The goal of these procedures is to evaluate sperm membranes integrity using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analysis. These techniques might help answer key questions in the field of reproduction such as prediction of fertilization potential and quality of sperm sample. The main advantage of these techniques is that they enable precise evaluation of multiple sperm membranes which are known to associate with sperm fertilization potential.
The implications of these techniques extend toward diagnosis of ejaculate quality as it enables more accurate evaluation of sperm plasma and acrosome membrane integrity as well as mitochondrial membrane potential. To begin this procedure, obtain approximately one to six milliliters of bull semen in a 15 milliliter tube at room temperature. Add six milliliters of NKM buffer pre-warmed to 37 degrees Celsius to each one milliliter of semen.
Centrifuge at 600 times g for eight minutes. Repeat the centrifugation once or twice until the supernatant is clear. After this, immediately discard most of the supernatant, leaving approximately one centimeter of supernatant above the pellet.
Carefully lean the tubes at a 30 degree angle in the incubator and wait 20 to 30 minutes to allow the spermatozoa to swim up. Using a micropipette, carefully remove the remaining one milliliter of supernatant which now contains the motile spermatozoa and transfer it to a fresh 1.5 milliliter tube. Keep the sperm at 37 degrees Celsius until ready to use.
First, prepare all needed stock solutions as outlined in the text protocol. Next, transfer 133 microliters of the motile spermatozoa at a concentration of 25 million sperms per milliliter to a new 1.5 milliliter tube. Add 17 microliters of the DAPI working solution and incubate at 37 degrees Celsius for 10 minutes.
Centrifuge at 1, 000 times g for five minutes. Discard the supernatant and add 100 microliters of NKM buffer to the pellet. Next, add 50 microliters of FITC-PSA, two microliters of JC-1, and three microliters of PI.Incubate at 37 degrees Celsius for 10 minutes.
Centrifuge at 1, 000 times g for five minutes. Discard the supernatant and resuspend the pellet in 40 microliters of NKM buffer. Then transfer 10 microliters of the sample to a glass slide.
Smear the sample and add a coverslip. After this, immediately begin visualizing the sample by epifluorescence microscopy with a 40X objective and a triple filter as outlined in the text protocol, using a digital camera to capture a separate image for each filter. Use the merge option in the camera software to merge the three images received from the filters, saving the new file in JPEG format.
Open the merged image with the paint tool and use the brush option to mark the counted spermatozoa. Next, classify the spermatozoa based on the fluorescence emitted from each probe, making sure to evaluate at least 200 spermatozoa per slide. All cells should appear blue from the DAPI.
Count the dead cells, which appear purple, to evaluate the viability. Then use the patterns of fluorescence staining to evaluate the acrosome status. Lastly, evaluate the mitochondrial membrane potential by distinguishing the spermatozoa with high mitochondrial membrane potential which exhibit a red-stained midpiece from the spermatozoa with low mitochondrial membrane potential which exhibit a green-stained midpiece.
Count red and green midpieces separately and calculate their ratio. To begin the plasma membrane integrity evaluation, take the desired number of wells from the viability and concentration kit. Transfer the wells to the working base and cover them with a flexible lid to protect them from light.
Add 199 microliters of buffered solution for cytometry to each well. Then add one microliter of homogenous semen at a concentration of 57 million cells per milliliter to each well and homogenize by pipetting. Cover the plate with the lid and incubate at 37 degrees Celsius for 10 minutes.
After this, run the sample through the flow cytometer using the viability setting. To begin assessing the mitochondrial membrane potential, take out the desired number of wells from the mitochondrial activity kit. Transfer them to the working base.
Add 10 microliters of absolute ethanol to each well and use a pipette to resuspend the powder present within the well. Add 190 microliters of PBS per well and homogenize by pipetting. Add 0.75 microliters of homogenous semen at a concentration of 57 million cells per milliliter to each well and homogenize by pipetting.
Cover the plate with the lid and incubate at 37 degrees Celsius for 30 minutes. After this, run the sample through the flow cytometer using the mitochondrial activity setting. To begin assessing the acrosomal membrane integrity, take out the desired number of wells from the viability and acrosome integrity kit.
Transfer the wells to the working base and cover them with a flexible lid to protect them from light. Add 200 microliters of buffered solution for cytometry to each well. Then add 0.7 microliters of homogenous semen at a concentration of 57 million cells per milliliter to each well and homogenize by pipetting.
Cover the plate with the lid and incubate at 37 degrees Celsius for 45 minutes. After this, run the sample through the flow cytometer using the insight setting. In this study, semen quality is evaluated using different techniques, one of which evaluated sperm membranes using fluorometric probes.
It is possible to evaluate the differences in sperm sample quality in terms of membrane integrity. For example, the ejaculate of bull number seven had a relatively low percentage of dead cells, a low proportion of sperm with pseudo-reacted acrosome, and higher mitochondrial membrane potential when compared to the ejaculate of bull number one. Samples are then evaluated for viability and mitochondrial activity.
The histograms for these representative examples represent the ungated spermatozoa and debris and the gated spermatozoa. These histograms also represent the viable and dead cells and the distribution of spermatozoa to polarized and de-polarized mitochondrial membrane. The acrosome integrity is then evaluated with a ready-to-use kit and read with flow cytometry, dividing the area into three marker areas that represent negligible low fluorescing cells with intact unstained acrosome, low fluorescing cells with residual stained part of the acrosome, and highly fluorescing cells with disrupted acrosome.
While attempting these procedures, it is important to make sure to prepare properly the initial sperm sample. These techniques can provide insight into sperm quality evaluation and can be applied to various organisms such as bovine, poultry, and human. Comparison between the two techniques, quadruple staining and flow cytometry indicated no significant differences for viability, mitochondrial membrane potential, and the acrosome integrity revealing that the two techniques produced matching results.
