Kinetics of spontaneous and induced acrosomal loss in human sperm incubated under capacitating and noncapacitating conditions

The kinetics of spontaneous and induced acrosomal loss have been studied in human sperm incubated in capacitating and noncapacitating media. Acrosomal status was quantitated using indirect immunofluorescence with a monoclonal antibody. The response of sperm to induction by calcium ionophores was time dependent reaching a maximum after 6 hours of incubation under capacitating conditions. The inducible population slowly decreased in size through the balance of a 24-hour incubation. The time-dependent development of ionophore responsiveness by sperm exposed to capacitating conditions corroborates the idea that only capacitated cells can respond to undergo acrosomal loss in response to ionophore. In contrast, only a small, constant percentage of sperm incubated under noncapacitating conditions responded to ionophore. Substitution experiments involving the addition or deletion of human serum albumin suggest that albumin is not absolutely required for capacitation but is essential for the maintenance of motility. Polyvinyl alcohol can be substituted for serum albumin, but it does not support capacitation or motility as well as HSA. These studies may provide a basis for optimizing capacitating conditions for human sperm in vitro as well as for diagnosing fertility or fertility potential based on measurements of spontaneous and ionophore induced acrosomal loss under defined culture conditions.     

Effect of ambient temperature on losses of volatile nitrogen compounds from stored laying hen manure

Eight one tonne lots of laying hen manure were stored in individual, environmentally controlled chambers for 18-weeks at one of four constant temperatures: 12, 15, 20 and 25 degrees C. The losses of volatile nitrogen compounds, ammonia, and changes in dry matter and pH were measured during the storage period. There was a linear (P < 0.001) loss of nitrogen from the manure over the 18-week storage period. This loss represented approximately 60% of the initial nitrogen present in the manure. The rate of nitrogen loss increased non-linearly (P < 0.05) with increasing storage temperature. Poultry housing systems that provide low temperature storage of manure are recommended to reduce the volatile nitrogen losses from egg-laying enterprises.     

Nitrogen storage and remobilization in ash (Fraxinus excelsior) under field and laboratory conditions

 Storage and remobilization of nitrogen (N) were studied in ash trees (Fraxinus excelsior) under both field and greenhouse conditions. Experiments in the greenhouse providing ¹⁵N labelled fertilizer to the trees showed that the major quantity of N remobilized during subsequent spring was from the roots, and only a small amount from the stem. This corresponded with a loss of soluble N (proteins and low-molecular-weight compounds) from both roots and stem. On the two field sites, which differed in water availability, there was a decrease in bark N content during leaf growth, but on the dry site net N export from the bark was sustained throughout the whole vegetation period. Remobilized N was derived from soluble proteins and low-molecular-weight compounds on the moist site, which was demonstrated by the seasonal dynamics of a 56 kDa polypeptide in bark and wood. On the dry site, lower contents of soluble proteins were associated with smaller amounts of N remobilized compared to the moist site. Uptake studies of ¹⁵N labelled fertilizer indicated a higher contribution of current uptake to leaf N increment during spring at the dry site compared to the moist site. Differential N availability during the season had a decisive effect on the nitrogen storage dynamics at the two sites. Thus the influence of current N supply on N remobilization and storage as found in the greenhouse-grown plants could be verified under field conditions. Received: 28 July 1995 / Accepted: 17 July 1996     

Effect of rice plants on nitrification-denitrification loss of nitrogen under greenhouse conditions

Summary Rice is unique among cereal crops in its ability to tolerate the anaerobic environment of waterlogged soils, but little is known about the influence of these plants on nitrogen loss by nitrification-denitrification. This problem was approached by loss of urea-N in cores with and without rice plants, using the acetylene inhibition method. Considerably greater denitrification was observed for surface-applied urea as compared to subsurface application in all cases. Regardless of the application point, however, the planted system yielded greater N₂O+N₂ accumulation in the first two days than from the nonplanted soil. After 4–6 days from fertilization no difference was observed in denitrification loss between planted and nonplanted systems. Inorganic NH ₄ ⁺ levels were observed to decrease rapidly in planted soils. Initial enhancement of gaseous N accumulation may occur because of the oxidized rice-root rhizosphere, however the appreciable denitrification in non-planted soil suggests that other N loss mechanisms are more important than the losses occurring in the root rhizosphere.     

The role of cattle in the volatile loss of nitrogen from a shortgrass steppe

The cycling and volatile loss of N derived from cattle urine at upland and lowland sites within the shortgrass steppe of eastern Colorado was studied, using¹⁵N-labelled urea as an N source. Losses of NH ₀ ³ were determined by direct measurement and by difference. Losses were higher from coarse (27% summer, 12% winter) than from fine textured (0–2%) soils. Immobilization and plant uptake of N accounted for significant amounts of added N. Extrapolating our plot measurements to a typical pasture, using spatially and temporally stratified urine deposition data, losses from upland sites were calculated to be 0.016 g N · m^-2 · y^-1, while losses from lowland sites were negligible. This resulted in an average loss of 0.011 g N · m^-2 · y^-1 for a pasture divided 70:30 between uplands and lowlands. The loss of urine N calculated assuming no spatial stratification would be sevenfold higher (0.076 g N · m^-2 · y^-1). Losses of NH ₀ ³ from urine, animal biomass removal, and NH₂O loss totaled only 0.07 g N · m^-2 · y^-1 , or about 25% of wet deposition input. We calculated a potential loss of NH ₀ ³ from senescing vegetation of 0.26 g N · m^-2 · y^-1, an order of magnitude larger than all other losses combined.     

Growth of phototrophic biofilms from limestone monuments under laboratory conditions

In the current study, five phototrophic biofilms from different Southern Europe limestone monuments were characterised by molecular techniques and cultivated under laboratory conditions. Phototrophic biofilms were collected from Orologio Tower in Martano (Italy), Santa Clara-a-Velha Monastery and Ajuda National Palace, both in Portugal, and Seville and Granada Cathedrals from Spain. The biofilms were grown under laboratory conditions and periodically sampled in order to monitor their evolution over a three-month period. Prokaryotic communities from natural samples and cultivated biofilms were monitored using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments in conjunction with clone sequencing and phylogenetic analysis. DNA-based molecular analysis of 16S rRNA gene fragments from the natural green biofilms revealed complex and different communities composition with respect to phototrophic microorganisms. The biofilms from Orologio Tower (Martano, Italy) and Santa Clara-a-Velha Monastery (Coimbra, Portugal) were dominated by the microalga Chlorella. The cyanobacterium Chroococcidiopsis was the dominating genus from Ajuda National Palace biofilm (Lisbon, Portugal). The biofilms from Seville and Granada Cathedrals (Spain) were both dominated by the cyanobacterium Pleurocapsa. The DGGE analysis of the cultivated biofilms showed that the communities developed differently in terms of species establishment and community composition during the three-month incubation period. The biofilm culture from Coimbra (Portugal) showed a remarkable stability of the microbial components of the natural community in laboratory conditions. With this work, a multiple-species community assemblage was obtained for further stone colonisation experiments.     

Use of compartmental analysis to study ruminal volatile fatty acid metabolism under steady state conditions in Holstein heifers

Four rumen-cannulated Holstein heifers were used to quantify volatile fatty acid (VFA) kinetics in the rumen using ¹³C stable isotopes. Heifers were fed either a low (L) or high (H) concentrate diet with level of concentrate of 250 or 700 g/kg dry matter, respectively. Isotopic tracers, Na-1-¹³C-acetate (Ac), -propionate (Pr), or -butyrate (Bu) were infused as a bolus into the rumen. Three days prior to sampling, cows were fed equal meals every 4 h for 2 days followed by 2 h feeding intervals 1 day prior to and on the day of sampling. The rumen was sampled 26 times (3 times prior to tracer infusion; background samples and 23 times during 8 h after tracer infusion). Isotopic enrichment was expressed as Tracer/(Tracee + Tracer) or F value. F values were corrected for skewness and graphed against time. A three-compartment model was adequate to describe VFA kinetics in the rumen. It was estimated that there was a flux of Ac to the Pr pool [0.05 g/min (L) versus 0.03 g/min (H)], resulting in an average conversion fraction of Ac to Pr of 0.051 in both groups. The model predicted that the fraction of Ac which contributed to the Pr pool was 0.198 (L) versus 0.145 (H). Although the fraction of the Ac conversion to Bu ranged from 0.367 (L) to 0.448 (H), only a small fraction [0.0137 (L) and 0.0160 (H)] of the Bu was converted to Ac. A major portion of the Bu was coming from Ac in both diet groups. The portion of Bu coming from Ac in the rumen varied between 0.95 and 0.65 in (L) and (H) diets, respectively. Turnover time of all VFA was calculated to be longer in H. Results indicate that Ac and Bu exchange, and that Ac contributes to the Pr pool. However, Pr does not contribute to the Ac or Bu pools. This study demonstrates the usefulness of ¹³C isotopes to study VFA kinetics and metabolism in cattle.     

Use of radiolabeled tracers in dilution grazing experiments to estimate bacterial growth and loss rates

Dilution grazing experiments were conducted to determine growth and loss rates of glucose-metabolizing and total bacteria. Bacterial growth rates were low and losses to grazers negligible in samples collected from the Celtic Sea in June 1986. Growth and loss rates of glucose-metabolizing bacteria were higher than growth and loss rates of total bacteria in a sample collected from the North Sea in October 1986.     

The relationship between the specific absorption rate and extremely low ambient nitrate concentrations under steady-state conditions

Some water-culture experiments were carried out to measure the ambient nitrate concentration under conditions of exponential addition of nitrate. Juvenile plants ofPlantago lanceolata were therefore grown on an agitated nutrient solution. Nitrate was continually added to the culture vessels during the experimental period. The amount added per plant increased exponentially with time, in accordance with the formula: $$NO_3 - N = N_0 .(e^{(RAR.t)} - 1)$$ (N₀=initial content of N in the seedling, RAR=relative addition rate, t=time in days). There were three treatments corresponding to RAR's of 0.20, 0.15 and 0.10 d^?1. All of them were suboptimal forP. lanceolata. Nitrate concentrations of the culture solution were measured weekly. In all cases plants were in steady state after two weeks. The relative growth rate was about equal to the RAR within treatments. The root weight ratio was inversely proportional to the RAR, and the internal N concentration proportional. Ambient nitrate concentrations were relatively high during the first two weeks. Ambient concentrations in the second half of the culture periods ranged from 0.35 to 1.8 μM. Linear and multiple regressions of the external nitrate concentration on the specific absorption rate (SAR), and on SAR and some other variables, respectively, were carried out. Most of its variation could be explained by the SAR (r²=0.9827); root size had a minor effect. The experimental results lead to the conclusion that ambient concentrations in culture experiments with exponential nutrient-addition are a result of the treatment. They also indicate which external concentrations may be expected to create strongly suppressed growth. Furthermore, the experiments suggest that the lower limit for nitrate absorption is a zero concentration.     

A comparison of specific growth rates of periphytic diatoms of varying cell size under laboratory and field conditions

Diatom species grown under non-limiting nutrient availability in multispecific biofilms were sampled from glass substrates immersed in the field and in experimental freshwater microcosms, and their growth responses were determined. The major species that developed on the substrates were common to both experiments and the specific growth rates (k) ranged from 0.06 to 0.41 division day⁻¹. An inverse relationship between k and cell sizes was observed, which is in accordance with allometry results reported by several authors. Although found in lower amounts, the large, slow-growing species accounted significantly in total community biovolume, underlying their significance for ecological purposes. From growth characteristics data, life history strategies were drawn for the dominant species recorded, from small pioneer species to large taxa that are more favoured by high resource supply. Kinetic data measured in both laboratory and in situ experiments stressed that the difficulties to mimic the field in laboratory experiments may have a strong impact on growth kinetics. Handling editor: J. Padisak     

Microbial characteristics of sandy soils exposed to diazinon under laboratory conditions

Changes in microbiological characteristics in response to diazinon, applied at three different dosages of 7, 35 and 700 mg kg^?1 soil, were studied in pots filled with sandy soils of different texture. The insecticide dosages corresponded to the maximum predicted environmental concentration (PEC) in field conditions and five or hundred times this rate, respectively. To ascertain these changes, activities of selected soil enzymes, numbers of some microbial populations and bacterial community structure (r/K-strategists) were determined. Acid phosphatase activity and concentrations of ammonium ions either increased or were unaffected by the diazinon dosages. In contrast, nitrate ions, alkaline phosphatase, urease and especially dehydrogenase activities decreased in both soils treated with the higher dosages of the insecticide. The diazinon treatments increased the numbers of culturable bacteria and fungi; however, N-fixing bacteria and nitrifiers (but not denitrifiers) were decreased in both soils. When the high concentration of diazinon (100-times PEC) was added to the loamy sand soil (LS), simulating an undiluted chemical spill, the bacterial community structure shifted towards domination of K-strategists over time. However, r-strategists dominated in the community exposed to the same dosage of the insecticide in the sandy loam soil (SL). Generally, SL soil characteristics were more suitable for microbial activity and growth than those of LS soil were. The results indicate that except a situation of undiluted spill diazinon seems to be a non-hazardous chemical for the culturable soil microorganisms when applied at recommended concentrations.     

Maintenance of previously uncultured freshwater archaea from anoxic waters under laboratory conditions

Culture conditions for the maintenance of previously uncultured members of the Archaea thriving in anoxic water layers of stratified freshwater lakes are described. The proposed enrichment conditions, based on the use of defined medium composition and the maintenance of anoxia, have been proven effective for the maintenance of the archaeal community with virtually no changes over time for periods up to 6 months as revealed by a PCR-DGGE analysis. Phylotypes belonging to groups poorly represented in culture collections such as the Deep-Sea Hydrothermal Vent Euryarchaeota (DHVE) and the Miscellaneous Crenarchaeotic Group (MCG) were maintained and selectively enriched when compared to the correspondent indigenous planktonic archaeal community.     

Composition and diversity of microbial communities recovered from surrogate minerals incubated in an acidic uranium-contaminated aquifer

Our understanding of subsurface microbiology is hindered by the inaccessibility of this environment, particularly when the hydrogeologic medium is contaminated with toxic substances. In this study, surrogate geological media contained in a porous receptacle were incubated in a well within the saturated zone of a pristine region of an aquifer to capture populations from the extant communities. After an 8-week incubation, the media were recovered, and the microbial community that developed on each medium was compared to the community recovered from groundwater and native sediments from the same region of the aquifer, using 16S DNA coding for rRNA (rDNA)-based terminal restriction fragment length polymorphism (T-RFLP). The groundwater and sediment communities were highly distinct from one another, and the communities that developed on the various media were more similar to groundwater communities than to sediment communities. 16S rDNA clone libraries of communities that developed on particles of a specular hematite medium incubated in the same well as the media used for T-RFLP analysis were compared with those obtained from an acidic, uranium-contaminated region of the same aquifer. The hematite-associated community formed in the pristine area was highly diverse at the species level, with 25 distinct phylotypes identified, the majority of which (73%) were affiliated with the beta-Proteobacteria. Similarly, the hematite-associated community formed in the contaminated area was populated in large part by beta-Proteobacteria (62%); however, only 13 distinct phylotypes were apparent. The three numerically dominant clones from the hematite-associated community from the contaminated site were affiliated with metal- and radionuclide-tolerant or acidophilic taxa, consistent with the environmental conditions. Only two populations were common to both sites.     

Cytochrome c oxidase activity in mitochondria of cardiomyocytes from isolated cardiac tissue incubated under long-term hypoxic conditions

Electron microscopic histochemistry based upon the oxidative polymerization of 3,3′-diaminobenzidine (DAB) was applied to identify cytochrome c oxidase activity. We found that the incubation of isolated small pieces of cardiac tissue over 72 h under hypoxic conditions caused changes in the mitochondrial ultrastructure and disorders in the functional activity of mitochondria, particularly in the IV complex of respirator chain. Small, electron-dense mitochondria appeared inside electron-light mitochondria (“mitochondria inside mitochondria”) stained positively for cytochrome c oxidase activity along the full length of crista. The results are discussed in connection with the concept of intracellular regeneration and mitochondria structural transformations during mitoptosis.     

Estimation of nitrogen fixation and transfer from alfalfa to associated grasses in mixed swards under field conditions

Fixation and transfer of nitrogen (N) from alfalfa (Medicago sativa L.) to different grass species including timothy (Phleum pratense L.) and bromegrass (Bromus inermis Leyss) were studied under field conditions, using the¹⁵N dilution technique.The percentage of alfalfa N derived from fixation (%NF) increased throughout the growing seasons and ranged from 62 to 83%. Nitrogen transfer (NT) from alfalfa to associated grasses was evident and contributed 26,46 and 38% of the total annual N yield of associated grasses or represented absolute amounts of 5, 20 and 19 kg N ha^–1 during the first, second and third year, respectively. The gradual and consistent percentage of NT that occurred before first harvest indicated that this transfer is a result of a direct excretion of N compounds from alfalfa root systems. Decomposition of root and nodule debris seems to contribute to the NT from alfalfa to associated grasses in the later cuts. All grass species benefitted similarly from alfalfa, although earlier maturing species with greater competitive ability were slightly more responsive.Contribution No. 1159 from the Plant Research Centre     

Lack of glyphosate resistance gene transfer from Roundup Ready soybean to Bradyrhizobium japonicum under field and laboratory conditions

A field study was conducted at the Russell E. Larson Agricultural Research Center to determine the effect of transgenic glyphosate-resistant soybean in combination with herbicide (Roundup) application on its endosymbiont Bradyrhizobium japonicum. DNA of bacteroids from isolated nodules was analysed for the presence of the transgenic 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS) DNA sequence using polymerase chain reaction (PCR). To further assess the likelihood that the EPSPS gene may be transferred from the Roundup Ready (RR) soybean to B. japonicum, we have examined the natural transformation efficiency of B. japonicum strain 110spc4. Analyses of nodules showed the presence of the transgenic EPSPS DNA sequence. In bacteroids that were isolated from nodules of transgenic soybean plants and then cultivated in the presence of glyphosate this sequence could not be detected. This indicates that no stable horizontal gene transfer (HGT) of the EPSPS gene had occurred under field conditions. Under laboratory conditions, no natural transformation was detected in B. japonicum strain 110spc4 in the presence of various amounts of recombinant plasmid DNA. Our results indicate that no natural competence state exists in B. japonicum 110spc4. Results from field and laboratory studies indicate the lack of functional transfer of the CP4-EPSPS gene from glyphosate-tolerant soybean treated with glyphosate to root-associated B. japonicum.     

Barley response to nitrogen and non-nutritional benefits of legume green manure

Green manure application may benefit subsequent crops not only by improving nitrogen (N) fertility but also via non-nutritional mechanisms. The quantification of the latter effect, however, is complicated by the confounding effect of N fertility. Two experiments were conducted in controlled environments to partition the yield response of barley to green manure between N and non-nutritional effects. Each experiment included a factorial of fertilizer N application rates and green manure application rates. The fertilizer was labelled with ¹⁵N to facilitate discrimination between N sources. Approximately 24% of the N applied in green manure was assimilated by barley after 45 days (Experiment 1) and 32% was recovered by barley grown to maturity (Experiment 2). Apparent recovery of green manure-N by barley was not appreciably affected by fertilizer application. Regression analysis of the relationship between dry matter yield and plant N uptake demonstrated that yield responses to green manure application were not entirely attributable to improved N fertility. For a given amount of N assimilated by the crop, yields were higher in green manure-amended treatments than in those receiving no green manure. In barley grown to maturity, barley response to N and non-nutritional effects were estimated to be 5.3 and 2.2g pot⁻¹, respectively. The relationship between dry matter yield and N uptake is suggested as a method for distinguishing nutritional and non-nutritional yield responses. This approach assumes that no other nutrient is limiting growth. The presence of non-nutritional benefits observed in this study demonstrates that the agronomic value of green manure is not limited to N release and casts doubt on the assumptions inherent to calculation of fertilizer equivalents. Contribution 3879132 Contribution 3879132