Effects of coated cysteamine hydrochloride on muscle fiber characteristics and amino acid composition of finishing pigs

Objective This experiment was designed to determine the effects of coated cysteamine hydrochloride (CC) on muscle fiber characteristics, amino acid composition and transporters gene expression in the longissimus dorsi muscle (LDM) of finishing pigs. Methods Two hundred and sixteen Duroc/Landrace/Yorkshire cross-bred male finishing pigs were fed with a corn-soybean basal diet supplemented with 0, 70, and 140 mg/kg cysteamine. Each group contained eight replicates of nine pigs per replicate. After 29 days, one pig was randomly selected from each replicate and slaughtered. Blood and LDM samples were collected and analyzed. Results The results showed that supplemental dietary CC increased (p<0.05) the muscle fiber density. And CC supplementation also up-regulated (p<0.05) the expression of myosin heavy chain 1 (MyHC1) and MyHC2x mRNA levels, and down-regulated (p<0.05) MyHC2b expression in the LDM. Additionally, supplemental dietary CC reduced (p<0.05) the concentration of total cholesterol in the plasma and enhanced (p<0.05) the concentrations of essential amino acid and total amino acid in the LDM. The relative expression levels of chloramphenicol acetyltransferase 2, b0,+ amino acid transporter, and y+-L-type amino acid transporter 1 were up-regulated (p<0.05) in the LDM when pigs were fed with the dietary CC of 70 mg/kg. Conclusion Cysteamine supplementation could increase fiber density and distribution of fiber types. It also improved the deposition of protein in the LDM by up-regulated the expression of amino acid transporters.


INTRODUCTION
Meat quality is an important economic trait in meat industry, with the freshness and nutri tional value of meat being the key factors that governs consumers' buying decision [1]. However, with the intensive selection for greater growth rate and lean percentage of pigs, meat quality also has deteriorated [2]. Therefore, identifying new ways to improve meat quality and nutritive value is important for human health and animal productivity. Meat quality is affected by the interaction between many factors such as muscle structure, postmortem environmental con ditions, meat processing, packaging, and storage conditions, etc. [3]. Muscle fiber characteristics are the basic and direct factors to affect meat quality [4]. Muscle fiber composition is received to distinguish red and white muscles, which predicts biochemical changes and consequently meat quality [5].
Meat protein value has been assessed according to its amino acids composition. The amino acid composition is considered as an important index for nutritional value of each food. Numerous studies have verified that dietary supplementation with extra amino acids can influence the amino acid profile of tissues, particularly muscles [6]. Additionally, different amino acids are transported by specific amino acid transporters (neutral, acidic, or basic) in the muscle. The difference of quan titative expression of amino acid transports reflects the process of amino acid metabolism.
Cysteamine, as a novel feed additive in animal production, is used in the form of coated cysteamine hydrochloride (CC). Numerous researches have investigated its biological activity, which induces the regulation of mTOR signaling for modifying protein metabolism and antioxidative activities [6,7]. Using the commercial pig model, the previous study has shown sup plemental dietary cysteamine improved the stability of pork color by regulating oxidation [8]. Cysteamine is a physiological performance promoter, which regulates protein synthesis and protein degradation in pigs [9]. The influence of cysteamine supplementation on protein digestion and absorption may be due to its improving jejunal amino acid transportation [10]. However, the effects of cysteamine on the regulation of amino acids metabolism in the muscle has not been investigated. The ability of cysteamine to enhance the muscle fiber characteristics of finishing pigs remains unexplored. A coating product pre vents its dissolution or disintegration in the gastric environment. Therefore, based on these previous studies, it was hypothesized that supplementation with CC might influence muscle fiber characteristics, muscular deposition of protein and gene ex pression of transporters in the longissimus dorsi muscle (LDM) of finishing pigs thereby improving its meat quality and nu tritive.

Animals and sampling
The animal experiment protocol (NO. 20160712) was approved by the animal welfare committee of the Institute of Subtropical Agriculture, The Chinese Academy of Sciences, Changsha, China.
Two hundred and sixteen crossbred (Duroc/Landrace/York shire) castrated pigs with an average initial weight of 88.3±0.3 kg were used in this study. Pigs were individually divided into three treatment groups, and each group was replicated eight times with nine pigs per replicate. The basal cornsoybean meal diet was respectively supplemented with 0 (control, CON), 70 mg/kg (CC70), and 140 mg/kg (CC140) cysteamine sup plied by coated CC (basal dietary composition is presented in Table 1). The basal diet was formulated to meet the nutritional requirement of finishing pigs recommended by the NRC [11]. Pigs had free access to feeds and drinkingwater through out the experimental stage. The experiment lasted for 29 d. Additionally, coated CC, supplied by Hangzhou King Techina Technology Co., Ltd (Hangzhou, China), contained 270 g/kg CC.
After a 29day period feeding experiment, one pig randomly selected from each pen was fasted overnight and the morning for 12 h and the killed by exsanguination after electrical stun ning (250 V, 0.5 A, for 5 to 6 s). Fresh blood samples from external jugular vein were collected and kept in vacuum tubes with ethylenediaminetetraacetic acid. Plasma was obtained by centrifugation at 3,500×g for 15 minutes at 4°C, and the super natant stored at -20°C for analyzing biochemical indices. Muscle samples were carefully removed from the LDM at between 6th and 7th thoracic vertebrae within 45 min postmortem. The LDM samples were manually trimmed into 0.5×0.5×1.0 cm pieces, promptly fixed in 10% formalin for microsection manufacture. Subsequently, about 20 g LDM samples were rapidly excised and weighted, then freezedried for amino acid analysis. Moreover, the extra LDM sample was packaged in foil, promptly frozen in liquid nitrogen, and stored at -80°C for molecular analysis.

Plasma biochemical indices
Plasmas were centrifuged at 3,500×g for 15 minutes at 4°C, and the supernatants were used to determine biochemical indices by an Automatic Biochemical Analyzer (F. Hoffmann La Roche Ltd, Basel, Switzerland). These plasma biochemical indices, containing the concentrations of total protein, trigly ceride, total cholesterol, glucose, urea nitrogen, and the activities of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase, were analyzed according to the com mercial reagents form Roche Diagnostics GmbH (Roche Diagnostics GmbH, Mannheim, Germany) and manufac turer's instructions [12].

Muscle fiber characteristics measurement
Muscle fiber density and diameter were measured by the tra ditional staining of hematoxylin and eosin (HE). Samples of the LDM were transferred form 4% paraformaldehyde to 70% ethanol. Before dehydrated through a serial alcohol gradient, individual sections of muscle biopsy material were placed in processing cassettes. The sections embedded into paraffin wax blocks were cut into 5μm thickness and dewaxed in xylene, then rehydrated through lower concentrations of ethanol. After washed in phosphate buffer saline, sections were stained with HE and dehydrated through increasing concentrations of etha nol and xylene. The morphology of the LDM was observed at 200 times under a light microscope [13]. The Imagepro plus6.0 pathologica system was applied for imaging analysis, and three views were selected for each section. Muscle fiber characteristics were determined by measuring muscle fiber density and diameter. Approximately 10 views were counted to estimate the crosssectional area of the muscle fiber. Muscle fiber density was expressed as the ratio of total number of fibers counted to the total area of muscle fiber measured. Muscle fiber diameters were analyzed using the ImagePro plus 5.1, which was an image analysis system (Media Cybernetics Inc., Rockville, MD, USA) [14].

Amino acid profile in muscle
Amino acid compositions (hydrolytic amino acids) of the LDM were determined by an amino acid analyzer (L8900, Hitachi, Tokyo, Japan). Amino acids were extracted from tissue following the technique described by Hu et al [15] with slight modifica tions. About 0.1 g of freezedried muscle samples was hydrolyzed with 10 mL of hydrochloric acid solution (HCl, 6 M) in a sealed ampoule bottle for 22 h at 110°C. Then all hydrolysate was di luted with doubledistilled water in volumetric flask of 100 mL, subsequently 1 mL of hydrolysate diluent was passed through a 0.22 mm membrane filter. Samples were analyzed for amino acids contents through comparing peak profiles of the squid samples with standard amino acid profiles.

Real-time quantitative polymerase chain reaction analysis
Total RNA was extracted from LDM samples using a Trizol Reagent (Invitrogen, Carlsbad, CA, USA) following manufac turer's instructions and dissolved in diethylpyrocarbonate (DEPC)treated water. The extracted RNA was quantified by measuring optical density at 260 and 280 nm using an Eppen dorf Biophotometer (Eppendorf AG, Hamburg, Germany) and its integrity verified by electrophoresis on a 1% agarose gel. The cDNA was reversetranscribed from 1 μg of total RNA using the DNase I treatment (Takara, Otsu, Japan), then diluted and used for evaluating gene expression. All primer sequences employed herein were developed previously for amplification of mRNA sequences of Sus scrofa (Table 2). These target genes Amplification of the housekeeping gene (glyceraldehyde 3phosphate dehydrogenase) and target genes was performed in a 10μL reaction volume including 1 μM of each forward and reverse primer, 2 μL of cDNA, 2 μL of DEPCtreated water, and 5 μL of SYBR Premix Ex Taq (Takara Bio Inc., Japan). The relative expression levels of genes were performed using Light cycler480II system (Roche Diagnostics GmbH, Germany). The realtime polymerase chain reaction (RTPCR) protocol was strictly carried out with forty cycles of amplification, with each cycle consisting of predenaturation at 95°C for 10 s; de naturation at 95°C for 5 s; annealing at 60°C for 30 s; and elongation at 52°C to 60°C for 30 s, followed by a melting curve analysis. Cycle threshold (Ct) values were employed to deter mine expression levels of genes and calculated of triplicate measurements. The mRNA expression levels were analyzed and expressed as the relative values to those for control pigs. The comparative Ct value method was descried by Tan et al [16].

Statistical analysis
The experimental data were analyzed statistically by the one way analysis of variance method using SAS (SAS Institute Inc., Cary, NC, USA; 2002) and expressed as means±standard error of the mean. Based on adjusted degrees of freedom solution, this data about muscle fiber characteristics measurements and amino acid composition were calculated using mixed proce dure for repeated measures. Duncan's multiplerange test was performed for indicating differences between significant mean values. The statistical differences were declared significant at p<0.05 in all analyses.

Plasma biochemical indices
Plasma biochemical indices in finishing pigs were tested and shown in Table 3. CC70 markedly reduced (p<0.05) the con centration of total cholesterol in the plasma when compared with the control and CC140 groups. However, compared with CC140 group, supplemental dietary 70 mg/kg CC significantly reduced (p<0.05) alanine aminotransferase activity in the plasma.

Muscle fiber characteristics
The morphology of LDM fiber is shown in Figure 1. Based on fiber counting methods, the muscle fiber characteristics of finishing pigs in each group were measured and the data were  analyzed, as shown in Table 4. In the same field of vision, there were more muscle fibers in the LDM in pigs fed the CC70 diet. Compared to control treatment and CC140, CC70 had a higher muscle fiber density (p<0.05) in the LDM.

Gene expression levels of myosin heavy chain
As shown in Figure 2

Amino acid profile in muscle
The amino acid profile that indicates protein metabolism in the LDM is detailed in Table 5. The concentrations of essen tial amino acid and total amino acid (TAA) were calculated based on the analyzed muscle amino acid contents in this study. Compared with CC140, supplemental dietary 70 mg/kg CC markedly improved (p<0.05) the muscle TAA. Leucine, iso leucine and valine are known as branchedchain amino acids. A significant increased isoleucine (p<0.05) was observed with pigs fed CC70 diet. Moreover, compared to control group, CC70  significantly enhanced (p<0.05) the concentration of histidine in the LDM. In the neutral amino acids, CC70 increased (p< 0.05) the content of phenylalanine. Dietary 70 mg/kg CC sup plemented pigs had higher (p<0.05) asparagine content in the LDM than control and 140 mg/kg CC.

Gene expression levels of amino acid transporters
To further explore the different influences of dietary CC on muscle amino acid composition, we measured the expression of key amino acid transporters The relative mRNA expression levels of CAT2, y + LAT1 and b 0,+ AT were upregulated (p<0.05) by 70 mg/kg CC supplementation of the diet compared to the control diet ( Figure 3C, 3D, 3F). However, no significant dif ference (p>0.10) was observed on ASCT2, CAT1, and LAT2 expression in the LDM between groups ( Figure 3A, 3B, 3E).

DISCUSSION
In livestock production dietary supplementation with certain nutrients is supposed to be an effective way of improving meat quality and nutrition [17]. Numbers of studies in vivo and in vitro have focused on the growthpromoting and antioxidant functions of cysteamine [18,19]. However, the previous studies have demonstrated that supplemental dietary CC had no sig nificant effect on meat pH and drip loss, but increased meat color values and the deoxymyoglobin content by improving glutathione levels and antioxidant activity [20]. Meanwhile, this present study is first to indicate that dietary cysteamine supplementation can improve meat quality of finishing pigs through changing muscle fiber density and type distribution and promoting amino acids deposition and transportation. Muscle fiber, as an important component of muscle, directly affects meat quality. Muscle fiber characteristics have been cor related with various meat quality traits including muscle pH, tenderness, drip loss, meat color, and intramuscular fat con tent [21]. Moreover, muscle fiber number, size and fiber type composition are closely interrelated and were implicated in muscle fiber characteristics [22]. The fiber density was positively related to tenderness of cooked pork [23,24]. In the present study, dietary CC supplementation increased muscle fiber den sity in finishing pigs which indicates improving the sensory quality of meat.
Additionally, muscle fiber types can determine the size, num ber and density of muscle fiber. In general, muscle fibers are Table 5. Effects of dietary coated cysteamine hydrochloride (CC) on amino acid composition in the longissimus dorsi muscle of finishing pigs (n = 8)

Items (mg/kg) Dietary level of CC 1) p-value CON CC70 CC140
divided into four types considered as slowoxidative type I, fast oxidoglycolytic type IIA, and fast glycolytic types IIX and IIB, and expressed by MyHC isoforms genes identified as MyHC1, MyHC2a, MyHC2b, and MyHC2x [25]. According to Kim et al [26], the content of myoglobin and fiber color increases in the rank order I>IIA>IIX>IIB, but the fiber size is in the oppo site order. A higher type 1 fiber proportion might be positively correlated with muscle fiber density. Previous researches also reported that low percentages of type 2A and 2B fibers in the meat resulted in increased lightness (L*) and redness (a*). This is agreed with our previous study, which suggested that pigs fed dietary CC had increased a* in the LDM which promoted the stability of pork color [19]. This was related to the myoglo bin content of different fiber types. Furthermore, it has been demonstrated that antioxidant status is positively correlated with a* or color stability in the LDM of beef [27]. Cysteamine, as an excellent scavenger of oxidants hydroxyl radical and hypo chlorous acid, can protect against body oxidation. A muscle fiber type transition toward more oxidative muscle fibers such as MyHC1 can be induced by antioxidant [28]. This present study firstly provides the results that dietary CC supplemen tation improves MyHC1 and MyHC2x expression and decreases MyHC2b mRNA expression, which may partly explain the mechanism of cysteamine on the improvement of meat quality. Intramuscular amino acid composition and metabolism are determined to evaluate the biological value of muscle pro tein nutrition contributing to meat quality. This present study indicated that dietary CC supplementation enhances the mus cle amino acids deposition of finishing pigs. Consistent with previous studies, cysteamine supplementation resulted in a marked stimulation of protein deposition in skeletal muscle [6,29]. Importantly, plasma metabolism profile is reflected by changes in physiological and metabolic activities in response to dietary manipulations [30]. In the present study, serum total cholesterol was decreased, and glucose was increased by di etary supplemented with CC, indicating the inhibition of lipid metabolism and acceleration of glucose metabolism. Cyste amine could accelerate glucose and amino acid into muscle for protein synthesis, which may be associated with an increase in insulinlike growth factor 1 consequently stimulating pro tein synthesis [31].
Additionally, specific transporters plays an important role for transporting amino acids into the cell and in response to amino acid availability [32]. Previous studies have found that amino acid transporters changed in small intestine, skeletal muscle, and other tissues once dietary nutrient content was varied [33]. At present, little information about the impact of cysteamine on amino acid transporters in the LDM is avail able. However, the branchedchain amino acids (Ile), alkaline amino acids (His) and neutral amino acids (Phe and Ans) con tents were increased in the LDM in the present study. Different amino acid transporters unquestionably play a critical role in transportation with different mechanisms and featuring with unique substrate specificities [34]. The cationic amino acid transporters (CAT) have an important role in the transpor tation of arginine, lysine, histidine, and ornithine to regulate their homeostasis, and are widely distributed in tissues [35]. The ASCT2, b 0,+ AT, y + LAT1, and LAT2 are neutral amino acids transporters, and are mainly involved in the transport of branchedchain amino acids and some small neutral amino acids including asparagine and glutamine [36]. In addition, b 0,+ AT, and y + LAT1 are Na + independent transporters [37]. This agreed with Zhou et al [10], who reported that cyste amine supplementation in finishing pigs enhanced protein digestion and absorption via upregulating the expression of amino acid transporters (y + LAT1 and b 0,+ AT) in the jejunum. Thus, it is possible that there is an inevitable correlation be tween muscle and intestine for transportation and absorption of nutrition. In addition, the present study has demonstrated that administration of 70 mg/kg CC had a greater influence on muscle fiber characteristics and amino acid metabolism than 140 mg/kg CC supplemented in finishing pigs. This might be relative to the fact that continuous addition of a high dose of CC can cause gastrointestinal ulcer [38].

CONCLUSION
Dietary coated cysteamine increased the fiber density, upregu lated the expression levels of MyHC1 and MyHC2x and down regulated expression of MyHC2b. In addition, coated cysteamine supplementation improved the deposition of amino acids by promoting amino acid transporters expression in the LDM. Above all this study presented the most appropriate dose of dietary 70 mg/kg CC supplementation, which will be useful in livestock industry to improve meat quality and nutritive value. Importantly, this study adds to the knowledge of cysteamine function but further research about underlying mechanisms are required.