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Effect of incubation and analysis temperatures on sperm kinematics and morphometrics during human semen analysis

IntroductionHuman semen analysis must be performed after the liquefaction of the ejaculate. This takes place about 30min after ejaculation and samples must be maintained in the lab during this time. The temperatures for this incubation and the final analysis of motility are crucial but seldom taken into account. This study aims to examine the effect of these temperatures on various sperm parameters both manually (sperm count, motility, morphology, viability, chromatin condensation and maturation and DNA fragmentation) and CASA (kinematics and morphometrics, using an ISAS®v1 CASA-Mot and CASA-Morph systems, respectively) analyzed. MethodsSeminal samples from thirteen donors were incubated for 10min at 37°C followed by additional 20min at either room temperature (RT, 23°C) or 37°C and then examined following WHO 2010 criteria. ResultsThe data obtained show that there were no significant differences (P>0.05) in the subjective sperm quality parameters with incubation temperature. On the other hand, the head sperm morphometric parameters were significantly higher after room temperature incubation showing, in addition, lower ellipticity (P<0.05). Furthermore, kinematic parameters were evaluated both at RT and 37°C for the two incubation temperatures. In general, the four temperature combinations showed that kinematic parameters followed this order: RT-RT<RT-37<37-37<37-RT (incubation and analysis temperatures respectively). ConclusionsOur results showed that temperature control during both incubation and analysis is needed for accurate semen analysis, recommending the use of 37°C during the entire process.

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Characterisation of European eel (Anguilla anguilla) spermatozoa morphometry using Trumorph tool in fixed and non-fixed samples

Currently, the techniques used to analyse fish spermatozoa head morphology are not only subjective and time-consuming, but also, variable due to alteration of real spermatozoa size during sample manipulation. Thereby, it is important to develop a new method to obtain accurate and objective results. So far, computer-assisted systems for morphometry analysis were developed and validated for mammals, although they could also be adapted to fish spermatozoa. The present study aimed to characterise the European eel spermatozoa morphometry, comparing the measurements (area and perimeter) obtained by computer-assisted spermatozoa between pre-treated or non-treated eel sperm samples (n = 5) with fixation solution. In both protocols, the TruMorph® tool was used to apply a constant force to extend the cells in a thin layer. Images of spermatozoa were captured using a 40× negative phase contrast objective and analysed with the ISASv1 CASA-Morph system. Sperm head morphometry showed significant differences in area and perimeter comparing both protocols. Besides, the head size analysis using TruMorph® without fixation along time showed that the sperm membrane remains intact with the use of this technique, preserving the semipermeable condition. Considering the fact that fixation solution produces dehydration/hydration that could affect the real spermatozoa size, the simple use of TruMorph® without fixation combined with CASA-Morph analysis could allow more realistic measurements of eel spermatozoa head.

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Kinematic Sub-Populations in Bull Spermatozoa: A Comparison of Classical and Bayesian Approaches.

The ejaculate is heterogenous and sperm sub-populations with different kinematic patterns can be identified in various species. Nevertheless, although these sub-populations are statistically well defined, the statistical differences are not always relevant. The aim of the present study was to characterize kinematic sub-populations in sperm from two bovine species, and diluted with different commercial extenders, and to determine the statistical relevance of sub-populations through Bayesian analysis. Semen from 10 bulls was evaluated after thawing. An ISAS®v1 computer-assisted sperm analysis (CASA)-Mot system was employed with an image acquisition rate of 50 Hz and ISAS®D4C20 counting chambers. Sub-populations of motile spermatozoa were characterized using multivariate procedures such as principal components (PCs) analysis and clustering methods (k-means model). Four different sperm sub-populations were identified from three PCs that involved progressiveness, velocity, and cell undulatory movement. The proportions of the different sperm sub-populations varied with the extender used and in the two species. Despite a statistical difference (p < 0.05) between extenders, the Bayesian analysis confirmed that only one of them (Triladyl®) presented relevant differences in kinematic patterns when compared with Tris-EY and OptiXcell®. Extenders differed in the proportion of sperm cells in each of the kinematic sub-populations. Similar patterns were identified in Bos taurus and Bos indicus. Bayesian results indicate that sub-populations SP1, SP2, and SP3 were different for PC criteria and these differences were relevant. For velocity, linearity, and progressiveness, the SP4 did not show a relevant difference regarding the other sperm sub-populations. The classical approach of clustering or sperm subpopulation thus may not have a direct biological meaning. Therefore, the biological relevance of sperm sub-populations needs to be reevaluated.

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Standardization of sperm motility analysis by using CASA-Mot for Atlantic salmon (Salmo salar), European eel (Anguilla anguilla) and Siberian sturgeon (Acipenser baerii)

It is essential to define an optimized standard method to assess the fish sperm quality to minimize the differences between the results obtained by different laboratories. Only this optimization and standardization can make them useful from academia to industry. This study presents the validation of sperm motility assessment using a CASA-Mot system for three endangered diadromous fish species: European eel (Anguilla anguilla), Atlantic salmon (Salmo salar) and Siberian sturgeon (Acipenser baerii). To attain this goal, different technical and data processing methods were tested: 1) magnification lens (×10 and ×20), 2) Spermtrack® reusable chambers (10 and 20 μm depth) and 3) different frame rates (50 ≥ FR ≤ 250). The results suggested that the sperm motility assessment for eel, salmon and sturgeon should be performed at 200, 250 and 225 frames s−1, respectively. Moreover, to obtain a high number of analysed spermatozoa in less time and a natural movement of the sperm cells, it is recommended to use ×10 objective and 20 μm depth. In conclusion, different technical settings influence sperm kinetic parameters and should be validated for each fish species to allow the comparison of results between laboratories.

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Effect of counting chamber depth on the accuracy of lensless microscopy for the assessment of boar sperm motility.

Sperm motility is one of the most significant parameters in the prediction of male fertility. Until now, both motility analysis using an optical microscope and computer-aided sperm analysis (CASA-Mot) entailed the use of counting chambers with a depth to 20µm. Chamber depth significantly affects the intrinsic sperm movement, leading to an artificial motility pattern. For the first time, laser microscopy offers the possibility of avoiding this interference with sperm movement. The aims of the present study were to determine the different motility patterns observed in chambers with depths of 10, 20 and 100µm using a new holographic approach and to compare the results obtained in the 20-µm chamber with those of the laser and optical CASA-Mot systems. The ISAS®3D-Track results showed that values for curvilinear velocity (VCL), straight line velocity, wobble and beat cross frequency were higher for the 100-µm chambers than for the 10- and 20-µm chambers. Only VCL showed a positive correlation between chambers. In addition, Bayesian analysis confirmed that the kinematic parameters observed with the 100-µm chamber were significantly different to those obtained using chambers with depths of 10 and 20µm. When an optical analyser CASA-Mot system was used, all kinematic parameters, except VCL, were higher with ISAS®3D-Track, but were not relevant after Bayesian analysis. Finally, almost three different three-dimensional motility patterns were recognised. In conclusion, the use of the ISAS®3D-Track allows for the analysis of the natural three-dimensional pattern of sperm movement.

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Comparison of sperm motility subpopulation structure among wild anadromous and farmed male Atlantic salmon (Salmo salar) parr using a CASA system.

Atlantic salmon (Salmo salar) is an endangered freshwater species that needs help to recover its wild stocks. However, the priority in aquaculture is to obtain successful fertilisation and genetic variability to secure the revival of the species. The aims of the present work were to study sperm subpopulation structure and motility patterns in wild anadromous males and farmed male Atlantic salmon parr. Salmon sperm samples were collected from wild anadromous salmon (WS) and two generations of farmed parr males. Sperm samples were collected from sexually mature males and sperm motility was analysed at different times after activation (5 and 35s). Differences among the three groups were analysed using statistical techniques based on Cluster analysis the Bayesian method. Atlantic salmon were found to have three sperm subpopulations, and the spermatozoa in ejaculates of mature farmed parr males had a higher velocity and larger size than those of WS males. This could be an adaptation to high sperm competition because salmonid species are naturally adapted to this process. Motility analysis enables us to identify sperm subpopulations, and it may be useful to correlate these sperm subpopulations with fertilisation ability to test whether faster-swimming spermatozoa have a higher probability of success.

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