1. Introduction
For the pork industry, fat and fatty acids (whether in muscle or adipose tissues) contribute importantly to various aspects of meat quality (e.g., flavor, taste) and are central to nutritional value [
1]. However, fat-type pigs exhibiting excessive amounts of subcutaneous adipose tissues have been recognized as detrimental to carcass quality [
2]. Moreover, imbalanced fatty acid composition is even harmful to the consumer [
3]. Therefore, increasing attention is mainly focused on safer, healthier, and taster of meat. Indeed, the use of nutritional approaches to optimize meat fatty acid composition has been a popular research topic, for example, supplying specific additives in diets such as linseeds, plant extracts [
4,
5]. The application of
N-carbamylglutamate (NCG) as a dietary supplement for the health of humans and animals also has gained increasing interest [
6,
7,
8].
NCG, as an effective and metabolically stable analog of
N-acetylglutamate, promotes the synthesis of endogenous arginine [
9,
10]. What’s more, NCG facilitates muscle protein synthesis [
11], protects the small intestinal morphologic [
12] and improves reproductive performance [
13,
14]. Moreover, new evidence indicated that NCG can enhance the antioxidant capability in the plasma, spleen, liver, and jejunum of rats [
12,
15,
16]. Our previous study also proved that NCG is a non-toxic substance with no genotoxicity in rats [
17]. However, few studies regard the effect of NCG on meat quality of pigs, and whether fatty acid metabolism may be involved in the regulation of the process. Advances in seabass demonstrated that NCG alleviates liver metabolic disease and hepatic inflammation via inhibiting ERK1/2-mTOR-S6K1 signaling pathway, and the ameliorated function is closely associated with the improved lipid metabolism indices, for example, lower plasma very low-density lipoprotein and hepatic triglyceride and non-esterified fatty acid accumulation [
10]. Ningxiang pig, as one of Chinese indigenous fat-type breeds, exhibits early sexual maturity, tender succulent flavor, strong adaptability and resistance, plays an increasingly significant role in the pork industry [
18]. Given the foregoing, we hypothesized that dietary NCG may affect meat quality traits of Ningxiang pigs through influencing lipid metabolism. Therefore, the purpose of the present study was to evaluate the effects of dietary NCG on carcass traits, meat quality, and fatty acid profiles in different tissues of Ningxiang pigs.
3. Results
Table 2 presents that NCG has no significant differences in carcass traits including slaughter yield, straight/oblique length and loin muscle area. In addition, pH values and muscle colors (i.e., L*, a*, and b*) were not affected by any of the dietary treatments (
p > 0.05). However, drip loss was significantly increased by 30.32% (
p = 0.044), while shear force was significantly decreased by 29.51% (
p = 0.004) after NCG supplementation.
The effects of NCG supplementation on amino acid profiles in the longissimus dorsi muscle of Ningxiang pigs are listed in
Table 3. NCG tended to increase the concentration of phenylalanine (Phe) (
p = 0.066) in the longissimus dorsi muscle. Moreover, increased levels of oleic acid (C18:1n9c) (
p = 0.009), paullinic acid (C20:1) (
p = 0.004), α-linolenic acid (C18:3n3) (
p < 0.001) and docosahexaenoic acid (C22:6n3) (
p = 0.082), while significant reduction in the proportions of arachidonic acid (C20:4n6) (
p < 0.001) and polyunsaturated fatty acid (PUFA) (
p = 0.017) were observed in the longissimus dorsi muscle of pigs fed NCG when compared with pigs fed the control diet (
Table 4).
As for adipose tissues,
Table 5,
Table 6 and
Table 7 present the effects of NCG supplementation on the fatty acid profiles in dorsal subcutaneous adipose (DSA), abdominal subcutaneous adipose (ASA), and perirenal adipose (PA) respectively. The C20:1 (
p = 0.045) proportion in DSA and C20:4n6 (
p = 0.070) in ASA, as well as the stearic acid (C18:0) (
p = 0.018) and C20:1 (
p = 0.063) levels in PA decreased in the pigs that were fed the NCG diet compared with those of the control diet. In contrast, the margaric acid (C17:0) (
p = 0.043) and C18:3n3 (
p = 0.071) proportions in PA were increased. Moreover, the NCG diet produced these adipose tissues with a greater proportion of total PUFAs (
p < 0.1) (particularly linoleic acid (C18:2n6c) (
p < 0.1)) compared with those produced by the control diet.
4. Discussion
New research shows that NCG may improve lipid metabolism with decreased plasma very low-density lipoprotein, hepatic triglyceride and non-esterified fatty acid accumulation, down-regulated fatty acid and cholesterol synthesis, and simultaneously increased lipolysis gene mRNA levels of fish [
10]. However, few studies regard the effect of NCG on meat quality of pigs. The present study for the first time reported the use of NCG as a feed additive for Chinese local pigs, to determine whether it could impact or even improve fatty acid profiles in different tissues. Fatty acids are essential components of membrane phospholipids, and many of them have been associated with cardiovascular, metabolic and neuropsychiatric disorders [
3].
No significant differences were observed in carcass traits of Ningxiang pigs between groups under the conditions of our study, which contradicted previous findings that NCG is effective to increase longissimus dorsi muscle area and decrease back fat accretion [
25]. The possible reasons for this discrepancy could be attributed to the diet factors (regular vs. reduced protein level) or the type of pigs (fat genotype vs. lean phenotype) used in studies. As mentioned previously, there is increasing interest in meat quality for consumers, particularly in tenderness and juiciness [
3]. Among them, tenderness is critically important from a sensory viewpoint. In the present study, pork from the NCG diet had a lower shear force value than from the control diet, indicating a more tender texture. Thus, NCG supplementation in the swine diet may be a good nutritional strategy for tender pork production. Drip loss, another quality measure of pork, is a natural phenomenon encountered during refrigerated storage of fresh meat. Generally, meat with a high drip loss percentage would lead to unattractive appearance and low consumer acceptance, which eventually reduce economic benefits [
26]. Another major finding from the present study was that dietary NCG had a significant adverse influence on drip loss compared to the control group. The moisture retention potential of fresh pork muscle is ostensibly related to some specific fatty acidss [
27]. It appears that total saturated fatty acids (SFAs) may be negatively associated with drip loss, suggesting that decreased SFA in the longissimus dorsi muscle may be an influencing factor for drip loss. The precise mechanism underlying this effect currently requires further investigations.
Muscle is the largest reservoir of amino acids in the body, and essential amino acids in meat can offer high nutritional values [
24,
28]. In addition, amino acid composition determines the flavor of meat, which is also an important source of essential amino acids in human diets [
29]. Recent research demonstrated NCG promotes intestinal absorption and transport of amino acids or peptides in suckling lambs via regulating the mTOR signaling pathway [
30]. Indeed, NCG could increase protein synthesis in skeletal muscle [
11]. In the present study, NCG increased the concentration of Phe in the longissimus dorsi muscle slightly, which was consistent with the previous result obtained by Liu et al. (2016) that the Phe content is increased by NCG intake in rat plasma [
31]. Phe is an essential amino acid for humans, and of great relevance to assessing the nutritional value of meat. One study reports that NCG could significantly decrease homogentisate, an intermediate of the metabolic breakdown of Phe [
32]. Besides, the coordinated activity of certain amino acid transporters in the cellular membranes may partially response to the intracellular presence of available amino acids [
33]. These transporters can sense the availability of amino acids, relay nutrient signals to the cell interior, move amino acid in or out of the cells, and launch a series of cascade responses, thus exhibiting a dual transporter and receptor function [
34]. A study by Yang et al. (2013) found that NCG ameliorates the absorptive capacity of weaned piglets by increasing mRNA expression of Slc6a19, Slc7a9 and protein abundance of ASCT2, B
0AT1 and b
0,+AT in the jejunum. These altered transporters involved in mediating the transfer of Phe may contribute to Phe increment in the longissimus dorsi muscle of Ningxiang pigs [
35].
A certain amount of fat in pork meat is favorably related to the palatability of the juiciness, odor, and flavor of pork meat when it is cooked as a roast or chop. Accordingly, the fatty acid composition of muscle seals the nutritional quality of pork, for example, PUFA content is positively correlated with meat off-flavor [
36,
37]. Various studies have demonstrated that NCG supplementation could affect lipid and energy metabolism (such as acetoacetate, acetone, lactate, creatine) in rats [
31,
32]. These results indicate that NCG may have beneficial effects on the taste and tenderness of pork since these meat characteristics are closely related to fatty acid composition [
1]. In the present study, the percentage of each fatty acid respect to all fatty acids within the fraction was calculated, and the NCG diet produced the longissimus dorsi muscle with a greater concentrations of C18:1n9c, C20:1, C18:3n3, and C22:6n3, and with a lower level of total PUFAs (particularly pro-inflammatory factor C20:4n6) compared with those produced by the control diet, indicated the reassignment of these fatty acids. These findings are partly consistent with the previous result obtained by Ye et al. (2017) that the muscular C20:4n6 content is decreased by NCG intake in finishing pigs fed the reduced protein diet [
25]. The possible reasons could be attributed that NCG could increase endogenous NO production, which accelerates the synthesis of eicosanoids, and results in the C20:4n6 proportion decrease [
19]. C18:1n9c is the most abundant showing levels of 90% total monounsaturated fatty acids (MUFAs) and positively correlated with flavor, and also described as a regulator of immune function and cholesterol levels [
38,
39]; whereas C20:4n6 is capable of being converted into numerous inflammatory mediators and stimulating the pathogenesis through the prostacyclin pathway [
40,
41]. Notably, the percentage of C18:3n3 and C22:6n3 were increased in the present study. C18:3n3 and C22:6n3 are both types of n-3 series fatty acids and have been well studied for their roles in reducing the risk factors of disordered lipid metabolism, suggesting that these changes may be beneficial in inhibiting fat accumulation [
42].
The effect of NCG on fatty acid composition was only evaluated in the longissimus muscle [
25], limited information is available on the fatty acid composition of adipose tissues. Indeed, lipid synthesis mainly occurs in adipose tissues of pigs. Subcutaneous and visceral adipose tissues with different anatomical locations show specific development and deposition, especially in de novo synthesized fatty acids due to desaturation and elongation [
43,
44]. NCG is involved in regulating the metabolism of energy substrates through nitric oxide production [
19,
45]. Nitric oxide, as a signaling molecule, stimulates glucose and fatty acid oxidation, enhances lipolysis, and inhibits lipogenesis in subcutaneous adipose tissues [
46,
47]. It seems that tissue-specific manner of dietary NCG on fatty acid composition in adipose tissues of Ningxiang pigs exists. Our results showed NCG supplementation resulted in an increased amount of C18:2n6c in these adipose tissues, which mainly explained the higher PUFA percentage. This shift towards greater unsaturation in adipose tissues and an increase in C18:2n6c could lead to stimulating lipid oxidation of the pork fat, and have a hypocholesterolemic effect and thereby slow the development of atherosclerosis for the consumer [
48]. However, such depot fats exhibiting a high content in C18:2n6c are often soft with a decrease in their storage capacity and their technological quality, C18:2n6c also elongates and desaturates to form C20:4n6 in the body, a precursor to pro-inflammatory compound that can have detrimental effects on health [
49]. Given the complexity of the nutritional role of linoleic acid, an appropriate level of intake should be considered [
50]. It is interesting to note that the decrease in C18:0 proportion was of greater magnitude in PA than in subcutaneous fat, and the opposite occurred for C16:0, thus indicating different regulatory effects of NCG. Besides, the percentage of C20:1 in DSA and PA was decreased, but further investigations into the potential mechanism of NCG on fatty acid metabolism are, therefore, warranted. Consequently, feeding NCG may be useful in modifying pork fatty acid composition to meet market demands (i.e., for either lower SFA and specific MUFA, or increased PUFA).