Effects of Staphylococcus aureus mastitis after endotoxin application on milk yield and composition during subsequent lactation of guinea-pigs.

The effects of Staphylococcus aureus mastitis on milk yield and composition throughout subsequent lactation in lactating guinea-pigs and the role of endotoxin pretreatment on these phenomena were investigated. Primiparous lactating guinea-pigs were intramammarily inoculated with sterile saline (group 1), S. aureus strain UC 6097 (group 2), or with S. aureus UC 6097 after endotoxin pretreatment (group 3). Clinical signs and survival rate were monitored. During the second lactation, daily milk yield was measured and milk composition was determined. In primiparous inoculated guinea-pigs, moderate (group 3)-to-severe acute mastitis (group 2) was produced. During subsequent lactation, milk yield in the control group peaked on day 5 and then decreased. Concentrations of Na+ and Cl- in milk, and concentrations of fat, gradually increased, but lactose and K+ decreased. After an early decrease, NAGase in milk increased towards the end of lactation. Except for higher NAGase concentrations in group 3, milk yield and composition during the second lactation did not differ significantly between the mastitis and the control groups. Endotoxin pretreatment only plays a role in the determination of the severity of the infection.

Effect of somatotropin on changes in milk production and composition during coliform mastitis in periparturient cows.

The potential protective and therapeutic effects of bST during coliform mastitis in periparturient cows were evaluated. In a first study, 19 cows, classified as moderate or severe responders based on the respiratory burst activity of blood neutrophils, were treated with recombinant bST or vehicle 48 h after intramammary inoculation of Escherichia coli. Clinical status and changes in milk production and composition were compared in the four groups. In a second study, 8 cows received bST or vehicle 7 d before bacterial challenge. During mastitis, losses in milk production and compositional changes were most pronounced in infected glands and in severe responders. Milk production of bST cows recovered better than that of placebo cows. Recovery of milk components was accelerated in severe responders treated with bST, but not in moderate responders. Pretreatment of severe responders with bST enhanced milk production before infection, protected the mammary glands from excessive loss of milk during the subsequently induced coliform mastitis, and accelerated normalization of milk composition. In conclusion, the beneficial effects of bST upon normalization of milk production and composition in periparturient cows suffering from coliform mastitis seem to be restricted to the severe responders. In severe responders that had been treated with bST, changes observed during mastitis resembled those in moderate responders treated with the placebo.

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 cow's sensitivity to Esch. coli mastitis.

Partial prostaglandin-mediated mechanism controlling the release of cortisol in plasma after intravenous administration of endotoxins.

In a first series of experiments we studied the influence of E. coli endotoxins or lipopolysaccharides (LPS) administered either intravenously (i.v.) or intramammarily (i.mam.) to lactating goats on plasma cortisol and rectal temperature (RT). Differences in the magnitude of the cortisol release and shape of the fever curve were observed. In both models maximal pyrexia and fever index (FI) were comparable. In a second series of experiments the influence of LPS on the plasma cortisol and RT was studied after i.v. injection of increasing doses of LPS:low (25 ng/kg), moderate (200 ng/kg) and relatively high (500 ng/kg). Although the cortisol response was dose dependent, the effect was not correlated with FI. The administration of flurbiprofen, a non steroidal antiinflammatory drug (NSAID), resulted in a complete inhibition of fever at all LPS doses and the cortisol release after administration of low doses LPS. This indicates a prostaglandin mediation. With moderate and high doses LPS the cortisol release was only partially inhibited and delayed suggesting a non prostaglandin mediated mechanism. In a third series of experiments the influence of flurbiprofen on fever and cortisol release was studied after i.mam. LPS administration. The observed increase of plasma cortisol and RT were completely abolished after flurbiprofen treatment. It is concluded that: 1) the increase of plasma cortisol after LPS administration in lactating goats is not related to hyperthermia per se, 2) the control of fever and cortisol release may, to some extent, differ according to the LPS dose and method of administration, 3) the cortisol release observed after moderate and high doses of LPS is probably controlled by two phenomena. The first being induced by cyclo-oxygenase metabolites, the second by intermediary mediators other than prostaglandins or by LPS itself. 4) Although an eight-fold higher dose of LPS was given i.mam., a cortisol release comparable to the lowest dose of LPS (25 ng/kg) was observed. These differences in cortisol release can be ascribed to 1) a detoxification of LPS at the level of the mammary gland or 2) a slower resorption of LPS from the mammary gland.

Role of prostaglandin-mediated mechanisms during experimentally induced endotoxin fever in the lactating goat.

The effects of endotoxin (LPS) on the cortisol, glucose, NEFA (non-esterified fatty acids), STH (somatotropin) and oxytocin levels in plasma of goats are described. The changes in plasma cortisol, STH and NEFA, as well as in RT (rectal temperature) were compared after i.v. and i.mam. administration of endotoxin. The other parameters, glucose and oxytocin, were followed only after i.v. endotoxin administration. The observed metabolic and hormonal alterations in plasma were also studied after pretreating the goats with the non-steroidal anti-inflammatory and antipyretic drug flurbiprofen in order to evaluate the possible involvement of prostaglandin in these phenomena. After i.v. administration of LPS a biphasic temperature curve for the highest dose of LPS with peak maxima at 1h and 4h after LPS challenge, was observed. Intramammary administration of endotoxin induces a monophasic fever response, with a latency time of approximately 3h, and peak values after 6h. The onset of the fever response in the i.v. experiments coincided with the oxytocin maximum and with early hyperglycemia. Intravenous endotoxin in goats also induces an increase in plasma NEFA, cortisol and STH. The early increase in NEFA, with a maximum after 2h and occurring before the fever peak, is followed by a significant rise in cortisol with peak effects after 3 h. The increase in plasma STH coincided with the decrease in plasma NEFA returning to control levels again. Peak concentrations in plasma STH occurred after 4 h. All the changes observed after the i.v. administration of endotoxin are dose-dependent. Pretreating goats with flurbiprofen completely abolished fever response, as well as the early hyperglycemia and the oxytocin release to i.v. LPS, indicating that these changes were prostaglandin-mediated and might be a reflexion of an activation of the sympathetic adrenomedullary system. The LPS-induced changes in plasma cortisol, NEFA and STH are only partly depressed and delayed by flurbiprofen. The residual hormonal responses to high doses of endotoxin suggest that an additional direct action of circulating endotoxins on the hypothalamus cannot be excluded. Intramammary LPS administration in goats only induced a very weak increase in plasma cortisol. The complex interplay of hormones and metabolic substances in the homeostasis of the inflammation reaction is discussed.

Relationship between numbers of alpha 2- and beta 2-adrenoceptors in teat tissue and blood cells and milkability of primiparous cows.

Milking characteristics were measured on 14 cows at morning milkings. After collection of blood, cows were slaughtered and teats were removed. beta 2-Adrenoceptors on teat tissue and lymphocyte membranes were identified by binding of [3H]dihydroalprenolol. For the determination of alpha 2-adrenoceptors on teat tissue and blood platelet membranes, [3H]rauwolscine was used. In teat tissue and blood cells, except for the amount of milk collected during the 3rd min, all other milking parameters were highly correlated with the ratio of beta 2-adrenoceptors to alpha 2-adrenoceptors. Good milkability appears to be associated with low ratio of beta 2-adrenoceptors to alpha 2-adrenoceptors densities in teat tissue, and a high ratio of beta 2- to alpha 2-adrenoceptors densities in blood cells. We postulated earlier that a low ratio of beta 2- to alpha 2-adrenoceptors in teat tissue of fast milking cows probably reflects changes of mainly prejunctional adrenoceptors. From our results on blood cells, we are now hypothesizing that in teat tissue prejunctional and extra junctional alpha 2- and beta 2-adrenoceptors might have been identified together, whereas alpha 2- and beta 2-adrenoceptors identified on blood platelets and lymphocytes, respectively, are reflecting by preference extra junctional adrenoceptors are activated primarily by circulating epinephrine. The high ratio of beta 2- to alpha 2-adrenoceptors observed in blood cells of fast milking cows might indicate a decreased vasomotor tone in the teat and an increased dilatation of the teat sphincter in these animals.

Changes in N-acetyl-beta-D-glucosaminidase activities in relation to other milk components throughout normal lactation in the guinea pig.

Changes in milk production, NAGase activities, Na, Cl, K, lactose, total N, and fat were followed throughout normal lactation in 26 healthy guinea pigs. Appropriate microassays were used and all determinations were performed on the same small skim milk sample (microhematocrit tube). A very sensitive and rapid spectrofluorimetric determination of NAGase in 10-microliters skim milk samples was evaluated. Four days after parturition, daily milk production reached a maximum of about 20 ml. Thereafter milk yield decreased progressively to about 7 ml on d 15. During the entire course of lactation, a progressive increase in NAGase, total N, fat, Na, and Cl was observed. However, K and lactose decreased. Highly significant positive correlations between NAGase and Na, Cl, fat, and total N were evident, whereas correlations were negative between NAGase and K, lactose, and milk production. These changes can be explained on basis of alterations that occur at the level of the blood-milk barrier. The short lactation period of the guinea pig, the easy handling and milking practices, together with the available sensitive micromethods can lead to the establishment of a convenient guinea pig mastitis model.

Comparison of fat and cream content in normal and mastitic milk of cows.

Two methods for estimating fat and cream content in cow's milk were compared in normal and mastitic milk. Fat content in milk from periparturient cows was estimated simultaneously by the method of Gerber and by simply spinning small samples of untreated milk in a haematocrit centrifuge. Mastitis was experimentally induced by inoculating mammary glands with Escherichia coli. On the average, cream content in milk from normal, and uninfected mammary glands measured by the haematocrit method was 20 to 23% higher than milk fat measured by the method of Gerber. In mastitic milk from infected mammary glands haematocrit readings were 40% higher than those of Gerber. Milk fat values as measured by both methods were highly correlated. It is concluded that the haematocrit method can be used for rapid estimation of milk fat content in normal milk of cows. The greater difference between cream and fat content in mastitic milk compared to normal milk can probably, at least partly, be ascribed to the presence of large clots of cells and fat in the milk during mastitis. During centrifugation of the viscous mastitic milk samples, milk cells and fat probably are not completely separated. This results in overestimated readings of milk cream content with the haematocrit method. It therefore seems advisable to use the Gerber method for accurate measurements of milk fat in mastitic milk.

Comparison of the milk leakage potencies of adrenergic agonists in lactating cows.

The activities of epinephrine (2-6 micrograms) and of norepinephrine (20-60 micrograms) on the relaxation of teat sphincter muscles were compared by measuring milk leakage from the full udder of 5 lactating cows. Substances were injected into the external pudic artery after pretreatment with an alpha-adrenoceptor blocking agent (prazosin or phentolamine). Milk leakage was induced by creating a vacuum (-30 cm of water) in a plethysmographic chamber enclosing one teat and volumes of milk loss were measured every min. In a second series of experiments the activities of the beta 2-adrenoceptor selective agonist zinterol (0.5-2.5 micrograms) and of the nonselective beta-agonist isoproterenol (3-22.5 micrograms) on sphincter muscle relaxation were compared. Epinephrine was found to be more potent than norepinephrine in inducing relaxation of sphincter muscles. Zinterol was more active than isoproterenol. It is concluded that there is a predominance of beta 2-adrenoceptors in the teat sphincters.

Relationship between milkability and adrenoceptor concentrations in teat tissue in primiparous cows.

Milking characteristics were measured on 19 primiparous cows of the Red Pied breed at morning milkings. Measurements included maximum and average rate of flow, yield of milk through the 1st, 2nd, and 3rd min, amount of milk through the first 2 and first 3 min of milking, and milking time. Subsequently cows were slaughtered and teats immediately removed. Adrenoceptors on membranes isolated from teat tissue were identified by radiolabeled antagonists: [3H]dihydroalprenolol for beta 2-adrenoceptors, [3H]rauwolscine for alpha 2-adrenoceptors, and [3H]prazosin for alpha 1-adrenoceptors. Measurements of milking characteristics were highly repeatable within cow. Cows showed five distinct and significantly different milk flow patterns, which were characteristic for fast (type I, II), relatively fast (type III), and slow (type IV, V) milking cows. Covariance analysis for the five different types revealed an average regression between alpha 2-adrenoceptor density and milking rate 1st min as well as between beta 2:alpha 2-adrenoceptor ratios and milking rate 2nd min and milking time. A hypothesis is presented to explain these observations. During milking, tone of efferent sympathetic nerves in the teat is low. This phenomenon is more pronounced in fast milking cows. The typical presynaptic adrenoceptor pattern in the teat of fast milkers (low beta 2:alpha 2-adrenoceptor ratio) results in a decline of norepinephrine release by feedback mechanisms.

Effects of valerate and isobutyrate on fatty acid secretion by the isolated perfused mammary gland of the lactating goat.

The isolated mammary glands of six lactating goats were perfused with heparinized and oxygenated blood for 8 to 11 h. Adequate quantities of glucose, acetate and amino acids (including valine) were added to the perfusate. Either unlabelled valerate or unlabelled isobutyrate was added in excess to the perfusate of one gland, while the respective symmetrical gland was used as a control. After the administration of valerate, the proportions of the odd-numbered fatty acids (C11:0, C13:0, C15:0) in the milk fat, collected every hour during perfusion, rose progressively after 5 h until the end. The synthesis of milk fatty acids from valerate is discussed. After isobutyrate was added to the perfusate, isoC12:O, isoC14:0 and isoC16:0 in the milk fat increased as compared to the control. The effect of isobutyrate indicated that valine acted as a precursor of milk iso-branched fatty acids after its metabolisation to isobutyryl-CoA. During perfusion in the presence of the complete substrate mixture, the proportion of certain major milk fatty acids (C10:0, C12:0, C14:0 and C16:0) increased, whereas the proportion of C18:0 and C18:1 decreased. These effects have been ascribed to the presence of acetate and beta-hydroxybutyrate in the substrate mixture.

Action of epinephrine on the function of the teat sphincter in the lactating cow.

Under continuous IV infusion of oxytocin, milk leakage was elicited in 6 lactating cows with full udders by induction of a vacuum (-30 cm of water) around 1 mammary papilla (teat) with a plethysmographic apparatus. The volumes of milk loss were measured every minute. Epinephrine injected either into the jugular vein (20, 100, and 500 micrograms) or into the external pudic artery (1.5, 7.5, and 37.5 micrograms) induced an increase in milk leakage in 5 cows. This effect could be antagonized by the beta-blocking agent sotalol hydrochloride (0.5 to 4 mg) injected into the external pudic artery. In 1 cow, however, epinephrine given intra-arterially exerted a biphasic effect, small doses inducing stimulation, and large doses evoking inhibition of milk loss. The stimulating effects were blocked by sotalol (2 mg). The inhibiting effects were antagonized by the alpha-blocking agent prazosin (2 mg).

Effect of prazosin on the function of the teat sphincter in lactating cows.

Oxytocin was infused for 60 min into the jugular vein of 8 lactating cows with full udders before milking. Under these circumstances milk loss occurred spontaneously in 3 cows and was evoked in 5 cows by reducing pressure around one teat (-30 cm water) using a plethysmographic chamber. Milk loss was measured continuously. The postsynaptic alpha-blocking agent prazosin ( PRZS ) injected either into the udder artery (2 or 5 mg) or into the jugular vein (200 mg) consistently induced a significant increase in milk leakage. It is suggested that PRZS effectively inhibits alpha-adrenoceptors in the teat sphincter muscles and thus promotes the action of beta-adrenoceptors, in this way inducing relaxation of the teat sphincter.

Effect of hypothalamic implantation of perphenazine on milk yield in goats.

The effects of perphenazine on galactopoiesis were studied in 52 goats in the phase of declining lactation (September-December). At the beginning of September, perphenazine (1 mg) was stereotaxically implanted into the hypothalamus, sham-implantations being performed on control animals. After the operation milk yield decreased and stabilized at a new starting level in most animals of both groups. The average weekly milk yield of the sham-implanted goats gradually fell during the experimental period. This progressive decline in yield was greatly reduced if perphenazine had been implanted in the median eminence (ME). No galactopoietic effects were observed when the implantation was localized outside the ME.

Effect of intracerebroventricular injection of nicotine and acetylcholine on renal water and salt excretion in conscious goats.

In hydrated, conscious goats nicotine was administered into the 3rd ventricle and its effects on water and electrolyte excretion were studied. Acetylcholine (ACh.) in combination with eserine (Es.) was injected by the same route in goats pretreated with atropine. Nicotine as well as large doses of Ach + Es. induced an antidiuresis of the pituitary type. The responses to ACh. were dose-dependent. Intracerebroventricular (icv.) pretreatment with hexamethonium blocked the responses to both nicotine and ACh., whereas atropine and propranolol were ineffective. It appears likely that nicotine receptors are involved inthe ACh.-induced release of antidiuretic hormone. Phentolamine (icv.) completely blocked the effects of ACh. + Es. and partly those of nicotine, indicating that catecholamines might be involved to some extent.

Effect of intracerebroventricular administration of opioid peptides on urinary function in the conscious goat.

The effects of methionine(met)-enkephalin, leucine(leu)-enkephalin, beta-endorphin and blocking substances upon renal function were studied in conscious goats. Injections were made through a permanent cannula into the 3rd ventricle. Leu- and met-enkaphalin, as well as beta-endorphin induced an antidiuretic response to the pituitary type. The responses to beta-endorphin were found to be dose-dependent. Pretreatment with naloxone, either into the 3rd ventricle or into the jugular vein, antagonised the antidiuretic responses to injected opioid peptides with the magnitude of the inhibition being dependent upon the dose. Atropine, hexamethonium or phentolamine did not interfere with the antidiuretic activity of beta-endorphin. Injection of naloxone alone into the 3rd ventricle of goats with a normal water balance, induced both a diuretic response and an increase in free water clearance. It is suggested that the opioid peptides are acting selectively on opiate receptors to influence the release of antidiuretic hormone.