Early identification of bovine pregnancy status and embryonic mortality†

Abstract

Bovine interferon-tau (bIFNT) is produced by the trophectoderm cells in the bovine conceptus as early as Day 12 following fertilization. It was hypothesized that IFNT detection in blood, milk, and/or cervical secretions could be used to diagnose pregnancy in lactating cows. Recombinant bovine (rb) IFNT was generated to produce goat and rabbit anti-rbIFNT polyclonal antibodies, and an enzyme-linked immunosorbent assay (ELISA) was developed for bIFNT using these reagents. The IFNT ELISA did not cross-react with other type I or II IFNs and had a limit of detection of 50-100 pg/ml. The IFNT ELISA detected IFNT in external ostium (os) cervical swabs from Days 15 to 25 post-AI, but did not detect IFNT in serum, plasma, or milk. The time for most accurately detecting IFNT in cervical fluid was Days 16-19 after AI. A custom bovine swab device used to collect cervical secretions reduced false-negative rates to 5.5% (94.5% sensitivity) in dairy cows on Day 17 and 0% to 3.4% (100% and 96.6% sensitivity) in beef cows on Days 18 or 16, respectively. In summary, the detection of IFNT in cervical fluid by ELISA provides an accurate indication of pregnancy status in lactating dairy cows. Early identification of the non-pregnant cow allows re-insemination on Day 21 compared to waiting until ultrasound (US) on Day ~32-39. In addition, the detection of IFNT on Day 17 followed by the loss of pregnancy detected by US on Day 32 provides a novel research tool for studying pregnancy loss caused by embryonic mortality.

Keywords: bovine; conceptus; diagnostic; interferon-tau; pregnancy.

Graphical Abstract

Figure 1

Description of the commercial equine swab (ES) device (left), external os location of cervical sampling (middle), and development of a custom bovine swab (BS) device (right). The custom BS device was constructed with an inner swab (S) that had superior absorbing and releasing features compared to the ES. It was stabilized using a stronger inner tube (I), which allowed entrance into the external os of the cervix and did not bend compared to the equine device. A similar outer protected sheath (O) to the ES was used to prevent the swab from picking up any other fluid when navigating from the vulva to the vagina to the cervix. The fully assembled (A) device was pressed against the cervical external os and opened to collect fluid just inside of the os and prior to the first cervical ring.

Figure 2

Determination of IFNT antigen specificity, IFN profiles, and accuracy estimates when detecting pregnancy status based on IFNT concentrations (open and pregnant cows) on days indicated compared to Day 32+ US. (A) IFNT ELISA antigen specificity. The bovine IFNT ELISA detected only IFNT and no other related type I IFNs or the more distant type II IFNG. (B) Detection of IFNT in blind vaginal swabs (n = 28 cows). There was 100% accuracy in specificity and FN calls on day of AI (Day 0). The OCT is based on the lack of detection of IFNT on days that IFNT is not expected to be present (Days 0–14). It is apparent from this study that IFNT is not present in blind vaginal swabs until about Day 16 following AI. There were no FP cows on Days 0–15 for this reason (Day P < 0.0001, Pregnancy status P < 0.0058, Day × Pregnancy status P < 0.0001). (C) IFNT concentrations in transrectal guided cervical external os samples and accuracy data using the equine swab (ES) device on Days 15–21 following AI (n = 37 cows) (Day P < 0.05, Pregnancy Status P < 0.001, Day × Pregnancy Status P > 0.05). (D) IFNT concentrations in transrectal guided external cervical os ESs in 34 lactating dairy cows on Days 13 to 31 following AI. This was a qualitative IFNT analysis. The line represents the highest concentration (500 pg/ml) in the linear range of the assay. Values above this line are not precise estimates of IFN concentrations. For accuracy estimates expanded on Day 17, see Table 1. Values represent average IFNT concentrations ± SE. The blue “*” represents a t-test at P < 0.05 when protected by the significant Day × Pregnancy Status (Day P < 0.0002, Pregnancy Status P < 0.0001, Day × Pregnancy Status P < 0.0001) (^*P < 0.05, ^**P < 0.01, ^***P < 0.001, †P < 0.10). Note that the actual absorbance value from the ELISA was used in these studies. There were also a few FP cows (detected IFNT concentrations greater than LOD on days tested but not pregnant (open in the graphs) at US. This is why there may appear to be a slight increase in IFNT concentrations after Day 17 in the cows determined to be open by US in Panels B and C. This was done in this figure to illustrate how FP cows can influence IFNT levels prior to US. For this reason, in all subsequent analyses, any cows with IFNT levels at or below the LOD on days tested were designated as 0 pg IFNT/ml.

Figure 3

IFNT concentrations using the custom BS device in external cervical os (Os Cx) compared to mid-cervix (Mid Cx, passed the first ring) on Day 17 open and pregnant cows (n = 99) (A) and distribution of IFNT concentrations and median values in FP compared to TP cows based on US on Day 32 (B). Swab samples were collected from the same cows. The external swab sample is inside of the os but prior to the first ring. The mid-cervical swab sample is a sample after the first cervical ring. The external swab sample was collected first and then the mid-cervical swab sample was collected using two separate BS devices. IFNT concentrations were 49,210 ± 21 261 pg/ml for Os Cx-Pregnant cows and 151,299 ± 34 479 pg/ml for Mid Cx-Pregnant cows. (B) Distribution of IFNT concentrations in external cervical os swab samples (Os Cx) on Day 17 in FP (FP/embryo mortality) and TP dairy cows called by US (BS; Table 1). Notice the high and low clusters of IFNT concentrations in both groups but also the much lower median concentration with 75% of values in the lower quadrant in FP pregnancies. Median concentrations of IFNT are shown by arrows for FP and TP cows (^**P < 0.01). The line shows the separation between high and low clusters. The LOD for this assay was 64 pg/ml. Data are quantitative. Values represent the mean ± SE.

Figure 4

Concentrations of IFNT and progesterone in TN (A and B), FN (C), TP (D), and FP (E) cows. The FP cows experienced embryonic mortality any time between OCT with a detectable IFNT concentration on Day 17 and US diagnosis on Day ≥32.

Figure 5

IFNT “Violin” profiles on Day 17 in TN, TP, FN, and FP cattle in the studies described in Tables 1 and 2. Columns represent the analysis of IFNT on Day 17 using either the equine swab device or the new BS. Notice that most of the FP dairy cows had very high IFNT levels indicative of presence of a conceptus at the time of sample but then lost the pregnancy (embryo mortality) by the time of US at 32 days. The line represents the highest concentration in the linear range of the assay. Values above this line are not precise estimates of IFN concentrations.