Sperm samples were collected by masturbation following 2–7 days of abstinence. Samples were allowed to liquefy for at least 30 min at 37°C and semen analysis was performed during the next hour. A macroscopic evaluation was first performed: The Sperm Class Analyzer CASA System (Microptic S.L., Barcelona, Spain) was used to determine sperm concentration, total sperm count and motility. Sperm vitality was established using an eosin/nigrosine coloration (FertiPro, Beernem, Belgium). Air-dried smears followed by a Romanowsky coloration were prepared according to the manufacturer’s recommendations to evaluate sperm morphology using Kruger’s strict criteria (Sperm stain, Microptic S.L., Spain). A minimum of 200 sperm were examined for each test.
Sperm class analyzer casa system
Manufactured by Microptic
Sourced in Spain
The Sperm Class Analyzer CASA System is a laboratory equipment designed for the assessment and analysis of sperm motility and other parameters. It utilizes computerized technology to provide objective and standardized measurements of sperm characteristics.
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7 protocols using sperm class analyzer casa system
1
Semen Analysis Protocol for CASA
2
Sperm Motility Analysis with CASA
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The influence of H2O2 exposure on sperm motility parameters was assessed using the Sperm Class Analyzer CASA System (Microptic S L, Barcelona, Spain) with SCA® v6.2 software (Microptic S L, Barcelona, Spain). Samples and controls (2 µL) were loaded into individual chambers of Leja Standrat Count 4 chamber slide 10 µm depth (Leja Products B.V., Nieuw-Vennep, The Netherlands) which were pre-heated at 37 °C. This temperature was maintained while at least 1000 sperm cells/measurement were evaluated.
3
Semen Cooling Analysis: Motility and DNA
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Semen was examined before and after cooling at different times, and total and progressive motilities (%), kinetic parameters, and DNA integrity (%) were evaluated as described below.
Moreover, semen analysis was performed by using the Sperm Class Analyzer CASA system (SCA; Microptic, Spain). Briefly, a 10-µL aliquot of semen sample was placed in a Makler chamber on a heated stage at 37℃, after pre-incubation at 37℃ for 10 min. At least 200 spermatozoa were counted to evaluate the percentages of motile (MOT) and progressively motile (PMOT) spermatozoa, as well as rapid (RAP), medium (MED), slow (SLOW), and static (STATIC) spermatozoa, and sperm kinetic parameters, i.e., curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN = VSL/VCL), and straightness (STR = VSL/VAP). The SCA settings were adjusted according to the manufacturer's instructions. All manual and SCA measurements were performed by trained and licensed medical technologists. The SCA results were used when the difference between manual and SCA values was less than 20%. In cases where the difference was greater than 20%, the manual results were reported [17 (link)].
Moreover, semen analysis was performed by using the Sperm Class Analyzer CASA system (SCA; Microptic, Spain). Briefly, a 10-µL aliquot of semen sample was placed in a Makler chamber on a heated stage at 37℃, after pre-incubation at 37℃ for 10 min. At least 200 spermatozoa were counted to evaluate the percentages of motile (MOT) and progressively motile (PMOT) spermatozoa, as well as rapid (RAP), medium (MED), slow (SLOW), and static (STATIC) spermatozoa, and sperm kinetic parameters, i.e., curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN = VSL/VCL), and straightness (STR = VSL/VAP). The SCA settings were adjusted according to the manufacturer's instructions. All manual and SCA measurements were performed by trained and licensed medical technologists. The SCA results were used when the difference between manual and SCA values was less than 20%. In cases where the difference was greater than 20%, the manual results were reported [17 (link)].
4
Sperm Motility Analysis with CASA
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Aliquots (5 μl) of the sperm suspensions were taken after 30 min incubation in BWW solutions with or without the chloride channel blockers and viewed on prewarmed (37°C) sperm motility analysis chambers (Makler® Counting Chamber, 10 μm deep; Sefi Medical Instruments, Haifa, Israel) using a negative phase contrast objective (×10). Several fields of view were video recorded over approximately 10 s (with a total magnification of ×100) and analyzed using a CASA system (Sperm Class Analyzer® CASA system, Microptic, Barcelona Spain). At least 200 spermatozoa were tracked for measuring progressive motility (PR), curvilinear velocity (VCL), straight-line velocity (VSL) and average path velocity (VAP). Measurements were made on 50 frames (frame rate, 50 Hz; minimum contrast, 60; minimum size, 3; minimum track points, 25; minimum VAP, 10 μm sec−1).
5
Comprehensive Sperm Quality Assessment
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The concentration, total and progressive motility of sperm was determined with the use of the Sperm Class Analyzer CASA system (Microptic SL, Barcelona, Spain) and the Nikon Eclipse E200 microscope (Nikon Instruments Inc., Melville, NY, USA) at 37°C [19 (link)]. At least 500 spermatozoa were evaluated in a minimum of five fields of view.
The sperm viability was evaluated using fluorescence microscopy. The ejaculate sample was mixed with the Hoechst 33258 dye in a1:1 ratio and then evaluated using the Olympus BX51 fluorescence microscope (Olympus Corporation, Tokyo, Japan) at 400× magnification [18 (link)]. The minimum number of evaluated spermatozoa was 200.
Smears for the morphological evaluation of the sperm were dyed using the Farelly method and evaluated through the Euromex BioBlue microscope (Euromex Microscopen bv, Arnhem, Netherlands) at 1,000× magnification using immersion oil. The minimum number of evaluated spermatozoa was 200. The abnormalities evaluated at this step were immature sperm cells with protoplasmic droplet, head defects, defects of midpieces, tail defects, and defects of acrosome [20 ].
The sperm viability was evaluated using fluorescence microscopy. The ejaculate sample was mixed with the Hoechst 33258 dye in a1:1 ratio and then evaluated using the Olympus BX51 fluorescence microscope (Olympus Corporation, Tokyo, Japan) at 400× magnification [18 (link)]. The minimum number of evaluated spermatozoa was 200.
Smears for the morphological evaluation of the sperm were dyed using the Farelly method and evaluated through the Euromex BioBlue microscope (Euromex Microscopen bv, Arnhem, Netherlands) at 1,000× magnification using immersion oil. The minimum number of evaluated spermatozoa was 200. The abnormalities evaluated at this step were immature sperm cells with protoplasmic droplet, head defects, defects of midpieces, tail defects, and defects of acrosome [20 ].
6
Cryopreservation of Canine Sperm: CASA Analysis
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CASA software imaging system (Sperm Class Analyzer® CASA system, MICROPTIC,
Nikon Ci-L, Tokyo, Japan) was employed to assess the kinematic parameters of post-thawed
sperm. In brief, sperm samples (5 µl) were placed on a counting slide for assessment and
for each sample, 5 different fields were randomly examined. For each sample at least 200
sperm were tracked for 1 sec at 25 Hz. The kinematic parameters analyzed included the
percentage of motile sperm, progressive motility, curvilinear velocity (VCL), average path
velocity (VAP), straight-line velocity (VSL), straightness, linearity, and amplitude of
lateral head displacement (ALH). The analysis of each sample was repeated 12 times for
increasing precisions of measurements. Sperm survival was considered on the basis of
plasma membrane integrity, assessed using the eosin-nigrosin staining procedure. Briefly,
sperm were stained 1:1 with the staining reagent and then smeared on a warm slide and
air-dried. At least 200 sperm per slide were examined at 1,000× magnification (Eclipse Ts
2, Nikon) to assess sperm survival. Sperm stained pink were considered non-viable, and
unstained sperm were considered viable. The optimal RSV concentration required for
cryopreservation of dog sperm was determined based on the results of parameters related to
motion characteristics and percentage of live sperm.
Nikon Ci-L, Tokyo, Japan) was employed to assess the kinematic parameters of post-thawed
sperm. In brief, sperm samples (5 µl) were placed on a counting slide for assessment and
for each sample, 5 different fields were randomly examined. For each sample at least 200
sperm were tracked for 1 sec at 25 Hz. The kinematic parameters analyzed included the
percentage of motile sperm, progressive motility, curvilinear velocity (VCL), average path
velocity (VAP), straight-line velocity (VSL), straightness, linearity, and amplitude of
lateral head displacement (ALH). The analysis of each sample was repeated 12 times for
increasing precisions of measurements. Sperm survival was considered on the basis of
plasma membrane integrity, assessed using the eosin-nigrosin staining procedure. Briefly,
sperm were stained 1:1 with the staining reagent and then smeared on a warm slide and
air-dried. At least 200 sperm per slide were examined at 1,000× magnification (Eclipse Ts
2, Nikon) to assess sperm survival. Sperm stained pink were considered non-viable, and
unstained sperm were considered viable. The optimal RSV concentration required for
cryopreservation of dog sperm was determined based on the results of parameters related to
motion characteristics and percentage of live sperm.
7
Sperm Kinematics Analysis Using CASA
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Sperm kinematics were measured in fresh and frozen-thawed samples using a computer-aided sperm analyzer (Sperm Class Analyzer® CASA System, Microptic; Barcelona, Spain). Aliquots of 5 µL sperm/sample were placed on a Makler chamber (10 µm depth; Haifa Instruments, Haifa, Israel) for each time interval and then observed in a microscope (Nikon Eclipse 80i, Tokyo, Japan) equipped with a warmed stage (37 °C) and a Basler A302fs digital camera (Basler Vision Technologies, Ahrensburg, Germany). Evaluations were made at 10× magnification and at least ten fields or 200 spermatozoa were recorded for each sample. Settings were adjusted to ram spermatozoa (20–90 µm2 for head area and 25 frames/s for image sampling frequency). Recorded parameters were curvilinear velocity (VCL, μm/s), straight line velocity (VSL, μm/s), average path velocity (VAP, μm/s), linearity (LIN, %), straightness (STR, %), wobble (WOB, %), lateral head displacement (ALH, μm), beat cell frequency (BCF, Hz) and total motility (%). VCL, VSL, STR, LIN, VAP, ALH, WOB and BCF were only calculated in motile spermatozoa.
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