When diagnosing pregnancy via transrectal palpation, reproductive structures are felt for positive signs of pregnancy. Most large animal veterinarians can accurately diagnose pregnancy as early as 30–35 days after insemination, but the level of accuracy is highly dependent on the expertise of the veterinarian and generally increases when examination is delayed to 40–50 days after breeding.³ Concerns over early embryonic mortality as a result of transrectal palpation, particularly via fetal membrane slip, have been expressed and has been the subject of multiple studies. The risks associated with transrectal palpation for diagnosis of pregnancy are fully addressed in Chapter 34.

Through transrectal ultrasound, pregnancy can be consistently and accurately diagnosed as early as 25 days after insemination. Maximum sensitivity in diagnosing a pregnancy is reached at day 26 and day 29 in heifers and cows respectively.^4,5 For this reason, transrectal ultrasound is often used as the “gold standard” when evaluating other means of pregnancy diagnosis. Although 25 days after insemination provides a diagnosis earlier than transrectal palpation, transrectal ultrasound still does not allow diagnosis of nonpregnancy before the next expected estrus and, like transrectal palpation, requires that the veterinarian has a great deal of technical skill in order to yield consistently accurate diagnoses. Furthermore, this method of pregnancy determination requires substantial inputs to purchase and maintain necessary equipment, and these costs must be passed on to the producer.

The ideal pregnancy test would accurately diagnose both pregnancy and nonpregnancy a short time after fertilization, have excellent sensitivity (the female that tests pregnant is indeed pregnant), specificity (the female that tests nonpregnant is indeed open), and accuracy (the proportion of true results). Furthermore, the ideal pregnancy test would be noninvasive in that there would be no risk of causing embryonic or fetal death through testing. In addition, it would be minimally expensive, require little or no technical skill, and could be quickly and easily performed chute-side. An ideal pregnancy test for the bovine would be very similar to the human chorionic gonadotropin (hCG) test for pregnancy in women.

The hCG test is a simple ELISA (enzyme-linked immunosorbent assay) that detects the presence of hCG protein in the urine of pregnant women. It can easily and inexpensively be performed in the home, requiring virtually no technical skill. Commercially available hCG tests boast extremely high accuracy (>99%), and some can be performed as early as 8–10 days after conception.⁶ Unfortunately, cows and heifers do not produce hCG or any analogous molecule that could be tested in a comparable fashion. Instead, tests have been developed to ascertain the presence of certain pregnancy-associated molecules such as elevated progesterone concentration, pregnancy-associated glycoproteins (PAGs), early pregnancy factor (EPF), and interferon-stimulated genes (ISG).² Additionally, as technology and scientific knowledge of early pregnancy advance, there are a number of other molecules that may one day be used as pregnancy biomarkers in the bovine.

An important distinction that producers should be mindful of when opting to utilize biochemical methods to determine pregnancy status is that progesterone, PAG, EPF, and ISG assays are more accurate in detecting nonpregnant females than in correctly identifying pregnancy. Consequently, endocrine-based pregnancy tests often have excellent specificity (>90%) but slightly less desirable sensitivity. This is of significant economic importance because females in intensive production scenarios that are falsely identified as open will often be administered prostaglandin in an attempt to initiate a new cycle and reduce time to next insemination. This injection of prostaglandin will of course induce abortion in pregnant females and since the average cost of a pregnancy loss in a dairy female in the United States is estimated at $555, there is significant incentive to minimize the incidence of iatrogenic embryonic and fetal loss.⁷

Similarly, another confounding factor when attempting to identify pregnancy soon after fertilization is natural pregnancy loss. Bovine fertilization rates are usually high (>90%), and therefore the main contributor to reproductive wastage is embryonic or fetal death.^8,9 Early embryonic death, occurring prior to day 17 of pregnancy, accounts for 20.5–43.6% of loss when coupled with frequency of failure of fertilization.¹⁰ However, late embryonic death, which represents loss from approximately 17 to 42 days of gestation, is estimated to be, on average, 12.8% in dairy cows and approximately 6.5% in beef females.^11–13 It has been estimated that pregnancy losses from fertilization to term in high-producing dairy cows may approach 60%.¹³ When critically evaluating the reliability of endocrine tests performed early in gestation, it is customary to confirm pregnancy diagnosis by transrectal palpation or ultrasound days or weeks after the initial test was performed. This creates a window in which embryonic or fetal loss can occur, thus skewing the data by incorrectly labeling some results as false positives as the female was indeed pregnant when first tested but lost the pregnancy before the confirmatory pregnancy evaluation.

Progesterone concentration

Serum and milk progesterone levels steadily increase following ovulation as a functional corpus luteum (CL) grows and develops. In nonpregnant females, progesterone declines as the CL regresses and the cow returns to estrus. In pregnant females, however, progesterone levels should remain elevated during the period of the next expected estrus as the CL continues to produce progesterone in order to maintain the pregnancy.

Progesterone concentration testing functions on the premise that serum and/or milk progesterone concentration will remain elevated at the time of the next expected estrus if the female were pregnant. If the female is open, progesterone concentration will decrease to baseline as the CL regresses and a new dominant follicle develops³ (see Chapters 23 and 24).

Discrepancies in progesterone testing may be largely caused by natural variation between individuals in luteal lifespan and estrous cycle length as progesterone levels remain high while a CL is present during diestrus.¹⁴ Furthermore, embryonic mortality between the time of progesterone testing and pregnancy confirmation via transrectal palpation or transrectal ultrasound examination may further contribute to perceived test error.³

A potential drawback to the use of progesterone testing in production settings is that the breeding or insemination date must be known in order to obtain reliable results from a single sample, and therefore the applicability of progesterone testing is somewhat limited.^2,3 Typically, a milk or serum sample is taken 21–24 days after breeding and is analyzed using either radioimmunoassay (RIA), ELISA or, less frequently, latex agglutination (LA). A number of progesterone ELISAs for serum and milk are available to producers and are marketed as cowside tests.

Progesterone testing is very reliable for identifying nonpregnant females, as cows with blood progesterone levels less than 1 ng/mL 21 days after insemination cannot be pregnant, but it is slightly less reliable for positively diagnosing pregnancy because of natural variations in estrous cycle lengths between individual animals.² Detection rates of 95–100% have been reported in detecting nonpregnancy. Pregnancy was correctly identified 68–97% of the time, with study designs varying in the day of gestation the test was performed, the standard it was compared against, and the type of assay used (ELISA, RIA, or LA).^15–20

Pregnancy-specific protein B

Ruminant trophoblasts release proteins as a part of maternal recognition of a viable conceptus. These proteins, known as PAGs, are aspartic peptidases and have not been found to be proteolytically active. The first PAG to be discovered was pregnancy-specific protein B (PSPB), formerly known as pregnancy-associated glycoprotein 1. This protein is produced by trophoblast binucleate cells in the bovine placenta around the time of implantation, though its exact role in pregnancy maintenance is not yet fully understood. It can be found in maternal circulation from the time of implantation throughout gestation, often persisting well into the postpartum interval.^21,22

As its name suggests, PSPB is a pregnancy-specific molecule and is not found in the serum of virgin heifers or open cows, thus demonstrating that the protein is produced solely as a response to a conceptus and not in response to luteal activity during the estrous cycle.²³ Unlike equine chorionic gonadotropin (eCG) and hCG, PSPB is not detectable in urine, and it is only found in milk during intervals when PSPB is at very high concentration in plasma, for example shortly after parturition.²¹

An RIA capable of reliably detecting PSPB in maternal serum as early as 24 days after conception was developed by Sasser et al.²³ in 1986. A then unique advantage of PSPB testing was that, unlike progesterone assays, insemination dates are not needed to confirm pregnancy in PSPB testing as the protein is pregnancy specific and present throughout gestation, peaking around the time of parturition.²³

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