Enzyme Immunoassays for the Determination of Ovine LH and FSH

The development of competitive enzyme immunoassays for ovine plasma LH (oLH) and FSH (oFSH) is described. Standards and plasma samples were preincubated with diluted antiserum to oLH or oFSH and the reacted solution (100 μl per well) was transferred to plates previously coated with oLH or oFSH, respectively. The second antibody used was anti‐rabbit IgG horseradish peroxidase. The measuring range was 0.39–50 ng/ml for each hormone and the 50% relative binding sensitivity was 9 ng/ml for oLH. The respective value for oFSH was 3.5 or 34 ng/ml with different hormone and antibody preparations used for the assay. The enzyme immunoassays were used to determine oLH and oFSH levels in plasma from ewes of two breeds during the oestrous cycle. The assays detected the first FSH surge coincident with the LH surge, the second FSH surge about 24 h later and the periodic fluctuations of FSH concentrations during the luteal phase of the oestrous cycle. These enzyme immunoassays are an efficient and economic alternative to the established radioimmunoassays (RIA) for oLH and oFSH.     

Soluble organic nitrogen in agricultural soils

 The existence of soluble organic forms of N in rain and drainage waters has been known for many years, but these have not been generally regarded as significant pools of N in agricultural soils. We review the size and function of both soluble organic N extracted from soils (SON) and dissolved organic N present in soil solution and drainage waters (DON) in arable agricultural soils. SON is of the same order of magnitude as mineral N and of equal size in many cases; 20–30 kg SON-N ha^–1 is present in a wide range of arable agricultural soils from England. Its dynamics are affected by mineralisation, immobilisation, leaching and plant uptake in the same way as those of mineral N, but its pool size is more constant than that of mineral N. DON can be sampled from soil solution using suction cups and collected in drainage waters. Significant amounts of DON are leached, but this comprises only about one-tenth of the SON extracted from the same soil. Leached DON may take with it nutrients, chelated or complexed metals and pesticides. SON/DON is clearly an important pool in N transformations and plant uptake, but there are still many gaps in our understanding. Received: 10 June 1999     

Interleukin-4 and interferon-gamma gene expression in summer pasture-associated obstructive pulmonary disease affected horses

We hypothesised that horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD) react to an allergen or allergens in their summer environment that is either absent or present at lower levels in their winter environment; and that such allergens stimulate SPAOPD-affected horses to produce a different T helper lymphocyte cytokine profile from that of control horses. The primary objective of this study was to determine the cytokine mRNA profile of T helper lymphocytes obtained from summer pasture-associated obstructive pulmonary disease (SPAOPD) affected horses when 1) the horses were showing signs of disease (summer) and 2) they were in clinical remission (winter). A further objective was to determine the differences between cytokine mRNA T helper lymphocyte profiles of control and affected horses in the summer and winter seasons. Interleukin 4 (IL-4) and interferon-gamma (IFN-gamma) mRNA expression levels were increased in bronchoalveolar lavage fluid (BALF) and peripheral blood mononuclear cell (PBMC) samples of affected horses during disease expression. No significant amounts of IL-5 mRNA were detected in any of the samples. These results suggest that there is an allergic component to SPAOPD of horses and that appropriate manipulation of the immune system could offer hope for treatment and prevention of the disease in the future. Further research studies will be needed to determine the most appropriate treatments to use to alter the antigen-stimulated cytokine profile being expressed by SPAOPD-affected horses or to alter the effects that these cytokines produce.     

Spatial and temporal characteristics of dicarboximide-resistant strains of Monilinia fructicola and brown rot incidence in stone fruit

The spatial and temporal distribution of dicarboximide-resistant strains of Monilinia fructicola were investigated in six peach and nectarine orchard blocks in 1987–89 using a dispersion index (Lloyd's Patchiness Index, LPI), and spatial, temporal and spatio-temporal autocorrelation analyses (Moran's Coefficient, I ). The LPI values indicated that resistant strains were aggregated in all blocks in all years. Spatial correlations were not significant beyond one quadrat for any spatial proximity pattern in five of six blocks. Thus the spread of resistant strains was mostly restricted to the vicinity of the original focus. An absence of significant temporal correlation between years in five of the six blocks indicated poor persistence of resistant strains at specific locations. Only one significant temporal correlation was detected at one block and this could have arisen by chance. Significant spatio-temporal correlation was not detected, suggesting that there was no focus expansion or carry-over of resistant strain inoculum from the previous sampling date. Spatial, temporal and spatio-temporal autocorrelation analyses were consistent with previously reported laboratory results that resistant strains had not acquired all the necessary characteristics to remain in, or dominate, field populations. The spatial pattern of brown rot incidence was investigated at one block in a separate study in 1988 and 1989. There were no significant spatial correlations for brown rot incidence in 1988 for any of the spatial proximity patterns analysed. In 1989, however, significant correlations indicated ellipsoid aggregates of brown rot orientated along the orchard rows.     

The Control of Reactive Oxygen Species Influences Porcine Oocyte In Vitro Maturation

The aim of this study was to examine the effect of varying intracellular reactive oxygen species (ROS) levels during oocyte in vitro maturation with enzymatic ROS production systems (xanthine + xanthine oxidase or xanthine + xanthine oxidase + catalase), scavenger systems (catalase or superoxide dismutase + catalase) or cysteine on porcine oocyte maturation. Oocyte ROS levels showed an increase when H₂O₂ or O₂?^‐ production systems were added to the culture medium (p < 0.05). On the other hand, the presence of ROS scavengers in the maturation medium did not modify oocyte ROS levels compared with the control after 48 h of maturation, but the addition of cysteine induced a decrease in oocyte ROS levels (p < 0.05). The ROS production systems used in this work did not modified the percentage of oocyte nuclear maturation, but increased the decondensation of sperm head (p < 0.05) and decreased the pronuclear formation (p < 0.05). In turn, the addition of O₂?^‐ and H₂O₂ scavenging systems during in vitro maturation did not modify the percentage of oocytes reaching metaphase II nor the oocytes with decondensed sperm head or pronuclei after fertilization. However, both parameters increased in the presence of cysteine (p < 0.05). The exogenous generation of O₂?^‐ and H₂O₂ during oocyte in vitro maturation would not affect nuclear maturation or later sperm penetration, but most of the spermatozoa cannot progress to form the pronuclei after fusion with the oocyte. The decrease in endogenous ROS levels by the addition of cysteine would improve pronuclear formation after sperm penetration.     

Atypical fibrosarcoma in the skin of a Roborovski hamster (Phodopus roborovskii)

A 2.5‐year‐old intact male Roborovski hamster (Phodopus roborovskii) was presented with a large subcutaneous mass overlying the abdomen, affecting the animal's ambulation and access to different compartments of the cage through narrow tubing. Ultrasound examination delineated a well‐circumscribed mass in the subcutis of the caudoventral abdominal region. The mass was surgically excised and on cytologic examination showed, in a background of blood, a small population of individually arranged oval to spindle‐shaped cells that exhibited a moderate degree of anisokaryosis, coarsely stippled chromatin, one or more prominent nucleoli, and lightly basophilic well‐defined cytoplasmic processes. Histologically, the mass was composed of interlacing streams and bundles of pleomorphic spindle cells (ganglion‐like cells) with variable amounts of collagenous stroma. The neoplastic cells exhibited moderate features of malignancy. These cells stained intensely with vimentin, but not with any other markers, including antibodies to cytokeratin AE1/AE3, S100 protein, desmin, smooth muscle actin, synaptophysin, neurofilament, and androgen receptor. Based on histologic features, the mass was diagnosed as an atypical fibrosarcoma. This is the first report of an atypical fibrosarcoma in a Roborovski hamster and one of few reports of atypical fibrosarcoma in domesticated hamsters overall.     

Phosphofructokinase and Malate Dehydrogenase Participate in the In Vitro Maturation of Porcine Oocytes

Oocyte maturation depends on the metabolic activity of cumulus–oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work, the enzymes [phosphofructokinase (PFK) and malate dehydrogenase (MDH)] were tested to elucidate the metabolic profile of porcine COCs during the in vitro maturation (IVM). Enzymatic activity was expressed in U/COC and U/mg protein (specific activity) as mean ± SEM. In vitro maturation was performed with 2‐oxoglutarate (5, 10 and 20 mm ) or hydroxymalonate (30, 60 and 100 mm ) inhibitors of PFK and MDH, respectively. The PFK and MDH activities (U) remained constant during maturation. For PFK, the U were (2.48 ± 0.23) 10^?5 and (2.54 ± 0.32) 10^?5, and for MDH, the U were (4.72 ± 0.42) 10^?5 and (4.38 ± 0.25) 10^?5 for immature and in vitro matured COCs, respectively. The specific activities were significantly lower after IVM, for PFK (4.29 ± 0.48) 10^?3 and (0.94 ± 0.12) 10^?3, and for MDH (9.08 ± 0.93) 10^?3 and (1.89 ± 0.10) 10^?3 for immature and in vitro matured COCs, respectively. In vitro maturation percentages and enzymatic activity diminished with 20 mm 2‐oxoglutarate or 60 mm hydroxymalonate (p < 0.05). Viability was not affected by any concentration of the inhibitors evaluated. The U remained unchanged during IVM; however, the increase in the total protein content per COC provoked a decrease in the specific activity of both enzymes. Phosphofructokinase and MDH necessary for oocyte IVM would be already present in the immature oocyte. The presence of inhibitors of these enzymes impairs the meiotic maturation. Therefore, the participation of these enzymes in the energy metabolism of the porcine oocyte during IVM is confirmed in this study.     

The resistance of meat chickens vaccinated by aerosol with a live V4 Newcastle disease virus vaccine in the field to challenge with a velogenic Newcastle disease virus

Meat chickens housed on a commercial broiler farm in Australia were vaccinated once at 10 to 11 days-of-age by aerosol with live V4 Newcastle disease virus (NDV) vaccine. Groups of vaccinated and unvaccinated birds were flown to Malaysia, where they were challenged with a virulent strain of NDV. Survival rates in vaccinated chickens challenged 7, 14, 21 or 31 d after vaccination were 0.47, 0.77, 0.97 and 0.92, respectively. All unvaccinated chickens died due to Newcastle disease (ND) following challenge. Chickens in Australia and Malaysia were bled and the serums tested for haemagglutination-inhibiting (HI) antibody to NDV. Many vaccinated birds with no detectable antibody, and all birds with a log2 titre of 2 or greater, survived challenge. The results showed that this V4 vaccine induced protective immunity in a significant proportion of chickens within 7 d of mass aerosol vaccination. This early immunity occurred in the absence of detectable circulating HI antibody. Non-HI antibody mediated immunity continued to provide protection up to 31 d after vaccination. Almost all vaccinated birds were protected within 3 w of vaccination. It is concluded that the V4 vaccine is efficacious and could be useful during an outbreak of virulent ND in Australia.