Top Reproduction Studies of 2012-'13

Veterinarians and researchers with interests in equine reproduction gathered Aug. 7-10 in Louisville, Ky., for the 2013 American College of Theriogenology (ACT) Symposia and Conference. On the last day of the event presenters Mary Beth Stanton, DVM, Dipl. ACT, of Equine Veterinary Reproduction Specialists, in Ocala, Fla., and Audrey Kelleman, DVM, Dipl. ACT, lecturer in large animal reproduction at the University of Florida College of Veterinary Medicine, reflected on recent research and advances in the field. They each presented a handful of studies from 2012-13 that they found interesting and influential.

Female Equine Embryo Survival Austrian researchers tested whether there might be sex-related differences in insulinlike growth factor-1 (IGF-1, an important protein known to promote cell proliferation and development) expression during pregnancy. "We know that when the uterine environment has a higher nutrition plane, it tends to promote the survival of male embryos," Stanton explained. "There was a proposal that there might be a difference in IGF-1 expression between sexes that might utilize the survival of one or the other sexes."

After collecting 28 embryos from 15 mares and staining them for IGF-1, the research team determined that female embryos have a consistently higher IGF-1 expression. "The sex-related differences were likely due to the presence of two X chromosomes that were not activated at that time in gestation," Stanton said.

Because female embryos are believed to be more prone to spontaneous abortion than male embryos, Stanton said, "we think that IGF-1 expression might be a method to counteract embryonic death loss." For more, see

Aurich C, Beckelmann J, Budik S, et al. Sex-dependent insulin like growth factor-1 expression in preattachment equine embryos. Theriogenology 2013 Jan 1;79(1):193-9 2.

Fluconazole for Treating Infectious Endometritis "The objective of this study was to determine the plasma and endometrial tissue concentrations of orally administered fluconazole (an antifungal medication) and to determine if these tissue levels surpassed the minimum inhibitory concentration (MIC) for Candida spp. organisms (that can cause infectious endometritis) in the reproductive tract of the mare," Stanton said.

The researchers administered a loading dose (14mg/kg) of fluconazole followed by six maintenance doses (5mg/kg) to three mares and collected blood plasma and endometrial tissue samples before and after administration. They found that both plasma endometrial fluconazole levels 24 hours after administration were well above the recommended standard for effective therapy.

"Basically, what this says is there is a big distribution of the fluconazole in tissue and plasma," Stanton said. "In conclusion, oral administration of fluconazole well surpassed the MIC for Candida spp. It is also a cost-effective option for oral treatment of Candida spp."

Schofield D, Wittenberg L, et al. Equine endometrial tissue concentration of fluconazole following oral administration. J Equine Vet Sci. 2013 January;33(1):44-45 3.

Embryo Transfer in Noncycling Mares Stanton said the concept of this study was to use estradiol benzoate (the ester of a potent estrogen that prepares the uterus for implantation of the fertilized ovum) to prepare the mare's uterus to expect an embryo. "This study aimed to prepare mares in anestrus (noncycling) or in the transitional period (between estrus and anestrus) as embryo recipients," she said.

The researchers administered 45 embryo-recipient anestrus or transitional mares intramuscular estradiol benzoate followed by long-acting progesterone and found that 60% completed the pregnancy.

"Anestrus and transitional phase mares may be successfully prepared and impregnated (using this method) with comparable pregnancy rates to cycling mares," Stanton concluded.

Kaercher F, Kozicki LE, et al. Embryo transfer in anovulatory recipient mares treated with estradiol benzoate and long-acting progesterone. J Equine Vet Sci. 2013 March;33(3):205-209 4.

New Seminal Plasma Removal Method In this study the researchers compared the new "Sperm Filter" method of seminal plasma removal before freezing semen with the traditional method of centrifugation, which has the potential to cause mechanical damage to sperm. They found that they were able to recover more sperm from 31 stallions' ejaculate using the filter than with centrifugation.

"The Sperm Filter provides efficient technique for plasma removal," Stanton said. "I think this is a very practical method in practice."

Ramires Neto C, Monteiro GA, et al. New seminal plasma removal method for freezing stallion semen. Theriogenology 2013 April;79(7):1120-1123 5.

Stem Cell Delivery to the Uterus "This next paper I found interesting because I think it opens up a whole new avenue of things to look at in stem cell research in our industry," Stanton began. "The object of this paper was to look at methods of stem cell delivery to ensure wide distribution in the uterus (to help treat endometrosis)."

In the team's study results, three of four affected mares that received stem cells via a method similar to that used for deep horn artificial insemination had positive staining for florescence of stem cell proliferation, indicating they were able to not only survive but also divide and develop. "This could be a simple, noninvasive method of stem cell delivery resulting in a wide incorporation of stem cells," Stanton concluded.

Mambelli LI, Winter GH, et al. A novel strategy of mesenchymal stem cell delivery in the uterus of mares with endometrosis. Theriogenology 2013 March;79(5):744-50 6.

Uterine Torsion and Mare/Foal Survival In this study researchers "looked at 12 pregnant mares between ages 3 and 12 that presented with colic signs, uterine torsion was confirmed on rectal palpation, and a midline celiotomy (to correct the torsion) was performed," Stanton said. Foals were delivered by cesarean section, with only three born alive and only one of those surviving long-term.

The researchers concluded that "75% (9/12) of the mares survived, survival was poor closer to term, and the longer the duration of torsion the more compromised the mare and foal," Stanton explained."Seven of nine surviving mares were successfully rebred and went on to foal with no complications."

Saini NS, Mohindroo J, et al. Surgical correction of uterine torsion and mare-foal survival. J Equine Vet Sci. 2013 January;33(1):31-34 7.

Enrofloxacin as Treatment for Bacterial Endometritis In this study Kelleman said the researchers wanted to look at the safety of the commercially available solution of the antibiotic enrofloxacin when administered via uterine infusion (an off-label use). Veterinarians might use this treatment for mares with certain otherwise resistant pathogens. However, all the mares treated with the intrauterine enrofloxacin developed severe endometrial inflammation and fibrosis (scarring).

"Used in this fashion, intrauterine administration of enrofloxacin is certainly not recommended because of the severity of the resulting endometrial damage," Kelleman concluded. For more, see

Rodriguez JS, Han S, Nielsen S, et al. Consequences of intrauterine enrofloxacin infusion on mare endometrium. J Equine Vet Sci. 2012;32:106-111. 8.

Uterine Fungus' Susceptibility to Antifungals The researchers looked at fungal isolates' susceptibility over time to various antifungals in an effort to improve treatment efficacy in mares with fungal endometritis.

"Out of 102 isolates cultured from 92 uterine samples, yeast (69%) and mold (26%) were most common," Kelleman said. The researchers found that molds and yeasts were 95 to 100% susceptible to amphotericin B, natamycin, and nystatin; molds were resistant to fluconazole and increasingly to ketoconazole; and yeasts showed 90% susceptibility to ketoconazole but increased resistance to miconazole over time.

Kelleman said, "Use a fungal medium if you are trying to culture a yeast or fungus on your mare. Some resistance has been seen over time, but the big picture is most of the isolates were susceptible to what we would consider the old-fashioned drugs: amphotericin B, natamycin, and nystatin."

Beltaire KA, Cheong SH, Coutinho da Silva MA, Retrospective study on equine uterine fungal isolates and antifungal susceptibility patterns (1999-2011). Equine Vet J 2012 Dec;44 Suppl 43:84-7 9.

Antibiotic-Containing Semen Extender to Prevent CEM In this study the researchers looked at: 1) the ability of semen extender that had certain antibiotics added to cut down on growth of CEM (contagious equine metritis) in extended semen, and 2) if such antibiotic extended CEM-positive semen caused infection in inseminated mares. The team also inseminated several mares with CEM-positive semen without added antibiotics and looked for signs of disease.

"They found in the laboratory study, that semen extended with those antibiotics did, in fact, reduce CEM growth in culture," Kelleman explained. "When they looked at it practically with the mares they had inseminated with CEM-positive semen, those mares that received semen extended with the antibiotics did not get CEM, while the ones inseminated without added antibiotics became infected. With their method, they reduced transmission of CEM in mares inseminated with CEM-positive semen."

She noted that the United States is considered free of CEM, and any occurrences are reportable and a great risk to the country's equine community.

Klein C, Donahue JM, Sells SF, Effects of antimicrobial-containing semen extender on risk of dissemination of CEM. J Am Vet Med Assoc. 2012 Oct 1;241(7):916-21. 10.

Fertility and Survival Post-Cesarean Section This retrospective study looked at mare and foal survival, as well as foaling rates subsequent to cesarean section. Mare survival to discharge was 84% and foal survival 34%. Foaling rate was lowest in the year following the surgery.

"Their conclusions were that foaling rates post-cesarean section were quite acceptable in cases where dystocia lasted less than 90 min (68% foaled) and in those mares younger than 16 years of age," Kelleman said. For more, see

Abernathy-Young KK, LeBlanc MM, Embertson RM, et al. Survival rates of mares and foals and postoperative complications and fertility of mares after cesarean section: 95 cases (1986-2000). J Am Vet Med Assoc. 2012;241:927-934. 11.

Circulating Cell Free Fetal DNA (cffDNA) to Determine Fetal Gender "Because of the type of attachment of the equine fetal membranes to the maternal uterine surface, there is very little fetal DNA that crosses into the maternal circulation," Kelleman began. "The researchers wanted to know if they could determine the fetal gender with a blood specimen from the mare. They would be looking for the little tiny bits of fetal DNA that manage to get into the maternal circulation."

Specifically, the researchers were looking for the sex-determining section of the Y chromosome, commonly called SRY, which determines male pathways. If the mare's blood was SRY-positive, that meant she was carrying a male fetus.

In their results, the team correctly identified all nine female fetuses and eight of the 11 male fetuses. "This is the first known report of cffDNA to determine equine fetal gender," Kelleman concluded. For more information, see

de Leon PM, Campos VF, Dellagostin OA, et al. Equine fetal sex determination using circulating cell free fetal DNA (cffDNA). Theriogenology 2012 Feb;77(3):694-8 12.

Genetically Distinct Infectious Endometritis Pathogen This research team wanted to determine whether there was a certain subpopulation of Streptococcus equi subspecies zooepidemicus that caused endometritis than those normally found in mares' caudal reproductive tract.

The researchers "demonstrated for the first time that there was a genetically distinct Streptococcus equi subspecies zooepidemicus associated with infectious endometritis," Kelleman said. "Maybe this will lead us to a better understanding of why these particular bacteria are so problematic in uterine infections."

Rasmussen CD, Haugaard MM, Petersen MR, et al. Streptococcus equi subsp. zooepidemicus isolates from equine infectious endometritis belong to a distinct genetic group. Vet Res. 2013 Apr 18;44(1):26 13.

Pregnancy Outcome and Colic Surgery In this retrospective study, the researchers looked at the ability of the mares to carry a foal depending upon the gestational age at the time of colic surgery.

"In a nutshell," Kelleman said, "the prognosis for carrying a foal to term post colic surgery was significantly better for those mares younger than 15 years of age and for those mares carrying pregnancies greater than 40 days gestation at the time of surgery."

Drumm NJ, Embertson RM, Woodie JB, et al. Factors influencing foaling rate following colic surgery in pregnant Thoroughbred mares in Central Kentucky. Equine Vet J. 2013 May;45(3):346-9

Disclaimer: Seek the advice of a qualified veterinarian before proceeding with any diagnosis, treatment, or therapy.