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1.
本试验探讨了氟对小鼠附睾中成熟精子超微结构的影响,为氟的生殖毒性研究与检测提供依据。选取8周龄性成熟雄性昆明小鼠20只,随机分为4组,对照组小鼠饮用蒸馏水,低、中和高氟组小鼠分别饮用含25、50和100 mg/L氟化钠的蒸馏水,于45 d后断颈处死小鼠,取小鼠附睾尾,经2.5%戊二醛固定后,采用透射电镜观察小鼠附睾中成熟精子的超微结构变化。与对照组相比,低氟组小鼠精子头部质膜断裂,部分脱落,个别线粒体肿胀、形态模糊;中氟组精子头部质膜脱落,顶体部分缺失,线粒体形状不规则,嵴间腔扩大;高氟组精子头部质膜脱落,线粒体排列不规则,出现空泡化,嵴结构模糊。结果表明,氟暴露小鼠附睾中成熟精子头、尾部中段均有不同程度的结构改变,尤以线粒体出现较明显的异常,且氟浓度越高,精子超微结构损伤越严重。  相似文献   

2.
实验旨在研究冷冻稀释液中添加附睾尾液（Cauda Epididymal Fluid,CEF）对绵羊精液冷冻保存的影响。利用假阴道法收集4只湖羊精液并混合,以附睾尾液中总蛋白为基准,在冷冻稀释液中分别添加不同浓度CEF(0、140、280、420μg/mL)。精液经过冷冻后投入液氮保存,解冻后检测精子活力、运动参数,以及质膜完整率、顶体完整率、线粒体膜电位、37℃精子存活时间等指标。结果表明：冷冻-解冻后280μg/mL CEF组的精子活力、前向运动、平均路径速度、直线运动速度、直线度、线性度、头部横向位移幅度均显著高于其他各组（P<0.01）,曲线运动速度高于其他各组（P<0.05）;精子质膜完整率、顶体完整率也高于其他各组（P<0.05）;JC-1染色结果显示,280μg/mL CEF组精子线粒体膜电位高于其他各组（P<0.05）;280μg/mLCEF组精子在37℃环境下可存活14h,较对照组精子能多存活4h。可见,在冷冻稀释液中添加适量CEF可以提高精液的冷冻效果。  相似文献   

3.
为了研究牛血清白蛋白(BSA)对犬精液冷冻保存效果的影响,试验配制含有不同浓度(0、3%、5%、7%)BSA的精液冷冻稀释液用于保存犬精液,测定冷冻-解冻后精子活力、质膜完整率、顶体完整率、总抗氧化能力(T-AOC)、丙二醛(MDA)含量及线粒体膜电位(MMP)等指标。结果表明：3%和5%BSA组冷冻-解冻后精子活力、质膜完整率、顶体完整率和T-AOC均显著高于对照组(P<0.05);3%BSA组MDA含量显著低于其他各组(P<0.05),线粒体膜电位值显著高于其他各组(P<0.05);除精子活力外,7%BSA组和对照组之间各项指标均差异不显著(P>0.05)。说明在犬精液冷冻稀释液中添加BSA能有效改善冷冻-解冻后精液品质,对提高精子抗氧化能力及代谢能力都有显著效果,BSA的最适添加浓度为3%。  相似文献   

4.
精液体外孵育保存会使精子品质下降,抗氧化剂L-肉毒碱可促进激活的脂肪酸从细胞质到线粒体基质的转运,减少ROS产生和脂质过氧化反应的发生。研究分析了牛精液体外保存过程中不同浓度L-肉毒碱(LC)(0、2、10和15mmol/L)和不同孵育时间(2、4、6h)对牛精子活力、质膜完整性、线粒体活性和顶体完整性的影响。结果表明:与对照组相比,在牛精液体外保存稀释液中添加10mmol/L和15mmol/L的LC均可显著(P0.05)提高精子活力(33.0%vs 45.6%,42.4%)、质膜完整率(61.2%vs 73.6%,68.4%)和顶体完整率(81.4%vs 88.4%,86.3%),而线粒体活性则是10 mmol/L LC组效果最好;将筛选的最佳浓度10mM LC组于27℃体外孵育2、4、6h后,其精子的活力、质膜完整率和线粒体高膜电位百分率均显著高于对照组(P0.05),顶体完整率仅在孵育6h后与对照组差异不显著(P0.05)。由此可见,LC可保护精子功能和结构免受损伤,不仅能改善牛精液体外保存的品质参数,又可延长牛精液体外保存的时间。  相似文献   

5.
采集欧拉型藏羊的睾丸及附睾,对其不同区段内精子的形态指标进行观察。结果显示:睾丸及附睾不同区段的精子密度依次为:睾丸1.05╳10~7个/mL、附睾头4.31╳10~7个/mL、附睾体2.13╳10~7个/mL、附睾尾1.42╳10~8个/mL;精子畸形率分别为:睾丸14.18%、附睾头15.13%、附睾体15.7%、附睾尾15.20%;顶体完整率分别为:睾丸70.15%、附睾头70.09%、附睾体71.53%、附睾尾70.76%。结果表明,欧拉型藏羊睾丸、附睾中精子密度、顶体完整率较低,可能与藏羊所处环境中日粮、环境温度等有关,但其精子畸形率符合受精标准。  相似文献   

6.
本实验旨在研究二甲双胍对犬冷冻-解冻后精液品质的改善效果,用含有不同浓度(0、25、50、100μmol/L)二甲双胍的Tris-卵黄稀释液冷冻保存犬精液,解冻后测定精子活力、质膜完整率、顶体完整率、活性氧(ROS)含量、丙二醛(MDA)含量、线粒体膜电位状态及腺苷三磷酸(ATP)含量等指标.结果显示:二甲双胍添加组的...  相似文献   

7.
在BF5稀释液中分别添加不同水平的牛血清白蛋白(BSA)(0.5,1,1.5 g/L)、二甲基乙酰胺(DMA)(0.5%,1%,1.5%)、甲基-β-环糊精载胆固醇(CLC)(1,2.5,5 g/L),对成年健康猪精液进行冷冻保存,于不同温度(37,50,70℃)解冻后分别检测冷冻后精子的活率、活力、质膜完整性、项体完整率、线粒体活性.结果显示,0.5 g/L BSA组冷冻后精子活力、质膜完整率、顶体完整率和线粒体活性均高于其他组,但差异不显著(P0.05).1% DMA组冷冻后精子活率和活力显著优于1.5%DMA组(P＜0.05),同时也高于对照组(3%甘油)冻后精子活率和活力,但差异不显著.不同质量浓度CLC组冷冻后精子的活率、活力、质膜完整性和线粒体活性与对照组相比差异不显著.50℃和70℃解冻组精子的活率和线粒体活性显著高于37℃解冻组的精子;50℃解冻组精子的活力和质膜完整率显著高于其他2组.因此,在猪精液冷冻稀释液中,用DMA可以代替甘油作为渗透性保护剂,且以1%DMA,50℃解冻最佳.  相似文献   

8.
在版纳微型猪近交系（BMI）公猪精液冷冻保存稀释液中添加不同浓度甘油,并对不同解冻液配方和解冻方法进行研究,对比解冻后的精子活力、质膜完整率和顶体完整率,优化BMI公猪精液冷冻保存方法。结果表明,解冻后,甘油浓度为2%和3%组的精子活力、质膜完整率、顶体完整率显著高于1%、4%和5%组（P<0.05）;Ⅰ号解冻液解冻后精子活力、质膜完整率、顶体完整率均显著高于Ⅱ、Ⅲ和Ⅳ号解冻液（P<0.05）;在活力、质膜完整率和顶体完整率方面,40℃ 6 s和50℃ 6 s这两种程序的解冻效果都显著优于38℃ 30 s（P<0.05）。由此可见,2%或3%的甘油作为抗冻保护剂、Ⅰ号解冻液解冻、40℃ 6 s或50℃ 6 s水浴解冻是较为理想的BMI公猪精液冷冻保存方法。  相似文献   

9.
使用荧光染色和流式细胞术检测山羊精子   总被引：1，自引：0，他引：1  
分别使用PI(碘化丙碇),FITC-PSA(异硫氰酸荧光素络合豌豆凝集素)和RH123(罗丹明)对山羊冷冻精子进行染色,再使用流式细胞仪进行检测、分析。试验发现,对于山羊的冷冻精子,PI染色阴性的为质膜完整的精子;FITC-PSA染色阴性的可能为顶体完整的精子,染色FITC-PSA+和FITC-PSA++,可能分别为顶体反应中的精子和顶体破损精子;RH123+应为线粒体无活性精子,有绿色荧光可能是因为染料残留在细胞膜上导致,而RH123++可能应为线粒体有活性的活精子。不同个体羊的精子对于低温敏感度不一样。不同个体羊之间的冻精顶体完整率、质膜完整率、线粒体有活性的精子比率之间差异显著,据此应选择合适的山羊个体进行精液冷冻。  相似文献   

10.
将新采集且活力达标的湖羊精液添加到含不同质量浓度(0.00,0.25,0.50,1.00,2.00 mg/L,Ⅰ～Ⅴ组)纳米颗粒硒(nanoparticulate selenium, SeNPs)的冷冻基础稀释液中进行稀释,然后将稀释的精液分装入0.25 mL冻精细管,并在液氮中储存。7 d后解冻,对每个处理的精液进行精子质量参数评估,包括精子活力、运动参数、畸形率、顶体完整率、质膜完整性,并测定各组精液的总抗氧化能力(T-AOC)、过氧化氢酶活性(CAT)、活性氧(ROS)和丙二醛(MDA)含量以及相关抗氧化基因(SOD1、GCLC、GCLM和PRDX1)的表达。结果显示,与Ⅰ组相比,Ⅲ,Ⅳ组对解冻后精子的运动能力、顶体完整率和膜完整性均有积极影响(P<0.05),尤其是Ⅳ组精子冻后活力达48.68%,顶体完整率为80.51%,头部质膜完整率为52.54%,尾部质膜完整率为50.99%,均处于最高水平;SeNPs处理后的冷冻精液T-AOC和CAT活性显著增加(P<0.05),其中Ⅳ组T-AOC最高为(10.94±0.25) U/mL、CAT活性为(11.01±0.92)U...  相似文献   

11.
Background: During sperm maturation, there is a reorganization of fatty acids from plasmatic membrane of the spermatozoa, which allows higher membrane integrity and acquisition of sperm motility. However, the fatty acid profile during sperm maturation remains unclear in dogs. Thus, the aim of this study was to identify the fatty acids from the epididymal spermatozoa and plasma during the sperm maturation, and observed changes in the motility and plasmatic membrane parameters. Twenty one adult dogs were used, subsequently to bilateral orchiectomy and epididymal storage, sperm samples were collected from the different segments of the epididymis. Samples were evaluated for conventional microscopy, computer-assisted motility analysis, sperm plasma membrane permeability and the fatty acid analysis(lipids were extracted, transmethylated and analyzed by chromatography).Results: Caput and corpus sperm showed lower values for the motility variables evaluated and plasmatic membrane integrity, indicating different levels of the fatty acids organization. Saturated, monounsaturated and polyunsaturated fatty acids were in higher concentrations in the spermatozoa from epididymis cauda. Highlighting the presence of caprylic, stearic and docosahexaenoic acids.Conclusions: These findings demonstrate the influence of the fatty acid profile during sperm maturation, assigning physical and chemical changes in sperm cells, essential for fertilization.  相似文献   

12.
The effectiveness of the water test, short hypoosmotic swelling test with ultrapure water was examined in canine epididymal spermatozoa to evaluate tail membrane integrity. Spermatozoa during epididymal transit were also characterized. Sperm suspension obtained from cauda epididymis was diluted 1:4 with ultrapure water, and incubated for 5 min. The percentage of swollen spermatozoa in the water test was significantly correlated with both the sperm motility and the swelling value obtained by the conventional hypoosmotic swelling test. Canine spermatozoa collected from the caput epididymis were not motile, but revealed membrane integrity in a water test. The water test can be used as a simple and short hypoosmotic swelling test to evaluate the tail membrane integrity of canine epididymal spermatozoa.  相似文献   

13.

Background

During epididymal transit, functional and structural modifications leading to full maturation enable male gametes to reach, recognize and fertilize the oocytes. In dogs, little is known on the modifications of spermatozoa during the passage in the epididymis. The aim of this study was to describe the motility, morphology and acrosomal patterns of canine spermatozoa retrieved from the epididymis caput, corpus and cauda.

Results

After the dilution required for the collection of epididymal content, sperm motility was significantly higher (P <0.0001) in the cauda compared to corpus and caput.Proportions of spermatozoa with normal morphology were significantly higher in corpus (P =0.02) and cauda (P <0.0001) compared to caput. Overall morphological abnormalities of the head and neck/midpiece were similar in the three different epididymal regions. A significantly increased prevalence of tail defects, mainly represented by single bent tails, was observed in the corpus compared to caput (P <0.0001) and cauda (P =0.006).Numbers of immature sperm with cytoplasmic droplets decreased from the proximal to the distal region of the epididymis. Particularly, proximal cytoplasmic droplets were more frequently found in spermatozoa collected from the caput epididymis than in the corpus (P <0.0001) and in the cauda (P <0.0001), whereas the occurrence of distal cytoplasmic droplets was higher in the corpus than in the caput (P =0.0003) and in the cauda (P <0.05).Significantly higher proportions of spermatozoa with intact acrosomes were retrieved from the cauda epididymis than from the caput (P =0.03) and the corpus (P =0.008). This difference was mainly due to a lower proportion of spermatozoa with abnormal acrosomes (mainly swollen acrosomes) rather than with absent acrosomes.

Conclusions

Canine spermatozoa undergo several modifications in the epididymis. The acquisition of progressive motility, migration of the cytoplasmic droplet and acrosomal reshaping lead to mature spermatozoa which are then stored in the cauda epididymis. From this site, spermatozoa can be retrieved and used in assisted reproductive techniques as a valuable tool for propagating genetic traits of high value individuals that dies accidentally or undergoes orchiectomy for medical purposes. Further investigations should be also focused on the potential use of spermatozoa recovered from other epididymal regions.  相似文献   

14.
In order to improve boar sperm quality during frozen-thawed process, the influence of the presence of trehalose on success of cryopreservation of boar sperm were investigated. We evaluated freeze-thawing tolerance of boar spermatozoa in a base cooling extender with the addition of different trehalose concentrations (0, 25, 50, 100 and 200 m m ), and try to determine the optimum concentration of trehalose. We chose sperm motility, mitochondrial activity, acrosome integrity and membrane integrity as parameters to evaluate cryopreservation capacity of boar spermatozoa. We obtained the best results for 100 m m trehalose-supplemented extenders, with values of 49.89% for motility, 44.69% for mitochondrial activity, 66.52% for acrosome integrity and 44.61% for membrane integrity, while freeze-thawing tolerance diminished significantly for 200 . The synergic effect of trehalose and glycerol resulted in better cryosurvival of boar spermatozoa than that of a single cryoprotectant. In conclusion, when trehalose-supplementation was added up to 100 m m , trehalose confers a greater cryoprotective capacity to the extender, and the sperm motility, mitochondrial activity, membrane integrity and acrosome integrity parameters were significantly improved during frozen-thawed process.  相似文献   

15.
The epididymis is the site of post-testicular sperm maturation, which constitutes the acquisition of sperm motility and the ability to recognize and fertilize oocytes. The role of miRNA in male reproductive system, including the control of different steps leading to proper fertilization such as gametogenesis, sperm maturation and maintenance of male fertility where the deletion of Dicer in mouse germ cells led to infertility, has been demonstrated. The identification of miRNA expression in a region-specific manner will therefore provide valuable insight into the functional differences between the regions of the epididymis. In this study, we employed RNA-seq technology to explore the expression pattern of miRNAs and establish some miRNAs of significant interest with regard to epididymal sperm maturation in the CY epididymis. We identified a total of 431 DE known miRNAs; 119, 185 and 127 DE miRNAs were detected for caput versus corpus, corpus versus cauda and caput versus cauda region pairs, respectively. Our results demonstrate region-specific miRNA expression in the CY epididymis. The GO and KEGG enrichment for the predicted target genes indicated the functional values of miRNAs. Furthermore, we observed that the expression of miR-200a was downregulated in the caput, compared with cauda. Since the family of miR-200 has previously been suggested to contribute to the distinct physiological function of sperm maturation in epididymis of adult rat, we speculate that the downregulation of miR-200a in CY caput epididymis may play an important role of sperm maturation in the epididymis of CY. Therefore, our findings may not only increase our understanding of the molecular mechanisms regulated by the miRNA functions in region-specific miRNA expression in the CY epididymis, it could provide a valuable information to understand the mechanism of male infertility of CY.  相似文献   

16.
All epididymal regions are lined with multiple epithelial cell types, each with different functions to provide the luminal environment for spermatozoal maturation. Epithelial cells also create apical blebs, which are released from the apical surface via apocrine secretion and disintegrate in the lumen, thereby releasing epididymosomes. Epididymosomes transport proteins to spermatozoa and contain microRNAs. We hypothesized that epididymosomes also transfer miRNA from epididymal epithelium to spermatozoa. Quantitative real-time polymerase chain reaction was used to determine miRNA profiles of epididymal tissue from caput and cauda, epididymal spermatozoa from caput and cauda, and epididymosomes and from caput, proximal corpus, distal corpus, and cauda. Pathway analysis was performed using DIANA tools on the miRNA unique to caudal spermatozoa. We found 66 newly acquired miRNAs in spermatozoa located in the caudal epididymis. Predicted pathways targeted by these miRNAs suggest a role in cell motility and viability and factors in oocyte and embryo maturation and development. These findings suggest that miRNAs are transported to spermatozoa from epididymal epithelium via epididymosomes.  相似文献   

17.
为了阐明树鼩精子中是否存在丝氨酸/苏氨酸蛋白磷酸酶1γ2（PP1γ2）及其在附睾精子中的存在形式,进而探究PP1γ2对精子成熟和运动性的调控作用,本试验以树鼩为研究对象,采用Western blotting分析了不同条件下树鼩附睾头和附睾尾精子中PP1γ2的存在形式和磷酸化程度,探讨了双丁酰环腺苷酸（db-cAMP）、3-异丁基-1-甲基黄嘌呤（IBMX）或Ca2+对树鼩精子中PP1γ2磷酸化表达水平的影响,并进一步研究了磷酸酶抑制剂冈田酸（okadaic acid,OA）和花萼海绵诱癌素A （calyculin A,CA）对树鼩精子中PP1γ2磷酸化程度的影响及其对树鼩附睾头和附睾尾精子运动度的影响。结果显示,在树鼩附睾头和附睾尾精子中均存在PP1γ2,且在等量的附睾头和附睾尾精子蛋白中,PP1γ2在附睾尾精子的磷酸化程度远高于附睾头精子;db-cAMP、IBMX或Ca2+不改变PP1γ2的磷酸化水平;磷酸酶抑制剂OA和CA能明显提高附睾头和附睾尾中PP1γ2磷酸化的程度,且能显著提高精子（尤其是附睾头精子）的运动度（P<0.05）,OA和CA的最佳作用浓度分别为1 μmol/L和10 nmol/L,最佳作用时间分别为15、20 min。本研究结果表明,蛋白磷酸酶PP1γ2对树鼩精子成熟及运动性具有重要的调控作用,其主要通过磷酸化和去磷酸化的变化发挥作用。  相似文献   

18.
旨在对绵羊附睾头、体和尾部的精子进行蛋白质组学分析,获得差异表达蛋白,对数据进行功能富集分析,挖掘精子发生/成熟关键蛋白质。本研究选择12月龄左右健康的3只雄性湖羊为试验动物,分离附睾并按区域收集精子,3组样本(附睾头部组、附睾体部组和附睾尾部组),每组3个生物学重复,共计9例绵羊精子细胞样本。基于TMT标定定量蛋白质组学分析和R语言等工具,在获取的差异表达蛋白中进行GO和KEGG富集分析,并利用蛋白质免疫印迹(Western bolt)、免疫荧光(immunofluorescence)和流式细胞术(flow cytometry)试验验证结果的可靠性。从22 841个唯一性肽中鉴定到差异蛋白质616种,其中,尾vs头组鉴定出309个差异表达蛋白(上调213个,下调96个);尾vs.体组鉴定出167个差异表达蛋白(上调107个,下调60个);体vs头组鉴定出140个差异表达蛋白(上调88个,下调52个)。根据差异倍数与蛋白质功能,筛选出可能与精子成熟、核质物质转运相关的关键蛋白-KPNA4。本研究揭示了绵羊附睾不同部位精子的特点与差异,这些数据为研究雄性绵羊的生殖机制和精子成熟提供了丰富的资源。  相似文献   

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Our aim was to evaluate the effect of Sephadex filtration on respiratory activity of porcine spermatozoa and its relation with quality and functional sperm parameters. Samples were evaluated regarding oxygen uptake and sperm parameters: motility, plasma and acrosome membrane integrity, capacitation and acrosome reaction induction in vitro, plasma membrane functionality, determined by the hypo‐osmotic swelling test (HOST), and lipid peroxidation assessed by thiobarbituric acid assay. Sephadex filtration improved all routine quality parameters (motility, plasma and acrosome membrane integrity) and functional parameters (HOST, in vitro capacitation and true acrosome reaction levels) and produced a significant decrease in cryocapacitation and lipid peroxidation. Oxygen uptake increased in Sephadex samples (41 ± 7%) respect to single washing. Oxygen addition of carbonyl‐cyanide‐m‐chlorophenylhydrazone (CCCP) confirmed mitochondrial coupling in washed and Sephadex samples; showing an increase of 2.6 and 4.2 times for oxygen consumption in single washing and Sephadex ones, respectively. The increase in oxygen uptake with succinate addition with respect to basal oxygen uptake was significantly lower in Sephadex samples (63 ± 25%) than in the washed ones (183 ± 35%). Sephadex samples showed higher mitochondrial activity measured by oxygen consumption and improved quality and functional parameters. Our study recommends this protocol due to the fact that this filtration method removes dead or damaged spermatozoa allowing to obtain cryopreserved boar spermatozoa with optimized fertilizing capacity.  相似文献   

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