How a stallion owner/manager responds to and handles mare owner concerns regarding semen quality or fertility can make or break a relationship or reputation. This article aims to give stallion owners an overview of the factors involved and provide a systematic guide to troubleshooting a resolution if possible. In Part 1 of this article, published in last month’s newsletter, we discussed the stallion’s breeding history, the importance of a breeding soundness exam (BSE), and stallion management practices as well as semen quality and evaluation. This month in Part 2, we will review common problems identified after the semen evaluation, the relevance of the mare book and their reproductive status and discuss the topic as it relates to those stallions breeding with frozen semen.
Common Problems Identified with the Semen Evaluation
After troubleshooting the poor semen quality with the stallion manager it is often determined to be related to management factors that can be readily identified and resolved. For example (in addition to those that may have already been mentioned in Part 1):
- Excessive dirt or debris in the ejaculate
- Presence of blood or urine in the ejaculate
- The use of too much lube in the AV
- Washing equipment with spermicidal substances
- Heat-shocking or cold-shocking sperm
Other causes of poor semen quality may not be so easily resolved and in these situations there are usually morphological changes also.
- Heat stress (from high ambient temperatures, testicular insult or fever) as described in our article, Heat Stress and Equine Reproduction
- Use of anabolic steroids
- Age related testicular degeneration (see our article Declining Fertility in the Aged Stallion)
A stallion may be shedding a mare pathogen, like Klebsiella pneumonia or Pseudomonas aeruginosa, in his semen. This may require treatment of the stallion before further breeding. And there will be some stallions that just have inherently poor semen quality. They may be managed by adjusting the breeding methods employed (as described in Part 1) and/or they may benefit from sperm enrichment protocols that use density gradient centrifugation to enhance semen quality, e.g. Equipure. Stallions with poor semen quality should be subjected to a more formal breeding soundness exam in order to determine if there is a physiological reason for the poor semen quality.
Problems with Cooled Semen
In most cases a cooled semen evaluation determines that there are inadequate numbers of progressively motile sperm in the breeding dose (i.e. less than the industry recommended minimum of 500 million at 24hrs). This may be caused by one or both of the following:
- Low sperm concentration in the breeding dose
- Poor or marginal sperm motility at 24hrs of storage
Quite often this is due to either inaccurate analysis of the initial semen quality by the collection facility, or inappropriate dilution. You may be surprised at how many breeding farms do not count the sperm prior to shipping, or look at the motility under the microscope. Some may perform the same dilution on every ejaculate (regardless of the initial sperm concentration) and others may not have a centrifuge in order to concentrate sperm for the breeding dose when initial sperm concentration is low. See our article Processing Equine Semen for Cooled Transport for recommendations regarding cooled semen processing.
Sperm motility may improve in a different semen extender. There are many options available on the market, most are standard formulations based upon non-fat dried skim milk (NFDSM) and sugars, but they may vary in composition and supplementary ingredients. Extenders like INRA96 use purified milk caseins instead of NFDSM. Each stallion is an individual and has their own extender preference. A cooled semen evaluation should be performed using two to four different extenders to help determine the most appropriate choice for your stallion. Stallions with marginal semen quality may benefit from an extender that provides more support than a basic NFDSM-sugar formulation. At SBS we have an extender that is more like a freezing extender in composition; it is a balanced salt solution that contains NFDSM and egg yolk. For some stallions this is the only extender they can be shipped in successfully.
When reviewing the extender selection, one must also consider the antibiotic that is added to the extender. A stallion may have sensitivity to an antibiotic and motility may be improved by switching the antibiotic in the extender. Antibiotic sensitivity must be directly confirmed by repeated analysis utilizing a split ejaculate comparison of sperm motility, comparing motility in the extender alone, to that in the extender plus antibiotic. Just changing the extender to switch the antibiotic does not confirm the antibiotic as the problem, since the improvement may simply be due to the new extender. It is recommended all extender or treatment comparisons be performed using the split-ejaculate method, i.e. the treatments are compared within the same ejaculate. Processing one ejaculate in extender 1 and the next ejaculate in extender 2, then making a decision on which gave the better motility can be biased by the semen quality of each ejaculate, as one ejaculate may have had better sperm motility to begin with.
Additional Sperm Testing
Sperm motility is just one indicator of sperm function and there may be unrelated sperm abnormalities that could prevent normal sperm function. Additional tests that could be performed on semen in a laboratory include morphology (by basic or electron microscopy), membrane or DNA integrity, mitochondrial or acrosomal function, the ability of sperm to bind to the oocyte and the presence of anti-sperm antibodies. A useful test to perform is the sperm chromatin structure assay (SCSA) to detect DNA damage; results of this test have been previously correlated with fertility. If abnormal sperm function is suspected one can follow-up with a reproductive specialty lab for more information on these sperm function assay, as many depend upon advanced microscopy or flow cytometry.
Mare Book
When it comes to securing a pregnancy the stallion is just half of the equation; his fertility is dependent upon the reproductive status and fertility of the mare. For stallions that breed a small book of mares the pregnancy data can easily be skewed in one direction or another by the population of mares or just a series of bad luck and unfortunate events. Maybe there is a high percentage of older or barren mares in his book or a higher number of rebreeds (mares who didn’t settle the first season probably have a higher risk of not conceiving the second year compared to a “new” mare).
At large breeding farms there may be a stallion on the roster who is the “go to guy”, the one stud with great semen quality and fertility who becomes the default for all those “problem” mares who didn’t get in foal to other stallions at the farm. His previously stellar fertility might take a hit when biased with the addition of those mares.
Therefore, Knowing the reproductive status (age, maiden, barren, foaling etc.) of the mares booked to your stallion is helpful. As much information you can glean about how she was managed will help you determine if she was a potential contributing factor. Were there missed cycles due to anovulatory or hemorrhagic follicles, positive cultures that required treatment or semen delays due to weather or FedEx/airline issues. If it is still quite early in the season, maybe the results are being influenced by breeding to a number of transitional mares whose cycles are not responding as anticipated.
If your stallion has not had an issue settling his mares before, he passed his breeding soundness exam and has good semen quality and longevity as determined by an independent lab, yet his end of season pregnancy rate was unusually poor, then consider the mare population, especially if he is breeding <10-15 mares a season, or simply recognize that statistically there is just not enough mares bred to make any firm conclusions. Most likely next year will return to normal but in the meantime monitor his health, management and semen quality closely as well as be sure to perform a breeding soundness exam at the start of the following season.
Stallions Breeding with Frozen Semen
If your stallion is breeding by frozen semen only and he is not getting his mares pregnant then we have primarily one variable to investigate, his frozen semen quality. However, it is helpful to have some history on the stallion - is this his first season breeding with frozen semen? If not what has been the typical fertility results with his frozen semen previously? Do you have any idea of his inherent fertility prior to breeding with frozen semen, i.e. did he have good fertility with fresh/cooled semen? Although good fertility with fresh/cooled semen doesn’t automatically mean he will have good fertility with his frozen semen, if he had sub-fertility with fresh/cooled semen then we can expect similar or lower outcomes with his frozen semen. Unless of course the stallion was inappropriately managed for cooled semen, or it may be that the stallion has reduced semen quality and fertility with fresh/cooled semen due to advancing age. Whereas, frozen semen is available that was collected and frozen when he was young, which potentially could have better fertility. Again, one should consider the number of mares, the status of mares bred and the mare management when reviewing the pregnancy data for your stallion, as discussed above, when trying to determine potential causes of reduced pregnancy rates.
If this is your stallion’s first season breeding with frozen semen then the first step should be to consider the semen quality. Select Breeders Services recommends that frozen semen used in a commercial distribution program have a minimum post-thaw motility of 30% progressive and contain a minimum of 200 million progressively motile sperm (PMS)/dose. It is a good idea to have straws sent out to an independent and objective lab (e.g. Select Breeders Service or a university lab) for an independent post-thaw semen analysis that can give you unbiased information about the semen quality. This is particularly helpful if sperm motility was estimated visually and to confirm the final sperm concentration. At Select Breeders Service we also culture the frozen semen, to ensure no mare pathogens are present. The presence of a bacterial or fungal mare pathogen in the frozen semen may reduce fertility by causing infections in mares, post-breeding endometritis or fluid retention. See our article, Quality Control is at the Core of the SBS Difference.
If the frozen semen does not meet the recommended minimums for commercial distribution this may explain the reduced fertility. However, if the frozen semen is determined to be of acceptable quality, what then? Although the recommended minimum is 200 million progressively motile sperm/dose, each stallion has a different threshold for optimal fertility, and for some stallions fertility may be improved by increasing this number to 250-300 million PMS/dose (this can be achieved by increasing the # straws/dose). However, this is dependent upon whether the reduced fertility is due to a compensable sperm defect, i.e. one that can be overcome by providing more sperm. See our blog article, It Only Takes One... Right?
Alternatively, sperm motility is just one indicator of sperm function and there may be unrelated sperm abnormalities that could prevent normal sperm function. See the section on Additional Sperm Testing above.
We often recommend test breeding to owners of stallions breeding for the first time with frozen semen, especially if the semen is to be exported to another country, since this involves additional expense that is hopefully not wasted on subsequently unsuccessful breeding attempts. In addition, a positive outcome in a new market can go a long way to securing a stallion’s reputation. Please refer to our blog article Test Breeding as an Aid to Marketing Frozen Semen. Test breeding, to confirm that pregnancies can be obtained with the frozen semen is particularly helpful if the stallion has not bred mares before, has a limited mare book or if the lack of fertility is suspected to be related to a negative bias in mare status and/or mare management. A positive pregnancy outcome, coupled with acceptable semen quality can go a long way to providing the stallion owner confidence in the product they are marketing.
If your stallion has previously good fertility with his frozen semen, has something changed? Are you using frozen semen that was collected and processed in a different year than previous seasons, was this semen frozen by a different facility? Even if post-thaw sperm motility is comparable between years or facilities, sperm motility is just one indicator of sperm function and there may be undetectable (by currently available assays) sub-lethal damage that could prevent normal sperm function.
A useful test to perform is the sperm chromatin structure assay that can detect DNA damage. We have seen instances before where post-thaw sperm motility is comparable between two batches of frozen semen (from different collection facilities or years), whereas the amount of DNA damage is significantly different; the semen with increased levels of DNA damage having lower fertility than the semen that was within a normal range. Perhaps there was a testicular insult that occurred and influenced the DNA integrity of the semen frozen subsequent to this event.
If you are shipping the same semen (i.e. from the same freezing session) this season compared to years past, then has the semen quality been compromised during storage? Liquid nitrogen constantly evaporates from storage tanks. The nitrogen levels must be checked regularly and the tanks filled as appropriate. If storage tanks were not maintained, semen may have been compromised. Did you move your semen to a new storage location? In which case the potential may exist for the semen to have been compromised during transfer and shipment. These questions can be answered by confirming the semen quality currently in storage by performing a post-thaw analysis, either in house or by sending straws out to an independent lab (like Select Breeders Service), to ensure that the post-thaw motility matches the original result at the time of freezing. If it does not, i.e. if the semen quality has declined or is now all dead, then herein lies your problem.
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