Effect of Sterilized Rumen Fluid Supplementation Ways in Lactation Period on Rumen Microora of Calves after Weaning

Background: The presence of the rumen makes the ruminant special, and the ruminal microbes has an important inuence on the ruminant. The rumen have an important developed in young ruminants. This study want to investigate the ecological function of rumen uid environmen from high-yielding dairy cows and effect of feeding of rumen uid to suckling calves. For the experiment, 12 newborn Holstein male calves with the same feeding environment and similar ages were selected. They were randomly divided into 3 groups, with 4 in each group. The three treatments were: addition of sterilized rumen uid to milk (Group M), addition of sterilized rumen uid to starter feed (Group S), and control group fed milk and starter feed(Group C). The growth performance indices and blood indices were measured, and rumen uid samples were collected after weaning,which used for 16S rDNA sequencing and LC-MS metabolome detection were performed. Results: Compared with the control group, the growth performance of group S was signicantly increased. For the rumen uid microbiome and metabolome detection, both groups M and S showed signicant differences from group C. Conclusions: The microbes and metabolites were different due to different supplementary feeding numerical indicate that which promoting better D-ribulose 5-phosphate, ergothioneine, thymidine 5'-monophosphate, cytidine 5'-monophosphate, UDP-D-galactose, uracil, urocanic acid, xanthine, N- acetyl-L-glutamate, salidroside. The main metabolic pathways involved include: pyrimidine metabolism (ko00240), pentose phosphate pathway (ko00030), vitamin B6 metabolism (ko00750), riboavin metabolism (ko00740), carbon metabolism (ko01200), amino acid biosynthesis (ko01230), pentose and glucuronic acid (ko00040), histidine metabolism (ko00340), amino sugar and nucleoside sugar metabolism (ko00520), galactose metabolism (ko00052), pantothenate and CoA biosynthesis (ko00770), β-alanine metabolism (ko00410), purine metabolism (ko00230), 2-oxocarboxylic acid metabolism (ko01210), arginine biosynthesis (ko00220), tyrosine metabolism (ko00350). Metabolites with signicantly decreased levels include: 5-Aminopentanoic acid, and the major metabolic pathways involved: arginine and proline metabolism (ko00330), lysine degradation (ko00310).

types of vitamin B may not be able to meet the growth needs of some speci c bacteria [7]. Production of ammonia through decomposition of proteins and amino acids in the rumen is an important prerequisite for the synthesis of microbial proteins. Studies have shown that ammonia and butyric acid substances produced by Prevotella degradation of casein can be utilized by Ruminococcus albus [8]. Muscato conducted four tests to infuse different rumen uid in 6-week-old calves at a dose of 8 mL/day by adding rumen uid to milk. The rumen uid fed in the experiment included fresh rumen uid, supernatant of rumen uid, cells of rumen uid and rumen uid sterilized by high-pressure steam [9]. The results prove that the rumen contents can promote the normal development of the rumen.
Under the intervention of exogenous rumen micro ora and diet , the adult rumen micro ora had strong anti-interference ability and restored the original components [10], indicating that it product bene cial effects in adult life through early life intervention on microbes. In fact, the change of rumen microbes have successfully achieved long-lasting results through intervene diet of Young ruminant [11,12]. In the past, dietary intervention strategies was mainly used to alter the rumen microbes but it's ine cient for adult ruminants, which has led to increasing interest in understanding microbial interactions in the rumen of young ruminant [13]. It has been reported that restricting access to protozoa in the early life of lambs also changes rumen microbial composition and fermentation as well as urine metabolites [14]. Therefore, these studies suggest that changes in the early rumen microbiota may affect the microbial succession process, and change the host phenotype.

Results
Growth performance results Table 2 shows the effects of the supplemental sterilized rumen uid treatments on growth and feed intake of the calf. There was no signi cant difference in the growth index of the calves between the groups before the test (P>0.05). Within ten weeks, the daily weight gain of the S group was signi cantly higher than that of the C group (P<0.05). Table 3 shows the changes in the calf serum index in each group. Due to differences among the individual test animals, the calf serum index (supplementation 1) between the groups before the test was statistically different, and this difference could not be avoided. Therefore, the change in serum results was statistically analyzed. The numerical magnitude represents the amount of change at two points in time, positive or negative indicating an increase or decrease. A comparison was made between after treatment and after treatment for a period of time, the rst stage was from 7 days to 15 days of age; the second stage was from 15 days to 63 days of age. It can be seen from table 3 that the experimental treatment had signi cant effects for most of the indicators during the two stages, but the effect on TNF-α was not signi cant in the rst stage and IgG in the second stage. The serum IL-1β increased in all the experimental groups, but the changes in M group and S group were signi cantly lower than that in group C (P<0.05). In the second stage, the C group showed an increasing trend, whereas the M and S groups showed signi cant decreasing trends (P<0.05). Serum TNF-α showed an increasing trend in all the rststage trials, the second-stage M and S groups showed a decreasing trend, and the change was signi cantly different from that of the C group (P<0.05). The amounts of serum IL-4 in M and S groups during the rst stage were signi cantly lower than that in the C group(P<0.05). Serum IFN-γ decreased in all the groups in the rst stage, but the decrease in the M group was signi cantly lower than that in the C group. Serum LP showed an increasing trend for all treatment groups in the rst stage, while group C showed a downward trend; the amount of change was signi cantly different (P<0.05).

16S rDNA sequencing results
Single sample diversity analysis From Figure 1, we found that the dilution curves of all samples tended to be at, indicating that the samples were adequately sequenced and the depth covered almost all species in the sample.

OTU analysis
The tags were clustered at the 97% similarity level to obtain OTU, and OTU was classi ed based on the Silva (bacteria) taxonomy database to obtain the OTU number of each sample. A total of 355 OTUs were obtained from 12 samples. There were 337 OTUs in the control group, with 315 OTUs in groups M and S. It can be seen from the Venn diagram that 280 of the OTUs were shared between the three groups; 19 OTUs were only in group C, 1 OTUs was only in group M, and 3 OTUs were only in group S (Fig. 2).

Species annotation and taxonomic analysis
The OTU representative sequence was compared with the microbial reference database to obtain species classi cation information corresponding to each OTU. Furthermore, the composition of each sample community was counted at each level (phylum, class, order, family, genus, species), and the abundance for each species at different classi cation levels was obtained.
At the phylum level, 11, 11, and 10 phyla were detected in rumen uid of groups C, M, and S, respectively ( Figure 3). Group Figure 5, we can see the proportion and relationship of different species among the three groups. Proteobacteria had the largest proportion in group C and the smallest proportion in group S. Fibrobacteres and Bacteroidetes have the largest proportion in group S and extremely small in group M. Tenericutes has the largest proportion in group C and the smallest proportion in group M, whereas Firmicutes have the largest proportion in group M and the smallest proportion in group C.

Metabolite volcano map analysis
The volcano map can be used to quickly view the differences in metabolite expression levels between the two groups, as well as the statistical signi cances of these differences. The differential expression volcano is as follows: Compared with group C, group M had 42 signi cant metabolites in positive ion mode, of which, levels of 3 were signi cantly down and levels of 39 were signi cantly up; There were 5 signi cant metabolites in negative ion mode, of which the level of 1 was signi cantly down and levels of 4 were signi cantly up. Compared with group C, group S had 38 signi cant metabolites in positive ion mode, of which, levels of 9 were signi cantly down and levels of 29 were signi cantly up; There were 27 signi cant metabolites in negative ion mode, of which, levels of 10 were signi cantly down and levels of 17 were signi cantly up (Figure 7).

Discussion
Effect of feeding rumen uid on the growth of calves Muscato supplemented calves with different treatments of rumen uid from healthy cows. The results showed that supplemental feeding of rumen uid signi cantly increased calf body weight gain during the rst 2 weeks (P<0.05) [9]. Zhang Lihua supplemented lambs with rumen uid preparations, and increased the daily gain of lambs in the experimental group. James gave newborn yak calves duodenal juice along with the initial colostrum feeding. The results showed that feeding duodenal juice can increase the daily gain of 7-day-old yak after birth [15]. The results of ours experiment showed that supplemental feeding of rumen uid could improve the growth performance of calves to a certain extent. From the numerical point of view, supplemental feeding of rumen uid with milk is better than starter.

Effect of feeding rumen uid on calf serum
IgA cannot pass through the placenta, and there is no IgA antibody in the newborn calf serum, which will gradually increase after birth and account for a small amount of total serum immunoglobulin. Serum IgG is the major antibody component of serum and accounts for the majority of serum immunoglobulins. The content of IgA and IgG in serum can re ect the establishment of the animal's immune system. The rst phase of the test was from 7 days to 15 days, and at this stage, the calves are no longer passively immunized by colostrum; during this phase, IgG decreased in the control group, which may be related to this. In the experiment, supplementing with rumen uid may increase the serum IgG content at this stage. In the rst stage, TNF-α increased in each group, but the changes in groups M and S were greater than those in group C. Lipopolysaccharide (LPS) is a component of the cell wall of Gram-negative bacteria, and strongly stimulates production of TNF-α. TNF-α can directly kill tumor cells and is not toxic to normal cells. The rumen uid of group M and group S may contain LPS. IFN-γ has antiviral, immunomodulatory and antitumor properties, and its concentration positively correlates with viral load. In the rst stage, the IFN-γ of all experimental groups showed a downward trend; during the second stage, group M still showed a downward trend while the other groups showed an increasing trend. These test results show that in some indicators, the supplemental feeding and ways of the sterilized rumen uid will have certain in uence on the serum index, and may have a bene cial effect on calves, but the mechanism needs further research.

Effect of feeding rumen uid on microbes in rumen uid of calves
The main micro ora in the rumen uid samples of groups M, S and C were Proteobacteria, Bacteroidetes and Firmicutes, and these results are consistent with a previous calf study of rumen micro ora on days 15-83 [16]. The rumen bacteria of unweaned calves mainly consist of three phyla: Bacteroidetes, Firmicutes and Proteobacteria. After weaning, the gastrointestinal tract develops and matures, and the abundance of each phyla may vary with different stages; however, these three phyla remain the most abundant [17]. The relative abundances of Oxalobacter and Dialister in groups M and S were signi cantly higher than those in group C, and Oxalobacter was not detected in group C. It has been reported that the relative abundances of Oxalobacter and Dialister are signi cantly increased in patients with diarrhea treated with probiotics [18]. In a bacterial colonization treatment of patients with in ammatory bowel disease (IBD), four microbial phylotypes were found in the intestinal tract of a patient with Crohn's disease (CD) who experienced transient remission, including Dialister. This study also suggested that the absolute abundance of bacteria in fecal samples used for micro ora transplantation could indicate the success of transplantation rather than the overall abundance relative to the recipient [19]. Selenomona was not detected in group M and group S, while it could be detected in group C. Studies have shown that the addition of a composite antimicrobial peptide reduces the genus Selenomona, which is associated with the degradation of starch [20]. The abundance of Firmicutes in group M was signi cantly higher than that in group C. Firmicutes have been shown to have cellulose and hemicellulose degrading enzymes [21,22]. Firmicutes mainly hydrolyze carbohydrates and proteins. Bacteroidetes mainly act on steroids, polysaccharides and bile acids, which help the body to absorb polysaccharides and perform protein synthesis [23,24]. Studies have found that the abundance of Firmicutes increases after weaning of male calves [17]. The Rumencoccaceae family in group M was signi cantly higher than in group C.
The Rumencoccaceae family was originally a benign species inhabiting the cecum and colon [25] and has been shown to degrade a variety of polysaccharides and brils [26], and represents the main brinolytic bacteria in the rumen. Compared with normal rat fecal ora, Rumencoccaceae is signi cantly reduced in colon cancer rats [27]. Studies suggest that with increasing age, there is a signi cant increase in the abundance of p2534-18b5_gut_group in the rumen of northern shaanxi white cashmere goats [28].
Although adult cow rumen uid was introduced into calves of group M, the p2534-18b5_gut_group in our study was signi cantly lower than that in group C. 16S rRNA analysis found that the intracellular Campylobacter-like organism (ICLO) of porcine proliferative enteropathy is closely related to desulfurization of Desulfovibrio [29], which can also be obtained from the rumen of sheep [30] and the human intestine [31,32]. Desulfovibrionales in Group M were signi cantly lower than in Group C; it is suggested that Mitsuokella may inhibit the growth of Salmonella in the gut [33], and interestingly, Mitsuokella was signi cantly lower in group S than in group C. Prevotella is usually found in the rumen of herbivores such as cattle and sheep, and the most abundant strain is also found in the human gut. It is known to contain enzymes that degrade various polysaccharides (including xylan) [34] and is a starchdegrading bacterium. Prevotella also plays a role in the degradation of proteins and the absorption and fermentation of peptides in the rumen. The abundance of Prevotella_7 in group S was signi cantly higher than in group C. Lactobacillus paracasei LC01 can signi cantly improve the intestinal Ruminococcaceae_UCG014 in mice when introduced by gastric irrigation [35]. The relative abundance of Ruminococcaceae NK4A214 group and Ruminococcaceae UCG -014 increased with increasing feed NDF levels [36]. The abundance of Ruminococcaceae UCG-014 in group S was signi cantly higher than in group C, but the abundance of Ruminococcaceae NK4A214 group was signi cantly lower in group S than group C. Synergistetes is a strict gram-negative anaerobic bacterium, and the abundance of ruminal Syntrophococcus is signi cantly increased with rumen acidosis . Calves fed starter supplemented with rumen uid, which would enter the rumen. After weaning, the rumen uid microbes of the S group produced signi cant differences relative to group C in the abundances of some species. It is speculated that the rumen uid directly enters the rumen and directly contacts the internal microorganisms and the rumen, thus affecting the rumen microorganisms. These differential microbial species may become the focus of the "peripheral microbe" species which are affected by the environment. The rumen uid supplementation of calves in group M was added through milk. We believe that milk bypasses the rumen and directly enters the abomasum due to the oesophageal groove in calves; however, no studies have shown that the addition of other ingredients in milk affects the closure of the oesophageal groove. After weaning, the abundances of some rumen uid micro ora species in group M were also signi cantly different from those in group C, but these species were in complete accordance with those in group S. The results suggested that rumen uid entering the abomasum may have some effects on the host, and the changes in host metabolism may also act on the rumen, indirectly changing the micro ora in the rumen. These different microbial species may become the focus of studies on the "core microbial" species affected by the host. It can be seen from this experiment that the addition of high-yield cow rumen uid in milk and starter diet can positively affect growth on the basis of maintaining normal rumen microorganisms. However, due to the different paths the rumen uid entered the body, there were differences in microorganism populations enhanced by the two supplementary feeding methods.
Effects of rumen uid supplementation on rumen uid metabolites and metabolic pathways in calves After the addition of rumen uid in milk and starter, the metabolites related to nucleotide metabolism, amino acid metabolism, carbohydrate metabolism, lipid metabolism, coenzyme and vitamin metabolism in rumen uid metabolites of calves signi cantly increased, while some metabolites signi cantly decreased. Among them, the main effects involved nucleotide metabolism, amino acid metabolism and sugar metabolism pathways.Lipid metabolism pathway associated myristic acid and coenzyme vitamin metabolic pathway associated thiamine were signi cantly reduced in group M. In group S, no differential metabolites related to lipid metabolism pathway were found and the 5-aminopentanoic acid associated with the amino acid metabolic pathway was signi cantly reduced. In amino acid metabolism, the carbon framework remaining after amino acid deamination can be used for the synthesis of various volatile fatty acids, and bacterial deamination can serve as an energy source for these bacteria. Bladen was isolated from a group of strains with high ammonia-producing activity and polysaccharide degradation activity, which could be maintained by fermentation of amino acids [37]. Ciliate has a strong deamination effect and the deamination of ciliate is mainly targeted at some amino acids of lesser abundances, such as citrulline, arginine and ornithine. In addition, protozoa can degrade amino acids to produce other products, such as thio-methionine to alpha-aminovaleric acid. In the process of bacterial growth, different bacteria use different amino acids, which alters the mode of amino acid deamination mode when the animals eat different diets, and this relates to the bacterial species in the rumen. Mcallan did an in vitro experiment on the degradation of nucleic acids in rumen uid. Pyrimidine nucleotides can form uracil and thymine, and cytosine can become uracil after deamination. Purine nucleotides can form hypoxanthine nucleotides and xanthine [38]. Nucleotide degradation by protozoa mainly depends on nucleotides derived from rumen bacteria. Gs studies on protozoa found that Entodinium caudatum and Eudiplodinum maggii can convert adenine and guanine into hypoxanthine and jaundice, and Entodinium caudatum can absorb bacterial nucleotides [39]. Some rumen bacteria can use adenine and guanine as nitrogen sources, and this ability may be related to the deamination and utilization of the released ammonia. Many rumen bacteria also produce extracellular nucleases, and these may be involved in the degradation of DNA.
Carbohydrates in the rumen are degraded in two stages: initially, they are degraded into simple sugars by various enzymes; they are subsequently used by microorganisms in the rumen; nally, they are converted into acetone for fermentation through various metabolic pathways. The arginine metabolic pathway has been linked with many bacteria [40,41] and Pseudomonas [42,43]in previous studies. Involvement of a novel transaminase family in the arginine biosynthesis pathway has been previously reported [44]. In xanthomonas, N-acetylornithine is converted to N-acetylcitrulline by an enzyme encoded by arg F' [45]. Arg E deacetylates N-acetylcitrulline to form citrulline. Xylella, Bacteriodes, Cytophaga, and fungi contain homologs of arg F'. The Bacteroides fragilis Arg F' is essential for the biosynthesis of arginine [46].Through the combined analysis of microbial-metabolite-metabolic pathway, we found that l-citrulline (L-Citrulline) in group M was signi cantly higher than that in group C (P < 0.05). It was involved in the arginine biosanabolism pathway, and it was also related to the bacteria Prevotella_7 and Odoribacter in the gastric uid of tumor, both of which belong to bacteroides. Ergosine (EGT), which has immunomodulatory effects [47], can be synthesized by non-yeast fungi, some Actinomycetes bacteria and Cyanobacteria, but not in animals [48]. M. smegmatis can produce EGT gene (egtA, egtB, egtC, egtD, egtE). EgtD has been proved to be a histidine methyltransferase, which converts histidine into histidine trimethionine through S-adenosylmethionine [49]. Studies have shown that rumen bacteria degrade histidine, producing uric acid, N-formamide-glutamic acid, formamide and glutamic acid [50]. Uric acid is an intermediate in the normal catabolism of histidine. The idea that free amino acids are intermediates in the breakdown of proteins by rumen microorganisms has been widely accepted. In addition, there is a large amount of circumstantial evidence that the intermediates of the degradation process are also important substrates for biosynthetic processes of rumen microorganisms [51]. Many substances related to amino acids, saccharide and nucleotides were detected in rumen uid of calves in group M and group S at higher concentrations than those in group C. These amino acids can be used as substrates for the synthesis of microbial proteins, and carbohydrates can provide energy for the synthesis of microbial proteins. This suggests that calves may gain more microbial proteins due to increased metabolism in many metabolic pathways by rumen uid supplementation.

Conclusions
Sterilized rumen uid supplementation during the lactation period can promote the growth of calves after weaning. It is better to feed through starter than through milk. Both supplementation methods have an effect on the microbiome and metabolome of calf rumen uid, but the effects of the two methods are different. The two supplementation methods involved entry of rumen uid into different parts of the body, thereby having differential effects on microorganisms. Therefore, it can be seen that the host can affect the rumen microorganisms.

Test design
For the experiment, 12 newborn Holstein male calve of similar age were single column feed and accepted the same feeding regimen. They were randomly divided into 3 groups on average. The treatments were as follows: addition of sterilized rumen uid to milk (Group M), addition of sterilized rumen uid to starter feed (Group S), and fed only milk and starter as control group (Group C). They were fed twice a day and water free.The 20 mL sterilized rumen uid was added to their diet at the every day of the second week. The sterilized rumen uid was extracted from a adult cow with an oral sampler; it ltered through four layers of gauze, decanted into a sterile cryotube, placed in an ultra-low temperature freezer, and repeatedly frozen and thawed 10 times. The repeated freezing and thawing uses the ice crystals inside the microorganisms to break their cell walls through the mechanical action of freezing-thawing, releasing some cellular components but avoiding the destruction of rumen uid components by chemical reagents or high temperature. The processed rumen uid was added to the milk and the starter to supplement the calves in groups M and S, respectively.
All calves come from Jinan High speed pastures and they were single column feed.

Calves feeding management
After birth, the umbilical cord was soaked in 7-10% of iodine, and birthweight was determined. Highquality pasteurized colostrum was fed, for a total of 4L in the rst hour (8-10% of calf body weight), and an additional 3-4L of colostrum was fed in 6-8 hours to ensure that the calf has 6-8L colostrum in 12 hours; colostrum was provided at 36 to 40℃ temperature. Normal milk was fed 8 hours after feeding colostrum, and was provided twice daily at 7:00 am and 15:00 pm. Calves that could not drink milk properly were fed using a bottle. On the 4th day of birth, calves were fed from a milk bucket, which was provided throughout the day. Water was supplied to the calves on the third day after birth. The freely available drinking water was warm water in winter, and provided 24 hours a day. Calves are fed daily according to standard (Table 1).

Sample collection and processing
Feed intake During the test period, the amount of feed and the amount of feed remaining were accurately recorded every day.

Weight and body size measurement
The whole body size was measured by the same person before and after the trial . Cannon circumference: The circumference measured at one third of the forelimb cannon bone, measured with a tape measure.
Body weight was measured before and after the treatments.

Blood collection and sample preparation
The trial lasted 70 days. We collected 20 mL jugular venous blood in the morning at 7 , 15 and 60 days old per cow. After the blood was collected, it was placed in a coagulation tube and allowed to stand at room temperature for 30 min. When the serum was precipitated, it was centrifuged at 3000 r/min for 15 min in a low-speed centrifuge, and the serum was aspirated by a pipette and dispensed into a 1.5 mL centrifuge tube and stored at -20 ℃ .

Collection of rumen uid
Week 10 ended, squeezed rumen uid from the rumen chyme after slaughter, ltered through four layers of gauze, and placed in a 5 mL cryotube, placed at -80 °C for testing.

Sample analysis
Analysis of microbial diversity in rumen uid based on 16S rDNA V3+V4 region After extracting the total DNA of the sample, primers were designed according to the conserved regions; sequencing primers were added at the end of the primers. PCR ampli cation was carried out, and the products were puri ed, quanti ed and homogenized to form a sequencing library, which was sequenced on the Illumina HiSeq 2500. The sequences were clustered at a level of similarity of 97%.

Metabolomics analysis of rumen uid based on LC-MS
Samples were analyzed with the Agilent 1290 UHPLC Ultra High Liquid Meter and AB 5600 Triple TOF Mass Spectrometer. The results were analyzed by statistical methods using orthogonal projections to latent structures-disciplinant analysis (OPLS-DA). Differential metabolites were screened by combining the P value of the Student's t-test with the VIP value of the OPLS-DA model. The criteria for screening were P value <0.05 and VIP >1.5.

Data analysis
The raw data was statistically processed using EXCEL. The data was analyzed using the one-way ANOVA model in SAS 8.2 software and compared using Duncan's multiple range test. P<0.01 indicates that the difference is extremely signi cant, 0.01<P<0.05 indicates that the difference is signi cant, and 0.05<P<0.1 indicates that there is a tendency of difference, and the result is expressed in the form of This study was carried out in accordance with the principles of the Basel Declaration and recommendations of Laboratory Animals-Guideline ,Welfare and Health Committee of Shandong Agricultural University.The protocol was approved by the ethics committee of Shandong Agricultural University. All study participants were consent to proceed with this project verbally. We also obtained written informed consent to use the cows in the study from the owner of the cows.

Consent for publication
Not applicable Availability of data and materials All data generated or analysed during this study are included in this published article [and its supplementary information les].

Competing interests
The authors declare that they have no competing interests.      Figure 1 Sample-based rarefaction curve of observed species Shared OTU across different groups  C_vs_S.volcano

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