1. Introduction
Global population growth and improved living standards have driven increasing demand for aquatic products. Consequently, the demand for high-quality aquatic products is growing [
1]. The fishing of natural resources alone is insufficient to meet the human demand for aquatic products, necessitating expansion of the scale of aquaculture to ensure a sustainable supply of high-quality protein sources for humans [
2]. However, a high dependence on the feed industry is a significant constraint on the development of aquaculture [
3]. Large-scale aquaculture requires the consumption of large amounts of compound feed to meet the demand for aquaculture. Increased feed complexity necessitates more feed ingredients, especially fishmeal [
4]. The global scarcity of fishmeal has prompted the exploration of alternative land-based sources of raw materials. Researchers have made progress in the research and development of new aquatic feed [
5]. Additional studies are required to investigate the effects of replacing land-based raw materials in fish feed on factors such as palatability, growth, muscle quality, and immunity in fish.
Several studies have explored alternatives to fishmeal in aquatic animal feeds [
6,
7,
8]. Simultaneously, several issues have been brought to light. Several fishmeal replacements, especially plant-based raw materials, contain anti-nutritional factors that adversely affect digestion, absorption, and utilization in animals [
9,
10]. Therefore, carnivorous fish are commonly fed animal proteins, single-cell proteins, or processed plant-based proteins as alternatives to fishmeal. In largemouth bass (
Micropterus salmoides), animal proteins exhibit better apparent digestibility than plant proteins [
11]. Enzymatic hydrolysis of poultry by-products (EHPB) is the process of breaking down poultry by-products using enzymes. A complex enzyme, mainly papain, was added to the poultry by-products composed of poultry racks, feathers, and blood. EHPB was obtained by enzymatic hydrolysis at a certain temperature. Poultry by-products share a composition similar to fishmeal. Enzymolysis enhances the absorption and utilization of small peptides and other substances that are decomposed from poultry by-products [
12]. Enzymatic hydrolysis partially converts insoluble proteins in poultry by-products into a water-soluble form, which facilitates absorption in the intestine [
13]. Previous studies have confirmed that EHPB could improve the digestibility, immunity, and growth performance and antioxidant capacity of turbot (
Scophthalmus maximus) [
14]. Therefore, EHPB serves as an ideal protein source for reducing the use of fishmeal in aquatic feeds.
Muscles of aquatic animals serve as the primary protein source in aquaculture. Muscle quality determines the success of aquatic products to some extent. The muscle quality of aquatic animals is influenced by various factors, including the growth stage [
15], feed composition [
16], culture pattern [
17], and aquaculture environmental factors [
18]. Biochemical processes during muscle-to-meat conversion significantly affect the quality of fresh muscle. These modifications affect various distinct characteristics of muscle, including appearance, nutritional composition, and texture. Therefore, modifying the muscle fiber characteristics of living animals may be a viable approach for regulating fresh muscle quality [
19]. Recent studies have demonstrated that substitution of dietary protein sources can affect the muscle quality of various aquatic species, such as mirror carp (
Cyprinus carpio) [
20], tilapia (
Oreochromis niloticus) [
21], and largemouth bass (
Micropterus salmoides) [
22]. In addition, animal protein sources, particularly poultry by-products, can improve the muscle quality of aquatic animals, including protein and fatty acid content and chewability [
23,
24]. In the context of fishmeal replacement, there is still a lack of understanding of the influence of various new protein sources on the muscle quality of aquatic products. Currently, research on the effects of EHPB as a feed protein source on the muscle quality of aquatic animals is limited. Moreover, the effect of EHPB on the muscle quality of largemouth bass remains unclear. Therefore, there is a pressing need to investigate the effect of EHPB as a potential replacement for fishmeal in largemouth bass.
Largemouth bass (
Micropterus salmoides) is a carnivorous fish native to Canada and the United States. The global cultivation of this species is prevalent because of its delicate meat and delicious taste. Decreased intermuscular counts facilitate feeding. Fishmeal is a crucial component of largemouth bass farming, accounting for a significant portion of the feed costs [
25]. Several studies have investigated alternative feed options for largemouth bass [
26,
27,
28]. Furthermore, there is a growing concern regarding the muscle quality of largemouth bass [
29,
30]. To date, no study has examined the use of EHPB as a replacement for fishmeal in largemouth bass diets, and the effect of EHPB on largemouth bass muscle quality remains unexplored. Therefore, this study aimed to investigate the potential of EHPB as a replacement for fishmeal in largemouth bass and its effect on the muscle quality of fish.
4. Discussion
The replacement of fishmeal in aquaculture feed is a widely discussed topic for industrial advancement. Significant progress has been made in research on fishmeal replacements for aquatic feed [
6,
7,
8]. This study revealed that replacing 8.89% fishmeal with EHPB promoted the growth performance of largemouth bass to a certain extent (refer to
Table 4). However, as more fishmeal was replaced by EHPB, the growth performance of largemouth bass showed a significant decline. Similar to the results of this study, the partial replacement of fish meal in the Gibel carp (
Carassius auratus gibelio) diet with certain poultry by-products promoted growth performance to a certain extent, but growth performance decreased with the increase in substitution [
40]. Similarly, a small amount of replacement of fishmeal in the Florida pompano (
Trachinotus carolinus) diet with poultry by-products did not negatively effect growth performance [
41]. This confirms, to a certain extent, that higher levels of fishmeal substitution can lead to a decline in fish growth performance. Interestingly, in juvenile red drums (
Sciaenops ocellatus), poultry byproducts negatively affect their body growth [
42]. The observed discrepancy may be attributed to variations in the quantity of fishmeal replaced with poultry by-products in the formula and the specific type and size of the fish used in the study. Another possible explanation could be that enzymatic hydrolysis improves the absorption and utilization of poultry by-products. In conclusion, substituting 8.89% fishmeal with EHPB as a protein source could promote the growth of largemouth bass and reduce the feed coefficient rate. Plasma biochemistry serves as a crucial indicator for assessing the health status of fish, with any signs of fish being in an unhealthy condition reflected in their plasma [
43]. In the plasma levels of this study, EHPB1 significantly increased ALB, ALT, AST, TC, GLU, and TP (refer to
Table 5). Similar results were observed in largemouth bass fed protein derived from
Clostridium autoethanogenum instead of fishmeal [
44]. In contrast, levels of AST and TG in Pengze crucian carp (
Carassius auratus) remained relatively stable when fed hydrolyzed feather meal instead of fishmeal, but the GLU concentration decreased [
45]. Interestingly, the plasma biochemistry of olive flounder (
Paralichthys olivaceus) is unaffected by the partial replacement of fishmeal with silkworm pupae meal, promate meal, and meat and bone meal [
46]. One possible explanation is that the largemouth bass, which is naturally diabetic, experiences hyperglycemia and hyperproteinemia when fed a high-protein diet, resulting in increased levels of plasma TP and GLU, which subsequently causes an increase in other plasma markers [
47,
48]. One of the possible reasons for the change in plasma biochemical indexes was that EHPB contains more small molecules, which are more conducive to the digestion and absorption of fish, and thus was reflected in plasma [
14]. Additionally, elevated levels of plasma biochemical indices such as GLU and TP suggest a higher metabolic rate in fish. The increased metabolism of EHPB1 may contribute to the successful substitution of fishmeal. Further research is needed to investigate the effect of EHPB on fish, particularly in relation to protein, glucose, and lipid metabolism, as the observed alterations in plasma biochemistry suggest potential effects in these areas.
Protein is a crucial component of aquatic feed and significantly contributes to its overall cost. In the current research, EHPB supplementation had a significant effect on the muscle quality of largemouth bass. Muscle hardness is strongly correlated with muscle texture [
49,
50]. It can directly affect various texture parameters and intuitively reflect muscle quality. In the structural analysis of largemouth bass back muscle, replacing only 8.89% fishmeal with EHPB in the diet significantly decreased the hardness, gumminess, and chewiness of the muscle, after which there was a trend of recovery (refer to
Figure 2). In contrast, the texture parameters of Pengze crucian carp increased when they were fed hydrolyzed feather meal instead of fishmeal [
45]. In addition, an increase in muscle texture was observed in grass carp (
Ctenopharyngodon idellus) consuming paper mulberry, Atlantic salmon (
Salmo salar) fed northern krill as a replacement for fishmeal, and grass carp consuming novel protein sources [
51,
52,
53]. In this experiment, springing also shows a similar trend to the previous three. Although there is no significant effect, this similar trend seems to indicate that elasticity is closely related to hardness, gumminess, and chewiness. In our study, EHPB decreased cohesiveness and resilience. The lower texture was characterized by the presence of coarser muscle fibers and a reduced fiber count in certain sections (refer to
Figure 3 and
Figure 4). Similar results have been observed in tilapia fed broad beans [
54]. Thinner muscle fibers in this section exhibited higher textural properties. However, other studies have reported conflicting results. A simultaneous increase in muscle stiffness and muscle fiber was found in red sea bream (
Pagrus major) fed olive leaf powder and European sea bass (
Dicentrarchus labrax) fed defatted yellow mealworm (
Tenebrio molitor) larvae meal [
55,
56]. The findings of the present study indicate that largemouth bass supplemented with EHPB1 have larger fiber intervals in their dorsal muscle fibers (refer to
Figure 3 and
Figure 4). This could explain the decrease in texture parameters such as muscle hardness and mastication, among other factors. Furthermore, reduced muscle fiber density may also be a potential contributing factor to this result [
57]. Moreover, replacing 8.89% fishmeal with EHPB did not affect the amino acid and fatty acid composition of largemouth bass muscle (Refer to
Table 7). No significant effect was found on largemouth bass muscle composition by replacing 8.89% fishmeal with EHPB (refer to
Table 6). No adverse effects were observed in this study when 8.89% fishmeal was replaced in the diet. Previous research has shown that replacing fish meal with poultry by-products can alter the amino acid and fatty acid composition of aquatic animals [
58,
59,
60]. Enzymatic hydrolysis potentially increases the levels of free amino acids and small peptides, improving the utilization rate of raw materials in largemouth bass and mitigating the negative effects [
61]. In summary, the analysis of muscle tissue and sections revealed that replacing 8.89% fishmeal with EHPB could significantly improve the muscle quality of largemouth bass without causing a loss of amino acids and fatty acids.
Physiological changes in aquatic animals frequently correlate with genetic alterations. Dietary EHPB had significant effects on both muscle composition and texture, as well as gene expression in largemouth bass. The analysis of mRNA related to protein metabolism-related pathways revealed that the replacement of 8.89% fishmeal with EHPB in the feed did not significantly affect protein metabolism-related genes (refer to
Figure 5). This coincides with the results of muscle protein content (refer to
Table 7). However, only
igf-1 expression was found to be significantly reduced.
igf-1 regulates protein metabolism and muscle cell production [
62]. The results in this experiment suggest that downregulation of
igf-1 expression may contribute to the reduction in largemouth bass muscle cell count. Variations in the quantity of largemouth bass muscle fibers further affect their muscle tissue texture. To further explore the factors influencing muscle cell growth, we identified gene expression patterns associated with muscle production.
tgf-β and
smad-2 have inhibitory effects on muscle cell growth and stimulatory effects on muscle cell proliferation [
63,
64]. In this study (refer to
Figure 6), we observed a slight decrease in the expression levels of
tgf-β and
smad-2 in the EHPB1 group, which was statistically significant in the EHPB2 group. This could explain the reduction in muscle cell count and increase in the cross-sectional area in the EHPB1 group. The reduction in the muscle fiber count may have contributed to the alteration of the texture parameters in this study. Myogenic regulatory factors, including
myod-1,
myf-5, and
myog, are involved in the regulation of muscle production [
65,
66]. In the current study, no significant effect on
myog was observed when the amount of EHPB replacing fishmeal in feed was increased. The mRNA levels of
myod-1 and
myf-5 were significantly elevated in the EHPB2 and EHPB3 groups compared to those in the control group. This suggests that a higher content of EHPB should be included in the diet than EHPB1 to influence alterations in texture parameters across these three factors. In this study, EHPB1 supplementation significantly increased
paxbp-1 mRNA content and
syndecan-4 expression in the largemouth bass muscle (refer to
Figure 7).
paxbp-1 regulates the cell growth checkpoint that controls muscle cell hypertrophy [
67]. Simultaneously,
syndecan-4 overexpression induces muscle cell hypertrophy [
68]. This finding explains the reason for muscle cell enlargement in the muscle section samples of largemouth bass in the EHPB1 group. In addition, the expression levels of
murf-1 and
myos in the muscle of largemouth bass fed an EHPB1 diet were not significantly affected.
murf-1 mediates muscle cell protein degradation and regulates muscle cell reduction [
69].
myos is a potent inducer of muscle atrophy and can inhibit muscle development [
70]. This finding aligns with the results of our muscle-slice experiment.