Specific setup and methodology for computer-assisted sperm analysis (CASA) in evaluating elasmobranch sperm

Cryopreservation of Potamotrygon Stingrays’ Semen: Enhancing One Conservation Effort

“…The possibility that the tank water could play a role in freshwater stingray sperm activation, as demonstrated in marine rays [25], indicates the importance of preventing sperm contact with water during collections used for artificial insemination or cryopreservation. The sperm collection performed in this study was made with care to avoid water contact, drying the cloaca region with absorbent paper, and, at any sign of feces and urine elimination, the collection tube was moved away to avoid contact with the contaminants, as recommended by Cosson et al [34] Regarding the sperm motile assessment, the use of counting chambers (Leja) on the IVOS II CASA equipment, utilizing the specific setup for elasmobranchs developed by Jorge-Neto et al [16], proved to be very effective with high accuracy, allowing the cells free movement without external mechanical interferences that could alter motility. The use of this technique is crucial, considering that the use of cover slips that press the deposited material onto the slide by their own weight can compromise the sperm movement [35].…”

“…Samples were diluted with either INRA 96 or OptiXcell at ratios of 1:100 (M3; v/v), 1:400 (F3; v/v), and 1:200 (all other males; v/v). This dilution was based on visual assessment, aiming to achieve an optimal concentration of 20 × 10 sperm/mL (~60 spermatozoa per system screen), thereby minimizing cell overlap and enhancing the accuracy of the analysis [16]. After dilution, the samples were homogenized for 5 s by vortexing, and 3 µL was placed onto a 4-chamber Leja slide (IMV Technologies, France).…”

“…Total motility and progressive motility parameters were assessed using Computer Assisted Sperm Analysis (CASA) with IVOS II equipment (version MK5; Hamilton Thorne, Beverly, MA, USA) and Animal Breeders II software (version 1.11.9; Hamilton Thorne, Beverly, MA, USA). The analysis followed the base setup for elasmobranchs, as previously described by Jorge-Neto et al [16]. Analysis was performed immediately after dilution at room temperature and again 30 min post-incubation at 37 • C, with the latter time point designated as D0.…”

“…CASA assessment was performed with a dilution of thawed samples at a 1:1 (v/v) ratio with EasyBuffer B (IMV Technologies, France), except for F3 (1:2 v/v; highly concentrated) and M3 (undiluted; low concentration). The appropriate dilution ratio should not exceed 30 × 10 6 sperm/mL or fall below 10 × 10 6 sperm/mL [16]. The samples were then incubated at 37 • C for 30 min before undergoing analysis as previously described.…”