Effects of dissolved oxygen and dietary lysine levels on growth of grass carp
1,2
222,3222
2
1
Animal Science College,South China Agricultural University,Guangzhou,China;2School of Life Science,Sun Yat-Sen University,Guangzhou,China;3Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization,Ministry of Agriculture,Freshwater Fisheries Research Center,Chinese Academy of Fishery Sciences,Wuxi,China
In order to investigate the effects of lysine and dissolved oxygen on grass carp,the grass carp were fed with 13,15and 17g kg à1lysine diet at about 6mg L à1(high dis-solved oxygen,HO group)or 3.5mg L à1(low dissolved oxygen,LO group)dissolved oxygen level,for 8weeks.The ?sh were fed to apparent satiation by hand.The results showed that apparent digestibility of protein,energy and dry matter were decreased signi?cantly when grass carp were fed at 3.5mg L à1dissolved oxygen,and feed intake (FI)was also inhibited by low dissolved oxygen (P <0.05).Weight gain,protein retention,protein ef?-ciency,feed conversion ratio and amino acid retention of ?sh at 6mg L à1dissolved oxygen level were signi?cantly improved at 3.5mg L à1dissolved oxygen level (P <0.05).Weight gain,protein and amino acid retention,and feed ef?ciency of grass carp at the two dissolved oxygen levels were signi?cantly improved by lysine supplementation (P <0.05).The dietary lysine level and dissolved oxygen of water had an interaction effect on feed conversion ratios (P <0.05).Grass carp fed at low dissolved oxygen level showed lower liver protein and fat contents.Plasma aspar-tate aminotransferase (AST)activity of grass carp fed at 3.5mg L à1dissolved oxygen level was signi?cantly increased compared to 6mg L à1dissolved oxygen level (P <0.05).Our results show that low dissolved oxygen level of water is harmful to the liver of grass carp.
KEY WORDS :Ctenopharyngodon idella ,lysine,oxygen
Received 28December 2011;accepted 14November 2012
Correspondence:Yong-Jian Liu,Institute of Aquatic Economic Animals,School of Life Science,Sun Yat-sen University,135Xin’gang Xi Road,Guangzhou 510275,China.E-mails:edls@https://www.sodocs.net/doc/9311353771.html,;ganlian@https://www.sodocs.net/doc/9311353771.html,
Grass carp (Ctenopharyngodon idella )has a long history in aquaculture and is one of the most important species cultured in inland water bodies in China.After silver carp,grass carp currently has the largest production in freshwater aquaculture globally.It constitutes 7.18%of the world aquaculture production (FAO 2010).Low dis-solved oxygen is a type of stress frequently found in grass carp farms characterized by high ?sh densities and pol-luted fresh waters in China.Dissolved oxygen (DO)is the most important factor controlling growth,and a long con-stant DO concentration below a critical level is considered to depress FI,growth and food conversion ef?ciency (Thetmeyer et al.1999;Buentello et al.2000;Pichavant et al.2000;Foss et al.2003;Tran-Duy et al.2008).Fish try to maintain oxygen delivery in the face of reductions in water oxygen levels.If oxygen delivery is compromised and tissue oxygen levels fall,then energy expenditure is reduced and anaerobic metabolism is up-regulated.So metabolism was changed when ?sh was exposed to acute environmental hypoxia (Dunn &Hochachka 1986;Bouti-lier et al.1988;Gracey et al.2001,2011;Delaney &Kle-sius 2004).
Lysine is the most limiting amino acid in plant protein meal such as canola meal and cotton meal,which are important feed stuffs locally available for formulating grass carp diets.Lysine levels can no longer satisfy the require-ments in the commercial diets because plant protein meal
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a2013Blackwell Publishing Ltd
2013doi:10.1111/anu.12030
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was used as a major protein resource for grass carp(Yang et al.2010).When lysine was de?cient,the growth perfor-mance of grass carp was depressed,and the feed conversion ef?ciency was very poor(Wang et al.2005).
Although dietary lysine and dissolved oxygen of water are very important for cultured species,most grass carps were fed with lysine-de?cient diets in low dissolved oxygen ponds in commercial farms of China.However,the rela-tionship between?sh nutrition and environmental factors, especially between dietary lysine and dissolved oxygen of water,is still less studied.So our objective was to determine whether there is any interaction effect between dissolved oxygen and dietary lysine levels in grass carp.
Three diets were prepared(Table1).The basal diet con-tained the minimum level of lysine,13g kgà1DM,from soybean meal,canola meal,cotton meal,rice bran meal and wheat?our(Zhuhai Shihai Feed Corporation Ltd, Zhuhai,China).Lysine concentration was increased in two steps of approximately2g lysine kgà1DM each,with lysine originating from L-lysine HCL(78%L-lysine;CJ Co.,Ltd,Liaocheng,China).The maximum intended lysine concentration was17g kgà1DM,which is optimally required by grass carp.Lysine supplementation was carried out by substituting L-glutamic acid for L-lysine HCL. Methionine was supplied to meet the requirement along with MHA.Ca(84%,Novus International Inc.,Zhibo, China).Amino acid composition of experimental diets is shown in Table2.All diets were isoenergetic.
All dry ingredients were?nely ground,weighed,mixed manually for5min and then transferred to a Hobart mixer (A-200T Mixer Bench Model unit,Resell Food Equipment Ltd,Ottawa,ON,Canada)for another15-min mixing.Soya lecithin was added to a preweighed premix of soy oil and mixed until homogenous.The oil mix was then added to the Hobart mixer slowly while mixing was still continuing. All ingredients were mixed for another10min.Then,dis-
tilled water(about300g kgà1diet)was added to the mix-ture to form dough.The wet dough was placed in a pelletizer(Institute of Chemical Engineering,South China University of Technology,Guangzhou,China)and pelleted through a1.25-mm die.The diets were dried with forced air at20°C for24h,and the moisture was reduced to about100g kgà1.The dry pellets were placed in plastic bags and stored in a deep freezer atà20°C until used.Grass carp juvenile from our facilities were used in this exper-
iment,and their initial wet weights were4.11?0.03g.Before
the experiment,the?sh were acclimated to the experimental
conditions for2weeks and fed a commercial diet containing
300g kgà1protein and40g kgà1lipid to satiation.Twenty-
?ve healthy?sh were randomly distributed to each of18 experimental?breglass tanks(98L948W942H cm,
water volume of200L)connected to a recirculation system.
Table1Formulation and approximate composition of practical
diets for grass carp
Diet
Lysine level(g kgà1diet)131517
Ingredient(g kgà1diet)
Soybean meal1303030
Canola meal1320320320
Cotton meal1200200200
Rice bran meal1186.5186.5186.5
Wheat?our1205205205
Glutamic acid 5.2 2.60
Mineral mix2555
Vitamin mix3555
Soy oil1555
Choline chlorine(50%)1222
Monocalcium phosphate1202020
L-Lysine.HCL40 2.6 5.2
84%MHA.Ca5 5.2 5.2 5.2
Y2O360.10.10.1
Phospholipid1101010
VC ascorbic acid111
Total100010001000
(DM)Approximate composition
(g kgà1dry matter)
Moisture81.388.088.2
Crude protein306308314
Crude fat31.231.031.8
Ash76.075.074.4
Gross energy kJ gà1
18.418.618.7
1Zhuhai Shihai Feed Corporation Ltd.
2Mineral mix(mg kgà1of diet):MgSO
4
.7H2O,315;ZnSO4.7H2O,
285;CaHPO4.2H2O,250;FeSO4.7H2O,200;MnSO4.H2O,25;
CoSO4.7H2O,25;CaIO3,25;CuSO4.5H2O,15;Na2SeO3,10(Gu-
angzhou Chengyi Aquatic Technology Ltd,Guangzhou,China).
3Vitamin mix(mg kgà1of diet):thiamine,3;ribo?avin,8;vitamin
A,1500IU;vitamin E,40;vitamin D3,2000IU;menadione,6;pyr-
idoxine,4;cyanocobalamin,2;biotin,2;calcium pantothenate,
25;folic acid,2;niacin,12;inositol,50(Guangzhou Chengyi Aqua-
tic Technology Ltd,Guangzhou,China).
4
L-Lysine.HCL contained L-lysine!78%(CJ Co.,Ltd.)
5MHA.Ca contained DL-HMTBA(2-hydroxy-4-methylthiobutanoic
acid)!84%(Novus International Inc.)
6Y
2
O3(Yttrium oxide),analytical pure(Weibo Chemical Ltd,Gu-
angzhou,China).
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Aquaculture Nutritiona2013Blackwell Publishing Ltd
The study was carried out at two levels of dissolved oxy-gen.Dissolved oxygen treatments were a high DO and a low DO.Dissolved oxygen levels inside the tanks were established by means of aeration and water?ow rates through the tanks.Each tank assigned to the high DO level was aerated with one air stone.Tanks assigned to the low DO level were not aerated.After the?sh were allocated to the tanks,water?ow rates were set at2L minà1.Then,the ?ow rates in the tanks assigned to the low DO level were gradually reduced to0.22L minà1within the next5days. Dissolved oxygen concentrations and temperature at the inlet,inside(central point)and outlet of each tank were measured six times per day,at around7:00,9:00,11:00, 13:00,15:00and17:00using oxygen meters(YSI500;USA). The dissolved oxygen concentration during the feeding per-iod is shown in Fig.1.On26May,2June,9June,16June, 23June,30June,7July and14July,DO concentrations were also measured every2h from17:30to17:30the next day to assess the diurnal variation in DO concentration(See Fig.2).Water samples were collected for total ammonia nitrogen analyses every2days.The dead?sh also was col-lected and weighed to correct?nal total?sh body weight. The water was oxygenated and passed through arti?cial sponge(3cm thickness),coral sand(25cm thickness)and active carbon?lter(25cm thickness)to remove chlorine. During the trial period,the diurnal cycle was12-h light/12-h dark.Water quality parameters monitored weekly were as follows:temperature,29.1?2.4°C;pH,7.9?0.09, respectively.
The?sh were fed manually thrice per day to apparent satiation for8weeks.Faeces were collected daily during the last2weeks as described by Wang et al.(2005).Faeces tankà1was dried at105°C and stored atà70°C for the determination of digestibility with Y2O3as indicator.
At the beginning of the feeding trial,18?sh were randomly sampled from the initial?sh and killed for analyses of whole-body composition.At the end of the56-day experi-ment,10?sh from each tank were randomly collected for proximate analysis,4for the analysis of whole-body com-position and6were anaesthetized with tricaine methane sulphonate(MS222)(50mg Là1)for blood collection and to obtain weights of individual whole body,viscera,liver and intraperitoneal fat.White muscle from both sides of the?llets without skin and liver were dissected and frozen immediately in liquid nitrogen and stored atà70°C until
Table2Amino acid composition of experimental diets for grass carp(g kgà1dry diets)
Diet
Lysine level(g kgà1diet)131517
Essential amino acids
Lysine13.015.217.6 Methionine11.311.411.0 Phenylalanine12.813.113.3 Histidine 6.957.067.12 Tryptophan0.000.000.00 Arginine19.620.220.0 Threonine9.439.879.84 Isoleucine10.711.011.1 Leucine17.918.218.0 Valine14.714.915.2 Non-essential amino acids
Serine9.509.929.89 Proline15.916.416.7 Cystine 1.70 1.73 1.70 Tyrosine 6.57 6.98 6.76 Aspartic acid21.822.422.4 Glutamic acid56.756.655.7 Glycine13.013.213.3 Alanine11.511.911.7∑AA253260261 Tryptophan is not detected.
As an analog of methionine,MHA.Ca cannot be detected by the amino acid analyser,so methionine value was analysed as the sum of MHA.Ca and methionine.
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Aquaculture Nutritiona2013Blackwell Publishing Ltd
used.The plasma was separated by centrifugation and also stored atà70°C until analysed.
Diets and?sh samples(including white muscle and liver) were analysed in triplicate for proximate composition. Crude protein,crude lipid,moisture,crude ash and gross energy were determined following standard methods (AOAC1984).Crude protein(N96.25)was determined by the Kjeldahl method after acid digestion using an Auto Kjeldahl System(1030-Auto-analyzer;Soxtec System,Tecator AB,Sweden).Crude lipid was determined by the ether extraction method using a Soxtec System HT(Soxtec System HT6,Tecator).Moisture was determined by oven drying at 105°C for24h.Crude ash was determined by incineration in a muf?e furnace at550°C for24h.Gross energy was determined using an adiabatic bomb calorimeter.Amino acids were analysed following acid hydrolysis using high-pressure liquid chromatography(HPLC;Hewlett Packard 1090,Palo Alto,CA,USA).The concentrations of dietary and faecal Y2O3were determined by inductively coupled plasma atomic emission spectrophotometer[ICP;model: IRIS Advantage(HR),Thermo Jarrel Ash Corporation, Boston,MA,USA)after perchloric acid digestion(Bolin et al.1952).The concentrations of total plasma protein (TP),albumin(ALB),cholesterol(CHO),triacylglycerol (TG),glucose(GLU),aspartate aminotransferase(AST), alanine aminotransferase(ALT),high-density lipoprotein (HDL),low-density lipoprotein(LDL),urea-N and gluta-mate dehydrogenase(GLDH)were determined using an automatic blood analyser(Hitachi7170A,Hitachi7170A, Hitachi Ltd,Japan)from a clinical laboratory.
All data are presented as means?SEM.The SPSS software version13.0for Windows of GLM procedure(SPSS Inc., Chicago,IL,USAVer13.0,USA)was used to conduct fac-torial ANOVA to determine the effects of dietary protein con-tent,crystal amino acid supplementation and interaction between the two factors.When interaction between protein level and amino acid supplementation was statistically sig-ni?cant for a particular response,differences among pro-tein levels within each diet type were determined using Tukey’s mean separation.Treatment effects and interac-tions were considered signi?cant at P<0.05.
Mean total ammonia nitrogen concentrations under HO and LO groups were0.61?0.15mg Là1and 0.59?0.11mg Là1respectively.Total ammonia nitrogen concentrations in high oxygen tanks were higher than in
low oxygen tanks,but there was no difference.Fish readily
accepted the experimental diets,and survival rate was very
high during the56-day feeding trial.There were no signi?-
cant differences in survival among?sh fed all the diets
(Table3).After8weeks’feeding trial,the?nal body
weight,weight gain(WG),speci?c growth rate(SGR),
nitrogen retention(NR),lipid retention(LR)and protein
ef?ciency ratio(PER)of grass carp were signi?cantly depressed by the low dissolved oxygen(P<0.001)and also
were signi?cantly improved with lysine supplementation
(P<0.05)except lipid retention.Feed intake was signi?-
cantly increased with an increase in dissolved oxygen
(P<0.001),and not affected by dietary lysine content.
Feed conversion ratio(FCR)also was improved with an
increase in lysine and dissolved oxygen(P<0.05),but interaction was found between dissolved oxygen and diet
lysine level(P<0.05).
The proximate compositions of whole body,white mus-
cle and liver of the grass carp are shown in Table4.No
effects of dissolved oxygen on whole-body crude protein, moisture,lipid and ash contents of the?sh were found.
The whole-body lipid content was signi?cantly decreased
when?sh was fed diets with lysine supplementation
(P<0.001).The liver protein and lipid contents were sig-
ni?cantly reduced when?sh were fed at the low dissolved
oxygen(P<0.05).The composition of white muscle was
not affected by dissolved oxygen and lysine level.
Condition factor,hepatopancreasomatic index(HSI), intraperitoneal fat(IPF)and viscerosomatic index(VSI)of
grass carp fed experimental diets are presented in Table4.
VSI and IPF decreased with increasing dietary lysine levels
among diet treatments.VSI,IPF and HSI of grass carp at
the low dissolved oxygen were signi?cantly higher than
those at high dissolved oxygen(P<0.05).
Apparent digestibilities of dry matter,protein and energy
provided in Table5were signi?cantly affected by dissolved
oxygen concentration(P<0.001),but not dietary lysine
level.The results indicated that apparent digestibilities of
dry matter,protein and energy were signi?cantly reduced
at the low dissolved oxygen concentration.No interaction
was detectable between the two experimental factors with
regard to digestibility.
Plasma biochemical parameters are provided in Table6.
No effects were found with increasing dietary lysine level.
The results indicated that AST,ALT,TG,GLU,urea and
GLDH of grass carp fed at low dissolved oxygen were sig-
ni?cantly higher than those of grass carp fed at high dis-..............................................................................................
Aquaculture Nutritiona2013Blackwell Publishing Ltd
T a b l e 3E f f e c t o n g r o w t h a n d n u t r i e n t s r e t e n t i o n i n g r a s s c a r p w i t h s u p p l e m e n t a l l y s i n e a t d i f f e r e n t o x y g e n l e v e l s
O x y g e n l e v e l L O
H O
P r >F
L y s i n e l e v e l g k g à1
13
1517131517O x y g e n L y s i n e O x y g e n *L y s i n e
I B W 4.06?0.024.11?0.034.10?0.034.11?0.024.14?0.014.12?0.02F B W 18.9?1.1721.3?0.1924.1?0.7632.0?1.0834.6?0.8836.1?1.560.0000.0040.821L O < H O S u r v i v a l 85.6?7.2993.3?5.0996.7?3.3394.4?2.2296.7?3.3398.9?1.110.1890.2180.707F C R 1.48?0.08B 1.27?0.02A B 1.10?0.05A 1.15?0.03a 1.07?0.05a 1.05?0.04a 0.0000.0010.046L O >H O N R 28.7?4.4233.5?1.3740.0?3.1939.9?0.4343.0?2.5143.9?1.950.0020.0400.383L O ?4.36183?11.96188?10.96250?12.46244?25.71220?22.890.0010.7860.501L O < H O M e a n s ?S E M o f t h r e e r e p l i c a t e s ,p r o b a b i l i t y a s s o c i a t e d w i t h t h e F s t a t i s t i c f o r t h e f a c t o r i a l A N O V A .W i t h i n a r o w ,c a p i t a l l e t t e r s i n d i c a t e d i f f e r e n c e s w i t h i n L O a n d l o w e r c a s e l e t t e r s i n d i c a t e d i f f e r e n c e s w i t h i n H O a t P <0.05w h e n i n t e r a c t i o n s o c c u r r e d f o r t h e f u l l m o d e l .I B W (g ?s h à1),i n i t i a l b o d y w e i g h t .F B W (g ?s h à1),?n a l b o d y w e i g h t .F I ,f e e d i n t a k e (%d à1)=g r a m s o f d r y f e e d c o n s u m e d 9100/???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????eI B W ?I n i t i a l f i s h n u m b e r T?eF B W ?F i n a l f i s h n u m b e r tD e a d f i s h b o d y w e i g h t Tp /56d a y s . S u r v i v a l (%)=1009(?n a l ?s h n u m b e r )/(i n i t i a l ?s h n u m b e r ).W G (w e i g h t g a i n ,%)=1009(?n a l b o d y w e i g h t -i n i t i a l b o d y w e i g h t )/i n i t i a l b o d y w e i g h t .S G R (s p e c i ?c g r o w t h r a t e ,S G R ,%d a y à1)=1009(l n ?n a l w t -l n i n i t i a l w e i g h t )/56d a y s . F C R ,f e e d c o n v e r s i o n r a t i o =F e e d c o n s u m e d /(F B W 9?n a l ?s h n u m b e r –I B W 9i n i t i a l ?s h n u m b e r +d e a d ?s h b o d y w e i g h t ).N R ,n i t r o g e n r e t e n t i o n =1009r e t a i n e d n i t r o g e n (g )/n i t r o g e n f e d (g ).P E R ,p r o t e i n e f ?c i e n c y r a t i o =(?n a l b o d y w e i g h t -i n i t i a l b o d y w e i g h t )/n i t r o g e n f e d (g ).L R ,l i p i d r e t e n t i o n =1009r e t a i n e d l i p i d (g )/l i p i d f e d (g ). .............................................................................................. Aquaculture Nutrition a2013Blackwell Publishing Ltd T a b l e 4B o d y c o m p o s i t i o n a n d m o r p h o m e t r y i n d e x o f g r a s s c a r p f e d e x p e r i m e n t a l d i e t s f o r 56d a y s O x y g e n l e v e l L O H O P r >F L y s i n e l e v e l g k g à1 13 1517131517O x y g e n L y s i n e O x y g e n *L y s i n e W h o l e -b o d y C o m p o s i t i o n (g k g à1)1 M o i s t u r e 738?4.9740?1.5750?1.7734?1.3737?2.5748?1.40.1640.0010.915P r o t e i n 142?1.6140?0.4142?2.6142?1.3143?1.0143?0.20.1060.8820.595L i p i d 90.6?9.477.7?1.969.6?0.489.6?2.581.7?3.873.7?3.80.5420.0060.825A s h 29.1?0.229.9 ?0.428.3?0.428.6?0.429.2?0.227.7?0.30.066 0.003 0.968 M u s c l e M o i s t u r e 807?3.1806?1.5801?1.4797?2.3804?4.7791?19.40.3070.5900.843P r o t e i n 172?2.8171?0.9174?1.6173?3.8172?3.5173?11.50.9210.9160.985L i p i d 11.4?1.411.1?1.5 10.6?1.512.5?2.110.2?1.79.7?2.9 0.852 0.581 0.813 L i v e r M o i s t u r e 615?5.1630?7.7644?0.3598?4.7623?26.9621?14.30.1760.1620.829P r o t e i n 83.9?4.785.7?2.382.5?6.0111?1.6108?6.3109?3.10.0000.9250.862L O 95.0?0.4147?11.6138?15.9 145?12.3 0.017 0.337 0.187 L O M o r p h o m e t r y 2V S I 11.1?0.2710.4?0.2010.3?0.1910.5?0.239.93?0.279.28?0.250.0010.0000.471L O >H O H S I 3.04?0.182.90?0.172.99?0.172.25?0.142.09?0.092.14?0.110.0000.9700.590L O >H O I P F 2.71?0.192.57?0.132.45?0.183.25?0.122.76?0.152.48?0.140.0440.0050.255L O >H O C F 1.96?0.03 2.02?0.021.95?0.031.96?0.042.00? 0.03 2.02?0.03 0.418 0.309 0.298 V S I ,v i s c e r o s o m a t i c i n d e x =1009v i s c e r o s o m a t i c w e i g h t (g )/b o d y w e i g h t (g ). H S I ,h e p a t o p a n c r e a s o m a t i c i n d e x =1009l i v e r w e i g h t (g )/b o d y w e i g h t (g ).I P F ,i n t r a p e r i t o n e a l f a t r a t i o =1009i n t r a p e r i t o n e a l f a t w e i g h t (g )/b o d y w e i g h t (g ).C F ,c o n d i t i o n f a c t o r =1009b o d y w e i g h t (g )/b o d y l e n g t h (c m )3.1M e a n s ?S E M o f t h r e e r e p l i c a t e s ,p r o b a b i l i t y a s s o c i a t e d w i t h t h e F s t a t i s t i c f o r t h e f a c t o r i a l A N O V A . 2M e a n s ?S E M o f 18r e p l i c a t e s . .............................................................................................. Aquaculture Nutrition a2013Blackwell Publishing Ltd solved oxygen(P<0.001).High-density lipoprotein and LDL were signi?cantly decreased when grass carp were fed at low dissolved oxygen(P<0.05).No interactions were found. Amino acid retentions are provided in Table7.The results indicated that amino acid retention except methio-nine were not signi?cantly affected by dietary lysine level, but it showed an increasing trend with increasing lysine level.Threonine,proline,glycine,alanine,cystine,valine, methionine,isoleucine,leucine,phenylalanine and histidine retention of grass carp at low dissolved oxygen were signi?-cantly lower than those at high dissolved oxygen (P<0.05).No interaction was found. When grass carp was fed at low dissolved oxygen,apparent digestibility of grass carp was signi?cantly reduced.Axels-son&Fritsche(1991)showed that acute hypoxia imposed an increased visceral vascular resistance in the blood?ow in Gadus morhua,leading to a reduction in the coeliac and mesenteric artery blood supply.Similar results were found in Dicentrarchus labrax(Axelsson et al.2002).It may be expected that a reduction in blood?ow can depress the digestion of?sh.Tran-Duy et al.(2011)had demonstrated that apparent digestibility of Oreochromis niloticus fed at low dissolved oxygen was reduced. Decreased oxygen availability is also considered a major factor in determining food intake.Grass carp showed a reduced appetite and growth performance when fed at low dissolved oxygen.Similar results have been obtained from Oncorhynchus mykiss(Pedersen1987;Glencross2009), https://www.sodocs.net/doc/9311353771.html,brax L(Thetmeyer et al.1999),Scophthalmus maxi-mus(Pichavant et al.2000),Anarhichas minor Olafsen (Foss et al.2003),Ictalurus punctatus(Buentello et al. 2000),O.niloticus(Tran-Duy et al.2008),Morone saxatilis(Brandt et al.2009),Hippoglossus hippoglossus L(Thorarensen et al.2010);all these?sh experienced reduced growth.It is not surprising that?sh show poor appetite during long hypoxia.When dissolved oxygen avail-ability of water drops to a level that cannot support aero-bic metabolism,?sh will shift to anaerobic pathways for energy production.Subsequently,metabolic depression occurs to minimize energy expenditure.Fish reduce or stop feeding completely during hypoxic conditions,presumably because food digestion is energetically demanding.Acquisi-tion of food and its digestion and assimilation are major energy expenditures(up to60%)of?shes(Van Dam& Pauly1995). Feed ef?ciency was affected by the dissolved oxygen,and ?sh always showed good feed ef?ciency when fed at enough dissolved oxygen water(Bergheim et al.2006).In our study,FCR of grass carp was signi?cantly reduced when dissolved oxygen was increased,and the lipid,protein retention,protein ef?ciency ratio and amino acid retention also were signi?cantly improved.Blood urea and GLDH activity were major indicators of amino acid metabolism (Kim et al.1987;Regnault1987;Encarnac?~a o et al.2004). The blood urea level and the activity of GLDH were signif-icantly higher when grass carp was fed at low dissolved oxygen level.It indicated that protein synthesis of grass carp was inhibited by low dissolved oxygen.Smith et al. (1996)found that when crucian carp were exposed to48-h anoxia,there was more than a56%reduction in protein synthesis rate in liver,52%in red muscle and56%in white muscle. Although there was a controversy about utilization of crystal amino acids for?sh,the growth performance and amino acid retention of grass carp fed at both dissolved oxygen levels were improved with lysine HCL supplementa-tion in our study.The same result was obtained by Yang et al.(2010).Some researchers also have demonstrated that Labeo rohita(Mukhopadhayay&Ray1999;Sardar et al. 2009),O.niloticus(Furuya et al.2004),O.mykiss(Cheng Table5Apparent digestibility of nutrients and energy of juvenile grass carp fed with lysine supplementation at different oxygen levels Oxygen level LO HO Pr>F Lysine level g kgà1131517131517Oxygen Lysine Oxygen*Lysine Dry matter74.4?0.8674.3?0.7374.9?0.3678.8?0.3477.5?0.5178.3?0.300.0000.3880.601LO Energy80.2?0.9780.8?0.3280.3?0.4983.5?0.3882.5?0.5083.7?0.100.0000.8530.217LO .............................................................................................. Aquaculture Nutritiona2013Blackwell Publishing Ltd et al.2003),Pagrus major (Takagi et al.2002)and Piarac-tus mesopotamicus (Abimorad et al.2009)fed diets supple-mented with crystal lysine and methionine had better growth performance.In our study,FCR of grass carp also was signi?cantly reduced with increasing lysine,and there was an interaction effect between lysine level and dissolved oxygen.It indicated that high dissolved oxygen and bal-anced amino acid diet were helpful to obtain low FCR and good growth performance of ?sh. Hepatopancreasomatic index of grass carp fed at low dis-solved oxygen was signi?cantly higher than that of those fed at high dissolved oxygen,but the protein and lipid contents of liver showed an opposite trend.It indicated that the liver of grass carp was impaired when fed at low dissolved oxy-gen.Aspartate aminotransferase and alanine aminotransfer-ase are usually used as general indicators of the functioning of vertebrate liver (Wr o blewski &Ladue 1956).High AST and ALT generally indicate the damage or weakening of normal liver function.ALT and AST were often used as markers of hepatocellular injury (Seymen et al.1999;O’Bri-en et al.2000;Pan et al.2010).And in our study,plasma ALT and AST of grass carp fed at low dissolved oxygen were signi?cantly increased,and it demonstrated that liver was impaired when grass carp was cultured at low dissolved oxygen.The same conclusion was obtained in Hyphessobry-con callistus Boulenger (Pan et al.2010). Blood glucose level was signi?cantly higher when grass carp were fed at low dissolved oxygen level.Acute hypoxia was known to increase the levels of catecholamines,activat-ing glycogenolysis and gluconeogenesis with a net result of increasing plasma glucose levels (Wright et al.1989).Simi-lar results were found on Sparus aurata (Henrique et al.1998),Nephrops norvegicus (Schmitt &Uglow 1998),O.nil-oticus (Delaney &Klesius 2004)and I.punctatus (Thomas L et al.2007).The glucose concentration increment was related to the mobilization of energy storage under stressful conditions of low oxygen availability,as a source of fuel for anaerobic metabolism.During exposure to acute hypoxia,cardiac ATP concentration of Tilapia (Oreochr-omis hybrid sp.)was unchanged compared with normoxia and anaerobic glycolysis contributed to ATP supply as evi-denced by considerable accumulation of lactate in the heart and plasma (Speers-Roesch et al.2010).When Gillichthys mirabilis was exposed to short hypoxia,Gracey et al.(2001)showed that genes involved in the glycolytic meta-bolic pathway,muscle contraction and locomotion of G.mirabilis are all down-regulated in the muscle cells.On the other hand,several genes involved in gluconeogenesis were up-regulated in the liver during hypoxia. T a b l e 6B i o c h e m i c a l c o m p o s i t i o n s o f p l a s m a f r o m g r a s s c a r p w i t h s u p p l e m e n t a l l y s i n e a t d i f f e r e n t o x y g e n l e v e l s O x y g e n l e v e l L O H O P r >F L y s i n e l e v e l g k g à1 13151713 15 17 O x y g e n L y s i n e O x y g e n *L y s i n e A S T u L à1 121?3.25134?15.6122?10.3536.5?4.7527.7?2.1947.6?2.890.0000.7920.199L O >H O A L T u L à1 3.60?0.553.93?0.643.67?0.641.67?0.292.27?0.241.95?0.050.0010.6330.959L O >H O T P g L à1 24.0?1.2724.1?1.5524.5?1.1824.8?0.5623.4?1.0123.9?1.240.8830.8650.788A L B g L à1 7.27?0.678.67?0.848.23?0.139.80?0.129.07?1.309.00?0.800.0720.9030.362C H O m m o l L à1 3.17?0.243.16?0.393.53?0.32 4.99?0.184.34?0.304.71?0.390.0000.4470.514L O 3.11?0.313.07?0.333.22?0.132.70?0.112.50?0.132.70?0.100.0130.6970.929L O >H O H D L m m o l L à1 0.57?0.050.60?0.040.77?0.111.13?0.040.98?0.061.10?0.070.0000.1610.227L O 0.24?0.070.25?0.040.41?0.111.02?0.040.82?0.110.94?0.100.0000.2800.321L O 13.6?1.3217.8?1.3313.9?2.5111.1?0.2311.0?0.4111.5?2.110.0090.3980.328L O >H O U r e a -N u L à1 2.17?0.092.27?0.181.93?0.231.13?0.091.17?0.120.93?0.030.0000.1470.936L O >H O G L D H u L à1 28.2?4.1426.8?2.3331.9?3.9810.8?1.11 12.8? 1.0211.9 ?2.260.0000.6460.558 L O >H O M e a n s ?S E M o f t h r e e r e p l i c a t e s ,p r o b a b i l i t y a s s o c i a t e d w i t h t h e F s t a t i s t i c f o r t h e f a c t o r i a l A N O V A . .............................................................................................. Aquaculture Nutrition a2013Blackwell Publishing Ltd The results of the present study showed that FI and growth of grass carp were depressed when fed at low dissolved oxygen,and the liver may be impaired.The growth perfor-mance of grass carp was improved when it was fed a diet with lysine supplementation. Abimorad,E.G.,Favero,G.C.,Castellani,D.,Garcia,F.&Carneiro, D.J.(2009)Dietary supplementation of lysine and/or methionine on performance,nitrogen retention and excretion in pacu Piaractus mesopotamicus reared in cages.Aquaculture,295,266–270. AOAC(1984)Of?cial Methods of Analysis.pp.1141.Association of Of?cial Analytical Chemists,Arlington,VA. Axelsson,M.&Fritsche,R.(1991)Effects of exercise,hypoxia and feeding on the gastrointestinal blood?ow in the Atlantic cod Gadus morhua.J.Exp.Biol.,158,181–198. 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Table7Amino acid retention of grass carp with supplemental lysine at different oxygen levels Oxygen level LO HO Pr>F Lysine level g kgà1131517131517Oxygen Lysine Oxygen*Lysine Aspartic acid39.2?3.8145.5?4.0851.0?2.8649.0?3.2053.8?8.2854.9?7.630.1240.2890.851 Threonine51.1?7.4156.6?1.9863.6?6.4367.3?1.5268.1?4.1667.2?3.110.0090.3490.336LO Glutamic acid20.5?1.8825.4?2.2028.8?2.2926.8?1.4629.8?2.7430.6?3.200.0530.0680.661 Proline23.9?3.7626.8?1.0631.1?1.9033.0?0.8133.4?2.1233.7?1.630.0040.2170.330LO Phenylalanine28.7?2.7732.9?2.4039.1?2.7938.0?1.5840.8?4.4841.1?4.430.0330.1580.514LO Histidine28.1?3.6331.8?1.1436.4?3.0235.9?0.9338.7?2.9039.7?4.610.0310.1650.743LO Means?SEM of three replicates,probability associated with the F statistic for the factorial ANOVA. *Amino acid retention=1009retained amino acid(g)/amino acid fed(g). .............................................................................................. 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