Equine digital veins are more sensitive to superoxide anions than digital arteries.

This work was designed to investigate (i) the effect of superoxide dismutase (SOD) inhibition on endothelial function and (ii) the free radical-induced endothelial dysfunction in equine digital veins (EDVs) and equine digital arteries (EDAs) isolated from healthy horses. EDV and EDA rings were suspended in a 5 ml organ bath containing Krebs solution. After a 60 min equilibration period, EDV and EDA rings were contracted with phenylephrine. Then, cumulative concentration-response curves (CCRCs) to acetylcholine were performed. In both EDVs and EDAs, acetylcholine (1 nM to 10 µM) produced concentration-dependent relaxation. We investigated the influence of SOD inhibition by diethyldithiocarbamate (DETC; 100 µM), a CuZnSOD inhibitor, on EDAs and EDVs relaxant responses to acetylcholine. Acetylcholine -mediated relaxation was impaired by DETC only in EDVs. SOD activity assayed by a xanthine-xanthine oxidase method was higher in EDAs compared with EDVs (P<0.05). CCRCs to acetylcholine established in the presence of pyrogallol (30 µM) or homocysteine (20 µM), two superoxide anions generating systems showed that in both EDVs and EDAs, the acetylcholine-mediated relaxation was significantly impaired by pyrogallol and homocysteine. This impairment was more pronounced in EDVs than in EDAs. Moreover, the pyrogallol-induced impairment of acetylcholine-mediated relaxation was potentiated by DETC to a greater extent in EDVs. We concluded that due to the lower activity of SOD, EDVs are more sensitive to superoxide anions than EDAs. So, any alteration of superoxide anions metabolism is likely to have a more important impact on venous rather than arterial relaxation.

Vasorelaxant effect of propentofylline in isolated equine digital veins.

We evaluated the vasorelaxant effect of propentofylline (PPF), a methylxanthine derivative, and its mechanism of action in equine digital veins (EDVs). Cumulative concentration-response curves to PPF (1 nM-300 µM) were recorded in phenylephrine-precontracted EDV rings under different experimental conditions. PPF-induced relaxation was partially inhibited by endothelium removal, but was unaltered by CGS-15943 (an adenosine receptor antagonist; 3 µM). PPF-induced relaxation was partially inhibited in the presence of L-NAME (a nitric oxide (NO) synthase inhibitor; 100 µM), ODQ (an inhibitor of soluble guanylyl cyclase; 30 µM) or Rp-8-Br-PET-cGMP-S (a protein kinase G inhibitor; 3 µM). It was not modified by indomethacin (a non-selective cyclooxygenase (COX) inhibitor; 10 µM), and was slightly potentiated by H-89 (a protein kinase A inhibitor; 2 µM). In endothelium-intact EDVs, PPF-induced relaxation was associated with a 2.4- and 24.1-fold increase in the tissue cGMP and cAMP content respectively. PPF (100 µM) did not shift the concentration-response curve to phenylephrine (1 nM-300 µM) but reduced the maximal effect. To investigate whether PPF can affect cAMP- and cGMP-induced relaxations, relaxation curves to forskolin (an activator of adenylate cyclase) and to sodium nitroprusside (SNP, a NO donor) were recorded in EDV rings pretreated with PPF (100 µM). PPF only slightly potentiated the forskolin-induced relaxation without affecting the SNP-induced relaxation. We demonstrated that PPF-induced relaxation in EDVs is partially endothelium-dependent. The PPF-induced relaxation partially occurred via NO release and both cAMP and cGMP generation, through COX-independent mechanisms but could also result from the inhibition of cAMP-phosphodiesterase activity for the highest concentrations.

Caprine arthritis encephalitis virus (CAEV) replicates productively in cultured epididymal cells from goats.

The transmission of CAEV from male goats has not been well studied and the target cells that support viral replication are not well characterized. Epididymal epithelial cells (EECs) are important and play a key role in the fertility and motility of spermatozoa. During their transit, spermatozoa incorporate several EEC-produced proteins into their plasma membranes to stabilize them and prevent premature acrosomal reaction. This intimate interaction between spermatozoa and EECs may increase the likelihood of the infection of semen with CAEV if epididymal tissue is productively infected and sheds the virus into the duct. The aim of this study was to examine whether goat EECs are susceptible to CAEV infection in tissue culture. Cells were isolated from epididymides obtained from goats that were sampled from a certified-CAEV-free herd. Cultured cells were then inoculated with a molecularly-cloned isolate of CAEV (CAEV-pBSCA). Inoculated cells developed cytopathic effects (CPE), showing numerous multinucleated giant cells (MGC) in cell-culture monolayers. Expression of CAEV proteins was detected by immunofluorescence using an anti-p28, Gag-specific antibody. The culture medium of inoculated cells was shown to contain high titers (10(6) tissue culture infectious doses 50 per ml (TCID50/ml)) of infectious, cytopathic virus when assayed using indicator goat synovial membrane (GSM) cells. Our findings clearly demonstrate that cells of the buck genital tract are targets of CAEV and are thus a potential reservoir that sheds infectious CAEV into the semen of infected animals. These data suggest the use of sperm from CAEV-free goat males for artificial insemination in genetic selection programs to minimize CAEV dissemination.

Autoantibodies against cardiac ß(1)-adrenoceptor do not affect the low-affinity state ß(1)-adrenoceptor-mediated inotropy in rat cardiomyocytes.

Circulating autoantibodies directed against the 2nd extracellular loop (EL-2) of ß(1)-adrenoceptors (ß(1)-AABs) have been detected in the serum of patients with various cardiovascular pathologies. ß(1)-AABs induce agonistic, positive inotropic effects via ß(1)-adrenoceptors (ß(1)ARs). In the mammalian heart, ß(1)-AR can exist in 2 distinct activated configurations (the so-called high- and low-affinity states). The aim of the present study was to investigate whether the action of ß(1)-AAB is dependent on the affinity state of ß(1)AR in isolated ventricular cardiomyocytes of adult Wistar rats. Immunoglobulin G (IgG) containing ß(1)-AAB obtained from animals immunized with a peptide corresponding to the EL-2 of human ß(1)-AR, caused a dose-dependent increase in cell shortening. Isoproterenol-induced inotropy was significantly reduced in cardiomyocytes that had been preincubated with IgG containing ß(1)-AAB and in cardiomyocytes isolated from immunized rats. The negative effects of preincubation with IgG containing ß(1)-AAB on the response to isoproterenol was inhibited in the presence of bisoprolol. CGP 12177A and pindolol-induced inotropy was not affected by IgG preincubation or immunization. No detectable inotropic effect of cell shortening was obtained with IgG containing ß(1)-AAB in the presence of propranolol and 3-isobutyl-1-methylxanthine. The present study demonstrates that ß(1)-AABs have no agonist/antagonist-like effects upon low-affinity state ß(1)-ARs. This result indicates that ß(1)-AABs recognize and stabilize the high-affinity state, but are unable to stabilize and (or) induce the low-affinity state receptor.

Goat uterine epithelial cells are susceptible to infection with Caprine Arthritis Encephalitis Virus (CAEV) in vivo.

The aim of this study was to determine, using immunofluorescence and in situ hybridization, whether CAEV is capable of infecting goat uterine epithelial cells in vivo. Five CAEV seropositive goats confirmed as infected using double nested polymerase chain reaction (dnPCR) on leucocytes and on vaginal secretions were used as CAEV positive goats. Five CAEV-free goats were used as controls. Samples from the uterine horn were prepared for dnPCR, in situ hybridization, and immunofluorescence. The results from dnPCR confirmed the presence of CAEV proviral DNA in the uterine horn samples of infected goats whereas no CAEV proviral DNA was detected in samples taken from the uninfected control goats. The in situ hybridization probe was complementary to part of the CAEV gag gene and confirmed the presence of CAEV nucleic acids in uterine samples. The positively staining cells were seen concentrated in the mucosa of the lamina propria of uterine sections. Finally, laser confocal analysis of double p28/cytokeratin immunolabelled transverse sections of CAEV infected goat uterus, demonstrated that the virus was localized in glandular and epithelial cells. This study clearly demonstrates that goat uterine epithelial cells are susceptible to CAEV infection in vivo. This finding could help to further our understanding of the epidemiology of CAEV, and in particular the possibility of vertical transmission.

Seroprevalence of Neospora caninum antibodies and its consequences for reproductive parameters in dairy cows from Dakar-Senegal, West Africa.

The aim of this study was to determine the seroprevalence of Neospora caninum antibodies and its effects on reproductive parameters in cows in intensive dairy herds in Dakar. Randomised blood samples were taken for serology from 196 cows in four herds with a history of sporadic abortion. All of the sera were assayed for antibodies against N. caninum, Candida guillermondii, Coxiella burnetii, and Chlamydophila sp. The associations between serostatus and reproductive parameters (abortion, number of inseminations to conception, and calving to conception interval) were assessed over a period of 5 years (2004-2008). The seroprevalence of N. caninum antibodies in dairy cattle was 17.9%. The local Gobra breed and crossbreeds had higher levels of N. caninum antibodies than exotic breeds (p < 0.05). For the most recent pregnancies, seropositive cows required more inseminations to establish conception than seronegative cows (p < 0.05). The results indicate that dairy cattle from Dakar are exposed to N. caninum. Neosporosis should, therefore, be systematically considered as a cause when the calving to conception interval is prolonged.

Modifications of bull spermatozoa induced by three extenders: Biociphos, low density lipoprotein and Triladyl, before, during and after freezing and thawing.

The success of artificial insemination with frozen semen implies the reduction of the deleterious effects on the cells induced by this technique. These effects can occur as early as during the first dilution in an extender, as well as at any step, during or after the freezing process. In this work, we have compared the modifications induced by Triladyl, low density lipoproteins (LDL) and Biociphos extenders, after dilution and cooling to 4 degrees C for 1, 4 and 24 h. Alterations in the cell structures were visualized by electron microscopy (EM). More than 80% of spermatozoa were injured after incubation for 4 h in Triladyl, while 3% and 47% were counted in LDL and Biociphos respectively. This latter extender was deleterious to cell membrane integrity after incubation for 4 h or longer. The ultrastructure of frozen spermatozoa was studied by EM of cryofixed-cryosubstituted samples obtained from regular 0.5 ml French straws frozen using our usual protocol. The main differences between samples concerned the size and appearance of the frozen extender veins, while very few cell defects were found to be added by the freezing process at any depth in the straws. After thawing, semen motility was twofold higher (P < 0.05) in Biociphos (64%) and LDL (61%) than in Triladyl (32%) and the cells were less altered in LDL. We concluded that the LDL extender offers a better protection for storage of frozen spermatozoa, and can probably also be used for the preservation of fresh semen for short periods.

Effects of glutamine on post-thaw motility of stallion spermatozoa: an approach of the mechanism of action at spermatozoa level.

The cryoprotective effect of l-glutamine and an approach of its mechanism of action, in preserving motility of stallion spermatozoa during the freezing-thawing process, were studied. In Experiment 1, thirty-six ejaculates were collected from six stallions (two good, two middle, and two of poor sperm freezability) and were diluted with 10 different freezing media derived from INRA 82 medium supplemented with 20 mM HEPES and 2% (v/v) centrifuged egg yolk (BM). After thawing, sperm motility was evaluated by a computer-assisted semen motility analyser. The effects of glutamine and glycerol at different concentrations on post-thaw sperm motility were studied. A possible interaction between medium and semen freezability was investigated. Only the 50 mM glutamine + 2.5% glycerol medium significantly improved sperm motility compared to classical freezing medium (2.5% glycerol). The presence of glutamine at 50 mM was not sufficient to offset the need to use glycerol in the freezing extender. The use of glutamine at a higher concentration >100 mM in the presence of 2.5% of glycerol was toxic. Reducing the glycerol proportion from 2.5% to 2 or 1.5% in the presence of glutamine at 50, 75, and 100 mM had no influence on post-thaw motility of semen of middle and good freezability. Moreover, the substitution of 2.5% glycerol by 50 mM glutamine in BM, did not significantly change the post-thaw motility of semen of good freezability. In Experiment 2, 3H-glutamine and 3H-glycerol were used to study the kinetics of penetration of glutamine and glycerol in sperm cells. The radioactivity of each radio-labelled semen pellet was measured at different times (0, 15, 30, 60, 90, 120 min), by using a Packard tri-carb 4530 apparatus. The percentages of incorporated radioactivity (%IRA) in semen pellets were calculated at different times. The %IRA of 3H-glycerol in semen pellets were significantly higher than the %IRA of 3H-glutamine. The %IRA of 3H-glycerol in semen pellets increased greatly from time 0 to 60 min, and then it is stabilized from 60 to 120 min. In contrast, the %IRA of 3H-glutamine in semen pellets increased slightly from 0 to 60 min, then it stabilized until 120 min. These experiments demonstrate that glutamine has a synergistic cryoprotective effect with glycerol on cryopreservation of stallion spermatozoa, and suggest that glutamine acts at the extra-cellular level, independently of glycerol.