What are the risks of ET?
ET is generally a safe and well-established assisted reproduction technique in mares with no detrimental effects on the mare’s future fertility.
Repeated cycles of breeding and embryo flushing, however, may predispose embryo donor mares to uterine inflammation and infection by the third to fifth cycle. These affected mares tend to accumulate fluid in their uterus, have inflammatory cells present on uterine cytology and show growth of bacterial organisms on uterine culture. Consequently, close attention is paid to the uterine health of individual donor mares and appropriate treatment initiated when needed.
Furthermore, rectal tears are a small, yet serious risk of every rectal palpation and can be life-threatening for the mare. Depending on the degree of the tear, conservative treatment or surgical management may be warranted. Overall prognosis for horses with rectal tears depends on size, grade and location of the tear, time between occurrence and treatment, and first aid measures applied.
All the veterinarians at Equiception B.V. are experienced and work with caution during rectal examinations and to increase safety, young inexperienced and fractious mares will be sedated just prior to the examination.
Is ET suitable for mares in athletic competition?
One of the big advantages of embryo transfer is that mares in athletic competition can remain in performance and still produce offspring. However, disruption to training and competition schedules, for some mares at least, may be necessary to optimise embryo recovery rate as exercise, heat (including exercise-induced heat) and stress (including increased cortisol levels caused by transportation) can disrupt ovarian function. These factors should especially be considered in mares from which there is a repeated failure of embryo recovery in the absence of clinically detectable reproductive abnormality. To optimise embryo recovery, owners may have to accept that alterations to management will be necessary and potentially training as well as competition may have to cease entirely during the periovulatory period and the gap between insemination and flushing. Having said this, in many cases mares manage to conceive repeatedly and donate embryos despite the stresses of training, competition and transportation. This is very fortunate, nonetheless it is important for mare owners to have realistic expectations and realise caution is warranted when it comes to deciding to breed from competition mares.
What are the costs of ET?
The approximate costs of ET at Equiception B.V. (excluding 21% VAT) are the following: flush procedure: €267,50, excluding required drugs: approximately: €15,-, transfer procedure: €267,50, excluding required drugs: approximately €50,-, packaging and transportation of the embryo (to Equiception B.V. recipient mare site): €167,50 and lease of recipient mare: €2890,-.
For example, your donor mare is flushed twice as the first attempt does not result in an embryo recovery. The second embryo flush is successful; this embryo is transferred, resulting in a pregnant recipient. 2 x flush procedure and 1 x transfer procedure (including drugs) is €852,50. Total costs including packaging and transport of the embryo and the lease of the recipient mare add up to a total of €3.910,-
Depending on factors like the fertility of the donor mare and the semen and recipient mare factors, more or less flush and/or transfer attempts will be needed to result in a pregnant recipient. At Equiception B.V. the overall costs for a successfully pregnant recipient mare by ET are on average €4.700,-
Please refer to Equiception B.V. current pricelist (hyperlink) for all up to date costs.
What results can be expected with ET?
The ET pregnancy rate per donor mare cycle is dependent on both the embryo collection rate and the recipient pregnancy rate after transfer. In order for the whole process to be successful, management of the donor and recipient mare must be optimal and the techniques of both embryo collection and transfer well understood.
The embryo collection rate can vary significantly per cycle, depending on the fertility of the donor mare and stallion and whether fresh, transported chilled or frozen semen is used. The recipient pregnancy rate after transfer can also vary, depending on quality of the embryo, factors associated with the recipient mare and the experience of the person performing the transfer. Also worth considering is the 8 to 10% of pregnancy loss after first detection of pregnancy, this rate being similar between embryo transfer recipient mares and mares bred to carry their own pregnancy. However, pregnancy loss may be higher in recipient mares carrying embryos from older donor mares.
Throughout the season Equiception B.V. sits at a pregnancy rate of 80% of embryos transferred into our own recipient mares. There is a significant variation of results associated with time of season, with a noted decline in pregnancy rates in periods of high temperature.
What is the role of the recipient mare?
One of the most important components of a successful embryo transfer is the recipient mare.
Recipient mares should be between 3 and 15 years of age and within a 150kg weight range of the donor mare, in good physical condition and easy to handle. A potential recipient mare should be critically evaluated before making the decision to use her as a surrogate. A general physical examination should be performed, followed by a thorough reproductive evaluation to determine if the mare has any condition that may affect her ability to become pregnant after transfer or prevent her from carrying a foal to term. Recipients may receive 2 or maximally 3 embryos in a given year. If they fail to come pregnant or keep a pregnancy after these 2 or 3 opportunities, they should no longer be used as embryo recipients.
Equiception B.V. owns a large number of quality recipient mares that have all been critically evaluated before making the decision to use them as surrogates. These mares are available as surrogate mares for the embryos from our clients’ donor mares. It is not unusual, however, for clients to want to use their own mare(s) as the recipient of their donor mare’s embryo. This is understandable and can be an acceptable option, but it is recommended to discuss this with your own veterinarian to decide if the mare would make a suitable surrogate.
Equiception BV. will not be held accountable for the possible consequences of using a less than ideal client-owned recipient mare.
Can the embryos be frozen?
Equine embryos can be frozen/vitrified successfully, however this is not a straightforward procedure and because of the significant challenges involved Equiception B.V. currently does not offer this service.
Only small equine embryos (<300μm) seem to be frozen successfully, freezing larger embryos is not a viable technique as evidenced by the low pregnancy rates. Yet managing mares to recover embryos when they are <300μm requires accurate timing of ovulation so that an early flush is performed for embryo recovery exactly 6,5 days later. The equine embryo leaves the oviduct and enters the uterus around day 5,5. However, embryos that have a delay in entering the uterus may not be retrieved with a flush on day 6,5 and are subsequently missed and it can be decided to perform a second flush on the mare several hours later. Increasing the difficulty is the rapid growth of an equine embryo at this stage of development. Even being 8-12 hours late with a flush can be the difference of recovering an appropriate sized embryo versus one too large to freeze.
Hence there is a very small window for when to perform these early flushes, making these currently logistically impossible to be performed by Equiception B.V.
The larger embryos, which are regularly flushed on days 7 to 9 post-ovulation, need to be ‘collapsed’ prior to the freezing process. This technique has been developed in the laboratory using microscopes with manipulators, embryo holding capabilities, and specialized drills to make small holes in the embryo. This equipment is expensive and operating it requires considerable expertise.
What is OPU & ICSI in short?
OPU (Ovum Pick Up) and ICSI (Intracytoplasmic Sperm Injection) are two different things, but are inextricably linked. The one always follows the other, but both represent the different steps of the complete process that will now be shortly explained (more detailed information can be found here hyperlink).
OPU -> ICSI -> development of an embryo in the laboratory -> slow freezing of the embryo -> thawing of the embryo -> transfer of the embryo into a recipient mare -> pregnancy check (approximately 1 week post-transfer)
OPU = the procedure that involves puncturing, emptying and re-filling ovarian follicles (fluid filled structures that contain the oocytes (egg cells)) via repeated suction and scraping in order to collect the oocytes.
- The mare’s tail base is shaved and an epidural injection is administered
- The mare is sedated
- Antibiotics, an anti-inflammatory and a rectal relaxant is administered
- An ultrasound probe, with a needle guide attached, is inserted into the vagina
- The ovary is held rectally and brought in contact with the ultrasound probe to visualize the follicles
- Each follicle (larger than ~ 1 cm diameter) is separately punctured, drained, scraped and flushed multiple times to collect the oocyte
- The retrieved flush fluid from the follicles is filtered and assessed in the laboratory for the presence of oocytes
- The oocytes are collected and stored in special medium to stimulate further maturation
- Only matured oocytes will be fertilized
The complete OPU procedure will take approximately 1 hour (depending on the amount of follicles) and can be performed in any stage of the oestrous cycle, including anoestrus. The mare will be ready to go home on the same day.
ICSI = the technique where a single sperm cell is injected into a matured oocyte with specialized equipment. To date, we have sent the oocytes to Avantea, a laboratory for advanced technologies in animal reproduction in Italy, where they perform the ICSI, embryo development and freezing
Development of the embryo – after successful fertilisation of an oocyte, the early embryo develops for 7-9 days in the laboratory.
Freezing of the embryo – cultured embryos of good quality are then frozen. Due to the small size of the embryos they handle the freezing process remarkably well.
Transplantation of the embryo – the frozen embryo can be stored indefinitely then carefully thawed when a suitable recipient mare is available.
Why choose for OPU & ICSI (and not ET)?
The recovery of embryos via embryo transfer (ET) has several limitations, it requires the donor mare to have a functional reproductive tract (at least to the point that sperm can reach the oviduct, the mare ovulates normally, and the oviduct and uterus can support fertilization and development of the embryo until day 7 or 8 after ovulation), breeding the donor mare requires a full insemination dose of good-quality semen and only the potential embryo resulting from ovulation of the one dominant follicle (or sometimes two if the mare double-ovulates) is available. As explained elsewhere (hyperlink), use of ET in mares in competition can be complicated by the effects of stress. Lastly, repeated manipulation of the mare’s uterus can be associated with induction of endometritis. Taking this into account, OPU and ICSI may be more successful for some mares and/or certain mare-stallion combinations.
The technique of oocyte recovery and intracytoplasmic sperm injection bypasses the uterus, oviducts and abnormalities in ovulation and can be the solution for the production of foals from mares with chronic uterine infections, suspected oviductal pathology, or mares that repeatedly have haemorrhagic anovulatory follicles. However, ICSI can be less helpful if the cause of the mare’s subfertility is due to a genetic anomaly.
In addition, for clients with limited access to semen (deceased stallion, sub-fertile stallion, limited supplies of frozen semen available), ICSI derived embryos may be a reasonable alternative, as only one sperm is needed for fertilisation. One straw of frozen semen can generally be used for up to 10 ICSI sessions In case of untimely death of a mare, or impending euthanasia, oocytes can be harvested from the ovaries post-mortem and used for ICSI as well.
A big advantage of OPU and ICSI is that these techniques can occur at any stage of the reproductive cycle of the mare (including anoestrus), and thus all year round, as the embryo is frozen and can be stored indefinitely then thawed when a suitable recipient mare is available.
Lastly, OPU is a stand-alone procedure for the mare without hormonal intervention and costs less time in comparison to ET (which involves tracking the mare’s cycle, breeding her and then flushing her a week later), making it a potentially more desirable procedure for mares in athletic competition.
It is good to realize that the OPU procedure has to be performed in a well-equipped clinic and so Equiception B.V. does NOT offer this as a mobile service. Donor mares will have to be brought to Equiception B.V. clinic location in Zegveld.
What are the risks of OPU?
There are risks associated with many veterinary procedures. This is also the case with OPU. Possible complications that may occur include: bleeding (rectal, vaginal and ovarian), rectal tears, rectal and ovarian abscesses and peritonitis, which in the worst case, can lead to the death of the mare. At Equiception B.V. we only work with experienced professionals who assess these risks and if they think it is necessary, will stop the procedure. Nevertheless, mare owners must be aware that transvaginal follicle aspiration carries a risk for the donor mare, it is not a benign procedure. The transvaginal probe is exposed to bacteria as it passes through the vestibule when it is inserted into the vagina. Thus, the needle transits through a potentially non-sterile environment as it is extended from the guide through the vaginal wall into the peritoneum and ovary.
Each OPU procedure is time limited to 45 minutes and the mare is then allowed time to wake up and is walked back to her stable. Usually mares are fit to travel back the same afternoon after completing the procedure. The evening and day after the treatment, some mares can be quiet with a reduced appetite and an elevated temperature. In some cases they are lethargic for a couple of days, but this rare.
Does OPU affect the future fertility of my mare? Will she run out of eggs?
The physical effects of OPU are short lasting, as described earlier. Within 3 days the ovary itself is more or less recovered and if the mare is cycling, will develop a new wave of follicles and go on to ovulate normally. OPU has no impact on subsequent chances of pregnancy or embryo retrieval as the oviducts and uterus are not affected. Even after repeated OPU procedures there are no significant changes to the structure or function of the ovary.
A mare is born with all the primary oocytes (eggs) that she will ever have – these remain at a microscopic size in the tissue of the ovary. Groups of these oocytes are recruited every cycle and begin to develop an antrum or fluid filled follicular space as they mature and then if selected – ovulate. There is constant hormonal communication involving feedback from the brain and cells lining each follicle. At every stage the cells lining the follicle change in sensitivity to the hormones they receive and produce, and slowly follicles fall out of the group selection, becoming atretic and die. This means that toward the end of the cycle there is usually only 1 dominant follicle remaining to be ovulated. The others were always destined to fail. With OPU we simply harvest as many of the oocytes as possible from the follicles large enough to puncture. It can be said, we are actually SAVING the oocytes, that would have been lost anyway, without touching the dormant microscopic follicles for future cycles.