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Chemical changes and influences of rapeseed antinutritional factors on lamb physiology and performance. 3. Antinutritional factors in plasma and organs
Animal Feed Science and Technology 85 (2000) 111±120
Chemical changes and in¯uences of rapeseed antinutritional factors on lamb physiology and performance. 3. Antinutritional factors in plasma and organs Nicolas Mabona, S.N.M. Mandikib,*, G. Deryckeb, J.-L. Bisterb, J.-P. Watheleta, M. Marliera, R. Paquayb a
Faculte universitaire des Sciences agronomiques de Gembloux, 2, Passage des DeÂporteÂs, B-5030 Gembloux, Belgium b FaculteÂs universitaires Notre-Dame de la Paix, 61, Rue de Bruxelles, B-5000 Namur, Belgium Received 5 May 1999; received in revised form 20 January 2000; accepted 10 February 2000
Abstract The impact of a high level of rapeseed meal in the diet on the concentration of antinutritional factors in plasma and organs was studied in 66 Texel, Suffolk or crossbred lambs. From 1 month of age till slaughter (13221 days), they were fed ad libitum with concentrates containing 0% or 25% of rapeseed meal obtained either SamouraõÈ or Honk rapeseed varieties. Lambs were weaned at 888 days of age and 245 kg live weight. Blood and organ samples were taken fortnightly for the determination of antinutritional factors. In plasma and organs, the thiocyanate concentrations were systematically higher in the SamouraõÈ and Honk lots than in the control. After weaning, the SCNÿ concentration in plasma increased up to 490 mmol lÿ1 in the SamouraõÈ lot. The goitrogen 5-vinyl-1,3-oxazolidine-2-thione (5-VOT) was determined in muscle, organs (thyroid, liver, kidney and lung) and biological ¯uid (plasma). The 5-VOT was present only after the ingestion of rapeseed meal but not to the same extent for the two varieties. The Honk rapeseed meal induced a 5-VOT level signi®cantly (p<0.05) higher in the target organs such as lung and thyroid than SamouraõÈ. Very low levels were found in muscle, liver, kidney, plasma and lung. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Rapeseed meal; Lambs; Thiocyanate ions; 5-Vinyl-1,3-oxazolidine-2-thione *
Corresponding author. Tel.: 32-8172-4377; fax: 32-8172-4420. E-mail address: [email protected] (S.N.M. Mandiki) 0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 7 - 8 4 0 1 ( 0 0 ) 0 0 1 2 2 - X
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1. Introduction Under the action of an endogenous enzyme, myrosinase (E.C.3.2.3.1), or the bacterial ¯ora in animals, the glucosinolates contained in rapeseed can be broken down and to some degradation products with antinutritional effects (Astwood et al., 1949; Mawson et al., 1993). The most important of these by-products are the 5-vinyl-1,3-oxazolidine-2thione (5-VOT) and the thiocyanate ions (SCNÿ) because of their interactions with the thyroid gland, which in turn, cause strong metabolic disturbances (Fenwick et al., 1983; Mawson et al., 1994; Gutzweiler, 1996). The 5-VOT is responsible for the reduction of animal performance (Maheshwari et al., 1979). It is transferred to serum, milk, muscular tissues and to certain organs: liver; lung; kidney; and mainly thyroid. Moreover, the precursor of the 5-VOT (known as goitrine), the 2-hydroxybut-3-enylglucosinolate or progoitrin, is still found in high proportion in most of the `00' varieties of rapeseed and remains in meal after oil extraction. Therefore, despite the decrease of the glucosinolates in rapeseed meal in Europe or Canada, their consumption can decrease yield and the meat quality. Levels of 5-VOT were in biological ¯uids and tissues of ruminants ranged from 3 to 8 mg kgÿ1 in milk, 15 to 200 mg kgÿ1 in plasma, 80 to 250 mg kgÿ1 in urine (De Brabander and Verbeke, 1982), 70 mg kgÿ1 in thyroid and <0.5 mg kgÿ1 in liver or kidney (De Brabander and Verbeke, 1986). The thiocyanate ions are degradation products from indol-3-ylmethylglucosinolates or other glucosinolates which give a carbonium ion and the thiocyanate ion in nonenzymatic and enzyme-catalysed reactions. When absorbed into the blood, the thiocyanate ions can be transferred to the milk (Papas et al., 1978, 1979; Laarveld et al., 1981b; Emanuelson et al., 1993) or transformed by liver enzymes. They may also competitively inhibit the transfer of iodide to the thyroid hormones in the thyroid gland (Laarveld et al., 1981a; Ahlin et al., 1994). The thiocyanate ions and iodide were thus considered to be important in relation to studies of the quality of rapeseed meal (Danielsen et al., 1994) and studies of antinutritional effects of glucosinolates, more precisely indol-3-ylmethylglucosinolates (Loft et al., 1992). In the two ®rst parts of this work, we studied the effect of the incorporation of rapeseed meal on animal performance and the histology of the thyroid gland as well as on the concentration of various plasma hormones (Derycke et al., 1999) and the effect on endocrine function and thyroid activity (Mandiki et al., 1999). In this part we studied the two main antinutritional factors, the 5-VOT and the SCNÿ ions, both during the process of oil extraction and during the preparation of the diets. Afterwards, the concentrations of 5-VOT and the SCNÿ ions were evaluated in samples of liver, kidney, lung, muscle, thyroid and plasma from lambs fed with diets containing 0 or 25% of rapeseed meal. 2. Experimental 2.1. Animals and management The general description of animals and management was given in the ®rst part of this series (Derycke et al., 1999).
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Table 1 Glucosinolate pro®le in the rapeseed SamouraõÈ, the corresponding meal and the diet containing 25% of rapeseed meal (mmol/g DM) Glucosinolates
Seed
Meal
Diet
Glucoiberin Progoitrin Epiprogoitrin Napoleiferin Glucoalyssin Sinalbin Gluconapin 4-OH Glucobrassicin Glucobrassicanapin Glucobrassicin Gluconasturtiin 4-Methoxyglucobrassicin Neoglucobrassicin Total
0.10 2.40 0.06 0.00 0.00 0.04 1.51 3.06 0.11 0.89 0.15 0.04 0.08 8.44
0.08 3.09 0.08 0.00 0.00 0.05 2.00 3.59 0.14 0.57 0.19 0.03 0.09 9.91
0.02 0.71 0.00 0.00 0.00 0.01 0.49 0.77 0.03 0.13 0.04 0.01 0.02 2.23
First, we determined the glucosinolate level in seven rapeseed varieties in order to select two, according to their total glucosinolate level and their glucosinolate pro®le. The glucosinolate concentration was determined in the seeds, the corresponding meals and diets (Tables 1 and 2), and veri®ed at each stage of production. The SCNÿ ions and the 5VOT were quanti®ed in the meals and diets (Table 3). In summary, 66 lambs allocated to three groups received ad libitum concentrates containing 0% (control) or 25% of rapeseed meal obtained from either the SamouraõÈ
Table 2 Glucosinolate pro®le in the rapeseed Honk, the corresponding meal and the diet containing 25% of rapeseed meal (mmol/g DM) Glucosinolates
Seed
Meal
Diet
Glucoiberin Progoitrin Epiprogoitrin Napoleiferin Glucoalyssin Sinalbin Gluconapin 4-OH Glucobrassicin Glucobrassicanapin Glucobrassicin Gluconasturtiin 4-Methoxyglucobrassicin Neoglucobrassicin Total
0.06 7.44 0.09 0.15 0.17 0.05 3.14 4.28 0.43 0.85 0.23 0.08 0.08 17.05
0.07 9.81 0.11 0.19 0.21 0.06 4.08 5.46 0.53 0.71 0.30 0.09 0.09 21.71
0.02 2.26 0.02 0.04 0.05 0.01 0.95 0.59 0.13 0.10 0.07 0.02 0.02 4.28
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Table 3 SCNÿ and 5-VOT level in the rapeseed meals, and the diets containing 25% of meal Meal
Diet
SCNÿ(mmol/g D.M.) SamouraõÈ Honk
0,48 0,24
0,59 1,01
5-VOT (mmol/g) SamouraõÈ Honk
0,10 0,10
0,16 0,29
variety or the Honk variety. The lambs were weaned at 888 days of age (245 kg live weight), and were slaughtered when their fattening state was estimated to be satisfying. In addition to the evaluation of animal performance and the analysis of plasma substances at slaughter, blood samples were taken fortnightly by venipuncture for the determination of the SCNÿ ions and the 5-VOT. Samples were immediately centrifuged (2000g for 15 min) and plasma was stored at ÿ208C until assayed. The muscle sample and organ samples (such as thyroid, liver, kidney and lung) were collected and stored at ÿ208C until assayed. 2.2. Analysis 2.2.1. Glucosinolates The glucosinolates were quanti®ed using the of®cial method ISO 9167-1. 2.2.2. 5-Vinyl-1,3-oxazolidine-2-thione As the analytical method proposed by Quinsac et al. (1992) caused some problems with complex matrices such as thyroid and liver, it has been improved by Mabon et al. (1999). 2.2.3. Thiocyanate ions The analytical method proposed by Johnston and Jones (1966) has been adapted to the different biological matrices. 2.2.3.1. Apparatus. Spectrophotometer UV±VIS Ultrospec 4000 (Pharmacia, Uppsala, Sweden), polystyrene measure cells, 10 mm optical path (Brand, Germany). 2.2.3.2. Reagents. Trichloro-acetic acid (TCA) (Merck, Darmstadt, Germany) solution 20% (m/v) in distilled water. Ferric reagent I: Ferric nitrate (Merck, Darmstadt, Germany) solution 0.8 M [320 g Fe(NO3)39H2O] in distilled water and nitric acid (d1.42, 125 ml) (UCB, Leuven, Belgium), made up to 1 l with water and ®ltered through a 0.45 mm Nylon ®lter (Gelman, Michigan, USA). Ferric reagent II: Five percent (m/v) mercuric chloride (Merck, Darmstadt, Germany) dissolved in ferric reagent I.
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Standards: 0±0.6 mmol mlÿ1 SCNÿ (Merck, Darmstadt, Germany) in distilled water. All the reagents used were of analytical grade. 2.2.4. Method 2.2.4.1. Sample preparation. Immediately after removal, the organs were frozen and stored at ÿ208C. Before analysis, the samples were defrosted and blended in a baby-food mixer (10 s for 40 g sample) in order to obtain a homogeneous paste. 2.2.4.2. Proteins precipitation. The sample (2 g of liver, kidney, lung, muscle, thyroid or 2 ml of plasma) was placed in a 16100 mm glass tube with Teflon-coated screw cap. Then, 1 ml of the aqueous solution of TCA was added. The dispersal of the tissue was achieved by vigorous manual shaking. It was necessary to wait at least 30 min and then to centrifuge for 10 min at 1000g. 2.2.4.3. Measurement. One milliliter of the supernatant was placed in a measuring cell, 0.50 ml of reagent I was added and the cell was stirred gently. The absorbance measurement was achieved and 0.10 ml of reagent II was added in the cell and the mixture stirred. After 5 min, the absorbance was measured. The SCNÿ was quantitated by the variation of the absorbance at 460 nm. 2.3. Statistical analysis Statistical analysis of the results was made by the methods described in the ®rst part of this work (Derycke et al., 1999). 3. Results 3.1. Glucosinolates and breakdown products in feed The glucosinolate concentration in the SamouraõÈ variety is ca. 8.4 mmol gÿ1 of dry matter (mmol gÿ1 DM) (Table 1) but in the Honk variety it is ca. 17.1 mmol gÿ1 DM (Table 2). Moreover, the main glucosinolates of rapeseed, progoitrin and 4-OH glucobrassicin formed, respectively, 28.4 and 36.3% of the total glucosinolate amount in the SamouraõÈ variety but 43.6 and 25.1% in the Honk variety. The glucosinolate concentration was determined in the seeds, the corresponding meals and diets (Tables 1 and 2), and veri®ed at each stage of production. After oil extraction, the glucosinolate level in the meal and in diets should be higher than the original values. Suggesting that a part of the glucosinolates was degraded during the processes of crushing and diet preparation. As we expected a degradation of the glucosinolates, we decided to quantify the SCNÿ ions and the 5-VOT in the meals and diets (Table 3). At each step, the amount of the increased SCNÿ ions was close to the indolyl glucosinolates degraded. Moreover, the amount of the 5-VOT found was close to the progoitrin which disappeared.
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3.2. Glucosinolates and breakdown products in biologic matrices 3.2.1. Intact glucosinolates In order to provide preliminary information the concentration of intact glucosinolates in the biological samples, ewe was fed for 3 months exclusively with the rapeseed meal Honk. At slaughter, samples of excrements, plasma, muscle and organs such as thyroid, liver, kidney and lung were collected but no intact glucosinolate was found in the following detection limits: 3.5 nmol gÿ1 for the thyroid, 35 pmol gÿ1 for muscle and kidney, 25 pmol gÿ1 for liver and lung, 20 pmol gÿ1 for plasma and excrements. We concluded that all the ingested glucosinolates were degraded and that only the two main breakdown products, the thiocyanate ions and the 5-VOT, had to be quanti®ed. 3.2.2. Thiocyanate ions Changes in the SCNÿ concentration in the plasma of the three groups are shown in the Fig. 1. The lambs were weaned at 888 day of age (245 kg of live weight). The values increased in the SamouraõÈ and Honk lots after weaning, up to 0.49 mmol mlÿ1 in the SamouraõÈ lot. The SCNÿ concentrations determined at slaughter are shown in Fig. 2 and were signi®cantly (p<0.05) higher in rapeseed fed ruminants than in the control. A low level was found in muscle and higher concentrations in liver, thyroid, plasma, kidney and lung. The SCNÿ concentration was not signi®cantly (p<0.05) different between SamouraõÈ and Honk varieties. 3.2.3. 5-Vinyl-1,3-oxazolidine-2-thione At slaughter, 5-VOT level was determined in muscle, organs (thyroid, liver, kidney and lung) and biological ¯uid (plasma). Values are shown in Fig. 3. The 5-VOT was present exclusively after the ingestion of rapeseed meal but not to the same extent for the two
Fig. 1. Evolution of the thiocyanate ions concentration in the plasma of growth-fattening lambs (n10).
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Fig. 2. Thiocyanate ions concentration in organs and plasma of lambs (n10).
varieties. The Honk rapeseed meal induced a 5-VOT level signi®cantly (p<0.05) higher in the target organs such as lung and thyroid than SamouraõÈ. 4. Discussion In the ®rst paper of this series (Derycke et al., 1999) we described the animal performance and morphological effects including 25% of rapeseed meal obtained from two varieties in the diet of lambs at the growth-fattening stage. The two rapeseed varieties were very different in their content and distribution of glucosinolates. The diets containing 25% of rapeseed meal were offered ad libitum to lambs from the end of their 1st month of age and were the main component of the diet from weaning to the end of the experiment apart from a low allowance of hay (0.4 kg per day). Moreover, rapeseed meal was almost the only protein supplement for the two experimental concentrates. Despite these conditions, no difference was observed in animal performance and carcass quality, apart from a slight improvement in the quality of perirenal fat and hyperthyroidism in animals fed with rapeseed meal. The second paper was focused on the impact of 25% of the rapeseed meal on hormones and physiological substances. Apart from the secretion of thyroid hormones, the incorporation of a high amount of rapeseed meal in the diet had no negative effect on either blood substances (insulin, transaminases) or the hormones secreted by the main
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Fig. 3. 5-VOT concentration in organs and plasma of lambs (n10).
endocrine glands implicated in the metabolic process (GH by the pituitary, cortisol by the subrenal). The results described in the current paper show that no intact glucosinolate was found within our detection limits in samples of plasma, muscle, thyroid, liver, kidney and lung collected at slaughter. We concluded that the ingested glucosinolates were degraded by the action of micro-organisms in the digestive system as reported by Fiems and Buysse (1985) and Mawson et al. (1994). Thus, the two main breakdown products were quanti®ed. The analyses reveal the presence of SCNÿ in plasma, muscle and some target organs from lambs fed with diets containing 25% of rapeseed meal. A high intake of rapeseed meal is re¯ected in the SCNÿ concentration in plasma, muscle and several organs, in agreement with Emanuelson et al. (1993). The SCNÿ concentration increased signi®cantly with the ingestion of rapeseed meal but not to the same extent for the two varieties. It can be correlated with the indolyl glucosinolates ingested. The 5-VOT was concentrated in lung and thyroid. The SamouraõÈ rapeseed meal induced a higher SCNÿ level in each analysed tissue, either in plasma or muscle and organs. This difference could be explained by the difference in the glucosinolate pro®le between the two rapeseed meals. The indolyl glucosinolates, precursor of SCNÿ ions, were in higher concentration in the SamouraõÈ rapeseed. It is interesting to notice that lungs and kidneys have the trend to accumulate thiocyanate. Kidneys could be considered as a route for SCNÿ elimination. In the future, a determination of thiocyanate in urine could test this a con®rmation of this
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hypothesis. The SCNÿ concentration was signi®cantly (p<0.05) different between lung and plasma but not between kidney and plasma. The 5-VOT is present only after the ingestion of rapeseed meal (Mawson et al., 1993) but not to the same extent for the two varieties. The Honk rapeseed meal induced a 5VOT level signi®cantly (p<0.05) higher in the target organs such as lung and thyroid than SamouraõÈ. This difference could be once again due to the glucosinolate pro®les of the two rapeseed meals. The progoitrine (2-hydroxybut-3-enyl glucosinolate), precursor of the 5VOT, was in higher concentration in the Honk variety. The results obtained in this experiment show that a high intake of rapeseed meal induces an accumulation of 5-VOT in lung and mainly in thyroid but in very low levels in muscle, liver, kidney and plasma. The 5-VOT concentration was signi®cantly (p<0.05) higher in thyroid than in lung. It is interesting to note that 5-VOT has a very high af®nity for thyroid. This behaviour can perhaps explain the morphological and the physiological changes in this organ (Mandiki et al., 1999). Acknowledgements This work has been undertaken with the ®nancial support of the `Direction geÂneÂrale, Recherche et DeÂveloppement du MinisteÁre des Classes moyennes et de l'Agriculture' and of the `Direction geÂneÂrale des Technologies, de la Recherche et de l'Energie du MinisteÁre de la ReÂgion wallone' of Belgium. The authors are grateful to Prof. P. Rollin (LCBA, Universite d'OrleÂans, France) for supplying the 4,4-dimethyl-1,3-oxazolidine-2-thione, to M. Hardenne of the Department of General and Organic Chemistry of Gembloux and to M.-A. Bouckoms-Vandermeir and P. Ghys of the Laboratory of Animal Physiology of Namur for their technical assistance. References Ahlin, K., Emanuelson, M., Wiktorson, H., 1994. Rapeseed products from double-low cultivars as feed for dairy cows: effects of long-term feeding on thyroid function, fertility and animal health. Acta Vet. Scand. 35, 37. Astwood, E.B., Greer, M.A., Ettlinger, M.G., 1949. L-5-vinyl-2-thiooxazolidone, an antithyroid compound from yellow turnip and from brassica seeds. J. Biol. Chem. 181, 121±130. Danielsen, V., Eggum, B.O., Jensen, S.K., SoÈrensen, H., 1994. Dehulled protein-rich rapeseed meal as a protein source for early weaned piglets. Anim. Feed Sci. Technol. 46, 239±248. De Brabander, H.F., Verbeke, R., 1982. Determination of oxazolidine-2-thiones in biological ¯uids in the ppb range. J. Chromatogr. 252, 225±239. De Brabander, H.F., Verbeke, R., 1986. Determination of 5-vinyloxazolidine-2-thione residue in ruminant tissues. Proceedings of the 32th European Meeting of Meat Research Workers. Gent 1, 45±48. Derycke, G., Mabon, N., Mandiki, S.N.M., Bister, J.-L., Wathelet, J.-P., Marlier, M., Paquay, R., 1999. Chemical changes and in¯uences of rapeseed antinutritional factors on lambs physiology and performances. 1. Zoothechnical performances and histological study. Anim. Feed Sci. Technol. 81, 81±91. Emanuelson, M., Ahlin, K.-A., Wiktorsson, H., 1993. Long-term feeding of rapeseed meal full-fat rapeseed of double low cultivars to dairy cows. Livest. Prod. Sci. 33, 199±214. Fenwick, G.R., Heaney, R.K., Mullin, W.J., 1983. Glucosinolates and their breakdown products in food and food plants. CRC Crit. Rev. Food Sci. Nutr. 18, 123±201.
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Fiems, L.O., Buysse, F.X., 1985. Aperc,u de l'utilisation de tourteaux de colza en tant que source de proteÂines dans les rations pour ruminants. Revue de l'Agriculture (Belgium) 2 (38), 261±274. Gutzweiler, A., 1996. Effets des glucosinolates du colza sur l'organisme. Revue suisse Agric. 28 (3), 129±132. Johnston and Jones, 1996. Variations in the thiocyanate content of Kale varieties, J. Sci. Fd Agric., Vol. 17, Feburary. Laarveld, B., Brockman, R.P., Christensen, D.A., 1981a. The effects of Tower and Midas rapeseed meals on milk production and concentration of goitrogens and iodide in milk. Can. J. Anim. Sci. 61, 131±139. Laarveld, B., Brockman, R.P., Christensen, D.A., 1981b. The effects of the level of iodine in canola meal concentrate on milk iodine and thiocyanate content and thyroid function in dairy cows. Can. J. Anim. Sci. 61, 625±632. Loft, S., Otte, J., Poulsen, H.E., SoÈrensen, H., 1992. In¯uence of intact and treated indolyl glucosinolates on the metabolism in vivo of metronidazole and antipyrine in the rat. Food Chem. Toxicol. 30, 927±935. Mabon, N., Wathelet, J.-P., Marlier, M., 1999. Analysis of 5-vinyl-1,3-oxazolidine-2-thione in complex matrices at ppb level. Talanta 49, 199±206. Maheshwari, P.N., Stanley, D.W., Gray, J.I., van de Voort, F.R., 1979. An HPLC method for simultaneous quantitation of individual isothiocyanates and oxazolidinethione in myrosinase digest of rapeseed meal. J. Am. Oil Chem. Soc. 56, 837. Mandiki, S.N.M., Mabon, N., Derycke, G., Mandiki, S.N.M., Bister, J.-L., Wathelet, J.-P., Paquay, R., Marlier, M., 1999. Chemical changes and in¯uences of rapeseed antinutritional factors on lambs physiology and performances. 2. Plasmatic substances and activity of the thyroid. Anim. Feed Sci. Technol. 81, 93±103. Mawson, R., Heaney, R.K., Piskula, M., Kozlowska, H., 1993. Rapeseed meal-glucosinolates and their antinutritional effects. Part I. Rapeseed production and chemistry of glucosinolates. Die NaÈhrung 37, 336±344. Mawson, R., Heaney, R.K., Zdunczyk, Z., Kozlowska, H., 1994. Rapeseed meal-glucosinolates and their antinutritional effects. Part IV. Goitrogenicity and internal organs abnormalities in animals. Die NaÈhrung 38, 178±191. Papas, A., Ingalls, J.R., Caus®eld, P., 1978. Effect of Tower and 1821 rapeseed meals and Tower gums on milk yield, milk composition and blood parameters of lactating dairy cows. Can. J. Anim. Sci. 58, 671±679. Papas, A., Ingalls, J.R., Campbell, L.D., 1979. Studies on the effects of rapeseed meal on thyropid status glucosinolates and iodine content of milk and other parameters. J. Nutr. 109, 1129. Quinsac, A., Ribailler, D., Rollin, P., Dreux, M., 1992. Analysis of 5-vinyl-1,3-oxazolidine-2-thione by liquid chromatography. J. Assoc. Off. Anal. Chem. 75 (3), 529±536.
Chemical changes and in¯uences of rapeseed antinutritional factors on lamb physiology and performance. 3. Antinutritional factors in plasma and organs Nicolas Mabona, S.N.M. Mandikib,*, G. Deryckeb, J.-L. Bisterb, J.-P. Watheleta, M. Marliera, R. Paquayb a
Faculte universitaire des Sciences agronomiques de Gembloux, 2, Passage des DeÂporteÂs, B-5030 Gembloux, Belgium b FaculteÂs universitaires Notre-Dame de la Paix, 61, Rue de Bruxelles, B-5000 Namur, Belgium Received 5 May 1999; received in revised form 20 January 2000; accepted 10 February 2000
Abstract The impact of a high level of rapeseed meal in the diet on the concentration of antinutritional factors in plasma and organs was studied in 66 Texel, Suffolk or crossbred lambs. From 1 month of age till slaughter (13221 days), they were fed ad libitum with concentrates containing 0% or 25% of rapeseed meal obtained either SamouraõÈ or Honk rapeseed varieties. Lambs were weaned at 888 days of age and 245 kg live weight. Blood and organ samples were taken fortnightly for the determination of antinutritional factors. In plasma and organs, the thiocyanate concentrations were systematically higher in the SamouraõÈ and Honk lots than in the control. After weaning, the SCNÿ concentration in plasma increased up to 490 mmol lÿ1 in the SamouraõÈ lot. The goitrogen 5-vinyl-1,3-oxazolidine-2-thione (5-VOT) was determined in muscle, organs (thyroid, liver, kidney and lung) and biological ¯uid (plasma). The 5-VOT was present only after the ingestion of rapeseed meal but not to the same extent for the two varieties. The Honk rapeseed meal induced a 5-VOT level signi®cantly (p<0.05) higher in the target organs such as lung and thyroid than SamouraõÈ. Very low levels were found in muscle, liver, kidney, plasma and lung. # 2000 Elsevier Science B.V. All rights reserved. Keywords: Rapeseed meal; Lambs; Thiocyanate ions; 5-Vinyl-1,3-oxazolidine-2-thione *
Corresponding author. Tel.: 32-8172-4377; fax: 32-8172-4420. E-mail address: [email protected] (S.N.M. Mandiki) 0377-8401/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 7 - 8 4 0 1 ( 0 0 ) 0 0 1 2 2 - X
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1. Introduction Under the action of an endogenous enzyme, myrosinase (E.C.3.2.3.1), or the bacterial ¯ora in animals, the glucosinolates contained in rapeseed can be broken down and to some degradation products with antinutritional effects (Astwood et al., 1949; Mawson et al., 1993). The most important of these by-products are the 5-vinyl-1,3-oxazolidine-2thione (5-VOT) and the thiocyanate ions (SCNÿ) because of their interactions with the thyroid gland, which in turn, cause strong metabolic disturbances (Fenwick et al., 1983; Mawson et al., 1994; Gutzweiler, 1996). The 5-VOT is responsible for the reduction of animal performance (Maheshwari et al., 1979). It is transferred to serum, milk, muscular tissues and to certain organs: liver; lung; kidney; and mainly thyroid. Moreover, the precursor of the 5-VOT (known as goitrine), the 2-hydroxybut-3-enylglucosinolate or progoitrin, is still found in high proportion in most of the `00' varieties of rapeseed and remains in meal after oil extraction. Therefore, despite the decrease of the glucosinolates in rapeseed meal in Europe or Canada, their consumption can decrease yield and the meat quality. Levels of 5-VOT were in biological ¯uids and tissues of ruminants ranged from 3 to 8 mg kgÿ1 in milk, 15 to 200 mg kgÿ1 in plasma, 80 to 250 mg kgÿ1 in urine (De Brabander and Verbeke, 1982), 70 mg kgÿ1 in thyroid and <0.5 mg kgÿ1 in liver or kidney (De Brabander and Verbeke, 1986). The thiocyanate ions are degradation products from indol-3-ylmethylglucosinolates or other glucosinolates which give a carbonium ion and the thiocyanate ion in nonenzymatic and enzyme-catalysed reactions. When absorbed into the blood, the thiocyanate ions can be transferred to the milk (Papas et al., 1978, 1979; Laarveld et al., 1981b; Emanuelson et al., 1993) or transformed by liver enzymes. They may also competitively inhibit the transfer of iodide to the thyroid hormones in the thyroid gland (Laarveld et al., 1981a; Ahlin et al., 1994). The thiocyanate ions and iodide were thus considered to be important in relation to studies of the quality of rapeseed meal (Danielsen et al., 1994) and studies of antinutritional effects of glucosinolates, more precisely indol-3-ylmethylglucosinolates (Loft et al., 1992). In the two ®rst parts of this work, we studied the effect of the incorporation of rapeseed meal on animal performance and the histology of the thyroid gland as well as on the concentration of various plasma hormones (Derycke et al., 1999) and the effect on endocrine function and thyroid activity (Mandiki et al., 1999). In this part we studied the two main antinutritional factors, the 5-VOT and the SCNÿ ions, both during the process of oil extraction and during the preparation of the diets. Afterwards, the concentrations of 5-VOT and the SCNÿ ions were evaluated in samples of liver, kidney, lung, muscle, thyroid and plasma from lambs fed with diets containing 0 or 25% of rapeseed meal. 2. Experimental 2.1. Animals and management The general description of animals and management was given in the ®rst part of this series (Derycke et al., 1999).
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Table 1 Glucosinolate pro®le in the rapeseed SamouraõÈ, the corresponding meal and the diet containing 25% of rapeseed meal (mmol/g DM) Glucosinolates
Seed
Meal
Diet
Glucoiberin Progoitrin Epiprogoitrin Napoleiferin Glucoalyssin Sinalbin Gluconapin 4-OH Glucobrassicin Glucobrassicanapin Glucobrassicin Gluconasturtiin 4-Methoxyglucobrassicin Neoglucobrassicin Total
0.10 2.40 0.06 0.00 0.00 0.04 1.51 3.06 0.11 0.89 0.15 0.04 0.08 8.44
0.08 3.09 0.08 0.00 0.00 0.05 2.00 3.59 0.14 0.57 0.19 0.03 0.09 9.91
0.02 0.71 0.00 0.00 0.00 0.01 0.49 0.77 0.03 0.13 0.04 0.01 0.02 2.23
First, we determined the glucosinolate level in seven rapeseed varieties in order to select two, according to their total glucosinolate level and their glucosinolate pro®le. The glucosinolate concentration was determined in the seeds, the corresponding meals and diets (Tables 1 and 2), and veri®ed at each stage of production. The SCNÿ ions and the 5VOT were quanti®ed in the meals and diets (Table 3). In summary, 66 lambs allocated to three groups received ad libitum concentrates containing 0% (control) or 25% of rapeseed meal obtained from either the SamouraõÈ
Table 2 Glucosinolate pro®le in the rapeseed Honk, the corresponding meal and the diet containing 25% of rapeseed meal (mmol/g DM) Glucosinolates
Seed
Meal
Diet
Glucoiberin Progoitrin Epiprogoitrin Napoleiferin Glucoalyssin Sinalbin Gluconapin 4-OH Glucobrassicin Glucobrassicanapin Glucobrassicin Gluconasturtiin 4-Methoxyglucobrassicin Neoglucobrassicin Total
0.06 7.44 0.09 0.15 0.17 0.05 3.14 4.28 0.43 0.85 0.23 0.08 0.08 17.05
0.07 9.81 0.11 0.19 0.21 0.06 4.08 5.46 0.53 0.71 0.30 0.09 0.09 21.71
0.02 2.26 0.02 0.04 0.05 0.01 0.95 0.59 0.13 0.10 0.07 0.02 0.02 4.28
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Table 3 SCNÿ and 5-VOT level in the rapeseed meals, and the diets containing 25% of meal Meal
Diet
SCNÿ(mmol/g D.M.) SamouraõÈ Honk
0,48 0,24
0,59 1,01
5-VOT (mmol/g) SamouraõÈ Honk
0,10 0,10
0,16 0,29
variety or the Honk variety. The lambs were weaned at 888 days of age (245 kg live weight), and were slaughtered when their fattening state was estimated to be satisfying. In addition to the evaluation of animal performance and the analysis of plasma substances at slaughter, blood samples were taken fortnightly by venipuncture for the determination of the SCNÿ ions and the 5-VOT. Samples were immediately centrifuged (2000g for 15 min) and plasma was stored at ÿ208C until assayed. The muscle sample and organ samples (such as thyroid, liver, kidney and lung) were collected and stored at ÿ208C until assayed. 2.2. Analysis 2.2.1. Glucosinolates The glucosinolates were quanti®ed using the of®cial method ISO 9167-1. 2.2.2. 5-Vinyl-1,3-oxazolidine-2-thione As the analytical method proposed by Quinsac et al. (1992) caused some problems with complex matrices such as thyroid and liver, it has been improved by Mabon et al. (1999). 2.2.3. Thiocyanate ions The analytical method proposed by Johnston and Jones (1966) has been adapted to the different biological matrices. 2.2.3.1. Apparatus. Spectrophotometer UV±VIS Ultrospec 4000 (Pharmacia, Uppsala, Sweden), polystyrene measure cells, 10 mm optical path (Brand, Germany). 2.2.3.2. Reagents. Trichloro-acetic acid (TCA) (Merck, Darmstadt, Germany) solution 20% (m/v) in distilled water. Ferric reagent I: Ferric nitrate (Merck, Darmstadt, Germany) solution 0.8 M [320 g Fe(NO3)39H2O] in distilled water and nitric acid (d1.42, 125 ml) (UCB, Leuven, Belgium), made up to 1 l with water and ®ltered through a 0.45 mm Nylon ®lter (Gelman, Michigan, USA). Ferric reagent II: Five percent (m/v) mercuric chloride (Merck, Darmstadt, Germany) dissolved in ferric reagent I.
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Standards: 0±0.6 mmol mlÿ1 SCNÿ (Merck, Darmstadt, Germany) in distilled water. All the reagents used were of analytical grade. 2.2.4. Method 2.2.4.1. Sample preparation. Immediately after removal, the organs were frozen and stored at ÿ208C. Before analysis, the samples were defrosted and blended in a baby-food mixer (10 s for 40 g sample) in order to obtain a homogeneous paste. 2.2.4.2. Proteins precipitation. The sample (2 g of liver, kidney, lung, muscle, thyroid or 2 ml of plasma) was placed in a 16100 mm glass tube with Teflon-coated screw cap. Then, 1 ml of the aqueous solution of TCA was added. The dispersal of the tissue was achieved by vigorous manual shaking. It was necessary to wait at least 30 min and then to centrifuge for 10 min at 1000g. 2.2.4.3. Measurement. One milliliter of the supernatant was placed in a measuring cell, 0.50 ml of reagent I was added and the cell was stirred gently. The absorbance measurement was achieved and 0.10 ml of reagent II was added in the cell and the mixture stirred. After 5 min, the absorbance was measured. The SCNÿ was quantitated by the variation of the absorbance at 460 nm. 2.3. Statistical analysis Statistical analysis of the results was made by the methods described in the ®rst part of this work (Derycke et al., 1999). 3. Results 3.1. Glucosinolates and breakdown products in feed The glucosinolate concentration in the SamouraõÈ variety is ca. 8.4 mmol gÿ1 of dry matter (mmol gÿ1 DM) (Table 1) but in the Honk variety it is ca. 17.1 mmol gÿ1 DM (Table 2). Moreover, the main glucosinolates of rapeseed, progoitrin and 4-OH glucobrassicin formed, respectively, 28.4 and 36.3% of the total glucosinolate amount in the SamouraõÈ variety but 43.6 and 25.1% in the Honk variety. The glucosinolate concentration was determined in the seeds, the corresponding meals and diets (Tables 1 and 2), and veri®ed at each stage of production. After oil extraction, the glucosinolate level in the meal and in diets should be higher than the original values. Suggesting that a part of the glucosinolates was degraded during the processes of crushing and diet preparation. As we expected a degradation of the glucosinolates, we decided to quantify the SCNÿ ions and the 5-VOT in the meals and diets (Table 3). At each step, the amount of the increased SCNÿ ions was close to the indolyl glucosinolates degraded. Moreover, the amount of the 5-VOT found was close to the progoitrin which disappeared.
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3.2. Glucosinolates and breakdown products in biologic matrices 3.2.1. Intact glucosinolates In order to provide preliminary information the concentration of intact glucosinolates in the biological samples, ewe was fed for 3 months exclusively with the rapeseed meal Honk. At slaughter, samples of excrements, plasma, muscle and organs such as thyroid, liver, kidney and lung were collected but no intact glucosinolate was found in the following detection limits: 3.5 nmol gÿ1 for the thyroid, 35 pmol gÿ1 for muscle and kidney, 25 pmol gÿ1 for liver and lung, 20 pmol gÿ1 for plasma and excrements. We concluded that all the ingested glucosinolates were degraded and that only the two main breakdown products, the thiocyanate ions and the 5-VOT, had to be quanti®ed. 3.2.2. Thiocyanate ions Changes in the SCNÿ concentration in the plasma of the three groups are shown in the Fig. 1. The lambs were weaned at 888 day of age (245 kg of live weight). The values increased in the SamouraõÈ and Honk lots after weaning, up to 0.49 mmol mlÿ1 in the SamouraõÈ lot. The SCNÿ concentrations determined at slaughter are shown in Fig. 2 and were signi®cantly (p<0.05) higher in rapeseed fed ruminants than in the control. A low level was found in muscle and higher concentrations in liver, thyroid, plasma, kidney and lung. The SCNÿ concentration was not signi®cantly (p<0.05) different between SamouraõÈ and Honk varieties. 3.2.3. 5-Vinyl-1,3-oxazolidine-2-thione At slaughter, 5-VOT level was determined in muscle, organs (thyroid, liver, kidney and lung) and biological ¯uid (plasma). Values are shown in Fig. 3. The 5-VOT was present exclusively after the ingestion of rapeseed meal but not to the same extent for the two
Fig. 1. Evolution of the thiocyanate ions concentration in the plasma of growth-fattening lambs (n10).
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Fig. 2. Thiocyanate ions concentration in organs and plasma of lambs (n10).
varieties. The Honk rapeseed meal induced a 5-VOT level signi®cantly (p<0.05) higher in the target organs such as lung and thyroid than SamouraõÈ. 4. Discussion In the ®rst paper of this series (Derycke et al., 1999) we described the animal performance and morphological effects including 25% of rapeseed meal obtained from two varieties in the diet of lambs at the growth-fattening stage. The two rapeseed varieties were very different in their content and distribution of glucosinolates. The diets containing 25% of rapeseed meal were offered ad libitum to lambs from the end of their 1st month of age and were the main component of the diet from weaning to the end of the experiment apart from a low allowance of hay (0.4 kg per day). Moreover, rapeseed meal was almost the only protein supplement for the two experimental concentrates. Despite these conditions, no difference was observed in animal performance and carcass quality, apart from a slight improvement in the quality of perirenal fat and hyperthyroidism in animals fed with rapeseed meal. The second paper was focused on the impact of 25% of the rapeseed meal on hormones and physiological substances. Apart from the secretion of thyroid hormones, the incorporation of a high amount of rapeseed meal in the diet had no negative effect on either blood substances (insulin, transaminases) or the hormones secreted by the main
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Fig. 3. 5-VOT concentration in organs and plasma of lambs (n10).
endocrine glands implicated in the metabolic process (GH by the pituitary, cortisol by the subrenal). The results described in the current paper show that no intact glucosinolate was found within our detection limits in samples of plasma, muscle, thyroid, liver, kidney and lung collected at slaughter. We concluded that the ingested glucosinolates were degraded by the action of micro-organisms in the digestive system as reported by Fiems and Buysse (1985) and Mawson et al. (1994). Thus, the two main breakdown products were quanti®ed. The analyses reveal the presence of SCNÿ in plasma, muscle and some target organs from lambs fed with diets containing 25% of rapeseed meal. A high intake of rapeseed meal is re¯ected in the SCNÿ concentration in plasma, muscle and several organs, in agreement with Emanuelson et al. (1993). The SCNÿ concentration increased signi®cantly with the ingestion of rapeseed meal but not to the same extent for the two varieties. It can be correlated with the indolyl glucosinolates ingested. The 5-VOT was concentrated in lung and thyroid. The SamouraõÈ rapeseed meal induced a higher SCNÿ level in each analysed tissue, either in plasma or muscle and organs. This difference could be explained by the difference in the glucosinolate pro®le between the two rapeseed meals. The indolyl glucosinolates, precursor of SCNÿ ions, were in higher concentration in the SamouraõÈ rapeseed. It is interesting to notice that lungs and kidneys have the trend to accumulate thiocyanate. Kidneys could be considered as a route for SCNÿ elimination. In the future, a determination of thiocyanate in urine could test this a con®rmation of this
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hypothesis. The SCNÿ concentration was signi®cantly (p<0.05) different between lung and plasma but not between kidney and plasma. The 5-VOT is present only after the ingestion of rapeseed meal (Mawson et al., 1993) but not to the same extent for the two varieties. The Honk rapeseed meal induced a 5VOT level signi®cantly (p<0.05) higher in the target organs such as lung and thyroid than SamouraõÈ. This difference could be once again due to the glucosinolate pro®les of the two rapeseed meals. The progoitrine (2-hydroxybut-3-enyl glucosinolate), precursor of the 5VOT, was in higher concentration in the Honk variety. The results obtained in this experiment show that a high intake of rapeseed meal induces an accumulation of 5-VOT in lung and mainly in thyroid but in very low levels in muscle, liver, kidney and plasma. The 5-VOT concentration was signi®cantly (p<0.05) higher in thyroid than in lung. It is interesting to note that 5-VOT has a very high af®nity for thyroid. This behaviour can perhaps explain the morphological and the physiological changes in this organ (Mandiki et al., 1999). Acknowledgements This work has been undertaken with the ®nancial support of the `Direction geÂneÂrale, Recherche et DeÂveloppement du MinisteÁre des Classes moyennes et de l'Agriculture' and of the `Direction geÂneÂrale des Technologies, de la Recherche et de l'Energie du MinisteÁre de la ReÂgion wallone' of Belgium. The authors are grateful to Prof. P. Rollin (LCBA, Universite d'OrleÂans, France) for supplying the 4,4-dimethyl-1,3-oxazolidine-2-thione, to M. Hardenne of the Department of General and Organic Chemistry of Gembloux and to M.-A. Bouckoms-Vandermeir and P. Ghys of the Laboratory of Animal Physiology of Namur for their technical assistance. References Ahlin, K., Emanuelson, M., Wiktorson, H., 1994. Rapeseed products from double-low cultivars as feed for dairy cows: effects of long-term feeding on thyroid function, fertility and animal health. Acta Vet. Scand. 35, 37. Astwood, E.B., Greer, M.A., Ettlinger, M.G., 1949. L-5-vinyl-2-thiooxazolidone, an antithyroid compound from yellow turnip and from brassica seeds. J. Biol. Chem. 181, 121±130. Danielsen, V., Eggum, B.O., Jensen, S.K., SoÈrensen, H., 1994. Dehulled protein-rich rapeseed meal as a protein source for early weaned piglets. Anim. Feed Sci. Technol. 46, 239±248. De Brabander, H.F., Verbeke, R., 1982. Determination of oxazolidine-2-thiones in biological ¯uids in the ppb range. J. Chromatogr. 252, 225±239. De Brabander, H.F., Verbeke, R., 1986. Determination of 5-vinyloxazolidine-2-thione residue in ruminant tissues. Proceedings of the 32th European Meeting of Meat Research Workers. Gent 1, 45±48. Derycke, G., Mabon, N., Mandiki, S.N.M., Bister, J.-L., Wathelet, J.-P., Marlier, M., Paquay, R., 1999. Chemical changes and in¯uences of rapeseed antinutritional factors on lambs physiology and performances. 1. Zoothechnical performances and histological study. Anim. Feed Sci. Technol. 81, 81±91. Emanuelson, M., Ahlin, K.-A., Wiktorsson, H., 1993. Long-term feeding of rapeseed meal full-fat rapeseed of double low cultivars to dairy cows. Livest. Prod. Sci. 33, 199±214. Fenwick, G.R., Heaney, R.K., Mullin, W.J., 1983. Glucosinolates and their breakdown products in food and food plants. CRC Crit. Rev. Food Sci. Nutr. 18, 123±201.
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