猪精子冷冻损伤研究进展

综述了猪冻存精子顶体、质膜和线粒体的形态学变化及其对精子受精能力的影响,同时分析了冻融精子中所发生的蛋白质、DNA等生物大分子的变化及其可能对冻存精子质量及受精能力的影响,指出冷冻保存过程对精子蛋白质、DNA等生物大分子质和量的影响是精子冷冻损伤的实质,应用先进的蛋白组学进行猪精子冷冻损伤机理研究,有助于深刻揭示冷冻损伤的分子机理,为推动猪精液冷冻保存技术研究取得突破性进展提供理论依据。     

猪精液冷冻影响因素的研究进展

由于在经济生产中的重要意义,猪精液冷冻保存技术已成为研究的焦点。文章综述了国内外猪精液冷冻技术的研究成果,主要阐述精液的冷冻保存原理,并从冷冻稀释液和冷冻保护剂、冷冻方法和解冻方法等方面阐述了猪精液冷冻的影响因素。     

冷冻—解冻猪精子的超微结构观察

自从50年代初Polge发现甘油有卓越的防冻作用以后,动物精液冷冻技术发展很快。现在,奶牛和羊冷冻精液已广泛应用于人工授精和体外受精中,效果与鲜精接近,而猪冷冻精液在人工授精后虽也能产仔,但妊娠率与每窝产仔数均下降(Johnson等,1981);体外受精率可达85％以上,但精子浓度需用5—6×10_7精子/ml,比鲜精用量提高25—100倍(Wang等,1991)。顶体形态正常精子的减少是造成冷冻精液受精率低的重要原因之一(Pursel,1979),用光镜不能清楚地观察顶体的形态变化,但运用电镜技术对猪冷冻-解冻精子的顶体形态的超微结构研究报道不多(Courtens等,1985;Hashizume,1990)。针对猪冷冻—解冻精子体内/体外受精能力降低的问题,我们用透射电镜对冷冻的猪精子解冻0 h及4 h后精子顶体与头部质膜的超微结构变化进行了观察。     

大熊猫精液超低温冷冻的比较

对卧龙自然保护区大熊猫研究中心的9只雄性大熊猫电刺激采精,比较研究了稀释液中甘油含量、精液离心、不同稀释液和冷冻方法对大熊猫精液超低温冷冻保存后的活力、运动状态和顶体的影响。稀释液中甘油的含量为4％－5％较好,冻精解冻后的活力和顶体的正常率能保持鲜精的一半。离心和未离心的精液经超低温冷冻,解冻后的活力和运动状态都较接近。TEST和SFS两种稀释液的效果没有明显的差异。细管的冷冻过程较颗粒方便、快捷,时间容易控制,是一种较好的超低温冷冻精液的方法。     

白鹤冷冻精液人工授精实验

为了探索鹤类精液冷冻保存和使用技术,2003～2005年,进行了白鹤(Grus leucogeranus)的冷冻精液保存及人工授精实验。使用Beltsville家禽精液稀释液作为白鹤精液稀释液,12%的二甲基亚砜(DMSO)为冷冻液。精液样本冷冻经过三个阶段的降温,最后保存在液氮中。成功保存了编号93001雄性白鹤精液36 支（0.2 ml/支）。冷冻精液在0~4℃冰水中解冻3~5 min,解冻后白鹤精液精子活率为29.3%±15.5%（n=16）,2004和2005年分别为92101号雌鹤产的两窝卵进行人工授精实验,2年共产卵5枚,其中1枚卵受精并成功孵化。实验发现在雌鹤产卵前一周和产卵期间每天输精,并增加每次输精量,同时在产完1枚卵后4 h内完成一次输精,效果最佳。     

兰州鲇精液超低温冷冻保存技术研究及细胞损伤检测

为保护兰州鲇(Silurus lanzhouensis Chen)种质资源,促进新品种选育,文章开展了兰州鲇精液超低温冻存抗冻剂筛选及程序保存技术研究,并通过精子解冻激活率、核DNA检测、扫描电镜和透射电镜检测技术对冻存效果和冻存损伤情况进行了评测。结果表明:兰州鲇精液以10%DMSO为抗冻剂, 4℃平衡20min,–80℃平衡30min后立即置于液氮超低温保存,并于40℃水浴解冻时精子冻存效果较佳,冻精激活率达(75.56±3.91)%。SCGE检测DNA损伤结果显示超低温冻存10d、20d及30d兰州鲇精液的彗星率和损伤系数无显著差异。扫描电镜检测显示兰州鲇精子明显分头部,中段及尾部3部分,属于单鞭毛型,无顶体,无侧鳍,中心粒相互垂直呈"T"形,鞭毛为典型的"9+2"微管结构。冻存结构损伤主要表现为膜损伤,细胞质膜与染色质膜发生破损、折皱、囊泡化或整体脱落,细胞核膜与质膜空隙加大,核发生变形,核质疏散,线粒体结构弥散,线粒体内容物外流,中心粒复合体易位,鞭毛外膜变形脱离,中段位置断裂,微管结构基本完好。研究筛选的超低温冷冻保存技术可长期有效保存兰州鲇精液,为兰州鲇种质资源保护及今后...     

滇南小耳猪精液的直接冷冻保存

目的建立滇南小耳猪精液冷冻保存方法,加速云南省特有小型猪种的小型化选育。方法利用脉冲电刺激模式诱导公猪输精管自助收缩排精。利用直接冷冻新技术（DFM）研究不同冷冻方案对精子的运动度、精子顶体完整性和体内授精胚胎发育能力的影响。结果在直接冷冻方法中,3%甘油防冻剂的作用下,60 s植冰时间和1.5 mm/s的冷冻降温参数对精子运动度保护良好,精子运动复苏率达到61.7%。但是,3%乙二醇虽然与甘油一样对精子的顶体完整性都有很好的保护作用,对精子运动度保护能力较差。此外,3%甘油、60 s植冰、1.5mm/s冷冻速度的直接冷冻的冻精解冻,移植到超数排卵的母猪子宫颈口实施人工授精,获得卵的受精和胚胎发育潜能尚可。结论玻璃管直接冷冻可以完成滇南小耳猪精子的冷冻保存。     

激光辐射奶牛冷冻精液对精子诱变效应的研究

为了探索激光辐射精子的诱变效应,本试验采用ＴＳＴ（Ｔｒｉｐｌｅ Ｓｔａｉｎ Ｔｅｃｈｎｉｑｕｅ,即精子三重染色法）、ＳＰＡ（Ｓｐｅｒｍ Ｐｅｎｅｔｒａｉｔｉｏｎ Ａｓｓａｙ,即精子穿透分析法和ＣＡＳ（Ｃｈｒｏｍｏｓｏｍａｌ Ａｎｅｕｐｌｏｉｄｙ ｉｎ Ｓｐｅｒｍａｔｏｚｏａ,即精子单倍染色体制备技术）,三项有关精子的现代分析技术,对激光辐射精子的受精学、遗传学效应进行了研究。结果表明：Ｈｅ－Ｎｅ激     

Cryopreservation of Boar Semen

The present review summarizes information concerning the methods available to cryopreserve boar semen, covering the historical background, cryobiology and cryoprotecting considerations, technological developments and recent advances in cryopreservation methodologies. Successful methods for cryopreservation of boar semen have not been achieved despite numerous efforts world wide. Improvements in semen preservation technologies have been deterred by lack of in vitro methods that can accurately predict in vivo fertilizing capacity of frozen boar semen. The cell membrane is of crucial importance with regard to freeze-thaw survival of spermatozoa. It is important to optimize the survival of the plasma membrane as this is a non homogenous entity both in structure and function. The boar sperm membrane exhibits extreme sensitivity to freezing treatment. Freezing and thawing results in considerable changes in electrolyte dynamics and damages have mainly been associated with alterations in the head membranes especially at thawing. To date fruitless efforts have been carried out to find a cryoprotectant for the spermatozoa membranes and glycerol still continues to be used despite its harmful effects to the membranes.     

Influence of Boar and Semen Parameters on Motility and Acrosome Integrity in Liquid Boar Semen Stored for Five Days

Ninety ejaculates from a total of 76 AI boars were extended in Beltsville Thawing Solution (BTS). Boar identity, breed, weight of the ejaculate and sperm concentration were registered. Motility and acrosome integrity were assessed after storage at 16–18°C for 6, 30, 54, 78, and 102 h. Storage time had a significant influence on both motility (p < 0.01) and acrosome integrity (p < 0.001). The Least Square Means for percentage of motility showed a small decline from 79.8% after 6 h of storage to 78.4% at 102 h. Motility at 78 and 102 h was significantly different from motility at 6 h (p < 0.05). The percentage of sperm cells with normal acrosomes declined throughout the experiment. The Least Square Means for 6, 30, 54, 78, and 102 h of storage were 93.9%, 90.6%, 88.0%, 84.8%, and 78.2%, respectively. The decrease in acrosome integrity from one storage time to the next was highly significant throughout the trial (p < 0.001). There was a significant influence of boar (p < 0.001) and sperm concentration (p < 0.01) on motility, while acrosome integrity was affected only by boar (p < 0.001). Breed of the boars and weight of the ejaculate did not influence the dependent variables.     

植物低氧胁迫伤害与适应机理的研究进展

不良的通气条件导致了正常生长发育的植物生理性缺氧,低氧胁迫是高等植物主要的非生物胁迫因素之一。本文综述了低氧胁迫对植物生长、植株形态的影响,低氧胁迫对植物内部水分、养分吸收的变化,呼吸代谢途径的变化、激素代谢的变化,氧化系统的变化的影响,以及低氧胁迫过程中植物体内信号的传导、基因的表达、蛋白质的合成等,在不同层面分析了低氧胁迫对植物的伤害及植物对低氧逆境适应机理的最新研究成果。     

秦岭大熊猫精液的细管冷冻保存

2008年3月1日至4月27日和2009年3月3日至5月1日,在陕西省珍稀野生动物抢救饲养研究中心对处于繁殖期内的4只雄性秦岭大熊猫(Ailuropoda melanoleuca qinlingensis)精液进行了细管冻精实验。比较组成不同的4种稀释液:葡萄糖-果糖-柠檬酸三钠-卵黄-甘油-双抗(稀释液1)、葡萄糖-蔗糖-柠檬酸三钠-卵黄-甘油-双抗(稀释液2)、葡萄糖-柠檬酸三钠-卵黄-甘油-双抗(稀释液3)和美国进口的TEST(加入3.5%甘油),以及直接降温平衡法(方法 1)与逐级降温平衡法(方法 2)2种冷冻保存操作方法,对秦岭大熊猫精液进行细管冷冻保存后精子活力和顶体完整率的影响。结果表明:稀释液1的精子活力为46.25%±11.67%,顶体完整率为80.75%±7.89%,TEST的精子活力为48.75%±8.54%,顶体完整率为84.50%±7.59%,两者的精子活力和顶体完整率均无明显差异(P0.05),但是都明显高于稀释液2(P﹤0.01)和稀释液3(P﹤0.01);采用方法 1冷冻保存秦岭大熊猫精液,解冻后精子的活力和顶体完整率分别为45.67%±10.54%和81.37%±8.42%,都显著高于方法 2(P﹤0.01);方法 1解冻后畸形率为23.50%±3.51%,明显低于方法 2(P﹤0.01)。经比较确定,方法 1(用稀释液1)是一种较好的细管冷冻保存秦岭大熊猫精液的方法。     

Sperm Cell Population Dynamics in Ram Semen during the Cryopreservation Process

Background

Sperm cryopreservation has become an indispensable tool in biology. Initially, studies were aimed towards the development of efficient freezing protocols in different species that would allow for an efficient storage of semen samples for long periods of time, ensuring its viability. Nowadays, it is widely known that an important individual component exists in the cryoresistance of semen, and efforts are aimed at identifying those sperm characteristics that may allow us to predict this cryoresistance. This knowledge would lead, ultimately, to the design of optimized freezing protocols for the sperm characteristics of each male.

Methodology/Principal Findings

We have evaluated the changes that occur in the sperm head dimensions throughout the cryopreservation process. We have found three different patterns of response, each of one related to a different sperm quality at thawing. We have been able to characterize males based on these patterns. For each male, its pattern remained constant among different ejaculates. This latter would imply that males always respond in the same way to freezing, giving even more importance to this sperm feature.

Conclusions/Significance

Changes in the sperm head during cryopreservation process have resulted useful to identify the ability of semen of males for freezing. We suggest that analyses of these response patterns would represent an important tool to characterize the cryoresistance of males when implemented within breeding programs. We also propose follow-up experiments to examine the outcomes of the use of different freezing protocols depending on the pattern of response of males.     

Cationic Synthetic Peptides: Assessment of Their Antimicrobial Potency in Liquid Preserved Boar Semen

Various semen extender formulas are in use to maintain sperm longevity and quality whilst acting against bacterial contamination in liquid sperm preservation. Aminoglycosides are commonly supplemented to aid in the control of bacteria. As bacterial resistance is increasing worldwide, antimicrobial peptides (AMPs) received lively interest as alternatives to overcome multi-drug resistant bacteria. We investigated, whether synthetic cationic AMPs might be a suitable alternative for conventional antibiotics in liquid boar sperm preservation. The antibacterial activity of two cyclic AMPs (c-WWW, c-WFW) and a helical magainin II amide analog (MK5E) was studied in vitro against two Gram-positive and eleven Gram-negative bacteria. Isolates included ATCC reference strains, multi-resistant E. coli and bacteria cultured from boar semen. Using broth microdilution, minimum inhibitory concentrations were determined for all AMPs. All AMPs revealed activity towards the majority of bacteria but not against Proteus spp. (all AMPs) and Staphylococcus aureus ATCC 29213 (MK5E). We could also demonstrate that c-WWW and c-WFW were effective against bacterial growth in liquid preserved boar semen in situ, especially when combined with a small amount of gentamicin. Our results suggest that albeit not offering a complete alternative to traditional antibiotics, the use of AMPs offers a promising solution to decrease the use of conventional antibiotics and thereby limit the selection of multi-resistant strains.     

牛精子和卵母细胞低温保存研究进展

牛的精子和卵母细胞的低温保存对于畜牧业的发展有重要价值。该文综述了近年来牛精子和卵母细胞低温保存的研究进展,包括保存方法、与低温保存有关的细胞特性、存在的问题和改进的方法等内容。     

果树种质资源超低温保存研究进展

综述了国内外在超低温保存果树种质资源方面的最新研究成果,从材料选择、材料预处理、冰冻保护剂、冰冻方法及超低温保存对组织超微结构的影响等几个方面总结了果树超低温保存的原理、影响因素及关键操作技术,展望了果树种质资源超低温保存技术的应用前景.