title: Using real-time PCR to identify pregnancy-associated glycoprotein 2 (PAG-2) in water buffalo (Bubalus bubalis) blood in early pregnancy.
abstract: This study investigates for the first time mRNA pregnancy-associated glycoprotein 2 (PAG-2) expression in blood cells during early pregnancy in water 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 analyzed-17 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 = 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(+)D40(+) group; the females diagnosed pregnant at D28 but not confirmed pregnant at D40 were defined as D28(+)D40(-) group; and the females that were diagnosed as nonpregnant on either days were defined as D28(-)D40(-) group. PAG-2 mRNA at Day 0 was not observed in any groups. The D28(+)D40(+) group showed the highest expression, starting on Day 18 and increasing progressively up to Day 75. PAG-2 mRNA was also expressed on Day 18 in both D28(+)D40(-) and D28(-)D40(-) groups, but their levels were lower than those of D28(+)D40(+) 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(+)D40(+) 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 mRNA was still present in blood circulation although at lower levels, as found in the D28(+)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.
authors.affiliation: Department of Veterinary Medicine, University of Perugia, Perugia, Italy. Electronic address: olimpia.barbato@unipg.it.
authors.forename: O
authors.initials: O
authors.lastname: Barbato
authors.affiliation: Department of Veterinary Medicine, University of Perugia, Perugia, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Guelfi
authors.affiliation: Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria (CREA) - Animal Production Research Centre, Monterotondo, Rome, Italy.
authors.forename: V L
authors.initials: VL
authors.lastname: Barile
authors.affiliation: Department of Veterinary Medicine, University of Perugia, Perugia, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Menchetti
authors.affiliation: Department of Veterinary Medicine, University of Perugia, Perugia, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Tortiello
authors.affiliation: Department of Veterinary Medicine, University of Perugia, Perugia, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Canali
authors.affiliation: Department of Veterinary Medicine, University of Perugia, Perugia, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Brecchia
authors.affiliation: Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Traina
authors.affiliation: Laboratory of Animal Endocrinology and Reproduction, Faculty of Veterinary Medicine, University of Liege, Liege, Belgium.
authors.forename: J F
authors.initials: JF
authors.lastname: Beckers
authors.affiliation: Laboratory of Animal Endocrinology and Reproduction, Faculty of Veterinary Medicine, University of Liege, Liege, Belgium.
authors.forename: N
authors.initials: N
authors.lastname: Melo de Sousa
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000097:blood* / Q000235:genetics; D002020:Buffaloes / Q000097:blood / Q000502:physiology*; D004973:Estrus Synchronization; D005260:Female; D007315:Insemination, Artificial; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011258:Pregnancy Tests / Q000662:veterinary; D011270:Pregnancy, Animal / Q000097:blood*; D012333:RNA, Messenger / Q000032:analysis*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*
chemical_list: D012333:RNA, Messenger; D016282:Aspartic Acid Endopeptidases; C095584:pregnancy-associated glycoprotein 2
keywords: Polymerase chain reaction; Pregnancy; Pregnancy-associated glycoprotein 2; Water buffalo