Fertilisation facts

Part 2

Following on from our article in Issue 161b, where we discussed live covering and artificial insemination, we return to look at the roles of intracytoplasmic sperm injection (ICSI), ovum pick-up (OPU) and embryo transfer. Once you understand all of these options, you're pretty close to becoming a breeding pro!

IVEP

The combination of ICSI and OPU together is known as In Vitro Embryo Production, or IVEP for short. In order to understand IVEP, we need to understand each technique individually and see exactly how they work together.

ICSI

ICSI, or Introplasmic Sperm Injection, is a modern breeding technique where a single sperm cell is injected directly into the cytoplasm of a single mature egg/oocyte to fertilise it.

ICSI

OPU

OPU is Ovum Pick Up, or Oocyte Aspiration.

The process

The mare will be sedated to prepare for the pick-up process; the procedure usually lasts two hours. Before the aspiration, a vet or breeding technician assesses the ovaries to ensure the mare's suitability. A suitable candidate will have a minimum of fifteen follicles with a diameter of less than one cm.

Oocytes are too small (125 - 150mm) to be seen via ultrasound, but follicles can be viewed. One follicle contains one oocyte. An ultrasound probe with a needle guide is placed into the vagina. The ovary is then grasped via the rectum and held against the vaginal wall. The fluid of each follicle is aspirated and then flushed several times to increase the oocyte recovery success. Once this has been completed, the recovery fluid is filtered. The filtered liquid is then searched for the presence of oocytes. OPU has a 75% success rate.

Oocytes are then safely transported and allowed to cultivate until maturity. Once maturity is reached, typically within 24 hours, ISCI will take place, and then the division to become embryos will begin just 24-30 hours post-injection. The development of an embryo takes between seven and nine days. Once an embryo has developed, breeding technicians will correctly freeze the embryo for when there is a suitable donor.

WHAT NEXT AFTER IVEP?

There are three types of transfer available:

1. Fresh transfer of embryos into the recipient mares

The embryo produced in the ICSI lab can be transferred into a synchronised recipient mare.

2. Auto-transfer back to donor mare

If the donor mare is young and healthy, she may be able to carry her ICSI-produced embryo. This transfer takes place primarily when the mare is healthy, fertile, and has no history of dystocia* or problem pregnancy.

3. Embryo vitrification and storage of embryo

Embryo vitrification is a means of cryopreserving (freezing) embryos to allow them to be kept indefinitely and utilised later. Once vitrified, the embryo is kept in nitrogen until use.

Advantages

ISCI is considered highly beneficial for both mares and stallions. With stallions, if there is little sperm available, ISCI is excellent, as you only have to utilise a minimal amount of frozen semen. Similarly, with mares with fertility issues, ICSI has proven very successful.

IVEP, as a combined procedure, is also convenient as it provides a variety of options for the transfer of the embryo and allows the mare to continue competing if this is desired.

Disadvantages

The OPU procedure is relatively invasive, and both ICSI and OPU are expensive.

*Definition of dystocia = a difficult or obstructed labour.

EMBRYO TRANSFER OR SURROGATE PREGNANCY

Both the donor and recipient mares will undergo a pre-breeding check.

The donor mare is then inseminated using artificial insemination. Once the embryo is formed, it is flushed out of the uterus and implanted into the recipient mare.

Before starting with the process, it is essential to have a recipient or surrogate ready. Many people suggest having a few mares available. When selecting surrogates, it is vital to remember the mare will need to look after the foal once it is born, so her behaviour will play a massive role in how the foal develops. Therefore, it is recommended that you find a mare or mares with pleasant temperaments who are easy to work with, between the ages of 4-12 years, and of a similar size to the donor. Many believe a smaller surrogate may create a smaller foal.

The process

The process of embryo transfer can be broken down into six steps:

Step 1: Synchronisation

Both the recipient mare and donor mare will be synchronised using hormonal medication. To have the recipient 'ready', she will be synced to ovulate either three days after or at the same time as the donor. Synchronising is to ensure that both uteri are at the same stage. Regular scanning will take place to monitor both mares’ progress.

Step 2: Insemination

The insemination process will be the same as regular AI (see Issue 161b to learn more). It is vital to know the date of insemination for the subsequent steps.

Step 3: Flushing

A vet or breeding technician will flush the donor mare's uterus with a special medium six to eight days post-ovulation. This process is not painful for the mare. Then, the fluid will be drained with an embryo filter and the embryo will be isolated visually or microscopically.

Step 4: Washing

Once the embryo is isolated, it is 'washed' to ensure there is no residual contamination from the donor mare that could be passed onto the recipient mare.

Step 5: Transferring

Immediately after 'washing', the embryo is placed into an insemination pipette, and during that process, the recipient mare is checked via ultrasound to ensure she is 'ready'. The embryo will then be transferred. The success rate for the transfer is around 57%.

Step 6: Scanning

Seven to nine days post-transfer, the recipient will be scanned to confirm a pregnancy. If there is a viable pregnancy, the mare will be placed on Regumate (progesterone supplement) until day 28, when the heartbeat scan will take place. A final scan will take place at the 45-day mark. Many vets suggest the surrogate stay on Regumate until day 120.

Advantages

The significant advantage of using this method is that top mares can still be competitive while increasing the gene pool. Unlike top-level stallions who can breed while still competing, mares are unable to compete while heavily in foal and while weaning their foal. In many cases, having a foal takes the mare out of the sport for almost two years. Another advantage is that embryo transfer allows mares with physical issues that prevent them from carrying a foal, to have a foal using a surrogate mare.

Disadvantages

Although ET has helped the breeding game immensely, it is not accessible to everyone due to the high cost and the fact that multiple mares are commonly required. For these reasons, it is usually only stud farms that use this technique.

Take home message

There are many breeding options available out there, and with all the complexities, it is best left up to the experts to decide the best option in each case. In our next issue, we'll be looking at the pregnancy process, so check back to find out more.