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.

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 1.2mM fatty acids (FA) to induce metabolic stress. Furthermore, hepatocytes were treated with 50µM 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, 50µM 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.

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.

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.

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.

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

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

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 cyto-lipotoxicity 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 (NEB) is not fully understood. Particularly regarding its involvement in lipolysis and autophagy. The objective of the present study was to assess the impact 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 NEB.

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.

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.

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.

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

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

Effects of perinatal stress on the metabolites and lipids in plasma of dairy goats.

Dairy goats experience metabolic stress during the peripartal period, and their ability to navigate this stage of lactation is related to the occurrence and development of metabolic diseases. Unlike dairy cows, there is a lack of comprehensive analysis of changes in the plasma profiles of peripartal dairy goats, particularly using high-throughput techniques. A subset of 9 clinically-healthy dairy goats were used from a cohort of 96 primiparous Guanzhong dairy goats (BCS, 2.75 ± 0.15). Blood samples were collected at seven time points around parturition (d 21, 14, 7 before parturition, the day of kidding, and d 7, 14, 21 postpartum), were analyzed using untargeted metabolomics and targeted lipidomics. The orthogonal partial least squares discriminant analysis model revealed a total of 31 differential metabolites including p-cresol sulfate, pyruvic acid, cholic acid, and oxoglutaric acid. The pathway enrichment analysis identified phenylalanine metabolism, aminoacyl-tRNA biosynthesis, and citrate cycle as the top three significantly-altered pathways. The Limma package identified a total of 123 differentially expressed lipids. Phosphatidylserine (PS), free fatty acids (FFA), and acylcarnitines (ACs) were significantly increased on the day of kidding, while diacylglycerols (DAG) and triacylglycerols (TAG) decreased. Ceramides (Cer) and lyso-phosphatidylinositols (LPI) were significantly increased during postpartum period, while PS, FFA, and ACs decreased postpartum and gradually returned to antepartum levels. Individual species of FFA and phosphatidylcholines (PC) were segregated based on the differences in the saturation and length of the carbon chain. Overall, this work generated the largest repository of the plasma lipidome and metabolome in dairy goats across the peripartal period, which contributed to our understanding of the multifaceted adaptations of transition dairy goats.

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

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

Intermediate metabolites and molecular correlates of one­carbon and nutrient metabolism differ in tissues from Holstein fetuses.

Methionine and folate cycles along with transsulfuration comprise the one­carbon metabolism (OCM) pathway. Amino acids and other nutrients feed into OCM, which is central to cellular function. mRNA abundance, proteins (Western blotting), and metabolites (GC-MC) associated with OCM were used to characterize these mechanisms in fetal tissues. Liver, whole intestine, and semitendinosus muscle were harvested from fetuses in 6 multiparous Holstein cows (37 kg milk/d, 100 d gestation). Data were analyzed using PROC MIXED (SAS 9.4). Protein abundance of BHMT was greatest (P < 0.01) in liver suggesting active remethylation of homocysteine to methionine. This idea was supported by the greater (P < 0.05) mRNA of CBS, BHMT, MTR, SHMT1, and MAT1A (encoding OCM enzymes) in liver. The antioxidant protein GPX3 had greatest (P < 0.05) abundance in liver, whereas the glutathione-transferase GSTM1 was 5-fold greater (P < 0.05) in intestine than liver and muscle. Greatest concentrations of glycine, serine, and taurine along with lower cysteine underscored the relevance of OCM in fetal liver. Phosphoethanolamine concentration was greatest (4-fold, P < 0.05) in intestine and along with the greatest (P < 0.05) mRNA of SLC44A1 (choline transporter), CHKA, and CEPT1 underscored the importance of the CDP-choline pathway. Greatest (P < 0.05) mRNA of PPARA, CPT1A, and HMGCS2 along with lower PCK1 in liver highlighted a potential reliance on fatty acid oxidation. In contrast, greater (P < 0.05) concentration of myo-inositol in muscle and intestine suggested both tissues rely on glucose as main source of energy. Future research should address how environmental inputs such as maternal nutrition alter these pathways in fetal tissues and their phenotypic outcomes.