BACKGROUND
domestic buffalo are considered to have low reproductive efficiency, characterised by late attainment of puberty and maturity, seasonality of calving, long postpartum anoestrus, poor expression of oestrus signs, low conception rate and long calving intervals  <cit> .
furthermore, there is some evidence of a high rate of embryonic loss, in particular during the critical phase of embryonic attachment  <cit> .
thus, an accurate early distinction of pregnant and non-pregnant animals is essential for improvement of reproductive efficiency in buffalo, particularly when breeding techniques such as “out of breeding season mating” or artificial insemination  are applied.
moreover, the study of embryonic mortality through the detection of pregnancy markers could support researchers aiming to improve oestrus synchronisation and fixed time ai programs in buffaloes.
since the early 1980s, a large family of placental proteins without known biological activity  or pregnancy-specific protein b ) has been purified from ruminant placenta  <cit> .
some of them are detected in the peripheral circulation of pregnant females, and are used as a tool to investigate placental function in ongoing or endangered pregnancies  <cit> .
pags are expressed in mono- and binucleate trophoblastic cells of the outer epithelial layer in the synepitheliochorial cotyledonary placenta  <cit> .
using an antiserum  raised against bovine pag , carvalho et al.
<cit>  confirmed that there is a strong homology between water buffalo and ruminant binucleate cells concerning cell morphology, protein expression, glycosylation pattern and characteristics of cell migration and fusion.
buffalo binucleate cells migrate toward the maternal epithelium and fuse with a uterine epithelial cell to form a trinucleate cells  <cit> .
maternal hybrid trinucleate cells can also further fuse with adjacent cells, resulting in the formation of a multinuclear syncytium  <cit> .
however, larger syncytia, with more than three nuclei, are much less frequent than trinucleate cells in buffalo placentas  <cit> .
pag molecules belong to the aspartic proteinase  superfamily  <cit>  and originated from an ancient pag-like precursor by duplication and positive selection approximately  <dig> million years ago  <cit> .
it was estimated that cattle, sheep, and probably other pecoran mammals possess many, possibly  <dig> or more, pag genes  <cit> .
to date,  <dig> different complementary dna  of pag genes  have been identified in species with a synepitheliochorial placenta.
in bovine species,  <dig> pag genes  have been cloned and fully sequenced  <cit> .
the number of identified pag polypeptide precursors is lower in ovine   <cit> , caprine   <cit> , cervid   <cit>  and water buffalo species .
molecular biology techniques have allowed for huge progress in understanding the phylogenetic diversity of pag molecules.
however, these techniques are not adequate to obtain purified and semi-purified pag preparations necessary for the development of radioimmunoassay  and enzyme-linked immunosorbent assay  protocols.
fortunately, since the 1990s, the selection of the most convenient chromatography procedures allowed for the isolation of an important number of native purified pag.
at present, forty pag isoforms have been isolated from cotyledons of the cow  <cit> , ewe  <cit> , goat  <cit> , buffalo  <cit> , bison  <cit> , moose and elk  <cit> .
some of them were used to immunise rabbits and the antisera obtained allowed the immunolocalisation of pag in placental tissue  <cit>  and the determination of secretory pattern measured in peripheral maternal blood .
in buffalo species, despite efforts made on the purification of pag molecules, the quantity of purified pag has not been sufficient to raise new pag antisera.
so far, pag concentrations in buffalo species were determined by heterologous pag-ria systems based on antisera raised against bovine or caprine pag  <cit> .
interestingly, by using these systems, concentrations of pag were remarkably distinct from those measured in cattle, increasing gradually from the 6th week of gestation to parturition , and reaching relatively low peripartum levels  <cit> .
here, we describe the successful isolation and characterisation of new buffalo pag molecules.
these newly purified proteins were used for the production of three specific antisera to develop a new pag-ria system .
finally, its ability to discriminate between pregnant and non-pregnant females was evaluated in italian mediterranean buffalo cows.
RESULTS
isolation and characterisation of water buffalo pag
figure  <dig> schematically shows the protocol used to isolate wbpag from fetal cotyledons.
amounts of total protein  and equivalent immunoreactive pag contents  in the different steps of wbpag purification are summarised in table  <dig>  highest immunoreactivity was observed at the deae  <dig> mm nacl  when compared to deae  <dig> mm nacl  and deae  <dig> mm nacl .
sephadex g <dig> peaks exhibiting the highest pag/tp ratios  were loaded onto vva lectin-affinity chromatography.
the vva chromatographies of sephadex g75-d <dig> peak iii, g75-d <dig> peak ii and g75-d <dig> peak i and ii resulted in pag/tp ratios higher than 100% .
aeight hundred milligrams from each deae were loaded on the sephadex g <dig>
tp: total protein.
equivpag: pag concentrations measured by heterologous ria.
adetermined by radioimmunoassay.
bobtained by weighing each fraction.
capparent molecular masses  were estimated after sds-page and western blot .
table  <dig> summarises the different purification steps used to obtain the eleven n-terminal sequenced water buffalo pag molecules , as well as their apparent molecular masses as determined after coomassie staining of the pvdf membrane  and western blot .
the apparent molecular masses of immunoreactive pag isoforms from vva peaks  ranged from  <dig> to  <dig> kda.
these molecular masses were systematically slightly lower than those revealed after coomassie staining of the pvdf membrane .
proteins were submitted to edman degradation based on their availability .
the protein sequence data described are available in the uniprot knowledgebase under the accession numbers p <dig> to p <dig>  isolated wbpag showed highly conserved amino acid residues at the beginning of the n-terminal extremity , with edman sequencing failing to give any signal on cycle  <dig> .
buffalo sequenced n-termini contained the consensus plr .
when compared, the percentage of amino acid identity of newly sequenced wbpag ranged from 80%  to 100%.
as shown in table  <dig>  in their sequenced part, wbpag65kda_e, wbpag58kda_f and wbpag73kda_i were identical, as well as wbpag67kda_a, wbpag76kda_d, wbpag76kda_g, wbpag63kda_h and wbpag60kda_k.
screening of the embl and swiss-prot data banks revealed 100% identity between four pag n-termini characterised here  and those deduced from cdna from buffalo species .
finally, the micro-sequence comparison also revealed that some wbpag forms  are unique compared to other purified proteins forms isolated from bovine, ovine, caprine and bison species .
development and validation of a new pag ria
figure  <dig> shows displacement of standard  inhibition curves  tested using three different antisera raised against buffalo pag .
all tested antisera gave very similar slopes.
highest dilutions of primary antisera were obtained with as# <dig> .
therefore, this system  was chosen for measuring concentrations of pag in water buffalo cows.
concerning ria- <dig> validation, mdl was  <dig>  ng/ml.
parallelism between standard curve and serial dilutions from a pregnant buffalo female is demonstrated in figure  <dig>  reproducibility measured as the coefficients of variation intra- and inter-assay was  <dig> %  and  <dig> % , respectively.
regarding specificity, the presence of different concentrations  of placental proteins, sugars and other plasmatic compounds did not alter the binding of radio-labelled pag.
finally, regarding recovery, it ranged from  <dig> to 110% when concentrations of  <dig>  and  <dig>  ng/ml were added to a sample containing low pag concentrations.
concentrations of pag in plasma samples of pregnant and non-pregnant buffalo cows
from a total of  <dig> buffalo females used,  <dig> became pregnant after ai, as detected by both rectal palpation and ria- <dig>  as shown in table  <dig>  in non-pregnant animals, mean pag concentrations remained under  <dig>  ng/ml at days  <dig>   <dig> and  <dig> after ai.
two non-pregnant females exhibited pag concentrations of  <dig>  ng/ml: one female at day  <dig>  and the other at day  <dig> after ai.
in the pregnant group, two females exhibited pag concentrations under  <dig>  ng/ml at day  <dig> after ai.
in this group, concentrations increased significantly from day  <dig> to  <dig> and from day  <dig> to  <dig>  when the best threshold for discrimination between pregnant and non-pregnant animals was observed.
pag concentrations are expressed as mean ± sem.
ai: artificial insemination.
values with similar superscripts in the same column are statistically different .
significant differences between pag concentrations from pregnant and non-pregnant females are indicated by asterisks .
discussion
purified native pag preparations are required for the development of specific and/or more sensitive immunoassay techniques that are currently used for pregnancy diagnosis and physiopathological investigations in ruminant species  <cit> .
our work describes the isolation and characterisation of different pags from buffalo placentas.
some proteins were used for production of polyclonal antisera, allowing the development of new radioimmunoassay systems.
in parallel, our work confirmed the large heterogeneity of pag molecules for both molecular mass and n-terminal amino acid sequence.
heterogeneity of the pag subfamily was evoked as early as  <dig>  when butler et al.
<cit>  estimated molecular mass of bovine pspb to range from  <dig> to  <dig> kda.
however, after several years, it was not clear if such diversity was due to the expression of distinct pspb/pag forms in ruminant placenta, to an extensive post-translational processing in placental tissue , or to both phenomena.
the identification of a high number of n-terminal sequences and cdna corresponding to different ruminant pag and the demonstration of an extensive glycosylation mechanism in bovine and ovine placenta  <cit>  confirmed that both factors contribute to the pag diversity described here and in previous works  <cit> .
for instance, different numbers of n-glycosylation sites of asparagines have been observed in pag isoforms identified in placentas of cattle, sheep and goats  <cit> .
in cattle, the dominant bopag67kda form has been shown to present  <dig> potential glycosylation sites and 10% of oligosaccharides content  <cit> .
multi-antennary oligosaccharides have been shown to represent  <dig> % of the relative molecular mass of ovpag  <cit> .
the number of potential sites of glycosylation of buffalo pag had not yet been described in the literature.
however, as previously reported, n-terminal sequences of wbpag failed to give any signal on cycle  <dig>  this blank cycle being followed by the consensus sequence l-t that is a characteristic of the n-glycosylation site.
our results confirm earlier findings of klisch et al.
<cit> , barbato et al.
<cit>  and kiewiesz et al.
<cit> , who reported that vva chromatography can be very useful to enrich placental glycoproteins produced by binucleate cells.
interestingly, despite several pag sequences have been obtained in buffalo species, none of them corresponded to the cdna sequence of buffalo pag- <dig> .
in bovine species , when using the classical purification protocol, a single major protein  was identified in placental extracts  <cit> .
on the other side, a large heterogeneity of pag molecules could be identified in placental extracts from small ruminants, european and american bison  <cit> .
concerning analysis of n-terminal amino acid sequences of newly purified buffalo pags, the interpretation was limited by the relatively low number of residues clearly identified .
three of them  were identical to wbpag62kda, previously isolated from mid-pregnancy  buffalo placenta  <cit> .
five other sequences  were 100% identical to proteins previously characterised in buffalo   <cit> , ovine   <cit>  and bison  placentas  <cit> .
our work describes for the first time the production of polyclonal antisera raised against pag molecules isolated from buffalo placenta.
proteins issued from three different fractions  were used to immunise rabbits resulting in the production of three distinct antibodies: as# <dig>  as# <dig> and as# <dig>  respectively.
these three obtained antisera gave very similar displacement of standard inhibition curves.
this finding was not surprising because pag molecules expressed in the same species can exhibit high sequence identities  <cit> , and thus probably share common epitopes  <cit> .
the highest dilution of primary antiserum  was obtained with as# <dig>  the remarkable parallelism between serial dilutions of pregnant buffalo samples and the standard curve indicates that ria- <dig> is a good immunoassay allowing distinguishing subtle quantitative differences in wbpag concentrations.
ria- <dig> also proved to be very repeatable for measurement of pag concentrations .
finally, it was observed that mdl of ria- <dig> was equivalent  to that previously described for ria- <dig>  <cit> , ria- <dig>  <cit>  and ria-pool  <cit> .
the use of pag/pspb ria systems are considered as reliable method for early pregnancy diagnosis and follow-up of trophoblastic function in ruminant species .
measurements of pag/pspb concentrations in the peripheral circulation of pregnant and non-pregnant buffalo cows have been performed using different ria methods  <cit> .
recently, barbato et al.
<cit>  compared ria- <dig>  ria- <dig> and ria- <dig>  for detecting pag molecules in pregnant buffalo females.
they reported that analogous pag antigens were better recognised using ria- <dig> from week  <dig> of gestation onwards.
by using the same pag-ria system, karen et al.
<cit>  described that pregnancy diagnosis reached 100% between days  <dig> and  <dig>
an additional feature of the present study was the observation of a rapid increase in pag concentrations from day  <dig>  to day  <dig> of pregnancy .
interestingly, a rapid increase in pag concentration during pregnancy is characteristic of ovine  <cit>  and caprine species  <cit> .
however, it differs largely from those described in bovine species, in which pag concentrations increase slowly during the first trimester of gestation  <cit> .
in egyptian buffalo cows, karen et al.
<cit>  and el-battawy et al.
<cit>  described mean pag concentrations ranging from  <dig>  to  <dig>  ng/ml  at around day  <dig> of pregnancy.
those values were higher than mean concentrations  measured by ria- <dig> at day  <dig> in italian mediterranean females .
egyptian and mediterranean buffalo cows are quite different in several aspects.
mediterranean females have a much higher milk yield than egyptian females   <cit> .
this observation of low pag levels in high producing milk females agrees with previous findings described by lopez-gatius et al.
<cit>  who reported that there is a strong negative correlation between milk production and pag concentrations during the first trimester of pregnancy.
differences in pag concentrations could also be partially due to other factors such as breed, placental mass, age of female or even farm   <cit> .
moreover, as previously described in bovine and ovine species, pag expression is complex throughout pregnancy.
some pag molecules are expressed early, while others only as pregnancy progresses.
other pag are also expressed throughout the whole pregnancy period  <cit> .
pag concentrations were detectable in peripheral plasma of all italian mediterranean pregnant buffalo cows at day  <dig>  at day  <dig>   <dig> out of  <dig> pregnant females were already positive by ria- <dig>  in dairy and beef cows, pag concentrations can be detected as early as at day  <dig> after ai  <cit> .
however, pregnancy diagnosis is only recommended from day  <dig> to  <dig> because of important individual variation on pag appearance in peripheral blood  <cit> .
according to different authors, expression of pag family members as early as day  <dig> after fertilisation suggests their potential role in cellular growth and differentiation, elongation, apposition, attachment and placentogenesis processes  <cit> .
on the other hand, in the present study, pag concentrations were higher than  <dig>  ng/ml in one non-pregnant female at days  <dig> and  <dig>  recently, karen et al.
<cit>  described pregnancy diagnosis in egyptian buffalo cows by using ria- <dig> .
the authors also reported 3/ <dig> and 2/ <dig> incorrect pregnancy diagnoses between days 31– <dig> and 36– <dig>  respectively.
the presence of detectable concentrations in non-pregnant females can be explained by the existence of extra-placental sources of pag, as suggested by zoli et al.
<cit> .
indeed, the presence of antigens immunologically related to pag has been demonstrated in testicular and in ovarian extracts, justifying, also, the adjective “associated” and not “specific” given to this family of placental glycoproteins  <cit> .
CONCLUSIONS
we have described for the first time the use of antisera raised against buffalo pag for ria development and pregnancy detection in buffalo cows.
moreover, we showed that ria- <dig> was quantitative, precise, accurate and sensitive in measuring pag concentrations.

 domestic buffalo are considered to have low reproductive efficiency, characterised by late attainment of puberty and maturity, seasonality of calving, long postpartum anoestrus, poor expression of oestrus signs, low conception rate and long calving intervals. furthermore, there is some evidence of a high rate of embryonic loss, in particular during the critical phase of embryonic attachment. thus, an accurate early distinction of pregnant and non-pregnant animals is essential for improvement of reproductive efficiency in buffalo, particularly when breeding techniques such as “out of breeding season mating” or artificial insemination are applied. moreover, the study of embryonic mortality through the detection of pregnancy markers could support researchers aiming to improve oestrus synchronisation and fixed time ai programs in buffaloes. since the early 1980s, a large family of placental proteins without known biological activity or pregnancy-specific protein b ) has been purified from ruminant placenta. some of them are detected in the peripheral circulation of pregnant females, and are used as a tool to investigate placental function in ongoing or endangered pregnancies. pags are expressed in mono- and binucleate trophoblastic cells of the outer epithelial layer in the synepitheliochorial cotyledonary placenta. using an antiserum raised against bovine pag, carvalho et al. confirmed that there is a strong homology between water buffalo and ruminant binucleate cells concerning cell morphology, protein expression, glycosylation pattern and characteristics of cell migration and fusion. buffalo binucleate cells migrate toward the maternal epithelium and fuse with a uterine epithelial cell to form a trinucleate cells. maternal hybrid trinucleate cells can also further fuse with adjacent cells, resulting in the formation of a multinuclear syncytium. however, larger syncytia, with more than three nuclei, are much less frequent than trinucleate cells in buffalo placentas. pag molecules belong to the aspartic proteinase superfamily and originated from an ancient pag-like precursor by duplication and positive selection approximately million years ago. it was estimated that cattle, sheep, and probably other pecoran mammals possess many, possibly or more, pag genes. to date, different complementary dna of pag genes have been identified in species with a synepitheliochorial placenta. in bovine species, pag genes have been cloned and fully sequenced. the number of identified pag polypeptide precursors is lower in ovine, caprine, cervid and water buffalo species. molecular biology techniques have allowed for huge progress in understanding the phylogenetic diversity of pag molecules. however, these techniques are not adequate to obtain purified and semi-purified pag preparations necessary for the development of radioimmunoassay and enzyme-linked immunosorbent assay protocols. fortunately, since the 1990s, the selection of the most convenient chromatography procedures allowed for the isolation of an important number of native purified pag. at present, forty pag isoforms have been isolated from cotyledons of the cow, ewe, goat, buffalo, bison, moose and elk. some of them were used to immunise rabbits and the antisera obtained allowed the immunolocalisation of pag in placental tissue and the determination of secretory pattern measured in peripheral maternal blood. in buffalo species, despite efforts made on the purification of pag molecules, the quantity of purified pag has not been sufficient to raise new pag antisera. so far, pag concentrations in buffalo species were determined by heterologous pag-ria systems based on antisera raised against bovine or caprine pag. interestingly, by using these systems, concentrations of pag were remarkably distinct from those measured in cattle, increasing gradually from the 6th week of gestation to parturition, and reaching relatively low peripartum levels. here, we describe the successful isolation and characterisation of new buffalo pag molecules. these newly purified proteins were used for the production of three specific antisera to develop a new pag-ria system. finally, its ability to discriminate between pregnant and non-pregnant females was evaluated in italian mediterranean buffalo cows.  isolation and characterisation of water buffalo pag figure  schematically shows the protocol used to isolate wbpag from fetal cotyledons. amounts of total protein and equivalent immunoreactive pag contents in the different steps of wbpag purification are summarised in table  highest immunoreactivity was observed at the deae mm nacl when compared to deae mm nacl and deae mm nacl. sephadex g peaks exhibiting the highest pag/tp ratios were loaded onto vva lectin-affinity chromatography. the vva chromatographies of sephadex g75-d peak iii, g75-d peak ii and g75-d peak i and ii resulted in pag/tp ratios higher than 100%. aeight hundred milligrams from each deae were loaded on the sephadex g tp: total protein. equivpag: pag concentrations measured by heterologous ria. adetermined by radioimmunoassay. bobtained by weighing each fraction. capparent molecular masses were estimated after sds-page and western blot. table  summarises the different purification steps used to obtain the eleven n-terminal sequenced water buffalo pag molecules, as well as their apparent molecular masses as determined after coomassie staining of the pvdf membrane and western blot. the apparent molecular masses of immunoreactive pag isoforms from vva peaks ranged from to kda. these molecular masses were systematically slightly lower than those revealed after coomassie staining of the pvdf membrane. proteins were submitted to edman degradation based on their availability. the protein sequence data described are available in the uniprot knowledgebase under the accession numbers p to p isolated wbpag showed highly conserved amino acid residues at the beginning of the n-terminal extremity, with edman sequencing failing to give any signal on cycle. buffalo sequenced n-termini contained the consensus plr. when compared, the percentage of amino acid identity of newly sequenced wbpag ranged from 80% to 100%. as shown in table  in their sequenced part, wbpag65kda_e, wbpag58kda_f and wbpag73kda_i were identical, as well as wbpag67kda_a, wbpag76kda_d, wbpag76kda_g, wbpag63kda_h and wbpag60kda_k. screening of the embl and swiss-prot data banks revealed 100% identity between four pag n-termini characterised here and those deduced from cdna from buffalo species. finally, the micro-sequence comparison also revealed that some wbpag forms are unique compared to other purified proteins forms isolated from bovine, ovine, caprine and bison species. development and validation of a new pag ria figure  shows displacement of standard inhibition curves tested using three different antisera raised against buffalo pag. all tested antisera gave very similar slopes. highest dilutions of primary antisera were obtained with as#. therefore, this system was chosen for measuring concentrations of pag in water buffalo cows. concerning ria- validation, mdl was ng/ml. parallelism between standard curve and serial dilutions from a pregnant buffalo female is demonstrated in figure  reproducibility measured as the coefficients of variation intra- and inter-assay was % and %, respectively. regarding specificity, the presence of different concentrations of placental proteins, sugars and other plasmatic compounds did not alter the binding of radio-labelled pag. finally, regarding recovery, it ranged from to 110% when concentrations of and ng/ml were added to a sample containing low pag concentrations. concentrations of pag in plasma samples of pregnant and non-pregnant buffalo cows from a total of buffalo females used, became pregnant after ai, as detected by both rectal palpation and ria- as shown in table  in non-pregnant animals, mean pag concentrations remained under ng/ml at days and after ai. two non-pregnant females exhibited pag concentrations of ng/ml: one female at day and the other at day after ai. in the pregnant group, two females exhibited pag concentrations under ng/ml at day after ai. in this group, concentrations increased significantly from day to and from day to when the best threshold for discrimination between pregnant and non-pregnant animals was observed. pag concentrations are expressed as mean ± sem. ai: artificial insemination. values with similar superscripts in the same column are statistically different. significant differences between pag concentrations from pregnant and non-pregnant females are indicated by asterisks. discussion purified native pag preparations are required for the development of specific and/or more sensitive immunoassay techniques that are currently used for pregnancy diagnosis and physiopathological investigations in ruminant species. our work describes the isolation and characterisation of different pags from buffalo placentas. some proteins were used for production of polyclonal antisera, allowing the development of new radioimmunoassay systems. in parallel, our work confirmed the large heterogeneity of pag molecules for both molecular mass and n-terminal amino acid sequence. heterogeneity of the pag subfamily was evoked as early as when butler et al. estimated molecular mass of bovine pspb to range from to kda. however, after several years, it was not clear if such diversity was due to the expression of distinct pspb/pag forms in ruminant placenta, to an extensive post-translational processing in placental tissue, or to both phenomena. the identification of a high number of n-terminal sequences and cdna corresponding to different ruminant pag and the demonstration of an extensive glycosylation mechanism in bovine and ovine placenta confirmed that both factors contribute to the pag diversity described here and in previous works. for instance, different numbers of n-glycosylation sites of asparagines have been observed in pag isoforms identified in placentas of cattle, sheep and goats. in cattle, the dominant bopag67kda form has been shown to present potential glycosylation sites and 10% of oligosaccharides content. multi-antennary oligosaccharides have been shown to represent % of the relative molecular mass of ovpag. the number of potential sites of glycosylation of buffalo pag had not yet been described in the literature. however, as previously reported, n-terminal sequences of wbpag failed to give any signal on cycle this blank cycle being followed by the consensus sequence l-t that is a characteristic of the n-glycosylation site. our results confirm earlier findings of klisch et al., barbato et al. and kiewiesz et al., who reported that vva chromatography can be very useful to enrich placental glycoproteins produced by binucleate cells. interestingly, despite several pag sequences have been obtained in buffalo species, none of them corresponded to the cdna sequence of buffalo pag-. in bovine species, when using the classical purification protocol, a single major protein was identified in placental extracts. on the other side, a large heterogeneity of pag molecules could be identified in placental extracts from small ruminants, european and american bison. concerning analysis of n-terminal amino acid sequences of newly purified buffalo pags, the interpretation was limited by the relatively low number of residues clearly identified. three of them were identical to wbpag62kda, previously isolated from mid-pregnancy buffalo placenta. five other sequences were 100% identical to proteins previously characterised in buffalo, ovine and bison placentas. our work describes for the first time the production of polyclonal antisera raised against pag molecules isolated from buffalo placenta. proteins issued from three different fractions were used to immunise rabbits resulting in the production of three distinct antibodies: as# as# and as# respectively. these three obtained antisera gave very similar displacement of standard inhibition curves. this finding was not surprising because pag molecules expressed in the same species can exhibit high sequence identities, and thus probably share common epitopes. the highest dilution of primary antiserum was obtained with as# the remarkable parallelism between serial dilutions of pregnant buffalo samples and the standard curve indicates that ria- is a good immunoassay allowing distinguishing subtle quantitative differences in wbpag concentrations. ria- also proved to be very repeatable for measurement of pag concentrations. finally, it was observed that mdl of ria- was equivalent to that previously described for ria-, ria- and ria-pool. the use of pag/pspb ria systems are considered as reliable method for early pregnancy diagnosis and follow-up of trophoblastic function in ruminant species. measurements of pag/pspb concentrations in the peripheral circulation of pregnant and non-pregnant buffalo cows have been performed using different ria methods. recently, barbato et al. compared ria- ria- and ria- for detecting pag molecules in pregnant buffalo females. they reported that analogous pag antigens were better recognised using ria- from week of gestation onwards. by using the same pag-ria system, karen et al. described that pregnancy diagnosis reached 100% between days and an additional feature of the present study was the observation of a rapid increase in pag concentrations from day to day of pregnancy. interestingly, a rapid increase in pag concentration during pregnancy is characteristic of ovine and caprine species. however, it differs largely from those described in bovine species, in which pag concentrations increase slowly during the first trimester of gestation. in egyptian buffalo cows, karen et al. and el-battawy et al. described mean pag concentrations ranging from to ng/ml at around day of pregnancy. those values were higher than mean concentrations measured by ria- at day in italian mediterranean females. egyptian and mediterranean buffalo cows are quite different in several aspects. mediterranean females have a much higher milk yield than egyptian females. this observation of low pag levels in high producing milk females agrees with previous findings described by lopez-gatius et al. who reported that there is a strong negative correlation between milk production and pag concentrations during the first trimester of pregnancy. differences in pag concentrations could also be partially due to other factors such as breed, placental mass, age of female or even farm. moreover, as previously described in bovine and ovine species, pag expression is complex throughout pregnancy. some pag molecules are expressed early, while others only as pregnancy progresses. other pag are also expressed throughout the whole pregnancy period. pag concentrations were detectable in peripheral plasma of all italian mediterranean pregnant buffalo cows at day at day out of pregnant females were already positive by ria- in dairy and beef cows, pag concentrations can be detected as early as at day after ai. however, pregnancy diagnosis is only recommended from day to because of important individual variation on pag appearance in peripheral blood. according to different authors, expression of pag family members as early as day after fertilisation suggests their potential role in cellular growth and differentiation, elongation, apposition, attachment and placentogenesis processes. on the other hand, in the present study, pag concentrations were higher than ng/ml in one non-pregnant female at days and recently, karen et al. described pregnancy diagnosis in egyptian buffalo cows by using ria-. the authors also reported 3/ and 2/ incorrect pregnancy diagnoses between days 31– and 36– respectively. the presence of detectable concentrations in non-pregnant females can be explained by the existence of extra-placental sources of pag, as suggested by zoli et al.. indeed, the presence of antigens immunologically related to pag has been demonstrated in testicular and in ovarian extracts, justifying, also, the adjective “associated” and not “specific” given to this family of placental glycoproteins.  we have described for the first time the use of antisera raised against buffalo pag for ria development and pregnancy detection in buffalo cows. moreover, we showed that ria- was quantitative, precise, accurate and sensitive in measuring pag concentrations. 
in addition, the ria- system was shown to be sensitive, linear, reproducible, accurate and specific in measuring pag concentrations in buffalo plasma samples from day of gestation onwards. three polyclonal antisera were raised in rabbits against distinct wbpag fractions. a specific radioimmunoassay was developed for early pregnancy diagnosis in buffalo species. our results confirmed the multiplicity of pag expression in buffalo placenta. here, we describe the purification of several pregnancy-associated glycoproteins from water buffalo placenta.
 pregnancy-associated glycoproteins 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. a specific radioimmunoassay was developed for early pregnancy diagnosis in buffalo species.  amino-terminal microsequencing of immunoreactive placental proteins allowed the identification of eleven wbpags sequences. three polyclonal antisera were raised in rabbits against distinct wbpag fractions. a new ria was developed and used to distinguish between pregnant and non-pregnant water buffalo females.  our results confirmed the multiplicity of pag expression in buffalo placenta. in addition, the ria- system was shown to be sensitive, linear, reproducible, accurate and specific in measuring pag concentrations in buffalo plasma samples from day of gestation onwards. water buffaloplacentapregnancy-associated glycoproteinpurificationradioimmunoassaypolyclonal antiserumpregnancy diagnosisn-terminal amino acid sequence 