栉江珧和近江牡蛎的精子超低温冷冻保存

栉江珧(Atrina pectinata)和近江牡蛎(Crassostrea rivularis)是我国重要的海产经济贝类,建立其精子冷冻保存技术及精子冷冻库,对种质资源的保护和遗传育种具有重要意义.本研究对其精子冷冻保存过程中冷冻剂种类和浓度的选择、冷冻程序、平衡时间、保存体积、精液稀释方式、保存时间等多个影响因素进行了比较筛选,并利用显微镜直接检测和曙红Y(eosin Y)染色检测结合的方式检验精子存活率.结果表明,二甲亚砜(dimethyl sulfoxide,DMSO)作为抗冻剂的保存效果要明显优于甘油(glycerol,Gly),终浓度为8％～10％的二甲亚砜保存效果最好,其栉江珧和近江牡蛎精子活率均可达60％以上,而甘油保护下精子活率小于40％.精液的稀释方式采用将抗冻液等分为3份,依次与精液混合,每次间隔8 min,整个过程约30 min;此种精液稀释方式与一次性混合方式相比,精子活率可提高约30％.稀释后精液需要在4℃下平衡15～25 min,平衡样品与直接冷冻样品相比,精子活率提高约50％.精液降温程序为在样品投入液氮前,于液氮面以上15 cm处停留5 min,5 cm处停留10 min,精子活率可达60％以上;而未经此降温程序直接投入液氮中的样品精子活率接近0.与此降温程序相对应的样品冷冻体积在1.0～1.4 mL范围内为宜.冷冻180 d内复苏的精子活率由90％左右(鲜精)下降至60％左右,180 d后精子活率下降至56％左右.研究结果为栉江珧和近江牡蛎精子超低温冷冻保存、双壳类种质保护和遗传育种提供了理论和技术依据.     

Effect of active immunization against GnRH on testosterone concentration,libido and sperm quality in mature AI boars

Background

The aim of the present study was to investigate the efficacy of the Improvac on testosterone concentration in blood serum, sexual behavior and sperm quality in matured AI boars. A total of nine Danish Landrace AI boars were included in the analysis.

Methods

The trial period lasted for 15 weeks and was divided into four periods: Control period: three weeks before vaccination; Period I – four weeks after first vaccination; Period II – four weeks after second vaccination, Period III – four weeks after third vaccination. Blood and sperm samples were collected at weekly intervals. Freshly collected sperm samples were analyzed.

Results

Testosterone concentration correlated with libido (r = 0.531; p < 0.001), volume of ejaculate (r = 0.324; p < 0.001) and the percentage of morphologically normal spermatozoa (r = 0.207; p < 0.05). Testosterone concentration rised significantly (p < 0.05) in 5–6 week of trial, e. i. after the first dose of Improvac and after this peak the level of testosterone further progressively decreased (p < 0.05).

Conclusions

Results from this study indicate that active immunization of sexually matured boars against GnRH has negative impact on testosterone concentration, sexual behavior, volume of ejaculate and total number of normal spermatozoa in ejaculate.     

超低温保存对藏獒犬精子质量及超微结构的研究(英文)

[目的]该研究旨在观察超低温保存对藏獒犬精子质量及超微结构的影响。[方法]通过精子运动度、膜完整率及顶体完整率评价4种冷冻保护剂、冷冻时距液氮面3种不同高度及添加物(SDS和Vc)对精子超低温冷冻效果的影响;在此基础上,在扫描电镜和透射电镜下观察超低温冷冻对精子超微结构的影响。[结果]试验1表明,EG作为冷冻保护剂获得了最好的冷冻效果,解冻后精子运动度、膜完整率及顶体完整率分别为36.3%、38.0%和42.0%。但EG和甘油作为冷冻保护剂并无显著差异存在(P>0.05)。试验2表明,距离液氮面5cm冷冻时冷冻效果最好。但除顶体完整率外,冷冻高度2和5cm间不存在显著差异(P>0.05)。试验3表明,稀释液中单独或共同添加十二烷基磺酸钠(SDS)和维生素C(Vc)均能改善精子冷冻-解冻效果。单独添加SDS或Vc时,除单独添加Vc组表现精子冷冻-解冻后运动度更低外(P<0.05),其它指标并无显著差异存在(P>0.05)。共同添加SDS和Vc时,精子冷冻效果最好,精子解冻后运动度、膜完整率和顶体完整率分别为44.1%、48.0%和48.2%。超微结构显示超低温保存引起藏獒犬精子不同程度的损伤,顶体损伤更严重,如顶体肿胀、囊泡化,甚至顶体消失。[结论]冷冻保护剂EG、距液氮面5cm高度及共同添加SDS和Vc可提高精子超低温保存效果,但超低温对精子超微结构仍具有一定损伤作用。     

不同稀释液对猪精液常温保存效果的影响

为研究适宜于大白猪精液常温保存的稀释液配方,以精子活率、质膜完整率、顶体完整率和呼吸强度为测定指标,分析4种不同配方的稀释液常温下保存大白猪精液的效果。结果表明,经48h保存后,稀释液Ⅰ和Ⅱ保存的精子活率、质膜完整性和顶体完整率分别达到54%、58%和88%,显著优于其他组(P<0.05)。经稀释液Ⅰ和Ⅱ保存的精子的呼吸强度分别为7.25min和8.25min,显著优于稀释液Ⅲ、Ⅳ和对照组(P<0.05),但稀释液Ⅰ和Ⅱ差异不显著(P>0.05)。因此,稀释液Ⅰ和Ⅱ是大白猪精液常温保存的适宜稀释液。     

单细胞凝胶电泳检测家蚕冷冻精子的DNA损伤

采用单细胞凝胶电泳法检测家蚕冷冻精子DNA损伤,结果未加冷冻保护剂(含10%DMSO的Grace s me-dium)的部分精子SCGE图呈明显的彗星状,其DNA损伤程度达56%,与对照组相比差异显著,表明DNA受到了一定程度的损伤;而添加冷冻保护剂的精液无论在-80℃下保存4个月还是在液氮中保存1年,其精子的SCGE图仍然呈圆形,DNA损伤程度与对照无显著性差异。     

磁场对低温保存鸡精液品质的影响

[目的]探讨磁化处理对低温保存鸡精液品质的影响。[方法]将5 m l新鲜精液平均分为5份,每份按1∶4（V/V）进行稀释,其中1份为对照组,不作磁化处理,其余4份分别放入自制磁化器中磁化6、12、24、48 m in,5组均置于2-4℃的冰箱中保存,定时观测精子活率、存活时间、生存指数和畸形率。[结果]精液经磁化后活率提高,并且有效生存指数明显优于对照组,尤以磁化24 m in效果最明显,比对照组提高了7.75%,而精子的畸形率磁化组低于对照组。[结论]磁化处理可有效提高冷冻保存鸡精液的品质。     

精子获能鉴定方法研究进展

精子获能是哺乳动物受精过程和体外受精技术中的关键环节。本文主要对获能精子的超激活运动、顶体反应、去能因子及呼吸能变化等鉴定方法进行了综述。     

Morphological study of boar sperm during their passage through the female genital tract

Once deposited in the female tract, sperm face a series of challenges that must be overcome to ensure the presence of an adequate normal sperm population close to the site of fertilization. Our aim was to evaluate the influence of the uterine milieu on boar sperm morphology. In experiment 1, sperm morphology was evaluated in the backflow (60 min after insemination) and within the uterotubal junction (UTJ) (collected ~24 h after insemination) following intrauterine sperm deposition (n = 6) and compared with the morphology of the sperm in the insemination dose. In experiment 2, the influence of the uterine fluid (UF) on sperm morphological modifications was evaluated. For this purpose, ejaculated (n = 4) and epididymal (n = 4) sperm were in vitro incubated with or without UF for 2 and 24 h. In both experiments, sperm were classified as normal, having a cytoplasmic droplet (proximal or distal) or having tail defects. The results of experiment 1 pointed to an increase in morphologically abnormal sperm collected in the backflow (27.70%) and a reduction of the same in the UTJ (2.12%) compared with the insemination dose (17.75%) (P < 0.05). In experiment 2, incubation of ejaculated sperm with UF did not provoke any morphological modifications; however, when epididymal sperm were incubated with UF, a pronounced increase in the percentage of normal sperm was evident after 24 h compared with the initial dose (from 25.77% to 53.58%, P < 0.05), mainly due to distal cytoplasmatic droplet shedding (53.22 vs. 20.20%). In conclusion, almost all the sperm that colonize the UTJ had a normal morphology, with part of the abnormal sperm having been discarded in the backflow and part selected/modified on their way to the oviduct. UF seems to influence cytoplasmic distal droplet removal, as demonstrated previously in seminal plasma.     

中国松干蚧属MATSUCOCCUS精子超微结构的进一步研究

本文主要研究了中国的四种松干蚧:海松干蚧M.koraiensis;樟子松干蚧M.dahuriensis;日本松干蚧M.matsumurae;马尾松干蚧M.massonianae的精子超微结构。研究表明,不同种松干蚧在精子超微结构上具有明显的差异,主要表现在精子核区与非核区微管的排列方式、微管的数量、微管列所跨的角度以及精子、精子束的长、直径等方面。     

大菱鲆不同繁殖期的精子质量分析

本研究运用计算机辅助精子分析（CASA）系统软件,针对大菱鲆繁殖期不同阶段的精子质量进行了检测分析.结果表明,在42d的实验过程中,精子活动比例、密度、产精量、直线运动速度、曲线运动速度均呈现不同的变化规律.其中,产精量由2.50ml降至2.23ml;密度由1.08×108个/ml降至0.46×108个/ml;精子活动比例变化范围为83.4％～36.9％;直线运动速度和曲线运动速度比值由0.69降低到0.35.繁殖期间各质量指标变化规律及幅度大小为鱼类繁殖和精液采集等操作提供了参考依据.