In vitro differentiation of bovine bone marrow‐derived mesenchymal stem cells into male germ cells by exposure to exogenous bioactive factors

Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to self‐renew and give rise to differentiated progeny. Previous studies have reported that MSC may be induced in vitro to develop into different types of specialized cells including male gametes. In vitro gamete derivation technology has potential applications as an alternative method for dissemination of elite animal genetics, production of transgenic animals and conservation of endangered species. This study aimed at investigating the in vitro effect of BMP4, TGFβ1 and RA on the potential for germ cell (GC) differentiation of bovine foetal MSC (bfMSC) derived from bone marrow (BM). The effect of BMP4, TGFβ1 and RA was analysed on the expression of pluripotent, GC and male GC markers on bfMSC during a 21‐day culture period. bfMSC cultured under in vitro conditions expressed OCT4, NANOG and DAZL, but lacked expression of mRNA of VASA, STELLA, FRAGILIS, STRA8 and PIWIL2. Treatment with exogenous BMP4 and TGFβ1 induced a transient increase (p < .05) in DAZL and NANOG mRNA levels, respectively. However, exposure to RA was more effective in increasing (p < .05) expression of DAZL and regulating expression of OCT4 and mRNA levels of NANOG. These data suggest that bfMSC may possess potential for early GC differentiation, where OCT4, NANOG and specially DAZL may play significant roles in controlling progression along the GC lineage.     

Vesicles Cytoplasmic Injection: An Efficient Technique to Produce Porcine Transgene‐Expressing Embryos

The use of vesicles co‐incubated with plasmids showed to improve the efficiency of cytoplasmic injection of transgenes in cattle. Here, this technique was tested as a simplified alternative for transgenes delivery in porcine zygotes. To this aim, cytoplasmic injection of the plasmid alone was compared to the injection with plasmids co‐incubated with vesicles both in diploid parthenogenic and IVF zygotes. The plasmid pcx‐egfp was injected circular (CP) at 3, 30 and 300 ng/μl and linear (LP) at 30 ng/μl. The experimental groups using parthenogenetic zygotes were as follows: CP naked at 3 ng/μl (N = 105), 30 ng/μl (N = 95) and 300 ng/μl (N = 65); Sham (N = 105); control not injected (N = 223); LP naked at 30 ng/μl (N = 78); LP vesicles (N = 115) and Sham vesicles (N = 59). For IVF zygotes: LP naked (N = 44) LP vesicles (N = 94), Sham (N = 59) and control (N = 79). Cleavage, blastocyst and GFP+ rates were analysed by Fisher's test (p < 0.05). The parthenogenic CP naked group showed lower cleavage respect to control (p < 0.05). The highest concentration of plasmids to allow development to blastocyst stage was 30 ng/μl. There were no differences in DNA fragmentation between groups. The parthenogenic LP naked group resulted in high GFP rates (46%) and also allowed the production of GFP blastocysts (33%). The cytoplasmic injection with LP vesicles into parthenogenic zygotes allowed 100% GFP blastocysts. Injected IVF showed higher cleavage rates than control (p < 0.05). In IVF zygotes, only the use of vesicles produced GFP blastocysts. The use of vesicles co‐incubated with plasmids improves the transgene expression efficiency for cytoplasmic injection in porcine zygotes and constitutes a simple technique for easy delivery of plasmids.     

Evaluation of action thresholds for chronic rice insect pests in the Philippines. I. Less frequently occurring pests and overall assessment

Action thresholds as decision tools for insecticide application were developed and tested against the major insect pests of rice at four sites in the Philippines over a 13-year period. Action threshold treatments were compared to the farmers' practice, prophylactic insecticide usage, and an untreated check. Yield loss data using the insecticide check method partitioned yield losses over three crop growth stages in the same test fields. Chronic pests that exceeded action thresholds in 79% of fields were whorl maggot Hydrellia philippina Ferino (Diptera: Ephydridae), defoliators Naranga aenescens Moore and Rivula atimeta (Swinhoe) (Lepidoptera: Noctuidae), leaffolders Cnaphalocrocis medinalis (Guenée) and Marasmia patnalis Bradley (Lepidoptera: Pyralidae), and stemborers Scirpophaga incertulas (Walker) and S. innotata (Walker) (Lepidoptera: Pyralidae). Minor chronic pests reached threshold levels in only one site each: rice bug Leptocorisa oratorius (F.) (Koronadal), whitebacked planthopper Sogatella furcifera (Horvath) (Zaragoza) and green leafhopper Nephotettix virescens (Distant) (Guimba); brown planthopper Nilaparvata lugens (Stål) did not exceed a threshold in any field. Stemborers were the most important pest group in terms of yield loss. Despite the insecticide check method underestimating losses, a mean crop loss of 0.62 t/ha was measured which showed ample scope for corrective action. But loss was evenly distributed across crop growth stages (0.15?–?0.24 t/ha) reducing the impact of insecticides. Action threshold treatments overall outyielded the untreated check, more so in the two sites with highest pest density. The benefit of thresholds was to reduce insecticide usage, as a cost saving. However all the practices showed poor economic returns including the farmers' practice. Farmers' practice employed low insecticide dosages and timing was not consistent with pest damage, but yields were often similar to threshold treatments. Farmers appear to use insecticide more for risk aversion than for profit. The best threshold characters when evaluated against resulting pest density and yield loss criteria showed accuracies >?90% correct decisions. Future work is needed to improve the insecticide response rather than monitoring tools. Thresholds need to be incorporated into improved crop management, which was often found suboptimal by farmers, to take advantage of the high levels of tolerance in modern high tillering cultivars. Crop husbandry practices which improve yield potential such as selection of longer maturing varieties and nitrogen fertilizer may be a more effective pest management strategy than insecticides.     

Dermatoses caused by infestations of immature Ixodes spp. on dogs and cats in Sydney, Australia

Infestations of larval and nymphal Ixodes spp. were identified in 16 dogs and 16 cats from several small animal clinics in Sydney. Cases occurred in late summer or autumn, peaking in February, and were seasonally recurrent in some individuals. Clinical signs of infestation included a papular dermatitis and irritation or pruritus that ranged from severe to mild or absent. The distribution of tick attachment tended to be cranial and ventral, with the face, legs, axillae and ventrum the most commonly affected sites. The estimated number of ticks in each infestation varied from less than 10 to more than 100. Basic morphological examination of ticks collected from affected animals was performed by attending veterinarians using light microscopy, and larvae and nymphs belonging to the Ixodes genus were identified. Ticks collected from 17 animals and submitted to the Department of Medical Entomology, Westmead Hospital were putatively identified as I. trichosuri (57%) and I. holocyclus (25%) larvae. Histopathological samples of attachment sites collected from three dogs and one cat were characterised by ticks attached in well-demarcated invaginations of the skin ('tick craters') associated with variable epidermal and/or dermal necrosis, focal eosinophilic intraspinous pustules, mild to marked eosinophilic and neutrophilic, superficial to deep, dermal perivascular to interstitial inflammation, and moderate to marked superficial dermal oedema and red cell extravasation. A range of topical acaricidal preparations, including fipronil and synthetic pyrethroids, were used for treatment.     

Comparative study of electron microscopical techniques for the detection of scrapie-associated fibrils

Samples of cervical spinal cord and four anatomical regions of the brains of 12 sheep with natural scrapie and six control sheep were examined by electron microscopy, after the tissues had been stored at 4°C and −20°C. The tissues were tested for the presence of scrapie-associated fibrils by a centrifugal extraction technique and by a touch-grid technique. The touch-grid technique was no better than the centrifugal extraction technique for the detection of fibrils. Structures which could have been classified as tubulofilaments were detected in touch-grid preparations without detergent treatment. With the centrifugal extraction technique there was a significant reduction of the fibril scores in some of the tissue extracts stored at −20°C, but not in any of the extracts stored at 4°C. There was, however, a reduction in the fibril scores when the final extracted pellets were stored at 4°C. The stability of the fibrils on the test grids was unaffected by six months storage at room temperature but the clarity of their ultrastructure did deteriorate. Poor hydrophilic spread of the sample on the test grids did not have a significant effect on the fibril scores.     

Effect of forskolin on voltage-gated K+ channels is independent of adenylate cyclase activation

Forskolin is commonly used to stimulate adenylate cyclase in the study of modulation of ion channels and other proteins by adenosine 3',5'-monophosphate (cAMP)-dependent second messenger systems. In addition to its action on adenylate cyclase, forskolin directly alters the gating of a single class of voltage-dependent potassium channels from a clonal pheochromocytoma (PC12) cell line. This alteration occurred in isolated cell-free patches independent of soluble cytoplasmic enzymes. The effect of forskolin was distinct from those of other agents that raise intracellular cAMP levels. The 1,9-dideoxy derivative of forskolin, which is unable to activate the cyclase, was also effective in altering the potassium channel activity. This direct action of forskolin can lead to misinterpretation of results in experiments in which forskolin is assumed to selectively activate adenylate cyclase.     

Restoration of inactivation in mutants of Shaker potassium channels by a peptide derived from ShB

Site-directed mutagenesis experiments have suggested a model for the inactivation mechanism of Shaker potassium channels from Drosophila melanogaster. In this model, the first 20 amino acids form a cytoplasmic domain that interacts with the open channel to cause inactivation. The model was tested by the internal application of a synthetic peptide, with the sequence of the first 20 residues of the ShB alternatively spliced variant, to noninactivating mutant channels expressed in Xenopus oocytes. The peptide restored inactivation in a concentration-dependent manner. Like normal inactivation, peptide-induced inactivation was not noticeably voltage-dependent. Trypsin-treated peptide and peptides with sequences derived from the first 20 residues of noninactivating mutants did not restore inactivation. These results support the proposal that inactivation occurs by a cytoplasmic domain that occludes the ion-conducting pore of the channel.