AAEP Reproduction Coverage: Reproductive Highlights of 2022

Mare and foal on springtime pasture. Mare is standing, foal is resting. Taken in springtime in Sande, Friesland, Lower Saxony, Germany, Europe

The 2022 AAEP Convention Kester News Hour equine reproduction update was led by Maria Schnobrich, VMD, DACT. She is with Rood and Riddle Equine Hospital in Lexington, Kentucky. Schnobrich reviewed four studies, relaying the clinical relevance of each to the session attendees.

Study 1: Hydrops Bodes Poorly for Foal, Good for Mare

After reviewing 10 years’ worth of data on hydrops, researchers concluded that if hydrops is recognized early, then there is a good prognosis for the affected mare with little impact on her future reproductive performance (Lemonnier, L.C.; Wolfsdorf, K.E.; Kreutzfeldt, N.; et al. Factors affecting survival and future foaling rates in Thoroughbred mares with hydropsJ Equine Vet Sci 2022;113:103941).

(Editor’s note: “The condition in the mare usually develops during the last trimester of pregnancy and is characterized by the excessive accumulation of allantoic—hydrops allantois—or amnionic fluid—hydrops amnion.” References from How to Manage Hydrops Allantois/Hydrops Amnion in a Mare by Nathan M. Slovis, DVM, DACVIM, CHT; Kristina G. Lu, VMD, DACT; Karen E. Wolfsdorf, DVM, DACT; and Walter W. Zent, DVM, DACT (hon) from 2013 AAEP Proceedings.)

Scnobrich felt this 2022 study was relevant because it helps answer questions that owners frequently ask when faced with hydrops: Will she carry to term? Will she survive? What does her future fertility look like? Will the hydrops recur in subsequent pregnancies? 

In total, 30 cases of hydrops were identified during the study period. Although only 10% of foals survived to hospital discharge, 90% of mares survived. Further, 95% of mares went on to have a future foal, including 75% the following year. 

None of the 30 mares had future episodes of hydrops. 

“This study shows that the future reproductive performance of mares with hydrops is good, but veterinarians should expect and be prepared for complications—metritis, retained fetal membranes, hypovolemic shock, hemorrhage and laminitis,” relayed Schnobrich.

This study was conducted for mares managed at a referral hospital. Whether similar results should be expected in a field situation remains unclear.

Study 2: News on Nocardioform Placentitis 

This is an emerging form of placentitis. Schnobrich mentioned the following studies, showing the expanding geographic range of Nocardioform placentitis and relaying the economic relevance of this disease:

Nocardioform Placentitis Presentation

Schnobrich further explained that Nocardioform placentitis has a unique presentation. It is characterized by a mucoid placentitis associated with lesions of the chorioallantois. The lesions are associated with, but not necessarily caused by, Actinomyces (bacteria), including Amycolatopsis spp, Crosiella equi, Streptomycesspp., Nocardia spp. and others. 

“We still don’t know what causes this disease as we’ve so far been unsuccessful at re-creating this disease artificially,” Schnobrich said. “We do know that it is not an ascending placentitis and that cases of Nocardioform placentitis often follow dry, hot summers (August and September).”

Schnobrich then referred to this study published by scientists at the Gluck Equine Research Center at the University of Kentucky:

ELISA Test

The goal of the study was to develop an assay that could potentially use the antibodies against these organisms to see if there was an increased seroprevalence in affected mares. They collected serum samples from mares, where they confirmed infection at foaling with either Crossiella equi or Amycolatopsis spp. and developed an enzyme-linked immunosorbent assay (ELISA) using antibodies from infected mares. 

The ELISA test for Crosiella equi exposure did have significantly higher values near foaling. But there was no Amycolatopsis.

They also noted an increase in titers for Crosiella equi in June and late September. According to Schnobrich, this could potentially help with identifying when there is exposure. 

“The take-home from this is that Nocardioform is having a wider distribution than previously thought and a more detrimental effect,” concluded Schnobrich. “The development of this serum ELISA for Crosiella equi may help identify affected mares.”

Study 3: Taking Advanced Reproductive Techniques to the Next Level

Intracytoplasmic sperm injection (ICSI) used for in vitro embryo production yields approximately two embryos from 12 immature oocytes. Due to this high success rate, ICSI is becoming an increasingly desired procedure

“The reason we have to do ICSI is because when simply put in a petri dish, stallion sperm will not fertilize an egg,” relayed Schnobrich. “There is no capacitation, and we aren’t sure why. The sperm seem to be ‘quiet quitting.’ They’re just not interested in doing what they’re supposed to do.”

To briefly review, Schnobrich said that ICSI is an assisted reproductive technique where veterinarians aspirate immature oocytes directly from a mare’s ovary. Those oocytes are subsequently allowed to mature. Then, they are fertilized by injecting a spermatozoan directly into the oocyte. The resultant blastocytes (embryos) are then transferred to the recipient mare. 

Alternative to ICSI for Equine Reproduction

Portrait of a mare and foal. Nice intimate moment as the mare protecting her curious foal. Canon Eos 1D MarkIII.

As an alternative to ICSI, a team headed by the University of Pennsylvania’s Katrina Hinrichs, DVM, PhD, DACT, found that if they take fresh semen and “pretreat” it, then capacitation-like changes of the sperm occur that allow it to achieve fertilization and produce embryos without ICSI. Due to time constraints and the complexity of the technique, Schnobrich did not delve into the details of the pretreatment. More details are available in the full-length study (Felix, M.R.; Turner, R.M.; Dobbie, T.; et al. Successful in vitro fertilization in the horse: production of blastocysts and birth of foals after prolonged sperm incubation for capacitationBiol Reprod 2022;107(6):1551-64). 

“This was the really exciting repro paper of the year,” shared Schnobrich.

She explained that traditional ICSI boasts 100% of eggs fertilized, a maturation rate of 60%, and 20-25% embryos produced. Overall, traditional ICSI results in 1.5 embryos produced from 10 oocytes.

“Hinrichs’ new technique, however, has a 90% fertilization rate and a 74% blastocyst rate, producing 6.6 embryos per 10 oocytes,” Schnobrich enthused. “This is awesome.”

Clinical Relevance of Hinrichs’ Technique for Equine Reproduction

The relevance to clinical practice, Schnobrich said, was that Hinrichs’ technique could:

  • Replace ICSI and result in more efficient ways to fertilize oocytes;
  • Increase the success of in vitro fertilization methods and efficiency; and 
  • Make in vitro fertilization more available and cost-efficient.  

Currently, only fresh semen has been used. Does the question remains whether these high embryo rates be achieved using cooled or frozen semen.

Hinrichs then provided additional personal comments via a prerecorded video. She said that IVF, where the egg and sperm are simply put into a petri dish together, is a more natural form of fertilization than ICSI. The sperm is going to “choose itself.” There are 50,000 sperm in a droplet with the oocyte and one sperm is the successful one. It is biologically a sperm that is successful in performing fertilization. 

“It’s going to undergo the acrosome reaction; it’s going to go through the outer coverings of the oocyte, the zona pellucida; and it’s going to fuse with that oocyte in the way that it would do in vivo,” explained Hinrichs. “And that is very different from what happens during ICSI when we pick up a sperm, rupture its membrane with our pipette, and then inject it with its acrosome and most of its membrane intact into an oocyte. In that case we’re selecting the sperm, but we don’t know if that is the best sperm of the bunch.” 

According to Hinrichs, this is a simpler procedure than ICSI and one that many more centers in the United States should be able to use. 

Next Steps: Frozen Semen

In practice, though, Hinrichs pointed out that we need to be able to get this to work with frozen-thawed semen. (This study was performed with fresh semen.) 

“So, that’s what we’re working on right now in our lab,” Hinrichs explained. “We’re doing research on methods to use frozen-thawed semen for this. It is a sensitive procedure, and the procedure we developed [means] the sperm has to live for a long time in the droplet to get ready to fertilize the oocyte. Frozen sperm doesn’t have that kind of longevity. So, we are looking at different ways to maintain the frozen sperm while it gains the capacity to fertilize.” 

Emphasizing the importance of this work, Eric Muller, DVM, PhD, DACVS, the field correspondent for the Kester News Hour, added, “This research has the potential to change how we breed horses moving forward.”

Study 4: Genetic Defect Causing Impaired Acrosomal Exocytosis (IAE)

Race horse on the grass track.

For Schnobrich’s fourth and final segment of the Kester News Hour, she chose a study on impaired acrosome exocytosis (IAE), an infrequent but interesting cause of subfertility. 

Sperm from affected stallions, for whatever reason, don’t undergo the same rate of acrosome reaction as normal, fertile stallions. 

“What is scary about these stallions is that they have normal seminal parameters on a breeding soundness examination, but when they breed, they are likely going to be subfertile because the sperm can’t undergo the acrosome reaction,” relayed Schnobrich.

Over the past few years, Schnobrich shared, work has been ongoing in this field to find the gene responsible for this, including the following article: 

Testing for IAE

“You can test for the gene for IAE, which is FKBP6 on chromosome 13, and the susceptible genotype is A/A-A/A. This genotype is particularly high in Thoroughbreds—about 4% compared to 0.7% in the general population,” Schnobrich relayed. 

“This genetic test can potentially screen stallions for IAE,” said Schnobrich. “Also, flow cytometry can be used to assess these stallions’ ability to acrosome react.”

The goal of that study was to describe the clinical characteristics of subfertile stallions diagnosed with IAE and identify the prevalence of subfertility due to IAE in stallions evaluated at a referral center. In total, they identified 21 stallions out of 1,128 with normal seminal parameters that were subfertile. They tested those stallions using flow cytometry and used the genetic test.

“It really was a small number, 1.8% of their population, that fit the subfertile but ‘normal’ seminal parameters, but 38.1% had evidence of IAE and 90% of them had the A/A-A/A genotype. All were Thoroughbreds,” relayed Schnobrich.

Genotype for Identifying Acrosome Dysfunction

This means that the genotype is very specific to identifying acrosome dysfunction. They identified a subset of horses that had the genotype but did not display pronounced subfertility. That group of horses had pregnancy rates of 20-30% per cycle versus 2-15% per cycle in horses with the genotype and subfertility.  

The relevance is that IAE is a cause of subfertility not identified as part of the regular breeding soundness exam (BSE).

“So, if you’re working with Thoroughbreds, this may be something people are requesting,” explained Schnobrich.

The test is about $100 to screen for the FKBP6 and $600 for the genotype plus acrosome tests. 

“This may be something that, in the future, will be required in Thoroughbreds prepurchase or on a BSE. But be aware that the genotype is a marker, not a cause of the condition,” concluded Schnobrich.

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