Identification of RSVP14 and RSVP20 components by two-dimensional electrophoresis and Western-blotting.

We have already shown that RSVP14 and RSVP20, two ram seminal plasma (SP) proteins postulated to be involved in sperm capacitation and gamete interaction can protect spermatozoa against cold-shock. In this study, we use two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for the analysis of SP proteins of Rasa Aragonesa rams, using enhanced protein solubilization in the presence of tributyl phosphine (TBP) and a polyacrylamide linear gradient gel with a narrow pH range (4-7). The image analysis of the 2D map detected 195 protein spots, with isoelectric points (pIs) ranging from 4.5 to 6.6, and molecular weight (M(r)) from 11.7 to 90.4. Staining of 2D gels with Pro-Q Emerald 300 Glycoprotein Stain revealed that most significant proteins in ram SP are glycosylated. The removing of protein N-linked oligosaccharides improved the gel resolution. 2D-PAGE analysis of the whole fraction 6 (F6) separated from ram SP by exclusion chromatography showed six main protein spots, four (a, b, c, d) in the 14 kDa and two (e, f) in the 20 kDa region. Western-blot analyses indicated that the anti-P14 antibody recognized four spots on the SP map, 4, 5, 6 and 7, that matched with spots a, b, c, d of F6 map. The anti-P20 antibody recognized spots 13 and 14 of SP map that corresponded to spots e, f of F6 map. The deduced sequences by de novo sequencing evidenced that protein spots 7 and 13 have significant similarities to BSP family, while protein spots 4 and 14 did not appear to be homologous with any reported protein in the current mammalian Proteinbank databases.     

Effect of Chemical or Electrical Activation of Bovine Oocytes on Blastocyst Development and Quality

Activation of in vitro‐matured (IVM) oocytes is essential for successful embryo production following nuclear transfer (NT) or intracytoplasmic sperm injection (ICSI). This study was designed to compare the rates of blastocyst production and embryo quality (as measured by numbers of viable cells) following parthenogenetic activation with electrical pulse or the use of two different calcium ionophores, A23187 (CA) or ionomycin (IO), with or without the addition of bovine serum albumin (BSA). IVM oocytes with a first polar body were randomly allocated to five treatment groups: CA (5 μm CA, 5 min; n = 88), CA + BSA (5 μm CA, 5 min; BSA, 5 min; n = 90), IO (5 μm IO, 5 min; n = 91), IO + BSA (5 μm IO, 5 min; BSA, 5 min; n = 86) and EL (two pulses of 1.5 kV/cm, 20 μs; n = 120). Blastocyst rates were higher (p < 0.05) for CA (54.4%), IO (51.4%) and EL (54.5%) than for IO + BSA (18.3%). Treatment CA + BSA (39.8%) did not differ from the others. There was no difference (p > 0.05) among treatments in total number of cells. However, the percentage of viable cells was reduced in CA (49.9%), CA + BSA (45.8%), IO (64.9%), IO + BSA (50.9%) compared with EL (82.7%). In summary, the addition of BSA to the IO treatment had an adverse effect on blastocyst production rates. Although there was no difference between electrical stimulation and chemical activation on blastocyst production rates, electrical activation resulted in blastocysts with a higher percentage of viable cells.     

Cross-protective immunity and the serological classification system for Bacteroides nodosus

The relationship between the serological classification system for serogroup B and for serogroup H of Bacteroides nodosus and cross-protection between subgroups within these serogroups was examined. Protection against ovine footrot following vaccination was achieved against other subgroup strains provided sufficient cross-reactive antibody was induced by shared pilus antigens. Within serogroup B, better cross-protection against one subgroup was obtained with a pili vaccine than a whole cell vaccine which correlated with higher pilus antibody titres induced by the former. For serogroup H, a lack of cross-protection and serological reactivity between subgroups was demonstrated, which indicates that the prototype strain of subgroup H2 should be designated a new serogroup.     

Relationship of blood corticosterone, immunoglobulin and haematological values in young crocodiles (Crocodylus porosus) to water temperature, clutch of origin and body weight

Objective To examine whether sub-optimal temperature induced stress and immunosuppression in farmed saltwater crocodile (Crocodylus porosus) hatchlings. Design A clinico-pathological study. Animals A total of 140 hatchlings were used. Procedure Body weight and length, plasma corticosterone and immunoglobulin concentrations and total and differential white blood cell counts were measured in 140 hatchlings from five clutches divided between five water temperature treatment groups. Initially all groups were housed at 32°C for 10 weeks, then two groups (L, LC) were changed to low temperature (28°C) and two groups (H, HC) to high temperature (36°C), while one group (C) remained at 32°C. The LC and HC groups were maintained at these temperatures for 10 days, after which the water temperature of both groups was returned to 32°C. Blood samples were collected twice (at 6 and 9 weeks of age) before the initial temperature change, and at 10 days and 4 weeks after the initial temperature change (at 11.5 and 14 weeks of age). Results Except for an increase in plasma corticosterone in the HC group and a decrease in the L group when the temperature change was first introduced, changes in plasma corticosterone were not significant. There were no significant changes in immunoglobulin concentrations. There were, however, significant decreases in the total white cell and lymphocyte counts in the LC group after the temperature was decreased to 28°C, and an increase in these counts after water temperature was returned to 32°C. Clutch of origin had significant effects on body weight and length gains, and there were negative relationships between body weight and corticosterone concentrations and between body weight and immunoglobulin concentrations. Conclusions As haematological changes indicative of stress were not associated with significant changes in serum corticosterone, immunosuppression in young crocodiles may be independent of the hypothalamic-pituitary-adrenal cortical axis.     

Lamprey GnRH‐III Releases Luteinizing Hormone but not Follicle Stimulating Hormone in Pigs

The effect of exogenous administration of lamprey GnRH‐III (IGnRH‐III) on gonadotropin secretion was evaluated in pigs. Six crossbred barrows (82.4 ± 3.5 kg body weight) were assigned randomly to a replicated 3 × 3 Latin square design to evaluate the effect of 0.1, 1.0 or 10.0 μg/kg body weight of exogenous IGnRH‐III on LH and FSH secretion. To facilitate blood collection and infusion of IGnRH‐III, barrows were catheterized in the jugular vein 1 day before initiation of experiments. Blood samples were taken at 10‐min intervals for 6 h, starting 2 h before treatments were applied. Relative concentrations of LH and FSH were calculated by obtaining the ratio of the average concentration of each hormone 2 h after infusion divided by the average concentration during the 2 h before infusion. Relative concentrations of FSH after IGnRH‐III infusion did not influence mean concentration of FSH at any of the doses; yet 10.0 μg/kg body weight had a significant effect on LH secretion (p < 0.01). Relative concentrations of LH averaged 1.2, 1.0 and 3.0 ng/ml (for doses of 0.1, 1.0 and 10.0 μg/kg body weight of IGnRH‐III respectively). Only a dose of 10 μg/kg body weight elicited a significant LH increase that was associated with exogenous IGnRH‐III infusion. We conclude that IGnRH‐III is a weak GnRH agonist and at high doses, IGnRH‐III has the ability to release LH but not FSH in barrows.     

Relation between plant nutrition, hormones, insecticide applications, bacterial endophytes, and Candidatus Liberibacter Ct values in citrus trees infected with Huanglongbing

Intensive insecticide and nutrient management have been attempted worldwide to reduce citrus huanglongbing (HLB) symptom development and yield loss. However, effects of insecticide and nutrient applications on HLB have been poorly understood. Leaf nutrients, jasmonic and salicylic acid contents, cycle threshold (Ct) values of Ca. Liberibacter asiaticus (Las), and community structure of endophytic α-proteobacteria were evaluated after insecticide treatment, ‘nutrition’ treatment (including systemic resistance inducing agents), or both in comparison with a control in a two-factor field experiment in 2008–2012. Leaf N, Mn, Zn and B significantly increased whilst Cu decreased after nutrient applications. Salicylic acid significantly increased in old leaves treated with insecticides, nutrients or both, and in young leaves treated with nutrients only. The jasmonic acid concentration was highest after the nutrition treatment in both old and young leaves. Ct values of Las and leaf area and weight significantly increased after long-term nutrient applications in 2011 and/or 2012. Redundancy analysis of the endophytic α-proteobacteria community structure indicated that the communities were mainly separated according to nutrient applications, which were positively associated with Ct values of Las and Ca, Mn, Zn, B, Mg, and Fe contents in leaf samples collected in 2012. Thus, effects of insecticides on HLB were significant in the early 2-year period whilst nutrients had significant effects on Las content and leaf size and weight after at least 3 years of application.     

Hemodynamic effects of sevoflurane in spontaneously breathing cats

Sevoflurane has recently been introduced in feline anesthesia. However, its cardiovascular effects have not, to our knowledge, been reported in this species. Six healthy cats, aged 1.81 ± 0.31 years (mean ± SEM) and weighing 3.47 ± 0.11 kg, were studied. Anesthesia was induced and maintained with sevoflurane in oxygen. Body temperature was maintained between 38.5 and 39.55 °C. After instrumentation, end‐tidal sevoflurane concentration was randomly set at 1.25, 1.5, and 1.75 times the individual minimum alveolar concentration (MAC), determined in a previous study, according to a Latin Square Design. Thirty minutes of stabilization was allowed after each change of concentration. ECG and heart rate, systemic and pulmonary arterial pressures, central venous pressure (CVP), and core body temperature were continuously monitored and recorded. Inspired and end‐tidal oxygen, carbon dioxide, and sevoflurane concentrations were measured using a Raman spectrometer, calibrated every 80 minutes with three calibration gases of known sevoflurane concentration (1, 2, and 5%). Moreover, at selected times, pulmonary artery occlusion pressure and cardiac output (thermodilution) were measured, and arterial and mixed venous blood samples were collected for pH and blood gas analysis, hemoglobin concentration, hemoglobin oxygen saturation, packed cell volume (PCV) and total protein determination, and lactate concentration measurement. Cardiac index (CI), stroke index (SI), systemic and pulmonary vascular resistance indices, rate‐pressure product, left and right ventricular stroke work indices (LVSWI and RVSWI, respectively), arterial and mixed venous oxygen contents, oxygen delivery, oxygen consumption, and oxygen utilization ratio were calculated. Data were analyzed by a Repeated Measure Latin Square Design followed by a Tukey's test for 2 × 2 comparisons. Arterial pH significantly decreased from 7.40 ± 0.05 to 7.29 ± 0.07 with the administration of increasing concentrations of sevoflurane. Similarly, LVSWI decreased from 3.72 ± 0.60 to 2.60 ± 0.46 g m^?2. Mean arterial pressure, PaO₂, mixed venous pH, CI, SI, and oxygen delivery tended to decrease dose‐dependently, whereas CVP, PaCO₂, Pv CO₂, PCV, and arterial and mixed venous hemoglobin concentrations tended to increase dose‐dependently with the administration of sevoflurane. However, these trends did not reach statistical significance, possibly because of the limited number of animals studied. Sevoflurane seemed to induce dose‐dependent cardiovascular depression in cats.