Manipulated Mouse Embryos as Bioassay System for Water Quality Control

Mouse pronuclear stage embryos with intact slit zona pellucida (manipulated) were cultured in vitro until the hatched blastocyst stage in simplex optimized medium with higher K⁺ concentration (KSOM) prepared with three different water types: tap, deionized reverse osmosis (D‐O) water and Milli‐Q system (M‐Q) water. The culture media were supplemented with or without protein and ethylenediaminetetraacetic acid (EDTA, disodium salt). The rates of hatched blastocysts were significantly affected (p < 0.01) by micromanipulation, protein supplement and water source. The water source has no influence (p > 0.05) on development in EDTA‐supplemented protein‐free culture media, whereas in EDTA‐ and protein‐free culture media, the water quality significantly (p < 0.001) affected the rates of development, with higher rates in media prepared with M‐Q water. The micromanipulated embryos showed higher sensitivity to the water quality (p < 0.01). It worth mentioning that the rates of hatched blastocysts in protein‐free culture media were very low (0–7.5%). Furthermore, the three different water types were analysed by measuring the electrical conductivity, inorganic ions, total organic carbon and endotoxins to evaluate the purity. M‐Q water showed the lowest levels of inorganic ion, total organic carbon and endotoxin concentrations. We concluded that manipulated mouse embryos are good system to evaluate the quality of water used in biological system.     

Validation of the specificity and sensitivity of species-specific primers that provide a reliable molecular diagnostic for <Emphasis Type="Italic">Xiphinema diversicaudatum</Emphasis>, <Emphasis Type="Italic">X. index</Emphasis> and <Emphasis Type="Italic">X. vuittenezi</Emphasis>

Xiphinema diversicaudatum and X. index are vector nematode species of economic importance in viticulture regions as they can transmit Arabis Mosaic, Grapevine Fanleaf and Strawberry Latent Ringspot viruses to grapevine. Wang et al. (2003) designed species-specific diagnostic primers from ribosomal genes for both these vector species as well as a vector and a non-vector species X. italiae and X. vuittenezi, respectively. Our study aimed to confirm the specificity and determine the sensitivity and reliability of the primers for the two vector species, X. diversicaudatumand X. indexwhen challenged with closely related longidorid species and general nematode communities typical of vineyard soil. With one exception, no PCR product was observed when the primers were tested against six Longidorus, one Paralongidorus and one Xiphinema non-target species. Occasionally (three out of eight replicate PCR reactions) a weak PCR product was noted when primers for X. index were tested with L. elongatus. Furthermore, when challenged with a range of non-target nematode species comprising the nematode community typical of viticulture soil, no PCR product was amplified. An experimental dilution series of extracted DNA rigorously demonstrated that DNA from an equivalent single specimen of the target virus-vector species, X. diversicaudatum and/or X. index, could be detected amongst 1000 equivalent non-targetX. vuittenezi. Also, extracted DNA from an equivalent single target specimen was detected when added to DNA extracted from the overall soil nematode community. The primers were assessed further by using serial mixtures of actual nematodes rather than extracted DNA to simulate field soil. Using this method, a single target nematode could be detected amongst 200 non-target specimens. Given their specificity, sensitivity and reliability, it appears that these diagnostic primers will be of great benefit to phytosanitary/quarantine services related to the viticulture industry.     

ACE inhibition in goats under fixed‐time artificial insemination protocol increases the pregnancy rate and twin births

To assess the effect of the angiotensin‐converting enzyme (ACE) inhibition on the efficiency of the fixed‐time artificial insemination (TAI), 69 goats were divided randomly into two groups: enalapril (n = 35) and control (n = 34). In the experiment, all animals underwent the protocol of fixed‐time artificial insemination for 12 days. Enalapril group received enalapril maleate dissolved in saline (Enalapril, Lab Teuto Ltda) subcutaneously at the following doses: 0.2 mg/kg/day in D0‐D2; 0.3 mg/kg/day in D3‐D6 and 0.4 mg/kg/day in D7‐D11. The control group received the corresponding volume of 0.9% saline solution. We performed a single insemination 36 hr after sponge removal using frozen semen from two adult male goats with recognized fertility. The ultrasound pregnancy diagnosis was 30 days after the artificial insemination (AI). There was significant increase in pregnancy rates and twinning as well as a decrease in foetal loss in animals receiving enalapril (p < .01). The use of ACE inhibitors during the TAI protocol was shown to be a promising alternative to increase the efficiency of such reproductive biotechnology.     

Gonadotropin‐induced Puberty Does Not Impair Reproductive Performance of Gilts over Three Parities

The aim of this study was to evaluate the reproductive performance of three parities of gilts treated or not treated with gonadotropin to induce puberty. Sixty gilts received 600 IU of equine chorionic gonadotropin (eCG) followed by 2.5 mg of porcine luteinizing hormone (LH) 72 h later. Fifty‐nine other gilts were exposed only to a mature boar for 15 min twice daily. Artificial insemination (AI) was performed at 0, 12 and 24 h after the detection of oestrus, and gestation was confirmed by ultrasound after 35 days. Sows were inseminated at the first post‐weaning oestrus. The total numbers of piglets born, piglets born alive, stillborn, mummified foetuses, as well as pregnancy and farrowing rates were evaluated for each of the three parities. Culling rates, farrowing intervals and weaning‐to‐oestrous intervals (WEI) were also analysed. Mean age at puberty and oestrous manifestation were not significantly different between treatments (p = 0.0639; 179.20 ± 17.52 compared with 173.96 ± 16.94, 91.66% compared with 94.92%) across the experimental period. However, females that underwent puberty induction showed modest increases both in the number of total pigs born and in the number of piglets born alive. In conclusion, puberty induction through exogenous gonadotropin administration in field conditions did not induce a more concentrated first oestrous manifestation, but trended to a modest increase in the number of pigs born alive in the first parity and a reduced culling rate during the first gestation.     

Control of early viral and bacterial distribution and disease by natural antibodies

Natural antibodies are often dismissed from immunological analysis as "background," but they may play an important role in conferring immunity against infections. In antibody-free mice infected with various viruses or with Listeria monocytogenes, viral or bacterial titers in peripheral organs, including the kidney and brain, were 10 to 100 times greater than in antibody-competent mice (and enhanced their susceptibility to some infections), and titers in secondary lymphoid organs were 10 to 100 times lower than in antibody-competent mice. Thus, natural antibodies play a crucial role by preventing pathogen dissemination to vital organs and by improving immunogenicity through enhanced antigen-trapping in secondary lymphoid organs.     

Virus-induced acquired immune suppression by cytotoxic T cell-mediated immunopathology.

Acute infection with lymphocytic choriomeningitis virus causes a severe immune suppression in immunocompetent mice by triggering a T cell-mediated immunopathology, probably directed against infected macrophages and dendritic cells. An important role of gamma interferon may be to protect lymphohaemopoietic cells from virus infection and destruction by cytopathic effects of either the virus or of anti-viral immunity.     

Development and validation of species-specific primers that provide a molecular diagnostic for virus-vector longidorid nematodes and related species in German viticulture

The nematode species Longidorus attenuatus, L. elongatus, L. macrosoma and Paralongidorus maximusare economically important pests to the viticulture industry due to their ability to vector two nepoviruses (Raspberry Ringspot Virus and Tomato Black Ring Virus) to grapevines. In Germany, these species occur in vineyard soil with other non-vector but morphologically similar longidorid species, L. helveticus, L.profundorum and L. sturhani. Species-specific primers were designed from ribosomal DNA for all seven species to facilitate taxonomic identification for non-specialists. Primers were assessed for their reliability by screening, where possible, a number of populations of each species. Furthermore, their selectivity and sensitivity were determined when challenged with closely related longidorid species and general nematode communities typical of vineyard soil. A multiplex approach using a common forward primer combined with species-specific reverse primers enabled three target nematode species to be detected in the same PCR reaction. All primers were highly specific, detecting all nematode developmental forms from disparate populations and were sufficiently sensitive to detect a single target nematode within a whole nematode community typical of a vineyard soil comprising of a range of non-target species. Given their specificity, sensitivity and reliability, these diagnostic primers should be of great benefit to both phytosanitary/quarantine services related to the viticulture industry and also as a decision management tool for growers.     

ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors

Cells must amplify external signals to orient and migrate in chemotactic gradient fields. We find that human neutrophils release adenosine triphosphate (ATP) from the leading edge of the cell surface to amplify chemotactic signals and direct cell orientation by feedback through P2Y2 nucleotide receptors. Neutrophils rapidly hydrolyze released ATP to adenosine that then acts via A3-type adenosine receptors, which are recruited to the leading edge, to promote cell migration. Thus, ATP release and autocrine feedback through P2Y2 and A3 receptors provide signal amplification, controlling gradient sensing and migration of neutrophils.