I; In ‘ l x \‘ } HHII rH'lUIHII 1 1 Hill ( I » HI 40—; 1000 [I (DCDCD THIS Ab TiFéCiAL RUAfiEN AS A TECE‘WKQUE "OF? EVALUATE‘%36 33R? MAWER DiGES?-i§i§.i7‘f O? 2311:3335 FG‘RAGES Tbs—33:5 For five Degrac a? 5’s. 3. MEMGAN STAYE UNEVERSSTY Fg‘afik V. @anéwfi E95? THESlS L [B R .4 R Y I“. Mi: hignn State University J‘m‘i d'rfih-w¢yo ~ -'~w- - “r“ m- THESM THESIS THE ARTIFICIAL RUKEN AS A TECHNIQUE FOR EVALUATING DRY MATTER DIGESTIBILITY CF PASTURE FCRAGES By FRANK V. GENDRON 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 1959 Approved THESIS ACKNOWLEDGMENTS The author wishes to express his sincere appreci- ation to Dr. C. A. Lassiter, Associate Professor in Dairy- ing, Dr. R. S. Emery, Assistant Professor in Dairying, and Dr. C. F. Huffman, Professor in Dairying, for their super- vision, counsel, and guidance throughout this study and for their critical reading of this manuscript. The writer is grateful to Dr. N. P. Ralston, Professor in Dairying, for the opportunity and provision of facili- ties used in this study. The author is also indebted to Mr. T. R. Lewis, Re- search Instructor in Dairying, for his technical assist- ance and to Dr. C. E. Meadows, Associate Professor in Dairy Extension, for his suggestions during the writing of this paper. Thanks are due to Mr. E. S. Smiley, W. B. Hutchinson, Ralph Reid, and the others who assisted in caring for the animals and arranging the facilities used in this study. THESIS TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . . . . Techniques in Forage Digestibility Evaluation Lignin Ratio Technique . . . . Nitrogen Ratio Technique . . . . Iron Oxide Technique . . . . . . Equations for Predicting Digestibility Plant Pigment Technique . . . . Chromic Oxide Ratio Technique. . Artificial Rumen Technique . . . EXPERIHENTAL PROCEDURE . . . . . . . . . . Part I. . . . . . . . . . . . . . . . Part II o o o o o o o o o o o o o o 0 Preparation of Forage for Fermentation Micro-Artificial Rumen Technique Inocula . . . . . . . . . . . . Analytical Methods . . . . . . . . . RESULTS AND DISCUSSION . . . . . . . . . . SUMMARY} . . . . . .. . . . . . . . . . . . LITERATURE CITED . . . . . . . . . . . . . Page CDCPU‘IW #wmm n) ro F4 +4 F1 +4 |4 F3 F4 I4 FJ a) \J \o (r a) v1 U1 \n \N xx 0 THESI LIST OF TABLES Table Page I Moisture of Pasture Forages . . . . . . . . 19 II Dry Matter Intake Per Cow . . . . . . . . . 21 III Per Cent Dry Matter Digested. . . . . . . . 22 IV Comparison of Duplicate Artificial Rumen Runs 0 O 0 I O O O O O O O O O O O O O O O O 23 V Comparison of Estimated Dry Matter and Anthrone Carbohydrate Digestibilities . . . 25 INTRODUCTION A simple and accurate technique for rapidly evalu- ating pasture forage quality is seriously lacking. Such a technique would be of great value to the plant breeder and others, in providing a rapid and inexpensive procedure for estimating forage digestibility and quality. Digestion trials with ruminants provide information regarding the nutritive value of forage in a satisfactory manner, however they are expensive in terms of time, labor, and equipment and require large amounts of forage. Fur- thermore the investigator is working with a dynamic system, in which the quality of the forage may change considerably during the experimental period. Recent reports on the use of the artificial rumen as a means of evaluating forage quality have indicated that this method may prove to be a satisfactory and reliable technique. Therefore the principle purpose of this study was to evaluate the use of the artificial rumen as a technique for determining pasture forage quality. REVIEW OF LITERATURE The first studies on the digestibility of the dry matter of forages were made by Reamur (1752), Stevens (1777), and Spallanzani (1784), as cited by Huffman (1958). However, the earliest known metabolism trials were con- ducted with a milk cow in 1839 by Boussingault. According to Schneider (1955) in 1864 Henneberg and Stohman, pioneer German workers in the nutrition of farm animals, conducted digestibility trials, which have changed but little since their adoption. Techniques in Forage Digestibility Evaluation The evaluation of pasture forage, until recently, was primarily accomplished by two methods. One consisted of calculating the nutrients the cow received from an area of pasture forage, on the basis of her milk production and change in body weight. This method did not prove satis— factory because of the many variables involved. The second method consisted of harvesting the forage and feeding it to cows in digestion stalls. This method provided more accurate information on forage intake and digestibility, however the results seldom agreed with those obtained from grazing cows. McCullough (1952) in his review pointed out that lit- tle progress was made in determining pasture forage quality by either of these two methods. Therefore, the natural constituents and chemical com- position of forage plants, indigestible "external" indica- tors and other techniques adaptable to grazing animals have been studied as a means of evaluating pasture forage quality. Lignin Ratio Technique Lignin, a natural constituent of plant material, has been studied as a possible plant indicator of digestibility. Ellis, Matrone and Maynard (1946), using the lignin ratio technique and the standard total fecal collection technique, found the digestibility to be similar in both techniques using cows, sheep, and rabbits. Hale, Duncan, and Huffman (1940) also reported that lignin may be used as an indi- cator for estimating cellulose digestion in the rumen. Kane (1950) found no significant difference between the lignin and Chromic oxide techniques as compared to the con- ventional collection technique of evaluating digestibility coefficients with dairy cattle. Cook and Harris (1951) showed a variation in dry matter digestibility using the 4 lignin ratio technique. Richards qt g1. (1958) reported considerable doubt on the predictive value of lignin as an indirect indicator of dry matter digestibility. Nitrogen Ratio Technique The calculation of forage digestibility by the forage and fecal nitrogen technique has been reported in a number of studies. Gallup and Briggs (1948) reported that the total fe- cal nitrogen excretion of steers was related to dry matter intake and that a constant relationship existed. They re- ported, that feed consumption of grazing animals could be determined by taking advantage of this relationship to determine dry matter intake and that a close relationship existed between protein content and digestibility of nu- trients in the hay. Lancaster (1949) presented a formula for evaluating pasture forage digestibility using fecal nitrogen as an indicator. Soni qt 31. (1954) reported that the fecal-nitrogen method appears to be as reliable as the chromogen method for determining dry matter digesti- bility and found no diurnal variation in nitrogen excre- tions when using Lancaster's formula. Forbes (1949) re- ported data to show that no constant relationship existed between dry matter intake and the excretion of fecal nitrogen. Iron Oxide Technique Iron oxide, as an indicator of the digestibility of feed, has been prOposed by Bergeim (1926). Gallup (1929) obtained favorable results using the iron oxide technique with rats. Heller qt g1. (1928) used the normal iron content of a ration fed to rats as an indicator for determining the digestibility of the ration. Knott (1956) compared the iron ratio technique using the natural iron content of feeds and reported it was not adaptable to ruminants. Hale (1940) in a study with alfalfa hay found the results highly variable and not reliable for use with ruminants. Druce and Willcox (1949) reported unreliable results with this method because of the low recovery of iron oxide. Equations for Predicting Digestibility Schneider 23 gl. (1951) published equations for esti- mating the digestion coefficients of nutrients in feeds fed to cattle and sheep and in concentrates fed to swine. They reported that the use of these equations will result in prediction error variances which are 25 to 45 per cent below those incurred if tabled average digestion coeffi- cients are employed. 'Equations were presented for pre- dicting the digestion coefficients for organic matter, crude protein, crude fiber, nitrogen free extract, ether extract and the content of total digestible nutrients in feed, using their percentages of crude protein, crude fiber, nitrogen-free extract, and ether extract as pre- dicting factors. These equations were reliable for only those feeds for which digestibility data were available. However, Schneider g3 g1. presented equations to estimate the digestion coefficients of crude protein, crude fiber, nitrogen-free extract and ether extract and the content of total digestible nutrients in feeds for which few or no digestibility data were available. These equations should be distinguished from those designed for use with feeds for which sufficient digestible data are available as they do not yield as precise estimates. Plant Pigment Technique Indigestible pigments, occurring naturally in plants, which are fully recoverable from the feces can be reliable indicators of forage intake and digestibility. Reid qt g1. (1950) first reported the use of the plant pigment tech- nique (chromogen ratio technique) using steers, calves, and wethers. Eighty-five per cent acetone was used as the sol- vent for extracting the plant pigment or pigments from the feed and feces and the diluted extracts were read at 406 mu using a Beckmann spectrOphotometer. The authors reported nearly 100 per cent recovery of the plant pigment from the feces in a large number of trials. The calculated digestion coefficients from the plant pigment technique revealed close agreement with conventional total fecal collection techni- ques. l. (1951) compared the lignin and chromogen Cook gt techniques with sheep on mature winter range pasture and reported negative digestion coefficients and low estimates of dry matter intake. However, when lignin and chromogen techniques were used with alfalfa hay they reported no significant difference between the two methods. It would therefore appear that the chromogen technique may not work with all species of forage. Kane gt g1. (1955) in a study with lactating cows on orchard grass also found that the chromogen ratio technique did not agree with conventional digestion trials. Reid gt g1. (1952) reported that there was a close relationship between forage chromogen concentration and fecal chromOgen concentration and presented regression equations for the predication of forage digestibility. Different relationships were found to exist for dry hays and their corresponding fecal chromogen concentration than that reported with fresh grasses. Soni (1954) pre- sented favorable resultsusing Reid's 33 gl. (1952) for- mula to determine dry matter digestibility of pasture forages with grazing sheep and cattle. Noller 33 gl. (1951) used the chromogen ratio tech- nique with milk cows on pasture to determine forage intake and digestibility. Chromic Oxide Ratio Technique Chromic oxide, an inert tracer material, has been one of the most widely used "external" indicators of digesti- bility. Edin (1918) first reported the feeding of chromic oxide, impregnated in blotter paper as a tracer substance and reported almost complete recovery. Kane 33 gl. (1950, 1955a, 1955b, 1957) reported no significant difference between digestion coefficients using chromic oxide and the total collection method with cows. Smith and Reid (1955), Crampton and Lloyd (1951) and Put- nam 22 gl. (1957) also reported satisfactory results with the chromic oxide ratio when used with cows. In other studies satisfactory results were reported with steers, by Brannon gt g1. (1954) and Hardison and Reid (1955); with sheep by Anderson (1954), Woolfolk gt g1. (1955), Skulmowski 93 gl. (1943), and Pigden and Brisson (1956); with goats by Chanda gt 3;. (1951); and with swine by Schurch 33 gl. (1952) and Lloyd (1951). Archibald gt g1. (1958) reported chromic oxide gave more uniform results than lignin in determining digesti- bility of forage with cows. 9 Crampton and Lloyd (1951) and Schurch (1952) reported a low recovery of chromic oxide when fed to sheep on an all roughage ration; however, when a concentrate was added to the ration satisfactory results were obtained. Variability in the recovery of chromic oxide from feces of cattle have been reported by Kane gt Q1. (1950, 1952), Reid (1952), Lancaster and Coup (1955), Hardison gt 3;. (1955), Oldfield gt al. (1956), Putman gt al. (1957), and Murdock qt g1. (1957). Smith and Reid (1955) and Hardison (1956) reported the time chromic oxide was given did not affect the excre— tion curves with cows. Hardison (1956) reported twice a day administration of chromic oxide to cows resulted in a more even rate of excretion. Reid gt g1. (1952) reported greater diurnal variation when using gelatin capsules than when chromic oxide was given in the feed. Kameoka (1956) reported results which showed that when sampling the feces twice a day for several consecu- tive days it was adequate for obtaining a representative sample. He also reported daily variation of chromic oxide excretion a normal occurrence amounting to 10 per cent. Davis, Byers and Luber (1958) reported considerable variation in the excretion of chromic oxide at various hours of the day regardless of when administered. However, 10 they indicated this variable could be overcome in digestion studies by sampling the feces for a ten-day period. Putnam gt g1. (1958) concluded that time of chromic oxide admin- istration was of primary importance in respect to time- concentration relationships of fecal chromic oxide, and that diurnal effects were of little importance. They sug- gested that a 12-hour sampling procedure would be more ac- curate than unequal fecal sampling times. Bloom qt g1. (1957) and Putnam (1958) reported no significant effect of prOportion of hay and grain intake on chromic oxide excretion curves for cows. Kane gt g1. (1953a, 1955b) reported favorable results were obtained in estimating pasture digestibility and dry matter intake when using chromic oxide in conjunction with the chromogen ratio technique. Artificial Rumen Technique One of the first serious attempts to duplicate the conditions of the rumen was made by Marston (1948), in studying cellulose digestion. Louw qt gl. (1949), Bur- roughs gt g1. (1950), and Huhtanen gt g1. (1952) further developed the artificial rumen and thus several types were sufficiently improved for use in attempting to evalu- ate forage quality. ll Numerous studies have been reported on the modifi- cation and develOpment of the artificial rumen and the reader is referred to Pigden (1955) and Salsbury (1955) for a more complete review. Baumgardt and Hill (1956) reported on a study of dry matter digestibility of various forages in the arti- ficial rumen and sources of error or bias. Dry matter losses noted were: Ladino clover, 65 per cent; alfalfa (bud stage), 58 per cent; Kentucky bluegrass (mature), 40 per cent; and wheat straw, 50 per cent. Kamstra gt g1. (1958) reported on the effect of stage of maturity upon plant cellulose digestion in the artificial rumen. Orchard grass, alfalfa, and timothy were harvested at 5 stages of maturity with orchard grass decreasing as much as 50 per cent in rate of cellulose digestion, and timothy and alfalfa 50 per cent between the first and third stage of maturity. Quicke gt g1. (1959),in a study of milling by- products, reported higher digestibility ig thgq, than in a conventional sheep digestion trials. However, they at- tributed this to the rapid passage of the feed through the rumen and therefore lower cellulose digestion in the rumen. Kamstra (1955) in a preliminary experiment found that in a 24-hourq1g vitro digestion period on orchard grass- l2 alfalfa mixture, a pelleted feed mixture, and a corn and cob meal-poor hay mixture cellulose digestion of 60.55, 48.00, and 51.66 per cent, respectively. These same feeds showed £2 yglq digestion coefficients with cattle of 68.17, 46.10, and 41.61, respectively. Pigden and Bell (1955) reported the TDN values estimated, by the use of a formula applied to the ;q XAEEQ results of 11 forages, fermented for 48 hours, com- pared favorably with the TDN values obtained gg zlyq with sheep. EXPERIMENTAL PROCEDURE The purpose of this study was to evaluate the use of the artificial rumen in estimating pasture forage digesti- bility. The experiment was designed in two parts. Part I was designed to evaluate the pasture forage digestibility, with dairy cows on pasture, calculating forage intake and dry matter digestibility with the chromic oxide technique of Hardison gt g1. (1955) and the chromOgen (5) ratio of Reid gt g1. (1952). Part II consisted of calculating the dry matter digestibility of these same forages using the artificial rumen technique as modified by Pigden (1955). Part I Nine pasture forages differing in botanical composi- tion or stage of maturity were compared. The nine pasture forages were as follows: Trial I Alfalfa-Ladino-Brome. 6 to 8 inches high. Preliminary period started May 22, 1956. Trial II June Grass. 8 to 10 inches high. Pre- liminary pasture period started May 22, 1956. Trial III Alfalfa-Brome (bud stage). 14 to 16 inches high. Preliminary pasture period started June 5, 1956. - 13 _ 14 Trial IV June Grass (heading out). 15 to 17 inches high. Preliminary pasture period started June 5, 1956. Trial V Alfalfa-Ladino-Brome. (Clipped). 6 to 8 inches high. Preliminary pasture period started June 29, 1956. Trial VI Ladino Clover. (Recovered). 6 to 8 inches high. Trial VII Sudan Grass. 14 to 18 inches high (weedy). Preliminary pasture period started July 12, 1956. Trial VIII Kentucky bluegrass (clipped). 4 to 6 inches high. Preliminary pasture period started July 19, 1956. Trial IX Rye (poor condition). 8 to 12 inches high. Preliminary pasture period started July 19, 1956. Three, mature, dry, Holstein cows were used on each pasture forage studied, using a seven-day preliminary period and five-day experimental, or collection period for each trial. The cows received no grain or feed sup- plements throughout the trial. Each cow received five grams of chromic oxide in a gelatin capsule administered orally with a balling gun twice daily at 5:50 a.m. and 5:50 p.m. Rectal "grab" samples were taken from each cow at twelve-hour intervals, at 5:50 a.m. and 5:50 p.m. each day during the five-day experimental period and composited. The fecal samples were stored at 4 to 5° C. until the com- pletion of the experimental period, when a total aliquot sample for each cow was frozen until analyzed. 15 Part II Plucked forage samples were collected from each trial pasture on the second and fourth day of the experi- mental period, stored in sealed polyethylene bags and frozen immediately after collection. Every attempt in the collection of each forage sample was made to duplicate the kind and quality of forage consumed by the cows. Prqparation of Forage for Fermentation Considerable difficulty was experienced in reducing the green frozen forage to a uniform particle size, adapt- able for use in the artificial rumen and in securing dup- licate forage samples. The technique which appeared to give the best results consisted of a hand model food chOp- per, maintained at or below a temperature of 0° C. Grind- ing of the frozen forage was completed in a walk-in freezer, to eliminate the thawing of the forage or the loss of plant juices and to facilitate a uniform mixing of the two-pound composite forage samples. Micro-Artificial Rumen Technique A description of the procedure as adOpted as the stan- dard procedure in the fermentation of the nine pasture for- ages is as follows: l6 Fifty ml. Erlenmeyer flasks, each equipped with a rubber stOpper and a hypodermic needle plugged with cot- ton were employed as the fermentation vessels. Five grams of green ground forage was weighed into each flask. Five ml. of distilled water was added to each flask and autoclaved at 20 pounds pressure for 15 minutes. The flasks were allowed to cool and 0.5 grams of calcium car- bonate and 5.7 ml. of complex salt solution were added to each. The flasks were then placed in a constant tempera- ture water bath and aerated with carbon dioxide for thirty minutes. During this gassing process the flasks were connected in series and equipped with stOppers with gas inlet and outlet tubes. The flasks were swirled gently every eight to twelve hours to remove any forage particles which were deposited on the sides during the active fermentation period. At the completion of the 48-hour fermentation period the flasks were placed in a drying oven at 105° C. to drive off the moisture. When the residues were nearly dry they were stirred thoroughly with a glass rod to prevent the residual calcium carbonate from caking on the bottom of the flask. Drying was then completed at a lower temper- ature of 50 to 60° C. When the fermentation residues were dry they were hydrolyzed with 20 m1. of 72 per cent sulfuric 17 acid for two hours at room temperature. Acid and residue were carefully mixed with a glass rod three times, once when the acid was first added and twice more during the first hour. At the end of the hydrolysis period the con- tents of each flask were diluted to 500 ml. and allowed to set for approximately one hour to allow the insoluble resi- due to separate out. Anthrone carbohydrate determinations were then made on the supernatant. Two extra flasks of each forage were prepared and dried immediately before fermentation occurred to obtain the carbohydrate present in each forage. These flasks were hydrolyzed with acid and analyzed for carbohydrate with their corresponding fermentation residues. The breakdown of carbohydrate in the fermentations was expressed as percentage carbohydrate fermented of the total initially present. The composition of the complex salt solution was as follows: Gm./2000 m1. Ingredients . Distilled Water Sodium phosphate (monobasis) 52.50 Sodium bicarbonate 52.50 Potassium chloride 7.50 Sodium chloride 7.50 Magnesium sulphate 2.25 Calcium chloride 0-75 18 Ferrous chloride 0.15 Manganous sulphate 0.08 Zinc sulphate 0.08 Copper sulphate 0.04 Cobalt chloride 0.02 P’“ Inocula The rumen fluid,obtained for the first run with the artificial rumen, was obtained from a fistulated Holstein cow fed a ration of June grass pasture. Rumen fluid from the same cow was obtained for the second run, however she had been returned to an alfalfa hay ration three days be- fore collection of the fluid. Analytical Methods The method used to determine chromic oxide of the fecal samples was that of Gehrke and Baker (1954) and the Chromogen (s) technique was that of Reid gt g1. (1952). The chromogen unit was standardized against Reid's chromo- gen unit used in his laboratory and the samples were ex- amined for light absorption with a Beckman B Spectrophoto— meter at 406 mu. The anthrone carbohydrate determinations were made as used by Pigden (1955). A standard anthrone carbohydrate curve was prepared with glucose using a Beckman Model B Spectrophotometer at a wave length of 540 mu. RESULTS AND DISCUSSION The moisture content of the nine pasture forages used in this study are shown in Table I. There is a possibil— ity that the low moisture values recorded for some of the forages may be due to the loss of moisture and dehydration EVVMI' of the forage samples during storage. Table I Moisture of Pasture Forages Wu". - Trial No. Forage % Moisture I Alfalfa-Ladino-Brome 74.5 II June grass 68.5 III Alfalfa—Brome 80.4 IV June grass 71.5 V Alfalfa-Ladino-Brome 77.1 VI Ladino clover 80.5 VII Sudan grass 65.4 VIII Kentucky bluegrass 68.7 IX Rye 76.0 The coefficients of digestibility of the forages in digestion trials with dairy cows as estimated by the - 19 _ THES! 2O chromogen ratio technique of Reid q; 31. (1952) was em- ployed as the criteria for evaluating the results ob- tained by the artificial rumen technique. For the pur- poses of this study the assumption was made that a close relationship exists between the per cent anthrone carbo- hydrate digested and the per cent dry matter digested. The dry matter intake as determined by a combination of the chromOgen and the chromic oxide technique is re- ported for each cow in Table II. The per cent digestible dry matter as determined by the chromogen technique for each forage is reported in Table III for each cow. The variability between cows on the same forage is less than the expected individual cow variation of 5 to 8 per cent generally reported. Duplicate trials using the micro-artificial rumen technique were run for each forage and the results are recorded in Table IV. A high correlation coefficient was found to exist between runs for the anthrone carbohydrate present at the zero hour (r = 0.822) and 48 hour (r = 0.975) fermentation periods. 0n the basis of this close correla- tion the laboratory techniques employed in determining anthrone carbohydrate were considered satisfactory and re— liable. The correlation coefficient between runs for the per cent anthrone carbohydrate digested for each forage was not ' ‘3; 19.1." x“: Table II Dry Matter Intake Per Cow 21 Cow Number Pounds D.M./Day Trial I 105 15.2 Alfalfa-Ladino-Brome 19 17.5 106 25.6 Trial II 20 21.4 June Grass 80 26.6 107 2996 Trial III 19 16.8 Alfalfa-Brome (Bud stage) 105 25.5 106 25.1 Trial IV 107 24.8 June Grass (Heading out) 20 28.9 80 26.0 Trial V 19 24.0 Alfalfa-Ladino—Brome (Clipped) 107 27.7 105* Trial VI 20 24.5 Ladino Clover (Recovered) 107 51.4 154 51.0 Trial VII 19 26.2 Sudan Grass 106 25.9 24 20.8 Trial VIII 154 25.8 Kentucky Blue Grass (Clipped) 20 26.5 106 28.1 Trial IX 94 2495 Rye (Poor condition) 22 18.0 20 17.1 * Removed from experiment due to calving. significant (r = 0.598). This low correlation between runs must in part be attributed to an accumulation of errors re— sulting from the laboratory procedures employed in deter- r‘uu_:.l (.1 THEE Table III Per Cent Dry Matter Digested 22 Cow Number % Digestion Average Trial I 19 65.8 Alfalfa-Ladino-Brome 105 65.5 66.24 106 69.5 Trial II 20 66.6 June Grass 80 71.0 69.91 107 72.1 Trial III 19 66.1 Alfalfa-Brome 105 69.1 67.69 106 67.9 Trial IV 20 59.9 June Grass 80 64.6 62.65 107 65.6 Trial V 19 70.1 Alfalfa-Ladino-Brome 107 72.2 71.15 105* Trial VI 20 71.9 Ladino Clover 107 72.9 72.44 154 72.6 Trial VII 19 69.5 Sudan Grass 24 67.6 68.76 106 69.4 Trial VIII 20 62.5 Kentucky Blue Grass 106 62.5 60.90 154 58.0 Trial IX 20 65.5 Rye 22 65.4 64.71 94 65.2 ‘ Removed from experiment due to calving. mining anthrone carbohydrate; however, the variability of the digestion coefficients observed between runs of the same forage must also reflect the errors incurred in 25 Table IV Comparison of Duplicate Artificial Rumen Runs First Run Second Run A verage Trial Hours Average‘ ug % Average‘ ug % IQ Vitro Fermented Anthrone Digest. Anthrone Digest. (0000) (0000) 1 48 57 55 54 80 66 0 79 166 2 48 145 44 148 52 58 0 258 219 5 48 52 42 45 52 47 0 9O 9O 4 48 152 24 145 35 29 0 200 217 5 48 88 47 78 52 49 0 167 161 6 48 59 60 26 78 69 0 97 118 7 48 155 42 150 49 46 0 254 256 8 48 98 55 115 47 51 0 219 214 9 48 26 84 46 64 74 0 166 129 * The values in this column represent the average ug of anthrone carbohydrate from duplicate flasks. obtaining uniform five gram forage samples and the errors resulting from the variability of the anthrone carbohy- drate digested in the micro-artificial rumen. A major source of this error and one that apparently varies between different forages is the variability due to different lots 23 Table IV Comparison of Duplicate Artificial Rumen Runs First Run Second Run A , verage Trial Hours Average* ug % Average‘ ug % IQ Vitro Fermented Anthrone Digest. Anthrone Digest. (0000) (0000) 1 48 57 55 54 80 66 0 79 166 2 48 143 44 148 32 58 0 258 219 5 48 52 42 45 52 47 0 90 90 4 48 152 24 145 55 29 0 200 217 5 48 88 47 78 52 49 0 167 161 6 48 59 60 26 78 69 O 97 118 7 48 155 42 150 49 46 0 254 256 8 . 48 98 55 115 47 51 O 219 214 9 48 26 84 46 64 74 0 166 129 * The values in this column represent the average ug of anthrone carbohydrate from duplicate flasks. obtaining uniform five gram forage samples and the errors resulting from the variability of the anthrone carbohy- drate digested in the micro-artificial rumen. A major source of this error and one that apparently varies between different forages is the variability due to different lots 24 of inoculum used in each trial. The difference between runs of the same forage, in the per cent anthrone carbo- hydrate digested, ranged from 4.5 to 26.4 per cent, with a mean difference for the nine trials of 12.7 per cent. Whether the repeatibility of the digestion coeffi- cients estimated by the artificial rumen can be improved can only be determined by further studies and the develop- ment of more accurate and reliable techniques. The percentage of dry matter digested (chromogen technique) is compared with percentage of anthrone carbo- hydrate fermented at 48 hours lg 11339 and is reported in Table V. The percentages recorded for the chromogen method represents the average per cent dry matter digested by the three cows for each forage and the la lipgq results represent the average per cent anthrone carbohydrate fer- mented in the two runs. The relative difference in per cent digestibility for each forage between the two methods would be of minor significance provided they were in the same relative direction; however, it is evident from Table V that this difference is of major importance as the le- vels of digestibility between the two methods for each forage varies significantly. The correlation coefficient between the two methods was not significant (r = 0.158) and substantiated this observation. The significant dif- ference observed between methods may be due to several possible factors. 25 The lack of repeatibility between lg 1§§£q runs must be considered as a major source of this error. As dis- cussed previously, the forage sampling and grinding pro- cedures, and the use of different lots of rumen inoculum, may account for the low correlation which existed between the la vitro runs of the same forage. Table V Comparison of Estimated Dry Matter and Anthrone Carbohydrate Digestibilities % Dry Matter Estimated % Trial Name of Forage Digested Anthrone (Chromogen)‘ Digested** I Alfalfa-Ladino-Brome 66.2 66.5 II June Grass 70.0 58.4 III Alfalfa-Brome (bud stage) 67.7 47.2 IV June Grass (heading out) 62.7 28.7 V Alfalfa-Ladino-Brome 71.1 49.4 VI Ladino Clover 72.4 69.1 VII Sudan Grass 68.8 45.7 VIII Kentucky Blue Grass 60.9 51.2 IX Rye 64.7 74.5 ‘ Average of three cows. ** Average of duplicate artificial rumen runs. However, another source of error between the two methods may be due to the selective grazing of the cows, VLQh". 13.24. > ' 26 and the type and quality of the forage samples collected from the pasture for the la thgq studies. It can be observed (Table V, Trials I and VI) that when the type of pasture limited the selective grazing of the forage by the cows, a closer relationship appeared to exist be- tween the digestion coefficients. f“”“ The results observed in this study however indicate that the major sources of procedural error must be over— come before the micro-artificial rumen technique can be considered as a reliable and accurate technique for the ;W,_ evaluation of pasture forage quality. SUM l‘IIARY Three mature Holstein dairy cows using the chromogen ratio technique of Reid 33 gl. (1952) were used to estimate the dry matter digestibility of nine pasture forages. The a? resulting digestibilities were used as criteria for evalu- fiJmK ating the results obtained from the same pasture forage "J ‘0; t" ‘. o. _ i-Av using the micro-artificial rumen technique of Pigden .‘.V".. (1955) with minor modifications. Duplicate lg zitgq runs with the same forage failed éq; to show a significant correlation between runs. The major source of error was attributed to the preparation of for- age, the forage sampling procedures, and to the variation in anthrone carbohydrate digeStion between runs of the same forage. No significant correlation was found to exist between the two methods studied. It was therefore concluded that under the conditions of the present experiment that the micro-artificial rumen technique was not a satisfactory technique for evaluating pasture forage quality. _ 27 _ LITERATURE CITED Anderson, A. C. 1954. Cited by Kane, E. A., R. E. Ely, W. C. Jacobson, and L. A. Moore. 1955. A comparison of various digestion trial techniques with dairy cat- tle. J. Dairy Sci. 56:525. Archibald, J. G., D. F. Owen, Jr., H. Fenner, and H. D. Barnes. 1958. Comparison of chromium ratio and lig- nin ratio techniques for determination of digestibil- ity of hays. J. Dairy Sci. 41:1100. Baumgardt, B. 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