Identification of mink (Neovison vison) fecal proteins during embryonic diapause and placental pregnancy for non-invasive pregnancy diagnosis in wildlife

Abstract

Currently, there is no method to diagnose pregnancy non-invasively in most wildlife species that experience delayed implantation and pseudopregnancy, either during embryonic diapause or placental pregnancy. The aim of this study was to utilize farm-raised mink (Neovison vison) as a model species to evaluate changes in the fecal proteome associated with pregnancy. Specific objectives were to: 1) determine if fecal peptides were differentially abundant in parturient versus non-parturient mink and; 2) identify proteins of interest. Samples (n=12) were selected retrospectively from mink (n=6) that were parturient (n=3) or non-parturient (n=3) and were collected from parturient females during embryonic diapause and placental pregnancy or, on the same calendar dates from non-parturient females. Following protein extraction, two-dimensional differential in-gel electrophoresis was utilized to assess differences in protein spot abundance among samples. The mean number of spots per gel was 2107±62.2 and spots meeting specific criteria (student's t-test; P<0.10; >2.5 fold change between groups) were selected for identification via matrix-assisted laser desorption/ionization- time-of-flight mass spectrometry. During diapause, six spots (angiotensin-converting enzyme 2, interleukin-36 receptor antagonist, carboxypeptidase A1 (two spots), carboxypeptidase A2, and chymotrypsin-like protease CTRL-1) were higher in parturient and one spot (intestinal fatty acid-binding protein) was higher in non-parturient. During placental pregnancy, seven spots (cytosol aminopeptidase (three spots), calcium-activated chloride channel regulator 1, carboxypeptidase A1 (two spots), and chymotrypsin) were higher in parturient and two (ovalbumin and protein PRR14L) were higher in non-parturient. This is the first description of the mink fecal proteome related to pregnancy and of changes in specific fecal proteins during embryonic diapause in any species.