Translation Series.No by Toru Ota, and Minoru Yamada

Transcription

1 FISHERIES RESEARCH BOARD OF CANADA iu Translation Series.No 2250 Lipids of Masu salmon Oncorhynchus masou. I. Variations of the lipid content and the fatty acid composition of juvenile Masu salmon during the period of smolt-trahsformation and on the influence of light upon:those variations, by Toru Ota, and Minoru Yamada Original title: SakUramasu Oncorhynchus masou no shishitsu dai-ippo hentai juki ni okeru sakuràmasu yogyono shishitsu ganryo.oyobi shibosan, sose1 po hendo'to sorera ni oyobosu hikari ho eikyo ni tsuite From: Hokkaido Daigàku Suisangakubu - Kenkyu Iho (Bulletin -of the Faculty of Fisheries,.Hokkaido University),. 22(2): , 1971 Translated by the Translation Bureau(YC) Foreign Languages. Division Department of the Secretary of State of.canada' Department of the,environment Fisheries Researchq3bard of Canada Halifax Laboratory Halifax, N. S pages typescript

2 DEPARTMEte OF THE SECRETARY OF STATE TRANSLATION BUREAU MULTILINGUAL SERVICES DIVISION mr\.b5'yg ff CANADA fr--/(\,) SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES 5-b TRANSLATED FROM - TRADUCTION DE AUTHOR - AUTEUR Japanese Toru OTA nt-id Minoru YeADA INTO - EN English TITLE IN ENGLISH TITRE ANGLAIS Lipid s of Ma su Se lm on ONCORHYNCHUS MA SOU 1. Variations of the Lipid Content and the Fatty Acid Composition of Juvenile Masu Salmon during the Period of ic.knolt-transfolmation., and on the Influence of Light upon those Variations SAKURAMA SU ON YNCHUS MA SOU NO SHISHITSU DAI-IPPO HTAI M JUKI NI OKERU n SUReA MM O NO SHISHITSU GN A RY0 =PI SHIBOSAN SOSEI NO HENPO TO SORERA NI OYOBOSU MARI NO EIKYO NI TSUITE TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGÉRE (TRANSCRIRE EN CARACTÈRES ROMAINS) REFERENCE IN FOREIGN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE FOREIGN CIIARACTERS. RÉFÉRENCE EN LANGUE ÉTRANGÉRE (NOM DU LIVRE OU PUBLICATION), AU COMPLET, TRANSCRIRE'EN CARACTÈRES ROMAINS. 1-10KUDAI SUI SAN II-10( Bulletin of -the Faculty of Fisheries, Hokkaido University) REFERENCE IN ENGLISH - RÉFÉRENCE EN ANGLAIS Bulletin of the Faculty of Fisheries, Hokkaido University PUBLISHER- ÉDITEUR Faculty of Fisheries, Hokkaido Universi4 PLACE OF PUBLICATION LIEU DE PUBLICATION YEAR ANNÉE DATE OF PUBLICATION DATE DE PUBLICATION VOLUME ISSUE NO. NUMÉRO Hokkaido, Japan. 18 PAGE NUMBERS IN ORIGINAL NUMÉROS DES PAGES DANS L'ORIGINAL e NUMBER OF TYPED PAGES NOMBRE DE PAGES DACTYLOGRAPHIÉES REQUESTING DEPARTMENT Tr. MINISTÈRE-CLIENT bnvironment BRANCH OR DIVISION DIRECTION OU DIVISION Fisheries Bervice Office of the Editor TRANSLATION BUREAU NO. NOTRE DOSSIER re TRANSLATOR (INITIALS) TRADUCTEUR (INITIALES) YCP PERSON REQUESTING John Carrtp AUG DEMANDÉ PAR YOUR NUMBER VOTRE DOSSIER NO DATE OF REQUEST DATE DE LA DEMANDE (REV. 2/ UNEDITED TRANSLATION For informalion only TRADUCTION NON nievi-52e

3 7530, DEPARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU 'MULTILINGUAL SERVICES DIVISION Erihm seed' CANADA SECRÉTARIAT D'ÉTAT, BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES CLIENT'S NO. DEpARTMENT DivisION/BRANCH CITY No Du CLIENT MINIsTiRE DIVISION/DIRECTION VILLE Environment Fisheries Service Ottawa. Office of the Editor BUREAU No. LANGuAGE TRANSLATOR (INITIALS) No DU BUREAU LANGUE TRADUCTEUR (INITIALES) Japanese YCP AyA ne2 n r ULIII Lipids of Masu Salmon ONCOMYNCIR3S MASOU. Report No. I. Variations of the Lipid Content and the Fetty Acid Composition of Juvenile Masu Salmon during the Period of Smolt- Transformation, and on the Influence of Light upon those Variations By Toru OTA and Minoru YAMADA Abstract The purpose of this \vork was to investigate the variations of the lipid content and the fatty acid composition in the muscle of the cultivated juvenile Masu salmon during the period of smolt-transformation,.and the influence of light upon those variations. Tho lipid content of the control group decreased rapidly at the mid-smolt stage, but remained almost unchanged from the mid-smolt stage to the postsmolt stage. On 'the contrary, the lipid content of the illuminated group was held constant from the pre-smolt stage to the mid-smolt stage, but decreased gradually from the mid-smolt stage to the post-smolt stage and it was smaller than that of the control group at the post-smolt stage. The contents of the total saturated and monoenoic acids decreased from the pre-smolt stage to the post-smolt stage in both groups. Especially, the rate of the decrease of the total monoenoic acids was greater in the illuminated group than in the control group at the post-smolt stage. :Et was estimated that hexadecanoic acid and octadecenoic acid were mainly used qis the source of energy during the period of -smolt- transformation. The fact that light accelerates transformation of the juvenile Masu salmon at the early stage, but delays it at the latter stage in the period of smolt-transformation» also became clear from the variations of the lipids in the muscle of the juvenile Masu sahnon. SOS UNEDITED TRANSLATION For information only TRADUCTION NON REVISI1E Information szulonlont

4 2 Introduction Throughout the life of Masu Salmon (Oncorhynchus Masou), its life phases cb,.n be divided into several ecological stages in accordance with its morphology and (the development of) physiological functions, and a name is given to each of these stages, as is done with the other members of.the salmon-trout families. Already, many researchers have reported on the function of lipids as source of energy in a living beings body, and it is conceivable that the lipid content in the Masu salmon also undergoes qualitative as well as quantitative changes during its gtowth process, as its physiological functions undergo changes. * Hokkaido Daigaku Suisan-gakubu Gyoyu Kagaku Koza (Laboratory of Chemistry of Fish Oil, Faculty of Fisheries, Hokkaido University). 152) Espectally, it is knon_that during the period when the juvenile Masu salmon undergoes smolt-transformation and ḇegins seaward migration from fresh water into saline water, it has a capacity to adjust to salinity, enabling it to adapt to the - environment.' Conceivably, a change in physiological function such as this would also cause some changes in the lipids in the body. Also, the external, environmental factors generally influence the development of the fish in a significant manner. Among the main environmental factors, one may enumerate the energy generated by the light, water temperature, food, etc.( 1 ) There factors are considered to be also the factors that influence the lipid content

5 in the body of fishes. Reiser et al, (2) Knipprath et al, (3 ) and Toyom izu et al (if)- have already reported on the influence of water temperature and food. Light is a factor that significantly affects the ripening of reproductive cells of the fish. In experiments using the river trouts (Salvelinus fontinalis Mitchell), the ripening process was accelerated in the group of fish that received strong illumination, compared to the group that received normal light. However, after strongly stimulating the development of the gonad, the light may have the'effect of suddenly lowering,the pace of the development.( 1 ) In his culturing experiment, Kubo (5 ) divided masu slamons into two groups, one receiving normal daylight and the other receiving additional artificial light during a given peric;d of time at night. It was observed that during the enolt transformation of the juvenile masu salmon, illumination contributed to the acceleration of the transformation process at first, and then to the slowing-down of that process at a later stage. Thus, illumination may be considered to have a certain given effect on the physiological function of fishes, and it interesting to consider what kind of changes in the lipid content of the body are simultaneously brotight about by illumination..for the purpose of solving the above problem, the authors used masu salmon an specimen and examined the changes in their lipid contents during the period of enolt-transformation. The influence of illumination

6 4 on these changes are also examined. The results are reported here. Method of Experiment Materials Used In The Experiment: The juvenile masu salmon used' in these experiments were cultured at the.nanameshi fish-culture experimental. station of the Dept. of Fisheries, Hokkaido University..Seventy six fish, all of which were estimated to undergo nnolt transfonnation in December 1965, were selected. Then thirty eight fish each - were put in two ater tanks (length: 5.4 m, width: 1.8 m.,depth 60 cm.), then one of the tanks was put under natural light while the water sur.r. face of the other tank was subjected to six hours of nighttime illumination under artificial light (One fluorescent lamp, 20 W), in addition to the same natural light that the first group received. Both»groups were fed with diet shown in Table 1. During the period before the beginning of mnolt transfonnation, i.e. Mar-ch-May, 1963, a few fish were taken at random from the two groups_every 20 days, to be used as the materials for studies. In the following, the group cultured under artificial light will be called the illuminated group, and the group. that did not receive artificial light will be called un-illuminated group, for the purpose of abbreviation. Extraction of Lipids: Crude lipids were extracted from the muscles of the fish using solvents such as aceton, ether etc. In order to

7 prevent any changes in the characteristics of the lipids, extraction and concentration were done in the carbondioxide current, under reduced pressure. The preparation of fatty acid mixture methylester: The acetonsoluble lipids were separated by treating the crude lipids with acetone. From these aceton-soluble lipids, mixed fatty acids were prepared by an ordinary method. Further, a 5% HCI-methanol solution was used to make, the mixed fatty acid into methylester. Gaschramatography: The analysis of the composition of fatty acids was conducted under conditions shown in Table 2, using as the material the mcthylester prepared in the above-described fashion, adopting the some method used by Ito et al (6), by means of two kinds of polyester columns, enploying the yanagimoto Gaschrumatograph model GOG-2. The identification of each component was made either by comparing it with standard substance, or by the linear correlation between the number of carbon atoms and the logarythmic value of relative staying time (stearic acid methylester = 100.0). The values obtained by cuantitativér, anaiysi.s were expressed by the percentages of the peak arecs calculated.by medium-value range method. Results and Observations Variations in Lipid Contents: Table 3 shows the body-lengths and

8 lipid contents of esch individual fish from the illuminated group and un-illuminated group. In both groups, decline in their lipid content was seen to accompany the progress of unolt transformation, but somewhat different tendency would be recognized in the degree of the decline. In the un-illuminated group, cultured under normal sunèhine, their lipid content fell rapidly during the period fram the beginning of the enolt transformation to its middle stage, from 3.8% to 2.3%. But after that, their lipid content showed virtually no change until the final stage of smolt transformation. It seems that this kind of variation in lipid content is related to the changes in the physiological functions of the fish body. This phenomenon seems to suggest that, during the period fram the beginning to the middle of smolt-transformation process e ( 153 Table 1. Composition' of basal diet (%) Artificial feed A* Artificial feed B** Dried ncomysis Bran (Lipid content 5.5%) * Nihon Haigo Shiryo K.K ** Nihon Kagalar Shiryo K.K Table 2. Operating conditions of gas-liquid clu omatography Instrument: Colutmi: Column temp.: Carrier gas: Detector: Yanagirnoto Gas Chromatograph CCG-2 1) 20% PolydiethvIene glycol succinate on Colite 545 (60-80 mesh) ' 2) 15% Poly-propylene glycol succinate on Colite 545 (60-80 mesh) 5 min i.d.x 2m. 215 C Helium, Flow rate 60 iii/min TCD, Sens. 2mV, Bridge Curr. 170 mà

9 7 the degree to which lipids are consumed is large because it is used as the source of energy for the purpose of carrying out smolt transfonnation..thus, even though the feeding activity during this period is the - same as that in normal times, it does not adequately replenish the lipids that are consumed. On the other hand, unlike the un-illuminated group, the lipid content of the illuminated group remained almost unchanged, at approximately 4%, from the early stage of enolt transformation throughout to the middle stage. However, from the'middle state to the final. stage, (the lipid.content of the illuminated group) declined gradually, and in the final period, showed a value which was 1% lower than that of the unilluminated group. Kubo (7) investigated the influence of the prolonged sunlight illumination on the growth of the body of the masu sâlmon during its enolt transformation period. The treatment by the prolongation of sunlight-illuminating time was achieved by the use of artificial light. It was observed that from the early stage through to the middle stage of unolt transformation, the growth of the body was good, but from the middle to the final stage, the growth tended to stagnate. The variation of the amount of lipid content may be considered to demonstrate a tendency similar to that of the changes in bodyirowth. In other words, during the earlier stages of enolt transformation the illuminating treatment has a positive effect on the functioning of the fish's body, leading to increased feeding

10 8 activities. If follows that body growth luld be satisfactory during this period, and the amount of lipids accumulated would be sufficient to compensate for the amount that would be consumed in this period. Conversely, from the middle to the final stage, as illumination suppresses the functioning Of the body, the accumulated lipids would be consumed, resulting in the reduction of lipid contents in the_muscles. The Composition of Fatty Acids: Table 4 shows the compositions fatty acids and the diet given to the juvenile masu salmon during the early and final stages of their molt transformation. Among the fatty acids that constituted the lipids in the juvenile masu salmon, about 20 kinds of saturated and unsaturated fatty acids were separated, ranging frm those with 12 carbon atms to those with 24 carbon atoms. Of these, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:5, 22:6 etc were found to exist in largest amounts. When the food given to. the juvenile masu salmons was compared to their fatty acid composition, it was found that the contents of 18:0, 16:1, 22:6 etc were the highest in the juvenile Masu Salmon while the content of 18:2 and 20: were low. Since this studyis original purpose is not in investigating the influence of food, the fatty acid composition of the juvenile masu salmons before their receive'd the basal diets was not examined. However, judging from the fact that the fatty acid composition in (the bodies of) Masu Salmon are different from that of the food, it may be reasonable to deduce that, when the fish is cultured for a long period of time under the same

11 diet, it will gradually form a fatty acid composition that is in tune with the fish's physiological and envixon mental conditions, althoue food does have certain influence cover the fatty acid composition of the fish). Changes in Fatty Acid,Composition: Fig. 1, Fig. 2, and Table 5 show the changes in the amounts of fatty acid and totally-saturated, monoenoic and polyenoic acids contained (in the body of the juvenile masu salmon), as well as the relative fatty acid content, frau the early stage to the final stage of smolt teansformation. As for the changes in the content of the fatty acids that were camnon to both groups, there was a tendency for 16:0, among the saturated acids, to decline. Among the monoenoic acids, in both groups, the content of 16:1 remained in the neighborhood of 10% from the early stage to the final stage of enolt trnasformation, demonstrating virtually no change. The proportion of 181 content showed decline in both groups: in the un-illuminated group it fell from 27.9% to 24.3%, and in the illuminated group,it fell from 28.5% to 23.2%. In the polyenoic acid, tlere were great deal of scatter in the variation of each individual fatty acid, and. (1,5

12 10 Table 3. Body length and lipid content of juvemle Mast& salmon Control group Illuminated group Date Stage Body Lipid Body Lipid Sex length. content Sex length content (cm) (%) (cm) (%) F* F F M Mar. 10 Pre-smolt F F M** Aver. 3.8 Aver. 4.1 P F F F Apr. 1 Mid-smolt F à 8 M F F M Aver. 2.3 Aver. 4.0 F F F F F F Apr. 22 Mid-smolt F F M F F Aver Aver. 3.1 F F M M May 7 Post-smolt F F F P P Aver. 2.5 Aver F 12. M M M May 22 Post-smolt M F F F F F Aver. 2.4 Aver. 1.4 * Female ** Male

13 11 1 it was not possible to recognize any fatty acid that is common to both groups which demonstrated common and consistent tendency to rise or fall. In the variations of the contents of totally saturated, monoenoic and polyenoic acids, totally-saturated acid and total monoenoic acid contents declined, and conversely, total polyenoic acid content,increased. As described earlier, the amount of lipid content declined during the period fram the early stage to the final stage of smolt transformation. It is surmised that this decline is due to the fact that lipids are consumed as the source of energy when the physiological function of the fish (body) changes (undergoes transformation). Judging fram the observation of the variation in fatty acid (contents) shown in this experiment, the consumption of fatty acids consisted mainly of 16:0 and 18:1. On the Influence of Light over Fatty Acid Composition: The differences between the two groups in their fatty acid composition, measured in terms of fatty ecid content ratio, were examined (Table 5). The value of was always higher for the illuminated group throughout the entire experimentation period. This seans to be caused by the fact. that the 18: content was higher for the illuminated group. Also, at the final stage of anolt transformation, the illuminated group showed a analler content of 16:1 and 18:1 (Table 4). As for the amount of total monoenoic acid and total polyenoic acid, the total monoenoic (acid content was smaller in the illuminated group

14 12 (155 Masu. salmon Fat-ty acid Basal diet Contro group Illuminated group Saturated acids Table 4. Fatty acid composition of acetone soluble lipids of basal diet and juvenile ilias;c salmon (%) 12:0 tr 14:0 15: O. 5 16: :0* Total 25.5 Monoenoic acids 141 tr 15:1 tr 16:1 4.6 tr 18: : :1 tr Total 32.2 Polyenoiç acids 18: : :2 tr 20:3 tr : :5 tr 22:6 4.5 Total 42.3 * Includes 16:2 ** Other acid may be present Mar. 10 May 22 Mar. 10 May , tr tr tr tr , tr tr tr tr tr tr

15 throughout the entire experimentation period. On the contrary, the total polyenoic acid content was greater (in the illuminated group). The difference between the two groups became great especially during the final stage of enolt transformation, compared to the early and middle stages. Further when the variations in the polyenoic acid (content) in the illuminated group from May 7 to May. 22 were examined, 18:22 changed from 12.8% to 12.2%, 18:3 changed from 6.6% to 3.8%, both showing decline. In contrast, 205 changed from 4.7% to 5.7%, and 22:6 changed from 7.7% to 9.4%, both showing increase. Both 18:2 and 18:3 are fntty acids that açe subject to external influences, and when the reductions in 18:2 and 18:5 are considered together with the reduction in total monoenoic acid content, the causal relation of these changes may be deduced as follows: illumination causes the feeding activities of the fish to decline during the final stage of molt transformation. At the saine time, the degree to which the lipids in the muscle are consumed as the souces of energy increases. The result is the decline in the fatty acid contents. On the bther hand, the increase in 20:5 and 22:6 may be related to the reduction in 18:3, when the mutual transformation, from 18:3 to 22:6 and vice versa, is considered. However, from the present experimentation, it is difficult to draw any conclusion on the degree to which illumination influences the mutual transformation between unsaturated fatty acids. The reasons are that the materials used in this experiment consisted of fish that are not under normal mode of life: i.e. They were undergoing emolt transformation. The fact that

16 14 ( o o 18:1 Control group Illuminated group :0' : :6 16: :0 20:5 00 8o c r?-0 o, 14:0. 0 ' 0 ' Mar. ' Apr. Apr. May May Mar. Apr. Apr. May May Date I Date Fig. 1. Variations of saturated and tno- Fig. 2. Variations of polyenoic acids noenoic acids in acetone soluble lipids of in acetone soluble lipids of juvenile Maszt juvenile ileau salmon salmon I_ 1, Total polyenoic acids Total monoenoic acids Total saturated acids 25 k / U, _..« Mar. Apr. Apr. May May Date Fig. 3. Variations of total saturated, monoenoic and polyenoic acids in acetone soluble lipids of juvenile Masu salmon

17 15 (157 ; Table 5. Variations of several ratios of fatty acids in acetone soluble lipids. of basal diet and juvenile illasu salmon Ratios 16 :0/Total sat. acids 16 :1/Total mono. acids 18 :1/Total mono. acids 16 :0/1'otal sat. acids 16:1/Total mono. acids 18 :1/Total mono. acids 1,13/X.6 Basal diet : Mar Mau. salmon Apr. Apr. 1 May May Control group Illuminated group : there are several elements.that are related to variations in the substances contained in the living body of a fish, also contributes to the difficulty. Consequently, in order to find. out what the ensral relationships between illumination and the lipid metabolism of the fish are, it seems necessary to carefully consider the physiological state of the fish and then examine the matter further. In the present experiment, juvenile cultured masu salmon was Used. as the material, and only the variation in lipids were examined, in terms of lipid content and fatty acid composition. In the future, it is necessary to study the masu salmon that grow in natural rivers, and the lipid compositions must be classified for all developmental stages, and not just the smolt transformation period.

18 1 6 The variation in tri-glyceride, free fatty'acid, and phosphorous lipids must also be examined. No doubt the accumulation of these data will provide e clue to the solution of the problems of masu salmon resource conservation. Summary 1) Cultured juvenile masu salmon (Oncorhynchus Masou) was used as the material for an investigation of lipid content and fatty acid composition in their muscle during the period of smolt transformation, as well ès the influence of illuminatjon on them. 2) In the un-illuminated group (control group), their lipid content sharply declined from 3.8% to 2.3% fram the early stage to the middle stage of smolt transformation. changes until the final stage. But after that there was almost no In. contrast, the lipid content in the illuminated group showed no change at 4% from the early to the middle stage of smolt transformation, but gradually declined fram the middle to the final stage and by the final stage showed a*value which was - lower than the unilluminated group by 1%. 3) The content of total saturated acid and total monoenoic acid in both groups declined as smolt transformation progressed, but the degree of decline in total monoenoic acid content at the final stage of smolt transformation was greater in the illuminated group. It is surmised that these phenomena are the result of the influence of illumination,

19 1 7 which lowered the feeding activities of the fish and at the same time caused the consumption of lipids in the muscle as the source of energy. 4) Among the lipids in the juvenile masu salmon, the major fatty acids that consumed as the sources of energy during the period of its enolt transformation are estimated to be 16:0 and 18:1. 5) Kubo (5) made clear in his report that during juvenile masu salmonts enolt transformation, illumination accelerates the transformation in the earlier stages but delays it in the later stages. It - was possible to confirm this fact frcel in terms of variations in lipid composition. In conducting these experiments, we received many advices from Mr. Tatsuro Kubo, Assistant Professor of the Dept. of Fisheries, Hokkaido University, and Mr. Jun Kosaka of Tohoku Regional Fisheries Research Station, and wish to express our deep appreciation. (157 1) NICHOLSKY (1965): GYOBUI SEITAI GAKU (Kamei Kenzo Yaku), 516P,YONEGO, SHINKAGAKU BUNKEN KANKO-KAI. (Fi.sh Ecology, translated by Kenzo Kamei, 516P. Published by the Association of New Scientific Liberature Pu Iblishing, Yonego.) 2) 3)

20 18 4) TO YOMIZU MASAMIOHI, KAWASAKI KENJI, TOMIYASU YUKIO (1963), NICCUISHI 29 (10) (biesamichi Toyomizu, Kenji Kawasaki, Yukio Tomiyasu (1963), Journal of Japanese Fisheries, 29(1O) ) 5) KUBO TATSURO (1959), HOKKAIDO SAKE.MASU FURAJO KENKYU HOKOKU. (14), (Tatsuro Kubo (1959), Research Report of the Hokkaido Salmon-Trout Hatchery. (14), 15-19) 6) _ ITO SUIKE:TAKA, FUKUZUVI KAZUO (1963),..)YUKAGAKU.. (Suketaka Ito, Kazuo Fukuzumi (1963), Oil Chemistry 12(5) ) 12 (5) ) KUBO TATSURO (1964), NIP:=ON SUISAN GAKKAI HOKKAIDO-SHIBU T.AIKAI KOEN YOSHI. (Tatsuro Kubo (1964), Summary of the Speach at the General Meeting of the Hokkaido Branch of the Japanese Fisheries Association. 1) :-.- )1,7 k (1963). ff,i3mw (eeer,-_-e). 316p. 2) Reiser, 11., Stevenson, B., Kayama, M., Choudhury, R. B. R. & Hood, D. W.(1963). J. Am. Oil Ch.em. Soc. 40(10), ) Knipprath, W. G. & Mead, J. F. (1966). Lipids. 1(2), ) 112mm plermt; germ (1963). HAtedg 29(10), ) Mk:es (1959). 1LekiU13' (14), ) fewer& imu xt (1963). 12(5), ) A'aile (1964). El*7Jceee4teillee-.-ezeuzW.