A new urinary biomarker reflecting the progesterone excretion pattern identified through serum steroid and urinary progesterone metabolite analysis during pro-oestrus and oestrus in female dogs (Canis familiaris)

In female dogs, the optimal breeding time is estimated by an increase in the serum progesterone hormone concentration. The method is not only invasive and unpleasant to dogs but also laborious and requires professional laboratory expertise. The main aim of this study was to explore potential urinary progesterone metabolites for non-invasive oestrus monitoring. Additionally, thirteen steroid hormones, such as sex steroids and corticosteroids, were determined in serum samples from nine female dogs via multiple reaction monitoring liquid chromatographymass spectrometry (LCMS). In addition, various urinary steroid metabolites from 22 dogs were analysed via nontargeted liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis. The serum oestrone, oestradiol, and progesterone concentration profiles were aligned with the existing literature. When the 17β-oestradiol secretion peak (49-83 pg/mL) during pro-oestrus was considered as Day 0, 17α-hydroxyprogesterone, testosterone, and androstenedione concentrations peaked on Days 0-3 (P-values 0.004, 0.015 and 0.007, respectively). Results of corticosteroids unveiled novel insights into steroid secretion during the breeding season of dogs. Parallel urine samples revealed that progesterone was excreted into the urine as the unidentified pregnanetriol glucuronide "PtGX" instead of the common metabolite pregnanediol-3-glucuronide (PdG) among mammals. The excretion profiles of pregnanetriol glucuronide support its usability as a biomarker in non-invasive oestrus monitoring in female dogs. The findings presented herein lay the foundation for a more animal-friendly and streamlined approach to monitoring the oestrus cycle of dogs, with substantial implications for dog breeding and advancing animal welfare practices.