Dynamic ROS Production and Gene Expression of Heifers Blood Neutrophil in a Oligofructose Overload Model.
ABSTRACT: Alimentary oligofructose (OF) overload can induce several diseases in cattle, such as ruminal acidosis, laminitis, and synovitis. The role of blood polymorphonuclear neutrophil (PMN) remains unclear during OF overload. The aim of this study was to investigate the dynamic changes in reactive oxygen species (ROS) production and the expression profile of genes in blood PMN in a model of OF overload. Twelve clinically healthy and non-pregnant Chinese Holstein heifers, aged between 18 and 26 mo, weighing 335-403 kg, BCS (5-point scale) ranges 2.7-3.3 were used for the experiments. OF heifers (n = 6) received 17 g/kg of BW oligofructose dissolved in 2 L/100 kg of BW tap water and the CON heifers (n = 6) received 2 L/100 kg of BW tap water. Blood PMN was isolated for each heifer 0, 6, 12, 18, 24, 36, 48, 60, and 72 h after administration. PMN was analyzed either by endogenous and phorbol myristate acetate (PMA)-induced ROS production or by quantitative real-time PCR. After 12 h, PMA-induced ROS production decreased, which was sustained until 48 h. The expressions of inflammation markers (IL1?, IL1?, IL6, IL10, TNF?, STAT3, TLR4, MMP9, and HP) and eicosanoids (ALOX5, ALOX5AP, and PLA2G4A) were upregulated. The expression of adhesion and migration (CXCR2, CXCL8, CD62L, ITGA4, ITGAM, and ITGB2) in OF heifers was increased compared with CON heifers. The expression of oxidative stress (SOD2 and S100A8) was upregulated, while SOD1 and MPO were downregulated. In metabolism and receptor genes, the expressions of GR? and INSR decreased after 12 h, while Fas increased until 6 h and then decreased at 18 h. The expression of LDHA and PANX1 did not show any differences after OF overload. These findings indicate that OF overload induced systemic activation of PMN, which provides a step toward a better understanding of the role of innate immune responses in response to oral OF administration.
Project description:BACKGROUND:Acute ruminal acidosis (ARA) is a metabolic disease of cattle characterized by an aseptic synovitis. ARA is the result of an increased intake of highly fermentable carbohydrates that frequently occurs in dairy cattle subjected to high production requirements. In human joint diseases such as rheumatoid arthritis and gout, several pro-inflammatory molecules are increased in the synovial fluid, including cytokines, prostaglandin E2 (PGE2), metalloproteinases, and neutrophil extracellular traps (NETs). The aim of this study was to identify the presence of proinflammatory mediators and neutrophils in the synovial fluid of heifers with ARA, induced by an oligofructose overload. Five heifers were challenged with an oligofructose overload (13 g/kg BW) dissolved in water. As a control, a similar vehicle volume was used in four heifers. Synovial fluid samples were collected from the tarso-crural joint and PGE2, IL-6, IL-1β, ATP, lactate dehydrogenase (LDH), albumin, glucose, matrix metalloproteinase-9 (MMP-9), cellular free DNA, NETs, and serpin B1 were analyzed at 0, 9, and 24 h post treatment. RESULTS:At 9 h post oligofructose overload, an increase of IL-1β, IL-6, PGE2, serpin B1 and LDH was detected in the joints when compared to the control group. At 24 h, the synovial fluid was yellowish, viscous, turbid, and contained abundant neutrophils. An increase of DNA-backbone-like traps, histone 3 (H3cit), aggregated neutrophil extracellular traps (aggNETs), and serpin B1 were observed 24 h post treatment. Furthermore, albumins, LDH, ATP, MMP-9, IL-6, and IL-1β were increased after 24 h. CONCLUSIONS:The overall results indicate that IL-1β, IL-6 and PGE2, were the earliest proinflammatory parameters that increased in the synovial fluid of animals with ARA. Furthermore, the most sever inflammatory response in the joint was observed after 24 h and could be associated with a massive presence of neutrophils and release of aggNETs.
Project description:Acute ruminal acidosis (ARA) is the result of increased intake of highly fermentable carbohydrates, which frequently occurs in dairy cattle and is associated with aseptic polysynovitis. To characterise the metabolic changes in the joints of animals with ARA, we performed an untargeted gas chromatography-mass spectrometry (GC-MS)-based metabolomic analysis of synovial fluid. Seven heifers were challenged with an intraruminal oligofructose overload (13 g/kg of body weight [BW]) dissolved in water. Synovial fluid samples were collected at 0, 9 and 24 h post-overload. Metabolome analysis revealed the presence of 67 metabolites. At 9 h post-overload, glyceric acid, cellobiose, fructose and lactic acid were all increased, whereas at 24 h, sorbitol, lactic acid and fructose levels were all increased >10-fold. At 24 h, citric acid and threonine levels were significantly reduced. We detected increased L- and D-lactate, and the presence of interleukin-6 (IL-6) in synovial fluid. Furthermore, using bovine fibroblast-like synoviocytes, we observed that D-lactate induces IL-6 synthesis. Our results suggest that ARA produces severe metabolomic changes in synovial fluid, including disturbances in starch and sucrose metabolism, and increased lactate levels. These changes were observed prior to the appearance of synovitis, suggesting a potential role in the onset of polysynovitis.
Project description:The objectives of this study were to examine the clinical response, changes in ruminal bacterial microbiota, and inflammatory response in lamellar tissues during oligofructose-induced laminitis. Ten fistulated sheep were randomly assigned into a control group ( = 5) and a treatment group ( = 5). The treatment group was infused with oligofructose (21 g/kg BW) by rumen cannula, and the control group was sham-treated with saline. Results showed that all 5 sheep treated with oligofructose developed anorexia and diarrhea 8 to 12 h after the administration of oligofructose. By 12 to 24 h after treatment, the treatment group developed lameness and roach back. Compared with the control group, oligofructose administration decreased ( < 0.001) the rumen pH and concentrations of total VFA and increased ( < 0.001) the level of lactic acid in the rumen. Microbial data analysis revealed that oligofructose infusion increased the abundance of ( = 0.009) and ( = 0.008) and decreased the percentage of unclassified Christensenellaceae ( = 0.028), unclassified Ruminococcaceae ( = 0.009), ( = 0.016), unclassified Lachnospiraceae ( = 0.009), and ( = 0.009) compared with the control group. Oligofructose infusion decreased the ACE ( = 0.047) and Shannon ( = 0.009) indices compared with the control group. The histomorphology analysis revealed that oligofructose overload resulted in damage to the dermoepidermal junction in the lamellar tissue of sheep. Quantitative real-time PCR results showed that compared with the control group, the mRNA expression of membrane-type metalloproteinase-1 ( = 0.049) was downregulated whereas the expression of proinflammatory IL-6 ( = 0.004) and matrix metalloprotease-9 ( = 0.037) was upregulated in the lamellar tissues of the oligofructose treatment group. In general, the present study provides the foundation for a sheep model of oligofructose-overload-induced acute laminitis that could be used in later experiments. Our findings suggest that intraruminal infusion of oligofructose altered ruminal microbiota and resulted in acute laminitis and that the inflammatory damage to the lamellae tissue may be related to the upregulation of matrix metalloprotease-9. The information generated will provide more insight into the systemic effects of lameness caused by oligofructose overload in sheep.
Project description:This study aimed to characterize oligofructose-induced laminitis in zebu cattle and comparatively evaluate four different diagnostic methods for laminitis. A total of 29 rumen-cannulated Nelore heifers, weighing 474.5 ± 58.5 kg were used. Laminitis was experimentally induced by intraruminal administration of 0.765 g/kg oligofructose twice daily for three consecutive days, followed by a single dose of 10.71 g/kg oligofructose on the fourth day. The animals were evaluated before administration of the highest dose of oligofructose (basal) and every six hours for up to 24 hours (6, 12, 18, 24 hours) and thereafter, every 12 hours for up to 72 hours (36, 48, 60, 72 hours) post-induction. The following diagnostic methods were used: hoof pain sensitivity test (hoof-testing), locomotion scoring, hoof infrared thermography, and force platform. Diagnosis of laminitis was confirmed after two positive responses to hoof pressure testing. Using a receiver operator characteristic (ROC) curve, we defined the appropriate cut-off for infrared thermography and force plate as 30 °C and < 24%, respectively. From the 29 heifers, 27 developed laminitis (93.1%) which occurred between 24 h to 72 h in the digits from two limbs, with more frequent sensitivity in the lateral digits. Locomotion analysis detected twenty-eight heifers with laminitis and showed that a greater (P = 0.006) number of animals had lameness in two limbs (n = 13; 56%). Using hoof-testing as gold standard for the diagnosis of laminitis the locomotion score displayed 100% sensitivity, 97% specificity and 98% accuracy; infrared thermography showed 96% sensitivity, 63% specificity, and 75% accuracy whilst force plate had 76% sensitivity, 82% specificity and 79% accuracy. This suggests that, for the diagnosis of laminitis in cattle, pain evaluation is more efficient. Considering the difficult to evaluate pain sensitivity in Nelore animals, filmed locomotion score, infrared thermography and force plate methods can be indicated for non-invasive lameness detection in beef farms.
Project description:This experiment evaluated the effects of postweaning body weight (BW) gain of replacement beef heifers on their reproductive development and productivity as primiparous cows. Seventy-two Angus × Hereford heifers were ranked on day -6 of experiment (17 d after weaning) by age and BW (218 ± 1.6 d of age and 234 ± 3 kg of BW), and assigned to receive 1 of 3 supplementation programs from days 0 to 182: 1) no supplementation to maintain limited BW gain (LGAIN), 2) supplementation to promote moderate BW gain (MGAIN), or 3) supplementation to promote elevated BW gain (HGAIN). Heifers were maintained in 2 pastures (36 heifers/pasture, 12 heifers/treatment in each pasture) with free-choice alfalfa-grass hay, and supplements were offered individually 6 d per week. Heifer shrunk BW was recorded on days -6 and 183 for average daily gain (ADG) calculation. Blood samples were collected for puberty evaluation via plasma progesterone weekly from days 0 to 182. On day 183, heifers were combined into a single group and received the same nutritional management until the end of the experimental period (day 718). From days 183 to 253, heifers were assigned to a fixed-time artificial insemination program combined with natural service. Average daily gain from days 0 to 182 was greater (P < 0.01) in HGAIN vs. MGAIN and LGAIN (0.78, 0.60, and 0.37 kg/d, respectively; SEM = 0.02), and greater (P < 0.01) in MGAIN vs. LGAIN heifers. Puberty attainment by the beginning of the breeding season was also greater in HGAIN vs. MGAIN and LGAIN (87.5%, 62.5%, and 56.5%, respectively; SEM = 7.1) but similar (P = 0.68) between MGAIN vs. LGAIN heifers. A treatment × day interaction was detected (P < 0.01) for calving rate, as HGAIN heifers calved earlier compared with MGAIN and LGAIN heifers. Ten heifers per treatment were assessed for milk production via weigh-suckle-weigh at 56.8 ± 1.5 d postpartum, followed by milk sample collection 24 h later. No treatment differences were detected (P ? 0.16) for milk yield and composition. However, mRNA expression of GLUT1 in milk fat globules was less (P ? 0.02) in LGAIN vs. MGAIN and HGAIN heifers, and expression of GLUT8 mRNA was also less (P = 0.04) in LGAIN vs. HGAIN heifers. No treatment differences were detected (P ? 0.44) for offspring weaning BW. Collectively, results from this experiment indicate that HGAIN hastened the reproductive development of replacement heifers, without negatively affecting their milk productivity and offspring weaning weight as primiparous cows.
Project description:The literature lacks studies investigating the performance of supplemented replacement heifers grazing on intensively managed warm-season pasture. Our objective was to evaluate the effects of supplement composition (energetic or protein) on the performance, muscle development, thermogenisis, nutrient intake, and digestibility of replacement Holstein heifers grazing Mombaça grass. Eighteen Holstein heifers with an average age and initial body weight (BW) of 12.57 ± 2.54 mo and 218.76 ±47.6 kg, respectively, were submitted to a randomized block design, with six replicates on a rotational grazing system of Panicum maximum cv. Mombaça pasture. Treatments were: control (CON; mineral salt ad libitum); energy supplement (ENE; corn meal as supplement, 8% CP and 3.78 Mcal/kg DE); and protein supplement (PRO; corn and soybean meal, 25% CP and 3.66 Mcal/kg DE). Supplements were individually fed at 0.5% BW. The experiment lasted 120 days, subdivided into three periods. Titanium dioxide and indigestible neutral detergent fiber (iNDF) were used to estimate the intakes and digestibility of the nutrients. BW, wither height, thoracic circumference, body length, and ultrasound of ribeye fat thickness measurements were taken once per period. Body condition score (BCS) was assessed twice during the experiment. The MIXED procedure of SAS, including period as a repeated measure, was used and significance was declared at P ? 0.05. Dry matter intake (DMI), CP intake (CPI) and DE intake were greater in heifers fed PRO compared to CON and ENE. Heifers supplemented with ENE had the lowest DMI. Treatment affected pasture intake/BW; it was similar between PRO and CON heifers, and lower for the ENE treatment. A treatment × period interaction was observed for NDF intake (%BW), in which heifers fed PRO and CON had the greatest NDF intake and ENE had the lowest. The digestibility of DM was the greatest in PRO-supplemented heifers and the lowest in CON heifers. Heifers fed ENE had decreased CP digestibility compared to PRO and CON heifers. Average daily gain (ADG) and thoracic circumference gain were greatest in the PRO treatment. BCS was greater in PRO compared to CON and ENE heifers. Supplementing Holstein heifers at 0.5% BW using PRO supplementation resulted in better animal performance, primarily greater ADG, than feeding ENE or not supplementing (CON). In conclusion, our results indicate that dairy heifers should be fed a protein supplement when grazing intensively managed Mombaça grass pasture.
Project description:In general, beef cattle long-distance transportation from cow-calf operations to feedlots or from feedlots to abattoirs is a common situation in the beef industry. The aim of this study was to determine the effect of rumen-protected methionine (RPM) supplementation on a proposed gene network for muscle fatigue, creatine synthesis (CKM), and reactive oxygen species (ROS) metabolism after a transportation simulation in a test track. Angus × Simmental heifers (n = 18) were stratified by body weight (408 ± 64 kg; BW) and randomly assigned to dietary treatments: 1) control diet (CTRL) or 2) control diet + 8 gr/hd/day of top-dressed rumen-protected methionine (RPM). After an adaptation period to Calan gates, animals received the mentioned dietary treatment consisting of Bermuda hay ad libitum and a soy hulls and corn gluten feed based supplement. After 45 days of supplementation, animals were loaded onto a trailer and transported for 22 hours (long-term transportation). Longissimus muscle biopsies, BW and blood samples were obtained on day 0 (Baseline), 43 (Pre-transport; PRET), and 46 (Post-transport; POST). Heifers' average daily gain did not differ between baseline and PRET. Control heifer's shrink was 10% of BW while RPM heifers shrink was 8%. Serum cortisol decreased, and glucose and creatine kinase levels increased after transportation, but no differences were observed between treatments. Messenger RNA was extracted from skeletal muscle tissue and gene expression analysis was performed by RT-qPCR. Results showed that AHCY and DNMT3A (DNA methylation), SSPN (Sarcoglycan complex), and SOD2 (Oxidative Stress-ROS) were upregulated in CTRL between baseline and PRET and, decreased between pre and POST while they remained constant for RPM. Furthermore, CKM was not affected by treatments. In conclusion, RPM supplementation may affect ROS production and enhance DNA hypermethylation, after a long-term transportation.
Project description:Ruminants play an important role in food security, but there is a growing concern about the impact of cattle on the environment, particularly regarding greenhouse gas emissions. The objective of this study was to examine the effect of humic substances (HS) on rumen fermentation, nutrient digestibility, methane (CH4) emissions, and the rumen microbiome of beef heifers fed a barley silage-based diet. The experiment was designed as a replicated 4 × 4 Latin square using 8 ruminally cannulated Angus × Hereford heifers (758 ± 40.7 kg initial BW). Heifers were offered a basal diet consisting of 60% barley silage and 40% concentrate (DM basis) with either 0- (control), 100-, 200- or 300-mg granulated HS/kg BW. Each period was 28 d with 14 d of adaptation. Rumen samples were taken on day 15 at 0, 3, 6, and 12 h postfeeding. Total urine and feces were collected from days 18 to 22. Blood samples were taken on day 22 at 0 and 6 h postfeeding. Between days 26 and 28, heifers were placed in open-circuit respiratory chambers to measure CH4. Ruminal pH was recorded continuously during the periods of CH4 measurement using indwelling pH loggers. Intake was similar (P = 0.47) across treatments. Concentration of ammonia-N and counts of rumen protozoa responded quadratically (P = 0.03), where both increased at H100 and then decreased for the H300 treatments. Apparent total tract digestibility of CP (P = 0.04) was linearly increased by HS and total N retention (g/d, % N intake, g/kg BW0.75) was improved (P = 0.04) for HS when compared with the control. There was no effect of HS on CH4 production (g/d; P = 0.83); however, HS decreased the relative abundance of Proteobacteria (P = 0.04) and increased the relative abundance of Synergistetes (P = 0.01) and Euryarchaeota (P = 0.04). Results suggest that HS included at up to 300 mg/kg BW may improve N retention and CP digestibility, but there was no impact on CH4 production.
Project description:A 3 yr study evaluated the effects of three preweaning injections of bovine ST, administered 14 d apart, on growth and reproductive performance of Bos indicus-influenced beef heifers. On d 0 of each year, suckling Angus × Brangus heifers (n = 15 heifers/treatment/yr) were stratified by BW (147 ± 20 kg) and age (134 ± 11 d) and randomly assigned to receive an s.c. injection of saline (SAL; 5 mL; 0.9% NaCl) or 250 mg of sometribove zinc (BST; Posilac, Elanco, Greenfield, IN) on d 0, 14, and 28. Heifers and respective dams were managed as a single group on bahiagrass (Paspalum notatum) pastures from d 0 until weaning (d 127). From d 127 to 346, heifers were grouped by treatment, allocated to bahiagrass pastures (1 pasture/treatment/yr) and fed a molasses-based supplement (2.9 kg/heifer daily; DM basis) until d 346. Blood samples were collected on d 0, 14, 28, 42, and then every 9-10 d from d 179 to 346. In yr 3, liver biopsy samples were collected on d 0, 42, and 263. Heifers were exposed to mature Angus bulls from d 263 to 346. Growth performance and physiological parameters were analyzed using the MIXED procedure, whereas reproductive variables were analyzed using the GLIMMIX procedure of SAS. Effects of treatment × year and treatment × year × time were not detected for any variable measured in this study (P ? 0.14), except for calving percentage (P = 0.03). Heifers assigned to BST injections had greater overall plasma concentrations of IGF-1 and ADG from d 0 to 42 (P ? 0.05), less ADG from d 42 to 127 (P = 0.04), but had similar BW at weaning and postweaning ADG (P ? 0.25) compared to SAL heifers. Heifers assigned to BST tended to achieve puberty 26 d earlier (P = 0.10), had greater percentage of pubertal heifers on d 244, 263, 284, and 296 (P ? 0.04), tended to have greater overall pregnancy percentage (P = 0.10), and had greater (P ? 0.05) calving percentages in yr 1 and 2 (but not yr 3; P = 0.68) compared to SAL heifers. Liver mRNA expression of GHR-1B and IGF-1 on d 0 and 42 did not differ between treatments (P ? 0.15), but was greater for BST vs. SAL heifers on d 263 (P ? 0.02). Hence, administering three injections containing 250 mg of sometribove zinc at 14 d intervals before weaning (between 135 and 163 d of age) induced long-term impacts on liver gene expression and may be a feasible management practice to enhance puberty and pregnancy attainment in B. indicus-influenced replacement beef heifers.
Project description:Myeloid-derived suppressor cells (MDSC) are induced by and accumulate within many histologically distinct solid tumors, where they promote disease by secreting angiogenic and immunosuppressive molecules. Although IL1? can drive the generation, accumulation, and functional capacity of MDSCs, the specific IL1?-induced inflammatory mediators contributing to these activities remain incompletely defined. Here, we identified IL1?-induced molecules that expand, mobilize, and modulate the accumulation and angiogenic and immunosuppressive potencies of polymorphonuclear (PMN)-MDSCs. Unlike parental CT26 tumors, which recruited primarily monocytic (M)-MDSCs by constitutively expressing GM-CSF- and CCR2-directed chemokines, IL1?-transfected CT26 produced higher G-CSF, multiple CXC chemokines, and vascular adhesion molecules required for mediating infiltration of PMN-MDSCs with increased angiogenic and immunosuppressive properties. Conversely, CT26 tumors transfected with IL1?-inducible molecules could mobilize PMN-MDSCs, but because they lacked the ability to upregulate IL1?-inducible CXCR2-directed chemokines or vascular adhesion molecules, additional PMN-MDSCs could not infiltrate tumors. IL1?-expressing CT26 increased angiogenic and immunosuppressive factors of tumor-infiltrating MDSCs, as did CT26 tumors individually transfected with G-CSF, Bv8, CXCL1, or CXCL5, demonstrating that mediators downstream of IL1? could also modulate MDSC functional activity. Translational relevance was indicated by the finding that the same growth factors, cytokines, chemokines, and adhesion molecules responsible for the mobilization and recruitment of PMN-MDSCs into inflammatory CT26 murine tumors were also coordinately upregulated with increasing IL1? expression in human renal cell carcinoma tumors. These studies demonstrated that IL1? stimulated the components of a multifaceted inflammatory program that produces, mobilizes, chemoattracts, activates, and mediates the infiltration of PMN-MDSCs into inflammatory tumors to promote tumor progression.