<abstract xmlns="http://www.w3.org/1999/xhtml"><p>Early diagnosis of pregnancy is important in livestock production, but there is no reliable technology used for pregnancy diagnosis within the first three weeks after insemination. During early pregnancy, the expression of interferon-stimulating genes (ISGs) in peripheral blood leukocytes (PBL) is significantly increased. However, due to different strains, detection sample types, detection methods, threshold value, etc. the specific effectiveness of early pregnancy diagnosis using ISGs is worth further study. The purpose of this study was to test interferon-stimulated protein 15 (<italic>ISG15</italic>), 2'–5'-oligoadenylate synthetase 1 (<italic>OAS1</italic>) and radical S-adenosyl methionine domain containing 2 (<italic>RSAD2</italic>) for early pregnancy diagnosis in dairy cows. The expression of <italic>ISG15</italic>, <italic>OAS1</italic>, and <italic>RSAD2</italic> in PBL of pregnant and non-pregnant heifers on days 0, 14, 18, 21 and 28 after artificial insemination (AI) was detected by fluorescence quantitative polymerase chain reaction (PCR). The sensitivity and specificity of the pregnancy diagnosis was analyzed using expression of these three genes separately or in combination by receiver operating characteristic curve. The combination with the highest accuracy used probe primers and duplex fluorescence quantitative PCR. The single quantitative PCR results showed that expression of <italic>ISG15</italic>, <italic>OAS1</italic> and <italic>RSAD2</italic> on day 18 after AI was significantly higher in pregnant than in non-pregnant cows. When these three genes were used separately, or in combination, for early pregnancy diagnosis, the sensitivity for the <italic>RSAD2</italic> gene was 100%, and the combination of <italic>ISG15</italic> with <italic>RSAD2</italic> was 94.7%. The duplex quantitative PCR showed that, although the sensitivity of <italic>ISG15</italic> alone was 100%, its specificity was only 88.2% (cut-off value 1.402). The sensitivity of <italic>RSAD2</italic> alone was 89.5%, and the specificity was 88.2%; however, when the two genes were used in combination, the sensitivity, specificity and diagnostic cut-off value were consistent with the results of single quantitative PCR. These results indicated that a duplex quantitative PCR assay system for early pregnancy diagnosis in cows using <italic>ISG15</italic> and <italic>RSAD2</italic> was established. Simultaneous detection of expression of <italic>ISG15</italic> and <italic>RSAD2</italic> by duplex quantitative PCR can effectively improve the diagnostic accuracy for dairy cows.</p></abstract>

Early diagnosis of pregnancy is important in livestock production, but there is no reliable technology used for pregnancy diagnosis within the first three weeks after insemination. During early pregnancy, the expression of interferon-stimulating genes (ISGs) in peripheral blood leukocytes (PBL) is significantly increased. However, due to different strains, detection sample types, detection methods, threshold value, etc. the specific effectiveness of early pregnancy diagnosis using ISGs is worth further study. The purpose of this study was to test interferon-stimulated protein 15 (ISG15), 2'–5'-oligoadenylate synthetase 1 (OAS1) and radical S-adenosyl methionine domain containing 2 (RSAD2) for early pregnancy diagnosis in dairy cows. The expression of ISG15, OAS1, and RSAD2 in PBL of pregnant and non-pregnant heifers on days 0, 14, 18, 21 and 28 after artificial insemination (AI) was detected by fluorescence quantitative polymerase chain reaction (PCR). The sensitivity and specificity of the pregnancy diagnosis was analyzed using expression of these three genes separately or in combination by receiver operating characteristic curve. The combination with the highest accuracy used probe primers and duplex fluorescence quantitative PCR. The single quantitative PCR results showed that expression of ISG15, OAS1 and RSAD2 on day 18 after AI was significantly higher in pregnant than in non-pregnant cows. When these three genes were used separately, or in combination, for early pregnancy diagnosis, the sensitivity for the RSAD2 gene was 100%, and the combination of ISG15 with RSAD2 was 94.7%. The duplex quantitative PCR showed that, although the sensitivity of ISG15 alone was 100%, its specificity was only 88.2% (cut-off value 1.402). The sensitivity of RSAD2 alone was 89.5%, and the specificity was 88.2%; however, when the two genes were used in combination, the sensitivity, specificity and diagnostic cut-off value were consistent with the results of single quantitative PCR. These results indicated that a duplex quantitative PCR assay system for early pregnancy diagnosis in cows using ISG15 and RSAD2 was established. Simultaneous detection of expression of ISG15 and RSAD2 by duplex quantitative PCR can effectively improve the diagnostic accuracy for dairy cows.

Duplex Quantitative Polymerase Chain Reaction of <italic>ISG15</italic> and <italic>RSAD2</italic> Increases Accuracy of Early Pregnancy Diagnosis in Dairy Cows

Key Laboratory of Agricultural Animal Genetics

Institute of Animal Science and Veterinary Medicine, Wuhan Academy of Agricultural Science and Technology

Annals of Animal Science

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Duplex Quantitative Polymerase Chain Reaction of  and  Increases Accuracy of Early Pregnancy Diagnosis in Dairy Cows

Early diagnosis of pregnancy is important in livestock production, but there is no reliable technology used for pregnancy diagnosis within the first three...

Duplex Quantitative Polymerase Chain Reaction of ISG15 and RSAD2 Increases Accuracy of Early Pregnancy Diagnosis in Dairy Cows

Published Online: May 02, 2019

Page range: 383 - 401

Received: Aug 21, 2018

Accepted: Jan 03, 2019

DOI: https://doi.org/10.2478/aoas-2019-0001

Keywords
cow, pregnancy diagnosis, interferon-stimulating gene, duplex quantitative PCR

© 2019 Lei Cheng et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Duplex Quantitative Polymerase Chain Reaction of ISG15 and RSAD2 Increases Accuracy of Early Pregnancy Diagnosis in Dairy Cows

Published Online: May 02, 2019

Page range: 383 - 401

Received: Aug 21, 2018

Accepted: Jan 03, 2019

DOI: https://doi.org/10.2478/aoas-2019-0001

Keywordscow, pregnancy diagnosis, interferon-stimulating gene, duplex quantitative PCR

© 2019 Lei Cheng et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Keywords
cow, pregnancy diagnosis, interferon-stimulating gene, duplex quantitative PCR