Cross-Oxygen Gradients Transcriptomic Comparison Revealed the Central Role of MAPK and Hippo in Hypoxia-Mediated Mammary Proliferation Inhibition.

The role of hypoxia in terms of affecting mammary epithelial cells (MECs) proliferation is closely associated with the milk synthesis of lactating mammals. Primary bovine MECs were cultured at 1, 6, 11, 16, and 21% O2 for 24 h. The results showed that cell proliferation decreased linearly, and hypoxic inducible factor (HIF)-1α expression increased linearly along with the declining O2. The linear increase in oxidative stress resulted in the accumulation of malondialdehyde and reactive oxygen species and decreased antioxidant enzyme activities following the reduced O2. Concerning mitochondria, the dynamin-related protein 1 showed improved expression, and optin atrophy protein 1 decreased along with the decreasing O2 gradient, which led to decreased mitochondrial mass and mitophagy emerging under 1% O2. Oxygen concentration-trend RNA-seq analysis was conducted. Specifically, HIF-1-MAPK (1% O2), PI3K-Akt-MAPK (6% O2), and p53-Hippo (11 and 16% O2) were found to primarily regulate cell proliferation in response to hypoxia compared with normoxia (21%), respectively. In conclusion, our study suggests that bMEC proliferation is suppressed in low-oxygen conditions, and is exacerbated following the reduced oxygen supply. The cross-oxygen gradient comparisons suggest that MAPK and Hippo, which are core pathways of mammary cell proliferation, are repressed by hypoxia via oxidative-stress-dependent signals.

Progress of Conjugated Linoleic Acid on Milk Fat Metabolism in Ruminants and Humans.

As a valuable nutrient in milk, fat accounts for a significant proportion of the energy requirements of ruminants and is largely responsible for determining milk quality. Fatty acids (FAs) are a pivotal component of milk fat. Conjugated linoleic acid (CLA) is one of the naturally occurring FAs prevalent in ruminant dairy products and meat. Increasing attention has been given to CLA because of its anti-cancer, anti-inflammatory, immune regulation, and lipid metabolism regulation properties, and these benefits potentially contribute to the growth and health of infants. In breast milk, CLA is present in trace amounts, mainly in the form of cis-9, trans-11 CLA. Notably, cis-9, trans-11 CLA improves the milk fat rate while trans-10, cis-12 CLA inhibits it. Apart from having multiple physiological functions, CLA is also a pivotal factor in determining the milk quality of ruminants, especially milk fat rate. In response to growing interest in green and healthy functional foods, more and more researchers are exploring the potential of CLA to improve the production performance of animals and the nutritional value of livestock products. Taken together, it is novel and worthwhile to investigate how CLA regulates milk fat synthesis. It is the purpose of this review to clarify the necessity for studying CLA in ruminant milk fat and breast milk fat.

Heat stress affects dairy cow health status through blood oxygen availability.

BACKGROUND: Rises in global warming and extreme weather occurrence make the risk of heat stress (HS) induced by high ambient temperatures more likely in high-yielding dairy cows, resulting in low milk quality and yield. In animals, oxygen is involved in many physiological and metabolic processes, but the effects of HS on oxygen metabolism remain unclear. Thus, the current study aimed to investigate how oxygen metabolism plays a role in health status of dairy cows by measuring the milk yield, milk composition, and blood biochemical variables of cows under different levels of HS: none (No-HS), mild (Mild-HS), and moderate HS (Mod-HS). RESULTS: The HS significantly increased rectal temperature (Ptreat < 0.01) and respiration rate (Ptreat < 0.01). Under Mod-HS, greater Na+ (P < 0.05) and lower total CO2, and pH (P < 0.05) were observed relative to those under No-HS and Mild-HS. Oxygen concentrations in both coccygeal artery and mammary vein (Ptreat < 0.01) were lower under Mod-HS than under No-HS. Coccygeal vein concentrations of heat shock protein 90 (HSP90) (P < 0.05) increased during Mod-HS compared with those in cows under No-HS. Malondialdehyde increased during Mod-HS, and glutathione peroxidase (P < 0.01) increased during Mild-HS. Coccygeal vein concentrations of vascular endothelial growth factor (P < 0.01), heme oxygenase-1 (P < 0.01), and hypoxia-inducible factor 1α (P < 0.01) were greater in cows under Mod-HS than those under No-HS. Red blood cell count (P < 0.01) and hemoglobin concentration (P < 0.01) were lower in the coccygeal vein of dairy cows under Mild- and Mod-HS than those of cows under No-HS. CONCLUSIONS: Exposure to HS negatively impacts the health status and lactation performance of dairy cows by limiting oxygen metabolism and transportation. However, the specific mechanism by which HS affects mammary function in cows remains unclear and requires further exploration.

Transformation of Lung Squamous Cell Carcinoma to Small Cell Lung Cancer After Immunotherapy Resistance: A Case Report.

The transformation of lung adenocarcinoma to small cell lung cancer (SCLC) following treatment with epidermal growth factor (EGFR) receptor tyrosine kinase inhibitors (TKIs) is a relatively common phenomenon. However, transformation of non-small cell lung cancer (NSCLC) to SCLC following treatment with immunotherapy is very rare. Here, we report a case of a 56-year-old patient diagnosed with driver gene mutation-negative lung squamous cell carcinoma (SCC). He received four cycles of immunotherapy with sugemalimab and chemotherapy with albumin paclitaxel in combination with carboplatin, and a partial response was achieved. Subsequently, the patient received 5 cycles of immunotherapy with sugemalimab. However, he developed rapid progression of mediastinal lymph nodes, and biopsy results showed transformation to SCLC. His tumor did not respond to the next line of carboplatin combined with etoposide, and he died six months after the discovery of SCLC transformation. In conclusion, SCLC transformation is also an important resistance mechanism for lung SCC patients treated with immunotherapy and predicts a very poor outcome. Repeat biopsy is needed for advanced lung SCC that has progressed with immunotherapy.

Antioxidant hepatic lipid metabolism can be promoted by orally administered inorganic nanoparticles.

Accumulation of inorganic nanoparticles in living organisms can cause an increase in cellular reactive oxygen species (ROS) in a dose-dependent manner. Low doses of nanoparticles have shown possibilities to induce moderate ROS increases and lead to adaptive responses of biological systems, but beneficial effects of such responses on metabolic health remain elusive. Here, we report that repeated oral administrations of various inorganic nanoparticles, including TiO2, Au, and NaYF4 nanoparticles at low doses, can promote lipid degradation and alleviate steatosis in the liver of male mice. We show that low-level uptake of nanoparticles evokes an unusual antioxidant response in hepatocytes by promoting Ces2h expression and consequently enhancing ester hydrolysis. This process can be implemented to treat specific hepatic metabolic disorders, such as fatty liver in both genetic and high-fat-diet obese mice without causing observed adverse effects. Our results demonstrate that low-dose nanoparticle administration may serve as a promising treatment for metabolic regulation.

Hypoxia-inducible factor-1α is involved in the response to heat stress in lactating dairy cows.

Hypoxia-inducible factor-1α (HIF-1α) is important in maintaining cellular oxygen homeostasis and cellular heat tolerance. To explore the role of HIF-1α in the response to heat stress (HS) in dairy cows, 16 Chinese Holstein cows (milk yield: 32 ± 4 kg/d, days in milk: 272 ± 7 d, parity: 2-3) were used to collect coccygeal vein blood and milk samples when cows were under mild (temperature-humidity index = 77) and moderate HS (temperature-humidity index = 84), respectively. Compared to cows under mild HS, the respiratory rate (P < 0.01), rectal temperature (P < 0.01), and blood concentrations of heat shock protein (HSP)70 (P < 0.01) and HSP27 (P < 0.01) were higher, but oxygen saturation (P = 0.02) and hemoglobin (P < 0.01) were lower in cows under moderate HS. Blood HIF-1α concentration was greater (P < 0.01) during moderate HS, indicating that HIF-1α is involved in lactating cows' response to HS. To confirm these findings, we collected coccygeal vein blood and milk samples from 59 dairy cows under moderate HS. The HIF-1α levels were correlated with the levels of heat shock transcription factor (HSF) (r = 0.7857, P < 0.01), HSP70 (r = 0.4543, P < 0.01) and HSP27 (r = 0.8782, P < 0.01). A comparison of 15 cows with higher HIF-1α (>482 ng/L) and 15 cows with lower HIF-1α levels (<439 ng/L) showed that reactive oxidative species were higher (P = 0.02), but superoxide dismutase (P < 0.01), total antioxidation capacity (P = 0.02) and glutathione peroxidase (P < 0.01) were lower in higher HIF-1α cows. These results suggested that HIF-1α may be indicative of the risk of oxidative stress in heat-stressed cows and may participate in the response of cows to HS by synergistically activating the expression of the HSP family with HSF.

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.

Partial Replacement of Oat Hay with Whole-Plant Hydroponic Barley Seedlings Modulates Ruminal Microbiota and Affects Growth Performance of Holstein Heifers.

The dairy industry is facing challenges in balancing forage supply and crop production. Therefore, forage supply based on a farm land-saving approach should be developed to overcome the human−livestock competition on farmland. The objective of this study was to learn the potential impact of partially replacing oat hay with whole-plant hydroponic barley seedlings (HBS) produced via a land-saving hydroponic method on growth performance, digestibility, and rumen microbiota in Holstein dairy heifers. In total, 39 Holstein heifers were randomly divided into 13 blocks based on age and body weight for an 8-week experimental period. The heifers within each block were randomly allocated to one of three diets group: (1) 0% HBS and 16% oat hay (CON); (2) 4% HBS and 12% oat hay (25% HBS); and (3) 8% HBS and 8% oat hay (50% HBS). Compared to CON, feed intake, growth performance, and body N retention were similar to those in cows fed 25% HBS but lower in 50% HBS-fed animals (p < 0.05). Reduced digestibility (crude protein (CP) and organic matter (OM)) was observed in 50% HBS animals (p < 0.05). Compared to the control, the levels of Lachnospiraceae_XPB1014_group, Bacillus, and Colidextribacter were higher, but the levels of Sphaerochaeta and Ruminiclostridium were lower in 50% HBS animals (p < 0.05). Additionally, the digestibility of CP (p < 0.01, r = −0.61) and ether extract (EE) (p < 0.01, r = −0.58) was negatively correlated with Lachnospiraceae_XPB1014_group. The digestibility of OM (p = 0.01, r = −0.55), neutral detergent fiber (NDF) (p = 0.01, r = −0.56), acid detergent fiber (ADF) (p = 0.02, r = −0.52), and CP (p < 0.01, r = −0.61) was negatively correlated with Bacillus. The digestibility of NDF (p = 0.02, r = −0.52) and ADF (p = 0.03, r = −0.50) was negatively correlated with Colidextribacter. The digestibility of OM (p = 0.03, r = 0.50), NDF (p = 0.03, r = 0.50), and ADF (p = 0.03, r = 0.49) was positively correlated with Ruminiclostridium. The digestibility of OM (p = 0.04, r = 0.47), CP (p < 0.01, r = 0.58), and EE (p = 0.03, r = 0.49) was positively correlated with unclassified_f_Rikenellaceae. The digestibility of CP was positively correlated with Sphaerochaeta (p = 0.02, r = 0.53). In conclusion, the current study suggests that HBS could replace oat hay in a ratio-dependent manner. The reduced growth performance could be caused by lower feed intake and digestibility, which may be attributed to the alteration in the rumen's microbial population. Further exploration of the inhibiting factors of HBS would broaden the application of hydroponic feed in the future.

Multiple-Vessel-Based Blood Gas Profiles Analysis Revealed the Potential of Blood Oxygen in Mammary Vein as Indicator of Mammary Gland Health Risk of High-Yielding Dairy Cows.

The blood gas profile is a routine method in the rapid disease diagnosis of farm animals, yet its potential in evaluating mammary health status of dairy cows remains to be investigated. This study was conducted to learn the potential of the blood gas parameter regarding the mammary gland health status in lactating dairy cows. Twenty animals were divided into two groups, the H-SCC group (milk SCC > 122 k/mL) and L-SCC group (milk SCC < 73.8 k/mL), to compare blood gas profiles from different blood vessels and to identify the key parameters associated with milk somatic cell count. H-SCC cows are higher in malondialdehyde content, but lower in SOD and T-AOC activities in the milk, compared to the L-SCC group. In terms of blood gas parameters, most differ across the three vessels, including K+, CO2 pressure, O2 pressure, HCO3−, base excess in the extracellular fluid compartment, and saturation of O2. The Pearson correlation analysis showed that oxygen-related variables in the mammary vein, including oxygen concentrations, O2 pressure, and saturation of O2, are negatively correlated with levels of malondialdehyde, lactate dehydrogenase, and plasmin in the milk. Our study revealed that oxygen-related variables in the mammary vein can be a marker in suggesting mammary-gland health status in high-yielding cows.

Mammary Leukocyte-Assisted Nanoparticle Transport Enhances Targeted Milk Trace Mineral Delivery.

Nanoparticles are applied as versatile platforms for drug/gene delivery in many applications owing to their long-retention and specific targeting properties in living bodies. However, the delivery mechanism and the beneficial effect of nanoparticle-retention in many organisms remain largely uncertain. Here, the transport and metabolism of mineral nanoparticles in mammary gland during lactation are explored. It is shown that maternal intravenous administration of iron oxide nanoparticles (IONPs; diameter: ≈11.0 nm, surface charge: -29.1 mV, surface area: 1.05 m2 g-1 ) provides elevated iron delivery to mammary gland and increased iron secretion into breast milk, which is inaccessible by classical iron-ion transport approaches such as the transferrin receptor-mediated endocytic pathway. Mammary macrophages and neutrophils are found to play dominant roles in uptake and delivery of IONPs through an unconventional leukocyte-assisted iron secretion pathway. This pathway bypasses the tight iron concentration regulation of liver hepcidin-ferroportin axis and mammary epithelial cells to increase milk iron-ion content derived from IONPs. This work provides keen insight into the metabolic pathway of nanoparticles in mammary gland while offering a new scheme of nutrient delivery for neonate metabolism regulation by using nanosized nutrients.

Constraints on the utilization of cereal straw in lactating dairy cows: A review from the perspective of systems biology.

Cereal straw, a human inedible crop byproduct, can be used as a roughage source in ruminants. However, the nutrition density and palatability are very low, limiting its efficient utilization in animal production. This review aims to systematically provide an overview of the limitations of cereal straws, which is crucial for developing new strategies to enhance the efficient use of cereal straws by lactating dairy cows. Evolutionary molecular biology makes it possible to comprehensively understand the limitations of using cereal straw as a roughage source in dairy cows by different techniques, e.g., multi-omics. Main constraints for utilization of cereal straw and stover in lactating dairy cows include low contents of easily fermented carbohydrates (pectin) and essential amino acids (Met, Phe, and branched-chain amino acids), high content of lignin and silica, and low nutrient digestibility. These cause insufficient supply of the precursors for milk synthesis and result in increased loss of nutrients in feces and urine. Several molecular mechanisms are revealed by multi-omics techniques, including changed amino acid and glucose metabolism, altered rumen microbial composition and function, and differential expression of miRNAs, mRNA, and protein in multi-organs that are associated with milk synthesis. These can be targets of approaches to improve the utilization of cereal straw by dairy cows. In addition, much attention should be given to the efficient countermeasures, including pretreatments by fibrolytic enzymes or steam explosion, dietary formulations such as supplement of pectin, methionine, and branched-chain amino acids, and feeding with other functional feedstuffs, which may improve the feeding and economic value of cereal straw for lactating dairy cows. The newly revealed functional genes (such as BAG3 in the rumen, PC in the liver, CSN1S2 in the mammary gland) and biomarkers (hippuric acid) as well as the integrative signaling and metabolic pathways (phenylalanine metabolism) related to the shortages of cereal straws could be used as nutritional or genetic regulatory targets to improve dairy cow production.

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.

Nano-sized zinc addition enhanced mammary zinc translocation without altering health status of dairy cows.

This study aimed to evaluate role of nano-sized zinc (Zn) on lactation performance, health status, and mammary permeability of lactating dairy cows. Thirty multiparous dairy cows with similar days in milk (158 ± 43.2) and body weight (694 ± 60.5 kg) were chosen based on parity and milk production and were randomly assigned to 3 treatment groups: basal diet (control, 69.6 mg/kg of Zn adequate in Zn requirement), basal diet additional Zn-methionine (Zn-Met, providing 40 mg/kg of Zn), and basal diet additional nano-sized Zn oxide (nZnO, providing 40 mg/kg of Zn). The study lasted for 10 wk, with the first 2 wk as adaptation. Feed intake, milk yield and the related variables, and plasma variables were determined every other week. Blood hematological profiles were determined in the 8th week of the study. We found that feed intake, milk yield, and milk composition were similar across the 3 groups. The nZnO- and Zn-Met-fed cows had greater milk Zn concentrations in the milk (3.89 mg/L (Zn-Met) and 3.93 mg/L (nZnO)) and plasma (1.25 mg/L (Zn-Met) and 1.29 mg/L (nZnO)) than the control cows (3.79 mg/L in milk and 1.21 mg/L in plasma). The nZnO-fed cows had higher Zn concentrations in plasma but not in milk compared to Zn-Met-fed cows. The Zn appearance in milk was greater in nZnO-fed (area under curve during the first 4 h post-feeding for milk Zn: 16.1 mg/L) and Zn-Met-fed cows (15.7 mg/L) than in control cows (15.0 mg/L). During the first 4 h post-feeding, milk to blood Zn ratio was greater in nZnO-fed animals but lower in Zn-Met-fed cows compared with control cows. Oxidative stress-related variables in plasma, blood hematological profiles, and mammary permeability related variables were not different across treatments. In summary, lactation performance, Zn concentrations in milk and plasma, hematological profiles, mammary permeability were similar in cows fed nZnO and Zn-Met. We therefore suggested that nZnO feeding can improve Zn bioavailability without impairing lactation performance, health status, and mammary gland permeability in dairy cows.

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.

Prognostic Value of Ki-67 Expression in Advanced Lung Squamous Cell Carcinoma Patients Treated with Chemotherapy.

BACKGROUND: The relationship between the Ki-67 expression level and chemotherapy response and survival prognosis in advanced lung squamous cell carcinoma (SCC) remains unclear. METHODS: A total of 101 patients were included in the study. All patients received systemic first-line platinum-based chemotherapy. The Ki-67 expression level was determined by immunohistochemistry analysis. RESULTS: The Ki-67 expression level was positively correlated with an increase in tumor T stage (P = 0.0140), N stage (P < 0.0001), and M stage (P < 0.0001) in advanced lung SCC. High Ki-67 expression could predict chemotherapy response (area under the curve = 0.7524, P < 0.0001). Patients with tumors that expressed high levels of Ki-67 had shorter overall survival (OS) (18.8 months vs 25.5 months, P = 0.0002) and progression-free survival (PFS) (4.8 months vs 6.7 months, P < 0.0001). Cox analysis found Ki-67 expression to be an independent prognostic biomarker of shortened OS (P = 0.009) and PFS (P = 0.008). CONCLUSION: Ki-67 expression may affect chemotherapy response and thus has prognostic value. Ki-67 expression may be a promising prognostic biomarker for advanced lung SCC.

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.

Integrative network analysis revealed molecular mechanisms of urine urea output in lactating dairy cows: Potential solutions to reduce environmental nitrate contamination.

BACKGROUND: The enriched nitrogenous compounds in the dairy farms negatively affect the surrounding soil quality and air condition. The objective of this study is to investigate the transcriptomes of five key tissues involved in nitrogen metabolism and their changes under different diets to elucidate the molecular regulatory mechanisms of urine urea nitrogen (UUN) yield, one of the indicators of nitrogenous compound secretion of dairy cows. RESULTS: Cows fed high quality forage-based diet had lower UUN content and UUN yield, compared to those fed low quality forage (crop byproducts) based diets. From the transcriptomes of rumen, duodenum, jejunum, liver and udder, key driver genes and their UUN yield-associated functional gene networks were identified. In addition, the functional networks and expression of key drivers in various tissues (such as S100A8, CA1 and BPIFA2C in the duodenum; A2ML1, HMGCS2 and S100A12 in the jejunum; CYP2B6 and GLYCAM1 in the liver; APOE in the udder) changed in the cows fed crop byproducts based diet, which might be the predominant molecules to drive the increase UUN yield in these cows. CONCLUSION: The information suggested that gut, liver and udder play important roles in regulating UUN yield, which could regulate nitrogen excretion waste. These findings provide fundamental information on future nutritional intervention strategies to reduce the UUN yield from dairy cows fed human inedible crop byproducts, which is vital for a sustainable and environmentally friendly dairy industry.

Altered protein S-glutathionylation depicts redox imbalance triggered by transition metal oxide nanoparticles in a breastfeeding system.

Nanosafety has become a public concern following nanotechnology development. By now, attention has seldom been paid to breastfeeding system, which is constructed by mammary physiological structure and derived substances (endogenous or exogenous), cells, tissues, organs, and individuals (mother and child), connecting environment and organism, and spans across mother-child dyad. Thus, breastfeeding system is a center of nutrients transport and a unique window of toxic susceptibility in the mother-child dyad. We applied metabolomics combined with redox proteomics to depict how nanoparticles cause metabolic burden via their spontaneous redox cycling in lactating mammary glands. Two widely used nanoparticles [titanium dioxide (nTiO2) and zinc oxide (nZnO)] were exposed to lactating mice via intranasal administration. Biodistribution and biopersistence of nTiO2 and nZnO in mammary glands destroyed its structure, reflective of significantly reduced claudin-3 protein level by 32.1% (P < 0.01) and 47.8% (P < 0.01), and significantly increased apoptosis index by 85.7 (P < 0.01) and 100.3 (P < 0.01) fold change, respectively. Airway exposure of nTiO2 trended to reduced milk production by 22.7% (P = 0.06), while nZnO significantly reduced milk production by 33.0% (P < 0.01). Metabolomics analysis revealed a metabolic shift by nTiO2 or nZnO, such as increased glycolysis (nTiO2: fold enrichment = 3.31, P < 0.05; nZnO: fold enrichment = 3.68, P < 0.05), glutathione metabolism (nTiO2: fold enrichment = 5.57, P < 0.01; nZnO: fold enrichment = 4.43, P < 0.05), and fatty acid biosynthesis (nTiO2: fold enrichment = 3.52, P < 0.05; nZnO: fold enrichment = 3.51, P < 0.05) for tissue repair at expense of lower milk fat synthesis (35.7% reduction by nTiO2; 51.8% reduction by nZnO), and finally led to oxidative stress of mammary glands. The increased GSSG/GSH ratio (57.5% increase by nTiO2; 105% increase by nZnO) with nanoparticle exposure confirmed an alteration in the redox state and a metabolic shift in mammary glands. Redox proteomics showed that nanoparticles induced S-glutathionylation (SSG) modification at Cys sites of proteins in a nanoparticle type-dependent manner. The nTiO2 induced more protein SSG modification sites (nTiO2: 21; nZnO:16), whereas nZnO induced fewer protein SSG modification sites but at deeper SSG levels (26.6% higher in average of nZnO than that of nTiO2). In detail, SSG modification by nTiO2 was characterized by Ltf at Cys423 (25.3% increase), and Trf at Cys386;395;583 (42.3%, 42.3%, 22.8% increase) compared with control group. While, SSG modification by nZnO was characterized by Trfc at Cys365 (71.3% increase) and Fasn at Cys1010 (41.0% increase). The discovery of SSG-modified proteins under airway nanoparticle exposure further supplemented the oxidative stress index and mammary injury index, and deciphered precise mechanisms of nanotoxicity into a molecular level. The unique quantitative site-specific redox proteomics and metabolomics can serve as a new technique to identify nanotoxicity and provide deep insights into nanoparticle-triggered oxidative stress, contributing to a healthy breastfeeding environment.

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.

Excessive supply of glucose elicits an NF-κB2-dependent glycolysis in lactating goat mammary glands.

During lactation, an improper glucose supply often threatens mammary gland (MG) health. However, information is limited on the metabolic trajectories and molecules that regulate lactating MGs with an excessive glucose supply. Based on the network analysis of transcriptome and microRNAs, we found that the oversupply of glucose-induced severe glucose metabolic disorders in MGs of lactating goats, shifting lactose synthesis to acute fermentative glycolysis which caused increased flux of glucose metabolism into lactate. Moreover, NF-κB2 played a key role in regulating glycolysis, exhibiting a metabolic shift when MGs had an excessive supply of glucose. In primary mammary epithelial cells, fermentative glycolysis, and intracellular concentration of reactive oxygen species (ROS) were reduced by ganoderic acid A through blocking NF-κB2, while activation of NF-κB2 with phorbol myristate acetate (PMA) upregulated fermentative glycolysis and increased cellular ROS accumulation under excessive glucose. Thus, we established an NF-κB2-targeting method to reform the metabolic shift toward glycolysis caused by glucose oversupply by integrating NF-κB2 blockade and intracellular ROS scavenging.