In vitro Evaluation of in vivo Fertilizing Ability of Frozen Rabbit Semen

The present work studied different spermatozoa parameters and the ability of frozen rabbit spermatozoa to fertilize, in vitro, in vivo‐matured oocytes, as a test to predict their in vivo fertility and prolificacy. Semen from rabbit bucks was frozen using two freezing protocols [in a freezer at ?30°C or in liquid nitrogen vapour (LNV)]. For the in vivo trial, females were inseminated with frozen‐thawed spermatozoa. Oocytes used for in vitro testing were recovered 14 h after ovulation induction from donors and co‐incubated with 2 × 10⁶ frozen‐thawed spermatozoa during 4 h at 37°C in Tyrode's medium under an atmosphere of 5% CO₂ in air with maximal humidity. After co‐incubation period, presumptive zygotes were cultured in TCM199 supplemented with 20% foetal bovine serum (FBS), under the same conditions described above. Although no statistical differences were observed between freezing protocols in seminal parameters [motility rate: 40 and 35%, VCL: 35 and 46 μm/s, amplitude of lateral head displacement (ALH): 1.7 and 2.4 μm, for semen frozen at ?30°C and in LNV, respectively], significant differences were noted in the fertilizing ability in vivo and in vitro. Semen frozen at ?30°C showed the highest fertilizing ability in vitro (26.7% vs 6.2 and 8.7% for semen frozen at ?30°C, in LNV and fresh semen, respectively) and the lowest fertility rate in vivo (21.7% vs 64.2% and 70.6% for semen frozen at ?30°C, in LNV and fresh semen, respectively). Sperm frozen at ?30°C seemed to be more capacitated.     

Autumn frost damage: clonal variation in Sitka spruce

Current-year needles of Sitka spruce (Picea sitchensis) can be injured by frost after hardening of stem and buds in the autumn, and in Scotland trees of southern provenances are most susceptible to damage. In October 1993 a frost of −5°C caused needle damage to clonal plants of Alaskan, Queen Charlotte Islands, and Washington provenances of Sitka spruce that had been selected for variation in the onset of root dormancy. Visual damage assessment revealed significant differences among clones in the severity of damage, and this damage was positively correlated with the lateness of root dormancy. Measurement of electrolyte leakage from shoot samples confirmed the observed differences between clones. The large clonal differences in frost hardiness found in this study demonstrate a potential for selecting frost tolerant trees from within southern provenances of Sitka spruce.     

Development of a widely applicable positive control strategy to support detection of infectious salmon anaemia virus (ISAV) using Taqman real-time PCR

Real-time PCR assays are being increasingly applied to the detection of fish pathogens due to their sensitivity, specificity and potential for high throughput sample processing. Such assays allow for the ready and efficient inclusion of appropriate quality controls which are fundamental to scientific integrity and to satisfying the demands of diagnostic test accreditation. In this article, we report development of a universal positive control strategy for real-time PCR assays, which has been used to support and improve a previously published method for detection of infectious salmon anaemia virus (ISAV). The strategy employed uses an RNA mimic template, which is based on the ISAV segment 8 target sequence but includes an artificial universal positive control sequence. Inclusion of this sequence, which is targeted by a second specific probe carrying a different fluorophore to the primary assay, allows for convenient screening of all real-time PCR reactions for the presence of contaminating positive control material. The development of readily distinguishable artificial positive control material offers distinct advantages to real-time PCR assays over using control material derived from clinical material.     

Molecular epidemiology of clinical isolates of Pseudomonas aeruginosa isolated from horses in Ireland

Clinical isolates (n = 63) of Pseudomonas aeruginosa obtained from various sites in 63 horses were compared using ERIC2 RAPD PCR to determine their genetic relatedness. Resulting banding patterns (n = 24 genotypes) showed a high degree of genetic heterogeneity amongst all isolates examined, indicating a relative non-clonal relationship between isolates from these patients, employing this genotyping technique. This study characterised 63 clinical isolates into 24 distinct genotypes, with the largest cluster (genotype E) accounting for 10/63 (15.9%) of the isolates. ERIC2 RAPD PCR proved to be a highly discriminatory molecular typing tool of P. aeruginosa in isolates recovered from horses. With the adoption of several controls to aid reproducibility, this technique may be useful as an alternative to PFGE, particularly in epidemiological investigations of outbreaks where speed may be a significant parameter. This is the first report of clonal heterogeneity amongst P. aeruginosa from horses and demonstrated that ERIC RAPD PCR is a rapid method for the examination of this species in horses, which may be useful in outbreak analysis.     

Serological survey of pre-weaned New Zealand fur seals (Arctocephalus forsteri) for brucellosis and leptospirosis

AIM: To conduct a longitudinal serological survey for evidence of Brucella spp and Leptospira spp infection of pre-weaned New Zealand fur seals in a colony on the Otago Peninsula.

METHODS: Seal pups were repeatedly captured on a monthly basis from February through to July 2001. Pups were tagged at first capture and a blood sample was taken at each capture event. A total of 163 sera were collected from 118 seal pups. Where sufficient volume was collected, the sera were tested for leptospirosis using the microscopic agglutination test (MAT), and for brucellosis using the competitive enzyme-linked immunosorbent assay (ELISA) for Brucella abortus.

RESULTS: None of 128 sera from 101 seals tested positive to the ELISA for B. abortus. All tests for Leptospira interrogans serovars Grippotyphosa, Copenhageni, Bratislava and Leptospira borgpetersenii serovar Ballum were negative at a cut-off of <1/100 dilution. Positive or suspicious titres were found to L. interrogans serovars Canicola and Pomona and L. borgpetersenii serovar Hardjo. The highest titres (12,800) were found to serovar Pomona. The titre to serovar Pomona in one seal rose from <1/50 in March to 12,800 in April and was <1/50 when re-sampled in July. The titre to serovar Pomona in another seal dropped from 12,800 in May to <1/50 in June. These seals also had titres to serovar Hardjo, which rose or fell in the same manner. All suspicious or positive titres occurred in late April and early May, when the pups were approximately 4–5 months old. In June and July, all seals tested were negative.

CONCLUSIONS: There was no serological evidence of Brucella infection in the pre-weaned fur seals at the colony. Positive titres to serovars Pomona, Hardjo, or Canicola suggest that a Leptospira species was present at the colony, however isolation or visualisation of the organism is required to confirm this. Care should be exercised when handling New Zealand fur seals to prevent human infection or inadvertent transfer of leptospirosis to another marine mammal species.     

Direct Radiative Forcing by Smoke from Biomass Burning

Airborne measurements in smoke from biomass burning in Brazil have yielded optical parameters that permit an improved assessment of the effects of smoke on Earth's radiation balance. The global-mean direct radiative forcing due to smoke from biomass burning worldwide is estimated to be no more than about -0.3 watt per square meter (cooling), compared with +2.45 watts per square meter (warming) due to anthropogenic greenhouse gases. On regional scales, direct radiative forcing due to smoke can be large and might indirectly affect global climate.     

Earth-Based Radio Tracking of the Galileo Probe for Jupiter Wind Estimation

Although the Galileo probe was designed to communicate only to the orbiter, the probe radio signal was detected at two Earth-based radio observatories where the signal was a billion times weaker. The measured signal frequency was used to derive a vertical profile of the jovian zonal wind speed. Due to the mission geometry, the Earth-based wind estimates are less sensitive to descent trajectory errors than estimates based on probe-orbiter Doppler measurements. The two estimates of wind profiles agree qualitatively; both show high wind speeds at all depths sampled.     

Geomicrobiology of subglacial ice above Lake Vostok, Antarctica

Data from ice 3590 meters below Vostok Station indicate that the ice was accreted from liquid water associated with Lake Vostok. Microbes were observed at concentrations ranging from 2.8 x 10(3) to 3.6 x 10(4) cells per milliliter; no biological incorporation of selected organic substrates or bicarbonate was detected. Bacterial 16S ribosomal DNA genes revealed low diversity in the gene population. The phylotypes were closely related to extant members of the alpha- and beta-Proteobacteria and the Actinomycetes. Extrapolation of the data from accretion ice to Lake Vostok implies that Lake Vostok may support a microbial population, despite more than 10(6) years of isolation from the atmosphere.