Plasma 13,14-dihydro-15-keto-prostaglandin F2α concentrations in prepubertal dairy heifers challenged with oxytocin

Twenty-nine prepubertal Holstein heifers were assigned by age to one of three age groups to determine if the prepubertal bovine uterus could respond to an oxytocin stimulus. Group 1 heifers were 6 to 7 months of age (AGE1; n = 11), group 2 heifers were 8 to 9 months of age (AGE2; n = 11) and group 3 heifers were 10 to 11 months of age (AGE3; n = 7). Blood samples were collected via an indwelling jugular catheter. Four samples were collected at 15-min intervals prior to oxytocin administration to determine basal 13,14-dihydro-15-keto-prostaglandin F2 alpha (PGFM) concentrations. Each heifer received 100 IU of oxytocin i.v., blood sampling continued at 5 min intervals for the next 30 min and for an additional 90 min at 15-min intervals. Heifers were considered responders to oxytocin if mean PGFM concentrations increased at least 1.5 times the SD of their basal PGFM concentration. Age of the heifer (P less than .0001) and responder status (P less than .05) affected plasma PGFM. Plasma PGFM was higher in AGE1 and AGE3 heifers than AGE2 (P less than .0001). The number of responders was greatest at AGE3 (P less than .03) with AGE1 and AGE2 being similar. Mean basal PGFM was lower (P less than .04) at AGE2 than AGE1 with AGE3 being intermediate. In addition, basal PGFM at AGE1 tended to be lower (P less than .08) in the responders than in the non-responders, while AGE2 basal PGFM did not differ between responders and non-responders (P greater than .10).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of constant infusion of oxytocin on luteal lifespan and oxytocin-induced release of prostaglandin F2α in heifers

Two experiments were conducted to determine whether constant infusion of oxytocin would prolong the luteal phase and inhibit uterine prostaglandin F2 alpha (PGF2 alpha) secretion in heifers. In Experiment 1, twelve heifers, treated with saline (SAL) or oxytocin (OXY) via jugular cannulae infusions (INF) or osmotic minipumps (OMP), were allotted at estrus into four treatment groups (n = 3). Treatments were: SAL-INF, SAL-OMP, OXY-INF and OXY-OMP. Physiological saline or oxytocin was given from Days 10 to 23 (Day 0 = estrus) of the estrous cycle. Method of treatment (jugular cannula infusion or osmotic minipump) had no effect (P greater than 0.05) on estrous cycle length or pattern of secretion of progesterone; therefore, data were pooled. Estrous cycle lengths were extended (P less than 0.01) for heifers which received oxytocin (25.3 +/- 0.4 d) compared to saline (20.5 +/- 0.4 d). Luteolysis did not occur in oxytocin-treated heifers until after treatment ceased. Experiment 2 was designed and conducted identically to Experiment 1 with the addition of a "challenge" injection of oxytocin (100 IU oxytocin, i.v.) given on Day 16 of the estrous cycle. Treatment of heifers with oxytocin extended (P less than 0.05) estrous cycle length by an average of 3 d compared to heifers treated with saline. The "challenge" injection induced (P less than 0.05) secretion of PGF2 alpha (as measured by the stable PGF2 alpha metabolite, 15-keto-13,14-dihydro-PGF2 alpha) in saline-treated but not oxytocin-treated heifers. In both Experiment 1 and 2, serum concentrations of FSH were elevated (P less than 0.05) in oxytocin-treated heifers. No increase was observed for LH or prolactin. The rise in estradiol-17 beta at luteolysis was not affected (P greater than 0.10) by treatment. In summary, constant infusion of oxytocin extended luteal lifespan, prolonged secretion of progesterone, and inhibited oxytocin-induced secretion of PGF2 alpha. Constant infusion of oxytocin did not affect serum concentrations of estradiol-17 beta, LH or prolactin; however, serum concentrations of FSH were elevated during the oxytocin treatment period.     

Effects of GnRH in combination with PGF2α on the dynamics of follicular and luteal cells in post-pubertal Holstein heifers

Holstein heifers were randomly allotted by weight, age and body condition score to one of three treatments to test the hypothesis that GnRH administration concurrent with PGF_2α injection would advance follicle or corpus luteum (CL) development parallel to an induced luteolysis of the pre-existing CL. Heifers in the control group (n = 14) received two treatments of PGF_2α(25 mg, im) given 10 days apart. Groups 2 (n = 14) and 3 (n = 14) received an additional treatment of GnRH (100 μg, im) after the first and second PGF_2α respectively. Estrus detection began immediately after PGF_2α and continued for 80 h. Blood sampling was initiated 7 days prior to the first PGF_2α (day − 7) and continued on days 0, 7, 10 (prior to the second PGF_2α), 17 and 24. Heifers were artificially inseminated after the second PGF_2α and pregnancy diagnosed at 60 days. There was a trend (P < .10) toward a lower estrus response in group 3 when compared to the other groups. Pregnant heifers in group 2 had lower progesterone (0.44 ± 0.09 vs. 1.72 ± 0.56 ng/ml) a week after the second PGF_2α than the non-pregnant animals in that group (P < .05). Similar results were observed in the control group but only within the responding heifers (0.61 ± 0.08 vs. 0.93 ± 0.03 ng/ml; P < .05). Progesterone in heifers in group 2 remained high on day 0, 7, and 10 (1.48 ± 0.37, 1.23 ± 0.39, 1.96 ± 0.36 ng/ml) in spite of the treatment with PGF_2α. This data suggest that administration of GnRH following PGF_2α alters bovine luteal and/or follicular cell function.     

The effect of prostäglandin F2α and oxytocin on porcine myometrium in vitro *

Acht geschlechtsreife Jungsauen wurden ungefähr 24 Stunden nach Eintritt der Brunst geschlachtet und 8 trächtige Jungsauen 30–60 Tage nach der Besamung geschlachtet. Von deren Uteri wurden Uterusstreifen präpariert und im Durchlaufbad eingespannt. Die Motilität der Muskelstreifen war bei den nicht trächtigen Uteri nach 2 Stunden regelmäβig mit 1–2 Kontraktionen pro Minute, während die spontane Motilität des Myometriums bei der Frühträchtigkeit sehr unregelmäβig war und manchmal ganz fehlte. PGF₂α und Oxytocin ergaben immer eine Stimulanz der Motilität in den Muskelstreifen bei den nicht trächtigen als auch bei den trächtigen Jungsauen.     

Analysis of monoclonal antibodies that recognize γδ T/null cells

Thirty two monoclonal antibodies (mAbs) from the first round of analysis in the Second International Swine CD Workshop were placed together with additional mAb derived from the first workshop in the null cell panel for further evaluation. Preparations of peripheral blood leukocytes, concanavalin A stimulated peripheral blood mononuclear cells, and spleen cells were used in flow cytometric analyses. Nineteen mAbs identified molecules that were not expressed on null cells, not lineage specific, or recognized activation molecules. Sixteen mAbs including control mAbs were identified that were specific for null cells. One of the latter mAbs, 041 (PGBL22A), that recognizes a determinant on a constant region of porcine γδ TcR established the majority of null cells are γδ T cells. Use of this mAb in further comparisons demonstrated the γδ T cell population is comprised of two major subpopulations, one negative and one positive for CD2. Two color analyses demonstrated that 11 of the mAbs formed a broad cluster that included control mAbs 188 (MAC320) that defined the CD2 negative SWC6 cluster in the first workshop and mAb 122 (CC101) that might recognize an orthologue of bovine WC1 and nine mAbs that recognize determinants on one or more molecules with overlapping patterns of expression on subsets of CD2⁻ γδ T cells. Two groups of mAbs formed the previously identified subset clusters SWC4 and SWC5. Two new mAbs formed a third subcluster. Three mAbs did not form clusters. Three mAbs predicted to recognize TcR in the first workshop (020 [PT14A], 021 [PT79A], and 022 [MUC127A]) and mAb PGBL22A were shown to immunoprecipitate a 37, 40 kDa heterodimer.     

Effects of α2-adrenergic drugs on small intestinal motility in the horse: An in vitro study

The effects of selective α₂-agonists (xylazine, detomidine and medetomidine) and antagonists (yohimbine and atipamezole) on in vitro small intestine motility in the horse were evaluated. Samples of equine jejunum were placed in isolated organ baths and drug-induced modifications of motility were measured by means of an isotonic transducer. All tested α₂-agonists dose-dependently reduced both spontaneous and electrically-evoked phasic contractions. Conversely, α₂-antagonists were ineffective when tested alone, and showed a heterogeneous and dose-independent ability to inhibit agonist activity. In particular, the antagonism exerted by higher concentrations of both yohimbine and atipamezole against α₂-agonists was weaker than when lower concentrations were used. The data are indicative of the presence of both pre- and post-synaptic α₂-adrenoceptors with inhibitory activity on equine jejunum motility, and support a possible therapeutic utility of these drugs in horse intestinal disorders associated with hypermotility.     

Studies of maternally-acquired antibodies in the foal to equine influenza A1 and A2, and equine rhinopneumonitis

Serum and colostrum were collected from 50 mares at parturition. Pre- and post-nursing serum samples were obtained from their foals. Bi-weekly serum samples were obtained from 25 of the foals for eight weeks. Hemagglutination-inhibiting (HAI) antibody titers to equine influenza viruses A₁ and A₂ (EIVA₁ and EIVA₂) and serum-neutralizing antibody titers to equine herpes virus 1 (EHV₁) were measured in serum and colostrum samples. IgG levels in serum and colostrum were determined.No antibody was detected in any foal's pre-nursing serum sample. Foal post-nursing antibody and IgG levels were equivalent to those measured in their dam's sera (EHVA₁ p=0.86; EHVA₂ p=0.54; EHV₁ p=0.91; IgG p=0.58). The half-life of maternally-acquired serum antibody in the foals was determined to be: EIVA₁=28.88 days (26.4 to 31.7 days); EIVA₂=29.1 days (26.7 to 32.1 days); EHV₁=31.0 days (28.1 to 34.8 days). Colostrum contained antibody and IgG at levels ranging from 2 to 8 times higher (4.3 average) than those detected in the mare's serum.     

Effects of oxytocin,lipopolysaccharide (LPS), and polyunsaturated fatty acids on prostaglandin secretion and gene expression in equine endometrial explant cultures

Increased secretion of prostaglandin F₂α (PGF₂α) within the uterus because of uterine inflammation can cause luteolysis and result in early embryonic loss. Supplementation with polyunsaturated fatty acids (PUFAs) has been shown to influence PG production in many species, although the effects on the mare remain unknown. The present study aimed to determine fatty acid uptake in equine endometrial explants and evaluate their influence on PG secretion and expression of enzymes involved in PG synthesis in vitro. Equine endometrial explants were treated with 100 μM arachidonic acid, eicosapentaenoic acid, or docosahexaenoic acid and then challenged with oxytocin (250 nM) or lipopolysaccharide (LPS; 1 μg/mL). Production of PGF₂α and PG E₂ (PGE₂) was measured, and mRNA expression of enzymes involved in PG synthesis was determined with quantitative real-time PCR. Media concentrations of PGF₂α and PGE₂ were higher (P < 0.0001) from endometrial explants challenged with oxytocin or LPS compared with controls despite which fatty acid was added. Only DHA lowered (P < 0.0001) media concentrations of PGF₂α and PGE₂ from explants. Endometrial explants stimulated with oxytocin had increased expression of PG-endoperoxide synthase 1 (PTGS1; P < 0.02), PG-endoperoxide synthase 2 (PTGS2; P < 0.001), PG F₂α synthase (PGFS; P < 0.01), PG E₂ synthase (PGES; P < 0.01), and phospholipase A₂ (PLA₂; P < 0.005) compared with controls and regardless of fatty acid treatment; whereas stimulation with LPS increased expression of PTGS2 (P < 0.004), PGFS (P < 0.03), PGES (P < 0.01), and PLA₂ (P < 0.01) compared with controls and regardless of fatty acid treatment. Treatment with PUFAs, specifically DHA, can influence PG secretion in vitro through mechanisms other than enzyme expression.     

Characterisation of equine T helper cells: demonstration of Th1- and Th2-like cells in long-term equine T-cell cultures

The aim of this study was to characterise CD4+T-cells in equines, as these cells are pivotal in establishing immune responses or regulating established ones. Peripheral blood mononuclear cells from a pony immunised with ovalbumin were cultured in vitro in the presence of the specific antigen and autologous antigen presenting cells. During the antigen starvation phase, cells were maintained on recombinant equine IL-2. After 35 days of culture, most of the cells were CD4+, CD8-and sIg-. Cells proliferated specifically in the presence of antigen, as tested on day 42 of culture. These cells were analysed by in-situ hybridisation to detect m RNA for IL-2 and IL-4, the presence of which suggested the existence of of Th1- and Th2-like cells.     

Bactrian camel (Camelus bactrianus) integrins αvβ3 and αvβ6 as FMDV receptors: Molecular cloning, sequence analysis and comparison with other species

Integrins are heterodimeric adhesion receptors that participate in a variety of cell–cell and cell–extracellular matrix protein interactions. Many integrins recognize RGD sequences displayed on extracellular matrix proteins and the exposed loops of viral capsid proteins. Four members of the αv integrin family of cellular receptors, αvβ3, αvβ6, αvβ1 and αvβ8, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro, and integrins are believed to be the receptors used to target epithelial cells in the infected animals. To analyse the roles of the αv integrins from a susceptible species as viral receptors, we have cloned Bactrian camel αv, β3 and β6 integrin cDNAs and compared them to those of other species. The coding sequences for Bactrian camel integrin αv, β3 and β6 were found to be 3165, 2289 and 2367 nucleotides in length, encoding 1054, 762 and 788 amino acids, respectively. The Bactrian camel αv, β3 and β6 subunits share many structural features with homologues of other species, including the ligand binding domain and cysteine-rich region. Phylogenetic trees and similarity analyses showed the close relationships of integrin genes from Bactrian camels, pigs and cattle, which are each susceptible to FMDV infection, that were distinct from the orders Rodentia, Primates, Perissodactyla, Carnivora, Galliformes and Xenopus. We postulate that host tropism of FMDV may in part be related to the divergence in integrin subunits among different species.     

Effects of polymyxin‐B on TNF‐α production in equine whole blood stimulated with three different bacterial toxins

Polymyxin‐B is used to treat equine systemic inflammation. Bacterial toxins other than lipopolysaccharide (LPS) contribute to systemic inflammation but the effects of polymyxin‐B on these are poorly defined. Whole blood aliquots from six healthy horses diluted 1:1 with RPMI were incubated for 21 hr with 1 μg/ml of LPS, lipoteichoic acid (LTA) or peptidoglycan (PGN) in the presence of increasing concentrations of polymyxin‐B (10–3000 μg/ml). A murine L929 fibroblast bioassay was used to measure TNF‐α activity. Polymyxin‐B significantly inhibited the effects of all three bacterial toxins. Analysis of variance showed the IC₅₀ value for polymyxin‐B for TNF‐α inhibition caused by LTA (11.19 ± 2.89 μg/ml polymyxin‐B) was significantly lower (p = .009) than the values for LPS (46.48 ± 9.93 μg/ml) and PGN (54.44 ± 8.97 μg/ml). There was no significant difference in IC₅₀ values between LPS and PGN (p > .05). Maximum inhibition of TNF‐α was 77.4%, 73.0% and 82.7% for LPS, PGN and LTA, respectively and was not significantly different between toxins. At the two highest concentrations of polymyxin‐B, TNF‐α began to increase. These data suggest that polymyxin‐B may inhibit the effects of bacterial toxins other than LPS and might be a more potent inhibitor of LTA than LPS or PGN.     

Effects of Ethanol on Synthesis of Prostaglandin F2α in Bovine Females

Ethanol stimulates the production of prostaglandins in many species. The purpose of this study was to verify the effect of ethanol on the production of prostaglandin F2α (PGF2α) and luteolysis in bovine females. In the first experiment, Holstein cows at day 17 of the oestrous cycle were treated with 100% ethanol (0.05 ml/kg of body weight, IV; n = 5), saline (0.05 ml/kg of body weight, IV; n = 4) or synthetic prostaglandin (150 μg of D‐cloprostenol/cow, IM; n = 4). The plasma concentrations of 13, 14‐dihydro‐15‐keto PGF2α (PGFM; the main metabolite of PGF2α measured in the peripheral blood) were assessed by radioimmunoassay (RIA). There was an acute release of PGFM in response to ethanol comparing to other treatments (p ≤ 0.05). However, only cows treated with PGF2α underwent luteolysis. In the second experiment, endometrial explants of cross‐bred beef cows (n = 4) slaughtered at day 17 of the oestrous cycle were cultured for 4 h. During the last 3 h, the explants were cultured with medium supplemented with 0, 0.1, 1, 10 or 100 μl of 100% ethanol/ml. Medium samples were collected at hours 1 and 4 and concentrations of PGF2α were measured by RIA. Ethanol did not induce PGF2α production by the endometrium. In conclusion, ethanol does not cause luteolysis in cows because it stimulates production of PGF2α in extra‐endometrial tissues.     

α- and β-adrenoceptors in the sheep urinary bladder

A study was undertaken to determine the presence and distribution of alpha- and beta-adrenoceptors in the sheep bladder body and base. In the bladder body, noradrenaline and isoproterenol induced relaxation which was significantly inhibited by propranolol, pafenolol and butoxamine. In the presence of propranolol (10(-5) M), noradrenaline induced a small contraction, as well as phenylephrine, but B-HT 920 failed to cause any effect on the bladder body. In the bladder base, noradrenaline caused a contraction that was significantly inhibited by prazosin but not by yohimbine. Phenylephrine also induced a contractile response in this structure which was inhibited by prazosin. Isoproterenol caused a relaxation that was significantly inhibited by propranolol and pafenolol but not by butoxamine. Relaxation was mediated by both beta 1 and beta 2-adrenoceptors in the detrusor muscle and by beta 1-adrenoceptors in the bladder base. Alpha 1-adrenoceptors contributed to maintain the detrusor tone and contract the bladder base.     

FC‐33 Expression of IL‐12 receptor β2 gene in atopic dogs

Atopic dermatitis (AD) is very common in dogs, but its pathogenesis is not yet fully understood. It has been suggested that a Th2‐dominant status may be associated with the occurrence of canine AD. IL‐12 is thought to be important for the differentiation of Th1 cells. The IL‐12 receptor β2 (IL‐12Rβ2) gene is considered to play a critical role in signal transduction and is attracting attention as one of the causative genes of AD in humans. The purpose of this study was to investigate the relationship between IL‐12Rβ2 gene expression and canine AD. The canine IL‐12Rβ2 gene was cloned by RT‐PCR and its nucleotide sequences were determined. Canine IL‐12Rβ2 showed 76.8% homology at the amino acid level with human IL‐12Rβ2, and its structural motifs were well conserved. cDNA with a 91 bp deletion including the transmembrane region was also cloned, which consequently produced a frame shift and an early stop codon. The deletion region corresponded to exon 14 of the human IL‐12Rβ2 gene on chromosome 1. The expression of deleted canine IL‐12Rβ2 mRNA in phytohemagglutinin‐stimulated peripheral blood mononuclear cells was examined in seven healthy dogs and 11 AD dogs. Both deleted and intact mRNAs were expressed at constant ratios in healthy and AD dogs. The results indicate that the deletion of the transmembrane region is not associated with the occurrence of AD, and that the expression of the deleted mRNA may be constitutive and produced by alternative splicing. Funding: Self‐funded.     

Expression of fibronectin and its integrin receptor α5β1 in canine mammary tumours

Fibronectin and its integrin receptor α5β1 were studied by immunohistochemical methods in five normal canine mammary glands, four dysplastic glands and 18 mammary tumours. The aim of the study was to evaluate the possible changes in the α5β1 integrin receptor and its ligand fibronectin in relation to the metastatic capacity of canine mammary neoplasms. The immunostaining of α5β1 was very uniform in the hyperplastic glands but uneven in the mammary tumours. The expression of α5 and β1 was diminished in metastatic tumours but there were some α5-positive cells with pronounced features of malignancy and immaturity. Stromal fibronectin was increased in most cases and cytoplasmic staining of fibronectin was observed in epithelial and myoepithelial cells in mammary neoplasms but not in normal or dysplastic mammary tissue. There was no relationship between the content of α5β1 and the expression of fibronectin in canine mammary tumours.