' '..' ’_ , ...r ’ ‘. v V V , all; 5 ya ‘ ' 7 K I "\ W’m"'7”t fr II"~‘Im \O-bUOPOl-‘th-J 0):?- [—1 O. OOOOOQPOOO) OOUit-‘mOl-‘bOH \oox Source: Weis (1983); * Includes mustard oil meal Area and production of oilseeds in Nepal from the fiscal years 1975-76 through 1984-85 are presented in Table 4. TABLE 4~AREA AND PRODUCTION OF MUSTARD SEEDS IN NEPAL Fiscal Area Oilseed Mustard Mustard oil cake year (ha) (mt) (mt) (mt) 000' 000' 000' 000' 1975-76 1,13 68.5 58.0 36.0 1976-77 1,08 61.0 52.0 32.0 1977-78 1,33 78.5 67.0 41.0 1978-79 1,44 92.5 79.0 49.0 1979-80 1,18 63.0 53.0 33.0 1980-81 1,22 77.0 66.0 41.0 1981-82 1,14 79.0 67.0 42.0 1982-83 1,10 70.0 59.0 37.0 1983-84 1,10 73.0 62.0 39.0 1984-85 1,28 84.0 71.0 44.0 Source: Agricultural Statistics Division, Food Uses 9; mustard and rapeseeds and their products: and Agricultural Marketing Services (Nepal,1985) Mustard seed is used as a spice in curry, pickles and sauces. It believed to act against the action of molds and yeasts. The major commercial products of mustard seeds are mayonnaise, mustard paste and mustard flour. Brassica nigra is a source of condiments , although it has been largely replaced by Brassica juncea (Vaughan, 1977). Mustard seed oil is the primary vegetable oil used in cooking and also in preserving pickles in Nepal. 1” Oils: Rapeseed and mustard seeds yield 24 to 40 percent of fatty oils depending upon variety, growing conditions and season. These oils are rich in erucic acid ( 22 cal). The oils from cruciferous seeds are composed of about 95-97% triacylglyceroles ( or triglycerides ) and only 0.3-0.5% free fatty acids (Appelqvist, 1971).The fatty acid composi- tion of mustard oil is given in Table 5. TABLE 5. FATTY ACID COMPOSITION (%) OF MUSTARD SEED OIL Fatty acids B; compestris B; juncea Toria Sarson Rai C 16 2.65 1.67 4.56 C 16:1 0.25 0.27 0.18 C 17 trace trace trace C 17:1 II II II I! II N C 18 1.43 1.22 1.37 C 18:1 11.77 12.33 10.74 C 20 7.82 6.96 8.41 C 20:1 0.85 1.64 2.25 C 22 1.22 2.14 --- C 22:1 45.70 48.71 32.22-44.72 C 24 0.51 0.81 0.99 C 24:1 1.96 1.53 2.92 Source: Dutta and Ghosh ( 1970 ) 2. Meals: Sixty to sixty-two percent of the oilseed is made into oil cake. Residual meals are rich source of protein (33- 40 %). These meals are excellent sources of 10 protein for livestock. However, due to the toxic problems associated with these meals, the full potential has not yet been exploited. Expeller-processed MOM contains approximate- ly 10-15% residual oil (Panda and Shrivastava, 1978). Composition of MOM and.RSM Appelqvist ( 1971 ) observed that the existence of crop types with the group names rapeseed and mustard seed is one reason for the greater compositional variation in com- mercial samples of these meals. Composition of MOM and RSM varies with species and within a species among the varie- ties. Protein and oil content of several species of the genus Brassica are given in Table 6. Superior protein quali- ty of E; alba compared to §;;juncea and.§; compestris has been reported (Pathak et al., 1973). Prasad (1978) observed that MOM from i j unce_a_ was little lower in protein com- pared to B; compestris varieties. Double-low cultivars of rapeseeds yield excellent quality protein ( Eggum, 1981 ). TABLE 6. OIL AND PROTEIN CONTENT OF BRASSICA OILSEEDS Percent( dry basis) Brassica oilseeds Oil Protein( N*6.25 ) (3:) Whole seed Defatted meals Brassica napus 44.7 23.8 43.1 Brassica campestris 42.7 23.8 41.5 Brassica juncea 37.2 27.5 43.8 Brassica hirta 28.1 31.2 43.4 Brassica nigra 32.4 31.2 46.2 Source : Miller, et a1. (1962). 11 Gross chemical composition: Extensive reviews of chemical composition and proper- ties of proteins and glucosinolates in RSM and MOM are available 1J1 the literature (Appelqvist, 1971; Shankarnarayana et al., 1972; Bell, 1984). Marquard et al. (1979) observed that various types of mustard (B; juncea, B; nigra and Sinapis alba) grown in different ecological loca- tions differed greatly in yield, content and composition of fats and protein. Proximate analysis of mustard oil meal is given in Table 7. Meals contain approximately 25% hull which lowers the nutritive value of canola (Mitaru et al., 1983). TABLE 7. PROXIMATE ANALYSIS OF MOM Component B; comp. B; jun. Dry matter 91.9 91.80 Crude protein 35.8 33.20 Crude fiber 8.11 9.79 Ether extract 14.43 10.98 Total ash 7.59 9.03 Calcium 0.79 0.75 Phosphorus 1.91 1.79 Source : Prasad (1978) Udaysekharrao and Srinivasrao (1980) found the crude protein and fat content of mustard seeds ( B; juncea ) to be 22.3% and 32%, respectively, which was Similar to the results obtained by Miller et a1. (1962). Zombade et a1. (1980) found crude protein to be 33.9% in MOM. Bell and Jeffers (1976) found the protein content of RSM to be 35.9%. 12 Minerals and their availability: MOM is a relatively rich source of calcium. Although it is also rich in phosphorous, availability is affected by phytic acid and high fiber content. Retention of all mine- rals are adversely affected by phytic acid and crude fiber .(Nwokolo and Bragg, 1977). RSM has been found to bind more zinc compared to SBM ( 83.6% vs. 50.3 ). Removal of soluble polyphenols significantly decreased the zinc binding capaci- ty of RSM (Seth et al., 1975a ). Rapeseed protein concen- trates contain 5¢}#L5% phytic acid assuming 28% phosphorous in phytic acid.(Erdman, 1979L.Shah et al.(1976) observed that adverse effect of Brassica protein concentrates contai- ning high levels of phytic acid on zinc metabolism of young rats can be overcome by zinc supplementation. Metabolizable energy: Lodhi et al. (1969a) found that the available carbo- hydrate in RSM was only 15% compared to 23.6% for SBM using chemical and biological assays. This seemed to affect the ME value of MOM. Muztar and Slinger (1982) using a rooster assay determined the TME of 3‘marieties of canola meals and found that TME ranged from 2.31 - 2.54 cal/g. However, their samples contained only 1,96 to 2.99 % ether extract. Low ME values in RSM are due to its low available carbohydrate (11%) ( Lodhi et al., 1969b; Zombade et al., 1980; Blair and Reichart, 1984), lower protein content , low 13 nitrogen absorbability (Rao and Clandinin, 1972) and high fiber content ( Vose, 1974; Sarwar et al, 1981). Differences in ME values of RSM for different stages of growth of chic- kens have been reported (Lodhi et al, 1969). Expeller processed MOC (13% ether extract) gave ME value of 3017 cal/kg 1J1 ‘white leghorn layers ( Shrivastava et al., 1976b). The ME value of RSM was not found to be affected by progoitrin (Rao and Clandinin, 1970). Significant improve— ment in the ME value of RSM was achieved upon removal of tannins (Yapar and Clandinin, 1972); however, removal of gum had no effect on TME values for chickens (Sibbald, 1977). Levels of inclusion of RSM, age of chicks and the duration of feeding had no effect on the ME values in chicks and roosters ( Bayley et al., 1974 ). ME value ranged from 1340 to 2000 kcal/ kg. ME value for canola meal were found to be 1900 kcal/kg for young chicks and 2000 kcal/kg for adult birds ( Clandinin.and Robblee, 1981 L.Ipdhi et al.(1970a) found ME for RSM to be 1880 kcal/kg for 4 week old chicks and 1800 kcal/kg for layers. Amino acid profile Varietal differences in amino acid composition of rapeseed, turnip rape and soybean have been reported (Soluski and.Sarwar, 1973L.The amino acid compositions of selected Brassica species are given in Table 8. TABLE 8. AMINO ACID PROFILE OF SELECTED BRASSICA SPECIES Amino acids 2; comp. E; hirta E; juncea B; napus B; nigra Lysine 6.048* 5.792 5.360 5.824 4.384 Methionine 1.920 1.552 1.664 1.776 1.504 Cystine 2.432 1.984 2.544 2.432 2.368 Isoleucine 3.792 3.312 3.776 3.648 3.760 Leucine 6.464 6.592 6.320 6.320 6.256 Phenylalanin 3.776 3.728 3.840 3.536 3.744 Tyrosine 2.736 3.296 2.672 2.624 2.640 Threonine 3.984 2.736 4.016 3.840 3.712 Valine 4.912 4.800 4.736 4.816 4.608 Histidine 2.720 2.624 2.576 2.576 2.528 Arginine 5.920 5.392 6.416 5.616 7.552 Source: Miller et a1. (1962). * gm amino acid/16 gm N. Protein quality ip RSM and MOM Protein quality of a feedstuff depends on many factors including the amino acid balance and availability (Wolzak et al., 1981 ). It is essential to consider amino acid balance as well as the quantity of the limiting amino acid proportionality patterns to evaluate dietary proteins. Kies (1981) concluded that bioavailability is secondary to amino acid proportionality patterns in determining the quality of a food protein. The optimum ratio between protein level and the most limiting amino acid is important ( Fisher et al., 1960 ). Anwar and Clandinin (1971) observed that nitrogen solubility in saline or acidic solutions can be used as a measure of relative protein availability'in.different.RSM samples. Solubility of nitrogen of RSM was significantly greater in 6N HCl than in 0.5% NaCl solution ( Seth and Lodhi (1970) observed that nitrogen Clandinin, 1975 ). 15 absorbability was 79.9% for RSM and 85.4% for SBM. Goh et al.(1980) suggested that the solubility of RSM protein in 0.2% KOH is not a good measure of protein quality. They suggested that dye binding capacity of protein was signifi- cantly correlated to TPE values. Haq et a1. (1982) observed that free fatty acids increase rapidly when stored under room temperature, therefore expeller processed oil cakes should be hexane extracted at 40 to 70 C to preserve protein quality. Amino acid availability: Weight gain is a function of the absolute intake of the first limiting amino acid ( Netke et al., 1969 ). As most feed grains are known to have lysine as a first limi- ting amino acid, it is essential to estimate availability values for lysine. RSM is low in lysine and also moderately low in arginine (Klain et al., 1956). Lodhi et a1. (1974) observed that amino acid balance of MOM was better than GNC. Nwokolo et a1. (1976) observed that all essential amino acids except methionine (78.4%) showed high availability, although significantly lower than in SBM; Availability of lysine was 94.4%. Vaidya et al. (1975) found available lysine in MOM to be 3.51 g/16 gm nitrogen. Zombade et a1. (1980) found lysine content to be 3.97 g/16 g N in 14 samples of MOM analyzed. Udaysekharrao and Srinivasrao (1980) observed that Brassica juncea was low in tryptophan (0.5%). Glucosinolates in RSM have no effect on amino acid 16 availability (Salo, 1982; Rao and Clandinin,1978). Available lysine ina.doubLe low cultivar (tower rapeseed) for pig was 79.9% compared to 91.4% for SBM (Pearson and Bowland , 1976). Leslie and Summers (1975) found that arginine is the limiting amino acid in RSM for maximum chick growth, and the addition of arginine and methionine improved chick growth. Purified diets require more arginine compared to practical corn-soy type diets (Snyder et al., 1956). Leslie et a1. (1976) found that in RSM the ratio of arginine to lysine was about 111:1. It was also suggested that arginine is less available from RSM due to the fact that some of the arginine is utilized in the metabolic process involved in tannic acid excretion. Interaction of arginine, methionine and tannic acid has been suggested. It was demonstrated that arginine deficiency could be alleviated by narrowing the protein energy ratio in an arginine deficient diet for chickens (Scott and Forbes, 1958). Procedures 39 determine available lysine The traditional method of determining protein quality in feedstuffs has been digestibility determination. Biologi- cal assays are needed to establish protein bioavailability. However, they are expensive and time consuming. It also requires considerable quantity of samples which are not always available.'Pherefore, chemical methods have been dev- eloped to evaluate protein quality (Wolzak et al., 1981). A mathematical model useful in predicting available lysine due 17 to heat processing was developed ( Wolf et al, 1983). Chick growth assay: A chick growth assay technique for amino acids was developed for the cases in which standard material cannot be fed free from the test material (Hill et al., 1966). Amino acid imbalances can be created using semipurified basal diets (casein - corn starch) and amino acid mixtures (low in one or several.of the limiting amino acid$ or'protein supplements like gelatin (Harper, 1959). Dietary lysine level had no effect on feed intake and relative amino acid balance was without any effect on animal performance ( Cieslak and Benevenga, 1984).The need of correction forin- creased protein levels, to measure available lysine in pro- tein supplements has been stressed (Grau, 1948). However, Gupta(l958) did not observe an appreciable effect on lysine availability by the changes in the levels of test protein. Netke and Scott (1970) used a purified basal diet using crystalline amino acid mixtures deficient only in lysine to study the lysine availability in test ingredients. Dean and Scott (1965) developed a purified amino acid reference diet with and without added casein that gave similar growth as practical corn-soy diet. Carpenter et aL.(1963 used a sesame seed meal basal diet to assay available lysine using chick growth assay. Chick growth assay was more reliable than FDNB ( Flouro—di nitro-benzene ) method. 18 Plasma amino acid level: It was demonstrated that the plasma level of the first limiting amino acid remains at a very low level in the blood irrespective of the severity of the deficiency (Zimmerman and Scott, 1965L.It.was also shown that arginine accumulates in plasma when the dietary level of lysine and valine is low. It was suggested that the plasma level of amino acids may be a good criterion to estimate availability of dietary amino acids. Rapid assay using roosters: Sibbald (1976) developed a method of determining true metabolizable energy (TME) in feedstuffs using adult rooster. In this method adult roosters are fasted for a given period of time (24 - 36 h) and force fed a known quantity of test ingredient and excreta is collected for 24 to 36 h. A pair of roosters are used for endogenous excretion. Feed intake, excreta collected from the roosters on test diets and from fasted roosters (endogenous excre- tion) are used to calculate TME. This method is simple and rapid. The same roosters can be used repeatedly. The TME method has been used for determining amino acid availability in feedstuffs (Likuski, 1978; Muztar et al.,l980). Farrell (1978) developed a rapid bioassay using adult roosters simi- lar to Sibbalds' TME method to determine metabolizable energy. Campbell et a1. (1981) observed negative nitrogen balance in TME determination using roosters. Available ly- sine value for RSM was 83 to 94% similar to SBM (Muztar et 19 al., 1980). Heat damage to SBM protein was detected by rapid assay using adult roosters ( Wallis and Balanave, 1984 ). Chemical assays: The TNBS(2,4,6-trinitribenzenesulfonic acid) method was comparable to FDNB method in the determination of available lysine ( Kakade and Liener, 1969). Good correlation between the values obtained by the FDNB method and the growth assay procedures for available lysine in raw or unheated feedstuffs were observed.IHowever, the FDNB method failed to take into account the rate of digestion and digestible peptides in heat treated proteins. Boctor and Harper (1968) found good correlation between growth assay and FDNB method for determining available lysine. Dye binding assay: A dye binding assay using acid orange 12 before and after treatment with propionic anhydride was rapid, inexpensive and highly reproducible ( Addo and Hill, 1982 ). Goh and Clandinin (1978) found good correlation between dye binding capacity of protein and Kjeldahl's protein values. Goh et al.(1979a) found highly negative correlations bet- ween the dye binding capacity of the protein of RSM and the duration of autoclaving. Hurrell and Carpenter (1979) found that the dye binding technique is sensitive enough to detect the range of first 15% damage in feedstuffs. Goh et al. (1979b) modified the procedure developed by Goh and Clandinin (1978) and recommended it's use in conjunction 20 with Kjeldahl's nitrogen analysis to detect inferior RSM which are severely heat damaged. Goh et al. (1979c) found high correlations between dye binding capacity of RSM and total protein efficiency values of the meals. Anti-nutritive components Bell (1984) reviewed nutrients and toxicants of RSM. Glucosinolates Glucosinolates are a class of secondary compounds present mostly in cruciferous plants.‘General formula for glucosinolates was revised by Ettlinger and Lundeen ( 1956) and then confirmed by them in 1957. Glucsinolates originate from amino acids and are derivatives of thiohydroxamic acids. Excellent review on its biosynthesis and catabolism was done by Underhill (1980). Comprehensive reviews on glu- cosinolates are available in the litrature ( Tookey, 1980: Larsen, 1981; Kjaer, 1981) and specific reviews on analysis are also available ( Rodman, 1978: Olsen and Sorensen, 1981). Glucosinolates are stored in the parenchymal cells of the cotyledons of seeds. They are present in all parts of the plants, however seeds have highest concentration. More than 80 glucosinolates have been identified ( Rodman,1978). All the 300 species of 1500 crucifers analyzed contain 1 to 7 glucosinolates. These differ from one another through a structure of the organic radical or aglucon, the frequency of their presence and the properties of their hydrolysis 21 products ( Maheshwari et al., 1981; Olsen and Sorensen, 1981 ). The majority of cultivated crucifers which contain gluco- sinolates are the source of food and condiments. So far 10 of the 80 identified glucosinolates have been found in rapeseeds. Nomenclature and the presence of glucosinolates are given in Table 9. TABLE 9. NOMENCLATURE AND THE PRESENCE OF GLUCOSINOLATES BRASSICA SEEDS Nomenclature of aglucones Trivial names Presence 3-propy1-(ally1) sinigrin E; jun.I 1B; pigga, g; abyss., B; hirta 3-buteny1 gluconapin B. camp., E; nap., g; abyss. 4-penteny1 glucobrassic- anapin B; camp., B; na . 4-methylsu1finylbutyl glucoallyssin B; camp. G-methylsulfinylpropyl glucoiberin E; nap. 5-methylsulfinylpentyl glucoraphanin By cam . 2-pheny1ethy1 gluconasturtin B; nap., C. abyss. B; camp. (R)-2-hydroxy-3butenyl progoitrin B; camp., B; nap. (S)-2-hydroxy-3-butenyl epi-progoitrin g; abyss. 2-hydroxy-4-penteny1 napoleiferin B; camp., B; na . p-hydroxybenzyl sinalbin By hirta, B; na . Sinapis alba 3-indolyl-methyl glucobrassicin B; nap. Source : Meith, et al. (1983a) In E; compestris , gluconapin and glucobrassicanapin constitute 35-45% of‘ each and progoitrin constitutes about 20%, where as in _B_:_ paws, the major glucosinolate is pro- goitrin (60-70%) and gluconapin and glucobrassicanapin constitute about 15-20% each. Traces of other glucosinolates may be present. _B_. Ma, E; pig and l_3_._ M as well as Crambe abyssinica contain exclusively'allylglucosinolate (sinigrin). The proportion of glucosinolates in species and 22 varieties of Brassica differs considerably and is condi- tioned by environmental factors and changes in the course of vegetation periods. Varietal differences in goitrogenic properties of RSM were observed (Clandinin et a1” 1959 L In general B; papps contain on average, more glucosinolates (4-8%) than B; ggmpgspgis (3-6%). Summer varieties of B; pgpps have lesser glucosinolates compared to the varieties‘ grown in winter. Hemingway and Vaughan (1960) found that mustard seed samples from Nepal contained only allyl isothiocyanate and was identified as B; jppgga. Kirk et a1. (1964) and Jensen et al. (1953b) confirmed that B; jppgea contained only allylglucosinolate (sinigrin). B; jppgea contain quantitatively higher amount of single glucosinolate, however B; ggmpestris and B; pappg contain several types of glucosinolates (Rahaman and Quddus, 1979). Gland et a1. (1981) analyzed 700 genotypes of Brassica and observed that B; papps did not contain sinigrin, where as B; ggmpespgis contain sinigrin. Glucosinolate contents of some cultivars of Brassica are presented in Table 10. In Brassica pappg the main constituent was found to be 3-buteny1isothiocyanate (Jensen et a1” 1953aL 23 TABLE 10. GLUCOSINOLATE CONTENTS OF RAPESEED MEALSa Brassica species and cultivars Total glucosinolates (mg/gm) B; napus Target* 9.50 Tower * 1.29 B. campestris dichotoma( pusa kalyan ) 9.70 T-g, Toria** 17.80 T-42, Yellow sarson** 6.06 B; juncea T-63, Brown mustard** 15.10 Source: Srivastava et al. (1976); a In dry defatted meal; * Canadian cultivars; ** Indian cultivars. Enzyme:(Myrosinase) Myrosinase (Thioglucoside glucohydrolase, EJL3.2¢L1) is a glycoprotein with two peptide chains with a molecular weight of approximately 130,000 daltons ( Tookey, 1973 L Myrosinase in Brassica seeds is stored inaaspecial cell called idioblasts. Bjorkman and Johonson (1972) identified 3 isoenzymes of myrosinase in Sinapis alba. It is now widely believed that myrosinase is a single enzyme system. Myrosinase from Crambe abyssinica and B; napus were stimulated by ascorbic acid (Tookey, 1973; Srivastava and Hill, 1974; West et al., 1977 ) whereas from Sipapi§.alba, it was not responsive to ascorbic acbd( Schwimmer, 19617 Srivastava and Hill , 1974). The enzyme can be extracted along with protein fractions with sodium chloride followed by (NH4)ZSO4 fractionation (Rao et a1” 1978). 24 Aglucons: Aglucones are formed due to enzymatic hydrolysis (Olsen and Sorensen, 1980; Leeson, 1984), when myrosinase front idioblasts and glucosinolates from parenchymal cells come in contact at proper moisture and temperature. Bell et a1. (1971) reported that oriental mustard (E; jppgga) con- tains only allyl isothiocyanate and yellow sarson ( B; ,ggmpggpgig contains butenyl isothiocyanate. Autolysis of most rapeseeds produce nitriles and small amounts of goitrin. However , upon incubation of heat treated seed meals with myrosinase produced only small amounts of nit- riles (Paik, et al., 1980). The detailed description of enzymatic and non- enzymatichydrolysisisgiveninFigure 1 (page 27L In the gastrointestinal tract of laying hens, different products of hydrolysis of goitrin results under different conditions (Smith and Campbell, 1976). Astwood et al. (1949) determined substances in RSM having equal potency of thiouracil for antithyroid activity in man as L-5-viny1-2-thiooxazolidone. Major hydrolysis products of B; jppgga and §;_pig£a plants were allyl and 2-pheny1 isothiocyanates ( Cole, 1976). It was also observed that autolysis of glucosinolates in several crucifers produced 1-cyano-2,3-epithiopropane and 1-cyano-3,4-epithiopentenes, in addition to the l-cyano- 3,4-epithiobutane ( Cole, 1975). In B; pigga and B; jppgga traces of 1-cyano-epithioalkanes were also found. 25 Kameoka and Hashimoto (1980) analyzed steam volatile oils from 1.31 11.1.2222. E. hirta and 131 22222512315 and identified total of fifty five compounds. However the major compounds were allylisothiocyanate, dimethyl trisulfide, 3- butenyl isothiocyanate, 3-pheny1 propionitrile and benzyl isothiocyanate. Matsumoto et a1. (1975) found that B; napus type rapeseed meal contains more oxazolidinethione (OZT) and isothiocyanate (ITC) compared to B; compestris type RSM. Daxenbichler et al.(1979) identified organic nitrile, 1- cyano-2-hydroxy-3-butane in cabbage by autolysis. Mixed samples of B_. compestris and _B_. juncea oil seed cakes con- tained 1.11 to 2.61 mg of OZT/g of meal and 3.18 to 4.85 mg of ITC/g of meal (Vaidya et al” 1975a). Matsumoto et a1. (1975) suggested that antithyroid activity of OZT was much stronger than ITC. Glucosinolate determination: A good review on advances in glucosinolate analysis is available in the literature (Olsen and Sorensen, 1981). Most of the earlier methods used in quantifying glucosinolates in RSM and MOM have been based on the measurement of aglucons produced by enzymatic or non-enzymatic hydrolysis of the glucosinolates. McGhee et a1. (1965) used a sulfur ion balance method based on the principle described by Shaw (1959) to quantify total glucosinolates in mustard seeds. This procedure is simple and.a way to measure total thioglucoside with good 26 accuracy. Spectrophotometric methods have been used to quantify thioglucosides by measuring oxazolidinethione ( Wetter, 1957; Wetter and Youngs, 1976 ) and allylisothiocyanate ( Devani et a1” 1976; Devani et a1” 1978; Mukhopadhyay and Bhattachrya, 1983). Titrimetric method has been used by Croft (1971) to measure total thioglucoside in RSM. Argenti- metric methods have been used in measuring allylisothiocya- nate to estimate the total thioglucosides ( Wetter, 1955; Vangheesdale and Bichot, 1970). Enzymatically released glucose as a: measure of glucosinolates in many crucifers have been used ( VanEtten et al., 1974: vanEtten et al., 1976; vanEtten and Daxenbichler, 1977; Daxenbichler et a1” 1979; Carlson et a1. 1981). More recently chromatographic techniques have been used to quantitatively identify glucosinolates ( Youngs and Wetter, 1967; Daxenbichler et al., 1970; Underhill and Kirkland, 1971; VanEtten et al., 1976; Thies, 1976). Newer methods using HPLC and GLC in combination, and GLC-MS are available to measure intact glucosinolates ( Olsen and Sorensen, 1981). While these methods are effi- cient for isolation, separation and quantification of gluco- sinolates they require more sophisticated equipment. Choice of the method depends upon the purpose of analysis and degrees of accuracy desired, in addition to the accessability. 27 RI R" OH R" / 330+ \ // CH--CH2--C ------- > -------- > CH--CH2--C +HONH3 +sto4 / | | Acidic solution / \ R' | \ S--Glucose R' OH | \ Carboxylic acid I Basic Soln.(OH) + | \ ° Thioglucose Myrosinases Naber type rearrangement | \ | + | \ R" NH3 l \ | | CH--CH--COO- Amino acids l / RI R" \ CH-CH2-N=C=S With unsaturated side chain cyano-epithioalkanes:- R' (Isothiocyanates) ---------------------------- R" N \ ||| CH-CH2-S-C=N C / I R' (Thiocyanates) HO- CH2-H R" | Epiprogoitrin \ / \ CH-CHZ-C=N /___s / R' (Nitriles) N R" III C CH-CHZ-NHZ | / CH2 R' (Amines) | With R" or R'=OH; B-hydroxylated H - C - OH Glucosinolates. | Progoitrin / \ Isothiocyanates upon cyclization /___S give rise to:- Oxazolidinethiones N CH2 III / \ C / \ l R - CH NH CH2 \ / l \ / CH2 0 ----- C | Gluconapin \\ / \ S S Figure 2. Enzymatic and non-enzymatic degradation of Glucosinolates; Adapted from Olsen and Sorensen(1980) 28 Nutritional Implications: The goitrogenic effect of the OZT is due to the fact that it inhibits the synthesis of thyroid hormones which can not be overcome by iodine supplementation ( Gmelin and Virtanen, 1960 ).tGlucosinolates per se are not toxic but the hydrolysis products due to the action of enzyme myrosinase yield toxic allyl isothiocyanates, goitrins and organic nitriles (Ieeson, 1984). In general, effects of glucosinolates on broiler chicks are reduced growth rate, reduced feed intake, and enlarged thyroids and reduced egg production, liver haemor- rhage, enlarged thyroids, reduced egg iodine content and egg tainting in layers (Leeson, 1984L.Toxicity’is closely re- lated to the residual amount of glucosinolates (Elnokrashy et al., 1975). Myrosinase inactivated dehusked RSM was found to contain low fiber and high quality of protein ( Agren and Ekmund, 1977). Bille et a1. (1983) suggested that myrosinase aggravates the toxic and antinutritional effects of some glucosinolates. Akiba and Matsumoto (1979) observed that there is very little contribution of microorganisms in the enlargement of the thyroid in chicks. ' Toxicity in RSM may be attributed to the production of nitriles during hydrolysis of the raw meals ( Lo and Hill, 1972). Josefsson and Munck (1968) found that growth was depressed in mice only when levels of potential isothio- cyanates and oxazolidinethiones exceeded 1 mg/g in the ‘-. m 29 diets. Brak and Henkel (1978) found good correlation between content of nitriles in RSM and repression of feed intake. Weight gain of rats and chicks were inversely proportional to the levels of nitrile rich RSM (Shrivastava, et al., 1975). Allyl isothiocyanate contents of the meals from B; jpncea, & compestris and B; alba were found to be 0.45%, 0.26% and 0.22% respectively ( Pathak, 1973 ). Lodhi et al. ( 1980 ) suggested the presence of goitrogenic compounds that are not reversed by iodine supplementation. Lodhi et al. (1970b) found that up to 0.054 to 0.069% OZT in the diet had no effect on ME for 4 or 6 week old chicks, however at 0.09% level, ME value slightly decreased. Erucic acid CH3(CH2)7CH==CH(CH2)llCOH Erucic acid is an isomer of docosanoic acid and is a characterstic acid of mustard and rapeseed oils. Fogerty et a1. (1978) confirmed that rapeseed oil contains only the cis form of 13-docosanoic acid (erucic acid). Oleic acid is a precursor of eicosenoic and erucic acids, in their biosyn- thesis (Downey and Craig, 1964; Rahaman and Quddus, 1979). Conventional varieties of rapeseed and mustard seed yield oils containing 40-50% erucic acid (C22:1) and 8-10% eicosenoic acid (C20fin ( Dutta and Ghosh, 1970: Spencer, 1975: Srivastava et al., 1976b;Prasad and Rao, 1977). The amount of residual oil left in the meals depends upon the 30 Inethod of oil extraction used. A higher percentage of oil in the meal may mean higher percentage of erucic acid in the meal, however varietal differences are to be taken into account. Usually 10-15% residual oil is left in the meals by expeller process ( Panda and Shrivastava, 1978 ), which amounts to 4 to 6% of erucic acid. Ruckemann (1978) developed a rapid method of determining erucic acid in oils similar to the method prescribed in AOAC (1985). Nutritional implications: Nutritional properties of rapeseed oil have been extensively reviewed by Slinger (1977) and effect of dietary erucic acid by Prasad and Rao ( 1977 ). Feeding of rapeseed oil with high erucic acid as a major source of dietary energy resulted in reduced growth and undesirable histopathological changes in the cardiac and skeletal muscles in rats ( Abdellatif and Vles, 1970; Bhatnagar and Yamashiro, 1979) and ducklings ( Abdellatif and Vles, 1970; Abdellatifet al.,1972).The main deleterious factor in rapeseed oil is erucic acid ( Chand and Sadgopan, 1980). Erucic acid in mustard and rapeseed oils may inhibit mitochondrial oxidation of other fatty acids causing accumu- lation of triglycerides and cholesterol esters. Lipidosis is the acute effect of mustard oil feeding in rats( Ray et a1” 1979 ). Incorporation of erucic acid in the test diets at the levels provided by 20 and 30% expeller processed MOM adversely affected the performance of chicks in as much the 31 same way and almost to a similar extent as the MOM diets (Prasad et al., 1978a). Similar results were obtained by Prasad and Rao (1977). It was suggested that the primary factor limiting the utilization of MOC in poultry is the presence of erucic acid (Prasad, et al., 1978a). Prasad and Rao (1982) suggested that the erucic acid may be an impor- tant deleterious agent adversely affecting chick performan— ce. They observed improvement in chick performance equiva- lent to GNC fed chicks upon hexane extraction and recconsti- tution of mustard oil cake. Lall and Slinger (1973) found that when B. napus or B. compestris meals were fed to the layers, erucic acid content in the egg yolk was 0.6%, whereas low erucic acid rapeseed oil produced eggs contai- ning only 0.2% erucic acid in the egg yolk. Mustard seed of Czech origin was found to contain significantly lower amounts of erucic acid compared to its fatty acids of glycerides (Pokorny and Zeman, 1971). Appre- ciable variation in erucic acid content among the varieties of mustard seeds from India was reported by Kharchenko (1969). Phenolic compounds The presence of 'phenolic fractions' is a characterstic feature of all plant tissues. They all posses an aromatic ring bearing a hydroxyl substituent. The major phenolic constituents of Brassica and Sinapis oilseeds is sinapine, a choline ester of sinapic acid ( Kozlowska et 32 al., 1983). Smaller amounts of p-coumaric acid, ferrulic acid, p-hydroxybenzoic acid and caffeic acid are also present in rapeseed ( Durkee and Thievierge, 1975). Free phenolic acid was not found in mustard and rapeseed meals. Chlorogenic acid in B; compestris has also been identified. Structures of some of the phenolic acids are given in Figure 2. Ismail and Eskin (1979) measured sinapine in rapeseed protein concentrates using colorimeter requiring titanium tetrachloride. MeO ______ \_____ HO___// \\__CH=CHC02H HO__// \\__CH=CHC02H \______/ \_____/ / MeO ( sinapic acid ) ( p-coumaric acid ) HO MeO \_____ \_____ HO // \\ CH=CHC02H HO // \\ CH=CHC02H ( Ferrulic acid ) ( Caffeic acid ) HO \_____ HO // \\__COZH \ / ( p-hydroxybenzoic acid ) Figure 3. Some of the phenolic acids present in RS and MS Tannin is an important polyphenol present in rapeseed and mustard” There are two types of tannin viz. condensed tannins (derivatives of flvanols; (C6-C3-C6)n) and hydroly- zable tannins ( esters of a sugar, usually a glucose with one or more trihydroxybenzene carboxy aciw (Gross, 1981). Hydrolyzable tannins are mixtures of gallic acid, megallic acid, trigallic acid and galloylated glucose. The chemistry 33 of tannin is most complex and non uniform ( Harborne, 1980 ). Vaidya et al.(1975a) found that MOM contained 2.05 to 3.74% of tannic acids. Nutritional implications Phenolic acids in the seeds cause deterioration in taste, odor and color of protein concentrates. Oxidized phenolic compounds bind with essential amino acids such as lysine and methionine forming complexes which are unassimi- lable in the gastrointestinal tract of animals and men. sinapine level more than 1% in the diet was found to adversely affect palatability ( Josefsson and Uppstrom, 1976). sinapic acid contributes to the bitter taste of rapeseed flour ( Rutkowski and Kozlowska, 1979 ). Tainted eggs ( fishy odor in eggs ) laid by some strains of layers laying brown shelled eggs have been linked to feeding of RSM in the diets (Hobson-Frohock et al., 1977; Pearson et al., 1979 ). Fishy odor is produced by trimethylamine ('FMA ). Feeding of RSM in the diets of tainters resulted in tainted eggs and deletion of the meal from the diets halted the production of tainted eggs ( Overfield and Elson, 1975). Hobson-Frohock et a1. (1975) devised a method to select the tainters from nontainters to study the effect of RSM feeding on egg taints. Goh et a1. (1983) found that when constant amount of sinapine is fed to the layers, OZT not the total glucosinolate tend to increase %age of fishy eggs. 34 Hepatic TMA oxidase is the enzyme that oxidizes TMA in itfls excretory form. Hepatic TMA oxidase activity was found to be lowered by feeding RSM to the hens (Pearson et al., 1979a). It was demonstrated that OZT in RSM is an important factor in producing tainted eggs in sensitive hens, along ‘with thyroid enlargement (Pearson et al., 1979b). Pearson et al. (1980a) demonstrated that even the feeding of low gluco- sinolate RSM ( cv. tower ) could depress TMA oxidation in sensitive hens and result in tainted eggs. Pearson et al. (1980b) ruled out that sinapine is involved.in.theidepres- sion of TMA.oxidation, when RSM is fed.as a sole source of TMA in the diet. It was observed that unabsorbed choline in the intestine is associated with TMA production by intesti- nal bacteria ( March and McMillan, 1980 ). Lee et al. (1982) could not confirm that the tainters are less efficient at excreting TMA.oxide..It was concluded that progoitrin is the major constituent of B; compestris meals for the potentials of tainting ( Pearson , 1983). Addition of antimicrobial drugs to laying rations containing RSM is unlikely to reduce TMA content of egg produced by the tainters (Goh, et al., 1982a). Growth was inhibited more by hydrolyzable tannins than by condensed tannins (Joslyn and Glick, 1969). Gallic acid and catechin depressed growth, however ellagic acid was without any effect. None of these compounds effected fecal nitrogen excretion (Glick and Joslyn, 1970a). Toxicity of tannin decreased with increase in age and weight of rats 35 (Glick and Joslyn, 1970b). When 5% tannic acid was in the diet, supplementation with choline or methionine did not improve growth whereas supplementation of casein improved growth indicating that effect of tannins may be reduced at higher protein levels. Prasad and Rao (1980) did not find any adverse effect of tannin components, on chick growth and feed efficiency singly or in combinations, at the levels present in 20 and 30% of MOM in the diets. As low as 0.5% dietary tannic acid caused a depression in growth of chic- kens and 70% mortality occured in 7 to 11 days at the 5% dietary level (Vohra et al., 1966). They also found that ME was also reduced along with reduced nitogen retention. Cho- lesterol level was also increased. Egg production and health of hens were not adversely affected by sinapine content in the diet ( Overfield and Elson, 1975 ) nor was growth depressed in the hens fed RSM ( Clandinin, 1961a). Prasad and Rao (1982a) found that tannic component had little or no effect on the utilization of MOM in chicks. Other toxic factors An anti thiamine factor was identified as 3,5- dimethoxy-4-hydroxy cinnamic acid ( methyl sinapate )knr Bhattacharya and Chaudhary ( 1974 ).Zemen et a1. ( 1964 ) observed the evidence of the presence of an unknown component presumably an unsaturated hydroxy acid in RSM. 36 Detoxification 9; MOM and RSM: Extensive review on physiochemical, microbiological and enzymatical treatments of brassica seeds on antinutritional components is available in the literature (Meith et al., 1983b). The techniques thus far developed can be broadly classified under the following categories (Maheshwari et al., 1981): J" Inactivation of the enzyme myrosinase The intact glucosinolates are not removed during oil extraction but toxicity prevails only if they are hydrolyzed 531theldigestive‘tract. Myrosinase inactivation starts at temperatures above 70 C if the moisture content is 6 to 10%. 2. Hydrolysis and removal of hydrolytic products Volatile isothiocyanates can be steam stripped however, oxazolidinethione and other non volatile products of autolysis ( enzyme hydrolysis ) remain in the meal. Autolysis starts at 45 to 50 C in the presence of high moisture content. 3. Removal of glucosinolates and their hydrolytic products: Glucosinolates are highly soluble ixi‘water. diffusion extraction from whole seeds prior to hydrolysis by myrosinase is effective in detoxification of RSM ( Rutkowski and Kozlowska, 1979 ). Dry heat treatment at 80 to 90 C for 15 to 30 minutes inactivates myrosinase and leaves intact glucosinolates in tflua meal. After inactivation of mW'uaa-fi-w ‘— 37 :myrosinase, treatment with 0.01 N NaOH at 60 C removes most of the glucosinolates (Soluski, 1972). Five to six 1-hr diffusion extraction of intact seeds of rapeseed also removes most of the glcosinolates. Inactivation of myrosinase by boiling water was effective. However, there is nutrient loss whenever there is aqueous leaching. 4.P1ant breeding techniques to breed out glucosinolates: In Canada and European countries newer varieties of rapeseed have been developed with low levels of glucosinolates and low levels of erucic acid. They are called double low cultivars. Varieties of rapeseed that are low in erucic acid ( <1% ) are available however varieties having zero glucosinolates are not yet available. Effect 9: processing: Processing techniques influence the bioavailability of protein, some positively and some negatively (Kies, 1981). Solvent processed meals supported 15% higher weight gain and 10% more efficient feed to gain ratio in rats (Goering et al., 1960). Prepress solvent processed RSM is nutritionally superior to expeller processed meal which may be due to the fact that lysine damage is spared in solvent processing (Clandinin, 1967). The nutritive properties of rapeseed meals are considerably improved if the glucosinolates are removed by autolysis followed by solvent extraction ( Mukherjee et a1” 1976 ). When 70% actone extraction and autolysis were 38 compared, both gave satisfactory results, however, acetone washing was more effective (Mukherjee, et al., 1979). Excessive heat treatment during the oil extraction adversely affected nutritional value of resulting meals (Clandidnin et al., 1959). Yule and Mcbride (1978) observed that solvent processed meal was nutritionally superior to expeller processed RSM. Cooking temperature of rapeseed should be such that at least 6% residual oil is left in the resultant meals to get good quality meals (Clandinin and Tajcner, 1961b). Removal of glucosinolates prior to oil extraction gives good quality meals ( Mustakas et al., 1962 ). There was a 30.69% reduction in lysine and 22.92% reduc- tion in arginine by open pan roasting of mustard seeds prior to oil extraction ( Capper et al., 1982 ). Effect of open pan roasting on essential amino acid composition of mustard seeds prior to oil extraction in is given in table 11. Eapan et a1. (1968) found that wet heating of RS prior to oil extraction produced better quality of both oil and meal. Dry heating was unsatisfactory in inactivating myrosinase and oil color; Lower temperature cooking (98.8 C for 30 min) gave higher lysine content and higher fat content in the resultant meals (Clandinin and Tajcner, 1961b). Two hour heat treatment of RS at 90 C caused no change in glucosinolate whereas a 12.38% reduction occured when heated at 150 C for 2 h (Akiba and Matsumoto, 1979b). No adverse effect of treating mustard seeds at 125 C for 30 39 min prior to oil extraction was observed, on metabolizable energy value for the growing chicks (Vaidya et al., 1975b). TABLE 11..AMINO ACID COMPOSITION OF ROASTED AND UNROASTED MUSTARD SEED CAKE Amino acids Unroasted Roasted Arginine 2.40 1.85 Lysine 2.15 1.49 Histidine 1.01 0.85 Isoleucine 1.74 1.49 Leucine 2.60 2.31 Phenylalanine 1.51 1.35 Methionine + Cystine 1.01 0.82 Threonine 1.86 1.49 Tryptophan 0.46 0.50 Valine 1.98 1.81 Source: Capper, et al. (1982); *DMB Heat treatments: Jensen et al. (1975) suggested that it is the effec- tiveness of heat penetration that is important in the inac- tivation of antigrowth factors in soybeans rather than the moisture levels. Higher temperature of oil extraction is responsible for the impared digestibility in pigs fed expeller proces- sed RSM ( Salo, 1982 ). Mild heat treatment are more effective in detoxification than severe treatment (Garcha et al., 1976). Heat treatment of the seeds prior to the oil extraction did not show any variation in the tannin content and OZT, however ITC content was lowered.(‘Vaidya et a1” 1979 ). 40 Wet heat treatment: Many researchers have shown that various types of heat treatments improve the nutritive quality of plant proteins (Olsen et al., 1975; Bayley and Summers, 1975; Srihara and Alexander, 1983; Srihara and Alexander, 1984). Thermal processing affects protein quality by rendering the basic amino acids especially lysine unavailable ( Wolzak et al., 1981). Increasing moisture content.of RSM from 12 to 40% before autoclaving improved chick growth ( Hijikuro and Takemasa, 1979a ). Goitrin content was almost eliminated and the destruction of lysine and arginine were also low, when RSM was autoclaved at 120 C for 60 min at 30% moisture level (Hijikuro and Takemasa, 1979b). Myrosinase activity of rapeseed was completely destroyed by the heat treatment at 90 C for 15 minutes at 8% moisture level (Appelqvist and Josefsson, 1967). Progressive decline in available lysine value in RSM was reported upon autoclaving at 120 C from 15 to 120 minutes ( Rayner and Fox, 1976 ). Steam heating of RSM reduced nutritive value, However, there was lesser destruc- tion of nutritive value when compared to ammoniation (Lipinska et alu,1978). Steam heating of RSM ( cultivar start ) supported growth similar to SBM diets ( Bock et al., 1981 ). Chickens fed CM desolventized at 100 C (after oil extraction) suppor- ted faster growth rate compared to chickens fed CM desolven- tized at 25 (Shires et al., 1982). Steam volatalization of 41 ITC after enzyme hydrolysis gave satisfactory RSM ( Goering et al., 1960 ). Steaming at 100 C for 30 minutes ensured no loss in available lysine and good quality RSM ( Rauchberger et a1” 1979 ). Wet heating of the rapesseds prior to oil extraction produce better quality of the meals and oils, whereas dry heat treatment was unsatisfactory in inactivating myrosinase (Eapan et al., 1968). Autolysis of RSM at 37 C for 1-hr followed by steaming at atmospheric pressure for 1-hr im- proved the feeding value (Srivastava and Hill, 1976). . Autoclaving of RSM at 15 psi improved chick growth independent of lysine destruction and total lysine was reduced by 24-36%, whereas dry heating at 115—120 C showed only little damage in lysine (Gray, et al., 1957). Dry heat treatment: Heat treatment of RSM at 150 C for twohours reduced physiologically active goitrin from 20.32 mg/100 gm of meal (in unheated RSM) to 5 mg/100 gm of meal (in heated meal). At 90 C no glucosinolate reduction occurred ( Akiba and Matsumoto, 1979a). Results of thermal detoxification are given in Table 12. Severe over heating of protein supplemental feeds result in seriously depressed availabi- lity of all amino acids , however lysine is more sensitive to heat ( Meade , 1972 ). Prolonged heating of MOM at higher temperatures and lower moisture contents were detrimental to the nutritive 42 value ( Mustakas et al., 1965 ). Optimum nutritive value of low glucosinolate RSM was obtained by heat treatment of the seeds with moisture content of 8% at 100 to 110 C for 15 to 60 minutes (Josefsson; 1975). Heat treatment of RSM at 100 C was more effective than at 90 C for chick growth, however, at 10 to 15% moisture level most of the lysine was destroyed and at higher moisture ( 30 - 40% ) methionine was also destroyed ( Josefsson and Munck, 1972 ). Hot water treatment: Hot water treatment of RSM removed a goitrogenic factor from the meals (Nakaya and Tagannis, 1965). Feeding hot water treated RSM up to the 30% level had no adverse effect on chick growth and no adverse effect on iodine content in the thyroids ( Nakaya, 1965 ). Microwave treatment: Microwave treatment of dehulled RSM for 1m5 minutes with 10 or 13% added moisture in the meals reduced some glucosinolates, however, goitrogenicity of the meal was not different from untreated RSM ( Maheshawri et al., 1980 ). Optimum moisture contents for the microwave treatment for the inactivation of myrosinase was found to be 14 to 16% (Medeiros et al., 1978). Marked improvement in the nutri- tive value of whole rapeseed by microwave treatment occurred at 329-399 C for 5-15 seconds ( Woodly et al., 1972 ). 43 Water treatment: Two extractions of RSM in water did not improve PER in rats nor was there any change in liver histology , however thyroid histology was changed (Ballester et al., 1977). Double water extraction with water was found to be more effective in reducing OZT and ITC ( Ballester et al., 1970b ). Diffusion extraction of glucosinolates was found to be effective in reducing glucosinolates ( Bock et al., 1979 ). After aqueous ethanol extraction of rapeseed, hot water washing of RSM three times reduced the glucosinolate content of the meal by 95% of raw untreated meal( Ekmund et al” 1971a ). Protein efficiency ratio of such meal was found to be 2.9 compared to 2.1 for the meal washed only once ( Ekmund et al., 1971b ). Jones described a water extraction procedure that removes 90% glucosinolates and the resultant meal was also rich in amino acids. Mustakas et al. (1976) described a procedure to remove glucosinolate by water was- hing. Kozlowska et al. (1972a) found that aqueous extraction of glucosinolates from RSM resulted in loss of soluble nitrogen and other nutrients. At 20% MOM dietary level, chick growth improved upon MOM treatment with hot water, whereas cold water treatment of MOM depressed growth. At 30% level, however, both the treatments depressed growth signi- ficantly (Prasad and Rao, 1978). 44 TABLE 12. DETOXIFICATION OF MOM BY THERMAL TREATMENT Treatments Conditions of Treatment Reduction in Temp. Moisture Time Myr. ITC OZT [ C ] [ % ] [ min] % % % 1.Heating of 80 3.6 15 100 defatted 90 3.6 15 92 meal in a 80 6.4 15 34 closed system 90 6.4 15 6 in presence of 80 8.4 15 31 water 90 8.4 5 4 80 7.4 15 40 2.Heating of deftd. meal in open syst. 105 7.9 60 83 in presence of 130 7.9 60 36 water 3.Thermal treatmt. 90 5 10 0.8 1.2 of deftd. meal 90 120 4 0.8 1.0 in presence of 95 5 10 0.9 1.1 8% moisture 95 120 0 0.7 0.9 100 5 4 0.9 1.1 100 120 0 0.6 0.8 105 5 7 0.9 1.1 , 105 120 1 0:5 0.8 4.Toasting or steaming of 105 30 0.58 0.39 deftd meal 135 30 (L34 0.13 105 90 0.56 0.07 135 90 0.27 tr. 105* 30 0.37 0.33 135* 90 0.13 0.23 5.Autoc1ave trt. of deftd. meal 121 20 6.2mg/ ( toria 29mg 100 g per 100 g ) 121 30 2.3mg/ 100 g 6. Hot water trt. (sarson 20.4 mg 100 20 3.1 mg/ per 100 g ) 100 g 100 30 4.4 mg/ 100 g 7.Roasting of deftd. 105 0.27 0.51 meal ( OZT=0.88; 115 0.24 0.46 ITC=O.61) 125 0.16 0.38 135 0.04 0.31 Sources:1 & 2, 7,Meith et a1 (19 Applequist and Josefsson (1967); 3, Josefsson (1975); 4, Meith (1983);5 & 6, Garch et a1 (1976) 83). * Steaming 45 Chemical treatments: MOM treated with ferous sulfate reduced oxazolidinethione content by 88% and isothiocyanate by 74% ( Daghir and Mian, 1976 ) and the ME value was also improved ( Daghir and Charlambous, 1978 ). Bell et al. (1971) found that ferous sulfate treated mustard had no allylisothio- cyanate. Mild alkali or acid treatment of oilseed meals were more effective in detoxification than severe treatments ( Garcha.et a1” 1976 L.Sodium.hydroxide treatment elimi- nates myrosinase activities.( Bhatty and Soluski, 1972 ). Autolysis or washing with 70% acetone were satisfactory in removing glucosinolates (Mukherjee et a1. 1979). RSM treated with 2% soda ash supported significantly higher weight gain in pigs compared to pigs fed untreated RSM at 5%leve1, of inclusion in the diets (Newman, et al., 1973). Beneficial effect of treating mustard meal with 3.8% sodium carbonate in swine and mice diets were reported (Sarwar and Bell, 1980). They attributed this improvement to the removal of sinapine. Ammoniating RSM in the presence of steam in the solventization process decreased sinapine and oxazolidinet- hione contents in the resulting meals (Goh et al., 1982b). .Ammoniation of mustard meal improved chick performance up to the 15% dietary level. However, at the 20% level there was a need to maintain lysine levels to improve performance (Blair, 1984). Ethanol treatment of RSM at 60 C completely destroyed myrosinase and more glucosinolates were removed (Josefsson and Munck, 1972). Kirk et a1. (1971) observed 46 that salts of iron and copper and to some extent zinc, promoted destruction of epi-progoitrin in crambe seed meal. Ammoniation is believed to reduce glucosinolates in RSM (Keith and Bell, 1982). Feeding values pf MOM and RSM Ip ruminants: Increase in thiocyanate ions in milk from cows fed RSM was reported (Rutkowski, 1971; Iwarsson, 1973; Papas et al., 1979a). Thyroid weight was increased and 24 hr thyroid radio-iodine uptake as well as lowered serum protein bound iodine.(PBI) occured in rats fed milk.from cows fed RSM in the diet (Iwarsson and Nilsson, 1973). Iodine concentration in milk of cows fed RSM was also observed ( Iwarsson, 1973). Depression in appettite and off flavour in milk from cows fed RSM at the rate of 1,2 kg/day was reported (Eggum, 1981). RSM at 14% level in grain mixture fed ad libitum to lactating cows reduced feed intake without depressing milk production, however, at the 19% level in the grain mixture RSM significantly depressed milk production (Ingalls and Sharma, 1975). A case of toxicity in bull (Allikutty, 1976) and in a she buffaloe (Shreemannarayana, 1976) have been reported due to over consumption of mustard seeds. Iwarsson et al. (1973) found enlarged thyroids in bulls fed 15% RSM which was significantly different from the control group. 47 Lindell (1981) observed no adverse effect of 10% RSM in lactating cows. Moss (1972) suggested that SBM can be replaced by RSM in the grain diets without any serious problems for dairy cows. Varietal differences in RSM were observed in calf rations (Schingoethe, et al., 1974). Fee- ding of 3% rapeseed gum had no adverse effect on apparent digestibility of nutrients, rate of gain, feed intake or feed efficiency of steers (Mathison, 1978). Wintering beef cattle can tolerate replacement of SBM by mustard meal in their diets, in lamb however, MOM feeding decreased wool growth (Peterson and Thomas, 1970L Addition of 20 to 30 % RSM in potato silage resulted in high quality silage as bacteria and yeasts were inhibited (Czarnocka-Roczniakowa et al., 1972; Kozlowska et al” 1972a). It was also suggested that glucosinolates in RSM were substantially reduced by lactic acid in silage (Kozlowska et a1” 1972bL Formaldehyde treatment of RSM was found to be beneficial on growth when fed to cross bred calves (Bedi and Jain, 1978). Palatability of RSM as a protein source in grain mixtures, depends on the levels of inclusion of RSM, the type and levels of glucosinolates, the process to which RSM has been subjected and also the age of the animals ( Thomke, 1981 ). Ten percent high-glucosinolate RSM and up to 25% low-glucosinolate RSM could be incorpo- rated in dairy rations without any adverse effect ( Thomke, 1981). 48 1p nonruminants: Swine: Feed intake was inversely proportional to glucosinolate content of the diet in swine feeding ( Lee and Hill, 1983 ). Regent rapeseed (full fat) were found to be a little better than candle seed for swine ( Salo, 1980a ). ME for regent was 4841 kcal/kg of DM and for candle it was 4473 kcal/kg. Replacement of SBM by erglu cultivar of RSM in pig rations did not affect growth (Peterson and Sculz, 1976). Pearson and Bowland (1976) concluded that double low culti- vars of rapeseed yield RSM that can substantially replace SBM in the diets. Singam and Lowerence (1979) observed that tower and erglu RSM were less acceptable than SBM, when offered abruptly to the young growing pigs. Candle RSM was 30% better in nutritive value for pigs compared to span- torch variety of rapeseed ( Salo, 1980b ). Palatability in young pigs was found to be a problem when 20% RSM is incor- porated in swine growing rations (Salo, 1982). Rundgren (1983) found that 10% low glucosinolate RSM can be incorpo- rated in sow rations, 50% in growing finishing ration and only 5% in growing finishing rations. It was also observed that palatability is the main limiting factor in starter rations. Eggum (1981) observed that pigs were less affected by the glucosinolates in double low cultivars than poultry or ruminants. Depressed serum thyroxin level and possibly thyroid dysfunction in pigs fed high and low glucosinolate RSM, 49 were observed however, it was much severe in the case of the group fed high - glucosinolate RSM (McKinnon and Bowland, 1979). No adverse effect of incorporation of 10% rapeseed gum in a 15% gum free RSM was incorporated in swine diets (McCuaig and Bell, 1981). Upto 15% RSM (cultivar candle) can be incorporated in pig rations without any adverse effect (Bell, et al., 1981). Bayley et al.(1969) suggested that prepressed solvent processed RSM may be fed at 11% level in a corn-soy diets supplemented with methio- nine and lysine in finishing swine without any adverse effect. Ammoniation is generally believed, to reduce glucosinolates, however no advantage was observed in feeding ammoniated RSM to pigs (Keith and Bell, 1982)..Ammoniated canola was inferior to steam treated RSM (Keith and Bell, 1983 ). Poultgy: Growing chicks: There is lack of unanimity in the replacement value of RSM or MOM for SBM or GNC in poultry rations. Five or ten percent of MOM (sarson) heated or unheated, did not adverse- ly affect chick growth, however at 15% level both the treat- ments lowered the gain significantly compared to GNC-refere- nce diet (Vaidya.and Panda, 1974). MOM at 20% or more in chick starter diets adversely affected the performance of chicks and it was suggested that erucic acid may be the primary factor limiting the 50 utilization of MOM in poultry diets ( Prasad et al., 1978a). Incorporation of MOM or RSM at the 10% level had no adverse effect on chick growth (Srivastava and Prasad, 1976; Marangos et al., 1974; Leeson et al., 1978: Matsumoto et al., 1979). Replacement of GNC by MOM up to 80% level had no adverse effect on growth and feed efficiency of growing chicks (Benerjee et al., 1975; Benerjee et al., 1976). No adverse effect of replacing GNC by MOM in chick starter diets was reported (Prasad et al., 1974; Ballester et al., 1970; Mandel et al., 1981). Good quality of RSM protein was suggested by Ballester et al. (1970a). Replacement of upto 50% of SBM by RSM had no adverse effect on chick growth (Hijikuro and Takemasa, 1979c). Significant decrease in slaughter weight of broiler chicks fed 10% RSM was observed (Kozlowski et al., 1981). Up to 15% of high-glucosinolate RSM and up to 20% of low-glucosinolate RSM have been recommended (Clandinin and Robblee, 1981). Incorporation of 1.5% gum in the diet did not affect chick growth ( Summers and Leeson, 1977; March and Soong, 1978 ). Replacement of 35% SBM by RSM in chick diets depressed growth by 25% ( Kratzer, et al., 1954 ). Yule and Mcbride (1978) observed that even 10% ( expeller processed ) or 15% ( solvent processed ) RSM depressed growth and decreased feed efficieny in growing chicks. Replacement of GNC by expeller processed MOC at 50 or 100% in chick rations lowered feed 51 intake and chick growth (Sadgopan et al., 1983). Even inclusion of heat treated RSM at 20% level resulted in lower rate of gain compared to 10% heated RSM in broiler ration (Olomu et al., 1973). Disagreement in toxic effect of sinigrin in MOM was put forward by Panda and Pradhan (1966). They observed significantly better growth in chicks fed MOM diet as compared to chicks fed GNC. Progressive decline in growth of chicks upon increa- sing levels of RSM in the diet was reported (Klain et al., 1956). Incorporation of 20% Midas variety of RSM in the diet decreased growth rate and it was lesser for heat treated meal (Paik, et al., 1981). Incorporation of 15% Tower rapeseed meal did not adversely affect the performance of broiler chicks (Summers and Leeson, 1978). Upto 5% canola seed bulls in broiler diets did not adversely affect growth and feed effeciency (Mitaru, et al., 1983). Internal Lesions: Haemorrhagic liver syndrome in white rock chicks fed diets supplemented with 50% RSM and 50% full fat rapeseed rere reported (Yamashiro et al., 1977). Perotic hock joints n young turkeys, fed 25% RSM ( cv. target ) in the diet- ere reported (Moody et al., 1978). Perotic effect on .icks fed 30% RSM is attributed to factor(s) other than itrogens (Holmes and Roberts, 1963). Thyroid size for chicks fed Midas was 4 to 20 mes greater than for controls,~whereas Tower rapeseed 52 resulted in only a 2 to 3 times greater thyroid size Toxic effects of MOM may be confined to young poultry (Prasad, et a1” 1973L Responses to RSM feeding may vary from one genotype of chicken to another'(Proudfoot et a1” 1983 ). Palatability: Summers et a1. (1982) suggested that palatability could be the part of the problem of inferiority of canola seed compared to soybean meal diets. Palatability is the problem in young birds and they adapt to RSM feeding after about 3 weeks ( Woodly et a1” 1972 L Laying Eggs: Egg production: No adverse effect of replacing 100% of crude protein of GNC in layers ration by RSM ( Brahmakshetriya et a1” 1975 ). Shrivastava et al. (1975b) observed significant reduction in egg production even at 10% level of MOC inclusion in the diets. There was progressive decline in egg prodution by increasing levels of MOC. RSM in layers reduced egg production (Summers, et a1” 1971; Marngos and Hill, 1976). Complete replacement of GNC by MOC in layers diet reduced egg production, increased the numbers of days for the first egg laid and decreased the percent hatchability from the fertile eggs (Agarwala, 1984). Incorporation of 1.5% gum did not affect the egg production in laying hens (Summers et al., 1978). Egg 53 production was depressed by feeding 12% RSM in the diet (Kubota, et al., 1972). RSM (i papifi) at 12% level in layer ration depressed egg production and reduced egg white (Marangos and Hill, 1976). Even 10% MOM reduced egg produc- tion significantly in layer rations (Shrivastava et al., 1976a). Significant reduction in egg production by replace- ment of GNC from GNC-based diet by 10 or 20% MOM. along with deterioration of internal quality of egg was also reported (Singh and Agarwala, 1975). RSM up to 17% in the diet of starting and growing pullets could be fed without any adver- se effect in growth and subsequent egg production (March, et al., 1975). Five to 10% RSM (cv. candle) can be incorpo- rated in layer diets without any adverse effect in egg production (Slinger, et al., 1978). Double low cultivars of rapeseeds (low in glucosinolates and low in erucic acid) yield excellent quality rapeseed proteins (Eggum, 1981). No adverse effects of replacing GNC by MOC at 50 to 100 % level were observed in pullets whereas in layers egg production and feed efficieny was depressed (Sadgopan, et al., 1983). Internal Lesions : Haemorrhagic liver syndrome occurred in laying hens fed 15% RSM (cv. span) in the diet (Leeson et al.,1976). Histological studies of thyroid gland revealed that thyroxine biosynthesis was inhibited by feeding RSM to 54 chickens (Summers et al., 1971, Summers et al., 1978) and it was suggested that growth depression and hyperthyroidism in birds fed RSM in the diet may not simply be due to reduction in serum thyroxine concentration (Summers et a1" 1977). Hypertrophy of thyroids were reported when 12% RSM was incorporated in layer rations (Marangos and Hill, 1976). Intact glucosinolates in hen diets produced moderate in- crease in thyroid size, which further increased in size upon addition of myrosinase (Papas, et al., 1979b). Palatability: The presence of an appetite depressing factor has been suggested as there was a significant decrease in feed consu- mption upon increasing levels of run: in the diets (Srivastava et al., 1975b). Palatability was not a problem in laying and breeding hens (O'neil, 1957). Layers adapted to a 20% level of RSM after adjusting to the initial phase of RSM feeding'oJacksonq 1969L.Protein.supplement.contai- ning 10% RSM was satisfactory in turkey breeder diets (McGregory 1960. Justification for proposed study: Feeding value: Thereis no agreement amongst the researchers as to how much of MOM can be satisfactorily fed to livestock. In the past it was believed that the toxicity due to MOM was limited to the monogastric animals. However, recent litera- ture reveals that there is a toxic effect in ruminants as ‘well. In this study the model for studying the antinutritive characteris of MOM was growing chicks. Some researchers suggested that MOM can replace 80'- 100% of GNC from a GNC based broiler rations. However, others have found that even 35 % replacement could lead to reduced growth. Some suggested that upto 10 % of MOM may be incorporated in chick starter rations, wheras others found severe growth depression upon feeding even 10 % MOM. These differences may possibly be attributed to the varietal dif- ferences, agroclimatic conditions and processing techniques. Processing technique: Expeller processed meal contains high amounts of residual oil (10 - 15 %), which may contain high amounts of erucic acid. Prepress solvent processed or solvent processed residual meal contains virtually no residual oil and thus devoid of erucic acid. Temperature and moisture level at the time of processing 55 56 influences the quality of MOM. At proper temperature and moisture conditions, enzymatic hydrolysis of the glucosino- lates yield toxic aglucons that enhance the toxicity prob- lem of MOM. Heat treatment of mustard seeds prior to oil extraction has been found to be beneficial for oil extrac- tion and also improves general feeding value. However, mustard nutrient loss due to heat treatment needs considera- tion. Open pan roasting of mustard seeds prior to oil extra- ction reduced lysine content by 36 %. and the availability of lysine was reduced from 94 % to 76 %. Detoxification: Inactivation of endogenous enzyme (myrosinase), removal of the glucosinolates or itfis hydrolytic products reduce toxicity associated with the glucosinolates. Chemicals like ethanol, ammonia, acetone, sodium carbonate, sodium hydroxide and other similar compounds have been found to reduce toxicity in MOM, however, they are rather costly processes. Diffusion extraction of the glucosinolates from the intact seeds prior to oil extraction has been found to be effective as the gluCosinolates are highly water soluble. Different types of heat treatment of seeds or meal have also been found to be effective in reducing toxicity in MOM. Overall nutritive value of MOM was found improved by wet heat treatment and dry heat treatment at high temperatures (121 C) has negative effective effect on nutritional quality 57 of MOM. However, effective practical systems of heat and moisture treatments have not been devised. Most of the detoxification techniques described in the literature also induce some nutrient loss. The degree of loss in the nutrients vary from one technique to another. Short of plant breeding techniques, compromise has to be made between the losses occurring due to detoxification and the gains due to improved nutitive value as a result of the treatments. With this background, the proposed study is being undertaken to chemically characterize MOM, to partition the effects of antinutritive components, to determine the effects of vrious types of heating system on the nutritive value, the effcts of temperature, moisture and heating period on lysine availability and the effects of amino acid supplementation on chick growth. This study examines the following aspects of MOM feeding: 1. Partitioning the antinutrient components: Limited information is available on the effect of various components of MOM. In this study we propose to partition the antinutritive effects due to erucic acid, the glucosinolates and tannic acids as these three make up the major antinutrive components. 2.Improvement in nutritive quality of MOM by heat treatment: Effect of moisture, temperature and the duration of 58 heat treatment influence the nutritional quality of MOM. In the literature there is abundance of information on laboratory heating techniques such as autoclaving, static oven etc. There is lack of information on the nutritive value of MOM, when it is heated while in motion. There is also a derth of information on availability of lysine in MOM upon heat treatment at lower temperature and high moisture levels. In this study it is proposed.to test.a practical type of heating system (rotating drum heating system) with flexi- bility in use of source of heat, to study the impact of lower temperature and high moisture level on the antinut- rient composition and lysine availability in MOM. 1. EXPERIMENTAL PART I Part I consists of the following experiments: Experiment 1 : Chemical characterization of mustard oil meal (MOM) Experiment 2 : Effects of wet heating (autoclaving) and dry heating (oven) and the levels of mustard oil meal CMOM) on chick growth Experiment 3 : Chick growth assay Comparison of autoclaved and hot water treated mustard oil meal (MOM) Experiment 4 : Effects of heating and water treatment of mustard oil meal (MOM) on amino acid composition Experiment 5 : Effects of autoclaving defatted and undefatted mustard oil (MOM) on chick growth at different levels of intake Experiment 6 : Partitioning the effects of erucic acid and other antinutritive components of mustard oil meal (MOM) using chick growth assay 59 PART Ii EXPERIMENT 1: CHEMICAL CHARACTERIZATION OF MUSTARD OIL MEAL (MOM) INTRODUCTION Information is limited on the chemical characterstics of Nepalese mustard oil meal (MOM) . Capper et al. (1982) analyzed MOM from Nepal produced from Brassica nigra. Hemingway and Vaughan (1960) analyzed several samples of mustard seeds (Brassica juncea) from Nepal and all of them contained only allylisothiocyanate. It is essential to know the chemical characteristics of MOM to study its nutritive value. In this section chemical characteristics of MOM are described. Materials and Methods: During the period of these studies, three batches of mustard oil cake (MOC) were shipped from Nepal on three different occasions. Upon arrival the cake was ground and vacum packed in plastic bags and stored in a freezer at below -40 C till needed for feeding trials. Samples were taken for laboratory analysis after grinding . Proximate analyses, mineral analyses and essential amino acid analyses are presented in Tables 13, 14 and 15, respectively. 60 61 TABLE 13. PROXIMATE ANALYSES (%) OF MOM* Components Batch 1 Batch 2 Batch 3 Dry matter 91.20 90.70 87.50 Crude protein 39.58 37.82 38.16 Ether extract 10.34 11.40 13.18 Crude fiber 6.36 6.95 7.43 Ash 12.61 7.72 8.00 NFE 22.31 26.81 20.73 * Dry matter basis (DMB) TABLE 14. MINERAL ANALYSES OF MOM* Elements Batch 1 Batch 2 Batch 3 Calcium (%) 1.85 0.63 0.62 Phosphorus (%) 1.55 0.88 1.01 Cobalt (ppm) <2 na 3. Copper (ppm) 24 na 6. Magnesium (%) 0.76 na 0.44 Manganese (ppm) 74 na 42. Potassium (%) 1.21 na 1.22 Sodium (ppm) 66 na 40. Zinc (ppm) 78 na 55. Iron (ppm) 759 na 569. na = not analyzed; All mineral analyses were at Supersweet Research Farm, Ehe courtesy of Multifood International Inc. As fed basis done Courtland MN, through 62 TABLE 15. AMINO ACID ANALYSIS (%) OF MOM* Amino acid Batch 1 Batch 2 Batch 3 Lysine 1.69 “7 2.31 2.94 Methionine 0.91 0.70 0.71 Cystine 0.46 0.54 0.67 Arginine 4.08 2.27 2.76 Histidine 1.05 1.05 1.26 Isoleucine 1.59 1.59 1.97 Leucine 2.76 2.75 3.37 Phenylalanine 1.68 1.50 1.84 Tyrosine 1.20 1.18 1.21 Threonine 2.10 1.76 2.11 Tryptophan 0.61 0.46 0.46 Valine 2.10 2.08 2.59 * Amino acid analysis were done at Supersweet research farm, Courtland MN, by the courtesy of Multifood International Inc.;a DMB Gross energy was determined by adiabatic bomb calorimeter (AOAC, 1980). Reactive lysine values were deter- mined.by a method modified.in our laboratory (unpublished data, appendix A-I, page 136) that was similar to the procedure described by Hurrell and Carpenter (1979). Metabo- lizable energy was determined.by a modified.TME method.of Sibbald (1977). The procedure for ME determination is given in appendix A-VII (page 144). Erucic acid was determined as l3-cis-docosenoic acid after methylation (AOAC, 1980). The procedure is given in appendix A-III (page 139). Total fat extraction for erucic acid determination was done by the method described by Folch et al. (1957). The procedure is given in appendix A-II (page 138). Total glucosinolate was determined by the method described by McGhee et a1. (1965). The procedure is given in 63 appendix A-IV (page 141). Allylisothiocyanate was determined after steam- volatilization by a GC method.(AOAC, 1984). The procedure is given in appendix A-V (page 142). Tannin was determined as quercitannic acid by the method described in AOAC (1984). The procedure for the determination of tannin is given in appendix ArVI (page 143). GE, ME, RL, erucic acid, total glucosinolates, allyl isothiocyanate and tannin content of raw MOM from batch 3 are presented in Table 16. TABLE 16. NUTRITIVE AND ANTINUTRITIVE COMPONENTS OF MOM Components - Amount Gross Energy (cal/kg) 4653d Metabolizable energy (cal/kg) 3295a Reactive Lysine (g/kg CP) 46.16a Erucic acid ( % in oil on DMB) 37.05 Total glucosinolates (%) 4.75b Allyl isothiocyanate (%) tr Tannins (as quercitannic acid) (%) 2.86b 4 DMB: b Fat free DMB Results and discussion: There were no appreciable differences in protein concentrations in three batches of MOM (Table 13). There were slight differences in fat concentrations in MOM. Batch 1 was markedly higher in calcium and phosphorus content compared to the other two batches (Table 14). MOM from batch 1 was lower in lysine compared to the other two batches. However, arginine was remarkably 64 higher in batch 1 than in batch 2 and 3. Methionine and tryptophan were also a little higher in batch 1 than in batch 2 and 3. Lysine content of samples from Nepal ranged 4.95 - 7:71 g/l6 g N which is much higher than the values of 2.91 g/16 g N reported by Prasad (1978) for MOM from India. Values reported in this study are closer to the values reported by Miller et a1. (1962) The only known background about.the MOM recieved, were that it belonged to the genus Brassica, and that it was expeller processed. Not knowing what it had gone through prior to oil extraction.or how long and.how it was stored, it was difficult to explain the discrepancy in lysine content. The lower lysine value in batch 1 cannot be ex- plained by possible-heat damage during oil extraction as the arginine value was rather high compared to the other batches. The ME value for MOM using the TME method of Sibbald was higher than the value obtained by Shrivastava et a1. (1976). They reported a value of 3017 kcal/kg as opposed to 3295 kcal/kg found in this study. Reactive lysine in MOM was within the range published for RSM. The available lysine value was 77%. Capper et al. (1982) reported the availability of lysine in MOM to be 94%. However, they found a severe decrease in availability of lysine (to 74%) when mustard seeds were open pan roasted for 20 to 30 min prior to oil extraction. Erucic acid in the residual oil was within the range 65 reported by Dutta and Ghosh (1970). Total glucosinolate content on a fat-free dry matter basis was within the range reported by Appelqvist (1971) for Brassica juncea(3 - 6%). Tannin content of lflnd was'similar to the value reported by Vaidya et al. (1975a). Allylisothiocyanate in MOM could not be detected by the method described by AOAC (1984). A possible explanation for the failure to detect this compound is that the enzyme myrosinase may have been completely destroyed during the extraction of oil. Appelqvist and Josefsson (1967) repor- ted that myrosinase was completely destroyed at 8% moisture level and at 90 C for 15 minutes . Moisture content in MOM analyzed for allylisothiocyanate was 12.5%. PART Ii EXPERIMENT 2: EFFECTS OF WET HEATING (AUTOCLAVING) AND DRY HEATING (OVEN) AND THE LEVELS OF MUSTARD OIL MEAL (MOM) ON CHICK GROWTH INTRODUCTION Wet heat treatment of RSM improves its nutritive value to chickens (Rauchberger et al., 1979: Bock et al., 1982). Improvement in quality of RSM upon wet heat treatment was observed by Eapan (1968). Chick growth was improved upon feeding RSM autoclaved at 15 psi (Gray et al., 1957) , which was independent of lysine destruction..Autoclaving'of RSM for 60 min at 120 C and 30% moisture level eliminated goit- rin (Hijikuro and Takemasa, 1979b). Limitation of use of mustard oil meal (MOM) and rapeseed meal (RSM) have been reported by several authors ( Agarwala, 1964; Kubota et al., 1972; Srivastava and Prasad, 1976; Marangos et al., 1978; Matsumoto et al., 1979; Hijikuro and Tkemasa,1979b; Kozlowska et al., 1981). It was attributed to the content of glucosinolates (El-nokrashy et al., 1975) in RSM and erucic acid in MOM (Prasad et a1” 1978). Replacement of GNC by MOM at the 80% level (Benergy, et al., 1974), at the 100% (Prasad et al., 1974; Mandal et al., 1981) and at the 100% level of RSM (Ballester et al., 1974) did not affect chick growth. March at al. (1975) did not find 66 67 any adverse effct of feeding RSM as the sole source of protein to layers. Hij ikuro and Takemasa (1979c) replaced 50% SBM by RSM without any adverse effet in chick starter diets. This experiment was designed to investigate the effect of dry and wet heat treatments on the nutritive value of MOM. Effect of incremental levels of MOM in chick growth was also investigated. Materials And Methods: MOM used in this trial was from batch 1. Heat treatments: 1) Autoclaving: MOM was thinly spread in an aluminium pan and placed in an autoclave and steam heated for 30 min at 121 C. After the treatment, the pan was left overnight to equilibrate with the atmosphere. 2) Oven heating: MOM was thinly spread in an aluminium pan and placed in an oven ( preheated to 121. C ) and heated for 30 min. After the treatment, the pan was left overnight to equilibrate with the atmosphere. Diet pgeparation: Basal component: In all the diets, the basal component provides 70 % with the remaining 30% containing combinations of MOM and substitute component which simulates MOM. The composition of the basal component and substitute component 68 are presented in Table 17. Combinations of these components are given in Table 18. All the diets were equicaloric and equinitrogenous (ME: 2531 Kcal/kg; CP: 18.00%L. TABLE 17. COMPOSITION OF BASAL AND SUBSTITUTE COMPONENTS* Ingredients** Basal Substitute Corn starch 14.02 ______ Wheat middlings 45.44 11.50 Yellow corn 36.56 ...... Corngluten meal ------ 80.50 Dical 0.76 1.57 Limestone 0.33 3.38 Coomon salt 0.71 ...... Mineral mix 0.04 ...... Vitamin mix2 0.43 ...... Lysine HCL (78.4%) 0.37 1.06 Methionine HC1(98%) 0.19 ------ Arginine ( 99.9% ) ------ 2.00 Sand 1.15 ...... * As fed basis;** Detailed composition in appendix B-1,2 (page 145 ): l & 2 appendix B-3,4 (page 146) TABLE 18. DIETARY COMBINATIONS # Diets % MOM % Substitute % Basal 1 Raw MOM 30 0 , 70 2 Dry heated 30 0 70 3 Autoclaved 30 0 70 4 Raw MOM 20 10 70 5 Dry heated 20 10 70 6 Autoclaved 20 10 70 7 Raw MOM 10 20 70 8 Dry heated 10 20 70 9 Autoclaved 10 20 70 10 Control 0 30 70 Experimental pgpcedure: This experiment was designed as a 2 X 3 factorial plus control (Gill, 1978). Twelve day old meat-type chicks, previously raised on commercial chick starter rations in a 69 battery brooder were wing banded, weighed and ranked by weight in groups of 8 chicks. One chick from each weight group was randomly selected and assigned to one of the twenty pens. Each pen had eight chicks representing eight weight groups. Ten dietary treatments were randomly assigned to twenty pens (2 replicates/dietary treatment). Weight and feed consumptions were recorded daily for two weeks. Chicks were fed ad libitum and fresh water was available at all times. Results and discussion: Initial weight, two week weights of chicks on experimental diets, feed consumption and feed to gain ratios are given in Table 19. TABLE 19. PERFORMANCE OF CHICKS IN EXPERIMENT 2 Diet Initial Final Feed F/G # weight weight consumption (Ratio) (9) (g) (g) 1 87.1 205.4 298.2 2.5 2 84.7 201.5 289.5 2.4 3 85.8 217.7 308.4 2.3 4 85.9 216.9 288.6 2.2 5 83.3 213.9 276.0 2.1 6 87.6 234.4 319.9 2.2 7 84.9 201.3* 243.8 2.1 8 86.9 206.3* 263.8 2.2 9 90.4 228.1* 296.1 2.1 10 85.9 204.6 251.2 2.1 * Excluded from statistical analysis; SE of treatment mean = +- Weight gain was subjected to ANOVA page 147). 5.69 (Appendix C-2, Autoclaving significantly improved growth com- 70 Weight gain was subjected to ANOVA (Appendix C-2, page 147). Autoclaving significantly improved growth com- pared to oven heat treated MOM (P<.05). Autoclaving of MOM supported higher growth rates compared to other treatments at all levels of incorporation. IHowever, the differences were more prominent at lower levels of incorporation. In Figures 3, 4 and 5, weekly weights of chicks at 10, 20 and 30% in the diets are presented. These results show that a progressive decline in growth occurred with increasing levels of MOM (P<.05). However, the decline was more severe for the raw and dry heated MOM diets. 71 240 * Autoclaved MOM O Oven heated MOM 0 Raw MOM 200 (0 :E160 < a '4 (D 12 -'120 p. I: 9. u: . 3 so I 0 7 TIME IN DAYS Figure 3. weekly weight of chicks fed autoclaved, oven heated and raw MOM at 10% level in the diets 72 240 _ *Autoclaved MOM QRaw MOM OOven heated MOM 200 u U) :E‘IESII _ '< I: , o J 12 ‘ 1 20 — [— II ‘2 In B 80 7 TIME IN DAYS Figure 4. Weekly weight of chicks fed autoclaved, oven heated and raw MOM at 20% level in the diets ‘—¥ 14 73 240 * Autoclaved MOM <3 Oven heated MOM 0 Raw MOM 200 a) 160 2 .( I: (9 :2 - 120 p. I: 9. Ifl ; so I 0 7 14 TIME IN DAYS Figure 5. Weekly weight of chicks fed autoclaved, oven heated and raw MOM at 30% level in the diets PART Ii EXPERIMENT I: CHICK GROWTH ASSAY TO COMPARE THE NUTRITIVE VALUE OF AUTOCLAVED AND HOT WATER TREATED MUSTARD OIL MEAL (MOM) INTRODUCTION Autoclaving has been shown to improve nutritive value of MOM by (Gray et al., 1957), and also in experiment 2, part I of this study. Autoclaving also destroys lysine, an important amino acid..Autoclaving is not practical except in laboratory conditions. Hot water treatment of RSM was beneficial in improving its nutritive value (Prasad and Rao, 9178). Hot water extraction of RSM reduced toxicity in growing chicks (Nakaya, 1965a; Nakaya, 1965b; Nakaya and Tagannis, 1965: Lo and Hill, 1971). Olomu et al. (1974) observed improvement in the quality of the product upon autoclaving or hot water treatment of rapeseed. It was observed in previous experiments that higher rates of incorporation of MOM without heat treatment impaired growth in growing chicks. Heating of MOM improved growth even at higher levels of incorporation. It seems that wet heating has a beneficial effect on chick growth. This experiment was designed to compare the nutritive value of autoclaved MOM and hot water treated MOM 74 75 at two levels of dietary incorporation. Materials and methods: MOM from batch 2 was used in this study. Hot water treatment: Water in a large pan was brought to boiling and then MOM was added to the boiling water, with continued heating for 30 minutes. It was occasionally stirred. Thermocouples were placed at different depths to measure the product temperature during the process. After the heat treatment the slurry was spread on shallow pans and dried in forced draft oven for 18 hours at 45 C. The slurry formed a hardend crust, so it was ground.in a Wiley mill. The meal was left in pans over night to equilibirate with the atmosphere. Autoclave treatment: See experiment 2, part 1 (page 67). Diet preparation: A corn-soy basal diet was formulated and soybean meal was replaced by mustard oil meal at two levels of replacement. All the diets were equicaloric and equinitrogenous ( ME: 3200 Kcl/kg; CP: 23.0% ). Trace mine- rals and vitamins were added according to NRC (1977) recom- mendations. Composition of experimental diets are given in table 20. 76 TABLE 20. COMPOSITION OF EXPERIMENTAL DIETS* Ingredients** Diets 1 2 3 4 5 SBM ( 44% CP 42.60 20.00 20.00 31.00 31.00 MOM ( 34.9% CP ) ---- 30.00 30.00 15.00 15.00 Yellow Corn 45.00 36.00 36.00 41.00 41.00 Corn oil 7.95 9.60 9260 8.67 8.67 Dical 3.13 2.66 2.66 3.07 3.07 Limestone 0.29 ---- ---- ---- ---- Salt 0.50 0.50 0.50 0.50 0.50 Vitamin Mix 0.30 0.30 0.30 0.30 0.30 Mineral Mix 0.03 0.03 0.03 0.03 0.03 Methionine 0.20 0.08 0.08 0.10 0.10 Lysine ---- 0.17 0.17 0.04 0.04 Sand ---- 0.66 0.66 0.29 0.29 * As fed basis; ** Detailed composition in Appendix B-1,2 (page 145); 1 & 2 in appendices B-3 and B-4 (page 146) Materials and methods: The experiment was designed in a 2 X 2 factorial plus control (Gill, 1978). Fifty, 12 day old Barred Rock meat type chicks previously raised on commercial broiler starter ration were selected from a batch of 200 birds. Birds weig- hing 101 gm to 110 gm were selected and were randomly assig- ned to 10 pens ( 5 birds/pen ). Five dietary treatments were randomly assigned to ten pens ( 2 pens/treatment ). Birds were fed ad libitum and fresh water was available at all times. They were housed in a battery brooder. Weight gain and feed consumption were recorded at 3 day intervals. Results and Discussion: Weight gain, feed consumptions and feed to gain ratio are presented in Table 21. Final weight was subjected to ANOVA (appendix C-37 page 148). 77 TABLE 21. PERFROMANCE OF CHICKS IN EXPERIMENT 3 Diet Initial Wt. Final Wt. Feed Consumption F/G # in grams in grams* in grams ratio 1. 105.10 219.00 236.14 2.07 2. 104.30 203.00 234.64 2.37 3. 103.40 194.10 211.51 2.33 4. 103.80 207.80 277.01 2.18 5. 101.70 204.90 230.00 2.22 * 9 day weight gain; SE of treatment means = +-2.606 Increasing levels of MOM in the diets resulted in lower rates of growth.(P5; page 149). Figure 7 shows the effect of levels of treated and untreated MOM in the diets. Figure 8 shows the weekly weight of chicks fed diets supplemented with mustard oil or corn oil. There was significant improvement due to autoclaving of MOM (P<0.009). Mean response to the levels of MOM in the diets were found significant (P<0JHH for the levels from 86 10% to 30%. Because of mild interaction between defatting and levels, effect of defatting was tested at 3 levels of dietary incorporation using Studentized t- test (Gill, 1978). At 10 and 20 % levels, defatted MOM supported signi- ficantly higher growth rate compared to undefatted MOM (P< (L05). However at 30% level of MOM there was no significant difference in growth of chicks fed defatted or undefatted MOM. Figures 9, 10 and 11 are presented to clearly indicate the differences in weight gain pattern at 10, 20 and 30% levels of MOM in the diets respectively. TABLE 25.FINAL WEIGHT, FEED INTAKE AND FEED/GAIN RATIOS OF CHICKS IN EXPERIMENT 5 Description MOM Init. Weight Feed F/G of diets % Wt. gain consumed ratio (9) (9) (9) Raw MOM diet 10 46.2 136.0 179.2 2.0 Autoclaved MOM " 44.0 155.9 190.1 1.7 Acetone washed " 45.2 149.0 209.0 2.1 Acet. Wd. Auto. MOM " 44.7 159.5 225.1 1.9 Raw MOM 20 45.2 124.7 189.1 2.4 Autoclaved MOM " 38.0 134.9 210.9 2.2 Acetone washed MOM " 43.4 143.5 208.0 1.9 Acet. Wd. Auto. MOM " 43.8 146.9 215.6 2.2 Raw MOM 30 45.8 120.0 178.6 2.4 Autoclaved MOM " 41.9 120.6 159.7 2.0 Acetone washed MOM " 44.4 116.2 173.6 2.4 Acet. Wd. Auto. MOM " 43.4 119.6 186.1 2.4 SE for treatmenets = +— 3.18 In Table 26 the effect of addition of mustard oil to the standard referance diet is presented. The results indicate 87 that there is detrimental effect of adding mustard oil in the diets. Significant weight reduction occurred due to mustard oil ( P<0.05L.‘Weekly weight of chicks fed diets l3, 14, 15 and 16 are presented in figure 11..ANOVA for two week weight is presented in appendix C-6 (page 150). TABLE 26. PERFORMANCE OF CHICKS FED MUSTARD OIL ADDED DIETS IN EXPERIMENT 5 Diet Treatment Initial Final Feed Feed/gain # weight weight consumption ratio 13. Corn-soy std diet 40.4 157.5 191.2 1.6 14. Corn-MOM control 44.0 109.3 179.2 2.7 15. #1 +Corn oil 43.7 160.6 217.4 1.9 16. #1 +Mustard oil 44.7 140.6 198.4 2.1 SE of treatment means = +- 6.69 Diets l4 and 16 are equal in erucic acid content. However, the growth decline in chicks fed diet 16 was less than due to diet 14 (P<0.05). It is reasonable to believe that there are still antinutritive factors in MOM, in addi- tion to erucic acid. 88 180 A Acet . Wd. & auto. 0 Acet. wd . 15° . Autoclaved (auto) ---- E3 Izaivv D4()ld \““ 3140 I ‘ . I O E 120 I- I . 3 3 100 80 l 0 2 0 . 3 0 [.SVBH 0? I0. II T" DIETS Figure 7. weight gain of chicks fed raw, acetone washed, acetone washed and autoclaved and autoclaved mustard oil meal (MOM) at 3 levels of intake 89 16° " 1:] Control + Corn oil ‘k Control 1% Control + Mustard oil 140 "’ 0 MOM basal diet , 20 WEIGHT INdGRAMS .. on G 40 7 14 TIM E IN DAYS Figure 8. Weekly weight of chicks fed mustard oil added standard reference diet 160 140 120 IN GRAMS on 3 Q Q WEIGHT 40 90 ‘ Control ‘fi'Acetone washed and autoclaved MOM C) Acetone washed MOM D Autoclaved MOM ‘3 Raw MOM . Raw MOM basal diet 7 14 TIME IN DAYS Figure 9. Weekly weight of chicks fed experimental diets at 10% level of treated or untreated MOM IN GRAMS WEIGHT 91 160 A Control 1% Acetone washed and autoclaved MOM O Acetone washed MOM 140 C] Autoclaved MOM (3 Raw MOM ‘D Raw MOM basal diet 120 100 80 60 40 0 7 TIME IN DAYS Figure 10. Weekly weight gain of chicks fed experimental diets containing 20% treated or untreated MOM * 14 160 140 120 IN GRAMS on 3 G’ c: WEIGHT 40 Figure 11. Weekly weight of chicks fed experimental diets 92 Control Acetone washed and autoclaved MOM Acetone washed MOM Autoclaved MOM Raw MOM Raw MOM basal diet .0004» 7 TIME IN DAYS at 30% level of treated or untreated MOM 14 PART _I_: EXPERIMENT é: PARTITIONING THE EFFECT OF ERUCIC ACID AND OTHER ANTINUTRITIVE COMPONENTS IN MUSTARD OIL MEAL (MOM) USING THE CHICK GROWTH ASSAY INTRODUCTION Deleterious effect of erucic acid in MOM has been evidenced by several researchers ( Abdellatif and Vles, 1970a; Abdellatif and Vles, 1970b; Abdellatif et al., 1972: Ray et al., 1979; Prasad and Rao, 1982). In experiment 4 of part 1 of this manuscript it was observed that acetone extraction of MOM resulted in better growth performance of the chicks compared to the chicks fed raw MOM in the diets. In experiment 5, part 1, it was observed that mustard oil addition to standard reference diet reduced growth signifi- cantly. However,corn-MOM diets, which contained equivalent erucic acid, supported significantly lower growth. The re- sults suggested that in MOM, in addition to erucic acid other antinutritive factors are present ( glucosinolates, tannins etoJ This experiment was designed to partition the effect of the oil faction (erucic acid ) and the meal fraction ( combined effects of glucosinolates, tannins etc. ). Materials and Methods: In this experiment MOM from batch 3 was used. 93 94 Oil extraction: A Soxhlet type extracter was used to extract oil from MOM. Hexane was used as organic solvent. After the extrac- tion the mixture was evaporated under the hood till no odor remained. Mustard oil was stored in brown glass bottles and kept in the refrigerator until used. Acetone washing: Mukherjee et a1. (1979) removed glucosinolates by washing RSM with 70% acetone. Pure acetone was mixed in a vat with defatted MOM at the ratio of 3:1, in a vat, stirred occasionally for 2 to 3 hours and let stand for 24 hours under the hood. The supernatant liquid was siphoned off into a large beaker and evaporated under the hood until the smell of acetone was completely gone. Diet preparation: Combinations of dietary treatments are given in Table 27 and composition of the diets are given in Table 28..All the diets are equicaloric (ME; 3132 Kcal/kg) and equinitrog- enous (CP: 21.32%). Defatted MOM, defatted and acetone was- hed MOM, and canola meal contribute crude protein in the diets equivalent to the percent crude protein contributed by the raw MOM in the corresponding diets. 95 TABLE 27. COMBINATION OF DIETARY TREATMENTS Dietary treatments Effects to be studied l. Corn-soy diet 2. Corn-soy diet + Mustard oil E 3. Corn-soy diet + Act. wd. rd. (G+O) 4. Raw MOM diet E+G+O 5. Defatted MOM (G+O) 6. Acetone washed defatted MOM (G+O) 7. Canola meal (6+0) G= Glucosinolate; E= Erucic acid; O= Others; TABLE 28. DIETARY COMPOSITIONS Ingredients Diet #s 1 2 3 4 5 6 7 Yellow corn 54.56 54.56 54.16 47.95 47.95 47.95 49.38 SBM 37.00 37.00 37.00 --- -- --- -- CGM --- --- --- 6.32 6.32 6.32 5.70 Corn oil 4.46 --- 4.46 2.25 6.07 6.52 5.30 Mustard oil --- 4.46 --- --- --- - --- --- Acetone Wd. residue --- --- 0.30 --- --- --- --- MOM --- --- --- 39.04 --- --- --- Defatted MOM —-- --- --- --- 35.53 --- --— Deftd. & Ac. Wd. MOM --- --- --- --- --- 34.59 --- Canola Meal --- --- --- --- --- --- 35.71 Dical 1.57 1.57 1.57 1.37 1.37 1.37 1.41 Limestone 1.43 1.43 1.43 1.20 1.20 1.20 1.17 Common Salt 0.40 0.40 0.40 0.40 0.40 0.40 0.40 Trace Min. Mix. 0.03 0.03 0.03 0.03 0.03 0.03 0.03 Vitamin Mix. 0.30 0.30 0.30 0.30 0.30 0.30 0.30 Dl-Meth. 0.25 0.25 0.25 0.10 0.10 0.10 0.08 Lysine-HCL --- --- --- 0.02 0.02 0.02 0.26 L-Arginine --- --- --- 0.12 0.12 0.12 0.26 Solkaflok --- --- --- 0.90 0.52 1.08 --- Experimental: Day-old Hubbard broiler male chicks were wing banded, weighed and grouped in four weight groups. Upper and lower weight groups were discarded. From the two selected 96 groups, equal numbers of chicks were randomly assigned to 21 pens(10 birds/pen) in a battery brooder. Seven dietary trea- tments were randomly assigned to 21 pens (3 pens/ treat- ments). Chicks were fed ad libitum and fresh water was available at all times. Weekly weight gains and feed consum- ptions were recorded. Results and discussion: Initial weight, final weight, feed consumption and feed to gain ratio are given in Table 29.Growth patterns during the two week growth assay period are shown inFigure 12. Final weight was subjected to ANOVA (Appendix C-8; page 151) . TABLE 29. PERFORMANCE OF CHICKS IN EXPERIMENT 6 Diet Initial Final Weight Feed F/G % Nitrogen # Weight Weight Gain Intake Ratio Absorbability 1 40.0 344.3 304.3 420.0 1.4 51.8 2 39.8 300.3 260.5 379.9 1.5 47.4 3 39.8 321.2 281.3 402.6 1.5 46.9 4 40.5 249.4 208.8 349.6 1.7 53.8 5 40.0 272.0 232.0 343.9 1.5 52.2 6 40.1 283.8 243.7 368.2 1.5 46.1 7 40.1 303.4 263.3 381.2 1.4 52.6 SE = +- 8.43; 95% CI for group means = Y +- 18.08 The addition of mustard oil equivalent to the amount in diet 4 to the standard reference diet significantly reduced growth of chicks (P