Colostrum plays a fundamental role in survival in the neonatal period of the dog, as well as in its future development as an adult [1, 3]. To date, despite knowledge of the nutritional and immunological components of colostrum in dogs [4, 5], there has been no study on biological nanostructures, such as exosomes, their cargo and biological functions.
As far as we know, this is the first study that describes and characterizes the presence of exosomes from CCM and evaluates their interaction with canine MSCs and fibroblasts.
Ultracentrifugation techniques allowed the isolation of abundant exosomes from CCM, similar to that already described for exosomes isolated from other canine species [27, 29]. The presence of CCM exosomes was confirmed by TEM, size determination and western blot analysis of the expression ALIX, Hsp70 and TSG101 exosomal markers, according to the recommendation of the International Society for Extracellular Vesicles [40, 41].
Exosomes play a key role in cell-to-cell communication and contain different specific proteins depending on their cellular origin. Nevertheless, exosomes share a subset of essential proteins for vesicular biogenesis, structure and distribution [34, 42, 43]. Through proteomic analysis, we identified 892 proteins mainly related to functions such as transport, metabolism, regulation of different biological functions, cell differentiation, organization and biogenesis. These results coincide with colostrum milk exosomes of other species [7, 43], which suggest the evolutionary importance of these vesicles in regulating different cellular functions in newborns [6, 13, 14, 25, 44], which is shared between different species of mammals .
When we compared the canine proteomic profile of colostrum exosomes with exosomes from different canine mesenchymal sources already described by our team , we found that they share 11 proteins with common functions, confirming that exosomes are carriers of certain proteins with basic functions within the same species. Among these proteins, those related to functions such as angiogenesis, growth, inflammation, metabolism and cell signalling stand out (additional file 3). Undoubtedly, more studies are needed to understand the functioning of exosomes in canine species.
MSCs play a major role in homeostasis and tissue repair; however, very little is known about the factors that may influence them in early neonatal stages. Evidence suggests that the loss or malfunctioning of stem/progenitor cells necessary for normal cell differentiation and tissue repair may underlie the pathobiology of some diseases .
Evaluating MSCs as the target of colostrum exosomes, we found interesting results that depend on the cellular source. CCM exosomes co-cultured with MSCs demonstrated a statistically significant increase in cAd-MSC proliferation, whereas this effect was not observed in cBM-MSCs.
We suggest that colostrum exosomes can play a very interesting role in the development of fat reserves in dog. The percentage of adipose tissue in newborns is low and increases rapidly during the first month of life, a critical process for avoiding the risk of neonatal mortality, which does not appear to be related to breeding size [1, 46, 47].
Adipose tissue, besides being an energy reservoir, represents a natural defence against hypothermia and fulfils metabolic, endocrine and regulatory functions, both with systemic and local effects [48, 49]. MSCs are exerted through a large diversity of secreted adipokines with complex autocrine and paracrine effects . MSCs are multipotent postnatal progenitors, with adipose tissue being the main source of this cell type [29, 51]. MSC fat residents are generally the principal source of adipocytes during postnatal growth and the maintenance of adipose tissue . Therefore, the increased proliferation of MSCs would help increase fat reserves.
In this study, we demonstrated that canine colostrum exosomes lead to changes in the secretory profile of both types of canine MSCs studied, but in a very different way. Of the 13 analytes evaluated, we found a significant increase in the production of 5 of analytes in cAd-MSCs (IL-8, MCP-1, IFN-γ, TNF-α and NGF-β) and 6 analytes in cBM-MSCs (IL-12p40, IL-6, IL-8, MCP-1, SCF and NO).
Both cell types showed an increase in the secretion of IL-8 and MCP-1, which are factors related to migration, chemotaxis and angiogenesis.
IL-8, also known as CXCL8, has been shown to have potent pro-angiogenic properties, promoting vein endothelial cell proliferation, migration, tube formation and the ability to attract and activate neutrophils . MCP-1, one of the factors associated with the immunomodulatory effects of MSCs, reduces apoptosis and plays a direct mediating role for angiogenesis, which is manifested by the formation of new blood vessels  that are necessary for the development and growth process.
cBM-MSCs stimulated with CCM exosomes specifically increase the production of factors related to immunity (IL-6, IL-12p40, NO) and regulation and the mobilization of haematopoiesis (SCF). IL-6 is a pleiotropic cytokine with a key role in different biological processes, such as regulation of the immune response, inflammation, haematopoiesis, apoptosis, cell survival and cell proliferation . IL-12p40 plays an important role in the development of T cells and enhances the production of immune factors . NO is a highly immunosuppressive soluble factor that decreases the proliferation and modulation of T cells and promotes apoptosis of immune cells .
In contrast to cBM-MSCs, colostrum exosomes in cAd-MSCs, in addition to stimulating their proliferation, demonstrated a change in their secretory profile by increasing the release of proinflammatory cytokines (TNF-α and IFN-γ). TNF-α is a pleiotropic cytokine with important but sometimes contradictory functions in numerous physiological processes related to immunity and inflammation . IFN-γ intervenes in macrophage activation, induces the expression of MHC class II molecules, increases cytotoxic potential and favours, together with TNF-α, the development of the fundamental Th1 cell responses to control viral infections [59, 60].
In addition, we found that colostrum exosomes increased the secretion of factors related to neurogenesis (NGF-β), most notably in cAd-MSCs. NGF plays a crucial role in the peripheral and central nervous systems; regulates the growth, differentiation and survival of neurocytes; improves cognitive functions; and shows potential to induce angiogenesis under physiological and pathological conditions [61, 62].
Although both MSC types demonstrate secretory similarity in terms of their functions related to angiogenesis, migration and chemotaxis of immune cells, the different behaviour of each cell type would confirm the importance of their cellular niche in the different biological functions of individuals. Thus, while adipose tissue MSCs show important endocrine and metabolic potential in adipose tissue development and neurogenesis, the response of BM-MSCs is more consistent with immunity, cell mobilization, angiogenesis and haematopoiesis.
Newborns, because of their immature antioxidant capacity, are more prone to oxidative stress than adults [35, 63-65], leading to an increase in the risk factors that trigger inflammation, infection and ischemia and resulting in damage to multiple organs, which plays a key role in the pathogenesis of several perinatal diseases [66–68].
Fibroblasts are an abundant cell type in the body, and their role is to produce the extracellular matrix necessary for the formation and maintenance of structural integrity at very important stages in the maturation of certain vital organs, such as the lung [69, 70]; therefore, they suffer the effects of free radicals. This is the justification for using this cell type to evaluate the antioxidant capacity of CCM exosomes.
Colostrum is known to be essential in the antioxidant mechanism of the neonate [71-74]; however, to date, the antioxidant potential of canine colostrum exosomes against fibroblasts has not been described. We demonstrate the important role that exosomes play in avoiding the effects of free radicals on fibroblasts and intervene in the maturation and development of the puppy.
Therefore, the results presented in our study aid in understanding how colostrum functions through its exosomes, its interrelationship with MSC and its antioxidant role [8, 16, 42].
Although our study obviously had limitations due to the small sample size of colostrum donors and the restrictions posed by the lack of specific reagents available for the canine species, we believe that our work is the first step in this direction. However, a more in-depth investigation of exosome functions with a focus on miRNA cargos, gene regulation, immunity and metabolism may be an interesting line of research.