Résumé — Comparaison entre l’électroéjaculation et les techniques de récolte de sperme épididymaire chez les étalons. Le but de cette étude était d’évaluer 2 méthodes de récolte de semence qui pourraient être utilisées comme techniques terminales chez des étalons atteints d’affections irrécupérables telles que fractures ou coliques. L’électroéjaculation a été tentée sous anesthésie générale. Quarante-huit heures plus tard, les poneys ont été castrés et 2 différentes techniques de récolte de sperme épidinymaire ont été essayées, soit une méthode de rinçage et une méthode de flottaison. De plus, l’effet d’une supplémentation en plasma séminal a été évalué. Expérimentalement, l’électroéjaculation s’est révélée une méthode sécuritaire mais inefficace de récolte terminale de sperme. Des spermatozoïdes viables ont été récupérés avec les 2 méthodes de prélèvement épididymaire. La méthode de flottaison était plus simple et avait tendance à être supérieure à celle du rinçage en terme de motilité et de pourcentage de cellules passant au travers d’un filtre de laine de verre/ séphadex. Cependant, les différences n’étaient pas significatives. L’addition de plasma séminal aux spermatozoïdes épididymaires avant la cryoconservation n’avait pas d’effet. En conclusion, les deux méthodes de récolte de sperme épididymal constituent une façon valide de prélèvement terminal de semence.

(Traduit par Docteur André Blouin)

Traumatic injuries or colic conditions can prematurely end the breeding careers of many stallions. In such circumstances, owners may request a final semen collection to allow propagation of valuable genetics. Little documented research exists on appropriate collection techniques for use in terminal cases. Our goal was to develop a model for terminal sperm cell collection in stallions.

Historically, little value has been placed on the use of electroejaculation in horses. The use of electroejaculation in standing or sedated horses is discouraged due to their temperament and the likelihood of trauma to the horse, the operator, or the handlers. The technique has been used with some success in chemically immobilized Przewalski horses (1). However, death following electroejaculation of a sedated, terminally ill domestic stallion has been reported (2). The potential use of electroejaculation under general anesthesia has been discussed, but its use has not been reported in a clinical setting (3,4). Electroejaculation under general anesthesia would simplify semen collection from stallions with acute irreparable conditions.

Successful epididymal sperm collections with resulting pregnancies have been reported in a number of species, including goats, red deer, dogs, and humans (5–9). Similarly, frozen equine epididymal spermatozoa have been proven to be fertile (10,11); however, recent attempts at achieving pregnancies have been challenging (11). While individual stallion variation exists, overall single cycle pregnancy rates following the use of frozen ejaculated semen range from 30% to 50.6% (12,13). The pregnancy rates following the use of frozen epididymal sperm are anecdotally extremely low using standard insemination techniques. However, a recent study achieved rates of 17% to 30% by depositing the semen onto the papillae of the uterotubal junction by using hysteroscopy (11). Possibly, the poor functional quality of frozen epididymal sperm is related to the absence of seminal plasma, a normal component of ejaculated semen. Seminal plasma may have important roles in sperm motility, membrane integrity, transport through the uterus, and the avoidance of phagocytosis (14,15).

In this study, electroejaculation and 2 epididymal collection techniques were evaluated, as well as the effect of adding low levels of seminal plasma to semen samples for storage in liquid nitrogen.

We considered that electroejaculation under general anesthesia could be a viable option for semen collection, although there was concern about retrograde ejaculation and urine contamination, which could hinder the fertility following freezing. By comparing electroejaculation under general anesthesia with epididymal collection techniques our goal was to identify a procedure that would allow efficient and efficacious recovery of semen for use in cases of acute terminal injury or illness. We utilized 2 postcastration techniques in an attempt to develop an efficient method of semen collection from the distal epididymis and vas deferens. Furthermore, we wanted to investigate the use of seminal plasma and its effect on post-thaw motility and acrosomal membrane integrity when added to epididymal spermatozoa.

Seven intact sexually mature male ponies between 2 and 4 y of age and weighing between 160 and 250 kg were used. All ponies had 2 palpably normal descended testicles. The project was approved by the University of Minnesota Institutional Animal Care and Use Committee.

Before the electroejaculation procedure was performed, the manure was evacuated from the rectum.

The self-contained RE uses a sine wave pattern at a frequency of 25 to 30 Hz, with a maximum output of 6 volts and 0.2 amps. Stimulations with this probe were commenced in a pulsatile pattern with approximately 2 s of stimulation per burst and 1 s rest between bursts. As ponies failed to respond, the length of each burst was gradually increased to a maximum of 10 s.

The PII also uses a sine wave pattern and has a maximum output at 25 Ω resistance of 45 volts and 1 amp. Stimulation was commenced at the lowest setting and increased in a stepwise fashion until ejaculation was achieved, or all 8 power levels had been used. At each power setting, the stimulus was started at zero and then increased to maximum where it was maintained for 2 to 3 s before returning to zero. Approximately 4 stimuli were given at each power setting. If the pony was actively ejaculating, the stimulus was continued at that power setting until the ejaculation was complete. If an attempt was unsuccessful, it was repeated after a short break. The maximum total time for electrostimulation attempts in any animal was 15 min.

During the procedure, all 7 ponies had their heart rate, respiratory rate, and depth of anesthesia monitored continuously; 6 of the ponies were monitored by electrocardiogram (ECG) (ECG-103; Brentwood, Danbury, Connecticut, USA) in a base apex lead, and 5 by direct arterial pressure. Evidence of elevated heart rate, respiratory rate, nystagmus, or change in anesthetic depth was noted. The anesthetic record was later evaluated along with electrocardiogram strips obtained before, during, and after electrical stimulation. The ponies were maintained at a surgical plane of anesthesia.

The ejaculate was collected into a sterile test tube and analyzed for volume, sperm concentration, progressive motility, and morphology. The level of urine contamination was evaluated by gross examination and pH value, with 8.0 or above indicating the presence of urine in the sample.

Following both successful and unsuccessful electroejaculation attempts, the penis was cleansed and a sterile urinary catheter was passed into the pony’s bladder, which was then lavaged with 500 mL of lactated Ringer’s solution. The lavage fluid was collected and evaluated for the presence of semen by allowing it to sediment over 4 h, after which the top half of the fluid was decanted. The remaining fluid was centrifuged at 400 × g and the resultant pellet was analyzed under a light microscope at 400 × for the presence of sperm.

Only 3 of the 7 ponies had any evidence of semen during attempted electroejaculation. Two of these samples were achieved at the highest setting of the probe and resulted in small volumes with high levels (grossly visible) of urine contamination. The semen from these 2 ponies had no discernible motility when evaluated within 5 mins of collection. The 3rd pony ejaculated a very small volume of semen without gross urine contamination, but the motility was poor with no progressively motile sperm. Two ponies passed a small amount of urine during the procedure with no evidence of semen. Despite achieving erections, the remaining 2 ponies did not show any evidence of ejaculation as a result of electrical stimulation. The larger probe with 6 ring electrodes trical did not result in an ejaculation.

Fluid collected from the urinary bladder revealed the presence of semen in the bladders of 4 of the 7 ponies, including the 2 ponies that produced urine contaminated semen during electroejaculation. The number of sperm in the urine collected from the bladder was not determined due to the large volume of flush used, but the mined quantity was subjectively graded as moderate to large in comparison with urine debris and crystals.

There were no significant effects of recovery method or seminal plasma addition on any of the post-thaw parameters examined (Table 1).

Table 1 Effect of recovery method and seminal plasma addition on post-thaw parameters of cryopreserved epididymal spermatozoa (mean, s)

The relationship between progressive motility before and after cryopreservation is shown in Table 2. Within recovery technique, there were significant decreases in progressive motility (P < 0.05) following cryopreservation, except for sperm recovered by the flotation technique and frozen without additional seminal plasma, where significance was approached. The only correlation that approached significance was for the flotation recovery technique without the addition of seminal plasma (r = –0.71; P = 0.07).

Table 2 Relationship between prefreeze and post-thaw progressive motility of epididymal sperm recovered by flushing or floatation and cryopreserved with or without the addition of seminal plasma (mean, s)

Our results suggest that preservation of genetics from valuable stallions by using terminal collection techniques is possible. However, since our subjects were healthy, the effects of severe disease processes on the success of these procedures remains to be determined and could certainly alter the recovery rates of semen.

Electroejaculation, while proving to be safe for the operator and patient, was not an effective method of obtaining viable semen under the conditions of this experiment. When ejaculation was achieved, the level of urine contamination was significant, reducing the potential of obtaining viable semen. The one grossly uncontaminated sample did not have sufficient motility, volume, or concentration to be used as an acceptable dose of semen. In one case report, in which the stallion underwent cardiac arrest following successful collection by using the electroejaculation probe, the motility was 10% compared with 65% motility following castration and flushing the ductus deferens (2). Urine contamination may have contributed to the low motility with ejaculation compared with epididymal collection. Urine contamination has been cited as one of the reasons that electroejaculation in horses may not be an acceptable method of collection (4). The use of ring type electrodes worn on the gloved hand of the operator may reduce this contamination and provide improved sperm recovery by allowing more targeted stimulation of the reproductive tract. This technique may be worthy of investigation in suitably sized subjects.

The presence of sperm in the urinary bladder in 4 of 7 ponies would suggest that retrograde ejaculation might be common in ponies when electroejaculation is attempted under general anesthesia. Retrograde ejaculation in conscious horses is considered rare but has been documented (19), and it is commonly associated with electroejaculation in other species (20–22). The presence of sperm in the urethra during catheterization can yield small numbers of sperm in the urinary bladder (19); however, the apparent large quantity of sperm isolated from the bladders of ponies in this study suggests that retrograde ejaculation was occurring during electrical stimulation. In dogs, an increase in the occurrence of retrograde ejaculation has been linked to the administration of xylazine (23). In contrast, in stallions, xylazine is used as part of the protocol to obtain ex-copula ejaculations (24). To our knowledge, there is no documentation on the frequency of retrograde ejaculation in these stallions. It is possible that the administration of xylazine for premedication in the anesthetic protocol or the combination of anesthetic drugs contributed to the occurrence of retrograde ejaculation. Tricyclic antidepressants, such as imipramine, have been used as therapy for urospermia in stallions in an effort to increase tone of the bladder sphincter (25). Brinsko (19) hypothesized that these drugs could be used to decrease the frequency of retrograde ejaculation. Such therapy might have improved the grade recovery of sperm during electroejaculation in this experiment. However, such treatment has been found to be ineffective for cases of urospermia in a controlled trial (26).

Options for removal of semen from the distal epididymis include aspiration (9), flushing (7), and flotation (8,27) or any combination of techniques (3,5,28). In this study epididymal sperm were successfully harvested by both the flushing and flotation techniques. The collection of spermatozoa was more rapidly achieved by the flotation technique than by the flushing technique, as the lumen of the vas deferens or epididymis did not have to be catheterized. The ease of the flotation technique accelerated the active collection process while preserving the viability of resultant sperm. The flotation technique produced slightly higher prefreezing total and progressive motility and number of sperm collected over the flushing technique, although the counts were not statistically significant. Due to repeated incisions and subsequent incubation of the cauda epididymis and vas deferens, there was more blood contamination with this procedure; however, this did not appear to be detrimental to the sperm under the conditions of our experiment. The flotation collection method may be selective for the more functional spermatozoa that are able to swim out of the epididymis. Alternatively, flushing the spermatozoa from the epididymis may be more traumatic to zoa individual sperm cells. However, there was essentially no difference in the post-thaw motility between the 2 recovery methods. Individual stallion variation existed within groups, although there was no discernible pattern to assist in predicting this trend in a clinical setting. In the absence of significant differences between the recovery techniques, either method would appear to be acceptable.

Progressive motility before freezing in this study ranged from 4% to 62% with a mean of 33%, somewhat lower than the results of Braun et al (29), who obtained prefreezing motility rates of 25% to 75% following collection after normal stallion castrations. However, the lection stallions in that study were on a regular collection schedule prior to castration, whereas ours were sexually rested. Progressive motility following thaw of cryopreserved epididymal sperm was 1% to 43% (mean 16.3%), comparable with 4% to 49% obtained by Braun et al (29).

Compared with sperm obtained at ejaculation, epididymal sperm are more resistant to cold shock, making them potentially more successful candidates for freezing and thawing than sperm mixed with accessory sex gland fluids during ejaculation (28). In a direct comparison with ejaculated sperm, epididymal sperm were found to have superior post-thaw motility (30). Volkmann et al (31) reported superior post-thaw progressive motility for epididymal sperm frozen without seminal plasma compared with both epididymal sperm with seminal plasma added prior to cryopreservation and frozen ejaculated sperm from the same stallions. However, similar to our results, other workers have found no significant effect of seminal plasma on post-thaw motility of epididymal sperm (32). The same large variations between stallions in post-thaw motility of ejaculated sperm have been shown to exist in epididymal sperm and the two are highly correlated (29,33). This lead Braun et al (29) to suggest that the freezability of equine spermatozoa is determined when it leaves the epididymis, prior to mixing with the seminal plasma.

A puzzling result of our study is the apparently negative correlation between prefreeze and post-thaw motilities for semen recovered by flotation and frozen without additional seminal plasma (r = –0.71, P = 0.07). This was driven by the change in stallion ranking between prefreeze and post-thaw motility. The stallions with the 3 highest prefreeze progressive motility rankings dropped to be in the bottom 4 post-thaw rankings, while the 3 with the lowest prefreeze motilities became the highest ranked on post-thaw motility. While this may be a reflection of the individual stallion variation in response to freezing, we do not currently have an explanation as to why this should have occurred in this treatment group and not the others. Further investigations are required to determine whether this is a sampling effect for this group of stallions or whether a true trend exists.

Plasma membrane and acrosomal integrity is critical to fertility, and the negative impact of cooling and cryopreservation on the fertility of sperm is mediated largely through damage to these structures (34). With GWS filtration, we found no significant effect of either recovery method or the addition of seminal plasma on membrane integrity following cryopreservation. The treatment rankings were highest for the flotation method of recovery, irrespective of seminal plasma addition, and lowest for the flush method of recovery without the addition of seminal plasma. Most other tendencies within this study also favored the flotation method of recovery.

Semen quality is dependent on many factors, including season of the year, previous sexual activity, and individual stallion variability, especially when pertaining to cryopreservation (35). Therefore, success in terminal collections with limited amounts of semen may be challenging. To our knowledge, terminal collection and cryopreservation of epididymal sperm in horses has resulted in few documented pregnancies and poor pregnancy rates (10,11). Further advances in technology are required to improve the utilization of terminally collected sperm.

The authors thank Dr. Erin Malone for surgical assistance, Dr. Lynelle Graham for anesthetic assistance, and Dr. Carl Jessen for statistical advice and assistance. CVJ