Impact of animal density on cattle nutrition in dry Mediterranean rangelands: a faecal near-IR spectroscopy-aided study.

In the context of determining the sustainable carrying capacity of dry-Mediterranean herbaceous rangelands, we examined the effect of animal density on cattle nutrition, which is fundamental to animal performance and welfare. The effects on dietary components of low (0.56 cows/ha; L) and high (1.11 cows/ha; H) animal densities were monitored for three consecutive years in grazing beef cows. In the dry season (summer and early autumn), cows had free access to N-rich poultry litter (PL) given as a dietary supplement. In each season, near-IR spectroscopy (NIRS) was used to predict the chemical composition of herbage samples (ash, NDF, CP, in vitro dry matter digestibility (IVDMD) and metabolizable energy (ME) content from IVDMD). Near-IR spectroscopy was applied also to faecal samples to determine the chemical composition of the diet selected by the animal, as well as the contents of ash, NDF and CP in the faeces themselves. A faecal-NIRS equation was applied to estimate the dietary proportion of PL. Seasonal categories were green, dry without PL supplementation and dry with it. We found no effects of animal density on nutrition during the green season but effects were apparent when cows consumed dry pasture. Ash content predicted by faecal NIRS was higher in the diet than in plant samples clipped from pasture, which infers that cows ingested soil. Dietary and faecal ash contents were higher (P<0.05) at the H, implying greater soil intake in these animals. During the dry period, dietary contents of ME were higher in L than in H (P<0.05). Poultry litter supplementation was associated with a marked increase (P<0.01) in dietary and faecal CP contents. Poultry litter represented 0.45 and 0.59 of the diet in treatments L and H, respectively (P<0.05). Consequently, treatment H had higher faecal protein (P<0.05). A tendency of higher dietary protein (P=0.08) and lower dietary NDF (P=0.10) in treatment H was probably related to greater PL ingestion. Given that high and sustained rates of poultry litter consumption are detrimental to animal health, the above results cast doubts on the long-term sustainability of the higher of the animal densities tested. Although it may be sustainable vis-à-vis the vegetation, treatment H may have exceeded the boundaries of what is acceptable for cow health. Chemical information revealed with NIRS can be used to evaluate whether animal densities are compatible with animal health and welfare standards and can play a role in determining the carrying capacity of Mediterranean rangelands.

Single-molecule spectroscopy of the beta(2) adrenergic receptor: observation of conformational substates in a membrane protein.

Single-molecule studies of the conformations of the intact beta(2) adrenergic receptor were performed in solution. Photon bursts from the fluorescently tagged adrenergic receptor in a micelle were recorded. A photon-burst algorithm and a Poisson time filter were implemented to characterize single molecules diffusing across the probe volume of a confocal microscope. The effects of molecular diffusion and photon number fluctuations were deconvoluted by assuming that Poisson distributions characterize the molecular occupation and photon numbers. Photon-burst size histograms were constructed, from which the source intensity distributions were extracted. Different conformations of the beta(2) adrenergic receptor cause quenching of the bound fluorophore to different extents and hence produce different photon-burst sizes. An analysis of the photon-burst histograms shows that there are at least two distinct substates for the native adrenergic membrane receptor. This behavior is in contrast to one peak observed for the dye molecule, rhodamine 6G. We test the reliability and robustness of the substate number determination by investigating the application of different binning criteria. Conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes. These studies provide insight into the conformational heterogeneity of G protein-coupled receptors in the presence and absence of a bound agonist.

Nonlinear optical measurement of membrane potential around single molecules at selected cellular sites.

Membrane potential around single molecules has been measured by using the nonlinear optical phenomenon of second harmonic generation. This advance results from the interaction between a highly dipolar molecule with a selectively directed highly polarizable 1-nm gold particle. With this approach, a second harmonic signal, which is enhanced by the nanoparticle, is detected from a volume of nanometric dimensions. This present work clearly shows that functional cellular imaging around single molecules is possible by selectively directing an antibody with a 1-nm gold label to a specific membrane protein. The results of this work open the way for three-dimensional, high resolution functional imaging of membrane electrophysiology in cells and cellular networks.

An optical submicrometer calcium sensor with conductance sensing capability.

The identification of chemical species and the measurement of their concentrations with high (submicrometer) spatial resolution are of considerable importance in cell biology. In this article we report the first successful development of a > or = 0.1-micron Ca2+ sensor based on a pulled micropipet, filled with a conducting porous sol-gel glass which was doped with the fluorescent calcium green 1 Ca2+ indicator. Such sensors are potentially capable of measuring Ca2+ concentrations as low as 10(-8) M, in confined volumes, with a three-dimensional resolution which exceeds approximately 0.1 micron. A major advantage of the sensor is its capability to be integrated into a multifunctional probe which will measure chemical analyte concentrations and ion conductance.

Infrared nonlinear optical measurements of membrane potential in photoreceptor cells.

In the past it has not been possible to measure optically the membrane potential of cells and collections of cells that are either naturally photosensitive or that can be activated by photolyzable caged transmitter molecules. This paper reports on a unique application of nonlinear optics that can monitor the potential of cellular membranes with a near-infrared source. Among many other singular advantages, this nonlinear optical approach to measuring membrane potential does not activate light sensitive cells or cell suspensions and cellular networks surrounded with photolyzable molecules. To demonstrate this capability we show that the technique can be applied to living photoreceptor cells that are very sensitive to visible light. These cells are ideal for characterizing such a new technique, not only because of their unmatched sensitivity to light, but also because their electrical responses have been extensively characterized (Minks and Selinger, 1992).