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Exploration of the Relationship between Intestinal Colostrum or Milk, and Serum Metabolites in Neonatal Calves by Metabolomics Analysis Yunxia Qi, Xiaowei Zhao, dongwei huang, xiaocheng pan, huiling zhao, Han Hu, Guanglong Cheng, and yongxin yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01621 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 25, 2018

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Journal of Agricultural and Food Chemistry

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Exploration of the Relationship between Intestinal Colostrum or Milk, and Serum

2

Metabolites in Neonatal Calves by Metabolomics Analysis

3 4

Yunxia Qi a †, Xiaowei Zhao a†, Dongwei Huang a, Xiaocheng Pan a, Yongxin Yang a*,

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Huiling Zhao a, Han Hu b, Guanglong Cheng a

6

a

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Sciences, Hefei 230031, China

8

b

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Agriculture, Chinese Academy of Agricultural Sciences, Beijing 100093, China

Institute of Animal Science and Veterinary Medicine, Anhui Academy of Agricultural

Institute of Apicultural Research/Key Laboratory of Pollinating Insect Biology, Ministry of

10

†

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* Corresponding author: Yongxin Yang

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E-mail: [email protected]

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Tel.: +86 551 65146065.

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Fax: +86 551 62160275.

These authors contributed equally to this work.

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1 ACS Paragon Plus Environment


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Abstract

17

In contrast to colostral immunoglobulins, changes in metabolite composition of ingested

18

colostrum in the gut have received little attention. Here, we characterized the metabolite

19

profiles of colostrum and milk, ingested colostrum and milk, and serum of neonatal calves by

20

LC–MS and GC–MS metabolomics approaches. Colostrum and milk underwent similar

21

changes in metabolite profiles in the gut after being ingested. These changes were

22

characterized by increase in methionine, glutamate, thymine and phosphorylcholine. After

23

ingestion, colostrum concentrations of several metabolites, such as γ-aminobutryic acid,

24

glutamate, cinnamic acid, and thymine increased, whereas concentrations of D-ribose, and

25

arginine decreased. These increases and decreases occurred in a time-dependent manner, and

26

were associated with alanine, aspartate, glutamate, and pyrimidine metabolism, and valine,

27

leucine and isoleucine biosynthesis, respectively. Meanwhile, similar changes in serum

28

metabolites were also observed in neonatal calves fed colostrum, which implies that

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colostrum metabolites are transported across the small intestine and into the bloodstream. In

30

addition, several metabolites of ingested milk were detected in the gut, and were also

31

transferred to the bloodstream. These metabolites were related to phenylalanine, tyrosine,

32

tryptophan, valine, leucine and isoleucine biosynthesis, the citrate cycle, and histidine

33

metabolism. These findings reveal that the serum metabolome of neonatal calves’ changes as

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a result of ingesting colostrum, which can provide health-related benefits in early life.

35 36

Keywords: Colostrum, Intestine, Metabolomics, Neonatal calves, Serum


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INTRODUCTION

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Colostral immunoglobulins (Igs) are important for the passive transfer of immunity in the

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first 24 h of life in ruminant neonates. 1, 2 In addition to Igs, colostrum also contains potential

40

immune components that enhance host defense against infection and promote intestinal

41

maturation.

42

instead of milk or formula. 5, 6 In calves fed colostrum, insulin-like growth factor-I and -II and

43

insulin are present in colostrum and blood and their receptors are expressed in the intestine. 7,

44

8

45

after colostrum intake, contributing to their metabolic and immune functions.

46

concentrations of total free essential amino acids, and several individual amino acids, such as

47

lysine, methionine, threonine, and glutamate, increased in serum within 12 h after calves

48

received colostrum.

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components were associated with changes in blood parameters of neonatal calves. To reveal

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the relationship between colostrum and serum metabolites, it is necessary to explore the

51

changes in metabolite composition of colostrum ingested in the gastrointestinal tract of

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neonatal calves. However, little is known about the changes in composition of small

53

molecules in ingested colostrum. A previous proteomics study of protein components of

54

colostrum and intestinal colostrum in piglets showed that their levels were altered by uptake

55

in the gut.

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components and of digestive enzymes in the gastrointestinal tract of the neonatal calves.

57

These results provide clues for a better understanding of the colostrum components

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metabolized and absorbed in the small intestine of neonatal calves.

3, 4

This has been demonstrated by studies in which calves were fed colostrum

Fat-soluble vitamin concentrations were found to be higher in the blood of neonatal calves

12

10, 11

9

In addition,

Taken together, these results demonstrated that several colostral

Colostrum intake has also been shown to affect the transport of other colostral


13


59

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Metabolomics is the global assessment of the complement of small molecules in biological 14

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samples

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platforms have been developed. These are: nuclear magnetic resonance spectroscopy, liquid

62

chromatography tandem-mass spectrometry (LC–MS) and gas chromatography-mass

63

spectrometry (GC–MS).

64

methods, as well as having a good dynamic range. In addition, the LC–MS procedure is

65

largely applied to the characterization of non-volatile, and high-molecular-weight metabolites,

66

whereas the GC–MS procedure provides complementary data to LC–MS analysis, and can be

67

applied to small volatile metabolites.

68

approaches have been widely used to detect metabolites in milk, blood, and other biofluids. 17,

69

18

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while colostrum contains more metabolites than milk.

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biofluids (milk, serum, rumen fluid, and urine) from lactating cows fed alfalfa hay and corn

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stover using GC–MS based metabolomics, and found differential metabolites involved in

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tyrosine, phenylalanine, glycine, serine, and threonine metabolism pathways. The authors

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suggested that differential metabolites and their related pathways may serve as biomarkers for

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higher milk yield and better milk protein quality.

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sensitive metabolomics methods could provide useful information for understanding the

77

metabolites of colostrum and their corresponding metabolites in the serum of neonatal calves.

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Based on previous studies, we hypothesized that changes in colostrum metabolized in the

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gastrointestinal tract and serum metabolites could be revealed by metabolomics approaches.

80

To address this issue, LC–MS and GC–MS based metabolomics techniques and multivariate

via

high-throughput

15

techniques,

and

three

major

analytical

Of these, LC–MS and GC–MS are highly sensitive and specific

16

Recently, LC–MS and GC–MS based metabolomics

Milk metabolites include amino acids, carbohydrates, lipids, vitamins, and nucleotides,

18

19

17

Recently, Sun et al. analyzed four

These results indicated that highly


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statistical analysis were used to investigate changes in serum and intestinal colostrum

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metabolite profiles of neonatal calves. Our results could provide insights into colostrum

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components ingested in the gastrointestinal tract and transported into the bloodstream of

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neonatal calves, which in turn can improve knowledge on current calf rearing practices.

85 86

MATERIALS AND METHODS

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Chemicals

88

Standard

DL-o-chlorophenylalanine

was purchased from GL Biochem Ltd. (Shanghai,

89

China). HPLC-grade acetonitrile, formic acid, and methanol were purchased from Merck

90

(Darmstadt, Germany). Methoxyamine hydrochloride, bis-(trimethylsilyl) trifluoroacetamide,

91

and pyridine were supplied by CNW (Shanghai, China). Ultrapure water used in LC– and

92

GC–MS was obtained from Milli-Q Ultrapure Water Systems (Merck, Darmstadt, Germany).

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Animals and experimental procedures

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Twenty-seven male Holstein calves with birth weight 40 ± 2 kg were used in this study.

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Animal care and use procedures were approved by the Animal Care Advisory Committee of

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the Anhui Academy of Agricultural Sciences (number A11-CS16). After birth, calves were

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separated from their mothers to prevent ingestion of colostrum. Colostrum from the first and

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second milking were pooled, and bulk milk from a herd was collected. Samples were pooled

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in large tubs, and the aliquoted into plastic bottles and stored at -20 °C. We used pooled

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colostrum from the first and second milking because previous studies demonstrated that

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colostrum from the first and second secretion from the mammary gland after birth was

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appropriate for IgG absorption in calves.

20

Before being given to the calves, colostrum or



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milk was incubated in a water bath at 40 °C and fed at approximately 8.0% body weight via a

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stomach tube, as described in previous studies. 21 It is widely accepted that gut closure occurs

105

approximately 24 h after birth in neonatal calves, and passive immunity transfer may take

106

place within 24 h after birth.

107

calves started to decrease from 6 to 12 h, and remarkably declined at 12 h after birth.

108

Thus, to investigate serum changes corresponding to colostrum ingestion and absorption in

109

the gut of the neonatal calves around the time of immunity transfer, calves were classified

110

into different experimental groups. Six calves were not fed colostrum or milk and were

111

defined as the control group (Ct group). Six calves received one colostrum meal at 1–2 h after

112

birth and were defined as the CI group. Six calves received two colostrum meals at 1–2 and

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10–12 h after birth and were defined as the CII group. Six calves received three colostrum

114

meals at 1–2, 10–12 and 22–24 h after birth and were defined as the CIII group. Three calves

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received two milk meals at 1–2 h and 10–12 h after birth, and were defined as milk group (M

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group, for a diagram of the workflow, see Figure S1).

22

Previous studies have found that colostral IgG absorption in 1, 23

117

Blood samples without anticoagulant were collected from the jugular vein approximately 2

118

h after birth for control calves, 8 h after birth for the CI group, 24 h after birth for the CII

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group, 36 h after birth for the CIII group and 24 h after birth for the milk group. Samples

120

were stored overnight at 20 °C and centrifuged at 3000 g for 15 min. Serum was collected,

121

and aliquoted into 1.5 mL tubes, and then frozen at -20 °C. After the blood samples were

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collected, the calves were slaughtered. Mid-duodenum, -jejunum and -ileum segments were

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separated within 30 min after opening the abdominal cavity.

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milk samples were collected from the mid-jejunum segment, frozen in liquid nitrogen, and


24

Ingested colostrum and/or

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then stored at -80 °C until analysis. As such, blood and colostrum samples were collected

126

approximately 2, 8, 24 and 36 h after birth, and used to characterize developmental changes

127

in serum and colostral components ingested in the gut of neonatal calves. Calves not fed at all

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and slaughtered approximately 2 h after birth were used as controls. In addition, calves in the

129

milk group were considered a positive control group for calves in the CII group, and this

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group was used as a positive control group to characterize the differences in colostrum and

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milk ingested in the gut.

132

Sample preparation

133

Three pooled colostrum samples, three bulk tank milk samples, and ingested colostrum or

134

milk samples from individual calves were thawed at room temperature, and centrifuged at

135

4000 g for 30 min at 4 °C. Skim milk was collected. Serum samples from individual calf

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were thawed at room temperature. Of these samples, one ingested colostrum sample from the

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CI, CII, and CIII groups each and one serum sample from a calf in the control group were too

138

small in volume for metabolomics analysis, and were excluded from the study. Samples of

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serum or skim milk (100 µL) were mixed with 300 µL methanol and 10 µL of 2.9 mg/mL

140

DL-o-chlorophenylalanine

141

and the supernatant was collected. For quality control (QC) samples, we pooled equal

142

volumes of skim milk from the colostrum and milk and their ingested products, and equal

143

volumes of serum from all samples. Prior to GC–MS analysis, the supernatant was first

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derivatized with 30 µL of 20 mg/mL methoxyamine hydrochloride in pyridine for 90 min at

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37 °C, and then with trimethylsilylated with 30 µL of bis-(trimethylsilyl) trifluoroacetamide

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for 1 h at 70 °C. Pooled samples were injected every several samples to evaluate

by vortexing. The mixture was centrifuged at 12000 g for 15 min



147

experimental reproducibility.

148

LC–MS analysis

149

Supernatants were subjected to LC–MS (Ultimate 3000LC Orbitrap Elite; Thermo Fisher

150

Scientific, Waltham, MA, USA), which was performed in both positive and negative ion

151

modes. For the former, spray voltage was set to 3.0 kV, heater temperature was 300 °C, and

152

capillary temperature was 350 °C. For the latter, spray voltage was set to 3.2 kV, heater

153

temperature was 300 °C, and capillary temperature was 350 °C. The scanning range of the

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full scan was set to 50–1000 m/z, with a scan time of 0.2 s and an inter-scan time of 0.02 s.

155

After equilibrating the column with 95% (v/v) buffer A (0.1% formic acid in water), sample

156

separation was performed with buffer B (0.1% formic acid in acetonitrile) using a Hypergod

157

C18 column (100 × 4.6 mm, 3 µm; Agilent Technologies, Santa Clara, CA, USA) at 40°C.

158

The gradient was 5%–95% buffer B at a flow rate of 300 nL/min for 15 min. The data were

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subjected to feature extraction and processed with SIEVE software (Thermo Fisher

160

Scientific). For each sample, duplicate technical repeats were performed. For colostrum and

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milk, 3783 and 2485 features were obtained in positive and negative ion modes, respectively.

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For serum, 1845 and 2309 features were obtained, respectively.

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GC–MS analysis

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The derivatized samples were subjected to GC–MS (6890A/5975C; Agilent Technologies)

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with a HP-5MS capillary column (30 m × 0.25 mm × 0.25 µm). The column temperature was

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held at 80 °C for 2 min, and then increased at 10 °C/min to 320 °C. The temperatures of the

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injector, ion source, and quadrupole rod were set to 280 °C, 230 °C, and 150 °C, respectively.

168

Highly pure helium (99.999%) was used as the carrier gas. The injection mode was set to


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splitless, and the injected volume was 1.0 µL. The column effluent was scanned in the mass

170

range from 50 to 550 m/z. Data were subjected to feature extraction with the XCMS

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procedure in R statistical software (R version 3.3.3; www.r-project.org). For each sample,

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duplicate technical repeats were performed. A total of 911 features were obtained for

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colostrum and milk, and 1669 for serum.

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Data analysis

175

Mass spectrometry data were normalized with respect to the total ion intensity to generate

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a two-dimensional matrix that included retention time, compound molecular weight,

177

observations (samples), and peak intensity for all tested samples. These matrix data were

178

imported into the SIMCA-P 13.0 software (Umetrics AB, Umeå, Sweden) for multivariate

179

statistical analysis. Principal component analysis (PCA) was used to visualize global

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clustering and trends in the samples from the studied groups. A supervised orthogonal partial

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least squares discriminant analysis (OPLS-DA) model was performed to check potential

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differential metabolites between the studied groups. In this model, parameters of R2X and Q2

183

were used to evaluate model quality and predictive ability, respectively. Values > 0.5

184

indicated that models were robust and had good predictive reliability.

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OPLS-DA were applied to visualize the separation between the studied groups, and loading

186

plots were used to find candidate biomarkers responsible for the separation. These

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biomarkers were therefore considered as differentiating metabolites that were selected on the

188

basis of the variable influence on projection (VIP) value > 1.0 as a threshold. In addition, an

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independent t-test was applied to examine the differences in relative intensity of candidate

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biomarkers obtained from OPLS-DA model using R software. Metabolites with VIP value >


25

Score plots of the


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1.0 and P value < 0.05 were defined as significantly different. Signals derived from

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differentially represented metabolites were searched against the Metabolite and Tandem MS

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database (www.metlin.scripps.edu) and the Human Metabolome Database (www.hmdb.ca).

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Pathways related to the differentially represented metabolites were predicted using

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MetaboAnalyst v.3.0 software (www.metaboanalyst.ca).

196 197

RESULTS

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LC–MS analysis for identifying differential metabolites

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To evaluate the quality of LC–MS data, total ion chromatograms of QC colostrum and

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milk samples were overlapped in positive and negative ion modes, respectively (Figure S2).

201

The retention time and peak intensity of metabolites from QC samples were stable and

202

reproducible. Additionally, eight QC samples clustered within a small area of the PCA score

203

plot in positive and negative ion modes (Figure S3). Similarly, total ion chromatograms and

204

the PCA score plot of QC serum samples (Figures S4 and S5) indicated that the LC–MS data

205

had good reproducibility.

206

The unsupervised PCA revealed the clustering of colostrum and milk samples and their

207

ingested products. Differences in metabolites between the studied groups were investigated in

208

greater detail by supervised PLS-DA and OPLS-DA. Score plots generated with the

209

OPLS-DA method in positive ion and negative ion modes are shown in Figure S6 and S7,

210

respectively. The Q2 value, an index of the predictive ability of a model, was at least 0.90 in

211

both modes. The differential metabolites in colostrum and milk and/or the other groups with

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VIP values > 1.0 and P values < 0.05 are listed in Tables S1 (positive ion mode) and S2


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(negative ion mode).

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Serum metabolite patterns in control calves and those fed colostrum or milk were

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completely separated by PCA. The OPLS-DA method was used to investigate metabolites

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that were differentially represented between studied groups; their score plots are shown in

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Figures S8 and S9. These models had robust predictive reliability, with a Q2 value of at least

218

0.90 in both positive and negative ion models. Differential metabolites with a VIP value > 1.0

219

and P value < 0.05 are listed in Tables S3 (positive ion mode) and S4 (negative ion mode).

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GC–MS analysis for identifying differential metabolites

221

To evaluate the quality of GC–MS data, total ion chromatograms of QC samples from the

222

colostrum/milk and serum groups were overlapped (Figure S10). Additionally, QC samples

223

categorized within a small area of the score plot were subjected to PCA analysis (Figure S11).

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The results indicated that the GC–MS data were robust and reproducible.

225

The OPLS-DA method was used to evaluate the differential metabolites in the

226

colostrum/milk groups; their score plots are shown in Figure S12. Groups that showed high

227

predictive reliability had Q2 values of at least 0.97. Differentially represented metabolites

228

were selected according to a VIP value > 1.0 and P value < 0.05; these are listed in Table S5.

229

OPLS-DA score plots for the serum group are shown in Figure S13. These models showed

230

robust predictive reliability, with a Q2 value of at least 0.89. Differential metabolites were

231

identified based on a VIP value > 1.0 and P value < 0.05 (Table S6).

232

Differential metabolites and their metabolic pathways between colostrum and milk before

233

and after ingestion

234

The results of the metabolomics analysis revealed that the concentrations of several



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metabolites including glutamate, ornithine, uric acid, stearidonic acid, and citric acid were

236

higher in milk than in colostrum, whereas concentrations of leucine, taurine, glycocholic acid,

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acetoacetic acid, and uridine were higher in colostrum than in milk. These differential

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metabolites were related to aminoacyl-tRNA biosynthesis, alanine, aspartate, glutamate, and

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butanoate metabolism, the citrate cycle; and histidine metabolism pathways that were

240

analyzed with MetaboAnalyst v.3.0 software (Figure 1). The concentrations of some

241

metabolites differed significantly between colostrum and ingested colostrum collected from

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the gastrointestinal tract over the course of digestion. In ingested colostrum, concentrations of

243

glutamate, leucine, glutamine, proline, γ-aminobutryic acid, cinnamic acid, thymine, and

244

phosphorylcholine were higher than in un-ingested colostrum, whereas those of

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glycine, arginine, sphingosine, nicotinic acid, D-galactose, 5-methylcytidine, and citric acid

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were lower in ingested colostrum than in un-ingested colostrum, and concentrations increased

247

or decreased in a time-dependent manner since ingestion (Table S7). Metabolites that differed

248

between colostrum and ingested colostrum (CI, CII, and CIII groups) were related to the

249

aminoacyl-tRNA biosynthesis, alanine, aspartate, glutamate, pyrimidine, histidine, and

250

nitrogen metabolism, and phenylalanine, tyrosine, tryptophan, valine, leucine, and isoleucine

251

biosynthesis pathways (Figure 2). Compared to un-ingested milk, glutamate, taurine,

252

methionine, lysine, γ-aminobutryic acid, taurocholic acid, and uridine concentrations were

253

higher in ingested milk, whereas citric acid, glycine, sphingosine, and acetylcarnitine

254

concentrations were lower in milk collected from the gastrointestinal tract than in un-ingested

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milk. The metabolic pathways associated with metabolites of milk and ingested milk are

256

shown in Figure 3, and were found to be similar to those of ingested colostrum. Colostrum


D-ribose,

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and milk underwent similar changes in metabolite profiles after ingestion by neonatal calves.

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In addition, changes in metabolites of ingested milk from calves in the milk group, relative to

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ingested colostrum from CII group were observed. That is, concentrations of thymine,

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stearidonic acid, nicotinamide, adenosine, guanine, and acetoacetic acid increased, whereas

261

concentrations of nicotinic acid, glucose 6-phosphate, threonine, and arachidonic acid

262

decreased in ingested milk. Differential metabolites were associated with aminoacyl-tRNA

263

biosynthesis, alanine, aspartate, glutamate, proline and arginine metabolism, the citrate cycle,

264

and biosynthesis of unsaturated fatty acids pathways (Figure S14). These pathways with a P

265

value no greater than 0.5 are shown in Supplementary Table S9.

266

Differential serum metabolites and their metabolic pathways between control calves and

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calves fed colostrum or milk

268

Compared to control calves, calves fed colostrum (CI, CII, and CIII groups) had lower

269

serum concentrations of cytosine,

270

glutamine, and higher serum concentrations of thymidine, asparagine, methionine, glutamate,

271

histidine, pentanedioic acid, palmitic acid, and linoleic acid. Serum concentrations of

272

glutamate, methionine, histidine, thymidine, palmitic acid, and linoleic acid increased in a

273

time-dependent manner in calves that were fed colostrum (CI, CII, and CIII groups) (Table

274

S8). Metabolic pathway analyses indicated that these metabolites were associated with the

275

biosynthesis of unsaturated fatty acids, aminoacyl-tRNA biosynthesis, and nitrogen and

276

galactose metabolism (Figure 4). We also found that serum linoleic acid, tyrosine, carnitine,

277

and cholic acid concentrations were higher, whereas serum concentration of tryptophan,

278

nicotinamide, D-fructose, D-sorbitol, and citric acid were lower in calves fed milk than in

D-ribose,

eicosatrienoic acid, glycodeoxycholate, and



279

control calves. The serum metabolites that differed between calves fed milk and controls

280

were assigned to the biosynthesis of unsaturated fatty acids, phenylalanine metabolism,

281

phenylalanine, tyrosine, tryptophan, valine, leucine, and isoleucine biosynthesis, the citrate

282

cycle, and histidine metabolism (Figure 5). Additionally, serum concentrations of glutamate,

283

glycocholic acid, lysine, and D-ribose were higher, whereas serum concentrations of linoleic

284

acid, thymine, and octadecanedioic acid were lower in calves fed colostrum in the CII group

285

as compared to the calves fed milk. These metabolites were categorized as pantothenate and

286

coenzyme A biosynthesis, aminoacyl-tRNA biosynthesis, and biosynthesis of unsaturated

287

fatty acids, and arginine and proline metabolism pathways (Figure S15). These pathways had

288

a P value no greater than 0.5 (Supplementary Table S10).

289 290

DISCUSSION

291

Changes in metabolites from colostrum and ingested colostrum

292

In the present study, differences in metabolite concentrations between colostrum and

293

ingested colostrum, and the corresponding serum metabolites in neonatal calves were

294

characterized by GC–MS and LC–MS based metabolomics approaches. In the ingested

295

colostrum samples collected from the gastrointestinal tract, changes in several metabolites

296

related to amino acids, lipids, carbohydrates, and nucleosides were observed in a

297

time-dependent manner. Regarding amino acids, increased glutamate and leucine

298

concentrations were possibly associated with the ingested colostral proteins, in which

299

glutamine and glutamate were the most abundant, and leucine was the second most abundant

300

protein-bound amino acid.

26

Major milk proteins, including caseins and beta-lactoglobulin,


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are likely to be digested as colostrum passes through the gastrointestinal tract of piglets,

302

according to proteomics analysis.

303

gastrointestinal tract of neonatal calves were induced by feeding calves colostrum.

304

Importantly, glutamate and glutamine function as primary contributors of intestinal energy

305

generation.

306

glutaminase, and it may serve as a specific precursor for the biosynthesis of bioactive

307

molecules, including glutathione, proline, and arginine. Glutathione, in particular, acts as a

308

major intracellular antioxidant in all tissues.

309

contributes to the production of γ-aminobutryic acid via glutamate decarboxylase, which is

310

the primary inhibitory neurotransmitter in mammalian central nervous systems.

311

Furthermore, γ-aminobutryic acid was found to be widely involved in the modulation of

312

immune cell activity and inflammatory responses associated with enteric inflammatory

313

conditions.

314

for protein synthesis, but also a nutrient signal to stimulate protein synthesis through the

315

mammalian target of rapamycin (mTOR) pathways in various tissues or cells.

316

piglets, leucine was involved in enhancing protein synthesis in skeletal muscle via mTORC1

317

activation,

318

whole-body growth. 34 Furthermore, concentrations of cinnamic acid and 2-hydroxycinnamic

319

acid, a derivative of cinnamic acid, were also higher in the ingested colostrum. Cinnamic acid

320

and its derivatives have been found to play a role in multiple biological functions including

321

anti-inflammatory, anti-oxidative, and anti-microbial activities.

322

information is available concerning the biosynthesis of cinnamic acid as colostrum is digested

28, 29

31

33

12

Furthermore, digestive enzyme activities in the 5, 27

Glutamate is also the immediate product of glutamine metabolism by

30

It is worth mentioning that glutamate also

30

Leucine, a member of the branched-chain amino acids, is not only a substrate

32

In neonatal

and supplementing piglets with leucine improved intestinal development and


35, 36

However, little


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323

in the gastrointestinal tract. In contrast, we found that concentrations of

324

D-ribose-5-phosphate,

325

It is well known that D-ribose is the structural backbone of purines and pyrimidines of genetic

326

material, and is a constituent of numerous cofactors, especially, adenosine triphosphate (ATP).

327

37

328

of the ingested colostrum may contribute to meeting the energy requirement for intestinal

329

ATP-dependent metabolic processes, and help to improve the intestinal maturation and

330

immune function as a compensatory mechanism to provide multiple protection for neonatal

331

calves against challenges in the extrauterine environment. In this regard, further

332

investigations on the impact of colostral metabolites on the immune function of neonatal

333

intestine are necessary.

334

Changes in serum metabolites between control calves and calves fed colostrum

D-ribose

and

a metabolic product of D-ribose, were lower in the ingested colostrum.

Based on these previous studies, and our results, we believe that changes in the metabolites

335

Changes in metabolites were observed in the serum of the calves that were fed colostrum.

336

Interestingly, some of the changes in serum amino acids, lipids, carbohydrates, and

337

nucleosides were similar to changes in these metabolites in ingested colostrum sampled from

338

gastrointestinal tract. Regarding amino acids, serum concentrations of glutamate, histidine,

339

methionine, proline, and asparagine were higher in calves fed colostrum, with increases

340

occurring in a time-dependent manner. Our results were partly in agreement with the results

341

of a previous study, in which serum concentrations of threonine, tryptophan, and methionine

342

increased in a time-dependent manner in calves after colostrum intake.

343

shown that serum methionine, threonine, histidine, and glutamate concentrations increased in

344

calves within 0–2 h of colostrum administration, while leucine, isoleucine, valine, and


38

It was recently

Page 17 of 42


11

345

histidine concentrations increased within 24–25 h relative to control calves.

346

noteworthy that most of the colostral components were digested and absorbed by the neonatal

347

intestine

348

several amino acids from ingested colostrum may be transported into the bloodstream, and

349

have health benefits for neonatal calves. For example, methionine is an oxidation-susceptible

350

amino acid, involved in mediating oxidative stress through inhibiting nitric oxide synthase, 40

351

and enhancing immune responses through its metabolic products.

352

antioxidant, and may play a role in activating the intracellular redox environment, and the

353

protection of mammalian cells against reactive oxygen species. 42 However, concentrations of

354

glutamine, Nα-acetyl-L-glutamine, and valine were lower in serum of calves fed colostrum

355

than in serum from control calves. Several previous studies found that serum glutamine

356

concentrations were lower in neonatal calves fed colostrum. 10, 11 Previous results suggest that

357

glutamine derived from blood acts as an important fuel substrate for the intestinal epithelium

358

in rats, and the intestine serves as a major site for glutamine metabolism.

359

amino acids and their derivatives, we observed differences in vitamin, lipid, and nucleoside

360

concentrations in the serum of neonatal calves that were fed colostrum. Colostrum contains

361

higher concentrations of vitamins A and E than milk, and concentrations of these vitamins

362

increased in the serum of neonatal calves following colostrum intake, and the serum vitamins

363

A and E concentrations were higher in calves fed colostrum within 8h after birth than in

364

calves in which colostrum intake was delayed one day after birth. 43, 44 In our study, the serum

365

vitamin E concentration increased, whereas vitamin E concentration decreased in ingested

366

colostrum in a time-dependent manner in calves fed colostrum. This phenomenon may be

It is

39

. Based on our results and the previous studies discussed above, we believe that


41

Proline served as an

28

In addition to


367

related to the incorporation of colostral vitamin E into chylomicrons within enterocytes that

368

traverse the intestine through the lymphatic pathway to reach the bloodstream. 45 It is widely

369

accepted that vitamin E is involved in the development of the newborn immune system, and

370

plays a role in the protection against oxidative stress.

371

concentrations showed that several fatty acids (including palmitic, oleic, and linolenic acids)

372

were increased in neonatal calves that received colostrum.

373

concentrations of pentanedioic acid was found to be positively correlated with IgG

374

concentration in the serum of neonatal calves by GC–MS. 38 Consistent with previous reports,

375

we found here that the concentrations of several fatty acids, such as pentanedioic, palmitic,

376

linolenic, and oleic acids were increased in the serum of neonatal calves. In contrast, these

377

fatty acids decreased in ingested colostrum in a time-dependent manner after calves were fed

378

colostrum. Furthermore, we found that the serum concentration of thymidine was increased,

379

whereas that of cytosine was decreased in neonatal calves that were fed colostrum, compared

380

to control calves. Based on previous studies and our results, we believe that some of the

381

metabolites in the ingested colostrum may be transported into the blood, and contribute to

382

improving the immune function of neonatal calves. However, the transport of colostrum

383

components across the small intestine and into the bloodstream, as well as their physiological

384

functions, need further investigation.

385

Changes in metabolites from milk and ingested milk

44, 45

An analysis of plasma fatty acid

43

More recently, a change in

386

We also observed changes in milk metabolites following ingestion. Unexpectedly, similar

387

changes in milk and colostrum metabolite profiles after ingestion were observed, according to

388

PCA analysis. These results were unexpected for two reasons: first, previous studies have


Page 18 of 42

Page 19 of 42


389

shown that colostrum is a unique diet for neonatal calves, and feeding calves milk instead of

390

colostrum resulted in calves experiencing severe diarrhea, and even death. 6 Second, previous

391

studies have shown that digestive enzyme activities of the neonatal intestine are induced by

392

feeding calves colostrum.

393

colostrum and milk, several differential metabolites, such as adenosine, thymine, linolenic

394

acid, and linoleic acid, were higher in the ingested milk. It is well known that adenosine is

395

implicated in a wide variety of biological functions, including nucleotide biosynthesis and

396

cellular energy metabolism. Adenosine is produced in response to metabolic stress and cell

397

damage,

398

response in the intestine.

399

improving epithelial integrity following mucosal injury.

400

gastrointestinal tract to digest milk may presumably be related to the intestinal development,

401

and may contribute to immune function through protected mucosal integrity.

402

Changes in serum metabolites between calves fed milk, and not fed or fed colostrum

46

5, 27

Although metabolite profiles were similar in ingested

and also serves as an important regulator of the immune and inflammatory 47

Linolenic and linoleic acids are thought to play a role in 48

Thus, the ability of the

403

The concentrations of some metabolites were altered in the serum of calves fed milk, and

404

the changes were like those observed in calves fed colostrum. For example, concentrations of

405

tyrosine, histidine, and linoleic acid were higher in the serum of calves fed milk. However,

406

serum IgG concentrations in calves fed milk were very low, and did not differ from the

407

concentrations in control calves (data not shown), which is in agreement with a previous

408

study.

409

metabolites into the bloodstream. We also found that the concentrations of citric, oxoglutaric,

410

and aconitic acids (which are associated with the citric acid cycle) were lower in calves fed

6

These results imply that IgG absorption is not coupled to the release of milk



Page 20 of 42

411

milk, implying that energy-associated metabolism was attenuated relative to control calves.

412

In addition, serum concentrations of amino acids such as glutamate, histidine, leucine, and

413

asparagine; lipids such as glycocholic acid; and nucleosides such as D-ribose and thymidine

414

were higher in calves fed colostrum than in calves fed milk. These amino acids are precursors

415

for protein synthesis mediated by the aminoacyl-tRNA biosynthesis pathway.

416

an essential amino acid that is used by histidine decarboxylase to form histamine, which is

417

necessary for inflammation.

418

then enters the citric acid cycle. 51 Thus, milk ingested in the gut led to calves being unable to

419

take up adequate nutritional and bioactive substrates for defense and development.

420

However, the functional significance of the observed changes in metabolite profiles from

421

ingested colostrum in the gastrointestinal tract to the bloodstream requires further

422

investigation.

50

49

Histidine is

Asparagine is degraded into oxaloacetate by asparaginase and

6, 10

423 424

In summary, metabolite profiles of serum and ingested colostrum or milk in the

425

gastrointestinal tract of calves were mapped using LC–MS and GC–MS based metabolomics

426

approaches (Figure 6). Concentrations of several differential metabolites, such as

427

γ-aminobutryic acid, glutamate, leucine, glutamine, proline, cinnamic acid, and thymine

428

increased, whereas concentrations of D-ribose, glycine, and arginine decreased in colostrum

429

ingested in the gastrointestinal tract. These changes occurred in a time dependent manner, and

430

were associated with alanine, aspartate, glutamate, and pyrimidine metabolism, and valine,

431

leucine and isoleucine biosynthesis. Meanwhile, concentrations of several serum metabolites,

432

such as glutamate, histidine, methionine, proline, oleic acid, vitamin E, and thymidine


Page 21 of 42


433

increased, whereas concentrations of glutamine and cytosine decreased in neonatal calves fed

434

colostrum, and these changes occurred in a time-dependent manner. Based on the changes in

435

serum and colostrum metabolites, several metabolites of ingested colostrum are likely to be

436

transported into the bloodstream of calves fed colostrum. We also found that several

437

metabolites of ingested milk could be transported from the gut into the bloodstream,

438

independent of IgG absorption. These differential metabolites were related to the

439

phenylalanine, tyrosine, tryptophan, valine, leucine, and isoleucine biosynthesis, the citrate

440

cycle, and histidine metabolism. These findings provide new insights into the digestion of

441

colostrum components in the gastrointestinal tract of calves, and the corresponding serum

442

metabolites, which in turn may provide clues to explore the absorption of nutritional and

443

bioactive substrates of colostrum by neonatal intestines, and the associated health benefits for

444

neonates.



445

Funding

446

The project was supported by the National Natural Science Foundation of China (31572434),

447

the Special Fund for Agro-scientific Research in the Public Interest (201403071), and the

448

Anhui Academy of Agricultural Sciences Project (14C0403).

449


Page 22 of 42

Page 23 of 42


450

Conflicts of Interest

451

The authors have no conflict of interest to declare.

452



453

Supporting information

454

The supplemental materials are available free of charge via the website http://pubs.acs.org:

455

Table S1. The differential metabolites in colostrum and milk, and ingested colostrum and

456

milk in positive ion mode of LC–MS data.

457


458

milk in negative ion mode of LC–MS data.

459

Table S3. The differential metabolites in serum from studied groups in positive ion mode of

460

LC–MS data.

461

Table S4. The differential metabolites in serum from studied groups in negative ion mode of

462

LC–MS data.

463


464

milk of GC–MS data.

465

Table S6. The differential metabolites in serum from studied groups of GC–MS data.

466

Table S7. The differential metabolites between un-ingested colostrum and ingested colostrum

467

(groups CI, CII, and CIII) collected from the gastrointestinal tract.

468

Table S8. The differential metabolites in serum between control calves and calves fed

469

colostrum (groups CI, CII, and CIII).

470

Table S9. The metabolic pathways of differential metabolites from colostrum and milk, and

471

ingested colostrum and milk with P values no greater than 0.5.

472

Table S10. The metabolic pathways of differential metabolites from serum between studied

473

groups with P values no greater than 0.5.

474


Page 24 of 42

Page 25 of 42


475

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599

Figure captions:

600

Figure 1. Pathway analysis of differential metabolites between colostrum and milk with

601

MetaboAnalyst. (a) Aminoacyl-tRNA biosynthesis; (b) Alanine, aspartate and glutamate

602

metabolism; (c) Butanoate metabolism; (d) Citrate cycle; (e) Arginine and proline

603

metabolism; (f) Beta-Alanine metabolism; (g) Pyrimidine metabolism; (h) Valine, leucine and

604

isoleucine biosynthesis; (i) Histidine metabolism; (j) Biosynthesis of unsaturated fatty acids;

605

(k) Nitrogen metabolism; (l) Phenylalanine metabolism; (m) Phenylalanine, tyrosine and

606

tryptophan biosynthesis. Pathway impact on the horizontal axis is the impact value calculated

607

from pathway topology analysis; -log(p) on the vertical axis is the negative logarithm

608

transformation of p value that calculated from the pathway enrichment analysis.

609

Figure 2. Pathway analysis of differential metabolites between colostrum and ingested

610

colostrum with MetaboAnalyst. (a) Aminoacyl-tRNA biosynthesis; (b) Pyrimidine

611

metabolism; (c) Alanine, aspartate and glutamate metabolism; (d) Pantothenate and CoA

612

biosynthesis; (e) Nitrogen metabolism; (f) Phenylalanine metabolism; (g) Beta-Alanine

613

metabolism; (h) Phenylalanine, tyrosine and tryptophan biosynthesis; (i) Valine, leucine and

614

isoleucine biosynthesis; (j) D-Glutamine and D-glutamate metabolism; (k) Arginine and

615

proline metabolism; (l) Histidine metabolism. Pathway impact on the horizontal axis is the

616

impact value calculated from pathway topology analysis; -log(p) on the vertical axis is the

617

negative logarithm transformation of p value that calculated from the pathway enrichment

618

analysis.

619

Figure 3. Pathway analysis of differential metabolites between milk and ingested milk with

620

MetaboAnalyst. (a) Aminoacyl-tRNA biosynthesis; (b) Beta-Alanine metabolism; (c) 31 ACS Paragon Plus Environment


621

Pyrimidine metabolism; (d) Butanoate metabolism; (e) Arginine and proline metabolism; (f)

622

Valine, leucine and isoleucine biosynthesis; (g) Biosynthesis of unsaturated fatty acids; (h)

623

Pantothenate and CoA biosynthesis; (i) Phenylalanine metabolism; (j) Alanine, aspartate and

624

glutamate metabolism; (k) Phenylalanine, tyrosine and tryptophan biosynthesis. Pathway

625

impact on the horizontal axis is the impact value calculated from pathway topology analysis;

626

-log(p) on the vertical axis is the negative logarithm transformation of p value that calculated

627

from the pathway enrichment analysis.

628

Figure 4. Pathway analysis of differential metabolites in serum from control calves and

629

calves fed colostrum with MetaboAnalyst. (a) Biosynthesis of unsaturated fatty acids; (b)

630

Aminoacyl-tRNA biosynthesis; (c) Nitrogen metabolism; (d) Galactose metabolism. Pathway

631

impact on the horizontal axis is the impact value calculated from pathway topology analysis;

632

-log(p) on the vertical axis is the negative logarithm transformation of p value that calculated

633

from the pathway enrichment analysis.

634

Figure 5. Pathway analysis of differential metabolites in serum from control calves and

635

calves fed milk with MetaboAnalyst. (a) Biosynthesis of unsaturated fatty acids; (b)

636

Phenylalanine metabolism; (c) Phenylalanine, tyrosine and tryptophan biosynthesis; (d)

637

Valine, leucine and isoleucine biosynthesis; (e) Citrate cycle; (f) Histidine metabolism.

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Pathway impact on the horizontal axis is the impact value calculated from pathway topology

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analysis; -log(p) on the vertical axis is the negative logarithm transformation of p value that

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calculated from the pathway enrichment analysis.

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Figure 6. An overview of metabolomics analysis of colostrum or milk, and serum

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metabolites in neonatal calves. QC shows quality control; IC-CI, IC-CII and IC-CIII show 32 ACS Paragon Plus Environment

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ingested colostrum collected from neonatal calves approximately at 8, 24 and 36 h after birth,

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respectively; IM shows ingested milk; Ct shows serum derived from control calves; S-CI,

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S-CII and S-CIII mean serum derived from the neonatal calves approximately at 8, 24 and 36

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h after birth, respectively; S-M shows serum derived from calves fed milk group.



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Supplemental Figure S1. An overview of the strategy used to reveal colostrum metabolites

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metabolized in the small intestine and then transfer into the bloodstream of neonatal calves.

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Supplemental Figure S2. The total ion chromatogram of QC samples from colostrum and

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milk, and ingested colostrum and milk groups in positive-mode (a) and negative-ion mode (b)

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of LC–MS.

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Supplemental Figure S3. The PCA scores plot of all samples from colostrum and milk, and

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ingested colostrum and milk groups in positive-mode (a) and negative-ion mode (b) of LC–

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MS.

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Supplemental Figure S4. The total ion chromatogram of QC samples of serum from studied

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groups in positive-mode (a) and negative-ion mode (b) of LC–MS.

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Supplemental Figure S5. The PCA scores plot of serum from studied groups in positive-mode

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(a) and negative-ion mode (b) of LC–MS.

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Supplemental Figure S6. The score plots of orthogonal partial least square discriminant

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analysis of the colostrum and milk, and ingested colostrum and milk groups derived from

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LC–MS data in positive-ion mode.

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analysis of the colostrum and milk, and ingested colostrum and milk groups derived from

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LC–MS data in negative-ion mode.

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analysis of serum from the studied groups derived from LC–MS data in positive-ion mode.

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analysis of serum from the studied groups derived from LC–MS data in negative-ion mode.


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Supplemental Figure S10. The total ion chromatogram of QC samples of GC–MS data from

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the studied colostrum/milk groups (a) and serum groups (b).

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Supplemental Figure S11. The PCA score plot of the GC–MS data from the studied

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colostrum/milk groups (a) and serum groups (b).

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analysis of colostrum and milk, and ingested colostrum and milk groups derived from GC–

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MS data.

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analysis of serum the studied groups derived from GC–MS data.

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Supplemental Figure S14. Pathway analysis of differential metabolites between colostrum

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and milk ingested in the gastrointestinal tract with MetaboAnalyst.

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Supplemental Figure S15. Pathway analysis of differential metabolites in serum from calves

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fed colostrum and milk with MetaboAnalyst.

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Figure 2

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Figure 3

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Figure 4



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Figure 5

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Figure 6

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metabolomics analysis of relationship between intestinal colostrum or milk, and serum metabolites 142x74mm (150 x 150 DPI)

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Exploration of the Relationship between Intestinal ... - ACS Publications

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