Aquaculture protein levels

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1 Ž. Aquculture Whole-ody mino cid pttern of F4 humn growth hormone gene-trnsgenic red common crp ž Cyprinus crpio/ fed diets with different protein levels Cuizhng Fu, Yio Cui, Sils S.O. Hung, Zuoyn Zhu,) Stte Key Lortory of Freshwter Ecology nd Biotechnology, Institute of Hydroiology, The Chinese Acdemy of Sciences, Wuhn, Huei , People s Repulic of Chin Deprtment of Animl Science, UniÕersity of Cliforni, One Shield AÕenue, DÕis, CA , USA Received 27 July 1999; received in revised form 28 Mrch 2000; ccepted 28 Mrch 2000 Astrct F genertion of humn growth hormone Ž hgh. 4 gene-trnsgenic red common crp, nd the non-trnsgenic controls were fed for 8 weeks on purified diets with 20%, 30% or 40% protein. Anlysis of whole-ody mino cids showed tht the proportions of lysine, leucine, phenyllnine, vline nd lnine, s percentges of ody protein, incresed significntly, while those of rginine, glutmic cid nd tyrosine decresed, with increses in dietry protein level in t lest one strin of fish. Proportions of the other mino cids were unffected y the diets. The proportions of lysine nd rginine were significntly higher, while those of leucine nd lnine were lower in the trnsgenics thn in the controls in t lest one diet group. Proportions of the other mino cids were unffected y strin. The results suggest tht the whole-ody mino cid profile of trnsgenic crp, when expressed s proportions of ody protein, ws in generl, similr to tht of the non-trnsgenic controls. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Trnsgenic fish; Amino cid composition; Growth hormone; Cyprinus crpio ) Corresponding uthor. Tel.: q ; fx: q Ž. E-mil ddress: zyzhu@ih.c.cn Z. Zhu r00r$ - see front mtter q 2000 Elsevier Science B.V. All rights reserved. Ž. PII: S

2 288 ( ) C. Fu et l.raquculture Introduction Trnsgenic fish hve een produced in vriety of species using different foreign gene constructs Ž Zhu, 1992; Hckett, 1993; Devlin et l., Mny investigtors hve reported tht the growth hormone Ž GH. -trnsgenic fish exhiited higher growth rtes thn the controls ŽZhu et l., 1986, 1989; Chourrout et l., 1986; Dunhm et l., 1987; Rokkones et l., 1989; Zhng et l., 1990; Du et l., 1992; Devlin et l., However, informtion on ody composition of trnsgenic fish is limited ŽChtkondi et l., 1995; Cui et l., 1996; Fu et l., 1998., nd there hs een only one report on the ody mino cid composition of trnsgenic fish, which suggested tht the contents of 14 of the 18 muscle mino cids were higher in the trnsgenic crp contining rinow trout GH gene thn in the controls Ž Chtkondi et l., In previous pper, we reported the growth, feed utiliztion nd proximte ody composition of F4 genertion of humn growth hormone Ž hgh. trnsgenic crp fed diets with different protein levels in n 8-week growth tril Ž Fu et l., The study showed tht trnsgenic crp hd higher growth rtes, protein retention, nd contents of ody dry mtter nd protein thn the controls. The purpose of the present study ws to compre whole-ody mino cid pttern etween the F4 hgh-trnsgenic nd the non-trnsgenic red common crp. 2. Mterils nd method Detils of fish, experimentl systems nd procedures were descried in Fu et l. Ž P0 trnsgenic fish were produced y microinjection of the hgh gene cpped with mouse metllothionein promoter into the fertilized eggs of common crp Ž Cyprinus crpio red vriety.. The F4 trnsgenic crp were produced in 1996 y reeding the F3 genertion following spwning induction. A tch of non-trnsgenic control crp Ž red vriety. ws lso produced on the sme dy. The polymerse chin rection Ž PCR. method sed on the procedure of Li et l. Ž ws used to detect the trnsgene-positive individuls. Results of PCR on 90 trnsgenic crp reveled tht 100% of the tested fish were positive. At the ge of 4 weeks, 300 trnsgenic nd 300 control crps were trnsferred to the lortory nd fed n equl mixture of the three experimentl diets. The growth experiment ws initited when the fish were pproximtely 8 weeks old nd weighed 1.75 g. Three isoenergetic purified diets, with crude protein levels of 20%, 30% nd 40%, nd crohydrte levels of 50%, 40% nd 30%, respectively, were formulted Žsee Tle 1., nd proximte composition nd gross energy of the diets were presented in Fu et l. Ž For mino cid nlysis, triplicte smples of ech diet were hydrolyzed with 6 N HCl for 24 h t 100 8C in tues seled fter nitrogen flushing. After dilution nd filtrtion, smll liquot ws derivitized with phenyisothiocynte. The derivitized mino cids were nlyzed y reverse phse liquid chromtogrphy using Pico-tg Amino Acid Anlysis System Žincluding Wters 510 pump, 717 utosmpler, 996 photodiode rry detector, 470 scnning fluorescence detector nd 2010 work sttion; Millipore, Milford, MA, USA.. Tryptophn ws not nlyzed in this study. The mino cid compositions of the diets re presented in Tle 1.

3 ( ) C. Fu et l.raquculture Tle 1 Formultion of the experimentl diets Ingredients Ž %. Dietry protein Ž % Csein Geltin Cod liver oil Soyen oil Dextrin Others Amino cid composition (% dry weight) Essentil Lys Met Thr Leu Ile His Arg Phe Vl Non-Essentil Cys Ser Gly Pro Al Glu Asp Tyr Ž. Others: 1% vitmin premix nd 4% minerl premix Fu et l., 1998, 16% cellulose, 2% croxymethylcellulose nd 1% chromic oxide. The growth experiment ws conducted in 18 circulr fierglss tnks Ždimeter 70 cm, wter volume 90 l., which were prt of recircultion system. Ech tnk hd flt ottom with centrl drin. The wter temperture vried from 268C to298c Žmen 27.58C, SE 0.9 8C.. Photoperiod ws controlled y rtificil lighting with the light period etween 07:00 nd 19:00. Thirty individuls of either trnsgenic or control crp were weighed nd put into ech tnk, nd three tnks were ssigned rndomly to ech comintion of strin nd diet. The growth experiment lsted for 8 weeks nd the fish were fed to stition twice dy t 08:00 nd 16:00. At the end of the experiment, the fish were not fed for 1 dy nd weighed. Twenty fish from ech tnk were killed nd used for the nlysis of ody composition. Amino cid nlysis of fish ody ws s descried for the diets. All nlyses were crried out in triplictes. The effects of diet nd fish strin were nlyzed y two-wy nlysis of vrince Ž ANOVA.. Multiple comprisons were mde sed on lest-squre mens ŽCui et l., Differences were regrded s significnt when P

4 290 ( ) C. Fu et l.raquculture Results nd discussion In generl, the mino cid profiles of ody protein in oth the trnsgenic crp nd the controls Ž Tles 2 nd 3. were similr to those reported for common crp or other cyprinids Ž Schwrz nd Kirchgessner, 1988; Gtlin, Of the essentil mino cids, regrdless of strin, the proportions of lysine, leucine, phenyllnine nd vline s percentges of ody protein, incresed significntly, while tht of rginine decresed, with increses in dietry protein level. Proportions of other essentil mino cids were unffected y the diets Ž Tle 2.. Of the non-essentil mino cids, the proportion of glutmic cid decresed with increses in dietry protein level in oth the trnsgenics nd controls. The proportion of lnine in the trnsgenics incresed with increses in dietry protein level, nd the proportion of tyrosine in the controls decresed with increses in dietry protein level. Proportions of the other non-essentil mino cids were unffected y the diets Ž Tle 3.. Few ppers hve reported the effect of dietry protein levels on the ody mino cid composition in fish. Schwrz nd Kirchgessner Ž reported tht dietry protein level hd no ovious influence on the ody mino cid pttern in common crp. The results were somewht different from those of the present study. Severl differences in experimentl conditions my prtly ccount for the different results etween the two studies. In Schwrz nd Kirchgessner s Ž study, fish were housed t men wter temperture of 248C nd received fixed feeding rte. The initil weight of the fish ws 170 g. In the present study, fish were fed to stition twice dy t men wter Tle 2 Whole ody essentil mino cid pttern Ž % protein. of control nd trnsgenic crp fed diets with different protein levels Ž men"se, ns3. Dietry protein Ž %. ANOVA Ž P-vlue effect Control Trnsgenics Control Trnsgenics Control Trnsgenics Overll Diet Strin Diet= strin Lys X 7.4"0.0 Y 7.7"0.0 X 7.5"0.1 Y 7.9"0.1 X 8.0"0.1 Yc 8.4" Met 2.9" " " " " " Thr 4.3" " " " " " Leu X 7.5"0.1 Y 6.7" " "0.2 X 8.9"0.5 Y 8.0" Ile 4.3" " " " " " His 2.8" " " " " " X Y Arg 7.9" " " " " " Phe 4.0" " " " " " Vl 4.8" " " " " " Totl essentil mino cids 45.8" " " " " " Letters fter ech vlue indicte results of pir-wise comprisons. Different upper cse letters Ž XY. indicte significnt differences etween fish strin within diet; different lower cse letters Ž c. indicte significnt differences etween diets within strin s rnked y lest squre mens.

5 ( ) C. Fu et l.raquculture Tle 3 Whole ody non-essentil mino cid pttern Ž % protein. of control nd trnsgenic crp fed diets with different protein levels Ž men"se, ns3. Dietry protein Ž %. ANOVA Ž P-vlue effect Control Trnsgenics Control Trnsgenics Control Trnsgenics Overll Diet Strin Diet= strin Cys 0.4" " " " " " Ser 4.6" " " " " " Asp 6.4" " " " " " Glu 17.1" " " " " " Pro 5.5" " " " " " Gly 8.9" " " " " " Al X 7.3"0.1 Y 6.7"0.1 X 7.6"0.2 Y 6.9" " " Tyr 4.0" " " " " " Totl non-essentil mino cids 54.2" " " " " " Letters fter ech vlue indicte results of pir-wise comprisons. Different upper cse letters Ž XY. indicte significnt differences etween fish strin within diet; different lower cse letters Ž. indicte significnt differences etween diets within strin s rnked y lest squre mens. temperture of 27.58C. The initil weight of the fish ws only 1.75 g. Formultions of the experimentl diets were lso different. Of the essentil mino cids, the proportion of lysine ws significntly higher in trnsgenics thn in controls regrdless of diets. The proportion of leucine ws significntly lower in the trnsgenics thn in the controls fed with 20% nd 40% protein diets, ut ws unffected y strin in fish fed with the 30% protein diet. The proportion of rginine ws significntly higher in the trnsgenics thn in the controls fed with 40% protein diet, ut ws unffected y strin in fish fed with diets tht hve lower protein levels. Proportions of the other essentil mino cids nd totl essentil mino cids were unffected y strin Ž Tle 2.. Of the non-essentil mino cids, the proportion of lnine ws significntly lower in the trnsgenics thn in the controls fed with 20% nd 30% protein diets. Proportions of the other non-essentil mino cids nd totl non-essentil mino cids were unffected y strin Ž Tle 3.. Chtkondi et l. Ž reported tht the contents of 14 of the 18 muscle mino cids were higher in the trnsgenic crp contining rinow trout GH gene thn in the controls. In the present study, only 4 of the 17 ody mino cids nlyzed were significntly different etween the trnsgenics nd the controls. Amino cid composition ws expressed s grms per 100 g of muscle in Chtkondi et l. Ž 1995., nd s grms per 100 g of protein in the present study. As the ody protein content ws significntly higher in the trnsgenics thn in the controls in the present study, the contents of most mino cids, when expressed s grms per unit ody weight, would e expected to e higher in the trnsgenics thn in the controls.

6 292 ( ) C. Fu et l.raquculture In conclusion, results of the present study suggested tht the whole-ody mino cid profile of the trnsgenic crp, when expressed s proportions of ody protein, ws in generl, similr to tht of the non-trnsgenic controls. Acknowledgements We would like to thnk T. Storekken for providing the vitmin nd minerl premix, nd S. Xie, Z. Cui, W. Lei, Y. Yng nd Y. Wng for their help during the experiment. This work ws supported y The Ntionl Nturl Science Foundtion of Chin through grnts to Y. Cui Ž project no nd Z. Zhu Ž , nd the 863 High Technology Project of Chin through grnt to Z. Zhu. References Chtkondi, N., Lovell, R.T., Duncn, P.L., Hyt, M., Chen, T.T., Powers, D.A., Weete, J.D., Cummins, K., Dunhm, R.A., Body composition of trnsgenic common crp, Cyprinus crpio, contining rinow trout growth hormone gene. Aquculture 138, Chourrout, D., Guyomrd, R., Houdeine, L.-M., High efficiency gene trnsfer in rinow trout ŽSlmo girdneri Rich.. y microinjection into egg cytoplsm. Aquculture 51, Cui, Y., Hung, S.S.O., Zhu, X., Effect of rtion nd ody size on the energy udget of juvenile white sturgeon. J. Fish Biol. 49, Cui, Z., Zhu, Z., Cui, Y., Li, G., Xu, K., Food consumption nd energy udget in MThGH-trnsgenic F red crp Ž Cyprinus crpio L. red vr.. 2. Chin. Sci. Bull. 41, Devlin, R.H., Yeski, T.Y., Bigy, C.A., Donldson, E.M., Swnson, P., Chn, W.K., Extrordinry slmon growth slmon growth. Nture 371, Du, S.J., Gong, Z., Fletcher, G.L., Shers, M.A., King, M.J., Idler, D.R., Hew, C.L., Growth enhncement in trnsgenic Atlntic slmon y the use of n ll fish chimeric growth hormone gene construct. Biotechnology 10, Dunhm, R.A., Esh, J., Askins, J., Townes, T.M., Trnsfer of metllothionein-humn growth hormone fusion gene into chnnel ctfish. Trns. Am. Fish. Soc. 116, Fu, C., Cui, Y., Hung, S.S.O., Zhu, Z., Growth nd feed utilistion y F4 humn growth hormone trnsgenic crp fed diets with different protein levels. J. Fish Biol. 53, Gtlin, D.M. III, Whole-ody mino cid composition nd comprtive spects of mino cid nutrition of the goldfish, golden shiner nd fthed minnow. Aquculture 60, Hckett, P.B., The moleculr iology of trnsgenic fish. In: Hochchk, P., Mommsen, R. Ž Eds.., Biochemistry nd Moleculr Biology of Fishes vol. 2 Elsevier, Amsterdm, pp Rokkones, E., Alestrom, P., Skjervold, H., Gutvik, K.M., Microinjection nd expression of mouse metllothionein humn growth hormone fusion gene in fertilized slmonid eggs. J. Comp. Physiol. 158, Schwrz, F.J., Kirchgessner, M., Amino cid composition of crp Ž Cyprinus crpio L.. with vrying protein nd energy supplies. Aquculture 72, Zhng, P., Hyt, M., Joyce, C., Gonzlez-Villsenor, L.I., Lin, R.A., Dunhm, R.A., Chen, T.T., Powers, D.A., Gene trnsfer, expression nd inheritnce of prcv-rinow trout-ghcdna in the common crp Cyprinus crpio. Mol. Reprod. Dev. 25, Zhu, Z., Genertion of fst growing trnsgenic fish: methods nd mechnisms. In: Hew, C.L., Fletcher, G.L. Ž Eds.., Trnsgenic Fish. World Scientific Press, Singpore, pp Zhu, Z., Xu, K., Li, G., Xie, Y., He, L., Biologicl effects of humn growth hormone gene microinjected into the fertilized eggs of loch Misgurnus nguillicudtus. KeXue TongBo 31, , Ž Science Bulletin, Acdemi Sinic.. Zhu, Z., Xu, K., Xie, Y., Li, G., He, L., A model of trnsgenic fish. Sci. Chin, Ser. B 2,