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ABSTRACT: The objective was to unravel the peripartum immune and metabolic changes associated with metritis in Holstein cows. Holstein cows (n = 128) had blood collected at −14, 0, 3, and 7 d relative to parturition (DRP). Flow cytometry was used to evaluate blood leukocyte counts, proportions, and activation. Total cells, live cells, single cells, monocytes (CD172α+/CD14+), polymorphonuclears (CD172α+/CD14-/SSChigh), B-cells (CD21+/MHCII+), CD4+ T-cells (CD4+), CD8+ T-cells (CD8+), and γδ T-cells (γδTCR+) were evaluated. Both CD62L and CD11b were used as markers of cell activation. Major histocompatibility complex class II was used as a marker of antigen presentation in monocytes. A Milliplex Bovine Cytokine/Chemokine 08-plex kit was used to evaluate plasma concentrations of IFN-γ, IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, and tumor necrosis factor-α. The body weight (BW) change prepartum was calculated as the difference between calving BW and prepartum BW divided by the number of days between measurements. Plasma fatty acids (FA) were measured at −14 and 0 DRP using untargeted gas chromatography with time-of-flight mass spectrometry. Data were analyzed by ANOVA for repeated measures. Cows that developed metritis (n = 57) had greater prepartum BW, prepartum BW loss, and greater FA concentrations at calving. Plasma FA at calving was positively correlated with IL-1β. Cows that developed metritis had persistent systemic inflammation, which was demonstrated by greater B-cell activation, greater pro-inflammatory cytokine concentrations, and greater cell damage pre- and postpartum. Postpartum, we observed greater polymorphonuclear cell activation and extravasation but lesser monocytes and CD4+ T-cells activation and extravasation, which suggests postpartum immune tolerance. Greater prepartum adiposity in cows that developed metritis may lead to systemic inflammation pre- and postpartum and immune tolerance postpartum, which may lead to failure to prevent bacterial infection, and development of puerperal metritis.

ABSTRACT: Objectives were to determine the effects of supplementing rumen-protected choline (RPC) on hepatic composition and secretion of triacylglycerol-rich lipoprotein when cows were subjected to feed restriction to develop fatty liver. It was hypothesized that RPC reduces hepatic triacylglycerol by enhancing secretion of hepatic lipoprotein. Pregnant, nonlactating parous Holstein cows (n = 33) at mean (± standard deviation) 234 ± 2.2 d of gestation were blocked by body condition (3.79 ± 0.49) and assigned to receive 0 g/d (CON), 25.8 g/d choline ion from a RPC product containing 28.8% choline chloride (CC; treatment L25.8), or 25.8 g/d of choline ion from a RPC product containing 60.0% CC (H25.8). Cows were fed for ad libitum intake for the first 5 d and restricted to 41% of the net energy for lactation required for maintenance and pregnancy from d 6 to 13. Intake of metabolizable methionine was maintained at 18 g/d during feed restriction by supplying rumen-protected methionine. Hepatic tissue was sampled on d 6 and 13 and analyzed for triacylglycerol and glycogen, and mRNA expression of hepatic tissue was investigated. On d 14, cows were not fed and received a 10% solution of tyloxapol intravenously at 120 mg/kg of body weight to block hydrolysis of triacylglycerols in very low density lipoprotein (VLDL). Blood was sampled sequentially for 720 min and analyzed for concentration of triacylglycerol and total cholesterol. Lymph was sampled 6 h after tyloxapol infusion, and analyzed for concentrations of fatty acids, β-hydroxybutyrate, glucose, triacylglycerol, and total cholesterol. A sample of serum collected at 720 min after tyloxapol was assayed for the metabolome composition. The area under the curve (AUC) of serum triacylglycerol, VLDL cholesterol, and total cholesterol were calculated. Orthogonal contrasts evaluated the effect of supplementing RPC (CON vs. [1/2 L25.8 + 1/2 H25.8]) and source of RPC (L25.8 vs. H25.8). Least squares means and standard errors of the means are presented in sequence as CON, L25.8, H25.8. During feed restriction, supplementation of RPC reduced hepatic triacylglycerol (9.0 vs. 4.1 vs. 4.5 ± 0.6%) and increased glycogen contents (1.9 vs. 3.5 vs. 4.1 ± 0.2%). Similarly, supplementation of RPC increased the expression of transcripts involved in the synthesis and assembly of lipoproteins (MTTP), cellular autophagy (ATG3), and inflammation (TNFA), and reduced the expression of transcripts associated with mitochondrial oxidation of fatty acids (HADHA, MLYCD) and stabilization of lipid droplets (PLIN2). After infusion of tyloxapol, RPC increased the AUC for serum triacylglycerol (21,741 vs. 32,323 vs. 28,699 ± 3,706 mg/dL × min) and VLDL cholesterol (4,348 vs. 6,465 vs. 5,740 ± 741 mg/dL × min) but tended to reduce the concentrations of triacylglycerol in lymph (16.7 vs. 13.8 vs. 11.9 ± 1.9 mg/dL). Feeding RPC tended to increase the concentrations of 89 metabolites in serum, after adjusting for false discovery, including 3 acylcarnitines, 1 AA-related metabolite, 11 bile acids, 1 ceramide, 6 diacylglycerols, 2 dihydroceramides, 1 glycerophospholipid, and 64 triacylglycerols compared with CON. Feeding 25.8 g/d of choline ion as RPC mediated increased hepatic triacylglycerol secretion to promote lipotropic effects that reduced hepatic lipidosis in dairy cows.

ABSTRACT: The objective of this study was to identify metabolites associated with metritis and use them for identification of cellular mechanisms affected during transition into lactation. Holstein cows (n = 104) had blood collected in the prepartum period (d −14 ± 6 relative to calving), at calving (d 0), and at the day of metritis diagnosis (d 7 ± 2 after calving). Cows with reddish or brownish, watery, and fetid discharge were diagnosed with metritis (n = 52). Cows with metritis were paired with herdmates without metritis (n = 52) based on days in milk. The metabolome of plasma samples was evaluated using untargeted gas chromatography time-of-flight mass spectrometry. Univariate analyses included t-tests and fold change analyses. Metabolites with false discovery rate adjusted P ≤ 0.10 on t-tests were used for partial least squares discriminant analysis coupled with permutational analysis using 2,000 permutations. Metabolites with false discovery rate adjusted P ≤ 0.10 on t-tests were also used for enriched pathway analyses and identification of cellular processes. Cows that developed metritis had affected cellular processes associated with lower amino acid metabolism in the prepartum period, greater lipolysis, cell death, and oxidative stress at calving and at metritis diagnosis, and greater leukocyte activation at calving, but lower immune cell activation at metritis diagnosis. In summary, cows that developed metritis had plasma metabolomic changes associated with greater lipolysis, oxidative stress, and a dysregulated immune response which may predispose cows to metritis development.

ABSTRACT: 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.

Intramammary 25-hydroxyvitamin D3 (25D) and 1,25-dihydroxyvitamin D3 (1,25D) treatments stimulate immune defenses of the mammary gland. We hypothesized 25D treatment, in contrast to 1,25D, would exert activity in the mammary gland without affecting serum calcium. The objective was to determine the effect of dose and source of intramammary vitamin D treatments on milk somatic cell gene expression and serum calcium. Twenty lactating Holstein cows with somatic cell count

ABSTRACT: The objectives of this experiment were to determine the effects of supplementing 25-hydroxyvitamin D3 (calcidiol, CAL) compared with vitamin D3 (cholecalciferol, CHOL) at 1 or 3 mg/d in late gestation on production outcomes of dairy cows. One hundred thirty-three parous and 44 nulliparous pregnant Holstein cows were enrolled in the experiment. Cows were blocked by parity and previous lactation milk yield (parous) or genetic merit (nulliparous) and assigned randomly to receive 1 or 3 mg/d of CAL or CHOL in a 2 × 2 factorial arrangement of treatments (CAL1, CAL3, CHOL1, and CHOL3). Treatments were provided to individual cows as a top-dress to the prepartum diet from 250 d in gestation until parturition. The prepartum diet had a dietary cation-anion difference of −128 mEq/kg of dry matter. Production and disease were evaluated for the first 42 d in milk, and reproduction was evaluated to 300 d in milk. Incidence of postpartum diseases did not differ among treatments. Feeding CAL compared with CHOL increased yields of colostrum and colostrum fat, protein, and total solids, resulting in an increased amount of net energy for lactation secreted as colostrum (CHOL = 7.0 vs. CAL = 9.0 ± 0.7 Mcal). An interaction between source and amount was observed for milk yield: CAL3 increased milk yield compared with CHOL3 (CHOL3 = 34.1 vs. CAL3 = 38.7 ± 1.4 kg/d) but milk yield did not differ between CAL1 and CHOL1 (CHOL1 = 36.9 vs. CAL1 = 36.4 ± 1.4 kg/d). Concentrations of serum calcidiol on day of calving and average serum Ca from d 2 to 11 postpartum were positively associated with milk yield in the first 42 d in milk. Interactions between source and amount of vitamin D were also observed for pregnancy after first AI: the percentage of cows receiving CHOL1 and CAL3 that became pregnant was smaller than that of cows receiving CHOL3 and CAL1. However, pregnancy per AI and pregnancy by 300 d in milk did not differ among treatments. Overall, CAL3 increased milk yield compared with CHOL3, whereas in cows fed 1 mg/d (CAL1 and CHOL1), the source of vitamin D generally had no effect. The effect of CAL3 may be explained in part by serum CAL concentrations and postpartum serum Ca, which were associated with milk yield.

ABSTRACT: The objectives were to test the effects of dietary vitamin D3 [cholecalciferol (CHOL)] compared with 25-hydroxyvitamin D3 [calcidiol (CAL)] on vitamin D status and response to an endotoxin challenge. Forty-five Holstein bull calves (5 ± 2 d of age) were blocked into weekly cohorts, fed a basal diet that provided 0.25 µg/kg body weight (BW) CHOL, and assigned randomly to 1 of 5 treatments: control [(CON) no additional vitamin D], 1.5 µg/kg BW CHOL (CHOL1.5), 3 µg/kg BW CHOL (CHOL3), 1.5 µg/kg BW CAL (CAL1.5), or 3 µg/kg BW CAL (CAL3). Calves were fed milk replacer until weaning at 56 d of age and had ad libitum access to water and starter grain throughout the experiment. Treatments were added daily to the diet of milk replacer until weaning and starter grain after weaning. Measures of growth, dry matter intake, and serum concentrations of vitamin D, Ca, Mg, and P were collected from 0 to 91 d of the experiment. At 91 d of the experiment, calves received an intravenous injection of 0.1 µg/kg BW lipopolysaccharide (LPS). Clinical and physiological responses were measured from 0 to 72 h relative to LPS injection. Data were analyzed with mixed models that included fixed effects of treatment and time, and random effect of block. Orthogonal contrasts evaluated the effects of (1) source (CAL vs. CHOL), (2) dose (1.5 vs. 3.0 µg/kg BW), (3) interaction between source and dose, and (4) supplementation (CON vs. all other treatments) of vitamin D. From 21 to 91 d of the experiment, mean BW of supplemented calves was less compared with CON calves, but the effect was predominantly a result of the CHOL calves, which tended to weigh less than the CAL calves. Supplementing vitamin D increased concentrations of 25-hydroxyvitamin D in serum compared with CON, but the increment from increasing the dose from 1.5 to 3.0 µg/kg BW was greater for CAL compared with CHOL (CON = 18.9, CHOL = 24.7 and 29.6, CAL = 35.6 and 65.7 ± 3.2 ng/mL, respectively). Feeding CAL also increased serum Ca and P compared with CHOL. An interaction between source and dose of treatment was observed for rectal temperature and derivatives of reactive metabolites after LPS challenge because calves receiving CHOL3 and CAL1.5 had lower rectal temperatures and plasma derivatives of reactive metabolites compared with calves receiving CHOL1.5 and CAL3. Supplementing vitamin D increased plasma P concentrations post-LPS challenge compared with CON, but plasma concentrations of Ca, Mg, fatty acids, glucose, β-hydroxybutyrate, haptoglobin, tumor necrosis factor-α, and antioxidant potential did not differ among treatments post-LPS challenge. Last, supplementing vitamin D increased granulocytes as a percentage of blood leukocytes post-LPS challenge compared with CON. Supplementing CAL as a source of vitamin D to dairy calves was more effective at increasing serum 25-hydroxyvitamin D, Ca, and P concentrations compared with feeding CHOL. Supplemental source and dose of vitamin D also influenced responses to the LPS challenge.

ABSTRACT: The objectives of the experiment were to determine the effects of supplementing 2 amounts of 25-hydroxyvitamin D3 (calcidiol; CAL) compared with equal amounts of vitamin D3 (cholecalciferol; CHOL) on serum concentrations, absorptions, and retentions of Ca, Mg, and P in periparturient dairy cows. One hundred seventy-seven (133 parous and 44 nulliparous) pregnant Holstein cows were enrolled in the experiment. Cows were blocked by parity and previous lactation milk yield (parous) or genetic merit for energy-corrected milk yield (nulliparous) and assigned randomly to receive 1 or 3 mg/d of CAL or CHOL in a 2 × 2 factorial arrangement of treatments. Treatments were provided to individual cows as a top-dress to the prepartum diet from 250 d gestation until parturition. The prepartum diet had a dietary cation-anion difference of −128 mEq/kg of dry matter. All cows were fed a common postpartum diet containing 46 μg of vitamin D3/kg of dry matter without further supplementation of treatments. Concentrations of vitamin D metabolites, Ca, Mg, and P in serum were measured pre- and postpartum, in addition to total-tract digestibility and urinary excretion of Ca, Mg, and P in the prepartum period. Feeding 3 mg compared with 1 mg of CAL increased serum 25-hydroxyvitamin D3 (CAL1 = 94 vs. CAL3 = 173 ± 3 ng/mL). In comparison, the increment in serum 25-hydroxyvitamin D3 from feeding 3 mg compared with 1 mg of CHOL was small (CHOL1 = 58 vs. CHOL3 = 64 ± 3 ng/mL). Feeding CAL increased prepartum concentration of P in serum compared with CHOL (CHOL = 1.87 vs. CAL = 2.01 ± 0.02 mM), regardless of the amount fed, but neither source nor amount affected prepartum Ca or Mg in serum. Feeding CAL increased serum Ca and P for the first 11 d postpartum compared with CHOL (CHOL = 2.12 vs. CAL = 2.16 ± 0.01 mM serum Ca; CHOL = 1.70 vs. CAL = 1.78 ± 0.02 mM serum P) but the amount of vitamin D did not affect postpartum concentrations of Ca, Mg, and P in serum. Feeding CAL increased prepartum apparent digestibility of Ca compared with CHOL (CHOL = 26.6 vs. CAL = 33.5 ± 2.8%) but treatments did not affect Ca retention prepartum. Neither source nor amount of vitamin D affected Mg and P apparent digestibility, but CAL decreased the concentration of P excreted in urine during the prepartum period (CHOL = 1.8 vs. CAL = 0.8 ± 0.3 g/d). Calcidiol tended to increase the amount of Ca secreted in colostrum (CHOL = 9.1 vs. CAL = 11.2 ± 0.9 g/d) and Ca excreted in urine postpartum (CHOL = 0.4 vs. CAL = 0.6 ± 0.1 g/d) compared with CHOL. Collectively, feeding CAL at 1 or 3 mg/d compared with CHOL in the last 24 d of gestation is an effective way to increase periparturient serum P concentration and postpartum serum Ca of dairy cows fed a prepartum diet with negative DCAD.

ABSTRACT: The objectives were to investigate whether supplementation with rumen-protected choline (RPC) during late pregnancy in Holstein cows affects offspring immunity and growth, and whether effects are utero-placental, colostrum dependent, or both. A total of 105 multiparous Holstein cows were assigned randomly to a prepartum diet (1.54 Mcal of NEL/kg of DM, and 15.8% CP) without (control) or with added RPC (12.9 g/d of choline ion). Calves (n = 111) were blocked by sex and assigned randomly to colostrum from control cows or colostrum from RPC cows, resulting in 4 treatments in a 2 × 2 factorial arrangement: (1) calves born and fed colostrum from non-supplemented dams (NN; n = 33); (2) calves from non-supplemented dams and fed colostrum from RPC-fed cows (NC; n = 25); (3) calves from RPC-supplemented dams and colostrum from non-supplemented cows (CN; n = 28); and (4) calves from RPC-supplemented dams and colostrum from RPC-fed cows (CC; n = 25). Growth, intakes, and immunity of females were evaluated up to 56 d of age. Growth and intake of male calves was evaluated up to 35 d of age, and physiological and immune responses to intravenous LPS challenge were evaluated from 21 to 35 d of age. Effects of prenatal and colostrum treatments and interactions between treatments were analyzed using mixed models. Calves fed colostrum from RPC-supplemented dams had a 17.4% increase in apparent efficiency of absorption of IgG compared with calves fed colostrum from control dams (27.4 vs. 23.3%). Incidence of fever in the first 21 d of age tended to be less in females born from RPC-supplemented dams compared with females born from control dams (31 vs. 58%). Prenatal RPC females had increased hematocrit and concentrations of red blood cells, leukocytes, neutrophils, and lymphocytes in blood compared with prenatal females born from control dams. Compared with prenatal control females, prenatal RPC females had greater intake of milk replacer (704 vs. 748 ± 9.9 g/d) and starter (45.4 vs. 60.2 ± 5.9 g/d) during the first 21 d of age. In male calves, mean intake of DM was greater (1,074 vs. 976 ± 45 g/d) after the LPS challenge (0 to 8 d) by calves born from dams fed RPC compared with males born from control dams. Calves born from RPC-fed dams also had lower mean rectal temperature (39.0 vs. 39.2°C) and mean respiration rate (35.6 vs. 39.3 breaths/min) compared with males born from control dams. Moreover, serum concentrations of metabolites (i.e., β-hydroxybutyric acid, fatty acids, and glucose), cytokines (i.e., tumor necrosis factor-α) and acute phase proteins (i.e., serum amyloid A) were consistent with less-severe inflammatory response to LPS in males born from dams fed RPC compared with control. Source of colostrum and interaction between prenatal and colostrum treatments had minimal effects on calf responses to LPS. Overall, maternal RPC supplementation during late gestation suggests a positive effect on immunity, in that colostrum from RPC-fed dams increased efficiency of IgG absorption and maternal supplementation with RPC during late gestation, regardless of colostrum source, attenuated responses to LPS.