Effects of Confluent, Roscovitine Treatment and Serum Starvation on the Cell-cycle Synchronization of Bovine Foetal Fibroblasts

The present study was designed to examine the effects of cell-cycle synchronization protocols, such as confluent, roscovitine treatment and serum starvation, in bovine foetal fibroblasts on synchronization accuracy at G0/G1, viability, apoptosis, necrosis and ploidy for use as a nuclei donor. The cells in 5-10 passages were randomly allocated into three treated groups. Cells were cultured either in Dulbecco's modified Eagle's medium (DMEM) + 10% foetal bovine serum (FBS) until 90% confluent (group 1, confluent), in DMEM + 10% FBS + 30 microM roscovitine for 12 h (group 2, roscovitine), or in DMEM + 0.5% FBS for 5 days (group 3, serum starvation). Most of the cells (>80%) in all groups were arrested at the G0/G1 stage. Although the rates did not differ, cells in group 1 showed an increased cell population arrested at the G0/G1 phase. Significantly (p < 0.05) higher rates of apoptosis occurred in group 3 than in group 1 and 2 (10% vs 6% and 6%, respectively). No differences in chromosomal abnormality were observed among groups. However, by increasing the number of cell culture passages up to 15, significantly (p < 0.05) higher chromosomal abnormality was observed than in 5 and 10 passages (39% vs 28% and 23%, respectively) in group 1. The results clearly indicated that bovine foetal fibroblasts could be effectively synchronized at G0/G1 stages by all the three different treatments, confluent, roscovitine and serum starvation. However, cells in confluent showed reduced apoptosis and necrosis when they underwent 5-10 passages, exhibiting increased percentage of cells with stable chromosome diversity. Hence, cells in confluent merit further studies before they could be used as nuclear donors.     

Characterization and taxonomic reassessment of the box blight pathogen Calonectria pseudonaviculata,introducing Calonectria henricotiae sp. nov.

Calonectria pseudonaviculata, the causal agent of the disease of Buxus spp. known as ‘box blight’, was first detected in the mid‐1990s in the UK and New Zealand. Since then, the geographic range of box blight has rapidly expanded to at least 21 countries throughout temperate regions of the world, causing significant losses in nurseries, gardens and wild boxwood populations. This study determined the genetic diversity in a collection of 234 Calonectria isolates from diseased Buxus plants, originating from 15 countries and four continents. Two genetic clades, G1 and G2, were identified within this sample using multilocus phylogenetic analysis. The application of genealogical concordance phylogenetic species recognition criteria using four independent nuclear loci determined that the Calonectria isolates in these two clades are separate phylogenetic species. The isolates in the G1 clade were upheld as C. pseudonaviculata sensu stricto. Based on phylogenetic distinctiveness and the lack of mating, a new species is proposed, Calonectria henricotiae sp. nov., for the Calonectria isolates in the G2 clade. A PCR‐RFLP assay and real‐time PCR assays were developed to easily and reproducibly differentiate these species. To assess the practical implications of the identification of the two species, their physiology, fungicide susceptibility and pathogenicity were compared. No differences in pathogenicity were observed. However, C. henricotiae isolates exhibited greater thermotolerance and reduced sensitivity to specific triazole as well as strobilurin fungicides. The identification of a second phylogenetic species causing box blight may have a substantial impact on the epidemiology and control of this destructive disease.     

Field testing the influence of sperm competition based on sperm mobility in breeder turkey toms

1. Commercial reproduction of turkeys relies on pooling of semen from multiple males for inseminations. Understanding how sperm characteristics influence paternity under commercial breeding conditions is important to improving production efficiency. 2. The objective of this study was to evaluate progeny production of individual toms following commercial practices of pooling semen to determine if sperm mobility influences progeny production in field conditions. 3. A total of 104 toms were evaluated for sperm mobility. A subset of 10 toms were housed together and semen was collected, pooled and used to inseminate hens (n = 28). Hens were inseminated at 30 weeks of age and weekly thereafter. 4. Ejaculates from each tom were evaluated on two separate days for sperm mobility. Semen from each tom was diluted and layered upon 6% (wt/vol) Accudenz solution. The sperm suspension was incubated at 41 degrees C for 5 min and absorbance was measured with a spectrophotometer. 5. Toms were ranked by absorbance and categorised as high or low if mobility score was +/- 1 SD from the flock mean (average). 6. For parentage determination, DNA was extracted from tom, hen and poult blood. Poult parentage (n = 276) was determined at one day of age or at 14 weeks by analysis of marker genotypes that were generated by polymerase chain reaction (PCR) amplification of genomic DNA with selected microsatellite markers. 7. Sperm mobility differed across males with absorbance values ranging from 0.147 to 0.366. 8. Findings demonstrate differences in poult production among individual toms when semen from multiple males was pooled and inseminated. Toms classified as high, average and low produced 55, 41 and 4% of the offspring, respectively. 9. It appears that sperm mobility is a trait that influences sperm competition among toms under field conditions where sperm numbers inseminated from individual toms are not controlled or constant and that toms with low sperm mobility produce few offspring.     

Monensin controlled-release intraruminal capsule for control of bloat in pastured dairy cows

Monensin, a polyether ionophore antibiotic, is potentially an important agent for bloat relief in dairy cows grazing temperate legume-based pasture. A series of studies was undertaken to determine the effect of monensin, when delivered continuously in the rumen of lactating dairy cows by means of controlled-release capsules (monensin CRC). Such devices release approximately 300 mg/head/day for 100 d. A short-term pilot study made at Ruakura, New Zealand, tested monensin CRC in cows selected for high susceptibility to bloat and grazing lucerne (Medicago sativa) or red clover (Trifolium pratense). Treatment significantly reduced the incidence of bloat, while milk yield and protein yield were increased. There was no effect on fat yield. Following the pilot study, 6 large-scale field experiments involving a total of 368 lactating dairy cows, were made in Australia and New Zealand to confirm the effectiveness of monensin CRC for bloat control and to measure the effect of such treatment on milk production and composition. A severe bloat problem occurred in 2 experiments, mild bloat occurred in 2 others, while no visual signs of bloat were observed in the remaining 2 experiments. Bloat was significantly (P less than 0.05) reduced by monensin CRC treatment when data was pooled over the 4 experiments in which bloat occurred. Daily milk yield was increased in all experiments from a mean of 17.7 in untreated groups to 18.8 kg/head/day (P less than 0.05) in monensin CRC-treated cows. Protein percentage was not affected by treatment, while there was a decrease from 4.29 to 4.10% fat, although total fat yield was not affected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of a headspace sorptive extraction method for the analysis of volatile components in South African wines

A headspace sorptive extraction (HSSE) in combination with thermal desorption gas chromatography-mass spectrometry (TD-GC-MS) method for the analysis of volatile components (alcohols, esters, carbonyls, acids, phenols and lactones) in wine samples was developed. Extraction conditions such as salting-out effects, sorption time, stirring speed, phase ratio, extraction temperature, and effect of pH were thoroughly evaluated as part of method validation. The method was very sensitive with LODs and LOQs between 50 pg/L to 299 microg/L and 0.2 ng/L to 0.996 microg/L, respectively. Repeatability for all the compounds was between 3 and 22%. The intermediate repeatability was obtained within the acceptable range. Out of 39 volatile compounds selected, 37 were detected and quantitated. The method was found to be simple, cost-effective, sensitive, and use a small sample volume. The method was successfully applied for the routine analysis of 79 young red and white wine samples from various South African districts.     

Sorting of mannose 6-phosphate receptors mediated by the GGAs

The delivery of soluble hydrolases to lysosomes is mediated by the cation-independent and cation-dependent mannose 6-phosphate receptors. The cytosolic tails of both receptors contain acidic-cluster-dileucine signals that direct sorting from the trans-Golgi network to the endosomal-lysosomal system. We found that these signals bind to the VHS domain of the Golgi-localized, gamma-ear-containing, ARF-binding proteins (GGAs). The receptors and the GGAs left the trans-Golgi network on the same tubulo-vesicular carriers. A dominant-negative GGA mutant blocked exit of the receptors from the trans-Golgi network. Thus, the GGAs appear to mediate sorting of the mannose 6-phosphate receptors at the trans-Golgi network.