Bacillus subtilis fmbj ameliorates lipopolysaccharide-induced intestinal dysfunction in broilers by enhancing the SIRT1/PGC1α pathway.

This study aimed to explore the impact of dietary Bacillus subtilis fmbj (BS) supplementation on acute intestinal dysfunction induced by lipopolysaccharide (LPS) in broilers. One hundred and eighty 1-day-old male Arbor Acres broilers were randomly divided into three treatment groups, each comprising ten replicates of 6 birds. On d 20, LPS-challenged (LPS group and LPS-BS group) and LPS-unchallenged (CON group) broilers received intraperitoneal injections of 1 mg/kg body weight LPS solution and an equivalent volume of sterile saline, respectively. Compared to the CON group, LPS disrupted (P < 0.05) the morphology of the small intestine (jejunum or ileum), exacerbated (P < 0.05) serum, small intestinal, and small intestinal mitochondrial antioxidant capacity, induced (P < 0.05) small intestinal oxidative damage, and altered (P < 0.05) the expression of genes and proteins related to antioxidants, cell adhesion, and mitochondrial function in the jejunum. The LPS-BS group exhibited a tendency towards improvement in small intestinal morphology, serum, small intestinal, and small intestinal mitochondrial antioxidant capacity, small intestinal oxidative damage, and the expression of genes and proteins related to antioxidants, cell adhesion, and mitochondrial function in the jejunum when compared to the LPS group. In conclusion, BS supplementation may confer protection against LPS-induced acute intestinal dysfunction in broilers by enhancing the activation of SIRT1/PGC1α, suggesting its potential as a valuable additive for the poultry industry.

TMEM241 is a UDP-N-acetylglucosamine transporter required for M6P modification of NPC2 and cholesterol transport.

Accurate intracellular cholesterol traffic plays crucial roles. Niemann Pick type C (NPC) proteins NPC1 and NPC2, are two lysosomal cholesterol transporters that mediate the cholesterol exit from lysosomes. However, other proteins involved in this process remain poorly defined. Here, we find that the previously unannotated protein TMEM241 is required for cholesterol egressing from lysosomes through amphotericin B-based genome-wide CRISPR-Cas9 KO screening. Ablation of TMEM241 caused impaired sorting of NPC2, a protein utilizes the mannose-6-phosphate (M6P) modification for lysosomal targeting, resulting in cholesterol accumulation in the lysosomes. TMEM241 is a member of solute transporters 35 nucleotide sugar transporters family and localizes on the cis-Golgi network. Our data indicate that TMEM241 transports UDP-N-acetylglucosamine (UDP-GlcNAc) into Golgi lumen and UDP-GlcNAc is used for the M6P modification of proteins including NPC2. Furthermore, Tmem241-deficient mice display cholesterol accumulation in pulmonary cells and behave pulmonary injury and hypokinesia. Taken together, we demonstrate that TMEM241 is a Golgi-localized UDP-GlcNAc transporter and loss of TMEM241 causes cholesterol accumulation in lysosomes because of the impaired M6P-dependent lysosomal targeting of NPC2.

Exhaustion-associated cholesterol deficiency dampens the cytotoxic arm of antitumor immunity.

The concept of targeting cholesterol metabolism to treat cancer has been widely tested in clinics, but the benefits are modest, calling for a complete understanding of cholesterol metabolism in intratumoral cells. We analyze the cholesterol atlas in the tumor microenvironment and find that intratumoral T cells have cholesterol deficiency, while immunosuppressive myeloid cells and tumor cells display cholesterol abundance. Low cholesterol levels inhibit T cell proliferation and cause autophagy-mediated apoptosis, particularly for cytotoxic T cells. In the tumor microenvironment, oxysterols mediate reciprocal alterations in the LXR and SREBP2 pathways to cause cholesterol deficiency of T cells, subsequently leading to aberrant metabolic and signaling pathways that drive T cell exhaustion/dysfunction. LXRß depletion in chimeric antigen receptor T (CAR-T) cells leads to improved antitumor function against solid tumors. Since T cell cholesterol metabolism and oxysterols are generally linked to other diseases, the new mechanism and cholesterol-normalization strategy might have potential applications elsewhere.

Milk yield variation partially attributed to blood oxygen-mediated neutrophil activation in lactating dairy goats.

Blood oxygen is an essential component for numerous biological processes of mammalian animals. Milk production of ruminants largely relies on the supply of nutrients, such as glucose, amino acids and fatty acids. To define the regulatory role of blood oxygen availability in regard to milk production, seventy-five healthy Guanzhong dairy goats with similar body weight, days in milk and parities were selected. For each animal, milk yield was recorded and milk sample was collected to determine compositions. Milk vein blood was collected to determine parameters including blood gas, physio-biochemistry and haematology. Another blood sample was prepared for transcriptome and RT-qPCR. Results showed that both pressure of oxygen (pO2) in the milk vein (positively) and numbers of neutrophils in mammary vein (negatively) were associated with milk yield of the animals. To learn the role of pO2 in blood cell functionality, twelve animals (six with higher yield (H-group) and six with lower yield (L-group)) from seventy-five goats were selected. Compared with animals in L-group, goats in H-group were higher in pO2 but lower in pCO2, lactate, lactate dehydrogenase activity and neutrophil abundance in milk vein, compared with L-group. The blood transcriptome analysis suggested that compared with L-group, animals in H-group were depressed in functionality including neutrophil activation and metabolic pathways including glycolysis, NF-κB and HIF-1. Our result revealed that lower milk production could be associated with neutrophil activation responding to low pO2 in the mammary vein. In the meantime, we highlighted the potential importance of blood oxygen as a milk yield regulator.

Blood neutrophil extracellular traps: a novel target for the assessment of mammary health in transition dairy cows.

BACKGROUND: Mammary health is important for transition dairy cows and has been well recognized to exert decisive effects on animal welfare. However, the factors influencing mammary health are still unclear. Differential somatic cell count (DSCC) could reflect the mastitis risk since it is the percentage of neutrophils plus lymphocytes in total somatic cells and could be reflective of mammary health of dairy cows. This work aimed to investigate the assessment and prognosis of the health of transition cows based on blood neutrophil extracellular traps (NETs). RESULTS: Eighty-four transition Holstein dairy cows were selected. The serum was sampled in all the animals at week 1 pre- and postpartum, and milk was sampled at week 1 postpartum. Based on the DSCC in milk at week 1, cows with lower (7.4% ± 4.07%, n = 15) and higher (83.3% ± 1.21%, n = 15) DSCCs were selected. High DSCC cows had higher levels of red blood cell counts (P < 0.05), hemoglobin (P = 0.07), and hematocrit (P = 0.05), higher concentrations of serum oxidative variables [(reactive oxygen species (P < 0.05), malondialdehyde (P < 0.05), protein carbonyl (P < 0.05), and 8-hydroxy-2-deoxyguanosine (P = 0.07)], higher levels of serum and milk NETs (P < 0.05) and blood-milk barrier indicators, including serum ß-casein (P = 0.05) and milk immunoglobulin G2 (P = 0.09), than those of low DSCC cows. In addition, lower concentrations of serum nutrient metabolites (cholesterol and albumin) (P < 0.05) and a lower level of serum deoxyribonuclease I (P = 0.09) were observed in high DSCC cows than in low DSCC cows. Among the assessments performed using levels of the three prepartum serum parameters (NETs, deoxyribonuclease I and ß-casein), the area under the curve (0.973) of NETs was the highest. In addition, the sensitivity (1.00) and specificity (0.93) were observed for the discrimination of these cows using NETs levels with a critical value of 32.2 ng/mL (P < 0.05). CONCLUSIONS: The formation of NETs in blood in transition dairy cows may damage the integrity of the blood-milk barrier and thereby increase the risk for mastitis in postpartum cows.

Treatment of immune dysfunction in intrauterine growth restriction piglets via supplementation with dimethylglycine sodium salt during the suckling period.

This study aimed to investigate the mechanism of small intestinal immune dysfunction in intrauterine growth restriction (IUGR) newborn piglets and relieve this dysfunction via dimethylglycine sodium salt (DMG-Na) supplementation during the suckling period. Thirty sows (Duroc × [Landrace × Yorkshire]) were selected, and 1 male newborn piglet with normal birth weight (NBW) and 1 male newborn piglet with IUGR were obtained from each sow. Among them, 10 NBW and 10 IUGR newborns were euthanized without suckling. The other 20 NBW newborns were allocated to the group named NCON, which means NBW newborns fed a basic milk diet (BMD) (n = 10), and the group named ND, which means NBW newborns fed BMD supplemented with 0.1% DMG-Na (n = 10); the other 20 IUGR newborns were assigned to the group named ICON, which means IUGR newborns fed BMD (n = 10), and the group named ID, which means IUGR newborns fed BMD supplemented with 0.1% DMG-Na (n = 10). The newborns were fed BMD from 7 to 21 d of age and euthanized at 21 d of age to collect serum and small intestinal samples. The growth performance, small intestinal histological morphology and sub-organelle ultrastructure, serum immunoglobulin, small intestinal digestive enzyme activity, inflammatory cytokine level, and jejunum mRNA and protein expression of the toll-like receptor 4 (TLR4)/nucleotide-binding oligomerization domain protein (NOD)/nuclear factor-κB (NF-κB) network deteriorated in the ICON group compared to that in the NCON group. The small intestinal histological morphology and sub-organelle ultrastructure, serum immunoglobulin, small intestinal digestive enzyme activity, and inflammatory cytokine level improved (P < 0.05) in the ID group compared to those in the ICON group. The jejunum mRNA and protein expression of the TLR4/NOD/NF-κB network improved (P < 0.05) in the ID group compared to that in the ICON group. In conclusion, the activity of the TLR4/NOD/NF-κB pathway was inhibited in the IUGR newborns, which in turn led to their jejunum immune dysfunction and reduced their performance. By ingesting DMG-Na, the IUGR newborns activated the TLR4/NOD/NF-κB pathway, thereby improving their unfavorable body state during the suckling period.

Supplementation of Dimethylglycine Sodium Salt in Sow Milk Reverses Skeletal Muscle Redox Status Imbalance and Mitochondrial Dysfunction of Intrauterine Growth Restriction Newborns.

The current study sought to understand the mechanism underlying skeletal muscle dysfunction brought on by intrauterine growth restriction (IUGR) and to explore the treatment benefits of applying dimethylglycine sodium salt (DMG-Na) in sow milk to newborns during the suckling period. Each of the 10 sows delivered one newborn with a normal birth weight (NBW) and one with an IUGR. Additionally, two NBW and two IUGR newborns were collected per litter of another 10 sows. The 20 NBW newborns were divided between the N (sow milk) and ND (sow milk + 0.1% DMG-Na) groups, while 20 IUGR newborns were divided between the I (sow milk) and ID (sow milk + 0.1% DMG-Na) groups. The skeletal muscle histomorphology, redox status, and levels of gene and protein expression were worse (p < 0.05) in the I group than in the N group. In addition, supplementation with DMG-Na (ND and ID groups) improved (p < 0.05) those parameters compared to the unsupplemented groups (N and I groups). Inhibited nuclear factor erythroid 2-related factor 2 (Nrf2)/sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptorγcoactivator-1α (PGC-1α) activity resulted in decreased redox status, skeletal muscle structural damage, skeletal muscle mitochondrial function impairment, and decreased performance in IUGR newborns. Supplementation of DMG-Na in sow milk activated the Nrf2/SIRT1/PGC-1α in IUGR newborns, thereby improving their skeletal muscle performance.

Dietary dimethylglycine sodium salt supplementation alleviates redox status imbalance and intestinal dysfunction in weaned piglets with intrauterine growth restriction.

There are few studies on the mechanism of redox status imbalance and intestinal dysfunction in intrauterine growth restricted (IUGR) newborn piglets. Here, we investigated the mechanism of jejunum dysfunction in weaned piglets with IUGR and the mechanism through which dimethylglycine sodium salt (DMG-Na) supplementation improving the imbalance of their redox status. In this work, a total of 10 normal birth weight (NBW) newborn piglets and 20 IUGR newborn piglets were obtained. After weaning at 21 d, they were assigned to 3 groups (n = 10/group): NBW weaned piglets fed standard basal diets (NBWC); one IUGR weaned piglets fed standard basal diets (IUGRC); another IUGR weaned piglets from the same litter fed standard basal diets plus 0.1% DMG-Na (IUGRD). The piglets in these 3 groups were sacrificed at 49 d of age, and the blood and jejunum samples were collected immediately. The growth performance values in the IUGRC group were lower (P < 0.05) than those in the NBWC group. Jejunum histomorphological parameters, inflammatory cytokines, and digestive enzyme activity as well as serum immunoglobulin were lower (P < 0.05) in the IUGRC group than those in the NBWC group. Compared with these in the NBWC group, the redox status of serum, jejunum, and mitochondria and the expression levels of jejunum redox status-related, cell adhesion-related, and mitochondrial function-related genes and proteins were suppressed in the IUGRC group (P < 0.05). However, compared with those in the IUGRC group, the growth performance values, jejunum histomorphological parameters, inflammatory cytokines, digestive enzyme activity, serum immunoglobulin, redox status of serum, jejunum, and mitochondria, and the expression levels of jejunum redox status-related, cell adhesion-related, and mitochondrial function-related genes and proteins were improved (P < 0.05) in the IUGRD group. In conclusion, dietary DMG-Na supplementation alleviates redox status imbalance and intestinal dysfunction in IUGR weaned piglets mainly by activating the sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptorγcoactivator-1α (PGC1α) pathway, thereby improving their unfavorable body state.

Dimethylglycine Sodium Salt Alleviates Intrauterine Growth Restriction-Induced Low Growth Performance, Redox Status Imbalance, and Hepatic Mitochondrial Dysfunction in Suckling Piglets.

This study aimed to investigate the mechanism of redox status imbalance and hepatic mitochondrial dysfunction induced by intrauterine growth restriction (IUGR) and relieve this condition through dimethylglycine sodium salt (DMG-Na) supplementation during the suckling period. Thirty normal birth weight (NBW) and 30 IUGR newborns were selected from 20 sows. Briefly, 1 NBW and 1 IUGR newborn were obtained from each litter of 10 sows, and 10 NBW and 10 IUGR newborns were obtained. Additionally, 2 NBW and 2 IUGR newborns were obtained from each litter of another 10 sows, and 20 NBW newborns were allocated to the N [basic milk diets (BMDs)] and ND (BMDs+0.1% DMG-Na) groups. Furthermore, 20 IUGR newborns were assigned to the I (BMDs) and ID (BMDs+0.1% DMG-Na) groups. The results revealed that the growth performance, serum and hepatic redox status, and hepatic gene and protein expression levels were lower (P < 0.05) in the I group compared to the N group. Additionally, supplementation with DMG-Na (ND and ID groups) improved (P < 0.05) these parameters compared to the non-supplemented groups (N and I groups). In conclusion, the activity of Nrf2/SIRT1/PGC1α was inhibited in IUGR newborns, and this led to their hepatic dysfunctions. Supplementation with DMG-Na activated Nrf2/SIRT1/PGC1α in IUGR newborns, thereby improving their performance.

Formation of Blood Neutrophil Extracellular Traps Increases the Mastitis Risk of Dairy Cows During the Transition Period.

The current study was conducted to analyze the functions of blood neutrophils in transition cows and their association with postpartum mastitis risk as indicated by somatic cell counts (SCCs) in milk. Seventy-six healthy Holstein dairy cows were monitored from Week 4 prepartum to Week 4 postpartum. Five dairy cows with low SCCs (38 ± 6.0 × 103/mL) and five with high SCCs (3,753 ± 570.0 × 103/mL) were selected based on milk SCCs during the first three weeks of lactation. At Week 1 pre- and postpartum, serum samples were obtained from each cow to measure neutrophil extracellular trap (NET)-related variables, and blood neutrophils were collected for transcriptome analysis by RNA sequencing. The serum concentration of NETs was significantly higher (P < 0.05) in cows with high SCCs than in cows with low SCCs (36.5 ± 2.92 vs. 18.4 ± 1.73 ng/mL). The transcriptomic analysis revealed that the transcriptome differences in neutrophils between high- and low-SCC cows were mainly in cell cycle-related pathways (42.6%), including the cell cycle, DNA damage, and chromosomal conformation, at Week 1 prepartum. The hub genes of these pathways were mainly involved in both the cell cycle and NETosis. These results indicated that the formation of NETs in the blood of transition dairy cows was different between cows with low and high SCCs, which may be used as a potential indicator for the prognosis of postpartum mastitis risk and management strategies of perinatal dairy cows.

Dimethylglycine sodium salt activates Nrf2/SIRT1/PGC1α leading to the recovery of muscle stem cell dysfunction in newborns with intrauterine growth restriction.

The objectives of this study were focused on the mechanism of mitochondrial dysfunction in skeletal muscle stem cells (MuSCs) from intrauterine growth restriction (IUGR) newborn piglets, and the relief of dimethylglycine sodium salt (DMG-Na) on MuSCs mitochondrial dysfunction by Nrf2/SIRT1/PGC1α network. In this study, six newborn piglets with normal birth weight (NBW) and six IUGR newborn piglets were slaughtered immediately after birth to obtain longissimus dorsi muscle (LM) samples. MuSCs were collected and divided into three groups: MuSCs from NBW newborn piglets (N), MuSCs from IUGR newborn piglets (I), and MuSCs from IUGR newborn piglets with 32 µmol DMG-Na (ID). Compared with the NBW group, the IUGR group showed decreased (P < 0.05) serum and LM antioxidant defense capacity, and increased (P < 0.05) serum and LM damage. Compared with the N group, the I group showed decreased (P < 0.05) MuSCs antioxidant defense capacity, mitochondrial ETC complexes, energy metabolites, and antioxidant defense-related and mitochondrial function-related gene and protein expression levels. The antioxidant defense capacity, mitochondrial ETC complexes, energy metabolites, and antioxidant defense-related and mitochondrial function-related gene and protein expression levels of MuSCs were improved (P < 0.05) in the ID group compared to those in the I group. The MuSCs of IUGR newborns activate the Nrf2/SIRT1/PGC1α network by taking in DMG-Na, thereby neutralizing excessive generated O2•- that may help to improve their unfavorable mitochondrial dysfunction in skeletal muscle.

Supplementing N-carbamoylglutamate in late gestation increases newborn calf weight by enhanced placental expression of mTOR and angiogenesis factor genes in dairy cows.

The objective of this study was to investigate whether supplementation with N-carbamoylglutamate (NCG) to cows during late gestation alters uteroplacental tissue nutrient transporters, calf metabolism and newborn weight. Thirty multiparous Chinese Holstein cows were used in a randomized complete block design experiment. During the last 28 d of pregnancy, cows were fed a diet without (CON) or with NCG (20 g/d per cow). The body weight of calves was weighed immediately after birth. Placentome samples were collected at parturition and used to assess mRNA expression of genes involved in transport of arginine, glucose, fatty acid and angiogenesis factors, as well as the mammalian target of rapamycin (mTOR) pathway. Blood samples of calves before colostrum consumption were also collected for the detection of plasma parameters, amino acids (AA) and metabolomics analysis. The newborn weight (P = 0.02) and plasma Arg concentration of NCG-calves was significantly higher (P = 0.05) than that of CON-calves, and the plasma concentrations of urea nitrogen tended to be lower (P = 0.10) in the NCG group. The mRNA abundance of genes involved in glucose transport (solute carrier family 2 member 3 [SLC2A3], P < 0.01), angiogenesis (nitric oxide synthase 3 [NOS3], P = 0.02), and mTOR pathway (serine/threonine-protein kinase 1 [AKT1], P = 0.10; eukaryotic translation initiation factor 4B pseudogene 1 [EIF4BP1], P = 0.08; EIF4EBP2, P = 0.04; and E74-like factor 2 [ELF2], P = 0.03) was upregulated in the placentome of NCG-supplemented cows. In addition, 17 metabolites were significantly different in the placentome of NCG-supplemented cows compared to non-supplemented cows, and these metabolites are mainly involved in arginine and proline metabolism, alanine, aspartate and glutamate metabolism, and citrate cycle. In summary, the increased body weight of newborn calves from the NCG supplemented dairy cows may be attributed to the increased angiogenesis and uteroplacental nutrient transport and to the activated mTOR signal pathway, which may result in the increased nutrient supply to the fetus, and improved AA metabolism and urea cycle of the fetus.

Effect of Yellow Wine Lees Supplementation on Milk Antioxidant Capacity and Hematological Parameters in Lactating Cows under Heat Stress.

Fifteen multiparous lactating Chinese Holstein dairy cows were used in a replicated 3 × 3 Latin Square Design to evaluate the effect of total mixed rations (TMR) containing unfermented and fermented yellow wine lees (YWL) on the oxidative status of heat-stressed lactating cows and the oxidative stability of the milk and milk fatty acids they produced. Cows were fed with three isonitrogenous and isocaloric diets as follows: (1) TMR containing 18% soybean meal, (2) TMR containing 11% unfermented YWL (UM), and (3) TMR containing 11% fermented YWL (FM). The rectal temperature (at 1300 h) and respiratory rate were higher in control cows than in cows fed UM or FM. Both types of YWL were greater in total phenolic and flavonoid contents, reducing power, and radical scavenging abilities than soybean meal. Cows fed UM or FM had higher blood neutrophil, white blood cell, and lymphocyte counts, as well as lower plasma malondialdehyde level, higher plasma superoxide dismutase, glutathione peroxidase, and 2,2-diphenyl-1-picryl-hydrazyl-hydrate levels, and higher total antioxidant capacity in the plasma than those fed control diet. The proportion of milk unsaturated fatty acids was higher and that of saturated fatty acids was lower in UM- and FM-fed animals than in the control animals. Milk from UM- and FM-fed cows had lower malondialdehyde content but higher 2,2-diphenyl-1-picryl-hydrazyl-hydrate content than the control cows. In conclusion, feeding TMR containing UM and FM to cows reduced both the oxidative stress in heat-stressed cows and improved the oxidative capacity of their milk.

Dietary dimethylglycine sodium salt supplementation improves growth performance, redox status, and skeletal muscle function of intrauterine growth-restricted weaned piglets.

Few studies have focused on the role of dimethylglycine sodium (DMG-Na) salt in protecting the redox status of skeletal muscle, although it is reported to be beneficial in animal husbandry. This study investigated the beneficial effects of DMG-Na salt on the growth performance, longissimus dorsi muscle (LM) redox status, and mitochondrial function in weaning piglets that were intrauterine growth restricted (IUGR). Ten normal birth weight (NBW) newborn piglets (1.53 ± 0.04 kg) and 20 IUGR newborn piglets (0.76 ± 0.06 kg) from 10 sows were obtained. All piglets were weaned at 21 d of age and allocated to the three groups with 10 replicates per group: NBW weaned piglets fed a common basal diet (N); IUGR weaned piglets fed a common basal diet (I); IUGR weaned piglets fed a common basal diet supplemented with 0.1% DMG-Na (ID). They were slaughtered at 49 d of age to collect the serum and LM samples. Compared with the N group, the growth performance, LM structure, serum, and, within the LM, mitochondrial redox status, mitochondrial respiratory chain complex activity, energy metabolites, redox status-related, cell adhesion-related, and mitochondrial function-related gene expression, and protein expression deteriorated in group I (P < 0.05). The ID group showed improved growth performance, LM structure, serum, and, within the LM, mitochondrial redox status, mitochondrial respiratory chain complex activity, energy metabolites, redox status-related, cell adhesion-related, and mitochondrial function-related gene expression, and protein expression compared with those in the I group (P < 0.05). The above results indicated that the DMG-Na salt treatment could improve the LM redox status and mitochondrial function in IUGR weaned piglets via the nuclear factor erythroid 2-related factor 2/sirtuin 1/peroxisome proliferator-activated receptorγcoactivator-1α network, thus improving their growth performance.

Dietary supplementation with N-carbamoylglutamate initiated from the prepartum stage improves lactation performance of postpartum dairy cows.

The objective of this study was to investigate the effects of supplementing N-carbamoylglutamate (NCG), an Arg enhancer, on amino acid (AA) supply and utilization and productive performance of early-lactating dairy cows. Thirty multiparous Chinese Holstein dairy cows were randomly divided into control (CON, n = 15) and NCG (CON diet supplemented with NCG at 20 g/d per cow, n = 15) groups at 4 wk before calving. Diets were offered individually in tie-stalls, and NCG was supplemented by top-dress feeding onto total mixed ration for the NCG group. The experiment lasted until wk 10 after calving. Dry matter intake tended to be higher (P = 0.06), and yields of milk (P < 0.01), milk protein (P < 0.01), and milk fat (P < 0.01) were higher in the NCG-cows than in the CON-cows. Plasma activities of aspartate aminotransferase (P < 0.01), alanine aminotransferase (P = 0.03), and plasma level of ß-hydroxybutyrate (P = 0.04) were lower in the NCG-cows than in the CON-cows, whereas plasma glucose (P = 0.05) and nitric oxide (NO, P < 0.01) concentrations were higher. Coccygeal vein concentrations of Cys (P < 0.01), Pro (P < 0.01), Tyr (P = 0.05), most essential AA except Thr and His (P < 0.01), total essential AA (P < 0.01), and total AA (P < 0.01) were higher in the NCG-cows than in the CON-cows. The arterial supply of all AA was greater in the NCG-cows than in the CON-cows. The NCG-cows had higher mammary plasma flow of AA (P = 0.04) and clearance rate of Cys (P < 0.01), Pro (P < 0.01) and Asp (P < 0.01), and higher ratios of uptake to output of Met (P = 0.05), Lys (P < 0.01), Cys (P = 0.01), Pro (P = 0.03), and Asp (P = 0.01). In summary, addition of NCG initiated from the prepartum period improved the lactation performance of postpartum dairy cows, which might attribute to greater Arg and NO concentrations, as well as improved AA supply and utilization, liver function, and feed intake in these cows.

Establishment of Deep-Eutectic-Solvent-Assisted Matrix Solid-Phase Dispersion Extraction for the Determination of Four Flavonoids in Scutellariae Radix Based on the Concept of Quality by Design.

BACKGROUND: Sample preparation is the most crucial step in analytical schemes. Micro-matrix solid-phase dispersion, as a method for microextraction of analytes, has prevailed recently for its low sample and extraction solvent consumption. However, small amounts of adsorbent or sample, or a short extraction time, always bring uncertainty to the result when using this method. OBJECTIVE: The aim was to develop a simple and reliable method of deep-eutectic-solvent-assisted ultrasonic-synchronized matrix solid-phase dispersion microextraction for the analysis of four flavonoids in Scutellariae Radix based on the concept of quality by design. METHOD: The ZSM-5 molecular sieve was used as a new adsorbent in the micro-matrix solid-phase dispersion process. Single-factor and Box-Behnken designs were used to construct the design space. RESULTS: Verification of the experiment demonstrated that the design space is robust. Under optimal conditions, all analytes showed good linearity (R2 > 0.999), high reproducibility (RSD < 2.24%) and stability (RSD < 2.87%), and satisfactory recoveries (95.90-102.31%), which indicated that the established method is reliable and reproducible. Moreover, it has been successfully applied to determine the flavonoids in nine batches of Scutellariae Radix. CONCLUSIONS: The results indicate a great potential for analyzing complicated samples especially with small amount and helping to promote the quality control of the sample preparation process for traditional Chinese medicines. HIGHLIGHTS: A systematic approach using a facile deep-eutectic-solvent-assisted ultrasonic-synchronized matrix solid-phase dispersion extraction coupled with HPLC for the analysis of flavonoids in Scutellariae Radix has been developed based on the concept of quality by design.

A Simple and Sensitive Dispersive Micro-Solid-Phase Extraction Coupled with High-Performance Liquid Chromatography for Quantification of Honokiol and Magnolol in Complex Matrices.

BACKGROUND: Honokiol and magnolol were considered as markers for the analysis of Cortex Magnoliae Officinalis, its related Chinese Patent Medicines and their metabolites. However, the determination of these two analytes in a water-soluble sample is difficult and therefore requires a more efficient method. OBJECTIVE: To develop a sensitive method for the determination of honokiol and magnolol in a water-soluble sample for better quality control of Cortex Magnoliae Officinalis and its related Chinese Patent Medicines. METHOD: In this work, a combination of dispersive micro-solid-phase extraction (DMSPE) and high-performance liquid chromatography (HPLC) has been developed for simultaneous preconcentration and determination of honokiol and magnolol in complex bio-samples. Several experimental factors affecting the extraction efficiency were optimized by single factor test. RESULTS: Under the optimized extraction conditions, the proposed method exhibited good linearity of not less than 0.9998, satisfactory precision with relative standard deviation of less than 1.3%, and acceptable mean recoveries of 97.3% and 101.5% for honokiol and magnolol, respectively. Furthermore, the method exhibits extremely high sensitivity with detection limits of 0.0097 and 0.0231 ng/mL, which is even more sensitive than those methods developed by MS. CONCLUSIONS: The method established in this study is fast, economic, accurate, easy to operate, and importantly well suited to the extraction and analysis of honokiol and magnolol in a real complex sample matrix.

Prognostic potential of pre-partum blood biochemical and immune variables for postpartum mastitis risk in dairy cows.

BACKGROUND: Mastitis is the most frequent diseases for transition cows. Identification of potential biomarkers for diagnosis of mastitis is important for its prevention. Thus, this study was conducted to investigate blood variables related to lipid metabolism, oxidative stress and inflammation, and serum variables that are related to health in postpartum cows. RESULTS: Seventy-six healthy Holstein dairy cows at week 4 before calving were selected to collect blood samples from weeks - 4 to 4 weekly relative to calving, respectively. Milk yield and composition were recorded weekly. According to the cut-off of somatic cell counts (SCC) for diagnosis of mastitis, 33 cows with SCC ≥ 500,000 cells ml- 1, 20 cows with 200,000 cells ≤ SCC < 500,000 cells ml- 1, and 23 cows with SCC < 200,000 cells ml- 1 were defined as high, middle, and low SCC, respectively. Serum concentrations of ß-hydroxybutyrate were higher (P < 0.01) during all weeks, and non-esterified fatty acids were higher in high SCC than in low SCC cows from weeks - 3 to 2 relative to calving. Higher serum concentrations of superoxide dismutase (P < 0.01) and lower malondialdehyde levels (P < 0.01) in low SCC than in high SCC cows indicate that the latter suffered from oxidative stress. The difference analysis of the three groups suggested that none of the above-mentioned variables can be used as potential prognostic candidates. On the other hand, high SCC cows exhibited higher blood neutrophil to lymphocyte ratio (NLR, P < 0.01) and platelet to lymphocyte ratio (PLR, P < 0.01) than low SCC cows, with a higher NLR (P < 0.01) in middle SCC than in low SCC cows. The high SCC cows had lower levels of anti-inflammatory factors including IL-10 (P = 0.05), but higher levels of proinflammatory factors such as IL-6 (P < 0.01), TNF-α (P < 0.05), and PSGL-1 (P < 0.01) than low SCC cows. CONCLUSIONS: The significantly different NLR and PLR pre-partum between the middle and low SCC cows suggest their prognostic potential for postpartum mastitis risk.

Simultaneous determination of seven compounds in Chinese patent medicines Chenxiangqu by matrix solid-phase dispersion coupled with ultra high performance liquid chromatography and quadrupole time-of-flight mass spectrometry.

A simple, efficient, and sensitive strategy by coupling matrix solid-phase dispersion with ultra high performance liquid chromatography quadrupole time-of-flight mass spectrometry was proposed to extract and determine three types of components (including seven analytes) in Chinese patent medicines Chenxiangqu. The highly ordered mesoporous material Fe-SBA-15 synthesized under weakly acidic conditions was selected as a dispersant in matrix solid phase dispersion extraction for the first time. Several parameters including the mass ratio of sample to dispersant, the type of dispersant, the grinding time, and the elution condition were investigated in this work. Under the optimized conditions, 20 compounds were identified by quadrupole time-of-flight mass spectrometry and seven analytes were quantified. The results demonstrated that the developed method has good linearity (r > 0.9995), and the limits of detection of the analytes were as low as 0.55 ng/mL. The recoveries of all seven analytes ranged from 97.6 to 104.6% (relative standard deviation < 3.4%). Finally, the improved method was successfully applied to determination of five batches of Chenxiangqu samples, which provided a robust method in quality control of Chinese patent medicines Chenxiangqu. The developed strategy also shows its great potential in analysis of complex matrix samples.

Online preconcentration by electrokinetic supercharging for sensitive determination of berberine and jatrorrhizine in biological samples.

Electrokinetic supercharging, a convenient and powerful online preconcentration technique in capillary electrophoresis, was introduced and evaluated for the determination of two alkaloids, berberine and jatrorrhizine, in mice fecal samples for the first time. The method depended on using a bare fused silica capillary (50 cm × 50 µm i.d.) and applying the voltage of 25 kV with UV detection at 205 nm. Parameters that affect the separation and preconcentration efficiency have been optimized. The optimum conditions used were as follows: background electrolyte consisting of 40mM sodium dihydrogenphosphate containing 30% methanol (v/v); hydrodynamic injection of 20mM KCl (50 mbar × 150 s) as the leading electrolyte; electrokinetic injection of the sample (+15 kV, 120 s) followed by the hydrodynamic injection of 30mM dodecyl trimethyl ammonium chloride (50 mbar × 12 s) as the terminating electrolyte. The results showed that the detection sensitivity of berberine and jatrorrhizine was, respectively, improved up 2740- and 2928-fold compared with normal injection, providing limits of detection lower than 3 ng/mL with good repeatability in areas (relative standard deviation < 3%). In summary, the developed method proved its ability in analyzing trace alkaloids in complicated biological samples.