AAEP 2023: Kester News Hour Reproduction Segment 
Four standout studies highlight important advances in equine reproduction.
Kester News Hour presentation at the 2023 AAEP Convention
Dr. Maria Schnobrich (second from left) presented the top equine theriogenology studies from the past year. | Courtesy AAEP

Maria Schnobrich, VMD, Dipl. ACT, from Rood and Riddle Equine Hospital in Lexington, Kentucky, returned as the reproduction anchor during the popular and highly acclaimed Kester News Hour at the 2023 AAEP Convention. Schnobrich reviewed four of her favorite studies published within the last year, relaying their clinical relevance and how the studies contribute to advancing equine theriogenology. 

Intrauterine Ozone for Treating Endometritis 

Photomicrographs of endometrial biopsies in horses obtained before and after ozone treatment.
Photomicrographs (HE staining) of endometrial biopsies obtained before treatment  (A–C)  and after treatment  (D–F). (A)  Endometrium of mare 3 before treatment with ozone (age 12, category III, moderate signs of endometritis).  (B)  Endometrium of mare 15 before treatment with trimethoprim-sulfadimethoxine (age 10, category III, moderate signs of endometritis).  (C)  Endometrium of mare 22 before treatment with air (age 9, category III, mild signs of endometritis).  (D)  Endometrium of mare 3 after intrauterine insufflation of ozone showing mild signs of endometritis and an intact surface epithelium.  (E)  Endometrium of mare 15 after systemic treatment with trimethoprim-sulfadimethoxine showing mild signs of endometritis and an intact surface epithelium.  (F)  Endometrium of mare 22 after intrauterine insufflation of air showing mild signs of endometritis and an intact surface epithelium. | Courtesy M Köhne et al., Front. Vet. Sci., Jan. 2023 

Mares with endometritis often receive antibiotic therapy, either locally or systemically. In the spirit of embracing antimicrobial stewardship, veterinarians are seeking antibiotic alternatives to treat this highly prevalent condition.  

“Ozone has three oxygen molecules and has antibacterial and biofilm-disrupting properties due to its powerful oxidizing effect,” explained Schnobrich.  

Researchers in Germany conducted a study to determine the efficacy of intrauterine ozone administration and systemic trimethoprim sulfadimethoxine (TMS) in resolving bacterial endometritis. The study included 30 mares with bacterial endometritis that were positive for bacteria on culture and had inflammatory cytology.  

“Medical-grade ozone was mixed with air and administered via intrauterine insufflation two times 48 hours apparent in 10 mares,” said Schnobrich. “TMS was administered at a dose of 30 mg/kg by mouth twice daily for five days and was the only treatment for 10 mares. In the final group of 10 mares, only intrauterine insufflation of sterile air was administered.” 

Surprisingly, she said, all treatments—including the sterile air insufflation—resulted in a decreased number of positive cultures. For Gram-positive bacteria, TMS was more effective than either ozone or air. For Gram-negative bacteria, ozone and TMS were more effective than air.  

The take-away message from this study was that insufflated ozone had no observed adverse effects and might reduce bacterial growth when used in isolation. None of the three treatments resulted in complete removal of intrauterine bacteria.  

“This is an exciting study, as we are looking for new agents that address bacterial endometritis,” concluded Schnobrich.  

The study, “Comparison of systemic trimethoprim-sulfadimethoxine treatment and intrauterine ozone application as possible therapies for bacterial endometritis in equine practice” was published by Köhne et al. in Frontiers in Veterinary Science in 2023. 

GnRH Vaccine in Stallions Is Reversible 

The next study Schnobrich described was “Re-stimulation of testicular function in GnRH-vaccinated stallions by daily GnRH agonist treatment,” published by Gautier et al. in Theriogenology.  

The researchers wanted to determine if the gonadotropin-releasing hormone (GnRH) vaccine used to block stallion reproductive function was reversible. Temporarily blocking testicular function in stallions is of interest because it leaves the possibility of breeding open while potentially facilitating management and training by reducing typical stallion behavior prior to their breeding career. The GnRH vaccine produces antibodies that prevent GnRH from binding to GnRH receptors in the pituitary gland. According to the researchers, this results in a loss of gonadal stimulation, decreases testicular steroidogenesis, and reduces sexual behavior and spermatogenesis. 

In their study, the researchers divided six Shetland stallions into two groups. Half were vaccinated two times four weeks apart. The other group remained unvaccinated. The researchers collected ejaculates for semen analysis and assayed blood for testosterone concentration and GnRH antibody titers. When testosterone dropped below 0.3 ng/mL, a hemicastration was performed on all stallions. The vaccinated stallions were then treated with the GnRH agonist buserelin (4 mg/day for four weeks, then 8 mg/day). The remaining testicle was removed from all stallions when testosterone concentration exceeded 0.5 ng/mL. 

“The results showed that the vaccine did increase GnRH antibody levels, which peaked about five weeks after vaccination,” said Schnobrich. “Total sperm count decreased and didn’t really recover to similar levels as control animals after buserelin administration. Decreased sperm motility was appreciated, but it did return to control levels after buserelin. Membrane integrity of the spermatozoa similarly decreased then recovered.”  

In addition, testis volume decreased after GnRH vaccination and began to increase following buserelin treatment. 

These results indicate the GnRH vaccine adversely affects seminal characteristics, but some of these adverse effects might be reversible.  

“Why is this interesting? We come across stallions that have been vaccinated, and we’re not sure if they’ll recover,” Schnobrich said. “It can be very detrimental to testes function in the future if the stallion was vaccinated while still of pubertal age. It would be nice to see what happens to vaccinated horses that aren’t given the agonist. Would they recover on their own? Buserelin might also be useful for low-libido stallions or stallions where vaccination has occurred.” 

Are Ovarian Issues to Blame for Poor Mare Behavior? 

Horse granulosa cell tumor
Researchers urged veterinarians to test AMH to diagnose GTCTs before recommending surgery for mares behaving poorly. | Courtesy Dr. Pat McCue

“This paper really caught my eye because the AAEP conducted a survey several years ago reporting that estrus negatively affected performance, and this is very controversial,” said Schnobrich. “We need to ask, ‘What is the evidence behind this?’ Also, it can be problematic that owners often perceive the ovaries as a cause of behavioral ‘problems’ and recommend or want an ovariectomy to remove the ovaries in animals where this is not the issue. This article seemed to look deeper into owner, trainer, and veterinarian perception of behavior following ovariectomy. But what we still don’t know is, what is the evidence behind this?” 

Researchers in Italy recently investigated these questions. They retrospectively evaluated the long-term follow-up of ovariectomized mares, focusing on the owner’s perception of the mare’s behavior following surgery. 

The most frequent owner complaints leading them to believe the mare had ovarian issues were increased sensitivity of the flanks, general riding problems, and kicking other horses. 

“But really, general riding problems can be due to so many things,” said Schnobrich.  

Medical intervention prior to referral for ovariectomy resulted in 25-50% of horses’ behaviors improving, but those behaviors returned after medication stopped. 

The study included 11 mares. Bilateral ovariectomy was performed in 5/11 cases (45%). Six of 11 mares had histopathology results available. Nine ovaries were histologically examined from six mares, and five granulose cell tumors/granulose thecal cell tumors (GCT/GTCTs) were diagnosed.  

Following surgery, behavior scores were lower (i.e., fewer poor behaviors) even if no pathology was identified. This prompted 91% of owners to recommend ovariectomy as a treatment for difficult behaviors. 

“This is really important because it questions whether ovariectomy is really a good solution to bad behavior. Shouldn’t we rule out other causes of pain before going to ovary? This problem of blaming poor performance on the ovary will continue to be an issue,” said Schnobrich. 

She added, “Hormonal testing would be a really nice precursor to surgery. AMH is 98% sensitive for diagnosing GTCTs, and it would better to have positive hormonal testing before recommending surgery for mares behaving poorly.” 

The study, “Behavioral disorders in mares with ovarian disorders, outcome after laparoscopic ovariectomy: A case series,” was published in Veterinary Science in August 2023 by  

Straticò et al. from the Department of Veterinary Medicine, University of Teramo, Italy. 

Lack of Progesterone Early in Pregnancy Negatively Affects Pregnancy, Foaling Rates 

Finally, Schnobrich summarized a study by an Austrian research group titled, “Low progesterone concentration in early pregnancy is detrimental to conceptus development and pregnancy outcome in horses.” It was published in 2023 in Animal Reproduction Science by Wagner et al.  

The rationale for the study was that high progesterone concentrations in the early luteal phase support pregnancy, but low (subphysiologic) progesterone concentrations delay embryonic development. The corpus luteum on the ovary produces progesterone in the early part of pregnancy. Later, around 100-120 days, placental progestogens take over and progesterone decreases. However, delayed progesterone production by the corpus luteum early in pregnancy might have detrimental effects. So, the researchers attempted to look at the long-term effects of low progesterone early in pregnancy. 

“Low progesterone in the early period of pregnancy can be caused by inflammation from infection or abnormal uterine environment or systemic stress or inflammation as seen in colic or severe illness,” Schnobrich said. 

The study’s purpose was to determine to what extent subphysiologic plasma progesterone concentration in the early postovulatory phase affects fetal development and pregnancy outcome. The research team measured conceptus growth throughout gestation, embryonic and fetal loss, and foal characteristics in Shetland ponies. Those ponies received prostaglandin F2α to induce luteolysis (to decrease progesterone production by the corpus luteum) on Days 0 to 3 of ovulation after being bred.  

The study included 12 Shetland mares that were bred every 48 hours until time of ovulation following a comprehensive breeding soundness exam.  

“The pregnancy rate was lower in the treatment group than the control group, with 75% of treated ponies diagnosed as pregnant versus 100% in the control group,” relayed Schnobrich.  

Conceptus and embryo size differed between the treatment and control groups, with the treatment group being smaller. Those size differences decreased over time, with no difference noted between the two groups.  

Forty-two percent of the ponies in the treatment group had live foals, whereas 89% of untreated pony mares foaled.  

The study authors concluded, “Reduced progesterone concentration in the early luteal phase leads to delayed conceptus growth beyond placentation and increased pregnancy loss.” 

“The take-home message was that low progesterone levels in that window between ovulation and progesterone production by the corpus luteum have far-reaching effects,” said Schnobrich. “This study suggests there may be an adverse effect in both pregnancy rate and fetal development.  

“This makes me ask, should we be looking early if they have conditions that induce luteolysis?” she added. “Should we be measuring progesterone earlier in pregnancy, around Day 5 post-ovulation? And would progestogen supplementation correct for these adverse effects?”  

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