Postmortem collection and cryopreservation of epididymal stallion sperm
Mouhamadou Diaw, DVM, MSc, Dipl. ACT, Theriogenology adjunct professor
In the event of sudden death or emergency castration of a valuable stallion, it is possible to collect and cryopreserve spermatozoa from the cauda epididymis to salvage indefinitely valuable genetics which would have been otherwise lost.
Spermatozoa are produced in the testicles. The process, known as spermatogenesis, is initiated within the seminiferous tubules at puberty. It takes 57 days for diploid primary sperm cells to undergo meiosis and form haploid spermatozoa. The immotile spermatozoa are released from the seminiferous tubules into the luminal fluid, transported passively to the rete testis, and then pass through the efferent ducts before starting a 7-10 days journey in the convolutedly coiled epididymis. During their transport, spermatozoa will go through a maturation process that will give them the ability to fertilize the ovum and the potential for mobility, albeit suppressed until the time of ejaculation. A total of up to 54 billion spermatozoa (Amann et al., 1979) will then be stored in the cauda epididymis and vas deferens of both testicles in a quiescent metabolic stage to prevent premature activation (Guimaraes et al., 2012).
This is the feature that is exploited to allow sperm recovery in dead, injured or castrated stallions. When this situation happens, it is very important to communicate as soon as possible with a referral center that has the personnel and equipment required to harvest and cryopreserve sperm from the epididymis.
REMOVING AND TRANSPORTING THE TESTICLES
When euthanasia is the ultimate option, testicles should be removed if possible before euthanasia, as we do not know the effects of euthanasia compounds on sperm viability. It is also recommended not to inject local anesthetics in the testicles before castration, to prevent negative local effects on spermatozoa (Morris et al., 2002). The testicles are removed using normal castration procedures, and it is very important to leave: (1) the testicle and epididymis intact, (2) as much of the vas deferens as possible attached to the epididymis (10 cm distal to its junction with the cauda epididymis). The deferent duct needs to be ligated with a surgical suture to prevent sperm cells from leaking out (fertile sperm cells are isolated from the cauda epididymis but also from the deferent duct) and blood contamination of the semen. Following their removal, the testicles are then rinsed with a sterile isotonic saline solution and stored individually in clean zip plastic bags, palpation sleeves, or other types of containers. Collected testicles are then shipped to a referral center in a cool environment (4-6°C). This is obtained by placing the container with the testicles wrapped in a towel on the top of cooling devices or ice packs. It is important to make sure that the testicles are NOT in direct contact with the source of cold to prevent freezing. A shipping container like an Equitainer™, a Styrofoam box™, or other insulated shipping boxes can be used. To optimize recovery and cryopreservation of epididymal spermatozoa, the shipment needs to be processed within 24 hours of castration.
HARVESTING EPIDIDYMAL SPERM
Upon arrival at the referral center and at room temperature, the epididymis and vas deferens are carefully separated from the rest of the testicle, and the cauda epididymis is dissected, removing all connective tissues and fascia. The sperm cells contained in the vas deferens and the cauda epididymis are then harvested using two methods: flotation (or float-up) and retrograde collection (or flushing). (1) In the flotation method, the cauda epididymis and the proximal duct deferens are sliced using a scalpel blade, then suspended in 5 mL of a pre-warmed (37°C) extender. The mixture is gently agitated for approximately 10 minutes. (2) In the retrograde collection method, flushing of the cauda epididymis is achieved with pressure generated by a syringe connected to the vas deferens via a needle, a catheter, or a pipette and containing approximately 10 mL of a pre-warmed (37°C) extender. In our hands, the retrograde collection method allows a greater recovery of spermatozoa, and contamination with blood and debris has not been observed.
Please follow your discharge instructions for the time you can start riding or training again. Remember: your horse is unfit and the abdominal incision is still gaining in streght. You should begin a regimen of steadily increasing exercise over several months.
HOW MANY DOSES CAN I EXPECT TO OBTAIN?
Typically, up to 20 billion sperm cells can be obtained using the retrograde flushing technique (Bruemmer, 2006), which is much higher than the 4-12 billion obtained on average from mature stallions in a single collection using an artificial vagina (Muradas et al., 2006; Brinsko et al., 2009) or the 4-5 billion sperm cells obtained using the flotation method (Bruemmer, 2006). Ejaculation frequency before harvesting can also influence total sperm numbers recovered from the epididymis: a stallion regularly collected before epididymal harvest would have less sperm in the epididymal reservoir than if he had been sexually rested. The quality and motility of epididymal sperm are usually good for reproductively normal stallions, but semen characteristics can decrease drastically if the stallion has had a prolonged illness or suffered an age-related reduction in semen quality and/or fertility before death. Time and temperature of transport of the testicles can also affect the outcome (Skaife, 2014). Epididymal sperm cells obtained are then evaluated and processed for freezing using standard techniques.
INSEMINATION USING EPIDIDYMAL SPERM
The first equine pregnancy reported using frozen-thawed semen resulted from insemination with epididymal sperm (Baker and Gandier, 1957). Since then, live foals have been obtained from frozen epididymal sperm using either a conventional insemination method (semen is deposited in the body of the uterus) or Assisted Reproductive Techniques (ART) such as the deep horn insemination technique and Intra Cytoplasmic Sperm Injection (ICSI). When performing the deep horn insemination technique, frozen-thawed epididymal sperm is deposited either (1) deeply into the uterine horn ipsilateral to the ovary where ovulation will take place or has taken place, using a long flexible insemination catheter, or (2) at the utero-tubal junction right in the tip of the uterine horn ipsilateral to the ovary where ovulation will take place or has taken place, using a video endoscope (hysteroscopic insemination). The second ART that can be used to maximize pregnancy when using epididymal sperm is ICSI. With this procedure, one spermatozoon is injected into a mature oocyte.
RESULTS
Although there is not an abundance of data on fertility, good results have been obtained with frozen-thawed epididymal sperm. Artificial insemination using frozen-thawed epididymal sperm should be performed close to ovulation. If routine insemination in the body of the uterus resulted in pregnancy (Tiplady et al., 2002), better pregnancy rates were obtained using the deep horn insemination technique (Squires et al., 1999; Morris et al., 2002; Alvarenga et al., 2002). When using ICSI, pregnancy rates for frozen epididymal semen and frozen ejaculated semen are equivalent.
TAKE HOME MESSAGE
It is possible to harvest and freeze sperm cells from the epididymis of the stallion after death or injury that will prevent further semen collection. Epididymal sperm can also be collected and cryopreserved following elective castration. Good pregnancy rates can be obtained when using frozen-thawed epididymal sperm but might require ART like the deep horn insemination technique or ICSI. However, it is important to understand that the use of epididymal sperm is unlikely to be successful if the stallion has had a prolonged illness or suffered age-related reduction of fertility before death.
REFERENCES
Alvarenga, M.A., Leao, K.M., 2002. Hysteroscopic insemination of mares with low number of frozen-thawed spermatozoa selected by percoll gradient. Theriogenology 58, 651–653.
Amann, R.P., Thompson, D.L. Jr., Squires, E.L., Pickett, B.W., 1979. Effects of age and frequency of ejaculation on sperm production and extragonadal sperm reserves in stallions. J Reprod Fertil Suppl. 27, 1-6.
Baker, C.A.V., Grandier, J.C.C., 1957. Pregnancy in the mare resulting from frozen epididymal spermatozoa. Can J Comp Med Vet Sci. February; 21(2), 47–51.
Brinsko, S et al. Manual of equine reproduction, third edition, 2009. St. Louis, MO: Mosby/Elsevier.
Bruemmer, J.E., 2006. Collection and freezing of epididymal stallion sperm. Vet. Clin. North Am. 22(3), 677–682.
Guimarães, T., Lopes, G., Ferreira, P., Leal, I., Rocha, A., 2012. Characteristics of stallion epididymal spermatozoa at collection and effect of two refrigeration protocols on the quality of the frozen/thawed sperm cells. Animal Reproduction Science 136 (1/2) 85– 89.
Morris, L., Tiplady, C., Allen, W.R., 2002. The in vivo fertility of cauda epididymal spermatozoa in the horse. Theriogenology 58, 643–646.
Muradás, P.R., Weiss, R.R., Kozicki, L.E., Granemann, L.C., Santos, I.W., Pimpão, C.T., 2006. Some viability parameters from equine spermatozoa harvested by artificial vagina and epididymal tail washing. Arch. Vet. Sci. 11, 69–74.
Squires, E.L., Pickett, B.W., Graham, J.K., Vanderwall, D.K., Mccue, P.M., Bruemmer, J.E., 1999. Cooled and frozen stallion semen. Fort Collins, Animal Reproduction and Biotechnology Laboratory Bulletin No. 9.
Skaife J. Harvesting and Freezing Equine Epididymal Sperm, The select breeder’s blog, 2014
Tiplady, C., Morris, L.H.A., Allen, W.R., 2001. Stallion epididymal spermatozoa: pre-freeze and post-thaw motility and viability after 3 treatments. Havemeyer Foundation Monograph Series, vol. 5, pp. 63–65.