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 farmraised 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, twodimensional 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/ ionizationtime-of-flight mass spectrometry. During diapause, six spots (angiotensinconverting 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 nonparturient. 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.