Abstract

   Data on the evolution of blood metabolites and metabolic markers in
   neonatal piglets are scarce, although this information is vital to
   detect physiological aberrations from normal development. We aimed to
   characterize age- and nutrition-related changes in the plasma
   metabolome and serum biochemistry of suckling and newly weaned piglets
   and assess metabolite patterns as physiological markers for the two
   phases. In two replicate batches (n = 10 litters/group), piglets either
   received sow milk alone or were additionally offered creep feed from
   day 10 until weaning (day 28). Blood was collected from one
   piglet/litter on days 7, 14, 21, 28, 31 and 35 of life, totaling five
   females and five males/group/day. Signature feature ranking identified
   plasma triglycerides (TG) as discriminative for age and nutrition
   during the suckling phase. Influential TG 20:4_36:5, TG 17:0_34:2 and
   TG 18:2_38:6 were higher in creep-fed piglets on days 14, 21 and 28 of
   life, respectively, compared to only sow milk-fed piglets. Metabolites
   belonging to pathways within histidine, D-glutamine and D-glutamate
   metabolism as well as hippuric acid were distinctive for the
   postweaning compared to the suckling period. In conclusion, plasma
   lipid profiles especially corresponded to the type of nutrition in the
   suckling phase and showed a strong weaning effect.

   Keywords: plasma metabolome, neonatal piglets, triglycerides, hippuric
   acid, creep feeding, weaning

1. Introduction

   Metabolomics approaches have been used to detect markers for a variety
   of health and physiological conditions in animals [[40]1,[41]2]. While
   there is an extensive body of research on weaned and growing pigs
   [[42]3,[43]4], the developmental patterns in the blood metabolome of
   neonatal piglets from the suckling to early postweaning phase have been
   incompletely described. Knowledge about the ranges of metabolites and
   metabolic markers used in routine diagnostics (e.g., total protein,
   alanine transaminase (ALT), aspartate transaminase (AST) and alkaline
   phosphatase (ALP)) at this age can provide valid data about the
   physiological state and health of piglets. Metabolite ranges can be
   different in infants and adults, requiring the establishment of
   appropriate reference ranges [[44]5]. Therefore, to understand
   alterations in the metabolic phenotype [[45]6], knowledge on the normal
   changes in the plasma metabolome in different life phases is
   fundamental.

   Piglets from modern hyperprolific breeds more than quadruple their
   birth weight during the 28-day-long suckling phase. This growth spurt
   demands a high turnover of nutrients. Concurrently, the energy
   metabolism shifts from being glucogenic to becoming ketogenic
   postpartum [[46]7]. Late colostrum and transient milk are especially
   rich in fat [[47]8], which is matched by the secretion of potent
   lingual, gastric and pancreatic lipases by the piglet, ensuring maximum
   digestive utilization of the fat [[48]9]. After weaning, the piglet is
   transferred to a high-starch, low-fat diet, shifting the metabolism
   back to glucogenic mode and respective metabolites. Moreover, the low
   feed intake in the first days after weaning and the upregulation of the
   hypothalamic–pituitary–adrenal stress response increase catabolic
   processes and mobilization of body tissue [[49]10], which may be
   noticeable in the plasma metabolome. However, conclusive metabolome
   profiles for the suckling and early postweaning phase in pigs are
   missing.

   Aside from sow milk, piglets are fed highly palatable and digestible
   creep feed as early as day 2 of life to ease the transition from liquid
   to solid feed after weaning. In addition, creep feeding can ensure
   adequate nutrition when sow milk production cannot adequately meet the
   needs of her litter which has become more important due to big litter
   sizes [[50]11]. The chemical composition of creep feed differs from
   porcine milk, containing a considerable amount of starch and less fat
   than porcine milk which can be attributed to ingredients like cow dairy
   by-products, such as whey powder and lactose, plant protein (e.g., soy
   protein hydrolysate), starches and plant oils. Consequently, if the
   consumption of creep feed is high enough, creep feed may alter the
   profiles of metabolites and metabolic markers in blood, which has not
   been sufficiently described before but may need to be considered
   especially in relation to routine diagnostics in veterinary practice.
   Piglets are changed to the cereal-based prestarter diet in the late
   suckling phase, which is then fed until after weaning, again modulating
   the intestinally and metabolically available nutrient composition.
   However, little information is available on whether blood metabolite
   profiles, when determined during the suckling and early postweaning
   period, are indicative for the nutrition of the piglets preweaning.

   We hypothesized that the introduction of creep feed would modulate the
   age-related metabolome profile, especially influencing the utilization
   of lipids as energetic substrate. Assessing this hypothesis could
   provide insights into age-related changes in the associated biochemical
   processes, and possibly reveal physiological biomarkers for the
   nutritional state of piglets. We further hypothesized that blood
   metabolite patterns in the early days postweaning would shift
   dramatically due to the removal of porcine milk and lower feed intake
   in the first days after weaning but still would allow distinguishing
   which piglets received creep feed. Therefore, the objective of the
   present study was to characterize age- and nutrition-related changes in
   the plasma metabolome as well as in additional parameters for clinical
   chemistry in the serum of suckling and newly weaned piglets.

2. Materials and Methods

2.1. Animals, Housing and Feeding

   The animal experiment was conducted under practical conditions at the
   pig facility of the research and teaching farm ‘VetFarm’ of the
   University of Veterinary Medicine Vienna (Vienna, Austria) in two
   replicate batches, with 10 sows per replicate batch. The sow handling
   and feeding were similar in both replicate batches. The sows, as part
   of the University sow herd, were fed and handled similarly during
   gestation and lactation according to standard procedures at the farm.
   Five days before farrowing, sows were moved into free-ranging farrowing
   pens (BeFree, Schauer, Agrotonic, Prambachkirchen, Austria; 2.3 × 2.6
   m) including a bowl drinker, feeder and hayrack for the sow and bowl
   drinkers and a nest with heated flooring for the piglets in one
   farrowing room. Sows were not constrained for farrowing. The farrowing
   pens comprised a nest for the piglets with heated flooring.
   Cross-fostering was minimized and applied only on day 1 of life to keep
   litter size at a maximum of 13 piglets. Mainly small birth weight
   piglets were cross-fostered to sows that were not included in the
   experiment. The other piglets remained with their mother sows
   throughout lactation.

   Piglets were individually weighed and identified with an ear tag after
   birth. Piglets received an iron injection on day 4 of life (2 mL
   Ferriphor 100 mg/mL, OGRIS Pharma Vetriebs-GmbH, Wels, Austria) and
   vaccination on day 17 of life (1 mL Ingelvac Circoflex plus 1 mL
   Ingelvac MycoFLEX, Boehringer Ingelheim RCV GmbH und Co KG, Vienna,
   Austria). Male piglets were castrated on day 11 of life after sedation
   (Stresnil 40 mg/mL, 0.025 mL/kg body weight, Elanco Tiergesundheit AG,
   Basel, Switzerland and Narketan 100 mg/mL, 0.1 mL/kg body weight,
   Vetoquinol Österreich GmbH, Vienna, Austria). On day 28 of life, the
   sows were first removed from the farrowing room, before piglets were
   transferred to the weaner pig room. Piglets were kept in groups of a
   maximum of 20 piglets (pen size 3.3 m × 4.6 m), whereby piglets from
   two to three litters of the same dietary group were housed together.
   Pens were equipped with a piglet nest, nipple and bowl drinkers and one
   round feeder. Straw was provided as bedding material. Sows and piglets
   had free access to water.

   Throughout lactation, sows were fed a commercial cereal–soybean
   meal-based lactation diet ([51]Table S1). Piglets from all litters
   suckled freely during the 28-day suckling period. The experimental
   outline is shown in [52]Figure 1. The piglets from five litters per
   replicate batch suckled only sow milk (sow milk group). The piglets
   from the other five litters per replicate batch additionally had free
   access to a commercial milk replacer as creep feed from day 10 of life
   (creep feed group). The dry powder was mixed 1:5 (wt/vol; 200 g/L) with
   warm water (45 °C) to achieve a thin liquid according to the
   manufacturer’s instructions that was manually served and offered in
   piglet feed troughs at least twice daily (0800 and 1500 h). On days 24
   and 25 of life the creep feed was gradually mixed with the prestarter
   diet ([53]Table S1), after which the piglets from the creep feed group
   were fed 100% of the prestarter diet. After weaning on day 28 of life,
   the prestarter diet was fed to 100% until day 35 of life, which marked
   the end of the experiment. Litters in the sow milk group (n =
   5/replicate batch) did not receive the prestarter diet before weaning
   and were offered the prestarter diet from day 28 to 35 of life. Piglets
   in both feeding groups had free access to the prestarter diet
   postweaning. All diets were commercial complete feeds and met the
   current recommendations for nutrient requirements [[54]12].

Figure 1.

   [55]Figure 1
   [56]Open in a new tab

   Outline of the experimental period. Ten litters only had access to sow
   milk during the suckling period (group 1 = sow milk group), whereas the
   other ten litters received additional creep feed from day 10 of life
   (group 2 = creep feed group). * On days 24 and 25 of life the creep
   feed was gradually mixed with the prestarter diet after which the
   piglets from the creep feed group were fed only the prestarter diet.

2.2. Body Weight Measurements and Blood Sampling

   Body weight (BW) of the piglets was measured on days 1, 3, 6, 13, 20,
   27, 30 and 34 of life. In each of the two replicate batches, blood
   samples were collected from ten piglets each at day of life 7, 14, 21,
   31 and 35. On each of the five sampling days, five piglets per diet and
   sex were selected based on average body weight development across
   litters. Blood was taken from the heart on days 7, 14, 21, 28, 31 and
   35 of life after deep sedation of the piglets (Stresnil 40 mg/mL, 0.025
   mL/kg body weight, Azaperone, Elanco Tiergesundheit AG, Basel,
   Switzerland and Narketan 100 mg/mL, 0.1 mL/kg body weight, Ketamine,
   Vetoquinol Österreich GmbH, Vienna, Austria). The blood was collected
   into plasma and serum tubes (Vacuette Röhrchen K3E K3EDTA and Vacuette
   Röhrchen CAT Serum, Greiner Bio-One International GmbH, Kremsmünster,
   Austria) for metabolomics and clinical biochemistry, respectively.
   After inverting the blood tubes, they were stored on crash ice until
   centrifugation at 3000× g for 20 min at 4 °C (Eppendorf Centrifuge 5810
   R, Eppendorf, Hamburg, Germany). Serum and plasma were aliquoted and
   stored at −80 °C until analysis.

2.3. Metabolomics

   Solvents and reagents for metabolomics analysis were of analytical
   grade or higher and obtained from VWR International GmbH (Vienna,
   Austria), Sigma-Aldrich (Vienna, Austria), and Merck (Darmstadt,
   Germany). Water was purified with an Arium^® pro Ultrapure Lab Water
   System (Sartorius, Göttingen, Germany). Reference standards for all
   analytes were purchased from Sigma-Aldrich (Vienna, Austria), VWR
   (Vienna, Austria), Cayman Chemicals (Ann Arbor, MI, USA) and Avanti
   Polar Lipids (Birmingham, AL, USA, products available through Merck
   KGaA, Darmstadt, Germany). ^13C-labelled internal standards were
   obtained from Sigma-Aldrich, deuterated bile acids from Sigma-Aldrich
   and Avanti Polar Lipids, and a deuterated cell-free amino acid mix
   containing 20 amino acids at different ratios was purchased from
   Eurisotop (Tewksbury, MA, USA).

   Metabolites in plasma were quantitatively determined by three reversed
   phase high-performance liquid chromatography tandem mass spectrometric
   (RP-HPLC-MS/MS) methods and one anion exchange chromatography
   high-resolution mass spectrometric (AIC-HR-MS) method. Amino acids,
   biogenic amines and most lipid classes were measured by tandem mass
   spectrometry in positive ionization mode after RP-HPLC separation,
   whereas bile acids as well as medium and long chain fatty acids were
   determined by RP-HPLC-MS/MS in negative ionization mode. Carboxylic
   acids, sugar phosphates and nucleotides were measured by AIC-HR-MS. An
   overview of the investigated compounds and the analyses methods used
   for their determination is given in [57]Tables S2–S6.

   For all compounds measured by RP-HPLC-MS/MS, sample preparation was
   performed in 96-well plates using a modified protocol based on
   Biocrates’ MxP^® Quant 500 kit (Biocrates, Innsbruck, Austria) that
   includes derivatization of amino acids and amines with phenyl
   isothiocyanate (PITC). Aliquots of 10 µL of plasma or different volumes
   of calibration solutions containing between 0.009 and 16 mg/L of
   analytes and 25 µL of internal standard solution (8.3 mg/L of
   ^13C-putrescine, 740 mg/L of labelled amino acid stock solution,
   0.5–2.0 mg/L of deuterated bile acids) were pipetted into a 96-well
   plate and evaporated to dryness in a Centrivap vacuum concentrator
   (Labconco, Kansas City, MO, USA) at 30 °C. Afterwards, 50 µL of
   derivatization reagent (ethanol/water/pyridine/PITC 31.7/31.7/31.7/5.0,
   v/v/v/v) was added and the covered plate was shaken for 20 s and placed
   in the dark at ambient temperature for derivatization of amino acids
   and amines. After 1 h, the derivatization reagent was evaporated in the
   Centrivap concentrator. Finally, analytes were extracted into 300 µL of
   methanol containing 4.9 mM ammonium acetate by shaking for 30 min and
   the extracts were centrifuged. One aliquot of the extracts was used
   directly for LC-MS/MS measurement in the negative ionization mode,
   while another aliquot was diluted 1 + 4 (v + v) with methanol for
   determination of amino acids, amines and lipids in the positive
   ionization mode.

   The RP-HPLC-MS/MS analysis was performed on an Agilent 1290 UHPLC
   system (Agilent Technologies, Waldbronn, Germany) coupled to a QTrap
   6500+ mass spectrometer equipped with an IonDrive TurboV source (SCIEX,
   Foster City, CA, USA). Chromatographic separation was carried out on a
   Kinetex C18 column (50 × 2.1 mm, 1.7 μm particle size, Phenomenex,
   Aschaffenburg, Germany) at a temperature of 50 °C. Three different
   chromatographic and mass spectrometric methods were applied for
   analysis of amino acids and amines (scheduled selected reaction
   monitoring mode, sSRM, positive ionization mode), bile acids and fatty
   acids (SRM, negative ionization mode) and lipids (SRM, positive
   ionization). The chromatographic methods are summarized in
   [58]Supplementary Tables S2–S6. The ion source parameters for MS
   detection in positive mode were as follows: source temperature 500 °C,
   ion spray voltage 5500 V, curtain gas 45 psi, ion source gas 1 60 psi
   and ion source gas 2 70 psi. In negative mode, the ion spray voltage
   was reduced to −4500 V. SRM transitions are given in [59]Supplementary
   Table S2. Analyst software version 1.6.3 (SCIEX, Framingham, MA, USA)
   was employed for instrument control and data analysis. Quantitative
   analysis was performed on the basis of linear or quadratic calibration
   curves in the range between 0.6 and 3000 ng/mL of derivatized reference
   standard compounds in measurement solution. Internal standards were
   used for recovery correction.

   In addition to quantitative determination of compounds with a reference
   standard, semi-quantitative analysis of several lipid classes without
   an available reference standard (acyl carnitines, phosphocholines,
   ceramides, cholesterol esters, sphingomyelins, diglycerides and
   triglycerides) was also performed. To that end, the LC method used for
   quantification of lipids with a reference standard was paired with SRM
   transitions of various lipid classes without a reference standard. The
   transitions used were the same as in the flow injection part of
   Biocrates’ MxP^® Quant 500 kit (Biocrates, Innsbruck, Austria).
   Semi-quantitative analysis was achieved on the basis of molar
   calibration curves for compounds of the same lipid class with available
   reference standard.

   Sample preparation for AIC-HR-MS consisted of shaking 20 µL of plasma
   with 10 µL of AIC internal standard solution (5 mg/L of fully
   ^13C-labelled acetic acid, propionic acid and butyric acid) and 470 µL
   of acetonitrile/water (80/20, v/v) at 4 °C for 10 min and
   centrifugation at 14,350× g for 10 min. The AIC-HR-MS measurements were
   carried out on a Dionex Integrion HPIC system coupled to a Q Exactive
   Orbitrap mass spectrometer (both Thermo Scientific, Waltham, MA, USA)
   as described in our previous work [[60]13]. Analytes were quantified on
   the basis of pure solvent calibration curves established between 0.1
   and 9000 ng/mL for all analytes. Hexoses and disaccharides that were
   co-eluting under the chosen conditions were quantified as sum
   parameters using glucose and sucrose, respectively, as reference
   standard.

2.4. Clinical Biochemistry

   The serum content of calcium, potassium, sodium, chloride, phosphate,
   total protein, ALT, AST and ALP were determined with enzymatic
   colorimetric assays using an autoanalyzer for clinical chemistry (Cobas
   6000/c501; Roche Diagnostics GmbH, Rotkreuz, Switzerland).

2.5. Statistical Analysis

   The statistical analysis of the plasma and serum metabolite data
   involved two parts. First, metabolomics datasets were analyzed using
   several modules, including the statistical, quantitative enrichment,
   pathway enrichment and biomarker analysis modules, of the web-based
   open access platform MetaboAnalyst 5.0 [[61]14]. Second, differential
   analysis of the metabolomics and clinical biochemistry parameters was
   performed using SAS (version 9.4, SAS Inst. Inc., Cary, NC, USA). As
   the different feeding of the suckling piglets only started from day 10
   of life, effects of the feeding were only analyzed for the time points
   thereafter. In each of the modules of MetaboAnalyst 5.0, the sample
   data were normalized to adjust for systematic differences among
   samples. In doing so, samples were normalized by sum and log
   transformed. After normalization the data were subjected to
   chemometrics analysis, specifically sparse Partial Least
   Squares-Discriminant Analysis (sPLS-DA), within the statistical
   analysis module. The ten most discriminant metabolites for each
   component were identified and pairwise score plots were used to
   identify characteristic trends or groupings among days of life. Based
   on the sPLS-DA results, the obtained metabolites were plotted according
   to their importance in separating the days of life based on the
   variable importance in the projection (VIP) scores and visualized in
   loading plots. The VIP score is a weighted sum of squares of the
   sPLS-DA loadings, whereby scores suggest that the selected variable is
   significantly involved in the separation of groups [[62]15]. Only the
   features for the first component are presented here. The metabolites
   were ranked by the absolute values of their loadings. In total, three
   sPLS-DA were conducted, one for the whole dataset, and one each for the
   data subsets of lipids and metabolites other than lipids. This was done
   due to the targeted metabolomics approach, featuring plasma lipids,
   whereas the metabolites other than lipids covered a broad range of
   metabolic pathways. Biomarker analysis was performed using
   MetaboAnalyst 5.0. Samples were filtered by determining the relative
   standard deviation (RSD = standard deviation/mean) and normalized as
   described for the statistical module. To identify important features,
   receiver operating characteristic (ROC) curve analysis based on partial
   least squares discriminant regression was performed as a classification
   and feature ranking method. Results for the 15 most important features
   and the abundance between the feeding groups on individual days of life
   were displayed as signature features. Afterwards, pathway enrichment
   analysis was performed using the respective module. Pathway analysis
   used the KEGG Pathway Database ([63]www.genome.jp/kegg/pathway.html)
   for Homo sapiens as a reference pathway library because no respective
   database was available for Sus scrofa. Previously, Lefort et al.
   [[64]16] manually cross-checked the Homo sapiens database to confirm
   the relevance of the human pathways for the pigs.

   Prior to the differential analysis in SAS, the residuals of the data
   for serum biochemistry and metabolome were tested for normal
   distribution using the Shapiro–Wilk test. If residuals were not
   normally distributed, they were converted using the Boxcox method and
   the Transreg procedure in SAS, before data were subjected to ANOVA
   using the MIXED procedure using repeated and random models. The first
   model for repeated measurements were used to assess whether differences
   in blood data existed over time. Fixed effects in this model included
   day of life, dietary treatment, sex and replicate batch and their two-
   and three-way-interactions. The experimental unit was piglet nested
   within litter. For many parameters, differences for sex and dietary
   treatment were not observed and not included in the final model for the
   respective parameter. Degrees of freedom were approximated by the
   Kenward–Roger method. Multiple pairwise comparisons among least-square
   means were performed using the probability of difference option in SAS.
   Data were expressed as least squares means ± standard error of the mean
   (SEM). Significance was defined at p ≤ 0.05 and trends were discussed
   at 0.05 < p ≤ 0.10.

3. Results

   The RP-HPLC-MS/MS and IC-HR-MS analyses identified a total of 345
   unique metabolites in plasma samples across all sampling time points
   and ages of piglets, of which about two-thirds represented metabolites
   from phospholipid, sphingomyelin and triglyceride metabolism. The
   majority of the metabolites could be detected in plasma at all age
   stages. Nevertheless, some triglycerides were not present in the plasma
   of piglets after weaning that received creep feed during the suckling
   period.

   In order to categorize the metabolites with the highest discriminant
   power between days of life, we performed three separate sPLS-DAs. As
   the lipid fraction dominated the most discriminant metabolites, besides
   analyzing the whole data set, we analyzed the datasets for plasma
   lipids and water-soluble metabolites separately in order to distinguish
   important metabolites from the various pathways. Irrespective of
   piglets’ nutrition during the suckling phase, orthogonal projections of
   the sPLS-DA showed distinguishable metabolite profiles for the
   different days of life ([65]Figure 2), with the greatest discrimination
   in plasma profiles between metabolomes before and after weaning.

Figure 2.

   [66]Figure 2
   [67]Open in a new tab

   Orthogonal projections of the sparse partial least squares-discriminant
   analysis for component 1, 2 and 3 of plasma metabolomes in piglets for
   the different days of life [Day of life (DoL) 7, 14, 21, 28, 31 and 35;
   n = 20/DoL].

   The sources of variation for the ten most influential metabolites were
   displayed based on their VIP scores (VIP > 1), and are presented
   separately for the whole dataset and the two subsets of metabolites
   ([68]Figure 3a–c). The VIP for the whole metabolite dataset showed that
   seven plasma triglycerides, hippuric acid and one phosphatidylcholine
   with diacyl residue sum C32:0 (PC aa C32:0) were the most influential
   metabolites ([69]Figure 3a). Triglycerides predominated as the most
   discriminant lipids in the respective dataset ([70]Figure 3b). For the
   water-soluble compounds, hippuric acid was the most influential
   metabolite ([71]Figure 3c). Other major influential water-soluble
   metabolites were alpha- and beta-amino acids, including
   alpha-aminobutyric acid (AABA), taurine, proline, tyrosine, ornithine,
   serine, beta-alanine and arginine ([72]Figure 3c).

Figure 3.

   [73]Figure 3
   [74]Open in a new tab

   Visualization of results from partial least squares-discriminant
   analysis in loading plots according to their importance for the
   individual days of life based on the variable importance in the
   projection (VIP) scores. Most discriminant plasma metabolites in
   piglets among (a) all metabolites; (b) lipids; and (c) water-soluble
   metabolites. Piglets were weaned on day 28 of life. AABA,
   alpha-aminobutyric acid; PC aa, phosphatidylcholine diacyl; TG,
   triglyceride.

   As the next step, biomarker analysis using multivariate ROC curve based
   exploratory analysis with PLS-DA as classification method was applied
   to identify marker metabolites for the two nutritional groups for day
   14, 21, 28, 31 and 35 of life. Signature feature ranking identified
   mainly plasma triglycerides as being characteristically different
   between the two nutritional groups from day 14 to 35 of life
   ([75]Figure 4a–e), whereas water-soluble metabolites were less
   represented among the 15 marker metabolites at each day of life.
   Although triglycerides (TG) were among the top metabolites, the actual
   TG differed between the two feeding groups on the various days of life.
   Accordingly, plasma levels of TG 20:4_36:5, TG 17:0_34:2, TG 18:2_38:6
   on days 14, 21 and 28 of life, respectively, were indicative for and
   higher in creep-fed piglets compared to piglets only receiving sow milk
   ([76]Figure 4a–c). Postweaning, marker analysis showed that TG
   C17:1_C34:2 and TG C18:0_C32:1 on day 31 and 35 of life, respectively,
   were indicative for piglets that received sow milk only ([77]Figure
   4d,e). From the water-soluble metabolites, it is noteworthy that
   serotonin was among the top ten metabolites on day 28 of life and two
   bile acids on day 35 of life; all three metabolites were lower in creep
   fed piglets compared to piglets that were fed only sow milk during the
   suckling piglets.

Figure 4.

   [78]Figure 4
   [79]Open in a new tab

   Biomarker analysis using multivariate receiver operating characteristic
   curve-based exploratory analysis with PLS-DA as classification method
   was applied to identify marker plasma metabolites for the two
   nutritional groups for the different days of life. Creep feed was fed
   from day 10 of life until weaning (day 28 of life). Most discriminant
   metabolites for day (a) 14; (b) 21; (c) 28; (d) 31; and (e) 35 of life.
   ADMA, asymmetric dimethylarginine; Met-SO, methionine sulfoxide; THDCA,
   taurohyodeoxycholic acid; TCDCA, taurochenodeoxycholic acid; TG,
   triglyceride.

   Enrichment pathway analysis was conducted for water-soluble metabolites
   in plasma only; first to identify enriched metabolite sets and pathways
   ([80]Figure 5, [81]Figure 6 and [82]Figures S1–S4). The major pathways
   that were enriched on day 7 of life and declined towards day 14 of life
   were purine metabolism, propanoate metabolism, tricarboxylic acid cycle
   (TCA) and pyrimidine metabolism ([83]Figure 5a). Compared to the
   suckling period, metabolites belonging to pathways within histidine
   metabolism, nitrogen metabolism and D-glutamine and D-glutamate
   metabolism were higher during the postweaning phase ([84]Figure 5b).
   The most affected pathways by creep feeding were similarly but
   separately investigated for the suckling and postweaning period. During
   the suckling period, plasma samples of creep-fed piglets distinguished
   by higher concentrations of metabolites within pathways related to
   pyruvate metabolism, lysine degradation and tryptophan metabolism
   ([85]Figure 6a). Postweaning, plasma of creep-fed piglets was enriched
   in metabolites related to pyrimidine metabolism, D-glutamine and
   D-glutamate metabolism and nitrogen metabolism ([86]Figure 6b).
   Influential metabolites from the three top enriched pathways are
   provided in [87]Figures S1–S4.

Figure 5.

   [88]Figure 5
   [89]Open in a new tab

   Enriched pathways (top 25) in discriminant plasma metabolites for
   sparse partial least squares-discriminant analysis. Pathway analysis
   used the KEGG Pathway Database for Homo sapiens as a reference pathway
   library because no respective database was available for Sus scrofa.
   Most discriminant pathways for (a) day 7 versus day 14 of life; and (b)
   suckling versus postweaning period. Piglets were weaned on day 28 of
   life.

Figure 6.

   [90]Figure 6
   [91]Open in a new tab

   Enriched pathways (top 25) in discriminant metabolites for sparse
   partial least squares-discriminant analysis. Pathway analysis used the
   KEGG Pathway Database for Homo sapiens as a reference pathway library
   because no respective database was available for Sus scrofa. Most
   discriminant pathways for (a) creep feeding effect during the suckling
   period (only sow milk versus additional creep feeding); and (b) creep
   feeding effect during the postweaning period (only sow milk versus
   additional creep feeding in suckling period). Creep feed was fed from
   day 10 of life until weaning (day 28 of life).

   Results for the differential analysis of the plasma metabolome using
   mixed models can be found in [92]Tables S7–S9. The influential plasma
   metabolites that were extracted by the sPLS-DA and pathway enrichment
   analysis were also different in the mixed models. For the majority of
   water-soluble metabolites, their plasma concentrations were mainly
   affected by age of the piglet, increasing or decreasing depending on
   the metabolite ([93]Table S7). For instance, differential analysis
   showed that primary and secondary bile acids increased in plasma from
   day 7 to day 28 of life and dropped postweaning. Likewise, the amino
   acid metabolome showed elevated levels of many proteinogenic amino
   acids as well as of taurine, citrulline and ornithine on day 7 and/or
   day 14 of life which declined afterwards. Concentrations of almost all
   lipids measured in plasma changed with age (p < 0.05; [94]Tables S8 and
   S9). Specifically, the concentrations of TG in plasma dropped after
   weaning; many of them were below the detection limit postweaning,
   whereas effects of the feeding during the suckling period were small.
   Acyl-carnitines were mostly higher in creep-fed piglets from day 14 of
   life compared to piglets that only received sow milk. Creep feeding in
   the suckling phase lowered several phosphatidylcholines, sphingomyelins
   and one lysophosphatidylcholine on day 35 of life compared to piglets
   that only received sow milk.

   The measured serum parameters of the clinical biochemistry were all
   affected by age (p < 0.05; [95]Table 1). Serum concentration of total
   protein remained stable throughout the suckling phase but decreased
   postweaning (p = 0.016). The effect of feeding in the suckling period
   indicated that creep-fed piglets had lower serum total protein from day
   28 of life and especially postweaning (p = 0.004). Serum levels of ALP
   decreased from day 7 to day 31 of life and remained at this level on
   day 35 of life. Serum ALT and AST mainly declined from day 7 to 14 of
   life and remained at this level (p < 0.05). Serum electrolytes (i.e.,
   calcium, phosphate, sodium, potassium and chloride) were stable during
   the suckling phase and mainly dropped on day 31 of life to recover on
   day 35 of life (p < 0.05). Creep-fed piglets had lower serum calcium (p
   = 0.003) and sodium (p = 0.020) on day 31 of life compared to piglets
   that received only sow milk.

Table 1.

   Differences in age-related development of clinical biochemistry
   parameters in serum of suckling and newly weaned piglets receiving only
   sow milk or additionally creep feed from day of life 10 *.
   Day of Life (DoL) 7 14 21 28 31 35 p Value
   Feeding (Feed) Sow Milk Sow Milk Creep Feed Sow Milk Creep Feed Sow
   Milk Creep Feed Sow Milk Creep Feed Sow Milk Creep Feed SEM DoL Feed
   DoL × Feed
   Total protein (g/dL) 4.64 4.45 4.45 4.48 4.24 4.48 4.06 4.32 3.70 4.48
   3.79 0.19 0.016 0.004 0.225
   ALP (U/L) 1582 1084 1155 836 756 504 434 312 254 277 224 182.1 <0.001
   0.653 0.998
   AST (U/L) 80 42 35 34 36 57 36 31 27 35 34 9.18 <0.001 0.701 0.447
   ALT (U/L) 54 31 29 31 28 35 35 43 46 36 38 4.78 <0.001 0.668 0.887
   Calcium (mmol/L) 2.81 2.78 2.77 2.67 2.65 2.69 2.53 2.28 2.08 2.46 2.29
   0.06 <0.001 0.003 0.482
   Phosphate (mmol/L) 2.94 2.94 3.07 2.81 2.80 2.75 2.73 2.25 2.03 2.18
   2.23 0.09 <0.001 0.996 0.540
   Sodium (mmol/L) 137 136 135 137 134 136 133 130 125 135 132 1.88 <0.001
   0.020 0.848
   Potassium (mmol/L) 5.67 5.06 5.23 4.74 4.81 4.94 4.95 4.42 4.39 5.00
   5.07 0.25 <0.001 0.483 0.987
   Chloride (mmol/L) 99 99 98 100 97 99 97 96 93 99 98 1.56 0.023 0.082
   0.821
   [96]Open in a new tab

   * Values are least squares means ± standard error of the mean (SEM).
   Piglets were weaned on day 28 of life. ALP, alkaline phosphatase; ALT,
   alanine aminotransferase; AST, aspartate aminotransferase.

   Body weight development of the piglets showed similar growth between
   the two feeding groups during the suckling phase, whereas on day 34 of
   life piglets that received creep feed during the suckling phase weighed
   1.8 kg less than the piglets that only suckled sow milk during the
   suckling phase (p = 0.005; [97]Figure 7).

Figure 7.

   [98]Figure 7
   [99]Open in a new tab

   Body weight development of piglets during the suckling and early
   postweaning period. Piglets were weaned on day 28 of life (n =
   5/feeding group and sex). * p < 0.05.

4. Discussion

   Normal development in the early neonatal period is essential for
   life-long performance and health in pigs [[100]17,[101]18]. Piglet’s
   development is driven by intrinsic (i.e., genetical programming) and
   extrinsic factors (e.g., nutrients) throughout the early life phases.
   Here, we provide missing data for postnatal concentrations of
   metabolites and metabolic markers in plasma and serum from day 7 of
   life until after weaning, which represent useful reference values for
   the practice to assess the nutritional, physiological and health status
   of very young piglets. In terms of the interpretation of the data, it
   should be noted that the presented plasma metabolome values are the net
   sum from intestinal absorption and hepatic and peripheral metabolism;
   hence, the summation from anabolic and catabolic reactions. In using
   targeted metabolomics, we had a strong focus on lipids and specific
   metabolic pathways related to sugar/energy, bile acid and amino
   acid/nitrogen metabolism, whereas other physiological pathways may have
   been underrepresented. Moreover, as no reference pathway library was
   available for Sus scrofa, the human KEGG pathway database was used to
   predict metabolome pathway enrichments. Although the similarity between
   the human and porcine genome amounts to about 90%, and despite the fact
   that the relevance of the human pathways for pigs was confirmed
   previously [[102]16] (Lefort et al., 2020), differences in the
   annotations may exist, which is a weakness when applying the pathway
   enrichment analysis for species other than humans and has to be kept in
   mind when interpreting the present results.

   The orthogonal projections of sPLS-DA provided the first idea that
   distinguishable metabolite profiles for the suckling and postweaning
   period could be obtained for the various days of life. Although
   metabolomes of the consecutive days in the suckling period overlapped,
   a certain trend for diverging plasma metabolomes was discernible, which
   was confirmed by the differential analysis and by protruding the VIP
   scores from the sPLS-DA models for the various days of life. The
   present age-related profiles showed evidence that the piglets’
   metabolism altered within the first two weeks of life and pinpointed
   markers for the developmental stage of the suckling and newly weaned
   piglets. The sPLS-DA supported the importance of lipids in the
   metabolism of the neonatal piglets, identifying several TGs as the most
   influential metabolites with elevated concentrations in the early
   suckling phase, probably being indicative for the role of fat to meet
   their energy demand for thermogenesis and ATP generation. In addition,
   the identified lipids (i.e., TGs) and phosphatidylcholine
   [phosphatitylcholine diacyl (PC aa) C32:0] are important building
   blocks for membranes, hormones and body insulation. The three most
   influential, TG 18:0_30:0, TG 14:0_34:1 and TG 16:0_34:0 according to
   the VIP scores, may be useful marker metabolites for the suckling phase
   that largely drop postweaning. Part of the identified lipids were
   probably endogenously synthesized in hepatocytes. Nevertheless, it
   needs to be considered that the milk fatty acid profile and thus the
   nutrition of the gestating and lactating sow probably influenced the
   detected fatty acid profiles in the piglet plasma [[103]19,[104]20].
   Therefore, the discriminant nature of the identified lipids from this
   experiment needs to be verified in further studies. Weaning largely
   affected the lipid profile. Many TG, acyl-carnitines and phospholipids
   dropped in their concentrations postweaning, often stronger on day 35
   than on day 31 of life; probably being indicative for the lower feed
   and fat intake in the first week postweaning. Biomarker identification
   showed that TG 17:1_34:2, TG 17:0_36:1 and TG 18:0_32:1 were
   distinctive for the postweaning days and may be marker candidates for
   this period. However, similar to the identified lipids for the suckling
   phase, there is the dependence of the plasma lipid profile from the
   dietary lipid intake. Consequently, their validity needs to be proven
   in a further set of piglets being fed a different prestarter diet.

   Our targeted approach revealed strong age-related concentration
   patterns of water-soluble metabolites in the first two weeks of life
   which matched the high anabolic rate, energy demand for thermogenesis
   and tissue growth in the piglets. Indeed, piglets from modern hybrid
   lines, like the present ones, double their body weight within the first
   two weeks of life. From the identified enrichment pathways, metabolites
   of the TCA (i.e., pyruvic acid, fumaric acid, succinic acid, aconitic
   acid and citric acid) and purine metabolism (i.e., urea and uric acid)
   as well as 2-hydroxybutyric acid, beta-alanine and histamine, were
   discriminated as key metabolites, being raised in plasma on day 7
   compared to day 14 of life. The elevated levels of many proteinogenic
   amino acids together with the urea cycle metabolites arginine,
   citrulline and ornithine on day 14 of life may point towards a higher
   muscle protein turnover and deamination and thus higher utilization of
   amino acids as energy substrates. By contrast, concentrations of
   primary and secondary bile acids were indicative for the later suckling
   period. Although bile acid concentrations were probably the result of
   the higher milk and consequently milk fat intake on days 21 and 28 of
   life, they were also a sign for digestive maturation. Weaning appeared
   not to impact bile acid concentrations and composition because they
   were still high on day 31 of life. The question arises whether the drop
   in bile acid concentrations on day 35 of life was a delayed weaning
   effect due to the lower feed intake and/or the shift in diet
   composition with less fat and mainly plant-based lipids. Plasma glycine
   and taurine concentrations needed for the conjugation of primary bile
   acids were lower on day 35 of life, supporting a lower delivery of
   nutrients. In addition, the secondary bile acids (e.g., hyodeoxycholic
   acid) originate from microbial metabolism of bile salts [[105]21] and
   were thus representative of microbial activity in the piglets’ gut,
   supporting high microbial activity on day 28 of life before weaning.
   Many bacterial taxa along the small and large intestines are capable of
   modifying the conjugated primary bile acids [[106]22]. Therefore, it is
   difficult to pinpoint the drop in secondary bile acids to the bile salt
   hydrolase and/or bile acid-inducible enzyme activity of a specific
   taxon and gut site. Microbial modification of bile salts is essential
   for bile acid homeostasis [[107]22] and their lower levels postweaning
   may have implications for mucosal functioning and inflammatory
   signaling [[108]23]. Likewise, plasma acetate was rising from day 7 to
   28 of life, suggesting increased intestinal fermentation of milk
   glycans [[109]24] with the increasing milk intake during the suckling
   phase. Contrary to the observation for bile acids, weaning seemed not
   to impair intestinal fermentation as acetate concentration in plasma
   continued to rise postweaning. Propionate and butyrate were only
   detectable in plasma after weaning, demonstrating the importance of
   substrate (plant-based diet) for their intestinal production and
   subsequent systemic appearance. Results from differential analysis and
   of the VIP scores from the sPLS-DA models identified benzoic acid and
   its glycine-conjugate hippuric acid as discriminant metabolites
   postweaning. Since plasma benzoic acid and hippuric acid originate from
   the intake of plant polyphenols [[110]25], their plasma concentrations
   may be useful markers for piglets’ plant intake postweaning. Indeed,
   their values would support increasing feed intake from day 31 to 35 of
   life. By contrast, largely raised plasma concentrations of
   phenylacetylglycine and alpha-aminobutyric acid on day 31 of life were
   indicative of catabolic reactions (i.e., phospholipid catabolism and
   methionine and threonine catabolism, respectively) [[111]26] and hence
   body tissue mobilization. The enrichment pathway analysis further
   emphasized glutamate and glutamine as key metabolites within the most
   influential pathways related to amino acid turnover, which were
   elevated in plasma postweaning compared to the suckling period.
   Concurrently, urea cycle metabolites dropped postweaning. On the one
   hand, this could mean that deamination pathways were not upregulated
   for use of amino acids as energetic substrate postweaning. This is
   contrasted with the elevated concentration of alpha-aminobutyric acid
   on day 31 of life, which indicated body tissue mobilization. Therefore,
   our observation of lower urea cycle metabolites may be a sign of low
   availability of the precursors, i.e., arginine and ornithine, due to
   lower feed intake to form sufficient citrulline and urea [[112]26],
   leading to the accumulation of glutamate and glutamine as key
   regulatory elements [[113]27].

   In modern pig breeding, weaning takes place earlier than naturally,
   shifting the piglet back to a glucogenic metabolism due to the
   carbohydrate-rich weaner diet. Creep feeding may have the same effect
   due to its high carbohydrate content. Therefore, we also characterized
   the impact of nutrition during the suckling phase as cause for
   variation in plasma metabolite concentrations, as this is relevant
   information for using the plasma metabolome data in practice. The
   commercial creep feed that we used in this study contained 29.4% starch
   and 26.8% sugar on dry matter basis, whereas the fat amounted to only
   7.8% on a dry matter basis. Although the actual amount consumed by the
   individual piglets probably varied, creep feeding altered the fat and
   carbohydrate metabolism as indicated by the different development of
   plasma acyl-carnitines in creep-fed and sow milk-only-fed piglets.
   Acyl-carnitines are key factors regulating the balance of intracellular
   sugar and lipid metabolism [[114]28]. Specifically, the plasma levels
   of free and acetyl-carnitine increased with creep feeding; suggesting
   that the higher rate of glycolysis due to the higher starch/glucose
   intake in the creep-fed piglets produced more acetyl-CoA which was then
   transported in the blood compared to piglets that only received sow
   milk. The enrichment pathway analysis further supported this
   assumption. Plasma concentrations of metabolites belonging to the
   pyruvate and glycolysis-gluconeogenesis pathways were elevated in the
   suckling period due to creep feeding. Likewise, results from
   differential analysis and signature features implied major alterations
   in plasma lipid fractions due to creep feeding. This was supported by
   the biomarker analysis which confirmed that the plasma lipid
   composition, especially TG, could distinguish which piglets received
   creep feed during the suckling period, e.g., TG 20:4_36:5, TG 17:0_34:2
   and TG 18:2_38:6 on days 14, 21 and 28 of life, respectively. The
   identified TG were distinctively elevated by the creep feeding compared
   to only sow milk nutrition. Moreover, the differential analysis
   supported that creep-fed piglets may have used less fat as fuel,
   favoring glucose as an energy substrate, as most TG were elevated from
   day 14 to 28 of life in creep-fed piglets. Although plasma glucose
   levels were not different, the postprandial insulin response in the
   piglets was presumably higher after ingesting the starch-rich creep
   feed and prestarter diet, promoting hepatic lipid synthesis and rising
   blood triglyceride levels [[115]29]. The creep feeding effect was not
   only detectable during the suckling phase, when the creep feed was
   provided, but lasted until after weaning; again major changes were
   noticeable in the postweaning TG profile. The ketone levels, i.e.,
   2-hydroxybutyrate and 2-keto isovaleric acid, were higher postweaning,
   particularly on day 31 of life, but not different between the feeding
   groups, supposing similar body fat mobilization due to low feed intake
   irrespective of the preweaning nutrition. However, the higher body
   weight on day 35 of life indicated that piglets that suckled only sow
   milk may have recovered faster from weaning stress and started eating
   more feed than piglets from the creep feed group. This is an
   interesting finding which rejects our hypothesis that the piglets from
   the creep feed group should have had an advantage by being adapted to
   consume dry feed and to the prestarter diet before weaning. Differences
   in feed intake may explain the diverging plasma lipid levels of
   creep-fed and only sow milk-fed piglets postweaning, becoming depleted
   in certain TG in the creep-fed piglets that were still detected in the
   plasma of only sow milk-fed piglets. Assumingly, the depletion may be
   related to a higher body fat mobilization in creep-fed piglets compared
   to sow milk-fed piglets.

   In terms of clinical biochemistry parameters that are regularly used in
   routine diagnostics, developmental patterns were discernible for serum
   total protein, liver enzyme activities and electrolytes, demonstrating
   again the importance for specific reference values for neonatal
   piglets. The serum enzymes ALP, AST and ALT showed especially strong
   developmental patterns with high activities on day 7 of life which then
   declined to day 14. This observation likely reflected the high
   metabolic rate of the hepatocytes at this age, thereby supporting the
   findings for the plasma metabolome profile at this age, and not liver
   dysfunction. Plasma total protein and electrolytes were relatively
   stable during the suckling phase, whereas weaning and creep feeding
   were more influential, with postweaning serum levels being generally
   lower. Interestingly, creep-fed piglets showed a much stronger
   postweaning drop in serum total protein, ALP, calcium and sodium
   especially on day 31 of life, which, from a clinical perspective, may
   be interpreted as malnutrition [[116]30] and may be additional useful
   markers for piglets feed intake postweaning.

   In conclusion, the present plasma metabolome and serum biochemistry
   data provide conclusive information for the postnatal evolution of
   lipids and water-soluble metabolites and metabolic markers, which can
   be used as reference values for the practice to assess the nutritional,
   physiological and health status of neonatal piglets. Plasma lipid
   profiles in particular corresponded to the type of nutrition in the
   suckling phase and showed a strong weaning effect. Potential marker
   metabolites for age, creep feeding and weaning were identified.
   Influential TG 20:4_36:5, TG 17:0_34:2 and TG 18:2_38:6 were higher in
   creep-fed piglets on days 14, 21 and 28 of life, respectively, compared
   to only sow milk-fed piglets. Metabolites belonging to pathways within
   histidine, D-glutamine and D-glutamate metabolism as well as hippuric
   acid were distinctive for the postweaning compared to the suckling
   period. Selected lipids should be verified in future work due to their
   dependency of the dietary fatty acid composition.

Acknowledgments