Not All Frozen Semen is Created Equally

June 20, 2012

Following up on our Blog article of last month Should the US Adopt Stricter Controls on Cooled and Frozen Semen Production Facilities, we thought it might be interesting to present some data to demonstrate the variability in the quality of frozen semen being imported into the US. For the purposes of this blog article we combined the data from client samples submitted for post-thaw analysis in the last 1-3 yrs. A post-thaw analysis is one of the services we offer to our clients importing frozen semen; we evaluate post-thaw motility by computer assisted sperm analysis (CASA), sperm concentration using the Nucleocounter and we perform a bacterial culture of the semen to check for mare pathogens.

The results of the post-thaw analysis enable the semen owner to adjust the number of straws per dose to ensure an appropriate breeding dose containing at least the industry recommended minimum of 200 million progressively motile sperm per dose, thus optimizing their chance of conception. In addition, to be distributed by SBS, imported frozen semen must meet our recommended minimum of 30% post-thaw progressive motility.

The data represents 21 Warmblood stallions whose semen was imported into the US from Europe. In most cases post-thaw analysis was performed on >1 ejaculate per stallion, with the average number of ejaculates analyzed per stallion being 4. Although there was variability between ejaculates in post-thaw quality, the mean value for motility and concentration was used to represent each stallion in the data set.

Method of Analysis

The protocol for post-thaw analysis was standardized across all samples, this is very important because variations in protocol can significantly impact the results. This is why it can be difficult to compare post-thaw results from two different labs, since it is likely that the two labs used different extenders, incubation times and methods, see our FAQ: Can’t Post-Thaw Motility Vary with the Laboratory Performing the Analysis? Although potentially difficult to implement, a standardized protocol for post-thaw analysis would be of great benefit for the equine frozen semen industry.

Immediately after thawing (30sec, 37°C) the contents of each straw were emptied into a pre-warmed sterile test tube from which bacterial cultures were taken. After bacterial culture, the semen was mixed and an aliquot removed for sperm concentration and motility analysis. In most cases the contents of two straws from each ejaculate were pooled for analysis.

Thawed semen was diluted with a pre-warmed skim milk-glucose extender and incubated at 37°C for 30 min prior to motility analysis on a 37°C heated stage by CASA. 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.

Sperm Concentration
Sperm concentration was determined by the ChemoMetec 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.

Bacterial Culture
TSA II 5% blood agar plates were streaked using a 10µL inoculation loop. A swab of the semen was then taken for subsequent submission to New Bolton Center, University of Pennsylvania for secondary culture and identification. The plates were incubated at 37°C and checked for growth at 24 and 48hr.


Post-Thaw Motility
The average post-thaw progressive motility across all stallions analyzed was 31%, with a range from 11 to 53%. Basically half (52%; 11/21) of the stallions had a post-thaw motility equal to/or greater than the industry recommended minimum of 30% progressive.

The average concentration across all stallions analyzed was 240 million/mL, with a range from 60 to 540 million/mL.

Recommended Number of Straws per Dose
The mean number of progressively motile sperm per 0.5mL straw was 37, with a range of 9 to 111. The average number of straws required to provide the industry recommended minimum of 200 million progressively motile sperm per dose was 8. This is of particular significance since in most cases the semen from these stallions is typically sold at 2-6 straws per dose. Only 5 of the 21 stallions would fulfill the recommendation of 200 million progressively motile sperm/dose with 4 straws per dose.

Of the 21 stallions, 18 (86%) had bacterial contamination of at least one ejaculate. When looking at the incidence across all post-thaw samples, 70% (57/82) had bacterial contamination and almost half (48%) of these samples had a heavy level of bacterial growth. Therefore if a stallion had bacterial contamination it was generally evident in either all or the majority of samples representing this stallion in the data set. Only three stallions (14%) had no bacterial growth evident in the frozen semen samples that were submitted for QC. Bacteria known to be mare pathogens were isolated from the semen of 2 stallions (11%). A high incidence (84%) of bacterial contamination in frozen semen samples in Europe was also reported by Corona and Cherchi (2009).

Just for comparison the incidence of bacterial contamination in samples frozen by SBS affiliate labs is <10%, and this is usually observed when we are freezing semen on the mobile lab and don’t have immediate control over the collection environment. The incidence of heavy bacterial growth in SBS frozen semen is <1% and we have not isolated a mare pathogen in the last 3yrs.

Select Breeders is one of the few laboratories in the industry to perform a bacterial culture on every ejaculate we freeze to confirm the absence of mare pathogens. We also do not recommend distribution of semen with significant bacterial contamination.

There are two potential sources of bacterial contamination in semen, from the stallion, i.e. originating with the semen at the time of ejaculation or contamination from the environment during semen collection and processing, i.e. contributed by poor hygiene protocols and/or dirty equipment and supplies. Semen extenders contain antibiotics which should eliminate any bacteria originating from the semen; however, if the bacterial load is particularly large, it may overwhelm the level of antibiotics in the extender. The high incidence of bacterial contamination in these imported samples reflects an overall lack of quality control during semen collection and processing.

As with the manufacturing of any commercial product, institution of quality control protocols improves the overall quality of the product and most importantly provides consistency in production. For this reason we instituted a Quality Control and Assurance Program throughout our SBS Affiliate Laboratory Network.

Certain bacteria, e.g. Pseudomonas aeruginosa, Klebsiella pneumoniae, E. Coli, Taylorella equigenitalis and β-hemolytic streptococci, are known to cause infection in the mare and may influence fertility. The presence of contaminating, i.e. non-pathogenic, bacteria in semen is not considered to impact fertility in the mare; however caution may be merited in those mares susceptible to post-breeding uterine infection or inflammation. In these cases, post insemination intra-uterine antibiotic therapy may be indicated. Although there has been limited research with stallions, reports in other species (ram, bull, boar) demonstrate a decline in semen quality and viability with bacterial contamination. Research by Ortega-Ferrusola et al (2009) suggests bacteria in the equine ejaculate may be responsible for some of the sub-lethal damage that occurs to sperm during freezing. The influence of bacterial contamination on semen quality and fertility requires further investigation in the horse.

In Conclusion

The quality of frozen semen being imported into the US is highly variable and we report that semen from half of the stallions in this study did not meet the industry recommended minimum of 30% post-thaw progressive progressive motility. In the Warmblood community, most of this semen is sold by the dose with no guarantee; therefore the mare owner takes on all the risk of breeding her mare, often at considerable expense. When importing several doses or investing a significant amount of money on the purchase of frozen semen we recommend having an independent post-thaw evaluation performed on the semen, so as to confirm the absence of mare pathogens and to determine the appropriate number of straws per dose to use with each breeding. This could save you significant expense, emotional stress and heart-ache in the long term.

You may also be interested in the following FAQs and articles:
What fertility should I expect with frozen semen?
Does Post-thaw Motility Correlate to Fertility?
Can you get a mare in foal with semen <30% progressive?
It Only Takes One…Right?

A. Corona and R. Cherchi. Microbial quality of equine frozen semen. Anim Reprod Sci 2009; 115(1-4):103-109.
C Ortega-Ferrusola, L Gonzalez-Fernandez, A Muriel, B Macıas-Garcıa, H Rodrıguez-Martınez, JA Tapia, JM Alonso and FJ Pena. Does the Microbial Flora in the Ejaculate Affect the Freezeability of Stallion Sperm? Reprod Dom Anim 2009; 44, 518–522.