Salvaging a horse’s epididymal sperm: “It’s not too late”

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To begin epididymal sperm extraction, the stallion has one or both testes removed using standard castration techniques.
To begin epididymal sperm extraction, the stallion has one or both testes removed using standard castration techniques.

Post-castration epididymal sperm extraction is a pioneering method of semen collection that permits semen extraction where other options don’t allow. Referring to the removal of semen from the epididymis of the testes, the procedure can be used following routine castration, upon death of the animal or where the testes have suffered a severe trauma or testicular torsion; the latter unplanned terminal/traumatic situations the pinnacle of its use.

Sudden death and semen extraction

The loss of a breeding stallion is undoubtedly a distressing and emotional time for stallion owners, but it can also have devastating financial implications. Customarily the breeding potential of a stallion is lost with death, nevertheless advances in cryopreservation techniques have meant that stallions can now have genetic material collected and stored indefinitely. In ordinary circumstances, valuable breeding stallions have semen collected and cryopreserved during periods of competing rest, or when fresh breeding programmes have concluded. But if this is not possible, and when stallion death is sudden and unanticipated, semen can now be salvaged and cryopreserved retrospectively via epididymal semen extraction.

The epididymis

The epididymis is a long, highly convoluted tubular duct that lies alongside the testes. Consisting of a head, body and tail, the epididymis acts as a storage area and site of final maturation for sperm cells prior to ejaculation.

Cross-section of equine testicle.
Cross-section of equine testicle.

The production of spermatozoa (spermatogenesis) takes about 57 days in the stallion. Sperm cells formed in the testes drain into the head of the epididymis before passing along the epididymal duct into the body and tail of the epididymis. As they do so, they undergo a series of crucial final maturation changes and gain the ability to move and fertilise. At any point, up to 62% of sperm cells in the epididymal duct can be found in the tail of the epididymis, providing a significant reservoir of potentially fertile semen that can be harvested post castration of the stallion.

Testes removal

For the procedure, the stallion has one or both testes removed using standard castration techniques, leaving the tail of the epididymis and as much of the ductus deferens as possible intact. The deferent duct is then ligated with a suture material to prevent sperm from leaking in transit. Each testis is washed with saline and placed in a sterile plastic bag.

Correctly tied off ductus deferens.
Correctly tied off ductus deferens.
Shipping of the testicles

Following castration, the testicles should be transported to the place of semen harvest as rapidly as possible to maximise the chances of successful sperm extraction. Semen can be extracted up to 24 hours after castration, but success rates can be considerably lower. Same day processing, ideally within a couple of hours of their removal, is therefore the preferable situation.

During shipping, the testes must be kept chilled to aid in semen preservation. They should therefore be packaged in an insulated shipping container with ice packs. It is vital the testicles are just chilled and are not allowed to freeze. Bubble wrap or newspaper should therefore be placed between the ice packs and testicles which should be wrapped themselves.

Correctly wrapped and packaged testicles.
Correctly wrapped and packaged testicles.
Extraction of semen from the epididymis

Upon arrival to the semen collection centre, the testicles are washed and the epididymis and adjoining vas deferens are removed and washed again.

Left: Washing of the testicle. Right: Stage 1 - Removal of the epididymis.
Left: Washing of the testicle. Right: Stage 1 – Removal of the epididymis.

The sperm cells are then harvested from the epididymis using a combination of techniques including aspiration, flushing and flotation. The procedure is repeated for both testes and the extracted semen is pooled together.

From left: Cannulation in preparation for semen extraction; Stage 2 of semen extraction; and semen being flushed from the epididymis.
From left: Cannulation in preparation for semen extraction; Stage 2 of semen extraction; and semen being flushed from the epididymis.
Left: The epididymis. Right: The epididymus elongated.
Left: The epididymis. Right: The epididymis elongated.

The sample is then centrifuged, and a density calculation is carried out before final dilution. The semen is then packaged and cryopreserved using standard laboratory techniques for normal ejaculated semen.

The final product: Cryopreserved semen.
The final product: Cryopreserved semen.
Health testing

Although epididymal semen extraction is normally carried out at short notice and often under difficult circumstances for the stallion owner, it is essential that biosecurity is not overlooked to prevent future disease outbreak. Stallions must therefore be tested for Equine Infectious Anaemia (EIA), Equine Viral Arteritis (EVA), Contagious Equine Metritis (CEM) and Klebsiella and Pseudomonas in accordance with the semen collection centre regulations. In cases of planned castration and euthanasia, these samples should be taken in advance of testicle removal and the results sent to the semen collection centre in preparation for the procedure. In unplanned situations, samples can be taken at the point of castration.

Doses and semen quality

The amount of semen harvested is highly variable between stallions and dependent upon numerous factors including age, testicular size and condition and ejaculation frequency prior to semen extraction. On average, the total dose number can range from 10 to 60 doses.

Semen quality is usually good for reproductively normal stallions but is dependent upon the stallion’s inherent semen quality prior to the procedure. Stallion related factors such as age, health status and reproductive history and extrinsic factors such as time from castration to semen extraction and temperature the testes were maintained at post castration are highly influential to the semen quality produced.

Fertility rates can be lower with epididymal semen versus ejaculated semen due to lack of exposure of the semen to components of the ejaculate that continue to mature the semen in preparation for fertilisation of the egg cell. The addition of a synthetic version of this component helps to mitigate the effects of this and should be added to the semen before freezing.

Intra-cytoplasmic sperm injection.
Intra-cytoplasmic sperm injection.

For the future, Intracytoplasmic Sperm Injection (ICSI) may be the preferred method of use for epididymal semen. This procedure requires only one sperm cell to be injected into an oocyte and therefore would help to conserve spermatozoa banks. When using ICSI to obtain a viable pregnancy, epididymal semen produces the same pregnancy rates as that of ejaculated frozen spermatozoa.

Epididymal versus ejaculated semen – a preferable alternative?

Whilst epididymal semen extraction is a highly beneficial method of extracting semen in non-planned, emergency situations, it is not recommended as an alternative to ejaculated semen freezing. Due to the factors that can affect the semen quality, it should be viewed as a last-chance method of semen harvesting. If situations permit for collection and freezing of ejaculated semen before castration of the stallion, this should always be recommended as the primary course of action. In this way, semen quality can be assessed before testes removal and actions can be taken to improve the semen quality if needed e.g.,nutritional supplementation of the stallion, extender testing of the semen, sexual rest of the stallion to permit increased maturation of the testes, etc.

The laboratory at Stallion AI Services. SAIS has carried out more than 70 epididymal semen extractions.
The laboratory at Stallion AI Services. SAIS has carried out more than 70 epididymal semen extractions.
Summary

Epididymal semen extraction is undoubtedly a pioneering method of semen collection that permits semen extraction where other options don’t allow. Britain’s Stallion AI Service has carried out more than 70 extractions, banking and saving the genetics and breeding potential of stallions that would otherwise have been lost.

Using its state-of-the-art laboratory, the latest technology in equine breeding and advanced semen processing techniques, it provides the highest quality service in epididymal semen extraction, providing semen with the best chance of successful cryopreservation.

For more information, call 01948 666 295 or email lab@stallionai.com.

References

Collection, storage and freezability of equine epididymal spermatozoa (Falmoet al., 2016).
Collection and freezing of equine epididymal spermatozoa (Roelset al., 2013).
Collection and freezing of epididymal stallion sperm (Bruemmer, 2006).

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