| Cells can communicate with neighboring or distant cells through complex packets stuffed with selected proteins,lipids,and nucleic acids,called exosomes.Exosomes are synthesized by a variety of cells and secreted into blood,urine,saliva,milk,amniotic fluid,ascites and cerebrospinal fluid,helping extensively regulate the body’s physiological and pathological processes.Recent studies have suggested that the mi RNAs in exosomes of cow blood and milk could be used as potential molecular markers of diseases,such as rumen acidosis and mastitis.At the same time,some scholars pointed out that milk exosomes contained many proteins,which conformed to the definition of food active ingredients.However,there are few reports on cow exosomes in China.We have shown that non-forage fiber sources(NFFS)could change the rumen microflora of Holstein cows,significantly reducing the ratio of acetate and propinate.And this study further explored the effects of NFFS diets on the blood exosomal mi RNA,as well as the milk bioactive exosomal components in Holstein cows.Twelve healthy Holstein cows(6 cows in each group)were randomly divided into the basal diet group and NFFS diet group(8.97% alfalfa hay and 2.51% corn silage were replaced by 5.72% whole cottonseed and 4.73% soybean hulls in the basal diet group),decreasing the forage neutral detergent fiber from 20.92% to 15.67%.The pre-feeding period was 15 days and the test period was 30 days.Blood and milk were collected before and after the test period.Firstly,blood exosomes were extracted by immunoaffinity adsorption method,and identified by nanoparticle tracking analysis and transmission electron microscopy.Then,the effects of NFFS diets on blood exosomes mi RNAs were analyzed by high-throughput sequencing.Secondly,the effects of NFFS diets on the milk exosomes mi RNAs was investigated by the same methods as that in blood after the exosomes were isolated by precipitation method.In addition,the effects of NFFS diet on exosome-proteins in milk were studied by isotope labeling relative and absolute quantitative techniques.Transmission electron microscopy showed that bovine serum and milk exosomes were rounded,wrapped by biofilm.Nanoparticle tracking analysis showed that the diameters of serum and milk exosomes were mainly distributed in 40-200 nm.RNA-Seq showed that bovine serum and milk exosomes contained a variety of small RNA,including mi RNA,sn RNA,ti RNA,pi RNA,cis-regulatory elements,etc.And mi RNA accounted for about 82% of the small RNA in serum exosomes,while the mi RNA ratio decreased to 22% in milk exosomes.A total of 359 mi RNAs were identified in the serum exosomes,among which,the expression level of bta-mi R-126-5p,bta-mi R-199a-3p,bta-mi R-2285 bc,bta-mi R-2288,bta-mi R-3613 a,bta-mi R-450 b and bta-mi R-500 significantly changed responding to the NFFS substitution.A total of 276 mi RNAs were identified in milk exosomes,among which,the expression level of bta-mi R-10167-3p,bta-mi R-103,bta-mi R-11973,bta-mi R-29 c,bta-mi R-302 d,bta-mi R-328,bta-mi R-383,bta-mi R-760-3p and bta-mi R-885 were significantly changed responding to the NFFS substitution.In addition,functional analysis of the top 20 highly expressed serum exosomal mi RNAs showed that about 80% of the mi RNAs were immune related.Similarly,about 76% of the mi RNAs in bovine milk were immune related.At the same time,bta-mir-26 a,bta-let-7b,bta-let-7a-5p,bta-mir-99a-5p,bta-mir-30 d,bta-mir-191,bta-mir-151-5p,bta-let-7f,bta-mir-423-5p,bta-mir-486 and bta-mir-125 b were highly expressed in both blood and milk exosomes,implying that some milk exosomal mi RNA mighe be derived from blood.A total of 488 proteins were identified in the milk exosomes,among which 40 proteins were significantly down-regulated and 25 proteins were significantly down-regulated.The functions of differentially expressed proteins are mainly enriched in complement and coagulation cascades,chemokine signaling pathway,phagocytosis and resistance to bacterial infection.In addition,57 proteins with high and stable expression cross species and body fluids were identified in this study,mainly enzymes and enzyme regulators,cytoskeleton proteins and transportation-related proteins.These proteins reflected the commonness of exosomes and were independent of species and different cell sources.The blood and milk exosomes of Holstein cows are successfully isolated and identified in this study,which provides a complete workflow reference for follow-up studies on cow exosomes.In addition,this study found that milk exosomes containe a large number of immune-related mi RNA and proteins,revealing the immune mechanism of milk from the perspective of exosomes.Cows’ blood and milk exosomal mi RNA can be affected by NFFS diets,suggesting that dietary factors should not be ignored when selecting exosomal mi RNA as disease diagnostic markers.This study provides scientific references for broadening the diagnostic markers of cow disease and new information on milk bioactive ingredients. |
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