High‐throughput Cryopreservation of Sperm from Sex‐reversed Southern Flounder,Paralichthys lethostigma

The Southern flounder, Paralichthys lethostigma, is a valuable aquaculture fish with established markets in the USA. All‐female production in this species is an important technology for aquaculture because the females usually have body sizes twice those of males at the same age, and sex‐reversed males (genotypic XX neomales) are used for all‐female production by crossing with genetically normal females. However, sperm volume from the neomales is usually small (<0.5 mL) and limits their application for all‐female fish production. Cryopreservation of sperm from these sex‐reversed neomales will provide access on demand with increased efficiency to extend the application of neomales. The goal of this study was to develop a protocol for cryopreservation of sperm from the Southern flounder by using an automated high‐throughput processing system. The objectives were to: (1) determine the effect of osmolality on activation of sperm motility; (2) evaluate the effect of extender solutions on sperm motility capacity; (3) evaluate the acute toxicity of cryoprotectants (dimethyl sulfoxide [DMSO], propylene glycol, and polyethylene glycol) on sperm motility, and (4) estimate the effect of cooling rate on sperm cryopreservation and post‐thaw fertilization. Sperm motility was activated when osmolality was 400 mOsmol/kg or higher. Of the three extender buffers tested, HEPES4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) at 300 mOsmol/kg resulted in better protection for sperm motility than did Hanks' balanced salt solution and Mounib solution at 300 mOsmol/kg during 7 d of refrigerated storage. After 30 min equilibration with the cryoprotectant of 15% DMSO, sperm motility was 24 ± 21% (fresh sperm motility without any cryoprotectants was 42%). After cooling at a rate of 20 C/min, post‐thaw sperm motility was 8 ± 5% and fertilization was 63 ± 40% evaluated at the 32–64 cell stage (5 × 10⁵ sperm per egg). Overall, a protocol was developed for sperm cryopreservation in the Southern flounder with high‐throughput processing, which provides a tool to preserve the valuable genetic resources from neomale flounders, and enables germplasm repository development for the Southern flounder.     

Histomorphometry and quantification of nucleolar organizer regions in bovine thyroid containing methylthiouracil residues

In order to study the morphology and morphometry and to characterize and quantify the nucleolar organizer regions (NORs) of bovine thyroids containing methylthiouracil (MTU) residues, five animals were orally treated with a suspension of MTU (5 g/animal/day) for 20 days (group A). This treatment protocol was interrupted 5 days before the the animals were slaughtered. Six animals receiving placebos composed group B. A third group (group C) was composed of normal thyroids obtained from a slaughterhouse. All glands were previously assessed for detection of antithyroid residues by chromatography, and only those glands from MTU-treated animals were positive. Follicles of glands from group A showed wide variation in size and shape. There was a predominance of small follicles covered by multiple layers of columnar cells, sometimes forming papillary projections into the lumen, characterizing severe interfollicular and intrafollicular adenomatosis. Many follicles had vacuolated cells with nuclei showing karyolysis or pyknosis and reduced amounts of a low-density and very excavated colloid. They also showed higher follicular epithelia and larger proportions of their structural components when compared with glands of groups B and C. In the thyroids from group A, the argyrophilic nucleolar organizer region-associated proteins (AgNORs) were greater in number, with small ones scattered all over the nucleus. Although the size of AgNORs in thyroids from groups B and C was variable, these AgNORs were fewer and larger than were those in glands from group A. In conclusion, the MTU induces proliferation and regressive changes in follicular cells, and the AgNOR technique is efficient to distinguish different degrees of thyroid hyperplasia.     

SDS-PAGE and Western blot of urinary proteins in dogs with leishmaniasis

Canine leishmaniasis is an endemic disease in the Mediterranean area caused by the protozoan Leishmania infantum, which usually produces renal failure. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot using antibodies to IgG and IgA from dogs were carried out in the urine of 22 dogs with leishmaniasis diagnosed by ELISA and confirmed by PCR, and 20 healthy dogs. The results were compared to renal function laboratory tests and to those from a histopathological study of the kidneys from sick animals that died naturally or were euthanized. Five different bands with molecular weights ranging from 10 to 110 kDa were obtained from the electrophoresis of the urine of healthy dogs. 33.5% of total proteins corresponded to low molecular weight proteins and the other proteins had middle and high molecular weights. However, in the group with leishmaniasis, a maximum of 11 different bands with molecular weights ranging from 10 kDa to 150 kDa were displayed in the electrophoresis of the urine. The urine electrophoretic pattern in the sick dogs was classified as mixed (proteins with high and low molecular weights) because low molecular weight proteins made up 57.9% and the rest of the proteins had middle and high molecular weights. In Western blot, none of the healthy dogs showed excretion of IgG and/or IgA, whereas IgG and IgA were detected in the Western blot of urine of 68% and 55% respectively of dogs with leishmaniasis. The results obtained in the leishmaniasis group agreed with glomerular and tubular damage, which were confirmed by the histopathological findings.     

Effects of low-intensity pulsed ultrasound on wound healing in corneas of dogs following keratoplasty

The effects of low-intensity pulsed ultrasound on wound healing were evaluated at the graft-cornea transition in dogs following lamellar keratoplasty using tunica vaginalis preserved in 98% glycerin. Twenty-one dogs were subdivided into three groups of seven animals. The first group (W/US) received daily treatment of low-intensity pulsed ultrasound (20 mW/cm2) for 15 min for the first 10 days post surgery. The second group (N/US) was submitted to the same procedure but with the ultrasound apparatus turned off. The third group, the control (CO), underwent the surgical procedure only. The animals were clinically evaluated during the initial (1-15 days), intermediate (16-30 days) and late (31-120 days) postoperative period. The corneas were evaluated by light microscopy at 1, 3, 7, 15, 30, 60 and 120 days after surgery. Clinically, there were no differences which would promote an advantage to any of the treatments. Light microscopy, however, revealed more extensive vascularization and more advanced wound healing in the W/US group, as well as a tendency towards early graft incorporation. Based on the present results, low-intensity pulsed ultrasound shows advantages, especially in situations where trophic support is a mandatory condition, facilitating better graft incorporation and rapid recovery of stromal organization.     

Herbicide resistance in Aster squamatus conferred by a less sensitive form of acetolactate synthase

A biotype of Aster squamatus (Sprengel) Hieronymus with suspected resistance to the ALS-inhibiting herbicide imazapyr was detected in a chicken farm in the province of Seville, Spain, which had been treated once a year with imazapyr for 10 years. Resistance to imazapyr in this biotype was studied using dose-response experiments, absorption and translocation assays, metabolism studies and ALS activity assays. The rate of imazapyr required to inhibit A squamatus growth by 50% (ED50) was 15 times higher for the R (resistant) than for the S (susceptible) biotype. Cross-resistance existed for the ALS-inhibitors imazamox, imazethapyr, amidosulfuron, nicosulfuron, rimsulfuron, triasulfuron and tribenuron, but not for bensulfuron. Control of A squamatus using alternative herbicides was poor with clopyralid, intermediate with quinclorac, amitrole and MCPA, and excellent with 2,4-D, glufosinate and glyphosate. Absorption of [14C]imazapyr increased over time for both the R and S biotypes, and translocation from the treated leaf to shoots and roots was similar in both biotypes, with most of the radioactivity remaining in the treated leaf. No metabolites of imazapyr were detected in either biotype. Sensitivity of the ALS enzyme (target site) to imazapyr was lower for the R biotype (I50(R) = 4.28 x I50(S)). The mechanism of imazapyr resistance in this R biotype appears to be an altered ALS conferring decreased sensitivity to imazapyr at the whole-plant level.     

A follow-up of Beagle dogs intradermally infected with Leishmania chagasi in the presence or absence of sand fly saliva

In this study, we compare the development of infection and/or disease in Beagle dogs intradermally infected with Leishmania chagasi, in the presence or absence of Lutzomyia longipalpis saliva, with those of intravenously infected animals.Spleen samples of all the animals inoculated with parasites had positive polymerase chain reaction tests for Leishmania DNA. Positive spleen cultures for Leishmania were detected earlier (P < or = 0.018) and were more frequent (five out of the five animals) in intravenously infected animals than in the intradermally infected animals, in presence (two out of the six animals) or absence (three out of the five animals) of salivary gland lysate of L. longipalpis. Significant increase in serum antibodies against Leishmania was observed only in the intravenously infected group (P = 0.004). In addition, dogs with infection confirmed by isolation of amastigotes or detection of parasite DNA were, nevertheless, negative for anti-Leishmania antibodies up to 5 months or more after infection. Only animals of the intravenously infected group developed progressive decreases in hematocrit (Pearson r = -0.8076, P = -0.0026) and hemoglobin (Pearson r = -0.8403, P = 0.0012) during the infection period. No significant difference in the course of infection was observed between groups of intradermally infected animals. The data presented herein confirms that the intradermal inoculation of dogs with Leishmania produces an asymptomatic form of infection. It also fails to show an advantage in using L. longipalpis saliva as an infection-enhancing agent in experimental canine leishmaniasis.     

Disseminated pythiosis in three horses

Three cases of equine subcutaneous pythiosis with dissemination to the internal organs were investigated. The subcutaneous lesions were observed on the mammary gland, nostrils and limbs of the infected horses. Histopathological analysis of the infected tissues revealed a strong eosinophilic reaction, with macrophages, mast cells and giant cells. Sparsely septated hyphal filaments of 4-6 microm diameter were identified in the center of the eosinophilic areas. Specific fluorescent antibody against Pythium insidiosum confirmed the hyphae in the infected tissues in three examined horses. In one of the three cases, the DNA sequences amplified from the infected subcutaneous tissues and internal organs, revealed that P. insidiosum's 18S SSU rDNA amplicons shared 100% identity with those sequences deposit in GenBank. This is the first report confirming by immunochemical and genetic techniques that P. insidiosum can disseminated from superficial to deep structures.     

The attenuated sopB mutant of Salmonella enterica serovar Typhimurium has the same tissue distribution and host chemokine response as the wild type in bovine Peyer's patches

Salmonella enterica serovar Typhimurium is an important cause of enteric infections in farm animals and it is one of the most frequent food borne infections worldwide. Serovar Typhimurium lacking the sopB gene is attenuated for induction of host inflammatory response and fluid accumulation into the intestinal lumen, which correlates with clinical diarrhea. SopB is an inositol phosphate phosphatase, but its exact role in the pathogenesis of salmonellosis is still unclear. We employed the bovine ileal ligated loop model to compare the tissue distribution of a sopB mutant and its wild type parent serovar Typhimurium. Sections of the Peyer's patches were histologically processed and immuno-stained for detection of serovar Typhimurium. In addition, samples were processed for transmission electron microscopy, and the profile of expression of host chemokine and cytokine responses was assessed. Ultrastructurally both strains had the same ability to invade intestinal epithelial cells. No differences were detected in the tissue distribution of the sopB mutant and the wild type organism and both strains elicited the same profile of chemokines and pro-inflammatory cytokines. In conclusion, our results indicate that the attenuation of the sopB mutant is associated with pathogenic mechanisms other than invasion and distribution in host intestinal tissues.     

Evaluation of metabolic detoxifying enzyme activities and insecticide resistance in Frankliniella occidentalis

The western flower thrips Frankliniella occidentalis (Pergande) is a very significant pest of a number of different agricultural crops in the south-east of Spain. The importance of thrips as a pest is not due mainly to the direct damage inflicted on the plant, but to the loss in commercial value which occurs as a consequence of the development of dark spots caused by the tomato spotted wilt virus (TSWV) which they transmit. The economic threshold is therefore almost zero, which enhances the problems of resistance management. The present work is part of a global project that attempts to evaluate the status of insecticide resistance in field populations of thrips obtained from several agricultural crops. We have studied, in either individual or pooled insects, some enzyme systems classically related to detoxification of insecticides: esterase and glutathione-S-transferase (GST). The activity of these enzymes from laboratory populations selected with various classes of insecticides has also been measured using several appropriate substrates. An increase in GST mean activity was found in two field-collected strains. Differences in frequency distributions of esterase and GST activities were found for both field-collected strains and for a laboratory strain selected with acrinathrin. These activities were compared with those of a wild-type reference strain.