When breeding with live cover—which is rarely done today in the sport horse industry—successful pregnancy can be thwarted by any number of factors. Those can include poor stallion fertility, endometritis issues and increasing mare age. To improve fertility and pregnancy rates, assisted reproductive technologies (ARTs) can mean dramatic improvements. Now, thanks to those ARTs, we can produce more than one foal from one mare in a single year.
In vitro embryo production via ovum pick-up (OPU) and intracytoplasmic sperm injection (ICSI) is the most advanced of the ARTs currently utilized in clinical practice. They were developed in the 1990s and have undergone slow development. However, many hurdles have been overcome. This technique has enjoyed remarkable innovations in the past several years and is now a “fan favorite” among Warmblood breeders as well as those in the sport horse sector.
While OPU-ICSI is used in dual-purpose mares (breeding and competition), this is not its typical use.
“OPU-ICSI is also beneficial for circumventing various fertility problems in both mares and stallions that other breeding techniques cannot remedy,” said Tom Stout, VetMB, PhD, of the Department of Equine Sciences at Utrecht University in The Netherlands.
Some of the reasons that theriogenologists select OPU-ICSI as their assisted reproductive technology of choice includes repeated endometritis, a torn cervix, and ovulation failure (i.e., when mares are sub-fertile using other breeding techniques).
“We get a lot of old or problems mares presenting for OPU-ICSI,” affirmed Stout.
This article briefly describes the OPU-ICSI process and how this technology might be integrated into the management of mares with inhospitable uterine environments (e.g., caused by endometritis) and older mares that might have declining fertility.
The process of producing in vitro embryos seems outwardly straightforward, but it actually is an incredibly intricate and delicate process.
First, oocytes are recovered from the ovary via transvaginal ultrasound-guided follicular aspiration (TVA), which is synonymous with OPU. Those oocytes are then transported to a laboratory capable of performing oocyte maturation, ICSI, and culturing the embryos to the blastocyst stage. The resultant embryos can then be implanted directly into the recipient mare. However, it is far more common to cryopreserve or vitrify the resultant embryos for later use.
Tamara Dobbie, DVM, DACT, an associate professor of Clinical Reproduction at the University of Pennsylvania’s New Bolton Center, added, “We can aspirate oocytes at any stage of the estrous cycle, but we like to perform the procedure when there are many, at least 10, small follicles between 10 and 25 mm. We get the best recovery rates when the follicles are small.”
Collecting those oocytes from the follicles is tricky. According to Dobbie, the oocyte is tightly attached to the wall of the follicle in mares. Oocyte recovery depends on aspirating the follicular contents as well as actually scraping the wall of the follicle with the needle.
Dobbie explained that, “We use a 12-gauge double lumen needle. We insert the needle into the follicle using ultrasound guidance and aspirate the contents of the follicle first. Once the follicle has collapsed, we vigorously scrape the wall of the follicle with the needle and then refill the follicle to its original size with flush media. We repeat this procedure five to 10 times for each follicle on the ovary.”
In Dobbie’s hands, the oocyte recovery rate is generally between 60% and 70%.
“As the follicle gets larger (more than 25 mm), it’s harder to recover the oocyte because it’s impossible to scrape the entire inside of the follicle with the needle,” she said.
After collection, the oocytes are held in media at room temperature overnight and subsequently evaluated by the ICSI laboratory team the following day. If collected at a distant facility, the oocytes can easily be shipped overnight at room temperature to the ICSI laboratory.
“At the ICSI lab, nondegenerate oocytes are placed into maturation media for approximately 30 to 35 hours,” said Dobbie. “Oocytes that resume development in culture will undergo ICSI, which is the procedure whereby a single, normal, motile sperm is injected into the oocyte under the microscope.”
Blastocysts, or embryos, can develop in six to 10 days following the ICSI procedure. Fertilized oocytes that develop into blastocysts in six to seven days are considered the best quality because development is occurring in a rapid fashion. Development is considered delayed if it takes longer to reach the blastocyst stage, but even Day 8 and Day 9 embryos can still yield acceptable pregnancy rates.
“Pregnancy and foaling rates are lower for blastocysts that take 10 days to develop,” stated Dobbie.
All embryos, regardless of the number of days it takes to reach the blastocyst stage, are considered a Day 6 embryo when they are transferred into a recipient mare. From there, recipient mares are examined seven days later with the hope of seeing an embryo consistent with a Day 13 pregnancy.
Complications and Benefits
With the degree of skill required to perform TVA/OPU-ICSI, it’s not surprising that each step of embryo production is costly, labor-intensive and wrought with complications that can decrease the success rate.
As one could imagine based on the above description of flushing, scraping and aspirating the oocytes, TVA/OPU is not a benign procedure—complications can occur. According to Dobbie, the most common complication is that the mare develops an elevated temperature and mild colic following the procedure, but this occurs in 1% or less of all TVA/OPU cases. Rectal irritation can also occur (≤5% of cases).
“Other mild complications include minor rectal tears, ovarian inflammation and mild peritonitis,” said Dobbie. “More severe complications occur in approximately 1 in 500 procedures and include ovarian abscesses, peritonitis, bleeding and more serious rectal tears. Death occurs in approximately 1 in 1,000 procedures.”
Despite potential complications and disappointing outcomes, the benefits of OPU-ICSI include being able to produce embryos at any time of the mare’s cycle and during any season. Those embryos can be vitrified (or frozen), which means the embryos can be thawed and transferred into a recipient mare at any time in the future.
According to Dobbie, TVA/OPU and ICSI are often the only way to get pregnancies from mares with chronic bacterial or fungal endometritis.
“Despite our best efforts, in some situations it is not possible to rid the mare’s uterus of bacteria and fungi, making even embryo transfer impossible,” she said. “TVA and ICSI have become the only alternative for obtaining embryos and pregnancies from these mares.”
Endometritis as a Cause of Infertility
Endometritis is widely viewed as a major cause of mare infertility. Inflammation is a normal mechanical and immunological response to the introduction of semen into the mare’s uterus during breeding. If the resultant fluid persists for longer than 48 hours, the mare is said to have a pathologic condition called persistent mating-induced endometritis (PMIE). If not addressed appropriately, it will markedly reduce the likelihood of pregnancy and could lead to persistent infections with bacteria or fungi—all of which are far more challenging to treat than PMIE.
“When faced with a mare suspected of having a bacterial endometritis, the first thing we do is get an accurate diagnosis,” Dobbie said. “It’s important to be clean when obtaining uterine samples for culture. We want our samples to be representative of the uterine environment and not contaminants.”
Dobbie said it’s easy to contaminate uterine samples during collection, especially in the field.
“Unfortunately, the isolation of bacteria alone does not mean the mare has bacterial endometritis,” Dobbie noted. “There should be other evidence that the mare has a problem. Does the mare have echogenic fluid in the uterus? Is there discharge from the vulva? Does the mare have edema during diestrus?”
She said veterinarians should only treat mares that have uterine infections. Dobbie reminded practitioners that treating mares that aren’t infected is not benign.
“Treatment is expensive, and it can accelerate the development of antibiotic-resistant bacteria,” she reminded. “Unnecessary intrauterine antibiotic therapy can cause secondary bacterial or even fungal endometritis, and systemic antibiotic therapy always carries the risk of inducing a colitis.”
She also pointed out that most uterine infections are caused by a single organism. If you isolate two or more organisms from a uterine culture, odds are the culture has been contaminated. The only exception would be the recovery of Streptococcus equi zooepidemicus and Escherichia coli. These bacteria can be isolated together in the case of uterine infection.
Dobbie said that interpreting uterine culture results is challenging.
“I often struggle to figure out if a mare is infected or not,” she admitted. “I therefore encourage veterinarians to reach out to other colleagues when they encounter a case they are unsure about. We need to be better stewards of antibiotics and not treat every mare that cultures positive.”
Some mares with bacterial or fungal endometritis can be treated successfully with either intrauterine or systemic antimicrobial therapy. Unfortunately, the treatment is not always successful. For those mares, TVA/OPU and ICSI becomes the only way to produce embryos and pregnancies. Veterinarians warn that you must be careful when performing TVA/OPU on mares with active endometritis.
“We still prefer that the uterus is clean before doing TVA/OPU,” Stout said. “Results are worse in mares with an active endometritis. And you need to be more careful not to take bacteria from the vagina into the abdomen/ovary.”
Bacterial or fungal organisms harbored in the vagina can be translocated into the abdomen and ovary, setting the mare up for peritonitis or an ovarian abscess, respectively.
Adding in Mare Age
As mares age and get “superaged,” they can run into reproductive problems. Pregnancy rates and embryo recovery rates decrease, and pregnancy loss rates increase as a mare ages. The age-related decrease in fertility is believed to be due to reduced oocyte quality.
In 2019, Stout and colleagues conducted a retrospective study designed to evaluate if maternal age affected the efficiency of various ARTs as determined by the proportion of mares that yielded at least one Day 45 pregnancy.
In total, reproductive records from 289 mares bred by natural mating, 328 mares bred by AI, 205 embryo donor mares (i.e., artificial insemination/embryo flushing/embryo transfer; AI-EF-ET), and 473 OPU-ICSI-ET mares were examined.
The researchers found that increasing maternal age significantly reduced the reproductive efficiency of all breeding techniques except OPU-ICSI-ET.
For example, the efficiency of AI-EF-ET mares decreased steadily from 50.6% Day 45 pregnancy per mare in the group of mares 1 to 7 years old to only 35.3% in mares 20 years or older (P=0.04). This was an overall decrease of 15.3%.
For OPU-ICSI-ET, the Day 45 pregnancy per mare in 1 to 7 years old was 38.2%, which remained steady with advancing age. In mares 20 years or older, the rate remained stable at 31.1% (P=0.07).
Stout and his colleagues concluded, “It is reasonable to advise horse owners that the breeding technique of choice for advanced age mares is OPU-ICSI.”
Dobbie noted that, “TVA/OPU and ICSI is the best way to get embryos from older mares. Older mares produce fewer follicles, so the number of oocytes recovered will be less. Fortunately, the developmental competence of those oocytes is maintained so the quality of embryos produced is similar to that of younger mares.”
Anticipated Outcomes by TVA/OPU and ICSI
In a 2022 study co-authored by Stout and Anthony Claes, the following success rates were reported:
- 13.8 immature oocytes from 25.9 antral follicles;
- One or more blastocysts (range, 0-13) from 78% of ICSI procedures (so 22% of ICSI procedures did not produce any blastocysts);
- A mean of 2.12 blastocysts produced per session (i.e., ICSI and the following incubation period to see which blastocysts develop to days 6-9);
- The likelihood of pregnancy after embryo transfer was 77.7%; and
- 60% of transferred embryos resulted in a live foal.
Stout clarified those statistics by saying, “Per OPU, there is only a 75% chance of getting anything. If you do, then we expect around 2.5 blastocysts per OPU-ICSI session. From there, about 75% result in a pregnancy and 60% in a foal. So, in 25% of mares/sessions you will get nothing. In the other 75% there is a high chance of at least one foal. However, there is still a lot of individual mare variation.”
Dobbie added, “At our facility, pregnancy rates following transfer of frozen thawed ICSI embryos are approximately 65% and embryonic loss is roughly 15%. Unfortunately, TVA/OPU and ICSI is still a numbers game. The more follicles you aspirate, the more oocytes mature, and the higher the chances are of getting an embryo.”
How do you increase the chances of success?
“Perform a TVA/OPU when the mare has many small follicles,” recommended Dobbie. “The spring transition (February, March and April for mares not maintained under lights) is often a good time to aspirate as this is a time when mares have large numbers of small follicles. Extending the time between aspirations (greater than 14 days) can also increase follicle numbers.”
She noted that the veterinarian might have to aspirate a mare more than one time to produce an embryo.
Stout agreed and emphasized that it is prudent to wait until the mare has more follicles.
“If you get nothing when you tried with 10 follicles, then wait longer than two weeks between aspirations until she has more,” he said.
In addition, Stout said, “Make sure the mare is in good health/body condition.”
Obesity and Fertility
According to D’Fonseca et al. (2021), “Obesity has been associated with altered reproductive activity in mares and may negatively affect fertility.”
In that study, Shetland pony mares were fed a high-energy diet containing 200% net energy requirements over the course of a three-year study period. Day 7 embryos were flushed from donor mares and transferred to recipients. On Day 21 after embryo transfer, the conceptuses were collected and their development assessed.
The researchers found that conceptuses collected from obese mares fed the high-energy diet were more likely to succumb to early embryonic death. The research team also found an extended period of annual cyclicity in the obese mares compared to the control group with a healthy body condition score. The period of cyclicity was calculated based on the number of days between the first and last ovulation in each year of the study.
When itemizing the negative effects of obesity on reproductive efficiency, the researchers relayed the following: Obesity might increase the likelihood of mares developing a hemorrhagic anovulatory follicle (HAF), which does not release an oocyte and therefore reduces fertility. Further, a persistent luteal structure might develop from an HAF that could extend the inter-ovulatory interval.
Reduced oocyte quality and altered embryonic development have been reported in women. Overweight/obese women have significantly lower clinical pregnancy and live birth rates and a significantly higher risk of miscarriage following in vitro fertilization compared to women of normal body-mass index
Finally, if no embryos are produced after two ICSI sessions, then it might be time to consider switching stallions.
Home Run, Not a Grand Slam
ICSI is an amazing technology, allowing us to produce embryos from mares that can no longer produce embryos any other way, such as those with endometritis or age-related fertility issues. Nonetheless, veterinarians need to help owners have realistic expectations for each TVA/OPU and ICSI session as it remains a highly variable procedure, all too frequently yielding results that owners find disappointing.