Elsevier

Theriogenology

Volume 84, Issue 1, 1 July 2015, Pages 76-81
Theriogenology

Research article
Accuracy of diagnosing double corpora lutea and twin pregnancy by measuring serum progesterone and bovine pregnancy-associated glycoprotein 1 in the first trimester of gestation in dairy cows

https://doi.org/10.1016/j.theriogenology.2015.02.014Get rights and content

Abstract

Progesterone (P4) and bovine pregnancy-associated glycoprotein 1 (bPAG-1) concentrations during gestation are dependent on the number of CL and fetuses, respectively. The objective of this present study was to measure and evaluate the usefulness of measuring the P4 and bPAG-1 concentrations in cases of single versus twin pregnancies and one versus two CL at the first 4 months of gestation. We hypothesized that both the number of the CL and the number of fetus might have an effect on P4 and pregnancy protein concentrations, and we can set up clinically useful threshold levels to predict twin gestations. Eighty-four Holstein-Friesian dairy cows were enrolled in this prospective observational clinical trial. Blood was collected at time point 1: between Days 29 and 42, time point 2: between Days 57 and 70, time point 3: between Days 85 and 98, and time point 4: between Days 113 and 126 of gestation, and bPAG-1 and P4 concentrations were measured. Binary logistic regression analyzing serum P4 concentrations differed at time point 2 compared with baseline level, but the area under the curve (AUC) had low sensitivity. The bPAG-1 concentrations were statistically different at each time point of gestation. The AUC cutoff values of serum bPAG-1 concentrations were sufficiently sensitive to differentiate between twin gestations from singleton ones. At time points 3 (cutoff value of 3.4 ng/mL) and 4 (cutoff value of 56.5 ng/mL), statistically significant differences with low sensitivity, high specificity, and a high AUC were found. On the basis of these results, the diagnosis of twin pregnancy using pregnancy protein measurements is clinically insufficient before Day 85 of gestation; however, the ability to confirm the early twin pregnancy diagnosis with bPAG-1 measurements appears to be promising. To achieve high sensitivity, further studies are required.

Introduction

Several pregnancy diagnosing methods are intensively used worldwide to diagnose whether a pregnant cattle is carrying multiple fetuses or not. The different rectal palpation techniques (palpation of the uterine fluctuation, amniotic vesicle, or fetal membrane slip) [1] are useful and widespread; however, they are really dependent on the skill of the examiner. An experienced veterinarian or technician even from Day 35 with the fetal membrane slip technique is able to set up pregnancy diagnoses [2]. However, the manual techniques are limited in diagnosing twins: Usually only the bilateral twins can be diagnosed accurately. The prediction accuracy of unilateral twinning is low [3].

Ultrasonography in the early period (around Day 30) of gestation is also widely used with high accuracy to diagnose early pregnancy. At this time, usually, twin pregnancies are also visible. When dealing with twin pregnancies at a herd level, we always have to take into consideration the phenomenon of late embryonic or early fetal mortality [4]. This can influence the accuracy of our diagnostic tests.

Another possibility for diagnosing early pregnancy in the field is the measurement of pregnancy specific proteins such as bovine pregnancy-associated glycoprotein 1 (bPAG-1), bovine pregnancy-specific protein B, or pregnancy serum protein, which are secreted by the binucleate cells originating from the bovine trophoblast. In contrast to progesterone (P4) measurements, they are probably good indicators of the presence of an alive conceptus [5], [6], [7]. Several assays have been developed, and nowadays not only blood but milk tests can be used for an accurate pregnancy diagnosis [8]. Numerous factors are influencing the levels of these proteins throughout gestation; twin pregnancy is one of these factors [9], [10]. There are several studies [11], [12] focusing on the measurements of pregnancy proteins in case of bovine twin pregnancies. Regarding these studies, the question is still open: whether we can determine a cutoff value for the pregnancy proteins at different time periods of gestation by which twin pregnant animals can be distinguished from singleton carrying ones.

Progesterone assays are also widely used to measure the blood P4 concentration even at a herd level. The disadvantage of these tests is that the P4 itself is nonspecific for pregnancy; therefore, performing pregnancy diagnoses by means of P4 measurement is not resulting in an accurate sensitivity and specificity [4], [5], [6].

We have hypothesized that serum P4 concentrations will be different between the single and twin pregnancies, and this will be a sensitive and specific clinical indicator for the prediction of the number of CL. Furthermore, we have also hypothesized that serum bPAG-1 concentrations will be different between single and twin pregnant groups at each time points and thus will serve as a sensitive and specific clinical indicator for the prediction of the number of fetuses.

The objectives of this study were to compare serum P4 (1) and bPAG-1 (2) concentrations at different time points of gestation in cattle with the accurate determination of one or two CL; to assess whether or not serum P4 concentration is a useful clinical indicator for the determination of the number of CL at any of the previously mentioned time points (3); and to assess whether or not serum bPAG-1 concentration is a useful clinical indicator for the determination of the number of fetuses at any of the previously mentioned time points (4).

Section snippets

Animals

Altogether 84 Holstein-Friesian dairy cattle were included in our study with a confirmed pregnancy by means of ultrasonography at the late embryonic period between Days 29 and 42 of gestation (time point 1). Animals were divided into three groups: the first group (TWIN group, n = 29) had two viable embryos in the uterine horns and two mature CL on the ovaries at the time of pregnancy diagnosis. Animals with one viable embryo in the pregnant uterine horn and two CL on the ovaries formed the

Analysis of serum P4 and bPAG-1 concentrations

The descriptive data of serum P4 and the bPAG-1 concentrations of the groups at different time points are given in Table 1.

The general linear model analyzing bPAG-1 concentrations revealed significant differences (P < 0.001) within groups and at every sampling time points. Regarding serum P4 concentrations, the general linear model revealed statistically significant differences (P < 0.005) between the time points 1 versus 4 and 2 versus 4.

Association between serum P4 concentrations and the number of CL

Cows were regrouped on the basis of the number of the CL

Discussion

In this study, the concentrations of P4 and bPAG-1 were measured in cases of singleton and twin gestations with one or two CL. Due to the fact that almost 95% of twin pregnancies are dizygotic [19], it was worth testing that double CL might produce more P4 at different stages of gestation. Because generally from around Day 150 of gestation, the placenta provides a marked amount of P4, it was important to test the animals before this time point to achieve a clinically reliable diagnosis. Several

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