What Exactly is a Dose of Frozen Semen?

August 21, 2013

Is a dose a breeding? Is a straw a dose? How many straws in a dose? How many sperm in a dose? These are often questions we hear from our clients when purchasing or using frozen semen. There is so much variability in the format of doses sold throughout the world that it can be confusing at times. To ensure you have the best opportunity of success when breeding your mare it is important to understand all the elements that determine an adequate dose of frozen semen. Here we review frequently asked questions that relate to what constitutes a dose of frozen semen.

What is a Dose of Frozen Semen?

A dose of frozen semen is an insemination unit of “x” number of straws. The straws contain a specified concentration and volume of semen such that when thawed and combined, the “dose” of semen constitutes one breeding insemination for a mare. The “dose” of semen should include enough viable sperm to provide a reasonable chance of obtaining a pregnancy in an appropriately managed, reproductively sound mare.

How Many Sperm Should be in a Dose?

There are currently no universally accepted standards for the production of quality semen by AI centers in North America, see our blog article Should the US Adopt Stricter Controls On Cooled and Frozen Semen Production Facilities. However, it is generally accepted that a dose of frozen semen should contain a minimum of 200 million progressively motile sperm. A European based organization called the World Breeding Federation of Sport Horses (WBFSH) several years ago published recommended guidelines for semen production and quality standards for its member breeding stations; the details of these guidelines can be found here.

At Select Breeders Service we also recommend that frozen semen have a minimum post-thaw progressive motility of 30% to be used in a commercial distribution program.

However, one must remember that although motility is an indicator of relative cell health, fertilization is a complex process that requires numerous functional attributes of both sperm and egg. Therefore the true fertility of any frozen semen can only be determined by properly timed insemination of reproductively healthy mares.

See also FAQs:
Does post-thaw motility correlate to fertility?
Can you get a mare in foal with semen < 30% progressive?
In our blog next month we review “What is Progressive Motility?”

How do I Calculate the Number of Progressively Motile Sperm in a Dose?

To calculate the number of progressively motile sperm in a dose you need to know the following information:

  • The total number of sperm in the dose
  • The post-thaw progressive motility for that production lot (i.e. collection date) of frozen semen

The total number of sperm in the breeding dose can be calculated by multiplying the sperm concentration by the volume of semen in the breeding dose. Let’s take the following as an example:

A breeding dose of 8 x 0.5mL straws consists of a total of 800 million sperm and a post-thaw progressive motility of 35%.
The dose volume is 4mL (8 x 0.5mL straws)
The sperm concentration is 200 million/mL. We know this because the total sperm in the dose is 800 million, so 800 divided by the volume of 4mL = 200 million sperm/mL

The progressively motile sperm (PMS) per dose is calculated by multiplying the total number of sperm by the progressive motility.

In this example we have 800 million total cells, but only 35% are moving in a progressive manner. Therefore 35% of 800 is 280 million progressively motile sperm per dose (800 x 35/100 or 0.35).

All too often you may not receive information on the sperm concentration or on the number of PMS in the dose, and sometimes the motility quoted is the average motility across several lots of semen, not for that collection date in particular. It is therefore important to ask the appropriate questions when purchasing frozen semen, so you can determine if the dose you are about to purchase contains an acceptable amount of sperm. See our blog Questions Every Mare Owner Should Ask Before Breeding with Frozen Semen?

A breeding dose prepared by an SBS Affiliated Laboratory is standardized to comprise of eight 0.5mL straws. Semen is frozen at a concentration of 200-250 million/mL, resulting in a total of 800 million to 1 billion sperm in a final dose volume of 4mL. Given our minimum recommended post-thaw motility for commercial distribution of 30% progressive, each breeding dose therefore also exceeds the industry recommended minimum of 200 million progressively motile sperm per dose after thawing.

Can’t the Analysis of Semen Quality Vary with the Laboratory Processing the Semen?

Yes, this is a very important factor to consider. Post-thaw motility may vary depending upon the method of analysis, the time and temperature of incubation and the extender used for dilution. Consequently a post-thaw analysis performed at one laboratory may provide completely different results from a post-thaw analysis from another laboratory. See the FAQ: Can’t post-thaw motility vary with the laboratory performing the analysis?

Sperm concentration can also be determined by many different methods of variable accuracy. Also the point at which the cell concentration is determined can influence the true concentration of cells in the breeding dose. Some labs may only perform a count on the raw semen; they assume 70-80% recovery of sperm after centrifugation and calculate the final resuspension volume of freezing extender based upon this “assumed” recovery. If there was an error in the initial sperm count or recovery after centrifugation was not as anticipated, the number of sperm in the dose will be compromised. A more appropriate approach is to count the sperm cells after centrifugation so you know what the true recovery was; you can then accurately dilute the cells in freezing extender to the desired final concentration.

At SBS we determine concentration after centrifugation, by either hemocytometer or nucleocounter, and then dilute the sperm to 200-250 million/mL in freezing extender before filing the straws. We perform a post-thaw evaluation on 2 pooled straws from every ejaculate frozen. After thawing, the semen is cultured to confirm the absence of bacterial growth and sperm motility is evaluated after the thawed semen is diluted in extender and incubated at 37°C for 30 minutes.

What Straw Sizes are Used for Frozen Semen?

Straws are the typical packaging unit for frozen semen, it is also possible although unusual, to receive frozen semen in pellets and packets. The most common straw size in use today is the 0.5mL straw, although you may occasionally come across 0.25mL straws. In the past semen was frozen in 4mL or 5mL straws, also known as macro-tubes. In the last 10 years, SBS has opted to use 0.5mL straws because a more consistent freezing rate within the straw can be achieved, as the straw diameter is considerably less than that of the macro straw. When the freezing rate of all of the sperm within the straw is consistent, then the overall quality of the semen is maximized once they are thawed and ready for insemination. Another benefit achieved from using 0.5 mL straws, is that they can be used with mechanical filling and sealing machines which ensure consistent filling of straws, a secure seal and a reduced processing time. Furthermore, 0.5mL straws lend themselves to several different methods of convenient storage in either large or small goblets or on canes. Plus post-thaw evaluation of 1 or 2 (0.5mL) straws from each batch represents only 1/8 to 1/4 of a breeding dose, whereas for macrotubes one straw often constitutes one dose, so when performing a post-thaw evaluation on one straw, one dose of semen is lost.

It is important to pay particular attention to the volume of the air bubble within the straw, it should not be excessive, because the more space taken up by air, the less space there is for semen. Since an adequate dose of semen is dictated by the sperm quality, concentration and volume, insufficient dose volumes may compromise your breeding dose. For this reason we prefer clear or translucent colored straws that enable one to visualize the volume of semen within the straw. If straws are observed to float in liquid nitrogen it is likely that they have large air bubbles and are partially filled. You should notify the semen owner/agent if you receive straws that float in LN2 or have large air bubbles.

How Many Straws are in a Dose?

The number of straws in a breeding dose can be highly variable within the industry; it may range from 1 to 10+ straws per dose. In the case of 5mL macro-tubes one straw may constitute one dose, but this may not always be the case. It is important that your breeding contract and/or thawing instructions specify exactly how many straws constitute a dose. Please note that the number of straws per dose may not always be the same for a particular stallion. Because the industry recommended minimum for a breeding dose is 200 million progressively motile sperm, semen owner/agents often adjust the number of 0.5mL straws per dose depending upon the post-thaw progressive motility for that lot (i.e. collection date) of frozen semen. Therefore a stallion may have doses on the market that are 2, 3 or 4 straws/dose.

Another factor to consider with respect to the number of straws per dose is the cell concentration. For a stallion with a post-thaw progressive motility of 30% frozen at a concentration of 200 million sperm/mL, the progressively motile sperm (PMS) in an 8 x 0.5mL straw dose is 200 million. However, if the stallion is frozen by another production laboratory, and they freeze him at 400 million sperm/mL and have the same post-thaw progressive motility of 30%, then only 4 x 0.5mL straws are needed to fulfill the requirement of 200 PMS/dose.

Another common practice with semen owner/agents is to cut the number of straws per dose if good fertility results were obtained. e.g. if a stallion started his first frozen semen breeding season with a 4 straw dose and at the end of the year had acceptable pregnancy rates, the following year the semen owner/agent may reduce the number of straws per dose to 3, if acceptable pregnancy rates are observed again, they may reduce to 2 straws the following year. This does not mean that for all stallions you could split the dose in half and get acceptable fertility results, because it depends upon the total number of PMS in the original dose and also the inherent fertility of that particular stallion, see the blog article “It Only Takes One….Right?”. We will tackle the question of splitting doses in a future blog article, in the meantime you may find the comments on this blog article helpful, “The Pros and Cons of 1 or 2 Dose Insemination Protocols”.

If the straws per dose aren’t standardized for a stallion and you have not been provided with sufficient information about the dose to determine this for yourself, you have no way of knowing if you are receiving an acceptable breeding dose or not. To address this issue we have standardized all doses prepared by SBS Affiliated Laboratories to be comprised of 8 x 0.5mL straws, regardless of the post-thaw motility. This way when you receive 8 straws from an SBS Affiliate lab you know you have a full dose. SBS semen is frozen at a concentration of 200-250 million/mL, resulting in a total of 800 million to 1 billion sperm in a final dose volume of 4mL. Given our minimum recommended post-thaw motility for commercial distribution of 30% progressive, each breeding dose therefore also exceeds the industry recommended minimum of 200 million progressively motile sperm per dose after thawing.