大熊猫精液品质的研究Ⅰ.精液质量的动态观察

本文用11只大熊猫作了41次人工采精。其精液质量表明,在6—20岁的雄兽发情季节里,只要采精技术得当,均能采到可用作人工授精的精液。鲜精在0—4℃保存61小时,或冷冻精液超低温(-196℃)保存1—6年,解冻后在37℃培养90分钟,其精子活力仍在30％以上,用作人工授精,仍能达到受孕、产仔效果。 将大熊猫与几种猫科及食草动物的精液质量进行比较,大熊猫的精子畸形率(6—59％,平均29.74％)比其他动物高,这是大熊猫自然交配或人工授精后受孕率仍然很低的重要原因之一。对大熊猫进行科学饲养管理,可以提高大熊猫的性欲和自然交配能力。     

猪精液冷冻保存影响因素的研究进展及展望

由于在经济生产中的重要意义,猪精液冷冻保存技术成为研究的焦点。早期的研究多集中在对冷冻保护剂和冷冻方法的筛选与优化上,为猪精液冷冻保存进行了深入的探索。但研究表明,无论采用何种冷冻保护剂、何种冷冻方法都损害了精子的受精能力,并伴随有蛋白质的丢失、表达量的改变、功能活性的增减等,这种研究现状迫使该领域的研究向充分揭示冷冻对精子损伤的机理方向转移,希望通过揭示冷冻保存所导致的蛋白质结构和功能改变的实质,充分阐明冷冻损伤的机理,为猪精液冷冻保存提供理论依据,并最终促进猪精液冷冻保存技术的快速发展。     

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。     

大熊猫人工授精的研究

繁殖大熊猫的目的是圈养种群的自我维持和保持遗传多样性。由于圈养种群中有自然交配能力的雄兽很少 ,人工授精则成为有效的遗传管理的重要手段。本研究的目的是确定单纯人工授精的效率。 1 998年至 2 0 0 0年期间 ,在中国保护大熊猫研究中心 ,对 7只大熊猫进行了人工授精 (每只连续 2d) ,精液通过人工采精方法从 6只不同的雄兽中获得 ,使用鲜精、冷藏精液和冻精多种方法进行人工授精。 6只雄兽的精液平均值是 :采精量 3.3±0 .5ml;精子密度 1 ,42 9.8± 2 35 .4× 1 0 6/ml;活力 81 .7± 2 .1 % ;运动状态 ( 0～ 5 ,5 =最好 )3 1± 0 .1 ;精子正常率 79 3± 9.2 %。对 7只大熊猫进行的 1 4次人工授精中 ,使用的精液体积为 2 4± 0 3ml;活力是 73.5± 2 .9% ;运动状态为 2 .5± 0 .1 ;每次人工授精总活动精子数是 684.2± 1 1 8.2× 1 0 6。 7只大熊猫有 4只受孕 ( 5 7.1 % ) ,共产 5仔。平均孕娠期 1 31 .5±9.7d ,每胎平均 1 .3± 0 .3仔。同时 ,运用自然交配与人工授精相结合的方法 ,进行了 1 8只次实验 ,成功 1 2只次 ,产仔 2 0只 ,繁殖成功率为 66.7%。本研究结果表明 ,人工授精能有效地使不能自然交配的雄性大熊猫参与繁殖 ,提高繁殖率 ,增加圈养大熊猫的遗传多样性。     

应用生殖免疫方法制作性别化冷冻精液对奶牛性别控制的研究

本实验在种公牛站精液生产过程中应用生殖免疫技术方法 ,制作性别化冷冻精液 ,结合人工授精技术进行奶牛性别控制的研究实验。根据精子DNA含量存在的差异 ,利用荧光染料与精子DNA结合 ,通过蔗糖溶液密度梯度离心法 ,分离出和鉴别出牛精液中的X与Y精子作为抗原。经与小鼠免疫后 ,制成H Y抗血清IgG ,再经酶联免疫吸附法 (ELISA)析测表明 ,获得了具有一定纯度和工作效价的阳性抗Y精子的H Y抗血清IgG ,H Y抗血清对性别化精液冷冻前 ,解冻后活力的影响与对照组无显著差异。配种受胎试验结果表明 ,性别化冷冻精液在获得与正常冷冻精液相似的情期受胎率的同时 ,对后代性别比率有显著影响 ,奶牛产母犊率可达 60 7% ,比自然产母犊性比率理论值提高 1 0 7个百分点 (P <0 0 5 )。     

圈养大熊猫冷冻精液对其种群遗传多样性的作用

在过去34年的圈养大熊猫种群保护工作中,我们成功建立了全球最大的大熊猫精子库,目前已保存50只大熊猫个体总计7 000余支细管冷冻精液(冻精)。冷冻精液一方面可以使物种的遗传资源得到长久保存,另一方面可以通过人工授精的方式促进种群繁育。但是,圈养大熊猫冷冻精液对其种群遗传多样性的作用尚未有明确报道。本研究首先根据成都大熊猫繁育研究基地2000—2014年冷冻精液人工授精数据,对比分析了冻精人工授精个体和圈养种群的遗传多样性。结果显示,冻精人工授精个体遗传多样性均高于同年圈养种群的平均遗传多样性,表明在繁殖年份中冻精人工授精可以显著提高圈养大熊猫种群的遗传多样性。统计精子库中所有冻精个体的平均血缘系数并与圈养种群进行对比分析,探究冷冻精液对圈养种群遗传多样性的潜在作用。结果显示,精子库中有21只已死亡个体的精液,其中有66.67%的个体平均血缘系数低于圈养种群;有14只20岁以上个体的精液,其中有50.00%的个体平均血缘系数低于圈养种群;另有15只20岁以下个体的精液,其中有53.33%的个体平均血缘系数低于圈养种群,表明冷冻精液对圈养种群遗传多样性的保护具有重要价值。综上所述,冷冻精液不但有效保存了大熊猫遗传资源,而且在保护圈养种群遗传多样性方面具有积极的促进作用。     

不同解冻温度对大熊猫冷冻精子复苏的影响简报

温度是精子最敏感的外界因素之一。长期以来,人们沿用家畜冷冻精液的解冻温度(40℃)解冻大熊猫冷冻精液颗粒。大熊猫冷冻精液适应的解冻温度范围以及最佳解冻温度,还不清楚。为此,了解不同解冻温度对大熊猫冷冻精子复苏的影响,找出适宜大熊猫冷冻精液的最佳解冻温度,是本试验的目的所在。     

非人灵长类动物精子冻存技术的研究进展

随着生物医学技术的发展,应用非人灵长类动物模型进行基础科学研究日益广泛。与此同时,由于栖息地破坏、狩猎和基因隔离,许多非人灵长类动物濒临灭绝。因此,改进非人灵长类动物精子冻存技术对物种遗传资源的保藏具有重要的意义。本文概述了非人灵长类动物的精液特征,介绍了精液液化和冷冻精子质量评估的方法,分析了冷冻保护剂、冷冻稀释液及冷冻方法等因素对精子冻存效果的影响,总结了目前非人灵长类精子冷冻常用的冷冻保存液和冷冻方法,并对相关精子参数进行了比较,同时探讨了非人灵长类动物精子冻存研究面临的困境,并提出了可行的方案。总之,本文综述了近年来非人灵长类动物精子冻存的重要研究成果,对开发新的冷冻保护剂及改进冷冻技术具有一定的参考价值。     

大熊猫冷冻精液解冻速度和解冻液中添加Pentyoxyfilline对其解冻后活力的影响

建立大熊猫的精子库,进行远距离圈养大熊猫种群间的人工授精和遗传物质的转运,维持遗传多样性,是目前大熊猫遗传管理的优先方法。要成为最有效的工具,精子库保存的精子解冻后的活力必须很好。本文对大熊猫冷冻精液的解冻速度和解冻液中添加化学激活剂Pentyoxyfilline(PF)后的精子活力进行了试验。试验用的精液采自11只成年大熊猫,精液冷冻速度为每分钟-40℃～-100℃。试验Ⅰ:将冷冻精液放入3种不同温度的水浴中解冻:(1)22℃(慢速解冻);(2)37℃(中速解冻)(3)50℃(快速解冻)。将冷冻前精子活力(78 1±2 9%)和解冻后的平均精子活力进行比较,快速解冻后的精子活力(57 5±5 4%)显著地降低(P<0 05),而中速解冻的精子活力(67 5±3 1%)和慢速解冻的精子活力(73 33±2 1%)与冷冻前的活力接近。试验Ⅱ:使用中速解冻方法解冻精液后,分别加入最终浓度为0mM、1mM、5mM和10mM的PF,然后分别保温15min和24h。在PF(0mM、1mM、5mM和10mM)中分别孵育15min的解冻精子活力,运动状态,活率和顶体正常率在试验期的90min内都很相似(P>0 05)。在1mMPF中孵育24h的精子活力没有变化(P>0 05)。在5mM和10mMPF中孵育过的精子活力(5mM:24 0±4 7%;10mM19 5±3 6%)比没有加PF的对照组的精子活力(38 3±5 2%)显著地低(P<0 05)。而且,在10mM     

两种冷冻保护剂和冷冻程序对兔精子冷冻效果的比较

目的比较不同冷冻保护剂和冷冻程序对兔精子冷冻保护的影响,以期提高兔精子冷冻保存的效果和效率。方法用三步降温法（程序Ⅰ）和两步降温法（程序Ⅱ）两种冷冻程序与终浓度分别为2%,3%,4%,5%的甘油和乙酰胺两种冷冻保护剂配合进行精液冷冻保存,统计精子复苏率。结果使用程序Ⅱ添加3%乙酰胺的冷冻保护剂实验组的精子复苏率较高,同其它组比较差异有显著性意义（P〈0.05）;程序Ⅱ比程序Ⅰ节省约70%的时间,同种浓度冷冻保护剂的不同冷冻程序组之间精子复苏率差异无显著性意义（P〉0.05）。结论程序Ⅱ与3%乙酰胺配合可以取得良好的冷冻保存效果;用程序Ⅱ进行兔精液冷冻保存可以大幅缩短操作时间。     

New aspects of boar semen freezing strategies

Although cryopreserved boar semen has been available since 1975, a major breakthrough in commercial application has not yet occurred. There is ongoing research to improve sperm survival after thawing, to limit the damage occurring to spermatozoa during freezing, and to further minimize the number of spermatozoa needed to establish a pregnancy. Boar spermatozoa are exposed to lipid peroxidation during freezing and thawing, which causes damage to the sperm membranes and impairs energy metabolism. The addition of antioxidants or chelating agents (e.g. catalase, vitamin E, glutathione, butylated hydroxytoluene or superoxide dismutase) to the still standard egg-yolk based cooling and freezing media for boar semen, effectively prevented this damage. In general, final glycerol concentrations of 2-3% in the freezing media, cooling rates of -30 to -50 degrees C/min, and thawing rates of 1200-1800 degrees C/min resulted in the best sperm survival. However, cooling and thawing rates individually optimized for sub-standard freezing boars have substantially improved their sperm quality after cryopreservation. With deep intrauterine insemination, the sperm dose has been decreased from 6 to 1x10(9) spermatozoa without compromising farrowing rate or litter size. Minimizing insemination-to-ovulation intervals, based either on estimated or determined ovulation, have also improved the fertility after AI with cryopreserved boar semen. With this combination of different approaches, acceptable fertility with cryopreserved boar semen can be achieved, facilitating the use of cryopreserved boar semen in routine AI programs.     

The role of antioxidants in sperm freezing: a review

Cryopreservation of spermatozoa is becoming more important because of new clinical requirements and current clinical practice. Despite the success of sperm cryopreservation this routinely used procedure induces serious detrimental changes in sperm function. Some researchers believe that cryopreservation is associated with DNA fragmentation and DNA single strand breaks in sperm. Mechanisms of cryodamage to human spermatozoa are thought to be multifactorial including: cold shock, osmotic stress, intracellular ice crystal formation, oxidative stress, and combinations of these conditions. Additives showing antioxidative properties reported to reduce the impact of ROS-induced and cold shock damages. Many studies exist as regards the effects of antioxidants on the cryopreservation aimed at improving the quality of post-thaw semen. Hence, this review will clarify results of recent applications of various antioxidants used in numerous research efforts to improve cryopreservation of spermatozoa. This review is to increase the understanding of the roles of these antioxidants concerning mechanisms which enhance resistance to cryodamage of spermatozoa.     

大熊猫精液品质的研究：精子和非精子的细胞成分超微结构观察

本文对刚接近性成熟大熊猫的精液在电子显微镜下进行精液品质鉴定,观察结果,除少量完成变态的精子外,多数是处在精子发生的各阶段并发育不正常,精液中伴随着大量巨噬细胞和被吞噬的衰亡精子及其碎片。镜检结果不仅说明这只雄性大熊猫性尚未成熟,其生殖系统可能还伴有生理或病理现象,这样的精液建议不能用于配种。     

Cryopreservation of semen from endangered pheasants: the first step towards a cryobank for endangered avian species

In order to improve the genetic management of bird species within the European Endangered Programs (EEP), a research project on artificial insemination and cryopreservation of Galliformes semen has been developed. The aim of the program is to create a sperm cryobank for threatened bird species. During this study, semen was collected from 17 pheasant species and specific characteristics of ejaculates were analyzed (volume, sperm concentration, motility, pH). Artificial insemination with fresh semen was performed in nine species and with frozen semen in eight species. Inseminations with frozen and thawed semen were made in 17 species. Viability of fresh and frozen semen was assessed in vitro using double stains, eosin and nigrosin. The effect of pH (7-8.5) on viability of fresh and frozen/thawed spermatozoa was also studied. Chicks hatched in eight and three species after insemination with fresh and frozen/thawed semen, respectively. Species varied widely in semen viability: 1-30% of spermatozoa survived freezing and thawing. There was a negative correlation between the viability of frozen spermatozoa and semen pH. In our experimental conditions, the pH of diluents had no effect on semen viability. However, semen with the highest pH had the lowest quality after freezing and thawing. These experiments demonstrated the feasibility of using a very simple and inexpensive method to achieve artificial insemination and cryopreservation of semen in endangered pheasant species.     

Prise en charge de l’infécondité dans les troubles de l’éjaculation: avis conjoints de l’andrologue, du biologiste et du gynécologue

It is estimated that eighty percent of men who present ejaculatory disorders would like to procreate but only 5–10% from them can do it. Ejaculation can be achieved by assisted-techniques used in association with pharmacological treatment. In cases of failure of ejaculation and azoospermia, epididymal and testicular chirurgical sperm extraction allows obtaining spermatozoa. Less numerous than those obtained in ejaculated sperm, these chirurgical spermatozoa offer the advantage to be not infected by bacteria and by seminal plasma. The quality of sperm is evaluated by conventional spermiologic methods and it is important to analyze presence of spermatozoa in the urine together with that in ejaculated sperm. In ejaculatory disorders, the quality of sperm is often impaired and the mobility and vitality of spermatozoa are abnormal. Numerous bacteria and leucocytes are also present. These changes seem to be related to many factors located in the seminal plasma rather than central and testicular damages. As in fertile and infertile man, cryopreservation causes a decrease in conventional variables. However, taking into account the difficulty to obtain sperm, all patients with ejaculatory disorders would benefit from semen cryopreservation. In order to achieved successful pregnancy, several fertility treatment are available: home insemination with semen obtained by vibroejaculation, intrauterine insemination, in vitro fecondation, or intracytoplasmic sperm injection. Counselling couples undergoing such treatment program needs coordinated efforts of different specialities, which may involve andrology, biology and gynaecology.     

Assessment of motility,acrosomal integrity,and viability of giant panda (Ailuropoda melanoleuca) sperm following short‐term storage at 4°C

Short‐term cool storage of semen affords many of the same benefits as cryopreservation, without the extensive cryoinjury to sperm associated with freezing. Semen storage for artificial insemination (AI) is an integral part of giant panda captive breeding programs. AI functions to generate offspring from males that have never bred, are underrepresented, or are unable to breed with genetically desirable females due to geographic separation. In the present study, semen was collected by electroejaculation from six giant pandas and extended in four media (TEST with 0% or 5% glycerol, or SFS with 0% or 5% glycerol). Subsequently, initial motility (MOT), speed of progression (SOP), percent live (% L), and percent normal acrosome (% NAR) values were recorded. These parameters were assessed again at 4, 8, 24, and 48 hr of incubation at 4°C in each medium. Motility scores (MS) were calculated as MOT×SOP². The MS of each sample was also recorded after the addition of 20 mM caffeine. Results were expressed as a percent of the initial value (% I). Although all three parameters decreased over time, the MS decreased at a faster rate than either the % L or the % NAR. There were significant differences between individual pandas for each measured parameter, while only % IMS was significantly affected by medium (P=.0006). The addition of caffeine increased % IMS at all time periods and reduced the differences between media to nonsignificant levels. The addition of glycerol was not beneficial for short‐term semen storage. These data indicate that storage of giant panda semen at 4°C for up to 48 hr maintains sperm viability at a level sufficient for use in an AI program. Zoo Biol 22:529–544, 2003. © 2003 Wiley‐Liss, Inc.     

Dynamics of sperm DNA fragmentation in domestic animals II. The stallion

The mixed success of equine artificial insemination programs using chilled and frozen-thawed semen is most likely associated with the variable response of the sperm cell to the preservation process and the fact that stallions are not selected on the basis of reproductive performance. We propose that the traditional indicators of sperm viability do not fully account for male factor infertility in the stallion and that knowledge of sperm DNA damage in the original semen sample and during semen processing may provide a more informed explanation of an individual stallion's reproductive potential. This study reports on the validation of a sperm DNA fragmentation test based on the sperm chromatin dispersion test (SCD) for stallion spermatozoa and on its application to semen that was chilled (4 degrees C; n=10) or frozen-thawed (n=13). Semen samples were collected by artificial vagina and the proportion of sperm with fragmented DNA determined. Seminal plasma was then removed by centrifugation and the sperm pellet re-suspended in commercial extenders prior to being chilled or cryopreserved using standard industry protocols. Chilled semen was cooled slowly to 4 degrees C and stored for 1h before commencing the analysis; cryopreserved semen was thawed and immediately analyzed. Following chilling or cryopreservation, the semen samples were incubated at 37 degrees C and analyzed for SCD after 0, 4, 6, 24 and 48 h storage. The results of this investigation revealed that there was no significant difference in the sperm DNA fragmentation index (sDFI) of sperm evaluated initially after collection compared to those tested immediately after chilling or cryopreservation. However, within 1h of incubation at 37 degrees C, both chilled and frozen-thawed spermatozoa showed a significant increase in the proportion of sDFI; after 6h the sDFI had increased to over 50% and by 48 h, almost 100% of the sperm showed DNA damage. While the sDFI of individual stallions at equivalent times of incubation was variable, an analysis of the rate of change of sDFI revealed no difference between stallions or the way in which the semen was preserved. In terms of sperm DNA fragmentation dynamics, the highest intensity of sperm DNA damage occurred in the first 6h of incubation. We suggest that the SCD test can be used as a routine assessment tool for the development and refinement of preservation protocols designed to reduce stallion sperm DNA damage.     

精子冷冻保存技术及研究进展

精子冷冻是辅助生殖技术的基础,能够有效的保存有价值的基因资源。文章回顾了近些年来国内外精子冷冻保存的重要研究成果,分析了精子的来源、冷冻预处理、冷冻和解冻方法、防冻剂的选择及其加入去除方式的选择等因素对精子冷冻效果的影响。文章还总结了精子冷冻效果的评价方法,展望精子冷冻技术的发展方向和应用前景。     

Artificial insemination in South American camelids and wild equids

An overview of the present status of the use of artificial insemination (AI) in South American camelids and wild equids is offered. Technical aspects of semen collection, dilution and cryopreservation have limited the development and use of AI in camelid and equid species. To-date, efficiency is low but progress has been made and viable offspring have been produced through the use of AI in domestic South American camelids using both fresh and frozen semen. The origin, composition, and function of the viscous component of camelid seminal plasma remain a mystery and an obvious area for future research. A better understanding of the normal constituents of seminal plasma will enable the rational design of semen extenders suitable for camelids. Post-thaw sperm viability is very low, and studies are needed to address questions of optimal freezing and thawing procedures as well as the insemination dose. The basis for differences in reported pregnancy rates with sexed and frozen semen in domestic equids, and the ultimate success of AI in wild equids will require continued research into the "stallion effect", extenders and cryoprotectants, optimal volume and number of spermatozoa, temperatures during handling, processing an transport, and insemination techniques. In both camelids and equids, research on domestic species under controlled conditions provides and excellent opportunity to develop effective semen handling techniques for application in wild and endangered species of the respective families.