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Background: Increased embryo loss as mares become older is a major consideration for breeders as older animals are currently used for reproduction in the equine industry. Lower embryo quality has been pointed out as partly responsible for this reduced fertility. Here, the effect of mare's age on blastocysts' gene expression was explored. Day 8 post ovulation embryos were collected by uterine flushing from multiparous young (YM, 6-year-old, N = 5) and older (OM, > 10-year-old, N = 6) non-nursing Saddlebred mares, inseminated with the semen of one stallion. Pure (TE_part) or inner cell mass enriched (ICMandTE) trophoblast were obtained by embryo bisection and paired end, non-oriented RNA sequencing (Illumina, NextSeq500) was performed on each hemi-embryo. To discriminate gene expression in the ICM from that in the TE, deconvolution (DeMixT R package) was used on the ICMandTE dataset. Differential expression was analyzed (DESeq2) with embryo sex and diameter as cofactors using a false discovery rate < 0.05 cutoff. Functional annotation and classification of differentially expressed genes and gene set enrichment analysis were also performed. Results: Maternal aging did not affect embryo recovery rate, embryo diameter nor total RNA quantity but modified gene expression in equine D8 embryos. The expression of genes in the ICM seemed to be more altered by maternal aging than in the TE. In both compartments, the expression of genes involved in mitochondrial function, translation and transcription due to chromatin modification were disturbed by maternal age. Mitosis, signaling and adhesion pathways and embryo development were particularly decreased in the ICM hemi-embryos from old mares. Finally, in TE, ion movement related genes were affected. Conclusions: This is the first study showing an effect of maternal age on gene expression in the equine blastocyst at Day 8 post ovulation. Maternal age, even for mares as young as 10 years old, disturbs mitosis, translation and transcription, cell signaling and adhesion as well as mitochondrial function and cell commitment of horse embryos. These perturbations may affect further embryo development and contribute to decreased fertility due to aging.

Although puberty can occur as early as 14–15months of age, depending on breed and use, the reproductive career of mares may continue to advanced ages. Once mares are used as broodmares, they will usually produce foals once a year until they become unfertile, and their productivity can be enhanced and/or prolonged through embryo technologies. There is a general consensus that old mares are less fertile, but maternal age and parity are confounding factors because nulliparous mares are usually younger and older mares are multiparous in most studies. This review shows that age critically affects cyclicity, folliculogenesis, oocyte and embryo quality as well as presence of oviductal masses and uterine tract function. Maternal parity has a non-linear effect. Primiparity has a major influence on placental and foal development, with smaller foals at the first gestation that remain smaller postnatally. After the first gestation, endometrial quality and uterine clearance capacities decline progressively with increasing parity and age, whilst placental and foal birthweight and milk production increase. These combined effects should be carefully balanced when breeding mares, in particular when choosing and caring for recipients and their foals.

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