Gradual weaning does not improve performance for calves with low starter intake at the beginning of the weaning process.

The weaning process may cause intense stress for dairy calves, even when low volumes of liquid diet are fed. Management tools that increase the intake of solid feeds, such as gradual weaning, can provide better physiological and metabolic conditions through better ruminal development, leading to better adaptation to ruminant metabolism and aiding in stress mitigation. The objective of this study was to evaluate the effects of 2 weaning protocols and 2 levels of concentrate intake on the performance and physiological and behavioral variables related to stress in dairy calves. Thirty-six newborn male Holstein calves were used in a randomized block design with a 2 × 2 factorial arrangement: 2 weaning strategies, abrupt or gradual, and 2 levels of concentrate intake at 5 wk of age, high (>350 g/d) or low (≤350 g/d). Calves were equally managed until they were 5 wk of age and then grouped according to concentrate intake. Statistical analyzes were performed using the MIXED procedure of SAS software (SAS Institute Inc., Cary, NC), and no significant interaction was observed between studied factors (weaning method and starter intake level); therefore, we considered each factor separately and their interactions with age. The highest dry matter intake and concentration of ß-hydroxybutyrate were recorded for animals with a high level of starter intake independent of the weaning method. Structural growth (cm/wk) and average daily gain were superior for calves with high starter intake, but weaning method had no effect. The gradual weaning protocol increased the time eating starter, regardless of the level of concentrate intake. Even animals with low concentrate intake that were weaned abruptly showed levels of cortisol and acid-soluble glycoprotein within normal physiological levels. Apparently, other factors besides the milk supply affect the starter intake level of calves in a conventional feeding program. The adoption of gradual weaning is not effective in improving performance when a calf has low intake 3 wk before weaning is complete, but it reduced vocalization on d 2 postweaning.

Intensive liquid feeding of dairy calves with a medium crude protein milk replacer: Effects on performance, rumen, and blood parameters.

The objective of this study was to evaluate the effects of different liquid-feeding systems using a medium crude protein milk replacer on performance, rumen, and blood parameters. Thirty newborn Holstein calves were blocked according to birth weight and date of birth, and randomly distributed to different liquid-feeding systems: conventional (4 L/d), intensive (8 L/d), or step-up/step-down (wk 1, 4 L/d; wk 2 to 6, 8 L/d; wk 7 and 8, 4 L/d). The commercial milk replacer (12.5% solids, 20.2% crude protein, 15.6% fat) was fed twice daily (0700 and 1700 h) until calves were weaned, at 8 wk of age. Calves were individually housed in wood hutches, with free access to water and starter concentrate, and to hay only after weaning. They were followed through 10 wk of age. Milk replacer and starter intake were inversely affected by feeding system. After weaning, starter intake and hay intake were similar among feeding systems. Total dry matter intake was higher during the liquid-feeding period for calves on the intensive system compared to calves on the conventional system, but conventional feeding resulted in the highest dry matter intake after weaning. Feed efficiency was similar among feeding systems before and after weaning. Average body weight and daily gain were not affected by feeding system before or after weaning. During liquid feeding, diarrhea occurrence was lower for calves on the conventional system; however, when calves on the step-up/step-down system were fed lower volumes of liquid feed, diarrhea occurrence was similar to that of calves on the conventional system. Plasma concentrations of ß-hydroxybutyrate were higher for calves on the conventional system, reflecting starter intake. Rumen pH, short-chain fatty acids, and N-NH3 were not affected by feeding system. Feeding higher volumes of milk replacer with a medium crude protein content had no beneficial effect on the performance of calves up to 10 wk of age.

Performance and Metabolism of Calves Fed Starter Feed Containing Sugarcane Molasses or Glucose Syrup as a Replacement for Corn.

The aim of this study was to evaluate the effect of replacing corn grain for sugar cane molasses (MO) or glucose syrup (GS) in the starter concentrate on performance and metabolism of dairy calves. Thirty-six individually housed Holstein male calves were blocked according to weight and date of birth and assigned to one of the starter feed treatments, during an 8 week study: i) starter containing 65% corn with no MO or GS (0MO); ii) starter containing 60% corn and 5% MO (5MO); iii) starter containing 55% corn and 10% MO (10MO); and iv) starter containing 60% corn and 5% GS (5GS). Animals received 4 L of milk replacer daily (20 crude protein, 16 ether extract, 12.5% solids), divided in two meals (0700 and 1700 h). Starter and water were provided ad libitum. Starter intake and fecal score were monitored daily until animals were eight weeks old. Body weight and measurements (withers height, hip width and heart girth) were measured weekly before the morning feeding. From the second week of age, blood samples were collected weekly, 2 h after the morning feeding, for glucose, ß-hydroxybutyrate and lactate determination. Ruminal fluid was collected at 4, 6, and 8 weeks of age using an oro-ruminal probe and a suction pump for determination of pH and short-chain fatty acids (SCFA). At the end of the eighth week, animals were harvested to evaluate development of the proximal digestive tract. The composition of the starter did not affect (p>0.05) concentrate intake, weight gain, fecal score, blood parameters, and rumen development. However, treatment 5MO showed higher (p<0.05) total concentration of SCFAs, acetate and propionate than 0MO, and these treatments did not differ from 10MO and 5GS (p>0.05). Thus, it can be concluded that the replacement of corn by 5% or 10% sugar cane molasses or 5% GS on starter concentrate did not impact performance, however it has some positive effects on rumen fermentation which may be beneficial for calves with a developing rumen.

Regulation of intracellular cardiomyocyte calcium stores by peptides: a new approach to cardiac protection.

The control of cytosolic calcium is a major determinant not only of cardiac function, but also of the capability of myocardial tissue to survive damage. Increase of diastolic calcium leads rapidly to cell injury, and may be induced by a wide range of causes. In this review we describe the major points of calcium control in cardiac myocytes, mainly in mammalian ventricle, focusing on mechanisms of intracellular calcium influx during excitation, voltage gated channels of the sarcolemma and ryanodine receptors of the sarcoplasmic reticulum (SR), and efflux during relaxation, principally the sodium/calcium exchanger in membrane and the SR calcium complex. Mitochondria also depend on calcium concentration while also participating in its control. Moreover, we will outline receptor check points and their roles in physiology and pathology. We will focus on some new aspects of potential protective mechanisms that have been recently described and that involve peptide ligands and that in the case of the Neuregulin1beta/ErbB pathway are already reaching the clinical trial relevance.

Endothelium dependent cardiovascular effects of the Chromogranin A-derived peptides Vasostatin-1 and Catestatin.

The involvement of Chromogranin A (CgA) in the cardiovascular function regulation is attributed to its function as a prohormone. Several studies indicated that CgA-derived peptides, particularly Vasostatin-1 (VS-1) and Catestatin (CST), exert signaling effects in numerous organs/systems, including the cardiovascular system. This review focuses on the recently described signaling pathways activated by VS-1 and CST, giving insights into the mechanisms at the basis of their cardiac negative inotropic action, their vasodilator effects and their cardioprotective role observed in different experimental conditions. Accumulated evidences provided convincing support for VS-1 and CST as vasoactive peptides indirectly acting on cardiomyocytes through a Ca(2+)-independent/PI3-K-dependent NO release from endothelial cells. This pathway is supposed to be triggered by the interaction of these peptides with the plasma membrane. The premise of these studies grounds on the biochemical features of VS-1 and CST, which are structurally characterized by amphipathic properties and the ability to interact with mammalian and microbial membranes. On the other hand, recent data obtained in both isolated heart and isolated cardiomyocytes suggest that the VS-1 and CST-mediated cardioprotective effects are primarily direct on the myocardium, rather than endothelium-dependent. Anyway, both direct and indirect pathways seem to be characterized by the absence of specific membrane receptors on target cells, highlighting intriguing novelties in the topic of cell signaling, in particular respect to an hypothetical receptor-independent eNOS activation.

The homologous rat chromogranin A1-64 (rCGA1-64) modulates myocardial and coronary function in rat heart to counteract adrenergic stimulation indirectly via endothelium-derived nitric oxide.

Chromogranin A (CGA), produced by human and rat myocardium, generates several biologically active peptides processed at specific proteolytic cleavage sites. A highly conserved cleavage N-terminal site is the bond 64-65 that reproduces the native rat CGA sequence (rCGA1-64), corresponding to human N-terminal CGA-derived vasostatin-1. rCGA1-64 cardiotropic activity has been explored in rat cardiac preparations. In Langendorff perfused rat heart, rCGA1-64 (from 33 nM) induced negative inotropism and lusitropism as well as coronary dilation, counteracting isoproterenol (Iso) - and endothelin-1 (ET-1) -induced positive inotropic effects and ET-1-dependent coronary constriction. rCGA1-64 also depressed basal and Iso-induced contractility on rat papillary muscles, without affecting calcium transients on isolated ventricular cells. Structure-function analysis using three modified peptides on both rat heart and papillary muscles revealed the disulfide bridge requirement for the cardiotropic action. A decline in Iso intrinsic activity in the presence of the peptides indicates a noncompetitive antagonistic action. Experiments on rat isolated cardiomyocytes and bovine aortic endothelial cells indicate that the negative inotropism observed in rat papillary muscle is probably due to an endothelial phosphatidylinositol 3-kinase-dependent nitric oxide release, rather than to a direct action on cardiomyocytes. Taken together, our data strongly suggest that in the rat heart the homologous rCGA1-64 fragment exerts an autocrine/paracrine modulation of myocardial and coronary performance acting as stabilizer against intense excitatory stimuli.

Role of endothelial cells in modulation of contractility induced by hexarelin in rat ventricle.

The synthetic growth hormone (GH) secretagogue hexarelin has important cardiac effects, that include a reduction of dysfunction in ischemic-reperfused hearts from GH-deficient rats after a chronic treatment and an increase of ejection fraction in acutely treated men. To investigate the mechanisms of its cardiac activity, we studied the effects of hexarelin (1-10 microM) on contractility of rat papillary muscles. We observed, in hexarelin treated papillary muscles, an improved recovery of contractility after anoxia. Hexarelin induced time- and frequency-dependent inotropic effects on papillary muscle. These effects were a transient increase in contractile force, abolished by propranolol (0.2 microM), followed by a reduction at low (60-240/min), but not at high (400-600/min) beating frequencies. The typical negative force-frequency relationship present in rat papillary muscles was therefore modified, and a minor increase in diastolic tension occurred after a sudden increase in stimulus frequency. Blockade of NO synthesis with 1 mM L-NAME, partially altered the response to hexarelin. MK-677 (1 microM), a non peptidyl GH secretagogue, reduced contractility, but did not alter the force-frequency relationship. The remaining effects of hexarelin were absent in papillary muscles pre-treated with indomethacin (1 microM), or after removal of endocardial endothelium with 0.5% triton X-100. The release of the prostacyclin metabolite 6-keto-PGF1alpha was increased during reoxygenation after a period of anoxia in hexarelin treated papillary muscles. Hexarelin had no significant effect on calcium transients and on I(Ca) measured in isolated ventricular cells. These findings suggest that the effects of hexarelin are mainly due to endothelium-released PGI2.

Regulation of cardiac calcium current by NO and cGMP-modulating agents.

Several effects of nitric oxide (NO) on the control of L-type calcium current (ICa) and of calcium handling in cardiomyocytes have been described. Cardiomyocytes have been shown to express in different conditions all types of nitric oxide synthases (NOS), but the role of NO in the regulation of calcium current remains controversial. Previously, we have shown in guinea pig ventricular cells a stimulatory effect of NOS inhibitors on ICa. Here we investigate the intracellular mechanisms involved in the putative inhibitory role of NO on basal ICa in ventricular cells. The stimulatory effect of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) (1 mM) was present also in calcium transient measurements, but only after a preincubation with L-arginine (L-arg, 0.1 mM). The nitric oxide scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO, 0.5 mM) increased peak ICa in a similar manner to NOS inhibitors in whole-cell voltage-clamp experiments. Also ODQ (1H-[1,2,4]oxidiazolo[4,3-a]quinoxaline-1-one, 0.1 mM), a specific inhibitor of a target of NO, the soluble guanylate cyclase, was able to stimulate ICa. The block of type II phosphodiesterase (cGMP-activated) by EHNA (erythro-9-[2-hydroxy-3-nonylladenine, 30 microM) exerted a similar effect on ICa as PTIO and ODQ. Carbachol (CCh, 1 microM) was able to revert the stimulatory effect on ICa observed with PTIO, ODQ, and EHNA. We propose that the increase of basal ICa in guinea pig cardiomyocytes previously observed with L-NMMA depends on the removal of a tonic NO inhibition. This increase of ICa is mimicked by blocking at different steps the cGMP-cascade activated by NO, suggesting a NO-guanylate cyclase mechanism in the basal control of ventricular calcium current.

Modulation of guinea-pig cardiac L-type calcium current by nitric oxide synthase inhibitors.

1. Electrophysiological (whole-cell clamp) techniques were used to study the effect of NO synthase (NOS) inhibitors on guinea-pig ventricular calcium current (ICa), and biochemical measurements (Western blot and citrulline synthesis) were made to investigate the possible mechanisms of action. 2. The two NOS inhibitors, NG-monomethyl-L-arginine (L-NMMA, 1 mM) and NG-nitro-L-arginine (L-NNA, 1 mM), induced a rapid increase in ICa when applied to the external solution. D-NMMA (1 mM), the stereoisomer of L-NMMA, which has no effect on NOS, did not enhance ICa. 3. Western blot experiments gave no indication of the presence of inducible NOS protein (iNOS) in our cell preparation, neither immediately after dissociation nor after more than 24 h. Statistically, there was no significant difference between electrophysiological experiments performed on freshly dissociated cells and experiments performed the next day. Moreover cells prepared and kept in the presence of dexamethasone (3 microM), to inhibit the expression of iNOS, gave the same response to L-NMMA as control cells. 4. The stimulatory effect of L-NMMA (1 mM) on basal ICa was reversed by competition with higher doses (5 mM) of externally applied L-arginine, the natural substrate of NOS. The effect of L-NMMA was also eliminated by L-arginine in the patch pipette solution. 5. Intracellular perfusion with GDP beta S (0.5 mM), which stabilizes the G-proteins in the inactive state, did not affect the L-NMMA-induced stimulation of ICa. 6. Carbachol (1 microM) reduced the ICa previously stimulated by L-NMMA, and intracellular cGMP (10 microM) prevented L-NMMA enhancement. 7. Simultaneous treatment with L-NMMA and isoprenaline (1 microM) induced a non-cumulative enhancement of ICa that could not be reversed by carbachol (1 microM). 8. NO synthesis, measured by the formation of [3H]citrulline from L-[3H]arginine during a 15 min incubation, showed a relatively high basal NO production, which was inhibited by L-NMMA but not affected by carbachol. 9. These results suggest that inhibitors of NOS are able to modulate the basal ventricular ICa in the absence of a receptor-mediated pathway, and that NO might be required for the muscarinic reduction of ICa under isoprenaline stimulation, even if NO production is not directly controlled by the muscarinic pathway.

Differential effects of paclitaxel and derivatives on guinea pig isolated heart and papillary muscle.

Paclitaxel (Taxol) is an anticancer agent with clinical activity against various human cancer types. Paclitaxel blocks cell division by stabilizing microtubules, a mechanism that also underlies its major side effects (neutropenia and neurotoxicity). Paclitaxel can also alter cardiac function, and to elucidate the mechanism of this activity, we tested the mechanical and electrical effects of paclitaxel and a series of analogs (docetaxel, taxol B, taxol C and N-methyltaxol C; 5-20 microM) on two different cardiac preparations, the isolated coronary perfused heart and the papillary muscle of the guinea pig. Paclitaxel and N-methyltaxol C induced conduction arrhythmias and reduced coronary flow and left ventricular systolic pressure in the isolated heart, whereas the other taxol derivatives tested had no significant effect. Moreover, paclitaxel blocked the vasodilator effect of bradykinin in the isolated heart. Paclitaxel and N-methyltaxol C produced a positive inotropic effect in papillary muscle, without alterations in the action potential. In the latter preparation, no significant variations were observed after treatment with the other taxol derivatives. The in vitro cardiodepressant and arrhythmogenic activity of paclitaxel is similar to that reported after its clinical administration and might be due to coronary vasoconstriction. The precise role of microtubules as modulators of intracellular calcium in cardiac and smooth muscle cells is at present unclear, because docetaxel and other taxol analogs, though they exhibited similar activity on tubulin, lacked cardiac effects.

Effects of yew alkaloids and related compounds on guinea-pig isolated perfused heart and papillary muscle.

The mechanical and electrical effects of selected yew alkaloids were studied on two different cardiac preparations: the isolated coronary perfused heart and the isolated papillary muscle of the guinea-pig. In the isolated heart, the Winterstein acid type alkaloids 1, 2 and 3 induced electrical and mechanical effects similar to those reported after yew intoxication (negative inotropic effect, block of atrio-ventricular conduction), but the coronary flow was unchanged. Taxine B (1), the most potent compound of this group, reduced cardiac contractility and the maximum rate of depolarisation of the action potential in the isolated papillary muscle, acting as a class I antiarrhythmic drug. In the isolated heart, the cinnamates 4 and 5, corresponding to the degradation products of 1 and 3, exerted arrhythmogenic effect due to a reduction of coronary flow. No alterations in electrical and contractile activities were in fact recorded after perfusion of the isolated papillary muscle with 4. Taxine A (6) and the taxane alcohol 7, corresponding to the terpenoid core of 3 had no significant cardiac effect. Our results suggest that the poisonous properties of the yew tree are probably due to the combined activity of alkaloids of the Winterstein acid type and their corresponding cinnamtes, which can reduce both the excitability and the coronary flow of the heart.

Guanylate-cyclase-mediated inhibition of cardiac ICa by carbachol and sodium nitroprusside.

We studied the role of cyclic guanosine monophosphate (cGMP) as a mediator of the reduction of L-type calcium current (ICa) induced by muscarinic receptor stimulation and by nitric oxide in isolated guinea-pig ventricular cells using the whole-cell patch-clamp technique. Our results show that when the level of cyclic adenosine monophosphate was increased by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX), stimulation of a pertussis-toxin (PTX)-sensitive muscarinic receptor by carbachol (1 microM) reduced the calcium current increase from 80.6 +/- 23.5% to 19.8 +/- 9.6% over the control and this effect was prevented by methylene blue (10 microM), an inhibitor of the soluble guanylate cyclase. Pipette solution containing 10 microM cGMP reduced the enhancement of ICa by IBMX from 121.9 +/- 11.6% to 14.2 +/- 5.4% above the control. Sodium nitroprusside (10 microM), a spontaneous donor of nitric oxide, and consequently a stimulator of soluble guanylate cyclase, also reduced IBMX-stimulated ICa from 115.2 +/- 13.2% to 32.2 +/- 6.9% above control and the sodium nitroprusside effect was also suppressed by methylene blue. The latter two reagents were ineffective on basal ICa.

M1 muscarinic receptors increase calcium current and phosphoinositide turnover in guinea-pig ventricular cardiocytes.

1. Physiological and molecular evidence for the presence and functional role of M1 muscarinic cholinergic receptors (mAChRs) in adult guinea-pig ventricular cells is presented. 2. Whole-cell clamp measurements of the L-type calcium current (ICa) in isolated myocytes were performed. Caesium was used to suppress potassium currents. ICa was increased by the muscarinic agonist carbachol in cells pretreated with pertussis toxin which blocked the M2 mAChR-triggered cascade of intracellular signalling, while it was not changed in untreated cells. 3. If the M2-mediated regulation of ICa was blocked by directly saturating the cell with cyclic adenosine monophosphate (cAMP) through the patch pipette, application of carbachol induced a further small increase of the current above the level reached after cAMP perfusion. This increase was more pronounced in cells pretreated with pertussis toxin. 4. The carbachol-induced increase of ICa was blocked by the selective M1 mAChR antagonist pirenzepine. 5. The application of high concentrations of carbachol increased the accumulation of [3H]inositol monophosphate up to 240% above control levels. This increase was reduced by application of pirenzepine. 6. The expression of M1 receptor mRNA in ventricular cardiocytes was shown by reverse transcriptase-polymerase chain reaction. 7. These results suggest that M1 mAChR regulation of ICa can be a component of the paradoxical positive inotropism induced by high concentrations of muscarinic agonists.