Ovine Pregnancy-associated Glycoprotein Research Articles

Plasmatic Profile of Pregnancy-Associated Glycoprotein (PAG) during Gestation and Postpartum in Sarda and Lacaune Sheep Determined with Two Radioimmunoassay Systems.

Simple SummaryNowadays the need to optimize and maximize the productivity of dairy sheep leads farmers to plan lambing in specific periods in order to avoid economic loss due to nonproductive animals. The goal is to diagnose pregnancy at early gestation in order to minimize the costs of unproductive animals and to properly formulate rations for the energy requirements of gestating or lactating animals at the same time. Moreover, early pregnancy diagnosis, as well as the possibility of distinguishing between single and multiple gestations, enables farmers to plan the management of lambing well in advance thus minimizing manpower requirements. This study showed, for the first time, the plasmatic profile of PAG (pregnancy-associated glycoproteins) in Sarda and Lacaune ewes during gestation and the postpartum period using two different radioimmune assay (RIA) systems, thus enhancing our knowledge regarding PAG concentrations in domestic ruminants. Moreover, it showed that for both breeds these RIA systems are capable of distinguishing pregnant from nonpregnant ewes starting from day 18 of gestation. Furthermore, the rapid disappearance of PAG concentration following lambing did not require the use of a cut-off limit in postpartum animals as a means for detecting a new pregnancy.This study was carried out to determine ovine pregnancy-associated glycoprotein (oPAG) levels in the plasma of Sarda and Lacaune ewes throughout gestation and in the first month postpartum, using two heterologous radioimmunoassays (RIA-706 and RIA-srPool) and to study the correlations between PAG levels and fetal gender and number. On Day 18 of pregnancy, PAG concentrations were detected in 90.1% and 80.8% of Sarda pregnant ewes with RIA-706 and RIA-srPool, respectively; and in 90% and in 75% of Lacaune pregnant ewes with RIA-706 and RIA-srPool, respectively. From Day 30, PAG concentrations were detected in all pregnant ewes by using both RIA methods. In the postpartum period, the PAG concentrations in Sarda ewes decreased rapidly reaching minimal levels (<1 ng/mL) on day 28 using both RIA-706 and RIA-srPool. In Lacaune ewes, PAG-706 levels were higher than PAG-srPool from parturition until the last day of observation (Day 28 postpartum). It was also observed that mean concentrations were higher in multiple than in single pregnancies in Sarda and Lacaune ewes. Moreover, due to the rapid disappearance of PAG concentration following lambing, a cut-off limit in postpartum animals was not required as a means for detecting a new pregnancy.

Pregnancy-associated glycoprotein (PAG) concentration in plasma and milk samples for early pregnancy diagnosis in Lacaune dairy sheep

In the present study, four RIA systems (RIA-1 to -4) based on two antisera raised against ovine pregnancy-associated glycoproteins (ovPAGs), combined with an ovine or a bovine PAG tracer were used to measure PAG concentrations in plasma and milk samples of dairy ewes. Blood and milk samples were collected on different days of gestation: 0, 18, 20, 22, 25, 28, 32, 42, and 49. From day 20 onward, the PAG in plasma could be detected in all pregnant ewes using the four RIA systems. By using milk, except for RIA-1, the other systems showed a sensitivity of 100% from day 28 of gestation onward. In plasma, PAG concentrations were higher in multiple than in single pregnancies, while no clear relationship was observed in milk. In conclusion, milk is a good alternative to plasma for early pregnancy diagnosis in sheep from day 28 to day 42.

Biomarkers coding for ovPAG-1 mRNA expression and pregnancy status in Dohne Merino ewes at an abattoir

The objective of the present study was to determine the pregnancy status of Dohne Merino ewes at slaughter. This was done by collecting blood samples from the ewes (n = 60) during exsanguination to assay for pregnancy biomarkers using radio-immunological and TaqMan reverse transcription polymerase chain reaction techniques. The expressions of ovine pregnancy-associated glycoprotein within a range of 1.068(-09) to 8.977(-07) indicated 43.33 % ewes with pregnancy (ΔmRNA) signals at the point of slaughter. The assay validation showed that half of these ewes were truly pregnant, and most of the ewes that exhibited 'true positives' were within 56-60 kg live weight and were about 30 months old. The biomarkers used in this study, therefore, showed that approximately 22 % Dohne Merino ewes were truly pregnant at the point of slaughter.

Extraction and isolation of Ovine Pregnancy-Associated Glycoprotein (ovPAG) from cotyledon placenta of Garut sheep

Pregnancy-associated glycoprotein (PAG) structurally related to aspartic protease, expressed in the outer epithelial cell layer (trophectoderm) of ungulate placenta. Ovine PAG (ovPAG) synthesized by mono- and binucleic trophoblast before complete implantation at Day 14-15. Of this, ovPAG could be used as a marker for early pregnancy. The objective of study was to extract and isolate PAG from placenta of Garut Sheep collected at term and to characterize their molecular weight. The procedures included extraction of protein at neutral pH (cotyledon was thawed, minced, added PBS, blended and centrifuged), acidic (H 3 PO 4 1M, pH 4,5; centrifuged) and ammonium sulfate (40% and 80% (NH 4 )SO 4 , centrifuged) precipitation; gel filtration (Sephadex-G75), anion exchange chromatography (DEAE- cellulose). Cotyledon extract was subjected to Sephadex-G75 and DEAE cellulose, and their fractions were measured their absorbances. Absorbances of Sephadex-G75 and DEAE fractions at peak were assayed for protein concentration (Bichinconinic protein assay). Continuously, these fractions were subjected to monogel SDS-PAGE and stained by Commassie Brilliant Blue. It was four different molecular weights isolating from cotyledon of Garut Sheep , namely 68.8, 36.04, 32.39 and 12.18 kDa. However, after chromatography anion exchange (DEAE-cellulose), only three bands exist, those were 71,67; 33,64 and 30,86 kDa. Key words : Garut sheep, Pregnancy-Associated Glycoprotein (PAG), Cotyledone, SDS-PAGE, DEAE-cellulose

Measurement of Ovine Pregnancy‐Associated Glycoprotein (PAG) During Early Pregnancy in Lacaune Sheep

This study describes ovine pregnancy-associated glycoprotein (ovPAG) concentrations in 20 Lacaune sheep during early pregnancy. Measurements were performed by using semi-purified ovPAG as standard, tracer and immunogens for antibody production in rabbits. Antisera R780 (against ovPAG(57+59kDa)) and R805 (against ovPAG5(58+61kDa)) were used respectively in RIA-780 and RIA-805. Blood samples were collected at days 0, 18, 20, 22 and 25 after artificial insemination. From day 18 after breeding onward, the mean ovPAG concentration was significantly higher (p < 0.001) in plasma samples from pregnant ewes (n = 17) than in non-pregnant ones (n = 3). The specific activity of the tracer was 11 760 Ci/mmol in RIA-780 and 14 900 Ci/mmol in RIA-805. The minimal detection limits for RIA-780 and RIA-805 were 0.2 ng/ml and 0.3 ng/ml, respectively. The intra-assay CV of samples with low (1.0 ng/ml), medium (2.5 ng/ml) and high (4.0 ng/ml) PAG concentrations were 3%, 6% and 9% for RIA-780 and 8%, 9% and 5% for RIA-805. The inter-assay CV in the same samples were 13%, 12% and 7% for RIA-780 and 13%, 11% and 5% for RIA-805. The recovery was higher than 95% in both assays. No cross-reaction was observed with members of aspartic proteinase family as well as with other tested proteins. In both RIA-780 and RIA-805, inhibition of the binding of the tracer by antisera was parallel between standard curve and serial dilutions of pregnant ewe samples. In conclusion, the two homologous RIA systems are suitable for early quantification of ovPAG concentrations in ewe plasma samples from day 18 after breeding.

Comparison of accuracy of transabdominal ultrasonography, progesterone and pregnancy-associated glycoproteins tests for discrimination between single and multiple pregnancy in sheep

The aim of the present study was to evaluate and, compare the accuracy of transabdominal ultrasonographic (US) and the progesterone (P4-RIA) and ovine pregnancy-associated glycoprotein (ovPAG-RIA) tests for the discrimination between single and multiple pregnancy in sheep. One hundred pregnant Awassi × Merino ewes were scanned by transabdominal ultrasonography (3.5 MHz linear-array transducer) at Days 43–56 and 81 of these ewes were scanned at Days 76–87 of gestation. The ewes were scanned in dorsal recumbency at the bare area of the inguinal regions (without pre-scanning shaving of the ventral abdominal wall). After each scan, blood samples were withdrawn from the jugular vein to estimate the levels of P4 and ovPAG by radioimmunoassay. At lambing, 61 ewes gave birth to single lambs and 39 ewes gave birth to multiples. The sensitivity of the transabdominal US, the P4-RIA and the ovPAG-RIA tests for determining ewes carrying multiples was 54, 64.1 and 64.1% at Days 43–56. At Days 76–87 of gestation these accuracies were 60.0, 66.7 and 76.6% for the US, P4-RIA and PAG-RIA tests, respectively. The specificity of the transabdominal US, the P4-RIA and the ovPAG-RIA tests for determining ewes carrying singles, was 78.6, 60.7 and 62.3% at Days 43–56 and 78.4, 64.7 and 70.6% at Days 76–87 of gestation, respectively. It is concluded that the accuracy of transabdominal ultrasonographic (without pre-scanning shaving of the ventral abdominal wall), the P4- and the ovPAG-RIA tests for determination of the fetal numbers in Awassi × Merino crossbred ewes is too low to be used in the field.

A new test for early pregnancy diagnosis in sheep: determination of ovine pregnancy associated glycoprotein (OVPAG) concentration by means of a homologous radioimmunoassy

A new test for early pregnancy diagnosis in sheep: determination of ovine pregnancy associated glycoprotein (OVPAG) concentration by means of a homologous radioimmunoassy

142DETECTION OF LATE EMBRYONIC MORTALITY BY MEANS OF OVINE PREGNANCY ASSOCIATED GLYCOPROTEINS

In sheep, conception rates of 80% are obtained after first mating. Some of the ewes that were not pregnant may have conceived, but underwent embryonic mortality. Embryonic mortality can be distinguished as early (EEM) or late (LEM) embryonic mortality. When EEM occurs, sheep return to estrus 17±2 days after mating, which makes the detection of EEM very difficult. Because determination of ovine pregnancy-associated glycoprotein (ovPAG) concentration can be used for pregnancy diagnosis from Day 25 of conception, with a specificity and sensitivity of 100% and 99%, respectively, it possibly could be used for detection of LEM (Verberckmoes et al., 2003. Pregnancy diagnosis in sheep by means of ultrasound and ovine pregnancy associated glycoproteins (ovPAG), 10th International Symposium on Veterinary Epidemiology and Economics (ISVEE 10), 17–21 November, 2003.). Therefore, in this study the prevalence of LEM and the usefulness of ovPAG concentration for the detection of LEM were examined. In a field trial, serum samples of 160 Texel and 127 Suffolk ewes were collected at the moment of estrus synchronization (T0), and at 25 (T25), 35 (T35), and 45 days (T45) after mating. Concentrations of ovPAG were determined by means of homologous RIA using ovine PAG as standard and tracer, and rabbit antibodies against ovPAG as antiserum. Ewes were considered as being pregnant when ovPAG concentrations were higher than 2ngmL−1. The F-test in a mixed model with ewe as random effect was used to compare the mean PAG concentrations. Of the 287 ewes examined, 5 Suffolk and 2 Texel ewes showed an abnormal PAG profile which was indicative of LEM. The average ovPAG concentration of the 7 former ewes at T25 was 5.6±2.0ngmL−1, which indicates that they were pregnant. However, in contrast to the normal pregnant ewes, they had significantly lower ovPAG concentrations at T35 (2.8±1.5ngmL−1 v. 9.5±2.3ngmL−1) and T45 (0.9±0.3ngmL−1 v. 9.0±1.9ngmL−1) (P&amp;lt;0.01), and they did not lamb at 145±5 days after mating. In conclusion, late embryonic mortality occurred in 2% of the ewes. Determination of the ovPAG concentration by means of homologous RIA is a useful tool for the detection of LEM in sheep.

141EFFECT OF NUMBER OF LAMBS, THEIR SEX AND BIRTH WEIGHT ON OVINE PREGNANCY-ASSOCIATED GLYCOPROTEIN (ovPAG) CONCENTRATIONS

Early pregnancy detection and prediction of the number of lambs would be profitable for sheep breeders, as this enables nourishment of pregnant ewes to their individual needs. Nonpregnant ewes can be separated and sold before winter. Prediction of sex is interesting for the curiosity of impatient breeders. Early pregnancy detection (before 45 days after mating) by means of ovine pregnancy-associated glycoproteins (ovPAGs) has proved to be a more reliable test than the commonly used transrectal ultrasound. The concentration of ovPAG has been claimed to be related to the number of lambs, their sex and birth weight, but contradictory results were obtained in different small-scale studies. In this study the correlation between number of lambs, their sex and birth weight and the ovPAG concentration in the serum of the ewes was examined. In a field trial, 160 and 70 ewes of the Texel and Suffolk breed, respectively, housed on 6 different farms, were synchronized by means of intravaginal sponges impregnated with 40mg cronolone (Chronogest®, Intervet, Boxmeer, The Netherlands). At the moment of synchronisation (T0) and at 25 (T25), 35 (T35) and 45 (T45) days after mating or artificial insemination, blood samples were taken by jugular vein puncture. A heterologous radioimmunoassay (RIA) was used to determine the ovPAG concentration. In the heterologous RIA boPAG molecules were used as tracer, and ovPAG molecules as standard, while rabbit antibodies against ovPAG were used as antiserum. At parturition, number, sex and birth weight of the lambs were registered and their effect on ovPAG concentrations was statistically evaluated. No significant difference in ovPAG concentration was found between ewes pregnant of a single male or female lamb (P=0.29). ovPAG concentrations were affected by the total birth weight (P&amp;lt;0.05). In ewes pregnant with multiples, the ovPAG concentrations were higher (P&amp;lt;0.01) than in ewes pregnant with singles at T25, T35 and T45, respectively. Because ovPAG concentrations were affected by the farm, it was impossible to predict the number of lambs by the ovPAG level. In conclusion, ovPAG concentrations were not affected by the sex of the lamb. However, ovPAG concentrations were related to the total birth weight. Within the farm, increased ovPAG concentrations may indicate pregnancy of multiples.

Isolation and partial characterization of three pregnancy-associated glycoproteins from the ewe placenta.

Pregnancy-associated glycoproteins (PAGs) are synthesized in the outer epithelial layer of the placenta in artiodactyls. In this work, three novel ovine PAGs were isolated from late-pregnancy fetal cotyledons and characterized biochemically. The isolation procedure included acid and ammonium sulfate precipitations and anion and cation exchange chromatographies. The isolated PAGs have different NH(2)-terminal amino acid sequences (RGSXLTILPLRNMRDIVY, ISRVSXLTIHPLRNIMDML, and RGSNLTIHPLRNIRD) and apparent molecular masses (55, 57, and 59 kDa). Each shows several isoforms with different pI values. The three proteins share high sequence identity with each other and with other ovine, bovine, and caprine PAGs. They have not been described previously. The ovPAG-59 sequence differs from the previously identified ovPAG-4 sequence (determined by DNA cloning and sequencing) at only one position among the 15 N-terminal residues. The newly characterized ovPAGs and the procedure used to isolate them will be helpful in producing new antisera for investigating PAG secretion in pregnant ewes.

Application of Ultrasonographic Examination of the Ovine Fetus in Normal Sheep and those Presenting with Obstetrical Problems

In the present study, four RIA systems (RIA-1 to -4) based on two antisera raised against ovine pregnancy-associated glycoproteins (ovPAGs), combined with an ovine or a bovine PAG tracer were used to measure PAG concentrations in plasma and milk samples of dairy ewes. Blood and milk samples were collected on different days of gestation: 0, 18, 20, 22, 25, 28, 32, 42, and 49. From day 20 onward, the PAG in plasma could be detected in all pregnant ewes using the four RIA systems. By using milk, except for RIA-1, the other systems showed a sensitivity of 100% from day 28 of gestation onward. In plasma, PAG concentrations were higher in multiple than in single pregnancies, while no clear relationship was observed in milk. In conclusion, milk is a good alternative to plasma for early pregnancy diagnosis in sheep from day 28 to day 42.

Plasma pregnancy-associated glycoprotein and progesterone concentrations in pregnant Assaf ewes carrying single and twin lambs

Ovine pregnancy-associated glycoprotein (oPAG) and progesterone (P4) concentrations were monitored weekly during the last three months of gestation and the first month postpartum in plasma of twelve Assaf ewes. The oPAG levels were determined with a heterologous RIA using bovine PAG as standard and tracer and rabbit antiserum against oPAG. The P4 levels were measured with a radioimmunological procedure with a sensitivity to less than 0.1 ng ml −1. The oPAG profiles in ewes carrying a single lamb and in those carrying twins were very similar from week 9 of gestation to lambing. Ewes which had twins presented numerically higher oPAG concentrations from week 12 to lambing, but that difference was only significant ( P < 0.05) at week 21. Ewes carrying twins presented higher ( P < 0.05) P4 concentrations from weeks 12 to 20 of gestation than those carrying a single lamb, but afterwards there was no difference. No correlation was found between P4 and oPAG concentrations at each stage of gestation studied in any group. After lambing, P4 levels dropped rapidly in one week, whereas oPAG decreased in four weeks to basal values in both groups of ewes. No relationship ( P > 0.05) was found between oPAG concentrations and lamb birth-weight at any week of gestation considered in this study. However, the lamb birth-weight was positively correlated ( P < 0.10) with the P4 concentrations from weeks 9 to 19 of gestation. Results indicate that number of fetuses could influence the oPAG production in ewes during the last three months of gestation.

Plasmatic profiles of pregnancy-associated glycoprotein and progesterone levels during gestation in Churra and Merino sheep

This study was carried out to determine ovine pregnancy-associated glycoprotein (oPAG) and progesterone (P4) levels in the serum of Churra and Merino ewes throughout gestation and the first month post partum. The oPAG levels were determined with an heterologus RIA using bovine PAG as standard and tracer and rabbit antiserum against oPAG, sensitivity was 4.0 ng/ml. The P4 levels were measured with a radioimmunological procedure, including a specific extraction step with petroleum ether (bp 60-80 degrees C) with a sensitivity of less than 0.1 ng/ml. There were no differences (P<0.10) in the oPAG profile between breeds from Weeks 1 to 18. From Week 18 to lambing, oPAG concentrations increased rapidly in Churra ewes (on average, from 250 to 650 ng/ml) while remaining relatively constant in the Merino ewes (around 250 ng/ml). No significant differences (P>0.05) were observed for mean weekly P4 levels between the 2 breeds. In both breeds, P4 increased throughout the whole length of gestation, with the highest level measured at Weeks 19-20, then declined 2 wk before parturition. No correlation was found between P4 and oPAG concentrations during gestation in either of the breeds. After lambing, oPAG and P4 levels decreased rapidly in 4 wk to basal values. In both breeds the oPAG concentrations at Weeks 19, 20 and 21 of gestation in ewes carrying male fetuses were higher than in those carrying female fetuses. From the results, we conclude that the breed and sex of the fetus could influence the production of oPAG.