile for milk protein synthesis, but whether this ratio is suitable for milk protein synthesis under HS needs to be further studied. Methods: To evaluate the molecular mechanism by which HS and Lys to Met ratios affect mammary cell functional capacity, an immortalized bovine mammary epithelial cell line (MAC-T) is incubated with 5 doses of Met while maintaining a constant concentration of Lys. The MAC-T cells was treated for 6 h as follow: Lys: Met 3.0: 1 (control 37°C and IPAA 42°C) or treatments under HS (42°C) with different ratios of Lys: Met at 2.0: 1 (LM20), 2.5: 1 (LM25), 3.5: 1 (LM35) and 4.0: 1 (LM40). RNA sequencing was used to assess transcriptome-wide alterations in mRNA abundance. Results: The significant difference between control and other groups was observed base on PCA analysis. A total of 2048 differentially expressed genes (DEGs) were identified in the IPAA group relative to the control group. Similarly, 226, 306, 148, 157 DEGs were detected in the LM20, LM25, LM35 and LM40 groups, respectively, relative to the IPAA group. The relative mRNA abundance of HSPA1A was upregulated and anti-apoptotic genes (BCL2L1 and BCL2) was down-regulated in the IPAA group, compared to the control group (p < 0.05). Compared with the IPAA group, the relative mRNA abundance of anti-apoptotic genes and casein genes (CSN1S2 and CSN2) was up-regulated in the LM25 group (p < 0.05). The DEGs between LM25 and IPAA groups were associated with the negative regulation of transcription RNA polymerase II promoter in response to stress (GO: 0051085, DEGs of BAG3, DNAJB1, HSPA1A) as well as the mTOR signaling pathway (ko04150, DEGs of ATP6V1C2, WNT11, WNT3A, and WNT9A). Several DEGs involved in amino acids metabolism (AFMID, HYKK, NOS3, RIMKLB) and glycolysis/gluconeogenesis (AFMID and MGAT5B) were up-regulated while DEGs involved in lipolysis and beta-oxidation catabolic processes (ALOX12 and ALOX12B) were down-regulated. Conclusion: These results suggested that increasing Met supply (Lys: Met at 2.5: 1) may help mammary gland cells resist HS-induced cell damage, while possibly maintaining lactation capacity through regulation of gene expression.

Role of hypoxia-inducible-factor-1α (HIF-1α) in ferroptosis of adipose tissue during ketosis.

Postpartum cows experience lipolysis in adipose tissue due to negative energy balance (NEB), and accumulation of free fatty acids (FFA) leads to metabolic stress in adipose tissue. Ferroptosis is a type of cell death triggered by excessive buildup of iron-dependent lipid peroxides, which is involved in the occurrence and development of various metabolic diseases in nonruminants. However, it is still unclear whether ferroptosis occurs in the adipose tissue of ketotic cows and the regulatory mechanisms behind ferroptosis. Despite multiple studies demonstrating the significant involvement of hypoxia-inducible-factor-1α (HIF-1α) in regulating cellular dysfunction, its specific function in adipose tissue of ketotic dairy cows remains uncertain, particularly its regulation of oxidative stress and ferroptosis. The study aimed to explore the impact of HIF-1α on oxidative stress and ferroptosis in bovine subcutaneous adipose tissue and isolated adipocytes. The adipose tissue of clinical ketosis cows (n = 15) with a serum BHB concentration of 3.13 mM (interquartile range = 0.14) and healthy cows (n = 15) with a serum BHB concentration of and 0.58 mM (interquartile range = 0.13) was collected. The results showed that the concentrations of lipid peroxidation malondialdehyde (MDA), reactive oxygen species (ROS), Fe2+ and total iron were increased in adipose tissue of cows with ketosis, while the contents of glutathione (GSH) were reduced. Furthermore, the protein levels of HIF-1α, heme oxygenase 1 (HMOX1), catalase (CAT), superoxide dismutase 1 (SOD1), acyl-CoA synthetase 4 (ACSL4), and nuclear factor erythroid-derived 2-like 2 (NFE2L2) exhibited higher abundance in adipose tissue obtained from cows with ketosis, whereas the protein abundance of solute carrier family 7 member 11 (SLC7A11), glutamate cysteine ligase catalytic subunit (GCLC), kelch-like ECH-associated protein 1 (KEAP1), glutamate cysteine ligase regulatory subunit (GCLM) and glutathione peroxidase 4 (GPX4) were lower. To simulate the ferroptosis state of adipose tissue in ketotic cows, primary bovine adipocytes were isolated from the adipose tissue of healthy cows and cultured with erastin to construct ferroptosis model. Adipocytes were cultured with either an adenovirus overexpressing HIF-1α or small interfering RNA targeting HIF-for 48 h, followed by exposure to erastin (1 µM) for 24 h. Treatment with erastin led to higher protein abundance of CAT, SOD1, NFE2L2 and HMOX1, while it inhibited the protein expression levels of GCLC, SLC7A11, GCLM, GPX4 and KEAP1. Furthermore, erastin treatment elevated the levels of ROS, MDA, Fe2+, total iron and reduced the content of GSH. The overexpression of HIF-1α reversed the erastin-induced decreases in the protein abundance of GPX4 and SLC7A11, as well as the levels of MDA, ROS, Fe2+ and total iron, while significantly increasing protein abundance and content of CAT, SOD1, NFE2L2, HMOX1, GCLC, GCLM, GPX4, SLC7A11 and GSH. Conversely, the silencing of HIF-1α further exacerbated the erastin-induced levels of MDA, ROS, Fe2+ and total iron, while inhibiting the upregulation of SOD1, CAT, NFE2L2 and HMOX1. Collectively, these findings suggest that activation of HIF-1α may function as an adaptive mechanism to mitigate ferroptosis and alleviate oxidative stress in adipose tissue.

Effects of reduced levels of organic trace minerals in proteinate forms and selenium yeast in the mineral mix on lactation performance, milk fatty acid composition, nutrient digestibility, and antioxidant status in dairy goats.

The objective was to evaluate the effects of replacing inorganic trace minerals (ITM) with reduced levels of organic trace minerals (OTM) in proteinate forms and selenium yeast (Se-yeast) in the mineral premix of prepartal and lactating dairy goats on lactation performance, milk fatty acid (FA) composition, nutrient digestibility, and antioxidant status. Xinong Saanen dairy goats (n = 40) were blocked by parity and body weight, and randomly assigned to either ITM or OTM treatments from 4 wk prepartum to 8 mo of lactation. Both groups received the same basal diet except for the trace mineral supplement. The ITM supplement included Fe, Cu, Zn, and Mn as sulfates, and Se as selenite to meet the recommendations. The OTM supplement included Fe, Cu, Zn, and Mn as proteinates at 50% of ITM supplement levels, and Se as Se-yeast at 100% of ITM supplement level. Sampling and measurements were performed in the first, second, fourth, and eighth month of lactation. Data were summarized by month and treatment, and analyzed using the Mixed Model of SPSS with repeated measures. OTM group showed lower milk fat (P = 0.02) and higher milk Se (P = 0.03) with no compromised effects on milk yield and milk protein compared to ITM group. Furthermore, OTM decreased the content of C6:0, C8:0, and C10:0 (P < 0.05) and increased the content of odd- and branched-chain FAs in milk fat due to greater content of C15:0 (P = 0.01) and anteiso C15:0 (P = 0.07). OTM led to greater total tract digestibility of dry matter (P = 0.03), crude protein (P = 0.07), ether extract (P = 0.03), and acid detergent fiber (P = 0.05). OTM goats showed less fecal excretion of Fe (P = 0.01), Cu (P < 0.01), and Zn (P = 0.08) compared to ITM goats. There was a tendency for greater serum GSH-Px activity (P = 0.09) with OTM. Overall, the long-term substitution of reduced levels of OTM for ITM can change milk fat and FA composition while maintaining milk yield, digestibility, and antioxidant status.

Lipids play important roles in the physiochemical properties of milk and dairy products. For example, specific milk fatty acids (FAs), such as those with 8- and 10-carbon chains, influence the flavor of goat milk. Additionally, certain odd- and branched-chain (OBCFA) exhibit anticarcinogenic effects in vitro. Studies in dairy cows have demonstrated organic trace minerals (OTM) can enhance lactation performance, nutrient digestibility, and antioxidant status. In this study, substituting OTM for inorganic trace minerals (ITM) in the diet of dairy goats decreased milk fat without negatively impacting milk yield, nutrient digestibility, and serum antioxidant status. Feeding OTM reduced the content of C6:0, C8:0, and C10:0 FAs while increasing the content of OBCFA in milk fat. The data suggest that replacing ITM with reduced levels of OTM in proteinates and selenium yeast can alter milk FA composition without compromising milk yield, nutrient digestibility, and antioxidant status in dairy goats.

m6A Methylation Mediates the Function of the circRNA-08436/miR-195/ELOVL6 Axis in Regards to Lipid Metabolism in Dairy Goat Mammary Glands.

The nutritional value of goat milk is determined by the composition of its fatty acids, with particular importance placed on the role of unsaturated fatty acids in promoting human health. CircRNAs have been known to affect fatty acid metabolism through different pathways. In this study, high-throughput sequencing was employed to construct expression profiles of mammary tissue harvested during the dry period and peak lactation stages of dairy goats. Differentially expressed circRNAs and mRNAs were screened, revealing significantly higher expression levels of circRNA-08436 and ELOVL6 during the peak lactation period compared with the dry period. Thus, circRNA-08436 and ELOVL6 were chosen for subsequent studies. The findings demonstrated that circRNA-08436 not only promotes the synthesis of triglyceride (TAG) and cholesterol in goat mammary epithelial cells (GMECs), but also increases the concentrations of saturated fatty acids in the cells. Through the utilization of software prediction, the dual luciferase reporter system, and qRT-PCR, it was observed that circRNA-08436 binds to miR-195, with its overexpression reducing the expression levels of miR-195 and inhibiting TAG synthesis. In addition, circRNA-08436 upregulated the expression levels of the miR-195 target gene ELOVL6. The data also revealed that YTHDC1 facilitated the transport of circRNA-08436 from the nucleus to the cytoplasm, while YTHDC2 in the cytoplasm functioned as a "reader" to identify and degrade circRNA-08436. Taken together, these findings contribute to a better understanding of the molecular regulation of fatty acid metabolism in the mammary glands of dairy goats, thus offering a sound theoretical basis for the production of high-quality goat milk.

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.

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.

ß-sitosterol alleviates high fatty acid-induced lipid accumulation in calf hepatocytes by regulating cholesterol metabolism.

A significant reduction in plasma concentration of cholesterol during early lactation is a common occurrence in high-yielding dairy cows. An insufficient synthesis of cholesterol in the liver has been linked to lipid accumulation caused by high concentrations of fatty acids during negative energy balance (NEB). As ruminant diets do not provide quantitative amounts of cholesterol for absorption, phytosterols such as ß-sitosterol may serve to mitigate the shortfall in cholesterol within the liver during NEB. To gain mechanistic insights, primary hepatocytes were isolated from healthy female 1-day old calves for in vitro studies with or without 1.2 mM fatty acids (FA) to induce metabolic stress. Furthermore, hepatocytes were treated with 50 µM ß-sitosterol with or without FA. Data were analyzed by one-way ANOVA with subsequent Bonferroni correction. Results revealed that calf hepatocytes treated with FA had greater content of non-esterified fatty acids (NEFA) and triacylglycerol (TAG), and greater mRNA and protein abundance of the lipid synthesis-related SREBF1 and FASN. In contrast, mRNA and protein of CPT1A (fatty acid oxidation) and the cholesterol metabolism-related targets SREBF2, HMGCR, ACAT2, APOA1, ABCA1 and ABCG5 was lower. Content of the antioxidant-related glutathione (GSH) and activities of superoxide dismutase (SOD) also was lower. Compared with FA challenge alone, 50 µM ß-sitosterol led to greater mRNA and protein abundance of SREBF2, HMGCR, ACAT2 and ABCG5, and greater content of GSH and activity of SOD. In contrast, compared with the FA group, the mRNA and protein abundance of SREBF1 and ACC1 and the content of TAG and NEFA in the ß-sitosterol + FA group were lower. Overall, ß-sitosterol can promote cholesterol metabolism and reduce oxidative stress while reducing lipid accumulation in hepatocytes challenged with high concentrations of fatty acids.

Disturbances of Ruminal Microbiota and Liver Inflammation, Mediated by LPS and Histamine, in Dairy Cows Fed a High-Concentrate Diet.

The ecosystem of ruminal microbiota profoundly affects the health and milk production of dairy cows. High-concentrate diets are widely used in dairy farms and evoke a series of metabolic disorders. Several studies have reported the effects of high-concentrate diets on the ruminal microbiome, while the effect of changes in ruminal microbial flora, induced by high-concentrate diet feeding, on the liver of dairy cows has not been studied before. In this study, 12 mid-lactating Holstein Friesian cows (weight of 455 ± 28 kg; parities of 2.5 ± 0.5; starting milk yield of 31.59 ± 3.2 kg/d; DMI of 21.7 ± 1.1 kg/d; and a DIM at the start of the experiment of 135 ± 28 d) were fitted with ruminal fistulas, as well as with portal and hepatic vein catheters. All cows were randomly divided into 2 groups; then, they fed with low-concentrate diets (LC, concentrate: forage = 40:60) and high-concentrate diets (HC, concentrate: forage = 60:40) for 18 weeks. The forage sources were corn silage and alfalfa hay. After the cows of two groups were euthanized over two consecutive days, ruminal microbiota; the concentration of LPS in the rumen content; cecum content; the levels of blood and histamine in rumen fluid, blood, and the liver; the histopathological status of the rumen and cecum; and the inflammatory response of the liver were assessed in dairy cows under conditions of subacute ruminal acidosis (SARA). These conditions were caused by high-concentrate diet feeding. All data were analyzed using the independent t-test in SPSS. The results showed that high-concentrate diet feeding increased the concentration of LPS and histamine in the rumen and plasma of veins (p < 0.05). The abundance of Bacteroidetes at the phylum level, and of both Bacteroidetes and Saccharibacteria at the genus level, was decreased, while the abundance of Firmicutes at the phylum level and Oscillibacter at the genus level was increased by high-concentrate diet feeding. The decreased pH values of ruminal contents (LC = 6.02, HC = 5.90, p < 0.05) and the increased level of LPS in the rumen (LC = 4.921 × 105, HC = 7.855 × 105 EU/mL, p < 0.05) and cecum (LC = 11.960 × 105, HC = 13.115 × 105 EU/mL, p < 0.01) induced the histopathological destruction of the rumen and cecum, combined with the increased mRNA expression of IL-1ß (p < 0.05). The histamine receptor H1R and the NF-κB signaling pathway were activated in the liver samples taken from the HC group. In conclusion, the elevated concentrations of LPS and histamine in the gut may be related to changes in the ruminal microbiota. LPS and histamine induced the inflammatory response in the ruminal epithelium, cecum epithelium, and liver. However, the cause-effect mechanism needs to be proved in future research. Our study offers a novel therapeutic strategy by manipulating ruminal microbiota and metabolism to decrease LPS and histamine release and to improve the health of dairy cows.

Potential Role of Lauric Acid in Milk Fat Synthesis in Chinese Holstein Cows Based on Integrated Analysis of Ruminal Microbiome and Metabolome.

The composition and metabolic profile of the ruminal microbiome have an impact on milk composition. To unravel the ruminal microbiome and metabolome affecting milk fat synthesis in dairy cows, 16S rRNA and internal transcribed spacer (ITS) gene sequencing, as well as ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) methods were used to investigate the significant differences in ruminal bacterial and fungal communities as well as metabolome among Chinese Holstein cows with contrasting milk fat contents under the same diet (H-MF 5.82 ± 0.41% vs. L-MF 3.60 ± 0.12%). Another objective was to culture bovine mammary epithelial cells (BMECs) to assess the effect of metabolites on lipid metabolism. Results showed that the acetate-to-propionate ratio and xylanase activity in ruminal fluid were both higher in H-MF. Microbiome sequencing identified 10 types of bacteria and four types of fungi differently abundant at the genus level. Metabolomics analysis indicated 11 different ruminal metabolites between the two groups, the majority of which were lipids and organic acids. Among these, lauric acid (LA) was enriched in fatty acid biosynthesis with its concentration in milk fat of H-MF cows being greater (217 vs. 156 mg per 100 g milk), thus, it was selected for an in vitro study with BMECs. Exogenous LA led to a marked increase in intracellular triglyceride (TG) content and lipid droplet formation, and it upregulated the mRNA abundance of fatty acid uptake and activation (CD36 and ACSL1), TG synthesis (DGAT1, DGAT2 and GPAM), and transcriptional regulation (SREBP1) genes. Taken together, the greater relative abundance of xylan-fermenting bacteria and fungi, and lower abundance of bacteria suppressing short-chain fatty acid-producing bacteria or participating in fatty acid hydrogenation altered lipids and organic acids in the rumen of dairy cows. In BMECs, LA altered the expression of genes involved in lipid metabolism in mammary cells, ultimately promoting milk fat synthesis. Thus, it appears that this fatty acid plays a key role in milk fat synthesis.

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 BHB in the 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; BCS, 3.1 ± 0.45). All plasma and milk samples (n = 90) were subjected to liquid chromatography-MS-based metabolomic analysis. Statistical analyses were performed using GraphPad Prism 8.0, MetaboAnalyst 4.0, and R 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, as well as lower milk protein, lactose, solids-not-fat, 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 versus SCK, CON versus CK, and SCK versus 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, and steroid hormone biosynthesis were identified as important 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, and fatty acid degradation were identified as important 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.

Preliminary Results on the Effects of Soybean Isoflavones on Growth Performance and Ruminal Microbiota in Fattening Goats.

Soybean isoflavones (SIFs), a group of secondary metabolites, have antioxidant, anti-inflammatory, and hormone-like activities. Supplementation with SIFs in the diet was reported to promote lactation performance in ruminants. The present study was performed to further decipher the effect of various concentrations of SIFs on growth and slaughter performance, serum parameters, meat quality, and ruminal microbiota in fattening goats. After a two-week acclimation, a total of 27 5-month-old Guanzhong male goats (18.29 ± 0.44 kg) were randomly assigned to control (NC), 100 mg/d SIF (SIF1), or 200 mg/d SIF (SIF2) groups. The experimental period lasted 56 days. The weight of the large intestine was greater (p < 0.05) in the SIF1 and SIF2 groups compared with the NC group. Meat quality parameters indicated that SIF1 supplementation led to lower (p < 0.05) cooking loss and shear force (0.05 < p < 0.10). The 16S rRNA sequencing analysis demonstrated that SIF1 supplementation led to lower (p < 0.05) proportions of Papillibacter and Prevotellaceae_UCG-004 but greater (p < 0.05) CAG-352 abundance in the rumen; these responses might have contributed to the improvement in production performance. In conclusion, meat quality and ruminal microbiome could be manipulated in a positive way by oral supplementation with 100 mg/d of SIFs in fattening goats. Thus, this study provides new insights and practical evidence for the introduction of SIFs as a novel additive in goat husbandry.

Maternal protein supplementation during mid-gestation improves offspring performance and metabolism in beef cows.

This study examined the impact of maternal protein supplementation during mid-gestation on offspring, considering potential sex-related effects. Forty-three pregnant purebred Tabapuã beef cows (20 female and 23 male fetuses) were collectively managed in a pasture until 100 d of gestation. From 100 to 200 d of gestation, they were randomly assigned to the restricted group [(RES) - basal diet (75% corn silage + 25% sugar cane bagasse + mineral mixture); n = 24] or control group [(CON) - same basal diet + based-plant supplement [40% of crude protein, 3.5 g/kg of body weight (BW); n = 19]. From 200 d of gestation until parturition, all cows were equally fed corn silage and mineral mixture. During the cow-calf phase, cows and their calves were maintained in a pasture area. After weaning, calves were individually housed and evaluated during the backgrounding (255 to 320 d), growing 1 (321 to 381 d), and growing 2 (382 to 445 d) phases. Offspring's blood samples were collected at 210 and 445 d of age. Samples of skeletal muscle tissue were collected through biopsies at 7, 30, and 445 d of age. Muscle tissue samples were subjected to reverse-transcription quantitative polymerase chain reaction analysis. Prenatal treatment and offspring's sex (when pertinent) were considered fixed effects. The significance level was set at 5%. At mid-gestation, cows supplemented with protein reached 98% and 92% of their protein and energy requirements, while nonsupplemented cows attained only 30% and 50% of these requirements, respectively. The RES offspring were lighter at birth (27 vs. 31 kg), weaning (197 vs. 214 kg), and 445 d of age (398 vs. 429 kg) (P ≤ 0.05). The CON calves had greater (P < 0.05) morphometric measurements overall. The CON offspring had ~26% greater muscle fiber area (P ≤ 0.01). There was a trend (P = 0.06) for a greater Mechanistic target of rapamycin kinase mRNA expression in the Longissimus thoracis in the CON group at 7 d of age. The Myogenic differentiation 1 expression was greater (P = 0.02) in RES-females. Upregulation of Carnitine palmitoyltransferase 2 was observed in RES offspring at 445 d (P = 0.04). Expression of Fatty acid binding protein 4 (P < 0.001), Peroxisome proliferator-activated receptor gamma (P < 0.001), and Stearoyl-Coenzyme A desaturase (P < 0.001) was upregulated in CON-females. Therefore, protein supplementation during gestation enhances offspring growth and promotes favorable responses to lipogenesis, particularly in females.

In tropical conditions, beef cows on pasture often experience protein restriction during mid-to-late gestation, potentially impacting offspring development negatively. To address this, we investigated the effects of strategic protein supplementation for pregnant beef cows fed low-quality forage during mid-gestation on the postnatal growth trajectory of their offspring. The supplementation program, implemented during mid-gestation, increased dry matter intake by addressing nitrogen deficiency in the rumen, resulting in meeting 98% and 92% of protein and energy requirements in supplemented cows. In contrast, nonsupplemented cows met only 30% and 50% of these requirements, respectively. Consequently, protein supplementation positively influenced the postnatal growth trajectory of the offspring, attributed to beneficial changes in secondary myogenesis and hypertrophy processes. Supplementing cows with crude protein also stimulated lipogenesis, potentially contributing to intramuscular fat deposition, particularly in females. Therefore, this study emphasizes the importance of nutritional interventions for pregnant beef cows fed low-quality forage.

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.

Total Replacement of Soybean Meal with Sundried Soymilk Residue in the Total Mixed Ration has a Negative Impact on Intake, Digestibility, and Milk Production in Dairy Goats.

This study aimed to evaluate whether total replacement of soybean meal (SBM) with sundried soymilk residue (SSR) in a total mixed ration (TMR) affects intake, digestibility, milk production, and blood metabolites in dairy goats. A total of 12 healthy Saanen dairy goats (40.12 ± 5.80 kg of BW) in midlactation (31.23 ± 10.12 days) were used in a randomized complete design (n = 4 goats/group). Dietary treatments were based on a TMR as follows: control TMR without SSR (CON) or SBM-based TMR with 50% or 100% of SSR replacing SBM (SSR-50 and SSR-100, respectively). All goats had ad libitum access to feed and clean water throughout the experiment. The dry matter (DM) intake decreased (p < 0.05) with the increasing replacement ratio of SBM and was lowest in the SSR-100 group. Similarly, organic matter (OM) digestibility was lowest (p < 0.05) in the SSR-100 group. However, the digestibility of DM, CP, NDF, and ADF did not change (p > 0.05) by dietary treatments. Compared with CON, the milk yield decreased significantly (p < 0.05) with increasing replacement ratio of SBM. In contrast, milk composition such as total solids, solids-not-fat, milk fat, lactose, protein, and pH were not influenced (p > 0.05) by feeding dietary SSR. Compared with other treatments, blood glucose concentration was lower (p < 0.05) in the SSR-100 group. In contrast, packed cell volume, glucose, and plasma urea nitrogen concentrations did not differ (p > 0.05). The results indicated that SSR could replace SBM in a TMR at less than 50%. Thus, the present study provides support for further investigation to enhance the utilization of soybean waste as an alternative protein source in the TMR for dairy goats and potentially other ruminants.

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.

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.

Inoculation of Newborn Lambs with Ruminal Solids Derived from Adult Goats Reprograms the Development of Gut Microbiota and Serum Metabolome and Favors Growth Performance.

Microbial transplantation in early life was a strategy to optimize the health and performance of livestock animals. This study aimed to investigate the effect of active ruminal solids microorganism supplementation on newborn lamb gut microbiota and serum metabolism. Twenty-four Youzhou dark newborn lambs were randomly divided into three groups: (1) newborn lambs fed with sterilized goat milk inoculated with sterilized normal saline (CON), supernatant from ruminal solids (SRS), or autoclaved supernatant from ruminal solids (ASRS). Results showed that SRS increased gut bacterial richness and community, downregulating the Firmicutes/Bacteroidetes ratio, and increased the abundance of some probiotics (Bacteroidetes, Spirochaetota, and Fibrobacterota), while reducing the abundance of Fusobacteriota, compared to the CON group. SRS also improved the plasma metabolic function, such as arachidonic acid metabolism, primary bile acid biosynthesis, and tryptophan metabolism and then actively promoted the levels of ALP and HLD. Our study indicated that inoculation with active ruminal solids significantly affected the intestinal microbial communities and metabolic characteristics, and these changes can improve the growing health of the newborn lamb. These findings provided an experimental and theoretical basis for the application of ruminal solid-attached microorganisms in the nutritional management of lambs reared for human consumption.