Differential metabolomic effects of progestagen-based estrus synchronisation in South African Dohne Merino ewes

This study represents one of the first to comprehensively profile the homeostatic cervicovaginal metabolome of South African Dohne Merino ewes using liquid chromatography high resolution mass spectrophotometry (LC-HRMS) and compare that metabolome immediately after estrus synchronisation using progestagen intravaginal pessaries. As the use of assisted reproductive technologies gains traction in various livestock production systems, characterising their effects using novel omics approaches is crucial to physiology, welfare and production. An ewe has well-developed metabolomic cascades that are homeorhetically responsive to the stress caused by assisted reproductive treatments in order to maintain homeostasis. The cervicovaginal fluid of sixty ewes was sampled by transvaginal FLOQSwabs before pessary insertion as well as immediately post-pessary removal. These swabs, along with field, solvent and swab blanks, underwent extraction in acetonitrile and drying under nitrogen prior to reconstitution. Using one-way ANOVA with Tukey's HSD post-hoc analysis and PERMANOVA, this study found no significant metabolomic features prior to treatment (P > 0.05). However, after pessary removal, 32 features were reported to be different between the CIDR-, sponge-treated and control group (P < 0.005 and False Discovery Rate < 0.05). Features were identified at four levels including at verification with pure chemical standards. Progesterone was found to be highest in the CIDR-treated group (P < 5.0 × 10^- 48) while linolenic acid, an immune modulator, was highest in the sponge-treated group (P < 5.0 × 10^- 3). These findings present a novel understanding of the metabolomic fluctuations that occur within the cervicovaginal microenvironment in response to progestagen synchronisation protocols, which are a routine part of management in the production systems of sheep and various other species of livestock. These stark metabolomic differences highlight endocrine and immune changes that are triggered by the CIDR or sponge and warrant further characterisation of the physiological and welfare implications of these technologies.