Equiception - EN

Milandweg 69
3474 KK Zegveld
Nederland

info@equiception.com
OPU Filler 600x959 - Equiception - EN

Two separate procedures, which are inextricably linked

OPU & ICSI

The topics OPU and ICSI have been discussed frequently amongst horse breeders over the previous years. Everyone has an opinion on this matter. But what exactly does it entail, how do the different procedures take place and what are the pros and cons? Here we would like to further explain these relatively new reproductive procedures, which are now offered at Equiception BV.

First of all, it is good to realise that OPU and ICSI are two separate procedures, which are inextricably linked.

Knowledge base

FAQ

OPU & ICSI

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).

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 a 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 at 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 partnered with a world class laboratory which performs 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.

There are risks associated with many veterinary procedures. This is 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 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. Some mares can be quiet with a reduced appetite and an elevated temperature the evening and day after the treatment. In some cases they are lethargic for a couple of days, but this rare.

The recovery of embryos via embryo transfer (ET) has several limitations. These limitations are:

  • 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 here in further detail, 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. If there is an 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 does NOT offer this as a mobile service. Donor mares will have to be brought to Equiception’s clinic location in Zegveld.

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.

OPU - Equiception - EN

Ovum Pick-Up

OPU

OPU stands for Ovum Pick-Up, meaning the recovery (flushing) of oocytes, and is the first step in the complete process.

The detailed preparation of the mare usually takes as long as the actual OPU-procedure, and is necessary for a successful result. The mare is sedated and an anti-inflammatory drug, pre-procedural antibiotics and intestinal relaxant are administered. A urinary catheter is placed and finally the mare receives an epidural so that the procedure is as safe and pain-free as possible.

During the OPU-procedure, the veterinarian will rectally locate and re-position the ovary. A specialised vaginal ultrasound probe, with a needle guide attached, is inserted into the mare’s vagina. The ovary is retracted carefully so that it communicates with the vaginal probe, only being separated from it by the vaginal wall. The complete ovary can now be visualized properly. The follicles (containing the oocytes) are then individually punctured, drained, scraped and flushed multiple times with the help of a long needle attached to the vaginal probe. The procedure is repeated for the other ovary. The retrieved fluid is taken to the laboratory to be filtered and examined for the presence of oocytes. The identified oocytes are ‘washed’, assessed and packaged for transport to our partner Avantea in Italy, a specialised laboratory for advanced veterinary reproductive procedures. Further maturation of the oocytes, the ICSI-procedure and the development of the embryos all take place at Avantea.

Foto stalling zorg 600x1000 - Equiception - EN

THE MARE

EFFECT ON THE MARE

The actual OPU-procedure will last no longer than 45 minutes. Usually mares are fit to travel back home the same day after completing the procedure.

The evening and day after the treatment, some mares can be quiet with a reduced appetite and have an elevated temperature.

It is important to realise that there are risks associated with many veterinary procedures, as 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. Of course Equiception BV only works with experienced professionals who assess these risks and if they think it is necessary, will stop the procedure.

Lastly, it should be said that depending on how many follicles a mare develops and how quickly she recovers from the procedure, an interval of at least 3 weeks between two OPU sessions on the same mare is regularly achieved. Mares are also often bred and flushed for an embryo in between two OPU sessions; OPU has no negative impact on the natural fertility of the mare.

ICSI - Equiception - EN

Intra Cytoplasmatische Sperma Injectie

ICSI

ICSI stands for Intra Cytoplasmic Sperm Injection: the injection of one single sperm cell into a matured oocyte, after which an embryo may develop. This is an advanced form of IVF.

After successful fertilisation of an oocyte, the early embryo develops in the laboratory for 7-9 days, after which it will developmentally be the equivalent to a ‘normal’ 5-6 day embryo.

This means ICSI-embryos are very small and thus tolerate the freezing procedure remarkably well. The frozen embryos are stored at Equiception BV. and can be thawed and subsequently transferred into a suitable recipient mare whenever it is desired.

ADVANTAGES

OPU and ICSI has many advantages; it can occur at any stage of the reproductive cycle of the mare (including winter-anoestrus) and year round as the embryo is frozen and can be stored indefinitely, then thawed when a suitable recipient mare is available (for example early in the breeding season).

Synchronisation of multiple recipient mares with the donor mare, as with embryo transfer (ET), is no longer needed, making it easier to also utilize a client-owned recipient mare.

With OPU and ICSI, embryos can often still be obtained from mares that do not become pregnant or do not give embryos via ET.

Also, in case of a sudden death or planned euthanasia of a mare, it is often possible to retrieve for ICSI suitable oocytes post-mortem.

Furthermore, OPU is a stand-alone procedure for the mare without hormonal intervention, possibly interesting for mares in athletic competition.

And finally, as only 1 sperm cell is needed for every injected oocyte, this technique offers great advantage when the supply of semen is limited, as is the case for deceased stallions, sub-fertile stallions and, last but not least, for extremely costly and exclusive frozen semen. A single straw of frozen semen can be used for up to 10 ICSI sessions.

RESULTS TO BE EXPECTED

CHANCE OF AN EMBRYO

At Equiception we reach a fairly consistent oocyte recovery rate of 65%, meaning that we recover an oocyte out of ± 65% of the punctured follicles. Nonetheless, these results will vary as some mares will give a lower yield (30%) and others occasionally reach a recovery of 100%.

After maturation and assessment, approximately 60% of the oocytes will be injected with a sperm cell. The overall chance of one or more frozen embryos after a single OPU session is approximately 60%. The average amount of embryos per successful OPU session is 1,9.

In summary, this means that on average out of 10 different OPU-ICSI sessions, 4 attempts will be unsuccessful; on the other hand, the other 6 successful attempts will have yielded 12 embryos total.

CHANCE OF A PREGNANCY

After thawing and transferring the ICSI-embryo into a recipient mare, approximately 80% will result in a pregnancy. The early embryonic mortality after transfer of ICSI-embryos is also slightly higher than with a ‘natural’ pregnancy, namely approximately 15%.

DONOR MARE REQUIREMENTS

REQUIREMENTS

Due to the many steps in the process and the cumulated fall-out associated, enough follicles should be present to puncture to increase the chance of producing embryos. We recommend that the mares that are brought to us for OPU have at least a total of 15 follicles (≥ 1 cm diameter) on their ovaries. Multiple smaller follicles on the ovaries is desirable for a successful outcome of the OPU-procedure.

To avoid disappointment, the mare must be checked and have her follicles counted by a veterinarian at home before coming to Equiception BV. for the OPU-procedure.

In addition, prior to the procedure the mare needs to be tested (and proved negative) for EIA (Equine Infectious Anaemia) and CEM (Contagious Equine Metritis). These are export requirements before the oocytes can be shipped to Italy. Both these results have to be known three days prior to the planned date of the OPU-procedure.