tanchivis Translation Series No Original title: Ricerche sugli effetti di una dieta comprendente olio di colza nel sumo 15 pages typescript

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1 tanchivis FISHERIES AND MARINE SERVICE Translation Series No Effects of a diet containing rapeseed oil in pig by L. Rampichini, M. Severini, A. Belgiomini S. Ranucci, D. Rutili, and E. Di Antonio Original title: Ricerche sugli effetti di una dieta comprendente olio di colza nel sumo Fr om : Arch. Vet. Ital. 27(5-6): , 1976 Translated by the Translation Bureau (MB) Multilingual Services Division Department of the Secretary of State of Canada Department of Fisheries and the Environment Fisheries and Marine Service Halifax Laboratory Halifax, N.S pages typescript

2 Ci" ty1 M Li I 3 DEPARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS MULTILINGUAL SERVICES DIVISION CANADA DIVISION DES SERVICES MULTILINGUES TRANSLATED FROM - TRADUCTION DE AUTHOR - AUTEUR Italian L. Rampichini et al. INTO - EN English TITLE IN ENGLISH - TITRE ANGLAIS Effects of a diet contahline, rapeseed oil in pig. TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE étrangère (TRANSCRIRE EN CARACTÈRES ROMAINS) Ricerche sugli effetti di 'Ina dieta comprendente olio di criza nel suinr. REFERENCE IN FOREIGN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE FOREIGN CHARACTERS. RÈ' FÈ' RENCE EN LANGUE itrangére (NOM DU LIVRE OU PUBLICATION), AU COMPLET, TRANSCRIRE EN CARACTÈRES ROMAINS. Archfvio,reterinario italianr REFERENCE IN ENGLISH - FddRENCE EN ANGLAIS Itanan 7reterinary Archives PUBLISHER - iditeur DATE OF PUBLICATION DATE DE PUBLICATION PAGE NUMBERS IN ORIGINAL NUMe.ROS DES PAGES DANS L'ORIGINAL PLACE OF PUBLICATION LIEU DE PUBLICATION YEAR ANNÈ' E VOLUME ISSUE NO. NUMÈ.R0 1 '70 NUMBER OF TYPED PAGES NOMBRE DE PAGES DACTYLOGRAPHIÈ'ES 1Q REQUESTING DEPARTMENT MINISTÈRE-CLIENT 4- Envl.rnnrlenu TRANSLATION BUREAU NO NOTRE DOSSIER N A4 6 8 BRANCH OR DIVISION DIRECTION OU DIVISION Fis'.1eries TRANSLATOR (INITIALS) TRADUCTEUR (INITIALES) er PERSON REQUESTING Dr. ' - C. Abkl-an DEMANDE PAR YOUR NUMBER VOTRE DOSSIER N 0 DATE OF REQUEST DATE DE LA DEMANDE SOS G (REV. 2/08)

3 f Secretary Secrétariat of State d'état TRANSLATION BUREAU MULTILINGUAL SERVICES DIVISION F ty, ei 113 BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES _ CLIENT S NO. DEPARTMENT DIVISION/BRANCH CITY NO DU CLIENT MINISTE' RE DIVISION/DIRECTION VILLE Environment Fisheries Ottawa BUREAU NO. LANGUAGE TRANSLATOR IINITIALs) Nce OU BUREAU LANGUE TRADUCTEUR (INITIALES) Italian M. Butler Sept. 2, 1977 RESEARCH ON THE EFFECTS OF A DIET CONTAINING RAPESEED OIL IN PIG L, Rampichini, M. Severini; A. Belgiomini; S. Ranucci; D. Rutili and E. Di Antonio. The autors report about 'results of research work on pigs kept for and 100 days on a diet contaizzing 13% of rapeseed-oil (40% of erucic acid) or 13% of olive-oil. - The animals fed with rapeseed- oit showed an early accunitilatièri Of the `értlbiè"*ocicrin the heart and skeletal muskle, but there was no early and notable lipidosis in the heart. Slight morfological changes in the rnyocard were found in all the animals of both groups, but they were more accentuated even if retarded in the animals fed with rapeseed-oit. KEY WORDS : Pig, rapeseed oit, erueic acid, heart,, Skeletal muscle. INTRODUCTION In a previous note (Rampichini et al., 1976 a), we demonstrated that pigs fed on a diet containing 11.5% rapeseed oil (with 35% erucic acid) had not shown typical functional and morphological changes during the first twenty days. This finding has created a problem of interpretation as regards the actual toxicity of erucic acid for pigs. (*) Some results of the present research were communicated at the XXIX Convention of the Italian Society of Veterinary Sciences (Begliomini et al., 1975), Portoferraio. The authors wish to thank Messrs. O. Cortona and M. Spaccini for their technical assistance. SEC 5-25T (6/76)

4 2 In the present work, we shall describe the results of further research conducted on pigs fed for a longer period of time on a diet containing a high percentage of rapeseed oil richer in erucic acid. For this experiment, we used the same rapeseed oil which, in the rat, in lesser doses,was found to be capable of inducing significant morphological changes, consisting of severe early lipidosis at the level of the myocardium and the kidneys (Rampichini et al., 1976). PERSONAL OBSERVATIONS Materials and Methods Sixteen pigs of the Large White breed, all females about 3 months old, having an average weight of 25 kg at the beginning of the experiment. The subjects were fed for ten days on SM 1 PETRINI feed, containing no methionine or antibiotics, in order to adapt them to the experimental diet. Subsequently, they were divided into three groups: the first group comprised nine pigs to which feed combined with rapeseed oil (containing 40% erucic acid) in a ratio of 13% by weight (27 cal. %) was administered: the second group comprised 5 subjects administered feed to which olive oil (containing no erucic acid) had been added in a ratio of 13% by weight (27 cal. %); the third sroup comprised 2 pigs to which feed only was administered. All the subjects received a constant dose of feed equal to 40 g/kg of live weight/pro die. The pigs were sacrificed after the following time intervals: Days of treatment No. of subjects - 1st group No. of subjects - 2nd group No. of subjects - 3rd group All the subjects underwent electrocardiographic, enzymatic e post-mortem, histological, histochemical and ultrastructural examinations. The electrocardiographic examinations were conducted at the beginning and end of the adaptation diet and at the end of the experimental diet, immediately prior to slaughter, according to the methods already described (Rampichini et al., 1976).

5 3 The serum values of the aldolase (ald.), creatine phosphokinase (CPK), glutamic oxalectic transaminase (GOT), glutamic pyruvic transaminase (GPT) and lactic dehydrogenase (LDH) were determined according to the methods already described (Tampichini et al, 1976). The chemical analyses for the evaluation of the water content, raw lipids and erucic acid were conducted on samples of myocardium and quadriceps femoris muscle according to the methods already described (Rampichini et al. 1976). The post-mort= was carried out immediately after slaughter with a captive bolt device and total bleeding of the subject by jugulation after twelve hours' fasting. The histological examinations were carried out on samples taken from the encephalon, thyroid, lung, heart, spleen, liver, pancreas, kidney, adrenal gland, stomach, large and small intestine, lymph glands of the chest and abdominal organs and skeletal muscles of the hind legs, fixed in formalin and included in paraffin. The prepared sections were stained with hematoxylin and eosine for the general morphology. The myocardium sections were further stained with the Van Gieson method for the connective tissue. The histochemical examinations were conducted on samples taken from the myocardium, the liver and the kidney, frozen in isopentane cooled with liquid nitrogen and sectioned under a cryostat. The sections were stained with Sudan III for the fats. The liver sections were further used to determine the activity of the glucose-6-phosphatase using the method of Wachstein et al. (1957). The ultrastructural examinations were carried out on samples of ventricular myocardium placed in 2% glutaraldehyde in ph 7.4 phosphate buffer for 2 hours, then fixed in osmium tetroxide and included in Epon 812. The ultrathin sections, contrasted with lead hydroxide and uranyl acetate, were observed with a Philips 300 M.E.

6 4 RESLTLTS Throughout the experiment, the subjects under study showed no pathological manifestations which were clinically detectable. The examination of the basal electrocardiographs and those carried out immediately prior to slaughter, in the subjects of all the groups, showed an essential superimposition of characteristics and the absence of pathological alterations, though within a certain range of variability as regards the shape and amplitude of the waves in the individual pigs. The data relative to the serum enzymes and those relative to the water and raw lipid content of the myocardium and skeletal muscle, together demonstrate an essential homogeneity of values among the pigs of the various groups. (Table No. 1). TABLE No. 1 Pig. gg Oil Md CPK GOT GPT LOH H2 0 Fat H2 0 Fat no. diet admin. mu/mi.mtuml m0/1111 mu/m1 mtliml heart heart muscle muscle (1) (1) 03 O) 1 7 RapeSeed , Rapeseed , Rapeseed ) Rapeseed , Rapeseed , , Rap.es.eed SD Rap:es-eed , Rapeseed 3, , , ' Rapeseed , , Olive , , Olive ,60 203,40 70,25 5,69 76, rive , ,27 3, Olive 3, , , Olive , , ,35 1h , , , , M. grams/100g of fresh tissue.

7 5 Erucic acid was found to be present in the myocardium and the skeletal muscle of subjects administered feed and rapeseed oil as early as 7 days from the start of the diet and its accumulation became more marked after the twentieth day (Table No. 2). In all the subjects the post-mortem examinations revealed the presence of lobular alterations in the lung (enzootic pneumonia). In the pigs fed on rapeseed oil or olive oil, the kidney had a somewhat pale appearance and sometimes the muscles of the hind legs as well. In some of these subjects, a small quantity of liquid discharge was noted in the pericardial cavity. In two subjects fed for 20 and 40 days on the diet containing rapeseed oil, a catarrhal gastritis was noted, limited to the mucosa. The histological examination revealed constant alterations only at the level of the heart, liver, kidney, intestine and lung. In the myocardium of all the pigs fed with rapeseed oil, the alterations, through diffuse, affected only a very limited area in all. In the subjects sacrified within the first 20 days of the diet, these consisted in the presence of rare, isolated fibres with homogenous,intensely eosinophile cytoplasm lacking TABLE No Pigs: erucic acid content of the heart qnd skeletal muscles in mg/kg of fresh tissue. 7 E :4 40 'Legend:---Heart le grovp ---Skeletal - muscie 1st group '----- Ijeart and skeletal muscle, ' 2nd and 3rd groups

8 6 visible transverse striae and showing a thick pycnotic nucleus; of thin fibres with paler granulose cytoplasm with visible transverse striae and a certain number of small interstitial infiltrates of histocyte cells. In the pigs sacrificed after 40 days, rare and isolated eosinophile fibres were observed, as well as a slightly higher number of fibres showing granular degeneration and diffuse histocyte infiltrates, generally small and localized in the interstice. Infiltrates of significant size or which tended to replace degenerated fibres were found only rarely (Fig. 1). In the myocardium of the subject sacrificed after 100 days of diet, the eosinophile fibres were practically absent, while fibres showing granular degeneration or small infiltrates of histocyte cells replacing them were clearly evident (Fig. 2). *".,' 27. _.1 r.,r-. '-"' Fig. No. 1 - Pig fed for 40 days with rapeseed oil. Staining: hematoxylin and eosine. x 280. Fig. No. 2 - Pig fed for 100 days with rapeseed oil. Staining: hematoxylin and eosine. x 140.

9 7 In the myocardium of pigs fed with olive oil, alterations more or less identical to the less serious ones observed in the animals fed on a diet with rapeseed oil were present, but the time of onset was different. After 7 days, in fact, the only thing noted was numerous small histocyte infiltrates at the interstitial level and after only 15 days, rare isolated eosinophilefibres with clear granulose cytoplams appeared, while the infiltrates became less numerous and were still interstitial. These alterations remained constant in the pigs sacrificed at a later date, though a slight accentuation of the processes of granular degeneration was observed in some fibres. In the liver of all the pigs examined, we noted some phenomena of microvacuolization of the liver cells close the centrolubular areas, which was slightly more accentuated and diffuse in subjects fed with rapeseed oil and in those fed for more than 7 days on the diet with olive oil, and in the kidney, slight degenerative alterations, with significant differences between the animals of the three groups. In the intestine of almost all the pigs fed with rapeseed oil or olive oil, slight catarrhal enteritis was noted. In the lung of all the subjects of the various groups, lesions characteristic of enzootic pneumonia were noted. Sporadically, alterations were observed in other organs. In the pancreas of 2 pigs fed with rapeseed oil or olive oil for 7 days, necroses of the exocrine glands were visible. In the stomach of 2 subjects fed on the diet containing rapeseed oil, respectively for 20 and 40 days, mild catarrhal gastritis was noted at the level of the mucosa.

10 8 Staining with Sudan III revealed the presence of a very few fibres containing minute drops of fat in the myocardium of 2 subjects fed for 15 and 20 days with rapeseed oil and in one fed with simple feed. In the liver of all the subjects administered rapeseed oil or olive oil for more than 7 days,acertain accumulation of fat was noted in the liver cells of the centrolobular areas. This alteration reached a high level of diffusion and severity only in one pig fed for 20 days on the diet with rapeseed oil. In the liver of the animals of the first two groups sacrificed after 7 days and in those of the third group, sudanophile drops were noted in a very limited number of isolated and irregularly distributed liver cells. In the kidney of all the subjects fed for more than 7 days on a diet containing rapeseed oil or olive oil, small drops of fat were found at the level of the epithelium of the convoluted tubules. The affected tubules were more or less isolated and rare, and only occasionally were there 3 or 4 contiguous ones. The histochemical reactions to reveal the activity of the glucose-6- phosphatase permitted us to confirm the presence of similar patterns in the liver ofth e of all three groups. Enzymatic activity was present in the cytoplasm of pigs all the liver cells in the form of minute dark brown-colored granules. The ultrastructural examinations revealed, in the myocardium of pigs fed with rapeseed or olive oil for 7, 15 and 20 days, some structural alterations and a slight accumulatibn of fat in a rather low percentage of fibres. The structural alterations of the fibres consisted in the presence of small light-colored cytoplasmic areas, for the most part in the vicinity of the nuclei, with no myofibrils and with mitochondria dispersed among finely granulose material (Fig. 3); of mitochrondria with reduced or dissolved crests; sometimes containing small vescicles or reduced themselves to vescicles (Figs ) and of dilations of the sarcotubular system, which

11 9 Fig. No. 3 - Pig fed for 7 days with rapeseed oil. Contrast: lead hydroxide and uranyl acetate x i ,._...-a A, Fig. No. 4 - Pig fed for 7 days with rapeseed oil. Contrast: lead hydroxide and uranyl acetate. x Fig. No. 5 - Pig fed for 7 days with rapeseed oil. Contrast: lead hydroxide and uranyl acetate. x 8800.

12 : t..,t Fig. No. 6 - Pig fed for 7 days with olive oil. Contrast: lead hydroxide and uranyl acetate. x Fig. No. 7 - Pig fed for 7 days with rapeseed oil. Contrast: lead hydroxide and uranyl acetate. x Contrast: lead hydroxide

13 11.. ;. -,. _..1 **1. ( --c...,..k s e, ,.--..., ,.5,,. :-..,...*. -, A.,: ,,..:.:, i.. '. 171:e..t1 :' ' ' *:...-e,-..,..-3 ''..r. J.: eh. ' Fig. No. 9 - Pig fed for 7 days with rapeseed oil. Contrast: lead hydroxide and uranyl acetate. x Fig. No Pig fed for 7 days with rapeseed oil. Contrast: lead hydroxide and uranyl acetate. x ; ; _ e\-. C ; ',, *. s... ',7 t à. Fig. No Pig fed for 100 days with rapeseed oil. xide and uranyl acetate. x 1900 Contrast: lead hydro-

14 12 Fig. No Pig fed for 100 days with rapeseed oil. Contrast: lead hydroxide and uranyl acetate. x in some cases had the appearance of mascicles. (Figs ). Significant accumulations ofmitochondriawere found occasionally between the myofibrils, in the vicinity of the nuclei (Fig. 4). The fatty accumulation was characterized by the presence of small, slightly electron dense drops, without a clear delimiting membranelle, isolated and localized between thenitochondria in the intermyofibrillar spaces or under the arches at the periphery of the cells (Figs ). The myocardium of pigs fed for 40 days on diets including rapeseed oil or olive oil showed the same characteristics described above, with the exception of the fatty accumulation; in fact, a very few isolated lipid droplets were detected only in a few rare fibres. The structural alterations were slighly more serious and diffuse, in particular in subjects fed rapeseed oil. The percentage of altered myocardial fibres was substantially unvaried in the pigs sacrificed after 100 days; the lesions were somewhat more accentuated in the animals that had received olive oil, while they showed indications of extreme gravity in those that had received rapeseed oil. In the myocardium of these latter subjects, there were in fact some fibres showing large cytoplasmic areas emptied of the normal structures and containing only

15 13 amorphous residues of cellular material alternating with small areas containing fragmented, but apparently preserved, myofibrils, and mitochondria with altered or swollen crests or lacking crests, between the still partially visible sections of the sarcotubular system (Figs ). Occasionally, some isolated histiocytes were detected in the interstitial spaces. No structural alterations were found in the myocardium of pigs receiving feed only; only rare fibres containing isolated drops of fat were observed. CONCLUSIONS AND CONSIDERATIONS The results obtained show that in the pig a diet including 13% rapeseed oil (with 40% erucic acid) causes an early progressive accumulation of erucic acid at the level of the heart and skeletal muscles, but causes no significant modifications in the electrocardiogram, nor any quantitative variations in serum enzymes (Ald., CPK, GOT, GPT, LDH), nor severe cardiac lipidosis such as that found in rats fed with a lower dose of the same oil (Rampichini et al., 1976b) and considered by numerous authors to be a specific lesion (Adbellatif et al., 1970; Beare-Rogers et al., 1972; Vodovar et al., 1972; Adbellatif et al., 1973; Beare-Rogers et al., 1974; Rauber et al., 1974; Cluzan et al., 1975; Dallocchio et al., 1975; Svaar et al., 1975; Vodovar et al., 1975). This diet, however, induces some slight constant morphological alterations in the heart, liver, kidneys and intestine and a slight fatty accumulation in the myocardium (detectable only with the M.E.) and in the liver (visible in sections stained with Sudan III). A diet including 13% olive oil causes essentially the same alterations, though the cardiac alterations throughout time appear to be less

16 14 severe than those caused by the diet which includes rapeseed oil. In fact, in the myocardium of pigs fed this oil for 40 and 100 days, we noted an accentuation of the degenerative processes in the individual fibres and a tendency of the histocytes to replace the most injured fibres. In essence, the results of our research demonstrate a certain sensitivity of the pig to hyperlipidic diets, perhaps somewhat increased by the presence in the diet of rapeseed oil rich in erucic acid, while they lead us to exclude the possibility that this oil has a toxic effect in this species causing the onset of severe, specific functional and morphological alterations. These data generally agree with those of some authors, accordings to whom diets containing a high percentage of fatty substances induce inflammatory lesions (Rome et al., 1960) and small necroses (Svaar et al., 1975) at the level of the heart, quite independent of the presence of rapeseed oil in the diet. However, they differ from those reported by other authors, who noted the onset of characteristics cardiac alterations, including an early severe lipidosis, in pigs that had received a percentage of rapeseed oil equal to or lower than that administered by us (Beare-Rogers et al., 1972; Vodovar et: al., 1973; Vodovar et al., 1975). An organic comparison with the results of these latter researchers is still difficult since they neglect or do not touch sufficiently upon the morphological aspects which can be revealed by an examination under the optic microscope, but describe symptoms observed only through staining with Sudan III or with the electron microscope. In conclusion, we feel that the toxic action of rapeseed oil (containing a high percentage of erucic acid) cannot be considered a definitely proven fact generalizable to all animalspacies since, on the basis of our

17 15 experiments, the administration of this oil in feed, even under extremely unfavorable test conditions, does not seem to cause any alterations in the pig different from or more serious than those induced by a diet containing olive oil. SUMMARY RESEARCH ON THE EFFECTS OF A DIET INCLUDING RAPESEED OIL IN PIG. NOTE II. The authors describe the results of a research conducted on pigs fed for 7, 15, 20, 40 and 100 days on a diet including 13% rapeseed oil (40% eurcic acid) or 13% olive oil. The diet with rapeseed oil caused an early accumulation, of erucic acid at the level of the heart and skeletal muscle, but not the onset of severe early cardiac lipidosis. Modest morphological alterations in the myocardium were noted in subjects fed on both diets and only later did these became more accentuated in those fed with rapeseed oil. BIBLIOGRAPHY (1) Abdellatif, A.i M. M. & Vies, R. O. (1970) NKr_ Metabol., 12, 285. (2) Abdellatif, A. M. M. & Vies, R. O. (1973) Nutr.. Metabol., 15, 219., (3) Beare-Rogers, J. L. & Nera, E. A. (1972) Comp. Biochenz. Physiol., 41 13, 793. (4) Beare-Rogers, J. L.; Nera, E. A. & Heggtveit, 11. A. (1974) Nutr. Metabol., 17, 213. (5) Begliomini, A.; Di Antonio, E.; Rampichini, L.; Ranucci, S.; Rutili, D. & Severini, M. (1976) Atti Soc. Ital. Sc. Veter. (in corso di stampa). (6) Cluzan, R. & Levillain, R. (1975) INSERM, Paris, pag,. 85. (7) Dallocchio, M.; Lamle, T.; Rabaud, M.; Razaka, G.; Crockett, R. 8c Bricaud, H. (1975) INSERM, Paris, pag (8) Rampichini, L.; Begliomini, A.; Di 'Antonio, E.; Ranucci, S.; Rutili, D. & Severini, M. (1976) Àrchivio Veterinario Italian On corso di stampa). (9) Rampichini, L.; Severini, M. & Begliornini, A. (1976) Archipio Veterinario Italian (in corso di siam oa). (10) Rauber, G.; Chcrrier. P. & Debry, G. (1974) C.R. Soc. B;ol., 168, 247. (11) Rauber, G.; Cherrier, P. & D:.bry, G. (1974) C.R. Soc. Biol., 168, 253. (12) Roine. P.; Uksila, E.; Teir, H. &.Rapola, (1960) Z. Ernahrungsiciss., 1, 118. (13) Svaar, H. & Langmark, F. T. (1975) INSERM, Paris, pag (14) Vodovar, N.; Desnoyers, F.; Cruzan, R.; Marson, A. M. & Lcvillain, R. (1972) C.R. Acad. Sc. Paris, 274, (15) Vodovar, N.; Desnoyers, F.; Levillain, R. & Cluzan, R. (1973) C.R. Acad. Sc. Paris, 276, (16).Vodovar, N. & Desnoyers, F. (1975) INSERM, Paris,. pag (17) Wachstein, M. & Meisel, E. (1957) Am. I. Clin.. Path., 27, I.