Wl Wllfllflllh N L 1 l w is: “WWWWlWlHl1LlLUlll‘\l 752 MLYETHYLENE GLYCOt AS AN NDECATOfi QF DIGESTEBELEW (N DAIRY” CQWS Thesis For “:19 Degree og M. S. MECHIGAN STATE UNIVERSETY Lloyd E, Christie 1957 THFSW} LIBRARY Michigan State University 'w-V‘Irw POLYETHYLENE GLYCOL AS AN INDICATOR OF DIGESTIBILITY IN DAIRY COWS By LLOYD E. CHRISTIE AN ABSTRACT Submitted to the College of Agriculture of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Dairy 1957 Approved (ca/{AA ABSTRACT LLOYD E. CHRISTIE Eight mature dairy cows on hay-grain rations were used to evaluate the use of polyethylene glycol (PEG) as an inert reference substance in ruminant digestion studies. Molecular weights of h,000, 6,000, 9,000, and 20,000 were tested, using a turbidimetric procedure to analyze the feces for PEG. Fifteen gm. of PEG was administered daily to each cow. Recoveries of PEG after passage through the digestive tract were: Pm-h,ooo, 77.7% (71.h-82.9%) ; I’m-6,000, 60.7% (Sis-66.0%); PEG-9,000, 5&.7%; and Pas-20,000, 50.1%. Excretion curves representing 2h-hour periods revealed a definite excretion pattern with lowest concentrations of PEG at h:00 p.m. and highest concentrations at 11:00 p.m. Average recovery of PEG added to dried feces was 103.7%. When PBS was added to fresh feces, 78.7% of PEG-h,000 and 66.8% of PEG-9,000 was recovered. Essentially a linear relationship was found between weights of dried feces added to aqueous PEG solutions and weights of PEG re- moved from the solutions. The most plausible explanation for the low recoveries observed, in view of the data presented, would seem to be adsorption of the PEG on the feces. Under the conditions of the present ex;eriment, PEG was not found to be a satisfactory indicator of digestibility. POLYETHYLENE GLYCOL AS AN INDICATOR OF DIGESTIBILITY IN DAIRY COWS By LLOYD E. CHRISTIE A THESIS Submitted to the College of Agriculture of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE ’ Department of Dairy 1957 /—— 7")“3 @275! ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Doctor C. A. Lassiter, Associate Professor in Dairying, and Doctor C. F. Huffman, Research Professor in Dairying for their counsel, guidance, and critical reading of the manuscript, and to Doctor N. P. Ralston, Professor in Dairying for the award of the Graduate Assistantship and provision of the facilities necessary for conducting the study. The author wishes to express his gratitude to Doctor J. R. Brunner, Associate Professor in Dairying, and Hr. A. D. McGilliard and Mr. G. F. Fries, Graduate Assistants in Dairying, for their technical contribu- tions, Hr. C. W. Duncan, Research Professor in Agricultural Chemistry for his suggestions, and Hr. Ralph Reid, herdsman, for care of the animals. The author is also indebted to Dr. G. L. Crenshaw of the Dow Chem- ical Company, Midland, Michigan for the polyethylene glycols used in this experiment. TABLE OF INTRODUCTION . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . Silicon . . . . . . . . . . . IronOxide ......... Titanium Dioxide . . . . . . BariumSulfate ....... Dyes . . . . . . . . . . . . Chronic Oxide . . . . . . . . Lignin ........... Methoxyl Groups . . . . . . . Normal Acid Fiber . . . . . . Fecal Nitrogen . . . . . . . Plant Pigments . . . . . . . Polyethylene Glycol (PEG) . . EXPERIMENTAL PROCEDURE . . . . . . RESULTS AND DISCUSSION . . . . . . 22.2132 Recovery of PEG . . . EMRecovery of PH} . . Excretion Curves of PEG . . . Possible Mode of Loss of PEG SUMMARY ............. LITERATURE CITED .-. . . . . . . . CONTENTS Page \J'lU'LE‘PWUJU 10 10 10 10 11 1h 18 19 22 2h 26 28 29 LIST OF TABLES Page Table I Percent Recovery of Polyethylene Glycol-h,000 after Passage through the Digestive Tract-- Trial 10 O O O O C O O O O O O O O O O O O O O O O C 20 Table II Percent Recovery of Polyethylene Glycol after Passage through the Digestive Tract--Trial II. . . . 21 Table III Recovery of Polyethylene Glycol-h,000 from Dried Feces O O C O C O O O O O O O O I O O O O O O 22 Table IV Recovery of Polyethylene Glycol added to Fresh Feces o o o o o o o o o o o o o o o o o o o o o o o 23 Table V Change in Concentration of Polyehylene Glycol Solution with Addition of Dried Feces . . . . . . . 2h LIST OF FIGURES Page Figure 1 Excretion Curves of Polyethylene G1ycol-h,000 . . . 25 INTRODUCTION The need for a reliable indicator of digestibility has long exist- ed. For many years experimenters have searched for an inert substance which would pass through the digestive tract in such a manner that digestion coefficients could be determined indirectly through ratio techniques. Recent mounting interest in the measurement of forage di- gestibility with the grazing animal has increased the significance of indicators in nutrition work. Indicators may be used in large, group-feeding experiments since no Special feeding or collecting equipment is normally required. In- direct methods therefore may be less expensive than direct methods of determining digestibility. In the conventional approach of estimating digestibility; % appar- ent digestibility - 100 - 100 % nutrient in feces X feces weight 1 nutrient in feed feed weight The digestibility formula, when using indicators becomes; Z appar- ent digestibility - 100 - 100 Z nutrient in feces X. % indicator in feed gInutrient in feed iIindicator in Igces Digestibility indicators may be divided into two categories; "ex— ternal" and "internal“ indicators. External indicators are those not normally present in the feedstuff, while internal indicators are nat- ural feed constituents. To give a valid estimation of digestibility an external indicator substance must (1) be quantitatively recovered in the feces (not pro- duced, absorbed, broken down, or adsorbed in the digestive tract); (2) be non-toxic to the animal and the microorganisms of the digestive tract; (3) have no effect on the digestibility of the ration; (h) pass through the digestive tract in a manner identical to the nutrient or nutrients being studied; (5) be readily analyzed by physical or chemical methods; and (6) have a distinct excretion pattern over the 2h-hour day. It is desirable that the external indicator substance (1) be in- expensive, and (2) reach an equilibrium rapidly in the digestive tract. Since no material has thus far been shown to satisfy all these re- quirements, the present experiment was conducted to further evaluate polyethylene glycol as an indicator substance. REVIEW OF LITERATURE The earliest known balance trials were conducted with a milk cow in 1839 by Boussingault. Digestibility trials were conducted in Germany by Henneberg and Stohman in 186b, according to Schneider 33 El. (1955). Just 10 years later, the first indicator of digestibility was used. Silicon. ‘Wildt (187D) made use of silicon to indirectly study the digestibility of hay and straw by sheep and Obtained 86.1-91.6% recovery of the indicator. Close approximations of digestibilities were observed by Skulmowski gt 21° (19h3) with sheep and horses, and Bruce and Willcox (19h9) with rabbits, using silicon ratios. Gallup (1929) obtained di- gestibilities of protein by rats using the silicon ratio method which varied.by only 0.01-0.50% from those obtained by the conventional method. Knott'gEIEI. (1936) concluded that the silicon ratio technique was unreliable due to contamination of the feed with soil. Gallup and Kuhl- man (1936) observed wide variations in silica content of silage and recovered only 85% after passage through the digestive tract of cows. Mineral balance studies by Forbes gt 51. (1916) indicated the existence of extensive metabolism of silicon by the cow. Gallup 22.3i' (19h5) recovered 105.6-111.6% of silica fed to sheep. Gallup and Kuhlman (1936) observed variations in feces concentration of silicon during the day Vim COWS. Iron Oxide. Bergeim (1926) proposed the use of iron oxide as an indigestible reference material. hamilton st 51. (1927) found close agreement between the results of iron oxide ratios and the conventional digestibility trial technique with steers. Favorable results were also reported by Gallup (1929) with rats. Low digestibility estimates were reported using iron oxide ratios by Gallup (1928) and heller gt El. (1928) with rats, Moore and'Winter (193h) with cows, and Druce and Willcox (19149) with rabbits. The use of naturally occurring iron in the feedstuff as an indicator was suggested by Heller 23.3i° (1928). Knott 22.2l! (1936) pointed out that iron occurred in feeds in such small amounts that determination methods were inadequate. Hale 23.5i! (19h0), working with cows, con- cluded that the iron ratio technique was unreliable due to accumulation of iron in the rumen in some instances and passage from the rumen.ahead of the ingesta at other times. Titanium Dioxide. Fournier gt 31. (195M reported that titanium dioxide passed through the digestive tract of the rat at a rate similar to that of phOSporous and was useful in absorption studies of that ele- ment. Lloyd'gt'gl. (1955) reported 99.8% recovery of titsnium.dioxide over a 7-day period with rats. gerigm_5ulfate. ‘Brown (195h) found great variations in the concen- tration of barium sulfate from day-to-day in the feces of cows. Muller (1953) encountered difficulties in chemical determination of this indi- cator from the feces of chickens. Radioactive barium sulfate was tested by Van Der Kley (1956), who reported large variations in excretion rate and large day—to-day variations with steers. EE' Anthraquinone violet was found to be rapidly absorbed from doubly-ligated rumens of open-abdomen calf preparations and rumen-fistula calves by Flatt _e_t_. 31. (1956). An average recovery of 72.11% was found after 1 hour. Corbin and Forbes (1951) obtained an average recovery of 100.5% of this dye and observed a variation in feces concentration dur- ing the day with sheep. Monastral blue (copper phthalocyanin) was tested by Coup and Lan- caster (1952) by addition to cow feces. The average recovery was 99.6%. Lambourne (1957s) obtained 101-1091 recovery of a single dose of this dye in 72 hours using sheep. Slightly lower recoveries were observed when administered in a hard gelatin mixture than in powder form. Higher recoveries of this dye were found with morning than evening dosage. ”011 Red 0" was only 81.6% recovered from lambs by Corbin and Forbes (1951). Chronic 21.1.9.3.“ The most widely used external indicator of digest- ibility is chronic oxide (chromium sesquioxide). Edin (1918) recovered chronic oxide almost completely when fed in the ration impregnated in blotting paper. Edin (1926) reported that a 2-day collection period using chronic oxide ratios gave as accurate digestion coefficients as a standard 7-day trial. Acceptable recoveries and digestion coefficients using chronic oxide ratios were obtained by Kane e_t _a_1_. (19149, 1953a, 1953b, 1957), Crampton and Lloyd (1951), Smith and Reid (1955), and Putnam gt 5;. (1957) work- ing with cows; Hardison and Reid (1953) and Brannon at 21; (1951:) With steers; and Armstrong and Preston (1951;) with calves. The chronic oxide ~6- technique was considered valid by Andersen (l93h), Skulmowski (19h3), Raymond and Minson (1955), Woolfolk gt g_1_. (1955), and Pigden and Brisson (1956) using sheep. Satisfactory recoveries of chronic oxide were also reported by Skulmowski (1951) with horses; Schurch (1952) and.Schurch 23.5l' (1952) with swine; Chanda 3_t_ 31. (1951) with goats; Lloyd and McCay (195A) with dogs; Eriksson (1953) and Yoshida and Horimoto (1957) with hens; Huang (195M with rabbits; Schurch £39 31. (1950) and Schurch (1952) with rats; and Kreula (191:7), Irwin and Crampton (1950), Virtanan (1950), and Schurch (1952) with humans. Edin (195h) reported favorable results with chronic oxide using fox and mink. Chronic oxide was used successfully in conjunction with chromogens in estimating pasture digestibility and dry matter intake by Kane st 31. (1953a, 1953b) with cows and Brannon st 31. (19510 with steers. Kane .22.§i' (1957) reported satisfactory results using radioactive methods of analyzing feces for chronic oxide. Even though.Lancaster and Coup (1953) recovered loo-102% of the chronic oxide fed, they found a coefficient of variation over a 5-day period of 10% with cows. Linkous gt 31. (195h) noted large between-cow variations. Murdock 2} 31. (1957) noted low recoveries of chronic oxide with heifers which were in estrus. Low recoveries were found by Cramp- ton and Lloyd (1951) when chronic oxide was administered to cows in oats-molasses pellets. Hardison gt‘gl. (1953) recovered 106% of the indicator fed over a 7-day period with steers. Lassiter and.Davis (195h) failed to obtain satisfactory results in one of three trials. Also working with steers, Oldfield 23:51. (1956) found 93.2% recovery of the indicator. 'Woolfolk gt Elf (1950) found the chromic oxide ratio tech- nique to be inaccurate due to variable recovery from the feces of calves. Pigden (1953) failed to Obtain complete recovery of chromic oxide from sheep. Schurch (1952) found that results were not satisfactory with sheep unless large amounts of concentrates were fed. Lambourne (1957a) recovered 83-10h% of single doses of chromic oxide from sheep in 72 hours. Only 67-90% of single doses of chromic oxide given to sheep in a hard gelatin mixture was recovered. Lambourne (1957b) was able to estimate fecal output of sheep over a 10-day period within only 8% by the chromic oxide technique. Bernicoat (19h5) recovered 75-95% of the chronic oxide fed to lambs, wethers, calves, and pigs. Schurch (1952) observed higher recovery of chromic oxide with sheep when larger doses were given. Lambourne (1957a) obtained a higher recovery from a morning dose than from one given in the evening to sheep. An average recovery of 90% of single doses of chromic oxide fed to horses was found by Olsson (1950). Kameoka (1956) observed 10% daybto- day variations in chromic oxide excretion with goats. Olsson gt El‘ (1919), Dansky and Hill (1952), and Mueller (1956) each obtained approx- imately 95% recovery of chromic oxide with chickens. Muller (1953) ob- tained only 88% recovery of this indicator with chickens. Mueller (1956) found higher chromic oxide recoveries with chickens fed a more finely ground ration. Definite diurnal patterns of chromic oxide excretion have been re- ported'by many workers, including Kane 33 31. (1950, 1952), Hardison gt El. (1953), Reid (1952), Lancaster and Coup (1953), Oldfield e_t 51. (1956), Murdock 33 g. (1957), and Putnam _e_i_; 31. (1957) with cattle; -6- and Muller (1953) and Mueller (1956) with chickens. A diurnal variation necessitates care in establishing a grab- sampling regime. The most valid sampling times have not been establish- ed, however, since the various workers do not agree as to the time of day of highest and lowest feces concentrations of chromic oxide. Andersen and'Winther (193h) and woolfolk‘gt‘gl. (1955) failed to find a definite pattern of chromic oxide excretion during the day with sheep. Raymond and Minson (1955) observed similar results with sheep and pointed out possible errors in sampling brought about by patterns of chromic oxide excretion due to grazing habits of animals on pasture. Bernicoat (19h5) using sheep, calves, and pigs, and Clawson gt El. (1955) using pigs reported results more nearly approximating the standard col- lection method when more samples were taken during the day than the usual two. Satisfactory results with a randompsampling program were ob- tained by Huang (195h) with rabbits and Schurch st 21. (1950) with rats. Time of day of chromic oxide administration was found to have no effect upon excretion curves of cows by Smith and Reid (1955) or Hardison (1956) working with cows, Hardison (1956) noted a more even excretion of chromic oxide with twice-daily than with once-daily dosing. Irwin and Crampton (1950) found the same effect of dosing frequency with humans. Kameoka (1956) working with goats, found that more frequent dosing shifted the excretion curve and gave a more definite peak excretion. Pigden and.Brisson (1956) found no diurnal excretion pattern.with sheep dosed six times daily. Linkous £2.51. (195h) Observed that the intervals between highest concentrations of chromic oxide in feces of cows during a 2b-hour period corresponded to the intervals between dosing times. \ Kameoka (1956) found less range of feces concentration during the day when larger amounts of chromic oxide were administered to goats. No effect of age of cattle upon the excretion curves was found by Hardison (1956). Bloom.gt 31. (1957) found little effect of level of hay and grain feeding on chromic oxide excretion curves of cows. Makaffey gt El' (195h) reported greater ranges of chromic oxide concentrations in the feces of steers during the day with more frequent feeding. Little effect of hay:grain ratio on chromic oxide excretion curves of cows was found by Bloom 33 g}. (195?). According to Barnicoat (195) the fibrous nature or fineness of the feed did not alter excretion curves of sheep, calves, or pigs. Crampton and.Lloyd (1951) found large var- iations in daily levels of chromic oxide excreted when administered to cows in oats-molasses pellets. Makaffey st 21. (195h) reported a smaller range of fecal concentra- tions when chromic oxide was administered to steers in pure form or dried ‘with collodion than in gelatin suSpension or baked flour paste with grain. Raymond and Minson (1955) were unable to reduce diurnal varia- tion by the administration of chromic oxide in a bentonite solution to cows. A greater diurnal variation was observed when using gelatin cap- sules than when administering chromic oxide in the feed, according to Reid (1952). Barnicoat (195), however, failed to find this difference with.sheep, calves, and.pigs. Radiography studies by Corbett and Benzie (1953) showed the passage of chromic oxide in gelatin capsules directly into the omasum or aboma- sum of sheep in some cases. Similar observations were reported.by Belch gt 3;. (1957) who found that as much as 60% of the chromic oxide administered to rumen-fistula steers in gelatin capsules passed to the omasum.in 30-60 minutes. Lambourne (1957) Observed fairly even mixing of chromic oxide in the rumen before passage to the remainder of the digestive tract of sheep. lflfifléflr The use of the lignin.portion of the plant as an indicator of digestibility was proposed by E1115.22.§l° (19h6). Using a 72% sul- furic acid determination method, they found the lignin ratio technique to compare closely to conventional methods of estimating digestibility in cows, sheep, and rabbits. Bondi and Meyer (19b3) found lignin dig- estibilities of 35.1-6h.0% in green forages containing 15.1-22.8% lig- nin. Ely 23.2l' (1953) observed 86.0-96.2% recovery of lignin from cows fed hay of varying degrees of maturity. Methoxyl Grogps. Richards and.Reid (1952) suggested the use of the methoxyl groups of lignin as a digestibility indicator. Ely st 31. (1953) found 20.3-31.61 digestibility of methoxyl groups of hay using sheep. Normal Acid Fiber. "Normal acid fiber" ratios (15% sulfuric-acid method) were reported by Raymond gt.§1. (1955) to closely approximate digestibilities found in the conventional manner with fresh forage fed indoors to cows. Fecal Nitrogen. The total fecal nitrogen excretion of steers was reported by Gallup and Briggs (19h8) to be related to dry matter intake in such a manner that either being known, the other could.be closely approximated with an appropriate factor. Forbes (19h9) found no appar- ent constancy between fecal nitrogen excretion and dry matter intake of steers. glagt Pigments. Reid 23 31, (1950) developed the "chromogen" ratio technique using steers, calves, and wethers. Spectral examination of 85% acetone extracts of feeds and feces at h06 mu revealed 100.5% re- covery of certain plant pigments in 36 trials. Irvin gt 31. (1953) found pigment nitrogens or porphyrins to be satisfactory as digestibility indicators in chromatographic and spectro- photometric studies with alcohol extracts of forages and feces. Kane and Jacobson (l95h) proposed the use of pheophytin ratios for the determination of digestibility. No significant differences between the chromogen and pheophytin ratios and conventional methods of deter- mining digestibility were found by Grainger and'Woolfolk (l95h) with 13 “b8. Polyethylene glyggl'(§§§). The way was opened for the use of a different sort of inert reference substance when Shaffer and Critchfield (19h7a) developed a gravimetric and a colorimetric method for the de- termination of solid.polyethylene glycols in blood and urine. In termp inal experiments with rats, Shaffer and Critchfield (19h7b) found approx- imately 2% intestinal absorption of PEG-1,000 (PEG of molecular weight 1,000) and PEG-1,5b0 in 5 hours, but no absorption of PEG-b,000 or PEG- 6,000. ‘With human subjects, no significant absorption of PEG-h,000 was detected, but an absorption of 8% of PEG-1,000 was estimated. All mol- ecular weights of PEG from 1,000 to 6,000 were found to be readily -12- excreted by the kidney through glomerular filtration. Hyden (1955b) Obtained recoveries of single drench doses of PEG- h,000 (3,500-h,200) from a rumen-fistula cow of 92.3% and an abomasal- fistula cow of 95.9%. In sheep, the recovery was 92.1%, hens 97%, and man 96.6%. The low recovery of 79.2% with the rabbit was attributed to delayed passage through the cecum. Almost perfect recovery was Obtained when PEG was added to the excreta of these subjects, and in terminal experiments with a cow and goats in which the total digestive tract contents were analyzed. The Shaffer and Critchfield (19h7a) method of determination was used. Recoveries of lie-707.; of the PEG-h,ooo fed to cows in grass-meal cubes over 5-day periods were reported by Corbett‘gt‘gl. (1956). Using PEG-h,000 (3,000-3,700) Sperber st 31. (1953) concluded that there was no significant precipitation, uptake, or adsorption of the indicator by rumen contents, on the basis of comparisons between centrifuged and non- centrifuged samples. Corbett gt_gl, (1956), however, conducted $2.31352 trials which suggested adsorption. No microbial breakdown or toxicity to rumen microorganisms in 31552 in 2h hours was found by Sperber EE.§l° (1953) using glucose utilization as an indication of microbial actibity. These findings are in agreement with those of Corbett st 31. (1956). No destruction of PEE-h,000 was found over short periods of time in stored samples by Hyden (1955b). However, recovery results suggested destruction in the digestive tract over longer periods of time. Doses up to 500 gm. of PEG-h,000 per day produced no signs of toxicity in cows according to Sperber st 31. (1953). No passage of PEG-h,000 through the rumen wall was believed to take -13... place by these workers or by Corbett st 31. (1956) as none was detected in the urine. Since the methods of Shaffer and Critchfield (19b78) were found to be time consuming and impractical for routine determinations of PEG, Hyden (1955a) introduced a simplified turbidimetric procedure for the determination of PEG in biological compounds. The method checked favor- ably with known concentrations of PEG in rumen contents and feces, and with the Shaffer and Critchfield (197ha) gravimetric method. Corbett .22.El° (1956) also tested the method and found that it checked favorably with the Shaffer and Critchfield (l9h7a) colorimetric method. -1b- EXPERIMENTAL PROCEDURE The po1yethy1ene glycols are long-Chain, highly polymerized hydro- carbon compounds with the general formula H0-CH2(CH2-O-CH2)n-CHZ-OH. The solid polyethylene glycols are waxelike compounds of varying degrees of hardness, soluble in water, aromatic hydrocarbons, acetone, and ethyl alcohol, and slightly alkaline. This experiment was initiated to evaluate a wide range of molecular weights of PEG for use as digestibility indicators. The experiment was divided into two trials. PEG-h,000 was tested in Trial I, using four mature, healthy Holstein cows; A-llO, K-239, K-139, and A-Yl. In Trial II, cows K-302 and A-71 were used with PEG-6,000, K-239 with PEG-9,000, and K-139 with PEG-20,000. The ration fed in Trial I consisted of 20 1b. of alfalfa hay and 6 1b. of a grain mixture. In Trial II, 20 lb. of alfalfa hay was fed with 8 1b. of grain mixture. The rations were fed at 8:00 a.m. and 2:00 p.m. A lh-day preliminary feeding period was allowed in both trials. In both trials, 15 gm. of PEG was administered orally with a ball- ing gun to each cow daily in two 22 mm. X 75 mm. gelatin capsules, each containing 7.5 gm. of PEG. The capsules were administered at 6:30 a.m. in Trial I and at 8:15 a.m. in Trial II. No loss of capsules due to regurgitation or other causes was detected. Total collection of feces was made in both trials with mechanical digestion stalls, beginning at the time of the first administration of -15- PEG. In Trial 1, total collections were made for 16 days and Trial II for 11 days. Aliquot samples were obtained from each day's collection. In Trial 1, rectal grab samples (approximately 1h oz.) were taken from cows K-239 and A-71 at 2-hour intervals from 6:00 a.m. of the 11th day of the trial through 6:00 a.m. of the 12th day, and from 6:00 a.m. of the 13th day to 6:00 a.m. of the lhth day of the trial. Feces samples were stored at h-SOC. until analyzed, with the excep- tion of the total collection aliquots of the first 5 days of Trial 1, which were held at -2o°c. Analyses on all feces samples were completed within 2 weeks follow- ing the end of the collection period in Trial I, and on the last day of collection in Trial II. Daily total collection aliquots were analyzed for PEG content to obtain an estimate of the recovery of PEG after pass- age through the digestive tract. The rectal grab-samples were analyzed to Obtain excretion curves for PEG for the 2h-hour periods. Analyses of PEG content of the feces were made by the turbidimetric method of Hyden (1955a). The only modification was the use of approx- imately 10 gm. samples instead of 1 gm. samples of feces. After macerat- ing in water, the samples were allowed to equilibrate for 15-30 minutes before being diluted to 50 ml. The diluted feces samples were then centrifuged at 2,500 r.p.m. for 30 minutes to Obtain a supernatant liquid for precipitation and filtration. A 5 ml. graduated syringe pipette was used to add the trichloroacetic acid-barium chloride solut- ion to develop turbidity. Duplicate determinations were made on all samples. A standard curve was plotted for each group of filtrates ana- lyzed o -16.. Since a slight yellow color was observed in the filtrates, absorp- tion and transmission curves were determined with a Beckman Model DK-2 Spectrophotometer. Maximum absorption was observed at a wave length of 300‘mp.and maximum transmission at 750 mu. Maximum transmission and absorption occurred at the same wave length with or without turbidity being developed in the samples. 0n the basis of these observations, turbidity was read in a Beckman Model B Spectrophotometer at 750 mu, using a red photosensative tube with no filter. Analyses for dry matter were conducted in duplicate on all rectal grab samples for excretion curves. Samples were dried to constant weight in porcelain crucibles at 100°C. in a convection oven. To determine if PEG could be quantitatively recovered from biolog- ical materials under the analytical conditions of this experiment, PEG- h,000 was added to dried feces to give a concentration of 0.39 mg. PEG per gm. and mixed thoroughly in a mechanical mixer. Samples of the dried feces weighing 2 gm. were diluted to 50 m1. and allowed to equilibrate for 1 hour before centrifuging. In a trial more closely approximating ig‘zivg conditions, PEG-h,000 and PEG-9,000 were added to duplicate 300 gm. samples of slightly diluted fresh feces in Waring blendors to give concentrations of 0.67 mg. PEG per gm. These samples were mixed 3-h minutes three times daily for 5 days and analyzed for PEG in the uSual manner. Another’iguxitpg trial was conducted to determine if PEG was lost in the presence of fecal material. One set of samples of dried feces of weights 0.5 gm., 1.0 gm., 1.5 gm., 2.0 gm., and 2.5 gm. were placed in flasks containing 50 ml. each of 0.1 mg. per m1. aqueous solution of -17- PEG-h,000, and a set of duplicates were placed in flasks containing 50 ml. each of 0.1 mg. per n1. aqueous solution of PEG-9,000. The samples were allowed to set for 1 day with occasional shaking. The supernatant liquid was analyzed in the same manner as that from centrifuged, diluted feces samples to detect any change in concentration of the PEG solutions. RESULTS AND DISCUSSION Solubilities of the PEG compounds used in this experiment expressed as percent by weight in water were approximately as follows: PEG-h,000, 125%; PEG-6,000, 90%; PEG-9,000, 85%; and PEG-20,000, 55%. The com- pound PEG-h,000 (3,000-3,700) used by Sperber 23 51. (1953) was report- ed to be 60% soluble by weight in water. The use of a water-soluble compound as an indicator would seem to eliminate some of the difficul- ties encountered with high Specific gravity substances due to complica- tions in passage through the digestive tract. Curves plotted from optical density readings on identical concen- trations of PEG-h,000, 6,000, 9,000, and 20,000 all fell on the same line. Thus it was considered valid to use the simplified turbidimetric method of Hyden (1955a) for determination of this range of molecular ‘weights. Hyden (1955a) found very similar curves for PEG-h,000 and.PEG- 6,000 when comparing effects of trichloroacetic-acid concentrations. However, it was found that molecular weights lower than h,000 had con- siderably lower Optical densities at a given trichloroacetic-acid con- centration. Hyden (1955a) reported the s10pe of the calibration curve to be stable. This was true in the present experiment with 0.05-0.10 mg. per ml. aqueous solutions of PEG until the solutions were h-6 weeks old. At that time, the calibration curve was found to shift upward and take on a greater slope. The high stability of PEG in urine reported by Hyden (1955b) was for a 6-day period only. .‘,- ‘w ..c ‘h The yellow color observed in the filtrates was felt to be due to the low feceszwater ratio required to attain levels of PEG in the fil- trates which could be detected with accuracy. Since Hyden (1955a, 1955b) used large doses of PEG, this problem was not encountered in their work. _I_n_ 33.33 Recovegy 3f PE. The recovery percentages of all molecular weights of PEG tested, as shown in Tables I and II were low and approx- imately within the b0-70% recovery range reported by Corbett 33 31. (1956). Recoveries of all molecular weights tested were lower than the 93% (82.9-102.0%) recovery of single, large doses given cows by Hyden (1955a). An inverse relationship was observed between the molecular weight of the PEG compound and the percent recovery. There was no uni- form tendency for daily recoveries to increase or decrease as the trial progressed with PEG-h,000 and only between the first and second collec- tion days with the higher molecular weights. Thus it appears that PEG reaches a rapid equilibrium in the digestive tract, however the daily excretions Observed were erratic with no constant day-to-day level of PEG excretion being reached. The highest feces concentration of PEG attained with the 15 gm. daily doses used in this experiment was 0.99 mg. PEG per gm. of feces. Complete recovery was represented by approximately 0.6-0.E mg. PEG per gm. of feces. Corbett 32 21. (1956) reported feces concentrations of 0.55-0.80 mg. PEG per gm. of faces with 25 gm. per day administration of PEG to cows. These workers found that increasing feces concentrations of PEG gave increasing, but still incomplete recovery. They suggested a feces concentration of 2.50 mg. PEG per gm. of feces, which would involve -20- Table I Percent Recovery of Polyethylene Glycol-h,000 after Passage through the Digestive Tract Cow A-llO Wax A-71 Day 1 52.7 62.7 53.1 60.3 2 61.6 57.1 105 .5 811.3 3 67.1 89.h 83.9 7h.3 n 61.0 1111.3 117.3 116.1 5 136.2 80.1 116.9 69.0 6 79.8 80.9 81.5 88.u 7 60.2 93.1 65.1 70.6 8 73.7 96.8 83.3 6u.1 9 103.7 92.5 105.9 78.3 10 .98.9 69.8 99.3 128.6 11 62.8 69.9 65.1. 61.14 12 75 .2 85.8 50.1; 59.h 13 85.1 96.6 6h.5 82.6 11, 113.6 62.6 87.3 16 .3 15 128.9 5h.2 56.9 20.8 16 65,1 61.8 00.0 39.1 Av. Recovery 82.9 79.2 77.3 71.h Std. Dev. .1 25.9 117.3 130.1 126.6 -21- Table II Percent Recovery of Polyethylene Glycol after Passage through the Digestive Tract H M01. Wt. PEG 6,000 6,000 9,000 20,000 Cow x-302 1-71 10-239 x-139 Day 1 2h.8 27.1 00.0 00.0 2 . 57.2 1414.1 88.7 1.3.9 3 55.5 1.1.2 1.8.3 38.1 h 38.2 35.9 31.3 115.11 S llh.6 7b.? 65.8 76.8 6 51.9 73.2 67.8 514.2 7 51.2 hh.8 53.1. 118.6 8 h8.2 S9.h 82.7 h6.8 9 62.7 L6.9 73,6 38.1 10 69.14 611.9 69.3 53.9 11 110.9 70.11 85.7 58.9 Av. Recovery” 66.0 55.5 58.7 50.1 Std. Dev. 1 26.0 111“; 116.5 111.2 ”Excluding lst day's recovery. -22- administration of 80-100 gm. of PEG per day. Sperber st 51. (1953) gave doses of 100-500 gm. of PEG per day and Hyden (1955b) administered 100-200 gm. of PEG per day to cows. The writer feels that these higher levels of PEG might be impractical for use in prolonged studies. In Vitro Recovery 9f PEG. The calculated recovery of PEG added to dried feces (Table III) was assumed to be within the range of experi- Table III Recovery of Polyethylene G1ycol-h,000 from Dried Feces Sample Recgvery 1 101.8 2 92.3 3 113.1 8 101.8 5 89.5 6 123.6 Average Recovery - 103.7% mental error due to the technical difficulties involved. The flaky nature of the PEG could very likely result in an uneven distribution of the marker leading to sizeable errors when using small dried feces samples. The results indicate that PEG may be recovered quantitatively with the alterations of the determination method imposed in this experi- ment. Recoveries of PEG-h,000 and PEG-9,000 mixed with fresh feces (Table IV) were very similar to the recoveries of these compounds after passage through the digestive tract. In contrast, Hyden (1955a, 1955b) reported almost complete recovery of PEG added to the feces of cows and sheep. Table IV Recovery of Polyethylene Glycol added to Fresh Esces __: M01. Wt. Sample Recovery Av. Recovery 3% % la 7h.6 PEG lb 73.1 78.7 h,000 2a 85.0 2b 82.1 38 68 . 7 PEG 3b 67.2 66.8 9,000 ha . 68.2 Hb 67.2 As shown in Table V, an essentially linear relationship was found between the weight of dried feces added to aqueous solutions of PEG- h,000 and PEG-9,000 and the weight of PEG removed from the solutions. The removal rate of PEG from the solutions was higher with the higher molecular weight of PEG. This observation is in agreement with the results observed with varying molecular weights in the studies on recov- ery of PEG after passage through the digestive tract and recovery from fresh faces. ~28- Excretion _C_1_1_r_v_e_s. The PEG excretion curves (Figure 1), drawn by plotting mg. PEG per gm. of feces dry matter against time, reveal a definite excretion pattern with the average low concentration of PEG at about 11:00 p.m. and the average high concentration at about 11:00 p.m. The high concentrations observed during the night can partially be ac- counted for by the higher dry matter content of the fecal samples taken during that part of the day. Table V Change in Concentration of Polyethylene Glycol Solution with Addition of Dried Feces W I Mol. Wt. Dry Feces Added PEG/ml. PEG Lost/gm. Feces Added gm. mg. mg./ml. 0 0.10h -- 0.5 0.102 0.008 PEG 1.0 0.099 0.005 8,000 1.5 0.096 0.005 2.0 0.098 0.005 2.5 0.085 0.008 0 0.108 -- 0.5 0.095 0.017 PEG 1.0 0.092 0.012 9,000 1.5 0.088 0.011 2.0 0.085 0.010 2.5 0.078 0.011 MG. PEG/GM. FECES DRY MATTER 4 K-239 IITH K-239 I3 TH .5 OJ N A-7I IITH .5 01 N A-7l ISTH L A IO ‘20! 2 4 GP“ 8 IO |2N 2 4 6 TIME 6A“ 8 FIG. I. excas‘rnou cuavss 0F POLYETHYLENE swoop-4,000 -26- Possible E233.2£ E2§§.3£.£§9' The loss of PEG.from the solutions With the addition of dried feces could be due either to adsorption of the PEG on the feces or to removal from the centrifugation supernatant in the precipitation process. No reliable quantitative method was found to determine if adsorp- tion of PEG on the feces took place. Studies in which centrifuged, diluted feces samples were compared with diluted samples which were allowed to sediment without centrifugation revealed higher apparent concentrations of PEG in the centrifuged samples. However, due to the smaller amount of foreign material remaining in the supernatant liquid of the centrifuged samples, the differences were not considered con- clusive. 0n the basis of similar tests with rumen fluid, Sperber gt 3.1: (1953) concluded that there was no significant adsorption, precipita- tion, or uptake of PEGeh,OOO by rumen contents. Attempts to carry through the sulfate-protein precipitation process with pure aqueous solutions of PEG, to determine if PEG is precipitated in the process, failed due to the incomplete filtration of barium sul- fate. The result was a turbid filtrate which could not be used. If PEG-were precipitated by the sulfate-protein precipitating rea- gents, a constant weight of PEG would be expected to be lost. Since very small concentrations of PEG may be detected (Hyden, 1955b), the loss due to this type of interaction could not account for enough PEG loss to eXplain the low recoveries observed. The high recoveries of PEG from dried feces are also in Opposition to this theory. Since the loss of PEG from the aqueous solutions to which dried feces were added was a function of the weight of feces added, it would seem that adsorption on the feces is the most likely mode of loss of PEG. Precipitation of PEG with the proteins or sulfates from the super- natant liquid due to adsorption to these components would allow the above linear relationship to hold, as well as adsorption on other feces constituents which.wou1d not allow the PEG to be released to the super- natant liquid for analysis. It may therefore be concluded that the most likely mode of loss of PEG in the digestive tract is through some type of adsorption on the feces. The use of large doses of PEG, as by Hyden (1955b) and as suggest- ed by Corbett gt 31. (1956) would make the losses of PEG observed in this experiment less significant in terms of total recovery of the indi- cator. This could explain the high apparent recoveries of PEG observed by Hyden (1955b). If the large amounts of PEG did not prove to be pro- hibitive, the practice of administering large doses might solve the major problems involved in the use of PEG as an indicator. -28- SUMMARY Eight mature dairy cows on hay-grain rations were used to evaluate the use of polyethylene glycol (PEG) as an inert reference substance in ruminant digestion studies. Fifteen gm. of PEG was administered daily. Molecular weights of 8,000, 6,000, 9,000, and 20,000 were tested, using the simplified tur- bidimetric method of Ryden(1955a) to analyze the feces for PEG. Recoveries of PEG after passage through the digestive tract were: PEG-8,000, 77.7% (71.11-82.95); PEG-6,000, 60.7% (Sis-66.0%); PEG-9,000, 58.7%; and PEG-20,000, 50.1%. Ekcretion curves representing 2h-hour periods revealed a definite excretion pattern with lowest concentrations of PEG at h:00 p.m. and highest concentrations at 11:00 p.m. Average recovery of PEG added to dried feces was 103.7%. ‘When PEG was added to fresh feces, 78.7% of PEG-h,000 and 66.8% of PEG-9,000'was recovered. Essentially a linear relationship was found between weights of dried feces added to aqueous PEG solutions and weights of PEG removed from the solutions. The most plausible explanation for the low recoveries observed in ‘view of the data presented, would seem to be adsorption of the PEG-on the feces. Under the conditions of the present eXperiment, PEG was not found to be a satisfactory indicator of digestibility. LITERATURE CITED Andersen, A. c. 1938. Cited.by Kane, E. A., R. E. Ely, w. c. Jacobson, and L. A. Moore. 1953. A comparison of various digestion trial techniques with dairy cattle. J. Dairy Sci. 36:325. Andersen, A. C., and J. E. Winther. l93h. 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