Isotope-edited nuclear magnetic resonance: novel methodologies for investigating metabolism

While the use of NMR and stable isotopes in metabolism studies is hardly new, it is only recently that isotope-edited NMR spectroscopy has been applied in kinetic studies of glyphosate metabolism of soil microbes. NMR can detect multiple species simultaneously and non-destructively, yielding valuable information on structural identification of metabolites. T riple R esonance I sotope ED ited spectroscopy (TRIED), [²H]NMR, and [²H–¹³C] INEPT (I nsensitive N ucleus E nhancement through P olarization T ransfer) are three isotope-edited techniques which have been used in combination to examine the microbial degradation of glyphosate (N-phosphonomethylglycine). Using ¹³C- and ¹⁵N-labeled glyphosate, TRIED can detect multiple metabolites in crude matrices at submicrogram levels, an improvement over earlier techniques where milligrams were needed. It can detect 500 nanograms of ¹³C–¹⁵N-labeled compound in a crude sample (1 : 1400 mass ratio), only a few hours work being required. [²H]NMR and [²H–¹³C]INEPT were also used as complementary techniques to further examine metabolites whose ¹³C–¹⁵N bond has been cleaved. The three-isotope-edited methods produced results consistent with both radioactivity and HPLC analyses. Accordingly, we are able to detect minute levels of metabolites in the presence of complex mixtures, minimizing the costs and time of sample purification. ©1999 Society of Chemical Industry     

Density‐ and species‐dependent errors in single‐pass timed electrofishing assessment of riverine salmonids

Electrofishing techniques are widely used for the estimation of the size of stream‐dwelling fish populations for both fishery management and scientific study. In contrast to multiple pass, population depletion methods, single‐pass catch‐per‐unit‐effort (CPUE) methods are less time‐consuming and labour‐intensive. A possible issue with the commonly used fixed total time CPUE protocol is that it does not differentiate between the time spent actively fishing and the time incurred while not actively fishing (e.g. removing fish from nets and navigating the site). This issue is likely related to fish density. This study compared two methods of CPUE electrofishing and tested the hypothesis that time spent handling fish and navigating a site can be a source of error in the commonly used fixed total time electrofishing method. Seventy‐one sites were sampled across three subcatchments in the Foyle catchment in Northern Ireland. We found a difference in the catch per unit time between the two methods and that this difference increased with fish density. The fixed‐time CPUE method also failed to detect a species presence in low‐density sites.     

Dissolved organic matter leaching in some contrasting New Zealand pasture soils

Leaching of dissolved organic matter (DOM) from pastoral soils is increasingly seen as an important but poorly understood process. This paper examined the relationship between soil chemical properties, microbial activity and the losses of dissolved organic carbon (DOC) and nitrogen (DON) through leaching from six pasture soils. These soils differed in carbon (C) (4.6–14.9%) and nitrogen (N) (0.4–1.4%) contents and in the amount of organic C and N that had accumulated or been lost in the preceding 20+ years (i.e. −5131 to +1624 kg C ha⁻¹ year⁻¹ and −263 to +220 kg N ha⁻¹ year⁻¹, respectively). The paper also examined whether between‐soil‐type differences in DOC and DON leaching was a major explanatory factor in the observed range of soil organic matter (SOM) changes in these soils. Between 280 and 1690 kg C ha⁻¹ year⁻¹ and 28–117 kg N ha⁻¹ year⁻¹ leached as DOC and DON, respectively, from the six soils in a lysimeter study, with losses being greater from two poorly drained gley soils. Losses of C and N of this magnitude, while at the upper end relative to published data, could not fully explain the losses at Rawerawe, Bruntwood and Lepperton sites reported by Schipper et al. (2007) . The study highlights the leaching of DOM as a significant pathway of loss of C and N in pasture soils that is often ignored or given little attention in predictive models and nutrient budgeting. Leaching losses of DOC and DON alone, or in combination with slightly increased respiration losses of SOM given a 0.2°C increase in the mean annual soil temperature, do not fully explain long‐term changes in the SOM observed at these sites. When soils examined in the present study were separated on the basis of drainage class, the losses of DOC by leaching were correlated with both total and hot‐water extractable C (HWC), the latter being a measure of the labile SOM fraction. Basal microbial CO₂ respiration rates, which varied between 1 and 3.5 µg CO₂‐C g⁻¹ soil hour⁻¹ in surface soils (0–75‐mm depth), was also linked to HWC and the quantities of C lost as DOC. Adoption of the HWC method as an approach that could be used as a proxy for the direct measurement of the soil organic C lost by leaching as DOC or respired needs to be examined further with a greater number of soils. In comparison, a poor relationship was found between the hot‐water extractable N (HWN) and loss of DON by leaching, despite HWN previously being shown to be a measure of the mineralizable pool of N in soils, possibly reflecting the greater competition for N than C in these soils.     

Improvement of Fertility in Artificially Inseminated Ewes Following Vaginal Treatment with Misoprostol Plus Terbutaline Sulphate

The effect of vaginal administration of misoprostol plus terbutaline sulphate 6 h prior to artificial insemination (AI) upon the site of AI (vaginal or cervical) and fertility was studied using a total of 87 estrous synchronized Serra da Estrela ewes (control n = 42 and treated n = 45). Artificial insemination was performed using refrigerated semen at 54–55 h after sponge removal. Lambing rate (fertility) and prolificacy were compared between control and treated ewes. The effect of the site of semen deposition on fertility was also evaluated. Prolificacy rate was not different between control (1.5) and treated (1.59) ewes. The proportion of cervical AI achieved in control (45.2%) and treated (37.8%) ewes was not significantly different. Overall, fertility was significantly lower in control than in treated ewes (42.9% vs 64.4%; p < 0.04). Fertility following vaginal AI was significantly lower for control for than treated ewes (30.4% vs 60.7%; p < 0.03) but the difference was smaller and not significant for cervical AI (control 57.9% vs 70.6%). It was concluded that vaginal administration of misoprostol plus terbutaline sulphate 6 h prior to artificial insemination did not affect the proportion of cervical inseminations but significantly improved the fertility of treated ewes. Although needing confirmation, it was hypothesized that drugs might have induced local secretory modifications leading to an increase of cervical ability to retain more viable spermatozoa for fertilization.     

Effect of storage regimes on pectolytic enzymes,pectic substances,internal conductivity and gel breakdown in cold stored ‘Songold’ plums

Summary

Plums (Prunus salicina cv. Songold) were cold stored according to a single-temperature regime comprising 28 d at ?0.5°C, and a dual-temperature regime comprising 10 d at ?0.5°C followed by 18 d at 7.2°C. After cold storage, the plums were ripened for 8 d at 10°C. Pectolytic enzyme activity, pectic composition, internal conductivity and gel breakdown were determined at seven stages during storage and ripening. Although not exposed to chilling temperatures prior to harvest, approximately 10% of the plums exhibited gel breakdown at harvest, indicating that the disorder cannot be classified solely as a cold-storage chilling disorder. The higher temperatures of the dual-temperature regime resulted in higher polygalacturonase activity than with the single-temperature fruit. Consequently, protopectin degradation and the concomitant production of water- soluble pectins were greater in the dual-temperature fruit. Single-temperature storage resulted in higher pectinmethylesterase activity during the latter stages of storage and during ripening. Increases in temperature after 10 d and 28 d in dual- and single- temperature fruit, respectively, were associated with significant increases in the viscosity of water-soluble pectins, internal conductivity and gel breakdown. The significant positive correlation between internal conductivity and gel breakdown suggested that membrane integrity is closely associated with development of gel breakdown.     

Effect of the Medium Replacement Interval on the Viability,Growth and In Vitro Maturation of Isolated Caprine and Ovine Pre‐Antral Follicles

The aim of the present study was to evaluate the effects of different medium replacement intervals on the viability, antral cavity formation, growth and in vitro maturation (IVM) of oocytes from caprine and ovine pre‐antral follicles. Pre‐antral ovarian follicles (≥150 μm) were isolated from the ovarian cortex of goats and sheep and were individually cultured for 24 days using two different medium replacement intervals [2 days (T₁) or 6 days (T₂)]. Follicle development was evaluated on the basis of antral cavity formation, increases in follicular diameter and the presence of healthy cumulus oocyte complexes and fully grown oocytes. For caprine species, results showed a higher percentage (p < 0.05) of viable follicles in T₁ than T₂ from day 6 until the end of the culture. In addition, when comparing both treatments after the same culture duration, the rate of antrum formation was significantly higher in T₁ than in T₂ from day 12 onwards. Yet, in ovines, when both treatments were compared on day 24 of the culture, there were more viable follicles in T₂ than in T₁ (p < 0.05). In the caprine species, percentages of fully grown oocytes (≥110 μm) acceptable for IVM after 24 days of culture were significantly higher in normal follicles cultured in T₁ (30.0%) than in T₂ (6.7%; p < 0.05). On the other hand, in ovines, at the end of the culture, the percentage of oocytes destined for IVM was higher in T₂ than in T₁ (23.5% vs 2.9%; p < 0.05). In conclusion, under the same conditions, the frequency of medium replacement significantly affected the in vitro development of caprine and ovine pre‐antral follicles. To improve the efficiency of the culture system, the medium must be replaced every 2 and 6 days for goat and sheep pre‐antral follicles, respectively.