EFFECT OF ALKALI TREATMENT ON THE DIGESTIBILITY OF ...
54
EFFECT OF ALKALI TREATMENT ON THE DIGESTIBILITY OF MESQUITE by HUEI-HSUAN WENDY CHIU YANG, B.S. A THESIS IN FOOD AND NUTRITION Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE IN HOME ECONOMICS f August, 1976 TEXAS TECH LIBRARY
Transcript of EFFECT OF ALKALI TREATMENT ON THE DIGESTIBILITY OF ...
EFFECT OF ALKALI TREATMENT ON THE
DIGESTIBILITY OF MESQUITE
by
HUEI-HSUAN WENDY CHIU YANG, B.S.
A THESIS
IN
FOOD AND NUTRITION
Submitted to the Graduate Faculty of Texas Tech University in
Partial Fulfillment of the Requirements for
the Degree of
MASTER OF SCIENCE
IN
HOME ECONOMICS
f
August , 1976
TEXAS TECH LIBRARY
73
I\l0- ^j^ ACKNOWLEDGMENTS
cot The author wishes to express her sincere gratitude
and earnest appreciation to Dr. S. P. Yang for his guidance,
advice, patience, and encouragement in the development of
this project and for his assistance in the preparation of
this manuscript. Sincere appreciation is also extended to
Dr. Donald W. Thayer, Dr. Mary A. Kenney, Dr. Robert C.
Albin and Dr. Sujit Kumar Roy for their evaluations and
helpful suggestions.
The assistance of Mr. Harvey Harris, Miss Jayne
Krook, and Mr. David Bernanke in English composition and
proofreading is gratefully appreciated.
The author also wishes to express her sincere ap
preciation to her husband Huei-Hsiung Yang for his help in
the animal feeding experiment, for his encouragement, and
his devotion to the completion of this project.
11
TABLE OF CONTENTS
ACKNOWLEDGMENTS ii
LIST OF TABLES iv
LIST OF ILLUSTRATIONS vi
Chapter
I. INTRODUCTION 1
II. LITERATURE REVIEW 3
Factors Affecting Digestibility 3
The Effect of Alkali Treatment 4
Usage of Mesquite 5
III. MATERIAL AND METHOD 8
Mesquite 8
Preliminary Trial 8
Animal Trial 10
IV. RESULTS AND DISCUSSION 17
Preliminary Trial 17
Animal Feeding 28
V. SUMMARY AND CONCLUSIONS 42
LITERATURE CITED 44
111
LIST OF TABLES
Table Page
1. Composition of Rations 11
2. Chemical Composition of Roughages 29
3. Chemical Composition of Rations 30
4. Effect of Sodium Hydroxide Treatment on the Digestibility of.Rations Containing Mesquite 3 2
5. Analysis of Variance of Crude Protein Digestibilities of Cottonseed Hulls, and Raw or Treated Mesquite Rations . . . . 33
6. Duncan's New Multiple Range Test of Crude Protein Digestibilities of Cottonseed Hulls, and Raw or Treated Mesquite Rations 33
7. Analysis of Variance of Dry Matter Digestibilities of Cottonseed Hulls, and Raw or Treated Mesquite Rations . . . . 34
8. Analysis of Variance of Digestible Energy Digestibilities of Cottonseed Hulls, and Raw or Treated Mesquite Rations 34
9. Analysis of Variance of Organic Matter Digestibilities of Cottonseed Hulls, and Raw or Treated Mesquite Rations . . . . 35
10. Analysis of Variance of Nitrogen Retentions of Cottonseed Hulls, and Raw or Treated Mesquite Rations 36
11. Duncan's New Multiple Range Test of Nitrogen Retentions of Cottonseed Hulls, and Raw or Treated Mesquite Rations . . . . 36
12. Analysis of Variance of Hemicellulose Digestibilities of Cottonseed Hulls, and Raw or Treated Mesquite Rations . . . . 37
IV
V
13. Analysis of Variance of Cellulose Digestibilities of Cottonseed Hulls, and Raw or Treated Mesquite Rations 38
14. Duncan's New Multiple Range Test of Cellulose Digestibilities of Cottonseed Hulls, and Raw or Treated Mesquite Ration 38
15. Analysis of Variance of Lignin Digestibilities of Cottonseed Hulls, and Raw or Treated Mesquite Rations 40
16. Duncan's New Multiple Range Test of Lignin Digestibilities of Cottonseed Hulls, and Raw or Treated Mesquite Rations 40
LIST OF ILLUSTRATIONS
Figure Page
1. Separation of Fiber Fraction 14
2. Effect of Sodium Hydroxide Treatment with or Without Auteclaving on the Hemicellulose Content of Mesquite 18
3. Effect of Sodium Hydroxide Treatment with or Without Auteclaving on the Cellulose Content of Mesquite 19
4. Effect of Sodium Hydroxide Treatment with or Without Auteclaving on the Lignin Content of Mesquite 20
5. Effect of Sodium Hydroxide Treatment with or Without Auteclaving en the Dry Matter Disappearance (Rumen Digestibility) of Mesquite 23
6. Relationship Between Lignin Content of Mesquite and Dry Matter Disappearance (Rumen Digestibility) 24
7. Relationship Between Hemicellulose Content of Mesquite and Dry Matter Disappearance (Rumen Digestibility) 25
8. Relationship Between Cellulose Content of Mesquite and Dry Matter Disappearance (Rumen Digestibility) 26
VI
CHAPTER I
INTRODUCTION
In order to provide an adequate diet for feeding the
increasing world's population, many attempts have been made
to increase the global food supply. One of the novel ap
proaches is to produce animal feed from waste materials,
which in turn may relieve the grain being used by animals
for human consumption (1).
Mesquite (genus Presopis) infests about 70 million
acres of grassland in the United States (2). For many years
the control of mesquite has been a serious problem on many
farms and ranches throughout the Southwestern United States.
Mesquite is a possible source of bulk in rations for
ruminant animals when ether roughages are scarce and rela
tively high in price; it may prove to be a dependable source
of roughage during drought (2). However, at the present time,
the low digestibility has been the primary factor for limit
ing greater feed utilization of mesquite.
The effect of alkali treatment in improving the
digestibility of weeds or wheat straw have been reported by
many researchers. In 1921, Beckmann (3) reported that the
results of studies in which straw with 1.5% sodium hydroxide
solution produced a twofold increase in the amount of crude
fiber utilized by ruminant animals. Tarkow and Feist (4)
stated that the minimum ratio, 5 to 6 g of sodium hydroxide
per 100 g of wood, for the maximum digestibility appeared
to be independent of weed species. The iji vitro maximum
digestibility with aspen was about 50% and with red oak,
about 15%. Wilson and Pigden (5) determined that the alkali
treatment on wheat straw and poplar weed up to about 9%
caused marked increases in their iTi_ vitro digestibilities.
The present study was conducted to determine the
effect of alkali treatment on the digestibility and utili
zation of mesquite as a roughage for ruminant animals.
CHAPTER II
LITERATURE REVIEW
Interest was expressed in Germany at the turn of
the century, in the possibility of using wood as feedstuff.
Beckman (6) compared the feed value of wood with that of
straw by chemical analysis. In analyzing several different
woods for nitrogen, fat, starch, and ash he found their value
were much lower than those obtained from straw. Haberlandt
(7) discussed the possibility of using the starch, oil, and
in seme case the protein and glucose stored in sapwood as a
feed source. He found this wood, being highly indigestible,
had to be ground very fine before horses and cattle could
utilize it to any degree.
Bissell and Wier (8), using sheep and deer with
alfalfa hay as a control, tested the digestibility of chamise
(Adenostema fasticulatum) as sources of protein and energy.
They found chamise to be an inferior source of protein and
energy when compared with alfalfa.
Factors Affecting Digestibility
The relationship between the cellulose or lignin
content of feeds and their digestibility has been demon
strated by Crampton and Maynard (9). They found that the
digestibility of cellulose varied inversely with the lignin
content as orchard grass matured had been suggested by
Ely et. a]^. (10) . They also showed that the lignin in the
orchard grass had digestion coefficients between 7.5 and
19.8. Later, Sullivan (11) confirmed the findings of Ely
et al. and recorded in his work that depending upon the
feedstuff the coefficients of digestibility for cellulose
were between 56 - 89 and for lignin about 10. Tomlin e_t al.
(12) in_ vitro studies also found that the cellulose digestion
and lignin content of grasses and legumes were inversely
correlated.
The Effect of Alkali Treatment
Since the herbivores are unable to fully utilize the
carbohydrate of wood and wheat straw, many investigators
have studied the effects of various treatments en the di
gestibility of wood.
Beckmann (3) treated the wheat straw with dilute
sodium hydroxide, and improved its digestibility. However,
Lampila (13) treated wheat straw with alkali and found that
the nutritional value of the treated straw as an energy
source for sheep fed urea as the nitrogen source was not
improved when compared with the untreated sample. Stone
et al. (14) found that the cellulose in bagasse treated
with 1.5% sodium hydroxide was mere available to cellulolytic
enzymes than that in the untreated samples. Waiss et al.
(15) reported that the digestibility and nutritive value of
straw can be improved by treatment with aqueous ammonia at
room temperature in a confined system. Ground oat straw
(16) was treated with a 13.3% sodium hydroxide solution,
neutralized with 50% acetic acid, and then dried. The alkali
treatment resulted in a significant increase in energy avail
ability of the straw. Feist e_t a_l. (17) found that alkali
treatment improved the digestibility of hardwoods by rumen
microorganisms.
The Beckmann (3) process proved popular in small
scale farm operations. Due to the large volume of dilute
sodium hydroxide solutions required, the tedious washing
operation to remove residual alkali, and the losses of solu
ble nutrients caused by washing, this process has net been
used for large scale operations. Wilson and Pigden (5)
studied the modification of the chemical delignificatien
procedures with a view to their wider application in lew-
quality forage improvement scheme. They suggested that
treating 100 g wood by mixing with 6 g sodium hydroxide in
30 ml water without washing with water increased the in vitro
digestibility. This newer modification which tended to over
come the use of excess chemicals and water had been demon
strated to be as effective in increasing the digestibility
as the Beckmann older procedures.
Usage of Mesquite
In the early days of settlement of southwest the
mesquite was of great value to both men and animals as feed.
shelter, and land cover (18). However, as the number of
settlers increased and ranching and farming were widely
practiced, the mesquite became a problem. The methods for
control and possible utilization of mesquite are vitally
important at the present time (19).
In 1957, Deelin (20) fed a ration containing mes
quite meal to cattle en his Rio Vista ranch in Dimmit
County, Texas. He used a mixture of 454.2 kg of mesquite
meal, 227.2 kg of molasses, 90.9 kg of grain and 90.9 kg
of cottonseed meal as a maintenance ration for his herd.
Marion et. aj^. (2) fed yearling steers a ration containing
3.2 kg per head daily (79.20%) of ground mesquite weed.
The steers gained 1.0 kg per head daily in a 140 day feeding
trial. Similar steers receiving cottonseed hulls instead of
the mesquite meal gained 1.04 kg per head daily. The steers
fed the mesquite meal made a higher net return based on the
price of $10 per ten for ground wood and $18 per ton for
cottonseed hulls. This experiment was followed by a trial
feeding yearling steers a ration containing 5.56 kg ground
mesquite wood per head daily for 112 days. The steers made
an average daily gain of 1.15 kg compared to 1.23 kg for
those fed a sumac silage ration. The mesquite fed steers
had a 32<: per head marketing advantage ever the silage group
A chemical analysis shewed that the mesquite meal
had a higher fiber and calcium content (2). Yang (21) also
found that mesquite wood has a protein content higher than
that of cottonseed hulls (8.94% versus 4.10%, respectively).
Since the mesquite had a high protein content and lower cost,
it seemed highly desirable to study the effect of chemical
treatment on the delignification and the digestibility of
mesquite wood.
CHAPTER III
MATERIAL AND METHOD
The purpose of this study was to investigate the
effect of sodium hydroxide treatment on the utilization of
mesquite weed as a feedstuff for ruminant animals. After
determining the digestibility of sodium hydroxide treated
mesquite by the nylon bag technique (22, 23, 24), a sheep
feeding trial was conducted to determine the utilization of
sodium hydroxide treated mesquite as the sole source of
roughage in the ration.
Mesquite
The mesquite was collected from brush and weed con
trol experimental field of the Department of Range and Wild
life Management of Texas Tech University. The trees were
cut with a hand saw. The materials were dried in the field,
and then ground through a hammer mill equipped with a 3/8
inch screen.
Preliminary Trial
Chemical and Auteclaving Treatment
In preliminary trials, the effect of two alkali
treatments en mesquite digestion were compared. In first
trial, the mesquite was treated with 4, 8, 12, 16, 20, 24,
28, 32, 36, or 40 g of sodium hydroxide with 60 g water per
8
100 g of mesquite at room temperature for 24 hours; while
the second trial under same levels of sodium hydroxide
treated samples were auteclaved at 121° C and 15 psi for
2 hours. After these treatments, samples were neutralized
with 50% acetic acid to pH 7.
Rumen Digestibility
The effect of each treatment on the digestibility
of mesquite samples was determined by a nylon bag technique
(22, 23, 24). The bags were filled with 5 g of even dried
(105° C) alkali-treated mesquite sample and tied 8.9 cm
from the bottom of the bag with nylon fishline. Each bag
was tied at 2.5 cm intervals to an iron chain. This kept
the bags in the ventral sac of the rumen where the most rapid
.digestion occurred. All bags were incubated in a fistulated
steer maintained en alfalfa hay for 24 hours prior to the
test. Two replicates were used for each level of alkali
experiment. Upon removal from the steer the bags were washed
by dipping in water until the water remained clear. Prior
to filling and after incubation in the rumen the bags were
dried in an air oven at 105° C, cooled in a desiccator and
weighed with an analytical balance to 0.1 mg. The percent
age of dry matter disappearance was calculated by weight
difference. The mesquite samples were also determined for
cellulose, lignin, and hemicellulose contents according to
procedures of Goering (25). All the data were calculated
10
en the sodium acetate free basis. The asymptotic regres
sion analysis (31) was used to determine the relationship
between the predicted and observed values for dry matter
disappearance and hemicellulose, cellulose or lignin. The
simple linear regression (32) was used to test the relation
ship between fiber content of mesquite and the dry matter
disappearance.
Animal Trial
A. Selecting of Sodium Hydroxide Level for Animal Feeding Experiment
The sodium hydroxide level chosen for the animal
feeding trial was based on (1) economic justification (2)
limited quantities of sodium hydroxide and water for easy
operation and (3) excessive alkali removal either by wash
ing or, alternatively, if neutralized by acid, the resul
tant sodium levels must net overload the animals' abilities
to maintain acid-base equilibrium.
Gharib (38) reported that near maximum in. vitro
digestibility of poplar bark was obtained with 12 g sodium
hydroxide per 100 g poplar bark for a 1-day reaction time.
Singh ejt al . (39) stated that with the sodium hydroxide
treatment, rumen digestibility (dacron bag technique) in
creased linearly with increasing quantities of sodium
hydroxide up to a level of 10 g per 100 g of roughage (maize
cobs, wheat straw, paddy straw, sorghum stover, or sugar
caneteps) and levelled of thereafter.
11
After review of the findings cited above and the
in vivo nylon bag digestibility trial herein reported, it
was decided to prepare mesquite for in. vivo animal feeding
trial by treating with 12 g sodium hydroxide and 60 ml water
per 100 g of mesquite without auteclaving. This level of
sodium hydroxide treatment had the highest effect en the
rumen digestibility of mesquite.
B. Preparation of Ration
As shown in Table 1, the concentrates which included
molasses, protein supplement and sorghum were the same for
the three rations, but cottonseed hulls, raw mesquite and
treated mesquite were used as the roughage of Rations 1, 2,
3, respectively.
TABLE 1
COMPOSITION OF RATIONS
Cottonseed hulls Raw mesquite Treated mesquite Molasses Protein supplemen Rolled sorghum
t
Ration (%)
25
5 6 64
1 Ration (%)
25
5 6
64
2 Ration 3 (%)
25 5 6
64
^Contained 10% dehydrated alfalfa, 39.5% sorghum, 27.5% cottonseed meal, 5.1% calcium carbonate, 3.8% defluerinated rock phosphate, 8.5% urea, 5% salt, .05% trace mineral pre-mix, .05% sulfurfleur, .33% vitamin A (30,000 lU/g), and .14% chlortetracyline (50 g/lb).
12
C. Animal
Eight 7-menth-old feeder lambs were confined to
individual digestion stalls designed to facilitate the total
collection of feces and urine. During the 21-day feeding
period, the first 14 days were used for the adjustment to
the ration and stress, while the digestibilities of the
rations were measured during the last 7 days.
In the first feeding trial two lambs were assigned
to Ration 1 and three lambs each to Ration 2 and 3. The
second trial had three lambs receiving Ration 1, and two
lambs each receiving Ration 2 and 3. Total number of ani
mals for each experimental ration was five. The animals
randomly arranged in the first feeding trial were reassigned
in the second trial. All the animals were first fed the
prospective experimental rations twice daily en a limited
basis (1% of body weight daily). The daily allowance was
gradually increased during the first 7-day adjustment period
with the maximum consumption limited to 0.82 kg and 0.91 kg
per animal daily for first and second trial, respectively.
The rations were fed in equal portions twice daily and water
was available at all times.
D. Collection of Samples
Rations were sampled at each feeding. Total wet
feces were weighed daily. Composite samples were collected
daily and stored in a freezer. The total daily urine
13
excretion was diluted to a constant volume and stored in a
sample bag. Each day, 5 ml of concentrated hydrecholeric
acid was put in the urinary collecting container to prevent
microbial growth. After collection the bags were stored in
a refrigerator until analyzed.
E. Chemical Analysis
1. Moisture and Crude Protein
The moisture content of feed and feces was deter
mined gravimetrically by drying the samples in a forced air
oven at 105° C for 48 hours. Total crude protein content
was determined by a macre-Kjeldahl method (29) and ex
pressed as N X 6.25.
2. Gross Energy
Gross energy value of feed and feces were determined
by the use of a bomb calorimeter. Samples of air dried feed
and feces were made into small pellets ranging in size from
0.5 - 1.0 g. The pellets were weighed to the nearest tenth
of a milligram. The operation of the bomb calorimeter was
carried out in accordance with the recommended operating
procedures set forth by the manufacturer (26).
3. Fiber
The separation of each fiber fraction of the feed
and feces is illustrated in Figure 1. Air dried samples
were ground to pass through a 20 to 30 mesh (1 mm) sieve.
14
EXTRACT
1 CELL CONTENT
DRIED SAMPLE
i NEUTRAL DETERGENT
RESIDUE
1 CELL WALL
r HEMICELLULOSE
r CELLULOSE
r ACID DETERGENT
I 24 N H2SO4 (72%)
4,
X
I CELLULOSE LIGNIN, ASH
I
1 LIGNIN, ASH
I ASH (muffle furnace 550° C, 8 hours)
1 ASH LIGNIN
Fig. 1.—Separation of fiber fraction
15
Each fiber fraction of the sample was determined by the
procedure described in the Agriculture Handbook No. 379
(25) . The neutral detergent procedure for cell wall con
stituents is a rapid method for analyzing the total fiber
in feed and feces.
The acid detergent fiber (ADF) procedure provides
a rapid method for lignecellulose determination in feed
and feces. The ADF residue consists of cellulose, lignin,
cutin and acid-insoluble ash (mainly silica). Treatment
with 72% sulfuric acid dissolves the cellulose in ADF
residue. Ashing of the 72% sulfuric acid reaction residue
produces the value for crude lignin fraction including cutin.
4. Mineral
Five g of dried ground sample were ashed in a muffle
furnace at 600° C overnight. After the samples cooled to
room temperature, 10 ml of 6 N HCl was added and each sample
was brought to a gentle boil for 5 minutes. The aliquete
was then filtered through Whatman No. 1 filter paper and
diluted to 100 ml with water.
For determining the calcium content, 3 ml of ethylene-
diaminetetraacetate (EDTA) was added to the 1 ml of the ash
extract and the pH was adjusted to approximately 13 by adding
5 ml of 3 N KOH. The solution was titrated with 0.005 M
•"•The concentration was 5.58 g EDTA in 1000 ml water.
16
CaCl2 to the end point which is the persistence of fluores
cence by adding fluorescein-iminodiacetic acid indicator
(27) .
The phosphate content of the ash extract was deter
mined by measuring a 13 ml aliquot which contained 1 ml of
1/10 dilution of the ash extract, 9 ml of water, 1 ml of
0.064 M ammonium melybdate solution ((NH4)gMe7024 . 4 H2O),
1 ml of 7.5 N H2SO4 and 1 ml of ferrous sulfate solution at
650 m^ on a colorimeter Spectrenic 20 (28). The ferrous
sulfate solution was made as 8 g FeS04 . 7 H2O, and 2 ml of
7.5 N H2SO4 per 100 ml solution.
F. Digestibility
The apparent digestibility of each nutrient was
calculated as the difference between nutrient intake and
fecal excretion (30).
For example, the digestibility of protein (%) was
calculated as follows:
dry wt. of ^ % protein _ dry wt. of ^ % protein -.QQ ration eaten in ration feces voided in feces
dry wt. of ration eaten x % protein in ration
G. Statistical Analysis
Analysis of variance was used to test the differ
ences in digestibility of the experimental rations. Dun
can's New Multiple Range Test was used to determine the
heterogeneity-homogeneity of different means (33) .
CHAPTER IV
RESULTS AND DISCUSSION
Preliminary Trial
A. Fiber Content of Treated Mesquite
The effect of experimental conditions and concen
trations of sodium hydroxide en the composition of mesquite
wood is presented in Figures 2, 3, and 4.
The asymptotic regression analysis (31), Figure 2,
shows a reverse relationship between the sodium hydroxide
levels and the hemicellulose contents of the treated mes
quite wood. When the wood was treated with 4, 8, or 12 g
of sodium hydroxide per 100 g wood, the hemicellulose con
tent decreased sharply. The results also indicate that the
sodium hydroxide treatment at 121° C, 15 psi is mere effec
tive than that at room temperature in reducing the hemi
cellulose content of mesquite.
The relation of cellulose content to the sodium
hydroxide levels is shewn in Figure 3. The cellulose con
tent is inversely related to the sodium hydroxide concen
tration. The curve, Y = 26.88 + 17.05 x (0.97) , shews
that the greatest effect of the level of sodium hydroxide
on cellulose content is between 0 and 12 g of sodium hy
droxide per 100 g of wood; above 20 g of sodium hydroxide
there is less effect in decrease cellulose content.
17
24 .
o
X—X NaOH a Auteclaving Y « 1 0 4 0 4 913 x( 0-91)^
0—-o NaOH Y« 11.57+ 11.41 x( 095)^
21
18 -
^
0> CO o
a> o
15
•= 12 . E
9 -
8 16 24 32
g NaOH per 100 g Mesquite
18
^
Fig. 2.—Effect of sodium hydroxide treatment with or without auteclaving on the hemicellulose content of mesquite.
V \
19
o
o
8«
0> CO
o
9 o
45 .
40 .
3 35 -
30
0
r\
V ' \ \
- \
1
X
. -J
X X NaOHaAutoclaving Y • 26.88 +17.05x(0.97)^
0 0 NoOH Y • 33.96+ 10.51 x( 0.91)^
X
X
0
0
1 1
0 0 0
^ X
^ ^ X 0 ^ ^
^ x^^^Jk 0
1 1 1 1 1
e
X
1 8 16 24 32 g NaOH per 100 g Mesquite
40
Fig. 3.—Effect of sodium hydroxide treatment with or without auteclaving on the cellulose content of mesquite
20
25 X—X NaOHaAutoclaving Y« 0.58 + 2068x(0.97)^ 0- -O NaOH Y- 8.16+ IO.I8x( 0.95)^
g NoOH per 100 g Mesquite
Fig. 4.—Effect of sodium hydroxide treatment with or without auteclaving on the lignin content of mesquite.
21
In Figure 4, the asymptotic regression analysis
shows the relation of lignin content of the wood to the
sodium hydroxide levels used in the treatment. The curve,
Y = 8.16 + 10.18 X (0.95)^, shows that the lignin content
of the wood is decreased with increasing levels of sodium
hydroxide treatment. The curve, Y = 0.58 + 20.68 x (0.97)^,
which includes auteclaving treatment is very similar to the
one without the auteclaving treatment. With auteclaving
treatment the most effective level is between 4 and 12 g of
sodium hydroxide per 100 g of weed.
The present findings en the effects of sodium hy
droxide en the constituents of mesquite are very similar to
those reported by other investigators. Wangaard (34) found
that the most significant chemical changes in weed follow
ing treatment with alkali were reductions in the hemicellu
lose and lignin contents. According to Wise and Jahn (35) ,
caustic solutions at room temperature dissolved part of the
hemicellulose of wood and converted a portion of the lignin
to a soluble alkali-lignin complex. Millett et a]^. (36)
found that when aspen wood was treated with 6 g or mere of
sodium hydroxide per 100 g of wood that the yield was de
creased because the hemicellulose became soluble in the
sodium hydroxide solution. Ololade and Mowat (37) reported
that the barley straw treated with sodium hydroxide had
lower cell wall constituents, but similar in ADF, lignin,
and cellulose contents as untreated straw.
22
B. Rumen Digestibility
The data obtained in the rumen digestibility studies
are presented in Figure 5. Dry matter disappearance from a
nylon bag into the rumen of a fistulated steer was used to
estimate the rumen digestibility. The curve, Y = 60.40 -
57.91 X (0.98) , shows that the dry matter disappearance
values markedly increased with the level of alkali up to
28 g of sodium hydroxide per 100 g of wood beyond which
less improvement occurred. Data en the improvement of rumen
digestibility of mesquite samples indicated that the sodium
hydroxide process altered the structure of the mesquite,
allowed the ruminant enzymes to attack the nutrients of the
mesquite more easily, and thus increased the digestibility.
The curve, Y = 55.32 - 46.65 x (0.95)^, which in
cludes additional auteclaving treatment shews slightly
superior responses than the curve obtained with only the
sodium hydroxide treatment (Y = 60.40 - 57.91 x (0.98) ).
Wilson et. aj^. (5) showed similar results when wheat straw
and poplar weed were treated with increasing levels of
sodium hydroxide.
C. Relationship between Structure Change of Mesquite and Rumen Digestibility
The simple linear regression (32) was applied to
analyze the relationship between in, vivo rumen digestibility
and structure change of mesquite. The results are presented
in Figures 6 to 8.
23
60
S 5 0
)i a 0) o. o. a jn O 4 . «>
^ »
o
40
30
20
10
X—X NaOHaAutoclaving Y • 5 5 . 3 2 - 4 8 6 5 x (0.95) ^
0 0 NoOH Y « 6 0 . 4 0 - 5 7 9 1 x ( 0.98)^
8 16 24 32
g NaOH per 100 g Mesquite
Fig. 5.—Effect of sodium hydroxide treatment with or without auteclaving en the dry matter disappearance (rumen digestibility) of mesquite.
24
60
#
r 50 0> o
o o. CL a CO
4 0
:;:: 30
•S 20
10
\
\
X
X ^
\N \
xN
X X NaOHaAutocIa^ Y • 7 0 8 1 - 2.87 X
0 - - 0 NoOH Y « 7 5 . 6 2 - 4 . 0 3 X
x V X
\ ^
\ ^
\
0 0 Nq
0
XSv X
/ing
\
\
\
\
x X
10 15
Lignin, %
20
Fig. 6.—Relationship between lignin content of mesquite and dry matter disappearance (rumen digestibility)
25
X — X NaOH a Autoclaving Y •81.23- 3.73 X
0 — 0 NaOH Y • 72.15 - 2.92 X
Q 10 -
Hemicellulose, %
Fig. 7.—Relationship between hemicellulose content of mesquite and dry matter disappearance (rumen digestibility) .
26
X — X NaOHaAutoclaving Y « 150.63- 3.20 X
0 — 0 NaOH Y •116 .47 - 2.53 X
35 40 Cellulose, %
45
Fig. 8.—Relationship between cellulose content of mesquite and dry matter disappearance (rumen digestibility)
27
In Figure 6, the linear regression equation of
lignin content (X) and the dry matter disappearance (Y) is
Y = 75.62 - 4.03X. The coefficient of determination (r^)
is 0.94 and the linear relationship is significant at the
0.01 level. The re&u^lts show that as the lignin content
of the treated mesquite decreases, the dry matter dis-
appearance^ncreases accordingly. This suggests that the
utilization of mesquite by rumen organisms can be improved
by delignification by sodium hydroxide treatment. There is
a linear relation, Y = 70.81 - 2.87X (r^ = 0.91, p<0.01)
between the lignin content of mesquite treated by sodium
hydroxide with auteclaving and the dry matter disappearance
(Figure 6). The lignin content is negatively correlated
with the rumen digestibility.
The dry matter disappearance of treated mesquite
was markedly influenced by the amount of hemicellulose con
tent in the mesquite. In Figure 7, the linear relation
between hemicellulose (X) and dry matter disappearance (Y)
is Y = 72.15 - 2.92X (r^ = 0.78, p<0.01). The equation,
Y = 81.21 - 3.73X (r^ = 0.75, p<0.01), suggests that the
digestibility of sodium hydroxide - auteclaved mesquite is
also influenced by the hemicellulose content.
Tarkow et. aj^. (4) observed the chemical changes that
occurred when woods were treated with diluted sodium hy
droxide. T*he chemical reaction was a saponification of
esters of 4 - 0 - methyl-gluconic acid attached to xylan
28
chain which in turn increased the digestibility of hemi
cellulose.
The statistical analysis of the results presented
in Figure 8 indicates that as the cellulose content of
treated mesquite increases, the dry matter disappearance
decreases. The linear relation, Y = 116.47 - 2.53X (r^ =
0.69), was significant at the 0.01 level. Identical to
the mesquite treated with sodium hydroxide and autoclaving,
the cellulose-dry matter disappearance relationship was
significant at 0.01 level (Y = 150.63 - 3.20X, r^ = 0.86).
The disappearance of dry matter increases as the
cellulose content decreases. This decrease may be due to
the hydrolysis of cellulose in weed by the sodium hydroxide
and as a result increases cellulose digestibility.
2 The coefficient of determination (r ) also suggests
that the lignin content is mere highly correlated to the
rumen digestibility than the cellulose or hemicellulose.
Animal Feeding
A. Rations
The control and testing rations contained 25% of
roughage (cottonseed hulls or raw or treated mesquite).
The data on the chemical analysis of these roughages are
shewn in Table 2. Dry matter of the treated mesquite was
about 4% less than that of the raw mesquite or cottonseed
hulls. The nitrogen content of the treated mesquite was
X:
29
0.3% lower than the raw mesquite sample, and 0.39% higher
than the value of the cottonseed hulls. The ash content of
treated mesquite was 14% higher than the raw mesquite sam
ple or the cottonseed hulls. This suggests that the alkali
treatment increases the mineral content of the mesquite.
Gross energy content of the treated mesquite was lower than
the values of the raw mesquite and cottonseed hulls. The
lower caloric value of the treated mesquite sample is due
to decreased dry matter content and increased mineral con
tent.
TABLE 2
CHEMICAL COMPOSITION OF ROUGHAGES
v\
Dry matter %
Nitrogen %
Crude protein %
Ash %
Gross energy (Kcal/g)
Calcium %
Phosphorus %
Cellulose %
Hemicellulose %
Lignin %
Neutral detergent % fraction
Cottonseed hulls
91.09
0.95
5.94
3.00
4.41
0.38
0.07
37
31
13
82
Raw mesquite
94.90
1.34
8.37
3.29
4.44
3.29
0.05
37
27
18
84
Treated mesquite
90.78
1.04
6.50
17.31
4.01
2.78
0.05
32
14
12
60
\
30
Raw mesquite contained more calcium than did the
treated sample. There was no change on phosphorus content
during sodium hydroxide treatment. The cellulose, hemi
cellulose, lignin and non-digestible fiber values of the
treated mesquite were lower than the raw mesquite and the
cottonseed hulls samples. it demonstrates that the sodium
hydroxide treatment changes the fiber structure of mesquite
AS showed in Table 3, the differences of each component
among the three rations are markedly decreased due to the
adding of concentrate (Table 1).
TABLE 3
CHEMICAL COMPOSITION OF RATIONS
Nutrients
Dry Matter %
Nitrogen %
Crude protein %
Ash %
Calcium %
Phosphorus %
Gross energy (Kcal/g)
Hemicellulose %
Cellulose %
Lignin %
Neutral detergent fraction %
Cottonseed Hulls Ration
93.65
1.79
11.19
2.46
0.87
0.28
4.28
37
15
4
55
Raw Mesquite Ration
94.61
1.89
11.81
3.52
1.60
0.27
4.29
36
14
5
55
Treated Mesquite Ration
93.58
1.82
11.37
7.00
1.47
0.27
4.18
33
13
4
49
31
B. Digestibility by Animals
The apparent digestibilities of dry matter, crude
protein, digestible energy, organic matter, hemicellulose,
cellulose, lignin, and cell wall (neutral detergent frac
tion) are shown in Table 4. Data on the nitrogen balance
are also presented in the same table. Each nitrogen bal
ance is the average obtained from 5 lambs. The one-way
analysis of variance was used to test the differences in
digestibility values among the three experimental groups.
Duncan's New Multiple Range Test was used to determine which
of the treatment means were different from the others.
It was decided to test the hypothesis that there
were no significant differences among the experimental groups
in their effect en the digestibility of crude protein in the
rations. The treatments consisted of an equal number of
observations, and the results are shown in Table 5. iTie
"F" test indicates that a significant difference exists
among the crude protein digestion of the rations. The re
sults of the Multiple Range Test are shown in Table 6. It
indicates that there are no significant differences between
the raw and treated mesquite samples.
Among raw and treated mesquite and cottonseed hulls
rations, the differences of digestibilities of dry matter,
digestible energy, and organic matter were tested by the
analysis of variance. The results are presented in Tables
7, 8, and 9. They indicate that three groups were a
32
TABLE 4
EFFECT OF SODIUM HYDROXIDE TREATMENT ON THE DIGESTIBILITY OF RATIONS CONTAINING MESQUITE
Cottonseed Raw Mesquite Treated Hulls Group Group Mesquite Group
Apparent Digestibility of
Dry Matter
Crude Protein
Gross Energy
Organic Matter
Hemicellulose
Cellulose
Lignin
Neutral detergent fraction
%
82.05
65.56
80.67
82.92
89
70
23
79
%
80.01
74.47
78.72
81.00
87
59
41
75
%
81.42
74.23
80.06
82.29
90
59
29
77
Nitrogen Balance
N retained 49 44 38
33
TABLE 5
ANALYSIS OF VARIANCE OF CRUDE PROTEIN DIGESTIBILITIES OF COTTONSEED HULLS, AND RAW OR TREATED MESQUITE RATIONS
Source of Variation
Between Groups
Within Groups
Total
DF
2
12
14
Sum of Squares
257.40
313.45
570.85
Mean Square
128.70
26.12
F Value
4.93*
Significant (p < 0.05)
TABLE 6
DUNCAN'S NEW MULTIPLE RANGE TEST OF CRUDE PROTEIN DIGESTIBILITIES OF COTTONSEED HULLS, AND
RAW OR TREATED MESQUITE RATIONS
Treatment
Cottonseed Hulls Group
Raw Mesquite Group
Treated Mesquite Group
Mean
65.56*
74.47'
74.23
Means with the same superscript were not significantly
different from each ether (p<0.05).
34
TABLE 7
ANALYSIS OF VARIANCE OF DRY MATTER DIGESTIBILITIES OF COTTONSEED HULLS, AND RAW OR
TREATED MESQUITE RATIONS
Source of Variation
Between Groups
Within Groups
Total
DF
2
12
14
Sum of Squares
10.84
76.21
87.05
Mean Square
5.42
6.35
F Value
0.85
TABLE 8
ANALYSIS OF VARIANCE OF DIGESTIBLE ENERGY DIGESTIBILITIES OF COTTONSEED HULLS, AND RAW OR TREATED MESQUITE RATIONS
Source of Variation
Between Groups
Within Groups
Total
DF
2
12
14
Sum of Squares
9.99
101.84
111.83
Mean Square
4.99
8.49
F Value
0.59
35
TABLE 9
ANALYSIS OF VARIANCE OF ORGANIC MATTER DIGESTIBILITIES OF COTTONSEED HULLS, AND RAW OR TREATED MESQUITE RATIONS
Source of Variation
Between Groups
Within Groups
Total
DF
2
12
14
Sum of Squares
9.72
79.05
88.77
Mean Square
4.86
6.59
F Value
0.74
homogeneous set. There are no significant differences among
the three groups for the digestibilities of dry matter, di
gestible energy, and organic matter.
Data on the statistical analysis of the nitrogen
balances are presented in Table 10. The results indicate
that there are significant differences among the three ex
perimental groups. Duncan's New Multiple Range Test was
used to test the difference between the means of nitrogen
balances of all possible treatment pairs, and the results
are presented in Table 11. It indicates that there are two
homogeneous subsets. One is between cottonseed hulls and
raw mesquite groups; while the other is between raw and
treated mesquite groups. The nitrogen retention values
indicate that the utilization of nitrogen of the treated
mesquite ration is lower than that of the cottonseed hulls
ration.
36
TABLE 10
ANALYSIS OF VARIANCE OF NITROGEN RETENTIONS OF COTTONSEED HULLS, AND RAW OR TREATED MESQUITE RATIONS
Source of Variation
Between Groups
Within Groups
Total
DF
2
12
14
Sum of Squares
324.31
282.57
606.88
Mean Square
162.16
23.55
F Value
•k
6.89
*Significant (p<0.05)
TABLE 11
DUNCAN'S NEW MULTIPLE RANGE TEST OF NITROGEN RETENTIONS OF COTTONSEED HULLS, AND RAW OR TREATED MESQUITE RATIONS
Treatment Mean
Cottonseed Hulls Group 49^
Raw Mesquite ^^-^ Group 44
Treated Mesquite j Group 3^
Means with the same superscript were net significantly
different from each other (p<0.05).
37
Analysis of variance was used to test the digesti
bilities of crude fiber among three groups. The results
presented in Table 12 indicate that the hemicellulose di
gestibilities are not significantly different among the
three experimental rations, but there are significant dif
ferences in the cellulose digestibilities among the three
experimental groups as shewn in Table 13. Duncan's New
Multiple Range Test presented in Table 14 indicates that
the digestibility of cellulose is net significantly dif
ferent between the raw and the treated mesquite rations.
TABLE 12
ANALYSIS OF VARIANCE OF HEMICELLULOSE DIGESTIBILITIES OF COTTONSEED HULLS, AND RAW OR TREATED MESQUITE RATIONS
Source of Variation
Between Groups
Within Groups
Total
DF
2
12
14
Sum of Squares
21.05
47.37
68.42
Mean Square
10.52
3.95
F Value
2.67
38
TABLE 13
ANALYSIS OF VARIANCE OF CELLULOSE DIGESTIBILITIES OF COTTONSEED HULLS, AND RAW OR
TREATED MESQUITE RATIONS
Source of Variation
Between Groups
Within Groups
Total
DF
2
12
14
Sum of Squares
366.88
298.86
665.74
Mean Square
183.44
24.91
F Value
•k
7.37
*Signifleant (p<0.05).
TABLE 14
DUNCAN'S NEW MULTIPLE RANGE TEST OF CELLULOSE DIGESTIBILITIES OF COTTONSEED HULLS, AND RAW OR TREATED MESQUITE RATION
Treatment Mean
Cottonseed Hulls Group 70^
Raw Mesquite , Group 59
Treated Mesquite , Group 59
Means with the same superscript were not significantly different from each other (p<0.05).
39
Data on the analysis of variance of lignin digesti
bilities are shown in Table 15. They indicate that there
are significant differences among the three experimental
rations. The results on Duncan's New Multiple Range Test
presented in Table 16 indicate that there are two homoge
neous subsets. Observation of the subsets shows there is
no significant difference between the cottonseed hulls
ration and the treated mesquite ration or the treated and
raw mesquite ration.
Chandra et. al . (40) found that the digestibility of
wheat straw increased linearly up to 3.3% of sodium hydrox
ide treatment. According to the rumen digestibility data
obtained in the present studies, the dry matter disappear
ance increases as the alkali level increases. However in
the animal feeding studies, the digestibility of alkali-
treated mesquite did not show the same results as in the
preliminary trial. It is possible that the increasing
amounts of residual alkali exerted an adverse effect on the
rumen fermentation. This effect would net appear when the
digestibility was determined by the nylon bad technique
which had only 1 to 5 g sodium hydroxide in the rumen of
steer as compared to 22 to 24 g consumed daily by the lambs
receiving treated mesquite in the animal feeding experi
ment.
40
TABLE 15
ANALYSIS OF VARIANCE OF LIGNIN DIGESTIBILITIES OF COTTONSEED HULLS, AND RAW OR
TREATED MESQUITE RATIONS
Source of Variation
Between Groups
Within Groups
Total
DF
2
12
14
Sum of Squares
795.73
936.40
1732.13
Mean Square
397.86
78.03
F Value
5.10
Significant (p < 0.05)
TABLE 16
DUNCAN'S NEW MULTIPLE RANGE TEST OF LIGNIN DIGESTIBILITIES OF COTTONSEED HULLS, AND
RAW OR TREATED MESQUITE RATIONS
Treatment Mean
Cottonseed Hulls Group
Raw Mesquite Group
Treated Mesquite Group
23*
41^
29 a,b
Means with the same superscript were not significantly
different from each other (p<0.05).
41
Table 2 indicates that ash content of treated mes
quite is 5 times greater than that of untreated mesquite;
and in Table 3, the ash content of treated mesquite ration
is twofold greater than that of raw mesquite ration. This
suggests that the low content of neutral detergent fraction
(including cellulose, hemicellulose, and lignin) is caused
by the increased sodium acetate content of the ration and
not by the alkali hydrolysis en the mesquite.
In summary, it is suggested that in further studies,
the samples should be washed after a sodium hydroxide treat
ment.
It is suggested that other chemicals also may be
tried on mesquite. Andersen (41) reported that NH^OH gave
statistically (p<0.05) greater digestibilities than that
of untreated ryegrass straw. The nitrogen content of NH4OH-
treated straw was significantly (p<0.05) greater than for
the untreated straw. The benefit for NH^OH treatment is
nitrogen added to the straw which would be usable by rumi
nants.
CHAPTER V
SUMMARY AND CONCLUSIONS
The objectives of the present study were to test the
effect of sodium hydroxide levels en the iri vivo rumen di
gestion of mesquite and the nutritive value of mesquite for
lambs.
The sodium hydroxide treatment with or without auto
claving markedly influenced the mesquite structure. There
was an inverse relationship between the sodium hydroxide
level and the lignecellulose content of mesquite.
In vivo rumen digestibility of mesquite increased
asymptotically as the sodium hydroxide level increased.
The digestibility of mesquite was negatively correlated to
the crude fiber content (cellulose, hemicellulose, and
lignin).
The digestibilities of crude protein and lignin of
treated and untreated mesquite rations were significantly
higher than those of the cottonseed hulls ration.
There were no significant differences between the
treated and untreated mesquite samples in the digestibili
ties of crude protein, cellulose, lignin and nitrogen bal
ance.
The digestibilities of dry matter, digestible energy,
organic matter, and hemicellulose among the three experi
mental groups were net significantly different.
42
43
The nitrogen balance and digestibility of cellulose
of treated mesquite ration were significantly lower than
those of the cottonseed hulls ration at the 0.05 level.
LITERATURE CITED
1. Asplund, J. M. and W. H. Pfander. 1972. "Production of single-cell protein from solid wastes" Alternative Sources of Protein for Animal Production. Printing and Publishing Office, National Academy of Science. 2101 Constitution Avenue, N.W. Washington, D.C. 20418.
2. Marion, P. E., C. E. Fisher, and E. D. Robinson. 1957. Ground mesquite as roughage in rations for yearling steers. Texas Agr. Exp. Sta. Progress Report No. 1972.
3. Beckmann, E. 1921. Conversion of grain straw and lupins into feeds of high nutrient value. Festschrift Kaiser Wilhelm Ges. F6rderung Wiss. Zehnjahrigen Jubilaum 18 (Chem. Abstr. 16: 765, 1922) .
4. Tarkow, H. and W. C. Feist. 1969. "A mechanism for improving digestibility of lignecellulesic materials with dilute alkali and liquid ammonia." In Robert F. Gould (ed.) Celluloses and Their Applications. Advances in Chemistry Series, No. 95 Amer. Chem. Sec, Washington, D.C.
5. Wilson, R. K. and W. J. Pigden. 1964. Effect of a sodium hydroxide treatment in the utilization of wheat straw and poplar (Populus alba) wood by rumen microorganisms.
6. Beckman, E. 1915. Determination of food value of wood and straw. Sitzb. Kgl. Press. Akad. Wiss. 638 (Chem. Abstr. 9: 3309, 1915).
7. Haberlandt, G. 1915. The food value of wood. Sitzb.
Kgl. press. Akad. Wiss. 243 (Chem. Abstr. 9: 1516, 1915) .
8. Bissell, H. D. and W. C. Weir. 1957. .^^^^^f^^ibili-ties of interior live oak and chamise by deer and sheep. J. Anim. Sci. 16: 476.
9. crampton, E. W. and L. A. Maynard. 1938 ^^^^^^^f °^ of cellulose and lignin content to the nutritive value of animal feeds. J. Nutri. 15: 3BJ.
44
45
10. Ely, R. E., E. A. Kane, W. C. Jacobson, and L. A. Moore. 1953. Studies on the composition of lignin isolated from orchard grass hay cut at four stages of maturity and from the corresponding feces. J. Dairy Sci. 36: 346.
11. Sullivan, J. T. 1955. Cellulose and lignin in forage grasses and their digestion coefficients. J. Anim. Sci. 14: 710.
12. Tomlin, D. C , R. R. Johnson, and B. A. Dehority. 1965. Relationship of lignificatien to in vitro cellulose digestibility of grasses and legumes. J. Anim. Sci. 24: 161.
13. Lampila, M. 1963. Experiments with alkali straw and urea. Ann. Agric. Fenn. 2: 105 (Nutri. Abstr. Rev. 34: 575, 1964).
14. Stone, E. J., R. E. Gireurd, Jr., J. B. Frye, Jr. 1965. Chemical pretreatment of roughage I. The effect of alkali and enzymes en cellulose content of bagasse. J. Dairy Sci. 48: 814.
15. Waiss, A. C. Jr., J. Guggolz, G. 0. Kohler, H. G. Walker, Jr., and W. N. Garrett. 1972. Improving digestibility of straws for ruminant feed by aqueous ammonia. J. Anim. Sci. 35: 109.
16. Donefer, E. , I. O. A. Adeleye, and E A. 0. C Jones. 1969 "Effect of urea supplementation on tne nutritive value of NaOH-treated oat straw." In Robert F. Gould (ed.) Celluloses and Their Applications. Advance in Chemistry Series, No. 95 Amer. Chem. S e c Washington, D.C.
17 Feist W C , A. J. Baker and H. Tarkow. 1970. Alkali r;quirei;;nts for improving digestibility of hard-weeds by rumen microorganisms. J. Anim. Sci. 30: 832.
1R Tanaferd, R. A. 1969. Uses of mesquite. In J. L. 18. ^^^g^g^^;,^; %d.) Literature on the Mes^u^^^ProsoHls
L ) of North America. Special Report No. 26 ICASALS, Texas Tech University, Lubbock, Texas.
19 Noxious Brush and Weed Control Research Highlights. 19. Noxio^^^^ international Center for Arid and Semi-
Arid Land Studies. Special Report No. 51.
46
20. D e e l i n , C. E. 1956. Feeding mesqui te t o c a t t l e . Farm Family Vol. 14 No. 4 pp. 3.
21. Yang, S. P. 1972. Personal communication.
22. Wayne Figreid, W. H. Hale and Brent Theurer. 1966. An evaluation of the nylon bag technique for estimating rumen utilization of grains. Proceedings, Western Section, American Society of Animal Science 17: 265.
23. Van Keuren, R. W. and W. W. Hienemann. 196 2. Study of a nylon bag technique for in_ vivo estimation of forage digestibility. J. Anim. Sci. 21: 343.
24. Hayes, B. W. , C. 0. Little and G. E. Mitchell, Jr. 1964. Influence of ruminal, abomasal and intestinal fistulatien of digestion in steers. J. Anim. Sci. 23: 764 (abstract).
25. Goering, H. K. and P. J. Van Soest. 1970. Forage Fiber Analysis (Appartus, Reagents, Procedures, and Seme Applications) U.S.D.A. Agricultural Handbook No. 379.
26. Manual No. 117. Parr Oxygen Bomb Calorimeters and Oxygen Bomb Sulfur Apparatus. Parr Instrument Company, Moline, Illinois.
27. Hunt, J. R. 1963. Rapid determination of calcium in feedstuffs. Journal of Agricultural and Food Chemistry 11: 346.
28. Sumner, B. J. 1944. A method for the colorimetrie determination of phosphorus. Science 100:413.
29. A.O.A.C. Official and Tentative Methods of Analysis. 1970. 10th ed. Washington, D.C. Association of Official Agriculture Chemists.
30. Crampton, E. W. and L. E. Harris. 1969. Applied Animal Nutrition. Second ed. San Francisco. W. H. Freeman.
31. Dixon, W. J. 1973. "Asymptotic regression" BMD-Biomedical Computer Programs. Third ed. University of California Press. Berkeley. Los Angeles. London.
47
32. Dixon, W. J. 1973. "Simple linear regression" BMD-Biomedical Computer Programs. Third ed. Uni^JiF-London^ California Press. Berkeley. Los Angeles.
33. Dixon, W. J. 1973. "Multiple range test" BMD-Biomedical Computer Procframs. Third ed. University of California Press. Berkeley. Los Angeles. London.
34. Wangaard, F. F. 1966. Resistance of weed to chemical degradation. Forest. Prod. j. 16: 53.
35. Wise, L. E. and E. C. Jahn. 1952. Weed Chemistry. Reinhold Publishing Co., New York.
36. Millet, M. A., A. J. Baker, W. C. Feist, R. W. Mellen-berger and L. D. Satter. 1970. Modifying wood to increase its iri vitro digestibility. J. Anim. Sci. 31: 781.
37. Ololade, B. G. and D. N. Mowat. 1969. In, vitro digestibility of sodium hydroxide treated straw. J. Anim. Sci. 29: 167.
38. Gharib, F. H., J. C. Meiske, R. D. Goodrich and A. D. Elserafy. 1975. In. vitro evaluation of chemically treated poplar bark. J. Anim. Sci. 40: 734.
39. Singh, M. and M. G. Jackson. 1971. The effect of different levels sodium hydroxide spray treatment of wheat straw on consumption and digestibility by cattle. J. Agri. Sci. 77: 5.
40. Chandra, S. and M. G. Jackson. 1971. A study of various chemical treatments to remove lignin from coarse roughages and increase their digestibility. J. Agri. Sci. 77: 11.
41. Anderson, D. C. and A. T. Ralston. 1973. Chemical treatment of ryegrass straw: In vitro dry matter digestibility and compositional changes. J. Anim. Sci. 37: 148.
