A fast and simple method for the polymerase chain reaction-based sexing of livestock embryos.

Embryo sexing is a powerful tool for livestock producers because it allows them to manage their breeding stocks more effectively. However, the cost of supplies and reagents, and the need for trained professionals to biopsy embryos by micromanipulation restrict the worldwide use of the technology to a limited number of specialized groups. The aim of this study was to couple a fast and inexpensive DNA extraction protocol with a practical biopsy approach to create a simple, quick, effective, and dependable embryo sexing procedure. From a total of 1847 sheep and cattle whole embryos or embryo biopsies, the sexing efficiency was 100% for embryo biopsies, 98% for sheep embryos, and 90.2% for cattle embryos. We used a primer pair that was common to both species and only 10% of the total extracted DNA. The whole protocol takes only 2 h to perform, which suggests that the proposed procedure can be readily applied to field conditions. Moreover, in addition to embryo sexing, the procedure can be used for further analyses, such as genotyping and molecular diagnosis in preimplantation embryos.

Stimulation of ouabain binding to Na,K-ATPase in 40% dimethyl sulfoxide by a factor from Na,K-ATPase preparations.

In 40% dimethyl sulfoxide (Me2SO) high-affinity ouabain (O) binding to Na,K-ATPase (E) is promoted by Mg2+ in the absence of inorganic phosphate (Pi) (Fontes et al., Biochim. Biophys. Acta 1104, 215-225, 1995). Furthermore, in Me2SO the EO complex reacts very slowly with Pi and this ouabain binding can therefore be measured by the degree of inhibition of rapid phosphoenzyme formation. Here we found that, unexpectedly, the ouabain binding decreased with the enzyme concentration in the Me2SO assay medium. We extracted the enzyme preparation with Me2SO or chloroform/methanol and demonstrated that the extracted (depleted) enzyme bound ouabain poorly. Addition of such extracts to assays with low enzyme concentration or depleted enzyme fully restored the high-affinity ouabain binding. Dialysis experiments indicated that the active principle had a molecular mass between 3.5 and 12 kDa. It was highly resistant to proteolysis. It was suggested that the active principle could either be a low-molecular-weight, proteolysis-resistant-peptide (e.g., a proteolipid) or a factor with a nonproteinaceous nature. A polyclonal antibody raised against the C-terminal 10 amino acids of the rat kidney gamma-subunit was able to recognize this low-molecular-weight peptide present in the extracts. The previously depleted enzyme displayed lower amounts of the gamma-proteolipid in comparison to the native untreated enzyme, as demonstrated by immunoreaction with the antibody.

Perspectives of using Euphorbia splendens as a molluscicide in schistosomiasis control programs.

A total of 1,426 species of plants had been tested in order to find out a vegetal-derived molluscicide against the vectors of Schistosoma mansoni. Euphorbia splendens is one of the most promising molluscicides. In this article we briefly present the main aspects that have to be considered to use this plant as a molluscicide, which include some agricultural aspects and operational methods for application. This can be done by the extraction of the active principles (millaminas) present in the latex or in the raw juice obtained through total maceration of plants. The culture of E. splendens, which spreads out by vegetative sprouts, showed an annual average growth of 30 cm. After 1.5 years the average productivity of crude latex with a DL90 of 12 ppm concentration, was 1 liter by 8m2 by cultivated area. This amount is enough to treat a volume of water of 97,200 liters. A comparative analysis between the cost of snail control through synthetic molluscicides was carried out based upon the data presented by Jobim (1979) for six controlling methods in several countries, and some cost aspects for E. splendens. This analysis was done considering a geographic parameter expressed by the index cubic meters of treated habitat per square kilometer, from which we can obtain an estimate dimension of the geographic area to be cultivated with E. splendens necessary to treat the same volume of water treated by synthetic molluscicides.

Evaluation of the molluscicidal property of Euphorbia splendens var. Hispolii (N.E.B.) (Euphorbiaceae): 2. Investigation in lotic habitat

The latex of Euphorbia splendens var. hispolii in 12 ppm concentration (12 mg/l) caused 100% mortality for a Biomphalaria tenagophila population in a lotic habitat after 9 hr of dripfeed application. For sentinel snails, in cages placed at the water surface and buried under 0.10 m, the mortality rates varied with the distance from the application point and were: 100% (0 m); 92.6% (50 m) and 94.7% (100). No lethal effects were observed for the other living species in this habitat (Pomacea haustrum and Poecilia reticulata)

Evaluation of temporal, seasonal and geographic stability of the molluscicidal property of Euphorbia splendens latex.

Laboratory tests with aqueous solutions of Euphorbia splendens var. hislopii latex have demonstrated seasonal stability of the molluscacidal principle, with LD90 values of 1.14 ppm (spring), 1.02 ppm (fall), 1.09 ppm (winter), and 1.07 ppm (summer) that have been determined against Biomphalaria tenagophila in the field. Assays on latex collected in Belo Horizonte and Recife yielded LD90 values similar to those obtained with the reference substance collected in Rio de Janeiro (Ilha do Governador), demonstrating geographic stability of the molluscacidal effect. The molluscacidal action of aqueous dilutions of the latex in natura, centrifuged (precipitate) and lyophilized, was stable for up to 124 days at room temperature (in natura) and for up to 736 days in a common refrigerator at 10 to 12 degrees C (lyophilized product). A 5.0 ppm solution is 100% lethal for snails up to 13 days after preparation, the effect being gradually lost to almost total inactivity by the 30th day. This observation indicated that the active principle is instable. These properties together with the wide distribution of the plant, its resistance and adaptation to the tropical climate, its easy cultivation and the easy obtention of latex and preparation of the molluscicidal solution, make this a promising material for large-scale use in the control of schistosomiasis.