熊精子顶体反应的检测

用Ionophore A23187(I—A)和咖啡因诱发熊精子的顶体反应,采用台盼兰—姬姆萨染色进行检测。根据染色结果可将精子分为四类:a)有正常顶体的活精子,b)无正常顶体的活精子,c)有正常顶体的死精子,d)无正常顶体的死精子。其中B类精子的百分率与仓鼠卵穿透率呈强正相关(r=0.9426,n=6.P<0.01)。     

诱发和检测牛精子顶体反应的一种简易方法

将冷冻保存的公牛精子解冻,冲洗,以高浓度悬浮于K-3培养液中。K-3溶液含有NaCl,CaCl_2和NaHCO_3。将此悬液预先孵育在39.5℃密闭玻璃管中,然后以台盼兰-姬姆萨(简称TG)染色,用田鼠试验以确定发生了顶体反应的精子。TG染色的涂片显示出四种类型不同着染的精子。发现仅在顶体后区未着色或淡兰色,以及顶体区未着色或部分浅红—深红着色的这两种精子的百分率与去透明带田鼠卵的穿透率之间有显著的正相关关系(r=0.732,P<0.05,n=12)。这样的精子被认为是发生了顶体反应的。公牛A与B,其精子样品预先分别在K-3培养液中孵育2小时和1～2小时,得到了很高的精子顶体反应的百分率(A 37％,B 43.8％～49.2％)和较高的卵子穿透率(A91.7％,B85.4％～97.7％)。这一结果表明公牛精子培养在只含NaCl_2、CaCl_2、NaHCO_3的培养液中,密封玻璃管中39.5℃孵育,即可发生顶体反应。TG方法可用于鉴定发生了顶体反应的精子。     

公牛冻精品质检测方法比较

<正> 1.材料与方法①精液样本:中国荷斯坦牛颗粒冻精(山西省种牛站)。②染色原理:利用活精子对特定染料不着色而死精子着色的特点来区分死精子和活精子,从而得出存活率指标。通过油镜检精查子的形态和顶体,统计出畸形率和顶体完整率指标。③染色试剂:刚果红、苯胺兰、伊红、美兰、10%葡萄糖、柠檬酸钠、姬姆萨染液(姬姆萨原液:磷酸缓冲液:蒸馏水=2:3:5)、甲醇。④制片方法:先将冻精用2.9%的柠檬酸钠解冻,并置于37℃温度下,孵育2小时染片。方法1(常规染色法):(1)存活率:采用伊红法,取精液和5%伊红溶液各一滴,置于清洁载玻片一端,迅速混合均匀,制成抹片,在400～600倍显微镜下观察。     

检测活精子顶体变化的简易染色法

为了测定活精子的顶体状况,试验将猪、羊精子用常用的苯胺黑—四溴荧光素(曙红)法(NE)和姬姆萨法(G)相结合,进行染色评定。结果,公猪精子用NE和NEG染色后,其中活精子比例间差异不显著。将经NEG染色评定的活精子比例与羧基双乙酸盐(carboxyfluorescein diacetate CFDA)/propidi-     

猪精液保存过程中精子顶体的变化

家畜精子在受精过控中,顶体起着十分重要的作用。因此,顶体形态、结构变化及功能的研究一直比较活跃。至今仍有许多问题有待揭示。一、结构与功能精子结构的研究始于上个世纪。随着技术的不断改进,方法不断更新,研究亦不断深入。但直到本世纪50年代初,对精子顶体结构的认识仍未能最后统一。Hancock(1952)采用多种染色方法,研究了多种不同处理后精子的顶体,并用电镜照像比较精子顶体的形态、结构。他发现无帽精子数与死精子数呈正比;精子头部的裸露与精子死亡密切相关。而所有的活精子顶体都与头部紧密相连。只有死精子才有松散     

使用荧光染色和流式细胞术检测山羊精子

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。     

公牛冻精品质检测方法比较

精液品质检测中，精液的存活率、精子畸形率、顶体完整率是判定精液品质优劣的常用指标。在以往检测过程中，通常采用常规染色法，即每测一个指标时进行一种染色操作，这样操作过程繁琐、耗时、耗力、耗材。本试验旨在对不同染色方法进行比较，从中寻找出一种操作简便、染色效果较佳的染色方法，这样即可省去许多步骤，也可减少多次实验操作造成误差。１材料与方法１．１精液样本：中国荷斯坦牛颗粒冻精（山西省种牛站）。１．２染色原理：利用活精子对特定染料不着色而死精子着色的特点来区分死精子和活精子，从而得出存活率指标。通过油镜…     

肝素和钙离子载体诱导猪射出精子体外获能的研究

本文采用肝素和钙离子载体诱导猪射出精子体外获能,根据对去透明带仓鼠卵的穿透情况评价获能效果,并用台盼兰·姬姆萨染色观察精子的死活及顶体状态。结果表明,未经获能处理的猪精子不能穿透仓鼠卵,肝素和I-A均能诱导猪精子体外获能并引起顶体反应。经获能处理的精子,在授精后2小时穿透卵子,4小时开始形成雄原核,6小时形成发育良好的雄原核。肝素处理以100μg效果最好,穿透率达77.8%,有原核卵百分率达47.2%;I-A处理以0.3uM为宜,穿透率达66.7%,有原核卵百分率达42.4%。咖啡因与肝素和I-A具有协同作用,能提高精子的穿透能力。有顶体反应的活精子百分率与穿透率呈强正相关(肝素组:r=0.97,P<0.01;I-A组:r=0.94,P<0.01)。     

肝素或钙离子载体诱导牛、羊冻精体外获能的研究

本文采用肝素或钙离子载体(I-A)诱导牛、羊冻精体外获能,根据对去透明带仓鼠卵的穿透情况评定获能效果,并用台盼蓝·姬姆萨(TG)染色观察了精子的死活及顶体状态.结果发现,牛、羊冻精经肝素或I-A处理后与卵子一起培养,2 h穿透仓鼠卵,4 h开始形成雄原核,6 h形成发育良好的雄原核.牛冻精用50μg/mL肝素及0.1μmol/L I-A处理效果最好,穿透率分别为72.8％和92.3％;羊则以20μg/mL肝素及0.3μmol/L I-A为宜,穿透率分别为73.1％和68.O％.咖啡因与I-A或肝素并用均有协同作用,能提高精子的穿透能力,并能促进卵内原核的发育.肝素和I-A均能在体外诱导牛、羊冻精的顶体反应,有顶体反应活精子百分率与穿透率呈强正相关,羊冻精比较脆弱,获能处理后活力较低.     

中性红、龙胆紫和尼罗蓝在精子活率测定中的应用研究

采用不同浓度梯度的中性红、龙胆紫及尼罗蓝染液对长白公猪精子染色,根据染色现象及精子活率,探究3种染料较为理想的作用时间和浓度。结果:0.01%中性红染色2 min后活精子着红色,死亡精子不着色;0.05%龙胆紫染色1 min后活精子着淡蓝色,死亡精子深紫色;0.1%尼罗蓝染色2 min后活精子着淡蓝色,死亡精子不着色。3种情况下精子的存活率与空白对照组比较无显著差异(P0.05)。试验表明,以上3种染色条件能清晰地区分存活精子和死亡精子,可用于测定精子活率。     

Effects of protein phosphatase inhibitor calyculin a on the postacrosomal protein serine/threonine phosphorylation state and acrosome reaction in boar spermatozoa incubated with a cAMP analog

In order to reveal the involvement of the sperm postacrosomal region in the acrosome reaction, we examined the effects of the protein phosphatase inhibitor calyculin A on the postacrosomal protein serine/threonine phosphorylation state and acrosome morphology in boar spermatozoa incubated with a cAMP analog. Proteins were highly phosphorylated on the serine/threonine residues only in the postacrosomal region before incubation. After 90-min incubation without calyculin A, the protein phosphorylation state declined in the postacrosomal region irrespective of the capacitation state while it remained under the detectable level in the other regions of the sperm head. However, addition of calyculin A effectively suppressed the decline in protein phosphorylation state and increased an inactive form of protein phosphatase 1 in the postacrosomal region. On the other hand, this inhibitor had no influence on the protein phosphorylation state in the acrosome and equatorial segment. After incubation without calyculin A for 180 or 360 min, many spermatozoa exhibited acrosomal changes and loss that indicated occurrence of the acrosome reaction. However, addition of calyculin A significantly blocked these events. These results are consistent with our suggestion that postacrosomal serine/threonine-phosphorylated proteins are involved in suppression of the acrosome reaction in boar spermatozoa in vitro.     

Fine surface structure of bovine acrosome-intact and reacted spermatozoa observed by atomic force microscopy

The atomic force microscope (AFM) provides nanometer resolution, topographic data of the natural surface structure of materials. We studied the topology of the surface structure of bovine sperm heads during the acrosome reaction by AFM. In addition, we numerically analyzed the areas of the median sagittal plane of the sperm heads. Bovine frozen-thawed spermatozoa were washed, capacitated by heparin, and incubated with lysophosphatidylcholine (LPC) to induce the acrosome reaction, smeared on a cover glass, air-dried, and observed with AFM using the dynamic force (tapping) mode. AFM analysis of spermatozoa showed the clear surface structure of acrosomes, equatorial segments, postacrosomal regions and necks. Although AFM images of spermatozoa capacitated by heparin had complete acrosomes, most spermatozoa treated with LPC had no acrosomal caps as shown by AFM. These observations coincided with those obtained by light microscopy after staining with naphthol yellow S and erythrosin B. Furthermore, numerical analysis of AFM images indicated that areas of the median sagittal plane of the anterior portions of acrosome-reacted sperm heads (2679 +/- 616 pixels) were approximately 40% less than those of intact heads (4535 +/- 174 pixels, P<0.05). These results indicate that AFM can usefully observe and numerically analyze the fine surface structures of bovine spermatozoa.     

Effect of Albumin and Polyvinyl Alcohol on the Vitality, Motility and Acrosomal Integrity of Canine Spermatozoa Incubated in vitro

Sperm culture media used for in vitro fertilization (IVF) procedures are important factors concerning the viability, motility and acrosomal integrity of spermatozoa. The aim of this study was to investigate the effects of three different sperm diluting media, tissue culture medium (TCM‐199), sperm culture medium (Sp‐TALP) and human tubular fluid (HTF) supplemented with varying concentrations of bovine serum albumin (1, 4 and 6%) or polyvinyl alcohol (0.8%) on the acrosomal integrity, motility and viability of canine spermatozoa. Ejaculates collected from four dogs were diluted in all media and spermatozoa were separated from seminal plasma by the swim‐up technique. Sperm progressive motility was assessed using a phase contrast microscope. Viability and acrosomal integrity were evaluated using a dual stain technique (Giemsa–Trypan blue). The results demonstrated that the number of live canine spermatozoa was similar in culture media supplemented or not supplemented with macromolecules. A minimal concentration of albumin (1%) in the three media showed similar effects on vitality, motility and acrosomal integrity, as had higher concentrations (4 and 6%). The percentage of acrosome‐intact spermatozoa was markedly higher after HTF (94.1%) than after TCM‐199 (70.1%) or Sp‐TALP (71.0%) without supplementation. It is concluded that serum bovine albumin, irrespective of the concentration, preserved sperm viability and function, and HTF is the most suitable medium for preserving the acrosome in canine spermatozoa prepared for in vitro manipulation through short incubation.     

Influence of wet fixation, staining techniques, and storage time on bull sperm morphology

The influence on sperm morphology of different methods for preparation of semen and of storage in a fixative solution was examined in 27 beef bulls subjected to a regular breeding health examination. Sperm head morphology under light microscopy did not differ between smears of fresh semen stained with carbol-fuchsin-eosin (Williams staining) or Nigrosin-Eosin. Nor was there any difference between samples stained immediately after collection and those stained after 1 month of storage at + 4 degrees C in buffered formal-saline solution. Formol-saline fixed spermatozoa examined in wet preparations under phase contrast microscopy had a higher prevalence of acrosome defects and cytoplasmic droplets than stained smears of fresh semen under light microscopy. One month of storage in formol-saline did not affect the prevalence of acrosome defects or cytoplasmic droplets. There was no influence of fixation method (wet or dry), staining, examination technique, or storage time on midpiece or sperm tail morphology. The affinity of spermatozoa to eosin at staining with Nigrosin-Eosin ("live and dead count") did not differ between fresh semen and spermatozoa that had been stored in formol-saline for 1 month. It is concluded that bull semen can be stored for at least 1 month at + 4 degrees C in buffered formal-saline without major changes in sperm morphology. Furthermore, examination of wet preparations of fixed spermatozoa under phase contrast microscope is likely to yield the most accurate results for morphological characteristics like acrosome morphology and cytoplasmic droplets.     

Induction and characterization of acrosome reaction in equine spermatozoa

Equine spermatozoa were incubated in a chemically defined medium for 8 hours. The medium preserved spermatozoal viability, as assessed by total spermatozoal motility, progressive spermatozoal motility, and spermatozoal exclusion of eosin stain. Effects of time and divalent cation ionophore, A23187, on the occurrence and character of the spermatozoal acrosome reaction were determined. Two light microscopic assays, a triple-stain technique and a chlortetracycline fluorescence assay, were calibrated with transmission electron microscopy for detection of the acrosome reaction. Incubation time and A23187 addition increased the percentage of acrosome reactions in sperm populations (P less than 0.05).     

Viability assessment of spermatozoa in large falcons (Falco spp.) using various staining protocols

Viability assessment is an important part of semen analysis, and various live/dead staining protocols have been used in semen of avian species. Results of live/dead count differed between dyes, staining protocols and bird species, impeding comparability between studies and requiring species-specific comparisons of viability stains. In raptor semen, similar comparisons are absent. Thus, the aim of the present study was to compare eight conventional viability stains. Eosin blue 2% [EB], eosin blue 2% with the addition of 3% sodium citrate [EB2], eosin blue–nigrosin 5% [EBN5], eosin yellow–nigrosin 5% [EYN5], eosin yellow–nigrosin 10% [EYN10], eosin blue–aniline blue [EBA], eosin yellow–aniline blue [EYA] and bromophenol blue–nigrosin [BBN] were evaluated in comparison with the fluorescence stain SYBR^® Green–propidium iodide [SYBR-PI] in spermatozoa of falcons. The comparison was performed using conventional light microscopy which is applicable in breeding centres, veterinary practices and field studies. Additionally, live/dead stains were correlated to motility values of the same samples to validate sperm viability. Light microscopy using EB and using SYBR-PI enabled an effective and clear differentiation between alive and dead spermatozoa of falcons. Motility values correlated significantly and strongly with EB only (r = .629; p < .001), but not with any other stain used in the study. Therefore, our results suggest EB as the most suitable stain for viability assessment in the semen of large falcons.     

Evaluation of Coomassie blue staining of the acrosome of equine and canine spermatozoa

OBJECTIVE: To evaluate Coomassie blue staining of the acrosome of equine and canine spermatozoa. SAMPLE POPULATION: Spermatozoa of 5 mixed-breed male dogs and 3 Thoroughbred stallions. PROCEDURE: Various proportions of intact and acrosome-damaged spermatozoa were fixed in 2% phosphate-buffered formaldehyde or 4% paraformaldehyde, smeared onto glass slides, and stained with Coomassie blue stain. Acrosomal status (damaged vs intact) was also assessed by use of flow cytometry after staining with fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and propidium iodide. Comparisons were made between percentages of expected and observed acrosome-intact spermatozoa in different proportions of live and flash-frozen samples; the percentages of acrosome-intact spermatozoa as determined by use of Coomassie blue staining and flow cytometry were also compared. RESULTS: Strong correlations were found between the expected and observed distributions of acrosome-intact spermatozoa when fixed in 4% paraformaldehyde (r2 = 0.93 and 0.89 for canine and equine spermatozoa, respectively) as well as between Coomassie blue-stained cells and those stained with FITC-PSA and assessed by use of flow cytometry (r2 = 0.96 and 0.97 for canine and equine spermatozoa, respectively). However, in canine samples that were fixed in 2% phosphate-buffered formaldehyde, these correlations were weak. CONCLUSIONS AND CLINICAL RELEVANCE: Staining with Coomassie blue stain was a simple and accurate method to evaluate the acrosome in equine and canine spermatozoa after fixation in 4% paraformaldehyde. This assay should be useful in routine evaluation of semen samples from these species.     

Participation of phosphofructokinase,malate dehydrogenase and isocitrate dehydrogenase in capacitation and acrosome reaction of boar spermatozoa

The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well‐known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential–interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/10¹⁰ spermatozoa, the activity of NAD‐ and NADP‐dependent IDH was 0.111 ± 0.005 U/10¹⁰ and 2.22 ± 0.14 U/10¹⁰ spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/10¹⁰ spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate‐induced capacitation and follicular fluid‐induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa.     

Effect of relaxin on acrosome reaction and utilization of glucose in boar spermatozoa

Relaxin is a peptide hormone found in seminal plasma that has a physiological influence on sperm motility in some species. There are no reports on the effect of relaxin on acrosome reaction and utilization of glucose in boar spermatozoa. In this study, to investigate the effects of relaxin on sperm motility, acrosome reaction, and incorporation and oxidation of labeled glucose, boar spermatozoa were washed and preincubated for swim-up and then incubated (0-6 h) with 0, 20, or 40 ng/ml relaxin in mTALP medium. The results indicated that the addition of relaxin stimulated sperm motility significantly (P<0.05) during 1-4 h of incubation. The percentage of acrosome-reacted live spermatozoa was higher (P<0.05) when the spermatozoa were treated with 20 or 40 ng/ml relaxin. The rate of incorporation, and oxidation of glucose were also greater (P<0.05) in the spermatozoa incubated with relaxin compared to the control spermatozoa. The rate of incorporation and oxidation of (14)C-glucose were increased in correlation with acrosome reaction up to 4 h of incubation and then decreased in line with the increasing incubation period. In conclusion, the present study demonstrates that relaxin accelerates not only motility but also the acrosome reaction and utilization of glucose in boar spermatozoa.     

Dog sperm DNA: Raw semen evaluation with Toluidine blue stain

The toluidine blue (TB) stain has been used in different species to evaluate the degree of chromatin condensation. The objectives of this study were as follows: simplify the TB stain to evaluate sperm in canine raw semen, verify the staining patterns for this species using this simplified technique and establish a protocol for using dithiothreitol (DTT) as a positive control for TB staining in dogs. Twenty‐one ejaculates were collected from 7 adult male dogs; semen was extended, fixed with ethanol 96° and stained with TB using 2 staining times: 15 and 30 min. In addition, 3 incubation times with 1% DTT were assayed (2, 5 and 30 min). Three staining patterns were established: light blue colouring (TB negative, normal chromatin condensation), light violet (TB intermediate, some degree of chromatin decondensation) and dark blue‐violet (TB positive, high degree of chromatin decondensation). No significant differences (p > 0.05) were observed between the staining times (15 and 30 min) for any of the TB patterns. All DTT incubation times (2, 5 and 30 min) showed 100% sperm positive to TB. To conclude, it was possible to simplify the TB stain and determine the different patterns in canine spermatozoa. Also, DTT can be used both as a positive control for the stain and to evaluate individual susceptibility to decondensation in vitro.