Title: Effect of molybdenum and copper in forage on nitrate reduction in ruminants /
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Title: Effect of molybdenum and copper in forage on nitrate reduction in ruminants /
Physical Description: vii, 95 leaves : ill. ; 28 cm.
Language: English
Creator: Buchman, David Thomas, 1933-
Publication Date: 1966
Copyright Date: 1966
 Subjects
Subject: Feeds   ( lcsh )
Animal nutrition   ( lcsh )
Nitrogen -- Metabolism   ( lcsh )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
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Thesis: Thesis (Ph. D.)--University of Florida, 1966.
Bibliography: Bibliography: leaves 85-95.
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by David Thomas Buchman.
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Bibliographic ID: UF00097850
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000401977
oclc - 24680624
notis - ACE7835

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EFFECT OF MOLYBDENUM AND COPPER

IN FORAGE ON NITRATE REDUCTION

IN RUMINANTS



















By
DAVID THOMAS BUCHMAN I;[ (










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, 1966













ACKNOWLEDGMENTS


The writer is sincerely grateful to Dr. R. L. Shirley, Chairman

of his supervisory committee, for his guidance and patience throughout

the experimental investigation and preparation of this dissertation;

and also to Drs. G. K. Davis, G. B. Killinger, J. E. Moore, J. A. Olson

and H. Smith for serving as members of the supervisory committee and

for their time and advice given toward the completion of this work.

For the many helpful suggestions of Jason Outler, I am thankful.

Special thanks for much kind assistance is due John F. Easley and

Charles W. Burgin.

The author is greatly appreciative of the labor, equipment and

land put at his disposal by Dr. Charles J. Wilcox of the Dairy Research

Unit. Without the fine cooperation extended by Dr. Wilcox, Mr. John P.

Boggs, Dairy Farm Manager, and others of the Dairy staff, the investi-

gation would have been almost impossible.

The assistance of his fellow graduate students, professors in the

Animal Science Department, laboratory technicians and secretaries at

the Nutrition Laboratory is greatly appreciated.

Special thanks go to Dr. Kenneth Smith of the Department of Dairy

Science for the many helpful suggestions in the analysis of the data.

The author is especially grateful and thankful for the eight years

of unswerving assistance, understanding, support and encouragement given


fI









by his wife, Maureen, without which this dissertation would surely never

have been written. For her unselfish help and devotion to his work he

is greatly appreciative. To her this work is dedicated.














TABLE OF CONTENTS


Page

ACKNOWLEDGMENTS ............................................... ii

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

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

LIST OF FIGURES ............................................. vii

CHAPTER

I. INTRODUCTION ........................................... 1

II. LITERATURE REVIEW ..................................... 3

III. ANALYSIS OF FLORIDA WATER FOR NITRATE ................ 21

IV. THE EFFECT OF A NITRATE DRENCH ON SHEEP CONSUMING A
PURIFIED DIET ......................................... 24

V. SUPPLEMENTARY MOLYBDENUM AND THE REDUCTION OF NITRATE
IN THE RUMEN OF STEERS FED MILLET SOILAGE CONTAINING
HIGH LEVELS OF NITRATE ............................... 33

VI. THE EFFECT OF MILLET GROWN WITH HIGH LEVELS OF NITRATE,
WITH AND WITHOUT MOLYBDENUM AND COPPER, WHEN FED TO
SHEEP AND CATTLE ....................................... 37

VII. THE EFFECT OF MOLYBDENUM AND COPPER ON NITRATE
REDUCTION IN VITRO ................................... 51

VIII. GENERAL DISCUSSION ....... ................... ......... 62

IX. SUMMARY ............................................... 67

APPENDIX .......................................... ........... 70

REFERENCES CITED ............................................. 85













LIST OF TABLES


Table Page

.1. Chlorine, Nitrate and Nitrite of Water Samples from
Central Florida ....................................... 22

2. Composition of Experimental Ration ..................... 25

3. Methemoglobin of Sheep Fed 2% Sodium Nitrate in Diet ... 26

4. Methemoglobin in Sheep Drenched with 16.8 gm. Nitrate
(Trial 2) .............................................. 28

5. Methemoglobin in Sheep Drenched with 16.8 gm. Nitrate
(Trial 3) .............................................. 29

6. Methemoglobin in Sheep Drenched with 33.6 gm. Nitrate
(Trial 4) .............................................. 31

7. Design for Testing Influence of Molybdenum on Nitrate
Reduction in the Rumen of Steers Eating Millet with High
Levels of Nitrate ...................................... 34

8. Methemoglobin in Steers Fed High Nitrate Millet Soilage
with and without Molybdenum ............................ 35

9. Composition of Millet, 1964 ............................ 38

10. Composition of Millet, 1965 ............................ 41

11. The Ammonia and Nitrate of Rumen Fluid and the Blood
Ammonia and Methemoglobin of Sheep, 1964 ............... 43

12. The pH, Nitrate, Ammonia and Carbon Dioxide Content of
Rumen Fluid from Steers, 1965 .......................... 45

13. Average Daily Intake of Millet by Steers ............... 46

14. Average Rumen'Volatile Fatty Acid Ratios from Steers,
1965 ................................................... 50

15. Composition of Fermentation Media ...................... 52













LIST OF APPENDIX TABLES


Table Page

16. Summary of Analysis of Variance of Slopes of Ammonia
Concentration in Rumen of Sheep, 1964 ................. 71

17. Individual Values of the pH of Rumen Fluid from Steers,
1965 .................................................. 72

18. Individual Values of the Nitrate Content of Rumen Fluid
from Steers, 1965 ..................................... 74

19. Individual Values of the Ammonia Content of Rumen Fluid
from Steers, 1965 ..................................... 76

20. Individual Values of the Carbon Dioxide Content of Rumen
Fluid from Steers, 1965 ................................ 78

21. Summary of Analysis of Variance of Nitrate Found in
Rumen of Steers, 1965 ................................. 80

22. Averaged Rumen Ammonia Levels Between One and Two Hours
After Feeding ......................................... 81

23. Summary of Analysis of Variance of pH Found in Rumen
Fluid of Steers, 1965 .................................. 81

24. Summary of Analysis of Variance of Total Carbon Dioxide
Found in Rumen Fluid of Steers, 1965 .................. 82

25. Summary of Analysis of Variance of Total Volatile Fatty
Acid Concentration and Ratios in the Rumen Fluid of
Steers, 1965 .......................................... 83

26. Volatile Fatty Acids Found in the Rumen Contents of
Steers Two Hours After Consuming High Nitrate Millet .. 84













LIST OF FIGURES


Figure Page

1. Rate of Nitrate Disappearance From Rumen Fluid of Steers
Drenched With 62 gm. of Nitrate 30 Minutes After Feeding ... 36

2. Summary of Effect of Molybdenum Forage Treatments on
Concentrations of Carbon Dioxide and pH of Rumen Fluid ..... 48

3. In vitro Nitrate Levels With and Without Nitrate and
Molybdenum ................................................. 55

4. In vitro Nitrate Disappearance With Varying Amounts of
Molybdenum ................................................. 56

5. In vitro Nitrate Levels With Molybdenum and With and Without
Tungsten ................................................... 57

6. In vitro Nitrate Levels With Molybdenum and With and Without
Tungsten ................................................... 58

7. In vitro Nitrate Levels With and Without Copper ............ 60

8. In vitro Nitrite Levels With and Without Copper ............. 61


vii













CHAPTER I


INTRODUCTION


Nitrate poisoning of livestock caused by an excessive intake of

nitrate from plants or water has been recognized for over 70 years. The

symptoms of nitrate poisoning in livestock include rapid, shallow breath-

ing, wobbly gait, decreased milk production, abortion and death. Nitrate

is the major nutrient form in which nitrogen is absorbed by plants and

in the great majority of cases, it is assimilated so rapidly that its

concentration rarely rises above 0.03%. When conditions occur which

retard growth of the plant, the nitrate content may rise to 5% or higher

on the dry matter basis. Water containing nitrate has also been re-

sponsible for poisoning cattle and sheep.

The native muck soils of Florida contain high levels of molybdenum

and nitrate but very low levels of copper. Molybdenum toxicity of cattle

grazing forages grown on these soils has been recognized and its allevi-

ation by supplementation of cattle with copper has been established.

Feed-lot trials with young dairy bull calves fed levels of molybdenum

as high as 100 and 200 ppm mixed in their rations for 22 weeks had no

ill effects. This indicated that molybdenum toxicity was more complex

than was generally recognized. Nitrate reductase contains molybdenum,

and copper has a similar relation to nitrite reductase. With high con-

centrations of nitrate in the forages grown on the muck soils of Florida,

the rate of nitrate reduction to nitrite in the rumen may be related to








the concentration of molybdenum present. The nitrite may accumulate

due to lack of copper and thereby increase the hazard of a moderate

concentration of nitrate. The nitrite, upon absorption into the blood,

may have contributed to the molybdenosis symptoms reported in cattle on

these muck soils by oxidizing the iron of hemoglobin to the ferric state

forming methemoglobin.

The purpose of the present study was to determine if different

levels of molybdenum and copper were practical factors in the rate at

which nitrate in forage is reduced in the rumen of cattle and sheep.

Cattle and sheep were fed forages especially grown to contain

high nitrate and varying levels of molybdenum and copper. The reduction

of nitrate with varying concentrations of molybdenum and copper was also

studied in vitro.













CHAPTER II


LITERATURE REVIEW


The Toxic Effects of Nitrate on Animals

The first authentic and detailed account of nitrate toxicity of

livestock was that of Mayo (1895). Corn (Zea mays L.) was grown in an

abandoned hog yard in the dry year of 1895. This corn fodder was fed

to cattle late one afternoon and about six hours later some of the

cattle were dead while others had collapsed. Potassium nitrate salt

was isolated from the corn stalks in amounts as high as 18.8% of the

dry matter. He called this toxicity "potash poisoning" and in so doing

inadvertently placed a stigma on the cation component which has re-

mained to the present time (Allaway, 1963).

Bradley et al. (1940) showed that the toxic factor of "potash

poisoning" was the nitrate ion as distinguished from its potassium or

sodium salt. They extracted poisonous oat hay with water and it pro-

duced typical nitrate intoxication symptoms when administered to calves.

They also found methemoglobin in the blood of these calves.

The following is a list of symptoms which have been caused by

excessive dietary nitrate.


Acute Toxicity

Initial symptoms

1. Unsteadiness, wobbly gait and muscle tremor.







2. Severe anoxia.

3. Severe cyanosis marked by rapid, shallow breathing and
weak, rapid pulse.

4. Cows apparently blind.

5. Mania.

6. Methemoglobin concentration increased in blood.

7. Dehydration and diuresis with high concentration of
nitrate in the urine.

8. Frequent drinking.

9. Gray-brown discoloration of white skin and mucous
membranes.

Terminal symptoms

1. Motor spasms (convulsions) progressing to death.

2. Abortion of fetus if present.

3. Death.

Chronic Toxicity

1. Decreased milk production.

2. Rough hair coat.

3. Poor growth and appetite.

4. Lowered conception rate.

5. Small offspring.

6. Irritable.

7. Increased heart and respiration rate.

8. Moderate levels of methemoglobin.

9. Vitamin A deficiency symptoms.

10. Degeneration and fatty infiltration of liver, degeneration
of vascular tissues of brain, heart, lungs, kidneys, and
testes.







Nitrate Accumulation by Plants

The chemically combined nitrogen absorbed by plants is almost

entirely in the nitrate form. Nitrate accumulation by plants thus

implies that the rate of utilization has not kept pace with the rate

of absorption. The accumulation is often only temporary, due to dry

weather, cloudy skies, or any condition that slows photosynthesis.

The nitrogen of the nitrate ion undergoes an 8-electron change of

valence, from +5 to -3 as shown in the following outline by Nason

(1962):

N03-- NO2---(HNO), NO2 NH2 or H2N202 --> NH20H -- NH3

+5 +3 +1 -1 -3

The nitrate is presumably built into amino acids by one of the two

methods described by Wood (1953):

NH3^
NH keto acid
NO3 -- NO2 ---> NH201 "r amino acid
ket o acid

corresponding oximino acid

In 1935 Eggleton studied the absorption of inorganic nitrogenous

salts by grasses. When plants, which had been growing on unmanured

soil, were given a solution of NaN02, NaN03, (NH4)2SO4 and K2S04, nitrite

ion could be detected in the plants two hours later. He stated that this

accumulation of nitrite was undoubtedly conditioned by rapid absorption

of nutrients and low intensity of solar radiation similar to the situ-

ation prevailing in spring, fall and under heavy shade in summer. Olson

and Moxon (1942) found that if oat (Avena sativa L,) hay and red root

(Amaranthus sp.) hay, which contained high levels of nitrate, were

moistened with water and held at room temperature for several days, high







levels of nitrite would be formed. Bacillus subtilis was isolated from

the red root hay and was found to be very effective in reducing nitrate

to nitrite. These are two of the very few reports in the literature

where nitrite was found in forage. Wright and Davison (1964) reported

that nitrite was found in very few plants containing high nitrate. Smith

(1963) completed numerous studies with corn that was fertilized with high

levels of nitrogen and although the nitrate ion content was as high as

4.8% in some samples, only negligible amounts of nitrite were found in

any of the forages he examined.

In 1958 Kretschmer found up to 4.7% nitrate ion in oats which were

over-seeded in St. Augustine.grass (Stenotaphrum secundatum (Walt.) Kuntze)

which had been sprigged in the summer on virgin peaty-muck at the Ever-

glades Experiment Station in Florida. Griffith (1958, 1960), Crawford

and Kennedy (1960), Perez and Story (1960), Cullison et al. (1962) and

Breniman (1963) have reported that high levels of nitrate in forages

were caused by high rates of nitrogen fertilization.

Another factor which caused nitrate accumulation in forages was

excess shading by high mountains (Gilbert et al., 1946) and by high

plant populations (Jordan et al., 1963; Gordon et al., 1962). Shaded

forages were relatively low in carbohydrate content due to a decreased

rate of photosynthesis. Gordon et al. (1962) explained that utilization

of nitrate within plants apparently depended on reduced nicotinamide

adenine dinucleotide (NADH) derived from carbohydrate respiration. There-

fore, any factor which decreased the production of NADH would enhance the

accumulation of nitrate. The converse of the above situation was studied

by Hamner (1936). Hamner (1936) washed the roots of 6-in. tomato

(Lycopersicon esculentum Mill.) plants and put them in sand containing








no nitrate. The starch content of the leaves rose to a "high level" and

there was little or no nitrate present. Nitrate added to the soil was

present at the top of the plant six hours later. Following nitrate

application the respiration rate was appreciably increased. The treated

plants were greener and larger 14 hours after the addition of nitrate.

The application of herbicides has been found to increase the

nitrate content of certain forages. Swanson and Shaw (1954) observed

a slight increase in nitrate level of young growing sudan grass (Sorghum

vulgare var. sudanense (Piper) Hitchc.) when 2,4-D was applied. Berg

and McElroy (1953) applied 2,4-D to oats, bromegrass (Bromus inermis

Leyss.), timothy (Phleum pratense L.), alfalfa (Medicago sativa L.),

red clover (Trifolium pratense L.), sweet clover (Melilotus alba Med.)

and white Dutch clover (Trifolium repens L.) and found no significant

increase in nitrate levels. However, they did find high nitrate content

in Canadian thistle (Cirsium arvense (L.) Scop.), dandelion (Taraxacum

officinale (L.) Weber), lambs quarter (Chenopodium album L.), red root

pigweed, Russian pigweed (Axyris amaranthoides L.) and Russian thistle

Salsula kali L.) when 2,4-D was applied. Frank and Grigsby (1957) also

found excess nitrate accumulation due to herbicides in many'of the weeds

that they studied.

Sund and Wright (1957) and Simon et al. (1959) found increased

numbers of abortions when cattle grazed excessively weedy pastures.

They found that red-berried elder (Sambucus pubens Michs.), goldenrod

(Solidago sp.), stinging nettle (Urtica sp.), boneset (Eupaturium per-

foliatum L.), red root pigweed, lambs quarter, burdock (Arctium minus

Schk.), Canadian thistle and bull thistle (Cirsium vulgare (Savi) Airy-

Shaw) accumulated much higher levels of nitrate than plants of other







species growing in the same field. When these weeds were eliminated

from a low-land pasture which had caused abortions for many years, no

more abortions occurred.

There may be a varietal difference in nitrate accumulation by

forages (Gul and Kolp, 1960). They planted several varieties of oats

at Laramie and Archer, Wyoming. Variety, as well as location and stage

of growth, had a significant effect on nitrate concentration. They

stated that it may be possible to select and breed oat varieties which

would have lower levels of nitrate. This problem was discussed at the

Conference on Nitrate Accumulation and Toxicity in New York City, April

15-16, 1963. At this meeting Dr. Griffith stated that he thought selec-

tion and breeding should not be made on the basis of resistance to

accumulation of nitrate but rather on the potentiality for production

of water-soluble carbohydrate since the latter is inversely related to

nitrate content and is an inherited character. Hageman's (1963) work

with corn hybrids, which contained high and low levels of nitrate, indi-

cated that certain crosses produced different amounts of nitrate reduc-

tase. Severe drought in an area increased nitrate levels of forages

according to Muhrer et al. (1955) and Crawford et al. (1961).

A lack of soil molybdenum has been responsible for nitrate accumu-

lation in higher plants (Spencer and Wood, 1954; Candela et al., 1957;

Stout and Meagher, 1948; Hewitt and Jones, 1947). Hewitt and Jones

(1947) showed that molybdenum deficient plants do not efficiently reduce

nitrate after it has been absorbed and translocated to the upper parts

of the plant. Tomato plants accumulated up to 12% of their dry weight

as nitrate ion. Forty-eight hours after the addition of molybdenum to

these tomato plants, the nitrate ion level was down to 1% and additional








chlorophyll had been formed as shown by the green color of these previ-

ously chlorotic plants.


Nitrate in Water Supplies

Walton (1951) found nitrate in drinking water supplies in 17

states. The levels were as high as 440 ppm nitrate and in some cases

even higher. Many of the states had reported appreciable levels of

methemoglobin and even some deaths of babies due to consumption of this

water. Very young babies are unusually susceptible to nitrate poisoning

because fetal hemoglobin is more easily oxidized than mature type hemo-

globin (Knotek and Schmidt, 1964). Emerick et al. (1965) found that one-

week-old pigs injected intravenously with 0.03 gm. sodium nitrite per kg.

of body weight developed a lower degree of methemoglobinemia than the

same pigs when similarly treated at approximately three months and five

and one-half months of age. Seerley et al. (1965) showed that as much

as 1330 ppm nitrate or 330 ppm nitrite in water had no effect on the

weight gains, thriftiness and reproductive performance of swine. The

high concentration of nitrite gave small increases in methemoglobin.

There were no effectson liver vitamin A values after 105 days on treat-

ment. Seerley et al. (1965) allowed sheep to drink natural water con-

taining up to 3300 ppm nitrate and observed methemoglobin levels as high

as 16% of the total hemoglobin. These high levels of nitrate had no

adverse effect on the performance of the lambs.


Metabolism of Nitrate in Ruminants

Nitrate in the rumen is rapidly reduced to nitrite by microorganisms

(Wright and Davison, 1964) provided the animal is on a diet containing

sufficient molybdenum (Tillman et al., 1965). If the rumen is not








overloaded with nitrate and there is sufficient energy, the nitrate is

probably reduced to nitrite and then through hydroxylamine to ammonia.

This was reported to be the reaction sequence in soybean leaves, Esch-

erichia coli and Neurospora by Nason (1962). The nitrate ion can be

used by the bacteria in two ways; (1) nitrate assimilation and (2)

nitrate respiration. Nitrate assimilation represents the "biological

reduction of nitrate to ammonia or the amino level with the products

being used for the biosynthesis of nitrogen-containing cell constituents,

for example, proteins and nucleic acids. The transformation of nitrate

to nitrite in the course of nitrate assimilation is the initial step in

the enzymatic pathway of the 8-electron change required to attain the

oxidation level of nitrogen as represented by the nitrogen of ammonia,

amino acids and proteins" (Nason, 1962). Nitrate used in this manner

would be a source of nonprotein-nitrogen for the animals. In nitrate

respiration, "nitrate is used as the terminal electron acceptor in place

of oxygen under anaerobic or partially anaerobic conditions. The reduc-

tion products, which may include nitrite, nitric oxide, nitrous oxide,

molecular and other oxidation stages of nitrogen, are apparently not

further utilized and are for the most part excreted into the surrounding

medium" (Nason, 1962).

Muhrer et al. (1955) showed that nitrite was 10 times more toxic

to rats and sheep than an equivalent amount of nitrate. This difference

is probably due to the animal having time to detoxify the nitrite as it

is slowly formed by reduction of the nitrate in the rumen. Both nitrate

and nitrite are highly water soluble and are readily absorbed into the

bloodstream. Bloomfield et al. (1962a) suggested that nitrate is

actively absorbed by the rat stomach. Nitrite, but not nitrate, oxidizes







the ferrous iron of hemoglobin to ferric iron. Nitrate is not reduced

to nitrite in the blood (Winter, 1962) and is for the most part excreted

in the urine. It has been speculated that the reduction of nitrate to

nitrite proceeds at a faster rate than the reduction of nitrite to

ammonia and that the excess nitrite is absorbed (Wright and Davison,

1964).

Ruminants are especially susceptible to nitrate toxicity because

metabolism'and absorption of nitrate and its reduction products occur in

the rumen prior to entering the stomach. When massive doses of nitrate

are placed directly into the rumen of cattle, it quickly disappears and

can be found in large quantities in the blood. But if equal amounts of

nitrate are sprayed on hay, the levels of nitrate that appear in the

blood are substantially lower (Wright and Davison, 1964). These lower

levels of blood nitrate were explained by the slower rate of administra-

tion that permitted the animal to reduce more of the nitrate in the rumen

or to excrete it via the urine. Once nitrate reaches the blood and is

apparently in equilibrium with the extra-cellular fluids (Garner et al.,

1961), it is probably metabolized and excreted in a manner similar to

that observed in non-ruminants (Kearley et al., 1962). The nitrate is

distributed throughout the body and as much as 90% may be recovered in

the urine (Wright and Davison, 1964). Setchell and Williams (1962)

drenched sheep with nitrate and recovered only 1% to 14% in the urine.

Holtenius (1957) speculated that the vasodilator effect of nitrite

might be one aspect of nitrate toxicity in sheep. He found that he gave

temporary relief to nitrate poisoned animals when he injected vasocon-

strictory drugs. Holtenius (1957) did not measure the blood pressure of

these animals but Wright and Davison (1964) found that the blood pressure







of cattle fed 440 to 660 mg. nitrate per kg. of body weight did not

differ from control animals. Asbury and Rhode (1964) injected calves

with 44 to 66 mg. sodium nitrite per kg. body weight and although there

was a small initial drop in blood pressure, they concluded that "the

clinical signs of nitrite toxicosis and death were not due to vascular

collapse."

Cattle and sheep have been observed to survive the conversion of

about 90% of their hemoglobin to methemoglobin (Holtenius, 1957). Hol-

tenius (1957) was unable to demonstrate anoxia in the tissues of sheep

when as much as 65% of the hemoglobin was converted to methemoglobin.

Also, ruminants adapted themselves to prolonged high levels of nitrate

feeding with an increase in their red cell volume and a resultant increase

of hemoglobin (Jainudeen et al., 1963; 1964).

Diven et al. (1964) found that pretreatment of sheep with potassium

nitrite increased the lethal dose of nitrite by a factor of 1.25 and de-

creased the amount of methemoglobin formed when given a sublethal dose.

This partial immunity was not due to increased hemoglobin levels since

the hemoglobin actually decreased from 11 gm. to 10 gm. per 100 ml. blood

after four weeks on treatment.

An increase in heart beat may also be a compensatory response of

the animal to the lowered oxygen carrying capacity of the blood. Prewitt

and Merilan (1958) discovered that the heart beat rate of bull calves

rose from 80 beats per minute to 160 beats per minute when methemoglobin

rose from 0 to 76% of the hemoglobin. Potassium nitrate had been admin-

istered to these calves by'gelatin capsule or dissolved in skim milk.

Two of the calves were given 33 gm. potassium nitrate per 100 kg. body

weight per day for eight days. These calves gained 1 kg. per day and had








less than 10% methemoglobin. Two other calves were given 66 gm. potassium

nitrate per 100 kg. body weight per day. These calves had 35% to 50%

methemoglobin and did not gain in weight.

It has been suggested that nitrate may interfere with the iodine

metabolism of cattle and sheep since it has been shown that nitrate

affects iodine metabolism in rats through suppression of thyroxine forma-

tion. Wyngaarden et al. (1952) studied the ability of nitrate and nitrite,

among other anions, to displace iodide from, or to block iodide uptake by,

the thyroid of rats. Although nitrate was the least potent inhibitor of

all the anions tested, his data have been cited as evidence that nitrate

interferes with thyroid function (Bloomfield et al., 1961, 1962b). Wyn-

gaarden's findings with rats have since been substantiated (Welsch et al.,

1961; Yadav et al., 1962; Bloomfield et al., 1961). When nitrate is

fed to sheep, it does not have this same blocking effect on iodine.

Bloomfield et al. (1961, 1962a) discovered that the thyroid uptake of

1131 and the concentration of plasma protein-bound 1131 was increased in

sheep fed 0.92% nitrate in their diet. The author has been unable to

find experimental reports in the literature to indicate a decreased

activity of the thyroid gland of ruminants fed nitrate.

Since an active thyroid had been reported to increase the intestinal

absorption and conversion of carotene to vitamin A in rats (Johnson and

Bauman, 1957), it seemed that nitrate could exert an effect on vitamin A

nutrition through the thyroid. Depletion of liver vitamin A stores has

beenreported in sheep fed nitrate (Goodrich et al., 1962, 1964; Hatfield

et al., 1961) or nitrite (Holst et al., 1961). More recently Ascarelli

et al. (1964) showed that there were no adverse effects on carotene

utilization when thiouracil was fed to chicks. Numerous other investi-







gators have failed to show that dietary nitrate depleted liver vitamin A

stores in cattle or sheep (Hale et al., 1961; Jordan et al., 1963; Weich-

enthal et al., 1961; Smith et al., 1962; Cline et al., 1962; Sokolowski

et al., 1961; Zimmerman et al., 1963; Wallace et al., 1964). In experi-

ments at Cornell (Jainudeen et al., 1965) no thyroid abnormalities were

observed in cattle fed nitrate nor was there any indication of impaired

vitamin A nutrition. Nitrate has been found to have little or no effect

on intestinal destruction or utilization of carotene (Miller et al., 1965;

Olson et al., 1963; Davison and Seo, 1963; Davison et al., 1964; Keating

et al., 1963). Little et al. (1965) found no effect of nitrate on pre-

intestinal destruction of vitamin A in steers. Thomas (1963) found that

nitrate incubated with homogenized rabbit or calf duodenal tissue de-

creased the amount of vitamin A converted from carotene.


Effects of Nitrate on Growth

Bradley et al. (1940) in an early experiment with long-term feeding

of nitrate, concluded that chronic nitrate toxicity did not exist in

cattle fed nitrate for more than two months. Clark and Quin (1951) fed

diets of "poor quality" grass hay and molasses supplemented with 4% sodium

and ammonium nitrate in their search for forms of nitrogen that could be

fed to sheep. No toxicity was observed. When 6% and 8% ammonium nitrate

or sodium nitrate was offered to sheep, consumption decreased greatly and

these diets were discontinued. The addition of 1% to 2% potassium nitrate

to corn silage or hay did not reduce gains of cattle nor did additions of

4% potassium nitrate to corn silage affect gains of sheep (Smith et al.,

1962). Fattening lambs have been fed up to 4% potassium nitrate in mixed

rations high in concentrate with no effect on rate of gain (Cline et al.,







1962, 1963). Nonpregnant and pregnant dairy heifers were fed oat hay

containing 1.23% nitrate (Crawford, 1960) and up to 660 mg. nitrate per

kg. body weight daily on chopped hay (Davison et al., 1963, 1964) with-

out reduced body weight gains. Work by Wallace et al. (1964) supports

the above observations.

A few experiments have been conducted where nitrate feeding has

reduced gains. Additions of 1% potassium or sodium nitrate to grain

reduced feed consumption and rate of gain of fattening cattle (Hale

et al., 1961; Weichenthal et al., 1961). Sokolowski et al. (1961)

observed reduced gains when 4% potassium nitrate was mixed in a grain

ration for fattening lambs. Although Weichenthal et al. (1963) found

that sodium nitrate depressed gains of cattle about k kg. daily, liver

and plasma vitamin A or carotene were not affected and clinical symptoms

of nitrate toxicity were not evident.

From the above reports it would seem that when nitrate is added to

roughage no effect on gain is observed unless a coincident decrease in

consumption occurs. If nitrate is given in the grain, performance may

be adversely affected. This may be attributed to the difference in rate

of ingestion of the nitrate.


Effects of Nitrate on Reproduction

There is no doubt that abortions have been caused by nitrates; but

the minimum amount that must be consumed cannot be stated precisely. The

case histories of abortions cited by Bradley et al. (1940) and Garner

(1958) were probably due to a much greater consumption of nitrate than

has been described as dangerous to pregnant animals by Muhrer et al.

(1956). Simon et al. (1959) produced abortions experimentally by placing

about 62 gm. nitrate as potassium nitrate directly into the rumen of

pregnant heifers.







Davison et al. (1964) fed 45 dairy heifers nitrate at levels of

0, 440, and 660 mg. per kg. body weight daily beginning three estrous

cycles before breeding or at 40, 150, or 240 days of pregnancy continuing

until they were killed 30 days after parturition. The estrous cycle re-

mained unchanged, but a decreased conception rate was noted in those fed

660 mg. nitrate per day. One abortion occurred in those fed the lower

level of nitrate while two abortions and two deaths occurred in those fed

the higher level. The gestation periods, the placentas, the birth weight

and appearance of the calves, vitamin A and carotene nutrition and milk

production were similar for all groups.

Fifteen gm. of potassium nitrate daily as a drench or sprayed on

hay given to sheep produced no abortions but reduced birth weight of

lambs (Sinclair and Jones, 1964).


Factors Affecting the Severity of Nitrate Poisoning

SThe rate and quantity of nitrate consumption, energy level or

adequacy of the diet, type of forage, adaptation, health, pregnancy and

species all affect the susceptibility of an animal to nitrate poisoning.

Smaller amounts of nitrate are required to cause death when given suddenly,

as by drenching, than when it is incorporated in the diet and thus admin-

istered slowly. Also, well fed animals may safely graze forages on which

hungry cattle have been poisoned. Hungry cattle eat faster than well fed

cattle (Kretschmer, 1958). Sheep and cattle maintained on inadequate

diets have been found to be more susceptible to nitrate than those fed

adequate diets (Holtenius, 1957; Crawford and Kennedy, 1960). Rumen

microorganisms obtained from animals fed alfalfa reduced nitrate and

nitrite at faster rates than those obtained from sheep fed grass hays

(Sapiro et al., 1949; Pfander et al., 1957). The addition of carbo-







hydrates such as glucose, lactate and similar compounds has been shown to

increase the rate of reduction of both nitrate and nitrite by rumen organ-

isms in vitro (Lewis, 1951; Barnett and Bowman, 1957; Emerick et al.,

1965). Sapiro et al. (1949) observed that feeding glucose enabled sheep

to withstand higher levels of nitrate.


Factors Reauired for the Complete Reduction of Nitrate

The first step in nitrate reduction is catalyzed by the molybdo-

flavoprotein nitrate reductase. This enzyme was first characterized

from Neurospora (Nason and Evans, 1953) and soybean (Glycine max Merrill)

leaves (Evans and Nason, 1953). A similar or closely related nitrate

reductase has since been reported in a variety of higher plants (Candela

et al., 1957; Hageman and Flesher, 1960; Hageman et al., 1962; Barnett,

1953; Tang and Wu, 1959; Spencer and Wood, 1954). Hewitt and Jones (1947)

were the first to suggest that molybdenum was required for the reduction

of nitrate in higher plants. Nicholas and Nason (1954) proved that molyb-

denum is the metal in the Neurospora enzyme when it "(1) increased nitrate

reductase activity, (2) decreased during dialysis against cyanide with a

concomitant decrease in enzyme activity, (3) reactivated the cyanide

dialyzed enzyme and (4) increased the nitrate reductase activity during

growth." Molybdenum has been found in nitrate reductase from Agrobacterium

tumefaciens (Ramakrishna Karup and Vaidyanathan, 1963) and in E. coli

(Taniguchi and Itagaki, 1960). Molybdenum is also required for nitrate

reduction by higher plants (Spencer and Wood, 1954; Candela et al., 1957).

The following equations illustrate our present knowledge of the mechanism

of action of nitrate reductase (Nason, 1962; Nicholas and Stevens, 1955):








NADH + H + FAD1--- FADH2 + NAD

FADH2 + Mo+6-- Mo+5 + FAD

Mo+5 + NO ---- NO- + Mo+6
3 2
This same sequence and mechanism has also been demonstrated in nitrate

reductase from soybean leaves (Nicholas and Nason, 1955a; Evans and Hall,

1955) and Escherichia coli (Nicholas and Nason, 1955b).

Iron (Fewson and Nicholas, 1960) and vitamin K (Medina and Heredia,

1958) have-also been shown to be required by E. coli for the reduction of

nitrate. The iron requirement is probably due to the need for iron in

cytochrome C which is in the electron chain when nitrate is used as a

terminal electron acceptor by E. coli (Fewson and Nicholas, 1960). Iron

and copper were found to be required for nitrite reduction in the systems

employed by Walker and Nicholas (1960), Chung and Najjar (1956) and

Nicholas et al. (1960). Lazzarini and Atkinson (1961) could not demon-

strate that E. coli needed any cofactors. These workers suggested that

the reduction of nitrate to ammonia was catalyzed by a single enzyme and

that hydroxylamine was not an obligate intermediate.

Nicholas (1959) suggested that magnesium and manganese were also

required for nitrite reduction in higher plants but more recent investi-

gations have not confirmed this (Walker and Nicholas, 1960; Cresswell

et al., 1962; Hageman et al., 1962).

Tillman et al. (1965) found that the blood and rumen contents of

sheep on a purified diet with no added molybdenum contained significantly

more nitrate ion than the blood and rumen contents of sheep on the same




Flavin adenine dinucleotide (FAD)








diet with 1 ppm molybdenum added. Ten ppm of copper and 100 ppm of iron

in the ration had no significant effect on the amounts of nitrite found

in the blood.


AdaDtive Nature of Nitrate Reductase

Higher plants such as rice (Oryza sativa L.) (Tang and Wu, 1959),

cauliflower (Brassica oleracea L. var Botrytis L.), white mustard

(Brassica hirta Mvench.) and sunflower (Helianthus annuus L.) (Hewitt

and Afridi, 1959) contain the enzyme nitrate reductase in large amounts

only when it is required. When these plants are grown with nitrate as

the only source of nitrogen, nitrate reductase activity is at its highest

level but when ammonium ion is the source of nitrogen, nitrate reductase

activity is minimal. Pateman et al. (1964) working with mutants of

Asoergillus nidulans which cannot reduce nitrate, presented a theory in

which an essential cofactor for nitrate reduction (and xanthine dehy-

drogenase activity) is also the repressor for nitrate reductase synthesis.

They state that "this is the first time that a cofactor seems to also

have a regulatory function." The cofactor acts in the normal way to re-

press nitrate reductase synthesis in the absence of nitrate ion. However,

when nitrate is in the medium it combines with the cofactor in such a way

as to prevent its "repressor" function. They also found that there is a

nitrate induced NADPH22--cytochrome C reductase which, after purifying 70

times, had the same behavior as nitrate reductase. They suggested that

these two enzymes were activities of the same protein. The essential

features of their theory were as follows:

1. A common cofactor is essential for both nitrate reductase and
xanthine dehydrogenase activity-in Aspergillus nidulans.




2
Reduced nicotinamide adenine dinucleotide phosphate (NADPH2)


~







2. The nitrate reductase protein also possesses NADPH2 linked
cytochrome C reductase activity and cofactor is not necessary
for this latter function.

3. The presence of cofactor represses the synthesis of the
nitrate reductase protein in the absence of nitrate. Nitrate
interacts with cofactor in such a way as to prevent its
"repressor" function.

More recent work by Sorger (1965) supports this single enzyme theory.

Sorger (1965) suggests that the enzyme is an aggregate of two polypeptides;

one transports electrons from NADH to FAD to cytochrome C and the other

accepts electrons from reduced FAD in the first polypeptide, passes them

to molybdate andfrom there to nitrate.


Nitrate Reductase Activity in Animal Tissues

The ability of animal tissues to reduce nitrate was first demon-

strated in liver preparations of variousispecies by Bernheim and Dixon

(1928). They showed that nitrate was enzymatically reduced by function-

ing as a hydrogen acceptor of aldehyde oxidase instead of oxygen. However,

the more recent studies of Omura (1959) and Omura and Takahashi (1959) have

led to the suggestion that the nitrate reductase activity of animal cell

preparations may not necessarily be due to the aldehyde oxidase and xan-

thine oxidase activities. Walters and Taylor (1964, 1965) have found

that minced fresh pig muscle will reduce nitrite to gaseous nitric oxide

which indicates that nitrite is able to compete with other electron ac-

ceptors for the reductive processes of mammalian respiratory enzyme systems.













CHAPTER III


ANALYSIS OF FLORIDA WATER FOR NITRATE


Water has been shown to contain appreciable amounts of nitrate

(Walton, 1951). A survey of some of the rivers, lakes and wells of

Florida was undertaken to determine if they were sufficiently high in

nitrate to cause a toxicity problem in the state.


Experimental procedure

Water samples were collected in polyethylene bottles from 22 loca-

tions in Central Florida. The samples were stored at 50C. until they

were analyzed for nitrate and nitrite (A. P. H. A., 1960).


Results and discussion

The results of the above analyses are shown in Table 1. Metzler

and Stoltenberg (1950) concluded that water containing 10 to 20 ppm

nitrate was unsafe for infants. Most of the samples listed in Table 1

were relatively low in nitrate but the sample from the well at the corral

at the Everglades Correctional Institution (E. C. I.) and the Hillsborough

River sample may have been high enough in nitrates to have had some sub-

clinical effect on human infants. These levels of nitrate in water would

probably have had no adverse effect on sheep or swine (Seerley et al.,

1965). Two of the samples obtained from Belle Glade contained relatively

high levels of nitrite (Well TR 859 and canal beside office) which could









TABLE 1

CHLORINE, NITRATE AND NITRITE OF WA'

Location

St. Johns' River, Sanford

St. Johns' River, Palatka

Kissimmee River, Okeechobee

Lake Tohopekaliga

Small unnamed river at Junction
U.S. 441 and St, 60

Fisheating Creek, Palmdale

Taylor Creek at Rt. 441

Canal near office, E.E.S., Belle Glade

Peace River, Zolfo Springs

Paynes Prairie

Well near corral, R.C.E.S., Ona

Orange Lake, McIntosh

Canal by corral, E. C. I., Belle Glade

Dr. Haines' well, at home, Belle Glade

Well TR 852, E. C. I., Belle Glade

Well TR 859, E. C. I., Belle Glade

Well at corral, E. C. I., Belle Glade

Caloosahatchee River, Moore Haven

Lake Harris, Leesburg

Lake Okeechobee, Clewiston

Hillsborough River, Tampa

Withlacoochee River, Dunnellon


TER SAMPLES FROM CENTRAL FLORIDA

Cl" (ppm) NO3 (ppm) NO2 (ppb)

272 3.7 16.5

226 2.2 trace

28 0.9 16.5

0.7 15.7


16

23

112

209

17

24

24

17

212

77

228

169

529

75

14

64

258

15


0.6

0.6

1.9

2.8

1.8

0.8

0.7

1.0

1.3

1.4

1.1

0.4

4.8

1.4

1.4

1.9

5.9

1.9


trace

trace

7.5

153.0

3.4

5.5

trace

2.0

10.5

7.5

7.5

610.0

73.5

16.5

0

5.5

76.5

12.5




23


have been potentially harmful if used as a source of drinking water for

cattle or humans.

Since knowledge of the chloride content of these water samples was

a prerequisite to analyzing them for nitrate, the chloride values are

given in Table 1. Several of the samples were high in chloride and the

water from the well at the corral, E. C. I., was quite salty to the

taste.














CHAPTER IV


THE EFFECT OF A NITRATE DRENCH ON
SHEEP CONSUMING A PURIFIED DIET


Sosa (1964) at the University of Florida fed yearling native ewes

a purified diet containing 2% sodium nitrate. The purpose of the nitrate

was to accelerate a vitamin A deficiency. These ewes were continued on

the purified diet and blood methemoglobin values were determined by the

author. The sheep then had the nitrate deleted from their diets and

equivalent amounts of nitrate were given daily as a drench. This was

done to compare the effect of two types of oral nitrate intake on the

rate and extent of methemoglobin formation in sheep.


Experimental procedure

Trial 1.--Six Florida native ewes approximately one year of age

were fed the purified diet given in Table 2. Blood from the jugular vein

was obtained nine times during a period of two months and analyzed im-

mediately each time for methemoglobin by the method of Evelyn and Malloy

(1938).

Trial 2.--The six sheep used in Trial 1 were fed the same purified

diets except nitrate was deleted in Trial 2. After one month on feed,

the sheep were drenched with 16.8 gm. of nitrate as the sodium salt at

9:00 a.m. Blood samples were obtained just prior to drenching and at

approximately 3-hour intervals during the day. The samples were immedi-

ately analyzed for methemoglobin. The surviving sheep were again

drenched and sampled for six consecutive days.

24










TABLE 2

COMPOSITION OF EXPERIMENTAL RATION

Ingredient Percent

Cellulose (Solka-floc) 20
Casein (90% protein) 20
Corn Starch 23
Corn Sugar 23
Corn Oil 4
NaNO3 2
Trace Mineral Premix B 0.5
Minerals A 6.5
Vitamin Premix C 1.0

100.0

A. Minerals Gm./100 kg. Feed

CaHPO4,2H20 3,177
K2C05.1H20 2,047
MgSO4.3H20 697
NaC1 481

B. Mineral Premixa Gm./100 kg. Feed

FeSO4.2H20 87.6
Na2B407.10H20 12.3
MnSO4.H20 6.4
ZnSO4.7H20 17.6
KI 1.9
CuSO4 2.6
2CoCO3.Co(OH)2 0.035
MoO3 0.051

C. Vitamin Premixb Gm./1000 kg. Feed

Vitamin A (250,000 I. U./gm.)c 26.4
Vitamin D (3,000 I. U./gm.) 220.0
Vitamin E (100,000 I. U./454 gm.) 176.0
Choline Chloride (25% choline) 4,400.0
Total 4,822.4

aOne-half kg. mixed with each 100 kg. feed.
bOne kg. mixed with each 100 kg. feed.
c Add 24 gm. of corn sugar to the vitamin A deprived ration.







Trial 3.--Trial 3 was conducted to provide more frequent sampling

and hemoglobin values. Four more sheep were obtained that had been on

the purified diet containing no nitrate or vitamin A for two months.

These four sheep, in addition to the two sheep that survived Trial 2,

were drenched with 16.8 gm. of nitrate as the sodium salt and blood

samples were obtained hourly. Hemoglobin (Hawk et al., 1954) and met-

hemoglobin levels were immediately determined. The sheep that survived

were drenched and sampled for five consecutive days.

Trial 4.--The five sheep that survived Trial 3 were drenched with

33.6 gm. of nitrate and were bled hourly. This amount of nitrate was

twice as much as they had received in Trials 2 and 3. Hourly blood

samples were obtained. Three sheep died the first day and the two re-

maining sheep were drenched with 33.6 gm. of nitrate the following day.


Results and discussion

Trial l.--The results of Trial 1 are shown in Table 3. Detectable

levels of methemoglobin were observed in only four of the six sheep.


TABLE 3'
METHEMOGLOBIN OF SHEEP FED 2% SODIUM NITRATE IN DIET

Sheep No. Date Sample Was Obtained in 1963

3/26 3/27 3/29 4/3 4/11 4/15 4/24 5/11 5/22
94 0a 0 0 0 0 0 0 0 0
93 0 0 0 0 0 0 0.3 '0 0
86 0 0 0 0.3 0 0 0 0 0
87 0 0 0 0 0 0.3 0 0 0
96 0 0 0 0 0 0 0 0 0 0
W75 0 0.3 0.2 0.2 0 0 0 0 0

a Gm. per 100 ml. blood.








Sinclair and Jones (1964) found that sheep that consumed hay on which

potassium nitrate had been sprayed to a final level of 0.93% nitrate had

no methemoglobin initially or after several months. Hoist et al. (1961)

fed wethers mixed feed which contained from 0.1% to 0.75% nitrite for

several months but no methemoglobin was found in the blood of these

sheep.

Feeding ewes approximately 17 gm. of nitrate per day did not pre-

cipitate nitrate poisoning symptoms or even raise blood methemoglobin

level very high, These ewes were then drenched with the same amounts

of nitrate that they had consumed daily in their feed and their responses

to this type of dosage were studied.

Trial 2.--The results of Trial 2 are shown in Table 4. The sheep

that died were examined by Dr. F. C. Neal of the Department of Veterinary

Science and the cause of death was given as nitrate poisoning in every

case.

The results of drenching ewes with 16.8 gm. of nitrate were much

more drastic (Table 4)'than when the same amount of nitrate was consumed

in the feed (Table 3). This was probably'due to the faster rate of intake

when the nitrate was given as a drench. With a drench, the rumen bacteria

do not have time to reduce the nitrate beyond the highly toxic nitrite

form. When the animal is drenched with nitrate the initial reduction of

nitrate to nitrite proceeds at a faster rate than the reduction of nitrite

to ammonia and nitrite may accumulate and be absorbed by the blood (Wright

and Davison, 1964).

Trial 3.--The results of Trial 3 are shown in Table 5. The first

day of drenching there was little methemoglobin formed. The second day

sheep 86 and 92 had over half of their hemoglobin converted to methemo-










TABLE 4

METHEMOGLOBIN IN SHEEP DRENCHED WITH 16.8 gm. NITRATE (TRIAL 2)


Sheep No. and Date Time After Drenching
7/23/63 0 Hour 2 Hours 4 Hours 8 Hours

94 Oa 0 -
93 0 2.5 (died at about 5 hours)
86 0 0 -
87 0 0 -
96 0 0.4 -
W75 0.4 0 -

7/24/63

94 0 1.4 4.1 (died at about 5 hours)
86 0 0 0
87 0 0 1.4 (died the following night)
96 0 0.2 0 -
W75 0 0 0

7/25/63

86 0 0 0
96 0 0 0
W75 0 0.5 2.5 -

7/26/63

86 0 0 1.1 -
96 0 0 0
W75 0 1.6 5.9 (died at 5 hours)

7/27/63


1.7
0


3.9
0.8


7/28/63

86 0 1.2
96 0 0.3


Gm. per 100 ml. blood










TABLE 5

METHEMOGLOBIN IN SHEEP DRENCHED WITH 16.8 gm. NITRATE (TRIAL 3)

Sheep No. and Date Time After Drenching
0 Hours 3 Hours 6 Hours 9 Hours 12 Hours 15 Hours
8/12/63 gm.a % gm. % gm. % gm. % gm. % gm. %

86 0 0 0 0 0.9 9.4 0.4 3.2 -
88 0 0 0 0 0 0 0 0 -
89 0 0 0 0 0 0 0 0 -
90 0 0 0 0 0 0 0 0 -
92 0 0 0 0 0 0 0.6 6.5 -
96 0 0 0 0 0 0 0 0 -

8/13/63

86 0 0 1.1 11.1- 4.6 40.8 7.0 56.4 6.4 59.6 -
88 0 0 0 0 0.4 3.1 1.0 7.5 0.4 2.8 -
89 0 0 0.5 4.5 0.7 6.0 0 0 -
90 0 0 0 0 0 0 0 0 -
92 0.4 2.8 0.5 4.3 3.4 27.3 6.5 52.4 5.8 46.9 -
96 0 0 0 0 1.0 10.4 0 0 -

8/14/63

86 0 0 0.8 8.4 1.3 12.3 1.7 15.1 2.4 20.1 0.7 6.7
88 0 0 0.7 5.4 0.5 3.8 0.5 4.1 0 0 -
89 0 0 1.4 12.2 3.2 27.7 3.3 26.3 0.5 4.8 -
90 0 0 0 0 0.2 2.1 0 0 0 0 -
92 0.4 2.8 0.7 5.8 1.9 15.3 4.2 33.2 3.0 21.8 0.7 5.8
96 0 0 3.0 32.4 5.0 49.2 2.5 24.9 0.8 7.4 -

8/15/63

86 0 0 1.1 9.4 4.0 33.6 6.9 56.3 5.8 47.5 -
88 0 0 1.1 10.1 1.6 12.9 1.8 13.6 0.5 4.2 -
89 0 0 0.9 8.2 3.0 24.7 2.2 19.7 0.6 5.6 -
90 0 0 0.5 4.2 0 0 0 0 -
92 0 0 0.6 5.0 1.8 13.8 0.8 7.0 0.4 3.0 -
96 0.2 2.3 4.4 45.2 5.9 58.7 (at death)

8/16/63

86 0.2 2.4 1.1 10.2 3.6 31.6 5.7 44.6 2.5 23.2 0.6 5.8
88 0 0 2.8 23.3 5.6 39.4 6.4 44.5 4.6 35.7 0.9 7.7
89 0 0 1.2 10.4 3.4 32.8 5.3 44.4 1.4 13.0 0 0
90 0 0 0.3 2.5 0.9 8.5 0.6 5.6 0 0 -
92 0 0 1.2 10.3 5.3 46.1 2.8 22.3 0.9 8.0 0 0


a Gm. methemoglobin
b1


per 100 ml. blood.


Methemoglobin as percent of hemoglobin.


u







globin while the other sheep had only small amounts or none converted.

The third day sheep 86, 88 and 92 did not reach the high levels of met-

hemoglobin attained on day 2, but the other three sheep had higher

values. On day 4 the methemoglobin values were about the same as on

previous days except for number 96 which died five hours after drenching.

It had 58.7% methemoglobin at the time of death.

The fifth day of drenching, sheep 86 did not reach as high a level

of methemoglobin as on the fourth day but the other sheep had more than

double the methemoglobin levels of the day before. Overall, the sheep

that survived regenerated their hemoglobin by the next morning. However,

each day it was apparent that more methemoglobin was formed, which indi-

cated that the sheep gradually lost their capacity to resist the toxic

nitrate. Sokolowski et al. (1960) suggested that lambs were apparently

able to counteract nitrate toxicity for about 48 hours due to detoxifi-

cation by specific microbes with the eventual elimination of these

microbes. Another explanation of the increasing daily levels of met-

hemoglobin would be an impaired ability of the blood and tissues either

to reduce nitrite or to eliminate it via the urine.

Trial 4.--The results of Trial 4 are shown in Table 6. Three of

the five sheep died the first day. Of the two remaining sheep, one died

the second day. The other, at the terminal stage of nitrate poisoning,

was administered intravenously a 3% solution of methylene blue and it

recovered. The amount of hemoglobin converted to methemoglobin at the

time of death varied between 51.5% and 70.6%. This is quite a wide

range and is lower than some of the high values reported in the liter-

ature (Holtenius, 1957; Diven et al., 1964; Setchell and Williams, 1962

and Stewart and Merilan, 1958). Since Diven et al. (1964) found 93%












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methemoglobin in sheep, death of the sheep reported herein at compara-

tively low levels of methemoglobin may have been caused by the unusual

amount of handling associated with drenching and frequent bleeding.

There seems to have been no immunity imparted by feeding nitrate

as suggested by Diven et al. (1964) since four of the sheep that had

been fed nitrate previously died during Trial 2 and only one of the sheep

that had not previously been fed nitrate died during Trial 3. The sheep

that had been fed nitrate previously were not fed the nitrate for approxi-

mately one month prior to nitrate drenching, thus any immunity from

previous treatment may have been lost during this time. If these sheep

had had more red blood cells due to nitrate feeding as suggested by

Jainudeen et al. (1963), the red blood cell volume could have adjusted

to normal during the one month preliminary period (Jamieson, 1958).

These trials with purified diets and nitrate feeding and drenching

demonstrated the susceptibility of sheep to nitrate poisoning. However,

it was believed that studies involving the influence of molybdenum and

copper on nitrate toxicity should be done using natural forage rations

containing various concentrations of molybdenum and copper and high levels

of nitrate.












CHAPTER V


SUPPLEMENTARY MOLYBDENUM AND THE REDUCTION OF NITRATE IN THE RUMEN OF
STEERS FED MILLET SOILAGE CONTAINING HIGH LEVELS OF NITRATE


In the spring of 1964 the University of Florida Dairy Research

Unit, Hague, had several fields of millet (Pennisetum glaucum L,) which

contained between 2% and 4% nitrate on the dry matter basis. This

millet was chopped daily and fed to the milking herd, dry cows, springing

heifers and to some two-year-old dairy steers. The high levels of nitrate

had no observable adverse effects on these animals. Normally all of the

animals at the Dairy Research Unit are fed minerals. However, the dairy

steers had inadvertently received no minerals for several months. It

was thought that if these steers were given molybdenum, the nitrate

would be reduced to nitrite at a faster rate than the nitrite could be

reduced and nitrate poisoning symptoms would be detected.


Experimental procedure

Two-year-old Guernsey and Jersey steers were assigned to the treat-

ments presented in Table 7. Two steers were fed alfalfa hay with added

molybdenum to check on the possible toxicity of molybdenum. The two

steers on each treatment were fed in open lots and observed several times

daily for signs of nitrate poisoning. Blood samples were obtained the

eighth day on feed and analyzed for methemoglobin.

After three weeks on treatment, one steer receiving millet plus

molybdenum and one steer receiving millet with no supplementary molybdenum








were allowed to eat millet for 30 minutes. They were then drenched with

62 gm. nitrate as sodium nitrate. Rumen samples were obtained periodi-

cally by stomach tube for three hours and analyzed for nitrate.


TABLE 7

DESIGN FOR TESTING INFLUENCE OF MOLYBDENUM ON NITRATE REDUCTION IN THE
RUMEN OF STEERS EATING MILLET WITH HIGH LEVELS OF NITRATE


Steer No. Treatment

486, 487 High nitrate millet plus 0.5 gm.
NaMo04-2H20a

912, 933 High nitrate millet

80, 256 Alfalfa hay plus 0.5 gm. NaMo04-2H20

a The 0.5 gm. of NaMoO4.2H20 was mixed in a handful of citrus pulp and
fed every other day.


Results and discussion

No toxicity symptoms were observed in the steers during the first

eight days of the experiment. Blood samples were obtained on the eighth

day and analyzed for methemoglobin (Table 8). The steers consuming the

high-nitrate millet had very low levels of blood methemoglobin while

those receiving the alfalfa had no methemoglobin. Nothing conclusive

could be said concerning the molybdenum treatment.

When nitrate was given as a drench, the steer that received no

supplementary molybdenum had higher levels of nitrate throughout the

sampling period as shown in Figure 1. These preliminary studies sug-

gested that molybdenum may have been responsible for an increased rate

of nitrate disappearance from the rumen.

















TABLE 8

METHEMOGLOBIN IN STEERS FED HIGH NITRATE MILLET SOILAGE WITH
AND WITHOUT MOLYBDENUM


Steer Treatment Methemoglobina


486 Millet + Mo 0.7

487 Millet + Mo 0.1

912 Millet 0.3

933 Millet 1.1

80 Alfalfa + Mo 0

256 Alfalfa + Mo 0


a Gm. per 100 ml. blood.





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CHAPTER VI


THE EFFECT OF MILLET GROWN WITH HIGH LEVELS OF NITRATE, WITH AND WITHOUT
MOLYBDENUM AND COPPER, WHEN FED TO SHEEP AND CATTLE


In order to determine the effect of varying concentrations of

molybdenum and copper in forage on the rate of nitrate reduction in the

rumen, the concentrations of nitrate and ammonia in rumen contents and

methemoglobin and ammonia in blood were determined. The pH, carbon

dioxide and volatile fatty acid changes in the rumen were also observed.

Star millet was raised in 1964 with high levels of nitrogen ferti-

lizer, with and without added molybdenum and copper. This was an effort

to produce forage with high levels of nitrate, molybdenum and copper.

The forage was irrigated when required, harvested, chopped and dried at

the pre-bud stage of maturity. The forage was fed to yearling wethers.

Gahi millet was raised in 1965 and harvested in approximately the

same manner as that in 1964. This forage was fed yearling Hereford steers

in a 4x4 Latin Square experiment. The steers were equipped with rumen

fistulas.


Experimental procedure

Trial 1 (1964).--On April 1, 1964 Star millet was sowed and ferti-

lized at the rate of 198 kg. per acre with 8-8-8 fertilizer. The field

was divided into four plots and the following applications were made:

Plot A, no supplemental copper or molybdenum; Plot B, 11.35 kg. copper

sulfate per acre at planting and 11.35 kg. copper sulfate per acre on








June 2; Plot C, 0.91 kg. sodium molybdate at planting and 0.91 kg. sodium

molybdate on June 2; Plot D, both copper and molybdenum at the rates

given for Plots B and C above. The supplemental copper and molybdenum

were carefully weighed and mixed with the fertilizer applied to each

plot. All four plots were top-dressed on April 27 at the rate of 173 kg.

ammonium nitrate per acre. The plots were again top-dressed on May 5 and

June 2 at the same rate for a total of 189 kg. of actual nitrogen per

acre for the season. The plots were irrigated when necessary.

The millet was cut May 19 and the forage was removed but was not

used experimentally. The second cutting of millet was made at the pre-

bud stage of maturity with a forage harvester on June 9. The chopped

forage was loosely bagged in large loose-weave burlap bags and dried

artificially at Mixons' Crop Drying Service, Williston, Florida. The

analysis of the second-cut forage is shown in Table 9.


TABLE 9

COMPOSITION OF MILLET, 1964

Plot Crude Inorganic
(Treatment) N03(%) N03-N(%) Protein(%) Cu(ppm) Mo(ppm) Sulfate(%)

A (-Cu, -Mo) 1.76 0.40 23.3 4.2 1.68 0.32

B (+Cu, -Mo) 1.95 0.44 24.1 78.3 0.56 0.40

C (-Cu, +Mo) 2.11 0.48 23.9 5.2, 17.68 0.46

D (+Cu, +Mo) 1.64 0.37 24.2 98.7 27.26 0.35




Nitrate content was determined by the method of Woolley et al.

(1960), molybdenum and copper by methods given by Sandell (1959) and







sulfate by Steinbergs' (1953) method. Crude protein was determined by

Kjeldahl method (A.O.A.C., 1960).

Sulfate was not an experimental variable and its concentration in

the forages was relatively uniform. The high levels of copper obtained

in Treatments B and D were probably due to contamination with the second

application of copper sulfate which was applied one week before harvest.

The millet was irrigated two times with overhead sprinklers between the

last fertilization and cutting but the supplemental copper could have

been trapped in the swirls of the millet plants. The same conditions

may have been true for the molybdenum.

The millet grown in 1964 was fed, along with water ad libitum, to

24 yearling wether sheep which weighed from 23 to 38 kg. The sheep were

housed in 4' x 8' pens with three sheep per pen and were randomly allotted

to treatments with six sheep per treatment. Each treatment forage was

fed continuously to each group throughout the two month duration of the

trial. Two days after the trial began, one wether on Treatment B acci-

dentally strangled and two wethers on Treatment A died due to unknown

causes. After one month on feed jugular blood and rumen samples were

taken from one animal on each treatment at 8:00 a.m. Feed had been with-

held the previous night. The sheep were then allowed access to feed for

one hour and blood and rumen samples were obtained. The sheep were then

drenched with 20 gm. potassium nitrate as a 20% solution which was approxi-

mately 0.66 gm. per kg. body weight. Rumen contents were sampled, with a

stomach tube, at the following intervals after drenching: 10, 30, 50, 70,

90 minutes, 2, 3, 4 and 6 hours. Blood samples were taken at the follow-

ing intervals after drenching: 1, 2, 3, 4 and 6 hours. The above sampling








procedure was repeated once a week until four sheep from each treatment

had been sampled.

The pH of the rumen samples was lowered to 1 to 1.5 with concen-

trated hydrochloric acid. They were stored frozen until analyzed for

ammonia by the microdiffusion method of Conway (1957), after which the

samples were filtered through analytical filter aid (Johns-Manville,

Celite) with vacuum and analyzed for nitrate by the method of Woolley

et al. (1960).

The blood samples were collected in lithium citrate to prevent

clotting and analyzed for ammonia (Conway, 1957) and methemoglobin

(Evelyn and Malloy, 1938).

S Trial 2 (1965).--On April 6, 1965, Gahi millet was planted in a

field at the Dairy Research Unit, Hague, which had been cleared only five

years before and had purposely not been fertilized with trace minerals.

Three hundred and thirty-six kg. of 10-10-10 fertilizer per acre were

applied at planting time. The field was divided into four equal-sized

plots and the following applications were made: Plot A, no supplemental

copper or molybdenum; Plot B, 12.5 kg. copper sulfate per acre; Plot C,

1 kg. sodium molybdate per acre; Plot D, both copper and molybdenum at

the rates given for Plots B and C above. All four plots were top-dressed

with 45.4 kg. nitrogen per acre as liquid ammonia on April 28 and with

200 kg. ammonium nitrate per acre on May 12 for a total of 146 kg. actual

nitrogen per acre for the season. "

The millet was harvested on May 21 and made into hay in the same

manner as described for 1964. Table 10 shows the analysis of this forage.








TABLE 10 rt k, \3

COMPOSITION OF MILLET, 1965


Plot Crude Inorganic
(Treatment) N03(%) NO3-N(%) Protein(%) Cu(ppm) Mo(ppm) Sulfate(%)

A (-Cu, -Mo) 0.75 0.17 11.5 13.74 0.58 0.21

B (+Cu, -Mo) 0.39 0.09 12.7 11.71 0.57 0.21

C (-Cu, -+Mo) 0.33 0.08 13.2 9.17 3.00 0.16

D (+Cu, +Mo) 0.49 0.11 13.2 10.66- 4.34 0.32



The millet grown in 1965 was fed to four rumen-fistulated yearling

Hereford steers in a 4x4 Latin Square. The steers were stabled in indi-

vidual pens eight feet square with an 8' x 20' exercise yard. Water was

supplied in automatic drinking cups. The hay was offered to the steers

for c-. hour at 8:00 a.m. and for one hour at 4:00 p.m. It was thought

that by allowing the steers only a limited time to eat each day, that

they would eat faster and get the nitrate of the millet into the rumen as

quickly as possible. There should have been a greater chance of observing

nitrate poisoning symptoms under these conditions.

There was a 14 day preliminary feeding period before each collec-

tion. Rumen pumps (Moore et al., 1964) were installed in the fistulas

the evening of the 14th day and the drinking water was removed. The

morning of the 15th day rumen samples were obtained from each steer.

This was designated the 0-time sample. Feed and water were offered for

one hour. Rumen samples were then obtained at the following intervals:

1, 1.33, 1.67, 2, 2.5, 3, 4, 5, 6, 7 and 8 hours. The pH of the rumen

samples was determined immediately. An aliquot was preserved by adding








concentrated hydrochloric acid to pH 1 to 1.5 and frozen for nitrate

(Woolley et al., 1960) and ammonia (Conway, 1957) analysis. Another

aliquot was preserved with mercuric chloride and frozen for volatile

fatty acid determination. Volatile fatty acids were analyzed by gas-

liquid chromatography. Additional samples were placed in serum bottles,

quickly stoppered and immediately analyzed for total rumen fluid carbon

dioxide concentration (bicarbonate plus carbonic acid-carbon dioxide,

Moore et al., 1964).


Results and discussion

Trial 1 (1964).-- Results of the analysis of samples taken from the

sheep are shown in Table 11. There was so much individual variation with-

in treatments that no trends for a different rate of disappearance of

nitrate from the rumen could be detected in any of the treatments. Levels

of rumen ammonia were not different among treatments (Table 16). The con-

centrations ranged between 10 and 30 mg. of ammonia nitrogen per 100 ml.

rumen fluid which is within a range considered normal for animals on good-

quality hay by Barnett and Reid (1961). Although there were relatively

high levels of protein and 1.6% to 2.1% nitrate in the hay, both of which

would be potential sources of rumen ammonia, the ammonia concentrations

were not extraordinarily high.

There were only very small differences among treatments in blood

ammonia. The range was between 0.30 and 1.1 mg. of ammonia nitrogen per

100 ml. blood serum. This is within a normal range for blood ammonia

(Barnett and Reid, 1961).

Two sheep, one on Treatment B and one on Treatment C, collapsed

about four hours after drenching and were treated with methylene blue.




















































































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The sheep on Treatment B died a few minutes later; the sheep on Treat-

ment C survived. Since methylene blue had been administered, meaningful

methemoglobin values were not obtained.

Blood methemoglobin values gradually increased and reached a maxi-

mum about four hours after the nitrate dose. This was several hours

earlier than when the highest level of methemoglobin was observed in the

sheep fed purified diets (Chapter IV). The most extreme toxicity symptoms,

due to a potassium nitrate drench to lambs, were observed at 4 hours by

Sokolowski et al. (1960). Lewis (1951) observed a peak in methemoglobin

values seven hours after a lamb was dosed with 25 gm. of sodium nitrate.

The period of time required for the peak levels of methemoglobin to appear

is apparently quite variable and is probably due to many factors including

the amount of nitrate ingested, the carbohydrate levels of the ration and

immediate history of nitrate ingestion. A possible explanation of the

difference in time required to reach maximum methemoglobin concentrations

among the sheep reported in this research would be the amount of carbo-

hydrates in the diet. The purified diets fed to sheep reported in Chapter

IV contained high levels of fermentable carbohydrates (Table 2) but the

sheep in this trial (1964) received millet hay with no grain.

Trial 2 (1965).--The averaged results of analysis of the samples

taken from the steers in Trial 2 are shown in Table 21. Individual

values are given in Tables 17 through 20 and 26. Steers that consumed

the four forage treatments in 1965 showed no statistical differences in

rumen nitrate levels four hours after they had eaten the forage. There

were also no statistical differences at four hours in micrograms of

nitrate per 5 ml. rumen fluid per gm. of nitrate fed. There were no

differences in the rate of nitrate reduction due to treatment














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as determined by statistical analysis of the slopes when rumen nitrate

concentration was plotted against time after feeding (Table 21). A

possible explanation for finding no differences in the rate of nitrate

disappearance from the rumen due to molybdenum is that the control

forage, which had not been fertilized with molybdenum, contained over

0.5 ppm of the element. This low level of molybdenum was apparently

sufficient for the rumen microorganisms to build an effective nitrate

reducing system. Tillman et al. (1965) found that the addition of 1 ppm

of molybdenum to a purified diet provided for a faster rate of nitrate

disappearance from the rumen than did a diet to which no molybdenum had

been added. Nine hours after feeding, these workers reported little

difference in the amounts of nitrate in the rumen. The molybdenum con-

tent of the control ration was not reported.

__7> The high molybdenum forages, Treatments C and D, were consumed in

smaller (P< .01) amounts than were the forages from Treatments A and B

(Table 13). Reduced feed intake is not generally considered a symptom

of molybdenum toxicity (Ferguson et al., 1943; Underwood, 1962).


TABLE 13
AVERAGE DAILY INTAKE OF MILLET BY STEERS

Plot
(Treatment) Average Daily Intake (kg.)


A (-Cu, -Mo) 4.5

B (+Cu, -Mo) 4.8

C (-Cu, +M6) 2.6a

D (+Cu, +Mo) 3.5

a Significantly less (P<.01).







However, Lesperance and Bohman (1961) reported that molybdenum added to

alfalfa or grass hay and cottonseed meal rations at 100 ppm decreased

consumption by heifers fed these rations from weaning to one year of age.

Although the level of molybdenum in the forage fed to the steers in Trial

2 was only 3 to 4.3 ppm, it seemed to be responsible for a highly signifi-

cant decrease in forage consumption. Intake data were not obtained in

1964 when relatively high levels of molybdenum were fed to sheep. How-

ever, since.the sheep in all four treatment groups lost an average of

between 1 and 2.7 kg. of weight during a 2-month feeding period, there

was probably no great difference in consumption of the forage due to

molybdenum content.

There was no treatment effect on ammonia concentration of rumen

contents. There was a significant effect (P< .05) on rumen ammonia

levels due to period (Table 22). Overall, the rumen ammonia levels in-

creased from the first to the fourth period and coincided with the in-

creased intake of the millet. Since the ammonia would come from the

fermentation of digesta, more rumen ammonia would be present when the

animals were consuming greater amounts of millet. The pH of the rumen

contents was statistically examined separately for the periods between

one and eight hours and between two and eight hours (Table 23). There

were no differences detected in either case due to treatment, animal or

period. The rumen fluid total carbon dioxide concentration in steers

that received the molybdenum fertilized forages (Treatments C and D) was

reduced (P<.05) as shown in Figure 2. However, when the rumen fluid

carbon dioxide levels were adjusted to account for forage intake, that is

m Mol. total carbon dioxide per liter of rumen fluid per kg. of millet





































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consumed, the differences between the molybdenum treatments disappeared

(Table 24).

Correlation coefficients were calculated for rumen pH against

carbon dioxide levels. The coefficient for the treatments having no

supplemental molybdenum (A, B) was 0.66 which indicated that 44.6% of

the variation in rumen carbon dioxide levels could be explained by

variations in pH. The coefficient for the molybdenum treatments (C, D)

was 0.87 which means that 76.3% of the variation in rumen carbon dioxide

levels was due to pH. Although the treatments did not significantly

affect pH, there was a trend to higher pH values with higher carbon

dioxide levels. This may have been caused by the increased levels of

alkaline salts present with the higher levels of carbon dioxide.

There were no significant differences due to treatment in the total

volatile fatty acid concentration in the rumen fluid (Table 25). The

volatile fatty acid concentrations found in the rumen are shown in

Table 26. Dobson (1961) said that about 1 molecule of bicarbonate

appeared in the rumen for every 2 molecules of fatty acid absorbed.

There was more of the low molybdenum forage consumed and presumably

more fatty acids produced, yet there was no difference in the amount

of fatty acids found in the rumen. If the presumed extra production

of fatty acids was absorbed, more bicarbonate would be found in the rumen

contents. This would explain the increased carbon dioxide concentration

observed when the steers consumed the low molybdenum forage. Since the

production of fatty acids is probably a function of intake, when the

total carbon dioxide concentration in the rumen was put on the basis

of units of forage intake, the difference between treatments disappeared.









Increased salivation with its high concentration of bicarbonate, ac-

companying the greater intake could also have contributed to the higher

carbon dioxide levels in the rumen.

An increased (P <.05) acetic:butyric acids ratio was observed in

the steers fed the molybdenum fertilized forages (Table 14). There was

no difference in the acetic:propionic acids ratio or in the propionic:

butyric acids ratio.


TABLE 14

AVERAGE RUMEN VOLATILE FATTY ACID RATIOS FROM STEERS, 1965

Treatment A/P A/B P/B

-Mo (plots A & B) 3.54 8.82 2.50

+Mo (plots C & D) 3.66 10.44a 2.88

a Significantly higher (P <.05).














CHAPTER VII


THE EFFECT OF MOLYBDENUM AND COPPER ON NITRATE
REDUCTION IN VITRO


The in vivo studies indicated that the nitrate reducing systems

of the rumen were relatively insensitive to added molybdenum and copper.

In vitro studies wore conducted whore greater control over the con-

stituents of the fermenting media was possible. These studies were

conducted to investigate (1) the effect of several levels of molybdenum

on the rate of nitrate reduction, (2) the effect of tungsten on nitrate

reduction and (3) the effect of added copper on nitrite reduction rates.


Experimental procedure

Fermentation flasks of 250 ml. capacity were placed in a water bath

maintained at 390C. for in vitro studies. Carbon dioxide gas was bubbled

through the fermenting media by way of a glass tube opening at the bottom

of the flask. Each flask contained 200 ml. of fermentation media with

the composition shown in Table 15 (Quicke et al., 1959).

The inoculum was prepared in the following manner: representative

rumen samples were obtained from a mature Angus steer with a rumen

fistula. The steer had been maintained for at least two years on Bermuda

grass hay fed ad libitum, 900 gm. soybean meal daily, trace mineralized

salt and vitamins A and D. Whole rumen contents were withdrawn from the

fistula and strained through four layers of cheesecloth. The filtrate













TABLE 15

COMPOSITION OF FERMENTATION MEDIA


Constituent Quantity/200 ml.


Urea 252 mg.

Na2CO3 400 mg.

FeC13'6H20 8.8 mg.

CaC12 10.6 mg.

Na2HPO4 226 mg.

NaH2PO4 218 mg.

KC1 86 mg.

NaC1 86 mg.

MgS04.7H20 23.3 mg.

Na2SO4 30 mg.

Valeric acid 50 mg.

Biotin 40 mcg.

PABAa 100 mcg.

Bermuda grass 4 gm.

a
Para-aminobenzoic acid.








obtained was centrifuged at 250 times gravity for 3 minutes to sediment

coarse particles and protozoa. Eighty ml. of the supernatent solution

were used to inoculate each flask. Flasks were prepared in duplicate

having the following composition:

Trial 1

1. 60 ml. H20 (control)

2. Sodium molybdate to bring the concentration in the final
volume to 40 ppm of molybdenum.

3. Potassium nitrate so as to have a final concentration of
0.003% nitrate ion.

4. Combination of #2 and #3 above.

Trial 2

5. 60 ml. H20 (control)

6. Molybdenum at 2 ppm.

7. Molybdenum at 10 ppm.

8. Molybdenum at 50 ppm.

9. Molybdenum at 2 ppm plus 200 ppm of tungsten.

10. Molybdenum at 10 ppm plus 1000 ppm of tungsten.

Trial 3

11. 60 ml. H20 (control)

12. Copper sulfate to final concentration of 2 ppm of copper.

There was a final volume of 200 ml. in each flask.

These flasks were allowed to ferment for a preliminary period of

4 hours at which time 15 ml. samples were withdrawn from each flask,

mixed with ml. concentrated hydrochloric acid and immediately frozen

for nitrate analysis. All samples were handled in this manner except

those obtained from Treatments 11 and 12. These samples were collected








and frozen immediately for nitrate and nitrite analysis. After the

samples were obtained at 4 hours, 0.6 gm. of nitrate was added as a

solution of potassium nitrate to each flask to bring the final concen-

tration to approximately 0.03% of nitrate. The contents were sampled

periodically for 7 to 8 hours.

The nitrate was added to Treatments 3 and 4 in an effort to

stimulate the synthesis of a nitrate reducing system by the bacteria

during the'4-hour preliminary period. Treatments 5 through 8 were an

effort to determine the effect, if any, of varying levels of molybdenum

on the rate of nitrate disappearance. Treatments 9 and 10 were efforts

to determine the effect of tungsten, a molybdenum antagonist (Ramakrishna

Karup and Vaidyanathan, 1963) on nitrate disappearance indirectly through

molybdenum. Treatments 11 and 12 would indicate the effect that additional

copper, at a level of 2 ppm, had on the disappearance rate of nitrite.


Results and discussion

The results from the first four treatments are shown graphically in

Figure 3. Nitrate was added and it appeared that the molybdenum may haye ...

caused an increased rate of nitrate disappearance during the first hour

of fermentation but this advantage was not apparent during the period

from 2 to 8 hours. Preincubation with nitrate (Treatments 3 and 4) had

no consistent effect on nitrate disappearance. Figure 4 shows the nitrate

levels from Treatments 5, 6, 7 and 8. There was no increase in nitrate

disappearance due to molybdenum. The higher the molybdenum levels the

more nitrate was present at the end of 8 hours of fermentation. As can

be seen from Figures 5 and 6, tungsten had no effect on nitrate reduction

in these fermentation flasks.













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Figures 7 and 8 show the levels of nitrate and nitrite from Treat-

ments 11 and 12 respectively. Copper at 2 ppm had no apparent effect

on the rate of nitrate and nitrite disappearance. As the nitrate dis-

appeared the nitrite increased. However, the nitrate decreased about

90 mcg. per ml. in 8 hours while the nitrite increased approximately

30 mcg. per ml. in the same period. This indicated that the nitrite

was an intermediate compound in the nitrate to ammonia reduction chain.

A possible explanation for these results is that extremely low

levels of molybdenum and copper are required for the nitrate and nitrite

enzyme reducing systems. These very low levels of molybdenum and copper

could have been carried into the system with the bacteria, as a contami-

nant in the media or in the Bermuda grass hay. This suggests that the

bacteria should be washed several times to rid them of all excess

molybdenum and copper before inoculating the media.

Generally, the in vitro studies support the in vivo work in that

the nitrate reducing system_sgeems to be indifferent to high level s_-L

molybdenum and copper. Tungsten, in amounts 100 times those of molyb-

denum, had no antagonistic effect on molybdenum as determined by the rate

of nitrate reduction.





60





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CHAPTER VIII


GENERAL DISCUSSION


The nitrate ion occupies a position of primary importance in the

normal metabolism of higher plants. However, nitrate may accumulate

within plants to very high levels with results disastrous to animals

consuming these plants or to animals and humans exposed to the gaseous

decomposition products. Acute poisoning has occurred in livestock and

many cases of chronic poisoning have been reported. "Silo fillers

disease" is a condition caused by inhalation of gases coming from silos

recently filled with high-nitrate forage. Most of the early research

concerned with nitrate toxicity was stimulated by sporadic but heavy

losses of livestock. This early research was mainly in the form of

surveys of the nitrate content of plants and reports of responses of

animals when they were dosed with nitrate or nitrite salts. Recently

more physiological and biochemical aspects have been investigated.

Molybdenum toxicity has been considered a problem involving copper

and the sulfate ion with the molybdenum somehow making the copper un-

available to the tissues. Copper supplementation overcame the toxicity.

Sulfate will decrease the molybdenum in the blood and tissues and in-

crease its excretion in the urine. Under conditions of high molybdenum

and high sulfate intake, the blood copper will increase rapidly and copper

deficiency symptoms will be apparent (Dick, 1956). Skipper (1951) fed







young dairy bull calves 100 and 200 ppm of molybdenum per day for five

months and Cox et al. (1960) fed up to 400 ppm of molybdenum to bull

calves for three to six months in the feedlot without any toxic effects.

These experiments suggested that some factor or factors may be present

in pasture that were not present in the hay and concentrate fed these

animals and that the toxicity was not due to high molybdenum levels

per se.

The present investigation concerned the possibility that high

levels of nitrate in forages were involved in the molybdenum-copper

relationship. About the time these experiments were culminated, Tillman

et al. (1965) at Oklahoma reported that purified diet to which 1 ppm of

molybdenum had been added, reduced nitrate at a faster rate than the

same purified diet to which no molybdenum had been added. The molybdenum

content of the control ration was not reported.

The present research indicated that rumen bacteria reduced nitrate

at a maximum rate with 0.6 ppm of molybdenum. The minimum molybdenum

requirement cannot be more accurately estimated from the results of this

study. The purified diets used by the Oklahoma workers may be presumed

to have contained less than 0.6 ppm of molybdenum unless the molybdenum

used in their diets was less available to the rumen bacteria than was the

molybdenum in the millet fed in the present experiments. The lack of

sensitivity to molybdenum by cattle in the studies of Skipper (1951) and

Cox et al. (1960) suggested that inorganic supplemental molybdenum did

not act in the rumen like molybdenum naturally occurring in forage.

Recent data published by Cook et al. (1966) indicated that there was

very little difference in availability of the two types of molybdenum.








The nitrate reducing enzyme system did not respond to added molyb-

denum when rumen contents were incubated in vitro. Neither did the

activity of the enzyme system decrease when tungsten was added. The

failure of tungsten to interfere with molybdenum by replacing the latter

in nitrate reductase did not concur with reports that have established

tungsten as a molybdenum antagonist (Higgins et al., 1956; Ramakrishna

Karup and Vaidyanathan, 1963). In the present study it is possible that

the nitrate reductase was present in the bacterial cell and was not

synthesized to any appreciable extent during the four-hour preliminary

fermentation period. This prior formation of nitrate reductase may have

prevented the tungsten from effectively competing with molybdenum for

incorporation into the enzyme. Prior formation of the enzyme would also

explain the lack of effect on the rate of nitrate reduction when molyb-

denum was added to the in vitro system in amounts as high as 50 ppm.

However, if nitrate reductase is an induced enzyme in rumen bacteria

as it is in higher plants (Tang and Wu, 1959; Hewitt and Afridi, 1959),

and E. coli and Neurospora (Nicholas, 1959), it is difficult to explain

why the enzyme should have been present in maximal amounts before the

addition of nitrate.

The forages high in molybdenum decreased the total carbon dioxide

and increased the acetic:butyric acid ratios in the rumen contents but

the relationship of these observations to the nitrate toxicity problem

is not apparent.

The present research demonstrated that a nitrate drench compared

to an equal amount of nitrate mixed in the feed produced more drastic

effects on the same sheep consuming identical diets under a similar

system of management.









The difficulty of growing experimental forages containing high

levels of nitrate has been reported (Davison et al., 1965). The nitrate

content of the millet grown for experimental purposes in 1964 and 1965

was much lower than the nitrate content of the millet grown at the Dairy

Research Unit for routine feeding of the dairy herd. In future attempts

to raise high nitrate forage, the author would recommend using much

higher levels of nitrogen fertilizer with a minimum amount of irrigation.

This work suggests that the activity of nitrate reductase should be

determined on rumen contents from cattle or sheep grazing forages con-

taining different levels of nitrate, copper and molybdenum. Such a study

would demonstrate how nitrate reductase activity was influenced by these

three factors.

It would also be of interest to find the exact molybdenum require-

ment of cattle for maximum levels of nitrate reductase. This might be

done using purified diets such as those used by Sheriha et al. (1962).

In vitro studies using washed bacteria would also be of use in this type

of determination.

The present study has demonstrated that there is little likelihood

that a deficiency or an excess of molybdenum would be a practical factor

in nitrate toxicity insofar as nitrate reduction to nitrite in the rumen

is concerned. However, the present study did not demonstrate the impor-

tance of copper in nitrate toxicity. If the copper were too low or un-

available for the normal activity of nitrite reductase, a lack of copper

would be of great significance as nitrite might accumulate to toxic

levels. It may be that this accounts for the observed benefit of inor-

ganic copper supplementation in salt mixtures when cattle graze pastures





66



which contain apparently adequate levels of copper. The author believes

that further work relating to nitrate toxicity should be directed more

toward the role of copper than that of molybdenum.














CHAPTER IX


SUMMARY


Molybdenum has been established as a component of nitrate reductase

and copper-functions in nitrite reductase activity. Since molybdenum

toxicity symptoms can be alleviated with copper supplementation, it was

considered that nitrate toxicity may have contributed to the molybdenosis

symptoms. The purpose of this investigation was to determine if different

levels of molybdenum and copper in forages were practical factors in the

rate of nitrate disappearance from the rumen.

Millet was raised during 1964 and 1965 with high levels of nitrogen

fertilization with and without supplemental molybdenum and copper. Sodium

molybdate added at the rate of 0.9 kg. per acre increased the molybdenum

content of forage from an average of 0.8 ppm in the control forage to 4

to 27 ppm. Copper sulfate applied at the rate of 11.35 kg. per acre

resulted in copper levels of 10 to 11 ppm, except in a few samples which

had surface contamination. Copper values on the unsupplemented forage

ranged from 4 to 13 ppm.

Levels of forage molybdenum ranging from 0.6 ppm to 27 ppm had no

consistent effect on the rate of nitrate reduction in the rumen of sheep

and cattle, indicating that rumen microorganisms have an effective nitrate
. . . m -

reducing enzyme system with as little as 0.6 ppm molybdenum The high

levels of molybdenum in the forage significantly decreased consumption








of the millet. There were increased acetic:butyric acid ratios and

decreased carbon dioxide levels in the rumen contents of the steers fed

the high molydenum millet. The treatments had no significant effect on

pH or ammonia concentration in rumen fluid. There were also no treat-

ment differences in the ammonia or methemoglobin levels in the blood.

Since additional molybdenum and copper in forage had no consistent

effect on the rate of nitrate disappearance from the rumen, it can be

concluded that the rumen nitrate reducing system does not depend on high

levels of these two elements to function.

In vitro studies indicated that added molybdenum, tungsten or

copper had no consistent effect on the rate of nitrate or nitrite reduc-

tion. These studies supported the in vivo studies in that the nitrate

reducing system seemed to be relatively insensitive to additions of

copper and molybdenum.

Sheep that consumed a purified diet were drenched with 16.8 gm.

nitrate daily and the levels of methemoglobin were determined for as

long as 15 hours after drenching. Although the sheep had consumed 16.8

gm. nitrate per day for two months preceding the drenching with no ill

effects, when they were drenched with nitrate, methemoglobin rose to

high levels and some of the sheep succumbed. The peak levels of met-

hemoglobin generally rose from the first to the fifth day which indicated

that there was a gradual loss of efficiency of the nitrate reducing

system.

Water samples obtained from wells and rivers in Central Florida

were analyzed for nitrate, nitrite and chloride content. The sample from

one of the wells at Belle Glade and the Hillsborough River sample were





69



relatively high in nitrates but the rest of the samples contained only

low levels of nitrate. Two samples from Belle Glade contained rela-

tively high levels of nitrite which would have been potentially dangerous

if used as a source of drinking water for livestock. Chloride levels

were quite high in a few cases but most of the samples were low in

chloride.





































APPENDIX














TABLE 16

SUMMARY OF ANALYSIS OF VARIANCE OF SLOPES OF AMMONIA
CONCENTRATION IN RUMEN OF SHEEP, 1964


Average Slopes


Treatment A

-1.0308

Source


Treatment B

2.7990


d.f.


Among Treatments

A,B vs C,D

Residual


Error


Total


Treatment C

3.3888


M.S.

1.75

5.18

0.08

0.98


Treatment D

-0.6644

Sig.


1.79

5.30

0.04
























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TABLE 21
SUMMARY OF ANALYSIS OF VARIANCE OF NITRATE FOUND IN RUMEN OF


STEERS, 1965


Sources

Animals

Treatment

Period

Error

Total


Nitrate Found in Rumen at 4 Hours

d.f. M.S. F

3 14 0.04

3 23 0.07

3 1043 3.17

6 329

15


Nitrate Found


Sources

Animals

Treatment

Period

Error

Total


in Rumen at 4 Hours per gm.
d.f. M.S.

3 10


11

16

3.33


Nitrate Fed
F

3.0

3.3

4.8


Slope of Curve of Nitrate Concentration Against Time After Feeding

Sources d.f. M.S. F. Sig

Animals 3 17.4 1.01 NS

Treatment 3 30.6 1.77 NS

Period 3 0.2 0.01 NS

Error 6 17.2

Total 15


NSi

NS

NS
NS


Sig
NS

NS

5%









TABLE 22

AVERAGED RUMEN AMMONIA LEVELS BETWEEN ONE AND TWO HOURS AFTER FEEDING

Sources d.f. M.S. F Sig.

Animals 3 9.33 0.95 NS

Treatment 3 9.53 0.97 NS

Period 3 57.20 5.83 5%

Error 6 9.80

Total 15




TABLE 23
SUMMARY OF ANALYSIS OF VARIANCE OF pH FOUND IN RUMEN FLUID OF STEERS, 1965

Average pH of Rumen Contents Between 1-8 Hours After Feeding
Sources d.f. M.S. F Sig.

Animals 3 0.083 2.52 NS

Treatment 3 0.027 0.82 NS

Period 3 0.077 2.33 NS

Error 6 0.033

Total 15


Average pH of Rumen Contents Between 2-8 Hours After Feeding
Sources d.f. M.S. F Sig.
Animals 3 0.09 2.16 NS

Treatment 3 0.043 1.00 NS

Period 3 0.10 2.32 NS

Error 6 0.042

Total 15













TABLE 24

SUMMARY OF ANALYSIS OF VARIANCE OF TOTAL CARBON DIOXIDE
FOUND IN RUMEN FLUID OF STEERS, 1965


Total Carbon Dioxide

Sources

Animals

Treatment

AB vs CD

Residual

Period

Error

Total


Total Carbon Dioxide
consumed) in Steers,

Sources

Animals

Treatment

AB vs CD

Residual

Period

Error

Total


(mMol. CO2/liter rumen fluid) in Steers, 1965

d.f. M.S. F Sig.

3 187. 5.23 5%

3 191. 5.34 5%

1 448. 12.51 5%

2 63. 1.76 NS

3 149. 4.17 NS

6 35.8

15


(mMol. CO2/liter rumen fluid per kg. millet
1965

d.f. M.S. F Sig.

3 803. 4.0 NS

3 414. 2.08 NS


305.

468

218.

199.


1.54

2.35

1.10


NS

NS

NS


]




83






TABLE 25

SUMMARY OF ANALYSIS OF VARIANCE OF TOTAL VOLATILE FATTY ACID
CONCENTRATION AND RATIOS IN THE RUMEN FLUID OF STEERS, 1965


The Total Volatile Fatty Acid Concentration (m equiv./L. rumen fluid)
in Steers

Sources d.f. M.S. F Sig.

Animals 3 464 2.91 NS
Treatment 3 355 2.23 NS
Period 3 248 1.55 NS
Error 6 159
Total 15


The Acetic:Propionic Acids Ratio in Steers

Sources d.f. M.S. F Sig.
Animals 3 1.54 25.67 1%
Treatment 3 0.07 1.08 NS
Period 3 0.01 0.15 NS
Error 6 0.065
Total 15


The Propionic:Butyric Acids Ratio in Steers

Sources d.f. M.S. F Sig.
Animals 3 0.56 0.187 NS
Treatment 3 0.28 0.094 NS
Period 3 0.10 0.033 NS
Error 6 2.99
Total 15


The Acetic:Butyric Acids Ratio in Steers

Sources d.f. M.S. F Sig.
Animals 3 3.14 2.91 NS
Treatment 3 6.42 5.94 5%
AB vs CD 1 10.59 9.80 5%
Residual 2 4.34 4.01 NS
Period 3 0.62 0.57 NS
Error 6 1.08
Total 15





















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