BACKGROUND
the degree of endogamy or consanguinity in the offspring increases proportionally to the genetic similarity of its ancestors, in contrast to individuals that are randomly selected from a given population.
the endogamy coefficient of an individual indicates the probability that a pair of randomly selected genes of the same locus is completely identical.
therefore, endogamic individuals can only result from mating between closely related ancestors  <cit> .
the study of endogamy in cattle is extremely important, particularly due to the widespread use of artificial insemination, which allows a high selection pressure and rapid genetic gain, resulting in thousands of descendants from one single sire.
despite the unquestionable genetic gain and consequent improvement in productive traits, extensive use of a few bulls may lead to reduced genetic variability and potential deleterious effects caused by endogamy  <cit> .
endogamy decreases the frequency of heterozygotes in the population, increasing the risk of deleterious effects of recessive genes, which may negatively influence the average productive performance in the population, a phenomenon known as endogamy depression  <cit> .
therefore, the primary effect of endogamy is an increase the frequency of homozygous genes, favoring manifestation of several recessive genes, which usually cause impairment on the average individual phenotypic merit.
possible causes for declining of phenotypic value as a consequence of endogamy includes the fact that favorable genes tend to be dominant or partially dominant  <cit> .
deleterious effects of endogamy on the reproductive ability of cows and heifers have been reported  <cit> .
however, whether endogamy influences the immune system remains to be investigated.
importantly, some studies report an association between low genetic diversity and increased susceptibility to diseases  <cit> .
evaluation of mitocondrial dna allows separation of american zebu cattle, according to its maternal lineage ancestry, into two groups: one with only bos taurus indicus mtdna  and other with bos taurus taurus mtdna   <cit> .
studies to evaluate the effect of mitochondrial dna on productive and reproductive traits in gir and guzerat have been conducted and show that the mitochondrial origin  significantly affects the age at first calving  <cit>  and it does not seem to have significant effects on lactation milk yield, days in milk, or calving interval  <cit> .
considering the scarcity of studies regarding the effect of endogamy and mtdna on immunological parameters, the goal of this study was to evaluate the relationship association between endogamy and the immune system.
our focus was to understand variations of immune parameters, including cd4+ t cells, cd8+ t cells, γδ t cells, and b cells  in peripheral blood, that may be associated to endogamy in guzerat  cattle.
RESULTS
immunological parameters are influenced by endogamy
multiple regression models including milk yield, age, mtdna, and endogamy coefficient for cows, and mtdna and endogamy coefficient for calves in relation to levels of lymphocyte subsets in the peripheral blood were performed and the results are summarized in tables  <dig> and  <dig>  the endogamy coefficient was significantly associated with increased cd21+ levels .
in calves, multiple regression analysis indicated that endogamy was significantly associated with increased cd8+ t and cd21+ cells, and with significantly decreased γδhigh t cells and t/b cell ratio .
importantly, the age of cows was significantly associated with increased cd4+ t cells, cd4+/cd8+ and t/b cell ratio, and with significantly decreased cd21+ and γδhigh t cells .
<dig>
cd4
+
cd21
+
γδ
high
γδ
low
cd4
+
/cd8
+
*significant differences at p <  <dig>  are highlighted in italic format.
cd8+, and γδ t lymphocytes did show any significant correlation.
<dig>
*significant differences at p <  <dig>  are highlighted in italic format.
cd4+, γδ , γδlow t lymphocytes and cd4+/cd8+ cell ratio did not show any significant correlation.
regression analysis was also performed to verify possible associations between the endogamy coefficient and immunological parameters.
in cows, there were no statistically significant correlations.
however, in calves there was a positive correlation between endogamy and cd8+ t lymphocytes  and cd21+ b cells , and a negative correlation with γδhigh t cells  and t/b cell ratio .
endogamy alters the immune profile of guzerat cattle
based on endogamy coefficients, cows and calves were grouped into high, medium, and low endogamy as described in methods.
the immune profiles of these cattle were then analyzed by flow cytometry .
these results indicated that cows with medium and low endogamy had a decreased percentage of b lymphocytes when compared to cows with high endogamy .
furthermore, cows with lower endogamy had a higher percentage of γδlow t cells when compared with cows with medium endogamy .
there was also a clear influence of endogamy on the immunological profile of calves.
calves with high endogamy had an increased percentage of cd8+ t lymphocytes when compared to calves with medium and low endogamy .
in contrast, calves with low endogamy had an increased percentage of γδt cells  and γδhigh t cells  as well as the cd4+/cd8+ t cell ratio  and t/b ratio  when compared to calves with high endogamy .
calves with low endogamy had a decreased percentage of γδlow t cells only in comparison to those with medium endogamy .
european mtdna in guzerat is associated with low endogamy and differences in immunological parameters
all cattle used in this study were mtdna genotyped.
this analysis allowed us to assess whether individual zebu cattle  remained with no introduction of maternal dna of b. taurus taurus origin, thus tending to have higher levels of endogamy  <cit> .
our results demonstrated that 42%  of the cows had mtdna from b. taurus taurus, and 58%  had only b. taurus indicus  mtdna.
the same result was observed in calves, which was expected since these calves were the offspring of those same cows mentioned above.
after grouping cows and calves according to the presence or absence of b. taurus taurus  mtdna, which were classified into “bt-mtdna” or “bi-mtdna”, respectively, the profile of leukocytes in peripheral blood was analyzed by flow cytometry.
in cows, the percentages of b lymphocytes, γδt cells, and γδhigh t cells were decreased in individuals with bi-mtdna when compared to individuals with bt-mtdna .
in calves, individuals with bi-mtdna had a decreased percentage of γδt cells and γδhigh t cells when compared to individuals with bt-mtdna .
as expected, the endogamy coefficient was lower in calves with bt-mtdnawhen compared to calves with bi-mtdna .
discussion
here we demonstrated a clear effect of endogamy on immune parameters of guzerat cattle.
this is the first evidence that endogamy may affect the immunological parameters in cattle.
the main consequences of endogamy increase are the reduction of genetic variability within inbred lines and increased frequency of homozygosity, which may be desirable for alleles with favorable effects.
however, a small fraction of undesirable deleterious recessive alleles may have an enhancement of their manifestation under these conditions.
therefore, endogamy tends to increase expression of undesirable recessive alleles  <cit> .
deleterious effects of endogamy have been demonstrated, especially on reproductive traits  <cit> .
a study of inbreed and outbreed populations of several different species of captive ungulates indicates that the rate of neonatal mortality is higher in endogamic when compared to non endogamic animals  <cit> .
in humans, consanguinity is an important risk factor for susceptibility to infectious diseases  <cit> .
however, direct associations of endogamy with the immune system have not been previously investigated in animals.
higher levels of endogamy clearly affected some of the immunological parameters, particularly γδ t cells.
these cells are the major circulating lymphocyte subset population in ruminants, especially in calves.
these cells play a role in the transition between innate and adaptive immune  <cit> .
γδ t lymphocytes can recognize and be activated by pathogen-associated molecular patterns  or danger-associated molecular patterns  in absence of other stimuli  <cit> , supporting the notion of an innate immune function of these cells.
however, other studies demonstrated an important role of γδ t lymphocytes in adaptive response since they recognize antigens presented on the surface of antigen-presenting cells , and they have cytotoxic effect mediated by expression of granulysin  <cit>  and perforin  <cit> .
these cells play an important role during infection with mycobacterium bovis, the causative agent of bovine tuberculosis  <cit> .
recent studies have also demonstrated the role of γδ t lymphocytes as apcs  <cit>  so they may play a role in developing protective immune responses following vaccination  <cit> , and are important for host defense during brucella abortus infection, when these cells enhance macrophage function via ifn-γ  <cit> .
although resistance or susceptibility to disease involves multifactorial mechanisms, it has been reported that zebu cattle tend to have a higher degree of natural resistance to diseases when compared to european cattle  <cit> .
analyzing the profile of peripheral blood leukocytes of different bovine breeds, we recently demonstrated that guzerat cattle comparatively has increased levels of circulating leukocytes, especially monocytes, eosinophils, t lymphocytes  and non-b/non-t  lymphocytes, with a decreased percentage of b lymphocytes, and consequent increase in the t/b and cd4+/cd8+ ratio  <cit> .
in another study, we compared the macrophage function of zebu cattle with that of macrophages from european cattle.
macrophages from zebu are more efficient in controlling intracellular replication of brucella abortus.
furthermore, zebu macrophages generate higher levels of nitric oxide and cytokines such as il- <dig> and tnf-α  <cit> .
therefore, increasing lines of evidences support the notion that resistance of zebu cattle may be due to quantitative and qualitative differences in immune cells.
in this study, we addressed for the first time whether endogamy have any influence on the profile of immune cells in the peripheral blood of zebu cattle.
here we demonstrated that age affected most of the immunological parameters of cows.
the influence of age on the immune system has already been demonstrated in both animals and humans, affecting both innate and adaptive immune systems  <cit> .
age-related decrease in b cell activity, potentially related to the decline in helper t cell activity, and decreased cytolytic and delayed-type hypersensitivity responses have been described  <cit> .
the age-related alteration in helper t cell activity appears to be due to intrinsic or functional changes within the cells, as well as to a shift in subset proportions of cd4+ and cd8+ t cell populations, with increases in the memory phenotype and corresponding decreases in the naive phenotype  <cit> .
CONCLUSIONS
in conclusion, our data indicate that endogamy influences the immune system of cattle since it is associated with significant changes in the profile of immune cells in the peripheral blood, particularly decreased levels of γδ t lymphocytes, an important subpopulation of lymphocytes in cattle, especially in calves.

 the degree of endogamy or consanguinity in the offspring increases proportionally to the genetic similarity of its ancestors, in contrast to individuals that are randomly selected from a given population. the endogamy coefficient of an individual indicates the probability that a pair of randomly selected genes of the same locus is completely identical. therefore, endogamic individuals can only result from mating between closely related ancestors. the study of endogamy in cattle is extremely important, particularly due to the widespread use of artificial insemination, which allows a high selection pressure and rapid genetic gain, resulting in thousands of descendants from one single sire. despite the unquestionable genetic gain and consequent improvement in productive traits, extensive use of a few bulls may lead to reduced genetic variability and potential deleterious effects caused by endogamy. endogamy decreases the frequency of heterozygotes in the population, increasing the risk of deleterious effects of recessive genes, which may negatively influence the average productive performance in the population, a phenomenon known as endogamy depression. therefore, the primary effect of endogamy is an increase the frequency of homozygous genes, favoring manifestation of several recessive genes, which usually cause impairment on the average individual phenotypic merit. possible causes for declining of phenotypic value as a consequence of endogamy includes the fact that favorable genes tend to be dominant or partially dominant. deleterious effects of endogamy on the reproductive ability of cows and heifers have been reported. however, whether endogamy influences the immune system remains to be investigated. importantly, some studies report an association between low genetic diversity and increased susceptibility to diseases. evaluation of mitocondrial dna allows separation of american zebu cattle, according to its maternal lineage ancestry, into two groups: one with only bos taurus indicus mtdna and other with bos taurus taurus mtdna. studies to evaluate the effect of mitochondrial dna on productive and reproductive traits in gir and guzerat have been conducted and show that the mitochondrial origin significantly affects the age at first calving and it does not seem to have significant effects on lactation milk yield, days in milk, or calving interval. considering the scarcity of studies regarding the effect of endogamy and mtdna on immunological parameters, the goal of this study was to evaluate the relationship association between endogamy and the immune system. our focus was to understand variations of immune parameters, including cd4+ t cells, cd8+ t cells, γδ t cells, and b cells in peripheral blood, that may be associated to endogamy in guzerat cattle.  immunological parameters are influenced by endogamy multiple regression models including milk yield, age, mtdna, and endogamy coefficient for cows, and mtdna and endogamy coefficient for calves in relation to levels of lymphocyte subsets in the peripheral blood were performed and the results are summarized in tables  and the endogamy coefficient was significantly associated with increased cd21+ levels. in calves, multiple regression analysis indicated that endogamy was significantly associated with increased cd8+ t and cd21+ cells, and with significantly decreased γδhigh t cells and t/b cell ratio. importantly, the age of cows was significantly associated with increased cd4+ t cells, cd4+/cd8+ and t/b cell ratio, and with significantly decreased cd21+ and γδhigh t cells. cd4 + cd21 + γδ high γδ low cd4 + /cd8 + *significant differences at p <  are highlighted in italic format. cd8+, and γδ t lymphocytes did show any significant correlation. *significant differences at p <  are highlighted in italic format. cd4+, γδ, γδlow t lymphocytes and cd4+/cd8+ cell ratio did not show any significant correlation. regression analysis was also performed to verify possible associations between the endogamy coefficient and immunological parameters. in cows, there were no statistically significant correlations. however, in calves there was a positive correlation between endogamy and cd8+ t lymphocytes and cd21+ b cells, and a negative correlation with γδhigh t cells and t/b cell ratio. endogamy alters the immune profile of guzerat cattle based on endogamy coefficients, cows and calves were grouped into high, medium, and low endogamy as described in methods. the immune profiles of these cattle were then analyzed by flow cytometry. these results indicated that cows with medium and low endogamy had a decreased percentage of b lymphocytes when compared to cows with high endogamy. furthermore, cows with lower endogamy had a higher percentage of γδlow t cells when compared with cows with medium endogamy. there was also a clear influence of endogamy on the immunological profile of calves. calves with high endogamy had an increased percentage of cd8+ t lymphocytes when compared to calves with medium and low endogamy. in contrast, calves with low endogamy had an increased percentage of γδt cells and γδhigh t cells as well as the cd4+/cd8+ t cell ratio and t/b ratio when compared to calves with high endogamy. calves with low endogamy had a decreased percentage of γδlow t cells only in comparison to those with medium endogamy. european mtdna in guzerat is associated with low endogamy and differences in immunological parameters all cattle used in this study were mtdna genotyped. this analysis allowed us to assess whether individual zebu cattle remained with no introduction of maternal dna of b. taurus taurus origin, thus tending to have higher levels of endogamy. our results demonstrated that 42% of the cows had mtdna from b. taurus taurus, and 58% had only b. taurus indicus mtdna. the same result was observed in calves, which was expected since these calves were the offspring of those same cows mentioned above. after grouping cows and calves according to the presence or absence of b. taurus taurus mtdna, which were classified into “bt-mtdna” or “bi-mtdna”, respectively, the profile of leukocytes in peripheral blood was analyzed by flow cytometry. in cows, the percentages of b lymphocytes, γδt cells, and γδhigh t cells were decreased in individuals with bi-mtdna when compared to individuals with bt-mtdna. in calves, individuals with bi-mtdna had a decreased percentage of γδt cells and γδhigh t cells when compared to individuals with bt-mtdna. as expected, the endogamy coefficient was lower in calves with bt-mtdnawhen compared to calves with bi-mtdna. discussion here we demonstrated a clear effect of endogamy on immune parameters of guzerat cattle. this is the first evidence that endogamy may affect the immunological parameters in cattle. the main consequences of endogamy increase are the reduction of genetic variability within inbred lines and increased frequency of homozygosity, which may be desirable for alleles with favorable effects. however, a small fraction of undesirable deleterious recessive alleles may have an enhancement of their manifestation under these conditions. therefore, endogamy tends to increase expression of undesirable recessive alleles. deleterious effects of endogamy have been demonstrated, especially on reproductive traits. a study of inbreed and outbreed populations of several different species of captive ungulates indicates that the rate of neonatal mortality is higher in endogamic when compared to non endogamic animals. in humans, consanguinity is an important risk factor for susceptibility to infectious diseases. however, direct associations of endogamy with the immune system have not been previously investigated in animals. higher levels of endogamy clearly affected some of the immunological parameters, particularly γδ t cells. these cells are the major circulating lymphocyte subset population in ruminants, especially in calves. these cells play a role in the transition between innate and adaptive immune. γδ t lymphocytes can recognize and be activated by pathogen-associated molecular patterns or danger-associated molecular patterns in absence of other stimuli, supporting the notion of an innate immune function of these cells. however, other studies demonstrated an important role of γδ t lymphocytes in adaptive response since they recognize antigens presented on the surface of antigen-presenting cells, and they have cytotoxic effect mediated by expression of granulysin and perforin. these cells play an important role during infection with mycobacterium bovis, the causative agent of bovine tuberculosis. recent studies have also demonstrated the role of γδ t lymphocytes as apcs so they may play a role in developing protective immune responses following vaccination, and are important for host defense during brucella abortus infection, when these cells enhance macrophage function via ifn-γ. although resistance or susceptibility to disease involves multifactorial mechanisms, it has been reported that zebu cattle tend to have a higher degree of natural resistance to diseases when compared to european cattle. analyzing the profile of peripheral blood leukocytes of different bovine breeds, we recently demonstrated that guzerat cattle comparatively has increased levels of circulating leukocytes, especially monocytes, eosinophils, t lymphocytes and non-b/non-t lymphocytes, with a decreased percentage of b lymphocytes, and consequent increase in the t/b and cd4+/cd8+ ratio. in another study, we compared the macrophage function of zebu cattle with that of macrophages from european cattle. macrophages from zebu are more efficient in controlling intracellular replication of brucella abortus. furthermore, zebu macrophages generate higher levels of nitric oxide and cytokines such as il- and tnf-α. therefore, increasing lines of evidences support the notion that resistance of zebu cattle may be due to quantitative and qualitative differences in immune cells. in this study, we addressed for the first time whether endogamy have any influence on the profile of immune cells in the peripheral blood of zebu cattle. here we demonstrated that age affected most of the immunological parameters of cows. the influence of age on the immune system has already been demonstrated in both animals and humans, affecting both innate and adaptive immune systems. age-related decrease in b cell activity, potentially related to the decline in helper t cell activity, and decreased cytolytic and delayed-type hypersensitivity responses have been described. the age-related alteration in helper t cell activity appears to be due to intrinsic or functional changes within the cells, as well as to a shift in subset proportions of cd4+ and cd8+ t cell populations, with increases in the memory phenotype and corresponding decreases in the naive phenotype.  in conclusion, our data indicate that endogamy influences the immune system of cattle since it is associated with significant changes in the profile of immune cells in the peripheral blood, particularly decreased levels of γδ t lymphocytes, an important subpopulation of lymphocytes in cattle, especially in calves. 
multiple regression analysis indicated that endogamy in calves was associated with increased cd8+ t and cd21+ b lymphocytes, and decreased γδhigh t cells in peripheral blood. calves with higher endogamy had higher levels of cd8+ t lymphocytes, whereas calves with lower endogamy had lower levels of γδlow t cells. mitochondrial dna allows the separation of american zebu and evaluate the effect of mitochondrial dna on productive traits of cattle. however, the effect of endogamy and mitochondrial dna on the immune system remains unclear. cows with medium and lower endogamy had a lower percentage of b lymphocytes and γδlow t cells and cows with lower endogamy had higher levels of γδ t cells and γδhigh t cells, as well as the cd4+/cd48+ cell ratio. age, endogamy, milk yield, and origin of mtdna were measured and their influence on immunological parameters was evaluated. the aim of this study was to evaluate the association between endogamy, mtdna and immune parameters. these results demonstrated for the first time that endogamy influences the immune system of cattle. older cows had increased cd4+ t cells, decreased cd21+ and γδhigh t cells as well as increased cd4+/cd8+ and t/b ratio.
 endogamy increases the risk of manifestation of deleterious recessive genes. mitochondrial dna allows the separation of american zebu and evaluate the effect of mitochondrial dna on productive traits of cattle. however, the effect of endogamy and mitochondrial dna on the immune system remains unclear. the aim of this study was to evaluate the association between endogamy, mtdna and immune parameters.  a total of cattle were used in this study. age, endogamy, milk yield, and origin of mtdna were measured and their influence on immunological parameters was evaluated. older cows had increased cd4+ t cells, decreased cd21+ and γδhigh t cells as well as increased cd4+/cd8+ and t/b ratio. multiple regression analysis indicated that endogamy in calves was associated with increased cd8+ t and cd21+ b lymphocytes, and decreased γδhigh t cells in peripheral blood. cows with medium and lower endogamy had a lower percentage of b lymphocytes and γδlow t cells and cows with lower endogamy had higher levels of γδ t cells and γδhigh t cells, as well as the cd4+/cd48+ cell ratio. calves with higher endogamy had higher levels of cd8+ t lymphocytes, whereas calves with lower endogamy had lower levels of γδlow t cells.  these results demonstrated for the first time that endogamy influences the immune system of cattle. endogamycattlemtdnaflow cytometry 