Seminal plasma improves cryopreservation of Iberian red deer epididymal sperm

We tested the protective action of seminal plasma on epididymal spermatozoa from Iberian red deer, especially considering cryopreservation, as a means for germplasm banking improvement. We obtained seminal plasma by centrifuging electroejaculated semen, and part of it was thermically inactivated (denatured plasma; 55 degrees C 30 min). Epididymal samples (always at 5 degrees C) were obtained from genitalia harvested after regulated hunting, and pooled for each assay (five in total). We tested three seminal plasma treatments (mixing seminal plasma with samples 2:1): no plasma, untreated plasma and denatured plasma; and four incubation treatments: 32 degrees C 15 min, 5 degrees C 15 min, 5 degrees C 2h and 5 degrees C 6h. After each incubation, samples were diluted 1:1 with extender: Tes-Tris-Fructose, 10% egg yolk, 4% glycerol; equilibrated for 2h at 5 degrees C, extended down to 10(8) spz./mL and frozen. Sperm quality was evaluated before 1:1 dilution, before freezing and after thawing the samples, assessing motility (CASA) and viability (percentage of viable and acrosome-intact spermatozoa; PI/PNA-FITC and fluorescent microscopy). Plasma treatment, both untreated and denatured, rendered higher viability before freezing and higher results for most parameters after thawing. The improvement was irrespective of incubation treatment, except for viability, which rendered slightly different results for untreated and denatured plasma. This may be due to the presence of thermolabile components. We still have to determine the underlying mechanisms involved in this protection. These results might help to improve the design of cryopreservation extenders for red deer epididymal sperm.     

Post mortem time and season alter subpopulation characteristics of Iberian red deer epididymal sperm

We have studied the effect of post mortem time and season on sperm subpopulation pattern and characteristics. We used epididymal samples from free-ranging Iberian red deers harvested during the hunting season. We studied samples at different moments of the year (rut, transition period and post-rut), and at different times post mortem (up to 4 days). Sperm were extracted from the cauda epididymis and their motility was evaluated by means of a CASA system. A principal component and clustering analysis were carried out to identify subpopulations. Post mortem time caused a significant decrease in motility quality, and a general deterioration in subpopulation characteristics. We found three subpopulations the first day, and the one indicating good sperm quality decreased with post mortem time until it disappeared on the fourth day. This may indicate considerable impairment of the samples after 72 h post mortem, which could compromise their use in AI programs. With regard to season, subpopulation pattern and characteristics were better in the transition and post-rut periods. Moreover, we found one subpopulation formed by mature spermatozoa, which increased from rut to post-rut. This might be a negative fact, because samples collected after the rut may undergo hypermaturation, which possibly impairs fertility. Our results are of interest for the management of wildlife germplasm banks based on post mortem sperm recovery.     

Objective assessment of goat sperm head size by computer-assisted sperm morphometry analysis (ASMA)

The aim of the present study was to identify different objective categories of sperm head size evaluation, using both morphometric and statistical analyses. For this purpose, semen samples (n = 16) were collected from 4 Florida male goats and assessed using a computer-assisted sperm morphometric analysis (ASMA) to obtain sperm head size measurements. The sperm heads were grouped into categories according to 25th and 75th percentiles (the lower and upper quartile, respectively) of their area values. Thereafter, a discriminant analysis was implemented on all the sperm morphometric parameters assessed, to obtain a classification matrix for sperm head size. Sperm heads by this method were classified into 3 categories depending on their size (small, medium and large), with a globally correct assignment of 95.5%. Moreover, significant differences (p < 0.001) were recorded between individual animals for all the sperm head morphometric parameters assessed. In conclusion, by using both statistical and morphometric analyses, it was possible to recognize the three categories of sperm head size. It is expected that research could be useful in defining the relationship between sperm head size measurements and actual fertility data.     

Comparison of two methods for obtaining spermatozoa from the cauda epididymis of Iberian red deer

We have compared two methods for salvaging epididymal sperm from post-mortem samples from Iberian red deer. Of each pair of testicles (29 samples), one cauda epididymis was processed by means of cuts (sperm was immediately diluted with extender) and the other was detached from the corpus and flushed from the vas deferens with 1 mL of extender. Sperm was processed for cryopreservation, and analyzed just after recovery, pre-freezing and post-thawing. Total spermatozoa recovered, contamination (concentration of epididymal cells and red blood cells (RBCs)) and quality (motility by CASA, and acrosomal status, viability and mitochondrial status by flow cytometry) were used to compare both methods. The number of recovered spermatozoa was similar for both methods. Contamination was higher for the cuts method, but when considering the final dilution before freezing, only RBCs concentration was significantly higher. Motility was similar just after extraction, but higher for both pre-frozen and post-thawed flushed sperm. Pre-freezing acrosomal status (P < 0.05) and viability (P < 0.1) were better for flushing; however post-thawing results were similar for the two methods. A clustering analysis using CASA data showed that the subpopulation pattern of motile sperm was different depending on the method, being better for flushing. With regard to yield, lower contamination (especially RBCs) and, in general, better quality results, flushing seems to be a more recommendable method for post-mortem sperm recovery. The cuts method may be more practical on certain occasions, but care must be taken in order to achieve rapid extension of the sample and to avoid contamination in order to improve sample condition.     

Comparison among different cryoprotectants for cryopreservation of epididymal sperm from agouti (Dasyprocta leporina)

We verify the effects of different cryoprotectants on the cryopreservation of agouti (Dasyprocta leporina) epididymal sperm. We used 16 pairs of testes–epididymis complexes of sexually mature animals. We immediately evaluated epididymal sperm obtained by retrograde flushing for concentration, motility, vigor, viability, osmotic response, and morphology. Samples were extended in a coconut water extender plus 20% egg yolk, containing glycerol, ethylene glycol, dimethylsulfoxide – DMSO, or dimethylformamide. Finally, samples were stored in 0.25 mL straws, frozen in liquid nitrogen, and thawed after one week, being reevaluated and assessed for membrane integrity using fluorescent probes. The higher values for postthawing sperm motility, vigor, and membrane integrity were achieved by the usage of glycerol, when compared to ethylene glycol and dimethylformamide (P < 0.05); however, no differences were found between glycerol and DMSO (P > 0.05). All cryoprotectants provided a similar effect on the preservation of sperm morphology, osmotic response, and viability (P > 0.05). Therefore, here onwards, there was testing of glycerol and DMSO at 3 and 6% concentrations using the same freezing–thawing protocol reported previously. As the main result, DMSO at 6% concentration provided a decrease in sperm parameters, as well as in the chromatin integrity and in the binding capability of sperm. In conclusion, glycerol 3 or 6% and DMSO 3% can be used as alternative cryoprotectants for agouti epididymal sperm cryopreservation.     

中华稻蝗血细胞染色方法的比较

通过Giemsa、Wright和Giemsa-Wright混合染色3种方法,对中华稻蝗Oxya chinensis(Thunberg)的血细胞进行了染色和血细胞的形态学观察,其结果是Giemsa染液染色时间较长,对细胞核染色效果较好,细胞质界限清晰,但对细胞质中的颗粒染色较差;Wright染液染色时间较短,对细胞核和细...     

Comparison between three different LCIA methods for aquatic ecotoxicity and a product environmental risk assessment

Background and Objective  In the OMNIITOX project 11 partners have the common objective to improve environmental management tools for the assessment of (eco)toxicological impacts. The detergent case study aims at: i) comparing three Procter &c Gamble laundry detergent forms (Regular Powder-RP, Compact Powder-CP and Compact Liquid-CL) regarding their potential impacts on aquatic ecotoxicity, ii) providing insights into the differences between various Life Cycle Impact Assessment (LCIA) methods with respect to data needs and results and iii) comparing the results from Life Cycle Assessment (LCA) with results from an Environmental Risk Assessment (ERA). Material and Methods  The LCIA has been conducted with EDIP97 (chronic aquatic ecotoxicity) [1], USES-LCA (freshwater and marine water aquatic ecotoxicity, sometimes referred to as CML2001) [2, 3] and IMPACT 2002 (covering freshwater aquatic ecotoxicity) [4]. The comparative product ERA is based on the EU Ecolabel approach for detergents [5] and EUSES [6], which is based on the Technical Guidance Document (TGD) of the EU on Environmental Risk Assessment (ERA) of chemicals [7]. Apart from the Eco-label approach, all calculations are based on the same set of physico-chemical and toxicological effect data to enable a better comparison of the methodological differences. For the same reason, the system boundaries were kept the same in all cases, focusing on emissions into water at the disposal stage. Results and Discussion  Significant differences between the LCIA methods with respect to data needs and results were identified. Most LCIA methods for freshwater ecotoxicity and the ERA see the compact and regular powders as similar, followed by compact liquid. IMPACT 2002 (for freshwater) suggests the liquid is equally as good as the compact powder, while the regular powder comes out worse by a factor of 2. USES-LCA for marine water shows a very different picture seeing the compact liquid as the clear winner over the powders, with the regular powder the least favourable option. Even the LCIA methods which result in die same product ranking, e.g. EDIP97 chronic aquatic ecotoxicity and USES-LCA freshwater ecotoxicity, significantly differ in terms of most contributing substances. Whereas, according to IMPACT 2002 and USES-LCA marine water, results are entirely dominated by inorganic substances, the other LCIA methods and the ERA assign a key role to surfactants. Deviating results are mainly due to differences in the fate and exposure modelling and, to a lesser extent, to differences in the toxicological effect calculations. Only IMPACT 2002 calculates the effects based on a mean value approach, whereas all other LCIA methods and the ERA tend to prefer a PNEC-based approach. In a comparative context like LCA the OMNIITOX project has taken the decision for a combined mean and PNEC-based approach, as it better represents the ‘average’ toxicity while still taking into account more sensitive species. However, the main reason for deviating results remains in the calculation of the residence time of emissions in the water compartments. Conclusion and Outlook  The situation that different LCIA methods result in different answers to the question concerning which detergent type is to be preferred regarding the impact category aquatic ecotoxicity is not satisfactory, unless explicit reasons for the differences are identifiable. This can hamper practical decision support, as LCA practitioners usually will not be in a position to choose the ’right’ LCIA method for their specific case. This puts a challenge to the entire OMNIITOX project to develop a method, which finds common ground regarding fate, exposure and effect modelling to overcome the current situa-tion of diverging results and to reflect most realistic conditions.     

Comparison of the Papanicolaou and Feulgen staining methods for DNA quantification by image analysis.

The possibility of using archival cytology material to study the evolution of neoplastic disease with regard to DNA content abnormalities was investigated. The accuracy of measuring the integrity optical density (OD) of nuclei that correlates to DNA amounts of those nuclei, on slides stained by the Papanicolaou method, was assessed and compared with a standard Feulgen method. Our data on rat liver nuclei peritoneal washings from patients with ovarian cystadenofibromas and ovarian cystadenocarcinomas suggested that analysis of cytological material using the Papanicolaou method is not reliable and that destaining the slides followed by Feulgen staining provides an optimal and reliable method of DNA quantification.     

Taking advantage of the use of supervised learning methods for characterization of sperm population structure related with freezability in the Iberian red deer

Using Iberian red deer as a model, this study presents a supervised learning method, the Support Vector Machines (SVM), to characterize sperm population structure related with freezability. Male freezability was assessed by evaluating motility, membrane status and mitochondrial membrane potential of sperm after a freezing-thawing procedure. The SVM model was generated using sperm motility information captured by computer-assisted sperm analysis (CASA) from thawed semen, belonging to six stags with marked differences on their freezability. A total of 1369 sperm tracks were recorded for seven kinematic parameters and assigned to four motility patterns based on them: weak motile, progressive, transitional and hyperactivated-like. Then, these data were split in two sets: the training set, used to train the SVM model, and the testing set, used to examine how the SVM method and three other unsupervised methods, a non-hierarchical, a hierarchical and a multistep clustering procedures, performed the sperm classification into subpopulations. The SVM was revealed as the most accurate method in the characterization of sperm subpopulations, showing all the sperm subpopulations obtained in this way high significant correlations with those sperm parameters used to characterize freezability of males. Given its superiority, the SVM method was used to characterize the sperm motile subpopulations in Iberian red deer. Sperm motile data from frozen-thawed semen belonging to 25 stags were recorded and loaded into the SVM model. The sperm population structure revealed that those males showing poor freezability were characterized by high percentages of sperm with a weak motility pattern. In opposite, males showing good freezability were characterized by higher percentages of sperm with a progressive and hyperactivated-like motility pattern and lower percentages of sperm with a weak motile pattern. We also identified a sperm subpopulation with a transitional motility pattern. This subpopulation increased as the freezability of males improved, and may be used as indicative of overall sperm motility.     

Comparison of different methods for decision-making in bioequivalence assessment

If the regulatory requirements are symmetrical, the use of symmetrical confidence intervals as a decision rule for bioequivalence assessment leads, as shown by simulations, to better level properties and an inferior power compared to a rule based on shortest confidence intervals. A choice between these two approaches will have to depend on a loss function. For asymmetric regulatory requirements, symmetrical confidence intervals should not be used; however, a decision can still be based on posterior probabilities, pr (theta epsilon [r1, r2]/x), or shortest confidence intervals. For purposes of inference, presentation and interpretation of results, we think that the use of symmetrical confidence intervals alone can be misleading and we therefore recommend that the posterior probabilities and densities, or at least the shortest confidence intervals, be given.     

New staining methods for sperm evaluation estimated by microscopy and flow cytometry

New staining methods and automated instruments are now available to evaluate the sperm cell in vitro. Individual compartments of the sperm cell, such as the nucleus and the plasma and acrosomal membranes, may be investigated, as well as the cell function as shown by mitochondria activity and capacitation. Various probes are used and they can be analyzed by direct light or fluorescent microscopy or by flow cytometry. The automated instruments allow objective and accurate analysis and quantification as well as the ability to evaluate large population of cells in a shorter time, thus providing accurate evaluation of sperm quality. However, before these test can be recommended for routine clinical and investigational use, in the stallion, they need to be confirmed on a larger number of stallions and their correlation with traditional semen parameters and with stallion fertility has to be demonstrated.     

Assessment of chromatin status (SCSA) in epididymal and ejaculated sperm in Iberian red deer, ram and domestic dog

Abnormal chromatin condensation is not detected using classical techniques for sperm analysis. SCSA has demonstrated its usefulness in sperm chromatin analysis in several species (human, bull, stallion and boar). In this work, we studied sperm samples from red deer, ram and dog to analyze the differentiation of chromatin structure applying SCSA in epididymal and ejaculated spermatozoa. Epididymal samples were obtained from the caput, corpus and cauda by means of cuts, and ejaculated ones were obtained by electroejaculation (deer), artificial vagina (ram) and digital manipulation (dog). SCSA results suggested different critical points in sperm maturation (spermatozoa with loose chromatin to more condensed chromatin) among species: from corpus to cauda in ram and from caput to corpus in deer and dog. Moreover, we also detected differences in ruminants and dog, reflected in the appearance of SCSA plots. Indeed, ram and deer samples rendered two peaks within the sperm main population (sperm with condensed chromatin), whereas only one was detected in dog. Although some differences were observed between cauda and ejaculated samples, SCSA parameters indicated good chromatin condensation, making these samples suitable for germplasm banking. Some species-dependent modifications in the analysis of the results may be necessary to take full advantage of its analytical power.     

Effects of exposure to static magnetic fields on the morphology and morphometry of mouse epididymal sperm

Morphologic and morphometric sperm characteristics of mouse epididymal extracts from animals exposed to static magnetic fields were evaluated. For this purpose, animals were exposed for 35 days to a field of 0.7 T generated by a commercial permanent magnet for either 1 or 24 h per day. The values of morphometric parameters were obtained using the morphometric module of the Sperm Class Analyzer® computerized image analysis system, and percentages of abnormalities were calculated. The size of sperm heads was unaffected by exposure to static magnetic fields. Lack of hook was a sperm head abnormality found significantly more frequently in animals exposed continually than in nonexposed animals, showing a possible alteration to the spermatogenic process after exposure to static magnetic fields. The percentage of sperm with coiled tails or of sperm with abnormal midpiece or tail was not altered by exposure. Bioelectromagnetics 19:377–383, 1998. © 1998 Wiley-Liss, Inc.     

Utilisation of different ages of heather on three Scottish moors by red grouse, mountain hares, sheep and red deer

Utilisation of heather at three Scottish moors, by red grouse, mountain hares, sheep and red deer, was measured by collecting their faecal droppings regularly from plots on heather patches of different ages. Rates of heather growth differed between moors, and preferences of the four species, all grazers of heather, were related more closely to its height than to its age. Thus hares spent most time on ground with heather less than 15 cm high, sheep less than 20 cm, grouse preferred heather 10–30 cm high and deer spent most time on heather over 25 cm high.
Differences between outer, middle and inner plots on patches at one of the moors in dicated that grouse are reluctant to move far from cover into areas of newly burned ground, and reluctant to move far into patches of tall, dense heather, when these are next to shorter material. Hares preferred the middle of patches on newly burned ground and very young heather.
At two of the moors, but not the third, there was evidence that heavy grazing by hares in winter reduced the amount of young heather available to hares and grouse in spring. This potential reduction in the quality of the spring diet of grouse is discussed In relation to previously reported associations between numbers of hares and grouse at those two moors.     

检测聚丙烯酰胺凝胶中蛋白质的3种染色方法比较

用考马斯亮蓝染色、银染色、铜染色等 3种方法对同一种蛋白质染色的灵敏度、快速性进行比较 ,得出 3种蛋白质染色方法的优缺点 ,为蛋白质电泳染色合理选用不同方法提供依据     

Comparison of three different methods for measurement of tissue platinum level

We attempted to make a comparison of three methods for tissue platinum; atomic absorption spectrometry (AAS), inductively coupled plasma atomic emission spectrometry (ICP-AES), and inductively coupled plasma mass spectrometry (ICP-MS). The determination lim its were 0.05 ng/mL on ICP-MS, 50 ng/mL on ICP-AES, and 200 ngJmL on AAS, and the recovery rates were 97.7 ± 6.9% on ICP-MS, 69.0 ± 3.0% on ICP-AES, and 102.4 ± 4.0% on AAS, respectively. Plat inum was detected by ICP-AES and ICP-MS in human vertebrae, but the level was higher by ICP-AES than by ICP-MS. In the mouse kid ney treated with cisplatin, platinum was detected by ICP-MS, but not by ICP-AES. As cadmium gives the absorption peak close to plat inum, cadmium was measured together with platinum by ICP-AES in the vertebrae. From these, ICP-MS is the most sensitive for measure ment at tissue platinum. The sensitivity of ICP-AES looks worse for measuring the tissue platinum, and it is necessary to take care of the contaminant of metals, especially cadmium. AAS is not suitable for measurement of tissue platinum as in the vertebrae and kidneys, because platinum was not detectable by AAS.     

Comparison of specific radioactivities of human alpha-lactalbumin iodinated by three different methods

Radioiodination provides an extremely sensitive method for the detection of low levels of proteins. In the development of a sensitive radioimmunoassay for human alpha-lactalbumin (alpha-LA), the protein was labeled to high specific activity (approaching 2000 Ci/mmol) with lactoperoxidase, chloramine-T, and Iodogen. Despite high specific activities of the labeled protein by each method, there was a considerable difference in their binding affinity with monoclonal anti-human alpha-LA antibodies due to varying degrees of protein damage. Iodination of human alpha-LA with Iodogen resulted in labels of the highest specific activity and immunoreactivity with the monoclonal antibodies used.     

Comparison of different staining procedures for the flow cytometric analysis of U-937 cells infected with different Leishmania-species.

The human macrophage cell line U-937 infected with different Leishmania species, Leishmania mexicana amazonensis (Lma), Leishmania donovani (Ld) and Leishmania infantum (Li), was analyzed by flow cytometry (FCM). Leishmania spp. were labeled with different stains prior to the infection of the U-937 cells (BCECF-Am, PKH2-GL and SYTO 17) or after the infection (AO, FITC-conjugated monoclonal antibodies, PI). Infected cells were analyzed by flow cytometry, fluorescence microscopy and in parallel microscopically after Giemsa staining. The data obtained by these two methods were compared to decide which method is mostly appropriate for detection and estimation of the infection rate. Three fluorescent stains were suitable: BCECF-Am, SYTO 17 and FITC-conjugated MoAb with 0.02% digitonin. None of the vital stains gave evaluable results after 3 days of incubation.