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1.
用曙红 Y、台盼蓝染料对马氏珠母贝(Pinctada martensii)精子进行染色,通过对不同制片方法、不同的染色时间和不同染料浓度的染色效果进行比较,筛选出可通过染色特征来检测精子死活及活精率的适宜方法,结果显示:(1)经染色后,死、活精子呈现出明显不同的特征:活精子均不着色,头部呈梨形、无色透明状;死精子着红色(曙红Y染色)或蓝色(台盼蓝染色),体积变大,头部近圆形。(2)曙红Y、台盼蓝最适染色浓度分别为0.04%和0.2%;最佳染色时间均为15min。(3)检测到的精子死亡率即实测精子死亡率,与“理论死亡率”之间有明显的相关性,相关系数为R=0.99(P<0.01),并且两种染色法的观察结果一致,表明这些检测结果客观可信;(4)染料对活精子毒性的检测显示,染色45min内,精子死亡率无显著性增加(P>0.05),说明在该时间范围内,染液对精子的毒性较小,精子存活率的检测结果稳定可靠。这些研究表明,曙红 Y、台盼蓝两种染色法准确灵敏,均适宜用于马氏珠母贝精子质量的检测与评价。  相似文献   

2.
目的探讨4℃保存的条件下,苯甲基磺酰氟(phenylmethylsulfonyl fluoride,PMSF)对犬精子的保护作用。方法犬精子以含不同浓度PMSF的Tris-卵黄液(Tris-egg yolk,EYT)处理后,于4℃低温保存,在24、48、72、96 h分别以伊红、瑞-吉和吖啶橙染色检测精子活率、顶体膜和DNA完整性。结果低温保存24 h后,吖啶橙染色检测DNA断裂率(%):对照组(5.93±0.32),实验组:20μg/mL(4.50±0.38)、40μg/mL(4.20±0.25)、80μg/mL(4.21±0.46)、160μg/mL(3.87±0.67),P〈0.05;各浓度组之间没有明显差异;72 h后,犬精子活率仍〉30%;96 h内,对照组和实验组犬精子在活率、顶体膜完整性上未见明显差异。结论PMSF于4℃低温保存条件下对犬精子具有保护作用,24 h内能有效保护精子DNA双链的完整性,4℃低温保存72 h后,犬精子仍符合人工授精要求。  相似文献   

3.
该文研究了丙烯酰胺在体外对人成熟精子功能的影响。不同浓度(0,2,10,50,250,1 000μmol/L)的丙烯酰胺溶液在体外处理人成熟精子,利用伊红苯胺黑染色、计算机辅助精子分析系统、金霉素染色、精子单细胞钙成像等方法检测精子活力、运动、获能、顶体反应和胞内钙动员等生理功能。结果显示,不同浓度的丙烯酰胺溶液对人成熟精子活率、运动、获能、顶体反应以及胞内钙动员等生理功能均未有显著影响。推测丙烯酰胺体外急性染毒在短时间内并不抑制人成熟精子功能。  相似文献   

4.
将采自23头成年圈养黑熊的精液,分别用3种稀释液(Ⅰ:Tris-乳-果-卵;Ⅱ:柠-葡-蔗-卵;Ⅲ:Tris-柠-果-葡-卵)稀释并在4℃下保存,通过检测精液在不同稀释液稀释条件下的保存时间,筛选出最适稀释液用于精液的冷冻保存;从精液解冻后精子的活率、活力、畸形率、顶体完整率4个指标,分别从3种冷冻保护剂(甘油3%、3.5%、4%)、两种冷冻方法(两步冷冻法和自动冷冻法)两个方面进行了比较试验.结果表明:精子活力在0.3以上时,稀释液Ⅲ保存时间为175.42±3.04 h,显著高于稀释液Ⅰ和稀释液Ⅱ(P﹤0.01),稀释液Ⅱ保存时间也明显高于稀释液Ⅰ(P﹤0.01);含3.5%甘油浓度的稀释液解冻后精子活率(41.75±3.46%)、活力(32.63±5.27%)和顶体完整率(85.62±4.58%)显著高于其他两组(P﹤0.01),并且精子畸形率(29.32±8.22%)明显低于其他两组(35.95±8.04%,36.07±7.72%)(P﹤0.01);采用自动冷冻法冷冻保存圈养黑熊精液,解冻后精子活率、活力和顶体完整率分别为41.75±3.46%、32.63±5.27%和85.62±4.58%,都明显高于两步冷冻法(P﹤0.01);解冻后畸形率为29.32±8.22%,明显低于两步冷冻法(P﹤0.01).  相似文献   

5.
OEP及卵黄浓度对蓝狐冻融精子质量的影响   总被引:6,自引:0,他引:6  
人工采取 6只优质芬兰雄性蓝狐精液 ,利用不同OEP及卵黄含量的Tris 果糖 -柠檬酸钠稀释液稀释 ,制成细管冻精 ,透射电镜下观察精子冷冻前后质膜和顶体超微结构 ,荧光免疫方法检测不同培养时间冻融精子的质量。结果表明 ,蓝狐精子顶体外膜双层膜的厚度为 0 0 2 0 μm ,冷冻 -解冻过程中易发生质膜膨胀、顶体外膜融合现象。顶体产生的囊泡分两种类型 ,一种是体积较大的中空囊泡 ,平均直径为 1 2 5 μm。另一种是体积较小的实体囊泡 ,内充满顶体内容物 ,平均直径为 0 83μm ,两种囊泡的数量不定。OEP能有效抑制顶体囊泡形成 ,影响顶体囊泡类型、体积大小及囊泡数量 ,添加适宜剂量OEP能使顶体囊泡的体积明显缩小 ,囊泡的总数及中空囊泡的数量显著降低。蓝狐冻融精子质量与OEP及卵黄剂量有关 ,在卵黄存在的前提下 ,OEP有利于维持冻融过程中质膜 (5 6 3% )、顶体的完整性 (5 7 8% ) ,显著提高冻融精子活力 (5 4 7% )。在蓝狐精液稀释液中 ,OEP、卵黄的适宜含量分别为 1 %、 2 0 %  相似文献   

6.
目的:为了更清晰地显示胰岛的A细胞、B细胞和D细胞。方法:zenker液固定,分别用苏木精-伊红和改良的Mallory氏法染色。结果:苏木精-伊红染色法,不易区分胰岛内三种细胞,而采用改良的Mallory氏染色方法,能够清晰地显示A细胞、B细胞和D细胞。结论:改良的Mallory氏染色法能够清楚的显示胰岛内三种细胞结构,为实验教学提供了优良的切片标本。  相似文献   

7.
目的:由于冷冻保存的猪精液在解冻后会出现低成活率和低繁殖率的情况,本项目研究猪精液的低温长期保存和保存后的精子质量评定。方法:本实验选用目前常用的BTS,ANDROHEP,KIEV,ZORLESCO等四种稀释剂,将猪新鲜精液进行稀释(1:1)后分别于17℃保存15天,每天取样对精子的质量进行评价,利用荧光染料Hoechst 33258和SYBR-14/碘化丙碇(PI)分别检测精子死活;利用活体荧光染料JC-1检测精子线粒体活性;利用考马斯亮兰染色检测精子顶体状态;利用金霉素(CTC)检测精子是否获能。结果:SYBR-14/PI和Hoechst 33258两种方法检测精子死活都很灵敏,前检测的活精子比例较后稍高,但都低于JC-1检测的线粒体有活性的精子比例,说明精子线粒体活性损失要慢于质膜完整性的破坏;活精子比例和线粒体活性在保存的前8天都是缓慢减少,获能比例和顶体状态分别在第9-14天和第11天变化较大,利用AN和ZO保存13天,BTS保存10天以及KIEV保存9天的精液中精子的活率,顶体完整率及线粒体有活性的比例仍高于50%,提示这些精液仍可用于人工授精或体外受精,这四种稀释剂保存15天后的精子获能比例接近70%,Hoechst33258,SYBR-14,JC-1,考马斯亮兰检测结果亮度相关。结论:这四种稀释剂对精子的低温保存效果依此为:AN>ZO>BTS>KIEV。  相似文献   

8.
长毛对虾精子发生的研究:Ⅰ.精子的形态结构   总被引:18,自引:4,他引:14  
洪水根  陈细法 《动物学报》1993,39(3):239-243
利用电子显微镜技术结合细胞化学染色方法,研究长毛对虾精子形态结构,结果显示:长毛对虾精子由圆球状球体部和钉子状棘突部组成;精子全长约7μm;棘突及与之相联的球体表面平滑无突起,而与棘突相对的球体底面有指状突起。透射和冷冻蚀刻电镜显示:长毛对虾精子棘突包括顶体锥和顶体帽两部分;顶体锥向外突出形成精子的棘突,顶体帽覆盖在球体部的胞质及核区上方。成熟精子胞质极度退化,仅靠顶体帽边缘可见1—2个线粒体。球体的中央区域为近球形,呈Feul-gen阳性反应,为精子的核区。其结构松散,电子密度低,属非浓缩型,内布许多絮状物质,外由许多长短不一的膜性结构不规则排列成不连续的核膜结构。研究结果认为:长毛对虾精子属不动无鞭毛精子类型,其棘突部并非鞭毛结构而是顶体的位置。  相似文献   

9.
圆斑星鲽精子的超微结构及核前区特殊结构   总被引:15,自引:0,他引:15  
分别以扫描电镜和透射电镜方法研究了圆斑星鲽 (Veraspervariegates)精子的超微结构。头部呈圆形、中段不发达 ,具有多个未分化的圆形小线粒体、鞭毛结构极简单 ,具有 9 2型的轴丝等特征 ,均表示圆斑星鲽的精子属于简单的原始类型 ,与多数其它高等鱼类精子结构相似。但是 ,圆斑星鲽精子核前部的凹陷 ,即不含染色质的电子透明区 ,以及核前区的囊泡结构却未见在其它新鳍鱼类精子中报道。圆斑星鲽精子核前凹陷开口于核前端稍偏处 ,凹陷呈不规则状 ,可深入到核的中央。凹陷处与染色质区无界膜分隔。凹陷区的前部及其开口处常有多个小的具单层界膜的囊泡 ,有时可见在凹陷区的开口处汇集成一个大泡。在某些处理过程中 ,例如在冷冻 -解冻过程中 ,囊泡可能丢失 ,而在精子顶端留下一个凹坑。尽管顶体在现存最早的新鳍鱼类 ,如雀鳝鱼及弓鳍鱼中已经完全消失 ,但考虑甚至在某些纯真骨鱼 ,如鲑形目中的螈鱼精子上 ,顶体仍然存在 ,而在其它一些纯真骨鱼精子或精子细胞中也存在顶体或顶体遗迹等事实。我们推测在圆斑星鲽精子中的上述特殊结构也是顶体的一种遗迹 ,但也不排除其是向外释放核内物质的一种途径的可能性  相似文献   

10.
目的以C群脑膜炎奈瑟菌(Neisseria meningitidis,简称C群脑膜炎球菌)为研究对象,探索一种新的细菌荚膜染色方法。方法制备C群脑膜炎球菌悬液,分别以Anthony法和新的荚膜染色法进行染色,新的荚膜染色法首先使荚膜与相应的抗血清进行免疫反应,生成易被染色剂染色的物质,再进行染色制片。光学显微镜下观察,对荚膜染色效果进行评价。结果 Anthony法染色后,仅菌体着色,为深紫色;荚膜透明不着色,表现为一圈白色的光环;荚膜与背景呈一定色差,但不明显;荚膜附着于细菌表面,边界不清晰,无法准确判断荚膜厚度。新的荚膜染色法染色后,菌体被染成深紫色,荚膜呈蓝绿色,与背景的色差明显;荚膜形状完整,均匀地附着于菌体表面,边界清晰,能够准确判断荚膜厚度。结论新的荚膜染色法染色效果好、操作简单、易于掌握,可用于C群脑膜炎球菌荚膜的染色。  相似文献   

11.
A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2–4 h Giemsa staining at 25–40° C is recommended for stallion semen.  相似文献   

12.
A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2-4 h Giemsa staining at 25-40° C is recommended for stallion semen.  相似文献   

13.
A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2-4 h Giemsa staining at 25-40 degrees C is recommended for stallion semen.  相似文献   

14.
Determination of the percentage of live cells with intact acrosomes and no morphologic aberrations could be a practical index of semen quality. We applied viability and acrosome staining techniques, originally described for bull, boar and rabbit sperm, to mouse spermatozoa. The viability stain was either trypan blue or Congo red. The stain was precipitated by neutral red in the fixative. The acrosome was stained by Giemsa. Sperm morphology, including cytoplasmic droplets, could be evaluated as well. The staining method described here is a useful routine tool for simultaneous evaluation of the plasma membrane integrity of different sperm subdomains, the status of the acrosome, and cellular morphology.  相似文献   

15.
ABSTRACT Seven staining techniques were modified and tested for differentially staining the live and the dead sperm cells for the honey bee (Apis mellifera L.). The eosin Y staining method was found to be a simple technique by which the live cells stain bluish purple whereas the dead cells stain bright yellow to greenish yellow. Therefore, it produces a strong contrast between the dead and live sperm cells, and appears to be the most suitable supravital staining method for evaluating the viability of honey bee sperm cells. The significance of supravital staining techniques in assessing the quality of sperm cells during cryopreserving sperm cells is discussed.  相似文献   

16.
Batrachochytrium dendrobatidis (Bd) has been implicated in hundreds of amphibian declines and is the focus of a vast amount of research. Despite this, there is no reported efficient way to assess Bd viability. Discriminating between live and dead Bd would help determine the dose of live Bd zoospores and whether factors have lethal or sublethal effects on Bd. We tested whether trypan blue, a common stain to discriminate live and dead cells, could be used to assess Bd viability. We show that the proportion of live zoospores (zoospores that excluded the trypan blue dye) matched the proportion of known live zoospores added to cultures. In contrast, all of the zoosporangia stages of Bd stained blue. These results demonstrate that trypan blue can be used to determine the viability of Bd zoospores but not zoosporangia. We recommend using trypan blue to report the number of live zoospores to which hosts are exposed.  相似文献   

17.
A simple dual staining procedure for detecting the true acrosome reaction in dried smears of buffalo spermatozoa is described. Trypan blue is used first to differentiate live from dead spermatozoa and the dried smears which have been prepared are stained with Giemsa for acrosome evaluation. Four categories of spermatozoa were recognized: A) live, intact acrosome (acrosome pink, postnuclear cap clear); B) dead, intact acrosome (acrosome pink, postnuclear cap blue); C) live, detached acrosome (acrosome clear, postnuclear cap clear); and D) dead, detached acrosome (acrosome clear, postnuclear cap blue). The procedure is simple, rapid and convenient for assessing true acrosome reaction in buffalo spermatozoa. Simultaneous assessment of sperm viability and its acrosomal status in dried smears makes this procedure attractive because the true acrosome reaction can be studied thoroughly at a later state after the incubation period.  相似文献   

18.
A simple dual stain procedure (DS) for simultaneously determining sperm viability and acrosomal status is described. The DS includes the use of the vital stain trypan blue to detect live and dead spermatozoa and Giemsa to detect the presence or absence of an acrosome. For staining, spermatozoa are washed, incubated with trypan blue, washed, dried onto slides, and subjected to Giemsa. Dead spermatozoa stain blue in the postacrosomal region while live spermatozoa remain unstained. The acrosome stains light purple–dark pink while acrosome-free sperm remain unstained. This staining pattern enables differentiation of spermatozoa which have undergone a true acrosome reaction (TAR) from those which have undergone a false acrosome reaction (FAR). Incubation of bull, boar, ram, and stallion spermatozoa for 60 minutes at 37°C in the presence of calcium ionophore A23187 increased the proportion of spermatozoa undergoing a TAR in all species except the stallion. Incubation of bull spermatozoa for up to 24 hours at 37°C resulted in a decrease over time in the percentage of live acrosome-intact spermatozoa and a simultaneous increase in the percentage of spermatozoa categorized as having undergone a TAR and FAR. The DS could be a useful technique in evaluating sperm viability and acrosomal status in fertilization and clinical studies.  相似文献   

19.
During routine evaluation of trypan blue-Giemsa stained semen smears, sperm cells can be found with unstained heads and with stained tails. It was hypothesized that these cells were immotile and should not be considered as live. Sperm motility was determined in isoosmotic, and presumably isotonic trypan blue-stained wet preparations. Bull, ram and boar semen smears were stained with hypoosmotic trypan blue-Giemsa to compare the relationship between the percentage of stained sperm tails and the percentage of sperm tails remaining straight under hypoosmotic conditions. Actively moving spermatozoa with unstained heads, but with stained tails were never observed in wet preparations. The correlation coefficient found between the percentage of sperm with stained tails and the percentage with straight tails was 0.81, 0.94 and 0.85 for bull, ram and boar spermatozoa, respectively. Results of this study show that sperm cells with an intact head membrane, but a stained and presumably membrane-damaged tail are not motile. Therefore these cells should be included in the dead category rather than alive in the usual live-dead studies with vital stains.  相似文献   

20.
The live/dead fluorescent assay provides a quick method for assessing the proportion of live and dead cells in cell culture systems or tissues and is widely used. Dead cells are detected by the fluorescence produced when propidium iodide (PI) binds to DNA; PI and similar molecules are excluded from live cells but can penetrate dead cells because of their loss of membrane integrity. Here we investigated the effect of serum in the culture medium on the reliability of the method. We assessed viability of chondrocytes with/without serum using both a live/dead assay kit and also trypan blue staining. We found that after 2 days of culture, the DNA-binding dye PI could no longer detect dead cells if serum was present but they were readily detected in serum-free medium or if an inhibitor to DNase I was added to the serum-containing medium. Dead cells could be detected by trypan blue staining in all cultures. Hence dead cells are no longer detected as the DNase I present in serum degrades their DNA. DNA-binding dyes may thus not give a reliable estimate of the number of dead cells in systems that have been cultured in the presence of serum for several days.  相似文献   

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