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Comparison of ultrasound and pregnancy associated glycoproteins to diagnose and
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follow ongoing or failing pregnancy in pregnant heifers on the market
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N. Prvanović1, J.F. Beckers2, J. Sulon2, A. Tomašković1, M. Cergolj1,T. Dobranić1 ,J.
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Grizelj1, M.Samardžija1,S: Vince1 ,P. Kočila3
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Belgium; 3Veterinary station Čakovec, Croatia
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Faculty of veterinary medicine Zagreb,Croatia, 2Faculty of veterinary medicine, Liege,
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Abstract:
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The objective of the present study was to compare transrectal ultrasonography and
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pregnancy associated glycoprotein (PAG) tests for determination of pregnancy in 72
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Holstein-friesian heifers imported from Holland to Croatia. All heifers came to Croatia
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with written confirmation of pregnancy. During caranteen we preformed rectal and
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ultrasound exploration of heifers and simultaneusly collected blood samples from each
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heifer. PAG concentrations were determined by a RIA imunoassay using bovine PAG
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67 KDa as a tracer and standard. In our study 68 heifers were confirmed to be pregnant
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by both methods and 4 cows were confirmed for abortion by PAG test and nonpregnant
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according to ultrasound checking. Transrectal ultrasonography is accurate test for
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pregnancy diagnosis in pregnant heifers but it is impossible to say only on basis of
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ultrasound examination if heifers conceived and aborted or they failed to conceive in the
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first place. PAG RIA test is not only highly accurate and reliable method to distinguish
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between pregnant and nonpregnant heifers on the market. It is also usefull to determine
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whether heifers were pregnant and embryo died and was absorbed during the trasfer or
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heifers failed to conceive. It is possible because level of PAG persists in circulation
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after abortion or embrional death. In doubtfull cases it can be used as a proof of
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pregnancy existence in heifers later confirmed to be nonpregnant.
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Key words:
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mortality
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Zusammenfassung:
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Das Ziel der Arbeit ist der Vergleich der transrektalen Ultraschalluntersuchung mit dem,
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im Labor durchführendem PAG Test, mit der Absicht der Diagnostik und Überwachung
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des Ablaufes, so wie der Befund der Gravidität in trächtigen Färsen auf dem Markt. In
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der Arbeit wurden 72 Färsen der Sorte Holstein, die nach Kroatien importiert wurden,
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überwacht, mit schriftlichem Nachweis, dass alle 70-90 Tage trächtig sind. Während der
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Quarantäne wurden die Färse mittels Ultraschall untersucht und im deren Blut wurde
pregnant heifer, ultrasonography, PAG, pregnancy testing, embrionic
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die Höhe von PEG bestimmt. Aufgrund des Ultraschallbefunds wurde festgestellt, dass
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68 Färse trächtig sind und 4 nicht trächtig sind. Aufgrund des PAG-Tests wurde
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festgestellt, dass 68 Färse trächtig sind und 4 erlitten Fehlgeburten, wahrscheinlich
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während des Transports, denn aufgrund des PAG-Profils wurde festgestellt, dass es
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weniger als 40 Tagen von der Fehlgeburt vergangen sind. Aufgrund der späteren Daten
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über der Geburt ist es sichtbar, dass beide Methoden 100% sicher sind, wobei die PAG
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die Methode der Wahl ist, denn sie ermöglicht den Unterschied zwischen Färsen, die
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eine Fehlgeburt erlitten haben von denen die nicht trächtig sind.
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Stichworte:trachtig Farse, Ultraschall, PAG, Schwangerschaft Diagnostik, Fehlgeburt
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Introduction
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Dairy heifers on the market should be sold pregnant to avoid any later complications
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with conception and to make the whole breeding more easier and profitable. Nobody
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would like to buy heifer unable to conceive. On the other side, pregnant animals are
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quite sensitive to many chalenges that are exposed to as change of environment, transfer
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to another farm or even country and many other stressfull events that ussually follows.
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Keeping in mind all mentioned facts it appears clear that we need not only to perform
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early pregnancy diagnostic but also later confirmations of pregnancy to see whether all
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necesarry changes and stresfull events had or didn't have influence on development of
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pregnancy. None method of early pregnancy diagnostics is 100% accurate so we need
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to combine methods to get even more accurate results. Good combination shuold be
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transrectal ultrasound checking (rapid, easy and accurate) and PAG determination
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(possible for retrograde checking and determination of embrionic mortality). Real time
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B-mode transrectal ultrasonography (5 MHz) is a simple, rapid and noninvasive
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technique applied on the farm. It can detect early pregnancy in form of anechoic
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intrauterine fluid as early as day 17 after conception but embryo is properly visible
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around days 21-34, and heart rate is detectible around day 24. However, the accuracy of
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transrectal ultrasonography to detect pregnant heifers and cows varies depending on
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timing of examination, but it consists 98,7% after day 24 (visible heart rate) (1). In the
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same time it is possible to detect pregnancy on basis of PAG and BPSP level in blood.
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Furthermore, proteins secreted by the placenta (2) when detected in the peripheral
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circulation of the mother, can be useful indicators of both pregnancy and feto-
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trophoblast well being. These glycoproteins (either PSPB-pregnancy specific bovine
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proteins or PAG-pregnancy associated glycoproteins) could be detected in the maternal
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circulation at around the time when the trophoblast forms definitive attachment to the
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uterine wall (3). In the veterinary practice radioimunoassays (RIAs) of PAG or/and
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PSPB in plasma samples are helpful to confirm pregnancy diagnoses established by
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rectal palpation or ultrasonography (4). Moreover, the follow-up of both bPAG and
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BPSP during pregnancy permits to keep track of the viability of the fetoplacental unit.
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However due to large variations in bPAG/PSPB concentrations of the maternal blood,
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only the marked decrease (or disappearance) of the serum concentrations of these
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proteins can be an umbiguous predicitive sign of embrionic or fetal death (5).
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Furthermore level of PAG decreases slowly till day 35 after embrionic mortality and it
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could be used as proof of existence and death of embryo in cows later confirmed to be
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nonpregnant (5).
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Material and methods
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We performed our study on 72 Holstein-friesian heifers imported from Holland to
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Croatia. All heifers came to Croatia with written confirmation of pregnancy. During
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caranteen we preformed rectal and ultrasound examination of heifers and simultaneusly
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collected blood samples from each heifer. We collected blood two times in interval od
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72 days and analysed all sera samples in duplicate. In the same time we perfoormed
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ultrasound scanning. We used real time B-mode transrectal ultrasound probe (5 MHz)
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to detect pregnancy and collected blood using vacutainer from v. jugularis into SST
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tubes. All blood samples were centrifuged immidiatly (1500xg for 20 minutes) and
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stored in eppendorf kivetes (-20°C) till tests. PAG concentrations were determined in
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duplicate by a RIA imunoassay using bovine PAG 67 KDa as a tracer and standard.
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Statistical analysis included sensitivity, specificity,, positive predictive and negative
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predicitve values. The sensitivity and specificity of the transrectal ultrasonographic and
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PAG tests were compared by Binomial exact test.
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Results
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All heifers in our study were confirmed to be pregnant 70 to 90 days. Rectal and
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ultrasound examination together with blood sampling were performed 35 days after the
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data on writen confirmation of pregnancy. Later data about calving rate have shown that
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positive diagnosis of pregnancy was correct in 100% using both PAG and
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ultrasonography.In our study 68 heifers (95%) were confirmed to be pregnant by both
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methods and 4 heifers (5%)f were confirmed for abortion by PAG test and nonpregnant
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according to ultrasound checking. Sensitivity consisted 100% for both methods.
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Specificity of both tests was extremely high due to fact that all heifers were in second
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trimester of pregnancy
80
70
60
50
40
30
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0
pregnant
nonpregnant
before transfer
after transfer
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Discussion
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In our study 68 heifers (95%) were confirmed to be pregnant by both methods and 4
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heifers (5%) were confirmed for abortion by PAG test and nonpregnant according to
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ultrasound checking. Later data about calving rate have shown that positive diagnosis of
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pregnancy was correct in 100% using both PAG and ultrasonography, similar as
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described in Goddard (1994).
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Transrectal ultrasonography is accurate test for pregnancy diagnosis in pregnant heifers
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but it is impossible to say only on basis of ultrasound examination if heifers conceived
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and aborted or they failed to conceive in the first place. PAG RIA test is not only highly
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accurate and reliable method to distinguish between pregnant and nonpregnant heifers
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on the market. It is also usefull to determine whether heifers were pregnant and embryo
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died and was absorbed during the trasfer or heifers failed to conceive. It is possible
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because level of PAG persists in circulation after abortion or embrional death as
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described by Beckers et al (2001). In doubtfull cases it can be used as a proof of
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pregnancy existence in heifers later confirmed to be nonpregnant.
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References
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(1994)
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2. Beckers J.F. , Zarrouk A., Batalha, E.S., Garbayo, J.M., Mester L. and Szenci
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O. Endocrinology of pregnancy: Chorionic Somatomammotropins and
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pregnancy Associated Glycoproteins: Review.Acta veterinaria hungarica 46,pp
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175-189(1998)
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3. Beckers J.F., P.V. Drion, J. M.Garbayo,, Zs Perenyi, A. Zarrouk, J. Sulon, B.
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Remy
and
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4. Szenci O. , Taverne M.,A.M., Beckers J.F.,, Sulon J.,, Varga J, Borszonyi L,
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Hanzen Ch., and Schekk, Gy: Evaluation of false ultrasonographic diagnoses
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in cows by measuring plasma levels of bovine pregnancy associated
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glycoprotein, Veterinary Record 142, pp 304-306 (1998)
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Varga J, Bajszy and Taverne M.A. M.
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conceptus proteins in cows with spontaneus embryonic/fetal mortality as
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diagnosed by ultrasonography, The Veterinary Journal50, 77-80 (1999)
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Plasma profiles of progesterone and