Characterization of mast cell numbers and subtypes in biopsies from the gastrointestinal tract of dogs with lymphocytic-plasmacytic or eosinophilic gastroenterocolitis

It has been suggested but not proven that hypersensitivity type I reactions are involved in the pathogenesis of canine inflammatory bowel disease (IBD). The main effector cells in type I hypersensitivity reactions are mast cells (MCs). Canine MCs, as human MCs, can be subdivided into three subtypes according to their content of mast cell-specific proteases: tryptase (MC_T), chymase (MC_C), or tryptase and chymase bearing MCs (MC_TC). In this study, numbers and subsets of mast cells were investigated in biopsies from the gastrointestinal tract of dogs with histopathologically confirmed lymphocytic-plasmacytic enteritis (LPE) (n = 4), lymphocytic-plasmacytic colitis (LPC) (n = 1) and eosinophilic gastroenterocolitis (EGE) (n = 11). Paraffin sections of formalin-fixed samples from the stomach, small intestine (duodenum, jejunum, ileum) and colon were stained by using a metachromatic staining method (kresylecht-violet; KEV) and a combined enzyme histochemical and immunohistochemical technique for chymase and tryptase. Additionally, immunohistochemistry with antibodies against T cells (CD3), macrophages (myeloid/histiocyte antigen) and IgA, IgG and IgM bearing cells was conducted. Quantitative evaluation of mast cells and semiquantitative scoring of immunohistochemically stained cells were performed. Between the two histopathologically defined groups clear differences concerning mast cell numbers were detected. In most affected intestinal tissue locations of dogs with LPE/LPC a decrease in metachromatically (kresylecht-violet) stained granule-containing MCs and immunohistochemically stained MC_T,C,TC was found. This reduction could be due to mast cell degranulation, a T helper cell 1 dominated reaction pattern or a “thinning out” due to increasing T cells, IgA and IgG bearing cells. Dogs with EGE displayed higher variability in mast cell numbers but most of the affected large and small intestinal locations had increased numbers of MCs. In these cases, T cells, IgA bearing cells and macrophages also increased. Increased numbers of MCs and eosinophils seen in the intestinal mucosa of dogs with EGE could indicate the presence of a type I hypersensitivity reaction (T helper cell 2 pattern) in response to dietary antigens. Changes in cell numbers occurred also in unaffected locations of dogs with LPE/LPC and EGE which showed reduced MC_T,C,TC, increased KEV positive cells and partially increased leucocytes and macrophages.     

Functional characterization of the muscarinic receptors involved in endothelium-dependent relaxation in isolated canine uterine artery

Acetylcholine interacts with endothelial muscarinic receptors releasing nitric oxide and causing vasodilatation. To identify the receptor subtype responsible for acetylcholine-induced relaxation in canine uterine artery, the usual organ bath method for in vitro investigation on isolated blood vessels was applied. Using a range of muscarinic receptor antagonists such as atropine (nonselective), pirenzepine (M₁-selective), methoctramine (M₂-selective) and p -fluoro-hexahydro-sila-difenidol ( p -FHHSiD) (M₁/M₃) and determining pA2 value of those antagonists through Shild analysis, we aimed at establishing a precise receptor mechanism underlying acetylcholine-induced relaxation in isolated canine uterine artery. The relaxation of uterine arterial rings in response to acetylcholine in the presence or absence of selective muscarinic receptors antagonists was calculated using concentration response curves. Acetylcholine induced concentration-dependent and endothelium-dependent relaxation of arterial rings precontracted with phenylephrine (pEC₅₀ = 6.90 ± 0.02). Muscarinic receptors antagonists atropine, pirenzepine, methoctramine and p -FHHSiD competitively antagonized the response to acetylcholine and obtained pA₂ values were 9.91 ± 0.06, 6.60 ± 0.04, 6.21 ± 0.08 and 8.05 ± 0.1, respectively. This study showed that acetylcholine induced endothelium-dependent relaxation of canine uterine artery by stimulation of muscarinic receptors localized on the endothelial cells. On the basis of differential antagonist affinity, we suggest that the muscarinic receptors involved in the acetylcholine-induced relaxation of canine uterine artery are predominantly of M₃ subtype.     

Effect of Progesterone and Ionomycin on Domestic Cat Sperm Motility Patterns and Acrosome Reaction

This study was aimed at assessing the changes in sperm motion patterns and the percentage of acrosome reaction (AR) in domestic cat semen after treatment with either ionomycin or progesterone (P₄). Ten ejaculates were collected from five tomcats using an artificial vagina, and were diluted, centrifuged and resuspended in a capacitation medium. Samples were evaluated and divided into seven equal aliquots and, after 2 h at 25°C, were incubated for 30 min at 38°C in 5% CO₂ and then analyzed. Computer-assisted sperm analysis and a combination of three fluorescent probes were used to assess sperm plasma, acrosomal membrane integrity and mitochondrial transmembrane potential. Thirty minutes after the start of incubation, P₄ was added (10 μg/ml) to the P1 group. Groups P2 and P3 were supplemented with P₄ (10 and 20 μg/ml, respectively) only after 2 h of incubation, and groups I1 and I2 were supplemented with ionomycin (4 and 8 μ m , respectively) 2 h after incubation. Group E was supplemented with ethanol (0.6%) at 2 h after incubation and group C received no supplementation. Ionomycin and P₄ treatments led to a hyperactivation-like sperm motion and an increase (p < 0.05) in the percentage of AR. Although a higher (p < 0.05) percentage of AR was obtained in group I2 when compared with all P₄ groups, a decrease (p < 0.05) in total and progressive motility was observed in I2 group. As I1 group was similar to I2 to induce AR without diminishing sperm motility, we can conclude that ionomycin at 4 μ m seems to be more suitable to trigger AR in domestic cat sperm.     

The Influence of Oxygen Tension on Cumulus Cell Viability of Canine COCs Matured in High-Glucose Medium

High in vitro oxygen (O₂) tensions are associated with enhanced levels of reactive oxygen species and cumulus oocyte complex (COC) apoptosis. The objective of this study was to determine the influence of O₂ tension on cumulus cell (CC) viability from canine oocytes. Cumulus oocyte complexes were distributed into three groups (CG, T20 and T5) and two O₂ tension levels (20% and 5%). The control group (CG) was matured in vitro in a humidified atmosphere with 5% CO₂ in air in TCM199 with 26.19 m m sodium bicarbonate, 10% (v/v) foetal calf serum (FCS), 0.10 m m gentamicin, 0.20 m m pyruvic acid, 20 μg/ml oestradiol, 0.5 μg/ml follicle-stimulating hormone, 0.03 IU/ml human chorionic gonadotropin, and 1.0 μg/ml human somatotropin. Groups T20 and T5 were matured under 20% or 5% O₂ tensions respectively in a high-glucose medium, without FCS. T20 and T5 were as CG, and supplemented with 0.1% Polyvinyl Alcohol, and 5.5 m m glucose. After 48 h of IVM, CCs from COCs were stained with propidium iodide (1.50 m m ). The results showed that viability of CCs (cytoplasmic features and nuclear morphological integrity) was different for the three groups. Rates of apoptosis were at 57.9% (521/900) for CG, 54.4% (490/900) for T20 and 38.9% (350/900) for T5 (p < 0.001). Predominant features in apoptotic cells (n = 1361) were DNA nuclear fragments (94.0%). It was concluded that CCs of canine COCs cultured in high-glucose medium showed significantly less apoptosis than those cultured in medium with FCS. Low O₂ tension was efficient in reducing apoptosis in canine CCs.     

Immunohistochemical and histochemical stains for differentiating canine cutaneous round cell tumors

Immunohistochemical and histochemical stains are useful adjunct techniques in the diagnosis of canine cutaneous round cell tumors, which can appear histologically similar. We applied a panel of monoclonal antibodies (recognizing tryptase, chymase, serotonin for mast cells; CD1a, CD18, MHC class II for histiocytes; CD3 for T lymphocytes; CD79a for B lymphocytes and plasma cells) and one histochemical stain (naphthol AS-D chloroacetate for chymase activity) to formalin-fixed, paraffin-embedded sections of canine cutaneous mast cell tumors, histiocytomas, lymphosarcomas, plasmacytomas, and unidentified round cell tumors. Of 21 tumors with a histologic diagnosis of mast cell tumor, 7/7 (100%) grade I, 6/7 (85.7%) grade II, and 3/7 (42.9%) grade III tumors were diagnosed as mast cell tumors based on positive staining for tryptase antigen and chymase activity. Mast cells were positive for both tryptase antigen and chymase activity, indicating equal efficacy of tryptase immunohistochemistry and chymase histochemistry. Chymase was detected immunohistochemically in both tumor and nontumor cells, while serotonin was not detected in most mast cell tumors, and thus, neither was useful in the diagnosis of mast cell tumors. Immunohistochemistry to detect CD18 and MHC class II was equally effective in staining histiocytomas, although lymphosarcoma must be ruled out through the use of CD3 and CD79a immunohistochemistry. Immunohistochemistry using three different monoclonal antibodies to human CD1a showed no cross-reactivity in canine histiocytomas and was not useful. A final diagnosis was obtained for 4/5 (80%) of the unidentified tumors, indicating the usefulness of multiple stains in poorly differentiated round cell tumors.     

Possible involvement of phosphatidylinositol 3-kinase in the maintenance of metaphase II attest in porcine oocytes matured in vitro

It has been reported that phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB) pathway plays a crucial role in the meiotic resumption and progression to the metaphase II (MII) stage of oocytes. However, the role of this pathway in meiotic arrest at the MII stage (cytostatic activity) is not well understood. In this study the effect of a PI3K inhibitor, LY294002, on the MAPK and p34^cdc2 kinase activities of matured porcine oocytes was examined. After maturation culture, both the MAPK and p34^cdc2 kinase activities in the oocytes were gradually decreased in a time-dependent manner. Although 25 µmol/L LY294002 did not affect either the MAPK or p34^cdc2 kinase activities, 50 µmol/L LY294002 suppressed the PKB phosphorylation and slightly decreased MAPK activity, but not the p34^cdc2 kinase activity. Therefore the effect of 10 µmol/L Ca²⁺ ionophore which was reported as inducing a transient decrease of p34^cdc2 kinase but not MAPK activities, was also examined in LY294002-treated oocytes. By additional treatment with LY294002 after Ca²⁺ ionophore, both the MAPK and p34^cdc2 kinase activities were decreased in a time-dependent manner, concomitantly with improvement of pronuclear formation. Therefore, we concluded that PI3K is involved in the maintenance of MAPK activity in matured porcine oocytes.     

Effects of Mycobacterial Cell Wall‐DNA Complex (MCC), Alendronate and Pamidronate on Canine Osteosarcoma Cell Lines

Introduction:  MCC, a cell wall composition prepared from Mycobacterium phlei ., inhibits the proliferation and induces apoptosis in a wide range of tumor cells. Bisphosphonates have been reported to inhibit the proliferation of canine osteosarcoma cell lines. In this study, we have determined the activity of MCC alone and in combination with the bisphosphonates alendronate and pamidronate on canine osteosarcoma cell lines.
Methods:  Canine osteosarcoma cell lines D17 and D22 were incubated with different concentrations of MCC (0.01–100 μg/ml) and suboptimal concentrations of alendronate and pamidronate for 72 hours. Cellular proliferation was measured by MTT reduction. Nuclear DNA condensation was determined using with Hoescht 33258 staining, and apoptosis by flow cytometry using active‐caspase‐3/PE and anti‐cleaved‐PARP/FITC antibodies.
Results:  MCC inhibited the proliferation of both canine osteosarcoma D17 and D22 cell lines in a concentration‐dependent manner. The IC₅₀ for D17 cells was 3.9 μg/ml and for D22 cells 44.4 μg/ml. Cells incubated with 100 μg/ml MCC were positive for Hoescht staining, active caspase‐3 and cleaved PARP, indicative of cell death by apoptosis. The addition of alendronate or pamidronate was found to potentiate the apoptosis‐inducing activity of MCC. Maximal activity was observed when 5 μM alendronante or 10 μM pamidronate were used in combination with 100 μg/ml MCC.
Conclusion:  MCC inhibits the proliferation and induces apoptosis in canine osteosarcoma cell lines in vitro . This anticancer activity can be potentiated by the use of alendronate and pamidronate. These data support the development of MCC as a therapeutic agent for the treatment of canine osteosarcoma.     

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.     

Regional specialization of the Ganglion cell density in the retina of the Ostrich (Struthio camelus)

In this study, retinal whole-mount specimens were prepared and stained with 0.1% cresyl violet for the ganglion cell study in the Ostrich (Struthio camelus) . The total number, distribution, and size of these cells were determined in different retinal regions. The mean total number of ganglion cells (three retinas) was 1 435 052 with an average density of 652 cells/mm². The temporo – nasal area of the retina with high cell density were identified with the peak of 7525 cells/mm² in the central area. The size of most ganglion cells ranged from 113–403 µm², with smaller cells predominating along the temporo-nasal streak above the optic disc and larger cells comprising more of the peripheral regions. The average thickness of the retina was 196 µm. The central area was the thickest area (268.6 µm), whereas the peripheral area was the thinnest area. Thus, the specialization of ganglion cell densities, their sizes and the thickness of the retina support the notion that the conduction of visual information towards the brain from all regions of the retina is not uniform, and suggests that the temporo – nasal streak is the fine quality area for vision in ostriches.     

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.     

Effects of Straw Volume and Equex-STM® on Boar Sperm Quality after Cryopreservation

The present experiments were designed to study the effect of adding the detergent Equex-STM^® to freezing extender, and of straw volume (0.25 ml vs 0.5 ml), on boar sperm quality after cryopreservation. Three ejaculates from each of four purebred boars (three Landrace and one Yorkshire) were collected and frozen with a lactose-egg yolk extender containing glycerol with or without 1.5% Equex-STM^®. The extended semen was loaded into either 0.25- or 0.5-ml straws. The straws were placed in liquid nitrogen (LN₂) vapour approximately 3 cm above the level of LN₂ for 20 min and then were plunged into LN₂. Thawing was achieved in warm water at 50°C for 12 s and then was incubated in a 38°C water-bath for 30 min before evaluating sperm quality. Results showed that the individual motility, viability and acrosomal normal apical ridge (NAR) were improved (p < 0.001) when Equex-STM^® was added to the freezing extender. There was no difference (p   =   0.48) in sperm motility between 0.25- and 0.5-ml straws when Equex-STM^® was added. The percentages of viable and of NAR sperm in 0.5-ml straws were higher than those in 0.25-ml straws (p   =   0.02, p   =   0.0003 respectively). The percentages of membrane intact sperm evaluated using the short hypo-osmotic swelling test were not affected by straw volume or the adding of Equex-STM^® (p   >   0.05). The results of these investigations suggested that Equex-STM^® exerts a beneficial effect on the quality of cryopreserved boar semen and this cryopreservation protocol was favourable for a 0.5-ml straw.     

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.     

Pharmacokinetics of a new long acting endectocide formulation containing 2.25% ivermectin and 1.25% abamectin in cattle

The objective of this study was to determine the kinetic parameters of a new formulation that contained 2.25% ivermectin combined with 1.25% abamectin in bovine plasma. The results for 2.25% ivermectin: C _max (37.11 ng/mL ± 7.42), T _max (16 days ± 5.29), T _1/2 (44.62 days ± 53.89), AUC (928.2 ng·day/mL ± 153.83) and MRT (36.73 days ± 33.64), and for 1.25% abamectin: C _max (28.70 ng/mL ± 9.54), T _max (14 days ± 4.04), T _1/2 (15.40 days ± 11.43), AUC (618.05 ng·day/mL ± 80.27) and MRT (20.79 days ± 8.43) suggest that this combination of 2.25% ivermectin + 1.25% abamectin possesses properties that give this pharmaceutical formula a longer activity time than two of the commercial products tested (1% ivermectin and 1% abamectin), and showed similarity to 3.15% ivermectin.     

Generation and characterization of bone marrow-derived cultured canine mast cells

Disorders of mast cells, particularly mast cell tumors (MCTs), are common in dogs. There now is evidence that many of these disorders exhibit breed predilections, suggesting an underlying heritable component. In comparison to humans and mice, little is known regarding the biology of canine mast cells. To facilitate the study of mast cell biology in other species, bone marrow-derived cultured mast cells (BMCMCs) often are used because these represent a ready source of large numbers of cells. We have developed a protocol to successfully generate canine BMCMCs from purified CD34(+) cells. After 5-7 weeks of culture with recombinant canine stem cell factor (rcSCF), greater than 90% of the cell population consisted of mast cells as evidenced by staining with Wright's-Giemsa, as well as production of chymase, tryptase, IL-8 and MCP-1. These cells expressed cell surface markers typical of mast cells including Kit, Fc epsilonRI, CD44, CD45 and CD18/CD11b. The canine BMCMCs were dependent on rcSCF for survival and proliferation, and migrated in response to rcSCF gradients. Cross-linking of cell surface-bound IgE induced the release of histamine and TNFalpha. Histamine release could also be stimulated by ConA, compound 48/80, and calcium ionophore. In summary, canine BMCMCs possess phenotypic and functional properties similar to mast cells found in vivo. These cells represent a novel, valuable resource for investigating normal canine mast cell biology as well as for identifying factors that lead to mast cell dysregulation in the dog.     

Fertilizing Capacity of Frozen Epididymal Sperm Collected from Dogs

The collection of epididymal sperm may be a valuable tool for canine reproduction especially since it can enable collection of cells after death of a valuable dog. The aim of the present study was to evaluate the viability of epididymal sperm after freeze-thawing. Epididymides were obtained from four adult dogs by elective orchiectomy. The caudal portion of the epididymides and part of the deferential ducts were squeezed by means of an anatomic clamp into a Petri dish containing either 0.9% saline solution (Group 1) or Ringer solution without lactate (Group 2). Samples were centrifuged at 800 ×  g for 10 min, the supernatant was removed and the pellet was diluted in one step with a Tris/citric acid/OEP (Orvus Es Paste) extender containing 7% glycerol and subjected to semen freezing. Oocytes were obtained from canine ovaries, after ovariohysterectomy. Only oocytes that were approximately 100 μm in diameter, with a dark ooplasm surrounded by three- or four-well formed cumulus cell layers were used for sperm testing. Frozen semen samples were thawed in a water bath at 70°C for 8 s and analysed at room temperature for sperm motility and velocity. Oocytes were incubated with spermatozoa in humidified atmosphere containing 5% CO₂ at 38°C for 18 h. Morphological and functional characteristics of spermatozoa were similar in both groups. However, the percentage of sperm cells bound to oocytes was significantly higher in Group 2 than in Group 1. This result suggests that the Ringer solution without lactate was a more suitable medium for collecting epididymal canine sperm than 0.9% saline.     

Affinity of detomidine, medetomidine and xylazine for alpha-2 adrenergic receptor subtypes

α₂-Adrenergic receptor agonists are widely used in veterinary medicine as sedative/hypnotic agents. Four pharmacological subtypes of the α₂-adrenergic receptor (A, B, C and D) have been identified based primarily on differences in affinity for several drugs. The purpose of this study was to examine the affinities of the sedative agents, xylazine, detomidine and medetomidine at the four α₂-adrenergic receptor subtypes. Saturation and inhibition binding curves were performed in membranes of tissues containing only one subtype of a₂-adrenergic receptor. The K_D for the α₂-adrenergic receptor radioligand, [³H]-MK-912, in HT29 cells (α_2A-), neonatal rat lung (α_2B-), OK cells (α_2C-) and PC12 cells transfected with RG20 (α_2D-) were 0.38 ± 0.08 n m , 0.70 ± 0.5 n m , 0.07 ± 0.02 n m and 0.87 ± 0.03 n m , respectively. Detomidine and medetomidine had approximately a 100 fold higher affinity for all the α₂-adrenergic receptors compared to xylazine but neither agonist displayed selectivity for the α₂-adrenergic receptor subtypes. These data suggest that available sedative/hypnotic α₂-adrenergic receptor agonists can not discriminate between the four known α₂-adrenergic receptor subtypes.     

Reduced mucosal injury of SPF chickens by mast cell stabilization after infection with very virulent infectious bursal disease virus

Recent studies here have demonstrated that increased mast cell populations and tryptase activity contribute to lesion formation in regions of immune organs in special-pathogen-free chickens after infection with very virulent infectious bursal disease virus (vvIBDV). Mast cells and their mediators have been implicated in acute inflammatory injury after vvIBDV infection, but their precise role in this process remains elusive. In this study, the role of mast cells in the vvIBDV infection process was examined using ketotifen, a mast cell membrane stabilizer. On days 1, 2, and 3 postinfection, the bursa of Fabricius (BFs) were collected to quantify mast cells, tryptase and histamine contents by cytochemistry, immunohistochemistry and fluorospectrophotometry analyses, respectively. The results showed that the mast cell populations, tryptase expression, and histamine released increased significantly in the BFs (p < 0.01) of infected birds compared to controls, and acute inflammatory responses were observed in the former. In contrast, in infected chickens pretreated with ketotifen, mast cells, tryptase, and histamine were markedly decreased (p < 0.01) and probably as a result, the BFs remitted significantly. The overall results suggest that mast cells are positively involved in BF injury induced by vvIBDV infection. Inhibition of mast cell degranulation and concurrent mediator release may represent a novel strategy to modulate this process. This study, thus, advances the understanding of the acute inflammatory injury mechanisms triggered by vvIBDV infection and the contribution of mast cell activity in this process.     

Gelatinized to non-gelatinized starch ratio in the diet of Labeo rohita: effect on digestive and metabolic response and on growth

A 60-days experiment was conducted to study the optimum gelatinized (G) to non-gelatinized (NG) starch ratio in the diet of Labeo rohita juveniles with respect to digestive and metabolic response and on growth. Two-hundred and thirty-four juveniles (avg. wt 2.53 ± 0.04 g) were randomly distributed in six treatment groups with each of three replicates. Six semipurified diets either containing NG and/or G corn starch viz., T₁ (100% NG, 0% G starch), T₂ (80% NG, 20% G starch), T₃ (60% NG, 40% G starch), T₄ (40% NG, 60% G starch), T₅ (20% NG, 80% G starch) and T₆ (0% NG, 100% G starch) were prepared. The dry matter digestibility and carbohydrate digestibility were highest (p < 0.05) in T₆ group and lowest in T₃ and T₄ groups. The amylase activity in intestine increased as G:NG level increased in the diet. Protease activity in intestine was highest in T₆ group and lowest in T₁ group. Similar trend was recorded for specific growth rate, protein efficiency ratio and apparent net protein utilization. Liver glycogen, hepatosomatic index and blood glucose level increased linearly with the increasing level of G starch in the experimental diet. The results indicate that higher nutrient digestibility and growth was recorded either at low (20% G starch, T₂) or high (100% G starch, T₆) G starch fed group. But high G starch fed group (T₆) exhibits higher liver glycogen and blood glucose level, which may lead to stress due to long-term feeding. Hence, it is suggested that 20% G and 80% NG starch is optimum for better nutrient digestibility and growth in L. rohita juveniles.