Biomarkers of inflammation in cattle determining the effectiveness of anti-inflammatory drugs

Myers, M. J., Scott, M. L., Deaver, C. M., Farrell, D. E., Yancy, H. F. Biomarkers of inflammation in cattle determining the effectiveness of anti-inflammatory drugs. J. vet. Pharmacol. Therap. 33 , 1–8.
The impact of nonsteroidal anti-inflammatory drugs (NSAID) on prostaglandin E₂ (PGE₂) production and cyclooxygenase 2 (COX-2) mRNA expression in bovine whole blood (WB) cultures stimulated by lipopolysaccharide (LPS) was determined, using the blood from six Holstein dairy cattle in various stages of lactation. Peak production of PGE₂ occurred 24 h after LPS stimulation but did not result in detectable concentrations of thromboxane B₂ (TXB₂). The NSAID indomethacin, aspirin, flunixin meglumine, and 4-[5-phenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzene sulfonamide (PTPBS; celecoxib analogue), along with dexamethasone, were all equally effective in reducing the concentration of PGE₂ in the bovine WB culture supernatants. Bradykinin exhibited peak supernatant concentrations 1 h after LPS stimulation. Dexamethasone and the NSAID used in this study were equally effective at inhibiting bradykinin production. Peak induction of COX-2 mRNA occurred 3 h post-LPS stimulation. However, neither dexamethasone nor any of the NSAID used in this study altered COX-2 mRNA concentrations. In contrast, aspirin, flunixin meglumine, and PTPBS reduced tumor necrosis factor-alpha (TNFα) mRNA concentration. These results demonstrate that bovine blood cells respond to NSAID therapy like other mammalian cells with respect to inhibition of PGE₂ production and suppression of TNF mRNA induction, but do not inhibit induction of COX-2 mRNA.     

Establishing and functional testing of a canine corneal construct

Purpose  To provide a model to be used for in vitro studies on drug effects in dogs, this study was conducted to establish a protocol for the construction of a three-dimensional corneal construct. Primary canine corneal cells and a rabbit corneal epithelial (RCE) cell line were used in comparison.
Methods  The corneal construct was assembled step by step in membrane inserts of a six-well plate over a total of 5 weeks, including culture at the air–liquid interface to allow a differentiation of the epithelial cells. The constructs were studied histologically.
Single cell cultures of canine corneal cells as well as RCE cells were stimulated with lipopolysaccharides (LPS) and sodium dodecyl sulfate (SDS). Treatment with different concentrations of dexamethasone was used to test its effects on the cellular prostaglandin E₂ (PGE₂) production. The same experiments were repeated with the corneal constructs and the reactions compared. Expression of the glucocorticoid receptor (GR) in the constructs was studied using immunohistochemistry.
Results  A protocol for the construction of a vital corneal construct was established and the morphological similarity to the canine cornea in vivo shown. The GR protein was detected in all three cell types of the constructs. Stimulation with LPS and SDS led only in the corneal constructs to a significantly increased PGE₂ production, which could be reduced by dexamethasone.
Conclusions  The corneal construct is an interesting system to test drug effects on corneal cells. It allows studies on a cornea-like system including all three major cell types.     

Characterization of a sterile soft-tissue inflammation model in Thoroughbred horses

This paper describes the use of subcutaneously-placed tissue chambers as a sterile soft-tissue inflammation model in Thoroughbred horses. Acute, nonimmune inflammation was initiated by injecting a sterile lambda carrageenan solution into a tissue chamber. This model was used to study the temporal changes in oxygen and carbon dioxide tensions, pH, bicarbonate, protein, albumin, prostaglandin E₂ (PGE₂) and leukotriene B₄ (LTB₄) concentrations, cell counts and differential counts in tissue fluid from inflamed tissue chambers and control chambers. Skin temperatures over control and inflamed chambers were also compared. Carrageenan-induced inflammation resulted in significant increases in tissue-fluid carbon dioxide tension, leucocyte count, albumin, and PGE₂ and LTB₄ concentrations. It also resulted in a significant decrease in tissue fluid pH and HCO₃- concentration. Inflammation did not result in significant changes in tissue-fluid protein concentration, differential cell counts or skin temperature over the chambers. The use of this type of tissue chamber is wellsuited for studying the pathophysiology of a self-contained, non-immune inflammatory process. The model described in this paper could prove to be very useful in studies of the distribution of anti-inflammatory drugs and the effects of such drugs on various aspects of the inflammatory process.     

Reactivity of Equine Palmar Digital Arteries and Veins to Vasodilating Agents

Palmar digital arteries and veins removed surgically from healthy horses under general anesthesia were cut into 4 mm vascular rings, suspended in tissue baths, and attached to force displacement transducers for continuous measurement of vascular tension. In vitro vascular responses were determined for acetylcholine, acepromazine, isoxsuprine hydrochloride (isoxsuprine), prostaglandin E₂ (PGE₂), and prostaglandin I₂ (prostacyclin). After preconstriction with norepinephrine hydrochloride (norepinephrine), or prostaglandin F₂ alpha (PGF₂ alpha), the concentrations needed to produce 50% maximum relaxation (EC₅₀) and the maximum percentage of relaxation were determined for each drug.
Acetylcholine was the most potent arterial vasodilator (smallest EC₅₀ value) and PGE₂ was the least potent. Prostacyclin was the least potent venodilator (highest EC₅₀ value); there were no differences between acetylcholine, acepromazine, isoxsuprine, and PGE₂. Isoxsuprine produced greater arterial relaxation than all other agents. Isoxsuprine and acepromazine produced significantly greater venous relaxation than did acetylcholine and PGE₂. Prostacyclin produced minimal vasodilation of arteries or veins. Acepromazine and isoxsuprine relaxed the veins significantly more than the arteries. When PGF₂ alpha was used instead of norepinephrine to preconstrict the arteries and veins, the potency and effectiveness of acepromazine and isoxsuprine to produce vasodilation were significantly decreased. Results indicate that acepromazine and isoxsuprine can relax the equine digital vasculature but their effectiveness varies depending on the origin of the constriction.     

Piroxicam Therapy in 34 Dogs With Transitional Cell Carcinoma of the Urinary Bladder

Thirty-four dogs with histopathologically confirmed, measurable, nonresectable transitional cell carcinoma of the urinary bladder were treated with piroxicam (0.3 mg/kg PO sid) and were evaluated for tumor response and drug toxicity. Dogs were evaluated at the Purdue University Veterinary Teaching Hospital by means of physical examination, thoracic and abdominal radiography, cystography, complete blood count, serum biochemistry profile, and urinalysis. In selected cases, prostaglandin E₂ (PGE₂) concentrations in plasma and in supernatants of stimulated monocytes, and natural killer cell activity were quantified, Dogs were evaluated before therapy and at 28 and 56 days after initiation of therapy. Dogs with stable disease or remission at 56 days remained on the study and were evaluated at 1 to 2 month intervals. Tumor responses were 2 complete remissions, 4 partial remissions, 18 stable diseases. and 10 progressive diseases. The median survival of all dogs was 181 days (range, 28 to 720+ days), with 2 dogs still alive. Piroxicam toxicity consisted of gastrointestinal irritation in 6 dogs and renal papillary necrosis (detected at necropsy) in 2 dogs. Monocyte production of PGE₂ appeared to decrease with therapy in dogs whose tumors were decreasing in size, and increased in dogs with tumor progression. A consistent pattern in natural killer cell activity was not observed. In vitro cytotoxicity assays against 4 canine tumor cell lines revealed no direct antitumor effects of piroxicam. In summary, antitumor activity, which was not likely the result of a direct cytotoxic effect, was observed in dogs with transitional cell carcinoma of the bladder treated with piroxicam.     

Pharmacokinetics and pharmacodynamics of meloxicam in piglets

The pharmacokinetics and pharmacodynamics of meloxicam in piglets (16–23 days old) were studied using a stratified parallel group design. One group ( n  = 13) received 0.4 mg/kg meloxicam intravenously, while the other group ( n  = 12) received physiological saline solution. A carrageenan-sponge model of acute inflammation was used to evaluate the effects of meloxicam. The plasma clearance was low (0.061 L/kg/h), the volume of distribution was low (0.19 L/kg) and the elimination half-life was short (2.7 h). At most time points, the mean concentration of meloxicam in plasma exceeded the concentrations in exudate indicating a limited accumulation of the drug at the site of the inflammation. There were significant differences between the groups in the exudate prostaglandin E₂ (PGE₂) concentration, but the inhibition of PGE₂ in the meloxicam group was limited. The inhibition of thromboxane B₂ (TXB₂) production in serum in the meloxicam group was extensive, but of shorter duration than the PGE₂ inhibition in exudate.     

Specific binding of dl-cloprostenol and d-cloprostenol to PG F2α receptors in bovine corpus luteum and myometrial cell membranes

Prostaglandin F_2α receptors (PGF_2α Rs) were measured in bovine corpus luteum and myometrial cell membranes using a radiometric method. The inhibition of labelled PGF_2α binding exerted by d-cloprostenol, dl-cloprostenol, PGF_2α and PGE₁ (l0–¹¹ M to 10^–4 M) was evaluated in vitro. Results strongly suggest that cloprostenol binding to PGF_2αRs is stereospecific. d-Cloprostenol and PGF_2α were equipotent, about 150 times more potent than d-cloprostenol (P < 0.05) and approximately 280 times more potent than PGE, (P < 0.05) in inhibiting [3H]PGF_2α binding to corpus luteum cell membranes. Such differences were less evident in myometrial cell membranes, where d-cloprostenol and PGF_2α were about 10 times more potent than d-cloprostenol (P < 0.05) and approximately 95 times more potent than PGE_l (P < 0.05).     

Induction of Chemoresistance by Transfection of the Full‐Length Canine mdr1 Gene in Canine Cell Lines

Introduction:  In the chemotherapy for treatment of lymphoid tumors in dogs, myelosuppression is a frequently encountered dose‐limiting factor. One possible approach to overcome myelosuppression is induction of chemoresistance in hematopoietic stem cells through expression of the mdr1 gene. A full‐length canine mdr1 cDNA clone was isolated in our laboratory. The present study was carried out to assess whether it confers multidrug resistance in canine cell lines.
Materials and methods:  The full‐length canine mdr1 cDNA was inserted into an expression plasmid vector. A canine mammary tumor cell line (CIPP) and osteosarcoma cell line (OOS) were transfected with the canine mdr1 expression vector. Expression of P‐gp was examined by immunoblotting. Function of ATP‐dependent drug efflux was measured by flow cytometric analysis using Rhodamine 123. Sensitivity to chemotherapeutic drugs was shown by estimation of 50% inhibitory concentrations (IC₅₀) of vincristine or doxorubicin.
Results:  Immunoblotting of the transfected cells revealed a strong band of P‐gp detected by a monoclonal antibody directed to P‐gp. Rhodamine 123 efflux test showed an apparent drug efflux activity in the transfected cells. From the IC₅₀ of the chemotherapeutic agents, the transfected cells showed a remarkable increase (20 to 60‐fold) in the resistance to vincristine or doxorubicin.
Conclusion:  Transfection of canine mdr1 gene induced P‐gp expression and strong drug resistance in canine cell lines.     

Role of Eicosanoids in the Regulation of the Periparturient Period in Cattle

Contents: In this review, the role of eicosanoids in regulation of parturition and the postpartum period was described with special emphasis on the bovine species. The metabolism of arachidonic acid and the production of eicosanoids during the peripartum period was discussed. Prostaglandin E₂ and F_2α (PGE₂, PGF_2α) play an important role in mechanisms controlling parturition. They are involved in luteolysis, uterine contractions and dilation of the cervix. Eicosanoids also seem to influence the loosening processes of the fetal membranes. However, in the literature, conflicting results were found. Many investigations suggested that retained placental membranes could be related to low PGF_2α production and/ or an imbalance of arachidonic acid metabolism in the uterus. The possible role of the lipoxygenase pathway metabolite 15-hydroxy-eicosatetraenoic acid (15-HETE) in expulsion of the fetal membranes was also discussed. As far as the postpartum period is concerned, a relationship between postpartum PGF_2α release and the involution of the uterus was found. Cows with undisturbed uterine involution had higher PGF_2α production than cows with delayed involution. In contrast to the positive effect of PGF_2α on uterine involution, PGE₂ seems to delay the involution processes. Further experiments are necessary in order to study the function of eicosanoids in mechanisms regulating parturition, release of the fetal placental membranes and involution of the uterus.     

Differential inhibition of cyclooxygenase isoenzymes in the cat by the NSAID robenacoxib

Robenacoxib is a new nonsteroidal anti-inflammatory drug (NSAID) developed for use in companion animal medicine. The objectives of this study were: to quantify the inhibitory actions of robenacoxib on cyclooxygenase (COX) isoenzymes in feline whole blood assays; to establish blood concentration–time profiles of robenacoxib after intravenous and subcutaneous dosing in the cat and; to predict the time courses of inhibition of COX isoforms by robenacoxib. COX-1 and COX-2 activities in heparinized feline whole blood samples were induced with calcium ionophore and lipopolysaccharide, respectively. Inhibition of thromboxane B₂ provided a marker of both COX-1 and COX-2 activities and a nonlinear parametric mixed effects modelling approach was used to establish the pharmacodynamic parameters describing this inhibition. Mean values (and prediction intervals) of IC₅₀ were 28.9 (16.4–51.1) μ m (COX-1) and 0.058 (0.010–0.340) μ m (COX-2). These parameters were used to compute several selectivity indices. Selectivity IC ratios (COX-1:COX-2) were 502.3 (IC₅₀/IC₅₀), 451.6 (IC₉₅/IC₉₅) and 17.05 (IC₂₀/IC₈₀). Based on a clinically recommended dosage regimen of 2 mg/kg, it was predicted that the corresponding mean robenacoxib blood concentration over the first 12 h after drug administration corresponded to 5% inhibition of COX-1 and 90% inhibition of COX-2.     

Pharmacological evaluation of sheep lung strip

Isolated sheep lung parenchymal strips responded to histamine > carbachol > PGF_2a > 5-HT with contractions, and to isoproterenol (Isop), and to large doses of epinephrine (E), norepinephrine (NE) and phenylephrine (PE) with relaxations. PGF_2a-contracted lung strip responded to PGE₁ and PGE₂ with relaxation. The strips which were partially contracted to histamine, PGF_2a, 5-HT and carbachol also responded to isop, E and NE with relaxations. Histamine responses were not modified by metiamide (an H₂-receptor antagonist). Mepyramine and atropine selectively antagonized contractions to histamine and carbachol, respectively. After β-blockade with propranolol, lung strips responded to NE > E > PE > isop with contractions, which were inhibited or reversed by phentolamine and dibenzyline. It is concluded that H₁ receptors are present in sheep peripheral airway smooth muscles, and that a- and β-adrenoceptors mediate contraction and relaxation, respectively, in sheep lung strips.     

Comparative study of the action of flunixin meglumine and tolfenamic acid on prostaglandin E2 synthesis in bovine inflammatory exudate

An acute non-immune inflammation model was used to compare the action of two non-steroidal anti-inflammatory drugs, flunixin meglumine and tolfenamic acid, on prostaglandin E₂, (PGE₂) synthesis in bovine inflammatory exudate. The tissue cage model used involves subcutaneous implantation of polypropylene cages and subsequent stimulation by carrageenan injection of the granulation tissue which develops within the cage. Twelve calves were randomly assigned to three groups receiving placebo, flunixin meglumine and tolfenamic acid, respectively. Inflammatory exudate was sampled 30 min after carrageenan injection and at seven subsequent time points. PGE², levels were determined by radioimmunoassay. At each time point post-carrageenan injection, flunixin meglumine inhibited PGE₂, synthesis to a greater extent than tolfenamic acid. At 4, 8,12 and 24 h these differences were statistically significant.     

A pharmacodynamic and pharmacokinetic study with vedaprofen in an equine model of acute nonimmune inflammation

The pharmacodynamics and enantioselective pharmacokinetics of vedaprofen were studied in six ponies in a two period cross-over study, in which a mild acute inflammatory reaction was induced by carrageenan soaked sponges implanted subcutaneously in the neck. Vedaprofen, administered intravenously at a dosage of 1 mg/kg, produced significant and prolonged inhibition of ex vivo serum thromboxane B₂ (TXB₂) synthesis and short-lived inhibition of exudate prostaglandin E₂ (PGE₂) and TXB₂ synthesis. Vedaprofen also partially inhibited oedematous swelling and leucocyte infiltration into exudate. Vedaprofen dis-played enantioselective pharmacokinetics, plasma concentrations of the R(–) enantiomer exceeding those of S(+) vedaprofen. The plasma concentration ratio, R:S, increased from 69: 31 at 5 min to 96: 4 at 3 h and plasma mean AUC values were 7524 and 1639 ng.h/mL, respectively. Volume of distribution was greater for S(+) vedaprofen, whilst elimination half-life (t_½β) and mean residence time were greater for R(–) vedaprofen. The penetration of vedaprofen into inflammatory exudate was also enantioselective. For R(–) and S(+) veda-profen maximum concentration (C_max) values were 2950 and 1534 ng/mL, respectively, and corresponding AUC values were 9755 and 4400 ng.h/mL. Vedaprofen was highly protein bound (greater than 99%) in both plasma and exudate. The significance of these data for the therapeutic use of vedaprofen is discussed.     

前列腺素E2和F2α对LPS刺激后奶牛子宫内膜上皮细胞COX-1和COX-2表达的影响

试验旨在阐明前列腺素E₂（prostaglandin E₂,PGE₂）和F_2α（prostaglandin F_2α,PGF_2α）对体外培养的奶牛子宫内膜上皮细胞中环氧合酶-1（cyclooxygenase-1,COX-1））与环氧合酶-2（cyclooxygenase-2,COX-2）表达的影响。培养奶牛子宫内膜上皮原代细胞和传代细胞,第4代细胞以1×10⁶个/孔接种于6孔板,以10^-7mol/L PGE₂和PGF_2α分别预处理细胞24 h,以100 ng/mL细菌脂多糖（lipopolysaccharides,LPS）刺激细胞4、8和12 h后分别提取RNA和总蛋白质,采用实时荧光定量PCR与Western blotting等技术检测COX-1与COX-2 mRNA和蛋白质的表达量。结果表明,与对照组相比,COX-1 mRNA表达量在PGE₂单独作用4、8和12 h后显著上调（P<0.05）;COX-2 mRNA表达量在PGE₂单独作用4和12 h后显著上调（P<0.05）,PGE₂单独处理使COX-1、COX-2蛋白表达量均显著上调（P<0.05）。与对照组相比,LPS刺激8和12 h时COX-1 mRNA表达量显著下调（P<0.05）,LPS刺激后COX-1蛋白表达量无显著变化（P>0.05）;LPS刺激后4、8和12 h时COX-2 mRNA表达量显著上调（P<0.05）,LPS刺激后COX-2蛋白表达量显著上调（P<0.05）。与LPS单独处理组相比,LPS+PGE₂处理组在8和12 h时COX-1和COX-2 mRNA表达量均显著上调（P<0.05）,同时COX-1和COX-2蛋白表达量也显著上调（P<0.05）。PGF_2α在LPS未刺激和刺激后对COX-1和COX-2 mRNA的表达无显著影响（P>0.05）,仅在PGF_2α单独处理8和12 h后COX-1 mRNA表达量上调（P<0.05）。两种激素联合处理与各自单独处理及LPS单独刺激相比,对COX-1和COX-2 mRNA表达具有一定的协同诱导作用。     

Concentrations of thyroid hormones and iodothyronine binding proteins in serum of the koala (Phascolarctos cinereus)

Objective To examine circulating total and free thyroid hormone (T₃ and T₄) concentrations, determine serum iodothyronine binding characteristics and estimate thyroid stimulating hormone (TSH) activity in sera of coastal and inland koalas.
Design A prospective study.
Procedure Koala serum T₃ and T₄ were measured by radioimmunoassay. T₄ binding parameters were determined by radioligand binding and electrophoresis. Koala TSH values were determined by bioassay.
Results Mean total T₄ concentrations were 3.2 ± 2.1 nM although values were significantly higher in inland-dwelling females in comparison to coastal-dwelling males. Free T₄ was 3.3 ± 2.1 pM. Total and free T₃ were 0.4 ± 0.2 nM and 1.4 ± 0.9 pM respectively, although these values were at the lower end of the assay detection limit and should be viewed with reservation. Electrophoresis of [¹²⁵I]-T₄-labelled serum revealed only two proteins of electrophoretic mobility similar to human transthyretin (TTR) and albumin. Scatchard analysis of T₄ binding to serum gave a curvilinear plot, which could be resolved into two binding sites with affinities identical to that of TTR and albumin but both of low concentration. The bioactivity of the TSH present in the sera was measured using a cell line (JP09) transfected with the human TSH receptor. The mean level of stimulation found in the sera corresponded to a bovine TSH activity of less than 10 mU/L.
Conclusion These results suggest that the serum concentrations of free and total thyroid hormones in koalas are low compared to other marsupials and very low compared to eutherian mammals. The mechanism of maintenance of euthyroidism in this species remains to be determined.     

Effects of Estrogen on COX-1 and COX-2 Expression in Bovine Oviduct Smooth Muscle

The aim of this study was to clarify the effects of estrogen (E₂) on cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) gene expression in the cow oviduct tissue in vitro.Quantitative Real-time PCR and Western blotting were used to analyze the mRNA and protein expression of COX-1 and COX-2 at different time points after different concentrations of E₂ treatment.The results indicated that the relative expression of COX-1 and COX-2 mRNA reached the highest levels with 10^-11 mol/L E₂ treatment,the relative expression of COX-1 mRNA reached the highest level at 8 h after treatment;The relative expression of COX-2 mRNA reached the highest level at 2 h after treatment.The relative expression of COX-1 protein increased significantly at the range from 8 to 48 h after E₂ treatment.However,we did not detect COX-2 protein expression.     

雌激素对奶牛输卵管组织COX-1和COX-2表达的影响

试验旨在阐明雌激素(E₂)对体外培养的奶牛输卵管组织中环氧合酶-1(cyclooxygenase-1,COX-1)与环氧合酶-2(cyclooxygenase-2,COX-2)基因表达的影响。分离培养奶牛输卵管组织,用不同浓度的E₂作用于奶牛输卵管组织,采用实时荧光定量PCR与Western blotting检测COX-1与COX-2 mRNA和蛋白的表达量。结果表明,E₂作用浓度为10^-11 mol/L时奶牛输卵管组织中的COX-1与COX-2 mRNA相对表达量达到高峰;E₂作用时间为8 h时COX-1 mRNA的相对表达量达到高峰,E₂作用时间为2 h时COX-2 mRNA的相对表达量达到高峰;浓度为10^-11 mol/L的E₂作用不同时间后,COX-1蛋白的相对表达量在8~48 h显著升高;没有检测到COX-2蛋白的表达。     

Soy Protein Isolate versus Meat-Based Low-Protein Diet for Dogs with Congenital Portosystemic Shunts

Background: Both presurgical preparation and long-term support of nonoperable dogs with congenital portosystemic shunts (CPSS) require optimal dietary management. Studies suggested that protein source may play an important role, with vegetable and dairy protein sources having better effects on hepatic encephalopathy (HE) than meat proteins.
Objectives: Determine whether a low-protein test diet with soy as its main protein source results in better scores than a control diet with the same composition but with poultry as its main protein source in dogs with CPSS.
Methods: In a double-blind cross-over study, 16 dogs received each diet for 4 weeks. Dogs in group T first received the test diet and then the control diet, whereas dogs in group C were fed the diets in the opposite order. Different variables (body weight, body condition score, HE score, fecal score, CBC, plasma tests of liver function including NH₃, and coagulation tests) were measured at the start of the study and after completion of each diet.
Results: One-way repeated measures ANOVA was performed. Plasma NH₃ was significantly lower after the test diet than after the control diet. The test diet also resulted in significantly higher fibrinogen concentrations and lower prothrombin times. The HE score improved with both diets, with no significant difference between the 2 diets.
Conclusions: Both diets achieved a significant improvement in HE score. The influence of the soy-based diet on plasma NH₃ concentration and coagulation parameters suggests that such a diet decreases the risk for HE and gives better support of liver function.     

Vitamin B12 responses to cobalt pellets in beef cows

Objective To assess the effectiveness of cobalt pellets in maintaining adequate vitamin B₁₂ in beef cows on pasture of low cobalt content.
Design A field experiment in a herd grazing cobalt deficient pasture.
Animals Mature Murray Grey cows.
Procedure Cows were given a single oral dose of 0, 1, 2 or 4 cobalt pellets (30 g pellets containing 30% by weight cobaltic oxide) with a selenium pellet and a grub screw. Samples of blood, liver, faeces and milk for chemical analyses were collected at intervals over a period of 2 years after treatment.
Results A single cobalt pellet raised liver vitamin B₁₂ concentration of cows above that of untreated cows for at least 28 weeks, and 2 or 4 pellets for 57 weeks. Plasma vitamin B₁₂ concentration was an unreliable indicator of the effectiveness of cobalt pellet therapy. Milk vitamin B₁₂ and faecal cobalt concentrations increased in response to cobalt pellet therapy.
Conclusion These studies show that one cobalt pellet will prevent vitamin B₁₂ inadequacy in beef cows for between 28 and 57 weeks; two or four pellets will prevent inadequacy for 57 to 75 weeks. Milk vitamin B₁₂ concentration may be a useful indicator of the effectiveness of cobalt pellets in increasing the vitamin B₁₂ supply in lactating cows.     

Effects of Exogenous Tumour Necrosis Factor-α on the Secretory Function of the Bovine Reproductive Tract Depend on Tumour Necrosis Factor-α Concentrations

The aim of study was to correlate tumour necrosis factor-α (TNF) infused doses used with the TNF concentrations achieved and with the secretory function of both the ovary and the uterus in cows. We evaluated the concentrations of progesterone (P4), prostaglandin (PG)F_2α, PGE₂ nitric oxide (NO) and TNF in the jugular vein and vena cava caudalis as parameters of exogenous TNF action on the female reproductive tract. Aortae abdominalis of cows (n = 18) were infused with saline or two doses of TNF (luteolytic – 1 μg or luteotrophic – 10 μg). In the peripheral blood, 1 μg TNF concentrations achieved within the range of 30–45 pg/ml, and 10 μg TNF provoked a sharp increase in achieved concentrations at a range of 250–450 pg/mL). The TNF concentrations achieved in vena cava caudalis were five to six times higher than that in peripheral blood (p < 0.001). One microgram TNF increased PGF_2α and NO (p < 0.001) and decreased P4 (p < 0.05). The higher TNF dose stimulated P4 and PGE₂ (p < 0.01). TNF infusion at luteolytic dose achieved its concentrations at the physiological range previously observed in cows. Luteotrophic TNF dose achieved the concentrations in vena cava caudalis that are much higher than physiological level and were previously noted in pathological circumstances (i.e. mastitis , metritis ).