How to Collect, Handle, and Process Post-Mortem Epididymal Sperm for Breeding or Assisted Reproductive Techniques

1. Introduction

Recent advances in assisted reproductive techniques in the horse allow for new and essential techniques for the practitioner and reproductive specialist. The unfortunate and unexpected loss of a breeding or potentially fertile stallion because of terminal colic or traumatic injuries can be devastating. The question of how to maintain valuable genetic lines is a problem that may be overcome with harvesting epididymal sperm for cryopreservation either before euthanasia or immediately post-mortem. Options for obtaining spermatozoa for cryopreservation in a terminal animal include pharmacologic induced ejaculation, electroejaculation, epididymal sperm aspiration, and post-mortem epididymal sperm collection [1-3]. Post-mortem epididymal sperm harvest and preservation is a technique that can be performed with minimal tools and expertise. The use of post-mortem harvested cryopreserved epididymal sperm has successfully resulted in equine pregnancy and pregnancy in multiple other species, including humans [4-8]. This paper will discuss and explain the proper post-mortem handling of testes, collection of epididymal sperm, and processing of epididymal sperm.

2. Materials and Methods

Testes are obtained from the stallion using a sterile open or closed castration method. It is not known whether drugs currently used for general anesthesia or euthanasia in horses exert an adverse effect on spermatozoa. It has been shown that halothane anesthesia in stallions does not have a negative effect on the post-thaw motility of epididymal sperm cells [9]. Likewise in rats, different methods of euthanasia did not affect sperm motility [10]. We recommend the use of general anesthesia with the animal being humanely euthanized after castration. We do recognize that general anesthesia is not always a feasible option because of the situation, and each must be treated individually. Special care must be taken to completely remove the epididymis without trauma to the tissues. Once the testes are removed from the stallion, it is crucial to ligate or clamp the vasculature to prevent contamination of the epididymal sperm with blood. The ductus deferens should also be identified and ligated separately and as proximal as possible. After harvest and ligation, the testicle should be cleaned and rinsed thoroughly with warmed isotonic solution to remove any remaining blood, tissues, or contaminants. The cleaned testes should be placed in sterile plastic sealable bags with minimal air.

Often tragic or fatal events will occur at suboptimal times and locations; therefore, the proper handling of the testes allows for delayed processing of sperm when necessary. Delays in processing of up to 24 h does not seem to have a negative affect on post-thaw motility in the horse [11,12] or on fertility in other species [13]. This delay can also be advantageous, because the blood that remains in the testis will have sufficient time to clot and reduce contamination during further processing. When processing is not immediate, the testes should be appropriately cooled to 5°C and sent to a facility for processing. Inappropriate temperature control is shown to have an adverse effect on the spermatozoa [14]. The testes should be placed into a slow cooling container [a] for shipment or transport. If transport is not necessary, the protected testes can be submerged in 37°C water and placed in the refrigerator that allows for proper cooling at a slow and controlled rate.

Once the testes are received at the processing laboratory, they should be handled in a sterile and aseptic manner. Both testes are placed on a sterile surface at room temperature. Dissection begins by separating the tail of the epididymis and ductus deferens from the testicle - an epididymodeferentectomy. A clamp is maintained on the proximal end of the ductus deferens, and a second clamp is placed at the junction of the tail and the body of the epididymis. The epididymis can be completely separated from the testicle at this point by cutting the epididymis just distal to the clamp at the junction of tail and body. The excess connective tissue and fascia is removed with careful dissection to promote surface area and decrease the tortuousness of the epididymis. Once dissection is complete, the remaining testes can be disposed of, and harvested epididymis is thoroughly rinsed with sterile isotonic fluid. Figure 1 is an example of a completely prepared distal ductus deferens and tail of the epididymis.

Figure 1. Isolated ductus deferens and tail of the epididymis prepared for flushing or float up sperm collection.

There are two reported techniques to process the epididymis to obtain the epididymal sperm: the retrograde flush methods and float-up method [2]. Both of these methods are easily performed without special tools and will be discussed here. The retrograde flush technique requires a catheter, a commercial semen extender, and a sterile collection device. When deciding to use a freezing extender [b] versus a milk-based semen extender [c] for the flush or the float-up method, we found it more feasible to use a milk-based semen extender even though the harvested sperm cells are destined for cryopreservation. Milk-based semen extenders are less expensive and much less viscous than freezing extenders, which allows for an easier flush, float up, and identification of individual sperm. A 3.5-F Tom-Cat catheter [d] is used to catheterize the lumen of the ductus deferens after removal of the clamp. The distal end of the tail of the epididymis is cut above the second clamp, and retrograde flush is applied with a commercial extender. If the flush is not productive, the lumen diameter of the epididymis may be too small to flush and needs to be cut just proximal (1 mm) to the distal end. The flush should begin initially with mild pressure, and the high resistance will decrease with continued flushing and emptying of the epididymis. Pressure is applied with the handler’s fingers to the outside of the ductus deferens around the catheter to prevent normograde efflux and loss of sperm. Attention must be paid not to rupture the epididymis because of the high pressure when initiating the flush and also not to slide down the catheter and contaminate the collection cup. The number of flushes and amount of extender used is based on size of epididymis, ease of flush, and gross density of subsequent flushes. We have found that a total volume of 50 ml is sufficient. Figure 2 shows the retrograde flush technique.

The float-up method requires a commercial semen extender and a sterile cup with a tight sealing lid. The cleaned epididymis is placed on the lid, and multiple incisions [10-15] are made throughout the tail, opening the lumen. The epididymis can also be sliced in individual 3-mm pieces. The number of incisions and pieces per epididymis are dependent on the size of the tail of the epididymis; mature breeding stallions and larger breed horses often require an increased number of cuts and pieces to expose sufficient lumen. Once the cuts are made, the tissue is placed in the sterile cup with 30 ml of semen extender, and the lid is applied. Figure 3 is an example of the float up method.

Figure 2. Retrograde flush of the ductus deferens and tail of the epididymis with tom-cat catheter fed retrograde through the ductus lumen and gentle pressure applied with flushing solution. To view click on figure

Figure 3.The lid is reinforced with temporary tape or sealant to prevent leakage. To view click on figure

The lid is reinforced with temporary tape or sealant to prevent leakage. The sample is allowed to sit in semen extender while being agitated occasionally for 10 - 15 min. The sample is strained with a nylon mesh filter [e] to remove tissue. The tissue may again be lavaged in the mesh filter with 10 - 20 ml fresh semen extender over the final collection cup. Our laboratory has retrieved similar numbers of total sperm with both methods. Although the flush method is more tedious, it results in less cellular contamination that can have a negative effect on the sperm. Post-thaw motility of spermatozoa recovered from float-up and flush methods seems to be similar [2]. There are no reports comparing the flush and the float-up method with respect to the fertilizing capacity of the harvested sperm.

With either method, the next step is determining the total number of sperm that has been recovered. The total number of sperm harvested is calculated (volume x concentration). A hemocytometer is used to determine the concentration. A spectrophotometer is not a valid method for determining the concentration, unless a non-refractory semen extender was used with the semen collection. For subsequent cryopreservation in 0.5 ml straws, we routinely use a final concentration of 400 x 106 sperm/ml to have a total of 200 x 106sperm per 0.5 ml straw. If a freezing extender has been used during the harvesting process, the concentration has to be adjusted either by adding more freezing extender if the concentration is too high or by centrifugation and removal of excess extender if the concentration is too low. Our laboratory routinely uses the milk-based extender for harvesting epididymal sperm, so that the first step of cryopreservation involves removal of the milk-based extender by centrifugation. The collected spermatozoa is centrifuged for 10 - 15 min at 400 x g (up to 900 x g if possible), the supernatant is removed, and the sperm pellet is resuspended with freezing extender to the appropriate concentration of 400 x 106 sperm/ ml. The final resuspension should be ≈ 90% cryopreservative extender; this ensures the appropriate dilution of glycerol is added to protect the vulnerable sperm membrane during the cryopreservation process [16].

It has been discussed whether the addition of seminal plasma to epididymal sperm has a beneficial effect on sperm cell motility and freezability. Reports are controversial on the effects of the addition of seminal plasma and its effects on cryopreserved sperm [2,16-18]. The addition of seminal plasma is ≈ 5 - 10% of the final volume. The source of seminal plasma should be a proven stallion with excellent semen quality. An ejaculate from a donor stallion should be obtained before processing the harvested epididymal sperm. This ejaculate should be centrifuged at high power to ensure all sperm from the proven stallion are removed from the seminal plasma. Immediately after centrifugation, the supernatant containing the seminal plasma is recovered and added before epididymal sperm before cryopreservation. Further research is needed to determine the effect on fertility with the addition of seminal plasma.

After the sperm are diluted appropriately with all additives to the concentration of 400 x 106 sperm/ ml, they should be loaded into 0.5 ml straws for freezing. The technique used to freeze the epididymal sperm can be through an automated freezing unit or by cooling the straws with liquid nitrogen vapors and submersing them in liquid nitrogen after cooling [19]. Once epididymal sperm are cryopreserved, they can be shipped to a facility that is experienced in assisted reproductive techniques or placed in storage for future decisions.

3. Results

Sperm recovery from epididymal collections is a function of the stallion and processing. We have collected a range of sperm from 5 to 18 billion per testicle. The dose is typically >200 x 106 progressively motile sperm when using frozen-thawed sperm [20]. The use of frozen thawed post mortem epididymal sperm for artificial insemination (AI) results in lower fertility rates compared with AI with fresh, cooled semen, or frozen thawed sperm, although no large-scale studies have been performed. Fertility rates with frozen thawed epididymal sperm are lower when performed with conventional AI techniques, but the use of hysteroscopic-guided AI does improve fertility rates [5,21]. It is feasible to use new techniques with frozen thawed epididymal sperm for producing offspring. These techniques include intracytoplasmic sperm injection (ICSI), gamete intra-fallopian transfer (GIFT), hysteroscopic insemination, and deep horn intrauterine insemination.

4. Discussion

This paper is aimed to guide the practitioner in preserving genetic materials for the client in the case of an acute death of a potential breeding animal. Although the harvested epididymal sperm may not be a candidate for traditional frozen thawed semen breeding, it can be used with other advanced reproductive techniques or preserved for the future when our techniques and expertise are further improved. With proper collection, handling, and processing of post-mortem epididymal sperm, we can provide alternatives for potential offspring when a life-threatening event has occurred to a stallion. To ensure optimal quality of the harvested sperm, one has to pay particular attention to the following parameters:

• minimize blood contamination (ligating/clamping vessels during castration);
• adequate temperature control of the specimen until processing;
• and timely processing, within 24 h.

Footnotes

[a] Equitainer, Hamilton Research, Inc., South Hamilton, MA 01982.
[b] Equi-Pro Cryoguard, Minitube of America, Verona, WI 53593.
[c] Equi-Pro, Minitube of America, Verona, WI 53593.
[d] Kendall Healthcare Products Co., Mansfield, MA 02048.
[e] Animal Reproduction Systems, Chino, CA 91710.