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Evidence for a Difference in Toxicity Between Common and Hairy Vetch Seed for Chicks12
271
RESEARCH NOTES
In view of these high correlations, it would appear that a linear relationship as shown in Figure 1 could be used to predict or derive one of the unknown measures from a reported measure. It is of interest to note, however, that the difference between the sums of squares due to regression when the third degree polynomial curve was fitted leads to a significant cubic term. This greater variation "explained" by the cubic curve indicates that a curve of this nature is the best fit for this data and should be used when converting from one of these measures to the other. If the linear relationship is used for conversion, the points close to the midpoint of the line
would perhaps be closely predicted. Conversion at the extremes of this curve, however, would likely lead to greater deviations from the true relationships. This third degree polynomial curve is valid only for comparisons within the specific gravity range reported in this paper, since a wider range may tend to alter the end points and thereby modify the curve. REFERENCES Godfrey, G. F., and R. G. Jaap, 1949. The relationship of specific gravity, 14-day incubation weight-loss and egg shell color to hatchability and egg shell quality. Poultry Sci. 28: 874-889. Olsson, N., 1934. Studies on specific gravity of hen's eggs. Otto Harrassowitz, Leipzig.
EVIDENCE FOR A DIFFERENCE IN TOXICITY BETWEEN COMMON AND HAIRY VETCH SEED FOR CHICKS1 G. H. ARSCOTT AND J. A. HARPER Department of Poultry Science, Oregon State University, Corvallis, Oregon (Received for publication October 7, 1963)
Recently, Kienholz et al. (1962) reported hairy vetch seed, when comprising 93.4 percent of the chick's diet, appeared exceedingly toxic resulting in 100 percent mortality at 3 weeks of age. In contrast, Harper and Arscott (1962) reported no toxicity as measured by mortality from feeding up to 40 percent of hairy vetch seed, Vicia villosa, var. glabrescens Koch, in rations for chicks. However, 20 to 40 percent of common vetch seed, V. sativa L. var. Willamette proved exceedingly toxic. In view of possible misinterpretation of these reports, experiments were conducted involving higher levels of hairy (HVS) and common (CVS) vetch seed2 than had pre1
Technical paper no. 1735, Oregon Agricultural Experiment Station. 2 The authors are indebted to Prof. H. A. Schoth and Dr. Te M. Ching, Dept. of Agronomy for assistance in taxonomic identification of the vetches used in these experiments.
viously been used in our investigations. In the first experiment duplicate lots of 12-day old White Vantress X Nichols 108 chicks, equalized for sex, were raised in electrically heated batteries, in a forcedraft ventilated room, with 24-hour lights. In the second experiment, two separate trials involving duplicate lots of 12 fourweek old White Vantress X Nichols 108 chicks, equalized for sex, that had been on a previous unrelated experiment, were used. These chicks were distributed into 10 groups in such a manner that prior treatment effects were without effect. The chicks were raised in finishing-type batteries and housed under conditions similar to those noted above. The chicks were weighed at the beginning and end of the 4-week period with daily mortality data recorded. In both trials feed and water were supplied ad libitum. The composition of the diet used has
272
RESEARCH NOTES
been reported previously as ration 1 by Arscott (1963). The experimental outline is provided in Table 1. Where 30 percent of vetch was used it replaced an equal amount of corn by weight, and where 84.8 percent of vetch was used it replaced all the corn and soybean meal in the diet. The results for experiments 1 and 2 are given in Table 1. When 30 percent of CVS was included in the diet 100 percent mortality resulted with an average survival time of 13.3 days. In the presence of 84.8 percent of CVS 100 percent mortality was noted with an average survival time of 4.2 days. Chicks fed either level exhibited a high degree of excitability, muscular incoordination, respiratory difficulty, and violent convulsions before death. In the presence of 30 percent of HVS no mortality was observed, although a decrease in body weight gain was evident at 4 weeks of age. When 84.8 percent of HVS was fed, 100 percent mortality occurred with an average survival time of 10.8 days. In no instance did the birds that died show neurological symptoms similar to chicks fed CVS. In contrast these chicks appeared to have starved to death. The results of the second experiment involving birds 4-8 weeks of age show 97.9 and 100 percent mortality with 30 and 84.8 percent of CVS with 11.3 and 8.8 days of survival time, respectively. The neurological symptoms previously noted with CVS were also evident at this age. With 30 percent HVS no mortality was observed while with 84.8 percent 27 percent died with a survival time of 25.4 days. The birds that died when a high level of HVS was included in the diet again displayed no neurological symptoms. However, a loss in body weight gain was evident during the 4 week period and it was obvious these birds were not eating sufficiently. These results serve to explain the apparent discrepancy noted in the two reports previously cited. When 30 percent HVS is
TABLE 1.—Effect of different levels of hairy (HVS) and common (CVS) vetch seed on growth, mortality and survival time of chicks
1 2 3 4 5
gm. % Experiment 1 (0-4 wks.) Control 440(22)' 8.3 30%HVS 281(23) 4.2 84.8% HVS — 100. 30% CVS — 100. 8 4 . 8 % CVS — 100.
1 2 3 4 5
Experiment 2 (4-8 wks.) control 953(47) 2.1 30% HVS 713(48) 0. 8 4 . 8 % HVS - 3 3 ( 3 5 ) 27.1 30% CVS 124(1) 97.9 84.8% CVS — 100. 1
days 27.0 27.4 10.8 13.3 4.2 27.9 28. 25.4 11.3 8.8
Figures in ( ) represent survivors.
fed no mortality occurs. On the other hand, it is quite evident that when HVS is included at 84.8 percent of the diet mortality occurs, although older birds appear less sensitive. Whether or not the mortality is caused by a toxin per se, the presence of growth inhibitors or due merely to palatability can not be presently determined although both groups of investigators have shown beneficial effects from autoclaving. In the studies reported here mortality symptoms between chicks fed the two vetches do not appear the same. In addition chicks fed CVS die sooner than those fed HVS. No description of such symptoms was made by Kienholz et al. (1962). As further evidence for a difference between the two vetches, Arscott and Harper (1963) have recently shown that an ethanol extract of CVS was highly toxic from the standpoint of mortality while a similar extract of HVS was not. In summary these results show both CVS and HVS caused chick mortality when high enough levels were used in the case of HVS. However, a difference in the symptoms noted prior to death was evident with neurological symptoms observed with CVS but not with HVS.
RESEARCH NOTES REFERENCES Arscott, G. H., 1963. Use of barley in high-efficiency broiler rations. 6. Influence of small amounts of corn on improvement of barley. Poultry Sci. 42: 301-304. Arscott, G. H., and J. A. Harper, 1963. The relationship of 2,5-diamino-4, 6-diketopyrimidine, 2,4-diamino-butyrio acid and a crude preparation of beta-cyano-L-alanine to the toxicity of com-
273
mon and hairy vetch seed fed to chicks. J. Nutrition, 80: 2S1-2S4. Harper, J. A., and G. H. Arscott, 1962. Toxicity of common and hairy vetch seed for poults and chicks. Poultry Sci. 4 1 : 1968-1974. Kienholz, E. W., L. S. Jensen and J. McGinnis, 1962. Evidence for chick growth inhibitors in several legume seeds. Poultry Sci. 4 1 : 367-371.
ANALYSIS OF EGG YOLK AND POULTRY TISSUES FOR CHLORINATED INSECTICIDE RESIDUES A. R. STEMP, B. J. LISKA, B. E. LANGLOIS AND W. J. STADELMAN Department o) Animal Sciences, Purdue University, Lafayette, Indiana (Received for publication October 11, 1963) INTRODUCTION
Numerous methods for the analysis of animal and poultry products for chlorinated insecticide residues have appeared in the literature. Most methods lack sensitivity, are specific for one insecticide, or require excessive time for analysis. A semi-quantitative method described by Mills (1959) is sufficiently sensitive and could be used to detect several insecticides, but it requires considerable sample preparation prior to actual analysis. Later, Watts and Klein (1962) reported on the use of electron capture gas chromatography (ECGC) for the detection of nanogram quantities of certain chlorinated insecticide residues. These authors used the Mills (1959) procedure for clean up of samples to prevent detector contamination. There is a need for a rapid clean up procedure for fatty animal product samples. Langlois et al. (1963) reported on a one step florisil column procedure for clean up of dairy products prior to ECGC analysis of selected chlorinated insecticide residues. Since the method was so successful with dairy products, a study was made to determine whether the method could be used to analyze egg yolk and
poultry tissue samples for chlorinated insecticide residues. PROCEDURE A two gram sample of egg yolk, or a one gram sample of poultry tissue or fat, was used for analysis. Either raw or cooked samples were analyzed with the method. Egg yolks were separated from the egg whites prior to sampling. Meat tissues were cut as finely as possible with a scapel prior to weighing a sample for analysis. The samples were ground with 25-30 grams of florisil until a free flowing powder was obtained. A 30 gram portion of florisil was placed in a 20 mm. X 600 mm. pyrex chromatographic column plugged at one end with glass wool. The sample-florisil mixture was placed in the chromatographic column as the top layer. A mixture of 20% methylene chloride in petroleum ether was used as the eluant. From 150 to 650 ml. of eluant were required, depending on the insecticides present. The eluant was evaporate at 50-60°C. in a water bath. Insecticide residues were transferred to calibrated test tubes with hexane. Insecticide residues were analyzed by electron capture gas chromatography as described by Langlois
RESEARCH NOTES
In view of these high correlations, it would appear that a linear relationship as shown in Figure 1 could be used to predict or derive one of the unknown measures from a reported measure. It is of interest to note, however, that the difference between the sums of squares due to regression when the third degree polynomial curve was fitted leads to a significant cubic term. This greater variation "explained" by the cubic curve indicates that a curve of this nature is the best fit for this data and should be used when converting from one of these measures to the other. If the linear relationship is used for conversion, the points close to the midpoint of the line
would perhaps be closely predicted. Conversion at the extremes of this curve, however, would likely lead to greater deviations from the true relationships. This third degree polynomial curve is valid only for comparisons within the specific gravity range reported in this paper, since a wider range may tend to alter the end points and thereby modify the curve. REFERENCES Godfrey, G. F., and R. G. Jaap, 1949. The relationship of specific gravity, 14-day incubation weight-loss and egg shell color to hatchability and egg shell quality. Poultry Sci. 28: 874-889. Olsson, N., 1934. Studies on specific gravity of hen's eggs. Otto Harrassowitz, Leipzig.
EVIDENCE FOR A DIFFERENCE IN TOXICITY BETWEEN COMMON AND HAIRY VETCH SEED FOR CHICKS1 G. H. ARSCOTT AND J. A. HARPER Department of Poultry Science, Oregon State University, Corvallis, Oregon (Received for publication October 7, 1963)
Recently, Kienholz et al. (1962) reported hairy vetch seed, when comprising 93.4 percent of the chick's diet, appeared exceedingly toxic resulting in 100 percent mortality at 3 weeks of age. In contrast, Harper and Arscott (1962) reported no toxicity as measured by mortality from feeding up to 40 percent of hairy vetch seed, Vicia villosa, var. glabrescens Koch, in rations for chicks. However, 20 to 40 percent of common vetch seed, V. sativa L. var. Willamette proved exceedingly toxic. In view of possible misinterpretation of these reports, experiments were conducted involving higher levels of hairy (HVS) and common (CVS) vetch seed2 than had pre1
Technical paper no. 1735, Oregon Agricultural Experiment Station. 2 The authors are indebted to Prof. H. A. Schoth and Dr. Te M. Ching, Dept. of Agronomy for assistance in taxonomic identification of the vetches used in these experiments.
viously been used in our investigations. In the first experiment duplicate lots of 12-day old White Vantress X Nichols 108 chicks, equalized for sex, were raised in electrically heated batteries, in a forcedraft ventilated room, with 24-hour lights. In the second experiment, two separate trials involving duplicate lots of 12 fourweek old White Vantress X Nichols 108 chicks, equalized for sex, that had been on a previous unrelated experiment, were used. These chicks were distributed into 10 groups in such a manner that prior treatment effects were without effect. The chicks were raised in finishing-type batteries and housed under conditions similar to those noted above. The chicks were weighed at the beginning and end of the 4-week period with daily mortality data recorded. In both trials feed and water were supplied ad libitum. The composition of the diet used has
272
RESEARCH NOTES
been reported previously as ration 1 by Arscott (1963). The experimental outline is provided in Table 1. Where 30 percent of vetch was used it replaced an equal amount of corn by weight, and where 84.8 percent of vetch was used it replaced all the corn and soybean meal in the diet. The results for experiments 1 and 2 are given in Table 1. When 30 percent of CVS was included in the diet 100 percent mortality resulted with an average survival time of 13.3 days. In the presence of 84.8 percent of CVS 100 percent mortality was noted with an average survival time of 4.2 days. Chicks fed either level exhibited a high degree of excitability, muscular incoordination, respiratory difficulty, and violent convulsions before death. In the presence of 30 percent of HVS no mortality was observed, although a decrease in body weight gain was evident at 4 weeks of age. When 84.8 percent of HVS was fed, 100 percent mortality occurred with an average survival time of 10.8 days. In no instance did the birds that died show neurological symptoms similar to chicks fed CVS. In contrast these chicks appeared to have starved to death. The results of the second experiment involving birds 4-8 weeks of age show 97.9 and 100 percent mortality with 30 and 84.8 percent of CVS with 11.3 and 8.8 days of survival time, respectively. The neurological symptoms previously noted with CVS were also evident at this age. With 30 percent HVS no mortality was observed while with 84.8 percent 27 percent died with a survival time of 25.4 days. The birds that died when a high level of HVS was included in the diet again displayed no neurological symptoms. However, a loss in body weight gain was evident during the 4 week period and it was obvious these birds were not eating sufficiently. These results serve to explain the apparent discrepancy noted in the two reports previously cited. When 30 percent HVS is
TABLE 1.—Effect of different levels of hairy (HVS) and common (CVS) vetch seed on growth, mortality and survival time of chicks
1 2 3 4 5
gm. % Experiment 1 (0-4 wks.) Control 440(22)' 8.3 30%HVS 281(23) 4.2 84.8% HVS — 100. 30% CVS — 100. 8 4 . 8 % CVS — 100.
1 2 3 4 5
Experiment 2 (4-8 wks.) control 953(47) 2.1 30% HVS 713(48) 0. 8 4 . 8 % HVS - 3 3 ( 3 5 ) 27.1 30% CVS 124(1) 97.9 84.8% CVS — 100. 1
days 27.0 27.4 10.8 13.3 4.2 27.9 28. 25.4 11.3 8.8
Figures in ( ) represent survivors.
fed no mortality occurs. On the other hand, it is quite evident that when HVS is included at 84.8 percent of the diet mortality occurs, although older birds appear less sensitive. Whether or not the mortality is caused by a toxin per se, the presence of growth inhibitors or due merely to palatability can not be presently determined although both groups of investigators have shown beneficial effects from autoclaving. In the studies reported here mortality symptoms between chicks fed the two vetches do not appear the same. In addition chicks fed CVS die sooner than those fed HVS. No description of such symptoms was made by Kienholz et al. (1962). As further evidence for a difference between the two vetches, Arscott and Harper (1963) have recently shown that an ethanol extract of CVS was highly toxic from the standpoint of mortality while a similar extract of HVS was not. In summary these results show both CVS and HVS caused chick mortality when high enough levels were used in the case of HVS. However, a difference in the symptoms noted prior to death was evident with neurological symptoms observed with CVS but not with HVS.
RESEARCH NOTES REFERENCES Arscott, G. H., 1963. Use of barley in high-efficiency broiler rations. 6. Influence of small amounts of corn on improvement of barley. Poultry Sci. 42: 301-304. Arscott, G. H., and J. A. Harper, 1963. The relationship of 2,5-diamino-4, 6-diketopyrimidine, 2,4-diamino-butyrio acid and a crude preparation of beta-cyano-L-alanine to the toxicity of com-
273
mon and hairy vetch seed fed to chicks. J. Nutrition, 80: 2S1-2S4. Harper, J. A., and G. H. Arscott, 1962. Toxicity of common and hairy vetch seed for poults and chicks. Poultry Sci. 4 1 : 1968-1974. Kienholz, E. W., L. S. Jensen and J. McGinnis, 1962. Evidence for chick growth inhibitors in several legume seeds. Poultry Sci. 4 1 : 367-371.
ANALYSIS OF EGG YOLK AND POULTRY TISSUES FOR CHLORINATED INSECTICIDE RESIDUES A. R. STEMP, B. J. LISKA, B. E. LANGLOIS AND W. J. STADELMAN Department o) Animal Sciences, Purdue University, Lafayette, Indiana (Received for publication October 11, 1963) INTRODUCTION
Numerous methods for the analysis of animal and poultry products for chlorinated insecticide residues have appeared in the literature. Most methods lack sensitivity, are specific for one insecticide, or require excessive time for analysis. A semi-quantitative method described by Mills (1959) is sufficiently sensitive and could be used to detect several insecticides, but it requires considerable sample preparation prior to actual analysis. Later, Watts and Klein (1962) reported on the use of electron capture gas chromatography (ECGC) for the detection of nanogram quantities of certain chlorinated insecticide residues. These authors used the Mills (1959) procedure for clean up of samples to prevent detector contamination. There is a need for a rapid clean up procedure for fatty animal product samples. Langlois et al. (1963) reported on a one step florisil column procedure for clean up of dairy products prior to ECGC analysis of selected chlorinated insecticide residues. Since the method was so successful with dairy products, a study was made to determine whether the method could be used to analyze egg yolk and
poultry tissue samples for chlorinated insecticide residues. PROCEDURE A two gram sample of egg yolk, or a one gram sample of poultry tissue or fat, was used for analysis. Either raw or cooked samples were analyzed with the method. Egg yolks were separated from the egg whites prior to sampling. Meat tissues were cut as finely as possible with a scapel prior to weighing a sample for analysis. The samples were ground with 25-30 grams of florisil until a free flowing powder was obtained. A 30 gram portion of florisil was placed in a 20 mm. X 600 mm. pyrex chromatographic column plugged at one end with glass wool. The sample-florisil mixture was placed in the chromatographic column as the top layer. A mixture of 20% methylene chloride in petroleum ether was used as the eluant. From 150 to 650 ml. of eluant were required, depending on the insecticides present. The eluant was evaporate at 50-60°C. in a water bath. Insecticide residues were transferred to calibrated test tubes with hexane. Insecticide residues were analyzed by electron capture gas chromatography as described by Langlois