Priming as a Mechanism in Induced Systemic Resistance of Plants

Induced systemic resistance is a plant defence state that is associated with an enhanced ability – the so-called priming – to resist pathogen attack by stronger activation of cellular defence responses. So far, however, priming has not been widely appreciated when studying induced plant disease resistance. During the past several years, it has been demonstrated that pre-treatment of cultured parsley cells with inducers of systemic resistance, salicylic acid or a benzothiadiazole, leads to the direct activation of a set of defence-related genes and also primes the cells for stronger elicitation of another set of defence genes including those encoding phenylalanine ammonia-lyase. From these results, it was concluded that the resistance inducers have at least a dual role in plant defence-gene activation. When elucidating whether priming plays a role in induced systemic resistance of Arabidopsis, pre-treating plants with benzothiadiazole was found to augment the subsequent activation of phenylalanine ammonia-lyase genes by Pseudomonas infection, wounding and osmotic stress and also to enhance wound/osmotic stress-induced callose production. The augmentation of phenylalanine ammonia-lyase gene activation or/and callose deposition was not seen in the Arabidopsis non-expresser of pathogenesis-related genes1 mutant which is compromised in induced resistance, while it was present, without benzothiadiazole pre-treatment, in the constitutive expresser of pr genes1 and 5 mutants in which induced resistance is constitutive. Together these studies point to priming as an important cellular mechanism in induced systemic resistance of plants which requires the intact non-expresser of pathogenesis-related genes1 gene.     

The Effect of Oxidative Stress on Thawed Bulk‐Sorted Red Deer Sperm

The aims of this study were to assess the effects of the sex‐sorting process on post‐thaw sperm quality as well as on induced oxidative stress damage (H₂O₂ 0 mm  = H000; H₂O₂ 50 mm  = H050; H₂O₂ 100 mm  = H100) and the protective action of reduced glutathione (GSH) and Trolox, when comparing sorted (BSS) and non‐sorted (NS) red deer spermatozoa incubated at 37°C. Sperm samples from three stags were collected by electroejaculation and frozen. Immediately after thawing, sperm motility was higher (p < 0.05) for NS (59% ± 3.3) than BSS (36.9% ± 5.8) sperm. Furthermore, the percentage of apoptotic sperm was higher (p < 0.05) for BSS (21.6% ± 5.0) than NS sperm (14.6% ± 1.2). The presence of H₂O₂ increased DNA damage in NS (H000 = 4.1% ± 0.9; H050 = 9.3% ± 0.7; and H100 = 10.9% ± 2.3), but not in BSS sperm. However, in the presence of oxidant, GSH addition improved (p < 0.05) sperm motility in both groups of sperm samples as compared to their controls (NS: 44.5 ± 4.8 vs 21.1 ± 3.9 and BSS: 33.3 ± 8.1 vs 8.9 ± 1.8). These results demonstrate that the sperm‐sorting process induces sublethal effects, albeit selecting a sperm population with a chromatin more resistant to oxidative stress than that in non‐sorted sperm. Moreover, addition of GSH at 1 mm may be a good choice for maintaining the quality of stressed sperm samples, unlike Trolox, which inhibited sperm motility.     

An In Vitro Evaluation of Biochemical Processes Involved in Lead‐Induced Changes on Ram Spermatozoa

Lead (Pb²⁺) is a toxic heavy metal which interferes with several physiological processes regulated by Ca²⁺, including those characterized by changes of the membrane stability and the motility of spermatozoa necessary for the fertilization of the oocyte. In this study, ejaculated sperm from six rams (Ovis aries) have been incubated in vitro with or without 50 ng Pb²⁺/ml during 30 min and in the presence or absence of three different potential modulators of the effects of Pb²⁺ on changes in the sperm membrane before fertilization: charybdotoxin, quinacrine and staurosporine. Sperm samples incubated with Pb²⁺ have shown significant reductions in acrosome integrity and sperm viability and an increase in progressive movement. None of the studied potential modulators had a protective effect against Pb²⁺ action. On the contrary, Pb²⁺‐incubated sperm in the presence of staurosporine had lower acrosome integrity, and lower sperm viability was observed when spermatozoa were incubated with Pb²⁺ + charybdotoxin. Quinacrine was the only tested substance capable of increasing the concentration of Pb²⁺ in spermatozoa; thus, the enhancement of Pb²⁺ effects produced by staurosporine and charybdotoxin was not produced by an increased uptake of Pb²⁺ by spermatozoa. However, the increase of intracellular Pb²⁺ in those spermatozoa incubated with quinacrine did not result in an adverse effect on sperm motility or viability although the acrosome integrity was negatively affected.     

Selection of red deer spermatozoa with different cryoresistance using density gradients

The objective of sperm selection media is selecting the best spermatozoa and to remove seminal plasma and diluent for using them in assisted reproductive techniques. It is known that individuals show different cryoresistance in response to the same freezing procedure. Our hypothesis was that the efficacy of selection media could be dissimilar for samples with different sperm quality after thawing. Epididymal sperm samples from mature Iberian red deer were collected and frozen. Males were classified as with high post‐thaw sperm quality when sperm motility (SM) ≥ 70%, or as with low post‐thaw sperm quality when SM ≤ 69%. Samples were centrifuged using the following density gradients (DG): Percoll^®, Puresperm^® and Bovipure^?, and several functional sperm parameters were assessed after sperm selecting and washing. Males classified with high sperm quality had higher post‐thawing values (p > .05) for all parameters evaluated, except for linearity index, than those categorized as low sperm quality. After selection, some sperm characteristics improved (viability, apoptosis and mitochondrial activity) for both groups, showing the males with high sperm quality higher values in all sperm parameters except for kinematic traits and DNA fragmentation index (%DFI), regardless of DG. Bovipure^? yield lower values of sperm motility, viability, apoptosis and mitochondrial activity in relation to Percoll^® and Puresperm^® considering both quality groups. There was an interaction between the type of DG and sperm quality group for sperm viability (p = .040) and apoptosis (p = .003). Thus, Percoll^® selected less live and more apoptotic spermatozoa than Puresperm^® and Bovipure^? for males with low sperm quality. In conclusion, the DG are more efficient selecting spermatozoa from samples with high sperm quality, acting differently depending on initial sperm quality.     

Luteolytic Effect of Prostaglandin F2α on Bovine Corpus Luteum Depends on Cell Composition and Contact

Prostaglandin F_2α (PGF_2α) is a main luteolytic factor in vivo; however, its direct luteolytic influence on steroidogenic cells of bovine corpus luteum (CL) is controversial and not fully understood. The aim of the study was to clarify PGF_2α action on bovine CL in different in vivo and in vitro conditions and to examine whether the contact among all main types of CL cells is necessary for luteolytic PGF_2α action. In experiment 1, the bovine CL (day 15 of the oestrous cycle) was perfused using in vivo microdialysis system with dinoprost (an analogue of PGF_2α) for 0.5 h. Dinoprost caused a short‐time increase in progesterone (P4), whose concentration decreased thereafter (at 6‐, 10‐, 12‐ and 24‐h after treatment). In experiment 2, the direct effect of PGF_2α on P4 accumulation in CL steroidogenic cells cultured in monolayer (day 15 of the cycle) was determined. PGF_2α after 24 h of incubation increased P4 accumulation in steroidogenic CL cells. In experiment 3 steroidogenic, endothelial CL and immune cells (day 15 of the cycle) were incubated with PGF_2α in cocultures for 24 h in glass tubes and the levels of P4, stable metabolites of nitric oxide (NO) and leukotriene (LT) C₄ were determined. Although PGF_2α treatment increased P4 secretion in homogeneous steroidogenic CL cell culture, the decrease in P4 secretion in cocultures of all types of CL cells was observed. The secretion of NO and LTC₄ increased after the treatment of PGF_2α both in pure cultures of CL cells and in cocultures. The interactions between endothelial and immune cells with steroidogenic CL cells are needed for luteolytic PGF_2α action within the bovine CL. Our results indicate that the cell coculture model, including the main types of CL cells, is the most approximate to study PGF_2α role in vitro.     

A Study on Postpartum Metritis in Iraqi Buffalo Cows: Bacterial Causes and Treatment

The objectives of the present study were to determine the relationship between bacteriological findings, clinical signs and histopathological changes in postpartum metritis. Evaluation of the treatment efficiency of using systemic or intra‐uterine infusion of antibiotics with some hormonal preparations for the treatment of postpartum metritis. Data were collected from 50 buffalo cows with history of calving of more than 1 month. All buffaloes were subjected to detailed clinical examination including external inspection, vaginoscopy and transrectal palpation of the cervix, uterus and ovaries. Swabs for bacteriology and biopsies for histopathology were collected from uterine lumen from each buffalo included in the present study. Bacteria identified using API systems following aerobic and anaerobic cultures. Vaginal mucus scored for character, odour and estimation of polymorphonuclear cells (PMNs). Treatment conducted using oxytetracycline in local intrauterine infusion or systemically with hormonal treatment including prostaglandinF2α (PGF2α) and oestradiol benzoate. Results revealed that the most predisposing factor for postpartum uterine infection was retained placenta and toxic puerperal metritis. The most prevalent bacteria in uterine lumen were Escherichia coli, Archanobacterium pyogenes, Bacteroides fragilis and Fusobacterium necrophorum the most prevalent bacteria in buffaloes with postpartum metritis. A. pyogenes and F. necrophorum were an important pathogens causing severe uterine inflammation as found in histopathological examinations. Buffaloes with postpartum metritis showed good clinical cure when oxytetracycline injected systemically with PGF2α. Intrauterine infusion of oxytetracycline had no advantage for the treatment of uterine infection in buffalo cows with postpartum metritis. PGF2α improved clinical cure of buffaloes with postpartum metritis.     

Serial Transrectal Ultrasonography for Monitoring the Reproductive Activity of the Asiatic Black Bear (Ursus thibetanus ussuricus)

This study evaluated the structural changes in the reproductive tract of Asiatic black bears using serial transrectal ultrasonography. In addition, the ultrasonographic observations were compared with the results of vaginal cytology and hormonal analyses. The collection of blood for hormonal analysis, vaginal cytology and transrectal ultrasonography was performed in two bears (Bears 1 and 2) from June 2011 to August 2013 without mating and in a third bear (Bear 3) from April to December 2012, allowing natural mating. Serial ultrasonographic observations showed cyclic changes in ovarian structures (e.g. emergence of small follicles, growth and ovulation of dominant follicles and corpus luteum (CL) formation) during the reproductive cycles of the three bears. The diameter of the uterine horns remained similar throughout the reproductive cycle in Bears 1 and 2, and it remained similar from April until October, but an enlargement containing foetuses was observed in Bear 3 in December. The ultrasonographic observations were consistent with the data obtained through vaginal cytology and progesterone analysis during the reproductive cycle. An average of 4.0 (±0.4) dominant follicles was observed during the oestrous stage (May‐August), during which the superficial cells accounted for >90% of the total vaginal cells. In addition, the detection of an average of 2.6 (±0.2) CL was associated with increased plasma progesterone concentrations (3.0 ± 0.4 ng/ml) between June and December (near hibernation). In conclusion, serial transrectal ultrasonography demonstrated yearly oestrous (ovulation) cycles via follicular dynamics and CL formation on ovaries, accordingly with vaginal cytology and hormonal level in the Asiatic black bear.     

Competitive direct enzyme-linked immunosorbent assay for detection of sulfamethazine residues in swine urine and muscle tissue

A sensitive assay for the detection of sulfamethazine in swine urine and muscle tissue using a direct competitive enzyme-linked immunosorbent assay (ELISA) has been developed. Undiluted urine or a phosphate-buffered saline extract of pork muscle tissue is mixed with an enzyme-labeled conjugate of sulfamethazine and horseradish peroxidase. The mixture is added to wells of a microtiter plate coated with antibody to sulfamethazine. After the test system is incubated, washed, and re-incubated with substrate and the reaction is stopped, the absorbance is measured at 405 nm. Levels of sulfamethazine as low as 20 ng sulfamethazine/g muscle tissue and 10 ng sulfamethazine/mL swine urine were detected and estimated.     

红松不同嫁接方法成活率及生长势初探

在同一生长季节对红松进行了髓心形成层贴接法和劈接法嫁接对比试验。结果表明:采取髓心形成层贴接法嫁接平均成活率为62%,劈接法为88%。髓心形成层贴接法嫁接成活后接穗平均生长量为5.9 cm,劈接法为11.8 cm,较髓心形成层贴接法提高了100%。经方差分析,两种方法嫁接成活率及成活后接穗平均生长量差异均极显著,说明红松采取劈接法嫁接优于髓心形成层贴接法。