HI 1 I THE USE OF CHROMIUM-Sl {N DEGESTWE STUDSES OF AVIAN SPECIES IUD—3 _cnoor\3 ['qu Thesis for the Degree of M. S. MICE~iEGAN SEATE UNWERSITY DONALD L. INMAN 1968 THESIS *' " 1L11713.51¢F1 5L.1t' Michigan State University ; *ixTQ'T "' BINDING av "' HUAB & SBN? ABSTRACT THE USE OF CHROMIUM-Sl IN DIGESTIVE STUDIES OF AVIAN SPECIES BY Donald L. Inman Chromium-51 was further evaluated as an inert food marker for studying the digestive characteristics of birds. Previous work using this isotope for studies of the ring- necked pheasant (Phasianus_golchicus) indicated that there might be as much as a ten percent discrepancy between 51Cr-ratio metabolizability coefficients determined by the technique and the conventional total—collection technique (Duke, 1967). Pheasant feeding rates were measured using Turkey 51 Breeder Pellets evenly dosed with CrCl and supplied fig 3 libitum. These data were compared to the total-collection of feces to enable the simultaneous calculation of daily metabolizability coefficients by both total-collection and ratio techniques. Although intestinal feces became marked with 51Cr soon after feeding, low levels of 51Cr character- istically prevailed in cecal defecations during the first 24 hours after ingestion of labelled foods. A valid calcu- lation of the coefficient by any technique, therefore, must Donald L. Inman require that more than 24 hours pass in order for the cecal as well as the intestinal feces to become defecations of the test food eaten. With the 51 Cr ratio method, feces which were residues of the test foods were characterized by a con— stant level of isotope concentration. For all present studies, the average metaboliza- bility coefficient for pheasants was 53.49 percent as de- termined by the 51Cr-ratio technique and 60.40 percent by the traditional total-collection procedure. The average per— 51 centage recovery of Cr for all tests was 89.48. Further trials with chickens (Gallus domesticus) and ruffed grouse (Bonasa_umbellus) were made to investigate the disappearance of 51Cr from birds as well as to obtain digestive data of comparative value. Loss of isotope on pellet crumbs during feeding was found to average 5.87 percent of total isotope ingested for chickens and zero for grouse. Retention of the isotope in the gastrointestinal tracts of chickens following continuous-dose feeding trials was found to occur, but it did not exceed 3.26 percent of the total isotope ingested. "Flaking" and retention of the isotope combined did not ac- count for all the 51Cr which disappeared. The average metabolizability coefficient for chickens fed Turkey Breeder Pellet was 56.52%.as determined by the ratio technique and 62.80% by the total-collection method. Donald L. Inman The average metabolizability coefficient for eight continuous- dose tests with ruffed grouse fed Turkey Growena Number One feed was 57.07 percent by the ratio technique and 61.31 per— cent by the total collection technique. Although averages for the two methods were in closer agreement for the grouse than for either the pheasant or chicken, day to day vari- ations were high even for the same specimens. Ingestion rates for grouse were variable, however, and the passage rate of foods was slower for grouse than for pheasants. These factors seemed to account for daily differences between the two methods but must be further appraised. Single doses of 51Cr allowed ready determination of the time required for the passage of foods through the di— gestive tract. For ruffed grouse, the average maximum passage rate for foods not receiving cecal digestion was 14.5 hours. The average time for this final appearance of radioactive cecal droppings was 53.2 hours. Relative concen- trations of isotope in the droppings, indicated 55.56% of the food eaten by ruffed grouse passed through the cecum. Two tests were completed using the radioactive com- pound 51Cr203 with ruffed grouse. The metabolizability co- efficients as determined by the ratio and total collection techniques were 62.69 and 64.10 respectively for test one and 66.89 and 67.49 respectively for test two. These results Donald L. Inman may indicate that Cr203, the traditional chemical for metabolizability studies, may be more suitable for such in- vestigations than is CrC13. No reason is apparent as yet for this discrepancy but, especially because of the diffi- culty of handling even the stable compound Cr203, it is essential that this matter be tracked to a final solution. THE USE OF CHROMIUMFSI IN DIGESTIVE STUDIES OF AVIAN SPECIES BY Donald Lvanman A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Fisheries and Wildlife 1968 ACKNOWLEDGMENTS My wife, Nancy, deserves an expression of gratitude for her constant encouragement and support. This study was supported by U. S. Atomic Energy Com- mission contract No. AT(ll-l)-1534. Dr. G. A. Petrides of the Fisheries and Wildlife De- partment served as principal advisor and Dr. R. K. Ringer of the Poultry Science Department served as co-advisor. I wish to thank them for many useful suggestions and for critically reading the manuscript. Dr. L. D. Fay of the Michigan Conservation Department provided some test birds and valuable advice for keeping sanitary conditions in the laboratory. Dr. D. E. Ulrey served as a consultant in various phases of the study. Discussions with William W. Mautz, doctoral candi- date at Michigan State University, were invaluable in the completion of my study. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 METHODS . . . . . . . . . . . . . . . . . . . . . . . 6 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 10 Continuous-dose Trials . . . . . . . . . . . . . . 10 Cecal Defecations . . . . . . . . . . . . . . . . 11 51Cr Body Burdens . . . . . . . . . . . . . . . . 12 "Flaking" . . . . . . . . . . . . 14 Variation in Ingestion Rates . . . . . . . . . . . 15 Tests of 51Cr203 versus 51CrC13 . . . . . . . . . 17 Comparative Avian Physiology . . . . . . . . . . . 18 Conclusions . . . . . . . . . . . . . . . . . . . 21 LITERATURE CITED . . . . . . . . . . . . . . . . . . . 39 iii Table LIST OF TABLES Hourly excretion pattern of a ring-necked pheasant fed Turkey Breeder Pellets dosed with 182 counts per minute of 51Cr per dry gram of feed. Test number 5. Michi- gan State University. November, 1967 Digestibility information for pheasan s fed Turkey Breeder Pellets dosed with 5 CrCl3 on a continuous-dose basis. Michigan State University. November, 1967 Metabolizability coefficients for chickens fed Turkey Breeder Pellets, calculated by several methods and corrected for isotope- flaking. Michigan State University. December, 1967 . . . . . . . The degree of loss of 51Cr by surface- flaking when Turkey Breeder Pellets are fed to chickens. Michigan State Uni- versity. December, 1967 . . . . . . 51Cr recovered before and after autopsy with three chickens fed Turkey Breeder Pellets on a continuous-dose basis. Michigan State University. December, 1967 . Digestibility information for four ruffed grouse fed Turkey Growena Number One feed treated with 5 CrCl3 on a continuous-dose basis. Michigan State University. February, 1968 . . . . . . . . Digestibility information for Turkey Growena Number One dosed with 51Cr20 and fed 3g libitum to ruffed grouse. Michigan State University. April, 1968 iv Page 28 3O 31 32 33 34 35 Table Page 8. Digestibility information for ruffed grouse as indicated by 51 CrCl single- dose feeding trials using Turgey Growena Number One feed. Michigan State Uni- versity. January, 1968 . . . . . . . . . . 36 9. A comparison of digestibility data for ruffed grouse and pheasants gained using lCr techniques. Michigan State Uni- versity. February, 1968 . . . . . . . . . . 37 10. Percentage metabolizability data derived from CrCl3 continuous-dose trials for ruffed grouse fed Turkey Growena Number One. Michigan State University. February, 1968 . . . . . . . . . . . . . . . 38 Figure 1. LIST OF FIGURES Page Excretion rate and pattern for a ring-necked pheasant fed Cr—51 dosed Turkey Breeder Pellets on a continuous-dose basis. Test No. 5, Michigan State University. November, 1967. . 25 Defecation rate and pattern fgr a ruffed grouse fed a single dose of 5 CrC13 on Turkey Growena Number One feed. Michigan State University. January, 1968 . . . . . . . . . . . 27 vi INTRODUCTION The development of techniques for the simple and ef- ficient determination of food digestibility percentages is essential in nutrition studies. Previous researchers (Bergeim, 1926; Hoelzel, 1930; Forbes and Garrigus, 1948) used indigestible solid markers or dyes for determining the length of time required for a food to pass through the gastro- intestinal tract as well as to determine the percentage of food digested. Chromic oxide (Cr ) has been used as a 203 food marker with apparent success in poultry and livestock (Irwin and Crampton, 1951; Schurch.§tu_l., 1950; Elam_§t-§l., 1962). The analysis of food and feces in order to quantify this marker, required an involved procedure (Dansky and Hill, 1952). More recently with wild vertebrates radioactive chromium has been used as a digestive marker (Duke, 1967; Mautz and Petrides, 1967; Petrides and Stewart, 1968). 51Cr has a short half-life and is relatively non-hazardous both to the animal species under investigation and to the experi- menter. Its gamma emission enables rapid quantitative de- tection of the isotope in food, in the experimental animal, and in excretory products. The length of time required for a food item to pass completely through the digestive tract of white-tailed deer (Qdocoileus virginianus) and ring—necked pheasants (Phasianus 51 colchicus) was efficiently determined using and Petrides, 1967; Duke, 1967). 51CrC13 also enabled de— terminations of the extent and rate of digestion in the ceca CrCl3 (Mautz (Duke, 1967). Using 51CrC13, the percentage of a particular food which was absorbed as it passed through the digestive tract also was accurately ascertained in the deer (Mautz and Petrides, 1967), cotton rat (Petrides and Stewart, 1968), and other mammals (Petrides, 1964). When this technique was applied to birds, however, descrepancies were encountered in comparing the results of the isotope technique to standard procedures. The conventional method for ascertaining the digesti- bility of a food necessitates measurements of the quantities of all food eaten and of all feces eliminated over a con- siderable period.' Usually data collected over a number of days enable the accurate calculation of dry-weight averages for a 24-hour period. The percentage of food absorbed is feces weight) then equal to 100 (l - food weight The ratio method using 51CrCl3 is less tedious, requiring only the continuous .gg libitum feeding of isotope-dosed food for 48 hours, or so, until the radioactive indicator reaches a stable level in the feces. Only the ratio between the amount of indicator present per unit weight of food and that present per unit weight of feces is required to enable the calculation of the 51 Cr/gram food ) 51 ° digestibility coefficient as 100(1 - Using Cr/gram feces the 51 Cr ratio method, the time and labor-consuming col- lection and measurement of all excreta is avoided. Re- quiring only random samples of food and feces, the determi- nation of isotope levels in the feces is simpler than analy— sis for stable chemical markers such as Chromic oxide (Cr203). Close correlations of the percentages of food ab- sorbed calculated simultaneously by ratio and total coln 1ection techniques were found for both the white-tailed deer (Mautz and Petrides, 1967) and cotton rat (Sigmodon hispidus) (Petrides and Stewart, 1968) using 51CrCl3. But when the ratio technique employing 51CrCl3 was applied to avian species, the percentages of food absorbed as calculated by the ratio method were about ten percent lower than those calculated by the conventional total collection technique (Duke, 1967). Duke (1967) could find no evidence rationale to explain this discrepancy and suggested the need for further research on this point. This study was directed toward additional evaluation of the 51CrCl3-ratio method for determining the percentage of food metabolized in avian species, using a standard diet. Efforts were made to ascertain what metabolic mechanisms were involved in pheasants to explain the discrepancy between the methods and what this indicated in terms of bird versus mammal metabolism. Special regard was placed on the per- centage recovery of 51Cr following a continuous-dose feeding, and attempts were made to locate any "lost" isotope materials. After autopsy, no evidence of digestive absorption of the marker was found in tissues of either birds or mammals (Duke, 1967; Mautz and Petrides, 1967). Visek_gt.al. (1953) reported that "less than 0.5 percent of the dose was absorbed from the gastrointestinal tract as indicated by tissue distribution studies" when the isotope was admin- istered orally as 51CrCl3 to white rats. Absorption of 51Cr203 from the gut of the chicken was found (Vohra and Kratzer, 1967) to be less than 1 percent. 51Chromium in the hexavalent state evidently is ab- sorbed to a greater extent than is the trivalent atom. Roche gt_al. (1957) found an average of 4.8 percent of the total dose of sodium chromate associated with urinary ex— cretions, while Downes and McDonald (1964) reported a maximum of 4.7 percent of 51Cr-EDTA appearing in the urine of sheep. MacKenzie__t__1. (1959) stated that ten times more hexa- valent chromium than trivalent chromium was absorbed when fed to both fasted and nonfasted rats. In the present study, the number of days required for the collection of excreta in order to obtain valid coefficients by total collection and ratio techniques were tested. Ruffed grouse (Bonasa umbellus) were studied in order to collect previously unknown comparative data for an— other gallinaceous species. METHODS Trivalent chromium—51 as 51CrCl3 was used as the digestive marker in this study. Normally inert in the di— gestive tract of animals in this valence state (Roche__t_al., 1957), this isotope has a half-life of 27.8 days. As received from the New England Nuclear Corporation, the 51Cr employed had a specific activity of 47.9 to 181 microcuries per milligram. A Nuclear—Chicago Well Scintil- lation Detector System (DS-202V) with an 8725 Analyzer/ Scaler was used to measure radioactive levels in food and feces. The ring-necked pheasants and the ruffed grouse were supplied by the Michigan Department of Conservation. Chickens were provided by the Poultry Science Department of Michigan State University. All investigations with caged birds were completed in the same room under controlled conditions of heat and light. Constant conditions with the temperature near 78 de- grees Fahrenheit and 14 hours of light per day were maintained. The pens in which the birds were held were wire cages that were l4x14x14 inches. All birds were placed in the test pens at least a week before feeding tests began to insure that they were accustomed to the conditions. The feces-collecting device was a roll of freezer paper 24 inches wide drawn beneath the cage at a constant speed, allowing collection and separation of feces on an hourly basis. The standard diet of the pheasants and chickens was Turkey Breeder Pellets made by the King Milling Company. The guaranteed analysis of this ration was: wheat middlings, 100, yellow corn meal, 1,120, ground oats, 100, 45% soybean oil meal, 200, 17/20% dehyalfalfa, 100, 50% meat/bone scraps, 100, 60% fish meal menhaden, 100, dried whey, 50, brewers dried yeast, 40, iodized salt, 10, dicalcium phOSphate, 20, ground limestone, 38% calcium, 50, Mr4 Vit. trace mineral premix, 5, carbosep, 2 parts by weight. The test ration used for all tests with ruffed grouse was Turkey Growena Number one manufactured by Purina. Its labelled analysis was: crude protein not less than 23.0%, crude fat not less than 3.0%, crude fiber not more than 6.5%, and added minerals not more than 4.0%. Water and foods (whether Cr—labelled or not) were offered_§g libitum in all trials. The techniques used for dosing foods with 51CrCl3 were the same as those developed by Duke (1967) and Mautz and Petrides (1967). Single-dose studies involved only one pellet of the standard diet labelled with 51Cr. The isotope was applied using a lambda pipette at a level large enough to make the food passage rate independent of dose level. Approximately ten lambdas of isotOpe solution were used, containing approximately 10,000 counts per minute of de- tectable radiation. For continuous-dose trials, a simple squeeze-type atomizer was used to apply SlCr to larger amounts of food. As found by Duke (1967), the level of isotope application which allowed satisfactory detection of radioactivity in small normal samples of excreta was approximately 100 counts per minute per gram of food. After spraying with the atom- izer, the food was allowed to air dry for one to two hours. The uniformly-labelled food was then counted carefully, either as samples or as a whole, to determine the average count per minute per gram of food. Labelled foods were presented to the test birds in the morning and the collection device was started then. Excrement samples were collected hourly and removed for study at 12-hour intervals. Collections were continued until no detectable radioactivity occurred in the feces. Samples of excreta for counting were placed in tubes and oven-dried at 100 to 1100 C for 24 hours, or until no further weight losses occurred. After drying, the samples were weighed on a Mettler balance and scaled to ascertain the counts per gram of feces. Counts that were lower than twice the background were not used. All sample counts were corrected for decay and background. The weight of food eaten over the duration of the test was determined. For each trial, food samples were oven-dried at 100 - 1100 C and all food eaten by the birds was recorded on a dry-weight basis. RESULTS AND DISCUSSION Continuous-dose Trials Studies were completed to determine whether the unex- pected non—conforming data of Duke (1967) were due to tech— nique or to differences between avian and mammal digestive processes. Collecting, drying, weighing and counting of radio- active excreta from ring-necked pheasants fed a continuous— dose of 51Cr yielded data on the hourly excretion pattern (Table 1) until a constant count per minute (cpm) per gram of excreta was reached (Figure 1). This "plateau" period was defined by Duke (1967) as the result of each contributing defecation being thoroughly mixed with approximately the same amount of isotope due to the ingestion of uniformly 5J'Cr— labelled foods. The beginning and end of the plateau marked the period that the bird ingested the labelled food. Cecal droppings were normally heavier and also con- tained higher concentrations of isotope than did intestinal droppings over the same 24—hour period. These varied from day to day (Table 1) so that a constant ratio between the two types of droppings did not occur. Because of this, it was necessary to collect all excreta, even when using the 10 11 ratio technique. The average cpm per gram of intestinal droppings and of cecal droppings were calculated and weighted appropriately for a 24-hour period. This value and the average cpm per gram of food as measured previous to feeding were used to calculate the metabolizability coefficients by the ratio method (Table 2). SimultaneouSly, the foods eaten were measured and their weights compared with that of all excreta over the plateau period thus enabling the calculation of metabolizability coefficients by the total collection method (Table 2). The metabolizability coefficients as determined by the ratio method did not parallel the metabolizability coef- ficients as determined by the total collection method. For ring—necked pheasants and chickens (Tables 2,3), statistical analysis using a paired t—test showed that the metaboliza- bility coefficients as determined by the ratio method for those days after the first day of the test were significantly lower than the coefficients as determined by the total col- lection technique at a level between 2% and 5%. Further tests were performed to evaluate this discrepancy. Cecal Defecations Duke (1967) stated that a continuous-dose test of one-day duration was sufficient to allow cecal and intestinal droppings to be marked properly with 51Cr and thus to reach a constant level of isotope excretion. But the two pheasants 12 used in this study displayed slower cecal digestion rates than did his birds. In the present study, the cecal droppings which were excreted within the first 20-24 hours after the dosed food had been presented contained a much lower level of 51Cr than did those cecal droppings excreted beyond one day of ad libitum feeding (Figure l). The metabolizability coef- ficient determined by the ratio technique for the first 24 hours following 51Cr-feeding therefore was lower than the ratio-method metabolizability coefficient determined for the succeeding second or third day of the test (Table 2). At least for those specimens, two days of continuous-dose feeding were required in order for marked cecal feces to at- tain a saturated level of 51Cr. Similar results were ob- tained with ruffed grouse and chickens. Therefore, the data of the first day following presentation of dosed foods did not allow the determination of valid metabolizability coef- ficients. Only the data for days following the first day were used in the remaining 51Cr results. 51Cr Body Burdens The average percentage recovery of 51Cr in the continuous-dose tests with pheasants was 89.48 and much vari- ation in percent recovery from test to test was observed (Table 2), including some recoveries of over 100%» No evi— dent rationale could be given for recoveries above 100%. 13 These findings prompted new investigations to ascertain the fate of the missing isotOpe. The loss of isotOpe evidently was the cause of the discrepancy between metabolizability coefficients determined by ratio and total collection techniques. Duke (1967) had sampled various pheasant tissues for the presence of radioactivity with negative results. A simi— lar search was made in this study to ascertain whether any isotope remained in the digestive tracts of several chickens which became available for sacrifice. After continuous-dose trials were completed, the entire digestive tracts were re— moved, carefully cut into pieces, placed in test tubes with the related food contents, and counted. The dosed food had been discontinued 49 hours before sacrifice for bird A and 25 hours before sacrifice for bird B. Only 2.66 percent of the total 51 Cr ingested was found in the tract of bird A and 0.93 percent in the tract of bird B. Three additional chickens were tested in the same manner, except that the continuous-dose levels of 51Cr- labelled foods were made especially high to insure easier detection of the presence of the isotope in the digestive tract. The dosed foods were removed after three days of ad libitum feeding and all excreta were continuously collected and counted until detectable radioactivity had declined to a level in the cecal droppings that was twice that of the background count. It was then presumed that all 51Cr had 14 been excreted. Upon sacrifice which followed 11—12 hours of chromium-free feeding, the results again showed very low levels of 51Cr remaining in the tracts. In these cases, the percentages of the original dose of 51Cr ingested for the three days found were 3.26, 2.30, and 0.94. Apparently, residues of 51Cr might remain in the digestive tracts of chickens for more than 12 hours after removal of dosed—foods, yet percentage residues were too low to account for the entire discrepancy in results by the two methods. "Flaking" It seemed that since pelleted foods were sprayed with an atomizer to accomplish the dosing process, some 51Cr may have fallen with crumbs from the outer surfaces of the food pellets as the bird ate. To investigate this matter the particles of food remaining at the bottom of the feed dish following completion of a continuous—dose test were separated from the remaining whole pellets and counted for 51Cr levels. In relation to the total 51Cr ingested, "flaking" was found to be a small factor in reducing the average percentage re- covery of the isotope with chickens (Table 4). Only an average of 5.87 percent of the total isotOpe ingested flaked from the food. After allowing for the lower cpm per gram of food ingested, and recalculating the metabolizability coef- ficients by the ratio method, the corrected values were 15 increased on the average by only 1.84 percent (Table 5). When flaking was investigated in ruffed grouse through six continuous-dose tests, essentially none was found to occur. Probably this was because of the differences in feeding habits between the chickens and grouse. The chickens hit the food forcefully with their beaks whereas the grouse picked up each pellet individually and carefully. It was felt that neither the discrepancy between total- collection and ratio techniques nor the less-than—lOO per- cent recovery of the isotope was the sole result of 5lCr flaking from the pelleted diet. Even the combination of di- gestive tract residues and flaking did not account for all "lost" 51Cr (Table 6). Variation in Ingestion Rates In the latest continuous-dose tests with ruffed grouse, the comparison of average metabolizability coef- ficients by total collection and ratio techniques yielded closer results than for pheasants or chickens, yet high day— to-day variations were observed. (A paired t-test showed the average coefficients to be significantly different at a level between 2% and 5%, identical with that calculated for pheasants and chickens.) In these tests, care was taken to 51 count all the Cr-food presented and not just samples of it. 16 Since the detail of counting all food had no effect on the results and since neither the isotope materials re- maining in the digestive tract nor the flaking of the iso- tope seemed to be a main cause of discrepancies between total-collection and 51Cr-ratio metabolizability coefficients, it was thought that perhaps variation in results from test to test or from day to day may have been due to the variable level of food intake. The weight of dry food eaten for each day of the continuous-dose trials was measured (Table 6). Because of the time-lag due to food passage through the digestive tract, which was found to be greater in this Species (see beyond), variable food intake rates may have resulted in failure to associate the proper quantities of food with the consequent fecal residues. This may have affected the daily digestive coefficients of the total-collection technique. The ratio method, however, involved only a constant isotope level in the food and therefore variations in food intake should have had little or no effect, and the computed metabolizability coefficient should have been correct. The percentage recovery of 51Cr was highly variable in ruffed grouse. In fact, recoveries well above 100% were observed in two tests (Table 6) and no rational basis could be assigned for such discrepancies. Dansky and Hill (1952) reported similar recoveries with Cr203. When the metaboliza- bility coefficients as determined by the ratio method were l7 recalculated on the basis of 100% recovery of the isotope, the average for the ratio procedure paralleled that for the total collection method. And when a paired t—test was ap— plied in order to compare the recalculated coefficients for the ratio and total collection methods, the differences were not significant at the 1% level. This indicated that the reliability of the ratio method directly depended upon full recovery of the radioactive marker. a Continuous-dose tests where food levels are standard— ized should be completed to ascertain whether closer day-to- !F _.—_. day correlations between total collection and 51Cr—ratio techniques would result. Tests of 51Cr 0 51 2 3 versus CrCl3 Since Powdered stable Cr203 had been used success— fully in digestibility studies for many years (Edin, 1917), a comparison between 51CrCl3 and 51Cr203 was made in order to evaluate further the 51Cr technique. 51Cr203, yielding 150 counts per minute per gram, the approximate dose of 51Cr when administered as 5J'CrClB, was mixed together with stable Cr203 in order to give a I% level of Cr203 (Schurch, 33 $1., 1950) in Turkey Growena Number One Feed. One grouse each was fed for two and three days, respectively, on this diet. Although the isotope was incorporated into a different compound, the test conditions and methods for 51Cr analysis 18 were the same as in previous tests using 51CrCl3. Even the same two grouse were used for this 51Cr203 trial. The metabolizability coefficient was determined over the same digestive period simultaneously by total collection and by 51Cr203-ratio techniques (Table 7). The 51Cr203 was harder to handle in the dosing pro- cedure than the liquid 51CrC13. Cr2 properties (Vohra and Kratzer, 1967) and separates from the 03 develops electrostatic food during sampling. Excess Cr203 was observed in the feed dish at the test's end during both experiments, i.e. "flaking," yet the metabolizability coefficients determined . 51 W1th Cr203 lower than by the total collection method. This was not by the ratio technique were only about 1% true when 51CrCl was used. This seemed to indicate some 3 difference between 51CrCl3 and 51Cr203 in avian metabolism. It may be that only the use of 51Cr203 is justified in di- gestive trials with avian species. Yet, no reason was ob- vious for requiring the use of this material. Comparative Avian Physiology In order to determine the rate of food passage in ruffed grouse, a single-dose of 51Cr was fed and feces col- lected mechanically. The defecation pattern found for ruffed grouse was similar to that reported (Duke, 1967) for pheasants (Figure 2). As described by Duke (1967), the initial phase where the isotope in excreta increased in concentration l9 relates to a stage during which food materials present in the crop and/or stomach are being mixed with incoming labelled food. A peak of isotope concentration in the feces corresponds to the time when all foods present become thoroughly mixed with 51Cr. A "purging" phase (Duke, 1967) then results as unlabelled foods enter and dilute the remain— ing 51Cr. r From least squares calculations (Ostle, 1965:164), . . ‘.i J! h". the slope of a calculated regression line (Figure 2) indi- cates the proportion of the isotope defecated per hour, re- [i flecting the percentage of food passed per hour (Table 8). The mixing phase and cecal defecations were not used in the computation of this line (Figure 2). The manner in which the isotope was purged from the ceca was similar in pattern to that for the intestine but the rate was slower for the former. The first and last appearances of the isotope in defecations enabled the calculation of minimum and maximum passage rates for Turkey Growena Number One. The average maximum intestinal passage rate for this diet in ruffed grouse was 14.5 hours, whereas for the similar Turkey Breeder Pellet with pheasants (Duke, 1967) it was nine hours (Table 9). The maximum passage rate for food undergoing cecal di- gestion was 53.2 hours for ruffed grouse and 39.4 hours for pheasants. Although the food was somewhat different, the 20 data seem to indicate mainly bird-species rather than food differences. The single-dose technique also enabled determination of the relative amount of food which entered the ceca of grouse. As described by Duke (1967), the percentage cecal influence is 100 times the counts per minute recovered in all radioactive cecal defecations divided by the total counts per minute recovered from all "hot" excreta. The average percentage of cecal influence (Table 9) was 55.56 for grouse and only 13.31 for pheasants (Duke, 1967). The increase in metabolizability of Turkey Growena Number One due to cecal digestion also was calculated. This was accomplished by determining the metabolizability coef- ficient by the ratio technique, using only the average cpm per gram of intestinal excreta during the plateau period. The resulting metabolizability figure was then subtracted from the metabolizability coefficient determined by using the average cpm per gram for all plateau feces on a weighted basis. The average percentage increase in metabolizability due to cecal digestion on a continuous-dose was thus de- termined as 34.93 (Table 10). This was about three times higher than the 10% reported for the pheasant (Duke, 1967). It was expected that cecal efficiency in digestion would be higher in a bud-eating species such as the grouse, where coarse foods are eaten, but not to the degree that it was observed with the standard diet. 21 Conclusions Despite the evident values of 51Cr in digestive studies of mammals, the 51Cr-ratio technique for determining the metabolizability coefficient of a standard diet was not satisfactorily precise for at least some of the avian species tested when 51CrCl3 was used. In both pheasants and chickens, “5 the average coefficient as determined by total collection was z‘j Jml? ' J on the order of 7% higher than the coefficient yielded by the ratio method. Based on the observed low recovery of the iso- \ ' b.1".’ tope, this discrepancy seemed to be a deficiency of the ; SlCrCl3 rather than of the ratio technique. Retention of residual amounts of 51 Cr in the digestive tract and loss of the isotope on food crumbs were insufficient in any of the species studied to account for all of the unrecovered isotOpe ingested in continuous-dose tests. In ruffed grouse, the averages of the metaboliza— bility coefficients as determined by ratio and total col— lection techniques were in close agreement. High day—to-day variation was observed, however, and this may have been in- duced by the highly variable daily ingestion rates observed combined with the slow food passage rates found in this species. Average metabolizability coefficients determined by the 51CrC13-ratio technique were more valid for the grouse as compared with total collection methods, than were those calculated for the pheasant and chicken. Possibly this 22 occurred because no food—flaking was found and the number of tests and birds were greater for the grouse. The number of days needed for collection of excreta in order to obtain valid coefficients by total collection and ratio techniques was established using 51CrClB. The ap- pearance of low, increasing levels of isotope in cecal feces i‘j during the first 24 hours after the ingestion of evenly and continuously-dosed foods proved that those feces were not entirely composed of residues from foods eaten during the first day. The final appearance of a constant level of iso- tope in feces established the total time required for the collection of Cr-marked excreta. The intervening plateau level was first achieved after the first 24 hours. Whether 51Cr method or the total collection method is used, it the became evident that time should be allowed for cecal as well as for intestinal feces to become entirely made up of the test food materials being ingested. In the several species tested, it was found that at least 24 hours should pass be— tween the first feeding of 51Cr-marked food and the first collection. 51Cr was useful in the efficient measurement of di— gestive passage rates for a given diet. Using the single- dose 51Cr procedure, the first and last appearance of iso- tope in cecal and intestinal excreta indicated the minimum and maximum times required for food to pass through the di- gestive tract. 23 The relative levels of isotope in cecal and in- testinal feces allowed calculation of the percentage of di— gestion which occurred in the intestine as contrasted with that which involved the ceca. Comparisons of data for grouse and pheasant indicated that the ceca played a large role in the former species, much greater than in the pheasant. F71 Continuous-dose tests using 5J'Cr203 with ruffed grouse seemed to indicate that this compound yielded more ac- curate metabolizability coefficients by the ratio technique than did 51CrCl . Percentage digestibilities determined by 3 L..— total collection and ratio methods using 51Cr203 showed less than.a 1% discrepancy. Although there are annoying electro- static qualities of Cr203, it is suggested that (a) this ma- terial be used to test further its suitability for determi- nations of metabolizability coefficients, and (b) a search be made for a more convenient 51Cr liquid compound which 51 51 would combine the advantages of CrCl3 with those of Cr203. 24 .homa .quE0>oz .hufimum>flcb wumum cmmflsoaz .m .02 umma .mHmMQ mmOplmsoscflucoo m co muwaamm umpmwnm hexane pmmoc amino 6mm beammmzm pmxomCImcHn m How cumuumm pom mums coflumuoxm .H ousmflm coflummmCH umumm mnsom mmaflmmoup amowu mmcHQQOHU Hmcflummucfl 25 OH ‘OOH mb/mdo woooH Figure 2. 26 Defecation rate and pattern for a ruffed grouse fed a single dose of 51CrCl3 on Turkey Growena Number One feed. Michigan State University. January, 1968. 27 . = intestinal drOppings 0,000. o = cecal drOppings <3 "3 G i 0 ! L. . 1,000 - ' Q E ~$ E O m o . 0 100- I 5 10 15 20 25 3b 3'5 40 45 so 55 60 Hours after Ingestion I d 28 Table l. Hourly excretion pattern of a ring—necked pheasant fed Turkey Breeder Pellets dosed with 182 counts per minute of 51Cr per dry gram of feed. Test number 5. Michigan State University, November, 1967. Hours Counts per After Dry Grams Minute per Inges ion of Gram of Average Cpm/gm of Cr Excreta Excreta Intes. Cecal Combined 1 0 0 2 .317 104 3 .216 380 4 .013 + *4c .914 162 5 .342 278 6 1.317 175 7 .273 271 8 .945 243 9 .804 228 9c .665 929 10 .389 360 11 .679 342 12 .833 292 13 .102 333 14 .522 230 15 0 0 16 .699 263 17 . 0 0 18 .762 315 19 .807 302 20 0 0 21 .443 201 22 .397 232 23 0 0 24 1.158 254 Day 1 267 546 301 25 .195 + 26 0 0 26c .810 1,306 27 .578 109 28 .581 169 29 .659 349 30 .645 333 31 .626 310 32 .814 280 33 .446 209 33c .544 1,267 34 .735 352 35 .194 186 t J". A"!k.,.. In... t 29 Table l--Continued J ’7 1 I Hours Counts per After Dry Grams Minute per Ingestion of Gram of Average Cpm/gm of 51Cr Excreta Excreta Intes. Cecal Combined 36 1.378 238 37 .105 371 38 .288 174 39 .593 231 40 .370 268 41 l 0 42 .764 275 43 .501 232 44 .847 197 45 .776 249 46 0 0 47 .865 224 48 1.765 261 Day 2 251 1,287 345 49 .306 219 50 .312 90 51 . .401 77 52 .523 228 52c 1.568 1,203 53 .563 229 54 .559 318 55 .540 331 56 .613 294 57 .501 285 57c .650 1,288 58 1.040 312 59 .889 229 60 .850 289 61 .465 249 62 .317 221 63 0 0 64 0 0 65 1.214 294 66 .410 239 67 .469 156 68 1.068 267 69 0 0 70 0 0 71 1.928 209 72 .863 261 Day 3 240 1,246 331 hour c = cecal excreta + = count below twice the background count 30 Table 2. Digestibility information for pheasants fed Turkey Breeder Pellets dosed with 51CrC13 on a continuous- dose basis. Michigan State University. N0vember, 1967. Metabolizability Coefficients Total— Percentage Day of Ratio Collection Reco ery Test Bird Test Method Method of 5 Cr 25% g . 1 Mrl 1 37.40 44.82 108.12 i 2 M71 1 40.30 58.74 97.81 Q 3 Mel 1 42.23 54.37 92.14 i 4 M21 1 46.62 71.05 t 2 55.83 59.11 3 60.82 59.48 91.62 5 Mel 1 39.54 63.57 2 47.25 65.44 3 52.73 62.39 4 55.29 55.29 5 49.02 60.69 74.84 6 F-l 2 .ééléé .91ng 72.34 AVERAGES 47.99 59.70 89.48 Averages ex- cluding day 1 53.49 60.40 of each test 31 Table 3. Metabolizability coefficients for chickens fed Turkey Breeder Pellets, calculated by several methods and corrected for isotope-flaking. Michigan State University. December, 1967. Metabolizability Coefficients Determined by 51 F Day of CrCl3 Corrected Total 1 . Bird Test Ratio Ratio Collection 1 F—2 2 56.80 57.70 63.40 ' 3 53.75 54.72 62.15 F-3 2 50.89 52.85 63.81 5-i 3 58.82 60.47 62.59 F-4 2 54.10 56.85 64.93 3 53.73 56.54 59.94 AVERAGES 54.68 56.52 62.80 32 Table 4. The degree of loss of 51Cr by surface-flaking when Turkey Breeder Pellets are fed to chickens. Michigan State University. December, 1967. *1 Grams of Excess Isotope It? Crumbs Isotope on % of Total Bird Collected Crumbs* Cpm** Dose Eaten Mrl .63 816 729 1.96 J F-l 3.30 1,348 883 2.89 L; F-2 3.15 2,593 1,974 4.34 F—3 4.10 5,693 4,759 8.12 F-4 13.50 9,038 6,206 1249; AVERAGE 5.87 *In counts per minute on all crumbs remaining at the end of the test. **Counts above the average for dosed whole pellets be- fore the test began. 33 Table 5. 51Cr recovered before and after autopsy with three chickens fed Turkey Breeder Pellets on a continuous- dose basis. Michigan State University, December, 1967. Isotope Detected (cpm) Total Gastrointestinal Percent Bird Eaten Excreta tract Crumbs Recovered F—2 45,533 36,154 1,289 2,127 86.90 F-3 58,575 46,030 1,349 4,759 89.01 F-4 51,522 39.724 482 6,206 90.08 Asmmumm coHuwmmcH CH GOHHMHHm>sv uxoa mom t AsmcoHumomme Hmooos "axes oomv UwUMHsono uoz + 34 hv.Nm No.Ho Hm.Ho no.hm mmwm NN.n©* mo.mo mo.mm mm.¢v Nm.oH N + + mm.0H H Him m em.vm mo.mm 0N.nm Hm.om Hm.¢H N + + mw.h H mix 5 mN.>© Ho.No Nm.mo 0N.¢H m mn.vm HH.¢© mo.Nm hH.Nm mm.MH N + + mm.HH H Him 0 mm.mm OH.oo on.Hm mp.0H m wH.¢m mm.¢m om.¢¢ mH.m¢ mH.o N + + hm.¢H H m1: m em.mot NN.mo 0H.oo oo.m¢ mh.mH N + + ew.¢H H N12 e mm.vm mm.mm «m.@m 0H.m¢ HN.>H N + + m¢.mH H H1: m om.Nm N5.N> mn.mm mo.mH m oo.mHHt Hm.oo om.m© om.©o mm.MH N + + hm.m H N12 N Hm.mNH« no.mo n¢.mm em.mn em.mH N + + Nm.MH H H12 H mnm>ooom 0m0u0mH mo >Hm>oomm pocuoz ponumz coumm umoB anm puma HOHm mo XboH mo mHmma map so coHuooHHou oHumm poom mum mo awn mommucmouom 0Humm pwumHsonomm Hmuoa mo mEmuw mosoeoemmooo SeHHHneNHHonmeoz .momH .mumsunwm .muHmno>HcD wumum cmchon .mHmmn mmOpnmooscHueoo m so mHoHo suHa pmummuu 608m 0:0 Hensoz mcmaouw Nexusa 60m mmsoum common meow How cmmmeuomcH NuHHHbHummmHo .o 0Hhme 35 Table 7. Digestibility informatgin for Turkey Growena Number One dosed with Cr203 and fed_ag libitum to ruffed grouse. Michigan State University. April, 1968. Metabolizability,Coefficients Day of 51er203 Total Collection Bird Test Ratio Method Method % Recovery M—3 3 62.69 64.10 87.07 F-l 2 66.89 67.49 100.55 36 P .15". 9:21.235": 2. .41.... 21.20.!— .mump mumHmEoucH u + 00.00H mm.mv om.mm N.mm m.hH m.vH h.N mm0<fim>¢ 3.1.: flm 3.3 INN Ila! IImIH. w. T: Ho.HNH OH.mo Hm.N¢ 00 m m m H12 mm.m0H wb.m¢ mm.vm we ¢N NH m N12 mm.¢0H Nh.m¢ hm.nv he NH MH N HLE mo.mm Hm.©m om.m© he vN vH m N12 ¢¢.mm >¢.mv om.mm om wN HH N H12 mm.mm + + + NH ON m N12 mm.mw mN.mm + + NH mH m H1: HOHm Hsom mom mocmsHmcH Esemez EseHcHZ EsEmeZ EBEHCHS pHHm mo mum>oomm mmmwwmm poom Hmuwo Hmumo HmcHummHCH mmmucmonom ommucmouom ommpcmouom 1| kusomv mmwmm wwmmmmm .momH amumSCmb .munHo>HCD mmmpm cmmHonS .pmmm 0:0 Monasz mCoBOHO hmxuse mchs mHMHHu mchmmm 0wOplmHmch HOHOHm ma poumoHch mm omsoum Ummmsu Mom coHumEHomcH muHHHQHumomHQ .m oHQmB 37 .HhomH .mxsnv mumHHmm Hmpwwum >0HH59 pom N .oco Hmnfisz mcm3ouw mmxnse pmmH Nm.HH om.mm Hm.mH o.mm 0.0 o.m H.H ucmwmmsz mm.vm mm.©v om.mm N.mm m.nH m.¢H h.N wmsoum pwmmsmH coHummmHn HHmcHummuch Amowo mnu .xmz .CHS .xmz .CHS Hmomu on man “now mom mCHkuco HmmE Hmomo HmcHumoHCH huHHHQMNHHOQmuwz mmmmmmm poom HMDOH mo “G50Emv Amnsomv wumm CH mmmeUCH wmmucwouom mocmsHHCH Hmowo mmmmmmm mmmum>4 wmmucooumm wmmum>¢ wmmucwouom ommwm>¢ mmmnwkd .eomH .Namsunom .muHmHm>HCD mumum cmmHsoHS UoQHmm wpcmmmwna pom wmsomm powwow How mump NHHHHQHpmmep mo COmHHmQEoo 4 .mosUHccowu HUHm mchs .m mHnt 38 Table 10. Percentage metabolizability data derived from CrC13 continuous-dose trials for ruffed grouse fed Turkey Growena Number One. Michigan State University. February, 1968. Metabolizability Coefficients Day of Intestinal Cecal Intestinal Plus Bird Test Feces Feces Cecal Feces Mel 2 38.22 35.12 73.34 Mr2 2 35.82 31.08 66.90 3 46.63 13.12 59.75 M-1 2 22.94 26.16 49.10 M-2 2 17.25 32.41 49.66 Mr3 2 5.67 42.52 48.19 3 13.05 38.65 51.70 F—l 2 30.00 32.17 62.17 3 35.78 29.74 65.52 Mr3 2 27.22 29.29 56.51 F-l 2 00.00 44.88 44.88 Mk3 3 9.10 53.59 62.69 F-l 2 21.47 _4§;42 66.89 AVERAGES 23.32 34.93 58.25 1? Bergeim, Curran, Dansky, Downes, Duke, G. Edin, H. Elam, L. Forbes, Hill, F. LITERATURE CITED 0. 1926. Intestinal chemistry IV. A method for the study of food utilization or digestibility. J. Biol. Chem. 70:29-33. M. K., J. D. Leaver, and E. W. Weston. 1967. A note on the use of Chromic oxide incorporated in a feed to estimate fecal output in ruminants. Animal Pro- duction. 9(4):561-564. L. 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Med. 84:610-615. 1'11 17: 111' 2046 E w N U E T A! ummsuum