The motility of cold-stored sperm preserved in preservation medium at 4°C was evaluated using a computer-assisted sperm analyzer (CASA, IVOS Sperm Analyzer, Hamilton-Thorne Research, Beverly, MA, USA) [26 (link)]. Epididymal sperm were transferred and incubated in a 100-μL drop of cTYH for 60 min. The sperm suspension was applied to a cell counting chamber (Hamilton-Thorne Research). Sperm motility was denoted by movement at a velocity of 5 μm/s in any direction. Progressive motility denoted a path velocity >50 μm/s and a straightness ratio >50%. In addition, the average path velocity, progressive velocity, track speed, lateral amplitude, and beat cross frequency were evaluated. In each experiment, 500–1000 sperm were analyzed. The experiments were independently conducted four to eight times.
Ivos sperm analyzer
Manufactured by Hamilton Thorne
Sourced in United States
The IVOS Sperm Analyzer is a laboratory instrument designed to analyze semen samples. It provides objective and quantitative measurements of sperm concentration, motility, and other parameters.
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8 protocols using ivos sperm analyzer
1
Evaluating Cold-Stored Sperm Motility
2
Sperm Motility Analysis with IVOS
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A computer-assisted sperm analyzer (IVOS Sperm Analyzer; Hamilton Thorne Inc., Beverly, MA, USA) was used to evaluate sperm motility. Cold-stored sperm were diluted to a final concentration
of 1,000–1,500 sperm/µl, and cultured in 200 µl mHTF at various concentrations of BSA (0–60 mg/ml) drops covered with paraffin oil for 2 h at 37°C and 5% CO2. Next, 10 µl of the
sperm suspension was added to the analysis chamber (MicroCell, 20 µm chamber depth, Cat No. 126575, Vitrolife K.K., Tokyo, Japan) for measurements of motility and other parameters. Sperm
motility was calculated as the ratio of sperm that moved at a speed of ≥ 5 µm/sec. Progressive motility was calculated as the percentage of total spermatozoa that progressed ≥ 50 μm/sec and
had a progressiveness ratio of ≥ 50%. The average path velocity (VAP) was calculated as the average velocity of motile sperm. Progressive velocity (VSL) was calculated as the average
velocity of motile sperm measured in a straight line from the start to the endpoint. The lateral amplitude (ALH) was calculated as the swing of the sperm head moving forward. The beat
frequency (BCF) was calculated as the frequency of the sperm head crossing relative to the sperm migration path. Straightness (STR) was calculated as the “straightness” of each sperm’s
average migration path. Between 500 and 1,000 sperm were analyzed in each experiment.
of 1,000–1,500 sperm/µl, and cultured in 200 µl mHTF at various concentrations of BSA (0–60 mg/ml) drops covered with paraffin oil for 2 h at 37°C and 5% CO2. Next, 10 µl of the
sperm suspension was added to the analysis chamber (MicroCell, 20 µm chamber depth, Cat No. 126575, Vitrolife K.K., Tokyo, Japan) for measurements of motility and other parameters. Sperm
motility was calculated as the ratio of sperm that moved at a speed of ≥ 5 µm/sec. Progressive motility was calculated as the percentage of total spermatozoa that progressed ≥ 50 μm/sec and
had a progressiveness ratio of ≥ 50%. The average path velocity (VAP) was calculated as the average velocity of motile sperm. Progressive velocity (VSL) was calculated as the average
velocity of motile sperm measured in a straight line from the start to the endpoint. The lateral amplitude (ALH) was calculated as the swing of the sperm head moving forward. The beat
frequency (BCF) was calculated as the frequency of the sperm head crossing relative to the sperm migration path. Straightness (STR) was calculated as the “straightness” of each sperm’s
average migration path. Between 500 and 1,000 sperm were analyzed in each experiment.
3
Sperm Motility Assessment from Vas Deferens
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Vas deferens were harvested from adult males, and placed in a puddle of (500 μL) in vitro fertilization (IVF) media (Research Vitro Fert K-RVFE-50; Cook Medical, Inc. USA). The semen was squeezed out by the needle and the sperm was allowed to swim out for 10 min at 37 °C. A 150 μL drop of sperm suspension was placed into the same volume of pre-warmed IVF media (37 °C). Then the sperm were moved to a prewarmed glass slide for motility assessment on an IVOS SpermAnalyzer (Hamilton-Thorne Research, Beverly, Mass.).
4
Comprehensive Sperm Quality Analysis
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The right epididymis for each mouse was dissected out for the analysis of sperm quality. Eleven parameters of mice sperm including sperm concentration (×109/ml), sperm motility (%), progressive (%), VAP (μm/s), VSL (μm/s), VCL (μm/s), ALH (μm), BCF (Hz), STR (%), LIN (%) and sperm activity (including rapid, medium, slow and static, %) were measured by IVOS sperm Analyzer (Hamilton Thorne Biosciences, USA). All analysis was run in a blinded fashion by one colleague.
5
Sperm Motility Analysis with IVOS
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The motility of fresh and cold-stored sperm was evaluated using a computer-assisted sperm analyser (IVOS Sperm Analyzer, Hamilton-Thorne Research Co. Ltd., USA). Fresh sperm were diluted to a final concentration of 500 sperm/µL and cultured in 200 µL of mHTF drops covered with paraffin oil for 2 h at 37 °C and 5% CO2. Next, 10 µL of the sperm suspension was collected and added to the measurement chamber to measure motility and other parameters. Sperm motility was calculated as the ratio of sperm that moved at a speed of ≥ 5 µm/s. Progressive motility was calculated as the percentage of total sperm that progressed ≥ 50 μm/s and had a progressiveness ratio of ≥ 50%. VAP was calculated as the average velocity of the motile sperm. Progressive velocity (VSL) was calculated as the average velocity of motile sperm when measured in a straight line from the start point to the end point. Lateral amplitude (ALH) was calculated as the swing of the sperm head as it moved forward. Beat frequency (BCF) was calculated as the frequency of sperm head crossings relative to the sperm migration path. Straightness (STR) was calculated as how close to a straight line the average migration path of sperm was. Between 500 and 1000 sperm were analysed in each experiment.
6
Sperm Motility Analysis using IVOS
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Sperm motility was assessed using a computer-assisted sperm analyser (IVOS Sperm Analyzer, Hamilton-Thorne Research Co. Ltd., USA)39 (link). Sperm were incubated in cTYH for 60 min at 37 °C under 5% CO2 in air. Sperm suspensions were diluted 50 times in mHTF, incubated for 5 min at 37 °C and sorted using the microfluidics chip cell sorting system described previously. After sorting, sperm suspensions were placed in a disposable sperm analysis chamber (Hamilton-Thorne Research) and analysed using the IVOS system. We analysed the following sperm motility parameters: percentage of motile sperm (motile sperm moved more than 5-µm/s), percentage of motile sperm with progressive motility (motile sperm with progressive motility were denoted by a path velocity> 50 µm/s and a straightness ratio> 50%) and a marker of hyperactivation [lateral amplitude of head (ALH): this is the average value of the maximum swing width of the sperm head]. In addition, path velocity (VAP), progressive velocity (VSL), track speed (VCL), beat frequency (BCF), straightness (STR), Linearity (LIN) and Elongation were measured. During motility analysis, the sperm analysis chamber was warmed at 37 °C. To analyse the motile parameters, 200–1000 sperm were examined in each experiment. The experiments were independently from 7 to 10 times.
7
Mouse Sperm Quality Evaluation
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At the end of the administration period, the animals were executed, and the sperm cells obtained from the cauda epididymis were collected into 1 mL of M199 culture medium containing 0.1% bovine serum albumin. Sperm motility was assessed after incubation for 30 min at 37° C. A sperm aliquot was placed on a pre-warmed 100-μm-deep counting chamber slide and analyzed using an IVOS sperm analyzer (HamiltonThorne Biosciences). The indicators used to monitor the quality of mouse sperm were recommended by the manufacturer and included average path velocity (VAP), straight line velocity (VSP); continuous line velocity (VCL), and amplitude of lateral head displacement (ALH).
8
Sperm Motility and Count Measurement
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Sperm motility and count were measured using a CASA system as described previously41 (link). Mature sperm were isolated from the cauda epididymis dissected from sexually mature mice. The tissue was incised at four sites and placed in 0.5 mL of PBS for 15 min in a 37 °C incubator. Debris was removed, and 20 μL aliquots of sperm were placed in an 80-μm-deep chamber for the assessment of sperm motility, and the sperm count was obtained by CASA using an IVOS Sperm Analyzer (Hamilton Thorne Biosciences).
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