Changes in the Relationship between Ionized and Total Calcium in Clinically Healthy Dairy Cows in the Period around Calving

We aimed to establish a model for prediction of iCa from tCa, using multivariable regressions with diverse blood constituents. Blood was taken from 14 cows at days −2, 0, 2, 4, 7, and 14 relative to parturition. Cows were clinically healthy, and no hypocalcaemia prophylaxis and treatment were applied. Total calcium and further parameters were determined from frozen serum. Ionized calcium, blood gases, and electrolytes were determined from heparin-stabilized blood samples. Linear regression between iCa and tCa was estimated. Precision improved only slightly using a multivariable model. Best precision was achieved when estimating the iCa:tCa ratio from other blood constituents. To identify the reason behind the poorly predictive value of tCa for iCa, the relative changes of iCa and tCa around calving were calibrated to the respective values of day −2 (=100%) for each cow. An increase in the iCa:tCa ratio was observed from 0.43 at day −2 to 0.48 at day 0, followed by a gradual decrease towards 0.43 at day 7. We conclude that routine measurement of iCa should be implemented in the diagnosis of hypocalcaemia. An optimized estimate of iCa from tCa with non-esterified fatty acids (NEFA), beta-hydroxybutyric acid, cholesterol, and phosphorous as co-predictors is still poorly satisfying

Dietary supplementation of essential oils in dairy cows: evidence for stimulatory effects on nutrient absorption

Results of recent in vitro experiments suggest that essential oils (EO) may not only influence ruminal fermentation but also modulate the absorption of cations like Na+, Ca2+ and NH4+ across ruminal epithelia of cattle and sheep through direct interaction with epithelial transport proteins, such as those of the transient receptor potential family. The aim of the current study was to examine this hypothesis by testing the effect of a blend of essential oils (BEO) on cation status and feed efficiency in lactating dairy cows. In the experiment, 72 dairy cows in mid-to-end lactation were divided into two groups of 36 animals each and fed the same mixed ration with or without addition of BEO in a 2×2 cross-over design. Feed intake, milk yield and composition, plasma and urine samples were monitored. Feeding BEO elevated milk yield, milk fat and protein yield as well as feed efficiency, whereas urea levels in plasma and milk decreased. In addition, plasma calcium levels increased significantly upon BEO supplementation, supporting the hypothesis that enhanced cation absorption might contribute to the beneficial effects of these EO

The bovine TRPV3 as a pathway for the uptake of Na+, Ca2+, and NH4+

Absorption of ammonia from the gastrointestinal tract results in problems that range from hepatic encephalopathy in humans to poor nitrogen efficiency of cattle with consequences for the global climate. Previous studies on epithelia and cells from the native ruminal epithelium suggest functional involvement of the bovine homologue of TRPV3 (bTRPV3) in ruminal NH4+ transport. Since the conductance of TRP channels to NH4+ has never been studied, bTRPV3 was overexpressed in HEK-293 cells and investigated using the patch-clamp technique and intracellular calcium imaging. Control cells contained the empty construct. Divalent cations blocked the conductance for monovalent cations in both cell types, with effects higher in cells expressing bTRPV3. In bTRPV3 cells, but not in controls, menthol, thymol, carvacrol, or 2-APB stimulated whole cell currents mediated by Na+, Cs+, NH4+, and K+, with a rise in intracellular Ca2+ observed in response to menthol. While only 25% of control patches showed single-channel events (with a conductance of 40.8 ± 11.9 pS for NH4+ and 25.0 ± 5.8 pS for Na+), 90% of bTRPV3 patches showed much larger conductances of 127.8 ± 4.2 pS for Na+, 240.1 ± 3.6 pS for NH4+, 34.0 ± 1.7 pS for Ca2+, and ~ 36 pS for NMDG+. Open probability, but not conductance, rose with time after patch excision. In conjunction with previous research, we suggest that bTRPV3 channels may play a role in the transport of Na+, K+, Ca2+ and NH4+ across the rumen with possible repercussions for understanding the function of TRPV3 in other epithelia

Effects of dietary menthol-rich bioactive lipid compounds on zootechnical traits, blood variables and gastrointestinal function in growing sheep

Background The present study aimed at investigating the influence of 90% menthol-containing plant bioactive lipid compounds (PBLC, essential oils) on growth performance, blood haematological and biochemical profile, and nutrient absorption in sheep. Twenty-four growing Suffolk sheep were allotted into three dietary treatments: Control (without PBLC), lower dose of PBLC (PBLC-L; 80 mg/d) and higher dose of PBLC (PBLC-H; 160 mg/d). Sheep in all groups were fed meadow hay ad libitum plus 600 g/d of concentrate pellets for 28 d. Results Average daily gain was not affected by treatment. Feeding of PBLC increased hay and total feed intake per kg body weight (P < 0.05). Counts of total leucocytes, lymphocytes and monocytes were not different among treatments. However, neutrophil count decreased (P < 0.05) in PBLC-H with a similar trend in PBLC-L (P < 0.10). Concentrations of glucose, bilirubin, triglycerides, cholesterol, urea and magnesium in serum were not different among sheep fed different doses of PBLC. However, serum calcium concentration tended to increase in PBLC-H (P < 0.10) and serum concentrations of aspartate & asparagine (P < 0.01) and glutamate & glutamine (P < 0.05) increased linearly with increasing PBLC dose. In ruminal epithelia isolated from the rumen after killing, baseline conductance (Gt; P < 0.05) and short-circuit current (Isc; P < 0.01) increased in both PBLC groups. Ruminal uptakes of glucose and methionine in the presence of Na+ were not affected by the dietary PBLC supplementation. In the absence of Na+, however, glucose and methionine uptakes increased (P < 0.05) in PBLC-H. In the jejunum, Isc tended to increase in PBLC-H (P < 0.10), but baseline Gt was not affected. Intestinal uptakes of glucose and methionine were not influenced by PBLC in the presence or absence of Na+. Conclusion The results suggest that menthol-rich PBLC increase feed intake, and passive ion and nutrient transport, the latter specifically in the rumen. They also increased serum concentrations of urea precursor amino acids and tended to increase serum calcium concentrations. Future studies will have to show whether some of these findings might be commonly linked to a stimulation of transient receptor potential (TRP) channels in the gastrointestinal tract

TRPV3 and TRPV4 as candidate proteins for intestinal ammonium absorption

Aim: Absorption of ammonia from the gut has consequences that range from encephalitis in hepatic disease to global climate change induced by nitrogenous excretions from livestock. Since patch clamp data show that certain members of the transient receptor potential (TRP) family are permeable to NH4+, participation in ammonium efflux was investigated. Methods: Digesta, mucosa and muscular samples from stomach, duodenum, jejunum, ileum, caecum and colon of pigs were analysed via colourimetry, qPCR, Western blot, immunohistochemistry and Ussing chambers. Results: qPCR data show high duodenal expression of TRPV6. TRPM6 was highest in jejunum and colon, with expression of TRPM7 ubiquitous. TRPM8 and TRPV1 were below detection. TRPV2 was highest in the jejunum but almost non-detectable in the colon. TRPV4 was ubiquitously expressed by mucosal and muscular layers. TRPV3 mRNA was only found in the mucosa of the caecum and colon, organs in which NH4+ was highest (>7 mmol·L−1). Immunohistochemically, an apical expression of TRPV3 and TRPV4 could be detected in all tissues, with effects of 2-APB and GSK106790A supporting functional expression. In symmetrical NaCl Ringer, removal of mucosal Ca2+ and Mg2+ increased colonic short circuit current (Isc) and conductance (Gt) by 0.18 ± 0.06 µeq·cm−2·h−1 and 4.70 ± 0.85 mS·cm−2 (P < .05, N/n = 4/17). Application of mucosal NH4Cl led to dose-dependent and divalent-sensitive increases in Gt and Isc, with effects highest in the caecum and colon. Conclusion: We propose that TRP channels contribute to the intestinal transport of ammonium, with TRPV3 and TRPV4 promising candidate proteins. Pharmacological regulation may be possible

Original recordings of two bTRPV3 cells filled with NMDG-gluconate solution.

<p>a) As before, replacement of chloride by gluconate resulted in a decrease in outward current at positive potentials, suggesting expression of chloride channels. Both addition of Ca²⁺ (10 mmol∙l⁻¹) and replacement by Ca-gluconate₂ (60 mmol∙l⁻¹) had a strong blocking effect, with washout to levels higher than before. The current could be partially blocked by Mg²⁺, arguing against a rupture of the seal. Note the current kinetics with amplitude decreasing after a hyperpolarising pulse suggesting that Mg²⁺ is being drawn into the channel pore by the negative membrane voltage. Conversely, a depolarising voltage pulse leads to a relief of the block. In both cases, the currents appear curved in contrast to the linear kinetics of currents in pure NMDG-gluconate solution. b) Second cell, showing a spontaneous activation of inward and outward current amplitude by a factor of about four between the first set of pulses in NMDG-gluconate EGTA solution (1) and a second set (2) that was run 100 seconds later. Current amplitude continued to increase both after addition of 10 mmol∙l⁻¹ Ca²⁺ to the NMDG-gluconate solution, and after replacement of NMDG-gluconate by Ca-gluconate₂. Both the current kinetics and the tail currents indicate a partial voltage-dependent block. The arrows above the first set of pulses indicate the interval used for the determination of mean current amplitude for the current-voltage plot, which is depicted in c), showing rectification as a further sign of a voltage-dependent block. The reversal potential in 60 mmol∙l⁻¹ Ca-gluconate₂ solution was 7.1 mV, revealing permeability to Ca²⁺.</p

Immunoblots and fluorescence microscopy images of HEK-293 cells expressing bTRPV3.

<p>a) Immunoblot of HEK-293 cell lysates 24 hours after transfection with a pIRES2-AcGFP1 vector. The marker lane (M) is followed by a lane of control cells transfected with the empty pIRES2-AcGFP1 vector (no band), followed by lane of strep-tagged bTRPV3-pIRES2-AcGFP1 cells. A strong band appears at ~ 90 kDa after staining with strep-antibody. b) Fluorescence microscopy image shows HEK-293 cells reseeded at a lower concentration for patch clamp measurements after 48 hours of incubation with bTRPV3-pIRES2-AcGFP vector. Green fluorescence indicates a successful expression of the bTRPV3-pIRES2-AcGFP vector. c) Immunoblot of HEK-293 cell lysates 24 hours after transfection with bTRPV3-pcDNA5/TO vector.</p

Inside-out measurements from cells expressing bTRPV3 in symmetrical solutions.

<p>Panels (a to d) show recordings from the same patch and identical scaling in a) symmetrical NaCl solution, b) NaCl solution with 1.5 mmol∙l ⁻¹ MgCl₂, c) Na-gluconate solution without MgCl₂ and d) return to symmetrical NaCl solution. The shift in the baseline at 0 mV pipette potential in (c) reflects the impact of the liquid junction potential. e) Patch from a second cell in symmetrical NH₄Cl solution. For clarity, this graph only shows currents at every other voltage step of pulse protocol III. f) Current-voltage plot of the unitary current amplitudes of the data from (a to e) (linear fit).</p

Current-voltage plots of whole cell measurements from Fig 2.

<p>The figures give the mean current values (± SEM) of control cells (a, c) and cells expressing bTRPV3 (b, d) in Na-gluconate (a, b) or NH₄-gluconate (c, d) bath solutions with (●) and without (○) divalent cations, as indicated. (*: p < 0.05, tested for −100 mV and +100 mV).</p

Response to various agonists of bTRPV3.

<p>Cells were filled with a CsCl pipette solution and stimulated with the continuous pulse protocol I. a) Original recording of a cell expressing bTRPV3, showing the response to application of menthol, thymol (both 1 mmol∙l⁻¹), and 2-APB (0.3 mmol∙l⁻¹) in NaCl bath solution. In response to 2-APB, the current rose dramatically, requiring an interruption of the measurement to readjust the gain (arrow). b) Original recording of a control cell transfected with the empty vector.</p