Open Access

Growth, digestive and absorptive capacity and antioxidant status in intestine and hepatopancreas of sub-adult grass carp Ctenopharyngodonidella fed graded levels of dietary threonine

Journal of Animal Science and Biotechnology20156:34

https://doi.org/10.1186/s40104-015-0032-1

Received: 19 December 2014

Accepted: 2 July 2015

Published: 8 August 2015

Abstract

Background

This study was carried out to investigate effects of threonine levels on growth, digestive and absorptive capacity and antioxidant status in intestine and hepatopancreas of sub-adult grass carp (Ctenopharyngodonidella).

Results

Weight gain, specific growth rate, feed intake and feed efficiency were significantly improved by dietary threonine (P < 0.05). Intestinal activities of trypsin, chymotrypsin, alpha-amylase, lipase, alkaline phosphatase, γ-glutamyl transpeptidase and creatine kinase took the similar trends. Contents of malondialdehyde and protein carbonyl in intestine and hepatopancreas were significantly decreased by dietary optimal threonine supplementation (P < 0.05). Anti-superoxide anion capacity, anti-hydroxyl radical capacity, glutathione content and activities of superoxide dismutase, catalase and glutathione-S-transferase in intestine and hepatopancreas were enhanced by dietary threonine (P < 0.05).

Conclusions

Dietary threonine could improve growth, enhance digestive and absorptive capacity and antioxidant status in intestine and hepatopancreas of sub-adult grass carp. The dietary threonine requirement of sub-adult grass carp (441.9-1,013.4 g) based on weight gain was 11.6 g/kg diet or 41.5 g/kg of dietary protein by quadratic regression analysis.

Keywords

Antioxidant status Grass carp Intestinal enzyme activity Threonine

Background

Threonine (Thr) is an indispensable amino acid for fish [1]. Dietary threonine deficiency has been shown to cause poor growth and feed conversion in juvenile Japanese flounder (Paralichthysolivaceus) [2], as well as low protein deposition in fingerling Indian major carp (Cirrhinusmrigala) [3]. It is well known that fish growth is greatly influenced by food digestion and nutrient absorption [4]. To date, there is only one report regarding the effects of dietary threonine on the digestive and absorptive capacity of fish, which showed that diet threonine improved the activities of trypsin, lipase and alpha-amylase in hepatopancreas and intestine of juvenile Jian carp (Cyprinuscarpio var. Jian), as well as the activities of intestinal enzymes related to absorption, including alkaline phosphatase (AP), γ-glutamyl transpeptidase (γ-GT) and Na+/K+-ATPase [5]. However, the digestive and absorptive capacity of fish varies with its feeding habit [6]. Generally, herbivorous fish have a higher digestive capacity in starch than that of omnivorous and carnivorous species [7]. While relative to omnivorous and carnivorous fish species, the herbivorous fish show a poor digestive capacity in protein and fat [6, 8]. Therefore, effects of dietary threonine on digestive and absorptive capacity may be different among fish with different feeding habits. The present study focused on the effects of threonine on digestive and absorptive capacity of herbivorous grass carp (Ctenopharyngodonidella).

The function of fish digestive organ is correlated with its development [9]. Threonine has been shown to improve intestinal folds height in juvenile Jian carp [5], as well as anterior intestinal villus height and serosa thickness in juvenile grass carp [10]. On the other hand, the growth and function of the digestive organs are usually correlated with its antioxidant status [11]. Our laboratory studies indicated that the function of digestive organs of juvenile Jian carp was positively related to antioxidant status by methionine hydroxy analogue [12]. However, no studies have been conducted to investigate the relationship between threonine and antioxidant status of tissues and organs in fish. Generally, reactive oxygen species (ROS) are produced during normal aerobic cellular metabolism [13]. When ROS generation rate exceeds that of their removal, oxidative stress occurs which may induce deleterious effects on cells, such as lipid peroxidation and protein oxidation [13]. Huang et al. [14] reported that free transition metal ions, such as iron, copper and manganese, could induce the formation of hydroxyl radicals via the Fenton-Haber Weiss reaction in biological systems. Chelating iron ions could reduce the formation of hydroxyl radicals in stomach of rats [15]. Threonine chelated with iron and copper ions in vitro biochemical assays [16, 17] and manganese ions in liver of rats [18]. Thus, threonine might be able to reduce the formation of hydroxyl radicals in living organisms. On the other hand, pig stomach mucins, which were rich in threonine, could scavenge hydroxyl radicals induced by iron ions in vitro biochemical assays [19]. It was found that intestinal mucins of common carp (Cyprinuscarpio L.) were rich in threonine [20]. Based on these data, threonine might be able to improve the function of fish digestive organs by increasing free radical scavenging ability.

In fish, ROS are scavenged by non-enzymatic antioxidants and antioxidant enzymes [21]. Glutathione (GSH) is an important non-enzymatic antioxidant compound of fish [22]. However, no studies have been conducted to investigate the relationship between threonine and GSH content in tissues and organs of fish. In rats, GSH synthesis takes place mainly in the liver, which needs the participation of ATP [23]. Ross-Inta et al. [24] reported that threonine increased liver ATP level in rats. As with other aerobic organisms, fish developed diverse antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx) [25]. To date, information regarding the effect of threonine on activities of antioxidant enzyme is not available in fish. Sidransky and Rechcigl [26] reported that dietary threonine increased CAT activity in liver and kidney of rats. E2 p45-related factor 2 (Nrf2) regulates a number of antioxidant enzyme genes in bone marrow stromal cells of mice, including SOD, CAT, GST and GR [27]. It was demonstrated that the phosphorylation of Nrf2 at the threonine residue was involved in Nrf2 activation in lung of mice [28]. Nrf2 was found to exist in zebrafish [29]. Based on these observations, threonine may influence the antioxidant defense of fish digestive organs, which warrants investigations.

Grass carp is one of the most important freshwater fish species in the world [30]. Nowadays grass carp is mainly dependent on aquaculture [31]. The threonine requirement of juvenile grass carp was estimated to 13.7 g/kg diet, corresponding to 36.0 g/kg of dietary protein [10]. However, nutrient requirements may vary with the growth stage of fish. Studies showed that the threonine requirement of fingerling India major carp was higher than that of juvenile India major carp [3, 32]. To date, except for juveniles, the threonine requirement for grass carp at other growth stage has not been estimated. Therefore, it is necessary to evaluate the threonine requirement of sub-adult grass carp.

The principal objective of this research was to determine effects of threonine on growth, digestive and absorptive capacity and antioxidant status in intestine and hepatopancreas of sub-adult grass carp. The optimum dietary threonine requirement for the sub-adult grass carp was also evaluated.

Materials and methods

Experimental design and diets

The composition of the basal diet is given in Table 1. Fish meal, casein and gelatin were used as intact protein sources. Fish oil and soybean oil were used as dietary lipid sources. According to Abidi and Khan [33], the amino acid profile of whole chicken egg protein was chosen. Crystalline amino acids were used to simulate the amino acid profile with 280 g/kg whole chicken egg protein, except for threonine. L-threonine was added to the basal diet to provide graded concentrations of 3.9 (unsupplemented diet), 6.4, 8.9, 11.4, 13.9, and 16.4 g threonine/kg diet. According to the method of Ahmed et al. [3], diets were made iso-nitrogenous by adjusting crystalline L-glycine. The pH of diets was adjusted to 7.0 with 6.0 N NaOH, as described by Li et al. [34]. According to Shiau and Lo [35], pellets were produced and stored at −20 °C until used. Threonine concentrations in diets analyzed by HPLC were 3.3 (unsupplemented diet), 5.9, 8.4, 10.9, 13.1 and 15.8 g threonine/kg diet, respectively.
Table 1

The composition and nutrient content of the basal diet

Ingredients

Content, g/kg

Nutrient contentf

Content, g/kg

Fish meal

68.0

Crude protein

280.6

Casein

30.0

Crude lipid

46.8

Gelatin

39.9

Available phosphorus

6.0

Crystalline AA mixa

146.4

n-3

10.0

Threonine premixb

50.0

n-6

10.0

Glycine premixc

100.0

  

α-starch

280.0

  

Corn starch

34.6

  

Fish oil

22.8

  

Soybean oil

18.9

  

Mineral premixd

20.0

  

Vitamin premixe

10.0

  

Ca(H2PO4)2

22.9

  

Choline chloride (500 g/kg)

6.0

  

Microcrystalline cellulose

150.0

  

Ethoxyquin (300 g/kg)

0.5

  

aAmino acid mix: lysine, 15.99 g; methionine, 8.18 g; tryptophan, 3.27 g; arginine, 11.80 g; histidine, 7.23 g; isoleucine, 11.82 g; leucine, 18.99 g; valine, 14.24 g; phenylalanine, 12.53 g; tyrosine, 10.00 g; glutamate, 32.32 g

bThreonine premix: Per kilogram of threonine premix composition from diet 1 to 6 was as follows (g/kg): L-threonine 0 g, 51.02 g, 102.04 g, 153.06 g, 204.08 g, 255.10 g, and corn starch 1, 000.00 g, 948.98 g, 897.96 g, 846.94 g, 795.92 g, 744.90 g, respectively

cGlycine premix: Per kilogram of glycine premix composition from diet 1 to 6 was as follows (g/kg): L-glycine 524.58 g, 508.66 g, 492.75 g, 476.83 g, 460.92 g, 445.00 g, and corn starch 475.42 g, 491.34 g; 507.25 g; 523.17 g; 539.08 g; 555.00 g, respectively

dPer kg of mineral premix: FeSO4•H2O (300 g/kg Fe), 25.00 g; CuSO4•5H2O (250 g/kg Cu), 0.60 g; ZnSO4•7H2O (345 g/kg Zn), 4.35 g; MnSO4•H2O (318 g/kg Mn), 2.04 g; KI (50 g/kg I), 1.10 g; NaSeO3 (10 g/kg Se), 2.50 g; MgSO4•H2O (150 g/kg Mg), 230.67 g. All ingredients were diluted with corn starch to 1 kg

ePer kg of vitamin premix: retinyl acetate (500, 000 IU/g), 0.80 g; cholecalciferol (500, 000 IU/g), 0.48 g; DL-α tocopherol acetate (500 g/kg), 20.00 g; menadione (230 g/kg), 0.22 g; cyanocobalamin (10 g/kg), 0.10 g; D-biotin (20 g/kg), 5.00 g; folic acid (960 g/kg), 0.52 g; thiamine hydrochloride (980 g/kg), 0.12 g; ascorhyl acetate (930 g/kg), 7.16 g; niacin (990 g/kg), 2.58 g; meso-inositol (990 g/kg), 52.33 g; calcium-D-pantothenate (900 g/kg), 2.78 g; riboflavin (800 g/kg), 0.99 g; pyridoxine hydrochloride (980 g/kg), 0.62 g. All ingredients were diluted with corn starch to 1 kg

fCrude protein and crude lipid contents were measured value. Available phosphorus, n-3 and n-6 contents were calculated according to NRC (1993)

Feeding trial

All experimental protocols were approved by Animal Care Advisory Committee of Sichuan Agricultural University. Sub-adult grass carp were obtained from the Bai-long Lake Fisheries (Sichuan, China). After acclimatized to the experimental condition for 2 weeks, a total of 600 fish with an average weight of 441.9 ± 2.6 g were randomly distributed into 30 cages (1.4 m × 1.4 m × 1.4 m, a gauze disc (diameter, 0.8 m) was placed on the bottom of each cage to collect uneaten feed). Fish were fed to apparent satiation 4 times per day for 8 weeks. According to Cai et al. [36], uneaten feed was removed at 30 min after feeding, air-dried and weighted to measure feed intake. Water temperature, dissolved oxygen and pH were 25 ± °C, 5.0 ± 0.3 mg/L and 7.5 ± 0.3, respectively.

Sample collection and analysis

Fish in each cage were weighed at the beginning and the end of the feeding trial. After 12 h of fasting, 15 fish from each treatment were anaesthetized in benzocaine bath (50 mg/L), as described by Berdikova Bohne et al. [37] with a minor modification. The intestine, hepatopancreas and muscle of the fish were quickly removed, weighed and stored at −70 °C until analyzed. Intestine, hepatopancreas and muscle samples were homogenized on ice in ten volumes (w/v) of ice-cold physiological saline solution and centrifuged at 6000 g for 20 min at 4 °C, and then the supernatant was conserved at −70 °C for determinations of the protein content and enzyme activities.

The protein content was analyzed according to the procedure described by Bradford [38]. Activities of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) were determined by methods of Bergmeyer and Bernt [39, 40], respectively. Trypsin and chymotrypsin activities were detected according to Hummel [41]. Alpha-amylase and lipase were assayed according to Furne et al. [42]. AP, γ-GT, creatine kinase (CK) and Na+/K+-ATPase activities were determined by the procedure described by Bessey et al. [43], Rosalki et al. [44], Tanzer and Gilvarg [45] and Weng et al. [46], respectively. Contents of malondialdehyde (MDA) and protein carbonyl (PC) were determined by the procedure described by Zhang et al. [47] and BaltacIoglu et al. [48], respectively. The anti-superoxide anion (ASA) capacity and anti-hydroxyl radical (AHR) capacity were analyzed by using the superoxide anion free radical detection Kit and hydroxyl free radical detection Kit (Nanjing Jiancheng Bioengineer Institute), respectively. GSH contents were determined according to the method of Vardi et al. [49]. GR activity was determined according to Lora et al. [50]. SOD and GPx activities were detected according to Zhang et al. [47]. Activities of CAT and GST were determined according to Aebi [51] and Lushchak et al. [52], respectively.

Statistical analysis

Results were present as means ± SD. Data were analyzed with one-way analysis of variance (ANOVA). Differences among dietary treatments were determined using the Duncan’s multiple-range test at the level of P < 0.05 through SPSS 18.0 for windows. Growth parameters with significant differences were subjected to second-degree polynomial regression analysis. According to Abidi and Khan [33], quadratic regression analysis was used to estimate optimum dietary threonine requirement of sub-adult grass carp.

Results

Growth performance

Effects of graded levels of dietary threonine on growth parameters are given in Table 2, weight gain (WG), specific growth rate (SGR) and feed intake (FI) were significantly improved as dietary threonine levels increased from 3.3 to 10.9 g/kg diet (P < 0.05), and decreased thereafter (P < 0.05). Fish fed the basal diet (unsupplemented control group) showed the lower feed efficiency (FE) and protein efficiency ratio (PER) compared to those fed threonine-supplemented diets (P < 0.05). Regression analysis showed that SGR, FI, FE and PER quadratically responded to increased dietary threonine levels (YSGR = −0.011X2 + 0.277X - 0.160, R2 = 0.989, P < 0.05; YFI = −8.729X2 + 200.8X - 113.1, R2 = 0.989, P < 0.05; Y FE = −0.172X2 + 4.152X + 28.43, R2 = 0.914, P < 0.05; Y PER = −0.006X2 + 0.148X + 1.013, R2 = 0.915, P < 0.05). As shown in Figure 1, the dietary threonine requirement of sub-adult grass carp (441.9-1,013.4 g) established by quadratic regression analysis based on WG was 11.6 g/kg diet, corresponding to 41.5 g/kg of dietary protein (Y = −5.284X2 + 123.0X - 169.5, R2 = 0.986, P < 0.05).
Table 2

Effects of dietary threonine levels on the growth performance of sub-adult grass carpg

Item

Dietary Thr levels, g/kg diet

3.3

5.9

8.4

10.9

13.1

15.8

IBW, g/fish

442.0 ± 5.1a

441.6 ± 2.3a

442.6 ± 1.7a

442.2 ± 1.3a

441.6 ± 3.0a

441.4 ± 2.0a

FBW, g/fish

623.4 ± 20.4a

817.4 ± 11.1b

911.8 ± 26.3c

1,013.4 ± 35.2e

969.0 ± 28.2d

896.6 ± 41.7c

WG, g/fish

181.4 ± 19.1a

375.8 ± 10.8b

469.2 ± 26.4c

571.2 ± 34.2e

527.4 ± 27.8d

455.2 ± 42.8c

SGRh, %/day

0.61 ± 0.05a

1.10 ± 0.02b

1.29 ± 0.05c

1.48 ± 0.06d

1.40 ± 0.05d

1.26 ± 0.09c

FI, g/fish

464.4 ± 21.3a

762.0 ± 14.9b

926.1 ± 49.0d

1,074.5 ± 29.5f

1,023.0 ± 10.6e

872.7 ± 5.4c

FEi, %

38.98 ± 2.44a

49.35 ± 2.20b

50.83 ± 4.80b

53.22 ± 3.94b

51.57 ± 2.90b

52.17 ± 5.06b

PERj

1.39 ± 0.09a

1.76 ± 0.08b

1.81 ± 0.17 b

1.90 ± 0.14b

1.84 ± 0.10 b

1.86 ± 0.18 b

IBW: Initial body weight, FBW: Final body weight, WG: Weight gain, SGR: Specific growth rate, FI: Feed intake, FE: Feed efficiency, PER: Protein efficiency ratio

a,b,c,d,e,fMeans in the same row without a letter in common are significantly different (P< 0.05)

gValues are mean ± SD (n = 5)

hSpecific growth rate =100 × {[ln (mean final body weight)-ln (mean initial body weight)]/days}

iFeed efficiency (%) =100× weight gain (g)/diet intake (g)

jProtein efficiency ratio = weight gain (g)/protein intake (g)

Fig. 1

Quadratic regression analysis of weight gain (WG) for sub-adult grass carp (Ctenopharyngodon idella) fed diets containing graded levels of threonine for 8 weeks

Activities of GOT and GPT in muscle and hepatopancreas

As shown in Table 3, activities of GOT in muscle and hepatopancreas were improved with increasing of dietary threonine levels up to 5.9 g/kg diet (P < 0.05). The GPT activity in muscle showed a similar trend with that of muscle GOT activity, and the highest value was obtained when threonine level was 10.9 g/kg diet (P < 0.05). However, the GPT activity in hepatopancreas was decreased with increasing of dietary threonine levels up to 8.4 g/kg diet (P < 0.05).
Table 3

Effects of dietary threonine levels on activities of GOT and GPT in muscle and hepatopancreas of sub-adult grass carpf

Item

Dietary Thr levels, g/kg diet

 

3.3

5.9

8.4

10.9

13.1

15.8

Muscle

 GOT, U/g protein

12.34 ± 0.89b

13.73 ± 1.14c

12.58 ± 1.21bc

12.72 ± 1.03bc

8.77 ± 0.89a

8.24 ± 0.81a

 GPT, U/g protein

6.26 ± 0.55a

11.61 ± 0.53c

11.86 ± 0.39c

14.41 ± 0.23e

13.13 ± 0.47d

9.61 ± 0.21b

Hepatopancreas

 GOT, U/g protein

27.00 ± 2.32a

34.35 ± 2.70b

34.68 ± 3.23b

35.24 ± 2.10b

27.03 ± 2.03a

27.09 ± 2.08a

 GPT, U/g protein

17.38 ± 0.82c

16.44 ± 0.89b

14.26 ± 0.51a

14.78 ± 0.65a

14.55 ± 0.62a

14.87 ± 1.06a

GOT: Glutamate oxaloacetate transaminase; GPT: Glutamate pyruvate transaminase

a,b,c,d,eMeans in the same row without a letter in common are significantly different (P< 0.05)

fValues are mean ± SD (n = 6)

Intestine and hepatopancreas growth

As shown in Table 4, the intestinal length and weight were significantly increased with increasing dietary threonine levels up to 10.9 g/kg diet (P < 0.05). Relative gut length (RGL) was not influenced by graded levels of dietary threonine (P > 0.05). The intestosomatic index (ISI) of fish fed the basal diet was significantly lower than that of fish fed threonine-supplemented diets (P < 0.05). The intestinal protein content (IPC) also followed a similar pattern to that as observed with intestinal length. The hepatopancreatic weight, hepatosomatic index (HSI) and hepatopancreatic protein content (HPC) were significantly improved with the supplementation of dietary threonine (P < 0.05), and the maximum values were obtained when threonine levels were 10.9, 8.4 and 8.4 g/kg diet, respectively.
Table 4

Effects of dietary threonine levels on IL, RGL, IW, ISI, IPC, HW, HSI, and HPC of sub-adult grass carp

Item

Dietary Thr levels, g/kg diet

 

3.3

5.9

8.4

10.9

13.1

15.8

ILf, cm /fish

58.1 ± 5.9a

58.5 ± 2.9a

61.8 ± 3.2ab

63.9 ± 3.5b

61.7 ± 3.2ab

59.6 ± 5.0a

RGLf, %

149.0 ± 18.1a

144.4 ± 7.6a

144.6 ± 8.1a

145.5 ± 7.7a

145.3 ± 7.7a

147.3 ± 10.8a

IWf, g /fish

6.3 ± 0.8a

9.8 ± 1.6bc

10.9 ± 1.6cd

14.0 ± 2.7e

11.8 ± 1.5d

9.2 ± 1.0 b

ISIf, %

1.04 ± 0.13a

1.24 ± 0.21b

1.16 ± 0.12b

1.26 ± 0.18b

1.25 ± 0.15b

1.16 ± 0.12b

IPCg, %

8.33 ± 0.63 a

9.57 ± 1.01bc

10.21 ± 1.02c

8.90 ± 0.73ab

8.71 ± 0.89ab

8.49 ± 0.90ab

HWf, g /fish

12.1 ± 2.1a

16.9 ± 2.5b

23.7 ± 5.7c

26.3 ± 7.1c

23.4 ± 3.6c

16.5 ± 3.3b

HSIf, %

1.98 ± 0.27a

2.14 ± 0.35ab

2.51 ± 0.54c

2.37 ± 0.48bc

2.49 ± 0.39c

2.08 ± 0.36ab

HPCg, %

9.46 ± 0.50a

9.12 ± 0.68a

11.61 ± 0.79c

10.60 ± 0.65b

11.15 ± 0.42bc

10.98 ± 0.97bc

IL: Intestinal length; RGL: Relative gut length; IW: Intestinal weight; ISI: Intestosomatic index; IPC: Intestinal protein content; HW: Hepatopancreatic weight; HSI: Hepatosomatic index; HPC: Hepatopancreatic protein content

a,b,c,d,eMeans in the same row without a letter in common are significantly different (P< 0.05)

fValues are mean ± SD (n = 15)

gValues are mean ± SD (n = 6)

Activities of intestinal enzymes

As shown in Table 5, intestinal activities of trypsin and alpha-amylase were significantly improved with increasing dietary threonine levels up to 10.9 and 8.4 g/kg diet, respectively (P < 0.05), and plateaued thereafter (P > 0.05). The highest intestinal activities of chymotrypsin and lipase were obtained when the threonine level was 8.4 g/kg diet.
Table 5

Effects of dietary threonine levels on activities of tryspin, chymotrypsin, alpha-amylase and lipase in intestine of sub-adult grass carpd

Item

Dietary Thr levels, g/kg diet

 

3.3

5.9

8.4

10.9

13.1

15.8

Trypsin, U/g protein

177.2 ± 10.2a

239.9 ± 12.5b

252.6 ± 12.1b

291.2 ± 16.8c

286.1 ± 10.0c

294.0 ± 10.6c

Chymotrypsin, U/g protein

1.03 ± 0.09a

1.00 ± 0.08a

2.29 ± 0.15c

1.58 ± 0.14b

1.45 ± 0.14b

1.48 ± 0.12b

alpha-amylase, U /mg protein

2.46 ± 0.12a

2.51 ± 0.19a

2.84 ± 0.09b

2.75 ± 0.06b

2.73 ± 0.04b

2.77 ± 0.04b

Lipase, U/g protein

19.11 ± 1.29ab

20.97 ± 1.77bc

22.71 ± 2.00c

22.55 ± 2.41c

21.06 ± 2.34bc

17.93 ± 1.26a

a,b,cMeans in the same row without a letter in common are significantly different (P< 0.05)

dValues are mean ± SD (n = 6)

As shown in Table 6, activities of AP in proximal intestine (PI), mid intestine (MI) and distal intestine (DI) were significantly improved with the supplementation of dietary threonine (P < 0.05), and the highest AP activities were observed for fish fed diets containing 13.1, 8.4 and 5.9 g threonine/kg diet, respectively. Fish fed the basal diet had significantly lower activities of γ-GT in PI and MI compared to those fed threonine-supplemented diets (P < 0.05). The highest activity of γ-GT in DI was obtained in fish fed the diet containing 10.9 g threonine/kg diet. Activities of CK in PI, MI and DI were significantly improved with the supplementation of dietary threonine (P < 0.05), and the maximum values were obtained when fish fed the diet containing 10.9 g threonine/kg diet. Na+/K+-ATPase activities in PI and MI were significantly improved with increasing dietary threonine levels up to 5.9 g/kg diet (P < 0.05), and gradually decreased thereafter (P < 0.05). However, the activity of Na+/ K+-ATPase in DI was not affected by dietary threonine levels (P > 0.05).
Table 6

Effects of dietary threonine levels on activities of AP, γ-GT, CK and Na+/K+-ATPase in intestine of sub-adult grass carpf

Item

Dietary Thr levels, g/kg diet

 

3.3

5.9

8.4

10.9

13.1

15.8

AP, mmol of nitrophenol released g/protein per h

   

 PI

69.5 ± 7.3a

84.4 ± 8.1b

102.8 ± 10.1cd

108.0 ± 10.7d

125.0 ± 10.4e

92.8 ± 9.1bc

 MI

110.1 ± 7.0c

120.3 ± 13.0cd

125.8 ± 4.9d

117.6 ± 8.2cd

91.9 ± 8.6b

71.3 ± 7.4a

 DI

72.2 ± 6.9c

80.3 ± 7.5d

69.0 ± 5.4c

54.7 ± 5.3b

53.8 ± 2.6b

35.8 ± 2.8a

γ-GT, mmol of 5-amino-2-nitrobenzoate released g/protein per min

 

 PI

1.82 ± 0.16a

2.27 ± 0.15b

2.59 ± 0.28c

2.52 ± 0.15c

3.00 ± 0.16d

2.24 ± 0.23b

 MI

2.12 ± 0.16a

2.89 ± 0.26b

3.05 ± 0.21b

3.09 ± 0.17b

2.93 ± 0.19b

2.94 ± 0.28b

 DI

0.73 ± 0.08a

0.87 ± 0.11b

1.20 ± 0.07c

1.23 ± 0.12c

1.13 ± 0.11c

0.76 ± 0.06ab

CK, μmol of phosphorus released g/protein per h

 

 PI

2.62 ± 0.25c

2.72 ± 0.19c

2.74 ± 0.24c

3.11 ± 0.29d

1.52 ± 0.13b

1.14 ± 0.13a

 MI

1.36 ± 0.09a

2.40 ± 0.18c

2.35 ± 0.17c

4.47 ± 0.44d

4.28 ± 0.34d

1.77 ± 0.17 b

 DI

6.69 ± 0.73a

7.26 ± 0.44a

6.96 ± 0.67a

9.35 ± 0.45b

8.86 ± 0.70b

7.15 ± 0.75a

Na+/K+-ATPase, μmol of phosphorus released g/protein per h

 

 PI

0.37 ± 0.06b

0.42 ± 0.05c

0.41 ± 0.02c

0.36 ± 0.03b

0.37 ± 0.03b

0.30 ± 0.03a

 MI

0.48 ± 0.04c

0.62 ± 0.04d

0.47 ± 0.03bc

0.43 ± 0.04ab

0.39 ± 0.03a

0.39 ± 0.04a

 DI

0.35 ± 0.02a

0.34 ± 0.02a

0.35 ± 0.02a

0.36 ± 0.01a

0.35 ± 0.03a

0.35 ± 0.02a

AP: Alkaline phosphatase; γ-GT: γ-Glutamyl transpeptidase; CK: Creatine kinase; PI: Proximal intestine; MI: Mid intestine; DI: Distal intestine

a,b,c,d,eMeans in the same row without a letter in common are significantly different (P< 0.05)

fValues are mean ± SD (n = 6)

Antioxidant status in intestine and hepatopancreas

As listed in Table 7, contents of MDA in intestine and hepatoancreas were significantly decreased with increasing threonine levels up to 8.4 and 10.9 g/kg diet (P < 0.05), and thereafter increased (P < 0.05). The lowest PC contents in intestine and hepatoancreas were observed in fish fed the diet containing 13.1 g threonine/kg diet. ASA capacities in intestine and hepatoancreas were the highest in fish fed diets containing 13.1 and 10.9 g threonine/kg diet, respectively. The AHR capacities in intestine and hepatoancreas were observed in fish fed the diet containing13.1 g threonine/kg diet (P < 0.05).
Table 7

Effects of dietary threonine levels on MDA, PC, AHR and ASA in intestine and hepatoancreas of sub-adult grass carpf

Item

Dietary Thr levels, g/kg diet

 

3.3

5.9

8.4

10.9

13.1

15.8

Intestine

 MDA, nmol/mg protein

2.97 ± 0.23d

1.98 ± 0.19c

1.33 ± 0.11 a

1.51 ± 0.07 b

1.53 ± 0.11b

1.57 ± 0.10b

 PC, nmol/mg protein

4.37 ± 0.34c

3.41 ± 0.25ab

3.52 ± 0.23b

3.19 ± 0.29ab

3.10 ± 0.18a

3.34 ± 0.30ab

 AHR, U/mg protein

120.5 ± 9.9a

175.2 ± 12.1b

192.9 ± 14.1c

266.9 ± 11.1e

278.3 ± 12.8e

227.6 ± 17.7d

 ASA, U/g protein

243.5 ± 14.7b

253.2 ± 18.3b

252.2 ± 17.6b

255.9 ± 22.3b

278.1 ± 17.2c

208.5 ± 14.5a

Hepatopancreas

 MDA, nmol/mg protein

2.38 ± 0.19e

2.17 ± 0.15d

1.57 ± 0.13b

1.37 ± 0.13a

1.95 ± 0.18c

1.92 ± 0.10c

 PC, nmol/mg protein

6.08 ± 0.41c

6.21 ± 0.41c

4.99 ± 0.50b

5.11 ± 0.36b

4.24 ± 0.38a

5.90 ± 0.52c

 AHR, U/mg protein

214.6 ± 14.8a

217.2 ± 11.6a

218.9 ± 5.4a

222.4 ± 7.0a

266.1 ± 10.5b

224.2 ± 13.3a

 ASA, U/g protein

220.8 ± 13.8a

224.0 ± 19.2a

237.2 ± 16.1a

264.9 ± 22.8b

241.4 ± 22.0a

230.4 ± 12.2a

MDA: Malondialdehyde content; PC: Protein carbonyl content; AHR: Anti-hydroxyl radical capacity; ASA: Anti-superoxide anion capacity

a,b,c,d,eMeans in the same row without a letter in common are significantly different (P< 0.05)

fValues are mean ± SD (n = 6)

As listed in Table 8, the supplementation of threonine to certain levels increased GSH contents in intestine and hepatoancreas (P < 0.05), the highest GSH contents in intestine and hepatoancreas were observed in fish fed diets containing 13.1 and 10.9 g threonine/kg diet, respectively. Antioxidant enzyme activities in intestine and hepatoancreas were significantly affected by graded levels of dietary threonine (P < 0.05). GR activity in the intestine was decreased with increasing the dietary threonine levels up to 8.4 g/kg diet (P < 0.05), However, the trend of hepatopancreatic GR activity was opposite to that in intestinal GR. Fish fed the basal diet had a significantly lower activity of intestinal SOD compared to those fed threonine-supplemented diets (P < 0.05). The highest activity of SOD in hepatoancreas was found in fish fed the diet containing 5.9 g threonine/kg diet (P < 0.05). CAT activities in intestine and hepatoancreas were significantly improved with increasing threonine levels up to 10.9 and 8.4 g/kg diet, respectively (P < 0.05), and decreased thereafter (P < 0.05). GST activity followed a similar pattern to that as observed with intestinal SOD activity, The highest activity of GST in hepatoancreas was found in fish fed the diet containing 5.9 g threonine/kg diet (P < 0.05). The intestinal GPx activity was lower in fish fed the diet containing 15.8 g threonine/kg diet than the other five treatment groups (P < 0.05).The GPx activity in hepatoancreas was improved with increasing the threonine levels up to 5.9 g/kg diet (P < 0.05), and decreased to a plateau thereafter.
Table 8

Effects of dietary threonine levels on GSH content and activities of GR, SOD, CAT, GST and GPx in intestine and hepatoancreas of sub-adult grass carpe

Item

Dietary Thr levels, g/kg diet

 

3.3

5.9

8.4

10.9

13.1

15.8

Intestine

 GSH, mg/g protein

1.81 ± 0.16a

1.83 ± 0.13a

2.56 ± 0.19b

3.00 ± 0.19c

5.63 ± 0.22d

2.98 ± 0.16c

 GR, U/g protein

23.15 ± 1.22b

24.57 ± 1.95b

17.05 ± 1.36a

19.27 ± 1.78a

27.65 ± 2.82c

33.59 ± 3.11d

 SOD, U/mg protein

19.84 ± 1.71a

23.87 ± 2.33b

23.62 ± 1.13b

23.41 ± 2.19b

24.83 ± 1.44b

23.45 ± 1.84b

 CAT, U/mg protein

11.70 ± 1.03a

16.65 ± 1.48c

16.77 ± 1.31c

22.59 ± 1.94d

18.06 ± 1.26c

14.41 ± 1.46b

 GST, U/mg protein

7.08 ± 0.75a

17.41 ± 1.58b

16.82 ± 1.48b

16.13 ± 1.41b

17.36 ± 1.33b

17.41 ± 1.58b

 GPx, U/mg protein

105.4 ± 9.7b

109.4 ± 6.6b

109.3 ± 4.1b

107.4 ± 9.3b

104.9 ± 5.7b

87.7 ± 8.8a

Hepatopancreas

 GSH, mg/g protein

6.13 ± 0.30b

7.07 ± 0.41c

7.06 ± 0.31c

7.83 ± 0.39d

6.08 ± 0.29b

5.67 ± 0.31a

 GR, U/g protein

4.08 ± 0.31a

13.22 ± 1.02c

7.74 ± 0.80b

7.91 ± 0.59b

7.36 ± 0.73b

7.58 ± 0.75b

 SOD, U/mg protein

83.16 ± 8.65b

100.42 ± 6.19c

80.39 ± 7.06b

84.66 ± 4.00b

78.72 ± 5.17b

66.57 ± 3.08a

 CAT, U/mg protein

55.67 ± 5.95a

59.11 ± 5.39a

79.98 ± 7.11c

69.65 ± 7.34b

57.94 ± 6.91a

56.51 ± 5.61a

 GST, U/mg protein

22.51 ± 2.05b

45.73 ± 2.73d

44.27 ± 3.78d

25.80 ± 2.68c

25.77 ± 2.48c

17.34 ± 1.66a

 GPx, U/mg protein

961.5 ± 79.2a

1,205.9 ± 44.5c

1,041.0 ± 55.3b

1,053.9 ± 57.1b

1,026.2 ± 46.4ab

995.7 ± 47.2ab

GSH: Glutathione; GR: Glutathione reductase; SOD: Superoxide dismutase; CAT: Catalase; GST: Glutathione-S-transferase; GPx: Glutathione peroxidase

a,b,c,dMeans in the same row without a letter in common are significantly different (P< 0.05)

eValues are mean ± SD (n = 6)

Discussion

In the present study, the growth performance of sub-adult grass carp was significantly influenced by dietary threonine levels. WG, SGR, FI and FE of sub-adult grass carp were significantly improved by dietary threonine, which were in agreement with reports for juvenile grass carp [10], juvenile Jian carp [5] and fingerling Indian major carp [3]. In this study, the improved fish growth may be partly attributed to the promotion of amino acid utilization. It is well known that GOT and GPT play an important role in amino acid metabolism, whose activity can be used to evaluate the utilization of essential amino acids in fish [53]. Results here showed that GOT and GPT activities in muscle, as well as GOT activity in hepatopancreas were significantly increased with optimal threonine supplementation. This was consistent with our previous study [5]. However, GPT activity in hepatopancreas was decreased with the increment levels of dietary threonine up to a certain point. The reason for this result may attribute to the enhanced hepatic gluconeogenesis induced by threonine deficiency. It was reported that threonine deficiency increased hepatic gluconeogenesis in rats [54]. GPT is a rate-limiting enzyme in the conversion of protein to carbohydrate, whose activity in rat liver can be enhanced by the increased hepatic gluconeogenesis [55]. However, this hypothesis needs further investigation in fish. Additionally, the trend of GPT activity in hepatopancreas was opposite with that of our previous study in juvenile Jian carp [5]. The reason for these results is not clear. A possible explanation might be related to the differences in threonine metabolism in different growth stage, as described in terrestrial animals [56, 57]. Further research is needed to clarify this hypothesis. Based on the quadratic regression analysis for WG, the requirement of threonine for sub-adult grass carp (441.9-1,013.4 g) was estimated to be 11.6 g/kg diet, corresponding to 41.5 g/kg of dietary protein.

Fish growth relies on nutrient utilization, which is related to the digestive and absorptive capacity [4]. Generally, fish digestive and absorptive capacity can be reflected by digestive organ growth and development, as well as activities of intestinal enzymes related to digestion and absorption [58]. In our study, there were significant improvements in intestinal length, intestinal weight, ISI and IPC, as well as hepatopancreatic weight, HSI and HPC content. Meanwhile, activities of trypsin, chymotrypsin, alpha-amylase, lipase, AP, γ-GT and CK in whole intestine, as well as Na+/K+-ATPase in PI and MI were improved by dietary threonine. All these data above suggested that threonine improved the digestive and absorptive capacity of sub-adult grass carp, which were in agreement with our previous study of juvenile Jian carp [5]. It is well known that digestive enzymes in intestinal lumen are mainly secreted from pancreas [59]. Hokin [60] reported that threonine was necessary for the pancreatic alpha-amylase synthesis in pigeons. Meanwhile, threonine increased pancreatic secretion of trypsin, alpha-amylase and chymotrypsinogen in chicks [61]. Besides, threonine was found to be served as an essential component of the active center in γ-GT of rats [62] and CK of chicken [63]. However, the mechanism which threonine improved the digestive and absorptive capacity of fish needs further study.

In fish, the normal function of the digestive organ is correlated with its antioxidant status [11]. The contents of products of lipid peroxidation and protein oxidation, such as MDA and PC, can reflect the antioxidant status of living organisms [64]. In the present study, contents of MDA and PC were decreased with increasing dietary threonine levels up to certain values in both intestine and hepatopancreas, suggesting depressions of the lipid peroxidation and protein oxidation. To date, there were no studies about the effect of threonine on the lipid peroxidation and protein oxidation in fish. Using biochemical in vitro assays, it was demonstrated that threonine reduced autoxidation rates of safflower oil in liquid emulsions [65]. As we all know, the lipid peroxidation and protein oxidation are induced by ROS, among which superoxide and hydroxyl radicals are most strongly involved in oxidative damages [13, 66]. In our study, both ASA capacity and AHR capacity in intestine and hepatopancreas were enhanced by dietary threonine, suggesting the improved scavenging abilities against superoxide anion and hydroxyl radicals. To date, information on the relationship between dietary threonine levels and capacity of ASA and AHR has not yet been reported in fish. A possible reason for the improved capacity of AHR might be that threonine enhanced mucin synthesis. Studies showed that intestinal mucin synthesis in piglets [67] and rats [68] were increased by threonine. Meanwhile, in vitro biochemical assays, pig stomach mucins could scavenge hydroxyl radicals [19]. Besides, the increased AHR capacity might be also related to the ability of threonine to chelate metal ions. In living organisms, the formation of hydroxyl radicals could be induced by free transition metal ions, such as iron, copper and manganese, via the Fenton-Haber Weiss reaction [14]. In the stomach of rats, the formation of hydroxyl radicals was reduced by chelating iron ions [15]. Threonine was found to chelate with manganese ions in the liver of rats [18] and iron and copper ions in vitro biochemical assays [16, 17]. Thus, threonine might be able to decrease lipid peroxidation and protein oxidation in fish digestive organ by improving radical scavenging abilities in these organs, which warrants further study.

In fish, free radicals can be scavenged by non-enzymatic antioxidants, such as vitamin C, vitamin E and GSH [21]. GSH is a direct free radical scavenger in fish [22]. In the present study, both intestinal and hepatopancreatic GSH contents of sub-adult grass carp were increased with optimal threonine supplementation. To date, information on the relationship between dietary threonine levels and GSH contents is limited in fish. Generally, cellular GSH homeostasis is maintained through de novo GSH synthesis, glutathione disulfide (GSSG) reduction and uptake of extracellular GSH [69]. In this study, the increased intestinal GSH contents by dietary threonine might be related to the increased uptake of extracellular GSH. It was reported that biliary GSH, which was secreted by liver, was one of the major sources of intestinal GSH in rats [70]. Lauterburg et al. [71] found that an increase in liver GSH content was associated with increased intestinal GSH contents in rats. In terrestrial animals, luminal GSH was uptake by intestine epithelial cells in two ways: (1) be transported intact into cells; (2) be cleaved into glutamate and cysteinylglycine by γ-GT, and then γ-GT transported the cysteinylglycine into the cell for re-synthesis of GSH [72, 73]. In our study, intestinal GSH content was positively related to the γ-GT activity in PI (r = + 0.838, P < 0.05), which might suggest that luminal GSH was mainly uptake by intestine epithelial cells of sub-adult grass carp in the second pathway. However, this hypothesis needs further investigation. Liver is the primary site for de novo GSH synthesis in rats, which requires the participation of ATP [23]. Ross-Inta et al. [24] reported that dietary threonine increased the liver ATP level of rats. However, whether this ATP synthesis promotion effect of threonine also exists in fish needs study. In the present study, the increased hepatopancreatic GSH content may also be attributed to the promotion of GSSG reduction. GR catalyses the reduction of GSSG back to GSH [74]. Threonine improved GR activity in hepatopancreas of sub-adult grass carp, indicating the improved GSSG reduction. However, the trend of intestinal GR activity was opposite with that in hepatopancreas. A possible reason for this result is that intestinal GR activity was inactivated by GSH. Ogus and Ozer [75] reported that human intestinal GR activity was inactivated by GSH in vitro. The reason for GSH not inhibiting GR activity in hepatopancreas might be that GSH in the liver is maintained mainly in the reduced state, and which is highly dependent on GR activity, as it was reported by Kaplowitz et al. [76]. However, further studies are needed to test this hypothesis.

Aside from the antioxidants, antioxidant enzymes, such as SOD, CAT, GST and GPx, also play an important role in protecting cells against free radical damages [13]. The present study showed that threonine enhanced intestinal and hepatopancreatic activities of SOD, CAT and GST, suggesting the improved enzymatic antioxidant ability. To date, few studies have evaluated effects of threonine on activities of antioxidant enzymes in fish. It has been demonstrated that expressions of SOD, CAT and GST are controlled by Nrf2-ARE system in bone marrow stromal cells of mice [27]. Meanwhile, the threonine phosphorylation was involved in Nrf2 activation in lung of mice [28]. Furthermore, the conserved threonine residue was essential for the structure stabilization of Nrf2 in HEK-293 T cells [77]. Kobayashi et al. [29] found that Nrf2 existed in zebrafish. Thus, beneficial effects of threonone on antioxidant enzyme activities might be partly attributed to the enhanced activation of Nrf2. However, this hypothesis needs further investigations. GPx protects cells from excessive levels of H2O2 and intracellular lipid peroxides by formation of GSSG [78]. In our study, threonine enhanced hepatopancreatic GPx activity of sub-adult grass carp. However, in the intestine, GPx activity was not improved by dietary threonine, but was decreased by excess threonine intake. A possible reason for this phenomenon might be the reduced intestinal mucin synthesis by excess threonine intake. Wang et al. [79] reported that excessive level of dietary threonine reduced mucin synthesis in small intestine of pigs. A decreased content of pig stomach mucins was associated with a decrease of hydroxyl radical scavenging ability in vitro biochemical assays [19]. Tabatabaie and Floyd [80] found that GPx of bovine erythrocytes was inactivated by hydroxyl radicals in vitro. However, further studies are needed to determine this hypothesis in fish.

Conclusions

Diets containing the appropriate amount of threonine improved growth, increased digestive and absorptive capacity, and enhanced intestinal and hepatopancreatic antioxidant defense of sub-adult grass carp. Based on the quadratic regression analysis for WG, the requirement of threonine for sub-adult grass carp (441.9-1,013.4 g) was estimated to be 11.6 g/kg diet, corresponding to 41.5 g/kg of dietary protein.

Abbreviations

AHR: 

Anti-hydroxyl radical

AP: 

Alkaline phosphatase

ASA: 

Anti-superoxide anion

CAT: 

Catalase

CK: 

Creatine kinase

DI: 

Distal intestine

FE: 

Feed efficiency

FI: 

Feed intake

GOT: 

Glutamate oxaloacetate transaminase

GPT: 

Glutamate pyruvate transaminase

GPx: 

Glutathione peroxidase

GR: 

Glutathione reductase

GSH: 

Glutathione

GSSG: 

Glutathione disulfide

GST: 

Glutathione-S-transferase

γ -GT: 

γ -glutamyl transpeptidase

HPC: 

Hepatopancreatic protein content

HSI: 

Hepatosomatic index

IPC: 

Intestinal protein content

ISI: 

Intestosomatic index

MDA: 

Malondialdehyde

MI: 

Mid intestine

Nrf2: 

E2 p45-related factor 2

PC: 

Protein carbonyl:

PER: 

Protein efficiency ratio

PI: 

Proximal intestine

RGL: 

Relative gut length

ROS: 

Reactive oxygen species

SGR: 

Specific growth rate

SOD: 

Superoxide dismutase

Thr: 

Threonine

WG: 

Weight gain

Declarations

Acknowledgements

The authors would like to thank National 973 Project of China (2014CB138600), National Department Public Benefit Research Foundation (Agriculture) of China (201003020), Science and Technology Support Programme of Sichuan Province of China (2014NZ0003), and Major Scientific and Technological Achievement Transformation Project of Sichuan Province of China (2012NC0007 and 2013NC0045) for their financial support.

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Animal Nutrition Institute, Sichuan Agricultural University
(2)
Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University
(3)
Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University
(4)
Sichuan Academy of Animal Science, Animal Nutrition Institute
(5)
Chinese Academy of Sciences, Institute of Hydrobiology

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