The metabolic differences of anestrus, heat, pregnancy, pseudopregnancy, and lactation in 800 female dogs.

Introduction: Reproduction causes major hormonal and physiological changes to the female body. However, the metabolic changes occurring during canine reproduction are scarcely studied. Methods: In this cross-sectional study, we assessed the metabolic effects of canine reproductive status using a 1H NMR metabolomics platform optimized and validated for canine use. The study population consisted of a total of 837 healthy, intact female dogs in breeding age, of which 663 dogs were in anestrus, 78 in heat, 43 were pseudopregnant, 15 were pregnant, and 38 were lactating. The differences in metabolite profiles between these states were studied by the Kruskal-Wallis test with post-hoc tests performed using the Dunn's test, and visualized by box plots and a heatmap. The ability of the metabolite profile to differentiate pregnant dogs from non-pregnant ones was assessed by creating a multivariate Firth logistic regression model using forward stepwise selection. Results: Lactation, pregnancy and heat all were associated with distinct metabolic changes; pregnancy caused major changes in the concentrations of glycoprotein acetyls, albumin and creatinine, and smaller changes in several lipids, citrate, glutamine, and alanine. Pseudopregnancy, on the other hand, metabolically largely resembled anestrus. Lactation caused major changes in amino acid concentrations and smaller changes in several lipids, albumin, citrate, creatinine, and glycoprotein acetyls. Heat, referring to proestrus and estrus, affected cholesterol and LDL metabolism, and increased HDL particle size. Albumin and glycoprotein acetyls were the metabolites included in the final multivariate model for pregnancy detection, and could differentiate pregnant dogs from non-pregnant ones with excellent sensitivity and specificity. Discussion: These results increase our understanding of the metabolic consequences of canine reproduction, with the possibility of improving maternal health and ensuring reproductive success. The identified metabolites could be used for confirming canine pregnancy.