Acute phase reaction to lipopolysaccharide-induced mastitis in early lactation dairy cows fed nitrogenic, glucogenic, or lipogenic diets.

The availability of certain macronutrients is likely to influence the capacity of the immune system. Therefore, we investigated the acute phase response to intramammary (i.mam.) lipopolysaccharide (LPS) in dairy cows fed a nitrogenic diet (n = 10) high in crude protein, a glucogenic diet (n = 11) high in carbohydrates and glucogenic precursors, or a lipogenic diet (n = 11) high in lipids. Thirty-two dairy cows were fed one of the dietary concentrates directly after calving until the end of trial at 27 ± 3 days in milk (mean ± standard deviation). In wk 3 of lactation, 20 µg of LPS was i.mam. injected in one quarter, and sterile NaCl (0.9%) in the contralateral quarter. Milk samples of the LPS-challenged and control quarter were taken hourly from before (0 h) until 9 h after LPS challenge and analyzed for milk amyloid A (MAA), haptoglobin (HP), and IL-8. In addition, blood samples were taken in the morning, and composite milk samples at morning and evening milkings, from 1 d before until 3 d after LPS challenge, and again on d 9, to determine serum amyloid A (SAA) and HP in blood, and MAA and HP in milk. The mRNA abundance of various immunological and metabolic factors in blood leukocytes was quantified by quantitative reverse-transcription PCR from samples taken at -18, -1, 6, 9, and 23 h relative to LPS application. The dietary concentrates did not affect any of the parameters in blood, milk, and leukocytes. The IL-8 was increased from 2 h, HP from 2 to 3 h, and MAA from 6 h relative to the LPS administration in the milk of the challenged quarter and remained elevated until 9 h. The MAA and HP were also increased at 9 h after LPS challenge in whole-udder composite milk, whereas HP and SAA in blood were increased only after 23 h. All 4 parameters were decreased again on d 9. Similar for all groups, the mRNA abundance of HP and the heat shock protein family A increased after the LPS challenge, whereas the mRNA expression of the tumor necrosis factor α and the leukocyte integrin ß 2 subunit (CD18) were decreased at 6 h after LPS challenge. The glucose transporter (GLUT)1 mRNA abundance decreased after LPS, whereas that of the GLUT3 increased, and that of the GLUT4 was not detectable. The mRNA abundance of GAPDH was increased at 9 h after LPS and remained elevated. The acute phase protein response was detected earlier in milk compared with blood indicating mammary production. However, immunological responses to LPS were not affected by the availability of specific macronutrients provided by the different diets.

Effect of different dietary regimens at dry-off on performance, metabolism, and immune system in dairy cows.

Concentrate withdrawal and feed restriction are commonly used to reduce milk production and to facilitate dry-off, but may impair immune function in dairy cows. We investigated the effect of feed rations providing different amounts of nutrients in combination with feed restriction on performance, endocrine, and metabolic responses, as well as on leukocyte function before and after abrupt dry-off. Forty-three cows were studied from d 12 before until d 6 after dry-off (56 d before scheduled calving). Cows were fed experimental concentrates rich in crude protein (nitrogenic, n = 14), glucogenic precursors (glucogenic, n = 14), or lipids (lipogenic, n = 15). On d 3 before dry-off, total feed allowance was restricted to 50% in half of the animals of each dietary group, whereas feed allowance remained unchanged in the other animals. Performance parameters (milk yield, milk composition, and dry matter intake) were recorded, and daily blood and milk samples were taken and analyzed for various metabolic and endocrine parameters. Additionally, activity and mRNA abundance of several genes in leukocytes were measured at selected time points before and after feed restriction and dry-off, respectively. Feed restriction immediately resulted in a negative energy balance and decreased milk production. Concomitantly, concentrations of nonesterified fatty acids increased, whereas insulin, insulin-like growth factor-1, and glucagon decreased. After dry-off, energy balance turned positive and plasma nonesterified fatty acids decreased. Plasma glucose, insulin, and insulin-like growth factor-1 concentrations increased in all groups after dry-off. Glucose, insulin, and glucagon concentrations in plasma were higher in nonrestricted compared with restricted animals after dry-off. The experimental concentrate types marginally affected the investigated metabolic and endocrine factors, with the exception of elevated milk and plasma urea concentrations in cows fed the nitrogenic concentrate. Chemotactic and phagocytic activity of leukocytes were not affected by diets, feed restriction, or dry-off. Likewise, blood leukocyte mRNA abundance encoding for tumor necrosis factor α (TNF), heat shock protein family A (HSP70), and the glucose transporters (GLUT) 1 and 3 remained unchanged throughout the study period. Overall, the short-term negative energy balance induced by feed restriction was temporarily accompanied by metabolic adaptations, but did not alter the studied factors related to the immune system. Metabolic and endocrine adaptations supporting milk synthesis were continued during the first days after dry-off despite cessation of milking. Thus, the abrupt dry-off resulted in a short-term increase of glucose and triglyceride concentrations, with a delayed endocrine response to re-establish nutrient homeostasis in blood.

Invited review: The role of the blood-milk barrier and its manipulation for the efficacy of the mammary immune response and milk production.

The intact blood-milk barrier (BMB) prevents an uncontrolled exchange of soluble and cellular components between blood and milk in the mammary gland. It enables the sustainability of the optimal milk composition for the nourishment of the offspring. Endothelial cells, connective tissue, the basal membrane, and mainly the epithelial cells provide the semipermeability of this barrier, allowing only a selective transfer of components necessary for milk production. The epithelial cells are closely connected to each other by different formations, in which the tight junctions are the most critical for separating the milk-containing compartments from the surrounding extracellular fluid and vasculature. During mastitis, the integrity of the BMB is reduced. This facilitates the transfer of immune cells and immune factors such as antibodies from blood into milk. Simultaneously, the transfer of soluble blood constituents without an obvious immune function into milk is promoted. Furthermore, a reduced BMB integrity causes a loss of milk constituents into the blood circulation. Different mechanisms are responsible for the barrier impairment including tight junction opening, but also cell degradation. To promote the cure of mastitis, the targeted manipulation of the BMB permeability may be a tool to optimize the immune function of the mammary gland. An intensified opening of the BMB supports the antibody transfer from blood into milk, which is supposed to increase the contribution of the specific immune system in the immune defense. On the contrary, a fast closure of the BMB during the recovery from mastitis can accelerate the normalization of milk composition and milk yield. Various agents have been experimentally shown to either open (e.g., pathogens and pathogen-associated molecular patterns, several nonsteroidal anti-inflammatory drugs, oxytocin, calcium chelators) or close (e.g., glucocorticoids, nonsteroidal anti-inflammatory drugs, natural anti-inflammatory drugs) the BMB.

Effects of local or systemic administration of meloxicam on mammary gland inflammatory responses to lipopolysaccharide-induced mastitis in dairy cows.

Nonsteroidal anti-inflammatory drugs (NSAID) are commonly used in combination with antimicrobial mastitis treatments to reduce pain. Little is known about whether meloxicam, an NSAID designed for the preferential inhibition of cyclooxygenase-2 over cyclooxygenase-1, affects the mammary immune response. The objective of this study was to analyze the mammary immune response to intramammary (local) or intravenous (systemic) administration of meloxicam with or without immune activation by lipopolysaccharide (LPS). We challenged 108 quarters of 30 cows with or without a low or high dose of LPS from Escherichia coli (0.1 or 0.2 µg/quarter), with or without meloxicam via intramammary administration (50 mg/quarter) or intravenous injection (0.5 mg/kg of body weight; ~300 mg/cow). Intramammary administration of meloxicam alone did not trigger an acute inflammatory response, verified by unchanged somatic cell count (SCC) and lactate dehydrogenase (LDH), BSA, and IgG concentrations in milk, which are normally augmented during mastitis due to an opening of the blood-milk barrier. Similarly, intramammary meloxicam did not change the mRNA abundance of inflammatory factors in mammary gland tissue. As expected, quarters challenged with either dose of LPS showed increased leukocyte infiltration (SCC); increased LDH, BSA, IgG, Na, and Cl concentrations; and diminished K concentrations in milk. In contrast to our hypothesis, the addition of intramammary or intravenous meloxicam did not reduce these markers of mastitis in milk. Instead, intramammary meloxicam appeared to accelerate the SCC response to LPS, but only at the lower LPS dose. Moreover, the mRNA expression of inflammatory factors in mammary tissue was not modified by the intramammary application of meloxicam compared with the contralateral quarters that were challenged with LPS only. We demonstrated for the first time that intramammary meloxicam at a dose of 50 mg/quarter did not trigger an immune response in the mammary glands of dairy cows. At the doses we used, meloxicam (intramammary or systemic) did not lower inflammatory responses. The intramammary administration of meloxicam seemed to stimulate leukocyte recruitment into the milk in quarters challenged with a low dose of LPS. The integrity of the blood-milk barrier was not protected by meloxicam in LPS-stimulated quarters. This study provides the first indications that meloxicam does not limit the inflammatory response in the mammary gland, although it does not impair the mammary immune system.

Nonsteroidal anti-inflammatory drugs affect the mammary epithelial barrier during inflammation.

During inflammation of the mammary gland, the blood-milk barrier, which is predominantly composed of mammary epithelial cells, loses its integrity and gradients between blood and milk cannot be maintained. Nonsteroidal anti-inflammatory drugs (NSAID) are commonly used systemically in combination with local administration of antimicrobials in mastitis treatments of dairy cows to improve the well-being of the cow during the disease. However, the knowledge about their effects on the blood-milk barrier is low. This study aimed to investigate effects of different NSAID, with different selectivity of cyclooxygenase-inhibition, on the transepithelial electrical resistance (TEER) and capacitance, cell viability, and expression of tumor necrosis factor α of bovine mammary epithelial barriers in vitro. Primary mammary epithelial cells of 3 different cows were challenged with lipopolysaccharide (LPS) from Escherichia coli with or without addition of ketoprofen (1.25 mg/mL or 4 mM), flunixin meglumine (1.0 mg/mL or 4 mM), meloxicam (0.25 mg/mL, 0.75 mg/mL, or 4 mM), diclofenac (0.75 mg/mL or 4 mM) or celecoxib (0.05 mg/mL) for 6 h. Concentrations were adapted to comparable relations of the recommended dosage for systemic application. Additionally, a similar molar concentration of all NSAID was used. Lipopolysaccharide with or without NSAID induced a decrease in TEER within 5 h, which returned to control level within 14 h. Viability of cells challenged with LPS only was not affected. However, the cell viability was decreased with increasing concentrations of NSAID and this effect was amplified with simultaneous LPS challenge. Ketoprofen at both dosages, flunixin meglumine at 1.0 mg/mL, and meloxicam at 0.75 mg/mL accelerated the recovery of TEER in comparison to LPS only (return to control level within 9 h). The comparison of NSAID effects at the same molecular quantity of 4 mM showed different effect on the barrier in which ketoprofen accelerated the recovery after LPS-induced barrier opening, whereas meloxicam and diclofenac slowed down the recovery (return to control level after 24 h). In conclusion, NSAID do not prevent the mammary epithelial barrier opening by LPS; however, ketoprofen, flunixin meglumine, and meloxicam obviously support the re-establishment of the barrier integrity. Used in mastitis therapy at an optimized dosage the tested NSAID would likely support the recovery of milk composition. However, an overdose of NSAID would likely cause tissue irritation and in turn, a delayed recovery of the barrier permeability.

Metabolic status is associated with the recovery of milk somatic cell count and milk secretion after lipopolysaccharide-induced mastitis in dairy cows.

Infections of the mammary gland in dairy cows are commonly accompanied by reduced milk production and feed intake and poor milk quality. The metabolic status of early-lactating cows is known to affect immune response to pathogens and imposed immune challenges. We investigated the extent to which metabolic status before an intramammary lipopolysaccharide (LPS) challenge (LPS-CH) is associated with immune response, milk production, and feed intake and the recovery thereof. In 15 Holstein cows, weekly blood sampling and daily recording of dry matter intake, milk yield, milk composition, and body weight (to calculate energy balance) was started immediately after parturition. In wk 4 after parturition, cows underwent an intramammary LPS-CH (50 µg of LPS into 1 quarter). Blood and milk samples were taken in parallel at 30- and 60-min intervals, respectively, until 10 h after the LPS application. Plasma concentrations of glucose, nonesterified fatty acids, ß-hydroxybutyrate (BHB), cortisol, and insulin were analyzed. In milk, serum albumin, IgG concentration, somatic cell count (SCC), and lactate dehydrogenase (LDH) activity were determined. Dry matter intake and milk yield were recorded for an additional 6 d. Milk of the LPS-treated quarter was sampled at every milking for 8 d after the challenge. Based on plasma glucose concentrations in wk 1 to 4 after parturition before the LPS-CH, cows were retrospectively grouped into a high-glucose group (HG; 3.34-3.93 mmol/L, n = 7) and a low-glucose group (LG; 2.87-3.31 mmol/L, n = 8). Data were evaluated using mixed models with time, group, and time × group interaction as fixed effects and cow as repeated subject. Glucose was lower and BHB was higher in LG compared with HG before LPS-CH, whereas dry matter intake, energy balance, and SCC did not differ. During LPS-CH, SCC and LDH increased similarly in HG and LG, body temperature increased less in HG, and BHB and nonesterified fatty acids were higher in LG compared with HG. Dry matter intake declined in both groups during the day of the LPS-CH but recovered to prechallenge values faster in HG. Milk yield recovered within 2 d after the LPS-CH with no differences in morning milkings, whereas evening milk yield increased faster in HG. During 8 d after LPS-CH, SCC, LDH, IgG, and serum albumin in milk were lower in HG compared with LG. In conclusion, the level of circulating glucose and BHB concentrations in cows was associated with metabolic responses during an LPS-CH as well as the recovery of udder health and performance thereafter.

Methane emission, metabolism, and performance of Holstein dairy cows with low, medium, and high lymphocyte proliferation during transition.

This study aimed to identify interactions between state of lactation (dry or early lactating) and immune responder group (low, medium, or high) for energy metabolism traits as well as metabolic and immunological traits in dairy cows. In early lactation, when the energy priority of cows shifts toward the mammary gland, the energy available to be partitioned toward the immune system may differ among individuals. The equilibrium between energy supply from feed, digestion, and body reserve mobilization and energy expenditure with milk, immune system, methane, and heat production is delicate in this stage. Seventeen Holstein cows entering their second to fifth lactation were kept under comparable feeding, housing, and management conditions and were studied from 14 ± 6 d before calving to 11 ± 3 d after calving. Feed intake, milk yield, body condition, blood metabolites, and cortisol as well as gaseous exchange in respiration chambers were measured. The latter was used to quantify methane emission and to calculate resting metabolic rate and heat production. Subsets of blood leukocytes and peripheral blood mononuclear cells (PBMC) were monitored. Activation and proliferation of the PBMC in response to the mitogen phytohemagglutinin ante- and postpartum were assessed using the oxygen consumption rate (24-h cell culture assay) and the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay (72-h cell culture assay). Cows were classified based on the in vitro proliferative response of the PBMC measured postpartum in low (n = 6), medium (n = 5), and high (n = 6) responders. We found no interaction of state of lactation with responder group for feed intake, milk yield, efficiency, metabolic traits, and immune cell activation ante- and postpartum. However, after calving, low-responder cows produced less methane per unit of body weight and per unit of energy-corrected milk compared with the other cows. This might be indicative of a low rumen fermentation intensity. Low responders might therefore suffer from a lower availability of digestible energy in early lactation and not be able to sustain the shift from immune cell activation to proliferation. If so, the selection of environmentally friendly low-methane emitters could promote phenotypes with a compromised immune response in the critical early lactation.

Meloxicam affects the inflammatory responses of bovine mammary epithelial cells.

Nonsteroidal anti-inflammatory drugs are used as supportive therapy with antimicrobial treatments for mastitis in cows to alleviate pain of the inflamed mammary gland. They act mainly by inhibition of cyclooxygenases. Meloxicam (MEL) is a drug designed for cyclooxygenase-2 selectivity, which is upregulated upon inflammation, acting as a key enzyme for the conversion of arachidonic acid to prostaglandins. Although some studies in dairy cows showed positive results in recovery from mastitis when MEL was added to the treatments, direct effects of MEL on the immune system of mastitic cows are unknown. The aim of this study was to investigate effects of MEL on the immune response of bovine mammary epithelial cells (MEC) with or without simultaneous immune stimulation by pathogen-associated molecular patterns of common mastitis pathogens. Mammary epithelial cells from 4 cows were isolated and cultured. To evaluate dose effects of MEL, MEC were challenged with or without 0.2 µg/mL lipopolysaccharide (LPS; serotype O26:B6 from Escherichia coli) with addition of increasing concentrations of MEL (0, 0.25, 0.5, 1.0, 1.5, or 2.0 mg/mL). The addition of MEL prevented the increase of mRNA expression of key inflammatory factors in LPS-challenged MEC in a dose-dependent manner. To investigate the effects of MEL on pathogen-specific immune responses of MEC, treatments included challenges with LPS from E. coli and lipoteichoic acid from Staphylococcus aureus with or without 1.5 mg/mL MEL for 3, 6, and 24 h. Meloxicam prevented the increase of mRNA abundance of key inflammatory mediators in response to LPS and lipoteichoic acid, such as tumor necrosis factor, serum amyloid A, inducible nitric oxide synthase, and the chemokines IL-8 and CXC chemokine ligands 3 and 5. The prostaglandin E2 synthesis in challenged and nonchallenged cells was reduced by MEL within 24 h. Furthermore, MEL reduced the viability and consequently the total RNA yield of the cells. However, mRNA abundance of apoptosis-related enzymes was not affected by any treatment. Meloxicam had clear dose-dependent effects on the immune response of MEC to pathogen-associated molecular patterns of common mastitis pathogens by preventing increased expression of important factors involved in inflammation. This nonsteroidal anti-inflammatory drug also has detrimental effects on cell viability. How these effects would influence the elimination of pathogens from an infected mammary gland during mastitis therapy with meloxicam needs to be further investigated.

Technical note: Effects of attachment of hind teats before cleaning and attachment of front teats on milking characteristics in automatic milking systems.

Milking characteristics differ between the 4 quarters of a dairy cow udder. In particular, milking time is mostly prolonged in hind quarters compared with front quarters because of the usually higher amount of stored milk. The standard milking routine (STDMR) in both conventional and automatic milking systems (AMS) consists of teat preparation of all 4 quarters, followed by attachment of the 4 teat cups, regardless of the distribution of milk between quarters. In the current study, an alternative teat preparation and milking routine (ALTMR) in AMS was tested, which consisted of cleaning and starting the milking of hind teats before cleaning and attachment of front teats. The hypothesis was based on the fact that hind quarters have usually a longer milking time than front quarters. Starting the milking of hind quarters while the front teats are being cleaned may reduce the difference in the end of milking between front and hind quarters and thus reduce total milking time. Both routines were tested on 5 Swedish dairy farms equipped with AMS in a 4-wk experiment in which treatments were alternated weekly. Total milk yield did not differ between treatments. Machine-on time (MOT) was longer in ALTMR than in STDMR because the difference in milking time between hind and front quarters was less than the time needed to prepare the front teats. However, the longer MOT in ALTMR was compensated by a shorter total preparation time, including the attachment of the first teat cup, as only the hind teats (instead of all 4 teats) were cleaned before milking was started. This resulted in a similar total milking time from start of cleaning of the first quarter until the end of milking of the last quarter in both treatments. Because of the prolonged MOT, average milk flow rate was lower in ALTMR than STDMR. Peak flow rate was higher in ALTMR than STDMR, but only in teat cups 1 (first attached, hind quarter) and 3 (third attached, front quarter), whereas main milk flow was higher in ALTMR than STDMR in both front quarters. In conclusion, splitting teat cleaning and the start of milking between hind and front quarters does not prolong total milking time, including teat cleaning. The partially positive effect on peak and main milk flow indicates that the ALTMR is a suitable milking routine in AMS. In herds with a greater difference of milk stored in hind compared with front quarters, a reduced total milking time can be expected for ALTMR.

Acute phase response in lactating dairy cows during hyperinsulinemic hypoglycaemic and hyperinsulinemic euglycaemic clamps and after intramammary LPS challenge.

The link between energy availability, turnover of energy substrates and the onset of inflammation in dairy cows is complex and poorly investigated. To clarify this, plasma inflammatory variables were measured in mid-lactating dairy cows allocated to three groups: hyperinsulinemic hypoglycaemic clamp, induced by insulin infusion (HypoG, n = 5); hyperinsulinemic euglycaemic clamp, induced by insulin and glucose infusion (EuG; n = 6); control, receiving a saline solution infusion (NaCl; n = 6). At 48 h after the start of i.v. infusions, two udder quarters per cow were challenged with 200 µg of E. coli lipopolysaccharide (LPS). Individual blood samples were taken before clamps, before LPS challenge (i.e. 48 h after clamps) and 6.5 h after. At 48 h, positive acute phase proteins (posAPP) did not differ among groups, whereas albumin and cholesterol (index of lipoproteins), negative APP (negAPP), were lower (p < 0.05) in EuG compared to NaCl and HypoG. The concentration of IL-6 was greater in EuG (p < 0.05) but only vs. HypoG. At 6.5 h following LPS challenge, IL-6 increased in the NaCl and EuG clamps (p < 0.05), while TNF-α increased (p < 0.05) in the EuG only. Among the posAPP, haptoglobin markedly increased in EuG (p < 0.05), but not in NaCl (p = 0.76) and in HypoG; ceruloplasmin tended to decline during LPS challenge, the reduction was significant when all animals were considered (p < 0.05). Conversely, all the negAPP showed a marked reduction 6.5 h after LPS challenge in the three groups. In conclusion, EuG caused an inflammatory status after 48-h infusion (i.e. decrease of negAPP) and induced a quicker acute phase response (e.g. marked rise of TNF-α, IL-6) after the intramammary LPS challenge. These data suggest that the simultaneous high availability of glucose and insulin at the tissue-level makes dairy cows more susceptible to inflammatory events. In contrast, HypoG seems to attenuate the inflammatory response.

Lipopolysaccharide enhances apoptosis of corpus luteum in isolated perfused bovine ovaries in vitro.

Lipopolysaccharide (LPS), the endotoxin of Gram-negative bacteria, has detrimental effects on the structure and function of bovine corpus luteum (CL) in vivo. The objective was to investigate whether these effects were mediated directly by LPS or via LPS-induced release of PGF2α. Bovine ovaries with a mid-cycle CL were collected immediately after slaughter and isolated perfused for 240 min. After 60 min of equilibration, LPS (0.5 µg/ml) was added to the medium of five ovaries, whereas an additional six ovaries were not treated with LPS (control). After 210 min of perfusion, all ovaries were treated with 500 iu of hCG. In the effluent perfusate, concentrations of progesterone (P4) and PGF2α were measured every 10 and 30 min, respectively. Punch biopsies of the CL were collected every 60 min and used for RT-qPCR to evaluate mRNA expression of receptors for LPS (TLR2, -4) and LH (LHCGR); the cytokine TNFA; steroidogenic (STAR, HSD3B), angiogenic (VEGFA121, FGF2), and vasoactive (EDN1) factors; and factors of prostaglandin synthesis (PGES, PGFS, PTGFR) and apoptosis (CASP3, -8, -9). Treatment with LPS abolished the hCG-induced increase in P4 (P≤0.05); however, there was a tendency (P=0.10) for increased release of PGF2α at 70 min after LPS challenge. Furthermore, mRNA abundance of TLR2, TNFA, CASP3, CASP8, PGES, PGFS, and VEGFA121 increased (P≤0.05) after LPS treatment, whereas all other factors remained unchanged (P>0.05). In conclusion, reduced P4 responsiveness to hCG in LPS-treated ovaries in vitro was not due to reduced steroidogenesis, but was attributed to enhanced apoptosis. However, an impact of luteal PGF2α could not be excluded.

Intramammary lipopolysaccharide infusion alters gene expression but does not induce lysis of the bovine corpus luteum.

Data from various studies indicate that the ovarian function in dairy cows can be compromised during intramammary infections. Therefore, in this study, we investigated if an experimentally induced mastitis has an effect on corpus luteum (CL) function in 14 lactating cows. On d 9 of the estrous cycle (d 1=ovulation), cows received a single dose of 200 µg of Escherichia coli lipopolysaccharide (LPS; dissolved in 10 mL of NaCL; n=8) or 10 mL of saline (control; n=6) into one quarter of the mammary gland. Measurements included plasma cortisol, haptoglobin, and progesterone (P4) concentrations, as well as luteal size (LTA) and relative luteal blood flow (rLBF). Sampling was performed on d 1, 4, and 8. On d 9, the main examination day, sampling was performed immediately before (0 h), every 1h (or at 3-h intervals for LTA and rLBF) until 9 h, as well as 12 and 24 h after treatment. Thereafter, measurements were taken on d 12, 15, 18, and then every 2 d until ovulation. Luteal tissue was collected for biopsy 24 h before and 6 h after treatment. Quantitative real-time PCR was applied to assess mRNA expression of steroidogenic factors (STAR, HSD3B), caspase 3, toll-like receptors (TLR2, -4), tumor necrosis factor α (TNFA), and prostaglandin-related factors (PGES, PGFS, PTGFR). Intramammary LPS infusion caused considerable inflammatory responses in the treated udder quarters. No decrease in plasma P4 concentrations was noted after LPS-challenge, and P4 levels did not differ between LPS-treated and control cows. Furthermore, LTA and rLBF values were not decreased after LPS challenge compared with the values obtained immediately before treatment. However, LPS infusion increased plasma levels of cortisol and haptoglobin compared with the control group. In the CL, mRNA abundance of TLR2 and TNFA was increased in cows after LPS-challenge (but not in control cows), whereas TLR4, steroidogenic, and prostaglandin-related factors remained similar to the mRNA abundance before treatment. In conclusion, intramammary LPS challenge induces systemic inflammatory reactions which alter the luteal mRNA abundance of TLR2 and TNFA but does not induce lysis of the CL.

Repeated intrauterine infusions of lipopolysaccharide alter gene expression and lifespan of the bovine corpus luteum.

Inflammation of the uterus is associated with disturbed ovarian function and reduced reproductive performance in dairy cows. To investigate the influence of endometritis on the bovine corpus luteum, 8 heifers received intrauterine infusions with either phosphate-buffered saline (PBS; 9mL) or Escherichia coli lipopolysaccharide (LPS; 3µg/kg of body weight diluted in 9mL of PBS) at 6-h intervals from 12h before and until 9d after ovulation during 2 cycles in a random order (ovulation=d 1). An untreated cycle was examined before and after PBS and LPS cycles, and the mean values from both untreated cycles were used as control. In all cycles, blood sampling and ultrasonography of the ovaries were performed on d 0, 1, 2, 4, 6, 8, 9, 10, 12, 15, 18, and then every 2d until ovulation. Endometrial cells were collected for cytology and quantitative real-time reverse transcriptase PCR on d 0, 6, and 9, and on d 0 and 6, respectively, and luteal tissue was collected for quantitative real-time reverse transcriptase PCR on d 6 and 9. Both, PBS and LPS infusions induced subclinical endometritis, which was accompanied by increased endometrial mRNA abundance of proinflammatory cytokines IL1ß, IL8, and tumor necrosis factor α. Additionally, LPS challenge induced premature luteolysis, which was characterized by increased plasma concentrations of PGF2α metabolite, decreased plasma progesterone concentrations, and reduced luteal size and blood flow compared with the control. The luteal mRNA expression of the LPS receptor TLR4, PGE synthase, and the apoptosis-related factor CASP3 were higher, and those of steroidogenic factors STAR and HSD3B, the PGF receptor, and the angiogenic factor VEGFA121 were lower after LPS challenge compared with the control. In conclusion, repeated intrauterine LPS infusions during the first 9d of the estrous cycle alter gene expression and shorten the lifespan of the bovine corpus luteum.

Colostrogenesis during an induced lactation in dairy cattle.

Colostrum immunoglobulin G (IgG) is of major importance for the newborn calf because epitheliochorial placentae do not provide transport in utero. The formation of colostrum occurs in the later stages of pregnancy. Our objectives were to induce lactation in non-pregnant dairy cows and (i) to determine the changes of IgG in serum and mammary secretions during the induction process and (ii) to establish α-lactalbumin (αLA) and prolactin (Prl) alterations to monitor the changing mammary epithelial tight junction status and development pattern. Estradiol-17ß (E2) and progesterone (P4) injections in a 1-7 days series were combined with a 3-day injection series (day 21-23) of dexamethasone (DEX). Blood and both front quarter secretion samples were collected daily. Milking started 24 days after the start of the experiment. Results show that the mammary secretory IgG1 was increased at >7 days after the start of steroid injections and depicted a bimodal pattern reaching a high of 16 mg/ml at 21 day compared with 3.2 mg/ml in the serum. There was a small increase in secretory IgG2 that did not correlate with tight junction status, but never reached the serum concentration. The injections of DEX resulted in constriction of tight junctions. Secretory αLA was immediately increased with steroid injections, dropped precipitously after 7 days and then began a steady increase until the start of milking. Changes in serum αLA are related to mammary tight junctions while serum Prl gradually increased from 30 to >60 ng/ml after the steroid injections stopped. These results provide insights into the mechanisms and timing of colostrogenesis during an induced lactation protocol.

Glucose transport and milk secretion during manipulated plasma insulin and glucose concentrations and during LPS-induced mastitis in dairy cows.

In dairy cows, glucose is essential as energy source and substrate for milk constituents. The objective of this study was to investigate effects of long-term manipulated glucose and insulin concentrations in combination with a LPS-induced mastitis on mRNA abundance of glucose transporters and factors involved in milk composition. Focusing on direct effects of insulin and glucose without influence of periparturient endocrine adaptations, 18 dairy cows (28 ± 6 weeks of lactation) were randomly assigned to one of three infusion treatments for 56 h (six animals each). Treatments included a hyperinsulinemic hypoglycaemic clamp (HypoG), a hyperinsulinemic euglycaemic clamp (EuG) and a control group (NaCl). After 48 h of infusions, an intramammary challenge with LPS from E. coli was performed and infusions continued for additional 8 h. Mammary gland biopsies were taken before, at 48 (before LPS challenge) and at 56 h (after LPS challenge) of infusion, and mRNA abundance of genes involved in mammary gland metabolism was measured by RT-qPCR. During the 48 h of infusions, mRNA abundance of glucose transporters GLUT1, 3, 4, 8, 12, SGLT1, 2) was not affected in HypoG, while they were downregulated in EuG. The mRNA abundance of alpha-lactalbumin, insulin-induced gene 1, κ-casein and acetyl-CoA carboxylase was downregulated in HypoG, but not affected in EuG. Contrary during the intramammary LPS challenge, most of the glucose transporters were downregulated in NaCl and HypoG, but not in EuG. The mRNA abundance of glucose transporters in the mammary gland seems not to be affected by a shortage of glucose, while enzymes and milk constituents directly depending on glucose as a substrate are immediately downregulated. During LPS-induced mastitis in combination with hypoglycaemia, mammary gland metabolism was more aligned to save glucose for the immune system compared to a situation without limited glucose availability during EuG.

Induced hyperketonemia affects the mammary immune response during lipopolysaccharide challenge in dairy cows.

Metabolic adaptations during negative energy and nutrient balance in dairy cows are thought to cause impaired immune function and hence increased risk of infectious diseases, including mastitis. Characteristic adaptations mostly occurring in early lactation are an elevation of plasma ketone bodies and free fatty acids (nonesterified fatty acids, NEFA) and diminished glucose concentration. The aim of this study was to investigate effects of elevated plasma ß-hydroxybutyrate (BHBA) at simultaneously even or positive energy balance and thus normal plasma NEFA and glucose on factors related to the immune system in liver and mammary gland of dairy cows. In addition, we investigated the effect of elevated plasma BHBA and intramammary lipopolysaccharide (LPS) challenge on the mammary immune response. Thirteen dairy cows were infused either with BHBA (HyperB, n=5) to induce hyperketonemia (1.7 mmol/L) or with a 0.9% saline solution (NaCl, n=8) for 56 h. Two udder quarters were injected with 200 µg of LPS after 48 h of infusion. Rectal temperature (RT) and somatic cell counts (SCC) were measured before, at 48 h after the start of infusions, and hourly during the LPS challenge. The mRNA abundance of factors related to the immune system was measured in hepatic and mammary tissue biopsies 1 wk before and 48 h after the start of the infusion, and additionally in mammary tissue at 56 h of infusion (8h after LPS administration). At 48 h of infusion in HyperB, the mRNA abundance of serum amyloid A (SAA) in the mammary gland was increased and that of haptoglobin (Hp) tended to be increased. Rectal temperature, SCC, and mRNA abundance of candidate genes in the liver were not affected by the BHBA infusion until 48 h. During the following LPS challenge, RT and SCC increased in both groups. However, SCC increased less in HyperB than in NaCl. Quarters infused with LPS showed a more pronounced increase of mRNA abundance of IL-8 and IL-10 in HyperB than in NaCl. The results demonstrate that an increase of plasma BHBA upregulates acute phase proteins in the mammary gland. In response to intramammary LPS challenge, elevated BHBA diminishes the influx of leukocytes from blood into milk, perhaps by via modified cytokine synthesis. Results indicate that increased ketone body plasma concentrations may play a crucial role in the higher mastitis susceptibility in early lactation.

Hyperketonemia during lipopolysaccharide-induced mastitis affects systemic and local intramammary metabolism in dairy cows.

Hyperketonemia interferes with the metabolic regulation in dairy cows. It is assumed that metabolic and endocrine changes during hyperketonemia also affect metabolic adaptations during inflammatory processes. We therefore studied systemic and local intramammary effects of elevated plasma ß-hydroxybutyrate (BHBA) before and during the response to an intramammary lipopolysaccharide (LPS) challenge. Thirteen dairy cows received intravenously either a Na-DL-ß-OH-butyrate infusion (n = 5) to achieve a constant plasma BHBA concentration (1.7 ± 0.1 mmol/L), with adjustments of the infusion rates made based on immediate measurements of plasma BHBA every 15 min, or an infusion with a 0.9% NaCl solution (control; n = 8) for 56 h. Infusions started at 0900 h on d 1 and continued until 1700 h 2 d later. Two udder quarters were challenged with 200 µg of Escherichia coli LPS and 2 udder quarters were treated with 0.9% saline solution as control quarters at 48 h after the start of infusion. Blood samples were taken at 1 wk and 2h before the start of infusions as reference samples and hourly during the infusion. Mammary gland biopsies were taken 1 wk before, and 48 and 56 h (8h after LPS challenge) after the start of infusions. The mRNA abundance of key factors related to BHBA and fatty acid metabolism, and glucose transporters was determined in mammary tissue biopsies. Blood samples were analyzed for plasma glucose, BHBA, nonesterified fatty acid, urea, insulin, glucagon, and cortisol concentrations. Differences were not different for effects of BHBA infusion on the mRNA abundance of any of the measured target genes in the mammary gland before LPS challenge. Intramammary LPS challenge increased plasma glucose, cortisol, glucagon, and insulin concentrations in both groups but increases in plasma glucose and glucagon concentration were less pronounced in the Na-DL-ß-OH-butyrate infusion group than in controls. In response to LPS challenge, plasma BHBA concentration decreased in controls and decreased also slightly in the BHBA-infused animals because the BHBA concentration could not be fully maintained despite a rapid increase in BHBA infusion rate. The change in mRNA abundance of citrate synthase in LPS quarters was significant between the 2 treatment groups. The results indicate that elevated circulating BHBA concentration inhibits gluconeogenesis before and during immune response to LPS challenge, likely because BHBA can replace glucose as an energy source.

Concomitant lipopolysaccharide-induced transfer of blood-derived components including immunoglobulins into milk.

During a mammary immune response, the integrity of the blood-milk barrier is negatively affected and becomes leaky. The aim of the present study was to demonstrate the blood origin, and to investigate changes in the concentration, of various constituents including immunoglobulins in blood and milk during the early phase of lipopolysaccharide (LPS)-induced mastitis. Five lactating dairy cows received continuous ß-hydroxybutyrate (BHBA) clamp infusions to maintain elevated BHBA blood concentrations (1.5 to 2.0 mmol/L) from 48 h before and 8h after LPS administration. One udder quarter was infused with 200 µg of Escherichia coli LPS. A second quarter served as control. Milk and blood samples were taken hourly for 8h postchallenge (PC). The somatic cell count in LPS-challenged quarters was increased from 4h PC to the end of the experiment compared with control quarters. In LPS-challenged quarters, l-lactate, BHBA, lactate dehydrogenase (LDH), IgG(1), and IgG(2) were increased at 3h PC and remained elevated until the end of experiment (8h PC) compared with control quarters. In addition, the optical density values in milk in a nonquantitative ELISA for antibodies directed against bluetongue virus (used as a measure of nonspecific antibody transfer; all animals were vaccinated) increased and, thus, indicates an increase in these antibodies in response to LPS treatment. l-Lactate concentration also increased in blood 2h PC and in the milk of control quarters during the experiment from 3h PC. A second experiment was conducted in vitro to investigate a possible contribution from destructed milk cells to l-lactate concentration and activity of LDH in milk. Aliquots of milk samples (n=8) were frozen (-20°C) or disrupted with ultrasound, respectively. Freeze thawing and ultrasound treatment increased LDH in milk samples, but had no effect on l-lactate concentrations. Results suggest that intramammary infusion of LPS induces a systemic response, as evidenced by an elevation of blood l-lactate concentration. The concomitant changes of all investigated components suggest that they were blood derived. However, the increase in blood components in the milk is not necessarily supportive of the mammary immune system, and likely a side effect of reduced blood-milk barrier integrity.

Short communication: differential immunoglobulin transfer during mastitis challenge by pathogen-specific components.

Mastitis induced by Escherichia coli is often characterized by severe clinical signs, indicating a more powerful combat of the immune system against the pathogen compared with Staphylococcus aureus infections, which are often represented by chronic and subclinical diseases. The aim of this study was to test the major pathogenic component lipopolysaccharide (LPS) from E. coli and lipoteichoic acid (LTA) from Staph. aureus for their effects on blood-milk barrier integrity and the related transfer of immunoglobulins and lactate from blood into milk. A similar somatic cell count (SCC) increase was achieved by intramammary challenge of 1 quarter of 5 cows with 20 µg of LTA, and 8 cows with 0.2 µg of LPS (maximum log SCC/mL: 7). Milk IgG(1) concentrations increased in LPS- but not in LTA-challenged quarters. Milk IgG(2) concentrations were increased in treated quarters at 3h after LPS, and 6h after LTA challenge. Higher maximum levels of IgG(2) were reached in milk of LPS-treated quarters (173 ± 58 µg/mL) than of LTA-challenged quarters (62 ± 13 µg/mL). Immunoglobulin G(1) and IgG(2) levels did not change in control quarters. l-Lactate concentrations in milk increased 4h after LPS and 5h after LTA challenge and reached higher maximum levels in LPS- (221 ± 48 mg/L) than in LTA-treated quarters (77 ± 18 mg/L). In conclusion, a mammary inflammation on a quantitatively similar level based on SCC increase achieves a more efficient transfer of blood components such as IgG(2) via the blood-milk barrier if induced by LPS from E. coli than by LTA from Staph. aureus. This pathogen-specific difference may play an important role in the cure rate of the respective intramammary infection, which is usually lower in Staph. aureus- than in E. coli-induced mastitis.

Long-term elevation of ß-hydroxybutyrate in dairy cows through infusion: effects on feed intake, milk production, and metabolism.

Elevation of ketone bodies in dairy cows frequently occurs in early lactation, usually concomitantly with a lack of energy and glucose. The objective of this study was to induce an elevated plasma ß-hydroxybutyrate (BHBA) concentration over 48 h in mid-lactating dairy cows (i.e., during a period of positive energy balance and normal glucose plasma concentrations). Effects of BHBA infusion on feed intake, metabolism, and performance were investigated. Thirteen cows were randomly assigned to 1 of 2 infusion groups, including an intravenous infusion with Na-dl-ß-OH-butyrate (1.7 mol/L) to achieve a plasma concentration of 1.5 to 2.0 mmol/L of BHBA (HyperB; n=5), or an infusion of 0.9% saline solution (control; n=8). Blood was sampled before and hourly during the 48 h of infusion. In the liver, mRNA transcripts related to gluconeogenesis (pyruvate carboxylase, glucose 6-phosphatase, mitochondrial phosphoenolpyruvate carboxykinase), phosphofructokinase, pyruvate dehydrogenase complex, and fatty acid synthesis (acetyl-coenzyme A carboxylase, fatty acid synthase) were measured by real-time PCR. Glyceraldehyde-3-phosphate dehydrogenase and ubiquitin were used as housekeeping genes. Changes (difference between before and after 48-h infusion) during the infusion period were evaluated by ANOVA with treatment as fixed effect, and area under the curve of variables was calculated on the second day of experiment. The plasma BHBA concentration in HyperB cows was 1.74 ± 0.02 mmol/L (mean ± SE) compared with 0.59 ± 0.02 mmol/L for control cows. The change in feed intake, milk yield, and energy corrected milk did not differ between the 2 experimental groups. Infusion of BHBA reduced the plasma glucose concentration (3.47 ± 0.11 mmol/L) in HyperB compared with control cows (4.11 ± 0.08 mmol/L). Plasma glucagon concentration in HyperB was lower than the control group. All other variables measured in plasma were not affected by treatment. In the liver, changes in mRNA abundance for the selected genes were similar between 2 groups. Results demonstrate that intravenous infusion of BHBA decreased plasma glucose concentration in dairy cows, but this decrease could not be explained by alterations in insulin concentrations or key enzymes related to gluconeogenesis. Declined glucose concentration is likely functionally related to decreased plasma glucagon concentration.