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Effects of dietary dl-methionyl-dl-methionine (Met-Met) on growth performance, body composition and haematological parameters of white shrimp (Litopenaeus vannamei) fed with plant protein–based diets

Published on: 12/3/2019
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A 10-week feeding trial was conducted to evaluate the effects of supplementing dif- ferent levels of dl-methionyl-dl-methionine (AQUAVI® Met-Met) in plant protein– based diets on Litopenaeus vannamei. The positive control (PC) and negative control (NC) diets were designed with 20% and 8% fishmeal respectively, and other six diets were formulated with graded levels of Met-Met from 0.05% to 0.30% with a 0.05% increment on the basis of NC diet (MM 0.05–MM 0.3). Six replicates were randomly assigned to each diet with 50 shrimp each having initial weight of 0.98 ± 0.02 g. The variation of FM concentration from 20% to 8% and supplemented with graded levels of Met-Met did not affect the survival rate, feed conversion ratio, protein efficiency ratio, whole body and muscle proximate compositions (p > 0.05). However, diets with ≤0.20% Met-Met supplementation resulted in significantly increased weight gain and specific growth rate, after which both parameters reached plateau. Shrimp fed the NC diet showed significantly lower total essential amino acid (EAA) content in muscle (p < 0.05). Supplementation of Met-Met significantly improved apparent digestibility coefficients of dry matter, crude protein, lipid, phosphorus and EAAs (p < 0.05). Based on broken-line analysis, the methionine requirement for white shrimp was estimated to be 0.87% when using Met-Met as methionine source.

KEYWORDS fishmeal reduction, growth performance, Litopenaeus vannamei, Met-Met, plant protein


White shrimp (Litopenaeus vannamei) is an important economic shrimp species cultured worldwide and has unique advantages in intensive culture system, such as rapid growth rate, and tolerance to a wide range of salinity and temperature (Xia & Wu, 2018; Zhou, Wang, Wang, & Tan, 2013). Fishmeal (FM) is commonly used as protein source for white shrimp. It has palatable taste and is an excellent source of nutrients because of its high protein level, favourable amino acid profile, essential fatty acid and unidentified growth factors which can meet the requirements of shrimp. Normally, 25%–50% FM are used in commercial feed formulations for shrimp, representing the most expensive part in the ingredients (Hernandez, Sarmientopardo, Gonzalez-Rodriguez, & Iadela, 2015; Samocha, Davis, Saoud, & Debault, 2004; Suárez et al., 2009). However, with the stronger global demand and limited FM production, the price of FM will keep increasing. FM will be widely used at lower levels as a strategic ingredient in the future (FAO, 2009).

Many studies have been conducted in terms of replacing FM with alternative protein sources such as poultry by-product, meat bone meal, brewer's yeast, soybean products and peanut meal (PM). Plant sources hold great promise due to their lower price, worldwide and sustainable production. However, these plant protein sources are not very suitable for aquaculture due to the existence of antinutrients and unfavourable essential amino acid (EAA) profile. Reducing antinutritional factors and supplementing amino acids to improve the nutritional profile are effective ways to achieve FM replacement by plant protein (FAO, 2009; Tacon, 1995; Tacon, Hasan, & Subasinghe, 2006). In shrimp feed, PM and soybean meal (SBM) are mostly used, while methionine is thought to be the first limiting amino acid (Gu, Zhang, Bai, Mai, & Xu, 2013). Methionine supplementation in Met-limited SBM diets was demonstrated as an efficient strategy for replacing FM for hybrid tilapia (Oreochromis niloticus × Oreochromis mossambicus) (2015, & Kiriratnikom, 22015), Japanese seabass (Lateolabrax ja- ponicus) (Zhang et al., 2016), red sea bream (Pagrus major) (Takagi, Shimeno, Hosokawa, & Ukawa, 2001), large yellow croaker (Pseudosciaena crocea R.) (Mai et al., 2006) and white shrimp (Xie et al., 2017).

Different forms of methionine are available in the market such as l-methionine (l-Met), dl-methionine (dl-Met), dl-2-hydroxy-4- methylthiobutanoic acid or methionine hydroxyl analogue calcium salt. The bioavailabilities of these free-form crystalline methionine sources were estimated by different studies (Niu et al., 2017; Vázquezañón et al., 2006; Vedenov & Pesti, 2010). Compared with crystalline methionine, methionine peptides may process a specific transport mechanism in the intestine resulting in faster and more efficient absorption rates (Adibi, 1997; Li, Zhao, Yang, Johnson, & Thacker, 1999; Mamauag et al., 2012; Zambonino Infante, Cahu, & Peres, 1997). A novel dipeptide dl-methionyl-dl-methionine (“Met-Met”, branded as AQUAVI® Met-Met) has special physical and chemical characteristics compared with other free-form crystalline methionine sources selected by Evonik's Aqua R&D Group. AQUAVI® Met-Met is the mixture of four different methionine stereoisomers (dl-Met-Met, ld-Met-Met, dd-Met-Met and ll-Met- Met) which could be efficiently cleaved by digestive enzymes to free d- and l-methionine, and the higher bioavailability value than dl-Met has already been demonstrated in white shrimp (Façanha, Oliveira-Neto, Figueiredo-Silva, & Nunes, 2016; Niu et al., 2017; Xie et al., 2017). As shrimp are benthos feeders with slow feeding behaviour, part of supplemented methionine could be lost by leaching before ingestion (Gu et al., 2013). However, Met-Met was demonstrated to have lower leaching loss in water than dl-Met (Xie et al., 2017).

This study aimed to evaluate the effects of supplementing graded levels of Met-Met on growth performance, feed utilization, body composition and haematological parameters of white shrimp fed with plant protein–based diets.


2.1 | Experimental diets

In this experiment, FM, SBM, PM and brewer's yeast were used as the main protein sources. Eight iso-energetic (17.84 kJ/g) and iso-nitrogenous (387 g/kg crude protein) diets were designed according to the nutritional requirements of white shrimp (Table 1). The positive control (PC) diet was formulated with 20% FM level, whereas the negative control (NC) diet had 8% FM level. Met-Met was supplemented to the NC diet from 0.05% to 0.30% with a 0.05% increment and named as MM 0.05–MM 0.3. Iso-nitrogenous and iso-energetic diets were made by adjusting the non-EAAs (NEAAs) glycine and aspartic acid (1:1). The actual methionine levels of the eight diets were 0.60%, 0.54%, 0.59%, 0.67%, 0.70%, 0.80%, 0.84% and 0.95% respectively. Essential amino acid composition of the experimental diets is presented in Table 2. All dry ingredients were finely ground through a 178-μm mesh, weighed and thoroughly mixed. 0.1% Y2O3 was mixed with the mash as the indicator for apparent digestibility. Fish oil, soy oil, soybean lecithin and VC-phosphate were added slowly and thoroughly mixed by hand. The dough was further homogenized and extruded through a 1.2 mm moudle of pelletizer (Modle HKJ-218; HUARUI, Wuxi, China). The pellets were cooked by steaming for 10 min and then dried at 24? for 72 hr using an air conditioner and electric fan, then collected in bags and stored in a refrigerator at −20?. The sample of each diet was taken for proximate analysis.

2.2 | Feeding trial

White shrimp were obtained from the Marine Fisheries Research Institute of Zhejiang Province in Zhoushan, China. The feeding trial was conducted in the Xixuan Fishery Science and Technology Island in Zhoushan, China. Before the feeding trial, all shrimp were acclimated to the experimental conditions and fed with a commercial feed for 2 weeks. After 24 hr of starvation, 2,400 healthy white shrimp (initial body weight: 0.98 ± 0.02 g) were randomly distributed into 48 fibreglass tanks (350 L water volume, blue cylindrical tank). The tanks were equipped with a flowing system. Seawater was obtained from the nearby seashore of the island, and pumped into each tank after sediment and sand filtration. One-third of the water was replaced daily. Each treatment randomly had six replicates with 50 shrimp in each tank. Shrimp were reared under natural photoperiod. The temperature, ammonia-nitrogen, and salinity of the seawater in tanks were 28 ± 1?, 0.02–0.06 mg/L and 28 ± 1 g/L respectively. pH was 7.6–7.8 and dissolved oxygen concentrations were maintained above 5.0 mg/L during the entire experimental period by using air stones with continuous aeration. The shrimp were fed experimental diets to apparent satiation three times daily (06:00, 12:00 and 18:00 hours) for 10 weeks. Residual feed and faeces were thoroughly cleaned 3 hr after the final feeding of the day.

2.3 |   Sampling

Before the feeding trial, 30 shrimp were sampled for whole body composition analysis. At the termination of the 10-week experiment, all shrimp were anaesthetized (MS-222 60 mg/L; Sigma, St Louis, MO) after a 24 hr fasting, then counted and weighed individually. Five shrimp from each tank were stored at −20? for subsequent whole body proximate composition analysis. Blood samples were drawn from the heart of 30 shrimp of each tank with a 1 ml syringe, then stored at 4? for 2 hr. The blood samples were centrifuged at 10 000 g for 15 min to obtain serum, then stored at −20? for chemical and enzyme analyses. The remaining shrimp were dissected and hepatopancreas were taken out carefully and weighed. Muscle sample was obtained from the shelled shrimp and stored at −20? for muscle composition analysis. From week 6, faeces were collected using the method described by Zhou, Xiao, Hua, Ngandzali, and Shao (2011). Faeces of each tank were sampled at 06:00 hours every morning before the next feeding, using a faecal collection column similar to the one described by Cho and Kaushik (1990) and the collected faeces were stored at −20? for further analysis.

2.4 | Chemical analysis

Chemical analysis was conducted following the standard laboratory procedures of AOAC (1995). Moisture concentration was determined by drying minced samples at 105? for 24 hr in a forced air oven. Ash content was analysed by incinerating the sample at 600? for 4 hr in a muffle furnace. Crude protein was determined as Kjeldahl-ni- nitrogen using boric acid to trap released ammonia. Lipid concentration was estimated by soxhlet extraction with petroleum ether. The content of Y2O3 in the diets and faeces was determined by inductively coupled plasma atomic emission spectrometer. Biochemical parameters in serum were measured using a diagnostic reagent kit purchased from Nanjing Jiancheng Bioengineering Institute according to the manufacturer's instructions. Biochemical measurements were conducted for albumin (ALB), glucose (GLU), total protein (TP) and total cholesterol (TC). The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined at 505 nm against distilled water. Phenoloxidase (PO) activity was measured according to the method described by Zhou et al. (2013). The amino acid compositions of experimental diets (Table 2), faeces (Table 5) and muscle (Table 6) were analysed following acid hydrolysis using an automatic amino acid analyser (Hitachi 835-50, Tokyo, Japan) with a column (Hitachi custom ion exchange resin no. 2619) in the Amino Lab of Evonik Degussa (China).

2.5 | Calculations and statistical analysis

Growth performance and feed utilization were expressed as follows:

Specific growth rate ( SGR,%per day) =100×(ln final body weight−ln initial body weight) /days

Weight gain ( WG,%) =100×(final body weight−initial body weight) /initial body weight

Condition factor ( CF,g/cm3 ) =final body weight/final body length3 ×100

Hepatosomatic index ( HSI) =100×(liver weight/body weight)

Survival rate ( SR,%) =100×(final shrimp number/initial shrimp number)

Feeding rate ( FR, %BW/d ) =dry weight of feed×100/ [ (initial body weight+final body weight) /2 ] /culture period

Feed conversion ratio ( FCR) =dry diet fed/weight gain

Protein efficiency ratio ( PER) =weight gain/protein intake

Protein productive value ( PPV,%) =tissue protein deposition/protein intake×100

Apparent digestibility coefficients of dry matter ( ADC,%) =100×[1− ( dietary Y2O3)/faecal Y2O3 ]

The results were presented as means ± SD. All data were tested for normality and homogeneity of variances by Kolmogorov– Smirnov and Levene's tests respectively. Four uniform growth data of each treatment were selected for broken-line analysis following the method described by Robbins, Norton, and Baker (1979). The data were analysed by one-way ANOVA followed by Ducan's test using spss 20.0 (SPSS Inc., Chicago, IL). The level of significance was set at p < 0.05.


Growth performance parameters and morphological indices of white shrimp fed different levels of Met-Met are presented in Table 3. In the present study, survival rate (SR) and condition factor (CF) were unaffected by dietary treatments after 10 weeks' feeding trial. There was no significant difference in weight gain (WG) and specific growth rate (SGR) in NC and MM 0.05 diets ( p > 0.05), but the WG and SGR of shrimp were significantly increased at ≤0.2% Met-Met and then plateaued, with shrimp fed the PC diet obtaining the highest value (p < 0.05). Broken-line analysis between dietary methionine level and WG were y = 634.96 + 31.28 x ( R 2 = 0.79) and y = 894.13 + 1.56 x ( R 2 = 0.85) (Figure 1). The optimum dietary methionine level based on WG was estimated to be 0.87% of the dry diet (2.03% of the dietary protein). A significantly higher hepatosomatic index (HSI) was obtained in shrimp fed the MM 0.25 diet ( p < 0.05).The supplementation of different levels of Met-Met in plant protein–based diets did not affect feeding rate (FR), feed conversion ratio (FCR), protein efficiency ratio (PER) and protein productive value (PPV) ( p > 0.05).

The moisture, crude protein, crude lipid and ash contents in whole body and muscle are presented in Table 4. The whole body and muscle composition data showed no significant difference among all dietary treatments ( p > 0.05).The moisture, crude protein, ash and crude lipid contents in whole body were 75.60%–76.28%, 16.75%–16.99%, 3.07%–3.23% and 2.59%–3.03% respectively.

The moisture, crude protein, ash and crude lipid contents in muscle were 74.71%–75.74%, 20.94%–21.57%, 1.40%–1.54% and 2.11%–2.78% respectively. The apparent digestibility of juvenile white shrimp among all dietary treatments is shown in Table 5. In this study, shrimp fed the NC diet had the lowest ADC of dry matter and crude protein ( p < 0.05) and other treatments had no significant differences between them ( p > 0.05). The ADC of crude lipid and phosphorus increased with increasing Met-Met levels and the highest values were obtained in shrimp fed the MM 0.3 diet ( p < 0.05).The ADC of EAAs increased with increasing Met-Met levels and shrimp fed the NC diet had the lowest value among all treatments ( p < 0.05).

The highest ADC values of methionine, valine, leu - cine, phenylalanine and histidine were obtained in shrimp fed the MM 0.3 diet, which were higher than that of the PC diet ( p < 0.05). The muscle EAA contents of juvenile white shrimp among all dietary treatments are shown in Table 6. Dietary treatments did not affect valine, leucine, histidine and arginine contents in muscle (p > 0.05). Methionine content in muscle of shrimp fed the NC diet was significantly lower than that of MM 0.1–0.25 diets ( p < 0.05). Isoleucine and tryptophan levels exhibited a trend similar to that of methionine levels. Shrimp fed MM 0.1 and MM 0.15 diets had the highest threonine and lysine levels in muscle, whereas shrimp fed the NC diet had the lowest content (p < 0.05), and the remaining treatments showed no significant difference (p > 0.05). Phenylalanine level in shrimp fed the NC diet was significantly lower than that of the MM 0.15 diet (p < 0.05). Shrimp fed the NC diet showed significantly lower total EAA content in muscle than shrimp fed the MM 0.05–0.15 diets (p < 0.05), but without significant differences be- tween the other treatments (p > 0.05).

The haematological parameters of white shrimp among all dietary treatments are presented in Table 7. Serum TP content of shrimp fed the MM 0.3 diet was significantly higher than that of shrimp fed the NC and MM 0.05 diets (p < 0.05). However, ALB content of shrimp fed the NC diet was significantly higher than that of shrimp fed the MM 0.3 diet (p < 0.05). The activities of serum AST, ALT and PO, as well as contents of serum GLU and TC were not significantly influenced by different dietary Met-Met treatments (p > 0.05).


The main objective of this study was to evaluate the effects of graded levels of dietary Met-Met in plant protein–based diets on white shrimp. According to the obtained results, the high level of dietary plant protein significantly inhibited the growth performance of white shrimp. This might possibly be due to low digestibility, reduction in palatability, feed intake and imbalanced amino acid of plant protein–based diets (Amaya, Davis, & Rouse, 2007; Hernández, Olvera-Novoa, Aguilar-Vejar, González-Rodríguez, & Parra, 2008). Methionine is known to be the first limiting amino acid in SBM-based diet (Baker, 2006; Figueiredo-Silva et al., 2015). In rainbow trout (Oncorhynchus mykiss), dietary methionine inclusion increased feed intake due to improvement of palatability in FM reduction diets (Mambrini, Roem, Carvã¨Di, Lallã¨S, & Kaushik, 1999). Improved FCR was also obtained in red seabream (Pagrus major) fed supplemented dl-Met or Met-Met in diets containing 25% soy protein isolate (Mamauag et al., 2012). In the present study, FR and FCR were not influenced among dietary treatments. Therefore, imbalanced amino acid, especially methionine, and lower digestibility might be the main factors that inhibited growth in the NC diet. Besides, this study also demonstrated that FM can be considerably replaced by plant protein by supplementing Met- Met in white shrimp diet. Methionine is required for normal growth and works as a precursor of many body components. Low dietary methionine content induced decreased growth performance of shrimp (Mamauag et al., 2012). Supplementing methionine in plant protein–based diets improved growth performance in many studies among different species (Browdy, Bharadwaj, Venero, & Nunes, 2012; Façanha et al., 2016; Mamauag et al., 2012; Zhang et al., 2016).The SRs were not significantly influenced by different treatments; this result is in accordance with the study on dietary threonine requirement of white shrimp (Mingyan et al., 2009). However, the values were not high enough (around 80%), but no disease was found during the feeding trial. That might be because the shrimp were small (initial weight: 0.98 ± 0.02 g) and had low ability to adapt to high-density culture and the change of feed. Huai et al. (2010) reported that high inclusion of synthetic amino acids can enhance the stress tolerance of shrimp and improve SR, which was not found in the present study. Many studies have investigated dietary methionine requirement of white shrimp. Lin et al. (2015) estimated the methionine requirement of white shrimp based on different size and the methionine requirement ranged from 0.91% (initial body weight: 0.55 g) to 0.66% (initial body weight: 9.77 g) of the diet. Niu et al. (2017) found the optimal dietary methionine level for best growth performance ranged from 0.71% to 0.81% (1.92%–2.19% of dietary crude protein), whereas Fox et al. (2010) estimated that the dietary methionine requirement was 0.74% (3.70% of dietary crude protein). In the present study, dietary methionine requirement was estimated to be 0.87% of the dry diet (2.03% of dietary crude protein) when using Met-Met as methionine source, which is similar to previous reports. Some studies have demonstrated that crystalline amino acids and protein-bound amino acids had similar bio-efficiency value (Hu et al., 2008; Zarate, Lovell, & Payne, 2015). Furthermore, Met-Met has a higher absorption rate than protein-bound methionine and has a higher bioavailability value than dl-Met (Niu et al., 2017; Xie et al., 2017), so the supplementation of Met-Met in plant-protein based diets could effectively improve the growth performance of shrimp. Highest SGR and WG were obtained in shrimp fed the PC diet, which was significantly higher than shrimp fed the MM 0.3 diet. In contrast, a study found that diet supplemented with 0.1% Met-Met in the 10% FM diet showed similar growth performance as the 26% FM diet in white shrimp (Xie et al., 2017). A possible reason could be the lower FM level (8%) in the NC diet in the present study. Normally, 12% FM is considered as the minimum amount to ensure suitable content of amino acids and other nutrients for normal growth and flesh quality in aquatic feed (Suárez et al., 2009). However Fox, Lawrence, and Smith (2004) did not find compromising growth performance, when white shrimp were fed diets containing 30%–35% crude protein with 7.5%–12.5% FM.

Met-Met levels higher than 0.2% did not further improve growth performance, and growth inhibition caused by excessive methionine was also not observed in this study. In the high methionine diet, some methionine is rapidly absorbed into plasma and some may not be absorbed by the tissues because the cells might have reached the plateau for protein synthesis. Consequently, methionine might be used as an energy source by donating its carbon chain during the oxidation process that eventually is related to a poor growth rate (Nunes, Sá, Browdy, & Vazquez-Anon, 2014; Tesser, Terjesen, Zhang, Portella, & Dabrowski, 2005). Fox et al. (2010) found reduced growth performance in white shrimp fed diets containing covalent methionine levels above 0.62%. Diets supplemented with 0.3% Met-Met also induced growth reduction in white shrimp (Niu et al., 2017). Excess of methionine also negatively influenced feed intake (NRC, 2011). In Atlantic salmon (Salmo salar), excessive dietary methionine reduced SR, growth performance and feed intake due to its toxic effect (Marit, Ernstm, Bjørn, Andreas, & Adel, 2008). In the present study, the MM 0.2–MM 0.3 diets contained 0.88%– 1.05% methionine, which were higher than the dietary methionine requirement level (0.87% dry diet), but may not reach the maximum level of methionine absorption. Thus higher levels of Met-Met supplementation with suitable FM level could result in a better growth performance. The PPV ranged from 27.48 to 30.08 and were not significantly influenced by dietary treatments. The low PPV can be attributed to part of dietary protein being used for providing energy rather than growth during the 10-week feeding trial. Besides, 4% blood meal used in the present study might cause low digestibility and utilization of protein, which has been reported in some fish species (El-Sayed, 1998; Roghayeh & Faghani-Langroudi, 2015), as well as in white shrimp (Liu, Ye, Kong, Wang, & Wang, 2013).

Dietary Met-Met treatments did not affect proximate compositions of whole body and muscle. Experimental diets were designed as iso-ni - trogenous and iso-energetic, thus shrimp received equal protein and energy amount in all diets. This result is in accordance with  Mamauag et al. (2012), as body proximate composition was not affected when red sea bream were fed with different forms of dietary methionine. Similar results were also obtained in protein reduction diets supplemented with methionine and lysine for grass carp (Ctenopharyngodon idella) (Gan et al., 2012), and Met-Met–supplemented diets in white shrimp (Façanha et al., 2016). However, a high plant protein diet supplemented with dl-Met or Met-Met affected the protein content of muscle while whole body moisture, lipid and ash contents were unaffected in white shrimp (Xie et al., 2017). Also, supplementation of crystalline methionine or oligo-methionine in plant protein–enriched diets significantly influenced whole body moisture, protein and lipid levels in white shrimp (Gu et al., 2013). These differences among different studies may be attributed to the differences in experimental design, ingredients, experimental conditions, species, size, age and other factors.

In hepatic tissue, methionine serves as the donor of methyl groups and is related to sulphur metabolism (Mato, Alvarez, Ortiz, & Pajares, 1997). In the present study, shrimp fed the lower Met-Met level diets had relatively lower HSI. Luo et al. (2005) observed a similar trend as juvenile grouper (Epinephelus coioides) fed the lowest methionine diet showed the lowest HSI and CF. This finding is in agreement with our present shrimp's HSI. However, Coloso, Murillo- Gurrea, Borlongan, and Catacutan (1999) observed that dietary methionine levels did not have a significant impact on HSI. Conversely, low methionine resulted in significantly higher HSI in Atlantic salmon (Marit et al., 2008). Gan et al. (2012) concluded that deficiency in amino acid results in excess energy deposition (e.g. fat in the liver, fillet or abdominal cavity), which is contrary to the present result.

Shrimp fed the Met-Met–supplemented diets showed higher total muscle EAA content than shrimp fed the NC diet, which was prob- ably because methionine is the first limiting amino acid in NC diet. Restriction of one EAA in diet causes more rapid oxidation of other EAAs and NEAAs, which inhibits protein synthesis and deposition (Luo et al., 2005; Ozório, Booms, Huisman, & Verreth, 2002; Rønnestad, Conceição, Aragão, & Dinis, 2000). Similar results were also obtained in hybrid tilapia fed SBM-based diets supplemented with different levels of dl-Met (Figueiredo-Silva et al., 2015). However, supplementing with dl-Met or Met-Met in the plant protein–containing diets did not have a significant influence on whole body amino acid contents in larvae and juvenile red sea bream (Mamauag et al., 2012). Methionine can be partially spared by cysteine (Cys). Normally, Cys excess of 3 g/ kg in the diet has no methionine-sparing effect (NRC, 2011; Nunes et al., 2014). All experimental diets in this study provided more than 3 g/kg of Cys, so the methionine-sparing effect was not considered. In the present study, higher level of Met-Met did not result in higher muscle methionine deposition. This result is in accordance with some previous studies which also found an insignificant relation between dietary amino acid level and tissue composition (Façanha et al., 2016; Mingyan et al., 2009; Zhou et al., 2013).

Diets supplemented with Met-Met improved the apparent digestibility of dry matter, crude protein, lipid, phosphorus and EAAs compared with the NC diet. Interestingly, supplementation of 0.3% Met-Met resulted in an even higher apparent digestibility of total phosphorus, methionine, valine, leucine, phenylalanine and histidine than the PC diet. Dietary methionine improved the use of other EAAs mainly by reducing the oxidation rate of other amino acids (Nunes et al., 2014), and the MM 0.3 diet may have better amino acid profile than the PC diet (Table 2). Besides, the increased digestibility may be attributed to enhanced intestinal function by Met-Met supplementation. The activities of intestinal proteolytic enzymes were improved by methionine supplementation in red sea bream (Mamauag et al., 2012) and white shrimp (Xie et al., 2017). Tang et al. (2009) reported that methionine-supplemented diets can improve intestinal growth and enzyme activities, as well as promote the growth of beneficial bacteria and inhibit the growth of harmful bacteria. Furthermore, methionine-supplemented diets can increase liver trans-sulphuration and produce more taurine, which could be conjugated to bile acids and enhance biliary secretion, and eventually improve the digestibility of amino acids (Espe, Liaset, Hevrøy, & El-Mowafi, 2010). In Atlantic salmon, all plant protein–based diets with 0.12%–0.72% crystalline methionine had higher digestibility of amino acids than the FM-based diet (Marit et al., 2008). Improvement in dietary amino acid utilization was also reported in low-protein diets by supplementing EAA in rainbow trout (Yamamoto, Sugita, & Furuita, 2005). Coated methionine also improved the apparent digestibility of amino acids, crude protein and lipid in white shrimp (Chi et al., 2015).

AST and ALT are two important enzymes in amino acid metabolic processes and are usually present within cell membranes, cytoplasm and mitochondria. Generally, the function of shrimp hepatopancreas can be presented as the activities of AST and ALT (Chaplin, Huggins, & Munday, 1967; Zhou et al., 2013). No significant difference was found in these two enzymes among dietary treatments. A similar result was also obtained in the study for assessing the optimum threonine level of white shrimp (Mingyan et al., 2009). Zhou et al. (2013) observed that shrimp fed 2.3% threonine diet had higher AST and ALT values, which indicated that poor shrimp health might be induced by high dietary threonine intake. Among serum constituents, shrimp fed the MM 0.3 diet had the highest TP concentration, whereas the lowest value was observed in shrimp fed the NC and MM 0.05 diets. Some studies in teleosts found that serum protein concentrations were influenced by dietary lysine level (Wang et al., 2005; Zhou, Wu, Chi, & Yang, 2007) and methionine level (Luo et al., 2005). Cholesterol and GLU were variable and not related to dietary treatment, with a similar result also obtained for assessing dietary methionine requirement of juvenile grouper at a constant dietary Cys level (Luo et al., 2005). Besides, blood total cholesterol content was also not significantly influenced in red sea bream fed with Met-Met or dl-Met diets (Mamauag et al., 2012). PO is a copper-containing enzyme with oxygenase activity. In this study, the serum PO activity was not affected by dietary Met-Met treatments, whereas the activity was significantly increased with the increasing threonine level in white shrimp (Wang & Jiang, 2004; Zhou et al., 2013).


In summary, plant protein–based diets supplemented with Met-Met could effectively improve the growth performance of white shrimp. Different levels of Met-Met did not affect of muscle and whole body composition but significantly improved the total EAA content in muscle. Met-Met supplementation also improved the apparent digestibility of dry matter, protein, lipid and phosphorus. High level of Met-Met did not induce growth inhibition, suggesting the possibility of application of a higher level of Met-Met. However, the optimal levels of Met-Met and FM should be explored in the future in order to obtain a better growth performance.


This research was supported and funded by the Project from Evonik Degussa GmbH of Germany (03 86 15004). We thank the China-Norwegian Joint Laboratory of Nutrition and Feed for Marine Fish (Xixuan Island, Zhoushan City, Zhejiang Province), and the Key Lab of Mariculture and Breeding of Zhejiang Province for supplying the experimental rearing system and for providing logistical support during the growth trial. We also thank Evonik Industries AG, Germany for funding and Met-Met® supply for this study. We are grateful to Fangping Yu, Huilai Si, Bai Lv, Shengli Tong and Kalhoro Hameeda for their assistance with husbandry, sampling and other related help.


All data generated or analysed during this study are included in this published article.

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