Identification of the SP22 Sperm Protein in Santa Inês and Dorper Rams

The sperm membrane protein referred to as SP22 has been identified in different species and, at least in rats, is highly correlated with fertility. The goals of this study were to identify and to quantify the SP22 protein on spermatozoa from adult rams (Dorper and Santa Inês breeds), and to correlate its levels to morphological and kinematics parameters. SP22 on ram sperm was effectively quantified by both enzyme‐linked immunosorbent assay (ELISA) and fluorescein isothiocyanate immunostaining analysis and the two methods were significantly correlated (R² = 0.70). Clustering analysis of motility parameters obtained by computer‐assisted semen analysis system was used to establish that three distinct kinematic subpopulations with different vigour and progressiveness coexistent within ejaculate. While there were significant differences in the distribution of the three subpopulations in the rams, there was no significant correlation between the proportion of each subpopulation in the rams and the SP22 levels. Quantification of SP22 immunostaining intensity was not correlated with any of the sperm parameters. However, SP22 levels obtained by ELISA were negatively correlated with morphological abnormalities and positively correlated with membrane integrity (three variable R² = 0.47). Future breeding studies are now needed to validate that this protein is a biomarker of fertility in this species.     

Diagnosis of bovine brucellosis by skin test: conditions for the test and evaluation of its performance.

Brucellergene OCB (Rh?ne-Mérieux) was used as an allergen to define the intrinsic parameters of a skin test and to compare its properties with serology for the diagnosis of bovine brucellosis. The skin test was also evaluated for its capacity to solve problems associated with false positive reactions in serological tests. The optimal reading delay for the skin test was 72 hours. The brucellosis allergic reaction was two to three times less intense than the tuberculosis allergic reaction. An increase of 1.1 mm or more in the skin thickness was therefore considered to be an adequate cut-off. The specificity calculated for 1192 brucellosis-free animals (including animals from brucellosis-free herds in which false positive serological reactions had been reported) was 99-83 per cent (95 per cent confidence interval [CI] 99-40 to 99-98 per cent). The sensitivity determined from 27 experimentally infected heifers ranged from 93 per cent (95 per cent CI 76 to 100 per cent) to 78 per cent (95 per cent CI 58 to 91 per cent) when measured respectively one and six months after the infection. Allergic reactions could be detected in vaccinated animals up to four-and-a-half years after the vaccination. On the other hand, no sensitisation was recorded in na?ve animals after up to eight monthly injections of the allergen. The skin test gave valuable information, in combination with the serological tests, in both acute and chronic brucellosis. The skin test discriminated brucellosis clearly from false positive serological reactions due to infections with Yersinia enterocolitica O9.     

Microbial respiration and substrate weight loss—I: A general model of the influences of abiotic variables

A general model of the influences of abiotic variables on microbial respiration and substrate weight loss is presented. The model, a complex hypothesis relating microbial respiration to temperature. moisture, O₂ and substrate, “explains” 71–96% of the variation in aerobic respiration rates measured from a variety of natural substrates. The model can project seasonal patterns of weight loss and thus permits evaluation of laboratory respirometry against field measures using litter bags. Using data from several countries simulated losses due to microbial respiration are 70–90% of the measured litter bag loss.     

Antibiotics for the preservation of koala (Phascolarctos cinereus) semen

Aim To determine the normal microbial flora of the koala ejaculate and prepuce in order to select appropriate antibiotics for addition into diluents designed for the preservation of semen.
Procedure Bacteriological samples of the koala prepuce (n = 12) and ejaculate (n = 20) were submitted for microbial culture and sensitivity testing. Microbial flora of ejaculates collected by electroejaculation and artificial vagina were compared. The effects of varying concentrations of penicillin G and gentamicin on sperm motility and on the growth of bacteria in diluted semen stored at room temperature and 16°C over a 24 h period were investigated.
Results A range of bacteria was isolated from the koala prepuce and ejaculate. The predominant organisms in semen collected by electroejaculation and artificial vagina were Corynebacterium spp, none of which could be assigned to any recognised species. The addition of penicillin G and gentamicin to a PBS-based diluent at dose rates of 1000 to 2000 IU/mL and 100 to 200 m g/mL respectively, resulted in no adverse effect on sperm motility over a 24 h incubation period. Penicillin G (1000 IU/mL) and gentamicin (100 m g/mL) prevented growth of bacterial contaminants in diluted koala semen.
Conclusion By controlling the growth of bacteria in extended koala semen, penicillin G and gentamicin are likely to lengthen the period by which spermatozoa can be stored at 16°C and reduce the possibility of disease transmission during artificial insemination procedures.     

Ketoprofen pharmacokinetics of R‐ and S‐isomers in juvenile loggerhead sea turtles (Caretta caretta) after single intravenous and single‐ and multidose intramuscular administration

Ketoprofen is a nonsteroidal anti‐inflammatory and analgesic agent that nonselectively inhibits cyclooxygenase, with both COX‐1 and COX‐2 inhibition. Recent studies on COX receptor expression in reptiles suggest that nonselective COX inhibitors may be more appropriate than more selective inhibitors in some reptiles, but few pharmacokinetic studies are available. The goal of this study was to determine single‐ and multidose (three consecutive days) pharmacokinetics of racemic ketoprofen administered intravenously and intramuscularly at 2 mg/kg in healthy juvenile loggerhead turtles (Caretta caretta). The S‐isomer is the predominant isomer in loggerhead sea turtles, similar to most mammals, despite administration of a 50:50 racemic mixture. Multidose ketoprofen administration demonstrated no bioaccumulation; therefore, once‐daily dosing will not require dose adjustment over time. S‐isomer pharmacokinetic parameters determined in this study were C_max of 10.1 μg/ml by IM injection, C₀ of 13.4 μg/ml by IV injection, AUC of 44.7 or 69.4 μg*hr/ml by IM or IV injection, respectively, and T½ of 2.8 or 3.6 hr by IM or IV injection, respectively. Total ketoprofen plasma concentrations were maintained for at least 12 hr above concentrations determined to be effective for rats and humans. A dose of 2 mg/kg either IM or IV every 24 hr is likely appropriate for loggerhead turtles.     

Determination of 5-methyltetrahydrofolic acid and folic acid in citrus juices using stable isotope dilution-mass spectrometry

A stable isotope liquid chromatography-mass spectrometry (LC-MS) method was developed for the quantitative determination of 5-methyltetrahydrofolic acid (5-MTHFA) and folic acid in a variety of commercial citrus juices. Folates were extracted from juices, and the polyglutamyl side chain of 5-MTHFA was cleaved to the monoglutamate form using rat plasma conjugase. The folates were purified on a Bond-Elut column and analyzed by LC-MS with electrospray ionization. The analytes were quantified using the (13)C(5) analogues of 5-MTHFA and folic acid as internal standards. The relative standard error of the method was 3.35% based on replicate analyses (n = 4). This method was then applied to the determination of 5-MTHFA and folic acid in a variety of citrus juices obtained from local supermarkets. It was observed that although both "store" brands and "national" brands of fresh (nonfrozen) juices contained similar concentrations of 5-MTHFA, the "store" brands of fresh juices had on average >5-fold the amount of folic acid compared to the "national" brands. In addition, the "total" folate concentrations were generally below values listed on the food label.     

Novel fluorometric assay for hydroxyl radical prevention capacity using fluorescein as the probe

A novel fluorometric method has been developed to evaluate hydroxyl radical prevention capacity using fluorescein (FL) as the probe. The hydroxyl radical is generated by a Co(II)-mediated Fenton-like reaction, and the hydroxyl radical formation under the experimental condition is indirectly confirmed by the hydroxylation of p-hydroxybenzoic acid. The fluorescence decay curve of FL is monitored in the absence or presence of antioxidant, the area under the fluorescence decay curve (AUC) is integrated, and the net AUC, which is an index of the hydroxyl radical prevention capacity, is calculated by subtracting the AUC of the blank from that of the antioxidant. Gallic acid is chosen as a reference standard, and the activity of sample is expressed as gallic acid equivalents. The method is rigorously validated through linearity, precision, accuracy, and ruggedness. A wide range of phenolic antioxidants is analyzed, and the hydroxyl radical prevention capacity is mainly due to the metal-chelating capability of the compounds.     

Microbial activity in soils frozen to below −39 °C

Recent research on life in extreme environments has shown that some microorganisms metabolize at extremely low temperatures in Arctic and Antarctic ice and permafrost. Here, we present kinetic data on CO₂ and ¹⁴CO₂ release from intact and ¹⁴C-glucose amended tundra soils (Barrow, Alaska) incubated for up to a year at 0 to −39°C. The rate of CO₂ production declined exponentially with temperature but it remained positive and measurable, e.g. 2-7 ng CO₂-C cm⁻³ soil d⁻¹, at −39 °C. The variation of CO₂ release rate (v) was adequately explained by the double exponential dependence on temperature (T) and unfrozen water content (W) (r²>0.98): v=A exp(λT+kW) and where A, λ and k are constants. The rate of ¹⁴CO₂ release from added glucose declined more steeply with cooling as compared with the release of total CO₂, indicating that (a) there could be some abiotic component in the measured flux of CO₂ or (b) endogenous respiration is more cold-resistant than substrate-induced respiration. The respiration activity was completely eliminated by soil sterilization (1 h, 121 °C), stimulated by the addition of oxidizable substrate (glucose, yeast extract), and reduced by the addition of acetate, which inhibits microbial processes in acidic soils (pH 3-5). The tundra soil from Barrow displayed higher below-zero activity than boreal soils from West Siberia and Sweden. The permafrost soils (20-30 cm) were more active than the samples from seasonally frozen topsoil (0-10 cm, Barrow). Finding measurable respiration to −39 °C is significant for determining, understanding, and predicting current and future CO₂ emission to the atmosphere and for understanding the low temperature limits of microbial activity on the Earth and on other planets.