The Order of Limiting Amino Acids in Ladino Clover Leaf Protein Concentrate Fed to Chicks

Transcription

1 227 The Order of Limiting Amino Acids in Ladino Clover Leaf Protein Concentrate Fed to Chicks Hiroshi UEDA and Mitsuaki OHSHIMA Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa-ken From the point of view of the nutritional and physiological differences between livestocks, it is one of useful means to extract protein concentrates from forages and feed them to mono-gastric animals and fibrous residues after the extraction to ruminant animals. OHSHIMA and his associates have examined the nutritive value of leaf protein concentrate (LPC) extracted from ladino clover as a protein source for rats1) or pigs2) and that of the remaining fibrous fraction as a roughage for sheep3). OHSHIMA and OOUCHI1) reported that the nutritive value of ladino clover LPC as a sole source of protein for rats was not so high but was drastically improved by supplementing methionine, first limiting amino acid, which were indicated by the biological values of 21 and 77, respectively. However, the nutritive value of ladino clover LPC for chicks has not been investigated yet. The objective of the present study was to determine the order of limitation of amino acids in ladino clover LPC as a sole source of protein fed to chicks and the supplementary effect of the limiting amino acids. Materials and Methods LPC was prepared from ladino clover (Trifolium repens L.) harvested in the year of Soon after being harvested, the herbage was chopped into 1 to 2cm length, disintegrated in a pulper and pressed using a presser with an oil jack. The expressed juice finely ground using a ball mill. This LPC was composed of 53.8% crude protein. Ladino clover LPC was used as a sole source of protein to provide 10% or 18% crude protein content. The composition of the 18% protein diet is given in Table 1. To prepare the 10% protein diet, extra LPC was replaced by the equal amount of cornstarch. The composition of amino acids in the 18% protein diet is shown in Table 2. Methionine and cystine were determined as methionine sulfone and cysteic acid, respectively, by a formic acid-hydrogen peroxide oxidation method and the other amino acids by a standard chromatographic procedure4) using an automatic amino acid analyser (Hitachi KLA-5, Hitachi Co. Ltd., Tokyo). Tryptophan was not determined. Table 2 shows that the basal diet was deficient in only sulfur-containing amino acids as compared with the amino acid requirement recommended by the National Research Council5). To determine the first to third limiting amino acids in the ladino clover LPC fed to chicks, three feeding trials were carried out. In the first 2 trials, the 18% protein diet Received February 2, 1982.

2 228 Japan. Poultry Sci., 19 (4), 1982 Table 1. Composition of basal diet containing 18% crude protein from ladino clover leaf protein concentrate (LPC) Table 2. Amino acid composition of the 18% protein diet 1 UEDA et al. (9). 1 NRC requirement (5). was used. In Experiment 1, graded levels of L-methionine from 0% to 0.4% in 0.1% increments were added and in Experiment 2, 0.37% L-lysine hydrochloride alone or together with 0.24% L-threonine or 0.42% L-arginine hydrochloride were added to the diet already supplemented with 0.3% L-methionine. In Experiment 3, the 10% protein diet was supplemented with 0.3% L-methionine alone or together with 0.37% L-lysine hydrochloride or 0.24% L-threonine, or both. In all experiments, the supplementation of amino acids to the respective basal diets was made at the expense of equal amounts of cornstarch and the level of the individual amino acids supplemented in the last 2 trials was equal to 0.3% methionine on the mole basis. Day-old Single Comb White Leghorn male chicks were reared on a commercial chick starter ration for 7 days in electrically heated battery brooders. On day 8, the necessary numbers of chicks were selected from the stock on the basis of the uniformity of their body weights and they were housed in stainless steel cages in pairs (Experiments 1 and 2) or individually (Experiment 3). Four replicates were randomly allotted into each of the dietary treatments and feed and water were supplied for 8 days (Experiments 1 and 2) or for 12 days (Experiment 3). Excreta were collected during the last 3 days of the experimental period at 24-hour interval in a tray sprayed with 5% hydrochloric acid. They were homogenized with an for 48 hours and analysed for nitrogen content by a Kjeldahl procedure. Nitrogen utilization was calculated as a ratio of retained nitrogen to nitrogen intake. After the body weight was measured at the end of the feeding trials, blood was taken from the individual chicks by heart puncture using a heparinized syringe and blood plasma was obtained by centrifugation at 3,000r.p.m. for 10 minutes. Equal volumes of the plasma samples within a replicate were pooled and plasma uric acid concentration was determined by the phosphotungustate reduction methods6) in all feeding trials. Plasma amino acid concentrations were determined in the first experiment by a standard chromatographic procedure4) using Hitachi KLA-5 amino acid analyser after the plasma was deproteinized with the 5-fold amounts of

3 saturated picric acid. UEDA, OHSHIMA: Ladino clover LPC and methionine 229 Data were analysed using the analysis of variance technique and multiple comparisons among treatment means were made by the TUKEY'S multiple procedure7). Regression analysis7) was also used when it was necessary. Results and Discussion The effects of supplementation of graded levels of methionine on performance, nitrogen utilization and plasma uric acid concentration in chicks fed the 18% protein diets were presented in Table 3. The supplementation of methionine up to 0.2% resulted in linear increases in body weight gain and gain/feed ratio (r=0.955 and 0.961, respectively, p< 0.01). Feed intake was also increased by 0.1% methionine supplement and reached constant above this level. Nitrogen utilization was linearly (r=0.764, p<0.01) increased and, in turn, plasma uric acid concentration was linearly (r=0.745, p<0.01) decreased with increasing the supplemental methionine level up to 0.2% beyond which they were unaffected by the supplemental methionine. Table 4 shows the plasma amino acid concentrations in the chicks fed graded levels of Table 3. Effects of the supplementation of graded levels of methionine on body weight gain, feed intake, gain/feed ratio, nitrogen utilization and plasma uric acid concentration in chicks fed the 18% protein diet (Experiment 1) 1 Values are mean of 4 replicates of 2 chicks each. Means not sharing common superscript letters are significantly different at 5% level. Table 4. Effect of the supplementation of graded levels of methionine on plasma amino acid concentrations in chicks fed the 18% protein diet (Experiment 1) 1 See footnotes 1 and 2 in Table 3.

4 230 Japan. Poultry Sci., 19 (4), 1982 methionine in Experiment 1. As the supplemental methionine level was increased up to 0.3%, methionine was linearly (r=0.919, p<0.01) accumulated in the plasma. The regression equation was Y=29.4X+6.1, where Y was plasma methionine concentration in 0.4% methionine supplement, the plasma methionine concentration was much greater than the value expected from the above equation. The result of Experiment 1 showed that the first limiting amino acid in the ladino clover LPC fed to chicks was methionine as well as in the case of rats1,8). The beneficial effect of the supplemental methionine on chicks resulted from both the increase in feed intake, by which energy utilization was probably improved9), and the improvement in nitrogen utilization. The 18% protein diet contained 0.50% the sulfur-containing amino acids so that the requirement recommended by the NRC5) was met by 0.1% methionine supplement. However, the maximal growth rate was not obtained by this level but by above 0.2% methionine supplements. The increase in growth rate by increasing the supplemental methionine level from 0.1% to 0.2% was accompanied by the improvement in nitrogen utilization and the decrease in plasma uric acid concentration but not by the increase in feed intake. Similar results were reported previously9) and by SOLBERG et al.10). In general, whether the supplemental methionine facilitates or not the feed intake seems to be dependent on the level of the sulfurcontaining amino acids in a basal diet11). The effect of 0.4% methionine supplement on chick performance, nitrogen utilization and plasma uric acid concentration was comparable to that of 0.2% or 0.3% supplement, but more methionine was accumulated in the plasma at 0.4% methionine supplement. According to ZIMMERMAN and SCOTT12), when a limiting amino acid (lysine, valine or arginine in their experiment) was added at superoptimal levels, the corresponding amino acid was rapidly accumulated in the plasma. A similar result was observed with chicks fed excess methionine9). Thus, the supplementation of 0.4% methionine seemed to be slightly excess and that of 0.2% to 0.3% to be adequate. That the first limiting amino acid was methionine was indicated by comparing the amino acid composition with the chick requirements (Table 2). The comparison further indicated that lysine and arginine ranked next to methionine. OHSHIMA and OOUCHI1) reported that the second limiting amino acid in the ladino clover LPC fed to rats was lysine but the third was threonine on the basis of the biological value and plasma urea concentration. As shown in Table 4, graded increments of dietary methionine level progressively lowered the plasma concentrations of lysine and threonine (r=0.937 and 0.832, respectively, p<0.01) but unaffected that of arginine. A similar result was reported by OHSHIMA and OOUCHI1). As shown in Table 5, however, the supplementation of lysine alone or together with threonine or arginine to the 18% protein diet containing the additional methionine did not affect chick performance, nitrogen utilization and plasma uric acid concentration. This ineffectiveness seemed to be attributed in part to the dietary protein content, since the 18% protein diet satisfied the requirement of the indispensable amino acids other than the sulfurcontaining amino acids (Table 2). Thus, in Experiment 3, the dietary protein content was reduced from 18% to 10% and the feeding period was prolonged from 8 days to 12 days. The results of Experiment 3 in which the supplementary effects of lysine and threonine

5 UEDA, OHSHIMA: Ladino clover LPC and methionine 231 Table 5. Effects of the supplementation of lysine alone or together with threonine or arginine on body weight gain, feed intake, gain/feed ratio, nitrogen utilization and plasma uric acid concentration in chicks fed the 18% protein diet containing additional methionine (Experiment 2) 1 Values are mean of 4 replicates of 2 chicks each. 2 See footnote 2 in Table 3. Table 6. Effects of the supplementation of methionine alone or together with lysine or threonine, or both on body weight gain, feed intake, gain/feed ratio, nitrogen utilization and plasma uric acid concentration in chicks fed 10% protein diet (Experiment 3) 1 Values are mean of 4 chicks each. 2 Not determined. 3 See footnote 2 in Table 3. as well as methionine were examined are presented in Table 6. Body weight gain, feed intake and gain/feed ratio were drastically improved and plasma uric acid concentration was significantly decreased by adding methionine. The supplementation of lysine in combination with methionine further increased body weight gain and gain/feed ratio although feed intake, nitrogen utilization and plasma uric acid concentration were not affected significantly. However, by replacing lysine with threonine, body weight gain, feed intake, gain/feed ratio and nitrogen utilization were significantly decreased. These results indicated that the second limiting amino acid in the ladino clover LPC diet was lysine, but the supplementary effect of lysine was much small as compared with that of methionine. The beneficial effect of threonine was obscure when it was supplemented in combination with methionine and lysine. As like as lysine or threonine, the plasma concentration of glycine plus serine was significantly lower in the chicks fed the 0.3% or 0.4% methionine-supplemented diet than those fed the diets containing lower levels of methionine (Table 4). However, the supplementary effect of glycine and serine was not examined because the ladino clover LPC was

6 232 Japan. Poultry Sci. 19 (4), 1982 rich in these amino acids as shown in Table 2. Plasma concentration of phenylalahine plus tyrosine was significantly increased by adding methionine above 0.2%, whereas those of other essential amino acids such as histidine, valine, isoleucine and leucine were unaffected by the dietary methionine level. As a conclusion, the amino acid composition of the ladino clover LPC was nutritionally well-balanced as a protein source for chicks if an adequate amount of methionine was fortified Summary The order of the limitation of amino acids in the ladino clover leaf protein concentrate (LPC) as a sole source of protein fed to chicks and the supplementary effect of the limiting amino acids were investigated. The feeding experiments were carried out in which chicks were fed 18% or 10% protein diet without or with supplementary amino acids. The first limiting amino acid in the ladino clover LPC was methionine. The supplemental methionine markedly improved growth rate in both protein levels resulting from the increase in feed intake and the improvement in nitrogen utilization which in turn lowered the plasma uric acid concentration. The second limiting amino acid was lysine the plasma concentration of which was progressively decreased with increasing the dietary methionine level. However, the supplementary effect of lysine in combination with methionine was not so great as compared with that of methionine alone, since the obvious effect was observed only when the 10% protein diet was fed. Acknowledgement We wish to thank Mr. R. TANIGUCHI and Mr. K. KONDOU for their excellent technical assistance. Literature 1) OHSHIMA, M. and K. OOUCHI: Nutr. Rep. Int., 14, 611, ) OHSHIMA, M., M. TAMAI and H. VEDA: ibid, 24, 1233, ) FUJIHARA, T. and M. OHSHIMA: J. Japan. Grassl. Sci., 26, 94, ) MORIMOTO, H., supervised: Experimental Methods in Animal Nutrition (in Japanese), Yokendo, Tokyo, Japan, ) National Research Council: Nutrients Requirements of Domestic Animals. I. Nutrients Requirements of Poultry, Nat. Acad. Sci. Res. Council, Washington, D. C., U.S.A., ) HENRY, R. J., C. SOBEL and J. KIM: Am. J. Clin. Path., 28, 152 (1957). 7) YOSHIDA, M.: Design of Experiments for Animal Husbandry (in Japanese), Yokendo, Tokyo, Japan, ) HORIGOME, T. and S. UCHIDA: Jpn. J. Zootech. Sci., 51, 429, ) UEDA, H., H. YOKOTA, M. OHSHIMA and I. TASAKI: Nutr. Rep. Int., 24, 85, ) SOLBERG, J., P. J. BUTTERY and K. N. BOORMAN: Br. Poult. Sci., 12, 297, 1971, 11) TASAKI, I, and M. YANAKA: Jpn. J. Zootech. Sci., 51, 632, ) ZIMMERMAN, R. A. and H. M. SCOTT: J. Nutr., 87, 13, 1965.

7 UEDA, OHSHIMA: Ladino clover LPC and methionine 233