Freezing Semen

Freezing semen from your stallion at the Goulburn Valley Equine Hospital

We offer two services for freezing semen.

Firstly, we can collect semen from your stallion at our hospital. This is the most popular service and gives us total control of all the management of the semen from collection to freezing. Your stallion can be housed in special stallion boxes or large turn out yards/paddocks with extremely safe large diameter pipe fences. In this system we most commonly freeze the stallions semen at every other day intervals. The reasons for this will be explained later.
Alternatively, we can freeze semen from your stallion after it has been collected on farm and sent to us cooled. This has been made possible by the development of some new equipment and extenders. We will only do this when the semen has been collected by another veterinarian and certified as being from the correct stallion (from papers). This system is expected to become more popular as the stallion does not have to leave your farm. The primary disadvantage is that a few stallions that would have frozen well immediately after collection have not survived as well this way.

What is involved with freezing semen?
Most importantly we try to use semen that is not old or stored in the stallions reproductive tract. We try to have the stallions sperm reserves depleted. That means that all the sperm ejaculated are the ones that have been just produced and therefore are more able to handle the stresses of centrifuging, freezing and then thawing. If the stallion is left without breeding for a week or so he may start to store semen and adversely affect the quality of our collection. In addition, the stallion produces sperm relatively constantly and spermatozoa produced take about 2 days to totally fill the extra-gonadal (outside the testicle) storage system after each ejaculation. This means that we only need to collect the stallion's semen every other day. We will not get more semen by collecting the semen more frequently, however, by waiting longer than 2 days we may waste semen.

The steps involved in the freezing process are listed below.

Collection:
We collect semen using an artificial vagina (AV) that is filled with water and held warm at about 44^oC. The stallion can mount either a mare in standing heat or a phantom (a immovable mounting device). We like the phantom as the stallion can not get hurt as is possible if a mare misbehaves. At our facility most stallions can be successfully trained  to mount a phantom within 1-2 days.

Centrifugation:
This process concentrates the semen into a little pellet. If we didn't do this then the volume necessary to give the right number of sperm would be extremely high (for most stallions). In addition we dilute and get rid of seminal plasma (the extra fluids that the stallion ejaculates with the collection) which is toxic to sperm if left mixed with it.

Adding the cryoprotectant:
After the sperm have been centrifuged they are re-suspended in the mixture of protective solutions ready for freezing. Two important components of this solution are 1) Egg yolk and 2) Glycerol. Without these the sperm would surely perish.

Freezing:
We are able to freeze semen two ways. Our most common method is to use a computer controlled program. This device can be seen in the picture of our semen freezing laboratory. The advantage of this unit is that it is extremely accurate in the cooling steps. Nothing is left to chance and the design ensures that all the sperm is subject to identical temperatures. This will minimise the commonly noted differences between different straws of the same freezing batch. The unit is Australian made and designed. If you are interested in CryoLogic's Freeze Control  Equipment visit their web site.
Another nice thing about the Freeze Control units is that the progress of the freeze can be monitored using a graphical interface that tells you how much time is left until the freeze is finished. Those of you who have frozen semen with us before will know that we try many different freezing curves (see picture to the left) and also some different cryoprotectant formulas, until we decide on what looks best for your stallion. That particular program is recorded on the computer under your stallions name.

Another  method of freezing semen is to use a vapour freeze. This is quite a bit less sophisticated. With this system the sperm is placed on a rack in straws at a specific height above a pool of liquid nitrogen. The technique is very quick and works quite well for some stallions.

After freezing stallions semen for many years the most important conclusions that we have come to is;

• that some stallions freeze very well and the method of freezing doesn't really affect the outcome,

• some stallions only freeze well under specific conditions and

• some stallions do not freeze at all no matter what tricks we might try
  

Storage:
After the freezing program is finished the semen is stored in liquid nitrogen a -196⁰C. This temperature is so cold that there is hardly any movement of atoms of the cells frozen. This means that the lifespan of these cells (spermatozoa) is greatly prolonged and stallion semen has been frozen for over 20 years and has been found to be just as viable as the day it was frozen. Bull semen has been frozen for even longer.

Your stallions semen can then be either,

• sent to you in a special shipping container
• stored by us for  later use or,
  
• stored at a  facility that is designed for long term, high volume storage

Costs:
Our charges are between $20-$40 per breeding dose (depends on how many does the stallion would normally provide). An average number of doses per ejaculate would be 10-12 for a mature large horse (Warmblood) when collected every other day. An English Riding Pony may only provide 4 doses. Freezing semen is in general less expensive than breeding with cooled transported semen. That may be difficult for some to believe, but when breeding with cooled transported semen there are some charges that people do not often factor into their calculations such as the:

• Costs of the collection (around $100).
• Costs of transport may vary from $60 (normal) to $300 (for a weekend shipment plus couriers).
• Difficulty in the semen always arriving when expected.
• Some mares have ovulated when the semen arrives and they then need to be recycled.
• Some mares do not ovulate when they are expected to and then another shipment of semen needs to be arranged.

 On occasion we have to freeze semen for international export which means that the stallion and any in-contact mares need to be isolated and tested before and during freezing for a multitude of diseases. Guidelines for different countries can be accessed through the Australian Quarantine Inspection Service (AQIS) website.

Thawing:
Mostly we thaw semen at 37⁰C in a water bath. There are variations and we try to look at them all. After the semen is thawed we look at the individual spermatozoa  to get an idea of how well they survived.

Success:
From our experience (and also supported by others) we can make some general conclusions:

• 30% of stallions in the general population will freeze well
• 40% will be probably be classified as satisfactory
  
• sperm from the remaining 30% will not survive the freezing process with a level of motility sufficient to be used for AI.

Tests Breeding:
We offer a service to our clients that allows them to test breed with their stallions frozen semen. We breed 10 mares and charge $100/ mare. We only use our own fertile research mares for this test (not the clients mares). The results from this test breed, whilst not always totally indicative of the stallions real frozen semen fertility, can be used to plan either further freezing, or to safely advertise the breeding with frozen semen from that stallion.