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Detection of twin pregnancies in ewes by pregnancy-associated glycoprotein assay and transabdominal ultrasonography
Journal Pre-proof Detection of twin pregnancies in ewes by pregnancy-associated glycoprotein assay and transabdominal ultrasonography Hasan Alkan, Mehmet Bugra Kivrak, Fatma Satilmis, Mustafa Agah Tekindal, Dursun Ali Dinc PII:
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DOI:
https://doi.org/10.1016/j.domaniend.2019.106399
Reference:
DAE 106399
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Domestic Animal Endocrinology
Received Date: 10 June 2019 Revised Date:
23 August 2019
Accepted Date: 11 September 2019
Please cite this article as: Alkan H, Kivrak MB, Satilmis F, Tekindal MA, Dinc DA, Detection of twin pregnancies in ewes by pregnancy-associated glycoprotein assay and transabdominal ultrasonography, Domestic Animal Endocrinology (2019), doi: https://doi.org/10.1016/j.domaniend.2019.106399. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Inc. All rights reserved.
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Detection of twin pregnancies in ewes by pregnancy-associated glycoprotein assay
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and transabdominal ultrasonography
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Running Title: Determination of twin pregnancies in sheep
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Hasan Alkana*, Mehmet Bugra Kivrakb, Fatma Satilmisa, Mustafa Agah Tekindalc, Dursun
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Ali Dinca
8 9
a
Selcuk University Faculty of Veterinary Medicine Department of Obstetrics and
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Gynecology, Konya, Turkey.
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b
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Gynecology, Sivas, Turkey.
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c
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Turkey.
Cumhuriyet University Faculty of Veterinary Medicine Department of Obstetrics and
Selcuk University Faculty of Veterinary Medicine Department of Biostatistics, Konya,
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*
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Medicine Department of Obstetrics and Gynecology, Alaeddin Keykubat Campus, 42003,
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Selcuklu/Konya, Turkey, Phone: +90 537 580 21 54, E-mail: [email protected]
Corresponding author: Hasan ALKAN, Selcuk University Faculty of Veterinary
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Abstract
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The objective of the present study were to evaluate and compare the accuracy of
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transabdominal ultrasonography and pregnancy-associated glycoprotein (PAG) assay in the
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diagnosis of twin pregnancies in ewes and to evaluate the utility of the PAG assay for
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predicting fetal gender in singleton pregnancies. The animals in the study consisted of 179
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pregnant ewes. The number of fetuses in the ewe was determined using transabdominal
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ultrasonography between days 40 and 60 (on days 40, 45, 50, 55, and 60). Blood samples
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were collected from all the ewes on the same day to determine the PAG concentrations.
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The results found were high sensitive for twin pregnancies detection by transabdominal
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ultrasonography. The accuracy of transabdominal ultrasonography in detecting twin
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pregnancies was found to be higher on day 60 than on other days (P < 0.05). The
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sensitivities of PAG assay in detecting twin pregnancies on days 40, 45, 50, 55, and 60
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were 91.67%, 66.67%, 81.82%, 88.89%, and 33.33%, respectively. The accuracies of the
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PAG assay in detecting twin pregnancies on days 40, 45, and 50 were found to be
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statistically significant higher than other days (P < 0.05). The PAG assay had low
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sensitivity, specificity for predicting fetal gender. It was concluded that twin pregnancies in
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ewes can be diagnosed with high accuracy using transabdominal ultrasonography on day
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60 of pregnancy and as well as using the PAG assay during the early days of pregnancy
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(on days 40, 45, 50). The PAG assay is not useful for predicting fetal gender.
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Keywords: Fetal Gender, Multiple pregnancy, Ultrasonography, PAG, Sheep.
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1. Introduction
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Nutritional requirements are shown significantly different among the non-pregnant,
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pregnant and multiple pregnant ewes. Multiple pregnant ewes require special care in terms
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of maintenance and nutrition during the last days of pregnancy [1]. The detection of the
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number of fetuses is of particular importance for appropriate transfer and maintenance of
2
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ewes, to minimize the mortality rate in ewes and lambs, to reduce the incidence of
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dystocia, and to prevent metabolic diseases, such as pregnancy toxemia [2-4].
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Ultrasonography is the most common method used to determine the number of
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fetuses in pregnant ewes. Studies have also attempted to determine the number of fetuses
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using other methods, such as the measurement of progesterone concentrations,
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radiographic investigations, and doppler ultrasonography. However, controversies exist
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regarding the accuracy and applicability of these methods [4-6]. Therefore, inexpensive
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and less labor-intensive methods are preferred in field conditions [7]. The method to be
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used for ultrasonographic examination varies depending on the pregnancy period,
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ultrasound probe used, working environment, and experience of the operator [7]. Multiple
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pregnancies can be diagnosed with high accuracy by transrectal ultrasonography between
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days 25 and 40 and by transabdominal ultrasonography between days 45 and 100 [6,7].
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However, the animals must have been fasting for at least 8 hours prior to transrectal
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ultrasonography, and the rectum must be emptied. Transabdominal ultrasonography from
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the ventral abdominal wall is difficult especially in wool breeds. The need for shaving this
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area may be time consuming and labor intensive [3, 8].
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The pregnancy-associated glycoprotein (PAG) assay can be used to diagnose
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pregnancy [9] and determine the number of fetuses in pregnant ewes [10]. PAG is of
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placental origin and often synthesized in the binucleate cells, although it is expressed in
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both the mono- and binucleate cells of the ruminant’s trophectoderm. PAG can be detected
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in the maternal blood at the time when trophoblastic binucleate cells begin migrating and
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fusing with endometrial cells [11,12]. Therefore, PAG is a good indicator of pregnancy and
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for determining fetomaternal status. PAG concentrations can be determined using
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radioimmunoassay (RIA) and enzyme immunoassay (EIA) [13]. The plasma PAG
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concentration in ewes begins to increase between 3 and 4 weeks of gestation with the
3
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increasing trend continuing until 9 weeks and the concentration becoming constant
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between 9 and 16 weeks of gestation [14].
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The objectives of the present study were to evaluate and compare the accuracy of
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transabdominal ultrasonography and the PAG assay for the diagnosis of twin pregnancies
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in ewes and to evaluate the utility of the PAG assay in predicting fetal gender in singleton
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pregnancies.
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2. Materials and Methods
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All procedures in this study were approved by the Experimental Animal Breeding
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and Research Center Ethics Committee of Selcuk University Faculty of Veterinary
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Medicine (2018/100).
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2.1. Animals, estrus and mating
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The animals in the study consisted of 179 pregnant Kangal-Akkaraman ewes aged 2–
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6 years and weighing 60–75 kg, with at least one parturition. All procedures were
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performed between September and December, which is the breeding season. The ewes
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were housed in an establishment (39°26'40. 5"N 37°02'27. 7"E), which belongs to the
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Sheep and Goat Breeders’ Association in the province of Sivas. The farm houses a total of
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1200 Kangal-Akkaraman sheep. They were fed with alfalfa hay, straw, corn silage, and
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concentrated feed.
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The ewes at natural estrus were mated with fertile rams using hand mating. For this
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purpose, teaser rams were used to find the ewes in estrus. Then, all ewes were separated
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into a paddock to mate with a certain ram determined beforehand, and the mating was
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monitored. The day of mating was considered as day 0.
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2.2. Determination of the number of fetuses using transabdominal ultrasonography
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The number of fetuses in pregnant ewes was determined using transabdominal
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ultrasonography (5 MHz, convex transducer, Mindray DP50, China) at five different time
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points between days 40 and 60 after mating (on days 40, 45, 50, 55, and 60).
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Ultrasonographic examination (USG) was performed in the standing position. After the gel
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was applied onto the abdominal probe, the transducer was placed on the hairless region just
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above the udder in the ventral side of the right paralumbar fossa. This inguinal region was
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fully screened up to the dorsal–caudal regions of the udder if no signs of pregnancy were
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observed. During this examination, the diagnosis of twin pregnancies was established if
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multiple parts relating to fetus were observed, such as two fetal heads, fetal heartbeats, or
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entire bodies.
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2.3. Blood Sampling
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Following transabdominal ultrasonography of all ewes, 10 ml of blood was
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withdrawn from the jugular vein into a tube without anticoagulants. Blood samples were
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centrifuged at 3000 × g for 15 minutes immediately after withdrawal. The serum were then
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stored at −20 °C until the analysis of PAG concentrations. Blood samples were collected
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on the same days when ultrasonographic examinations were performed.
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2.4. Lambing Records
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The ewes that were determined to be pregnant were monitored until lambing. All the
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ewes were examined during the lambing. The number and gender of lambs were recorded.
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2.5. Determination of PAG concentrations
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Serum samples were analyzed using the IDEXX Pregnancy Test. This test uses
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monoclonal antibodies directed against PAG, which are set on a microplate and will bind
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to PAG present in the sample. Secondary antibodies coupled with a signal amplification
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system were used for detection. The enzyme substrate 3,3′,5,5′-tetramethylbenzidine was
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used as a colored indicator to detect PAG concentrations in the sample. After stopping the
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reaction, the optical density of each well was read at the wavelength of 450 nm. The results
5
125
were calculated and expressed as sample-negative. For serum samples, if the result was
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≥0.3 ng/ml, the samples were considered as positive (pregnant), and if the result was <0.3
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ng/ml, they were considered as negative (not pregnant) [15].
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2.6. Statistical Analysis
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The SPSS 25 software package (IBM Corp. Released 2017. IBM SPSS Statistics for
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Windows, Version 25.0. Armonk, NY: IBM Corp.) was used to evaluate the data. Firstly,
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descriptive statistics of continuous values were presented as mean ± standard deviation and
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frequency (percentage). The conformity of data with repeated-measures analysis of
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variance (ANOVA) was evaluated by the Mauchly’s sphericity test and Box-M variance
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homogeneity test. Factorial repeated-measured ANOVA was used in the comparison of
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mean values. Either the Greenhouse–Geisser [16] with the correction for degrees of
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freedom or the Huynh–Feldt [17] test was used if the requirements for parametric tests
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were not met (factorial repeated-measured ANOVA). A Bonferroni test was used in case of
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multiple comparisons. The variables were evaluated after testing if they met the
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prerequisites for normality and homogeneity of variances (Shapiro–Wilk and Levene’s
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tests). In the data analysis, sensitivity, specificity, positive predictive value, negative
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predictive value, positive and negative likelihood ratio, and accuracy were calculated. The
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relationships between categorical variables were analyzed using the Fisher’s exact test and
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Chi-square test. In cases where the expected frequencies were less than 20%, Monte Carlo
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simulation was used for the inclusion of these frequencies in the analysis. The performance
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of a test can be defined by the diagnostic capability of the test, or the capacity to accurately
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distinguish subjects from subgroups (Twin or Singleton / Sheep, etc.). The cut-off scores of
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PAG values in twin/singleton and female/male births were evaluated by ROC analysis.
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AUC Value, Sensitivity, Specificity values were calculated. Cutting points were evaluated
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according to Youden Index. P values <0.05 and <0.01 were considered statistically
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significant.
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3. Results
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A total of 179 Kangal-Akkaraman ewes were examined for fetal number
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determination in the different days of pregnancy. The day of pregnancy and the number of
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sheep examined are shown in table 1.
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3.1. Lambing Records
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According to the lambing records, it was observed that 117 ewes singleton (65.36%)
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and 62 ewes twin (34.63%) born. Fetal number of the ewes are shown in table 1 in the
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different days of pregnancy.
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The gender of the offspring of the ewes born singleton were 52 (44.44%) female and
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65 (55.55%) male, and is given in table 2 according to the different days of pregnancy.
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3.2. Transabdominal ultrasonography results
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Of the 29 ewes examined by transabdominal ultrasonography on day 40, singleton
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pregnancies were detected in 18 (62.06%) ewes, and twin pregnancies were detected in 11
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(37.93%) ewes. Of the 38 ewes examined on day 45, singleton pregnancies were detected
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in 32 (84.21%) ewes, and twin pregnancies were detected in 6 (15.78%) ewes. Of the 33
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ewes examined on day 50, singleton pregnancies were detected in 27 (81.81%) ewes, and
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twin pregnancies were detected in 6 (18.18%) ewes. Of the 42 ewes examined on day 55,
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singleton pregnancies were detected in 31 (73.80%) ewes, and twin pregnancies were
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detected in 11 (26.19%) ewes. Of the 37 ewes examined on day 60, singleton pregnancies
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were detected in 26 (70.27%) ewes, and twin pregnancies were detected in 11 (29.72%)
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ewes. Sensitivity, specificity, positive predictive value, negative predictive value, positive
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likelihood ratio, negative likelihood ratio, and accuracy of transabdominal ultrasonography
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during different periods of pregnancy in diagnosing twin pregnancies are shown in Table 3.
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The accuracy of transabdominal ultrasonography in diagnosing twin pregnancies was
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found to be higher on day 60 than on the other days (P < 0.05).
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3.3. PAG results
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PAG concentrations of single and twin pregnancies on days 40, 45, 50, 55, and 60
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are presented in Figure 1. The PAG concentrations increased with rising gestational age.
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The PAG concentrations in the ewes with twin pregnancies were higher than in those with
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singleton pregnancies. This difference in PAG concentrations was statistically significant
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on days 40, 45, and 50 of pregnancy (P < 0.05).
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Different cut-off values were determined for all PAG concentration measurements
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performed on different days of pregnancy for the diagnosis of twin pregnancies.
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Accordingly, the cut-off values determined on days 40, 45, 50, 55, and 60 were 0.684,
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1.277, 1.205, 1.111, and 1.99 ng/ml, respectively (Figure 2). Based on these results,
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sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and accuracy of
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the PAG assay in diagnosing twin pregnancies are presented in Table 4. The sensitivity and
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specificity values of the PAG concentration measurements during the early periods of
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pregnancy (days 40, 45, and 50) were found to be high. Therefore, it was concluded that
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the PAG assay can be used for early diagnosis of twin pregnancies.
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PAG concentrations were also evaluated to predict fetal gender during different
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periods of pregnancy in ewes with a singleton pregnancy (Figure 3). Accordingly, the cut-
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off values determined on days 40, 45, 50, 55, and 60 of pregnancy were 0.58, 0.54, 0.96,
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0.75, and 1.91 ng/ml, respectively (Figure 4). The sensitivity, specificity, positive
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likelihood ratio, negative likelihood ratio, and accuracy of the PAG assay for predicting
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fetal gender are presented in Table 5. It was concluded that PAG assay is not useful for
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predicting fetal gender.
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4. Discussion
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In recent years, many studies have investigated the PAG assay for the diagnosis of
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pregnancy in cows, sheep, and goats [9,15,18,19]. Moreover, some studies involving ewes
201
have attempted to detect multiple pregnancies using PAG concentration measurements [10,
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12, 20]. In this study, PAG concentration measurement and transabdominal
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ultrasonography were performed simultaneously on different days of pregnancy and the
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optimum day of examination was investigated for the diagnosis of twin pregnancies.
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Ledezma-Torres et al. [20]
reported that plasma PAG concentrations begin to
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increase between weeks 3 and 4 of pregnancy with the upward trend sustaining until week
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9, after which PAG concentrations remain constant until week 19. Therefore, the present
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study evaluated the PAG concentrations between days 40 and 60 of pregnancy. There was
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a statistically significant difference between ewes with singleton and twin pregnancies in
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terms of PAG concentrations on days 40, 45, and 50 (P < 0.05). However, no statistically
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significant difference was found between the PAG concentrations on days 55 and 60 (P >
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0.05). El Amiri et al. [12] have also reported higher plasma PAG concentrations during the
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early periods of pregnancy in ewes with multiple pregnancies. In their study, Robert et al.
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[21] found that the PAG concentration 4 weeks before parturition was higher in ewes with
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twin lambs than in those with a single lamb. So, PAG concentrations during pregnancy
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may vary depending on the number of fetuses [10, 22]. The variability in PAG
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concentrations is associated with the number of binucleate cells in fetal trophectoderm
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[21]. Ranilla et al. [10] suggested that the reason for higher PAG concentration in ewes
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with twin pregnancies was the presence of more attachment points. They reported that
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ewes with two placentas had a higher secretory activity. In this study, it was speculated
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that the reason for high PAG concentrations in ewes with twin fetuses is the increased
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number of binucleate cells and increased PAG secretion due to larger placental size.
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In previous studies, multiple pregnancies in ewes were detected by measuring
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progesterone concentrations [4], by ultrasonography [8, 23], by measuring PAG
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concentrations [10, 12, 21], and by measuring placental diameters [24]. The present study
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attempted to diagnose twin pregnancies by simultaneously performing transabdominal
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ultrasonography and PAG concentration measurements on different days of pregnancy. It
228
was noted that transabdominal ultrasonography was more accurate in diagnosing twin
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pregnancies with increasing pregnancy period, whereas PAG concentration measurement
230
was more useful for diagnosing twin pregnancies during early periods of pregnancy. Dias
231
et al. [23] reported higher accuracy for transabdominal ultrasonography performed on day
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35 in ewes with a singleton pregnancies compared to those with twin pregnancies (P <
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0.05). Furthermore, the accuracy of this method was reported to vary depending on the
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experience of the operator [23]. Jones et al. [8] reported that the accuracy of
235
transabdominal ultrasonography before day 45 of pregnancy decreased with increasing
236
number of fetuses. In singleton, twin, and triplet pregnancies, the accuracy was reported to
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be 100%, 87%, and 31%, respectively. The accuracy of transabdominal ultrasonography in
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detecting the number of fetuses was 78%. Hence, transabdominal ultrasonography could be
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used to determine the number of fetuses. However, it has been reported that the
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determination of multiple pregnancies can be improved by taking into account the early
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placental development and fetal findings [8]. In this study, it was observed that the most
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appropriate day for determining twin pregnancies with transabdominal ultrasonography
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with high accuracy is day 60 of pregnancy. Yotov [4] compared estimation of progesterone
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concentrations and transrectal ultrasonography for determining the number of fetuses in
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ewes. It was reported that progesterone concentration measurement should be performed
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on day 60 and transrectal ultrasonography should be performed on day 40. However, the
10
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results of the study by Yotov [4] may differ from the results of this study because
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transrectal ultrasonography was used in the former study.
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The present study attempted to diagnose twin pregnancies by measuring PAG
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concentrations on different days of pregnancy. The accuracies of the receiver operating
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characteristic (ROC) analysis performed on day 40, 45 and 50 of pregnancy for
252
determining twin pregnancies was found to be statistically significant higher than other
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days. However, it was found that the accuracy of the test decreased with increasing
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pregnancy period. Karen et al. [3] found that the sensitivity of the PAG-RIA test in
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detecting twin pregnancies was 64.1%, and the specificity was 62.3% between days 43 and
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56 of pregnancy. In this study, the sensitivity was 91.67% and the specificity was 58.82%
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on day 40. The sensitivity values were higher than those reported by Karen et al. [3].
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Therefore, PAG concentration was found to be adequate in determining twin pregnancies
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on day 40 of pregnancy. The sensitivity, specificity, and accuracy of the test on day 50 of
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pregnancy were found to be 81.82%, 77.27%, and 59%, respectively. The accuracy of the
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test on day 50 is similar to that reported by Karen et al. [3]. However, our sensitivity and
262
specificity values were higher than those reported by Karen et al. [3], who stated that the
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efficacy of the PAG assay was low in detecting twin pregnancies. On the contrary, Roberts
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et al. [21] suggested that the PAG concentration measurement could be used effectively as
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a marker of the number of fetuses in crossbreed ewes. Ranilla et al. [10] reported that the
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number of fetuses affects PAG production during the last 3 months of pregnancy and that
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PAG concentration measurement could be effective in determining twin pregnancies
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during this period. Barbato et al. [25] reported that PAG concentrations were higher in
269
multiple pregnancies than in singleton pregnancies between days 24 and 50. However,
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because of individual differences, it was suggested that new studies should be performed in
271
order to determine multiple pregnancies using this measurement. In the present study, it
11
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was concluded that the PAG assay could be used during the early periods of pregnancy (on
273
days 40, 45, and 50) for determining twin pregnancies.
274
Placental development varies according to the number, gender, size, and weight of
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the fetus. The weight and size of male fetuses are larger than that of female fetuses. For
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this reason, the placenta develops to a greater extent in mammals with male fetuses, and
277
thus, PAG production increases [26-28]. Ranilla et al. [11] found that PAG concentration
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was higher in ewes carrying male fetuses than in ewes carrying female fetuses during
279
weeks 19, 20, and 21 of pregnancy. According to these results, the gender of the fetus has
280
been reported to affect PAG production. However, the present study found no statistical
281
difference between PAG concentrations in ewes carrying male and female fetuses during
282
different periods of pregnancy. It was thought that the difference between Ranilla et al.
283
[11] and this study was related to different pregnancy periods. According to the ROC curve
284
analysis, the accuracy of the PAG assay was found to be very low in this study. Therefore,
285
it was concluded that the prediction of fetal gender is not possible based on the PAG
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concentration measurement.
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In conclusion, it was observed that twin pregnancies could be determined with high
288
accuracy by transabdominal ultrasonography in ewes on day 60 of pregnancy. On the
289
contrary, the PAG assay can detect twin pregnancies with high accuracy during the early
290
stages of pregnancy (on days 40, 45, and 50). Besides, increasing the number of animals
291
was recommended to achieve more significant results. And it was also concluded that the
292
PAG concentration is not affected by fetal gender, and hence, it cannot be used for
293
predicting fetal gender.
294
Acknowledgements
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This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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Conflict of Interest Statement
298
The authors declare no conflicts of interest.
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ultrasound scanning, progesterone, and pregnancy-associated glycoprotein assays.
353
Theriogenology 2004;62:1108–1115.
354
[20] Ledezma-Torres RA, Beckers JF, Holtz W. Assessment of plasma profile of
355
pregnancy-associated glycoprotein (PAG) in sheep with a heterologous (anti-
356
caPAG55+59) RIA and its potential for diagnosing pregnancy. Theriogenology
357
2006;66:906–912.
358
[21] Roberts JN, May KJ, Veiga-Lopez A. Time-dependent changes in pregnancy-
359
associated glycoproteins and progesterone in commercial crossbred sheep.
360
Theriogenology 2017;89:271–279.
361
[22] Serrano B, López‐Gatius F, Santolaria P, Almería S, García‐Ispierto I, Bech‐Sabat G,
362
Sulon J, De Sousa NM, Beckers JF, Yániz JL. Factors affecting plasma
363
pregnancy‐associated glycoprotein 1 concentrations throughout gestation in
364
high‐producing dairy cows. Reprod Domest Anım. 2009;44(4):600-605.
365
[23] Dias LMK, Souza JCD, Assis RDM, Raymundo CDM. Pregnancy diagnosis, fetal
366
quantification and gender estimation by ultra-sonography in ewes. Ciênc agrotec
367
2009;33(3):911-916.
368
[24] Kaşikçi G, Yilmaz OT, Gündüz MC, Kirşan I. Comparison of placentome diameters
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in single and twin-pregnant sheep by ultrasonographic method. Turkish J Vet Anim
370
Sci. 2011;35:187–191.
371
[25] Barbato O, Sousa NM, Debenedetti A, Canali C, Todini L, Beckers JF. Validation of a
15
372
new pregnancy-associated glycoprotein radioimmunoassay method for the detection
373
of early pregnancy in ewes. Theriogenology 2009;72(7):993-1000.
374 375 376 377
[26] Echternkamp SE. Relationship between placental development and calf birth weight in beef cattle. Anim Reprod Sci. 1993;32:1–13. [27] Ferrell CL. Factors influencing fetal growth and birth weight in cattle. 1993. https://digitalcommons.unl.edu/hruskareports/132. Date of access: 20.06.2018
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[28] Lampl M, Gotsch F, Kusanovic JP, Gomez R, Nien JK, Frongillo EA, Romero R. Sex
379
differences in fetal growth responses to maternal height and weight. Am J Hum Biol.
380
2010;22(4):431-443.
381
16
1
TABLES
2
Table 1. Number of singleton and twin lamb of sheep examined on different pregnancy days
3
according to lambing records. Pregnancy days 40 days
45 days
50 days
55 days
60 days
Total
Single (%)
17 (58.62)
29 (76.31)
22 (66.66)
24 (57.14)
25(67.56)
117(65.36)
Twin (%)
12 (41.37)
9 (23.68)
11 (33.33)
18 (42.85)
12(32.43)
62 (34.63)
Total (n)
29
38
33
42
37
179
4 5
Table 2. Gender of singleton lambs of sheep examined on different pregnancy days according
6
to their lambing records. Pregnancy days 40 days
45 days
50 days
55 days
60 days
Total
Female (%)
8 (47.05)
9 (31.03)
12 (54.54)
15 (62.5)
8 (32.0)
52 (44.44)
Male (%)
9 (52.94)
20 (68.96)
10 (45.45)
9 (37.5 )
17 (68.0)
65 (55.55)
Total (n)
17
29
22
24
25
117
7
8
Table 3. Sensitivity, specifity, positive predictive value (PPV), negative predictive value
9
(NPV), positive likelihood ratio (+LR), negative likelihood ratio (-LR) and accuracy of the
10
transabdominal ultrasonography on different days of pregnancy
Sensitivity (%) Specificity (%) PPV (%) NPV (%) +LR -LR Accuracy
11
40 day 76.92 66.66 71.42 72.72 2.30 0.34 0.720
45 day 96.55 55.55 87.5 83.33 2.17 0.06 0.8684
Pregnancy days 50 day 90.90 36.36 74.07 66.66 1.42 0.25 0.7272
55 day 100 61.11 76.66 100 2.57 0 0.8292
60 day 96.15 91.66 96.15 91.66 11.53 0.04 0.9473
12
Table 4. Optimum cut-off PAG values and respective sensitivity, specificity, area under the
13
curve (AUC), standard error (SE), likelihood ratio (LR) and accuracy of the twin prediction in
14
different days of pregnancy in sheep. Pregnancy days
PAG concentrations Cut-off (ng/ml)
AUC
SE
40 day
0.684
0.760
0.0912
45 day
1.277
0.766
0.107
50 day
1.205
0.798
0.0988
55 day
1.411
0.764
0.0749
60 day
1.99
0.543
0.109
95% CI for AUC 0.5660.898 0.6010.888 0.6220.917 0.6080.881 0.3720.708
Sensitivity (95% CI)
Spesificity (95% CI)
+LR
-LR
Accuracy
P
91.67
58.82
2.23
0.14
0.5049
0.004
66.67
89.66
6.44
0.37
0.5632
0.013
81.82
77.27
3.60
0.24
0.5909
0.002
88.89
58.33
2.13
0.19
0.4722
0.065
33.33
92
4.17
0.72
0.2533
0.692
15
16
Table 5. Optimum cut-off PAG values and respective sensitivity, specificity, area under the
17
curve (AUC), standard error (SE), likelihood ratio (LR) and accuracy of the fetal gender
18
prediction in different days of pregnancy in sheep.
19 20
Pregnancy days
PAG concentrations Cut-off (ng/ml)
AUC
SE
40 day
0.58
0.618
0.145
45 day
0.54
0.572
0.114
50 day
0.96
0.558
0.131
55 day
0.75
0.522
0.130
60 day
1.91
0.511
0.123
95% CI for AUC 0.3560.837 0.3760.753 0.3440.767 0.3110.728 0.3050.714
Sensitivity (95% CI)
Spesificity (95% CI)
+LR
-LR
Accuracy
P
55.56
75.0
2.22
0.59
0.3056
0.41
25.0
100
-
0.75
0.2500
0.52
80.0
50.0
1.60
0.40
0.3000
0.65
33.33
80.0
1.67
0.83
0.1333
0.86
76.47
-
0.76
-
0.2353
0.92
1
FIGURES
2 3
Figure 1. PAG concentrations belong to sheep delivered singleton and twin on different days of
4
pregnancy (*P<0.05, each differs from singleton and twin).
5 6 7 8 9
10
11
12 13
Figure 2. ROC analysis of the PAG concentrations in the determination of twin pregnancies on 40, 45,
14
50, 55 and 60 days of pregnancy.
15 16
Figure 3. PAG concentrations belong to ewe carrying male and female fetus on different days of
17
pregnancy.
18
19
20
21
22
23
24
25
26
27
28 29
Figure 4. ROC analysis of the PAG concentrations in the determination of gender of the fetus on 40,
30
45, 50, 55 and 60 days of pregnancy
31
S0739-7240(19)30077-3
DOI:
https://doi.org/10.1016/j.domaniend.2019.106399
Reference:
DAE 106399
To appear in:
Domestic Animal Endocrinology
Received Date: 10 June 2019 Revised Date:
23 August 2019
Accepted Date: 11 September 2019
Please cite this article as: Alkan H, Kivrak MB, Satilmis F, Tekindal MA, Dinc DA, Detection of twin pregnancies in ewes by pregnancy-associated glycoprotein assay and transabdominal ultrasonography, Domestic Animal Endocrinology (2019), doi: https://doi.org/10.1016/j.domaniend.2019.106399. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Elsevier Inc. All rights reserved.
1
Detection of twin pregnancies in ewes by pregnancy-associated glycoprotein assay
2
and transabdominal ultrasonography
3 4
Running Title: Determination of twin pregnancies in sheep
5 6
Hasan Alkana*, Mehmet Bugra Kivrakb, Fatma Satilmisa, Mustafa Agah Tekindalc, Dursun
7
Ali Dinca
8 9
a
Selcuk University Faculty of Veterinary Medicine Department of Obstetrics and
10
Gynecology, Konya, Turkey.
11
b
12
Gynecology, Sivas, Turkey.
13
c
14
Turkey.
Cumhuriyet University Faculty of Veterinary Medicine Department of Obstetrics and
Selcuk University Faculty of Veterinary Medicine Department of Biostatistics, Konya,
15 16
*
17
Medicine Department of Obstetrics and Gynecology, Alaeddin Keykubat Campus, 42003,
18
Selcuklu/Konya, Turkey, Phone: +90 537 580 21 54, E-mail: [email protected]
Corresponding author: Hasan ALKAN, Selcuk University Faculty of Veterinary
19 20 21 22 23 24 25
1
26
Abstract
27
The objective of the present study were to evaluate and compare the accuracy of
28
transabdominal ultrasonography and pregnancy-associated glycoprotein (PAG) assay in the
29
diagnosis of twin pregnancies in ewes and to evaluate the utility of the PAG assay for
30
predicting fetal gender in singleton pregnancies. The animals in the study consisted of 179
31
pregnant ewes. The number of fetuses in the ewe was determined using transabdominal
32
ultrasonography between days 40 and 60 (on days 40, 45, 50, 55, and 60). Blood samples
33
were collected from all the ewes on the same day to determine the PAG concentrations.
34
The results found were high sensitive for twin pregnancies detection by transabdominal
35
ultrasonography. The accuracy of transabdominal ultrasonography in detecting twin
36
pregnancies was found to be higher on day 60 than on other days (P < 0.05). The
37
sensitivities of PAG assay in detecting twin pregnancies on days 40, 45, 50, 55, and 60
38
were 91.67%, 66.67%, 81.82%, 88.89%, and 33.33%, respectively. The accuracies of the
39
PAG assay in detecting twin pregnancies on days 40, 45, and 50 were found to be
40
statistically significant higher than other days (P < 0.05). The PAG assay had low
41
sensitivity, specificity for predicting fetal gender. It was concluded that twin pregnancies in
42
ewes can be diagnosed with high accuracy using transabdominal ultrasonography on day
43
60 of pregnancy and as well as using the PAG assay during the early days of pregnancy
44
(on days 40, 45, 50). The PAG assay is not useful for predicting fetal gender.
45
Keywords: Fetal Gender, Multiple pregnancy, Ultrasonography, PAG, Sheep.
46
1. Introduction
47
Nutritional requirements are shown significantly different among the non-pregnant,
48
pregnant and multiple pregnant ewes. Multiple pregnant ewes require special care in terms
49
of maintenance and nutrition during the last days of pregnancy [1]. The detection of the
50
number of fetuses is of particular importance for appropriate transfer and maintenance of
2
51
ewes, to minimize the mortality rate in ewes and lambs, to reduce the incidence of
52
dystocia, and to prevent metabolic diseases, such as pregnancy toxemia [2-4].
53
Ultrasonography is the most common method used to determine the number of
54
fetuses in pregnant ewes. Studies have also attempted to determine the number of fetuses
55
using other methods, such as the measurement of progesterone concentrations,
56
radiographic investigations, and doppler ultrasonography. However, controversies exist
57
regarding the accuracy and applicability of these methods [4-6]. Therefore, inexpensive
58
and less labor-intensive methods are preferred in field conditions [7]. The method to be
59
used for ultrasonographic examination varies depending on the pregnancy period,
60
ultrasound probe used, working environment, and experience of the operator [7]. Multiple
61
pregnancies can be diagnosed with high accuracy by transrectal ultrasonography between
62
days 25 and 40 and by transabdominal ultrasonography between days 45 and 100 [6,7].
63
However, the animals must have been fasting for at least 8 hours prior to transrectal
64
ultrasonography, and the rectum must be emptied. Transabdominal ultrasonography from
65
the ventral abdominal wall is difficult especially in wool breeds. The need for shaving this
66
area may be time consuming and labor intensive [3, 8].
67
The pregnancy-associated glycoprotein (PAG) assay can be used to diagnose
68
pregnancy [9] and determine the number of fetuses in pregnant ewes [10]. PAG is of
69
placental origin and often synthesized in the binucleate cells, although it is expressed in
70
both the mono- and binucleate cells of the ruminant’s trophectoderm. PAG can be detected
71
in the maternal blood at the time when trophoblastic binucleate cells begin migrating and
72
fusing with endometrial cells [11,12]. Therefore, PAG is a good indicator of pregnancy and
73
for determining fetomaternal status. PAG concentrations can be determined using
74
radioimmunoassay (RIA) and enzyme immunoassay (EIA) [13]. The plasma PAG
75
concentration in ewes begins to increase between 3 and 4 weeks of gestation with the
3
76
increasing trend continuing until 9 weeks and the concentration becoming constant
77
between 9 and 16 weeks of gestation [14].
78
The objectives of the present study were to evaluate and compare the accuracy of
79
transabdominal ultrasonography and the PAG assay for the diagnosis of twin pregnancies
80
in ewes and to evaluate the utility of the PAG assay in predicting fetal gender in singleton
81
pregnancies.
82
2. Materials and Methods
83
All procedures in this study were approved by the Experimental Animal Breeding
84
and Research Center Ethics Committee of Selcuk University Faculty of Veterinary
85
Medicine (2018/100).
86
2.1. Animals, estrus and mating
87
The animals in the study consisted of 179 pregnant Kangal-Akkaraman ewes aged 2–
88
6 years and weighing 60–75 kg, with at least one parturition. All procedures were
89
performed between September and December, which is the breeding season. The ewes
90
were housed in an establishment (39°26'40. 5"N 37°02'27. 7"E), which belongs to the
91
Sheep and Goat Breeders’ Association in the province of Sivas. The farm houses a total of
92
1200 Kangal-Akkaraman sheep. They were fed with alfalfa hay, straw, corn silage, and
93
concentrated feed.
94
The ewes at natural estrus were mated with fertile rams using hand mating. For this
95
purpose, teaser rams were used to find the ewes in estrus. Then, all ewes were separated
96
into a paddock to mate with a certain ram determined beforehand, and the mating was
97
monitored. The day of mating was considered as day 0.
98
2.2. Determination of the number of fetuses using transabdominal ultrasonography
99
The number of fetuses in pregnant ewes was determined using transabdominal
100
ultrasonography (5 MHz, convex transducer, Mindray DP50, China) at five different time
4
101
points between days 40 and 60 after mating (on days 40, 45, 50, 55, and 60).
102
Ultrasonographic examination (USG) was performed in the standing position. After the gel
103
was applied onto the abdominal probe, the transducer was placed on the hairless region just
104
above the udder in the ventral side of the right paralumbar fossa. This inguinal region was
105
fully screened up to the dorsal–caudal regions of the udder if no signs of pregnancy were
106
observed. During this examination, the diagnosis of twin pregnancies was established if
107
multiple parts relating to fetus were observed, such as two fetal heads, fetal heartbeats, or
108
entire bodies.
109
2.3. Blood Sampling
110
Following transabdominal ultrasonography of all ewes, 10 ml of blood was
111
withdrawn from the jugular vein into a tube without anticoagulants. Blood samples were
112
centrifuged at 3000 × g for 15 minutes immediately after withdrawal. The serum were then
113
stored at −20 °C until the analysis of PAG concentrations. Blood samples were collected
114
on the same days when ultrasonographic examinations were performed.
115
2.4. Lambing Records
116
The ewes that were determined to be pregnant were monitored until lambing. All the
117
ewes were examined during the lambing. The number and gender of lambs were recorded.
118
2.5. Determination of PAG concentrations
119
Serum samples were analyzed using the IDEXX Pregnancy Test. This test uses
120
monoclonal antibodies directed against PAG, which are set on a microplate and will bind
121
to PAG present in the sample. Secondary antibodies coupled with a signal amplification
122
system were used for detection. The enzyme substrate 3,3′,5,5′-tetramethylbenzidine was
123
used as a colored indicator to detect PAG concentrations in the sample. After stopping the
124
reaction, the optical density of each well was read at the wavelength of 450 nm. The results
5
125
were calculated and expressed as sample-negative. For serum samples, if the result was
126
≥0.3 ng/ml, the samples were considered as positive (pregnant), and if the result was <0.3
127
ng/ml, they were considered as negative (not pregnant) [15].
128
2.6. Statistical Analysis
129
The SPSS 25 software package (IBM Corp. Released 2017. IBM SPSS Statistics for
130
Windows, Version 25.0. Armonk, NY: IBM Corp.) was used to evaluate the data. Firstly,
131
descriptive statistics of continuous values were presented as mean ± standard deviation and
132
frequency (percentage). The conformity of data with repeated-measures analysis of
133
variance (ANOVA) was evaluated by the Mauchly’s sphericity test and Box-M variance
134
homogeneity test. Factorial repeated-measured ANOVA was used in the comparison of
135
mean values. Either the Greenhouse–Geisser [16] with the correction for degrees of
136
freedom or the Huynh–Feldt [17] test was used if the requirements for parametric tests
137
were not met (factorial repeated-measured ANOVA). A Bonferroni test was used in case of
138
multiple comparisons. The variables were evaluated after testing if they met the
139
prerequisites for normality and homogeneity of variances (Shapiro–Wilk and Levene’s
140
tests). In the data analysis, sensitivity, specificity, positive predictive value, negative
141
predictive value, positive and negative likelihood ratio, and accuracy were calculated. The
142
relationships between categorical variables were analyzed using the Fisher’s exact test and
143
Chi-square test. In cases where the expected frequencies were less than 20%, Monte Carlo
144
simulation was used for the inclusion of these frequencies in the analysis. The performance
145
of a test can be defined by the diagnostic capability of the test, or the capacity to accurately
146
distinguish subjects from subgroups (Twin or Singleton / Sheep, etc.). The cut-off scores of
147
PAG values in twin/singleton and female/male births were evaluated by ROC analysis.
148
AUC Value, Sensitivity, Specificity values were calculated. Cutting points were evaluated
6
149
according to Youden Index. P values <0.05 and <0.01 were considered statistically
150
significant.
151
3. Results
152
A total of 179 Kangal-Akkaraman ewes were examined for fetal number
153
determination in the different days of pregnancy. The day of pregnancy and the number of
154
sheep examined are shown in table 1.
155
3.1. Lambing Records
156
According to the lambing records, it was observed that 117 ewes singleton (65.36%)
157
and 62 ewes twin (34.63%) born. Fetal number of the ewes are shown in table 1 in the
158
different days of pregnancy.
159
The gender of the offspring of the ewes born singleton were 52 (44.44%) female and
160
65 (55.55%) male, and is given in table 2 according to the different days of pregnancy.
161
3.2. Transabdominal ultrasonography results
162
Of the 29 ewes examined by transabdominal ultrasonography on day 40, singleton
163
pregnancies were detected in 18 (62.06%) ewes, and twin pregnancies were detected in 11
164
(37.93%) ewes. Of the 38 ewes examined on day 45, singleton pregnancies were detected
165
in 32 (84.21%) ewes, and twin pregnancies were detected in 6 (15.78%) ewes. Of the 33
166
ewes examined on day 50, singleton pregnancies were detected in 27 (81.81%) ewes, and
167
twin pregnancies were detected in 6 (18.18%) ewes. Of the 42 ewes examined on day 55,
168
singleton pregnancies were detected in 31 (73.80%) ewes, and twin pregnancies were
169
detected in 11 (26.19%) ewes. Of the 37 ewes examined on day 60, singleton pregnancies
170
were detected in 26 (70.27%) ewes, and twin pregnancies were detected in 11 (29.72%)
171
ewes. Sensitivity, specificity, positive predictive value, negative predictive value, positive
172
likelihood ratio, negative likelihood ratio, and accuracy of transabdominal ultrasonography
7
173
during different periods of pregnancy in diagnosing twin pregnancies are shown in Table 3.
174
The accuracy of transabdominal ultrasonography in diagnosing twin pregnancies was
175
found to be higher on day 60 than on the other days (P < 0.05).
176
3.3. PAG results
177
PAG concentrations of single and twin pregnancies on days 40, 45, 50, 55, and 60
178
are presented in Figure 1. The PAG concentrations increased with rising gestational age.
179
The PAG concentrations in the ewes with twin pregnancies were higher than in those with
180
singleton pregnancies. This difference in PAG concentrations was statistically significant
181
on days 40, 45, and 50 of pregnancy (P < 0.05).
182
Different cut-off values were determined for all PAG concentration measurements
183
performed on different days of pregnancy for the diagnosis of twin pregnancies.
184
Accordingly, the cut-off values determined on days 40, 45, 50, 55, and 60 were 0.684,
185
1.277, 1.205, 1.111, and 1.99 ng/ml, respectively (Figure 2). Based on these results,
186
sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and accuracy of
187
the PAG assay in diagnosing twin pregnancies are presented in Table 4. The sensitivity and
188
specificity values of the PAG concentration measurements during the early periods of
189
pregnancy (days 40, 45, and 50) were found to be high. Therefore, it was concluded that
190
the PAG assay can be used for early diagnosis of twin pregnancies.
191
PAG concentrations were also evaluated to predict fetal gender during different
192
periods of pregnancy in ewes with a singleton pregnancy (Figure 3). Accordingly, the cut-
193
off values determined on days 40, 45, 50, 55, and 60 of pregnancy were 0.58, 0.54, 0.96,
194
0.75, and 1.91 ng/ml, respectively (Figure 4). The sensitivity, specificity, positive
195
likelihood ratio, negative likelihood ratio, and accuracy of the PAG assay for predicting
196
fetal gender are presented in Table 5. It was concluded that PAG assay is not useful for
197
predicting fetal gender.
8
198
4. Discussion
199
In recent years, many studies have investigated the PAG assay for the diagnosis of
200
pregnancy in cows, sheep, and goats [9,15,18,19]. Moreover, some studies involving ewes
201
have attempted to detect multiple pregnancies using PAG concentration measurements [10,
202
12, 20]. In this study, PAG concentration measurement and transabdominal
203
ultrasonography were performed simultaneously on different days of pregnancy and the
204
optimum day of examination was investigated for the diagnosis of twin pregnancies.
205
Ledezma-Torres et al. [20]
reported that plasma PAG concentrations begin to
206
increase between weeks 3 and 4 of pregnancy with the upward trend sustaining until week
207
9, after which PAG concentrations remain constant until week 19. Therefore, the present
208
study evaluated the PAG concentrations between days 40 and 60 of pregnancy. There was
209
a statistically significant difference between ewes with singleton and twin pregnancies in
210
terms of PAG concentrations on days 40, 45, and 50 (P < 0.05). However, no statistically
211
significant difference was found between the PAG concentrations on days 55 and 60 (P >
212
0.05). El Amiri et al. [12] have also reported higher plasma PAG concentrations during the
213
early periods of pregnancy in ewes with multiple pregnancies. In their study, Robert et al.
214
[21] found that the PAG concentration 4 weeks before parturition was higher in ewes with
215
twin lambs than in those with a single lamb. So, PAG concentrations during pregnancy
216
may vary depending on the number of fetuses [10, 22]. The variability in PAG
217
concentrations is associated with the number of binucleate cells in fetal trophectoderm
218
[21]. Ranilla et al. [10] suggested that the reason for higher PAG concentration in ewes
219
with twin pregnancies was the presence of more attachment points. They reported that
220
ewes with two placentas had a higher secretory activity. In this study, it was speculated
221
that the reason for high PAG concentrations in ewes with twin fetuses is the increased
222
number of binucleate cells and increased PAG secretion due to larger placental size.
9
223
In previous studies, multiple pregnancies in ewes were detected by measuring
224
progesterone concentrations [4], by ultrasonography [8, 23], by measuring PAG
225
concentrations [10, 12, 21], and by measuring placental diameters [24]. The present study
226
attempted to diagnose twin pregnancies by simultaneously performing transabdominal
227
ultrasonography and PAG concentration measurements on different days of pregnancy. It
228
was noted that transabdominal ultrasonography was more accurate in diagnosing twin
229
pregnancies with increasing pregnancy period, whereas PAG concentration measurement
230
was more useful for diagnosing twin pregnancies during early periods of pregnancy. Dias
231
et al. [23] reported higher accuracy for transabdominal ultrasonography performed on day
232
35 in ewes with a singleton pregnancies compared to those with twin pregnancies (P <
233
0.05). Furthermore, the accuracy of this method was reported to vary depending on the
234
experience of the operator [23]. Jones et al. [8] reported that the accuracy of
235
transabdominal ultrasonography before day 45 of pregnancy decreased with increasing
236
number of fetuses. In singleton, twin, and triplet pregnancies, the accuracy was reported to
237
be 100%, 87%, and 31%, respectively. The accuracy of transabdominal ultrasonography in
238
detecting the number of fetuses was 78%. Hence, transabdominal ultrasonography could be
239
used to determine the number of fetuses. However, it has been reported that the
240
determination of multiple pregnancies can be improved by taking into account the early
241
placental development and fetal findings [8]. In this study, it was observed that the most
242
appropriate day for determining twin pregnancies with transabdominal ultrasonography
243
with high accuracy is day 60 of pregnancy. Yotov [4] compared estimation of progesterone
244
concentrations and transrectal ultrasonography for determining the number of fetuses in
245
ewes. It was reported that progesterone concentration measurement should be performed
246
on day 60 and transrectal ultrasonography should be performed on day 40. However, the
10
247
results of the study by Yotov [4] may differ from the results of this study because
248
transrectal ultrasonography was used in the former study.
249
The present study attempted to diagnose twin pregnancies by measuring PAG
250
concentrations on different days of pregnancy. The accuracies of the receiver operating
251
characteristic (ROC) analysis performed on day 40, 45 and 50 of pregnancy for
252
determining twin pregnancies was found to be statistically significant higher than other
253
days. However, it was found that the accuracy of the test decreased with increasing
254
pregnancy period. Karen et al. [3] found that the sensitivity of the PAG-RIA test in
255
detecting twin pregnancies was 64.1%, and the specificity was 62.3% between days 43 and
256
56 of pregnancy. In this study, the sensitivity was 91.67% and the specificity was 58.82%
257
on day 40. The sensitivity values were higher than those reported by Karen et al. [3].
258
Therefore, PAG concentration was found to be adequate in determining twin pregnancies
259
on day 40 of pregnancy. The sensitivity, specificity, and accuracy of the test on day 50 of
260
pregnancy were found to be 81.82%, 77.27%, and 59%, respectively. The accuracy of the
261
test on day 50 is similar to that reported by Karen et al. [3]. However, our sensitivity and
262
specificity values were higher than those reported by Karen et al. [3], who stated that the
263
efficacy of the PAG assay was low in detecting twin pregnancies. On the contrary, Roberts
264
et al. [21] suggested that the PAG concentration measurement could be used effectively as
265
a marker of the number of fetuses in crossbreed ewes. Ranilla et al. [10] reported that the
266
number of fetuses affects PAG production during the last 3 months of pregnancy and that
267
PAG concentration measurement could be effective in determining twin pregnancies
268
during this period. Barbato et al. [25] reported that PAG concentrations were higher in
269
multiple pregnancies than in singleton pregnancies between days 24 and 50. However,
270
because of individual differences, it was suggested that new studies should be performed in
271
order to determine multiple pregnancies using this measurement. In the present study, it
11
272
was concluded that the PAG assay could be used during the early periods of pregnancy (on
273
days 40, 45, and 50) for determining twin pregnancies.
274
Placental development varies according to the number, gender, size, and weight of
275
the fetus. The weight and size of male fetuses are larger than that of female fetuses. For
276
this reason, the placenta develops to a greater extent in mammals with male fetuses, and
277
thus, PAG production increases [26-28]. Ranilla et al. [11] found that PAG concentration
278
was higher in ewes carrying male fetuses than in ewes carrying female fetuses during
279
weeks 19, 20, and 21 of pregnancy. According to these results, the gender of the fetus has
280
been reported to affect PAG production. However, the present study found no statistical
281
difference between PAG concentrations in ewes carrying male and female fetuses during
282
different periods of pregnancy. It was thought that the difference between Ranilla et al.
283
[11] and this study was related to different pregnancy periods. According to the ROC curve
284
analysis, the accuracy of the PAG assay was found to be very low in this study. Therefore,
285
it was concluded that the prediction of fetal gender is not possible based on the PAG
286
concentration measurement.
287
In conclusion, it was observed that twin pregnancies could be determined with high
288
accuracy by transabdominal ultrasonography in ewes on day 60 of pregnancy. On the
289
contrary, the PAG assay can detect twin pregnancies with high accuracy during the early
290
stages of pregnancy (on days 40, 45, and 50). Besides, increasing the number of animals
291
was recommended to achieve more significant results. And it was also concluded that the
292
PAG concentration is not affected by fetal gender, and hence, it cannot be used for
293
predicting fetal gender.
294
Acknowledgements
295 296
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
12
297
Conflict of Interest Statement
298
The authors declare no conflicts of interest.
299
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300
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1
TABLES
2
Table 1. Number of singleton and twin lamb of sheep examined on different pregnancy days
3
according to lambing records. Pregnancy days 40 days
45 days
50 days
55 days
60 days
Total
Single (%)
17 (58.62)
29 (76.31)
22 (66.66)
24 (57.14)
25(67.56)
117(65.36)
Twin (%)
12 (41.37)
9 (23.68)
11 (33.33)
18 (42.85)
12(32.43)
62 (34.63)
Total (n)
29
38
33
42
37
179
4 5
Table 2. Gender of singleton lambs of sheep examined on different pregnancy days according
6
to their lambing records. Pregnancy days 40 days
45 days
50 days
55 days
60 days
Total
Female (%)
8 (47.05)
9 (31.03)
12 (54.54)
15 (62.5)
8 (32.0)
52 (44.44)
Male (%)
9 (52.94)
20 (68.96)
10 (45.45)
9 (37.5 )
17 (68.0)
65 (55.55)
Total (n)
17
29
22
24
25
117
7
8
Table 3. Sensitivity, specifity, positive predictive value (PPV), negative predictive value
9
(NPV), positive likelihood ratio (+LR), negative likelihood ratio (-LR) and accuracy of the
10
transabdominal ultrasonography on different days of pregnancy
Sensitivity (%) Specificity (%) PPV (%) NPV (%) +LR -LR Accuracy
11
40 day 76.92 66.66 71.42 72.72 2.30 0.34 0.720
45 day 96.55 55.55 87.5 83.33 2.17 0.06 0.8684
Pregnancy days 50 day 90.90 36.36 74.07 66.66 1.42 0.25 0.7272
55 day 100 61.11 76.66 100 2.57 0 0.8292
60 day 96.15 91.66 96.15 91.66 11.53 0.04 0.9473
12
Table 4. Optimum cut-off PAG values and respective sensitivity, specificity, area under the
13
curve (AUC), standard error (SE), likelihood ratio (LR) and accuracy of the twin prediction in
14
different days of pregnancy in sheep. Pregnancy days
PAG concentrations Cut-off (ng/ml)
AUC
SE
40 day
0.684
0.760
0.0912
45 day
1.277
0.766
0.107
50 day
1.205
0.798
0.0988
55 day
1.411
0.764
0.0749
60 day
1.99
0.543
0.109
95% CI for AUC 0.5660.898 0.6010.888 0.6220.917 0.6080.881 0.3720.708
Sensitivity (95% CI)
Spesificity (95% CI)
+LR
-LR
Accuracy
P
91.67
58.82
2.23
0.14
0.5049
0.004
66.67
89.66
6.44
0.37
0.5632
0.013
81.82
77.27
3.60
0.24
0.5909
0.002
88.89
58.33
2.13
0.19
0.4722
0.065
33.33
92
4.17
0.72
0.2533
0.692
15
16
Table 5. Optimum cut-off PAG values and respective sensitivity, specificity, area under the
17
curve (AUC), standard error (SE), likelihood ratio (LR) and accuracy of the fetal gender
18
prediction in different days of pregnancy in sheep.
19 20
Pregnancy days
PAG concentrations Cut-off (ng/ml)
AUC
SE
40 day
0.58
0.618
0.145
45 day
0.54
0.572
0.114
50 day
0.96
0.558
0.131
55 day
0.75
0.522
0.130
60 day
1.91
0.511
0.123
95% CI for AUC 0.3560.837 0.3760.753 0.3440.767 0.3110.728 0.3050.714
Sensitivity (95% CI)
Spesificity (95% CI)
+LR
-LR
Accuracy
P
55.56
75.0
2.22
0.59
0.3056
0.41
25.0
100
-
0.75
0.2500
0.52
80.0
50.0
1.60
0.40
0.3000
0.65
33.33
80.0
1.67
0.83
0.1333
0.86
76.47
-
0.76
-
0.2353
0.92
1
FIGURES
2 3
Figure 1. PAG concentrations belong to sheep delivered singleton and twin on different days of
4
pregnancy (*P<0.05, each differs from singleton and twin).
5 6 7 8 9
10
11
12 13
Figure 2. ROC analysis of the PAG concentrations in the determination of twin pregnancies on 40, 45,
14
50, 55 and 60 days of pregnancy.
15 16
Figure 3. PAG concentrations belong to ewe carrying male and female fetus on different days of
17
pregnancy.
18
19
20
21
22
23
24
25
26
27
28 29
Figure 4. ROC analysis of the PAG concentrations in the determination of gender of the fetus on 40,
30
45, 50, 55 and 60 days of pregnancy
31