1. Introduction
Protein is the most expensive ingredient in aquafeed and profoundly impacts the growth and health of aquatic animals. However, the shortage of traditional protein sources, such as fishmeal and soybean meal, hinders the development of the global feed industry [
1]. Cottonseed protein plays an essential role in aquaculture as a plant protein source [
2]. However, cottonseed contains gossypol and other anti-nutritional factors, limiting its inclusion level in a diet [
2]. Through a series of processing, including dehairing, dehulling, low-temperature oil extraction, and solvent extraction, the cottonseed is processed into cottonseed protein concentrate (CPC), which significantly reduce the anti-nutritional factors [
3]. Currently, researchers are extensively investigating the potential of CPC as alternative materials in diets [
3,
4,
5], aiming to alleviate protein shortages. CPC can replace 40–45% of fishmeal in largemouth bass (
Micropterus salmoides) diets without affecting largemouth bass growth performance [
3,
4]. Likewise, CPC can replace 24% of the fishmeal in pearl gentian grouper (
Epinephelus fuscoguttatus♀ ×
Epinephelus lanceolatu♂) and promote growth performance [
5]. However, nutritional trials using CPC as the sole protein source are uncommon, which is desirable to evaluate the biological value of CPC.
Grass carp (
Ctenopharyngodon idellus) is herbivorous and feeds on certain water plants in its natural environment [
6]. The grass carp culture shares a long history and the largest freshwater aquaculture production in China due to the euryphagic feeding habit and delicious meat of grass carp, which is one of the “four major Chinese carps”. Grass carp has been introduced into more than 100 countries around the world for food, weed control, or research [
7]. According to FAO statistics, the global aquaculture production in 2020 was 87.5 million tons, among which grass carp production was 5.79 million tons (the top three countries in production are China, Bangladesh, and Iran), accounting for about 6.62% [
8]. Nowadays, grass carp culture relies mainly on feeding with compound feed to meet the market demand. Previous studies have determined protein requirements for grass carp based primarily on semi-purified feeds using fishmeal or casein, which indicated that the optimal protein level vary according to the protein materials and fish growth stages [
9,
10,
11,
12]. Moreover, precise nutrition for aquatic animals requires accurate knowledge regarding the protein sources and their optimal inclusion levels, which has progressively attracted attention from scholars. Therefore, evaluating the optimal dietary protein level in grass carp diets based on CPC as a protein source must be emphasized under the circumstance of precision nutrition.
Flesh quality has always been the most valued part by consumers. The indicators for evaluating flesh quality include cooking loss, texture properties (including hardness, cohesiveness, gumminess, springiness, resilience, and chewiness), pH, and antioxidant capacity [
13]. Muscle growth is a dynamic process, including muscle hyperplasia and hypertrophy [
14], which is affected by multiple regulatory factors such as muscle regulatory factors (MRFs), insulin-like growth factors (IGFs), fibroblast growth factor 6 (
fgf6), myostatin (
mstn), and target of rapamycin (
tor) [
15,
16]. There have been many achievements in regulating flesh quality by dietary nutrients in mammals, but the regulation of flesh quality in aquatic animals still needs to be investigated in depth. Existing studies have shown that amino acid supplementation at an appropriate level can improve the flesh quality of grass carp [
13] and hybrid bagrid catfish (
Pelteobagrus vachelli♀ ×
Leiocassis longirostris♂) [
15]. Grass carp that ate fava beans gained better flesh quality, mainly due to increased muscle hardness [
17,
18]. Furthermore, a previous study showed that replacing rapeseed meal and cottonseed meal with DDGS induced the myosin isoforms transformation of grass carp, altering the muscle texture properties, histological muscle properties, and gene expression of MRFs [
16,
19]. A recent study showed that feeding grass carp (6.80 ± 0.10 g) with graded protein level (soybean meal) diets improved flesh quality at the appropriate protein level [
20]. Likewise, in a study of large grass carp (264.11 ± 0.76 g), optimal protein levels (fish meal, casein and gelatin) also improved muscle texture and antioxidant capacity [
12]. Therefore, the effect of varying dietary protein levels from CPC on the muscle histology, myosin heavy chain (
myhc) gene expression levels, and muscle texture properties deserves investigation, for a more comprehensive nutritional value evaluation of CPC.
The homeostasis of intestinal microbes regulated by nutrients has crucial guiding significance for human health [
21]. Rapid, low-cost, and precise DNA sequencing methods, such as those offered by Illumina (San Diego, CA, USA), are becoming increasingly popular and extensively used to examine intestinal microbial composition [
21]. Recently, the importance of intestinal microbes in aquatic animals has been paid more attention to by researchers [
4,
5]. However, previous studies focused on the diversity in intestinal microbial composition after fishmeal replacement or exposure to specific factors [
3,
22]. Research on the effect of protein level on fish intestinal microbiota has only been reported for Songpu mirror carp (
Cyprinus carpio) [
23]. In addition, the mammalian gut–muscle axis also have received attention [
24], but these studies are still in their infancy in aquatic animals. In this regard, a crosstalk analysis between the homeostasis of intestinal microbes and the growth and health status will beneficial, to illustrate the regulative mechanism of the CPC protein level.
To sum up, given the importance of protein resources, the urgent need of farmers for the rapid and healthy growth of fish, and the pursuit of superior flesh quality by consumers, it is necessary to evaluate the nutritional value of CPC as a single protein source and to quantify the optimal protein level for grass carp. Using CPC as a protein source, six treatments were designed containing a gradient of protein levels, subsequently conducting an 8-week feeding trial to investigate the effects of these protein levels on the growth, health, flesh quality, and intestinal microbiota of grass carp.
4. Discussion
The present study showed that the optimal dietary protein levels improved grass carp SGR and FE, while high protein levels (CPC5 and CPC6) showed relatively poor growth performance. This may be due to the depression in feeding rate and more energy expenditure on processing excess protein for deamination, increasing the nitrogen metabolism burden and affecting fish growth [
28]. Based on SGR and FE, the optimal dietary protein levels for juvenile grass carp (4.68 ± 0.01 g) were estimated to be 38.61 and 38.66% in this study, respectively, which was highly consistent with the 38.63% protein requirement of grass carp (6.80 ± 0.10 g) [
20]. Moreover, the result of this study was close to the 40% requirement for juvenile grass carp (4.27 ± 0.01 g) reported by Jin et al. [
10], but slightly lower than the 41–43% requirement for grass carp fry (0.15–0.20 g) reported by Dabrowski [
9]. This variation in dietary protein requirements is strongly associated with different life-history stages, as reported in Abdel-Tawwab et al. [
27], as heavier fish reduces the protein requirements. In other words, the present study indicates that CPC is a potential material in juvenile grass carp diet, with similar nutritional value for fish growth compared with soybean meal and other traditional materials.
In this study, PER decreased with increased protein levels, and was significantly lower in the CPC6 group compared with other groups, which was consistent with previous reports in Nile tilapia (
Oreochromis niloticus) [
27] and Songpu mirror carp [
23]. Improved CF indicates good health and growth performance [
10]. CF peaked in the CPC4 group in the present study, suggesting an optimal dietary protein level may promote grass carp growth by improving morphology. Previous studies have shown that HSI and MFI tend to decrease as dietary protein levels increase [
10,
27]. In this study, HSI and MFI first showed an increasing and then decreasing trend, which may be due to CPC use. Similar morphological parameters were reported when CPC was used to replace fishmeal in largemouth bass [
4] and pearl gentian grouper [
5]. The present study showed that high and low protein diets decreased dry matter digestibility, which further explains the depressed growth. In contrast, protein digestibility increased linearly, which indicates that a non-fishmeal diet (based on CPC) is tolerable for grass carp.
There was a significant negative correlation between net protein utilization (NPU) and urea levels [
29]. The current study found that urea nitrogen increased linearly with the dietary protein level, suggesting that excess dietary protein can lead to protein waste. Previous studies on rainbow trout (
Oncorhynchus mykiss) [
30] and Nile tilapia [
27] have shown that serum lipids tend to increase with dietary protein levels. Consistently, serum biochemical indices of triglyceride and LDL showed a trend of increasing first and then decreasing as dietary protein levels increased in this study. These serum parameters may be elevated due to converting excess protein to lipids and carbohydrates. Complement 3 and immunoglobulin M are important antibacterial compounds related to the immune response of teleost fish [
11]. In this study, the content of C3 and IgM in the serum of fish showed quadratic and linear models, respectively, in response to the dietary protein level, which indicated that the CPC4 group had an improved immune ability. Consistently, the optimal protein levels could improve the immune function of the grass carp gut [
11]. Similar results were also observed in mirror carp (
Cyprinus carpio), in that the optimal protein level enhanced the C3, C4, and IgM of mirror carp at different water temperatures [
31]. The immune-enhancing effect of appropriate dietary protein can be explained by increasing protein intake and feed utilization with appropriate dietary protein levels [
10,
27].
Oxidative damage is closely related to antioxidant enzymes and can be expressed using MDA content [
32]. The current study demonstrated that optimal dietary protein levels increased the T-AOC content and GSH-Px activity while it reduced the MDA content in the hepatopancreas of grass carp, indicating that optimal dietary protein levels could protect grass carp from oxidative damage. However, the dietary protein levels did not significantly affect the hepatopancreatic SOD and CAT activities. Similarly, Xu et al. [
11] reported that the optimal protein levels reduced the MDA contents but did not alter the CAT activities of grass carp in the mid intestine and distal intestine. ALT and AST are essential amino acid metabolizing enzymes and the improvement of ALT and AST reflects the vigorous activity of amino acid metabolism in fish [
11,
27]. In the current study, hepatopancreatic ALT and AST activities were improved in the CPC4 group, implying that amino acid metabolism was enhanced at the optimal dietary protein levels. These results suggest that the optimal protein levels for improved grass carp growth may be partly due to enhanced amino acid metabolism and improved antioxidant capacity. The hepatopancreas is an essential organ for metabolism in fish, and histological changes are considered a crucial indicator in evaluating nutritional status [
19]. In the present study, fish fed CPC3 and CPC4 showed normal polygonal-shaped hepatocytes with large, clear nuclei centrally located without noticeable swelling or atrophy. These results indicate that the appropriate dietary protein level plays an essential role in promoting the antioxidant capacity and maintaining the health of the hepatopancreas. Noticeable, CPC5 and CPC6 diets depressed the hepatopancreatic ALT and AST activities and damaged the microstructure of the hepatopancreas, which indicated a reverse effect on fish metabolism and health, and possibly contributed to the depressed growth.
This study is the first to report the effect of protein levels on flesh quality when CPC was used as the protein source. Cooking loss, texture characteristics (including hardness, springiness, cohesiveness, gumminess, chewiness, resilience), and pH value are crucial parameters for evaluating muscle sensory quality [
13,
18]. The increasing cooking loss represents a decreased muscle water-holding capacity [
12]. In this study, muscle cooking loss increased linearly with protein level, suggesting that a high-protein diet had poor muscle water-holding capacity. In addition, this study showed that the muscle pH value linearly decreased as the protein level increased. The high dietary protein group (CPC6 group) had a reduced pH value compared to the low dietary protein group (CPC1 group), which may be related to the production of more lactic acid in the muscle [
12,
13]. This study showed that the optimal protein level (CPC4 group) improved the texture characteristics of grass carp, such as hardness, cohesiveness, gumminess, chewiness, and resilience. Recent studies show that muscle shear force or hardness was maximized at the optimal dietary protein levels (fishmeal and casein) [
12].
Flesh quality is often closely related to muscle fiber diameter, showing a negative correlation between the muscle fiber diameter and the hardness [
14,
16,
19]. The larger myofiber diameters (class50, 60, and 70) accounted for more of the myofibers of fish from both the low protein level (CPC1 group) and the high protein level (CPC6 group). In contrast, the optimal protein level (CPC4 group) had smaller muscle fibers (class20, 30, and 40). Further statistical analysis showed that CPC4 had a smaller mean diameter of myofibers and a greater density of myofibers. The histological results of this study also confirmed the negative correlation between muscle fiber diameter and hardness.
In the current study, T-AOC was elevated at optimal protein levels, and the MDA content was significantly reduced, suggesting that the optimal protein levels can maintain muscle structural integrity by inhibiting oxidative damage. Further research found that the enhanced antioxidant capacity was partly attributable to the improved GSH content and SOD activity of the muscle. Differences in antioxidant capacity between treatments at different protein levels obtained in a previous study further corroborate the findings of this study [
12].
After myoblasts initially form skeletal muscle, satellite cells provide additional nuclei required for skeletal muscle expansion [
15]. MRFs regulate satellite cells,
myod and
myf5 regulate satellite cell activation and proliferation, and
myog and
mrf4 act on cell differentiation [
15]. In addition,
myhc plays a vital role in fish muscle growth by promoting myofiber proliferation and hypertrophy [
16,
19]. In this study, MRFs,
myhc-1, and
myhc-4 were upregulated at the optimal dietary protein levels, indicating that appropriate dietary protein levels can effectively promote grass carp muscle growth. Moreover, optimal protein levels in this study significantly upregulated
fgf6a and downregulated
mstn. As reported in grass carp, the
fgf6a plays a vital role in muscle growth regulation [
16]. Besides,
mstn has been shown to inhibit teleost muscle growth [
15]. These results also confirmed that the optimal protein levels in this experiment could promote grass carp muscle growth by regulating MRFs,
fgf6a, and
mstn. A previous study showed that transcriptome analysis of broad bean-fed grass carp (higher hardness) muscle detected upregulation of
myog, which plays a crucial role in promoting the formation of new muscle fibers [
17]. Likewise, a recent study found that optimal dietary protein (soybean meal) levels promoted the expression of genes
myhc-1 and
myhc-4 by regulating a family of MRFs and contributed to flesh quality improvement [
20]. Optimal dietary protein levels in this investigation may improve flesh quality through the same pattern.
Tor regulates phosphorylation of its downstream effectors, ribosomal S6 kinase 1 (
s6k1) and eukaryotic translation initiation factor 4 e-binding protein 1 (
4e-bp1), which ultimately promote protein synthesis in fish and can affect
nrf2 expression as an upstream regulator of antioxidant capacity [
13]. Recent studies have shown that dietary protein levels can enhance the antioxidant capacity of grass carp muscle by upregulating
tor and
s6 k1 [
12]. The optimal dietary protein level in this study may promote the muscle antioxidant capacity through the same pattern. In addition, it has been demonstrated that
igf-I and
igf-II promote muscle growth in hybrid catfish [
15] by binding to
igf1 r, which may indicate that dietary protein may promote muscle growth through IGFs. Still, the specific mechanism needs to be further studied.
The intestinal microbiome has profound effects on human well-being, including host metabolism, physiology, nutrition, and immune function, and is even referred to as a “metabolic organ” [
21]. Recently, studies on the regulation of intestinal microbes by dietary nutrients have received increasing attention [
3,
23]. In this study, the results of 16 S amplicon sequencing showed that the dietary protein decreased the intestinal microbiota diversity of grass carp, estimated using the Shannon and Simpson indices. The PCoA of the weighted UniFrac distances further revealed that CPC1, CPC4, and CPC6 were separated, and the weighted UniFrac-based ANOSIM revealed significant differences in microbiota structure between different protein levels. These findings suggest that protein levels may alter the intestinal microbiota structure of grass carp. In addition, this study identified the dominant phyla of grass carp were
Proteobacteria,
Fusobacteriota,
Actinobacteria,
Firmicutes, and
Bacteroidota, which is consistent with the previous study on cyprinids [
6,
23]. Abundant
Proteobacteria can be used to characterize intestinal microbial homeostasis, as dysregulation of homeostasis when the
Proteobacteria abundance rises often leads to metabolic disturbances or inflammation [
21]. A previous study of Songpu mirror carp reported that the abundance of
Proteobacteria increases with protein levels [
23]. This is partly consistent with the result between the CPC4 and CPC6 groups. The abundance of
Proteobacteria was reduced at the optimal protein level (CPC4), which may benefit the homeostasis of intestinal microbiota.
More and more attention has been paid to studying the abundance ratio (F/B ratio) of
Firmicutes and
Bacteroides [
33]. In humans and mice, increased F/B ratios are often associated with obesity, diabetes, and metabolic disorders [
33]. In this study, the F/B value of the CPC4 group decreased significantly and showed good growth performance and health status, which provided a reference for studying the relationship between F/B and health. A decrease in the F/B ratio with increasing protein levels was consistently observed in Songpu mirror carp [
23].
Firmicutes can extract energy from food [
33]. Increased dietary protein levels in the present study promoted the abundance of
Firmicutes, suggesting that the better weight gain in CPC4 and CPC6 groups than the low-protein group (CPC1 group) may be attributed to increased energy generation and utilization. The LEfSe analysis showed that the abundance of
Bacteroides increased in response to the protein levels and contributed significantly to the differences in the CPC6 group. It is reported that
Bacteroidetes have carbohydrate-related enzymes, and
Bacteroidetes and
Firmicutes play an essential role in the energy metabolism and glucose metabolism of organisms [
34]. Combined with the apparent digestibility data, these results may imply that more energy is available for growth in the CPC4 group than in the CPC1 group. Still, the increase in ADC
p in the CPC6 group and the decrease in ADC
d indicate that less energy is available for growth in the CPC6 group than the CPC4 group.
At the genus level,
Cetobacterium was the most abundant in the gut of grass carp through community composition maps, consistent with previous studies on freshwater fish [
3,
23].
Cetobacterium is reported to produce vitamin B12 and can ferment peptides and carbohydrates, and inhibit the growth of harmful bacteria [
35].
Akkermansia represents a novel biomarker of intestinal metabolic health coupling and is essential for treating metabolic syndrome [
36]. Spearman’s correlation analysis showed that
Cetobacterium and
Akkermansia were strongly associated with growth performance while negatively correlated with muscle MDA content. These results well demonstrate the excellent growth performance of CPC4 in this study and explain its possible assistance to the antioxidant capacity of muscle. In contrast,
Microbacterium and
Pseudomonas exhibited a negative correlation with SGR, muscle hardness, and muscle GSH content while they were strongly positively correlated with muscle MDA content. These results confirmed that
Microbacterium is one of the most closely related cornerstone genera of other bacteria and plays a crucial role in the growth and health of fish [
37], and
Pseudomonas is one of the most important opportunistic pathogens of grass carp [
6]. In this study, in addition to being associated with growth and health, these genera also affected muscle hardness and antioxidant capacity. The interrelationship between intestinal microbiota and growth, health, and flesh quality requires more research to elucidate its specific regulatory mechanisms.
Intestinal microbiota affects the host mainly through its metabolites. The phylum
Fusobacterium can metabolize carbohydrates to butyrate [
34,
38], which mediates the regulation of intestinal inflammatory processes, atherosclerosis, and immune system maturation [
39]. In addition, among
Firmicutes, the
Lachnospiraceae,
Lactobacillaceae, and
Ruminococcaceae species hydrolyze starch and other sugars to produce butyrate and other short-chain fatty acids (SCFAs) [
39], which inhibit the growth of harmful bacteria. In this study,
Fusobacterium and
Lachnospiraceae were significantly enriched in the CPC4 group, suggesting that optimal protein levels may further contribute to the health of grass carp by enhancing the abundance of SCFA-producing bacteria. However, the mechanism of their specific interactions needs further study.
The functional prediction of grass carp intestinal microbial communities based on Tax4 Fun revealed that, interestingly, at KEGG pathway level 3, ko00480 (Glutathione metabolism) and ko00620 (Pyruvate metabolism) showed significant differences between the CPC1 and CPC6 groups. Glutathione metabolism is part of amino acid metabolism and contains important antioxidant molecules to protect the body from oxidants [
22]. A previous study showed that grass carp fed low protein levels had a lower intestinal GSH-Px and GSH content than the high protein levels [
11]. Similarly, ko00480 (Glutathione metabolism) was significantly downregulated in the low protein level treatment group in this study, suggesting that a diet below the optimum protein requirement may impair amino acid metabolism functions and the antioxidant system. Cells convert glucose to pyruvate in the cytoplasmic matrix through glycolysis. Pyruvate can produce a large amount of adenosine triphosphate (ATP) under aerobic conditions. In contrast, pyruvate can produce lactate and a small amount of ATP through anaerobic glycolysis under anoxic conditions [
40]. The significant enrichment of the ko00620 (Pyruvate metabolism) pathway in the CPC6 group may indicate that grass carp respond to a high-protein diet by enhancing glucose metabolism to obtain more energy to meet the needs of metabolizing protein. However, the mechanism of action between animal intestinal microbiota and metabolic function requires more studies.
Microcrystalline cellulose is the most commonly used filler and binder in fish feed [
41]. Previous studies on the dietary protein requirements of red drum (
Sciaenops ocellatus) [
42] and dietary carbohydrate-to-lipid ratios of channel catfish (
Ictalurus punctatus) [
43] reached 15.56 and 40.61% microcrystalline cellulose use, respectively, which suggest that the use of a varying amount of cellulose in protein requirement studies is acceptable. In addition, a recent study also showed that using 1.84–31.84% microcrystalline cellulose in grass carp diets did not produce adverse effects [
41]. This study was conducted to meet the demand for extruded feed in the aquatic feed market by producing extruded feed, and varying starch levels may disable the production of extruded diets at high starch levels, so microcrystalline cellulose was used as a filler to make the extruded feed. This might result in a relatively high optimal protein level. However, when comparing the optimal protein level with other results in grass carp, this study is acceptable.