Motility and viability, assayed as plasma membrane integrity, were sequentially analyzed using an IVOS II CASA system (Hamilton Thorne, Beverly, MA, USA) on the same five fields under phase contrast and fluorescent illumination (using the Viadent filter), respectively [39 (link)]. For viability analysis, samples were previously stained using the VIADENT™ stain biz-benzamide trihydrochloride (Hoeschst 33258, 5 μg/mL; Hamilton Thorne, Beverly, MA, USA) which penetrates only in cells with a damaged membrane and attaches to the DNA, emitting fluorescence.
Ivos 2 casa system
Manufactured by Hamilton Thorne
Sourced in United States
The IVOS II CASA system is a computer-assisted semen analysis (CASA) instrument designed to evaluate sperm motility, concentration, and other parameters. It provides automated analysis of semen samples to assist in the assessment of male fertility.
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Lab products found in correlation
7 protocols using ivos 2 casa system
1
Sperm Motility and Viability Assay
2
Sperm Motility Evaluation by CASA
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Sperm concentration, motility and kinematics were assessed using an IVOS II CASA system driven by the software version 1.10.1 (Hamilton Thorne Inc., Beverly, U.S.A.). A pre-warmed (38 °C) 20 μm-deep 4-chamber Leja slide (IMV Technologies; L’Aigle, France) was filled with 6 μL of semen, and a minimum of 1000 cells were analyzed in at least eight randomly selected fields with 30 frames acquired per field at a frame rate of 60 Hz. Sperm with straightness ≥70% and average path velocity ≥ 50 μm/s were considered progressively motile. In each sample the percentage of progressively motile sperm (progressive motility) was recorded.
3
Sperm Motility Analysis in Mice
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For computer-assisted sperm analysis (CASA), epididymal sperms from adult male mice were collected by swim-out procedure from the epididymis in standard HS buffer [135 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 20 mM Hepes, 5 mM glucose, 10 mM dl -lactic acid, and 10 mM pyruvic acid (pH 7.4)]. Mouse sperm were washed twice in HS medium (5 min at 300g) and resuspended to a final concentration of 1 × 106 cells/ml in HS buffer or HSB buffer, containing 15 mM HCO3−. To both media, 5% bovine serum albumin (BSA) was added to reduce unspecific binding of sperm head to the glass surface. For analysis, 30 μl of the sperm suspension was loaded into a 100-μm-deep chamber slide (Leja Products B.V.), and sperm motility parameters were determined using the IVOS II CASA System (Hamilton Thorne). The parameters measured were the sperm concentration (cells/ml), average velocity, straight-line velocity, and curvilinear velocity in micrometers per second.
4
Assessing Melatonin's Impact on Sperm Kinematics
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In order to assess the effects of melatonin on in vitro sperm kinematics and motion parameters at different incubation periods, we used the IVOS® II CASA system (version 1.10, Hamilton Thorne, Beverly, USA). Prior to evaluation, the samples were gently homogenized. Then, 10 μL of the sample was placed in a Makler chamber and analyzed. Instrument settings of the CASA system are shown in S1 Table
All measurements were performed in triplicate, each analyzing five capture frames. The mean of the three replicates was used for statistical evaluation of average CASA parameters. The CASA parameters included total motility (%), progressive motility (%), straight line velocity (VSL: μm/s), curvilinear velocity (VCL: μm/s), average path velocity (VAP: μm/s), amplitude of lateral head displacement (ALH: μm), beat cross frequency (BCF: Hz), linearity (LIN: %), straightness (STR: %) and wobble (WOB: %):
All measurements were performed in triplicate, each analyzing five capture frames. The mean of the three replicates was used for statistical evaluation of average CASA parameters. The CASA parameters included total motility (%), progressive motility (%), straight line velocity (VSL: μm/s), curvilinear velocity (VCL: μm/s), average path velocity (VAP: μm/s), amplitude of lateral head displacement (ALH: μm), beat cross frequency (BCF: Hz), linearity (LIN: %), straightness (STR: %) and wobble (WOB: %):
5
Sperm Characteristics of Bull with 1-bp Deletion
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Ejaculates from a bull homozygous for the 1-bp deletion were collected at 14, 16, 17 and 20 months of age using an artificial vagina. Sperm concentration, total sperm count, and sperm motility were determined with an IVOS II CASA system (Hamilton Thorne Inc., Beverly, USA) using Leja 2-chamber slides (Leja, Nieuw-Vennep, the Netherlands). Semen was fixed in buffered formol saline solution (Na2HPO4 4.93 g, KH2PO4 2.54 g, 38% formaldehyde 125 mL, NaCl 5.41 g, distilled water qs 1000 mL) and smears were prepared according to Hancock [29 ] for morphological examination. At least 200 sperm were subsequently evaluated by phase contrast microscopy using oil immersion (Olympus BX50, UplanF1 100×/1.30, Olympus, Wallisellen, Switzerland). Morphologically abnormal sperm were assigned to defect categories according to Blom [30 (link)].
Sperm viability was assessed using the eosin-nigrosin staining method [31 ]. At least 200 sperm were evaluated on stained slides under oil immersion using a light microscope (Olympus BX50, UplanF1 100×/1.30, Olympus, Wallisellen).
The bull that was homozygous for the 1-bp deletion was slaughtered at 21 months at a regular slaughterhouse. Testis tissue was preserved in formalin, embedded in paraffin and subsequently stained with hematoxylin–eosin for histological evaluation.
Sperm viability was assessed using the eosin-nigrosin staining method [31 ]. At least 200 sperm were evaluated on stained slides under oil immersion using a light microscope (Olympus BX50, UplanF1 100×/1.30, Olympus, Wallisellen).
The bull that was homozygous for the 1-bp deletion was slaughtered at 21 months at a regular slaughterhouse. Testis tissue was preserved in formalin, embedded in paraffin and subsequently stained with hematoxylin–eosin for histological evaluation.
6
Evaluating MitoQ Toxicity in Bull Sperm
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To verify the potential toxicity of MitoQ, stored straws from four Simmental bulls (four straws/bull) were thawed in a water bath (38 ˚C, 30 s). Thawed semen samples from each bull were homogenized in Tyrode's solution (1:1, v:v) and aliquoted individually into five treatments supplemented with 40 μg/ml Hoechst 33342, for discrimination of sperm from non‐sperm particles, and various concentrations of MitoQ: Control (no MitoQ), 0.2, 2, 20 and 200 nM.
Semen was examined by CASA, as reported (Ibanescu et al., 2020 (link)), using the same settings. Briefly, after 15 min of incubation at 38˚C, 6 µl of the sample mix was loaded into a pre‐warmed (38°C) 4‐chamber Leja slide (IMV Technologies), and the kinematic parameters from a minimum of 1000 sperm in no less than five randomly selected fields were assessed using an IVOS II CASA System (version 1.10.1; Hamilton Thorne Inc., Beverly, MA). Percentages of total and progressive motility were recorded for analysis.
Semen was examined by CASA, as reported (Ibanescu et al., 2020 (link)), using the same settings. Briefly, after 15 min of incubation at 38˚C, 6 µl of the sample mix was loaded into a pre‐warmed (38°C) 4‐chamber Leja slide (IMV Technologies), and the kinematic parameters from a minimum of 1000 sperm in no less than five randomly selected fields were assessed using an IVOS II CASA System (version 1.10.1; Hamilton Thorne Inc., Beverly, MA). Percentages of total and progressive motility were recorded for analysis.
7
Sperm Motility Analysis using CASA
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Sperm motility and kinematics were assessed using an IVOS II CASA system driven by software version 1.10.1 (Hamilton Thorne Inc., Beverly, MA). A prewarmed (38°C) 20-μm-deep 4-chamber Leja slide (IMV Technologies) was filled with 6 μL of semen, and a minimum of 1,000 cells was analyzed in at least 8 randomly selected fields with 30 frames acquired per field at a frame rate of 60 Hz. Sperm with straightness ≥70% and average path velocity ≥50 μm/s were considered progressively motile, whereas sperm with average path velocity ≥50 μm/s were classified as rapidly motile. In each sample the percentage of total motile (total motility), progressively motile (progressive motility), and rapidly motile sperm (rapid motility) was recorded.
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