Short communication: A note on the correction for the effect of freezing on the outcome of pregnancy-associated glycoprotein measurement in blood and serum of cows

Early pregnancy detection is a measure of considerable economic relevance for dairy cattle breeders, and analysis of pregnancy-associated glycoprotein (PAG) values in blood is one of the methods implemented in practice. Starting from d 30 postconception, cows are considered to be pregnant at PAG levels of 2.0 ng of PAG/mL of blood and higher. However, little is known about preanalytic sources of errors that might affect PAG values. Based on blood samples from 65 dairy cows, the present study showed that freezing of samples, such as may be the case during shipping in wintertime, will lower PAG values considerably. Therefore, a Bland-Altman analysis was used to derive a correction factor. Overall, the mean differences (+/- standard deviation) between frozen and respective fresh samples was -5.5 +/- 7.4 ng of PAG/mL of blood and 0.9 +/- 6.1 ng of PAG/mL of serum. However, the Bland-Altman plot revealed a concentration-dependent effect of freezing on PAG values with higher variability and larger declines at higher FAG levels. Therefore, to minimize chances of false-negative results, different correction factors are suggested for different levels of FAG (e.g., based on the upper bound of the 95% confidence interval 0.67 for PAG levels between 2.0 and 3.9 ng of PAG/mL and 0.25 for PAG levels between 4.0 and 7.9 ng of PAG/mL). With these concentration-dependent correction factors, implementation into practice will be possible. The accuracy is adequate because no quantitative information but qualitative results (pregnant vs. nonpregnant) are required. However, due to larger chances of false-negative results, the application of the correction factor should only be a last resort if temperature exposure of a sample is unknown.