TEMPERATURE AND SEMEN ELECTROLYTES
Lake, P. E., 1957. Fowl semen as collected by the massage method. J. Agric. Sci. 49: 120-126. Lorenz, F. W., 1959. Reproduction in the domestic fowl: Physiology of the male. Vol. 1, pp. 343-390 In: H. H. Cole and P. T. Cupps. Reproduction in Domestic Animals. Academic Press. New York, N.Y. Ostle, B., 1966. Statistics in Research, 2nd Ed. Iowa State University Press, Ames, Iowa. Parker, J. E., and B. J. McSpadden, 1943. Seasonal variation in semen production in domestic fowl.
861
Poultry Sci. 22: 142-147. Salisbury, G. W., and J. R. Lodge, 1962. Metabolism of spermatozoa. Adv. Enzymology, 24: 35-104. Salisbury, G. W., and N. L. Van Demark, 1961. Physiology of Reproduction and Artificial Insemination of Cattle. Freeman Co., California. Chap. 21, pp. 527-530. Singh, Bhupal, B. B. Mabapatro and D. P. Sadhu, 1969. Chemical composition of cattle and buffalo spermatozoa and seminal plasma under different climatic conditions. J. Reprod. Fer. 20: 175-178.
Rapeseed Meal Glucosinolates: Metabolism and Effect on Performance in Laying Hens T. K. SMITH1 AND L. D. CAMPBELL
Department of Animal Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 (Received for publication June 23, 1975)
ABSTRACT Two experiments were conducted with Hyline Leghorn hens to study the metabolism and detrimental effects of rapeseed meal (RSM) glucosinolates. Raw Target RSM was force fed to 12 hens which were killed after varying time intervals (15 min., 30 min., 60 min.) and the contents of areas of the digestive tract (crop; proventriculus and gizzard; duodenum and ileum) were analyzed for the presence of hydrolysis products of progoitrin. Nitrile compounds were found to be present in all areas of the digestive tract in much larger relative amounts than was oxazolidinethione. When commercially prepared RSMs of varying glucosinolate content were fed to laying hens at a 50% level of dietary inclusion, high glucosinolate-content RSM depressed egg production (P < 0.05) more than low glucosinolate-content RSM but did not cause a greater frequency of liver hemorrhage. Histological examination of liver tissues from hens suffering liver hemorrhage revealed a severe reticulolysis. POULTRY SCIENCE 55: 861-867, 1976
INTRODUCTION
R
APESEED and related Cruciferae have long been known to contain glucosinolates. The compounds may be hydrolyzed by the enzyme thioglucoside glucohydrolase (EC 3.2.3.1.) to produce a variety of potentially toxic products. The predominant glucosinolate in rapeseed of the Brassica napus type is progoitrin which may be hydrolyzed to a mixture of l-cyano-2-hydroxy-3-butene (CHB), diasteriometic l-cyano-2-hydroxy-3, 4-epithiobutanes (CHE) and 5-vinyloxazolidine-2-thione (OZT) (Daxenbichler et al., 1966, 1967). The relative amounts of these
1. Present address: Department of Poultry Science, Cornell University, Ithaca, N.Y.
products vary primarily with the pH of the hydrolysis medium. In a biological evaluation of crambe seed meals and derived products by rat feeding, Van Etten et al. (1969) determined that a mixture of CHB and CHE was more toxic ( = 8 x ) than OZT. Rapeseed meal (RSM) is used in Canada as a protein supplement for poultry feeds although its use has been limited because of the presence of antinutritional or toxic factors. Jackson (1969) determined that the feeding of RSM for nine 28-day periods at levels of up to 20% of the diet to laying hens produced high mortality as a result of massive liver hemorrhage. Hall (1972) observed similar pathology in commercial laying flocks believed to have been fed RSM. Marangos et al. (1974) reported that RSM of high
862
T. K. SMITH AND L. D. CAMPBELL
glucosinolate content caused significantly greater mortality among laying hens than did low glucosinolate-content RSM. The current studies were undertaken to determine the effect of conditions in the laying hen digestive tract on the hydrolysis of progoitrin and to determine the relationship between levels of RSM glucosinolates and the frequency of liver hemorrhage. MATERIALS AND METHODS Experiment 1. Two RSMs were fed in this experiment. One was a commercial meal prepared from Target rapeseed and supplied by Co-op Vegetable Oils Ltd., Altona, Manitoba while the other was prepared in the laboratory from Target rapeseed provided by the Department of Plant Science, University of Manitoba. In the latter case seed was coarsely ground in a Wiley mill and then extracted with hexane for 24 hr. in a Soxhlet apparatus. The extracted product was re-
ground to give a fine powder which was referred to as "raw Target RSM." Twelve 32-week old Hyline White Leghorn hends were randomly selected over a twoweek period in May, 1974 for use in this experiment. Feed (a commercial laying hen diet) was withdrawn from each bird for a period of 18 hr. previous to the test feeding. For each bird consecutively, a sample (30 g.) of raw Target RSM was blended evenly with H 2 O (60 ml.) and transferred to a 100-ml. plastic syringe with a plastic needle. The mixture was injected as deeply as possible into the throat of the test birds. Four hens (three fed raw Target RSM and one the commercial meal) were killed by cervical dislocation at intervals of 15, 30 or 60 min. after force feeding. The digestive tracts of the birds were excised and the digesta from three areas (crop; proventriculus and gizzard; duodenum and ileum) were removed and placed in sealed containers containing methylene chlo-
TABLE 1.—Composition of diets fed in Experiment 21 Diets Ingredient (%) Bronowski rapeseed meal 1788 rapeseed meal Target rapeseed meal Barley Wheat Herring meal Soybean meal Calcium carbonate Oyster shell Deflourinated rock phosphate Soybean oil Vitamin mixture 2 Mineral mixture 3
Bronowski
1788
Target
0.00 0.00 0.00 75.60 0.00 5.00 10.00 2.50 2.50
50.00 0.00 0.00 0.00 37.25 0.00 0.00 2.50 2.50
0.00 50.00 0.00 0.00 41.10 0.00 0.00 2.50 2.50
0.00 0.00 50.00 0.00 37.87 0.00 0.00 2.50 2.50
2.40 0.50 1.00 0.50
2.40 3.85 1.00 0.50
2.40 0.00 1.00 0.50
2.40 3.23 1.00 0.50
100.00
100.00
100.00
100.00
Control
'Calculated energy content for all diets was 2,600 kcal. per kg. 2 Supplied per kilogram of diet: 7,150 I.U. vitamin A, 818 I.C.U. vitamin D 3 , 3.50 I.U. vitamin E, 11.0 meg. vitamin B, 2 , 7.7 mg. riboflavin, 2.2 mg. pantothenic acid, 3.3 mg. niacin, 5.5 mg. choline, 250 mg. santoquin. 'Supplied per kilogram of diet: 80.45 mg. manganese, 44.19 mg. zinc, 4.8 g. sodium chloride.
863
RAPESEED MEAL GLUCOSINOLATES
ride. Samples were mechanically agitated (60 min.) and filtered (Whatman 541). Residues were washed twice with methylene chloride and all washings combined with the extract. Samples were concentrated by flushing with purified N 2 and dried over anhydrous N a 2 S 0 4 (18 hr.). The resultant solution was centrifuged (15 min., 48,000 g) and stored (5°) until analyzed by gas chromatography. Experiment 2. One hundred sixty 22-week old Hyline White Leghorn hens were randomly distributed to 20 raised wire laying cages with eight hens per cage. Four diets (Table 1) were fed for three 28-day periods. The RSMs fed were 1788 (an unnamed low glucosinolate cultivar) and Bronowski (supplied by the Rapeseed Association of Canada) and Target (previously described, commercially-prepared). A standard barley-soybean diet served as a control. Feed consumption and weight gains were determined for each period while egg production was measured on a weekly basis. Initially, hens were exposed to 11 hr. of light daily and this was increased 30 min. at weekly intervals for six weeks. At the conclusion of the experiment all surviving hens were killed and the livers examined for the presence of non-fatal hemorrhage and from birds killed at the termination of the experiment were stained with hematoxylin and eosin and impregnated with silver by the method of Gordon and Sweets (Drury et al., 1967).
Analyses. Hydrolytic products from progoitrin were measured by a modification of the technique of Daxenbichler et al. (1970). An F and M Scientific 700 gas chromatograph equipped with a H 2 flame ionization detector was used. The column was glass (244 cm. x 6.35 mm. o.d.) and packed with \% EGSSX on 100-120 mexh Gas-Chrom Q. Operation conditions were: He flow, 30 ml./min.; air pressure, 20 p.s.i.; H 2 pressure, 16 p.s.i.; injection port temperature, 165° C ; detector block, 250° C. The usual temperature program was 115° C. for 10 min., 115° C. to 180° C. at 2° C./min., 180° C. to 210° C. at 5°/min., 210° C. for the remainder of the analysis. Peaks on sample chromatograms were identified by comparison of retention times with those of the chemically pure standard compounds: (s)-l-cyano-2-hydroxy-3butene; (2s,3s)-l-cyano-2-hydroxy-3,4-epithiobutane; (R)-5-vinyloxazolidine-2-thione. Glucosinolate contents of the RSMs were determined according to Youngs and Wetter (1967). Data were analyzed by the analysis of variance as described by Snedecor (1956). Significantly different means were separated using Tukey's honestly significant difference test (Steel and Torrie, 1960). RESULTS Experiment 1. The relative amounts of the hydrolytic products from progoitrin found in the digestive tracts of raw Target RSM-fed
TABLE 2.—Relative amounts (%) of the hydrolytic products from progoitrin found in the digestive tracts of hens fed raw target RSM Crop 30 12.56 52.36
Proventriculus, gizzard 30 15 60 7.81 9.87 6.87 36.76 30.71 32.18
15 4.53 31.53
ileum 30 8.14 31.37
60 4.98 32.24
SE 0.55 1.82
27.91
28.19
27.34
28.71
25.86
0.82
31.51
32.76
36.60
31.78
36.92
1.14
Product 152 60 OZT 9.75 10.61 CHB 64.03 59.97 CHE (threo) 12.28 17.32 14.50 27.24 CHE (erytho) 13.08 17.76 15.78 28.19 'Standard error of the mean. 2 Interval between feeding and killing (min.).
]Duodenum,
864
T. K. SMITH AND L. D. CAMPBELL
TABLE 3.—Glucosinolate content of rapeseed meals used in experiment 2 Component1 3-butenylisothiocyanate 4-pentenylisothiocyanate 5-methyl sulphinyl-pentylisothiocyanate 2-phenylethylisothiocyanate Oxazolidinethione Total 'All values expressed as mg. per g. 2 Standard deviation of the mean.
Rapeseed meal 1788 0.12 + 0.02 0.02 ± 0.01
Bronowski 0.19 ± 0.012 0.10 ± 0.01
0.00 ± 0.00 0.00 ± 0.00 0.20 ± 0.04 0.49 of meal on an "as fed'
hens are given in Table 2. Only OZT was significantly affected by the time interval between feeding and killing, it being significantly greater (P < 0.05) after 30 min. than after either 15 min. or 60 min. Relative amounts of OZT and CHB were significantly greater in the crop than in either of the other digestive tract areas examined (P < 0.01) between which there were no significant differences (P > 0.05). The diastereometric CHE were found in significantly smaller relative amounts in the crop than in the other areas of the digestive tract between which there were again no significant differences (P > 0.05). Relative amounts of hydrolytic products from progoitrin in the digestive tracts of hens fed the commercial Target RSM could not be determined since all products except CHB were present in concentrations below the limit of detection. Peaks corresponding to the various hydrolytic products were observed, however, and the amount of CHB present was much greater in the crop than in the other digestive tract areas. In all cases the summation of the relative amounts of the three nitrile products greatly exceeded the relative amount of OZT. Experiment 2. Bronowski and 1788 RSMs were determined to contain the same amount of glucosinolates within the bounds of experimental error while Target RSM contained substantially higher levels (Table 3). The
Target 1.02 ± 0.13 0.39 ± 0.03
0.00 ± 0.00 0.00 ± 0.00 0.20 ± 0.04 0.34 basis.
0.05 ± 0.02 0.03 ± 0.01 2.13 ± 0.13 3.62
levels observed for all meals, however, are low and it is possible that some prior hydrolysis to nitrile compounds had occurred. Nitrile compounds are not detected by the method of analysis employed. Furthermore, the values for Target RSM may be disproportionateTABLE 4.—Average weight gains1 of hens consuming RSM of varying glucosinolate content during the three 28-day periods in experiment 2 Period Diet Control Bronowski 1788 Target Mean SE =
Mean 2
2
3
0.055
0.00
0.027
0.027a
0.050 0.082 0.014 0.050a 0.0093
-0.036 -0.023 -0.009 -0.018b
0.014 0.014 0.00 0.014c
0.0O9a,b 0.023a,b 0.000b
1
'Expressed in kg. per hen. Means with the same letter are not significantly different (P > 0.05). 'Standard error of the mean for diets. 2
TABLE 5.—Average feed consumption1 of hens during the three 28-day periods in experiment 2 Period Diet
1
2
3
Mean 2
Control Bronowski 1788 Target Mean SE
2.77
2.84
2.83
2.81a
2.71 2.43 2.41 2.58 0.O43
2.76 2.55 2.50 2.66
2.87 2.74 2.76 2.80
2.78a 2.57b 2.55b
'Expressed in kg. per hen. Means with the same letter are not significantly different (P > 0.05). 'Standard error of the mean for diets. 2
865
RAPESEED MEAL GLUCOSINOLATES
c as V
vo >n t-^
TABLE 7.—Hens suffering either fatal or non-fatal liver hemorrhages while consuming rapeseed meals of varying glucosinolate content (experiment 2)
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ment
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© • 0.05). Hens fed the control diet laid significantly more eggs than did hens fed any other diet (P < 0.05) (Table 6). There were no significant differences (P > 0.05) in the frequency of fatal or non-fatal liver hemorrhages between hens fed any of the RSM diets (Table 7). Hematoxylin and eosin staining of liver tissues from hens that died of liver hemorrhage showed diffuse edema, massive parenchymal hemorrhages and thrombosis. Similar tissues impregnated with silver demonstrated an almost complete lack of reticulin fibres. These birds generally suffered more severe damage to the liver than did birds that were killed at the end of the experiment. DISCUSSION
•i-* CO
5 2 S 5 00 U CO
££5
The results of Experiment 1 demonstrate that when progoitrin is hydrolyzed under conditions existing in the laying hen digestive
866
T. K. SMITH AND L. D. CAMPBELL
tract, nitrile compounds are the predominant product. This is of note when one considers that the effects of feeding nitrile compounds to poultry have received very little attention compared to the effects of feeding isothiocyanates. The lack of effect of the time between feeding and killing on the relative amounts of hydrolytic products is thought to reflect a lack of significant hydrolytic activity of microbial origin. The three areas of the digestive tract that were investigated represent three distinct pH ranges. It has been shown (Daxenbichler et al., 1966) that pH has a major effect on the pathway of glucosinolate hydrolysis and this is demonstrated by the increased amounts of OZT and CHB and reduced amounts of CHE found in the crop. This pattern was reversed in the more acidic proventriculus and gizzard. Tookey (1973a, b) has recently shown that in Crambe abyssinica an "epithio specifier protein" acts with myrosinase to form a two-protein system. The specific protein determines the relative amounts of the various nitrile products formed during progoitrin hydrolysis. The activity of this protein may be influenced by pH and if present in RSM the alteration of its activity could have produced the observed results. The lack of difference between the small intestine and the gizzard and proventriculus is thought to indicate that hydrolysis was complete by the time this area was reached. Van Etten et al. (1969) have shown that CHB and CHE are acutely toxic to the rat. These same compounds may also have been responsible for the decreased egg production noted in hens fed Target RSM in Experiment 2. The lack of response of the frequency of fatal and non-fatal liver hemorrhages to the dietary levels of glucosinolates may have been accented by the feeding of high levels of RSM. Marangos et al. (1974) reported that laying hens fed medium- and high-glucosinolate RSM suffered significantly greater mortality than did hens fed low-glucosinolate
RSM. These results were complicated by the observation of a similar high mortality among hens fed a mustard seed meal found to be free of glucosinolates by the method of Appelquist and Josef sson (1967). This method of analysis does not determine all possible hydrolytic products of glucosinolates, however, and the importance of doing this is illustrated by the results of the current experiments and is also stressed by Lo and Hill (1972). The destruction of hepatic reticulin fibres seen in the current experiment is similar to that noticed by Hall (1972). It is interesting to speculate that the nitrile hydrolytic products might have an influence in the development of the connective tissue matrix of the liver and hence predispose these birds to liver hemorrhage. ACKNOWLEDGEMENTS This project was supported by a grant from the Rapeseed Utilization Assistance Program. Histological work was carried out by Dr. B. R. Boycott, Veterinary Services Branch, Manitoba Dept. of Agriculture, Winnipeg. Chromatographic standard compounds were generously provided by Dr. M. E. Daxenbichler, Agricultural Research Service, United States Dept. of Agriculture, Northern Regional Research Laboratory, Peoria, Illinois 61604, U.S.A.
REFERENCES Appelquist, L. A., and E. Josefsson, 1967. Method for quantitative determination of isothiocyanates and oxazolidinethiones in digests of seed meals of rape and turnip rape. J. Sci. Food Agr. 18: 511-519. Daxenbichler, M. E., G. F. Spencer, R. Kleiman, C. H. Van Etten and I. A. Wolff, 1970. Gas-liquid chromatographic determination of products from the progoitrins in crambe and rapeseed meals. Anal. Biochem. 38: 373-382. Daxenbichler, M. E., C. H. Van Etten, W. H. Tallent and I. A. Wolff, 1967. Rapeseed meal autolysis. Formation of diasteriometric (2R)-l-cyano-2-hy-
RAPESEED M E A L GLUCOSINOLATES
droxy-3,4-epithio-butanes from progoitrin. Can. J. Chem. 45: 1971-1974. Daxenbichler, M. E., C. H. Van Etten and I. A. Wolff, 1966. (S)- and (R)-l-Cyano-2-hydroxy-3-butene from myrosinase hydrolysis of epi-progoitrin and progoitrin. Biochemistry, 5: 692-697. Drury.R. A. B.,andE. A. Wallington, 1967. Carleton's histological technique. Oxford University Press, New York, New York. Finlayson, A. J., J. Krzymanski and R. K. Downey, 1973. Comparison of chemical and agronomic characteristics of two Brassica Napus L. cultivars, Bronowski and Target. J. Amer. Oil Chem. Soc. 50: 407-410. Hall, S. A., 1972. Lysis of hepatic reticulin: an unusual lesion in laying fowls possibly associated with rapeseed meal. Vet. Rec. 91: 495. Jackson, N., 1969. Toxicity of rapeseed meal and its use as a protein supplement in the diet of two hybrid strains of caged laying hens. J. Sci. Food Agr. 20: 734-740. Lo, M. T., and D. C. Hill, 1972. Glucosinolates and their hydrolytic products in intestinal contents, feces, blood and urine of rats dosed with rapeseed meals. Can. J. Physiol. Pharmacol. 50: 962-966. Marangos, A., R. Hill, B. M. Laws and D. Muschamp,
867
1974. The influence of three rapeseed meals and a mustard seed meal on egg and broiler production. Br. Poultry Sci. 15: 405-414. Snedecor, G. W., 1956. Statistical Methods. 5th ed. Iowa State Univ. Press, Ames, Iowa. 291-328 pp. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc., New York, New York, p. 109. Tookey, H. L., 1973a. Solubilization and selected properties of crambe seed thioglucosidase (thioglucoside glucohydrolase, EC 3.2.3.1.). Can. J. Biochem. 51: 1305-1310. Tookey, H. L., 1973b. Crambe thioglucoside glucohydrolase (EC 3.2.3.1.): Separation of a protein required for epithiobutane formation. Can. J. Biochem. 51: 1654-1660. Van Etten, C. H., W. E. Gagne, D. J. Robbins, A. N. Booth, M. E. Daxenbichler and I. A. Wolff, 1969. Biological evaluation of crambe seed meals and derived products by rat feeding. Cereal Chem. 46: 145-155. Youngs, C. G., and L. R. Wetter, 1967. Microdetermination of the major individual isothiocyanates and oxazolidinethione in rapeseed. J. Amer. Oil Chem. Soc. 44: 551-554.
NEWS AND NOTES (Continued from page 851) New York Poultry Society that had conferred a complimentary membership upon the author. MANITOBA NOTES Dr. Allan C. Cox is the new Chief of the Feed Analysis Service, Animal Industry Branch, Manitoba Department of Agriculture. Dr. Cox was Research Nutritionist at the Agriculture Canada Research Station, Kentville, Nova Scotia. ALBERTA NOTES Miss Dora Graham was appointed Home Economist and Products Promotion Officer for the Alberta Egg & Fowl Marketing Board, Calgary, Alberta, Canada, in September. She is a native of Lethbridge, Alberta, and graduated in Household Economics from the University of Alberta, Edmonton. C.I.S.P. NOTES On November 4, the President, President-Elect, and
Immediate Past-President of the American Society of Animal Science, the American Dairy Science Association, and the Poultry Science Association, met in Champaign, Illinois, and finalized an organizational statement for the Council of Inter-Society Presidents (C.I.S.P.). The Council is composed of the President, President-Elect, and Immediate Past-President of each of the member societies. The Chairman of the Council will be on a rotational basis and the Council will meet annually. The declared purposes are: 1. to provide a mechanism whereby the Presidents of the three societies can coordinate efforts on items of mutual concern, 2. to strengthen and lend support to concerns relating to the animal sciences and technology in extension, research, and teaching and to other matters on which the member societies can function more effectively as a single unit, and 3. to foster meaningful industry-society interrelationships and to convey to the consuming public the
(Continued on page 873)
Lake, P. E., 1957. Fowl semen as collected by the massage method. J. Agric. Sci. 49: 120-126. Lorenz, F. W., 1959. Reproduction in the domestic fowl: Physiology of the male. Vol. 1, pp. 343-390 In: H. H. Cole and P. T. Cupps. Reproduction in Domestic Animals. Academic Press. New York, N.Y. Ostle, B., 1966. Statistics in Research, 2nd Ed. Iowa State University Press, Ames, Iowa. Parker, J. E., and B. J. McSpadden, 1943. Seasonal variation in semen production in domestic fowl.
861
Poultry Sci. 22: 142-147. Salisbury, G. W., and J. R. Lodge, 1962. Metabolism of spermatozoa. Adv. Enzymology, 24: 35-104. Salisbury, G. W., and N. L. Van Demark, 1961. Physiology of Reproduction and Artificial Insemination of Cattle. Freeman Co., California. Chap. 21, pp. 527-530. Singh, Bhupal, B. B. Mabapatro and D. P. Sadhu, 1969. Chemical composition of cattle and buffalo spermatozoa and seminal plasma under different climatic conditions. J. Reprod. Fer. 20: 175-178.
Rapeseed Meal Glucosinolates: Metabolism and Effect on Performance in Laying Hens T. K. SMITH1 AND L. D. CAMPBELL
Department of Animal Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2 (Received for publication June 23, 1975)
ABSTRACT Two experiments were conducted with Hyline Leghorn hens to study the metabolism and detrimental effects of rapeseed meal (RSM) glucosinolates. Raw Target RSM was force fed to 12 hens which were killed after varying time intervals (15 min., 30 min., 60 min.) and the contents of areas of the digestive tract (crop; proventriculus and gizzard; duodenum and ileum) were analyzed for the presence of hydrolysis products of progoitrin. Nitrile compounds were found to be present in all areas of the digestive tract in much larger relative amounts than was oxazolidinethione. When commercially prepared RSMs of varying glucosinolate content were fed to laying hens at a 50% level of dietary inclusion, high glucosinolate-content RSM depressed egg production (P < 0.05) more than low glucosinolate-content RSM but did not cause a greater frequency of liver hemorrhage. Histological examination of liver tissues from hens suffering liver hemorrhage revealed a severe reticulolysis. POULTRY SCIENCE 55: 861-867, 1976
INTRODUCTION
R
APESEED and related Cruciferae have long been known to contain glucosinolates. The compounds may be hydrolyzed by the enzyme thioglucoside glucohydrolase (EC 3.2.3.1.) to produce a variety of potentially toxic products. The predominant glucosinolate in rapeseed of the Brassica napus type is progoitrin which may be hydrolyzed to a mixture of l-cyano-2-hydroxy-3-butene (CHB), diasteriometic l-cyano-2-hydroxy-3, 4-epithiobutanes (CHE) and 5-vinyloxazolidine-2-thione (OZT) (Daxenbichler et al., 1966, 1967). The relative amounts of these
1. Present address: Department of Poultry Science, Cornell University, Ithaca, N.Y.
products vary primarily with the pH of the hydrolysis medium. In a biological evaluation of crambe seed meals and derived products by rat feeding, Van Etten et al. (1969) determined that a mixture of CHB and CHE was more toxic ( = 8 x ) than OZT. Rapeseed meal (RSM) is used in Canada as a protein supplement for poultry feeds although its use has been limited because of the presence of antinutritional or toxic factors. Jackson (1969) determined that the feeding of RSM for nine 28-day periods at levels of up to 20% of the diet to laying hens produced high mortality as a result of massive liver hemorrhage. Hall (1972) observed similar pathology in commercial laying flocks believed to have been fed RSM. Marangos et al. (1974) reported that RSM of high
862
T. K. SMITH AND L. D. CAMPBELL
glucosinolate content caused significantly greater mortality among laying hens than did low glucosinolate-content RSM. The current studies were undertaken to determine the effect of conditions in the laying hen digestive tract on the hydrolysis of progoitrin and to determine the relationship between levels of RSM glucosinolates and the frequency of liver hemorrhage. MATERIALS AND METHODS Experiment 1. Two RSMs were fed in this experiment. One was a commercial meal prepared from Target rapeseed and supplied by Co-op Vegetable Oils Ltd., Altona, Manitoba while the other was prepared in the laboratory from Target rapeseed provided by the Department of Plant Science, University of Manitoba. In the latter case seed was coarsely ground in a Wiley mill and then extracted with hexane for 24 hr. in a Soxhlet apparatus. The extracted product was re-
ground to give a fine powder which was referred to as "raw Target RSM." Twelve 32-week old Hyline White Leghorn hends were randomly selected over a twoweek period in May, 1974 for use in this experiment. Feed (a commercial laying hen diet) was withdrawn from each bird for a period of 18 hr. previous to the test feeding. For each bird consecutively, a sample (30 g.) of raw Target RSM was blended evenly with H 2 O (60 ml.) and transferred to a 100-ml. plastic syringe with a plastic needle. The mixture was injected as deeply as possible into the throat of the test birds. Four hens (three fed raw Target RSM and one the commercial meal) were killed by cervical dislocation at intervals of 15, 30 or 60 min. after force feeding. The digestive tracts of the birds were excised and the digesta from three areas (crop; proventriculus and gizzard; duodenum and ileum) were removed and placed in sealed containers containing methylene chlo-
TABLE 1.—Composition of diets fed in Experiment 21 Diets Ingredient (%) Bronowski rapeseed meal 1788 rapeseed meal Target rapeseed meal Barley Wheat Herring meal Soybean meal Calcium carbonate Oyster shell Deflourinated rock phosphate Soybean oil Vitamin mixture 2 Mineral mixture 3
Bronowski
1788
Target
0.00 0.00 0.00 75.60 0.00 5.00 10.00 2.50 2.50
50.00 0.00 0.00 0.00 37.25 0.00 0.00 2.50 2.50
0.00 50.00 0.00 0.00 41.10 0.00 0.00 2.50 2.50
0.00 0.00 50.00 0.00 37.87 0.00 0.00 2.50 2.50
2.40 0.50 1.00 0.50
2.40 3.85 1.00 0.50
2.40 0.00 1.00 0.50
2.40 3.23 1.00 0.50
100.00
100.00
100.00
100.00
Control
'Calculated energy content for all diets was 2,600 kcal. per kg. 2 Supplied per kilogram of diet: 7,150 I.U. vitamin A, 818 I.C.U. vitamin D 3 , 3.50 I.U. vitamin E, 11.0 meg. vitamin B, 2 , 7.7 mg. riboflavin, 2.2 mg. pantothenic acid, 3.3 mg. niacin, 5.5 mg. choline, 250 mg. santoquin. 'Supplied per kilogram of diet: 80.45 mg. manganese, 44.19 mg. zinc, 4.8 g. sodium chloride.
863
RAPESEED MEAL GLUCOSINOLATES
ride. Samples were mechanically agitated (60 min.) and filtered (Whatman 541). Residues were washed twice with methylene chloride and all washings combined with the extract. Samples were concentrated by flushing with purified N 2 and dried over anhydrous N a 2 S 0 4 (18 hr.). The resultant solution was centrifuged (15 min., 48,000 g) and stored (5°) until analyzed by gas chromatography. Experiment 2. One hundred sixty 22-week old Hyline White Leghorn hens were randomly distributed to 20 raised wire laying cages with eight hens per cage. Four diets (Table 1) were fed for three 28-day periods. The RSMs fed were 1788 (an unnamed low glucosinolate cultivar) and Bronowski (supplied by the Rapeseed Association of Canada) and Target (previously described, commercially-prepared). A standard barley-soybean diet served as a control. Feed consumption and weight gains were determined for each period while egg production was measured on a weekly basis. Initially, hens were exposed to 11 hr. of light daily and this was increased 30 min. at weekly intervals for six weeks. At the conclusion of the experiment all surviving hens were killed and the livers examined for the presence of non-fatal hemorrhage and from birds killed at the termination of the experiment were stained with hematoxylin and eosin and impregnated with silver by the method of Gordon and Sweets (Drury et al., 1967).
Analyses. Hydrolytic products from progoitrin were measured by a modification of the technique of Daxenbichler et al. (1970). An F and M Scientific 700 gas chromatograph equipped with a H 2 flame ionization detector was used. The column was glass (244 cm. x 6.35 mm. o.d.) and packed with \% EGSSX on 100-120 mexh Gas-Chrom Q. Operation conditions were: He flow, 30 ml./min.; air pressure, 20 p.s.i.; H 2 pressure, 16 p.s.i.; injection port temperature, 165° C ; detector block, 250° C. The usual temperature program was 115° C. for 10 min., 115° C. to 180° C. at 2° C./min., 180° C. to 210° C. at 5°/min., 210° C. for the remainder of the analysis. Peaks on sample chromatograms were identified by comparison of retention times with those of the chemically pure standard compounds: (s)-l-cyano-2-hydroxy-3butene; (2s,3s)-l-cyano-2-hydroxy-3,4-epithiobutane; (R)-5-vinyloxazolidine-2-thione. Glucosinolate contents of the RSMs were determined according to Youngs and Wetter (1967). Data were analyzed by the analysis of variance as described by Snedecor (1956). Significantly different means were separated using Tukey's honestly significant difference test (Steel and Torrie, 1960). RESULTS Experiment 1. The relative amounts of the hydrolytic products from progoitrin found in the digestive tracts of raw Target RSM-fed
TABLE 2.—Relative amounts (%) of the hydrolytic products from progoitrin found in the digestive tracts of hens fed raw target RSM Crop 30 12.56 52.36
Proventriculus, gizzard 30 15 60 7.81 9.87 6.87 36.76 30.71 32.18
15 4.53 31.53
ileum 30 8.14 31.37
60 4.98 32.24
SE 0.55 1.82
27.91
28.19
27.34
28.71
25.86
0.82
31.51
32.76
36.60
31.78
36.92
1.14
Product 152 60 OZT 9.75 10.61 CHB 64.03 59.97 CHE (threo) 12.28 17.32 14.50 27.24 CHE (erytho) 13.08 17.76 15.78 28.19 'Standard error of the mean. 2 Interval between feeding and killing (min.).
]Duodenum,
864
T. K. SMITH AND L. D. CAMPBELL
TABLE 3.—Glucosinolate content of rapeseed meals used in experiment 2 Component1 3-butenylisothiocyanate 4-pentenylisothiocyanate 5-methyl sulphinyl-pentylisothiocyanate 2-phenylethylisothiocyanate Oxazolidinethione Total 'All values expressed as mg. per g. 2 Standard deviation of the mean.
Rapeseed meal 1788 0.12 + 0.02 0.02 ± 0.01
Bronowski 0.19 ± 0.012 0.10 ± 0.01
0.00 ± 0.00 0.00 ± 0.00 0.20 ± 0.04 0.49 of meal on an "as fed'
hens are given in Table 2. Only OZT was significantly affected by the time interval between feeding and killing, it being significantly greater (P < 0.05) after 30 min. than after either 15 min. or 60 min. Relative amounts of OZT and CHB were significantly greater in the crop than in either of the other digestive tract areas examined (P < 0.01) between which there were no significant differences (P > 0.05). The diastereometric CHE were found in significantly smaller relative amounts in the crop than in the other areas of the digestive tract between which there were again no significant differences (P > 0.05). Relative amounts of hydrolytic products from progoitrin in the digestive tracts of hens fed the commercial Target RSM could not be determined since all products except CHB were present in concentrations below the limit of detection. Peaks corresponding to the various hydrolytic products were observed, however, and the amount of CHB present was much greater in the crop than in the other digestive tract areas. In all cases the summation of the relative amounts of the three nitrile products greatly exceeded the relative amount of OZT. Experiment 2. Bronowski and 1788 RSMs were determined to contain the same amount of glucosinolates within the bounds of experimental error while Target RSM contained substantially higher levels (Table 3). The
Target 1.02 ± 0.13 0.39 ± 0.03
0.00 ± 0.00 0.00 ± 0.00 0.20 ± 0.04 0.34 basis.
0.05 ± 0.02 0.03 ± 0.01 2.13 ± 0.13 3.62
levels observed for all meals, however, are low and it is possible that some prior hydrolysis to nitrile compounds had occurred. Nitrile compounds are not detected by the method of analysis employed. Furthermore, the values for Target RSM may be disproportionateTABLE 4.—Average weight gains1 of hens consuming RSM of varying glucosinolate content during the three 28-day periods in experiment 2 Period Diet Control Bronowski 1788 Target Mean SE =
Mean 2
2
3
0.055
0.00
0.027
0.027a
0.050 0.082 0.014 0.050a 0.0093
-0.036 -0.023 -0.009 -0.018b
0.014 0.014 0.00 0.014c
0.0O9a,b 0.023a,b 0.000b
1
'Expressed in kg. per hen. Means with the same letter are not significantly different (P > 0.05). 'Standard error of the mean for diets. 2
TABLE 5.—Average feed consumption1 of hens during the three 28-day periods in experiment 2 Period Diet
1
2
3
Mean 2
Control Bronowski 1788 Target Mean SE
2.77
2.84
2.83
2.81a
2.71 2.43 2.41 2.58 0.O43
2.76 2.55 2.50 2.66
2.87 2.74 2.76 2.80
2.78a 2.57b 2.55b
'Expressed in kg. per hen. Means with the same letter are not significantly different (P > 0.05). 'Standard error of the mean for diets. 2
865
RAPESEED MEAL GLUCOSINOLATES
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TABLE 7.—Hens suffering either fatal or non-fatal liver hemorrhages while consuming rapeseed meals of varying glucosinolate content (experiment 2)
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© • 0.05). Hens fed the control diet laid significantly more eggs than did hens fed any other diet (P < 0.05) (Table 6). There were no significant differences (P > 0.05) in the frequency of fatal or non-fatal liver hemorrhages between hens fed any of the RSM diets (Table 7). Hematoxylin and eosin staining of liver tissues from hens that died of liver hemorrhage showed diffuse edema, massive parenchymal hemorrhages and thrombosis. Similar tissues impregnated with silver demonstrated an almost complete lack of reticulin fibres. These birds generally suffered more severe damage to the liver than did birds that were killed at the end of the experiment. DISCUSSION
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The results of Experiment 1 demonstrate that when progoitrin is hydrolyzed under conditions existing in the laying hen digestive
866
T. K. SMITH AND L. D. CAMPBELL
tract, nitrile compounds are the predominant product. This is of note when one considers that the effects of feeding nitrile compounds to poultry have received very little attention compared to the effects of feeding isothiocyanates. The lack of effect of the time between feeding and killing on the relative amounts of hydrolytic products is thought to reflect a lack of significant hydrolytic activity of microbial origin. The three areas of the digestive tract that were investigated represent three distinct pH ranges. It has been shown (Daxenbichler et al., 1966) that pH has a major effect on the pathway of glucosinolate hydrolysis and this is demonstrated by the increased amounts of OZT and CHB and reduced amounts of CHE found in the crop. This pattern was reversed in the more acidic proventriculus and gizzard. Tookey (1973a, b) has recently shown that in Crambe abyssinica an "epithio specifier protein" acts with myrosinase to form a two-protein system. The specific protein determines the relative amounts of the various nitrile products formed during progoitrin hydrolysis. The activity of this protein may be influenced by pH and if present in RSM the alteration of its activity could have produced the observed results. The lack of difference between the small intestine and the gizzard and proventriculus is thought to indicate that hydrolysis was complete by the time this area was reached. Van Etten et al. (1969) have shown that CHB and CHE are acutely toxic to the rat. These same compounds may also have been responsible for the decreased egg production noted in hens fed Target RSM in Experiment 2. The lack of response of the frequency of fatal and non-fatal liver hemorrhages to the dietary levels of glucosinolates may have been accented by the feeding of high levels of RSM. Marangos et al. (1974) reported that laying hens fed medium- and high-glucosinolate RSM suffered significantly greater mortality than did hens fed low-glucosinolate
RSM. These results were complicated by the observation of a similar high mortality among hens fed a mustard seed meal found to be free of glucosinolates by the method of Appelquist and Josef sson (1967). This method of analysis does not determine all possible hydrolytic products of glucosinolates, however, and the importance of doing this is illustrated by the results of the current experiments and is also stressed by Lo and Hill (1972). The destruction of hepatic reticulin fibres seen in the current experiment is similar to that noticed by Hall (1972). It is interesting to speculate that the nitrile hydrolytic products might have an influence in the development of the connective tissue matrix of the liver and hence predispose these birds to liver hemorrhage. ACKNOWLEDGEMENTS This project was supported by a grant from the Rapeseed Utilization Assistance Program. Histological work was carried out by Dr. B. R. Boycott, Veterinary Services Branch, Manitoba Dept. of Agriculture, Winnipeg. Chromatographic standard compounds were generously provided by Dr. M. E. Daxenbichler, Agricultural Research Service, United States Dept. of Agriculture, Northern Regional Research Laboratory, Peoria, Illinois 61604, U.S.A.
REFERENCES Appelquist, L. A., and E. Josefsson, 1967. Method for quantitative determination of isothiocyanates and oxazolidinethiones in digests of seed meals of rape and turnip rape. J. Sci. Food Agr. 18: 511-519. Daxenbichler, M. E., G. F. Spencer, R. Kleiman, C. H. Van Etten and I. A. Wolff, 1970. Gas-liquid chromatographic determination of products from the progoitrins in crambe and rapeseed meals. Anal. Biochem. 38: 373-382. Daxenbichler, M. E., C. H. Van Etten, W. H. Tallent and I. A. Wolff, 1967. Rapeseed meal autolysis. Formation of diasteriometric (2R)-l-cyano-2-hy-
RAPESEED M E A L GLUCOSINOLATES
droxy-3,4-epithio-butanes from progoitrin. Can. J. Chem. 45: 1971-1974. Daxenbichler, M. E., C. H. Van Etten and I. A. Wolff, 1966. (S)- and (R)-l-Cyano-2-hydroxy-3-butene from myrosinase hydrolysis of epi-progoitrin and progoitrin. Biochemistry, 5: 692-697. Drury.R. A. B.,andE. A. Wallington, 1967. Carleton's histological technique. Oxford University Press, New York, New York. Finlayson, A. J., J. Krzymanski and R. K. Downey, 1973. Comparison of chemical and agronomic characteristics of two Brassica Napus L. cultivars, Bronowski and Target. J. Amer. Oil Chem. Soc. 50: 407-410. Hall, S. A., 1972. Lysis of hepatic reticulin: an unusual lesion in laying fowls possibly associated with rapeseed meal. Vet. Rec. 91: 495. Jackson, N., 1969. Toxicity of rapeseed meal and its use as a protein supplement in the diet of two hybrid strains of caged laying hens. J. Sci. Food Agr. 20: 734-740. Lo, M. T., and D. C. Hill, 1972. Glucosinolates and their hydrolytic products in intestinal contents, feces, blood and urine of rats dosed with rapeseed meals. Can. J. Physiol. Pharmacol. 50: 962-966. Marangos, A., R. Hill, B. M. Laws and D. Muschamp,
867
1974. The influence of three rapeseed meals and a mustard seed meal on egg and broiler production. Br. Poultry Sci. 15: 405-414. Snedecor, G. W., 1956. Statistical Methods. 5th ed. Iowa State Univ. Press, Ames, Iowa. 291-328 pp. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc., New York, New York, p. 109. Tookey, H. L., 1973a. Solubilization and selected properties of crambe seed thioglucosidase (thioglucoside glucohydrolase, EC 3.2.3.1.). Can. J. Biochem. 51: 1305-1310. Tookey, H. L., 1973b. Crambe thioglucoside glucohydrolase (EC 3.2.3.1.): Separation of a protein required for epithiobutane formation. Can. J. Biochem. 51: 1654-1660. Van Etten, C. H., W. E. Gagne, D. J. Robbins, A. N. Booth, M. E. Daxenbichler and I. A. Wolff, 1969. Biological evaluation of crambe seed meals and derived products by rat feeding. Cereal Chem. 46: 145-155. Youngs, C. G., and L. R. Wetter, 1967. Microdetermination of the major individual isothiocyanates and oxazolidinethione in rapeseed. J. Amer. Oil Chem. Soc. 44: 551-554.
NEWS AND NOTES (Continued from page 851) New York Poultry Society that had conferred a complimentary membership upon the author. MANITOBA NOTES Dr. Allan C. Cox is the new Chief of the Feed Analysis Service, Animal Industry Branch, Manitoba Department of Agriculture. Dr. Cox was Research Nutritionist at the Agriculture Canada Research Station, Kentville, Nova Scotia. ALBERTA NOTES Miss Dora Graham was appointed Home Economist and Products Promotion Officer for the Alberta Egg & Fowl Marketing Board, Calgary, Alberta, Canada, in September. She is a native of Lethbridge, Alberta, and graduated in Household Economics from the University of Alberta, Edmonton. C.I.S.P. NOTES On November 4, the President, President-Elect, and
Immediate Past-President of the American Society of Animal Science, the American Dairy Science Association, and the Poultry Science Association, met in Champaign, Illinois, and finalized an organizational statement for the Council of Inter-Society Presidents (C.I.S.P.). The Council is composed of the President, President-Elect, and Immediate Past-President of each of the member societies. The Chairman of the Council will be on a rotational basis and the Council will meet annually. The declared purposes are: 1. to provide a mechanism whereby the Presidents of the three societies can coordinate efforts on items of mutual concern, 2. to strengthen and lend support to concerns relating to the animal sciences and technology in extension, research, and teaching and to other matters on which the member societies can function more effectively as a single unit, and 3. to foster meaningful industry-society interrelationships and to convey to the consuming public the
(Continued on page 873)
