75
_________________________ Revista Cientíca, FCV-LUZ / Vol. XXXIII, Supl. Esp., 75 - 81, 2023, https://doi.org/10.52973/rcfcv-wbc010
PREGNANCY-ASSOCIATED GLYCOPROTEINS IN BUFFALO:
ORIGINS, FUNCTIONS AND CLINICAL APPLICATION FOR
PREGNANCY FOLLOW-UP
Glicoproteínas asociadas a la preñez en búfala: orígenes, funciones y aplicación clínica
para el seguimiento de la preñez
Olimpia Barbato1, Vittoria Lucia Barile2
1Department of Veterinary Medicine, University of Perugia, Perugia, Italy
2Research Centre for Animal Production and Aquaculture, Consiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria
(CREA), Monterotondo (Rome) Italy
*Corresponding e-mail: Barbato, Olimpia (olimpia.barbato@unipg.it).
ABSTRACT
Reproductive eciency represents an essential aspect of farm
management as the productivity and protability of a farm de-
pend on it, particularly in dairy farming. Early detection of preg-
nancy and identication of non-pregnant bualo cows is funda-
mental in the management of reproduction because it allows
an early resynchronization and rebreeding of the non-pregnant
animals, shortening the calving-conception interval. Dierent
pregnancy diagnostic tools are currently available; among
these, the utilization of pregnancy-associated glycoproteins
(PAGs) contributed signicantly to the improvement of repro-
ductive management. PAGs are secreted by the placenta of
several ruminant species, including bualo, and can be detect-
ed in the maternal blood. In addition to their utilization as diag-
nostic tools to identify pregnant or non-pregnant animals, PAGs
help identify the presence of vital embryos and predict future
embryo mortality during early gestation. This paper presents
an overview of the PAGs origins, function, pregnancy trend,
and their use to improve reproductive management in bualo
farming.
Keywords: pregnancy-associated glycoproteins, bualo, preg-
nancy, reproductive management.
RESUMEN
La eciencia reproductiva representa un aspecto esencial de
la gestión ganadera ya que de ella dependen la productivi-
dad y la rentabilidad de una explotación, especialmente en la
producción lechera. La detección temprana de la gestación y
la identicación de búfalas no gestantes es fundamental en
el manejo de la reproducción porque permite una resincroni-
zación temprana y una nueva reproducción de los animales
no gestantes, acortando el intervalo parto-concepción. Ac-
tualmente se encuentran disponibles diferentes herramientas
de diagnóstico de la preñez; entre ellos, la utilización de gli-
coproteínas asociadas a la preñez (PAGs) la cual contribuye
signicativamente a la mejora del manejo reproductivo. Las
PAGs son secretados por la placenta de varias especies de
rumiantes, incluida la búfala, y pueden detectarse en la san-
gre materna. Además de su utilización como herramientas de
diagnóstico para identicar animales preñados o no preñados,
las PAGs ayudan a identicar la presencia de embriones via-
bles y predecir la mortalidad futura de los embriones durante
la gestación temprana. Este artículo presenta una descripción
general de los orígenes, la función, la tendencia de preñez y
su uso de los PAG para mejorar el manejo reproductivo en la
cría de búfalos.
Palabras clave: glicoproteínas asociadas al embarazo, búfa-
lo, el embarazo, manejo reproductivo.
INTRODUCTION
In dairy production, reproductive performances and prof-
itability are strongly associated. Declining fertility is a worldwide
recognized problem that represents a major source of econom-
ic loss and culling of large ruminants [1]. Many factors con-
tribute to the decline in reproductive eciency; among these,
embryonic mortality (EM) represents the major cause of repro-
ductive failure. The EM following day 28 of pregnancy has a
greater economic impact on dairy herd management because
the delay in resynchronization and re-breeding of animals pro-
76
13th World Bualo Congress ~ 13er Congreso Mundial de Búfalos / Lectures / Reproduction ______________________________________
longs their intercalving. Identication of early embryonic mor-
tality improves reproductive eciency and pregnancy rate by
decreasing the interval between AI services, thereby increasing
the AI service rate [2]. So, identifying EM can help in resched-
uling new inseminations of non-pregnant animals, reducing the
environmental impact of the cow’s unproductive period.
In the last decades, new technological approach in di-
agnostic methods has contributed to the development of the
earlier diagnosis of pregnancy in cattle, including bualo [3,4].
Pregnancy can be monitored using various methods, in-
cluding direct methods such as rectal palpation or ultrasonog-
raphy or indirect methods using biochemical markers that can
be detected in the maternal blood of pregnant animals. Among
these, pregnancy-associated glycoproteins (PAGs) have be-
come a valuable tool for early pregnancy diagnosis and pre-
dicting embryonic mortality [4]).
PAGS ORIGINS AND FUCTIONS
PAGs are a family of glycoproteins belonging to the sub-
class of aspartic proteases [5,6], proteolytic enzymes with an
acid pH. They are characterized by the presence of aspartic
acid residues surrounding the recognition sites and are similar
to pepsin, renin, cathepsin D and E, and chymosin [7]. These
proteins have a sequence that is 50% like pepsin [8], but a
substitution of amino acids in the active site renders them en-
zymatically inactive [9]. Other authors successively showed
that PAGs conserved features are typically found in functionally
aspartic proteins; therefore, some possess proteolytic activity
[10].
PAGs are expressed in the outer epithelial layer (cho-
rion/trophectoderm) of the placenta in the eutherian species
of Cetartiodactyla order (even-toed ungulate) [11]. They are
synthesized by the mononucleate and binucleate trophoblastic
cells, some of which are secreted into the maternal blood when
the conceptus becomes more closely attached to the uterine
wall and placentome formation begins [12,13]. From a phyloge-
netic point of view, PAGs can be grouped as “ancient” (originat-
ed about 87 million years ago), expressed in both mono- and
binucleate trophoblastic cells (PAG-2 group), and “modern”
(originated about 52 million years ago), expressed only in bi-
nucleate cells (PAG-1 group) [14]. Bovine PAG-2 coexists with
Bovine PAG-1 in the trophectoderm [15]; while PAG-1 is pro-
duced in binucleate cells of both inter cotyledonary and cotyle-
donary chorion, PAG-2 molecules are produced in both mono-
nucleate and binucleate trophoblastic cells [13]. In ruminants,
the PAGs gene family is particularly large and complex. It was
estimated that cattle, sheep, and other pecoran mammals pos-
sess 100 or more PAG genes [16]. To date, 74 dierent com-
plementary DNA (cDNA) of PAG genes (diering by at least 5%
in nucleotide sequence) have been identied in species with a
synepitheliochorial placenta. In bovine species, 22 PAG genes
(boPAG-1 to boPAG-22) have been cloned and fully sequenced
[11,17], and many also in water bualo species (wtPAG-1 [18];
wtPAG-2 to wtPAG-19 [15].
Not all PAGs are present in the same stage of gestation,
as some appear earlier and others later. Green et al. [11] have
highlighted the existence of their spatially and temporally dis-
tinct expression patterns during pregnancy. Some PAGs are
entirely expressed in the trophectoderm, while others are local-
ized mainly in binuclear cells: among the latter, some are pres-
ent from mid to late pregnancy, while others already appear at
day 25 but are absent in the advanced stages [11]. The dierent
PAGs temporal expressions opened the way for their practical
use and speculation of their potential physiological role.
Given that the peculiar characteristic of PAGs is the con-
stant increase along the course of gestation, it is reasonable to
think that this is related to their biological function. Since PAGs
and PSPBs were identied, several authors have searched for
a relationship between these molecules or their concentration
prole and a local or systemic immunological function. Telogu
et al. [10,19] having highlighted that some PAGs possess pro-
teolytic activity, it seems possible that proteolytically active
PAGs present at the placenta-uterine interface could process
latent growth factors that are known to be present at that loca-
tion [20]. Another possibility proposed by Wooding et al. [21] is
that PAGs accumulating at the placenta–uterine interface might
play a role in adhesion by acting as bridging molecules. Sev-
eral works correlate the high concentrations of PAGs with the
decrease in the activity of polymorphonuclear neutrophils [22],
suggesting that the trophoblast PAGs production, inuencing
the maternal immunological status, could be a mechanism by
which the conceptus protects itself from rejection. Austin et al.
[23] attribute a hormonal role to PAGs in inducing the release of
granulocyte chemotactic protein-2 (GCP-2), an alpha chemok-
ine whose synthesis is induced by interferon-tau (IFN-tau) in
early pregnancy. In support of a possible luteotropic role of the
PAGs, the studies by Del Vecchio et al. [24] and Weems et
al. [25] showed how these glycoproteins induce the release of
prostaglandin (PG) E2 and progesterone from luteal cells and
PGE2 from endometrial cells grown in vitro. For this reason, it
was considered to represent one of the luteotropic factors of
the placenta of ruminants [9].
Many authors have highlighted the relationship between
PAGs synthesis and fetal well-being [7,26]. The relationship
between PAGs and fetal well-being is clearly understood if we
consider that these glycoproteins are secreted by the mono-
and binucleate cells of the trophectoderm, which migrate from
the fetal to uterine tissue and fuse with maternal uterine epi-
thelial cells to form the hybrid feto-maternal trinucleate cells,
which are responsible for the releasing of glycoproteins in the
maternal organism. This active process presupposes the pres-
ence of healthy trophoblastic tissue and, therefore, of a healthy
embryo. If this condition fails, the source of production of the
proteins themself is missing. Furthermore, thanks to this “active
migration”, PAGs would play an essential role in remodeling
fetal membranes during pregnancy [7].
77
________________________________________________________ Revista Cientíca, FCV-LUZ / Vol. XXXIII, Supl. Esp., 75 - 81, 2023
The use of the PAGs is helpful in advancing a pregnancy
diagnosis and obtaining information on embryonic and/or fetal
well-being. The concentration decline in maternal circulation is
linked to a reduction in the activity of trophoblast cells. There-
fore, PAGs can be considered a marker of placental function
and a direct tool to monitor the vitality of the conceptus.
PAGS IN REPRODUCTIVE MANAGEMENT
In bovine, PAGs have been detected in maternal blood
as early as Days 15 to 22 after fecundation [27]. The detection
results are more accurate from Days 28 to 30 onwards [28].
Their concentrations then triple, more precisely between 20
and 10 days prepartum, up to their maximum increase, which
occurs about 5 - 10 days before calving [12]. Thus, detecting
PAGs in the maternal circulation can be a helpful tool for early
pregnancy diagnosis. After calving, the PAGs concentrations
begin to disappear from maternal blood to return to the baseline
levels but persist in appreciable concentration for 80-100 days
[12,29]. Therefore, to diagnose pregnancy using the PAGs test
in the early stage of the postpartum in cows, the interval be-
tween calving and AI should be at least 60 days [29]. In this
case, post-calving interference with the residual PAGs in the
peripheral circulation during the postpartum period can be min-
imal.
Recently, many authors have shown the relationship be-
tween PAGs level and embryonic losses [30-32], suggesting
that determining PAGs concentration besides pregnancy diag-
nosis can also help to determine embryonic or fetal mortalities.
Unlike the bovine, bualo species are characterized by
reproductive seasonality that aects the eciency of breeding
programs, particularly during the spring and summer seasons
with the daylight lengthening period, which corresponds to the
low breeding period for bualo [33,34]. It was observed a high-
er incidence of embryo loss (20 to 40%) in bualoes that con-
ceive during the daylight lengthening period, whereas a lower
incidence (7%) was observed during decreasing daylight length
[35,36].
PAGs determination in maternal blood represents one
strategy for improving reproductive performance aimed at di-
agnosing pregnancy and shortening the calving-conception
interval by rapidly identifying embryo losses and rebreeding
non-pregnant bualo cows. The rst RIA system adopted for
detecting PAGs molecules in bualo was RIA-706, which uses
antisera raised against caprine. This system was proved to be
accurate for detecting pregnant bualoes from days 28 and
after mating [37,38]. More recently, the isolation and purica-
tion of PAGs from bualo placenta allowed for developing a
specic RIA system for bualo (RIA 860) [39-42]. This system
described bualo plasma PAGs proles during pregnancy and
postpartum (FIG. 1) [41]. During gestation, PAGs concentra-
tion increased to day 105 and remained constant until parturi-
tion. PAGs concentrations decrease rapidly in the postpartum
period, reaching minimum values (<1 ng/mL) on day 30. The
rapid decrease in PAGs concentration during the postpartum is
essential when using PAGs as an appropriate marker of preg-
nancy just after calving. Moreover, unlike bovines, they do not
require a cut-o limit in postpartum animals to detect a new
pregnancy.
As demonstrated by dierent authors [42-46], PAGs
assay can be a reliable biomarker for early pregnancy detec-
tion and EM in bualo (FIG. 2). Recently, Barile et al. [4] have
shown that PAGs permitted the discrimination between bua-
lo that experienced embryonic mortality and those that main-
tained pregnancy starting from 25 days of gestation, dening
the optimal cut-o value for predicting mortality in 1,1 ng/mL
and 2.2 ng/mL at day 25 and 28 respectively, post articial in-
semination (AI). Since PAGs reect embryo well-being, the re-
FIGURE 1. Pregnancy-associated glycoprotein (PAG) plasma prole during pregnancy and post-partum period in bualo
cows; adapted from Barbato et al [41].
78
13th World Bualo Congress ~ 13er Congreso Mundial de Búfalos / Lectures / Reproduction ______________________________________
duction of its circulating concentrations is a prognostic sign of
pregnancy failure.
Therefore, PAGs could be utilized as a diagnostic tool to
improve farm reproductive management through pharmacolog-
ical intervention for animals at risk for embryo loss. In this case,
a single injection of GnRH at day 35 after AI increased PAGs
concentration and recovery of embryo well-being, allowing a
reduction of pregnancy loss in the early pregnancy in bualo
at risk of EM [47]. GnRH treatment enhances embryo survival
rates by delaying the luteolytic mechanism [48] that could occur
due to failure in maternal recognition of pregnancy.
LATEST RESEARCH ON EARLY PREGNANCY
MARKERS EXPRESSION
The peri-implantation period is an extremely delicate
time in the establishment of pregnancy in which various
mechanisms are involved in maintaining the corpus luteum
(CL). One of the rst molecules involved in early maternal rec-
ognition in ruminants is the interferon tau (IFNt) [49]. This pro-
tein is secreted by the trophectodermal cells of the blastocysts
around days 16-25 in bualo [50] and increases with the elon-
gation of the conceptus. IFNt prevents secretion of PGF2α by
uterine epithelium, prolonging CL life span and maintaining
the progesterone production for the maternal recognition of
pregnancy. IFN-τ enters circulation, triggering the expression
of a group of molecules known as Interferon Stimulated Genes
(ISGs). The members of ISGs, such as interferon-stimulated
protein 15 (ISG15), myxovirus resistance protein 2 (MX2),
and 2’-5’oligoadenylate synthetase (OAS1), are hypothesized
to regulate endometrial receptivity to implantation as well as
survival, growth, and development of the conceptus [51]. In
addition to IFNt, it is hypothesized that PAGs participate in im-
mune modulation at the conceptus–maternal interface during
early pregnancy [23]. The correlation between ISGs and IFNt
expression and PAGs plasma concentration during peri-im-
plantation and early pregnancy in bualo cows [52] supports
the possible immunomodulatory role for these glycoproteins
at the maternal-fetal level, necessary for the establishment
and the maintenance of the maternal-fetal unit histocompat-
ibility, and to prevent luteolysis by inhibiting PGF2α release,
resulting in the maintenance CL function. When PAG-1, IFNt
and ISGs expression were used as diagnostic and prognostic
markers of maternal-fetal cellular interaction in bualo cows,
ISGs proved to be a more reliable peripheral biomarker for
the prediction of pregnancy and embryonic mortality in bua-
lo during the peri-implantation period [53]. The results of this
study showed that MX2 and MX1 were the best genes to dis-
criminate pregnancy or embryonic mortality, respectively, at
19 days post-AI.
Studies on PAG expression in the maternal subset
of blood leukocytes at the peri-implantation period in bualo
showed that the quantication of PAG-2 mRNA could be used
to dierentiate pregnant and non-pregnant bualoes starting
from the second-week post-AI [54,55].
At the moment, PAG-2, as well as ISGs expression,
could be a valuable marker for studies on early pregnancy and
embryonic mortality but not for use in the eld because of the
single analysis’s cost and procedure.
CONCLUSIONS
In veterinary practice, the PAGs measurement is a fea-
sible method used for early pregnancy diagnosis, pregnancy
conrmation, and follow-up of the embryo vitality. The rst as-
pect can help breeders manage reproduction, allowing an early
resynchronization and rebreeding of the non-pregnant animals,
aiming to shorten the calving-conception interval.
FIGURE 2. Pregnancy-associated glycoprotein (PAG) plasma concentrations in bualo cows experienced embryonic
mortality; adapted from Barbato and Barile [45]
79
________________________________________________________ Revista Cientíca, FCV-LUZ / Vol. XXXIII, Supl. Esp., 75 - 81, 2023
The identication of subjects at risk of embryonic mor-
tality can inuence management decisions for the recovery
of those animals that would experience pregnancy loss, i.e.,
through pharmacologic intervention strategies for the mainte-
nance of pregnancy.
Finally, quantifying PAG mRNA expression could be a
helpful tool to understand better the mechanisms involved in
embryonic mortality, supporting studies on embryonic implan-
tation and pregnancy failures.
REFERENCES
[1] Deka, R.P.; Magnusson, U.; Grace, D.; Randolph, T.F.;
Shome, R.; Lindahl, J.F. Estimates of the economic cost
caused by ve major reproductive problems in dairy. Ani-
mals 2021, 11, 3116.
[2] Fricke, P.M. Scanning the future – ultrasonography as a
reproductive management tool for dairy cattle. J Dairy Sci
2002, 85, 1918–26.
[3] Fricke, P.M.; Ricci, A.; Giordano, J.O.; Carvalho, P.D.
Methods for and implementation of pregnancy diagno-
sis in dairy cows. Vet. Clin. Food Anim. Pract. 2016, 32,
165–180.
[4] Barile, V.L.; Menchetti, L.; Casano, A, B.; Brecchia, G.;
Sousa, N.M.; Zelli, R.; Canali, C.; Beckers, S.F.; Barbato,
O. Approaches to Identify Pregnancy Failure in Bualo
Cows. Animals 2021, 1, 481.
[5] Xie, S.C.; Low, B.G.; Nagel, R.J.; Kramer, K.K.; Anthony,
R.V., Zoli, A.P.; Beckers, J.F. & Roberts, R.M. Identica-
tion of the major pregnancy-specic antigens of cattle
and sheep as inactive members of the aspartic proteina-
se family. PNAS 1991, 88, 10247–10251.
[6] Green, J.A.; Xie, S.; Roberts, R.M. Pepsin-related mole-
cules secreted by trophoblast. Reviews of Reproduction
1998, 3, 62–69.
[7] Beckers, J.F.; Drion, P.V.; Garbayo, J.M., Perenyi Z.; Za-
rrouk, A. Sulon, J.; Remy, B.; Szenci, O. Pregnancy-as-
sociated glycoproteins in ruminantes: inactive members
of the aspartic proteinase family. Acta Veterinaria Hunga-
rica 1999, 47, 461-469.
[8] Roberts, R.M.; Xie, S.; Mathialagan, N. Maternal rico-
gnition of pregnancy. Biology of Reproduction 1996, 54,
294-302.
[9] Xie, S.; Low, B.G.; Nagel, R.J.; Beckers, J.F.; Roberts,
R.M. A novel glycoprotein of the aspartic proteinase gene
family expressed in bovine placental trophectoderm. Biol
Reprod 1994, 51, 1145-1153.
[10] Telugu, B.P.; Palmier, M.O.; Van Doren, S.R.; Green,
J.A. An examination of the proteolytic activity for bovine
pregnancy-associated glycoproteins 2 and 12. Biological
Chemistry 2010, 391, 259–270.
[11] Green, J.A.; Xie, S.; Quan, X.; Bao, B.; Mathialagan, N.;
Beckers, J.F.; Roberts, R.M. Pregnancy-associated bo-
vine and ovine glycoproteins exhibitspatially and tempo-
rally distinct expression patterns during pregnancy. Biol
Reprod 2000, 62, 1624-1631.
[12] Zoli, A.P.; Guilbault, L.A.; Delahaut, P.; Ortiz, W.B.;
Beckers, J.F. Radioimmunoassay of a bovine pregnan-
cy-associated glycoprotein in serum: its application for
pregnancy diagnosis. Biology of Reproduction 1992, 46,
83–92.
[13] Touzard, E.; Reinaud, P.; Dubois, O.; Guyader-Joly, C.;
Humblot, P.; Ponsart, C.; Charpigny, G. Specic expres-
sion patterns and cell distribution of ancient and modern
PAG in bovine placenta during pregnancy. Reproduction
2013, 146, 347–362.
[14] Hughes, A.L.; Green, J.A.; Garbayo, J.M.; Roberts, R.M.
Adaptive diversication within a large family of recent-
ly duplicated, placentally-expressed genes. Proc. Natl.
Acad. Sci. USA 2000, 97, 3319–3323.
[15] Wallace, R.M.; Pohler, K.G.; Smith, M.F.; Green, J.A.
Placental PAGs: Gene origins, expression patterns, and
use as markers of pregnancy. Reproduction 2015, 149,
R115–R126
[16] Xie, S.; Green, J.A.; Bao, B.; Beckers, J.F.; Valdez, K.E.;
Hakami, L.; Roberts, R.M. Multiple pregnancy-associa-
ted glycoproteins are secreted by day 100 ovine placen-
tal tissue. Biol Reprod 1997, 57, 1384–1393.
[17] Garbayo, J.M.; Serrano, B.; Lopez-Gatius, F. Identica-
tion of novel pregnancyassociated glycoproteins (PAG)
expressed by the perimplantion conceptus of domestic
ruminants. Anim Reprod Sci 2008, 103, 120–134
[18] Jerome, A.; Singh, S.K.; Agarwal, S.K.; Mohini, S.; Raut
A. Characterization and In Silico analysis of pregnan-
cy-associated glycoprotein-1 gene of bualo (Bubalus
bubalis). Genet Res Int 2011, 436138.
[19] Telugu, B.P.; Green, J.A. Characterization of the pepti-
dase activity of recombinant porcine pregnancy-associa-
ted glycoprotein-2. Journal of Biochemistry 2008, 144,
725–732.
[20] Moussad, E.E.; Rageh, M.A.; Wilson, AK.; Geisert, R.D.;
Brigstock, D.R. Temporal and spatial expression of con-
nective tissue growth factor (CCN2; CTGF) and tran-
sforming growth factor beta type 1 (TGF-b1) at the ute-
ro-placental interface during early pregnancy in the pig.
Journal of Clinical Pathology: Molecular Pathology 2002,
55, 186-192
[21] Wooding, F.B.; Roberts, R.M.; Green, J.A. Light and
electron microscope immunocytochemical studies of
80
13th World Bualo Congress ~ 13er Congreso Mundial de Búfalos / Lectures / Reproduction ______________________________________
the distribution of pregnancy-associated glycoproteins
(PAGs) throughout pregnancy in the cow: possible fun-
ctional implications. Placenta, 2005, 26, 807–827.
[22] Dosogne, H.; Burvenich, C.; Freeman, A.E.; Kehrli Jr,
M.E.; Detilleux, J.C., Sulon, J., Beckers, J.F.; Hoeben,
D. Pregnancy-associated glycoprotein and decreased
polymorphonuclear leukocyte function in early post-pr-
tum dairy cows. Vet Immunology and immunopathology
1999, 67, 47-54.
[23] Austin, K.J.; King, C.P.; Vierk, J.E.; Sasser, R.G.; Han-
sen, T.R. Pregnancy-specic protein B induces release
of an alpha chemokine in bovine endometrium. Endocri-
nology 1999, 140, 542–545.
[24] Del Vecchio, R.P.; Sutherland, W.D.; Sasser, R.G. Bovine
luteal cell production in vitro of prostaglandin E2, oxyto-
cin and progesterone in response to pregnancy-specic
protein B and prostaglandin F2a. Journal of Reproduction
and Fertility 1996, 107, 131–136.
[25] Weems, Y.S.; Lammoglia, M.A.; Vera-Avila, H.R.; Ran-
del, R.D.; King, C.; Sasser, R.G.; Weems, C.W. Eect
of luteinizing hormone (LH), PGE2, 8-Epi- PGE1, 8-EPI-
PGE2, trichosanthin, and pregnancy specic protein B on
secretion of progesterone in vitro by corpora lutea (CL)
from nonpregnant and pregnant cows. Prostaglandins &
Other Lipid Mediators 1998, 55, 27–42.
[26] Kornmatitsuk, B.; Veronesi, M.C.; Madej, A.; Dahl, E.;
Ropstad, E.; Beckers, J.F.; Forsberg, M.; Gustalsson, H.;
Kindhal, H. Hormonal measurements in late pregnancy
and parturition in dairy cows-possible tools to monitor fe-
tal well being. Anim Reprod Sci 2002, 72, 153-164.
[27] Giordano, J.O.; Guenther, J.N.; Lopes, G. Jr; Fricke, P.M.
Changes in serum pregnancy-associated glycoprotein,
pregnancy-specic protein B, and progesterone concen-
trations before and after induction of pregnancy loss in
lactating dairy cows. Journal of Dairy Science 2012, 95,
683–697.
[28] Karen, A.; De Sousa, N.M.; Beckers, J.F.; Bajcsy, A.C.;
Tibold, J.; Madl, I.; Szenci, O. Comparison of a commer-
cial bovine pregnancy- associated glycoprotein ELISA
test amd pregnancy-associated glycoprotein radioimmu-
noassay test for early pregnancy diagnosis in dairy cattle.
Anim Reprod Sci 2015, 159, 31-37.
[29] Haugejorden, G.; Waage, S.; Dahl, E.; Karlberg, K.;
Beckers, J.F.; Ropstad, E. Pregnancy associated glyco-
proteins (PAG) in postpartum cows, ewes, goats and
their ospring. Theriogenology 2006, 66, 1976-1984.
[30] Franco, G.A.; Peres, R.F.G.; Martins, C.F.G.; Reese,
S.T.; Jose Luiz Moraes Vasconcelos, J.L.M.; Pohler, K.J.
Sire contribution to pregnancy loss and pregnancy-asso-
ciated glycoprotein production in Nelore cows. J. Anim.
Sci. 2018, 96, 632–640.
[31] Reese, S.T., Geary, T.W.; Franco, G.A.; Moares, J.G.N.;
Spencer, T.E.; Pohler, K.G. Pregnancy associated glyco-
proteins (PAGs) and pregnancy loss in high vs sub fertili-
ty heifers. Theriogenology 2019, 135, 7-12.
[32] Pohler, K.G.; Reese, S.T.; Franco, G.A.; Oliveira, R.V.;
Paiva, R.; Fernandez, L.; de Melo, G.; Vasconcelos,
J.L.M.; Cooke, R.; Poole, R.K. New approaches to diag-
nose and target reproductive failure in cattle. Anim. Re-
prod. 2020, 17. [
[33] Barile, V.L. Improving reproductive eciency in female
bualoes. (Review). Liv. Prod. Science, 2005, 92, 183-
194.
[34] Barile, V.; Terzano, G.; Pacelli, C.; Todini, L.; Malfatti,
A.; Barbato, O. LH peak and ovulation after two dierent
estrus synchronization treatments in bualo cows in the
daylight-lengthening period. Theriogenology 2015, 84,
286–293.
[35] Baruselli, P.; Visintin, J.; Barnabe, V.; Barnabe, R.; Ama-
ral, R.; Souza, A. Early pregnancy ultrasonography and
embryonic mortality occurence in bualo. In Proceedin-
gs of the V World Bualo Congress,1997. Caserta, Italy,
13–18 October 1997; pp. 13–16.
[36] Campanile, G.; Neglia, G.; D’Occhio, M.J. Embryonic
and fetal mortality in river bualo (Bubalus bubalis). The-
riogenology, 2016, 86, 207-213.
[37] Karen, A.; Darwish, S.; Ramoun, A.; Tawfeek, K.; Van
Hanh, N.; De Sousa, N.; Sulon, J.; Szenci, O.; Beckers,
J.-F. Accuracy of ultrasonography and pregnancy-asso-
ciated glycoprotein test for pregnancy diagnosis in bua-
loes. Theriogenology 2007, 68, 1150–1155.
[38] El-Battawy, K.A.; Sousa, N.M.; Szenci, O.; Beckers, J.F.
Pregnancy-associated glycoprotein prole during the rst
trimester of pregnancy in Egyptian bualo cows. Reprod
Domest Anim Zuchthyg 2009, 44, 161–166.
[39] Barbato, O.; Sousa, N.M.; Klisch, K.; Clerget, E.; Debe-
nedetti, A.; Barile, V.L.; Malfatti, A.; Beckers, J.F. Isolation
of new pregnancyassociated glycoproteins from water
bualo (Bubalus bubalis) placenta by Vicia villosa anity
chromatography. Res Vet Sci 2008, 85, 457–466.
[40] Barbato, O.; Melo de Sousa, N.; Barile, V.L.; Canali,
C.; Beckers, J.-F. Purication of pregnancy-associated
glycoproteins from late-pregnancy Bubalus bubalis pla-
centas and development of a radioimmunoassay for pre-
gnancy diagnosis in water bualo females. BMC Vet Res
2013, 9, 89.
[41] Barbato, O.; Menchetti, L.; Sousa, N.M.; Malfatti, A.;
Brecchia, G.; Canali, C.; Beckers, J.F.; Barile, V.L. Preg-
nancy-associated glycoproteins (PAGs) concentrations
in water bualoes (Bubalus bubalis) during gestation and
the postpartum period. Theriogenology 2017, 97, 73–77.
81
________________________________________________________ Revista Cientíca, FCV-LUZ / Vol. XXXIII, Supl. Esp., 75 - 81, 2023
[42] Barbato, O.; Menchetti, L.; Sousa, N.M.; Brecchia, G.;
Malfatti, A.; Canali, C.; Beckers J.F.; Barile, V.L. Corre-
lation of two radioimmunoassay systems for measuring
plasma pregnancy-associated glycoprotein concentra-
tions during early pregnancy and postpartum periods in
water bualo. Reprod Domest Anim 2018, 53, 1483-149.
[43] Karen. A.; Darwish, S.; Ramoun, A.; Tawfeek, K.; Van
Hanh, N.; Sousa, N.M.; Sulon. J.; Szenci, O.; Beckers,
J.F. Accuracy of ultrasonography and pregnancy-asso-
ciated glycoprotein test for pregnancy diagnosis in bua-
loes. Theriogenology 2007, 68, 1150–1155.
[44] Nguyen, V.H.; Barbato, O.; Bui, X.N.; Beckers, J.-F.; de
Sousa, N.M. Assessment of pregnancy-associated glyco-
protein (PAG) concentrations in swamp bualo samples
from fetal and maternal origins by using interspecies an-
tisera. Anim Sci J 2012, 83, 683–689.
[45] Barbato, O.; Barile, V.L. The pregnancy diagnosis in buf-
falo species: laboratory methods. J Bualo Sci 2012, 1,
157-162.
[46] Barbato, O.; Chiaradia, E.; Barile, V.L.; Pierri, F.; Sousa,
M.N.; Terracina, L.: Canali, C.; Avellini, L.; Investigation
into omocysteine, vitamin E and malondialdeyde as indi-
cators of successful articial insemination in synchroni-
zed bualo cows (Bubalus bubalis). Research in Veteri-
nary Science 2016, 104, 100-105.
[47] Pacelli, C.; Barile, V.L.; Sabia, E.; Casano, A.B.; Braghie-
ri, A.; Martina, V.; Barbato, O. Use of GnRH Treatment
Based on Pregnancy-Associated Glyco-Proteins (PAGs)
Levels as a Strategy for the Maintenance of Pregnancy
in Bualo Cows: A Field Study. Animals 2022, 12, 2822.
[48] Mann, G.E; Lamming, G.E.; Fray, M.D. Plasma oestra-
diol and progesterone during early pregnancy in the cow
and the eects of treatment with buserelin. Anim. Re-
prod. Sci. 1995, 37, 121–131.
[49] Thatcher, W.W.; Meyer, M.D.; Danet-Desnoyers, G. Ma-
ternal Recognition of Pregnancy. J. Reprod. Fertil.-Suppl.
1995, 49, 15–28.
[50] Saugandhika, S.; Sharma, V.; Malik, H.; Saini, S.; Bag,
S.; Kumar, S.; Singh, N.K.; Mohanty, A.K.; Malakar, D.
Expression and Purication of Bualo Interferon-Tau and
Ecacy of Recombinant Bualo Interferon-Tau for in Vi-
tro Embryo Development. Cytokine 2015, 75, 186–196.
[51] Rocha CC, Silveira JC, Forde N, Binelli M, Pugliesi G.
Conceptus-modulated innate immune function during
early pregnancy in ruminants : a review. Anim Reprod.
2021,18(1) :e20200048.
[52] Casano, A.B; Barile, V.L.; Menchetti, L.; Guel, G.; Brec-
chia, G.; Agradi, S.; De Matteis, G.; Scatà, M.C.; Grando-
ni, F.; Barbato, O. Interferon Tau (IFNt) and Interferon-Sti-
mulated Genes (ISGs) Expression in Peripheral Blood
Leukocytes and Correlation with Circulating Pregnan-
cy-Associated Glycoproteins (PAGs) during Peri-Implan-
tation and Early Pregnancy in Bualo Cows. Animals,
2022, 12, 3068.
[53] Casano A.B. Menchetti L., Trabalza-Marinucci M., Riva
F, De Matteis G., Brecchia G., Inglesi A, Rossi E., Si-
gnorelli F., Barile V.L., Barbato O. Gene expression of
pregnancy-associated glycoproteins-1 (PAG-1), interfe-
ron-tau (IFNt) and interferon stimulated genes (ISGs) as
diagnostic and prognostic markers of maternal-fetal cel-
lular interaction in bualo cows. Theriogenology, 2023,
209, 89-97.
[54] Barbato O, Guel G, Barile VL, Menchetti L, Tortiello C,
Canali C, Brecchia G, Traina G, Beckers JF, Melo de
Sousa N. 2017. Using real-time PCR to identify pregnan-
cy-associated glycoprotein 2 (PAG-2) in water bualo
(bubalus bubalis) blood in early pregnancy. Theriogeno-
logy 2017, 89, 106-113.
[55] Barbato, O.; Guel, G.; Menchetti, L.; Brecchia, G.;
Sousa, M.N.; Canali, C.; Grandoni, F.; Scatà, M.C.; De
Matteis, G.; Casano, A.B.; Beckers, J.F.; Barile, V.L. In-
vestigation of PAG2 mRNA Expression in Water Bualo
Peripheral Blood Mononuclear Cells and Polymorphonu-
clear Leukocytes from Maternal Blood at the Peri-Implan-
tation Period. Veterinary Sciences 2019, 6, 8.
