Pronuclear Embryo Yield in Canindé and Saanen Goats for DNA Microinjection

The objective of this study was to examine the effect of donor breed on pronuclear‐stage embryo yield to be used for DNA microinjection in a transgenesis goat program. Twelve Canindé and twelve Saanen goats were heat synchronized using a progestagen‐cloprostenol treatment. Forty‐eight hours before the sponge removal, superovulation was induced with a total administration of 4.4 mg/kg bodyweight NIH‐FSH‐P1, given twice daily in decreasing doses over 3 days. In addition, goats received 100 μg of GnRH and they were hand‐mated at 36 and 48 h after progestagen removal. Embryo recovery was performed by oviduct flushing at 72 h after sponge removal. Embryos were microinjected with a DNA construct and noticeable swelling of the nuclei was the criterion for successful microinjection. The total diameter, cytoplasm diameter, zona pellucida thickness and pronuclei diameter were measured for each microinjected embryo. A higher (p < 0.05) percentage of fertilized ova was observed in Canindé (89.9%) than Saanen (36.2%) goats. In addition, Canindé donors produced a higher percentage of pronuclear embryos when compared with Saanen: 72.5% vs 20.6% (p < 0.05), respectively. Successful microinjection was verified in 96.7% and 73.3% of times in Canindé and Saanen embryos, respectively (p < 0.05). Significant differences were observed for all morphometric parameters except for cytoplasm diameter. In conclusion, under our study experimental conditions, Canindé were more efficient than Saanen goats concerning the pronuclear embryo yield and manipulation. The use of Canindé goats in transgenesis programs could be increase the interest in their breeding and could be contribute to saving them from extinction.     

Methods for Cercospora coffeicola protoplast isolation and genetic transformation with the green fluorescent protein

Cercospora coffeicola is the causal agent of brown eye spot on coffee leaves. Although the disease has significant importance, few molecular studies have been done with C. coffeicola. Here we report a protocol for isolating protoplasts as well as development of a genetic transformation system using Green Fluorescent Protein. High yields of protoplasts (≈10⁸/ml) were obtained from mycelial cultures from five isolates of C. coffeicola. One isolate was transformed with a vector encoding hygromycin resistance and Green Fluorescent Protein. Out of 43 hygromycin-resistant transformants obtained, Green Fluorescent Protein was highly expressed in one (2.3 %).     

Appraisal and standardization of curvilinear velocity (VCL) cut‐off values for CASA analysis of Japanese quail (Coturnix japonica) sperm

One of the basic steps in objective analysis of sperm motility is the subdivision of a motile sperm population into slow, medium and rapid categories based on their velocity. However, for CASA analysis of quail sperm, the velocity values for categorization of slow, medium and rapid sperm have not yet been standardized. To identify the cut‐off values of “velocity curvilinear” (VCL) for quail sperm categorization, we captured and analysed 22,300 tracks of quail sperm using SCA^®‐CASA. The median and mean VCL values were 85 and 97 μm/s. To define the VCL cut‐off values, we used two methods. In the first, we identified the upper (rapid sperm) and lower (slow sperm) cut‐off values using: (i) median VCL ± 25% or ± 50% or ± 75% of median VCL value; (ii) first and third quartile values of VCL data (i.e. 25% cut‐off setting); and (iii) 33% and 66% of VCL data. Among these settings, sperm categories and their corresponding motility characteristics recorded using the “25%” setting (i.e. slow ≤36 ≤ medium ≤154 ≤ rapid) were found the most realistic and coherent with male ranking by fertility. In the second method, we calculated heteroscedasticity in the total VCL data using PCA and the two‐step clustering method. With this approach, the mean of the high and low clusters was 165 and 51 μm/s, respectively. Together, the mean from two methods suggested that, for SCA^®‐CASA categorization of quail sperm, sperm should be classed as “rapid” at VCL ≥160 μm/s and “slow” at VCL ≤45 μm/s.     

Analysis of genetic diversity and discrimination of Oil Palm DxP populations based on the origins of pisifera elite parents

A total of 251 Dura cross Pisifera (DxP) hybrid palms from six populations descending from six parental African Pisifera origins and involving 12 progenies were analyzed with 19 selected Simple Sequence Repeats (SSR) markers. A total of 110 alleles were produced, ranging from three to eight per SSR, with a mean of 5.8 alleles per SSR locus. Of these, 68.5% were considered shared alleles by more than one population and the remaining 31.5% were population specific alleles. They generated between six and 21 haplotypes in all populations, and depending on the SSR marker, between one and 10 haplotypes within populations. Various parameters for analyzing genetic variability, differentiation and genetic structure were computed using GenAlEx, Structure and Darwin software. The obtained results confirmed microsatellites as a robust, feasible and trustful method for obtaining DNA fingerprints, tracing the source of oil palm samples. With respect to the authenticity of materials or for solving legitimacy issues, accession belonging to each population by SSR markers could be distinguished, but additional SSR should be screened for improving this process.     

Thermoluminescence dates and palaeoenvironmental information of the late Quaternary sand deposits, Tierra de Pinares, Central Spain

A large amount of Quaternary palaeo-environmental evidence lies within the terrestrial aeolian sediments filling the Duero basin in central Spain. Previous work has identified a number of stratigraphical units although the absolute age and origin of some of these is equivocal. The uppermost unit, the focus of this paper, is a widely distributed sand which covers most of the Tierra de Pinares, north of Segovia. Detailed investigations of an exposure at Burgomillodo reveal over 35 m of laminated and cross-bedded sand from which seven thermoluminescence dates were obtained. The bedding structures indicate a northwesterly palaeowind direction, different from the present dominating westerly winds. Two phases of sand deposition are identified at the site with evidence of erosion and reactivation phases. An upper unit of ca. 7 ka during the warm-aridity of the Atlantic period and a lower unit between 12.5–11 ka coinciding with the cold-aridity of the Younger Dryas event. The latter, in which at least 25 m of sand were deposited at the site, is the dominant phase and is coincident with what has been identified elsewhere in Spain from biological data as a period which was both cold and dry. The Burgomillodos site, therefore, represents the first sedimentological evidence for this arid phase in the Younger Dryas in central Spain.     

Dispharynx nasuta (Nematoda:Acuariidae) infection causing proventricular lesions and death in three captive rosellas (Psittaciformes:Psittacidae)

Two adult male Crimson rosellas (Platycercus elegans) and an adult female eastern rosella (Platycercus eximius) were found dead in their cages in a private aviary in March 2009, April 2010, and February 2010, respectively, without premonitory signs. Their postmortem examination showed pectoral muscle atrophy and a distended proventriculus. The proventricular mucosal surface was covered with a gelatinous, whitish material, where a large number of nematode parasites identified as Dispharynx nasuta were present. A histologic examination revealed a hyperplastic mucosa, spirurid nematodes in the mucosal layer and inflammatory mononuclear cells in the lamina propria. This is the first report of a D. nasuta infection associated with proliferative proventriculitis and subsequent death in psittacine birds.     

Evaluation of acibenzolar-S-methyl and other low-toxicity products for control of rose powdery mildew (Podosphaera pannosa) in greenhouses

Abstract

In an attempt to find alternative products to classical fungicides, several products with low toxicity were tested against powdery mildew of roses. These products included resistance inducers (Bion, BABA, and ROS), potassium salts (Resistim, monopotassium phosphate), and seed extracts. The best results were obtained with acibenzolar-S-methyl (Bion). The utilization of Bion as prophylactic treatment, watered at a concentration 0.1–0.2 mg/ml, together with good cultural practices can be enough to effectively control powdery mildew on roses. Treatments with Resistim reduced the disease incidence, but not always significantly compared to the controls. None of the other products had effect on powdery mildew.     

Biomimetic Pathways for Assembling Inorganic Thin Films

Living organisms construct various forms of laminated nanocomposites through directed nucleation and growth of inorganics at self-assembled organic templates at temperatures below 100°C and in aqueous solutions. Recent research has focused on the use of functionalized organic surfaces to form continuous thin films of single-phase ceramics. Continuous thin films of mesostructured silicates have also been formed on hydrophobic and hydrophilic surfaces through a two-step mechanism. First, under acidic conditions, surfactant micellar structures are self-assembled at the solid/liquid interface, and second, inorganic precursors condense to form an inorganic-organic nanocomposite. Epitaxial coordination of adsorbed surfactant tubules is observed on mica and graphite substrates, whereas a random arrangement is observed on amorphous silica. The ability to process ceramic-organic nanocomposite films by these methods provides new technological opportunities.     

An insight on Octopus vulgaris paralarvae lipid requirements under rearing conditions

In this study, two new alternative preys: Grapsus adscensionis zoeae (as sole prey) and Palaemon elegans zoeae (in cofeeding with Artemia sp.), as well as, Artemia sp. juveniles were used as feed for octopus paralarvae, as a way to understand its lipid requirements. Total lipid (TL) content, lipid class (LC) and fatty acid (FA) profiles of preys, octopus hatchlings and 9‐day‐old paralarvae were analysed. Growth and survival of the paralarvae were also determined. Regardless the prey provided, a notable shift in the lipid profile of paralarvae was registered after 9 days of rearing. The highest index of growth rate (IGR) recorded when decapod crustacean zoeae were supplied might have some relation with levels of 20:4n‐6 (ARA) and DHA/EPA ratio observed. In this sense, Grapsus adscensionis zoeae leaded to a higher content of ARA and a lower content of EPA, which may indicate a possible competition between these two FA. For that a balanced EPA/ARA ratio might be significant in this species nutrition without disconsidering DHA levels as an essential fatty acid. Finally, the changes observed in paralarvae FA profile might not only be related to prey FA profile, but also with changes occurring in the lipid classes contents.     

Flower emasculation accelerates ovule degeneration and reduces fruit set in sweet cherry

Flower emasculation is commonly used to make flowers unattractive to pollinating insects and to carry out controlled pollinations. In sweet cherry, we have observed recurrent low fruit set after flower emasculation and compatible pollination without apparent causes. This led us to evaluate its effect on the progress of the reproductive phase and on fruit set in this species. Flower emasculation reduced by more than a half the fruit set obtained in crosses made during two consecutive years. This effect could be traced back to the first week after anthesis where weight increase of pistils from emasculated flowers was smaller and ovule degeneration was accelerated compared to pistils from non-emasculated flowers. Pollen tubes, which behaved similarly at the stigma-style level in emasculated and non-emasculated flowers, lost their directionality in the area close to the degenerated ovule in the ovary. While flower emasculation is valid to evaluate pollen tube performance in the style and to determine incompatibility relationships, the lower fruit set registered after emasculation alerts on its use in fruit set experiments and breeding programs.