Transcriptomics of circulating neutrophils in dairy cows with subclinical hypocalcemia.

Hypocalcemia is closely associated with inflammatory diseases in dairy cows. Recent research has underscored the key role of calcium in the adaptations of the innate immune system during this period. The main objective in the present study was to compare the transcriptome profiles and analyze differences in the expression of neutrophil (PMNL) immune function-related genes and calcium binding-related genes in hypocalcemic cows. At 2 days postpartum, a concentration >2.10 mmol Ca2+/L was used to classify cows as controls (CON), and a concentration <2.00 mmol Ca2+/L used to classify cows as low-calcium (LCAL) (n = 8 in each group). A routine medical examination was conducted by the attending veterinarian to ensure there were no other complications and that the blood ß-hydroxybutyrate was <1.2 mmol/L. Blood was collected from the tail vein (20 mL) to isolate PMNL, and 5 cows in each group were used for RNA sequencing and statistical analysis of gene expression differences. Transcriptome RNA-seq sequencing analysis was via omicsstudio using the R package edgeR. GO and KEGG enrichment analysis were used for bioinformatics. The remaining 3 cows in each group were used for validation of RNA sequencing data via quantitative PCR, which confirmed the observed responses. Compared with CON, 158 genes in LCAL were significantly up-regulated and 296 genes were down-regulated. The downregulation of Interleukin-12 (CXCL12), Tubulin beta chain (TUBB1), L1 cell adhesion molecule (L1CAM), and Myeloperoxidase (MPO) indicated a decrease in immune function of PMNL in LCAL cows. The decreased expression of calcium-binding pathway-related genes in PMNL of LCAL cows indicated a decrease in immune function of PMNL likely related to calcium ions. For example, cartilage acid protein 1 (CRTAC1) and calcium/calmodulin-dependent kinase 4 (CAMK4) were significantly reduced in LCAL cows. The upregulation of Cyclin dependent kinase inhibitor 1A (CDKN1A), Perforin 1 (PRF1), and Homeodomain interacting protein kinase 3 (HIPK3) indicated that LCAL led to greater cell apoptosis and senescence. Overall, the analyses indicated that the reduction in PMNL immune function during hypocalcemia is associated with downregulation of intracellular Ca2+ related genes and upregulation of genes controlling apoptosis and senescence. Together, these alterations contribute to an immunosuppressive state during the transition period.

The Early Prediction of Common Disorders in Dairy Cows Monitored by Automatic Systems with Machine Learning Algorithms.

We use multidimensional data from automated monitoring systems and milking systems to predict disorders of dairy cows by employing eight machine learning algorithms. The data included the season, days in milking, parity, age at the time of disorders, milk yield (kg/day), activity (unitless), six variables related to rumination time, and two variables related to the electrical conductivity of milk. We analyze 131 sick cows and 149 healthy cows with identical lactation days and parity; all data are collected on the same day, which corresponds to the diagnosis day for disordered cows. For disordered cows, each variable, except the ratio of rumination time from daytime to nighttime, displays a decreasing/increasing trend from d-7 or d-3 to d0 and/or d-1, with the d0, d-1, or d-2 values reaching the minimum or maximum. The test data sensitivity for three algorithms exceeded 80%, and the accuracies of the eight algorithms ranged from 65.08% to 84.21%. The area under the curve (AUC) of the three algorithms was >80%. Overall, Rpart best predicts the disorders with an accuracy, precision, and AUC of 81.58%, 92.86%, and 0.908, respectively. The machine learning algorithms may be an appropriate and powerful decision support and monitoring tool to detect herds with common health disorders.

Effects of different fatty acids on BRL3A rat liver cell damage.

To evaluate the effects of fatty acids on endoplasmic reticulum (ER) stress, oxidative stress, and lipid damage. We treated BRL3A rat liver cells with, linoleic (LA), linolenic, oleic (OA), palmitic (PA), palmitoleic (POA), or stearic (SA) acid for 12 hr. The characteristics of cell lipid deposition, oxidative stress indexes, ER stress markers, nuclear factor κB p65 (NF-κB p65), lipid synthesis and transport regulators, and cholesterol metabolism regulators were analyzed. Endoplasmic chaperones like glucose-regulated protein 78, CCAAT-enhancer-binding protein, NF-κB p65, hydrogen peroxide, and malonaldehyde in PA- and SA-treated cells were significantly higher than in other treated cells. Deposition of fatty acids especially LA and POA were significantly increased than in other treated cells. De novo lipogenesis regulators sterol regulatory element-binding protein 1c, fatty acid synthase, and acetyl-coenzyme A carboxylase 1 (ACC1) expression were significantly increased in all fatty acid stimulation groups, and PA- and SA-treated cells showed lower p-ACC1 expression and higher scd1 expression than other fatty acid groups. Very low-density lipoprotein synthesis and apolipoprotein B100 expression in free fatty acids treated cells were significantly lower than control. PA, SA, OA, and POA had shown significantly increased cholesterol synthesis than other treated cells. PA and SA showed the lower synthesis of cytochrome P7A1 and total bile acids than other fatty acids treated cells. Excess of saturated fatty acids led to severe ER and oxidative stress. Excess unsaturated fatty acids led to increased lipid deposition in cultured hepatocytes. A balanced fatty acid intake is needed to maintain lipid homeostasis.

Ketotic cows display a different serum nonesterified fatty acid composition.

The experiments reported in this research communication aimed to compare the serum nonesterified fatty acid (NEFA) composition in ketotic cows and healthy cows during the perinatal period. NEFAs play significant roles in etiology and pathology of ketosis. We hypothesized that ketotic cows will display a different serum NEFA composition compared to healthy controls, and fatty acid related indicators for ketosis prediction can be screened. Pre-partum healthy cows were recruited, and blood samples were collected on -7, 3, 7, 14 and 21 d postpartum. Cows were further divided into a healthy control group (C group, n = 6) and a ketosis group (K group, n = 6) if blood ß-hydroxybutyric acid levels exceeded 1.2 mm during the experiment. NEFA composition was then analyzed by means of Gas Chromatography-Mass Spectrometer (GC-MS). Only C12 : 0% was significantly higher in C group than K group on 7 d pre-partum (P < 0.05), when the cows were not diagnosed with ketosis. Five fatty acids displayed statistical differences in composition between C and K group (P < 0.05), namely C12 : 0, C16 : 0, C17 : 0, C18 : 1n9 and C22 : 1n9. Saturates%, unsaturates%, mono-unsaturates% and saturates/unsaturates were also different between C and K group (P < 0.05). Of note, C18 : 1n9/C12 : 0 and C18 : 1n9/C22 : 1n9 in K group were significantly higher than those in controls on 7 d pre-partum (P < 0.05). It is suggested that the ratios show potential as indicators for prediction of ketosis.

Influence of oleic acid in different solvent media on BRL 3A cell growth and viability.

Oleic acid (OA) is widely used in pathology studies of hepatocellular lipid deposition. Identifying the effects of different solvents on OA-induced liver lipid deposition would be beneficial for studies on hepatocytes. We treated BRL 3A cells with OA dissolved in different solvents. After 12 h incubation, cell viability was assessed using MTT assays. Reactive oxygen species (ROS), triglyceride (TG) and total cholesterol (TC) counts, and the expression level of glucose regulated protein (GRP78), sterol regulatory element binding protein (SREBP-1C) and fatty acid synthase (FAS) were analyzed. Water, PBS and DMSO were disadvantageous to the dissolution of OA and did not cause an OA-induced response in hepatocytes. In the alcohol+OA-treated cells, the severe ER stress, oxidative stress and cellular fat deposition were significantly increased. BSA promoted cell growth and the cells treated with 1.2% BSA+OA showed a lower grade TG and endoplasmic reticulum stress compared with KOH+OA and alcohol+OA treatments. KOH had no significant influence on BRL 3A cells viability. When treated with OA dissolved in KOH, BRL 3A cells showed a typical hepatocyte damage. KOH was considered the suitable choice for an OA solvent for BRL 3A cells in hepatic lipidosis research.