Research articleDifferent computer-assisted sperm analysis (CASA) systems highly influence sperm motility parameters
Introduction
Recently, computer-assisted sperm analysis (CASA) systems have become very popular for practical analyses of sperm motility in many species and have mostly replaced subjective or “manual” motility estimation methods used in the past [1], [2]. Fish exhibit large variability in sperm motility behaviors and parameters, which is particularly species-dependent. Short movement duration and quick changes in main sperm motility descriptors during the motility period [3] make them difficult to evaluate by CASA, which was originally designed for mammalian spermatozoa. CASA settings and the technical conditions for recordings (i.e., local optical contrast, sperm concentration, type of microscopy, objective lens or camera) are very different, and each specific parameter requires careful attention and investigation. Although comparisons and evaluations of different CASA systems have been studied in mammals, to the best of our knowledge, such issues have not been addressed for fish milt to date.
CASA systems are generally thought to provide objective and repeatable results, and one should be cautious when interpreting data, as the results could be critically affected by the CASA instrument itself as well as the settings of the tool and conditions under which the motility was recorded. Each CASA system requests minimal validation tests, and it has been predicted that the results are highly dependent on the settings of each system, which could lead to biased conclusions based on a combination of factors [4]. Several authors have described artifacts that could affect results obtained from a CASA, such as the focal position inside the open drop of swimming sperm [5], field of observation location [6], frame rate of recordings [7], and type of CASA used [8].
In this study, we compared the results obtained from three different CASA systems (CRISMAS CASA, Hobson Sperm Tracker, and Image J CASA plug-in) in order to detect the potential differences between CASA instruments, evaluate the amplitude of these differences, and postulate on the origin of such differences. Thus, the same exact recordings (25 and 50 Hz frame rate) were used to eliminate any possible influence originating from the recordings themselves as well as sample preparation quality on the obtained results. The frame rates (25 or 50 Hz) chosen in this study were those that were commonly used in analog video cameras from Europe (standard PAL system, ITU-R BT.470-6).
Sperm motility records were obtained from three taxonomically distant fish species (sterlet, Acipenser ruthenus L.; common carp, Cyprinus carpio L.; and rainbow trout, Oncorhynchus mykiss Walbaum), which were selected as three species with significantly different spermatozoa behaviors during the motility period. Sterlet sperm are characterized by a markedly long duration of motility that lasts several minutes. In contrast, the duration of sperm motility in rainbow trout is extremely brief (usually 20–30 seconds). Spermatozoa of common carp are characterized by an intermediate duration of motility (∼1 minute). These differences in motility duration are accompanied by unique sets of species-specific motility parameters values related to the speed and trajectory of movement [3], [9].
The main goal of this study was to distinguish possible influence of different CASA systems on values of motility parameters studied on different species. We hypothesized that these three CASA systems, which were initially based and designed on different technical approaches with various options for manipulating the main settings, would result in differences in the parameters describing sperm motility. Moreover, the differences would likely depend on species-specific spermatozoa movement characteristics.
Section snippets
Fish and sperm sampling
Samples of rainbow trout and common carp milt were collected by stripping (abdominal massage), whereas samples of sterlet milt were collected by inserting a syringe attached to polyethylene rigid tubing into the urogenital opening.
Three-year-old rainbow trout (n = 5) were raised at the Department of Salmonid Research, Rutki, Poland. The fish were stocked in concrete ponds (150 m3) supplied with water from the Radunia river. The temperature of water was 10 °C to 12 °C.
Milt of common carp was
Results
Main effects and interactions were significant for studied parameters with P < 0.01. That is why we apply Tukey's honestly significant difference (HSD) test to compare the individual differences between groups. In the text below, we will discuss and present the P value only for HSD test.
Influence of frame frequency on sperm motility parameters
Our results obtained after analyses of sperm motility corresponding to a 12-second video recording indicated an increase in the measured sperm velocity at a frame frequency of 50 Hz compared with 25 Hz. Importantly, these results are in agreement with those previously described [7] and are related to the side-to-side motion of spermatozoa heads during progressive motility. This could be also explained by the fact that BCF (frequency of head trajectory crossing VAP) parameter also increased.
Acknowledgments
The authors acknowledge the projects CZ.1.05/2.1.00/01.0024, GACR P502/11/0090, and GACR P502/12/1973. This research was partially supported by funds appropriated to Institute of Animal Reproduction and Food Research in Olsztyn. The authors greatly appreciate the valuable help from Jacky Cosson during the manuscript preparation.
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