Referring to the removal of semen from the epididymis of the testes, the epididymal semen extraction 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 a distressing time for stallion owners; it can also have devastating financial implications. Customarily, the breeding potential of a stallion is lost with death; however, 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. 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 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 (Figure 1).
The production of spermatozoa (spermatogenesis) takes approximately 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 percent 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 and shipping
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 (Figure 2). Each testis is washed with saline and placed in a sterile plastic bag.
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 24hrs after castration; however, 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/newspaper should therefore be placed between the ice packs and testicles, which should be wrapped themselves (Figures 3 and 4).
Extraction of semen from the epididymis
Upon arrival at the semen collection centre, the testicles are washed and the epididymis and adjoining vas deferens are removed and washed again (Figures 5 to 8).
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 (Figures 9 to 11).
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 (Figure 12).
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 prior to freezing helps to mitigate the effects of this.
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 (Figures 13 and 14).
Epididymal versus ejaculated semen
Whilst epididymal semen extraction is a highly beneficial method in non-planned, emergency situations, it is not recommended as an alternative to ejaculated semen freezing. Due to the factors that can affect semen quality, it should be viewed as a last chance method of semen harvesting. If situations permit for collection and freezing of ejaculated semen prior to castration of the stallion, this should always be recommended as the primary course of action. This way, semen quality can be assessed prior to testes removal and actions can be taken to improve the semen quality if necessary, eg nutritional supplementation of the stallion, extender testing of the semen, sexual rest of the stallion, etc.
Stallion AI Services has carried out over 70 extractions, banking and saving the genetics and breeding potential of stallions that would otherwise have been lost. Epididymal semen extraction is undoubtedly a pioneering method of semen collection that permits semen extraction where other options don’t allow.
A full reference list is available on request