Isolation and differentiation of bovine mammary gland progenitor cell populations

OBJECTIVE: To isolate bovine mammary gland cells with stem cell characteristics. SAMPLE POPULATION: Monolayers of bovine mammary gland cells. PROCEDURE: Mammary gland cell populations were separated by use of selected media supplements. Phenotypic characteristics were examined via light and transmission electron microscopy. Cellular expression of casein and connexin 43 was identified immunohistochemically. A scrape-loading and dye transfer assay was used to examine the mammary gland cell populations for homogenous gap junctional intercellular communication (GJIC). RESULTS: Subpopulations of mammary gland cells grown in vitro are classified on the basis of their distinct morphologic features and ability to communicate via gap junctions. Ultrastructurally, 2 morphologically distinct cell types were classified as type I and II cells. Type I cells were small light undiffertiated cells and large light undifferentiated cells that were deficient in functional gap junctions (as is characteristic of stem cells). Type II cells included large light differentiated cells and terminally differentiated cells; GJIC was functional in type II cells. Type II cells had cytoplasmic expression of connexin 43, whereas, type I cells did not. All cells expressed casein. CONCLUSIONS AND CLINICAL RELEVANCE: Subpopulations of bovine mammary gland cells with stem cell characteristics were identified. Phenotypic differences are observed among type I bovine mammary gland cells with stem cell characteristics. Gap junctional intercellular communication may be necessary for the differentiation of stem cells. Characterization of bovine mammary gland stem cells and their progeny may provide a new tool with which to study mammary gland health.     

Production of Wild Buffalo (Bubalus arnee) Embryos by Interspecies Somatic Cell Nuclear Transfer Using Domestic Buffalo (Bubalus bubalis) Oocytes

The objective of this study was to explore the possibility of producing wild buffalo embryos by interspecies somatic cell nuclear transfer (iSCNT) through handmade cloning using wild buffalo somatic cells and domestic buffalo (Bubalus bubalis) oocytes. Somatic cells derived from the ear skin of wild buffalo were found to express vimentin but not keratin and cytokeratin‐18, indicating that they were of fibroblast origin. The population doubling time of skin fibroblasts from wild buffalo was significantly (p < 0.05) higher, and the cell proliferation rate was significantly (p < 0.05) lower compared with that of skin fibroblasts from domestic buffalo. Neither the cleavage (92.6 ± 2.0% vs 92.8 ± 2.0%) nor the blastocyst rate (42.4 ± 2.4% vs 38.7 ± 2.8%) was significantly different between the intraspecies cloned embryos produced using skin fibroblasts from domestic buffalo and interspecies cloned embryos produced using skin fibroblasts from wild buffalo. However, the total cell number (TCN) was significantly (p < 0.05) lower (192.0 ± 25.6 vs 345.7 ± 42.2), and the apoptotic index was significantly (p < 0.05) higher (15.1 ± 3.1 vs 8.0 ± 1.4) for interspecies than that for intraspecies cloned embryos. Following vitrification in open‐pulled straws (OPS) and warming, although the cryosurvival rate of both types of cloned embryos, as indicated by their re‐expansion rate, was not significantly different (34.8 ± 1.5% vs 47.8 ± 7.8), the apoptotic index was significantly (p < 0.05) higher for vitrified–warmed interspecies than that for corresponding intraspecies cloned embryos (48.9 ± 7.2 vs 23.9 ± 2.8). The global level of H3K18ac was significantly (p < 0.05) lower in interspecies cloned embryos than that in intraspecies cloned embryos. The expression level of HDAC1, DNMT3a and CASPASE3 was significantly (p < 0.05) higher, that of P53 was significantly (p < 0.05) lower in interspecies than in intraspecies embryos, whereas that of DNMT1 was similar between the two types of embryos. In conclusion, these results demonstrate that wild buffalo embryos can be produced by iSCNT.     

Vacuum Squeezing of Solids: Macroscopic Quantum States Driven by Light Pulses

Femtosecond laser pulses and coherent two-phonon Raman scattering were used to excite KTaO3 into a squeezed state, nearly periodic in time, in which the variance of the atomic displacements dips below the standard quantum limit for half of a cycle. This nonclassical state involves a continuum of transverse acoustic modes that leads to oscillations in the refractive index associated with the frequency of a van Hove singularity in the phonon density of states.     

Low-Temperature Nonoxidative Activation of Methane over H-Galloaluminosilicate (MFI) Zeolite

Conversion of methane to higher hydrocarbons by its low-temperature activation without forming undesirable carbon oxides is of great scientific and practical importance. Methane can be highly activated, yielding high rates of conversion to higher hydrocarbons and aromatics (10 to 45 percent) at low temperatures (400° to 600°C), by its reaction over H-galloaluminosilicate ZSM-5 type (MFI) zeolite in the presence of alkenes or higher alkanes. The methane activation results from its hydrogen-transfer reaction with alkenes.     

Virulence profile and genetic structure of a North Dakota population of Pyrenophora teres f. teres, the causal agent of net form net blotch of barley

A Pyrenophora teres f. teres population in North Dakota was analyzed for virulence variation and genetic diversity using 75 monospore isolates that were collected across a 4-year period (2004 to 2007) from two North Dakota State University agricultural experiment stations at Fargo and Langdon. Pathogenicity tests by inoculation onto 22 barley differential lines at seedling stage revealed 49 pathotypes, indicating a wide range of pathogenic diversity. Two-way analysis of variance of disease ratings revealed a significant difference in the virulence among isolates and in the resistance among barley lines, as well as in the interactions between the two. 'CI5791', 'Algerian', and 'Heartland' were three barley lines showing a high level of seedling resistance to all North Dakota isolates tested; however, many previously reported resistance genes have been overcome. Forty multilocus genotypes were identified from this set of isolates by genotyping at 13 simple-sequence repeat loci. High percentages of clonal cultures were detected in the samplings from 2005 and 2007 in Fargo and 2005 in Langdon. Using a clone-corrected sample set, the mean gene diversity (h) was estimated to be 0.58, approximately the same for both locations. The calculated Wright's F(ST) value is small (0.11) but was significantly >0, indicating a significant differentiation between the Fargo and Langdon populations. In the gametic disequilibrium test, only 3 of 78 possible pairwise comparisons over all isolates showed significant (P < 0.05) nonrandom association, suggesting a random mating mode. Our results suggest that the populations from the two locations are derived from a common source and undergo frequent recombination. This research provides important information for barley breeders regarding development and deployment of cultivars with resistance to net form net blotch in this region.     

Impact of molybdenum on the copper status of red deer (Cervus elaphus)

AIM: To determine the effect of increasing molybdenum (Mo) intakes on serum and liver copper (Cu) concentrations and growth rates of grazing red deer (Cervus elaphus).

METHODS: Molybdenum- and Cu-amended fertilisers were applied to six 1.1-ha paddocks in a 3 × 2 design. Three levels of Mo were applied on two paddocks at each level in mid April (designated Day 1); levels were: none (control), 0.5 (medium) and 1.0 (high) kg Mo/ha as sodium molybdate. In late May (Day 39), two levels of Cu (none and 3.0 kg Cu/ha, as copper sulphate) were applied to each of the three levels of Mo-treated paddocks. Pasture Mo, Cu and sulphur (S) concentrations were measured at about fortnightly intervals. In late June (Day 74), ten 6-month-old red deer hinds were placed on the six experimental pastures, and serum and liver Cu concentrations were monitored at about monthly intervals for 102 days. The hinds were weighed on four occasions during the trial.

RESULTS: Mean pasture Mo concentrations on Day 56 were 2, 4.6 and 11.3 mg/kg dry matter (DM) for the untreated control, medium and high Mo-treated pastures, respectively. Pasture Cu concentration was 95 mg/kg DM on Day 59, 53 mg/kg DM on Day 90, and 9 mg/kg DM by Day 153. Mean S concentration in pasture was 3.3 (range 3.03–3.45) g/kg DM. Copper application to pasture had no significant effect on serum and liver Cu concentrations in deer so data were pooled within Mo treatment. Mean initial (Day 74) serum Cu concentration was 9.2 µmol/L. In the deer grazing the control Mo pasture, this increased to 10.3 µmol/L on Day 112, before decreasing to 6.4 µmol/L on Day 176. In deer grazing the medium and high Mo-treated pastures, mean serum Cu concentrations were 3.8 and 3.9 µmol/L, respectively, on Day 112, and 2.5 and 3.3 µmol/L, respectively, on Day 176. Mean initial (Day 74) liver Cu concentration was 131 µmol/kg fresh tissue. In the deer grazing the control Mo pasture, this declined to 120 and 52 µmol/kg on Days 112 and Day 176, respectively. In deer grazing the medium and high Motreated pastures, liver Cu concentrations decreased to 55 and 52 µmol/kg fresh tissue, respectively, on Day 112, and 21 and 20 µmol/kg fresh tissue, respectively, on Day 176. Mean serum and liver Cu concentrations were not significantly different between deer grazing the medium and high Mo-treated pastures, and were lower (serum p=0.003, liver p<0.001) in those groups than in deer grazing the untreated control pastures. No clinical signs of Cu deficiency associated with lameness were observed. Deer grazing pastures that had Mo concentrations >10 mg/kg DM had lower (p=0.002) growth rates (100 vs 130 g/day) than those on pastures containing <2.4 mg Mo/kg DM.

CONCLUSION: Increasing pasture Mo concentrations from 2 mg/kg DM to ≥4.6 mg/kg DM significantly reduced serum and liver Cu concentrations in grazing deer. Reduced growth rate was observed at pasture Mo concentrations >10 mg/kg DM.