Evaluation of a Test for Equine Early Conception Factor

The lateral flow dipstick test for equine Early Conception Factor (ECF) [a] shows a sensitivity of 53.8% and a specificity of 71% in its ability to detect the barren mare at 7 - 8 days after ovulation. The predictive value of a negative test is 71% accurate at 7 - 8 days after ovulation.

1. Introduction

The early detection of pregnancy in mares has been limited to either recovery of an embryo intended for transfer or the use of ultrasonography at 12 - 14 days after ovulation. In an embryo-transfer program, embryos are flushed from donor mares 7 - 8 days after ovulation. An accurate and reliable test for pregnancy at this stage of embryonic development would have great economic value to the equine breeding industry, because it would determine the need for the embryo flush. Furthermore, an accurate test for early conception would prove valuable for identifying mares that conceive and lose the pregnancy by the time of ultrasound examination.

In the serum of many mammalian species, the mare included, an Early Pregnancy Factor (EPF) can be reliably detected hours after fertilization has occurred by the rosette inhibition test (RIT) [1-6]. An EPF-like factor has been produced in vitro as well from cell cultures of both fertilized bovine ova [7] and mouse ovaries and oviducts exposed to estradiol and progesterone [8]. EPF has been identified as an immunosuppressive glycoprotein [9] believed to be involved in suppression of the lymphocytic immune response in the mare [10]. Although highly accurate, the RIT is difficult to maintain in clinical practice. Therefore, the objective of this study was to evaluate a commercially available "stall-side" early pregnancy test that is designed to identify the barren mare by detecting the presence of an equine early conception factor (ECF). Unlike some mammalian EPF that has been isolated, purified, and characterized based on its molecular weight [11], ECF has yet to be described in scientific literature.

2. Materials and Methods

Mares in estrus (n = 68) were examined daily by transrectal palpation and ultrasound examination. Test mares (n = 46) were bred by natural cover or artificial insemination 1 - 2 days before ovulation (day 0). Control mares (n = 22) were not bred. A test for ECF was performed on the fresh serum from all 68 mares on day 7 or 8 after ovulation.

The horse ECF test is designed as a lateral flow assay where both monoclonal and polyclonal antibodies are incorporated into a nitrocellulose membrane that uses colloidal gold conjugate as the indicator of ECF glycoprotein. Following the manufacturer’s instructions, the ECF test (Fig. 1) was performed by adding one drop of serum and two drops of buffer solution (using the dropper provided by the manufacturer) to the round sample hole of the cassette. Then, the cassettes were incubated at room temperature until the results were recorded 2 h later. When only a single red line appeared in the control region "C", a negative result was recorded, indicating barren status. When two red lines appeared, one line in the control region "C" of the cassette and the other in the test region "T" of the cassette, a positive result was recorded, indicating "not barren" status (Fig. 2).

Figure 1. The ECF test kit.

Figure 2. A positive ECF test.

Pregnancy status was either determined by the results of an embryo flush at days 7 -8  after ovulation or a transrectal ultrasound examination 12 - 14 days after ovulation. The pregnancy results were compared with the results of ECF test. The sensitivity, specificity, and predicted value of the negative test were determined by the following formulas.

True Positive / True Positive + False Negative × 100 = Sensitivity (%)        (1)

True Negative / False Positive + True Negative × 100 = Specificity (%)        (2)

True Negative / False Negative + True Negative × 100 = Negative Predictive Value (%)        (3)

3. Results

The results of the study are shown in Table 1. The ECF test predicted that 42 of 68 mares were barren. Actual pregnancy diagnosis showed that 12 of 42 "barren" mares were pregnant (false negatives). All of the ECF tests from the 22 control mares (not bred) correctly identified the mares as barren.

Therefore, the predicted value of a negative test that identifies the barren mare is calculated to be 71%. The ECF test predicted that 26 of 68 mares were not barren. Actual pregnancy diagnosis determined that only 14 of those 26 mares were pregnant at the time of the embryo flush or ultrasound examination. Twelve mares were barren (false positives).

The sensitivity and specificity of the ECF test were calculated to be 53.8% and 71%, respectively.

4. Discussion

The objective of this study was to determine the accuracy of a stall-side test for the early detection of pregnancy. Not only would an accurate test allow prediction of presence of an embryo before uterine flushing, but it would also aid in identifying mares that lost an early conceptus; from this, appropriate treatment might be designed to enhance the possibility of live-foal production. Also, identification of an immunosuppressive glycoprotein secreted early in pregnancy may play an important role in the maternal recognition of pregnancy.

Although the RIT has shown high accuracy in detecting equine pregnancy as early as 2 days after ovulation, this test is difficult to maintain in most laboratories and private equine practices. Unfortunately, identification and purification of an early conception or pregnancy factor and the development of an assay that can predict the barren mare with a minimum 99% specificity has been elusive.

It should be noted that the sensitivity and specificity of such an assay for conception is likely to be compromised unless pregnancy immediately after conception can be determined. In evaluating early embryo loss rates, Ball et al. [12] found that in mares, conception rates at 2 days after ovulation were far higher than 14-day pregnancy rates. This suggests that conception occurs in most breedings but that the embryo is lost before 14 days after ovulation. It is not surprising that an early conception factor may be detectable in a significant number of bred mares that may be found barren at 14 days post-ovulation. Furthermore, in this study, pregnancy determination was based either on the recovery of an embryo at 7 - 8 days after ovulation or on ultrasonographic confirmation at 12 - 14 days after ovulation. It is quite possible that some pregnancies were lost between 8 and 14 days, thereby increasing the number of false-positive tests. Additionally, embryo recovery is not 100% efficient; some mares could have conceived, but the embryo was not recovered. All of these conditions compromise the specificity of this assay.

The true value of this assay, however, lies in its sensitivity. It must accurately and reliably predict the barren mare. For such a test to be useful in detecting conception, the sensitivity would have to approach 99%. The sensitivity of the ECF test was calculated in this study to be 53.8%, which may be falsely elevated because of the high number of control mares. Therefore, the negative predictive value, affected by sensitivity, may also be falsely elevated.

Reports that have evaluated ECF test in predicting "non-pregnancy" in the cow either by examining serum or milk have been conflicting. Threlfall and Bilderbeck [13] reported that the test correctly identified "non-pregnancy" in the cow 24 - 48 h after breeding in 94.6% of the tests based on rectal palpation for the determination of pregnancy 32 - 45 days after breeding. In contrast, Gandy et al. [14] reported that, relative to actual pregnancy rates, a negative ECF test was correct in only 38.5% (true negative) of the cases.

This study in mares indicates that the first generation of the ECF does not yet have the accuracy needed for commercial use in the equine breeding industry.

The authors thank Heather Brummond, Shelley Pruitt, and Brad Root for technical assistance.

Footnote

1. Concepto Diagnostics, Knoxville, TN 37914.