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Journal of Animal Science - Animal Nutrition

Plasma metabolomics indicates metabolic perturbations in low birth weight piglets supplemented with arginine1

 

This article in JAS

  1. Vol. 93 No. 12, p. 5754-5763
     
    Received: May 11, 2015
    Accepted: Sept 29, 2015
    Published: December 18, 2015


    2 Corresponding author(s): rdilger2@illinois.edu
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doi:10.2527/jas.2015-9293
  1. C. M. Getty*†‡,
  2. F. N. Almeida*,
  3. A. A. Baratta*‡ and
  4. R. N. Dilger 2*†§
  1. * Department of Animal Sciences, University of Illinois, Urbana 61801
     Division of Nutritional Sciences, University of Illinois, Urbana 61801
     College of Veterinary Medicine, University of Illinois, Urbana 61801
    § Neuroscience Program, University of Illinois, Urbana 61801

Abstract

Large profit losses in the swine industry can be attributed to morbidity and mortality of piglets before weaning, especially in the low birth weight (LBW) piglet. Recent evidence suggests sow’s milk contains insufficient concentrations of Arg to support optimal growth and health of piglets. Therefore, our objective was to assess global metabolomic profiles and the potential for Arg supplementation to promote growth of LBW (≤0.9 kg BW) and average birth weight (ABW; 1.3 to 1.5 kg BW) piglets. Piglets were selected in littermate pairs at processing to receive either l-Arg or an isonitrogenous control (l-Ala) and weighed daily to assess growth rate, and blood was collected at approximately 16 d of age for metabolomics analysis. In terms of growth, LBW and ABW piglets supplemented with Arg weighed 22.3 and 12.7% less, respectively, at d 16 compared with Ala-supplemented piglets of the same birth weight group. Overall, differences (P < 0.05) were observed among treatments for metabolic pathways involving energy (i.e., tricarboxylic acid cycle intermediates), AA, nucleotides, and fatty acids. Increased nucleotide turnover, indicative of an increase in DNA damage and cell death, was particularly noted in the LBW piglet. However, Arg supplementation reduced these effects to levels comparable to those observed in ABW piglets. Moreover, changes in glucose metabolism suggested a compromised ability to extract energy from dietary sources may have occurred in the LBW piglet, but these effects were partially recovered by Arg supplementation. We conclude that a reduction in the growth potential of LBW piglets may be associated with alterations in multiple metabolic pathways, and further reduction due to Arg supplementation may have resulted from perturbations in multiple metabolic pathways.



INTRODUCTION

Large profit losses in the swine industry can be attributed to morbidity and mortality of piglets before weaning, especially in the low birth weight (LBW) piglet. This condition may affect 15 to 20% of all pigs born each year in the U.S. swine industry (Wu et al., 2006). Importantly, LBW piglets still remain at risk for above-normal rates of morbidity and mortality during the preweaning period mainly due to underdeveloped function of the gastrointestinal tract (Wang et al., 2005, 2008).

A promising avenue to reduce losses due to LBW status is the provision of supplemental Arg. Arginine is a conditionally essential AA, particularly in growing mammals, for both protein and nonprotein functions. Epithelial cells in the gut of the neonate synthesize Arg from multiple sources, including citrulline, proline, and glutamine (Wilkinson et al., 2004; Haines et al., 2011); however, this endogenous source may not be sufficient to maximize BW gain. Furthermore, neonatal piglets cannot obtain adequate amounts of Arg from suckling, as sow’s milk is estimated to contain 40% or less of the amount required by the piglet (Wu et al., 2000; Kim and Wu, 2004). Arginine also plays an important role in ammonia excretion as a contributor to the urea cycle and in vasodilation as a precursor to nitric oxide (NO). As such, improved blood flow theoretically allows quantitatively greater distribution of nutrients throughout the body and may, therefore, aid in greater nutrient absorption and animal growth.

The driving hypothesis for this study was that Arg supplementation will improve growth trajectory of LBW piglets through positive changes in metabolism. As such, our objective was to determine whether daily oral dosing of Arg alters growth and global metabolite profiles of preweaning piglets in a commercially applicable setting.


MATERIALS AND METHODS

All procedures involving animals were approved by the Institutional Animal Care and Use Committee at the University of Illinois.

Characterization of Piglet Birth Weights

Farrowing records from the University of Illinois Imported Swine Research Laboratory farm (Champaign, IL) were used to create a database of birth weights from 8,207 piglets farrowed between July 8, 2008, and October 21, 2011. Three separate data sets were created based on ultimate disposition of piglets, as follows:

  1. all piglets born,

  2. piglets denoted as low viability plus piglets ultimately weaned, and

  3. piglets ultimately weaned.

Litter data were excluded if meeting any of several criteria, including 1) litter size smaller than 5 piglets, 2) dam of an underrepresented breed, or 3) less than 30% of pigs in a litter surviving through weaning (approximately 21 d of age). Any stillborn or piglets that were euthanized for reasons other than LBW were also excluded from the final data set.

Various statistical models were tested, and the best model (i.e., that with the smallest Akaike information criterion fit statistic) for characterizing piglet birth weights included the fixed effects of sire breed, dam breed, dam parity (with all 2- and 3-way interactions), and piglet sex, along with the random effects of record year and litter. Studentized residuals were generated to detect outliers, and any observation with an absolute studentized residual greater than 3 was removed from the data set. The process of detecting and removing outliers was repeated until the data sets stabilized and, ultimately, descriptive statistics were generated.

Experimental Treatment of Piglets

Thirty-two piglets (13 castrated male and 19 female piglets) were selected in littermate pairs within 24 h of birth based on BW; 15 separate litters were used. The LBW piglet was defined as weighing less than 0.9 kg (range 0.69 to 0.92 kg), whereas the average birth weight (ABW) piglet was defined as weighing 1.3 to 1.5 kg, as determined from the population characterization described above. A litter was selected only if it contained both LBW and ABW piglets. Efforts were made to choose LBW/ABW pairs of the same sex; however, the number of available litters made this impossible for every pair. Of the LBW/ABW pairs, only 3 of the 16 pairs were mixed sex. Within each birth weight group, piglets were assigned to an AA solution of either l-Arg HCl (treatment; 145.0 mg/kg BW per administration) or an isonitrogenous concentration of l-Ala (control; 245.5 mg/kg BW per administration). In total, there were 4 treatment groups (n = 8 piglets per treatment), including ABW-Ala, ABW-Arg, LBW-Ala, and LBW-Arg.

Amino acid solutions (final pH 6.6 to 6.7) were prepared fresh once per week by dissolving l-Arg HCl or l-Ala in sterile PBS to prevent gastrointestinal distress; solutions were always stored at 4°C between administration sessions. Piglets did not exhibit any abnormal stool or feeding behaviors throughout the duration of the experiment. For dosing, individual piglets were removed from the sow and weighed each morning to calculate the volume of AA solution to be dispensed. Every 12 h, piglets received half of the total daily volume of assigned AA solution by oral gavage and were immediately returned to the sow. Gavage of LBW and ABW piglets began at processing (approximately 1 d of age) and continued through the end of the study (16 to 17 d of age). Piglets were allowed ad libitum access to water and nursed as allowed by the sow but were not provided creep feed. At study termination, blood was collected from the cranial vena cava of individual piglets into evacuated tubes containing lithium heparin 6 h after the first daily AA dose. Blood was centrifuged at 1,300 × g for 20 min. at 4°C, and plasma was stored at –80°C pending analysis.

Biochemical Profiling

The metabolome is defined as the complete set of small-molecule metabolites present in any biological sample. Therefore, the simultaneous analysis and interpretation of metabolic effects due to treatment is referred to as metabolomics. Piglet blood samples were submitted to a commercial laboratory (Metabolon, Inc., Durham, NC) for global metabolite profiling. At the time of analysis, lithium heparin–treated blood samples were prepared using a proprietary series of organic and aqueous extractions to remove the protein fraction and allow maximum recovery of small biomolecules. The resulting extract was divided into 2 fractions for analysis on either the gas chromatography–mass spectrometry or liquid chromatography–mass spectrometry platforms. Thorough quality control steps were taken to ensure integrity of final data, including normalization of data when sample analyses occurred over multiple days. Semiquantitative methods were used to assess treatment effects on individual metabolites, and data were expressed as fold changes relative to the control calibrator group (ABW-Ala).

Blood Urea Nitrogen Analysis

Blood urea nitrogen (BUN) concentration of plasma was assayed using a colorimetric procedure (Urea Assay Kit, MAK006; Sigma-Aldrich, Inc., St. Louis, MO). The assay was run according to manufacturer instructions, and colorimetric results were determined using a plate reader (Biotek, Winooski, VT) set to an absorbance of 570 nm. Colorimetric results were plotted against a standard curve generated by subjecting serial dilutions of urea to the same protocols.

Statistical Analyses of Experimental Data

Piglet BW gain data were analyzed by ANOVA using the MIXED procedure of SAS 9.3 (SAS Inst. Inc., Cary, NC). The statistical model included the fixed effects of birth weight (LBW vs. ABW) and AA solution (Arg vs. Ala) as well as their interaction. Longitudinal piglet BW data were analyzed by a repeated measures ANOVA using the same statistical model described above, with day of age serving as the repeated measure. Piglet sex and sow parity (gilt vs. sow) were included as random effects. Statistical differences were considered significant at P ≤ 0.05; trends were accepted at 0.05 < P ≤ 0.10.

Metabolomics and BUN data were subjected to a 2-way ANOVA using SAS 9.3 using fixed effects of birth weight (LBW vs. ABW) and AA solution (Arg vs. Ala) as well as their interaction. Piglet sex and sow parity (gilt vs. sow) were included as random effects. Statistical differences were considered significant at P < 0.05. Considering the large number of metabolites that were compared, the possibility for false discovery does exist; however, no conclusions were drawn from changes in only a single metabolite.


RESULTS

Definition of Treatment Groups

A histogram of piglet birth weights, including those piglets successfully weaned and those euthanized for complications associated with being born with a LBW, is shown in Fig. 1. Based on the lowest 10th percentile of the data set, the LBW piglet was defined as weighing less than 0.9 kg, and the ABW piglet was defined as weighing 1.3 to 1.5 kg at birth.

Figure 1.
Figure 1.

Histogram of birth weights of piglets at the University of Illinois Imported Swine Research Laboratory (Champaign, IL; including years 2008 to 2011). All piglets that were weaned and those euthanized due to complications associated with low birth weight are included. Blue bars represent pigs defined as average birth weight (1.3 to 1.5 kg), and orange bars represent those pigs defined as low birth weight (≤0.9 kg) at approximately 1 d of age.

 

Growth Performance

For the dosing study, final BW of ABW piglets was greater (P < 0.001) than LBW pigs. Similarly, ADG was greater (P < 0.001) for ABW piglets (204 g/d) than for LBW piglets (129 g/d). Rates of BW gain for the LBW and ABW piglets were similar to previously reported values recorded over a 21-d period (Ramsay et al., 2010). In general, Arg supplementation reduced (P = 0.002) final BW compared with Ala supplementation, and Arg-supplemented pigs had lower (P = 0.002) ADG than Ala-supplemented piglets. The negative effects of daily Arg supplementation became evident within birth weight status starting around d 11 of the study (Fig. 2).

Figure 2.
Figure 2.

Effects of birth weight status and AA supplementation on growth performance of piglets. Values are means of 8 replicate pigs exposed to either average birth weight (ABW) or low birth weight (LBW) status and either Ala or Arg supplementation (e.g., ABW piglets administered Ala [ABW-Ala] as the control group). In general, Arg supplementation reduced (P < 0.001) final BW compared with Ala supplementation, and Arg-supplemented pigs had lower (P < 0.001) ADG than Ala-supplemented piglets. The negative effects of daily Arg supplementation became evident within birth weight group starting around d 11 of the study (*P < 0.05). ABW-Arg = ABW piglets administered Arg; LBW-Ala = LBW piglets administered Ala; LBW-Arg = LBW piglets administered Arg

 

Nitrogen Metabolism

Importantly, no differences were observed for plasma Ala concentrations; however, Arg levels were greater (P = 0.013) in piglets supplemented with Arg than those supplemented with Ala (Fig. 3; Supplemental Table S1 [see the online version of the article at http://journalofanimalscience.org]). Interestingly, an interaction (P = 0.027) between birth weight status and AA supplementation was observed for dimethylarginines (asymmetric dimethylarginine and symmetric dimethylarginine), where greater levels were observed for LBW-Arg pigs than for ABW-Ala, ABW-Arg, or LBW-Arg piglets, which were not different from each other (Fig. 3; Supplemental Table S1 [see the online version of the article at http://journalofanimalscience.org]). Plasma urea measured by metabolomics indicated that LBW piglets tended to have greater (P = 0.088) levels of urea compared with ABW piglets, regardless of AA supplementation status. However, when BUN was measured quantitatively using a colorimetric assay (Fig. 3), a similar pattern was observed, but in this case, the effect of birth weight status became significant (P = 0.043).

Figure 3.
Figure 3.

Effects of birth weight and AA supplementation status on relative plasma Arg and Ala concentrations. Values are means of 8 replicate pigs exposed to either average birth weight (ABW) or low birth weight (LBW) status and either Ala or Arg supplementation (e.g., ABW-Ala: average birth weight piglet administered alanine [ABW-Ala] as the control group) with blood samples collected at 16 to 17 d of age. (A) There were no effects of either birth weight or AA supplementation on plasma Ala concentrations (presented as fold change relative to the ABW-Ala treatment group). (B) There was a main effect of AA supplement on plasma Arg concentrations where those pigs provided supplemental Arg had greater (P = 0.013) levels than those provided Ala, regardless of birth weight status (plasma Arg levels presented as fold change relative to the ABW-Ala treatment group). (C) There was a main effect of birth weight on blood urea nitrogen (BUN) where LBW piglets had greater (P = 0.043) concentrations than ABW piglets, regardless of AA supplementation status. ABW-Arg = ABW piglets administered Arg; LBW-Ala = LBW piglets administered Ala; LBW-Arg = LBW piglets administered Arg.

 

Energy Metabolism

Energy metabolism, including compounds related to the tricarboxylic acid (TCA) cycle, was impacted by birth weight status and AA supplementation (Table 1; Supplemental Table S1 [see the online version of the article at http://journalofanimalscience.org]). In general, energy metabolism appeared to be restricted in LBW-Ala piglets but not in LBW-Arg piglets. Interactions between birth weight status and AA supplementation were observed for fumarate (P = 0.001), malate (P = 0.015), and succinate (P = 0.040) where ABW-Ala, ABW-Arg, and LBW-Arg piglets were not different but the LBW-Ala piglets had greater concentrations of each metabolite compared with all other treatment groups. A similar interaction (P = 0.014) was also observed for glucose. Interestingly, an interaction (P = 0.007) was also observed for sorbitol, where greater levels were observed for LBW-Ala piglets compared with ABW-Ala or LBW-Arg piglets but were not different from the ABW-Arg group.


View Full Table | Close Full ViewTable 1.

Effects of birth weight and AA supplementation status on plasma metabolomic profiles of 16-d piglets1

 
Treatment2
ABW
LBW
P-value3
Metabolite Pathway ALA ARG ALA ARG Pooled SEM BirthWt AA Interaction
Aspartate Amino acid 1.00 1.80 1.66 1.11 0.28 0.968 0.659 0.023
Citrulline Amino acid 1.00 1.09 1.21 1.25 0.11 0.094 0.576 0.817
Creatine Amino acid 1.00 1.31 0.99 1.25 0.21 0.877 0.191 0.900
Creatinine Amino acid 1.00 0.97 0.96 0.82 0.05 0.056 0.085 0.285
Dimethylarginines (SDMA + ADMA4) Amino acid 1.00a 0.92a 1.18b 0.86a 0.05 0.255 0.001 0.027
Glutamine Amino acid 1.00 0.87 0.90 0.96 0.11 0.947 0.656 0.216
Histidine Amino acid 1.00 1.01 1.03 1.08 0.06 0.375 0.595 0.661
Isoleucine Amino acid 1.00 0.94 1.13 1.04 0.05 0.022 0.133 0.812
Leucine Amino acid 1.00 0.98 1.00 1.04 0.07 0.639 0.907 0.667
Lysine Amino acid 1.00 1.05 1.14 1.16 0.11 0.261 0.748 0.889
Methionine Amino acid 1.00 0.92 0.94 0.86 0.06 0.344 0.204 0.922
Threonine Amino acid 1.00 1.16 0.97 1.17 0.10 0.918 0.045 0.846
Tyrosine Amino acid 1.00 0.85 0.89 0.93 0.10 0.820 0.473 0.220
Valine Amino acid 1.00 1.02 1.12 1.10 0.06 0.141 0.999 0.735
Urea Amino acid 1.00 1.02 1.59 2.36 0.54 0.088 0.477 0.497
Glucose Carbohydrate 1.00a 1.02a 1.16b 0.95a 0.04 0.332 0.045 0.014
Sorbitol Carbohydrate 1.00a 2.06ab 2.61b 1.25a 0.50 0.339 0.723 0.007
Alpha-ketoglutarate Energy 1.00bc 0.81ab 1.20c 0.49a 0.16 0.646 0.001 0.042
Citrate Energy 1.00 0.80 1.17 0.76 0.11 0.542 0.006 0.303
Fumarate Energy 1.00a 1.09a 1.51b 0.93a 0.16 0.064 0.012 0.001
Malate Energy 1.00a 1.26a 2.62b 1.27a 0.32 0.014 0.089 0.015
Succinate Energy 1.00a 1.07a 1.72b 1.11a 0.16 0.020 0.094 0.040
1-Docosapentaenoylglycerophosphocholine Lipid 1.00 0.66 0.80 0.29 0.21 0.121 0.022 0.634
1-Eicosadienoylglycerophosphocholine Lipid 1.00 1.07 0.93 0.46 0.20 0.054 0.251 0.123
1-Eicosatrienoylglycerophosphocholine Lipid 1.00 1.01 0.97 0.47 0.13 0.035 0.069 0.055
Docosadienoate (22:2n6) Lipid 1.00a 0.89a 0.99a 1.83b 0.27 0.017 0.054 0.015
Glycerol-3-phosphate Lipid 1.00ab 1.17b 1.28b 0.80a 0.14 0.628 0.115 0.003
Glycerophosphocholine Lipid 1.00 1.39 1.35 0.84 0.27 0.621 0.747 0.029
Hexadecanedioate Lipid 1.00 1.32 1.90 1.37 0.26 0.039 0.635 0.066
Octadecanedioate Lipid 1.00 1.20 1.83 1.10 0.32 0.145 0.282 0.070
Oleoylcarnitine Lipid 1.00 0.81 1.30 0.47 0.33 0.913 0.014 0.116
Palmitoylcarnitine Lipid 1.00 0.94 1.17 0.75 0.18 0.919 0.084 0.191
Propionylcarnitine Lipid 1.00 1.04 1.92 1.18 0.39 0.016 0.097 0.074
5,6-Dihydrouracil Nucleotide 1.00a 1.04a 1.59b 0.83a 0.16 0.251 0.036 0.021
a–cMeans within a row and without a common superscript differ (P < 0.05).
1Values are means of 8 replicate piglets born either LBW or ABW and supplemented with Arg or Ala (average birth weight piglets administered Ala [ABW-Ala] as the control group) with blood collected from piglets at 16 to 17 d of age. Data presented as fold change relative to average birth weight piglets administered Ala (ABW-Ala) group.
2ABW = average birth weight; ALA = Ala supplementation; ARG = Arg supplementation; LBW = low birth weight.
3BirthWt = main effect of birth weight status; AA = main effect of amino acid supplementation; Interaction = interactive effect of birth weight status and AA supplementation. Bold font = significant P-values.
4ADMA = asymmetric dimethylarginine; SDMA = symmetric dimethylarginine.

Lipid Metabolism

Metabolism of lipids was altered by birth weight status and AA supplementation (Table 1; Supplemental Table S1 [see the online version of the article at http://journalofanimalscience.org]). There are many examples where lysophosphatidylcholine species (i.e., metabolic products of phosphatidylcholine) were altered by both birth weight status and AA supplementation. Specifically, an interaction (P = 0.002) was observed for 1-palmitoylglycerophosphocholine where ABW-Ala, ABW-Arg, and LBW-Ala piglets were not different but LBW-Arg piglets exhibited concentrations that were lesser than each of the other treatment groups. Additionally, a similar interaction was observed for 1-eicosatrienoylglycerophosphocholine (P = 0.055) and 1-oleoylglycerophosphocholine (P = 0.029). There was also an interaction (P = 0.003) observed for glycerol-3-phosphate, a metabolite common to both phospholipid synthesis and degradation, where ABW-Ala, ABW-Arg, and LBW-Ala piglets were not different but LBW-Arg piglets were similar only to the ABW-Ala group. Additionally, a main effect of birth weight status was observed for palmitate, where LBW piglets had greater (P = 0.033) levels than ABW piglets.

Nucleotide Metabolism

In general, LBW piglets had altered plasma nucleotide levels, which was reversed by Arg supplementation (Fig. 4; Supplemental Table S1 [see the online version of the article at http://journalofanimalscience.org]). Specifically, an interaction between birth weight status and AA supplementation was observed for cytidine (P = 0.002), guanosine (P = 0.001), inosine (P = 0.003), and uracil (P = 0.001), where ABW-Ala and LBW-Arg piglets were not different but greater levels of these nucleotides were observed in ABW-Arg and LBW-Ala piglets. A similar interaction (P = 0.021) was observed between birth weight status and AA supplementation for 5,6-dihydrouracil, an intermediate breakdown product of uracil (Table 1).

Figure 4.
Figure 4.

Effects of birth weight and AA supplementation status on relative nucleotide concentrations. Values are means of 8 replicate pigs exposed to either average birth weight (ABW) or low birth weight (LBW) status and either Ala or Arg supplementation (e.g., average birth weight piglets administered Ala [ABW-Ala] as the control group) with blood samples collected at 16 to 17 d of age. (A) There was an interaction between birth weight status and AA supplementation where cytidine (P = 0.002) levels were greater in the average birth weight piglets administered Arg (ABW-Arg) and low birth weight piglets administered Ala (LBW-Ala) piglets, but in low birth weight piglets administered Arg (LBW-Arg) groups, levels returned to levels similar to the control group. There was a similar interaction between birth weight and AA supplementation status for (B) uracil (P = 0.001), (C) inosine (P = 0.003), and (D) guanosine (P = 0.001). (E) A similar trend for an interaction (P = 0.069) between birth weight status and AA supplementation was observed for guanine. (F) A main effect of AA supplementation was observed for thymidine, where Arg-supplemented piglets had lower (P = 0.044) concentrations compared with their Ala-supplemented counterparts.

 


DISCUSSION

This piglet study sought to evaluate the effects of LBW status on growth and metabolic outcomes in the piglet in concert with oral Arg supplementation to both ABW and LBW piglets. The following 3 main findings were observed: 1) objective definition of ABW and LBW piglet populations occurred, 2) Arg supplementation negatively impacted growth trajectories of both ABW and LBW piglets, and 3) both birth weight status and Arg supplementation influenced the metabolomic profiles of preweaned piglets.

Definition of the Low Birth Weight Piglet

Although LBW is the first and most notable sign of LBW status, there is great variability in these weights largely due to genetics, parity of the sow, and the number of littermates, with much of this problem relating to intrauterine growth restriction. Because of this variability, we first defined the LBW piglet in our production population. Defining the LBW piglet as weighing less than 0.9 kg and the ABW piglet weighing 1.3 to 1.5 kg, our findings were in good agreement with published values (Hegarty and Allen, 1978; Wang et al., 2010).

Arginine Supplementation Negatively Affects Growth of Average Birth Weight and Low Birth Weight Piglets

Growth depression due to Arg supplementation was unexpected as this AA has been shown to induce protein synthesis in in vitro studies using porcine cells (Bauchart-Thevret et al., 2010). Moreover, others have reported increased piglet growth when milk replacer formulas were supplemented with Arg (Kim and Wu, 2004). Importantly, the daily Ala and Arg dosages supplied to piglets in our study were based on approximately 50% of the Arg concentration found in sow’s milk as suggested by Wu et al. (2004), and oral dosage rates were chosen specifically to replicate those used a previous study (Wu et al., 2004). However, the concentration of Arg in sow’s milk varies (Mavromichalis et al., 2001; Renaudeau et al., 2003; Monaco et al., 2005).

Unfortunately, the Arg concentration of sow’s milk in the present study was not measured, which raises the possibility that depression in growth performance observed herein may have resulted of an imbalance in the dietary AA profile that pigs were receiving relative to specific AA requirements within each birth weight status. Although increased growth performance of piglets supplemented with Arg has been reported (Kim and Wu, 2004), this observation may be a result of an unusually low Arg intake in the milk provided to piglets in the control group (Ball et al., 2007). Importantly, piglets in our study were reared with their dams and littermates, thus providing application of these findings to practical industry settings.

Birth Weight and Arginine Supplementation Status Impact Metabolomic Profiles

Energy Metabolism.

Energy metabolism appeared to be restricted in the LBW-Ala piglet but was unhindered in the LBW-Arg piglet. Although the static nature of metabolomic profiling does not allow for the assessment of flux through a pathway, alterations in specific metabolites suggested the TCA cycle was altered by LBW status and AA supplementation. In the piglet dosing study, decreases in citrate and α-ketoglutarate concentrations were observed when both piglet birth weight statuses were supplemented with Arg, although this reduction was greater in LBW piglets compared with ABW piglets. Arginine is used to synthesize NO, which plays an important role in energy metabolism, and this gaseous molecule is known to stimulate expression of GLUT-4, a key glucose transporter, thus increasing glucose uptake by cells (Jobgen et al., 2006). Consequently, glucose oxidation may be expected to increase due to likely higher rates of glycolysis and flux through the TCA cycle. Indeed, although not significant, pyruvate was lower in LBW piglets supplemented with Arg compared with their LBW-Ala counterparts.

Nitrogen Metabolism.

In addition to altering energy metabolism, we speculate that LBW status resulted in AA requirements that are lower than for the ABW piglet. Lower rates of lean tissue accretion in LBW piglets, due to lower protein synthetic capacity, would result in reduced muscle growth and lower overall BW gain (Foxcroft et al., 2006). In this scenario, AA supplied in excess of physiological requirements would be deaminated to ultimately form urea, and this theory is corroborated by increased concentrations of blood urea detected in LBW piglets supplemented with Arg. Interestingly, this effect was observed only for piglets supplemented with Arg and not with Ala, suggesting that the additional Ala may have been more efficiently utilized compared with Arg.

Nucleotide Metabolism.

Changes in metabolites associated with nitrogen handling may also be related to increases in circulating concentrations of nucleotides. This observation is in agreement with Yamauchi et al. (2002), who observed that supplementation with Arg decreased the flux of glutamine to Arg in immortalized human intestinal cells in vitro, ultimately resulting in enhanced nucleotide synthesis. Our results support this observation as Arg-supplemented ABW piglets also exhibited a significant increase in blood nucleotides (e.g., cytidine, guanosine, and uracil). One possible reason for this observation is that NO has potential to induce cell apoptosis, thus increasing nucleotide turnover and circulating nucleotide concentrations as observed for Arg-supplemented ABW piglets. Other studies have observed that LBW status increases apoptosis of lymphocytes (Barg et al., 2004) in human infants and increased apoptosis of jejunal cells in piglets (Wang et al., 2010).

Lipid Metabolism.

Supplementation with Arg to LBW piglets had a significant impact on phosphatidylcholine metabolism. Phosphatidylcholine is an important phospholipid component of cell membranes, and several indicators of membrane phospholipid turnover (i.e., lysophospholipids and glycerophosphocholine) were decreased by LBW status or Arg supplementation in ABW pigs; however, this change was recovered by Arg supplementation in LBW piglets. These changes suggest that LBW status may lead to a lower rate of phospholipid breakdown or remodeling. Indeed, this suggestion is supported by the lower rate of growth in LBW piglets as compared with ABW piglets.

Decreased phospholipid turnover may indicate more subtle causality behind slower growth of the LBW piglet. Enzymes associated with phosphatidylcholine remodeling are also involved in the production of phosphatidylcholine-rich lung surfactants, which are required for proper lung function (Bridges et al., 2010). Additionally, lysophospholipids are immune cell stimulators (Takatera et al., 2007), and it has been shown that LBW human infants have lower total white blood cell and neutrophil counts compared with ABW counterparts (Troger et al., 2013). Therefore, Arg supplementation should have some impact on the health and performance of these LBW piglets, as Arg is a precursor for NO as a potent vasodilator (Loscalzo, 2000) and modulator of neonatal lymphocyte proliferation (Yu et al., 2014). Indeed, as described above, phosphatidylcholine and lysophosphatidylcholine species were decreased by LBW status or Arg supplementation in ABW piglets; however, this change was recovered by Arg supplementation in LBW piglets. Unfortunately, this study did not evaluate immune cell populations in the blood, so further study of this phenomenon is warranted.

In a fashion opposite to phospholipid metabolism, free fatty acid (FFA) oxidation was elevated in the LBW piglet. We observed elevated concentrations of fatty acids (both saturated and mono-/polyunsaturated) in LBW-Arg piglets when compared with ABW-Arg piglets. Arginine supplementation also resulted in decreased long-chain acylcarnitines in both birth weight groups, but the effect was more pronounced in LBW-Arg piglets. Acylcarnitine species (i.e., intermediates in FFA transport into the mitochondria) in the plasma reflect the amount of FFA utilization taking place (Eaton et al., 1996). These changes observed in FFA metabolism are similar to those previously described in LBW infants, where those born with LBW had higher rates of lipolysis and tended to contribute more of this lipid to energy metabolism when compared with ABW infants (Patel and Kalhan, 1992).

Elevations in the long-chain dicarboxylic acids (e.g., octadecanedioate and hexadecanedioate) in LBW-Ala piglets, as compared with ABW-Ala piglets, suggest that omega oxidation of FFA may be occurring in the endoplasmic reticulum. Omega oxidation is considered a minor pathway for fatty acid catabolism but has been previously shown to compensate for deficiencies in the β-oxidation pathway in human infants (Gregersen and Ingerslev, 1979). This alternative pathway may provide succinyl-CoA (i.e., the end product of omega oxidation) to be used in the TCA cycle for energy production. It is interesting to note that these changes were observed only in the LBW-Ala group, which, coupled with the observation of possible improvement of TCA cycle activity in the LBW-Arg group, suggests that provision of supplemental Arg may shift selection of fuel source away from fatty acids and toward carbohydrates, such as glucose.

In summary, our results indicate that Arg supplementation to LBW piglets did not improve growth performance as previously hypothesized. In fact, Arg supplementation reduced growth of piglets in both birth weight groups, which may reflect a dietary imbalance of AA intake. From the metabolomics analyses, we concluded that oral dosing of Arg altered the global blood metabolite profile of piglets, mainly involving metabolites associated with AA, energy, lipid, and nucleotide pathways. Although Arg supplementation was able to correct some of the metabolic abnormalities observed in LBW piglets, these effects appeared to be independent of growth mechanisms as piglets of both birth weight statuses were negatively impacted by daily dosing of this AA. Considering piglets in this study received AA supplements while remaining on the sow in a commercially applicable setting, these results provide industry-relevant information to better understand the implications of Arg supplementation in preweanling piglets of differing birth weight categories.

 

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