Technical note: Validation of a chemical pregnancy test in dairy cows that uses whole blood, shortened incubation times, and visual readout

Chemical pregnancy testing is an alternative to traditional methods of pregnancy diagnosis (either manual palpation or ultrasound) in postpartum dairy cows and heifers. The objective was to validate a chemical pregnancy test that confers the advantages of using whole blood, rapid incubation times, and visual readout. Blood and milk samples were collected from Holstein dairy cows [n = 320; 162 ± 62 (mean ± SD) d in milk] on a confinement farm in northeast Missouri at 28 d after artificial insemination (AI). The samples were assayed for pregnancy-associated glycoproteins (PAG) by using a rapid visual test as well as traditional plasma- and milk-based tests. Transrectal ultrasonography diagnosis for pregnancy at 35 to 38 d after AI was the reference (gold) standard for all PAG tests. One hundred fifty-nine cows were diagnosed as pregnant by the reference standard (pregnancies per AI = 49.7%). The tests were ELISA and either optical density (OD; measured with a microtiter plate reader; plasma, milk, and rapid visual tests) or visual readout (rapid visual test) were used to diagnose pregnancy. When OD was used, the percentage of pregnant cows classified correctly (sensitivity) for the plasma, milk, and rapid visual tests were 97 ± 1, 96 ± 2, and 95 ± 1% (±SE), respectively. The sensitivity of the rapid visual test when assessed visually was 98 ± 1%. The specificity (proportion of nonpregnant cows classified correctly) for the plasma, milk, and rapid visual was 94 ± 2%, 94 ± 2%, and 93 ± 2% when an OD was used. When read visually, the specificity of the rapid visual test was lesser (85 ± 3%) because some cows with faint visual signals yielded false positive diagnosis. The overall accuracy (proportion of pregnant and nonpregnant cows diagnosed correctly) was similar for all tests (plasma, milk, rapid visual OD, and rapid visual; 96 ± 1, 95 ± 1, 94 ± 1, and 92 ± 2%, respectively). In a second experiment, lactating Holstein cows (n = 291) from 4 commercial confinement dairy farms in western Kentucky were tested 25 to 95 d after AI using the rapid visual test. The OD of the rapid visual test followed the known profile for PAG in circulation during the first trimester of pregnancy. The conclusion is that the rapid visual test has equal sensitivity and accuracy as existing PAG tests. A slightly lower specificity was found when the rapid visual test was read visually.