Assisted reproductive technologies (ARTs) are breeding techniques used to produce foals from subfertile mares and stallions. Some techniques, such as oocyte transfer, are primarily used for mares who are unable to carry a pregnancy to term or even produce embryos for embryo transfer.
Other techniques, such as intracytoplasmic sperm injection (ICSI), benefit subfertile and even infertile stallions as they only require one viable sperm to successfully achieve pregnancy.
Generally, assisted reproductive techniques used in horses require a donor mare to supply an egg, a recipient mare to carry the resulting foal, and sperm from a desired stallion. Harvesting oocytes from donor mares is an invasive and costly procedure, so typically ARTs are only performed if repeated breeding attempts between the desired pair have been unsuccessful.
The main ART used today with subfertile stallions is intracytoplasmic sperm injection, but it can be used for infertility in both mares and stallions. In this procedure, the oocyte is injected with a single sperm in a laboratory setting, and the resulting embryo is placed into a recipient mare or frozen for future embryo transfer.
Assisted Reproductive Technologies for Breeding Horses
Assisted reproductive technology (ART) typically refers to a group of reproductive procedures that use eggs from a donor mare to produce offspring. The main ARTs in equine reproductive are: 
- Embryo transfer
- Oocyte transfer
- Intracytoplasmic sperm injection (ICSI)
- Gamete intrafallopian transfer (GIFT)
- In vitro fertilization (IVF)
Advantages and Disadvantages
Typically, the most advanced assisted reproductive techniques are used in one of two situations: 
- Mares with fertility issues that prevent them from producing embryos for embryo transfer or who are unable to carry a foal to term
- Using very poor-quality semen or semen in extremely limited supply, such as after the death of the stallion, to maximize pregnancy rates
The primary drawback of ARTs is that they require the invasive collection of oocytes (eggs) from the donor mare, demanding specialized expertise from the veterinarian. ART is also more expensive than more routine methods of breeding, because it requires specialized equipment for oocyte processing.
The first step for all assisted reproductive technologies is recovery of oocytes from the donor mare. The two main methods of oocyte recovery are transvaginal oocyte pickup and flank puncture. 
Transvaginal oocyte pickup (OPU) of immature oocytes is becoming more common, as it is less invasive and more flexible regarding the timing of the procedure and handling of the oocytes. 
Mature vs. Immature Oocyte Collection
As part of the ART protocol, the veterinarian must decide whether to collect mature or immature oocytes from the donor mare. The veterinarian will select the best option based on their own skillset, equipment available, and other factors.
A mature oocyte is a fully developed egg cell that is ready for fertilization. Mature oocyte harvesting occurs in the period right before ovulation, requiring the veterinarian to track the mare’s estrus cycle closely so that harvesting occurs at the correct time point. 
- May improved oocyte quality, as the mare’s hormones stimulate maturation rather than artificial maturation after harvesting
- Oocytes are easier to remove as they loosen from the follicle wall in the mature stage
Immature oocytes are undeveloped egg cells that have not yet completed the necessary stages of development to be fertilized. Immature oocytes must undergo a maturation procedure in the laboratory before they can be used for ICSI. 
The main disadvantage of using immature oocytes is that they can be more difficult to remove from the follicle because they tightly adhere to the follicle wall.  This can lead to ovarian tissue damage if the veterinarian has to scrape the follicle wall to extract the oocytes.
- The oocytes are less affected by temperature variation after harvesting
- This method does not require the veterinarian to track the mare’s estrus cycle
Flank Puncture Procedure
The flank puncture procedure is a surgical technique in which the ovary is accessed through the flank of the mare to retrieve oocytes.
Prior to oocyte recovery, mares receive a dose of gonadotropin, a hormone that stimulates the ovaries to produce 1 – 2 mature follicles that are ready for ovulation.  These follicles contain the oocytes harvested during the recovery procedure.
In horses, inducing “superovulation” (the maturation of multiple follicles) is less effective than in other species. The unique anatomy of the mare ovary usually only allows for the aspiration of only one or two mature follicles at a time per ovary per cycle. 
To perform a flank puncture, the veterinarian places a cannula in the flank of the mare. They then would manipulate the ovary into position via rectal palpation and a needle is passed through the cannula into the mature follicle. 
Once the follicle is targeted, its contents including the oocyte and surrounding fluid are drawn into a syringe through a process called aspiration. The collected oocyte then undergoes further examination for use in assisted reproductive techniques.
Transvaginal Oocyte Pickup (OPU) Procedure
Transvaginal Oocyte Pickup (OPU) is another type of recovery procedure where eggs are collected from the ovaries of a mare using a needle inserted through the vaginal wall. Typically, this procedure is used for gathering immature oocytes, but can also be used for mature oocytes
OPU does not require stimulation of the mare’s ovaries, and can be performed at any point in the mare’s estrus cycle.  Some large operations perform OPU procedures every 14 days on their mares, to maximize the oocyte recovery. 
In OPU, the veterinarian inserts a specialized ultrasound probe into the mare’s vaginal canal. The probe has a channel for a needle attached to a vacuum pump.  After placing the probe, the veterinarian manipulates the ovary into place through rectal palpation, until the ovary is visible on the ultrasound screen.
The veterinarian then insert the needle into a follicle and activates the vacuum pump to drain it.  The veterinarian may also choose to fill and then drain the follicle with sterile fluid several times to try and loosen the oocytes from the follicle wall. 
The procedure is repeated for each follicle that is accessible to the veterinarian.
Oocyte transfer is a reproductive technique where an oocyte (egg) recovered from a donor mare is transferred to a recipient mare’s oviduct for fertilization and subsequent development.
The oviduct, also known as the fallopian tube in humans, is the tube that connects the ovary to the uterus in female mammals. This is where fertilization of an egg by a sperm typically occurs in natural mating.
In this procedure, the recipient mare’s oocytes are first removed and replaced with the oocyte from the donor mare to ensure that any offspring produced have the donor’s genetic material.
Benefits of Oocyte Transfer
The main reason for using oocyte transfer is to produce foals from mares that cannot produce their own embryos. Common conditions in the donor mare that may require oocyte transfer include: 
- Chronic endometritis
- Damage to the uterus or cervix from a previous injury or dystocia (Difficult labour)
Drawbacks of Oocyte Transfer
- Requires good semen quality to maximize the chance of pregnancy
- There is a risk that not all of the recipient mare’s oocytes are removed, resulting in an embryo with the recipient mare’s genetics
- Only able to impla