Responses of the blood acid-base balance and blood plasma metabolomics of broiler chickens after change to diets with high free amino acid levels.

Free amino acids (AA) are needed to fulfill the AA requirements of broiler chickens in diets low in CP. This study investigated whether the acid-base balance and the blood plasma metabolome are affected immediately after a change to diets with high free AA levels. Male broiler chickens received a starter diet with 164 g CP/kg and 80 g soy protein isolate/kg until d 7 post-hatch. From this day on, birds were offered a diet almost identical to the starter diet (0FAA) or 2 diets with 50% (50FAA) or 100% (100FAA) of the digestible AA from soy protein isolate substituted with free AA. Blood was sampled to determine the acid-base status and for untargeted metabolomics analysis on d 0, 1, 2, 4, 7, and 14 and d 1, 7, and 14 after diet change, respectively (n = 14 birds/treatment). Compared to 0FAA, blood pH was decreased on d 4 and 7 for 100FAA and on d 4 for 50FAA (P ≤ 0.019). On d 4, 7, and 14, bicarbonate, base excess, and total carbon dioxide were lower for 100FAA than for 0FAA (P ≤ 0.006). The partial pressure of carbon dioxide was higher for 50FAA than for 0FAA on d 4 (P = 0.047). Compared to 0FAA, chloride was higher for 100FAA on d 1, 2, 4, 7, and 14, and for 50FAA on d 1, 2, and 4 (P ≤ 0.030). In the metabolomics assay, 602, 463, and 302 metabolites were affected by treatment on d 1, 7, and 14, respectively (P < 0.050), but they did not indicate that metabolic pathways were affected. Flavonoids were the most consistently affected category of metabolites. The results indicated a metabolic acidosis for 100FAA from d 4 to 7 and a respiratory acidosis for 50FAA on d 4 after diet change. These types of acidosis were compensated later on in the experiment. The metabolomics analysis did not indicate that high free AA inclusion affected metabolic pathways.

The influence of substituting dietary peptide-bound with free amino acids on nitrogen metabolism and acid-base balance of broiler chickens depends on asparagine and glutamine supply.

Reducing dietary crude protein (CP) concentration while maintaining adequate amino acid (AA) supply by free AA inclusion can contribute to attenuate the negative environmental effects of animal farming. This study investigated upper limits of dietary free AA inclusions without undesirable effects including the dependence on asparagine (Asn) and glutamine (Gln) supply. Ten broilers were allocated to sixty-three metabolism units each and offered nine experimental diets from day (d) 7-21 (n 7). One diet (167 g CP/kg) contained 80 g soya protein isolate (SPI)/kg. In the other diets, 25, 50, 75 and 100 % of the digestible AA from SPI were substituted with free AA. Digestible Asn+aspartic acid (Asp) and Gln+glutamic acid (Glu) were substituted with Asp/Glu or 50/50 mixes of Asp/Asn and Glu/Gln, respectively. Total excreta were collected from d 11-14 and from d 18-21. Growth and nitrogen accretion were unaffected by 25 and 50 % substitution without and with free Asn/Gln, respectively, but decreased at higher substitution (P ≤ 0·024). Circulating concentrations of Asp, Glu and Gln were unaffected by treatment, while Asn decreased at substitution higher than 50 % when Asn/Gln were not provided (P ≤ 0·005). Blood gas analysis on d 21 indicated a compensated metabolic acidosis at substitution higher than 50 and 75 % without and with free Asn/Gln, respectively (P ≤ 0·017). Results suggest that adding Asn/Gln increased an upper limit for proportion of dietary free AA from 10 to 19 % of dietary CP and enabled higher free AA inclusion without affecting the acid-base balance.

Clinical evaluation of the Immulite® 1000 chemiluminescent immunoassay for measurement of equine serum insulin.

Introduction: Accurate quantitative analysis of equine insulin in blood samples is critical for assessing hyperinsulinemia in horses. Although there are various laboratory methods for evaluating equine serum insulin, different immunoassays show significant discrepancies between the determined insulin concentrations and are often not comparable. The aim of this study was to evaluate the Immulite® 1000 chemiluminescent immunoassay (CLIA) to establish independent laboratory and assay-specific cut values to provide an accurate diagnosis of hyperinsulinemia in horses. Thus, the analytical and clinical performance of Immulite® 1000 CLIA in terms of precision (intra- and inter-assay coefficient of variance, CV) and recovery upon dilution were evaluated and compared with radioimmunoassay (RIA), which has been previously validated for use in horses. Material and methods: Archived serum samples (n = 106) from six Quarter horse mares enrolled in the glucose phase of a Frequently Sampled Insulin and Glucose Test (FSIGT) study were used to measure blood insulin. Results: The Immulite® 1000 CLIA had good precision with acceptable intra- and inter-assay CVs, adequate recovery on dilution, and a strong correlation with the RIA (r = 0.974, P < 0.0001), with constant bias resulting in consistently lower values. Discussion: On this basis, the Immulite® 1000 Insulin Assay is valid for measuring equine serum insulin for diagnostic and monitoring purposes when cut values are appropriately adjusted.

Pathological Features and Genomic Characterization of an Actinobacillus equuli subsp. equuli Bearing Unique Virulence-Associated Genes from an Adult Horse with Pleuropneumonia.

Actinobacillus equuli subsp. equuli is the etiological agent of sleepy foal disease, an acute form of fatal septicemia in newborn foals. A. equuli is commonly found in the mucous membranes of healthy horses' respiratory and alimentary tracts and rarely causes disease in adult horses. In this study, we report a case of a 22-year-old American Paint gelding presenting clinical signs associated with an atypical pattern of pleuropneumonia subjected to necropsy. The gross and histopathological examinations revealed a unilateral fibrinosuppurative and hemorrhagic pleuropneumonia with an infrequent parenchymal distribution and heavy isolation of A. equuli. The whole genome sequence analysis indicated that the isolate shared 95.9% homology with the only other complete genome of A. equuli subsp. equuli available in GenBank. Seven virulence-associated genes specific to the isolate were identified and categorized as iron acquisition proteins, lipopolysaccharides (LPS), and capsule polysaccharides. Moreover, four genes (glf, wbaP, glycosyltransferase family 2 protein, and apxIB) shared higher amino acid similarity with the invasive Actinobacillus spp. than the reference A. equuli subsp. equuli genome. Availability of the whole genome sequence will allow a better characterization of virulence determinants of A. equuli subsp. equuli, which remain largely elusive.

Yak rumen microbiome elevates fiber degradation ability and alters rumen fermentation pattern to increase feed efficiency.

Rumen microbes play an important role in ruminant energy supply and animal performance. Previous studies showed that yak (Bos grunniens) rumen microbiome and fermentation differ from other ruminants. However, little is understood about the features of the rumen microbiome that make yak adapted to their unique environmental and dietary conditions. This study was to investigate the rumen microbiome and metabolome to understand how yak adapt to the coarse forage and harsh environment in the Qinghai-Tibetan plateau. Nine female Qaidam yellow cattle (Bos taurus), 9 dzomo (hybrids of cattle and yak) and 9 female plateau yak (B. grunniens), about 5 to 6 years old, were used in this study. Rumen fermentation parameters, fibrolytic enzyme activities, and rumen metataxonomic were determined. Then 18 (6 samples per group) were selected for rumen metagenomic and metabolome analysis. Metataxonomic analysis revealed that the rumen microbiota was significantly different among plateau yak, Qaidam yellow cattle, and dzomo (P < 0.05). Metagenomic analysis displayed a larger gene pool encoding a richer repertoire of carbohydrate-active enzymes in the rumen microbiome of plateau yak and dzomo than Qaidam yellow cattle (P < 0.05). Some of the genes encoding glycoside hydrolases that mediate the digestion of cellulose and hemicellulose were significantly enriched in the rumen of plateau yak than Qaidam yellow cattle, but glycoside hydrolase 57 that primarily includes amylases was abundant in Qaidam yellow cattle (P < 0.05). The rumen fermentation profile differed also, Qaidam yellow cattle having a higher molar proportion of acetate but a lower molar proportion of propionate than dzomo and plateau yak (P < 0.05). Based on metabolomic analysis, rumen microbial metabolic pathways and metabolites were different. Differential metabolites are mainly amino acids, carboxylic acids, sugars, and bile acids. Changes in rumen microbial composition could explain the above results. The present study showed that the rumen microbiome of plateau yak helps its host to adapt to the Qinghai-Tibetan plateau. In particular, the plateau yak rumen microbiome has more enzymes genes involved in cellulase and hemicellulase than that of cattle, resulting higher fibrolytic enzyme activities in yak, further providing stronger fiber degradation function.

Effects of dietary branched-chain amino acid supplementation on serum and milk metabolome profiles in dairy cows during early lactation.

The 3 branched-chain AA (BCAA), Val, Leu, and Ile, are essential AA used by tissues as substrates for protein synthesis and energy generation. In addition, BCAA are also involved in modulating cell signaling pathways, such as nutrient sensing and insulin signaling. In our previous study, dietary BCAA supplementation was shown to improve protein synthesis and glucose homeostasis in transition cows. However, a more detailed understanding of the changes in metabolic pathways associated with an increased BCAA availability is desired to fine-tune nutritional supplementation strategies. Multiparous Holstein cows (n = 20) were enrolled 28 d before expected calving and assigned to either the BCAA treatment (n = 10) or the control group (n = 10). Cows assigned to BCAA were fed 550 g/d of rumen-protected BCAA mixed with 200 g/d of dry molasses from calving until 35 DIM, whereas the cows assigned to the control were fed only 200 g/d of dry molasses. Serum samples were collected on d 10 before expected calving, as well as on d 4 and d 21 postpartum. Milk samples were collected on d 14 postpartum. From a larger cohort, we selected 20 BCAA-supplemented cows with the greatest plasma urea nitrogen concentration, as an indicator for greater BCAA availability, for the metabolomics analysis herein. Serum and milk samples were subjected to a liquid chromatography-mass spectrometry-based assay, detecting and measuring the abundance of 241 serum and 211 milk metabolic features, respectively. Multivariable statistical analyses revealed that BCAA supplementation altered the metabolome profiles of both serum and milk samples. Increased abundance of serum phosphocholine and glutathione and of milk Val, Ile, and Leu, and decreased abundance of milk acyl-carnitines were associated with BCAA supplementation. Altered phosphocholine and glutathione abundances point to altered hepatic choline metabolism and antioxidant balance, respectively. Altered milk acyl-carnitine abundances suggest changes in mammary fatty acid metabolism. Dietary BCAA supplementation was associated with a range of alterations in serum and milk metabolome profiles, adding to our understanding of the role of BCAA availability in modulating dairy cow protein, lipid, and energy metabolism on a whole-body level and how it affects milk composition.

Changes of the liver metabolome following an intravenous lipopolysaccharide injection in Holstein cows supplemented with dietary carnitine.

BACKGROUND: Carnitine facilitates the flux of long-chain fatty acids for hepatic mitochondrial beta-oxidation, which acts to ameliorate the negative energy balance commonly affecting high-yielding dairy cows. Inflammation triggered by lipopolysaccharide (LPS) load can however pose a challenge to the metabolic integrity via the expression of pro-inflammatory mediators, leading to immune system activation and respective metabolic alterations. The effect of enhanced carnitine availability on hepatic metabolome profiles during an inflammatory challenge has not yet been determined in dairy cows. Herein, Holstein cows were supplemented with 25 g/d rumen-protected carnitine from 42 d prepartum until 126 d postpartum (n = 16) or assigned to the control group with no supplementation during the same period (n = 14). We biopsied the liver of the cows before (100 d postpartum) and after (112 d postpartum) an intravenous injection of 0.5 µg/kg LPS. Liver samples were subjected to a targeted metabolomics analysis using the AbsoluteIDQ p180 Kit (Biocrates Life Sciences AG, Innsbruck, Austria).  RESULTS: Multivariate statistical analyses revealed that hepatic metabolome profiles changed in relation to both the carnitine supplementation and the LPS challenge. Comparing the metabolite profiles on 100 d, carnitine increased the concentration of short- and long-chain acyl-carnitines, which may be explained by an enhanced mitochondrial fatty acid shuttle and hence greater energy availability. The LPS injection affected hepatic metabolite profiles only in the carnitine supplemented group, particularly altering the concentration of biogenic amines. CONCLUSIONS: Our results point to interactions between an acute hepatic inflammatory response and biogenic amine metabolism, depending on energy availability.

Sphingolipidome of plasma, liver, and adipose tissues and its association with insulin response to oral glucose testing in Icelandic horses.

Insulin dysregulation (ID) is a determinant of equine metabolic syndrome. Among the sphingolipids, ceramides contribute to the development of ID; however, the cross talk between the liver and adipose tissue (AT) depots and the variation among AT depots in terms of ceramide metabolism are not well understood. We aimed to characterize the sphingolipidome of plasma, liver, and AT (nuchal, NUAT; subcutaneous, SCAT; omental, OMAT; retroperitoneal, RPAT) and their associations with insulin response to oral glucose testing (OGT) in normoinsulinemic and hyperinsulinemic horses. Plasma, liver, and AT samples were collected from 12 Icelandic horses upon euthanasia and analyzed by liquid chromatography-mass spectrometry. Eighty-four targeted compounds were effectively quantified. Comparing the AT depots, greater (false discovery rate, FDR < 0.05) ceramide, dihydroceramide, and sphingomyelin concentrations and lower glucosyl- and galactosyl-ceramides were found in RPAT and OMAT than in NUAT and SCAT. Hyperinsulinemic response to OGT was associated with sphingolipidome alterations primarily in the RPAT and OMAT, whereas the NUAT sphingolipidome did not show signs of ceramide accumulation, which was inconsistent with the previously proposed role of nuchal adiposity in ID. The plasma sphingolipidome was not significantly associated with the liver or AT sphingolipidomes, indicating that plasma profiles are determined by an interplay of various organs. Furthermore, hepatic sphingolipid profiles were not correlated with the profiles of AT depots. Finally, statistically valid partial least square regression models predicting insulin response were found in the plasma (Q2 = 0.58, R2 = 0.98), liver (Q2 = 0.64, R2 = 0.74), and RPAT (Q2 = 0.68, R2 = 0.79) sphingolipidome, but not in the other adipose tissues.

Ceramide metabolism associated with chronic dietary nutrient surplus and diminished insulin sensitivity in the liver, muscle, and adipose tissue of cattle.

High dietary energy and protein supply is common practice in livestock nutrition, aiming to maximize growth and production performance. However, a chronic nutritional surplus induces obesity, promotes insulin insensitivity, and triggers low-grade inflammation. Thirty Holstein bulls were randomly assigned to two groups, low energy and protein (LEP), and high energy and protein (HEP) intake, provided from the 13th to the 20th month of life. Body weight, carcass composition, laminitis score, and circulating insulin and glucose concentrations were assessed. The expression and extent of phosphorylation of insulin signaling proteins were measured in the liver, muscle, and adipose tissue. The sphingolipid metabolome was quantified by a targeted liquid chromatography-mass spectrometry based metabolomics approach. The HEP bulls were obese, had hyperinsulinemia with euglycemia, and expressed clinical signs of chronic laminitis. In the liver, protein kinase B (PKB) phosphorylation was decreased and this was associated with a higher tissue concentration of ceramide 16:0, a sphingolipid that diminishes insulin action by dephosphorylating PKB. In the adipose tissue, insulin receptor expression was lower in HEP bulls, associated with higher concentration of hexosylceramide, which reduces the abundance of functional insulin receptors. Our findings confirm that diet-induced metabolic inflammation triggers ceramide accumulation and disturbs insulin signaling. As insulin insensitivity exacerbates metabolic inflammation, this self-reinforcing cycle could explain the deterioration of metabolic health apparent as chronic laminitis. By demonstrating molecular relationships between insulin signaling and sphingolipid metabolism in three major tissues, our data extend our mechanistic understanding of the role of ceramides in diet-induced metabolic inflammation.

Klebsiella pneumoniae infection is associated with alterations in the gut microbiome and lung metabolome.

Gut microbiota is involved in maintaining homeostasis, and intestinal dysbiosis may lead to opportunistic infections and diseases. Pathogens can disrupt the gut homeostasis and establish colonization, but how they modulate the microbiome and metabolome along the gut-lung axis warrants further investigation. In the present study, we used a classical low virulence Klebsiella pneumoniae (cKp) strain to address this question. We assessed the gut microbiome and lung metabolome in cKp-infected mice by 16S rRNA sequencing and untargeted liquid chromatography-mass spectrometry, respectively. Our data revealed that cKp infection reduced gut microbiota diversity and altered microbiome composition. Specifically, cKp infection increased the abundance of MWH-CFBk5 and Actinomadura and reduced the abundance of Lachnospiraceae_NK4A136_group, Clostridium sensu_stricto 1, Bifidobacterium, and Intestinimonas at the genus level. Notably, caffeine and caffeine metabolism were significantly affected in the lung by cKp infection. Moreover, Spearman correlation analysis revealed remarkable correlations of specific lung metabolites and bacteria species at the genus level. These findings suggest that cKp infection is linked to gut dysbiosis and alterations in the lung metabolome. This study is of significance for developing innovative gut microbiota-directed therapy for respiratory diseases.

In-depth characterisation of the urine metabolome in cats with and without urinary tract diseases.

INTRODUCTION: Our understanding of the urine metabolome and its association with urinary tract disease is limited in cats. OBJECTIVES: We conducted a case-control study to characterise the feline urine metabolome, investigate its association with chronic kidney disease (CKD) and feline idiopathic cystitis (FIC), and assess its compositional relationship with the urine microbiome. METHODS: The urine metabolome of 45 owned cats, including 23 controls, 16 CKD, and 6 FIC cases, was characterised by an untargeted metabolomics approach using high-performance chemical isotope labelling liquid chromatography-mass spectrometry. RESULTS: We detected 9411 unique compounds in the urine of controls and cases and identified 1037 metabolites with high confidence. Amino acids, peptides, and analogues dominated these metabolites (32.2%), followed by carbonyl compounds (7.1%) and carbohydrates (6.5%). Seven controls from one household showed a significant level of metabolome clustering, with a distinct separation from controls from other households (p value < 0.001). Owner surveys revealed that this cluster of cats was fed dry food only, whereas all but one other control had wet food in their diet. Accordingly, the diet type was significantly associated with the urine metabolome composition in our multivariate model (p value = 0.001). Metabolites significantly altered in this cluster included taurine, an essential amino acid in cats. Urine metabolome profiles were not significantly different in CKD and FIC cases compared with controls, and no significant compositional relationship was detected between the urine metabolome and microbiome. CONCLUSION: Our study reveals in-depth diversity of the feline urine metabolome composition, and suggests that it can vary considerably depending on environmental factors.

Modulation of Plasma and Milk Sphingolipids in Dairy Cows Fed High-Starch Diets.

Bovine milk is a significant source of sphingolipids, dietary compounds that can exert anti-inflammatory actions, and which can modulate the host's microbiome. Because sphingolipid synthesis can be modified by diet, we hypothesized that dietary conditions which reduced FFA availability may result in reduced sphingolipid synthesis. Twelve ruminally cannulated cows (120 ± 52 DIM; 35.5 ± 8.9 kg of milk/d; mean ± SD) were randomly assigned to treatment in a crossover design with 21-d periods. Treatments were (1) High starch (HS), (2) Control. The HS diet contained 29% starch, 24% NDF, and 2.8% fatty acids (FA), whereas the Control diet contained 20% starch, 31% NDF, and 2.3% FA. Plasma and milk samples were obtained on d 21 of each period and sphingolipids were quantified using targeted metabolomics. Univariate and multivariate analyses of generalized log-transformed and Pareto-scaled data included ANOVA (fixed effects of treatment) and discriminant analysis. The lipidomics analysis detected 71 sphingolipids across plasma and milk fat, including sphinganines (n = 3), dihydro-ceramides (n = 8), ceramides (Cer; n = 15), sphingomyelins (SM; n = 17), and glycosylated ceramides (n = 28). Followed by Cer, SM were the most abundant sphingolipids detected in milk and plasma, with a preponderance of 16:0-, 23:0-, and 24:0-carbon sidechains. Although no effects of HS diets were observed on plasma sphingolipids, we detected consistent reductions in the concentrations of several milk Cer (e.g., 22:0- and 24:0-Cer) and SM (17:0- and 23:0-SM) in response to HS. Discriminant analysis revealed distinct metabolite separation of HS and Control groups, with several Cer and SM being distinctively predictive of dietary treatment. We conclude that HS diets can reduce the secretion of milk Cer and SM, even in the absence of changes in circulating sphingolipids.

Increased plasma and milk short-chain acylcarnitine concentrations reflect systemic LPS response in mid-lactation dairy cows.

Lipopolysaccharides (LPS) challenge the metabolic integrity of high-yielding dairy cows, activating the immune system and altering energy metabolism. Fatty acid oxidation, a major energy-gaining pathway, can be improved by supplementary carnitine, facilitating the transport of fatty acids into mitochondria. The metabolic response to the LPS challenge could alter both the plasma and the milk metabolome. Plasma and milk samples collected from cows treated with (n = 27) or without (n = 27) dietary carnitine, before and after intravenous administration of LPS, were subjected to a targeted metabolomics analysis. Multivariate statistical analyses revealed that both plasma and milk metabolome changed in response to the LPS challenge in both the carnitine-supplemented and the control cows. Short-chain acylcarnitines (carbon chain length C2, C3, C4, and C5) and long-chain acylcarnitines (C14, C16, and C18) had the highest performance to indicate LPS response when testing the predictive power of single metabolites using receiver-operator characteristics (ROC) analysis. The maximum area under a ROC curve (AUC) was 0.93. Biogenic amines, including sarcosine, and amino acids such as glutamine and isoleucine had AUC > 0.80 indicating metabolic changes due to the LPS challenge. In summary, the metabolites involved in the LPS response were acylcarnitines C2 and C5, sarcosine, glutamine, and isoleucine in plasma, and acylcarnitines C4 and C5 in milk. The interrelationship of plasma and milk metabolome included correlation of acylcarnitines C2, C4, and C5 between plasma and milk.

Associations of plasma sphingolipid profiles with insulin response during oral glucose testing in Icelandic horses.

BACKGROUND: Sphingolipids modulate insulin sensitivity in mammals. Increased synthesis of ceramides is linked to decreased insulin sensitivity of tissues. Conversely, activation of the insulin signaling pathway can downregulate ceramide synthesis. Elucidating the association between sphingolipid metabolism and insulin response during oral glucose testing may help explain the pathophysiology of insulin dysregulation in horses. HYPOTHESES: Horses with insulin dysregulation will have a plasma sphingolipid profile characterized by increased ceramide concentrations. The plasma sphingolipid profile will have decreased ceramide concentrations after acute activation of the insulin signaling pathway by oral glucose testing. ANIMALS: Twelve Icelandic horses. METHODS: Horses were subjected to an oral glucose test (0.5 g/kg body weight glucose), with plasma insulin concentrations measured at 0, 30, 60, 120, 180, and 240 minutes postglucose administration. Plasma samples were collected at 0 and 120 minutes for sphingolipid profiling using a liquid chromatography-mass spectrometry-based metabolomics analysis. Eighty-three species of sphingolipids were detected, including 3-ketosphinganines, dihydroceramides, ceramides, dihydrosphingomyelins, sphingomyelins, galatosylceramides, glucosylceramides, lactosylceramides, and ceramide-1-phosphates. RESULTS: Glucose administration did not significantly alter plasma sphingolipid profiles. C22:0-ceramide, C24:1-ceramide, C23:0-ceramide, C16:1-sphingomyelin, C22:0-dihydroceramide, and C24:0-ceramide were positively correlated with the insulin response (area under the curve). CONCLUSION AND CLINICAL IMPORTANCE: Positive correlation between the insulin response and sphingolipid concentrations implies upregulated sphingolipid metabolism in insulin dysregulated horses. A high plasma ceramide concentration can indicate insulin dysregulation in horses.

Association between alterations in plasma metabolome profiles and laminitis in intensively finished Holstein bulls in a randomized controlled study.

Metabolic consequences of an energy and protein rich diet can compromise metabolic health of cattle by promoting a pro-inflammatory phenotype. Laminitis is a common clinical sign, but affected metabolic pathways, underlying pathophysiology and causative relationships of a systemic pro-inflammatory phenotype are unclear. Therefore, the aim of this study was to elucidate changes in metabolome profiles of 20 months old Holstein bulls fed a high energy and protein diet and to identify novel metabolites and affected pathways, associated with diet-related laminitis. In a randomized controlled feeding trial using bulls fed a high energy and protein diet (HEP; metabolizable energy [ME] intake 169.0 ± 1.4 MJ/day; crude protein [CP] intake 2.3 ± 0.02 kg/day; calculated means ± SEM; n = 15) versus a low energy and protein diet (LEP; ME intake 92.9 ± 1.3 MJ/day; CP intake 1.0 ± 0.01 kg/day; n = 15), wide ranging effects of HEP diet on metabolism were demonstrated with a targeted metabolomics approach using the AbsoluteIDQ p180 kit (Biocrates Life Sciences). Multivariate statistics revealed that lower concentrations of phosphatidylcholines and sphingomyelins and higher concentrations of lyso-phosphatidylcholines, branched chain amino acids and aromatic amino acids were associated with an inflammatory state of diet-related laminitis in Holstein bulls fed a HEP diet. The latter two metabolites share similarities with changes in metabolism of obese humans, indicating a conserved pathophysiological role. The observed alterations in the metabolome provide further explanation on the underlying metabolic consequences of excessive dietary nutrient intake.

Alterations of Serum Metabolites and Fecal Microbiota Involved in Ewe Follicular Cyst.

While the interactions of the gut microbiome and blood metabolome have been widely studied in polycystic ovary disease in women, follicular cysts of ewes have been scarcely investigated using these methods. In this study, the fecal microbiome and serum metabolome were used to compare between ewes diagnosed with ovarian cystic follicles and ewes with normal follicles, to investigate alterations of the fecal bacterial community composition and metabolic parameters in relation to follicular cystogenesis. Ewes from the same feeding and management system were diagnosed with a follicular cyst (n = 6) or confirmed to have normal follicles (n = 6) by using a B-mode ultrasound scanner. Blood serum and fresh fecal samples of all ewes were collected and analyzed. The α-diversity of fecal microbiome did not differ significantly between follicular cyst ewes and normal follicle ewes. Three genera (Bacteroides, Anaerosporobacter, and Angelakisella) were identified and their balance differentiated between follicular cyst and normal follicle ewes. Alterations of several serum metabolite concentrations, belonging to lipids and lipid-like molecules, organic acids and derivatives, organic oxygen compounds, benzenoids, phenylpropanoids and polyketides, and organoheterocyclic compounds, were associated with the presence of a follicular cyst. Correlation analysis between fecal bacterial communities and serum metabolites indicated a positive correlation between Anaerosporobacter and several fatty acids, and a negative correlation between Bacteroides and L-proline. These observations provide new insights for the complex interactions of the gut microbiota and the host serum lipid profiles, and support gut microbiota as a potential strategy to treat and prevent follicular cysts in sheep.

Influence of N-acetylcysteine on steroidogenesis and gene expression in porcine placental trophoblast cells.

N-acetylcysteine (NAC) is a widely used anti-inflammatory agent and antioxidant in vivo and in vitro. As a nutritional supplement, NAC can improve production and reproductive performances in animals through enhancing placental function and regulating hormone production. Trophoblast proliferation and steroid hormone production are two major functions in the placenta. We hypothesized that the effects of NAC on placental function is due to its direct and indirect effects on gene expression in placental trophoblast cells (pTr). To evaluate this hypothesis, we investigated the effects of NAC on steroidogenesis, gene expression, and cell proliferation in porcine pTr in vitro. pTr were treated with NAC in serum-free medium for 24 h with different concentrations (0, 0.1 µM, 1.0 µM, 10.0 µM, 0.1 mM, 1.0 mM, and 10.0 mM). Low-dose NAC (1 µM) stimulated pTr proliferation and decreased progesterone production, while increasing estradiol production (P < 0.05). High-dose NAC (10 mM) suppressed cell proliferation (P < 0.05), but had no effect on steroidogenesis. Low-dose NAC increased CCDN1 and decreased CASP3 and CASP8 mRNA levels (P < 0.05), whereas high-dose NAC decreased CDK4 and CCDN1 and increased CASP3 mRNA levels (P < 0.05). NAC had no effect on the mRNA abundance of StAR and HSD3B. Low-dose NAC upregulated CYP19A1 mRNA expression, and high-dose NAC downregulated CYP11A1 mRNA abundance (P < 0.05). Only low-dose NAC increased NOS3 mRNA abundance and tetrahydrobiopterin reduction (BH4/BH2 ratio). We conclude that NAC may act directly and indirectly on pTr with a dose-dependent manner and may regulate placental function by affecting pTr differentiation via regulating pTr steroid synthesis, cell proliferation, and apoptosis in sows.

Sphingolipid Profiling Reveals Different Extent of Ceramide Accumulation in Bovine Retroperitoneal and Subcutaneous Adipose Tissues.

Sphingolipids are bioactive lipids that can modulate insulin sensitivity, cellular differentiation, and apoptosis in a tissue-specific manner. However, their comparative profiles in bovine retroperitoneal (RPAT) and subcutaneous adipose tissue (SCAT) are currently unknown. We aimed to characterize the sphingolipid profiles using a targeted lipidomics approach and to assess whether potentially related sphingolipid pathways are different between SCAT and RPAT. Holstein bulls (n = 6) were slaughtered, and SCAT and RPAT samples were collected for sphingolipid profiling. A total of 70 sphingolipid species were detected and quantified by UPLC-MS/MS in multiple reaction monitoring (MRM) mode, including ceramide (Cer), dihydroceramide (DHCer), sphingomyelin (SM), dihydrosphingomyelin (DHSM), ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), galactosylceramide (GalCer), glucosylceramide (GluCer), lactosylceramide (LacCer), sphinganine (DHSph), and sphingosine (Sph). Our results showed that sphingolipids of the de novo synthesis pathway, such as DHSph, DHCer, and Cer, were more concentrated in RPAT than in SCAT. Sphingolipids of the salvage pathway and the sphingomyelinase pathway, such as Sph, S1P, C1P, glycosphingolipid, and SM, were more concentrated in SCAT. Our results indicate that RPAT had a greater extent of ceramide accumulation, thereby increasing the concentration of further sphingolipid intermediates in the de novo synthesis pathway. This distinctive sphingolipid distribution pattern in RPAT and SCAT can potentially explain the tissue-specific activity in insulin sensitivity, proinflammation, and oxidative stress in RPAT and SCAT.

Very Low Crude Protein and Varying Glycine Concentrations in the Diet Affect Growth Performance, Characteristics of Nitrogen Excretion, and the Blood Metabolome of Broiler Chickens.

BACKGROUND: The minimum to which dietary crude protein (CP) level for broiler chickens can be reduced without decreasing growth and the glycine equivalent (Glyequi) concentration required are not known. The plasma metabolome might reflect dietary influences on physiological processes. OBJECTIVE: The aim of this study was to investigate the effect of 3 low CP levels with 4 Glyequi concentrations on growth and characteristics of nitrogen excretion, and to identify plasma metabolome variations. METHODS: Male Ross308 broiler chickens were provided 1 of 12 dietary treatments in 84 metabolism cages (10/cage) from days 7 to 21. Three diets with 163 (CP163), 147 (CP147), and 132 (CP132) g CP/kg were formulated, each containing 12, 15, 18, and 21 g Glyequi/kg. Essential amino acid concentrations were the same in all diets. Animals and feed were weighed on days 7 and 21 to determine average daily gain (ADG) and gain:feed ratio (G:F). Excreta were collected from days 18 to 21 to analyze nitrogenous components, and blood was obtained on day 21 to conduct a metabolome analysis. RESULTS: Two-factor ANOVA showed significant interaction effects for ADG, G:F, and nitrogen efficiency (P < 0.001). Reduction of CP decreased ADG and G:F, and increased nitrogen efficiency. Glyequi supplementation increased ADG (by 7.9 g/d) and G:F (by 0.07 g/g) at CP132. The ADG (by 2.4 g/d) at CP147 and G:F (by 0.02 g/g) at CP147 and CP163 increased up to 15 g Glyequi/kg. Multivariate statistical analysis showed an influence of Glyequi on plasma acylcarnitine and lysophosphatidylcholine concentrations, and a decrease of plasma phosphatidylcholine and sphingomyelin concentrations with reduced CP. CONCLUSIONS: These results suggest that a nutrient other than Glyequi limited growth when CP was reduced from CP163 to CP147, and that the response of broiler chickens to Glyequi is dependent on the dietary CP level. Plasma metabolites indicate dietary influences on the physiological state of the animals.