Genetic and functional analysis of tocopherol biosynthesis pathway genes from rapeseed (Brassica napus L.)

Brassica napus L. is one of the most important oilseed crops in the world. Improving oil quality by increasing vitamin E content is a major target of rapeseed breeding. Vitamin E compounds, or tocopherols, are lipid‐soluble antioxidants that are essential nutrients for mammals. In this study, we report the characterization of rapeseed orthologs of the Arabidopsis tocopherol genes VTE1, VTE2 and PDS1. For each gene, at least two homologous sequences were found, and their expression was analysed in different tissues from three rapeseed genotypes. Genetic complementation experiments demonstrated that BnaX.VTE1.b and BnaA.VTE2.b homologs are capable of recovering seed γ‐tocopherol in Arabidopsis mutants. Overexpression of the genes in Arabidopsis Col‐0 shifted the seed tocopherol composition towards higher α‐tocopherol without increasing the total tocopherol content. To address the functionality of BnPDS1 sequences, we performed overexpression tests in Escherichia coli and enzymatic activity analyses. Overall, our results show that the identified sequences from rapeseed are functional orthologs of the Arabidopsis VTE genes and thus have considerable potential as molecular markers for selecting rapeseed with improved seed oil quality.     

Rumen-protected conjugated linoleic acid supplementation to dairy cows in late pregnancy and early lactation: effects on milk composition,milk yield,blood metabolites and gene expression in liver

Background

Conjugated linoleic acid (CLA) is a collective term for isomers of octadecadienoic acid with conjugated double-bond system. Thus, it was the objective to investigate whether milk composition and metabolic key parameters are affected by adding CLA to the diet of dairy cows in the first four weeks of lactation.

Methods

A study was carried out with five primiparous cows fed a CLA supplemented diet compared to five primiparous cows without CLA supplementation. CLA supplemented cows received 7.5 g CLA/day (i.e. 50% cis(c)9,trans(t)11- and 50% t10,c12-CLA) starting two weeks before expected calving and 20 g CLA/day (i.e. 50% c9,t11- and 50% t10,c12-CLA) throughout day 1 to 28 of lactation.

Results

The CLA supplement was insufficiently accepted by the animals: only 61.5% of the intended amount was ingested. Fed CLA were detectable in milk fat, whereas contents of c9,t11-CLA and t10,c12-CLA in milk fat were higher for CLA supplemented cows compared to the control group. On average over the entire treatment period, there was a decrease of saturated fatty acids (FA) in milk fat of CLA supplemented cows, combined with a higher content of monounsaturated and trans FA.Our study revealed no significant effects of c9,t11- and t10,c12-CLA supplementation either on milk yield and composition or on metabolic key parameters in blood. Furthermore the experiment did not indicate significant effects of c9,t11- and t10,c12-CLA-supplementation on gene expression of peroxisome proliferator-activated receptor-alpha (PPARα), PPARγ, sterol regulatory element-binding protein-1 and tumor necrosis factor-alpha in liver tissue.

Conclusions

Feeding c9,t11- and t10,c12-CLA during the first weeks after calving did not affect metabolic key parameters of blood serum or milk composition of fresh cows. Milk fatty acid composition was changed by feeding c9,t11- and t10,c12-CLA resulting in higher contents of these isomers in milk fat. High contents of long chain FA in milk fat indicate that CLA supplementation during the first four weeks of lactation did not affect massive peripheral lipomobilization.     

Disentangling complexity of fishing fleets: using sequence analysis to classify distinguishable groups of vessels based on commercial landings

Capturing the diversity of fishing fleets and identifying distinct subgroups is essential for effectively directing research and management efforts. In this study, the German Baltic gillnet fleet was split into distinguishable groups of vessels by applying a stepwise approach. Monthly landing profiles were classified using clustering techniques and arranged as annual landing sequences, as the basis to form groups of vessels with distinct annual landing sequences using sequence analysis. Commercial landings from 1243 vessels across 11 years (2008–2018) resulted in 8031 annual landing sequences, which were clustered into eight groups, each with a characteristic, annually recurring, seasonal landing pattern (cod group, cod-herring group, herring-flounder group, herring group, freshwater fish group, pikeperch group, eel group and port group). The results highlight the heterogeneity of the fleet and a strong adaptation to regional and seasonal resource availability. Studying sequences of landings instead of isolated events in time provides insight into the interlinkage and succession of landings and can aid at classifying fishing fleets and better targeting groups of vessels of interest.     

Degradation of transgenic Cry1Ab DNA and protein in Bt-176 maize during the ensiling process

Maize silage is commonly used as feed for farm animals. The aim of this study was to monitor the time-dependent degradation of non-recombinant chloroplast DNA (exemplified by the rubisco gene) in comparison with the recombinant cry1Ab gene in the course of the ensiling process. In parallel, the Cry1Ab protein content and fragment sizes were determined. Fragments of the rubisco (173, 896, 1197, 1753 and 2521 bp) and of the cry1Ab gene (211, 420, 727 and 1,423 bp) were selected to investigate the DNA degradation process. The detection of the Cry1Ab protein was performed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Rubisco gene fragments of 173 bp were still detectable after 61 days, while fragments of 1,197 and 2,521 bp were detectable up to 30 days and on the first day only respectively. Polymerase chain reaction (PCR) analyses revealed that fragments of the cry1Ab gene with sizes of 211 and 420 bp were detectable up to 61 days, fragments with sizes of 727 and 1,423 bp, 30 and 6 days respectively. The ELISA showed a decrease of the Cry1Ab protein in maize silage during the ensiling process. No marked degradation was observed during the first 43 h. Thereafter, a sharp decrease was measured. After 61 days, 23.6 +/- 0.9% of the initial Cry1Ab protein was still detectable. Immunoblotting confirmed the results of the ELISA showing a positive signal of approximately 60 kDa size for 8 days of ensiling; no further immunoactive fragments were detectable by immunoblotting. In conclusion, the ensiling process markedly decreases the presence of long functional cry1Ab gene fragments and full size Cry1Ab protein.     

Effect of N-modified lignite granulates and composted biochar on plant growth,nitrogen and water use efficiency of spring wheat

ABSTRACT

Organic soil amendments such as biochar are increasingly used to improve the fertility of degraded soils and marginal lands, plant growth, water retention and carbon sequestration. The performance of biochar depends on the parent material, the pyrolysis conditions and the nutrient enrichment process, accounting for the variability of the final products. Recently lignite granulates came into focus offering an alternative characterised by homogeneity, vast availability of the raw material and a standardised production process including nitrogen enrichment through oxidative ammonolysis. In a greenhouse experiment the effects of N-modified lignite granulates (NLG) and composted biochar (BC) on the growth of spring wheat (Triticum aestivum) in a sandy, carbon-free substrate were compared. Additionally, the effect of different NLG application rates (5, 7.5, 11, 15, 28 t ha^?1) was investigated. Yields as well as nitrogen and water use efficiency of the soil-plant system were determined at the end of the experiment. Both organic amendments increased yields relative to control plants. An increase in yield, nitrogen and water use efficiency for NLG even at low application rates and a better growth performance compared to BC were observed. Present findings, therefore, indicate, that such granulates offer an alternative to existing organic soil amendments.     

Cortisol levels in skimmed milk during the first 22?weeks of lactation and response to short-term metabolic stress and lameness in dairy cows

Background

Cortisol is secreted into blood in reaction to acute stress, but also in phases of diminished feed intake and changed animal behavior. As cows do not always show clear signs of discomfort, reliable diagnostic markers could be used to provide information regarding individual cows’ distress. The objective of this study was to establish an ether free immunoassay for the detection of cortisol and to determine values during the first 22 weeks of lactation. Furthermore, the response in milk cortisol levels was assessed during times of metabolic stress and pain associated symptoms of lameness.

Methods

Milk yield and composition, blood serum glucose, NEFA and BHBA as well as milk cortisol were determined in 24 multiparous Holstein-Friesian cows over the course of the first 22 weeks of lactation. Animals were further checked for signs of clinical diseases on a daily basis. Two feed restrictions over three days (FR; 70 % of precious ad libitum intake) were performed during the 4^th wk and the 21^st wk, respectively. An ELISA for cortisol measurement in easily accessible bovine skimmed milk was established and applied.

Results

On the last day of FR in early lactation, a reduction in milk yield and changes in serum metabolites compared to respective previous values were detected. The FR in mid-lactation resulted in no changes in milk production and serum metabolites. Milk cortisol was highest during first wk of lactation and remained on comparable levels thereafter. Milk yield and composition were not influenced by FR. Lameness resulted in enhanced milk cortisol levels.

Conclusion

Milk cortisol could be used as an indicator of painful symptoms such as lameness. Higher values of milk cortisol levels during first wk of lactation should be taken into account for interpretation.     

Chicken egg antibodies for prophylaxis and therapy of infectious intestinal diseases. III. In vivo tenacity test in piglets with artificial jejunal fistula

Large quantities of specific egg antibodies can be produced with little effort by immunization of laying hens. Several antibody containing egg preparations were subjected to a tenacity test against digestive activity taking piglets with artificial jejunum fistulas as a model. Even with high doses of orally administered yolk lyophilisate no antibody activity could be found in the distal jejunum. The additional feeding of egg white, however, showed a significant protective effect on the yolks antibodies during the digestive act. No difference between egg white of immunized hens and egg white of unimmunized hens could be detected. Buffering the acidic gastric environment with sodium bicarbonate increased the antibody activity in the intestine by an average of 40%. Eggs administered in hot (70 degrees C) beef stock had higher antibody titers than eggs that were boiled for 4 minutes. Tenacity in antibodies fed in pellets was significantly lower than in powdered feed.     

Chicken egg antibodies for prophylaxis and therapy of infectious intestinal diseases. IV. In vitro studies on protective effects against adhesion of enterotoxogenic Escherichia coli to isolated enterocytes.

The pathogenic effect of enterotoxogenic E. coli is mainly to be found in the jejunum. Hence, an effective immunoprophylaxis should be focussed at this site. This can be done effectively by an oral application of specific antibodies. The present study describes the effectiveness of specific antibodies against E. coli. The antibodies were gained from the yolk of E. coli immunized hens and tested for their in vitro activity. The tests showed a significantly reduced adhesion of enterotoxogenic E. coli onto isolated pig enterocytes as long as the bacteria had been incubated with the specific yolk antibodies beforehand. For this reason, yolk immunoglobulins from immunized hens prove to be an effective and handy source of specific antibodies which offers good protection against adhesion of E. coli onto pig enterocytes.     

The effectiveness and safety of an immunization against parvovirus and rabies in anesthetized puppies

The efficacy and safety of a vaccination during anaesthesia was examined in 20 puppies from 8 litters of one kennel. The animals were vaccinated in a state of reflex-free anaesthesia (neuroleptanalgesia in combination with halothane, nitrous oxide and oxygen). 20 puppies from the same litters were used as controls. A parvovirus living vaccine (Canimed) and a rabies vaccine from inactivated viruses (Rabisin) were used for the vaccinations. The titers of neutralizing antibodies against rabies virus were significantly lower in the group of anaesthetized animals compared to the control group on the 10th and 20th day p.vacc. The average titers of antibodies against parvovirus of both groups, however, do not allow any statistically significant statements. It is nevertheless remarkable that 6 of the anaesthetized puppies (three different litters) showed no increase in antibodies. The results of the investigations lead to the recommendation not to vaccinate anaesthetized puppies. The risk of reducing the efficacy of vaccinations with vaccines from inactivated agents is greater than with living vaccines.     

Use of the cell wall precursor lipid II by a pore-forming peptide antibiotic

Resistance to antibiotics is increasing in some groups of clinically important pathogens. For instance, high vancomycin resistance has emerged in enterococci. Promising alternative antibiotics are the peptide antibiotics, abundant in host defense systems, which kill their targets by permeabilizing the plasma membrane. These peptides generally do not act via specific receptors and are active in the micromolar range. Here it is shown that vancomycin and the antibacterial peptide nisin Z use the same target: the membrane-anchored cell wall precursor Lipid II. Nisin combines high affinity for Lipid II with its pore-forming ability, thus causing the peptide to be highly active (in the nanomolar range).