- Research article
- Open Access
Exploration of microRNAs in porcine milk exosomes
- Ting Chen†1,
- Qian-Yun Xi†1,
- Rui-Song Ye1,
- Xiao Cheng1,
- Qi-En Qi1,
- Song-Bo Wang1,
- Gang Shu1,
- Li-Na Wang1,
- Xiao-Tong Zhu1,
- Qing-Yan Jiang1 and
- Yong-Liang Zhang1Email author
© Chen et al.; licensee BioMed Central Ltd. 2014
- Received: 8 March 2013
- Accepted: 31 January 2014
- Published: 5 February 2014
Breast milk contains complex nutrients and facilitates the maturation of various biological systems in infants. Exosomes, membranous vesicles of endocytic origin found in different body fluids such as milk, can mediate intercellular communication. We hypothesized that microRNAs (miRNAs), a class of non-coding small RNAs of 18–25 nt which are known to be packaged in exosomes of human, bovine and porcine milk, may play important roles in the development of piglets.
In this study, exosomes of approximately 100 nm in diameter were isolated from porcine milk through serial centrifugation and ultracentrifugation procedures. Total RNA was extracted from exosomes, and 5S ribosomal RNA was found to be the major RNA component. Solexa sequencing showed a total of 491 miRNAs, including 176 known miRNAs and 315 novel mature miRNAs (representing 366 pre-miRNAs), which were distributed among 30 clusters and 35 families, and two predicted novel miRNAs were verified targeting 3’UTR of IGF-1R by luciferase assay. Interestingly, we observed that three miRNAs (ssc-let-7e, ssc-miR-27a, and ssc-miR-30a) could be generated from miRNA-offset RNAs (moRNAs). The top 10 miRNAs accounted for 74.5% (67,154 counts) of total counts, which were predicted to target 2,333 genes by RNAhybrid software. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses using DAVID bioinformatics resources indicated that the identified miRNAs targeted genes enriched in transcription, immunity and metabolism processes, and 14 of the top 20 miRNAs possibly participate in regulation of the IgA immune network.
Our findings suggest that porcine milk exosomes contain a large number of miRNAs, which potentially play an important role in information transfer from sow milk to piglets. The predicted miRNAs of porcine milk exosomes in this study provide a basis for future biochemical and biophysical function studies.
- Porcine milk exosomes
- Solexa sequencing
Milk, as the sole source of nutrition for infants, contains a potent mixture of diverse components such as milk fat globules (MFG), immune competent cells and soluble proteins, for instance IgA, cytokines and antimicrobial peptides, which can provide protection against infections in newborns. In addition, breast milk may have a role in tolerance induction and may protect infants from developing allergies.
Exosomes are small (30–100 nm) membrane vesicles of endocytic origin that are released into the extracellular environment upon fusion of multivesicular bodies (MVB) with the plasma membrane. Many cells have the capacity to release exosomes, including reticulocytes, dendritic cells, B cells, T cells, mast cells, epithelial cells and tumor cells. In addition, exosomes have been found in physiological fluids, such as saliva[13, 14], plasma, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid and synovial fluids. Several studies have suggested that exosomes, which contain proteins, mRNA and microRNA (miRNA), stimulate and transfer surface receptors to target cells[21–23], as well as serve as novel vehicles for genetic exchange between cells. As with other biological fluids, microvesicle-like particles are also present in mouse milk and human milk. Recent published studies have isolated mRNAs and miRNAs from bovine milk-derived microvesicles. One study via deep sequencing technology identified 602 unique miRNAs originating from 452 miRNA precursors (pre-miRNAs) in human breast milk exosomes and found that, out of 87 well-characterized immune-related pre-miRNAs, 59 (67.82%) were enriched in breast milk exosomes. Recently, porcine milk was reported to contain 180 pre-miRNAs, including 140 known and 40 novel porcine pre-miRNAs, altogether encoding 237 mature miRNAs.
MiRNAs are widespread among eukaryotes and represent key components of a conserved system of RNA-based gene regulation[30–33]. Many studies have demonstrated that miRNAs are key post-transcriptional regulators of gene expression and play important roles in a wide range of physiological and pathological processes, including development, differentiation, proliferation and immune responses. It is believed that about 60% of mammalian genes are regulated by miRNAs[35–39].
Aside from being important farm livestock, pigs are also model animals for medical research. In the present study, we investigated miRNAs in milk exosomes of Landrace pigs in order to provide new information for investigations into the physiological functions of porcine milk.
Porcine milk samples were collected between day 1 to 5 after parturition from healthy lactating Landrace female pigs bred in the breeding farm of the Livestock Research Institute (Guangzhou, China). Milk samples were frozen immediately after milking and were kept at-80°C until use.
Preparation of exosomes from milk
Porcine milk samples were centrifuged first at 2,000 × g for 30 min at 4°C to remove MFGs as well as mammary gland-derived cells. Defatted samples were then subjected to centrifugations at 4°C for 30 min at 12,000 × g to remove residual MFGs, casein and other debris. Subsequently, from the final supernatant (so-called whey or milk serum), the membrane fraction was prepared by ultracentrifugation at 110,000 × g for 2 h in an SW41T rotor (Beckman Coulter Instruments, Fullerton, CA).
Transmission electron microscopy (TEM)
The final fraction obtained as described above was diluted with 0.01 M PBS and ultracentrifuged again to recover microvesicles as pellets. Following fixation in 2% glutaraldehyde, microvesicles were negatively stained with uranyl acetate and observed by TEM (JEOL JEM2000EX, Tokyo, Japan).
RNA isolation and Solexa sequencing
Total RNA was isolated from samples collected after ultracentrifugation using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The quality of RNA was examined by 2% agarose gel electrophoresis and with a Biophotometer 6131 (Eppendorf, Germany), as well as further confirmed by using a Bioanalyzer (Agilent Technologies, Santa Clara, CA). Small RNAs (18–30 nt) were obtained from the total RNA, 5’ and 3’ adaptors were ligated to the small RNAs, then the adaptor-ligated RNAs were subsequently transcribed into cDNA by RT-PCR, and the samples were amplified by PCR using primers complementary to the two adaptors. The PCR products were purified and subjected to Solexa sequencing (Illumina, CA) at the Beijing Genomics Institute (BGI, Shenzhen, China).
Sequence data analysis
The raw reads obtained from Solexa sequencing were processed to obtain clean reads by summarizing data production, evaluating sequencing quality, calculating the length distribution of small RNA reads, removing low quality reads and adaptor sequences as described in previous paper. All the clean reads were aligned against non-coding RNAs from the GenBank and Rfam (11.0) (ftp.sanger.ac.uk/pub/databases/Rfam) database to annotate and classify rRNA, tRNA, snRNA and other ncRNA sequences using tag2 annotation software (developed by BGI). Then the selected sequences were mapped to the pig genome (sscrofa9, http://www.ensembl.org/Sus_scrofa/) using SOAPv1.11 software to analyze their expression and distribution. Subsequently, the miRNA candidates were further analyzed by miRDeep 2 against all known miRNAs and porcine miRNA precursors (miRBase 20.0). All remaining candidates who did not map to any miRNAs in miRBase 20.0 were considered as potential novel miRNAs. To further identify these potential novel miRNA candidates, software MIREAPv0.2 (http://sourceforge.net/projects/mireap) developed by BGI was used to predict novel miRNA by exploring the secondary structure, the Dicer cleavage site and the minimum free energy of the annotated small RNAs which could be mapped to genome. In briefly, the sequence length should be between 18–26 nt, maximal free energy allowed for a miRNA precursor was -18 kcal/mol, maximal space between miRNA and miRNA* was 35 nt, and flank sequence length of miRNA precursor should be 10 nt. Finally, all remaining novel miRNA candidates were further subjected to MiPred (http://www.bioinf.seu.edu.cn/miRNA/) to filter out pseudo-pre-miRNAs. The minimum free energy must be > -20 kcal/mol or P-value was >0.05, and their secondary structures were also checked using the Mfold3.2 software. All data for analysis in this study have been deposited in https://mynotebook.labarchives.com/share/allinchen/MTkuNXwxMzMxMS8xNS0yL1RyZWVOb2RlLzE1NzEyODU2fDQ5LjU= with a DOI:10.6070/H4DN432G.
PCR and qRT-PCR identification of known and novel miRNAs
PCR primers for miRNAs
miRNAs target prediction and plasmid construction
Leuciferase reporter assay
IPEC-J2 cells were maintained in DMEM/F12 (1:1) (GIBCO) and supplemented with 10% fetal bovine serum (FBS, GIBCO), 5 ng/ml EGF (peprotech, USA) and 5 ug/ml insulin (Sigma, USA). Lipofectamine 2000 (Invitrogen) was used for transfection. Cells (10,000) were plated in a 96-well plate. After 24 h cultivation, cells were transfected with a mixture including 500 ng pGLO-IGF-1R-3’UTR or pGLO-IGF-1R-3’UTR-delete construct and 30pM of miR-PC-86 or miR-PC-263 mimics (GenePharma, Shanghai, China). For control, 500 ng of pmirGLO-scramble including a scrambled sequence of the miRNA target sequence was used. Cells were collected 48 h after transfection, and luciferase activity was measured using a Dual-GLO luciferase reporter assay system (Promega). Statistical differences between treatment and control groups were determined using Student’s t-test, at P < 0.05.
Chromosomal localization and cluster analysis of miRNAs
Pre-miRNAs of all miRNAs (known miRNAs and novel miRNAs) were mapped to the porcine genome (sscrofa9, http://www.ensembl.org/Sus_scrofa/) according to their positions on the chromosomes. Pre-miRNA positions less than 10 kb apart were considered to belong to the same miRNA cluster.
Target prediction and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses
Porcine miRNA targets were obtained at the genome level. In brief, miRNA targets were predicted using the RNAhybrid software algorithm (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/) in 3’-UTR sequences of transcripts from the whole pig genome obtained from Ensembl Gene 66 database (sscrofa9, http://www.ensembl.org/Sus_scrofa/). Strict criteria (perfect match of 2–8 nt in the seed region; no more than -25 kcal/mol low free energy of miRNAs-mRNA binding) were applied to the target prediction procedure. GO and KEGG pathway analyses were performed using DAVID bioinformatics resources (http://david.abcc.ncifcrf.gov/).
Identification of exosomes
Porcine milk exosomes contain RNA
Solexa sequencing and analysis
Porcine novel miRNAs conserved in other species (miRBase release 20.0)
1nt add, 1nt sub
1nt sub, 1delete
2nt sub, 2nt delete
3nt delete, 1nt sub
Identification of miRNAs by PCR and sequencing
miRNAs matched to sequecing
Predict new miRNA
Target verification of miR-PC-86 and miR-PC-263 against 3’UTR of IGF-1R using luciferase report assay
To investigate whether the predicted miR-PC-86 and miR-PC-263 (Figure 1) were functional novel miRNAs, target genes were predicted, and miR-PC-86/ miR-PC-263 were found to directly target IGF-1R 3’UTR sequence. The full-length 3’UTR of IGF-1R mRNA was inserted downstream of the luciferase gene in the pmirGLO Dual-Luciferase miRNA Target Expression Vector reporter plasmid, and the seed sequence was also delete to disrupt miR-PC-86/ miR-PC-263 binding (Figure 1B). The wild-type (pGLO-IGF-1R-3’UTR) or delete (pGLO-IGF-1R-3’UTR-delete) plasmid was co-transfected with the miR-PC-86 and miR-PC-263 mimics into IPEC-J2 cells. Forty-eight hours after transfection, the luciferase activity of the miR-PC-86 and miR-PC-263 group were significantly lower than that of the NC group (P < 0.05) respectively, and the reduction was rescued in the delete group (Figure 1C). Thus, IGF-1R was initially confirmed as the target of miR-PC-86 and miR-PC-263.
Genomic localization of pre-miRNAs
In addition, we observed many mature miRNAs having multiple miRNA precursors located in the same or different chromosomes. Of the novel predicted miRNAs, 40 pre-miRNAs had two copies in the genome, 7 pre-miRNAs had 3 copies, 2 pre-miRNAs had 4 copies, 1 pre-miRNA had 8 copies and 249 pre-miRNAs were unique. With regard to known miRNAs, 4 pre-miRNAs had 3 copies, 22 pre-miRNAs had two copies and 149 pre-miRNAs had only one copy.
Pre-miRNAs and their corresponding mature miRNAs type
ssc-mir-24-1, ssc-miR-27a, ssc-mir-23a
ssc-let-7a-2, P-m0204, ssc-let-7f-2, ssc-let-7d
ssc-mir-99b, ssc-let-7e, ssc-mir-125a
P-m0346, P-m0347, P-m0348,P-m0349
ssc-mir-24-2, ssc-mir-27b, ssc-mir-23b
ssc-mir-17, ssc-mir-18a,ssc-mir-19b-1, ssc-mir-92a-1
P-m0024, P-m0025, P-m0026
P-m0125, P-m0126, P-m0127, P-m0128, P-m0114
P-m0136, P-m0137, ssc-mir-183
ssc-mir-363-1, ssc-mir-92a-2, ssc-mir-19b-2, P-m0370, sc-mir-20b-1/ssc-mir-20b-2
ssc-mir-363-1, ssc-mir-92a-2, ssc-mir-19b-2, P-m0371, ssc-mir-20b-1/ssc-mir-20b-2, ssc-miR-106a
ssc-let-7i, ssc-miR-98, ssc-let-7a,ssc-let-7f, ssc-let-7c, ssc-let-7 g, ssc-let-7e, ssc-let7d-5p
ssc-miR-1, ssc-miR-206, PC-117
ssc-miR-148b, ssc-miR-152, ssc-miR-148a
ssc-miR-106a, ssc-miR-17-5p, ssc-mir-20b-1/ssc-mir-20b-2
ssc-miR-181a, ssc-miR-181b, ssc-miR-181c, ssc-miR-181d-5p
ssc-miR-30e-5p, ssc-miR-30c, ssc-miR-30d, ssc-miR-30b-5p, ssc-miR-30a-5p
ssc-miR-363, ssc-miR-92b-3p, ssc-miR-92a
ssc-miR-497, ssc-miR-15b, ssc-miR-16, ssc-miR-15a
ssc-miR-99a, ssc-miR-99b, ssc-miR-100
In addition, many miRNA families showed low expression (count number <100) in milk exosomes, such as the miR-1, miR-130, miR-17, miR-10, miR-29, miR-374, mir-9, miR-15 and miR-491 families (Figure 12F), which are routinely expressed in specific tissues[53–56]. Interestingly, all 9 novel miRNAs families showed extremely low expression levels (Figure 12G, count number <50), indicating that these miRNAs may only be expressed in certain physiology processes and may be the reason for why these miRNAs have not been detected until now. MiR-148a was reported to be an important biomarker for milk exosome miRNAs[28, 57]. In this study, three members of this family (miR-148a, miR-148b and miR-152, Figure 12H) showed modest expression levels, suggesting that miR-148 may be a stably expressed miRNA in exosomes of most mammals including pigs.
GO and KEGG pathway analyses
Gene ontology analysis of potential targets of top10 miRNAs
regulation of transcription
antigen processing and presentation
regulation of RNA metabolic process
regulation of transcription, DNA-dependent
antigen processing and presentation of peptide or polysaccharide antigen via MHC class II
MHC class II protein complex
MHC protein complex
large ribosomal subunit
transcription regulator activity
sequence-specific DNA binding
ligand-dependent nuclear receptor activity
transcription factor activity
steroid hormone receptor activity
phosphatase regulator activity
protein phosphatase regulator activity
KEGG pathway analysis of potential targets of top10 miRNAs
ssc04514: Cell adhesion molecules (CAMs)
CADM3, CD4, CD40, F11R, LOC100521555, SELE, SELL, SELP, SLA, SLA-DMA, SLA-DOA, SLA-DOB, SLA-DRA, SLA-DRB1
CD40, SLA, SLA-DMA, SLA-DOA, SLA-DOB, SLA-DRA, SLA-DRB1
ssc04672: Intestinal immune network for IgA production
CD40, SLA, SLA-DMA, SLA-DOA, SLA-DOB, SLA-DRA, SLA-DRB1, TGFB3
ssc04330: Notch signaling pathway
DLL4, PTCRA, LOC733643, APH1A, NOTCH4
ssc04612: Antigen processing and presentation
CD4, LOC100152370, NFYB, PSME1, SLA, SLA-DMA, SLA-DOA, SLA-DOB, SLA-DRA, SLA-DRB1
AGPAT1, AGPAT4, |AGPAT6, GNPAT, LOC100152491, PCYT1B
ssc00020:Citrate cycle (TCA cycle)
ACO2,DLST, IDH2, LOC100157889, PCK1
Targets of miRNAs in IgA immune network
In the present study, a comprehensive miRNA expression profile of porcine breast milk exosomes was explored via a deep sequencing approach. We found in total 176 known miRNAs (miRBase 20.0) and 366 pre-miRNAs producing 315 mature miRNAs. Luciferase reporter assay was used to explore the targets of two predicted novel miRNAs in this study. Results indicated both of them down-regulated the luciferase expression by targeting 3’UTR of IGF-1R. All these pre-miRNAs were distributed in 30 clusters (11 novel and 19 known clusters), and the mature miRNAs could be assigned to 35 families (26 known and 9 unknown families). GO and KEGG pathway analyses show that those miRNAs may participate in many different immune-related processes. An analysis of the top 20 miRNAs showed that 14 of them may be involved in many regulatory aspects of the IgA immune network.
A recent study of exosome miRNAs in Yorkshire sow milk discovered 180 pre-miRNAs, including 140 known porcine pre-miRNAs and 40 novel pre-miRNAs, which encode 237 mature miRNAs (234 unique miRNAs) In the current study, we discovered 205 known porcine pre-miRNAs (176 mature miRNAs) and 366 novel pre-miRNAs (315 mature miRNAs), approximately 254 more mature miRNAs than were revealed in the former report. Therefore, our results substantially supplement the known pig miRNAs, particularly milk exosome miRNAs. Interestingly, most of the novel miRNAs were low in abundance (312 miRNAs with less than 100 reads and only 3 miRNAs with >100 reads), which is possibly the reason for why these miRNAs were not detected in a previous study by Gu et al.. Further comparison revealed that miR-191 and let-7a, which potentially play a vital role in immunity, were found in both that study and in the top 10 miRNAs of our study. Other miRNAs identified previously (miR-30a-5p, miR-25-3p, miR-182-5p, miR-200c-3p and miR-375-3p) were not detected in our study. Furthermore, miR-148a, a potential biomarker for quality control in bovine milk and human milk, which was found to be highly expressed throughout the lactation period of Yorkshire sows, was only detected at a moderate level in Landrace pigs in our study. MiR-148a has been reported to be a tumor metastasis suppressor in gastric cancer, and ectopic expression of miR-148a was shown to induce apoptosis and silence Bcl-2 in colorectal cancer cells. By bioinformatics analysis, miR-148a was determined to be possibly related to immunity and gastrointestinal health, but the underlying regulatory mechanism remains unclear.
MiR-92a belongs to the miR-17 ~92 cluster with seven miRNAs (miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-19b, miR-20a and miR-92a) and was first described as an oncogenic miRNA cluster involved in B-cell lymphoma. Recent studies indicated that the miRNA-17-92 (miR-17-92) cluster directly targets the TGFB pathway in cancer cell lines in the mouse embryo stage. In addition, the miR-17-92 cluster also participates in normal development of the heart, lungs and immune system. MiR-19 can promote leukemogenesis in Notch1-induced T-cell acute lymphoblastic leukemia (T-ALL) in vivo. Overexpression of the mir-17–mir-18a–mir-19b-1 cluster was shown to accelerate Myc-induced tumor development in a mouse B-cell lymphoma model. The combined results above imply that members of the cluster miR-363/92a/19b-2/20b/106a may be related to cell proliferation and development. In porcine milk, miR-363/92a/19b-2/20b (miR-363/92a/19b-2/20b/106a) and miR-17/18a/19b-1/92-1 were also detected. The miR-181 (181a/b/c/d) family is related to the development of different cells. It was reported that miR-181c/d can inhibit cell cycle and proliferation and that miR-181c regulates TNF-α. The miR-30(b/c/d/e) family regulates kidney development by targeting the transcription factor Xlim1/Lhx1 in Xenopus. The well-known let-7(a/b/d/f) family is involved in oncogene expression, and let-7/miR-98 family members are expressed late in mammalian embryonic development. Thus, these miRNAs mentioned above may participate in development of the piglet digestive tract.
Notably, some miRNAs among the top 10 identified here have been reported to be related to immunity (miR-320, miR-181a, miR-30a-3p, let-7a, let-7f and let-7c) and development (miR-193a-3p, miR-378 and miR-191). MiR-193a-3p was demonstrated to regulate cell proliferation, cell cycle progression in vitro and in nude mice. MiR-378 promotes osteoblast differentiation by targeting polypeptide N-acetylgalactosaminyltransferase 7 (GalNAc-T7 or GalNT7), and miR-191 regulates erythroid differentiation in mammals by up-regulating erythroid-enriched genes Riok3 and Mxi1. Meanwhile, miR-320 is able to inhibit HL-60 cell proliferation by suppressing receptor 1 (TfR-1; CD71), and miR-181a was believed to act as an intrinsic antigen sensitivity “rheostat” during T cell development. MiR-320, miR-181a, miR-30a-3p and let-7 were shown to be downregulated in colorectal cancer. Of course, further experimental evidence is needed to verify that these miRNAs are indeed related to immunity of the piglet digestive tract.
IgA is a major immunoglobulin in milk. Expression of the polymeric IgA receptor (pIgR) in mammary epithelial cells contribute much to the development of the immune system at the early stage of lactation. In the present study, some miRNAs were predicted to target genes (CD40, SLA, SLA-DMA, SLA-DOA, SLA-DOB, SLA-DRA, SLA-DRB1 and TGFB3) involved in processes of the intestinal immune network for IgA production in porcine milk. CD40 is a B-cell antigen activated during immune responses. CD40 and CD40 ligand (CD40L) expressed on activated T cells are essential to B cell proliferation and secretion of IgG, IgA and IgE. SLA Class I were found to be expressed in the epithelial and lamina propria cells of the intestine in adult pigs and to be involved in mother-newborn interactions. A study in humans showed that TGF-β acts as a specific switch for IgA present at early stages of development of B cells.
In the present study, the top 20 miRNAs were used for IgA network analysis. APRIL was the predicted target of miR-193a-5p, which is essential to triggering IgA2 class switch in human B cells. Intestinal epithelial cells (IECs) release APRIL after sensing bacteria through Toll-like receptors, and mucosal vaccines activate IECs to induce more effective IgA2 responses. The let7 family and miR-423-5p were predicted to target CCL25, a potent and selective chemoattractant for IgA antibody-secreting cells. CCL25 is known to selectively modulate immune responses, specifically the localization of T lymphocytes to the small-intestinal mucosa. CD80 and CD86, which are costimulators of T lymphocytes, were identified as possible targets of five miRNAs in our study. Let-7a, let-7c, miR-181b, miR-185, miR-378 and miR-423-5p were predicted to target the inducible co-stimulatory molecule (ICOS), which plays a key role in regulating T-cell differentiation, T-cell proliferation, and secretion of lymphokines, providing effective help for antibody secretion by B cells. We hypothesize that some miRNAs identified here in porcine milk regulate IgA production in the intestine of piglets, which may play an important role in mucosa immunity. However, their regulatory mechanisms warrant further study.
In conclusion, the present study revealed 176 known miRNAs and 366 (315 mature miRNAs) novel pre-miRNAs in porcine milk, most of which were predicted to be involved in regulation of digestive tract development and immunity of newborn piglets. These findings contribute to an increased understanding of the roles of miRNAs in porcine (S. scrofa) milk exosomes and to building the foundation for understanding their physiological functions and regulatory mechanisms.
All the supporting data has been deposited in https://mynotebook.labarchives.com/share/allinchen/MTkuNXwxMzMxMS8xNS0yL1RyZWVOb2RlLzE1NzEyODU2fDQ5LjU= with a DOI:10.6070/H4DN432G.
This work was supported by grants from the Key Project of Guangdong Provincial Nature Science Foundation (S2013020012766), National Basic Research Program of China (973 Program, 2011CB944200, 2009CB941600 and 2013CB127304), Natural Science Foundation of China program (31272529) and the Natural Science Foundation of Guangdong Province (S2013010013215). We thank the breeding farm of the Livestock Research Institute (Guangzhou, China) for providing milk samples.
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