Mammary uptake and excretion of prostanoids in relation to mammary blood flow and milk yield during pregnancy-lactation and somatotropin treatment in dairy goats

Mammary arterious − venous differences (A − V) and excretion into milk of four prostanoids were related to changes in milk yield and milk vein blood velocity (MBV) in goats at different stages of pregnancy and lactation, and during somatotropin (ST) treatment in mid-lactation. Arterial concentrations and mammary A − V for the vasodilators prostacyclin (PGI₂) and prostaglandin (PG) E₂ (measured as 6-keto-PGF₁ and bicyclic PGE₂, respectively) decreased from late pregnancy to lactation. A − V were negatively correlated to MBV (r = −0.32 to −0.34). Arterial concentrations of the vasoconstrictors PGF₂ and TXA₂ (measured as TXB₂) changed similarly, but no A − V across the mammary gland were found. The vasodilator to vasoconstrictor ratio in plasma was around 1:1, and in skimmed milk around 0.29–0.49 due to significantly higher TXB₂ levels in milk compared to plasma. Close linear correlations were established between milk yield and excretion of TXB₂ into milk (r = 0.80, P < 0.001), and between MBV and PGE₂ excretion into milk (r = 0.69, P < 0.001). ST treatment stimulated MBV and mammary prostanoid supply, and decreased prostanoid concentration in milk vein plasma. The high arterial levels of prostaglandins during pregnancy most likely reflected uterine synthesis. Our results support a role for PGI₂ and PGE₂ in local mammary blood flow regulation during lactation. Increased mammary uptake of these two prostanoids may be involved in the mammary blood flow response to ST. TXA₂ may be synthesized by mammary epithelial as well as vascular cells, and TXA₂ may be an important factor in regulation of mammary function.     

Tissue chamber model of acute inflammation in farm animal species

A tissue chamber model of acute inflammation for use in comparative studies in calves, sheep, goats and pigs has been established and validated. Tissue chambers were prepared from silicon rubber tubing, of inner diameter 12.7 mm, length 115 mm and volume 15 ml, with 10 holes, each of 6mm diameter, at each end. In each animal two or four chambers were inserted at subcutaneous sites. Six weeks after implantation an acute inflammatory reaction in a single cage was generated by the intracaveal injection of 0.5 ml of 1% carrageenan solution. Serial samples of exudate (injected chamber), transudate (non-injected chamber) and blood were collected for measurement of exudate and transudate leucocyte count, prostaglandin (PG)E(2) concentration in exudate and serum thromboxane (Tx)B(2) concentration. In addition, skin temperature changes over exudate and transudate chambers were recorded. In all four species, carrageenan induced an acute inflammatory response, indicated by increases to peak values followed by return towards baseline in skin temperature, leucocyte count and PGE(2) concentration. For each of these variables in calves, sheep and goats the increases were significantly greater for exudate than for transudate. The degree of intra-species variation in each variable was acceptable. Marked inter-species differences were recorded: skin temperature rise was greatest in calves and least in sheep and goats; exudate PGE(2) concentration was increased in the order sheep>goat>pig>calf; serum TxB(2) concentration was increased in the order calf>goat>sheep>pig and exudate leucocyte count was increased to a greater extent in the pig than in the three ruminant species. The model has advantages over some previously described tissue chamber models of inflammation and will be suitable for use in comparative studies of inflammatory mechanisms and the pharmacokinetics and pharmacodynamics of anti-inflammatory drugs.     

Xylooligosaccharide attenuates lipopolysaccharide-induced intestinal injury in piglets via suppressing inflammation and modulating cecal microbial communities

Xylooligosaccharide (XOS) has been considered to be an effective prebiotic, but its exact mechanisms remain unknown. This research was conducted to evaluate the effects of XOS on pig intestinal bacterial community and mucosal barrier using a lipopolysaccharide (LPS)-caused gut damage model. Twenty-four weaned pigs were assigned to 4 treatments in a 2 × 2 factorial design involving diet (with or without XOS) and immunological challenge (saline or LPS). After 21 d of feeding 0% or 0.02% commercial XOS product, piglets were treated with saline or LPS. After that, blood, small intestinal mucosa and cecal digesta were obtained. Dietary XOS enhanced intestinal mucosal integrity demonstrated by higher villus height, villus height-to-crypt depth ratio, disaccharidase activities and claudin-1 protein expression and lower crypt depth. XOS also caused down-regulation of the gene expression of toll-like receptor 4 and nucleotide-binding oligomerization domain protein signaling, accompanied with decreased pro-inflammatory cytokines and cyclooxygenase 2 contents or mRNA expression and increased heat shock protein 70 mRNA and protein expression. Additionally, increased Bacteroidetes and decreased Firmicutes relative abundance were observed in the piglets fed with XOS. At the genus level, XOS enriched the relative abundance of beneficial bacteria, e.g., Faecalibacterium, Lactobacillus, and Prevotella. Moreover, XOS enhanced short chain fatty acids contents and inhibited histone deacetylases. The correlation analysis of the combined datasets implied some potential connections between the intestinal microbiota and pro-inflammatory cytokines or cecal metabolites. These results suggest that XOS inhibits inflammatory response and beneficially modifies microbes and metabolites of the hindgut to protect the intestine from inflammation-related injury.     

In vitro effects of gonadotropin-releasing hormone (GnRH) on Leydig cells of adult alpaca (Lama pacos) testis: GnRH receptor immunolocalization, testosterone and prostaglandin synthesis, and cyclooxygenase activities

The main objective of this study was to examine the modulatory in vitro effects of gonadotropin-releasing hormone (GnRH) on isolated Leydig cells of adult alpaca (Lama pacos) testis. We first evaluated the presence of GnRH receptor (GnRHR) and cyclooxygenase (COX) 1 and COX2 in alpaca testis. We then studied the in vitro effects of buserelin (GnRH analogue), antide (GnRH antagonist), and buserelin plus antide or inhibitor of phospholipase C (compound 48/80) and COXs (acetylsalicylic acid) on the production of testosterone, PGE(2), and PGF(2α) and on the enzymatic activities of COX1 and COX2. Immunoreactivity for GnRHR was detected in the cytoplasm of Leydig cells and in the acrosomal region of spermatids. COX1 and COX2 immunosignals were noted in the cytoplasm of spermatogonia, spermatocytes, spermatids, Leydig cells, and Sertoli cells. Western blot analysis confirmed the GnRHR and COX1 presence in alpaca testis. The in vitro experiments showed that buserelin alone increased (P < 0.01) and antide and buserelin plus acetylsalicylic acid decreased (P < 0.01) testosterone and PGF(2α) production and COX1 activity, whereas antide and compound 48/80 counteracted buserelin effects. Prostaglandin E(2) production and COX2 activity were not affected by buserelin or antide. These data suggest that GnRH directly up-regulates testosterone production in Leydig cells of adult alpaca testis with a postreceptorial mechanism that involves PLC, COX1, and PGF(2α).     

Evaluation of cyclooxygenase-1 and cyclooxygenase-2 expression and the effect of cyclooxygenase inhibitors in canine prostatic carcinoma

Prostatic carcinoma occurs primarily in older castrated male dogs and is typically a fatal disease (most dogs die within few months after the initial diagnosis). Surgery, i.e., total prostatectomy, or radiation therapy is often not pursued due to risks of complications and a high rate of distant metastasis. Cyclooxygenase‐2 (Cox‐2) expression has been documented in several malignancies, including canine prostatic carcinoma. Cox‐2 inhibition has been reported to have preventative effects on several human malignancies and has therapeutic effects on both laboratory and spontaneous tumour models. The purpose of this retrospective study was to evaluate Cox expression and the effects of Cox inhibitors on survival in dogs with prostatic carcinoma. 94.1 and 88.2% of the tumours expressed Cox‐1 and Cox‐2, respectively. Furthermore, dogs treated with Cox inhibitors (piroxicam or carprofen) lived significantly longer than untreated dogs, 6.9 versus 0.7 months (P < 0.0001), suggesting that Cox inhibitors may have an important role in canine prostate cancer therapy.     

Effects of different cyclooxygenase inhibitors on prostaglandin E2 production,steroidogenesis and ovulation of bovine preovulatory follicles

Ovulation is an inflammation-like process, and cyclooxygenase-2 (COX-2)-dependent production of prostaglandin E₂ (PGE₂) is its key mediator. Balanced regulation of inflammatory processes in high-yielding dairy cows may be essential for physiological ovulation and fertility. This study aimed to elucidate the mechanisms underlying ovulation failure and cyst development after disturbing intrafollicular inflammatory cascades. Therefore, nonselective (indomethacin and flunixin-meglumine), COX-2 selective (meloxicam), and highly COX-2 selective (NS-398) inhibitors were injected into preovulatory follicles 16 h after administration of GnRH, and ovulation was monitored via ultrasound examination. Additionally, follicular fluid was collected after injection of indomethacin, meloxicam, and NS-398. Moreover, primary granulosa cell cultures from preovulatory follicles were prepared and treated with indomethacin, meloxicam, and NS-398. The concentrations of 17β-estradiol, progesterone, and prostaglandin E₂ (PGE₂) in the follicular fluid and cell supernatant were estimated. Indomethacin and flunixin-meglumine blocked ovulation, even at low doses, and led to ovarian cyst development. The selective and highly selective COX-2 inhibitors meloxicam and NS-398 were not effective in blocking ovulation. However, indomethacin, meloxicam, and NS-398 significantly and comparably reduced PGE₂ concentration in vivo and in vitro (P < 0.05) but had no effect on estradiol or progesterone production. This may contradict the generally accepted hypothesis that PGE₂ is a key mediator of ovulation and progesterone production. Our results suggest a connection between ovarian disorders and inflammatory actions in early postpartum cows.     

Intra‐articular opioid analgesia is effective in reducing pain and inflammation in an equine LPS induced synovitis model

Reasons for performing study: Intra‐articular administration of morphine as a local analgesic and anti‐inflammatory drug is widely used in human medicine. In equids, little is known about its clinical analgesic and anti‐inflammatory efficacy. Objectives: To use an inflammatory orthopaedic pain model to investigate the analgesic and anti‐inflammatory effects of intra‐articularly administered morphine as a new treatment modality in horses with acute arthritis. Methods: In a crossover study design, synovitis was induced in the left or right talocrural joint by means of intra‐articular injection of 0.5 ng lipopolyssacharide (LPS). The effect of 120 mg morphine, intra‐articularly administered at 1 h after induction of synovitis, was evaluated using both physiological and behavioural pain variables. Synovial fluid was sampled at 0, 4, 8, 28 and 52 h after induction of synovitis and analysed for total protein concentration, leucocyte count and for prostaglandin E₂, bradykinin and substance P concentrations by ELISA. Ranges of motion of metatarsophalangeal and talocrural joints were measured as kinematic variables with the horses walking and trotting on a treadmill under sound and lame conditions. Clinical lameness scores and several behavioural variables related to the perception of pain were obtained. Results: LPS injection caused marked transient synovitis, resulting in increased concentrations of inflammatory synovial fluid markers, clinical lameness, joint effusion and several behavioural changes, such as increased time spent recumbent, decreased limb loading at rest and decreased time spent eating silage. Intra‐articular morphine resulted in a significant decrease in synovial white blood cell count, prostaglandin E₂ and bradykinin levels and improvement in clinical lameness, kinematic and behavioural parameters, compared to placebo treatment. Conclusions: Intra‐articular morphine offers potent analgesic and anti‐inflammatory effects in horses suffering from acute synovitis. Potential relevance: Local administration of opioids may be useful for horses with acute inflammatory joint pain and offers possibilities for multimodal analgesic therapies without opioid‐related systemic side effects.     

Effect of maternal restraint stress during gestation on temporal lipopolysaccharide-induced neuroendocrine and immune responses of progeny

The impact of gestational dam restraint stress on progeny immune and neuroendocrine temporal hormone responses to lipopolysaccharide (LPS) challenge was assessed. Maternal stress (5-min snout snare restraint stress during days 84 to 112 of gestation) increased (P < 0.05) the magnitude of tumor necrosis factor (TNF)-α, interleukin-6, epinephrine (E), norepinephrine, and serum amyloid A (SAA) production following LPS infusion in the offspring. Moreover, these effects appear to be dependent on gender for TNF-α, E, and cortisol production. However, maternal stress did not affect (P > 0.05) the normalization of proinflammatory cytokines or neuroendocrine hormones produced following LPS. Collectively, these results indicate that maternal stress impacts aspects of the proinflammatory cytokine and stress hormone response in their progeny following LPS dosing of the offspring. This response is potentially responsible in part for the resultant changes to SAA production. Because several of the changes observed here are dependent on pig gender, these results are also the first evidence that inherent epigenetic factors coupled with maternal stress impact the cumulative response to stress and LPS in young pigs.     

Pharmacokinetic and pharmacodynamic interactions of tolfenamic acid and marbofloxacin in goats

Pharmacokinetic and pharmacodynamic properties in goats of the non-steroidal anti-inflammatory drug tolfenamic acid (TA), administered both alone and in combination with the fluoroquinolone marbofloxacin (MB), were established in a tissue cage model of acute inflammation. Both drugs were injected intramuscularly at a dose rate of 2 mg kg⁻¹. After administration of TA alone and TA + MB pharmacokinetic parameters of TA (mean values) were C_max = 1.635 and 1.125 μg ml⁻¹, AUC = 6.451 and 3.967 μg h ml⁻¹, t_1/2K₁₀ = 2.618 and 2.291 h, Vdarea/F = 1.390 and 1.725 L kg⁻¹, and ClB/F = 0.386 and 0.552 L kg⁻¹ h⁻¹, respectively. These differences were not statistically significant. Tolfenamic acid inhibited prostaglandin (PG)E₂ synthesis in vivo in inflammatory exudate by 53-86% for up to 48 h after both TA treatments. Inhibition of synthesis of serum thromboxane (Tx)B₂ ex vivo ranged from 16% to 66% up to 12 h after both TA and TA + MB, with no significant differences between the two treatments.From the pharmacokinetic and eicosanoid inhibition data for TA, pharmacodynamic parameters after dosing with TA alone for serum TxB₂ and exudate PGE₂ expressing efficacy (E_max = 69.4 and 89.7%), potency (IC₅₀ = 0.717 and 0.073 μg ml⁻¹), sensitivity (N = 3.413 and 1.180) and equilibration time (t_1/2K_e0 = 0.702 and 16.52 h), respectively, were determined by PK-PD modeling using an effect compartment model. In this model TA was a preferential inhibitor of COX-2 (COX-1:COX-2 IC₅₀ ratio = 12:1). Tolfenamic acid, both alone and co-administered with MB, did not affect leucocyte numbers in exudate, transudate or blood. Compared to placebo significant attenuation of skin temperature rise over inflamed tissue cages was obtained after administration of TA and TA + MB with no significant differences between the two treatments. Marbofloxacin alone did not significantly affect serum TxB₂ and exudate PGE₂ concentrations or rise in skin temperature over exudate tissue cages. These data provide a basis for the rational use of TA in combination with MB in goat medicine.     

The kinetics of inflammation and phagocytosis during bovine mastitis induced by Streptococcus agalactiae bearing the protein X

The protein X of Streptococcus agalactiae is a surface antigen borne by a high proportion of strains isolated from bovine mastitis. We have tested the capacity of two strains of X-bearing Streptococcus agalactiae to induce mastitis in dairy cows. The reference X-strain (411.07) produced an intramammary infection with local clinical signs in the three inoculated quarters. Another X-bearing strain (443.31) of bovine origin produced infection in all 11 quarters inoculated with only 25 or 85 colony-forming units. In naive cows, strain 433.31 induced less exudation of plasma into the milk, shedding of bacteria, macroscopic alteration, and a lower somatic cell count (SCC) than did the reference strain. Only one quarter spontaneously eliminated the infection before antibiotic treatment 9 days after inoculation.The serum of all the cows contained naturally acquired or induced antibodies to the challenge strain (443.31) and possessed opsonic activity. Before inflammation occurred, the milk was almost devoid of antibody or opsonic activities. The early phase of infection was characterized by rapid multiplication of streptococci in the milk, followed by a sharp drop in bacterial counts concomitant with the onset of inflammation.Three cows immunized with protein X displayed higher SCC and bactericidal activity in milk from the inoculated quarter at the onset of inflammation than non-immunized cows. Two of the three immunized cows underwent an early and transient febrile episode and eliminated the infection.     

Porcine mononuclear phagocyte subpopulations in the lung,blood and bone marrow: dynamics during inflammation induced by Actinobacillus pleuropneumoniae

Mononuclear phagocytes (MP) are cells of nonspecific immunity, playing an essential role in defense against bacterial pathogens. Although various MP subpopulations have been described in the pig, relations among these populations in vivo are unknown to date. The present study was aimed at describing porcine MP subpopulations infiltrating inflamed tissue of pigs under in vivo conditions. Actinobacillus pleuropneumoniae (APP) infection was used to induce an inflammatory response. CD172α, CD14, CD163, MHCII and CD203α cell surface molecules were used to identify MP by flow cytometry. Changes in MP subpopulations in the peripheral blood (PB) and bone marrow (BM) compartments along with the analysis of MP appearing in the inflamed lungs were assessed to elucidate the possible origin and maturation stages of the infiltrating MP. The MP population migrating to the inflamed lungs was phenotype CD14⁺ CD163⁺ CD203α^+/− MHCII^+/−. Concomitantly, after APP infection there was an increase in the PB MP CD14⁺ CD163⁺ CD203α⁻ MHC II⁻ population, suggesting that these cells give rise to inflammatory monocytes/macrophages. The CD203α and MHCII molecules appear on these cells after leaving the PB. In healthy animals, the BM MP precursors were represented by CD14⁻ CD163⁻ cells maturing directly into CD14⁺ CD163⁻ that were then released into the PB. After infection, an altered maturation pathway of MP precursors appeared, represented by CD14⁻ CD163⁻ CD203α⁻ MHCII⁻ MP directly switching into CD14⁺ CD163⁺ CD203α⁻ MHCII⁻ MP. In conclusion, two different MP maturation pathways were suggested in pigs. The use of these pathways differs under inflammatory and noninflammatory conditions.