IMR Press / FBL / Volume 27 / Issue 1 / DOI: 10.31083/j.fbl2701033
Open Access Original Research
Microarray analysis reveals an important role for dietary L-arginine in regulating global gene expression in porcine placentae during early gestation
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1 Departments of Animal Science, Texas A&M University, College Station, TX 77843, USA
2 Departments of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX 77843, USA
3 Departments of Poultry Science, Texas A&M University, College Station, TX 77843, USA

§The current address of Xilong Li is Institute of Feed Research, The Chinese Academy of Agricultural Sciences, 100081 Beijing, China.
The current address of Huaijun Zhou is Department of Animal Science, University of California at Davis, Davis, CA 95616, USA.
Academic Editor: Graham Pawelec

Front. Biosci. (Landmark Ed) 2022 , 27(1), 33; https://doi.org/10.31083/j.fbl2701033
Submitted: 22 November 2021 | Revised: 28 December 2021 | Accepted: 4 January 2022 | Published: 18 January 2022
Copyright: © 2022 The Author(s). Published by IMR Press.
This is an open access article under the CC BY 4.0 license.
Abstract

Background: Increasing the dietary provision of L-arginine to pregnant swine beginning at Day 14 of gestation enhances embryonic survival, but the underlying mechanisms are largely unknown. Objective: This study determined the effects of dietary supplementation with 0.8% L-arginine to gilts between Days 14 and 25 of gestation on the global expression of genes in their placentae. Methods: Between Days 14 and 24 of gestation, gilts were fed 2 kg of a corn- and soybean meal-based diet (containing 12.0% crude protein and 0.70% Arg) supplemented with 0.8% L-arginine or without L-arginine (0.0%; with 1.64% L-alanine as the isonitrogenous control). On Day 25 of gestation, 30 min after the consumption of their top dressing containing 8 g L-arginine or 16.4 g L-alanine, gilts underwent hysterectomy to obtain placentae, which were snap-frozen in liquid nitrogen. Total RNAs were extracted from the frozen tissues and used for microarray analysis based on the 44-K Agilent porcine gene platform. Results: L-Arginine supplementation affected placental expression of 575 genes, with 146 genes being up-regulated and 429 genes being down-regulated. These differentially expressed genes play important roles in nutrient metabolism, polyamine production, protein synthesis, proteolysis, angiogenesis, immune development, anti-oxidative responses, and adhesion force between the chorioallantoic membrane and the endometrial epithelium, as well as functions of insulin, transforming growth factor beta, and Notch signaling pathways. Conclusion: Dietary supplementation with L-arginine plays an important role in regulating placental gene expression in gilts. Our findings help to elucidate mechanisms responsible for the beneficial effect of L-arginine in improving placental growth and embryonic/fetal survival in swine.

Keywords
Amino acids
Metabolism
Nutrition
Pigs
Placenta
Pregnancy
1. Introduction

There is growing interest in the nutritional role of L-arginine (Arg) to enhance litter size in livestock species [1, 2, 3]. However, only a few studies have been conducted to explore the underlying mechanisms [4, 5, 6]. Thus, there is a limited understanding of regulatory functions of Arg in the placenta. Results of recent studies indicated that Arg is not only a building block for proteins, but also has multiple physiological roles in cell signaling and function [7, 8]. For example, Arg stimulates the production of nitric oxide (NO) and polyamines (key regulators of cell growth and development) by placental cells [9, 10], as well as the placental expression of aquaporins and the transport of water across the placentae [6]. In addition, Arg may influence the expression of genes related to amino acid transport, anti-oxidative responses, and protein synthesis in mammalian cells [9, 11]. As an approach to understanding how Arg acts on the placentae at the gene level, we used the 44-K Agilent porcine gene platform to determine changes in global gene expression in placentae at Day 25 of gestation from gilts receiving dietary Arg supplementation between Days 14 and 25 of gestation. This nutritional method is effective in enhancing placental growth and embryonic survival in swine [12].

2. Materials and methods
2.1 Animals and diets

The experimental design, including the diets of gilts before and after breeding, has been described by Li et al. [12]. Briefly, gilts (F1 crosses of Yorkshire × Landrace sows and Duroc × Hampshire boars) were checked daily for estrus with boars and bred 12 h and 24 h after the onset of the second estrus detected by the boars. Immediately after breeding, gilts were assigned randomly to one of the two treatment groups [0.0% Arg (with 1.64% L-alanine as the isonitrogenous control) or 0.8% Arg]. There were 10 gilts (individually penned) per treatment group. Between Days 14 and 23 of gestation, gilts were fed twice daily (07:00 h and 18:00 h) 1 kg of a corn- and soybean meal-based diet (containing 12.0% crude protein and 0.70% Arg) supplemented with 0.0% Arg (1.64% L-alanine; Control group) or 0.8% Arg (Arg group) [12]. The total feed intake of each gilt was 2 kg per day. On each day, L-alanine or Arg was mixed with cornstarch and then added to the basal diet as a top dressing consumed by each gilt. On Day 24 of gestation, gilts were fed once (08:00 h) with 2 kg of diet supplemented with either 0.8% Arg or 1.64% Ala. On Day 25 of gestation, 22 h after the last meal and 30 min after the consumption of their top dressing containing 8 g Arg or 16.4 g L-alanine, gilts were prepared for surgery and hysterectomized to obtain uteri and conceptuses (fetus and placenta). L-Alanine, rather than a mixture of amino acids, was used as the isonitrogenous control, because it is rapidly catabolized by pigs [5, 12], is not a substrate for arginine synthesis [5, 12], and does not affect any of the measured variables of reproductive performance on Day 25 of gestation (the number of corpora lutea; uterine, placental, and embryonic/fetal weights; the total number of fetuses, embryonic survival, and the number of live fetuses; and volumes of amniotic and allantoic fluids, compared with non-supplemented gilts [2, 3, 5, 6, 12]). This research was approved by Texas A&M University Animal Use and Care Committee.

2.2 Collection of placentae

Each placenta was obtained from a live fetus. A portion of the placenta was immediately snap-frozen in liquid nitrogen. All snap-frozen samples were stored at –80 C until analyzed. Eight gilts (three placentae from each gilt) in each group were selected randomly for the extraction of total RNA.

2.3 Total RNA isolation

Total RNA was isolated from the frozen placenta (approximately 30 mg) according to the manual of the RNeasy Mini Kit (Qiagen Inc., Valencia, CA) [4]. The quantity of the total RNA was measured by NanoDrop 1000 Spectrophotometer (Thermo Scientific, USA). The quality of total RNA was determined by 1% agarose electrophoresis. In addition, we determined the ratio of absorbance at 260 nm and 280 nm, which was used to assess the purity of RNA, was approximately 2.0 for the total RNA isolated from porcine placentae. The total RNA from 3 placentae from each gilt was combined at equal quantity to represent one biological replicate, and there were 8 biological replicates for each treatment group in the following microarray analysis.

2.4 Microarray analysis

Total RNA (400 ng) was reverse-transcribed to cDNA. T7 RNA polymerase-driven RNA synthesis was used for the preparation and labeling of cRNA with Cy3 or Cy5 dye. In each treatment group, 4 samples were treated with the Cy3 (green) dye, and 4 samples were treated with the Cy5 (red) dye. The labeled cRNA probes were purified with the RNeasy Mini Kit (Qiagen Inc., Valencia, CA). Purified cRNA was quantified with the NanoDrop 1000, and 825 ng of each was hybridized on the 44-K Agilent porcine gene expression microarray (Agilent, Santa Clara, CA). This array included 43,803 probes that were prepared using gene sources from RefSeq, UniGene, and TIGR. The slide format was printed using the Agilent’s 60-mer SurePrint technology. The hybridized slides were washed according to the manual of a commercial kit (Agilent Technology, Palo Alto, CA), followed by scanning with a Genepix 4100A scanner (Molecular Devices Corporation, Sunnyvale, CA) with the tolerance of saturation setting of 0.005%. A locally weighted linear regression (LOWESS) method was applied to normalize the data by the median of the signal intensity and local background values. SAS 9.1.3 program (SAS Institute Inc. Cary, NC) for the mixed model was used to analyze the normalized data [13]. Statistical significance to detect differentially expressed genes was determined by the approximate t-test for least-square means, where p < 0.05 was considered to be statistically significant. The false discovery rate (Q value) was calculated for each p-value using the R program [13]. Genes were annotated by the basic local alignment search tool (BLAST) in the database of the National Center for Biotechnology Information (NCBI) and the Institute for Genomic Research (TIGR). The database for annotation, visualization, and integrated discovery (DAVID) version 6.7 was used to generate specific functional annotations of biological processes for the differentially expressed genes [14].

2.5 Interaction pathways analysis for selected genes that were differentially expressed in the placentae of Arg-supplemented gilts

GO terms for biological processes (GO_TERM_BP) and KEGG pathways were identified for differentially expressed genes (both up- and down-regulated genes) using the database for DAVID version 6.8 [15, 16]. Ensembl gene IDs were converted to official gene symbols for input into DAVID using Ensembl’s Biomart, which is an open-source software and data service to the international scientific community (https://m.ensembl.org/info/data/biomart/index.html). Significance cutoff was p < 0.05.

2.6 Quantitative real-time PCR

Total RNA (1 μg) from each sample was used for cDNA synthesis with a random hexamer primer of a Thermoscript RT-PCR system kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The cDNAs were quantified by quantitative RT-PCR using the ABI Prism 7900HT system with SYBR Green PCR Master Mix (Applied Biosystems, Foster, CA) [4]. The primers for each gene were designed by using the Oligo6 program (www.oligo.net; Table 1). The cycling conditions of quantitative RT-PCR amplification were: 1 cycle at 95 C for 10 min, 40 cycles at 95 C for 15 s and optimal annealing temperature for 1 min. The porcine tubulin α gene was used as the housekeeping gene, and its expression was not affected by dietary Arg supplementation. Dissociation curves were performed at the end of amplification for validating data quality. For the RT-PCR analysis, slope values ranged from –3.51 and –3.32, which corresponded to the reaction efficiencies of 93% and 100%, respectively.

Table 1.Sequence and optimal annealing temperatures for primers used in quantitative RT-PCR analyses.
Accession No. Gene Primer sequence Product length (bp) Annealing Temp. (C )
NM_001001861 CXCL2 Forward: 5′- CACTGTGACCAAACGGAA -3′ 120 53
Reverse:5′- GTTGGCACTGCTCTTGTTT-3′
NM_214003 IGFBP2 Forward: 5′- GTGGATGGGAACGTGAACTT-3′ 111 56.8
Reverse:5′- GTGCTGCTCCGTGACTTTCT-3′
TC267605 PFKFB1 Forward: 5′- GCCTAAGATGACTCAAGAGA-3′ 187 53.3
Reverse:5′- CGTGGAGATGTAGGTCTTT-3′
NM_213963 PPARGC Forward: 5′- AACCCACAGAGACCCGAAAC-3′ 82 53
Reverse:5′- AAATGTTGCGACTGCGATTG-3′
AK231515 Presenilin 2 Forward: 5′- AAGGAGCACAGCGGACTCT-3′ 299 57
Reverse:5′- TGGGTACTGAACGGGTGTTT-3′
TC275071 RAG-2 Forward: 5′- ATGCCAGATCCTTAACCCAC-3′ 82 53
Reverse:5′- GCAGCAGAAATGAATCCAAC-3′
BI341657 RasGEF Forward: 5′- CTCCCATCTACAGCGAGGAA-3′ 104 56
Reverse: 5′- GAGCGTGGTCCTGAGGGTCT-3′
TC243513 RHBG Forward: 5′- GTGCCTACTTTGGGTTGGTC-3′ 103 56
Reverse:5′- ATGGCAAAGAGGTCCGAATG-3′
TC257543 RU2S Forward: 5′- CACTTCTGGAACCCTGCACT-3′ 103 53
Reverse:5′- TGATCCCACTGATTCAAGGC-3′
NM_001001863 TNNT3 Forward: 5′- CCTGTACCARCTGGAGATTG-3′ 78 51
Reverse: 5′- CTGAGGTTGATGATGTCGTA-3′
DQ225365 Tubulin α Forward: 5′-GCAGTGTTTGTAGACCTG GA-3′ 139 55
Reverse:5′-CAATGGTGTAGTGACCTCGG-3′
EU288086 MTOR Forward: 5′- GTCTCTATCAAGTTGCTGGC-3′ 126 53
Reverse: 5′- CTTTCGAGATGGCAATGGAA-3′
NM_001012613 SLC7A1 Forward: 5′- ACTCGACTCTCGTGGACCTT-3′ 134 54
Reverse:5′ GGTCAGTTGACTTTCTGCCT-3′
Primers were prepared using the oligo6 program (www.oligo.net).

All samples were run in triplicate and the average critical threshold cycle (Ct) was used to calculate the relative mRNA levels of target genes by the 2-ΔΔCT method [17]. We chose four significantly increased genes with a fold change more than 1.5, four significantly decreased genes with a fold change more than 2, and two genes with no change in mRNA levels based on microarray analysis to run quantitative real-time PCR for verifying the microarray data.

2.7 Statistical analysis

Data were analyzed by the unpaired t-test using the SPSS (Version 15.0, Chicago, IL). Gilt was considered as the experimental unit. Probability values <0.05 were considered statistically significant.

3. Results
3.1 Global change in placental mRNA levels based on microarray analysis

One hundred and forty-six (146) expressed sequence tags (ESTs) were up-regulated (Supplementary Table 1) and 429 ESTs were down-regulated (Supplementary Table 2) in response to dietary supplementation with 0.8% Arg between Days 14 and 25 of gestation. Some of the up-regulated and down-regulated genes with known physiological functions are summarized in Tables 2 and 3, respectively. Among the up-regulated genes in the placentae of Arg-supplemented gilts, the mRNA level of troponin T type 3 (TNNT3) was the greatest, followed by leucine-rich repeat-containing protein 51-like, calcitonin receptor, presenilin 2, ceroid-lipofuscinosis, and leucine-rich repeat-containing protein 18-like in descending order. Among the down-regulated genes in the placentae of Arg-supplemented gilts, the reduction in the placental mRNA for cytochrome b was the greatest, followed by Ras GEF domain 1A-similar gene, probable dolichyl pyrophosphate GMGGT-like gene, doublecortin domain-containing protein 2 (RU2S), acetyl-coenzyme A carboxylase alpha, and pig endogenous retrovirus group beta3 polymerase in descending order.

Table 2.Selected gene expression in the porcine placentae was up-regulated by dietary supplementation with 0.8% L-arginine between Days 14 and 25 of gestation in comparison with effects of the control diet.
Expressed sequence tag (EST; gene ID) Accession No. Gene name Fold change p-Value
BX918610 NM_001001863 Troponin T type 3 (TNNT3) 4.61 0.004
TC292911* XM_003129590 Leucine-rich repeat-containing protein 51-like 4.49 0.001
EW039857 NM_001742 Calcitonin receptor (CALCR) on chromosome 7 3.23 0.038
AK231515 EU287432 Presenilin 2 (PSEN2) 2.31 0.006
TC278497* NM_018941 Ceroid-lipofuscinosis, neuronal 8 2.23 0.010
TC289044* XM_001929300 Sus scrofa leucine-rich repeat-containing protein 18-like 2.10 0.020
PGM1 NM_001076903 Phosphoglucomutase 1 (PGM1) 1.86 0.030
TC275071* NM_000536 Recombination activating gene 2 (RAG-2) 1.76 0.006
TC275071* AB091391 Recombination activating gene 2 (RAG-2) 1.76 0.006
TC274023* NM_001097446 Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3F (APOBEC3F) 1.70 0.005
BX666795 XM_001924347 Similar to solute carrier organic anion transporter family member 3A1 (SLCO3A1) 1.67 0.003
TC278155* NM_214378 Rh blood group polypeptide 1.66 0.019
TC267605 NM_001143721 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 (PFKFB1) 1.55 0.025
EW660666 NM_001045886 Phenazine biosynthesis-like protein domain containing 1.54 0.013
TC246855* AY208121 Myostatin 1.54 0.022
AY610045 XM_001924474 Similar to androgen-induced 1 1.42 0.018
DY428406 NG_016762 Pyruvate dehydrogenase kinase isozyme 3 (PDK3) 1.40 0.042
CF361829 A9YMB8 NADH dehydrogenase subunit 2 1.34 0.012
AJ947745 NG_007956 Cytochrome P450 family 20 subfamily A polypeptide 1 (CYP20A1) 1.33 0.022
TC261962* EW422073 Hemoglobin subunit epsilon 1 (HBE1) 1.31 0.035
AK234630 XM_001927389 FK506 (Tacrolimus)-binding protein 1.28 0.009
AJ584674 NM_213757 Beta-Galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4) 1.27 0.000
BW980922 XM_001113023 dUTP pyrophosphatase isoform 2 transcript variant 4 (DUT) 1.27 0.021
AK239509 AB529869 Peroxisomal trans-2-enoyl-CoA reductase (PECR) 1.25 0.027
BX667232 XM_001925672 Similar to pecanex-like protein 1 1.23 0.030
CN155716 EU617320 Small calcium-binding mitochondrial carrier 1 1.23 0.038
EV880225 DQ629170 Ribosomal protein S6 (RPS6) 1.22 0.017
CK467702 NM_001035277 Cadherin 13 (CDH13) 1.22 0.013
CD572284 AJ009912 Proteolipid protein (PLP) 1.21 0.006
TC258084 NM_006690 Matrix metallopeptidase 24 (MMP24) 1.19 0.045
DN125568 GQ184633 Cell division cycle 2 (CDC2) 1.18 0.048
EW299999 XM_001498308 Similar to eukaryotic translation elongation factor 1 beta 2 (EF1 β2) 1.17 0.015
TC258796 XM_001928025 Calcineurin A protein transcript variant 2 1.14 0.049
SCYE1 NM_001114283 Aminoacyl tRNA synthetase complex-interacting multifunctional protein 1 (AIMP1) 1.12 0.026
*Sequence can be accessed on http://compbio.dfci.harvard.edu/cgi-bin/tgi.
Table 3.Selected gene expression in porcine placentae was down-regulated by dietary supplementation with 0.8% L-arginine between Days 14 and 25 of gestation in comparison with effects of the control diet.
Expressed sequence tag (EST; gene ID) Accession No. Gene name Fold change p-Value
AJ964783 O48246 Cytochrome b 0.15 0.001
BI341657 XM_001926447 RasGEF domain 1A 0.18 0.013
TC273367* XM_003129699 Probable dolichyl pyrophosphate GMGGT-like 0.20 0.010
TC257543* XM_001927988 Doublecortin domain-containing protein 2 (RU2S) 0.23 0.015
DN100844 FJ263680 Acetyl-coenzyme A carboxylase alpha 0.27 0.003
NP321728 AF274712 Pig endogenous retrovirus group Beta3 polymerase 0.29 0.014
BI360386 XM_003133904 Oncostatin-M-specific receptor subunit beta-like 0.31 0.009
TC238637* NM_214376 Amphiregulin 0.31 0.045
CF178669 AJ427478 Agouti signaling protein 0.33 0.023
CX061534 XM_003130350 Torsin-1A-interacting protein 1-like 0.40 0.007
TC301037* XM_003357826 Serine/threonine-protein kinase [doublecortin like kinase 1 (DCLK1)]-like 0.42 0.012
TC243513* NM_213996 Rh family, B glycoprotein (RHBG) 0.45 0.006
DN106254 NM_001098597 Osteocrin (OSTN) 0.47 0.025
AY577905 NM_001001861 Chemokine (C-X-C motif) ligand 2 (CXCL2) 0.49 0.013
TC278652* NM_214003 Insulin-like growth factor binding protein 2 0.49 0.002
BP443132 XM_864245.3 Cytochrome P450 family 2 subfamily C member 33 (CYP2C33) 0.50 0.037
AY198323 NM_214257 Dipeptidyl peptidase 4 (DPP4) 0.51 0.030
TC280345* XM_003122165 Golgin A1 0.51 0.018
TC290654* NM_001105290 Bone morphogenetic protein 7 (Bmp7) 0.55 0.030
CO989438 XM_001928917 Potassium large conductance calcium-activated channel, subfamily M, beta member 4 0.56 0.017
DQ836054 NM_001097442 Disabled-1(DAB1) 0.57 0.021
TC270858* AF228059 Decay-accelerating factor CD55 0.58 0.026
CV878027 XM_001926796 Sterile alpha motif domain containing 4A (SAMD4A) 0.58 0.018
TC290589* XM_003132094 Upstream binding protein 1 0.58 0.005
CA513725 XM_003129205 Heat shock 70kDa protein 4-like 0.58 0.016
EV881857 XM_003132080 Sodium bicarbonate cotransporter 3-like 0.59 0.009
TC266622* XM_003127574 Methylenetetrahydrofolate reductase (NAD(P)H), transcript variant 1 0.60 0.018
TC286353* NM_001243919 Coupling of ubiquitin conjugation to ER degradation (CUE) domain containing 1 0.60 0.007
TC250322* NM_001037965 Inhibitor of DNA binding 2 0.61 0.007
CN159399 NM_001128506 Charged multivesicular body protein 4b-like 0.61 0.012
AK230591 NM_001128488 Antizyme inhibitor 1 0.62 0.016
AK234300 XM_003125957 RIB43A-like with coiled-coils protein 2-like 0.63 0.005
TC247541* XM_003134192 Pericentriolar material 1 0.64 0.015
CF181641 XM_003128338 Dystonin, transcript variant 2 0.64 0.015
AK233736 XM_001927836 Similar to Down syndrome critical region gene 1-like 1 protein 0.65 0.033
DQ866834 DQ279926 Retinoid X receptor alpha (RXRalpha) 0.65 0.047
AB271924 NM_001099924 Fibroblast growth factor receptor 2 (FGFR2) 0.68 0.019
AY850382 NM_001011505 Kruppel-like factor 13 (KLF13) 0.68 0.006
AB116561 NM_213772 Interferon alpha and beta receptor subunit 1 (IFNAR1) 0.69 0.012
TC248589* NM_001077215 Regulator of differentiation 1 (ROD1) 0.70 0.025
AY610204 NM_214296 Rho family GTPase 3 (RND3) 0.70 0.039
BP142559 XM_001926474 A-kinase anchoring protein 13 (AKAP13) 0.70 0.016
TC257240* XM_001925375 Similar to positive regulatory (PR) domain containing 1, with ZNF domain transcript variant 2 0.71 0.042
AY284842 AY284842 Glycerol-3-phosphate acyltransferase (GPAT) 0.71 0.016
AK235700 NM_001078670 Interferon regulatory factor 9 0.71 0.024
AK235466 DQ105589S2 CDP-Diacylglycerol Synthase 2 (CDS2) 0.71 0.013
EU095967 NM_001105286 TNF receptor associated factor 6 (TRAF6) 0.71 0.023
BP444119 NM_214224 4-Hydroxyphenylpyruvate dioxygenase (HPD) 0.72 0.007
AY159788 NM_214266 5’-AMP-activated protein kinase catalytic subunit alpha-2 (PRKAA2) 0.72 0.025
AK235681 NM_213963 Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC-1) 0.72 0.032
AK240475 XM_001927539 Similar to general transcription factor IIH 0.73 0.006
BP446317 NM_001097440 Bridging Integrator 1 (BIN1) 0.73 0.036
CK461960 NM_001162401 lysophosphatidic acid receptor 2 (LPAR2) 0.73 0.048
BI184146 XM_001927725 Prostaglandin F2 receptor inhibitor (PTGFRN) 0.74 0.002
CV875504 XM_001926134 Similar to chloride channel 3 0.74 0.040
EU009401 NM_001098605 Patatin-like phospholipase domain containing 2 (PNPLA2) 0.74 0.014
TC261381 NM_213973 Heat-shock protein 90 (HSP90) 0.75 0.036
AK233668 NM_213830 Folate-binding protein (FBP) 0.75 0.029
AY609622 AY609622 Similar to small nuclear RNA activating complex 0.76 0.037
TC299692 NM_001025107 Homo sapiens adenosine deaminase RNA-specific (ADAR) 0.76 0.046
DY420532 NM_017902 Homo sapiens hypoxia inducible factor 1 alpha subunit inhibitor (HIF1AN) 0.76 0.047
AB254406 NM_001101814 Nuclear receptor subfamily 1 group H member 3 (NR1H3) 0.77 0.028
DN120475 XM_001927228 Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein 0.77 0.013
AY644721 NM_001009581 Peripherial benzodiazepine receptor associated protein (PAP7) 0.78 0.037
AJ955195 XM_001929149 Similar to transmembrane protein 77 0.79 0.036
AK237448 XM_001928092 Similar to Rab-1C 0.79 0.033
AK234427 XM_001928746 Similar to adenosine deaminase-like protein 0.79 0.046
TC278200* XM_001925656 Similar to procollagen 0.79 0.038
AK235686 XM_001925381 Similar to insulin-degrading enzyme 0.80 0.016
AK237044 XM_001499279 Similar to ubiquitin-conjugating enzyme E2Z 0.80 0.034
AK232486 NM_001159481 Pyruvate dehydrogenase kinase isozyme 2 (PDK2) 0.81 0.042
DN100853 AF339885 Mannose-6-phosphate/insulin-like growth factor II receptor 0.81 0.038
*Sequence can be accessed on http://compbio.dfci.harvard.edu/cgi-bin. CDP, cytosine diphosphate; RasGEF, Ras (rat sarcoma protein p21) guanine nucleotide exchange factor; GMGGT, Glc1Man9GlcNAc2 alpha-1,3-glucosyltransferase; RIB43A, ribbon protofilament protein 43A (43-kDa protein); TNF, tumor necrosis factor; ZNF, zinc finger.

The placental expression of mRNAs for the following enzymes or proteins related to amino acid metabolism did not differ (p > 0.05) between the control and 0.8% Arg-supplemented gilts: arginase I, ornithine carbamoyltransferase, acetylornithine and succinylornithine aminotransferase, pyrroline-5-carboxylate reductase 1, creatine kinase (mitochondrial) 2, guanidinoacetate N-methyltransferase, glutamine synthetase, glutaminase, glutamine:fructose-6-phosphate aminotransferase, carbamoyl-phosphate synthetase I, glutamine-dependent carbamoyl-phosphate synthase, glutamate decarboxylase, N-acetylglutamate synthase, aspartate aminotransferase 1 (glutamic-oxaloacetic transaminase 1, cytosolic), aspartate aminotransferase 2 (glutamic-oxaloacetic transaminase 2, mitochondrial), aspartate kinase, aspartate carbamoyltransferase, argininosuccinate synthetase, the glycine cleavage system H protein, glycine N-methyltransferase, lysine α-ketoglutarate reductase/saccharopine dehydrogenase, L-pipecolic acid oxidase, methionine-R-sulfoxide reductase B3, S-adenosylmethionine decarboxylase, S-adenosylhomocysteine hydrolase, betaine-homocysteine S-methyltransferase, methionyl-tRNA formyltransferase, prolyl 4-hydroxylase α subunit, prolyl 4-hydroxylase isoform c, threonyl-tRNA synthetase, serine racemase, homoserine kinase, kynurenine 3-monooxygenase (kynurenine 3-hydroxylase), mechanistic target of rapamycin (MTOR), selenocysteine-specific translation elongation factor, dopamine beta-hydroxylase, urate oxidase, monoamine oxidase B, D-amino acid oxidase, glutathione peroxidase 5, glutathione S-transferase, solute carrier family 7 member 1 (SLC7A1 encoding for the CAT-1 protein), and the branched-chain amino acid transport system carrier protein.

Dietary supplementation with 0.8% Arg did not affect (p > 0.05) the placental expression of mRNAs for the following enzymes or proteins related to glucose, fructose, fatty acid, and vitamin metabolism, as well as the Krebs cycle, the mitochondrial respiratory chain, hydroxylation, peroxidation, and water transport: glucose transporter 5, lactate dehydrogenases (A and C), hexokinase II, fructose-1,6-bisphosphatase, fructose-1,6-bisphosphate aldolase, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1, aldolase B, ketohexokinase isoform A, pyruvate dehydrogenase E1 component alpha subunit, dihydrolipoamide S-acetyltransferase (E2 component of pyruvate dehydrogenase complex), pyruvate kinase II, pyruvate dehydrogenase kinase isozymes (1 and 4), glucose-6-phosphate dehydrogenase, malate dehydrogenase 1 (NAD+, soluble), malate dehydrogenase (NADP+), 25-hydroxyvitamin D3 1α-hydroxylase, pyridoxine-5’-phosphate oxidase, NADPH oxidase 4, cytochrome oxidase subunits (I, II, and III), cytochrome c oxidases (I and III), methylmalonate-semialdehyde dehydrogenase, medium-chain acyl-CoA dehydrogenase, acyl-coenzyme A oxidase 2, peroxisomal, succinyl-CoA:α-ketoacid coenzyme A transferase, citrate synthase, isocitrate dehydrogenase 3 (NAD+) beta, isocitrate dehydrogenase (NAD+) subunit 1, cytosolic NADP+-dependent isocitrate dehydrogenase, succinate dehydrogenase subunits (A, B and D), NADH dehydrogenase (ubiquinone) 1 beta subcomplex, NADH dehydrogenase subunits (1, 3, 4 and 6), cytochrome oxidase subunit I, cytochrome P450 21-hydroxylase, cytochrome P-450 17a-hydroxylase, cytidine monophosphate-N-acetylneuraminic acid hydroxylase, peroxidase precursor, and aquaporins (1, 3, 4, 5, 7, 8, 9, 11, and 12).

3.2 DAVID analysis of differentially expressed genes in porcine placentae

The functional analysis by the DAVID program revealed that the genes with altered expression are related to nutrient transport, protein synthesis, protein degradation, polyamine synthesis, ion transport, glucose metabolism, fatty acid biosynthesis, immune development, inflammation, and anti-oxidative responses, as well as insulin, transforming growth factor beta, and Notch signaling pathways (Table 4). Changes in metabolic pathways were associated with alterations in the expression of single genes or a group of related genes.

Table 4.Pathway analysis for genes using the functional annotation of the DAVID program.
Gene name Species Database Pathway
5,10-methylenetetrahydrofolate reductase (NADPH) Homo sapiens KEGG_PATHWAY hsa00670:One carbon pool by folate
hsa00680:Methane metabolism
Acetyl-coenzyme A carboxylase alpha Homo sapiens KEGG_PATHWAY hsa00061:Fatty acid biosynthesis
hsa00620:Pyruvate metabolism
hsa00640:Propanoate metabolism
hsa04910:Insulin signaling pathway
Asparagine-linked glycosylation 8, alpha-1,3-glucosyltransferase homolog (S. cerevisiae) Homo sapiens KEGG_PATHWAY hsa00510:N-Glycan biosynthesis
Chemokine (C-X-C motif) ligand 2 Sus scrofa KEGG_PATHWAY ssc04062:Chemokine signaling pathway
Chromatin modifying protein 4B; similar to LOC616164 protein Bos taurus KEGG_PATHWAY bta04144:Endocytosis
Inhibitor of DNA binding 2 Sus scrofa KEGG_PATHWAY ssc04350:TGF-beta signaling pathway
Oncostatin M receptor Homo sapiens KEGG_PATHWAY hsa04060:Cytokine-cytokine receptor interaction
hsa04630:Jak-STAT signaling pathway
Potassium large conductance calcium-activated channel, subfamily M, beta member 4 Sus scrofa KEGG_PATHWAY ssc04270:Vascular smooth muscle contraction
Presenilin 2 Sus scrofa KEGG_PATHWAY ssc04330:Notch signaling pathway
ssc05010:Alzheimer’s disease
Recombination activating gene 2 Sus scrofa KEGG_PATHWAY ssc05340:Primary immunodeficiency
Ribosomal protein S6 (RPS6) Sus scrofa KEGG_PATHWAY Protein synthesis
Protein for ubiquitin conjugation Sus scrofa KEGG_PATHWAY Protein degradation
Antizyme inhibitor 1 Sus scrofa KEGG_PATHWAY Polyamine synthesis
Troponin T Sus scrofa KEGG_PATHWAY Cell growth and development
Cadherin 13 Sus scrofa KEGG_PATHWAY Cell–cell adhesion in tissues
Organic anion transporter Sus scrofa KEGG_PATHWAY Transport of organic anions
CYP20A1 Sus scrofa KEGG_PATHWAY Removal of xenobiotics
Heat shock 70kDa protein 4-like Sus scrofa KEGG_PATHWAY Inflammation and oxidative stress
Acetyl-coenzyme A carboxylase alpha Homo sapiens BIOCARTA Leptin Pathway:Reversal of Insulin Resistance by Leptin
5,10-Methylenetetrahydrofolate reductase (NADPH) Homo sapiens PANTHER_PATHWAY P02743:Formyltetrahydroformate biosynthesis
Doublecortin-like kinase 1 Homo sapiens PANTHER_PATHWAY P00031:Inflammation mediated by chemokine and cytokine signaling pathway
5,10-Methylenetetrahydrofolate reductase (NADPH) Homo sapiens REACTOME_PATHWAY REACT_11193:Metabolism of vitamins and cofactors
Acetyl-coenzyme A carboxylase alpha Homo sapiens REACTOME_PATHWAY REACT_1505:Integration of energy metabolism
REACT_602:Metabolism of lipids and lipoproteins
Pericentriolar material 1 Homo sapiens REACTOME_PATHWAY REACT_152:Cell cycle, mitotic
KEGG, a database resource for understanding the metabolic network and functions of the biological system; STAT, signal transducer and activator of transcription; TGF, transforming growth factor.
3.3 Interaction pathways analysis for selected genes that were differentially expressed in the placentae of Arg-supplemented gilts

Table 5 summarizes the results of the GO terms and KEGG interaction pathways for selected genes that were differentially expressed in the placentae of arginine-supplemented gilts. We noted that supplementing Arg to the diet of gestating gilts influenced the following interaction pathways: phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, regulation of circadian rhythm, glucagon signaling pathway, cell surface determinants, inflammation, osteoclast differentiation, Hippo signaling pathway, membranous septum morphogenesis, nitrogen utilization, mesenchymal cell differentiation, branching involved in salivary gland morphogenesis, ammonium transmembrane transport, organic cation transport, mesenchymal cell differentiation, beta-amyloid metabolic process, positive regulation of astrocyte differentiation, nutrient oxidation, extracellular space metabolism and remodeling, cell growth and development, regulation of gene transcription, and embryonic pattern specification.

Table 5.Interaction pathways analysis for selected genes that were differentially expressed in the placentae of arginine-supplemented gilts.
Term Count % p-Value Genes
ssc04151:PI3K-Akt signaling pathway 7 13.46154 0.001015 NM_213973, XM_001927228, NM_214266, NM_001099924
NM_001162401, NM_213772, XM_003133904
GO:0042752 regulation of circadian rhythm 3 5.769231 0.004521 NM_213963, NM_214266, NM_001037965
ssc04922:glucagon signaling pathway 4 7.692308 0.005019 XM_001928025, NM_213963, NM_001143721, NM_214266
GO:0009986 cell surface determinants 5 9.615385 0.008898 XM_001925381, NM_001114283, NM_214376, NM_214257
NM_001162401
ssc05160:inflammation 4 7.692308 0.011177 NM_001101814, NM_001078670, NM_001105286, NM_213772
ssc04380:osteoclast differentiation 4 7.692308 0.012663 XM_001928025, NM_001078670, NM_001105286, NM_213772
ssc04390:Hippo signaling pathway 4 7.692308 0.013715 NM_001105290, XM_001927228, NM_214376, NM_001037965
GO:0003149 membranous septum morphogenesis 2 3.846154 0.01435 NM_001099924, NM_001037965
GO:0019740 nitrogen utilization 2 3.846154 0.01435 NM_213996, NM_214378
GO:0060445 branching involved in salivary gland morphogenesis 2 3.846154 0.017907 NM_001105290, NM_001099924
GO:0072488 ammonium transmembrane transport 2 3.846154 0.02145 NM_213996, NM_214378
GO:0015695 organic cation transport 3 3.846154 0.024981 NM_213996, NM_214378, NM_214376
GO:0048762 mesenchymal cell differentiation 2 3.846154 0.024981 NM_001105290, NM_001099924
GO:0050435 beta-amyloid metabolic process 2 3.846154 0.0285 XM_001925381, NM_001078666
GO:0048711 positive regulation of astrocyte differentiation 2 3.846154 0.0285 NM_001097440, NM_001037965
GO:0014850 nutrient oxidation 2 3.846154 0.0285 NM_213963, NM_214266
GO:0005615 extracellular space metabolism and remodeling 7 13.46154 0.032712 XM_001925381, NM_001105290, NM_001114283, NM_001098597,
NM_214376, NM_001001861, NM_214003
GO:0060749 cell growth and development 2 3.846154 0.038982 NM_214376, NM_001037965
GO:0048557 regulation of gene transcription 2 3.846154 0.042451 NM_001099924, NM_001037965
GO:0009880 embryonic pattern specification 2 3.846154 0.042451 NM_001105290, NM_001099924
PI3K, phosphoinositide 3-kinase.
3.4 Change in placental mRNA levels based on RT-PCR analysis

Data from the RT-PCR analysis of selected genes largely confirmed results from the microarray analysis (Table 6). These genes were chemokine (C-X-C motif) ligand 2 (CXCL2), MTOR, presenilin 2, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 (PFKFB1), recombination activating gene 2 (RAG-2), Ras (rat sarcoma protein p21) guanine nucleotide exchange factor (RasGEF), Rh family B glycoprotein (RHBG), RU2S, SLC7A1, and TNNT3.

Table 6.Verification of microarray analysis data using quantitative RT-PCR analyses.
Accession No. Gene name Microarray analysis RT-PCR analysis
Fold change p-Value Fold change p-Value
NM_001001863 TNNT3 4.61 0.004 4.37 0.015
AK231515 Presenilin 2 2.31 0.006 1.77 0.021
TC275071 RAG-2 1.76 0.006 1.68 0.013
TC267605 PFKFB1 1.55 0.025 1.51 0.036
XM_001926447 RasGEF 0.18 0.013 0.22 0.019
NM_001001861 CXCL2 0.49 0.013 0.43 0.016
TC243513 RHBG 0.45 0.006 0.63 0.016
TC257543 RU2S 0.23 0.015 0.38 0.044
EU288086 MTOR 1.01 0.783 1.04 0.520
NM_001012613 SLC7A1 0.97 0.602 0.94 0.550
CXCL2, chemokine (C-X-C motif) ligand 2; MTOR, mechanistic target of rapamycin; PFKFB1, 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 1; RAG-2, recombination activating gene 2; RasGEF, Ras (rat sarcoma protein p21) guanine nucleotide exchange factor; RHBG, Rh family, B glycoprotein; RU2S, doublecortin domain-containing protein 2; SLC7A1, Sus scrofa solute carrier family 7 member 1 (CAT-1); TNNT3, troponin T type 3.
4. Discussion

The placenta plays a critical role in transporting amino acids from mother to fetus, thereby having an enormous impact on fetal survival, growth, and development [18]. The pig has true epitheliochorial placentation, meaning that the placenta is only superficially attached to the uterine luminal epithelium. Such a placental structure increases the efficiency of gas and nutrient exchanges between fetus and mother [19]. Consistent with the increased availability of Arg in the conceptus of Arg-supplemented gilts [4], results of this microarray analysis revealed that dietary supplementation with 0.8% Arg to gilts between Days 14 and 25 of gestation altered the expression of 575 genes in their placentae. To our knowledge, this is the first study of effects of dietary Arg supplementation on in vivo expression of placental genes in any animal species. The microarray assay provides a powerful molecular technology to allow for the simultaneous determination of the expression of thousands of genes (particularly unexpected ones) in a tissue. The results can facilitate the elucidation of mechanisms responsible for the effects of nutrients or other substances.

Polyamines are crucial for cell growth, migration, and proliferation, as well as angiogenesis [20]. We recently reported that dietary supplementation with Arg to gilts increased the activity of ornithine decarboxylase (ODC) and the synthesis of polyamines from ornithine in their placenta [4]. A novel and unexpected finding of the present study is that Arg supplementation reduced the placental expression of ODC antizyme inhibitor 1 (Table 3). This inhibitor protein binds to and destabilizes ODC, thereby suppressing ODC activity. Thus, a decrease in the expression of the ODC antizyme inhibitor 1 alleviates the inhibitory effect on ODC activity, leading to enhanced polyamine synthesis in placentae. This action of Arg is associated with an increase in the transmembrane transport of Ca2+ in the porcine placentae (Table 2), which further stimulates ODC activity in mammalian cells [21].

Results of our previous in vitro studies revealed that, as compared with 10 μM Arg, augmentation of Arg concentration in culture media from 50 to 350 μM dose-dependently increased protein synthesis and inhibited protein degradation as well as the proliferation of trophectoderm cells partly via a mechanism requiring MTOR activation [9]. Leucine and glutamine also activate the MTOR cell signaling pathways in placental cells and embryos [22, 23, 24, 25]. Other underlying mechanisms likely require the following six regulatory pathways. The first pathway is related to increases in the placental expression of aminoacyl tRNA synthetase complex-interacting multifunctional protein 1, ribosomal protein S6 (a component of the 40S ribosomal subunit for mRNA translation), eukaryotic translation elongation factor 1 beta 2, and cell division cycle 2 (Table 2), leading to increased protein synthesis. The second pathway may require an increase in the placental expression of dUTP pyrophosphatase (Table 2), which is critical for the fidelity of DNA replication and repair [26]. The third pathway involves decreases in the placental expression of ubiquitin-conjugating proteins, resulting in a reduction in intracellular proteolysis. Fourth, an increase in the expression of type-3 troponin may beneficially enhance the growth of the placenta and alter its structure, as reported for myogenesis [27], to allow for the efficient transfer of nutrients and oxygen from mother to fetus [5]. Fifth, the up-regulated expression of leucine-rich repeat-containing proteins in the placenta of Arg-supplemented gilts may facilitate gene transcription, as reported for other cell types [28] to enhance the receptivity of the organs to Arg or its metabolites in placental cells [29]. Sixth, in coordination with all these changes, down-regulated expression of insulin-like growth factor 2 (IGF-2) binding protein can enhance the availability of IGF-2 to promote placental cell growth and differentiation via phosphoinositide 3 (PI3) and mitogen-activated protein (MAP) kinase signaling pathways [30]. Thus, collectively, Arg regulates intracellular protein turnover to favor protein accretion in cells and their growth through multiple mechanisms.

Dietary Arg supplementation enhances placental angiogenesis (the growth of new blood vessels from the existing vasculature) partly via the generation of polyamines and NO [4, 5]. In addition, there is emerging evidence that glycans are novel activators of angiogenesis under physiological conditions due to changes in protein glycosylation [31]. Consistent with this notion, the expression of beta-galactoside alpha 2–3 sialyltransferase (a glycosyltransferase), a key enzyme that catalyzes protein glycosylation via the terminal sialylation of glycoproteins and glycolipids, was enhanced in the placentae of Arg-supplemented gilts as compared to the control group (Table 2). Likewise, calcitonin stimulated all phases of angiogenesis through the calcitonin receptor [32], and matrix metallopeptidases contributes to angiogenesis through the degradation of the vascular basement membrane and remodeling of the extracellular matrix [33]. Furthermore, calcineurin (a calcium- and calmodulin-dependent serine/threonine protein phosphatase) stimulates angiogenesis through Ca2+ and calmodulin signaling (including the synthesis of NO by endothelial NO synthase) in cells [34], whereas presenilin 2 helps to process intracellular proteins that transmit chemical signals (e.g., vascular endothelial growth factor) from the cell membrane into the nucleus [35]. In this regard, it is noteworthy that maternal Arg supplementation augmented the expression of calcitonin receptor, matrix metallopeptidase 24, calcineurin A, and presenilin 2 in porcine placentae (Table 2). Thus, Arg-induced angiogenesis in porcine placentae is supported by multiple mechanisms (Table 5).

Arg is known to alleviate inflammation [11, 36] and enhance immune responses [37] in animals but the underlying mechanisms are not fully understood. For example, dietary supplementation with Arg reduces risk for gastrointestinal infections and embryonic deaths in gestating gilts [38]. Interestingly, unexpected results of the present work revealed increases in the placental expression of the following key genes related to anti-oxidative and immune responses in gestating gilts supplemented with Arg (Table 3). These genes include: (a) the recombination-activating genes (RAGs), which encode part of a protein complex that plays important roles in the rearrangement and recombination of the genes for B-cell development and the production of immunoglobulins [39], as well as T-cell receptor molecules [40]; (b) leucine-rich repeat-containing proteins 51-like and 18-like, which promote the maturation of cells of the innate immune system [41, 42]; and (c) solute carrier organic anion transporter family member 3A1 (SLCO3A1), which encodes for a membrane protein in immune cells that mediates inflammatory processes in epithelial cells through the activation of the NF-kB cell signaling pathway [43]. Likewise, dietary supplementation with Arg to gestating gilts reduced the placental expression of mRNAs for heat shock protein 70, hypoxia inducible factor 1 alpha subunit inhibitor, decay-accelerating factor CD55 (that is involved in epithelial inflammation) [44], amphiregulin (a transmembrane glycoprotein that participates in cell inflammatory responses [45]), and CXCL2 (Table 3), indicating an improvement in cellular redox balance and a reduction in cellular inflammation.

There is much evidence that Arg regulates the metabolism of lipids and glucose in mammalian liver, skeletal muscle, and white adipose tissue [8, 46], as well as nutrient transport by the small intestine [47]. However, little is known about the roles of Arg in these biochemical processes in placentae. Results of the microarray analysis indicated, for the first time, that dietary supplementation with Arg altered the expression of some key genes in porcine placentae that are involved in: (a) glycolysis and glucose oxidation to CO2; (b) fatty acid synthesis and oxidation; (c) one-carbon unit metabolism; and (d) ion transport (Tables 2 and 3). The up-regulated genes include phosphoglucomutase, PFKFB1, pyruvate dehydrogenase kinase isozyme 3, NADH dehydrogenase subunit 2, peroxisomal trans-2-enoyl-CoA reductase, cytochrome P450 family 20 subfamily A polypeptide 1, apolipoprotein B mRNA editing enzyme, hemoglobin subunit epsilon 1 (for iron-storage), small calcium-binding mitochondrial carrier 1, and solute carrier organic anion transporter family member 3A1. The down-regulated genes include adenosine deaminase, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, pyruvate dehydrogenase kinase isozyme 2, acetyl-coenzyme A carboxylase alpha, glycerol-3-phosphate acyltransferase, cytosine diphosphate diacylglycerol synthase 2, sodium bicarbonate cotransporter 3-like, chloride channel 3, potassium large conductance calcium-activated channel subfamily M, and methylenetetrahydrofolate reductase. The changes in the expression of the metabolic enzymes were associated with those for cell signaling protein, including FK506 (Tacrolimus)-binding protein, retinoid X receptor alpha, Kruppel-like factor 13 (zinc finger transcription factor), insulin-degrading enzyme, Rho family GTPase 3, interferon alpha and beta receptor subunit 1, general transcription factor IIH, hypoxia inducible factor 1 alpha subunit inhibitor, and mannose-6-phosphate/insulin-like growth factor II receptor (Tables 2 and 3). Future metabolic studies involving isotope tracers are required to determine actual changes in the rates of placental nutrient transfer, synthesis, and catabolism in Arg-supplemented dams.

Another novel and important finding from the current work is that dietary Arg supplementation increased cadherin expression in porcine placentae (Table 2). Cadherin is a transmembrane protein that mediates cell–cell adhesion [48]. By regulating the stability of contacts between cells, cadherins play a crucial role in tissue morphogenesis and homeostasis. This is consistent with the analysis of interaction pathways for differentially expressed genes (Table 5) and the report that the apparent adhesion force between the chorioallantoic membrane and the endometrial epithelium was greater in Arg-supplemented gilts than control gilts [49]. Further analysis of the adhesion strength would require mechanical testing equipment.

5. Conclusions

Dietary supplementation with 0.8% Arg to gilts between Days 14 and 25 of gestation increased the expression of mRNAs for the syntheses of polyamines and protein, angiogenesis, cell-to-cell interactions, immune development, and antioxidative responses in placentae. Arginine supplementation reduced the placental expression of genes for protein degradation, inflammation, and cell injury. Furthermore, some of the key genes for glucose and fatty acid metabolism, ion transport, and cell signaling in placentae were differentially expressed between control and Arg-supplemented gilts to support placental growth and differentiation. Results from this microarray study will help to elucidate complex mechanisms responsible for the beneficial effects of Arg in improving conceptus growth, survival, and development in swine and possibly other mammals.

Author contributions

GW, FWB, and GAJ conceived and designed the study. XL, GW, FWB, GAJ, and RCB performed the experiment. XL and HZ analyzed the data. XL and GW summarized the results and wrote the manuscript. All authors contributed to data interpretation and manuscript revisions, and approved the final manuscript.

Ethics approval and consent to participate

This study was approved by The Institutional Animal Care and Use Committee of Texas A&M University. No consent to participate was applicable.

Acknowledgment

Not applicable.

Funding

This work was supported by Agriculture and Food Research Initiative Competitive Grants (2015-67015-23276) from the USDA National Institute of Food and Agriculture.

Conflict of interest

The authors declare no conflict of interest. GW is serving as one of the Editorial Board members of this journal. We declare that GW had no involvement in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to GP.

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