Optimizing injection time of GFP plasmid into sheep zygote

Microinjection of exogenous DNA into the cytoplasm of matured oocytes or zygotes is a promising technique to generate transgenic animals. However, the data about the microinjection time and procedure in sheep are limited and have not treated in detail. To obtain more in-depth information, the Sarda sheep oocytes from abattoir-derived ovaries were subjected to IVM and IVF. Then, the GFP plasmid as a reporter gene was injected into the cytoplasm of MII oocytes (n: 95) and zygotes at different post-insemination intervals (6–8 hpi, n: 120; 8–10 hpi, n: 122; 10–12 hpi, n: 110 and 12–14 hpi, n: 96). There were no significant differences in the cleavage rates between the groups. However, blastocyst rate of injected zygotes at all-time intervals was significantly lower than injected MII oocytes and control group (p < 0.05). Interestingly, the proportion of GFP-positive embryos was higher at 8–10 hpi compared with other injected groups (4 % versus 0 %, p  < 0.01). Among these, the proportion of mosaic embryos was high and two of those embryos developed to the blastocyst stage. In conclusion, we settled on the cytoplasmic microinjection of GFP plasmid at 8–10 hpi as an optimized time point for the production of transgenic sheep and subsequent experiments.     

Assessment of microbial biocontrol agent (BCA) viability to mechanical and thermal stress by simulating spray application conditions

Background

In order to improve the biological control agent (BCA) efficacy, stress factors threatening the viability of microorganisms during spray application need to be determined. The effect of spray mixture temperature and exposure time on Trichoderma harzianum T 22 and Bacillus amyloliquefaciens QST713 viability were tested. Concurrently the combined effect of mechanical and thermal stress effect on BCA viability were tested at two initial spray mixture temperatures (14 and 25 °C) by simulating a spray application using airblast sprayers featured by different tank capacity and a spray liquid circuit (without and with hydraulic agitation system). To assess the BCA microorganism viability, spray mixture samples were collected at time intervals along trials and plated to count the colony forming units (CFU).

Results

The critical temperature threshold that inhibited BCA viability was 35 °C with 30 min of exposure. The sprayer type, the initial temperature of the spray mixture and the temperature increment during the trials significantly decreased the number of CFU recovered. When simulating a spray application, the spray mixture temperature increase rate was determined mainly by the residual amount of spray mixture in the tank. Even if the tank capacity does not substantially affect the final temperature reached by the spray mixture, the higher residual spray mixture in bigger tanks can expose the BCAs for a longer time to critical temperatures.

Conclusions

Experimental trials allowed us to identify the effect of factors affecting the viability of tested BCAs, providing information about the actual chance to guarantee the biological efficacy of BCA treatments. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Enzyme Bioprospection of Marine-Derived Actinobacteria from the Chilean Coast and New Insight in the Mechanism of Keratin Degradation in Streptomyces sp. G11C

Marine actinobacteria are viewed as a promising source of enzymes with potential technological applications. They contribute to the turnover of complex biopolymers, such as pectin, lignocellulose, chitin, and keratin, being able to secrete a wide variety of extracellular enzymes. Among these, keratinases are a valuable alternative for recycling keratin-rich waste, which is generated in large quantities by the poultry industry. In this work, we explored the biocatalytic potential of 75 marine-derived actinobacterial strains, focusing mainly on the search for keratinases. A major part of the strains secreted industrially important enzymes, such as proteases, lipases, cellulases, amylases, and keratinases. Among these, we identified two streptomycete strains that presented great potential for recycling keratin wastes—Streptomyces sp. CHA1 and Streptomyces sp. G11C. Substrate concentration, incubation temperature, and, to a lesser extent, inoculum size were found to be important parameters that influenced the production of keratinolytic enzymes in both strains. In addition, proteomic analysis of culture broths from Streptomyces sp. G11C on turkey feathers showed a high abundance and diversity of peptidases, belonging mainly to the serine and metallo-superfamilies. Two proteases from families S08 and M06 were highly expressed. These results contributed to elucidate the mechanism of keratin degradation mediated by streptomycetes.     

Phenotypic and genotypic characterization of canine pyoderma isolates of Staphylococcus pseudintermedius for biofilm formation

Biofilm-forming ability is increasingly being recognized as an important virulence factor in several Staphylococcus species. This study evaluated the biofilm-forming ability of sixty canine derived clinical isolates of S. pseudintermedius, using three phenotypic methods, microtiter plate test (MtP), Congo red agar method (CRA) and tube adherence test, and the presence and impact of biofilm-associated genes (icaA and icaD). The results showed that icaA and icaD genes were detected concomitantly in 55 (91.7%) of 60 isolates. A majority (88.3%) of the strains screened had matching results by the tube adherence test, MtP and PCR analysis. Better agreement (95%) was found between the PCR-based analysis and the CRA. Results of the icaA and icaD gene PCRs showed good agreement with CRA results, with a kappa of 0.7. Comparing the phenotypic methods, the statistical analysis showed that the agreement among the phenotypical tests using categorical data was generally good. Considering two classes (biofilm producer and biofilm non-producer), the percentage of matching results between the CRA method and the tube adherence test and between the CRA method and the MtP was 93.3%. A concordance of 100% was revealed between the MtP and the tube adherence test. The results indicate a high prevalence of the ica genes within S. pseudintermedius isolates, and their presence is associated with in vitro formation of a biofilm. A combination of phenotypic and genotypic tests is recommended for investigating biofilm formation in S. pseudintermedius.     

Investigation of the Potential Health Benefits as Lipase Inhibitor and Antioxidant of <Emphasis Type="Italic">Leopoldia comosa</Emphasis> (L.) Parl.: Variability of Chemical Composition of Wild and Cultivated Bulbs

There is a great interest in the nutritional value of vegetables and fruits and how the habitat affects nutritive and biological properties. In vitro studies here reported were performed to evaluate the inhibitory activity of formulations from edible plant on pancreatic lipase. The aim of this study was also to evaluate the biovariability of L. comosa (L.) Parl. bulbs from Italy. The wild bulbs were compared with the same cultivated species that are commonly commercialized to identify samples with the best quality for a potential therapeutic application. Hydroalcoholic extract and polar fraction of wild bulbs showed a very important pancreatic lipase inhibitory activity, with IC₅₀ values of 0.166 ± 0.005 and 0.153 ± 0.005 mg/mL, respectively. In order to characterize the extracts, gas chromatography associated with mass spectrometry (GC/MS) analysis was performed, revealing the predominance of palmitic acid. Phenolic and flavonoid composition was also evaluated. L. comosa extract obtained from wild bulbs demonstrated both antioxidant and anti-obesity activities that might be attributed to a wide range of present phenolic compounds.