Fluorescent Stain Method for the Simultaneous Determination of Mitochondrial Potential and Integrity of Plasma and Acrosomal Membranes in Boar Sperm

The purpose of this study was to validate a technique for simultaneous evaluation of the plasma, acrosomal and mitochondrial membranes in boar spermatozoa, using an association of fluorescent probes: Propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and JC-1. Three ejaculates from each of four different boars, all showing motility >or=80% and abnormal morphology 相似文献   

Serological response to Actinobacillus pleuropneumoniae serovar 7 infection in a commercial pig herd

Serological responses to Actinobacillus pleuropneumoniae serovar 7 infection were monitored by enzyme-linked immunosorbent assay in a cohort of 66 pigs between weaning and market. Antibody concentrations were high (63/65 seropositive) at 4 weeks of age but declined to low levels from 8 to 12 weeks. Mean antibody concentrations rose significantly (p less than 0.001) between 12 and 23 weeks. Between 8 and 23 weeks of age, 33 (51.5%) of 64 surviving pigs seroconverted to A pleuropneumoniae serovar 7. Peak antibody concentrations in the seroconverting pigs usually (28/33) occurred at 23 weeks. Seroconversion to A pleuropneumoniae during the grower/finisher phase was not significantly associated (p greater than 0.05) with passive antibody concentrations at 4 weeks of age, lack of vaccination against Mycoplasma hyopneumoniae, or weaning weight. Pleuropneumonic lesions were evident at slaughter in 4 (6.3%) of 64 pigs. A pleuropneumoniae serovar 7 was isolated from 2 of 4 lungs with pleuropneumonia and from another lung with lesions considered typical of enzootic pneumonia.     

Practical Techniques for Bovine Sperm Simultaneous Fluorimetric Assessment of Plasma, Acrosomal and Mitochondrial Membranes

This experiment was performed to develop and validate practical techniques for simultaneous evaluation of the integrity of plasma and acrosomal membranes, as well as mitochondrial function in bovine spermatozoa using associations of fluorescent probes. Four protocols of fluorescent probes association were defined: protocol 1: propidium iodide (PI), fluorescein isothiocyanate‐conjugated Pisum sativum agglutinin (FITC‐PSA) and rhodamine 123; protocol 2: PI, FITC‐PSA and MitoTracker Green FM (MITO); protocol 3: PI, Hoechst 33342 (H342), FITC‐PSA and CMXRos; and protocol 4: PI, H342, FITC‐PSA and JC‐1. Three ejaculates from each of the four bulls (n = 12) were utilized, showing sperm motility ≥80% and abnormal morphology ≤10%. The semen was diluted in Modified Tyrode’s medium (TALP) (25 × 10⁶ spermatozoa/ml) and split into two aliquots, one sample was flash‐frozen in liquid nitrogen and thawed. Samples for three treatments were prepared with the following ratio of fresh semen : flash‐frozen semen: 100 : 0, 50 : 50 and 0 : 100. Samples were stained in all four protocols and evaluated by epifluorescence microscopy. Protocol 1 did not result in a satisfactory stain, so it could not be validated. Protocols 2, 3 and 4 were validated and showed high determination coefficient to plasma membrane integrity (R² = 0.95, 0.93 and 0.92, respectively), acrosome integrity (R² = 0.95, 0.92 and 0.91, respectively) and mitochondrial function (R² = 0.84, 0.93 and R² = 0.93, respectively). These techniques are efficient for the simultaneous integrity evaluation of plasma and acrosomal membranes and mitochondrial function in bovine spermatozoa. However, JC‐1 has an advantage over MITO and CMXRos, as it separates two cell populations with high and low mitochondrial membrane potential.     

Peripheral blood lymphocyte subsets in fleece rot-resistant and -susceptible sheep

Objective To compare haematological values and lymphocyte phenotypes in the peripheral blood of fleece rot-resistant and -susceptible sheep.
Procedure Experiments were conducted on 2- and 3-year-old Merino rams, flock 1 (17 rams) and flock 2 (32 rams), respectively. Within each flock, individual rams were classified as fleece rot-resistant or -susceptible, based on established criteria. Total and differential white cell counts, and indirect fluorescent antibody tests specific for B cells and T cells were performed on all sheep. The concentration of various subsets of circulating lymphocytes was then determined in each sheep.
Results There were no significant differences between fleece rot-resistant and -susceptible sheep from either flock in the mean total or differential white cell counts. However, fleece rot-resistant rams in flock 1 did have a significantly higher concentration of circulating SBU-T1⁺ cells than fleece rot-susceptible rams from the same flock. No such difference was noted in the rams from flock 2. While all rams in flock 1 were free of clinical fleece rot, 24 rams in flock 2 (comprising all 17 fleece rot-susceptible and 7 of 15 fleece rot-resistant animals) had clinical signs of the disease. Fleece rot-free rams in this flock (irrespective of their classification as fleece rot-resistant or -susceptible) had significantly higher concentrations of circulating SBU-T1⁺ cells compared with fleece rot-affected animals. They also had significantly higher concentrations of circulating B cells, and total lymphocytes.
Conclusions An examination of peripheral blood lymphocyte subsets in fleece rot-resistant and -susceptible sheep revealed a possible association between resistance to fleece rot and the concentration of circulating SBU-T1⁺ cells.     

Effect of Antioxidants During Bovine In Vitro Fertilization Procedures on Spermatozoa and Embryo Development

Increased amounts of reactive oxygen species (ROS) during in vitro fertilization (IVF) may cause cytotoxic damage to gametes, whereas small amounts of ROS favour sperm capacitation. The aim of this study was to investigate the effect of antioxidants [50 μ m β-mercaptoethanol (β-ME) and 50 μ m cysteamine (Cyst)] or a pro-oxidant (5 m m buthionine sulfoximine) on the quality and penetrability of spermatozoa into bovine oocytes and on the subsequent embryo development and quality when added during IVF. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes, and mitochondrial function, was diminished (p < 0.05) after 4-h culture in the presence of antioxidants. Oocyte penetration rates were similar between treatments (p > 0.05), but antioxidants adversely affected the normal pronuclear formation rates (p < 0.05). The incidence of polyspermy was high for β-ME (p < 0.05). No differences were observed in cleavage rates between treatments (p > 0.05). However, the developmental rate to the blastocyst stage was adversely affected by Cyst treatment (p < 0.05). The quality of embryos that reached the blastocyst stage, evaluated by total, inner cell mass (ICM) and trophectoderm cell numbers and ICM/total cell ratio was unaffected (p > 0.05) by treatments. The results indicate that ROS play a role in the fertilizing capacity in bovine spermatozoa, as well as in the interaction between the spermatozoa and the oocytes. It can be concluded that supplementation with antioxidants during IVF procedures impairs sperm quality, normal pronuclear formation and embryo development to the blastocyst stage.     

Lunar surface magnetic fields and their interaction with the solar wind: results from lunar prospector

The magnetometer and electron reflectometer experiment on the Lunar Prospector spacecraft has obtained maps of lunar crustal magnetic fields and observed the interaction between the solar wind and regions of strong crustal magnetic fields at high selenographic latitude (30 degreesS to 80 degreesS) and low ( approximately 100 kilometers) altitude. Electron reflection maps of the regions antipodal to the Imbrium and Serenitatis impact basins, extending to 80 degreesS latitude, show that crustal magnetic fields fill most of the antipodal zones of those basins. This finding provides further evidence for the hypothesis that basin-forming impacts result in magnetization of the lunar crust at their antipodes. The crustal magnetic fields of the Imbrium antipode region are strong enough to deflect the solar wind and form a miniature (100 to several hundred kilometers across) magnetosphere, magnetosheath, and bow shock system.     

Infection of Cranberry Flowers by Monilinia oxycocci and Evaluation of Cultivars for Resistance to Cottonball

ABSTRACT Infection of cranberry flowers by conidia of Monilinia oxycocci, the cottonball pathogen, was investigated using a squash-mount histological method. Conidia germinated on anthers, nectaries, petals, and stigmata, but not styles. The stigma was the only flower part penetrated by the fungus, but no specialized infection structures were noted. Both fungal and pollen germ tubes grew through the stylar canal and made contact with ovules and nucellar tissue by 72 h after inoculation and pollination. Cottonball incidence was greatest when stigmata were inoculated; the low level of cottonball that resulted from inoculation of other flower parts and in noninoculated flowers was attributed to contamination of stigmata. In greenhouse tests, cottonball incidence was 25, 28, 31, and 38% for cvs. Searles, Pilgrim, Ben Lear, and Stevens, respectively, and was greater for M. oxycocci isolate 593 than isolate 591. We conclude that the stigma is the sole floral infection court for conidia of M. oxycocci and that the most popular cranberry cultivars in Wisconsin do not differ in inherent resistance to cottonball. The relevance of these findings to the long-term management of cottonball is discussed.     

Utilization of deoiled castor cake for crop production

A screen house experiment was undertaken to study effect of time of application of deoiled castor cake on the yield, N and P uptake by wheat crop. The results indicate that the wheat growth decreased significantly with increasing dose of castor cake, when castor cake was applied just before sowing of wheat. When castor cake was applied 10 days before wheat sowing, the dry matter yield (DMY) of wheat increased up to 0.125% dose of castor cake and reduced thereafter. But when the castor cake was applied 20 days before wheat sowing, the DMY was at par under all the doses of castor cake and better than control indicating the degradation of the toxicants produced by the castor cake. Application of castor cake can also be helpful in reducing the cost of phosphatic fertilizer. Thus it can be concluded that castor cake should be applied at least 3 weeks before sowing of the crop and keeping the field moist for degradation of the toxicants.