Eddy Roets

Classification of newly calved cows into moderate and severe responders to experimentally induced Escherichia coli mastitis.

In the present study newly calved cows were tentatively classified as moderate and severe responders to experimentally induced Escherichia coli mastitis based upon the reactive oxygen species (ROS)-generating capacity of their blood neutrophils before infection. The groups differed in blood and milk composition prior to infection. This initial classification was supported by the corresponding variation in clinical symptoms and in the changes in milk production and composition measured during mastitis. Responses of newly calved cows to Esch. coli challenge varied from mild to severe symptoms of inflammation in infected glands and differed in the intensity of systemic disturbances and general illness. Losses in milk yield and compositional changes were most pronounced in inflamed glands and in severe responders. In inflamed glands milk yield and composition did not return to preinfection level in either moderate or severe responders. The yields of lactose, alpha-lactalbumin, casein and fat followed the same pattern as milk yield. It is concluded that the severe and long lasting systemic disturbances observed in severe responders can be ascribed to absorption of endotoxin from infected glands into circulation, indicating the important role of endotoxin in the pathology of coliform mastitis in periparturient cows. Evaluation of the ROS-generating capacity of blood neutrophils and blood and milk composition before infection might help to predict the cows sensitivity to Esch. coli mastitis.

Increased surface expression of CD11b receptors on polymorphonuclear leukocytes is not sufficient to sustain phagocytosis during Escherichia coli mastitis in early postpartum dairy cows.

Phagocytosis, CD11a and CD11b adhesion receptor expression, O2-production and maturity of circulating polymorphonuclear leukocytes (PMN) were studied during acute coliform mastitis in early postpartum dairy cows to obtain a better insight in the role of neutrophils in the pathology of this disease. The mammary gland of twelve newly calved high-yielding dairy cows was experimentally infected with Escherichia coli. Variability in clinical signs of mastitis and inhibition of milk production among cows was very high. There was a significant negative correlation between the number of circulating neutrophils immediately before infection and severity of mastitis represented by the decrease in milk production of non-infected quarters two days after infection. Pre-infection phagocytosis of E. coli, CD11a and CD11b receptor expression, phorbol myristate acetate (PMA)-induced O2-production and maturity of neutrophils on a per cell basis were not related to severity of mastitis. However, significant correlations between severity of mastitis and the total number of phagocytic PMN and mature PMN in blood immediately before infection were found. PMN characteristics responded differently to mastitis depending on the severity of the disease. Neutrophil functions from cows classified as severe (S) and moderate (M) responders to infection of the mammary gland were compared. Surface expression of CD11a receptors on PMN was decreased in all cows 24 h after infection, and this decrease was long-continued in S responders. A biphasic upregulation of the number of CD11b receptors on PMN was observed with a more pronounced response in S cows. PMN phagocytosis was decreased 12 h after infection in S cows and 18 h after infection in S and M cows and was normalized 24 h post-infection. The decrease of phagocytosis coincided with the first peak of CD11b receptor expression. Phorbol myristate acetate (PMA)-induced production of O2-by PMN was decreased for three days after infection in S responders compared to only one day in M responders and was followed by an upregulation. These data demonstrate a complexity in alterations of PMN functions during mastitis and suggest the involvement of differences in systemic factors dependent on severity of mastitis.

Variations in the concentrations of free amino acids in the plasma of the dairy cow at parturition

The plasma levels of individual amino acids were studied in 6 dairy cows from 4 days before to 3 days after calving. During this sampling period, the concentrations of 13 amino acids showed significant changes. The levels of several amino acids were depressed markedly in the sample collected immediately before calving. Following parturition, the concentration of most amino acids gradually returned to values obtained 3 days before calving. The glutamine and alanine contents of the plasma rose to a peak value 1 day after calving and subsequently decreased. The mean concentrations of glycine and α-aminobutyric acid did not change before parturition but rose significantly thereafter. These observations are discussed in terms of amino-acid utilization for milk protein synthesis and gluconeogenesis at the onset of lactation. The changes in plasma amino acid levels appear to be synchronized with those reported for prolactin and progesterone in the 24 h before parturition. This may indicate an important influence of both hormones on the lactogenic process in the cow. The highly significant correlations obtained between the concentrations of 14 individual amino acids are discussed.

Metabolism of [U-14C; 2, 3-3H]-L-valine by the isolated perfused goat udder

Two lactating mammary glands excised from 2 goats were perfused for several hours in the presence of [U-14C; 2,3-3H]-L-valine and received adequate quantities of glucose, acetate and amino acids. In the synthesized milk 96 and 89% respectively of the casein valine was derived from free plasma valine. Valine was extensively catabolized by mammary tissue, resulting in a considerable 14CO2 production and in the incorporation of 14C into milk citric acid and to a lesser extent into casein aspartic acid and glutamic acid. About 30% of the valine molecules which were taken up by the mammary gland were oxidized to CO2 and 70% were incorporated in casein as valine residues. About 10% of the plasma valine molecules were reversibly transaminated during one passage through the udder. An important amount of radioactivity of plasma was present in unknown metabolites. Only 7% of this activity was localized in isobutyrate. The radioactivity of total milk fat was very low. Mainly iso-14:0, iso-16:0 and 15:0 were labelled.

Metabolism of leucine by the isolated perfused goat udder

Seven lactating goat mammary glands from 6 goats were perfused for several hours in the presence of [U-14C]L-leucine (4 experiments) or [2-3H; 1-14C]DL-leucine (3 experiments) and received adequate quantities of glucose, acetate and amino acids. Radioactivity in casein was mainly recovered in leucine and 90% of casein leucine was derived from free plasma leucine. About 64% of the leucine molecules were used for casein synthesis. Up to 12% of the molecules were channelled into lipid synthesis, while the remaining (up to 24%) were metabolized to CO2. From the 3H/14C ratio of casein and casein leucine, it was calculated that 70-80% of the leucine molecules were reversibly transaminated before their incorporation into milk protein. However, only 4-8% of the plasma leucine molecules were transaminated during passage through the udder. Different pools for oxidation and for protein synthesis may be present in the goat mammary gland.

The influence of ageing on muscarinic receptors, β-adrenoceptors and adenylate cyclase activity in the bovine lung

Muscarinic and β-adrenoceptors were identified in airway epithelium, smooth muscle and lung parenchyma from Holstein-Friesian calves and cows and were characterized with [3H]quinuclidinyl benzilate and [3H]dihydroalprenolol, respectively. The muscarinic receptor density in the smooth muscle of cows (Bmax=4803±245 fmol/mg protein) was 33% greater (p<0.01) than in calves. Low receptor numbers were detected in the epithelium and parenchyma. In both calves and cows, the density of epithelial β-adrenoceptors was twice as high as in smooth muscle and parenchyma. The quantity of β-adrenoceptors in the tracheal epithelium (Bmax=994±83 fmol/mg protein) and smooth muscle (Bmax=492±41 fmol/mg protein) in cows was respectively 37% (p<0.001) and 35% (p<0.01) lower than in calves. Adenylate cyclase (AC) assays indicated that the basal and the (−)-isopropylnoradrenaline- (ISO-) stimulated cAMP production were not significantly different between the calves and cows. After stimulation with NaF, significantly higher cAMP production was found in all tissues from cows. Significant correlations were found between absolute AC responses to NaF and β-adrenoceptor density in epithelium (r=−0.75,p<0.001) and smooth muscle (r=−0.63,p<0.01). It seems that, in older animals, the production of cAMP is independent of the number of receptors, indicating the presence of fully active compensatory mechanisms.

Relationship between numbers of α2- and β2-adrenoceptors on blood cells of bulls and milkability of their daughters

Milk yield and milking time mere measured on one occasion for several daughters (n = 6-44) from 16 bulls at morning milkings. Blood from the bulls was collected, and platelets and mononuclear leucocytes were isolated. The alpha(2)-adrenoceptors on platelet membranes were identified by binding of [H-3]rauwolscine, whereas for the determination of beta(2)-adrenoceptors on intact mononuclear leucocytes, [H-3]CGP-12177 was used. It was found that mean milk flow rate was highly correlated (P < 0.001) with the alpha(2)-adrenoceptor densities on blood platelets. No correlation was found with the beta(2)-adrenoceptors on mononuclear leucocytes. It is concluded that estimation of the alpha(2)-adrenoceptors on blood platelets from bulls could eventually be used to investigate milking characteristics of cows, and might be useful in the future as a marker in genetic studies.

Effect of atropine on plasma amino acid levels and milk secretion of cows

Subcutaneous injections of 30 mg atropine into lactating cows induced a 20--40% decrease of free amino acid (AA) levels in arterial plasma. Minimum levels were observed after 30--50 min. The decline persisted for more than 6 h. The greatest fall in concentration was noted for tyrosine, methionine, lysine, arginine, phenylalanine and threonine. Arterial glucose levels remained unaffected. The effect of atropine on milk secretion was studied in 2 cows which were milked every hour with the aid of oxytocin. Maximal effects were observed after 3--4 h. They included reduction in concentration of casein and alpha-lactalbumin (alpha-la) and a decline in production of milk (20%), casein (35%), alpha-la (45%) and lactose (18%). Uptake by the lactating udder over a period of about 1 h after injection of atropine was studied in 2 cows. Mammary blood flow and glucose uptake remained unaffected. There was a positive correlation between arteriovenous differences of essential AA and arterial plasma concentrations. The uptake of essential AA decreased by approximately 50%. There was no evidence that atropine has a direct inhibiting effect on the udder. It is suggested that the decrease of alpha-la synthesis might induce an inhibition of lactose synthesis and milk production.

Determination of beta-adrenoceptors and camp in muscle from normal and splayleg Belgian landrace pigs.

Abstractβ-Adrenoceptors were identified and characterized by [3H]dihydroalprenolol ([3H]DHA) binding experiments in muscle membrane preparations from piglets. The [3H]DHA binding was rapid, reversible and stereoselective. Catecholamines competed for specific binding with a rank order of potency (-)-isopropylnoradrenaline > (-)-epinephrine » (-)-norepinephrine, indicating a β-subtype of adrenoceptor. Saturation binding experiments with [3H]DHA showed no significant difference for either the number of binding sites or the equilibrium dissociation constants in normal and splayleg pigs.Adenylate cyclase assays indicated that the basal adenylate cyclase activity and the prostaglandin E1 (PGE1)-, (-)-isopropylnoradrenaline (ISO)-, 5′-guanylyl-imidodiphosphate (GppNHp)- and sodium fluoride (NaF)-stimulated values were not significantly different in normal and splayleg pigs. In both groups, PGE1 did not affect basal activity, whereas ISO and GppNHp stimulated adenylate cyclase activity significantly (p<0.001) to about 40% above basal level. NaF induced a significant (p<0.001) increase of cAMP in normal and splayleg pigs amounting to 48% and 61% respectively. Significant correlations between absolute adenylate cyclase responses to ISO (r=0.83***), NaF (r=0.72**) and GppNHp (r=0.61*) and basal activity in the splayleg pigs were the most striking findings. In contrast, these correlations could not be detected in the normal pigs. Whether or not this observation reflects an alteration in the signal transduction system needs to be further investigated.To the best of our knowledge this is the first biochemical study which relates an altered β-adrenoceptor function and porcine splayleg.

Muscarinic receptor subtypes, β-adrenoceptors and cAMP in the tracheal smooth muscle of conventional and double-muscled calves

The total muscarinic (M1 + M2 + M3) and β-adrenergic receptors in the tracheal smooth muscle of conventional and double-muscled calves were identified and characterized with the non-specific antagonists [3H]quinuclidinyl benzilate ([3H]QNB) and [3H]dihydroalprenolol ([3H]DHA) respectively.Although the quantity of β-adrenoceptors in double-muscled calves was 25% lower (p<0.05) than in conventional calves (Bmax=327±89 fmol/mg protein), adenylate cyclase assays indicated that the basal adenylate cyclase activity and the (−)-isopropylnoradrenaline (ISO)- and sodium fluoride (NaF)-stimulated values were not significantly different between these calves. However, the density of muscarinic receptors in double-muscled calves was 40% higher (p<0.01) than in conventional calves (Bmax=2955±625 fmol/mg protein). Subtypes of muscarinic receptors were studied with [3H]telenzepine (M1-receptors), [3H]AF-DX 384 (M2-receptors) and [3H]4DAMP (M1 and M3-receptors). It was found that in both double-muscled and conventional calves about 40% of the receptors were of the M3-subtype, the remaining 60% being M2-receptors.From these results, it is suggested that inflammation of the respiratory tract in double-muscled calves may be complicated by an imbalance between the cholinergic bronchoconstrictor and the β-adrenergic bronchodilator components of the autonomic nervous system.