Increased maternal consumption of methionine as its hydroxyl analog promoted neonatal intestinal growth without compromising maternal energy homeostasis
- Heju Zhong†1,
- Hao Li†1,
- Guangmang Liu†1,
- Haifeng Wan1,
- Yves Mercier2,
- Xiaoling Zhang1,
- Yan Lin1,
- Lianqiang Che1,
- Shengyu Xu1,
- Li Tang1,
- Gang Tian1,
- Daiwen Chen1,
- De Wu1 and
- Zhengfeng Fang1Email authorView ORCID ID profile
© The Author(s). 2016
Received: 8 November 2015
Accepted: 18 July 2016
Published: 5 August 2016
To determine responses of neonatal intestine to maternal increased consumption of DL-methionine (DLM) or DL-2-hydroxy-4-methylthiobutanoic acid (HMTBA), eighteen primiparous sows (Landrace × Yorkshire) were allocated based on body weight and backfat thickness to the control, DLM and HMTBA groups (n = 6), with the nutritional treatments introduced from postpartum d0 to d14.
The DLM-fed sows showed negative energy balance manifested by lost bodyweight, lower plasma glucose, subdued tricarboxylic acid cycle, and increased plasma lipid metabolites levels. Both villus height and ratio of villus height to crypt depth averaged across the small intestine of piglets were higher in the DLM and HMTBA groups than in the control group. Piglet jejunal oxidized glutathione concentration and ratio of oxidized to reduced glutathione were lower in the HMTBA group than in the DLM and control groups. However, piglet jejunal aminopeptidase A, carnitine transporter 2 and IGF-II precursor mRNA abundances were higher in the DLM group than in the HMTBA and control groups.
Increasing maternal consumption of methionine as DLM and HMTBA promoted neonatal intestinal growth by increasing morphological development or up-regulating expression of genes responsible for nutrient metabolism. And increasing maternal consumption of HMTBA promoted neonatal intestinal antioxidant capacity without compromising maternal energy homeostasis during early lactation.
KeywordsEnergy metabolism Intestine Lactating sows Methionine nutrition Neonatal pigs
The gut plays a key role not only in the digestion, absorption and metabolism of nutrients, but also in the immune surveillance of the intestinal epithelial layer and regulation of the mucosal response to foreign antigens . There is growing evidence indicating that sulfur-containing amino acids (SAA), methionine and cysteine, play an important role in maintaining mucosal growth and antioxidant defense of neonatal intestine [2, 3]. The synthesis of the major cellular reductant, glutathione , and of the major extracellular reductant, cysteine , both depend on cysteine or its precursor, methionine . SAA deficiency suppressing intestinal epithelial growth has been demonstrated in a previous study in neonatal pigs . DL-methionine (DLM) and its hydroxyl analog, DL-2-hydroxy-4-methylthiobutanoic acid (HMTBA), are two methionine sources commonly used in commercial feed . Our recent studies indicated that increased inclusion of methionine as HMTBA in diets of sows or piglets promoted milk synthesis , attenuated the detrimental effect of early weaning on piglet growth and ameliorated intestinal antioxidant capacity . However, little is known about the association of pre-weaning intestinal growth status with maternal methionine nutrition.
The objectives of the present study were to determine whether neonatal intestinal growth could be promoted by increased maternal consumption of methionine as DLM or HMTBA. Sow plasma metabolites were analyzed by 1H nuclear magnetic resonance (NMR) spectroscopy method, and intestinal antioxidant capacity, morphology and expression of genes related to digestion, transport, metabolism, growth and immunity of piglets were also evaluated, which may provide a biological explanation for neonatal intestinal growth in response to maternal methionine nutrition.
Animals and diets
Ingredients and composition of the control diet of sowsa
Calculated protein and AA contents d
Soybean meal (CP 43 %)
Methionine + Cystine
Analyzed AA contents
Methionine + Cystine
Premix b, c
Choline chloride (50 %)
Milk collection of sows
The milk samples (20 mL) from each sow were collected at postpartum d0 and d7 before the morning meal as described previously . In brief, before manually milking functional pectoral and inguinal glands, piglets were separated from sows for 90 min firstly, then, each sow was injected with 10 I.U. oxytocin from the ear vein to collect milk samples which were stored at −20 °C until milk composition was analyzed.
Tissue collection of offspring
At postnatal d14, one female suckling piglet approaching average body weight of the litter, from each sow was slaughtered by exsanguination as described . After death, the abdomen was immediately opened and the entire intestine was rapidly removed, thoroughly flushed with ice-cold sterile saline to remove luminal chyme. Then, the intestine was dissected free of mesenteric attachments, and placed on a smooth and cold surface tray. Next, the middle site of duodenum, jejunum and ileum were obtained quickly as described . Several two-cm-long sections of tissues were taken at a pre-determined distance from the jejunum, and frozen in liquid nitrogen for subsequent glutathione (GSH) and glutathione disulfide (GSSG) analysis and RNA isolation. Two-cm-long segments of duodenum, jejunum and ileum were sampled and immediately fixed in phosphate-buffered paraformaldehyde (4 %, pH 7.6) for histological measurements.
Sow plasma metabolites by 1H NMR spectroscopy
Plasma samples were prepared by mixing 200 μL of plasma with 400 μL of saline containing 50 % D2O (for field frequence lock purposes). The proton NMR spectra of plasma were recorded at 298 K on a Bruker Avance DRX-600 spectrometer (Bruker Biospin, Rheinstetten, Germany) operating at a 1H frequency of 600.11 MHz with a triple-resonance, high-resolution probe. A water-presaturated Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence (recycle delay–90 o–(τ–180 o–τ)n–acquisition) was used to attenuate NMR signals from macromolecules. The spin-spin relaxation delay (2nτ) of 200 ms was employed. Typically, 90 o pulse was set to 10.0 μs and 32 transients were collected into 32 k data points for each spectrum with a spectra width of 20 ppm. For assignment purposes, five two-dimensional (2D) NMR spectra including 1H-1H J-resolved spectroscopy (J-Res), 1H-1H correlation spectroscopy(COSY), 1H-1H total correlation spectroscopy(TOCSY), 1H-13C heteronuclear single quantum coherence spectroscopy (HSQC) and 1H-13C heteronuclear multiple bond correlation spectroscopy (HMBC) were acquired for selected samples.
The free induction decays were multiplied by an exponential window function with a 1 Hz line-broading factor prior to Fourier transformation. All NMR spectra were initially phase adjusted, and then the baseline was corrected by using Mestrenova 7.0 software (Mestrelab Research SL, Spain). Chemical shift was referenced to the peak of the methyl proton of L-lactate at δ 1.33.
NMR spectra (δ 0.5–9.5) were integrated into regions of 0.002 ppm wide by using Mestrenova 7.0 software (Mestrelab Research SL, Spain). Regions distorted by imperfect water saturation were discarded with the regions containing urea signals. These regions were δ 4.47–5.18, δ 5.5–6.0 and δ 4.28–4.45. Subsequently, each integral region was normalized to the total sum of all integral regions for each spectrum prior to pattern recognition analyses.
Sow milk composition analysis
Frozen milk samples were thawed at 4 °C, and then 10 mL of each sample was used for milk fat, protein and solids-not-fat (SNF) analysis by a quick milk element analyzer (MILKYWAY-CP2, Hangzhou Simple Technology. Co., Ltd.).
Antioxidant capacity of neonatal jejunal tissue
The jejunum samples were thawed at 4 °C and grinded on ice in glass homogenizer with 20 volumes (wt/v) of ice-cold physiological saline. After that, homogenates were centrifuged at 4,000 × g and 4 °C for 20 min, and then supernatants were collected for GSH and GSSG analysis. The GSH plus GSSG levels of the samples were determined by using a method as described . Briefly, total glutathione was determined by following the rate of reduction of 5, 5′-dithiobis-2-nitrobenzoic acid (DTNB) by GSH at 412 nm and comparing this to a GSH standard curve. GSSG in the samples were detected by using the same method after treating samples with 4-vinylpyridine for 60 min. The GSH concentration was calculated using the total glutathione subtracted by twofold of GSSG.
Intestinal morphology analysis of neonates
Histomorphometric analyses were performed on H&E-stained tissue sections as described . In brief, the tissue samples were split along the mesentery and fixed smoothly then immersed in Davidson’s fixative (333 mL of 95 % ethanol, 220 mL of 37 % formaldehyde, 110 mL of glacial acetic acid, and 330 mL of distilled water) for 24 h. After that, the tissue samples were taken out from the fixative, cut into 1 cm2 sections, preserved in fresh fixative. Subsequently, they were cleared in xylene before being embedded in paraffin. Four cross-sections per tissue sample were stained with hematoxylin and eosin. Villus height and crypt depth were determined for 12 villi and crypts using the Nikon Eclipse 80I fluorescence microscope (Nikon company, Japan) equipped with an epi-fluorescence image analysis system. Villi and crypts were only measured when there was a complete longitudinal section of a villus and an associated crypt. The average villus height and crypt depth per slide was used as experimental observation .
RNA extraction and real-time qPCR in neonatal intestinal tissue
Accession number, primer sequence and product size of genes evaluated
Gene (accession number)
Primer sequence (5'–3')
Product size, bp
18S ribosomal RNA (NR_046261.1)
DNA-binding protein inhibitor ID-2 (NM_001037965.1)
IGF-II precursor (NM_213883.2)
Somatostatin precursor (NM_001009583.1)
Ubiquitin carboxyl-terminal hydrolase FAF-g (NM_004654.3)
Adenylate cyclase (NM_001114)
N-Acyl-D-glucosamine 2-epimerase (NM_213900.1)
Carnitine transporter 2 (NM_003060)
Oxysterol binding protein-related protein 10 (NM_017784)
Acyl-CoA dehydrogenase (NM_213897.1)
Aminopeptidase A (NM_214017.1)
Cathepsin F precursor (TC104654)
Leukocyte antigen related protein precursor (NM_130440.2)
Preprogalanin (NM_214234 · 1)
Sodium-and chloride-dependent creatine transporter 1 (NM_001177327.1)
Data are presented as means with pooled SEM, unless otherwise specified. The pen was considered the experimental unit for statistical analyses. Body weight, feed intake, milk composition, jejunal antioxidant and mRNA abundance data were analyzed by using the GLM procedures of SAS statistical package (version 8.1; SAS Institute, Inc.). The following statistical model was used: Y ij = μ + T i + e ij where Y is the analyzed variable, μ is the overall mean, T is the effect of treatment (i = 1…3), and e is the residual error (i = 1…3, j = 1…6). The least significant difference test was used to compare the group means when the F test in the analysis of variance table was significant. Meanwhile, body weight at postpartum d7 was compared with that at postpartum d0 by a two-sample paired t test. Multivariate data analysis was carried out on the normalized NMR datasets with the software package SIMCA-P+ (v11 · 0, Umetrics, Sweden). Principal component analysis (PCA) was performed to show an overview of intrinsic similarity/dissimilarity within the data sets. The orthogonal projection to latent structure-discriminant analysis (OPLS-DA) was further carried out using the unit-variance scaled (UV) NMR data as X-matrix and class information as Y-matrix . The quality of the model was assessed by the parameters R2X representing the total explained variations for X matrix and Q2 indicating the model predictability. The models were validated by two methods: a cross validation method and a permutation test [22, 23]. The models were interpreted by the coefficient coded loading plots. The loadings were backtransformed in Excel (Microsoft, USA) and plotted with color-coded absolute coefficient values (|r|) of the variables in MATLAB . The coefficient plot indicated the significance of variables (resonances) that contributed to the differentiation of classes of interest. The significant discriminatory metabolites were indicated in red color whereas blue color showed no significance. In the present study, appropriate correlation coefficients were used as the cutoff values (depending on the number of animals used) for the statistical significance based on the discrimination significance at the level of P < 0.05; such was determined according to the test for the significance of the Pearson’s product–moment correlation coefficient . P < 0.05 was considered statistical significance. As described by Littell et al. , the repeated-measures data for intestinal morphology was analyzed by using the MIXED procedures of SAS statistical package (version 8.1; SAS Institute, Inc.).
Body weight and feed intake of sows, and body weight of piglets
Milk composition of lactating sows
Multivariate data analysis result of NMR data
OPLS-DA coefficients derived from the NMR data of metabolites in plasma obtained from different treatments
CON-d7 (vs CON-d0)
DLM-d7 (vs DLM-d0)
HMTBA-d7 (vs HMTBA-d0)
Lipid, CH 3-(CH2)n-(LDL)
Lipid, CH 3-(CH2)n-(VLDL)
Lipid, CH3-(CH 2)n-(LDL)
Lipid, =CH-CH 2-CH=
Lipid, −CH = CH-
Antioxidant index of suckling piglets
Jejunal glutathione and glutathione disulfide concentration at postnatal d14 of suckling piglets
GSH (μmol/g protein)
GSSG (μmol/g protein)
Intestinal morphology of suckling piglets
Genes expression in suckling piglet jejunum
The first endpoint of this study was to determine how the sows respond to increased consumption of methionine as DLM or HMTBA during early lactation. Notably, milk fat content was found to be lower in the DLM group than in the HMTBA group at postpartum d7. However, it appeared that there was no difference either in body weight at postpartum d7 or in feed intake during postpartum wk1 among the three treatment groups. Considering that the coefficient of variation of bodyweight among sows within a treatment group was up to 16 %, the potential difference of bodyweight change might have been masked due to the relatively small replicate numbers. Therefore, a paired t test was further used to compare the body weight at postpartum d7 against at postpartum d0 in each treatment group. Intriguingly, only DLM-fed sows showed lost body weight, which might account for the difference in milk composition among treatment groups. Given that physiological metabolism is influenced in lactating sows using body reserve to satisfy lactation , the metabolomics of plasma at postpartum d7 against at postpartum d0 was further analyzed, which may provide further biochemical evidence for the change of body reserve. An important finding in the present study was that the DLM-fed sows had reduced plasma levels of betaine and creatinine, but increased plasma level of dimethylamine at postpartum d7 compared with those at postpartum d0. It has been indicated that creatine is involved in energy metabolism in vertebrates. The conversion of creatine to creatinine is a spontaneous and nonenzymatic process . Betaine is a methyl donor to facilitate methionine synthesis from homocysteine , thereby improving biosynthesis of creatine . However, methionine is also methyl donors of methylamine to form dimethylamine . Another significant finding was that there were decreased plasma levels of glucose, glycerol, myo-inositol and succinate but increased plasma levels of 3-hydroxybutyrate, lipoprotein and lipids in the DLM-fed sows at postpartum d7 compared with those at postpartum d0. It is well known that glucose can be completely catabolized as energy source via the tricarboxylic acid cycle, but it also generates glycerol to synthesize lipid . A previous study indicated that myo-inositol deficiency resulted in increased fatty acid mobilization from adipose tissue . Meanwhile, when triglycerides are presented as lipoproteins, it can be cleaved by endothelial lipoprotein lipase and converted into ketone body such as 3-hydroxybutyrate in liver . Skeletal muscle or other tissues will give priority to utilize ketone body as an energy source when glucose consumption is reduced . We can infer that the energy release from the tricarboxylic acid cycle is depressed by reduced concentrations of glucose and succinate. It was therefore proposed that the increased plasma levels of 3-hydroxybutyrate, lipoproteins and lipids and reduced glycerol and myo-inositol contents were a result of reduced level of glucose available for energy supply. Taken together, the compromised creatinine production and tricarboxylic acid cycle along with increased lipid catabolism and 3-hydroxybutyrate production illustrated that the DLM-fed sows might be in negative energy balance during the first wk postpartum. This notion was further supported by body weight loss of the DLM-fed sows at the first wk postpartum. In contrast, compared with postpartum d0, there was no change in plasma energy related metabolites and body weight of the HMTBA-fed sows at postpartum d7, suggesting that energy metabolism of lactating sows during early lactation was not affected by increased consumption of methionine as HMTBA.
Milk is the major nutrients source for suckling piglets, and the intestine plays a key role in the digestion, absorption and metabolism of nutrients . The HMTBA-fed sows appeared to have higher milk fat content at postpartum d7 than the DLM-fed sows and higher milk fat and lactose content at postpartum d14 than the DLM- and CON-fed sows . Accordingly, higher body weight of suckling piglets was observed in the HMTBA group at postnatal d14 . Thus the second endpoint of this study was to determine whether neonatal intestinal growth could be affected by increased maternal consumption of methionine as DLM or HMTBA. A previous study has indicated that carnitine deficiency was associated with carnitine transporter 2 deficiency, which give rise to a mitochondrial fatty acid oxidation problem thereby inhibiting lipid and energy metabolism [33, 34]. Piglets in the DLM group had up-regulated intestinal expression of carnitine transporter 2, suggesting enhanced energy expenditure in the intestine. In support of this view, there was extended expression of sodium- and chloride-dependent creatine transporter 1 which mediates intestinal uptake of creatine . Piglets in the DLM group also had higher expression of IGF-II precursor which regulates the expression of the IGF-II , and IGF-II is supposed to trigger intestinal growth . However, piglets in the DLM group did not show higher jejunal villus than those in the CON or HMTBA groups. It was therefore proposed that the up-regulated expression of genes responsible for energy metabolism might be a result of intestinal compensatory growth.
Glutathione is a momentous intracellular peptide with antioxidant defense, and the ratio of GSSG/GSH is shown to be a good measure of oxidative stress of an organism. It was observed in our study that piglets reared by the HMTBA-fed sows had lower jejunal GSSG content than those reared by the CON-fed sows and, moreover, piglets in the HMTBA group had the lowest GSSG/GSH, indicating improved antioxidant capacity of neonatal intestine by increased maternal consumption of methionine as HMTBA. Previous studies have shown that oxidative stress accelerates degeneration of the intestinal epithelium , but a reduced redox potential maintains the proliferative state of intestinal epithelium . Thus, we proposed that the increased jejunal villus height and the ratio of villus height to crypt depth in piglets reared by the HMTBA-fed sows were associated with the increased jejunal antioxidant capacity. In contrast, jejunal GSSG content and GSSG/GSH in piglets reared by the DLM-fed sows remained to be the same as that in the CON group. It appeared that increased maternal consumption of methionine as HMTBA could improve intestinal antioxidant capacity of suckling piglets. This might be associated with higher milk fat which could be used as an energy source to satisfy intestinal growth thereby benefiting intestinal health.
Aminopeptidase A, an important digestive enzyme , can split off protein and peptide . Noting that aminopeptidase A mRNA abundance was lower in the CON than in the DLM and HMTBA groups, we proposed that increased maternal consumption of methionine might promote degradation and subsequent absorption of milk protein by neonatal pigs. Oxysterol binding protein-related protein 10 has been implicated in sterol containing cholesterol transport [42, 43], and low level of cholesterol is considered good for small intestinal microvillus membrane fluidity . In this regard, decreased expression of oxysterol binding protein-related protein 10 following increased maternal consumption of methionine as DLM and HMTBA might suggest the facilitation of nutrients transport in the small intestine. However, given that galanin derived from preprogalanin inhibits intestinal smooth muscle activity directly [45, 46], increased maternal consumption of methionine as DLM might compromise neonatal intestinal motility due to the up-regulated expression of preprogalanin. N-acyl-D-glucosamine 2-epimerase is involved in bioconversions of N-acetyl-D-glucosamine to N-Acetyl-D-neuraminic acid , the derivatives of which play a prominent role in antiviral agents . The up-regulated expression of N-acyl-D-glucosamine 2-epinerase suggested that the intestine of piglets in the HMTBA group might have greater intestinal disease resistance capability than those in the CON and DLM groups.
The DLM-fed sows were in negative energy balance during the first week postpartum. Meanwhile, though increasing maternal consumption of methionine as DLM and HMTBA promoted neonatal intestinal growth by increasing morphological development or up-regulating expression of genes responsible for nutrient metabolism, suckling piglets in DLM group emerged intestinal compensatory growth and underpowered intestine motility, probably. In addition, increasing maternal consumption of methionine as HMTBA promoted neonatal intestinal antioxidant ability.
2D, two-dimensional; CON, control; COSY, correlation spectroscopy; CPMG, Carr-Purcell-Meiboom-Gill; DLM, DL-methionine; DTNB, 5, 5′-dithiobis-2-nitrobenzoic acid; GSH, glutathione; GSSG/GSH, the ratio of glutathione disulfide to glutathione; GSSG, glutathione disulfide; HMBC, heteronuclear multiple bond correlation spectroscopy; HMTBA, DL-2-hydroxy-4-methylthiobutanoic acid; HSQC, heteronuclear single quantum coherence spectroscopy; J-Res, J-resolved spectroscopy; LDL, low density lipoprotein; NMR, 1H nuclear magnetic resonance; NSF, solids-not-fat; OPLS-DA, orthogonal projection to latent structure-discriminant analysis; PCA, principal component analysis; PVHCD, percentage of villus height to crypt depth; SAA, sulfur-containing amino acids; TOCSY, total correlation spectroscopy; UV, unit-variance scaled; VLDL, very low density lipoprotein
The authors would like to thank the staff of the Key Laboratory for Animal Disease Resistance Nutrition of the Ministry of Education for their ongoing assistance.
This study was financially support from the Rhodimet Research Grant from Adisseo France S.A.S., Briand, Antony Cedex, France, the National Natural Science Fundation of China (31472109), Sichuan Province Science Foundation for Fostering Youths Talents (2011JQ0015), and Program for Changjiang Scholars and Innovative Research Team in University (IRT13083). All sources of funding were involved in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
Availability of data and materials
The datasets generated during and/or analysed during the current study are available in the HMDB repository, http://www.hmdb.ca/. All data generated or analysed during this study are included in this published article.
HZ performed the statistical analysis and drafted the manuscript. HL and HW participated in animal feeding, sample collection and analyses. GL participated in statistical analysis and writing of the manuscript. LT participated in sample collection and analysis. YM, XZ, YL, LC, SX, GT, DC and DW participated in study design and writing of the manuscript. ZF designed the study, performed the statistical analysis and participated in writing of the manuscript. All authors read and approved the final manuscript.
The financial support was partially from Adisseo, the producer of DLM and HMTBA. And Yves Mercier is the member of Adisseo. However, this does not alter our adherence to Journal of Animal Science and Biotechnology policies on sharing data and materials. All authors agreed to publish this paper.
Consent for publication
Ethics approval and consent to participate
All procedures involving animals were approved by the Animal Care and Use Committee of Animal Nutrition Institute, Sichuan Agricultural University.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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