Processing the Ejaculate
This section provides a summary of the steps required to process and package the semen for shipment in the Equitainer using the New Tube Style Isothermalizer.
(Instructions on packaging semen for the old cup style Isothermalizer are also available here.)
Immediately after the Collection
- Place the gel free ejaculate in the pre-warmed sterile polypropylene cup.
- Using the graduated marks on the side of the cup, note the ejaculate volume and record it in the collection record.
- Place the cup containing the semen into the water bath or the incubator.
- For best results, the semen should be extended, packaged and loaded into the Equitainer within 15 minutes of collection.
Determining the Concentration of the Raw Ejaculate
In order to extend the semen properly, the concentration of the raw ejaculate, in millions/milliliter (M/ml) must first be determined. There are several methods that may be used to do this including:
- Spectrophotometer: Prior to collection, calibrate the system with a cuvette containing only formalin. Add the required volumes of semen and formalin to the sample cuvette. Read the percent transmittance/absorbance and convert to M/ml (a conversion chart created specifically for your instrument is required).
- Manual Microscope Count: Using a fixed depth chamber, such as a hemacytometer, follow the manufacturers instructions on how to perform the count and calculate the sperm concentration.
Extending the Semen
For the best results, the semen should be extended within 10 minutes of collection. This means that transferring the semen from the collection pouch to the specimen cup and determining concentration must be performed quickly. This is also why it is vital that the extender is mixed ahead of time and warmed to 37°C.
Extend either the entire ejaculate or only a portion if a limited number insemination doses are needed (this will save extender). However, you should always extend enough semen so a sample can be maintained on site for a 24 hour and 48 hour motility check.
Packaging the Semen for the Tube Style Isothermalizer
Once the semen has been extended, it may stay at room temperature while packaging the insemination doses. Fill both Centrifuge Tubes to the TOP with either extended semen or water (both Isothermalizer wells must be filled to achieve the proper cooling rate).
Loading the Tube Style Isothermalizer
- Place a filled Centrifuge Tube (containing either extended semen or water) into each Isothermalizer well.
- Both Isothermalizer wells must be filled to achieve the proper cooling rate
- If using the Equitainer II, place the red cover on top of the Isothermalizer
Loading the Equitainer
- If using the Equitainer I, place two (2) freezer cans, right-side up, into the ziplock bag.
- Put the loaded Isothermalizer on top of the freezer can(s).
- Slide the ziplock bag into the center well of the Equitainer.
- Close the lid and secure the latch.
Assessing Sperm Motility
Take an aliquot of the extended semen, place it on a microscope slide, add a coverslip, and view the specimen under the microscope. It is important that the slide be maintained at 37°C during analysis for accurate motility analysis. (see the MiniTherm Stage Warmer). Estimate the percentage of motile sperm and the percentage of progressively motile sperm (moving forward, not in circles). Record the percent motile and progressively motile in the collection record. Once you get proficient at assessing motility, this may be performed before you package the semen.
A sample of each collection should be saved for evaluation at 24 and 48 hours post collection. By doing this you will be aware if there are any problems with the shipped semen. You should pack the sample in an extra Equitainer. If you do not have an extra Equitainer, you can mimic the cooling procedure of the Equitainer using a "slow-cool" container:
- To make a slow cool container, you will need a lidded, plastic container about 6 inches in diameter and an extra polypropylene cup.
- First, fill the plastic container about 3/4 full with water warmed to 37°C.
- Next, take a bag of extended semen, wrap it in the ballast bags (if available) and place into the polypropylene cup and close the lid.
- Place the polypropylene cup into the larger container of water and secure the lid.
- Place the entire container into a refrigerator set to 5°C.