I. II. UTILIZATION OF BIURET BY SHEEP EFFECT OF STARVATION AND SUBSEQUENT REFEEDING ON SOME IN VITRO ACTIVITIES OF RUMEN MICROORGANISMS By Jay C. Meiske AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State University of Agriculture and Applied Sciences in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Husbandry 1957 Approved ^9.^. 74 ProQuest Number: 10008384 Ail rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete m anuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest, ProQuest 10008384 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This w ork is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 ABSTRACT I. Utilization of Biuret by Sheep Four feeding trials involving 170 sheep were conducted to establish whether biuret could be utilized as a non-protein source of nitrogen for ruminants* Urea was used as a comparison in all trials. biuret was also tested* Crude In the first and second trials, the basal ration consisting of corn silage and ground corn was too high in crude protein to serve as a negative control. Thus, the addition of a non-protein nitro­ gen source to this basal ration did not increase the rate of gain signi­ ficantly. For the third and fourth trials a basal ration of approximately 7.1 percent crude protein was fed. The addition of either urea, biuret or crude biuret to the basal ration increased the average daily gain signi­ ficantly. No significant differences were noted among the lots fed the supplemental nitrogen sources. It is concluded that under the conditions of these experiments, urea, biuret and crude biuret are satisfactory sources of supplemental nitrogen for sheep. II. Effect of Starvation and Subsequent Refeeding on Some In Vitro Activities of Rumen Microorganisms. Samples of rumen fluid were collected at intervals from a fistulated steer treated in the following manner: (1) fed a high-roughage ration regularly, (2) no ration, starvation for either two or three days, (3) refed (following starvation) a high-roughage ration. The ability of rumen fluid to digest cellulose in vitro, and to reduce the viscosity of a stable suspension of carboxymethylcellulose (CMC) was determined. A fraction (S-l) of rumen fluid free of protozoa and plant material and a second fraction (S-2) free of bacteria, protozoa and plant material were also tested for activity on CMC. When the steer was starved for three days, the ability of rumen samples to digest cellulose decreased approximately 90 percent. The pH of the rumen fluid rose during the starvation periods from normal values between 6.30 and 6.90 to high values between 7.65 and 7.94. Rumen fluid taken one to three days after refeeding (following starvation) digested cellulose at normal rates. When the steer was fed regularly, the strained and S-l fractions reduced the viscosity of a CMC suspension more than did the S-2 frac­ tions, indicating the amount of enzyme free of the cell was not great. All fractions became progressively lower in their ability to reduce the viscosity of CMC as starvation progressed* The activities of all frac­ tions during the first two or three days of refeeding following starvation showed unusually high activity on CMC vindicating that the enzymes were largely extracellular at this time. The activities of the various frac­ tions returned to normal when the steer had been refed two to four days after starvation. No differences in the activities of rumen microorganisms could be attributed to the type of hay the steer received before or after the starvation period. Upon refeeding following starvation, the steer readily consumed all of his ration at each feeding. The ability of rumen fluid to digest cellulose in vitro and to reduce the viscosity of CMC suspensions was usually normal within two to four days after refeeding following starvation. I. II. UTILIZATION OF BIURET BY SHEEP EFFECT OF STARVATION AND SUBSEQUENT REFEEDING ON SOME IN VITRO ACTIVITIES OF RUMEN MICROORGANISMS By Jay C. Meiske A THESIS Submitted to the School of Graduate Studies of Michigan State University of Agriculture and Applied Sciences in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Husbandry 1957 dr / ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. J. A. Hoefer, Dr. R. W. Luecke and Dr. R. L. Salsbury for their personal interest and advice. Their ready assistance and stimulation have been invaluable. Thanks are due to Dr. Hoefer, Dr. Luecke, Dr. Salsbury and Dr. E. P. Reineke for their valuable suggestions in the preparation of this thesis. Grateful acknowledgment is due to Miss Betty Baltzer and Mr. Alvin Vander Kolk for their assistance in the in vitro determinations; also to Dr. E. R. Miller, Mr. Howard Stowe and Dr. S. C. Stothers for their timely assistance during the course of many of these ex­ periments. The author deeply appreciates the financial support of Michigan State University through an assistantship and is grateful to Dr. R. H. Nelson and Dr. Luecke for the use of the facilities of the Animal Husbandry and Agricultural Chemistry Departments. The writer is greatly indebted to his wife, Donna, for her constant interest and encouragement and for her assistance in one way or another. Jay C. Meiske candidate for the degree of Doctor of Philosophy Final Examination, May 17, 1957, 1:30 P.M., 101 Anthony Hall Dissertation: I. II. Utilization of Biuret by Sheep Effect of Starvation and Subsequent Refeeding on Some in vitro Activities of Rumen Microorganisms Outline of Studies: Major subject: Animal Husbandry— Nutrition Minor subjects: Biochemistry, Physiology Biographical Items: Born, June 22, 1930, Hartley, Iowa Undergraduate studies, Iowa State College, 1948-1952 Graduate studies, Oklahoma A & College, 1952-1953 Michigan State University, 1953-1957 Experience: Member: Graduate Assistant, Oklahoma A & M, 1952-1953 Graduate Teaching Assistant, Michigan State University, 1953-1954 Graduate Research Assistant, Michigan State University, 1954-1957 Delta Sigma Phi, Alpha Zeta, Gamma Sigma Delta, Phi Kappa Phi, Sigma Xi, American Society of Animal Production TABLE OF CONTENTS PART I. UTILIZATION OF BIURET BY SHEEP Page INTRODUCTION.......................................... 2 REVIEW OF LITERATURE In vitro Studies • • ........ • • • • • • • • • . * 4 Toxicity Studies • • • • • • • • • • • « • • • • • • 4 Digestion Trials • • .................. Feedlot Trials • •••• ••• •• • • . • • 5 ............... 6 ............. 8 TRIAL I Procedure • • • • • • • • • • • • • • Results and Discussion * • • • • • • • • • • • • • • 1 0 TRIAL II Procedure • • * • • • • • • • • • • • • • * • • • • • 1 2 Results and Discussion • • • • • • • ......... . • 13 TRIAL III Procedure • • • • • • • • • • • • • • • • • • • • • • 1 6 Results and Discussion •••• ....... ••••••18 TRIAL IV Procedure • • • • • • • • Results and Discussion .................. •••21 • • • • • • • • • • • • • • • 2 3 S U M M A R Y ............................................. 26 LITERATURE CITED......................................27 PART II. EFFECT OF STARVATION AND SUBSEQUENT REFEEDING ON SOME IN VITRO ACTIVITIES OF RUMEN MICROORGANISMS Page INTRODUCTION......................................... 30 REVIEW OF LITERATURE.................................. 32 PROCEDURE Sampling • •••••••• In vitro Studies • ••••• ....... ....... ••••••••37 •••••••37 Viscosimetric Studies • • • • • • • • • • • • • • • • 4 0 Experimental Rations • • • • • • • • . • • • • • • • 42 RESULTS AND DISCUSSION Trial I • • • • • • ....... Trial U • • • • • • • • • * • • ............ 44 47 Trial I I I .......................................50 Trial I V .......................................53 Trial V « * v « . « « . * « * * * * « . * * . . . . . 5 6 Trial V I ......... 59 Trial V I I ................................ General Discussion ......... •••••••••••65 S U M M A R Y .................................... LITERATURE CITED • . . 63 .... 69 ............................. 71 LIST OF TABLES PART I. UTILIZATION OF BIURET BY SHEEP Page 1. COMPOSITION OF CONCENTRATE MIXTURES (TrialI) . . . . 9 2. PROXIMATE ANALYSES OF CONCENTRATE MIXTURESAND CORN SILAGE (Trial I) ........................... 9 3. RESULTS OF TRIAL I ............................. 11 4. COMPOSITION OF CONCENTRATE MIXTURES (TrialII). . . 5. RESULTS OF TRIAL II . . . 6. COMPOSITION OF EXPERIMENTAL RATIONS (Trial III) . . . 7. PROXIMATE ANALYSES OF EXPERIMENTAL RATIONS (Trial III) 18 8. RESULTS GF TRIAL I I I ............................. 20 9. COMPOSITION OF EXPERIMENTAL RATION (Trial IV) . . . . 22 . 13 ....................... 15 17 10. PROXIMATE ANALYSES OF EXPERIMENTAL RATIONS(Trial IV) 22 11. RESULTS OF TRIAL I V ............... 24 PART II. EFFECT OF STARVATION AND SUBSEQUENT REFEEDING ON SOME IN VITRO ACTIVITIES OF RUMEN MICROORGANISMS 1. SALT SOLUTIONS USED FOR DJ VITRO S T U D I E S ........... 38 2. COMPOSITION OF THE STEER SUPPLEMENT................ 42 3. PROXIMATE ANALYSES OF FEEDS 4. RESULTS OF TRIAL I 5. RESULTS OF TRIAL I I ................................47 6. RESULTS OF TRIAL I I I .............................. 50 7. RESULTS OF TRIAL I V ................................54 8. RESULTS OF TRIAL V .................. 42 ............................. 44 ................................57 Page 9. RESULTS OF TRIAL VI 60 10. RESULTS OF TRIAL VII 63 LIST OF FIGURES PART II. EFFECT OF STARVATION AND SUBSEQUENT REFEEDING ON SOME IN VITRO ACTIVITIES OF RUMEN MICROORGANISMS 1. Change in viscosity of CMC substrate by the different fractions of rumen fluid taken during Trial I . • • 46 2. Change in viscosity of CMC substrate by the different fractions of rumenfluid taken during Trial II . . 49 3. Change in viscosity of CMC substrate by the different fractions of rumenfluid taken during Trial III • • 52 4. Change in viscosity of CMC substrate by the different fractions of rumenfluid taken during Trial IV . . 55 5. Change in viscosity of CMC substrate by the different fractions of rumen fluid taken during Trial V • • • 58 6. Change in viscosity of CMC substrate by the different fractions of rumenfluid taken during Trial VI . . 61 7. Change in viscosity of CMC substrate by the different fractions of rumen fluid taken during Trial VI (continuation of Figure 6) . . . . . . . . . . . . 62 8* Change in viscosity of CMC substrate by the different fractions of rumenfluid taken during Trial VII . • 64 UTILIZATION OF BIURET BY SHEEP - 2 - INTRODUCTION Urea can be utilized as a nitrogen source for ruminants; however, it is thought that the amount of urea used in rations has to be restricted because of possible toxic effects. Biuret, a condensation product of urea, has been found by Meiske et al, (1955) and Repp et al, (1955) to be less toxic than urea. The latter workers found that feeding of biuret in large amounts produced very little change in blood urea or ammonia indicating that ammonia was released very slowly, if at all, during digestion* They further indicated that the abscence of biuret toxicity may be due to a lack of amidase activity in the rumen. Urea toxicity has been explained on the basis of high urease activity of the rumen contents (Dinning et al*, 1948; Repp et al*, 1955). It has been shown by Hale and Kirg. (1955) that acute urea toxicity may not be due to ammonia but rather to ammonium car­ bonate. Biuret, however, is not attacked by urease, When biuret was added as the nitrogen source in determining cellulolytic activity of rumen microorganisms in vitro, the cellulose digested was only a fraction of that occurring when urea was added as the nitrogen source (Belasco, 1945; Salsbury, 1955). It was thought, however, that biuret offered promise as a nitrogen source in ruminant rations since it was non-toxic at high levels of intake and could therefore possibly be used in place of or to combine with urea to produce a more desirable nitrogen source. The purpose of this study was to determine whether biuret could be utilized for growth and fattening of sheep or lambs. also tested. Crude biuret was Crude biuret is made by the application of heat to urea - 3 - under suitable conditions resulting in a crude material containing about 41 percent biuret, 46 percent urea, 6.5 percent triuret and 6.5 percent cyanuric acid. 4 REVIEW OF LITERATURE Hatfield et al* (1956) pointed out that biuret is much less soluble than urea and, theoretically its nitrogen should become available to rumen microorganisms at a slow rate. Thus, nitrogen of biuret would be released less rapidly and therefore may be utilized more efficiently. At the same time, the slower release of biuret nitrogen should provide the rumen microorganisms with available nitrogen for a longer period of time after feed is consumed* Because of its relatively low solubility, biuret may also affect the palatability of rations less than urea does* In vitro studies: Using an in vitro technique, Belasco (1954) tested several nitrogen compounds as possible sources of nitrogen for rumen microorganisms* He reported that biuret was among those compounds that showed little, if any, available nitrogen. When biuret was used as the nitrogen source, cellulose digestion was only seven percent of that resulting when urea was used. Bacterial growth was also comparatively poor* Salsbury (195 5) has obtained similar results. Toxicity studies: Meiske ^t al. (1955) reported that lambs developed toxicity symptoms when given urea at the rate of 25 and 28*5 grams per 100 lb. of body weight. Lambs given 31 grams of biuret per 100 lb. of body weight showed no toxi­ city symptoms. Repp et al. (1955) studied the influence of oral administration of several non-protein nitrogen compounds upon blood ammonia and urea levels - 5 - in lambs. Biuret failed to produce any symptoms of toxicity when given at a rate of 60 grams per 100 lb. of body weight. Urea caused the death of several lambs when given at a rate of 40 grams per 100 lb. of body weight. These workers found no substantial increase in ammonia— N or urea levels of blood when biuret was given. Hatfield et al. (1956) drenched lambs with biuret at levels up to 275 grams per lamb. They also fed one ewe a daily allowance of ten pounds of a Mfattening-typew ration containing 375 grams of biuret* No toxicity symptoms were observed in any of the tests except for some urinary distress a few hours after the lambs were drenched with the highest levels of biuret. According to Berry et al. (1956), rats and poultry failed to develop toxicity symptoms when fed biuret at levels of 10 percent and 1 percent of the ration, respectively. Rats receiving daily injections of 150 milligrams of biuret developed no toxicity symptoms. Digestion trials: Hatfield et al. (1956) fed steers a ration supplemented with urea, biuret or soybean oil meal. They reported that the average digestion coefficient of nitrogen was lowered when biuret replaced urea or soybean oil meal in the ration. Crude fiber digestion was decreased when either urea or biuret was used in the ration in place of soybean oil meal. There were no significant differences in the average digestion coeffi­ cients for dry matter or ether extract resulting from the use of different nitrogen sources. When biuret was used as the nitrogen supplement, the - 6 - nitrogen retenticn was less than when either urea or soybean oil meal was used* Gaither et al* (1955) found that lambs retained nitrogen equally well when fed rations that contained either urea or biuret. In this study, urea or biuret supplied over 80 percent of the nitrogen in the ration. Welch et al* (1956) fed lambs a ration in which two-thirds of the nitrogen was supplied by urea, crude biuret or pure biuret. When crude biuret was used in the ration, the apparent digestibility of nitrogen was depressed but the digestibility of organic matter and utilization of nitrogen were not changed appreciably. When pure biuret was used in the ration, digestibility of both organic matter and nitrogen was reduced* The utilization of nitrogen was also reduced. Campbell et al* (1956) used lambs in their trials and fed rations that were similar to those fed by Welch et al. (1956). Two-thirds of the nitrogen in the ration was supplied by urea, crude biuret or a com­ bination of the two. The effect of diethylstilbestrol on digestion was also studied in this trial. as the trial progressed. Nitrogen utilization appeared to increase This was true whether or not diethylstilbestrol was fed* Feedlot trials: In a trial involving lambs, Berry et al. (1956) used rations con­ taining crude biuret, urea, a mixture of crude biuret and urea, or cottonseed oil meal as the supplemental nitrogen source. They noted - 7 - that lambs fed the rations containing crude biuret ate slower, consumed larger amounts of water and excreted greater quantities of urine than lambs receiving the rations containing urea or cottonseed oil meal* They suggested that crude biuret may be utilized as well as urea as similar gains were made by lambs receiving urea or crude biuret. They indicated that biuret was relatively inert when added to a ration that was adequate in protein. No toxicity symptoms or depression of appetite were noted when lambs were fed the rations containing crude biuret. These authors also fed a high-roughage, low-concentrate ration containing urea to 450-650 lb. steers. When crude biuret replaced part of the urea, a significantly lower gain resulted. It was concluded that biuret was not a dependable source of nitrogen for ruminants. Reporting on a long-term feeding trial involving ten lambs, Hatfield et al. (1956) compared urea and biuret as supplemental nitrogen sources in a low protein basal ration. After a year the sheep receiving biuret had gained as well as those receiving urea. The authors concluded that sheep can utilize nitrogen from biuret as well as nitrogen from urea. - 8 - TRIAL I PROCEDURE On April 1, 1954, twenty-five native yearling ewes were divided into five lots on the basis of weight and the gain made during a twoweek preliminary feeding period. imately 70 pounds each. They had an average weight of approx­ The rations consisted of corn silage and a concentrate mixture that was mixed with the silage at the time of feeding. The five concentrate mixtures differed in their sources of supplemental nitrogen (table 1), The basal concentrate contained no added nitrogen source and had an average crude protein content of 8*19 percent* It was made up of cracked corn, dicalcium phosphate, iodized salt and a trace mineralized premix for ruminants-^* Urea, biuret and methylenedi- urea were added to the basal mixture in lots 2, 3 and 4, respectively* din amounts sufficient to bring the crude protein content up to approxi­ mately 13 percent. Soybean oil meal replaced part mixture of lot 5, bringing the crude protein level of the corn in the up to about 15 percent. All rations were approximately equal in energy content. The silage was of good quality and had an average crude protein content of 2.78 percent. Proximate analyses for all concentrate mixtures and the corn silage are presented in table 2. The ewes were fed twice daily. Each lot had free access to water and the same mineral mixture as used in the concentrate mixture. was terminated at the end of the 84th day, June 24, 1954. J- Product of Calcium Carbonate Company The trial - 9 - Table 1. Lot Composition of Concentrate Mixtures 1 2 3 Percent 4 5 98.04 95.98 95.98 95.98 84.02 -- --- 14.02 Ingredients Cracked corn Soybean oil meal --- Urea -- 2.06 Biuret --- --- 2.06 --- --- Methylenediurea --- --- --- 2.06 --- Mineral mixturea 1.96 1.96 1.96 1.96 1.96 --- a Mineral mixture: 50 percent dicalcium phosphate, 47 percent iodized salt and 3 percent trace mineral premix for ruminants. Table 2. Proximate Analyses of Concentrate Mixtures and Corn Silage 1 2 Ration Basal Basal* Urea Moisture 14.75 14.62 14.66 Ash 2.64 2.81 Crude fiber 2.20 Ether extract 4 5 Basal* Basal* Methylene­ Basal* SB0M Biuret diurea Percent Silage 15.18 14.06 64.78 2.88 2.23 3.35 1.40 2.47 2.23 2.31 2.80 8.08 3.57 3.50 4.22 3.13 2.65 1.06 Crude protein 8.19 13.85 13.00 13.10 15.06 2.78 N-free extract 68.65 62.75 63.01 64.05 62.08 21.81 Lot 3 - 10 - RESULTS AND DISCUSSION The data presented in table 3 were analyzed by an analysis of vari­ ance (Snedecor, 1946) and multiple range and multiple F tests (Duncan, 1955). The results indicate that soybean oil meal added to the basal ration increased the average daily gains significantly (P less than 0.01). The increase in average daily gain resulting from adding biuret closely approached significance. The average daily gain of lot 5 was not sig­ nificantly greater than the average daily gain of lots 2, 3 or 4. When either urea, biuret or methylenediurea was added to the basal ration, an apparent increase of about 0.10 lb. in average daily gain resulted, but this was not statistically significant. The higher crude protein content and/or the likely presence of un­ identified growth factor(s) of the ration containing soybean oil meal may account in part for the increase in average daily gain of lot 5 over lots fed the non-protein nitrogen sources. There was no difference in feed consumption among lots. Adding either urea, biuret, methylenediurea or soybean oil meal appeared to increase the feed efficiency of lots 2, 3, 4 and 5, respectively, over the feed efficiency of lot 1. Lot 5, receiving soybean oil meal, ap­ peared to have a higher feed efficiency than the other lots. this may be due to the factors mentioned above. Again, - 11 - t• H S c c SO to CO P Q cd C/3 to P Q o • O l> • CO +1 00 • 00 05 CO • CO +1 00 CO • CO + 1 05 +1 ❖ rH r-f • HM rj1 CO • CO Tt< • rH CM CM • 05 05 • CO o 05 • rH rH cd © ' •H T3 0 H (9 a CO o cd H to P Q • o & P t- P tH 0 cd h CO CO •H £ P Q o • 05 00 CM o • +1 CO • CO CM r-f * o 05 05 rH • • CO CO CM to • rH o .a Table 3* Results of Trial I 0 IS IO P Q + o t- H cd cd 0 03 CO cd t=> P Q H o • to c— • rH 05 * • cd tP + H cd CO cd • CO *« CM • to P Q to +1 00 +1 • 00 CO • c00 to rO r—1 • CM +1 o • 05 CD to CO O • +1 CO CM • o • +1 CO CM • • +1 CO r-f • O * 05 t- • to CO • rH to 05 05 • CO • CO o CO to o 05 05 • CO to • to T —f w CO to +1 • rH o to • CO r-f CO * trH * • o CO ■«d* • rH .05 05 • CO o o • r*J H rH t- m o co CO & • X rH •» P O •H P • • O rH • O P O rH f-. 0 Pu P P *-5 O *rf P cd 0 0 > 0 • o 525 • rH P £ • P is .a r-f P •H cd b u Q rH cd *rl P •H rH cd •H *H > a • > P • • JO 0 H k 3 §f 0 3 fn CO ^ a o o •o p X *H B 0 0 0 P C fH cd t-l >s+> rH p •H 0 cd o *o P o * o > (3 « cTt< 0 C c Q cd rH •H (0 p u o o cd « uo 0 • P xt cd rH \ Tf 0 0 P P P 0 O

+ 3 , H t— t *H a m Table 5. Results of Trial II (Two replicates, 5 lambs each, per treatment) to at ^ cq co • o at if* <1, Jb CO tH !=> (4 CO^ at c m OQ • H + CO at P & H 03 w ^ at co pq • o at to at 41 • CO tH CM • IO 05 00 • CM IO • CO +1 tH +1 • CO t> • 05 05 LO • CM CO • CO 4*1 tH CO +1 CM o • 41 c- CM * CO tH ♦ CM CO 05 * o t> IO o 05 CM O • Hrt CO • CM CO • O • 41 CO CM « CM # tH • CM CM O • *M 00 CM CO CM tH +« •M4 • pq • CO 05 00 • CO * CM CM O • o CO to O IO • tH CM t- CO o IO CO t- 05 05 05 CM uo O O • 05 CO r-l CM to CM O LO CO CM CM co o o O H 4 CO 05 rjt co H4 O CO o o IO 05 o • O IO 05 CM • • +1 to *1 o o CM tH CO l> CO tH• CO o CM tH o CM • CO t> tH * tH H4 • CO CM 05 * CO 05 + 1 05 • O 05 CM • O at 00 * tH CM * CM CO tH • CM CM • CO 05 • O ct• 00 t• CO • CM CM CO • o LO to • • • 3 o tH s ts g • •H at aO °-a IQB ) 4®-> • % p c*-t at % •H P at 3 a o •pi *H P P4 tH • > < a •H £ •H at • > • > o tH 3 o • OO 5 3 o b -Og * (4 e bO <1) bQ at tH •H 05 T3 3 b (0 3 lb io CO • tH +1 tH 3 U O O ® 3 tH at A •H (0 t> • P. bO •3 H CO to Q> a to *®a o cct o f-i -H XJ m -oG CO o CO o o ixO o o o Co o Standard error of the mean* Two animals were withdrawn from lot 1 during the trial and results do not include data from these animals. Both were diagnosed as having developed enterotoxemia. - 15 - at x> - 16 - TRIAL III PROCEDURE Fifty western lambs were drenched with phenothiazine, vaccinated for enterotoxemia, and allotted on the basis of sex and weight into five equal lots* each* Each lot was divided into two groups of five lambs The lambs averaged 75 pounds in weight and were fed 73 days starting February 28, 1955. Wheat straw was used as the roughage in this trial so that the basal mixture would not supply enough protein to give optimum gains unless a nitrogen source was added* The two previous trials indicated that a basal ration consisting of corn silage, corn and minerals was too high in crude protein to serve as a negative control* The addition of either a non-protein nitrogen source or a preformed protein did not consistently increase the rate of gain* T^e basal ration used in the present trial consisted of ground wheat straw, ground corn, molasses, dehydrated alfalfa meal, trace mineralized salt and dicalcium phosphate (table 6). crude protein content of 7*13 percent* It had an average Blackstrap molasses and dehy­ drated alfalfa meal were incorporated into the ration as it has been shown that both stimulate gains when a poor quality roughage is fed (Bentley et al., 1952). It was thought that the molasses would also improve the palatability of the ration* The rations of lots 2, 3 and 4 were modified with regard to supplemental nitrogen source by the addi­ tion of either urea, biuret or crude biuret, respectively. The crude protein of these rations ranged between 9.08 and 9.11 percent. Soybean oil meal replaced part of the corn in the ration of lot 5, bringing the - 17 - Table 6. Composition of Experimental Rations Lot 2 1 Ingredients 3 4 5 Percent 31.67 31.67 31.67 31.67 31.67 6.67 6.67 6.67 6.67 6.67 51.33 51.33 51.33 51.33 45.53 Alfalfa meal (dehydrated) 8.33 8.33 8.33 8.33 8.33 Dicalcium phosphate 1*30 1.30 1.30 1.30 1.30 Trace mineralized salt 0.70 0.70 0.70 0.70 0.70 Soybean oil meal --- --- --- --- 5.90 Urea -- 0.85 --- --- Biuret --- 0.85 --- --- Crude biureta --- --- 0.85 --- ¥heat straw (ground) Molasses Corn (coarsely ground) --- a Crude biuret analysis: 41 percent biuret, 46 percent urea, 6.5 percent triuret and 6*5 percent cyanuric acid. crude protein level up to about 9*7 percent. mately equal in energy content* All rations were approxi­ The proximate analyses of the five rations is shown in table 7. Each lot was started on low quality hay which was gradually decreased, and the experimental rations gradually increased until at the end of two weeks all lambs were receiving the experimental rations* The lambs were fed twice daily an amount they would consume in approximately one hour. They had free access to water and a 1:1 mineral mixture of dicalcium phosphate and trace mineralized salt. - 18 - Table 7* Proximate Analyses of Experimental Rations Lot 1 2 Ration Basal Basal *Urea Basal* Biuret Percent Moisture 12.31 13.31 4.58 4 5 Basal + Cr. Biuret BasalSB0M 13.55 11.99 11.71 4.50 4.75 4.30 4.60 16.78 16.80 16.28 16.96 16.83 Ether extract 2.62 2.50 3.18 2.67 2.40 Crude protein 7.13 9.11 9.08 9.10 9.69 N-free extract 56.58 53.78 53.16 54.98 54.77 Ash Crude fiber 3 RESULTS AND DISCUSSION The results presented in table 8 indicate that the addition of the non-protein nitrogen sources or soybean oil meal increased the average daily gains significantly (P less than 0.05 in lot 4). than 0*01 in lots 2, 3 and 5; P less The average daily gain of lot 4 very closely approached the highly significant figure. The average daily gain of lots 2, 3 or 4 was not significantly different from the average daily gain of the sheep in lot 5 fed soybean oil meal. When either urea, biuret or crude biuret was added to the basal, an increase of about 0*10 lb. in average daily gain resulted. Feed efficiency was significantly higher in lots 2, 3 , 4 and 5 (P less than 0.01). The basal group (lot l) consumed an average of 14.95 lb. of feed per pound gain. Lots 2, 3, 4 and 5 consumed an aver­ age of 9.76, 9.86, 10.52 and 9.53 lb. of feed per pound of gain, respect­ ively. - 19 - There appeared to be little difference in dressing percentage and carcass grade among lots but the results all favored the lots fed the higher protein rations. - 20 - + tH ip «J to (S CQ S O PQ CO • CM +tH 1 • IP t> CM • CO +1 o• t> 05 $ 5t« CO o• Hrl o CO • o CM O • *1 IP CO • CM I C ~~ 'M* • +1 CO tp • 05 as o o• CO tH P TO 05 00 ■nt4 IO tH 0 no £ tH o + tH ■M1 W M &H to x £ PQ • £ O p £ 05 + P H 05 £ p CO as £ to £ td 05 (d *H PQ c q £ P £ tH 05 OS P. •H £ •* 6-i X CM O Cm tH (S •H P P £ o •H P 5 & • • X tH «N • P £ tH cd £ £ •H •rl • • > «m > < X X •» £ •H td bO £ •H id n£ • !> < & £ £ to £ o o no 0 05 cm 9 • > •8 X t o Cd 0 £ tH cd t> •rl W5 • X tH 0 0 C m • > no 0 to •H ttS £ P-i PH "=11 G 5= a X! P ♦ £ $ *H (S no CGm £ X p £ 0 O £ 0 Ph taO £ •H m oi 0 £ a XI -H O o •• to 0 no 0 td O •H £ *H no x 00 O *H X O p to O bo £ 0 • • • td o o o o Jz; ^ £ cS O • s n2o e Cm P § 8 o o o G G 05 05 a a IO tH 05 05 X x 4-» P •£d § p £ p to tcS «tt to -H p p £ p G £ CCS CCS o £ £ 0 csx •9 a •HO -HO S ° n0 •a tH «h Cm Cm § _ 05 d 00 d 00 4^ J 3 •H *H £ C/5 X 05 05 o I H4 . CO -frl +O1 CM CM • CM• 09 O • CM CM CM X rH • -H £ cHd -* rH cd o rH d +» Q M 0 (X • o d o •H H-» cd P3 •H d * «>t{ d •H C m • > at to • > at XJ • > < 1 1£ 0 d o bo d a) 13 d d ft ® bo d P4 cd 0 b o £ rH r*> >S 1 — 1 cd •H rH •H o d 0 (0 -H E- rH o o d O •dH tH 0 • X rH •> d •ri cd bo * X rH 'x ' 0 0 tf-c • > <3 cd ’O 43 •H d h 0)X 09 0 X! O H-» s o d 0 X +-» Cw 0 d S 0 at H-> 0t •d XdJ at *d 0 d01 xi H-» E-t CO XI o ^Significant at the 5 percent level ^■Significant at the 1 percent level, to ■4* d +» rH O 0 a> cd d - 25 - the rather dusty nature of the rations fed in this trial contributed to the low feed consumption. The sheep coughed and sneezed considerably during feeding. Frequently, a sheep would take several drinks of water during feeding* Perhaps pelleting would have made the physical form of the rations more acceptable to the sheep as Cate et al, (1955) have shown that pelleting rations containing low quality roughages increased consumption. As there was large variability in the type and quality of lambs within each lot, no data were collected regarding carcass grades and dressing percentages. - 26 - SUMMARY Four feeding trials involving 170 sheep were conducted to establish whether biuret could be utilized as a non-protein source of nitrogen for ruminants. Urea was used as a comparison in all trials. was also tested* Crude biuret In the first and second trials, the basal ration con­ sisting of corn silage and ground corn appeared to be too high in crude protein to serve as a negative control. Thus, the addition of a non­ protein nitrogen source to this basal ration did not increase the rate of gain significantly. For the third and fourth trials a basal ration of approximately 7.1 percent crude protein was fed. The addition of either urea, biuret or crude biuret to the basal ration increased the average daily gain significantly. No significant differences were noted among the lots fed the supplemental nitrogen sources. It is concluded that under the conditions of these experiments, urea, crude biuret and biuret are satisfactory sources of supplemental nitrogen for sheep. - 27 - LITERATURE CITED Belasco, I. J. 1954. New nitrogen feed compounds for ruminants— a laboratory evaluation. J. Ani. Sci. 13:601. Bentley, 0. G., R. R. Johnson, T. V. Hershberger,J. H. Clineand A. L. Moxon. 1955. Cellulolytic-factor activity of certain short-chain fatty acids for rumen microorganisms in vitro. J. Nutr* 57:389. Bentley, 0. G., R. R. Johnson, S. Vanecko and C. H. Hunt. 1954. Studies on factors needed by rumen microorganisms cellulose digestion in vitro. J. Ani. Sci. 13:581. Bentley, 0. G., E. W. Klosterman and A. L. Moxon. 1952. Supplements to poor quality hay for fattening cattle. J* Ani. Sci. 11:757. Berry, W. T. jr., J. K. Riggs and H. 0. Kunkel. 1956. The lack of toxicity of biuret to animals. J. Ani. Sci. 15:225. Campbell, C. D., G. A. McLaren, G. S. Smith, J. A. Welch, D.C.Shelton and G. C. Anderson. 1956* The influence of diethylstilbestrol, urea and biuret upon digestibility and nitrogen metabolism is lambs. J. Ani. Sci. 15:1264. (Abst.) Cate, H. A., J. M. Lewis, R. J. Webb, M. E. Mansfield and U. S. Garrigus. 1955. Theeffect of pelleting rations of varied quality on feed utilization by lambs. J. Ani. Sci. 14:137. Dinning, J. S., H. M. Briggs, K. D. Gallup, H. W. Orr and R. Butler. 1948. Effect of orally administered urea on the ammonia and urea concentration in the blood of cattle and sheep, with observations on blood ammonia levels associated with symptoms of alkalosis. Amer* J. Physiol. 153:41. Duncan, D. B. 11: 1. 1955. Multiple range and multiple F tests. Biometrics Gaither, W., U. S. Garrigus, R. M. Forbes and E. E. Hatfield. 1955. Biuret as a source of non-protein nitrogen for sheep. J. Ani. Sci. 14:1203. (Abst.) Hatfield, E. E., W. Gaither, U. S. Garrigus, £. M. Forbes and R. C. Ewan. 1956. Biuret vs. urea. Illinois Sheep Day Rpt. Hatfield, E. E*, R. M. Forbes, A. L. Neumann and U. S. Garrigus. 1955. A nitrogen balance study with steers using urea, biuret and soybean oil meal as sources of nitrogen. J. Ani. Sci. 14:1206. (Abst.) - 28 - Meiske, J. C., W. J. Van Arsdell, R. W. Luecke and J. A. Hoefer. 1955. The utilization of urea and biuret as sources of nitrogen for growingfattening lambs. J. Ani. Sci. 14:941. Repp, W. W., K. H. Hale, E. W. Cheng and W. Burroughs. 1955. The influ­ ence of oral administration of non-protein nitrogen feeding compounds upon blood ammonia and urea levels in lambs. J. Ani. Sci. 14:118. Salsbury, R. L. 1955. Unpublished data. Mich. Agr. Expt. Sta. Snedecor, G. W. 1946. Statistical Methods, Fourth edition, Ames, Iowa: Iowa State College Press. Welch, J. A., G. A. McLaren, G. S. Smith, D. C. Campbell, D. C. Shelton and G. C. Anderson. 1956. The relative value of biuret, creatine, soybean protein and other nitrogenous materials for lambs in digestion and nitrogen metabolism trials. J. Ani. Sci* 15:1265. (Abst.) EFFECT OF STARVATION AND SUBSEQUENT REFEEDING ON SOME IN VITRO ACTIVITIES OF RUMEN MICROORGANISMS 30 - INTRODUCTION Government regulations specify that cattle shipped for long distances must be rested, fed and watered in 28 hours* to 36 hours if the shipper signs a release. This period may be extended When feeding of transported cattle is resumed in the feedlot, it is probable that conditions in the rumen have a great effect on the ability of microorganisms to digest feed. For this reason it is common practice to start animals on feed by grad­ ually increasing the amount of feed offered. Activity of the rumen microorganisms may be influenced by the nature of the previous ration, rate of food and water intake and the pH. Effects produced are un­ doubtedly varied and may consist of changes in absolute concentration, in relative proportions of various types, and in metabolic rates of individual, microorganisms. The experiments reported here are preliminary studies on the possible effects that longer periods of starvation might have on the return of microorganisms to a normal rate of certain meta­ bolic activities. The possible effects of different roughages fed before and after starvation were also studied. Because of the complexity of the microbial population of the rumen, it is extremely difficult to measure differentially the causes and effects of metabolic changes. By identifying and counting various types of micro­ organisms, attempts have been made to determine the effects of feed consumption by a ruminant. Unfortunately, with present techniques, it is not possible to identify and count all types of rumen microorganisms. However, the results of Quin (1943), Johnson et al. (1944) and Bortree - 31 - jet al* (1946) indicate that changes in numbers and proportions of certain rumen bacteria do occur following food intake by the animal. Another approach is to measure the summation of all effects, namely, the overall metabolic rate* This can be estimated by determining the rate at which end products are produced from some added substrate by a sample of rumen fluid. Quin (1943) and Jacobson et al* (1942) used gas production as an index of metabolic activity in the rumen, while Phillipson (1942) used volatile fatty acid production. Coop (1949) estimated the overall metabolic activity by measuring the rate of volatile fatty acid production from glucose and production of free HCN from the cyanogenetic glucoside lotaustralin. In the work reported here, the ability of rumen microorganisms to digest cellulose in vitro and lower the viscosity of a stable suspension of a cellulose derivative, carboxymethylcellulose, was used as a measure of their activities in samples taken at different times* 32 - REVIEW OF LITERATURE Jacobson et al* (1942) reported that only one-fourth of the ingesta of a dairy cow remained in the rumen after feed had been withheld for 24 hours«and pH of the rumen had risen* duction by samples of ingesta taken The in vitro rate of gas pro­ at this time was very slow* Changes in dilution, temperature and pH of the samples, within ranges normally occurring in the rumen, had little effect on in vitro fermentation. Phillipson (1942) found that when sheep were fasted 48 hours the pH of the rumen had risen to 7.6 with a concomitant fatty acid content. fall in volatile This indicated that fermentation was subsiding but had not necessarily ceased. A fall in pH of the rumen ingesta was usually accompanied by an increase in the Volatile fatty acid content. Upon refeeding the sheep after fasting, the pH of the rumen ingesta fell to as low as 4.36, but the sheep were not ill and were eating normally the following morning. The fermentation rate appeared to be most rapid when cabbage and mangolds were fed, slower when bran and oats were fed and slowest when hay was fed alone. Using gas production from added glucose as an index of fermentation, Quin (1943) noted that gas production in vitro reached a peak within 30 minutes when the samples were obtained from sheep fed lucerne hay. When wheat straw was fed to sheep accustomed to lucerne, no gas production was noted in 90 minutes. slow rate. After that, gas production occurred at a very Introduction of glucose into the rumen of sheep fed lucerne resulted in rapid gas production. However, when glucose was introduced 33 - into the rumen of sheep that had been fed only a poor quality grass hay, gas production was delayed* Glucose fermentation in vitro by ingesta taken from sheep that had been starved for 65 hours was negligible. Quin (1943) concluded that starvation causes a rapid and practically complete suppression of the fermenting ability of rumen ingesta in vitro. Sheep which had been starved 65 hours required 3 days to attain their usual daily intake. The glucose fermenting ability of the ingesta re­ turned to normal only after the sheep had been refed for ten days. Coop and Blakely (1949) found that HCN was released by rumen micro­ flora from the cyanogenetic glucoside lotaustralin. They reported that when a normal sheep was dosed with lotaustralin, cyanide poisoning developed rapidly. But when a sheep was starved several days, then dosed with lotaustralin and given food, no symptoms of cyanide poisoning occurred for several hours, after which they developed rapidly. This observation was interpreted as showing that, when a sheep is starved, the metabolic activity of its microflora is at a low level. When food was given to the animal the microbial population increased and the medium for metabolic activity improved with increasing momentum to a point at which lotaustralin was rapidly hydrolyzed. Coop (1949) recorded pH changes, volatile fatty acid production and rates of glucose and lotaustralin fermentation by rumen ingesta obtained during and following several starvation periods. old Romney wether as his experimental animal. He used a four-year- When the sheep was receiving approximately 2.5 kg. of feed per day, rumen pH varied between 6.0 and 7.0, half-time for lotaustralin hydrolysis was between 3 and 18 minutes, - 34 - half-time for glucose fermentation was between 5 and 10 hours, and volatile fatty acid content of rumen samples was between 10 and 20 mM percent* When the sheep was starved for three days, rumen pH rose to between 7.5 and 8*1, half-time for lotaustralin hydrolysis increased to between 120 and 660 minutes, half-time for glucose fermentation rose to between 20 and 50 hours, and volatile fatty acid content decreased tobetween 3*8 and 5.6 mM percent. 12to 24 hours samples of ingesta reached approximately normal values. After the sheep had been refed for The data of Coop (1949) show that the metabolic activity of samples taken 12 to 24 hours after refeeding often surpassed the activity of samples taken when the sheep was regularly fed. However, Coop (1949) evidently assumed this to be recovery to a normal level of activity and did not report the activities of samples taken after this 24-hour period. No differences in the recovery rates could be correlated with the kind of hay or grass the animal received prior to or following the starvation period. It was noted, however, that the animal frequently took a longer time to consume its ration when it was refed following a period of starvation. Robertson and Thin. (1952) studied the effect of starvation on ewes and cows in various stages of lactation and gestation. starved for five or six days. The cows were Total volatile fatty acid content of rumen samples decreased as the starvation period progressed, but increased slightly on the third day. The pH of the rumen rose to 8.4 on the second day but fell to 7.4 on the fifth day. Milk yields of lactating cows fell - 35 - sharply when the cows were starved. Blood levels of ketone bodies of cows in late pregnancy increased as the starvation period progressed. the peak of their lactation developed ketonemia. Cows at Blood sugar values de­ creased on the first day of starvation but returned to onormal.v values after the second or third day. In the case of ewes, blood levels of ketone bodies increased until the third day of starvation. Complete starvation seemed to have little effect on the animals except those at the peak of their lactation. Stone (1956) determined the volatile fatty acid content and gas production of ingesta from normal and atonic bovine rumens. Gas production in vitro and volatile fatty acid content were much lower in samples of rumen ingesta taken at the end of a 48-hour fast. Rumen motility decreased markedly during the fast. White et-al* (1956) found that blood samples from sheep whose feed intake had been gradually decreased indicated no change as long as the sheep had an average daily intake of at least 500 gm. When average daily intake fell below 500 gm., levels of blood sugar and keto-acids calcium each showed successive slight falls. and serum When the sheep were starved for 9 days, blood sugar levels decreased, blood levels of ketone bodies increased 8.1 mg. percent, inorganic phosphorus content of the blood rose to 7*6 mg. percent, and serum calcium and magnesium levels decreased. The number of leucocytes decreased, but the percentage of neutrophils was higher. Hemoglobin and hematocrit values did not change significantly. T^e sheep were consuming their usual amount of feed two or three days after feeding was resumed. consumption was 460 gm. During starvation, the average daily water - 36 - Williams and Christian (1956) reported a highly significant positive correlation between the level of rumen volatile fatty acids and the level of feed intake of sheep* Twelve sheep had their average daily consumption of dried grass gradually reduced from 1000 gm* to 400 gm. The acetic: propionic acid ratio in the rumen was significantly higher when the sheep were receiving less feed* The pH, ammonia-N, protein-N and free microbial cotints of the rumen ingesta were unaffected by the decrease in feed intake* Dale et _al. (1954) found that only slight changes occurred in the concentration of principal inorganic anions and cations in the plasma of cattle which had been starved for 5 days* occurred in the urinary excretion* However, massive changes Most significant were the decreased excretions of cations and carbonate ions and the increased excretion of phosphate. Blood pH did not change appreciably. Concentration of ketone bodies in blood and urine increased and organic acid excretion in urine was decreased. - 37 - PROCEDURE Rumen Sampling The inocula for the laboratory studies were obtained from a fouryear-old Hereford steer fitted with a permanent rumen fistula. was kept in a stanchion. The steer Samples of the fluid portion of the ingesta were strained through a single layer of cheesecloth to remove the larger plant particles. The strained fluid was collected in a thermos flask that had been warmed with water and thoroughly gassed with carbon dioxide to pro­ duce anaerobic conditions. The pH of rumen fluid taken after the first trial was determined as soon as the samples were brought to the labora­ tory. In Vitro Studies The fermentation procedure used was a modification of the method described by Huhtanen et al. (1954) and is similar to that described by Salsbury et al. (1956). Eight gm. of Solka-Floc-*- were weighed into a 950 ml. brown glass reagent bottle and 800 ml. of the strained rumen fluid were added. To ensure an adequate supply available nitrogen two ml. of a solution containing 200 mg. urea per ml. were pipetted into the reagent bottle when the rumen fluid was added. The temperature of the mixture was maintained by placing the bottle in a water bath at 39 degrees C. Carbon dioxide was bubbled through the mixture fast enough to give efficient mixing; the bottle was swirled vigorously, and 25 ml. Solka-Floc BW-40, Brown Co., Berlin, N. H. - 38 - of the suspension was- pipetted into a cellophane sac2 by means of a pipette with a large orifice. The sac was then placed in a four- ounce, screw-capped bottle containing 100 ml. of salt solution (table 1) which had previously been warmed to 39 degrees C. and gassed with carbon dioxide. The incubation periods were 3, 6, 9 and 24 hours. The samples were all run in triplicate. However, itwas decided that only the results of the 24-hour fermentation periods would be used as they best showed the differences in sample activities at different times of sampling. Table 1. Salt Salt Solutions Used for in vitro Studies Concentration (gm./L.) NaHCOg 4.9 Na2 HP0 4 1.86 NaCl 0.24 KC1 0.29 CaCl2.2 H2 O 0.026 MgCl2.6H20 0.064 FeS04.7H20 0.04 ZnS04*7H20 0.002 Cu S04.5H20 0.001 MnS04.H20 0.001 2Made from cellophane tubing 32/32, Visking Corp., Chicago, 111. - 39 - Replicate 25 ml* samples were pipetted into 200 ml. Berzelius beakers at the time the incubated samples were pipetted. These were used to determine the actual amount of cellulose present at the start of the incubation. They were dried immediately at 105 degrees C. After incubation of the test samples, the salt solution was poured out and the samples frozen in a deep-freeze until determinations were run. Then the contents of the sacs were transferred to 200 ml. Ber­ zelius beakers and dried at 105 degrees C. The method of Crampton and Maynard (1938) with modifications (Salsbury ^t al., 1956) was used for determination of cellulose. Fifteen ml, of 80 percent acetic acid and 1.5 ml. of concentrated nitric acid were added to each beaker and the mixture was refluxed on a hot plate. cold water served as a condenser. A round-bottom flask filled with Each sample was transferred to another beaker using absolute ethanol and filtered through a Selas porcelain crucible of extra coarse porosity. Filtered samples were dried at 105 degrees C. overnight, cooled and weighed. Then the samples were ashed for two hours at 700 degrees C., cooled and weighed again. The differ­ ence in weight before and after ashing was taken as the cellulose content of the sample. The benzene and ether washes of the Crampton and Maynard method were omitted. The difference between the amounts of cellulose in the control samples and the amounts in the incubated samples was consi­ dered to be the amount of cellulose digested. This difference was expressed as a percentage of the cellulose of the control samples. - 40 - Viscosimetric Studies It has been shown that there are microorganisms present in the rumen which produce enzymes capable of attacking the substituted cellu­ lose derivative carboxymethylcellulose (Underkofler and Underkofler, 1954; King, 1956). al., 1953; Kitts Changes in viscosity of carboxy­ methylcellulose (CMC) suspensions have been used to demonstrate that a number of organisms produce these enzymes (Levinson and Reese, 1950; Reese et al., 1950; Jermyn, 1952). Consequently, it was decided to use a viscosimetric method to investigate the effect of starvation and re­ feeding upon the activity of the enzymes that act upon cellulose deriva­ tives* The method used involved measuring the change in viscosity of CMC suspensions brought about by rumen fluid fractions. Viscosity was measured by determining the time required for a definite volume of the CMC solution to flow through a calibrated capillary. The substrate used in these viscosimetric studies consisted of a 0.3 percent solution of carboxymethylcellulose (CMC)l. About 400 ml. of water were heated to 80 degrees C. and added, together with 1.5 gm. of CMC, to a Waring Blendor. It was mixed in the blendor for 25 minutes, transferred to a 500 ml. volumetric flask, cooled and made up to mark with distilled water. Eight ml* of 0.3 percent CMC substrate, 1 ml, of pH 6.9 phosphate buffer (ionic strength * 0.145), and 1 ml. of the rumen fluid fraction to be tested, were placed in a viscosity tube? in that order. The vis­ cosity tubes were suspended in a constant temperature viscosimeter water J-Hercules Powder Co., Wilmington, Delaware. ^Ostwald-Femske, ASTM, 200 series. Lot 5734 CMC 50. - 41 - bath* The temperature of the water bath was maintained at 37 degrees C. Each tube had been standardized by determining the flow time with dis­ tilled water (ranged between 8.3 and 9.7 seconds for the tubes used in these studies). The flow time of subsequent test solutions was divided by the flow time for distilled water to give a relative viscosity value. Densities of water and rumen fluid were not sufficiently different to appreciably affect results and hence, were not considered in the cal­ culations. The zero-time viscosity for test solutions was that obtained from a solution containing rumen fluid that had been boiled to inactivate the enzyme. Subsequent readings were taken at 10, 20, 30, 40 and 60 minutes after introduction of the unheated sample. However, it was decided that the difference between initial and 10 minute viscosity readings gave the best measure of the activity of the sample used. Data bn two fractions of rumen fluid were recorded. consisted of the strained material. One fraction The other fraction (hereafter called S-l) was prepared by centrifuging the strained rumen fluid in an Inter­ national centrifuge, equipped with a four-place head, at 1500 r.p.m. for 5 minutes. This removed the protozoa and plant debris from the inocula. The S-l was a cloudy yellowish fluid. During the last two starvation trials, and for the last sample from the second trial, a third fraction of rumen fluid was tested. This fraction (hereafter called S-2) was prepared by centrifuging the S-l fraction in a refrigerated Servall centrifuge (SS-1 head) at 12,000 r.p*m. for 110 minutes. This operation removed the bacteria from the S-l fraction and resulted in a cell-free preparation of rumen fluid. was a clear yellow solution# The S-2 - 42 - Experimental Rations During all feeding periods the steer was fed twice daily a ration of 10 pounds of hay and 1 pound of supplement. supplement is given in table 2, The composition of the The proximate analyses of the hays and the supplement used in these studies are given in table 3* Table 2. Composition of the Steer Supplement Ingredients Percent of mixture Corn cobs (ground) 30.0 Soybean oil meal (solvent extracted) 32.25 Dried molasses 10.0 Alfalfa meal (dehydrated) 15.0 Dicalcium phosphate 5.0 Trace minerals 0.2 Vitamin A & D concentrate 0.05 Trace mineralized salt 1.5 Urea 6.0 Table 3. Proximate Analyses of Feeds Steer Supplement Alfalfa Hay Percent Timothy Hay Moisture 12.03 11.52 12.02 Ash 10*02 8.25 3.97 Crude fiber 12*22 22.81 32.37 Ether extract 1.13 1.97 2.32 Crude protein 38.19 17.13 5.66 N-free extract 26.41 38.32 43.66 Feed - 44 - RESULTS AND DISCUSSION For convenience of comparison, results of in vitro.cellulose digestion and of viscosity studies are presented together* TRIAL I The steer received timothy hay before and after the 48-hour star­ vation period. Results of the laboratory tests are presented in table 4 and figure 1. In this and all following tables, ttS** and »RM indicate starvation or refeeding, respectively. The number just preceding the nSn or f,Rn indicates the hours of starvation or refeeding, respectively, that had elapsed at the time of sampling. The zero-hour of sampling was always just prior to the morning feeding of the steer, or about 15 hours after the last feeding of the preceding day* Table 4. Sample time, hrs. Fraction Strained 0 In 1-S vitro 77.0 — Results of Trial I 3-S 6-S cellulose 78.6 9-S 24-S digestion, 81.5 76.5 32-S 48-S 24-R 65.8 79.7 percent 71.3 Change produced in viscosity of CMC Strained 1.02 S-l 0*74a 1.18 0.89 1.03 1.11 0.90 0.84 0.39 1.08 0.66 0.48 — 0.24 0.19 0.10 0.72 aThis S-l includes a mixture of 0 and 1-S samples. - 45 - The results indicate that no appreciable change occurred in the ability of the samples to digest cellulose in vitro. The CMC activity of the strained and S-l fractions appeared to have decreased consider­ ably by the end of the starvation period. samples appeared approximately normal. Values obtained from the 24-R - 46 - 0 Strained sample • S-l fraction o •H ra -H •rt •H 00 O 24-R Time of Sampling Figure F. Change in viscosity of CMC substrate by the different fractions of rumen fluid taken during Trial 1* TRIAL II In this trial more samples were procured during the period of* re­ feeding following a 48-hour starvation period# Again, the steer received timothy hay before and after the starvation period# The results are shown in table 5 and figure 2# Table 5. Sample time, hr. 0 Fraction Strained Results of Trial II 6-S 24-S 48-S 3-R 6-R 9-R 24-R 48-R In vitro cellulose digestion, percent 76.6 84.4 83.3 52.2 73.6 80.6 81.5 86.1 85.9 Change produced in viscosity of CMC Strained 0.88 0.79 0.95 0.41 0.41 0.19 0.36 1.07 0.95 S-l 0.54 0.73 0.42 0.11 0.32 0.26 0.24 1.55 1.13 pH Strained S-l — — 7.1 7.6 — 7.1 7.1 6.6 6.55 6.75 6.70 7.5 7.9 7.15 7.2 7.15 6.7 6.6 The ability of the samples to digest cellulose in vitro was not reduced appreciably by the 48-hour starvation period. The pH of the samples during the starvation period were higher at the end of the star- . vation period but returned to normal approximately 24 hours after refeeding. The ability of the fractions to reduce the viscosity of the CMC solution was decreased by starvation. No explanation was evident for the continued - 48 - decreasing activity of strained rumen fluid collected shortly after refeeding not being shown in the activity of the S-l. When offered the regular ration after the 48-hour starvation period, the steer ate readily. - 49 - Change in Viscosity O Strained sample e S-l fraction 1. — m s Time of sampling Figure 2* Change of viscosity of CMC substrate by the different fractions of rumen fluid taken during Trial II* - 50 - TRIAL III The steer was starved 72 hours since it was felt that a longer starvation period might affect the rate of recovery. Timothy hay served as the roughage before and after the starvation period. The results are presented in table 6 and figure 3. Table 6. Sample time, hr. 0 24-S Fraction Strained Results of Trial III 48-S 72-S 3-R 6-R 9-R 24-R In vitro cellulose digestion, percent 79.0 — 50.4 25.0 23.0 34.6 48.5 48.0 Change produced in viscosity of CMC Strained 0.87 S-l 0.34 0.80 — 0.27 0.21 1.37 1.55 2.27 3.10 0.18 0.19 1.25 1.40 1.97 2.99 2.91 S-2 pH Strained 6.8 7.15 7.6 7.8 7.4 6.85 6.5 5.95 S-l 6.9 7.32 7.7 7.75 7.5 6.95 6.5 5.95 The activity of rumen fluid, as measured by cellulose digestion in vitro and by action on CMC, was much less at the end of the starvation period. percent. The ability to digest cellulose was reduced approximately 70 Samples taken during the refeeding period showed an unusually high activity on CMC. This was evident from the sample taken 3 hours - 51 - after refeeding* It was during this trial that the S-2 fraction was tested for action on CMC. Removing the bacteria from the rumen fluid did not greatly affect the action. From this, it seemed likely that the enzyme or enzymes acting upon CMC was largely extracellular. The ability to digest cellulose was low following the starvation period, but increases were noted from the results of samples taken 6 hours after re­ feeding. - 52 - O Strained sample • S-l fraction * S-2 fraction Change of Viscosity 2.0 1.0 24-R Time of Sampling Figure 3. Change of viscosity of Oiil CHC substrate by the different fractions of rumen fluid taken during Trial III. - 53 - TRIAL IV This trial was to have been a repeat of the previous 72-hour star­ vation trial. period. Timothy was again used before and after the starvation Results are given in table 7 and figure 4. The steer had consumed some of his bedding (wheat straw) during the last 24 hours of the starvation period. This could be detected from the inocula obtained, and was reflected in the results. There was no substantial rise in pH of samples taken after 48 hours of starvation, nor was there much change in either ability to digest cellulose in vitro or attack CMC. The animal consumed approximately five gallons of water during the last 24 hours of the starvation. During the previous trial, the steer had consumed no water the last 60 hours of the starvation period. It must be assumed, therefore, that this trial does not give a true picture of the effects of starvation. - 54 - p3 Ni LO CO rH o OS • IO CO oo LO H CO CO cm tH id I (M +» 3 o> o Results of Trial IV • 00 t> CO S3 o •H bJO *rl X3 0> c/a i CM t- • CO c- CO o rH 3 rH rH CO 4 o Al H-» *H co o o CO •c* tr­ S3 •H LO ee • CO CO 00 a rH • O c/a i 00 • O > 0 O 3 TO o 0 bO § 43 O . CM I> • o rH rH OS 00 • o co LO CO LO CO CO co CM O CM O CD CO LO OS OS LO LO 00 to LO 00 • 03 ft o co CO CM CM CD CD CM CM 00 CM OO * to OS o 00 U +* cd c/a rH 00 rH O CD CO CM 0) a •H CO rH cA CO 00 • 8 cd O 0 0• rH rH a u 00 O •o rl ft 00 o t- -P O P4 i LO 00 d> ft P4 I os LO r-1 "d* ft! os • 00 ID CM cd -P ua - 55 - *o •3 5J § o rH Ch O (0 £ o • H +» O Rj u Cm -H N G d) J CH s ,a G o ♦H HG O (0 o s a ChO +> (0 O O CO G •H d> uo N • o IO 03 rH • CO o • CO o o • CD oo o 00 o 1 CO a> Pi P4 i 03 Pi 1 CO •H EU -t «H O 03 -P i—I rs o Pi 0 CL, *S ci o *ri +» (0 <1> ttO •rl CO o rH 3 rH rH O o O W o rH • 05 CO Cm O >» -P •rl CO rH • to 03 o o > ■H > CO • 00 rH p; •H TD 4) O 3 • o IO IO OO • IO 03 t• CO tt• CO t> 00 • CO 00 • co CO 00 • C- m • rH CO co • o tCO • o LO • o W fa Oi CO rH • o b• t- LO IO rH • O • t- t- IO CO rH co 00 03 O o O CO t- t• tt- CO o • rH rH • O to to • t- • t- 'O T3 92 a •H ctf Pi •P O Pi fa 0) h/) ri ro O 1 1 M • o CO 1 1 03 o oo 05 o rH • O rH • oo P *| CO 1 • 03 03 2 -p •H o o • CO . ■ o • • o • « o • M •% i—| B 3 CO Pi o •rl -P o a u fa 4) a •rl cti Pi -P CO to 'O o ps •H rH 1 CO a u -p CO rH 1 to - 58 - 3.0 Strained sample S-l fraction Change in Viscosity 0 • 0 Figure 5. 24-S 48-S 72-S 24-R Time of Sampling 48-R 72-R Change of viscosity of CMC substrate by the different fractions of rumen fluid taken during Trial V. 96-R - 59 - TRIAL VI In this trial, the steer received alfalfa hay before and after starvation. The results are given in table 9 and figures 6 and 7. The results differed little from those obtained in the previous trial, except during the later part of refeeding. The ability to digest cellulose was reduced approximately 85 percent at the end of / the starvation period. After refeeding was initiated, in vitro diges­ tion of cellulose increased to approximately normal between two and four days* Samples taken four hours after refeeding indicated, as judged by pH of the rumen fluid, that'fermentation was underway in the rumen. However, pH continued to decline and was lowest at 96-R. The values for CMC action did not return to normal in three days. Samples taken a week later indicated a continuing high activity for all three fractions. Samples taken 11 and 19 days after refeeding indicated that the activities were still above normal though the activity of the S-l and S-2 fractions had decreased considerably. In vitro cellulose digestion was approximately normal at these times, however. Samples taken 25 and 30 days after refeeding indicated that the fractions fluctuated in activity. It was thought that perhaps the starvation had upset the usual microbial population and that it had not yet become re-established. The steer was given a transfusion of approximately three liters of rumen fluid from another steer that had been fed regularly. Samples taken 14 days after the rumen transfusion gave results of normal values. The appetite of the steer was good throughout the refeeding period. - 60 - Pl i p ID eo i i 1 1 O I n i i e d s O tH OO S-l Pd I CO 05 f tPi I oo CM Pd I Table 9. CM Pd i 05 ' ^5 Pi . 8 o p CO 00 O bo *CJ <0 CO rH 5H r Q O O J-i 5 a o Ch O P« 00 Q o P •H (0 O O CO •rl > CD • CM CD ID • LO 05 • CO •H TJ 0> o w P T3 O Sh Ph 0) c3 x; o Pd i LO rH C/5 CD CM t- rH I c/5 I 00 01 5 H * CM • CO 05 05 • CM CD ID • CM ttfr e> • rH CO o « rH fPi .8 -p o «s P* XOS .crj8 u p C/D o • rH & rH CO • o • CO « CO 00 05 • CM OO • CM CD CD • rH CO 05 • O * CO • ID • O CD CM 'd* O ID CO • CM CO CD • rH ID 05 • O Pr o CD CO CD 00 CD CD • JU CO tr­ ee IO • ID • 05 00 • CM ID 05 ID feH t> ID ID ID to 00 • ID ID ID O CD 00 • ID 00 05 rH ID CM CD t- 00 CD CO • CD ID CD oo CM 00 rH • O co * ID CO 00 00 rH • O fc- ID 00 O CD ■M* H rH • 0 0 CO CD CO rH • o • CM • O CM Pi IO ID CD IO t> CM • rH o 1 1 rH • o o O • H* o tH rH • O O O CD t> • CM CM • O C> CD O 05 O CD CD O rH • *