Xu Hong,Zhu Qing,Wang Chang-an,Zhao Zhi-gang,Luo Ling,Wang Lian-sheng,Li Jin-nan,and Xu Qi-you

Heilongjiang River Fisheries Research Institute of Chinese Academy of Fishery Sciences,Harbin 150070,China

Introduction

Glutamine (Gln) is the most abundant amino acid in the bloodstream,accounting for 30% to 35% of the amino acid nitrogen in the plasma and in the free amino acid pool in the body (Newsholme et al.,1985).Gln is an important precursor for the synthesis of the amino acids,nucleotides,amino sugars,proteins,and many other biologically important molecules (Souba,1993).Gln increases protein synthesis and inhibits protein degradation in skeletal muscle,thereby promoting a positive nitrogen balance under catabolic conditions (MacLennan et al.,1987).Gln is the main energetic substrate for rapidly proliferating cells such as intestinal enterocytes and activated lymphocytes (Calder and Yaqoob,1999) and influences mammalian target of the rapamycin (mTORC1) activity independent of its effects on the cell content of the leucine (Chiu,2012).The intestine is a major organ of Gln utilization and converts Gln into other amino acids (Windmueller and Spaeth,1980;Yoshida et al.,1995).A lack of Gln promotes mucosal atrophy,an increase in intestinal permeability and bacterial translocation,and a reduced synthesis of glutathione (Schroder et al.,1995;Blaauw et al.,1997).Gln is considered a conditionally essential amino acid in some species under inflammatory conditions,such as infection and injury (Newsholme,2001).

Dietary supplementation with Gln improves weight gain,feed efficiency,intestinal histological structures and/or digestive enzyme activities in juveniles of Jian carp,Cyprinus carpio var.Jian (Lin and Zhou,2006),and red drum,Sciaenops ocellatus (Cheng et al.,2011).However,Gln is unstable in aqueous solutions,particularly under acid conditions and during heating and storage,which leads to the formation of potentially toxic substances,such as pyroglutamic acid (Gerly et al.,2005).The above mentioned limitations resulted in an intensive search for alternative Gln sources and precursors.Among them,dipeptides appeared to be a reasonable candidates,because the dipeptide molecule with Gln at C-terminal position fulfills all chemical and physical criteria needed for approval by the authorities for the composition of the parenteral solutions (Filip,2008).Using this stable form of Gln,we examined the effects of the exogenous Ala-Gln on the growth performance,the intestinal development,the antioxidative defense status and the plasma nonspecific immune factors of juvenile Songpu Mirror carp.

Materials and Methods

Experimental design and diets

Young Songpu mirror carp (Cyprinus carpio L.) were divided into six treatments.Aln-Gln was incorporated into the basal diet at concentrations of 0,2.5,5.0,7.5,10.0 and 15.0 g · kg-1of the dry feed weight,respectively,the experimental diet formulation and the approximate composition are shown in Table 1.Diets were made isonitrogenous by adjusting the amounts of Aln-Gln and glycine.Aln-Gln (99.0% purity) was purchased from Shanghai Jiancheng Chemical Co.,Ltd (Shanghai,China).The other feed ingredients were purchased from Wangshui Feeds Co.,Ltd (Harbin,China).These ingredients were mixed with Aln-Gln at definite concentrations,the mixture was then extruded and air-dried at room temperature,and maintained at –20℃ until further use.

Table1 Formula and nutritional levels of experimental diets as g · kg-1 dry matter

Culture condition

Songpu Mirror carps were obtained from Songpu Station of Heilongjiang River Fisheries Research Institute,China.The fish were acclimated in tanks and fed with a basal diet 3 times daily for 2 weeks.Then,healthy carp with an average initial weight (12.97±0.18 g) were randomly assigned to 18 aquaria (150 L) with an initial stocking density of 15 fish per aquarium.The experiments were conducted in triplicate groups and cultured for 12 weeks.

For the feeding trail,fish were fed by hand,3 times daily at 8:00 a.m.,13:00 p.m.,and 18:00 p.m.,at a rate of 4.0% body weight per day.The fish in each aquarium were weighed at the start and every two weeks during the 12 weeks of the experimental period.Fish were starved for 24 h before weighing.Water temperature and dissolved oxygen during this period averaged (22.5±1.0)℃ and (7.52±0.76) mg · L-1,respectively,and ammonia-N concentrations were not in excess of 0.46 mg · L-1.

Sample collection and analysis

Fish were counted and weighed at the beginning and end of the experiment.The growth performance,feed conversion ratio,and protein efficiency ratio were determined according to Cho and Kaushik (1985) as the followings:

Weight gain rate (WGR)= (Final total weight–Initial total weight)/Initial total weight×100%;

Feed conversion ratio (FCR)=Feed fed (g)/Wet weight gain (g);

Protein efficiency ratio (PER)=Weight gain (g)/ Protein fed (g)

Before sampling,the fish were starved for 24 h,nine fishes were sampled from each experimental group and euthanized with an overdose of tricaine methane sulphonate (MS-222).Blood samples were extracted from the caudal veins with a syringe (5 mL) and centrifuged at 5 000 r · min-1for 10 min.The separated plasma samples were then stored at –80℃ until analyzed.After the blood sampling,the hepatopancreas,intestine and back muscles were removed from the same fish and immediately placed in liquid nitrogen and maintained at –80℃ for further using.Tissue samples were subjected to ice-cold homogenization by using an Ultra-Turrax homogenizer diluted in physiological saline solution (NaCl 0.86%) at a concentration of 1:9 (wet weight: volume).All the homogenates were centrifuged at 4 000 g for 10 min at 4℃.The supernatant samples were frozen and stored at–80℃ until analyzed.

Protease activity was measured by the method of Lowry et al.(1951),lipase activity was determined according to a modified method based on Gjellesvik et al.(1992),and amylase activity was quantified using a solution to detect non-hydrolyzed starch (Worthington,1993).The activity of Na+,K+-ATPase,superoxide dismutase (SOD),glutathione peroxidase (GPX),malondialdehyde (MDA),glutathione (GSH),lysozyme (LSZ) and protein content of the tissues were determined by the kit which made by Nanjing Jiancheng Bioengineering Institute (Nanjing,China) according to the manufacturer's instructions,respectively.Na+,K+-ATPase activity in tissues was expressed as μmol Pi per h per mg protein,1 U of Na+,K+-ATPase was defined as ATPase of per mg protein tissue resolved ATP into 1 μmol inorganic phosphorus per hour.SOD activity in tissues was measured by chemical colorimetry and was expressed as a nitrite unit (NU) per milligram protein,1 NU was defined as the amount of SOD that inhibited the rate of the cytochrome c reduction by 50%.GPX activity in tissues was measured by chemical colorimetry and expressed as a unit of activity (U) per milligram protein,1 U of GPX was defined as the production of 1 mg hydroxybenzene by reaction for 5 min at 37℃.MDA concentration was measured by an improved colorimetric method and was expressed as nanomoles per milligram protein.GSH concentration in tissues was measured by chemical colorimetry.LSZ activity was determined by adding 200 μL of the plasma sample to 1 800 μL suspension of Micrococcus lysodeikticus (0.2 mg · mL-1) in sodium phosphate buffer.The reaction was carried out at 37℃ for 15 min,and the absorbance of the suspension was measured at 530 nm by spectrophotometry.The total plasma levels of the humoral complement C3 and C4 were measured by nephelometric analyses (MD-100 nephelometer analyser,Japan).

Another six fishes were sampled from each experimental group and a 1 cm in the mid of each segment intestine was sampled (Lin,1998),and the intestinal sample fixed with formalin (10%),sliced and stained with HE dye for histological analyses.The morphometric variables analyzed in intestine mucosa included: fold height and number of folds.

Statistical analysis

An analysis of the variance was performed on the data using SAS ANOVA procedure.Duncan's Multiple Range Test was used to compare differences among individual means.Results were presented as the mean±SD.Differences were considered significant at the level of P<0.05.Broken line regression procedure was used to determine the breakpoint that represents the optimal dietary Aln-Gln (Robbins et al.,1979).

Results

Growth and feed efficiency

Supplementing diets with 7.5,10.0,or 15.0 g · kg-1of Aln-Gln caused a significant increase in WGR by 26.31%,28.91%,and 29.36% compared to fish fed the control diet (Table 2).PER also increased significantly (P<0.05) by 22.90%,25.23%,and 28.04% in fish fed diets supplemented with Aln-Gln at a concentration of 7.5,10.0,and 15.0 g · kg-1,respectively.However,FCR and survival of the fish were similar between treatments,and no significant differences were observed (P>0.05).

Table2 Effects of Aln-Gln supplementation on growth and feed efficiency of Mirror carp

Optimal Aln-Gln contents associated with WGR was based on broken-line regression model analysis.Associated optimal dietary Aln-Gln contents for WGR was 8.24 g · kg-1diet (Fig.1).

Intestine histological analysis and digestive enzyme activities

Intestinal histological analysis of the experimental carp is presented in Table 3.Intestine fold height and number were significantly enhanced with increasing dietary Aln-Gln supplementation up to 7.5 g · kg-1(P<0.05),and no further improvement was seen with Aln-Gln levels higher than 7.5 g · kg-1(P>0.05).

Intestinal digestive enzyme and Na+,K+-ATPase activities are presented in Table 4.Protease,lipase,amylase and Na+,K+-ATPase activities of all the intestinal segments increased significantly with increasing dietary supplementation with Aln-Gln concentrations from 0.0 to 7.5 g · kg-1(P<0.05),and there was no significant difference in Aln-Gln supplementation from 7.5 to 15.0 g · kg-1(P>0.05).

Plasma non-specific immune index

Dietary supplementation with different concentrations of Aln-Gln resulted in different results in the levels of the plasma LSZ activity,C3,and C4 (Table 5).Plasma LSZ activity increased significantly (P<0.05) with an increasing Aln-Gln concentrations from 0.0 to 7.5 g · kg,but there was no significant difference in Aln-Gln supplementation from 7.5 to 15.0 g · kg-1.The maximum plasma C3,C4 level was observed in fish fed with a diet supplemented with 5.0 g · kg-1Aln-Gln (P<0.05),but there was a gradual reduction in the level with an increase in the concentration of 7.5 (P<0.05),and 10.0,15.0 g · kg-1(P<0.01) Aln-Gln.

Fig.1 Optimal Aln-Gln levels for Mirror carp to keep higher WGR

Table3 Effects of Aln-Gln supplementation on intestinal morphological measurements of Mirror carp

Antioxidative indices

The effect of Aln-Gln supplementation on antioxidant levels are shown in Table 6.It was evident that the activities of the intestine,plasma,hepatopancreas and muscle GPX,GSH and SOD increased with an increase in relation to Aln-Gln supplementation in diets.Compared with control group,plasma GPX activity increased significantly by 6.23%,6.95%,and 63.93% when fish were supplemented with 7.5,10.0(P<0.05),or 15.0 g · kg-1(P<0.01) of Aln-Gln.SOD activity increased significantly (P<0.05) by 12.71%,15.80%,18.23%,and 18.61% when supplemented with 5.0 (P<0.05),7.5,10.0,and 15.0 g · kg-1(P<0.01),respectively.The intestinal GPX activity increased by 2.96% (P<0.05),5.17% (P<0.05),5.71% (P<0.05),and 5.81% (P<0.05) in fish fed the diets supplemented with 5.0,7.5,10.0 and 15.0 g · kg-1Aln-Gln,respectively.The intestine,plasma,hepatopancreas and muscle MDA levels significantly decreased,when supplemented with 5.0,7.5,10.0 (P<0.05),or 15.0 g · kg-1(P<0.01) Aln-Gln.

Table4 Effect of Aln-Gln supplementation on intestinal digestive enzyme and Na+,K+-ATPase activities of Mirror carp

Table5 Effects of Aln-Gln supplementation on plasma non-specific immune parameters of carp

Table6 Effects of Aln-Gln supplementation on antioxidation of Mirror carp

Continued

Discussion

It was clearly found that the administration of Aln-Gln via the basal diets had beneficial effect on young carp growth performance.WGR and PER values of fish all increased with increasing dietary Aln-Gln supplementation up to a point.Similar findings have been reported on sturgeon (Wang et al.,2011;Xu et al.,2011).It might be related to the beneficial effect of Aln-Gln that promoted the synthesis of the muscle protein and reduced its degradation.Millward et al.(1989) demonstrated that muscle Gln concentration correlated directly with the rate of muscle protein synthesis and RNA/protein ratio and was inversely correlated with the rate of protein degradation.Similarly,MacLennan et al.(1987) used a perfused rat hindlimb model to increase intramuscular Gln concentrations and found that increasing intramuscular Gln increased protein synthesis.

For stomachless fish such as carp,the intestine plays a key role as the site of nutrient digestion and absorption,and digestive function correlates with intestinal development.Compared to terrestrial animals,the mucosa structure is simple in aquatic species and form folds only (Lin and Zhou,2006).The intestinal folds in aquatic animals are presented instead of the intestinal villus in terrestrial animal.Intestinal fold height is regarded as a sign of the absorption ability in aquatic animals (Olli and Krogdahl,1994;Farhangi et al.,2001).The present study showed significant changes in the mucosal architecture in terms of increased fold height and number in fish fed with an Aln-Gln supplemented diet.The increase in fold height and number that were observed might indicate that the fish fed diets supplemented with Aln-Gln might have greater nutrient absorption and utilization because increases in fold height and number resulted in more surface area for nutrient utilization.The increase in surface area might also explain the significantly improved weight gains that were observed due to Aln-Gln supplementation.Wu et al.(1996) reported that fold height in the duodenum increased as dietary Gln level increased from 0.0 to 1.0%,the same results were reported on carps (Lin ＆Zhou,2006),sturgeon (Wang et al.,2011;Xu et al.,2011),rat (Tannuri et al.,2000),chickens (Bartell and Batal,2007),and pigs (Cabrera,2013).Increased villi height has been proposed to increase performance by improving nutrient absorption (Coates et al.,1954;Izat et al.,1989).Dietary supplementation of Gln might reduce bacterial utilization and degradation of host-derived Gln or modulate the utilization of AA by the host and gut bacteria,thereby helping to maintain gut integrity and function and also reduce occurrence of the bacterial invasion and infection (Dai,2013).

Digestion ability is positively correlated with digestive enzyme activity,as nutrients are digested by these enzymes.In this study,intestine digestive enzyme activity was positively correlated with dietary Aln-Gln supplementation levels.Lin and Zhou (2006) suggested that the enhancement of intestinal digestive enzyme activity after Gln supplementation was positively related to the development of hepatopancreas in carp.The addition of Aln-Gln with a certain content improved intestine digestive enzyme activity,which might consequently explain better growth performances observed due to Aln-Gln supplementation.Similar results were also observed by Rebecca et al (1998).

ATPase (adenosine triphosphatase) plays important roles in osmoregulation,cytosol and humoral fluids,and maintaining several ions at equilibrium physiological levels.Under physiologic conditions,Gln oxidation can account for as much as one-third of cellular ATP production in many cultured cells (Spolarics et al.,1991).Na+,K+-ATPase is responsible for the osmoregulation of Na+and K+ions by trans-ferring ions at the expense of the energy supplied by ATP decomposition.Some nutrients such as amino acids and glucose are absorbed in association with absorption of Na+by Na+,K+-ATPase (Lin and Zhou,2006).The activity of this enzyme indirectly reflects absorption ability (Rhoads et al.,1994).In the present study,the activity of Na+,K+-ATPase significantly increased with the increase of the dietary Aln-Gln level.Na+,K+-ATPase activity was high in the mid-gut,which suggested that Aln-Gln might promote intestinal absorption ability in fish.Similar results were also observed by Lin and Zhou (2006) on carps,Wang et al.(2011) on sturgeon and Xu et al.(2011) on sturgeon.

As the first line of the defense,various peptides,such as lysozyme,complement factors,and other lytic factors are present in plasma,where they prevent adherence and colonization of microorganisms (Alexandar and Ingram,1992),thus protecting the organism from infection and disease.Moreover,lysozyme contributes to the innate immunity of animals by their bactericidal and anti-inflammatory properties (Jolles and Jolles,1984).In the present study,plasma LSZ activity increased significantly with an increase of Aln-Gln levels.It is possible that Aln-Gln supplementation contributes to the lymphocyte proliferation and cytokine production by lymphocytes and macrophages,which promotes the secretion of the lysozymes.This indicates that the fish fed diets supplemented with Aln-Gln exerted a better barrier function because the fish had a higher plasma LSZ activity and thus may be more resistant to infection.However,these hypotheses must be further studied and evaluated.

The complement system is a major component of the innate immune system and it plays an important role in adaptive immunity (Morgan et al.,2005).C3 and C4 play a pivotal role in the activation of the complement system,a noncatalytic,but essential component of C3 convertases of the alternative and classical pathways,respectively (Muller,1988).They have a characteristic internal thioester bond that is exposed to the molecular surface upon activation and reacts with nearby hydroxyl or amino groups to form a covalent bond (Campbell et al.,1981).This covalent tagging of the foreign molecules by C3 or C4 seems to be the most critical step in complement activation,which leads to cytolysis through the activation of the late components,or phagocytosis through the binding to the complement receptors on phagocytes (Dodds and Law,1998).There are no studies on whether dietary Gln enhances plasma complement levels in animals.In the current study,addition of 5.0 g · kg-1Aln-Gln in the diet significantly enhanced the plasma C3 and C4 levels for carps under the current experimental conditions,the result was agreed with previous work,dietary Gln supplement tended to increase the concentration of the plasma IgA of weaned pigs (Lee,2008) and in the early starter stage of piglets (Hsu,2012).However,the values depressed with an increase in the concentration of the supplemented Aln-Gln beyond this level,which indicated that appropriate levels of the Aln-Gln could improve the plasma complement levels and that excessive supplementation had a detrimental effect on the alexinic factors.Bartell and Batal (2007) also reported that the chick fed on diets supplemented with 4% Gln had significantly lower plasma IgA concentrations by 21 days of age as compared with birds that were fed with the control diet and birds fed with diets supplemented with 1% Gln.However,there is no clear explanation for this effect,and the effect of Aln-Gln should be further investigated in relation to a molecular mechanism to evaluate its efficacy in modulating plasma complement levels.

Aerobic organisms continuously produce endogenous reactive oxygen species (ROS) in the process of metabolism,which could affect macromolecules,such as proteins,carbohydrates,nucleic acids,and lipids,in a process of the oxidative damage or oxidative stress (Finkel and Holbrook,2000).Therefore,mechanisms are in place to remove excessive ROS by an antioxidant defense system comprising an enzymatic system,such as SOD,CAT and GPX and non-enzymatic antioxidants,such as GSH,vitamins E and C (Wilhelm,1996).Adding Aln-Gln in the diets could enhance the activities of SOD and GPX,and GSH levels in intestine,hepatopancreas,plasma and muscles.Basivireddy et al.(2004) showed that pretreatment with Gln blocked the decrease of SOD and GPX activity in small intestinal mucosa of drugtreated rats.Chen et al.(2009) demonstrated that when enterocytes were treated with Gln in the presence of H2O2,the decrease in SOD,and GPX activities,as well as GSH concentration were completely prevented.MDA as the final product of lipid peroxidation is considered to be a biomarker for oxidative stress (Doyotte et al.,1997).In present study,the significant decrease in intestinal MDA content clearly showed that Aln-Gln inhibited lipid peroxidation and promoted cellular membrane restoration.Similar findings have been reported in other studies (Prabhu et al.,2003;Joon et al.,2003).This might suggest that the antioxidative effect of Aln-Gln may be attributed to its ability to enhanced free radical scavenging capacity.

Conclusions

The results of the study presented showed the positive effects of Aln-Gln supplementation on growth performance of carp,in addition to improvement in the development and function of the intestine,activity of the antioxidant defense system and the plasma nonspecific immunity of carps.In our research,optimal Aln-Gln level was 8.24 g · kg-1diet for WGR based on broken-line regression model analysis.

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