Storage of Equitainer Components Prior to Use
Coolant cans must be stored upside down in a deep freeze for at least 24 hours. Some small freezers are not always cold enough.
Centrifuge tubes, ballast bags, sterile cups and any equipment that comes into contact with the semen must be stored in an incubator set to 37°C at least 4 hours before to use.
The Equitainer and Isothermalizer may be left at room temperature.
Preparation for Collection
Mix the extender or thaw previously frozen extender. Add an appropriate antibiotic (if not already added).
Place the cup of extender in a water bath or incubator set to 37°C. It is critical that the extender is at 37°C before mixing it with semen.
Prepare the equipment necessary for measuring the motility and concentration of the semen. Slides and coverslips to be used for motility assessment should be warmed to 37°C.
Prepare the AV as usual for collection. Be sure to use a sterile filter, a non-spermidical lubricating gel (such as Priority Care), and avoid any types of contamination. Pay particular attention to the temperature of the AV, especially the tip and collection pouch during cold weather. Some AVs come with a separate insulated cover to keep the sample warm after collection. If this is not available, an oven mitt taped in place over the collection pouch makes a suitable substitute.
The stallion's penis should be washed gently with plain, warm water and then be allowed to air dry or patted dry with a paper towel. Washing with soaps or disinfectants upsets the natural balance of the bacteria and lets pathogens multiply.
After the semen is collected, immediately remove it from the collection pouch, place it in a sterile specimen cup and store at 37°C until ready to extend.
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.
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.
The recommended sperm concentration of the diluted semen is 40 million total sperm per ml (M/ml). By diluting to this concentration, you can then ship a volume of 50 ml to obtain the correct number of total sperm for a single inseminate dose (2 billion sperm).
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
Loading the Equitainer
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. 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.