Equine reproduction articles - CASA Screen

How Progressively Motile Are Those Sperm?

– A comparison of videos of CASA evaluations showing poor, good and excellent progressively motile sperm. A useful teaching aid for vets and breeders.

“Motility” evaluation – description and use.

A basic evaluation  of a stallion’s semen will include a review of samples under the microscope to determine if and how the sperm are moving. This is typically achieved at a 100x or 400x magnification. Situations where such an evaluation would be performed are prior to shipment of semen cooled; prior to (or following) insemination of semen; or before, during, or after freezing of semen. There are two categories of motility – total and progressively motile sperm, which are usually expressed as a percentage of all sperm. “Total motility” indicates the overall percentage of sperm that are moving in any direction, whereas “progressive motility” indicates the percentage overall of sperm moving in more or less
a straight line. A further category of non-motile is applied to sperm that are not moving.

How straight is “straight”?

Progressively motile sperm definition diagramIt is a generally accepted that for an equine sperm to be considered progressive, it should demonstrate 75% or greater straightness. To simplify: if a sperm were to travel 1 inch when going along in an averaged path of it’s actual convoluted track, a straight line drawn between the starting and end points would be no longer than ¾ of an inch long. With CASA (Computer Assisted Semen Analysis) a further figure is included in the equation, which is the speed of travel in those two parameters, so the full equation at which the percentage of “Straightness” (STR) is arrived measures the “Velocity Average Path” (VAP) and the “Velocity Straight
Line” (VSL) to produce the following formula:

Percentage straightness = VSL/VAP x 100

Essentially therefore the actual “smoothed” path of the sperm can be no more than one-third greater than that of the straight line drawn between the start and finished points. This means that the difference between progressive or not can be subtle and therefore difficult to determine when reviewed by a human through a microscope – especially when one considers that these sperm are (hopefully) not lazily moving along, but are in fact quite rapid!

Subjective evaluation under the microscope.

The first point to make note of is that unless the raw semen sample is not particularly concentrated, it is almost impossible to reliable determine percentages of progressive/overall motility without dilution. Furthermore, some stallions have seminal plasma that is so toxic to sperm that unless the sample is diluted, the sperm will rapidly lose motility and die. Dilution with at least an equal part of extender by volume is therefore an important first step. It also will make it easier if the sample to be evaluated for motility is diluted to a concentration of 25 million sperm/ml or lower. In particular when first starting our evaluating semen, a lower sperm concentration is essential – ideally with around 20 sperm being visible on the individual microscope field of view. Note that while adding a smaller drop of semen to the microscope slide can achieve the lower number, if too small a drop is used, the lack of depth of liquid between the cover slip and slide may negatively impact sperm motility.

Objective evaluation using CASA

Computer-Assisted Semen Analysis takes the guesswork out of determining motility, be it progressive or total. The CASA unit takes a very short video of the semen (typically about one-half second) and compares the position of all the sperm in each frame during that short time period. A computer then evaluates the differences in the sperm movement and provides a variety of different results including total and progressive motility. When one uses CASA, it quickly becomes apparent that for many people, their estimates – particularly of progressive motility – under the microscope is an over-estimate. One frequently hears stallion owners commenting “my stallion has excellent motility – he’s 70% progressive at 24 hours”. In fact, it is rare that one sees 70% progressive motility immediately after collection, never mind at 24 hours! Typically 50-60% progressive motility can be considered to be good – above that is exceptional!

Motility videos using CASA

Below we have a variety of videos of CASA evaluations. These have been recorded using one of our Hamilton-Thorne CEROS I Computer Assisted Semen Analysers. Each video starts by showing the sperm moving, then you will see the button being clicked to activate the CASA evaluation, then the results will show. After that on some of the videos, you will see a screen where you can view each sperm track. Different colours of track mean different things in those images: the blue/turquoise tracks are considered “progressive”; the green tracks are merely “motile” (i.e. not progressive); while the sperm that have a little red dot on them are considered non-motile. We would encourage you to start each video and determine your own estimate – pausing it before it reaches the CASA reading on order not to cheat (we give you about a 5 second warning)! If you need a little longer to get your own personal estimate, “rewind” the video back to the beginning and start again. We think you may well be surprised at the difference between your estimate and the actual CASA readings!

(You can go “full screen” by clicking the screen icon in the bottom right corner of each video when playing. Full Screen Button)

Average Progressively Motile Sperm

The above samples are representative of an “average” collection, but although you may estimate the total motility in the right range, it is quite likely that you will over-estimate the progressive motility! Note that we consider these to be “average”, and not “poor”!
Object lesson: It’s not the percentage progressive motility that is important; it’s the number of sperm that are progressively motile!!

Good Progressively Motile Sperm

As you can see, one of these samples appears to have lower sperm concentration than the other – perhaps somewhere around half. But guess what? Both samples came from the same container! The drop of semen placed on the slide on the right is about twice as thick as the one on the left, and consequently appears more concentrated – and remember that we are talking only microns of difference!
Object lesson: You cannot “guesstimate” concentration just by looking at a sample under the microscope!!!
This sample appears to have good motility, but the sample is so concentrated that even the CASA unit cannot obtain a motility reading.
Object lesson: You must dilute samples prior to evaluation of motility. Ideally the concentration once diluted will be 25 million sperm/ml or less

Poor Progressively Motile Sperm

These samples show poor motility. Note that not only is the progression poor, but the velocity (the speed at which the sperm is moving) is also an issue on a couple of them. Review the “tracks” – shown in green or turquoise – and compare it with the tracks for one of the samples with good motility above. Progressive motility is only part of the equation – there should be good sperm velocity present as well.
Object lesson: Progressive motility and velocity are important – but remember that those figures need to be applied to total sperm numbers to get a meaningful statistic!

Numbers not percentages!

We cannot stress this enough! Many people get “hung up” on percentage progressive motility and voice major concerns when they receive semen that is say 10% progressive. Well, of course one would prefer to receive semen with sperm that are more progressive than that, but without looking at the actual numbers of sperm involved, one is not seeing the whole picture. This is very clearly explained by looking at the following sets of figures:
% Progressive Motility Number of sperm in shipment Number of progressively motile sperm in shipment
60 250 million 150 million
10 2 billion 200 million
So as you can see from the above numbers, in fact the sample with 60% progressive motility doesn’t have as many progressively motile sperm as the sample with 10% progressive motility!
Object lesson: One should not concentrate solely on the percentages!
Another issue we frequently encounter is the concept that a cooled insemination dose containing anything fewer than 500 million progressively motile sperm is the kiss of death to fertility. We discuss this in greater detail in our article entitled “The Semen Looked Bad – Or Is It?“, but in essence, presuming insemination reasonably close and prior to ovulation (within 24 hours), the insemination dose can be as low as 100 million progressively motile sperm and one will see no reduction in fertility levels.

Key points To Consider

  • Be tough on your visual evaluations of sperm motility – in particular progressive
    motility! Remember that 60%+ progression is a rare entity in real
  • Do not attempt to determine motility levels with too concentrated a sample – it
    can’t be done with repetitive accuracy!
  • You cannot “guesstimate” sperm concentration using a plain microscope slide. If
    you are going to count it under a microscope, use a hemacytometer or counting chamber
    of some sort. Alternatively obtain an estimate using a mechanical sperm counter.
  • Don’t worry too much about the percentages – worry about the numbers! Multiply
    percentage motility by sperm numbers present to obtain the actual number of
    progressively motile sperm. That is a significant statistic.
  • Pay attention to insemination dose numbers, but don’t rely on old research!
    >100 million progressively motile sperm is enough to do the job presuming close
    proximity of insemination to ovulation.
  • Remember that progressive (or any) motility is not an absolute guarantee of
    fertility! While it’s a good start, it is still possible to have good progressive
    motility, poor or even absolutely zero fertility! This is particularly the case with
    frozen semen, where the cell may be negatively affected during the freezing/thawing

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