Worldwide reports suggest that infertility affects 15%– 20% of couples of reproductive age, and approximately 50% of these are accountable to the male partner. Although men with infertility represent a significant percentage of the infertile population, public awareness of this fact is limited at best. Literature and the other media have often neglected the male component of reproduction other than its sexual nature. Wider access to male infertility assessment may help to resolve this deficiency.
Determining the initial quality of a boar ejaculate is the first step in semen processing and should ensure that prior to further processing, a high quality artificial insemination dose of semen will be produced.
Consistent and accurate sampling of semen is a critical step in properly evaluating and processing swine semen. Common errors and inaccuracies while using pipettes can occur from a number of causes, including: improper technique when sampling, dirty or contaminated pipettes and inaccurately calibrated pipettes. The following manufacturer guidelines and recommendations are for maintaining accuracy using air-displacement pipettes when sampling body fluids.
Pipette Use – General Guidelines
- Check your pipette at the beginning of the day for dust and dirt on the outside. If needed, wipe with 70% ethanol.
- Set the volume within the range specified for the pipette.
- Hold the pipette so the ‘grippy finger rest’ rests on your index finger.
- To maximize accuracy, the pipette, tip and semen should be at the same temperature.
- Check that you are using tips recommended for this pipette. To ensure accuracy, use only high-quality tips made from contamination-free polypropylene.
- Tips are designed for single use. They should not be cleaned for reuse as their metrological characteristics will no longer be reliable.
- Pre-rinsing (1-3 times) the tip with the semen may improve accuracy, especially when using positive displacement tips.
- Avoid turning the pipette on its side when there is semen in the tip. Semen might flow into the interior of the pipette and contaminate the pipette.
- Avoid contamination to or from fingers by using the tip ejector and gloves.
- Store pipettes in an upright position when not in use. Pipette stands are ideal for this purpose.
Check the calibration of your pipette regularly, depending on the frequency of use and on the application, but at least twice a year. If used daily, a three-month interval is recommended. Follow the instructions for recalibration in the manufacturer’s instruction manual.
Properly Sampling Semen
The following instructions and diagram demonstrate the proper technique for sampling semen.
- Dip the pipette tip into the semen and press the operating button to the first stop. Make sure the tip is sufficiently below the surface, but not so deep that the pipette barrel is in the semen.
- Slowly release the operating button to the ready position. This action will fill the tip with semen. Do not remove the tip from the solution.
- Press the operating button to the first stop again and release slowly. This action pre-rinses and primes the tip. Repeat this process to load semen into the tip.
- Place the loaded tip into the test tube where the semen is to be diluted.
- Press the operating button to the first stop, then continue pressing to the second stop, which will completely empty the semen from the tip with a puff of air.
- Release the operating button to the ready position.
- Make certain that the pipette tip does not pull in solution from the second stop. This could result in solution coming into the barrel and contaminating the pipette.
At GenePro, we do not include the percent acrosomal damage in the viability calculation, the reason being is that we can’t determine if the cell was or is alive or dead.
We base this on the fact that living sperm cells are theoretically acrosomally okay. We are associating acrosomal damage with cell destabilization and preeminent death some time shortly there after. From a viability standpoint we do not want to count a dead cell and an acrosomal damaged cell twice. Doing that would be a falsely negative representation of number of actual viable cells. That is why we simply state that NAR was conducted on non-living cells and is not included in viability estimates.
It is a widely held scientific belief that the capacitation process which leads to an acrosome reaction is considered to be a controlled destabilization process which reduces the life span of sperm (Harrison, 1996.) No one knows exactly how the life span is affected, but we do know from frozen semen experiments (frozen-thawed sperm are generally capacitated or in the process) that this lifespan is between three and 12 hours-approximately after capacitation has occurred.
We don’t have a very good way to determine if a living cell has acrosome damage or not. The only way to theoretically do this is with a live dead stain and then one has to question if the staining or the smear had anything to do with the damage?
Long story short, we don’t put a lot of weight in acrosome evaluation of sperm cells and prefer sperm membrane viability assessment with stains because it is better, however a bit more costly. A number of years back I tested PNA stain (acrosome stain) with SYBR 14 and PI to see if I could find any “green” (living) cells with acrosome damage (these cells would be green with an orange cap) and there wasn’t a single cell that showed this in fresh pig semen. However, we did see it occur in frozen thawed samples (small amount). The bull guys are the ones who discovered this triple stain technique with frozen-thawed bull sperm.
If I took three people and showed them what a lifted or ruffled acrosome was, variation should be very small when one has great optics and the same sample. Variation can occur if you asked each individual to prepare a stained smear, or if they each had a different microscope. The key to looking at acrosomes is microsope optical quality. If you don’t have phase contrast and infinity corrected objectives you can’t see acrosomes very well, or at all. The other wild card is what some people call “pitted” acrosomes. This is difficult to see and can be quite often miss-read and would result in a large technician variance because of the interpretation involved. This is why membrane viability staining is better.
A lifted or ruffled acrosome is pretty obvious with good optics, and this is why there should be very little variance from one tech to another if evaluated correctly.
Like anything, if sodium citrate or extender get old it does not work as well.
Two weeks is the maximum for keeping a solution of sodium citrate in a bottle, after that there could be a problem with crystallization. You can use fresh extender as well, as long as it contains sodium citrate (sodium citrate is a clarifying agent) and you zero out the spectrophotometer to that reagent. To keep a fresh sodium citrate solution you can make it up in small batch sizes, like 500 ml.