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/lp/springer-journals/growth-digestive-and-absorptive-capacity-and-antioxidant-status-in-lidujmaM4d
- Publisher
- Springer Journals
- Copyright
- Copyright © 2015 by Hong et al.
- Subject
- Life Sciences; Agriculture; Biotechnology; Food Science; Animal Genetics and Genomics; Animal Physiology
- eISSN
- 2049-1891
- DOI
- 10.1186/s40104-015-0032-1
- pmid
- 26257911
- Publisher site
- See Article on Publisher Site
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 effects of dietary threonine on the digestive and absorp- Threonine (Thr) is an indispensable amino acid for fish tive capacity of fish, which showed that diet threonine [1]. Dietary threonine deficiency has been shown to improved the activities of trypsin, lipase and alpha- cause poor growth and feed conversion in juvenile Japa- amylase in hepatopancreas and intestine of juvenile Jian nese flounder (Paralichthysolivaceus) [2], as well as low carp (Cyprinuscarpio var. Jian), as well as the activities protein deposition in fingerling Indian major carp (Cir- of intestinal enzymes related to absorption, including al- rhinusmrigala) [3]. It is well known that fish growth is kaline phosphatase (AP), γ-glutamyl transpeptidase (γ- + + greatly influenced by food digestion and nutrient absorp- GT) and Na /K -ATPase [5]. However, the digestive and tion [4]. To date, there is only one report regarding the absorptive capacity of fish varies with its feeding habit [6]. Generally, herbivorous fish have a higher digestive * Correspondence: xqzhouqq@tom.com; zhouxq@sicau.edu.cn; fenglin@sicau.edu.cn capacity in starch than that of omnivorous and carnivor- Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan, China ous species [7]. While relative to omnivorous and car- Fish Nutrition and Safety Production University Key Laboratory of Sichuan nivorous fish species, the herbivorous fish show a poor Province, Sichuan Agricultural University, Chengdu 611130, Sichuan, China Full list of author information is available at the end of the article © 2015 Hong et al. Open Access This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 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. Hong et al. Journal of Animal Science and Biotechnology (2015) 6:34 Page 2 of 11 digestive capacity in protein and fat [6, 8]. Therefore, ef- [25]. To date, information regarding the effect of threo- fects of dietary threonine on digestive and absorptive nine on activities of antioxidant enzyme is not available capacity may be different among fish with different feed- in fish. Sidransky and Rechcigl [26] reported that dietary ing habits. The present study focused on the effects of threonine increased CAT activity in liver and kidney of threonine on digestive and absorptive capacity of herbiv- rats. E2 p45-related factor 2 (Nrf2) regulates a number orous grass carp (Ctenopharyngodonidella). of antioxidant enzyme genes in bone marrow stromal The function of fish digestive organ is correlated with cells of mice, including SOD, CAT, GST and GR [27]. It its development [9]. Threonine has been shown to im- was demonstrated that the phosphorylation of Nrf2 at prove intestinal folds height in juvenile Jian carp [5], as the threonine residue was involved in Nrf2 activation in well as anterior intestinal villus height and serosa thick- lung of mice [28]. Nrf2 was found to exist in zebrafish ness in juvenile grass carp [10]. On the other hand, the [29]. Based on these observations, threonine may influ- growth and function of the digestive organs are usually ence the antioxidant defense of fish digestive organs, correlated with its antioxidant status [11]. Our labora- which warrants investigations. tory studies indicated that the function of digestive or- Grass carp is one of the most important freshwater gans of juvenile Jian carp was positively related to fish species in the world [30]. Nowadays grass carp is antioxidant status by methionine hydroxy analogue [12]. mainly dependent on aquaculture [31]. The threonine However, no studies have been conducted to investigate requirement of juvenile grass carp was estimated to the relationship between threonine and antioxidant sta- 13.7 g/kg diet, corresponding to 36.0 g/kg of dietary pro- tus of tissues and organs in fish. Generally, reactive oxy- tein [10]. However, nutrient requirements may vary with gen species (ROS) are produced during normal aerobic the growth stage of fish. Studies showed that the threo- cellular metabolism [13]. When ROS generation rate ex- nine requirement of fingerling India major carp was ceeds that of their removal, oxidative stress occurs which higher than that of juvenile India major carp [3, 32]. To may induce deleterious effects on cells, such as lipid per- date, except for juveniles, the threonine requirement for oxidation and protein oxidation [13]. Huang et al. [14] grass carp at other growth stage has not been estimated. reported that free transition metal ions, such as iron, Therefore, it is necessary to evaluate the threonine re- copper and manganese, could induce the formation of quirement of sub-adult grass carp. hydroxyl radicals via the Fenton-Haber Weiss reaction The principal objective of this research was to deter- in biological systems. Chelating iron ions could reduce mine effects of threonine on growth, digestive and ab- the formation of hydroxyl radicals in stomach of rats sorptive capacity and antioxidant status in intestine and [15]. Threonine chelated with iron and copper ions hepatopancreas of sub-adult grass carp. The optimum in vitro biochemical assays [16, 17] and manganese ions dietary threonine requirement for the sub-adult grass in liver of rats [18]. Thus, threonine might be able to re- carp was also evaluated. duce the formation of hydroxyl radicals in living organ- isms. On the other hand, pig stomach mucins, which Materials and methods were rich in threonine, could scavenge hydroxyl radicals Experimental design and diets induced by iron ions in vitro biochemical assays [19]. It The composition of the basal diet is given in Table 1. was found that intestinal mucins of common carp Fish meal, casein and gelatin were used as intact pro- (Cyprinuscarpio L.) were rich in threonine [20]. Based tein sources. Fish oil and soybean oil were used as diet- on these data, threonine might be able to improve the ary lipid sources. According to Abidi and Khan [33], function of fish digestive organs by increasing free rad- the amino acid profile of whole chicken egg protein ical scavenging ability. was chosen. Crystalline amino acids were used to simu- In fish, ROS are scavenged by non-enzymatic antioxi- late the amino acid profile with 280 g/kg whole chicken dants and antioxidant enzymes [21]. Glutathione (GSH) egg protein, except for threonine. L-threonine was is an important non-enzymatic antioxidant compound of added to the basal diet to provide graded concentra- fish [22]. However, no studies have been conducted to tions of 3.9 (unsupplemented diet), 6.4, 8.9, 11.4, 13.9, investigate the relationship between threonine and GSH and 16.4 g threonine/kg diet. According to the method content in tissues and organs of fish. In rats, GSH syn- of Ahmed et al. [3], diets were made iso-nitrogenous by thesis takes place mainly in the liver, which needs the adjusting crystalline L-glycine. The pH of diets was ad- participation of ATP [23]. Ross-Inta et al. [24] reported justed to 7.0 with 6.0 N NaOH, as described by Li et al. that threonine increased liver ATP level in rats. As with [34]. According to Shiau and Lo [35], pellets were pro- other aerobic organisms, fish developed diverse antioxi- duced and stored at −20 °C until used. Threonine con- dant enzymes including superoxide dismutase (SOD), centrations in diets analyzed by HPLC were 3.3 catalase (CAT), glutathione-S-transferase (GST), gluta- (unsupplemented diet), 5.9, 8.4, 10.9, 13.1 and 15.8 g thione reductase (GR) and glutathione peroxidase (GPx) threonine/kg diet, respectively. Hong et al. Journal of Animal Science and Biotechnology (2015) 6:34 Page 3 of 11 Table 1 The composition and nutrient content of the basal diet [36], uneaten feed was removed at 30 min after feeding, Ingredients Content, g/kg Nutrient content Content, g/kg air-dried and weighted to measure feed intake. Water temperature, dissolved oxygen and pH were 25 ± °C, 5.0 ± Fish meal 68.0 Crude protein 280.6 0.3 mg/L and 7.5 ± 0.3, respectively. Casein 30.0 Crude lipid 46.8 Gelatin 39.9 Available 6.0 phosphorus Sample collection and analysis Crystalline AA mix 146.4 n-3 10.0 Fish in each cage were weighed at the beginning and the Threonine premix 50.0 n-6 10.0 end of the feeding trial. After 12 h of fasting, 15 fish Glycine premix 100.0 from each treatment were anaesthetized in benzocaine α-starch 280.0 bath (50 mg/L), as described by Berdikova Bohne et al. [37] with a minor modification. The intestine, hepato- Corn starch 34.6 pancreas and muscle of the fish were quickly removed, Fish oil 22.8 weighed and stored at −70 °C until analyzed. Intestine, Soybean oil 18.9 hepatopancreas and muscle samples were homogenized Mineral premix 20.0 on ice in ten volumes (w/v) of ice-cold physiological saline Vitamin premix 10.0 solution and centrifuged at 6000 g for 20 min at 4 °C, and Ca(H PO4) 22.9 then the supernatant was conserved at −70 °C for deter- 2 2 minations of the protein content and enzyme activities. Choline chloride 6.0 (500 g/kg) The protein content was analyzed according to the procedure described by Bradford [38]. Activities of glu- Microcrystalline 150.0 cellulose tamate oxaloacetate transaminase (GOT) and glutamate Ethoxyquin (300 g/kg) 0.5 pyruvate transaminase (GPT) were determined by methods of Bergmeyer and Bernt [39, 40], respectively. Amino 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, Trypsin and chymotrypsin activities were detected ac- 14.24 g; phenylalanine, 12.53 g; tyrosine, 10.00 g; glutamate, 32.32 g cording to Hummel [41]. Alpha-amylase and lipase were Threonine 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, assayed according to Furne et al. [42]. AP, γ-GT, creatine 204.08 g, 255.10 g, and corn starch 1, 000.00 g, 948.98 g, 897.96 g, 846.94 g, + + kinase (CK) and Na /K -ATPase activities were deter- 795.92 g, 744.90 g, respectively Glycine premix: Per kilogram of glycine premix composition from diet 1 to 6 mined by the procedure described by Bessey et al. [43], was as follows (g/kg): L-glycine 524.58 g, 508.66 g, 492.75 g, 476.83 g, Rosalki et al. [44], Tanzer and Gilvarg [45] and Weng 460.92 g, 445.00 g, and corn starch 475.42 g, 491.34 g; 507.25 g; 523.17 g; et al. [46], respectively. Contents of malondialdehyde 539.08 g; 555.00 g, respectively Per kg of mineral premix: FeSO � H O (300 g/kg Fe), 25.00 g; CuSO � 5H O 4 2 4 2 (MDA) and protein carbonyl (PC) were determined (250 g/kg Cu), 0.60 g; ZnSO � 7H O (345 g/kg Zn), 4.35 g; MnSO � H O (318 g/kg 4 2 4 2 by the procedure described by Zhang et al. [47] and Mn), 2.04 g; KI (50 g/kg I), 1.10 g; NaSeO (10 g/kg Se), 2.50 g; MgSO � H O 3 4 2 (150 g/kg Mg), 230.67 g. All ingredients were diluted with corn starch to 1 kg BaltacIoglu et al. [48], respectively. The anti-superoxide Per kg of vitamin premix: retinyl acetate (500, 000 IU/g), 0.80 g; cholecalciferol anion (ASA) capacity and anti-hydroxyl radical (AHR) (500, 000 IU/g), 0.48 g; DL-α tocopherol acetate (500 g/kg), 20.00 g; menadione capacity were analyzed by using the superoxide anion free (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 radical detection Kit and hydroxyl free radical detection acetate (930 g/kg), 7.16 g; niacin (990 g/kg), 2.58 g; meso-inositol (990 g/kg), Kit (Nanjing Jiancheng Bioengineer Institute), respectively. 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 GSH contents were determined according to the method diluted with corn starch to 1 kg of Vardi et al. [49]. GR activity was determined according Crude protein and crude lipid contents were measured value. Available to Lora et al. [50]. SOD and GPx activities were detected phosphorus, n-3 and n-6 contents were calculated according to NRC (1993) according to Zhang et al. [47]. Activities of CAT and GST were determined according to Aebi [51] and Lushchak et al. [52], respectively. Feeding trial All experimental protocols were approved by Animal Care Advisory Committee of Sichuan Agricultural University. Statistical analysis Sub-adult grass carp were obtained from the Bai-long Results were present as means ± SD. Data were analyzed Lake Fisheries (Sichuan, China). After acclimatized to the with one-way analysis of variance (ANOVA). Differences experimental condition for 2 weeks, a total of 600 fish among dietary treatments were determined using the with an average weight of 441.9 ± 2.6 g were randomly dis- Duncan’s multiple-range test at the level of P < 0.05 tributed into 30 cages (1.4 m × 1.4 m × 1.4 m, a gauze disc through SPSS 18.0 for windows. Growth parameters (diameter, 0.8 m) was placed on the bottom of each cage with significant differences were subjected to second- to collect uneaten feed). Fish were fed to apparent sati- degree polynomial regression analysis. According to ation 4 times per day for 8 weeks. According to Cai et al. Abidi and Khan [33], quadratic regression analysis was Hong et al. Journal of Animal Science and Biotechnology (2015) 6:34 Page 4 of 11 used to estimate optimum dietary threonine requirement Intestine and hepatopancreas growth of sub-adult grass carp. 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 Results gut length (RGL) was not influenced by graded levels of Growth performance dietary threonine (P > 0.05). The intestosomatic index Effects of graded levels of dietary threonine on growth (ISI) of fish fed the basal diet was significantly lower than parameters are given in Table 2, weight gain (WG), spe- that of fish fed threonine-supplemented diets (P <0.05). cific growth rate (SGR) and feed intake (FI) were signifi- The intestinal protein content (IPC) also followed a simi- cantly improved as dietary threonine levels increased lar pattern to that as observed with intestinal length. The from 3.3 to 10.9 g/kg diet (P < 0.05), and decreased hepatopancreatic weight, hepatosomatic index (HSI) and thereafter (P < 0.05). Fish fed the basal diet (unsupple- hepatopancreatic protein content (HPC) were signifi- mented control group) showed the lower feed efficiency cantly improved with the supplementation of dietary (FE) and protein efficiency ratio (PER) compared to threonine (P < 0.05), and the maximum values were ob- those fed threonine-supplemented diets (P < 0.05). Re- tained when threonine levels were 10.9, 8.4 and 8.4 g/kg gression analysis showed that SGR, FI, FE and PER qua- diet, respectively. dratically responded to increased dietary threonine levels 2 2 (Y = −0.011X + 0.277X - 0.160, R = 0.989, P < 0.05; SGR Activities of intestinal enzymes 2 2 Y = −8.729X + 200.8X - 113.1, R = 0.989, P < 0.05; Y FI As shown in Table 5, intestinal activities of trypsin and 2 2 = −0.172X + 4.152X + 28.43, R = 0.914, P < 0.05; Y FE alpha-amylase were significantly improved with increas- 2 2 = −0.006X + 0.148X + 1.013, R = 0.915, P < 0.05). As PER ing dietary threonine levels up to 10.9 and 8.4 g/kg diet, shown in Figure 1, the dietary threonine requirement respectively (P < 0.05), and plateaued thereafter (P >0.05). of sub-adult grass carp (441.9-1,013.4 g) established The highest intestinal activities of chymotrypsin and lipase by quadratic regression analysis based on WG was were obtained when the threonine level was 8.4 g/kg diet. 11.6 g/kg diet, corresponding to 41.5 g/kg of dietary pro- As shown in Table 6, activities of AP in proximal in- 2 2 tein (Y = −5.284X + 123.0X - 169.5, R =0.986, P <0.05). testine (PI), mid intestine (MI) and distal intestine (DI) were significantly improved with the supplementation of Activities of GOT and GPT in muscle and hepatopancreas dietary threonine (P < 0.05), and the highest AP activities As shown in Table 3, activities of GOT in muscle and were observed for fish fed diets containing 13.1, 8.4 and hepatopancreas were improved with increasing of dietary 5.9 g threonine/kg diet, respectively. Fish fed the basal threonine levels up to 5.9 g/kg diet (P < 0.05). The GPT diet had significantly lower activities of γ-GT in PI and activity in muscle showed a similar trend with that of MI compared to those fed threonine-supplemented diets muscle GOT activity, and the highest value was obtained (P < 0.05). The highest activity of γ-GT in DI was ob- when threonine level was 10.9 g/kg diet (P < 0.05). How- tained in fish fed the diet containing 10.9 g threonine/kg ever, the GPT activity in hepatopancreas was decreased diet. Activities of CK in PI, MI and DI were significantly with increasing of dietary threonine levels up to 8.4 g/kg improved with the supplementation of dietary threonine diet (P < 0.05). (P < 0.05), and the maximum values were obtained when Table 2 Effects of dietary threonine levels on the growth performance of sub-adult grass carp Item Dietary Thr levels, g/kg diet 3.3 5.9 8.4 10.9 13.1 15.8 a a a a a a IBW, g/fish 442.0 ± 5.1 441.6 ± 2.3 442.6 ± 1.7 442.2 ± 1.3 441.6 ± 3.0 441.4 ± 2.0 a b c e d c FBW, g/fish 623.4 ± 20.4 817.4 ± 11.1 911.8 ± 26.3 1,013.4 ± 35.2 969.0 ± 28.2 896.6 ± 41.7 a b c e d c WG, g/fish 181.4 ± 19.1 375.8 ± 10.8 469.2 ± 26.4 571.2 ± 34.2 527.4 ± 27.8 455.2 ± 42.8 h a b c d d c SGR , %/day 0.61 ± 0.05 1.10 ± 0.02 1.29 ± 0.05 1.48 ± 0.06 1.40 ± 0.05 1.26 ± 0.09 a b d f e c FI, g/fish 464.4 ± 21.3 762.0 ± 14.9 926.1 ± 49.0 1,074.5 ± 29.5 1,023.0 ± 10.6 872.7 ± 5.4 i a b b b b b FE , % 38.98 ± 2.44 49.35 ± 2.20 50.83 ± 4.80 53.22 ± 3.94 51.57 ± 2.90 52.17 ± 5.06 j a b b b b b PER 1.39 ± 0.09 1.76 ± 0.08 1.81 ± 0.17 1.90 ± 0.14 1.84 ± 0.10 1.86 ± 0.18 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,f Means in the same row without a letter in common are significantly different (P< 0.05) Values are mean ± SD (n =5) Specific growth rate =100 × {[ln (mean final body weight)-ln (mean initial body weight)]/days} Feed efficiency (%) =100× weight gain (g)/diet intake (g) Protein efficiency ratio = weight gain (g)/protein intake (g) Hong et al. Journal of Animal Science and Biotechnology (2015) 6:34 Page 5 of 11 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 fish fed the diet containing 10.9 g threonine/kg diet. Na capacities in intestine and hepatoancreas were observed /K -ATPase activities in PI and MI were significantly in fish fed the diet containing13.1 g threonine/kg diet improved with increasing dietary threonine levels up to (P < 0.05). 5.9 g/kg diet (P < 0.05), and gradually decreased thereafter As listed in Table 8, the supplementation of threonine + + (P < 0.05). However, the activity of Na /K -ATPase in DI to certain levels increased GSH contents in intestine and was not affected by dietary threonine levels (P >0.05). hepatoancreas (P < 0.05), the highest GSH contents in intestine and hepatoancreas were observed in fish fed di- Antioxidant status in intestine and hepatopancreas ets containing 13.1 and 10.9 g threonine/kg diet, respect- As listed in Table 7, contents of MDA in intestine and ively. Antioxidant enzyme activities in intestine and hepatoancreas were significantly decreased with in- hepatoancreas were significantly affected by graded creasing threonine levels up to 8.4 and 10.9 g/kg diet levels of dietary threonine (P < 0.05). GR activity in the (P < 0.05), and thereafter increased (P < 0.05). The low- intestine was decreased with increasing the dietary est PC contents in intestine and hepatoancreas were threonine levels up to 8.4 g/kg diet (P < 0.05), However, observed in fish fed the diet containing 13.1 g threo- the trend of hepatopancreatic GR activity was opposite nine/kg diet. ASA capacities in intestine and hepatoan- to that in intestinal GR. Fish fed the basal diet had a sig- creas were the highest in fish fed diets containing 13.1 nificantly lower activity of intestinal SOD compared to and 10.9 g threonine/kg diet, respectively. The AHR those fed threonine-supplemented diets (P < 0.05). The Table 3 Effects of dietary threonine levels on activities of GOT and GPT in muscle and hepatopancreas of sub-adult grass carp Item Dietary Thr levels, g/kg diet 3.3 5.9 8.4 10.9 13.1 15.8 Muscle b c bc bc a a GOT, U/g protein 12.34 ± 0.89 13.73 ± 1.14 12.58 ± 1.21 12.72 ± 1.03 8.77 ± 0.89 8.24 ± 0.81 a c c e d b GPT, U/g protein 6.26 ± 0.55 11.61 ± 0.53 11.86 ± 0.39 14.41 ± 0.23 13.13 ± 0.47 9.61 ± 0.21 Hepatopancreas a b b b a a GOT, U/g protein 27.00 ± 2.32 34.35 ± 2.70 34.68 ± 3.23 35.24 ± 2.10 27.03 ± 2.03 27.09 ± 2.08 c b a a a a GPT, U/g protein 17.38 ± 0.82 16.44 ± 0.89 14.26 ± 0.51 14.78 ± 0.65 14.55 ± 0.62 14.87 ± 1.06 GOT: Glutamate oxaloacetate transaminase; GPT: Glutamate pyruvate transaminase a,b,c,d,e Means in the same row without a letter in common are significantly different (P< 0.05) Values are mean ± SD (n = 6) Hong et al. Journal of Animal Science and Biotechnology (2015) 6:34 Page 6 of 11 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 f a a ab b ab a IL , cm /fish 58.1 ± 5.9 58.5 ± 2.9 61.8 ± 3.2 63.9 ± 3.5 61.7 ± 3.2 59.6 ± 5.0 f a a a a a a RGL , % 149.0 ± 18.1 144.4 ± 7.6 144.6 ± 8.1 145.5 ± 7.7 145.3 ± 7.7 147.3 ± 10.8 f a bc cd e d b IW , g /fish 6.3 ± 0.8 9.8 ± 1.6 10.9 ± 1.6 14.0 ± 2.7 11.8 ± 1.5 9.2 ± 1.0 f a b b b b b ISI , % 1.04 ± 0.13 1.24 ± 0.21 1.16 ± 0.12 1.26 ± 0.18 1.25 ± 0.15 1.16 ± 0.12 g a bc c ab ab ab IPC , % 8.33 ± 0.63 9.57 ± 1.01 10.21 ± 1.02 8.90 ± 0.73 8.71 ± 0.89 8.49 ± 0.90 f a b c c c b HW , g /fish 12.1 ± 2.1 16.9 ± 2.5 23.7 ± 5.7 26.3 ± 7.1 23.4 ± 3.6 16.5 ± 3.3 f a ab c bc c ab HSI , % 1.98 ± 0.27 2.14 ± 0.35 2.51 ± 0.54 2.37 ± 0.48 2.49 ± 0.39 2.08 ± 0.36 g a a c b bc bc HPC , % 9.46 ± 0.50 9.12 ± 0.68 11.61 ± 0.79 10.60 ± 0.65 11.15 ± 0.42 10.98 ± 0.97 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,e Means in the same row without a letter in common are significantly different (P< 0.05) Values are mean ± SD (n = 15) Values are mean ± SD (n =6) highest activity of SOD in hepatoancreas was found in fish utilization. It is well known that GOT and GPT play an fed the diet containing 5.9 g threonine/kg diet (P <0.05). important role in amino acid metabolism, whose activity CAT activities in intestine and hepatoancreas were signifi- can be used to evaluate the utilization of essential amino cantly improved with increasing threonine levels up to acids in fish [53]. Results here showed that GOT and 10.9 and 8.4 g/kg diet, respectively (P < 0.05), and de- GPT activities in muscle, as well as GOT activity in hep- creased thereafter (P < 0.05). GST activity followed a simi- atopancreas were significantly increased with optimal lar pattern to that as observed with intestinal SOD threonine supplementation. This was consistent with activity, The highest activity of GST in hepatoancreas was our previous study [5]. However, GPT activity in hepato- found in fish fed the diet containing 5.9 g threonine/kg pancreas was decreased with the increment levels of diet (P < 0.05). The intestinal GPx activity was lower in dietary threonine up to a certain point. The reason for fish fed the diet containing 15.8 g threonine/kg diet than this result may attribute to the enhanced hepatic gluco- the other five treatment groups (P < 0.05).The GPx activity neogenesis induced by threonine deficiency. It was re- in hepatoancreas was improved with increasing the threo- ported that threonine deficiency increased hepatic nine levels up to 5.9 g/kg diet (P < 0.05), and decreased to gluconeogenesis in rats [54]. GPT is a rate-limiting en- a plateau thereafter. zyme in the conversion of protein to carbohydrate, whose activity in rat liver can be enhanced by the in- Discussion creased hepatic gluconeogenesis [55]. However, this hy- In the present study, the growth performance of sub- pothesis needs further investigation in fish. Additionally, adult grass carp was significantly influenced by dietary the trend of GPT activity in hepatopancreas was oppos- threonine levels. WG, SGR, FI and FE of sub-adult grass ite with that of our previous study in juvenile Jian carp carp were significantly improved by dietary threonine, [5]. The reason for these results is not clear. A possible which were in agreement with reports for juvenile grass explanation might be related to the differences in carp [10], juvenile Jian carp [5] and fingerling Indian threonine metabolism in different growth stage, as de- major carp [3]. In this study, the improved fish growth scribed in terrestrial animals [56, 57]. Further research may be partly attributed to the promotion of amino acid is needed to clarify this hypothesis. Based on the Table 5 Effects of dietary threonine levels on activities of tryspin, chymotrypsin, alpha-amylase and lipase in intestine of sub-adult grass carp Item Dietary Thr levels, g/kg diet 3.3 5.9 8.4 10.9 13.1 15.8 a b b c c c Trypsin, U/g protein 177.2 ± 10.2 239.9 ± 12.5 252.6 ± 12.1 291.2 ± 16.8 286.1 ± 10.0 294.0 ± 10.6 a a c b b b Chymotrypsin, U/g protein 1.03 ± 0.09 1.00 ± 0.08 2.29 ± 0.15 1.58 ± 0.14 1.45 ± 0.14 1.48 ± 0.12 a a b b b b alpha-amylase, U /mg protein 2.46 ± 0.12 2.51 ± 0.19 2.84 ± 0.09 2.75 ± 0.06 2.73 ± 0.04 2.77 ± 0.04 ab bc c c bc a Lipase, U/g protein 19.11 ± 1.29 20.97 ± 1.77 22.71 ± 2.00 22.55 ± 2.41 21.06 ± 2.34 17.93 ± 1.26 a,b,c Means in the same row without a letter in common are significantly different (P< 0.05) Values are mean ± SD (n =6) Hong et al. Journal of Animal Science and Biotechnology (2015) 6:34 Page 7 of 11 + + f Table 6 Effects of dietary threonine levels on activities of AP, γ-GT, CK and Na /K -ATPase in intestine of sub-adult grass carp 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 a b cd d e bc PI 69.5 ± 7.3 84.4 ± 8.1 102.8 ± 10.1 108.0 ± 10.7 125.0 ± 10.4 92.8 ± 9.1 c cd d cd b a MI 110.1 ± 7.0 120.3 ± 13.0 125.8 ± 4.9 117.6 ± 8.2 91.9 ± 8.6 71.3 ± 7.4 c d c b b a DI 72.2 ± 6.9 80.3 ± 7.5 69.0 ± 5.4 54.7 ± 5.3 53.8 ± 2.6 35.8 ± 2.8 γ-GT, mmol of 5-amino-2-nitrobenzoate released g/protein per min a b c c d b PI 1.82 ± 0.16 2.27 ± 0.15 2.59 ± 0.28 2.52 ± 0.15 3.00 ± 0.16 2.24 ± 0.23 a b b b b b MI 2.12 ± 0.16 2.89 ± 0.26 3.05 ± 0.21 3.09 ± 0.17 2.93 ± 0.19 2.94 ± 0.28 a b c c c ab DI 0.73 ± 0.08 0.87 ± 0.11 1.20 ± 0.07 1.23 ± 0.12 1.13 ± 0.11 0.76 ± 0.06 CK, μmol of phosphorus released g/protein per h c c c d b a PI 2.62 ± 0.25 2.72 ± 0.19 2.74 ± 0.24 3.11 ± 0.29 1.52 ± 0.13 1.14 ± 0.13 a c c d d b MI 1.36 ± 0.09 2.40 ± 0.18 2.35 ± 0.17 4.47 ± 0.44 4.28 ± 0.34 1.77 ± 0.17 a a a b b a DI 6.69 ± 0.73 7.26 ± 0.44 6.96 ± 0.67 9.35 ± 0.45 8.86 ± 0.70 7.15 ± 0.75 + + Na /K -ATPase, μmol of phosphorus released g/protein per h b c c b b a PI 0.37 ± 0.06 0.42 ± 0.05 0.41 ± 0.02 0.36 ± 0.03 0.37 ± 0.03 0.30 ± 0.03 c d bc ab a a MI 0.48 ± 0.04 0.62 ± 0.04 0.47 ± 0.03 0.43 ± 0.04 0.39 ± 0.03 0.39 ± 0.04 a a a a a a DI 0.35 ± 0.02 0.34 ± 0.02 0.35 ± 0.02 0.36 ± 0.01 0.35 ± 0.03 0.35 ± 0.02 AP: Alkaline phosphatase; γ-GT: γ-Glutamyl transpeptidase; CK: Creatine kinase; PI: Proximal intestine; MI: Mid intestine; DI: Distal intestine a,b,c,d,e Means in the same row without a letter in common are significantly different (P< 0.05) Values are mean ± SD (n =6) quadratic regression analysis for WG, the requirement weight, ISI and IPC, as well as hepatopancreatic weight, of threonine for sub-adult grass carp (441.9-1,013.4 g) HSI and HPC content. Meanwhile, activities of trypsin, was estimated to be 11.6 g/kg diet, corresponding to chymotrypsin, alpha-amylase, lipase, AP, γ-GT and CK + + 41.5 g/kg of dietary protein. in whole intestine, as well as Na /K -ATPase in PI and Fish growth relies on nutrient utilization, which is re- MI were improved by dietary threonine. All these data lated to the digestive and absorptive capacity [4]. Gener- above suggested that threonine improved the digestive ally, fish digestive and absorptive capacity can be and absorptive capacity of sub-adult grass carp, which reflected by digestive organ growth and development, as were in agreement with our previous study of juvenile well as activities of intestinal enzymes related to diges- Jian carp [5]. It is well known that digestive enzymes in tion and absorption [58]. In our study, there were sig- intestinal lumen are mainly secreted from pancreas [59]. nificant improvements in intestinal length, intestinal Hokin [60] reported that threonine was necessary for Table 7 Effects of dietary threonine levels on MDA, PC, AHR and ASA in intestine and hepatoancreas of sub-adult grass carp Item Dietary Thr levels, g/kg diet 3.3 5.9 8.4 10.9 13.1 15.8 Intestine d c a b b b MDA, nmol/mg protein 2.97 ± 0.23 1.98 ± 0.19 1.33 ± 0.11 1.51 ± 0.07 1.53 ± 0.11 1.57 ± 0.10 c ab b ab a ab PC, nmol/mg protein 4.37 ± 0.34 3.41 ± 0.25 3.52 ± 0.23 3.19 ± 0.29 3.10 ± 0.18 3.34 ± 0.30 a b c e e d AHR, U/mg protein 120.5 ± 9.9 175.2 ± 12.1 192.9 ± 14.1 266.9 ± 11.1 278.3 ± 12.8 227.6 ± 17.7 b b b b c a ASA, U/g protein 243.5 ± 14.7 253.2 ± 18.3 252.2 ± 17.6 255.9 ± 22.3 278.1 ± 17.2 208.5 ± 14.5 Hepatopancreas e d b a c c MDA, nmol/mg protein 2.38 ± 0.19 2.17 ± 0.15 1.57 ± 0.13 1.37 ± 0.13 1.95 ± 0.18 1.92 ± 0.10 c c b b a c PC, nmol/mg protein 6.08 ± 0.41 6.21 ± 0.41 4.99 ± 0.50 5.11 ± 0.36 4.24 ± 0.38 5.90 ± 0.52 a a a a b a AHR, U/mg protein 214.6 ± 14.8 217.2 ± 11.6 218.9 ± 5.4 222.4 ± 7.0 266.1 ± 10.5 224.2 ± 13.3 a a a b a a ASA, U/g protein 220.8 ± 13.8 224.0 ± 19.2 237.2 ± 16.1 264.9 ± 22.8 241.4 ± 22.0 230.4 ± 12.2 MDA: Malondialdehyde content; PC: Protein carbonyl content; AHR: Anti-hydroxyl radical capacity; ASA: Anti-superoxide anion capacity a,b,c,d,e Means in the same row without a letter in common are significantly different (P< 0.05) Values are mean ± SD (n =6) Hong et al. Journal of Animal Science and Biotechnology (2015) 6:34 Page 8 of 11 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 carp Item Dietary Thr levels, g/kg diet 3.3 5.9 8.4 10.9 13.1 15.8 Intestine a a b c d c GSH, mg/g protein 1.81 ± 0.16 1.83 ± 0.13 2.56 ± 0.19 3.00 ± 0.19 5.63 ± 0.22 2.98 ± 0.16 b b a a c d GR, U/g protein 23.15 ± 1.22 24.57 ± 1.95 17.05 ± 1.36 19.27 ± 1.78 27.65 ± 2.82 33.59 ± 3.11 a b b b b b SOD, U/mg protein 19.84 ± 1.71 23.87 ± 2.33 23.62 ± 1.13 23.41 ± 2.19 24.83 ± 1.44 23.45 ± 1.84 a c c d c b CAT, U/mg protein 11.70 ± 1.03 16.65 ± 1.48 16.77 ± 1.31 22.59 ± 1.94 18.06 ± 1.26 14.41 ± 1.46 a b b b b b GST, U/mg protein 7.08 ± 0.75 17.41 ± 1.58 16.82 ± 1.48 16.13 ± 1.41 17.36 ± 1.33 17.41 ± 1.58 b b b b b a GPx, U/mg protein 105.4 ± 9.7 109.4 ± 6.6 109.3 ± 4.1 107.4 ± 9.3 104.9 ± 5.7 87.7 ± 8.8 Hepatopancreas b c c d b a GSH, mg/g protein 6.13 ± 0.30 7.07 ± 0.41 7.06 ± 0.31 7.83 ± 0.39 6.08 ± 0.29 5.67 ± 0.31 a c b b b b GR, U/g protein 4.08 ± 0.31 13.22 ± 1.02 7.74 ± 0.80 7.91 ± 0.59 7.36 ± 0.73 7.58 ± 0.75 b c b b b a SOD, U/mg protein 83.16 ± 8.65 100.42 ± 6.19 80.39 ± 7.06 84.66 ± 4.00 78.72 ± 5.17 66.57 ± 3.08 a a c b a a CAT, U/mg protein 55.67 ± 5.95 59.11 ± 5.39 79.98 ± 7.11 69.65 ± 7.34 57.94 ± 6.91 56.51 ± 5.61 b d d c c a GST, U/mg protein 22.51 ± 2.05 45.73 ± 2.73 44.27 ± 3.78 25.80 ± 2.68 25.77 ± 2.48 17.34 ± 1.66 a c b b ab ab GPx, U/mg protein 961.5 ± 79.2 1,205.9 ± 44.5 1,041.0 ± 55.3 1,053.9 ± 57.1 1,026.2 ± 46.4 995.7 ± 47.2 GSH: Glutathione; GR: Glutathione reductase; SOD: Superoxide dismutase; CAT: Catalase; GST: Glutathione-S-transferase; GPx: Glutathione peroxidase a,b,c,d Means in the same row without a letter in common are significantly different (P< 0.05) Values are mean ± SD (n =6) the pancreatic alpha-amylase synthesis in pigeons. capacity of ASA and AHR has not yet been reported in Meanwhile, threonine increased pancreatic secretion of fish. A possible reason for the improved capacity of AHR trypsin, alpha-amylase and chymotrypsinogen in chicks might be that threonine enhanced mucin synthesis. Stud- [61]. Besides, threonine was found to be served as an es- ies showed that intestinal mucin synthesis in piglets [67] sential component of the active center in γ-GT of rats and rats [68] were increased by threonine. Meanwhile, [62] and CK of chicken [63]. However, the mechanism in vitro biochemical assays, pig stomach mucins could which threonine improved the digestive and absorptive scavenge hydroxyl radicals [19]. Besides, the increased capacity of fish needs further study. AHR capacity might be also related to the ability of threo- In fish, the normal function of the digestive organ is nine to chelate metal ions. In living organisms, the forma- correlated with its antioxidant status [11]. The contents tion of hydroxyl radicals could be induced by free of products of lipid peroxidation and protein oxidation, transition metal ions, such as iron, copper and manganese, such as MDA and PC, can reflect the antioxidant status via the Fenton-Haber Weiss reaction [14]. In the stomach of living organisms [64]. In the present study, contents of rats, the formation of hydroxyl radicals was reduced by of MDA and PC were decreased with increasing dietary chelating iron ions [15]. Threonine was found to chelate threonine levels up to certain values in both intestine with manganese ions in the liver of rats [18] and iron and and hepatopancreas, suggesting depressions of the lipid copper ions in vitro biochemical assays [16, 17]. Thus, peroxidation and protein oxidation. To date, there were threonine might be able to decrease lipid peroxidation no studies about the effect of threonine on the lipid per- and protein oxidation in fishdigestive organ byimprov- oxidation and protein oxidation in fish. Using biochem- ing radical scavenging abilities in these organs, which ical in vitro assays, it was demonstrated that threonine warrants further study. reduced autoxidation rates of safflower oil in liquid In fish, free radicals can be scavenged by non-enzymatic emulsions [65]. As we all know, the lipid peroxidation antioxidants, such as vitamin C, vitamin E and GSH [21]. and protein oxidation are induced by ROS, among which GSH is a direct free radical scavenger in fish [22]. In the superoxide and hydroxyl radicals are most strongly in- present study, both intestinal and hepatopancreatic GSH volved in oxidative damages [13, 66]. In our study, both contents of sub-adult grass carp were increased with opti- ASA capacity and AHR capacity in intestine and hepato- mal threonine supplementation. To date, information on pancreas were enhanced by dietary threonine, suggesting the relationship between dietary threonine levels and GSH contents is limited in fish. Generally, cellular GSH the improved scavenging abilities against superoxide anion and hydroxyl radicals. To date, information on homeostasis is maintained through de novo GSH syn- the relationship between dietary threonine levels and thesis, glutathione disulfide (GSSG) reduction and Hong et al. Journal of Animal Science and Biotechnology (2015) 6:34 Page 9 of 11 uptake of extracellular GSH [69]. In this study, the in- threonine residue was essential for the structure creased intestinal GSH contents by dietary threonine stabilization of Nrf2 in HEK-293 T cells [77]. Kobayashi might be related to the increased uptake of extracellular et al. [29] found that Nrf2 existed in zebrafish. Thus, GSH. It was reported that biliary GSH, which was se- beneficial effects of threonone on antioxidant enzyme creted by liver, was one of the major sources of intes- activities might be partly attributed to the enhanced acti- tinal GSH in rats [70]. Lauterburg et al. [71] found that vation of Nrf2. However, this hypothesis needs further an increase in liver GSH content was associated with investigations. GPx protects cells from excessive levels of increased intestinal GSH contents in rats. In terrestrial H O and intracellular lipid peroxides by formation of 2 2 animals, luminal GSH was uptake by intestine epithe- GSSG [78]. In our study, threonine enhanced hepato- lial cells in two ways: (1) be transported intact into pancreatic GPx activity of sub-adult grass carp. However, cells; (2) be cleaved into glutamate and cysteinylgly- in the intestine, GPx activity was not improved by diet- cine by γ-GT, and then γ-GT transported the cysteinyl- ary threonine, but was decreased by excess threonine in- glycine into the cell for re-synthesis of GSH [72, 73]. In take. A possible reason for this phenomenon might be our study, intestinal GSH content was positively related the reduced intestinal mucin synthesis by excess threo- to the γ-GT activity in PI (r = + 0.838, P < 0.05), which nine intake. Wang et al. [79] reported that excessive might suggest that luminal GSH was mainly uptake by level of dietary threonine reduced mucin synthesis in intestine epithelial cells of sub-adult grass carp in the small intestine of pigs. A decreased content of pig stom- second pathway. However, this hypothesis needs further ach mucins was associated with a decrease of hydroxyl investigation. Liver is the primary site for de novo GSH radical scavenging ability in vitro biochemical assays synthesis in rats, which requires the participation of [19]. Tabatabaie and Floyd [80] found that GPx of bo- ATP [23]. Ross-Inta et al. [24] reported that dietary vine erythrocytes was inactivated by hydroxyl radicals threonine increased the liver ATP level of rats. However, in vitro. However, further studies are needed to deter- whether this ATP synthesis promotion effect of threo- mine this hypothesis in fish. nine also exists in fish needs study. In the present study, the increased hepatopancreatic GSH content Conclusions mayalsobeattributedtothe promotionofGSSG re- Diets containing the appropriate amount of threonine duction. GR catalyses the reduction of GSSG back to improved growth, increased digestive and absorptive GSH [74]. Threonine improved GR activity in hepato- capacity, and enhanced intestinal and hepatopancreatic pancreas of sub-adult grass carp, indicating the im- antioxidant defense of sub-adult grass carp. Based on proved GSSG reduction. However, the trend of the quadratic regression analysis for WG, the require- intestinal GR activity was opposite with that in hep- ment of threonine for sub-adult grass carp (441.9- atopancreas. A possible reason for this result is that 1,013.4 g) was estimated to be 11.6 g/kg diet, corre- intestinal GR activity was inactivated by GSH. Ogus sponding to 41.5 g/kg of dietary protein. and Ozer [75] reported that human intestinal GR ac- Abbreviations tivity was inactivated by GSH in vitro.The reason for AHR: Anti-hydroxyl radical; AP: Alkaline phosphatase; ASA: Anti-superoxide GSH not inhibiting GR activity in hepatopancreas anion; CAT: Catalase; CK: Creatine kinase; DI: Distal intestine; FE: Feed efficiency; FI: Feed intake; GOT: Glutamate oxaloacetate transaminase; might be that GSH in the liver is maintained mainly in GPT: Glutamate pyruvate transaminase; GPx: Glutathione peroxidase; the reduced state, and which is highly dependent on GR: Glutathione reductase; GSH: Glutathione; GSSG: Glutathione disulfide; GR activity, as it was reported by Kaplowitz et al. [76]. GST: Glutathione-S-transferase; γ -GT: γ -glutamyl transpeptidase; HPC: Hepatopancreatic protein content; HSI: Hepatosomatic index; However, further studies are needed to test this IPC: Intestinal protein content; ISI: Intestosomatic index; hypothesis. MDA: Malondialdehyde; MI: Mid intestine; Nrf2: E2 p45-related factor 2; Aside from the antioxidants, antioxidant enzymes, PC: Protein carbonyl:; PER: Protein efficiency ratio; PI: Proximal intestine; RGL: Relative gut length; ROS: Reactive oxygen species; SGR: Specific growth such as SOD, CAT, GST and GPx, also play an import- rate; SOD: Superoxide dismutase; Thr: Threonine; WG: Weight gain. ant role in protecting cells against free radical damages [13]. The present study showed that threonine enhanced Competing interests The authors declare that they have no competing interests. intestinal and hepatopancreatic activities of SOD, CAT and GST, suggesting the improved enzymatic antioxi- Authors’ contributions dant ability. To date, few studies have evaluated effects All authors made significant contributions to perform the research. Especially X-Q Z designed the study and drafted the initial manuscript. All authors read of threonine on activities of antioxidant enzymes in fish. and agreed the final manuscript. It has been demonstrated that expressions of SOD, CAT and GST are controlled by Nrf2-ARE system in bone Acknowledgements The authors would like to thank National 973 Project of China (2014CB138600), marrow stromal cells of mice [27]. Meanwhile, the National Department Public Benefit Research Foundation (Agriculture) of China threonine phosphorylation was involved in Nrf2 activa- (201003020), Science and Technology Support Programme of Sichuan Province tion in lung of mice [28]. Furthermore, the conserved of China (2014NZ0003), and Major Scientific and Technological Achievement Hong et al. Journal of Animal Science and Biotechnology (2015) 6:34 Page 10 of 11 Transformation Project of Sichuan Province of China (2012NC0007 and 20. Neuhaus H, Van der Marel M, Caspari N, Meyer W, Enss ML, Steinhagen D. 2013NC0045) for their financial support. 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Journal
Journal of Animal Science and Biotechnology – Springer Journals
Published: Aug 8, 2015