MSU LIBRARIES n RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES wiII be charged if booE is returned after the date stamped below. ' o A n r f 1“ ~12w1+ L? . i \ -' ‘. d . I.) J . JR 7 r - -~ . \ . J {;Lu I 5}“95 PARTITION, ANATOMY, CHEMISTRY AND DIGESTIBILITY OF ALFALFA AND BIRDSFOOT TREFOIL BY Simon-P. Guertin A DISSERTATION Submitted to Michigan State University in partail fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Scieces 1986 demos ABSTRACT PARTITION, ANATOMY, CHEMISTRY AND DIGESTIBILITY 0F ALFALFA AND BIRDSFOOT TREFOIL By Simon-P. Guertin Alfalfa (Médicago sativa L.) and birdsfoot trefoil (Lotus cornicu- Zatus L.) were grown in the greenhouse. The plants were collected at early bud, 102 bloom and full bloom. The morphological components of each legume were separated and several variables were evaluated so as to determine their level of changes along the stem and maturity and to compare their levels between both alfalfa and trefoil. In chapter I, alfalfa had a higher leaf-stem ratio than trefoil, particularly in the middle layer, at blooming stages. Within both spe- cies, the leaf-stem ratio declined down the stem. The greatest concen- tration of both leaf and stem was found in the median portion of the shoot of both alfalfa and trefoil. In chapter II, the percentage area of vascular bundles, sclerenchy- ma, xylem and phloem was greater in leaves of alfalfa and in trefoil. MeSOphyll cells area was greater in trefoil than in alfalfa. The amount of epidermis, sclerenchyma and mesophyll cells were correlated with the relative feeding value (RFV). The epidermis, bundle sheath, interfascicular xylem, vascular bund- le and pith parenchyma cells of stem were affected by the location along the shoot. Several tissue types such as pith parenchyma, bundle sheath, epidermis, vascular bundles, xylem were correlated with stem IVDMD and RFV. Simon-P. Guertin In chapter III, the trefoil leaf and stem had a higher level of NDF, ADF and wall protein than alfalfa. The levels of p-lignin and cellulose were higher in the leaf and stem of trefoil than in alfalfa. Cellular content in alfalfa was higher than in trefoil in both leaf and stem. Ba— sal leaves contained greater NDF, ADF and cellulose than the upper and me- dian leaves. In the stem, wall protein level was higher in the upper than in the two lower stem portions. In chapter IV, leaf digestibility of alfalfa was higher than trefoil, particularly at the two upper layers. Stem digestibility declined from top to bottom in both alfalfa and trefoil. In chapter V, digestion of most living tissue types in both leaf and stem was partial after 6 or 12 hours and showed visible degradation after 12 or 24 hours of fermentation. Digestion of leaf and stem of both spe- cies was characterized by the bacterial specificity, the surface potential of tissues and the interaction between bacteria and substratum. ACKNOWLEDGEMENTS The author expresses his deep gratitude to his parents for their great generosity and constant support. The author wants to extent his gratitude to Dr. Milo B. Tesar for making realisable the research work and the manuscript. The author wants to thank the Department of Animal Science, the Department of Animal Husbandry and the Department of Botany and Plant Pa— thology and also the Department of Plant Science of Laval University to have allowed him to use their facilities to do the research work and the analysis of the data. The appreciation of the author are extended to the members of his committee Dr. R. Emery, Dr. M. Yokoyama, Dr. C. Cress and Dr. S. Flegler and to the direction of Agriculture-Quebec for their service and support. TABLE OF CONTENTS ABSTRACT LIST OF TABLES LIST OF FIGURES PREFACE CHAPTER I MORPHOLOGICAL STUDY OF ALFALFA AND BIRDSFOOT TREFOIL .......... 1 Abstract.......... ....................................................... 2 Introduction ............................................................. 4 Methodology ........ . ..................................................... 6 Results and discussion ................................................... 9 References..... .......................................................... 29 CHAPTER II HISTOLOGICAL STUDY OF ALFALFA AND BIRDSFOOT TREFOIL ........... 31 Abstract ................................................................. 32 Introduction ............................................................. 34 Methodology ..... ... .......... . ........................................... 37 Results and discussion ................................................... 40 References ............................................................... 75 CHAPTER III CHEMICAL COMPOSITION OF ALFALFA AND BIRDSFOOT TREFOIL .......... 77 Abstract.. ............................................................... 78 Introduction ............................................................. 80 Methodology .............................................................. 83 Results and discussion ................................................... 85 References ............................................................... 132 CHAPTER IV DIGESTIBILITY STUDY OF ALFALFA AND BIRDSFOOT TREFOIL ........... 135 Abstract ......................... . ....................................... 136 Introduction ............................................................. 137 Methodology. ............................................................ 140 Results and discussion .................................................. 142 References .............................................................. 152 CHAPTER V RUMINAL DEGRADATION OF LEAF AND STEM OF ALFALFA AND BIRDSFOOT TREFOIL BY ELECTRON MICROSCOPY ............................... 155 Abstract.... ......... . .................................................. 156 Introduction.. .......................................................... 157 Methodology........ ..................................................... 160 Results and discussion ................... . ............................. . 162 References.... ....................... . .................................. 178 LIST OF TABLES TABLES CHAPTER I 1, Analysis of variance of the factors; maturity stages and plant development of both alfalfa and birdsfoot trefoil and the com- bined factor interactions on the leaf-stem ratio...... ............. 2. Variation in the partition of leaf to stem among the various mor— phological groups of two legumes at three different maturity sta- ges (Exp. I) ..... .0.0.000000. ....... 00.00000.00.00.0000.00.00 000000 3. The mean ratio leaf to stem of legume species at each maturity stage (Exp. I)................... ..... ........ ..... ....... ......... 4, Analysis of variance of factors; species, maturity stages and plant layers and their interactions on the ratio leaf to stem, on the distribution of leaves and stems along the shoot of plants with intermediate number of node (18) (Exp. II and III)............ 5. The mean leaf-stem ratio of each legume species with an interme- diate number of nodes at each studied maturity stages (Exp. 11).... 6. Vertical changes in the percentage contribution of flowers to the total plant stems fraction in both alfalfa and birdsfoot trefoil at three maturity stages (Exp. II)............... ........ .......... 7. Vertical changes in the percentage contribution of secondary—ter- tiary leaves to the total plant leaves of both alfalfa and birds- foot trefoil at three maturity stages (Exp. II)............. ....... 8. Vertical changes in the percentage contribution of secondary-ter- tiary stems to the total plant stems in both alfalfa and birds- foot trefoil at three maturity stages (Exp. II).............. ...... 9. Mean contribution of leaves (Z) and of stems (Z) of each plant layer to the whole plant leaf and stem of the bulk data of both leguminous species (Exp. III)...................................... 10. Vertical changes of the stem diameter of both alfalfa and birds- foot trefoil at three maturity stages................ ...... .. ...... CHAPTER II 11. Mean area of anatomical components in leaves of alfalfa and birds- foot trefoil and analysis of variance of the main factors: species, maturity stages and plant layers on the percentage area of anato- mical components (in plant with 18 nodes development).............. Page 10 11 12 14 15 19 20 21 25 28 41 12. Vertical distribution of the number of vascular bundles in leaf cross section of alfalfa and birdsfoot trefoil at three maturity stages .............................................................. 13. Vertical changes of the ratio leaf cross sectional length to leaf cross sectional width of both alfalfa and birdsfoot trefoil at three maturity stages ............................................... 14. Vertical changes of the mean area of anatomical components in leaf and in stem of both alfalfa and birdsfoot trefoil .............. 15. Vertical changes of the leaf thickness (leaf cross sectional width) of both alfalfa and birdsfoot trefoil at three maturity stages ...... l6. Correlation coefficient between the various anatomical components in leaf of each leguminous species and their respective leaf IVDMD (24 hours) and RFV .................................................. 17. Mean area of anatomical components in stems of alfalfa and birdsfoot trefoil and analysis of variance of the main factors: species, matu— rity stages and plant layers on the percentage area of anatomical com- ponents (in plant with 18 nodes development)............. ..... ...... 18. Vertical distribution of the number of vascular bundles in stem cross section of alfalfa and birdsfoot trefoil at three maturity Stages 00000000000000000000000000 0 0000000000000000000 0 000000000000000 19- Correlation coefficient between the various anatomical components in stem of each legume and their respective stem IVDMD (24 hours) and CHAPTER III 20. Analysis of variance of the factors: species(S), maturity stages (M) and morphological development(D) and their interactions on the level of various feeding value 'variables of the whole plant with node number ranging from 10 to 28, in experiment I.. ...... .... ...... 21. Mean level of feeding value variables in forage of both alfalfa and birdsfoot trefoil in experiment I ....... . ....................... 22. Correlation coefficient between the leaf—stem ratio and the fee- ding value variables as well as among the cell wall constituents of the bulked data of both studied legumes, in experiment 1.. ......... . 23. Correlation coefficient between the ratio of leaf to stem.and the fee- ding value 'variables as well as among the cell wall constituents in forage of each studied legume in Exp. I ....... ..... .............. 42 46 52 54 55 57 61 70 86 87 88 89 24. 25. 26. 27. 28. 29. 300 31. 32. 33. 34. 35. 36. Changes in the mean content of the various variables of feeding value in forage of alfalfa and birdsfoot trefoil with maturity, in experiment I ..................................................... Changes in the level of NDF among the various morphological groups of both alfalfa and birdsfoot trefoil, at early bud and at full bloom (Exp. I) ............. . ........................................ Changes in the level of cellular content among the various mor- phological groups of both alfalfa and birdsfoot trefoil, at ear- ly bud and at full bloom (Exp. I) .................. . ................ Changes in the level of ADF among the various morphological groups of both alfalfa and birdsfoot trefoil, at early bud and at full bloom (Exp. I) ......... . ................ . ........................... Changes in the level of hemicelluloses among the various morpholo- gical groups of both alfalfa and birdsfoot trefoil, at early bud and at full bloom (Exp. I)... ............. . ........... . ............. Changes in the level of p-lignin among the various morphological groups of both alfalfa and birdsfoot trefoil at early bud and at full bloom (Exp. 1) ................................................. Changes in the level of cellulose among the various morphological groups of both alfalfa and birdsfoot trefoil at early bud and at full bloom (Exp. I) ................................................. Changes in the level of wall crude protein among the various morpho- logical groups of both alfalfa and birdsfoot trefoil, at early bud and at full bloom (Exp. I)... ............. . ......................... Changes of the forage feed relative value among the various morpho- logical groups of both alfalfa and birdsfoot trefoil, at early bud and at full bloom (Exp. 1) ..................... . ..... . .............. Analysis of variance of the factors: species(S), maturity stages (M) and plant layers(P) and their interactions on the content of va- rious feeding 'variables in leaf of plants with 18 nodes development (EXP. II) 00000000000000000 o 0000000000000 .0... ooooooooooooooooooooooo Mean level of feeding ‘variables in leaf and stem of both alfalfa and birdsfoot trefoil (Exp. II) ............................ . ........ Vertical changes of the mean level of various feeding 'variables 111 leaf and in stem of both alfalfa and birdsfoot trefoil (Exp. 11).... Analysis of variance of the factors: species(S), maturity stages (M) and plant layers(P) and their interactions on the level of va- rious feeding variables in stem of plant with 18 nodes develOpment (Exp. II).... ...... . ............................... . .......... . ..... 91 92 93 94 95 97 98 99 100 101 102 106 107 37. Mean level of various feeding variables in leaf and in stem of both alfalfa and birdsfoot trefoil at the two studied maturity stages (Exp. II)........ ......... ....... ....... .......... ..... . ..... CHAPTER IV 38. 39. 40. 41. 42. Analysis of variance on the effects of factors species(S), matu- rity stages(M) and plant development(P), plant layers(L) and fer- mentation time(F) on the digestibility of forage legumes in expe- riment I and II.. .............. ... ..... ............ ................. Changes in in vitro dry matter digestibility (IVDMD) of different groups of plant develOpment at three different fermentation time, within early bud and full bloom of both alfalfa and birdsfoot tre- foil, in experiment I...................................... ......... Changes in in vitro dry matter digestibility (IVDMD) of leaf, ver- tically down the shoot of both alfalfa and birdsfoot trefoil at two growth stages and at two fermentation time, in experiment II ........ Changes in in vitro dry matter digestibility (IVDMD) of stem, ver- tically down the shoot of both alfalfa and birdsfoot trefoil, at two growth stages, and two fermentation time, in experiment II ...... Contribution(Z) of inflorescence to the total stem fraction of each layer on both alfalfa and birdsfoot trefoil at every maturity stage (Exp. II)..... ....... ......... ............... . ...................... 146 149 151 LIST OF FIGURES FIGURES CHAPTER I 1. Vertical and horizontal distribution of the leaf—stem ratio within both alfalfa and birdsfoot trefoil (Exp. 11).. ..................... 2. Vertical changes in the contribution of leaves to the whole plant leaves in alfalfa and birdsfoot trefoil at various maturity stages (Exp. III) ..... ................. .......... ................. ........ 3. Vertical changes in the contribution of stems to the whole plant stems in alfalfa and birdsfoot trefoil at various maturity stages (Exp. III). .......... .. ..... . ....... .......... ...... .. ...... . ...... CHAPTER 11 4. A vertical distribution of sclerenchyma in leaves of alfalfa and birdsfoot trefoil at various maturity stages.................. ..... 5. A vertical distribution of xylem in leaves of alfalfa and birds— foot trefoil at various maturity stages............................ 6. A vertical distribution of phloem in leaves of alfalfa and birds- foot trefoil at various maturity stages........................... 7. A vertical distribution of vascular bundle in leaves of alfalfa and birdsfoot trefoil at various maturity stages............ ....... 8. A vertical distribution of collenchyma in leaves of alfalfa and birdsfoot trefoil at various maturity stages...................... 9. A vertical distribution of mesophyll cells in leaves of alfalfa and birdsfoot trefoil at various maturity stages.................. 10. A vertical distribution of epidermis in leaves of alfalfa and birdsfoot trefoil at various maturity stages...................... 11. A vertical distribution of epidermis in stems of alfalfa and birdsfoot trefoil at various maturity stages...................... 12. A vertical distribution of bundle sheath in stems of alfalfa and birdsfoot trefoil at various maturity stages...................... 13. A vertical distribution of parenchyma cells in the pith of stems of alfalfa and birdsfoot trefoil at various maturity stages....... 17 23 26 43 44 45 47 48 49 51 60 63 64 14. A vertical distribution of interfascicular xylem in stems of a1- falfa and birdsfoot trefoil at various maturity stages .............. 65 15. A vertical distribution of xylem in stems of alfalfa and birdsfoot trefoil at various maturity stages .................................. 66 16. A vertical distribution of vascular bundle in stems of alfalfa and birdsfoot trefoil at various maturity stages ........................ 67 17. A vertical distribution of chlorenchyma in stems of alfalfa and birdsfoot trefoil at various maturity stages ........................ 68 18. A vertical distribution of collenchyma in stems of alfalfa and birdsfoot trefoil at various maturity stages ............. . .......... 69 19. A vertical distribution of sclerenchyma in stems of alfalfa and birdsfoot trefoil at various maturity stages .................. . ..... 72 20. A vertical distribution of phloem in stems of alfalfa and birds- foot trefoil at various maturity stages ............................. 73 21. A vertical distribution of parenchyma in xylem of vascular bundles of stem of alfalfa and birdsfoot trefoil at various maturity stages. 7A CHAPTER III 22. Vertical distribution of NDF in leaf of both alfalfa and birdsfoot trefoil at two maturity stages (Exp. 11)..... ....................... 104 23. Vertical distribution of NDF in stem of alfalfa and birdsfoot tre- foil at two maturity stages (Exp. 11)................‘ ....... . ....... 109 24. Vertical distribution of cellular content in leaf of both alfalfa and birdsfoot trefoil at two maturity stages (Exp. 11)..... ........ 112 25. Vertical distribution of cellular content in stem of both alfalfa and birdsfoot trefoil at two maturity stages (Exp.II)............... 113 26. Vertical distribution of ADF content in leaf of both alfalfa and birdsfoot trefoil at two maturity stages (Exp. II)............ ...... 114 27. Vertical distribution of ADF content in stem of both alfalfa and birdsfoot trefoil at two maturity stages (Exp. 11).... ......... ..... 115 28. Vertical distribution of hemicelluloses in leaf of both alfalfa and birdsfoot trefoil at two maturity stages (Exp. II).................. 117 29. Vertical distribution of hemicelluloses in stem of alfalfa and birdsfoot trefoil at two maturity stages (Exp. 11) .................. 118 30. Vertical distribution of p—lignin in leaf of both alfalfa and birdsfoot trefoil at two maturity stages (Exp. 11) ................ 31. Vertical distribution of p-lignin in stem of both alfalfa and birdsfoot trefoil at two maturity stages (Exp. 11).. ..... . ........ 32. Vertical distribution of cellulose content in leaf of both al- falfa and birdsfoot trefoil (Exp. 11) ............................. 33. Vertical distribution of cellulose in stem of both alfalfa and birdsfoot trefoil at two maturity stages (Exp. 11) ................ 34. Vertical distribution of the cell wall crude protein in leaf of both alfalfa and birdsfoot trefoil at two maturity stages (Exp. II)... 00000 .... 00000 .0 ....... ....000... ..................... 35. Vertical distribution of cell wall crude protein in stem of both alfalfa and birdsfoot trefoil at two maturity stages (Exp. 11).... 36. Vertical changes in the leaf indice of relative feeding value of both alfalfa and birdsfoot trefoil at two maturity stages (Exp. II)............ ............. ......................... ....... 37. Vertical changes of the stem relative feeding value of both alfal- fa and birdsfoot trefoil at two maturity stages (Exp. 11) ......... CHAPTER V 38. Leaf of alfalfa incubated 12 hours in a ruminal solution. x 400 . ...... . ....... . ......... 0 0000000 . ..... 0 ..................... 39. Leaf of birdsfoot trefoil incubated 12 hours in a ruminal solution 40. Leaf of alfalfa incubated 24 hours in a ruminal solution. X 400.. 41. Leaf of birdsfoot trefoil incubated 24 hours in a ruminal solution x4000... .......... 0000 ..... 000. ....... . 00000000000000 0 ..... ...... 42. Colonies of cocci sp. eroding the epidermis of the alfalfa leaf after 12 hours of fermentation. X 400.. .......... .......... ..... 43. Magnified view of colonies of cocci sp. eroding the epidermis of the alfalfa leaf. The bacteria seemed to be linked together by some lateral appendages (arrow) (12 hours of fermentation). X5000 000000000000 0.000000 ooooooooooooooooooooooooooooooooooooooo 120 121 122 125 129 130 131 163 163 164 164 165 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. The stomate is a location on the leaf epidermis where bacteria can penetrate to degrade the internal tissues (12 hours of fer- mentation). X5000 ........... . ......... ..... .................... 166 Bacteria with rod shape as well as with round shape showed atta— chement to the epidermis surface. They appeared to have some appendages that linked them (arrow) (12 hours of fermentation). X 20000 ...... . .................................................. 166 Population of ruminal bacteria degrading the epidermal cells in an eroded area of an alfalfa leaf. Bacteria showed ramifications between them which could be used to adhere to the tissue (24 hours of fermentation). X 5000 .................................. 167 Population of ruminal bacteria interspersed with fungi (mycellium arrow) on mesophyll cells of alfalfa leaf (12 hours of fermenta- tion). X 5000 .................................................. 167 Phloem in the main vein of an alfalfa leaf attacked by rod bacte- ria sp.. Several rod bacteria showed round shape bodies along their cell wall. These bodies had appendages to permit them to adhere to the rod bacteria (12 hours of fermentation). X 5000.. 169 The main vein xylem of alfalfa leaf attacked by a dense popula- tion of bacteria of rod shape at the periphery and at the middle lamella of the tissue (24 hours of fermentation). X 5000....... 169 Bacterial population on the epidermis of birdsfoot trefoil, after 24 hours of fermentation in a ruminal solution. X 5000 ......... 170 Fimbriae bacteria found in great numbers on the epidermis of the birdsfoot trefoil leaf (24 hours of fermentation). X 20000 ..... 170 Mesophyll cells of birdsfoot trefoil leaf incubated 12 hours in a ruminal solution. X 5000.. ..... . ..... ................... ..... 171 Mesophyll cells of birdsfoot trefoil incubated 24 hours in a ru- minal solution. X 5000. ..... .................... ......... ...... 171 Birdsfoot trefoil stem collected at the top portion after a fer- mentation period of 24 hours in a ruminal solution. X 400...... 173 Birdsfoot trefoil stem collected at the bottom portion, after a fermentation of 24 hours in a ruminal solution. X 400.......... 173 Trefoil stem incubated 6 hours in the rumen-buffer solution. x400.0.00000 ..... 0 000000000 O ........... .0.00.. ................. 174 57. 58. 59. 60. Trefoil stem incubated 24 hours in a rumen—buffer solution. X 400....... ......... .. ............... . ....... . ...... . .......... Bacterial activity on phloem of alfalfa stem after 6 hours of fermentation. The rod shape bacteria showed lateral link with other microorganisms of round shape. X 5000........ ....... Bacteria of rod shape sp. degraded (arrow) the middle lamella area of the xylem cell wall of trefoil upper stem (24 hours of fermentation). X 10000 ..................................... Cellulolytic bacteria attached to the internal wall of xylem in trefoil upper stem (24 hours of fermentation). X 20000........ 174 176 176 177 PREFACE Alfalfa (Medicago sativa L.) and birdsfoot trefoil (Lotus corni- cuZatus L.) are perennial legumes used primarily to supply forages to 1i— vestock. Alfalfa is considered by many to be the best forage crop although several factors such as soil fertility, acidity and moisture limit its use. Birdsfoot trefoil is a valuable alternative to alfalfa under some growth condition. The high feeding value of both forage legumes is well known. However, controversial reports have been found about the difference of the nutritive value of the hay of the legume species as they matured. For ins— tance, birdsfoot trefoil was found to retain more its digestibility than alfalfa with maturity but no explanation was given to support such an observation. The main objective of the present study is to compare changes in feeding parameters between both alfalfa and trefoil with maturity. Nine experiments were conducted and distributed into five distinct chap- ters. Four chapters were related respectively to changes in partition of plant parts, histology, chemistry, and digestibility of both species with maturity. The fifth chapter is on ruminal bacteria activity in the degradation process of tissue types of both leaf and stem. In section 1, three different aspects of partition of leaf and stem studied: 1) on selected groups of morphological development collec- ted at early bud, lOZ bloom and full bloom of the whole plant, 2) along the shoot of plants harvested at various growth stages, and 3) as contri- bution of each plant layers to the total amount of leaves and stems. In chapter II, a quantitative evaluation of anatomical components was done in leaf and in stem of both alfalfa and birdsfoot trefoil, at three maturity stages and at different positions along the stem. The degree of association between the amount of various tissue types in leaf and in stem and the in vitro dry matter digestibility (IVDMD) and the re- lative feeding value (RFV) of leaf and stem was determined on all data. Fibrous fractions and the cell wall constituents were determined in chapter 111. In the first experiment, the feeding parameters were de- termined in ten groups of plant development of plants harvested at early bud and at full bloom within each species studied. In a second experi— ment, the same parameters were evaluated vertically and horizontally in leaf and in stem from a selected population of plants with 18 nodes. Changes in the concentration of the feeding parameters were obtained on both species at different positions on the shoot from early bud and full bloom plants. In chapter IV, the digestibility of both alfalfa and trefoil fo- rage was investigated in order to determine the degree of variation in digestibility among three groups of plants having varing development wi— thin the same stage of maturity. Another experiment was performed on alfalfa and trefoil to study the changes of the IVDMD of leaf and stem within a selected population of plants with 18 nodes. In chapter V, the rate of degradation of the leaf and the stem tissue types from various position along the shoot was examined on a scanning electron microscope of both species collected at early bud, 10% bloom and full bloom. The association of the bacteria with the ul- trastructure of the various tissue types and the interrelationships bet— ween the ruminal bacteria was determined. CHAPTER I MORPHOLOGICAL STUDY OF ALFALFA AND BIRDSFOOT TREFOIL ABSTRACT Alfalfa (M@d%cago sativa L.) and birdsfoot trefoil (Lotus corni- cuZatus L.) were grown in the greenhouse to determine the partition of leaf and stem at early bud, 10% bloom and full bloom. Leaves and stems were studied: a) at the same maturity stage; b) in the top, middle and bottom of the plant; and c) in each of the three plant layers. The partition of leaf and stem fractions on alfalfa was affected by the level of plant development. Alfalfa with low number of nodes(10 to 16) had a mean leaf-stem ratio lower than those with a higher node number. Trefoil leaf-stem ratio was greatly affected by the stage of maturity, de- clining from 0.68 at early bud to 0.47 at full bloom. From early bud to blooming stages, the leaf to stem ratio decreased for plants with 18, 20 to 22 nodes and increased for plants with lower numbers. Alfalfa had a higher leaf-stem ratio at blooming stages than tre— foil, particularly in the middle layer. The leaf-stem ratio declined mar— kedly down the stem from 1.39 to 0.25 on alfalfa and from 1.26 to 0.19 on trefoil. The proportion of leaf to stem decreased from 0.67 at early bud to 0.43 at full bloom in trefoil and decreased from early bud (0.71) to 10% bloom (0.64) on alfalfa. The leaf-stem ratio at the upper layer declined from early bud (2.07) to full bloom (0.80) on alfalfa and from 1.98 to 0.75 on trefoil. The ratios of the basal segment on both species were unchanged with ma- turity. At the median layer, trefoil had a lower leaf-stem ratio of 0.56 at full bloom compared to 0.72 at early bud and 0.69 at 10% bloom. Alfalfa was more leafy with a ratio of 0.80 at early bud, 0.84 at 10% bloom and 0.96 at full bloom. The greatest concentration of both leaf and stem was found in the median portion of the shoot of both alfalfa and trefoil. Trefoil had more uniform leaf distribution among the upper layers than alfalfa in the upper layer while alfalfa was more leafy in the median portion. Trefoil had a higher proportion of stem in the upper layer and a lower proportion in the bottom layer than alfalfa. The species were similar in the median segment. INTRODUCTION In animal feeding, leaves are consumed in greater quantity than stems (Larendo and Minson, 1973). Leaves have the highest concen- trations of constituents important in nutrition (Sotola 1933, Mowat et a1 1965, Smith 1969, Smith 1970, Martin 1970, Sullivan 1973). Conse- quently, forage quality would be greatly influenced by any morphologi- cal changes that increase the proportion of leaves. Woodman and Evans (1935) found that leaf-stem ratio would be an important feature in the variations of some chemical components of legumes such as alfalfa. Since then, it has been well documented that the leaf-stem ratio would have an effect on nutritive value of legumes (MacDonald 1946, Reid et a1 1959, and Ulyatt 1973). Variations of the leaf—stem ratio could be influenced by several factors such as species, maturity stages, plant development, and location on the stem. Terry and Tilley (1964), Sullivan (1973), and Hoveland and Monson (1980) have studied the effect of maturity on the digestibility of alfalfa. All these authors reported a decrease in digestibility as the alfalfa matured; a reduction in leaf-stem ratio in maturing plants was partially responsible for their lower digestibility. Lowe (1981) reported the possibility of using the leaf—stem ratio of alfalfa to select genotypes which would have higher digesti- bility but no further research was conducted. Finally, there is no published research a) to document the relationship that exists between the leaf-stem ratio and morphological development of legumes horizontally and vertically on the stem and b) to establish the contribution of each morphological component: leaf and stem from various positions on the stem to the total leaves and total stems. METHODOLOGY Alfalfa (Medicago sativa L.) cultivar "Saranac" and birdsfoot trefoil (Lotus corniculatus L.) cultivar "Viking" were grown in the greenhouses from seed in polyethylene pots 25 cm in diameter and 23 cm deep with drainage holes in the bottom. Soil pH of the sterilized soil mixture was adjusted with limestone to 6.8. The plants were watered and fertilized as needed for optimum growth. Artificial light from cool white fluorescent tubes insured a minimum of 3230 lux at a level of 1.1 m above pot height with a 16 hours photoperiod. Temperature averaged 230C during the day and 160C at night. Four replications were used, each with six randomized treatments composed of 11 pots. Harvest was on the whole plant at appropriate matu- rity stages: early bud, 10% bloom and full bloom. The plants were cut about 1.5 cm from the crown. All the samples were frozen on dry ice and stored in a freezer at -200C until used. The number of nodes on each plant was counted, starting from the node of the first fully expanded leaf near the shoot apex. In experiment 1, plants were classified into ten groups of morphological development: 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28 nodes. Leaves and stems were separated on a piece of dry ice. The stem fraction included petioles and inflorescences wherever appropria- te. The plant parts of the whole plant were placed separately on alu— minum dishes and transfered into a cooler filled with dry ice. The frozen samples were lyophilized to remove the moisture inside the plant material, dried and placed in a dessicator. The dried sample of leaf and of stem were weighed. The ratio of leaf to stem of the whole plant was then determined on a dry weight basis. In experiment 11, plants with 18 nodes were selected to evaluate the rate of changes of the leaf—stem ratio according to position on the shoot and maturity stages within and between both legumes. In addition, this selected sampling was used to establish the distribution of leaf and stem according to vertical layers and the repartition of different leaf types as well as stem types and inflorescence along the shoot layers and inside of each layer. The selected plants represented the median groups of plant development that had a high frequency in a plant population dis- tribution at every maturity stage for both species. These plants were sectioned into three segments of six nodes each: top, middle and bottom. The vertical layer sampling procedure minimized variations due to uncontrollable experimental factors and insu- red the same relative maturity of each plant layer. These three layers were obtained by dividing the total nodes of the whole plant by three and starting to count from node of the first fully expanded leaf down the plant. The top, the middle and the bottom were, respectively, the first to sixth node, the seventh to twelfth node, and the thirteenth to eighteenth node. Branches made up of secondary-tertiary leaves and stems were cut, individually wrapped from the main shoot which represented the primary structure and placed in a freezer at -20°C. The various plant fractions (primary leaves, secondary-tertiary leaves, primary stems plus petioles, secondary—tertiary stems plus petioles, inflorescences) were separated on a piece of dry ice and placed separately into aluminum dishes that were transfered into a cooler of dry ice. The frozen samples were then lyophi- lized to remove the moisture inside of plant material. The dried material was removed from the lyophilizer, transfe- red into a dessicator and the dry weight was taken for each of the samples. Data were first reported as the leaf-stem ratio for each vertical layer. The leaf fraction was composed of primary, secondary-tertiary stem and in- florescences when appropriate. The petioles from the primary leaves and from the secondary-tertiary leaves were included in their corresponding stem fraction. Finally, the contribution of leaves and stems to the total plant leaves and stems was measured for each studied vertical layer. All repor- ted data are on a dry matter basis. The analysis of variance of all data in experiment I followed a 2 x 10 factorial. arrangement with two replications. In experiment 11 and III, a 2 x 2 x 3 factorial with four replications was used on each plant parts (leaf, stem) for the analysis of variance. A Duncan's Multiple Ran- ge test was used on data of both experiments in order to determine the dif- ference between the treatments. In addition, a t-test was used to deter- mine the difference among the various treatments between alfalfa and birds- foot trefoil. RESULTS AND DISCUSSION EXPERIMENT I Leaf-stem ratio vs studied variables The partition of the morphological components was affected diffe- rently by the level of plant development of each species (Table l). The leaf-stem ratio increased significantly for alfalfa but not for trefoil as the plant developed (Table 2). Under the experimental conditions in the greenhouses, the faster growth of alfalfa led to a highly developed alfal- fa aerial portion. Consequently, a greater variation in the partition of the leaf and stem occurred on alfalfa than on trefoil. Stages of maturity did not affect the leaf-stem ratio of alfal- fa but had a significant effect on trefoil (Table l). Leaf-stem vs maturity stages The leaf-stem ratio of trefoil declined significantly from early bud (0.68) to full bloom (0.47) (Table 3). The leaf-stem ratio decreased with age in plants with 18, 20, 22 nodes (Table 2). Younger plants with 10, 12, l4, 16 nodes had a higher ratio at the early bud at the two bloo- ming stages. Leaf-stem ratio vs plant development The mean leaf-stem ratio of both legumes at the two lowest groups of plant development(lO and 12 nodes) was lower than those of plants with the highest development (24, 26 and 28 nodes) (Table 2). Within the early bud stage, alfalfa with 26 nodes showed a leaf- stem ratio of 0.77, higher than those reported on plants with 12 to 16 no- des (Table 2). At 10% bloom alfalfa with 24 nodes had the higest leaf-stem ratio (0.88). Alfalfa in full flower had a higher leaf-stem ratio on plants 10 Table 1. Analysis of variance of the factors; maturity stages and plant development of both alfalfa and birdsfoot trefoil and the com- bined factor interactions on the leaf-stem ratio (Exp. 1). Sources Leaf-stem ratio Alfalfa Trefoil Maturity stages N.S. ** Plant development * N.S. Maturity stages x plant development N.S. N.S. The significance of factors was evaluated at 1 percent level and followed by ** and by * at 5 percent level and N.S. followed those which are not signifi- cant. 11 .umwu owns: vanguaaz 0.:oocsc 0:0 50 Ho>o~ acouuon 0 0:0 as acououuav haucouuuacwfiu up: ouo nouuoa mean 050 sua3 owuuo>o 0:» use «noun auuusuwa meow may easy“: mason» amouwoaosduoe on» >c< naaouw HoodonoznuoE naowun> as» 0:030 Ewan cu wood 00 cowuuuuun 0:» :« coauowuub .N oubmh 3‘ $6. 2: 26 < $6 Ba 26 22 35 a8 3.: 8.. $6 8 and a 3.0 a and 3.32;. a 00.0 a 00.0 a 00.0 a 00.0 c 00.0 a 00.0 a 50.0 a 00.0 a an.0 a an.0 ounuo>< a 50.0 a 00.0 a nn.0 an 00.0 an 00.0 no no.0 an 00.0 as 50.0 0 00.0 0 50.0 aooaa dash ~«00oua a 00.0 a 00.0 an 50.0 a 00.0 a 50.0 a 00.0 an 00.0 an 00.0 no nn.0 0 00.0 sooHA 50H 0 50.0 a 05.0 an 05.0 an 00.0 as «5.0 an 50.0 as 05.0 an 00.0 A H0.0 0. 00.0 030 Adams 0: 00.0 an 50.0 a 05.0 an 50.0 an 00.0 an 00.0 0 00.0 0 n0.0 0 50.0 0 H0.0 ouauu>< an 00.0 no H0.0 a 55.0 no 00.0 as 00.0 no n0.0 0a ~0.0 no H0.0 0 00.0 0 00.0 floods Hush uu~n0~< 0 n5.0 0 00.0 a 00.0 0 00.0 0 00.0 0 00.0 0 50.0 0 50.0 0 H0.0 0 00.0 30000 "ca an 05.0 n 55.0 an 05.0 an 05.0 an 05.0 no H5.0 0 00.0 0 00.0 0 00.0 an 00.0. 0:0 manna nouaun muuuaun: 05 05 05 55 05 0a 0a 0a «a 0A auquluauauqduuua. nouounm .AH .axmv nowauu auauauua acououuuv oousu um cassava can 00 12 . «« 50 coaqu00 mum Ho>mH ucwuuma H 050 um mason ,Imwwfin 0o305m uwnu mcmwe mnu 0cm ummulu wcu 5n mcov mum mmuumam :uon cmw3uwn mcomaumueoo .umou 0.000030 onu up Ho>oH unwouwa m mnu um ucmuwmwfiv hauamu«uacwfim uo: mum umuuma mEmm may nuqa momma mnu 00m wumuuma Hmu lemma 50 cacao mum mofioomm £000 00 mumc xaan msu you mmwmum xuausuma comaumn mcomwumaaoo ..ummu wwcmm oaaauaaz m.cmo::a onu 50 Ho>oH unmouoa m mnu um acouommfiv maucmowwficwfim uo: mum mouuwa meow mzu sags momma onu 00m muouuoa Hausa 50 csozm mum mmfiuwam oEmm mnu pow mowmum zuwuaums :oo3uon mcomfiu00500 ---- m mm.o m< Ho.o < mo.o mmmoo>< sm.o at u as.o we a sm.o «a m mo.o Huommoa mo.o a. a me.o «« m oe.o «a m Ha.o m0~m00< wmmum>< Eooam HHsm Eoon NOH 0:0 5Humm mmfiuomm .AH.0xmv owmum xuwusume comm um mmwomam oeswma mo Eoum cu mmma afiumu some one m «Hams 13 ratio (0.88). Alfalfa in full flower had a higher leaf-stem ratio on plants with 24 nodes than those with 10 and 12 nodes. Two hypothesis could be suggested to explain these results. Firstly, at the same ma- turity stage, as alfalfa increased its physiological age its leafiness enhanced following the development of numerous axillary buds. Secondly, the increasing leafiness of alfalfa with its advancing physiological age might be caused by the expansion of the existing secondary-tertiary leaves. EXPERIMENT II Leaf—stem ratio of plants with 18 nodes The leaf-stem ratio of plants with 18 nodes was affected by all factors studied (Table 4) and by the species x maturity stages interaction. Species The leaf-stem ratio was greater on alfalfa (0.66) than on trefoil (0.56) (Table 5), possibly reflecting the greater vigor of alfalfa under the experimental conditions. This statement was supported by the studies of Bjorkman and Holmgren (1963) and Rhykerd et a1 (1959). Bjorkman and Holmgren found that plants in a greenhouse showed particularities in their physiological as well as morphological responses to the low light intensi- ty. Rhykerd et a1 (1959) found that alfalfa and trefoil responded diffe— rently to given light intensities. Conversely, alfalfa showed a high leaf-stem ratio at low intensity but a lower ratio at high light inten— sities. They indicated that the lack of competiveness of trefoil could be associated with its low preportion of leaves under low light intensi- ties. 14 .uchHuacme Do: mum £0003 mwonu 0mzoaaow .m.z was Hm>mH ucwouma m um « 50 0am as 50 assoaaom 0am Hm>mH ucmuumm H um vmumsam>m mm: muouumm mo munmuamwfiwfim one «a «a .m.z muohma ucmaa x mowmum %u«u=ume x mmfiuwam «« «a a« muohma unmfla x mwwmum muwunumz as «« .m.z mumhma unmaa x mmwuonm .m.z .m.z .m.z mmwmum huwusume x mwfiommm as as «a muwhma uamam .m.z .m.z «« mowmum zufiusumz .m.z .m.z as mwfiummm msmum 00 coausnfiuumflo mm>mmH 00 cowusnfiuumfin oHumu Emuwlumma moanwaum> moousom .AHHH was HH .axmv Awav oven 00 “mass: oumwwwauouafi nuaa ucmHa 00 00050 onu macaw mEoum 06m mm>mma 00 sowusnfiuumww mnu so .8oum ou 0mmH 000mm mnu so maoauomuouca Hams» 00m mumxma udMH0 00w mommum augusuma .mmfiommm “muouomm mo oucmaum> mo mwmzama< .0 manna 15 .Hw>mH ucmouma H onu mm «« >0 was « >3 umsoHH00 mum Ho>ma unmouma m ocu um mummy Immwfic vmaonm umnu momma onu was ammqu man >0 mcov mum moaowdm nuon soosumn msomfiummsoo I .umou m.amusoa ozu >0 Hm>oH ucouuoa m onu um acmuomwfiv >Hucmoamwawfim uos mum nouuoa osmm ozu cuaz scams mnu 0cm muouuoa Hmu Ifimmo >0 csonm mum mmfiomam :uon mo oumw xaan on» How mmwmum >uHu=umE somzumn maomfiquEoo I .umou wwamm vanguasz m.smosso man >0 Hm>mH usmuuma m onu um acoumwmav >Hucmofimfiswfim uoc mum umuuoa 08mm mnu zufia momma mnu 00m muouuma Hamam >0 ozonm mum moaomam meow osu How mowmum >uau=uma comaumn msomfiumaaou I IIII m mm.0 m 00.0 0 00.0 mwmuo>< 00.0 r o mq.0 «a 0 00.0 «a m 50.0 afiowoua 00.0 a n m0.0 «« n 00.0 «a m H5.0 mMHMMH< mwmuo>< EOOHm aflam Eooam NOH 0:0 >Humm mofiuomm .AHH .axMV mmwmum >uwu=uma vowvsum Sumo um move: 00 Hogans mumfiwoeumucfi so saws mmaumdm oeawoa comm mo cauou EmumImmmH same one .0 manma 16 Maturity stages The leaf-stem ratio of alfalfa and trefoil decreased between early bud to 10% bloom (Table 4) but only trefoil showed a decline from 10% bloom (0.56) to full bloom (0.43). Most authors report a decline in the leaf-stem ratio of alfalfa with age due to the increase of steminess in the whole plant (Kiesselbach and Anderson 1926; Mac Donald 1946; Terry and Tilley 1964; Mo- wat et a1 1965). No information is available on the decrease of leaf-stem ratio of trefoil with increasing maturity but a similar explanation as for alfalfa is suggested. Vertical layers The leaf-stem ratio decreased from the top (1.39) to the bottom (0.25) on alfalfa and from 1.26 to 0.19 on trefoil (Fig. 1). The leaf—stem ratio declined for each species and for each maturity stage (Fig. 1). The increase in stem weight down the shoot was a major factor in the decline of the ratio along the shoot in earlier studies (Terry and Tilley 1964; Fuess and Tesar 1968; Smith 1970; Thom 1978). Falling leaves (Rhykerd et a1 1959; Verhagen et a1 1963; Taylor and Templeton 1966; Fuess and Tesar 1968; Carter and Scheaffer 1983) and small leaves in the shadow of the ca- nopy (Cooper and Qualls 1967) reduce the leaf fraction down the stem. Comparison of the leaf-stem ratio between species Full bloom alfalfa was 50% leafier with a leaf-stem ratio of 0.29 than trefoil (0.15) at the basal segment. In the upper layer, the leaf-stem ratio declined with maturity from 2.07 to 0.80 on alfalfa and 1.98 to 0.75 on birdsfoot trefoil (Fig. 1). Both legumes had, however, a similar ratio at the top shoot at every maturity stage. 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O. can >4¢mH museums H mnu um unmmeMfiv hauamuww .Hm>oH unmowma m use um ucwummmav haummofiuwcwwm moaumamuuoo s mN.0 + NN.0 + No.0 I mm.0 + 0N.0 + «« No.0 I s« No.0 + .>.m.m m0.0 I NH.0 + no.0 + HH.0 + m~.0 + HH.0 I H0.0 + QZQ>H mmamwad 00.0 + m0.0 I s Nq.0 I 0H.0 I HN.0 I «« 0m.o I as Ho.0 + mmfiumam no.0 + No.0 + ma.0 I 00.0 I 0H.0 + H0.0 + 00.0 + QZD>H Hsommte Beak“ Emoanm msksummumaow mavens umasowm> maknucwaaou wHHmo Hamzaommz maaummfiam mmahe mammHH .>.m.m mam Amazon «NV 020>H mmmH m>Huomammn ufimnu was mmfiomam m:o:a8:wma 50mm 00 mama a“ mucmcoasoo HmUHEOumsm msowum> was cmm3umn uanUMMMmoo cofiumamuuoo.0fl manme 5(5 the proportion of mesophyll cells in the leaf increased due to the ease of this material to be degraded and assimilated. STEMS Effect of species, maturity and vertical layers on anatomical components Several anatomical components of the cross section of the stem were statistically affected by the species. There was, however, no in- teraction between species and maturity stages and/or plant layers that si- gnificantly affected the percentage of the tissue types (Table 17). The percentage area of the epidermis, cortical parenchyma, in- terfascicular xylem, sclerenchyma and collenchyma were mainly affected by the species. In general, the stem of birdsfoot trefoil had relatively more epidermis, cortical parenchyma, sclerenchyma and interfascicular xy- lem than the stem of alfalfa (Table 17). In contrast, the area of collen- chyma in a stem was higher in alfalfa (3.3%) than in trefoil (0.7%). The larger cortical zone in trefoil stems compared to those in alfalfa may be due to the smallness stem diameter of trefoil (Table 10) or, less likely, as a need for a site for photosynthetic activity. Firstly, the smaller diameter of the trefoil than the alfalfa stem could have allowed a larger proportion of its cortical zone to be taken on the photomicrograph. A similar explanation could be apply to the larger amount of epidermis and interfascicular xylem in the trefoil than in the alfalfa stem. Secondly, the large amount of cortical parenchyma in the trefoil stem may be the site of some photosynthetic activity so as to compensate the lack of leafiness of trefoil as compared to that on a1- falfa (Chapter I) under the present experimental conditions. Table 17. 57 Mean area of anatomical components in stems of alfalfa and birdsfoot trefoil and analysis of variance of the main factors: species, maturity stages and plant layers on the percentage area of anatomical components (in plant with 18 nodes development). Tissue Types Species Factors Alfalfa Trefoil S M P (7.) (Z) Epidermis 5.0 ** 6.0 ** ** N.S. * Chlorenchyma 12.2 ** 18.1 ** ** N.S. N.S. Collenchyma 3.3 ** 0.7 ** ** N.S. N.S. Bundle sheath 3.0 2.9 N.S N.S. ** Vascular bundle 44.6 40.1 N.S N.S. ** Sclerenchyma 9.5 ** 12.3 ** * N.S N.S. Phloem 7.0 7.1 N.S N.S N.S Xylem 23.3 17.7 N.S N.S. ** Xylem parenchyma 4.8 3.0 N.S N.S. N.S. Xylem interfascicular 13.9 ** 17.9 ** ** N.S. * Pith parenchyma cells 18.1 13.6 N.S N.S. ** The significance of factors was evaluated at 1 percent level and followed by ** and by * at 5 percent level and N.S. followed those which are not significant. £58 Alfalfa and trefoil differed in their vascularization develop- ment of the stem. The ratio of xylemzphloem was 4:1 for alfalfa and 3:1 for trefoil. The above characteristics of the vascular system of the tre- foil stem would suggest a better adaptation of trefoil to dry periods than alfalfa. Thus, Nelson and Smith (1968) reported that trefoil dis- played a steady development during a dry period while alfalfa showed a decrease of development during a dry period while alfalfa showed a de- crease of development of new organs in similar conditions. However, some defects of trefoil, such as its slow growth and its susceptibility to lodge, could also be associated to its vascular system. Trefoil favors sclerenchyma for strenghtning the stem tissue in the cortex. The ratio of the amount of sclerenchyma to the amount of collenchyma in the stem averaged 12:1 in trefoil and 3:1 in alfalfa. In the alfalfa stem, the sclerenchyma were around the vascular vessels (separated of the collenchyma by the bundle sheath). The need of mechanical tissues was particularly important for alfalfa which had a larger stem as well as many vascular bundles. Thus, alfalfa stems averaged 0.133 cm in diameter and 16 vascular bundles while the trefoil stem had a mean diameter of 0.110 cm and nine vascu- lar bundles (Table 18). For both legumes, the number of vascular bund- les was positively correlated (p < 0.01) to the amount of sclerenchyma r:-90.6l and to the amount of collenchyma r=¢-0.71. Changes in stem tissue types in vertical layers and according to maturity The percentage of the epidermis, bundle sheath, interfascicular xylem, xylem, vascular bundle and parenchyma cells in the pith was sta— 59 tiscally affected by the stem position on the shoot but not by maturity (Table 17). Epidermis The mean percentage of epidermis area of the legume at both stages of maturity decreased from 6.2 to 5.0% down the shoot (Table 14). Stem diameter (Table 10) and the percentage of epidermis area (r:-0.75) and the number of vascular bundles and the percentage of epidermis area (r=-0.77) were negatively correlated (p<:0.01). It could then be hy- pothesized that the epidermal layer would become more elongated with thicker walls for strenthening the tissues as the internal structure of the stem increased in size. Reciprocally, the decrease of the epidermis area in the stem could result in its declining importance down the shoot compared to the area of some other tissue types such as vascular bundles. For instance, a significant negative association was found between the epidermis area and the vascular bundle area (r=-0.78, p < 0.01). The mean percentage of epidermis area in alfalfa declined from the upper (5.6%) to the median (4.8%) and basal stem (4.7%) (Fig. 11). The epidermis in trefoil gradually declined from the tOp (6.9%) to bot- tom (5.4%). The difference could be due to the variation in the influ- ence that exerted the number of vascular bundles (Table 18) on the a- mount of epidermis. Thus, there was a significant and negative asso- ciation of r:-0.53, p < 0.01 between the percentage of epidermis area and the number of vascular bundles on trefoil but not in alfalfa. The percentage of epidermis area along the shoot was constant in early bud trefoil and full bloom alfalfa, suggesting that both spe- .auuuuan use . so“: unouuua Hanan as can ”douomu you . so“: umuuwm Huufiawu as cases mun newsman sums canon: some sasa use mo uuoasa cassava wcosuuumaou .umOu mums: manuuasz u.:eo:=a ssu an ~m>0H unsousm n emu on “consumes no: one season seam one so“: mamas sea was magnum: :uzuus usuuoa Human A: van "usuouu casual summon amuuawu an amaze one common zuunsumfl 0:1 mush-H cesium; uncouuwalou W .nsmmum zuuusuma e=o«us> us Afiouomu uncumvnan vcm ounmuam uo ensue :q muahuvumo uo newusnuuueuv Houuuuo> < .AH onsmum Amman sandy souuon an Auto: :ro canvas u: Ammo: zuev ecu "h Ibex-H amuwuus> «552 some» a I h D .a h o. o .7 .7 9 9 G 9 .7 9 as 9 .7 .7 9 9 9 .7 .7 9 9 9 2. u.rrrnonr.run.rrvu..ru.om -2 ' 3 . a. u. n a v. u p m o ..a. v s u n M v n u a u u v a an- a FE on. -on of .2. on- .3 3453. zooms 5.5.. 200...... axe 0. can 553 as- .3 on- -on on. -e. (z) aaxs Isuoyzoas ssoxa parpnzs sq: J0 aSszuaoxsg 61 Table 18. Vertical distribution of the number of vascular bundles in stem cross section of alfalfa and birdsfoot trefoil at three maturity stages. Species Layers Maturity Stages Early Bud 10 2 Bloom Full Bloom Average Alfalfa Top 4 14 B (4th node) 15 ab 14 b 1 6 Middle 15 16 AB (9th node) 16 ab 16 ab ab Bottom 16 17 A (15th node) 31 ab 2 a __ ab _ Average 16 A 16 A 15 A 16 ** Trefoil Top 9 cde 8 e 8 de 8 D (4th node) Middle 10 ab 9 bed 10 abc 10 A (9th node) Bottom 11 a 10 abc 10 ab 10 A (15th node) Average 10 A 9 A 9 A 9 ** Average 13 A 12 A 12 A Comparisons between layers and maturity stages within each species are shown by small letters and the means with the same letter are not different at the 5 percent level by the Duncan's Multiple Range test. Comparisons between maturity stages and between layers within each species are shown by capital letter and the means with the same letter are not different at the 5 percent 1e- vel by the Duncan's test. Comparisons between both species (average) are done by the t-test and the means that shown difference at the 1 percent level are followed by ** . 62 cies may have either a differential process of aging of the shoot apical meristem or a differential response of the stem anatomy upon the shoot growth rate. Bundle sheath, parenchyma cells The combined data of both legumes showed that area of the bundle sheath and pith parenchyma cells area of the stem declined from top to mid- dle and, then remained, unchanged (Table 14). These results could be re- lated to the size of the vascular bundle which was influenced by the shoot age. As the vascular bundle became larger, the area of bundle sheath as well as of parenchyma cells in the pith became smaller (Fig. 12, 13). The area percentage of the bundle sheath was negatively correlated (p < 0.05) with the vascular bundle (r:-—0.67) and with the parenchyma cells in the pith and the vascular bundle area (r:-0.68). Interfascicular xylem, xylem and vascular bundles The percentage area of interfascicular xylem, xylem and vascular bundles of each legume increased from top to middle and then remained sta- ble toward the basal stem portion (Fig. 14, 15, 16), particularly for the xylem (Fig. 18). The percentage of the xylem and vascular bundle area seemed to be related to the rate of increase of the stem diameter. Association of the stem components with plant nutritive value The percentage of the cross sectional area of stem tissue types was related to their stem IVDMD. The percentage area of parenchyma cells in the pith, bundle sheath and epidermis were correlated with the stem IVDMD in both alfalfa and trefoil (Table 19). The chlorenchyma, collen- chyma and phloem of alfalfa were highly correlated with the IVDMD of the stem. 63 00. 0hr .euunuas uou . sag: auouuofi guess as was quuouu you . sou: nausea Hosanna an enema one awesome some easy“: some xgsa «so we such-H cassava escouuoasou .ueou muss: ouaauasz e.=sussa sea as dosed usuuuua n «so as massaccuv so: one usuuom some use sous asses «so use unusua- cusuws usuuou ”Hols as was _«oLouu susuus uuuuea Houuasu as :30:- ous some». huuusuel was amazon ssosusa moo-«usalou .aoweue meanness assuus> us «acuuuu uooLevuwn use suneuno mo ensue nu sneeze mucosa mo souusauuueuv “souuuo> < .NA shaman Ammo: zunav saunas "a Ammo: sumv campus A: Ammo: easy so» "a euoasa usuauuo> a u. h o c. h III III we no a. no no m. we we .a we on e. no w. .u no no a. we .a as we r. .c .s no my a. .6 e. .w .5 r. .w n. r. .2 a. n. .s I. my ”w my no I. m. my Anna"..»uuvvvuwuv.. n.... u 3 w0<¢w>< 20040 4.5... 20040 e? O. can >..¢(m 10p .0“ .0? .06 .00 105 r0. 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In alfalfa, a negative correlation occurred between the percentage of cross sectional area of vascular bundle, interfascicular xylem and the stem IVDMD. The previous data confirmed some the aspects of Shenk and Elliot (1971) who reported a negative influence of the stem percentage area of vascular bundles and xylem on the stem IVDMD of alfalfa. Besides the influence of the stem anatomy on the stem IVDMD, the level of intake digestible energy (RFV) of stems of both legume was highly correlated with the living tissues of epidermis, pith parenchyma cells and bundle sheath which change morphologically with the plant age and had a positive influence on the RFV of stems of both species (Table 19). The RFV of alfalfa was positively correlated (p < 0.05) with the percentage of cross sectional area of collenchyma (r:wr0.46), of chlo- renchyma (r: +0.41), and of phloem (r:-.O.4l). Conversely, the RFV of the trefoil stem was negatively corre- lated with the percentage of cross sectional area of supporting tissues such as vascular bundle (r=«-0.66, p < 0.01), xylem (r:-0.70, p < 0.01) and sclerenchyma (r=-0.40, p < 0.05). The RFV of the alfalfa stem was correlated (p < 0.01) with the percentage area of vascular bundle (r:— 0.56), of xylem (r=-0.69), and of interfascicular xylem (r=-0.54). 72 .eu4eu4e non . 444s enonno4 44ele 54 use 44onenn non . 4n43 nouno4 4en4aeu n4 ssose one eo4oeae soeo s4444s eneu 44:4 04» no eno>e4 soosno4 use-«nealoo .neou oases o4s4u4s: e.seussa 04n >4 4o>o4 usounom n us» be neonoun4u nos one nonuo4 osee use 4443 eseol 04» use en4en4e s44n4s nonuo4 44eIe >4 use 44ouonn s44n4s noouo4 4eu4meu >4 ssose one eoueue >n4nsnes use enoue4 soosuo4 esoe4neeiou .eomeue >u4nsuel eso4ne> ne 44oeonu nooneun44 use eu4eu4e no esoue s4 eshsusono4ue mo so4us44nue4u 4eo4nno> < .04 onsw4m Aouos 44n4v scuno4 "a Aouos snov o4uu4I ": Aouos 4440 men "a eno>e4 4eu4uno> < zoos. do... zooae 2. o. one 556 .3 .05 .0. (Z) sass tsuoruoas ssoaa parpnzs sq: Jo 3833033135 73 00.. 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RFV and mean level of feeding variables in forage of both alfalfa and birdsfoot trefoil (Exp. 1). Species Alfalfa Trefoil NDF 41.6 * 44.0 * Cellular content 58.4 * 56.0 * ADF 30.1 31.2 Hemicelluloses 11.7 12.0 p-Lignin 8.0 8.6 Cellulose 18.0 17.5 Wall crude protein 2.39 * 3.21 * R.F.V. 141.1 135.6 Comparisons between both species within each variable re- sult of t-test and the means that showed difference at 5 percent level are followed by * 88 .Hm>mH unmouoa H wnu um ufimummmap mauamofimacwwm :ofiumamuuoo «« .Hw>mH uamouwa m mnu um ucmummmfip kauamOHMHcmwm nowumamuuoo « ON.OI HH.OI HN.OI OH.O+ «%H¢.OI «*mm.o+ ¥¥mm.0l r mm.o+ .>.m.m Hm.OI *«mo.o+ HN.OI ¥«m¢.o+ «OM.OI «cm.o+ %« mQ.OI Amy GHmHOHQ HHQ3 wN.OI ««©N.O+ HH.O+ «mm.OI «mm.o+ « #m.¢l on mmOHSHHmU no.0: «smo.o+ «4um.0I «*5m.o+ « mm.O| Amy awawfigla ca.o+ ««oq.ou ¥«mq.o+ «« n¢.0l AqV mmmoaaaawofiawm ««w~.ou «awn.o+ «« oc.0| Amv ma< *«oo.fln ** so.o+ ANV unmuaou “maaaflmo we so.on AHV mnz Hfioumuuummflmma< ofiuwu Ase on Amv AqV Ame Ame AHV amumummmo mmfinmfiump mmsumam .H uaoefiummxm a“ .mmason pwfipsum cuon mo coop xasn mnu mo mucmouaumcoo HHm3 Haoo mnu wcosm mm Hams mm moanwfium> wafipoom msu pom owumu amumlmmwa mnu ammsumn ucoaofiwwwoo aofiumawuuoo .NN manna .Hu>u~ unmouoo H mzu um ucououuuv zuucmofiuucwum :ouumaouuou «a ~o>om unmouoa m us“ an accumuuqv >Hucuuuuucuum acuumaouuoo * NH.o+ mo.oI o~.oI ofl.oI a~.o+ «mm.c+ «mm.c I mq.o + Ame >mm m~.oI a~.o+ oe.oI «sm.o+ e~.oI q~.o + n~.o I any cacao»; “an: so.oI *«em.c+ o~.o+ «om.cI wom.o I «¢qe.o I Rev «moaaflfioo Ho.ou «ace.o+ emc.OI «mc.o + «oq.c I Amy cwcwuqna No.o+ ««~n.cI ««~n.c I ««oo.o I Ace mouoaafiaoUfiamz .«H~.cI ««~h.c + ««H~.o I any mn< «anm.o + ANV ucoucoo amusfiamo ««ma.o I a~v we: HaOWoue oo.o+ mm.OI so.o+ -.oI «m¢.oI aaq.o+ suq.o I «H.o I Amy >mm «oq.oI m~.o+ qq.oI -.oI H~.o+ -.o I mo.o I Rho csmuoua dam: ~«.OI ««o~.o+ ea.o+ an.oI an.o + o~.o I Ace umo~=~Huo a~.cI ««oe.o+ «as.oI «mq.c I -.o + Amv nonwsAIa ~c.c+ oe.oI c¢.o I o~.o I Ace momoaaaamUfiawx .aoo.oI «.om.o + an.c + Amv mo< «.oo.~ I ae.o I Amy acmuaou uaaasfimu ae.c + .AHV an: mcfiuCH< ouuou ahv on Any Age any ANV Adv smuquauo modamoum> mofiumam .H .axm :« maamwu vuuvauo sumo mo owouou cu aucoaufiumcoo Hum: Haoo osu woosu no Mama on monnoaua> o=~m> acupoou ago can soon ou mood uo aqua» ozu :oozuon ucuauwuumoo newowfiouuou .mN «Home 9() tion increased in trefoil and decreased in alfalfa (Table 24). Conver- sely, the cellular content of trefoil decreased and of alfalfa increased during the corresponding periods (Table 24). Two hypothesis could be pro- posed to explain the above results: a) the remobilization of nutrients through the inflorescences, in the case of trefoil, and b) the remobili- zation of nutrient through the leaf fraction. Alfalfa with 14, 16, 18, 20 and 28 nodes and trefoil ranging from 10 to 24 nodes showed similar trends of changes of NDF and cellular content with age (Table 25 and 26). Comparison between both legumes Early bud alfalfa with 16, 18 and 20 nodes had a greater NDF level and a lower cell solubles content than the corresponding stage of trefoil (Table 25, 26). At full bloom, trefoil had both a greater NDF level and lower cell solubles than alfalfa (Table 25 and 26). ALF The ADF fraction was not affected by the species, the maturi- ty and the morphological deve10pment (Table 20, 27). Hemicelluloses The level of hemicelluloses was affected by the stage of mor— phological development. Both species with high node numbers (24, 26 and 28 nodes) had lower hemicelluloses than in less deve10ped plants ( 24 nodes) (Table 28). Hemicelluloses were negatively correlated with the leaf-stem ratio of both species (r=-0.47, p < 0.01, Table 22), main- ly influenced by the trend in trefoil (r=-0.40, p < 0.01, Table 23). 91 4 >9 pmzoaaom mum Hm>mH ucoouma m may um mucouommwp vm3onm umsu momma wnu paw amoulu onu ha mcop mum mmwMum muauouwa :mmBuwn maomwuwnaoo 4 H.mmH 4 ~.mmH .>.m.m 4 hq.m 4 oo.~ cfimuoua mecca Hams H.mH a.oH mmoflsaamo 4 H.m 4 4.4 afiamfiaIa m.~4 0.44 mmmossflfimofismm 4 o.mm 4 m.m~ mq< 4 o.mm 4 H.wm ucmucou amazfiamo 4 H.04 4 $.44 maz Hfioumps 4 m.m44 4 w.om4 .>.m.m Hm.~ mm.~ :Hmuoua manna Ham: n.44 m.m4 mmofisflfloo H.m o.~ aficwfioIa m.HH o.~H mwmoasflamofiamm a.a~ 5.0m 49¢ 4 ~.am 4 o.mm ucwucou “masfifimo 4 m.oq 4 e.~s moz muHmuH< sooam Haas can sflumm mommum zuwuoumz mmHanum> mwflomam .AH.axmv huHHSums :ufi3 aflommuu pow mwammam mo mwmuow mo >hm msu paw m=Hm> wsapomm mo moanmaum> msoaum> mnu mo Auouums hub mo NV ucoucoo cams may a“ mowcmso .qm manna 92 .umou madam oaawuasz m.:mu:=o ago an Ho>oH unmouoa n u;u um ucououugv afiucmuauacwfiw no: mum nouuma oEmo o:u :uqa umouo>w any can ammuo muuuaume 05mm onu :«zuus mason» amoumoaonnuoe ozu >=< <.o.~q <.n.~q < e.~q < ~.ne < m.mc < a.nq < o.mq < m.~e < m.~e < H.Hq owmuo>< a n.ne m ~.mc w H.ne a n.4c u o.qq m m.ee a c.ec a ¢.cq a m.ec a e.n< omnum>< m m.mc pm “.44 no o.me pm «.04 no H.mc n o.hq n e.~c as a.cq nu H.h¢ m m.me eoofin Hash Hfiocous a ~.mc a c.44 m H.He a q.~c m a.o« m w.H« m q.Hc m e.~o a m.~< m c.~e can sfiuwm a w.~e m a.~q m ~.~e m H.~e a o.~¢ a m.~q a a.~q cm c.~a u m.om on o.wm owuuu>< a e.oq a o.~q an o.~e m m.~¢ can ~.Hc no o.Hc can m.oe unm ~.mm on o.mm o ~.wn soofln “Ham muficu~< vuom~.nq v ~.~c pop m.~< up ~.Hc no o.qq no m.nc a c.mq onu m.me up o.oe o m.wm van aauam momma» humusum: 2 ea 2 2 8 3 3 i 2 S 348% omwoum .aon>on .AH.mxmv hoods Haau van was hauuo um .auououu accumvuua van ouamufio goon mo mason» aaouono:auoa enouua> uzu mcoeu Auouuua muv mo NV maz mo Ho>o~ «so :« nowcnso .mN ofinmh 93 .uoou «mama 0454449: o.=uu==n 0&4 an 40>UH acouuoa m on» no acuuuuu4v saucmowuaswaa no: cum umuuoa mama «cu £443 ounuo>o usu can owuun huauauua mama usu casuaz mason» auoamoaosnuoa on» as< 4 4.44 < 4.44 < 4.44 < 4.44 4 4.44 < 4.44 4 4.44 < 4.44 4 4.44 4 4.44 4444444 4 4.44 4 4.44 4 4.44 4 4.44 4 4.44 4 4.44 4 4.44 4 4.44 4 4.44 4 4.44 4444444 4 4.44 44 4.44 44 4.44 44 4.44 44 4.44 4 4.44 4 4.44 44 4.44 44 4.44 44 4.44 E4444 4444 4444444 4 4.44 4 4.44 4 4.44 4 4.44 4 4.44 4 4.44 4 4.44 4 4.44 4 4.44 4 4.44 444 44444 44 4.44 44 4.44 44 4.44 44 4.44 4 4.44 4 4.44 4 4.44 44 4.44 44 4.44 4 4.44 4444444 444 4.44 4 4.44 44 4.44 4 4.44 444 4.44 44 4.44 444 4.44 444 4.44 44 4.44 4 4.44 44444 4444 4444444 44444.44 4 4.44 444 4.44 44 4.44 44 4.44 44 4.44 4 4.44 444 4.44 44 4.44 4 4.44 444 44444 oumoum 4444544: 4 44 44 44 44 44 44 44 44 44 44 444444 .4444444 44444 4 .44 .4444.aoo44 4444 44 444 444 44444 44 .4444444 444444444 444 4444444 4444 44 mason» HuoquHosnuoa uncauu> usu «scam acoucoo uu4344oo uo Ho>uu may :« nowcuso 44444 94 .unwu «man: manquaaz n.:nu:=a ozu ma Hu>uH ucouuua n way an ucuuuuuuv 444:404u4cw44 no: 444 nouuuu wean ozu :ua) owuuo>u use can «moan huausuua mama osu 64:44: museum unuumoaosnuoe can ac< < 4.~n < o.~n n< n.4m m< o.om n< w.m~ < c.4n < c.4n n< c.0n n< ~.cn m 4.o~ uwuuo>< a n.mm ~.Nn a 4.4m a ~.oM a q.a~ 4 «.mm a ~.Nn a 4.4m u n.4n a c.4n umuuo>< a O O O O a O O O O O O m mm m an a e mm a 4 4n 4 4n 4 ~ 4n n c an a 4 4n 4 m wm a a Na 50049 44:» Huouuuu nun: 4.4m a m.o~ a 4.w~ a n.4~ a N.om a 4.0n a 0.9m a «.0n n 4.o~ van 4444a an o.a~ w.4n a n.4n nu o.m~ an ~.on a n.4n an m.on an w.o~ 0: o.m~ o «.mw uwuuo>< cu o.m~ n.4m an 4.4M can w.a~ vuaaH.On can 4.4n mvu o.wN 0v «.mw mu o.m~ w ~.4N soon; Hush nuauu4< ulna 4.4m a 4.4m a 0.0m a n.0n a c.4n a w.~n a ~.4n a o.o~ a N.- can aauwm uuwnua auquaum: .AH .axmv BooHn Haau ya can can huuwu an .Huouuuu accuovu4n van numwudu :uoa mo museum anagwoaozauos.n:o«ua> mzu macaw hn< no Ho>- o£u :4 mowcnsu .NN 44444 95 .uomu owcwx vanguaaz n.6uucsa as» an Hu>o~ ucuuuua m wsu uu acououmuv xaucmuaudcmmm ac: mum nouuua 05mm wsu saw: mmuuv>u ozu van away» muuuauwa mama any :«zuut museum Hmuwwoaognuoe 03» xc< con.oH am.o 0¢.oH n< 0; H.o~ u o.a on D.HH o u.- an m.- o N.na an e.NH so m.~a u w.nH an ~.- owwuw>< O O O o I a O a O m 0 O O a O o a o H OH R a on c HH an e mm a n ma H «H n m Na on c mm a o ea n M NH 50 an ~H m ~«0uwuu sun: 0 m.o on m.o~ nu o.~H no o.HH a q.~H a n.- an o.- a a.- no o.~H van zfiuwm van o.o~ v H.oH cu o.oH can H.~H no; n.H~ a n.m~ an m.- on“ m.~H on ~.~H can c.- owwuu>< vu o.o~ v n.0H to o.o~ a o.- vu m.o~ an ~.- can ~.NH van H.HH vonmn.HH canaa.- Eooaa flush uuaaufi< In»: 0 w.m mv o.~H ova ~.HH won ¢.~H a N.¢H unw w.- an e.nm can n.- cop H.NH van zfiuau nmwaum >uuuauuz aw om cu Nu ON ma ca «a Na om mwwnum .mo~o>mn muwouaw .AH .axmv EooHn Hana us can van haunw um .HuOumuu accumvuua vcw uuamuao :uon mo museum Haufiwoaosauoe m:o«um> osu wcosaounofiaaaouaew: mo Ho>u~ any cu muwcmso .wN uflnaa £36 Permanganate-lignin (p-lignin) The level of p-lignin increased from early bud to full bloom, in most groups of plant development of trefoil (Table 29). In contrast, the p-lignin level generally remained unchanged in alfalfa with maturity. These results followed the changes in morphology, especially of the leaf to stem (Chapter I, Exp.I), that would affect the proportions of the va- rious types of cell wall present. At early bud, trefoil had a higher level of p-lignin in the wall of its plants with 12 and 18 nodes than in the wall of corresponding alfalfa (Table 29) but alfalfa with 20 and 26 nodes had a higher level of p-lignin than trefoil. At full bloom, alfalfa of nodal group 10, 12 and 16 contained less p-lignin than the corresponding group of birdsfoot trefoil (Table 29). Cellulose The cellulose level of the legumes was affected by the stage of development. Plants with the highest number of nodes had the lowest cellu- lose level (Table 31). Cellulose was negatively correlated to the leaf- stem ratio (r:- 0.34, p < 0.05, Table 22). Trefoil cellulose was the most affected by the proportion of plant parts (r=-0.64, p < 0.01, Table 23). These results indicated that the level of cellulose was generally close- ly influenced by the cellulose contained in the stem fraction. The leaves diluted the cellulose fraction of the total. Cell wall_protein The content of wall crude protein was influenced by the species. At every maturity stage, trefoil had a higher wall crude protein than al- 97’ .uouu macaw «HaHuHsz o.cmu:=o uzu an H0>~H unouuun m use an acouuuqu mHunuUHuHcmHm you can nauuuH can» mnu :uH3 uwuuo>c may vac owmun hUHuaqu use» may :HsuHa museum HoUHwOHosauoa 03» >c< < a w.o an n.» no a.w no e.w an H.m no n.w a H.oH n o.~ no c.m a ¢.oH EooHA HHau HHOWouH Inn: unm o.» a H.a on 0.5 o «.5 a H.a can o.w an w.m can o.n o M.“ van hHumm a n.w o a.m a a.» a ~.m a e.» a H.w u o.» a H.w a n.e a c.n umuuo>< nu m.» an o.m a c.m a ~.m no e.m on H.m on c.n up m.n u a.o on o.» EooHn HHsm uuHmuH< nun: a H.@ a H.» a ~.m m ~.m a H.w u 0.x a n.w a ~.c a m.m was mHuwm momma» zuHuaumz ooHuwam mm cw «N NN ON mH eH «H NH OH momuum .QOHo>oa .AH .axuv aooHa HHau an we. van sHuau “a HHoumuu uoouuuuaa can ouHccac ego» to nuaouu HuonoHosauoa msoHuw> ecu macaw :chHHla mo Hw>uH 0:» cH muwcmsu .mN mHan zHuchHMchHn yo: man nouuwH mean osu .uuou owcwm oHaHuHaz n.:nu:=n mag £uH3 omwu0>u uzu vac «noun auHuauna mama ozu an H0>¢H ucuuuon m usu an acuuouqu :HsuHD museum HuuHNOHonnuoe can ac< no o.n~ a m.n~ no ~.nH on< n.o~ on< ~.mH < «.mH non< as ~.cH u H.nH u n.mH an o.cH no c.5H a n.aH on ~.sH a. ~.oH an “.mH an “.mH omuuo>< u ~.oH u s.n~ up s.eH a a.oH can H.mH a s.o~ can c.oH no H.¢H an o.aH up“ H.wH aooaa Has“ HHOumHH ---- u a.sH u ~.s~ gnu N.NH can H.~H an ~.wH up n.nH can ~.~H an n.mH . a n.aH use sHuau a e.nH u o.nH no H.oH can c.mH an m.wH a n.c~ no c.aH can H.oH an n.aH was c.5H ouauo>< U OoWH up“ OohH U ¢oWH D” OoDH “up“ @omfi a Qamfi up” MoNH on“ OoNH OD“ GoNH Unw N00: EOOfinfl Hfiah “HHQHH< ---- u H.sH cu m.oa on «.5H can ~.oH a ~.HN no o.o~ one n.wH aw a.o~ can n.mH can sHuam («manquuuqmmmumz ow. oN s~ «N oN mH 0H «H NH cH aoaouam .AH .axmv sooHa HHau us can van >Hu¢u an HHOuouu accuoqup vac uuHuqu :uon mo unsoum HuonoHosauos usoHuu> may macaw ooOHaHHuu no Ho>oH as» aw muwcczo .on mHan .uaou omens «HaHuHaz u.:mo::a hHucaUHuchHm uo: mun umuuuH 06am ozu aqu owuuo>c onu vac owwuo auHuauua wean usu.>n Hm>uH acuuuoa m usu um acououqu usu :HsuHs mason» HuUHNOHosauos osu as< museum Huuwononnuoe oaouuu> OSu macaw ARV cHououa «capo HHwa no Hu>uH may cw mowcuzo .Hn wHamH < oe.n n< wo.n n< Hw.N a -.N n nn.~ a ~o.~ n ne.~ n ce.~ a mm.~ n no.~ owauo>< a om.n m nm.n a an.n a wH.n n a~.n a um.~ a oo.n a ow.~ u ~m.~ o «H.n awuuo>< w mm.c no m~.q on ¢¢.n a no.n van nq.m o ww.~ cvu oH.n u Hm.m ova co.n ow Ho.m EooHn HHam . 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The RFV and the mean levels (2 of dry matter) of feeding varia- bles in leaf and stem of alfalfa and birdsfoot trefoil (Exp. II). Plant parts Variables Species Alfalfa Trefoil Leaf NDF 16.4 ** 17.8 ** Cellular contents 83.6 ** 82.2 ** ADF 11.1 ** 12.7 ** Hemicelluloses 5.0 5.3 . p-Lignin 2.1 ** 4.2 ** Celluloses 6.9 * 6.1 * Wall crude protein 6.37 ** 10.30 ** R.F.V. 131.1 ** 135.1 ** Stem NDF 53.1 * 54.8 * Cellular contents 46.9 * 45.2 * ADF 36.9 ** 40.0 ** Hemicelluloses 16.5 15.4 p—Lignin 8.7 9.2 Celluloses 24.1 * 24.9 * Wall crude protein 1.96 ** 2.34 ** R.F.V. 88.3 90.1 Comparisons between both species are done by the t—test and the means that showed difference at 5 percent level are followed by * and by ** at the 1 percent level. 1()3 falfa (Table 21, 24). The levels of wall crude protein and lignin were correlated which would indicate the existence of a link between both cell wall components. The comparison of the wall crude protein between the various development groups of both species in the early bud stage showed higher level of nitrogen in almost all groups of trefoil than alfalfa (Table 31). In full bloom plants, trefoil with 20 or more nodes had a higher wall pro— tein fraction than alfalfa. The nitrogen in the cell wall seems to be highly influenced by the physiological age of the plant: the longer a plant delays in reaching a specific stage, the greater is its wall pro- tein. Relative Feeding Value The RFV of both species changed slightly with the stage of matu- rity but it was not affected by the stage of development as well as by the species (Table 24). EXPERIMENT II £23 Birdsfoot trefoil leaves had higher NDF (17.8%) than alfalfa (16.4%) Table 34. Trefoil leaves had a higher NDF content than alfalfa (Fig. 22) at most plant layers of every growth stage except that the ba- sal leaves of both early bud trefoil and alfalfa had a comparable NDF level. A possible association could exist between the NDF and the thickness of the epidermal layer and the thickness of the mesophyll cell wall. 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The RFV and the mean level of various feeding variables in leaf and in stem of alfalfa and birdsfoot trefoil at the two studied maturity stages (Exp. 11). Species Plant parts Variables Maturity Stages Early Bud Full Bloom Alfalfa Leaf NDF 16.8 16.0 Cellular content 83.2 84.0 ADF 11.4 10.8 Hemicelluloses S 1 4.9 pALignin 1.9 2.2 Cellulose 7.1 6.7 Wall crude protein 6 57 6.16 R.F.V. 132.3 * 129.9 * Stem NDF 54.9 ** 51.3 ** Cellular content 45.1 ** 48.7 ** ADF 38.2 * 35.7 * Hemicelluloses 16.9 16.1 p-Lignin 9.0 8.4 Cellulose 24.3 24.0 Wall crude protein 2.05 * 1.88 * R.F.V. 84.3 ** 92.5 ** Trefoil Leaf NDF 18.0 17.6 Cellular content 82.0 82.4 ADF 12.6 12.8 Hemicelluloses 5.8 ** 4.7 ** p-Lignin 4.4 3.9 Cellulose 5.9 6.3 Wall crude protein 10.46 10.15 R.F.V. 135.6 134.6 Stem NDF 54.3 55.3 Cellular content 45.7 44.7 ADF 40.1 40.0 Hemicelluloses 14.9 15.9 p-Lignin 9.2 9.3 Cellulose 24.2 ** 25.6 ** Wall crude protein 2.14 ** 2.54 ** R.F.V. 92.4 * 87.9 * Comparisons between maturity stages for each variable and for each plant part of each species are done by the t-test and the means that showed difference at the 5 percent are followed by * and by ** at the 1 percent level. 1(JO .uuHuuHu new . ..qu uuuuumH HHuIc an was 330..» new . 5H: .33.: H.323 .3 5.5:» a: uuHuoan soon 5an 32. 9:3 23 we who»: :33qu 2.5023500 .33 09.5— oHnHuHsz 3:35.: 0.: an Ho>oH anyone... n can an 330:2. ac: v.3 .533 as: 2: 5H: usual 23 can uuH-mHu 55H: ufluuoH HHS-u an E.- HHeuuu :HsuHI youuwH Haydn—mu .3 .59.» ohm sou-an 523:! can such-H .503qu neonHuualou .AHH .93.: .313: we woman: 95 u. :30: goo-«nun: was uuHauHa uo Eu: 5" .52 mo :oHusaHnuaHv Haunt; mu .3..— novo: :umHIMHV EOuuom um Ammo: LuNHns wHUmH: "x $50: .301: nah "a. mmmrj H m S h m S m S h_m I h 0H uncouon m ozu um uauuuuqu us: on: nouuuH slum «Au zuH: canal egg was IuHauHI cquH: nouuoH HHaln >3 can Haemouu cH:UH: uuuuuH HauHmnu an nabs. one woman» quususu van auoan cassava ocoanualou .HHH.axuv nowaua uansuQI can an HHOHouu unnunvan van um.nuHa soon we uuoH cH uaoueou uuHaHHou mo :oHu50HuuoHv HuuHuuo> on .aHm Hmwvo: :uaHunHv acuuon "n Hoots: zuNHluv ovaHs ".z Anovo: :u wqu ecu “P m¢m>¢q A «min—.3 douwch (HUSH; 4.0mm?— m ... p m a a m x » 0'1 91 ,. ”8 lllllllllllllllllll|||I|||||||l|||||||||||l|||||i m ""9 llll|l||IIIIIIIIIIIlllllllllllllllllllIllllllllllll v't'“ llllllllll|lllIIllllllll||l|lllllllllllllllllllll om.- EL m a 9 a w 8 a a a a a a a . in c ' n . c z z ' c z z I. I. . u o u c . . u . a B L C 9 C 0 C 9 9 0 l C O 9 . V M I I V V 3 I N m... M m 02- . a a a m0< EOOJD ...-3". 0.2.0 >J¢ n—1 on! mil 001 can aunuHs soon we lay. cH acouuou n-HaHHou mo :oHuanuunHv HHUHuuo> nu .mHm .wer llllllllllllllllllllllllll' “'0 IllIIII||||||l||IIIllllllllllllllllllllll -'M° IIIHll|Illll|||||lllll|||Ill|||||||||||l||l||||||||||IllIlllllll .uUHnuHu new . :uHI nuouuuH HHusu up was HHOuonu you . :uH: .unOu own-x anHUHax a.:uu::n osu ha Hu>0H unuuuun m 0:» 95»: .325 o.— use-Huang”. .AHH .axuv mow-us hanauul 0:» an HHOuonu anew-qun m0< h n 2 h az'ec[ ' O'll 2004a 443m 'L'LG I ("3534‘ 4.0mm”: 'G'LZ I h n I h Ch w 1 v . 9 T n a n 9 1 a Q can >4¢< IOOHG ...—D“. can >H¢ 5N .dwh Amoco: suaaumae ensues “a Agave: au~anae «Hues: u: Amoco: agenda and “a mumaaa a ("H—(“.12 ...Oumfl... 0 I h m I h u I ... D I .— < 300..” ...—DE 030 >J¢(w In— ..on In. r00 (I) am: 116 Hemicelluloses Hemicelluloses in the leaf cell wall were affected primarily by plant maturity. Leaf wall hemicelluloses in trefoil declined from 5.8% at early bud to 4.7% at full bloom (Table 37). Hemicellulose con- tent of alfalfa leaves remained near 5.0% over the two maturity stages. Hemicellulose level was higher in the basal leaves of full bloom alfal- fa than trefoil (Fig. 28). Hemicelluloses in the stem were affected by position on the shoot and by the interaction species x plant layers. The species and the stages of maturity were not determining factors in the content of hemicelluloses in the stem. Hemicellulose increased from 12.0% at the top to 17.5% at the middle and 18.1% at the bottom layer as an average species (Table 35). Hemicellulose in the upper stem was significantly lower than that of the two lower segments (p < 0.05). Hemicellulose levels in the stem were affected differently by species. In alfalfa, the level of hemicelluloses increased from 10.6% at the top to 21.3% at the bottom layer (Fig. 29). In trefoil, the highest level of hemicellulose was in the median segment (17.7%) while it was 15.0% in the basal and 13.5% in upper stem. The hemicellulose fraction between stem and leaf for the ave- rage of the species was 2:1 at the top, 3:1 at the middle, and 4:1 at the bottom layer. This partition progressed at an equal rate as the from top to bottom as the amount of supporting tissues such as the xylem and the sclerenchyma. 117 .suH-uHa uou . suH: auouuoH HHulo an vs. HHOuouu uOu . suHa uouuoH HuuHaou up cause oua uoHuoaa soso aHsuH: ouqv xHaa ogu we ouousH cooauoa.o=ooHu-aloo .u-ou oases oHAHust 0.:sucsn 0:» an Ho>oH unouuoa n onu an ueououqu use on: uouuoH old. osu zuHa canon 0:» van IuHauHI sH£UHa wouuoH HHula a: van HHOuouu cH:UH3 nouuoH HouHaou as sub:- oun sow-an huHusu-l vs. ouohoH sooauoa aaoaHuoalou .AHH.axmv nonsu- huHusuol oau an HHOuouu accuoqua vs- 815. .32. do on: 3 883:8?!— oo 83:353.. 3325 8 a: 382 52A: 832. 3 :32. 52-3 “5.3.... ”x 20.3: 5?: a8 a. 2:5 333., (“3534 4.93": <5< ...Ommc... .nshmapospos»oa»mlh o.“ m0<¢w>< EOOJG ...—Du— ODm >J¢oH uaoouoa n oau uu “aoaouqu no: on: nouuoH onus use :uH: nauol ozu van uuHauH- aHauH: nouuoH HH-Ia an van HHOuoau aHauHJ aouuoH HauHauu an asoao on: now-us auuuau-I vac anomaH aooiuoa oaonHu-alou .AHH .axmv mouuun hanauII can as HHOuouu uoounqun van auHauHa no soua aH oonoHaHHoquo: no aoHuanHuuoHv HauHuuo> .a~ .uHh Acne: gsma-nav season ”a Auuo: zu~H1aV waves: 1: ”one: :uo-Hv now ”a maua _ < 20040 1.4:“. 03m >J¢oH uaouuoa n oau an uaoaouqu uoa nun nouuoH olnn on» :uH: nanol oau van nanan aHauHa unuuoH HHnIn an van HHOuouu aHauHa uouuoH HnuHano an aaoan nun nounun huHuaunl van nuohnH aoosuoa naonHunaloo .HHH .axuv nomnun auHusunl can an HHOuoau uncunqun van nanan guns as unoH aH aHauHHua uo aoHuanuunHv HnuHuuo> On .uHm Amovoa aulenHv Ecuuoa "n Anovoa suNHluv ovaHl n: Anovoa :uOIHv any "a mmu»<4 H (“3534 (a 45...? maps...» ashes» exams» 00.0 00.0 S 0'.“ 2 (2) nIusI1-d 5 00.0 . § 00.0 3 3 00.“ _ m0<¢m>< 30040 ...—3“. 0:0 >a¢nH anoiuon naonHunalou .unou owanx oHaHuHax n.anua:a osu up Ho>oH uaooaoa n oau an uaououqu no: can houuoH olnn on» :an nanol oau van nanan aHsuH: nouuoH HHnln an van HHOuoau aH:uH> nouuoH Hnannu an aaozn oun nomnun auHaaunl van nuohnH,aooluon naonHunnlou .HHH .axmv HHOuouu unounvnHa van nanan soon we unoH aH uaouaou onoHnHHnu uo aoHuanHuunHv HnUHuuo> Nn . uHm Anovoa :umHIMHV acuuon "n Anovoa :uNHlnv ovaHl u: Anovoa auonHv any "H mmm><4 H<0thm> .5353. 3.95”: «SE: 5.01m": m I h m I h m I b m I b m I b n I h 06. so Nil INA v.“- .5 an- :3" 0.? 1 I ma! L L . . ..1 6.01 r n. .... 1. Ion J O I L a. 1 .n 1 V 1 .U II; I «at n n r u. . n. r 1 -«N . 9 . V . . .1 n to] 1 u. 46 0 Q a .. r m0<¢w>< IOOJDJHDu ODm>A¢5 gHafi> (“3534 4.0mm": 001 n01 99'“ lIIllllllllllllllllllllllIlllllllllllllllllllllllllll‘ M's: IllIllll|IlllIllllllllllllllllllllllllllF .nanan new . 59H: nuouuoH HHnIn an van HHOuouu new . auHa aouuoH HnuHanu an asoan van noHuoan aono aHauHa nunv xHan one no nuohnH annauoa naonHunalou .unnu nuanu oHaHust n.anoa:a man an HoanH uaoouoa n oau m0<¢m>< .HHH .axuv nounun huHununl can an anew unvaHa van nanan suns no noun aH onoHaHHou mo aoHuanuunHv HnoHuuo> an . (“35%; ...Oumch a c. h a. o u. h 4 top In— Al, Io“ .6 I8I,IL I ... ... n F n ... ...-a a O s "_r . 1.8 w . u r r v ... you can ...-D“. can zanw (1) 350101130 126 .nanan new . auH: nuouuoH HHnnn an van HHououu uou . auH: uouuoH HnuHanu an aaoan oun noHuoan aono aHauH) nunv anA nan no nunhnH aooauna naonHunanoo .unnu nuanu nHAHuHs: n.anua:a on» an HoooH announa n nan un uaououqu uoa nun nouuoH onnn nan 59H: nannl oau van nanan aHauH: uouuoH HHnIn up van HHOuouu aHsuHa unuunH HnuHano an asoan nun nounun huHuaunl van nuonnH announa nannHunanno .HHH .axmv nounun huHasunn one an HHOuoau uoounvaHa van nanan anon no unoH aH aHounaa ovaao HHna HHou on» we aoHuanuunHv HnuHuuo> an .uHm 32.2. .372 V 8:2. a 22.2. 52-: 02.3... a. $32. 5?: a8 a. «5:: .235? «54 3.9;...» 3.9.9.» 0 cc 5. O a. h D s. b. D c. h D c. hr 0 c. h 0.0 0.0 t t n c on V I w0<¢m>4 IOOJD ..de 030 >42¢m 0.0 0.0 (X) NIELOXJ 30083 11V0 1130 127 wall crude protein was found in the median level leaves of both alfalfa and trefoil. An increase (p‘< 0.05) in the level occurred in correspon— ding leaves of trefoil from early bud (7.77%) to full bloom (9.76%). The stem of trefoil had a higher level of cell wall protein (2.34%) than alfalfa (1.96%) probably due to its slow growth rate (Ta- ble 34). The alfalfa stem contained less crude protein in its cell wall at full bloom than at early bud (2.05%, Table 37) but the trefoil stem increased its cell wall crude protein as the plant matured (from 2.14 to 2.54%). A close and negative association existed between the propor— tion of stem and the crude protein in stem wall. The latter relationship corresponded, respectively, on alfalfa at r=-0.76, p < 0.01 and on tre- foil at r:- 0.47, p < 0.01, indicating that the increasing stem weight with age might have a diluting effect on the protein. The increase in stem weight may come from the enhancement of branches on alfalfa which diluted the wall protein level of the stem fraction or the enhancement of the wall thickeness on trefoil by accumulation of the structural compo: nets. The crude protein in the cell wall of a young stem segment was higher (2.65%) than in median (1.87%) and basal (1.90%) segments (Table 35). The level of crude protein in cell walls of leaves was four times higher than in stems for the two species average. The wall crude protein between stem and leaf was 1:3 at the top and 1:4 at the middle and at the bottom layer. The cell walls of alfalfa had a 1:3 ratio of stem to leaf crude protein while trefoil had a 1:4 ratio. 12!! Relative Feeding Value The RFV of leaves was affected by species and position on the shoot. The maturity stages and the combined factor interactions did not have any effect on the leaf RFV. The trefoil leaf had a similar RFV (135.1) as alfalfa (131.1, Table 34). The RFV of leaves of the two upper layers was similar but increased for the basal leaves (Table 35). A similar trend was noticed for each species at each maturity stage. except that the leaf RFV of ear- ly bud trefoil was stable from plant top to bottom (Fig. 36). The RFV of both species declined from an average 143.3 at the top to 41.4 at the bottom layer (Table 35). A similar decline in the stem RFV was noticed within each species at each maturity stage (Fig. 37). These results supported those of Terry and Tilley (1964) and Smith (1970). The upper stem of alfalfa had a greater RFV than of trefoil at corresponding layer at both maturity stages. Conversely, the basal stem of trefoil had a greater RFV than alfalfa, at both growth stages (Fig. 37). At the median segment, the stem RFV of alfalfa was greater than trefoil only at full bloom. 129 .nanan new . auHa nuouuoH HHnln ha van HHouoau new . :uH: aouuoH HnuHano an asoan oan noHuoan aono aHauH: nunv ana on» us nuohnH aooauna naonHunAIoo .unou ouanu oHaHanz n.anoa:a oau an HoboH uaoouoa n oau un uaoaouqu uoa oun nouuoH olnn on» auHa nanol oau van nanan aHauH:_uouunH HHnnn 53 van HHOVouu aHauH: nouuoH HnuHano an aaoan nun nownun huHuaunl van nuoanH annsuoa naonHunaloo .HHH .axuv nounun AUHusunl can an HHououu unnunqua van nanan anon «a noun aH aHouOua ovauo HHns HHoo no aoHuaaHuunHv HnuHuuo> nn .uHm Anovoa zumHlnHv IOquA “a Anovoa suNHIsv ovaHl-u: Anovoa aucIHv no» uh m¢mr $3534 gonna: b a a » “But-”a “amoeba away; mmmmmmmmmmm 1L” ..Ih nqdv - mmmwwm . ... WWW/”fl .... - WWW/fl” 3 .. HW/V/ , .3 r ...W7 W I...- u .- ... («1 M WI“ . / 8 m m .u ... .8 W .9 1‘“ 4... W w . . wm u w “WHY. J. no I n I runfifl" m [ n.4- - m .3. ‘10:. F.1d‘ ed- rod moacm: 300.3 .39. can 55:. (I) NIZIOIJ 30080 TTVH 1130 130 .nanan no“ . :uH> naouuoH HHnln an van HHOunuu new . auH: aouuoH HnuHanu an azoan nan noHuoan :uno aHauH: nunv ana «an no nuoanH anoiuoa naonHunalou . .unou unanm oHaHuHat n.anoa:n oz» an H0>0H uaouuoa n oau an uaoaomqu no: nan unuunH olnn nan auHi nannl ogu van nanan aHauH: unuunH HHnln an van HHOunau auauH: nouuoH HnuHanu an azoan nun nomnun auHuaunl van nun>nH aoosuoa naonHunaloo .AHH .axmv nomnun auHaaunl can an HHOuoau uocwnqua van nanan guns as oaHn> maHvoou o>HunHoa no oUHvaH unoH ozu aH nnuanao HnoHuun> .cn .th Anovoa sumHunHv Ibuuoa "a Anovoa zuNHunv ovaHI u: Anovoa :uoqu any «H mum><4 H < IOOJQJJDK ODD >J¢oH uaooaoa n nan an uaoaouuqv uoa nun aouuoH onnn oau saw: nannl oau van nanan aHAuH: nouuoH HHnnn an van HHOHouu aHauHJ aouuoH HnUHanu an asoan oan nonnun auHusunl van nuoanm anoauoa naonHunalno .HHH.anuv auHasunn mo nonnun can an HHouoau unaunvaHa van nanan ago; no oaHn> uaHvoou o>HunHoa noun ozu us nouanau HnuHuao> an .mHu Anovoa suothHv [canon "n Anovoaw—In~ ovaHl u: Anovoa :uoaHv may uh anarca A «55.: 5.9.2: . 333.1 5.935 0 s. a. n u. h o u. .h n .u h n c. h 0 .I h 0- _III 0 . .... .._.. on.- ..” n :3 .... an. . 9 l u u a ... fl. - .9 o n Agofil “W J r. I.!0¢ n m . n .o .. 0 . L _L _l .1 mm on? m... u .c .62 m M m .... m . r w u 33 monum>< 300.3 .33.. an: 5.2m can 301VA 0010333 3011'133 10 301001 132 REFERENCES Bailey, R.W., and M.J. Ulyatt. 1970. Pasture quality and ruminant nutri- tion. II. Carbohydrate and lignin composition of detergent extracted re- sidues from pasture grasses and legumes. N.Z.J. Agric. Res. 12: 591-604. Bailey, R.W., and D.I.H. Jones. 1971. Pasture quality and ruminant nutri- tion. III. Hydrolysis of ryegrass structural carbohydrates with carbohy- drases in relation to rumen digestion. N.Z.J. Agric. Res. 14: 847-857. Bailey, R.W. 1973. Structural carbohydrates. in G.W. Butler and R.W. Bailey (ed.). Academic Press. London pp. 157-211. Barnes, R.P. 1973. Laboratory methods of evaluating feeding value of her- bage. in G.W. Butler and R.W. Bailey (ed.). Chemistry and Biochemistry of herbage. Academic Press N.Y. pp. 179-214. Barnes, R.P., D.A. Rohweder, and N. Jorgensen. 1977. The proposed esta- blisment of hay standards. Proc. 34th Southern Pasture and Forage Crop Improvement Conference. Auburn, Alabama. April 12—14. pp. 120-128. Bhat, A.N., and B.R. Christie. 1975. Plant composition and in vitro di- gestibility in bromegrass genotypes. Crop Sci. 15: 656-679. Christian, K.R., D.B. Jones, and M. Freer. 1970. Digestibility and che- mical composition of fractions of lucerne during spring and summer. J. Agric. Sci. Camb. 75: 213-222. Coughman, J.F. 1960. The distribution of carbohydrates, nitrogen and lignin in soluble fractions of the stems and leaves of lucerne hay. J. Brit. Grassld. Soc. 15: 169-173. Daughtry, C.S.T., D.A. Holt, and V.L. Lechtenberg. 1978. Concentration, com- position and in vitro disappearance of hemicellulose in tall fescue and orchardgrass. Agron. J. 70: 550-554. Dehority, B.A. 1973. Hemicellulose degradation by rumen bacteria. Fed. Proc. 32: 1819. Goering, H.K., and P.J. Van Soest. 1970. Forage fiber analysis (apparatus, reagents, procedures and some applications). Agric. Handb. No. 379. U.S.D.A. A.R.S. Harkin, J.M. 1973. Lignin. In G.W. Butler and R.W. Bailey (ed.). Chemis- try and Biochemistry of Herbage. Academic Press. London. pp. 323-373. 133 Hirst, R.L., D.J. Mackenzie, and C.B. Wylam. 1959. Analytical studies on the carbohydrates of grasses and clovers. IX Changes in carbohydra— te composition during the growth of lucerne. J. Sci. Food Agric. 10: 19-26. Howarth, R.E., B.P. Goplen, A.C. Fesser, and S.A. Brandt. 1978. A possi- ble role for leaf cell rupture in legume pasture bloat. Crop Sci. 18: 129-133. Jarrige, R. 1963. The membrane constituents of forage plants. Ann. Biol. Anim. Biochem. Biophys. 3: 143-190. Jung, G.A., R.L. Reid, and J.A. Balasko. 1969. Studies on yield, manage— ment, persistence, and nutritive value of alfalfa in West Virginia. West VA. Agric. Exp. Stn. Bull. 581. pp. 1-80. Lees, G.L., R.E. Howarth, and B.P. Goplen. 1982. Morphological characte- ristics of leaves from some legume forages: relation to digestion and me- chanical strength. Can. J. Bot. 60: 2126-2132. Lopez, J.P., J.Q. Prestes and E. Magalhaes. 1966. The growth curve, con- tents of soluble and structural carbohydrates, lignin and protein and di- gestibility of Lotus corniculatus L. IX Int. Grassld. Congr. Brazil. 1: 851-857. Moir, R.W. 1982. In vitro gas production from plants fermented in rumen fluid: the influence of the plant cell wall. Lab. Pract. 31: 457-458. Smith, D. 1970. Influence of temperature on the yield and chemical com- position of five forage legume species. Agron. J. 62: 520-523. Sotola, J. 1933. The nutritive value of alfalfa leaves and stems. J. Agric. Res. 47: 919—945. Stobbs, T.H. 1973. The effect of plant structure on the intake of tro- pical pastures. I. Variation in the bite size of grazing cattle. Aust. J. Agric. Res. 24: 809-819. Terry, R.A. and J.M.A. Tilley. 1964. The digestibility of the leaves and stems of perennial ryegrass, coksfoot, timothy, tall fescue, lucerne and sainfoin, as measured by an in vitro procedure. J. Brit. Grassld. Soc. 19: 363-373. Theander, 0. and P. Xman. 1980. Chemical composition of some forages and various residues from feeding value determinations J. Sci. Food Agric. 31: 31.-37 n Tomlin, D.C., R.R. Jonhson and B.A. Dehority. 1965. Relationship of ligni- fication to in vitro cellulose digestibility of grasses and legumes. J. Anim. Sci. 24: 161-165. 134» Van Soest, P.J. 1963 (a). Use of detergents in the analysis of fibrous feeds. II. A rapid method for the determination of fiber and lignin. J. Assoc. Of.Agric. Chem. 46: 825-835. Van Soest, P.J. 1963 (b). Use of detergents in the analysis of fibrous feeds. I. Preparation of fibre residues of low nitrogen content. J. Assoc. Off. Agric. Chem. 46: 825- 829. Van Soest, P.J. 1964. Symposium on nutrition and forage and pastures: new chemical procedures for evaluating forages. J. Anim. Sci. 23: 838— 845. Van Soest, P.J. 1965. Symposium on factors influencing the voluntary intake of herbage by ruminants: voluntary intake in relation to chemical composition and digestibility. J. Anim. Sci. 24: 834-843. Van Soest, P.J., R.R. Wine, and L.A. Moore. 1966. Estimation of the true digestibility of forages by the in vitro digestion of cell walls. In Proc. 10th Int. Grassld. Congr. Helsinki pp. 438-441. Van Soest, P.J. 1967. Development of a comprehensive system of feed ana- lyses and its application to forages. J. Anim. Sci. 26: 119. Van Soest, P.J. 1973. Collaborative study of acid detergent fiber and lignin. J. Assoc. Off. Agric. Chem. 56: 781. Van Soest, P.J. 1975. Agriculture group symposium on the nutritive va- lue of plant fiber. J. Sci. Food Agric. 26: 1433-1438. Van Soest, P.J., and J. B. Robertson. 1980. Systems of analysis for evaluating fibrous feeds. in W.J. Pigden, C.C. Balch, and M. Graham (ed.) Standardization of analytical methodology for feeds. Proc. Int. Workshop, held in Ottawa, Canada 1979. Wilkinson, S.R., W.E. Adams, and W.A. Jackson. 1970. Chemical composition and in vitro digestibility of vertical layers of coastal bermudagrass (Cy- nodbn dbctylon L.) Agron. J. 62: 39—43. Wilman, D., M. Daly, A. Koocheki, and A.B. Iwoga. 1977. The effect of interval between harvests and nitrogen application on the proportion of lignified tissue in leaf cross-section in two perennial ryegrass varieties. J. Agric. Sci. 89: 53—63. Woodman, R.E., and R.E. Evans 1935. Nutritive value of lucerne IV. The leaf-stem ratio. J. Agric. Sci. 25: 578-597. CHAPTER IV DIGESTIBILITY STUDY OF ALFALFA AND BIDSFOOT TREFOIL 1136 ABSTRACT Alfalfa (Medicago sativa L.) and birdsfoot trefoil (Lotus corni- culatus L.),grown in a greenhouse, were collected at early bud and at full bloom to determine the variation of IVDMD among three groups of plant deve- lopment for each species and to compare the vertical and horizontal changes of IVDMD of each plant part. The IVDMD of the three groups (1: 10-14 nodes; 2: 18 nodes; 3: 24-28 nodes) within each growth stage did not differ at p < 0.05. Species and the stage of maturity did not have a significant effect on the forage IVDMD. Fermentation for 24 hours in the rumen was adequate to evaluate the IVDMD of both alfalfa and trefoil. Leaves and stems were collected from plants with 18 nodes. Leaf digestibility of alfalfa was higher than trefoil at every maturity stage. Alfalfa leaves was more digestible than trefoil at the two upper layers. Di- gestibility increased between 12 and 24 hours but was not affected by sta- ges of maturity and position on the stem. Stem digestibility was affected by the position on the shoot ra- ther than by species, maturity stages and fermentation time. The IVDMD of the stem decreased from plant top to bottom within alfalfa but trefoil showed some discrepencies in the pattern of changes of the stem IVDMD. 137 INTRODUCTION Feeding value of forage species for ruminants can be eva- luated by an in viva or in vitra rumen fermentation. The two sta- ge rumen fermentation in vitra proposed by Tilley and Terry (1963) and modified by both Goering and Van Soest (1970) and Marten and Bar- nes (1979) is considered the most reliable technique for the es- timation of digestibility in viva (Barnes 1966; Ademosum et al, 1968; Mayer et a1, 1971; Nelson et al, 1975; and Horton et al, 1980). For this reason the in vitra procedure of Tilley and Terry was chosen for the present study. The two stage technique in vitra measurement of forage diges- tibility attempts to estimate the digestible soluble fraction of fo- rage as well as the digestible fibrous fraction. The first stage involves substrate fermentation by rumen microorganisms incubated for several hours. A period of 24 hours of incubation is considered adequate to estimate the in viva digestibility of legumes (Hopson et al, 1963; Donefer 1970; Reid et al, 1960; and Chenost et al, 1966). The second stage is the digestion of the first stage resi- due with hydrochloric acid-pepsin for 48 hours. Marten and Barnes (1979) pointed out that this step was used to simulate the in vitra breakdown of feed and microbial protein by the digestible enzymes of the ruminant abomasum. Using Tilley-Terry's in vitra procedure, several authors reported a decrease in digestibility of the whole alfalfa plant and birdsfoot trefoil, with advancing maturity (Horrocks and Washko, 1238 1968; Barnes and Gordon, 1972; Hoveland and Monson, 1980; Wilman and Altimimi, 1984; Buxton et al, 1985). The latter trend was found to be related to the increase in the whole plants structural constituents ( Allinson et al, 1969). In addition, Walters et al (1967) suggested that aging plants showed less digestibility because the herbage con- tained a higher proportion of senescent and dead material. When the digestibility of alfalfa was compared to birdsfoot trefoil during ma- turation, Anderson et a1 (1973) reported a faster decline of alfalfa digestibility than trefoil. A larger canopy mass of trefoil over that of alfalfa in a late growth stage was suspected to be partially res- ponsible for the difference in digestibility between species. The high dry matter digestibility of leaf remained relative- ly constant during plant maturation whereas the stem digestibility decreased (Terry and Tilley, 1964; Mowat et a1, 1965; Chenost et al, 1970; Thom, 1978). Some authors reported that the digestibility of the plant parts, like the whole plants, frequently showed a differen- tial response in concentration among the species (Wilman and Altimi- mi, 1984; Buxton et al, 1985). According to Buxton et al, the diges- tibility of birdsfoot trefoil leaf was often significantly inferior to that of alfalfa. Conversely, the stem digestibility of trefoil was greater than alfalfa at a mature stage. Buxton et a1 (1985) related the difference in stem digestibility between trefoil and alfalfa to the greater amount of younger tissue in the stem fraction of trefoil. 1139 Within a particular forage species at a given stage of maturity, the digestibility of its plant parts may change vertically along the shoot. Terry and Tilley (1964), Christian et a1, (1970), Thom (1978), Wilman and Altimimi (1984), and Buxton et al, (1985) reported that the leaves of al- falfa showed little change in digestibility with their position on the shoot whereas the stem digestibility progressively declined from top to bottom. Little information is available on the vertical changes in diges- tibility of birdsfoot trefoil. The study of Buxton et al pointed out that birdsfoot trefoil seemed to follow a similar trend of digestibility as that of alfalfa. The purpose of the present study was to determine 1) if there was a variation in the digestibility of the dry matter of various seg— ments within the same species at the same growth stage, 2) the rate of digestibility of those segments with maturity, and 3) and compare the dry matter digestibility of segments of alfalfa and birdsfoot trefoil ver— tically down the shoot and with maturity. 14C) METHODOLOGY Two separate trials were performed on alfalfa and birdsfoot trefoil l) to evaluate the degree of variation in dry matter digestibi- lity between the segments of each species for the same stage of maturity and 2) to compare vertically and horizontally the dry matter digestibi- lity of each segment of both leguminous species. EXPERIMENT I The forage was collected at early bud and full bloom. The fo- rage was separated into three groups: a) group 1, plants with few nodes. Because of the limited amount of material for this group with 10 nodes, a subsample of equal weight was taken from each group of plants with 10, 12 and 14 nodes and pooled to constitute the sample; b) group 2, plants with an intermediate number of nodes (18); and c) group 3, plants with a high number of nodes. The limited amount of material among the plants with 24, 26 and 28 nodes led to a pool subsample similar to group one of the same species. The IVDMD test was done on the bulked sample. In vitra dry matter digestibility (IVDMD) was determined accor- ding to a modified two stage technique of Tilley and Terry (1963) amended by Marten and Barnes (1979). A 250 mg sample finely ground through a fi- ne screen (1 mm) of a cyclone mill and transferred into a 50 ml centri- fuge tubes. Twenty m1 of buffer was placed into the tubes to which was placed into the tubes to which was added 5 m1 of rumen fluid prepara- tion (as described in Chapter V). Incubation took place in a water bath with the temperature maintained at 390 C for 12, 24 and 48 hours. After each fermentation, the buffer inoculum solution was re- 141 placed by a pepsin-hydrochloride acid solution and incubated for 48 hours in a water bath maintained at 390 C. The residue was recovered into a ta- red filter paper Whatman no. 54. It represented the indigested dry matter. EXPERIMENT II The material was prepared as previously described in Chapter III. The period of fermentation were 12 and 24 hours. A 2 x 3 x 2 x 3 factorieal analysis of variance with two repli- cations was used for trial 2 of each plant part. A Duncan's Multiple Ran- ge test was used to compare the mean difference between treatments within species and maturity stages. A t-test was performed on the mean of each group studied to determine possible differences between species. 142 RESULTS AND DISCUSSION EXPERIMENT I Fermentation time was the only variable that statistically af— fected the forage IVDMD. Plant development, species and maturity stage did not affect the IVDMD of the material. In addition, no interaction of the combined factors was statistically significant at p<< 0.05 (Table 38). Fermentation time: The forage IVDMD of both species averaged 51% dry mat- ter, after a 12 hours fermentation period (Table 39). The rate of diges— tion of both legumes increased sharply between 12 and 24 hours of fermen- tation, from 51 to 64% of dry matter and then remained unchanged (67%) (Table 39). The change of IVDMD with the time of incubation was also noti- ced in each species at both early bud and full bloom stages. Thus, 48 hours of fermentation was generally not needed to estimate the IVDMD of both legumes. This decision was in full agreement with other studies such as Reid et al (1960), Hopson et a1 (1963), Chenost et a1 (1966) and Allinson et al (1969). Comparison between species: Alfalfa and birdsfoot trefoil averaged a comparable mean IVDMD with 61 and 57% dry matter, respectively (Table 39). A similarity in the forage digestibility of the whole plant alfal- fa and birdsfoot trefoil was also reported by Ingalls et a1 (1965), Sea- ney and Henson (1970) and Collins (1983). This experiment suggests that the digestibility of the whole plant was conditioned by the proportion of the stem fraction as well as of its digestibility, as reported by Ter- ry and Tilley (1964). Table 38.Analysis of variance mes in experiment 1 and 2. on the effects of factors species (S), maturity stages (M), plant development (P), plant layers (L) and fermentation time (F) on the digestibility of forage legu- Sources Experiment 1 Experiment 2 Leaf Stem Species (S) N.S. ** N.S. Stade of maturity (M) N.S. N.S. N.S. Plant development (P) N.S. - - Plant layers (L) - N.S. ** Fermentation time (F) ** ** N.S. S X M N.S. N.S. N.S. S X P N.S. - - S X L - N.S. N.S. S X F N.S. N.S. N.S. M X P N.S. - - M X L - N.S. N.S. M X F N.S. N.S. N.S P X F N.S. - - L X F - N.S. N.S. S X M X P N.S. - - S X M X L - N.S. N.S M X P X F N.S. - - M X L X F - N.S. N.S. S X M X P X F N.S. - - S X M X L X F - N.S. N.S The significance of factors was evaluated at 1 percent level and followed by ** and by * at 5 percent level and N.S. followed those which are not significant. 143 144 . i an vnaofigou nun ~n>nn uanouna n as“ an nuanunuuuv vnzosn unau manna nau van unnuuu nzu an naov nun nsfiu aowunuamsunw nsnn way an nnaunan :uon annaunn van munznfi uanaa annaunn maomuunaaoo I .ummu m.anoa:a may as ~n>o~ uanouna m man an uanunuuav aauanoumgawun uoa nun unuuna nsnn nau and: manna nag van nunuunfi anuaano x; a3oan nun zuuuaune uo nwnun nsnn may you mafia acuunuanaunm annzuna naonfiunaaou u .unnu nwanm nfiafiuaaz n.anoaaa man an Hn>oH uanuuna m nan an uanunuuav xuuanofimuawun uoa nun unuuna osnn nau auuz manna may van nunuuna Hanan an azoam nun .nnaonan aunn away“; nwnun unquauns nEnn may on van nnEHu acuunuanaunu auon an nun>n~ uanfia annauna naonuunasou I mm \ < mm. < mm. m.MN < mm. <.MM 9 mm. < mm. < mm. m.WN nwnun>< Ammlcmuemv n no n an a me n me n an « n a: n me n 00 a a Ca m naouo Amoco: may n me n co 3 me an an e an mm a Nm n co n no a me N aaouo afiownuh AqdnmauoHv n me n do a on n me n an a me n co n no a on H aaouo as \ < so < so 6 mm < he < No a em < no < so a mm owmum>< 1|. 1|. 1!. II. II. II .II II. II. Amnvoa mmlvmucwv n we on co o mm n Kc. n no a 9 mm n no n no a A mm m aaouu Annvoa mav an me an me o mm n no a n no 3 mm n cc n no u an N aaoau nuanu~< Amuse: «a-~a-0av on so a do u mm n no a on 3 mm n we n so A mm a macho no em NH an em NH we cm NH Amaaosv Anuaoav Anuaozv nwnun>< mega acaunuanEunm mafia aowunuanEunm eooam Haze ezm sauna nnwnum aumuaun: mnaouo nnuonam .H uamsqanaxn au .Huomnuu uoou Invuqa van nudnufin soon mo Eoofin ddau van van sauna aazuuz.mauu aofiunuansunm uanunmmuv nousu an uanan~n>nv uandn mo maaouw uanunumfiv uo Anza>uv zuuauauunnwwv unuunE >av oauva 2% a« nnwansu.om manna L45 In addition, the morphological composition may also exert an important influence on the digestibility of alfalfa and trefoil. The latter factor became very important in the nutritive value of the two legumes, since a difference in digestibility of leaves of both species has been demonstrated in Experiment II of the present chapter. Follo— wing this reasoning, it is understandable that full bloom alfalfa had a higher digestibility (65%) than full bloom trefoil (58%) (Table 39) because of its greater leaf-stem ratio as well as of its higher leaf di- gestibility. When the leafiness and the leaf digestibility are combi- ned to give superiority of one species over another, the feeding value elements of a forage counterbalanced the influence of the stem fraction upon the overall forage digestibility (Chapter 1, pp.13-18). EXPERIMENT II Leaf digestibility: The species as well as the fermentation period we- re the factors that statistically affected the level of leaf digestibi- lity. The other factors such as stages of maturity and leaf position on the shoot did not play any important role in the digestibility of the leguminous leaves. No interactions between the factors were found significant (Table 38). Species: Alfalfa averaged a leaf digestibility significantly greater (81%) than that of trefoil (73%) (Table 40). The latter trend between both alfalfa and trefoil occurred at each maturity stage (Table 40). Some chemical or anatomical features specific to each specie could be involved in the difference of leaf digestibility between alfalfa and birdsfoot trefoil. .~n>n~ uanouna m was an a an van «a x; vn3o_dou nun ~n>n~ uanuuna a was an noananuuuv vnzoan unau manna nag van unnqu nag >4 naov nan menu aoaunuansanu nEnn nau an nnfionan auon annzunn van nunand uan~a annzuoa aonuunneou I .ummu n.anoaan may >3 ~n>n~ uanouna m nag un uanunuuuv >~uanouuuamwn uoa nan banana nsnn nag sud: manna may van nanuun~ finuuano ha azozn nun zuuuauna mo nunun nann nay u0u mega acuunuaNEunu annauns acnqunasoo I .unnu nwanm manquda: n.anoa:a was an Hn>n~ canouna n may on uananmufiv aduanouuwawun uoa nun unuunm nann ms» and: manna nag van managed aanan >3 azoan nan .nnuonan zonn auauu3 nwnun xuuuauns nann was an van nnsuu aouunuanaunu auon an nunmna uanHa annauna naonaunaeou I 146 «« ma \ <.Wm a. m as «a < an a mo «« <.wm «« a so an ommum>< I... I II I Amnvoa waInmv m on a as « a Na 3 as m a“ a as season Amuse: Na-~o m on I a so « a on « a on an as a as maven: aaoemta Amuse: euav a ma « a as « a an a as a a a“ «« a so aoa «« an \ < mm 4* a ma «I c an m a“ «a < am *4 a an «« ommum>< Amnvoa m~Im~v an on o RN « on qw vo mm n an n ma souuom Annvoa -Imv nuanM#< n on « on aa a n Rm « v cu n on n mm n~vvax Amnvoa oIHv n mm a o m“ a an em vo a“ a n mm «a n NR nob «N Na «N Na «N Na Anuaozv Anuaozv Amuaozv nwnam>< nsau aoaunuansanm nsau anonuanean Eoofim dunk van >~unm nnunum >uuuaunz manana uanfia mnaonam .HH uansuunaxn a“ .neau aouunaansanu ozu an van mnwnum suaoaw ozu an ~‘ounuu uoounvuua van n-nu~n :uoa no acozn may aaov >-nUauun>.unn~ mo Anzo>~v xuudwpaumnwav anuune zav sigma :m a“ mnwanau.ov wanna 147' Jones and Lyttleton (1971), Howarth et a1 (1978) and Lees et a1 (1982) reported the occurrence of tannin vesicules in the mesophyll cells of birdsfoot trefoil while none of these structures were observed in the alfalfa leaf (Gutek et al, 1974; Sarkar and Howarth, 1976; Goplen and Howarth, 1977; and Rumbaugh 1979). Van Sumere et a1 (1975) and Sar— kar et a1 (1976) showed that condensed tannins inhibited cellulase and pectinase, limiting the leaf digestion by the rumen microorganisms. On the other hand, Howarth et al (1978) suggested that the mesophyll cells of a bloat-safe birdsfoot trefoil had cell walls with greater resistance to initial rupture by mechanical or rumen microrganisms than the meso- phyll cell walls of a bloat-causing alfalfa. Fermentation time: The leaf digestibility of both legumes was affected by the time of fermentation. A rumen fermentation of 12 hours gave a leaf IVDMD of 73% while 24 hours gave a IVDMD of 81%. Leaf digestibili- ty increased 8% for alfalfa and 15% for trefoil between 12 and 24 hours of fermentation (Table 40). Comparison of leaf digestibility between species: Some significant dif- ferences in the leaf IVDMD occurred between species. The mean IVDMD of the upper and median alfalfa leaves averaged 85 and 86% as compared to 75 and 76% of trefoil (Table 40). Alfalfa had a greater proportion of secondary-tertiary leaves than trefoil at the two upper layers at both growth stages (Table 40). These young leaves may have increased the digestibility of the alfalfa leaf of these two upper segments. The limited digestibility of the up- per layer of trefoil leaf might result from the presence of tannin in 148 the vesicules in the mesophyll cells. It was in early bud stage that the digestibility of the trefoil upper leaves was the most affected. Early bud trefoil leaves showed a gradual increase in di— gestibility from the top (71%) to the bottom of the plant (79%) (Ta- ble 40). Such an observation led to two hypothesis based on Cutler's (1978) potential function of tannin. The tannin in younger leaves might act as an ultraviolet light shield, preventing possible damage to the chloroplasts, or this polyphenolic compound could be regarded as an astringent agent that protects the young leaves from being eaten by insects. Median leaves of full bloom alfalfa had a higher IVDMD (87%) than those of trefoil (80%) (Table 40). Alfalfa had four times more easily degradable secondary-tertiary leaves than trefoil (Table 40). Stem digestibility: The IVDMD of the stem was affected by the position of the segment on the shoot. Other factors such as species, maturity stages and fermentation time did not have a statistical influence on the stem IVDMD (Table 38). Vertical layers: Alfalfa and trefoil declined from 75 to 48% in their stem IVDMD (24 hours) from top to bottom (Table 41), supporting the results of Terry and Tilley (1964) and Thom (1978) on alfalfa. They suggested that the decrease of stem IVDMD downward corresponded to the increase in structural material in the stem. In Chapter II (Table 14), the amount of supporting tissues such as sclerenchyma and xylem was si- gnificantly greater at the two lower layers (middle and bottom) than at the upper segments. The increased percentage of the above tissues down— ward in the shoot was more consistent in alfalfa than in trefoil. 149 . « >A vnzouaou nun ~n>n~ uanoana m nAu an noanunuufiv vnaoAn unAu manna nAA van unnqu 0A» »A naov nun mafia aofiunuanEAnu nsnn nAu an nnaonan AuOA annzunA van nanzvd uanaa annzunA naonwunasoo I .umwu n.anoa:: nAu aA ~n>n~ uanoana n nAu an uanunuuuv hnuanouuuaqu no: man unuuna nann 0:» Aug: manna nAu van nunuunu Anuuano >A azcan nun >uuuauna mo nunun nann nAu uOu naqu aouunuanaunm annzunA naomuunasoo I .uunu swans nmauuuax n.anoaao nAu >A ~n>n~ uanouna m 0AA an Aanunuuuv Anuanoauuawmn uoa nun unuuna nann 0AA Aug: manna nAu van nunuunu AAnan >A a3OAn nun .nnuonan aunn a3Auqa nmnun zuuuauna nann nAu an van nuauu aouunuanaunu AuOA an nun>n~ uanua annaunA naonuunaeoo I om \ < mm < mm. <.Im < mm 4 mm < mm omnuo>< Amnvoa wfiImfiv m an u on a we a on 0A Am a we season Annvoa NAINV o Am v mm An um oA mm oA Am oA mm navvqx Afiounue Ammvoa nIAV n no a A Go n no a An co a n «n A on may we \ < NM < mm < MM < WM < NM < mm nwnun>< Annvoa muInuv u me u on a An v we a an a on Bouuom Ammvoa NaIav A AA A on A on A on A no oA mm ndvvm: nuunua< Annvoa oImv n mm a n on n mm c n um a n om . n as ao9 cm NA «N NA cm «A Anuaosv Anuaozv AnuaoAv nwnan>< neg“ aofiunuansunm nEAA aofiunuanEana sooan Adam can sauna nnunum >uuuaunz nanan uan~m nnAUnam .Hu Aanawunaxn a“ .nequ aowunuanEAnu ozu an van.mnwnun Auzouw 03A un.-ounaA AoounvaAA van numnAAn Aqu no AooAn MAu a30v >-novupn> .Enun mo Anza>~v >AA_«AAunnw«v unuuns zav 919w: :w a“ nnwanAu .nq nHAnH 15C) The IVDMD (24 hours) of the upper stem of trefoil was greater (74 and 65%) than that of the basal stem (51 and 48%) at both stages and the median stem (56%) at the early bud stage (Table 41). Full bloom plants were similar in stem IVDMD at the top (65%) and middle (57%) layers. The occurrence of branches at the median and basal layers of early bud trefoil did not exert a positive influence in stem digestibili- ty of corresponding layers but the inflorescences affected the digestibi- lity at the upper and median layers of full bloom plants. Comparison of stem digestibility: Stem IVDMD of both legumes within each layer and each maturity stage was similar except in full bloom plants where the upper stem of alfalfa had a higher IVDMD (83%) than tre- foil (69%) (Table 41). Inflorescences composed 56% of the upper stem in alfalfa and 46% in trefoil (Table 42) which were found to have nutriti- ve value close to leaves (Woodman and Evans 1935). Comparison of the leaf and stem digestibility: With 24 hours fermenta- tion, leaves were 30% higher than stems in IVDMD (Table 41). The diffe- rence in digestibility between leaf and stem of both legumes increased down the shoot from a similar IVDMD at the top layer to a difference 40% in favor of the leaves at the middle layer and 67% at the bottom segment. Species reacted similarly except that the difference in IVDMD between leaf and stem was greater in alfalfa. 151 II NH o nwnun>< AA «A o nwnun>< Amnvoa malmav o o o aouuom Amuse: NHINV m H o navvwz Amnvoa oIHV am es 0 may Haonmaa oH AH o mwmum>< Amnvoa mHImav o o o Eouuom Amnvoa NHINV H o o navvfiz Amnvoa oIHV om mm 0 now nuanwa< ANV ANV ANV nwnun>< Eoon Hank Eoon NOH vam manna nnwnum mufiuaunz munzng mnfionam .AHH .axmv nwnum zuwuauna >un>n An Hfiomnuu uoomnuHA van annman AuOA ao unmna Aonn mo aowuonuw Enun Anuou nAu cu nuanonnuoamafi mo ANV aoHuaAHuuaoo .Nq nHAnH 152 REFERENCES Ademosum, A.A., B.R. Baumgardt, and J.M. Scholl. 1968. 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Hoveland (ed.) crop quality, storage and uti- lization. Chapter 6 Am. Soc. Agron. Madison. Howarth, R.E., B.P. Goplen, A.C. Fesser and S.A. Brandt. 1978. A possi- ble role for leaf cell rupture in legume pasture bloat. Crop Sci. 18: 129-133. Jones, W.T., and J.W. Lyttleton. 1971. Bloat in cattle: A survey of legume forages that do and do not produce bloat. N.Z. J. Agric. Res. 14: 101-107. Lees, G.L., R.E. Howarth and B.P. Goplen. 1982. Mbrphological characte- ristics of leaves from some legume forages: relation to digestion and mechanical strength. Can. J. Plant Sci. 60: 2126-2132. Marten, C.C., and R.F. Barnes. 1980. Prediction of energy digestibility of forages with in vitro rumen fermentation and fungal enzyme systems. In W.J. Pigden, C.C. Balch and‘M. Graham (ed.) Standardization of analy- tical methodology for feeds. Proc. Int. Werkshop held in Ottawa, Canada. 12-14. 154 Meyer, R.M., E.E. Bartley, F. Julius, L.R. Finees. 1971. Comparison of four in vitro methods for predicting in vitro digestibility of fo- rages. J. Anim. Sci. 32: 1030-1036. Mowat, D.N., R.S. Fulkerson, W.E. Tossell, and J.E. Winch. 1965. The In vitro digestibility and protein content of leaf and stem portions of forages. Can. J. Plant Sci. 45: 321-331. Nelson, B.D., C.R. Montgomery, P.E. Schilling and L. Mason. 1975. Ef- fects of fermentation time on in vitro / in vivo 'relationships. J. Dairy Sci. 59: 270-277. Reid, R.L., and al. 1959. Pasture quality as determined by in vitro and in vivo techniques. J. Anim. Sci. 18: 1537-1538. Rumbaugh, M.D. 1979. A search for condensed tannins in the genus Medicago. Agron. Abs.: 75. Sarkar, S.R., R.E. Howarth and B.P. Goplen. 1976. Condensed tannins in herbaceous legumes. Crop Sci. 16: 543-546. Terry, R.A. and J.M.A. Tilley. 1964. The digestibility of the leaves and stems of perennial ryegrass, cockfoot, timothy, tall fescue, lucerne and sainfoin, as measured by an in vitro procedure. J. Brit . Grassl. Soc. 19: 363-373. Tilley, J.M.A. and R.A. Terry. 1963. A two-stage technique for in_ vitro digestion of forage crops. J. Br. Grassl. Soc. 18: 104-111. Thom, E.R. 1978. Effect of stage of growth and season on yield of lucerne (Medicago sativa L.) and on vitro digestibility of the whole plant and its component parts. Proc. Agron. Soc. N.Z. 8: 43-46. Van Sumere, C.F., J. Albrecht, A. Dedonder, H. de Pooter and L. Fe. 1975. The chemistry and biochemistry of plant proteins In J.B. Harbone and C.F. Van Sumere (ed.) Academic Press. N.Y.: 245. Walters, R.J.K., G. Griffith, R. Hughes, D.I.H. Jones. 1967. Some fac- tors causing differences in digestibility of grasses measured by an in_ vitro method. J. Br. Grass. Soc. 22: 112-116. Wilman, D., and M.A.K. Altimimi. 1984. The in vitro digestibility and chemical composition of plant parts in white clover, red clover, and lucerne during primary growth. J. Sci. Food Agric. 35: 133-138. WOodman, R.E. and R.E. Evans. 1935. Nutritive value of lucerne IV. The leaf-stem ratio. J. Agric. Sci. 25: 578-597. CHAPTER V RUMINAL DEGRADATION OF LEAF AND STEM OF ALFALFA AND BIRDSFOOT TREFOIL BY ELECTRON MICROSCOPY 1565 ABSTRACT Leaves and stems were collected from alfalfa (Medicago sativa L.) and birdsfoot trefoil (Lotus corniculatus L.) at top, middle and bottom of of plants at early bud, 10% bloom and full bloom. Three aspects were stu- died: a) the ultrastructure of leaf and stem from various position along the shoot exposed to a ruminal fermentation; b) the pattern of degradation of the various tissue types of both legumes with maturity; c) the inter- relationships between the rumen bacteria and the various tissue types. Digestion of most tissue types in the leaf of both legumes was partial after 6 or 12 hours of fermentation. Mesophyll cells and the phloem showed visible degradation after 12 or 24 hours, at every position and at every maturity stage. Digestion of the leaf and stem of both legumes resulted in some bacterial eJents characteristized by a) the types of anatomical compo- nents attacked by the bacteria; b) the surface potential of the tissue; c) the types of microorganisms in the ruminal inoculum; d) the inter- relationships between bacteria on a particular substratum; and e) the interaction between the bacteria and the substratum . 157 INTRODUCTION The use of scanning electron microscope (SEM) has been a use- ful tool to explore the microanatomy of a specimen since it provides a highly magnified three dimensional image with a great depth of focus. This technique has been used to investigate rumen microorganisms degra- dation of structural components by Akin et al (1973) on tall fescue and coastal bermudagrass, Brazle et a1 (1979) on bluestem, Brazle and Harbers (1979) on alfalfa, Harbers and Thouvenelle (1980) on corn and sorghum, and Harbers et al (1981) on smooth bromegrass. Akin and Burdick (1975) characterized the differential degra- dation of various tissues in leaves during the ruminal digestion as fol- lows: the mesophyll cells and phloem are readily degraded, and lignified vascular tissues resist digestion. Consequently, the latter authors proposed that a forage with high amount of mesophyll and phloem would have a high digestibility. In the study of Brazle and Harbers (1979) on the digestion of alfalfa leaves, mesophyll cells were partially to completely degraded and vascular tissues were partially digested with undegraded external structure after 24 hours of fermentation. Alfalfa stems showed only a partial disappearance of a dense zone of cells beneath the cuticle, fol- lowing 24 hours of fermentation. After 48 hours of fermentation, the latter zone of cells beneath the cuticle completely disappeared. Most of vascular tissues as well as cuticle was still undegraded after 72 hours of fermentation. 15E! Akin et a1 (1977 a) reported that the digestion of the various anatomical components in leaves of coastal bermudagrass was affected by their position along the stem as well as by the growth stage. The meso— phyll cells were digested to a lesser extent at the bottom than at the top portion of the leaf sheath. However, the bottom leaf blades were di- gested as much as the tOp leaf blades. In a recent paper, Akin et a1 (1984) found in Panicum miZiaides and in Panicum Zaxum that the mesophyll cells in bottom lamina tended to be more readily degraded than those in the upper lamina (even after 48 hours of fermentation). The augmentation of lignification of parenchyma cells in stem limited their degradation with maturity on wheatgrass and bromegrass (Pig- den, 1953), and on coastal bermudagrass (Akin et al, 1977 b). In the process of digestion of the plant structural components, some interrelationship occurred between the rumen bacteria and the plant walls. Several authors (Akin and Amos, 1975, 1979; Akin, 1976 a, b, 1979; Amos and Akin, 1978; Harber and Thouvenelle, 1980; Brazle and Harbers, 1977) have reported attachement of some rumen microorganisms to plant tis- sues following observations with SEM. For instance, rumen bacteria like cocci appeared to need attachement to plant walls to degrade them (Akin et al, 1974; Akin and Amos, 1975). In addition, several bacilli also need to adhere to the substrate. Patterson et a1 (1975) found that the bacilli got attached to the substrate by using fibrous material surrounding the bacte- ria cell wall. Akin et a1 (1974) reported a change in the physical relation- 15S) ship between the rumen bacteria during the digestion and the plant tis- sue types. Thus, rumen bacteria appeared to adhere to the thick cell walls on the epidermis and of bundle sheath prior to degradation. However, bacteria attacking mesophyll cells and phloem showed no need of attachement (Akin et al, 1984). Rumen bacteria did not attach to the cuticle. The rumen bacteria favored three different ways to attack plant cell walls (Moore and Mott, 1973 and Collings, 1979). Both au- thors reported that rumen microorganisms attacked the plant cell wall by 1) invasion of the middle lamella of adjoining cells; 2) penetra- tion of the lumen of plant fibers; 3) invasion of the fractures which may result in the a) removal of the cuticle b) the mechanical breaks of fibers during the process of rumination, and c) the destruction of the internal structure leading to the collapse of the structure con- tributing to the increase of fractures. In the present study, the objectives were 1) to examine the ultrastructure of leaf and stem from various position along the shoot of both alfalfa and birdsfoot trefoil exposed to a ruminal fermentation; 2) to compare the pattern of degradation of the various tissue types of both legumes with maturity; and 3) to examine the interrelationship between the rumen bacteria and the various tissue types. 1,60 METHODOLOGY Alfalfa and birdsfoot trefoil were grown in the greenhouse as in Chapter 1. Samples were collected as in Chapter II. Fresh samples were sectioned in 3 mm lengths at the midpoint of the leaflet and sepa— rated in two equal portions so as to have one portion in buffer and one segment in the rumen inoculum. Stem specimens were cut in 3 mm lengths just above the selected node on the plants with 18 nodes (Chapter II) and separated in two equal segments for the same purpose previously men- tioned. The samples were placed on dry ice and stored in a deep freeze until used. Samples were allowed to reached room temperature progressively. Sections of specimens of each plant part were transfered into polyethyle— ne tubes (50 ml) containing Mc Dougall's buffer and rumen inoculum in a ratio of 1:4 and gassed with a stream of C02' A blank check was also used at each incubation time. Rumen fluid was collected from a fistula- ted Holstein cow fed twice daily on an alfalfa hay diet. Rumen fluid was collected, strained through four layers of cheesecloth and placed in a prewarmed thermos (39°C). The ruminal fluid was centrifuged once at 1000 r.p.m. for one minute to sediment foreign matter and protoza. The surpernantant was centrifuged at 5000 r.p.m. for 20 minutes. The resul- ting residue suspended in a warm phosphate-carbonate buffer (39°C) at pH 7.0 (as described by Cheng et al, 1955) served as a source of rumen bac- teria inoculum. After 6, 12, 18 and 24 hours of fermentation at 39°C, specimens were removed and fixed in 5% glutaraldehyde for several hours, washed 161 with a 0.1M cacodylate buffer at pH 7.0, and dehydrated in ethanol. Spe- cimens were then subjected to a critical point drying procedure, mounted on aluminum stubs, coated with about 300 X of gold in a film-vac sputter coater. Specimens were observed on a ISI-SUPER III SEM operated at 15 Kvolts. Photographs were on films negative-positive Polaroid 665. 162 RESULTS AND DISCUSSION .Leaf: Alfalfa and trefoil leaves from various positions on the stem showed partial degradation of the mesophyll cells and the phloem at the early incubation times. The epidermis and the vascular vessels were the site of bacterial activity but there was little visible evidence of de- gradation after 6 to 12 hours of fermentation (Fig. 38 and 39). With a 12 hours fermentation period, most of the above tissue types showed some degradation (Fig. 40 and 41). The epidermal layer of the adaxial side of the alfalfa leaf was attacked randomly by dense populations of cocci in cluster (Fig. 42 and 43). These bacteria had appendages that could cause them to adhe- re to the tissue (Fig. 44). Subsequently, some rod-shaped bacteria in- vaded the area of the cocci where the epidermal surface was eroded (Fig. 45). The degradation of epidermis cells appeared to occur after the rod-shaped bacteria were adhered on the tissue in a horizontal position (Fig. 45). The existence of ramifications that linked bacteria of the same species and/or of the different species together during digestion are shown in Fig. 45 and 46. The reasons for this type of association are unclear but it could be related to the need of the microorganism to be hold firmly to the tissue in order to be not easily removed. The degradation of mesophyll cells of alfalfa leaf seemed to involve, besides the bacteria, some other microorganisms such as fungi (Fig. 47), in support of the report of Lund (1974). The need of attach- ment to the tissue was not a requirement for all species of bacteria acting in the digestion. 163 Figure 38. Leaf of alfalfa incubated 12 hours in a ruminal solution. X400. Figure 39. Leaf of birdsfoot trefoil incubated 12 hours in ruminal solution. X400. 164 Figure 40. Leaf of alfalfa incubated 24 hours in a ruminal solution. X400. Figure 41. Leaf of birdsfoot trefoil incubated 24 hours in a ruminal solution. X400. 165 Figure 42. Colonies of cocci sp. eroding the epidermis of the alfalfa leaf after 12 hours of fermentation. X400. Figure 43. Magnified view of colonies of cocci sp. eroding the epidermis of the alfalfa leaf. The bacteria seemed to be linked together by some lateral appendages (arrow) (12 hours of fermentation). X5000. 166 54.42,;(3'I ‘ _ t ‘ 4+3, , . \J ". ’2’ .. 3)" f Figure 44. The stomate is a location on the leaf epidermis where bacteria can penetrate to degrade the internal tissues (12 hours of fermentation). X5000. Figure 45. Bacteria with rod shape as well as with round shape showed attachement to the epidermis surface. They ap- peared to have some appendages that linked them (arrow) (12 hours of fermentation). X20000. 167 Figure 46. Population of ruminal bacteria degrading the epidermal cells in an eroded area of an alfalfa leaf. Bacteria showed ramifi— cations between them which could be used to adhere to the tissue (24 hours of fermentation). X5000. Figure 47. Population of ruminal bacteria interspersed with fungi (mycellium; arrow) on mesophyll cells of alfalfa leaf (12 hours of fermentation). X5000. 165! Several species of rod-shaped bacteria were involved in the digestion of phloem tissue in alfalfa leaf (Fig. 48). The bacteria at- tacked the periphery of the tissue at an angle without visible attache- ment. Round-shaped bodies on the cell wall of rod-shaped bacteria were frequently attached to cell wall of the bacteria. Leaf vascular vessels such as xylem were attacked by the rod- shaped bacteria at the middle lamella (Fig. 49). The degradation of the middle lamella was at the periphery or at any exposed edges by bacteria. Consequently, the number of exposed edges that would be attacked by bac- teria (specially rod-shaped bacteria) would likely determine the degree of degradation of the tissue. This is confirmed to some extent by Akin and Amos (1975) on the leaf of coastal bermudagrass and Brazle et a1 (1979) on bluestem. Some cellulolytic cocci species preferred the inter- nal part of the xylem as location to degrade the cellulose (Fig. 49). Bacteria randomly attacked the epidermal layer on leaves of birdsfoot trefoil (Fig. 50). The bacterial population seemed to be lower than that on alfalfa leaves. Fimbriae bacteria showed preferen- tial growth on the epidermal layer of the trefoil leaf (Fig. 51). Mea- dows (1971) found that bacteria with pili adhered to surfaces having an electrostatic barrier. The high population of the bacteria on the leaf epidermis suggested that trefoil had a surface potential different than that on alfalfa. The digestion of trefoil mesophyll cells (Fig. 52) was initi- ally slower than that of alfalfa after 12 hours of fermentation. Little bacterial activity on the mesophyll cells of trefoil could explain this observation. However, the increase of the time of fermentation allowed Figure 48. Phloem in the main vein of an alfalfa leaf attacked by rod bacteria sp.. Several rod bacteria showed round shape bodies along their cell wall. These bodies had appendages to permit them to adhere to the rod bacteria (12 hours of fermentation). X5000. Figure 49. The main vein xylem of alfalfa leaf attacked by a den- se population of bacteria of rod shape at the periphery and at the middle lamella of the tissue (24 hours of fermenattion). X5000. 170 Figure 50. Bacterial population on the epidermis of birdsfoot trefoil, after 24 hours of fermentation in a ruminal solution. X5000. Figure 51. Fimbriae bacteria found in great numbers on the epidermis of the birdsfoot trefoil leaf (24 hours of fermenta— tion. X20000. 171 Figure 52. Mesophyll cells of birdsfoot trefoil leaf incubated 12 hours in a ruminal solution. X5000. Figure 53. Mesophyll cells of birdsfoot trefoil incubated 24 hours in a ruminal solution. X5000. 17:2 bacteria to adhere densely to the tissue to digest it (Fig. 53). The population of rod-shaped bacteria (Fig. 53) appeared to be more homogenous on mesophyll cells of trefoil than on of alfalfa Fig. 47). Very few ramifications linking bacteria together occurred on mesophyll cells of trefoil. The rod-shaped bacteria degraded the tissues along the cell wall of the microorganisms suggesting the occurrence of a slime around the bacteria cell wall containing hydrolitic enzymes. The latter structure also allowed the bacteria to firmly adhere on a hori- zontal position. The digestion of the phloem and the xylem in the trefoil leaf followed a similar pattern as previously mentioned for alfalfa (Fig. 48 and 49)- §£EEF The digestion of the stem of both legumes by the rumen bacteria followed a similar pattern as far as the degradation of the cor- tical parenchyma, phloem and at the partial degradation of the xylem, sclerenchyma and epidermis are concerned. The tissues in the upper stem of trefoil were digested to a greater extent (Fig. 54) than at the two lower stem segments (Fig. 55). Degradation of some tissue types in the stem of both legumes was noticed after 6 hours of fermentation. Thus, the parenchyma cells in cortex and the phloem started to be digested, as showed in the top stem of 10% bloom trefoil (Fig. 56). The digestion of both tissue types were almost complete after 24 hours of fermentation (Fig. 57). The vas- cular vessels, the sclerenchyma and the epidermis showed little visible changes in their degradation from 6 (Fig. 56) to 24 hours (Fig. 57) of incubation. This was also obvious for the other stem segments. 173 Figure 54. Birdsfoot trefoil stem collected at the top por- tion after a fermentation period of 24 hours in a ruminal so— lution. X400. Figure 55. Birdsfoot trefoil stem collected at the bottom portion, after a fermentation of 24 hours in a ruminal so- lution. X400. 174 Figure 56. Trefoil stem incubated 6 hours in the rumen— buffer solution. X400. Figure 57. Trefoil stem incubated 24 hours in a rumen- buffer solution. X400. 175 The stem epidermis, like the leaf epidermis, was randomly attac- ked by the bacteria. After 24 hours of fermentation, the stem epidermis was still intact (Fig. 57). The cortical parenchyma and the phloem were rapidly degraded. Twenty-four hours of fermentation was enough to bring the tissue diges- tion behind the depth of focus (Fig. 57). The rod-shaped bacteria adhe- red tightly to the tissues before starting the digestion (Fig. 58). The vascular vessels were not easy to degrade. They remained almost intact although a great bacterial activity was present during the 24 hours of fermentation period. Figure 59 shows bacteria of rod-shape attacking the middle lamella area of the upper stem trefoil. Some bac- teria were held on the xylem cell wall by an appendage occurring at one end of its body cell wall (arrow). At the other extremity of the bacteria (Fig. 59) an extracellular substance that would contain hydrolytic enzy- mes would digest the structural polysaccharides because of the changes in the depth of focus around this substance (corresponding to zones of degradation (arrow)). The above hydrolytic activity of the rod-shaped bacteria at the middle area of the cell wall could be needed so as to create breaches of the outer part of the cell walls. Consequently, the physical changes in the cell wall may allow cellulolytic bacteria to digest the cellulose layers located inside the cell wall (Fig. 60). The ease with which bac- teria would attack and cause breaches in the outer layer of the cell wall would determine, to some extent, the degree of digestion of the conducting water tissues. 176 Figure 58. Bacterial activity on phloem of alfalfa stem after 6 hours of fermentation. The rod shape bacteria showed lateral link with other microrganisms of round shape. X5000. Figure 59. Bacteria of rod shape sp. degraded (arrow) the middle lamella area of the xylem cell wall of trefoil upper stem (24 hours of fermentation). X10000. Figure 60. Cellulolytic bacteria attached to the internal wall of xylem in trefoil upper stem (24 hours of fermentation). X20000. The bacteria produced some bridging polymers that could be used to overcome the electrostatic repulsion barrier (arrow). 178 REFERENCES Akin, D.E., H.E. Amos, F.E. Barton II, and D. Burdick. 1973. Rumen micro- bial degradation of grass tissue, revealed by scanning electron microscopy. Agron. J. 65: 825-828. Akin, D.E., D. Burdick, and G.E. Michaela. 1974. Rumen bacterial interre- lationships with plant tissue during degradation revealed by transmission electron microscopy. Appl. Microbiol. 27: 1149-1156. Akin, D.E. and D. Burdick. 1975. Percentage of tissue types in tropical and temperate grass leaf blades and degradation of tissues by rumen micro- organisms. Crop. Sci. 15: 661-668. Akin, D.E. and H.E. 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The relation of lignin, cellulose, protein, starch, and ether extract to the "curing" of range grasses. Can. J. Agric. Sci. 33: 364-378. MICHIGAN STRTE UNIV. LIBRRRIES W"WI"IIIHIIW‘IIIHIIIIWIIHIIWII"WNWll 31293007025913