Effects of gonadotropin-releasing hormone agonist administered in microparticles on sperm quality and quantity, and plasma sex steroid levels in northern pike.

Artificial reproduction of northern pike Esox lucius is impeded by the likelihood of obtaining only a small volume of sperm of inconsistent quality. A controlled-release hormone delivery system has the potential to enhance sperm production while avoiding multiple injections The objective of this study was to investigate the effects of mammalian gonadotropin-releasing hormone agonist (mGnRHa) incorporated into poly(lactic-co-glycolic acid) (PLGA) microparticles on milt production, spermatozoon characteristics, and secretion of 17ß-estradiol (E2), 11-keto testosterone (11-KT), and testosterone in northern pike. Fish were divided into four groups and injected with 2 mg/kg BW carp pituitary extract (CPE), 20 µg/kg BW mGnRHa in PLGA microparticles, or 20 µg/kg BW mGnRHa plus 20 mg/kg BW metoclopramide (MET) in PLGA microparticles (PLGA + MET), along with a control group injected with 1 ml/kg 0.9% NaCl. At 48 h postinjection, the volume of milt produced was significantly greater in groups treated with CPE and PLGA + MET than in other groups. At 96 h postinjection, all hormone-treated groups exhibited significantly higher spermatozoon average velocity than recorded in the control group. Spermatozoon motility was significantly increased (P < 0.05) in the CPE and PLGA groups compared to baseline values. All treated groups showed significantly lower levels of 11-KT after the hormone injection compared to baseline values and to controls. Plasma testosterone levels increased in all hormone-treated groups. The use of PLGA microparticles, with or without metoclopramide, is suitable for use as a carrier of hormone treatments to regulate spermiation in mature northern pike.

Frass produced by the primary pest Rhyzopertha dominica supports the population growth of the secondary stored product pests Oryzaephilus surinamensis, Tribolium castaneum, and T. confusum.

Primary pests such as Rhyzoperta dominica may increase the contents of dockage, dust, and frass in grain mass. Although it has been suggested that frass can affect the population growth of stored product pests and ecological interactions among primary and secondary pests in stored grain, this has not been validated experimentally. Therefore, this work experimentally tested the hypothesis that R. dominica wheat frass may support population increases in secondary pests such as Tribolium confusum, T. castaneum, and Oryzaephilus surinamensis for the first time. The effect of frass on secondary pest performance was compared with the effects of various physical qualities of wheat grain (i.e., intact grain kernels, grain fragments, flour, grain + frass) and an artificially enriched control diet (milled wheat kernels, oat flakes, and yeast). The results showed that the clean intact grain kernels did not support the population growth of any tested species, and the nutrient-rich control diet provided the best support. Frass was a significantly better food medium for O. surinamensis and T. castaneum than flour or cracked grain, while T. confusum performed equally well on flour and frass. Our results showed that in terms of food quality and suitability for the tested species, frass occupied an intermediate position between the optimized breeding diet and simple uniform cereal diets such as cracked grain or flour. The results suggest that (i) the wheat frass of primary pest R. dominica is a riskier food source for the development of the tested secondary pests than intact or cracked wheat grain or flour; (ii) frass has the potential to positively influence interspecific interactions between R. dominica and the tested secondary pests; and (iii) wheat grain should be cleaned if increases in R. dominica populations and/or accumulated frass are detected.

Evaluation of contamination of packages containing cereal-fruit bars by eggs of the pest Indian meal moth (Plodia interpunctella, Lepidoptera) due to perforations in their polypropylene foil packaging.

The quality and type of food packaging affect the level of food protection against pests. This work first evaluated the effect of package perforations on the infestation of cereal-fruit bars by the eggs of the Indian meal moth (Plodia interpunctella). We measured the differential oviposition of moths on the unpackaged bars, empty packages, bars in packages and bars in perforated packages in choice and no-choice experiments. Almost 100% of the laid eggs were laid directly on the bars when they were placed in the enclosure without packaging. A low proportion of the eggs (0.4-3%) were laid on either the empty or non-perforated polypropylene foil packages. Plodia interpunctella efficiently located and infested the bars with eggs when 5 mm package perforations were present in no-choice test. In choice test P. interpunctella preferred to oviposit on open bars than on the packaged or perforated bars. After deposition on the bars the egg hatchability ranged between 40 and 74%. For the food industry, the most significant practical conclusions of this study are that non-perforated packages provide protection against oviposition of P. interpunctella, but small perforations enable the moth to infest the resource with eggs.

Microsatellite markers for Cryptolestes ferrugineus (Coleoptera: Laemophloeidae) and other Cryptolestes species.

Cryptolestes ferrugineus (Stephens, 1831) is an important insect pest of stored products. Due to its broad host range, short life cycle, and high reproductive capacity, this species has rapidly colonized temperate and tropical regions around the world. In this study, we isolated 18 novel polymorphic microsatellite loci from an enriched genomic library based on a biotin/streptavidin capture protocol. These loci will be useful tool to better understand the genetic structure and migration patterns of C. ferrugineus throughout the world. The genetic parameters were estimated based on 80 individual C. ferrugineus from two natural populations. The results revealed that 18 loci were different polymorphic levels. The numbers of alleles ranged from 3 to 12, and eleven loci demonstrated polymorphic information contents greater than 0.5. The observed (H O) and expected (H E) heterozygosities ranged from 0.051 to 0.883 and 0.173 to 0.815, respectively. Five locus/population combinations significantly deviated from Hardy-Weinberg equilibrium. We also demonstrated the potential utility of the C. ferrugineus microsatellites as population and species markers for four additional Cryptolestes species.

Splenic lipidosis in intensively cultured perch, Perca fluviatilis L.

Macroscopically visible lipid deposition varying in size from pinpoint to 8-mm diameter was found in spleens of a population of intensively farmed perch, Perca fluviatilis L. over a 24-month rearing period. Large agglomerates of adipocytes distinguishable from surrounding normal tissue occurred in all individuals with spleen lipidosis. Several affected fish presented total dystrophy of large clusters of hepatocytes. Prevalence of lipidosis was 5.0% at 12 months and 16.6% at 24 months. There was no significant difference between fatty acid profiles of liver or perivisceral fat of perch with and without lipidosis except for linoleic, myristic, γ-linoleic, cis-eicosatrienic, palmitooleic acid. Body weight and hepatosomatic, perivisceral fat and splenosomatic indices were not associated with lipidosis. There was no significant effect of lipidosis on mortality or growth.

Susceptibility of Two Strains of the Confused Flour Beetle (Coleoptera: Tenebrionidae) Following Phosphine Structural Mill Fumigation: Effects of Concentration, Temperature, and Flour Deposits.

In this study, we evaluated phosphine efficacy against two strains of the confused flour beetle, Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae), one laboratory strain, with no previous exposure to phosphine, and one field strain, originated from the flour mill on which we performed the fumigation. The standard Detia Degesh Phosphine Resistance Kit showed that the adults of the field strain of T. confusum required 4.4 × longer time to be knocked down than the laboratory strain. In order to assess the efficacy of phosphine in the field against these strains, aluminium phosphide (AlP) was applied in a flour mill in Czech Republic, in 2014. In this application, temperature among the six floors of the flour mill varied between 20 and 30°C, relative humidity (RH) between 44 and 78%, and phosphine concentration-time-products (CtP) between 24 and 38 g.m(-3).h(-1). Moreover, the insects were bioassayed in dishes that contained either no commodity or 1, 3, and 5 cm of flour. Nevertheless, despite these variations, all adults and larvae from both strains were dead, regardless of the floor, the quantity of the commodity, and the conditions prevailing. However, larval emergence from eggs that were used in the bioassays for both strains was recorded in some of the locations tested. In addition, larval emergence was negatively correlated with both temperature and RH. At the same time, emergence was generally similar between strains. The results of the present study illustrate that highly visible dead adults and larvae after the application of phosphine falsely imply good fumigation efficacy, given that a considerable number of eggs are very likely to survive in a wider range of conditions, and the concomitant larval emergence may result in rapid population grown right after the fumigation.

COI barcode based species-specific primers for identification of five species of stored-product pests from genus Cryptolestes (Coleoptera: Laemophloeidae).

Flat grain beetles of the genus Cryptolestes (Coleoptera: Laemophloeidae) are one of the economically most important stored-product pests which feed on many kinds of agricultural products, especially grains. Nine of more than 40 described Cryptolestes species are recognized as stored-product pests and two of the pest species have a cosmopolitan distribution. Given the rapid growth in global trade of food products, ecological barriers to the spread of pests are easily overcome. Therefore, development of reliable systems for routine quarantine inspection and early infestation detection is vital. In the present study, we established a new rapid and accurate cytochrome c oxidase subunit I-based system for molecular identification of five common stored-product Cryptolestes species, namely, Cryptolestes capensis, Cryptolestes ferrugineus, Cryptolestes pusilloides, Cryptolestes pusillus and Cryptolestes turcicus. Five species-specific primer pairs for traditional uniplex polymerase chain reaction assay are described and their specificity and sensitivity for the identification process is evaluated using larval samples of 12 different populations from three continents (Asia, Europe and North America).

DNA barcoding of five common stored-product pest species of genus Cryptolestes (Coleoptera: Laemophloeidae).

Several species of the genus Cryptolestes Ganglbauer, 1899 (Coleoptera: Laemophloeidae) are commonly found in stored products. In this study, five species of Cryptolestes, with almost worldwide distribution, were obtained from laboratories in China, Czech Republic and the USA: Cryptolestes ferrugineus (Stephens, 1831), Cryptolestes pusillus (Schönherr, 1817), Cryptolestes turcicus (Grouvelle, 1876), Cryptolestes pusilloides (Steel & Howe, 1952) and Cryptolestes capensis (Waltl, 1834). Molecular identification based on a 658 bp fragment from the mitochondrial DNA cytochrome c oxidase subunit I (COI) was adopted to overcome some problems of morphological identification of Cryptolestes species. The utility of COI sequences as DNA barcodes in discriminating the five Cryptolestes species was evaluated on adults and larvae by analysing Kimura 2-parameter distances, phylogenetic tree and haplotype networks. The results showed that molecular approaches based on DNA barcodes were able to accurately identify these species. This is the first study using DNA barcoding to identify Cryptolestes species and the gathered DNA sequences will complement the biological barcode database.

Sensory and textural attributes and fatty acid profiles of fillets of extensively and intensively farmed Eurasian perch (Percafluviatilis L.).

Sensory attributes, texture and fatty acid profiles of fillets of Eurasian perch (Percafluviatilis L.) reared under two conditions were compared. Perch were reared either in an extensive pond-based (EC) system in polyculture with carp, or intensively cultured (IC) in a recirculation system. Attributes of raw and cooked fillets of marketable perch (120-150g) were compared. No significant differences were found between groups for odour, flavour, aftertaste, or consistency in subjective evaluation of cooked fillets. The texture profile analysis (TPA) showed raw fillets from the EC group to exhibit higher values of hardness, springiness, cohesiveness, and gumminess than the IC group. Fish from the IC group had a lower content of saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA) and a higher content of monounsaturated fatty acids (MUFA) in comparison to EC perch. The proportion of iso- and anteiso-SFAs was 2.6% in the EC group and 0.75% in the IC group. The content of n-3 PUFA was lower in IC than in EC, while the content of n-6 PUFA was higher in IC than in EC. The ratio of n-3:n-6 PUFA was 1.42 for the IC group and 2.85 for the EC group.

Lymphocyte proliferative response to Helicobacter pylori antigens in H. pylori-infected patients.

Helicobacter pylori (Hp) contributes to the development of gastric and extra-gastric diseases such as autoimmune thyroiditis (AT), and causes persistent life-long infection despite local and systemic immune response. We determined the specific cellular immune response to Hp antigens and PWM (control mitogen) in two groups of Hp infected patients--group A (n = 21), involving patients with autoimmune thyroiditis and group B (n = 13) of patients without AT--using modified lymphocyte transformation test before and after eradication therapy in comparison with healthy controls (group C, n = 15). Immune reactivity to the majority of Hp antigens (aHp, hHp, HpAg, CagA) was significantly lower in group B before eradication therapy in comparison with healthy Hp negative controls. A significant increase in immune reactivity was observed in group B to certain Hp antigens after successful eradication. The same levels (but insignificant) of immune reactivity were shown in group A. Our results indicate that Hp can cause the inhibition of the specific cellular immune response in Hp infected patients with or without autoimmune diseases such as AT, which can be abrogated by successful eradication of Hp. Lymphocyte transformation test appears to be a good tool for detection of immune memory cellular response in patients with Hp infection.

Dynamics of ATP and movement in Eurasian perch (Perca fluviatilis L.) sperm in conditions of decreasing osmolality.

Repetitive activation of perch (Perca fluviatilis L.) sperm motility was investigated in this study. The first phase of sperm motility activation was initiated by dilution in a 260 mM glucose solution (75% motility). The second phase of motility was achieved by adding water to previously activated sperm, so that the glucose concentration dropped to 220 mM (24% motility). Finally, the third phase was obtained by further addition of water (down to 90 mM glucose) to the activated sperm suspension (15% motility). Parallel measurements of sperm ATP content were also made. The median value for nonactivated sperm was 43.9 nmol ATP/10(9) spermatozoa. The ATP concentration decreased significantly from 35 to 7 nmol ATP/10(9) spermatozoa after successive activations of motility in the above glucose solutions. Sperm velocity ranged in value from 25 to 330 microm/sec at 10 sec postactivation, from 10 to 290 microm/sec at 30 sec, and from 0 to 200 microm/sec at 45 sec. A model postulating several classes in the population of spermatozoa is developed, tentatively accounting for such successive activation. Possible further application of multiple sperm activation is discussed.

Mites and fungi in heavily infested stores in the Czech Republic.

Toxigenic and allergen-producing fungi represent a serious hazard to human food and animal feed safety. Ninety-four fungal species were isolated from mite-infested samples of seeds taken from Czech seed stores. Fungi were isolated from the surface of four kinds of seeds (wheat, poppy, lettuce, and mustard) and from the gut and external surface of five species of mites (i.e., Acarus siro L., 1758, Caloglyphus rhizoglyphoides (Zachvatkin, 1973), Lepidoglyphus destructor (Schrank, 1781), Tyrophagus putrescentnae (Schrank, 1781) and Cheyletus malaccensis Oudemans 1903) separately. Multivariate analysis of fungi complex composition showed that the frequency of fungal was species significantly influenced by the kind of seed. Fungal frequencies differed between mites gut and exoskeleton surface and between the surfaces of mites and seeds. Three groups of fungal species were recognized: 1) mite surface-associated fungi: Penicillium brevicompactum, Alternaria alternata, and Aspergillus versicolor; 2) mite surface- and seed-associated fungi: Aspergillus niger, Penicillium crustosum, Penicillium aurantiogriseum, Penicillium chrysogenum, and Aspergillus flavus; and 3) seed-associated fungi: Cladosporium herbarum, Mucor dimorphosporus f. dimorphosporus, Botrytis cinerea, Penicillium griseofulvum, and Eurotium repens. Mite-carried species of microfungi are known to produce serious mycotoxins (e.g., aflatoxin B1, cyclopiazonic acid, sterigmatocystin, ochratoxin A, and nephrotoxic glycopeptides) as well as allergen producers (e.g., A. alternata and P. brevicompactum). Storage mites may play an important role in the spread of some medically hazardous micromycetes. In addition, these mite-fungi associations may heighten the risk of occurrence of mycotoxins in food and feed stuffs and cause mixed contamination by fungal and mite allergens.

[Effect of heavy metals on immune reactions in patients with infertility]. / Vliv tezkých kovu na imunitní reakci u pacientu s prokázanou neplodností.

BACKGROUND: Heavy metals can negatively influence reproduction because in sensitive persons they are able to alter the immune reactions including autoantibodies production. The altered immune reaction can then cause infertility. METHODS AND RESULTS: The in vitro lymphocyte reaction after stimulation with metals, the production of interferon (IFN-gamma) and antisperm antibodies in supernatants after lymphocyte stimulation in patients with infertility and with the antisperm antibodies present in their serum were investigated. The cause of antisperm antibodies presence was not determined. The diagnosis of metal intolerance was performed by the proliferation method modified for metals (Melisa). In supernatants of tissue cultures of lymphocytes without the antigen stimulation and after stimulation with mercury chloride, the in vitro production of gamma interferon and antisperm antibodies was studied by Elisa. More than 50% of patients did not tolerate mercury, iron, aluminium and silver. When the lymphocyte reaction was compared in patients with and without mercury intolerance we found that lymphocytes of patients with mercury intolerance produced less gamma interferon and more antisperm antibodies in supernatants after mercury stimulation of lymphocytes. CONCLUSIONS: In patients with metal intolerance diagnosed by the Melisa test, metal ions released from the dental materials can represent a factor, that does not cause infertility but is able to influence it negatively.

HLA-association in patients with intolerance to mercury and other metals in dental materials.

A group of selected 25 patients with serious intolerance to heavy metals used for dental restoration were examined for HLA antigens. A significant increase for HLA -- B37, B47 and DR4 was found. The value of the relative risk is not significant after correction for the number of antigens tested and therefore further studies of more patients are needed.

In vitro lymphocyte proliferation in the diagnosis of allergy to phenoxymethylpenicillin.

BACKGROUND: The aim of this study was to investigate in vitro lymphocyte proliferation in the diagnosis of allergy to phenoxymethylpenicillin (PcV), comparing chemically reactive PcV, added to cell cultures in unconjugated form, to a PcV-PLL (poly-L-lysine) conjugate as antigens. Side-chain specificity of lymphoproliferative responses was investigated with reactive benzylpenicillin (PcG) and bacampicillin. METHODS: Seventeen patients with a history of hypersensitivity reactions in connection with PcV treatment were studied by means of the lymphocyte transformation test (LTT), the radioallergosorbent test (RAST), skin tests (prick and intracutaneous), and oral challenge with PcV. LTT was also performed in 20 control subjects exposed to PcV therapeutically, and in eight subjects with occupational exposure to this penicillin. RESULTS: Nine patients had a positive in vivo test to PcV (five by oral challenge, three by intracutaneous test, and one by both tests), and six were challenge-negative. When reactive PcV was used as antigen in LTT, positive LTT responses were observed in five of the nine patients with a positive in vivo test, and two of them were also side-chain specific. Positive LTT responses with reactive PcV also correlated with a positive RAST in five of seven subjects. None of the six patients with a negative challenge test, and only one of the 28 controls showed a positive LTT result with reactive PcV. Thus, the specificity of LTT with reactive PcV was 96%. In contrast, when PLL-conjugated PcV served as antigen, four challenge-negative subjects and 11 controls were LTT-positive. CONCLUSIONS: The results of this study indicate that LTT with chemically reactive PcV could be useful as an in vitro complement in the diagnosis of PcV allergy and as a tool to reveal the side-chain specificity of peripheral blood lymphocytes. A positive LTT to PLL-conjugated PcV may be an indicator of immunization, but not necessarily allergy, to the penicilloyl structure.

Mercury-specific lymphocytes: an indication of mercury allergy in man.

In this study, 18 patients with oral lichen planus (OLP), adjacent to amalgam fillings, were tested in vitro with an optimized lymphocyte proliferation test, MELISA (memory lymphocyte immunostimulation assay) and with a patch test. Twenty subjects with amalgam fillings but without oral discomfort and 12 amalgam-free subjects served as controls. The results show that patients with OLP have significantly higher lymphocyte reactivity to inorganic mercury, a corrosion product of amalgam, compared to control groups. Removal of amalgam fillings resulted in the disappearance of oral mucosal changes, thus indicating a causal relationship. Positive responses to phenylmercury (phenyl-Hg), a bactericidal agent in root fillings and in pharmaceutical preparations, were also noted in the oral lichen group but not in the control groups. Thus, low-grade chronic exposure to mercury may induce a state of systemic sensitization as verified by Hg-specific lymphocyte reactivity in vitro.