Biological characteristics of pregnancy in captive Yangtze finless porpoises revealed by urinary metabolomics†

Abstract

The Yangtze finless porpoises (Neophocaena asiaeorientalis a.) are an endemic and critically endangered species in China. Intensive captive breeding is essential for understanding the biology of critically endangered species, especially their pregnancy characteristics, knowledge of which is crucial for effective breeding management. Urine metabolomics can reveal metabolic differences, arising from physiological changes across pregnancy stages. Therefore, we used the urinary metabolomic technology, to explore urinary metabolite changes in pregnant Yangtze finless porpoises. A total of 2281 metabolites were identified in all samples, which including organic acids and derivatives (24.45%), organoheterocyclic compounds (20.23%), benzenoids (18.05%), organic oxygen compounds (7.73%), and phenylpropanoids and polyketides (6.48%). There were 164, 387, and 522 metabolites demonstrating differential abundance during early pregnancy, mid pregnancy, and late pregnancy, respectively, from the levels observed in nonpregnancy. The levels of pregnenolone, 17α-hydroxyprogesterone, and tetrahydrocortisone were significantly higher during all pregnancy stages, indicating their important roles in fetal development. The differential metabolites between nonpregnancy and pregnancy were mainly associated with amino acid and carbohydrate metabolism. Moreover, metabolic activity varied across pregnancy stages; steroid hormone biosynthesis was predominant in early pregnancy, and amino acid biosynthesis and carbohydrate metabolism were predominant in mid pregnancy and late pregnancy, respectively. Our results provide new insights into metabolic characteristics in the Yangtze finless porpoises' urine during pregnancy, and indicate that the differential levels of urine metabolites can determine pregnancy in Yangtze finless porpoises, providing valuable information for the husbandry and management of pregnant Yangtze finless porpoises in captivity.

Keywords: Yangtze finless porpoises; biomarker; pregnancy; urinary metabolomics.

Graphical Abstract

Figure 1

The pie chart of metabolite classification according to category information (Super class) from the HMDB 5.0 database. The different colors represent different HMDB categories. The metabolites with percentages lower than 2% were clustered into “other,” accounting for 13.05%.

Figure 2

Differentially abundant metabolite selection process. NP, nonpregnancy stage; EP, early pregnancy stage; MP, mid pregnancy stage; LP, late pregnancy stage. (A) PCA score plot shows the difference between the pregnancy and nonpregnancy groups. (B) OPLS–DA further revealed the differences among the comparable groups. (C) Overlap of metabolites between pregnancy periods (EP, MP, and LP) and the nonpregnancy period (NP). (D) Heatmap of the relative intensity of 12 potential biomarkers with pregnancy progression. (E) Time expression features of 12 potential biomarkers. Tetrahydrocortisone (ID: S5657), showed a different expression feature compared to the other metabolites, with increased levels in late pregnancy.

Figure 3

Pathway analysis summary histogram. Differential metabolites were classified by KEGG pathway enrichment and significance analysis. Histograms A, B, C, D, E, and F represent the comparisons of NP vs. EP, NP vs. MP, NP vs. LP, EP vs. MP, EP vs. LP, and MP vs. LP, respectively.