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Effects of various physical and chemical treatments on the in virtro rumen digestibility and chemical… Huffman, James Grant 1970

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THE EFFECTS OF VARIOUS PHYSICAL AND CHEMICAL TREATMENTS ON THE W VITRO RUMEN DIGESTIBILITY AND CHEMICAL COMPOSITION OF FOUR WOODS by JAMES GRANT HUFFMAN B.S.A. University of British Columbia, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Division of Animal Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 1970 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study . I f u r t h e r agree t h a t permiss ion fo r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department o r by h i s r e p r e s e n t a t i v e s . I t i s understood that copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada i ABSTRACT Samples of sawdust from poplar, alder and Douglas f i r , were ground past screens ranging in size from 2.21 mm to 0.25 mm (60 mesh). .A reduction in particle size significantly increased the in vitro rumen digestibility of poplar, but had l i t t le effect on the digestibility of alder or f i r . The above woods, plus sludge (a by-product of the pulping process) were treated with NaOH solutions of 2, 4 and 6%. These solutions were used at three treatment periods of 0.5, 1.0 and 1.5 hours, and all treatments were carried out at 1.05 kg/sq cm and 121° C. NaOH treatment significantly increased the in vitro cellulose digesti-bi l i ty of all woods except f i r . The optimum treatment conditions for increasing the in vitro cellulose digestibility of all woods were 4% NaOH at the 1.5 hour treatment period, except in the case of alder which was most digestible when treated with 2% NaOH for 1.5 hours. Gamma irradiation increased the in vitro cellulose digestibility of all woods. Alder and f i r were most digestible when treated with 8 2 x 10 rads, but poplar and sludge reached their peak digestibility 8 when exposed to 1 x 10 rads. Irradiation was found to decrease the cellulose and acid detergent fibre (ADF) content of all the woods studied. Acid detergent lignin (ADL) also decreased in response to irradiation in all woods except sludge. Irradiation had no effect on the ash content of any of the samples. 1i Three methods of cellulose analysis were used on both untreated and irradiated wood, and there was a significant difference shown among the methods. The lowest values were obtained using Van Soests' KMnO^  method, next were the results from Van Soests' 72% HgSO^  method, and the highest values were obtained using the Crampton and Maynard procedure for cellulose. Two lignin methods were also compared and i t was found that the KMnO^  lignin values were significantly higher than those obtained using the 72% H?S0. method for l ignin. i i i TABLE OF CONTENTS Chapter Page A INTRODUCTION • « o » » » » » » * » « » « « » » - o » * » « o 1 B LITERATURE REVIEW . . . . . . . . . . . 2 I. The importance of Class and Species of Tree . . . 2 II. Untreated Wood as a Feedstuff . . . . . . . . . . 3 III. Chemical and Physical Treatments of Wood . . . . 5 1. Physical Treatments . . . . . . . . . . . . . 6 (a) Fine Grinding . . . . . . . . . . . . . 6 (b) Irradiation . . . . . . . . . . . . . . 7 2. Chemical Treatment . . . . . . . . . . . . . 9 (a) Sodium Hydroxide 9 IV. In Vitro Evaluation and Chemical Analysis . . . . 11 C GENERAL METHODS . . . . . . . . . . . . . . . . . . . . . 17 I. Experimental Methods . . . . . . . . 17 II. Statistical Methods . . . . . . . . . . . . . . . 17 D EX PER I MENTAL o o o o » o o < > o o o o » o o © © o © » o o o 18 Experiment I. The effect of fine grinding on the in vitro rumen digestibility of wood . . . . . . 18 1. Materials and Methods . . . . . . . . . . . . 18 (b) Sample Preparation . . . . . . . . . . . 18 2. Results and Discussion 18 ) 1v Chapter Page Experiment II. The effect of NaOH steeping on the in vitro rumen digestibility and chemical composition of wood . . . . . . . . . . . . 23 1. Materials and Methods . . . . . . . . . . . 23 (s) Ssirnpl 6s • » » » i » o » o < » o . 23 (b) Sample Preparation . . . . . . . . . . 23 2. Results and Discussion . . . . . . . . . . 24 Experiment III. The effect of gamma irradiation on the in vitro rumen digestibility and chemical composition of wood . . . . . . . . . . . . 35 1. Materials and Methods . . . . . . . . . . . 35 (ct) Sdrnpl GS » a » » < > < i [ » 9 o o ( > s » o o 35 (b) Sample Preparation . . . . . . . . . . 35 (c) Experimental Methods . . . . . . . . . 35 2. Results and Discussion . . . . . . . . . . 36 E SUMMARY » « » « o » » » » » * » o © a » » » « » » » » » _ 53 8 IBL IOGRAPHY o o a o a o a o a o o a a o o a o o o o o o o o o 0 0 57 APPENDIX I Method for In Vitro Digestibility Studies . . . . . 64 II Statistical Models . . . . . . . . . . . . . . . . 69 III Standard Deviations for Procedures . . . . . . . . 75 IV Figures II-l to 111-8 77 i V LIST OF TABLES Table Page 1-1 The effect of grinding on the in vitro dry matter disappearance of three woods by rumen microorganisms . 19 1-2 The effect of grinding on the in vitro cellulose digestion of three woods by rumen microorganisms . . . 20 II-l The effect of NaOH treatment on the cellulose content of four woods as determined by the Crampton method . . . . 25 II-2 The effect of NaOH treatment on the ADF content of II-3 The effect of NaOH treatment on the ADL content of fOLiy* WOOClS o o o o o o o o o o o o o o o o o o a o o o 27 II-4 The effect of NaOH treatment on the in vitro dry matter disappearance of four woods by rumen microorganisms . . 30 I1-5 The effect of NaOH treatment on the in vitro cellulose digestion of four woods by rumen microorganisms . . . . 31 1 III-l The effect of irradiation on the cellulose content of four woods as determined by the Crampton method . . . . 37 II1-2 The effect of irradiation on the ADL content of fOUP WOOdS » 9 » * < » » » » o a » » 9 » » o a » a » » 0 37 III-3 The effect of irradiation on the ADF content of "TOUP WOOdS o o o o a a o o a a o a a o o o o s e o a o 39 III-4 The effect of irradiation on the ash content of four woods i . . 40 vi Table Page II1-5 The effect of irradiation on the in vitro dry matter disappearance of four woods by rumen microorganisms . ... 41 II1-6 The effect of irradiation on the in vitro cellulose digestion of four woods by rumen microorganisms . . . . 41 III-7 The effect of irradiation on the in vitro production of VFA's from four woods by rumen microorganisms (meq/g substrate) . . . . . . . . . . . . . . . . . 44 III-8 Comparison of three methods of cellulose determination using four irradiated woods (sample by method means). . 49 III-9 Comparison of three methods of cellulose determination using four irradiated woods (irradiation by method means 111-10 Comparison of two methods of lignin determination using four irradiated woods (sample by method means) . . . . 51 i V11 LIST OF FIGURES Figure Page II-1 The effect of NaOH treatment on the Crampton cellulose content of poplar and alder . . . . . . . . . . . . . . 78 II-2 The effect of NaOH treatment on the Crampton cellulose content of f i r and sludge . . . . . . . . . . . . 0 0 . 78 II- 3 The effect of NaOH treatment on the ADF and ADL content of poplar, alder, f i r , and sludge . . . . . . . . . . . 80 II-4 The effect of NaOH treatment on the in vitro dry matter disappearance of poplar, alder and sludge . . . . . . . 82 II-5 The effect of NaOH treatment on the in vitro cellulose digestion of poplar, alder, f i r and sludge . . . . . . 84 III-l The effect of gamma irradiation on the ADF, ADL and Crampton cellulose content of poplar and alder . . . . 86 III-2 The effect of gamma irradiation on the ADF, ADL and Crampton cellulose content of sludge and f i r . . . . . 88 III- 3 The effect of gamma irradiation on the in vitro dry matter disappearance of poplar, alder, f i r and sludge . . . . 90 III-4 The effect of gamma irradiation on the in vitro cellulose digestion of poplar, alder, f i r and sludge . . . . . . 92 III-5 The effect of gamma irradiation on the milliequivalents of VFA's produced in vitro per gram of poplar, alder, f i r and sludge . . . . . . . . . . . . . . . . . . . 94 Figure Page III—JB The effect of species on three methods of cellulose determination . . . . ' . ' . . . . • • • < • • • • • » • • 96 III-7 The effect of irradiation on three methods of cellulose determination . . . . . . • . . » • • ' • • • • • • • • • 96 III-8 The effect of species on two methods of lignin determination . . . • • » . 98 ACKNOWLEDGMENTS I wish to thank Dr. W. D. Kitts, Professor of Animal Science and Chairman of the Department of Animal Science, for his interest and guidance throughout the course of this study. I am also grateful to my fellow students for their constructive suggestions and valuable assistance. INTRODUCTION The rapid increase in world population makes i t imperative that greater emphasis be put on the efficient utilization of all potential human foodstuffs. To meet this challenge, new priorities regarding the use of cereal grains and other food will be necessary. Livestock, in order to remain a competitive and valuable source of human food, will accordingly have to be produced using increasing amounts of raw materials and industrial wastes which are not edible by man. The ruminant supports in its digestive tract a microbial popula-tion which ferments sugars, starches and cellulose to produce volatile fatty, acids. These acids are absorbed by the host animal and are used for energy and various synthetic functions. For this reason ruminants seem part icular ly well adapted for util izing cellulosic materials such as wood and converting them into foods acceptable to man. Wood and wood wastes consist primarily of cellulose, hemicellu-lose and l ignin, the latter being virtually undigested by ruminants. Lignin, because i t exists in close physical and/or chemical association with cellulose and hemicellulose, acts to impede the microbiological breakdown of these compounds. This project was designed to study the effects of fine grinding, as well as sodium hydroxide and gamma irradiation treatments on the chemical composition and in vitro rumen digestibility of the ligno-cellulose complex in wood.. B LITERATURE REVIEW I. The Importance of Class and Species of Tree The nutritive value of wood for rumen microorganisms is dependent on several important factors. The primary considerations are the class and species of tree being evaluated. Softwoods are usually less digest-ible than hardwoods (Stranks^). This is mainly due to the higher lignin content, (25-30% compared to 18-21% in hardwoods) and the type of lignin present in softwoods. The lignin in softwoods is derived mainly from sinapyl alcohol and shows more cinnamaldehyde and carbon-carbon bonding, making the lignin molecule less susceptible to chemical reaction (Sarkanen et at., and Freudeberg ). Hardwood lignin is derived from guiacyl alcohol and shows more free hydroxy! groups, making the ether bonds of the hardwood lignin more susceptible to chemical attack. The presence of various essential oils in softwoods which inhibit rumen microorganisms (Oh et at. J**) may also partially account for the digestibility difference between softwoods and hardwoods. Differences in the digestibility of woods by rumen micro-52 53 organisms within a class exist. Nehring and Schram ' have shown considerable differences between the in vivo digestibil it ies of several hardwoods. These differences may be due to the fact that the hardwoods 77 show more variation in lignin content and composition (Timell , Sarkanen et al.t, ), as well as hemicelluloses composition (Springer and Zoch^), than do softwoods. In contrast, softwoods are all 72 48 highly indigestible, both in vivo and in vitro (Stranks , Mater , and Liebscher^). The similar composition of all softwood lignin (Sarkanen et al.^) may explain the similar nutritive value of all softwoods. II. Untreated Wood as a Feedstuff There are several examples of untreated wood being used as a 47 maintenance feed for ruminants. Ludemann , used a ration of 1 inch hammermilled mopani leaves and/or twigs plus urea, molasses, and minerals to maintain steers during drought periods. Acacia twigs plus urea was also found to be capable of maintaining a steer's weight over a period of 9 weeks. It was shown that when 4 pounds of maize meal were added to 15 pounds of acacia twigs per day, the steers on test gained 1.6 pounds daily (Ludemann^). E l l is 2 5 5 studied the value of ground mesquite wood for maintaining gestating cows. The ration con-sisted of ground mesquite wood ad libitum (6.35 to 7.26 kg/day) plus 0.675 kg milo and 0.45 kg molasses per day. The animals were fed this ration for 125 days prior to calving. He concluded that this ration furnished enough energy to maintain the cows before calving, but did not provide sufficient energy for maintenance and milk produc-tion after calving without causing severe body weight loss. The number of cattle being fed in feedlots on high concentrate rations is rapidly increasing. It is generally recognized that these cattle require a roughage source in the ration to maintain normal rumen function. A roughage source has been found beneficial in alleviating such problems as bloat, rumen parakeratosis and liver abscesses in feedlot cattle. Various low feed value and non-nutritive roughage sources such as sand, oyster shel l , ground polyethylene, ground corn-15 cobs and cotton seed hulls have been used (Cooley and Burroughs , 34 90\ 14 Hughes et at./, Wise et al.3 ). Cody et al.3 used short leaf pine sawdust as an energy diluent and roughage source for calves fed a high concentrate ration. Normal rumen function and growth were shown by animals fed 25-45% wood in the ration. At levels of fibre below 25% there were occasional problems with bloat and parakeratosis. Anthony and Cunningham*, carried out a study to compare hardwood sawdust and oyster shell as roughage sources in all-concentrate rations. The experimental rations were, basal (no roughage), basal plus 2.5% oyster shel l , basal plus 2.5% sawdust and basal plus 10% sawdust. The 2.5% sawdust ration supported the highest gains and the 10% sawdust ration produced gains equal to the basal ration alone. The wood rations in both cases gave better gains than the oyster shell ration. No deleterious effects were noted from feeding the hardwood sawdust. Shelford 6 6 , studied the value of alder sawdust as a roughage source in a concentrate finishing ration. The rations consisted of equal amounts of basal ration, to which increments of alder sawdust were added (0, 13, 27, and 35%). The animals receiving sawdust in the 5 ration had a higher rate of gain than the group receiving no sawdust. The rations were found to have no significant effect on carcass grade or meat acceptability (tenderness and juiciness) as determined by a taste panel. The same rations were subjected to digestion tr ials using mature wethers. It was found that i f the digestibility of the basal ration was assumed constant, the wood had a digestion coefficient ranging from 46.5 to 13.5%. It was suggested that the variation in the digestibil ity of the sawdust was due to the increased utilization of the basal ration when the sawdust was present. In another t r i a l , Shelford used three roughage sources (hay, alder sawdust and extruded alder sawdust) to determine the effect of heat and pressure (extrusion) on the utilization of alder sawdust. He also compared the value of both untreated and treated alder sawdust to hay as roughage sources. The rate of gain of the animals on the hay rations was significantly greater than that of the animals on the wood rations. The effect of extruding the wood was not significant, a l -though the animals on the extruded wood diet had a slightly higher rate of gain than those on the untreated wood ration. III. Chemical and Physical Treatments of Wood The fact that cellulose is a valuable energy source for ruminants 51 has long.been recognized. Nehring et al.a found purified cellulose to be as good if not better source of energy for ruminants than sucrose. 6 The cellulose present in plant material is only partially 1 fi available to rumen microorganisms. Crampton and Maynard examined the relationship between the cellulose and lignin content of feeds and their digestibil ity. They found that the digestibility of the cellulose 78 varied inversely with the lignin content of the feed. Tom!in et al.3 used in vitro techniques to study the relationship between lignification n and cellulose digestion. Similarily Burdick and Sullivan found a positive correlation between ease of acid hydrolysis of hemicellulose and its digestibil ity, indicating the interference of lignin in the hydrolysis of hemicellulose. The fact that the carbohydrate portion of wood and straw is not fully utilized by herbivores led investigators to study the effects of treating 1ignocellulosic materials in various ways to increase their digestibil ity. The treatments to be discussed can be divided into two classes, physical (fine grinding and irradiation) and chemical (NaOH treatment). 1. Physical Treatments (a) Fine Grinding 21 DeHority and Johnson proposed the theory that the extent of deposition of lignin around the cellulose fibre (forming a physical barrier), rather than the total concentration of l ignin, is probably responsible for the decreased digestibility of forages as they mature. If such a protective lignin sheath exists, then physical rupture of the forage particles could increase the amount of cellulose available for 7 21 digestion. Data reported by DeHority and Johnson showed that balN milling bromegrass and orchardgrass for 72 hours increased their in vitro cellulose digestibi l ity, especially in more mature samples. Samples of less mature bromegrass and orchardgrass showed increased cellulose digestibil it ies of 43 to 76% and 54 to 83%, respectively. 85 8fi 87 Virtanen et al.3 * ' . studied the effect of particle size on the cellulose digestibility of various woods. They found that birch sawdust cellulose digestibility increased from 13 to 68%, aspen in-creased from 15 to 64%, and pine increased from 5 to 46% when ground to extreme fineness with emery paper. 59 Pew and Weyna have reported that ballmilling renders wood carbohydrates almost completely accessible to the cellulolytic enzymes of' Triahoderma vivide<, The cellulases showed l i t t l e activity with spruce and aspen wood of 40 mesh size, but could digest 70% of the spruce after 8 hours of ballmilling and 80% of the aspen after 5 hours of ballmilling. (b) Irradiation The chemical effects of gamma irradiation on wood have been 65 studied and reviewed in an extensive article by Seifert . He reported 6 5 that at dosages above 1 x 10 rads cellulose was decomposed at the rate of 1% per 2.5 x 10^ rads. Lignin was also decomposed at high dosage levels, but to a much lesser degree than cellulose. Long chain polymers such as cellulose were fragmented, therefore decreasing the degree of polymerization. Degradation of various wood components produced an increase in the alkali soluble fraction which was made up primarily of 65 xylobiose, glucuronic acid, cellotriose and xylotriose. Seifert also reported that there were small increases in pentosans at low levels of irradiation. These pentosans were degradation products of cellulose, but they themselves were further degraded at high levels of irradiation. A small weight loss as a result of irradiation was attributed to decarboxylation and also to the formation of volatile compounds during the demethoxylization of l ignin. Several studies have demonstrated that irradiation alters the structure of wood or straw in such a way that some of the insoluble 4 carbohydrates become available to rumen microorganisms. Lawton et al.3 exposed basswood to high energy cathode rays and measured the effect of such irradiation by subjecting the basswood to in vitro fermentation by rumen microorganisms. Weight loss of treated wood increased with in -creasing irradiation. The volatile fatty acid production reached a o peak at 2 x 10 Roentgens, and then began declining with increasing dosage. They suggested that the drop in VFA production and the con-tinued weight loss at higher dosage levels indicated that the carbo-hydrates of the wood had been converted to compounds not utilized by the rumen microorganisms, but soluble in the incubation medium. Similar data was obtained by Pritchard et a l . J * ' . They studied the effect of gamma irradiation on the utilization of wheat straw by 7 rumen microorganisms. Dosages of up to 1 x 10 rads caused only slight o increases in dry matter digestion, whereas exposure to 1 x 10 rads or more caused marked increases in digestion. The solubility of the straw in the fermentation medium also increased with dosages of 1 x 10 rads or more. However, increases in VFA production from in vitro fermenta-tions were found only up to dosages of 2.5 x 10 rads, indicating that higher levels of irradiation altered the carbohydrates so that they were no longer suitable substrates for rumen microorganisms. 2. Chemical Treatment (a) Sodium Hydroxide 74 Tarkow and Feist studied the physical and chemical effects of dilute NaOH on wood. They found that the swelling capacity of the wood was approximately doubled after NaOH treatment. The increase in swelling capacity resulted from the saponification of esters of 4-0-6 methyl glucuronic acid attached to the xylan chains. In the natural condition, these esters act as cross-links, limiting the swelling or dispersion of the polymer segments in water. The increased swelling capacity increased the pore size in the wood and therefore allowed larger molecules to penetrate the wood. Also, once the enzyme molecule had penetrated the wood structure, i t had more carbohydrate surface to react with. Similar conclusions about the importance of pore size and swelling have been reported by Stone et al.J®. Because of the few active centres on enzyme molecules, the enzymes must come into proper 74 stereo-relationship with the carbohydrate bonds. Tarkow and Feist suggested that the submicroscopic water pockets created due to the swelling may have facilitated the free rotation of the enzyme molecule and increased the likelihood of a successful interaction between the 10 enzyme and substrate. Their data indicated then, that delignification as such was not the mechanism whereby NaOH increased the digestibility of lignocellulosic materials. Rather i t was a modification of pore size that effected their increased susceptibility to bacterial degrad-ation. One of the most widely used alkali treatments has been the 9 Beckmann process for treating straw. Straw was steeped for 24 to 48 hours at normal or elevated temperatures with about 8 to 10 volumes of 1.5 to 2.0% NaOH solution. A twofold increase in crude fibre ut i l i za -tion was noted, but significant losses of soluble nutrients occurred due to the washing procedure. Wide scale use of the Beckmann procedure has been limited because of the large volume of dilute NaOH required, the tedious washing procedure, and the loss of soluble nutrients. In 89 order to overcome some of these problems Wilson and Pigden developed the "dry" process of alkali treatment. They treated 100 grams of poplar wood with 6 to 9 grams of NaOH dissolved in 30 mis of water. The product was then evaluated without washing. Wood in vitro dry matter digestibility increased from 5 to 45% when 9% NaOH was used. 22 Donefer et at., studied the effect of urea supplementation on the nutritive value of NaOH-treated straw. They treated ground oat straw with a 13.3% NaOH solution (at the rate of 60 l itres of solution per 100 kg. straw). It was then neutralized with 16.7 l itres of 50% acetic acid and dried. Untreated or treated straw, each with or without urea (2.5% of ration) and/or sucrose (3.5% of ration) was fed ad libitum to sheep. Alkali treatments showed a significant increase in energy digestibi l ity, but had no effect on voluntary feed intake unless urea was added. The urea supplementation of treated straw was shown to increase voluntary intake by 160% when compared to untreated, unsupple-mented controls. Sucrose had no significant effect on either digestibil ity or voluntary intake. NaOH treated, urea supplemented straw supplied 220% more digestible energy than the control ration. IV. In Vitro Evaluation and Chemical Analysis Feeding tr ials and in vivo digestion tr ials have been used extensiyely to evaluate roughage sources and roughage treatments. How-ever, these techniques are expensive, time consuming, and require considerable faci l i t ies to conduct. To overcome some of these limita-tions, in vitro (artif icial rumen) techniques and chemical analysis are often used to estimate the value of a roughage or roughage treatment. Many in vitro techniques are available, and though they follow the same pattern many modifications occur between laboratories on specific steps in the procedure. Barnes^ carried out a collaborative in vitro study in which he examined the differences in techniques among the major researchers in the area of in vitro feed evaluation. The main modifications are as follows: type of incubation vessel, source and preparation of inoculum-buffer system, fermentation time, sample size, and criteria for analysis of in vitro activity. The three main classes of incubation vessels are: the continuous yessel or chemostat, the semipermeable vessel and the closed vessel. 12 The continuous flow procedure refers to a system which is being continuously supplied with fresh buffer and nutrients, and where fermentation end products are washed out with the incoming nutrient 88 30 and buffer solutions, (Warner , Harbers and Tillman and Stewart et al.j ). The semipermeable incubation vessel consists of a dialysis bag containing the substrate and inoculum which is placed in a stationary buffer solution. This allows passage of nutrients and buffer into, and fermentation end products out of the fermentation area (Louw etat.^t Pettyjohn et al.,58). The closed system utilizes a nonpermeable glass or polyethylene incubation vessel. A gas release valve is usually used (Baumgardt et at., * Til ley and Terry , Chalupa and Lee ), or provision is made 20 37 38 for continuous gasing with COg (DeHority , Johnson et al.a )> No nutrients are added or end products removed during the fermentation period. 27 El Shazly et al.t compared all three systems and found no difference among them as far as digestibil it ies or bacterial counts were concerned. The closed system, because of its simplicity and repeat-abil ity has consequently become the most common type of incubation procedure. The collection and preparation of rumen fluid inoculum is one of the largest sources of error and variability among laboratories conduct-ing in vitro fermentation studies. Most workers use cattle as their donor animal (Barnes^) but studies by Troelsen and Hanal 7 9 , Barnes^, 13 13 Church and Peterson J reveal l i t t l e or no difference in inoculum effect among species or among animals within a species on in vitro cellulose or dry matter digestibil it ies with forages. The importance of a standard-ized collection time has been stressed by Johnson"^, Troelsen and Hanal 7 9 , 42 and Knipfel and Troelsen . Several forms of rumen fluid inoculum exist, such as whole 12 76 strained rumen fluid (Chalupa and Lee , Ti l ley et al.3 , and Oh et aZ. j 5 ^) , washed cell suspensions (Kamastra et al.3^)t phosphate buffer 38 23 6? extract (Johnson et al.3 , Donefer et al.s t and Quicke et al,3 )„ and pure cultures (Stranks 7 1, Dehor i ty 1 8 , 1 9 ) . Whole strained rumen fluid has found wide acceptance as an inoculum (Barnes4), due to its simplicity of preparation and the accuracy of the in vitro results obtained with its 5fi 7 use (Oh et al.3 , Baumgardt et al.3 ), The type of buffer used depends on the inoculum preparation 49 employed. Most laboratories use McDougall's buffer in combination with 4 whole strained rumen fluid (Barnes ). The use of cell suspensions, phosphate buffer extracts or pure cultures require that additional nutrients be supplied J Oas the nutrients in the rumen fluid are discarded in the inoculum preparation (Quicke et al.3 , Donefer et al.3 , Johnson et a i . , ^ ) „ The fermentation time used by various laboratories has varied from one week to 12 hours. The length of time varies according to the substrate being evaluated and the regression equations used in the particular laboratory (Karn et al.3^). A fermentation time of 48 hours appears to be most commonly used, although a longer time may be required when evaluating highly indigestible materials such as straw or wood (St ranks 7 1 , 7 2 , Dehority et a Z . J 8 , 1 9 ) . Sample size varies among laboratories from 0.25 grams to 1.0 grams. The effect of sample size is diff icult to assess, because the sample size 7 to inoculum ratio also differs among laboratories. Baumgardt et al.3 39 and Kamastra et al.3 , have determined that increasing the sample size beyond the point where substrate was limiting caused a decrease in cellulose digestion. The validity of comparing estimates of digestibility determined in different laboratories and based upon different in vitro procedures, is dependent on the criteria used for analysis. The types of in vitro analysis include: cellulose digestion, dry matter disappearance (DMD) (Baumgardt and 0h^, Bowden and Church^), succinic acid production 71 2 43 (Stranks ), VFA production (Asplund et al.3 , Lawton et al.3 ), reagent tests for carbohydrate analysis (Pigden and Bel l 6 ^ , Baumgardt et al.3^). Dry matter digestion and cellulose digestion appear to be the most accurate predictors of in vivo performances and are the most A commonly used criteria for analysis (Barnes )„ Chemical analysis as used in feed evaluation has consisted 32 primarily of the Weende system, (Henneberg and Stohmann ) whereby the carbohydrates of feeds are determined as two groups: crude fibre and nitrogen-free extract. Early chemists thought that crude fibre repre-sented the indigestible part of the feed and estimated nutritive value on that basis. Haubner shattered the theoretical model upon which the proximate system of analysis was based by his discovery of the 15 digestibil ity of fibre and cellulose by herbivores. The modern criticism of crude fibre is not based so much on its use to estimate nutritive value, as i t is upon the poorly defined chemical isolation and the variable composition of the product. Many attempts have been made to find an effective replacement for crude fibre. Lignin has been considered to be a candidate, since i t would retain the original ideal of crude fibre, i . e . , that i t is representa-tive of the indigestible portion of the feed. However, the complex chemistry of lignin and the diff icult ies associated with its determina-16 tion have presented problems not yet surmounted (Crampton and Maynard , E l l is et al.a26). Because the NaOH used in the Weende crude fibre procedure to remove protein was found to lead to a considerable loss of l ignin, (Norman5*4, and Hallsworth2 9), new methods for the determination of the fibrous portions of forages have been introduced by Van Soest and W i n e 8 0 , 8 1 , 8 2 , 8 3 , 8 4 . These methods util ize detergents in lieu of NaOH to remove nitrogen under milder conditions and thereby preserve the integrity of the lignin fraction. Ideally, the fibre as a chemical entity should contain the cel lu-lose and lignin with as l i t t l e admixture of nitrogenous substances as possible. The composition of crude fibre and acid detergent fibre (ADF) has been investigated by Kim et aZ..,41. Fibre was isolated from feces, silage and pellets by both AOAC crude fibre and ADF methods and analyzed for l ignin, pentosans and cellulose. The ADF retained virtually all the lignin and cellulose while the AOAC fibre retained the original 16 cellulose but lost 60 to 84% of the original lignin. Both methods lost 80% of the original pentosans. Correlations have been calculated between in vivo digestibility and the parameters of crude fibre, ADF and acid detergent lignin (ADL). The relationship between digestible dry matter and ADF was found to be closer than the relationship between digestible dry matter and the conventional crude fibre. Correlations of ADL with dry matter digesti-bi l i ty were comparable to that of crude fibre and digestible dry matter. Howeyer, i t is well known that important differences exist between 73 species in regard to lignin (Sullivan ). When correlations were calculated between ADL and digestibility for legumes and grasses separ-ately, considerably closer relationships were obtained. Because ADF is composed of l ignin, cellulose and insoluble minerals, a method of partitioning ADF with respect to its components 80 has been developed by Van Soest and Wine . This was done by using permanganate (KMn04) to dissolve the lignin in the ADF. The lignin was then estimated as the loss in weight after permanganate oxidation. The cellulose was determined as the loss upon ashing, leaving the mineral residue for further examination. c GENERAL METHODS I. Experimental Methods The in vitro rumen procedure for measuring dry matter disappear-ance (DMD) and cellulose digestion was developed by modifying the 75 50 methods of Til ley and Terry , and Mellenberger et al.3 . The method is outlined in detail in Appendix I. ' Cellulose, referred to as Crampton cellulose, was determined 16 according to the method of Crampton and Maynard , as modified by Donefer et al.P*. Acid detergent fiber (ADF), acid detergent lignin (ADL), KMn04 ADL, KMn04 cellulose, ADF-ADL cellulose and ash were al l determined according to the methods of Van S o e s t 8 ^ ' ^ ' 8 4 . II. Statistical Methods The data were subjected to analysis of variance (Steele and CO Torrie ) and the means of the sources of variation found significant (P<.01) were tested in sets by Duncan's24 new multiple range test at the 1% level of significance. All differences throughout the study, that are referred to as being significant, are so at the 1% level. The statistical models used for the various experiments are given in Appendix II. The standard deviations associated with the replication of procedures are listed in Appendix III. D EXPERIMENTAL -Experiment I. The Effect of Fine Grinding on the in vitro Rumen Digestibility of Wood Experiment I was designed to determine the effect of fine grind-ing on the in vitro dry matter disappearance (DMD) and cellulose digestibil ity of three woods by rumen microorganisms. 1. Materials and Methods (a) Samples The three woods used were alder (Alnus rubra Bong.), Douglas f i r (Pseudotsuga menziesii (Mirb.) Franco) and poplar (species unknown). (b) Sample Preparation Samples were prepared by grinding the wood past screens of the following sizes: 2.21 mm, 1.60 mm, 1.25 mm, 0.80 mm, 0.42 mm (40 mesh) and 0.25 mm (60 mesh). 2. Results and Discussion The results (Tables 1-1 and 1-2) showed a significant difference among woods regardless of grind, for both DMD and cellulose digestibi l i -ty. The samples in order of increasing digestibility were f i r , alder, TABLE 1-1 THE EFFECT OF GRINDING ON THE IN VITRO DRY MATTER DISAPPEARANCE OF THREE WOODS BY RUMEN MICROORGANISMS Percent Dry Matter Disappearance (Screen size X Sample Means from triplicate determinations) Screen Size (mm.) Poplar Sample Alder Fir 2.21 0.00 0.00 0.00 1.60 2.51 0.00 0.00 1.25 3.59 0.00 0.00 0.80 5.98 0.00 0.00 0.42 11.64 0.36 0.00 0.25 13.67 2.51 0.32 6.23A 0.48B 0.05B Sample*Means * Indicates sources of variation found significant (P<.01) in the analysis of variance. The means for these sources of variation were tested in sets by Duncan's (24) new multiple range test. Means within a set having different superscripts differ significantly (P<.01). TABLE 1-2 THE EFFECT OF GRINDING ON THE IN VITRO CELLULOSE DIGESTION OF THREE WOODS BY RUMEN MICROORGANISMS Percent Cellulose Digestion (Screen size X Sample Means from triplicate determinations) Screen Size (mm.) Poplar Sample Alder Fir 2.21 9.77 0.00 0.00 1.60 10.84 0.00 0.00 1.25 12.18 0.00 0.00 0.80 13.59 0.00 0.00 0.42 20.38 0.00 0.00 0.25 22.16 3.87 1.01 14.82A 0.65B 0.17B Sample*Means * Indicates sources of variation found significant (P<.01) in the analysis of variance. The means for these sources of variation were tested in sets by Duncan's (24) new multiple range test. Means within a set having different superscripts differ significantly (P<.01) and poplar. For both digestion parameters f i r and alder were s ignif i -cantly different from poplar, but not from each other. Similar variations in wood digestibilities have been reported by several re-71 55 52 53 searchers (Stranks , Oh et al.3 and Nehring and Schram 5 ). Grinding (decreasing particle size) did not have a significant effect on either DMD or cellulose digestion when the data for all woods were combined. However, the significance of the increase in poplar digestibil ity, in response to a reduction in particle size, was masked by the relative lack of response shown by f i r and alder. Although the experimental design does not allow a meaningful statistical analysis to be made on the f i rst order interaction between samples and particle size, the very apparent trend shown by poplar should not be overlooked. The data (Table 1-1, p. 19, and 1-2, p. 20) indicate that particle size reduction had a greater effect on poplar than on alder or f i r when either DMD or cellulose digestion were measured. Similar differential responses to fine grinding by different woods have been reported by 59 Pew and Weyna. Several workers using both forages and various woods as sub-strates have reported increases in substrate digestibility in response 21 to a decrease in their particle size. Dehority and Johnson decreased the particle size of orchardgrass and bromegrass by ballmilling, and noted significant increases in the forages' in vitro cellulose digesti-85 86 bi l i t ies as the extent of ballmilling increased. Virtanen et al.3 ' found similar increases in the cellulose digestibilities of birch and pine when they were ground to extreme fineness with emery paper. 22 The relative ineffectiveness of the coarser grinds on increasing cellulose and dry matter digestion as noted in Tables 1-1 (p. 19) and 1-2 (p. 20) has been reported by Pew and Weyna^. They observed that the cellulases of Trichoderma viride showed l i t t le activity on spruce •and aspen wood when not ground past at least a 40 mesh screen, but that these same samples when ballmilled for 5 to 8 hours exhibited cellulose digestibilities of 70 to 80%. It must be stressed that these data have been obtained using an in vitro system, and there are important physiological factors to be considered before such data can be meaningfully applied to the in vivo situation. Most of the fermentation of 1ignocellulose occurs in the rumen. These materials, to be of any nutritive value must be retained in the rumen long enough for the microorganisms to obtain the available energy from them. This is of particular significance because the rumen retention time of a given feed particle depends chiefly on its size and specific gravity (Balch ). Grinding not only increases surface area and fermentation, but may also reduce the particles to a sufficiently small size to allow passage through the reticulo-omasal orifice soon 3 after entering the rumen. Balch , remarking on this aspect suggested that the crit ical size is about 2 mm. Because wood has to be ground to extreme fineness to obtain any appreciable increase in in vitro digestibil ity, i t seems doubtful that fine grinding could be employed as a useful preparatory treatment for wood that is to be used in practical animal feeding. Experiment II. The Effect of NaOH Steeping on the In Vitro Rumen Digestibility and Chemical Composition of Wood Experiment II was conducted to study the effects of treating woods with various concentrations of NaOH under heat and pressure for different periods of time. The parameters measured were: in vitro DMD and cellulose digestion, as well as ADF, ADL and cellulose content. 1. Materials and Methods (a) Samples Four samples of wood were used: poplar, alder, Douglas f i r and sludge. Sludge is a by-product of the commercial pulping process and consists of residues from hemlock and silver f i r . It was dried and ground through a 2.21 mm screen before further treatment. (b) Sample Preparation The samples were all ground past a 0.25 mm (60 mesh) screen and then treated with the appropriate NaOH solution. There were three concentrations of NaOH used: 2, 4 and 6% on a w/v basis. The control solution (0% NaOH) consisted of disti l led water only. Each NaOH solu-tion was used at three different treatment intervals: 0.5, 1.0 and 1.5 hours. The wood was treated in a 9:1 ratio of NaOH solution to wood. Thirty grams of wood and 270 ml of the appropriate NaOH solution were placed in a one l i t re flask and autoclaved at 1.05 kg/sq cm pressure and 121°C for the specified time. The flask was then removed from the autoclave and allowed to cool. After cooling the slurry was strained through 8 layers of cheese cloth using a large Buchner funnel and a suction flask. The wood was subsequently washed with approximately 8 l itres of water, or until the f i l t rate was neutral. The samples were oven-dried at 40°C and left open to the air to equilibrate and then stored in plastic bags until used. 2. Results and Discussion Crampton cellulose determinations on control (0% NaOH) samples and those treated with 2, 4 and 6% NaOH showed that NaOH treatment had a significant effect on the percent cellulose content of all woods. The data (Table II-l and Figures II-l and II-2 (Appendix IV)) indicate that the control samples were significantly lower in apparent cellulose than any of the NaOH treated samples. There was no significant differ-ence among the samples treated with 2, 4 or 6% NaOH. Because the samples were washed until neutral after NaOH treatment, any wood con-stituents soluble in dilute NaOH were Tost. It is known that lignin and hemicelluloses are soluble to varying degrees in dilute NaOH (Hallsworth29 and Norman54). Because cellulose is not soluble, the percent increase in cellulose can be attributed to the losses of lignin and hemicelluloses. The cellulose content of the wood samples, as determined by the Crampton method were found to be significantly different from each other. In order of increasing cellulose content they were: alder, poplar f i r and sludge. The high cellulose content of sludge may be attributed 25 TABLE II-1 THE EFFECT OF NaOH TREATMENT ON THE CELLULOSE CONTENT OF FOUR WOODS AS DETERMINED BY THE CRAMPTON METHOD Percent Cellulose (means of triplicate determinations Treatment Length ) Sample % NaOH 0.5 1.0 1.5 Sample X NaOH*Means % NaOH Sample* Means Poplar Alder Fir SIudge 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 57.70 68.34 68.36 68.62 53.73 64.33 64.62 64.36 61.05 69.10 70.85 69.13 77.22 79.97 78.29 78.76 57.28 68.73 68.96 69.93 53.86 65.29 65.32 66.11 60.93 68.02 68.30 68.49 76.51 79.54 78.40 78.51 57.93 68.62 70.00 70.12 55.12 66.77 65.89 65.59 64.49 70.14 70.89 70.18 79.36 79.62 78.00 80.33 57.64' 68.561 69.101 b 66.21 69.55 b 54.249 65.46e 62.58 65.27* 65.34e 62.15 e 69.08 b 67.68h 70.00L 69.471 77.69" 79.71 d 78.23 cd 78.70 79.20 cd 68.40 68.38 69.60 62.93' 70.70 70.65 70.89 B Treatment Length*Means NaOH*Means * Indicates the sources of variation found significant (P<.01) in the analysis of variance. The means for these sources of variation were tested in sets by Duncan's (24) new multiple range test. Means within a set having different superscripts differ s ignif i -cantly (P<.01). to the fact that i t had previously been exposed to chemical treatments during the pulping process. Such treatments may have decreased the content of readily soluble, non-cellulose (hemicellulose and lignin) constituents thus increasing its proportion of cellulose. The influence of treatment length, regardless of wood type or NaOH concentration, on the percent apparent cellulose was found to be significant. The 1.5 hour treatment period produced samples that had a significantly higher proportion of cellulose than either the 0.5 or 1.0 hour treated samples. This effect seems reasonable in that the longer the NaOH was in contact with the wood, the greater its opportunity to solubilize the non-cellulose constituents of the wood. The significant sample by NaOH interaction (Table II - l , p. 25), indicates that NaOH treatment was more effective in solubilizing the hemicelluloses of some woods than others. The sample by NaOH means show that poplar, alder and f i r were all more responsive to NaOH treat-ment than was sludge. This is likely due to the previously stated fact that sludge is the product of severe chemical treatments which will have already caused chemical changes similar to those caused by dilute NaOH. ADF and ADL were determined on samples treated only for the one treatment interval of 1.5 hours. Therefore wood type and NaOH concen-tration were the only variables considered in this section. The variation in ADF content attributable to wood type (Table I1-2 and Figure I1-3 (Appendix IV)) shows that poplar and alder were significantly lower in ADF than either f i r or sludge. TABLE II-2 THE EFFECT OF NaOH TREATMENT ON THE ADF CONTENT OF FOUR WOODS Percent ADF (Sample X NaOH Means from duplicate determinations) Sample* Means % NaOH Sample 0 2 4 6 Poplar 75.71 81.86 83.99 86.27 81.95X Alder 73.67 83.77 85.47 86.05 82.24X Fir 82.57 92.67 93.28 93.96 90.61* Sludge 89.17 89.47 90.89 91.92 90.36* 80.27A 86.94B 88.408 89.55B NaOH*Means TABLE I1-3 THE EFFECT OF NaOH TREATMENT ON THE ADL CONTENT OF FOUR WOODS Percent ADL (Sample X NaOH Means from duplicate determinations) Sample* Means % NaOH Sample 0 2 4 6 Poplar 13.42 14.13 16.27 15.43 14.81 A B Alder 16.67 16.49 16.18 16.80 16.53A Fir 28.84 27.87 31.31 31.93 29.99C Sludge 13.63 11.60 10.66 11.01 11.728 Indicates the sources of variation found significant (P<.01) in the analysis of variance. The means for these sources of variation were tested in sets by Duncan's (24) new multiple range test. Means within a set having different superscripts differ s igni f i -cantly (P<.01). NaOH treatment significantly increased the proportion of ADF in all samples of wood, regardless of concentration. This response can be attributed to the solubilization and subsequent loss of hemicellu-loses. Lignin solubilization is not a factor which would increase ADF percent because lignin along with cellulose, make up ADF (Kim et al., 41). Although the experimental design does not allow the sample by NaOH interaction to be analyzed statist ical ly, there is an important trend evident. That i s , NaOH treatment caused a greater increase in the proportion of ADF in poplar, alder and f i r than i t did in sludge. The lesser response shown by sludge can again be attributed to its prior exposure to chemical treatment. The ADL data (Table II-3 (p. 27) and Figure II-3 (Appendix IV)) shows no significant difference between the ADL content of poplar and alder or between poplar and sludge, but alder and sludge were s igni f i -cantly different. Fir was significantly higher than all other woods in ADL. Sludge was the lowest in ADL, which supports the previous suggestion that the pulping treatments may have delignified the sludge to some degree. NaOH treatment did not have a significant effect on percent ADL, It is possible that the NaOH concentrations used were not high enough to cause a measurable loss of l ignin, or, i t may be that the loss of lignin did not show up as a percentage decrease because there was an equal i f not greater concomitant loss of hemicelluloses. A statistical analysis of the data (Tables II-4, I1-5 and Figures 11-4 and 11-5 (Appendix IV)) showed that the DMD and cellulose digesti-bi l i ty of all woods, regardless of treatment were significantly different from each other. For both parameters f i r was the least digestible, followed by alder, poplar and sludge. The NaOH concentrations, irrespective of sample or length of treatment, showed some significant effects on both DMD and cellulose digestion. In the case of DMD the NaOH concentrations giving the lowest to the highest DMD's were: control (0%), 6%, 4% and 2%. The 2% and 0% levels were significantly different from each other and also the other two treatment levels, while the 4 and 6% levels were not significantly different from each other. These data indicate that the 2% solution of NaOH was the optimum concentration for increasing the DMD of poplar and sludge. The 4% solution gave the maximum DMD for alder. At none of the NaOH concentrations used was f i r digestible in terms of dry matter. The data on cellulose digestion (Table 11-5) is slightly differ-ent in that the order of NaOH concentrations producing the lowest to the highest cellulose digestibilities were as follows: control (0%), 2%, 6%, and 4%. The control samples were significantly less digestible than any of the others. The 2% and 6% samples were not significantly different from each other. The 4% samples were significantly more digestible than the 2% samples, but not significantly different from the 6% samples. Samples treated with 4% NaOH, with the exception of alder, contained the most digestible cellulose. The optimum 30 TABLE IX-4 THE EFFECT OF NaOH TREATMENT ON THE IN VITRO DRY MATTER DISAPPEARANCE OF FOUR WOODS BY RUMEN MICROORGANISMS Percent Dry Matter Disappearance (means of triplicate determinations) Treatment Length Sample X NaOH*Means % NaOH Sample* Means Sample % NaOH 0.5 1.0 1.5 Poplar Alder Fir Sludge 0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 6 14.03 41.16 28.04 34.19 0.00 7.33 7.91 4.34 0.00 0.00 0.00 0.00 50.46 53.75 43.78 42.13 12.32 43.90 32.21 38.65 0.00 9.10 9.71 5.04 0.00 0.00 0.00 0.00 47.09 53.12 50.97 43.18 7.07 34.21 35.50 40.90 0.00 8.27 9.95 8.32 0.00 0.00 0.00 0.00 49.98 55.01 54.30 47.76 11.14e 39.76 be 30.18n 31.921 37.92 b 0.009 8.23£ 5.83" 9.19e 5.90° 0.00-0.009 0.00 9, Q.OQv 0.009 49.18 de 53.96' 49.96 e 49.29' 44.35 de 15.08" 25.49 22.70' 22.04 NaOH*Means Indicates the sources of variation found significant (P<.01) in analysis of variance. The means for these sources of variation were tested in sets by Duncan's (24) new multiple range test. Means within a set having different superscripts differ signifi -cantly (P<.01). the 31 TABLE II-5 THE EFFECT OF NaOH TREATMENT ON THE IN VITRO CELLULOSE DIGESTION OF FOUR WOODS BY RUMEN MICROORGANISMS Percent Cellulose Digestion (means of triplicate determinations Treatment Lenqth Sample X NaOH*Means ) % NaOH Sample* Means Sample % NaOH 0.5 1.0 1.5 0 2 4 6 Poplar 0 2 4 6 17.91 64.08 73.54 70.00 17.53 66.98 72.52 70.57 18.54 64.34 74.08 71.79 17.99 a e 65.139 73.38b 70.79 b f 56.82n Alder 0 2 4 6 2.75 23.41 16.61 12.91 2.48 25.19 18.87 14.98 4.66 27.89 20.38 18.01 3.30 c 25.50h 18.47a 15.30e 15.64x Fir 0 2 4 6 0.87 2.20 6.06 4.43 3.55 1.43 3.47 5.31 7.76 4.20 9.20 10.13 4.06 c d c 2.61 c 6.25 d 6.62d 4.88 y Sludge 0 2 4 6 68.84 70.00 72.95 72.90 69.26 70.29 73.78 72.85 67.88 72.88 74.66 75.38 68.52 f 71.05 b f . 73.80° 73.71b 71.77* 36.19A 36.82A 38.84Z 23.47C 41.01A 42.97B 41.60 A B Treatment Length*Means Na0H*Means * Indicates the sources of variation found significant (P<.01) in the analysis of variance. The means for these sources of variation were tested in sets by Duncan's (24) new multiple range test. Means within a set having different superscripts differ s igni f i -cantly (P<.01). concentration of NaOH in order to maximize the cellulose digestibility of alder was 2%. It can be seen (Tables I1-4 and I1-5 (pps. 30, 31)) that the NaOH treatments had a significantly greater influence on both the DMD and cellulose digestibility of some woods than others. The samples in order of increasing responsiveness to NaOH treatment were: f i r , sludge, alder and poplar. The effect of treatment length on DMD was not significant. How-ever, in the case of cellulose digestion, the 1.5 hour treatment produced samples that were significantly higher in digestible cellulose than either the 1.0 or 0.5 hour treatments. The data obtained in Experiment II show a positive relationship between ADF content and digestibility for all woods. That i s , as the ADF content increased in response to NaOH treatment, the digestibility 84 also increased. This is in contrast to data reported by Van Soest which indicates a negative relationship between ADF content and the digestibility of several forage species. Although the relationship between ADL and digestibility in this experiment is not as marked as the one between ADF and dry matter digestibil ity, the general trend indicates that digestibility increased as ADL increased. Again, this does not agree with the negative relation-84 ship between lignin and digestibility that Van Soest as well as many other workers have reported. 84 It must be stressed that Van Soest was studying the correlation of ADF and ADL with DMD among different forage species, whereas the data reported in this experiment are relating ADF and ADL to digesti-bi l i ty within a wood sample whose composition has been altered chemically. However, the different context within which the relation-ships were made does not completely explain the discrepancy between the two sets of data. Another aspect to be considered, is the way in which the samples were prepared. Because the samples were washed after treat-ment, most of the soluble components were lost. This caused a percentage increase in the content of water insoluble compounds such as cellulose, ADF and ADL. If the samples had not been washed i t is unlikely that any change in component proportions would have occurred, and there would not have been as close a positive correlation between chemical composition and digestibil ity. 74 Relative to the above is a paper by Tarkow and Feist in which they have reported that the increase in the digestibility of woods treated with dilute NaOH was not due to chemical changes as such, but to changes in physical structure. They reported that the important chemical reaction was the saponification of the esters of uronic acid associated with xylan chains. These esters bridge polymeric units, therefore the effect of saponification was to break crosslinks. The physical effect was to increase the moisture holding capacity of the cell walls. This was found to improve conditions for the diffusion of water soluble materials, as well as to provide improved enzyme substrate interactions. They also found indications that there was a small increase in the maximum molecular weight of 34 globular proteins capable of diffusing into the wood. In a similar study, Stone et al.J^ emphasized the importance of physical changes such as increased swelling capacity and pore size, to increasing the digestibility of cellulosic materials. By using sugars and dextrans of varying molecular sizes to determine wood pore size, they concluded that there was a direct relationship between pore size and cellulose digestibil ity. An increase in pore size and swelling capacity not only allowed larger molecules such as enzymes to penetrate the substrate, but also increased the surface area available for enzymatic reactions to take place. Stone et al.J® also found that pulps of the same chemical composition, but different in physical character, were very different in digestibil ity. They therefore pointed out that i f chemical composi-tion was in some cases a poor indicator of digestibility in pulps, the same may be true when comparing forages of different species, or even when comparing the same forage species at different stages of maturity. Experiment III. The Effect of Gamma Irradiation on the In Vitro Rumen Digestibility and Chemical Composition of Wood Experiment III was designed to determine the effects of gamma irradiation on the chemical composition and in vitro rumen digesti-b i l i ty of four woods. 1. Materi als and Methods (a) Samples Poplar, alder, Douglas f i r and sludge were the woods used in this experiment. (b) Sample Preparation All samples were ground past a 0.25 mm (60 mesh) screen before placing them in aluminium containers to be irradiated by a Cobalt-60 6 7 source* for sufficient time to provide dosages of 1 x 10 , 1 x 10 , 8 8 1 x 10 and 2 x 10 rads. The samples were then stored in plastic bags until used. (c) Experimental Methods Total volatile fatty acids (VFA) were determined by the method of Olmstead57 as modified by Ross 6 3 . At the end of each in vitro incuba-tion period, approximately 25 ml of the supernatant were collected from Gammacell 220 producing 10 rads per hour; product of Commercial Products Division, Atomic Energy of Canada, Ltd. , Ottawa, Ont. each incubation vessel, and used to determine total VFA concentration. This was done by steam dist i l l ing 5 ml of the supernatant plus 2 ml of 10 N H 2S0 4, and collecting 125 ml of the dist i l late. The disti l late was then titrated against standardized NaOH to the phenolpthalein end point. By relating the value obtained for 5 ml to the total volume, VFA production per gram of substrate was determined. 2. Results and Discussion 65 Seifert has reported that high dosages of irradiation degrade wood cellulose with the formation of pentosans, carboxylic acids and oligosaccharides. It can be seen from the data (Table III-l and Figures III-l and 111-2 (Appendix IV)) that the cellulose content of the four woods studied in Experiment III decreased in response to high levels of irradiation. Untreated (control) samples and those exposed to 1 x 10^ or 1 x 107 rads contained significantly more cellulose than 8 ' 8 those treated with either 1 x 10 or 2 x 10 rads. 65 Seifert also reported an indirect relationship between lignin content and irradiation level in wood. As the data (Table III-2 and Figures III-l and II1-2 (Appendix IV)) indicate, there was no signif i -cant difference in the ADL content of samples exposed to either 1 x 106 or 1 x 10 rads and those samples which were untreated, regardless of the kind of wood. There was a significant decrease in the ADL content 8 8 of all wood samples treated with 1 x 10 or 2 x 10 rads. The composition of ADF has been studied (Kim et al.^) and found to be made up primarily of lignin and cellulose. Since both of its TABLE IXI-1 THE EFFECT OF IRRADIATION ON THE CELLULOSE CONTENT OF FOUR WOODS AS DETERMINED BY THE CRAMPTON METHOD Percent Crampton cellulose (Sample X Irradiation Means from triplicate determinations) Rads Sample* Means Sample 0 1 x 106 1 x 107 1 x 108 2 x 108 Poplar Alder Fir Sludge 56.98 57.64 56.19 44.75 30.28 50.98 51.40 50.90 43.69 29.12 55.89 54.86 52.46 40.06 24.64 69.97 70.42 71.36 55.85 42.32 49.17 x 45.22* 45.58* 61.99* 58.46A 58.58A 57.73A 46.098 31.59C Irradiation*Means TABLE III-2 THE EFFECT OF IRRADIATION ON THE ADL CONTENT OF FOUR WOODS Sample Percent ADL (Sample X Irradiation Means from duplicate determinations) Rads 0 1 x 105 1 x 107 1 x 108 2 x 108 Sample* Means Poplar Alder Fir Sludge 13.13 13.19 12.41 10.20 8. 49 17.86 18.72 16.38 12.93 9. 53 30.83 29.43 27.46 23.81 22. 64 12.44 12.07 11.49 11.08 10. 18 18.56 A" 18.35' 16.93 AB 14.50 B C 12.71° Irradiation*Means 11.48' 15.08v 26.83' 11.45y Indicates the sources of variation found significant (P<.01) in the analysis of variance. The means for these sources of variation were tested in sets by Duncan's (24) new multiple range test. Means within a set having different superscripts differ s igni f i -cantly (P<.01). components have been shown to decrease in response to high levels of irradiation, i t would seem reasonable to expect ADF to decrease also. The data (Table III-3) shows that such a decrease did occur. Samples 8 8 treated with 1 x 10 or 2 x 10 rads were significantly lower in ADF 7 6 than the control samples or those exposed to 1 x 10 or 1 x 10 rads. A comparison of the ash content of the four woods and the effect of irradiation on i t is given in Table III-4. The ash content of sludge was significantly higher than in alder, poplar or f i r . Exposure to irradiation showed no significant effect on the ash content of any 65 of the woods. Similar data has been presented by Seifert . He reported no measurable change in the ash content of pine or beech wood when they were exposed to high levels of irradiation. The variation in DMD and cellulose digestion attributable to sample (Tables II1-5 and III-6, and Figures II1-3 and III-4 (Appendix IV)) followed a different pattern than that observed in Experiment II. Poplar, alder and f i r were not significantly different but sludge proved to be significantly more digestible than any of the other three samples. When either DMD or cellulose digestion were considered, the woods in order of increasing digestibility were as follows: f i r , alder, poplar and sludge. Irradiation caused the same proportional variation in both DMD and cellulose digestion. The untreated samples and those exposed to 1 x 10^ and 1 x 107 rads showed the lowest digestion coefficients, and they were not significantly different from each other. Both diges-tion parameters increased significantly in a direct response to the TABLE III-3 THE EFFECT OF IRRADIATION ON THE ADF CONTENT OF FOUR WOODS Percent ADF (Sample X Irradiation Means from duplicate determinations) Sample* Means Rads Sample 0 1 x 106 1 x 107 1 x 108 2 x 108 Poplar 73.45 73.86 71.49 51.65 35.59 61.21n Alder 71.47 70.57 68.43 56.85 38.23 61.11n Fir 78.41 75.64 72.48 57.80 44.40 65.74x Sludge 81.03 79.05 79.80 62.48 47.36 69.94* 76.09A 74.78A 73.05A 57.19C 41.39° Irradiation*Means * Indicates the sources of variation found significant (P<.01) in the analysis of variance. The means for these sources of variation were tested in sets by Duncan's (24) new multiple range test. Means within a set having different superscripts differ significantly (P<.01)a 40 TABLE XII-4 THE EFFECT OF IRRADIATION ON THE ASH CONTENT OF FOUR WOODS Percent Ash (Sample X Irradiation Means from duplicate samples) Sample* Means Rads Sample 0 1 x 106 1 x 107 1 x 108 2 x 108 Poplar 0.51 0.24 0.22 0.13 0.33 0.28X Alder 0.38 0.26 0.30 0.35 0.39 0.33 x Fir 0.50 0.50 0.31 0.25 0.18 0.35 x Sludge 3.79 3.43 3.29 3.15 3.78 3.49* * Indicates the sources of variation found significant (P<.01) in the analysis of variance. The means for these sources of variation were tested in sets by Duncan's (24) new multiple range test. Means within a set having different superscripts differ significantly (P<.01). TABLE XIX-5 THE EFFECT OF IRRADIATION ON THE IN VITRO DRY MATTER DISAPPEARANCE OF FOUR WOODS BY RUMEN MICROORGANISMS % Dry Matter Disappearance (Sample X Irradiation Means from triplicate determinations) Sample* Means Rads Sampl e 0 1 x TO6 1 x 107 1 x 108 2 x 108 Poplar 9.18 8.34 11.81 55.05 50.77 27.03* Alder 1.17 0.0 3.28 41.60 71.23 23.54* Fir 1.14 1.54 3.11 21.13 39.13 13.21* Sludge 47.51 46.13 47.55 59.19 66.05 53.29* 14.75A 14.00A 16.44A 44.24B 56.79B Irradiation*Means TABLE II1-6 THE EFFECT OF IRRADIATION ON THE IN VITRO CELLULOSE DIGESTION OF FOUR WOODS BY RUMEN MICROORGANISMS % Cellulose Digestion (Sample X Irradiation Means from triplicate determinations) Rads Sample* Means Sample 0 1 x 106 1 x 107 1 x 108 2 x 108 Poplar Alder Fir SIudge 18.43 18.55 22.54 70.72 49.25 0.64 0.13 4.90 49.92 77.21 4.07 1.17 4.17 23.79 37.89 69.85 69.22 69.07 74.18 73.91 35.90* 26.56* 14.22* 71.25* 23.25A 22.27A 25.17A 54.65 A B 59.57B Irradiation*Means * Indicates the sources of variation found significant (P<.01) in the analysis of variance. The means for these sources of variation were tested in sets by Duncan's (24) new multiple range test. Means within a set having different superscripts differ significantly (P<.01). 42 O Q increasing irradiation dosages of 1 x 10 and 2 x 10. rads, except in 8 the case of poplar. At the highest dosage of 2 x 10 rads, a decrease in the digestibility of poplar was noted. Although the experimental design does not allow the interaction between sample and irradiation dosage to be statistically analyzed, there is an evident trend which indicates that irradiation was more effective in increasing the digestibility of some woods than others. The data (Tables III-5 and II1-6 (p. 41)), show that irradiation in -creased the digestibility of poplar, alder and f i r to a greater extent than i t did in the case of sludge. A similar situation occurred in Experiment II in response to NaOH treatment. The difference in that experiment was that both f i r and sludge were unresponsive to the treatment. In this case, the digestibility of f i r was probably affected by the treatment because irradiation is a much stronger and more drastic process than NaOH steeping. It would seem that the digestibility of sludge was not affected by either treatment for the same reason. That i s , because sludge is the product of severe chemical processes used in pulping, i t has been purified in terms of containing relatively high levels of cellulose and low levels of lignin (Tables III-l and II1-2 (p. 37)) which make i t a highly digestible substrate. Due to its inherent high digestibi l ity, further treatments in the form of NaOH steeping or irradiation, did not produce correspondingly proportional increases in digestibil ity. High dosages of irradiation have been shown to increase the water soluble components of wood (Lawton et al.J^ and Sei fer t^) . Because i t is possible that these soluble products are not utilized by rumen microorganisms, DMD may not be a reliable measure of microbial digestion. As VFA's are the end products of microbial digestion, i t was decided to measure their production as well as cellulose digestion, neither of which are affected by substrate solubility. The variation in VFA production (Table 111-7, and figure II1-5 (Appendix IV)) from the different woods, ranked them in the same order of digestibility as the other digestibility parameters had done. There were significant-ly greater amounts of VFA's produced from sludge than from any of the other woods. There was no significant difference between poplar and alder or between alder and f i r , but poplar produced a significantly greater amount of VFA's than did f i r . 8 8 The irradiation dosages of 1 x 10 and 2 x 10 rads caused significantly higher VFA production in all woods than did the samples fi 7 treated with 1 x 10 or 1 x 10 rads or those which were untreated. As was noted in the DMD and cellulose digestion data, poplar, alder and f i r were more responsive to irradiation exposure than was sludge. Lawson et al.3^ and Pritchard et al.3^ noted a decrease in the VFA production from substrates exposed to levels of 2.5 x 10 rads and above, although DMD continued to increase. Both workers attributed this effect to a conversion of the carbohydrate fraction of the wood into compounds that could not be utilized by the rumen microorganisms, but which remained soluble in the incubation medium. 44 TABLE III-7 THE EFFECT OF IRRADIATION ON THE IN -VITRO PRODUCTION OF VFA'S FROM FOUR WOODS BY RUMEN MICROORGANISMS (meq/g substrate) meq VFA Produced/Gram of Substrate (Sample X Irradiation Means from t r ip l i cate determinations) Sample* Means Rads Sample 0 1 x 106 1 x 107 1 x 10 8 2 x 10 8 Poplar 2. 17 2.12 1.94 5.66 5.25 3.43 X Alder 0. 65 1.14 1.09 4.08 5.46 2 . 4 8 X y Fir 0. 33 0.29 0.69 2.15 2.41 1.17 y Sludge 4. 99 4.78 6.40 6.76 6.80 5.94* 2. 04 A 2.08 r t 2.53 A 4.66 B 4.98 B Irradiation*Means * Indicates the sources of variation found signif icant (P<.01) in the analysis of variance. The means for these sources of variation were tested in sets by Duncan's (24) new multiple range test. Means within a set having different superscripts d i f fer s ignif icant ly (P<.01). Similar decreases in VFA production did not occur in this study, except in the case of poplar, where VFA production did decrease from o samples exposed to 2 x 10 rads. Since both DMD and cellulose digestion also decreased, there appears to have been an experimental error made in the determination of the digestibility of that particular sample. If the VFA decrease had been due to a disintegration of nutrients then DMD would have increased, as solubility has been shown to increase 43 with increasing dosages of irradiation (Lawton et al.3 Pritchard et a l . j ^ and Seifert^ 5). The fact that VFA production did not decrease (except in the case of poplar which has already been explained) in samples treated with high levels of irradiation, does not prove that there was no con-version of carbohydrates into compounds not utilized by rumen micro-organisms, but i t does indicate that the high levels of irradiation produced a greater proportion of digestible nutrients than non-digest-65 ible compounds. Work by Seifert supports this proposal. He 8 reported that the degradation products of irradiation, up to 1.8 x 10 rads were made up largely of pentosans, oligosaccharides and carboxylic acids, all of which can be utilized by rumen microorganisms. Unlike the digestibility data obtained in the NaOH experiment, the data obtained in the irradiation experiment followed the trend 84 reported by Van Soest . That i s , as ADF and ADL decreased, digestibi l -ity increased. The difference in these two experiments with respect to the relationship between chemical composition and digestibility has already been explained, at least partially, by pointing out the different way in which the samples were prepared in regard to the washing out of the soluble components. A further aspect to consider is the apparent difference in the mechanisms by which NaOH treatment and irradiation increase the digestibility of wood. It has been fair ly well established (Stone et al.J® and Tarkow and Feist 7 4 ) that NaOH treatment increases the digestibility of wood by changing its physical structure in such a way as to allow cellulases greater access to the cellulose. From data collected in this studys no definite conclusions can be drawn as to the mechanism by which irradiation renders wood more digestible. It appears that i t alters the wood both chemically and physically. A substantial portion of the increase in DMD and VFA production in irradiated wood appears to be due to changes in chemical composition. As indicated in Table III-l (p. 37), cellulose was degraded at higher levels of irradiation. The majority of the degradation products as 65 identified by Seifert are compounds which can be utilized by rumen microorganisms. It therefore appears that irradiation induces chemical changes which result in increased digestibil ity. The significant increase in the digestibility of the cellulose remaining in the irradiated wood suggests that physical changes may haye occurred in this cellulose. Although no data has been collected on the physical characteristics of irradiated wood in this study, i t o may be noted that Becker and Burmester have reported a decrease in the o fibre saturation point and hygroscopicity of woods exposed to 1 x 10 rads. Since this effect is the opposite to the physical changes thought 47 to be responsible for the increased digestibility of NaOH-treated woods, no conclusions can be drawn in this regard. It is possible that a l -though there was a decrease in the fibre saturation point of irradiated wood as a whole, the fibre saturation point of the cellulose portion of the irradiated wood may have actually increased, thereby increasing 43 its digestibil ity. Lawton et al.3 have suggested that irradiation may break a particular cellulose-lignin bond which rumen microorganisms are unable to hydrolyze, thus making the cellulose available to them, but this theory has not been verified. It was noted that the cellulose values obtained using the modified Crampton and Maynard method, appeared slightly higher than 77 those reported by Timell for the same woods. Also, the cellulose isolated was very yellow in colour, which suggested that i t was not pure cellulose. For these reasons, i t was decided to determine cellulose in both untreated and irradiated woods by three different methods and to compare the results. The three methods used were: (a) the modified Crampton and May-nard 1 6 procedure, (b) VanSoests' 72% H2S04 method83, (c) the method of 80 Van Soest and Wine which utilizes permanganate (KMnO )^. The Crampton method is based on a simple digestion procedure using acetic acid and nitric acid to solubilize all material except cellulose. Both of Van Soests' methods uti l ize ADF as a starting material. In the 72% HgSO^  method, the acid is added to the ADF which hydrolyzes all the cellulose and leaves the lignin. Therefore the difference between the ADF and ADL is equal to the cellulose content. In the other procedure, KMn04 is added to the ADF to oxidize the lignin and leave the cellulose and ash which are partitioned by ashing. A statistical analysis of the data (Table II1-8) showed that when the method of analysis was the only source of variation considered, all methods were significantly different from each other. The KMn04 method gave the lowest results, followed by the 72% H2S04 (ADF-ADL) method, and the highest values were obtained using the Crampton method. The comparison between the Crampton method and the KMn04 method agrees 80 well with the data from a study done by Van Soest and Wine . They found the Crampton method cellulose values to be consistently higher than the KMn04 cellulose values. A possible explanation for the ADF-ADL values being significantly higher than the KMn04 values may be given 45 by the results of Loras and Loschbrandt . They have identified lignin fractions which are soluble in 72% HgSO^ This would mean that such lignin fractions would show up as cellulose instead of l ignin, and possibly increase the cellulose percentage enough to make i t s igni f i -cantly greater than the KMn04 cellulose. The significant sample by method interaction (Table III-8) is represented graphically (Figure II1-6 (Appendix IV)) by the lack of parallelism in the three lines which represent the three different methods. The interaction indicates that, (a) the fraction any of the methods isolated as cellulose from one wood, was not of the same composition as the fraction the same method was isolating as cellulose from another wood, or, (b) that each method was isolating different 49 TABLE II1-8 COMPARISON OF THREE METHODS OF CELLULOSE DETERMINATION USING FOUR IRRADIATED WOODS (SAMPLE BY METHOD MEANS) Percent Cellulose (Sample X Method*Means) Sample Methods* Means Methods Poplar Alder Fir Sludge Crampton 49.13c 44.95b 44.98b 61.901 50.24y ADF-ADL 49.73 c 46.03 f 38.91a 58.49h 48.29A KMn04 40.77e 38.93a 38.07d 52.679 42.61* TABLE II1-9 COMPARISON OF THREE METHODS OF CELLULOSE DETERMINATION USING FOUR IRRADIATED WOODS (IRRADIATION BY METHOD MEANS) Percent Cellulose (Irradiation X Method* Means) Rads Methods* Means Methods 0 1 x 106 1 x 107 1 x 108 2 x 108 Crampton ADF-ADL KMn04 58.35d 57.52 c d 52.79a 58.22d 56.43b 52.71a 56.89 b c 56.11b 50.71k 45.99 J 42.691 34.65h 31.759 28.68 f 22.18e 50.24y 48.29X 42.61* * Indicates the sources of variation found significant (P<.01) in the analysis of variance. The means for these sources of variation were tested in sets by Duncan's (24) new multiple range test. Means within a set having different superscripts differ significantly (P<.01). proportions of what i t defined as cellulose from different woods, or, (c) a combination of these two factors. The significant irradiation by method interaction (Table II1-9 (p. 49)) is represented graphically in Figure III-7 (Appendix IV). A l -though the three lines corresponding to the three different methods do not overlap as in Figure II1-6 (Appendix IV), they are not parallel and the interaction is therefore statistically significant. This indicates that the composition and/or proportion of cellulose isolated by a given method was significantly affected by the level of irradiation. As noted previously, Loras and Loschbrandt45 have identified lignin fractions which are soluble in 72% H2S04. In the light of this fact i t is possible that the ADL isolated by using 72% H2S04 may be lower than the true lignin value. The KMn04 method of Van Soest and 80 Wine overcomes the problem of lignin loss due to acid solubilization. They reported consistently higher lignin values using the KMnO^  pro-cedure when compared to values obtained using the 72% H2S04 method. It was suggested by them that KMn04 lignin may be closer to the true lignin value. Both lignin methods were used on untreated and irradiated wood samples (Table I11-10) to determine i f there was an appreciable differ-ence between the values obtained. Statistical analysis of the data showed that the KMnO^  lignin values were significantly higher than those obtained using the 72% H2S04 (ADF-ADL) method. Such a trend 80 agrees well with that noted by Van Soest and Wine . The significant sample by method interaction (Figure III-8 (Appendix IV)) indicates 51 TABLE XXI-10 COMPARISON OF TWO METHODS OF LIGNIN DETERMINATION USING FOUR IRRADIATED WOODS (SAMPLE BY METHOD MEANS) Percent (Sample X M< Sam Lignin =thod*Means) pie Methods* Means Methods Poplar Alder Fir Sludge H2S04 KMn04 11.48a 18.62c 15.08b 19.70d 26.84 f 25.859 11.45a 14.58b 16.21x 19.69* * Indicates the sources of variation found significant (P<.01) in the analysis of variance, The means for these sources of variation were tested in sets by Duncan's (24) new multiple range test. Means within a set having different superscripts differ significantly (P<.01). that either the composition or the proportion of lignin isolated by a method was significantly affected by different woods. It is also possible that both of these factors were occurring in combination to cause the significant interaction. The data collected in this study indicate that both sample type and sample treatment have to be considered when evaluating cellulose and lignin values obtained using different methods of determination. That i s , one particular method will not necessarily measure cellulose or lignin at a consistently higher or lower level than another method in different samples or in samples which have been irradiated at different levels. Since the KMnO^  method of Van Soest and Wine allows the determination of ADF, ADL, cellulose and ash in a single sample, and as i t is procedurally simple and gives values that are in close agreement with values reported using other more involved pro-77 cedures (Timell ), i t appears to be the method of choice among those studied for the determination of cellulose and lignin in a study of this kind. E SUMMARY The main conclusions to be drawn from these experiments are summarized below? 1. The in vitro digestibility of poplar wood by rumen micro-organisms increased in response to a reduction in particle size by grinding. Fir and alder wood showed only slight increases in their in vitro digestibility following grind-ing* 2. NaOH steeping significantly increased the in vitro digesti-bi l i ty of all woods except f i r . The woods in order of increasing responsiveness to NaOH treatment were: f i r , sludge, alder and poplar. The optimum NaOH concentration for maximizing digestibility was between 2 and 4%. The duration of the treatment had a significant effect on in vitro cellulose digestibil ity, as the 1.5 hour treatment produced samples whose cellulose was significantly more digestible than either the 1.0 or 0.5 hour treated samples. 3. The in vitro cellulose digestibility of all woods except poplar, increased in response to the irradiation dosages 8 8 of 1 x 10 and 2 x 10 rads. The digestibility of poplar 54 8 increased in response to the dosage of 1 x 10 rads, but o the dosage of 2 x 10 rads caused its digestibility to 8 decrease slightly, below the level at 1 x 10 rads. Irradia-tion caused a greater increase in the digestibility of poplar, alder and f i r than in sludge. 8 8 4. The two highest irradiation levels of 1 x 10 and 2 x 10 rads caused decreases in the cellulose and ADF content of al l woods when compared to the untreated samples or to those 6 7 exposed to 1 x 10 and 1 x 10 rads. There was a decrease in the ADL content of all woods except sludge, with each 7 increase in irradiation dosage above 1 x 10 rads. 5. Three different methods for determining the cellulose con-tent of-wood were compared, and a significant difference was found among their results. The KMn04 method gave the lowest results, then was the 72% HgSO^  (ADF-ADL) method, and the highest values were obtained using the Crampton method. Two methods of lignin analysis were compared and found to give significantly different results. The KMnO^  lignin values were significantly higher than those obtained using the 72% H^ SO^  method. 6. The four woods, regardless of the treatment or the digestibility parameter which was measured, remained in the same order of digestibil ity. In order of increasing digest-ib i l i t y , they were: f i r , alder, poplar and sludge. 55 7. Since the digestion data presented have been obtained using an in vitro system, there are important physiologic factors to be considered before such data can be meaningfully applied to the in vivo situation. It is doubtful i f the increase in the in vitro digestibility of poplar due to fine grinding would be evident under in vivo conditions. This would be due to the fact that fine grinding greatly decreases the time of passage in the ruminant, thus limiting the time the wood is available to the cellulolytic micro-organisms of the rumen. Although irradiation causes a dramatic increase in the in vitro digestibility of wood, there are economic limitations which would seriously limit its practical usefulness. To be effective, the dosage 8 requirement is approximately 1 x 10 rads. Considering the presently available faci l i t ies for gamma irradiation, this level is well above what is practically feasible for routine operations. Of the three treatments studied, NaOH steeping is l ikely the only one that will possibly find any application in practical ruminant feeding. Several investigations have been reported, in which large scale NaOH treatment has increased the in vivo digestibility of ligno-cellulosic materials such as straw and wood. This study has confirmed much of the data in the literature con-cerning the effects of fine grinding, NaOH steeping and irradiation on both the in vitro digestibility and chemical composition of wood. 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Chemical effects of high energy irradiation of wood. For. Prod. J . 7:208. 49. McDougall, E.I. 1948. Studies on ruminant saliva. I. The composi-tion and output of sheeps, saliva. Biochem. J . 43:556. 50. Mellenberger, R.W., M.A. Millet, A.J. Baker, L.D. Satter, and B.R. Baumgardt. 1968. Application of an in vitro forage evaluation test to wood and wood residue. J . Dairy Sci. 51:974 (Abstr.). 51. Nehring, K., R. Schiemann, L. Hoffman, W. Klippet, W. Jentsch. 1965. "Energy Metabolism," K.L. Blaxter, Ed., Academic Press, New York. 61 52. Nehring, K. and W. Schram. 1951. The composition and nutritive value of leaves and twigs. II) The digestibility of leav s and summer twigs. Il l) Nutritive value of autumn leaves and winter twigs. Archiv. Tierernahrung 1:264, 342. 53. Nehring, K. and W. Shram. 1949. Digestibility of various kinds of cellulose by ruminants. Tierzucht 1:11. 54. Norman, A.G. 1935. J . Agr. Research 25:529. 55. Oh, Hi Kon, M.B. Jones, and W.M. Longhurst. 1968. Comparison of rumen microbial inhibition resulting from various essential oils isolated from relatively unpalatable plant species. Appl. Microbiol. 16:39. 56. Oh, Hi Kon, B.R. Baumgardt, and J.M. Scholl. 1966. Evaluation of forages in the laboratory. V. Comparison of chemical analysis, solubility tests, and in vitro fermentation. J . Dairy Sci . 49:850. 57. Olmstead", W.H., W. M. Whitaker, and C. W. Duden. 1930. Steam disti l lation of the lower volatile fatty acids from a saturated salt solution. J . Biol. Chem. 85:109. 58. Pettyjohn, J .D. , J.M. Leatherwood, and R.D. Mochrie. 1964. Simplified technique for in vitro comparison of cellulose and dry matter digestibil it ies of forages. J . Dairy Sci. 47:1102. 59. Pew, J.C. and P. Weyna. 1962. Fine grinding, enzyme digestion, and the lignin-cellulose bond in wood. Tappi 45:247. 60. Pigden, W.J. and J.M. Bell . 1955. The art i f ic ia l rumen as a pro-cedure for evaluating forage quality. J . Animal Sci . 14:1239. 61. Pritchard, G.I., W.J. Pigden and D.J. Minson. 1962. Effect of gamma radiation on the utilization of wheat straw by rumen microorganisms. Can. J . Animal Sci. 42:215. 62. Quicke, G.V., O.G. Bentley, H.W. Scott and A.L. Moxon. 1959. Cellulose digestion in vitro as a measure of the digestibility of forage cellulose in ruminants. J . Animal Sci . 18:175. 63. Ross, J.P. 1967. Ration effects on blood metabolites in pregnant ewes. M.S.A. Thesis. U.B.C. Library, The University of British Columbia. 64. Sarkanen, K.V., How-Min Chang and G.G. Allan. 1967. - Species variation in lignins. II. Conifer lignins. III. Hardwood lignins. Tappi. 50:583,587. 62 65. Seifert, K. 1964. On the chemistry of gamma irradiated wood. Holz Roh-Werkstoff. 22:267. 66. Shelford, J.A. 1969. The utilization of alder sawdust by sheep and cattle. MSc. Thesis. U.B.C. Library, The University of British Columbia, 67. Springer, E.L. and L.L. Zoch. 1968. Hydrolysis of xylan in different species of hardwoods. Tappi. 51:214. 68. Steel, R.G.D. and J.H. Torrie. 1960. Principles and Procedures of Statistics. McGraw-Hill Co., New York, N.Y. 69. Stewart, D.G., R.G. Warner and W.W. Seely. 1961. Continuous culture as a method for the study of in vitro rumen fermenta-tion. Appl. Microbiol. 9:150. 70. Stone, J . E . , A.M. Scallan, E. Donefer, E. Ahlgren. 1969. Digesti-b i l i ty as a simple function of a molecule of a similar size to a cellulase enzyme. In Robert F. Gould (Ed.) Cellulases and their applications. Advances in Chemistry Series, No. 95, Amer. Chem. S o c , Washington, D.C. 71. Stranks, D.W. 1959. Fermenting wood substrates with a rumen cellulolytic bacterium. For. Prod. J . 9:228. 72. Stranks, D.W. 1961. Utilization of aspen wood residues. For. Prod. J . 11:288. 73. Sullivan, J.T. 1959. A rapid method for the determination of acid insoluble lignin in forages and its relationship to digestibil ity. J . Animal Sci. 18:1292. 74. Tarkow, H., and W.C. Feist. 1969. A mechanism for improving the digestibility of 1ignocellulosic materials with dilute alkali and liquid ammonia. In Robert F. Gould (Ed.). Cellulses and their applications. Advances in Chemistry Series, No. 95. Amer. Chem. S o c , Washington D.C. 75. T i l ley, J.M.A. and R.A. Terry. 1963. A two-stage technique for the in vitro digestion of forage crops. J . Brit. Grassland Soc. 18:104. 76. T i l ley, J.M.A., R.E.Deriaz, and R.A. Terry. 1960. The in vitro measurement of herbage digestibility and assessment of nutri-tive value. Proc of Eigth Intern. Grassland Cong. pp. 533. 77. Timell, T.E. 1957. Carbohydrate composition of 10 North American species of wood. Tappi. 40:569. 63 78. Tomlin, D.C., R.R. Johnson, B.A. Dehority. 1965. Relationship of lignification to in vitro cellulose digestibility of grasses and legumes. J . Animal Sci. 24:161. 79. Troelsen, J . E . , and D.J. Hanal. 1965. Ruminant digestion in vitro as affected by inoculum donor, collection day, and fermentation time. Can. J . Animal Sci . 46:149. 80. Van Soest, J.P. and R.H. Wine. 1968. Determination of lignin and cellulose in acid-detergent fibre with permanganate. J.A.O.A.C. 51:780. 81. Van Soest, J.P. and R.H. Wine. 1967. Use of detergents in the analysis of fibrous feeds. IV. Determination of plant c e l l -wall constituents. J.A.O.A.C. 50:50. 82. Van Soest, J.P. 1965. Use of detergents in analysis of fibrous feeds. III. Study of effects of heating and drying on yields of fibre and lignin in forages., J.A.O.A.C. 48:787. 83. . 1963. Use of detergents in the analysis of fibrous feeds. I. Preparation of fibre residues of low nitrogen content. J.A.O.A.C. 46:825. 84. . 1963. Use of detergents in the analysis of fibrous feeds. II. A rapid method for the determination of fibre and lignin. J.A.O.A.C. 46:829. 85. Virtanean, A.I. and J . Hukki. 1946. Thermophilic fermentation of wood. Suomen Kemistilehti. B. 19:4. 86. Virtanean, A. I . , and 0. E. Nikkila. 1946. Cellulose fermentation in wooddust. Suomen Kemistilehti. B. 19:3. 87. Virtanean, A. I . , 0. Koistinen, and V, Kuru. 1938. Fermentation of native cellulose in wood. Suomen Kemistilehti. B. 11:30. 88. Warner, A.C.I. 1956. Criteria for establishing the validity of in vitro studies with rumen microorganisms in so-called a r t i f i -cial rumen systems. J . Gen. Microbiol. 14:733. 89. Wilson, R.K. and W.J. Pigden. 1964. Effect of a sodium hydroxide treatment on the utilization of wheat straw and poplar wood by rumen microorganisms. Can. J . Animal Sci. 44:122. 90. Wise, M.B., R.W. Harvey, E.R. Barrick, and T.N. Blumer. 1967. Influence of adding limited amounts of various roughages to an all-concentrate ration for growing-finishing steers. N. Carolina St. Univ. Agr. Expt. Sta. ANS Report, 176. AH series 126. APPENDIX I METHOD FOR IN VITRO DIGESTIBILITY STUDIES 65 METHOD FOR IN VITRO DIGESTIBILITY STUDIES Materials Samples - All samples were air dried and ground past the 60 mesh screen of a laboratory Wiley mi l l . A sample size of 1 gram was used for all incubations. Incubation Vessels - Incubations were carried out in capped (sealed) 250 ml polycarbonate bottles* Buffer-Mineral Medium - The solution used was the same as that of Baumgardt et al.J. Before using the solution, i t was thoroughly saturated with COg until i t became clear (approximately one-half hour). Dispenser - A gravity operated dispenser was used for dispensing both the buffer solution and the rumen liquor inoculum. The reser-voir consisted of a 2000 ml glass bottle with a rubber tubing connection near the bottom. The bottle was wrapped with an electrical heating tape capable of maintaining the temperature of the contents at 39° C. The bottle was placed on a magnetic stirrer to keep the contents mixed. A self-zeroing burette was modified to deliver 60 ml of liquid from the reservoir. Incubator - A thermostatically controlled water bath set at 39°C was used as an incubator. It was capable of holding 24 bottle at one time. Rumen Fluid Inoculum - Rumen fluid was collected from a Herford steer fitted with a permanent rumen f istula. The steer was maintained on an alfalfa hay diet, supplemented with trace mineralized salt. Feed was not available to the animal for 12 hours prior to sampling. The rumen contents were immediately strained through 4 layers of 80 grade cheese cloth into pre-warmed vacuum flasks. The flasks were then taken to the laboratory where the strained rumen fluid was placed in 8, 250 ml centrifuge bottles and centri-fuged at 2000 R.P.M. for 5 minutes. The supernatant was decanted off and used as the inoculum. The plant material remaining in the bottom of the bottles was discarded. Procedure - Samples were weighed into the incubation bottles and 60 ml of the buffer medium added. The bottles were then put in the water bath prior to collecting the rumen sample, so that the medium was at 39° C when the inoculum was added. The rumen sample was collected and prepared as previously described. Sixty ml of the inoculum were added to the incubation bottles, and a fine stream of COg used to evacuate the air from them before sealing. They were then returned to the water bath for the 72 hour incubation period. All bottles were shaken 4 times daily to resuspend the substrate and to free trapped gas bubbles. It was found that the seal formed by the caps on the bottles allowed sufficient gas to escape to prevent any significant build-up of pressure in the bottles. At the end of the fermentation period the bottles were removed from the water bath and centrifuged at 2000 R.P.M. for 20 minutes. If total VFA analysis was to be done, approximately 25 ml of the supernatant was stored and the rest discarded. The residue was transferred quantitatively, with the aid of a wash bottle, into tared 100 ml beakers and dried at 100° C for 24 hours. The dried beakers and residues were weighed and dry matter disappearance calculated. The digestion procedure of the cellulose analysis was carried out in the same 100 ml beakers. This was done using the method of Crampton and May-16 23 nard as modified by Donefer et al.a . From this analysis cellulose digestion was calculated. A run consisted of 24 bottles, 6 experimental samples in tr ipl icate, a Solka Floe standard in tr ipl icate, plus a t r i p l i -cate of controls. The standard consisted of 1 gram of the purified cellulose (Solka Floe) plus 60 ml of medium and 60 ml of inoculum. In order to decrease the variation between runs, the cellulose digestion coefficient obtained for Solka Floe was used as the criteria for accepting or rejecting the data from a run. It was decided that only data obtained from trials where the cellulose digestion of Solka Floe had been 80% or greater would be accepted. The control bottles had no substrate added and were used to determine the dry matter, cellulose and VFA contribution made by the inoculum to the experimental bottles. The equation used to calculate percent dry matter (D.M.) disappearance in vitro was: (g substrate D.M.) - (g residual D.M. - g D.M. from inoculum) . . _ _ x 1 0 0 (g of substrate D.M.) The same equation was used for the calculation of percent cellulose digestion by substituting cellulose for dry matter. The total milliequivalents of VFA's produced per gram of sub-strate was calculated as follows: (meq/5 ml experimental supernatant - meq/5ml control supernatant) 120 . x  g substrate 5 In order to overcome any effect that incubation position or order of inoculum addition might have on microbial activity, a random-ized complete-block design was used. The 24 bottles were divided into 3 blocks; numbers 1 to 8, 9 to 16, and 17 to 24. Each treatment appeared once in each block, That i s , each block con-tained one control, one Solka Floe standard, and one of each experimental triplicate. Within each block, the bottles were assigned their position randomly. APPENDIX. II STATISTICAL MODELS 70 STATISTICAL MODELS Experiment I Yijk .-* + s 1 + V ( s 9 , 1 J + E1Jlc fijk * f * h Y... = k determination of the dependent variables in the J . U i t , j grind and i species, where the dependent variables were: in vitro DMD and in vitro cellulose digestion. y = population mean s. = effect of i species g. = effect of j grind (sg)i. = effect of the f i rst order interaction between the j grind and i species. This term was used as the residual term for testing all main effects. th th z• • k = random effect of the k determination in the j Mjk grind and i species, Experiment II (a) Y i j k l = y + s. + n j + t k + (sn)1 j + (s t ) 1 k + (nt) j k + ( sn t ) 1 j k + z i j k l Y i j k l =1 determination of the dependent variables in the. 71 ' i jkl k t h treatment interval, j t h NaOH level and i t h species, where the dependent variables were: in vitro DMD, in vitro cellulose digestion and Crampton cellulose content, y = population mean s. - effect of i ^ species n. = effect of j t h NaOH level J k (sn).. = effect of the f i rst order interaction between the t,, = effect of k treatment interval i t h species and j t h NaOH level (st )^ = effect of the f i rst order interaction between the i species and k treatment interval (nt)j^ = effect of the f i rst order interaction between the j t h NaOH level and k t h treatment interval. (sntj.jjk = effect of the second order interaction between the i species, j NaOH level and k treatment interval. This term was used as the residual term for testing all main effects and f i rst order inter-actions. s.-.-i.i = random effect of the 1 determination in the k treatment interval, j NaOH level and i species. <b> Y i j k = » + s i + n j + ( s n ) i j + E i j k ljk Y... - = k^ determination of the dependent variables in the j t h NaOH level and the i t h species, where the dependent variables were: ADF and ADL content. population mean effect of i ^ species, effect of j thNaOH level effect of the f i rst order interaction between the i species and j NaOH level. This term was used as the residual term for testing all main effects, random effect of the k**1 determination in the NaOH level and i t h species. Experiment III •„ + s. + r. + (sr) . . + E . . jk J . L . k determination of the dependent variables in the j irradiation dosage and the i species, where the dependent variables were: in vitro DMD, in vitro cellulose digestion, meq of volatile fatty acids produced per gram of substrate, ADF, ADL, KMnO^  ADL, KMnO^  cellulose, ADF-ADL cellulose and ash content, population mean t h effect of the i species. effect of the j irradiation dosage effect of the f i rst order interaction between the i species and the j irradiation dosage. This term was used as the residual term for testing main effects. , 73 tat, = random effect of the k determination of the j 'ijk J th irradiation dosage of i species. (b) Y . j R 1 = u + s. + r. + mk + (sr)1 j +" (sra)1k •+ (rm) . R + ( s ™ > i j k + E i j k l ^ijkl = determination of the dependent variables in the k method, j irradiation dosage and i species, where the dependent variables were: cellulose and lignin content, y = population mean s. - effect of the i species th r_. = effect of the j irradiation dosage th m,. = effect of the k method of determination = effect of the f i rs t order interaction between the i species and j irradiation dosage (sm)-k = effect of the f i rst order interaction between the •f* h + h i species and k method of determination ..th (rm).., = effect of the f i rst order interaction between the j J K th irradiation dosage and k method of determination (srm)..|, = effect of the second order interaction between the i t n species, j t n irradiation dosage and k t n method of determination. This term was used as the residual term for testing all main effects and f i rst order interactions. 74 E i j k l = r a n d o m e f f e c t o f t n e ^ determination in the k**1 method, j irradiation dosage and i species. The variance associated with triplicate or duplicate determinations was estimated from the z*^ or i . . ^ term in the preceding linear models, and are listed in Appendix III. APPENDIX III STANDARD DEVIATIONS FOR PROCEDURES 76 STANDARD DEVIATIONS FOR PROCEDURES Procedure Experiment I (Grinding) Experiment II (NaOH) Experiment III (Irradiation) % DMD ±0.61 ±3.67 ±1.16 % Cellulose Digestion ±0.63 ±1.58 ±1.14 % Crampton Cellulose ±1.22 ±1.14 % ADF ±0.93 ±0.62 % ADL ±0.47 ±0.61 meq VFA/g substrate ±0.63 % KMn04 ADL ±0.97 % KMn04 Cellulose ±0.86 % ADF-ADL Cellulose ±0.37 % Ash ±0.74 METHODS COMPARISON (ON IRRADIATED WOOD) % Cellulose - includes all three methods % Lignin - includes two methods ±0.61 ±0.81 APPENDIX IV FIGURES II-l to III-8 FIGURE II-l The effect of NaOH treatment on the Crampton cellulose content of poplar and alder FIGURE II-2 The effect of NaOH treatment on the Crampton cellulose content of f i r and sludge CO o -I _1 _J UJ o z o t-0 -< or o h-Z UJ O or. UJ a, 79 POPLAR - • 1 ALDER •0.5 HOURS — — I .0H0URS 1.5 HOURS 4 PERCENT NaOH UJ CO o _ l _J _J UJ o z o r-0 . < or a 8 0 SLUDGE FIR 70 6 0 0.5 HOURS — 1.0 HOURS • - — I . 5 H 0 U R S PERCENT NaOH FIGURE II-3 The effect of NaOH treatment on the ADF and ADL content of poplar, alder, f i r , and sludge ACID DETERGENT F I B R E • — P O P L A R — A L D E R — F I R — S L U D G E ACID DETERGENT LIGNIN PERCENT NaOH FIGURE II-4 The effect of NaOH treatment on the in vitro dry matter disappearance of poplar, alder and sludge 8: 0.5 HOURS U J o z < < UJ Q, Q. < GO o UJ h-< 2. >= or Q z UJ o or UJ a. — - 1 . 0 HOURS 0 — 1 . 5 HOURS PERCENT NaOH FIGURE I1-5 The effect of NaOH treatment on the in vitro cellulose digestion of poplar, alder, f i r and sludge PERCENT NaOH 86 FIGURE III-l The effect of gamma irradiation on the ADF, ADL and Crampton cellulose content of poplar and alder 88 FIGURE III-2 The effect of gamma irradiation on the ADF, ADL and Crampton cellulose content of sludge and f i r 90 FIGURE II1-3 The effect of gamma irradiation on the in vitro dry matter disappearance of poplar, alder, f i r and sludge LOG OF IRRADIATION DOSE (RADS) FIGURE II1-4 The effect of gamma irradiation on the in vitro cellulose digestion of poplar, alder, f i r and sludge LOG OF IRRADIATION DOSE (RADS) FIGURE III-5 The effect of gamma irradiation on the mi H i -equivalents of VFA's produced in vitro per gram of poplar, alder, f i r and sludge LOG OF IRRADIATION DOSE (RADS) FIGURE II1-6 The effect of species on three methods of cellulose determination FIGURE III-7 The effect of irradiation on three methods of cellulose determination 97 KMnOL C E L L U L O S E POPLAR ALDER FIR SLUDGE SPECIES LOG OF IRRADIATION DOSE (RADS) F I G U R E ni-a The effect of species on two methods of lignin determination 99 SPECIES 

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