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.

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.

Freshwater is not abundantly available in many parts of the planet. Drylands cover about 41.3% of the face of the earth and support more than 2 billion people. Drylands are susceptible to a wide variety of natural and/or human-driven disasters which could lead to human catastrophes such as famine and huge population displacements. Populated areas prioritize their interests by controlling and diverting the water resources available, consequently, they have negative effects on the delicate natural ecosystems in drylands. Moreover, global warming and desertification threaten the wildlife and force the remaining floral elements to face the dilemma of between migration and extinction. Cities, with their managed and irrigated green spaces are safe havens for many plants. In this paper, the necessity of turning cities in drylands to artificial protected areas is discussed and the various aspects of urban life to serve not only human population, but the endangered plant species are also addressed.

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.

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.

Semen analysis is a key factor when determining the fertility ability in males. South American camelids, and in particular the alpaca, have been studied very little when compared with other farm animals. The aim of this work was to perform the kinematic characterization of alpaca spermatozoa collected directly from the deferent duct by using CASA-Mot (Computer Assisted Semen Analysis for Motility) technology. Samples were obtained every three days throughout the reproductive season during two periods and with a break of seven days in the middle. During both periods, the quality of the sample's motility and kinematics increased over the first two days and then subsequently decreased. This pattern was similar in all animals. It was concluded that the introduction of resting times can be useful to improve sperm quality for artificial insemination purposes in natural conditions.

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.

For over 30 years, CASA-Mot technology has been used for kinematic analysis of sperm motility in different mammalian species, but insufficient attention has been paid to the technical limitations of commercial computer-aided sperm analysis (CASA) systems. Counting chamber type and frame rate are two of the most important aspects to be taken into account. Counting chambers can be disposable or reusable, with different depths. In human semen analysis, reusable chambers with a depth of 10µm are the most frequently used, whereas for most farm animal species it is more common to use disposable chambers with a depth of 20µm . The frame rate was previously limited by the hardware, although changes in the number of images collected could lead to significant variations in some kinematic parameters, mainly in curvilinear velocity (VCL). A frame rate of 60 frames s-1 is widely considered to be the minimum necessary for satisfactory results. However, the frame rate is species specific and must be defined in each experimental condition. In conclusion, we show that the optimal combination of frame rate and counting chamber type and depth should be defined for each species and experimental condition in order to obtain reliable results.

The science of reproductive biology has been a source of fascination for centuries and has been marked both by important landmarks of discovery and influential, but persistent, errors of fact. The first century physician and philosopher, Galen of Pergamum, carried out empirical investigations on both humans and animals and proposed that conception required a combination of both male and female ‘principles’…

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.