The Beltsville sperm sexing technology: high-speed sperm sorting gives improved sperm output for in vitro fertilization and AI

The Beltsville sperm sexing technology is currently the only effective means of altering the sex ratio of offspring in livestock. The method is based on the flow-cytometric separation of X- and Y-chromosome-bearing sperm based on X/Y DNA content difference. It is an effective means of producing progeny of predetermined sex in cattle, swine, sheep, and laboratory animals. The method involves treating sperm with a DNA-binding fluorochrome, Hoechst 33342, and flow-cytometrically sorting them into separate X and Y populations that can subsequently be used for surgical intratubal or intrauterine insemination, deep-uterine insemination, regular artificial insemination in some cases, in vitro fertilization to produce sexed embryos for transfer, and intracytoplasmic sperm injection of ova. Skewed sex ratios of 85 to 95% of one sex or the other have been repeatably achieved in most species. The method has been used worldwide to produce several hundred morphologically normal animal offspring of the predicted sex. It has also been validated in the laboratory using DNA reanalysis of the sorted sperm populations and by fluorescence in situ hybridization and PCR of individual sperm. We developed a new orienting nozzle that we have fitted to both conventional and high-speed cell sorters that have been modified for sperm sorting. Recently we completed the adaptation of the new orienting nozzle to a Cytomation MoFlo high-speed cell sorter modified for sperm. This adaptation of the nozzle has increased the overall production rate of sorted X and Y sperm from about .35 million/h to 5 or 6 million sperm/h (each population). Calves have been born from cows artificially inseminated using conventional technique and sexed sperm. In addition, numerous litters of pigs have been born after transfer of embryos produced from X or Y sorted sperm.     

Structure and expression of ovine complement receptor type 2

The structure of sheep complement receptor type 2 (CR2) was characterised by cDNA cloning, protein sequencing and immunoprecipitation. The primary structure of sheep CR2 is similar to known mammalian homologues but the higher-order structure is unusual. Two distinct CR2 isoforms occur, one of which is ubiquitinated in the cytoplasmic domain, and the two molecular forms are expressed at the cell surface as non-covalently associated dimers. The percentage of sheep B-cells that express CR2 changes during development and varies between different body compartments. CR2+ and CR2 B-cell subsets also differ in the expression of other surface markers and in functional properties. Differential expression of CR2 may, therefore, delineate B-cells that arose by alternative developmental pathways, or it could be a marker for B-cells at different phases of antigen exposure.     

Branch characteristics of widely spaced Douglas-fir in south-western Germany: Comparisons of modelling approaches and geographic regions

Models of Douglas-fir branch and whorl characteristics were developed from contrasting spacing experiments in southwest Germany. The dataset was based on 100 young (20–30 years old), unpruned and partially pruned trees from a 100, 200, and 1200 stems ha⁻¹ spacing experiment on Douglas-fir that was replicated 3 times across the region. The material was used to predict (1) the number of branches whorl⁻¹, (2) branch angle, (3) status (living/dead) of the branches within the living crown, (4) maximum branch diameter whorl⁻¹, and (5) relative diameter of branches within a whorl. For each of these models (except branch status), both a linear and nonlinear, generalised hierarchical mixed effects equation was developed. The comparison of the linear and nonlinear approaches showed that both had a relatively similar level of bias, but the nonlinear equations generally performed better (reduction in mean absolute error of 1.1–69.5%). Overall, individual branch and tree properties were sufficient to give logical and precise predictions of the branch characteristics for the models across the range of sampled stand densities. In addition, the models showed a similar behaviour compared to models on Douglas-fir crown structure from the Pacific Northwest, USA. This suggests that the allometric relationship between tree size and branch characteristics for a given species may be relatively consistent across regions, even ones with highly contrasting growing conditions like in this study. The models performed well across a range of stand conditions and now will be further integrated into an individual tree growth and yield simulations system.     

Site-occupancy of bats in relation to forested corridors

Although use of corridors by some wildlife species has been extensively examined, use by bats is poorly understood. From 1 June to 31 August (2004–2005), we used Anabat II detectors to examine bat activity and species occupancy relative to forested corridors on an intensively managed forest landscape in southern South Carolina, USA. We compared bat activity among corridor interiors, corridor edges, and stands adjacent to corridors. We also compared models relating occupancy of bat species to site-level characteristics using an information theoretic approach. We identified 16,235 call sequences of 8 species and detected bat presence at 89% (n = 320) of sites sampled. Our results indicate higher occupancy rates for bats along corridor edges compared to interior corridor or adjacent stands. Although we found few differences among species with respect to site-level characteristics, occupancy of all bat species was positively associated with corridor overstory height and negatively associated with adjacent stand age. The presence of roads adjacent to corridors positively influenced occupancy of Eptesicus fuscus, Lasiurus seminolus, and Perimyotis subflavus. Our results suggest management practices designed to create and enhance corridors may represent an ecologically sound method for maintaining important bat habitat features (i.e., edge) across managed forest landscapes.     

Progress in Mapping and Cloning Qualitative and Quantitative Resistance Against <Emphasis Type="Italic">Phytophthora infestans</Emphasis> in Potato and Its Wild Relatives

Cultivated potato is susceptible to many pests and pathogens, none of which is more of a threat to potato agriculture than the late blight disease, caused by the oomycete Phytophthora infestans (Mont.) de Bary. To date all efforts to thwart this most adaptive of pathogens have failed, and early attempts to deploy ‘R genes’ introgressed from the wild Mexican hexaploid Solanum demissum ended in abject failure. With the advent of facile gene mapping and cloning, allied to knowledge of plant resistance gene structure, renewed efforts are leading to mapping and isolation of new sources of late blight resistance in potato wild species, many of which are being performed under the auspices of the BIOEXPLOIT project (Sub-project 2). We document recent advances in late blight resistance gene mapping and isolation, and postulate how these genes, allied to knowledge of pathogen effectors and their recognition specificity, may greatly enhance our chances of halting the progress of late blight disease in potato crops worldwide.     

Does growing on a slope affect tree xylem structure and water relations?

Variations in slope, exposure, relief and substrate over a short distance and their influences on plant function are poorly understood. We investigated the influences of soil hydrological characteristics on internal stem structure and hydraulic properties of downy oak (Quercus pubescens Willd.) growing along a hill slope. Increment wood cores were extracted from the base and at breast height (BH) of tree stems. Relative wood water content (W(c)) and wood density (D(w)) were measured in the sapwood. Wood compression strength (delta) in the longitudinal direction was measured with a fractometer. Thin sections were cut from the transversal face of each core and vessel lumen area (V(A)) was measured and xylem theoretical hydraulic conductivity (L(th)) estimated over the sapwood. Topsoil volumetric water content (theta(v)) was determined around trees and the hydrological behavior of the slope was studied through field surveys. Data were used as input to a hydrological model to simulate topsoil water distribution along the slope. Results showed that theta(v) tends to decrease with increasing altitude. Groundwater levels were lower upslope than downslope, and results from the hydrological model confirmed these trends. Mean W(c) at the base of each tree decreased significantly with increasing altitude, whereas at BH, no differences were found along the slope. There was a significant positive relationship between W(c) measured at the tree base and theta(v) along the hill slope, but not for W(c) measured at BH. Values of D(w) and delta measured at both stem positions increased significantly with increasing altitude and decreasing theta(v). Significant negative relationships were found between delta and theta(v) measured at the stem base and at BH. At both stem positions, delta was closely related to D(w) and L(th). Vessel lumen areas at BH and the stem base were significantly regressed with altitude, theta(v), D(w) and delta. Xylem theoretical hydraulic conductivity at both stem positions was negatively related to altitude and soil theta(v), but only L(th) measured at the stem base was negatively regressed with D(w). The results are discussed in the context of how tree position along a hill slope influences water uptake and internal xylem structure.     

Development of a PCR-based selection assay for root-knot nematode resistance ( Rmc1) by a comparative analysis of the Solanum bulbocastanum and S. tuberosum genome

A PCR-based assay has been developed for marker assisted selection of root-knot nematode resistance ( Rmc1) in potato. To this end, a comparative genome analysis was carried out between Solanum bulbocastanum and S. tuberosum to identify PCR-based chromosome 11 alleles linked to Rmc1. The use of co-migrating AFLP markers, obtained by using primer combinations previously applied for AFLP analysis of the S. tuberosum genome, failed to align the AFLP map of the S. bulbocastanum genome with the S. tuberosum map. Apparently, the S. bulbocastanum genome is genetically too distantly related to the S. tuberosum genome for this type of analysis. Cleaved amplified polymorphic sequence (CAPS) markers were more readily applied for a comparative analysis within the region of interest. Rmc1 could be localized within a 4 cM interval between markers CT182 and M39b. It is demonstrated that the resistance spectrum of Rmc1 includes not only Meloidogyne chitwoodi and the related species M. fallax but also a genetically distinct population of M. hapla. The cost-efficiency of the CAPS markers applied for Rmc1 renders this approach as an attractive alternative for screening large segregating populations of potato for root-knot nematode resistance. This revised version was published online in July 2006 with corrections to the Cover Date.     

Peripheral and intracerebral T cell immune response in cats naturally infected with Borna disease virus.

Borna disease virus (BDV) is a neurotropic agent with capacity to cause encephalomyelitis in a wide range of animal species, including horses and cats. Recent studies also point to a link between BDV and human neuropsychiatric disorders. The pathogenesis of Borna disease (BD) has been proposed to be immune-mediated, mainly through the effects of cytotoxic T cells. We used flow cytometric analysis in order to characterize the peripheral and intracerebral T cell immune response in cats naturally infected with BDV. Our results show the presence of two different CD8+ cell populations (CD8+low and CD8+high) in the blood, spleen and brain of these cats. In the brain, CD8+low cells predominated over CD8+high cells. Since CD8+low cells have been suggested to represent a non-MHC-restricted T cell population, the recruitment of such cells to the brains of BDV-infected cats could possibly be of importance for the clearance of virus from neurones.     

Red operculum spots,body size,maturation and evidence for a satellite male phenotype in non‐native European populations of pumpkinseed Lepomis gibbosus

Carotenoid‐based pigmentation is a striking feature of many taxa, yet the function, if any, of colour traits is often unclear. Pumpkinseed Lepomis gibbosus, a widely introduced freshwater sunfish that exhibits alternative male mating strategies, express a striking, red operculum spot. To investigate the potential function of this red spot as a signal in this species' mating system, we determined the presence and measured the size of red operculum spots in fish collected from 12 populations in five European countries in which pumpkinseed is an established non‐native species. We subsequently related the presence and size of the red spot to body size and mating strategy, based on an analysis of relative gonad size (gonado‐somatic index, GSI), using a mixed modelling approach. The study demonstrated that the presence of a red operculum spot in pumpkinseed is associated with sexual maturation, with GSI frequency distributions suggesting that cuckolders in some non‐native populations comprised both sneaker and satellite males, the latter not having previously been reported for this species. Further, the size of red spot correlated strongly with body size in parental and cuckolder males, although there was no difference in the presence or size of the red operculum spot between male mating strategies. The function of a red operculum spot in females is not clear but may be partly mediated by pleiotropic genetic mechanisms. Red operculum spots appear to function as signals of male maturation and body size in pumpkinseed, irrespective of mating strategy.     

Agronomic and quality effects in winter wheat of a gene conditioning resistance to wheat streak mosaic virus

Wheat streak mosaic virus (WSMV) is one of the most important diseases limiting winter wheat (Triticum aestivum L.) production in the western Great Plains of North America. There is no known effective WSMV resistance within the primary gene pool of wheat. However, a resistance gene (Wsm1) has been transferred to wheat from a perennial relative, intermediate wheat-grass [Thinopyrum intermedium (Host) Barkworth & DR Dewey]. Nebraska-adapted winter wheat lines carrying Wsm1 were used to characterize the effects of this alien introgression on agronomic and quality traits. Sister-lines from six breeding populations were evaluated under virus-free conditions, and under a naturally occurring viral infection. In uninfected locations, no significant difference for grain yield was detected between resistant (R) and susceptible (S) lines, when averaged over populations, but resistant lines had significantly higher test weights. Within populations, significantly higher grain yield was observed only in population 1, while significantly higher test weights occurred in populations 1, 2, 5 and 6. At the infected location, resistant lines were significantly higher in yield in five of six populations. In two of six populations, susceptible lines were significantly higher in bread loaf volume and bake mix time, while in the remaining populations, no significant quality differences were observed. As the Wsm1 gene provided yield advantages under viral infection, and there was no yield detriment in the absence of the virus, its deployment in hard winter wheat cultivars merits consideration.Joint contribution of the United States Department of Agriculture, Agricultural Research Service and the Department of Agronomy, University of Nebraska-Lincoln as Journal Series Paper No. 15066. Mention of firm names or trade products does not imply that they are endorsed or recommended by the USDA or the University of Nebraska over other firms or products not mentioned.