猪精液冷冻损伤机理研究进展

阐明降温-升温过程精子损伤的原因是目前冷冻生物学的研究热点之一。借助分子生物学手段,猪精子冷冻损伤研究已深入到分子水平,对在冷冻过程中的损伤机理研究方面取得很大突破。在参考国内外文献的基础上,综述了猪精液冷冻损伤机理在近几年来的研究进展。     

猪卵母细胞体外成熟与冷冻附睾精子的体外受精

由屠宰母猪卵巢分离得到505枚卵母细胞,经成熟培养,其中468枚卵丘细胞扩张,成熟率为92.7％。采用冷冻的附睾精子授精,受精率为41.9％(57/136),其中单精受精率36.8％(50/136),多精受精率5.1％(7/136)。授精卵体外培养24和48小时后的卵裂率分别为5.3％(15/258)和12.9％(30/232)。把35枚早卵裂期胚胎和250枚未见卵裂的1-细胞期受精卵移入4头受体母猪的输卵管内,其中2头妊娠,分别于1990年4月10日和29日正常分娩,共生产仔猪21头。     

海藻糖对猪精子冷冻真空干燥保存效果的影响

猪精子经冷冻干燥后,在光学显微镜和电子显微镜下观察其超微结构,并借助辅助生殖技术将其注入猪卵母细胞后,进一步观察受精卵的发育情况。结果表明：海藻糖组雄原核形成率 (68.52％)、卵裂率 (59.17％) 和囊胚率 (19.16％) 优于EDTA组 (64.59％、56.26％和15.62％) 和对照组 (35.36％、52.33％和8.60％) (P＜0.05);海藻糖组的冷冻真空干燥猪精子分别在4℃下保存60、120、180 d,雄原核形成率、卵裂率和囊胚率均无显著差异 (P＞0.05);海藻糖组的冷冻真空干燥猪精子复水化后孵育1 h和2 h,卵裂率、卵裂率和囊胚率均差异显著 (P＜0.05);海藻糖处理组与EDTA处理组中的冷冻真空干燥猪精子分别在4℃和?20℃下保存后各处理组间精子形态差异不显著 (P＞0.05);海藻糖组中B级冷冻真空干燥精子百分数显著多于EDTA处理组 (P＜0.05)。超微结构分析表明,冷冻真空干燥猪精子的损伤主要表现在顶体和颈部的肿胀与缺损、尾部断裂。     

哺乳动物精子冷冻的抗氧化研究进展

人工授精是迄今为止应用最广泛并最有成效的辅助生殖技术,而高品质的精液是提高人工授精受胎率的关键。近年来在家畜精液冷冻保存技术中应用抗氧化剂的研究受到广泛关注,通过添加抗氧化剂降低了精子在冷冻保存过程中所遭受的氧化损伤,提高了冷冻精液质量和母畜的受胎率。可添加的抗氧化剂种类很多,通常有维生素类和酶类抗氧化剂等。针对目前抗氧化剂在大熊猫精液上应用研究甚少的现状,该文对哺乳动物精子的氧化损伤机制和常用的抗氧化剂进行综述,期望对大熊猫的相关研究提供理论依据和参考。     

中缅树鼩精子形态特征及冷冻损伤

树鼩作为一种新型的、接近灵长类的实验动物,在医学生物学上的应用受到越来越多的重视。精子的结构特性研究及冷冻后结构的完整性分析是精子生物学的主要内容,也有助于树鼩的实验室快速繁殖。该研究采用人工饲养的中缅树鼩(Tupaia belangeri chinensis),结果显示其睾丸占总体重的(1.05±0.07)%,总体积为(1.12±0.10)mL。附睾尾及输精管精子总量估计在2.2×107～8.8×107,其运动度和顶体完整率分别为(68.8±3.9)%和(90.0±2.1)%。利用扫描电子显微镜和透射电子显微镜对树鼩附睾精子的超微结构进行的观察和分析显示精子头部呈圆形或卵圆形;头部长度、宽度平均分别为6.65和5.82μm;精子尾部中段、主段、尾段和精子总长度平均分别为13.39、52.35、65.74和73.05μm;尾部中段的线粒体螺旋数量为48个,其轴丝结构为典型的"9+9+2"结构。冷冻解冻后的精子主要表现在顶体与质膜不完整、精子断裂、尾部扭曲和膨大。上述结果提示树鼩精子与其他哺乳动物精子的结构特征相似,但是精子大小和线粒体螺旋数目有明显的差别,且超微结构改变仍是冷冻精子运动和受精能力下降的主要原因。     

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。     

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。     

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。     

精子DNA损伤检测技术的研究进展

精子DNA完整性与男性生育力之间的关系是近些年来生殖医学研究领域的热点之一,精子DNA损伤已成为反映男性生育力的一个新指标。精子DNA的损伤原因有很多,有时可能是多种因素共同作用的结果。生殖系统疾病、环境污染、吸烟、微量元素及各种理化因素等原因都可能导致精子DNA完整性受损。常见的精子DNA完整性检测技术有原位末端标记法、精子染色体扩散实验、精子染色质结构分析试验、单细胞凝胶电泳、荧光原位杂交技术和8-羟基脱氧鸟苷测定法等。随着检验技术的不断发展,关于精子DNA损伤的检测技术也在不断更新改进。本文主要就近十年来精子DNA损伤机制、检测技术的相关研究进展作一综述,提示现有的精子DNA完整性检测技术尚不能满足临床和科研需要,急需找到一种理想的检测方法为男性不育的诊断和治疗提供重要依据。     

乙二醇降低超低温冷冻黄姑鱼精子的DNA损伤

为了解乙二醇(EG)为抗冻剂超低温冻存黄姑鱼(Nibea albiflora)精子的活力及DNA损伤情况,本研究以Hank′s盐溶液(HBSS)为稀释液,5%~30%乙二醇(EG)为抗冻剂,0.5 ml麦细管为冻存管,两步降温法超低温冷冻保存黄姑鱼精子,用显微观察法测定精子活力,用单细胞凝胶电泳技术(SCGE)检测精子的DNA损伤,用SPSS 11.5处理实验数据。黄姑鱼鲜精的激活率为85.67%±2.09%、运动时间为(318.67±6.11)s、寿命为(405.67±7.77)s。5%、10%、15%乙二醇(EG)组冻精的运动时间及寿命与鲜精相比差异不显著,其中10%乙二醇(EG)组冻精的激活率为84.67%±1.15%、运动时间为(319.00±12.12)s、寿命为(400.67±4.73)s;20%、25%、30%乙二醇(EG)组冻精的运动时间及寿命与鲜精相比差异显著。5%、10%、15%、20%乙二醇(EG)组冻精核DNA损伤状况与鲜精无显著差异,25%、30%乙二醇(EG)组冻精核DNA损伤状况与鲜精差异显著,且冻精核DNA的损伤程度与乙二醇(EG)浓度成正相关。分析认为,5%~15%乙二醇(EG)适宜作为黄姑鱼精子超低温冷冻保存用抗冻剂。     

Fertility of Deep Frozen Boar Spermatozoa

In the present investigation the results of two insemination trials with deep frozen boar spermatozoa are presented. The aim of the trials was to study the effect of different thawing diluents and to compare the fertility of deep frozen spermatozoa from four boars. The trials utilized a total of 139 gilts. The thawing diluents used were boar seminal plasma, protein free seminal plasma, the thawing diluent OLEP and isotonic glucose solution. The composition of OLEP was based on physical and biochemical analyses of boar seminal plasma. The electrolyte levels, pH and osmotic pressure of OLEP are similar to those of boar seminal plasma. From the results it is evident that thawing in boar seminal plasma, protein free seminal plasma and OLEP yielded equal results. Thawing in isotonic glucose solution yielded significantly poorer results concerning percentage of fertilized ova 24–48 hrs. after insemination and almost significantly poorer fertility results four weeks after insemination. The possible effects of the thawing diluents are discussed. With the freezing procedure applied, electrolyte levels, pH and osmotic pressure seem to be factors of importance for the survival of the frozen and thawed spermatozoa and for the maintenance of their fertilizing capacity. Almost significant differences were found in fertility of spermatozoa from different boars. These differences were reflected in pregnancy rates as well as ratio of foetuses to c. 1. in pregnant gilts. The differences were found to be independent of thawing diluent. The variation seems to be caused by differences in resistance of the spermatozoa to the freezing and thawing procedure. The need for laboratory methods for selection of boars with spermatozoa of good freezability is stressed.     

Discovery of Predictive Biomarkers for Litter Size in Boar Spermatozoa

Conventional semen analysis has been used for prognosis and diagnosis of male fertility. Although this tool is essential for providing initial quantitative information about semen, it remains a subject of debate. Therefore, development of new methods for the prognosis and diagnosis of male fertility should be seriously considered for animal species of economic importance as well as for humans. In the present study, we applied a comprehensive proteomic approach to identify global protein biomarkers in boar spermatozoa in order to increase the precision of male fertility prognoses and diagnoses. We determined that l-amino acid oxidase, mitochondrial malate dehydrogenase 2, NAD (MDH2), cytosolic 5′-nucleotidase 1B, lysozyme-like protein 4, and calmodulin (CALM) were significantly and abundantly expressed in high-litter size spermatozoa. We also found that equatorin, spermadhesin AWN, triosephosphate isomerase (TPI), Ras-related protein Rab-2A (RAB2A), spermadhesin AQN-3, and NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2) were significantly and abundantly expressed in low-litter size spermatozoa (>3-fold). Moreover, RAB2A, TPI, and NDUFS2 were negatively correlated with litter size, whereas CALM and MDH2 were positively correlated. This study provides novel biomarkers for the prediction of male fertility. To the best of our knowledge, this is the first work that shows significantly increased litter size using male fertility biomarkers in a field trial. Moreover, these protein markers may provide new developmental tools for the selection of superior sires as well as for the prognosis and diagnosis of male fertility.Prognosis and diagnosis of male fertility is a major concern in both animals and humans worldwide. In humans, about half of the fertility problems arise because of male factors. In addition, 50% of breeding system failures that are contributed by the sire lead to huge economic drawbacks in the animal industry (1 –5). Therefore, the development of new methods is needed to ensure more accurate prognosis and diagnosis of male fertility.Worldwide, artificial insemination (AI)¹ has been extensively performed in animal industries. Recent data revealed that more than 90% of the sows in Europe and the United States have been bred using AI during last three decades (6). The AI system sufficiently contributes to the achievement of high performance swine production through the selection of quality semen. Moreover, AI has been implemented extensively in swine industries for genetic up-grading (7, 8). However, the selection of high quality semen still depends on conventional sperm analyses such as the analysis of sperm morphology (9), motility (10), and sperm penetration assays (11, 12). Although these tests are commonly used to evaluate the male factor of fertility/infertility, the clinical value is still debated (13). Therefore, to evaluate the limits of conventional sperm analyses, the development of new methods to assess sperm function and fertility should be seriously considered for animal species of economic importance as well as for humans. Additionally, it is important to note that the optimization of sperm production will be possible when the methods to choose superior sires with greater efficiency become available. In this regard, the identification of global protein biomarkers using comprehensive proteomic tools represents a new method on the horizon that may facilitate the prediction of superior sires.Recently, several studies have reported that proteomics is an effective tool that has the potential to transform our understanding of spermatozoa (14 –16) by acquiring new biomarkers of male infertility and/or fertility. In addition, the development of mass spectrometry (MS) allows the potential identification of sperm proteins (17, 18). In fact, increased knowledge of the sperm proteome allows us to identify new molecular markers.In this study, we used high- and low-litter size boar spermatozoa to develop suitable biomarkers. First, sperm motility (%), motion characteristics, and capacitation status were evaluated by computer-assisted sperm analysis (CASA) and combined Hoechst 33258/chlortetracycline fluorescence assessment. Second, to find deferentially expressed proteins (>threefold) between high- and low-litter size boar spermatozoa, a 2-DE proteomic approach was applied following the identification of proteins by ESI-MS/MS and a MASCOT search. The 2-DE results were confirmed by a Western blot analysis that was performed with five commercially available antibodies. Third, to validate discovered markers for male fertility prediction, the expression levels of five proteins from 20 randomly selected boar spermatozoa with broad fertility ranges (i.e. litter size) were analyzed by enzyme-linked immunosorbent assay (ELISA), and the relationship between protein expression and male fertility was determined. Moreover, to represent the entire proteomic event, biological functions and interactions of the deferentially expressed proteins were schematized by a signaling pathway.     

Viability and Acrosome Staining of Bull, Boar and Rabbit Spermatozoa

A practical and reliable staining procedure was developed to distinguish the viability and acrosomal status of bull, boar and rabbit spermatozoa. The first stain with trypan blue or Congo red is rapid and avoids artifacts. This stain is precipitated by neutral red during the 2 min required for fixation. The precipitate gives a high contrast black color, resistant to the subsequent rinsings and persists during the time required for staining the acrosome with Giemsa. Ten classes of spermatozoa are distinguished (live or dead with intact acrosomes, loose acrosomes, damaged acrosomes, no acrosome, or with no acrosome and no postacrosomal ring). The intact acrosomes are purple, the loose acrosomes are dark lavender and the damaged acrosomes are pale lavender. The anterior part of the head of live spermatozoa with no acrosome is white or light pink and the same area of dead spermatozoa is white or pale gray. The postacrosomal ring is red. The postacrosomal area of the head of live spermatozoa is white or light pink and the same part of dead spermatozoa is black, dark violet or gray. The procedure did not give satisfactory results for stallion spermatozoa.     

On the Fertility and Survival of Deep Frozen Boar Spermatozoa Thawed in Skim Milk

Satisfactory conception rates of deep frozen boar spermatozoa were obtained, with insemination by way of the cervix, after thawing the deep frozen spermatozoa in boar seminal plasma, both in preliminary trials (Crabo & Einarsson 1971, Crabo et al. 1972 b) and in a large field trial (Einarsson et al. 1972). Fertility with pellet frozen boar spermatozoa, thawed without dilution, was reported by Graham et al. (1971 a, b) and Pursel & Johnson (1971).     

Fertility of Deep Frozen Boar Spermatozoa at Various Intervals between Insemination and Induced Ovulation

This study was performed to investigate the influence of boars and thawing diluents on the fertilizing capacity of deep frozen spermatozoa at various intervals between inseminations and ovulation. Forty-four Swedish crossbred gilts were inseminated following injection of HCG late in the prooestrus. Inseminations were performed 22, 28, 34 and 38 hrs. after injection of HCG. Ovulation was expected to occur 40 hrs. after injection of HCG. Two boars, previously tested for fertility with frozen semen, supplied the spermatozoa. Roar seminal plasma and OLEP were utilized as thawing diluents. The gilts were slaughtered 32–48 hrs. after estimated ovulation. The genital tracts were removed immediately after stunning and bleeding and the numbers of recent ovulations, recovered ova and fertilized ova were recorded. Additionally recovered ova were classified according to estimated numbers of spermatozoa attached to the zona pellucida. Similar fertilization rates were obtained when inseminations were performed 2 and 6 hrs. before estimated ovulation. A clear decline in fertility appeared when inseminations were performed earlier than 6 hrs. before expected ovulation. The results were influenced by the boars as well as by the thawing diluents. Seminal plasma yielded a higher fertilization rate than OLEP in inseminations performed 2 hrs. before estimated ovulation. The boars yielded similar fertility in inseminations performed 2 hrs. before estimated ovulation. With increasing intervals between inseminations and ovulation the difference between the boars increased. The single gilt in which fertilized ova were found after insemination 18 hrs. before ovulation was inseminated with spermatozoa from the superior boar, thawed in seminal plasma. The present results indicate that spermatozoa with low resistance to freezing-thawing have a short fertile life in the female genital tract after insemination.     

Influence of Boars on the Relationship between Fertility and Post Thawing Sperm Quality of Deep Frozen Boar Spermatozoa*

The aim of this investigation was to evaluate the possibility of selecting boars for deep freezing by means of laboratory tests on frozen-thawed spermatozoa. Thirty-one randomly selected frozen ejaculates from four boars were investigated by a thermoresistance test after thawing in boar seminal plasma and in OLEP. Extracellular ASAT activity was measured in samples from 30 of the ejaculates after thawing in OLEP and in isotonic glucose solution. Twenty of the ejaculates were utilized for fertility tests by artificial insemination of 37 gilts preceding the laboratory investigation. Three of the boars proved fertile with frozen semen. One of these boars seemed to yield superior fertility to the other two boars. No fertility was obtained with frozen spermatozoa from the fourth boar. Prior to the freezing trial this boar had been used for fresh semen inseminations giving higher pregnancy rates than the average of Swedish A.I.-boars. This boar was therefore considered a case of “low freezability”. In the laboratory tests the samples from this boar showed the lowest motility after 3 hrs.’ storage at 37°C, the highest relative decrease of motility during the thermoresistance test, the highest release of ASAT after thawing in OLEP and the highest relative release of ASAT. Analyses of variance indicated significant and almost significant variation among boars in relative decrease of motility during the thermoresistance test and in relative release of ASAT. The results indicate that the boars were the main cause of variation in fertility as well as in outcome of the laboratory tests. These results do not permit a complete evaluation of the relationship between fertility and outcome of the applied laboratory tests. However, the results indicate a possibility of detecting boars producing spermatozoa with low freezability by means of laboratory tests.     

III: Ultrastructure of Boar Spermatozoa Frozen Ultra-Rapidly at Various Stages of Conventional Freezing and Thawing

Ejaculated boar spermatozoa subjected to a conventional freezing and thawing process, were ultra-rapidly fixed, freeze-substituted and examined by electron microscopy to monitor the presence of real or potential intracellular ice and the degree of cell protection attained with the different extenders used during the process. Numerous ice crystal marks representing the degree of hydration of the cells were located in the perinuclear space of those spermatozoa not in proper contact with the extender containing glycerol (i.e. prior to freezing). The spermatozoa which were in proper contact with the extenders presented a high degree of preservation of the acrosomes, plasma membranes as well as the nuclear envelopes. No ice marks were detected in acrosomes before thawing, indicating that the conventional assayed cryopreservation method provided a good protection against cryoinjury. The presence of acrosomal changes (internal vesiculization, hydration and swelling) in thawed samples however, raises serious questions about the thawing procedure employed.     

猪精子中与卵透明带糖蛋白ZP3结合的蛋白质

依次经ＰＳＬ－Ｓｅｐｈａｒｏｓｅ亲和层析柱和纤维素ＣＭ－５２离子交换层析柱,从猪精子的ＣＨＡＰＳ抽提液分离得４个蛋白质组分。用固相透明带精蛋白结合试验（ＩＺＰＧＢＡ）检测;表明精子蛋白ＳＰ１和ＳＰ２具有结合透明带糖蛋白ＺＰ３的活性,ＳＰ２并显示凝集血球的活性。精子蛋白ＳＰ１与卵预温育明显抑制精卵结合,抑制活性与加入的精子蛋白的浓度呈正相关。用生物素标记的ＺＰ３和蛋白质印迹技术,证明ＳＰ１中的６８ｋＤ精子蛋白与ＺＰ３结合,提示６８ｋＤ精子蛋白参与精卵结合。     

野猪种源生态与杂交利用的研究进展

野猪是家猪的祖先,分欧洲野猪与亚洲野猪两大类,共有27个野猪亚种.野猪多柄息于山林近水地带,食性杂,主要以植物为食.夏季以夜间活动为主,冬季多为白天活动.野猪前躯大,后躯小,与家猪正好相反,体高与体长的比例也较家猪大,这种体型适于其野外生活与奔袭.冬季发情配种,春季产仔.野猪与家猪杂交能够产生具有正常繁殖力的后代.野猪具有耐粗饲、抗病力强、肌纤维细的优良特性.野猪与家猪杂交可改良家猪肉质,生产特色猪肉.     

Isolation and Characterization of Membrane Vesicles from Human and Boar Spermatozoa: Methods Using Nitrogen Cavitation and Ionophore Induced Vesiculation

Abstract

A method for isolation of plasma membrane vesicles from human and boar spermatozoa using nitrogen cavitation is described. The purity of the preparations were assessed by electron microscopy, marker enzyme assay and the sedimentation characteristics of fused plasma membrane-acrosomal membrane vesicles in sucrose gradients. PAGE-SDS profiles of plasma membrane polypeptides from boar spermatozoa were significantly different from those of human spermatozoa. Differences in electrophoretic profiles of polypeptides from different regions of the spermatozooon were also observed.