Induced endometritis in early lactation compromises production and reproduction in dairy cows

Objectives of this experiment were to study the effect of infusing utero-pathogenic bacteria to induce endometrial inflammation on productive performance in early lactation and subsequent reproduction. Although endometritis is associated with perturbed reproduction, numerous factors may contribute to the observed association. It was hypothesized that induced endometrial inflammation, resulting in localized and systemic inflammatory responses, compromises production and reproduction. Holstein cows without clinical disease and with less than 18% polymorphonuclear leukocytes (PMN) in endometrial cytology on d 31 ± 3 postpartum had their estrous cycle synchronized. Cows were blocked by parity and genomic breeding value for cow conception rate and, within block, assigned randomly to remain as untreated controls (CON; n = 37) or to receive an intrauterine infusion of 5.19 × 10⁸ cfu Escherichia coli and 4.34 × 10⁸ cfu Trueperella pyogenes during the luteal phase to induce endometrial inflammation (INF; n = 48). Endometrial cytology was taken on d 2 and 7 after treatment to evaluate the proportion of PMN. Rectal temperature, dry matter intake, and yields of milk and components were measured in the first 7 d after treatment. Blood serum was analyzed for concentration of haptoglobin. Leukocytes were isolated from blood on d 2 and 7 after treatment and on d 19 after artificial insemination (AI) and mRNA was quantified for a select group of genes. Cows received AI and reproduction was followed for 300 d postpartum. Bacterial infusion induced endometrial inflammation with increased proportions of PMN in the endometrial cytology on d 2 (4.4 ± 0.7 vs. 26.3 ± 2.8%) and 7 (10.9 ± 1.7 vs. 17.4 ± 2.1%) after treatment, resulting in increased mean prevalence of subclinical endometritis (>10% PMN; 23.3 ± 6.3 vs. 80.9 ± 5.1%). Rectal temperature did not differ between CON and INF, but the concentration of haptoglobin in serum tended to increase in INF compared with CON (113 ± 14 vs. 150 ± 16 µg/mL). Induced endometrial inflammation reduced yields of milk (44.9 ± 0.8 vs. 41.6 ± 0.8 kg/d), protein (1.19 ± 0.03 vs. 1.12 ± 0.03 kg/d), and lactose (2.17 ± 0.04 vs. 2.03 ± 0.04 kg/d) and tended to reduce dry matter intake (20.7 ± 0.5 vs. 19.4 ± 0.6 kg/d) in the first 7 d after treatment. Indeed, the reduction in milk yield lasted 4 wk. However, treatment did not affect yields of energy-corrected milk or fat because treatment with INF increased the concentration of fat in milk (3.54 ± 0.10 vs. 3.84 ± 0.10%). Induced endometrial inflammation reduced pregnancy per AI at all inseminations (33.4 ± 5.1 vs. 21.6 ± 3.7%) and the hazard of pregnancy (0.61; 95% CI = 0.36–1.04), which extended the median days open by 24 d. Blood leukocytes from INF cows had increased mRNA expression of the pro-inflammatory gene IL1B on d 2 and 7 after treatment, but reduced expression of the IFN-stimulated genes ISG15 and MX2 on d 19 after AI. Induced endometrial inflammation depressed production and caused long-term negative effects on reproduction in lactating dairy cows.     

Supplementation with N-carbamoylglutamate during the transition period improves the function of neutrophils and reduces inflammation and oxidative stress in dairy cows

The aim of this study was to investigate the effects of N-carbamoylglutamate (NCG) supplementation during the transition period on the functions of blood polymorphonuclear neutrophils (PMN), inflammation, and oxidative stress in dairy cows. Thirty multiparous Chinese Holstein dairy cows at wk 4 before parturition were blocked into 2 groups by parity, body weight, and milk yield of previous lactation, and randomly allocated to 2 dietary treatments of basal diet supplemented without (control, n = 15) or with 20 g/d per cow of NCG (NCG, n = 15). The supplementation was carried out from d ?21 to 21 relative to calving. Health incidents (mastitis, retained placenta, and lameness) were recorded, and blood samples were collected at d ?21, ?7, 0 (the calving date), 7, and 21 relative to parturition and analyzed for variables related to inflammation and oxidative stress. In addition, whole blood was collected at d 7 to isolate PMN and used for analysis of the expression of functional genes and from d ?21 to 21 for determination of weekly hematological parameters. The number of lymphocytes was greater at d 7 in the blood of NCG cows. The plasma level of malondialdehyde was lower in the NCG group, and blood reactive oxygen species were lower at d 7, whereas total antioxidant capacity tended to be greater in the NCG group and glutathione peroxidase tended to be higher at d 21 in cows fed NCG, suggesting that NCG supplementation improved antioxidation in cows. In addition, the concentration of serum amyloid A was lower in NCG-fed animals during the postpartum stage. Blood concentrations of IL6 and tumor necrosis factor-α were lower and tended to be lower in NCG-fed animals at d 7, respectively. Meanwhile, the concentrations of IL6 tended to be lower in NCG-fed animals at d 21. Furthermore, the expression of S100A9 and MMP9 in the PMN was lower and tended to be lower, respectively, whereas the expression of ITGB2, XBP1 tended to be higher and expression of CLEC6A was higher in NCG-fed cows. Overall, our results indicated that supplementation with NCG during the transition period showed the beneficial effects on animal health, by improving PMN functions and alleviating inflammation status and oxidative stress in dairy cows.     

Rumen-protected B vitamin complex supplementation during the transition period and early lactation alters endometrium mRNA expression on day 14 of gestation in lactating dairy cows

Greater metabolic demands in high-producing dairy cows are believed to be a cause of sub-fertility in these animals. Previously, supplementation with vitamin B complex molecules has shown benefits in improving milk production, health, and reproductive efficiency of dairy cows. The primary aim of this project was to determine the effects of rumen-protected vitamin B complex supplementation of 100 g of Transition VB (Jefo, St. Hyacinthe, QC, Canada) and 4 g of Lactation VB (VB; Jefo), during the transition and early lactation periods, respectively, compared with a control diet containing no supplementation on d 14 endometrial outcomes of pregnancy. In the vitamin B supplemented cows, we expect to see a change in the mark-up of endometrial genes important for embryo survival before implantation. Multiparous Holstein cows were enrolled into the study 3 wk before parturition and were randomly assigned to either the VB or control treatment. Twice-a-week blood samples, weekly milk samples, and daily feed intake were collected. Cows were enrolled onto a double-ovsynch protocol at 33 ± 3 d postpartum and inseminated by timed artificial insemination. Milk production and components, concentrations of BHB, haptoglobin, and progesterone in serum, and ovarian dynamics were also measured, but no treatment effect was observed. The uterus was flushed on d 14 after artificial insemination (around 72 DIM) for conceptus collection, and endometrial samples were collected at the same time. Overall, 42 cows were flushed and 13 embryos were collected. Analysis of mRNA expression of genes related to embryo development, immune system, adhesion, and regulation of vitamin B molecules showed that OXTR, MUC5B, MUC1, IL1B, SPP, TRD, FZD8, and FOLR1 genes were significantly upregulated in the VB group. Vitamin B supplementation had no effect on the size of the embryo and ovulatory follicle or corpus luteum diameter at embryo collection. In conclusion, the benefits of strategic dietary VB supplementation during the transition and early lactation might be directly linked to endometrial functions required for embryo survival during the peri-implantation period.     

Identification of potential embryokines in the bovine reproductive tract

Knowledge of the molecules used by the maternal reproductive tract to regulate development of the preimplantation embryo is largely incomplete. The goal of the present experiment was to identify candidates for this function. The approach was to assess expression patterns in the endometrium and oviduct of 93 genes encoding for hormones, growth factors, chemokines, cytokines, and WNT-related molecules. Results show that all of the genes were expressed in the reproductive tract. Expression in oviduct was affected by day of the estrous cycle for 21 genes with 11 genes having highest expression at estrus (CCL21, CTGF, CXCL10, CXCL16, DKK3, FGF10, IL18, IL33, IL34, PGF, and SFRP2), 1 gene at d 3 (WNT4), 8 at d 5 (BMP7, HGF, IL6, SFRP1, TGFB1, WIF1, WNT2, and WNT5A), and 1 at d 7 (IK). For endometrium, expression of 34 genes was affected by day of the estrous cycle with 11 having highest expression at d 0 (BMP7, CCL14, CCL21, CCL26, CTGF, CXCL12, IGF2, IL16, IL33, SFRP2, and WIF1), 2 at d 3 (HDGF, IL15), 14 at d 5 (CSF2, CX3CL1, CXCL3, FGF1, FGF2, GRO1, HGF, IGF1, IL1B, IL8, SFRP1, SFRP4, WNT5A, and WNT16), and 7 at d 7 (CXCL16, FGF13, HDGFRP2, TDGF1, VEGFB, WNT7A, and WNT11). Results are consistent with a set of genes regulated by estradiol early in the estrous cycle and another set regulated by progesterone later in the cycle. The cell-signaling genes identified here as being expressed in the oviduct and endometrium could serve to regulate early embryonic development in a stage-of-pregnancy-specific manner.     

Pegbovigrastim treatment affects gene expression in neutrophils of pasture-fed,periparturient cows

Treatment with granulocyte colony-stimulating factor has been reported to increase circulating neutrophil count and enhance neutrophil function in the periparturient cow. It was hypothesized that a commercially available recombinant bovine granulocyte colony-stimulating factor product (pegbovigrastim) affects gene expression profiles of neutrophils and supports neutrophil function in periparturient cows. Hence this study was undertaken to analyze expression of genes involved in neutrophil functions, including migration, interaction with pathogens, and cell survival. It also assessed the hypothesis that gene expression profiles in neutrophils are modulated by negative energy balance in the peripartum period. Holstein-Friesian, Jersey, and mixed-breed cows on pasture were blocked by expected calving date and body condition score and randomly assigned in a 2 × 2 factorial design. Cows were fed to exceed energy requirements prepartum (122%) or restricted to approximately 85% of prepartum energy requirements. At approximately 7 d before expected calving date, half the cows in each feed group were randomly assigned to be injected with pegbovigrastim or saline. Treatments were repeated within 24 h after calving. Blood samples were collected pretreatment approximately 7 d before calving (d −7). Blood, uterine flush, and milk samples were collected at 4 (d 4) and 7 d in milk (d 7) to measure the expression of a panel of 20 genes representing cell adhesion, pattern recognition, inflammation and cytokine response, antimicrobial capacity, and apoptosis functions in neutrophils using NanoString technology (NanoString Technologies Inc., Seattle, WA) to quantify RNA copy numbers. No effects were observed of prepartum feeding group or a feeding group × treatment interaction for any of the investigated genes. An effect was observed of time on expression of several genes in blood neutrophils. After calving, expression of 2 of 4 cell adhesion-related genes, 3 of 4 pattern recognition receptors, 2 of 4 inflammatory genes, 2 antimicrobial genes, and 2 of 4 cell survival genes was significantly greater at d 4 or 7 or both compared with before calving (d −7). Expression of ICAM1, TLR2, and PTGS2 was significantly higher in blood neutrophils from animals treated with pegbovigrastim compared with untreated controls, suggesting greater migration, pattern recognition, and inflammatory response ability. Pegbovigrastim also affected RNA expression in uterine cells with ICAM1, NOD1, CLEC6A, PTGS2, MPO, DEFB5, and CATHL6 being expressed at higher levels and SELL, ITGB8, IL8RB, and IL10 at lower levels. Milk somatic cells showed a similar pattern but with fewer significant changes. In contrast to the reported decline in neutrophil function in the transition period, neutrophil gene expression was increased for many of the genes studied, an apparent attempt to compensate for reduced neutrophil function. Treatment with pegbovigrastim further increased expression of several genes involved in these processes in blood neutrophils and changed uterine cells to a phenotype with increased antimicrobial capacity, typical for neutrophils that have migrated into their target tissue.     

Fetuin-A: A negative acute-phase protein linked to adipose tissue function in periparturient dairy cows

Fetuin-A (FetA) is a free fatty acid transporter and an acute-phase protein that enhances cellular lipid uptake and lipogenesis. In nonruminants, FetA is involved in lipid-induced inflammation. Despite FetA importance in lipid metabolism and inflammation, its expression and dynamics in adipose tissue (AT) of dairy cows are unknown. The objectives of this study were to (1) determine serum and AT FetA dynamics over the periparturient period and in mid-lactation cows in negative energy balance (NEB) after a feed restriction protocol and (2) characterize how an inflammatory challenge affects adipocyte FetA expression. Blood and subcutaneous AT were collected from 16 cows with high (≥3.75, n = 8) or moderate (≤3.5, n = 8) body condition score (BCS) at ?26 ± 7 d (far off) and ?8 ± 5 d (close up) before calving and at 10 ± 2 d after parturition (early lactation) and from 14 nonpregnant mid-lactation cows (>220 d in milk) after a feed restriction protocol. Serum FetA concentrations were 0.89 ± 0.13 mg/mL at far off, 0.96 ± 0.13 mg/mL at close up, and 0.77 ± 0.13 mg/mL at early lactation and were 1.09 ± 0.09 and 1.17 ± 0.09 mg/mL in feed-restricted and control cows, respectively. Serum and AT FetA contents decreased at the onset of lactation when lipolysis was higher. No changes in AT and serum FetA were observed after feed restriction induced NEB in mid-lactation cows. Prepartum BCS had no effect on serum FetA, but AT expression of AHSG, the gene encoding FetA, was reduced in periparturient cows with high BCS at dry-off throughout all time points. Circulating FetA was positively associated with serum albumin and calcium and with BCS variation over the periparturient period. The dynamics of AHSG expression were analogous to the patterns of lipogenic markers ABDH5, ELOVL6, FABP4, FASN, PPARγ, and SCD1. Expression of AHSG and FetA protein in AT was inversely correlated with AT proinflammatory markers CD68, CD44, SPP1, and CCL2. In vitro, bovine adipocytes challenged with lipopolysaccharide downregulated FetA protein expression. Adipocytes treated with FetA had lower CCL2 expression compared with those exposed to lipopolysaccharide. Overall, FetA is a systemic and local AT negative acute-phase protein linked to AT function in periparturient cows. Furthermore, FetA may support physiological adaptations to NEB in periparturient cows.     

A combination of lactic acid bacteria regulates Escherichia coli infection and inflammation of the bovine endometrium

Uterine function in cattle is compromised by bacterial contamination and inflammation after calving. The objective of this study was to select a combination of lactic acid bacteria (LAB) to decrease endometrium inflammation and Escherichia coli infection. Primary endometrial epithelial cells were cultured in vitro to select the most favorable LAB combination modulating basal tissue inflammation and E. coli infection. Supernatants were obtained to determine expression of pro-inflammatory cytokines, and E. coli infection was evaluated after harvesting the tissue and plate counting. The selected LAB combination was tested in uterus explants to assess its capacity to modulate basal and acute inflammation (associated with E. coli infection). The combination of Lactobacillus rhamnosus, Pediococcus acidilactici, and Lactobacillus reuteri at a ratio of 25:25:2, respectively, reduced E. coli infection in vitro with (89.77%) or without basal tissue inflammation (95.10%) compared with single LAB strains. Lactic acid bacteria treatment reduced CXCL8 and IL1B expression 4.7- and 2.2-fold, respectively, under acute inflammation. Ex vivo, the tested LAB combination reduced acute inflammation under E. coli infection, decreasing IL-8, IL-1β, and IL-6 up to 2.2-, 2.5-, and 2.2-fold, respectively. In the total inflammation model, the LAB combination decreased IL-8 1.6-fold and IL-6 1.2-fold. Ultrastructural evaluation of the tissue suggested no direct interaction between the LAB and E. coli, although pathological effects of E. coli in endometrial cells were greatly diminished or even reversed by the LAB combination. This study shows the promising potential of LAB probiotics for therapeutic use against endometrial inflammation and infection.     

Prevalence,risk factors,and effects on fertility of cytological endometritis at the time of insemination in Norwegian Red cows

The present study aimed to assess the occurrence of cytological endometritis (CYTO), a nonsymptomatic inflammation of the endometrium, at first artificial insemination (AI) postpartum in Norwegian Red cows. Further, risk factors for CYTO manifestation and its effect on reproductive success and late embryo loss were evaluated. In total 1,648 cows located in 116 herds were included in the study. On mainly spontaneous estrus, endometrial cytology samples were collected using a cytotape technique, and a total of 300 representative epithelial cells and polymorphonuclear neutrophils (PMN) were counted at 400× magnification. Vaginal mucus obtained by Metricheck (Simcro) and body condition score were recorded. Milk samples for progesterone analysis were collected at AI and 21 d later. Pregnancy was diagnosed by rectal palpation or analysis of pregnancy-associated glycoproteins. Based on the constructions of a receiver operator characteristics curve, the cut-off level for PMN defined as CYTO was set to 3.0%, representing the level at which the PMN occurrence affected pregnancy outcome, with the highest summation of sensitivity (32.4%) and specificity (74.9%). Three logistic models with herd included as random factor were constructed. The outcome for the first model was the likelihood for CYTO based on the endometrial samples, in the second model pregnancy to first AI, and in the third model embryo loss. The proportion of CYTO was 28.0% (461/1,648). The average interval in days to first AI was 71.7 d (standard error ± 0.7) and the overall pregnancy incidence to first AI was 59.8% (866/1,449). The likelihood for CYTO at first AI was associated with AI personnel, calving to first AI interval, vaginal mucus characteristics, amount of red blood cells in sample, season, and barn type. Pregnancy to first AI was lower in CYTO-positive cows (odds ratio = 1.51, confidence interval = 1.17–1.94). Other factors affecting pregnancy to first AI were AI personnel, test day milk yield, barn type, and obstetrical conditions or fertility treatments before first AI. The proportion of late embryo loss and abortion was 8.6% (82/948) and 2.8% (24/866), respectively. Late embryo loss was associated with treatment against fertility disorders before first AI, but not associated with CYTO. Overall, our results suggest that even if Norwegian Red cows show a fairly high prevalence of CYTO in the endometrium at first AI, it does not seem to have a major effect on the reproductive performance. The Norwegian Red breeding program has emphasized fertility and health for decades, and a genetically advantageous uterine immunology might be one of the preserved mechanisms.     

Hepatic nuclear factor kappa B signaling pathway and NLR family pyrin domain containing 3 inflammasome is over-activated in ketotic dairy cows

Ketosis is an important metabolic disease that can negatively affect the production efficiency of dairy cows. Earlier studies have revealed metabolic and inflammatory alterations in the blood associated with ketosis; however, a link between ketosis and hepatic inflammation has not been well documented. The objective of this study was to investigate whether the nuclear factor kappa B (NF-κB) signaling pathway and NLR family pyrin domain containing 3 (NLRP3) inflammasome were activated in the liver of ketotic cows. Liver and blood samples were collected from healthy (n = 15, control group) and ketotic (n = 15, ketosis group) cows that had a similar number of lactations (median = 3, range = 2 to 4) and days in milk (median = 6 d, range = 3 to 9 d). Results showed that serum levels of fatty acids, β-hydroxybutyrate (BHB), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were higher and glucose was lower in ketotic cows. Concentrations of serum proinflammatory cytokines IL18, tumor necrosis factor (TNF)-α, and IL1B were greater and the anti-inflammatory cytokine IL10 was lower in the ketosis group. Cows with ketosis had triacylglycerol accumulation in the liver. Upregulation of phosphorylated (p)-NF-κB and p-inhibitor of κB (IκB)α protein abundance in cows with ketosis indicated that the hepatic NF-κB signaling pathway was overactivated. The mRNA abundance of TNFA, inducible nitric oxide synthase (NOS2), IL18, and IL1B were greater and IL10 was lower in ketotic cows. More importantly, the mRNA and protein abundance of NLRP3 and caspase-1 (CASP1) along with CASP1 activity were greater in the liver of cows with ketosis. Overall, the data indicate that the onset of ketosis is accompanied by activation of the NF-κB signaling pathway and NLRP3 inflammasome, resulting in a state of inflammation.     

Far-off and close-up dry matter intake modulate indicators of immunometabolic adaptations to lactation in subcutaneous adipose tissue of pasture-based transition dairy cows

The common practice of increasing dietary energy density during the close-up dry period (last ～3 wk prepartum) has been recently associated with a higher incidence of metabolic disorders after calving. Despite these reports, over-feeding of metabolizable energy (ME) during the far-off, nonlactating period is a common management policy aimed at achieving optimum calving body condition score (BCS) in pasture-based systems, as cows are generally thinner than total mixed ration cows at the end of lactation. Our hypothesis was that both far-off and close-up overfeeding influence the peripartum adipose tissue changes associated with energy balance and inflammatory state. Sixty mid-lactation, grazing dairy cows of mixed age and breed were randomly allocated to 1 of 2 groups that were managed through late lactation to achieve a low and high BCS (approximately 4.25 and 5.0 on a 10-point scale) at dry-off. The low BCS cows were then overfed ME to ensure that they achieved the same BCS as the higher BCS group by calving. Within each rate of BCS gain treatment, cows were offered 65, 90, or 120% of their pre-calving ME requirements for 3 wk pre-calving in a 2 × 3 factorial arrangement of treatments (i.e., 10 cows/treatment). Subcutaneous adipose tissue was collected via biopsy at ?1, 1, and 4 wk relative to parturition. Quantitative PCR was used to measure mRNA and microRNA expression of targets related to adipogenesis and inflammation. Cows overfed in the far-off period had increased expression of miR-143 and miR-378 prepartum (?1 wk) indicating greater adipogenesis, consistent with their rapid gain in BCS following dry-off. Furthermore, the lower postpartum expression of IL6, TNF, TLR4, TLR9, and miR-145, and a higher abundance of miR-99a indicated lower body fat mobilization in early lactation in the same group. In the close-up period, feeding either 65 or 120% of ME requirements caused changes in FASN, IL1B, IL6R, TLR9, and the microRNA miR-143, miR-155, and miR-378. Their respective expression patterns indicate a tentative negative-feedback mechanism in metabolically compromised, feed-restricted cows, and a possible immune-related stimulation of lipolysis in apparently static adipocytes in overfed cows. Data from cows fed 90% of ME requirements indicate the existence of a balance between lipolytic (inflammatory-related) and anti-lipolytic signals, to prime the mobilization machinery in light of imminent lactation. Overall, results indicate that far-off dry cow nutrition influences peripartum adipose tissue metabolism, with neither strategy negatively affecting the physiological adaptation to lactation. Furthermore, to ensure a favorable transition, cows should be subjected to a small feed restriction in the close-up period, irrespective of far-off nutritional management.     

Free fatty acids promote degranulation of azurophil granules in neutrophils by inducing production of NADPH oxidase–derived reactive oxygen species in cows with subclinical ketosis

Subclinical ketosis (SCK) in dairy cows, a common metabolic disorder during the peripartal period, is accompanied by systemic inflammation. Excessive release of azurophil granule (AG) contents during degranulation of polymorphonuclear neutrophils (PMN) could contribute to systemic inflammation in SCK cows. Although the increase in blood free fatty acids (FFA) in SCK cows may promote AG degranulation from PMN, the underlying mechanisms are unclear. Thirty multiparous cows (within 3 wk postpartum) with similar lactation numbers (median = 3, range = 2–4) and days in milk (median = 6, range = 3–15) were classified based on serum β-hydroxybutyrate (BHB) level as control (n = 15, BHB < 0.6 mM) or SCK (n = 15, 1.2 mM < BHB < 3.0 mM). Cows with SCK had greater levels of serum haptoglobin, serum amyloid A, IL-1β, IL-6, IL-8 and tumor necrosis factor-α. These proinflammatory factors had strong positive correlations with myeloperoxidase (MPO), a marker protein of PMN AG, whose content was greater in the serum of SCK cows. Both the number of AG and the protein abundance of MPO were lower in PMN isolated from SCK cows. Additionally, we found a greater ratio of blood CH138A⁺/CD63^high cells and greater mean fluorescence intensity of CD63 on the PMN membrane, further confirming the greater degree of AG degranulation in cows with SCK. In vitro FFA dose response (0, 0.3, 0.6, 1.2, and 2.4 mM for 4 h) and time course (0, 0.5, 1, 2, and 4 h with 0.6 mM) experiments were performed on PMN isolated from control cows. The increase in MPO content in extracellular supernatant resulting from those experiments led to the selection of 0.6 mM FFA for 1 h duration as conditions for subsequent studies. After FFA treatment, release of intracellular MPO was increased along with increased levels of CD63 mean fluorescence intensity on the PMN membrane, confirming that FFA promoted degranulation of AG. In addition, FFA treatment increased reactive oxygen species (ROS) production by PMN, an effect that was attenuated by incubation with diphenyleneiodonium chloride (DPI), a NADPH oxidase-derived ROS inhibitor. The mitochondrial-derived ROS inhibitor carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) did not affect ROS in response to FFA treatment. Treatment with FFA increased p47 phosphorylation and mRNA abundance of NCF1, NCF2, and CYBB in PMN. Furthermore, DPI, but not FCCP, dampened the degranulation of PMN AG induced by FFA in vitro. These data suggested that the degranulation of AG in PMN induced by FFA was mediated by NADPH oxidase-derived ROS. As verified ex vivo, PMN from SCK cows had greater levels of ROS, phosphorylation of p47, and mRNA abundance of NCF1, NCF2, and CYBB. Overall, the present study revealed that high blood concentrations of FFA in SCK cows induce the production of NADPH oxidase-derived ROS, thereby promoting degranulation of AG in PMN. The stimulatory effect of FFA on the release of AG content during degranulation, especially MPO, provides a new insight into the systemic inflammation experienced by peripartal cows with SCK.     

Impaired hepatic autophagic activity in dairy cows with severe fatty liver is associated with inflammation and reduced liver function

The ability of liver to respond to changes in nutrient availability is essential for the maintenance of metabolic homeostasis. Autophagy encompasses mechanisms of cell survival, including capturing, degrading, and recycling of intracellular proteins and organelles in lysosomes. During negative nutrient status, autophagy provides substrates to sustain cellular metabolism and hence, tissue function. Severe negative energy balance in dairy cows is associated with fatty liver. The aim of this study was to investigate the hepatic autophagy status in dairy cows with severe fatty liver and to determine associations with biomarkers of liver function and inflammation. Liver and blood samples were collected from multiparous cows diagnosed as clinically healthy (n = 15) or with severe fatty liver (n = 15) at 3 to 9 d in milk. Liver tissue was biopsied by needle puncture, and serum samples were collected on 3 consecutive days via jugular venipuncture. Concentrations of free fatty acids and β-hydroxybutyrate were greater in cows with severe fatty liver. Milk production, dry matter intake, and concentration of glucose were all lower in cows with severe fatty liver. Activities of serum aspartate aminotransferase, alanine aminotransferase, glutamate dehydrogenase, and γ-glutamyl transferase were all greater in cows with severe fatty liver. Serum concentrations of haptoglobin and serum amyloid A were also markedly greater in cows with severe fatty liver. The mRNA expression of autophagosome formation-related gene ULK1 was lower in the liver of dairy cows with severe fatty liver. However, the expression of other autophagosome formation-related genes, beclin 1 (BECN1), phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3), autophagy-related gene (ATG) 3, ATG5, and ATG12, did not differ. More important, ubiquitinated proteins, protein expression of sequestosome-1 (SQSTM1, also called p62), and microtubule-associated protein 1 light chain 3 (MAP1LC3, also called LC3)-II was greater in cows with severe fatty liver. Transmission electron microscopy revealed an increased number of autophagosomes in the liver of dairy cows with severe fatty liver. Taken together, these results indicate that excessive lipid infiltration of the liver impairs autophagic activity that may lead to cellular damage and inflammation.     

CRISPR-Cas9–mediated knockout of TLR4 modulates Mycobacterium avium ssp. paratuberculosis cell lysate–induced inflammation in bovine mammary epithelial cells

Toll-like receptor 4 (TLR4) is a pattern-recognition receptor involved in the recognition of microbial pathogens and host alarmins. Ligation to TLR4 initiates a signaling cascade that leads to inflammation. Polymorphisms in bovine TLR4 have been associated with Mycobacterium avium ssp. paratuberculosis (MAP) susceptibility and resistance, the cause of Johne's disease, and milk somatic cell score, a biomarker of mastitis. Although the contribution of TLR4 to recognition of bacterial lipopolysaccharide (LPS) has been well characterized, its role in MAP recognition is less certain. Clustered regularly interspaced short palindromic repeats–Cas9 mediated gene editing was performed to generate TLR4 knockout (KO) mammary epithelial cells to determine if TLR4 expression is involved in the initiation of the host inflammatory response to MAP cell lysate (5 and 10 µg/mL) and Escherichia coli LPS (5 µg/mL). The absence of TLR4 in KO cells resulted in enhanced expression of key inflammatory genes (TNFA and IL6), anti-inflammatory genes (IL10 and SOCS3), and supernatant cytokine and chemokine levels (TNF-α, IL-6, IL-10, CCL3) in response to the MAP cell lysate (10 µg/mL). However, in response to LPS, the KO cells showed reduced expression of key inflammatory genes (TNFA, IL1A, IL1B, and IL6) and supernatant cytokine levels (TNF-α, IL-6, CCL2, IL-8) as compared with unedited cells. Overall, these results confirm that TLR4 is essential for eliciting inflammation in response to LPS; however, exacerbated gene and protein expression in TLR4 KO cells in response to MAP cell lysate suggests a different mechanism of infection and host response for MAP, at least in terms of how it interacts with TLR4. These novel findings show potential divergent roles for TLR4 in mycobacterial infections, and this may have important consequences for the therapeutic control of inflammation in cattle.     

Changes in gene expression in the rumen and colon epithelia during the dry period through lactation of dairy cows and effects of live yeast supplementation

The objectives of this study were (1) to use endoscopy to collect biopsies from the rumen and colon epithelia to describe changes in gene expression in these 2 tissues as cows move from a dry to a lactation ration and (2) to evaluate the potential influence that supplementation of live yeast could exert on these 2 epithelia. Twenty-one Holstein cows were split into 2 treatments and received either 300 g/d of corn containing 1 × 10¹⁰ cfu/d of live yeast (LY; n = 10) or 300 g/d of corn with no supplementation (control; n = 11) starting 21 ± 2.6 d (average ± SD) before until 21 d after calving. At 14 ± 2.6 d before the expected calving date, and exactly at 7 and 21 d after calving, rumen and colon biopsies were obtained from each cow using an endoscope. Total RNA was extracted from rumen and colon tissues, and the expression of IL10, TNFA, TLR4, IL1B, PCNA, MKI67, SGLT1, BAX, CASP3, OCLN, CLDN4, HSPA1A, HSPB1, DEFB1, and MCT1 (the latter only in rumen samples) was quantified by quantitative PCR. Overall, fluctuations in expression of the selected genes in the colon between the 2 stages of production and the 2 treatments were smaller than those found in the rumen. In the rumen epithelium, expression of TLR4 and DEFB1 was greatest before calving, with LY cows having a greater expression of TLR4 than control cows. Similarly, expression of IL10 was greatest in LY cows before calving. Expression of TNFA in the rumen epithelium of control cows was lowest at 21 DIM but in LY cows was kept steady among production stages. The expression of PCNA and MKI67 in the rumen epithelium was greatest at 7 DIM, indicating a high proliferation rate of this epithelium after calving. In the colon mucosa, expression of TLR4 and DEFB1 was greater than in the rumen, and DEFB1 expression was greater in LY cows than in control cows. The use of an endoscope allowed us to study the dynamics of rumen epithelium adaptation to increased supply of concentrate after calving, consisting of increased epithelia remodeling, reduction of the TLR4, and increased IL10 expression. Furthermore, the rumen epithelium of dry cows responded rapidly to live yeast, with changes in the expression of genes involved in the immune response becoming evident after 7 d of exposure to yeast. The expression of genes related to the immune response (mainly TLR4 and DEFB1) in the colon mucosa was greater than in the rumen, and the expression of DEFB1 was further stimulated by live yeast. It is concluded that the use of an endoscope allows the study of gene expression patterns in the rumen and hindgut epithelia. We report marked changes in the rumen wall and more modest changes in the colon when transitioning from a dry to a lactation ration. Furthermore, supplementation of live yeast fostered and increased expression of genes regulating inflammation and epithelial barrier in the rumen, and in the colon it increased the expression of DFEB1 coding for an antimicrobial peptide.     

Hot topic: Pregnancy-induced expression of interferon-stimulated genes in the cervical and vaginal mucosal membranes

In ruminants, IFN-tau (IFNT) is a pregnancy recognition signal secreted by the embryonic trophectoderm before implantation, and it induces the expression of IFN-stimulated genes (ISG) in the uterine endometrium and blood leukocytes. The expression of ISG in blood leukocytes could indicate the presence of a viable conceptus before return of the next estrus; however, expression levels have high variation for confirming pregnancy. We hypothesized that the secreted IFNT in the uterus would affect ISG expression in cervical and vaginal tissues because they are directly adjacent to the uterus. To prove the hypothesis, we investigated the expression of 3 ISG (ISG15, MX1, and MX2) in cervical and vaginal mucosal membranes collected from pregnant (n = 12) and nonpregnant (n = 11) lactating Holstein cows at 17 to 18 d after artificial insemination. Mucosal membrane samples of the cervical canal near the external os (cervix) and deep vaginal wall surrounding the external os (vagina) were collected separately by simply scraping with a curette on d 17 or 18 of pregnancy (d 1 = ovulation), at which time IFNT secretion into the maternal uterus is maximal. After pregnancy diagnosis on d 30 and 60, separately collected samples confirmed as pregnant and nonpregnant were used for evaluation of the expression of IFN-stimulated protein 15 kDa (ISG15) and myxovirus-resistance protein 1 and 2 (MX1, MX2) with quantitative real-time PCR. The collected mucosal membrane samples from cervix contained mostly cell clots showing membrane structure and a low content of blood cells. The expression levels of all 3 genes were significantly increased in pregnant cows compared with nonpregnant cows in both cervical and vaginal samples. These results suggest that increased expression of ISG in the cervix and vagina is a pregnancy-associated phenomenon and is highly affected by IFNT secreted from the conceptus through the uterus.