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• W2078791680 abstract "In dogs and cats, computer-assisted sperm analysis (CASA) was originally described almost 20 years ago. Subsequently, numerous CASA systems were validated and used for various applications in dogs and to a lesser extent in cats. CASA systems offer an accurate, rapid, objective and simultaneous assessment of different semen parameters allowing the visualization of subtle changes in sperm characteristics, which cannot be identified by conventional semen analysis. The main problems of these computerized measuring devices are the relatively high investment costs and the need for standardization and validation before any practical use is possible. In comparison with automated motility and concentration assessment, automated morphometry and morphology assessment is more complex and time-consuming. Once validated, CASA systems can be routinely used in veterinary centres for assessment of fertility and for the improvement of sperm diluters, cooling and cryopreservation procedures in dogs and cats. Furthermore, information obtained by CASA systems could also be important when monitoring for example the effect of environmental stress on spermatozoa and for toxicity studies. In cats, CASA is less documented, and most studies describe the characteristics of epididymal sperm, which is frequently used for in vitro fertilization in cats. Implementation of the CASA technique in cat reproduction could be interesting to further optimize assisted reproductive techniques in domestic cats and endangered wild felids. In the past, light microscopy was routinely used to evaluate the main sperm parameters, that is, concentration, motility and morphology. The main problems when using these methods are subjectivity and variability mainly owing to the observer's experience and the quality of the microscope. Additional drawbacks of these techniques are the low number of spermatozoa analysed, and the time needed to perform the evaluation. These drawbacks have led to the development of several computerized measuring devices. Computer-assisted sperm analysis (CASA) was originally described by Dott and Foster (1979) more than 30 years ago in a wide variety of species including cattle, horse, pig, rabbit, rat and sheep and has gained increasingly more interest in veterinary medicine during the last decades. In dogs and cats, CASA was first described almost 20 years ago by Günzel-Apel et al. (1993) and Stachecki et al. (1993), respectively. Subsequently, numerous commercial CASA systems including the CellSoft computer videomicrography, Strömberg-Mika Cell motion analyser, Hobson Sperm Tracker, Cell Track/s System, Hamilton-Thorne, Sperm Vision, Sperm Class Analyser were validated mainly in dogs and to a lesser extent in cats, both for research purposes and various clinical applications (Smith and England 2001; Verstegen et al. 2002; Rijsselaere et al. 2003; Schäfer-Somi and Aurich 2007; Dorado et al. 2011). The aim of this short review is to discuss the working mechanism, the main (dis)advantages and some possible applications of CASA systems in dogs and cats. Computerized measuring devices generally consist of a phase-contrast microscope, a camera, a minitherm stage warmer, an image digitizer and a computer to save and analyse the data. These devices operate as a cell motion analyser, reconstructing sperm trajectories from the position of sperm heads in successive frames and calculating various motility and concentration parameters simultaneously. A playback facility inserted in most of these devices allows projection of the video sequences of the last field scanned, providing an additional control to validate whether all sperm cells were identified and whether their trajectory was reconstructed correctly. Computer-assisted sperm analysis systems offer an accurate, rapid and simultaneous assessment of different semen parameters such as concentration, total and progressive motility, slow, medium and rapid moving spermatozoa, linearity of sperm movement, the beat cross frequency, the amplitude of the lateral head displacement and different velocity parameters (Verstegen et al. 2002; Rijsselaere et al. 2005). Therefore, these computerized measuring devices are valuable for the detection of subtle changes in sperm motion, which cannot be identified by conventional semen analysis. Moreover, high numbers of spermatozoa (several thousands) can be analysed individually within a short period of time (<1 min), which make these systems very practical for daily clinical use. Owing to the high concentration of fresh canine ejaculates, a predilution is usually required before the analysis by a CASA system can be performed. The main problems when using these computerized measuring devices are the relatively high investment costs and the need for standardization and validation of the system before any practical use is possible. The choice of internal image settings (e.g. minimum cell size, frame rate, analysis time), which is important to identify and reconstruct the trajectory of the different spermatozoa accurately, clearly influences the results obtained. Additionally, significant alterations of CASA measurements have been described owing to the dilution of the semen sample, the diluent used, the analysis temperature and the counting chamber (Verstegen et al. 2002; Rijsselaere et al. 2005). As a consequence, the different available CASA systems, the computer parameters selected by the user, the software used and the microscopy conditions might lead to a new source of subjectivity among laboratories. Owing to this lack of uniformity and owing to the use of different instruments, a definition of standard accepted values for motility and sperm velocity is difficult to determine in dogs. Standardization of the technical settings, however, could be important to compare results between laboratories considering the popularity of international exchange of cooled and frozen dog semen. It additionally highlights the importance of detailed descriptions of materials and methods in all studies involving CASA procedures. Moreover, it is important to consider that CASA settings for fresh semen may not be optimal for chilled or cryopreserved sperm as the latter media often contain egg-yolk particles. Once these systems have been standardized, computer-calculated motility, progressive motility and concentration were highly correlated with conventional light microscopic evaluation. However, both over- and underestimation of the sperm concentration by CASA have been described (Verstegen et al. 2002; Rijsselaere et al. 2003). A high degree of repeatability can be achieved with inter- and intra-assay coefficients of variation of <12% and 3%, respectively, for most parameters (Smith and England 2001). Automated sperm morphometric analysis (ASMA) allows for the identification of subtle sperm characteristics, which cannot be detected by light microscopic evaluation. Previously, detailed canine sperm morphology assessment has been attempted by means of scanning and transmission electron microscopy. Subsequently, several canine sperm head dimensions (length, width, area, roundness) were measured automatically using a Leica computer system. Subsequently, the Metrix Oval Head Morphology software was implemented in the Hamilton-Thorne CASA system and was validated for the evaluation of dog sperm morphometric dimensions and morphology. This system provides very detailed information on sperm head dimensions (length, width, area, roundness, perimeter), tail length and tail abnormalities (bent, coiled, absent). More recent CASA systems such as the SpermVision can additionally evaluate the viability of spermatozoa by fluorescent labelling with SYBR14/PI and using a fluorescence microscope that is an accessory part of these CASA systems (Schäfer-Somi and Aurich 2007). As for the automated motility and concentration assessment, validation and standardization are required as, for example, the magnification level of the objective, the sperm concentration, the number of evaluated spermatozoa and the staining method influence most of the morphometric dimensions obtained. High correlations were established for the percentage of normal spermatozoa assessed by light microscopic evaluation and by the HTR-Metrix (Rijsselaere et al. 2004). In comparison with automated motility and concentration assessment, however, ASMA appeared to be a more complex and time-consuming process, partly because it requires an additional step (i.e. staining of the semen sample before analysis) and partly owing to the need for evaluation at a higher magnification level which reduces the number of evaluated spermatozoa per microscopic field (Rijsselaere et al. 2004). Moreover, the staining technique used for ASMA was shown to be species specific. Several authors provided large data sets of CASA measurements obtained from proven fertile dogs, which could serve as reference values in dogs (Verstegen et al. 2002; Rijsselaere et al. 2007). However, it still needs to be determined which sperm movement characteristics are of clinical value for the prediction of in vivo fertility in dogs. Subtle changes in sperm motility characteristics and velocity patterns have been correlated with fertilizing ability in vitro and in vivo in several species, including rat, bull, stallion and human. In dogs however, CASA has not yet been documented to give a more accurate prognosis towards in- or subfertility cases. Moreover, in most cases, the evaluation of a single sperm feature is not likely to provide enough power for the prediction for fertilization outcome and in dogs generally good and highly concentrated sperm samples are collected in comparison with, for example, human sperm. In one study including 111 dogs, the body weight of the dog was significantly correlated with the total sperm output and negatively correlated with the curvilinear velocity, and significant differences were detected between fertile and subfertile dogs for most of the evaluated sperm quality parameters assessed by the Hamilton-Thorne analyser (Rijsselaere et al. 2007). Moreover, in a more recent data set analysing the sperm quality of more than 200 dogs by the same CASA system, differences in sperm quality owing to the age and the breed could be elucidated (Rijsselaere et al. 2012, personal observation). The capability of CASA systems to detect slight alterations in sperm movement proved to be very useful for objectively comparing different sperm diluters and for the improvement of cooling and cryopreservation procedures in dogs (Schäfer-Somi and Aurich 2007; Verstegen et al. 2002). CASA systems were successfully used for the comparison of several diluents for chilling which only differed in the type of sugar (Iguer-ouada and Verstegen 2001), for the comparison of ethylene glycol versus glycerol for dog sperm cryopreservation (Rota et al. 2006), for assessing the effect of post-thaw dilution with autologous prostatic fluid (Rota et al. 2007) and several motility enhancers (Milani et al. 2010) and for assessing the effect of addition of Equex STM (Nizanski et al. 2009). Additionally, CASA systems were used to identify canine sperm cell subpopulations which interestingly offered new insights in the differences in freezability of semen among dogs and which could to a great extent predict the outcome of a cryopreservation procedure of a given semen sample (Nuñez-Martinez et al. 2006). The capability of CASA systems to detect slight alterations in sperm movement proved to be very useful for objectively comparing different sperm diluters and for the improvement of cooling and cryopreservation procedures in dogs (Schäfer-Somi and Aurich 2007; Verstegen et al. 2002). CASA systems were successfully used for the comparison of several diluents for chilling which only differed in the type of sugar (Iguer-ouada and Verstegen 2001), for the comparison of ethylene glycol versus glycerol for dog sperm cryopreservation (Rota et al. 2006), for assessing the effect of post-thaw dilution with autologous prostatic fluid (Rota et al. 2007) and several motility enhancers (Milani et al. 2010) and for assessing the effect of addition of Equex STM (Nizanski et al. 2009). Additionally, CASA systems were used to identify canine sperm cell subpopulations which interestingly offered new insights in the differences in freezability of semen among dogs and which could to a great extent predict the outcome of a cryopreservation procedure of a given semen sample (Nuñez-Martinez et al. 2006). Moreover, CASA systems can assess the capacitation status and detect populations of hyperactivated spermatozoa, a process which is necessary for successful penetration of the zona pellucida. A significant increase in VCL and ALH and a decrease in linearity is generally regarded as an indication of hyperactivation (Schäfer-Somi and Aurich 2007; Verstegen et al. 2002). Furthermore, information obtained by CASA allows for the detection of subtle changes in sperm motion, which may follow exposure of the male dog to environmental insults, or for assessing the effect of drugs on sperm quality and especially sperm motion characteristics in dogs. Using ASMA, it was demonstrated that dog sperm heads were smaller than bull, ram, goat and rabbit sperm heads but, interestingly, were larger than, for example, horse sperm heads. However, large variations in the sperm morphometric dimensions were found among individual dogs and even among ejaculates of the same dog. Significantly, lower morphometric dimensions of the canine sperm head were described after cryopreservation. Nuñez-Martinez et al. (2005) described for the first-time sperm morphometric subpopulations in the canine ejaculate. Linear regression analysis revealed significant relationships among several sperm morphometric parameters and the percentage of denaturated DNA in each individual sperm cell (Nuñez-Martinez et al. 2005). Moreover, morphometry-derived indexes showed high correlations with the percentage of intact sperm membranes post-thaw (Nuñez-Martinez et al. 2006). In contrast with dogs, CASA is rather poorly documented in felids and studies, which actually validate these systems in cats, are very scarce. Stachecki et al. (1993) were the first to describe CASA in cats by comparing epididymal sperm motion characteristics from normozoospermic and teratozoospermic cats. Subsequently, the same authors showed that motility-stimulating agents, such as caffeine, pentoxifylline and 2′deoxyadenosine, could enhance several motility parameters of cryopreserved epididymal and ejaculated cat spermatozoa without deleterious effects on longevity. More recently, it was shown that ejaculated cat sperm exhibited hyperactivation-like motion parameters (i.e increase in ALH and BCF and a decrease in VSL and LIN) when stimulated with ionomycin or progesterone. Most CASA studies in cats, however, describe the characteristics of epididymal sperm, which are frequently used for in vitro fertilization in cats (Stachecki et al. 1993; Filliers et al. 2008). Recently, reference values for cat epididymal sperm samples obtained after gradient density centrifugation were described which can facilitate comparisons of sperm quality and in vitro fertilization among laboratories (Filliers et al. 2008). Improvement of biotechnical methods that result in fast and precise semen quality assessment would however be interesting to further optimize assisted reproductive techniques in domestic cats and endangered wild felids. Ghent University is acknowledged for the financial support throughout the years. All co-authors are acknowledged for their valuable contribution to the manuscript. None of the authors have any conflicts of interest to declare. Tom Rijsselaere, Ann Van Soom, Dominiek Maes and Wojtek Nizanski contributed to the manuscript writing and design. Dominiek Maes contributed and performed statistical analyses of the studies." @default.
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• W2078791680 title "Computer-Assisted Sperm Analysis in Dogs and Cats: An Update after 20 Years" @default.
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