The results presented here show that SLC-selected spermatozoa achieved fertilization and subsequent activation of the oocytes even after storage for up to 96 h at 6 °C. In fact, the SLC-selected spermatozoa fertilized oocytes up to an estimated 113 h after semen collection, as shown in Table 1. Two of the mares had already ovulated at the time of AI. Therefore, it is likely that they did not conceive because the oocytes would have been too old for fertilization by the time that spermatozoa were in the oviducts rather than because of a problem with the fertility of the SLC-sample. If these two mares are excluded from the total, the per cycle conception rate from SLC-selected sperm samples would be 11/14 or 79%. The per cycle conception rates for cooled sperm doses from these stallions, i.e. after AI at approximately 24 h after semen collection, and the number of mares, were as follows: stallion A 67% (24), stallion B 74% (23), stallion C 64% (97), stallion E 70% (10), stallion F 50% (20). The per season conception rate for Stallion D was 90% (the per cycle pregnancy rate is not available for this stallion).
Since the horses were kept at pasture, no examinations were carried out at night, when most ovulations actually occur . Thus the exact timing of ovulation relative to insemination was not known. Even so, the number of pregnancies followed a pattern, with most pregnancies occurring when the spermatozoa were less than 113 hours old at the time of insemination, although the fact that most of the AIs with spermatozoa >110 h coincidentally took place where there was a longer interval from AI to ovulation could indicate that the lack of pregnancy was not necessarily due to the age of the SLC-selected spermatozoa. Pregnancy was terminated before day 20 to provide as accurate a picture as possible of the sperm effects on pregnancy. If the embryo was already dead at the time of ultrasound examination, the yolk sac would most likely have been smaller in size than observed . The size of the embryonic vesicle was considered to be within the normal range for this time after AI [11, 12]. Although waiting longer before termination would have provided more information about the normality of embryo development, it would also have allowed non-sperm factors to be introduced. Now that we have established the proof of concept that the spermatozoa are capable of fertilization up to 112 hours after semen collection and SLC-selection, it is our intention to perform additional studies in which subsequent embryo development, among other factors, will be studied.
Other studies with stored semen have shown that spermatozoa from the first jet of the ejaculate, extended in Kenney´s extender and used for insemination after 70 h and 80 h of storage using a dose of 2x109 spermatozoa gave a pregnancy rate of 65% . However, this method of collection requires an open-ended artificial vagina, which is not readily available on most studs, and a certain amount of skill to use the technique. Furthermore, the insemination dose was very large in that study, four times the dose used in the study described here; such large doses are not practical for a busy stud. Using standard collection methods, storage of semen extended in INRA96 for 48 h did not influence pregnancy rate negatively compared to fresh semen doses [14, 15]. To our knowledge, this present study is the first to report conception rates after storing cooled SLC-selected sperm doses for long intervals (48-96 h) after semen collection. The stallions used for this trial were of known fertility and were selected at random from those available at the studs. In contrast, the reproductive histories of the mares were largely unknown and only one of them had carried a foal in the recent past. This might have had an adverse effect on the outcome of AI compared to using reproductively active mares of known fertility. However, it is interesting to note that satisfactory conception rates were achieved in such mares when SLC-selected spermatozoa were used. The pregnancies in this study were terminated at day 16–18. A full-scale AI trial with SLC-sperm doses subjected to prolonged cooled storage, in which the pregnancies are followed to their natural conclusion, is warranted to establish whether SLC could prolong the shelf-life of semen doses sufficiently to be useful for the equine breeding industry.
SLC has been used previously to obtain good quality sperm samples from problem stallion ejaculates . However, early studies with Percoll™ (polyvinylpyrrolidone-coated silica particles) showed that although centrifugation with Percoll™ improved the quality of poor semen samples, it did not improve sperm quality in “normal” ejaculates . The current study used Androcoll-E, a silica colloid optimized for stallion spermatozoa , which may explain why sperm quality was improved in SLC-selected samples from “normal” ejaculates. The SLC-technique is not difficult to perform and takes approximately 30 min per ejaculate, including the 20-min centrifugation, which would enable it to be incorporated into the stud´s normal semen processing routine. A fertility trial is currently underway to investigate whether the apparent improvement in sperm quality seen in laboratory assays in SLC-selected sperm samples compared to controls is reflected in an improvement in fertility when AI is performed at 24 h after semen collection, as is normally the case for cooled sperm doses.
In conclusion, SLC-selected sperm samples stored for 48-96 h at 6°C were capable of fertilization. The longest time interval between semen collection/SLC-processing and successful fertilization in this study was 112 h. Thus the proof of concept that SLC-selected spermatozoa are capable of fertilization after prolonged storage has been verified.