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Interferon stimulated genes as peripheral diagnostic markers of early pregnancy in sheep: a critical assessment

Published online by Cambridge University Press:  06 May 2016

V. Mauffré ,
B. Grimard ,
C. Eozenou ,
S. Inghels ,
L. Silva ,
C. Giraud-Delville ,
D. Capo ,
O. Sandra  and
F. Constant
V. Mauffré*
Affiliation:
Ecole Nationale Vétérinaire d’Alfort, UMR 1198 Biologie du Développement et Reproduction, Université Paris Est, 7 av du général de Gaulle, F-94704 Maisons-Alfort Cedex, France UMR BDR, INRA, Université Paris Saclay, 78350 Jouy en Josas, France
B. Grimard
Affiliation:
Ecole Nationale Vétérinaire d’Alfort, UMR 1198 Biologie du Développement et Reproduction, Université Paris Est, 7 av du général de Gaulle, F-94704 Maisons-Alfort Cedex, France UMR BDR, INRA, Université Paris Saclay, 78350 Jouy en Josas, France
C. Eozenou
Affiliation:
UMR BDR, INRA, Université Paris Saclay, 78350 Jouy en Josas, France
S. Inghels
Affiliation:
Ecole Nationale Vétérinaire d’Alfort, UMR 1198 Biologie du Développement et Reproduction, Université Paris Est, 7 av du général de Gaulle, F-94704 Maisons-Alfort Cedex, France UMR BDR, INRA, Université Paris Saclay, 78350 Jouy en Josas, France
L. Silva
Affiliation:
Ecole Nationale Vétérinaire d’Alfort, UMR 1198 Biologie du Développement et Reproduction, Université Paris Est, 7 av du général de Gaulle, F-94704 Maisons-Alfort Cedex, France UMR BDR, INRA, Université Paris Saclay, 78350 Jouy en Josas, France
C. Giraud-Delville
Affiliation:
UMR BDR, INRA, Université Paris Saclay, 78350 Jouy en Josas, France
D. Capo
Affiliation:
INRA, UE 1298 Unité Commune d’Expérimentation Animale, Domaine de Bressonvilliers, F-91630 Leudeville, France
O. Sandra
Affiliation:
UMR BDR, INRA, Université Paris Saclay, 78350 Jouy en Josas, France
F. Constant
Affiliation:
Ecole Nationale Vétérinaire d’Alfort, UMR 1198 Biologie du Développement et Reproduction, Université Paris Est, 7 av du général de Gaulle, F-94704 Maisons-Alfort Cedex, France UMR BDR, INRA, Université Paris Saclay, 78350 Jouy en Josas, France
*
† Email: [email protected]
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Abstract

We investigated the diagnostic reliability of pregnancy detection using changes in interferon stimulated gene (ISG) messenger RNA (mRNA) levels in circulating immune cells in ewes. Two different groups of ewes (an experimental group, experiment 1 and a farm group, experiment 2) were oestrus-synchronized and blood sampled on day 14 (D0=day of insemination in control animals, experiment 1) and day 15 (experiment 2). Real-time PCR were performed to evaluate the abundance of different ISG mRNAs. In the experimental group, peripheral blood mononuclear cells of 29 ewes born and bred in experimental facilities were isolated using a Percoll gradient method. Gene expression for Chemokine (C-X-C motif) ligand 10 (CXCL10), Myxovirus (influenza virus) resistance 1 (MX1) and Signal transducer and activator of transcription 1 (STAT1) mRNA were, respectively, 8.3-fold, 6.1-fold and 2.7-fold higher (P<0.001) in pregnant compared with non-pregnant ewes. The receiver operating characteristic (ROC) curves generated from the real-time PCR data demonstrated that a reliable cut-off could be established for CXCL10, MX1 and STAT1. In the farm group of animals, peripheral blood leucocytes of 37 cross-bred multiparous ewes bought from several herds were isolated using the PAXgene® procedure. This blood sampling procedure is achievable in farms, whereas the Percoll method is not. No significant differences (P>0.10) in CXCL10, STAT1, MX1, Myxovirus (influenza virus) resistance 2 (MX2) and ISG15 ubiquitin-like modifier (ISG15) mRNA expression were found between pregnant and non-pregnant ewes. The ROC curves and the hierarchical classification generated from the real-time PCR data failed to discriminate between pregnant and non-pregnant animals. In this group of animals, our results show a strong variability in ISG expression patterns: 17% of animals identified as non-pregnant by the five tests were in fact pregnant, only 52% of pregnant animals had at least two positive results (two genes above threshold), whereas up to five positive results (five genes above threshold) were needed to avoid misclassification. In conclusion, this study illustrates the high variability in ISG expression levels in immune circulating cells during early pregnancy and, therefore, highlights the limits of using ISG expression levels in blood samples, collected on PAXgene® tubes on farms, for early pregnancy detection in sheep.

Keywords

interferon stimulated genespregnancy diagnosisearly pregnancyimplantationsheep
Type
Research Article
Information
animal , Volume 10 , Issue 11 , November 2016 , pp. 1856 - 1863
Copyright
© The Animal Consortium 2016 

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