In the past two decades, much progress has been made in equine assisted reproduction, especially regarding the use of advanced and laboratory procedures for in vitro embryo production (Squires, 2020). According to the most recent International Embryo Technology Society annual report (Viana, 2023), the number of in vivo-derived equine embryos collected and transferred worldwide decreased by around 5% and 12%, respectively, compared to the previous year. Nevertheless, the reported number of in vitro-produced equine embryos increased by 23%, and 233% for transferred in vitro-produced embryos. Although the total annual reported number of in vivo-derived equine embryos (24 248) still surpasses the reported number of in vitro-produced equine embryos (14 242), if the current trends continue, the numbers of in vitro-produced embryos may surpass the numbers of in vivo-derived embryos, as has happened in the cattle industry since 2016 (Viana, 2023).
For production of in vitro-produced embryos, cumulus oocyte complexes are most commonly recovered from mares’ ovaries by transvaginal ultrasound-guided follicular aspiration. They can also be obtained by directly scraping follicles from ovaries harvested from deceased or ovariectomised mares (Carnevale, 2016). The first report of transvaginal ultrasound-guided follicular aspiration in mares dates back to more than 30 years ago (Brück et al, 1992). When transvaginal ultrasound-guided follicular aspiration started to be implemented in mares, dominant preovulatory follicles were aspirated for collection of maturing cumulus oocyte complexes (Squires, 2020). Recovery of cumulus oocyte complexes from dominant preovulatory follicles in mares is easier when compared to recovery of cumulus oocyte complexes from smaller secondary follicles because of granulosa and cumulus cell expansion associated with the maturation process (Carnevale, 2016). Additionally, cumulus oocyte complexes recovered from dominant preovulatory follicles often have better quality and developmental competence in comparison to cumulus oocyte complexes recovered from secondary follicles (Foss et al, 2013), although this difference is not consistently observed (Jacobson et al, 2010).
When collecting cumulus oocyte complexes from secondary follicles, transvaginal ultrasound-guided follicular aspiration is a more challenging procedure in mares than cows. This is because equine cumulus oocyte complexes are more adhered to the follicular walls (Hawley et al, 1995), thus requiring flushing and scraping of the inner walls of each follicle. These can be achieved through the use of a double lumen aspiration needle, allowing simultaneous aspiration of follicular contents and infusion of flush media, and repeated rotation of the aspiration needle (Stout, 2020). Important advantages of the transvaginal ultrasound-guided follicular aspiration of secondary follicles are recovery of more cumulus oocyte complexes per procedure, the ability to perform the procedure in a fixed schedule without having to serially monitor ovarian activity between aspirations and the ability to hold immature cumulus oocyte complexes at room temperature for up to 24 hours without impairing their quality, aiding in shipment and scheduling of laboratory procedures (Hinrichs, 2018). With these advantages, transvaginal ultrasound-guided follicular aspiration of secondary immature follicles has become an increasingly standard practice in equine reproduction (Stout, 2019). Although not required, monitoring ovarian activity prior to aspiration may be appropriate to select out mares with low follicle numbers.
In the equine industry, transvaginal ultrasound-guided follicular aspiration and in vitro production of embryos through intracytoplasmic sperm injection were initially developed to overcome fertility limitations of donor mares or stallions. When harvesting cumulus oocyte complexes from ovarian follicles, problems associated with the oviducts, uterus and cervix can be bypassed. This allows the production of embryos from mares who are unable to sustain early embryonic development as a result of reproductive tract pathologies (Carnevale, 2008). However, advancements in cumulus oocyte complex recovery and in vitro culture conditions during the past few years have allowed improvements in embryo rates per procedure, even exceeding the success rates of in vivo-derived embryo collections (Claes and Stout, 2022). With that, transvaginal ultrasound-guided follicular aspiration and intracytoplasmic sperm injection are now used widely in the industry, even though they are widely considered to be more invasive and costly procedures compared to traditional embryo collection and transfer.
Transvaginal ultrasound-guided follicular aspiration in mares is not an innocuous procedure and requires appropriate and intensive training to be performed safely (Carnevale, 2016). However, not many practitioners have access to appropriate training, or to a sufficient number of mares to use for training before performing it on client animals. This literature review summarises the reported potential complications associated with transvaginal ultrasound-guided follicular aspiration in mares, as well as the possible negative effects on future fertility. Even with the increasing popularity of this procedure in the equine industry worldwide, not many clinical reports or research studies have investigated in depth the potential harmful effects of transvaginal ultrasound-guided follicular aspiration in mares.
Reported complications
The reported prevalence of complications is generally low, but with a wide range (0.4–22%) and can extend from mild (rectal or vaginal bleeding) to severe clinical scenarios (rectal tears, peritonitis, acute haemorrhage). This emphasises that transvaginal ultrasound-guided follicular aspiration should be considered as an invasive procedure, in which the mare's health can be seriously compromised. Transvaginal ultrasound-guided follicular aspiration requires vigorous rectal ovarian manipulation and the passage of a moderate calibre needle (most commonly 12G) through the cranial vaginal wall and into the abdominal cavity and ovary of the mare. Under appropriate sedation, analgesia and anti-spasmodic therapy, signs of discomfort during the procedure are minimal and transient (Diego et al, 2016). Adequate restraint, sedation, cleanliness and communication during the procedure are important for its success and prevention of potential complications (Hatzel, 2019).
One of the most commonly reported complications of transvaginal ultrasound-guided follicular aspiration in mares is rectal bleeding. This can occur as a result of manual irritation of the rectal mucosa because of ovary grasping and manipulation, or by puncturing of the rectum with the aspiration needle. Rectal bleeding is most often limited to mucosal damage and not associated with rectal tears (Cuervo-Arango et al, 2019). In one research setting with transvaginal ultrasound-guided follicular aspiration performed repeatedly at approximately 14-day intervals, rectal bleeding incidences of 16% (25/153), 22% (29/131) and 9% (10/106) were reported over three consecutive years (Velez et al, 2012). These episodes were mostly mild and transient, with no clinical alterations evident in any of the mares. In a commercial setting with frequent transvaginal ultrasound-guided follicular aspiration procedures, 0.4% (2/515) of rectal bleeding occurrences associated with rectal mucosal irritation were reported (Claes and Stout, 2022), and no cases of Grade 3 or 4 rectal tears (Stout, 2020). In the authors’ university and private clinical programmes, a similar incidence of rectal bleeding (0.4% (4/983) and 0.9% (1/113), respectively) over the past 2–5 years was observed, including both client mares or uni-versity-owned mares used for transvaginal ultrasound-guided follicular aspiration training or research (Table 1). In both the university and private clinic, only one of the rectal bleeding cases was associated with a first-degree rectal tear. It is generally thought that the incidence of rectal bleeding and irritation is influenced by the experience of the veterinarian performing transvaginal ultrasound-guided follicular aspiration (Claes and Stout, 2022), but no complications were observed in a study that monitored transvaginal ultrasound-guided follicular aspiration performed by veterinarians with varying experience in equine reproduction (5–20 years) but no previous experience in equine transvaginal ultrasound-guided follicular aspiration (Rodriguez et al, 2021). The authors believe that lack of appropriate guidance during transvaginal ultrasound-guided follicular aspiration training, or difficulty in ovarian manipulation associated with mare factors (short mesovarium, obesity, excessive presence of air in the rectum) can contribute to higher incidences of rectal bleeding.
| Clinica | setting | |
|---|---|---|
| University | Private | |
| Period | Mar 2019 – May 2024 | Oct 2022 – May 2024 |
| Total transvaginal ultrasound-guided follicular aspiration procedures | 983 | 113 |
| Total number of mares | 340 | 39 |
| Mare age (mean and range) | 13 (2–27) | 13 (3–24) |
| Procedures/mare (mean and range) | 2.9 (1–28) | 2.9 (1–13) |
| Number of follicles aspirated (mean and range) | 14 (1–53) | 16 (1–35) |
| Number of recovered cumulus oocyte complexes (mean and range) | 10 (0–44) | 9 (0–31) |
| Cumulus oocyte complexes recovery rate (mean) | 71% | 56% |
| Incidence of rectal bleeding | 0.4% (4/983) | 0.9% (1/113) |
| Incidence of first-degree rectal tear | 0.1% (1/983) | 0.9% (1/113) |
| Incidence of colic or abdominal discomfort post transvaginal ultrasound-guided follicular aspiration | 0.1% (1/983) | 0.9% (1/113) |
Although precise incidences have not been reported in clinical or research settings, mild clinical signs including abdominal discomfort, inappetence, dullness and low-grade fever can be observed following transvaginal ultrasound-guided follicular aspiration procedures (Cuervo-Arango et al, 2019). A client survey of 913 transvaginal ultrasound-guided follicular aspiration procedures performed in a clinical setting over three years found that 24% of mares experienced some degree of discomfort the day after the procedure was performed in a clinical setting in three years, decreasing to 10% and 3% on days 2 and 3 following the procedure (Martin-Pelaez et al, 2022). Therefore, it is often recommended that client-owned mares are monitored for rectal temperature and changes in behaviour or appetite for 3 days following a transvaginal ultrasound-guided follicular aspiration procedure whenever possible (Carnevale, 2016). Additionally, owners of sport mares should be advised that training and competition may not be possible for few to several days post transvaginal ultrasound-guided follicular aspiration (Cuervo-Arango et al, 2019).
Clinical data are lacking, but some research studies have investigated potential complications associated with repeated transvaginal ultrasound-guided follicular aspiration procedures performed regularly in small groups of research mares. No apparent complications were observed in a study that performed repeated transvaginal ultrasound-guided follicular aspiration in five mares at intervals of 6 or 23 days for a total of 4–6 times (Brück et al, 1997). A second study saw transvaginal ultrasound-guided follicular aspiration performed in 11 mares at 14-day intervals for eight consecutive sessions (Jacobson et al, 2010). In both these studies, all follicles ≥5 mm in diameter were punctured and aspirated. In another study, transvaginal ultrasound-guided follicular aspiration was performed in four mares repeatedly (14–26 times per mare) over a period of 8 years, with puncture and aspiration of all follicles ≥5 mm in diameter (Bøgh et al, 2003). Ovarian follicular activity and corpus luteum formation assessed through transrectal ultrasound examination remained normal in all mares. Ovaries were harvested through ovariectomy or postmortem. Histological evaluation revealed a slightly increased amount of reparative fibrosis in the stroma of all ovaries, which did not seem to impact ovarian function. Peritoneal adhesions of one ovary to the abdominal wall and spleen were observed in one mare, and this same ovary had several foci of chronic apostematous oophoritis. No details regarding bacteria isolates are provided in this study. This mare did not exhibit any signs of discomfort, lack of appetite or pyrexia (Bøgh et al, 2003).
Formation of ovarian abscess without clinical signalment was also reported in a study that used 32 mares for repeated transvaginal ultrasound-guided follicular aspiration procedures over three consecutive years (Velez et al, 2012). Streptococcus equi subsp. Zooepidemicus was isolated from the ovarian abscess after postmortem evaluation. The incidence of ovarian abscess postrepeated transvaginal ultrasound-guided follicular aspiration reported in this study was <0.5% (1/390 transvaginal ultrasound-guided follicular aspirations performed), and no prophylactic antibiotics were used (Velez et al, 2012). A recent clinical case report documented resolution of two cases of ovarian abscess formed after repeated research transvaginal ultrasound-guided follicular aspiration for 12 months with no use of prophylactic antibiotics (Fernández-Hernández et al, 2024). Pyrexia, tachypnoea and tachycardia were observed in one of the mares the day following transvaginal ultrasound-guided follicular aspiration, while no clinical signs (other than ovarian abnormality observed during transrectal ultrasound) were noted in the other mare. In both cases in this report, Streptococcus equi Zooepidemicus were isolated. This bacterium is the most common isolate associated with infectious endometritis in mares (Li et al, 2021), suggesting a potential inoculation of bacteria present in the uterus and vagina into the ovary during transvaginal ultrasound-guided follicular aspiration. In this case report, the prevalence of ovarian abscesses fell from 3.33% (2/60) to 0% (0/216) after instituting use of regular prophylactic antibiotics after transvaginal ultrasound-guided follicular aspiration procedures (Fernández-Hernández et al, 2024). This observation emphasises that, despite the general concerns about indiscriminate use of prophylactic antibiotics, these may be important for the prevention of ovarian abscesses in mares undergoing repeated transvaginal ultrasound-guided follicular aspiration. This approach is performed by different clinical programs (Velez et al, 2012; Carnevale, 2016; Claes and Stout, 2022; Fernández-Hernández et al, 2024), with no occurrence of ovarian abscesses in >2200 transvaginal ultrasound-guided follicular aspirations (Stout, 2020). Additionally, transvaginal ultrasound-guided follicular aspiration should be avoided in mares with active endometritis, as a result of a higher predisposition to ovarian abscesses and peritonitis (Fernández-Hernández et al, 2024).
Severe and even fatal complications such as peritonitis and acute haemorrhage can also occur post-transvaginal ultrasound-guided follicular aspiration. One case report described the occurrence of acute haemorrhage after transvaginal ultrasound-guided follicular aspiration of two preovulatory follicles in a Paint mare (Vanderwall and Woods, 2002). In this report, the mare was found in lateral recumbency, tachypnoeic, with pale mucous membranes and unable to stand only 30 minutes following the procedure. The mare was euthanised with a presumptive diagnosis of severe internal haemorrhage. Necropsy examination revealed presence of 4–6 litres of free blood and clots in the abdominal cavity, and large haematomas in the broad ligament and surrounding soft tissues. The exact location of vascular injury that led to haemorrhage was not identified but appeared to be the left uterine artery (Vanderwall and Woods, 2002). This was the only complication observed post transvaginal ultrasound-guided follicular aspiration in a total of 388 procedures performed in 132 mares within 3 years by this group (Vanderwall and Woods, 2002). In a busy clinical setting with more than 3000 transvaginal ultrasound-guided follicular aspirations performed, severe internal haemorrhage has been reported in three mares (Stout, 2020; Claes and Stout, 2022). These mares developed clinical signs of acute blood loss (colic, pale mucous membranes, tachycardia and tachypnoea) either immediately after or the day after transvaginal ultrasound-guided follicular aspiration. All mares survived hospitalisation and medical treatment (Claes and Stout, 2022). However, minor internal haemorrhage may be much more common and most often unnoticed. Increased red blood cell counts have been noted in peritoneal fluid from all of six mares evaluated 3 days after transvaginal ultrasound-guided follicular aspiration and one of two mares evaluated 10 days post transvaginal ultrasound-guided follicular aspiration (Velez et al, 2012), suggesting that mild abdominal bleeding is induced by transvaginal ultrasound-guided follicular aspiration. This same study reported slightly increased peritoneal fluid white blood cell counts in two of six mares evaluated 3 days post transvaginal ultrasound-guided follicular aspiration, suggesting possible occurrence of subclinical peritonitis post transvaginal ultrasound-guided follicular aspiration (Velez et al, 2012). One case of post transvaginal ultrasound-guided follicular aspiration peritonitis and associated acute laminitis has been reported among thousands of transvaginal ultrasound-guided follicular aspiration procedures in a large clinical setting (Stout, 2020; Claes and Stout, 2022).
Effects on future fertility
Clients often ask whether transvaginal ultrasound-guided follicular aspiration can lead to ovarian damage that could impair future mare fertility, especially when performed repeatedly. Although long-term studies with large populations of mares are lacking, research reports have failed to demonstrate any major effect of repeated transvaginal ultrasound-guided follicular aspiration on future pregnancy rates. One study used 10 mares that underwent repeated transvaginal ultrasound-guided follicular aspiration (4–10) at 10–15-day intervals to breed them through artificial insemination with fresh semen at the first oestrus following the last transvaginal ultrasound-guided follicular aspiration (Mari et al, 2005). A 70% early pregnancy rate was reported, suggesting no negative effect of repeated transvaginal ultrasound-guided follicular aspiration in mare fertility (Mari et al, 2005). Another study reported a 58% (7/12) pregnancy rate after artificial insemination (fresh or cooled semen) or embryo transfer performed after the last of eight transvaginal ultrasound-guided follicular aspirations per mare conducted at 10–11-day intervals (Purcell et al, 2007). A larger study that used 82 mares assessed early pregnancy rates after artificial insemination (fresh or cooled semen) of mares that had preovulatory follicle transvaginal ultrasound-guided follicular aspiration performed zero, few (1–4) or several (5–11) times (Vanderwall et al, 2006). They reported no differences in early pregnancy rates between groups. Another study used 66 mares as embryo transfer recipients between 35–60 days post-transvaginal ultrasound-guided follicular aspiration procedure performed by training veterinarians (Rodriguez et al, 2021). The authors reported normal corpus luteum formation and good (85%) early pregnancy rates after embryo transfer (Rodriguez et al, 2021). Embryo recovery by uterine flush at day 7 post-ovulation did not seem to be affected by previous transvaginal ultrasound-guided follicular aspiration procedures (1–5 times), with a positive embryo flush on 4/5 mares (Velez et al, 2012).
In this same study, which used a total of 32 mares, the authors reported that follicle growth and oocyte viability appeared to be unaffected by repeated aspiration of all visible immature follicles (Velez et al, 2012). Follicle growth was apparently not affected by repeated transvaginal ultrasound-guided follicular aspiration of all visible follicles performed every 9–12 days for a total of eight times in a group of seven Standardbred mares, and all mares resumed normal ovarian activity and ovulation by 16–30 days after the last transvaginal ultrasound-guided follicular aspiration (Iacono et al, 2014). Similar findings were reported after eight transvaginal ultrasound-guided follicular aspiration procedures performed at 10–11-day intervals in 14 light horse mares (Purcell et al, 2007). A linear decrease in the number of follicles has been reported over 11 successive transvaginal ultrasound-guided follicular aspirations of all follicles ≥8 mm performed weekly in a small group of warmblood mares (n=3), but not in pony mares (n=3) that underwent the same repeated transvaginal ultrasound-guided follicular aspiration protocol (Duchamp et al, 1995), suggesting a potential effect of breed on follicular growth post-transvaginal ultrasound-guided follicular aspiration. In the authors’ clinical program, population of follicles in the mare's ovaries has not been observed, even after repeated transvaginal ultrasound-guided follicular aspiration procedures (up to 28 times) at varying time intervals (usually 2 weeks to 2 months). Because all the cumulus oocyte complexes collected in the clinical program are shipped to different intracytoplasmic sperm injection laboratories, the possible effect of repeated transvaginal ultrasound-guided follicular aspiration on oocyte quality is not known.
Conclusions
With the increasing popularity of transvaginal ultrasound-guided follicular aspiration for collection of cumulus oocyte complexes for in vitro embryo production in the equine reproduction industry, veterinarians should understand that appropriate training and several crucial steps need to be completed for safe conduction of transvaginal ultrasound-guided follicular aspiration in clinical practice. Crucial steps include appropriate mare restraint, sedation and rectal relaxation, as well as adequate cleanliness of equipment and perineal area, use of prophylactic antibiotics and sufficient number of personnel. Although complications of transvaginal ultrasound-guided follicular aspiration in mares do not appear to be common, they can have serious implications for mare health and fertility and can even be fatal. There is an important paucity of data in literature regarding incidence of transvaginal ultrasound-guided follicular aspiration complications and potential effects on future mare fertility. With the clear increase in numbers of in vitro-produced equine embryos, intracytoplasmic sperm injection laboratories and practitioners offering transvaginal ultrasound-guided follicular aspiration services worldwide, more reports are expected to reach the public and guide the industry. Prevention of complications with appropriate prior training and adequate steps when performing transvaginal ultrasound-guided follicular aspiration should be carefully considered to protect welfare, health, and fertility of the mare.