So How Long Does Frozen Equine Semen Remain Fertile?

Foal produced from 28 year-old frozen semenIt's a question periodically asked, and durations as long as 10-50,000 years have been suggested as answers, but although that sounds good, what is the actual known duration of viability of stored frozen equine semen?

Last year, Equine-Reproduction.com LLC was asked to assist in fertility evaluation of semen frozen from the great Olympic show jumper "Galoubet A". Galoubet himself had died in 2005, and the semen had been frozen in the mid to late 1980's. Two pregnancies were established, although one was lost between 45 and 60 days of gestation, despite having developed a heartbeat. The second pregnancy successfully proceeded to term and the filly shown at left - "Galoubelle d'Avalon", owned by Avalon Equine is the athletic result, seen here at just 2 days of age! It is thought that this foal is the only offspring of Galoubet A to have ever been produced by the use of frozen semen.

The dose of semen which resulted in the failed pregnancy had been frozen in 1987. We cannot of course determine if the loss was related to the semen, although the mare bred was a young mare with a sound reproductive evaluation. The semen which resulted in Galoubelle d'Avalon had been frozen prior to 1987, with the straws carrying no freeze date or having accompanying information. It is thought however to likely date from about 1985-86. From this, we can safely say that frozen semen if stored correctly will maintain fertility for at least 28-30 years!


ISER 12 - a Date to Save!

The 12th International Symposium on Equine Reproduction (ISER) has been scheduled for January 8-12, 2018 in Doha, Qatar. This meeting - which is held every 4 years - presents new research in the field from top researchers. Representatives from Equine-Reproduction.com were fortunate enough to be invited to attend in the past, and have been able to disseminate much of the practical information presented there since. The main meeting will be followed by the ISER Veterinary Practitioners Conference on Equine Reproduction, Genetics and Neonatology January 14-15, 2018 in the same location.

AQHA Cryopreserved Reproductive Material Rule Change Leaves Some Confused

In 2003, the AQHA rewrote a rule to allow indefinite use of cryopreserved sperm, embryos or oocytes with which to register a resulting foal following the donor's death or sterilization. The industry "scuttlebut" suggests that the owner of a stallion which had died contacted the AQHA and showed a willingness to pursue legal action if the then-existing rule was maintained. The rule which was re-written limited the registration of foals produced by frozen semen to those produced by use no later than the end of the year following the stallion's death. Whether such pressure truly was effected is unclear, but certainly the rule change appeared only shortly after a significant award of damages against AQHA had been made following a lawsuit brought regarding the restriction of a single embryo transfer foal registration per year.

Now the regulation regarding the use of the cryopreserved material - including both semen and embryos - has been changed back again.

At the AQHA annual convention this year, the decision to change the rule once again was made. The new (old) rules will be written as subsections to Reg 111 and 112, and will limit the use of the reproductive material to within the two years following the donor animal's death or sterilization. This however - and this is where some confusion has arisen - only applies to product obtained from animals which were born in 2015 or later. If one already has cryopreserved material stored from animals which were foaled last year (2014) or earlier, then that material can be used to register foals indefinitely. This does seem to present somewhat of a restriction for owners of animals produced this year or later, when their competition with already-stored product have the ability to indefinitely reproduce their animals in the event of an untimely (or even timely) demise or castration.

The change in the rule was made to allegedly address the possible reduction of the gene pool with the widespread use of frozen semen after a stallion's death. Veterinarian and AQHA President Dr. Glenn Blodget is reported as saying "This new rule points the Association in the direction of more responsible breeding practices and broadening the gene pool". As horse breeders are notoriously fickle in their stallion choice - more often than not seeking the current top producing stallion or "flavour of the month" - it is not clear how this is really going to make a huge impact in the long-run. It is generally seen and agreed that once a stallion dies, although there will be some mares bred using frozen semen after his death, the popularity of the stallion will decline as a son or some other horse completely takes his place in the limelight. It would appear possible that this rule amendment may in fact place extra strain on a popular stallion while he is still alive to produce even more foals per year, as the mare owners will know that there will be this limitation after his death. Similarly, it would seem a possibility that within those two years, there will a increased number of breedings as his popularity will not have diminished in that time frame, and the pressure will be increased to "get a last foal from..."! As a final possible negative point, there are situations where bloodlines or horse types which are beneficial end up being lost owing to changes in "fads", immediate demand, or simply unfortunate circumstances. Subsequently if it is discovered that those bloodlines are desirable and the availability of frozen semen from long-dead stallions is present, a resurrection of lineage can be obtained. It now seems unlikely that anyone will maintain semen for years after a stallion's death as this rule change will not allow registration of the resultant product of the old bloodlines.

One positive aspect with this rule change is that it will reduce the possibility of fraudulent use of semen from a clone of a dead stallion. While that possibility will of course still exist, it is now going to be limited in only being able to occur in the two years following the DNA donor's death or castration, which reduces the potential for "contamination" of the Stud Book, should such a practice occur.

Cloning Judgement Reversed - AQHA Able to Deny Registration of Cloned Quarter Horses

Upon appeal, the Fifth Circuit Court of Appeal has reversed the decision of the lower court which caused the AQHA to register clones or offspring of clones. This move will undoubtedly please some members of the Association - although evidence presented at the original hearings indicated that the majority of AQHA members were neither breeders nor interested - but it still leaves wide open the question of how the AQHA is going to avoid abuse of the Stud Book by the "illegal" use of semen from clones of stallions. One solution may be to require mitochondrial DNA recordation of all stallions, as unless a female sibling of the stallion's dam (or the dam herself) is used as the donor of the oocyte used in the cloning process, the mtDNA will not match the stallion's own mtDNA. Other breed registries have been involved in the investigation of mtDNA and rather surprising results have been presented, which indicate that maternal lines in some cases are not as "pure" as was thought! Hopefully this situation with the use of cloned semen will not arise in a hurry, nor will the AQHA's 75 year-old stud book be found to be compromised in the future if mtDNA evaluation becomes routine!

Australian Attempt to Modernize Thoroughbred Breeding Comes to an End

Four years ago, former Australian bookmaker Bruce McHugh started a crusade to attempt to end the Thoroughbred industry's ban on the use of artificial insemination as an accepted breeding technique. With Thoroughbreds, in order to be eligible for registration, a horse must have been produced using live-cover techniques. Among other matters, McHugh - and many others involved in the field of equine reproduction - argued that the restriction was archaic and not based upon sound scientific reasoning. It had been introduced in Australia in 1947 to avoid incorrect - either intentional or accidental - replication of bloodlines owing to the misuse of collected semen. With the advent of DNA testing, McHugh pointed out that it was easy to determine if the sire was indeed the sire of record. Most other breeds and registries that utilize AI require DNA testing, so parentage verification protocols are well-proven and clearly definitive.

As we have previously reported, the case which was heard in 2011 was finally adjudicated upon in December of 2012, at which time the Courts found for the Defendants, concluding that McHugh's arguments carried no - or insufficient - merit. Undaunted, McHugh appealed, but was again shot down with a judgement against him handed down in April of 2014. Not yet prepared to "roll over and play dead", McHugh sought special leave to appeal from Australia's High Court, but yesterday was denied that opportunity by the Court.

While McHugh acknowledges that his own attempts are at an end - after spending millions of dollars - he also observes that the subject is unlikely to die. It is an emotive subject, but one which carries a pretty clear scientific base in favour of AI, and would potentially prevent the need for "shuttle stallions" being transported from Northern Hemisphere to Southern and back for a year-round breeding season, as well as preventing the need for stallions to breed multiple mares day after day, both of which can be decidedly stressful on the animal.

The aspect of DNA-testing is clear cut as far as parentage verification is concerned, but even that issue may be more complicated for all breeds and registries if the subject of mitochondrial DNA-testing is considered. MtDNA looks solely at DNA inherited from the dam - which is only passed through the direct female line - and has been used recently to show that the likelihood of the famous 19th Century Thoroughbred racehorse Ben d'Or was in fact most likely not that horse, but in fact one called Tadcaster. While this supposed error was allegedly as a result of accidental foal-swapping, investigation of all MtDNA is quite likely to result in other errors being identified - and remember this was at a time when live cover was the only option - so one has to wonder at the continued arguments being offered. There is no doubt that "round two" with a different set of protagonists will occur, the only question being when.

New "Cloning" Research with an Interesting Twist Released

Nuclear vs mtDNA TransmissionUp until now, in most cases there has been a way to differentiate - using DNA profiling - between a donor animal and a clone of that animal. The differentiation can be made by looking at the Mitochondrial DNA of the animal in question. The important things to remember are that there are two forms of DNA to consider - Nuclear DNA and Mitochondrial DNA (mtDNA) - and that in respect to the latter, it can only be passed down to the next generation by the female. To the left are a pair of slides ("click" to enlarge) we used to demonstrate that at the lecture we gave at the American Hanoverian Society's AGM a few months ago. It's really very simple, but often causes confusion. The red lines in the diagram are the mtDNA being transmitted - the mares are to the right in the "pedigree" - while the blue lines are the Nuclear DNA. As you can see, both sire and dam transmit nuclear DNA, but only the dam can transmit the mtDNA. If the offspring to which the mtDNA is transmitted is male, then the transmission stops there.

So with cloning (the type of which that is used for the equine is technically called "Somatic Cell Nuclear Transfer") an oocyte - any suitable oocyte - is taken and "enucleated", or has the nuclear DNA removed. Without getting too technical, the mtDNA from the oocyte remains, but doesn't (as far as we know) provide any characteristics related to the next generation, but is primarily associated with energy for the whole cell division (or fertilisation) process. The donor DNA is then placed within that enucleated oocyte and undergoes a process to convince the cell that it's just been fertilised. Cell division starts and in due course we hope, along comes a (DNA) replica of the DNA donor - except, in most cases, for the mtDNA. Because the mtDNA came from a completely different source (the oocyte that was used) than did the original donor's mtDNA (which would have come from his Dam's oocyte), if the clone's mtDNA is looked at, then it will be different from the DNA donor's mtDNA, so the differentiation can be made.

Where it gets tricky is if the "new" clone is male. If that stallion goes on to breed, there is no way to differentiate between an offspring of the original DNA donor, and an offspring of the clone, because the mtDNA would - in that offspring - have come from its Dam, not the (clone) Sire. This is the reason why we strongly encourage breed registries to record clones in some way so that there is not a "ringer" throw into the mix at some point - but that's another discussion for a different day...

So on to the twist...

A paper that is due to be published in the Theriogenology Journal (Choi Y., Ritthaler J., Hinrichs K. Production of a mitochondrial-DNA identical cloned foal using oocytes recovered from immature follicles of selected mares, Theriogenology, Article in press May 2014) reports on work that has been done where the oocytes that were used for the cloning process were taken from close female maternal line relatives - in one instance a cousin (a female offspring of a female sibling of the DNA donor's dam) and in another instance, a second cousin (a female offspring of a female offspring of a female sibling of the DNA donor's dam). As a consequence, the resulting foal (ultimately there was only one) had identical DNA - both nuclear and mtDNA - to the original DNA donor.

This has several interesting implications. Obviously the first is that it is now possible to replicate an animal that - at this time - we cannot differentiate genetically from the original; the second is that it opens the door to further research as to what effect - if any - the mtDNA truly does have on phenotype and performance in the equine.

AQHA Required to Register Clones

In a ruling handed down today, U.S. District Judge Mary Lou Robinson required the AQHA to commence registering clones of registered American Quarter Horses and their offspring 30 days from the time the injunction is signed, which could occur this week. Plaintiffs Jason Abrahams and Gregg Veneklasen are delighted with the result which will allow them to register their 20 clones as well as future animals, however the AQHA is still resisting the decision and still intends to "continue to take any and all necessary legal action in seeking to have the verdict of the jury and any judgment entered by the Court in favor of plaintiffs reversed".

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Mares do NOT have a
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What was the duration in days of your mare's live-foal producing pregnancy?
Survey commenced in 2008 foaling season
Results © Equine-Reproduction.com
result319 Days or fewer:4.51%
result320-326 Days:7.05%
result327-332 Days:7.55%
result333-338 Days:9.59%
result339-344 Days:14.77%
result345-350 Days:12.73%
result351-356 Days:8.83%
result357-362 Days:5.86%
result363-370 Days:8.43%
result371 Days or greater:20.68%

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Equine reproduction topics covered in our articles section on this site include artificial insemination (A.I.); information about, and the use of frozen semen; stallion handling articles, including "phantom mare" training, and other semen collection methods; the collecting and processing of cooled transported semen; different equipment and supplies needed for semen collection and processing, and artificial insemination; managing the mare for breeding (including hormonal manipulation and the use of other drugs such as Oxytocin); and some articles relative to foals and foaling.

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