Postprandial transfer of colostral extracellular vesicles and their protein and miRNA cargo in neonatal calves

Extracellular vesicles (EVs) such as exosomes are key regulators of intercellular communication that can be found in almost all bio fluids. Although studies in the last decade have made great headway in discerning the role of EVs in many physiological and pathophysiological processes, the bioavailability and impact of dietary EVs and their cargo still remain to be elucidated. Due to its widespread consumption and high content of EV-associated microRNAs and proteins, a major focus in this field has been set on EVs in bovine milk and colostrum. Despite promising in vitro studies in recent years that show high resiliency of milk EVs to degradation and uptake of milk EV cargo in a variety of intestinal and blood cell types, in vivo experiments continue to be inconclusive and sometimes outright contradictive. To resolve this discrepancy, we assessed the potential postprandial transfer of colostral EVs to the circulation of newborn calves by analysing colostrum-specific protein and miRNAs, including specific isoforms (isomiRs) in cells, EV isolations and unfractionated samples from blood and colostrum. Our findings reveal distinct populations of EVs in colostrum and blood from cows that can be clearly separated by density, particle concentration and protein content (BTN1A1, MFGE8). Postprandial blood samples of calves show a time-dependent increase in EVs that share morphological and protein characteristics of colostral EVs. Analysis of miRNA expression profiles by Next-Generation Sequencing gave a different picture however. Although significant postprandial expression changes could only be detected for calf EV samples, expression profiles show very limited overlap with highly expressed miRNAs in colostral EVs or colostrum in general. Taken together our results indicate a selective uptake of membrane-associated protein cargo but not luminal miRNAs from colostral EVs into the circulation of neonatal calves.

One dietary source rich in EV-encapsulated miRNAs is milk [20]. Studies have shown that a large proportion of miRNAs in milk is localised in exosomes and exosome-like EVs [21].
Despite a significant reduction during processing or storage [22,23], milk miRNAs are readily detected in commercial dairy products and even milk powder [24]. More So far, the vast majority of experiments has focused on the potential uptake of miRNAs from commercially available bovine milk in adult non-bovine species, neglecting the unique advantages of newborn calves and colostrum as a model for transfer of dietary miRNAs. Colostrum not only contains significantly higher amounts of miRNAs compared to mature milk, it was also shown that these are largely associated with EVs [20]. Moreover, transfer of colostral protein such as immunoglobulins to the circulation has already been studied extensively in ruminants. Although the intestinal uptake can be receptor-mediated, it is mostly driven by unspecific pinocytosis and occurs exclusively during the first days after parturition [34]. Additionally, it stands to reason that an uptake of milkderived EVs is more likely to be observed between mother and direct offspring as opposed to a cross-species transfer to adult individuals.
The potential implications of a widespread absorption of dietary miRNAs are far-reaching and intimidating. Given the unique role miRNAs play in almost all physiological and pathological processes [35], it would cause no less than a paradigm shift in our perception of nutrition in general [36]. The objective of this study was to assess the potential transfer of colostral EVs and their cargo to the circulation of newborn calves by analysing colostrum-specific protein, miRNA and isomiR markers.

Results
Blood and colostrum from adult cows bear two distinct EV populations Particles with characteristical EV morphology and size were found in blood as well as colostrum EV preparations by transmission electron microscopy (TEM) (Supplemental Figure 1). No apparent size differences between tissues or sucrose density fractions could be detected and the majority of vesicles were less than 150 nm in diameter. Subsequent analysis by nanoparticle tracking analysis (NTA) confirmed these findings with mean and mode vesicle diameters ranging from 129.6 nm and 98.3 nm in 30 % sucrose density gradient (SDG) colostrum EV samples to 167.8 nm and 112.4 nm in 40-50 % SDG blood EV samples, respectively ( Figure 1A). Although particles showed little size heterogeneity, striking differences were found for particle concentrations. EV preparations from colostrum showed very high mean particle numbers per ml (4.59E10 ± 4.25E9 P/ml) and consisted predominantly of particles floating in 40-50 % SDG with only a very minor amount of particles originating from the 30 % SDG fraction (p ≤ 0.05, Figure 1B). Contrary to that, total particle numbers in cow blood were reduced by over 10-fold (2.97E9 ± 3.15E8 P/ml) and were significantly enriched in 30 % SDG particles (p ≤ 0.05). Additional information on particle size and concentration measurements from NTA can be found in Supplemental Table 1.
To further differentiate between blood and colostrum particles, EV-and milk-specific proteins as well as a negative marker were assessed by Western blot for 40-50 % SDG preparations ( Figure 2). Particles from both fluids were positive for CD63, a commonly used marker for vesicles, with EVs isolated from colostrum showing higher intensities, while no signals could be detected for calnexin, a marker for non-vesicular membrane contamination. A different pattern was observed for MFGE8 and BTN1A1, both of which were previously found to be highly associated with milk vesicles [37,38]. Colostrum EV isolates displayed very strong signals, whereas protein lysates from blood EV preparations were negative for these markers, which we therefore considered specific to milk EVs.
Calf plasma levels of EVs that share colostrum characteristics are increased after feeding Comparable to EV isolates from adult cows, particles in EV preparations from calf blood showed similar size ranges with mean and mode diameters ranging from 142.3 nm and 122.1 nm to 160.3 nm and 142.9 nm, respectively, and no significant size differences between sampling time points or SDG fractions ( Figure 3A). Particle concentrations for 30 % SDG were stable over all time points with no significant changes in particle numbers ( Figure 3B). Meanwhile, concentrations of vesicles isolated from 40-50 % SDG were consistently and significantly (p ≤ 0.05) increased for every time point compared to prefeeding samples, reaching a maximum concentration of 1.97E10 ± 7.33E9 P/ml at 9-12 h postprandial (p ≤ 0.05) and were significantly more abundant compared to 30 % SDG particles. Further similarities between EVs isolated from colostrum and calf plasma after feeding were found by Western blot analysis. CD63 and the colostrum-specific BTN1A1 were detected with increasing signal intensities with progressing time points, albeit with no or only weak expression for 0 h samples. Postprandial samples were positive for MFGE8 as well, but for no more than a single time point in each calf and only after 6 or 9-12 h ( Figure 2). Analysis of tissue-and compartment-specific small RNA profiles by next-generation sequencing Mean library sizes generated by small RNA sequencing differed within expected dimensions, ranging from 5.01E6 ± 1.30E6 in calf EV samples, potentially reflecting low input amounts for library preparation due to low RNA yield, to 1.34E7 ± 2.17E6 in colostrum cells. One sample each from whole colostrum, colostrum cells as wells as calf EV 0 h groups had to be excluded from further analysis, since they failed to amplify correctly during sequencing. Differences in small RNA profiles were assessed by aligning reads against miRNA and isomiR sequences as well as other major small RNA classes (rRNA, tRNA, snRNA, snoRNA) and plotting them together with unmapped and short reads (<16 nt) as percentages of total library size per group ( Figure 4). Highest enrichment for isomiRs including canonical miRNAs was seen in whole blood samples from cow and calf along with calf blood cells (>80 %) with little to no other small RNA species present. On the other hand, libraries from calf EVs were dominated by a large number of unmapped sequences not belonging to any of the major small RNA classes, and a comparatively low number of miRNAs. Similar to blood, colostrum-derived samples exhibited a clear distinction between extracellular, EV-associated samples and unfractionated or cellular groups, respectively. All colostrum samples displayed higher relative numbers of tRNA reads compared to blood samples, which was most pronounced in colostrum cells.
Unfractionated colostrum and colostrum EVs, on the other hand, contained the highest frequencies of short reads. Additionally, a strong relative enrichment of miRNA reads was observed in colostrum EVs compared to colostral cellular samples. Further information on alignment distributions and library sizes can be found in Supplemental Table 2.
Analysis of canonical miRNAs reveals an influence of colostrum feeding on expression profiles in calf EVs only Differential regulation of canonical miRNAs between colostrum and blood compartments along with postprandial time points within the same sampling group was assessed using DESeq2 and applying conservative filtering criteria (adjusted p-value ≤ 0.05, log 2 fold changes ≥ |1|, baseMean ≥ 50). Unfractionated colostrum differed minimally from colostrum EVs with only 9 significantly regulated miRNAs, while both groups showed a considerably different miRNA expression profile compared to colostrum cells (Table 1), reflecting RNA species distribution seen during alignment. The biggest expression changes in the data set could be found between all three colostrum-derived sample groups and unfractionated cow blood with over 150 differentially expressed miRNAs in each group (Table 1). Albeit expression profiles of calf blood-derived samples displayed high diversity between individual compartments with numbers of significantly regulated miRNAs ranging from 105 to 147 (Table 1), expression changes within different postprandial time points of blood compartments were scarce with the exception of calf EVs. Similar to calf blood samples, which exhibited no alterations at all over all time points, miRNA expression in unfractionated blood samples was very stable with a total of two significant regulations after 9-12 h ( Table 2). On the other hand, expression changes in postprandial calf EVs samples compared to pre-feeding samples ranged from two (after 1 h) to 24 (after 9-12 h) miRNAs and continually increased in magnitude with progressing time ( Table 2). Although changes were up regulated, 12 of those miRNAs showed significantly smaller abundances in colostral EV samples compared to calf EV 0 h samples. Furthermore, out of the top 15 most highly expressed miRNAs in colostral EVs only three showed a significant increase after feeding with all of them belonging to the canonical miR-200a/b/c family.
Differentially expressed miRNAs together with corresponding log 2 fold changes of key comparisons are provided in Supplemental Table 3. Table 1. Numbers of canonical miRNAs differentially regulated between unfractionated, cellular and EV-related sampling groups in colostrum and blood. Table 2. Numbers of differentially regulated canonical miRNAs between postprandial time points and pre-feeding samples (0 h) for calf blood-derived samples.
Hierarchical clustering analysis (HCA) of total miRNA expression confirmed expression profile changes detected in DESeq2 results by clearly separating specific unfractionated from cellular and EV-associated sample groups ( Figure 5). Gene expression differences within unfractionated calf blood, blood cells and, to a smaller degree, blood EVs displayed remarkable homogeneity, resulting in groups defined by individual animals rather than clusters of particular postprandial time points. Furthermore, miRNA expression in calf blood EVs resembled colostrum profiles much more closely than any other blood-derived sample from cow or calf. Continuing the pattern revealed in RNA species distribution and differential gene expression, colostrum cells could be clearly separated from unfractionated colostrum and colostrum EVs. The potential physiological impact of upregulated miRNAs in postprandial calf EVs was evaluated by enrichment analysis of KEGG pathways as proposed by Godard and van Eyl [39]. The most highly enriched pathway across all time points was insulin signaling, followed by TGF-beta signaling and cytokinecytokine receptor interaction, but no apparent overall regulation pattern could be discerned. Top 20 enriched pathways along with involved miRNAs can be found in Supplemental Table 4. isomiR expression profiles in postprandial calf EVs suggest non-colostral origin of up-regulated miRNA isoforms To assess whether miRNA regulation changes in calf EVs could be attributed to colostral EV cargo, the distribution of miRNA isoforms was analysed. Since isomiR expression is highly specific for tissues and developmental stages [7][8][9], it allows for more precise The ability to isolate reasonably pure vesicles is a prerequisite for any viable EV study [40,41]. Following well-established protocols for differential ultracentrifugation in combination with flotation into a density gradient [21,42] yielded particles in the size range of typical small EVs in all biofluids. Mean diameters of particles (~130 to 160 nm) were slightly above exosome-like size (30-100 nm) [43] and in line with previous findings on EVs from bovine or human milk and blood [25, 44, 45] ( Figure 1A). No significant differences in size could be detected between blood and colostrum samples or particles recovered from different sucrose density fractions, hinting at a high reproducibility of EV isolation methods and low inter-individual variability. Quantitative analysis of particles on the other hand, revealed prominent differences in particle composition and concentration in blood and colostrum ( Figure 1B). In adult animals, EV numbers were more than 10 times higher in colostral samples compared to blood samples. While this stands in contrast to the findings of Koh et al, who found concentrations of milk particles to be ~3.2 fold lower than plasma particles [45], it is corroborated by previous reports that have shown that miRNAs associated with colostrum EVs are highly enriched compared to milk from later lactation stages [20,21,24]. Furthermore, the majority of colostrum EVs were significantly denser (40-50 % SDG) than blood-derived EVs (30 % SDG), although all isolated particles generally fell in the density range expected for exosome-like vesicles (1.1270 -1.2296 g/ml) [43]. Predominant EVs of high densities in colostrum were in agreement with findings from Hata et al., showing highest content of vesicle-associated proteins and RNAs from density fractions corresponding to 1.20 g/ml compared to lower densities [21]. While density of particles from plasma was in line with numerous reports [46,47], this is to the best of our knowledge the first time that a significant shift in density between bovine EV populations of blood and milk has been reported. It should be noted, however, that concentration measurements of particles by NTA are prone to overestimation due to co-isolated contaminants such as lipoproteins and protein aggregates, especially in vesicle preparations of low purity [48][49][50]. Particularly preparations from milk or colostrum, with its high content of fat globules and senescent, ex-foliated epithelial cells seem prone to accumulate unwanted particles that can mimic EV properties for example by aggregation during ultracentrifugation [51]. Nevertheless, detected EV concentrations in adult blood and colostrum seem genuine as most contaminants should be discarded in SDG fractions of higher (protein aggregates) or lower (aggregated small fat globule membranes) densities [42]. Endosomal origin of membranous EV preparations as well as higher concentrations of particles in colostrum were further supported by Western blot analysis of positive (CD63) and negative (Calnexin) vesicle markers ( Figure 2). Positive expression of BTN1A1 in colostrum-derived EVs is in concordance with recordings on Vesiclepedia and ExoCarta [52,53], the biggest databases on molecular data of extracellular vesicles, which list BTN1A1 as exclusively associated with bovine and human milk EVs [37,38]. Expression of MFGE8 however, was reported to be widespread in EVs originating from a large variety of sources including B cells and platelets [54,55]. Although MFGE8 was to our knowledge never reported to be associated with bovine EVs apart from milk [38] and we considered absent expression in cow blood to be genuine, it could also not be excluded that a lack of signal originated from low starting input material due to the fact that only samples from 40-50 % SDG fractions were analysed in Western blots.
Differentiation between blood-and colostrum-derived samples and especially EVs was further driven by abundances of small RNA species analysed by Next-Generation Sequencing (NGS). High throughput sequencing has become the tool of choice for analysing nucleic acids due to its high precision in quantifying of single RNA sequences and its accurate detection of diverse RNA compositions. Clear differences were found in relative frequencies of miRNA and tRNA as well as reads shorter than 16 nt between whole blood and colostrum-derived samples in cows ( Figure 4). Higher incidences of short reads most likely stem from an increased number of degradational products tracing back to exfoliated, senescent epithelial cells present in colostrum, while enrichment of miRNAs in colostrum vesicles compared to the cellular fraction has been reported before by Sun et al [20]. Additionally, varying expression both between blood-and colostrum-derived samples and within cellular and extracellular fractions in colostrum were confirmed by differential analysis of canonical miRNAs (Table 1). In general, miRNA expression profiles in colostrum-derived samples were in concordance with previous reports with a substantial overlap in the most abundant miRNAs [21, 56,57]. Although miRNA expression in blood and colostrum was completely dissimilar, duplicating results obtained from milk and peripheral blood cells [58] as well as circulating miRNAs [59], differences between unfractionated colostrum and colostrum EVs were minuscule compared to colostrum cells, suggesting that the majority of miRNAs in colostrum is present in the extracellular compartment.
Taken together, quantitative and qualitative analyses of EVs in cows indicate distinct populations of vesicles in blood and colostrum, enabling the identification of a potential postprandial transfer of colostrum EVs into the blood circulation based upon EV concentrations, protein cargo and miRNA expression. Vesicles identified in calf blood after feeding indeed shared characteristics classified as specific for colostrum EVs (Figure 3).
Particle concentrations for preparations increased significantly in a time-dependent manner solely from 40-50 % SDG, from levels similar to cow blood to approximately half the abundance in colostrum. Contrary to adult cow blood EVs, calf particle numbers from the 30 % SDG fraction even at pre-feeding time point were significantly lower by an order of magnitude compared to high-density vesicles. Similar distributions of EVs as encountered in cow blood EVs could potentially be acquired later during development, but it is also conceivable that higher amounts of 40-50 % SDG EVs at time point 0 h are the product of autonomous but very limited feeding before first blood samples could be drawn even though all calves were under the supervision of a milker. Protein expression of highdensity particles was positive for both milk-specific markers, although only BTN1A1 followed the progressing pattern of increase indicated by particle concentrations.
Divergent expression of MFGE8 with only low abundances detectable after 6 or 9-12 h suggests either the existence of two milk EV subpopulations in 40-50 % SDG fractions, with greatly differing transfer efficiency through the intestinal epithelium and implying selective mechanisms for uptake, or a breakdown of milk EVs in epithelial cells followed by repackaging of protein cargo in a selective and directive manner.
Small RNA expression profiles and in-depth analyses of isomiR distributions in calf blood-derived samples however, painted a somewhat different picture of the bioavailability of dietary EVs and their cargo from colostrum. While miRNA expression contrasted significantly between all three blood-derived samples (Table 1), postprandial changes within sample groups could only be detected for calf EVs (Table 2). Absence from miRNA expression changes in unfractionated calf blood and blood cells was also apparent from hierarchical clustering analysis with sub-clusters within sample groups being defined by inter-animal differences rather than postprandial time points ( Figure 5). Even though a time-dependent influence of colostrum feeding on expression of miRNA and their isoforms in calf blood EVs was evident ( Table 2

Conclusions
In conclusion, our findings on the bioavailability of colostrum-derived EVs in the bloodstream of neonatal calves suggest an even more complex mode of uptake than previously assumed [10,30]. The unequal uptake of protein and miRNA cargo discourages the hypothesis of para-and transcellular transport of intact dietary EVs through the intestinal epithelium into the blood. The reason that we readily found colostrum-specific protein markers but could not detect any meaningful uptake of miRNAs could well be attributable to their respective localization in or on the EV. Postprandial expression of BTN1A1 as well as MFGE8, both membrane-associated proteins, is in line with studies on orally administered labeled exosomes that utilised a lipophilic membrane dye [33]. miRNAs, on the other hand, are thought to be incorporated into the lumen of the vesicle and, like us, a number of studies have failed to detect an uptake of dietary miRNAs so far [13,31,32]. One possible conclusion to this decoupling of membrane-associated cargo from luminal miRNAs, might be an uptake of dietary EVs into intestinal epithelial cells, followed by disassembly of the EV prior to repackaging of EV protein cargo and delivery into the blood. Evidence for this was recently given by a small study deducted by Manca et al., who showed distinct localization of EVs (mainly liver) and their miRNA cargo (mainly brain and kidney) [64]. Based on our experiments we cannot rule out the possibility that miRNAs from colostrum EVs were either not taken up at all, remained in the intestinal epithelium, or were directly transported to recipient tissues without extended circulation in the blood. To discern final destinations of miRNAs, further investigations including sampling from intestinal tissues as well as likely recipient organs such as liver or kidneys are needed.

Sample collection
Blood and colostrum were sampled from randomly selected, healthy, multiparous, pregnant Brown Swiss cows (n = 9) borne and raised at our affiliated research station Veitshof (TU Munich, Weihenstephan) on the day of parturition. Blood samples from calves were taken directly before the first ad libitum feeding with colostrum (0 h) as well as 1 h, 3 h, 6 h afterwards plus directly before second feeding (9-12 h). All blood samples were drawn from vena jugularis in 9 ml K3 EDTA-Vacuette tubes (Greiner bio-one) with singleuse needles (20G x 1'', Greiner bio-one). Plasma and blood cells were separated within half an hour after sampling by centrifugation at 1850 g for 20 min at 4 °C. Comparably, colostrum was centrifuged at 1850 g for 30 min at 4 °C within one hour post sampling.
After removal of the fat layer, skim milk and colostrum cells were collected. All samples including whole blood and colostrum were stored at -80 °C until further analysis.
Colostrum was collected as total quarter milk and fed within 2 h of parturition, and calves were monitored to prevent autonomous feeding before blood sampling. Animals were housed and fed according to good animal attendance practice under permanent surveillance of a veterinarian, and all efforts were made to minimize suffering (permission number 55.2-1-54-2531-5-08).

Isolation of extracellular vesicles
EVs were isolated from cell-free sample fractions (~ 4 ml of plasma and 66 ml of skim milk) by differential ultracentrifugation as described previously [21, 42] followed by flotation into a sucrose density gradient (SDG) [42]. In short, samples were diluted in phosphate buffered saline (PBS) if necessitated by uneven fill levels, and pre-cleared by low-speed centrifugation (12,000 g, 1 h, k-factor: 2335.3). Plasma EVs were pelleted from 12,000 g supernatant (100,000 g, 2 h, k-factor: 278.3). For skim milk samples, the 12,000 g supernatant was subjected to further centrifugation steps at 35,000 g (1 h, k-factor: 797.4) and 70,000 g (3 h, k-factor: 397.9) before EVs were pelleted at 100,000 g (1 h, k-

Data analysis
Sequencing data was processed using a self-compiled bioinformatic pipeline as described previously [67,68] with the added functionality of discovering and quantifying isomiRs. In short, 3'-end adaptor sequences were trimmed using Btrim [69], and length distribution and sequencing quality were monitored via FastQC [70]. To prevent false positive mappings to miRNAs and isomiRs, reads matching rRNA, tRNA, snRNA or snoRNA sequences obtained from RNAcentral [71] along with reads shorter than 16 nt were excluded from further analyses. Filtered reads were then aligned to a newly designed mapping reference consisting of all bovine miRNAs (miRBase v21) [72] and their respective miRNA isoforms. isomiR sequences were derived from canonical miRNA sequences by consecutive trimming of up to 6 nt or addition of 3 nt on 5'-and 3'-end, and included mismatch information was stored during mapping. Alignment was performed using Bowtie [73], allowing for a single mismatch over the whole sequence, and applying the 'best' algorithm. From the resulting SAM files, isomiR read count tables were generated by incorporating mismatch information that describes potential polymorphic isomiRs together with sequence additions and trimmings, and finally calling the sum of

RT-qPCR
The Exiqon miRCURY LNA Universal RT microRNA PCR system (Exiqon) was used to validate miRNAs selected from NGS results in a larger cohort comprised of 6 cows and their 8 calves including two twin births (total n=144). Reverse transcription and qPCR were performed according to the manufacturer's instructions with 10 ng of total RNA as starting input except for calf plasma EV samples, where 2 µl of undiluted sample was used due to their low yield. Assays for validation included: bta-miR-21-5p, bta-miR-26b, bta-miR-30a-5p, bta-miR-141-3p, bta-miR-144, bta-miR-146a, bta-miR-146b-5p, bta-miR-148a-3p, bta-miR-200a, bta-miR-200b, bta-miR-451 and bta-miR-2285t. Specificity of all assays and samples was ensured by using non-template and negative RT controls in representative sample pools from each fraction. All qPCR reactions were measured on a CFX384 Real-Time PCR Detection System (Bio-Rad). Statistical significance on geo-mean normalised data [83] was tested using F-test for normality and Student's t-test.

EV characterization
Isolated EV suspensions were further characterised in terms of morphology, particle size and concentration as well as protein cargo. Calf EV samples 1 h postprandial had to be omitted since plasma yield was very low and no EV isolations could be performed. A workflow for all EV characterization related analyses is provided in Figure 9.
Nanoparticle Tracking Analysis Transmission electron microscopy Diluted EVs were adsorbed onto glow-discharged, carbon-coated copper grids (Quantifoil) for 2 min before manually removing excess liquid by filter paper. Grids were negatively stained in 2 % uranyl acetate for 2 min and air-dried prior to imaging. All images were taken on a JEOL JEM 100CX electron microscope at 100 kV.
Western blot EV fractions were lysed in ice-cold detergent lysis buffer (0.1 % Triton X-100 in PBS) supplemented with protease inhibitors (cOmplete Mini Protease Inhibitor Cocktail, Roche).
To enhance rupture of membranes, lysates were sonicated for one minute in a water bath prior to protein quantification using BCA assay (Sigma Aldrich).

Consent for publication
Not applicable.

Availability of data and materials
The datasets generated and/or analysed during the current study are available in the ENA repository, under accession number PRJEB28002 (http://www.ebi.ac.uk/ena/data/view/ PRJEB28002).

Competing interests
The authors declare that they have no competing interests.        Schematic overview of sample groups for RNA extraction and transcriptomic analyses. Total RNA was extracted from unfractionated, cellular and EV compartments of colostrum and calf blood samples, as well as unfractionated blood from cows before first feeding and at four defined postprandial time points (n=3 each) before being profiled by small RNA-Seq. Expression of differentially regulated and highly abundant miRNAs was subsequently assessed by RT-qPCR in an expanded animal cohort (cows n=6, calves n=8).

Figure 9
Schematic overview of sample groups for EV characterization. EVs were isolated by differential ultracentrifugation and sucrose density gradient in colostrum and blood samples from calves and cows before first feeding and at three defined postprandial time points (n=3 each). EV morphology and protein cargo were characterized by Nanoparticle Tracking Analysis, Western blot and transmission electron microscopy.

Supplementary Files
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