Effect of prepartum energy balance on neutrophil function following pegbovigrastim treatment in periparturient cows

Treatment with granulocyte colony-stimulating factor (G-CSF) increases polymorphonuclear cell (neutrophil) count and enhances neutrophil function in the periparturient cow. Prepartum undernutrition was hypothesized to reduce the effect of a commercially available recombinant bovine G-CSF product (pegbovigrastim) on neutrophil count and function. Hence this study was undertaken to test the effect of undernutrition for approximately 1 mo before calving on the innate immune response to pegbovigrastim. Cows (n = 99) on pasture were blocked by expected calving date and body condition score and randomly assigned in a 2 × 2 factorial design. The first factor was that cows were fed to exceed energy requirements prepartum (full feeding) or restricted to approximately 85% of prepartum energy requirements (restricted feeding). The second factor was that at approximately 7 d before expected calving date, half the cows in each feed group were injected with pegbovigrastim and the remaining half were injected with saline. Treatments were repeated on the day of calving. Blood samples were collected pre- and postcalving for complete blood count, biochemistry, and in vitro assessment of neutrophil function including phagocytosis, myeloperoxidase release, and oxidative burst. Prepartum energy restriction resulted in lower body weight, a higher proportion of cows with elevated concentrations (i.e., >0.4 mmol/L) of fatty acids, and higher average β-hydroxybutyrate concentrations before calving relative to fully fed cows. Treatment with pegbovigrastim increased the total white cell, neutrophil, lymphocyte, and monocyte counts. Pegbovigrastim treatment resulted in increased release of myeloperoxidase by neutrophils. Prepartum feeding group did not have an effect, and no feeding group × treatment interaction was observed for any of the white cell counts or functional tests. We concluded that pegbovigrastim treatment results in significant increases in neutrophil count and enhances neutrophil function as indicated by increased myeloperoxidase release. The response to pegbovigrastim was not affected by restricted prepartum energy intake.     

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.     

Immunometabolic status and productive performance differences between periparturient Simmental and Holstein dairy cows in response to pegbovigrastim

In the present study, we aimed to investigate the side effects of pegbovigrastim, injected approximately 7 d before parturition and on the day of calving, on a panel of plasma biomarkers to evaluate energy, inflammatory, oxidative, and liver function status. We also addressed treatment responses in different breeds during the transition period. Holstein and Simmental cows were randomly assigned into 2 groups based on expected calving date and according to parity: the treated group (PEG; 14 Holstein and 12 Simmental cows) received pegylated recombinant bovine granulocyte colony stimulating factor (pegbovigrastim, Imrestor; Elanco Animal Health, Greenfield, IN), and the control group (CTR; 14 Holstein and 14 Simmental cows) received saline solution. The PEG or CTR treatments were administered via subcutaneous injection in the scapular region at approximately 7 d (mean 7.80 ± 5.50 d) before expected parturition and within 24 h after calving. Blood samples were collected at ?21, ?7 (before injection), 1, 3, and 28 d relative to calving. Milk production was recorded at 7, 15, 21, 30, and 42 d. A mixed model with repeated measures was fitted to the normalized data using Proc MIXED of SAS (SAS Institute Inc., Cary, NC). Simmental PEG cows showed higher plasma protein concentrations at 1 and 3 d after calving compared with Simmental CTR and Holstein PEG cows, whereas no differences were detected between Holstein PEG and CTR cows. Albumin was greater at 1 d in Simmental PEG compared with Simmental CTR cows. In contrast, γ-glutamyl transferase was higher overall (across breed) in PEG than in CTR. The PEG group had higher values throughout the postcalving period compared with CTR. Cows treated with pegbovigrastim had also higher alkaline phosphatase (ALP) activity at 1 and 3 d after calving. The Holstein PEG group had higher ALP activity at 3 d compared with the Holstein CTR and Simmental PEG groups, and higher ALP at 1 d compared with the Simmental CTR group. The PEG group had higher levels of IL-6 at 3 and 28 d but higher IL-1β only at 28 d after calving compared with the CTR group. Overall, Holstein cows were characterized by a greater response in the production of inflammation biomarkers (cytokines, haptoglobin, and ceruloplasmin). In addition, PEG cows had higher values of zinc at 1 and 3 d after calving compared with CTR cows. The response observed in plasma biomarkers for energy metabolism and liver functionality after pegbovigrastim treatment in Simmental and Holstein cows was not different from that in control cows. However, our data shed light on the different metabolic adaptations during the transition period between Simmental and Holstein cows, characterized by different energy, inflammatory, and oxidative pattern responses. For the first time, we have highlighted the effect of pegbovigrastim in maintaining stable cytokine levels during the first month after parturition, reflecting greater regulation of neutrophil recruitment, trafficking, and maturation during the inflammatory response. These results provide evidence of the immunomodulatory action of pegbovigrastim around parturition, when dairy cows are highly immunosuppressed. At the same time, these data demonstrate that increasing release of cytokines after parturition is not linked to exacerbation of a systemic inflammation evaluated based on haptoglobin and ceruloplasmin levels.     

Flow cytometric assessment of myeloperoxidase in bovine blood neutrophils and monocytes

Myeloperoxidase (MPO) is a lysosomal peroxidase enzyme mainly stored in the azurophilic granules of neutrophils playing an important role in innate immunity for first-line protection against microorganisms in many species including cattle. As such, determination of MPO has become of great interest for the diagnosis of infectious and inflammatory diseases in multiple species such as humans. In cattle, MPO determination is rarely done because methods to assess MPO in this species are limited: functional assays have been described earlier, but so far, the quantification of MPO at the single cell level has not been done yet. In the present paper, an innovative flow cytometric method to assess MPO in blood leukocytes of dairy cattle is described. A commercial anti-bovine MPO was used following density gradient separation to isolate polymorphonuclear (PMN) and mononuclear (MN) leukocytes from blood. Identification of PMN and MN, subdivided in monocytes and lymphocytes, was based on the expression of the surface markers CH138A and CD172A. The optimized protocol was subsequently evaluated on blood samples of 17 Holstein Friesian heifers. Myeloperoxidase expression was measured flow cytometrically and visualized by fluorescence microscopic imaging of sorted PMN and MN populations. We suggest this innovative method to be useful in the field for early detection of cows at higher risk for inflammatory diseases such as mastitis and metritis during the transition period.     

Changes in skeletal muscle thickness and echogenicity and plasma creatinine concentration as indicators of protein and intramuscular fat mobilization in periparturient dairy cows

High-producing dairy cows experience a state of negative energy balance in the periparturient period that is partially addressed by increasing the rate of fat and protein mobilization. Previous studies have focused on the rate of fat mobilization, and consequently the rate of protein mobilization has not been well characterized. The objective of this study was therefore to determine the change in indicators of muscle mass during early lactation using ultrasonographic measurement of muscle thickness and changes in plasma creatinine concentration. The maximum thickness of the gluteus medius and longissimus dorsi muscles of 106 Holstein cows (34 primiparous, 72 multiparous) was determined ultrasonographically on d ?3, 0, 3, 7, 14, 21, and 28 relative to the day of parturition. Plasma creatinine concentration was measured periodically during the same period. Mixed models analysis and Passing-Bablok regression were used to analyze the data. Gluteus medius thickness, longissimus dorsi loin thickness (LDLT), and longissimus dorsi thoracic thickness (LDTT) were decreased at 28 d postpartum compared with d 3 antepartum. Plasma creatinine concentration was weakly associated with gluteus medius thickness, LDLT, and LDTT (Spearman's rho = 0.31, 0.39, and 0.32, respectively). Plasma creatinine concentration in primiparous and multiparous cows at 28 d postpartum decreased by 0.24 and 0.30 mg/dL, respectively, compared with values 3 d antepartum. We concluded that ultrasonographic measurement of LDLT and LDTT and change in plasma creatinine concentration may provide practical methods for monitoring the rate of protein mobilization in periparturient dairy cows. Ultrasonographic examination of LDLT and LDTT therefore complements ultrasonographic measurement of backfat thickness and may be useful in the evaluation of energy reserve mobilization in periparturient dairy cows.     

Effects of supplementing a Saccharomyces cerevisiae fermentation product during the periparturient period on the immune response of dairy cows fed fresh diets differing in starch content

The objective of this study was to evaluate the effects of supplementing a Saccharomyces cerevisiae fermentation product (SCFP; NutriTek, Diamond V, Cedar Rapids, IA) during the periparturient period (d ?28 ± 3 to 44 ± 3 relative to calving) on mRNA abundance of genes in the rumen epithelium, inflammation indicators, oxidative status, and adaptive immunity of dairy cows fed diets with different starch content after calving. From d 28 ± 3 (± standard deviation) before the expected calving date to calving, Holstein cows (n = 38) received a common basal controlled-energy close-up diet (1.43 Mcal/kg, net energy for lactation; 13.8% starch) with (SCFP; n = 19) or without (CON; n = 19) SCFP, and cows within each treatment (CON or SCFP) were fed either a low- (LS; 22.1% starch) or high-starch (HS; 28.3% starch) diet from d 1 to 23 ± 3 after calving (fresh period). There were 4 treatment groups: LS + CON (n = 9), LS + SCFP (n = 10), HS + CON (n = 10), and HS + SCFP (n = 9). From d 24 ± 3 to 44 ± 3 after calving, all cows were fed the HS diets (post-fresh period). Animal assignment to treatments was balanced for parity, body condition score, and expected calving date. An interaction was observed between dietary starch content and SCFP on indices of oxidative stress; plasma concentrations of total antioxidant capacity tended to be reduced on d 21 after calving for SCFP compared with CON cows when a LS fresh diet was fed, but did not differ for cows fed HS fresh diets. Regardless of starch content, SCFP supplementation increased plasma concentrations of malondialdehyde at d 21 after calving compared with CON. Supplementing with SCFP reduced serum concentrations of haptoglobin on d 7 after calving, indicating reduced inflammation, and feeding LS fresh diets reduced mRNA abundance of IL receptor associated kinase-1 in rumen tissue at d 21 after calving, suggesting reduced immune activation in rumen tissue. Other than the anti-inflammatory effects indicated by lower serum haptoglobin concentration, no other effects of treatment on adaptive immunity were detectable. These results indicate that supplementing SCFP through the transition period and feeding low-starch diets during the fresh period may reduce inflammation.     

Effects of dietary vitamin C on neutrophil function and responses to intramammary infusion of lipopolysaccharide in periparturient dairy cows

Neutrophil function and the severity and incidence of mastitis in dairy cows is related to the intake of many antioxidant nutrients. Because vitamin C is the major water-soluble antioxidant in mammals, we examined the effect of dietary vitamin C on neutrophil function and responses to intramammary infusion of lipopolysaccahride (LPS) in periparturient dairy cows. At 2 wk before anticipated calving, Holstein cows were fed diets that provided 0 (16 cows) or 30 (15 cows) g/d of supplemental vitamin C (phosphorylated ascorbic acid). Treatments continued until 7 d after cows received an infusion of 10 μg of LPS into one quarter of the mammary gland (on average, this occurred 32 d postcalving). Supplementation of vitamin C increased plasma concentrations of vitamin C at calving, but no differences were observed in samples taken 24 h postinfusion. Concentrations of vitamin C in milk (24 h postinfusion) and in neutrophils (calving and 24 h postinfusion) were not affected by treatment, but vitamin C concentrations in neutrophils isolated from milk were about 3 times greater than concentrations in blood neutrophils. The LPS infusion did not alter concentrations of vitamin C in plasma or milk, suggesting that the LPS model did not produce the same effects as a bacterial infection of the mammary gland with respect to antioxidant effects. Supplemental vitamin C had no effect on neutrophil phagocytosis or bacterial kill. Dietary vitamin C reduced the milk somatic cell count but did not affect the febrile response or milk production following LPS infusion.     

The effect of calcareous marine algae,with or without marine magnesium oxide,and sodium bicarbonate on rumen pH and milk production in mid-lactation dairy cows

Two experiments were carried out to evaluate different dietary buffers and their influence on (1) rumen pH in dairy cows and (2) milk production in dairy cows. The supplements included were calcareous marine algae (CMA; Lithothamnion calcareum), with or without marine magnesium oxide (MM; precipitated magnesia derived from seawater), and sodium bicarbonate (SB). Dietary treatments in experiment 1 consisted of the control [32.9% starch and sugar, and 19.9% neutral detergent fiber from forage per kg of dry matter (DM)] including no dietary buffer (CON); the control plus 0.45% DM CMA (CMA); the control plus 0.45% DM CMA and 0.11% DM MM (CMA+MM); the control plus 0.9% DM SB (SB). Diets were formulated to a dry matter intake (DMI) of 18 kg per cow/d. Dietary treatments in experiment 2 also consisted of CON (28.3% starch and sugar, and 23% neutral detergent fiber from forage per kg of DM), CMA, CMA+MM, and SB and were formulated to achieve identical intakes of experimental ingredients (80 g of CMA, 80 g of CMA plus 20 g MM, and 160 g of SB per cow/d) with a DMI of 22.6 kg per cow/d. Experiment 1 used 4 rumen-cannulated dairy cows in a 4 × 4 Latin square design. Rumen pH was measured over five 2-h periods, following feeding, using rumen pH probes. In experiment 2, 52 multiparous and 4 primiparous cows (62.7 ± 3.4 d in milk) were assigned to 4 experimental treatments for 80 d. Both CMA treatments maintained a greater mean rumen pH than the CON during 4 of the 5 periods following feeding and the CON had a greater number of hours below rumen pH 5.5 compared with all other treatments. Dry matter intakes tended to be higher on the SB compared with CON. The CMA treatment increased the production of milk fat and protein yield (kg/d) compared with all other treatments. Both CMA and CMA+MM increased milk fat yield compared with CON but were similar to each other and SB. Protein yield was highest in the CMA treatment compared with CON, CMA+MM, and SB. All 3 buffer treatments increased milk fat concentration compared with CON but did not differ from each other. The SB treatment reduced milk protein concentration and milk production efficiency, energy-corrected milk per kilogram of DMI. Results indicate that the addition of CMA can benefit milk fat and protein production when included in diets based on typical feedstuffs of the northern European region. The use of CMA when compared with SB, in such diets, can increase milk protein production and milk production efficiency.     

Effects of the presence of the mammary gland on expression of neutrophil adhesion molecules and myeloperoxidase activity in periparturient dairy cows

Neutrophil function is reduced in periparturient dairy cows. Possible factors that reduce neutrophil function include endocrine changes associated with parturition and metabolic stresses associated with lactogenesis. In this study, mastectomized and intact cows were studied to specifically examine the effects of lactogenesis on neutrophil function in periparturient cows. Expression of adhesion molecules on neutrophils (L-selectin, mediating capture and rolling adhesion, and beta 2-integrin, mediating tight adhesion vital to egress) and neutrophil myeloperoxidase activity (an index of bactericidal activity) were assessed in mastectomized and intact cows. Expression of L-selectin decreased at parturition followed by rapid recovery to prepartum values in both intact and mastectomized cows. Expression of beta 2-integrins increased in intact cows at parturition but not in mastectomized cows. Expression of beta 2-integrins was greater in intact cows than in mastectomized cows throughout the study. Neutrophil myeloperoxidase activity decreased from baseline prepartum values as parturition approached in both intact and mastectomized cows, which suggests the endocrine changes associated with the act of parturition are predominant factors causing loss of neutrophil function. Myeloperoxidase activity recovered to prepartum values within a week of parturition in mastectomized cows; however, myeloperoxidase activity remained depressed in neutrophils obtained from intact cows throughout the first 20 d of lactation. The presence of the mammary gland and its attendant metabolic stresses slowed recovery of neutrophil function, which suggests that the metabolic stress of lactation exacerbated periparturient immunosuppression.     

Effect of recombinant bovine granulocyte colony-stimulating factor covalently bound to polyethylene glycol injection on neutrophil number and function in periparturient dairy cows

Dairy cows often experience decreased immune function around the time of calving, typified by impaired polymorphonuclear neutrophil (PMN) function and a transient neutropenia. This is associated with increased disease incidence, including mastitis, retained placenta, and metritis. In an attempt to improve PMN functional capacity during the periparturient period, we injected cows with recombinant bovine granulocyte colony-stimulating factor covalently bound to polyethylene glycol (PEG rbG-CSF) twice subcutaneously, about 6 d before calving and within 24 h after calving. Twenty-one cows in their second pregnancy were enrolled in this study and divided into 2 groups: PEG rbG-CSF treated (n = 11) and saline-treated controls (n = 10). The PMN numbers quickly and dramatically increased after PEG rbG-CSF administration and remained elevated through the end of the experiment (13 d after calving). Exocytosis of myeloperoxidase by stimulated PMN, which is generally decreased in periparturient cows, was markedly increased by PEG rbG-CSF after injection. Higher myeloperoxidase exocytosis persisted for at least 10 d after calving. The PMN superoxide anion release and phagocytosis activity did not differ between groups. Injection of PEG rbG-CSF was safe for cows, with no significant negative effects observed. The greatest single effect of PEG rbG-CSF administration was a dramatic increase in circulating numbers of PMN. The increased numbers of PMN ready to move to a site of infection early in the course of an infection may improve the ability of the cow to ward off clinical disease in the periparturient period.     

Effects of recombinant bovine interleukin-8 (rbIL-8) treatment on health,metabolism, and lactation performance in Holstein cattle I: Production and functional characterization of rbIL-8 in vitro and in vivo

In the present study, we standardized processes of cloning and purification of recombinant bovine interleukin-8 (rbIL-8) from bacterial culture and assessed its biological activity in Holstein cattle. Plasmid containing a subclone of bovine IL-8 was expressed using Escherichia coli BL21 and cell lysate was purified by chromatography. The presence of rbIL-8 was assessed by Western blot analyses and function was confirmed in vitro using a chemotaxis chamber. Based on optical density values, chemoattractant properties of rbIL-8 were 10-fold greater compared with control wells. Two in vivo studies were conducted to assess the biological activity of rbIL8. For study 1, one-year-old Holstein heifers (n = 20) were randomly allocated to receive a single intravaginal administration containing 1,125 µg of rbIL-8 diluted in 20 mL of saline solution (rbIL-8, n = 10) or a single intravaginal administration of 20 mL of saline solution (control, n = 10). For study 2, nonpregnant lactating Holstein cows (n = 31) were randomly allocated to receive an intrauterine administration with 1,125 µg of rbIL-8 diluted in 20 mL of saline solution (rbIL-8, n = 11), a positive control consisting of resin-purified lysate of E. coli BL21 not transfected with the plasmid coding for rbIL-8 diluted in 20 mL of saline solution (E. coli, n = 10), and a negative control administered with 20 mL of saline solution (control, n = 10). An increase in vaginal neutrophils was observed in heifers treated with rbIL-8 within 3 h of treatment, but not in control heifers. Additionally, intrauterine administration of rbIL-8 increased the proportion of PMN cells in uterine cytological samples from 3.5% before treatment to 75.8% 24 h later—an increase that was not observed in the negative control group and cows treated with resin-purified lysate of E. coli. To further evaluate the effect of local and systemic rbIL-8 stimulation on the dynamics of circulating white blood cells, a third study was conducted. In study 3, nonpregnant 8-mo-old Holstein heifers (n = 30) were randomly allocated into 1 of 3 treatment groups: intravenous rbIL-8 (1,125 µg of rbIL-8 diluted in 5 mL of saline solution, n = 10); intravaginal rbIL-8 (1,125 µg of rbIL-8 diluted in 20 mL of saline solution; n = 10); or intravaginal saline (20 mL of saline solution, n = 10). Intravenous injection of rbIL-8 resulted in a transient increase in rectal temperature, which was greater at 2 h after treatment compared with cows treated intravaginally with rbIL-8 or heifers treated with saline solution. Heifers treated with rbIL-8 intravenously displayed a marked reduction in neutrophils, basophils, lymphocytes, and monocytes within the first 4 h posttreatment compared with heifers treated intravaginally. However, at 6 h after treatment, heifers treated with rbIL-8 intravenously displayed a rebound in white blood cell counts caused by an increase in neutrophil counts. These results show that the presented purification method is effective and results in biologically active rbIL-8 that can be used safely to modulate immune responses in cattle.     

Effects of in vitro insulin and 2,4-thiazolidinedione on the function of neutrophils harvested from blood of cows in different physiological states

Neutrophils [polymorphonuclear neutrophilic leukocytes (PMNL)] were isolated from 26 Holstein cows in different physiological states (12 ± 1.7 d prepartum, n = 8; 7 ± 0 d postpartum, n = 9; 253 ± 25.2 d postpartum, n = 9) and incubated in vitro for 120 min in a factorial arrangement of treatments with 0, 1.5, or 15 ng/mL of bovine insulin and 0 or 300 μg/mL of the peroxisome proliferator-activated receptor-γ ligand 2,4-thiazolidinedione (TZD). Following the incubations, PMNL functional assays were performed to determine treatment effects on proxies for total, extracellular, and intracellular generation of reactive oxygen species (ROS), neutrophil extracellular trap formation, and phagocytic killing abilities. The ROS production of PMNL collected from cows at 7 d postpartum was reduced compared with that of PMNL from midlactation and prepartum cows, but neutrophil extracellular trap expression was 23 and 36% greater in PMNL from prepartum cows compared with that in PMNL from midlactation and postpartum cows, respectively. Insulin had no effect on PMNL functional assay results. In contrast, TZD inhibited a measurement of total ROS production by 89%, increased extracellular superoxide generation by 43%, but had no effect on the intracellular ROS measured. Interestingly, TZD did not alter the ability of the PMNL to release neutrophil extracellular traps and engulf or kill Staphylococcus aureus. These findings suggest a possible anti-inflammatory effect of TZD that may result in reduced extracellular oxidative damage with maintenance of PMNL antimicrobial activity.     

Meta-analysis of effects of inoculation with homofermentative and facultative heterofermentative lactic acid bacteria on silage fermentation,aerobic stability,and the performance of dairy cows

Forages are usually inoculated with homofermentative and facultative heterofermentative lactic acid bacteria (LAB) to enhance lactic acid fermentation of forages, but effects of such inoculants on silage quality and the performance of dairy cows are unclear. Therefore, we conducted a meta-analysis to examine the effects of LAB inoculation on silage quality and preservation and the performance of dairy cows. A second objective was to examine the factors affecting the response to silage inoculation with LAB. The studies that met the selection criteria included 130 articles that examined the effects of LAB inoculation on silage quality and 31 articles that investigated dairy cow performance responses. The magnitude of the effect (effect size) was evaluated using raw mean differences (RMD) between inoculated and uninoculated treatments. Heterogeneity was explored by meta-regression and subgroup analysis using forage type, LAB species, LAB application rate, and silo scale (laboratory or farm-scale) as covariates for the silage quality response and forage type, LAB species, diet type [total mixed ration (TMR) or non-TMR], and the level of milk yield of the control cows as covariates for the performance responses. Inoculation with LAB (≥10⁵ cfu/g as fed) markedly increased silage fermentation and dry matter recovery in temperate and tropical grasses, alfalfa, and other legumes. However, inoculation did not improve the fermentation of corn, sorghum, or sugarcane silages. Inoculation with LAB reduced clostridia and mold growth, butyric acid production, and ammonia-nitrogen in all silages, but it had no effect on aerobic stability. Silage inoculation (≥10⁵ cfu/g as fed) increased milk yield and the response had low heterogeneity. However, inoculation had no effect on diet digestibility and feed efficiency. Inoculation with LAB improved the fermentation of grass and legume silages and the performance of dairy cows but did not affect the fermentation of corn, sorghum, and sugar cane silages or the aerobic stability of any silage. Further research is needed to elucidate how silage inoculated with homofermentative and facultative heterofermentative LAB improves the performance of dairy cows.     

Dietary cation-anion difference and the health and production of pasture-fed dairy cows 2. Nonlactating periparturient cows

Anecdotal observations of reduced hypocalcemia due to small reductions in the precalving dietary cation-anion difference (DCAD) are widely reported in Australia and New Zealand. Diets offered to nonlactating, periparturient dairy cows in pasture-based dairy systems in southeastern Australia can vary in their cation-anion difference from 0 to +76 mEq/100 g. The effects of such a range in the DCAD on the health and production of cows, on a pasture-based diet, were examined in an indoor feeding experiment. Four groups of four cows were offered pasture-hay and freshly cut pasture, a periparturient diet typical of that associated with the grazing system in Australia and New Zealand. Varying levels of salt supplementation were used to alter the dietary cation-anion difference, which ranged from -12 to +69 mEq/100 g. Blood and urine pH and mineral concentrations and urine hydroxyproline were measured. The addition of anions to the diet, to produce a negative DCAD, resulted in a nonrespiratory systemic acidosis. With decreasing DCAD, the pH of blood and urine and the strong ion difference of urine decreased curvilinearly, blood bicarbonate decreased linearly and the urinary ratio of Ca to creatinine increased curvilinearly. Although systemic pH was not reduced at a DCAD of +16 mEq/100 g, urine Ca-to-creatinine ratio had begun to rise, probably indicating increased calcium absorption. The absorption and renal excretion of Mg increased with decreasing DCAD. No differences were observed in urine hydroxyproline concentrations and no significant differences in milk production were measured.     

Effect of experimental design on responses to 2 concentrations of metabolizable protein in multiparous dairy cows

The objective of this research was to characterize the implications of changing between diets formulated to be adequate (ADMP) or low (LOMP) in metabolizable protein in a Latin square (LSq) design or of feeding the same diets continuously in a randomized complete block experimental design (RCBD). Fifty-four multiparous early-lactation cows (initial average ± SD; parity 2.8 ± 0.9, 85.8 ± 31 d in milk, 715 ± 63 kg of body weight, 29.1 ± 2.7 kg of dry matter intake/d, and 57.7 ± 5.7 kg of milk yield/d) were blocked by parity and days in milk and were then randomly assigned to experimental design, with 16 cows assigned to LSq and 38 cows assigned to RCBD. Cows within blocks in LSq were randomly assigned to sequence in a 4-sequence, 4-period, 2-treatment LSq balanced for the effects of previous treatment carryover. Cows within blocks in RCBD were randomly assigned to dietary treatment, which was fed over the same four 28-d periods as the cows in LSq. Treatment diets were formulated to be similar in composition with the exception of exchanging an equal quantity of expeller soybean meal from ADMP (16.5% crude protein; 28.4% ash-free, amylase-treated neutral detergent fiber organic matter) for soybean hulls in LOMP (14.6% crude protein; 31.1% ash-free, amylase-treated neutral detergent fiber organic matter). Cows were individually fed treatment diets in a tiestall barn once daily for ad libitum consumption, milked 3 times daily, and administered recombinant bovine somatotropin every 14 d. Milk yield and feed offered and refused were measured daily; BW was recorded on 2 consecutive days each week; milk composition was measured at 6 consecutive milkings each week; and spot samples of feces, urine, and blood were collected during the last week of each period and a covariate period. Experimental designs were analyzed separately using results from wk 4 of each period with mixed effects modeling. Dry matter intake and milk fat yield were not affected by diet in either design, whereas milk and protein yields were greater for cows fed ADMP in both designs. Milk fat and protein percentage responses and milk energy output inferences were different between designs. Milk fat yield and percentage responses were affected by previous treatment carryover in LSq. Metabolic and digestibility inferences were very similar between designs. Under the conditions of this experiment, inferences on N metabolism and the majority of production measurements were not affected by experimental design, with the principal exceptions of milk fat and protein percentage and milk energy output.     

Short communication: Effects of an immunomodulatory feed additive on phagocytic capacity of neutrophils and relative gene expression in circulating white blood cells of transition Holstein cows

High-producing dairy cows typically experience immunosuppression with dysregulated neutrophil function (e.g., compromised phagocytosis) during the transition period (3 wk before to 3 wk after parturition), which is causally associated with increased risk of infections. Enhanced neutrophil immune competence has significant bearing with the wellbeing of transition dairy cattle. In the current study, we investigated the effect of OmniGen-AF (OG; Phibro Animal Health, Quincy, IL) and its dose in modulation of neutrophil function of transition cows. Forty-eight multiparous cows were stratified by parity, somatic cell count, and expected calving date and randomly assigned to 3 treatments: OG fed at 0 g/head per day (CON), 60 g/head per day (OG60; recommended dose), and 90 g/head per day (OG90; 1.5× recommended dose). The OG was added from dry off (61.8 ± 1.69 d before parturition) to 28 d in milk (DIM), and removed from all treatment groups at 29 to 35 DIM (the last week of the experimental period). Neutrophil phagocytic ability against Staphylococcus aureus and Escherichia coli was improved and tended to be improved, respectively, by OG from d 28 before parturition to 28 DIM. Cows in OG60 had higher neutrophil phagocytic ability against S. aureus and E. coli compared with CON cows from d 28 before parturition to 28 DIM. Neutrophil phagocytosis of S. aureus and E. coli was higher and tended to be higher for OG60 compared with CON on 35 DIM. The relative gene expression of CXCL8 and SELL were upregulated and tended to be upregulated by OG from 60 d before parturition to 28 DIM; this was due to cows in OG60 having greater SELL and CXCL8 gene expression than CON. Expression of SELL in circulating white blood cells of OG60-treated cows was greater than OG90 and the relative expression of CXCL8 gene tended to be greater for OG60 compared with CON on 35 DIM. In conclusion, feeding OG at the recommended dose of 60 g/head per day from dry off was effective in maintaining peripheral blood neutrophil function in transition dairy cows, and it is not necessary to feed OG beyond the recommended dose.     

Non-aureus staphylococci in fecal samples of dairy cows: First report and phenotypic and genotypic characterization

The aims of this study were to determine whether non-aureus staphylococci (NAS) are present in rectal feces of healthy dairy cows, and if so, to delineate species to which they belong and to study several phenotypic and genotypic traits as a first step toward determining the potential impact of fecal shedding of NAS on bovine udder health. Fecal samples were aseptically collected from the rectum of 25 randomly selected clinically healthy dairy cows in a commercial dairy herd using an automated milking system. Fecal NAS were isolated and then identified at the species level using transfer RNA-intergenic spacer PCR and sequencing of the 16S rRNA housekeeping gene. Strain typing was performed using random amplification of polymorphic DNA (RAPD)-PCR. The antimicrobial resistance profiles, biofilm formation, and growth and inhibitory characteristics of all NAS isolates were evaluated. Half of the cows were shedding NAS, resulting in 31 NAS isolates belonging to 11 different species. The most prevalent species were Staphylococcus rostri (23%, n = 7), Staphylococcus cohnii (16%, n = 5), and Staphylococcus haemolyticus (13%, n = 4) with all Staphylococcus agnetis, Staphylococcus chromogenes, and Staph. rostri isolates belonging to the same strain according to RAPD banding patterns. Acquired antimicrobial resistance was observed in 28 of the 31 NAS isolates, mainly due to β-lactamase production. Most of the isolates (84%, n = 27) had a weak biofilm-forming potential, but only 2 contained the bap gene. The ica and aap genes were not detected in any of the isolates. In vitro growth of Staphylococcus aureus and Streptococcus dysgalactiae was inhibited by Staph. agnetis isolates, and Staph. chromogenes isolates were able to inhibit the growth of Strep. dysgalactiae and Streptococcus uberis. All fecal isolates were able to grow when oxygen and iron were limitedly available, mimicking the growth conditions in the mammary gland.     

Differential phenotype of immune cells in blood and milk following pegylated granulocyte colony-stimulating factor therapy during a chronic Staphylococcus aureus infection in lactating Holsteins

Neutrophils are principal host innate immune cell responders to mastitis infections. Thus, therapies have been developed that target neutrophil expansion. This includes the neutrophil-stimulating cytokine granulocyte colony-stimulating factor (gCSF). Pegylated gCSF (PEG-gCSF; Imrestor, Elanco Animal Health, Greenfield, IN) has been shown to reduce the natural incidence of mastitis in periparturient cows in commercial settings and reduce severity of disease against experimental mastitis challenge. Pegylated gCSF stimulates neutrophil expansion but also induces changes in monocyte and lymphocyte circulating numbers, surface protein expression changes, or both. We hypothesized that PEG-gCSF modulates surface expression of monocytes and neutrophils and facilitates their migration to the mammary gland. We challenged 8 mid-lactation Holsteins with approximately 150 cfu of Staphylococcus aureus (Newbould 305) in a single quarter via intramammary infusion. All animals developed chronic infections as assessed by bacteria counts and somatic cell counts (SCC). Ten to 16 wk postchallenge, 4 of the animals were treated with 2 subcutaneous injections of PEG-gCSF 7 d apart. Complete blood counts, SCC, bacterial counts, milk yield, feed intake, neutrophils extracellular trap analysis, and flow cytometric analyses of milk and blood samples were performed at indicated time points for 14 d after the first PEG-gCSF injection. The PEG-gCSF-treated cows had significantly increased numbers of blood neutrophils and lymphocytes compared with control cows. Flow cytometric analyses revealed increased surface expression of myeloperoxidase (MPO) on neutrophils and macrophages in milk but not in blood of treated cows. Neutrophils isolated from blood of PEG-gCSF-treated cows had decreased surface expression of CD62L (L-selectin) in blood, consistent with cell activation. Surprisingly, CD62L cell surface expression was increased on neutrophils and macrophages sourced from milk from treated animals compared with cells isolated from controls. The PEG-gCSF-treated cows did not clear the S. aureus infection, nor did they significantly differ in SCC from controls. These findings provide evidence that PEG-gCSF therapy modifies cell surface expression of neutrophils and monocytes. However, although surface MPO⁺ cells accumulate in the mammary gland, the lack of bacterial control from these milk-derived cells suggests an incomplete role for PEG-gCSF treatment against chronic S. aureus infection and possibly chronic mammary infections in general.     

Automatic detection of feeding- and drinking-related agonistic behavior and dominance in dairy cows

Accurate assessments of social behavior and dominance relationships in cattle can be time consuming. We investigated whether replacements at the feed bunk and water trough—one type of agonistic interaction—can be used to automatically assess dominance relationships. Our study set out to (1) validate a replacement detection algorithm using combined data from electronic feed and water bins, and (2) investigate the applicability of this algorithm to identify individual dominance scores and group-level social hierarchy in freestall-housed dairy cows. We used 4 groups of lactating cows kept in different group sizes (11 to 20 cows) located at 2 research facilities. In both facilities, feed and water were provided via automated feeding systems. A trained observer recorded all agonistic interactions in the pen over multiple days using video. Data from the electronic feed and water bins for the same days were analyzed using an algorithm to detect replacements (i.e., visits where a receiver cow was competitively replaced by an actor cow). Most agonistic interactions at the feed bunk were replacements. These replacements were associated with a brief interval between the time the receiver cow left the bin and the actor cow took her place; the optimal threshold to detect these replacements varied from 22 to 27 s between groups, independent of stocking density. The recall and precision of an algorithm based upon this threshold was high (on average >0.8), comparable to that of trained human observers. We improved data preparation by controlling for detection errors and included filtering to reduce false positives. This resulted in a >20% decrease in false positives and an increase in precision of 0.043. The dominance hierarchy based upon algorithm-detected replacements was similar to that based upon total agonistic interactions observed in the pen; the Spearman rank correlation coefficient between these hierarchies varied among the groups from 0.81 to 0.96. We conclude that data from electronic feed and water bins can accurately estimate agonistic behavior and dominance relationships among dairy cows.     

Relationships between body condition,body condition loss,and serum metabolites during the transition period in primiparous and multiparous cows

In the transition period from late gestation to early lactation, dairy cows undergo tremendous metabolic changes. Insulin is a relevant antilipolytic factor. Decreasing serum concentrations of insulin and glucose, increasing serum concentrations of nonesterified fatty acids (NEFA) and β-hydroxybutyrate (BHB), and changes in body condition score (BCS) reflect the negative energy balance around calving. This study investigated peripartum metabolic adaptation in 359 primiparous and 235 multiparous German Holstein cows from a commercial dairy herd under field conditions. Body condition score was recorded and blood samples were taken 10 to 1 d prepartum, 2 to 4 d postpartum, and 12 to 20 d postpartum. Generalized mixed models and generalized estimation equations were applied to assess associations between prepartum BCS; BCS changes during the transition period; insulin, glucose, NEFA, and BHB serum concentrations; and milk yield, which was taken from an electronic milk meter from d 6 of lactation. Serum insulin concentrations of multiparous postpartum cows were lower compared with prepartum, and compared with primiparous cows. In general, primiparous cows had lower postpartum NEFA and BHB concentrations than multiparous cows. In primiparous cows, we identified a positive association between prepartum BCS and prepartum serum insulin concentration. Prepartum obese multiparous cows, but not primiparous cows, were characterized by higher postpartum serum NEFA and BHB concentrations and lower milk yield than other cows in the same parity class. Primiparous cows with a smaller degree of BCS loss during the transition period had higher postpartum insulin and lower NEFA concentrations and lower milk yield than other primiparous cows. In conclusion, primiparous cows had less lipolysis and lower milk yield than multiparous cows, associated with higher insulin concentrations. Avoiding high body condition loss during the transition period is a main factor in preventing peripartal metabolic imbalances of glucose and fat metabolism.