• TABLE OF CONTENTS
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 Title Page
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Introduction
 Literature review
 Experimental procedure
 Results and discussion
 Summary
 Appendix
 Literature Cited
 Biographical sketch
 Copyright














Title: interrelationship of phosphorus, sulfur, copper, and molybdenum on cellulose digestibility of rumen microorganisms and in rat metabolism
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Title: interrelationship of phosphorus, sulfur, copper, and molybdenum on cellulose digestibility of rumen microorganisms and in rat metabolism
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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
        Page iii
    Table of Contents
        Page iv
        Page v
        Page vi
    List of Tables
        Page vii
        Page viii
        Page ix
        Page x
    List of Figures
        Page xi
    Introduction
        Page 1
        Page 2
    Literature review
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
    Experimental procedure
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
    Results and discussion
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
        Page 61
        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
        Page 67
        Page 68
        Page 69
        Page 70
        Page 71
        Page 72
        Page 73
        Page 74
        Page 75
        Page 76
        Page 77
        Page 78
        Page 79
        Page 80
        Page 81
        Page 82
        Page 83
        Page 84
    Summary
        Page 85
        Page 86
        Page 87
        Page 88
    Appendix
        Page 89
        Page 90
        Page 91
        Page 92
        Page 93
        Page 94
        Page 95
        Page 96
        Page 97
        Page 98
        Page 99
        Page 100
        Page 101
        Page 102
        Page 103
        Page 104
        Page 105
        Page 106
        Page 107
        Page 108
    Literature Cited
        Page 109
        Page 110
        Page 111
        Page 112
        Page 113
        Page 114
        Page 115
        Page 116
        Page 117
        Page 118
        Page 119
        Page 120
        Page 121
        Page 122
        Page 123
        Page 124
        Page 125
    Biographical sketch
        Page 126
        Page 127
    Copyright
        Copyright
Full Text











The Interrelationship of Phosphorus, Sulfur, Copper,

and Molybdenum on Cellulose Digestibility by

Rumen Microorganisms and in Rat Metabolism










By
JOSEPH LISTON EVANS










A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY










UNIVERSITY OF FLORIDA
June, 1959













ACKNOWLEDGMENTS


The writer wishes to express his sincere apprecia-
tion to Dr. George K. Davis, chairman of his supervisory
committee, for his guidance, assistance, and patience

throughout the experimental investigation and preparation
of this dissertation, and to Drs. D. S. Anthony, R. B.
Becker, R. L. Shirley, and H. D. Wallace for aid and sug-

gestions and for serving as members of his graduate connit-
tee. He wishes to thank Dr. W. 0. Ash for his technical

assistance and cooperation with the statistical analyses.

He wishes to thank Drs. C. A. Ammerman, L. R. Arrington,
T. J. Cunha, J. P. Feaster, and J. T. McCall and Messrs.

J. F. Easley, J. E. Wing, graduate students, and other staff
of the Department of Animal Husbandry and Nutrition for their
technical assistance and cooperation. He wishes to thank
Dr. C. F. Simpson and r., J. II. Green for preparing the
rumen fistulas.

This investigation was supported by the Nutrition
Foundation, The National Institutes of Health, the Soft
Phosphate Research Institute, and the Sugar Research Founda-
tion. The writer wishes to acknowledge his appreciation for
this support.

He wishes to thank Brown Company, New York, New York,

for the Solka-Floc; Chas. Pfizer and Company, Terre Haute,


ii










Indiana,for the vitamin A; E. I. Du Pont De Nemours and
Company, Winter Park, Florida, for the urea; Hercules
Powder Company, Wilmington, Delaware, for the cotton liners;
and the United States Sugar Corporation. Clewiston- Florida,
for the sugar cane bagasse used in this experimental in-
vestigation.
The writer is especially indebted to his wife, Gail,
for her.. altist-ra assistance throughout the course of
his graduate study.
Sincere appreciation is also expressed to Mrs. E. P.
Christie for typing the manuscript.


iii













TABLE OF CONTENTS


Page
ACENOZ'LEDGIIENTS . . o . . . . i

LISTOF TABLES * * * * vii

LIST OF PIGURES .. . . .. xi
INTRODUCTION .. .. .. 1
LITFLATURE EVIE .. . . . 3

Digestion of Cellulose by Rumen Microorganisms ,. 3
Techniques for Studying the Activity of
Rumen Microorganisms .r . . 3
n vLvo techniques .. .. ,.. 3
Svitro techniques *. . . . 5
3Livo ~ bag technique . . . . 5
Effects of Minerals on the Activity of
Rumen Microorganisms... . .* . .* 6
Variability of Different Sources of Cellulose 7
Phenomena of the Rumen .. . 9
Effects of Phosphorus, Sulfur, Copper, and.
Molybdenum on Metabolic Processes., . . . 13
Phosphorus *. * .. 9 13
Sulfur . . . 15
Copper * 9 . . 19
Molybdenum . . . 21
Phosphorus, Sulfur, Copper, Molybdenum
Interrelationship . . . . 22

EXPERDIMETAL PROCEDURE . , , . . 26

Experiment i 4 . .2 . 26
Study of Cellulose Digestion in the Rumen
of Rumen-Fistulated Steers . . . . 2G
Study of steer variation (uniformity trial). 29
Study of the effect of phosphorus, sulfur,
and molybdenum additions to a steer ration 32
Experiment II # * ,# . . . 33
Study of Cellulose Digestion in the
Artificial numon . .. .* 33
The comparison of cellulose digestion by
rumen inoculum taken from different steers 33










TABLE OF CONTENTS--Continued


Page
The comparison of the digestion of cellu-
lose from different sources and from the
same source with different particle sizes 35
The effect'of different concentrations of
phosphorus, sulfur, copper, and molybdenum
on cellulose (Solka-Floc) digestion . . 35
Experiment III . . . . . 37
Study of the Effect of the Interrelationship
of mineralss on Growth and Bone Formation in
Rats * * . .* * . 37
Trial I. The effect of different concen-
trations of phosphorus, sulfur, copper,
and molybdenum on growth and bone
formation * . * . . . . 41
Trial II. The effect of sulfur and copper
additions to a ration containing 300 parts
per million of molybdenum on growth and
bone formation * * * . 42
Trial III. The effect of different concen-
trations of sulfur, copper, and molybdenum
on growth and bone formation . . 42
RESULTS AND DISCUSSION . . . . 44

Experiment I . *. . . . 44
Study of Cellulose Digestion in the Rumen
of Rumen-Fistulated Steers . . . 44
Study of steer variation (uniformity trial) 44
Study of the effect of phosphorus, sulfur,
and molybdenum additions to a steer ration. 47
Study of the effect of phosphorus, sulfur,
and molybdenum additions to a steer ration
on the rumen liquid concentrations of
phosphorus, sulfur, and copper . . 52
Experiment II *. . .6 . . .** 58
Study of Cellulose Digestion in the
Artificial Rumen .......... 58
Comparison of cellulose digestion by rumen
inoculumr taken from different steers con-
suring the same ration. .. .. .. . 58
Comparison of the digestion of cellulose
from different sources and from the same
source with different particle sizes . 58
Study of the effect of-different concen-
trations of phosphorus, sulfur, copper, and
molybdenum on cellulose digestion . . 60










TABLE OF CONThETS--Continued


Pare
Experiment III e r e n . * . . . . 0 4
Study of the Effect of the Interrelationship
of Minerals on Growth and Bone Formation in
Rats . . . . a . . . 64
Trial I. The effect of different concen-
trations of phosphorus, sulfur, copper, and
molybdenum on growth and bone formation. . 64
Trial II. The effect of sulfur and copper
additions to a ration containing 300 parts
per million of molybdenum on growth and
bone formation *. . * . *. .* 75
Trial III. The effect of different concen-
trations of sulfur, copper, and molybdenum
on growth and bone formation . . . 9
SPPENDIA . . . . . . . 85

APPENDIX . . . . . . 89

LITERATURE CITED . . . 109

BIOGRAPHICAL SKETCIIH * .. ... 126













LISTT 1OF TABLTrS


Table Page
1. Composition of Steer Ration Used for Studying
Cellulose Digestion by .an, Y -~l Bag Technique
in Experiment I and by an In Yjtr0 Technique in
Experiment II . .. .. . . . . 27

2. Study of Cellulose Digestion IL VIyo. Concen-
trations of Phosphorus, Sulfur, and Holybdenum,
Used to Supplement the Steer R~tion of Table 1. 28
3. Study of Cellulose Digestion In Yitro.L. Concen-
trations of Phosphorus, Sulfur, Copper, and
Molybdenum Used to Supplement the Medium for.
Rumen Microorganisms i . ,. *. 36

4. Composition of Hat :Ration Used for Studying the
Effect'of the Interrelationship of Phosphorus,
Sulfur, Copper, and Molybdenum on Growth and
Bone Formation in Experiment III . . 38
5. Study of Growth and Done Formation in Rats. "
Concentrations of Phosphorus, Sulfur, Copper,
and Holybdenum Used to Supplement the Rat
Ration of Table 4 . . . . . 40
6. Effect of Steer Variation on the Digestion of
Cellulose from Different Sources .* 45
7. Statistical Analysis of the Data Given in
Table 6 , . . . 46
8, Effect of Treatments Given in Table 2 on the
Digestion of Cellulose from Different Sources 48

9. Statistical Analysis of the Data Given in
Table 8 * . . . 49
104 E]ffect-of Steer Variation on the Phosphorus,
Sulfur, and Copper Concentrations of Hlumen
Liquid . . * . . . . . . 54

114 Statistical Analysis of the Data Summarized
in Table 10 . . . . . . . o. 55


vii










LIST OF TABLES--Continued


Table Page

12. Effect of Treatments Given in Table 2 on the
Phosphorus, Sulfur, and Copper Concentrations
of Rumen Liquid . . .* * *. * 50
13. Statistical Analysis of the Data Summarized
in Table 12 ,. . .. . . 57
14. Study of Cellulose Digestion In Viro. The
Comparison of Cellulose Digestion by Rumen
Inoculum Taken from Different Steers Consuming
Ration S-0 Given in Table 2 . GO
15. Study of Cellulose Digestion In Vitro. The
Comparison of the Digestion of.Cellulose from
Different Sources and from the Same Source with
Different Particle Sizes ,. . . . . 61

16. Study of Cellulose (Solka-Floc) Digestion In
Vitro. Effect of Different Concentrations of
Phosphorus, Sulfur, Copper, and Iolybdenum. 62
17. Trial I, Experiment III. Summary of 56-Day Rat
Weight Gains on Various Rations Given in
Table 5 , . . . 9, . 65
18. Trial I, Experiment III. Summary of Milligrams
Ash per Femur per Gram of Body Weight of Rats
on Various Rations Given in Table 5 . . 66
19. Trial I, Experiment III. Summary of Percentage
Ash of Femurs of Rats on Various Rations Given
in Table 5, . . . . . . 0 67

20 Trial I, Experiment III. Summary of Femur Ash
Concentrations of Rats on Various Rations
Given in Table 5 . . . . . .. 68
21. Trial I, Experiment III. Summary of Percentage
Phosphorus of Dry, Fat-Free Femurs of Rats on
Various MRtl~ns Given in Table 5 . 69
22. Trial I, Experiment III. Summary of Percentage
Phosphorus of Livers on a Dry Hatter Basis of
Rats on Various Rations Given in Table 5 . 70


viii










LIST OF TABLES--Continued


Table Page
23. Trial I, Experiment III. Summary of Copper
Concentrations of Livers on a Dry Matter
Basis of Rats on Various Rations Given in
Table 5 a. . . .... .... .71
24. Trial II, Experiment III. Summary of 56-Day
Weight Gains, MIlligrams Ash per Femur per
Gram of Body Weight, Percentage Ash of Femurs,
and Femur Ash Concentrations of Rats on
Various Rations Given in Table 5.. .. ... ... 76
25. Trial II, Experiment III. Statistical
Analysis of the Data Summarized in Table 24 77
2G. Trial III, Experiment III. Summary of 35-Day
Rat Weight Gains on Various Rations Given in
Table 5 o . . ,. ** , *... 80

27. Trial IIIi Experiment III. Summary of Percent-
age Ash of Femurs of Rats on Various Rations
Given in Table 5 .., . . .. 81
28, Trial III, Experiment III. Summary of Femur
Ash Concentrations of Rats on Various Rations
Given in Table 5 .. . .. .. 82
294 Trial IIIi Experiment III. Summary of
Hemoglobin Values of Rats on Various Rations
Given in Table 5 ..... . . 83
30. Study of Cellulose Digestion la Y"yg. Effect
of Different Concentrations of Phosphorus,
Sulfurj and 'olybdenum Given in Table 2 on,
the Digestion of Cellulose from Solka-Floc,
Cotton Linters, and Sugar Cane Dagasse. . 90
31, Analysis of Variance of the Effect of Different
Treatments on the Phosphorus, Sulfur, and Cop-
per Concentrations of Rumen Liquid on a Dry
Matter Basis .. * . . 94
326 Trial I, Experiment III. Ration, Number, Sex,
Starting Weight, Weight Gain, Dry Fat-Free Femur
Weight, and Ash Weight per Femur of Rats Used 95










LIST OF TABLES--Continued


Table Page
33. Trial I, Experiment III. Summary of the Analysis
of Variance of the Data Given in Table 32., .. 101
34. Trial II, Experiment III. Ration, Number, Sex,
Starting Weight, Weight Gain, Dry Fat-Free
Femur Weight, and Ash Weight per Femur of Rats
Used . .. . . . . .102
35. Trial III, Experiment III. Ration, Number,
Sex, Hemoglobin, Starting-Weight, Weight Gain,
Dry Fat-Free Femur Weight, and Ash Weight per
Femur of Rats Used . . . . . . 104
36. Trial III, Experiment III. Summary of the
Analysis of Variance of the Data Given in
Table 35 . . .. . . . . . 106
37. Experiment III. Feed Efficiency of Rats .107
38. Summary of Chemical Composition of Rations . 108













LIST OF FIGURES


Figure Page

1. The effect of the interrelationship of phos-
phorus, sulfur, and molybdenum on the digestion
of cellulose from different sources . .. 51

2. The partitioning of 56-day weight gains of male
rats of Trial I of Experiment III by two-week
periods * 9 . . 99

3. The partitioning of 56-day weight gains of female
rats of Trial I of Experiment III by two-week
periods * . * * . * 100
4. The partitioning of weight gains of female
rats of Trials II and III of Experiment III
by two-week periods *. * * *. . 103












INTRODUCTION


"In consideration of mineral nutrition, the inter-
relationships of the different elements are equally as im-

portant as the requirements of the individual elements. It
seems quite probable that these interrelationships may well

explain many of the differences obtained by different inves-

tigators of mineral nutrition and may indeed provide the key

for better nutrition for both humans and animals in the many
varied conditions which exist in the world today." (52)

Maynard and Loosli (116) have presented a map pre-
pared by Dr. K. C. Beeson listing the mineral-deficient

areas of the United States. From this map, it is evident
that many deficiencies exist in different parts of the United
States as well as in Florida. These deficiency problems
have stimulated research, and, as a result, recommended
mineral-mixture formulas have been provided for Florida cat-
tlemen by Becker g t a. (21).

When a mineral mixture is provided for livestock of
a given area, it is necessary that consumption of the mixture
as well as of other nutrients be attained. A review of the

literature suggests that in studies of mineral interrelation-
ships there has been little data acquired with respect to
the effect of mineral imbalances on consumption of feeds,
activity of rumen microorganisms, skeletal formation, and

1










metabolic processes within the body.
In the present research, consideration has been
given to the interrelationship of phosphorus, sulfur, cop-
per, aid molybdenum. Three experimental programs have been
carried out.
In the first group of experiments, a technique was
evaluated for studying cellulose digestion in animals with
rumen fistulas. Then, using this technique for measuring
cellulose digestion, studies were made to estimate the vari-
ability among fistulated steers within periods and among
periods, to study the effect of the mineral interrelation-
ship using the above technique on cellulose digestion by
ruLien microorganisms, to compare the digestion of cellulose
from different sources, and, at the same time, to acquire
some information for relating mineral content of the ration
to mineral content of the rumen ingesta.

In the second group of experiments, aIn~J vitro.
technique was used for studying the effect of the interrela-
tionship of phosphorus, sulfur, copper, and molybdenum, for
determining the effect of the particle size of the source and
the source of cellulose, and for comparing the effect of the
inoculum from different steers on cellulose digestion by
rumen microorganisms.
In the third group of experiments, rats wore used for
studying the effect of the interrelationship of phosphorus,
sulfur, copper, and molybdenum on growth and bone formation.













LITEaATUIE RIL'VIT


Digestion of Cellulose by Rumen I~crooranisms

Many reviews are available covering various aspects
of rumen function (13, 32, 60, 03, 65, 93, 94, 99, 116, 133,
135, 130, 144, 184). The present review of the literature

covers only those aspects of ruminology closely bearing on
the techniques for studying the activity of rumen micro-
organisms, the effects of minerals on the activity of rumen
microorganisms, the variability of different sources of

cellulose, and the phenomena of the rumen.

Techniques for Studying the Activity
of Rumen Microorganisms

In vivo techniques

When a comparison of seven different methods of
determining digestion coefficients was made, comparable
coefficients were secured with the standard ten-day con-
sumption-excretion method and the chromium and plant pigment
ratio techniques hen calculated with either the total col-
lection samples or the averages of partial collections of
three days (99). The crude lignin and the corrected lignin

ratio techniques using the total fecal collection or grab-
sampling did not give good results.










Other workers reported that the total variation in
digestion coefficients for crude fiber was higher than for
dry matter or crude protein (85, 86). For crude fiber and
dry matter, coefficients of digestion were significantly
different anong periods; however, period effects tended to
be small.
The digestion coefficients of feeds containing
various ratios of hay to grain by fistulated steers and
milking cows were significantly different at the 1 per cent
level (62). .llowover, in a later study, it was found that

digestion coefficients obtained with intact and fistulated
steers were not different.
The addition of crude fiber reduced the total amount
of protein and ether extract digested, but the most promi-
nent effect of added carbohydrate was to reduce the digest-
ible crude fiber and the digestible protein (1GG). Casein
increased the digestion of protein, ether extract, and
energy, and it decreased the digestion of crude fiber and
nitrogen-free-extract. Possibly, part of this could be ex-
plained by the fact that the variability in the digestion
of a given nutrient tends to be inversely related to the con-
tent of that nutrient in the feed (151),
When the hay to grain ratio of the rationfluctuated
widely, preliminary feeding periods of between sixteen and
thirty days were needed (53, 130). A period of seven days
appeared adequate when just the protein supplement varied (130).










3n vitro techniques
Sjlau or artificial rumen techniques (148) and
the nutritional .requirements. (97) for rumen microorganisms
have been reviewed.
Recently, experiments to set up criteria for estab-
lishing the validity of artificial rumen studies have been
completed (89,i 141, 178). :Of the, measurements used, cellu-
lose digestion appeared to be the most.closely related to
Ia yvi digestion data (16). A high correlation was found
between cellulose ,digestion i_ vIJvo and .i vitn o (90, 10),.
but differences were found due to inoculum from sleep and
steers and inoculum front the same species but on different
rations (106), Other workers found that when.different
forages were fed to a steer used as a source of inoculumt
the ji vitrg cellulose digestion of these forages was not
affected by the type of forage fed the steer (141)., Also,
the an vitro cellulose digestion coefficients showed less
variation than those from ain Lv studies. The cellulose
from legume hays but not from grass hays was found to have
significantly different cellulose digestion coefficients
when measured by i& 9-il and in itro methods.

In vive ba b techniane
This technique involves the suspension in the rumen
of a bag made from an indigestible material which contains
the product to be assayed* Several workers have used this








6
technique (14, 24, 103, 114, 117, 125, 135, liu, 174), This
technique allows random movement within the rumen limited
only by the length of string needed to secure the bag.
Umezu 1t al. (174) reported that maximum cellulose digestion
occurred after twenty hours. To determine this, silk baZs

were inserted through the mouth and removed at various
intervals. Using fistulated animals, it was found that 50
per cent of the cellulose in wheat and oat straws disap-
peared after three to six days and 30 to 40 per cent re-
nained after seven days (117).
Results demonstrated greater activity by rumen micro-
organisms with this a _ivo bag technique than with the
artificial rumen (125)* Also, the daily pattern of cellu-
lolytic activity appeared to vary more between days within
animals than between animals within days. Balch and
Johnson (14) reported an increased activity going from a
dorsal to a more ventral position by distances of six, twelve,
and eighteen inches from the rumen cannula.
Another modification has been the use of a porcelain
test tube placed inside an aluminum shield and suspended in
the rumen (72). However, these tubes wee not permeable to
bacteria.

Effects of Minerals on the Activity
of Rumen Microorganisms
Alfalfa ash has been reported to both improve diges-
tion (30, 165) and not to improve digestion (170). Others

have explained a part of this improvement from alfalfa ash










on the basis of the increased ruminal calcium requirement
when corn oil is added to the ration (9).
It has been demonstrated from ; "i ,ro experiments
that rumen microorganisms have a definite phosphorus re-
quiremont (7) and that the low phosphorus content of hay is
a limiting factor in its utilization (25, 180), Other
mineral requirements as well as toxic levels have been
proposed for rumen microorganisms jin itro (64, 91# 92, 120,
148). These reported required levels vere in a similar
ratio to the average composition of alfalfa ash (20).
Cardon (33) found no depression with either Ain yio
or n svitro cellulose digestion resulting from high sodium
chloride concentrations* Other results have shown a rela-
tionship between sodium and potassium (92).

Variability of Different Sources of Cellulose
Huffman (94) has concluded that probably the most
important factor affecting microbial digestion of celluloses
is the increased deposition of lignin in the vegetative part
of the plant as it matures. Stallcup (161) agreed with the
above, but Quicke and Bentley (139) concluded that in the
nature brome and orchard grass hays studied, the differences
in the lignin content of hays cut at different stages were
too small to account for all the observed differences in
Cellulose digestion. But, by separating the cellulose from
the lignin, cellulose digestion was ir.:proved JA vitro (98),










However, it appears that the lignin or cellulose content
may give a better evaluation of roughage than crude fiber
(162).
The amorphous regions of cellulose have a greater
surface area exposed than do the crystalline regions (42),
But the mechanical disintegration of cellulosic materials
increased their susceptibility to enzymatic hydrolysis nore
than can be accounted for by the increase in the surface
area alone. Bayley and Bishop (10) have proposed that the
variability in the crystallinity of cellulose is caused by
defects in the structure of the cellulose moloculos. ,At,
the same time, other work, quoted by T:astra '::af al. (98)1
slowed that a correlation existed between the increased
degree of crystallinity and the increased resistance against
enzymatic attack and that physical constants were not iden-
tical for celluloses from different sources.
The nutritive value of sugar cane bagasse has been
reviewed (73). Wayman, quoted by Fonseca (73), found very
low digestion coefficients for the fiber in rations contain-
ing sugar cane bagasse pith, and should the other constitu-
ents of the ration be given the value given them by Morrison,
the value for bagasse pith would be negative* Others con-
cluded that sugar cane bagasse was lacking in palatability
(138) and that it had a low digestion coefficient (157).
Rations containing wood-pulp and a balanced grain
mixture had an apparent total digestible nutrient content








9
exceeding 68 per cent (183). I vitro cellulose digestion
coefficients of 50, 70, 76, and 79 per cent were obtained
for cellulose from cotton linters, hardwood cellulose,
another wood cellulose, and softwood cellulose, respectively
(11). The digestion of cellulose in alfalfa meal showed a
more rapid initial rate than softwood cellulose (149).
~heat straw was characterized by a slow and uniform rate
throughout the incubation period.

Phenomena of the Rumen
In a review on ruminant digestion, Balch (13) has
given some tentative values for the percentage of crude
fiber digested in the reticulo-runen. The Proceedin.s of

the American Grassland Council of 1958 presented much data
on the production, preservation, and utilization of forages
(137). Crampton 11 al, (44) found no significant differences
in the digestion of two low-fiber rations by rats, guinea

pigs, sheep, swine, or human subjects, but a significant
difference was found amrong the digestibilities of the differ-
ent nutrients. Other workers concluded that crude fiber
digestion was possibly one of the more important factors
influencing feed efficiency (10). Soluble carbohydrates
have been reported to decrease the digestion of dry matter
(32) and crude fiber (74). Others reported no depression of
cellulose digestion from soluble carbohydrate (132).
For stimulating urea utilization, corn starch was








10
found to be superior to other forms of carbohydrate (23,
27) and a starch-fruetose mixture was superior to starch
alone (27). By increasing the energy content of the fer-
mentation medium, increased urea utilization resulted (32).
In sheep, a lowered bacterial population was found when
urea was fed without sulfur (75). The digestion of poor
quality roughage cellulose was improved with mineral sup-
plementation (32, 100). The trace mineral requirement for
cattle increased as the protein content of the ration in-
creased (100),
By using the lignin ratio, Phillipson (136) con-
cluded that 63 to 83 per cent of the digestible nutrients
disappeared in the rumen and the reticulum. Agrawala
AI.. (l) and Hale VI al. (87) reported that 58 and 48,4
per cent of the dry matter was removed in the reticulo-
rumen after six and fourteen hours, respectively. In addi-
tion, the latter reported that 78.9 per cent of the total
cellulose digestion occurred in the rumen. The period of
greatest activity was the second six hours after consump-
.tion, and the caecum digested an average of 11.6 per cent
of the cellulose in the ration. Tentative values given by
Balch (13) for crude fiber digestion in the reticulo-rumen
ranged from 25 to 49 per cent. Gray (82) concluded that 70
per cent of the digested cellulose was broken down in the
rumen. In a later report, Gray tI aL, (83) found the ex-
tent of cellulose digestion in the rumen of sheep fed on










wheaten hay and wheat straw to be about 30 per cent, on
wheaten hay about 40 per cent, and on alfalfa hay rore than
50 per cent.'
The efficiency of fiber digestion was greatest when
the moisture intake was hi&gest in relation to dry natter
intake (12), Usually, the water found in the runmen paral-
leled the level of feeding and the amount of water consumed
(31). When the plane of nutrition increased, the dry matter
tended to disappear from the rumen more rapidly shortly
after feeding. The depressions in the crude fiber digestion
of hay, known to occur when hay is finely ground or where
highly digestible carbohydrates are added to hay diets, were
not the result of changes in the rate of hay passage (12).
Wlhen the absorption of volatile fatty acids by way
of the rumen epithelium was checked, no significant dif-
ferences were found due to area (172). Ethanol, acetone,
and acetic, propionic, and butyric acids were absorbed at a
rapid rate (173)* Although glucose was absorbed at a slower
rate than the above, it was the only substance that was ab-
sorbed opposing the blood concentration. Elsden and
Phillipson (65) concluded that the size of the molecule
governs the rate of passage through the rumen epithelium;
however, butyrato was absorbed faster from the "miniature
rumen" than propionate (173).

Top rumen ingesta were higher in total nitrogen,








12
nonprotein nitrogen, organic nonprotein nitrogen, ammonia,
water-soluble sugars, volatile fatty acids, and crude fiber
but lower in ether extract, pH, and percentage of digestion
of added cellulose si _itro than bottom ingesta (158), There
was no difference in cellulose digestion occurring in the
rumen of animals fed two and ten times daily (142). The pH
was lower in the animals fed ten times per day. Earlier
results showed that the rate of fermentation and the speed
and extent of the pH variation in the rumen depend on the
diet (134),* Later, a significant positive correlation was
found to exist between the ash content of the in-esta and

the pH of the rumen (34). Bluegrass juice had a higher
buffering capacity than either ladino or alfalfa juice (5).
Sheep plasma was found to be equivalent to a 0.167
molar sodium chloride solution, and solutions in the rumen
have to be equivalent to approximately 0.180 molar sodium
chloride in order to prevent loss of water by absorption
(135). The chloride was absorbed against a concentration
gradient, but no evidence Was reported for the absorption
of sodium against a concentration gradient. Blood was found
to have a higher positive potential than rumen contents.
This potential enhanced the passage of anions but impeded
the passage of Cations from the rumen to the blood. At the
present, the movement of inorganic phosphate from the rumen
to the blood by way of the rumen epithelitu is controversial.
Possibly, a detailed study of the phosphate ion similar to








13
that carried out on the sodium and the chloride ions could
settle the question.:

Effects of Phosphorus, Sulfur. Coppler,. and
Molybdenum on Metabofic Processes
,Many reviews are available concerning different
phases of phosphorus, sulfur, copper, and molybdenun.fune-
tions (3, 4, 21, 47, 51, 52, 58, 59, 93, 116, 123, 175).
The present review of the literature concerns only those
metabolic processes closely related to the functioning of
phosphorus, sulfur, copper, molybdenum, and their inter-
relationships.

Phosphorus
Huffman (93) has listed the symptoms of a phosphorus
deficiency in their order of appearance The symptoms were
impaired feed utilization, reduced rate of gain, poor feed
consumption, depraved appetite, emaciation, rough hair coat,
stiffness in the hind legs, enlargement of knees and hocks,
and alteration of body conformation.
Beeson s, (8. (22) reported that the phosphorus re-
quirement for growing and fattening beef steers was between
0.15 and 0.19 per cent. However, the 0.15 per cent was not
adequate. Hay containing 0.10 per cent phosphorus produced
deficiency symptoms (180). But hay containing 0,15 per cent
phosphorus was apparently equal in feeding value to hay with
a higher phosphorus content. Other work has pointed out that








14
the phosphorus availability is greater in high-phosphorus
hay than in low-phosphorus hay (182).
When phosphorus was added as the orthophosphate,
an Vtro studies with rumen microorganisms:showed that cel-
lulose digestion levelled off between 20 anad 60 nmirograms
per milliliter of medium (7).
With a large excess of either calcium or phosphorus,
the other one tends to become tied up as the insoluble
tricalcium phosphate (116) Franklin ai ajg, quoted by
Huffmanr (93), produced hypocaleemia in sheep :by feedinglow-
calciunm, high-phosphorus rations. This hypocalcemia could
be prevented by supplementing .the ration with either legume
roughage or finely ground limestone, but not with bone meal*
With beef calves a critical Calcium:phosphorus ratio may
exist between 4.3:1 and 9.1:1 (61). The effect of ratios
ranging from 14:1 to 28:1 has been discussed for rats (70),
When rats received rations containing 0.014 and
0,25 per cent phosphorus, total phosphorus values of their
respective tissues indicated depletion in the femurs but
not in the soft tissues of the lower phosphorus group (70).
The effect of the calcium:phosphorus ratio and the phosphorus
level of rations for pullets has been discussed as to bone
formation and the development of reproductive organs (167),
Using guinea pigs, Maynard St a,. (115) found that a ration
containing 1.7 per cent phosphorus caused kidney damage.
Also, the findings suggested that it is the ratio of magnesium










to both calcium and phosphorus rather than to either one
alone that is of primary importance.
Clark (37) and Garton (80) found that the concen-
tration of water-soluble phosphorus ranked from 23 to 51 arid
from 28 to 81 milligrams per 100 milliliters of runen con-
tents, respectively. Clark (37) also concluded that when
cattle and sheep consume a very low level of phosphorus for
several months, "the water-soluble phosphorus content of
ruminal ingesta was maintained and that.the supplem1entation
with water-soluble phosphorus would not have any effect on
the ruminal function.
Results indicated no net absorption of inorganic
phosphorus from the ruimen, but it was absorbed from the
small intestine and orasum (37, 135, 150). Phosphorus may
enter the rumen either by way of the saliva (37, 135, 150,
155) or directly through the rumen wall (155). The runminal
epithelial linings of sheep and cattle showed structural
differences (156). With calves, a difference in phosphorus
secretion rates was found for contents of the digestive tract,
and with older cattle, a lower uptake rate on tissue phos-
phorus metabolism was demonstrated.

Sulfur
The metabolism of sulfur is usually considered a part
of the metabolism of the sulfur-containing ac:ino acids. But,
with the increased use of urea as a nitrogen source for the










ruminant, the study of the i-ietabolism of sulfur in the
runinant has increased.
Reviews have been written about the utilization of

protein and nonprotein nitrogen by the rui-minunt (35, 77,
88, 143).
'Starkls 'gJ L. (163) stated that sulfur deficiency
symptoms Core poor appetite, loss of 1ool or hair, lacrina-
tion, cloudy oyes, profuse salivation, dullness, iJealneess,
emaciation, and finally death.
toosli gt. a. (113) found that riunn nricroorganisnr
synthesized amino acids from urea. Urea increased fiber
digestion in the rumen of the sheep (71): 2Tethioninc (78,
7S, 101, 110, 112, 145) ana sulfate sulfur (79, 112, 1G9)
increased the feeding value of-urea for lai-bs. Additional
sulfur did not improve the feeding value of a ration.con-
taining 13.4 per cent protein ana 0,23 per cent total sulfur


As the level of urea increased in the rz.tion, blood
serum levels of nonprotein nitrogen increased significantly,
and the serum protein levels decreased significantly (104).
However, these rations contained 3, 5, and 7 per cent urea

and 0.171, 0.141, and 0.112 per cent sulfur, respectively,
Because there was a possibility of a sulfur deficiency in
the rations containing the higher levels of urea, additional
work vas imdertaken. This tile, the rations contained 0.171,
0.174, and 0.178 per cent sulfur, and 30, 50, and 70 per cent








17
of the nitrogen was added as urea, respectively (105). As
the level of urea increased, daily gains decreased. The
workers concluded that other factors were needed for proper
utilization of rations in which urea supplied up to 70 per
cent of the total nitrogen. However, in the latter report
nothing was said about the possibility of needing more
sulfur.
Serun inorganic-sulfate sulfur reflected the sulfur
intake (145, 179)* Serum inorganic-sulfate sulfur values
for sheep dropped from 2.0 to 4.0 milligrams to 0.2 milli-
gram per 100 milliliters as the sulfur content of the
ration dropped from 0.26 to 0.02 per cent.
Lambs consuming rations containing like amounts of
nitrogen ad 0.705 and 0,062 per cent sulfur were in positive
and negative balance, respectively, as to both nitrogen and
sulfur (163). Other lamb studies at Illinois showed that
the sulfur requirement increased when the sulfur source
changed from methionine to sodium sulfate to elemental sul-
fur (2, 164). In vitro results were in agreement (95, 171).
Other workers concluded that a sulfur content of
0.1 per cent of the total ration was adequate for milking
cows (96) and mature ewes (181). However, when the sulfur
content of a sheep ration was increased from 0.05 to 0.7 per
cent sulfur, the width of the loop of tension and relaxation
of the constant load-extension curve of wool fibers increased
with each sulfur addition (36).








18
Rumen microorganisms converted the different forms
of sulfur.to sulfide (6, 107). High concentrations of the
sulfide ion were absorbed through the rumen epithelium. (6),
While ih vitro studies showed that 30 micrograms of sulfite
sulfur per milliliter of medium inhibited cellulose diges-
tion (171), a concentration of 471 .micrograms of sulfur as
hydrogen sulfide per milliliter of rumen liquid was not
toxic to rumen bacteria in Yvjr (107). 1n vi~ro results
gave an optimum range for sulfur of from 10 to 500 micro-
grams per milliliter for rumen microorganisms (91). Radio-
active sulfate was not incorporated by the majority of
rumen microorganisms (66). Only three rumen microorganisms
were found that incorporated the sulfur from sodium sulfate
into proteins when cysteine was in the medium (07). One of
the three required both sulfate sulfur and cysteine sulfur.
Other workers found that a mutant of ECherichia cO l re-
quired both rcethionine and non-methionine :sulfur for maxi-
Kmtu growth (41).
Witli rat experiments, it was shown that the biologi
cal value of rumen ingesta was better than that of alfalfa
(159). For lambs, but not for. mature sheep,: the biological
value of alfalfa.was improved with cystine supplementation.
Work.with chicks indicated that they have a sulfur require-
ment per se (81), and a high sulfur-containing ration was

reported to raise the vitanin D requirements (153). Other

work with Irats showed that high levels of methionine








19
exerted its growth-depressing effect through the metabolism
of its homocysteine moiety (39). By reducing the protein
content of rat rations, the sulfur content was reduced in
all tissues, but the concentrations of glutathione in the
nuclear and the mitochondrial fractions were unchanged (15).

Copper
Becker fJ. al (1), Davis (51), and Underwood (175)
have discussed the following copper deficiency symptoms:
anemia, depressed growth, bone disorders, achromotrichia,
domyelination of the spinal cord, fibrosis of the myocardiunm,
anorexia, increased alkaline blood phosphatase values as
copper values decreased, diarrhea, and eventually death.
Other deficiency symptoms listed are an early, severe, and
progressive loss of cytochrome oxidase, an increased suscep-
tibility of mitochondria to aging, and a decreased rate of
phospholipide synthesis (76).

It was concluded that the copper level of feeds
controlled the level of copper in the blood and the liver
(49, 50, 54, 126). While calves did not show an ability to
correct their low-level copper stores through free-choice
selection of a copper-containing salt mixture (55), herbage
gave a more rapid recovery from copper deficiency in rats
than an equivalent quantity of copper as the ionic salt
(123). McCall (118) found that the concentration of body
copper depended upon an interaction between the copper and








20
the protein levels of the ration fed. The theory presented
was that the action of protein appears to be both in the
intestinal tract where there is a regulation of copper ab-
sorption. and in the liver where there is an increase in the
excretion of copper as the concentration approaches a toxic
level.
Baxter Aj al. (17, 18) found that the growth and the
development of copper-deficient dogs were normal except for
the bone changes. The bone disorder was characterized by
abnormally thin cortices, deficient trabeculae, and wide
epiphyses. There was an excessive absorption of bone in
addition to an increased destruction. Baxter je al, (18)
stated that the copper-deficient picture observed was
similar to an ascorbic acid deficiency which was also re-
ported by Davis (51). This included a derangement in the
osteoblastic activity, as far as the elaboration of bone
matrix was concerned, together with a cessation of the de-
struction of the calcified cartilaginous matrix while
chandroblastic activity was not impaired.
The copper requirement for rumen microorganisms stud-
iod A viAtro appeared to be between 0.36 and 1.0 microgram
per milliliter of medium (91, 148) while concentrations of
1.5 (91), 5.36 (148), and 10.0 (120) micrograms per millili-
ter reduced the activity of rumen bacteria.










Molybdenum
Becker t aL. (21), Davis (51), and Underwood (175)
have discussed the symptoms of molybdenum toxicity, Unlike

the other minerals discussed, molybdenum elicited different
symptoms from different species. In ruminants, the symptoms
were diarrhea, loss of weight, anorexia, anemia, achromo-
trichia, bone abnormalities, and an increased rate of excre-
tion of phosphorus from the body. In rats, rabbits, and
guinea pigs, the symptoms were retardation of growth, loss
of weight, and anorexia. But in young rabbits, the toxic
syndrome was characterized by anorexia, loss of weight,
alopecia, dermatosis, and anemia. Also, other studies with
young rabbits concerning bone formation showed that the dis-
order consisted of a diffuse osteoporosis and possibly no
disturbance in the calcification mechanism (147). Earlier,
it was found that a molybdenum toxicity caused irregular
redeposition patterns in bone (109).
Thomas and Moss (168) reported that in calves high
levels of molybdenum did not reduce feed consumption or
cause diarrhea after one to three weeks, Ellis et al (64)
using ;n v&Liv and Jn vitro studies found that a copper-

molybdenum supplementation improved cellulose digestion.
Other l vitro results showed that 20 micrograms (148) per
milliliter of medium reduced cellulose digestion and that
rumen bacteria would tolerate 100 to 1000 micrograms (120)
per milliliter of medium,








22

Underwood (175) has listed sore rcpcrts that indi-
cated that molybdenum may be required for animals in small
amounts. With this in mind, Richert and Westerfeld (146)
found that the aldehydc-oxidizing activity of a liver ho-
mocenate was nolybdenum dependent. Another article report-
ed that low concentrations of sodium Liolybdate inhibited
all phosphatases and did not have any effect on ita--
glUcosidases, sucrase, aiylases, lipases, ureaseo oxidases,
peroxidases, catalases, deliydroconases, or carboxylases
found in plant tissue extracts (8S).

Phosphorus Sulfur, Copper, Molybdenum
Interrelationship
The copper-molybdenum, the sulfur-moolybdeniu, the
copper-sulfur-molybdenum, the copper-rmolybdenum-phosphorus,
and other interrelationships will be reviewed in this sec-
tion.

Cox (43) found that high dietary levels of molybde-
num had no effect on copper storage when some of the rations
contained. 25 to 30 per cent casein. Reviews (43, 47, 52,
175) and other articles (3, 8, 46,, 48, 84, 102) have reported
conflicting results concerning copper metabolism which may
be explained partly by the copper-protein interaction re-
ported by HcCall (118). N eilands j .l (129) found that
when rations contained 18 per cent casein and 400 micrograms
of molybdenum per gram, approximately 97 ricrograms of cop-
per added as copper sulfate per gram of ration gave more








23
protection from the molybdenum than 77 micrograms of copper.
The activities of the enzymes, cytochrome c oxidase,
cytochrome c reductase, nitrate reductase, acid phosphatase,
and reduced triphosphopyridine nucleotide diaphorase, of
Neurosporn crassa were either enhanced or reduced by high

concentrations of copper or molybdenum (40).

Van Reen and Williams (176) found that sulfur con-
taining compounds alleviated the toxic effect of molybdenum
for liver and kidney alkaline phosphatase. Also, liver
sulfide oxidase was depressed in molybdenum toxicity (124).
In embryonic cartilage, ammonium molybdate inhibited sul-
fate fixation without inhibiting respiration (29).
The effect on molybdenum toxicity of various levels
of sulfur added as sodium sulfate have been studied in the
rat (122, 124),
Molybdenum appeared to reduce the sulfur content of
liver, muscle, and bone, bone weight (69), and the percentage
femur ash (122).
Dick (58, 59) and Allcroft and Lewis (4) have re-
viewed the effects of molybdenum toxicity in relationship to
copper and sulfur in sheep. When the molybdenum was increased
from 1 to 100 milligrams per day, the blood molybdenum level
increased until it levelled off, and the copper level of the
blood and the tissues did not change with an intake of 3.0
to 10.0 milligrams of copper and 0.5 gram or less of sulfate
per day. With an intake of 3.0 to 10.0 milligrams of copper










and 2.0 to 10.0 grams of sulfate per day, there was a de-
creased blood and tissue molybdenum concentration, a reduced
absorption of molybdenum from the intestinal tract, an
increased urinary excretion of molybdenum, and the effect of
blood and tissue copper depended upon the molybdenum intake.
Pertinent data for the above statements have been provided
by several articles (56, 57, 185, 186).,
When molybdenum and phosphorus were administered
simultaneously to rats, this resulted in an increased phos-
phorus uptake (51). But when molybdenum was fed prior to
phosphorus administration, a reverse effect on phosphorus oc-
curred. Shirley .Al (153) found a change in the phos-
phorus metabolism of steers on high molybdenum* Not only
was the pathway of normal excretion changed from urine to
feces, but also the rate of excretion, as exhibited by the
high phosphorus content of the feces and the very low con-
tent of the urine. The addition of copper sulfate and
sodium molybdate together resulted in less effect upon the
pathway of phosphorus excretion than when each was given
individually, but it did result in slightly lower tissue
phosphorus values* Also,, molybdenum and phosphorus decreased
the accumulation of copper in the liver (51).
Using calcium-45 and phosphorus-38, no failure was
found in the absorption of calcium and phosphorus in rabbits
receiving 0.20 per cent molybdenum (68). It appeared that
molybdenum was more toxic when superimposed on an 018 per
cent phosphorus ration than on a 0.40 per cent phosphorus








25
ration (121). However, differences were reported for the

liver copper concentrations, but a more rapid resorption
of calcium-45 from the site of original laydown in the

epiphysis of the long bones was proposed. A condition simi-
lar to a copper deficiency was reported, but pastures had
a high calcium:phosphorus ratio and were considered adequate
in copper (177).

Apparently, the effect of varying levels of copper,
molybdenum, and zinc in the ration of calves did not cause
any abnormalities in growth or in calcium, copper, phosphorus,
hemoglobin, hematocrit, or zinc blood values (154).
Vitamin E reduced the urinary excretion of phosphorus
caused by molybdenum, and it increased the retention of

molybdenum in the intestine, liver, and heart (128).

When a ration contained 7.6, 2.4, 91, and 300 micro-
grams of copper, molybdenum, manganese, and sulfate per gram,
respectively, no effect was found when the manganese was
increased (127). By increasing the sulfate to 0.55 per cent,
there was a reduction in liver copper and an increase in
serum inorganic sulfate. By increasing the molybdenum to
9.2 micrograms per gram of ration, there was a reduction in
the blood and the liver copper at the higher sulfate level.
But the lower sulfate level caused no definite effect.













EXPERIM-h1ENTAL PROCEDURE


Experiment I

Study of Cellulose Digestion in the Rumen
of Rumen-Fistulated Steers
Four rumen-fistulated Jersey steers were used in
this experiment. Steers designated by the numbers, 1, 2,
and 3, were twenty months of age and weighed 585, 570, and
620 pounds, respectively, at the beginning of the experi-
ment in June, 1958. Steer designated number 4 was approxi-
mately four years of age and weighed 1220 pounds at the
beginning of the experiment.

The steers were fed 1.9 pounds of the ration (dry
matter basis), given in Table 1, per 100 pounds of body
weight per day. This was fed in equal aotunts at 7:50 A.N.
and 4:30 PMl. Water was supplied ad libitum. The chemical
composition of the ration is given in the Appendix, Table
38, as determined by various methods (26, 45, 108, 119, 131),

This experiment was conducted during eight consecu-
tive periods. A period lasted for 24 days, For the first
four days, the steers were fed the ration given in Table 1

plus Treatment S-1, given in Table 2, which did not include
any phosphorus, sulfur, or molybdenum supplementation. From
Day 5 through Day 24, the steers were fed the ration given
in Table 1 plus one of the treatment combinations given in

26









TABLE 1

COMPOSITION OF STEER RATION USED FOR STUDYING CELLULOSE
DIGESTION BY AN a yMI BAG TECHNIQUE IN EXPERIMENT I
AND BY AN JN VITRO TECHNIQUE IN EXPERIMENT II


Ingredient t


Timothy Hay
Cotton Linters
Solka-Floc (Softwood)
Sugar Cane Bagasse
Cerelose
Starch
Corn Oil
Urea
Mineral Mixture2
0.20z Ca as 4
0.01l Mg as 3
0.002, Zn as
0.002 o Min as a
0.0001% Co as 4
0.00002% I as K:
(Variable) NaCI a'
of eac


36.0
5.0
5.0
6.3
21.0
21.0
4.0
2.0
0.7
LaCO3
1g003
6nC03
AnC03
CoCl12.6H2
I
Aided to make the added Na content
h ration equal 0.93; Na


1The chemical- composition of this ration can be
found in the Appendix, Table 38. Vitamin A was added as
the palmitate to give 5,000 I.U. of vitamin A per pound
of ration fed.
2The minerals are expressed as a percentage of the
total ration. The concentrations of phosphorus, sulfur,
copper, and molybdenum used in the in _ivj and the n vitro
studies are listed in Tables 2 and 3, respectively,









28

Table 2 which supplied various levels of phosphorus, sulfur,

and molybdenum to the ration.


TABLE 2

STUDY OF CELLULOSE DIGESTION IN YIo.
CONCENTRATIONS OF PHOSPHORUS, SULFUR, AND MOLYBDENUM
USED TO SUPPLEMENT THE STEER RATION OF TABLE 1


p
added
as
NaH2PO4 H2O


0.12


0.00
0.48
0.48
0.00
0.48


Added Mineral
S
added
as
Na2S04


0.00
0.00

0040
0.00


0.40
0.00
0.40
0.40
0.40


Tfo
added
Sas_
Na2MoO4- 2120

pop.m.1
0.50
0.00
0.00
0.00
25.00
0.00
25.00
25.00
25.00


1Parts per million.


For 30 days before Period 1, the steers were allowed

to adjust to the ration with Treatment S-1 added. Period

1 was used as a time of adjustment for the technique that

was used. During this period, all the steers received Treat-

ment S-0 which added 0.12 per cent phosphorus as NaII2P04*120

and 0.5 part per million of molybdenum as Na2Mo04.2H,0. In

addition, six parts per million of copper as CuC12*2H20 were

added to the ration from the fifth through the eighteenth

day.


5-0
S-1
S-2
S-3
S-4
S-5
S-6
S-7
S-8


. . . . . . Ir .


-1-- --


__ _










Once during the first three days of each period,
approximately 20 per cent of the rumen contents of each
steer were removed and mixed, and equivalent amounts were
returned to the rumen of each steer in order to encourage
as similar a microflora as possible in the various rumina.
Two types of rumen cannulae were used, The plastic
type was described and used by Sala (146), and the pneu-
matic cannula and bung type was manufactured by the Avon
India Rubber Company, London, England.

Study of steer variation (uniformity trial)
Periods 4 and 7 were used to evaluate a technique
for studying cellulose digestion in steers with rumen
fistulas. The four steers received Treatment S-0. Vari-
ability among steers within periods and between periods
for the digestion of cellulose from different sources using
the JA vLio bag technique and for the phosphorus, sulfur,
and molybdenum concentrations of rumen ingesta were deter-
mined. The sources of cellulose were Solka-Floc cotton






1Cellulose digestion coefficient is used to mean
the percentage cellulose of a given sample that is broken
down.
2The Solka-Floc SW-40A used was a purified cellulose
prepared from soft woods by the Brown Company, New York,
Uew York. The purification process involved a sulfite di-
gestion followed by bleaching.










linerss, and sugar cane bagasse

The Ijn vivo bag technique involved the suspension
in the rumen of a 2 X 5 inch nylon bag containing 1.8 to
3.2 grams of the source of cellulose to be assayed. This

bag was secured to the cannula with a nylon string. The
length of string from the cannula to the top of the bag was
80 per cent of the distance from the cannula to the most
ventral part of the rumen. A twenty-gram glass marble was

placed inside the bag as a weight. The nylon material was
permeable to bacteria and had 80 X 160 threads per inch.
The bags were put in the rumina before the morning
feeding on Days 19, 21, and 23 of each period. Usually,
nine bags, i.e., three per each of the three sources of
cellulose, were put in the rumen at one time. The bags
were removed 48 hours later. At this time, the larger par-

ticles of the rumen digesta were rinsed off the outside of
the bags with cool water, and the bags were suspended in
70 per cent ethanol for a minimum of one hour to stop the
bacterial action. The samples were transferred to a 100

1The cotton linters used were a purified cellulose
prepared from the seed hairs of cotton by the Hercules
Powder Company, Wilmington 99, Delaware. The purification
process involved an alkali digestion under pressure fol-
lowed by bleaching. The processing raised the crystal-
linity.
2The sugar cane bagasse used was an unpurified source
of cellulose prepared from the crushed stalks of sugar cane.
The crushed stalks were ground to pass a one-quarter inch
screen. This sugar cane bagasse contained 46.8 per cent
cellulose (dry matter basis) on analysis by the method of
Crampton and Maynard (45),










milliliter test tube for cellulose determination by the
method of. Crampton and Maynard (45), This procedure was
applied as follows: (a) the supernatant was removed from
the test tube containing the sample by centrifuging for
two to three minutes at approximately 1,200 times gravity,
(b) the supernatant was decanted, and twelve milliliters of
glacial acetic acid and two milliliters of concentrateda
nitric acid were added to each residue, (c) after mixing,
the tubes were heated in a boiling water bath for twenty
minutes, and the contents were filtered while hot through
a Gooch or a Selas Porcelain crucible# (d) after using hot
water in transferring the residue to the crucible, the:
residue was washed with hot 95 per cent ethanol, hot benzene,
hot 95 per cent ethanol, and ether in that order, (e) the
crucible containing the sample was dried at1050 Centigrade
for 24 hours, and (f) then, the cellulose was determined by
the weight loss on ignition in a muffle furnace at 6600
Centigrade for ten to twelve hours. This method was modified
from that of Crampton and Maynard (45) by filtering in steps
(c) and (d) above instead of centrifuging.
Sarples of rumen liquid were taken on the morning of
Day 25, or fifteen hours after the last feeding, for phos-
phorus (26), sulfur (108), copper (119), and moisture anal-
yses. For the moisture determinations, the samples were
dried for 24 hours at 1050 Centigrade, and the percentage
rioisture was determined by the weight loss.








32
To obtain the rumen liquid the ingesta were strained
first through four layers and then through eight layers of
cheesecloth of grade 50*
The.statistical analyses of the data followed the
methods of Snedecor (160) and Cochran and Cox (38).

Study of the effect of phosphorus, sulfur, and ,olybdenum
additions to a steer ration
A split-split plot in a 23 factorial experiment with
each of the two replications divided into two randomized1
blocks of four units each with the phosphorus-sulfur-iclyb-
denum interaction confounded was used to study the effect of
phosphorus, sulfur, and molybdenum on the digestion of cellu-
lose and on the phosphorus, sulfur, and copper concentrations
of the rumen liquid.
The treatments used are given in Table 2. The,
treatments used for a given steer during Periods 2, 3, 5,
and 6 are given in Tables 12 and 30. During Period 8,
Steers 2 and 3 received Treatments S-4 and S-2, respectively.
The sources of cellulose and the techniques used in
this trial were the same as those used in the uniformity
trial* In the breakdown of the treatment significance of
the analysis of variance, treatment is used to mean factorial
effect. However, because of the variation among steers as
shown by the uniformity trial, a correction of the digestion
coefficients for cellulose was made before the data of this
trial were analyzed statistically, This correction was based

1The randomization was controlled so that no steer
received the same treatment twice.










on the digestion coefficients of Solka-Floc and cotton
linters obtained during the uniformity trial. The diges-
tion coefficient for cellulose from sugar cane bagasso was
not included in the correction because of its low and more
variable response. The correction was calculated as fol-
lows: (a) the average digestion coefficients of Solka-
Floe and cotton liners as given in Table 6 were added for
each steer, (b) Steer 1 was assigned a value of.100.0 per
cent, and (o) with Steers 1, 2, 3, and 4 having values of
73.2, 94.1, 85.0, and 117.1, respectively, each value was
divided by 73,2. Thus, the digestion coefficients of cel-
lulose from Solka-Floc, cotton liners, and sugar cane
bagasse for Periods 2, 3, 5, and 6 as found in the Appendix,
Table 30, for Steers 1, 2, 3, and 4 were divided by 1.000,
1.28G, 1.16i, and 1.600, respectively.

ExperimentII

Study of Cellulose Digestion in the
Artificial Rumen

The comparison of cellulose di estion by rumen inoculum
taken from different steers
For this study the four fistulated steers were fed
the ration given in Table l plus Treatment S-O given in
Table 2 as previously described. Period 7 was extended five
days so that this experiment could be completed. Humen
inoculum was sampled every second day for five consecutive










times just before the morning feeding .
This Inoculum was first filtered through four
layers of cheesecloth into a beaker placed in a water bath

maintained at approximately 400 Centigrade. After the
sar:ple fror a steer was added to a beaker, it was covered
with Parafiln. Then the samples of inoculum were taken
to the laboratory. Here, they were refiltered through
eight layers of cheesecloth into a flask that had been
charged with carbon dioxide previously.

The previous day, a known amount of Solka-Floo,

approximately 0.28 gram, was added to the number of 75-
milliliter test tubes or artificial runina needed. Those
test tubes were placed in a.water bath maintained at 390
to 400 Centigrade overnight. Then 25 milliliters of inocu-

lumr were added to each tube; three tubes per source of
inoculum were used. The test tubes were then arranged
randomly in a series, and carbon dioxide was bubbled into
the mixture to Cid in mixin. and in maintaining anaerobic

conditions*

Samples of each inoculum were saved for cellulose
analyses. This was taken into consideration when the di-
gestibility of cellulose was calculated. The pH of the
samples of inoculum ranged from 6,6 to 6G8. The concentra-
tions of phosphorus, sulfur, and copper in the samples are

given in Table 10 for Period 7. All analyses for Experiment
II were made as described for Experiment I.








35

The comparison of the digestion of cellulose from different
sources and from theU sarie source with different partilel
Sizes
For this study, inoculum was taken from Steer 1.. The
steer was fed the ration given in Table 1 plus Treatment S-0
given in Table 2 as previously described., The procedure was
the same as previously described with the exception that 15
milliliters of.rumen inoculum were used per test ;bae.: Ten
milliliters of Treatment 17T,- given in Table 3, were added
to each tube. The sources of cellulose used were Solka-Floc,
cotton linters, sugar cane bagasso, and cotton liners and

sugar cane bagasse ground to pass a forty mesh screen.
The concentrations of phosphorus, sulfur, and copper

for the inoculum from Steer 1 for this trial are given in
Table 10 for Period 8.

Theeoffect of different concentrations of phosphorus, sul-
fur. cooper, and molybdenum on cellulose Solka-F oc)
digestion
A 24 factorial experiment in five completely ran-

domized replications was used in this study. The various

treatment combinations of phosphorus, sulfur, copper, and
molybdenum used are given in Table 3. The inoculum was ob-
tained from Steer 1, and the procedures used were the same
as those used in the previous trial. However, the number of
test tubes or artificial rumina used per day was increased
to a minimum of 34. Day 3 gave rather low values, possibly
because the carbon dioxide tank was almost empty which









TABLE 3
STUDY OF CELLULOSE DIGESTION fl ITROR
CONCENTRATIONS OF? PHOSPHORUS, SULPUR, COPPER, AND MOLYBDENUM
USED TO SUPPLEIAINT THE ~EDIUM FOR IIU'IEN M:IClOOGANISMS

Added Mineral1 (jg/ml2)
P S Cu Mo
Treatment added added ad added added
as as as as
NaH2PO4*H20 Na2SO4 CuC1221Hg0 Na2Mo 0 211g2

1T 0.00 0.00 0.00 0.00
2T 0.00 0.00 0.00 150.00
3T 0.00 0.00 1.50 0.00
4T 0,00 0.00 1,50 150.00
ST 0.00 750.00 0.00 0.00
GT 0.00 750,00 0.00 150,00
7T 0.00 750.00 1,50 0.00
8T 0.00 750.00 1.50 150.00
9T 750.00 0.00 0.00 0.00
10T 750.00 0.00 0.00 150.00
11T 750.00 0.00 1,50 0.00
12T 750.00 0.00 1.50 '150.00
13T 750.00 750.00 0.00 0.00
14T 750.00 750.00 0.00 150.00
15T 750,00 750.00 1.50 0.00
16T 750.00 750.00 1,50 150.00
17T 75.00 75.00 0.15 1.50

1Mixture added to each treatment to give 300 Bg/ml
Ca as CaClo, 80 ag/ml Hg-as Mg0lh, I p1g/ml Hn as lnC12,
3 ag/ml *e as FeC13*GI20, 300 gh /m K as KC1, 750 Jg/ml N
as urea, and Na as NaCI to give all treatments 1.668 ag
Na/m.
Micrograms per milliliter.








37
reduced the flow of carbon dioxide through the tubes and

possibly caused less mixing action.
In the breakdown of the treatment significance of
the analysis of variance, treatment is used to mean fao-
torial effect.

Experiment III

Study of the Effect of the Interrelationship of
1ianerals on Growth and Bone Formation
in Rats
All the rats used in the following experiments were
of the Long-Evans strain and were obtained at weaning from
the Animal Nutrition Laboratory stock colony. At weaning,
the rats were 20 to 24 days of age.
During the experimental periods, the animals were
housed in stainless steel cages. All rations were fed ad
libitum from earthenware feeders, and distilled water was
supplied from polyethylene bottles with a glass outlet. A
sheet of stainless steel was used to protect the bottles from
being gnawed by the animals.
The ingredients of the ration used are given in
Table 4. The various treatment combinations for Trials I,
II, and III are given in Table 5.
The procedures for the laboratory determinations
wore as follows. For the moisture determinations, the samples
were dried for 24 hours at 1050 Centigrade and the percentage
moisture was determined by weight loss. A twelve-hour










TABLE 4
COMPOSITION OF RAT RATION USED FOR STUDYING TIIU EFFECT OF THE
IITERIfELATIOI;SIIIP OF PH.OSPHIORUS, SULUR, COPPER. AND
MOLYBDE NU ON GROWTH AND BOiJE FORMATION IN
InPEI RIFIT III

Ingredient S'


Sucrose
Starch
Casein
Corn Oil
Vitamin Mixture2 (Nutritional Biochemicals Cop.)

Vitamin A Concentrate (200000 I.U./gm) C.50
Vitamin D.Concentrate (400,000 I,T./gn) 0.25
Alpha Tocopherol 5.00
Ascorbic Acid 45.00
Inositol 5.00
Choline Chloride 75,00
Henadione 2.25
Para-aminobenzoic Acid 5,00
Niacin 4,50
Riboflavin 1.00
Pyridoxine Hydrochloride 1.00
Thianine Hydrochloride 1 00
Calcium Pantothenate 3.00
Biotin 0.020
Folio Acid 0.090
Vitamin B-12 0.00135
Dextrose 847,39


Mineral Mixtu
0,450
0.03;7
0.007Th
0.00251'
0.00251,
0.0001'p
0.00002
0.0, 0.01,

0.0, 0.033
.0,0 0,3{3+


38.0
30.0
18.0
4.0
1.0 or 1*5















8.5 or 0.0


Ca as CaCO3
Mg as MCO3
Fe as FeC13*6H20
Zn as ZnCO3
Un as IInC1224UO1
Co as CoC12-*620
I as KI
Cu in Trials I and II; 0.0,0.003% Cu in Trial
III as CuCl2*H20O
No in Trials I and III; 0,03 Mo in Trial II
as Na2Mo04-21[0 -
S in Trial I1 0.0,0.165,0.30.6O. S in Trial
II; 0.0,0.OG-; S in Trial III as Na2SO4


res










TAILE 4--Continued


Ingredient f

0.08, 0,7.8o P added as KII PO4 (0.40o P) and NaINPO420
(0.38, P) in Trial I; 0.08,- P added as
NaHI2PO 'HoO in Trials II and III
(Variable) Na as nOa03 to nnl:o all rations equal in
Na content
(Variable) Cellulose as Solka-Floc to ahol 100%

1The chemical' composition of this ration can be
found in the Appendix, Table 38.

2, .was used for Trials I and II and 1,5' for
Trial III.
3The minerals are expressed as a percentage of the
total ration. The concentrations of phosphorus, sulfur,
copper, and molybdenum used in this study are listed in
Table 5.

anhydrous ether extraction with the Goldfisch apparatus
manufactured by the Laboratory Construction Company, Kansas
City,1Missouri, was used to remove the fat from the femurs.
After the dry, fat-free weight of a femur was determined,
the fear ash was determined by the weight loss on ignition
in a muffle furnace at 6GO Centigrade for ten to twelve hours.

The percentage ash of a femur was determined by dividing the
total femur ash weight by the total dry, fat-free femur
weight. The ash per femur in milligrams per gram of live
body weight was deterinced by dividing the total femur ash
weight in milligrams by the total live body weight in grams,
Feed efficiency data and data showing the paritioning of
weight gains of the rats, by two-week periods, are given in









TABLE 5
STUDY OF GROWTH AND BONE FORMATION IN RATS.
CONCENTRATIONS OF PHOSPHORUS, SULFUR, COPPER, AND HOLYBDEInUI
USED TO SUPPLEMENT THE RAT RATION OF TABLE 4

Treatment p .Added Minerall
addE r adted added aded ad eed
Experiment III a s as as as
Trial NaH24*12O Na2SO4 CuC12*2H0O Na2Mo04H20
I II III and KH2PO4. a

% % p.p.m. p.p.m.
1P1 3R1 0.08 0.00 0.0 0.0
1112 2R1 3R2 0.08 0.00 0.0 300.0
1R3 0.08 0.00 100.0 0.0
1R4 2R5 0,08 0.00 100.0 300.0
1R5 0.08 0.33 0.0 0.0
1R6 2R3 0.08 0.33 0.0 300.0
1R7 0.08 0.33 100.0 0.0
1R8 0.08 0.33 100.0 300.0
R89 0.78 0.00 0.0 0.0
1R10 0.78 0.00 0.0 300.0
1Rll 0.78 0.00 100.0 0.0
1R12 0.78 0.00 100.0 300.0
1R13 0.78 0.33 0.0 0.0
1R14 0.78 0.33 0.0 300.0
R115 0.78 0.33 100.0 0.0
1R16 0.78 0.33 100.0 300.0
1R17 0.22 0.11 6.0 1.0
2R2 0.08 0.165 0.0 300.0
2R4 0.08 0.66 0.0 300,0
3R3 0.08 0.00 30.0 0.0
314 0.08 0.00 30.0 300.0
3R5 0.08 0.06 0.0 0,0
3R6 0.08 0.06 0.0 300.0
3R7 0.08 0.06 30.0 0.0
318 0.08 0.06 30.0 300,0

Ini Trials I, II, and III of Experiment III enough
Na was added as Na2CO to mako the ration contain added Na
of 0.75, 0.75, and 0.34<, respectively, with 2R4 at 1.21%
added Na.
2In treatments 1R9 through 1R16, 0.38p P was'added
as NaH2PO4.H20 and 0.40% P as KH2P4 in ration 1R17, 0.10,
P was added as NaH2PO4*H20 and 0.12p P as KH2P04, and all
other P additions were made front NaRH2P04loO0.










the Appendix for each trial.
The statistical analyses of the data followed the
methods of Snedccor (100) and Cochran and Co. (38). In the
breakdown of the treatment significance of the analysis of
variance for Trials I and III, treatment is used to r.ie~n
factorial effect,

Trial 1. The effect of different concentrations of phos-
phomrus sulfur, connpcr, nrf ,ol bdenuJn on growth a'1d bone
foraetion
The weanling rats, 71 males and 73 females, were
I
randomly allotted to the Treatments 11 through 1R17 given
in Table 5. A 20 factorial experiment with four complete
replications was used in this study. Treatment 1R17 was
not a part of the 25 factorial but was used as a control,
The animals were weighed at the beginning of the
experiment and at Wveely intervals for the following eight
weeks. After 56 days, the 144 rats were sacrificed. All
the ferurs were taken for total ash and percentage ash
analyses. The liver and a femur were taken from two females
from each treatment for mineral analyses. Those animals
selected are shown in the Appendix, Table 32. The livers
were analyzed for phosphorus (26), sulfur (108), and copper
(119). On analysis, the sulfur concentration of the livers

1The randomization was controlled so that four male
and four female rats were allotted to each trreat:ent, and so
that no two rats from the same litter were allotted to the
save treatment. Because of an error in sexin-, three treat-
ments had a five-three split.










was approximately 1.35 per cent when expressed on a dry
matter basis. The various treatments did not appear to

affect the liver sulfur concentrations. The femurs were

analyzed for phosphorus (26).

Trial II. The effect of sulfur and copper additions to a
ration containing 300 parts per million of molybdenum on
growth and bone formation

Thirty female weanling rats were randomly allotted
to Treatments 2R1 through 2R5 given in Table 5.

After 56 days, the rats were sacrificed. Weekly
weights, percentage femur ash, total femur ash, milligrams
ash per femur per gram of live body weight, and the statis-

tical analyses of the data were determined as outlined for
Trial I.

Trial III. The effect of different concentrations of sulfur,
copper, and molybdenum on growthh and bone formation
Forty-eight female weanling rats were randomly

allotted to the Treatments 3R1 through 3R8 given in Table 5,
A 23 factorial experiment with six complete replications was

used in this study.

After 35 days, the rats were sacrificed. This experi-
ment was terminated after 35 days rather than after 56 days
because the animals were larger at weaning and the levels of
the minerals being studied were reduced. Thus, it was nec-

essary to collect the data at a more critical age because ihe

treatment effects were expected to be smaller,








43
Weekly weights, percentage femur ash, total femur
ash, milligrams ash per femur per gram of live body weight,
and the statistical analyses of the data were determined
as outlined for Trial I. The hemoglobin was determined
using the acid-hematin method of Cohen and Smith, quoted
by Cox (43).












RESULTS AND DISCUSSION


Study of Cellulose Digestion in the Rumen
of Rumen-Fistulated Steers

Study of steer variation uniformityt trial)
Average rumen digestion coefficients for cellulose
from different sources by different steers for two different
periods are summarized in Table 0. A summary of the statis-
tical analysis of these data is shown in Table 7. The aver-
ages of a limited number of rumen digestion coefficients
for cellulose from the Timothy hay used in the steer ration
were 32.3, 37.9, 35.4, and 49.3 per cent for Steers 1, 2,

3, and 4, respectively. The digestion of the Timothy hay
cellulose was determined by the same procedure that was used
for the other cellulose sources; however, the Timothy was
ground to pass a twenty mesh screen.
Steer 4, the largest and the oldest, had significantly
higher rumen cellulose digestion coefficients than the other
steers. It is felt that a larger number of animals should
be used to verify this observation because these data suggest
an increased efficiency in the utilization of cellulose and
possibly poor quality roughage as cattle grow older.










TABLE 6

EFFECT OF STEER VARIATION ON THE DIGESTION OF
CELLULOSE FROM DIFFERENT SOURCES1,2


Steer 1 2 3 4
Solka-Ploc
56.1 60.6 51.9 71.3
42.3 61.5 54.2 78.3
Average 49.2 61.1 53,1 74.8

Cotton Linters
25.1 30,3 30.2 39.6
23.0 35.6 33.6 46.7
Average 24.0 33.0 31.9 42.3
Surar Cane Barasse
9.0 16.9 3.4 23.1
0.6 11,4 5.6 12.6
Average 4.8 14.1 4,5 17.9

IThis is a summary of the data of Periods 4 and 7
listed in the Appendix, Table 30.
2All steers were fed Ration S-0 as given in Table 2.
The values for the amount of cellulose digested are given
as a percentage.

A highly significant difference for cellulose di-
gestion among sources was shown. The digestion of cellulose

from Solka-Floc was the highest; cotton liners, intermediate;
and sugar cane bagasse, lowest. The presence of lignin in

the sugar cane bagasse possibly accounts for a part of the
reduction in cellulose digestion. However, if lignin were

the only cause, the difference between the digestion of

purified cellulose from Solka-Floo and cotton linters is not

explained. Tomlin and Davis at the Nutrition Laboratory,

University of Florida, are presently working on the










TABLE 7
STATISTICAL ANALYSIS OF THE DATA
GIVEN IN TABLE 6


Source DF SS MS F

Total 7 12,440.20
Period 1 53.70 53,70
Steer (S) 3 11,437.68 3,812.56 12,05*
4 vs 1+ 2 +3 1 8,681.96 8;681.96 27.45*
2 vs 1+ 3 1 2,367.36 2,367.36 7.49
1 vs 3 1 388.36 388,36 1.23
rrror (a) 3 948.82 316.27

Total 16 17,073.24
Time (Ti) 2 728.55 364,28 0.88
Ti X S 6 '573.91 92.32 0.22
Error (b) 8 3,330.58 416.32

Total 48 108 445.56
Source (So)1 2 87,317.3443,658.67 493.54**
SCB vs SF + OL 1 62,076,07 63,076.07 701.74**
SF vs CL 1 25,241.27 25,241.27 285.34**
So X S 6 1,219.56 203.26 2.30
So X Ti 4 150.18 39.79 0.45
So X S X Ti 12 .553.24 46.10 0.52
Frror (c) 24 2,123.00 88.46

Total 215 123;111.54
Determination 144 14,065.98 101.85

*Significant probability less than 0.05.
**Highly significant probability less than 0.01.

1SCB stands for cellulose of sugar cane bagasse; SF,
Solka-Floc; and CL, cotton liners.








47

hypothesis that the degree of crystallinity of cellulose is

negatively correlated with cellulose digestion. Their re-

sults may explain a part or all of the difference in the
digestion of cellulose by rumen microorganisms between these
two purified cellulose sources. The possible effect of
particle size will be discussed in rxporirent II.
The size of the mean square for determination indi-
cates that triplicate determinations are adequate for de-
termining rumen cellulose digestion when using the technique
and the sources of cellulose used in this experiment.

Study of the effect of phosphorus, sufuer, nd molybdontmn
additions to a steer ration
The effectsof-different dietary levels of phosphorus,

sulfur, and molybdenum on average rumen digestion coeffi-
cients for cellulose from different sources for two dif-
ferent periods are summarized in Table 8. A summary of the
statistical analysis of these data is shown in Table 9.
With 0.54 per cent phosphorus in the ration, the
cellulose digestion :as significantly lowered but only by a
small amount, With 0.55 per cent sulfur in the ration, there
was a highly significant increase in cellulose digestion.
A part of this increase could be attributed to the correction
of the data; however, the increased cellulose digestion
caused by the sulfur supplementation is real. From these
data and the results presented by Lassiter pj l. (105), it
appears that when urea supplies 70 per cent of the nitrogen









TABLE 8


EFFECT OF TREATMENTS GIVEN IN TABLE 2 ON THE DIGESTION
OF CELLULOSE FROM DIFFERENT SOURCES1

Ration, Steer (S) S-1 S-2 8-3 S-4 S-5 8-6 S-7 S-8
and Digestion
Coefficient (DC), S DCS DCS D S DC S DC S DS DC S DC
S 'Solcka-Ploc
2 73.7 4 64.5 3 87.4 1 43.4 1 68.9 4 74,2 3 85.9 2 93.0
3 74.5 1 35.9 2 88.9 3 79.2 4. 97.1 2 32.7 1 96.8 4 80.0
Average 74.1 50,2 88.2 61.3 83.0 53.5 91.4 86.5
Uniformity Tria1-'
Average 57.1 62.0 57.1 51.2 62.0 68.0 51.2 68.0
Cotton winters
2 47.4 4 41.4 3 67.7 1 22.1 1 52.9 4 41.0 3 62.6 2 72.7
3 50*2 1 19*0 2 65.1 3 48*2 4 74.5 2 15.3 1 69.4 4 53.9
Average 48*8 30.2 66.4 35.2 63.7 28.2 GG.0 3.3
Uniformity Trial
nfAverage 32,5 33*6 32.5 28*0 33.6 38.1 28.0 38.1

SAurar Cane 3aDanes
,2 16*6 4 10.8 3 18*2 -1 5*9 1. 11.5 4 23.5 3 18.5 2 18.9
3 13*9 1 0.1 2 27*4 3 11.8 4 23.0 2 0*1 1 25.1 4 23.0
Average 15.3 5.5 22.8 8.9 17.3 11.8 21.8 21.0
Uniformity Trial 9
Average 9.3 11*4 9*3 4*7 11*4 16,0 4*7 16*0


1This is a summary of the
Table 30.


data of Periods 2, 3, 5, and 6 listed in the Appendix,


2This is the average digestion coefficient given in Table 6 for cellulose for
the different sources given by the two steers when both were fed actionn S-0.










TLTTLE 9


STATISTICAL A.YALYSIS OF TIWE DATA
GIVEII IN TJTLE 8


Source DF SS iMS F


Total
Period
Treatment (T)1
Phosphorus (P)
Sulfur (8)
PXS
P X S
Ilolybdenum (M)
PSX M
S'X M
Error (a)


24,108.55
-558.42
190220.72
5;535.36
12,757,70
81.00
3.36
43,78
307.42
4,329.41


-186.14
3;203.45
5;535.36
12,757.70
81.00
3.36
43.78
307.42
721.57


Total 32 2,800.51
Time (Ti) 2 800,17 400.08 5.56*
Ti X T 14 -849.44 60.67 0.84
Error (b) 16 1,150.90 71.93


Total 96
Source (So)2 2
SOB vs SF + CL 1
SP v. CL 1
So XT 14
So X Ti 4
So X Ti X T 28
Error (c) 48


860926.37
53;709.22
45; 290.46
8,418.76
3,355.69
240,83
-597.45
2,114.12


26;854.61
45;290.46
8,418,76
239.69
60,21
21.34
44.04


-609.77*
1,028.39**
191.16**
5.44**
1.37
0.48


*Significant probability less than 0.05,

**Highly significant probability less than 0.01l

lThe PSM interaction was confounded.
2SCD stands for cellulose of sugar canebaasse; SF,
Solka-Floc; and CLt cotton linters.

3For the possible significance of this interaction,
'see Figure 1,


4.44*
7.67*
17.68"*
0.11
0.01
0.06
0.43








50
in a cattle ration, the sulfur requirement is greater than

0.17 per cent. -I this o::pcrricit the stoors consumed the
ration containing 0.55 per cent sulfur in a somnewhlt sl91-

t~rlh manner. In contrast, the 0.54 per cent phosphorus
ration was consumed vigorously.
When Steers 1, 2, and 3 were fed the steer ration
plus Treatrment S-4, low phosphorus and high molyb'enun,
only Steer 1 would eat all that was offered. For Steer 1,
cellulose digestion was sonmehat depressed; however, for
Steers 2 and 3, molybdenuma posed a palatability problem.
Decker aL 1. (21), Davis (51), leade (121), Thomas and

Hoss (168), and Underwnood (175) reported that high intakes
of molybdenum caused diarrhea, but in this study, the addi-
tion of 25 parts per million of molybdentu to the ration
given in Table I for 20 days did not cause diarrhea.
However, the high molybdenum intakes as well as the other
less desirable rations appeared to reduce the liquid con-
tent of the rumen. Balch and Johnson (14) found that the
amono't of cellulose digestion in the rLrien was greatest
r:hen the surrounding rumen digosta had a lotw dry-natter
content.
The highly significant differences among sources
Gs:pport the results is c:ssed in L th previous trial. In

addition, in this trial, there is a sourco-treatneoit inter-
action. The corrected data are shown graphically in Figure
1. It appears that the addition of molybdonul to Treatments




















0 Cellulose
Di.aosted
(as Correcte-l
for
St.tistic:l'
Ar.'"lysis,
Table 9) 334,


Cellulose Source
Solka-Ploc
Cottoi Linters
Sua*I' Cane Bagasse


Treatrcont (Table 3) S1i
Ilineral Cozbination pswm


S-a
Psm


S-3
pS


S-4


S-5 SG 6 S-7
PaS PsM pSM


Figure 1. The effect of the interrelationship of phosphorus, sulfur,
and molybdenum on the digestion of cellulose from different sources.


X' X


x


X
X


S-8
PSII


I -~ -








52
S-2 and S-5, each of which included phosphorus at the 0.64
per cent level, increased the digestion of cellulose of
sugar cane bagasse and not of Solka-Floo and cotton liners.
If the statistical significance which assumes that the dif-
ferences are real, even though the variations are large,
is accepted, an explanation of the source-treatment inter-
action could be based on the sugar cane bagasse copper con-
centration which was eleven parts per million. This is
explained more fully by the ,n vjtro data given in Table 10
which shows a highly significant interaction among phosphorus,
copper, and molybdenum. The significance of this interaction
is that when either copper or molybdenum is present at a
level that will depress cellulose digestion, the addition
of the other in the presence of a high level of phosphorus
will lessen the severity of the depression.
The significance attributed to time in the statis-
tical analysis given in Table 9 shows that the cellulose
digestion coefficients decreased during the last six days of
a period. The effect was produced by the continued in-
creased depression from the less desirable treatment combina-
tions.

Study.of the effect of phosphorus, sulfur, and _olybdenun
additions to a steer ration-on the rumen liquid concentra-
tions of phosphorus, sulfur. and cooperr
Average rumen liquid concentrations of phosphorus,
sulfur, and copper for steers consuming various treatment








53

combinations are given in Tables 10 and 12. Suwmaries of

the statistical analyses of these data are shown in Tables
11 and 13, respectively.
The statistical analysis of the data, given in Table

10, shows that significant differences, although small, exist
;aong steers. For phosphorus, Steer 1 had a significantly
higher concentration than Steer 3; for sulfur, Steer 4 had a
significantly 1ger concentration than Steers 1, 2, and 3;
but for copper, Steer 4 had a significantly lower concentra-
tion than Steers 1, 2, and 3. The statistical analyses of

the data given in Table 12 show that phosphorus and sulfur
additions to the ration consumed by the steers increase phos-
phorus and sulfur rumon liquid concentrations, respectively.

Anderson gj al. (7), Hubbert .t al. (91, 92),

Johnson al*. (97), and Sala (148) have determined various
requirements for rumen microorganisms in vitro. However,
it is somewhat difficult to transpose their data to in viM
conditions. In order to make better use of in 1itro results,
a more efficient means of transposition would be desirable.
The work by Clark (37) suggests a cautious approach. He
concluded from his work that awen a phosphorus deficiency
caused a ruminant to dio, the water-soluble phosphorus con-

tent of the ruminal ingesta was maintained at a level that

was adequate for ruminal function. Smith et al~ (156)
found a slight effect with advancing age on tissue phosphorus
metabolism with a lower uptake rate in older animals. The
aging effect proposed by Smith et al. (156) could explain









TABLE 10

EFFECT OF STEER VARIATION ON THE PHOSPHORUS, S IUR,
AND COPPER CONCENTRATIONS OF RUMEN LIQUID *


Steer 1 2 3 4
'4 '


Phosphorus2
Period 1
4
7
8
Average

Sulfur2
Period 1
4
7
8
Average


.o
3.34
2.80
2,45
2.67
2.82


gg/ml
485
472
457
472
472


% Mg/al
0.225 32
0.193 33
0.300 56
0.259 48
0.244 42


jo
2.64
2.53
2.19


ag/ml1
420
432
370


2.45 407

% 9g/ml
0.215 33
0.209 36
0.287 49

0.237 39


% u /ml
2.06 340
1.34 260
2.19 465

1.86 355

% ug/mi
0.204 33
0.214 41
0.327 68

0,248 47


1.50
2.44
2.16
1,77
1.99


275
505
465
415
415


% Mg/ml
0.267 46
0.223 46
0.383 96
0.286 67
0.290 63


3
ConDer
Period 1
4
7
8
Average


ag/gm
4.8
7.1
1.1
7.3
5.1


ug/10(
ml
7
12
2
13
9


pg/gm
5.7
8.2
1.2

5.0


Bg/lO
ml
9
14
2


Mg/gm
3.6
5.2
0.9


8 3.2


ag/100
ml uj/gm
6 1.2
10 4.3
2 0.9
-- 4.7
6 2.8


1All steers were fed Ration S-0 as given in Table 2.

2The results are listed with the percentage of the
mineral on a dry matter basis.

3The results are listed with the concentration of
the mineral expressed on a dry matter basis.


ug/100
ml
2
9
2
11
6










TABLE 11

STATISTICAL ANALYSIS OF THE DATA SUMMARIZED IN TABLE 101

Phosphorus
Source DF SS IS F
Total 11 3.0679
Period 2 0.0579 0.0290
Steer 3 1.7612 0.5871 2.85
4 vs 1 +2+ 3 1 0.2450 0.2450 1.19
2 Vs 1 +3 1 0.0162 0.0162 0.08
1 vs 3 1 15000 1.5000 .7.28*
Error 6 1.2488 0.2061




Sulfur
Source DF SS MS F

Total 11 0.038513
Period 2 0.030329 0.015170
Steer 3 0.005716 0.001905 4.59
4 vs 1 +2 +3 1 0.005501 0.005501 13.26*
2 vs 1 +3 1 0.000093 0.000093 0.22
1 vs 3 1 0.000122 0.000122 0.29
Error 6 0.002468 0.000415




Copper
Source DF SS MS F
Total 11 74.58
Period 2 53.GO 26.85
Steer 3 14.55 4.85 4.58
4 S 1 +2 +3 1 9. 61 0.61 9.07*
2 vs 1+3 1 3.12 3.12 .2.94
1 vs 3 1 1.82 1,82 1,72
Error 6 6.34 1.06

*Significant probability less than 0.05.

1The percentage of the mineral on a dry matter
basis was used for the analysis.









TAMLE 12

EFFECT OF TREAT!ZENTS GIVEN IN TABLE 2 ON THE FPHOSPIORUS,
SULFUR, AND COPPER CONCENTIAATOKS OF IRUIEN LIQUID

Nation S-l 8-5 S-6 S-7
l . L i i i m J i J i, i i 1 i i i ]


Phospahorus1 a
Period 1.13
5 a3 090
Average 1.02


235 ,1
230 4
233


2.55
2,49
2.02


595 _4
527 2
561


fo.
2.87
3,50
3,19


Ug/Mal
592 3
560 1
576


0~.55
1.37
0.96


120
295
208


sulfur1
Period 2

Average

Co er
Period 2
:5

Average


I% g/ml
0,297 60 1
0.254 64 i
0'.276 62


- 9.6
3 3.9
6.8


0.394 .90
0'537 114 2
0.466 102


ug;/e0
aml ag/gm
20 9.0
10 4 1.4
15 5.2


Mi Bg//nA -, % g/ml
0.328 66 3 0.274 58
0.380 60 1 0.417 88
0.354 63 '0'346 73


ug/oo
UiS/100
ml Bg/gm
21 4 4.9
3 2 2,5
12 3.7


ug/100
ml Mg/gM
10 3 5.0
S4 1 3.7'
7 4.4


~g/0oo
mi
11
S' 8
10


Ration S-2 S-3 S-4 S-8


Period 3
6
8
Average

sulfur
Period 3
6
8
Average


3.33
3.82
2.08
3.08


ag/ml
577 3
585 2
400 -
521


% Mg/ml
0.264 46 3
0.345 51 2
0,258 50 -
0.289 49


0.40
0.73

0.57


120 1
182 3
151


% US/ml
0.323 95 1
0.482 116 3

0.403 106


0.85
0.71
1.23
0.93


Mg/ml %
145 &2 3.20
168 j 1.97
290 --
201 2.59


0% ug/mi
0.200 38 2
0.328 75 4
0.281 47 -
0.270 53


ug/iml
630
418

524


% Mg/mi
0.295 58
0.557 114
0.426 86
0.426 80


Copler2
Period 3
6
8
Average


ad/gm
- 8.3
14.6
3 8.9
5.3


ug/L00
ml ug/gim
43a1.7
7 23.6
17- --
9 2.7


/00oo
ml H g/gm
5 4.7
9 3 5,1
-- 2 5.1
7 5.0


ug/100
ml ug/gm
8 & 5.1
12 4 2.4
12 --
11 3.8


1The results are listed with the steer number under-
lined and the percentage mineral on a dry matter basis.

2The results are listed with the steer number under-
lined and the concentration of the mineral expressed on a
dry matter basis,


Mg/l00
ml
10
5

8









57

part of the variation in the rumen liquid concentrations

of minerals among the steors used in this experimeoit. Thus,

with variations in the concentrations of nutrients in ruuen

ingesta, the data presented here would suggest variations

in the efficiency of rurnen microorganisms in digesting

cellulose,

TABLE 13

STATISTICAL ANALYSIS OF THE DATA
SUMMARIZED IN TAsLE 121

Effect Hean
Treatment2 Phosphorus Sulfur Copper

(1) 1.998 0.3547 4.344
P 2.136** 0.0856 -0.638
S 0,481 0.1104** -0.713
PS 0,346 0.0062 1.G13
M 0.041 -0.0146 -0.337
PM -0.121 0.0190 1-0.263
SM 0,271 -0.0336 0.463

S**Highly significant probability less than 0.01.
1Tho percentage of the mineral on a dry r. tter basis
was used for the analysis. The results for Period 8 were
not used in the analysis.
S2The letters, P, S, and 1, stand for phosphorus,
sulfur, and molybdenum, respectively.
3This is the breakdown of the treatment significance
in the analysis of variance listed in the Appendix, Table
31.

Stallcup (102) found that the crude fiber fraction

of plants varied among species and that the determination of
lisnin and cellulose might give a better evaluation of.










roughage than crude fiber. In like manner, it appears
that the results of the experiments presented in this
dissertation substantiate the need for a more detailed

routine mineral analysis of feedstuffa.

Experiment II

Study of Cellulose Digestion in the
Artificial Rumen

Comparison of cellulose digestion by rumen inoculum taken
from different steers consuming the same ration
The data with their statistical analysis showing
the comparison of cellulose digestion by rumen inoculum
taken from different steers consuming Ration S-O, given in
Table 2, are given in Table 14.

The data demonstrate that when cellulose digestion
is used as a criterion, individual steers vary more from

day to day than several steers vary among themselves on the
sane day. Also it appears that the antibiotics, penicillin
and streptomycin, when nixed with the rumen contents of
Steer 1 five days before the an vitro studies were begun,
reduced cellulose digestion.

Comparison of the digestion of cellulose from different
sources and frr.nithe qane ,..rce with different particle
sizes

The data with their statistical analysis showing the
comparison of the digestion of cellulose from different
sources and from the same source with different particle









TABLE 14
STUDY OF CELLULOSE DIGESTION IN VYITRO
THE COMPARISON OF CELLULOSE DIGESTION BY RUMEN INOCULUM
TAKEN FROM DIFFERENT STEERS CONSUMING RATION
S-0 GIVEN IN TABLE 2

% Cellulose (Solka-Floc) Digested
Steer 1 2 3 4
Day 1 Determination 1 22,7 43.4 55,3 56.8
2 30.9 42,3 55.9 59.6
3 29.9 46,3 54.9 61.3
Average 27.8 44.0 55.4 59.2
2 Determination 1 20.4 38.9 44.9 45.8
2 24.0 40.7 47.0 46.3
3 21.8 41.3 49.0 46.6
Average 22.1 40.3 47,0 40.2
3 Determination 1 30.3 69.1 50.0 60;5
2 27,2 69.4 50.5 61;9
3 26.5 69,2 49,6 61.2
Average 28.0 69,2 50.0 61.2
4 Determination 1 23.5 57.4 44.6 55,8
2 17.8 57.8 45.0 50.0
3 22.6 5G.1 40.4 57;2
Average 21.3 57.1 43.3 56.3
5 Determination 1 21.4 66.7 57.4 63.3
2 17.3 65.1 60.6 59.6
3 18.8 64.0 60.4 58.4
Average 19.2 65.3 59.5 00.4
Average 23.7 55.2 51.0 56.7



Analysis of Variancel
Source DF SS MS F
Total 44 3i227.70
Day 4 1,724.86 431,22
Steer. 2 257.13 128*57 3092*
4 vs 2+3 1 128.17 128,17 3.91
2 vs 3 1 '128,96 128.96 3*93
Error 38 1,245.71 32*78

*Significant probability less than 0.05*
1The results from Steer 1 are not included in the
statistical analysis because it was given approximately 0.25
gram of streptomycin and 150,000 units of penicillin five
days before Day 1. The antibiotics were mixed with the.
rumen contents.










sizes are given in Table 15.
The significance of the difference among sources
was discussed in Experiment I. The grinding of the cotton
liners and sugar cane bagasse could have converted part
of the crystalline regions to amorphous regions. The blades
of the Wiley mill used for grinding were slightly dull.
Perhaps, this caused more of a crushing action similar to
ball milling which will convert crystalline cellulose to
amorphous cellulose (42). At the present time, with the
work, quoted by Karstra gE a, (98), showing that with a
reduction in crystallinity there was an increase in cell'u-
lose digestion, this appears to be the best explanation fo,
the beneficial effect of grinding of different sources of
cellulose on cellulose digestion by rumen microorganisms

in xitrao

Study of the effect of different concentrations of pohos rus.
sulfur, copper. and molvbdenum on cellulose digestion
The data with their statistical analysis showing the
effect of different concentrations and combinations of phos-
phorus, sulfur, copper, and molybdenum on cellulose digestion
are given in Table 16.
The optimum control or Treatment 17T which is not
included in the statistical analysis caused the rumen micro-
organisms to produce a nmuch higher cellulose digestion coef-
ficient, 35.7 per cent, than the average, 23.3 per cent, of
Treatments 1T through 16T. This would suggest that those









TABLE 15

STULY OF CELLULOSE DIGESTION V VITRO.
THE COMPARISON OF THE DIGESTION OF CELLULOSE FROM
.DWPFEENT SOURCES AND FROM TU SAIi SOURTC
WITH DIFFERENT PARTICLE SIZES

% Cellulose Digested
Source Solka- Cotton Sugar Cane
.Flo Linteers _Tiaasse
..21 21 1 12
Day 1 Determination 1 30.7 26.4 27.7 15.6 25.9
2 30.6 29.1 28.2 15.5 23.0
Average 30.7 27.8 28.0 15.6 24.5
2 Determination 1 39.7 23.1 33,6 10.4 20.2
2 29.4 27.8 30,5 16,3 20.4
Average 34.6 25.5 32.1 13.4 20.3
3 Determination 1 22.0 25.5 26.3 13.4 19.9
2 26.4 28.0 20.0 15.9 18.7
Average 24.2 26.8 26.4 14.7 19.3
4 Determination 1 26.2 20.8 49.8 13.2 18.4
2 33.5 30.2 52.4 12.8 19.8
Average 29.9 25.5 51.1 13.0 19.1
5 Determination 1 47.2 24.0 61.1 13.6 23,3
2 55.0 30,2 61.9 16.1 23.0
Average 51.1 27.1 61.5 14.9 23.2

Average 34.1 26.5 39.8 14.3 21.3




Analysis of Variance
Source DF SS S F


Total
Day
Source
SF vs Others
CL1 vs SC~
CL vs CL
SCB vs SCB*1
Error


49 7,182.00
4 1,02#681
4 4,073.83 1
1 -594.44
1 2,359.30 2
1 876.49
1 -243.60
41 2.081.36


*256.70
,018.46
594.44
,359.30
876,49
243.60
50.76


1 his material was ground to pass a forty


mesh screen.


20.06**
11.71**
46.48**
17.27**
4.80*


* Significant probability less than 0.05.

** Highly significant probability less than 0.01.


r









TABLLF 16

STUDY OF CELLULOSE (SOLKA-PLOC) DIGESTION iE VITRO.
EFFECT OF Dl(MrEILI:T CONCElTIATIOI;S OF PHIOSPMORLUS
SULFUiR COPPER, AND MOLYBD3 MEIn


Di. estion Coofficients
1 2 3 4 5 Avera


18.9 24.94 14.7 22.8 23.6
17.2 19.4 14.9 20.9 20.2
1014 12.6 7.5 17.7 14,3
6.1 13.9 5,6 16.1 10,4
28.9:27.2 9.8 36.1 41.0,
32.9 29.8 21.8 4:0.5 43.3
13.6 36.44 7.0 24.7 26.4
11.9 19,5 4,. 15.6 10.5
35.2 28.7 15.4 25.6 32.0
30.0 28.4 17,9 22.0 .24.6
18,7 18.3 4.8 19.9 17.3
21.0 23.8 5.3 19.2 19.3
42.0 41.1 25.7 40.7 60.1
31.9 29.5 22.5 45.G 55.7
20.5 17.7 7.0 33.0,22.0
27,2 28,3 9.4 30.5 40.0.
33.0 31,8 29.5 48.9 35.4


SStatistical Analvsis
,,e Treat- Effect
ment2 .ean-


21.0
18.5
12.5
10.4
.28.6
33.7
,21.6
13.6
27.4
,24.0
'15,8
17.7
41.9
37.0
,21.4
,27,1
35.7


(1)
PC

S
PS

.PCS.
S11

Cli
,PSM

,PSH
.CSM -
PCSi;


...23.30
6.59**~
-11.5i0**
0.66
9*63**
0.89
2.81*
0.21
0.95
0,90
0.32
3.49**
0.41
0.05
Q 0.95
2.42


Analysis of Variance
Source IF SS M F
J i i _________, ,_ _' '_ / _,, i. r i L '_ .. : _ ^ ^ .- - ... ....


Total
Day
Treatment
Error


10645.23
2,817.55
6G000.463
1,810.20


704.39
400 .57
30.32


*Significant probability less than 0.05.
**1Highly significant probability loss than
1These are listed in Table 3,


13.21**


0.01.


2The letters, P, C, S,-and iM, stand for phosphorus,
copper, sulfur, and molybdenum, respectively,.
3This is the breakdown of the treatment significance
in the analysis of variance.
4All values are averages of two or more values ex-
cept these.
5This is not included in the analysis of variance.


Day
Treat-
arnmt.+


IT1
2T
3T
4T
5T
6T
7T
8T
9T
10T
11T
12T
13T
14T
15T
16T
17T


--


-Vlu


--- - --- -- ~-- --I -- -~-I-~I--~----








63

factorial effects that are not significantly different from
the experimental mean are producing a depression on cellu-
lose digestion when compared with Treatment 17T.
The phosphorusI -molybdenuL interaction, dis-
cussed in Experiment I, shows that when either copper or
molybdenum is present at a level that will depress the
digestion of cellulose by rumen inoculum, the addition of
the other in the presence of a high level of phosphorus
will lessen the severity of the depression. The copper-
sulfur interaction shows that sulfur is reducing the se-
verity of the depression produced by copper. The actual
mechanism of the above interrelationships remains obscure.
Because of these existing interactions, the positive effects
of phosphorus and sulfur should be dealt with cautiously.
The effect of each will depend on the levels of the other
interrelated nutrients. The effects of phosphorus and
sulfur appear to be additive in this experiment. When
Treatment IT contained 283 and 25 micrograms per milliliter
of phosphorus and sulfur, respectively, the addition of 75
micrograms per milliliter each of phosphorus and sulfur to
Treatment IT, to give Treatment 17T, improved cellulose
digestion. The addition of 7500 micrograms per milliliter
each of phosphorus and sulfur to Treatment IT did not ap-
pear harmful when cellulose digestion was used as a cri-
terion of measurement.










Exeriment III

Study of the Effect of the Interrelationship of
Minerals on Growth and Bone Formation in- Rats ,

.r.ia I. The effect- of .iifforont concntgratiotn of phos-
phorus, sulfur, copper, and .olybdenur on ~p-rowth a~~nd bo
formation
The data with their statistical analyses showingef-
fectsof different concentrations and combinations of phos-
phorus, sulfur, copper; and rolybdenur on weight gains,
milligrams of ash per femur per gram of live body weight, per-
contage ash of the femur, femur ash, percentage phosphorus of
the femur, percentage phosphorus of the liver,,and percentage
copper of the liver of rats are given in Tables 17, 18, 19,
20, 21, 22, and 23, respectively.
The treatment combination used in Treatment 1R17
gave values that were higher than the experimental mean for

weight gains, percentage ash of the femur, femur ash, and
percentage phosphorus of the femur and of the liver. Per-
centage copper of the liver was lower, and the milligrams

of ash per femur per gram of live body weight were about the
same. From the data presented in this experiment, it appears
that the rats with the lower liver copper values gained
weight more efficiently and were supported by a skeletal
framework that contained a more concentrated mineral reserve.

Female rats were found to have statistically signifi-
cant lower weight gains and femur ash values than males.
However, the females h-.d a statistically significant higher









TABLE 17

TRIAL I, .iXPF DtET III.
SUIMARY OF 56-DAY RAT WEIGHT GAINS Nl.
VARIOUS RATIONS GIVEN IN TABLE 51

nation Sex Weight Gain Statistical Analvai
m Treatment Fffect rlean'
1 1 Hale 168e 4 (1) 121.30
Female 124 10 P -14'78*
1112 ale 125 15 C 1.34
Female 79 15 PC 0.84
1113 Male 173 32 S 30.06**
Female 109 19 PS 7-13
1R4 Male 85 16 CS 14.75**
Female 70 17 PCS O.C3
IR5 Male 194 18 H -24.03**
Female 136 12 PM 20.22**
1G MHale 118 47 CM 4.60
Female 77 34 PCM 6.53
1R7 Male 176 15 SIl 8.50J
Female 128 11 PSM 0.81
R18 Male 163 10 CSH 14.03**
Female 129 10 PCSTI 5.00
1n9 ;Iale 99 15 E -35.19**
Female 107 12 PE 7.88
1R10 Male 115 15 CE 2.75
Female 88 7 PCE 1,62
IRL Male 95 10 SE -11.97**
Female 107 18 PSE 9*97*
1R12 Male 83 6 CSE 1.59
Female 70 12 PCSE 178
1R13 Male 141 7 ME 2.12
Female 112 6 PME -12.69@*
1814 Male 157 27 CME 4.87
Female 92 38 PCIIE 187
1R15 Male 153 14 SME 1*22
Female 113 2 PSME 1.66
1R16 Male 178 6 CSME 3.60
Female 115 1 PCSIIE 4.03
1R175 Male 200 16
__ 1Female 135* 12
*Significant probability less than 0.05.
*Highly significant probability less than 0.01.
1The data summarized in this table can be found in
the Appendix, Table 32
2This is the-group mean tthe mean variation.
S3The letters, P,CS,o, and E, stand for phosphorus,
copper, sulfur, molybdenur, and sex, respectively. Females
are represented by E.
S This is the breakdown of the treatment significance
in the analysis of variance given in the Appendix, Table 33.
5This is not included in the statistical analysis.









TA3DLr. 18

TRIAL I, EXPEIhIENT III,
SUMMARY OF MILLIGRAN] ASH PER FMiUR PER GRAlI OF BODY WEIGHT
OF RATS ON VARIOUS RATIONS GIVEN, IN TABLE 51

Ration Sex MG Statitical Analysis
_Ashfenur/Gn Weiah5 Treatment effect Nea


181 Male
Female
1112 Male
Femalo
1R3 Male
Female
1R4 Male
Fenale
1R5 Male
Female
1R6 Male
Female
1R7 Male
Female
1R8 Nale
Female
119 Male
Female
IRIO Male
Female
IR11 Male
Female
IR12 Hale
Female
1113 Male
Female
1R14 Male
Female
1R15 Hale
Female
1R16 Male
Female
IR175 Male
Foiiiln P.


0,81+-
0.93'
0.84
0.98
0.83
0,99
0.95
0,94
0#77

0,77
0.98
0.66
0.79
0,79
0.88
0,83
0.91

1.05
0.89
0.91
0.97
1.03
0.94
0.92
1.00
1.05
0*92
1.02
0.94
0.97
0.90
1.05
0.90,
1 -09-
0,79
0#88
0083

0096
1105
0189
01,91
0,97
1.03

0,92
1.00
1.05

1*02
0.94
0.97
0690

0190
1.0t


0.04
0.03
0402


0.06
0,06
0.05
0,07

0.09
0.03
0.03
0.05
0.04
0.05
0.03
0.03
0.03
0.07
0.05
0.05
0.04
0.04
0.04
0.04
0.02
0,02
0.03
0.02
0.03
0.04
A.-01


(1)
0p
PC
S
PS
CS
PCS
PM



PSM
CSM


CE
PCE
SE
PSE
CSE
POSE
IM
PME

SME
PSI.E
CSME
PCSME


0.9170
0 .1027**
0.0136
-0 0252*
-0.0280*
0.0520**
-0.0083
-.00133
-0.0342**
-0.0211
0.0373**
-0.0108
-0.0067
0.0439**
0.0286**
-0.0220
0.0883**
-0.0311**
-0.0227
0.0107
0.0195
0.0039
0,0023
0.0130

0.0183
-0.0020
0.0080
0,0227
0,0177
0.0223
-0.0127


*Significant probability less than 0.05.
**Iiighly significant probability loss than 0.01.,
IThe- data summarized in this table can be found in
the Appendix, Table 32.
2This is the roup mean t the mean variation,
S3The letters, PC,S,M, and E, stand for phosphorus,
copper, sulfur, molybdenum, and sex, respectively. Females
are represented by E.
4T! is is the breakdown of the treatment significance
in the analysis of variance given in the Appendix, Table 33.
5This is not included in the statistical analysis.









TABLE 19

TRIAL I, 1E;PEMIE;:T III.
SUMMARY OF PERCENTAGr ASH OF FEJ3MURS OF, RATS
ON VARIOUS 11ATIOiS GIVI. IN TALE 51


Ration Sex


11 Male
Female
1R2 Male
Female
1113 iale
Female
1R4 Male
Female
1R5 Male
Female
IR6 Male
Female
1R7 Male
Female
IR8 Male
Female
1R9 Male
Female
1R10 Male
Female
IR11 Nale
Female
1R12 Male
Female
1R13 Male
Female
1R14 Male
Female
1R15 Male
Female
1R16 Male
Female
1R175 Mnale
BPtsma1a


__~_ _1


*Significant probability less than 0.05,
**Highly significant probability less than 0.01.
lThe data summarized in this table can be found in
the Appendix, Table 32,
2This is the-group mean +thel mean variation.
S3The letters, P,C,SII, and E, stand for phosphorus,
copper, sulfur, molybdenum, and sex, respectively, Females
are represented by E.
4This is the breakdown of the treatment significance
in the analysis of variance given in the Appendix, Table 33.
5This is not included in the statistical analysis.


1_1_~_11__~_
_


Femur Ash4

59.11 1.1
62.1 0.6
61.1 0,5
58.7 1,7
60.7 1.5
60.9 2.1
58.8 0.9
57.6 1.3
60.0 1.8
00.G 0.8
56.1 4.5
55.8 3.8
61.0 1.1
60.8 0.5
59.6 1.5
62.5 1.0
61.5 2,4
63.4 0,7
61.0 0.9
61.0 1.7
63.2 1.1
62,8 1,0
59.4 0.7
61.9 1.4
62,8 0.9
63.1 1.8
61.0 1.9
63.0 2.5
64,1 1.5
64.1 0.2
63.4 0.6
63.5 1,1
63.9 1.0
64,6t 0.8


Statistical Anal.avis
Treatment Effect IIearr-
(1) 61.067
P 2.713**
C 0.850G'
PC -0.178
S 0.494
PS 0.784
CS 1.147**
PCS -0.G38
M -1.588**
PM 0.303
CM 0.303
PCM -0.381
SM 0.106
PSM 0.400
CS11 0.975*
PCSM -0.647
E i 0.594
PE 0.259
CE -0.053
PCE -0.125
SE 0,147
PSE -0,300
CSE 0.106
PCSE -0,360
M. -0.141
PME 0.438
CME 0.694
PCHE -0,303
SME 0.569
PSM9E -0.553
CSME -0.428
PCSII~ -0.438









TABLE 20

TRIAL I, EXPERITOET III,
SUMMARY OF FEMUR ASH CONCEITRATIONS OFI HATS ON
VARIOUS IL\TIONS GIVEN IN TABLE 51


Ration Sex


..


i .......


'Significant probability less than 0,05.
**Highly significant probability less than 0.01.
1The- data summarized in this table can be found in
the Appendix, Table 32.
2Thlis is the-group mean t the mean variation.
S3The letters, P,C,S,M, and E, stand for phosphorus,
copper, sulfur, molybdenum, and sex, respectively. Females
are represented by E.
4This is the breakdown of the treatment significance
in the analysis of variance given in the Appendix, Table 33.
5This is not included in the statistical analysis.


- -- -- ------ -- --- ---
-- --- -- -- ---


R1 I Male
Female
12 Nlale
Female
13 Male
Female
1R4 Male
Female
1R5 Male
Female
1116 Male,
Female
1i7 Male
Female
1R8 Male
Female
1R9 Male
Female
1RIO Malo
Female
IR11 Male
Female
1H12 Male
Female
1R13 Male
Female
1R14 Male
Female
1R15 Male
Female
11116 Male
Female
1R175 Male
T?

Femur Ash'z

1641 7
145 10
135 15
102 12
175 39
138 30,
117 116
103 20
173 11
169 15
105 33
89 36
164 13
142 8.
160 10
147 4
184 17
149 16
130 17
106 5G
124 12
142 28
108 13
95 18
174 4B
153 15
178 23
132 26
176 12
138 3.
188 10
157 06
211 11
183- 21


Statistical Analy
Treatmentm fect Mea.7
(1) 140, 79
P 2.01
C 2.01
PC 4.01
S 24,89**
PS 15,42*
CS 9.42*
PCS 1.29
M -25.39**
PI 14.33**
CN 9.64*
PCI 6,89
SM 7.79
PSM 6.27
CSM 15.58**
PCSM 645
E -18,01**
PE 1.33
CE 1.11
PCE 1,23
SE 5.70
PSE -12.23 *
CSE 1.79*
PCSE 0.92'
ME 6.05,
PME 6.27
CME 7.23
PCIE 0.73'
SHE 3.27
PSIXI 4.92
CSIE 0.29
PCSUE 1.70


- ~U1Y









TABLE 21

TRIAL I, EXPERIMENT III.
SUMARY OF PERCENTAGE PHOSPHORUS OF DRY, FAT-FREE FEMURS
OF RATS ON VARIOUS RATIONS GIVEN IN TABLE 5

Ration Phosphorusl Statistical Analysis
Treat- Effect
12 Average nent2 _Hoan3

1R1 12.4 11.9 12.2 (1) 0.99
1R2 12,0 15.3 13,7 P -1.24**
183 10.5 12.0 11.3 C -0.88*
1R4 7.8 9.9 8,9 PC -0.33
I15 7.8 8.5 8.2 S -0.28
1R6 8.7 10.5 9.6 PS 1.44**
1117 10.0 10.4 10.2 CS Z.00OG
1R8 11.1 11.5 11.3 PCS -0.24
1119 11.3 11.4 11.4 1 -0.26
1R10 9.6 8.9 9.3 PM -0.62
1R11 10.5 7.5 9.0 CM -0.30
1812 6.3 4.8 5.6 PCM 0.79
1B13 8.8 11.1 10.0 SM 1.35**
1R14 10.0 8.7 9.4 PSM 0.54
1115 9.2 8.7 9.0 CSM 0.99*
IR16 11.9 10.8 11.4 PCSM 0.16
1R174 10.2 11.6 10.9



Analysis of Variance
Source DF SS M1S F
Total 31 126.38
Replication 1 0.50 0.50
Treatment 15 105.36 7.02 5.12**
Error 15 20.52 1.37

*Significant probability less than 0.05.
**Highly significant probability less than 0.01.
IDeterminations I-and 2 refer to the first and the
second rats, respectively, of each ration designated for
mineral analysis listed-in-the Appendix, Table 32.
2The letters, P, C, S' and M, stand for phosphorus,
copper, sulfur, and molybdenum, respectively.
3This is the breakdown of the treatment significance
in the analysis of variance.
nThis is not included in the statistical analysis.









TABLE 22

TRIAL 1l EXPfnIIIIENT III.'
SUMMARY OF PERCENTAGE PHOSPHORUS OF LIVERS ON A DRY LATTERR
BASIS OF RATS ON VARIOUS RATIONS GIVEN IN TABLE 5

Ration EI Phosphorus1 Statistical AnalysPi
Treat- Effect
1 2 Average ment" Mean

11 0.76 0.87 082 (1) 1.19
1R2 0 88 0,98 0.93 P 0.310**
1R3 0.06 0.93 0.95 C 0.040
1i4 0.92 1.06 0.90 PC -0.043
1R5 0,98 0.97 0.98 S 0.004
1R6 0,88 1.06 0.97 PS -0.084
1R7 1.10 0.97 1.04 CS -0.046
1R8 1.11 0.99 1.05 PCS -0.034
1n9 1.11 1.13 1,12 11 0,043
1R10 1432 1.54 1.43 PH 0.000
1111 1,75 1,21 1 8 CHi -0.050
1R12 1.37 1.08 1,23 PCM -0.038
11113 1,30 1430 1.30 SM -0,011
1R14 1.35 1,15 1.25 PS11 0,026
1n15 1.01 1.21 1.11 CSM 0.109*
1R16 1.28 1.27 1.28 PCSM 0.086
1R174 1.41 1.21 1.31



Analysis of Variance
Source DFSS MS F
Total 31 1.3904
Replication 1 0.0041 0.0041
Treatment 15 1.0849 0.0723 3.48*
Error 15 0,3114 0.0208

*Significant probability less than 0.05.
*HIIighly significant probability loss than 0.01,
iDeterminations l-and 2 refer to the first and the
second rats, respoc:tively, of each ration dosignated:for
mineral analysis listed-in-the Appendix, Table 32.
The letters, P C, S,"'a nd M, stand for phosphorus,
copper, sulfur, and nolybdenum, respectively.
3This is the breakdown of the treatmeilt significance
in the analysis of variance.
4This is not included in the statistical analysis.










TABLE 23
TRIAL I, rXPERI.IFNT III.
SUMMARY OF COPPER CONCENTRATIONS OF LIVERS ON A DRY HATTER
BASIS OF RATS ON VARIOUS RATIONS GIVEN IN TABLrE

Ration .--- Copper/Er Statistical Analysis
Treat- Effect
1 2 Average mernt Mean3
IR1 321 276 M2. (1) 306.41
1R2 223 268 246 P 128.81**
1R3 279 273 276 0 69.G9**
1114 303 314 309 PC 10G.69**
1R5 128 157 143 S -267.81**
1B6 263 448 356 PS -187.56**
1R7 151 129 140 CS 7744**
1R8 158 181 170 PCS 20.19
1R9 399 378 389 M 140.81**
11110 491 578 535 PM 85.31**
1R11 355 356 356 CM 54.44
1R12 1208 1023 1116 PCMI 78,93*:
1R13 79 89 84 SM 80.560*
1R14 154 93 124 PSM -146.31**
1R15 293 113 203 CS1I -120.44**
IR16 160 164 102 PCSN 53.19
IR174 106 118 112



Analysis of Variance
Source DF SS MS F
Total 31 1,880,749.72
Replication 1 -488.28 488.28
Treatment 15 1,821 370.22 121,424.68 30.93**
Error 15 58,891.22 3,920,08

**Highly significant probability less than 0.01.
1Determanations 1 and 2 refer to the first and
the second rats, respectively, of each ration designated
for mineral analysis listed in the Appendix, Table 32.
The-letters, P, C, 8, and M, stand for phosphorus,
copper, sulfur, and molybdenum, respectively.
3This is the breakdown of the treatment significance
in the analysis of variance.
4This is not included in the statistical analysis.









amount of femur ash per unit of live body weight, These
differences appear to be normal according to the work of
Sherman and Quinn, quoted by Willia is j al. (182), who found
that female rats which have not born young and are more than
21 days of age have more calcium, phosphorus, and total min-
eral per unit of body weight than male rats. Aubel it al,,
quoted by :laynard and Loosli (116), found that the additions
of 0.18, 0.33, and 0.59 per cent phosphorus to a swine ration
increased the percentage phosphorus in the femurs and humeri
of swine with each addition of dietary phosphorus.
Sex, phosphorus, sulfur, and molybdenum showed inter-
actions for their effects on weight gains, r'illigrams of ash
per femur per gram of body weight values, and femur ash
values. The addition of sulfur as sodium sulfate to Treat-
ment Ir1T stinrulated growth in both male and fomealo rats.
In the absence of the added sulfur, weight gains and fmcur
ash values were depressed much more in male than in female
rats by the addition of 0.78 per cent phosphorus. The addi-
tion of sulfur to this high phosphorus ration was effective
in counteracting the depression of weight gains and femur
ash values in male rats caused by the high level of phos-
phorus supplementation. The values for milligrams of ash
per femur per gram of live body weight show that the female
rat accumulates more phosphorus per unit of body weight than
the male rat. This should partly explain the greater depres-
sion caused by high phosphorus levels in male as opposed to
female rats. Also, this high dietary phosphorus level in-
creased liver phosphorus and decreased femur phosphorus for








73
female rats. The addition of sulfur decreased the phosphorus
concentration of the femur but not to the same degree as
that produced by the high level of phosphorus in the ration.
In .addition, the sulfur supplementation partially counter,
acted the lowered fcrrar phosphorus produced by the highu
phosphorus intake. Should the phosphorus of the high phos-
phorus rations be reacting with calcium to form an insoluble
tricalcium phosphate as described by laynard and Loosli
(116), then perhaps the fc:.:ur phosphorus metabolism was
limited by a.lack of calcium. The results given in Tables
17, 20, 21, and Appendix Th.blo 32 show that the high phos-
ph orus rations did not change the total femur aeh; but boid;-
weight gains, total fat-free, dry femur weights, and the

percentage phosphorus of the foeurs were reduced. There-
fore, had only percentage ash of the femur been used as a
criterion, erroneous conclusions may have been made.
The addition of molybdenum to various treatments
reduced xucigt gains, milligrams of ash per femur per gram
of body weight, femur ash, percentage femur ash, and per-
centage phosphorus of the femur and of the liver, but in-
creased liver copper values. In the presence of the high
phosphorus level nentionei above, the addition of molybdenum
depressed weight gains and femur ash values for female rats
and increased weight gains and femur ash values for male
rats. The addition of either copper as CuC12*2i20 or sulfur
as Na2SO4 failed to alleviate the depression produced by the

molybdenum. Underwood (175) concluded from data presented








74

by other workers that the role of copper in preventing or
overcoming the toxic effect of molybdenum had been estab-
lished beyond doubt. In these studies; copper was added as
copper sulfate and other minerals were added as the sulfate
which made it very difficult to attribute the correction of
a molybdenum' toxicity to copper per se. The effects of a
molybdenum toxicity in relationship to copper and sulfur in
sheep as reviewed by Dick (57, 58) and by Allcroft and Lewis
(4) were given previously in the Literature Review. In
reviews (43, 47, 51. 52, 102, 121, 123) and in other articles
(8,48, 49, 50, 84, 129), the conflicting findings concern-
ing molybdenum toxicity as related to copper, sulfur, and
other minerals have been discussed.-:
In the experiment reported here, the supplementation
with both copper andd sulfur was'necessary to reverse the
depressing effect of molybdenum, When 300 parts p.r million
of molybdenum were added to Ration 131 for which the compo-
sition is given in the Appendix, Table 38, the addition of
100 parts per million of copper plus 3300 parts per million
of sulfur reversed the depressing effect of molybdenum on
weight gains, milligrams of ash pr femur per gram of live
body weight, femur ash, percentage ash of the femur, per-
centage phosphorus of the femur and of the liver, and per-
centage copper of the liver,
McCall (118) concluded that the effect of the level
of protein in the ration is greater than the effect exerted










by either molybdenum, phosphorus, zinc, or copper on the
accumulation of copper in the livers of rats fed high levels
of copper. The data presented in Table 23 show that phos-
phorus, sulfur, copper, and molybdenum are interrelated in
mtny ways which will affect liver copper concentrations.
The additions of sulfur to the rations caused a lowering of
liver copper values while added phosphorus, copper, and
molybdenum caused an increase. These results suggest that
the sulfur of Na2SO4 was beneficial in regulating the cop-
per concentration of the liver,

Trial II. The effect of sulfur and copper additions to a
ration containing 300 parts per million of molvbdenum on.
growth and bone formation
The data summarized in Table 24 show the effect of
sulfur and copper additions to a ration containing 300 parts
per million of molybdenum on weight gains, milligrams of
ash per femur per gram of live body weight, percentage ash
of the femur, and femur ash. The statistical analyses of
the data are given in Table 25.
When 0*66 per cent sulfur was added to Ration 2L4,
it produced a very toxic effect. For this reason, the ex-
perimental results obtained from the rats consuming Ration
2R4 were not included in the statistical analyses. Of the
six rats allotted to this treatment, three died during the
experimental period, and two of the remaining three rats
were developing symptoms similar to those of the ones










that died. The symptoms which preceded death included a
loss of weight and a diarrhea which Cox (43) found could be
caused by a low-copper ration. Two other rats, one on
Treatment 1R6 of Trial I and one on Treatment 2R3 of this
trial consuming rations containing 0.33 per cent added sul-
fur, died during the experimental period after showing
similar symptoms to those mentioned previously. Perhaps
these symptoms resulted from the high level of dietary
sulfur producing acid intoxication or a greatly reduced
liver copper resulting in a copper deficiency.

TABLE 24
TRIAL II, PERIMTENT III.
SUMIARY OF 56-DAY WEIGHT GAINS, MIMIGRAMS ASH PER FEMlOR
PER GRAM OF BODY WEIGHT, PERCENTAGE ASH OF PEMURS,
AND FEIIUR ASH CONCENTRATIONS OF RATS ON VARIOUS
RATIONS GIVEN IN TABLE 51

Ration Weight Gain 1 Femur Ash. Femur Ash
gm Ash/Femur/Gm Weight2 g

2R1 119t 5 0.99Q 0,03 63.4: 0,4 146t 6
2B2 134 4 0.98 0.04 61.5 0.8 159 4
2R3 108 33 1.01 0.05 60.6 2,4 141 35
2R4 59 38 0,84 0 10 58.6 3.1 83 54
2R5 89t 8 1.03+ 0.06 63.0t 0.5 121+ 13

1The'data summarized in this table can be found in
the Appendix, Table 34.
2This is the group mean t the mean variation.









TABLE 25

TRIAL II, EXPEM2IEi;T III.
STATISTICAL ANALYSIS OF THE DATA SUlMMAIZED IN TABLE 24

Weight Gain1
Source DF SS INS F
Total 23 20521.34
Replication 5 3,240.34 648.07
Treatment 3 6,614,34 2,204.78 3.100
2R5 vs Others 1 4;480,89 4;480.89 6.301*
2R2 vs 281 + 2R3 1 1,792.12 1,792.12 2.520
2R1 vs 2R3 1 -341.33 341,33 0.480
.Error ... 15 .10.666,66 711.11]

a111ligrams Ashfoemur/Gram Weight1
Source DF SS MS F
Total 23 0.0825
Replication 5 0.0144 0.00288
Treatment 3 0.0107 0.00357 0.932
2R5 vs Others 1 0.0080 0.00800 2.088
2R2 Vs 2R1 + 2R3 1 0.0023 0.00230 0.601
211 vs 2R3 1 0.0004 0.00040 0.000
Error 15 ._0..0574 0.00383 .

Percentage Ash of Femurl
Source DlF SS MS F
Total 23 99.52
Replication 5 7.99 1.598
Treatment 3 30.63 10,210 2.515
215 vs Others 1 6.48 6.480 1.59G
2R1 vs 2R2 + 2R3 1 21.62 21,620 5.325*
2R2 Vs 2R3 1 2.53 2.530 0.623
Error 15 60.90 4.060

'''' ' Femur Ash i
Source .F 12 SS ._ F
Total 23 21,650.00
Replication 5 3,340,75 '668.15
Treatment. 3 4,529.49 1,509.83 1.643
2115 vs Others 1 3,514.03 3,514,03 3.825
2R2 vs 211 + 213 1 940.46 940.46 1.024
2R1 vs 2R3 1 75.00 75.00 0.082
Error ..._15 13,779.75 918.65
*Significant probability less than 0.05.
lRation 2R4 is not included in the statistical
analysis.








78
From the results of this and the previous trial, it
appears that in rats, sulfur is more beneficial in main-
taining the composition of the soft tissues of the body,
and copper is more beneficial in maintaining the composition
of the skeletal framework of the body especially in the
presence of high dietary levels of molybdenum. As in Trial
I and in this trial, the addition of 100 parts per million
of copper to a ration containing 300 parts per million of
molybdenum significantly reduced rat weight gains. The
results found in Trial I, in this trial, and in those re-
ported by McCall (118) suggest that an excessive amount of
dietary copper is rendering an essential nutrient or nu-
trients or perhaps a sulfur-containing amino acid unavail-
able to the rat or that the growing rat has a requirement
for sulfur per se as suggested for the chick by Gordon and
Sizer (81).
The addition of 100 parts per million of copper to
the rat ration given in Table 4 did not significantly reduce
femur ash and percentage ash of the femur for rats. The
addition of sulfur at either the 0*33 per cent level or the
0.165 per cent level lowered the percentage ash of the femurs.
However, after a closer inspection of the data, it appears
that sulfur also produced an increase, but not a statistically
significant one, in both the ash and the non-ash parts of the
femur.








'79
Triala III. The effect of different concentrations of sulfur,
copper and molybdenum on growth and bone formation
The data with their statistical analyses showing the
effectsof different concentrations and combinations of sul-
fur, copper, and molybdenum on weight gains, percentage ash
of the femur, femur ash, and hemoglobin values are given in
Tables 26, 27, 28, and 29, respectively.
In this trial, the addition of 30 parts per million
of copper to a ration containing 300 parts per million of
molybdenum did not have an additive effect on the depression
of weight gains and femur ash values caused by the molyb-
denum, but the addition of 100 parts per million of copper
in Trials I and II did have an additive effect.
The depression of weight gains, femur ash, and
hemoglobin values caused by the addition of molybdenum were
reversed by the simultaneous addition of 30 and 600 parts
per million of copper and sulfur, respectively. The findings
presented here support the results of Trial I concerning the
sulfur-copper-molybdenum interaction; however, the results
suggest that the levels of sulfur and copper used in Trial
III and perhaps even lower levels of one or both might cor-
reot the depression which 300 parts per million of molybdenum
effected on growth and bone formation in weanling rats.
Results reported by Miller &t aL. (122) and by Mills gi Zi.
(124) support the above findings. Miller l. al, (122) used
a 12 per cent crude casein ration which contained 4 parts










TABLE 26
TRIAL III, EKPER11IENT III.
SL-2MARY OF 35-DAY RAT WEIGHT GAINS ON VARIOUS
RATIONS GIVEN IN TABLE 51


Ration Sex Weight Gain2 Statistical Analypis
gm TreatmentU Effect Mean*

3R1 Female 100o 5 (1) 92.90
3R2 Female 63 18 C 9.29*
3R3 Female 99 8 S 13.04**
3R4 Female 84 18 CS 0.63
3R5 Female 97 6 M -13.29*"
3R6 Female 93 10 CM 7.71
3R7 Female 102 8 SM 13.13*
3R8 Female 106+ 8 CSM 3.71

*Significant probability less than 0.05.
**Highly significant probability less than 0.01.
1The data summarized in this table can be found in
the Appendix, Table 35.
2This is the group mean t the mean variation.
3The letters, C, S, and I, stand for copper, sulfur,
and molybdenum, respectively.
4This is the breakdown of the treatment significance
in the analysis of variance given in the Appendix, Table
36,










TABLE 27
TRIAL III, EXPFLIMENT III,
SUPJMMARY OF PERCENTAGE ASH OF FEJIURS OF RATS ON
VARIOUS RATIONS GIVEN IN TABLE 51


Ration Sex Femur Ash2 Statistica1 Analysis
% Treatmentu Effect Meax4

3M1 Female 58.9t 0.3 (1) 59823
3112 Fenalo 56.7 2.4 C 1.254
3R3 Female 59.4 1.0 S 0.83
3R4 Fen.nle 60.3 1.4 CS 0,84
3R5 Feri.l 59.7 0,6 M 0.04
3R6 Female 59.A 1.4 CM 1.30*
3R7 Female 59.0 0.8 SM 0.02
3R8 Female 60.7t 0.8 CSM 0.21

*Significant probability less than 0.05.
1The data sunm arized in this table can be found in
the Appendix, Table 35.
2This is the group mean t the mean variation.
3The letters, C, S, and H, stand for copper, sul-
fur, and molybdenum, respectively,
4This is the breakdown of the treatment significance
in the analysis of variance given in the Appendix, Table
36.,










TABLE 28


TaIAL III, EXPErInENT III;
SUIMMARY OP FENiUR ASH CONCrENTRATIOS;OS,OF.nATS Oil
VARIOUS RATIONS GIVE: IN TABLE 51


Ration Sex Femur Ash2 Statistical Analysis
.g Treatment3 Effect Mean4

31i Feaalo 119t 8 (1) 113.73
3R2 Female 81 21 0 10.46*
3R3 Female 118 12 S 14.G3**
3R4 Female 108 18 CS 2.71
3R5 Female 120 7 M 9.88
386 Female 115 15 CM 11.79*
3R7 Female 118 7 SM 14.13**
3R8 Female 132t 8 CS 2.88

*Significant probability less than 0.05,

**Highly significant probability less than 001.
1The"data summarized in thts table can be found in
the Appendix, Table 35.
2This is the group mean + the mean variation.
SThe letters, C, S, and My stand for copper, sulfur,
and molybdenum, respectively*
4This is the breakdown of the treatment significance
in the analysis of variance given in the Appendix, Table
3G.










TABLE 29

TRIAL III, FXPTUIEIINT III.
SUMIMAlR OF IZEOGLOBIN VALUES OF RATS ON
VARIOUS RATIONS GIVEN IN TABLE :5


Ration Sex Hemoglobin2 Statistical Analysis
xV/100 ml 'Treatmentd, Effect ean<

3R1 Female 14.41 0.7 (1) 14.21
3R2: Female 12.6 0,9 C 0.C61
3r3 Female 15.0 0.3 S 0.03**
3R4 Female 13.6 0.8 CS 0.18
3R5 Female 14.5 0.5 M 0.00D*
3R6 Female 14.1 0.7 CMI 0o15
3R7 Female 14.8 0.2 SM 0.6G**
3R8 Female 14,7+ 0,3 CSM 0.03

*Significant probability less than 0.05 .

**Highly significant probability less than 0.01.
1The'data summarized in this table can be found in
the Appendix, Table 35.
P2'his is the group nean + the mean variation.

3The letters, C, S, and M, stand for copper, sulfur,
and molybdentunm, respectively.
4This s s the breakdown of the treatment significance
in the analysis of variance given in the Appendix, Table
36,








84

per million of molybdenum. The addition of 497 parts per

million of sulfur as Na2SO4 to the above ration overcame
the growth inhibition caused by 75 parts per million of
molybdenum but not that caused by 300 parts per million of
molybdenum. In another study using the same basal ration,
the addition of 88 parts per million of sulfur did not over-
come the growth inhibition produced by the addition of 100
parts per million of molybdenum. Levels of 497 and 746
parts per million of sulfur appeared to be effective in
preventing the growth depressing effects of the molybednur,
but the control rats gained an average of only 16 grams per

week. Hills -i al. (124) used a 20 per cent casein ration
which contained 14.2 parts per million of copper. When

905 parts per million of sulfur were added to this ration,
800 parts per million of molybdenum depressed weight gains
during a five-week experimental period by 9*1 per cent,












S1NIARY


Three experimental programs have been carried out
to determine the effect of the interrelationship of phos-

phorus, sulfur, copper, and molybdenum on cellulose di-

gestion by rumen inoculum of runen-fistulated steers using
both Jn juvyA and jn vitro techniques and on growth and,
bone formation in weanling rats.

In the first group of experiment an rja vvo bag
technique was evaluated and found to give satisfactory

results for comparing the digestion of cellulose from dif-
ferent sources. The digestion of cellulose from Solka-Floo
was highest; cotton linters, intermediate; and sugar cane

bagasse, lowest. This injivo bag technique was used in
determining the effect of the interrelationship of phos-
phorus, sulfur, and molybdenum on the digestion of cellulose
from the three sources mentioned above. A total phosphorus
concentration in the steer ration of 0.54 per cent slightly
reduced cellulose digestion while an addition of 0.40 per
cent sulfur as sodium sulfate to the ration which contained
0.15 per cent sulfur and 1.46 per cent nitrogen of which two-
thirds was supplied by urea produced a highly significant
increase in cellulose digestion. Also, the results suggest
that as ruminants get older the rumen inoculum becomes more
efficient in cellulose digestion. This may possibly be
85








86

related to the mineral concentrations in the rumcen inoculum.
The supplementation of a steer ration with sulfur
or phosphorus produced a significant increase of that ele-
rent in the ruren liquid,

In the second group of experiments, an artificial

rumen technique was used to study cellulose digestion. As

in the in vivo data, the digestion of cellulose from Solka-
Floe was highest; cotton liners, intermediate; and sugar

cane bagasse, lowest. Grinding the cotton liners and the

sugar cane bagasse improved the digestion of .cellulose
from each. The addition of either sulfur or phosphorus to
runen inoculum improved cellulose digestion, and the im-
provements appeared to be additive. In the presence of a high

level of; phosphorus, when either copper or molybdenum was

present. at a level that depressed cellulose digestion by
runen inoculum, the addition of the other lessened the
severity of the depression. The depression produced by ex-

cessive copper was reduced by added sulfur. In the 24

factorial experiment which included phosphorus, sulfur,
copper, and molybdenum, the effects of rolybdenun and the
remaining first and second order interactions not mentioned
above were not different from the experimental mean but were

lower than the optimLu control.
In the third group of experiments, weight gains,

milligrams ash per femur per gram of live body weight, per-
centage ash of the fear on a dry, fat-free basis, and








87

femur ash were used as criteria to evaluate the effect of
the above mineral interrelationship on growth and ,bone-
formation in weanling rats. Hemoglobin, percentage phos-
phorus of the femur and of the liver and percentage cop-
per of the liver values wore dotermainod for a limited number
of female rats.
.Although female rats had a significantly higher
amount of femur ash per unit of live body weight than
males, the females had lower weight gains and femur ash
values. The addition of sulfur as sodium sulfate to a
basal ration containing 18 per cent casein stiun~lated growth
in male and female rats. In the absence of .the added sul-
fur, weight gains and femur ash values were depressed more
in male than in female rats by the addition of 0,78 per
cent phosphorus. The addition of sulfur to this high phos-
phorus ration was effective in counteracting the depression
caused by the high level of phosphorus supplementation.
This bigh dietary phosphorus level increased liver phos-
phorus and decreased femur phosphorus for female rats*- The
sulfur addition decreased the phosphorus concentration of
the femur but not to the same degree as that produced by
the high level of phosphorus. In addition the sulfur
supplementation partially counteracted the lowered fer.ur
phosphorus produced by the high phosphorus intake. In the
presence of this high dietary phosphorus level, the addi-
tion of molybdenum depressed weight gains and fem-r ash










values for female rats and increased weight gains and'
femur ash values for male rats. The addition of either

CuC12'2-i20 or Na2SO4 failed to alleviate the depression
produced by the molybdenum. However, the depression Caused
by 300 parts per million of molybdenm was reversed by the
simultaneous addition of 30 and 600 parts per riillion of
copper and sulfur, respectively. When 0.66 per cent sul-
fur was added to the rat basal ration, it was toxic.
Phosphorus, sulfur, copper, and molybdenum are
interrelated in many ways which effect liver copper con-
centrations. The addition of sulfur caused a lowering of
liver copper values while added phosphorus, copper, and
molybdenum caused an increase.




































APPENDIX




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