Fish meal (FM) has been used as a major protein source in aqua-feeds due to its relatively balanced amino acid pattern, high mineral and vitamin contents, and long-chain omega-3 fatty acids [1
]. Over the last 20 years, although the global FM production has remained relatively stable, it has not been able to match the rapid growth of the worldwide aquaculture industry. The limited supply of FM has led to a continuous rise of the price of commercial diets [2
]. Therefore, it is critical to seek alternative sources of protein to ensure a steady supply of commercial diets. Numerous researchers who use plant protein sources such as soybean meal, canola meal, pea protein, corn gluten meal, and so on to partially or completely replace FM have reported significant progress in different fish species [5
Compared with other plant proteins, soybean meal (SBM) is regarded as a nutritious feedstuff with a high crude protein content, wide availability, relatively balanced amino acid profile, and a stable supply [7
]. Some studies have demonstrated considerable success in the partial or complete replacement of FM with SBM in the diets of many fish species [10
]. However, some species have a limited capacity to use SBM. High dietary levels of soybean meal also significantly reduce the protease, amylase, and lipase activities in the digestive tract of Oreochromis niloticus
and Myxocyprinus asiaticus
]. Even in some fish, such as some salmonids, high levels of SBM can cause severe enteritis [13
]. Therefore, it is worth noting that the ability to utilize SBM as a protein source varies among different species.
As a non-proteinogenic essential amino acid or conditionally essential amino acid, taurine has received special attention from nutritionists [14
]. Taurine plays a vital role in regulating the physiological functions of fish, including growth promotion, immune response modulation, feeding stimulation, cellular osmoregulation, antioxidant action, and detoxification [15
]. Although most animals can synthesize taurine from methionine and cysteine, some species have a limited capacity to synthesize this sulfur-containing amino acid due to the lack of L-cysteine sulphinate decarboxylase [14
]. In addition, plant-based ingredients are severely deficient in cysteine, methionine, and serine and do not contain taurine, compared to animal-based ingredients [15
]. Thus, diets containing plant protein must provide sufficient taurine to meet the physiological needs of fish for optimal growth, health, and development. It is reported that taurine supplementation can increase growth performance and feed utilization efficiency in various fish fed diets containing high levels of plant proteins, such as Argyrosomus regius
], Diplodus sargus
], Oreochromis niloticus
], Acanthopagrus schlegelii
], and Oncorhynchus mykiss
]. These results suggest the potential for incorporating high levels of SBM into the diets with added taurine.
The spotted knifejaw, Oplegnathus punctatus,
is a carnivorous marine fish with high economic value due to its beautiful appearance and good taste [27
]. In recent years, the aquaculture of Oplegnathus punctatus
has increased due to the continuous expansion of the market demand in China. However, studies on the nutritional requirements of this species are limited. For example, the optimal lipid and protein requirements were estimated to be 10.46–12.83% and 42.92–46.44%, respectively [29
]. The current culture of Oplegnathus punctatus
mainly relies on the commercial diets of other species, for example, Pseudosciaena crocea
. It is incompatible with the development of the emerging aquaculture culture industry. There are currently no studies on alternative protein sources for this species, especially the impact of SBM on the growth, antioxidant capacity, and immunity of juvenile Oplegnathus punctatus
. Therefore, there is an urgent need to develop an effective diet that provides balanced nutrition for Oplegnathus punctatus
2. Materials and Methods
2.1. Experimental Diets
Seven isonitrogen (43% crude protein) and isoenergy (20.00 kJ g−1
) diets were prepared. FM protein was replaced with 0%, 30%, 40%, 50%, 60%, and 70% SBM protein, and 1.2% taurine was added on the basis of 50% SBM (designated as SBM0, SBM30, SBM40, SBM50, SBM60, and SBM50 + T, respectively). The composition and formula of the diets used in the experiment are listed in Table 1
. Table 2
shows the composition of amino acids in the diets. All dry ingredients were mixed thoroughly for 15 min using a mixer. Subsequently, oil (soybean oil and fish oil) and water were added to the dry mixture sequentially, which was mixed again for 15 min. Afterward, the diets (size, 1.5 mm) were obtained by a twin-screw extruder, air-dried in a 45 °C oven overnight, and then stored in a refrigerator at −20 °C until use.
2.2. Experimental Fish and Feeding Trial
The fish used in the experiments were provided by China Qingdao Mingbo Aquatic Products Co., Ltd., and the feeding experiment was carried out in the Key Laboratory of Mariculture and Improvement, Zhejiang Institute of Marine Fisheries, Zhoushan, China. Before starting the feeding experiments, fish were fed commercial diets for 20 days to adapt to the laboratory environment. After that, 21 circular tanks filled with 300 L of seawater were stocked with 18 fish (average initial body weight, 14.62 ± 0.02 g per). Fish were fed twice (8:30 and 16:30) per day for 8 weeks. During feeding, a root blower was used for uninterrupted aeration to guarantee that dissolved oxygen surpassed 6 mg L−1. The water temperature was maintained at 28.7 ± 1.4 °C, and the ammonia nitrogen concentration did not exceed 0.05 mg L−1. The salinity and pH value of the water were 24 ± 0.8 g L−1 and 7.5 ± 0.1 respectively. Experiments were performed under a natural photoperiod, and each tank’s lighting was kept consistent.
2.3. Sample Collection and Analysis
Before the feeding test, 18 fish were randomly chosen as initial samples and were frozen for the subsequent whole-body composition analysis. At the end of the culture experiment, all fish were starved for 24 h before sampling, and the total number and weight of fish in each tank were subsequently recorded. Three fish were randomly sampled from each tank for whole-body composition and total energy analyses. Blood samples were collected from the caudal vein with a hypodermic syringe and centrifuged at 4000× g (4 °C) for 10 min (CT15RE centrifuge, Hitachi, Japan). The viscera, intraperitoneal fat, liver, and intestine were extracted and weighed. All of these samples were processed using liquid nitrogen and then stored at −80 °C.
According to AOAC (1995), the analysis of the whole body and the tissues and the composition of the feed were determined. Moisture was detected by weight removal using a drying oven (diet, 105 °C) or a lyophilizer (LL1500, Thermo Scientific, Waltham, MA, USA) (fish and tissue, −110 °C). The crude protein of the sample was analyzed by an Auto Kjeldahl System (K355/K437, Buchi, Flawil, Switzerland) according to the Kjeldahl method. The ash content was determined in a muffle furnace at 550 °C for 12 h. Crude lipids were determined by ether extraction using a Soxhlet apparatus (E816, Buchi, Flawil, Switzerland). With the use of a calorimeter (HWR-15E, Shangli, Shanghai, China), the total energy of the diet and that of the whole body were assessed. To test the amino acid composition of the samples, the amino acid samples were sent to a professional laboratory for measurement using an automatic analyzer (L-8900, HITACHI, Tokyo, Japan).
The triglyceride (TG) and total cholesterol (T-CHO) contents in serum were evaluated through the method provided by [31
]. The activities of lipase (LPS) and amylase (AMS) in the intestinal tract were determined, as described by [32
]. Catalase (CAT) was analyzed using the methods provided by [33
]. Superoxide dismutase (SOD) activity was measured according to the method provided by [34
]. The concentration of malondialdehyde (MDA) was determined by the method provided by [35
]. All parameters above were measured by commercial test kits (Nanjing Jianchen Bioengineering Institute, Nanjing, China) and a microplate reader (Multiskan Go, Thermo Scientific, Waltham, MA, USA).
2.4. Gene Expression
Total RNA from liver was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and quantified using 1.0% agarose electrophoresis. Subsequently, according to the manufacturer’s scheme, 1 μg of RNA was used for cDNA synthesis using a PrimeScriptTM
RT Reagent kit (perfect Real Time; Takara, Dalian, China). The relative expressions of interleukin-8 (il-8
) and transforming growth factor β1 (tgf-β1
) genes were determined by a Real-Time PCR System (QuantStudioTM
6 Flex, Life Technologies, Carlsbad, CA, USA) according to [36
]. The β-atin
gene was chosen as an internal reference. The specific primers used in this study are shown in Table 3
. Relative expression levels were calculated based on the 2−△△CT
equation according to [37
]. Each treatment was performed in triplicate and was repeated three times per sample.
2.5. Calculation and Statistical Analysis
Weight gain (%) = 100 × (final body weight − initial body weight)/initial body weight.
Specific growth rate = 100 × (Ln (final body weight) − Ln (initial body weight))/days.
Feed efficiency = wet weight gain/dry feed consumed.
Protein efficiency ratio = wet weight gain/protein intake.
Daily feed intake = 100 × feed offered/average total weight/days.
Viscerosomatic index = 100 × (viscera weight/whole body weight).
Intraperitoneal fat ratio = 100 × (intraperitoneal fat weight/whole body weight).
Hepatosomatic index = 100 × (hepatosomatic weight/whole body weight).
Condition factor = 100 × (live weight/length3).
Average body weight (ABW) = (initial body weight + final body weight)/2.
Daily nitrogen intake = feed intake nitrogen/ABW × days.
Daily nitrogen gain = (final body weight × final body nitrogen −initial body weight × initial body nitrogen)/ABW × days.
Nitrogen retention = 100 × daily nitrogen gain/daily nitrogen intake.
Daily energy intake = feed intake energy/ABW × days.
Daily energy gain = (final body weight × final body energy − initial body weight × initial body energy)/ABW × days.
Energy retention = 100 × daily nitrogen gain/daily nitrogen intake.
Daily lipid intake = feed intake lipid/ABW × days.
Daily lipid gain = (final body weight × final body lipid −initial body weight × initial body lipid)/ABW × days.
Lipid retention = 100 × daily lipid gain/daily lipid intake.
Data were expressed as an average (n = 3) and analyzed statistically using SPSS 24.0 (IBM, Chicago, IL, USA). After Levene’s test was used to analyze the homogeneity of each treatment, one-way analysis of variance (ANOVA) was used to determine the treatment effect, and Duncan’s multi-range test was used to determine the treatment deviation. Linear and quadratic regression models were constructed to test the correlation between results and dietary SBM substitution FM levels. The statistical significance level was 5% (p < 0.05). Based on WG and FE, a polyline model was applied to determine the maximum substitution level of SBM for FM.
After an eight week feeding trial, the growth performance and feed utilization of Oplegnathus punctatus
were significantly affected by different dietary treatments. The highest WG values were observed in the SBM0 (264.26 ± 49.45%) and SBM30 (268.57 ± 24.37%) groups. Although not statistically different, the WG values of the SBM0 and SBM30 groups were numerically higher than those of the SBM40 and SBM50 groups and were significantly higher than those of the SBM60 and SBM70 groups. Similar results were also found in other fish species such as Takifugu rubripes
], Hemibagrus wyckioides
], and Channa striata
], with a maximum replacement level of 30%. In addition, the highest FE value occurred in the SBM30 group, which was significantly higher than that in the SBM40 and SBM50 groups. According to the broken line regression analysis of WG and FE, the optimal SBM replacement FM level for spotted knifejaw ranged from 24.07% to 25.31%. These values are similar to those reported for other fish species, such as 24% for juvenile Paralichthys olivaceus
], 25% for Oreochromis niloticus
], and 25% for Rhabdosargus sarba
]. Notably, the SGR and WG values of the SBM50 + T group (with 1.2% taurine added) were significantly higher than those of the corresponding SBM50 group but were not statistically different from those in the SBM30 group. This indicated that FM replacement levels could reach 50% using SBM with 1.2% taurine added to the diet without negatively affecting the growth performance of Oplegnathus punctatus
. The beneficial influences of dietary taurine on enhancing the efficacy of plant proteins have also been reported for other carnivorous fish such as Dicentrarchus labrax
] and Channa striata
When using high levels of SBM instead of FM in the diet, the decrease in feed intake (FI) caused by poor palatability was probably one of the key factors for the decline in the growth performance in some fish species. In this investigation, the FI values of all groups were not significantly different. Consistent with this, there were no significant differences in DNI values (from 1.52 to 1.79) among fish groups treated with increasing dietary SBM. In aquaculture practice, it was observed that the species actively feed on algae attached to the cage. Therefore, it can be concluded that palatability should not be a major factor in the decline of the growth performance of Oplegnathus punctatus
or can be ignored. Similar results were also found in studies on N. miichthioides
] and Lutjanus campechanus
SBM ingredients commonly contain various anti-nutritional factors (ANFS), such as protease inhibitors, phytate, saponins, lectins, and oligosaccharides. In the industrial production of SBM, the activity of some ANFs in soybean can be reduced by heat treatment, such as trypsin inhibitors and other heat-sensitive ANFs [48
]. However, this treatment is basically ineffective in destroying NSPs, lectins, saponins, and phytic acid [49
]. In fact, the main antigenic components existing in SBM, such as glycinin and β-conglycinin, may be mainly responsible for inducing abnormal intestinal structural changes and activating the immune system, leading to harmful inflammatory reactions [51
]. In Atlantic salmon, numerous studies reported that plant feedstuffs can result in changes in intestinal histomorphology and gene expression related to immune responses within four weeks [54
]. In the present study, after eight-week culture experiments, no symptoms of intestinal inflammation were observed histologically in all groups (data not provided). However, it was found that the mRNA levels of the pro-inflammatory cytokine interleukin-8 (il-8
) were higher in the livers of fish fed high replacement levels of SBM (SBM40 to SBM70) compared with the SBM0 and SBM30 groups, and the expression of the anti-inflammatory cytokine transforming growth factor β1(tgf-β1
) was suppressed. il-8
is an important pro-inflammatory cytokine that recruits and activates macrophages and neutrophils to clear cellular debris and invading microorganisms and promotes the regeneration of damaged tissues [58
]. Meanwhile, tgf-β
acts as an anti-inflammatory cytokine, counteracting the production of pro-inflammatory cytokines and limiting the inflammatory response [59
]. Significantly, the mRNA level of the pro-inflammatory cytokine il-8
in the SBM50 + T group was lower than that in the SBM40 to SBM70 groups, which was equivalent to that in the SBM0 and SBM30 groups, whereas the expression of the anti-inflammatory cytokine tgf-β1
was the opposite. Researchers [60
] have demonstrated that the primary role of taurine is to protect and maintain the homeostasis of cells involved in acute and chronic inflammation. Therefore, in this study, the supplementation of taurine in the SBM50 + T group may improve the health status of Oplegnathus punctatus
. However, [61
] found that changes in gene expression associated with immune responses were detected on the third day of a plant-based diet, which was much earlier than the signs of inflammation in histological assessment. Since different species have different levels of susceptibility to ANFs, the effect of supplementing taurine in SBM instead of FM on the digestive tract structure of Oplegnathus punctatus
needs long-term evaluation.
In this study, the content of most amino acids in whole body remained unchanged except proline, which is consistent with the fact that the composition of essential amino acids in the whole body is relatively stable and is hardly affected by fish size or dietary composition, as the biosynthesis of body protein is genetically determined [62
]. It is well known that the essence of amino acid balance is that the ratio of each amino acid constituting a protein should be appropriate. If one amino acid is deficient, it will adversely affect the synthesis of intact protein molecules and will affect the availability of other amino acids [64
]. The retention of protein and essential amino acids is considered to be the most sensitive indicator of insufficient amino acid supply [65
]. In the current research, the methionine content (1.77% to 1.41%) of the experimental diets gradually decreased with increasing levels of dietary SBM, which significantly affected the retention of essential amino acids. The retention of most amino acids was significantly higher in the SBM30 group than in the SBM50, SBM60, and SBM70 groups, except lysine. This was also confirmed by higher DNG and NR values in the SBM30 group compared to the SBM40 to SBM70 groups. These results suggest that methionine deficiency affected the utilization of other amino acids in the high SBM groups (SBM50 to SBM70). In these groups, due to the lack of methionine, there was a relative surplus of other amino acids, which were used for oxidative breakdown rather than synthesis, ultimately inhibiting growth. Therefore, the imbalance of amino acids in the diet is probably the key factor for the decreased growth performance of Oplegnathus punctatus
in the high SBM groups. A similar situation was observed in Pseudobagrus ussuriensis
], Epinephelus fuscoguttatus
], and Gadus morhua L.
] fed soybean meal-based diets. Interestingly, although not statistically different from the SBM50 group, the retention values of most essential amino acids were higher in the SBM50 + T group than in the SBM50 group. Moreover, the NR and SGR values of the SBM50 + T group were significantly higher than those of the SBM50 group. When taurine is insufficient in the diet, part of methionine may be converted into taurine to meet the physiological needs of fish, thus aggravating the lack of methionine and adversely affecting growth. After taurine addition, methionine is saved to a certain extent. More importantly, the saved methionine will be used for growth [15
The inclusion of SBM in the diet may have a negative impact on the digestibility of fish, for example, adding SBM to the diet decreased lipid deposition, digestible protein, and digestible energy in Salmo salar
]. In this study, although DNI and DEI were not significantly different in all treatment groups, DEG, DNG, DLG, ER, NR, and LR decreased notably with increasing levels of SBM. A similar situation was observed in Liza H.
]. In addition, the amylase (AMS) activity of the SBM0 and SBM30 groups was significantly higher than that of the SBM40 to SBM70 groups, which may partly be responsible for the decreased dietary digestibility after SBM replaced FM. Triglycerides (TGs) and total cholesterol (T-CHO) are mainly synthesized in the liver, and their changes reflect the lipid metabolism in body to a certain extent. Increased levels of TG and T-CHO indicate that the endogenous fat transport is active [75
]. Several studies have found that nonstarch polysaccharides (NSP) can reduce plasma and liver T-CHO levels in fish, possibly because they interfere with fat digestion and absorption [49
]. In this study, the levels of TG and T-CHO in the high SBM groups were lower than those in the low SBM and control groups. Therefore, the significant reduction in the whole body and muscle lipid content in the high replacement groups may be related to NSP. A similar result was seen for redlip mullet Liza haematocheila
]. Interestingly, the T-CHO and TG values of the SBM50 + T group were higher than those of the SBM50 group. Researchers [78
] showed that taurine has a beneficial effect on fat metabolism. However, there were no significant differences in the whole fish and muscle lipid content between the SBM50 + T and SBM50 groups. The specific reason is not clear, so the effect of taurine on lipid metabolism in Oplegnathus punctatus
needs further evaluation.
Antioxidant enzymes (such as SOD and CAT) in the antioxidant defense system can effectively remove excess reactive oxygen species and protect cells from oxidative damage [79
]. The plant protein in the diet may have a harmful influence on the antioxidant status of fish. For example, when feeding a diet containing high levels of SBM, the antioxidant capacity of Monopterus albus
decreased significantly [80
]. SOD activity was not significantly different among the groups in the study, but the SBM70 treatment group had the lowest SOD activity. As the SBM content in the diet increased, CAT activity decreased significantly. The results were similar to those of Ctenopharyngodon I.
]. In addition, malondialdehyde (MDA) produced by endogenous oxidative damage in vivo is one of the final metabolites of lipid peroxidation, which can reflect the extent of lipid peroxidation and the extent of cell injury [82
]. Our study found that the supply of high levels of SBM (SBM50 to SBM70) significantly increased the concentration of MDA in the liver of Oplegnathus punctatus
, indicating that the health status of Oplegnathus punctatus
could be affected by using SBM instead of FM. Many studies showed that taurine can have beneficial effects in diets containing plant proteins. For example, supplementation of taurine in plant protein diets significantly improved the growth performance as well as the feed conversion ratio of Diplodus sargus
]. In Totoaba macdonaldi
, supplementation of 1.2% taurine in a diet that replaced 60% FM with soybean protein concentrate restored the level of lipid peroxidation and increased the activities of catalase and the key enzymes of intermediate metabolism to normal levels [83
]. According to current results, taurine supplementation significantly improved the growth and feed efficiency of Oplegnathus punctatus
. Compared with the SBM50 group, the SOD and CAT enzyme activities were higher in the SBM50 + T group, while the MDA level was lower. Importantly, taurine has a wide range of biological effects, including membrane stability, neurotransmitter regulation, and antioxidant effects, especially in osmotic pressure regulation and hormone release [15
]. The effects of taurine added to a high-level SBM diet on the growth rate, feed efficiency, and antioxidant stress of Oplegnathus punctatus
should be studied further. These findings can significantly change the quantities and types of substitute proteins that could be effectively added to the diets of juvenile Oplegnathus punctatus
and could reduce the dependency of the industry on FM supplies.