Artificial insemination in horses: Factors influencing semen quality
Artificial insemination (AI) in horses has become a pivotal advancement in equine breeding, providing both convenience and enhanced genetic potential for breeders worldwide. With the ability to reduce the risk of injury during natural breeding, manage the reproductive cycles of mares more effectively, and optimize stallion performance, AI has revolutionized horse breeding. However, the success of AI in horses is influenced by many details, such as stallion related factors and processing.
Each stallion should be treated as an individual. Performing a test cool/freeze before shipping semen allows the stallion owner to determine the best protocol for processing that stallion’s ejaculate and what changes may need to be used to improve semen quality. As the stallion’s ejaculate may change with age and other circumstances, these test procedures should be done prior to each breeding season.
As with frozen semen, stallion specific needs should be considered in the choice of diluents/extender (for centrifugation as well es for final dilution) as well as centrifugation parameters (duration and speed).
Numerous authors have reported that it may be beneficial to store stallion semen at higher temperatures. However, these higher temperatures also favor bacterial growth. Minitube has successfully introduced a new extender, Beyond®, which is capable of storing liquid semen long-term, both at ambient temperature and chilled, while inhibiting bacterial growth reliably.
Beyond® allows long-term storage of up to 14 days at +5°C. These new options are possible due to the fully synthetic composition of this extender and a special formula that uses a different metabolic pathway in the sperm cells than other extenders.
There are numerous advantages to storing stallion spermatozoa in a synthetic medium for relatively longer periods without cryopreservation
Freezing and subsequent thawing has the greatest negative impact on sperm. While freezing reduces the number of progressively motile sperm cells, thawing results in structural damage of acrosomes, mitochondria and cell membranes. Around 20% of sperm cell heads get damaged during cryopreservation.
To protect the sperm cells, Glycerol is the most widely used cryoprotectant to freeze stallion spermatozoa. Despite its protective effect, it is also toxic to the sperm cells and negatively affects post thaw motility. The optimal concentration is still not clear, most conventional freezing extenders use concentrations between 2% and 6%.
Usually, stallions with a good initial semen quality are more likely to achieve acceptable semen after cryopreservation (>35% progressively motile sperm cells after thawing) than stallions with an already reduced quality directly after collection. Warmblood stallions seem to be suitable for semen freezing if raw semen has a concentration of 200 million sperm cells per ml, a progressive motility higher than 50% at more than 70% morphological normal sperms. Nevertheless, there is always a chance of having a so called “bad freezer” despite a good quality of the raw ejaculate. Studies show that approximately 20% of stallions are “bad freezers”.
As already mentioned, semen quality also depends on the age of the stallion, as freezing results depend on the initial quality of the raw semen, it also depends on the age of the stallion. While studies so far cannot agree if freezing stallions before six years results in acceptable semen quality or not, it is clear that quality of frozen semen decreases in stallions older than nine years.
Another factor influencing freezing results is the breed. Some breeds like Warmblood or Lipizzaners show more tolerance to cryopreservation than others, like Quarter and Icelandic Horses. The reason seems to be the genetic basis of these breeds resulting in different compositions of seminal plasma as well as sperm metabolism and membrane patterns.
Finally, and already mentioned, season has an influence on sperm quality and freezability. As it affects the composition of seminal plasma as well.
If a stallion has a low tolerance to freezing, it is advisable to retest freezability at a different season. Or, in case the stallion is at a very young age, to try it again when he is older.
As an alternative to freezing, Beyond®, a newly developed synthetic fresh semen extender, enables an unprecedented storage time, that allows the use of chilled semen in logistically challenging scenarios, potentially eliminating the need to freeze semen.
Literature:
Atroshchenko, Mikhail M., et al. "Conservation of genetic resources in horse breeding and major structural damages of sperm during semen cryopreservation in stallions." Nature conservation research. Заповедная наука 4.S2 (2019): 78-82.
Wiebke, M., et al. "Cooled storage of semen from livestock animals (part I): Boar, bull, and stallion." Animal Reproduction Science 246 (2022): 106822.
Aurich, Jörg, et al. "Efficiency of semen cryopreservation in stallions." Animals 10.6 (2020): 1033.
Bradecamp, Etta A. "How to process high-and low-quality semen for cooling and shipment." (2011): 19-23.
Stallion related factors
Effective stallion management is critical for maximizing the success of artificial insemination programs. It encompasses various aspects, from the stallion’s overall health and reproductive condition to the timing of semen collection as well as individual traits. Here’s an overview of stallion related factors influencing the success of AI.- Health and nutrition: A stallion’s physical condition significantly impacts semen quality. A balanced diet rich in essential nutrients, including proteins, vitamins, and minerals, is crucial for maintaining optimal sperm production. Regular veterinary checks and proper health management practices, such as vaccination, parasite control, and dental care, should be a priority.
- Training and handling: Stallions must be trained and handled in a way that minimizes stress and injury. They should be accustomed to semen collection procedures, which are typically performed using an artificial vagina (AV), a phantom and a teaser mare. Stress can reduce sperm quality, so proper training ensures that semen is collected efficiently without causing discomfort or anxiety to the stallion.
- Reproductive evaluation: A comprehensive reproductive evaluation of the stallion is necessary before starting an AI program. This includes assessing semen quality, scrotal circumference, testicular health, and sperm motility. A stallion’s fertility can fluctuate, so monitoring its reproductive health periodically is vital for ensuring high-quality semen is collected.
- Timing and collection: Timing plays a crucial role in maximizing the chance of fertilization. Stallions are usually collected once or twice a week during the breeding season, but collection frequency may vary based on sperm count and quality.
- Season: The composition of the ejaculate varies during the year. The volume and total number of sperm cells are lower during winter compared to breeding season. Motility is lowest during summer while concentration is lowest in springtime. Also, the composition of the seminal plasma varies throughout the year.
- Age: Stallions reach their sexual maturity by the age of six. This means that younger stallions might not have reached their peak of semen quality yet. Therefore, the best breeding age seems to be between six and eleven years. Various studies showed that semen quality decreases in older stallions.
- Seminal plasma: The majority of the raw ejaculate consists of seminal plasma, which negatively affects the storability of semen of all stallions but in some stallions is even toxic to the sperm cells.
Each stallion should be treated as an individual. Performing a test cool/freeze before shipping semen allows the stallion owner to determine the best protocol for processing that stallion’s ejaculate and what changes may need to be used to improve semen quality. As the stallion’s ejaculate may change with age and other circumstances, these test procedures should be done prior to each breeding season.
Key aspects of semen processing
Once semen is collected from the stallion, it must be processed and prepared for storage or immediate use. Semen processing is a delicate procedure that requires attention to detail to ensure that sperm viability is preserved. There are several important steps in semen processing:- Semen evaluation: After collection, semen is evaluated for motility (the ability of sperm to swim), concentration (the number of sperm per ml), and morphology (the shape and structure of the sperm). This evaluation is crucial for determining whether the semen is of sufficient quality for insemination.
- Dilution: Stallion semen is typically diluted with a specialized extender to increase its volume and protect the sperm from the damaging effects of freezing or storage. Extenders contain nutrients and buffers that help maintain sperm motility and protect against environmental stresses like temperature fluctuations.
- Centrifugation: This step is important to get rid of the seminal plasma, which negatively affects the storability of semen. It is necessary if semen is to be frozen and for some stallions also for chilling, but highly recommended for all kinds of semen preparations. For even better quality after centrifugation, this step can be done with a centrifugation cushion. Research has shown that the use of a centrifugation cushion can improve recovery rates of sperm while maintaining semen quality and minimizing the negative effects that centrifugation may have on sperm.
Liquid semen storage
Chilling semen to +5°C reduces microbial growth and sperm metabolism and consequently makes it storable for a longer period. However, between 19 and 8°C, stallion sperm cells are sensitive to cold shock. Liquid semen preservation at +5°C in the stallion so far was limited to 48h to 72h hours. At this relatively low temperature, centrifugation to remove seminal plasma and removal of oxygen rich air are proven to improve semen quality. However, the latter is often neglected in practice.As with frozen semen, stallion specific needs should be considered in the choice of diluents/extender (for centrifugation as well es for final dilution) as well as centrifugation parameters (duration and speed).
Numerous authors have reported that it may be beneficial to store stallion semen at higher temperatures. However, these higher temperatures also favor bacterial growth. Minitube has successfully introduced a new extender, Beyond®, which is capable of storing liquid semen long-term, both at ambient temperature and chilled, while inhibiting bacterial growth reliably.
Beyond® allows long-term storage of up to 14 days at +5°C. These new options are possible due to the fully synthetic composition of this extender and a special formula that uses a different metabolic pathway in the sperm cells than other extenders.
There are numerous advantages to storing stallion spermatozoa in a synthetic medium for relatively longer periods without cryopreservation
- less time-dependent synchronization of ovulation times for mares
- possibly reducing the usage of hormones
- possibly reducing semen collections per week for stallions
- no need of expensive freezing equipment or dry shippers
Cryopreservation
Freezing and subsequent thawing has the greatest negative impact on sperm. While freezing reduces the number of progressively motile sperm cells, thawing results in structural damage of acrosomes, mitochondria and cell membranes. Around 20% of sperm cell heads get damaged during cryopreservation.To protect the sperm cells, Glycerol is the most widely used cryoprotectant to freeze stallion spermatozoa. Despite its protective effect, it is also toxic to the sperm cells and negatively affects post thaw motility. The optimal concentration is still not clear, most conventional freezing extenders use concentrations between 2% and 6%.
Usually, stallions with a good initial semen quality are more likely to achieve acceptable semen after cryopreservation (>35% progressively motile sperm cells after thawing) than stallions with an already reduced quality directly after collection. Warmblood stallions seem to be suitable for semen freezing if raw semen has a concentration of 200 million sperm cells per ml, a progressive motility higher than 50% at more than 70% morphological normal sperms. Nevertheless, there is always a chance of having a so called “bad freezer” despite a good quality of the raw ejaculate. Studies show that approximately 20% of stallions are “bad freezers”.
As already mentioned, semen quality also depends on the age of the stallion, as freezing results depend on the initial quality of the raw semen, it also depends on the age of the stallion. While studies so far cannot agree if freezing stallions before six years results in acceptable semen quality or not, it is clear that quality of frozen semen decreases in stallions older than nine years.
Another factor influencing freezing results is the breed. Some breeds like Warmblood or Lipizzaners show more tolerance to cryopreservation than others, like Quarter and Icelandic Horses. The reason seems to be the genetic basis of these breeds resulting in different compositions of seminal plasma as well as sperm metabolism and membrane patterns.
Finally, and already mentioned, season has an influence on sperm quality and freezability. As it affects the composition of seminal plasma as well.
If a stallion has a low tolerance to freezing, it is advisable to retest freezability at a different season. Or, in case the stallion is at a very young age, to try it again when he is older.
As an alternative to freezing, Beyond®, a newly developed synthetic fresh semen extender, enables an unprecedented storage time, that allows the use of chilled semen in logistically challenging scenarios, potentially eliminating the need to freeze semen.
Literature:
Atroshchenko, Mikhail M., et al. "Conservation of genetic resources in horse breeding and major structural damages of sperm during semen cryopreservation in stallions." Nature conservation research. Заповедная наука 4.S2 (2019): 78-82.
Wiebke, M., et al. "Cooled storage of semen from livestock animals (part I): Boar, bull, and stallion." Animal Reproduction Science 246 (2022): 106822.
Aurich, Jörg, et al. "Efficiency of semen cryopreservation in stallions." Animals 10.6 (2020): 1033.
Bradecamp, Etta A. "How to process high-and low-quality semen for cooling and shipment." (2011): 19-23.
