BACKGROUND
the relationship between colostrum intake and newborn ruminant survival has been extensively characterized  <cit> .
colostrum is the first source of nutrition in neonatal ruminants, supplying not only nutrients, but having also a fundamental biological function, promoting immunoglobulin  transfer from the dam to the newborn.
moreover, colostrum has been described as having a mixture of diverse components, such as fat, lactose, vitamins and minerals that have a high nutritional importance  <cit> .
however, beyond the nutritional function, colostrum contains a complex mixture of proteins that actively participate in the protection of the neonate against pathogens and other post-partum environmental challenges  <cit> .
to date, a wide variety of colostrum and milk bioactive peptides and proteins have not only been linked to the passive immune transfer, such as lactoferrin  <cit> , lactoperoxidase  <cit>  or lysozymes  <cit> , but also promoting gastrointestinal growth and development of the newborn, such as insulin-like growth factors  or the transforming growth factor beta   <cit> .
as a consequence, colostrum intake and colostrum protein absorption play an essential role in passive immune transfer and ultimately in newborn survival  <cit> .
however, the absorbance conditions for intact proteins decrease during the first 48 h after birth, therefore colostrum feeding must take place during this period  <cit> .
it has been described how feeding newborn ruminants with colostrum, with a concentration of igs that is insufficient results in high mortality rates and low productive performances, with negative consequences for the economic benefits of farmers and breeders and severe consequences to animal welfare  <cit> .
proteomics has been used to characterize protein changes in the transition from colostrum to milk in cattle  <cit> .
additionally, the study of low abundance proteins from different body fluids such as blood plasma, colostrum or milk is becoming increasingly relevant  <cit> .
despite the previous proteomic studies in colostrum and milk, it is still not fully known which proteins are absorbed or increased in lamb blood plasma as a result of colostrum intake.
it is hypothesized that early colostrum intake modifies the proteome of newborn lamb blood plasma.
for this reason, the aim of this study was to analyze blood plasma low abundance proteins from colostrum-fed lambs compared to colostrum-deprived lambs, during the first  <dig> h after birth, in order to identify these plasma protein changes.
results from this study will contribute to understand the importance of colostrum on passive immune transfer and the lamb immune system development.
RESULTS
in this experiment we have used an approach based on the analysis of the proteome of low abundant proteins in plasma from newborn lambs using the proteominer®  technology that allows the removal of the higher-abundance proteins in the plasma, particularly albumin, igg and igm, followed by a 2-de dige analysis and protein identification using mass spectrometry.
with reference to this topic, many methodologies can be found in the market, however several authors have observed excellent results using proteominer® not only in the removal of high abundance proteins, but also in the high concentration and intensity of low abundance proteins  <cit> .
during this study, no body weight differences were observed between groups .
moreover, no evidences of illness were detected during health status monitoring.
nd means no detectable; bw means body weight; igg means immunoglobulin g; igm means immunoglobulin.
we evaluated the levels of igg and igm in non-proteominer® treated plasma samples in order to determine the presence or absence of colostrum proteins in both groups  at the two studied times .
results are shown also in table  <dig> where the concentration of the two igs in the two studied groups  and at  <dig> and 14 h after birth is presented.
at birth  animals from both groups had no detectable  igg concentration in blood.
however, when both groups were compared at 14 h after birth, igg concentration could be detected only in c group .
a similar pattern was observed when igm concentrations were analyzed, with no detection at 2 h after birth in any of the experimental groups and being detected in c group, but not in the dc group, at 14 h after birth.
several authors have observed a similar evolution in igs level, depending on the total amount of igs present in colostrum, in newborn blood from lambs  <cit> , calves  <cit>  and goat kids  <cit> .
as expected, these results confirm that the presence of colostrum igg and igm in the c group in blood at 14 h after birth is due to colostrum intake.
as shown in figure  <dig>  a total of  <dig> spots showing over-expression in lambs at 14 h after birth were detected in the c group.
these spot relative intensities were similar between groups  at 2 h after birth and did not increase in the dc group at 14 h .
of these  <dig> spots, we were able to identify a total of  <dig> spots, as presented in table  <dig>  the spots were identified as apolipoprotein a-iv , plasminogen , serum amyloid a  and fibrinogen gamma chain .
these proteins may play an important role either in the immune-system development or in the immune protection or even both at the early stages of life and will subsequently be described separately.
c 2 h and dc 2 h means colostrum group and delayed colostrum group at 2 h after birth, respectively; c 14 h and dc 14 h means colostrum group and delayed colostrum group at 14 h after birth.
a
b
c
anumber of peptides, matching the identified protein, whose sequence differs in at least one amino acid residue; bpercentage of the identified protein sequence covered by the matched peptides; cidentification score obtained with the mowse algorithm.
a result is considered to be significant when a score above  <dig> is attained.
apolipoprotein a-iv
the metabolic function of apolipoprotein a-iv  has not been fully established, however it has been suggested that apo a-iv plays an important role at early life, modulating the enterocyte lipid transport efficiency in fatty foods, namely colostrum  <cit> .
for this reason, the intestinal synthesis and secretion of apo a-iv increases during fat absorption  <cit> .
additionally, apo a-iv has antioxidant properties, acts as a postprandial satiety signal, and reduces gastric acid secretion  <cit> .
an increase in the expression of this protein in colostrum may play a role in the protection of the immunoglobulin molecule structure, reducing the gastric acid secretion in the stomach of the newborn lamb and increasing the total amount of intact igs absorbed in the intestine.
finally, apo a-iv has been described as having an immunomodulatory effect against external agents  in mice  <cit> .
consequently, an increase in the blood plasma levels of apo a-iv could also contribute to protect the newborn from infections at this early stage of life.
plasminogen
this glycoprotein is the precursor of plasmin, a fibrinolytic enzyme that plays an important role in the dissolution of fibrin blood clots in order to prevent thrombosis  <cit> .
nevertheless, this protein has been identified not only in blood, but also in colostrum and milk  <cit> .
in bovine, sheep and goat milk, plasmin and plasminogen forms are identical to those found in blood  <cit> .
in addition to its main role in the dissolution of fibrin blood clots, plasminogen is structurally similar to apo a-iv  <cit> .
apo a-iv, as with other apo a proteins, has the capacity to bind fibrin and proteins of endothelial cells and monocytes, and therefore may inhibit plasminogen binding and plasmin generation  <cit> .
the presence of this protein in colostrum and the increase of this protein in blood plasma could thwart the apo a-iv to bind fibrin in newborn lambs.
additionally, plasminogen has immune activity, as it contributes to neutrophil migration to an infection site  <cit> .
an increase of this protein expression in plasma promotes the neutrophil migration in blood and therefore may contribute to the immune response against potential infections in the newborn lamb.
in agreement with these findings, theodorou et al.
<cit>  found an increase of plasminogen concentration in blood and milk during acute mastitis in lactating dairy ewes.
therefore the colostrum intake seems to be an important factor that increases plasminogen concentration in blood at this stage of life.
plasminogen participates also in the regulation of cellular apoptosis  <cit> , specifically the apoptosis of adherent cells induced by disruption of integrin-mediated cell-matrix interactions.
this has been described under specific physiological conditions, such as the involution of the mammary gland after lactation and the renewal of intestinal epithelial cells.
the apoptotic processes in the latter tissue is of important relevance in the absorption of igs by newborn ruminants during the first hours after birth, as described by castro-alonso et al.
<cit> .
therefore, the increase of this protein in blood may delay the decrease of the apoptosis rate of intestinal epithelial cells during the early stage of life, increasing the available time for colostrum components absorption, including both igs and other proteins.
serum amyloid a
serum amyloid a  is normally found in different isoforms and complexes with lipoproteins, while its plasma concentration vary depending on the species  <cit> .
it is an apolipoprotein that takes part in the acute phase of inflammation  <cit>  and represents one of the most conserved proteins among mammals supporting the premise that it has a basic and essential role in the innate immune system.
however, this protein has also been identified in colostrum of several species, such as human  <cit> , horse, cattle and sheep  <cit> .
the importance of it in inflammatory processes has been fully monitored, showing that the circulating concentration of saa protein is increased by 1000-fold within  <dig> to 48 h after infection/inflammation from a basal level of  <dig>  ±  <dig>  μg/ml  <cit> .
the saa protein has numerous pro-inflammatory actions: it works as a chemoattractant to neutrophils, monocytes, and t lymphocytes, causing leukocyte infiltration and promoting neutrophil adhesion to endothelial cells  <cit> , stimulating neutrophils and monocytes to release not only cytokines  <cit> , but also matrix metalloproteinases  <cit> .
according to he et al.
<cit> , these findings suggest a key function for saa not only in the establishment, but also in the maintenance of inflammation, meaning that newborn lambs fed with colostrum at this early stage of life reportedly have a clear advantage, increasing this protein level in blood, and consequently producing a more efficient immune status.
fibrinogen gamma chain
the fibrinogen gamma chain  is one of the three components of fibrinogen which is the precursor of fibrin, the most abundant component in blood clots.
however, this protein also has a defensive function, having been demonstrated that fibrinogen concentration increases during acute-phase reactions  <cit> .
moreover, yamada et al.
<cit>  studied differences in low abundance proteins between bovine colostrum and milk, showing that some of them were only present in colostrum, such as fibrinogen, which could explain the plasma increase of this protein in animals that were fed with it.
additionally, several authors have described that fibrinogen can bind to integrins  <cit> , that are normally expressed on cells of the immune system, such as cd11b+/cd18+ monocytes.
the cd11b+/cd18+ integrin receptor  is a member of the β <dig> integrin family, which is in turn expressed on monocytes and macrophages.
when fibrinogen binds to cd11b+/cd18+, integrin causes an extensive array of cell signaling responses, namely the activation of the nuclear factor kappa-light-chain-enhancer of activated b cells  and mitogen-activated protein kinase /phosphatidylinositol 3-kinase .
this data indicates that fibrinogen can function not only as a substrate in the clotting cascade, but also as an important effector during the evolution of the innate immune response  <cit> .
therefore, intake of colostrum and the subsequent increase of fibrinogen in lamb blood plasma may benefit the newborn immune system efficiency.
CONCLUSIONS
in conclusion, early colostrum intake produced an increase of non-immunoglobulin proteins in lamb blood plasma, such as apoliprotein a-iv, plasminogen, serum amyloid a and fibrinogen.
these proteins have reported immune functions in other species, suggesting that colostrum provides not only igs, but also non-immunoglobulin proteins.
these proteins play a fundamental role in the activation and attraction of immune cells, the apoptosis rate of the enterocytes and the low gastric secretion, among other roles.
the results of this work contribute information about proteins with immune function that are increased after colostrum intake.
high plasma concentrations of these proteins may decrease lamb mortality and increase the economic benefit for farmers.
in the future, further proteomic studies will be necessary in order to increase the general knowledge about the role of colostrum in the passive immune transfer.
such studies could consist in the fully qualitative characterization of proteins present in colostrum, as well as samples from c and dc group at  <dig>   <dig> and 26 h. moreover, a quantitative study could be performed on samples from both groups  at  <dig>   <dig> and 26 h in order to detect if lambs, receiving a delayed colostrum meal  are able to reach a similar protein concentration than lambs fed with colostrum after birth  at 26 h after birth.

 the relationship between colostrum intake and newborn ruminant survival has been extensively characterized. colostrum is the first source of nutrition in neonatal ruminants, supplying not only nutrients, but having also a fundamental biological function, promoting immunoglobulin transfer from the dam to the newborn. moreover, colostrum has been described as having a mixture of diverse components, such as fat, lactose, vitamins and minerals that have a high nutritional importance. however, beyond the nutritional function, colostrum contains a complex mixture of proteins that actively participate in the protection of the neonate against pathogens and other post-partum environmental challenges. to date, a wide variety of colostrum and milk bioactive peptides and proteins have not only been linked to the passive immune transfer, such as lactoferrin, lactoperoxidase or lysozymes, but also promoting gastrointestinal growth and development of the newborn, such as insulin-like growth factors or the transforming growth factor beta. as a consequence, colostrum intake and colostrum protein absorption play an essential role in passive immune transfer and ultimately in newborn survival. however, the absorbance conditions for intact proteins decrease during the first 48 h after birth, therefore colostrum feeding must take place during this period. it has been described how feeding newborn ruminants with colostrum, with a concentration of igs that is insufficient results in high mortality rates and low productive performances, with negative consequences for the economic benefits of farmers and breeders and severe consequences to animal welfare. proteomics has been used to characterize protein changes in the transition from colostrum to milk in cattle. additionally, the study of low abundance proteins from different body fluids such as blood plasma, colostrum or milk is becoming increasingly relevant. despite the previous proteomic studies in colostrum and milk, it is still not fully known which proteins are absorbed or increased in lamb blood plasma as a result of colostrum intake. it is hypothesized that early colostrum intake modifies the proteome of newborn lamb blood plasma. for this reason, the aim of this study was to analyze blood plasma low abundance proteins from colostrum-fed lambs compared to colostrum-deprived lambs, during the first h after birth, in order to identify these plasma protein changes. results from this study will contribute to understand the importance of colostrum on passive immune transfer and the lamb immune system development.  in this experiment we have used an approach based on the analysis of the proteome of low abundant proteins in plasma from newborn lambs using the proteominer® technology that allows the removal of the higher-abundance proteins in the plasma, particularly albumin, igg and igm, followed by a 2-de dige analysis and protein identification using mass spectrometry. with reference to this topic, many methodologies can be found in the market, however several authors have observed excellent results using proteominer® not only in the removal of high abundance proteins, but also in the high concentration and intensity of low abundance proteins. during this study, no body weight differences were observed between groups. moreover, no evidences of illness were detected during health status monitoring. nd means no detectable; bw means body weight; igg means immunoglobulin g; igm means immunoglobulin. we evaluated the levels of igg and igm in non-proteominer® treated plasma samples in order to determine the presence or absence of colostrum proteins in both groups at the two studied times. results are shown also in table  where the concentration of the two igs in the two studied groups and at and 14 h after birth is presented. at birth animals from both groups had no detectable igg concentration in blood. however, when both groups were compared at 14 h after birth, igg concentration could be detected only in c group. a similar pattern was observed when igm concentrations were analyzed, with no detection at 2 h after birth in any of the experimental groups and being detected in c group, but not in the dc group, at 14 h after birth. several authors have observed a similar evolution in igs level, depending on the total amount of igs present in colostrum, in newborn blood from lambs, calves and goat kids. as expected, these results confirm that the presence of colostrum igg and igm in the c group in blood at 14 h after birth is due to colostrum intake. as shown in figure  a total of spots showing over-expression in lambs at 14 h after birth were detected in the c group. these spot relative intensities were similar between groups at 2 h after birth and did not increase in the dc group at 14 h. of these spots, we were able to identify a total of spots, as presented in table  the spots were identified as apolipoprotein a-iv, plasminogen, serum amyloid a and fibrinogen gamma chain. these proteins may play an important role either in the immune-system development or in the immune protection or even both at the early stages of life and will subsequently be described separately. c 2 h and dc 2 h means colostrum group and delayed colostrum group at 2 h after birth, respectively; c 14 h and dc 14 h means colostrum group and delayed colostrum group at 14 h after birth. a b c anumber of peptides, matching the identified protein, whose sequence differs in at least one amino acid residue; bpercentage of the identified protein sequence covered by the matched peptides; cidentification score obtained with the mowse algorithm. a result is considered to be significant when a score above is attained. apolipoprotein a-iv the metabolic function of apolipoprotein a-iv has not been fully established, however it has been suggested that apo a-iv plays an important role at early life, modulating the enterocyte lipid transport efficiency in fatty foods, namely colostrum. for this reason, the intestinal synthesis and secretion of apo a-iv increases during fat absorption. additionally, apo a-iv has antioxidant properties, acts as a postprandial satiety signal, and reduces gastric acid secretion. an increase in the expression of this protein in colostrum may play a role in the protection of the immunoglobulin molecule structure, reducing the gastric acid secretion in the stomach of the newborn lamb and increasing the total amount of intact igs absorbed in the intestine. finally, apo a-iv has been described as having an immunomodulatory effect against external agents in mice. consequently, an increase in the blood plasma levels of apo a-iv could also contribute to protect the newborn from infections at this early stage of life. plasminogen this glycoprotein is the precursor of plasmin, a fibrinolytic enzyme that plays an important role in the dissolution of fibrin blood clots in order to prevent thrombosis. nevertheless, this protein has been identified not only in blood, but also in colostrum and milk. in bovine, sheep and goat milk, plasmin and plasminogen forms are identical to those found in blood. in addition to its main role in the dissolution of fibrin blood clots, plasminogen is structurally similar to apo a-iv. apo a-iv, as with other apo a proteins, has the capacity to bind fibrin and proteins of endothelial cells and monocytes, and therefore may inhibit plasminogen binding and plasmin generation. the presence of this protein in colostrum and the increase of this protein in blood plasma could thwart the apo a-iv to bind fibrin in newborn lambs. additionally, plasminogen has immune activity, as it contributes to neutrophil migration to an infection site. an increase of this protein expression in plasma promotes the neutrophil migration in blood and therefore may contribute to the immune response against potential infections in the newborn lamb. in agreement with these findings, theodorou et al. found an increase of plasminogen concentration in blood and milk during acute mastitis in lactating dairy ewes. therefore the colostrum intake seems to be an important factor that increases plasminogen concentration in blood at this stage of life. plasminogen participates also in the regulation of cellular apoptosis, specifically the apoptosis of adherent cells induced by disruption of integrin-mediated cell-matrix interactions. this has been described under specific physiological conditions, such as the involution of the mammary gland after lactation and the renewal of intestinal epithelial cells. the apoptotic processes in the latter tissue is of important relevance in the absorption of igs by newborn ruminants during the first hours after birth, as described by castro-alonso et al.. therefore, the increase of this protein in blood may delay the decrease of the apoptosis rate of intestinal epithelial cells during the early stage of life, increasing the available time for colostrum components absorption, including both igs and other proteins. serum amyloid a serum amyloid a is normally found in different isoforms and complexes with lipoproteins, while its plasma concentration vary depending on the species. it is an apolipoprotein that takes part in the acute phase of inflammation and represents one of the most conserved proteins among mammals supporting the premise that it has a basic and essential role in the innate immune system. however, this protein has also been identified in colostrum of several species, such as human, horse, cattle and sheep. the importance of it in inflammatory processes has been fully monitored, showing that the circulating concentration of saa protein is increased by 1000-fold within to 48 h after infection/inflammation from a basal level of  ±   μg/ml. the saa protein has numerous pro-inflammatory actions: it works as a chemoattractant to neutrophils, monocytes, and t lymphocytes, causing leukocyte infiltration and promoting neutrophil adhesion to endothelial cells, stimulating neutrophils and monocytes to release not only cytokines, but also matrix metalloproteinases. according to he et al., these findings suggest a key function for saa not only in the establishment, but also in the maintenance of inflammation, meaning that newborn lambs fed with colostrum at this early stage of life reportedly have a clear advantage, increasing this protein level in blood, and consequently producing a more efficient immune status. fibrinogen gamma chain the fibrinogen gamma chain is one of the three components of fibrinogen which is the precursor of fibrin, the most abundant component in blood clots. however, this protein also has a defensive function, having been demonstrated that fibrinogen concentration increases during acute-phase reactions. moreover, yamada et al. studied differences in low abundance proteins between bovine colostrum and milk, showing that some of them were only present in colostrum, such as fibrinogen, which could explain the plasma increase of this protein in animals that were fed with it. additionally, several authors have described that fibrinogen can bind to integrins, that are normally expressed on cells of the immune system, such as cd11b+/cd18+ monocytes. the cd11b+/cd18+ integrin receptor is a member of the β integrin family, which is in turn expressed on monocytes and macrophages. when fibrinogen binds to cd11b+/cd18+, integrin causes an extensive array of cell signaling responses, namely the activation of the nuclear factor kappa-light-chain-enhancer of activated b cells and mitogen-activated protein kinase /phosphatidylinositol 3-kinase. this data indicates that fibrinogen can function not only as a substrate in the clotting cascade, but also as an important effector during the evolution of the innate immune response. therefore, intake of colostrum and the subsequent increase of fibrinogen in lamb blood plasma may benefit the newborn immune system efficiency.  in conclusion, early colostrum intake produced an increase of non-immunoglobulin proteins in lamb blood plasma, such as apoliprotein a-iv, plasminogen, serum amyloid a and fibrinogen. these proteins have reported immune functions in other species, suggesting that colostrum provides not only igs, but also non-immunoglobulin proteins. these proteins play a fundamental role in the activation and attraction of immune cells, the apoptosis rate of the enterocytes and the low gastric secretion, among other roles. the results of this work contribute information about proteins with immune function that are increased after colostrum intake. high plasma concentrations of these proteins may decrease lamb mortality and increase the economic benefit for farmers. in the future, further proteomic studies will be necessary in order to increase the general knowledge about the role of colostrum in the passive immune transfer. such studies could consist in the fully qualitative characterization of proteins present in colostrum, as well as samples from c and dc group at and 26 h. moreover, a quantitative study could be performed on samples from both groups at and 26 h in order to detect if lambs, receiving a delayed colostrum meal are able to reach a similar protein concentration than lambs fed with colostrum after birth at 26 h after birth. 
in conclusion, colostrum intake modified the low abundance proteome profile of blood plasma from newborn lambs, increasing the concentration of apolipoprotein a-iv, plasminogen, serum amyloid a and fibrinogen, demonstrating that colostrum is essential, not only for the provision of immunoglobulins, but also because of increases in several low abundance proteins with immune function. these proteins have not been previously described as increased in blood plasma of newborn ruminants by colostrum intake. colostrum intake by newborn lambs plays a fundamental role in the perinatal period, ensuring lamb survival. in this study, blood plasma samples from two groups of newborn lambs at and 14 h after birth were treated to reduce the content of high abundance proteins and analyzed using two-dimensional differential in gel electrophoresis and maldi ms/ms for protein identification in order to investigate low abundance proteins with immune function in newborn lambs. the results showed that four proteins were increased in the blood plasma of lambs due to colostrum intake. moreover, these proteins have been described as having an immune function in other species, some of which were previously identified in colostrum and milk.
 colostrum intake by newborn lambs plays a fundamental role in the perinatal period, ensuring lamb survival. in this study, blood plasma samples from two groups of newborn lambs at and 14 h after birth were treated to reduce the content of high abundance proteins and analyzed using two-dimensional differential in gel electrophoresis and maldi ms/ms for protein identification in order to investigate low abundance proteins with immune function in newborn lambs.  the results showed that four proteins were increased in the blood plasma of lambs due to colostrum intake. these proteins have not been previously described as increased in blood plasma of newborn ruminants by colostrum intake. moreover, these proteins have been described as having an immune function in other species, some of which were previously identified in colostrum and milk.  in conclusion, colostrum intake modified the low abundance proteome profile of blood plasma from newborn lambs, increasing the concentration of apolipoprotein a-iv, plasminogen, serum amyloid a and fibrinogen, demonstrating that colostrum is essential, not only for the provision of immunoglobulins, but also because of increases in several low abundance proteins with immune function. 