Pregnancy diagnosis in cattle using pregnancy associated glycoprotein concentration in circulation at day 24 of gestation

Abstract

Cattle producers are limited to day 28-30 of gestation as the earliest time point for accurate pregnancy diagnosis due to the effectiveness of ultrasound and chemical based methods, including commercially available pregnancy associated glycoproteins (PAG) tests. The objective of the current studies were to determine if early gestation circulating PAG concentrations at day 24 could be used to diagnose pregnancy in dairy cattle undergoing embryo transfer. In vitro produced embryos were transferred into Holstein x Gir cows and heifers on day 7 following ovulation. Study 1 utilized only cows (n = 101) determined to be pregnant on day 24 of gestation by progesterone concentration, as well as CL and PAG presence. In study 2, animals were not predetermined to be pregnant and both heifers (n = 111) and cows (n = 242) were used. In both studies, blood was collected at day 24 for PAG analysis as well as day 31. Final pregnancy confirmation occurred on day 60 via transrectal ultrasonography. Serum PAG concentrations were quantified using an in house PAG ELISA. Following timed embryo transfer (TET) in study 1, of the 101 cows diagnosed as pregnant on day 24, 80 cows were identified as still pregnant on day 31 of gestation (77%). Study 2 had a pregnancy rate at day 31 of 33.7% of total embryos transferred. Mean circulating PAG concentration at day 24 differed (P < 0.001) between animals diagnosed pregnant and non-pregnant at day 31 in both studies (study 1, 2.964 ± 0.262 ng/mL vs 0.946 ± 0.168 ng/mL and study 2, 1.962 ± 0.261 ng/mL vs 0.731 ± 0.109 ng/mL). Concentration of PAG between pregnant and non-pregnant cows in study 1 and 2 was significant, however, pregnant heifers in study 2 (1.562 ± 0.266 ng/mL) had concentration of PAGs that only had a tendency to differ compared to non-pregnant heifers (non-pregnant, 0.799 ± 0.290 ng/mL; P = 0.0669). Only animals that were pregnant at day 31 were analyzed in late embryo mortality analysis (heifers, n = 54; cows, n = 159), defined as pregnancy loss between day 31 and 60. Between day 31 and 60, 39 (12 in study 1 and 28 in study 2) animals experienced late embryo mortality. Circulating concentrations of PAG were not significantly different (P > 0.05), in either study, at day 24 of gestation in animals that maintained pregnancy until day 60 compared to animals that lost pregnancy between day 31 and 60 (late embryo mortality, LEM). In summary, early gestation circulating PAG concentration may have application in diagnosing pregnancy at day 24 of gestation and more work is needed to determine the potential of early gestation PAGs in predicting embryo loss in dairy.

New approaches to diagnose and target reproductive failure in cattle

“…In addition to the high rate of false positives, false negative diagnosis can be an issue as circulating PAG concentrations increase at different rates in individual cows. Reese et al (2018) reported up to a 55% false negative rate using a 90% confidence cutoff value; whereas, Middleton and Pursley (2019), using a different antibody-assay combination, observed a 6% false negative rate indicating that antibody selection towards early secreted PAGs is critical. One challenge is day 24 PAG concentrations exhibit increased variability compared to day 30 samples, making it more difficult to establish cutoff values for pregnancy success or embryonic mortality predictions.…”

Impact of fetal vs. maternal contributions of Bos indicus and Bos taurus genetics on embryonic and fetal development1

“…Two plasma controls containing moderate (2.0 ng/mL) and low (1.0 ng/mL) PAG concentrations were included in each plate. Plasma concentrations of PAG were also measured by a monoclonal-based PAG in-house ELISA (in-house) as described by Green et al (2005) and modified by Pohler et al (2016) and Reese et al (2018), as well as a second commercial assay (A2; Idexx, Westbrook, ME). The in-house assay uses a polyclonal antibody raised against first-secreted PAG (Ab 63; Reese et al, 2018), whereas the antigen used to create a detection antibody for A2 consisted of a mixture of PAG (PAG 4,6,9,16,18,and 19;US Patent no.…”

“…on recipient plasma concentrations of pregnancy-associated glycoproteins (PAG). Blood samples were collected weekly from days 28 to 91 of gestation and plasma concentrations of PAG were measured using two commercial PAG ELISA antibody combinations (A1 [Panel A] and A2 [Panel B]), and an in-house monoclonal-based PAG 63 ELISA (Panel C) as described by Green et al (2005) and modified by Reese et al (2018). A1: embryo breed × day (P < 0.01).…”

Characterization of Pregnancy-Associated Glycoprotein as a Biomarker of Pregnancy in Etawa Crossbred Goat.

“…PAG can be measured in maternal blood circulation (Shahin et al, 2013). Pregnancy is diagnosed using PAG test on day 24 of gestation (Reese et al, 2017). The knowledge of mechanisms involved in the production and control of PAG is beneficial in livestock breeding and facilitates diagnosis of pregnancy (Santos et al, 2018).…”