Recombinant Sheep Pregnancy-associated glycoprotein 60f

What is Pregnancy-Associated Glycoprotein (PAG) and what is its significance in ovine reproduction?

Pregnancy-Associated Glycoproteins (PAGs) are a family of proteins expressed by trophoblast cells in the placenta of ruminants. In sheep, these glycoproteins are secreted into maternal circulation during pregnancy and can be reliably detected in blood serum. PAGs belong to the group of aspartic proteases and have become important biomarkers for pregnancy diagnosis in sheep and other ruminants. Their measurement allows for non-invasive monitoring of pregnancy status, which is crucial for effective reproductive management in sheep flocks .

At what stage of pregnancy can PAG be detected in sheep?

According to recent studies, PAG can be detected as early as day 18 after mating in pregnant ewes. Using the sheep ELISA kit, PAG values become detectable in all pregnant ewes from day 18 onward . When using radioimmunoassay (RIA) methods, detection rates vary slightly. With RIA-706, PAG concentrations were detected in 90.1% of Sarda pregnant ewes and 90% of Lacaune pregnant ewes at day 18. Using RIA-srPool, detection rates were 80.8% for Sarda and 75% for Lacaune ewes at the same timepoint. By day 30, PAG concentrations were detectable in all pregnant ewes using both RIA methods .

What is the discrimination threshold for PAG-based pregnancy detection in sheep?

Research indicates that the discrimination value (cutoff point) for detection of pregnancy by PAG test is ˃0.915 ng/ml when using the ROC Curve analysis. This threshold allows for reliable differentiation between pregnant and non-pregnant ewes from day 18 after mating . The specific threshold values may vary depending on the detection methodology used and should be validated for each specific assay system implemented in research.

What are the comparative advantages of different PAG detection methods in ovine samples?

Multiple methods have been developed for PAG detection in sheep, with the most common being ELISA and radioimmunoassay (RIA).

The sheep ELISA kit offers advantages in terms of accessibility and ease of use in field conditions, making it suitable for practical applications in reproductive management. For research purposes, RIA methods may provide higher sensitivity for early detection but require specialized laboratory equipment and radioactive materials .

How do PAG concentrations change throughout pregnancy and postpartum in sheep?

PAG concentrations follow a distinct profile throughout pregnancy and the postpartum period:

During pregnancy:

• Detectable levels appear by day 18 after mating

• Concentrations progressively increase throughout gestation

• Different RIA methods (RIA-706 and RIA-srPool) show similar profiles but with different absolute values

During the postpartum period:

• PAG concentrations decrease rapidly after lambing

• In Sarda ewes, PAG levels reach minimal values (<1 ng/mL) by day 28 postpartum using both RIA-706 and RIA-srPool

• In Lacaune ewes, PAG-706 levels remain higher than PAG-srPool from parturition until day 28 postpartum

• The estimated half-lives of PAG in the postpartum period are 5.8 days and 5.3 days for PAG-706 and PAG-srPool in Sarda ewes, and 6.9 days and 6.3 days respectively in Lacaune ewes

This rapid disappearance of PAG following lambing means that a cut-off limit in postpartum animals is not required for detecting a new pregnancy .

How does fetal number affect PAG concentrations in maternal circulation?

Multiple studies have confirmed that PAG concentrations are significantly higher in ewes carrying multiple fetuses compared to those with single pregnancies. Research demonstrated that mean PAG values were significantly (P<0.05) higher in twin pregnancies than in single pregnancies in crossbred Iraqi ewes throughout the gestation period . This finding was consistent across different sheep breeds, with similar patterns observed in Sarda and Lacaune ewes .

The ability to differentiate between single and multiple pregnancies based on PAG concentrations provides valuable information for flock management and targeted nutritional interventions for ewes carrying multiple fetuses.

What factors should be controlled when designing PAG-based studies in sheep?

When designing studies involving PAG measurements in sheep, researchers should consider several factors that may influence results:

• Breed differences: PAG profiles show variations between sheep breeds (e.g., Sarda vs. Lacaune), potentially affecting reference ranges and detection thresholds

• Detection method: Different assays (ELISA vs. various RIA methods) yield different absolute values and may have varying detection sensitivities

• Sampling time: PAG concentrations change significantly throughout pregnancy and postpartum, requiring precise timing protocols

• Fetal number: Ewes with multiple fetuses show higher PAG concentrations than those with single pregnancies

• Fetal sex: Some studies indicate that PAG levels may differ based on fetal sex, with male single pregnancies showing higher levels than female single pregnancies

• Statistical analysis approach: The choice of statistical methods affects the interpretation of results. Mixed models with repeated measures considering breed, RIA method, sampling time, type of delivery, and sex of fetuses as fixed factors are recommended

What is the relationship between fetal sex and PAG concentrations in sheep?

Research has shown that fetal sex can influence PAG concentrations in maternal circulation. Studies in crossbred Iraqi ewes revealed that PAG values were significantly (P<0.05) higher in male single pregnancies compared to female single pregnancies from early gestation through to parturition . This difference suggests that the placental production of PAG may be influenced by fetal gender, possibly due to differences in placental development or function.

How should PAG concentration data be statistically analyzed for pregnancy diagnosis research?

For robust statistical analysis of PAG concentration data, researchers should consider the following approaches:

• Appropriate statistical models: Use the GLM (General Linear Model) procedure with mixed models incorporating repeated measures

• Fixed factors to include: Sheep breed, detection method (e.g., RIA type), sampling time, type of delivery (single or multiple), and fetal sex

• Random factors: Individual ewe should be considered as a random factor

• Interaction analysis: Examine interactions between main factors (e.g., breed × RIA method, sampling time × pregnancy type)

• Data representation: Express PAG and progesterone concentrations as least square means ± standard error of the mean (±SEM)

• ROC curve analysis: For determining optimal discrimination values (cutoff points) for pregnancy detection

• Half-life calculations: When studying postpartum PAG clearance, appropriate decay models should be applied

The data obtained can be analyzed using statistical software such as SAS, with appropriate significance thresholds (typically p<0.05) .

How can PAG data be used to evaluate sheep reproductive efficiency?

PAG measurements can serve as valuable tools for evaluating reproductive efficiency in sheep populations:

• Early pregnancy detection: PAG testing from day 18 enables earlier identification of non-pregnant ewes, allowing for more timely rebreeding decisions

• Fetal number determination: Significant differences in PAG levels between single and multiple pregnancies allow for identification of ewes carrying multiple fetuses

• Potential fetal sex determination: The correlation between PAG levels and fetal sex (in single pregnancies) may provide early information about lamb gender distribution

• Pregnancy loss monitoring: Sequential PAG measurements can detect pregnancy losses when concentrations decrease unexpectedly

• Breed-specific reproductive patterns: Comparing PAG profiles between breeds may reveal differences in placental function and development

Implementing PAG-based monitoring in research settings provides objective, quantitative data for evaluating reproductive interventions and management strategies .

What are the main challenges in PAG assay development for sheep models?

Researchers face several challenges when developing and implementing PAG assays for sheep:

• Assay specificity: The multigene family nature of PAGs means multiple PAG variants exist, requiring careful antibody selection for specific detection

• Cross-reactivity: Ensuring assays specifically detect ovine PAGs without cross-reactivity with other ruminant species

• Sensitivity requirements: Developing assays sensitive enough for early pregnancy detection (day 18) when PAG concentrations are still low

• Standardization: Establishing consistent standards and controls across different laboratories and studies

• Sample handling: Ensuring proper collection, processing, and storage of blood samples to maintain PAG stability

• Validation across breeds: Different sheep breeds may exhibit variations in PAG profiles, requiring validation across diverse genetic backgrounds

These challenges can be addressed through careful assay design, validation across multiple sheep populations, and rigorous quality control procedures.

How can PAG measurement be integrated with other pregnancy biomarkers for comprehensive research?

For comprehensive pregnancy research in sheep, PAG measurement can be integrated with other biomarkers:

• Progesterone analysis: Combining PAG with progesterone measurements provides complementary information, as progesterone reflects corpus luteum function while PAG indicates placental development

• Ultrasound examination: Correlating PAG levels with ultrasound findings enhances accuracy of pregnancy diagnosis and fetal number determination

• Estrus synchronization protocols: Studying PAG profiles in conjunction with different synchronization methods (e.g., CIDR, vaginal sponge) provides insights into early pregnancy establishment

• Placental development markers: Combining PAG with other placental markers (e.g., placental lactogen) creates a more comprehensive picture of placental function

• Metabolic parameters: Integrating PAG data with metabolic profiles helps understand the relationship between maternal metabolism and placental development

This multi-biomarker approach enables more robust research findings and deeper understanding of sheep reproductive physiology .