不同孵育时间对猪精子转染外源DNA效果的影响

建立稳定的精子载体法技术体系对家畜的转基因育种等研究具有重要意义.为获得公猪精子与DNA最佳共孵育时间,优化建立猪精子载体法技术体系,本研究通过定量PCR、荧光显微镜检测和精液常规分析方法等,研究分析猪精子与DNA不同共孵育时间对猪精子活力、活率、DNA转染率、吸附DNA和内化DNA量的影响.结果表明,多聚乙烯亚胺(polyethyleneimine,PEI)包裹的标记DNA与精子共孵育15、30、60和90 min后,随着孵育时间的延长,精子活力和活率有极显著下降(P＜0.01),而精子转染率呈极显著(P＜0.01)或显著(P＜0.05)上升,吸附外源DNA量和内化外源DNA量也呈上升趋势,但是30 min后不同孵育时间之间差异不显著(P＞0.05).此外,随着共孵育时间的延长,与精子转染率提高幅度相比,精子活力和活率的下降幅度更明显.综合考虑精子活力、活率、转染率、吸附DNA量和内化DNA量等参数,猪精子与外源DNA共孵育时间以30 min为最佳.     

Low reproductive success of leatherback turtles, Dermochelys coriacea, is due to high embryonic mortality

We examined the mechanism responsible for low reproductive success in leatherback turtles (Dermochelys coriacea) at Playa Grande, Costa Rica: low egg fertilization versus high rates of embryonic death. Leatherbacks at this beach had a high rate of fertility (=93.3%±2.5%, n=819). We incubated 10 eggs from every clutch encountered of 19 females during 3 months of the 1998-1999 nesting season. Fertility rate of some females decreased during the nesting season, but overall was high. Detection of fertility was difficult using standard methods because fertility rates cannot be determined accurately from nests excavated after hatching because of egg decomposition. Removal and incubation of eggs from nests provided a better estimate. Embryonic death, particularly in the beginning of incubation before embryos are visible to the unaided eye, was the cause of low hatching success in this population. Hatching success increased with increasing fertility and differed between females, with some mothers having 71-81% success and others 23-32%. Embryonic death and not low egg fertility drives poor recruitment at Playa Grande. Improved conservation of this species at Playa Grande will require a better understanding of the mechanism behind embryonic death.     

C signature of CO2 evolved from incubated maize residues and maize-derived sheep faeces

Analyses of the spatial and temporal variations in the natural abundance of ¹³C are frequently employed to study transformations of plant residues and soil organic matter turnover on sites where long continued vegetation with the C₃-type photosynthesis pathway has been replaced with a C₄-type vegetation (or vice versa). One controversial issue associated with such analyses is the significance of isotopic fractionation during the microbial turnovers of C in complex substrates. To evaluate this issue, C₃-soil and quartz sand were amended with maize residues and with faeces from sheep feed exclusively on maize silage. The samples were incubated at 15 °C for 117 days (maize residues) or 224 days (sheep faeces). CO₂ evolved during incubation was trapped in NaOH and analysed for C isotopic contents. At the end of incubation, 63 and 50% of the maize C was evolved as CO₂ in the soil and sand, respectively, while 32% of the faeces C incubated with soil and with sand was recovered as CO₂. Maize and faeces showed a similar decomposition pattern but maize decomposed twice as fast as faeces. The δ¹³C of faeces was 0.3‰ lower than that of the maize residue (δ¹³C −13.4‰), while the δ¹³C of the C₃-soil used for incubation was −31.6‰. The δ¹³C value of the CO₂ recovered from unamended C₃-soil was similar or slightly lower (up to −1.5‰) than that of the C₃-soil itself except for an initial flush of ¹³C enriched CO₂. The δ¹³C values of the CO₂ from sand-based incubations typically ranged −15‰ to −17‰, i.e. around −3‰ lower than the δ¹³C measured for maize and faeces. Our study clearly demonstrates that the decomposition of complex substrates is associated with isotopic fractionation, causing evolved CO₂ to be depleted in ¹³C relative to substrates. Consequently the microbial products retained in the soil must be enriched in ¹³C.     

Soil respiration is not limited by reductions in microbial biomass during long-term soil incubations

Declining rates of soil respiration are reliably observed during long-term laboratory incubations. However, the cause of this decline is uncertain. We explored different controls on soil respiration to elucidate the drivers of respiration rate declines during long-term soil incubations. Following a long-term (707 day) incubation (30 °C) of soils from two sites (a cultivated and a forested plot at Kellogg Biological Station, Hickory Corners, MI, USA), soils were significantly depleted of both soil carbon and microbial biomass. To test the ability of these carbon- and biomass-depleted (“incubation-depleted”) soils to respire labile organic matter, we exposed soils to a second, 42 day incubation (30 °C) with and without an addition of plant residues. We controlled for soil carbon and microbial biomass depletion by incubating field fresh (“fresh”) soils with and without an amendment of wheat and corn residues. Although respiration was consistently higher in the fresh versus incubation-depleted soil (2 and 1.2 times higher in the fresh cultivated and fresh forested soil, respectively), the ability to respire substrate did not differ between the fresh and incubation-depleted soils. Further, at the completion of the 42 day incubation, levels of microbial biomass in the incubation-depleted soils remained unchanged, while levels of microbial biomass in the field-fresh soil declined to levels similar to that of the incubation-depleted soils. Extra-cellular enzyme pools in the incubation-depleted soils were sometimes slightly reduced and did not respond to addition of labile substrate and did not limit soil respiration. Our results support the idea that available soil organic matter, rather than a lack microbial biomass and extracellular enzymes, limits soil respiration over the course of long-term incubations. That decomposition of both wheat and corn straw residues did not change after major changes in the soil biomass during extended incubation supports the omission of biomass values from biogeochemical models.     

孵化期间乌骨鸡种蛋水钙变化研究

孵化期间，鸟骨鸡种蛋内蛋黄和蛋清的含水量变化呈互补性，即蛋黄含水多时．蛋清含水少，而胚胎的含水量是逐渐减少的。12日龄前，蛋壳、蛋清、蛋黄内钙含量变化相对较小，12日龄后，蛋壳内的钙比蛋清、蛋黄内的钙变化大，且成为胚胎发育过程中的主要钙源。试验还发现，孵化前蛋黄内的水、钙含量和出壳时剩余蛋黄内的水、钙含量相差不大，但18日龄前后，蛋黄内的钙大幅度上升，证实蛋黄有富集钙的功能。鸟骨鸡胚胎的生长在9日龄前是缓慢的，9日龄以后是快速的，且胚胎的重量与孵化时问的关系呈指数式回归。     

Acceleration of N mineralization by release of enzymes and substrates from soil mineral particles with phosphates

A study was conducted to develop an improved method for measuring organic N (net) mineralization in which chemical extraction takes place in combination with suspension incubation in ammonia-absorbing membrane bottles. To obtain direct evidence of the extent to which extracted organic N is mineralizable, the extraction suspension was further incubated immediately after the extraction procedure with mild and selective extractants. In this ‘extraction incubation’ method, extraction continues during the incubation but only relatively easily mineralizable organic matter is released. Standard incubation is usually carried out in sealed N₂-flushed bottles. However, when phosphate or pyrophosphate soil suspensions are incubated, mineralization is much higher than in soil water suspensions. Further, accumulation of ammonia+(ammonium) and other gases, i.e. CO_2, can affect the reaction rate and final reaction equilibrium in the sealed incubation flask. It was to avoid these effects that the membrane method was developed. With this procedure, the flask is closed with an ammonia-absorbing membrane permeable to other gases. Water, phosphate and pyrophosphate suspensions were incubated at 37 °C in sealed bottles (SB), in sealed N₂ gas-flushed bottles (SBN₂), and in bottles with ammonia-trapping filters (MB). The maximum amount of released during 10 days' incubation was 133.0 mg kg⁻¹ in the water, 208.0 mg kg⁻¹ in the phosphate and 454.1 mg kg⁻¹ in the pyrophosphate suspension (soil total C content 6.2% and N 0.25%). During incubation in phosphate and pyrophosphate suspensions, the mobilization was nearly linear in membrane bottles. The variation between replicates was also smallest in these bottles. It was concluded that membrane bottles were best suited to incubation when mobilization reactions were accelerated with phosphate or pyrophosphate extractants. The method was easy to perform and gave results with good replicability.     

不同施肥处理对黄泥土微生物生物量碳氮和酶活性的影响

通过室内培育实验,研究了不同施肥处理对黄泥土微生物生物量C、N和脲酶、酸性磷酸酶、蔗糖酶活性的影响。培养过程中,单施化肥处理土壤微生物生物量C、N均呈下降趋势,施用有机肥处理的土壤微生物量C前期升高至一定水平后或保持稳定、或呈下降趋势,而微生物生物量N总体均呈下降趋势;不同施肥处理的脲酶活性呈前期上升后期下降趋势,而酸性磷酸酶和蔗糖酶则单施化肥处理呈上升趋势、配施有机肥处理呈下降趋势。与对照处理相比,单施化肥处理显著降低土壤微生物生物量C、N,高量施用化肥处理还显著降低土壤脲酶活性,但对酸性磷酸酶和蔗糖酶活性的影响并不明显。秸秆施用可显著提高土壤微生物生物量C、N和酶活性,特别是高量施用秸秆的效果更明显。施用猪粪由于同时带入了大量的活性养分,对土壤微生物生物量C、 N和酶活性的影响尚难评价。在高度集约农业利用下,继续保持较高的化肥施用量并不利于土壤生物质量的维护和提高,而秸秆直接还田才是保持土壤健康状态的有效措施。     

溴隐亭和克罗米芬对乌鸡生殖激素水平及产蛋性能的影响

研究溴隐亭和克罗米芬对乌鸡血液中生殖激素水平、就巢时间和产蛋性能的影响。将36只就巢的丝羽乌鸡随机分成3组,Ⅰ组为对照组;Ⅱ组第1～2天每只口服溴隐亭1.25mg/d,第3～7天每只口服克罗米芬12.5mg/d;Ⅲ组第1～2天每只口服溴隐亭0.625mg/d,第3～4天每只口服溴隐亭1.25mg/d,第5～6天每只口服克罗米芬25mg/d,第7天每只口服克罗米芬12.5mg。试验结果表明,Ⅱ组血液中雌激素水平显著高于Ⅰ组(P<0.05),Ⅲ组血液中雌激素水平极显著高于Ⅰ组和Ⅱ组(P<0.01);Ⅰ组平均就巢时间最长,为8.58d,Ⅱ组为6.67d,Ⅲ组为6.83d;Ⅰ～Ⅲ组处理后1个月内的平均产蛋率分别为37.72%、59.65%和53.51%,Ⅱ组和Ⅲ组的产蛋率显著高于对照组(P<0.05);3个组的平均蛋质量分别为43.86、44.65和44.16g,蛋形指数分别为1.306、1.312和1.323,蛋壳厚度分别为0.327、0.338和0.332mm,溴隐亭和克罗米芬对后3项指标的作用效果差异不显著(P>0.05)。     

风干土保存时间和湿土培育时间对黄淮海平原潮土酶活性的影响

以经历18年不同施肥管理的土壤为研究对象,阐明它们经过4个不同时间保存或处理后的土壤脲酶、转化酶、脱氢酶、及FDA酶活性的动态变化。处理包括:风干保存30天或鲜土状态、风干保存210天、风干土湿润至田间持水量(25℃)条件下分别培育15天和51天;同时评估这些酶活性的变化程度与土壤本身有机碳含量之间的关系。结果表明,风干土保存时间和风干土湿润后短期培育均对脲酶活性影响很小,但风干土湿润培养51天后其活性则显著降低;随风干土保存时间延长,转化酶活性显著降低;与鲜土相比,风干土湿润培养15天后,脱氢酶活性显著提高,但继续湿润培养至51天后,其活性又降至与鲜土相当,因此风干土湿润培育一定时间后测定的脱氢酶活性可用来代表其田间自然湿度时的状态;FDA酶活性的变异程度最大,与其从鲜土状态至风干状态的活性急剧下降有关。土壤本身有机碳含量与脲酶和脱氢酶的活性变化程度成显著负相关关系,说明土壤有机碳含量是决定它们随环境条件改变而变化的主要因素之一。另外,土壤NH4+-N、NO3--N和可溶性有机碳含量对上述4种处理的响应程度也存在差异。其中风干状态土壤经湿润培育处理后,NH4+-N含量呈先降后升趋势,正好与脲酶活性变化趋势相反;而NO3--N含量整体上呈上升趋势,可溶性有机碳含量则正好相反。     

孵化期间鸡胚蛋内蛋白质转移的研究

对不同日龄的广西黄鸡胚蛋的卵白、卵黄,胚胎中粗蛋白,尿囊液中的尿素氮含量进行了测定,结果表明,这些指标呈一定规律性变化:前期卵黄、卵白中蛋白质含量稳定,卵白中蛋白质在孵化的第9和12天胚龄含量较高,之后极显著下降。卵黄中的粗蛋白质含量呈逐渐增加的趋势。胚胎中的粗蛋白含量从9～12d胚龄显著降低;尿囊液中尿素氮的含量在12～21d胚龄迅速增加,21d胚龄达到最高水平。说明蛋白在中期转移最多,后期利用率较低。