Optimization of culture conditions for short-term maintenance,proliferation, and colony formation of porcine gonocytes

Background: Gonocytes give rise to spermatogonial stem cells, and thereby play an essential role in establishing spermatogenesis. Optimized culture conditions for gonocytes provide an opportunity for their study and in vitro manipulation for potential application in reproductive technologies. Using six experiments in a step-wise design, we examined the effects of several culture conditions on the maintenance, proliferation, and colony formation of porcine gonocytes. Testis cells from neonatal piglets were cultured for 7 d in DMEM supplemented with 10% fetal bovine serum. The examined culture conditions included using different cell seeding densities, gonocyte proportions, incubation temperatures, sampling strategies, and medium changing regimens.Results: Confluency of cel s was optimal(90% by ~6 d) when 3.0 × 104 testis cel s/cm2 containing ~40% gonocytes were used. Incubating the cel s at 35 °C or 37 °C resulted in similar cel number and viability at confluency, but incubation at 35 °C resulted in a delayed confluency. In the first 2 d of culture, gonocytes remained mostly floating in the medium and gradual y settled over the next 5 d. Consequently, not changing the medium for 7 d(as opposed to changing it every 2 d) led to a significant increase in the number of gonocyte colonies by reducing the loss of "floating gonocytes".Conclusion: We found that gonocytes require the presence of a critical minimum number of somatic cel s for settlement, and can proliferate and form growing colonies even in a basic medium. Large numbers of viable gonocytes remain floating in the medium for several days. The optimized culture conditions in the present study included seeding with 3.0 × 104 testis cel s/cm2 containing ~40% gonocytes, incubating at 37 °C, and without changing the medium in the first week, which can result in improved colony formation of porcine gonocytes.     

Effect of nitrogen fertilization on yield and nitrogen and water use efficiencies of winter wheat (durum and bread) varieties grown under conditions found in Central Anatolia

In order to evaluate the influence of different N rates on percent N derived from fertilizer (%Ndff) at different growth stages, on yields and the percent N use efficiency (%NUE) values of two winter wheat varieties (durum and bread), field experiments on fallow were carried out at four different locations in Central Anatolia, in the 1991–1992 and 1992–1993 growing seasons. At each site the rates of N (0, 40, 80 and 120?kg N/ha) were applied as ammonium sulphate [(NH₄)₂SO₄] using a Latin Square experimental design with four replicates. The total amounts of N fertilizer were applied once after seedling emergence at all experimental sites. Labelled (¹⁵NH₄)₂SO₄ fertilizer was applied to sub-plots from which %Ndff values were determined at tillering, booting, grain filling, and harvest stages. Yield sub-plots received unlabelled (NH₄)₂SO₄ from which total dry matter (seed and straw) and N yields were determined. Also the %NUE values were calculated by the ¹⁵N and "difference" methods at the harvest stage. Stored soil moisture at 0–90?cm depth, evapotranspiration and water use efficiency values were calculated as well. The results obtained showed that (1) Gerek-79 variety used both the applied N fertilizer and the available soil moisture more efficiently, (2) the percent NUE values obtained overall were generally less than 20 for both varieties and (3) with the ¹⁵N method, less variable %NUE values were obtained in comparison to the difference method.     

Current progress in studying blackleg disease (Leptosphaeria maculans and L. biglobosa) of canola in Iran: Where do we stand now?

Blackleg, caused by a complex of Leptosphaeria species (L. maculans and L. biglobosa), is a fungal disease on Brassica species, especially important in canola (Brassica napus). Since the first report of L. biglobosa in Iran in 2007 and L. maculans in 2008, both species are now of major importance in Iran affecting 10 provinces and 30 regions, with a higher prevalence in the northern provinces of Mazandaran and Golestan. Despite the rapid progression of the disease and the emergence of new Leptosphaeria races in Iran, the research into this pathogen has not progressed at the same rate and is limited to phenotypic characterization studies, pathogenicity research, and to a lesser extent, disease management research. Given the rapid increase in canola cultivation in Iran and changes in the genetic diversity of the pathogen populations, it is likely that blackleg disease will increasingly become a severe threat to Iran’s canola production. Therefore, systematic and prospective studies, along with fundamental research on the pathogen's biology, epidemiology, and genetic diversity, would provide critical information for the development of disease management strategies. Here, we review the research that has been carried out to date on blackleg disease in Iran and describe the extent of progress towards disease control, especially in disease-prone regions.     

氰氨化钙与秸秆还田协同处理对生菜土传病害防效及土壤质量的影响

 为明确氰氨化钙与秸秆还田协同处理对土传病菌的抑制效果以及对土壤微生物群落的恢复和土壤理化性质的影响,采用实时荧光定量PCR(Real-time quantitative PCR, qRT-PCR)、平板分离计数等方法测定土传病菌和微生物数量动态变化。生菜收获期测定植株农艺性状和产量,结合土壤理化性质开展土壤处理模式与土壤微生物互作相关分析。结果表明,氰氨化钙和玉米秸秆协同处理对生菜根腐病和生菜茎基腐病的防效分别达到56.81%和47.49%,且增产率(44.08%)较高,具有防病促生效果。氰氨化钙和玉米秸秆协同处理在消毒揭膜0 d微生物数量显著减少,定植期微生物数量逐渐恢复。此外,两种土壤处理均可提高土壤pH值,降低电导率含量。其中氰氨化钙和玉米秸秆协同处理可提高土壤有机质、有效磷和全氮含量。因此,氰氨化钙和玉米秸秆协同处理可抑制土传病菌增殖,增加生菜生物量,改善土壤理化性状,后期土壤微生物数量恢复且不会对微生物群落产生明显的扰动影响,对环境较安全。     

芸薹根肿菌单孢亚甲基蓝琼脂糖分离法及其对生理小种的鉴定

对传统芸薹根肿菌（Plasmodiophora brassicae）单孢分离的水琼脂法进行改进,确定了亚甲基蓝和琼脂糖的最佳浓度,将0.01%亚甲基蓝染色液与1.5%琼脂糖溶液按体积比1︰300混匀,制成亚甲基蓝琼脂糖凝层,在显微镜下可成功分离芸薹根肿菌单孢。大白菜单孢接种最佳苗龄为催芽24 h,接种后暗培养24 h,再移栽至育苗钵,发病率最高。利用该方法分离获得中国11个省十字花科（大白菜、甘蓝、油菜）芸薹根肿菌单孢系菌株79株,采用Williams鉴别系统鉴定出P2、P3、P4、P5、P6、P7、P8、P9、P10、P12、P13和P14等12个不同的生理小种类型,其中P4为优势生理小种,出现频率为49.37%。明确了中国11个省份根肿菌的生理小种类型及其分布,为十字花科蔬菜抗病育种提供重要资源。     

Repair of experimental plaque-induced periodontal disease in dogs

Forty mongrel dogs were used in this study for induction of periodontal disease by placing subgingival silk ligatures affecting maxillary and mandibular premolar teeth during a 12-month period. Experimental premolar teeth received monthly clinical, radiographic, and histometric/pathologic assessments. The results demonstrated significant increases in scores and values of periodontal disease parameters associated with variable degrees of alveolar bone loss. The experimental maxillary premolar teeth exhibited more severe and rapid rates of periodontal disease compared with mandibular premolar teeth. Histometric analysis showed significant reduction in free and attached gingiva of the experimental teeth. Histopathological examination of buccolingual sections from experimental premolar teeth showed the presence of rete pegs within the sulcular epithelium with acanthosis and erosive changes, widening of the periodontal ligament, and alveolar bone resorption. Various methods for periodontal repair were studied in 194 experimental premolar teeth exhibiting different degrees of periodontal disease. The treatment plan comprised non-surgical (teeth scaling, root planing, and oral hygiene) and surgical methods (closed gingival curettage, modified Widman flap, and reconstructive surgery using autogenous bone marrow graft and canine amniotic membrane). The initial non-surgical treatment resulted in a periodontal recovery rate of 37.6% and was found effective for treatment of early periodontal disease based on resolution of gingivitis and reduction of periodontal probing depths. Surgical treatment by closed gingival curettage to eliminate the diseased pocket lining resulted in a recovery rate of 48.8% and proved effective in substantially reducing deep periodontal pockets. Open root planing following flap elevation resulted in a recovery rate of 85.4% and was effective for deep and refractory periodontal pockets. Autogenous bone graft implantation combined with canine amniotic membrane as a biodegradable membrane was used in 18 premolar teeth and failed to improve advanced furcation defects in most teeth.     

The optimal immunization procedure of DNA vaccine pcDNA–TA4–IL-2 of Eimeria tenella and its cross-immunity to Eimeria necatrix and Eimeria acervulina

The immunization procedure of DNA vaccine pcDNA–TA4–IL-2 of Eimeria tenella, including route, dose, time of immunization and age of primary immunization of chicken, was optimized. The stability and the cross-species protection of the vaccine were also analyzed. Efficacy of immunization was evaluated on the basis of oocyst decrease ratio, lesion score, body-weight gain and the anti-coccidial index (ACI). Chinese Yellow chickens were randomly distributed into corresponding groups (30/group). The challenged, unchallenged and vector control groups were designed. The results illustrated that 25 μg was the optimal dose and intramuscular injection was the most effective route to induce protective immunity. There were no significant differences of ACIs between boosting and non-boosting groups. Storage time and temperature had little effect on the immunizing efficacy of the vaccine. The vaccine could provide partial cross-protection against the challenge with E. necatrix and E. acervulina, but not with E. maxima.