Using real-time PCR to identify pregnancy-associated glycoprotein 2 (PAG-2) in water buffalo (Bubalus bubalis) blood in early pregnancy

This study investigates for the first time mRNA pregnancy-associated glycoprotein 2 (PAG-2) expression in blood cells during early pregnancy inwater buffalo. The PAGs constitute a large family of glycoproteins expressed in the outer epithelial layer of the placenta in eutherian species. All PAGs are not concomitantly expressed throughout pregnancy; some of them are expressed in the earlier phases, whereas others appear later and are expressed over a shorter period. Twenty-one lactating buffaloes were analyzedd17 females were synchronized with PRID and artificially inseminated (AI), whereas four females were synchronized but not inseminated (control group). Blood was collected at Days 0, 18, 28, 40, and 75 from AI (AI ¼ Day 0). Expression of PAG-2 mRNA in blood samples was measured with real-time polymerase chain reaction. Pregnancy diagnosis was performed on Day 28 (D28) and Day 40 (D40) after AI by ultrasonography (US) and by PAG-1 RIA method. The females diagnosed pregnant at D28 and confirmed pregnant at D40 were defined as D28(þ)D40(þ) group; the females diagnosed pregnant at D28 but not confirmed pregnant at D40 were defined as D28(þ)D40() group; and the females that were diagnosed as nonpregnant on either days were defined as D28()D40() group. PAG-2mRNA at Day 0was not observed in any groups. The D28(þ)D40(þ) group showed the highest expression, starting on Day 18 and increasing progressively up toDay 75. PAG-2mRNAwas also expressed on Day 18 in both D28(þ)D40() and D28()D40() groups, but their levels were lower than those of D28(þ)D40(þ) group and almost constant over time. PAG-2 mRNA was never detected in the control group. The significant difference in the expression of PAG-2 mRNA between the D28(þ)D40(þ) group and the D28()D40() group, starting from Day 18, suggests that these animals might have conceived, but have experienced early embryonic loss; therefore, the PAG-2 mRNAwas still present in blood circulation although at lower levels, as found in the D28(þ)D40() group. In conclusion, this study shows that PAG-2 mRNA can be detected in peripheral maternal blood cells earlier than circulating PAG-1 molecules and could be useful for studies on early pregnancy and embryonic mortality.