Purification of pregnancy-associated glycoproteins from late-pregnancy Bubalus bubalis placentas and development of a radioimmunoassay for pregnancy diagnosis in water buffalo females

Abstract

BackgroundPregnancy-associated glycoproteins (PAGs) were first described as placental antigens present in the blood serum of the mother soon after implantation. Here, we describe the purification of several pregnancy-associated glycoproteins from water buffalo placenta (wbPAGs). A specific radioimmunoassay (RIA) was developed for early pregnancy diagnosis in buffalo species.ResultsAmino-terminal microsequencing of immunoreactive placental proteins allowed the identification of eleven wbPAGs sequences [Swiss-Prot accession numbers: P86369 to P86379]. Three polyclonal antisera (AS#858, AS#859 and AS#860) were raised in rabbits against distinct wbPAG fractions. A new RIA (RIA-860) was developed and used to distinguish between pregnant (n = 33) and non-pregnant (n = 26) water buffalo females.ConclusionsOur results confirmed the multiplicity of PAG expression in buffalo placenta. In addition, the RIA-860 system was shown to be sensitive, linear, reproducible, accurate and specific in measuring PAG concentrations in buffalo plasma samples from Day 37 of gestation onwards.

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

“…This is a similar trend to that described by Gajewski et al [46] for Berrichon ewes, by Ledezma-Torres et al [14] for various sheep breeds and by Ranilla et al [11,24] for Churra sheep, yet this trend was not observed for Merinos ewes whose PAG levels initially increased and then dropped to baseline concentrations around mid-pregnancy. Patterns of plasma ovine PAG concentrations differed from those reported for bovine [30], goats [47,48] and buffalo [20,49]. These differences could be explained by the ability of the antisera to distinguish between various epitopes [20].…”

“…These molecules are present in maternal plasma of sheep in concentrations detectable with RIA methods as early as 18-20 days after conception, [3,[11][12][13][14][15][16] and by the enzyme immune assay (EIA) method [17][18][19], as well as in other species of ruminants [1,[20][21][22]. Throughout pregnancy, PAG concentrations in these species differ according to the breed, fetal number, sex and birth weight [3,[23][24][25][26][27][28][29], as in other ruminants [30].…”

Correlation of two radioimmunoassay systems for measuring plasma pregnancy‐associated glycoproteins concentrations during early pregnancy and postpartum periods in water buffalo

“…Using various chromatographic procedures, some members of the PAG family have been isolated from the cotyledons of several species of the order Cetardiodactyla (Beriot, Tchimbou, Barbato, Beckers, & Sousa, 2014;Brandt, Parks, Killian, Ealy, & Green, 2007;El Amiri et al, 2004;Garbayo et al, 1998;Green et al, 2000;Majewska, Panasiewicz, Majewski, & Szafranska, 2006;Sousa et al, 2003;Szafranska et. al., 1995;Xie et al, 1997;Zoli et al, 1991) including water buffalo (wbPAG; Barbato, Melo de Sousa, Barile, Canali, & Beckers, 2013;Barbato et al, 2008).…”

“…Our research group (Barbato et al, 2016(Barbato et al, , 2013 reported using antisera raised against buffalo PAG molecules, purified from buffalo placenta, to develop a specific RIA system (RIA-860) for pregnancy diagnosis in buffalo. Using this system, the plasma PAG profiles of water buffalo during pregnancy and postpartum periods have been recently described (Barbato et al, 2017).…”

Investigation into omocysteine, vitamin E and malondialdehyde as indicators of successful artificial insemination in synchronized buffalo cows (Bubalus bubalis)

“…Transrectal ultrasonography (Aloka -SSD500 scanner equipped with 7.5 MHz linear-array transducer) confirmed pregnancy status and embryo viability (heartbeat visualization) on 28 d, 40 d and 56 d. All ultrasound scans were performed by the same operator. PAG RIA plasma concentrations were determined in duplicate, as previously described by Barbato et al (2013). The specific antibody was an anti buffalo PAG73kDa serum (AS#860) raised in rabbit.…”