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.     

Mesohabitat associations of the threatened San Marcos salamander (Eurycea nana) across its geographic range

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Conserving indigenous crayfish: stock assessment and habitat requirements in the threatened Austropotamobius italicus

• 1. As part of the Austropotamobius pallipes species complex, the crayfish Austropotamobius italicus is a species of community interest whose preservation requires the designation of Special Areas of Conservation (SACs) (Annex II, EU Habitats Directive). This study aimed at (1) assessing the conservation status of this threatened indigenous species by stock assessment in central Italy and (2) identifying some aspects of its elective habitat.
• 2. Surveys were conducted in nine streams harbouring A. italicus (streams WI) and in 10 streams where crayfish populations became extinct at least 5 years before the study (streams WO).
• 3. The results confirmed that A. italicus is a K‐selected species, with a relatively slow growth rate (males: 0.34; females: 0.37) and a long life expectancy (males: 8.2 years; females: 7.8 years). The extant populations are healthy, showing balanced sex‐ratios and well structured age‐class compositions. Mortality is mainly due to fishing, which is illegal in Tuscany.
• 4. Principal Components Analyses showed that the streams WI and WO differ in the abundance of allochthonous plant detritus but not in the taxonomic composition of their macroinvertebrate communities. Age classes were found to be spatially segregated, juveniles mainly using cobbles as substrates and adults seemingly avoiding them.
• 5. The loss of the pristine riverine landscape seems to have been responsible, together with illegal fishing, for the local extinction of the species. As a consequence, retaining, enhancing, and restoring the habitat and its complexity are required for the preservation of A. italicus.
• 6. The designation of SACs might help in this endeavour if accompanied by programmes aimed at publicizing the need for conservation of this species. Unfortunately, crayfish‐focused projects supported by LIFE in Italy since 1992 (4%) and the SACs involved (1.4%) have been relatively few, despite the poor conservation status of this species and its well recognized ecological role.

Copyright © 2008 John Wiley & Sons, Ltd.     

<Emphasis Type="Italic">In vitro</Emphasis> screening for sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis>L.) resistant calli to <Emphasis Type="Italic">Diaporthe helianthi</Emphasis> fungal culture filtrate

Diaporthe helianthi the causal agent of sunflower (Helianthus annuus) stem canker, causes significant reductions in yield and oil content in most sunflower-growing areas. With the aim of enhancing host resistance, we selected in vitro sunflower calli against culture filtrates of two pathogen isolates (7/96 and 101/96). This technique may be an effective and rapid tool to discriminate the most virulent D. helianthi isolate and to screen for host resistance in the early stage of a breeding programme. Further investigation on the mechanisms involved in defence pathways showed no induction of salicylic acid and pathogenesis-related proteins in calli, indicating that the host resistance is not associated with Systemic Acquired Resistance but probably other biochemical mechanisms.     

Chemical Characterization and Release of Polyphenols from Pecan Nut Shell [<Emphasis Type="Italic">Carya illinoinensis</Emphasis> (Wangenh) C. Koch] in Zein Microparticles for Bioactive Applications

The pecan nut [Carya illinoinensis (Wangenh) C. Koch] is a natural source of polyphenols with antioxidant properties. In this study, the encapsulation of aqueous and hydroalcoholic extracts of pecan nut shell were evaluated for the release of bioactive compounds and antioxidant potential in order to explore food applications using zein as encapsulating agent. The extracts showed high contents of total phenolics, condensed tannins and high antioxidant activity. Concentrations of proanthocyanidins were 9-fold higher in hydroalcoholic extracts. The LC-DAD analysis showed that catechins were the major phenolic compounds in samples, with epigallocatechin levels up to 138.62 mg mL^?1. Zein microparticles loaded with aqueous extract released 2.3 times more phenolic compounds than the hydroalcoholic extracts and the DSC thermograms showed that extracts of pecan nut shell remained thermally stable up to 240 °C. The zein microcapsules obtained in this study were efficiently encapsulated and represent an interesting additive due its high antioxidant capacity, physicochemical characteristics and morphology. The use of zein microparticles combined with natural extracts constitute a step forward in the improvement of current technology for delivering phenolic compounds with applications in functional foods and nutraceuticals.     

Chitosan associated with whole raw soybean in diets for Murrah buffaloes on ruminal fermentation,apparent digestibility and nutrients metabolism

This study aimed to investigate the effects of chitosan and whole raw soybean on nutrient intake, apparent digestibility, nitrogen utilization, microbial protein synthesis, blood metabolites, feeding behavior, ruminal fermentation, digesta kinetics, and reticular flow of nutrients of buffaloes. Four ruminally‐cannulated Murrah buffaloes (351 ± 15 kg of initial BW) were randomly assigned according to a 4 × 4 Latin square design. Treatments were arranged as 2 × 2 factorial arrangement: the first factor was whole raw soybean (WRS), and the second factor was chitosan (CHI) with or without their inclusion in diets. Intake and apparent digestibility of ether extract (p < .01; p = .04, respectively), non‐fiber carbohydrates intake (p = .03) and apparent ruminal digestibility of dry matter (p = .01) were affected by diets. An interaction effect or tendency was observed for microbial nitrogen (p = .09), concentrations, ruminal ammonia nitrogen (p = .05), total volatile fatty acid (p = .03). Association of chitosan with whole raw soybean has potential effects as a modulator of rumen fermentation; therefore, chitosan can be applied as an alternative non‐ionophore for Murrah buffaloes.     

The Production of Microbiologically Safe Effluents for Wastewater Reuse in the Middle East and North Africa

Wastewater treatment in waste stabilization ponds (WSP) is a very efficient, low cost, low maintenance process which is well able to produce an effluent quality that meets the World Health Organization's recommendations for wastewater reuse for crop irrigation. Treatment in anaerobic and facultative ponds is required for restricted irrigation, with further treatment in maturation ponds for unrestricted irrigation. However, it is shown that the land requirements for the latter are at least twice that for the former. Unrestricted irrigation should, therefore, only be selected if it is economically viable. The use of wastewater storage and treatment reservoirs (WSTR), after pretreatment in anaerobic ponds, is advantageous in that it permits the whole year's wastewater to be used for irrigation, so allowing a much greater quantity of crops to be produced. A hybrid WSP-WSTR system can be used which produces effluents safe for both restricted and unrestricted irrigation.     

Molecular evidence of Leishmania spp. in spider monkeys (Ateles geoffroyi) from The Tuxtlas Biosphere Reserve,Veracruz, Mexico

Veterinary Research Communications - The black-handed spider monkey (Ateles geoffroyi) is a platyrrhine primate distributed in southern Mexico, Central America, and part of South America. Two...     

Canine circovirus and Canine adenovirus type 1 and 2 in dogs with parvoviral enteritis

Veterinary Research Communications - Canine parvovirus type 2 (CPV-2) is one of the most relevant pathogens associated with enteritis in dogs and is frequently reported in association with the...