Analysis of Frozen-Thawed Equine Semen

April 13, 2017

Analysis of Frozen Thawed Semen - Microscope Analysis.jpgOnce equine semen is frozen it is recommended that a post-thaw analysis be performed in order to ascertain the semen quality. There is variability in how well sperm from different stallions respond to the cellular stress of freezing and thawing. It is important to determine how well the sperm from a particular stallion withstood the stress of cryopreservation in order to make informed decisions regarding the management of their frozen semen inventory and breedings. Also, when we freeze a stallion for the first time we do a test freeze comparing several different protocols. We then use the post-thaw motility to select the freezing protocol for subsequent collections which gives the best post-thaw result. There are industry recommended minimums for commercial distribution of equine frozen semen and therefore it is pertinent to know if your stallion’s frozen semen would qualify. Consequently, the analysis of frozen-thawed semen is a valuable tool for the semen freezing lab, but also for the veterinarian receiving the semen. Your vet may base the breeding management of your mare upon the post-thaw quality of the semen. Also, many vets take a look at the motility of the frozen semen at the time of insemination, for future reference if there are concerns should the mare not check in foal.

What is the Commercially Accepted Minimum?

SBS recommends that frozen semen used for commercial distribution contain > 200 million progressively motile sperm (PMS) after thawing with a minimum of 30% progressive motility. Our article, What is Progressive Motility?, provides additional information about the importance of knowing the progressive motility.

Does Post-thaw Motility Correlate to Fertility?

Fertility is inherent to each stallion and motility is just one attribute of sperm function. Fertilization is a complex process that requires numerous functional attributes of both sperm and egg. Therefore, the true fertility of any semen - fresh, cooled or frozen - can only be determined by properly timed insemination of reproductively healthy mares. Nonetheless, sperm motility is still one of the best indicators we have for predicting success in a breeding program. If the post-thaw motility is quite low (10-20%) then fertility more than likely would be low as well. However, fertility does not necessarily improve with increasing motility. A stallion with a post-thaw progressive motility of 35% may be equally as fertile as one with 55% progressively motile sperm.

Post-thaw motility is a good indicator of how well the sperm have withstood the stresses of being frozen and thawed and one should be cautious about using frozen semen with less than acceptable post-thaw motility. A significant reduction in progressive motility after thawing is indicative of sperm damage and there may even be sub-lethal damage to sperm that remain motile after thawing that would reduce fertility. The article, It Only Takes One…Right?, is a good resource for information about effects of the freezing process as well as why motility does not equal fertility.

How does SBS Perform a Post-thaw Analysis?

CASA We perform a thorough post-thaw evaluation on two pooled straws from EVERY ejaculate frozen by SBS. Two straws are combined to increase the probability that the results adequately represent the quality of all the straws in the ejaculate (as opposed to just a single straw). After thawing, the semen is cultured to confirm the absence of bacterial growth and sperm motility is evaluated using a Hamilton-Thorne CEROS model automated motility analyzer. We perform this motility evaluation after the thawed semen is diluted in extender and incubated at 37°C for 30 minutes. Computer assisted sperm analysis (CASA) allows objective evaluation of sperm motility and provides information on total and progressive motility as well as velocity parameters.

The volume of semen and the sperm concentration are also important factors in the formulation of a breeding dose. At SBS, we perform sperm counts using a NucleoCounter® SP-100™. This is a relatively new technology that counts cells based upon staining of their nuclear DNA with a fluorescent probe. It has advanced accuracy and high repeatability and allows sperm counts in semen extender. An alternative method for counting sperm would be to use a hemocytometer, a chambered slide commonly used for counting other cell types e.g. blood cells.

Is Post-Thaw Motility Information Equal Between All Facilities?

Reported post-thaw motility can vary significantly between the laboratory or technician performing the analysis. Therefore, when reviewing post-thaw motility results, it is important to understand and take into consideration how the post-thaw analysis was performed and by what method the sperm motility was analyzed. It is recommended you inquire with the facility which froze the semen to determine their process of analysis because as described in the article, Not All Frozen Semen is Created Equally

Post-thaw motility may vary depending upon:

  • How the straws are thawed
  • The method of analysis
  • The time and temperature of incubation
  • The extender used for dilution

In addition to the above variables reviewed in more detail below, the clarity of the sample and concentration of the sperm can affect the post-thaw analysis as well. Sperm concentration can influence the determination of motility, particularly if the semen sample is undiluted prior to analysis, since sperm can be frozen at concentrations from 100-400 million/mL. Sperm at very high cell densities are difficult to evaluate, and the same can be true when samples are over diluted, so it is important to ideally standardize the sperm concentration for motility analysis as reviewed below. Semen freezing extenders contain egg yolk, and it is preferable to utilize extenders that have been “clarified”, which means they have been processed by high speed centrifugation to remove any large egg yolk particles. Unclarified extenders can confound semen analysis, particularly on the CASA system, because the egg particles can be a similar size to sperm heads and the machine can experience difficulty differentiating them. In this case a visual motility estimate is required, but even still it can be hard to ascertain an accurate read of motility when analyzing sperm in unclarified extender also.      Analysis of Frozen Thawed Semen_Lab Set Up 1 Analysis of Frozen Thawed Semen_Lab Set Up 2

How Straws are Thawed

Straws of frozen semen usually come with instructions on the appropriate method for thawing. Thawing instructions can vary depending upon the size of straw and the method primarily used by the semen freezing lab. In order to confirm semen quality, when comparing to the original result, the technician would want to ensure the method of thawing is the same as recommended by the semen freezing laboratory, in order to eliminate this as a potential source of variability in the results. Instructions for thawing 0.5ml and 5ml straws prepared by SBS can be found here: 0.5 ml Thawing Instructions and 5 ml Thawing Instructions.

Method of Analysis

At SBS we test post-thaw motility objectively by computer assisted sperm analysis (CASA). Clinicians without a CASA system determine motility visually by looking into the microscope and estimating the percent motility. This method is subject to observer bias and shows poor consistency and accuracy between technicians. However, there are still opportunities for the user to introduce bias into a CASA analysis which include field selection and definition of the CASA settings. In our CASA system, a progressively motile sperm is defined as one that has an average path velocity (VAP) of > 50 microns/second AND is moving with a straightness value (STR) of > 75% (see photo above right). Since the thresholds for progressive motility can be set by the user it is important to understand that the clinician can increase the percentage of progressively motile sperm by lowering the thresholds for inclusion. It also means that two measures of motility by CASA cannot be definitively compared unless the machines operate with the same settings. 

Time and Temperature of Incubation

At SBS we test post-thaw motility after 30 mins incubation at 37°C. This is to provide a stress test to the frozen semen that may reveal latent damage to the sperm caused by the freezing process that may not be evident immediately after thawing. Some clinicians may only look at sperm motility immediately after thawing. This value may be higher than a sample evaluated 30 minutes later and does not give any indication of the longevity of the semen.

Temperature is equally important; sperm motility is best evaluated at 37°C, simulating the temperature within the mare’s reproductive tract. At SBS we pre-warm the tubes, extender and microscope slides used in the post-thaw analysis and evaluate the motility upon a heated microscope stage. Sperm analyzed at room temperature generally exhibit reduced velocity compared to a sample evaluated at 37°C.

The Extender Used for Dilution

In order to perform an accurate CASA analysis, the frozen semen must be diluted after thawing to an appropriate cell concentration. For semen analysis on the CASA we aim for a final concentration of 20-40 million sperm/ml. Since stallions are known to have extender preferences and sperm motility may vary between extenders, the extender selected for dilution of the thawed semen may influence the post-thaw motility. At SBS we dilute the frozen semen into the same extender that was used for centrifugation during the initial semen processing for freezing. We don’t recommend diluting the semen when using it to inseminate a mare and we explain why in the article Should Frozen-Thawed Semen Be Diluted Prior to Insemination?

Sperm Analysis at the Time of Insemination

Because of all the potential sources of variability described above, it is unlikely that your veterinarian is going to visualize the same post-thaw motility at the time of insemination as was reported by the semen freezing lab, because they are both evaluating the semen under quite different conditions. Nonetheless, a motility check at the time of insemination can provide valuable information even if the semen quality is defined simply as Poor, Fair, Good and Excellent;  Analysis of Frozen Thawed Semen_Straws in Water Bath providing the technician is consistent in their categorization. However, most vets feel comfortable assigning their own values for total and progressive motility based upon a visual assessment of sperm motion.

As a minimum, we would recommend using a microscope with a heated stage, and pre-warmed slides and coverslips. Ideally, the semen should be diluted with one part semen to four parts of an appropriate prewarmed extender (or to 20-40 M/mL if the sperm concentration is known and accurate methods of aliquoting semen and extender are available). This will provide a more accurate reflection of the sperm motility than a room temperature evaluation of an undiluted semen sample.

Unless the semen is incubated to test the longevity of the sperm motility, then it is important to note that the motility immediately upon thawing may not be retained. To determine sperm longevity, it is preferable to dilute the semen into a cooled semen extender, thereby reducing the concentration of sperm cells and glycerol in the freezing media. This will optimize the sperm motility during incubation, since sperm motility is sensitive to the presence of glycerol and high cell concentrations over time. However, it’s important to note that this “longevity test” is not necessarily indicating the longevity of the semen in the mare’s reproductive tract and is just a way to expose latent damage that may influence fertility.

In conclusion, post-thaw analysis and determination of post-thaw motility can vary significantly between technicians or semen freezing labs and the methods employed. However, it is a useful tool that can provide the veterinarian with valuable information that may influence the management protocol of the mare and influence the outcome of a frozen semen breeding.