Equiception - EN

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3474 KK Zegveld
Nederland

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Embryo transfer

Embryo transfer (ET) refers to the removal of an embryo from the uterus of the donor mare and the transfer of that embryo into the uterus of the recipient mare. The first foal produced by embryo transfer was born in 1974 and today embryo transfer is a common reproductive procedure in the equine breeding industry.

Knowledge base

faq

EMBRYOTRANSFER (ET)

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 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 will not be held accountable for the possible consequences of using a less than ideal client-owned recipient mare.

Equine embryos can be frozen/vitrified successfully, however this is not a straightforward procedure and because of the significant challenges involved Equiception does not currently offer this service.

Only small equine embryos (<300μm) seem to be able to be frozen and then successfully used to impregnate a mare. Using frozen larger embryos is not seen as a viable technique as it has resulted in 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. However, a second flush may be performed on the mare several hours later. Adding to what is already a difficult task 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 appropriately sized embryo versus one too large to freeze.

Given the very small window to perform these early flushes, Equiception is not able to logistically perform them for clients.

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 specialised drills to make small holes in the embryo. Successful operation of this equipment requires considerable expertise.

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 the quality of the embryo, factors associated with the recipient mare and the experience of the person performing the transfer. It is also important to be aware that there is an 8 to 10% chance 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.

Equiception has a pregnancy rate of 80% when transferring embryos to our own recipient mares. However, there is a significant variation of results associated with the time of season, with a noted decline in pregnancy rates in periods of high temperature.

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.

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.

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.

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ADVANTAGES

Potential advantages of embryo transfer are:

  • Mares can have multiple foals per year
  • Mares in athletic competition can remain in performance and still produce offspring
  • Medical risks associated with pregnancy and parturition are avoided for the (valuable) donor mare
  • Older mares can produce embryos to be carried in young recipient mares
  • Mares with certain reproductive problems are able to produce embryos to be carried by reproductively healthy recipient mares
  • Mares with a history of losing pregnancies can produce embryos to be carried in recipient mares
  • Mares with serious medical or musculoskeletal problems can produce embryos to be carried in recipient mares
  • Mares that foal late in the season or are still open for whatever reason can produce an embryo and then remain open to be bred early the following year

Embryos can be cooled and transported to another location for transfer

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POTENTIAL FOR SUCCESS

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 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.

EMBRYO COLLECTION PROCEDURE

FLUSH PROCEDURE

A nonsurgical, transcervical uterine lavage is the standard method of embryo collection in the mare. So called ‘Y-tubing’ connects a uterine lavage catheter to both a bag of special flush medium and to a holding cup with an embryo filter. All tubing is sterilized prior to use. Fluid will run from the bag into the uterus of the mare and back out again through the filter, with clamps on the ‘Y-tubing’ regulating in- and outflow. If an embryo is present, it will be flushed out of the mare’s uterus and will be retained in the cup as it not able to pass through the embryo filter.

During the procedure the donor mare is restrained in safe examination stocks. Most mares undergo the procedure well and do not require sedation to safely and effectively perform the uterine flush, however sedation may be warranted in young, excited or inexperienced mares. The mare’s tail is wrapped, manure removed from the rectum and the vulva and vestibule are cleaned thoroughly. The ‘Y-tubing’ is connected to the bag of flush medium, the uterine catheter and the filter cup and the entire system is preloaded with flush medium to prevent air from entering the mare’s uterus. The uterine catheter is then manually inserted into the mare’s vagina and through the closed cervix, into the uterus. The cuff of the lavage catheter is inflated with air and pulled back against the cervical opening on the inside of the uterus, hereby sealing off the uterus. Approximately 1 litre of warmed flush medium is infused into the uterus by gravity flow. The aim is to expand the uterine lumen sufficiently to allow fluid to effectively reach all parts of the uterus, including the area between the endometrial folds. Hence, the amount of fluid used for each flush is dependent on the size of the uterus; young maiden mares may only be able to hold 500 mls for example. The flush medium is then allowed to flow back out of the uterus through the catheter to the embryo cup and through the filter by gravity flow. This process is repeated a total of three to six times. Rectally massaging the mare’s uterus during infusion and recovery of the flush media ensures both horns fill and empty properly and can assist with recovery of the embryo. Furthermore, the drug Oxytocin may be administered to aid in fluid recovery by stimulating uterine contractions. After completing the lavage procedure, the uterine catheter’s cuff is deflated and the tubing is removed from the mare. Lastly, the remaining fluid in the tubing is allowed to flow through the embryo filter. The cup and its contents are then brought into the laboratory for further processing. A prostaglandin injection is administered to the mare before returning her to her stall. The goal is to allow the mare to return to oestrus by lysing the corpus luteum; hereby minimizing the chance of a bacterial infection and eliminating the possibility that a pregnancy will continue in the donor mare if in the unlikely event an embryo was left behind.

EMBRYO SEARCH, HANDLING AND EVALUATION

Sometimes a larger embryo may be directly visualized in the embryo filter cup, however, in most instances a microscope is needed to help identify the embryo. The contents of the filter cup are poured into a sterile plastic see-through search dish. A final rinse with flush medium is performed on the embryo filter and cup to ensure the embryo is not left behind. The search dish is then examined for the presence of an embryo with the aid of a microscope.

Once an embryo has been identified, it is removed from the search dish and is ‘washed’ by gently moving it through multiple drops of embryo holding medium and then placed in a dish containing embryo holding medium. The embryo is evaluated to determine embryo quality based on embryo morphology and a grade is assigned to the embryo. This grading is positively associated to pregnancy rate after transfer.

Most embryos collected from donor mares are good to excellent in quality, grades 2 and 1 respectively. Poor quality and dead embryos are likely retained in the oviduct and therefore will rarely enter the mare’s uterus, however they will still be recovered occasionally. Even though chances of a successful pregnancy are significantly lower for these grade 3 and 4 embryos, usually the decision to transfer them will still be made as without a transfer they will certainly not have any chance of survival.


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Donor mare management

Equiception performs the embryo collection and transfer procedures, however we rely on the client’s regular veterinarian to appropriately manage and breed the donor mare prior to the day of flushing. Therefore, a crucial part of producing pregnancies via ET is out of our hands, with increasing success when both parties know what is expected and communicate well with each other.

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EMBRYO TRANSFER PROCEDURE

Equine embryos are routinely transferred into a recipient mare using a non-surgical, transcervical procedure.

Usually a final evaluation of the chosen recipient mare is performed just prior to the transfer procedure. Besides the presence of a morphologically normal corpus luteum, special attention is also paid to the presence of good muscular tone in the uterus and cervix and the absence of uterine oedema, these are associated with an elevated endogenous progesterone level. In case several recipient mares are available, the most satisfactory one will be chosen and prepared for the transfer procedure.

The recipient mare is restrained in safe examination stocks. She is sedated 5 to 10 minutes prior to the procedure. A non-steroidal anti-inflammatory drug is administered to prevent the possible production of prostaglandins associated with cervical manipulation during the transfer. These prostaglandins could alter the quality of the corpus luteum and cause a decrease in progesterone levels.

The mare’s tail is wrapped and the vulva and vestibule are cleaned thoroughly. The embryo is loaded into the transfer instrument and the transferring veterinarian holds the tip of the transfer gun within a hand covered by a sterile obstetrical sleeve. The hand is inserted into the recipient mare’s caudal reproductive tract and then gently directed forward towards the cervix. The entrance to the cervix is located and the tip of the transfer instrument directed there. The protective chemise is penetrated as the transfer instrument enters the cervix. The transfer instrument is then gently passed through the closed cervix with as little manipulation of the cervix as possible. The embryo is then deposited in the lumen of the uterine body or horn.

Occasionally embryos are not discharged from the transfer instrument properly and can become caught in the steel tip of the instrument. After transfer the stainless steel tip of the disposable sheath of the transfer instrument is rinsed and the rinse fluid checked under a microscope for the presence of the embryo.

After transfer it is important to minimize (social) stress in the recipient mare and thus keep her in her original herd for a while before moving her to a new group of mares.

Alternatively, equine embryos can be transferred transcervically using a so-called Wilsher forceps. This set of specialized forceps are used to grasp the cervix to pull it back and straighten it, hereby facilitating the passage of the transfer instrument trough the cervical canal. To enable viewing of the cervix directly, a sterile vaginal speculum is used in combination with the Wilsher forceps.

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