Saliva and plasma metabolome changes during anoestrus, the oestrous cycle and early gestation in the mare: A pilot study.

Successful reproductive management of domestic mammals depends primarily upon timely identification of oestrous cycle stages. There is a need to develop an alternative non-invasive, welfare-friendly, accurate and reliable method to identify reproductive cycle stages. This is of particular interest for horse breeders, because horses are high-value farm animals that require careful management and individual monitoring. Saliva sampling is non-invasive, painless and welfare-friendly. Thus, we performed a metabolomic analysis of equine saliva during different reproductive stages to identify changes in the salivary metabolome during anoestrus, the oestrous cycle and early gestation. We compared the saliva and plasma metabolomes to investigate the relationship between the two fluids according to the physiological stage. We collected saliva and plasma samples from six mares during seasonal anoestrus, during the follicular phase 3 days, 2 days and 1 day before ovulation and the day when ovulation was detected, during the luteal phase 6 days after ovulation, and during early gestation 18 days after ovulation and insemination. Metabolome analysis was performed by proton-nuclear magnetic resonance spectroscopy. We identified 58 and 51 metabolites in saliva and plasma, respectively. The levels of four metabolites or groups of metabolites in saliva and five metabolites or groups of metabolites in plasma showed significant modifications during the 4 days until ovulation, ie 3 days prior to and on the day of ovulation. The levels of 11 metabolites or groups of metabolites in saliva and 17 metabolites or groups of metabolites in plasma were significantly different between the seasonal anoestrus and the ovarian cyclicity period. The physiological mechanisms involved in the onset of ovarian cyclicity and in ovulation induced modifications of the metabolome both in plasma and saliva. The metabolites whose salivary levels changed during the reproductive cycle could be potential salivary biomarkers to detect the reproductive stage in a welfare friendly production system. In particular, we propose creatine and alanine as candidate salivary biomarkers of ovulation and of the onset of ovarian cyclicity, respectively. However, extensive validation of their reliability is required. Our study contributes to extend to domestic mammals the use of saliva as a non-invasive alternative diagnostic fluid for reproduction in a welfare-friendly production system.

Horses can learn to identify joy and sadness against other basic emotions from human facial expressions.

Recently, horses and other domestic mammals have been shown to perceive and react to human emotional signals, with most studies focusing on joy and anger. In this study, we tested whether horses can learn to identify human joyful and sad expressions against other emotions. We used a touchscreen-based automated device that presented pairs of human portraits and distributed pellets when the horse touched the rewarded face. Six horses were trained to touch the sad face and 5 the joyful face. By the end of training, horses' performances at the group level were significantly higher than chance level, with higher scores for horses trained with the sad face. At the individual level, evidence of task learning varied among horses, which could be explained by individual variations in horses' ability to identify different human facial expressions or attention issues during the tests. In a generalization test, we introduced portraits of different humans than those presented during training. Horses trained with the joyful face performed better than chance, demonstrating generalization. Conversely, horses trained with the sad face did not. Horses also showed differences in learning performance according to the non-rewarded emotion, providing insights into horses' cognitive processing of facial expressions.

Chicory (Cichorium intybus) reduces cyathostomin egg excretion and larval development in grazing horses.

Cyathostomins are the most prevalent parasitic nematodes of grazing horses. They are responsible for colic and diarrhea in their hosts. After several decades of exposure to synthetic anthelmintics, they have evolved to become resistant to most compounds. In addition, the drug-associated environmental side-effects question their use in the field. Alternative control strategies, like bioactive forages, are needed to face these challenges. Among these, chicory (Cichorium intybus, Puna II cultivar (cv.)) is known to convey anthelmintic compounds and may control cyathostomins in grazing horses. To challenge this hypothesis, we measured fecal egg counts and the rate of larval development in 20 naturally infected young saddle horses (2-year-old) grazing either (i) a pasture sown with chicory (n = 10) or (ii) a mesophile grassland (n = 10) at the same stocking rate (2.4 livestock unit (LU)/ha). The grazing period lasted 45 days to prevent horse reinfection. Horses in the chicory group mostly grazed chicory (89% of the bites), while those of the control group grazed mainly grasses (73%). Cyathostomins egg excretion decreased in both groups throughout the experiment. Accounting for this trajectory, the fecal egg count reduction (FECR) measured in individuals grazing chicory relative to control individuals increased from 72.9% at day 16 to 85.5% at the end of the study. In addition, larval development in feces from horses grazed on chicory was reduced by more than 60% from d31 compared to control individuals. Using a metabarcoding approach, we also evidenced a significant decrease in cyathostomin species abundance in horses grazing chicory. Chicory extract enriched in sesquiterpenes lactones was tested on two cyathostomins isolates. The estimated IC50 was high (1 and 3.4 mg/ml) and varied according to the pyrantel sensitivity status of the worm isolate. We conclude that the grazing of chicory (cv. Puna II) by horses is a promising strategy for reducing cyathostomin egg excretion and larval development that may contribute to lower the reliance on synthetic anthelmintics. The underpinning modes of action remain to be explored further.