Graduate Student University of Florida MARIANNA, Florida, United States
Abstract Authors: Maria C. Lopez-Duarte1, Daniella Heredia1, Mauro Venturini1, Kamryn Joyce1, Araceli Maderal1, Federico Tarnonsky1, Angela Gonella-Diaza1 1. Department of Animal Sciences (NFREC), University of Florida, Marianna, FL.
Abstract Text: Cell-fee DNA (cfDNA) results from cell death and can help measure the processes of apoptosis in animals and humans. Previous evidence has suggested that cfDNA can be used as a biomarker and a liquid biopsy for cancer and fetal aneuploidy diagnosis in humans. We recently showed that plasma cfDNA concentration increases forty-eight hours after induction of luteolysis with Prostaglandin F2 alpha analog in beef cows. Natural luteolysis occurs when no pregnancy signaling occurs around day 16 –18 after estrus. We hypothesized that increments in plasma cfDNA concentrations could be used to diagnose early pregnancy in beef cattle at day twenty-two after Time Artificial Insemination (TAI). In super early resynchronization protocols, a Doppler ultrasound is performed on days 21–22 to diagnose a super early pregnancy by assessing luteal blood flow. This allows for a second-timed artificial insemination (TAI) on day 24. Angus heifers (n = 73) were synchronized using the 7-Day CoSynch + CIDR protocol, and TAI was performed with conventional semen. Twenty-two days later, blood samples were collected to measure plasma cfDNA concentration and interferon-stimulated gene (ISG) expression in peripheral blood mononuclear cells (PBMCs), color-Doppler ultrasonography was performed to measure the total area of the corpus luteum (CL) and to determine luteal blood perfusion (LBP = percentage of colored pixels within the CL-Area), both relative gene expression (ISG15, MX1, MX2 and OAS1Y) and LBP were used as an early pregnancy diagnosis approach. Non-pregnant heifers were re-synchronized, and a second TAI was performed. Final pregnancy diagnosis was conducted on day 30 by B-mode ultrasonography, and heifers were retrospectively classified as pregnant (PR) and non-pregnant (NP). Data were analyzed using Proc Mix of SAS. On day 22, thirty-three heifers were classified as NP and thirty-nine as PR by Doppler ultrasonography. Day 60 B-mode ultrasonography confirmed the classification (NP, n = 33 and PR, n = 39). There was no significant difference in plasma cfDNA concentration between PR and NP heifers (p = 0.44, PR = 232.32 ± 51.04 ng/μL, NP = 163.56 ± 59.55 ng/μL). On the contrary, transcript abundance of ISG15 (PR = 0.15 ± 0.01 AU, NP = 0.1 ± 0.02 AU; p < 0.006), OAS1Y (PR = 0.14 ± 0.01 AU, NP = 0.05 ± 0.01 AU; p < 0.0001), MX1 (PR = 0.54 ± 0.03 AU, NP = 0.31 ± 0.03 AU; p < 0.0001) and MX2 (PR = 0.17 ± 0.01 AU, NP = 0.07 ± 0.01 AU; p = < 0.0001) as well as CL total area (PR = 3.31 ± 0.2 cm2, NP = 1.82 ± 0.02 cm2; p < 0.0001,) and LBP% (PR = 16.48 ± 1.64%, NP = 7.82 ± 1.64%; p < 0.0001) where different between NP and PR heifers allowing us to confirm that the expression of ISG and luteal characteristics differ between NP and PR heifers. In conclusion, cfDNA concentration did not differ between PR and NP heifers at day 22 of pregnancy. In contrast, the expression of ISG15, OAS1Y, MX1, and MX2 in PBMCs, as well as LBP% and corpus luteum (CL) area, differed between the groups. Therefore, ISG and LBP% remain as the most reliable indicators for early pregnancy diagnosis before day twenty-eight.
.jpg "Maria Camila Lopez Duarte photo")