FISHERIES RESEARCH BOARD OF CANADA Translation Series No. 1413

Influence of ozidized oil and vitamin E on the culture of yellowtail.

By H. Sakaguchi and A. Hamaguchi .

Original title: Sanka yu tenka shiryo ni yoru hamachi no shiiku to bitamin E tenka no Koka.

From: Nihon Suisan Gakkai-Shi (Bulletin of the Japanese Society of Scientific Fisheries), 35 (12): 1207-1214, 1969.

Translated by the Translation Bureau (RK) Foreign Languages Division Department of the Secretary, of State of Canada

Fisheries Research Board of Canada Halifax Laboratory Halifax, N.S.

1970

22 pages typescript Fee

DEPARTMENT OF THE SECRETARY OF STATE SECRÉTARIAT D'ÉTAT . TRANSLATION BUREAU BUREAU DES TRADUCTIONS FOREIGN LANGUAGES DIVISION DES LANGUES DIVISION CANADA ÉTRANGÈRES

TRANSLATED FROM'- TRADUCTION DE

Japanese English

• AUTHoR - AUTEUR .

H.Sakaguchi Hamaguchi

TITLE IN ENGLISH«,.- TITRE ANGLAIS • • Sanka.yu tenkehiryo ni yoru.hamachi no shiiku to bitamin ' -d'tenka, no ttiie 'in foreign:44eguagia (tranàliterate foreign oharactera) :Treluence. oftjçieized Oil anct , Vitamin , E on the Culture of .

RVURENCE IN Foftgtori ‘,ANGUAgE (MARE OF ISOOK OR PUBLICATION) IN FULL. TRANSLITERATE FoREIGN CHARACTERS. •IREFERENCE EN LANGUE ETRANGERE (NOM Du LIVRE Ou PUBLICATION), Au COMPLET. TRANSCRIRE EN CARACTERES PHONETIQUEs. Nippon Suisan Gakkai-Shi

REOERENCE. IN ENGLISH - RÉFÉRENCE EN ANGLAIS Bulletin oftheJapanese Society of - Scientific'Fisheries,

PUBLISH ER - iorrEurt PAGE.NUMBERS IN ORIGINAL DATE OF PUBLICATION • • NumER0S DES PAGES DANS Nippàn sp,isan etkkal DATE DE PUBLICATION L , 0RIGINAL 1207-1214 YEfte ISSLIE.No. VOLUME ANNEE NUMERO • PLACE OF, PUBLICATION NUMBER OF TYPED PAGES . 0E4 . .DE PUBLICAtION NOMBRE DE PAGeS DACTYLOGRAPHIÉES 1969 35 12 22

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DEPARTMENT OF THE SECRETARY OF STATE SECRÉTARIAT D'ÉTAT TRANSLATION BUREAU BUREAU DES TRADUCTIONS FOREIGN LANGUAGES DIVISION DIVISION DES LANGUES ÉTRANGÈRES

CLIENT'S NO. DEPARTMENT DIVISION/BRANCH CITY N° DU CLIENT MINISTERE DIVISION/DIRECTION VILLE Fisheries Research Board Halifax Laboratory Halifax, N.S.

BUREAU NO. LANGUAGE TRANSLATOR (INITIALS) DATE • No DU BUREAU LANGUE TRADUCTEUR (INITIALES) 0056 Japanese M. K.

Bulletin of the Japanese Society of Scientific Fisheries Vol.35, No.12, 1969. 1207 Influence of Oxidized Oil and Vitamin E on the Culture of Yellowtail

Hiromi Sakaguchi and Akira Hamaguchi**

In the previous paper1) , the effect of antioxidant treated sand-eel .as food for yellowtail was reported. Thereafter, we investigated the effects of oxidized oil and vitamin E administration on yellowtail fed with artificial diets. Fish were fed for 93 days with diets containing 8.5% of fresh or oxidized cod liver oil to which DL-etocopheryl acetate was either added or not (Table 1).. The weight gains of the 'fish are shown in Fig. 1 and Table 2. The fish fed with oXidized oil, without the addition ! 0 ‘LU of vitamin E, showed a slightly poor appetite after about • • (")

(ji 40 days • — . Cy. of feeding, and at the termination of experiment a

(72. 1.7.: •••• i; leaning of the dorsal muscle was observed on about 10% of the

0 fish. These symptoms are probably due to muscle dystrophy as 0 reported with the carp12) (Fig.2). However such symptoms were ( : 1 not found e -- < in the vitamin E supplemented group. I. . The muscle of fish fed with oxidized oil but without

** (Fish. Exp. 5 . Hyogo Pref., Nakasaki-machi, Akashi, Japan)

SOS:-200-10-31 the addition Of vitamin E showed a sli,rhtly higher TBA value than that of the vitamin E supplemented fish, but there was no extreme difference in the TBA values of the hepatopancreas of these two groups (Table 3). At the termination of experiment, the glycogen contents in the hepatopancreas of groups 1, 2, and 4 were elevated to about twice_compared to its content at 70th . day. However, such elevation of the'glycogen level was not seen in the group fed with oxidized oil. The activities of transaminases (GOT, GPT) were higher in the fish fed with oxidized oil than the fish fed with fresh oil and these enzyme activities could not be reduced even by the administration of vitamin E. The alkaline phosphatase activity showed little, if any, difference between these groups (Table 4). 3.

Death believed caused by oxidized oil, as well as being observed in other fish, can be seen quite frequently. during the rearing of yellowtail . Sakaguchi et ai' investi- gated the effect of antioxidant-treated sand-eel as food for yellowtail and reported that there was no ill effect on the growth of yellowtail if the peroxide value of fat in the feed was below 50. Furukawa et al 2 used sand-eel which was kept improperly cooled for one year as feed to raise yellowtail. The result of their experiment showed that the oxidized oil poisoned yellowtail, however, the poisoning was prevented by the addition of vitamine E to the food. The author has raised yellowtail using oxidized cod liver oil in order to see the influence of oxidized oil on the growth of yellowtail. We have obtained severa l . observations as a result of the experiment, on the drawbacks caused by the oxidized oil and the effect of vitamine E on preventing the drawbacks caused by said oil.

Experiment Fish and the method of raising, The fish used were Seriola Quinqueradiate TEMMINCK et SCHLEGEL and they were obtained from the same group of fish as those used in the previous report. 3 The average weight and the average length of the fish at the beginning of the experiment were 43g and 13.2cm respectively. The experiment 4 was started on July 20, 1968 and continued for three months. The test groups were made up of four separate groups, namely the group fed sand-eel (group 1, comparative group), the group fed pure cod'liver oil added to the assorted feed

(group 2, standard feed group), the group fed assorted feed with oxidized cod liver oil added (group 3) end the group fed on the _same feed system as 3 except vitamine E is added

(group 4) The feed compositions are .shown in Table 1.

The combined feed was made up of fish meal, gluten, glucose, • vitamine mixture and mineral mixture with pure cod liver oil, oxidized cod liver oil, etc., have been added. The vitamine mixture was the same as the mixture used in the comparative group in the previous report 3 except vitamine E was not contained in the mixture used in this experiment. The cod liver oil used was the pure sample with a peroxide value of less than 13. This cod liver oil was used in the second group - without modifying the composition but the oil was oxidized by passing air through the oil at room temperature for 260-290 hours so that the resulting compound had a peroxide value of between

480-500 before the oil was used for groups 3 and 4. The vitamine content in the feed system of-the second and third groups prior to the addition of oil was 1.3me. The feed system of the fourth group was made up by adding vitamine E

(a-Tocopheryl acetate 25011)/g) to the feed system of the third . group. The amount of vitamine added to the feed system was kept at 0.6% for the first 71 days and the amount was changed 5.

Table 1. Composition of'experimental diets

Group Sand-eel Fish Gluten Glucose Cod liver Vitamin Eineral Jitamin No. meal oil mixture* mixture** E***

1 100

2 68 17 2.5 ' 8.5++ 3 1

3 68, 17 2.5 8.5+++ 3 1

4 68 17 2.5 8.5 1 ++ 3 1 0. 6 5+ 68 17 2.5 8.5++ 3 1

+ Fish from control group Of vitamin test. I.+ Fresh oil (POV 11 e-'13). +++ Oxidized oil (POV 4E0"500). * Same as previous one. In groups 2 to 4, DL-06-tocophery1 acetate was replaced by same amount ofa-starch. ** Saine as Halver's4) . *** .0.6 g(150 TU) of DL-e-tocopheryi acetate was added until 71st day, thereafter increased to 1.2 g,

to 1.2% from the 72nd day to the end of the experiment. The mineral mixture was according to Halver. 4 The amount of feed used per day was kept between 2.5-3% (dry weight base) of the weight of the fish tested. The feed was given once in the morning and once in the evening. The weight of the fish was checked after every 10 feedings and the amount of feed was adjusted accordingly. The testing pond was 3.5 x 1.2m and contained 10 tons of water. Four testing ponds of the same dimensions were used in this experiment. Each pond was filled with 150 fish. The water waS filtered with a rapid filtering machine and air was passed through it before passing it into the pond. The flow rate of the water was about 10 tons per hour. However, the flow rate was not controlled after September during the day and sand-filtered water was circulated during the night.

The measurement of growth rate and the treatment of test fish The measurement of the body length was done every 10 days. Twenty to thirty fish were taken out Of each group and after anesthetizing them with MS-220 (concentration of 1/10,000) the fish were weighed and the body length was measured. The fish taken oùt on the 41st, 70th and 93rd days were not only weighed.and measured but alsO blood samples and a specimen for chemical analysis were collected from them. The collecting of sample blood was done by cutting the tail part of the fish.

Chemical analyses of fish The fish from which the blood samples were taken were used for chemical analyses. The mixed material made up of five fish was used to measure the water content, the amount of fat and thiobarbituric acid value in the muscle. The water content, fat content, the amount of glycogen and thio- barbituric acid value of the hepatopancreas were measured using the mixed material made up of 7-10 fish. The blood samples collected from 7-10 fish were mixed and separated by the method mentioned in the previous report. 3 The measurements of the amount of protein, total cholesterol, alkaline phosphatase activity, glutamic-Oxalacetic transaminase and glutamic-pyruvic transaminase were done after the *separation process. The electrophoresis of serum protein was carried out on three separate fish. Analyses were also carried out on the fish from the comparative group in the previous report 3 the time of conclusion of the test on each of the above at items for the purpose of comparison. This will be designated as group 5. The usual method was used to analyse the amount of water and fat. The amount of glycogen was determined by Sam Seifter et alis anthrone method. 5 The Reitman-Frankel method6 was used for transaminase, Bessey-Lowry method 7 alkaline phosphatase, Zak- .enly method 8 for total for cholesterol. Bulet method 9 for the dete-rmination of protein and the cellulose acetate membrane methodl ° was used for electrophoresis. An "Atagoosmer" 7 densitometer was used for the graduation ratio of the phoresis image and in measuring the absorbency. The modified Zalkin et al methodll was used for measuring the TDA value. That is, 10 ml of distilled water was ' added to the ground material in a mortar (1 g of muscle or 5g of hepatopancreas) and stirred for 10 minutes. 10m1 of trichloroacetic acid (10%) was added to the mixture and it was filtered. 1 ml of 40% ethanol and 1.25 ml of thiobarbital

. (75%) solution were added to 3 ml of the filtrate and heated

. at 100 degrees C. for 10 minutes. The solution was cooled and the color was compared at 530 mu. The unit was shown by the absorbancy at 530 mp. 8.

Results Growth rate The growth rate of yellowtail in each group is shown in Figure 1 and Table 2. The average weight of the fish at the time of the completion of the experiment in the first group was.532g, 415g for the second group, 384 g.for the third grOup. and 414 g for the fourth group. The first uoup fed sand-eel had the best growth rate. The growth rate of the third group was slightly lower than the second group which was on the pure oil added feed system, the fourth group was on the vitamine enriched feed system. The degree of fatness was 21.6 for the first group, 21.5 for the second group, 20.1 for the third group and 20.6 for the fourth group. The third and fourth'groups on the feed system containing oxidized oil had slightly lower values compared to the rest. The rate of death was highest for the first group ,due to system trouble with the aerationicarp

9.

20 40 60 80 dys Fig. 1. Growth of.the yellowtail fed with experiMental diets.

Table 2. Growth rate of the yellowtail. _.- .. Wt. of hepatopancreas Group Body weight(g) Fatness x 10 Number of No. Wt . of bod y dead fish C* A B C A B C B C 1 43.5 335 532 15.9 21.9 21.6 1.25 1.37 1.89 26** 1 2 43.5 269 415 15.9 22.1 21.5 1.25 1.55 2.03 3 0 3 43.5 261 384 15.9 21.8 20.1 1.25 1.63 1.79 7 0 4 43.5 248 414 15.9 19.9 20.6 1.25 1.34 2.03 3 1 5 1 43.5 238 393 15.9 19.3 21.4 1.25 2.15 7*** 5* ** + Fish from control group of vitamin test. * A: Initial, B: Intermediate (66th day), C: Final (93rd day). ** Attributed to accident. *** Attributed to ichthyophthiriasis ,like disease.

and the number of fish with this sickness reached around 10% . at termination of the experiment (Figure 2).

The result of chemical analyses Chemical analyses on the muscle and hepatopancreas

are shown in Table 3.

1 0.

1,

Fig. 2. Yellowtails fed With oxidized oil supplemented diet to which vitamin E was added (upper figure) and not added. (middle and lower figures).

Table 3: Analysis of the muscle and hepatopancreas of the yellowtaiL

Group Fat Moisture Fat Moisture ClvcoLen No. TBA* (%) (0) TBA=, (>9) (%) mg/g

1 A** 4.6 74.5 8.9 73.2 B** 0.86 7.2 70.3 0.26 23.0 57.0 2 2.2 C* 0.80 14.5 63.5 0.21 33.0 49.4 39.6 2 A 1.3 76.3 9.3 71.2 B 0.65 3.0 74.3 0.17 15.2 65.2 22.3 c 0.64 6.8 70.4 0.16 22.9 57. 0 55.5 3 A 1.2 76.6 10.7 70.8 B 0.79 3.1 75.2 0.24 17.5 63.1 25.9 C 0.78 8.9 69.0 0.28 23.1 57.9 28.3 4 A 1.0 76.8 9.4 71.0 B 0.48 1 2.7 74.9 0.23 21.8 60.9 25.8 c 0.15 7.9 69.6 0.23 22.0 58.2 47.3 5 C*** 0.65 7.3 69.9 0.17 20.9 57.7 56.4 * Thiobarbituric acid value. Expressed in optical density at 530 mil. ** A: 41st day, B: 70th day, C: 93rd day. ,‘ *** Fish from control group of vitamin test, '.

The TBA value of the muscle was highest in the first u.oup at termination of . the experiment, however, the third group which was on the combined feed.system had 0.78. The 11, value was rowest at 0.15 for the fourth group - which was.on the vitamine E enriched feed system. The TBA value of the hepato- pancreas- was low for the second and fifth groups. The difference between the third and the fourth groups was only slight. The first group showed high fat content for both muscle and hepatopancreas. The fat contents of the fish in the groups on the combined feed system (groups 2-4) showed the following characteristics. There was a slight increase in oil content of muscle for the fish on the oxidized oil added feed system. No difference in the oil content of the hepatopancreas was observed among groups 2-4. •

The amount of glycogen in the hepatopancreas determined on the 70th day of the experiment showed a very small difference between fish In the groups, however, the amount increased to twice the amount obtained on the 70th day for groups 1, 2 and 4 at termination of the experiment. The third group which was on the oxidized oil added feed system, however, showed a very small increase. The values obtained for various chemical analyses of the fifth group of fish did not differ much from the results obtained for the fish in the second group (Table 3). The activity of each enzyme and the amount of protein in the blood serum are shown in Table 4.

The activity of serum GOT on the 70th day for the second group was 27.0 which was the lowest and 53.0 for the first group which was the highest. However, the activities of , the third and fourth groups became slightly higher than the activities of the first and second groups at termination of the 12.

experiment. The addition of vitamine E did not decrease

the activity. - The GOT activity of the fifth group was 57.6 at termination of the experiment. This group was 57.6 at termination Of the experiment. This value was almost the same as the value for the second group. There was a similar trend for the GPT activity. 0.35-0.36 were the values shown for the activity of alkaline phosphatase in the first group. This value was slightly higher than the other groups. The difference between the other groups was small. No changes in the activity due to the presence of oxidized oii in the feed system was observed. 643-738111g/di was the value obtained for the total amount of cholesterol for the first group which was fed sand-eel. This value was the highest for this experiment.

The values between 373 - 425mg/d1 were obtained for groups 2-4 which were on the cod liver oil added feed system. The second group (basic feed group) showed a slightly higher value than the remaining two groups.

The amount of sérum protein was 5.37 - 5.52g/di for the first group. The values for the rest of the groups were slightly lower than for the first group. The third group had a value of 4.28g/d1 at termination of the experiment, which was the lowest. The fraction ratios of electrophoresis of serum protein are shown in Table 5. If one arranges each fraction into I-IV fractions as in the previous report 3 , the fraction ratios of fraction I are 54.4% for the first group, 43.4% for the

13

Second group, 41.44 for the third group and 35.8 for the fourth group. .The change of fraction I is considerable.

Table 4. Activities of enzymes and contents of protein and cholesterol in serum of experimental fish. Glutamic- Glutamic- • Total Group oxalacetic pyruvic Alkaline Protein cholesterol No. transaminase** transaminase* *phosphatase*** g/dl mg/di 1 B* 53.0 18.5 0.35 5.52 738- C* 54.5 15.5 0.36 5.37 643 2 B 27.0 11.1 0.19 5.37 420 C 58.0- 13.3 0.25 5.00 425 3 B 45.3 15.0 0.19 5.00 397 C 63.5 16.7 0.24 4.28 373 4 B 37.5 12.7 0.25 4.70 380 C 67.1 16.0 0.20 4.75 379 5 C 57.6 12.6 0.18 4.94 * B: 70th day. 93rd day. ** Expressed in Karmen unit. *** Expressed in Bessey-Lowry unit.

Table 5. Composition of each fraction of serum protein separated by electrophoresis with cellulose acetate membrane.

Group Fraction (%) No. II TII 1V 1 49.2 20.9 8.2 9.0 range 58.2 26.2 10.8 15.4

mean* 54.4 23.9 9.8 11.4 40.3 25.4 14.5 9.8 range 45.9 33.9 16.9 13.6

mean* 41.4 29.1 15.9 11.6 3 35.9 23.9 14.3 11.9 range 45.2 35.7 16.7 16.4 mean* 43.4 28.6 15.8 14.2 4 34.7 28.0 19.3 12.3 range 36..8 31.6 20.5 16.0 mean* 35.8 30.3 19.9 13.9 5 42.0 27.7 13.5 10.1 range 44.5 28.3 17.5 16.1 mean* 43.8 26.0 15.8 14.2 * Mean of 3 individuals. 14

Discussion High death rate, anemia, contraction of muscles and incomplete growth were observed in carp 12 and rainbow trout13 when okidized oil was added tosthe feed system. It has also been reported that the growth rate of mice decreased when on a riboflavin-less oxidized oil added feed system. 14 In this report too the growth of group 3 which wa4 on the oxidized oil added feed system was the worst. 10% of fish in group 3 showed sickness similar to that found in carp's back, at termination of the experiment. The hinderance of the growth cailsed by oxidized oil in rainbow trout appeared at a relatively early stage, however, the hinderance of the growth of yellowtail did not appear until around the 45th day even though oil with a peroxide value of between 480-500 was used. The first sign of hinderance of the growth was the decrease of the feeding activity. From this, one can say that the appearance of growth hinderance caused by oxidized oil in yellowtail is later than in rainbow trout. However, according to Furukawa 2 et al , the signs of hinderance such as stoppage of feeding, swimming activityetc., appeared two weeks after the start of the experiment if there was no vitamine E contained in the feed system. That result differs considerably from the result of this experiment. One of the reasons for this difference is believed to be the amount of feed given to the fish. The type of feed used is believed to have caused the difference, because thé sand-eelS used »y Furukawa et al were kept frozen for one year under incomplete freezing conditions. If one 1 5

combines the result of Kaunitz et al 1 14 with the result of Furukawa et al, one can say the following. The appearance of the signs was quickened because of the multiple reactions caused by several factors such as the formation of oxidized oil, the decomposition of vitamines, the formation of poisonous amines, etc. Phenomena such as contraction of muscles, increase in TBA value and activity of transaminase can be observed in mouse 11 guinea pig,• 16 min k17, rabbit 18 , etc., caused by lack of vitamine E. Conditions such as decrease in the rate of . growth, protrusion of eyeballs and anemia are observed in spring salmon (Masunosuke)I 9 caused by lack of vitamine E. These phenomena did not appear in the fish in group 2 of this experiment which was fed on the standard feed system without vitamine E. This is believed to be mainly . due to the difference in the animals tested. Vitamine E contained in the standard feed system was sufficient for yellowtail to resume its normal course of growth. This theory can also be obtained from the results for group 2 and 5 (comparative group from the previous report 3 ) re TBA values of muscles and hepatopancreas, the amount of.glycogen, water and the . activity of serum transaminase, , protein, and fraction ratio of electrophoresis. The fact that similar values are obtained for both groups proves the correctness of the theory (Table 3-5). Muscle TBA value was lowest for .group 4 which was on the vitamine added feed system. . This is*believed to be caused by the antioxidizing reaction -within the muscle; a reaction similar to the one observed in 16

the muscle of carp . 12 However, I do not wish to talk about the effect of vitamine E just from this extent of TBA value. I hope the reader will wait until further. investigations are carried out on this subject in the future. . The 'amunt of glycogen in the hepatopancreas doubled at termination of the experiment as compared to the amount obtained on-the 70th day of the experiment for the fish in groups 1, 2 and 4 but the amount was quite small for the fish in group 3 fed on the oxidized oil added feed system.

Not only was the amount small but also the rate of increase was small for the fish in group 3. One et al 20 have observed the decrease of glycogen in livers of rainbow trout, caused by oxidized oi1. 20 It is believed that in yellowtail too oxidized oil has some unfavorable effect on the storage of glycogen in the liver. Generally the total amount of cholesterol in the blood serum changes according to the type of oil consumed by the fish. This-is believed to be the reaSon for the difference in the amount of total cholesterol in group 1 and groups 2-4 'since the oil contained in group 1 differed from the oil in the feed systems used for groups 2-4. It is also known that the amount of cholesterol in the serum decreases if the intake of unsaturated fatty acid increases. However, this trend was not observed. in yellowtail. The amount of cholesterol in group 2,which was fed on the standard feed system, was slightly higher than in groups 3 and 4 (Table 4). River trout too showed a trend similar to that of yellowtail. 1 7

According to Shimizu et a 1 22 the activity of alkaline phosphatase and the total cholesterol in the blood serum of cultivated yellowtail are 0.35-1.97 nitrophenol unitsand 11.8.6-157.5mg/d1 respectively. The values however change considerably according to .seasons. The value for the activity of alkaline phosphatase obtained in this experiment was slightly lower than-the value obtained by Shimizu et al but the total cholesterol obtained in this experiment had a higher value than that of Shimizu et al (Table 4). The total cholesterol content in the blood serum of river troUt differed according to the cultivation temperature. 21 For this reason, the differences between the values obtained by Shimizu et al and ours are believed to be caused by the difference in the type of feed used, the amount of feed used, cultivation temperature, cultivation equipment and cultivation conditions. There are still some needs for investigation on this subject. The amount of serum protein was slightly lower for group 3 when compared to the other groups (Table 4). It is said that yellowtail 12 and rainbow trout become anemic when oxidized oil is added to the feed system. Blood and blood protein of the anemic yellowtail decrease.'"-'e 24 However , the yellowtails in group 3 were it was not clear whether anemic or not in this experiment. 18

The fraction ratio of electrophoresis of the brood serum differed considerably between the groups fed sand-eel and the combined feed system. It is believed that this difference arose because of the difference in the nutrient values of sand-eel and the combined feed and the rate of digestion. The fraction ratio of the first fraction corresponding to albumin of group 4 was considerably lower than that of group,3. This is different from the report on 17 mink which showed an increase of albumin due to the presence of vitamine E in the feed system. The difference may be due to a difference in the reaction of yellowtail and mink toward vitamine E, may be due to the difference between male and female which was observed in ayu25 and in scorpion- 25 fish or may be due.to the excess addition of vitamine E. There is a need for future investigation on this subject in order to clarify this matter. The oxidized oil had an effect.on the TBA value of the muscle and the glycogen-storing ability of the hepato- pancreas. As for the sickness caused by oxidized oil, the reduction in feeding activity and sunken back sickness seen in carp12 was observed. The addition of vitamine E is believed to prevent the sunken back sickness, anemia and growth retardation which were observed in carp, 12 rainbow 13 trout as well as in yellowtail. We wish to make the effective amount of vitamine E to be added to the feed system our next subject for research. Very often the blame is placed on oxidized oil in the feed system when one talks 19

about disease . caused by the feed, however, tnere is a need for investigations on .the decomposition of vitamines caused by the oxidation of oil and the production of poisonous amines in the feed system together with the oxidation of oil. I wish to thank Dr. Okaichi of Kanagawa University and Prof. Hashimoto of Tokyo University for their valuable assistance inelectrophoresis of blood serum, Yoshikawa Oil Co. for providing the test oil, Nippon Nogyokogyo K.K. central research station for preparin the feed used, Mr. Iizawa and Mr. Fukuda of this experimental station for their valuable aid. • • • 20

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1 5 ) Fonso, KLITTIFiEtaiga. , O zaki, Yoneraura, frR1p2

(Oil chemistry) 16, 135-126 (1967), e • 21

16) J. W. C. Boio and N. A. B. SZABO: Proc. Soc. Exp. Biol. Med., 117, 395-350 (1964). 17) H. D. STOVE and C. K. WHITEHMa: Am. J. Vet. Res., 25, 1542-1549 (1964). 18) M. ALOISI, A. ASCENZI, and E. BoNETTI: Biochem. Biophys. Acta, 10, 70-76 (1953). 19) A. N. WOODALL, L. M. ASEILEY, J. E. HALVER, H. S. OLCOTT, and J. VAN DER VEEN: J. Nutrition, 84, 125-135 (1964).

20) T. ONO, F. NAGAYAMA, and T. MASUDA: J. Tokyo Univ. Fish., 46, 97-109 (1960). 21) T. H. MCCARTNEY: Cortland Hatchery Report, No. 33, for the year 1969, 35-41 (1965).

22) '3 12 -1_711.711, Yori (7) , , 1 - (1

23) Fujitni, rnsukahar:

of south ersterwi

2 11-) Ta'çahashi., Isa, rroolr:s : - 111 c.;n 17, (s. s. r i mult,i plicat on , l, -1‹ (1 0 ).

r 2) Suzu.ki , \ra e koen (The annual 1etïnE, of 4;'-le T Ti. ri AsSoc 1 a'- '1:arl)

(1969),

26) Yàmashita: This rrlagazi ,I, 1 - 10 ,-; ( 1