• TABLE OF CONTENTS
HIDE
 Title Page
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Introduction
 Literature review
 Experiment I
 Results and discussion
 Experiment II
 Results and discussion
 Experiment III
 General discussion
 Summary
 Appendix
 Literature Cited
 Biographical sketch
 Copyright














Title: Influence of dietary magnesium and sulfur upon phosphorus utilization, rumen function and appetite in ruminants
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Title: Influence of dietary magnesium and sulfur upon phosphorus utilization, rumen function and appetite in ruminants
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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
        Page iv
    List of Tables
        Page v
        Page vi
        Page vii
    List of Figures
        Page viii
    Introduction
        Page 1
    Literature review
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
    Experiment I
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
    Results and discussion
        Page 22
        Page 23
        Page 24
        Page 25
    Experiment II
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
    Results and discussion
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
    Experiment III
        Page 43
        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
    General discussion
        Page 57
        Page 58
        Page 59
        Page 60
        Page 61
        Page 62
    Summary
        Page 63
        Page 64
        Page 65
    Appendix
        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
        Page 85
        Page 86
    Literature Cited
        Page 87
        Page 88
        Page 89
        Page 90
        Page 91
        Page 92
        Page 93
        Page 94
    Biographical sketch
        Page 95
        Page 96
    Copyright
        Copyright
Full Text










INFLUENCE OF DIETARY MAGNESIUM AND

SULFUR UPON PHOSPHORUS UTILIZATION,

RUMEN FUNCTION AND APPETITE

IN RUMINANTS








By
JACK EDWARD MARTIN


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
April, 1963















ACKNOWLEDGEMENTS


The writer wishes to express his sincere appreciation to Dr.

L. R. Arrington, chairman of his supervisory committee, for his guid-

ance, assistance, and patience throughout the experimental investiga-

tion and preparation of this dissertation and to Drs. G. K. Davis,

R. L. Shirley, T. W. Stearns, C. B. Ammerman and J. E. Moore for aid

and suggestions and for serving as members of the writer's supervisory

committee. He wishes to especially thank Dr. J. E. Moore for his

assistance and for the teaching of the techniques and concepts of

ruminant nutrition investigations. He is grateful for the assistance

given him by many of the graduate students, faculty members, and lab-

oratory technicians of the Animal Science Department.

This investigation was supported in part by grants from the

Moorman Manufacturing Company and the National Institutes of Health.

The writer wishes to acknowledge his appreciation for this support.

He wishes to thank Monsanto Chemical Company, St. Louis,

Missouri, for the Santoquin and E. I. Du Pont De Nemours and Company,

Wilmington, Delaware for crystalline urea used in the research.

The writer is especially indebted to his wife, Norma, for her

inspiration, understanding and assistance during the course of this

investigation.

Sincere appreciation is also expressed to Mrs. Dorothy Hurd

for typing this dissertation and to Mr. R. W. Rainey for producing the

figures.
















TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS ............................................. ii

LIST OF TABLES .............. .......................... ........ v

LIST OF APPENDIX TABLES ...................................... vi

LIST OF FIGURES ............................................. viii

INTRODUCTION .............................. ................... 1

LITERATURE REVIEW ............................................ 2

The Effect of Magnesium and Sulfur on Phosphorus
Utilization ...................... ....... ... ............ 2
The Effect of Magnesium and Sulfur on Rumen Function .... 6

GENERAL EXPERIMENTAL PROCEDURE ............................... 11

EXPERIMENT I ................................................. 13

Effect of Magnesium and Sulfur on Phosphorus Utilization 13
Procedure: Trial one .............................. 13
Procedure:" Trial two .............................. 17

RESULTS AND DISCUSSION ....................................... 22

Trial one -- lambs ................................ 22
Trial two -- steers ................................ 23

EXPERIMENT II ................................................ 26

Effect of Magnesium and Sulfur on Cellulose Digestion and
Rumen Function in Steers ............................... 26
Rumen fistulation .................................. 26
Procedures for collecting samples .................. 27
Laboratory procedures for measurement of rumen
function ........................................... 29











TABLE OF CONTENTS--Continued


Page

RESULTS AND DISCUSSION ....................................... 33

In vivo cellulose digestion ........................ 33
In vitro cellulose digestion ........................ 35
Total steam volatile fatty acid concentration ...... 36
Trichloroacetic acid insoluble nitrogen ............ 38
Buffering capacity of rumen liquor ................. 39
Bacteriological observations ....................... 41

EXPERIMENT III ............................................... 43

Effect of magnesium on appetite in sheep and in
vitro cellulose digestion ....................... 43
Magnesium effect on appetite ...................... 43
Cellulose digestion ................................ 49
Magnesium requirement of cellulolytic microorganisms 52

GENERAL DISCUSSION ........................................... 57

SUMMARY ....................................................... 63

APPENDIX ..................................................... 66

LITERATURE CITED ............................................. 87















LIST OF TABLES


Table Page

1. Constituents of Purified Ration ......................... 14

2. Experimental Design for Study of Magnesium and Sulfur on
P-32 Absorption ........................................ 16

3. Experimental Design for the Study of Magnesium and Sulfur
on Phosphorus Utilization .............................. 18

4. Summary of P-32 Absorption in Lambs During Five Day
Collection Period...................................... 23

5. Summary of Phosphorus Utilization in Steers During Five
Day Collection Period ................................ 25

6. Effect of Magnesium and Sulfur upon in vivo Cellulose
Digestibility in Steers ............................... 34

7. Cellulose Digestion in vitro During Twenty-Four Hour
Fermentation Period~by Rumen Inoculae ................. 36

8. Total Steam Volatile Fatty Acid Concentration of Rumen
Fluid From Steers Fed Purified Rations With and Without
Magnesium and Sulfur .................................. 37

9. Trichloroacetic Acid Insoluble Nitrogen Concentration of
Rumen Fluid From Steers Fed Purified Rations With and
Without Magnesium and Sulfur .......................... 39

10. Buffering Capacity of Rumen Fluid From Steers Fed Puri-
fied Rations With and Without Magnesium..and Sulfur .... 41

11. Average Daily Feed Consumption of Lambs on Appetite
Recovery Trial With Magnesium Oxide Capsules .......... 46

12. Average Daily Feed Consumption of Lambs on Appetite
Maintenance Trial With Magnesium Sulfate Injections ... 46

13. Cellulose Digestion in vitro by Rumen Inoculum From Lambs
Fed Purified Rations With and Without Magnesium ....... 51

14. Grams Cellulose Digested by Day in vitro Using Transfer
Technique ................... ..... ... ........ ...... 53















LIST OF APPENDIX TABLES


Table Page

1. Per Cent of the Dose of P-32 Recovered in the Urine of
Lambs. Experiment I, Trial 1 ........................ 67

2. Per Cent of the Dose of P-32 Recovered in the Feces of
Lambs. Experiment I, Trial 1 ........................ 67

3. Total Urine Excreted in Five Days by Lambs on P-32
Study, Milliliters. Experiment I, Trial 1 ........... 68

4. Average Daily Feed Consumption for Lambs on P-32 Absorp-
tion Trial, Grams. Experiment I, Trial 1 ............ 68

5. Total Phosphorus Excreted in Urine by Steers in Five Day
Balance Period, Grams. Experiment I, Trial 2 ........ 69

6. Total Phosphorus Excreted in Feces by Steers in Five Day
Balance Period, Grams. Experiment I, Trial 2 ........ 69

7. Total Phosphorus Excreted by Steers During Five Day
Balance Period, Grams. Experiment I, Trial 2 ........ 70

8. Average Daily Voluntary Feed Intake of Steers on
Phosphorus Balance, Grams. Experiment I, Trial 2 .... 70

9. Total Five Day Phosphorus Intake of Steers on Balance
Study, Grams. Experiment I, Trial 2 ................ 71

10. Total Urine Excreted by Steers During Five Day Phos-
phorus Balance Study, Milliliters. Experiment I,
Trial 2 .............................................. 71

11. Total Feces Excreted by Steers During Five Day Phos-
phorus Balance Study, Grams. Experiment I, Trial 2 .. 72

12. Statistical Analysis of Fecal Phosphorus Excretion of
Steers. Experiment I, Trial 2 ....................... 73

13. Statistical Analysis of Urinary Phosphorus Excretion
of Steers. Experiment I, Trial 2 .................... 74










LIST OF APPENDIX TABLES--Continued


Table Page

14. Statistical Analysis of Total Phosphorus Excreted by
Steers. Experiment I, Trial 2 ........................ 75

15. Statistical Analysis for Apparent Phosphorus Absorption
by Steers. Experiment I, Trial 2 ..................... 76

16. Statistical Analysis of Apparent Phosphorus Retained
by Steers. Experiment I, Trial 2 ..................... 77

17. Total Cellulose Intake of Steers During Five Day
Collection Period, Grams. Experiment II .............. 78

18. Total Cellulose Excreted by Steers During Five Day
Collection Period, Grams. Experiment II .............. 78

19. Statistical Analysis of in vivo Cellulose Digestibility
of Steers Fed a Purified Ration, Experiment II ........ 79

20. Statistical Analysis of in vitro Cellulose Digestion of
Rumen Fluid From Steers Fed a Purified Ration.
Experiment II ......................................... 80

21. Statistical Analysis of Total Volatile Fatty Acid
Concentration of Rumen Fluid From Steers Fed a
Purified Ration. Experiment II ....................... 81

22. Statistical Analysis of Trichloroacetic Acid Insoluble
Nitrogen of Rumen Fluid From Steers Fed a Purified
Ration. Experiment II................................. 82

23. Statistical Analysis of Buffering Capacity of Rumen
Fluid From Steers Fed a Purified Ration. Experi-
ment II ................................................ 83

24. Cellulose Digested in vitro by Rumen Inoculae From
Lambs Fed a Purified Ration With and Without Magnesium,
Grams. Experiment III ................................. 84

25. Cellulose Digested in vitro Using Solka-Floc Substrate
and Rumen Inoculum from Lambs Fed a Complete Purified
Ration, Grams. Experiment III ........................ 85

26. Cellulose Digested in vitro Using Ration Substrate and
Inoculum from Lambs Fed a Complete Purified Ration,
Grams. Experiment III ................................ 86















LIST OF FIGURES


Figure Page

1. Funnel type pan designed for feeding test rations
through the ruminal fistula ........................... 20

2. Unassembled rumen cannula and tool designed to facilitate
assembling .................................. ............ 28

3. Daily feed consumption of lambs on appetite recovery
trial ................................................. 47

4. Daily feed consumption of lambs on appetite maintenance
trial ................................... .............. 48

5. In vitro cellulose digestion using transferal technique.. 55


viii















INTRODUCTION


The efficient utilization of dietary nutrients is essential for

.the maximum production of meat, milk and wool. In ruminant animals,

satisfactory production is dependent largely upon the fermentation

processes in the rumen which permit' utilization of roughage type feed-

stuffs that cannot be utilized efficiently by monogastric animals.

Numerous factors, both dietary and environmental, are known to affect

body function and thus efficiency of the conversion and utilization of

dietary nutrients. The element phosphorus is required by nearly all

living organisms and its optimum utilization may be altered by inter-

relationships with other dietary nutrients. The functioning of the

rumen in ruminant species involves a symbiotic relationship between

the host and microorganisms. Complex interactions among nutrients

have not been extensively studied in ruminants.

Among the factors which are known to affect phosphorus utiliza-

tion and rumen function are mineral intake and mineral interrelation-

ships, but their specific actions are not completely understood. In

order to study the effect of magnesium and sulfur on specific body

processes, three experiments were conducted with cattle and sheep. The

effects of the two mineral elements were determined on specific diges-

tive processes which could be measured by established in vivo and in

vitro techniques. The objectives were to identify the role and to

measure the effect of magnesium and sulfur on phosphorus utilization

and rumen function.















LITERATURE REVIEW


The Effect of Magnesium and Sulfur on Phosphorus Utilization

The major role of calcium and phosphorus in nutrition, the

dietary requirements, interrelationships of these elements and their

economic importance in animal feeding are generally well known. Less

is known, however, of the effects of other minerals on the utilization

of these essential dietary nutrients.

As early as 1928, Palmer et al. observed that magnesium sulfate

supplementation of mature cows receiving rations low in phosphorus had

a detrimental effect on calcium balance which could be improved with

phosphorus supplementation. The addition of 2.5 grams of magnesium per

100 pounds of body weight to one week old calves resulted in higher

terminal serum inorganic phosphorus and magnesium and lower calcium and

higher magnesium in the bone ash.(Dehority et al. 1961). Stewart and

Moodie (1956) found that heavy oral doses of magnesium sulfate increased

the blood phosphorus content and decreased the blood calcium of sheep.

However, these workers did not determine if this increase in blood phos-

phorus was a result of increased phosphorus absorption or a mobilization

of phosphorus from the bones and tissues. It would also be of interest

to speculate upon the effect that sulfate might have had on these experi-

ments. A recent report by O'Dell (1960) includes a comprehensive review

of the relation of magnesium to other dietary constituents.

The various studies of magnesium deficiency in relation to









magnesium tetany or "grass staggers" in cattle are of interest. The

development of the tetany tends to be seasonal, occurring usually in

the first week after grazing rapidly growing young cereal grains or

young spring pastures high in protein (Sjollema, 1930, Blaxter and

McGill, 1956). The application of nitrogen fertilizers has been asso-

ciated with the development of this condition (Sjollema, 1932, Allcroft,

1956). On the other hand, the application of dolomite or other sources

of magnesium has almost always reduced the incidence of magnesium tetany

(Jacob, 1958, Stewart and Reith, 1956). The condition, therefore, seems

to be the result of low magnesium and high crude protein level of the

forage and suggests that perhaps the ammonium content of the rumen may

be interfering with the utilization of magnesium (Ershoff, 1948).

It is interesting to note that bone magnesium is not depleted

under these conditions while in all other types of magnesium deficiency

a depletion of bone magnesium is a characteristic finding. The fact

that bone magnesium is not decreased would tend to support the hypoth-

esis that magnesium tetany is the result of a poor mobilization mech-

anism in the bone. With a decreased absorption of the dietary magnesium,

the animal is unable to meet the demands from increased physical activity

(Blaxter, 1956).

Evans (1959) reported that the addition of 0.78 per cent phos-

phorus, as sodium acid phosphate, to a rat diet deficient in sulfur

depressed body weight gains and femur ash as compared to the basal ra-

tion which contained 0.16 per cent phosphorus. The addition of sulfur

as sodium sulfate to this high phosphorus diet was effective in counter-

acting the depression of weight gain and femur ash. No additional









information on a possible interaction of sulfur and phosphorus has

appeared in recent literature.

Young rats which were fed a rachitogenic diet low in phosphorus

became rachitic but usually recovered after twenty-five to thirty days

(Roche, 1932). The addition of 2 per cent magnesium carbonate to the

diet resulted in a negative phosphorus balance until death. Some rats

lost as much as 50 per cent of the total body phosphorus in six months.

Similar results were reported by Villus (1932) and Buckner (1932).

Villus found that magnesium oxide given with sodium phosphate to rats

suffering from experimental rickets had an antagonistic and decalcify-

ing action. However, Gershoff and Andrus (1961) reported high magnesium

diets prevented calcium oxalate deposition in the urinary tracts of

pyridoxine deficient rats.

Different results were obtained with guinea pigs as the test

animal. House and Hogan (1955) reported that a phosphorus intake of

1.7 per cent of the diet was injurious to guinea pigs. This injurious

effect could be reduced remarkably with increased magnesium intake.

On the other hand, Maynard et al. (1958) reported that lowering only

the level of magnesium in a guinea pig diet which was relatively high

in phosphorus, calcium and magnesium was more effective in preventing

the deleterious effect of high calcium and phosphorus intakes than any

combined decrease in magnesium and calcium or magnesium and phosphorus.

An excessive intake of phosphorus was observed by O'Dell et al.

(1960) to accentuate the symptoms of magnesium deficiency in guinea

pigs. The injurious effect of phosphorus, when added to a diet low in

magnesium, was more marked than that of calcium, but the effect was

largely eliminated by adequate magnesium. A dietary magnesium level









of 0.06 per cent maintained near normal tissue composition in guinea

pigs fed a high phosphorus diet (Morris and O'Dell, 1961). These

workers concluded that a higher magnesium level is required to maintain

tissue composition than to support maximal growth.

Leichsenring et al. (1951) reported data collected from college

women receiving a basal diet containing 250 milligrams of magnesium,

300 milligrams of calcium and 800 milligrams of phosphorus. The diet

was supplemented with either 1200 milligrams of calcium or 600 milli-

grams of phosphorus. Using partial correlations as criteria, a sig-

nificant relationship was observed between fecal calcium and phosphorus,

but not between magnesium and calcium or phosphorus. Urinary excretion

of magnesium was significantly correlated (P <.05) with the intake

levels of both calcium and phosphorus. However, Carswell and Winter

(1931) reported that the intake of over one gram of magnesium daily,

improved calcium balances in humans provided the phosphorus level was

adequate. On the other hand, Tibbetts and Aub (1937) observed that

increasing the phosphorus intake increased magnesium excretion in the

urine of human subjects and suggested that intracellular magnesium can

be used for neutralizing excess acid in the urine.

In an early review of magnesium studies, Duckworth (1939)

stated that, under certain circumstances, the addition of magnesium

increased calcium retention. In some cases this result was probably

due to incidental changes in the balance between calcium and phosphorus

absorbed, or to simultaneous adjustments in the intake of these elements.

In other cases it may have been due to increased growth, if the original

diet was deficient in magnesium. On the other hand, large increases in









dietary magnesium resulted in a tendency to decalcify the skeleton un-

less compensatory adjustments were made in the calcium and phosphorus

intakes.

The results reported from these studies are varied and indicate

that the role of magnesium in phosphorus and calcium metabolism is not

well understood. Further work to help clarify this role of magnesium

is desirable. That sulfur as sulfate will affect phosphorus utiliza-

tion in a monogastric animal, such as the rat, is intriguing since

their only known requirement for sulfur is in the form of sulfur con-

taining essential amino acids. It seemed desirable, therefore, to

study further the effect of sulfur on phosphorus utilization in a ru-

minant animal which has been shown to be capable of utilizing elemen-

tal sulfur.


The Effect of Magnesium and Sulfur on Rumen Function

In the past, magnesium has received considerable attention in

studies on its function in monogastric animal nutrition. Considerable

work has also been conducted with ruminants in investigations of its

role in magnesium tetany or "grass staggers." However, little research

has been reported to study the effect of magnesium and sulfur on basic

in vivo and in vitro rumen function..,

Chamberlain and Burroughs (1962) reported results of a study

using washed cell suspensions of rumen microorganisms in a cellulose

digestion study. The omission of magnesium from the nutrient media

did not result in a significant reduction in cellulose digestion. Other

reports of studies of a specific effect of magnesium upon rumen micro-

organisms were not found in the literature.









The important role of sulfur in ruminant nutrition was empha-

sized with the work of Loosli et al.(1949) and since that time other

research has established its importance in ruminant nutrition. The

work of Loosli demonstrated the microbial synthesis of methionine from

inorganic sulfur. Thomas et al. (1951) and Starks et al. (1954) fur-

ther verified the importance of sulfur in ruminant nutrition in trials

conducted with lambs fed purified rations with and without sulfur. The

lambs receiving the sulfur deficient rations lost weight from the start

of the experiment and died after 53 to 140 days later. Studies of

samples of rumen fluid revealed marked changes in numbers and types

of microflora present.

That the absence of sulfur from the ration will alter rumen

microflora was further illustrated by Gall et al. (1951). Bacterio-

logical findings from three groups of lambs fed purified rations in-

dicated that sulfur had a pronounced effect on both the kinds and

numbers of bacteria present. The numbers present showed that the ra-

tion containing sulfur supported a rumen microbial population double

that of the sulfur deficient ration. The rumen of animals fed the

ration containing sulfur contained Gram-positive slender, curved rods

or Gram-positive large, flat rods. The sulfur deficient ration showed

Gram-negative medium rods.

The stimulation of vitamin B synthesis, cellulose digestion

and urea utilization in vitro by rumen microorganisms supplemented with

sulfur was reported by Hunt et al.(1954) and Trenkle et al. (1958).

Starch was used as a source of energy in these experiments and, with

sulfur, was shown to stimulate the synthesis of riboflavin, niacin,









pantothenic acid, cyanocobalamin and to increase the utilization of

urea. The activity of the rumen microorganisms was stimulated more by

sulfur as sodium sulfate or as methionine than by cystine or elemental

sulfur.

The stimulation of urea utilization when starch was used as a

source of energy was indicated by McDonald (1952) in studies where the

accumulation of rumen ammonia was reduced when starch was added to a

timothy hay and urea ration. The addition of starch suspensions to

the rumen 20 hours after feeding a purified diet was observed to lower

ammonia levels (Mills et al. 1942). On the other hand, Annison (1956)

found that the omission of starch and sucrose from a purified ration

resulted in an increased ammonia production. Foreman and Herman (1953)

showed that the addition of two pounds of molasses to a grain, alfalfa

hay ration fed to dairy cows apparently increased the digestibility of

crude protein, cellulose and crude fiber.

In vitro studies (Belasco, 1956, Bloomfield et al. 1958) have

shown starch to be superior to other forms of carbohydrates for the

stimulation of urea utilization. Arias et al. (1951) and Burroughs

and Hall (1954) reported that soluble carbohydrate increases urea util-

ization but decreases cellulose digestion. This decrease in cellulose

digestion due to a soluble carbohydrate has been reported by Balch et al.

(1954), Burroughs et al.(1949), Hamilton (1942), Kane et al. (1959),

Swift et al. (1947), Fontenot et al. (1955) and Louw and Van der Wath

(1943). However, the addition of a limited amount of a soluble carbo-

hydrate has been shown to increase cellulose digestion (Burroughs et al.

1950, Clark and Quinn, 1951, Foreman and Herman 1953 and Louw and Van

der Wath, 1943).









The stimulation of urea utilization from a limited amount of

soluble carbohydrate results in increased bacterial growth which may be

related to increased cellulose digestion. A large amount of soluble

carbohydrate in the ration would result in a competition for available

nutrients between the cellulolytic and amylolytic organisms. That this

competitive condition could alter the microbial population was reported

by Williams et al. (1953) and Burroughs et al. (1950) in studies where

the addition of starch to the ration resulted in a depression of total

bacteria numbers. However, Williams et al. (1953) found that by raising

the level of protein in the ration the depression in cellulose digestion

and bacteria numbers could be alleviated.

The interrelationship of starch, cellulose and urea has recently

been reported from in vitro studies by el-Shazly et al. (1961). They re-

ported that increased urea nitrogen levels reversed the inhibition of

cellulose digestion. Starch added to a hay ration for sheep inhibited

cellulose digestion as determined by the digestion of filter paper sus-

pended in the rumen inside nylon bags. The addition of urea through the

fistula alleviated the inhibition unless the estimated cellulose:starch

ratio was 1:5.

The addition of various substances (alfalfa ash, water extracts

of alfalfa, molasses ash, mineral mixtures, yeast, dried distillers

solubles, autoclaved water extract of cow manure and autoclaved rumen

fluid) have resulted in an increased in vitro cellulose digestion (Bentley

et al. 1954, Burroughs et al. 1950 and 1951) and in vivo cellulose diges-

tion (Bentley et al. 1951, Burroughs et al. 1948, and Klosterman et al.

1953).









The validity of in vitro rumen fermentation studies and their

usefulness as a measure for the digestibility of forages in ruminants

have been studied (Halliwell, 1957, Quicke et al. 1959 and Warner,

1956). In vitro cellulose digestion was shown to be more closely re-

lated to in vivo nutritive than other laboratory methods (Baumgardt

et al. 1958). Hershberger et al. (1956) and LeFevre and Kamstra (1958)

reported a high correlation between cellulose digestion in vitro and

in vivo but differences were found between sheep and steer inoculum

and inoculum from the same species but on different rations. When

different forages were fed to a steer used as a source of inoculum,

the in vitro cellulose digestion of these forages was not affected by

the type of forage fed to the steer according to Quicke et al. (1959).

Also, the in vitro cellulose digestion coefficients showed less vari-

ation than those from in vivo studies.

The interrelationships shown to exist between cellulose diges-

tion, urea utilization and starch fermentation is further complicated

by the effects and interrelationships of various minerals. These re-

lationships, if defined, would contribute greatly to our knowledge of

rumen function. The use of in vitro techniques provides a quick and

relatively inexpensive means of obtaining information on rumen function

as compared to in vivo methods (Nickelson et al. 1956).















GENERAL EXPERIMENTAL PROCEDURE


Studies of the effect of magnesium and sulfur upon certain

digestive processes were conducted with cattle and sheep representing

two ruminant species. Rations were prepared from purified ingredients

so that magnesium and sulfur could be omitted from or added to the ra-

tion. Measurements of the effects of the elements when omitted or

added were based upon established techniques which reflected metabolic

functions or the influence of the elements in certain digestive proc-

esses.

Phosphorus absorption and utilization were studied in sheep

and cattle using radioactive phosphorus in a balance trial with sheep

and stable phosphorus in a conventional balance experiment with steers.

Absorption and excretion of phosphorus weredetermined in both species

fed the purified rations with and without magnesium and sulfur.

The effects of the two elements upon rumen function were de-

termined by measurement of in vivo and in vitro cellulose digestibility

and by estimations of total steam volatile fatty acid concentration

(TVFA), trichloroacetic acid insoluble nitrogen (TCA-N) concentration

and buffering capacity of the rumen fluid. Microscopic examinations

of the rumen microorganisms were also conducted.

The purified ration used (Table 1) was the same for all experi-

ments. Individual ingredients of the ration were secured from commer-

cial sources and were of the quality used in experimental rations in

11







12

research. Mineral compounds used to supply minerals for the ration

were reagent grade.

The statistical analyses were based upon the Matrix-Vector

product method for calculating the analysis of variance of a Latin

Square design, by Brandt (1962).















EXPERIMENT I


Effect of Magnesium and Sulfur on Phosphorus Utilization

Experiment I was conducted in two separate trials. During trial

one the absorption of radioactive phosphorus in lambs, fed a purified

ration, served as the criterionfor measuring the treatment effect on

phosphorus utilization. In trial two the phosphorus status of fistu-

lated steers, fed a similar purified ration, was the criterion for de-

termining the treatment effect on phosphorus utilization. The phosphorus

status of the animals was determined through phosphorus balance studies.


Procedure: Trial One.

Six, ten month old wether lambs ranging in weight from 50 to

57 pounds with a mean weight of 54 pounds were used as experimental

animals. All the lambs were fed a purified ration (Table 1) and dis-

tilled water ad libitum until placed on experiment. Preceding the first

experimental period, a preliminary trial collection period was made to

cull any lambs that would not adapt to the metabolism crates. Two of

the original lambs were culled on this basis and the four remaining

lambs were then assigned to treatments.

The percentage composition of the complete purified basal ration

is given in Table 1. The four ration treatments were: basal, basal

minus magnesium, basal minus sulfur and basal minus magnesium and sulfur.

These rations will be referred to as complete, magnesium deficient, sul-

fur deficient and magnesium and sulfur deficient rations, respectively.

13









TABLE 1

CONSTITUENTS OF PURIFIED RATION

Ingredient

Cerelose

Corn starch

Cellulose (Solka-Floc)

Cottonseed oil

Crystalline urea

Mineral mixture

Vitamin A Palmitate (10,000 U.S.P./gram)

Vitamin D2 (9,000 I.U./gram)

d-alpha Tocopherol



MINERAL MIXTURE


CaHPO4.2H20

KC1

Na2S04

NaCI

FeC6H507. 2H20

MgO

MnCO3

CuC12.6H20

'"CO3


CC12.6H20


28.797

28.000

30.000

4.000

4.000

5.110

0.044

0.048

0.001
100.000


62.241

18.220

8.621

4.310

3.233

3.164

0.076

0.057

0.039

0.038

0.001
100.000








The rations contained an average of 0.56 per cent phosphorus. The com-

plete and sulfur deficient rations contained by analysis (Welcher, 1961)

an average of 950 parts per million magnesium while the magnesium defi-

cient and the magnesium and sulfur deficient rations were found to con-

tain no detectable magnesium. The complete and magnesium deficient ra-

tions were found to contain by analysis (Association of Offical Agri-

cultural Chemists, 1955) 970 parts per million of sulfur while the

sulfur deficient and the magnesium and sulfur deficient rations con-

tained no sulfur. The deficient rations were prepared by omitting mag-

nesium oxide and/or sodium sulfate from the mineral mixture. In order

to prevent the development of rancidity the'rations were stored in a

refrigerator at 4 degrees Centigrade.

The daily allowance for each lamb was 400 grams of the purified

ration. This was the level readily consumed by the lambs. The lambs

were fed twice daily, receiving one-half of their daily ration per feed-

ing. Feed refusals were collected and weighed prior to each successive

feeding.

The lambs were assigned to four treatments in a Latin Square

design (Table 2). Following each treatment and collection period the

lambs were immediately fed the next assigned ration. By the end of

each collection period, most of the radioactivity had been excreted in

the urine and feces. Residual excretion of radioactivity was determined,

prior to successive treatments, from a twelve hour collection of urine

and feces. This was taken into consideration in subsequent periods.

Each treatment period was 21 days in length. Individual feed-

ing stalls were employed the first 14 days of each treatment period.

this gave the lambs sufficient time to adjust to the new treatment.








During the remaining seven days they were confined to metabolism

crates, which had been thoroughly cleaned, for quantitative collection

of feces and urine. On the 16th day of each treatment period each

lamb was given an oral dose of 50 microcuries of radioactive phos-

phorus as phosphoric acid in weak hydrochloric acid. One milliliter

of a stock solution of radioactive phosphorus, which had been diluted

to provide 50 microcuries of radioactivity per milliliter, was placed

in one-fourth ounce gelatin capsules containing Solka-Floc. The cap-

sule was administered orally with a balling gun immediately following

the morning feeding.


TABLE 2

EXPERIMENTAL DESIGN FOR STUDY OF MAGNESIUM
AND SULFUR ON P-32 ABSORPTION


Lamb Number


Period 1 4 5 6


1 B1 A D C

2 A B C D

3 D C B A

4 C D A B


1A Complete ration
B Magnesium deficient ration
C Sulfur deficient ration
D Magnesium and sulfur deficient ration.


The collection of urine and feces was begun at 8:00 P. M. of

the second day of the collection period and was terminated 136 hours

later. Urinary and fecal collections were made at 12 hour intervals.








Total fecal collections were air dried and then ashed at 640

degrees Centigrade for 12 hours. The ash was taken up under heat with

concentrated hydrochloric acid and distilled water and then filtered

through Whatman No. 41 filter paper. The filtrate was collected in

100 milliliter volumetric flasks and brought up to volume.

The activity of the fecal solution and untreated whole urine

was determined with a dipping type Geiger tube and commercial scale.

A matching cup for the dipping tube was used to contain the solutions

counted. The matched cups were calibrated to contain seven milliliters

of the solution and constructed to provide duplication in sample counting.


Trial Two.

Six, grade yearling Angus steers ranging in weight from 400 to

560 pounds, with a mean weight of 430 pounds, were originally selected

for this study. During a five week adjustment period they were fed

pangolagrass hay ad libitum and three to five pounds of a concentrate

mix. During this period the steers were fitted with ruminal fistulas

described later. The steers were tied and brushed daily to accustom

them to close handling. Of the six steers in the original group, two

were culled. Of the two that were culled, one was found to be highly

nervous and unable to adapt to the environmental conditions, the other

steer was larger and did not conform to the other steers in body type.

The four selected fistulated steers were randomly assigned to ration

treatments in a Latin Square design with four treatments and four

periods (Table 3).

Each treatment period was 21 days in length. During the first

14 days the animals were maintained in individual box stalls and for








the last seven days they were confined to metabolism crates for quan-

titative collection of urine and feces. Following the collection

period, a seven day recovery period was allowed during which the com-

plete ration was fed. This recovery period was considered important

as evidenced by the results of trial one where one lamb died due to the

prolonged deficiency treatment effects. It was also considered desir-

able to maintain the steers on a constant intake of the experimental

ration to prevent variation due to intake which was evidenced in the

lamb trial by a loss of appetite on the deficient rations. To facil-

itate this, ruminal fistulated steers were used so the ration which

was refused could be placed in the rumen via the rumen fistula.


TABLE 3

EXPERIMENTAL DESIGN FOR THE STUDY OF MAGNESIUM AND
SULFUR ON PHOSPHORUS UTILIZATION


Steer Number


Period 5 6 7 8


1 C1 A D B

2 A B C D

3 D C B A

4 B D A C


1A Complete ration
B Magnesium deficient ration
C Sulfur deficient ration
D Magnesium and sulfur deficient ration.








The purified ration fed the steers was the same as the ration

given in Table 1. The four ration treatments were complete, magnesium

deficient, sulfur deficient and magnesium and sulfur deficient. The

deficient rations were prepared by omitting magnesium oxide and/or

sodium sulfate from the mineral mixture. The cottonseed oil used in

all treatments was stabilized with Santoquin to prevent the development

of rancidity.

The daily allowance for each steer was calculated to furnish

approximately 2000 Calories in excess of each steer's basal metabolic

77
requirement. This level was estimated from the formula 70WL (70 Cal-

ories/Kilogram body weight .73), as described by Brody (1945).

The steers were fed twice daily, receiving one-half of their

daily ration per feeding. Ration refusals were weighed and placed in

the rumen via the rumen fistula prior to each subsequent feeding. A

pan designed for feeding through the ruminal fistula is illustrated in

Figure 1. Distilled water in stainless or galvanized steel containers

was available at all times.

The steers were allowed an adjustment period for the first 14

days of each treatment period. This provided sufficient time for ad-

justment to the ration treatments. At the end of the 14 day adjustment

period, the steers were placed in the metabolism crates which had been

thoroughly cleaned. The collection of urine and feces was started at

8:00 A. M. and terminated at the same time five days later. Collections

were made daily. Total fecal excretion was weighed on a solution balance

and a 10 per cent aliquot taken. The aliquots were dried in an oven at

70 degrees Centigrade for 48 hours after which the daily aliquots for

each steer were composite. Total urine output was measured in a



































































Figure 1: Funnel type pan designed for feeding test rations through the
ruminal fistula.









graduated cylinder and one per cent of the total retained. The five

day urine collection for each steer was composite and stored in 250

milliliter polyethylene bottles and held in a food freezer until ana-

lyzed.

Total phosphorus was determined in the feed, fecal and urine

samples for measurement of phosphorus intake and excretion. The fecal

samples were first ground in a Wiley mill through an eight mesh screen

and thoroughly mixed. Duplicate one gram samples of feces and rations

were placed in crucibles and ashed at 640 degrees Centigrade for six

hours. Duplicate 10 milliliter samples of urine were pipetted into 50

milliliter beakers and brought to dryness over a hot plate. The dried

urine samples were then ashed for six hours at 640 degrees Centigrade.

the resulting ash from the feed, fecal and urine samples was taken up

under heat with concentrated perchloric acid and distilled water and

then filtered through Whatman No. 41 filter paper. The filtrate was

collected in 100 milliliter volumetric flasks and brought up to volume.

Phosphorus in the resulting solution was determined in duplicate by the

colorimetric method as described by Fiske and Subbarow (1925).
















RESULTS AND DISCUSSION


Trial One -- Lambs.

The percentage of the dose of radioactive phosphorus excreted

in the urine and feces of the lambs is shown in Tables 1 and 2, appendix.

A summary table of the mean urinary and fecal phosphorus excretion,

apparent phosphorus absorption, apparent phosphorus retention and daily

feed refusals per treatment are shown in Table 4. The average daily

feed refusals are given in Table 4, appendix.

The data in the summary table indicate that in the absence of

magnesium the apparent absorption and retention of phosphorus from the

alimentary tract was increased. In the absence of sulfur the apparent

absorption and retention of phosphorus was decreased.

On the 16th day of the fourth period the lamb on the sulfur

deficient ration was found dead. The lamb on the magnesium and sulfur

deficient ration was emaciated and stiff. The experiment was then

terminated. The loss of data from the fourth period prevented a valid

statistical analysis.

Throughout the experiment feed refusals occurred by the third,

fifth and sixth days from the beginning of each period for the magnesium

deficient, sulfur deficient and magnesium and sulfur deficient rations

respectively. The lambs receiving the deficient rations lost weight.

Since voluntary feed intake was greatly affected by treatment it would








seem reasonable that fecal excretion rate of radioactive phosphorus

would also be affected. However, this was not observed as illustrated

by a comparison of the average daily feed refusals with the average

period excretion of radioactive phosphorus.


TABLE 4

SUMMARY OF P-32 ABSORPTION IN LAMBS DURING
FIVE DAY COLLECTION PERIOD


Treatment

Mean Values1 Complete -Mg -S -Mg-S


Urinary P-32 6.60 11.21 0.73 14.40

Fecal P-32 45.12 18.77 56.73 27.59

Total 51.72 29.98 57.46 41.99

Apparent
absorption 54.78 81.23 43.27 72.41

Apparent
retention 48.28 70.02 42.54 58.01

Daily feed2
refusal 25.5 130.0 99.5 178.0


1Mean values expressed
recovered.
2Daily feed refusal in
daily.


as percent

grams, all


of dose of radioactivity

lambs received 400 grams


Trial Two -- Steers.

Data representing the grams of total phosphorus excreted by the

steers in the urine and feces during each five day collection period

are shown in Tables 5 and 6, appendix. The data for total grams of

phosphorus excreted in the urine and feces combined during the five day

collection periods and the average daily feed refusals are given in

Tables 7 and 8,-appendix. A summary showing the means of urinary










and fecal phosphorus excretion, phosphorus intake, apparent phosphorus

absorption, apparent phosphorus retention and daily feed refusals is

given in Table 5. The original data for grams of phosphorus intake,

total urine volume and total fecal volume are given in appendix Tables

9, 10 and 11, respectively. The statistical analyses for fecal phos-

phorus, urinary phosphorus, total grams of phosphorus excreted, apparent

phosphorus absorption and apparent phosphorus retention are given in

appendix Tables 12, 13, 14, 15 and 16, respectively.

The amount of phosphorus excreted in the feces was not statis-

tically affected by the ration treatment. ,However, the removal of

magnesium from the ration resulted in a numerical but non-significant

elevation of fecal excretion of phosphorus. This increase in phosphorus

excretion placed the steers in a negative phosphorus balance.

The effect of treatment on urinary phosphorus showed a low

order of probability (P <.10). A comparison of the urinary phosphorus

excretion data shows that the urinary phosphorus was consistently lower

in the sulfur deficient treatment as compared to the basal. This is in

complete agreement with trial one in which lambs receiving the sulfur

deficient ration had a low urinary phosphorus excretion rate.

Total phosphorus excreted was not statistically affected by

treatment. However, when the treatment effect was analyzed by individ-

ual degrees of freedom, the effect of magnesium in the ration exhibited

a low order of probability (P<.25).

Apparent phosphorus absorption was not statistically affected

by treatment. The effect of magnesium on apparent phosphorus retention

exhibited a low order of probability (P 4.25).









A comparison of the average daily feed refusals by the sheep

and the steers indicates a treatment effect on appetite of the same

magnitude in both species. Since the steers were maintained on a con-

stant intake by placing the feed refused in the rumen via the ruminal

fistula, it would seem that the difference in phosphorus absorption

and utilization between the lambs and steers could be accounted for

by the variation total feed intake. The low feed intake of the lambs

may have resulted in reduced rate of passage and an increase in phos-

phorus absorption.


TABLE 5

SUMMARY OF PHOSPHORUS UTILIZATION IN STEERS
DURING FIVE DAY COLLECTION PERIOD

Treatment

Mean ValuesI Complete -Mg -S -Mg-S


Urinary Phosphorus 18.11 17.02 8.95 21.91

Fecal Phosphorus 33.90 75.86 35.61 38.56

Total 52.01 92.88 44.56 60.47

Phosphorus
intake 56.75 57.10 57.10 57.20

Apparent
absorption 22.85 .,-18.76 21.49 18.64

Apparent
retention 4.74 -35.78 12.54 -3.27


1 Mean values expressed as total grams
collection period.


of phosphorus for five day















EXPERIMENT II


Effect of Magnesium and Sulfur on Cellulose Digestion
and Rumen Function in Steers

This experiment was designed to study the effects of magnesium

and sulfur upon in vivo cellulose digestibility, in vitro cellulose

digestion by rumen microorganisms, buffering capacity of the rumen

fluid and TVFA and TCA-N concentration of the rumen fluid. The design

of the experiment permitted simultaneous study of these various meas-

ures of rumen function.

The experiment was conducted concurrently with the phosphorus

balance study in Experiment I. Therefore, the design, treatments and

ration are the same as previously described.


Rumen Fistulation..

During the preliminary adjustment period the steers were fistu-

lated in a two-phase operation. Immediately preceding the first and

second phases, the steers were given an intramuscular injection of

tranquilizer (Promazine Hydrochloride) and two million units of Peni-

cillin. The operation area was then clipped, washed with Green soap,

painted with Merthiolate and desensitized with a local anesthetic.

In the first phase, a vertical incision approximately five

inches long was made through the skin and subsequent layers of tissue

to expose the peritoneum. When necessary some muscular tissue and

fascia were removed to facilitate the exclusion of these tissues from









an area approximately five inches in diameter. Stay-sutures were placed

through the skin, peritoneum and rumen wall excluding the muscular

tissue and fascia from an area five inches in diameter. The peritoneum

within this circular area was removed and the rumen wall scarred with

the point of a scalpel. The incision was closed by suturing, including

the rumen wall in the center sutures of the closure. A 14 day recovery

period followed to insure adhesion of the rumen wall and skin.

The second phase of the operation, which followed the recovery

period, consisted of surgically removing a section of the adhesion

approximately three inches in diameter from the center of the adhered

area. A polyvinylchloride pipe cannula three inches long with an in-

side diameter of two and one-fourth inches and adapted to receive a

thermos bottle cap and two threaded plexiglass flanges (Figure 2) was

fitted in the fistula. This was accomplished by inserting the cannula

and a plexiglass flange into the rumen and assembling. A rubber gasket

was placed on the cannula to separate the rumen wall from the plexiglass

flange. The cannula was then drawn through the fistula, another rubber

gasket placed on the cannula and the second plexiglass flange threaded

on. The thermos bottle cap was inserted into the cannula and snapped

tight. An instrument designed and constructed especially to facilitate

the assembling of the cannula is shown in Figure 2.


Procedures for Collecting Samples.

The steers were housed in individual pens for the first 14 days

of each treatment period. This provided sufficient time for adjust-

ment to the ration treatments. At the end of the 14 day adjustment

period, the steers were placed in the metabolism crates for quantitative




































































Figure 2: Unassembled rumen cannula and tool designed to facilitate
assembling. Two rubber gaskets not illustrated.








collection of feces. The daily collection of feces was started at

8:00 A. M. on the third day after the steers were placed in the metab-

olism crates and terminated at the same time five days later. Total

feces were weighed on a solution balance and a 10 per cent aliquot

taken. The aliquot was dried in an oven at 70 degrees Centigrade for

48 hours after which the daily aliquots for each steer were composite.

During the seven day collection period samples of rumen contents

were taken via the rumen fistula using a manual suction pump. The

samples were collected two hours after the morning feeding. The pH of

the rumen contents was immediately recorded. The sample was then trans-

ferred to the laboratory in sealed jars where it was strained through

four layers of cheesecloth. A portion of the rumen fluid obtained was

used to inoculate in vitro cellulose digestion tubes. Slides for micro-

scopic examination were prepared by making a smear of fresh rumen fluid

from each steer and staining with Gram-stain as described by Stahly and

Wiser (1951). The remainder of the sample was placed in plastic freezer

bags, frozen in a subzero freezer and transferred to a food freezer for

storage until further analyzed.


Laboratory Procedures for Measurement of Rumen Function.

The dried fecal samples were ground in a Wiley mill through an

eight-mesh screen and thoroughly mixed. Cellulose content was deter-

mined in duplicate on one gram samples of the feed and feces by the

method of Crampton and Maynard (1938). The results of the cellulose

determinations were used to calculate the in vivo cellulose digestibility

of the ration treatments.

Prior to the collection of rumen fluid to be used as inoculum









for in vitro cellulose digestion studies, 75 milliliter fermentation

tubes had been prepared. The substrate was BW-40 grade Solka-Floc1

in known amounts, approximately 0.5 grams per tube. k2wenty-two milli-

liters of a complete nutrient media described by Bentley et al. (1955)

or a similar media deficient in the nutrient corresponding to the ration

treatment was added. The tubes were placed in a water bath maintained

at 39 degrees Centigrade for two hours prior to inoculation and anaerobic

conditions were established and maintained by bubbling carbon dioxide

through the media. Twenty-five milliliters of inoculum were added to

each of three tubes per ration treatment.

Samples of each inoculum were saved for analysis of the cellulose

present. This was taken into consideration when the digestion of the

cellulose was calculated. The average initial pH values of the different

inoculae were 6.08, 5.91, 6.21 and 5.84 for the complete, magnesium de-

ficient, sulfur deficient, and magnesium and sulfur deficient, respec-

tively. The pH of the in vitro fermentation media was adjusted to 6.9

at eight hour intervals during the incubation period. The amount of

residual cellulose was determined on the entire tube contents at the

end of the 24 hour fermentation period by the method of Crampton and

Maynard (1938).

Buffering capacity of the rumen fluid was determined with the

use of an automatic constant rate burette coupled with a pH recording

adapter and a recorder. Seventy-five milliliters of rumen fluid were

titrated against 0.5 normal hydrochloric acid after adjusting the fluid

to pH 7.0 with 1.0 normal sodium hydroxide. The rumen liquor was adjusted



1Brown and Company, Berlin, New Hampshire.









to pH 7.0 and then titrated to pH 3.0, this was back titrated to pH 7.0

and returned to pH 3.0.

Total steam volatile fatty acid (TVFA) concentration of the

rumen fluid was determined on a sample. prepared by the following pro-

cedure. Rumen fluid was centrifuged for five minutes at approximately

250 times gravity, 25 milliliters of supernatant were transferred to

an Erlenmeyer flask, two milliliters of 25 per cent zinc sulfate and

three milliliters of 1.6 normal sodium hydroxide were added. The solu-

tion was mixed and centrifuged ten minutes at approximately 500 times

gravity. Five milliliters of the supernatant were transferred to a

25 milliliter volumetric flask and brought up to volume with triple

distilled water. Two milliliters of this solution plus one milliliter

of concentrated phosphoric acid were placed in a steam distillation

apparatus and 100 milliliters of distillate collected. The distillate

was titrated against 0.01 normal sodium hydroxide using Phenol Red

indicator.

Trichloroacetic acid insoluble nitrogen (TCA-N) was determined

by the method of Cline et.al. (1958). This method consisted of mixing

20 milliliters of rumen fluid and five milliliters of 50 per cent by

weight trichloroacetic acid in a 50 milliliter lusteroid tube and

allowing to stand 12 hours in a refrigerator. The precipitated protein

was sedimented by centrifugation for 15 minutes at approximately 1200

times gravity and the supernatant removed by suction. The precipitate

was washed by blowing 20 milliliters of 10 per cent trichloroacetic

acid from a pipette directly onto the precipitate. Centrifugation was

repeated and the supernatant removed by suction. The precipitate was






32

transferred to Kjeldahl flasks and nitrogen determined by the procedure

outlined by the Association of Official Agricultural Chemists (1955).














RESULTS AND DISCUSSION


In vivo Cellulose Digestion.

Values for per cent cellulose digestibility are recorded in

Table 6. The original data for grams of cellulose intake and grams

of fecal cellulose are given in Tables 17 and 18, appendix. F-ratios

obtained by analysis of variance and error mean squares are recorded

in Table 19, appendix.

The effect of treatment on cellulose digestibility showed a

high order of probability (P(.01). Most of the treatment difference

could be accounted for by the effect of sulfur (P<.001) and magnesium

(P <.05). The digestibilities of the cellulose in the magnesium de-

ficient, sulfur deficient and magnesium and sulfur deficient rations

were reduced 34.2, 69.5 and 71.9 per cent of that in the control ration,

respectively.

Other effects of the ration treatment were evident during the

experimental period. By the 14th day of each period the rumens of

the steers receiving the deficient rations were filled 'to near capacity

with ingesta. Removal of the cannula stopper resulted in spillage of

rumen ingesta from the fistula. The rumen of the steer fed the complete

ration was approximately half filled with ingesta as observed through

the fistula.

The consistency of the rumen ingesta was also affected by








treatment. Ingesta of the steers receiving the complete ration was

of a fluid consistency but it was quite difficult to separate the

liquid by straining through cheesecloth, indicating a high water bind-

ing capacity. The ingesta from the steers receiving the deficient

rations was "mealy" in texture and produced a large volume of easily

strainable rumen fluid. The greater volume of rumen contents and dif-

ferent consistency observed on the deficient rations was considered to

be due in part to a decrease in cellulose digestibility and not entirely

to a decreased rate of passage. Original data on total fecal excretion.

(Table 11, appendix) show: that total dry matter excretion was not af-

fected by treatment and suggests that rate of passage through the in-

testinal tract was not altered on an absolute basis.


TABLE 6

EFFECT OF MAGNESIUM AND SULFUR UPON IN VIVO
CELLULOSE DIGESTIBILITY IN STEERS


Treatment


Period Replications

1 1
2


1
2

1
2

1
2

Mean

1Values adjust


Complete -Mg -S
Per cent Cellulose Digested
71.3 53.01 40.5
71.5 53.01 40.3

69.2 51.5 11.9
76.3 50.2 12.5

74.6 49.0 22.8
75.3 49.0 22.8

55.5 25.6 6.8
54.6 25.1 8.5

68.5 44.5 20.8

ed for missing data.


-Mg-S

39.9
39.6

0.0
0.0

37.6
37.7

0.0
0.0

19.4









n vitro Cellulose Digestion.

The percentage cellulose digested in a 24 hour in vitro fermen-

:ation period is given in Table 7. Calculated F-ratios of these data

are recorded in Table 20, appendix.

The effect of treatment on in vitro cellulose digestion was

more pronounced than was observed in the in vivo studies. Inoculae

from steers fed the magnesium deficient, sulfur deficient and magnesium

and sulfur deficient rations digested respectively, 88.3, 95.2 and

82.9.per cent less cellulose in vitro then did inoculae from steers fed

the control ration.

The effect of treatment on cellulose digestion, when a nutrient

media corresponding to the ration treatment was used, showed a high order

of probability (P ~.01). An interaction of magnesium on sulfur and

sulfur on magnesium manifested itself with a high order of probability

(P <.01). The interaction indicates that the addition of magnesium to

a sulfur deficient ration and in vitro nutrient media or the addition

of sulfur to a magnesium deficient ration and in vitro nutrient media

has a deleterious effect on cellulose digestibility in vitro. However,

a comparison of the in vivo and in vitro cellulose digestion data indi-

cates that this interaction may have resulted from the variability within

treatments of the in vitro cellulose digestion trial.










TABLE 7

CELLULOSE DIGESTION IN VITRO DURING TWENTY-FOUR HOUR
FERMENTATION PERIOD BY RUMEN INOCULAE FROM STEERS



Complete -Mg -S -Mg-S
Period Replications Per cent Cellulose Digested
1 41.98 8.88 8.07 13.03
2 18.22 3.00 2.37 3.00

1 52.87 7.41 0.73 5.62
2 52.38 10.04 0.60 0.59

1 25.53 0.68 0.00 0.22
2 25.07 0.66 0.00 0.23

S1 48.36 .0.00 0.59 19.12
2 3.60 0.75 0.00 4.05

Mean 33.50 3.93 1.55 5.73



Total Steam Volatile Fatty Acid Concentration.

The importance of TVFA as an important intermediate end-product

of digestion was suggested by Hale et al. (1947).. McAnally and Phillip-

son (1942) and Phillipson and McAnally (1942) demonstrated that volatile

fatty acids are produced in and absorbed from the rumen. It has been

estimated that TVFA supply from 63 to 71 per cent of the energy require-

ment for maintenance (Stewart et al. 1958, and Carroll and Hungate,

1954). In the present study, TVFA concentrations were made as one of

the measures of rumen function.

Values for the millequivalent per cent of TVFA in the rumen

fluid from animals on the four ration treatments are recorded in Table

8. Calculated F-ratios of these data are recorded in Table 21, appen-

dix.








The concentrations of TVFA in the rumen fluid were affected by

the magnesium or sulfur. The differences were in a low order of prob-

ability (P (.25) and most of this difference could be accounted for by

the effect of sulfur (P .10). The concentration of TVFA in the rumen

fluid of the steers fed the magnesium deficient, sulfur deficient and

magnesium and sulfur deficient rations were respectively 3.69, 4.34

and 4.91 milliequivalents per cent lower than that in the rumen fluid

of steers fed the complete ration.

The experimental ration was composed of approximately 57 per

cent of a soluble carbohydrate which could be readily fermented to

lactic acid. The sampling of the rumen ingesta two hours following

the morning feeding would be expected to provide a sample rich in lac-

tic acid. The presence of lactic acid could account for the low pH

values even though TVFA levels were low in the rumen fluid of the

steers fed the deficient rations.


TABLE 8

TOTAL STEAM VOLATILE FATTY ACID CONCENTRATION OF
RUMEN FLUID FROM STEERS FED PURIFIED RATIONS
WITH AND WITHOUT MAGNESIUM AND SULFUR


Treatment
Complete -Mg -S -Mg-S
Period Replications Millequivalent per cent TVFA
1 1 9.21 5.67 5.64 7.20
2 9.60 5.67 5.61 7.05

2 1 9.96 7.20 5.94 2.04
2 10.05 7.29 6.15 .1.98

1 12.18 13.20 6.27 11.79
3 2 12.06 13.47 6.69 11.31

1 15.09 5.67 9.21 8.76
2 14.45 5.91 8.85 8.79

Mean 11.70 8.01 6.79 7.36









Trichloroacetic Acid Insoluble Nitrogen.

Trichloroacetic acid insoluble nitrogen (TCA-N) has been used

as an indication of microbial protein concentration. In this role it

has been a useful tool in in vitro fermentation studies (Cline et al.

1958 and Bentley et al. 1955). In the present study it was used as an

additional measure of the effect of magnesium and sulfur on rumen func-

tion. This technique was valid since the ration contained no protein

and any TCA-N would arise from bacterial protein synthesis.

The per cent TCA-N in 100 milliliters of rumen fluid from

animals on the different rations is given in Table 9. Calculated F-

ratios of these data are recorded in Table 22, appendix.

The effect of magnesium or sulfur on TCA-N concentration showed

a high order of probability (P (.05). The concentrations of TCA-N in

the rumen fluid of steers fed the magnesium deficient, sulfur deficient

and magnesium and sulfur deficient rations were respectively 8.7, 11.1

and 0.9 milligram per cent lower than in the rumen fluid of steers fed

the complete ration. The reduction in TCA-N when magnesium or sulfur

were deficient might be expected from the data obtained on TVFA deter-

minations. A comparison of the data indicates a direct relationship

between the amount of bacterial protein present, as estimated by TCA-N,

and the amount of cellulose digested by in vitro techniques except for

the magnesium and sulfur deficient ration.










TABLE 9

TRICHLOROACETIC ACID INSOLUBLE NITROGEN CONCENTRATION
OF RUMEN FLUID FROM STEERS FED PURIFIED RATIONS
WITH AND WITHOUT MAGNESIUM AND SULFUR


Treatment
Complete -Mg -S -Mg-S
Period Replications Milligram per cent
1 1 30.1 9.0 5.1 25.2
2 30.0 9.2 5.1 25.4

S1 18.6 9.2 10.9 19.7
2 18.8 9.0 11.1 19.5

3 1 7.9 11.4 4.7 7.9
2 7.9 11.0 5.1 9.0

1 16.1 9.0 7.7 16.5
2 16.6 9.2 7.7 16.7

Mean 18.2 9.6 7.1 17.3



Buffering Capacity of Rumen Liquor.

Total milliliters of 0.05 normal hydrochloric acid required to

lower 75 milliliters of rumen fluid from pH 7.0 to pH 3.0 are given in

Table 10. Calculated F-ratios are recorded in Table 23, appendix.

The effect of treatment on buffering capacity of rumen fluid

showed a high order of probability (P <.05). Most of the treatment

difference could be accounted for by the effect of sulfur (P<.01).

The buffering capacities of rumen fluid from steers fed the sulfur de-

ficient and magnesium and sulfur deficient rations were respectively

13.4 and 9.4 per cent lower than that from steers fed the complete ra-

tion, while the magnesium deficient ration did not affect buffering

capacity.

The principal buffer systems present in the rumen fluid are









the bicarbonate, phosphate and volatile fatty acid systems (Turner and

Hodgetts, 1955). In the present study, the bicarbonate system was appar-

ently eliminated during storage of the rumen fluid samples in the pres-

ence of air and by aeration from mechanical stirring of the sample prior

to and during titration. The absence of a bicarbonate system in these

samples was observed in a comparison of the duplicate titration curves

for each sample. The effectiveness of the phosphate buffer system is

regulated by the pK1, pK2 and pK3 values of phosphoric acid which are

2.1, 7.2 and 12.0,respectively (Pauling 1947). Within the pH range of

this study phosphate is most effective as a buffer near the pK2 value.

It ceases to be effective at pH values of less than 5.5, at which it is

almost totally ionized (Turner and Hodgetts, 1955). The buffering capac-

ity of rumen fluid between the pK1 and pK2 values of the phosphate sys-

tem is mainly attributed to the volatile fatty acid system. It was

observed from the titration curves obtained that the effect of treat-

ment on buffering capacity was most evident between pH 5.5 and pH 4.0.

At this pH range the buffering capacity of the rumen fluid would be

attributed to the volatile fatty acids.

The highly significant effect of sulfur on buffering capacity

could be expected since the effect of sulfur on cellulose digestion

both in vivo and in vitro was highly significant. With a decreased

rate of fermentation, in the absence of sulfur, the production of vola-

tile fatty acids could be reduced resulting in a decrease in buffering

capacity of the rumen fluid. The alternative production of lactic acid

would not contribute to buffering capacity in the pH range of this study.










TABLE 10

BUFFERING CAPACITY OF RUMEN FLUID FROM STEERS FED PURIFIED
RATIONS WITH AND WITHOUT MAGNESIUM AND SULFUR

Treatment

Period Replications Complete -Mg -S -Mg-S

1 1 22.051 26.40 19.40 20.60
S2 23.40 26.70 21.80 20.60

2 1 25.73 21.80 20.40 21.30
2 25.30 21.50 19.20 20.70

3 1 23.16 23.02 20.47 19.09
2 23.15 23.54 21.20 18.83

4 1 26.20 22.40 22.60 26.30
2 23.75 21.70 21.80 27.10

Mean 24.09 23.38 20.86 21.82

iValues shown in milliliters of 0.05N HC1 required to reduce
75 milliliters of rumen fluid from pH 7.0 to pH 3.0.


Bacteriological Observations.

Microscopic examination of the Gram-stained slides prepared from

the fresh rumen fluid revealed alterations in the microflora population

with the different mineral intakes. The complete ration supported a pop-

ulation of both Gram-positive and Gram-negative organisms. The Gram-

positive organisms were streptococci, slender rods and flat rods. The

Gram-negative organisms were predominantly cocci. The magnesium defi-

cient ration supported a predominant population of various Gram-negative

organisms. These Gram-negative organisms were mostly divided slender

rods. The organisms observed in the slides prepared from the sulfur de-

ficient ration were Gram-negative cocci and divided rods. The rumen

fluid from the steers fed the magnesium and sulfur deficient ration






42


contained predominantly small Gram-negative slender rods and small

cocci. The slides prepared from this fluid during the fourth period

contained both Gram-positive slender curved rods and Gram-negative

cocci. During the preceding three periods the organisms present were

predominantly Gram-negative and small, as compared with the organisms

obtained from the rumen fluid from steers fed the complete ration.

The appearance of the Gram-positive organisms in this period cannot

be explained from data obtained.
















EXPERIMENT III


Effect of Magnesium on Appetite in Sheep and in Vitro
Cellulose Digestion

The results of Experiment II indicated that magnesium defi-

ciency reduced appetite and the effect of magnesium may be specific

upon cellulose digesting microorganisms. Experiments were conducted

to further study the influence of magnesium upon appetite and micro-

bial cellulose digestion, and to determine if cellulolytic microorgan-

isms exhibit a specific requirement for magnesium.


Magnesium Effect on Appetite.

Appetite, as measured by voluntary feed intake in the previous

experiment, appeared to be related to cellulose digestibility. The

decrease in feed consumption was accompanied by poorer cellulose di-

gestibility and greater volume of rumen contents. It was not deter-

mined whether this relationship may be physical and related to rate of

disappearance of ingesta from the rumen or to some other effect of mag-

nesium.

Eight yearling wether lambs ranging in weight from 57 to 65

pounds with a mean.weight of 61 pounds were used as experimental ani-

mals. During an eight day preliminary period they were shorn, housed

in individual box stalls and fed the complete purified ration described

in Experiment II. The daily ration of 818 grams was fed in two equal

portions and tap water was supplied ad libitum. Following the eight








day adjustment period, four lambs were selected at random to receive

the magnesium deficient ration and the remaining four continued to

receive the complete ration.

On the third day the four lambs on the magnesium deficient

ration exhibited partial loss of appetite. Starting on the morning

of the fourth day two of the lambs on the magnesium deficient ration

were given 1.5 grams of magnesium oxide per head daily in one-fourth

ounce gelatin capsules with a balling gun. The effect of magnesium on

appetite was measured by the rate at which the lambs returned to normal

dietary intake.

Following completion of this study, six of the lambs were fed

the complete ration for 60 days recovery period and used in the study

of the effect of intravenous magnesium upon feed intake. Two lambs

were continued on the complete ration and four were fed the magnesium

deficient ration. Intravenous injections of 2.5 milliliters of 25 per

cent magnesium sulfate in physiological saline were given to two lambs

on the magnesium deficient ration. One lamb receiving the complete ra-

tion was given an intravenous injection of 2.5 milliliters of physio-

logical saline. Injections were made daily starting with the first day

on the experiment ration and continued for a six day period.

The magnesium sulfate solution was prepared by dissolving 25

grams of magnesium sulfate in 100 milliliters of physiological saline.

Daily intravenous injections of 2.5 milliliters of the prepared solution

were calculated to supply 200 milligrams of magnesium or approximately

two times the daily magnesium intake of the lambs receiving the complete

ration.








Values representing the amount of feed consumed by lambs with

and without supplemental magnesium and after oral administration of

magnesium are presented in Table 11. On the third day, lambs without

dietary magnesium consumed only 63.5 per cent of the amount of ration

consumed on the previous day. The consumption of the ration by the

lambs which did not receive supplemental magnesium continued at approxi-

mately this level until returned to a ration containing magnesium.

The oral administration ofmagnesium oxide starting on the

fourth day resulted in a marked improvement in feed intake on the

seventh day. By the eighth day, essentially normal appetite was re-

stored (Table 11). The recovery of appetite with magnesium supple-

mentation is illustrated in Figure 3.

Daily feed intakes of lambs on the second appetite study are

shown in Table 12. The feed intakes are graphically illustrated in

Figure 4. The significant decrease in feed consumption on the third

day by lambs receiving the ration without magnesium was again demon-

strated. The daily intravenous injection of magnesium sulfate did not

lessen the severity of this loss of appetite which consistently appeared

on the third day in previous trials. However, a slight improvement in

appetite of the two lambs receiving the magnesium sulfate injections

was noted on the fourth day.

The results obtained from the two trials on the effect of mag-

nesium on appetite indicate that the primary effect is one related to

microbial digestion and not magnesium per se. The oral administration

of magnesium oxide did not give an immediate response as might be ex-

pected if magnesium alone affected appetite. Appetite recovery following








TABLE 11


AVERAGE DAILY FEED CONSUMPTION OF LAMBS ON APPETITE RECOVERY TRIAL WITH MAGNESIUM OXIDE CAPSULES

Feed Consumed per Day, Grams
Ration Number 1 2 3 4 5 6 7 8 Total Per Cent
Treatment Lambs Consumed
-Mg 2 818 818 678 352 323 289 350 441 4069 61.2

-Mg 21 818 818 362 309 357 416 647 754 4481 68.5

+Mg 42 818 818 818 818 818 818 818 818 6544 100.0

2Lambs received magnesium oxide capsules from third day to termination.
Two of the four lambs received empty gelatin capsules from third day to termination.


TABLE 12

AVERAGE DAILY FEED CONSUMPTION OF LAMBS ON APPETITE MAINTENANCE
TRIAL WITH MAGNESIUM SULFATE INJECTIONS

Feed Consumed per Day, Grams
Ration Number 2 3 5 Per Cent
1 2 3 4 5 6 Total
Treatment Lambs Consumed
-Mg 2 818 818 382 353 300 351 3022 61.6
-Mg 2 818 818 403 510 540 558 3647 74.3
+Mg 2 818 818 818 818 818 818 4908 100.0

Lambs received intravenous injections of magnesium sulfate from start of experiment.
20ne of the two lambs received injections of physiological saline from start of experiment.













-- -- Complete ration

Mg deficient ration
+ MgO capsules
------ Mg deficient ration


<.0e
I--,


450


350


1 2 3 4 Days 5 6
Figure 3: Daily feed consumption of lambs on appetite recovery trial.









850 -



750 -



650 -



550-



450



350-



250

)1


- Complete ration

Mg deficient ration + MgSO4 injections

Mg deficient ration


Days 6
Figure 4: Daily feed consumption of lambs on appetite maintenance trial.


----, ,,









oral magnesium administration required a period of four days before

an increase in voluntary feed intake was recorded.

The loss of appetite which appears by the third day in rumi-

nants fed a magnesium deficient ration appears to be the first symptom

associated with a magnesium deficiency in these species when a puri-

fied or semi-purified ration is fed (McAleese et al. 1961, Evered,

1961). The intravenous injections of magnesium sulfate failed to pre-

vent the loss of appetite which consistently appeared on the third day

in the previous trials, nor did it lessen the severity of this loss of

appetite. The partial recovery in appetite observed on the fourth day

might be explained by the excretion of magnesium into the rumen through

the saliva. The daily injections of magnesium supplied approximately

two times the amount normally consumed in the complete ration and

probably would be several times greater than the amount absorbed from

the ration. Therefore, the saliva produced by the lambs given the

magnesium sulfate injections might'have been-especially rich in mag-

nesium.


Cellulose Digestion.

On the first, second and third days of both appetite recovery

and maintenance trials, using oral and intravenous magnesium, samples

of rumen ingesta were collected by stomach tube from the lambs in each

treatment for in vitro cellulose digestion studies. The ingesta was

collected two hours following the morning feeding, immediately trans-

ferred to the laboratory and strained through four layers of cheese-

cloth. Prior to inoculation, 250 milliliter fermentation bottles cali-

brated at 80 and 100 milliliter levels, had been prepared containing a








known amount of BW-40 grade Solka-Floc (approximately two grams) and

55 milliliters of the nutrient media corresponding to the ration treat-

ment as described in Experiment II. The fermentation bottles were

immersed in a water bath controlled at 39 degrees Centigrade two hours

prior to inoculation and anaerobic conditions were established and

maintained by bubbling carbon dioxide through the media. The fermen-

tation bottles were inoculated with 25 milliliters of strained rumen

fluid. The medium was adjusted to pH 6.9 at intervals during the fer-

mentation period. Following a 24 hour fermentation the bottles were

brought up to the 100 milliliter level, thoroughly mixed, and 25

'milliliter aliquots removed for the determination of cellulose con-

tent by the method used in Experiment II.

Data representing the effect of dietary magnesium intake upon

cellulose digestion are presented in Table 13 as grams and as per cent

of cellulose digested in vitro. The original data for grams of cellu-

lose digested by periods for each treatment are presented in Table 24,

appendix.

When the magnesium deficient ration was fed, there was a marked

reduction in cellulose digestion by the rumen inoculae on the third day.

This would indicate a carry-over of magnesium in the rumen which is de-

'pleted by the third day. An analysis of variance of the grams of cellu-

lose digested gave a significant treatment effect (P <.05).

The results indicate that magnesium is an essential dietary con-

stituent for the maintenance of cellulose digestion. The effect of a

magnesium deficiency on cellulose digestion may not manifest itself

until the residual carry-over in the rumen is depleted. When cellulose











TABLE 13


CELLULOSE


DIGESTION IN VITRO BY RUMEN INOCULUM FROM LAMBS FED
PURIFIED RATIONS WITH AND WITHOUT MAGNESIUM


Cellulose Digested per Day

1 2 3
Ration
.Treatment Replications % grams % grams % grams

1 82.01 .3712 78.0 .354 83.0 .378

Complete 2 97.5 .450 61.0 .280 44.0 .201

Mean 89.8 .411 69.5 .317 63.5 .290



1 82.0 .371 76.0 .341 31.0 .135

Magnesium 2 58.4 .268 56.6 .256 0.0 .000
Deficient
Mean 70.2 .320 66.3 .299 15.5 .068


1Values are the
2Mean values of


means of three fermentation vessels.
original substrate on dry weight basis was


.452 grams.









digestion by the rumen inoculae decreased there was a concurrent de-

crease in voluntary feed intake of the animals.


Magnesium Requirement of Cellulolytic Microorganisms.

The results of the previous trials in this experiment indi-

cated that the effect of magnesium on appetite is manifested through

ruminal fermentation. However, Chamberlain and Burroughs (1962) re-

ported that the addition of magnesium did not significantly increase

cellulose digestion in vitro with a washed cell suspension. Therefore,

a series of in vitro cellulose digestion studies were conducted to de-

termine if a specific requirement for magnesium could be demonstrated

in vitro.

The basic technique, as previously described, using 250 milli-

liter fermentation bottles was used in two trials with two treatments

each. In trial one, the substrate was-one gram of Solka-Floc with the

complete nutrient media and with a magnesium deficient media. In trial

two, the substrate was two grams of the basal test ration and complete

nutrient media, and two grams of the magnesium deficient nutrient media.

Duplicate fermentation bottles were used for each treatment and each

trial was replicated.

The fermentation bottles were inoculated with 25 milliliters of

whole rumen fluid obtained by stomach tube from a lamb receiving the

complete ration. The inoculum was prepared by straining the ingesta

through four layers of cheesecloth. The pH of the fermentation was

adjusted to 6.9 at intervals during the fermentation period.

Following a 24 hour fermentation period one-fourth of the fermen-

tation media were transferred to fresh fermentation media of similar

treatment. This served to inoculate the fresh media. The remainder of






53
the fermentation media was used for cellulose determination. Transferal

inoculations were made in this manner daily over a five day period so

that residual magnesium from the original rumen inoculum was progres-

sively diluted. The evaluation of a requirement for magnesium by the

cellulolytic microorganisms was based upon the cellulose digestion

under these conditions.

The average grams of cellulose digested in vitro, in trial one

and two, on the first day following inoculation with whole rumen inoc-

ulae and after four consecutive transferals are recorded in Table 14.

The original data for grams of cellulose digested by day for each

period are recorded in Tables 25 and 26, appendix.


TABLE 14

GRAMS CELLULOSE DIGESTED PER DAY IN VITRO
USING TRANSFER TECHNIQUE


Days
Trial Treatment Substrate 1 2 3 4 5

-Mg Solka-Floc .4101 .289 .284 .174 .097

+Mg Solka-Floc .422 .371 .324 .302 .329


-Mg Ration ..172 .039 .010 .000

+Mg Ration .306 .268 .243 .207


Average grams of cellulose digested in duplicate bottles in
duplicate periods.


In trial one, on the first day following inoculation, no dif-

ferences in cellulose digestion were noted. Since strained rumen fluid

was used as inoculum, a sufficient amount of magnesium was probably

added through this source to meet the requirement for cellulose digestion








during the 24 hour fermentation. With each successive fermentation

this source of magnesium was diluted by a factor of four. On the fourth

day following successive transfers a significant reduction in cellulose

digestion was noted. Since the nutrient media substrate and other con-

ditions were equal except for the decrease in magnesium due to dilution,

the reduction in cellulose digested was attributed to the decrease in

magnesium. Cellulose digestion was further decreased on the fifth day,

but was maintained near the original level by the complete nutrient

media which contained magnesium. The effect of magnesium on in vitro

cellulose digestion is graphically illustrated in Figure 5.

The digestion of the cellulose in the ration substrate without

magnesium was reduced on the first day following inoculations. On suc-

cessive days, complete inhibition of cellulose digestion was observed

in the magnesium deficient media. The presence of magnesium in the

ration substrate and nutrient media supported cellulose digestion, how-

ever, throughout the experimental period.

The results from trial one indicate that magnesium is essential

for cellulose digestion in vitro. It was also apparent that the contam-

ination of magnesium in the rumen fluid was sufficient to maintain cellu-

lose digestion over a three day period with serial dilutions (Table 14).

However, the addition of the ration'substrate, which contained 57 per

cent soluble carbohydrate, greatly decreased cellulose digestion in the

absence of magnesium in trial two. This may have resulted from compe-

tition between cellulolytic and amylolytic microorganisms for the mag-

nesium ion. Since the ration substrate contained nearly twice as much

soluble carbohydrate as cellulosethe amylolytic organisms would be in

a greater concentration than the cellulolytic organisms. Also, the













.-.---- Solka-Floc substrate + Mg

Solka-Floc substrate Mg

----Test ration substrate + Mg


S-**- *.- Test ration substrate Mg


Days
Figure 5: In vitro cellulose digestion using transferal technique.






56

rapidity in fermentation of soluble carbohydrates would tend to inhibit

cellulolytic organisms (Van der Wath, 1948, Swift et al. 1947, Burroughs

et al. 1949, and Kane et al. 1959). These conditions might help explain

the decreased in vivo cellulose digestion and the rapid loss of appetite

in the animals receiving the magnesium deficient ration.














GENERAL DISCUSSION


The data obtained with lambs in Experiment I indicated that

feeding the magnesium deficient ration resulted in increased absorption

and retention of phosphorus as compared to the complete rations. When

the sulfur deficient ration was fed an elevated fecal phosphorus excre-

tion but greatly reduced urinary phosphorus excretion was observed as

compared to the complete ration. The decrease in volume of ingesta

present in the digestive systems of the lambs receiving the deficient

rations, resulting from a loss of appetite, would be expected to favor

conditions for maximum absorption of dietary nutrients. This might ex-

plain the increased absorption of radioactive phosphorus observed with

the magnesium deficient ration. However, the reverse seems to be true

in the absence of sulfur. If the effect of sulfur on phosphorus ab-

sorption is manifested through protein synthesis, a complex interrela-

tionship between magnesium, protein and phosphorus exists. Sjollema

(1932) and Allcroft (1956) have suggested that high levels of dietary

protein reduce magnesium absorption. The results of the present study,

on the other hand, indicate that a high magnesium intake in the presence

of a low bacterial protein level reduced phosphorus absorption.

The experimental design of trial one did not include a recovery

period between treatments, and the lambs became progressively more emaci-

ated with each successive period. One lamb died in the latter part of

the fourth period at which time the experiment was terminated. The

severe emaciation resulted from a loss of appetite due to the ration








treatment and not from the type of ration fed since this ration has been

shown to give satisfactory gains in body weight in sheep (Meacham, 1962)

and the lamb receiving the complete ration during each experimental period

gained weight.

During trial two with steers, the variation due to a possible

carry-over of the treatment effect was minimized by using fistulated

steers which could be fed through the ruminal fistula. The data ob-

tained under these conditions indicated that the sulfur deficient ra-

tion reduced urinary phosphorus excretion and slightly increased fecal

phosphorus excretion as compared to the complete ration. The increased

fecal phosphorus excretion observed on the magnesium deficient ration

indicates that phosphorus absorption under these conditions is a func-

tion of the volume of ingesta present in the digestive system. These

results are in complete disagreement with work reported with rats

(Roche, 1932, Villus, 1932, and Buckner, 1932).

Although the magnesium deficient ration appeared to increase

absorption of radioactive phosphorus in sheep, differences among animals

and within treatment were variable. Data obtained with the steers in-

dicated an opposite effect, and the apparent improvement in phosphorus

utilization in the presence of magnesium appears more consistent with

the expected effect of magnesium. 'Magnesium is required with calcium

and phosphorus for optimum bone formation and the ion is also required

in the glycolytic cycle as an enzyme activator for the formation of high

energy phosphate bonds (Sallach and McGilvery, 1960). Although the effects

of magnesium and sulfur on phosphorus utilization cannot be clearly de-

fined from results of these experiments, the data suggests the need for

further study of the interrelationships which have been indicated.









The low urinary phosphorus excretions obtained from the lambs

and steers consuming the sulfur deficient rations were accompanied by

a decreased volume of urine excreted during the five day collection

periods. In addition, the fecal ash from the animals receiving the

sulfur deficient ration appeared to contain large amounts of carbonate.

The type of carbonate present was not determined. The changes in urinary

and fecal phosphorus excretion related to sulfur intake indicate that

sulfur may influence retention and excretion but not absorption of

certain elements. This function should be studied further with regard

to sulfur and to preformed protein on thirst motivation and renal and

fecal excretions of various ions.

In the in vivo and in vitro cellulose digestion studies of

Experiment II, the magnesium and/or sulfur deficient rations and nutrient

media significantly reduced cellulose digestion as compared to the com-

plete ration and media. This reduction in cellulose digestion apparently

resulted from competition between the amylolytic and cellulolytic micro-

organisms for nutrients such as the magnesium ion for enzyme activation,

or sulfate for the formation of bacterial protein.

The sulfur deficient ration significantly decreased TCA-N and

TVFA concentration and buffering capacity of the rumen fluid as compared

to the complete ration. These three measurements of rumen function are

closely related. TCA-N concentrations of the rumen fluid indicates the

amount of bacterial protein present when the.ration is made up of non-

protein nitrogen. A large concentration of rumen organisms, as measured

by TCA-N, could produce greater concentrations of TVFA, thereby contrib-

uting to the buffering capacity of the rumen fluid. The relationship









of sulfur to bacterial synthesis of protein from the ration urea has

been previously demonstrated (Loosli et al. 1949), Thomas et al. 1951,

Starks et al. 1954, and Gall et al. 1951).

The magnesium deficient ration reduced TVFA and TCA-N concen-

trations numerically. The ration, which was composed of approximately

57 per cent soluble carbohydrates, would be expected to produce high

levels of lactic acid under conditions favoring rapid fermentation.

An increase in lactic acid concentration with a corresponding decrease

in TVFA concentration would be expected. The low pH of the rumen fluid

from the magnesium deficient rations may have resulted from an increased

lactic acid concentration. Future studies on the effect of magnesium

on rumen function should consider lactic acid concentration as one of

the criteria.

In Experiment III, the effect of magnesium on appetite was

studied by oral doses of magnesium oxide and intravenous injections of

magnesium sulfate given to lambs fed the magnesium deficient ration.

The oral administration of magnesium oxide did not improve appetite

until after four days of treatment, after which time the recovery was

quite rapid. The daily intravenous injections of magnesium sulfate

failed to prevent or lessen the severity of the loss of appetite that

occurred on the third day. However, a slight recovery in appetite was

observed on the fourth day in the lambs receiving the magnesium sulfate

treatments. The control lambs receiving the magnesium deficient ration

without supplemental magnesium exhibited an improvement in appetite on

the sixth and seventh days of the trial period. This improvement in

appetite probably resulted from the cycling of magnesium into the rumen

from the body stores and helps verify that a poor mechanism for the








rapid mobilization of magnesium from the bone exists (Blaxter, 1956).

These results suggest that the effect of magnesium on appetite is one

related to rumen function, such as cellulose digestion or lactic acid

concentration, and not to magnesium metabolism in the body per se.

In order to study the relation of appetite loss to a decrease

in cellulose digestion, rumen fluid obtained on the first, second and

third days after the lambs were placed on the magnesium deficient ra-

tion was used as inoculum for in vitro cellulose digestion studies and

compared to inoculum from lambs receiving the complete ration. Cellu-

lose digestion was maintained at a high level by the inoculum prepared

on the first and second days of the feeding period. However, the in-

oculum prepared on the third day from the lambs receiving the magnesium

deficient ration supported very little cellulose digestion as compared

to the inoculum from the lambs receiving the complete.ration. This

loss in cellulose digestion on the third day corresponded with a loss

of appetite observed in the same lambs and suggests that appetite may

be related to the digestibility of the ration cellulose. Further

studies on appetite as affected by'the rate of cellulose digestion or

an alteration in TVFA and lactic acid concentrations in the rumen or

other end products of bacterial fermentation would be beneficial for

our understanding of hunger motivation in the ruminant animal.

In order to demonstrate a specific requirement for magnesium

by cellulolytic microorganisms, in vitro cellulose digestion was studied

with complete ration rumen fluid as inoculae for complete and magnesium

deficient media. The initial inoculation was made using rumen fluid

from a lamb receiving the complete ration; successive daily inoculations

were made from the previous day's fermentation. In this trial, with








Solka-Floc as the substrate, cellulose digestion was maintained at a

relatively constant level for a five day period with the complete media.

The magnesium deficient media supported cellulose digestion at a con-

stant level for three days, after which cellulose digestion decreased

rapidly. With the complete ration as the substrate, the presence of

magnesium maintained cellulose digestion throughout the experimental

period. However, when the magnesium deficient ration was used as sub-

strate in magnesium deficient nutrient media, cellulose digestion was

maintained only for the first day, after which a rapid loss in cellu-

lose digestion was observed.

These trials provided evidence that cellulolytic microorganisms

have a specific requirement for magnesium and that the addition of a

soluble carbohydrate will inhibit cellulose digestion in vitro. This

inhibition can apparently be reduced by the addition of magnesium to

the fermentation media. The initial carry-over of magnesium with the

complete rumen inoculum was sufficient, even with serial dilutions,

to maintain cellulolytic organisms for a three day period in the absence

of soluble carbohydrates. Magnesium present in the rumen inoculum was

not sufficient, in the presence of soluble carbohydrates, to maintain

cellulose digestion. The requirement of a mixed culture of cellulo-

lytic and amylolytic microorganisms' for magnesium in the presence of

soluble carbohydrates should be extensively studied for its practical

application.
















SUMMARY


Three experiments have been carried out to determine the effect

of magnesium and sulfur on phosphorus utilization and rumen function in

lambs and steers fed purified rations.

In the first experiment, the effect of magnesium and sulfur on

phosphorus utilization was studied using a radioactive tracer technique

in a balance study with lambs and using stable phosphorus in a conven-

tional balance technique with fistulated steers. The percentage of the

oral dose of radioactive phosphorus excreted by the lambs or the phor-

phorus status of the steers, as determined by total phosphorus excretion,

were used as criteria for determining the effect of magnesium and sulfur

on phosphorus utilization.

The magnesium deficient lamb ration increased apparent absorption

and retention of phosphorus while the sulfur deficient ration decreased

absorption but greatly reduced urinary excretion of phosphorus. When the

dietary intake of the ration was kept constant by forced feeding through

the rumen fistula in the steer study, the magnesium deficient ration had

an opposite effect and appeared to reduce phosphorus retention while the

sulfur deficient ration was again observed to reduce urinary phosphorus

excretion.

In the second experiment, yearling steers were fistulated and

used in a study to determine the effect of magnesium and sulfur on

cellulose digestion, rumen fluid buffering capacity, total steam volatile









fatty acid (TVFA) concentration, trichloroacetic acid insoluble nitro-

gen (TCA-N) concentration and the morphological changes in the rumen

microbial populations.

In vivo cellulose digestion was determined by total collection

technique and in vitro cellulose digestion by the in vitro rumen fermen-

tation technique. The magnesium and/or sulfur deficient rations signif-

icantly reduced in vivo cellulose digestion. This significant loss in

cellulose digestion was also observed in the in vitro studies.

Feeding the sulfur deficient ration resulted in a rumen fluid

of significantly lower buffering capacity and TVFA concentration. TCA-N

was significantly lowered when the magnesium or sulfur deficient rations

were fed.

Microscopic examination of the microbial population revealed an

alteration of the types of bacteria present. The complete ration sup-

ported a mixed population of Gram-positive and Gram-negative organisms

while the magnesium and sulfur deficient rations supported a population

composed primarily of Gram-negative organisms.

In the third experiment, wether lambs were used to determine

the influence of magnesium upon appetite. Also, concurrent in vitro

cellulose digestion studies were conducted to determine if cellulolytic

microorganisms exhibit a specific requirement for magnesium.

The administration of oral doses of magnesium to lambs suffering

from a loss of appetite due to a magnesium deficiency did not produce

an immediate increase in appetite but after a period of several days

appetite increased to near normal. Intravenous injections of magnesium

did not prevent nor lessen the severity of the characteristic loss of








appetite observed on the third day after the animals were placed on

the deficient ration, but did not result in an improvement in voluntary

feed intake in successive days. In vitro cellulose digestion studies

revealed a sharp drop in cellulose digestion by the rumen inoculum on

the third day after the magnesium deficient ration was fed to the

lambs. This loss in cellulose digestion corresponded with the loss

of appetite observed in the same lambs on the third day. These results

indicate that the effect of magnesium is one related to rumen function

and not to magnesium per se.

Using transferal inoculations of in vitro fermentation media

in order to serially dilute the carry-over'of magnesium, a specific

requirement for magnesium by cellulolytic bacteria was demonstrated.

The addition of a soluble carbohydrate to the fermentation substrate

decreased the rate of cellulose digestion which could be overcome by

added magnesium.


































APPENDIX











TABLE 1-A

PER CENT OF THE DOSE OF P-32 RECOVERED
IN THE URINE OF LAMBS
EXPERIMENT I, TRIAL 1


Treatment

Period Complete -Mg -S -Mg-S

1 10.87 6.65 0.33 14.19
2 1.10 4.96 0.18 3.50
3 7.84 22.02 1.67 25.51
41
Mean 6.60 11.21 \ 0.73 14.40

Incomplete period.





TABLE 2-A

PER CENT OF THE DOSE OF P-32 RECOVERED
IN THE FECES OF LAMBS
.EXPERIMENT I, TRIAL 1

Treatment

Period Complete -Mg -S -Mg-S

1 41.07 33.99 50.00 41.85
2 57.98 2 9.00 66.27 18.22
3 36.32 7.32 53.93 22.71
41
Mean 45.12 18.77 56.73 27.59

Incomplete period.












TABLE 3-A

TOTAL URINE EXCRETED IN FIVE DAYS BY
LAMBS ON P-32 STUDY, MILLILITERS
EXPERIMENT I, TRIAL 1


Treatment
Period Complete -Mg -S -Mg-S

1 6805 5655 580 3860
2 8840 1935 980 2045
3 4395 2780 1745 5735
41
Mean 6680 3457 \ 1102 3880

Incomplete period.





TABLE 4-A

AVERAGE DAILY FEED CONSUMPTION FOR LAMBS ON
P-32 ABSORPTION TRIAL, GRAMS
EXPERIMENT I, TRIAL 1

Treatment

Period Complete -Mg -S -Mg-S

1 4001 258 364 270
2 400 230 314 233
3 361 274 254 227
42 325 295 270 167
Mean 371 264 301 222

1400 grams of ration offered per head per day.
2Average daily feed consumption for the first fifteen days
of the fourth period.










TABLE 5-A

TOTAL PHOSPHORUS EXCRETED IN URINE BY STEERS
IN FIVE DAY BALANCE PERIOD, GRAMS
EXPERIMENT I, TRIAL 2


Treatment
Period Complete -Mg -S -Mg-S

1 7.36 16.271 3.96 17.89
2 29.18 18.63 25.55 22.21
3 17.72 13.24 3.43 30.90
4 18.19 19.96 2.86 16.64
Mean 18.11 17.02 8.95 21.91

Value adjusted for missing data.




TABLE 6-A

TOTAL PHOSPHORUS EXCRETED IN FECES BY STEERS
IN FIVE DAY BALANCE PERIOD, GRAMS
EXPERIMENT I, TRIAL 2


Treatment

Period Complete -Mg -S -Mg-S

1 31.45 54.691 35.68 12.29
2 17.74 40.00 21.15 55.92
3 53.92 28.86 42.50 12.84
4 32.50 179.89 43.12 73.20
Mean 33.90 75.86 35.61 38.56

1Value adjusted for missing data.












TABLE 7-A

TOTAL PHOSPHORUS EXCRETED BY STEERS DURING
FIVE DAY BALANCE PERIOD, GRAMS
EXPERIMENT I, TRIAL 2


Treatment

Period Complete -Mg -S -Mg-S

1 38.81 70.961 39.64 30.18
2 46.92 58.63 46.70 78.13

3 71.64 42.10 45.93 43.74
4 50.69 199.85 45.98 89.84
Mean 52.01 92.88 44.56 60.47

1Value adjusted for missing data.




TABLE 8-A

AVERAGE DAILY VOLUNTARY FEED INTAKE OF
STEERS ON PHOSPHORUS BALANCE, GRAMS
EXPERIMENT I, TRIAL 2


Treatment
Period Complete -Mg -S -Mg-S

1 2033 326 1163 552
2 1970 '1463 818 0
3 1970 572 1238 493

4 1992 719 617 573
Mean 1996 795 959 405












TABLE 9-A

TOTAL FIVE DAY PHOSPHORUS INTAKE OF
STEERS ON BALANCE STUDY, GRAMS
EXPERIMENT I, TRIAL 2


Period Complete -Mg -S -Mg-S

1 57.94 55.86 55.86 56.89

2 56.15 59.62 56.77 56.15
3 56.15 56.77 59.62 56.15

4 56.77 56.15 \ 56.15 59.62
Mean 56.75 57.10 57.10 57.20


TABLE 10-A

TOTAL URINE EXCRETED BY STEERS DURING FIVE DAY
PHOSPHORUS BALANCE STUDY, MILLILITERS
EXPERIMENT I, TRIAL 2


Treatment

Period Complete -Mg -S -Mg-S

1 9135 8300 11010
2 14150 22940 11680 11840
3 17719 13233 8125 10300

4 37500 9240 12440 18040

Mean 19626 15138 10136 12797





















TABLE 11-A

TOTAL FECES EXCRETED BY STEERS DURING FIVE DAY
PHOSPHORUS BALANCE'STUDY, GRAMS
EXPERIMENT I, TRIAL 2


Treatment


Complete -Mg


1 1850 2450 2210

2 1690 2650 3270 4030

3 2130 2280 3250 2400

4 2600 2700 3450 4800

Mean 2067 2543 3105 3360


Period


-S' -Mg-S














TABLE 12-A


STATISTICAL ANALYSIS OF FECAL PHOSPHORUS EXCRETION
EXPERIMENT I, TRIAL 2


OF STEERS


(M-V)2
Zc2 Divisor M-V Divisor


Period PI
P2
P3

Steer S1
S2
S3


-382.62
379.82
-395.78

-107.68
343.70
261.68


-263.88
-175.58
-370.88
339.24
312.06
448.72
-273.26
-107.18
-338.44


Treatment
Mg effect
S effect
Mg X S effect


Analysis of Variance
Source d_
Mg effect
S effect
Mg X S effect
:Error (


SS
4010.94
2532.94
3043.17
17054.47


359.26
-284.70
-312.06


MS
4010.94
2532.94
3043.17
2842.41


4574.94
4508.23
4895.05

362.34
3691.55
2139.88

2176.02
963.39
4298.50
3596.37
3043.17
6292.18
2333.47
358.99
3579.43


4010.94
2532.94
3043.17


F
1.41

1.07





74








TABLE 13-A

STATISTICAL ANALYSIS OF URINARY PHOSPHORUS EXCRETION OF STEERS
EXPERIMENT I, TRIAL 2

(M-V) 2
c2 Divisor M-V Divis
-Divisor

Period P1 16 32 -84.90 225.25
P2 16 32 -115.46 416.59
P3 16 32 36.22 41.00

Steer S1 16 32 \ 107.50 361.13
S2 16 32 44.30 61.33
S3 16 32 -7.74 1.87
P1 X Sl 16 32 -22.70 16.10
P1 X S2 16 32 63.50 126.01
Pl X S3 16 32 -70.18 153.91
P2 X Sl 16 32 -40.02 50.05
P2 X S2 16 32 -112.38 394.66
P2 X S3 16 32 -21.46 14.39
P3 X Sl 16 32 -59.02 108.86
P3 X S2 16 32 -35.50 39.38
P3 X S3 16 32 -83.42 217.47

Treatment
Mg effect 16 32 94.96 281.79
S effect 16 32 -34 24 36.64
Mg X S effect 16 32 112.38 394.66

Analysis of Variance
Source df SS MS F
Mg effect 1 281.79 281.79. 4.15
S effect 1 36.64 36.64
Mg X S effect 1 394.66 394.66 5.81
Error 6 407.74 67.96















TABLE 14-A


STATISTICAL ANALYSIS OF TOTAL PHOSPHORUS EXCRETED
EXPERIMENT I, TRIAL 2


Yc2 Divisor M.V M-V)2


Period P
P2
P3

Steer S1
S2
S3


16
16
16

16
16
16

16
,16
16
16
16
16
16
16
16


Treatment
Mg effect
S effect
Mg X S effect


Analysis of Variance
Source df
Mg effect 1
S effect 1
Mg X S effect 1
Error 6


SS-,
3905.86
2077.66
941.04
8584.28


-421.767
310.093
-313.837

k -45.933
433.733
208.177

-322.283
-66.353
-486.789
253.461
245.411
381.539
-377.837
-96.916
-467.607


499.975
-364.651
-245.411


MS
'3905.86
2077.66
941.04
1430.71


BY STEERS


2779.49
1502.46
1538.96

332.97
2939.44
677.15

1725.19
68.79
3702.56
1003.79
941.04
2274.56
2230.64
146.77
3416,50


3905.86
2077.66
941.04


F
2.73
1.45















TABLE 15-A

STATISTICAL ANALYSIS FOR APPARENT PHOSPHORUS ABSORPTION BY STEERS
EXPERIMENT I, TRIAL 2


Sc2 Divisor M-V DiV)ior
Divisor


379.35
-385.09
394.51


Period P1
P
2
P3

Steer S1
S2
83


Treatment
Mg effect
S effect
Mg X S effect


Analysis of Variance
Source
Mg effect
S effect
Mg X S effect
Error


SS--
1965.48
1307.63
1511.94
8577.67


89.46
-377.51
-241.47

268.47
178.57
368.29
-336.65
-311.07
-453.31
275.66
112.17
333.85


-354.67
289.29
311.07


MS
1965.48
1307.63
1511.94
1429.61


2248.54
2317.10
2431.84


125.05
2226.78
911.06

1126.19
498.24
2119.33
1770.83
1511.94
3210.78
1187.32
196.59
1741.49


1965.48
1307.63
1511.94


F
1.37

1.06














TABLE 16-A


STATISTICAL


ANALYSIS OF APPARENT PHOSPHORUS RETAINED BY STEERS
EXPERIMENT I, TRIAL 2


(M-V)2
c2 Divisor M-V Divisor


Period P1
P2
P3

Steer SI
82
S3


Treatment
Mg effect
S effect ]
Mg X S effect ]

Analysis of Variance
Source (
Mg effect
S effect
Mg X S effect
Error


SS
3180.82
1619.76
607.19
8856.70


461.81
-271.19
355.85

-20.47
-420.25
-231.29

289.61
117.50
440.03
-299.07
-197.12
-429.41
333.13
150.11
418.83


-451.19
321.97
197.13


MS
3180.82
1619.76
607.19
1476.11


3332.32
2298.25
1978.58

6.54
2759.53
835.85

1310.53
215.72
3025.41
1397.54
607.13
2881.14
1733.99
352.08
2740.91


3180.82
1619.76
607.19


F
2.15
1.10


-












TABLE 17

TOTAL CELLULOSE INTAKE OF STEERS DURING FIVE
DAY COLLECTION PERIOD, GRAMS
EXPERIMENT II


Treatment

Period Complete -Mg -S -Mg-S

1 2561.6 2515.0 2569.6 2515.0

2 2482.2 2636.0 2510.0 2482.2
3 2484.2 2510.0 2636.0 2482.2

4 2510.0 2482.2 2482.2 2636.0
Mean 2509.0 2535.8 2549.5 2528.8







TABLE 18
TOTAL CELLULOSE EXCRETED BY STEERS DURING
FIVE DAY COLLECTION PERIOD, GRAMS
EXPERIMENT II

Treatment

Period Complete -Mg -S -Mg-S

1 733 11851 1473 1516

2 676 '1301 2204 2539

3 621 1281 2035 1548
4 1128 1853 2291 3072
Mean 790 1405 2001 2171

1Value adjusted for missing data.












TABLE 19-A


STATISTICAL ANALYSIS OF IN VIVO CELLULOSE DIGESTIBILITY
FED A PURIFIED RATION
EXPERIMENT II


OF STEERS


Zc2 Divisor M-V (M-V)2
Divisor


Period P1
P2
P3

Steer Sl
S2
S3


Treatment
Mg effect
S effect
Mg X S effect


Analysis of Variance
Source
Mg effect
S effect
Mg X S effect
Error


SS
1295.41
10636 :'11
1023.78
1207.87


330.6
-54.8
135.4


103.4
21.8
58.8

-324.8
70.4
-236.6
10..6
-181.0
-132.0
-143.2
-53.2
462.2


-203.6
-583.4
181.0


MS
1295.41
10636.11
1023.78
201.31


3415.51
93.85
572.91

334.11
14.85
108.05

3296.72
154.88
1734.61
3.51
1023.78
544.50
640.82
88.45
6675.90


1295.41
10636.78
1023.78


F
6.43*
52.80***
5.09














TABLE 20-A

STATISTICAL ANALYSIS OF IN VITRO CELLULOSE DIGESTION OF RUMEN
FLUID FROM STEERS FED A PURIFIED RATION
EXPERIMENT II


yc2 Divisor M-V (M-V)
Divisor


Period P1
P2
P3

Steer S
S2
S3


Treatment
Mg effect
S effect
Mg X S effect


Analysis of Variance
Source
Mg effect
S effect
Mg X S effect
Error


-166.38
-23.76
298.80


51.32
45.00
232.34

-619.88
-68.28
-286.90
-151.30
-811.30
-147.72
172.50
187.54
715.08


-610.28
-724.68
811.30


SS
3879.60
9350.03
6856.33
2027.16


MS
3879.60
9350.03
6856.33
337.85


288.36
5.88
930.02

27.44
21.09
562.31

4002.62
48.56
857.41
238.46
6856.33
227.30
309.96
366.37.
5326.45


3879.60
5470.43
6856.33


F
11.48*
29.24**
20.29**













TABLE 21-A


STATISTICAL ANALYSIS OF TOTAL VOLATILE
OF RUMEN FLUID FROM STEERS FED
EXPERIMENT II


FATTY ACID CONCENTRATION
A PURIFIED RATION


Sc2 Divisor M-V Diviso
Divisor


Period PI
P2
P3

Steer SI
S2
S


16
16
16

16
16
16

16
16
16
16
16
16
16
16
16


Treatment
Mg effect
S effect
Mg X S effect

Analysis of Variance
Source
Mg effect
S effect
Mg X S effect
Error


14.27
-4.19
-58.45

28.03
S.-23.75
-11.45

-12.19
18.23
-9.31
-24.29
-34.07
-18.53
-10.15
-2.29
57.17


-24.95
-44.41
34.07


MS
19.45
S61.63
36.28
12.06


6.36
0.55
106.76

24.55
17.63
4.10

4.64
10.39
2.71
19.44
36.27
10.73
3.22
0.16
102.14


19.45
61.63
36.28


F
1.61
5.11
3.01


SS
19.45
61.63
36.28
72.34













TABLE 22-A


STATISTICAL ANALYSIS OF TRICHLOROACETIC
OF RUMEN FLUID FROM STEERS FED A
EXPERIMENT II


ACID INSOLUBLE NITROGEN
PURIFIED RATION


c2 Divisor M-V (M-V)2
Divisor


Period P
P2
P3

Steer Sl
S2
83


-13.01
58.03
92.63


-0.73
' -78.99
-13.27

-9.33
-87.19
-8.87
-29.69
-150.63
23.09
-30.09
-13.03
5.29


12.17
-26.79
150.63


Treatment
Mg effect
S effect
Mg X S effect


Analysis of Variance
Source
Mg effect
S effect
Mg X S effect
Error


If SS
1 4.628
1 22.428
1 709.044
6 542.279


5.289
105.233
268.135

0.017
194.982
5.502

2.720
237.566
2.459
27.546
709.044
16.660
28.290
5.306
0.874


4.628
22.428'
709.044


MS
4.628
22.428
709.044
90.379


F


7.85*













TABLE 23-A

STATISTICAL ANALYSIS OF BUFFERING CAPACITY OF RUMEN FLUID
FROM STEERS FED A PURIFIED RATION
EXPERIMENT II


Sc2 Divisor M-V (M-V)'
Diviso;


Period PI
P
2
P3

Steer S
S2
S3


-14.37
24.37
-7.43


32
32
32

32.
32
32
32
32
32
32
32
32


Treatment
Mg effect
S effect
Mg X S effect


Analysis of Variance
Source
Mg effect
S effect
Mg X S effect
Error


SS
0.2426
92.2080
11.1056
25.416


-18.07
-1.07
-5.05

-21.23
8.37
-36.81
-21.73
-13.33
-3.95
-3.57
36.63
15.21


1.97
-38.41
13.33


MS
0.2426
92.2080
11.1056
4.236


6.453(
18.6201
1.7252

10.2039
0.0358
0.7970

14.0848
2.1893
42.3430
14.7560
5.5528
0.4876
0.3983
41.9299
7.2295


0.2426
92.2080
11.1056


F

21.77**
2.62












TABLE 24-A


CELLULOSE DIGESTED IN
A PURIFIED RATION


VITRO BY RUMEN INOCULAE FROM LAMBS FED
WITH AND WITHOUT MAGNESIUM, GRAMS
EXPERIMENT III


Day
Period Treatment Replications 1 2 3


1 .3661 .354 .378
+Mg 2 .374. .348 .377
3 .372 .361 .379
Mean .371 .354 .378
I

-Mg 1 .373 \ .338 .128
2 .369 .347 .132
3 .370 .338 .145
Mean .371 .341 .135



+Mg 1 .448 .278 .199
2 .456 .297 .203
3 .447 .265 .202
Mean .450 .280 .201
II

1 .252 .231 .000
-Mg 2 .285 .263 .000
3 .268 .273 .000
Mean .268 .256 .000


Each fermentation vessel originally contained approxi-
mately .458 grams Solka-Floc on dry weight basis.

















TABLE 25-A

CELLULOSE DIGESTED IN VITRO USING SOLKA-FLOC SUBSTRATE AND RUMEN
INOCULUM FROM LAMBS FED A COMPLETE PURIFIED RATION, GRAMS
EXPERIMENT III

Day
Period Treatment Replications 1 2 3 4 5


-Mg 1 .3521 .296 .444 .204
2 .392 .304 .480 .208
I
1 .352 .596 .312 .388
+Mg 2 .392 .436 .308 .380

-M 1 .556 .124 .172 .056 .040
2 .528 .096 .132 .084 .032
II
+Mg 1 .528 .152 .180 .088 .368
2 .600 .252 .160 .320 .268

-Mg 1 .288 .440 .412 .272 .132
2 .268 .496 .420 .288 .184
III
1 .266 .536 .468 .372 .360
g 2 .280 .544 .460 .388 .320


Each fermentation vessel originally
.916 grams Solka-Floc on dry weight basis.


contained approximately




















TABLE 26-A


CELLULOSE DIGESTED IN VITRO USING RATION
FROM LAMBS FED A COMPLETE PURIFIED
EXPERIMENT III


SUBSTRATE AND INOCULUM
RATION, GRAMS


Period Treatment Replications 1 2 3' 4


1 .008 .000 .000 .000
-Mg 2 .016 .000 .000 .000
I
1 .172 .056 .144 .196
+Mg 2 .172 .072 .128 .208



1 .320 .016 .040
g 2 .344 .140 .000
II
1 .444 .476 .364
+Mg 2 .436 .468 .336














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