Effect of Dietary Betaine on Muscle Protein Deposition, Nucleic Acid and Amino Acid Contents, and Proteomes of Broilers

Simple Summary In animal production, growth trials have shown that betaine has a positive effect on growth performance, especially improving meat yield. Proteins are the largest unit comprising muscle cells except moisture. However, how betaine regulates muscle protein anabolism and abundance levels remains unclear. Therefore, this study was conducted to investigate the effect of dietary betaine on growth performance, muscle protein deposition, and proteome of broilers. The results suggested that betaine could improve growth performance and muscle protein deposition of broilers. Alterations in muscle nucleic acids, amino acids, and protein abundance levels were involved in this process. Proteomic analysis further revealed that 35 proteins in breast muscle were identified as differentially abundant proteins by betaine supplementation, which were mainly related to cell differentiation, small molecule metabolic process, and tissue development. Abstract To investigate the effect of betaine supplementation on growth performance, muscle protein deposition, muscle nucleic acid and amino acid contents, and muscle proteome of broilers, 160 one-day-old male partridge shank broiler chickens were randomly divided into 2 groups with 8 replicates of 10 broilers each. Broilers were fed a basal diet alone, or a basal diet supplemented with 1000 mg/kg betaine. Compared with the control group, the betaine group significantly increased (p < 0.05) the broilers average daily gain, the levels of serum insulin-like growth factor-1 (IGF-1), growth hormone (GH), total protein (TP), the contents of muscle absolute protein deposition, RNA, Ser, Glu, Met, and Phe, and the ratio of RNA/DNA, and decreased (p < 0.05) the feed conversion ratio and serum blood urea nitrogen content. Moreover, proteomic analysis revealed 35 differentially abundant proteins (DAPs) in the betaine group compared with the control group, including 27 upregulated proteins and 8 downregulated proteins (p < 0.05). These DAPs were mainly related to cell differentiation, small molecule metabolic process, and tissue development. In conclusion, diets supplemented with 1000 mg/kg betaine improved growth performance and muscle protein deposition of broilers. Increased serum GH, IGF-1, and TP contents, and alterations in muscle nucleic acids, amino acids, and protein abundance levels were involved in this process.


Introduction
Betaine is a trimethyl derivative of the amino acid glycine, which is widely distributed in nature [1]. Due to its chemical structure, betaine plays an important role in osmotic protection and transmethylation. As an organic osmolyte, betaine can maintain cell structure, and protect protein and enzyme activities against environmental stress. As a methyl donor, betaine can take part in the methionine cycle and donates its labile methyl group to synthesize numerous substances, such as carnitine, choline, and creatine [1,2]. In animal production, growth trials revealed the positive effects of betaine on growth performance, especially increasing meat yield [3][4][5][6]. In human nutrition, betaine has been used as an

Animals, Experimental Design, Diets, and Management
A total of 160 1-day-old male partridge shank broiler chickens with similar birth weights (39.0 ± 0.1 g) were randomly divided into 2 groups, with 10 birds in each group for 8 repetitions, and the feeding experiment was carried out for 52 days. The control group broilers were fed a basal diet (Table 1), and the experimental group birds were fed a basal diet supplemented with 1000 mg/kg anhydrous betaine (96%), which was obtained from Yixing Skystone Feed Co., Ltd. (Yixing, Jiangsu, China). The level of betaine was selected according to our previous study that 1000 mg/kg anhydrous betaine could improve growth performance and breast muscle yield of broilers [12]. Birds were allowed free access to mash feed and clean water under a 23 h light/1 h dark lighting program. The ambient temperature of experimental room was maintained at 33 • C for the first 3 days, then slowly decreased by 3 • C each week to a final temperature of 20 • C. At 52 days of age, broilers were weighed after a 12 h feed deprivation, and feed intake was recorded by replicate to calculate average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR).

Sample Collection
At 52 days of age, one broiler from each replicate (cage) was randomly selected, weighed, and euthanized by cervical dislocation. Blood samples were collected from wing veins and centrifuged at 3000× g for 15 min at 4 • C to separate serum, which were refrigerated at −20 • C until determination. Then, whole breast muscles were excised, weighed, and a part of samples collected from pectoralis major muscles were immediately stored at −80 • C for further analysis.

Muscle Protein Deposition and Nucleic Acids Contents Measurement
According to the AOAC (2000) method, the moisture and crude protein (CP) components in breast muscle samples were analyzed. Moisture content was determined by weight loss after 12 h in an oven at 105 • C, and CP composition was accurately measured using the Kjeldahl method. Absolute protein deposition was calculated at breast muscle weight × (1 − moisture%) × CP%. Relative protein deposition was calculated as absolute protein deposition divided by broilers weight (g/kg). The concentrations of DNA and RNA in breast muscle were extracted using DNAiso and RNAiso reagents (Takara Biotechnology, Dalian, Liaoning, China), respectively, in accordance with the manufacturer's instructions. The concentrations of nucleic acids were quantified by ND-1000 spectrophotometer (Nano Drop Technologies, Wilmington, DE, USA).

Muscle Amino Acids Contents Measurement
About 0.2 g of muscle sample was put into a sealed evacuated tube (filled with nitrogen), acidly hydrolyzed with 6 mol/L hydrochloric acid, and heated in an oven at 110 • C for 22 h. After cooling, the volume was made up to 50 mL with ultrapure water. Then, 1 mL of sample was evaporated to dryness in a rotary evaporator at 60 • C, and then 2 mL of hydrochloric acid solution (0.02 mol/L) was added to reconstitute it. After filtering with 0.22 µm disposable filter paper, the amino acid contents in the test samples were analyzed by an automatic amino acid analyzer (L-8900; Hitachi, Tokyo, Japan).

iTRAQ Experiments
In order to reduce individual differences, identical pieces of muscle tissue from 2 birds in the same group were combined into one biological replicate. Each group received 4 biological replicates. About 0.2 g of muscle samples were ground into powder in liquid nitrogen, and then SDT lysis buffer (4% SDS, 1 Mm DTT, 100 mM Tris-HCI, pH 7.6) was added. After that, samples were heated at 100 • C for 3 min, followed by ultrasonic treatment for 5 min, and then further heated at 100 • C for 3 min. The supernatant was collected by centrifugation at 14,000× g for 40 min at 20 • C, and the concentrations of extracted proteins were determined using the BCA protein assay kit (Thermo Scientific, Waltham, MA, USA).
Protein digestion was conducted through the FASP procedure [21]. Protein samples at 300 µg were added to dithiothreitol for a final concentration of 100 Mm, and heated at 100 • C for 5 min. After cooling to room temperature, 200 µL UA buffer (8 M Urea, 150 mM Tris-HCl, pH 8.0) was added to the samples and centrifuged, twice, at 12,000× g for 15 min in 10 kD ultrafiltration filters. Subsequently, samples were mixed with 100 µL IAA (50 mM iodoacetamide in UA) and incubated at room temperature for 30 min in the dark, followed by centrifugation at 12,000× g for 10 min. After that, 100 µL UA buffer was added to the samples and centrifuged at 12,000× g for 10 min, twice. Additionally, 100 µL of NH 4 HCO 3 buffer was added to the filter, followed by centrifugation at 12,000× g for 10 min, twice. Next, 40 µL of trypsin (6 µg trypsin in 40 µL NH 4 HCO 3 buffer) was added to protein suspension at 37 • C for 18 h, followed by centrifugation at 12,000× g for 10 min. Finally, the filtrate was collected and the thermo desalting spin column was desalted for peptide quantification.
Each sample containing 100 µg digested protein was labeled with the iTRAQ reagent-8 plex multiplex kit (Applied Biosystems, Grand Island, NY, USA), as per the manufacturer's instructions. Samples were mixed with equal volume, desalted, and dried by vacuum. The labeled peptides were fractionated using the pierce high pH reversed-phase peptide fractionation kit on a HPLC system (Thermo Scientific). Ten fractions were collected, vacuum dried, and reconstituted with 0.1% formic acid. The Easy nLC-1200 HPLC system (Thermo Scientific) coupled with a Q-Extractive HF-X mass spectrometer (Thermo Scientific) was used for LC-MC/MC analysis. Samples were first injected into a trap C18 column (100 µm × 20 mm, 5 µm) and then passed through an analyzed C18 column (75 µm × 150 mm, 3 µm) for gradient separation at a flow rate of 300 nl/min. The MC data acquisition parameters were set as follows: analysis time, 60 min; detection mode, positive ion; precursor ion scan range, 300-1800 m/z; primary MC resolution, 60,000 at 200 m/z; automatic gain control (AGC) target, 3 × 10 6 ; maximum ion accumulation time (MIT), 50 ms. Peptide secondary MC was collected according to the following methods: 20 highest collision energy dissociation fragment files collected per full scan (MS2 scan); resolution, 15,000 at 200 m/z; AGC target, 1 × 10 5 ; MIT, 25 ms; activation type, HCD; isolation window, 1.6 m/z; normalized collection energy: 32 eV.

Quantitative Real-Time PCR Validation
Total RNA was isolated from muscle samples using RNAiso Reagent (Takara), as per the manufacturer's instructions. The concentration and purity (OD260/OD280 > 1.8) of RNA were quantified by ND-1000 spectrophotometer (Nano Drop Technologies). Then, all RNA samples were diluted to a uniform concentration with diethyl pyrocarbonate-treated (DEPC) water and reverse transcribed into cDNA with the PrimeScript RT Reagent Kit (Takara). As shown in Table 2, the primer sequence for the test genes were designed using Primer 5.0 software and synthesized by Sangon Biotechnology Co., Ltd. (Shanghai, China). Quantitative real-time PCR was carried out on the ABI StepOnePlus Real-Time PCR System (Applied Biosystems) using the SYBR Premix Ex Taq II Kit (Takara), according to the manufacturer's protocols. The β-actin and glyceraldehyde 3-phosphate dehydrogena (GAPDH) were used as internal standard to calculate the relative mRNA levels of target genes according to the 2 −∆∆CT method [22]. The mRNA level of each target gene in the broilers fed with the basal diet was assigned a value of one.

Statistical Analysis
Data were analyzed by independent sample T tests using SPSS statistical software (Version 20.0 for Windows, SPSS Inc., Chicago, IL, USA). The differences were considered significant at p < 0.05. Results were presented as group means and standard errors of means.

Growth Performance
As shown in Table 3, diet supplemented with betaine significantly increased (p < 0.05) the ADG and decreased (p < 0.05) the FCR of broilers from 1 to 52 d of age when compared with the control group. However, a difference in ADFI was not observed between the two groups.

Serum Parameters
Compared with the control group, the concentrations of serum GH, IGF-1, and TP were significantly increased (p < 0.05), and BUN was significantly decreased (p < 0.05) by dietary betaine supplementation. However, other indicators did not differ between the two groups (Table 4).

Muscle Protein Deposition and Nucleic Acids Contents
Compared with the control group, the contents of absolute protein deposition, RNA, and the ratio of RNA/DNA in breast muscle were significantly increased (p < 0.05) by dietary betaine supplementation (Table 5).

Muscle Amino Acids Contents
As shown in Table 6, diet supplemented with betaine significantly increased (p < 0.05) the contents of Ser, Glu, Met, and Phe in breast muscle when compared with the control group.

Muscle Protein Identification and Quantification
A total of 1084 proteins were identified and quantified at the FDR of 1% or less through proteomics analysis. Following statistical analysis, 35 proteins were regarded as DAPs (p < 0.05) in the betaine group compared with the control group, including 27 upregulated proteins and 8 downregulated proteins ( Table 7). The results of the volcano plot and hierarchical cluster analysis for DAPs were shown in Figure 1.

Bioinformatics Analysis
The GO enrichment analysis of DAPs was performed to investigate the biological process (BP), cellular component (CC), and molecular function (MF). As shown in Figure  2, the enriched BP category expressed in breast muscle were related to cell differentiation, small molecule metabolic process, tissue development, and inorganic ion homeostasis; macromolecular complex, endoplasmic reticulum part, cytosolic ribosome, and protein−DNA complex were involved in the CC category; and protein binding, RNA binding, protein heterodimerization activity, and actin binding were the most abundant subcate-

Bioinformatics Analysis
The GO enrichment analysis of DAPs was performed to investigate the biological process (BP), cellular component (CC), and molecular function (MF). As shown in Figure 2, the enriched BP category expressed in breast muscle were related to cell differentiation, small molecule metabolic process, tissue development, and inorganic ion homeostasis; macromolecular complex, endoplasmic reticulum part, cytosolic ribosome, and protein−DNA complex were involved in the CC category; and protein binding, RNA binding, protein heterodimerization activity, and actin binding were the most abundant subcategories in the FM category.
Animals 2022, 12, 736 9 of 13 process (BP), cellular component (CC), and molecular function (MF). As shown in Figure  2, the enriched BP category expressed in breast muscle were related to cell differentiation, small molecule metabolic process, tissue development, and inorganic ion homeostasis; macromolecular complex, endoplasmic reticulum part, cytosolic ribosome, and protein−DNA complex were involved in the CC category; and protein binding, RNA binding, protein heterodimerization activity, and actin binding were the most abundant subcategories in the FM category.

Validation of Differentially Abundant Proteins
To validate the results obtained from iTRAQ experiments, 8 DAPs were randomly selected for transcript level analysis by real-time PCR. As indicated in Figure 3, the transcript levels of RPS15, BDH1, TST, ALDH1A1, SERP1NH1, and PRPSAP2 showed the same change pattern as their protein levels (p < 0.05). There were no significant differences in EPAS1 and MPST at the transcript level, but the variation trends were consistent with the results of iTRAQ experiments.

Validation of Differentially Abundant Proteins
To validate the results obtained from iTRAQ experiments, 8 DAPs were randomly selected for transcript level analysis by real-time PCR. As indicated in Figure 3, the transcript levels of RPS15, BDH1, TST, ALDH1A1, SERP1NH1, and PRPSAP2 showed the same change pattern as their protein levels (p < 0.05). There were no significant differences in EPAS1 and MPST at the transcript level, but the variation trends were consistent with the results of iTRAQ experiments. Verified the selected eight DAPs by real-time PCR. CON-control group; BET-betaine group; EPAS1-endothelial PAS domain protein 1; RPS15-ribosomal protein S15; BDH1-3-hydroxybutyrate dehydrogenase 1; MPST-mercaptopyruvate sulfurtransferase; TST-thiosulfate sulfurtransferase; ALDH1A1-aldehyde dehydrogenase 1 family member A1; SERPINH1-serpin family H member 1; PRPSAP2-phosphoribosyl-pyrophosphate-synthetase-associated protein 2. Results presented as means ± SEM (n = 8). * Bars marked with an asterisk were significantly different at p < 0.05.

Discussion
In animal feed, betaine has long been used as a nutritive additive due to its lipotropic and growth-promoting effects [2]. In this study, the ADG was higher and FCR was lower in the betaine group than those in the control group, which implied that betaine supplementation might have positive effects on growth performance of broilers. This result was similar to the data of Rao et al., who reported that the ADG of broilers was significantly increased by 800 mg/kg betaine supplementation during a 42 d feeding trial [23]. Moreo- Figure 3. Verified the selected eight DAPs by real-time PCR. CON-control group; BET-betaine group; EPAS1-endothelial PAS domain protein 1; RPS15-ribosomal protein S15; BDH1-3hydroxybutyrate dehydrogenase 1; MPST-mercaptopyruvate sulfurtransferase; TST-thiosulfate sulfurtransferase; ALDH1A1-aldehyde dehydrogenase 1 family member A1; SERPINH1-serpin family H member 1; PRPSAP2-phosphoribosyl-pyrophosphate-synthetase-associated protein 2. Results presented as means ± SEM (n = 8). * Bars marked with an asterisk were significantly different at p < 0.05.

Discussion
In animal feed, betaine has long been used as a nutritive additive due to its lipotropic and growth-promoting effects [2]. In this study, the ADG was higher and FCR was lower in the betaine group than those in the control group, which implied that betaine supplementation might have positive effects on growth performance of broilers. This result was similar to the data of Rao et al., who reported that the ADG of broilers was significantly increased by 800 mg/kg betaine supplementation during a 42 d feeding trial [23]. Moreover, some other experiments had also confirmed the growth-promoting effect of betaine in poultry and pigs [5,6,24,25]. This effect might be given by that betaine could improve intestinal health and nutrient digestibility by enhancing intestinal structural integrity and digestive enzyme activity which, in turn, improved the growth performance of broilers [2,5].
Serum biochemical parameters can reflect the nutritional metabolism and health status of animals [26]. GH is a pleiotropic hormone secreted by pituitary gland that coordinates an array of physiological processes, among which the growth-promoting effect on bone and muscle is most notable [27,28]. In the somatotrophic axis, GH can directly act on the cell itself or indirectly activate growth hormone receptor to stimulate IGF-1 synthesis, and then IGF-1 acts on the target cells to promote tissue growth [29,30]. Multiple studies showed that IGF-1 could promote skeletal muscle fibers differentiation and increase myotubes size, resulting in an increase in muscle protein accretion [11,30,31]. It had been reported that supplemental betaine could increase serum GH and IGF-1 levels in pigs and increase IGF-1 concentrations in serum and liver of laying hens [3,32]. Our study also found that the concentrations of GH and IGF-1 in serum of broilers were significantly increased by dietary betaine supplementation, and this might explain why broilers fed with betaine showed a better growth performance in the present study. Moreover, compared with the control group, a higher content of serum TP and a lower BUN content were observed in the betaine group, which were consistent with the previous studies [6,33]. Increased concentration of serum TP was related to the improvement of body protein anabolism [26]. BUN was a key end-product of protein metabolism in the animal body, and decreased serum BUN content implied that more nitrogen was used for protein synthesis [34]. Accordingly, the results of these serum parameters indicated that betaine might play an important role in promoting protein anabolism of broilers.
Skeletal muscle is the main product of protein deposition, which accounts for 35~45% of broilers carcass weight. It is known that proteins are synthesized through a process called translation, which takes place in the cytoplasm and involves DNA transcription into RNA, RNA translation into peptide chain, and peptide chain processing into protein. [13]. In this study, the contents of absolute protein deposition and RNA, and the ratio of RNA/DNA in breast muscle were higher in the betaine group than that in the control group. It was believed that the quantity of DNA in muscle cell was normally stable, but the content of RNA was closely related to the rate of protein synthesis [14]. Moreover, RNA/DNA ratio has been proven as a useful indicator of the nutritional status and growth of animals in relation to protein metabolism [14,35]. Therefore, these results further confirmed the promoting effect of betaine on protein synthesis. We speculated that betaine regulated muscle nucleic acids contents in two aspects. On the one hand, betaine could promote the secretion of IGF-1 to enhance the activity of RNA polymerase, thereby promoting DNA transcription into RNA and accelerating protein synthesis. On the other hand, betaine, acting as a methyl donor, could methylate RNA to increase its stability and prevent degradation. For example, promoting guanylate methyl reaction was conducive to the generation of 5 end cap of mRNA [13]. Amino acids are the basic components of peptide and protein. Betaine, a methyl donor, can participate in the regeneration cycle of Met, and then decompose into some secondary metabolites, such as Ser and Gly [36]. This may explain the increased Met and Ser contents in breast muscle of broilers fed betaine. Similar results were reported by previous studies that betaine supplementation could increase serum Ser and Met contents of goats, and linearly enhance the content of Met, Ser, and Glu in muscle tissue of ducks [37,38]. However, the reason why the contents of Glu and Phe were increased by betaine supplementation still needs further research. The changes of amino acids compositions will affect the structure and functional expression of protein.
Thus, we speculate that betaine may affect the abundance levels and function of skeletal muscle protein.
The iTRAQ technology combined with LC-MS/MS is now one of the most sensitive methods used for quantitative analysis of proteomes. In this study, a total of 1084 proteins were detected through proteomics analysis, in which 35 proteins were identified as DAPs in the betaine group compared with the control group, including 27 upregulated proteins and 8 downregulated proteins. These DAPs were mainly involved in cell differentiation, small molecule metabolic process, tissue development, inorganic ion homeostasis, and S-adenosylmethionine metabolic process. S-AdoMet_synt_C-domain-containing protein, as a methionine adenosyltransferase, participates in the formation of S-adenosylmethionine (SAM), which plays a vital role in transmethylation reactions and the transsulfuration pathway [39]. Thiosulfate sulfurtransferase (TST) and uncharacterized protein (MPST) are involved in transferring sulfur-containing groups. It had been reported that betaine could take part in the metabolism of sulfur-containing amino acids and DNA methylation [36,40]. DNA methylation was known to have regulatory effects on DNA transcription and chromosome structure, which might alter protein functions [41]. Our results indicated that betaine increased muscle Met content and regulated DNA methylation might be related to upregulated S-AdoMet_synt_C-domain-containing protein, TST, and MPST. Transcription regulation plays an important role in muscle growth and function. Phenylalanyl-tRNA synthetase beta subunit (FARSB) and asparagine-tRNA ligase (RCJMB04_13p14) are responsible for attaching L-phenylalanine and L-asparagine to the terminal adenosine of the appropriate tRNA, respectively, to take part in RNA binding and protein biosynthesis. PHD finger protein 20-like protein 1 (PHF20L1) regulate the transcription by RNA polymerase II, and 40 S ribosomal protein S15 (RPS15) is a key component in the assembly of the small ribosomal subunit, which is essential to RNA binding [42]. The upregulation of FARSB, RCJMB04_13p14, PHF20L1, and RPS15 indicated that betaine might play a positive role in RNA binding, which was consistent with the result that the content of RNA in breast muscle was increased by betaine supplementation in this study. Moreover, uncharacterized protein (LOC107050760) positive regulates protein localization to plasma membrane and plays an important role in skeletal muscle contraction and fiber development. Previous studies had reported that betaine could promote muscle growth of animals [4,10,12]. Our results further confirmed that betaine could affect the expression of some functional proteins in muscle. However, from the results of muscle proteomics, the number of DAPs affected by dietary betaine was not much, especially only 6 proteins were upregulated with fold change >1.5. This may be related to low abundance of many transport and signaling proteins which are below detection under the current experimental settings. It could be possible that many DAPs are regulated by post-translational modifications. So, future studies that multiple fractionation scheme and post-translation modifications (PTMs) studies, such as methylation, phosphorylation, and acetylation, are needed to provide comprehensive understanding of the effect of dietary betaine on muscle protein anabolism.
In order to verify the results obtained from iTRAQ experiments, 8 DAPs were randomly selected for transcript level analysis by real-time PCR. Our results showed that the mRNA levels of RPS15, BDH1, TST, ALDH1A1, SERP1NH1, and PRPSAP2 had the same change pattern as their protein levels, supporting the proteomic data. However, there were no significant differences in EPAS1 and MPST at the transcript level, which might be due to the RNA editing, post-translation modification, or other regulatory mechanisms.

Conclusions
In summary, diets supplemented with 1000 mg/kg betaine improved growth performance and muscle protein deposition of broilers, which were associated with increased serum GH, IGF-1, and TP contents, and alterations in muscle nucleic acids (RNA), amino acids (Ser, Glu, Met, and Phe), and protein abundance levels (27 upregulated proteins and 8 downregulated proteins).