By wrapping high-molecular-weight proteins (e.g., cytosolic, cytoskeletal, plasma membrane and heat shock proteins), lipids (e.g., sphingomyelin, cholesterol, phosphatidylserine and glycosphingolipids), and nucleotides, especially miRNAs, extracellular vesicles (EVs) can mediate intercellular communication in a novel way using these contents [1
]. EVs are heterogeneous in size (40–2000 nm) and include inward luminal budding of exosomes and outward budding of microvesicles, as well as apoptotic bodies [3
]. EVs are not only secreted from hematopoietic origins, particularly by cells of the immune system, including dendritic cells, macrophages, B cells and T cells, but also from non-hematopoietic origins, like neurons and epithelial cells [4
]. After being synthesized by these cells, EVs are released into various biological fluids such as blood, saliva, urine, amniotic fluid, and milk, which contribute to many mammalian physiological and pathological processes [5
]. Blood bathes all the organs in the body and collects and transports many types of biomolecules secreted, excreted, or discarded by different cells [6
]. Serum is an aqueous solution and abundant with dissolved albumin, globulin, enzymes, hormones, nutrients, metabolites and various complex organic molecules, including EVs [5
]. Enclosed by a strong phospholipid bilayer membrane, the cargos in serum EVs, especially miRNAs, can deliver at great distances without dilution or degradation. Therefore, various studies have regarded serum EVs as biomarkers with the ability to identify many diseases—such as cancer, immunological disorders, and other systematic disorders in different organs—due to their containing miRNAs [7
The 18–25 nt non-coding miRNAs can regulate gene expression and control protein synthesis at the post-transcriptional level. After the cleavage of the primary miRNAs (pri-miRNA) and yielding of hairpin precursor miRNAs (pre-miRNA), mature miRNAs capable of performing biological functions are produced [8
]. As the key regulatory factors for mammalian growth and development, miRNAs are involved in cell cycling, programmed cell death, cell proliferation, cell differentiation, sensing of nutrient stress and immune processes such as the development of B and T cells, the release of inflammatory mediators, the differentiation of dendritic cells and macrophages, and the proliferation of neutrophils and monocytes [8
]. Specific miRNAs are found in the majority of eukaryotic tissues and organs, and circulatory miRNAs, that are balanced across the entire body, can be free or vesicle-enclosed. Serum EVs are the major miRNA carriers, representing about 78% of total serum miRNAs [11
The rise of “nutrigenomics” in recent years, which explores the metabolism of nutrients using high-throughput technologies, elucidated another important role of diets besides providing nutrients, namely the regulation of gene expression [12
]. It has been proven that changes in nutrient intake induced by different diets such as fatty acids, carbohydrates, amino acids, vitamins, and phytochemicals could regulate the expression of host miRNAs, leading to the modification of host gene expression. Additionally, changing the expression of miRNAs through dietary change has been used as a therapeutic agent to treat some human diseases [13
]. Given that diets can change the expression of host circulating miRNAs, and that EVs are the major miRNA carriers in serum, we hypothesized that different diets could change bovine circulatory EV-miRNA expression and, accordingly, designed different feed formula. We partly replaced the alfalfa hay in the bovine feed formula with whole cotton seed and soybean hull to reduce the cost of forage due to high-quality alfalfa being in short supply and costly [14
]. On the other side, as high-fiber byproduct feeds, whole cotton seed and soybean hull are recycled forage, conforming to the concept of the sustainable development of agriculture. This study shows that bovine serum EVs were abundant with immune-related miRNAs, and that different diets did change the serum EV-miRNA profiling. It is a great possibility that people could implement effective dietary strategies to intervene in the physiological state of animals.
2. Materials and Methods
2.1. Ethics Statement
The Holstein cows involved in this experiment were supplied by the Beijing Dairy Cow Center. The study was approved by the State Key Laboratory of Animal Nutrition, the Institute of Animal Science, and the Chinese Academy of Agriculture Sciences, Beijing, China. All operations were performed strictly according to the Directions for the Caring of Experimental Animals from the Ministry of Science and Technology, China (no. 398, 2006). The experimental period was 30 days and the cows had access to water and feed ad libitum throughout the experiment.
2.2. Animals and Management
Six Holstein cows (n = 6101 ± 10 DIM, 574 ± 36 kg BW, 32 ± 2 kg milk/d) were utilized in the experimental period, while housed in 6 environmentally controlled chambers and exposed to a constant temperature of 20 °C, with 40% humidity. The control cows were fed with Total Mixed Ration (TMR), while the tested cows were fed with feed that partly replaced alfalfa hay with whole cotton seed and soybean hull. The TMR formula (Table 1
) for the two groups was designed to exceed the predicted requirements (NRC, 2001).
2.3. Serum Collection
The blood samples from each cow were collected at 07:00 h on the 30th day of the experimental period. A total of 10 mL of whole blood was collected using a blood collection tube, which should not contain anticoagulants such as EDTA or citrate. The serum was isolated at 3000× g for 10 min at room temperature. The serum samples were preserved at liquid nitrogen.
2.4. Preparation of EVs from Serum
The exoEasy Maxi Kit (catalog no. 76064, QIAGEN, Germany) was used for the purification of exosomes and other EVs from cattle serum, depending on a membrane-based affinity binding step. According to the instructions, 4 mL serum was taken as a response system. An amount of 4 mL buffer XBP was mixed with 4 mL sample and warmed to room temperature. The mixture was added to the exoEasy spin column and centrifuged at 500× g for 1 min and the flow-through was discarded. An amount of 10 mL buffer XWP was added to the column and centrifuged at 5000× g for 5 min and the flow-through was discarded. An amount of 400 μL Buffer XE was added to the membrane and incubated for 1 min. The liquid was centrifuged at 500× g for 5 min to collect the eluate, and then the centrifuging was repeated at 5000× g one more time.
2.5. Nanoparticle Tracking Analysis
Serum pools from the control and tested groups were chosen to carry out this test. Serum mixtures of 3 cows in the control group were pooled together and fed and treated equally. Similarly, 3 cows from the tested group were pooled together. The isolated EVs were analyzed using nanoparticle tracking analysis to identify their physical characterization. The operations were carried out in strict accordance with the operating procedures. The particle size was tested using a sCMOS Nanosight camera with a 405 nm laser (catalog no. NS300, Malvern, UK).
2.6. Transmission Electron Microscopy
Serum pools from the control and tested groups were chosen to carry out the test as before. The exosomes were resuspended with 2% paraformaldehyde (catalog no. 158127, Sigma, US) and 5 μL was dropped onto the copper wire and rested at room temperature for 20 min. After being rinsed with 1× phosphate buffered solution 3 times, it was fixed with 1% glutaraldehyde solution (catalog no. G6257, Sigma, US) for 5 min. Then, it was rinsed 10 times with distilled water and negative dye with 5.4% uranyl acetate (catalog no. 22400, Electron microscopy sciences, US) for 5 min. After drying, it was observed using a Transmission Electron Microscope (TEM) (catalog no. FEI Tecnai G2 F20 S-TWIN, US).
2.7. Small RNA Library Construction and Sequencing
The serum EV-miRNAs were isolated using the miRNeasy Serum/Plasma Kit (catalog no. 217184, QIAGEN, Germany) following the manufacturer’s instructions. The quality of small RNA was tested by 2% agarose gel electrophoresis and Agilent Bioanalyzer 2100 (catalog no. G2939A, Agilent, CA, US). TruSeq Small RNA Sample Prep Kits (catalog no. RS-200-0012, Illumina, US), Qubit dsDNA Assay Kit (catalog no. Q328520, Life Technologies, US), Qubit RNA Assay Kit (catalog no. Q32852, Life Technologies, US), SuperScript II Reverse Transcriptase (catalog no.18064-014, Invitrogen, US) and DNA loading dye (catalog no.LC6678, Invitrogen, US) were utilized in these steps. Briefly, 3’ and 5’ adapters were ligated to the end of the RNA molecules. Then, the libraries were reverse transcribed, and the polymerase chain reaction was subsequently amplified. The cDNA constructs were purified and recovered, and the libraries were checked and eventually normalized.
2.8. Bioinformatics Analysis
The basic reads were converted into raw data/reads by base calling. Low-quality reads, 5’ primer contaminants and poly (A) were removed, and the sequences between 15 and 41 nt were retained. FASTX Toolkit (version 0.0.13) (http://hannonlab.cshl.edu/fastx_toolkit/
) and NGS QC Toolkit (version 2.3.2) (https://omictools.com/ngs-qc-toolkit-tool
) were used to control the quality of Q20 and filter the reads with N bases. The RNAs were subjected to the GenBank databases (http://www.ncbi.nlm.nih.gov/genbank/
) to match the reads with the reference genome (Bos taurus
, RefSeq assembly: GCF_002263795.1). BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi
) and Rfam (version 10.1) (http://www.sanger.ac.uk/software/Rfam
) were used to remove transcripts, repeat sequences and annotate the non-coding RNAs as tiRNAs, rRNAs, small nuclear RNAs, small nucleolar RNAs, Cis-reg, other Rfam RNAs, etc. The known miRNAs and their structures were identified using miRBase (version 22.0) database (http://www.mirbase.org/
). The unannotated sequences were made use of definition new miRNAs with miRDeep2 (https://www.mdc-berlin.de/content/mirdeep2-documentation
) and the hairpin-structures were predicted using RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi
). The target genes of differentially expressed miRNAs were predicted by miRanda software (http://miranda.org.uk
). Gene ontology (GO) (http://geneontology.org
) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses (https://www.kegg.jp
) were performed with the predicted target genes. GO function enrichment took all gene transcriptions/lists as a background and the different miRNA target genes list as a filter of the candidate list. Hypergeometric tests and the Benjamini and Hochberg multiple inspection were utilized to calculate the representative p
value and their correction. The pathway analysis was conducted using the KEGG database (combined with KEGG annotation results, https://www.kegg.jp
), and the significance of enrichments in the pathways was calculated using a hypergeometric distribution test.
The forage-concentrate ratio is traditionally emphasized as a starting point for formulating dairy cattle rations. However, this metric is imprecise when applied to nonforage fiber sources (NFFS), which are high in fiber (like forages) but rapidly passed from the rumen (like concentrates) [13
]. NFFS include wheat bran, soybean hull, rice husk, whole cotton seed, and other particular fractions of plants, most often derived from wheat, soybean, rice, corn, oats, or barley [15
]. The high-fiber byproducts of the crops are disposable and of little value in most cases. The increase in land use, tighter supplies of food and higher prices of cereal grain result in dramatically higher costs of cattle feeding. In this case, the judicious utilization of NFFS provides a practical strategy for nutritionists to incorporate the feedstuffs into the diets of cattle [13
]. It was estimated that the replacement of about 30% of the dry matter grain with soybean hull has no negative effects, either on the fermentation or on the digestion of nutrients, nor on the performance of dairy cows [16
]. Ipharraguerre et al. found that the replacement of corn with varying amounts of soyhulls caused a decrease in dry matter intake, but an increase in the milk fat concentration of dairy cattle, with few effects on other production parameters [17
]. In this study, we partly replaced alfalfa hay and corn silage with whole cotton seed and soybean hull to reduce the breeding costs and maintain the production of dairy cattle. Besides the favorable price and high availability of soybean hull and whole cotton seed, the latter, as a potentially valuable protein and energy dietary supplement for dairy cows, had high protein, fiber, and oil contents [18
]. We found that the reasonable substitution of high-quality feed by high-fiber byproduct feed did not compromise the health and productivity of cattle, whilst sharply reducing feeding costs at the same time (unpublished).
Functioning as fuel and co-factors, micro- and macronutrients, viewed as ’dietary signatures’, can regulate gene and protein expression in the body and, accordingly, influence homeostasis. Emerging genomics tools—such as transcriptomics, proteomics and metabolomics—allow people to identify the regulatory pathways through which these dietary signatures influence homeostasis, being the foundation of nutrigenomics [19
]. The outputs of ruminant nutrigenomics studies could help improve the performance of high-yielding dairy cows and modify milk quality for the growing demand for “healthy” food [20
]. Toral et al. discussed the specific impact of nutrigenomics on the ruminant physiological processes of milk lipid synthesis [21
]. They suggested that a methionine-deficient diet could up-regulate 3% and down-regulate 1% of known miRNAs in mice livers, with miR-182, miR-183, miR-199a-3p, miR-705 and miR-1224 up-regulated, while miR-130a and miR-30c were down-regulated [22
]. The ingestion of essential amino acids could increase the expression of miR-499, miR-208b, and miR-23a in human skeletal muscle [23
]. High glucose increased the expression of miR-21, which could regulate Akt/TORC1 activity and diabetic kidney disease [24
]. N-3 polyunsaturated fatty acids modulated the expression of let-7d, miR-15b, miR-107, miR-191 and miR-324-5p in rat colons [25
]. In conclusion, dietary adjustment, including inducing changed intakes of nutrients such as amino acids, fatty acids, carbohydrates, vitamins, and phytochemicals, led to different miRNA expression profiles in various tissues and organs [13
The cardiovascular system is the first formative organ in vertebrate embryo and other organs start to develop adjacent to it, interacting with the preexisting vessels and receiving vascular signals [26
]. Therefore, blood is suggested, from the early stage of life development, as the mutual link among different tissues, and lays the foundation of homeostasis. Most, if not all, cell types from different organs can release EVs that then enter into the bio fluids, especially blood [27
]. Blood EVs are enriched in miRNAs, with special attributes of their parent cell, or the derived origin, and therefore serve as biomarkers of many diseases [28
]. At present, studies on cattle blood EV-miRNAs—which might act as promising early diagnostic biomarkers to improve the health issues in cows, such as mastitis, acidosis, metritis, as well as welfare issues—are very rare [11
]. This study showed about 245 miRNAs expressed in bovine serum EVs by RNA sequencing. Similarly, Zhao et al. found that there were about 260 miRNAs in bovine serum EVs using the same method [11
]. Moreover, bta-miR-148a, bta-miR-30d, bta-miR-191, bta-miR-423-5p, bta-miR-486, bta-miR-186 and bta-miR-92a were highly expressed in both studies, implying their possible function in the growth and development of cattle. As was predicted in our hypothesis, different diets did change the expression of bovine circulating EV-miRNAs. But the seven differently expressed miRNAs (bta-miR-126-5p, bta-miR-199a-3p, bta-miR-2285bc, bta-miR-2288, bta-miR-3613a, bta-miR-450b, and bta-miR-500) between the two groups were low-expressed on the whole, ranking below 100. To date, there is no information about the target genes of bta-miR-126-5p, bta-miR-2285bc, bta-miR-3613a and bta-miR-450b. The pathway analysis of the target genes of the differentially expressed miRNAs referred to thyroid hormone signaling pathways, insulin signaling pathways, and protein digestion and absorption, indicating that dietary change might lead to different nutrient metabolism and regulate homeostasis.
To date, little was known about the specific destination of the EV-miRNAs in the circulatory system. But we did know most miRNAs were evolutionarily conserved [29
]. Therefore, function studies from other species on highly conserved miRNAs might also be applied to cattle. Most highly expressed miRNAs in the serum EVs of this study were immune-related, as reported in bovine or other mammals before. The let-7a-5p and miR-423-5p, highly expressed in this study, were differently expressed in Mycobacterium bovis
infected cows, suggesting their role in tuning the complex interplay between pathogens and the host immune response [30
]. The highly expressed miR-191 in this study was a key regulator of naive, memory, and regulatory T cells, and thereby helped maintain the lymphocyte reservoir, which was necessary to mount productive immune responses [31
]. Similarly, miR-30d could control glycosylation by targeting N-acetylgalactosamine transferases, resulting in increased tumor invasion and immune-suppression [32
]. The overexpression of miR-125b-5 or miR-99a-5p could promote human γδ T cell apoptosis and inhibit γδ T cell activation, intervening in rapid response to infections as important components of innate immunity [33
]. All the findings supported the theory that immune function might be one of the destinations for peripheral blood EV-miRNAs.