CORRELATION BETWEEN BREAKDOWN PRODUCTS OF GLUCOSINOLATES AND THE AMOUNT OF RAPESEED MEAL INTRODUCED IN THE DIETS OF 80 LAMBS AND 32 BULLS. EVOLUTION OFá 5-VINYL-1,3-OXAZOLIDINE-2-THIONE (5-VOT) AND THIOCYANATE IONS IN BIOLOGICAL FLUIDS AND ORGANS
Nicolas Mabon1, Jean-Paul Wathelet1, Guy Derycke2, S. N. M. Mandiki2, Jean-Loup Bister2, Michel Marlier1 and Raymond Paquay2
1 FacultÚ universitaire des Sciences agronomiques, UnitÚ de chimie gÚnÚrale et organique
Passage des DÚportÚs, n░2, B-5030 Gembloux, Belgium
2 Les FacultÚs universitaires Notre-Dame de la Paix, Laboratoire de physiologie animale rue de Bruxelles n░61, B-5000 Namur, Belgium
A large-scale experimentation has been conducted in Belgium since 4 years in order to study the influence of antinutritional rapeseed factors and their degradation products in ruminants. Animals have been fed with iso-energetic, isolipidic and isoproteic diets containing various proportions of industrial rapeseed meal (lambs: 0% to 40%; bulls: 0% to 34%). The trials and the study of zootechnical, physiological, histological aspects were co-ordinated by the University of Namur. The University of Gembloux was concerned by chemical aspects (analysis of glucosinolates, aromatic choline esters and their respective degradation products such as 5-vinyl-1,3-oxazolidine-2-thione, nitriles, isothiocyanates, thiocyanate ions and trimethylamine). In this presentation we would like to point out the results concerning the evolution of the SCN- ions concentration in plasma, the correlation between the proportions of rapeseed meal in the diets of growing-fattening ruminants and SCN- ions, and 5-VOT content in muscle, some organs (thyroid, kidney, liver and lung), plasma and urine. Correlations (for lambs and for bulls) have been established between thiocyanate ions concentration and the percentage of rapeseed meal in the diets. SCN- ions were specially accumulated in kidney and lung although the 5-VOT is observed in high concentration in thyroid.
Brassicaceae, antinutritionnal factors, subproducts, analysis
Rapeseed is an important source of protein for animal feed. Under the action of an endogenous enzyme, the myrosinase (E.C.188.8.131.52), or the action of the bacterial flora in animals, the glucosinolates contained in rapeseed can be broken down and give some degradation products with antinutritional effects (Astwood et al., 1949). The most important of these by-products are the 5‑vinyl-1,3-oxazolidine-2-thione (5-VOT) and the thiocyanate ions (SCN-) because of their interactions with the thyroid gland, which in turn, cause strong metabolic disturbances (Fenwick et al., 1983).
The 5-VOT is responsible for the reduction of the zootechnic performances (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 goitrin), the 2-hydroxybut-3‑enylglucosinolate or progoitrin, is still found in moderate proportion in most of the "00" varieties of rapeseed and remains in meal after oil extraction. Therefore, despite the decrease of the glucosinolates quantity in rapeseed meal in Europe or Canada, their consumption can decrease yield and presents potential damages for the quality of animal growing. Experiments performed on ruminants showed that different levels of 5-VOT were found in biological fluids and tissues.
The thiocyanate ions are degradation products from indol-3-ylmethylglucosinolates or other indolyl glucosinolates. When absorbed from digesta to the blood, the thiocyanate ions are transferred to different organs (Emanuelson et al., 1993). It may also competitively inhibit the transfer of iodide to the thyroid hormones in the thyroid gland (Sch÷ne et al., 1993; Wemheuer, 1993; 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 (Michaelsen et al., 1991; Danielsen et al., 1994) and studies of antinutritional effects of glucosinolates (Bjergegaard et al., 1994) more precisely indol-3-ylmethylglucosinolates (Loft et al., 1992).
In this part we studied the two main antinutritional factors, the 5-VOT and the SCN- ions, in order to better understand their impact in the target organs and to correlate these results with the zootechnic performances. Afterwards, the concentrations of 5-VOT and the SCN- ions were evaluated in samples of liver, kidney, lung, muscle, thyroid, and plasma from lambs fed with diets containing 0, 5,10, 15, 20, 25, 30 or 40 % of rapeseed meal. Similar samples plus urine, from bulls fed with diets containing 0, 10, 20 or 34 % of rapeseed meal, have been analysed.
MATERIELS and METHODS
Animals and management
First, we determined the glucosinolate level in the industrial rapeseed meal and in the corresponding diets. The SCN- ions and the 5-VOT were quantified in the meals and diets.
In summary, 80 lambs allocated to eight groups received ad libitum concentrates containing 0 % (control), 5%, 10%, 15%, 20%, 25%, 30% and 40 % of rapeseed meal. The lambs were weaned at 50 ▒ 13 day of age and were slaughtered when their fattening state was estimated to be satisfying.
On an other hand, 32 bulls were allocated to four groups and received ad libitum concentrates containing 0 % (control), 10%, 20% and 34 % of rapeseed meal. The bulls were allotted at 300 ▒ 30 kg of live weight and were slaughtered when their fattening state was estimated to be satisfying.
To the evaluation of the plasmatic substances, blood samples were performed fortnightly by venipuncture for the determination of the SCN- ions and the 5-VOT. Samples were immediately centrifuged and plasma stored at ‑20░C until assayed. The muscle sample and organ samples were collected and stored at -20░C until assayed.
All chemicals were analytical grade obtained from commercial sources.
The glucosinolates were quantified using the official method ISO 9167-1.
The 5-vinyl-1,3-oxazolidine-2-thione was quantified in complex matrices with the protocol proposed by Mabon et al., (1998).
The thiocyanates ions were analysed in the fresh matrices with the method proposed by Johnston and Jones (1962).
RESULTS and DISCUSSION
Experiment with lambs
The evolution of the SCN- concentration in the plasma of the eight groups of lambsare shown in the figure 1.
The values increased in the different lots during the first tree weeks and stay relatively constant untill the end of the experimentation. The SCN- concentrations were determined at the slaughtering in several matrices and are shown in figure 2.
The lower level was found in muscle and the concentration was higher and higher in liver, thyroid, plasma, kidney and lung. In plasma, muscle and organs, the SCN- concentration was significantly (P < 0.05) higher in lots fed with diets containing rapeseed meal than in the control. It is interesting to notice that lung and kidney have the trend to accumulate thiocyanate. Kidneys can be considered as a way of SCN- elimination. In the experimentation with bulls, the determination of the thiocyanate level in urine gave a confirmation of this supposition. The ingestion of high amount of rapeseed meal is connected with the modification of the SCN- concentration in plasma, muscle and several organs. The SCN- concentration increased significantly with the percentage of rapeseed meal ingested. More precisely, it can be correlated with the indolyl glucosinolates ingested (precursor of the SCN- ions). At slaughtering, 5-VOT level was determined in muscle, organs (thyroid, liver, kidney and lung) and biological fluid (plasma). Values are shown in figure 3.
The 5-VOT is present exclusively with the ingestion of rapeseed meal. The rapeseed meal induced a 5-VOT level significantly (P < 0.05) higher in the target organs such as lung and thyroid than the control. This difference is once again due to the glucosinolate concentrations between the diets. The results obtained with lambs show that the ingestion of high amount 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 significantly (P < 0.05) higher in thyroid than in lung. It is interesting to notice that 5-VOT has a very high affinity for thyroid. This behaviour can perhaps explain the morphological and the physiological changes in this organ.
Experiment with bulls
The experimentation operated with bulls gave similar results than with lambs. The evolution of the SCN- concentration in the plasma of the four groups was followed each 48 h until a stability. We observed that the values increase regularly in the different lots during eight days and stay relatively constant till the end of the experimentation. The SCN- concentrations were determined at the slaughtering in several matrices. The lower level was found in muscle and the concentration was higher and higher in liver, thyroid, plasma, kidney and lung. In plasma, muscle and organs, the SCN- concentration was significantly (P < 0.05) higher in lots fed with diets containing rapeseed meal than in the control. The concentration of thiocyanates in urine is five times higher than in plasma. These results confirm that kidneys are able to concentrate actively the SCN- ions. The ingestion of high amount of rapeseed meal is connected with the modification of the SCN- concentration in plasma, muscle and several organs. The SCN- concentration increased significantly with the percentage of rapeseed meal ingested. Like with lambs, it can be correlated with the indolyl glucosinolates ingested.
At slaughtering, 5-VOT level was determined in muscle, organs (thyroid, liver, kidney and lung) and biological fluid (plasma). The 5-VOT is present exclusively with the ingestion of rapeseed meal. The rapeseed meal induced a 5-VOT level significantly (P < 0.05) higher in the target organs such as lung and thyroid than the Control. This difference is once again due to the glucosinolate concentrations between the diets. The results obtained with bulls show that the ingestion of high amount 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 significantly (P < 0.05) higher in thyroid than in lung.
The two rapeseed varieties were very different in their content and distribution of glucosinolates. The concentrates (for lambs or bulls) were offered ad libitum and were the main component of the diet to the end of the experiment apart a low quantity of hay.
Despite these conditions, no difference was observed for the animal performances and carcass quality, apart from an hyperthyroidism.
The analyses reveal the presence of the SCN- ionsá in plasma, muscle and some target organs from lambs or bulls fed with diets containing industrial rapeseed meal. The 5-VOT was concentrated in lung and in thyroid. In each case, the amount of SCN- ions and 5-VOT found in the target organs are proportional with the proportion of rapeseed meal introduced in the diets.
This work has been supported by the General Office of Research and Development of the Belgian Agricultural Ministery and by the General Direction of Technologies, Research and Energy of Ministery of "Region wallonne" in Belgium.
Ahlin K., M. Emanuelson and H. Wiktorson, 1994. Acta Veterina. Scandinavia., 35, 37.
Astwood E.B., M.A. Greer and M.G. Ettlinger, 1949. Journal of Biological Chemistry, 181, 121.
Bjergegaard C., P.W. Li, S. Michaelsen, P. M÷ller, J. Otte and H. S÷rensen, 1994. Bioactive Substances Food Plant Origin, 1,1.
Danielsen V., B.O. Eggum, S.K. Jensen and H. S÷rensen, 1994. Animal Feed Science and Technology, 46, 239.
Emanuelson M., K.-A. Ahlin and H. Wiktorsson, 1993. Livestock Production, 33, 199.
Fenwick G.R., R.K. Heaney and W.J. Mullin, 1983. CRC Critical Review of Food Science and Nutrition, 18, 123.
Loft S., J. Otte, H.E. Poulsen and H. S÷rensen, 1992. Food Chemistry and Toxicology, 30, 927.
Mabon N.,á J.-P. Wathelet and M. Marlier, 1998. Talanta (in press).
Maheshwari P.N., D.W. Stanley, J.I. Gray and F.R. van de Voort, 1979. Journal of Americal Oil Chemist Society, 56, 837.
Michaelsen S., K. Mortensen and H. S÷rensen, 1991. GCIRC Congress, Saskatoon, Canada, VI, 1890.
Sch÷ne F., G. Jahreis, G. Richter and R. Lange, 1993. Journal of Science and Food Agriculture, 61, 245.
Wemheuer W., 1993. Reproduction Domestical Animal 28, 385.