Onion (Allium cepa L.) peel extract (OPE) regulates human sperm motility via protein kinase C-mediated activation of the human voltage-gated proton channel.

Onion (Allium cepa L.) and quercetin protect against oxidative damage and have positive effects on multiple functional parameters of spermatozoa, including viability and motility. However, the associated underlying mechanisms of action have not yet been identified. The aim of this study was to investigate the effect of onion peel extract (OPE) on voltage-gated proton (Hv1) channels, which play a critical role in rapid proton extrusion. This process underlies a wide range of physiological processes, particularly male fertility. The whole-cell patch-clamp technique was used to record the changes in Hv1 currents in HEK293 cells transiently transfected with human Hv1 (HVCN1). The effects of OPE on human sperm motility were also analyzed. OPE significantly activated the outward-rectifying proton currents in a concentration-dependent manner, with an EC50 value of 30 µg/mL. This effect was largely reversible upon washout. Moreover, OPE induced an increase in the proton current amplitude and decreased the time constant of activation at 0 mV from 4.9 ± 1.7 to 0.6 ± 0.1 sec (n = 6). In the presence of OPE, the half-activation voltage (V1/2 ) shifted in the negative direction, from 20.1 ± 5.8 to 5.2 ± 8.7 mV (n = 6), but the slope was not significantly altered. The OPE-induced current was profoundly inhibited by 10 µm Zn2+ , the most potent Hv1 channel inhibitor, and was also inhibited by treatment with GF109203X, a specific protein kinase C (PKC) inhibitor. Furthermore, sperm motility was significantly increased in the OPE-treated groups. OPE exhibits protective effects on sperm motility, at least partially via regulation of the proton channel. Moreover, similar effects were exerted by quercetin, the major flavonoid in OPE. These results suggest OPE, which is rich in the potent Hv1 channel activator quercetin, as a possible new candidate treatment for human infertility.

Gq-activated fibroblasts induce cardiomyocyte action potential prolongation and automaticity in a three-dimensional microtissue environment.

Cardiac fibroblasts (CFs) are known to regulate cardiomyocyte (CM) function in vivo and in two-dimensional in vitro cultures. This study examined the effect of CF activation on the regulation of CM electrical activity in a three-dimensional (3-D) microtissue environment. Using a scaffold-free 3-D platform with interspersed neonatal rat ventricular CMs and CFs, Gq-mediated signaling was selectively enhanced in CFs by Gαq adenoviral infection before coseeding with CMs in nonadhesive hydrogels. After 3 days, the microtissues were analyzed by signaling assay, histological staining, quantitative PCR, Western blots, optical mapping with voltage- or Ca2+-sensitive dyes, and microelectrode recordings of CF resting membrane potential (RMPCF). Enhanced Gq signaling in CFs increased microtissue size and profibrotic and prohypertrophic markers. Expression of constitutively active Gαq in CFs prolonged CM action potential duration (by 33%) and rise time (by 31%), prolonged Ca2+ transient duration (by 98%) and rise time (by 65%), and caused abnormal electrical activity based on depolarization-induced automaticity. Constitutive Gq activation in CFs also depolarized RMPCF from -33 to -20 mV and increased connexin 43 and connexin 45 expression. Computational modeling confers that elevated RMPCF and increased cell-cell coupling between CMs and CFs in a 3-D environment could lead to automaticity. In conclusion, our data demonstrate that CF activation alone is capable of altering action potential and Ca2+ transient characteristics of CMs, leading to proarrhythmic electrical activity. Our results also emphasize the importance of a 3-D environment where cell-cell interactions are prevalent, underscoring that CF activation in 3-D tissue plays a significant role in modulating CM electrophysiology and arrhythmias.NEW & NOTEWORTHY In a three-dimensional microtissue model, which lowers baseline activation of cardiac fibroblasts but enables cell-cell, paracrine, and cell-extracellular matrix interactions, we demonstrate that selective cardiac fibroblast activation by enhanced Gq signaling, a pathophysiological trigger in the diseased heart, modulates cardiomyocyte electrical activity, leading to proarrhythmogenic automaticity.

Cavernosum smooth muscle relaxation induced by Schisandrol A via the NO-cGMP signaling pathway.

To evaluate the effect of Schisandrol A on rabbit corpus cavernosum smooth muscle and elucidate the potential mechanism. Penises were obtained from healthy male New Zealand White rabbits (2.5-3.0 kg). The pre-contracted penis with phenylephrine (Phe, 10 µM) was treated with accumulative concentrations of Schisandrol A (10-7, 10-6, 10-5 and 10-4 M). The change in intracavernosum pressure (ICP) and tension was recorded, cyclic nucleotides in the cavernosum tissue were measured by radioimmunoassay, mRNA level and expression of endothelial nitric oxide synthase (eNOS) and neuronal NOS (nNOS) were measured by real time PCR and western blot respectively. The corpus cavernosum smooth muscle relaxation induced by Schisandrol A was in a dose-dependent manner. Pre-treatment with NOS inhibitor (Nω nitro-L-arginine-methyl ester, L-NAME) or guanylyl cyclase inhibitor (1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, ODQ) significantly diminished the relaxation. The cyclic guanosine monophosphate (cGMP) level was significantly increased in the cavernosum tissue. Real time PCR and western blot showed the mRNA level and expression of eNOS and nNOS was also upregulated. Schisandrol A relaxes the cavernosum smooth muscle by activating NO-cGMP signaling pathway. It may be a new promising treatment for erectile dysfunction and cardiovascular disease.

Additive effects of Artemisia capillaris extract and scopoletin on the relaxation of penile corpus cavernosum smooth muscle.

The objective was to investigate the cellular effect and action mechanism of Artemisia capillaris extract (ACE) and its component, scopoletin, on penile corpus cavernosum smooth muscle (PCCSM). In vitro study with PCCSM, the precontracted PCCSM with phenylephrine was treated with ACE or scopoletin. Cyclic nucleotides in the perfusate were measured by radioimmunoassay and expression of protein and mRNA of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase in the perfused PCCSM were measured by western blot and real-time PCR, respectively. The interaction of ACE or scopoletin with udenafil was also evaluated. ACE and scopoletin exerted a significant and concentration-dependent relaxation in PCCSM. The perfusion with ACE or scopoletin significantly increased cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) and the perfusion with ACE or scopoletin increased the expression of eNOS mRNA and protein. Furthermore, ACE or scopoletin enhanced udenafil-inducing relaxation in PCCSM. ACE and scopoletin relaxed the PCCSM mainly by activating nitric oxide-cGMP system and cAMP pathway and they may be additive therapeutic candidates for ED patients who do not completely respond to udenafil.

Analysis of testicular-internal spermatic vein variation and the recreation of varicocoele in a Sprague-Dawley rat model.

Many laboratories tried to recreate the varicocoele model have met with varied success. To recreate a consistent varicocoele model by exploring the anatomic variability of the testicular-spermatic venous system in Sprague-Dawley (SD) rats. Seventy-two sexually mature SD male rats were randomly divided into three groups containing 24 rats per group. Partial ligation of the left renal vein and internal spermatic vein (ISV) communicating branches to common iliac vein and ISV communicating branches ligation (RVISVCBCIV) or partial ligation of the left renal vein and ISV communicating branches ligation (RVISVCB). The results showed that the mean diameter of the left ISV was significantly increased in the RVISVCBCIV group compared with the control and RVISVCB groups (p < 0.001). Using ISV as the reference, the sensitivity of varicocoele was 71.43%, and the specificity was 80%. In addition, the positive predictive value was 83.33%, and the negative predictive value was 66.67%. Sperm count, motility, Johnsen score and the spermatogenic cell density were lower in the RVISVCBCIV group compared with the control (p < 0.01). The apoptotic index was higher in the RVISVCBCIV group compared with control groups (p < 0.01). The RVISVCBCIV provides a more effective method for establishing a varicocoele-induced model.

Cleavage of BCR-ABL transcripts at the T315I point mutation by DNAzyme promotes apoptotic cell death in imatinib-resistant BCR-ABL leukemic cells.

The BCR-ABL fusion transcript encodes the BCR-ABL tyrosine kinase (TK), which causes chronic myelogenous leukemia (CML). Although the TK inhibitor imatinib mesylate, which targets the BCR-ABL protein, has been proven to be effective in controlling leukemic growth, imatinib resistance has been observed with disease relapse because of point mutations in the ABL gene that inhibit imatinib efficacy. In this study, we designed oligodeoxyribozymes (DNAzymes) that specifically target and cleave both the junction sequence and the site of the point mutation (T315I), conferring imatinib resistance in BCR-ABL mRNA. DNAzymes significantly induced apoptosis and inhibited proliferation in wild-type and T315I-mutant BCR-ABL-positive cells. Selective cleavage of T315I-mutant ABL mRNA by DNAzyme (T315I Dz) led to cell cycle arrest in G0/G1 phase, with induction of caspase-3/-7 in imatinib-resistant BCR-ABL-positive cells harboring the T315I mutation. Moreover, cotreatment with the DNAzyme targeting the T315I mutation and imatinib resulted in enhanced inhibition of proliferation and induction of apoptosis in T315I leukemic cells as compared with imatinib alone, thereby antagonizing imatinib resistance in CML cells bearing T315I-mutant BCR-ABL. Therefore, cleavage of T315I-mutant ABL mRNA by DNAzyme combined with imatinib treatment may be an alternative approach to overcoming imatinib resistance in leukemic cells.

Functional scaffold-free 3-D cardiac microtissues: a novel model for the investigation of heart cells.

To bridge the gap between two-dimensional cell culture and tissue, various three-dimensional (3-D) cell culture approaches have been developed for the investigation of cardiac myocytes (CMs) and cardiac fibroblasts (CFs). However, several limitations still exist. This study was designed to develop a cardiac 3-D culture model with a scaffold-free technology that can easily and inexpensively generate large numbers of microtissues with cellular distribution and functional behavior similar to cardiac tissue. Using micromolded nonadhesive agarose hydrogels containing 822 concave recesses (800 µm deep × 400 µm wide), we demonstrated that neonatal rat ventricular CMs and CFs alone or in combination self-assembled into viable (Live/Dead stain) spherical-shaped microtissues. Importantly, when seeded simultaneously or sequentially, CMs and CFs self-sorted to be interspersed, reminiscent of their myocardial distribution, as shown by cell type-specific CellTracker or antibody labeling. Microelectrode recordings and optical mapping revealed characteristic triangular action potentials (APs) with a resting membrane potential of -66 ± 7 mV (n = 4) in spontaneously contracting CM microtissues. Under pacing, optically mapped AP duration at 90% repolarization and conduction velocity were 100 ± 30 ms and 18.0 ± 1.9 cm/s, respectively (n = 5 each). The presence of CFs led to a twofold AP prolongation in heterogenous microtissues (CM-to-CF ratio of 1:1). Importantly, Ba(2+)-sensitive inward rectifier K(+) currents and Ca(2+)-handling proteins, including sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, were detected in CM-containing microtissues. Furthermore, cell type-specific adenoviral gene transfer was achieved, with no impact on microtissue formation or cell viability. In conclusion, we developed a novel scaffold-free cardiac 3-D culture model with several advancements for the investigation of CM and CF function and cross-regulation.

Anterior fusion alone compared with combined anterior and posterior fusion for the treatment of degenerative cervical kyphosis.

We evaluated the efficacy of anterior fusion alone compared with combined anterior and posterior fusion for the treatment of degenerative cervical kyphosis. Anterior fusion alone was undertaken in 15 patients (group A) and combined anterior and posterior fusion was carried out in a further 15 (group B). The degree and maintenance of the angle of correction, the incidence of graft subsidence, degeneration at adjacent levels and the rate of fusion were assessed radiologically and clinically and the rate of complications recorded. The mean angle of correction in group B was significantly higher than in group A (p = 0.0009). The mean visual analogue scale and the neck disability index in group B was better than in group A (p = 0.043, 0.0006). The mean operation time and the blood loss in B were greater than in group A (p < 0.0001, 0.037). Pseudarthrosis, subsidence of the cage, and problems related to the hardware were more prevalent in group A than in group B (p = 0.034, 0.025, 0.013). Although the combined procedure resulted in a longer operating time and greater blood loss than with anterior fusion alone, our results suggest that for the treatment of degenerative cervical kyphosis the combined approach leads to better maintenance of sagittal alignment, a higher rate of fusion, a lower incidence of complications and a better clinical outcome.

Properties of new, long-wavelength, voltage-sensitive dyes in the heart.

Membrane potential measurements using voltage-sensitive dyes (VSDs) have made important contributions to our understanding of electrophysiological properties of multi-cellular systems. Here, we report the development of long wavelength VSDs designed to record cardiac action potentials (APs) from deeper layers in the heart. The emission spectrum of styryl VSDs was red-shifted by incorporating a thienyl group in the polymethine bridge to lengthen and retain the rigidity of the chromophore. Seven dyes, Pittsburgh I to IV and VI to VIII (PGH I-VIII) were synthesized and characterized with respect to their spectral properties in organic solvents and heart muscles. PGH VSDs exhibited 2 absorption, 2 excitation and 2 voltage-sensitive emission peaks, with large Stokes shifts (> 100 nm). Hearts (rabbit, guinea pig and Rana pipiens) and neurohypophyses (CD-1 mice) were effectively stained by injecting a bolus (10-50 microl) of stock solution of VSD (2-5 mM) dissolved in in dimethylsulfoxide plus low molecular weight Pluronic (16% of L64). Other preparations were better stained with a bolus of VSD (2-5 mM) Tyrode's solution at pH 6.0. Action spectra measured with a fast CCD camera showed that PGH I exhibited an increase in fractional fluorescence, DeltaF/F = 17.5 % per AP at 720 nm with 550 nm excitation and DeltaF/F = - 6% per AP at 830 nm with 670 nm excitation. In frog hearts, PGH1 was stable with approximately 30% decrease in fluorescence and AP amplitude during 3 h of intermittent excitation or 1 h of continuous high intensity excitation (300 W Xe-Hg Arc lamp), which was attributed to a combination of dye wash out > photobleaching > dynamic damage > run down of the preparation. The long wavelengths, large Stokes shifts, high DeltaF/F and low baseline fluorescence make PGH dyes a valuable tool in optical mapping and for simultaneous mapping of APs and intracellular Ca(2+).

Phylogenetic analysis of protozoa in the rumen contents of cow based on the 18S rDNA sequences.

AIMS: To examine the diversity of protozoa in the rumen contents of cow. METHODS AND RESULTS: Protozoa that inhabit the rumen were detected by PCR using protozoan-specific primers. Libraries of protozoan rDNA sequences were constructed from rumen fluid, solid tissues and epithelium. Twenty-three clones isolated from rumen fluid fell into two genera identified as Entodinium (69.6% of clones) and Epidinium (31.4% of clones). Of the clones isolated from rumen fluid, a moderate number were unidentifiable (30.4%). CONCLUSIONS: The predominant protozoan genus identified in the whole rumen belonged to the Entodinium group (81.1%). Protozoa were not detected in the rumen epithelium. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that rumen fluid and solid tissues contain different protozoan populations that may play specific roles in rumen function. Quantitative PCR techniques and a more specific set of phylogenetic probes that distinguish between protozoan species are needed to determine the significance of newly identified groups and to determine the distribution of identified protozoan clusters in rumen microbial communities.

The distribution of refractory periods influences the dynamics of ventricular fibrillation.

The spatial and dynamic properties of ventricular fibrillation (VF) may be random or related to cellular electrical properties of the normal heart. Local activation intervals (AIs) in VF may depend on the local refractory period (RP), and sustained VF may require a steep action potential (AP) restitution curve. In guinea pig hearts, AP durations (APDs) and RPs on the epicardium are shorter at the apex and progressively longer toward the base, producing gradients of RPs that may influence the spatial organization of VF. In the present study, the influence of APDs on VF dynamics is investigated in perfused guinea pig hearts stained with a voltage-sensitive dye by comparing APD gradients to the dynamics of VF elicited by burst pacing. In VF, AIs had no clear periodicity, but average AIs were shorter at the apex (57.5+/-8.1 ms) than the base (76.1+/-1.5 ms, n=6, P<0.05) and had gradients similar to APD gradients (correlation coefficient 0.71+/-0.04). Analysis of local velocity vectors showed no preferential directions, and fast Fourier transform (FFT) power spectra were broad (10 to 24 Hz) with multiple peaks (n=6). However, the selective inhibition of delayed K(+) rectifying currents, I(Kr) (E4031; 0.5 micromol/L, n=3), shifted FFT spectra from complex to a lower dominant frequency (10 Hz) and altered repolarization but retained the correlation between mean AIs and RPs. Thus, VF dynamics are consistent with a multiple wave-make and wave-break mechanism, and the local RP influences VF dynamics by limiting the range of VF frequencies and AIs at each site. The full text of this article is available at http://www.circresaha.org.

Cholecystokinin mediates depression of feed intake in dairy cattle fed high fat diets.

Two experiments were conducted to investigate the effects of 1) different concentrations of dietary fat and 2) i.v. administration of a cholecystokinin receptor antagonist (MK-329) on feed intake and plasma concentrations of hormones and metabolites in dairy cattle. In Experiment 1, 4 lactating Holstein cows were used in a 4 x 4 Latin square design. Treatments were diets with 1) no fat added, 2) 30 g fat/kg feed (calcium salts of long-chain fatty acids as fat supplement), 3) 60 g fat/kg, and 4) 90 g fat/kg added. Cows were fed once daily a diet of concentrate, corn silage, alfalfa haylage, and alfalfa pellets. Dry matter intake decreased linearly with increasing concentrations of dietary fat (P < 0.0001). Overall plasma concentrations of nonesterified fatty acids (P < 0.0001), triacylglycerol (P < 0.0006), and cholecystokinin (P < 0.02), increased linearly with each level of dietary fat, but there was a linear decrease in plasma insulin (P < 0.0008). In Experiment 2, 4 nonpregnant and nonlactating Holstein heifers were used in a cross-over design in a 2 x 2 factorial arrangement of treatments. Treatments were diet (fatty acids, 27 g/kg vs 103 g/kg diet dry matter) and i.v. injections (MK-329 vs vehicle). Heifers were fed once daily a total mixed ration of corn silage, cracked corn and soybean meal with or without fat supplement. Diets were switched by period and either MK-329 (70 microg/kg body weight) or its vehicle was injected i.v. at 2 hr postfeeding. Daily dry matter intake was decreased by feeding the high fat diet (P < 0.02) but was not affected by injections. Injection of MK-329, however, increased dry matter intake by 92% in heifers fed the high fat diet during the first 2 hr postinjection compared to vehicle injection. Plasma pancreatic polypeptide concentration was increased by the high fat diet at 2 hr postfeeding (P < 0.02) but was lowered by MK-329 at 1 hr postinjection (P < 0.001). Plasma insulin was lowered by the high fat diet (P < 0.01) but was not affected by injections. The elevated plasma cholecystokinin concentration may have mediated depressed feed intake of dairy cattle fed the high fat diets.

Simultaneous maps of optical action potentials and calcium transients in guinea-pig hearts: mechanisms underlying concordant alternans.

1. The mechanisms underlying electro-mechanical alternans caused by faster heart rates were investigated in perfused guinea-pig hearts stained with RH237 and Rhod-2 AM to simultaneously map optical action potentials (APs) and intracellular free Ca2+ (Ca2+i). 2. Fluorescence images of the heart were focused on two 16 x 16 photodiode arrays to map Ca2+i (emission wavelength (lamdda;em) = 585 +/- 20 nm) and APs (lamdda;em > 715 nm) from 252 sites. Spatial resolution was 0.8 mm x 0.8 mm per diode and temporal resolution 4000 frames s-1. 3. The mean time-to-peak for APs and [Ca2+]i was spatially homogeneous (8.8 +/- 0.5 and 25.6 +/- 5.0 ms, respectively; n = 6). The durations of APs (APDs) and Ca2+i transients were shorter at the apex and progressively longer towards the base, indicating a gradient of ventricular relaxation. 4. Restitution kinetics revealed increasingly longer delays between AP and Ca2+i upstrokes (9.5 +/- 0.4 to 11.3 +/- 0.4 ms) with increasingly shorter S1-S2 intervals, particularly at the base, despite nearly normal peak [Ca2+]i. 5. Alternans of APs and Ca2+i transients were induced by a decrease++ in cycle length (CL), if the shorter CL captured at the pacing site and was shorter than refractory periods (RPs) near the base, creating heterogeneities of conduction velocity. 6. Rate-induced alternans in normoxic hearts were concordant (long APD with large [Ca2+]i) across the epicardium, with a magnitude (difference between odd-even signals) that varied with the local RP. Alternans were initiated by gradients of RP, producing alternans of conduction that terminated spontaneously without progressing to fibrillation.

Enhanced dispersion of repolarization and refractoriness in transgenic mouse hearts promotes reentrant ventricular tachycardia.

The heterogeneous distribution of ion channels in ventricular muscle gives rise to spatial variations in action potential (AP) duration (APD) and contributes to the repolarization sequence in healthy hearts. It has been proposed that enhanced dispersion of repolarization may underlie arrhythmias in diseases with markedly different causes. We engineered dominant negative transgenic mice that have prolonged QT intervals and arrhythmias due to the loss of a slowly inactivating K(+) current. Optical techniques are now applied to map APs and investigate the mechanisms underlying these arrhythmias. Hearts from transgenic and control mice were isolated, perfused, stained with di-4-ANEPPS, and paced at multiple sites to optically map APs, activation, and repolarization sequences at baseline and during arrhythmias. Transgenic hearts exhibited a 2-fold prolongation of APD, less shortening (8% versus 40%) of APDs with decreasing cycle length, altered restitution kinetics, and greater gradients of refractoriness from apex to base compared with control hearts. A premature impulse applied at the apex of transgenic hearts produced sustained reentrant ventricular tachycardia (n=14 of 15 hearts) that did not occur with stimulation at the base (n=8) or at any location in control hearts (n=12). In transgenic hearts, premature impulses initiated reentry by encountering functional lines of conduction block caused by enhanced dispersion of refractoriness. Reentrant VT had stable (>30 minutes) alternating long/short APDs associated with long/short cycle lengths and T wave alternans. Thus, optical mapping of genetically engineered mice may help elucidate some electrophysiological mechanisms that underlie arrhythmias and sudden death in human cardiac disorders.

Optical mapping of atrioventricular node reveals a conduction barrier between atrial and nodal cells.

The mechanisms responsible for atrioventricular (AV) delay remain unclear, in part due to the inability to map electrical activity by conventional microelectrode techniques. In this study, voltage-sensitive dyes and imaging techniques were refined to detect action potentials (APs) from the small cells comprising the AV node and to map activation from the "compact" node. Optical APs (124) were recorded from 5 x 5 mm (approximately 0.5-mm depth) AV zones of perfused rabbit hearts stained with a voltage-sensitive dye. Signals from the node exhibited a set of three spikes; the first and third (peaks I and III) were coincident with atrial (A) and ventricular (V) electrograms, respectively. The second spike (peak II) represented the firing of midnodal (N) and/or lower nodal (NH) cell APs as indicated by their small amplitude, propagation pattern, location determined from superimposition of activation maps and histological sections of the node region, dependence on depth of focus, and insensitivity to tetrodotoxin (TTX). AV delays consisted of tau 1 (49.5 +/- 6.59 ms, 300-ms cycle length), the interval between peaks I and II (perhaps AN to N cells), and tau 2 (57.57 +/- 5.15 ms), the interval between peaks II and III (N to V cells). The conductance time across the node was 10.33 +/- 3.21 ms, indicating an apparent conduction velocity (theta N) of 0.162 +/- 0.02 m/s (n = 9) that was insensitive to TTX. In contrast, tau 1 correlated with changes in AV node delays (measured with surface electrodes) caused by changes in heart rate or perfusion with acetylcholine. The data provide the first maps of activation across the AV node and demonstrate that theta N is faster than previously presumed. These findings are inconsistent with theories of decremental conduction and prove the existence of a conduction barrier between the atrium and the AV node that is an important determinant of AV node delay.

Sodium mercaptoacetate is not a useful probe to study the role of fat in regulation of feed intake in dairy cattle.

Inhibition of fatty acid oxidation by mercaptoacetate stimulates food intake of rats fed dietary fat. To study regulation of feed intake of ruminants fed fat, dry matter intake and plasma concentrations of insulin and metabolites were determined in eight nonpregnant Holstein heifers in a cross-over design with two 14-d feeding periods by using a 2 x 2 factorial arrangement of treatments. Treatments were combinations of diet (27 or 103 g fatty acids/kg food dry matter) and injection (mercaptoacetate or saline). Half the heifers were fed each diet in Period 1, and diets were reversed in Period 2. On d 10 of each period, two animals per treatment were injected intravenously with either mercaptoacetate (300 mumol/kg body weight 0.75) or saline at 2 h postfeeding. Injections were reversed on d 12. Dry matter intake was suppressed by the high fat diet. Intravenous injection of mercaptoacetate decreased dry matter intake to 25% that of the control during 4 h postinjection. Both the high fat diet and mercaptoacetate injection increased plasma non-esterified fatty acid concentration, whereas plasma beta-hydroxybutyrate concentration was lowered by the high fat diet and by mercaptoacetate injection. Plasma triglyceride concentration was increased by the high fat diet, but was decreased by mercaptoacetate injection. Mercaptoacetate elevated plasma glucose concentrations at 2 and 3 h postinjection, possibly because plasma insulin concentration was lower. Effects of mercaptoacetate on plasma insulin and metabolite concentrations may have been confounded by the effects of decreased feed intake. Therefore, direct effects of mercaptoacetate injection were not separated from effects of feed intake on plasma insulin and metabolite concentration. Because mercaptoacetate injection decreased dry matter intake it was not a useful probe to study mechanisms of feed intake regulation in dairy cattle fed fat.

High fat diets increase plasma cholecystokinin and pancreatic polypeptide, and decrease plasma insulin and feed intake in lactating cows.

High fat diets often decrease feed intake in dairy cows; however, mechanisms underlying fat-induced depression of feed intake are yet to be established. The postulate that high fat diets decrease feed intake by increasing concentrations of lipid metabolites or satiety hormones in blood was tested by using eight multiparous Holstein cows in a simultaneously replicated 4 x 4 Latin-square design. Treatments were control diet with 1) no fat added, 2) 30 g/kg calcium salts of long-chain fatty acids, 3) 60 g/kg calcium salts of long-chain fatty acids, and 4) 90 g/kg calcium salts of long-chain fatty acids. Cows were fed once daily a diet of concentrate, corn silage, alfalfa haylage and alfalfa hay (50:25:14:11 on a dry matter basis). Dry matter and energy intakes were decreased by inclusion of calcium salts of long-chain fatty acids >30 g/kg of total diet dry matter (P = 0.0001). Plasma nonesterified fatty acids and triglyceride concentrations were increased linearly by feeding increasing amounts of fat (P < 0.003 and P = 0.0001, respectively), whereas plasma beta-hydroxybutyrate and glucose concentrations were not influenced by supplemental fat. Fat supplementation increased postfeeding plasma cholecystokinin concentrations and linearly increased plasma pancreatic polypeptide concentrations. Highest concentrations of plasma cholecystokinin (P < 0.001) and pancreatic polypeptide (P < 0.05) were observed in cows fed the 90 g/kg fat supplement. Plasma insulin was lowered linearly by feeding fat (P = 0.0001). Increased concentrations of cholecystokinin and pancreatic polypeptide were associated with decreased intakes of feed and energy, whereas insulin may not be involved in the control of feed intake in cows fed fat.