Background: Methionine (Met) is usually the second or third limiting
amino acid in swine diets and plays vital roles in promoting the growth,
especially, the muscle growth of pigs. This research evaluated the effects of
dietary Met restriction on the growth performance, plasma metabolite
concentrations, and myogenic gene expression in growing pigs. Materials
and methods: Eight genes in two families (myogenic regulatory factor family and
myocyte enhancer factor 2 family) were selected for the analysis. Twenty
individually penned barrows (crossbred, 23.6
Known as 2-amino-4-methylthio butanoic acid in chemistry, methionine (Met) is usually the second or third limiting amino acid (AA) in typical swine diets [1]. Commercial product of crystalline DL-Met that consists of 50% D-Met and 50% L-Met is commonly added to the swine diets that are low in Met content [1, 2]. In addition to functioning as a building block for body protein biosynthesis [3, 4], Met has several other biological functions that include (1) protein translation initiation, (2) methyl donation, (3) sulfur source, (4) endogenous antioxidant, (5) precursor of bioactive compounds such as taurine, glutathione, choline, and betaine, and (6) an intermediary in the synthesis of cysteine (Cys) or cystine [1, 5]. Previous research has showed that either a deficiency or a surplus of dietary Met would depress the weight gain and feed efficiency in growing and finishing pigs [6]. The beneficial effects of dietary Met at an optimal level on the growth performance and meat yield of pigs have also been previously reported [5, 7, 8, 9]; however, the regulatory molecular mechanisms through which Met regulates the skeletal muscle formation and growth in pigs are still unclear [1, 2].
As is known, myogenesis is a biochemical process of muscle formation regulated by a broad spectrum of cell signaling molecules [10] which are affected by nutrient availability and nutrient metabolism [11]. Among the hierarchical interactions between those molecules and nutrient metabolites, the families of myogenic regulatory factors (MRF) and myocyte enhancer factor 2 (MEF2) for the transcription factor-mediated regulation, are key regulators of muscle growth and differentiation and have been a focus of many previous studies in humans and animals [12, 13]. However, until now little is known about the effects of nutrient Met, a functional AA, on the expressions of these factor genes in pigs.
The MRF family comprise myogenic differentiation 1 (MyoD or MyoD1; a.k.a. myoblast determination protein 1), myogenin (MyoG; a.k.a. myogenic factor 4, Myf4), myogenic factor 5 (Myf5), and myogenic factor 6 (Myf6; a.k.a. myogenic regulatory factor 4, Mrf4), while the MEF2 family comprise Mef2A, Mef2B, Mef2C, and Mef2D [10, 14]. Therefore, the objectives of this study were to evaluate (1) the growth performance, (2) the nutrient metabolite profile in the blood, and (3) the expression of these eight genes in skeletal muscle, of young growing pigs when a Met restricted diet was fed.
The experimental protocol involving caring, handling, and treatment of pigs was
approved by the Mississippi State University Institutional Animal Care and Use
Committee. Twenty crossbred young growing barrows (Yorkshire
A corn and soybean meal based diet (Diet 1, a Met-restricted diet) was formulated to meet or exceed the recommended requirements for energy, crude protein, essential AA, minerals, and vitamins, except for Met [4, 15]. Diet 2 (a Met-adequate diet) was produced by supplementing a commercial product of crystalline DL-Met (99% purity; Evonik Operations GmbH, Hanau-Wolfgang, Germany) to Diet 1 at the expense of corn to meet the requirement of pigs for Met [4, 15]. The diet composition and the calculated nutrient contents are both shown in Table 1 (Ref. [16]), which demonstrates that Diet 2 was a Met-adequate diet while Diet 1 was deficient in SID Met by roughly 40.5%.
Dietary treatment | |||
Item | Diet 1 | Diet 2 | |
Ingredient, % | |||
Corn | 79.03 | 78.88 | |
Soybean meal | 17.00 | 17.00 | |
Poultry fat | 0.01 | 0.01 | |
L-lysine HCl, 78.8% | 0.62 | 0.62 | |
DL-methionine, 99% | – | 0.15 | |
L-threonine, 98.5% | 0.27 | 0.27 | |
L-tryptophan, 98% | 0.09 | 0.09 | |
L-isoleucine, 96% | 0.10 | 0.10 | |
L-valine, 96.5% | 0.18 | 0.18 | |
L-cysteine HCl, 76.9% | 0.11 | 0.11 | |
Limestone | 0.81 | 0.81 | |
Dicalcium phosphate | 1.40 | 1.40 | |
Salt | 0.18 | 0.18 | |
Mineral premix |
0.10 | 0.10 | |
Vitamin premix |
0.10 | 0.10 | |
Total | 100.00 | 100.00 | |
Major nutrients, %, calculated | |||
Dry matter | 86.48 | 86.50 | |
Net energy |
2,545 | 2,547 | |
Crude protein | 15.42 | 15.50 | |
SID |
13.10 | 13.20 | |
SID lysine | 1.08 | 1.08 | |
SID methionine | 0.22 | 0.37 | |
SID methionine + cysteine | 0.52 | 0.67 | |
SID threonine | 0.72 | 0.72 | |
SID tryptophan | 0.22 | 0.22 | |
SID valine | 0.76 | 0.76 | |
SID isoleucine | 0.60 | 0.60 | |
SID leucine | 1.20 | 1.19 | |
Total calcium | 0.67 | 0.67 | |
STTD |
0.38 | 0.38 | |
Crude fiber | 2.26 | 2.26 | |
Ash | 2.02 | 2.02 | |
To confirm the contents of major nutrients, samples of the two diets were submitted to the Essig Animal Nutrition Laboratory at Mississippi Agricultural and Forestry Experiment Station for proximate and energy analyses, and to the Evonik’s chemical laboratory at Hanau-Wolfgang, Germany for AA analysis. For proximate analysis, the contents of dry matter, crude protein, crude fat, crude fiber, and ash were determined according to AOAC International (2000) [17] official methods 9340.01, 2001.11, 920.39, 92.09, and 924.05, respectively. Gross energy was determined using a Parr 1261 Isoperibol Bomb Calorimeter (Parr Instrument Company, Moline, IL, USA). Amino acids were analyzed using ion-exchange chromatography [18, 19]. Tryptophan was analyzed by high-performance liquid chromatography with fluorescence detection [20]. The determined compositions of selected nutrients contained in the diets are shown in Table 2 (Ref. [16]), which indicate that Diet 1 was a diet deficient in total Met by roughly 35.1%.
Dietary treatment | |||
Nutrient and energy |
Diet 1 | Diet 2 | |
Proximate analysis | |||
Dry matter | 87.35 | 87.67 | |
Gross energy, kcal/kg | 3,847 | 3,935 | |
Crude protein | 15.00 | 15.08 | |
Crude fat | 1.64 | 1.95 | |
Crude fiber | 1.66 | 1.76 | |
Ash | 4.23 | 4.42 | |
Amino acid, total | |||
Lysine | 1.17 | 1.15 | |
Methionine | 0.24 | 0.37 | |
Cysteine | 0.34 | 0.34 | |
Methionine + Cysteine | 0.59 | 0.70 | |
Threonine | 0.78 | 0.77 | |
Tryptophan | 0.23 | 0.23 | |
Arginine | 0.91 | 0.90 | |
Histidine | 0.39 | 0.39 | |
Leucine | 1.35 | 1.36 | |
Isoleucine | 0.66 | 0.65 | |
Valine | 0.82 | 0.83 | |
Phenylalanine | 0.72 | 0.71 | |
Tyrosine | 0.37 | 0.38 | |
Proline | 0.94 | 0.93 | |
Aspartic acid | 1.35 | 1.34 | |
Glutamic acid | 2.60 | 2.59 | |
Serine | 0.72 | 0.71 | |
Alanine | 0.82 | 0.82 | |
Glycine | 0.60 | 0.60 | |
Supplemented free amino acid | |||
Lysine | 0.43 | 0.45 | |
Methionine | 0.00 | 0.13 | |
Threonine | 0.23 | 0.25 | |
Valine | 0.16 | 0.17 | |
During the four-week feeding trial, pigs had ad libitum access to the experimental diets and fresh water. All feeders, waterers, and pigs were checked at least twice a day (0600 to 2100 hr) to ensure proper function of the facilities and healthy animal behavior. Feed refusals and spillage were collected and immediately returned to the feeders or reserved and weighed for feed intake calculation. Pigs’ BW were measured immediately before, and also at the end of, the four-week feeding trial. The average daily gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F) were then calculated accordingly.
Immediately before the beginning and at the end of the feeding trial, blood
samples (approximately 10 mL/pig) were collected by jugular venipuncture of
individual pigs [in a non-fasting state as in an industry setting where pigs are
not fasted] in early morning (between 0600 and 0800 hr). The remaining feeds in
all the feeders, however, were removed approximately 30 to 60 minutes before the
blood collection. Blood samples were kept on ice immediately after the collection
until plasma was separated within 30 to 60 minutes through centrifugation at 800
After blood collection, a muscle sample (about 200 mg/pig) was collected from
the middle portion (the left side) of longissimus dorsi muscle of each
pig using our standard aseptic biopsy protocol [21]. All muscle samples collected
were snap frozen in liquid nitrogen, and then transferred to a –80
The concentrations of plasma free AA were determined at the analytical laboratories of Ajinomoto Heartland, Inc. (Chicago, IL, USA) and Evonik Operations GmbH (Hanau-Wolfgang, Germany) using the official standard high-performance liquid chromatography methods [17]. The principles and procedures of the methods were briefly described by Regmi et al. [22] previously.
Batch analysis using the automated ACE Alera Clinical Chemistry System (Alfa Wassermann, West Caldwell, NJ, USA) was performed at the College of Veterinary Medicine Diagnostic Laboratory of Mississippi State University for determination of the concentrations of six representative plasma metabolites with six respective ACE reagents (Alfa Wassermann), and these six metabolites are urea nitrogen, albumin, total protein, glucose, triglycerides, and total cholesterol. The principles and procedures of these laboratory analyses were briefly described by Regmi et al. [23] previously.
The myogenic gene expression was analyzed by following our previously reported
protocols [24]. Briefly, the total RNA was extracted from approximately 50 mg of
muscle sample per pig using TRIzol Reagent (Invitrogen Corporation, Carlsbad, CA,
USA) following the manufacturer’s instructions. Briefly, a frozen sample was
homogenized in a 15-mL polypropylene centrifuge tube using a Polytron mixer (0.5
mL TRIzol per 50 mg tissue), and the homogenate was transferred to a 1.5-mL
micro-centrifuge tube. Chloroform (400
First-strand cDNA was reverse-transcribed from 1
The comparative
Data were subjected to statistical analysis with Student t-test using
the SAS software (version 9.4; SAS Institute Inc., Cary, NC) with pigs being the
experimental units. A P-value less than or equal to 0.05 was considered
as having a significant difference between treatment means, and a
P-value between 0.05 and 0.10 as having a tendency to be different. Each
value of the measurements is presented as mean
As shown in Table 3, there was no difference in the initial BW between the two
dietary treatment groups (P
Dietary treatment | |||
Item | Diet 1 | Diet 2 | P-value |
Initial body weight, kg | 23.54 |
23.64 |
0.935 |
Final body weight, kg | 47.44 |
50.55 |
0.113 |
Average daily gain, kg | 0.87 |
0.98 |
0.006 |
Average daily feed intake, kg | 1.94 |
1.96 |
0.849 |
Gain:feed ratio | 0.45 |
0.50 |
0.001 |
In order to further understand how dietary Met restriction could affect nutrient
metabolism in growing pigs, the concentrations of plasma free AA were analyzed.
As shown in Table 4, prior to the 4-week feeding trial there was no differences
(P
Dietary treatment | ||||
Amino acid, nmol/mL |
Diet 1 | Diet 2 | P-value | |
Total EAA | 1,243 |
1,178 |
0.659 | |
Methionine | 41.4 |
38.4 |
0.282 | |
Leucine | 185.0 |
161.3 |
0.182 | |
Histidine | 118.7 |
116.8 |
0.903 | |
Phenylalanine | 104.4 |
99.2 |
0.704 | |
Isoleucine | 138.2 |
125.7 |
0.440 | |
Threonine | 225.2 |
251.5 |
0.659 | |
Valine | 296.9 |
275.0 |
0.490 | |
Lysine | 65.0 |
50.8 |
0.192 | |
Tryptophan | 67.9 |
59.5 |
0.366 | |
Total NEAA | 3,678 |
3,664 |
0.972 | |
Arginine | 174.4 |
144.2 |
0.115 | |
Citrulline | 94.7 |
83.7 |
0.304 | |
Alanine | 644.6 |
613.3 |
0.661 | |
Glutamate | 270.9 |
357.9 |
0.190 | |
Glycine | 908.8 |
823.7 |
0.365 | |
Asparagine | 112.8 |
107.7 |
0.756 | |
Aspartate | 22.2 |
30.7 |
0.074 | |
15.5 |
18.8 |
0.244 | ||
Glutamine | 750.5 |
844.2 |
0.594 | |
Ornithine | 178.0 |
156.4 |
0.242 | |
Serine | 180.3 |
160.3 |
0.333 | |
Taurine | 213.2 |
216.8 |
0.943 | |
Tyrosine | 112.6 |
106.5 |
0.745 | |
Cysteine | 188.6 |
186.0 |
0.813 | |
Proline | 337.2 |
350.0 |
0.692 | |
Total AA | 4,921 |
4,843 |
0.882 | |
Dietary treatment | ||||
Amino acid, nmol/mL |
Diet 1 | Diet 2 | P-value | |
Total EAA | 2,258 |
1,697 |
0.146 | |
Methionine | 28.7 |
53.6 |
||
Leucine | 262.1 |
224.6 |
0.261 | |
Histidine | 119.5 |
84.1 |
0.033 | |
Phenylalanine | 88.7 |
71.4 |
0.115 | |
Isoleucine | 162.4 |
130.1 |
0.205 | |
Threonine | 574.7 |
372.5 |
0.154 | |
Valine | 422.5 |
358.6 |
0.376 | |
Lysine | 459.1 |
292.6 |
0.067 | |
Tryptophan | 140.4 |
111.3 |
0.178 | |
Total NEAA | 4,144 |
3,659 |
0.293 | |
Arginine | 169.4 |
146.8 |
0.427 | |
Citrulline | 75.8 |
63.5 |
0.211 | |
Alanine | 670.8 |
572.2 |
0.301 | |
Glutamate | 198.8 |
256.4 |
0.145 | |
Glycine | 1162.7 |
921.5 |
0.031 | |
Asparagine | 158.1 |
99.3 |
0.086 | |
Aspartate | 22.3 |
21.9 |
0.914 | |
24.2 |
26.7 |
0.474 | ||
Glutamine | 936.5 |
898.4 |
0.793 | |
Ornithine | 182.7 |
172.9 |
0.619 | |
Serine | 271.9 |
206.8 |
0.145 | |
Taurine | 113.4 |
142.1 |
0.049 | |
Tyrosine | 157.0 |
130.6 |
0.276 | |
Cysteine | 157.4 |
226.4 |
||
Proline | 327.8 |
346.1 |
0.427 | |
Total AA | 6,402 |
5,358 |
0.197 | |
The shift of plasma AA profile in pigs fed two different levels of dietary Met is generally in agreement with the results of Li et al. [26] and Tian et al. [27]. Li et al. [26] reported that the plasma concentrations of Met (numerically) and taurine were lower, while the plasma concentrations of lysine (numerically) was higher, in the sows fed a Met adequate vs. a Met excess diet. No data for Cys and glycine from these sows were reported by Li et al. [26]. Tian et al. [27] reported that the serum concentrations of Met and Cys (numerically) was lower, while the serum concentrations of lysine, histidine (numerically), and glycine were higher, in young growing pigs fed a Met deficient than fed a Met adequate diet. No data for taurine were reported by Tian et al. [27].
Obviously, the lower concentrations of plasma Met in pigs fed diets deficient or
low in Met can be attributed to the limited dietary supply of Met in these
studies. Given the fact that Cys and taurine are products of Met metabolism [28],
the lower Cys and taurine concentrations in the plasma of pigs fed a Met
restricted diet might be due to the limited dietary Met supply as well. The
higher plasma concentrations of lysine, histidine, and glycine associated with
the low dietary Met supply might be attributed to the fact that lysine,
histidine, glycine, asparagine, and Met share the B
A high plasma glycine concentration indicates that it was possible that less glycine was utilized for glutathione synthesis due to insufficient Met supply in the diet. Given the fact that both glutathione and taurine (derived from Cys) are essential antioxidants in pig body [1], the reduced plasma concentrations of Met, taurine, and possibly glutathione, might also be responsible for the reduced growth performance of pigs fed the Met-restricted diet. Although the parameters related to oxidative status were not measured in this study, the change in plasma AA concentration indicates that dietary Met restriction may negatively affect pig’s antioxidant capacity, health status, and, in consequence, growth performance.
In addition to the free AA, the plasma concentrations of six major metabolites
were also analyzed. As shown in Table 6, before the feeding trial, there were no
differences (P
Dietary treatment | ||||
Metabolite | Diet 1 | Diet 2 | P-value | |
Before the feeding trial | ||||
Urea nitrogen, mg/dL | 12.4 |
11.1 |
0.156 | |
Total protein, g/dL | 4.75 |
4.59 |
0.251 | |
Albumin, g/dL | 2.40 |
2.34 |
0.591 | |
Glucose, mg/dL | 111.7 |
106.2 |
0.251 | |
Total cholesterol, mg/dL | 95.4 |
87.0 |
0.208 | |
Triglycerides, mg/dL | 45.2 |
47.2 |
0.756 | |
After the feeding trial | ||||
Urea nitrogen, mg/dL | 6.6 |
4.2 |
||
Total protein, g/dL | 5.71 |
5.51 |
0.211 | |
Albumin, g/dL | 3.64 |
3.41 |
0.085 | |
Glucose, mg/dL | 108.8 |
115.0 |
0.250 | |
Total cholesterol, mg/dL | 77.6 |
84.4 |
0.180 | |
Triglycerides, mg/dL | 40.7 |
53.4 |
0.123 | |
The plasma urea nitrogen concentration is a reliable indicator of AA utilization efficiency by the animal [30, 31, 32]. The ideal amounts and ratios of all proteinogenic AA are essential for efficient nitrogen utilization, minimal AA catabolism, maximal protein synthesis, and thus the least plasma urea nitrogen concentration [11, 23, 33]. Data from this study showed that the plasma urea nitrogen concentration was greater in the Met-restricted pigs than in the Met-adequate pigs. The probable reason for this finding is that in the Met-restricted group, Met was the first limiting AA, and after the Met was exhausted, all other AA became “extra” and were catabolized to produce more urea nitrogen [30].
The plasma albumin concentration is also a good indicator of the effectiveness of dietary AA utilization, and of the liver capacity of protein synthesis [23, 34, 35]. The present study is in consistent with the result of Tian et al. [27] for young growing pigs. Our result is also supported by Litvak et al. [36] who reported that a low-level Met diet increased serum albumin concentration in growing pigs. It may also be speculated that increased hepatic albumin synthesis reflects increased need for endogenous Met to support the immune response since albumin is used to transfer Met [36].
There was no difference in the plasma total protein concentrations between the
pigs fed Diet 1 and Diet 2 (P
There was no significant difference (P
Before the feeding trial, the mRNA levels of the selected myogenic genes were
similar (P
Dietary treatment | |||||
Gene name |
Gene symbol | Diet 1 | Diet 2 | P-value | |
Before the feeding trial | |||||
Myogenic differentiation 1 | MyoD | 1.34 |
1.17 |
0.468 | |
Myogenin | MyoG | 0.95 |
1.13 |
0.446 | |
Myogenic factor 5 | Myf5 | 1.21 |
1.04 |
0.184 | |
Myogenic factor 6 | Myf6 | 1.56 |
1.23 |
0.524 | |
Myocyte enhancer factor 2A | Mef2A | 1.40 |
1.24 |
0.170 | |
Myocyte enhancer factor 2B | Mef2B | 2.19 |
2.25 |
0.961 | |
Myocyte enhancer factor 2C | Mef2C | 1.24 |
1.38 |
0.687 | |
Myocyte enhancer factor 2D, transcript variant X1 | Mef2D | 0.87 |
1.08 |
0.383 | |
After the feeding trial | |||||
Myogenic differentiation 1 | MyoD | 1.03 |
1.24 |
0.521 | |
Myogenin | MyoG | 1.16 |
1.11 |
0.855 | |
Myogenic factor 5 | Myf5 | 1.50 |
1.12 |
0.572 | |
Myogenic factor 6 | Myf6 | 0.76 |
1.10 |
0.079 | |
Myocyte enhancer factor 2A | Mef2A | 1.14 |
1.07 |
0.796 | |
Myocyte enhancer factor 2B | Mef2B | 1.17 |
1.39 |
0.590 | |
Myocyte enhancer factor 2C | Mef2C | 0.83 |
1.16 |
0.194 | |
Myocyte enhancer factor 2D, transcript variant X1 | Mef2D | 0.69 |
1.13 |
0.083 | |
As it is known, the MRF family are highly conserved and collectively expressed in skeletal muscle lineages [10, 14]. Assisted by the MEF2 family, MRFs coordinate the activities of a host of co-activators and co-repressors, resulting in a tight control of gene expression during myogenesis [41, 42]. Either MyoD or Myf5 is sufficient for skeletal muscle formation [43], but MyoG and Myf6 are directly involved in myotube differentiation [10, 44]. Therefore, the present data imply that Met may affect the myotube or muscle cell differentiation, but not the muscle cell formation. The functions of different MEF2 isoforms (activating the muscle structural genes) are difficult to distinguish because they are expressed in distinct but overlapping patterns [2, 45]. The reason why only Mef2D, but not the other isoforms, was affected by Met in this study are unknown.
A study conducted in pigs by Li et al. [2] showed that dietary Met supplementation increased the mRNA expression levels of MyoG, Mef2A, and Mef2D, but not of Myf6 (as in this study). The discrepancy between the results of Li et al. [2] and of this study might be mainly due to the difference in the animal models used. The low birth weight piglets were used by Li et al. [2] without reporting breed and sex, and there was no difference in the growth performance between the control and the Met supplemented pigs [2]. In this study, the muscle samples were collected when pigs were around 80 d of age, while Li et al. [2] collected their muscle samples when pigs were 180 d of age.
Dietary Met restriction reduced the plasma concentrations of Met, Cys and taurine, but increased or tended to increase the concentrations of histidine, glycine, lysine, and asparagine in growing pigs. The Met restriction also increased or tended to increase the plasma concentration of urea nitrogen and albumin. These results confirmed that insufficient amount of dietary Met as a protein building block was the primary reason for the compromised G:F and ADG of the pigs fed with Met restricted diets, and the compromised G:F and increased plasma concentration of urea nitrogen can be attributed to the reduced efficiency of AA utilization and body protein synthesis. The Met restriction tended to reduce the abundance of Myf6 and Mef2D mRNA in the longissimus muscle of the pigs, which suggests that the reduced efficiency of AA utilization and protein synthesis may be associated with a reduced level of myotube differentiation rather than muscle cell formation.
SFL and JKH conceived and designed the experiment; ZY, MSH, and RMH performed the experiment; ZY analyzed the data and prepared the first draft of the manuscript; SFL supervised the experiment and finalized the manuscript. All authors have read and approved the final manuscript.
The experimental protocol involving the caring, handling, and treatment of pigs for the experiment was approved by the Mississippi State University Institutional Animal Care and Use Committee (IACUC #16-016).
The significant assistance in sample collection, laboratory analyses, and data interpretation from Drs. Derris Burnett and Jean Feugang in the Department of Animal and Dairy Sciences, Mississippi State University is greatly appreciated. Donations of mineral and vitamin premixes from Nutra Blend, LLC (Neosho, MO, USA) and L-tryptophan and L-valine from Ajinomoto Heartland, Inc. (Chicago, IL, USA) are kindly acknowledged. Authors also wish to thank Ajinomoto Heartland for their laboratory analyses of feed and blood plasma amino acid contents.
This research was financially supported in part by the USDA National Institute of Food and Agriculture (Hatch/Multistate Project MIS-351130 1024665) and by Evonik Operations GmbH (Hanau-Wolfgang, Germany).
JKH is an employee at Evonik Operations GmbH (Hanau-Wolfgang, Germany), a commercial supplier of DL-methionine to the global feed industry. All other authors have no conflicts of interest regarding the publication of this article.
Met, methionine; ADG, average daily gain; ADFI, average daily feed intake; G:F, gain, feed ratio; AA, amino acids; Cys, cysteine; MRF, myogenic regulatory factors; MEF2, myocyte enhancer factor 2; MyoD or MyoD1, myogenic differentiation 1; MyoG, myogenin; Myf5, myogenic factor 5; Myf6, myogenic factor 6.