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Availability and utilisation of free lysine in rainbow trout (Oncorhynchus mykiss)
Aquaculture 187 Ž2000. 177–183 www.elsevier.nlrlocateraqua-online
Availability and utilisation of free lysine in rainbow trout žOncorhynchus mykiss / 2. Comparison of L-lysineP HCl and L-lysine sulphate Markus Rodehutscord ) , Friedeman Borchert, Zoltan Gregus, Ernst Pfeffer UniÕersity of Bonn, Department of Animal Nutrition, Endenicher Allee 15, 53115 Bonn, Germany Accepted 30 November 1999
Abstract A basal diet, aiming at a low lysine concentration Ž9 grkg DM., was formulated with wheat gluten as the sole source of protein. Lysine concentration was increased in two steps of approximately 4 grkg DM each by use of either L-lysineP HCl or L-lysine sulphate. Each diet was fed at a restricted rate to three groups of 20 rainbow trout for 56 days. Faecal samples were collected via a sedimentation system and availability was determined using acid-insoluble ash as a marker. Retention was measured via comparative whole body analysis. Faecal lysine excretion was not affected by lysine supplementation. Body weight gain and gainrfeed ratio, as well as the composition of body weight gain, were significantly affected by dietary lysine concentration but not by lysine source. The utilisation of supplemental lysine, calculated as DretainedrDdigested= 100, was, on average, 68% and was not significantly different between the two lysine sources Ž P s 0.62.. It is concluded that both lysine sources are equally available to rainbow trout. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Rainbow trout; Free lysine sources; Comparison; Utilisation; Digestibility; Availability
) Corresponding author. Universitat 06099 Halle, ¨ Halle-Wittenberg, Institut fur ¨ Ernahrungswissenschaften, ¨ Germany. Tel.: q49-345-55-227-16; fax: q49-345-55-271-24. E-mail address: [email protected] ŽM. Rodehutscord..
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 3 8 9 - 0
178
M. Rodehutscord et al.r Aquaculture 187 (2000) 177–183
1. Introduction Free lysine is a very common compound for pig and poultry diets which helps to save protein in diets with lysine being the first-limiting amino acid. With the pressure put on aquaculture industries to reduce nitrogen and phosphorus waste, free lysine becomes increasingly interesting for compound fish diets as well as a tool to optimise the dietary amino acid pattern. L-lysine monohydrochloride ŽL-lysineP HCl. is a very common form of free lysine. One alternative is L-lysine sulphate. Both are products of bacterial fermentation but the post-fermentation process differs for L-lysine sulphate; the fermentation broth is not separated from the bacterial biomass and not transferred to the hydrochloric salt ŽSchutte and Pack, 1994.. L-lysine sulphate showed equal efficacy as L-lysineP HCl in studies with chicken and pigs ŽSchutte and Pack, 1994.. Nothing is known about the efficacy of this lysine source for rainbow trout. It was the aim of this study, therefore, to compare L-lysineP HCl and L-lysine sulphate regarding its availability to trout. It should be a common principle that studies aiming to characterise different nutrient sources regarding their respective availability to an animal or to compare different sources of a certain nutrient regarding their capacities to promote performance need to be conducted at a deficient level of supply. Beginning with a very low dietary lysine concentration, the response of trout to increasing lysine concentration shows a phase which is almost linear but turns into a clearly curvilinear relationship, following the principle of diminishing returns and approaching an asymptote ŽRodehutscord and Pack, 1999.. If different lysine sources are available to a different extent, these differences are less likely to be identified as closer the selected lysine concentration is to this asymptote, which certainly is not affected by the lysine source. Availability studies always focus on the capacity given by a feedstuff and must, therefore, be conducted under conditions of marginal supply in order to force the fish to maximise utilisation.
2. Materials and methods Five diets were prepared ŽTable 1.. Wheat gluten Ž134 g Nrkg DM. with its low lysine concentration Ž1.7 gr16 g N. was chosen as the dietary protein to achieve a low basal lysine level Ž9 grkg DM.. Lower lysine concentrations must be expected to cause increased mortality ŽRodehutscord et al., 1997.. Other essential amino acids were contained at a level of at least 130% of the level required for high protein retention according to our previous results ŽRodehutscord et al., 1997.. Lysine concentration was increased in two steps of approximately 4 g lysinerkg DM each, with lysine originating either from L-lysineP HCl ŽEurolysine, with a minimum of 78% L-lysine. or L-lysine sulphate ŽBiolys w 60, Degussa-Huls, ¨ which contains at least 46.8% free L-lysine and an additional 0.5% lysine bound in biomass.. The maximum intended lysine concentration, therefore, was 17 grkg DM, which is below the optimum required for high protein retention Ž27.7 grkg DM; Rodehutscord et al., 1997.. Lysine supplementation was done on a nitrogen equivalent basis by substituting L-alanine for L-lysineP HCl and by
M. Rodehutscord et al.r Aquaculture 187 (2000) 177–183
179
Table 1 Composition of diets fed to rainbow trout Lysine level
9
13
L-lysine
–
HCl
Sulphate
HCl
Sulphate
Ingredients (g r kg) Basal mix a Wheat starch, gelatinised L-alanine L-lysinePHCl L-lysine sulphate Calculated lysine conc. Žgrkg DM.
916 74 10 – – 9.0
916 74 5 5 – 12.8
916 71 5 – 8 12.6
916 74 – 10 – 16.7
916 68 – – 16 16.4
Analysed (per kg DM) GE ŽMJ. DE ŽMJ. b N=6.25 Žg. Lipid Žg. Lysine Žg. Alanine Žg.
21.8 19.8 545 197 8.9 24.5
21.8 19.9 551 196 12.8 19.1
22.0 20.1 555 187 12.8 19.2
21.9 19.9 546 199 16.4 13.7
22.3 20.4 556 197 16.8 14.1
supplemented as
17
a Basal mix comprised Žin g.: wheat gluten 650, fish oil 100, mineral–vitamin premix ŽRodehutscord et al., 1995. 65, sunflower oil 25, beef tallow 25, inorganic binder ŽSipernat w 50S. 40, TiO 2 10 and L-lysinePHCl 1. b Gross energy analysed, digestibility of energy measured Žsee Table 2..
substituting L-alanine and starch for L-lysine sulphate. Intended lysine concentrations were confirmed by analysis ŽTable 1.. Lipids were supplied as fish oil, sunflower oil and beef tallow. An inorganic binder ŽSipernat w 50S. was included to allow for a better handling of the high lipid levels and to increase the concentration of acid-insoluble ash, which was used as indigestible marker. Concentrations of crude protein and digestible energy were approximately 550 grkg DM and 20 MJrkg DM, respectively. Each diet was fed to three groups of 20 rainbow trout Ž Oncorhynchus mykiss . for 56 days at a restricted rate Žapproximately 16 g DM per kg BW per day.. This allowed for a fast and complete intake even for the lowest dietary lysine level. Initial fish weight at the beginning of the experiment was, on average, 50 g. Spot samples of faeces were collected via a sedimentation unit throughout the experiment beginning 1 week after the start of the experiment. Acid-insoluble ash was used as a marker. ‘Availability’ of lysine is defined as the proportion of intake that is not excreted with faeces, often also referred to as ‘digestibility’. Retention of lysine, protein, lipid and energy was determined by comparative whole body analysis. Details of diet preparation, fish handling, faeces collection, data recording and analysis are given in the first communication ŽRodehutscord et al., 2000.. The treatments with L-lysineP HCl as the supplemental lysine source are identical with the ‘high protein’ treatments in the first communication. Results of BW gain, feed conversion and composition of gain were subjected to ANOVA procedure. The efficiency of utilisation of the two lysine sources was compared by regressing BW gain or lysine retention on lysine intake. The following equation was used: y s a q bn x n , with y the response criterion, a intercept, bn slope for lysine source n and x n lysine intake from supplement
M. Rodehutscord et al.r Aquaculture 187 (2000) 177–183
180
n. The parameters a, b HC l and b Sulphate were estimated simultaneously through GLM procedure, and slopes were compared by F-test of SAS ŽSAS Institute, Cary, NC..
3. Results and discussion One individual Žmedium L-lysineP HCl level. had to be removed from the experiment because it showed incidence for fungal infections. For none of the traits recorded in this experiment, a significant interaction between the lysine level and the lysine source occurred ŽTables 2 and 3.. 3.1. Digestibility Digestibility of crude protein, organic matter and, subsequently, energy were at a very high level as can be expected with this semipurified-type diet ŽTable 2.. The crude protein digestibility was similar to the one we previously found for wheat gluten with faeces obtained by stripping the fish ŽPfeffer et al., 1995.. Gelatinised wheat starch is almost completely digestible at the present level of inclusion ŽHenrichfreise and Pfeffer, 1992. and the lipids can also be regarded more than 90% digestible ŽSchulte, 1995.. Digestibility of crude protein, organic matter and energy were not significantly affected by the dietary lysine level. The lysine source significantly affected organic matter Ž P - 0.01. and energy Ž P s 0.02. digestibility. However, these effects were numerically low ŽF 0.4 percentage units.. For cost reasons, faecal lysine concentration was determined only in samples obtained from fish fed 9 or 17 g lysinerkg DM. According to unpaired t-test, lysine availability was higher Ž P F 0.05. for the diets containing 17 as compared to 9 g lysinerkg DM but lysine availability was not significantly different between the two lysine sources on the high lysine level Ž P s 0.35.. To obtain the availability of the supplemented lysine, the D values are needed for lysine intake and lysine excretion. They can be calculated from data for lysine intake and lysine availability given in Tables 2 and 3 and are 11.9 and 11.9 grtank for L-lysineP HCl and 12.1 and 12.0 grtank for L-lysine sulphate which means complete availability for both lysine sources. This is in
Table 2 Digestibility of energy, organic matter, crude protein, and lysine availability in rainbow trout fed diets differing in level and source of supplemented L-lysine Ž%. Ž ns 3 groups of 20 trout per treatment. Lysine level L-lysine
supplemented as
Energy Organic matter Crude protein Lysine a b
9
13
17
–
HCl
Sulphate HCl
Sulphate
90.9 89.0 98.4 96.6
91.2 89.2 98.6 n.d.a
91.4 89.6 98.3 n.d.a
91.5 89.5 98.4 97.9 b
91.1 89.2 98.5 98.1b
Pooled SE
P ŽANOVA. Level
Source
L=S
0.07 0.06 0.05 0.36
0.62 0.86 0.78
0.02 - 0.01 0.19
0.41 0.59 0.58
n.d.s not determined. Not significantly different with P s 0.35 according to unpaired t-test.
M. Rodehutscord et al.r Aquaculture 187 (2000) 177–183
181
Table 3 Intake, growth and composition of gained body mass in rainbow trout fed for 56 days diets differing in level and source of supplemented L-lysine Ž ns 3 groups of 20 trout per treatment. Lysine level L-lysine
9
supplemented –
13 HCl
17 Sulphate HCl
Pooled SE P ŽANOVA. Sulphate
Level
Source L=S
DM intake, grtank 1494 1515 1501 1535 1513 3.8 Lysine intake, grtank 13.3 19.4 18.9 25.6 24.8 1.20 Initial BW, grtank 998 1003 1003 1002 1000 1.3 BW gain, grtank 1283 1471 1456 1644 1606 35.3 BW gainrDM intake 0.86 0.97 0.97 1.07 1.06 0.022
- 0.01 0.27 - 0.01 0.60
0.62 0.75
Concentration in gained BW a Lysine, grkg 7.01 Protein, grkg 113 Lipids, grkg 247 Energy, MJrkg 12.7
- 0.01 - 0.01 - 0.01 - 0.01
0.67 0.55 0.55 0.22
a
8.99 133 210 11.6
8.90 135 205 11.6
10.6 150 175 10.7
10.2 150 177 11.1
0.36 3.7 7.6 0.19
0.52 0.43 0.77 0.11
Retention determined by comparative whole body analysis.
line with findings in pigs and poultry for crystalline lysine and other crystalline amino acids as reviewed by Baker Ž1994. and verifies the common assumption that free amino acids can be completely absorbed from the salmonids intestine. 3.2. Retention Body weight gain of trout and gainrfeed ratio significantly increased with increasing dietary lysine concentration ŽTable 3., which confirms results from previous dose–response studies ŽWalton et al., 1984; Lanari et al., 1991; Kim et al., 1992; Pfeffer et al., 1992; Rodehutscord et al., 1997.. Not only the amount, but also the composition of gained BW changed. While the concentration of protein increased Ž P - 0.01. with increasing dietary lysine concentration, the concentrations of lipid Ž P - 0.01. and energy Ž P - 0.01. decreased. The lysine source, however, did not effect amount or composition of gained body weight. A more sophisticated comparison of the two lysine sources is given by the comparison of slopes, when the response of trout is regressed on the quantitative lysine intake and where the slope describes the pure effect of the supplemented lysine. This is done in Fig. 1 for both BW gain and lysine retention as the respective response criterion. For BW gain and lysine retention Fig. 1 clearly shows that lysine supply was the limiting factor. In both cases, the slope of the regression line is numerically smaller for L-lysine sulphate than for L-lysineP HCl but these differences are not statistically significant for either BW gain Ž P s 0.70. or lysine retention Ž P s 0.62.. Based on this analysis, the availability of both lysine sources to trout can be regarded as equal, which is in agreement with observations made in pigs and poultry ŽRoth et al., 1994; Schutte and Pack, 1994.. This conclusion is favoured by the fact that the differences in slope between the two lysine sources are due mainly to differences in lysine retention on the highest level of supplementation. In fact, when t-test is applied to compare lysine retention measured of
182
M. Rodehutscord et al.r Aquaculture 187 (2000) 177–183
Fig. 1. Response of rainbow trout to supplementation of lysine in the form of either L-lysinePHCl Žinterrupted line. or L-lysine sulphate Žsolid line.. The slopes of the two respective regression lines are not significantly different for BW gain Ž P s 0.70. and lysine retention Ž P s 0.62.. Values in brackets show the standard error of the respective estimated parameter.
the highest lysine level only, the difference between the two lysine sources Ž1.0 g lysine retentionrtank. is significant Ž P s 0.03., whereas the difference on the low level of supplementation is only 0.2 g lysine retentionrtank Ž P s 0.73.. It is unclear whether these differences can be attributed to the sole difference between the two sources, which was the concentration of sulphate. Perhaps other residues of the fermentation process that are still contained in the biomass may have a growth-diminishing effect at high level of inclusion of this product. With regard to the application in practise, however, this appears irrelevant because such a high level of inclusion of the free amino acids is unnecessary when proteins with a more balanced amino acid pattern than wheat gluten are used. The availability or ‘bioavailability’ is often calculated to rank different sources of one nutrient based on the ratio of slopes like the one given in the upper panel of Fig. 1. This,
M. Rodehutscord et al.r Aquaculture 187 (2000) 177–183
183
however, can only give a relative value for the sources tested and requires a well-standardised reference if the values are to be used by the commercial feed compounder. In cases where lysine retention is determined and plotted against lysine intake, the slope is a direct measure of and gives an absolute value for the utilisation of the supplemented lysine source. Based on Fig. 1 Žlower panel., the utilisation of free lysine to rainbow trout is, on average, 68%.
Acknowledgements The authors gratefully acknowledge amino acid analysis done by Degussa-Huls ¨ and statistical advice by Dr. Ernst Tholen.
References Baker, D.H., 1994. Utilization of precursors for L-amino acids. In: D’Mello, J.P.F. ŽEd.., Amino Acids in Farm Animal Nutrition. CAB International, Wallingford, pp. 37–61. Henrichfreise, B., Pfeffer, E., 1992. Wheat and wheat starch as potential sources of digestible energy for rainbow trout Ž Oncorhynchus mykiss .. J. Anim. Physiol. Anim. Nutr. 67, 143–147. Kim, K.-I., Kayes, T.B., Amundson, C.H., 1992. Requirements for lysine and arginine by rainbow trout Ž Oncorhynchus mykiss .. Aquaculture 106, 333–344. Lanari, D., Tibaldi, E., Ballestrazzi, R., Tulli, F., 1991. Impiego di diete a moderato contenuto di farina di pesce per la trota iridea. Rivalutazione del fabbisogno di lisina della specie ed effetti sulle acque reflue. In: Atti IX Congresso Nazionale. pp. 1089–1098. Pfeffer, E., Al-Sabty, H., Haverkamp, R., 1992. Studies on lysine requirements of rainbow trout Ž Oncorhynchus mykiss . fed wheat gluten as only source of dietary protein. J. Anim. Physiol. Anim. Nutr. 67, 74–82. Pfeffer, E., Kinzinger, S., Rodehutscord, M., 1995. Influence of the proportion of poultry slaughter by-products and of untreated or hydrothermically treated legume seeds in diets for rainbow trout, Oncorhynchus mykiss ŽWalbaum., on apparent digestibilities of their energy and organic compounds. Aquacult. Nutr. 1, 111–117. Rodehutscord, M., Becker, A., Pack, M., Pfeffer, E., 1997. Response of rainbow trout Ž Oncorhynchus mykiss . to supplements of individual essential amino acids in a semipurified diet, including an estimate of the maintenance requirement for essential amino acids. J. Nutr. 127, 1166–1175. Rodehutscord, M., Borchert, F., Gregus, Z., Pack, M., Pfeffer, E., 2000. Availability and utilisation of free lysine in rainbow trout Ž Oncorhynchus mykiss .: 1. Effect of dietary crude protein level. Aquaculture 187, 163–176. Rodehutscord, M., Mandel, S., Pack, M., Jacobs, S., Pfeffer, E., 1995. Free amino acids can replace protein-bound amino acids in test diets for studies in rainbow trout Ž Oncorhynchus mykiss .. J. Nutr. 125, 956–963. Rodehutscord, M., Pack, M., 1999. Estimates of essential amino acid requirements from dose–response studies with rainbow trout and broiler chicken: effect of mathematical model. Arch. Anim. Nutr. 52, 223–244. Roth, F.X., Stangl, G.I., Kirchgessner, M., 1994. Biological efficiency of L-lysine base and L-lysine sulphate compared with L-lysinePHCl in piglets. Agribiol. Res. 47, 177–186. Schulte, H.-G., 1995. Bewertung verschiedener Fettarten in der Ernahrung von Regenbogenforellen. Doctoral ¨ thesis, University of Bonn, Shaker, Aachen. Schutte, J.B., Pack, M., 1994. Biological efficacy of L-lysine preparations containing biomass compared to L-lysine-HCl. Arch. Anim. Nutr. 46, 261–268. Walton, M.J., Cowey, C.B., Adron, J.W., 1984. The effect of dietary lysine levels on growth and metabolism of rainbow trout Ž Salmo gairdneri .. Br. J. Nutr. 52, 115–122.
Availability and utilisation of free lysine in rainbow trout žOncorhynchus mykiss / 2. Comparison of L-lysineP HCl and L-lysine sulphate Markus Rodehutscord ) , Friedeman Borchert, Zoltan Gregus, Ernst Pfeffer UniÕersity of Bonn, Department of Animal Nutrition, Endenicher Allee 15, 53115 Bonn, Germany Accepted 30 November 1999
Abstract A basal diet, aiming at a low lysine concentration Ž9 grkg DM., was formulated with wheat gluten as the sole source of protein. Lysine concentration was increased in two steps of approximately 4 grkg DM each by use of either L-lysineP HCl or L-lysine sulphate. Each diet was fed at a restricted rate to three groups of 20 rainbow trout for 56 days. Faecal samples were collected via a sedimentation system and availability was determined using acid-insoluble ash as a marker. Retention was measured via comparative whole body analysis. Faecal lysine excretion was not affected by lysine supplementation. Body weight gain and gainrfeed ratio, as well as the composition of body weight gain, were significantly affected by dietary lysine concentration but not by lysine source. The utilisation of supplemental lysine, calculated as DretainedrDdigested= 100, was, on average, 68% and was not significantly different between the two lysine sources Ž P s 0.62.. It is concluded that both lysine sources are equally available to rainbow trout. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Rainbow trout; Free lysine sources; Comparison; Utilisation; Digestibility; Availability
) Corresponding author. Universitat 06099 Halle, ¨ Halle-Wittenberg, Institut fur ¨ Ernahrungswissenschaften, ¨ Germany. Tel.: q49-345-55-227-16; fax: q49-345-55-271-24. E-mail address: [email protected] ŽM. Rodehutscord..
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 3 8 9 - 0
178
M. Rodehutscord et al.r Aquaculture 187 (2000) 177–183
1. Introduction Free lysine is a very common compound for pig and poultry diets which helps to save protein in diets with lysine being the first-limiting amino acid. With the pressure put on aquaculture industries to reduce nitrogen and phosphorus waste, free lysine becomes increasingly interesting for compound fish diets as well as a tool to optimise the dietary amino acid pattern. L-lysine monohydrochloride ŽL-lysineP HCl. is a very common form of free lysine. One alternative is L-lysine sulphate. Both are products of bacterial fermentation but the post-fermentation process differs for L-lysine sulphate; the fermentation broth is not separated from the bacterial biomass and not transferred to the hydrochloric salt ŽSchutte and Pack, 1994.. L-lysine sulphate showed equal efficacy as L-lysineP HCl in studies with chicken and pigs ŽSchutte and Pack, 1994.. Nothing is known about the efficacy of this lysine source for rainbow trout. It was the aim of this study, therefore, to compare L-lysineP HCl and L-lysine sulphate regarding its availability to trout. It should be a common principle that studies aiming to characterise different nutrient sources regarding their respective availability to an animal or to compare different sources of a certain nutrient regarding their capacities to promote performance need to be conducted at a deficient level of supply. Beginning with a very low dietary lysine concentration, the response of trout to increasing lysine concentration shows a phase which is almost linear but turns into a clearly curvilinear relationship, following the principle of diminishing returns and approaching an asymptote ŽRodehutscord and Pack, 1999.. If different lysine sources are available to a different extent, these differences are less likely to be identified as closer the selected lysine concentration is to this asymptote, which certainly is not affected by the lysine source. Availability studies always focus on the capacity given by a feedstuff and must, therefore, be conducted under conditions of marginal supply in order to force the fish to maximise utilisation.
2. Materials and methods Five diets were prepared ŽTable 1.. Wheat gluten Ž134 g Nrkg DM. with its low lysine concentration Ž1.7 gr16 g N. was chosen as the dietary protein to achieve a low basal lysine level Ž9 grkg DM.. Lower lysine concentrations must be expected to cause increased mortality ŽRodehutscord et al., 1997.. Other essential amino acids were contained at a level of at least 130% of the level required for high protein retention according to our previous results ŽRodehutscord et al., 1997.. Lysine concentration was increased in two steps of approximately 4 g lysinerkg DM each, with lysine originating either from L-lysineP HCl ŽEurolysine, with a minimum of 78% L-lysine. or L-lysine sulphate ŽBiolys w 60, Degussa-Huls, ¨ which contains at least 46.8% free L-lysine and an additional 0.5% lysine bound in biomass.. The maximum intended lysine concentration, therefore, was 17 grkg DM, which is below the optimum required for high protein retention Ž27.7 grkg DM; Rodehutscord et al., 1997.. Lysine supplementation was done on a nitrogen equivalent basis by substituting L-alanine for L-lysineP HCl and by
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179
Table 1 Composition of diets fed to rainbow trout Lysine level
9
13
L-lysine
–
HCl
Sulphate
HCl
Sulphate
Ingredients (g r kg) Basal mix a Wheat starch, gelatinised L-alanine L-lysinePHCl L-lysine sulphate Calculated lysine conc. Žgrkg DM.
916 74 10 – – 9.0
916 74 5 5 – 12.8
916 71 5 – 8 12.6
916 74 – 10 – 16.7
916 68 – – 16 16.4
Analysed (per kg DM) GE ŽMJ. DE ŽMJ. b N=6.25 Žg. Lipid Žg. Lysine Žg. Alanine Žg.
21.8 19.8 545 197 8.9 24.5
21.8 19.9 551 196 12.8 19.1
22.0 20.1 555 187 12.8 19.2
21.9 19.9 546 199 16.4 13.7
22.3 20.4 556 197 16.8 14.1
supplemented as
17
a Basal mix comprised Žin g.: wheat gluten 650, fish oil 100, mineral–vitamin premix ŽRodehutscord et al., 1995. 65, sunflower oil 25, beef tallow 25, inorganic binder ŽSipernat w 50S. 40, TiO 2 10 and L-lysinePHCl 1. b Gross energy analysed, digestibility of energy measured Žsee Table 2..
substituting L-alanine and starch for L-lysine sulphate. Intended lysine concentrations were confirmed by analysis ŽTable 1.. Lipids were supplied as fish oil, sunflower oil and beef tallow. An inorganic binder ŽSipernat w 50S. was included to allow for a better handling of the high lipid levels and to increase the concentration of acid-insoluble ash, which was used as indigestible marker. Concentrations of crude protein and digestible energy were approximately 550 grkg DM and 20 MJrkg DM, respectively. Each diet was fed to three groups of 20 rainbow trout Ž Oncorhynchus mykiss . for 56 days at a restricted rate Žapproximately 16 g DM per kg BW per day.. This allowed for a fast and complete intake even for the lowest dietary lysine level. Initial fish weight at the beginning of the experiment was, on average, 50 g. Spot samples of faeces were collected via a sedimentation unit throughout the experiment beginning 1 week after the start of the experiment. Acid-insoluble ash was used as a marker. ‘Availability’ of lysine is defined as the proportion of intake that is not excreted with faeces, often also referred to as ‘digestibility’. Retention of lysine, protein, lipid and energy was determined by comparative whole body analysis. Details of diet preparation, fish handling, faeces collection, data recording and analysis are given in the first communication ŽRodehutscord et al., 2000.. The treatments with L-lysineP HCl as the supplemental lysine source are identical with the ‘high protein’ treatments in the first communication. Results of BW gain, feed conversion and composition of gain were subjected to ANOVA procedure. The efficiency of utilisation of the two lysine sources was compared by regressing BW gain or lysine retention on lysine intake. The following equation was used: y s a q bn x n , with y the response criterion, a intercept, bn slope for lysine source n and x n lysine intake from supplement
M. Rodehutscord et al.r Aquaculture 187 (2000) 177–183
180
n. The parameters a, b HC l and b Sulphate were estimated simultaneously through GLM procedure, and slopes were compared by F-test of SAS ŽSAS Institute, Cary, NC..
3. Results and discussion One individual Žmedium L-lysineP HCl level. had to be removed from the experiment because it showed incidence for fungal infections. For none of the traits recorded in this experiment, a significant interaction between the lysine level and the lysine source occurred ŽTables 2 and 3.. 3.1. Digestibility Digestibility of crude protein, organic matter and, subsequently, energy were at a very high level as can be expected with this semipurified-type diet ŽTable 2.. The crude protein digestibility was similar to the one we previously found for wheat gluten with faeces obtained by stripping the fish ŽPfeffer et al., 1995.. Gelatinised wheat starch is almost completely digestible at the present level of inclusion ŽHenrichfreise and Pfeffer, 1992. and the lipids can also be regarded more than 90% digestible ŽSchulte, 1995.. Digestibility of crude protein, organic matter and energy were not significantly affected by the dietary lysine level. The lysine source significantly affected organic matter Ž P - 0.01. and energy Ž P s 0.02. digestibility. However, these effects were numerically low ŽF 0.4 percentage units.. For cost reasons, faecal lysine concentration was determined only in samples obtained from fish fed 9 or 17 g lysinerkg DM. According to unpaired t-test, lysine availability was higher Ž P F 0.05. for the diets containing 17 as compared to 9 g lysinerkg DM but lysine availability was not significantly different between the two lysine sources on the high lysine level Ž P s 0.35.. To obtain the availability of the supplemented lysine, the D values are needed for lysine intake and lysine excretion. They can be calculated from data for lysine intake and lysine availability given in Tables 2 and 3 and are 11.9 and 11.9 grtank for L-lysineP HCl and 12.1 and 12.0 grtank for L-lysine sulphate which means complete availability for both lysine sources. This is in
Table 2 Digestibility of energy, organic matter, crude protein, and lysine availability in rainbow trout fed diets differing in level and source of supplemented L-lysine Ž%. Ž ns 3 groups of 20 trout per treatment. Lysine level L-lysine
supplemented as
Energy Organic matter Crude protein Lysine a b
9
13
17
–
HCl
Sulphate HCl
Sulphate
90.9 89.0 98.4 96.6
91.2 89.2 98.6 n.d.a
91.4 89.6 98.3 n.d.a
91.5 89.5 98.4 97.9 b
91.1 89.2 98.5 98.1b
Pooled SE
P ŽANOVA. Level
Source
L=S
0.07 0.06 0.05 0.36
0.62 0.86 0.78
0.02 - 0.01 0.19
0.41 0.59 0.58
n.d.s not determined. Not significantly different with P s 0.35 according to unpaired t-test.
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181
Table 3 Intake, growth and composition of gained body mass in rainbow trout fed for 56 days diets differing in level and source of supplemented L-lysine Ž ns 3 groups of 20 trout per treatment. Lysine level L-lysine
9
supplemented –
13 HCl
17 Sulphate HCl
Pooled SE P ŽANOVA. Sulphate
Level
Source L=S
DM intake, grtank 1494 1515 1501 1535 1513 3.8 Lysine intake, grtank 13.3 19.4 18.9 25.6 24.8 1.20 Initial BW, grtank 998 1003 1003 1002 1000 1.3 BW gain, grtank 1283 1471 1456 1644 1606 35.3 BW gainrDM intake 0.86 0.97 0.97 1.07 1.06 0.022
- 0.01 0.27 - 0.01 0.60
0.62 0.75
Concentration in gained BW a Lysine, grkg 7.01 Protein, grkg 113 Lipids, grkg 247 Energy, MJrkg 12.7
- 0.01 - 0.01 - 0.01 - 0.01
0.67 0.55 0.55 0.22
a
8.99 133 210 11.6
8.90 135 205 11.6
10.6 150 175 10.7
10.2 150 177 11.1
0.36 3.7 7.6 0.19
0.52 0.43 0.77 0.11
Retention determined by comparative whole body analysis.
line with findings in pigs and poultry for crystalline lysine and other crystalline amino acids as reviewed by Baker Ž1994. and verifies the common assumption that free amino acids can be completely absorbed from the salmonids intestine. 3.2. Retention Body weight gain of trout and gainrfeed ratio significantly increased with increasing dietary lysine concentration ŽTable 3., which confirms results from previous dose–response studies ŽWalton et al., 1984; Lanari et al., 1991; Kim et al., 1992; Pfeffer et al., 1992; Rodehutscord et al., 1997.. Not only the amount, but also the composition of gained BW changed. While the concentration of protein increased Ž P - 0.01. with increasing dietary lysine concentration, the concentrations of lipid Ž P - 0.01. and energy Ž P - 0.01. decreased. The lysine source, however, did not effect amount or composition of gained body weight. A more sophisticated comparison of the two lysine sources is given by the comparison of slopes, when the response of trout is regressed on the quantitative lysine intake and where the slope describes the pure effect of the supplemented lysine. This is done in Fig. 1 for both BW gain and lysine retention as the respective response criterion. For BW gain and lysine retention Fig. 1 clearly shows that lysine supply was the limiting factor. In both cases, the slope of the regression line is numerically smaller for L-lysine sulphate than for L-lysineP HCl but these differences are not statistically significant for either BW gain Ž P s 0.70. or lysine retention Ž P s 0.62.. Based on this analysis, the availability of both lysine sources to trout can be regarded as equal, which is in agreement with observations made in pigs and poultry ŽRoth et al., 1994; Schutte and Pack, 1994.. This conclusion is favoured by the fact that the differences in slope between the two lysine sources are due mainly to differences in lysine retention on the highest level of supplementation. In fact, when t-test is applied to compare lysine retention measured of
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M. Rodehutscord et al.r Aquaculture 187 (2000) 177–183
Fig. 1. Response of rainbow trout to supplementation of lysine in the form of either L-lysinePHCl Žinterrupted line. or L-lysine sulphate Žsolid line.. The slopes of the two respective regression lines are not significantly different for BW gain Ž P s 0.70. and lysine retention Ž P s 0.62.. Values in brackets show the standard error of the respective estimated parameter.
the highest lysine level only, the difference between the two lysine sources Ž1.0 g lysine retentionrtank. is significant Ž P s 0.03., whereas the difference on the low level of supplementation is only 0.2 g lysine retentionrtank Ž P s 0.73.. It is unclear whether these differences can be attributed to the sole difference between the two sources, which was the concentration of sulphate. Perhaps other residues of the fermentation process that are still contained in the biomass may have a growth-diminishing effect at high level of inclusion of this product. With regard to the application in practise, however, this appears irrelevant because such a high level of inclusion of the free amino acids is unnecessary when proteins with a more balanced amino acid pattern than wheat gluten are used. The availability or ‘bioavailability’ is often calculated to rank different sources of one nutrient based on the ratio of slopes like the one given in the upper panel of Fig. 1. This,
M. Rodehutscord et al.r Aquaculture 187 (2000) 177–183
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however, can only give a relative value for the sources tested and requires a well-standardised reference if the values are to be used by the commercial feed compounder. In cases where lysine retention is determined and plotted against lysine intake, the slope is a direct measure of and gives an absolute value for the utilisation of the supplemented lysine source. Based on Fig. 1 Žlower panel., the utilisation of free lysine to rainbow trout is, on average, 68%.
Acknowledgements The authors gratefully acknowledge amino acid analysis done by Degussa-Huls ¨ and statistical advice by Dr. Ernst Tholen.
References Baker, D.H., 1994. Utilization of precursors for L-amino acids. In: D’Mello, J.P.F. ŽEd.., Amino Acids in Farm Animal Nutrition. CAB International, Wallingford, pp. 37–61. Henrichfreise, B., Pfeffer, E., 1992. Wheat and wheat starch as potential sources of digestible energy for rainbow trout Ž Oncorhynchus mykiss .. J. Anim. Physiol. Anim. Nutr. 67, 143–147. Kim, K.-I., Kayes, T.B., Amundson, C.H., 1992. Requirements for lysine and arginine by rainbow trout Ž Oncorhynchus mykiss .. Aquaculture 106, 333–344. Lanari, D., Tibaldi, E., Ballestrazzi, R., Tulli, F., 1991. Impiego di diete a moderato contenuto di farina di pesce per la trota iridea. Rivalutazione del fabbisogno di lisina della specie ed effetti sulle acque reflue. In: Atti IX Congresso Nazionale. pp. 1089–1098. Pfeffer, E., Al-Sabty, H., Haverkamp, R., 1992. Studies on lysine requirements of rainbow trout Ž Oncorhynchus mykiss . fed wheat gluten as only source of dietary protein. J. Anim. Physiol. Anim. Nutr. 67, 74–82. Pfeffer, E., Kinzinger, S., Rodehutscord, M., 1995. Influence of the proportion of poultry slaughter by-products and of untreated or hydrothermically treated legume seeds in diets for rainbow trout, Oncorhynchus mykiss ŽWalbaum., on apparent digestibilities of their energy and organic compounds. Aquacult. Nutr. 1, 111–117. Rodehutscord, M., Becker, A., Pack, M., Pfeffer, E., 1997. Response of rainbow trout Ž Oncorhynchus mykiss . to supplements of individual essential amino acids in a semipurified diet, including an estimate of the maintenance requirement for essential amino acids. J. Nutr. 127, 1166–1175. Rodehutscord, M., Borchert, F., Gregus, Z., Pack, M., Pfeffer, E., 2000. Availability and utilisation of free lysine in rainbow trout Ž Oncorhynchus mykiss .: 1. Effect of dietary crude protein level. Aquaculture 187, 163–176. Rodehutscord, M., Mandel, S., Pack, M., Jacobs, S., Pfeffer, E., 1995. Free amino acids can replace protein-bound amino acids in test diets for studies in rainbow trout Ž Oncorhynchus mykiss .. J. Nutr. 125, 956–963. Rodehutscord, M., Pack, M., 1999. Estimates of essential amino acid requirements from dose–response studies with rainbow trout and broiler chicken: effect of mathematical model. Arch. Anim. Nutr. 52, 223–244. Roth, F.X., Stangl, G.I., Kirchgessner, M., 1994. Biological efficiency of L-lysine base and L-lysine sulphate compared with L-lysinePHCl in piglets. Agribiol. Res. 47, 177–186. Schulte, H.-G., 1995. Bewertung verschiedener Fettarten in der Ernahrung von Regenbogenforellen. Doctoral ¨ thesis, University of Bonn, Shaker, Aachen. Schutte, J.B., Pack, M., 1994. Biological efficacy of L-lysine preparations containing biomass compared to L-lysine-HCl. Arch. Anim. Nutr. 46, 261–268. Walton, M.J., Cowey, C.B., Adron, J.W., 1984. The effect of dietary lysine levels on growth and metabolism of rainbow trout Ž Salmo gairdneri .. Br. J. Nutr. 52, 115–122.