Stallions show decreased sexual responses and activities during short days in winter. To evaluate the importance of sexual olfactory communication in horses, we tested whether sexual responses could be stimulated through various sexual olfactory stimulations in winter. To this end, we presented stallions with various olfactory stimulations (urine from mares at different stages of the reproductive cycle, urine from stallions or geldings, or chemically defined synthetic odorant) during the non-breeding season and measured their behavioral responses through (1) a test of olfactory investigation (olfactory investigation and flehmen behavior) and (2) a test of sexual activity in the context of semen collection for artificial insemination. It appears that the duration of olfactory investigation and flehmen behavior is longer after presentation of urine (stallion, gelding, anestrous, diestrous and estrous mare) than after presentation of water or synthetic odorant. By contrast, geldings showed reduced flehmen behavior that did not differ from that after water presentation. It is of interest that during the mounting test, mare estrous urine was associated with significantly reduced latency to ejaculation when spread in the nostril of the stallion, compared to anestrous mare urine or water. Anestrous mare urine seems to even inhibit stallion sexual motivation as measured through a longer latency to reach mounting and ejaculation. It appears therefore that during the season of sexual rest, stallion sexual motivation can be stimulated by mare estrous urine and inhibited by mare anestrous urine. These results also suggest that the physiological state of the mare influence the content of urinary chemosignals.

As sport career is a priority in most of equine breeds, mares are frequently bred for the first time at an advanced age. Both age and first gestation were shown to have a deleterious effect on reproduction outcomes, respectively on fertility and offspring weight but the effect mare’s parity in older mares on embryo quality has never been considered. The aim of this project was to determine the effect of old mare’s nulliparity on gene expression in embryos. Day 8 post ovulation embryos were collected from old (10-16 years old) nulliparous (ON, N=5) or multiparous (OM, 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. Although the expression of only a few genes was altered by mare’s nulliparity (33 in ICM and 23 in TE), those genes were related to nutrient exchanges and responses to environment signaling, both in ICM and TE, suggesting that the developing environment from these mares are not optimal for embryo growth. In conclusion, being nulliparous and old does not seem to be the perfect match for embryonic development in mares.Summary sentenceMare’s parity in old mares impacts the expression of genes related to development and molecule exchanges in ICM and TE of blastocysts suggesting an adaptation to an altered environment.

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