Targeting epigenetic nuclear reprogramming in aggregated cloned equine embryos.

Epigenetic perturbations during the reprogramming process have been described as the primary cause of the low efficiency of somatic cell nuclear transfer (SCNT). In this study, we tested three strategies targeting nuclear reprogramming to investigate effects on equine SCNT. First, we evaluated the effect of treating somatic cells with chetomin, a fungal secondary metabolite reported to inhibit the trimethylation on histone 3 lysine 9 (H3K9 me3). Second, caffeine was added to the culture medium during the enucleation of oocytes and before activation of reconstructed embryos as a protein phosphatase inhibitor to improve nuclear reprogramming. Third, we tested the effects of the histone deacetylase inhibitor trichostatin A (TSA) added during both activation and early embryo culture. Although none of these treatments significantly improved the developmental rates of the invitro aggregated cloned equine embryos, the first equine cloned foal born in Australia was produced with somatic cells treated with chetomin. The present study describes the use of chetomin, caffeine and TSA for the first time in horses, serving as a starting point for the establishment of future protocols to target epigenetic reprogramming for improving the efficiency of equine cloning. Cloning is an expensive and inefficient process, but has gained particular interest in the equine industry. In this study we explored different strategies to improve cloning efficiency and produced the first cloned foal born in Australia. Our data serve as a starting point for the establishment of future protocols for improving equine cloning efficiency.

Fetal Membrane Removal in the Mare: Proactive Versus Reactive Approaches.

Retained fetal membranes are the most common postpartum condition in mares. Although the incidence of retained fetal membranes is low, the consequences for the health of the mare can be severe (metritis, endotoxemia, laminitis, death). Oxytocin administration is often the first line of therapy for management of retained fetal membranes. Removal of fetal membranes using umbilical vessel infusion and manual membrane removal are effective tools for reducing risks associated with abnormally heavy membranes, retained membranes, or for mares that are geographically limited for veterinary care.

High ambient temperature and humidity associated with early embryonic loss after embryo transfer in mares.

Increased ambient temperatures has been identified to contribute to reproductive outcomes for several domesticated species, but its impact on equine reproduction has not been previously investigated. Therefore, this study aimed to examine the relationship between ambient climatic conditions, as measured by temperature-humidity index (THI) between day 7 and day 14 of gestation, and early embryonic loss (EEL) in recipient mares undergoing embryo transfer. The study examined records from 834 embryo transfers at an equine breeding facility in Victoria, Australia. Early embryonic loss was defined as a negative transrectal ultrasound on day 14 of gestation after an embryo was transferred on day 7. Both maximum THI on the day of transfer (day 7) and mean THI between day 7 and day 14 were investigated for association with the outcome of EEL using multivariable logistic regression, controlling for confounders including embryo age and quality, recipient mare quality and embryo transfer quality. EEL was observed in 21% of embryo transfers. A five-unit increase in maximum THI on the day of transfer was associated with an 18% increase in the odds of EEL (p = 0.01). Similarly, the odds of EEL increased by 25% for each five-unit increase in mean THI between day 7 and 14 (p = 0.003). As both single and cumulative episodes of high THI were associated with increased EEL in embryo transfer mares from this equine breeding facility, further studies are warranted to identify similar effects in a broader population, establish causality and evaluate possible mitigation strategies in anticipation of heat waves.

Evidence for anti-inflammatory effects of firocoxib administered to mares with experimentally induced placentitis.

PROBLEM: Minimal evidence exists supporting therapeutic selections for equine placentitis. The goal of this study was to characterize the anti-inflammatory effects of firocoxib when administered to mares with placentitis. METHODS: Mares (gestation D270-300) were assigned to: INFECT (n = 6; placentitis, no treatment), FIRO (n = 6; placentitis, firocoxib, 0.1 mg/kg, PO, daily), and NORM (n = 6; no infection/treatment). Allantoic fluid (8 hours, 24 hours, birth) and amniotic fluid (birth) were collected from mares after infection. Concentrations of IL-1ß, IL-6, TNF-α, IL-10, PGF2α , and PGE2 in fluids were measured by ELISA. mRNA expression of IL-1ß, IL-6, TNF-α, IL-8, IL-10, matrix metalloproteinases (MMPs) -1, 3, and 9 in fetal membranes/fetuses was quantified using real-time PCR. RESULTS: Allantoic TNF-α concentrations were lowest in FIRO at 8 hours and 24 hours post-infection; IL-6 concentrations were lower in FIRO than NORM at 8 hours, lower in FIRO than INFECT at 24 hours post-inoculation, and lower in NORM than FIRO or INFECT at birth. Marginal mean allantoic IL-ß and IL-10 concentrations were lower in FIRO and NORM than INFECT. Amniotic fluid cytokines were lowest in NORM with all measurements in that group being below the limit of detection. Allantoic PGF2α concentrations were lower in FIRO and INFECT than NORM at 8 hours post-inoculation, and lower in FIRO than INFECT or NORM at 24 hours post-inoculation. Allantoic PGE2 concentrations were lower in FIRO than INFECT. Amniotic PGF2α and PGE2 concentrations were lower in NORM than INFECT. In fetal membranes, group differences with respect to IL-1ß, IL-6, IL-8, and MMP1 were dependent on tissue type. CONCLUSIONS: Data suggest a suppressive effect of firocoxib administration on cytokine and prostaglandin production in mares with placentitis.