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After it has been zeroed, a raw sample of semen,180µL, is added to the cuvette. Then, the cuvette is placed in the machine and “zeroed” prior to including the semen. It should only be handled on the ribbed sides so fingerprints do not interfere with the light passing through the sample which could result in an inaccurate count. The cuvette is designed with two smooth sides and two ribbed sides.
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The following protocol is used for the ARS Densimeters: 3.42mL of 10% buffered formalin is aliquoted into the cuvette. This is because the liquid in the tube can dry out overnight and therefore, the amount of formalin pumped into the cuvette could be less than required to correctly run a sample. It is recommended to discard and do not use the first pump of the day from the formalin dispenser. It is also a good idea to have the pipettor cleaned and calibrated on a regular basis to ensure you are obtaining the most accurate data possible. The plastic cuvettes used in these systems are disposable and manufactured to be used for a single analysis. This allows the system to accurately determine the count of the cells.
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The formalin solution kills the sperm and renders them immobile. The dispenser aliquots the 10% buffered formalin into the cuvette to perform the sample analysis. Whereas the larger model, the ARS 591B Equine Densimeter is best used in a stationary environment but can also be mobile with the use of a 12-volt car adapter.īoth of these systems are self-calibrating but require additional supplies to determine the concentration such as a pipette, disposable pipettor tips, cuvettes and a formalin solution with a dispenser.
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The ARS 590B Equine Densimeter (photo below) is designed to be portable and has a built-in battery charger. Densimeter (aka The Blue Box)Īnimal Reproduction Systems (ARS) currently has two options of the Densimeter, aka The Blue Box, available. Though each of these machines is validated by the manufacturer, it is always a good idea to occasionally perform a hemocytometer count (discussed below) on a sample to test the calibration of the machine. For example, if a greater amount of light is transmitted then the concentration of sperm will be lower but if the amount of light is low then the concentration of sperm will be higher. The amount of light transmitted through the sample is inversely correlated to the concentration. When a sample is analyzed, an absorbance value is obtained by a signal from the photodetector which is given as a number for the concentration of sperm in the sample. These instruments measure the degree to which the light changes direction and provides us with the number of sperm per milliliter of the ejaculate being analyzed. Refraction of the light occurs when it enters the sample (diluted or raw), bounces off of the sperm cells and the light is made to change direction.
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Each system passes a beam of light through a sample of raw semen. Several instruments within the equine breeding industry utilize spectrophotometry to determine the number of sperm per milliliter. For the purpose of this article we are going to break them down into two types of methods, those which require raw semen for analysis vs those which can perform an analysis using either raw or diluted semen. However, the cost and skill level required for each method widely vary. If used properly, each method will result in the same information.
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The two most commonly used on breeding farms are spectrophotometric based machines and manual counting using the hemocytometer. There are several different methods that can be used to determine the concentration of sperm in a stallion’s ejaculate. This information is valuable so that any concerns can be addressed as soon as they arise. However, if the number of sperm in the ejaculate varies drastically, from collection to collection or a steady decrease in his total number of sperm over time, one may begin to ask what is causing the change. Slight variations are likely nothing to be concerned about. Also, keeping a record of each number can help stallion managers recognize if there are any changes in the reproductive health of the stallion. These two numbers (volume and concentration) are used to calculate insemination doses. The concentration of his semen, along with the volume of his ejaculate, is used to determine the total number of sperm he produces in a given collection. Knowing and keeping track of the concentration, or the number of sperm per milliliter, of each ejaculate for a stallion is very important for a variety of reasons.