人和哺乳动物精液的蛋白质组学研究进展

蛋白质组学技术是21世纪研究生命科学最有价值的技术之一,将其应用到精子和精浆蛋白质的研究后,已取得了许多成果。对蛋白质组、蛋白质组学、双向电泳、质谱、生物信息学等进行了简要介绍,并重点阐述了蛋白质组学技术在精子蛋白和精浆蛋白上的研究进展。     

Quality of Raw and of Cold-Stored Semen in Icelandic Stallions

The aim of the present study was to evaluate the quality of raw and cooled semen in Icelandic stallions. Experiments were performed using seven stallions aged between 3 and 19 years. From each stallion, six ejaculates were collected, and semen quality was determined. Thereafter, the semen was split into eight equal parts and processed with and without centrifugation using the extenders INRA 82-egg yolk, INRA 96, GENT, and Equi-Pro to a final concentration of 30 × 10⁶ sperm/mL. The extended semen was then cooled in an Equitainer, where it was stored for 24 hours, and subsequently refrigerated for another 24 hours at 5°C. Immediately after dilution as well as after 24 and 48 hours storage, sperm motility was analyzed using computer-assisted sperm analyzer, and viability was assessed after dual DNA staining with SYBR-14 in combination with propidium iodide. The results show that the stallion had a significant (P < .05) influence on all variables evaluated in raw semen, and mean (±SEM) values of 43.4 ± 4.3 mL for the volume, 193.0 ± 17.0 × 10⁶ sperm/mL for the concentration, 6.7 ± 0.5 × 10⁹ for total sperm and 73.5 ± 2.1% for total sperm motility, 48.7 ± 2.0% for progressive motility, and 65.3 ± 2.0% for rapid cells were measured. In the cold-stored semen, all variables were significantly (P < .05) influenced by the stallion, extender, and storage time (48 hours). Except for Equi-Pro, all extenders examined were suitable for cooled semen preservation. For storage of more than 24 hours, centrifugation and removal of the seminal plasma were advantageous for all extenders with the exception of Equi-Pro.     

Methods of Concentrating Stallion Semen

Removal of excess seminal plasma is sometimes necessary to increase the quality and the longevity of cooled equine semen; moreover, this procedure is an indispensable step aiming to concentrate the sperm cells before freezing equine semen. Typically, the removal of seminal plasma is achieved by centrifugation; however, studies have shown that the force and duration of centrifugation can damage sperm cells and reduce the sperm recovery rate. Recently, new methodologies, such as cushion and filtration, have been described that aim to decrease the mechanical damage of centrifugation to sperm cells. This study aims to compare different methods for concentrating stallion semen.     

Myeloperoxidase in Equine Semen: Concentration and Localization during Freezing Processing

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after lysis. Post-thaw semen contains MPO, and concentration of this enzyme is associated with decreased motility. The aim of this study was to determine kinetics of MPO concentration during freezing, its origin, and its impact on frozen-thawed semen. Forty ejaculates were used. Semen was frozen using the classical freezing procedure. MPO concentrations were assayed in fresh semen, after centrifugation, and after cooling down to 4°C. Post-thaw MPO assay results and spermogram characteristics were determined. MPO immunocytochemistry was performed on 4 different ejaculates at each step of freezing procedure. MPO concentration increased after cooling down to 4°C and thawing compared with fresh semen. As temperature decreased, MPO was higher or tended to be higher in post-thaw poor quality samples. Nonsperm cells showed various degrees of MPO immunostaining and were observed as epithelial cells with nuclear pyknosis and keratinization. MPO immunostaining increased in medium and decreased in nonsperm cells during freezing. Our study shows that MPO concentration in equine semen increases when temperature decreases. We hypothesize that nonsperm cells present in fresh semen could release MPO.     

精液对母兔子宫免疫及孕向发育的影响

在结扎子宫颈的情况下,在同一子宫的两侧子宫角分别注入生理盐水（对照组）和精液（试验组）以研究精液对子宫免疫和孕向发育的影响。试验共用母兔8只。子宫注入后48 h处死试验用兔,取子宫进行测量,并制片染色,用图像采集分析系统进行研究。结果显示：1）与对照组相比,兔子宫注入精液后可以显著（P〈0.05）增加子宫的长度、直径和子宫角指数;可明显增加子宫壁（P〈0.05）、黏膜上皮层（P〈0.01）和黏膜层（P〈0.05）的厚度;可显著增加黏膜中腺管（P〈0.05）和血管（P〈0.05）数量,可明显增加腺上皮的厚度（P〈0.05）。2）子宫注入精液后,子宫冲洗液中的细胞数（53.3±4.03×109/L）较对照组极显著增加（P〈0.01）;单位面积子宫黏膜中肥大细胞的数量（2.85±0.05个/mm2）较对照组（3.72±0.04个/mm2）显著减少（P〈0.05）。子宫冲洗液涂片染色检查发现,子宫冲洗液中的细胞主要为嗜中性粒细胞。本研究表明,兔精液确实能够促进母兔子宫的孕向发育,能够改变子宫的免疫状态。     

Effect of Centrifugation Technique on Post-storage Characteristics of Stallion Spermatozoa

Three ejaculates were collected from four stallions and used to compare the effects of three centrifugation methods on post-storage motility and recovery of available sperm. Two aliquots per ejaculate were diluted with skim milk-glucose (SKMG) extender to 50×10⁶ sperm/mL, placed in 50-mL conical bottom tubes, and centrifuged at either 700g for 15 minutes (700g) or 600g for 12 minutes (600g). A third aliquot was diluted 1:1 with SKMG, placed in 15-mL conical tubes, and centrifuged at 400g for 7 minutes (400g). Subsamples from each pre-treated diluted ejaculate were held at room temperature and evaluated for motility at the same time as the post-centrifugation pre-storage motility evaluation was made for treated aliquots. After centrifugation, samples from each aliquot were stored at 5°C for evaluation after 24 and 48 hours or frozen in liquid nitrogen. Percentage of available sperm harvested was higher (P ≤ .05) for aliquots centrifuged in 15-mL tubes at 400g versus 600g in 50-mL tubes. After centrifugation, total but not progressive motility of aliquots centrifuged at 700g was lower than that for noncentrifuged controls and sperm from aliquots centrifuged at 400g in 15-mL tubes. After cold storage, values for total but not progressive motility or velocity were higher (P ≤ .05) for aliquots centrifuged in 15-mL tubes at 400g compared with those centrifuged in 50-mL tubes at both 600g and 700g. Postthaw motility of frozen sperm was not different between centrifugation treatments. Poststorage percentages of intact acrosomes and detached heads did not differ because of centrifugation treatment.     

奶牛冷冻稀释液研究进展

为了提高冻精质量,人们对稀释液进行了大量科学试验,获得许多重要成果.本文综述了奶牛冷冻精液稀释液的研究进展,为进一步研究稀释液和精子之间的相互关系及新型冷冻稀释液的开发和利用提供参考.     

稀释液溶质、渗透压、pH值和缓冲指数与低温保存鸡精液效果的关系

将葡萄糖、氯化钠、氯化钾、柠檬酸钠四种物质分别配制成三个渗透压水平(278mOsm、328mOsm、378mOsm)的12种单方精液稀释液，在低温(1～4℃)下进行鸡精液稀释保存试验，在四种物质中各选出一种好的稀释液，再将所选四种单方稀释液按不同比例制成复方精液稀释液，再次进行保存试验。所有稀释液均测定渗透压、pH值、缓冲指数、精子存活时间、精子生存指数等指标。结果表明：①柠檬酸钠、葡萄糖对鸡精液的保存具有重要作用，而氯化钾在鸡精液稀释液中并不是必需的。②复方稀释液保存效果普遍优于单方稀释液。③低温下鸡精液稀释液渗透压应为等渗或接近等渗，且最适渗透压还与构成稀释液的物质种类和相互配比有关系。④稀释液pH值应接近中性，最好与精液pH值保持一致。⑤在缓冲指数为3．894—7．257的范围内，缓冲指数与精子存活时间和生存指数呈现显著的负相关。