Effect of nicotinic acid on the plasma membrane function and polyunsaturated fatty acids composition during cryopreservation in boar sperm

This study was conducted to evaluate the effect of nicotinic acid on plasma membrane integrity and fatty acid composition in frozen–thawed boar sperm. Boar semen was cryopreserved using freezing extender containing nicotinic acid (NA), then plasma membrane integrity, osmotic equilibration, lipid peroxidation and fatty acid were analysed. The plasma membrane integrity of frozen–thawed sperm was significantly higher in the 10 mM NA than in the 0 and 20 mM NA treatment groups (p < 0.05). Additionally, the osmotic equilibration ability was not different in treatment groups, but lipid peroxidation was significantly decreased in the 10 mM NA treatment group (p < 0.05). The saturated fatty acids were significantly decreased in the 10 mM NA treatment group, and C18:1n‐9, C18:2n‐6, C20:4n‐6, C22:5n‐6 and C22:6n‐3, and total polyunsaturated fatty acids (PUFAs) were significantly increased in the 10 mM NA treatment groups (p < 0.05). In summary, 10 mM NA improved plasma membrane integrity, inhibited lipid peroxidation and increased PUFAs in frozen–thawed boar sperm. These results suggest that NA may be useful to protect the plasma membrane and inhibit the loss of PUFAs for sperm cryopreservation in pigs.     

Cryodamage of plasma membrane and acrosome region in chicken sperm

Sperm plasma membrane is an essential structure of sperm resistance to freezing. Signs of cryodamage can be visible on the sperm plasma membrane. The aim of our study was to evaluate the appearance of plasma membrane and acrosome in fresh and frozen‐thawed chicken sperm using electron and fluorescence microscopy. Semen was collected from 12 sexually mature roosters of Ross PM3 heavy line, diluted with Kobidil⁺ extender with 16% of ethylene glycol (KEG; control) or with KEG in combination with one of following non‐permeating cryoprotectants: trehalose (KEG‐TRE) or glycine (KEG‐GLY). Fluorescence staining was used for detection of the membrane integrity, apoptotic changes and viability (Annexin V, Yo‐PRO‐1, PI, respectively). Ultrathin sections (70 nm) from samples were prepared to examine sperm head ultrastructure. Freezing process significantly worsened the status of the sperm plasma membranes. In all frozen groups, only about a quarter of the evaluated sperm were graded as class I quality. In the KEG and KEG‐GLY groups, about half of sperm had severe plasma membrane damages (III class). In sperm with extensively damaged membranes (III class), the acrosome–sperm head junction was mostly disturbed. The use of trehalose was more beneficial (p < 0.05) for sperm plasma membrane than the use of glycine. In contrast, a decrease (p < 0.05) in the apoptotic sperm ratio (Yo‐PRO‐1) was noted in the KEG‐GLY group when compared to other treatments. In conclusion, we identified different plasma membrane and acrosome damages in cryopreserved chicken sperm. The loss of acrosomes can contribute to diminishing of fertilization ability of cryopreserved chicken sperm.     

New strategies of boar sperm cryopreservation: Development of novel freezing and thawing methods with a focus on the roles of seminal plasma

Cryopreservation of boar spermatozoa offers an effective means of long‐term storage of important genetic material. Many researchers have investigated how to improve reproductive performance by artificial insemination (AI) using cryopreserved boar spermatozoa. Recently, we and other groups reported that high conception rates (70–80%) can be achieved by AI with frozen‐thawed boar spermatozoa using a modified temperature program during freezing, or a novel cryopreservation extender to improve sperm quality (including sperm survivability, motility, membrane status and fertilization ability) after thawing, or a novel sperm infusion method, deep intra uterine insemination. However, these techniques have not yet been used for commercial pig production. The variation in sperm freezability among boars or among ejaculations in an identical boar is one of the main reasons for this problem. In our previous study, it was revealed that some components of seminal plasma have a negative effect on the freezability of boar sperm. One of these factors is bacteria‐released endotoxin (lipopolysaccharide: LPS). LPS binds to Toll‐like receptor‐4 (TLR‐4) expressed on the sperm surface, resulting in induction of apoptosis. On the other hand, seminal plasma suppresses cryo‐capacitation induced by thawing stress. On the basis of these findings, we designed a novel protocol of AI using frozen‐thawed boar sperm.     

Spermatozoa DNA and plasma membrane integrity after pellet optimized processing for cryopreservation in meat type chicken breeders

1. Aim of this study was the development of an optimised cryopreservation pellet procedure for chicken semen and the assessment of DNA and membrane integrity in frozen/thawed spermatozoa in a Hubbard F15 meat type selected strain.

2. The following semen processing conditions were studied: spermatozoa working concentration (SWC), 1.5 vs 2 × 10⁹ cells/ml in pre-freezing extender; equilibration of diluted semen at 5°C, 20 vs 40 min; dimethylacetamide concentration, 6% vs 9%; dimethylacetamide equilibration time at 5°C, 1 vs 30 min; thawing at 60°C for 10 vs 50°C for 30 sec. Spermatozoa viability (EtBr exclusion procedure – stress test), mobility (Accudenz® swim-down test) and subjective motility were assessed in fresh and frozen-thawed semen.

3. The lower SWC (1.5 × 10⁹ cells/ml) and the higher dimethylacetamide concentration (9%) had positive significant effects on the recovery rate of motile (22% vs 16%) and viable spermatozoa (39 vs 34%), respectively.

4.Membrane (SYBR14-PI staining) and DNA integrity (comet assay) were assessed before and after freezing/thawing according to the optimised protocol.

5. Recovery rates of spermatozoa with undamaged plasma membrane and DNA were 41% and 76%, respectively. The distribution of spermatozoa in classes of DNA damage was also analysed and discussed.

6. It was concluded that pellet cryopreservation was a damaging process mainly for plasma membrane rather than nuclear DNA in chicken spermatozoa.     

唐古特白刺质膜Na+/H+逆向转运蛋白基因的克隆与表达分析

土壤中过多的Na⁺会导致植物产生盐害,严重影响植物的生长和发育。质膜型Na⁺/H⁺逆向转运蛋白SOS1介导植物细胞内过量的Na⁺外排,在植物抵御盐胁迫过程中起着重要的作用。唐古特白刺隶属于蒺藜科白刺属,是中国特有的盐生植物,主要生长于西北地区的荒漠或盐渍化荒漠,具有极强的抗逆能力。应用简并RT-PCR及RACE技术克隆获得唐古特白刺Na⁺/H⁺逆向转运蛋白NtSOS1基因(GenBank登录号KC292267)。序列分析显示,该 cDNA全长4 008 bp,包含3 492 bp的开放阅读框,编码分子量为127.59 kDa的蛋白质。疏水性分析预测NtSOS1基因编码蛋白N端具有11个跨膜结构域,C端具有一个面向胞质的长亲水尾部,包含保守环核苷酸结合区域、自我抑制基序及磷酸化位点等多个活性调控结构域。NtSOS1氨基酸序列与葡萄、霸王、拟南芥等植物的SOS1序列同源性较高,分别可达71.77%,68.94%,62.65%。系统发育分析表明NtSOS1基因为质膜型Na⁺/H⁺逆向转运蛋白,与液泡型Na⁺/H⁺逆向转运蛋白属于不同分支。半定量RT-PCR结果显示冷、热、盐和干旱胁迫都可以诱导NtSOS1基因的表达,同时,NtSOS1基因随着盐处理浓度增加呈现出先上升(0~300 mmol/L)后下降(400 mmol/L)的趋势。结合植物的生境特点和NtSOS1表达模式分析,该基因表达水平的升高可能在唐古特白刺适应恶劣生境的过程中发挥积极作用。本研究的开展为利用NtSOS1基因进行作物与牧草改良并深入研究唐古特白刺抗逆分子机制奠定了基础。     

酸和铁胁迫对紫花苜蓿根系质膜H＋-ATPase活性的影响

以紫花苜蓿品种WL-525为材料，研究了在pH4．5和pH6．0的环境下，缺Fe^2＋和富Fe^2＋胁迫对紫花苜蓿根系质膜H^＋-ATPase活性的影响。实验结果表明：紫花苜蓿根系质膜H＋-ATPase酶的活性在强酸胁迫、缺Fe^2＋和富Fe^2＋胁迫，以及酸、铁共同胁迫下均发生明显的变化，呈现出短时间胁迫，质膜H^＋-ATPase活性升高，然后随着处理时间的增加，酶活性呈现不同程度的下降趋势。研究结果说明，在强酸和Fe^2＋胁迫下，苜蓿根系质膜H＋-ATPase酶的活性会发生变化，以减轻苜蓿根系受到的伤害。     

Evaluation of epididymis storage temperature and cryopreservation conditions for improved mitochondrial membrane potential,membrane integrity,sperm motility and in vitro fertilization in bovine epididymal sperm

The maintaining of the epididymis at lower temperatures during storage and transport improves sperm quality. Our study aimed to test whether epididymis storage temperature (post‐mortem) and sperm cryopreservation affect sperm kinetics, membrane integrity, mitochondrial potential and fertility capacity. Thirty‐six epididymides were collected from 18 bulls after slaughter and divided into two groups: at 4 or 34°C for 2–3 hr. The sperm was collected from the epididymis cauda. The evaluation consisted of computer‐assisted sperm analysis (CASA), SYBR14/PI/JC1 to evaluate membrane integrity, mitochondrial membrane potential (MMP) and measurement of lipid peroxidation (TBARS). The sperm was then frozen using an automatic device. After thawing, sperm samples were evaluated by the same variables and further in vitro fertilization rates. Cryopreservation negatively affected sperm motility in samples stored at 4 and 34°C. Nevertheless, the 4°C samples yielded higher rates of blastocyst formation. Pre‐freeze sperm motility, progressive motility and velocity were higher in sperm from epididymis stored at 4°C while post‐thaw sperm motility, progressive motility and velocity remained the same among samples from epididymis stored at 4 or 34°C. However, with regard to the kinetic patterns, samples collected from epididymis stored at 34°C had lower values when compared to those stored at 4°C prior the cryopreservation process. Our results indicate that epididymis handling conditions after cryopreservation may affect sperm quality after thawing, especially due to compromised MMP in sperm collected from epididymis stored at higher temperatures.     

The comparison of two different extenders for the improvement of large‐scale sperm cryopreservation in common carp (Cyprinus carpio)

In our study, a traditionally used (Grayling, already used in cyprinid species) and a newly tested (Pike) extender was tested to avoid sperm agglutination phenomenon following thawing during carp sperm cryopreservation. A large‐scale (elevated volume of sperm) freezing method in a controlled‐rate freezer using 5 ml straw and 10 ml cryotube was also systematically established. In all experiments, the sperm cryopreserved in using Grayling extender (except only one sample) showed an agglutination phenomenon (damaged and intact cells adhered to each other) after thawing where Pike extender resulted the regular cell suspension. No significant difference was observed between the two cryopreserved groups (Pike and Grayling extender) in all motility parameters using the 0.5 ml straw and the polystyrene box. Similarly, motility parameters did not show a significant difference in the two frozen groups with the 5 ml straw, also in the polystyrene box. A significantly higher progressive motility (pMOT, Grayling: 54% ± 8%, Pike: 37% ± 5%), straight line velocity (VSL, Grayling: 50 ± 5 µm/s, Pike: 39 ± 4 µm/s) and beat cross frequency (BCF, Grayling: 20 ± 1 Hz, Pike: 17 ± 1 Hz) was observed in the case of the grayling extender by the 5 ml straw cryopreserved in a controlled‐rate freezer (CRF) compare to the pike extender. A significantly higher VSL (Grayling: 45 ± 3 µm/s, Pike: 38 ± 4 µm/s) was observed by the grayling extender using the 10 ml cryotube than with the pike extender. Despite the randomly occurring differences in a few parameters, our new controlled freezing method using the newly tested Pike extender, the 5 ml straw or the 10 ml cryotube can be a good solution for the preservation of elevated volume of carp sperm.     

在NaCl胁迫下羊草幼苗生理反应及外源钙的缓解效应

在不同浓度NaCl营养液模拟的土壤盐化胁迫条件下,研究盐分对羊草的生理和代谢反应,结果表明,羊草对Na⁺胁迫的适应方式以耐胁性为主,通过吸收无机离子和积累脯氨酸进行渗透调节。在Na⁺胁迫基础上进行钙处理,结果表明外源钙对Na⁺危害有-定的缓解效应。可为今后治理盐碱化草场提供新的途径。     

Sarcoplasmic Reticulum Ca2+-ATPase Activity and Glycogen Content in Various Fiber Types after Intensive Exercise in Thoroughbred Horses

To find a new parameter indicating muscle fitness in Thoroughbred horses, we examined time-dependent recovery of glycogen content and sarcoplasmic reticulum (SR) Ca²⁺-ATPase activity of skeletal muscle after intensive treadmill running. Two repeated 50-sec running sessions (13 m/sec) were performed on a flat treadmill (approximately 90%VO₂max). Muscle samples of the middle gluteal muscle were taken before exercise (pre) and 1 min, 20 min, 60 min, and 24 hr after exercise. Muscle fiber type composition was determined in the pre muscle samples by immunohistochemical staining with monoclonal antibody to myosin heavy chain. SR Ca²⁺-ATPase activity of the muscle and glycogen content of each muscle fiber type were determined with biochemical analysis and quantitative histochemical staining, respectively. As compared to the pre value, the glycogen content of each muscle fiber type was reduced by 15–27% at 1 min, 20 min, and 60 min after the exercise and recovered to the pre value at 24 hr after exercise test. These results indicate that 24 hr is enough time to recover glycogen content after short-term intensive exercise. The mean value of the SR Ca²⁺-ATPase activity showed a slight decrease (not significant) immediately after exercise, and complete recovery at 60 min after exercise. There were no significant relationship between the changes in glycogen content of each muscle fiber type and SR Ca²⁺-ATPase. Although further studies are needed, SR Ca²⁺-ATPase is not a useful parameter to detect muscle fitness, at least in Thoroughbred horses.     

Ontogeny of the stomach in yellow catfish (Pelteobagrus fulvidraco): detection and quantifictation of pepsinogen and H+/K+‐ATPase gene expression

Yellow catfish (Pelteobagrus fulvidraco) is an important commercial species with high aquaculture potential in China. To better understand the process of digestive functioning of gastric gland development during the larval from 1 dph (day post‐hatching) to 30 dph, real‐time PCR was used to detect and quantify the pepsinogen and H⁺/K⁺‐ATPase gene expression in P. fulvidraco. These data were also compared with the adult situation. The results showed that the expression of pepsinogen and H⁺/K⁺‐ATPase genes in P. fulvidraco larvae both started at 1 dph, though the expression level was very low until 3 dph. The quantification of pepsinogen gene expression increased significantly from 4 to 8 dph, increased fluctuantly from 8 to 23 dph and rose sharply from 23 to 30 dph. In comparison with adult fish, there were no significant differences with larvae at 5 and 23 dph. However, data of 10 and 30 dph larvae were obviously higher than those of adult group. H⁺/K⁺‐ATPase gene expression increased linearly from 1 to 30 dph. However, it was significantly lower than that of adult. The results show that P. fulvidraco larvae have an earlier functional stomach, though the function of the stomach is still not perfect. There is a gradual acidification environment within the stomach during the P. fulvidraco larvae development. Based on these results, we suggest that the weaning time for P. fulvidraco larvae would be much better after 23 dph.     

肉鸡腹水综合征患鸡右心组织钙沉积和Ca2+-ATPase活性变化

右心肥大衰竭是腹水综合征患鸡发病的重要环节之一,而心肌细胞内Ca^2＋浓度在调节心脏收缩和舒张功能及其生长方面都起着重要作用。本试验应用右心导管法测定AS患鸡右心压力变化情况,采用焦锑酸钾沉淀法、电镜酶细胞化学法研究AS患鸡右心组织Ca^2＋和钙泵（Ca^2＋-ATPase）活性变化及其精确定位。结果显示AS组肉鸡右心室舒张压极显著高于对照组（P〈0.01）,同时,右心室内压最大变化速率也极显著降低（P〈0.01）;对照组肉鸡右心组织偶见少量散在的Ca^2＋沉淀颗粒,低温诱发AS患鸡右心组织发生了明显的钙沉积;对照组肉鸡的右心组织Ca^2＋-ATPase以高电子密度颗粒分布于肌浆网、线粒体膜等处,AS患鸡心脏组织的Ca^2＋-ATPase的电子密度颗粒显著减少或缺失。本研究揭示,在低温条件下AS患鸡具有明显的右心舒张功能障碍,Ca^2＋浓度增高和Ca^2＋-ATPase功能抑制可能在其中起着重要作用。     

不同浓度Ca2+对塔里木马鹿冻融精子体外获能及蛋白酪氨酸磷酸化的影响

为探讨不同浓度Ca²⁺对马鹿精子体外获能的影响,本研究以塔里木马鹿冻融精子为试验材料,将精子分别悬浮于含不同浓度Ca²⁺（0、1.1、2.2、3.5、5.0 mmol/L）的台氏液（sp-TALP液）中,在培养0、2、4 h时,采用金霉素（CTC）染色法评价精子获能状态,采用SDS-PAGE分离精子膜蛋白,进行免疫印迹分析,检测酪氨酸磷酸化蛋白的表达水平。结果表明,Ca²⁺浓度为1.1、2.2 mmol/L有利于精子活力的维持（P<0.05）,精子获能率极显著高于对照组和高浓度组（3.5、5.0 mmol/L;P<0.01）,精子存活时间最长（P<0.01）,但高浓度Ca²⁺（5.0 mmol/L）对精子活力具有显著抑制作用（P<0.05）,精子获能率极显著低于低浓度组（P<0.01）,精子存活时间最短（P<0.01）;另外,随着培养时间的推移精子发生酪氨酸磷酸化蛋白的表达水平有所不同,培养2、4 h时,1.1 mmol/L组精子蛋白磷酸化水平极显著高于其他各组（P<0.01）,高浓度Ca²⁺（3.5、5.0 mmol/L）组酪氨酸磷酸化蛋白的表达水平极显著下降（P<0.01）。结果表明,塔里木马鹿精子体外获能所需的适宜Ca²⁺浓度为1.1 mmol/L,且获能过程中Ca²⁺的存在是必要的。     

Effects of crocin on frozen-thawed sperm apoptosis,protamine expression and membrane lipid oxidation in Yanbian yellow cattle

Glycerol is used as a bovine semen osmotic cryoprotectant that greatly improves the quality of frozen and thawed bovine sperm. However, high glycerol concentrations can have a toxic effect on frozen and thawed bovine sperm. Therefore, this experiment investigated the effect of replacing a portion of the glycerol in a cryoprotectant solution with crocin on the sperm apoptosis, protamine deficiency and membrane lipid oxidation of frozen and thawed Yanbian yellow cattle sperm. The experiment included a control group (6% glycerol) and four treatment groups: I (3% glycerol), II (3% glycerol +0.5 mM crocin), III (3% glycerol + 1 mM crocin) and IV (3% glycerol + 2 mM crocin). Computer assisted semen analysis was used to detect sperm motility, Hoechst 33,342, propidium iodide, and JC-1 staining were used to analyse sperm viability and mitochondrial membrane potential, chromomycin A3 staining was used to detect protamine deficiency and DNA damage, flow cytometry was used for sperm membrane lipid disorder detection and analysis, and real-time quantitative RT-qPCR was used to detect the mRNA expression levels of protamine-related genes (PRM2, PRM3), sperm acrosome-associated genes (SPACA3), oxidative stress-related genes (ROMO1) and apoptosis-related genes (BCL2, BAX). Compared to the control group, replacing a portion of glycerol with 1 mM crocin significantly improved sperm motility, plasma membrane integrity, membrane lipid disorders (p < .05) and viability, mitochondrial membrane potential, protamine deficiency (p < .01). The expression level of PRM2, PRM3, SPACA3 and BCL2 significantly increased (p < .05), while the expression levels of ROMO1 and BAX significantly decreased (p < .05). Accordingly, the BCL2/BAX ratio significantly increased (p < .05). In summary, the substitution of a portion of glycerol with crocin in cryoprotective solution improved the quality of Yanbian yellow cattle sperm after freezing and thawing.     

内毒素对肝脏p53表达及Ca2+浓度的影响

为探讨内毒素在致大鼠肝损伤过程中对肝脏p53表达及Ca2+浓度的影响,将48只SD大鼠随机分2组:Ⅰ组为NS对照组;Ⅱ组为ET致病组,2组大鼠分别于第3、4、8、12h每组各处死6只,采集肝脏分别用于p53表达的Western-blotting检测和Ca2+浓度的FCM检测。结果显示,Ⅱ组p53的表达明显高于Ⅰ组(P<0.01),且Ⅱ组一直呈上升趋势;Ⅱ组Ca2+的荧光指数明显高于Ⅰ组(P<0.01),且一直呈上升趋势。结果表明,ET能有效上调肝细胞p53蛋白的表达及Ca2+浓度。     

Involvement of Ca2+-ATPase in suppressing the appearance of bovine helically motile spermatozoa with intense force prior to cryopreservation

In cattle, cryopreserved spermatozoa are generally used for artificial insemination (AI). Many of these specimens exhibit helical movement, although the molecular mechanisms underlying this phenomenon remain unclear. This study aimed to characterize helically motile spermatozoa, investigate the involvement of Ca²⁺-ATPase in suppressing the appearance of these spermatozoa prior to cryopreservation, and examine the potential of helical movement as an index of sperm quality. In the cryopreserved semen, approximately 50% of spermatozoa were helically motile, whereas approximately 25% were planarly motile. The helically motile samples swam significantly faster than those with planar movement, in both non-viscous medium and viscous medium containing polyvinylpyrrolidone. In contrast, in non-cryopreserved semen, planarly motile spermatozoa outnumbered those that were helically motile. Fluorescence microscopy with Fluo-3/AM and propidium iodide showed that flagellar [Ca²⁺]_i was significantly higher in cryopreserved live spermatozoa than in non-cryopreserved live ones. The percentage of non-cryopreserved helically motile spermatozoa was approximately 25% after washing, and this increased significantly to approximately 50% after treatment with an inhibitor of sarcoplasmic reticulum Ca²⁺-ATPases (SERCAs), “thapsigargin.” Immunostaining showed the presence of SERCAs in sperm necks. Additionally, the percentages of cryopreserved helically motile spermatozoa showed large inter-bull differences and a significantly positive correlation with post-AI conception rates, indicating that helical movement has the potential to serve as a predictor of the fertilizing ability of these spermatozoa. These results suggest that SERCAs in the neck suppress the cytoplasmic Ca²⁺-dependent appearance of helically motile spermatozoa with intense force in semen prior to cryopreservation.     

桑树Ca~(2+)/H~+反向转运体基因MCAX-1的克隆及序列与表达分析

植物体内的Ca2+/H+反向转运体在Ca2+介导的营养和信号转导中发挥着重要作用。选择桑树幼叶cDNA文库中功能注释为Ca2+/H+反向转运体基因(CAX)的一条EST序列设计引物,通过cDNA末端快速扩增(RACE)与反转录PCR(RT-PCR)在桑品种育71-1的幼叶中克隆获得该基因的全长cDNA序列,命名为MCAX-1(GenBank登录号:JN716318)。序列分析显示:MCAX-1全长1 784 bp,包括197 bp的5'端非翻译序列和243 bp的3'端非翻译序列,含有一个1 344 bp的完整ORF,编码447个氨基酸残基,预测蛋白质分子质量为47.93 kD,等电点为5.67。构建的桑树和其它植物基于CAX蛋白氨基酸序列的系统进化树显示:桑树与盐地碱蓬(Suaeda salsa)、毛果杨(Populus trichocarpa)、蓖麻(Ricinus communis)等有较近的亲缘关系。半定量RT-PCR分析表明:MCAX-1在桑树幼芽、嫩叶、根和幼花中的表达量较高,在韧皮部、木质部和成熟果实中的表达量较低;MCAX-1在低温胁迫下的表达量减少,-1℃时几乎无表达,在干旱胁迫下的表达量随着胁迫时间延长逐渐达到最大值之后又迅速降低,在盐分胁迫初期的表达量无变化,但胁迫18 d时表达量明显降低。初步推测MCAX-1与桑树的抗逆性能有一定关联。     

H2O2胁迫下豌豆初生根及抗氧化酶系统对外源 Ca2＋的响应

以豌豆品种“陇豌一号”为材料,通过对豌豆种子萌发初期初生根生理特性的研究,探索提高豌豆初生根外源H2 O2胁迫条件下抗性能力的途径。运用生理生化方法,测定 H2 O2胁迫下豌豆初生根在外源 Ca2＋处理后的弯曲率和根系活力,并对豌豆初生根内的丙二醛（MDA）含量、相对膜透性、超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）和抗坏血酸过氧化物酶（APX）活性进行测定。结果显示,80 mmol／L 的 H2 O2处理下的豌豆初生根正常生长受到显著抑制,但是经过 Ca2＋处理后,根系生长抑制作用得到缓解,根系活力得以恢复。外施10 mmol／L Ca2＋初生根 MDA 值较 CK1降低了37．32％,并显著地提高了 POD,SOD,CAT 和 APX 的活性,其值分别为51．946 U／（mg·min）,865．174 U／g FW,1．9739 mmol／（L·g·min）和2．569μmol／（L·g·min）。总之,外源施加 Ca2＋能够有效降低 H2 O2造成的氧化胁迫,缓解对初生根细胞膜的伤害,降低质膜透性,增强初生根系抗氧化酶活性,达到抵抗逆境胁迫的目的。     

Ca2＋对植物乳杆菌LIP-1冷冻干燥抗性的影响及其在热饮中的应用

以具有降血脂益生特性的植物乳杆菌LIP-1为研究对象,通过在培养基中添加不同浓度CaCl2,探究Ca2＋对菌株冷冻干燥抗性及耐热性的影响,并比较未加钙菌粉、加钙菌粉和未加钙市售菌粉在不同热饮中的活菌数。结果表明：与未添加Ca2＋对照组相比,培养基中添加0.5 mmol/L Ca2＋不仅可以显著提高菌株的冷冻干燥存活率（P＜0.05）,并可以改善其耐热性;与市售菌粉相比,加钙菌粉在热饮中的植物乳杆菌LIP-1存活率显著提高,探究其内在机制发现,加钙菌粉的植物乳杆菌LIP-1细胞膜与细胞壁损伤程度显著低于其他组别（P＜0.05）,同时细胞膜不饱和脂肪酸含量显著升高。上述结果表明,在培养基中添加Ca2＋能够显著提高植物乳杆菌LIP-1对冷热的抗性。     

A comparative study of the effects of intramuscular administration of gonadorelin,mating and intrauterine infusion of either raw seminal plasma or seminal plasma purified β‐NGF on luteal development in llamas

The aim of this study was to compare the effect of the intramuscular administration of 50 μg of gonadorelin acetate versus natural mating, intrauterine infusion (i.u.) of a physiological relevant dose of either raw llama seminal plasma (SP) or purified beta‐nerve growth factor from seminal origin (spβ‐NGF) on ovulation rate and corpus luteum (CL) development and function in llamas. Females with a follicle (≥8 mm) were assigned to groups: (i) i.m. administration of 50 μg of gonadorelin acetate (GnRH; positive control; n = 4); (ii) single mating (mating; n = 6); (iii) i.u. infusion of 4 ml of llama SP (SP; n = 4); or (iv) i.u. infusion of 10 mg of spβ‐NGF contained in 4 ml of PBS (phosphate‐buffered saline) (spβ‐NGF; n = 6). Ovaries were examined by power Doppler ultrasonography at 0, 1, 3, 6, 12 and 24 hr after treatment to determine preovulatory follicle vascularization area (VA), and additionally every 12 hr until Day 2 (Day of treatment = Day 0) to determine ovulation. Afterwards, ovaries were examined every other day until Day 8 to evaluate CL diameter and VA. Blood samples were collected on Days 0, 2, 4, 6 and 8 to determine plasma progesterone (P4) concentration. Ovulation rate did not differ (p = .7) among groups, but treatment affected (p < .0001) preovulatory follicle VA. Neither treatment administration nor treatment by time interaction affected (p ≥ .4) CL diameter, VA and plasma P4 concentration. Mating tended (p = .08) to increase CL VA when compared to the seminal plasma group by Day 8. Intrauterine administration of seminal plasma or spβ‐NGF does not increase CL size and function when compared to i.m. GnRH treatment, suggesting that the administration route of spβ‐NGF influences its luteotrophic effect in llamas.