J.W. Blum

Tumor necrosis factor-α and nitrite/nitrate responses during acute mastitis induced by Escherichia coli infection and endotoxin in dairy cows

Concentrations of tumor necrosis factor-alpha (TNF-alpha) and of NO(x) (sum of nitrite and nitrate as indicators of endogenous nitric oxide production) in milk and blood plasma were measured in three mastitis models in dairy cows in early lactation. Escherichia coli P4:O37 bacteria or endotoxin O111:B4 were administered into both left quarters of 12 and 6 cows, respectively. Six of the E. coli-infected cows were treated with a bactericidal antibiotic (Enrofloxacin; Bayer AG, Leverkusen, Germany) i.v. at 10 hr and subcutaneously (sc) at 30 hr after infection. NO(x) concentrations transiently increased maximally 10- to 11-fold in milk of E. coli-infected quarters with or without antibiotic treatment at 24 hr and after endotoxin administration. NO(x) concentrations did not change in milk of unchallenged quarters and in blood plasma. Increases of NO(x) were proceeded by a transient (96- to 149-fold) rise of milk TNF-alpha concentrations, which in endotoxin-administered quarters was maximal at 6 hr and in infected quarters without or with Enrofloxacin treatment at 10 and 14 hr. In blood plasma TNF-alpha concentrations only moderately increased to peaks in endotoxin-administered cows at 6 hr and in E. coli-infected cows at 14 hr postchallenge. In one severely sick, nontreated E. coli-infected cow milk, TNF-alpha response at 14 hr was excessive and followed by a spectacular rise of NO(x) concentration in milk between 48 and 72 hr. In conclusion, a possible clinical relevance of nitric oxide production associated with a rise of intramammary and systemic TNF-alpha during acute mastitis by E. coli infection and endotoxin in lactating dairy cows is indicated, but could not be inhibited by antibiotic treatment.

Modulation of the inflammatory reaction and neutrophil defense of the bovine lactating mammary gland by growth hormone

This review is focused on the possible interactions of prolactin and somatotrope hormone in the modulation of inflammation of the mammary gland. Several different models are examined: Escherichia coli, Streptococcus uberis, and endotoxin mastitis. Subsequently, the release of growth hormone and insulin-like growth factor during fever and mastitis, the immunophysiological effects of GH on E. coli mastitis, S. uberis and endotoxin mastitis, the galactopoietic action of rBST on healthy and mastitis cows as well as the immunologic effects of GH on leukocytes in healthy and diseased cows are discussed. It can be concluded that the underlying regulation of the neuro-endocrine network is fundamental in the normal function of the immune system.

Detection and quantification of mRNA expression of α- and β-adrenergic receptor subtypes in the mammary gland of dairy cows

Adrenergic receptors are pharmacologically classified into the receptor types 1, 2, 1, 2, and 3. Structural differences and varying affinities in radioligand binding studies lead to a further classification of 1- and 2-receptors into subtypes which are termed 1A (formerly 1C), 1B, and 1D (formerly 1AD), and 2AD, 2B, and 2C, respectively. mRNA expression of all but one -adrenergic receptor subtypes and of all -adrenergic receptor types was measured quantitatively in total RNA extracted from mammary tissue of 10 lactating dairy cows by real-time reverse transcription (RT) polymerase chain reaction (PCR). mRNA expression of1-adrenergic receptors was highest for the 1A-subtype followed by 1B, whereas the 1D-subtype could not be detected. The highest mRNA expression of2-adrenergic receptors was found for the 2AD-subtype, followed by 2B and 2C. Within the -adrenergic receptors, the 2-receptor type was most highly expressed, followed by 1 and 3. In conclusion, eight of nine adrenergic receptors classified to date were detected and relatively quantified in the mammary gland of dairy cows.

mRNA of insulin-like growth factor (IGF) quantification and presence of IGF binding proteins, and receptors for growth hormone, IGF-I and insulin, determined by reverse transcribed polymerase chain reaction, in the liver of growing and mature male cattle

Plasma insulin-like growth factor-I (IGF-I) concentrations were related to hepatic levels of IGF-I mRNA measured by competitive reverse transcription polymerase chain reaction (PCR) (RT-PCR) in neonatal (8 d old) calves, veal calves, fattened castrated bulls and mature intact bulls. Furthermore, the presence of mRNAs of IGF-II and of receptors for IGF-I (IGF-IR), growth hormone (GHR) and insulin (IR), as well as mRNAs of IGF binding proteins (IGFBP-1, -2 and -3) were assessed by RT-PCR. Hepatic IGF-I mRNA levels and plasma IGF-I concentrations in veal calves, fattened castrated bulls and in intact bulls were 4 to 8 times higher than in 8-d old calves and were 2 to 3 times higher in calves fed colostrum than in calves fed only milk replacer. Hepatic IGF-I mRNA concentrations were closely correlated (r = 0.92) with plasma IGF-I concentrations, suggesting that hepatic IGF-I production largely determines plasma IGF-I levels. The presence of IGF II, IGF-IR, GHR, IR and IGFBP-1, -2 and -3 mRNA was confirmed in the liver of 8-d old calves and older cattle as well, and among newborn calves their presence was independent of differences in nutrition. In conclusion, the major hepatic components of the GH-IGF axis were present in neonatal calves, but the IGF-I expression and therefore also plasma IGF-I levels were relatively low.

Energy metabolism in the newborn farm animal with emphasis on the calf: endocrine changes and responses to milk-born and systemic hormones

Neonatal mammals need adaption to changes in nutrient supply because energy intake shifts from continuous parenteral supply of nutrients (mainly glucose, lactate, and amino acids) via the placenta to discontinuous colostrum and milk intake with lactose and fat as main energy sources. Besides ingested lactose, endogenous glucose production is essential in the neonate to assure sufficient glucose availability. Fetal endogenous glucose production is low, but endocrine changes (especially the prenatal rise of glucocorticoid production) promote maturation of metabolic pathways that enable marked glycogen synthesis before and enhanced gluconeogenesis after birth to establish an adequate glucose status during postnatal maturation. In preterm born farm animals gluconeogenic activity is low, mainly because of a low glucocorticoid and thyroid status. In full-term neonates, endogenous glucose production increases with age. Colostral bioactive components (such as growth factors, hormones, bioactive peptides, and cytokines) do not have a direct effect on endogenous glucose production. However, colostrum feeding stimulates intestinal growth and development, an effect at least in part mediated by bioactive substances. Increased nutrient and glucose absorption thus allows increased glucose supply and hepatic glycogen storage, which improves the glucose status. The improved energetic status of colostrum-fed neonates is reflected by an accelerated maturation of the somatotropic axis, leading especially to enhanced production of IGF-I in the neonate. Secretion and production of hormones involved in the regulation of glucose and fat metabolism in neonates depend on the developmental stage and the response to feeding. In addition, many such hormones have actions in the neonate that differ from adult animals. Endocrine action to support endogenous energy supply in neonates is probably not fully established, and therefore, needs postnatal maturation. Therefore, our knowledge on energy metabolism in the neonate needs to be extended to better understand the function and the failure and to assess endocrine responses during the neonatal period.

Effects of glucose, propionic acid, and nonessential amino acids on glucose metabolism and milk yield in Holstein dairy cows

Whole-body glucose rate of appearance (Ra) responses and milk lactose secretion were compared in dairy cows receiving duodenal infusions of glucose (Glc), a mixture of 5 nonessential amino acids (NEAAm), or ruminal infusions of propionic acid (C3). Four mid-lactation Holstein cows, fitted with both duodenum and rumen cannulas, were used in a 4 x 4 Latin square design with 14-d periods. Cows were fed a grass silage-based diet (Ctrl) that provided 88% of net energy of lactation and 122% of protein requirements. Concentrate was formulated with wheat (21.5%) and barley (20%) containing some starch. Isoenergetic infusions (5.15 Mcal/d of digestible energy) of Glc into the duodenum (7.7 mol/d), C3 into the rumen (14.1 mol/d), or NEAAm into the duodenum (in mol/d; Ala: 1.60; Asp: 0.60; Glu: 5.94; Gly: 1.22; Ser: 2.45) were given as a supplement to the Ctrl diet. During each period on d 13, [6,6-(2)H(2)]glucose was infused into one jugular vein and blood samples were taken from the other jugular vein to measure glucose enrichment and determine Ra. Dry matter intake decreased slightly with the infusions (6%), but did not differ among them. Whole body glucose Ra averaged 502, 745, 600, and 576 mmol/h for Ctrl, Glc, C3, and NEAAm, respectively. It increased with the increase in energy supply (Ctrl vs. infusions) and differed according to the nutrients infused. The Ra response was higher with Glc and C3 than with NEAAm and higher with Glc than with C3. Plasma concentrations of insulin were not affected, but insulin-like growth factor 1 increased with infusions. Plasma glucagon increased with NEAAm, which could favor the increased Ra. Overall, milk lactose yield (137, 141, 142, and 130 mmol/h for Ctrl, Glc, C3, and NEAAm, respectively) was not modified by the infusions, but was lower with NEAAm compared with Glc and C3. Changes in lactose yield did not parallel the increase in Ra, and therefore the ratio of lactose yield to Ra decreased with the infusions and was lower in Glc compared with C3, suggesting a shift of glucose utilization away from lactose synthesis toward other pathways, including mammary metabolism. Intestinal Glc was the most efficient nutrient in terms of increasing glucose Ra; however, there was no direct link between the increases in whole body glucose Ra observed with the 3 types of nutrients and milk lactose yield.

Limitations of high Alpine grazing conditions for early-lactation cows: effects of energy and protein supplementation

In each of 2 years (years A and B), the effects of three nutritional regimes were examined using 12 cows kept at pasture for 77 days at 2000 m above sea level. Two supplement formulations (1 and 2) were designed, both equally high in readily fermentable energy (14·6 MJ metabolizable energy (ME) per kg dry matter (DM)) but differing in crude protein content (53 and 193 g/kg DM). The treatments imposed were either pasture grass alone (control groups; both seasons) or grass and supplements provided at three levels relative to energy (E) and protein (P) maintenance requirements estimated for lowland conditions. These levels were (i) 1·0 E: 0·8 P provided by 4·6 kg of supplement 1 per day in both years; (ii) 0·5 E: 0·4 P provided by 2·2 kg of supplement 1 per day in year A; (iii) 1·0 E: 2·5 P provided by 4·4 kg of supplement 2 per day in year B. Intensive measurement periods, including food intake estimation by the double alkane technique, were carried out in weeks 3, 7 and 11 on Alpine pasture. Performance data, plasma levels of indicative blood traits and body condition were additionally monitored in the cows at a lowland site for 2 weeks prior to transport. Supplementing with a high energy/low protein concentrate gave no clear benefit in milk yield, which declined proportionately by 0·33 in the 11 weeks under Alpine conditions. Provision of additional supplementary protein (supplement 2) resulted in a proportionate decline in milk yield of only 0·20 over the 11-week period. Cows exhibited high substitution ratios of 1·4 to 2·6 kg herbage DM per kg concentrate DM and cows on all treatments were estimated to consume similar amounts of ME. A combination of reduced fibre intake and lower fibre digestibility with supplementation significantly reduced milk fat contents to low levels. Energy supplementation significantly reduced plasma β-hydroxybutyrate levels. However, live-weight and body tissue loss rates (based on ultrasonic scans of subcutaneous fat layer and longissimus dorsi muscle) were equally great with and without energy supplementation but less severe with extra protein (supplement 2). Adaptation to the high Alpine conditions was achieved in all treatments as indicated by increased blood haemoglobin, accompanied by a lower plasma level of insulin-like growth factor-I. Plasma thyroid hormone levels suggested that adaptation to energy deficiency and possibly to cold was more effective with supplementary energy. Other energy-dependent blood metabolites and insulin responded similarly in all treatments to high altitude grazing. Estimated maintenance energy requirement for Alpine conditions was 0·72 times greater than lowland maintenance requirement.

High constitutional nitrate status in young cattle.

Nitrate or nitrite can be ingested or endogenously produced from nitric oxide. They can cause intoxication and are of general concern for health because they relate to various diseases. Our goal was to study ontogenetic and nutritional effects on the nitrate+nitrite (NOx-) status in cattle, particularly calves. NOx- concentration in blood plasma, cerebrospinal fluid, saliva, and urine was measured based on nitrate conversion by added nitrate reductase to nitrite, which was then determined by the Griess reaction. Concentrations of nitrate were the result of the difference between NOx- and nitrite values. Nitrate in blood plasma, saliva and urine was > or =97% and in cerebrospinal fluid of calves was approximately 35% of NOx-. Preprandial plasma NOx- in calves born after shortened or normal lengths of pregnancy (277 and 290 days) was 470 and 830 micromol/l, respectively, decreased within 4-7 days to 40-60 micromol/l, remained in this range up to 4 months, was < or =5 micromol/l in heifers and no longer measurable in 3-8-year-old cows. Cerebrospinal NOx- in 8-day-old calves was 14 micromol/l and approximately 11-fold lower than in blood plasma. Salivary NOx- decreased postnatally from 600 to 200 micromol/l at 2 days and to 25 micromol/l at 4 weeks. Urinary NOx- excretion decreased from 125 or 16 micromol/l per kg x 24 h in 5-day-old calves to 45 or 8 micromol/kg x 24 h between 10 and 115 days of life and was undetectable in urine of heifers and cows. Feeding neonatal calves no or variable amounts of colostrum, delaying colostrum intake by 24 h after birth or feeding at different feeding intensity had no effect on the NOx- status. In conclusion, the high plasma, salivary and urinary NOx- concentrations especially in newborn calves, ingesting but insignificant amounts of nitrite or nitrate, indicated marked endogenous formation of nitrate, which decreased with age. The high nitrate status may contribute to enhanced susceptibility of young calves to exogenous nitrite+nitrite ingestion.

Messenger RNA Levels and Binding Sites of Muscarinic Acetylcholine Receptors in Gastrointestinal Muscle Layers from Healthy Dairy Cows

Acetylcholine interacts with muscarinic receptors (M) to mediate gastrointestinal (GI) smooth muscle contractions. We have compared mRNA levels and binding sites of M1to M5 in muscle tissues from fundus abomasi, pylorus, ileum, cecum, proximal loop of the ascending colon (PLAC), and external loop of the spiral colon (ELSC) of healthy dairy cows. The mRNA levels were measured by quantitative RT-PCR. The inhibition of [3H]-QNB (1-quinuclidinyl-[phenyl-4-3H]-benzilate) binding by M antagonists [atropine (M1 − 5), pirenzepine (M1), methoctramine (M2), 4-DAMP (M3), and tropicamide (M4)] was used to identify receptors at the functional level. Maximal binding (Bmax) was determined through saturation binding with atropine as a competitor. The mRNA levels of M1, M2, M3, and M5 represented 0.2, 48, 50, and 1.8%, respectively, of the total M population, whereas mRNA of M4 was undetectable. The mRNA levels of M2 and of M3 in the ileum were lower (P < 0.05) than in other GI locations, which were similar among each other. Atropine, pirenzepine, methoctramine, and 4-DAMP inhibited [3H]-QNB binding according to an either low- or high-affinity receptor pattern, whereas tropicamide had no effect on [3H]-QNB binding. The [3H]-QNB binding was dose-dependent and saturable. Bmax in fundus, pylorus, and PLAC was lower (P < 0.05) than in the ELSC, and in the pylorus lower (P < 0.05) than in the ileum. Bmax and mRNA levels were negatively correlated (r = -0.3; P < 0.05). In conclusion, densities of M are different among GI locations, suggesting variable importance of M for digestive functions along the GI tract.

Low-dietary protein intake induces problems with glucose homeostasis and results in hepatic steatosis in heavy milk-fed calves.

We studied effects of protein intake at two protein-free energy intake levels on plasma glucose and insulin concentrations, urinary glucose excretion and on liver and intestinal fat content in milk-fed veal calves. Two experiments were performed at body weights (BW) of 80-160 kg (mean 120 kg; Exp. 1) and 160-240 kg (mean 200 kg; Exp. 2). In each experiment, 36 calves were allocated to one of six protein intake levels, at each of two energy intake levels. Digestible protein intakes ranged between 0.90 and 2.72 g nitrogen (N)/(kg BW(0.75) x d) in Exp. 1 and between 0.54 and 2.22 g N/(kg BW(0.75)x d) in Exp. 2. The two energy intake levels were kept constant on a protein-free basis and were 663 and 851 kJ/(kg BW(0.75) x d) in Exp. 1 and 564 and 752 kJ/(kg BW(0.75)x d) in Exp. 2. Blood samples were taken between 5 and 6h post-feeding at 14-d intervals until calves reached target BW, and liver fat mass was determined at slaughter. Urinary glucose excretion was quantified at 120 and 200 kg BW in Exps. 1 and 2, respectively. Increased protein-free energy intake increased plasma glucose concentrations and urinary glucose losses in 200 kg calves, but not in 120 kg calves. Increasing protein intake decreased plasma glucose, urinary glucose and plasma insulin in both experiments. Liver fat content decreased with increasing protein intake. In conclusion, long-term low-dietary protein intake increased hyperglycemia, hyperinsulinemia, glucosuria and hepatic steatosis in heavy milk-fed calves, likely associated with increased insulin resistance.