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Effect of cobalt and/or vitamin B12 on performance and metabolic characteristics of lambs fed diets with or without ionophores
Vermeire, Drew Alan, Ph.D.
The Ohio State University, 1991
Copyright ©1991 by Vermeire, Drew Alan. All rights reserved.
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THIS REPRODUCTION IS THE BEST AVAILABLE COPY. EFFECT OF COBALT AND/OR VITAMIN B12 ON PERFORMANCE AND
METABOLIC CHARACTERISTICS OF LAMBS FED DIETS
WITH OR WITHOUT IONOPHORES
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Drew Alan Vermeire, B.S. Agr., M.S.
* * * * *
The Ohio State University
1991
Dissertation Committee: Approved by
J.H. Cline
S.R. Baertsche Q. Adviser B.A. Dehority Department of Animal Science
S .C . Loerch
C.F. Parker
W.J. Tyznik Copyright by Drew Alan Vermeire 1991 To Elaine ACKNOWLEDGMENTS
The following pages contain the names of those persons who have been involved with this research, and to whom I am deeply endebted. I have only been a guest conductor, responsible for the music, but the orchestra and its fine musicians were here before I arrived.
My mentor, Dr. Jack H. Cline has given me the inspiration for this work, countless hours of his time, and has been advisor, father, friend and fellow traveler.
I am deeply grateful for his support and dedication.
Dr. Steve Baertsche, co-advisor, has been a good friend as well as a good "boss". I am thankful for the opportunities that I've had working in Animal Science
Extension under him. The experience has been invaluable.
To the other members of my committee, Drs. Burk
A. Dehority, Steven C. Loerch, Charles F. Parker and
William J. Tyznik, I am grateful. These men have each contributed their time, resources and expertise to help me perform this work. They've made me feel like a partner, rather than a student. I appreciate their faith and confidence in me. I am also grateful to Dr. Donald
E. Becker who provided valuable insight and advice and
iii intended to serve on my committee prior to his leaving the
University.
I would like to thank Dr. Grady W. Chism III and
Dr. John B. Allred, Department of Food Science and
Nutrition, for their help with the lab analyses. Dr.
Chism opened his lab for my use and I couldn't have completed this work without the technical advice and assistance he provided.
I have been blessed by the opportunity to conduct my research at the Eastern Ohio Resource Development Center
(EORDC) at Belle Valley, Ohio. The Late Charlie Boyles, manager, was an inspiration to work with and a blessing to know. Bob McConnell, manager, has maintained the excel lence that Charlie had attained, and I'm thankful to Bob for his willingness to work together to meet the chal lenges which this research has presented. Jim Wells, Jim
Murphy, Raymond Wheeler, Glen Hague, Lester Smithberger and Dave Biedenbach comprise the rest of the farm crew at
EORDC. They work as a family and I'm thankful to each of them for adopting me. EORDC has been a second home over the last four years and I cannot repay these men for the innumerable times they've gone "above and beyond" the call of duty.
I would also like to thank the following people from the University: Ken McClure, OARDC, for use of facili ties, technical advice, and the opportunity to attend the
iv NC-111 technical committee meetings in 1985 and 1986;
Sharon Evans, Becky Johnston, Linda Price and Joyce
Knauerhase for their fine secretarial talents which were almost daily tested keeping track of me; Dan Cottle,
OARDC, for taking care of my sheep at Wooster; Fay E.
Smith, Patricia A. Tirabasso, Anthony P. Griffo, Jr. and
Peggy A. Hiltner, all of OARDC, and Dr. Tom Jenkins,
Clemsen University, for help with the laboratory analyses;
Frederick S. Ruland for help "above and beyond" the call of duty with the statistical analysis of my data;
Dr. James R. Holman, OARDC, for help setting up the statistical models and analysis; Mr. Jack Bardall, OARDC, for his help with the diets; Drs. Donald M. Rings, Peter
D. Constable, William D. Hueston, Garnard J. Boner,
Timothy D. Woodward and William F. Pope for help, technical assistance and information in collecting the biological samples; Cathy Palatinus, Dan Tonne, Ed
Kennedy, Shawn Thayer, Lorene Wurtz, and my fellow graduate students for help and support.
I have had the opportunity to meet many people from the agricultural community and I am thankful to them for their support and help with my research. To many of them,
I'm sure my request for products seemed insignificant, but
I greatly appreciate their generosity and goodwill. The following are industry people to whom I am grateful. I offer my thanks to them and the companies they represent:
v Drs. George Gunderson and Bill Rupe, Wellcome Research
Laboratories; Drs. Robert L. Stuart, Ted Frye, and L.
Arnold Petersen, Hoffmann-La Roche, Inc.; Drs. Emerson
Potter and Lee Watkins and Mr. Dwight Youngkin, Lilly
Research Laboratories; Mr. Scott Remington, Agri-Labs
Med-Tech, Inc.; Dr. Larry Chase, Cornell University;
Ms. Nancy Cook and Mr. Dennis Miller, Rocco Further
Processing, Inc.; Mr. Dale Flowers, Muskingum Livestock
Sales; Mr. John Niver, Countrymark, Inc.; Drs. Jack Taylor
and Jack Caldwell, US Food and Drug Administration,
Dr. William Thomson, Mr. Jerry Hill, Cindy Lyons, Linda
Thompson, Brenda Sager, Jim Leach, Ethyl Mark, and Aaron
Jones, Ross Laboratories division of Abbott Laboratories;
and Drs. Alitar and Vossburgh, USDA, Timberville, VA.
To my collegues with Allegro Feed Company, Inc., I
appreciate your support and enthusiasm. Your humorous
view of the world has made my task seem less difficult and
I thank all of you.
An endeavor of this magnitude would be impossible without the support, understanding and help of my family.
The sense of accomplishment and joy of completing this
degree is shared with them, for I couldn't have done it
alone. My mother, Susan D. Peters and husband Bill, my
dad, Dr. David A. Vermeire and wife Diane, and my in-laws,
Paul and Sally Dove, have been behind me and I'm moved by
the interest they have shown in my research and career. I am not permitted the space it would require to thank each
of you for the love, help and support you've each given me. You know how much you've helped me, and I hope you
realize how very much you mean to me and how much I
appreciate the help.
Finally, I thank my loving wife, Elaine and our daughter Bonnie. Elaine has been my number one fan, lab assistant, research farm helper, co-pilot, companion, constructive critic, and "buddy". Bonnie has allowed me to see the world in color, instead of just black and white. Both have made many personal sacrifices to enable me to obtain this degree, the least of which has been long, sleepless nights in the lab, and long nights alone while I've been in the lab or office. I am blessed to have them and eternally grateful for their love and support.
vii VITA
September 18, 1958..... Born-Sharon, Pennsylvania
September 1964 ...... Family moved to Kansas City, Kansas
June 1981...... B.S. Agriculture The Ohio State University Columbus, Ohio
March 1985...... M.S. Animal Science The Ohio State University Columbus, Ohio
October 1984-July 1988. Graduate Teaching Asso ciate, Graduate Research Associate. Department of Animal Science-Extension. The Ohio State University Columbus, Ohio
July 1988-December 1989 Nutritionist. Indiana Farm Bureau Cooperative Association, Inc. Indianapolis, Indiana.
January 1990-Present... Ruminant Nutritionist and President, Allegro Feed Company, Inc. Warsaw, Indiana.
viii PUBLICATIONS
Vermeire, Drew A. 1985. in vitro and in vivo evaluation of municipal tree leaves as a feedstuff for sheep. M.S. Thesis. The Ohio State University, Columbus.
Vermeire, Drew A. 1991. Management of Holstein Calves from Birth to 20 Weeks. Proc. Holstein Beef Prod. Symp. Harrisburg, PA Feb 13-15, NRAES, Cooperative Extension, Ithica NY.
Vermeire, D.A., S.R. Baertsche and J.H. Cline. 1986. Effect of lasalocid on the protein requirements of growing lambs. Sheep Industry Development Research Digest Vol. 3 No. 1 pp. 1-4.
Vermeire, D.A., S.R. Baertsche and J.H. Cline. 1986. Effect of lasalocid on the protein requirements of growing lambs. (Abstr. 134) Am. Soc. Anim. Sci. Mid-western Section DeMoines, Iowa.
Vermeire, D.A., J.H. Cline and S.R. Baertsche. 1990. Effect of cobalt on feedlot performance and propionate clearance of lambs with or without ionophores. J. Anim. Sci. Vol. 68, Supp. 1 (abstract 689)
Vermeire, D.A., W.J. Tyznik and J.H. Cline. 1985. Evaluating tree leaves as a feedstuff for sheep. Ohio Report 70:80
FIELD OF STUDY
Major Field: Animal Science
Studies in Ruminant Nutrition under Dr. Jack H. Cline
and Dr. Stephen R. Baertsche
ix TABLE OF CONTENTS
DEDICATION ...... ii
ACKNOWLEDGMENTS ...... iii
VITA ...... viii
LIST OF T A B L E S ...... xii
LIST OF FIGURES ...... xiv
CHAPTER PAGE
I. INTRODUCTION ...... 1
II. REVIEW OF LITERATURE ...... 7 Outline of review of literature ...... 8 Cobalt and vitamin B12 in animal nutrition . 10 I o n o p h o r e s ...... 58 Research question— hypothesis ...... 76
III. MATERIALS AND M E T H O D S ...... 93
Preliminary Studies 1984-85...... 95 EXPERIMENT 1-Cobalt, Monensin, and Lasalocid Feedlot Study ...... 102 EXPERIMENT 2-Vitamin B12 & Monensin Metabolism Study ...... 110 EXPERIMENTAL METHODS ...... 112
IV. RESULTS AND D I S C U S S I O N ...... 126
Preliminary Studies - 1984-85 130 EXPERIMENT 1 ...... 135 EXPERIMENT 2 ...... 143 General Discussion and Conclusions ...... 146
x APPENDIX ...... 157
FDA New Animal Drug Exemptions ...... 158
LITERATURE CITED ...... 166
xi LIST OF TABLES
1. Summary of metabolic effects of ionophores on the rumen fermentation...... 65
2. Biological effects on monensin in the rumen....66
3. Accepted or probable system modes of action of ionophores...... 67
4. Composition of diet for preliminary study...... 96
5. Treatment assignment for preliminary Study..... 97
6. Composition of creep feed...... 99
7. Composition of grower diet ...... 100
8. Composition of creep feed diet ...... 103
9. Composition of mineral supplement...... 104
10. Composition of concentrate mix ...... 105
11. Dietary Treatment Assignment for feedlot study...... 106
12. Composition of treatment diet ...... 107
13. ANOVA for experiment 1 ...... 109
14. ANOVA for experiment 2 ...... Ill
15. Results for preliminary study ...... 131
16. Effect of cobalt and vitamin B^2 on performance of Lambs...... 133
xii 17. Effect of Cobalt, Monensin and Lasalocid on Performance of Feedlot Lambs...... 136
18. Plasma propionate levels vs time...... 140
19. Effect of Monensin and Vitamin B±2 on Liver Vitamin B12 ...... 143
20. Cobalt analytical results for Experiment 1....149
xiii LIST OF FIGURES
1. Blood plasma propionate levels vs time, 139
xiv CHAPTER I
INTRODUCTION
Perhaps the most exciting chapters in the history of nutrition were those written about the discovery of cobalt and vitamin B12. The discovery of vitamin B12 ended the
"vitamin age" and was a labor of various researchers around the world over a period of one-hundred years. It is exciting, too, because the only known metabolic function of cobalt in mammals is as part of the vitamin
B12 molecule, and a similar vitamin-mineral interaction has yet to be discovered. Another intriguing aspect is that vitamin B12 requires intrinsic factor to be absorbed efficiently. Pernicious anemia is a condition in humans caused by the inability of some individuals to secrete intrinsic factor-thereby greatly reducing the amount of vitamin B12 absorbed from the intestines. The ruminant represents an interesting case, since the ruminant does not appear to have intrinsic factor and rumen microbes synthesize both vitamin B^2 and analogs of B12 (pseudo
B12) which are not biologically active. Rumen microbes may have a metabolic need for cobalt, as well.
1 2
The discoveries of cobalt, vitamin B12, and interrelationships between them brought together animal and human nutrition, agriculture and medicine, and international science in the solution of common problems.
Finally, the role of vitamin B12 mammals has but two known functions: as a coenzyme in methylmalonyl-CoA mutase-the enzyme involved in the conversion of methyl- malonyl-CoA to succinyl-CoA; and secondly, as a coenzyme in N5-Methyltetrahydrofolate homocysteine methyltrans- ferase-the enzyme involved in recycling homocysteine to methionine.
To summarize, the only known metabolic function of cobalt is as a component of vitamin B^2. Absorption of
B ^2 is enhanced by intrinsic factor which the animal secretes in the stomach. Animals which do not produce intrinsic factor have very poor absorption of vitamin B12.
Once absorbed, B^2 has but two known functions as cofactors of specific enzymatic steps in metabolism. To further complicate this, there are numerous B12 analogs produced in the rumen which are not biologically active, but which may compete with true vitamin B12 for absorption or utilization.
The hypothesis of this dissertation is based upon field observations that symptoms and physiological changes of the ruminant during cobalt deficiency are similar to the animal response to ionophore addition to high energy 3 diets. Field observations of reduced feed intake of high concentrate fed dairy cows (lactating), which respond to vitamin B ^2 injection or dietary cobalt additions strongly suggest cobalt availability is at least marginally deficient in nutritionally stressed animals. The mechanisms summarized in the last paragraph provide a logical explanation for the field observations which this research is designed to test.
The discovery of polyether acid ionophoric antibio tics, or ionophores, in 1951 was largely overlooked by the scientific community until discovery in 1968 that monensin was an effective drug against coccidia. This opened a multi-million dollar world-wide market for these drugs and during the 1970's and 80's, volumes of research were published on feeding these drugs to nearly all domestic livestock species. Today, the poultry industry mainly uses ionophores for coccidiosis control.
Also during the 1970's, ionophores were found to have a profound effect on the rumen microbial population.
Research into this area has led to widespread feeding of ionophores to beef cattle for improved feed efficiency and improved rate of gain. The feed intake response to the ionophore is inconsistent in ruminants, however. When the ionophore is added to a diet which has a high energy density (high concentrate), feed intake is usually reduced by 6-10% with no reduction in rate-of-gain. When the drug is added to a diet which has a low energy density (high roughage) , feed intake is unaffected and gain is improved. Both situations result in improved feed efficiency.
This raises an obvious question. Why is there a difference in the animal's feed intake response with the two types of diets when the ionophore is added? This question is the subject of this research. It would seem logical if feed intake depression was due to the ionophore interacting with some physiological or physicochemical receptor, the response would be independent of diet. The ionophore would consistently effect feed intake (either positively or negatively) in a similar manner on both high and low concentrate diets. The response, however, is not consistent as noted above. Even on high concentrate diets there is greater variability. In some research studies, animals fed an ionophore actually eat more than control animals which were not fed the ionophore.
Since feed intake depression generally occurs on high concentrate diets, the mode of action of the ionophore must be examined to explain the observed result. In both dietary situations there is a shift in ruminal volatile fatty acid (VFA) production with less acetate and butyrate being produced and a concomitant increase in propionate production. No net increase or decrease in total VFA production occurs as a result of feeding the ionophore. Total VFA production on high concentrate diets is
significantly greater than total VFA production on high
roughage diets.
The "regulatory enzyme" (biochemical control
mechanisms) in the conversion of propionate to glucose in
liver is propionyl-CoA carboxylase, a biotin-containing
enzyme. Hogue and Elliot (1964) showed that vitamin B12
increased feed intake in rats fed propionate, while biotin
had no effect. Rats fed propionate had reduced feed
intake relative to control rats not fed propionate. This
turns attention to the next enzyme in the conversion of
propionate to glucose, methylmalonyl-CoA mutase. Although
methylmalonyl-CoA mutase is not the "regulatory enzyme" in
the pathway from propionate to glucose, if the dietary
cobalt status were marginal cr deficient, conversion of methylmalonyl-CoA to succinyl-CoA would be the rate-
limiting step. Increased production of ruminal propionic
acid, which occurs as a result of ionophore addition to high concentrate diets, may exceed the liver capacity to
convert propionate to glucose if vitamin B12 is limiting.
Limitation of B12 reduces liver propionate clearance rate by retarding conversion of methylmanonyl Co A to succinyl
Co A. Increased blood propionate results from delayed propionate clearance rate, and feed intake is reduced.
This is the normal sequence of events in uncomplicated cobalt deficiency in ruminants (Marston et al. 1972). This research will investigate the response of growing lambs to ionophores in all concentrate diet and determine the effect of supplemental cobalt and vitamin
B12 on this response. If the addition of an ionophore to a high concentrate diet increases ruminal propionate production, metabolic need for vitamin B12 should also be increased. If vitamin B i 2 were limiting, the animal response to increased ruminal propionate would be reduced feed intake. Supplemental cobalt and/or vitamin B12 possibly could overcome this feed intake reduction and result in improved rate of gain. This dissertation discusses the research conducted to examine these questions. CHAPTER II
Review of Literature
Before one can fully understand and appreciate the problem which this research addresses, it is imperative that one have a working knowledge of cobalt, vitamin B12 and ionophores as they relate to animal nutrition and metabolism. This review is not intended to delve into every piece of research that has been done on these subjects. Rather, it will examine the historical record and discuss the important findings and major events.
The uniqueness of cobalt deficiency in ruminants, as well as the physiological and metabolic function of the cobalt-containing vitamin B ^ molecule will be discussed.
Manifestations of vitamin B12 deficiency in man and in livestock, and methods of controlling the deficiency will be examined. The discovery, function and usage of iono- phoric antibiotics will be addressed, as well as the effect of these compounds on the animal. Finally, the hypothesis of this research will be examined. Methods employed to test the hypothesis, and other research and evidence which either supports or contradicts the hypo thesis will be discussed.
7 OUTLINE OF REVIEW OF LITERATURE
Cobalt and vitamin B i 2 i-n animal nutrition
1. Cobalt deficiency in ruminants— a historical view (page 10)
2. Uniqueness of cobalt deficiency in ruminants (page 16)
3. Historical view of vitamin B12 (page 19)
4. Synthesis and absorption of vitamin B12 (page 23)
5. Metabolic role of vitamin B i 2 (page 41)
6. Summary-Cobalt/vitamin B12 in animal nutrition (page 56) IONOPHORES
1. Discovery (page 58)
2. Function of ionophores (page 60)
3. Use in ruminant nutrition (page 69)
RESEARCH QUESTION— HYPOTHESIS
1. Observations (page 78)
2. Methodology (page 84)
3. Other Research on this Question (page 90) 10
Cobalt and Vitamin B ^2 in Animal Nutrition
1. Cobalt Deficiency in Ruminants— A Historical View
Dietary deficiency of cobalt is a condition which
occurs uniquely in ruminant animals in many geographic
locations throughout the world. Nonruminants such as
swine and horses flourish, while cattle and sheep, grazing
in the same pasture, waste and die. The condition occurs
in specific geographical areas where the soil is deficient
in cobalt or where availability of cobalt is very low.
In 1933, Filmer published a "preliminary report"
which has become the monumental work in cobalt defi
ciency. This report described the sickness known as
"Denmark wasting disease" as a geographic problem that
could be reversed by moving affected animals to "sound"
country. The disease occurred primarily in younger
livestock although older cattle and sheep were also
affected. The highest incidence of death occurred during
the spring and early summer when pastures were most
luxuriant, and sex or breed of animals had no apparent
link to the disease.
Filmer noted the similarity between wasting disease and "bush sickness" of New Zealand and suggested the term
"enzootic marasmus" to replace wasting disease. Clinical manifestations were described and included progressive 11
emaciation and weakness with rough and long hair coat.
Infestation with lice was common and visible mucous membranes were blanched. Temperature, pulse, and
respiration were reported as normal at rest with pulse and
respiration accelerated on exercising. Cows had reduced milk production or a total suppression of lactation.
Estrous or conception rarely occurred, and abortion was
common between 6 and 8 weeks in cows which did conceive.
Calves were anorexic and often had diarrhea or constipa tion. A higher incidence of internal parasites, especially Ostertaqia nodules, Nemetodirus sp., Moniezia sp., and Chabertia ovina was observed in affected animals.
The descriptions of other metabolic changes such as histological and hemological changes were landmark as well. Fatty changes in the liver occurred to a varying degree in all affected animals. Red blood cell count was always low in affected animals. Anemia associated with wasting disease was described as hypochromic normocytic in lambs and probably a hypochromic microcytic anemia in calves.
In 1931, the Department of Agriculture leased an unsound farm for research. Filmer reported only one heifer had been raised to lactation since stocking the farm in June 1926, and several cows had died of marasmus.
Experiments on the farm failed to establish a cure. Perhaps the greatest contribution of Filmer's "Pre
liminary Report" was his insight and interpretation of
results. He recognized that wasting disease was the same
condition reported from around the world as "bush sick
ness", "nakuruitis", "pine", and "salt sick". He sug
gested all of these conditions be termed "enzootic marasmus" and detailed the metabolic changes associated with the disease. Most importantly, he recognized this
condition was not simply an iron deficiency, as had been
reported. He concluded some other mineral was responsible
for enzootic marasmus. He hypothesized the mineral
involved was a contaminant in iron sources which cured the disease, while this mineral was lacking in iron sources which failed to cure it. In his studies, slight amounts of iron cured the disease, while several times the estimated requirement failed to cure it when supplemented with iron of a different source. Filmer recognized similarity of marasmus to pernicious anemia in humans, and hypothesized the role of the unknown mineral in iron metabolism.
Within two years of Filmer's "preliminary report", enzootic marasmus was shown to be curable by feeding minute amounts of cobalt. This was reported simul taneously by Filmer and Underwood in Western Australia, and Marston and Lines in Southern Australia. Filmer and
Underwood (1934) showed iron was not the curative agent in 13
limonite since iron-free acid extract had curative
properties, while iron-rich residue did not. Secondly,
the initial acid extract had curative properties, but
subsequent extractions (containing equal amounts of iron)
lacked curative properties. This was demonstrated with both calves and lambs.
Underwood (1934) compared iron content of liver, kidney, and spleen for animals affected with enzootic marasmus and those not affected. Mean iron content in
liver of affected animals was considerably higher than mean values reported for normal animals. A similar pattern existed for spleen and kidney. This provided
further evidence that the metabolic problem associated with enzootic marasmus was not a deficiency of iron, but an inability to properly metabolize iron. Mean concentra tion in liver, spleen, and kidney was five, three, and
five times greater in affected lambs than normal lambs, respectively. Iron content of these organs were elevated
in the calf as well.
The culmination of Filmer and Underwood's work came with discovery that the biologically active component of limonite was cobalt (Underwood and Filmer, 1935). Their experiments showed the active compound was not iron.
Limonite extracts were fed to lambs and two control groups of lambs were also used; one which received no treatment and one which received limonite. Surprisingly, lambs fed iron or zinc, limonite, or extracts of limonite had similar results. Systematic testing of these elements from numerous sources, alone and in combination showed the active element was cobalt. Underwood and Filmer, however, acknowledge in November, 1934, personal communication from
H.R. Marston indicated cobalt was being used to cure
"coast disease" in experimental animals in South Aust ralia. Underwood and Filmer credit the workers at the
Animal Nutrition Division with being the first to use cobalt to treat a disease in domesticated animals.
Marston (1935) described "coast disease" of South
Australia with respect to geographical and soil condi tions, animal characteristics, etc., but was not nearly as detailed as the paper by Filmer discussed above. Marston fed lambs a salt mix containing manganese, zinc, cobalt and nickel in addition to "those elements usually considered necessary for higher animals". Control lambs were fed white salt instead of the mix. Both groups were kept on pastures known to produce the disease, but the group fed the salt mixture failed to develop the disease.
Further investigation under laboratory conditions in which lambs were fed forage from the "unsound" areas showed that cobalt cured the disease. Marston noted similarities and differences between "coast disease" and "bush sickness" of
New Zealand, "enzootic marasmus" of Western Australia,
"nakuruitis" of Kenya, "pine" of the Inner Hebrides" and 15
"salt sickness" of Florida. In some cases, there were
obvious geological differences between "sound" and
"unsound" areas, but in other cases, there were no easily
determined differences. Differences in pathology of the
diseases, were most likely due to confounding conditions
such as copper deficiency, rather than simple cobalt
deficiency.
A companion paper to Marston, above, was a prelim
inary note by Lines (1935) of the effect of minute
quantities of cobalt on sheep. This provided further
evidence "coast disease" was a dietary deficiency of
cobalt.
During the next 10-15 years, research from around the world demonstrated cobalt deficiency occurred in areas where soil was low in cobalt and could be cured by feeding supplemental cobalt or by applying cobalt to soil. 16
2. Uniqueness of cobalt deficiency in ruminants.
As was mentioned in the preceding section, Filmer
(193 3) made the keen observation that cattle and sheep were very susceptible to marasmus, horses were never affected and there was some doubt in regard to pigs.
Hastings (194 8) reiterated that dietary cobalt appeared to be required only by ruminants, but not by non-ruminants, rabbits or rats.
Though the mode of action of cobalt remained obscure, it was hypothesized that it was necessary for the rumen microorganisms. This was first suggested by McCance and
Widdowson (1944).
Gall et. al. (1949) compared bacterial numbers and types from stomach-tube samples from sheep receiving cobalt in the diet, cobalt injections, no cobalt, or fed cobalt but with restricted feed intake. The cobalt deficient sheep and sheep injected with cobalt had 30.2 and 30.7 billion bacteria per gram of rumen contents, respectively. This was far less than the sheep fed cobalt, and the sheep fed cobalt with restricted feed intake, which, respectively, had 54.6 and 56.3 billion bacteria per gram of rumen contents. This demonstrated that cobalt was only effective when fed, that cobalt was a necessary component for rumen bacteria, and reduction in bacterial numbers was not a function of feed intake. 17
The discovery by Rickes, et al. (1948) that vitamin
B12 contains cobalt resulted in research efforts to
determine if vitamin B12 was, in fact, the necessary
component for ruminants as well as non-ruminants. While
early studies, such as Becker et al. (1949) and Becker and
Smith (1951) concluded the necessary component was not
vitamin B12, S.E. Smith (1952) quickly reversed this
position and explained the failure of the initial studies
as having used too little vitamin B^2 to elicit a
response. This was confirmed when the liver fraction
responsible for remission of cobalt-deficiency symptoms
was found to be a concentrated source of vitamin B^2.
By the mid-1950s, a considerable volume of informa
tion was available on cobalt and vitamin B^2. Smith and
Loosli (1957) reviewed cobalt and vitamin B^2 in ruminant
nutrition and summarized the results of many research
studies. Since metabolic aspects of vitamin B12 are
covered in a later section of this literature review, only
the findings which relate to this section will be covered
at this point.
In addition to the need for cobalt or cobalt-contain
ing compounds by rumen microorganisms, it was clear the
ruminant has a metabolic need for vitamin B^2.
Presumably, rumen bacteria which synthesize B^2 have a metabolic need for cobalt (Gall et al., 1949). In
addition to true vitamin B^2, rumen microbes also syn 18 thesize compounds which are similar to Bi2, termed pseudo-vitamin B12 (or anti-vitamin B12)> which are not biologically active. Smith and Loosli (1957) cited work by Ford and Hutner (1955) which suggested 90% of vitamin
B]_2 in rumen contents is pseudo-vitamin B12, and only 10% true B^2 .
Absorption of vitamin B12 in sheep is inefficient.
Kercher and Smith (1955) concluded 3% of orally administered vitamin B12 was absorbed by lambs, since it required 35 times the Bi2 orally as was required by parenteral dose to alleviate deficiency symptoms.
The explanation for dietary requirement of cobalt by ruminants, but not by non-ruminants, is a combination of metabolic requirement of cobalt or cobalt-containing com pounds by ruminal microbes, poor absorption of vitamin B12 by ruminants, and inefficient synthesis of vitamin B^2 by rumen microbes. As will be later shown, swine, man, and other non-ruminants possess intrinsic factor which greatly enhances efficiency of absorption of B^2. Some species, such as the horse, practice coprophagy which allows for recycling of vitamin B12. 19
3. Historical view of vitamin B12
Although vitamin B12 was discovered in 1947, its role
in animal nutrition was not immediately recognized.
Pernicious anemia was believed to be a condition unigue to
humans. Research with vitamin B12 in humans and with
cobalt in ruminants was convergent, but was conducted
independently of each other. Castle (1975) provides a
fascinating historical account of vitamin B^2 from Combe's
earliest observations of pernicious anemia in 1824 through
biogenesis and synthesis of vitamin B^2 in the 1960's. To
briefly summarize this account, earliest observations were
of the human condition called pernicious anemia which was
usually fatal. This condition was recognized by workers
in the United States and Europe and thought to be in part
due to atrophy of secretory glands in the stomach. Many
cases were later observed in people who lack hydrochloric
acid in the stomach.
In the early 1900's, the concept of nutritional
deficiency became established and many human conditions were routinely treated with meat and liver. In pernicious
anemia, treatment with liver often gave favorable
results. Experiments were conducted to determine the active component of liver by workers throughout the world, and liver extracts became commercially available to treat pernicious anemia. It is interesting to note the 20
Norwegian workers LeLand and Klein purified vitamin B12 in
193 6 but were interrupted by the German invasion and World
War II. Nineteen years passed before their work was re-discovered. In the meantime, however, Vitamin B i 2 hac* been discovered by other workers elsewhere in the world.
Merck & Co., Inc. is credited with discovery of vitamin B12 in 1947 (Merck & Co., Inc, 1957). Working independently, E.L. Smith isolated B^2 in England (E.L.
Smith, 1952). By 1957, volumes of information were available on vitamin B12 in nutrition of man and animals.
Howe (1958) reported improvements in numerous parameters in growth-retarded children which were both deficient or apparently adequate in vitamin B12. Merck & Co.,
Inc. (1957) published a service bulletin which provided information on the role of vitamin B12 in the nutrition of animals, a history of vitamin B^2, product information, and the structure of the newly-discovered vitamin.
Work by Becker and Smith (1951) showed liver extract was effective in alleviating the symptoms of cobalt deficiency in lambs when injected, but not when fed. S.E.
Smith et al. (1951) found the liver extract with highest potency for curing cobalt deficiency symptoms was also high in vitamin B12. When given at higher levels, vitamin b12 injections were shown to cure symptoms of cobalt deficiency, and it was concluded vitamin Bi2 played an important intermediate role in cobalt metabolism. It was 21
later shown that many rumen bacteria require cobalt, while
some bacteria, all protozoa and mammals require vitamin
b12 • With elucidation of the structure of vitamin B ^ anc*
its analogs, researchers tested several forms for
biological activity. Vitamin B^2 (cyanocobalamin) and
B12b (hydroxocobalamin) were found to be equal in biological activity in sheep (Koch and Smith, 1951), and
in humans with pernicious anemia (Lichtman et al. cited by
Ellenbogen 1984). It was later recognized that the cyano-
form of B12 was not the naturally occurring form, but was a result of the isolation procedure. The cyano- form is quite stable, however, and cyanide is used in extraction of vitamin B^2 from biological matrices both to extract and to stabilize the vitamin (The Society for Analytical
Chemistry, 1956). The two known coenzyme forms of vitamin
B12 in mammals are methylcobalamin and adenosylcobalamin.
Cyanocobalamin is usually used to supplement the vitamin, and most research studies utilize cyanocobalamin to study the vitamin as well.
The metabolic requirement of some bacteria for vitamin B12-like compounds had been exploited to assay the vitamin (Skeggs, 1963; Guttman, 1963; Voigt and Eitenmil- ler, 1985; Association of Vitamin Chemists, Inc. 1966;
Chin, 1985; Ellenbogen, 1984). Bacteria which synthesize vitamin B12 apparently require cobalt and it is not 22
unreasonable that other microbes require cobalt or
cobalt-containing compounds as well. Durand and Kawashima
(1980) discuss both positive and negative effects of
cobalt on the rumen microbial system. Latteur (1962)
emphasizes the requirement of rumen protozoa for vitamin
B12 an(* that a regression of the microfauna, as evidenced by microscopic examination of rumen contents, is helpful
in early detection of sub-optimal cobalt intake.
Studies by MacPherson et al. (1976) suggest an
involvement of cobalt or vitamin B^2 in metabolism of thiamin and ascorbic acid. Their work suggests bacteria which synthesize thiamine also have a requirement for cobalt or vitamin B12•
Young (1979) discusses metabolic need and intolerance of bacteria to cobalt. Interaction of cobalt deficiency and susceptibility to Johnes disease or paratuberculosis in cattle is discussed by Latteur (1962).
Vitamin B i 2 sub-deficiencies discussed by Howe (1958) in man and Latteur (1962) in ruminants, as well as claimed benefits of vitamin B12 by authors in the popular press suggest benefits and effects of vitamin B^2 are yet to be fully documented.
The reader is directed to Ellenbogen in Handbook of
Vitamins edited by Machlin (1984) or the book Cobalamin: fiigghgmjstry and Pathophysiology edited by Babior (1975) for further information about vitamin B12. 23
4. Synthesis and absorption of vitamin B12
It is not the intent of this section to delve into
biosynthetic pathways by which bacteria produce
cobalamins. The reader is referred to Freidmann (1975)
for detailed information on biosynthesis of corrinoids and
other vitamin B12-like compounds.
This section will provide an up to date review of the
knowledge of synthesis of vitamin B12 as it applies to
ruminant nutrition. Specifically, those studies which
demonstrate differences in amount of ruminal B i 2 production on various diets, cobalt levels, etc. will be
examined. Studies in which absorption of vitamin B12 was determined will also be discussed.
Advances in understanding of production, absorption, utilization and excretion of vitamin B12 in sheep and cattle occurred primarily in the 1960's and 1970's. Gall et al. (1949) had reported the bacterial population in the rumen was greatly reduced in cobalt deficient lambs and cobalt-deficient lambs which had been given cobalt by
injection. Lambs fed cobalt or fed cobalt on restricted
feed intake had approximately 55 billion bacterial cells, while cobalt-deficient lambs had approximately 30 billion bacterial cells per gram of rumen contents. This demonstrated the profound effect of cobalt on the ruminal population, but with discovery of vitamin Bi2/cobalt 24 interaction, further work was not conducted. Smith and
Loosli (1957) lamented further studies had not been conducted in this area.
Ruminal synthesis of vitamin B12 was determined by
Hale et al. (1950) using fistulated lambs and chick assay method. Intake of cobalt in two lambs was reported to be approximately 0.03 mg and 1.7 mg. per day for deficient and supplemented lambs, respectively. They demonstrated ruminal vitamin B12 synthesis was limited in cobalt deficiency. Vitamin B^2 in rumen contents of a cobalt-supplemented lamb contained a minimum of 60 micrograms of vitamin B^2 per 100 gm. dried rumen contents. They presented three possible explanations for the role of cobalt and vitamin B12 in sheep. First, vitamin B12 is required by sheep and B i 2 synthesis is insufficient when cobalt is lacking in the diet.
Secondly, they proposed that certain rumen microbes require vitamin B12 and cannot utilize cobalt. Their final hypothesis was vitamin B12 is essential for formation of some unknown factor(s) which is essential for s h eep.
Rothery et al. (1953) fed radioactive cobalt (Co60) to deficient sheep for 3-4 months to study distribution of the labelled cobalt in sheep. Data from a 10 day balance trial indicated that 6-8 % of ingested cobalt was excreted in urine. The authors reported highest concentration of 25
cobalt was found in liver, and muscle contained an amount
of cobalt equal to liver, at a low concentration but a
large proportion of body mass. Their data also showed
small intestine was higher in cobalt concentration than any other section of gastrointestinal tract.
Kercher and Smith (1956) found the total content of vitamin B^2 was significantly higher in liver, rumen-
-reticulum, omasum-abomasum, duodenum-jejunum, ileum, cecum, large intestine, kidney, and pancreas of sheep fed cobalt than sheep which were cobalt-deficient. Vitamin
B12 content of cecum and large intestine was similar for cobalt-fed sheep and sheep which had been given cobalt by injection, however. This suggested that injected cobalt is excreted in bile, and vitamin B^2 synthesis may also occur in cecum and large intestine, rather than rumen only. This also suggests vitamin B12 is not absorbed in the hind-gut, since cobalt-injected sheep showed no signs of remission of deficiency symptoms.
In two experiments with sows, Frederick and Brisson
(1959) showed serum vitamin B12 levels were increased with increasing oral doses of B12 up to 100 micrograms per day and increased at a reduced rate up to 200 micrograms per day. Sows in the study had been fed a diet deficient in vitamin B i 2 for two years prior to the experiment. Their data suggested a dietary requirement of 200-300 micrograms
B12 per day for sows to maintain serum B^2 levels. Tissue vitamin B^2 concentration has been used as an indication of B12 synthesis and absorption in ruminants.
Johnson and Bentley (1958) discussed several studies by different workers which showed increased B12 content in liver, kidney, blood plasma and dried feces when cobalt was supplemented. Work was discussed which showed an increased concentration of B12 1° r\ra®n juice with cobalt supplementation of mature timothy hay. They cite work in which in vitro synthesis of B ^2 was increased by cobalt addition. An excellent discussion of Bi2-like substances synthesized by rumen bacteria in vitro and in vivo is also included. This discussion suggests liver contains approximately 75% B^2 and B12b and 25% other B12-like compounds. Approximately 50% and 52% of the B12-like compounds in rumen liquor and feces, respectively, of sheep were found to be B12 and B12b. These two forms contain 5,6-Dimethylbenzimidazole base, while the pseudo
B ^ 2 s contain adenine, 2-methyladenine, hypoxanthine,
2-methylhypoxanthine, or no nucleotide portion. Vitamin
B12 was later identified as cyanocobalamin, and Bi2b as hydroxocobalamin.
Dryden (1962) used pure cultures of 48 strains of rumen bacteria to determine synthesis of vitamin B12 using an £. coli mutant as an assay organism. Twenty-one species of rumen bacteria were represented in this study and it was concluded synthesis of B12 was primarily due to 27
8 strains which represented only 4 species of rumen
bacteria. Butvrivibrio fibrisolvens. Peotostreptococcus
elsdsnii, gelsnQmgna? ruminantiuro. and an unnamed group
B385-1 were the four species primarily responsible for vitamin B12 production. Assay with Q. malhamensis
revealed these species did not synthesize a large proportion of true vitamin Bi2 compared to analogs.
Effect of cobalt intake on plasma vitamin B^2 concentration was studied by Somers and Gawthorne (1969).
Diets contained 0.04, 0.06, 0.10, 0.18, and 0.34 ppm cobalt which provided 33, 50, 84, 150, and 284 micrograms of cobalt per day, respectively. Two 18-month- old Merino wethers were assigned to each treatment and fed for 39 weeks. Within 3 weeks of the initiation of feeding 0.04 and 0.06 ppm cobalt diets, plasma B^2 concentration had significantly dropped. Sheep fed the 0.10 ppm cobalt diet showed a slight decrease in plasma B12 concentration through the 39 week study. Sheep fed diets containing
0.18 or 0.34 ppm cobalt had an increase in plasma B i 2 level within 2 weeks, but had considerable variation in concentration over the remaining 37 weeks of the experiment. Sheep fed the two lowest cobalt levels had very little variation between sheep or over time throughout the experiment. During the 35th week of this experiment, diurnal plasma B12 concentration was determined for each treatment from blood samples taken at 28
4 hour intervals after feeding. Sheep fed the two lowest
cobalt diets had nearly unchanged plasma B12 levels over a
24 hour period, while sheep fed the other diets had more variable B i 2 concentrations. Sheep fed diets containing the highest levels of cobalt had decreased plasma vitamin
B12 concentration after feeding followed by an increase
and gradual decrease over a 24 hour-period. This study demonstrated plasma concentration of vitamin B^2, as determined by Eualena assay, is dependent upon level of cobalt in the diet and time after feeding in which the sample was taken. Cobalt requirement in sheep is estimated at 0.1 ppm (NRC, 1985).
Hedrich et al. (1973), using three sheep fitted with reentrant intestinal cannulas and rumen cannulas, de veloped multiple regression equations for predicting both production and absorption of vitamin B^2. They determined
B12 flow rates in sheep fitted with ileal and duodenal reentrant cannulas using chromium EDTA as a marker. The experiment was a 3 X 3 Latin Square design and had 3 week adjustment to each diet. Diets contained 0.06, 0.50, and
1.02 ppm cobalt and were pelleted corn/hay based diets.
No information is given about the mineral content of the diets, except calcium, phosphorus and salt. Their data showed that daily production of Bi2 (estimated by both radioisotope and Ochromonas malhamensis) increased significantly when dietary cobalt level was increased from 0.06 to 0.50 ppm. Their data also suggested a relationship between vitamin B-±2 production and cobalt intake, percentage of roughage in the diet, and digestible dry matter intake (R2=0.53). Predicted absorption of vitamin B12 was derived from apparent absorption data obtained from two sheep fitted with reentrant ileal and duodenal cannulas. Digesta flow rate, production and true B^2 vs* analogs all are cited as affecting the absorption of B-±2 and their predictions were reported to agree with previously reported research from other workers.
Rates and efficiencies of production and absorption of vitamin B12, and tissue distribution and excretion of
B12 was investigated by Smith and Marston (1970a). Of particular importance are numerous conclusions drawn by these researchers. Daily requirement for vitamin B12 bY sheep was estimated as about 11 micrograms per day from previous research and should be kept in mind by the reader. A mature ewe was fed a cobalt-deficient diet, but supplemented with 1 mg cobalt/d for seven months. Cobalt supplementation was discontinued, with the last supplement containing Co 60. Approximately 93% of the radioactive cobalt was excreted in feces within the first 5 days, with over 60% excreted within the first 2 days. Over the
14-day collection period, 95.1% of radioactive cobalt was recovered in feces and 2.5% was in urine. Ruminal production of vitamin B12 was determined using several assay methods including Ochromonas malhamensis. Escherichia coli (plate and tube assay), and
Lactobacillus leichmanii. It was found that only 4% of
B12 activity in rumen contents was in the fluid fraction, and even on the assumption all of the residual water in wet residue contained B^2 activity, 90% of activity in the rumen contents could be attributed to fibrous solids. The authors describe treatment methods employed in order to release B^2 from this fraction of rumen contents, and claim a 90% release of vitamin B i 2 using repeated mechanical shaking in McDougall's artificial saliva containing 0.005% KCN at pH 7.
Using a severely vitamin B12-deficient ewe, the authors provided daily oral treatment with 500 micrograms of cyanocobalamin. Response in feed intake and body weight was almost immediate. Feed intake increased from approximately 200 to over 1000 g/d within three weeks.
Positive weight gain continued until vitamin B12 supplementation was discontinued four months later. Very little vitamin B i 2 was apparently available for storage since feed intake was again reduced only seven weeks after oral treatment was discontinued. The ewe was then given
50 micrograms of cyanocobalamin by injection and recovery was again immediate. Absorption was calculated to 1-3 % of the 500 microgram dose or 6-12 micrograms per day. 31
Ruminal degradation of cyanocobalamin was determined using the four assay methods mentioned above. Over the first 10 hours, the ratio of Ochromonas vitamin B12 (true B12) to lignin declined rapidly and was accompanied by an increase in net £. coli (plate) vitamin B12 (includes analogs) to lignin ratio.
Ruminal production of vitamin B^2 was estimated by determining Ochromonas B12:lignin ratio 4 hours after feeding and multiplying by the daily lignin intake. The authors admit imperfection of this method, and state their estimates must be regarded as minimums, as concentration of vitamin B^2 in the rumen and B12:lignin tended to reach minimum values approximately 4 hours after feeding. The diet used in their determination contained 0.03 ppm cobalt and 11.1% lignin. Estimated minimum vitamin B12 production for two experimental sheep was 4 06 and 42 3 micrograms per day when supplemented with cobalt, and 46 and 92 micrograms per day without supplemental cobalt.
Production of vitamin B12 decreased from 600-750 to about
50 micrograms per day within 5 days of cobalt withdrawal.
This lower value reflected the B^2 production on the basal fodder which contained 0.01 ppm cobalt. Vitamin B12 production of 65 micrograms per day was reported for a diet containing 0.04 ppm cobalt. Comparison of data from cobalt-deficient sheep, cobalt-supplemented sheep, and grazing animals indicated proportion of B12 analogs 32 compared to true vitamin B12 increases with cobalt supplementation.
Daily urinary excretion of vitamin B12 was 0.76 micrograms from a ewe fed 1 mg cobalt per day compared to
0.07 micrograms from a cobalt-deficient ewe. Urinary excretion from a ewe given 50 micrograms of cyanocobalamin daily by injection was 15.2 micrograms compared to 0.09 micrograms B12 excreted per day by a pair-fed, cobalt-deficient ewe.
Ruminal vitamin B12 concentration of sheep decreased approximately 2 days after terminating daily oral cobalt supplementation. Efficiency of conversion of cobalt into true vitamin B12 decreased in a linear fashion from about
15% in the rumen of cobalt-depleted animals to about 3% in the rumen of sheep which had been supplemented with 1 mg cobalt per day.
Finally, distribution of daily, 500 microgram, subcutaneous injection of cyanocobalamin was reported.
Injections were continued for 20 days in a vitamin
B i 2-deficient sheep. Percentage of total administered dose for body tissues was as follows: liver, 16.5; kidneys, lungs, 3.1; 5.3; rumen, 10.5; reticulum, 1.9; omasum, 3.2; abomasum, 1.2; ileum, 3.8; cecum, 0.9; colon,
1.3; and rectum, 0.3. It is interesting to note a relatively high proportion of injected vitamin B12 which was recovered in the gastrointestinal tract. Excretion of 33 a [Co58] cyanocobalamin daily injection of 25 micrograms for 20 days was 19.9% in feces and 13.3% in urine. The authors suggested B12 was secreted into duodenum and reabsorbed in ileum. There was no evidence for absorption of B12 below small intestine. High content of vitamin B^2 in rumen wall was thought to be due to tissue propionate metabolizing ability and need for B12-containing methyl-malonyl Co A mutase for this process.
Jones and Anthony (1970) compared level of dietary cobalt to level of fecal vitamin B^2 in an attempt to use the latter parameter to predict cobalt intake in lambs.
Three lambs were used per treatment for each of four dietary treatments. Treatments consisted of 0, 50, 100, and 150 micrograms of cobalt per lamb per day in addition to the 51 or 57 micrograms of cobalt provided by the basal diet, depending on intake. The regression eguation developed (R2=.73) was Y=1.209+0.008X, where Y is micrograms B i 2 per gram of dry feces, and X is micrograms of cobalt intake. The equation developed for predicting oral cobalt intake from fecal vitamin B12 concentration is
Y=0.0779X-0.0757 where Y is oral cobalt intake expressed as ppm in dry feed and X represents fecal B^2 concentration expressed as micrograms vitamin B^2 per gram of dry feces. Sutton and Elliot (1972) studied effect of diet and
feed intake on ruminal production of vitamin B12 sheep using reentrant intestinal cannulas, rumen cannulas, and two assay methods. Three diets contained ratios of timothy hay and corn of 100:0, 70:30, and 40:60, and contained cobalt at the concentration of 0.62, 0.60 and
0.61 ppm, respectively. No diet was tested which had more than 60% corn. Production of B12 was significantly less
for sheep fed 40:60 timothy hay:corn when compared to the other diets. Production of analogs was not concomitantly decreased, therefore the proportion of true vitamin B12 to analogs was reduced. As digestible dry matter intake increased, production of true vitamin B^2 increased in a linear fashion, apparently at the expense of analogs of vitamin.
The reader is directed to detailed information on absorption and transport of cobalamin in humans by
Ellenbogen (1975). The author quotes Castle discussing intrinsic factor, cobalamin transport, and pernicious anemia in 1953: "Thus this disease would not develop if the patient could effect daily the transfer of a millionth of a gram of vitamin B12 the distance of a small fraction of a millimeter across the intestine and into the blood stream. This he cannot do, principally as a result of failure of his stomach to secrete into its lumen some essential but still unknown substance". It is now known that intrinsic factor is secreted by the fundic and cardiac stomach regions, binds to vitamin
B 1 2 , and facilitates absorption of B12 from the ileum.
Atrophy of the fundic, but not pyloric region, is observed in human patients with pernicious anemia. Ellenbogen cites work by Hoedemaeker, et al. (1964, 1966) which reports intrinsic factor has been isolated in the stomach of the cat, rabbit, monkey, and ox. Considerable research has been conducted using intrinsic factor from swine, which has also been used to treat pernicious anemia in man. Unlike man, however, the pyloric region has been shown to be the richest source of intrinsic factor. The rat has also been used extensively for intrinsic factor and cobalamin research. Ellenbogen cites work in which desiccated stomach parts were used to facilitate cobalamin transport in pernicious anemia patients as a means of detecting intrinsic factor in various animal species.
Animals in which intrinsic factor was detected in this manner includes: man, monkey, pig, rat, cow, ferret, rabbit, hamster, wild boar, silver fox, lion, tiger and leopard. Animals in which no intrinsic factor was detected in this manner include: dog, guinea pig, horse, sheep, elephant, giraffe, rhinoceros, hippopotamus, deer, chicken, toad, frog, black slug, edible snail, lobster, alligator, lizard, crocodile, eel and sea trout. It was proposed negative results may indicate specie specificity, 36 rather than a lack of intrinsic factor. This notion is supported by observations by Filmer in 193 3 of sheep and cattle being affected with marasmus, but never horses. It would appear from this observation sheep and cattle lack intrinsic factor, while horses have intrinsic factor.
Ellenbogen discusses isolation and assay of intrinsic factor, as well as structure, properties and specificity of the molecule. Basically, however, vitamin B12 is bound by intrinsic factor and in the bound state, both molecules are resistant to enzymatic degradation. Absorption of vitamin B12 facilitated by intrinsic factor and transport to tissues, through blood, is accomplished by transport proteins called transcobalamins. Although poorly understood, attachment of intrinsic factor- cobalamin complex is not energy demanding, but does require calcium. In contrast, however, absorption of this complex requires energy, takes 3-4 hours and is thought to be quite complex. Pancreatic secretions are also believed necessary and have been shown to play some role in the absorption of B^2* Intrinsic factor is not thought to be absorbed, and the cobalamins are bound to transport proteins (transcobalamins) when they enter the blood stream. Ellenbogen details the role of transcobalamins and the reader is referred to this paper for more specific information. Most importantly, absorption of vitamin B12, normally over 90%, is only 1-3% in patients which lack 37
intrinsic factor. This is similar to estimated absorption
of B12 reported in sheep by Kercher and Smith (1955), and
Smith and Marston (1970a).
Citrin et a l . (1957) studied absorption of B 12 in both
normal and pernicious anemia patients using an intestinal
delivery tube and radioactive (Co60) vitamin B12. When
intrinsic factor was provided, small doses of B12 (0.5
micrograms) were absorbed from the entire small
intestine. Doses of 0.5 to 3.0 micrograms were not
absorbed from colon with or without intrinsic factor
added. The authors suggest colon lacks transport
mechanisms necessary to absorb B12. It was also found
absorption was greater when B12 was instilled into
duodenum than when instilled into stomach. This
inhibition by stomach could be due to binding of B12 by proteins which do not possess intrinsic factor activity,
or due to effects of low pH on intrinsic factor and
subsequent impairment of vitamin B12 absorption.
Johnson et al. (1961) found that bile inhibited absorption of radioactive (Co60) vitamin B12 in the intact rat. Addition of an equivalent amount of gastric juice overcame this inhibition. The authors had no explanation of this phenomenon. It was reported that pH of bile was
9, and gastric juice was 1. This difference in pH and its effect on the absorption of B12 was not investigated. The authors cited other work which found that milligram 38
quantities of purified intrinsic factor was required to
catalyze absorption of microgram quantities of vitamin
B12. Inhibition of intrinsic factor by bile was offered
as a hypothesis and this work indicated that B^2 absorp
tion was impaired by bile, however, the mechanism of
inhibition was not investigated.
Studies by Abels and Schilling (1964) with rats
indicated vitamin B12 protected intrinsic factor from
peptic degradation in stomach. Studies with human gastric
juice jjj vitro showed B^2 binding capacity decreased over
time, but addition of B12 prevented this loss of binding
-apparently protecting intrinsic factor from degradation.
Experiments with pernicious anemia patients and human
gastric juice from normal patients showed absorption of
B12 was always enhanced when gastric juice was digested in
presence of vitamin B12. This again indicated intrinsic
factor was protected from degradation by B12. The vitamin
Bi2-binding ability of intrinsic factor was shown to be present, even when absorption of B12 was impaired by peptic digestion of up to two hours. In all cases, however, presence of vitamin B^2 during digestion
increased absorption of vitamin B12 and demonstrated Bi2 apparently protects intrinsic factor from degradation in the stomach.
In two experiments with pigs, Henderickx et al.
(1964) found that between 41.6 and 58.0% of radioactivity 39 of a 0.114 microgram dose of (Co57) vitamin B12 was absorbed from colon. The Co57 was recovered from feces, urine, liver, heart, kidney and spleen. Work cited earlier concluded absorption of B^2 from colon does not occur in sheep. This study raised interesting questions, but only radioactive cobalt was measured and it was not determined if B12 or Co57 was absorbed from hindgut.
In another study with the pig, Frederick (1965) suggested absorption of radioactive (Co60) vitamin B12 increased during late gestation and early lactation. An association between poor absorption of B^2 and reproductive failure is also suggested. This study had several technical difficulties discussed by the author, however, physiological state may influence absorption of vitamin B12 and is worthy of further investigation.
Schade and Schilling (1967) fed radioactive (Co57) vitamin B12 in egg to normal and pernicious anemia patients to determine effect of pepsin on absorption of
B12 and iron. Radioactive B12 was obtained from egg by injecting radioactive vitamin into the breast muscle of the hen, then collecting, boiling, and homogenizing egg.
It was digested with pepsin in vitro or with acid alone without pepsin to determine effect of pepsin on B^2 absorption. Their studies suggest that pepsin may have some involvement in absorption of vitamin B i 2, but further studies in this area are also needed. Absorption of B^2 discussed in detail in reviews by Glass (1963), Donaldson (1964), Herbert (1965), Spray
(1967), Ellenbogen (1975), and Donaldson (1975). These papers deal primarily with human research. Extensive research in ruminants has not been performed. Mechanisms of absorption and/or transport of cobalamins in the human may not apply to the ruminant since presence of intrinsic factor has not been fully documented. This information is presented here as a basis of knowledge for the reader, and further research needs to be performed in ruminants before these processes can be fully understood. 41
5. Metabolic role of vitamin B12
The metabolic role of vitamin B12 has been studied since its discovery in 1948. Metabolic reactions
involving various forms of vitamin B^2 are discussed in detail by Ellenbogen (1984), Poston and Stadtman (1975), and Babior (1975).
Methy lcobalamin is involved in synthesis of methionine, methane and acetate; while adenosylcobalamin
is involved in rearrangement reactions in which a single hydrogen atom is exchanged for another group on an adjacent carbon atom. Only two of the known enzymatic reactions which require vitamin B12 occur in mammalian tissues. The first is conversion of methylmalonyl-Co A to succinyl-Co A in which adenosylcobalamin functions as a coenzyme to methylmalonyl-Co A mutase. Methylmalonic acid
(MMA) is excreted in urine when B^2 is deficient. In non-ruminants, methylmalonyl-Co A is mainly derived from propionyl-Co A which in turn is produced from the breakdown of valine, isoleucine, methionine and threo nine. In ruminants, however, propionyl-Co A is formed primarily from ruminal fermentation which produces large quantities of propionic acid.
The second reaction involves transfer of a methyl group from N5-Methyltetrahydrofolate to cobalamin to form methylcobalamin, followed by transfer of the methyl group 42 to homocysteine to form methionine. Vitamin B12 deficiency results in formimino-L- glutamic acid (FIGLU) being excreted in urine when the N5-Methyltetrahydrofolate homocysteine methyl transferase (methionine synthetase) reaction is impaired. Urinary level of FIGLU may also be elevated due to folic acid deficiency. Very similar mechanisms occur in strictly anaerobic bacteria in which methanol and hydrogen are substrates and methane is produced or hydrogen and carbon dioxide are used to produce acetic acid.
Involvement of B^2 methionine synthesis is apparently present in some bacteria. Dehority and Scott
(1963) found an absolute requirement for B ^2 in some bacteria when casein hydrolysate was excluded from the growth medium. This B12 requirement was shown to be satisfied by B12, casein hydrolysate, or the amino acid methionine. This would suggest the involvement of B^2 in satisfying the methionine requirements of bacteria which were met by supplying the amino acid in media.
The reader is referred to literature cited above for detailed information on these reactions in mammals and in bacteria as the remainder of this section will address metabolic and physiological changes which occur in the ruminant during cobalt/vitamin B12 deficiency. It will be later shown how urinary MMA or FIGLU, propionate clearance 43 rates, and vitamin B12 status have been used to study B12 metabolism and its significance in animal physiology.
Marston et al. (1961) reported propionate metabolism was impaired in cobalt/vitamin B^2 deficiency in sheep.
This was demonstrated in vitro and in vivo. The site of impairment was shown to be conversion of methylmalonyl Co
A to succinyl Co A by methylmalonyl Co A mutase. The authors also observed an occasional, though rare, circum stance in which sheep fed cobalt deficient rations for two years, then supplemented with injections of vitamin B12 had an impairment of acetate metabolism as well. They reported a massive dose of folic acid restored ability of sheep to metabolize acetate normally. Upon removing B12 supplementation, propionate metabolism was again impaired, but acetate metabolism was normal. This demonstrated simple B12 deficiency, with rare acetate metabolism impairment due to complications of concomitant folic acid deficiency.
Important aspects of cobalt/vitamin B12 deficiency in the ruminant were summarized in this report. When ruminal cobalt concentration falls below 40 nanograms (ng) co/gm of rumen contents (20 ng/ml of the bacteria-free superna tant) , a dramatic change occurs in the rumen microbial population. Level of vitamin B i 2 production drops from
600-1000 micrograms (ug)/day to less than 50 ug/d. Since less than 3% of vitamin B12 is absorbed in the ruminant, 44
and such a small quantity of B12 i-s available for
absorption, vitamin B12 stores in liver are drained until
depletion occurs. When concentration of B12 in liver
falls below 0.15 ug/gm, there is a loss of metabolic
efficiency, feed intake is reduced, the animal loses weight, and death finally results. Primary metabolic
defect of cobalt deficiency is an impairment of propionate metabolism, however, abnormal rumen fermentation may occur
due to cobalt deficiency (McDonald and Suttle, 1986).
Frederick (1960), recognizing the trend to feed protein from plant sources rather than animal proteins to
swine, hypothesized that vitamin B^2 status could be in
jeopardy. Data suggested vitamin B^2 could be involved in reproductive failures in sows. By comparing the ratio of
serum B^2 levels before, and mean of 24 and 48 hr after
250 microgram of vitamin B12 by injection, a significant difference was detected between barren and fertile sows.
The author reports in the herd tested, sows should have a serum B12 ratio greater than 0.60 to have a 93% chance of producing living offspring. Frederick cites work in
Germany by Johnsen et al. (1952. Danske Dyrl. 63:1-11) in which sows were fed a B^2-deficient diet. One group was supplemented with 80 micrograms of B12 orally per day.
Ovulation rates were 12.3 and 11.7 ova for vitamin B^2 deficient and supplemented sows, respectively. Four weeks postbreeding, however, sows averaged 5.6 and 10.4 embryos 45 per sow, respectively for the same two groups. Nearly
100% fertilization rate was assumed and differences in embryonic mortality were attributed to vitamin B12 status. Frederick also cites work by Wokes (1958.
Soc. Arts 106:113-128) which suggests an association between low serum B12 levels and frequent menstrual disturbances in vegetarian women.
Schweigert (1961) discussed role of vitamin B 12 in nucleic acid synthesis. Although he cited work which showed a reduction in nucleic acid and DNA in rat liver when B12 was deficient, he concluded the role may be indirect. It is not unreasonable to conclude vitamin B12 deficiency could indirectly effect any metabolic process.
Synthesis of methionine, catabolism of valine and isoleucine, and gluconeogenesis from methylmalonyl Co A from a number of sources including propionate are all impaired in B12 deficiency.
Vitamin B^2 status may effect or be effected by status of other nutrients. Marchetti and Testoni (1963) discussed the relationship between vitamin Bg and vitamin b 12* of interest were conclusions that absorption of B12 was impaired when Bg was deficient and B^2 status was effected by Bg status, though the converse was not true.
Interaction of B12, thiamin and ascorbic acid has been suggested by MacPherson et al. (1976) as was discussed in a preceding section. Marston et al. (1961) noted inter 46
action in rare cases of B^2 deficient sheep becoming folic
acid deficient as well. Geographic areas which are
deficient in soil (therefore forage) cobalt, often are
also deficient in other nutrients such as copper.
Ruminants grazing such areas may also be deficient in
other minerals which confound or complicate deficiency of
vitamin B12•
Metabolism and physiology of vitamin B^2 deficiency
has been studied in many species. Newberne (1963)
reported birth weights of rats, taken by cesarian section
at day 21, were significantly less for vitamin B^2
deficient dams than for those fed adequate or excessive
amounts of B12. Reported average litter size was less
(4.33) for B^2 deficient rats than sufficient (5.75) or
excess B12 groups (5.76). This supports Johnsen's work
with sows discussed above. Embryos from Bi2 deficient
dams were immature and had tissue damage or morphologic
alterations in the spleen, liver, kidney, and bone
marrow.
Hogue and Elliot (1964) studied effect of dietary
propionate on dietary requirement for vitamin B12, biotin,
and folic acid in the rat. Control diet was formulated with no supplementation of these vitamins or sodium
propionate in a 24 factorial arrangement of treatments.
Growth rate was not improved in rats fed vitamin B i 2 , biotin, or folic acid or combination of these vitamins when fed control diet. However, when sodium propionate was added to the diet rats fed biotin had improved growth rates for the initial 18 d compared to those not fed biotin. Rats fed folic acid had similar growth rates to those fed diets without supplemental folic acid. Vitamin
B12 had a profound effect across all treatment combinations. Rats fed B12 had growth rates 30.8% greater than rats not fed B12. Feed intake was 6.3% greater in rats fed propionate when B12 was added compared with rats not supplemented with B12. Increased feed intake of 0.2% was attributable to B i 2 in rats fed control diet.
Although B12 increased feed intake in rats when propionate was included, intake was not as great as in rats fed control diet without propionate. Amount of supplemental
B i 2 could have been insufficient to fully compensate for increased need for B12 due to propionate inclusion in the diet. Compared to control diet, feed intake for biotin and folate supplemented rats was reduced 21.8% without B^2 and 16.9% when B12 was supplemented to the propionate diet. In a second experiment with 0, 3, or 6% sodium propionate, significant differences in growth rate and feed intake due to vitamin B12 were observed. Significant interaction between B12 and level of sodium propionate was also observed with B12 having a greater effect at higher propionate inclusion rate. Of great importance is the finding that vitamin B12 had negligible effect on growth 48
rate and feed intake of rats fed control diet, but when
sodium propionate was included in the diet, B^2
significantly increased growth rate and feed intake.
Kutzbach et al. (1967) studied interactions of
vitamin B12, methionine, and folic acid in the rat. They
concluded level of methionine synthetase was B12
dependent, and was decreased by methionine additions to
the diet. Rats fed diets low in methionine and deficient
in B 12 showed a marked drop in liver folate levels even
though there were high levels of folic acid in the diet.
Supplementation of either methionine or vitamin B^2
corrected this low level of liver folate.
The remainder of this section will deal exclusively
with ruminants. According to Elliot (1966), the dairy cow
is perhaps the most nutritionally stressed animal
studied. He estimates a cow producing 100 pounds of milk
is secreting 4.5 pounds of lactose in milk per day. This
is synthesized from blood glucose which, in turn, is
primarily derived from ruminal propionic acid through gluconeogenesis. Although great, this need for glucose
represents only part of total energetic need since cells
synthesizing milk need glucose for energy, as do other body tissues. He illustrated findings of Marston et al.
(1961) in which the primary defect in cobalt deficiency was found to be a limitation of methyl-malonyl-Co A mutase. Based upon this work in sheep, Elliot lists six factors which may contribute to impairment of propionate metabolism in lactating dairy cattle. First, ruminal synthesis of vitamin B^2 has not been studied according to level or type of feed. Secondly, a large proportion of cobalt-containing ruminal compounds are not vitamin B12 > but analogs of B^, which are not biologically active in mammals. Thirdly, absorption of B12 has been shown to be inefficient. Forth, colostrum contains relatively high amounts of vitamin B12 and losses in colostrum and milk represent a significant daily drain of total B12 supply.
Fifth, lactating cows, which most likely metabolize more propionate than other animals, have the lowest blood vitamin B12 level of species studied. Finally, the developing fetus in late gestation might require considerable quantities of vitamin B12 at a time when the cow could be on relatively low cobalt intakes. An initial experiment showed reduced blood and liver vitamin B ±2 levels during the first 7 weeks when compared to later in lactation. Liver homogenates from cows in early lactation tended to convert propionate to succinate at a slower rate in vitro than homogenates from cows later in lactation.
Rates of conversion were positively correlated with liver vitamin B12 values. Further studies need to be conducted in cows to determine synthesis, absorption and metabolism of vitamin B12. Metabolic evidence of vitamin B^2 deficiency in sheep was studied by Gawthorne (1968). Urinary excretion of methylmalonic acid (MMA) was significantly increased in
severely vitamin B±2 deficient sheep compared to pair-fed control animals, injected with 50 micrograms of B12 every
four days. Both groups were fed a cobalt-deficient diet.
Urinary output of MMA and formimino-L-glutamic acid
(FIGLU) was studied during induction and remission of vitamin B12 deficiency in a second experiment. Five stages of B^2 deficiency were defined. In stage 1, no clinical signs of B12 deficiency were observed and feed
intake was 813 gm/d (similar to controls). Feed intake voluntarily dropped to 500 gm/d in stage 2, and further dropped to 200 gm/d in stage 3, with apparent signs of severe deficiency. Vitamin B12 injections were initiated each fourth day, and within three weeks feed intake had increased to 400 gm/d and was termed stage 4. Finally, in stage 5, feed intake had increased to 813 gm/d and all clinical signs of B12 deficiency were remitted. Urinary excretion of MMA was significantly increased in stage 3 compared to stage 2. No significant difference in level of MMA was observed between stage 1 and stage 2.
Excretion of MMA was significantly increased only in profound vitamin B12 deficiency. Significant increase in
FIGLU excretion occurred between stage 1 and stage 2. No significant difference in FIGLU level between stage 2 and 51
stage 3 was observed. Increased urinary FIGLU can be used to aid in detecting early stages or mild B^2 deficiency, while increased urinary MMA can be useful in detecting more severe deficiencies. MMA levels of 16 and 60 micromoles/kg body weight/24 hr were reported for B12-suf-
ficient and B12-deficient sheep, respectively. FIGLU
levels increased from 11 to 33 micromoles/kg body weight/24 hr as vitamin B12 deficiency progressed from stage 1 to stage 2. This work provided metabolic evidence of impairment of methylmalonyl Co A mutase (increased MMA excretion) and N5-methyltetrahydrofolate homocysteine methyl transferase (increased FIGLU excretion) in sheep, and a method to detect vitamin B12 deficiency.
Sommers (1969) reported blood clearance rates of propionate and acetate were impaired in vitamin B12 deficiency. This research was conducted simultaneously with the same animals and facilities as the study by
Gawthorne, above. During deficiency stages, above,
Sommers fasted and denied water to sheep for 18 h. Sodium propionate or sodium acetate were infused into the blood at the rate of 3.5 millimoles per kg body weight. Blood samples were collected over the next 4 h. Half-time of propionate in stage one, two, and three was 9.0, 34.5, and
108 minutes, respectively, for B12-deficient sheep. Pair- fed control sheep had propionate half-times of 7.5, 12.8, and 18.8 minutes, respectively, for stage 1, 2, and 3. 52
Blood propionate returned to pre-infusion level within one hr for control sheep, but remained high during the four hr collection period in B i 2 deficient sheep. Blood propionate returned to pre-infusion level approximately four hours after B12_deficient sheep were given 300 micrograms of B^.
Acetate clearance was also impaired in B^2 deficient lambs. Half-times for acetate in stage one, two and three of deficiency was 14.3, 28.5, and 69.0 minutes, respectively, for B12-deficient sheep and 15.8, 18.8, and
22.5 minutes, respectively, for pair-fed control sheep which were not B12 deficient. Basal blood VFA level increased as deficiency progressed in B^2 deficient sheep, but decreased in sheep supplemented with B ^ • Sommers1 results are in agreement with Marston et al. (1961), concerning propionate clearance rates, but are at variance concerning acetate clearance rates. Marston et al. (1961) reported impaired acetate metabolism in only rare cases of folic acid deficiency.
Vitamin B i 2 content of liver, rumen epithelium and rumen wall was investigated by Elliot and Hughes (1976).
Since it has been suggested rumen epithelium metabolizes propionate, and B12 is necessary for conversion of propionate to succinate, it would be reasonable this tissue would contain B^2 • Vitamin B i 2 was measured by both non-specific (radioisotope) and true-B^ specific 53
(Ochromonas malhamensisl methods. B12 concentration was essentially equal by either method for liver, rumen wall and rumen mucosa. A high correlation was reported between rumen mucosa and liver B12 activity, however, correlation of B12 activity between rumen wall and liver was low and not significant. Level of B12 in rumen mucosa was approximately 10% of B12 content of liver. Since enzyme activities of mucosa and liver were not determined, B^2 content should not be interpreted to mean rumen mucosa has
10% of the propionate metabolizing activity of liver.
Liver is the major site of propionate metabolism, but rumen mucosa may play a significant role as well.
Smith and Marston (1970b) conducted experiments to determine if wasting associated with cobalt deficiency was due to metabolic changes, or solely a function of depressed feed intake. Animals fed cobalt-deficient rations maintained apparently normal health and feed intake for 20-35 weeks after daily oral cobalt drenches and intramuscular B^2 injections were discontinued. Feed intake decreased and animals lost weight, eventually dying in an emaciated condition after another 20-35 weeks if left untreated. Deficiency symptoms were attributed to metabolism of B^2 in the tissues, rather than presence of ruminal cobalt. The authors concluded loss of body weight was due to both reduced feed intake and decreased metabolic efficiency in cobalt/vitamin B^2 deficiency. Changes associated with loss of appetite in cobalt/- vitamin B12 deficiency were reported by Marston et al.
(1972). As an author aside, this paper was published posthumously by the senior author and represents the last study in a lifelong research career into metabolism of cobalt and vitamin B^. The reader is reminded Marston was credited as being the first to use cobalt to treat a disease in animals (Underwood and Filmer, 1935). Marston et al. (1961) also were first to report an impairment in propionate metabolism as the primary metabolic defect in
B12 deficiency. In the present paper, B^-deficient sheep consumed their ration over a 24 hr period, while pair-fed control sheep, supplemented with vitamin B12, consumed their ration in 1-2 h. After a 24 hr fast, deficient sheep would eat ravenously for a short time, then leave the remainder of its ration untouched for several hours.
This demonstrated an altered pattern of eating for vitamin
B ^2 deficient sheep. Animals with adequate vitamin B12 consumed the ration as a meal, while vitamin B^2 deficient sheep consumed the ration as small, frequent meals over a
24-hour period. Propionate clearance rates were retarded when anima\ls were vitamin B 12 deficient. Delay in acetate clearance was attributed to involvement of elevated blood propionate levels. Addition of cobalt to the cobalt-deficient diet had no effect on the molar proporti ons of acetate (60%), propionate (26%), or butyrate (14%) 55 in sheep having adequate liver B12 stores. Blood VFA levels tended to be higher in B12-deficient sheep than control sheep fed B12.
The role of vitamin B^2 in propionate metabolism has been recently reviewed by Elliot (1980). This work discusses metabolism of propionate by liver and rumen epithelial tissue, and other tissues, glucose synthesis, biochemical roles of propionate and B^2, and theoretical implications of vitamin B^2 in animal production. The reader is referred to this work for more detailed information. 56
6. Summary of cobalt and vitamin B12 animal nutrition.
To summarize above discussion, mammals have a metabolic need for vitamin B12 which functions as part of only two known enzymatic reactions. The animal must absorb vitamin B i 2 from dietary sources to meet this need. The ruminant meets its requirement for vitamin B i 2 by ingesting adequate dietary cobalt for rumen bacterial synthesis of vitamin B12. Bacteria are then digested and
B q_2 absorbed. Since the only known metabolic function of cobalt is as part of the vitamin B12 molecule, cobalt deficiency occurs uniquely in ruminants. Specifically, cobalt deficiency occurs in the rumen microbial population and may induce vitamin B12 deficiency in the ruminant.
When level of ruminal cobalt falls below a critical level, a great change occurs in the rumen population, and amount of vitamin B^2 synthesized drops dramatically.
Several analogs of vitamin B12 are also produced by rumen microbes which are not biologically active in the ruminant. When high concentrate diets are consumed, there is a greater proportion of analogs than true vitamin B12.
Absorption of B12 is very low in ruminants, approximately
3%, which is similar to selected non-ruminant animals which lack intrinsic factor. Metabolic need for vitamin
B12 is higher in ruminant than non-ruminant animals; 57 presumably due to increased demand for methylmalonyl-CoA mutase, an enzyme necessary for conversion of propionyl-CoA to succinyl-CoA in liver. This is a necessary step in conversion of propionic acid to glucose. Propionate is a major product of ruminal fermentation and primary substrate for gluconeogenesis in ruminants. Vitamin B12 i-s involved in methylation of homocysteine to form methionine. It has been suggested that B 12 is involved in disease processes in cobalt deficiency and sub-deficiency. Involvement of B12 in reproduction and DNA and RNA metabolism is fairly well documented, however, ketosis, and other metabolic processes have not been fully researched.
The reader is referred to various texts currently available on cobalt and vitamin B^2 for more specific information. Young (1979) provides comprehensive coverage of cobalt in plant, soil, and animal systems. Cobalt is also discussed in-depth in two books by Underwood (1977 and 1981) , and more recently by R.M. Smith in "Trace
Elements in Human and Animal Nutrition" edited by Walter
Mertz (1987). Volumes of information are available on
Vitamin B^2 including: "Vitamin B12 Animal Nutrition" by Merck & Co., Inc. (1957), "Cobalamin: Biochemistry and
Pathophysiology" edited by Babior (1975), and covered in
"Handbook of Vitamins: Nutritional, Biochemical and
Clinical Aspects" edited by Machlin (1984). 58
B. IONOPHORES
1. Discovery
Westley (1982) reports the earliest information on
ionophores were papers by Berger et al.(1951) and Harned et al. (1951). In these reports, X-206, lasalocid and nigericin were first identified and classified. It was suggested they constitute a new class of antibiotics due to their unique qualities. All were acidic and very soluble in organic solvents, but unlike other known antibiotics, addition of an aqueous base to organic solution of the antibiotic resulted in cations being extracted into the solvent phase, rather than extraction of the antibiotic anion. Upon evaporation, salt of the antibiotic resulted. These antibiotics were reported to be effective against gram-positive bacteria and mycobacteria in vitro and relatively toxic parenterally.
The fourth polyether antibiotic was discovered in
1958. Westley (1982) reports Lardy et al. (1958) used dianamycin in metabolic studies. They reported dianamycin, nigericin, and several other antibiotics were powerful inhibitors of the oxidation of pyridine-nucleo- side linked substrates. 59
According to Westley (1982), monensic acid (monensin) was first reported by Agtarap et al. in 1967, and was the
fifth ionophore discovered. In addition to reporting the structure of monensin, anticoccidial activity was reported. This provided incentive for discovery of over
forty new ionophores by 1978, compared to five from 1951-
1968. This number grew to 76 by 1982, and was over 100 by
1988 (Westley, 1988) .
The reader is referred to the text: Polvether
Antibiotics: Naturally Occurring Acid Ionophores. edited by John W. Westley (1982) for more information on iono phores. In this two-volume set, the biology and chemistry of ionophores are discussed. Classification, notation, biochemistry and various aspects of production and biosynthesis of ionophores are discussed.
Use of ionophores in ruminants began with monensin at
Eli Lilly & Co. (Raun Patent, 1974). The remaining section of literature review will be devoted to various aspects of ionophores in ruminant nutrition. This will include mode of action, effect on host animal, and various aspects of the use in ruminant nutrition. 60
2. Function of ionophores
The biochemical activity mechanisms of ionophores are discussed in detail by Taylor et al. (1982), and Reed
(1982). The reader is directed to these works for more
in-depth information on thermodynamics of complexing and transport of ions by these compounds. Use of ionophores in veterinary applications is discussed by Ruff (1982) and use of lasalocid and monensin in chickens is discussed by
Osborne et al. (1982) . The reader is directed to these works for more information on use of ionophores in non-ruminants.
To briefly summarize, the mode of action of iono- phoric antibiotics lies in their ability to effect transport of ions across cell membranes. The net result is an efflux of potassium ions and an influx of sodium ions. Precise mechanism depends upon ion affinity of the antibiotic. If affinity for sodium is greater than for potassium, as is the case with monensin or dianamycin, cellular changes include a pH change. As sodium ions are transported into the cell, hydrogen ions are transported out. To adjust pH, hydrogen ions then move into the cell and potassium ions are transported out. If affinity for sodium and potassium ions is equal, the influx of sodium ions is equal to the efflux of potassium ions with no hydrogen ion transport. With antibiotics such as 61
lasalocid or nigericin, affinity for potassium ions is greater than affinity for sodium ions. In this
circumstance, hydrogen ions are transported into the cell
and potassium ions are transported out. An adjustment in pH occurs as sodium ions are transported into the cell and hydrogen ions are transported out. The overall effect in all three circumstances is an influx of sodium ions and a concomitant efflux of potassium ions, however, transient changes in pH may occur. Affected rumen bacteria are
inhibited by uncoupling of the cell's energy system and ruminal microbial population is altered as unaffected bacteria predominate (Reed, 1982).
More recently, Russell (1987) has proposed the mechanism of action of monensin in Streptococcus bovis as an efflux of potassium ions rather than sodium ions due to the potassium gradient being greater than the sodium gradient. This resulted in an inversion of pH which the cell corrects by expelling H+ with an influx of Na+ occurring. This process requires ATP and would inhibit growth of the bacteria. The reader is referred to this work for more information.
Bergen and Bates (1984) describe ionophores as one class of compounds which can achieve the "long cherished dream of ruminant nutritionists.. .to manipulate and improve the efficiency of ruminal fermentation". This review represents the state of knowledge on ionophores at 62 the time it was first presented in August 1982. The reader is encouraged to read this work and subsequent journal articles from the symposium on "Monensin in
Cattle". Bergen and Bates (1984) cite three major areas of animal metabolism which account for improved perfor mance of growing and finishing cattle when ionophores are added to the diet: 1) increased efficiency of energy metabolism in the rumen and (or) animal; 2) improved nitrogen metabolism in the rumen and (or) animal; 3) the retardation of feedlot disorders, especially lactic acidosis (chronic) and bloat.
Observed effects of the ionophore on animal performance are often dependent upon the diet being consumed. When high levels of readily fermentable carbohydrates are being fed, feed intake is generally depressed, while average daily gain (ADG) is unchanged with ionophore supplementation. This results in an improvement in feed efficiency (feed/gain). With diets high in fiber (roughage) , ADG is increased without altering feed intake. This also results in improved feed efficiency (Bergen and Bates, 1984).
Feed intake is thought to be controlled by numerous factors, however, physical fill of the rumen is considered to limit feed intake when highly fibrous diets are fed
(high roughage). Intake on diets containing high amounts of rapidly fermentable carbohydrates (high concentrate) is 63
controlled by chemostatic mechanisms which regulate energy
intake (Forbes, 1980).
Ruminal production of volatile fatty acids (VFAs) is usually altered by addition of an ionophore. Molar proportion of propionic acid is increased (Allen and
Harrison, 1979) with a concomitant decrease in molar proportion of acetic and butyric acids (Bergen and Bates,
1984; Richardson, et al., 1975; Richardson, et al., 1978;
Schelling, 1984; Thonney, et al., 1981; Van Maanen, et al., 1978; and Wallace, et al., 1981). Theoretical and experimental consequences of this shift in VFA production are discussed by Bergen and Bates (1984).
The ionophore has an effect on nitrogen metabolism in both the rumen and ruminant. Several studies indicate requirement for dietary protein is reduced when an
ionophore is added to ruminant diets. Ruminal amino acid degradation is reduced, ruminal ammonia nitrogen production is decreased, rumen turnover rate is depressed, and ruminal protein by-pass is increased (Beede, et al.,
1985; Beede, et al., 1986; Bergen and Bates, 1984;
Faulkner, et al., 1985; Muntifering et al. 1980; Poos, et al. 1979; Schelling, 1984; Thompson and Riley, 1980; and
Vermeire, et al., 1986). Changes in ruminal nitrogen metabolism are associated with changes in bacterial population and fermentation. The protein-sparing effect of ionophores in ruminants is associated with improved rumen nitrogen metabolism, and/or theoretically less amino acid catabolism necessary to satisfy gluconeogenic needs when ruminal propionate is increased. Bergen and Bates
(1984) discussed reduction of feedlot disorders in cattle fed ionophores. Inhibition of gram positive organisms by ionophores, and lactic acid producers in particular, reduces incidence of lactic acidosis and bloat in feedlot cattle. Nagaraja, et al. (1981) reported intraruminal administration of monensin or lasalocid reduced incidence of experimentally induced lactic acidosis in cattle.
Metabolic effects of ionophores on ruminal fermenta tion are summarized in Table 1. These observations result from changes in rumen fermentation, rather than the ionophore's direct mode of action. 65
Table 1 Summary of metabolic effects of ionophores on the rumen fermentation
1. Shift in acetate-propionate ratio toward more propionate. 2. Some increase of lactate to propionate production via the acrylate pathway. 3. Decreased ruminal protein breakdown and deamination; lower ruminal ammonia-N. 4. Primary H+ or formate producers, gram positive organisms, are inhibited. 5. Decrease in methane production primarily due to lowered availability of H2 and formate and depressed interspecies H2 transfer. 6. Depression of lactic acid production under acidosis inducing conditions. 7. Gram negative organisms, of which many produce succinate (source of propionate) or possess capacity for the reductive tricarboxylic acid cycle to use bacterial reducing power, survive. 8. Some evidence for depressed rumen content turnover. 9. A mild inhibition of protozoa. 10. Decrease in rumen fluid viscosity in bloated animals. 11. Depressed growth yield efficiency of the ruminal microbes.
From; Bergen and Bates (1984)
Understanding metabolic changes in the rumen and responsive changes in the ruminant animal is imperative.
Improved animal performance and improved efficiency of animal production is the net result of numerous biological changes summarized in Table 2. Some improved animal performance can be attributed to effects not occurring in the rumen, but in other areas of the G.I. tract. Control of coccidiosis in sheep and other animals is an example. 66
Table 2 Biological effects of monensin in the rumen
1. Greater ruminal propionate concentration. 2. Lower ruminal acetate concentration. 3. Lower ruminal butyrate concentration. 4. Lower ruminal lactate in stressed animals. 5. Higher ruminal pH in stressed animals. 6. Less ruminal methane production. 7. Decreased intake of grain diets. 8. Increased intake of forage diets. 9. Increased ruminal forage fill. 10. Decreased ruminal rate of passage. 11. Increased dry matter digestibility. 12. Increased protein digestibility. 13. Decreased ruminal deamination. 14. Decreased ruminal proteolysis. 15. Protein sparing effect. 16. Modified ruminal escape of protein. 17. Modified ruminal escape of starch. 18. Modified rumen microbial population. 19. Increased body glucose turnover. 20. Modified substrate gluconeogenesis. 21. Reduced 3-methylindole production. 22. Earlier puberty in heifers. 23. Reduced fly pupae in feces.
From : Schelling (1984)
Schelling (1984) discussed types of inodes of action for Ionophores. The "basic mode of action" is modification of the movement of ions across biological membranes. Bergen and Bates (1984) provide an excellent discussion of this "basic mode of action" of ionophores.
The reader is referred to this work and to Taylor et al. (1982) for detailed information on transport of ions across biological membranes and significance of these phenomena upon rumen fermentation. 67
The "basic mode of action" produces numerous changes, many of which are summarized in Table 1 and Table 2.
Schelling (1984) terms these changes "system mode of action". The "system mode of action", such as the shift in ruminal VFAs, elicits various "animal responses", such as improved average daily gain. The system modes of action are summarized in Table 3 below.
Table 3 Accepted or probable system modes of action of ionophores
1. Modification of acid production. 2. Modified feed intake. 3. Change in gas production. 4. Modified digestibilities. 5. Change in protein utilization. 6. Modified rumen fill and rate of passage. 7. Other ruminal modes of action.
From: Schelling (1984)
These system modes of action are examined individual ly by Shelling (1984) and the reader is referred to this work for further information. Of particular relevance to this subject is the modification of acid production. As mentioned above, the most common observation is a shift in ruminal VFA production towards increased propionate, and a concomitant decrease in the molar proportion of acetate.
No net change in total VFA production typically occurs. The "basic mode of action" (transport of ions across cell membranes) also affects host animal physiology. This is reviewed by Bergen and Bates (1984), and discussed by
Donoho (1984), Todd et al. (1984), and Potter et al. (1984). The pharmacology of lasalocid is discussed by
Hanley and Slack (1982) and the reader is also directed to this work. 69
3. Ionophore use in ruminant nutrition
The ruminant animal holds a unique niche in that it can convert fibrous feeds into high-quality food and fiber. The ruminant animal also provides great oppor tunity to enhance utilization of feedstuffs by altering microbial fermentation of the rumen. At the same time, however, the rumen maintains a continuous nutrient source to the animal, primarily composed of microbial cells and fermentation products. The nutrient supply reaching the abomasum is of a fairly constant composition, despite the variability of feedstuffs presented to the rumen microbes. In order to enhance nutrient supply to the ruminant animal, one must either improve ruminal fermentation, or provide a high quality nutrient source which can escape ruminal degradation and be digested and absorbed in the abomasum and small intestine. Rumsey
(1984) discusses the potential impact of chemicals for improving animal agriculture and ionophores as a means to modify ruminal fermentation in particular. Through modification of ruminal fermentation, the efficiency of food production could be improved world-wide.
The ionophore monensin was approved in 1976, and was reportedly used by 80% of the U.S. feedlots by 1978 (Owens
1980). This wide spread and rapid acceptance is evidence of the efficacy and cost-effectiveness of feeding iono- 70
phores to feedlot beef cattle. Owens discusses the animal
response and effectiveness of narasin and lasalocid.
As of this writing (March 1991) lasalocid and monensin are the only ionophores approved for use in
cattle or sheep in the US. Several ionophores are in developmental stages including: narasin, salinomycin, and
tetronomycin.
Perry et al. (1976) reported the results of a 232 d
feeding study involving 72 growing-finishing beef steers.
In this study, monensin-fed cattle had identical rate of gain (0.89 kg/d), but consumed 90% of the DM per kg gain
as cattle not fed monensin (7.01 vs. 7.78 kg). Liver
condemnation for the control cattle (not fed monensin) was
8% vs. 56% in monensin-fed cattle. Rumen samples taken on
d 56 of the experiment showed a decrease in acetate (53.6 vs. 64%) and butyrate (7.8 vs. 14%), with an increase in propionate production (38.7 vs. 22%) in cattle fed monensin vs. control animals. Steers had similar carcass weights and slaughter weights, however, monensin-fed
cattle averaged one half quality grade lower than cattle
not fed monensin (5.8 vs. 6.9), and 0.35 yield grade lower
than the control cattle (3.05 vs. 3.4).
Goodrich et al. (1984) summarized data on 11,274 cattle from 228 trials to identify the influences on
animal performance. An increase (1.6 %) in average daily gain (ADG), decrease (-6.4 %) in dry matter intake (DMI), 71 and an improved (-7.5 % feed/100 kg gain) feed efficiency
(FE) was observed over all trials. The standard deviation for all of these variables was quite large and the authors developed regression equations in an attempt to explain the great variability. Using a number of variables, the model accounted for approximately 33, 35 and 55 % of the variability in ADG, DMI, and FE, respectively. Variables included in the model were control daily gain, metabolizable energy intake (MCal), density of monensin in the diet (mg/kg DM), sex, weight class, use of growth promotant, control daily feed intake, control feed efficiency, and metabolizable energy density (MCal/kg) of the diet. These regressions show factors unknown at present may account for 67, 65, and 45% of the variability, respectively, in ADG, DMI, and FE. Carcass characteristics summarized by Goodrich et al. (1984) showed little differences between cattle fed monensin and control cattle not fed monensin. Monensin fed cattle had slightly less fat as evidenced by a lower yield grade
(3.15 vs. 3.21), lower marbling score (5.22 vs. 5.34), and slightly less kidney heart and pelvic fat (2.91 vs. 2.93% of the carcass).
Several other observations were discussed by Goodrich et al. (1984). Among the most important were the observa tion of a protein-sparing effect of monensin; the observa tion that although monensin reduces heat generated in the 72 rumen, animal performance is not adversely affected in cold weather; the observation of a reduction of lactic acidosis in feedlot cattle; control of coccidia; inhibi tion of face and horn flies; reduction of bloat in feedlot cattle and alfalfa pasture-fed cattle; improved (10-15%)
ADG in pasture-fed cattle; and increased stocking rates in beef cows fed poor quality pasture.
Similar observations were reported in a summary by
Wagner (1984). Weighted means of 45 trials with monensin in feedlot cattle for ADG, DMI and Feed/Gain were +2.0,
-6.1 and -7.7%, respectively. Data reported reflects the variability discussed above. Response in ADG ranged from an increase of 10.8% in a study in Kentucky to a depres sion of 14.8% at Cornell. The DMI response ranged from an increase of 3.6% in a New Jersey trial to a decrease of
18.1% in a study conducted in Kansas. Feed/gain ranged from 0 response in Washington, to an improvement of 14.3% in a study in Florida.
Feedlot results with lasalocid showed an increase of
6.4% in ADG, a decrease of 4.6% in DMI, and an improvement of 9.9% in FE. For the 16 studies reported, ranges of response were 0.6 to 19.0% increase in ADG, increase of
3.2% to a decrease of 11.6% in DMI, and improvement of
1.7 to 15.0% for FE. With a limited number of feedlot trials reported, narasin and salinomycin showed improve ments in FE of 9.8 and 14.8%, respectively. All of these 73 antibiotics have similar "basic modes of action" and
"system modes of action". Although the means for variables measured are unequal for the different ionophores, the ranges of variability are similar for these compounds. Work by Bartle et al. (1988) showed similar effects of the ionophore tetronasin to monensin and lasalocid when fed to growing-finishing cattle.
Decreased feed intake, similar gain, and improved feed efficiency were observed in cattle fed tetronasin compared to control cattle which were not fed any drug.
Effects of ionophores have been widely reported for cattle and sheep in pasture settings (Sharrow et al.,
1981; Burris, 1978; Rumensin Technical Manual, 1978;
Bovatec Technical Manual, 1982) and in feedlot settings
(Kinsella and L'Estrange, 1980; Shqueir et al., 1982;
Rumensin Technical Manual, 1975; Rumensin Feedlot
Technical Manual Update I, 1978; Bovatec Technical Manual,
1982) . Comparisons to other drugs or in combination with other drugs have also been reported (Wittier et al., 1982;
Berger et al., 1981; Ricke et al., 1984).
Control of coccidiosis in cattle and lambs by use of monensin is discussed by Stockdale (1981). Inclusion rates of 5-30 gm/ton were reported to be effective at reducing oocysts in lambs. The effective rate for calves was reported to be 1 mg/kg/d beginning 10 d after inocula tion. 74
Lasalocid was approved for the control of coccidiosis in confined sheep in June 1984 and is the only approved use of an ionophore in sheep. Studies have reported using monensin to control coccidiosis and improve animal performance in lambs such as the work by Calhoun et al.,
(1979), and comparisons of lasalocid and monensin in the control of coccidiosis by Horton and Stockdale (1981).
Use of ionophores in the gestating and lactating ewe has been investigated, also (Ross, 1984). Theoretically, the increased ruminal propionate should help prevent pregnancy toxemia or ketosis in the ewe. Oddy et al.
(1978) found the inclusion of monensin into pregnant ewe diets increased the supply of glucose precursors in the body. Woodward (1988) found a highly significant decrease in the number of oocysts in the feces of lambs whose dams had been fed lasalocid compared to lambs whose dams had not been fed the ionophore. Ewes fed lasalocid had significantly less fecal oocysts than ewes not fed lasalocid. Presumably, ewes fed lasalocid provided a less contaminated environment for their lambs than ewes not fed lasalocid prior to lambing.
Finally, the use of ionophores has not been approved for the lactating dairy cow. Monensin and lasalocid are approved for use in pasture-fed growing dairy heifers for improved ADG, however. Feeding monensin to lactating dairy cows has been shown to increase the efficiency of milk production, increase the total milk production of the cow, but reduce the milk butterfat percentage (Potter, personal communication 1988). Altered ruminal production of propionate and acetate may reduce acetate available for butterfat synthesis by the mammary gland. The lactating cow provides great opportunity for using ionophores since improved feed efficiency for an animal consuming 4-5% of its body weight per day would have tremendous economic benefit. 76
C. RESEARCH QUESTION— HYPOTHESIS
In this section, the basis of research will be
discussed. The preceding pages were intended to provide a
foundation of understanding concerning cobalt, vitamin
B12, and ionophores as they relate to animal metabolism
and nutrition. Previous work in Ohio (Klosterman et
al. 1953) demonstrated a response to alfalfa ash, molasses
ash and a trace mineral mix in feedlot cattle fed ground
ear corn, mature timothy hay, soybean meal and minerals
(calcium, phosphorus and salt). These cattle were hand
fed to appetite twice per day and subsequent research
demonstrated the major response was due to cobalt in the
alfalfa, molasses and mineral mix (Lamb et al., 1958).
These cattle had increased gain of 0.2-0.3 lb/day and
increased feed intake. Hogue and Elliot (1964) showed
increased vitamin B12 requirement in rats fed 3 or 6% propionate in the diet. Vitamin B12 had a profound effect upon rate of gain (+30.8%) and feed intake (+6.3%) in rats
fed propionate. Therefore, because ionophores increase
the production of propionic acid which has been shown to
increase vitamin B12 requirements in rats, the purpose of
this research is to determine if the performance response to cobalt supplementation is effected when ionophores are
fed in all concentrate diets and to examine metabolic
indicators of vitamin B12 status. Later in this section, methods employed to test the hypothesis will be discussed. Other research addressing this question will also be presented, as will related metabolic theory and field observations. 78
1. Observations
Observations in the field with dairy cattle, beef cattle, and sheep raise questions this research is designed to answer. These field and research observations are presented here. Elliot (1966) estimated the synthesis of lactose in the lactating dairy cow to be 4.5 lb for a cow giving 100 lb per day and discussed the possible stress imposed on the vitamin B12 status of the cow. He presented research findings which show a decrease in serum vitamin B12 levels and liver B12 levels early in lacta tion. Rates of conversion from propionate to succinate were reduced in liver homogenates from early lactation compared to liver samples from later in lactation. This again suggests a marginal or deficient metabolic vitamin
B12 status in early lactation.
Vitamin B^2 involvement in metabolism has been discussed in detail in previous sections. Field observa tions have been made of ketotic lactating cows, where an injection of B^2 alleviated symptoms of ketosis and gets the cow back on feed. Improved milk production and persistence of lactation was also reported with weekly injections of 40,000 meg of vitamin B12 (Sanders, 1988).
Excretion of vitamin B12 in milk, coupled with feeding of high concentrate diets low in cobalt could stress the vitamin B12 status of the dairy cow. Elliot 79
(1980) cited work which showed a 41% increase in propionyl
Co A carboxylase in the liver of the lactating cow when fed an all-concentrate diet compared to a control diet of hay and concentrate. This would suggest the rate-limiting step in conversion of propionate to succinate may be dependent upon the B^2 status of the animal by affecting conversion of methylmalonyl Co A to succinyl Co A.
Activity of phosphoenolpyruvate carboxykinase was also increased when the all-concentrate diet was fed.
Young beef animals are typically "backgrounded" on high roughage diets for some time before moving to the feedlot. Corn silage contains 0.06 ppm cobalt, while grain contains 0.04 ppm (NRC, 1985). Vitamin B12 status of these animals is not known, but cobalt content is low in both the feedlot and backgrounding diet without cobalt supplementation.
Observation of a feed intake depression is common when an ionophore is added to the diet of feedlot beef cattle. This suppressed intake typically occurs early in the feeding period. Wagner (1984) summarizes 32 trials with monensin in which feed intake responses range from an increase of 3.6% in a New Jersey trial, to a decrease in feed intake of 18.1% in a trial in Kansas. The weighted mean of 45 trials was a decrease of 6.2% in feed intake.
He summarizes 14 trials with lasalocid in which feed intake response ranged from an increase of 3.2% to a 80 decrease of 11.6%. The weighted mean of these trials was a decrease of 4.6% in feed intake. Average daily gain was increased an average of 6.4% in these trials. This is presumably due to the increase in propionate relative to acetate, and the increase in metabolic efficiency afforded by this shift in VFA.
Under these circumstances, it is not unreasonable to postulate that the vitamin B i 2 status of the animal is stressed. Corn silage-based diets in the feedlot are quite low in cobalt, and the cattle may not have been fed cobalt supplementation while "backgrounded". An increase in propionic acid should require an increase in vitamin
B12 needed in the conversion of methylmalonyl Co A to succinyl Co A. If B^2 were deficient, a period of time with reduced feed consumption would be expected after each eating period since additional time would be needed to clear additional propionate (Marston et al. 1972).
Effect of location, soil cobalt content or dietary cobalt content on feed intake response with ionophore supplementation has not been studied. The reader is reminded Goodrich et al. (1984) developed regression equations which could account for only 33% of the variability of feed intake and 35% of the variability of
ADG when ionophores were fed to cattle.
Latteur (1962) recognized many regional problems associated with cobalt deficiencies and sub-deficiencies 81 in ruminants. These include classical vitamin B12 deficiencies which occur in specific areas where soil cobalt is extremely low, and marginal cobalt areas where symptoms are less obvious. One such area problem is increased susceptibility to Johnnes Disease in cattle.
Another area-related observation also involves dairy cattle. There have been conflicting reports in the literature concerning the influence of methionine-hydroxy analog (MHA) on ketosis and milk fat test in cows. In some studies, MHA is beneficial in reducing ketosis, while it has no effect in other studies (Shaw, 1946; Schultz,
1959; Schultz, 1971; Fox, 1971; McCarthy, et al., 1968).
One possible explanation of these findings involves vitamin B12• If it were marginal, feeding MHA would spare
B12 from the methionine synthetase reaction in which a methyl group is transferred to homocysteine from
N5-methyltetrahydrofolate to form methionine. This, in turn, would leave more vitamin B12 available for conversion of methylmalonyl Co A to succinyl Co A, and ultimately could result in increased glucose synthesis from gluconeogenesis. If vitamin B12 were adequate, or severely deficient, addition of MHA should have no effect on ketosis. Research in cobalt marginal areas may yield one result, which cannot be repeated in other areas.
In the 1957 review of cobalt and vitamin B^2 by Smith and Loosli, authors address claims of therapeutic value of cobalt for diseases such as sterility, milk fever, ketosis, brucellosis and others. Authors cite the 1947 paper by Henderson (Cornell Vet. 37:292) entitled "Ketosis in dairy cow with emphasis on treatment". Though Hender son reported successfully treating twelve cases of ketosis with cobalt sulfate, Smith and Loosli conclude: "these claims are based on faulty or inadequate experimental techniques, especially the lack of adequate controls. If cobalt plays some role in the prevention or cure of ketosis, it remains to be shown". Fox (1971), writing on the clinical diagnosis and treatment of ketosis, and a veterinarian at the Cornell Ambulatory Clinic, states his usual treatment is I.V. dextrose, plus oral propylene glycol with cobalt added. He reports excellent results from this treatment. Schultz (1959) also discusses cobalt/B12 interrelationships with ketosis.
Nutritional differences in cobalt intake affect the vitamin B12 status of the animal. Response in feed intake to ionophore supplementation is inconsistent and may be geographically influenced. Are these differences in cobalt status responsible for different feed intake responses observed when ionophores are added to high- concentrate diets for ruminants? Does the ionophore increase metabolic need for vitamin B^2 as well? Answers to these questions would be helpful in better 83 understanding the effects of ionophores, cobalt, and B12, on all or high concentrate fed ruminants.
Experiments to measure response in average daily gain and feed intake to supplemental cobalt and/or injected vitamin B12 will be conducted with lambs fed all concentrate diets as a preliminary step. Finally then experiments to investigate the response of growing lambs fed all concentrate diets to ionophores with and without supplemental cobalt will be conducted. Determinations of liver changes in vitamin B12 levels and estimates of propionate clearance time in blood will also be made on a representative sample of these animals. 84
2. Methodology
Young (1979), Underwood (1981), Smith and Marston
(1970a), Smith and Loosli (1957), Somers (1969), Gawthorne
(1968), Marston et al. (1972), Latteur (1962), and Filmer
(1933) have discussed relationships between soil cobalt, cobalt content of plants grown on that soil, and meta bolic, physiological and observed performance differences in animals fed plants of varied cobalt content. Wagner
(1984) summarized the changes in performance charac teristics due to ionophores in the diet, from numerous studies.
Observation of differences in feedlot performance has been a criterion in both cobalt/vitamin B^2 research and work done with ionophores. Clark et al. (1985) state "the most conclusive way of determining whether animals are deficient in a trace element is to measure production responses to supplementation in a field trial". Since a field trial is not always possible, an attempt is made to correlate tissue testing with trace element status in the animal. Metabolic evidence of effect of ionophores on B^2 metabolism could include excretion of formiminoglutamic acid and/or methylmalonic acid, blood propionate clearance rates, blood and liver B12 concentration, changes in B^2 concentration, and feedlot performance in a field trial. 85
Under both field conditions with cobalt, and research conditions using B i 2, combination of one or more of the above variables should give indication of the effect of ionophores on B12 status of the animal. Mills (1987) discusses physiological indicators of cobalt status, as does Marston (1935), and Constable (1985). The increased sensitivity of FIGLU versus MMA, as shown by Giorgio and
Plaut (1965), plus the availability of FIGLU assay kits made it the test of choice. Difference in B^2 level between liver biopsy samples taken prior to ionophore feeding and slaughter samples should quantify the effect of ionophores on liver B12 stores.
Among feedlot variables to be measured, feed intake, average daily gain (ADG), and feed efficiency (feed/gain) are most commonly reported. For the 56, 70 and 84 d trials herein reported, weights taken every 14 d should provide sufficient information to draw conclusions. Smith and Marston (1970b) discussed feed intake, body weight, and other variables relative to B12 status during the onset and remission of B i 2 deficiency.
Research animals should be rapidly growing, healthy animals, acclimated to concentrate feeding and not under excessive stress. For these reasons, lambs were weaned when consuming approximately 0.5-1.0 kg of creep feed and weighing approximately 13.5-18.1 kg. Lambs were provided with the creep diet ad lib. for 10-14 days after weaning 86 before being assigned to treatment diets and being moved into pens for the trial period. This provided an adjust ment period to reduce the influence of post-weaning stresses on the trial results.
Each lamb was weighed at birth and given 1/2 cc injection of Bo-Se selenium/vitamin E emulsion, 1 cc vitamin A and D, and care was taken to assure each lamb received colostrum from its dam or stored ewe colostrum within the first 12 hrs after birth. All lambs were de-wormed with TramisolR and vaccinated for enterotoxemia and tetanus with Covexin-8R per manufacturer's directions prior to initiation of the trial.
Most importantly, research studies should be designed to avoid confounding effects. In feedlot studies, lambs were allotted to provide an equal distribution of wether and ewe lambs, and single, twin, and triplet lambs in each pen. This provided equal distribution across treatment combinations. In metabolism studies, twin wether lambs were selected, having similar breeding, age, and weight in an attempt to reduce confounding effects.
Young growing lambs fed ground and pelleted all concentrate diets were chosen as a model for feedlot beef cattle. This feedlot situation should provide for maximum growth and, thus, maximum B12 stress. Work discussed in earlier sections showed an increase in the amount of pseudo-B12 relative to true B12 when high concentrate 87
diets were fed. This should further stress the vitamin
B12 status of the lambs and provide opportunity to measure
effects of vitamin B i 2, c°balt and ionophores.
Observation of reduced blood propionate clearance was
first reported by Marston et al. (1961) and impaired propionate metabolism is considered to be the primary manifestation of B12 deficiency in ruminants. The method
to determine propionate clearance was employed by Marston
et al. (1961), and later by Sommers (1969), and Marston et
al. (1972). It involved the infusion of sodium propionate
into the jugular vein at a rate of 3.5 mmole per kg body wt in a 1% solution. The tungstic acid plasma filtrate was then steam distilled and the distillate titrated to d e t e r m i n e v o l a t i l e fatty acid (VFA) concentration.
Sommers (1969) reported using gas-liquid chromatography to determine individual VFA and found the change in total VFA was relative to the acid infused. Ross and Kitts (1971)
reported an improved method of determining blood VFA which
involved steam distillation, but required 20 ml blood for each analysis. A procedure using 2 ml blood plasma and
freeze-drying for the determination of blood VFA is
recommended by Supelco, Inc. (Anonymous, 1975). Chase
(1985) further discussed this method for determining VFA
in blood. Jenkins (1989) provided a modification of the method used by Bjorkman and Forslund (1986) which was accurate, repeatable, and reasonably simple to perform. 88
The procedure extracts the VFA and denatures the protein
simultaneously using cold ethanol. VFA is stabilized with
sodium hydroxide and concentrated by drying the ethanol in
a vacuum oven. VFA is acidified and liquified in metaphosphoric acid immediately prior to injecting the
sample into the gas chromatograph.
Newer methods of assay for B i 2 such as radioassay
techniques have been shown less reliable than microbiological methods (Millar and Penrose 1980). The generous offer of Ross Laboratories division of Abbott
Laboratories to permit this author to assay liver samples
in their laboratory precluded other possibilities. Ross
Laboratories assay method used the test organism
Lactobacillus leichmannii. which would have been the method of choice for liver samples, had this offer not been made.
Liver vitamin B12 levels are indicative of the B12 status of the animal and represent true B12 under normal conditions (Sutton and Elliot, 1972). By taking a liver biopsy sample prior to treatment being administered, and comparing this B12 level to a liver sample obtained at slaughter, the treatment effect can be assessed.
Slaughter liver vitamin B12 level can also be used to assess the B12 status of the animal (Andrews et al., 1958). Young (1979) discussed the role of cobalt in soil, bacteria, plants, and animals. In mammals, the only known function is as part of the vitamin B12 molecule in two specific enzymatic reactions. Marston et al. (1961) reported the primary metabolic defect in B12 deficiency in sheep was an impaired propionate metabolism. This was demonstrated using blood propionate clearance rates after propionate infusion into the jugular vein as the indicator of choice. Somers (1969) and Marston et al. (1972) demonstrated the impaired propionate metabolism in the
B12-deficient sheep by showing delayed blood propionate clearance rates. Acetate clearance was reportedly delayed as well in the study by Somers (1969) , but Marston et al. (1972) reported impaired acetate metabolism was a secondary result of impaired propionate metabolism, rather than a primary result of B12 deficiency. 90
3. Other Research on this Question
Only a few other researchers have conducted studies with this research objective, and to date it has not been demonstrated under field conditions. Hembry et al.,
(1979) showed an increased ADG in cattle fed 33 ppm monensin plus 2 ppm cobalt compared to monensin alone.
Cattle fed monensin alone consumed less feed than cattle fed the control diet containing 0.45 ppm cobalt. Cattle fed 2 ppm cobalt plus monensin consumed more feed than cattle fed the control diet. Hill et al. (1980a,b) presented results of follow up studies conducted with beef cattle fed combinations of monensin and 2 ppm additional cobalt. ADG, dry matter intake, and feed efficiency were all slightly improved in cattle fed cobalt in addition to monensin when compared to cattle fed monensin without supplemental cobalt. Zinn et al. (1987) reported no difference in feedlot performance for steers supplemented with ten water soluble vitamins to a level equal on a body weight basis to a 35 kg pig. There was significantly
(P<.10) less sickness in vitamin supplemented than control animals (33.1 vs. 50.2 %). Cobalt was supplemented with
0.5% trace mineralized salt containing 0.068% cobalt.
This should have added 0.34 ppm Co to the total diet.
No additional research was conducted with beef cattle to examine this question. Georgievskii et al. (1979), however, report the accepted level of cobalt in diets of sheep and cattle (beef and dairy) in the Soviet Union is
1.0 ppm. Data are presented showing a 22.9% increase in milk production for the first two months of lactation when zinc, manganese, cobalt, and iodine were supplemented to
80, 60, 1.0, and 0.55 ppm, respectively. Copper was also supplemented. The initial level of cobalt was 0.24 ppm, and the level of vitamin B12 in the milk was increased from 3.77 to 4.75 ug/1 when the cobalt level was increased from 0.24 to 1.0 ppm, respectively. Toxic levels of cobalt were reported to be 100-200 ppm.
Experiments conducted in New Mexico by Daugherty et al. (1986) compared performance and metabolic character istics of lambs fed monensin with or without weekly injections of cyanocobalamin. No positive benefits were observed as a result of vitamin B i 2 injections in ADG, dry matter intake, or feed efficiency. No differences were observed in serum or liver B12 levels as a result of the treatment combination, however, monensin significantly decreased fumarate and malate formation by liver homo- genates. The basal diets were supplemented with 0.5% of a trace mineralized salt which contained 0.011% cobalt to simulate common feedlot conditions. This should have added 0.55 ppm cobalt to the diet. Cobalt content of the diets, however, was reported to be 0.25 ppm. Lambs used in these studies averaged 27.5 kg when weaned and were fed a 60% concentrate diet for 17 d prior to application of
the treatment combinations. The liver B12 data presented
by Daugherty (1984) is at variance with Wahle and Livesey
(1985) in which monensin increased serum and liver vitamin
B12 levels. Lindemans et al. (1986) suggested an
increased excretion of vitamin B i 2 in the rat when fed monensin, since monensin inhibits calcium-dependent
endocytosis, and the reabsorption of transcobalamin
II-bound cobalamin is a calcium dependent process. The
research and observations discussed above have led to the
studies herein presented. CHAPTER III
MATERIALS AND METHODS
93 OUTLINE OF MATERIALS AND METHODS
Preliminary Studies 1984-85
EXPERIMENT 1-
Cobalt, Monensin, and Lasalocid Feedlot Study
EXPERIMENT 2-
Vitamin B12 & Monensin Metabolism Study
EXPERIMENTAL METHODS 95
A. Preliminary Studies 1984-85
Several preliminary studies were conducted to learn methodology and answer questions relative to design and execution of the feedlot and metabolic studies reported in sections B and C. In the initial trial, four male Dorset lambs, weighing approximately 35.75 kg were fed a diet containing 54 ppm lasalocid and 0.1 ppm cobalt.
Composition of the pelleted, corn and soybean meal-based diet is shown in table 4. Analysis was done on composite samples.
Lambs were of similar age and born from ewes fed corn silage as a major constituent of their diet during late gestation and lactation. Lambs were assigned to treatment combinations with a 2 X 2 factorial assignment of cobalt and vitamin Bi2. One lamb was assigned to each treatment combination at random. The treatments consisted of either an injection of vitamin (1000 micrograms cyanocobalamin bi-weekly), or an injection of saline solution (1 cc bi-weekly). An intraruminal cobalt bullet2 and a grinder screw (16.5 X 11 mm) was administered to one pair of lambs receiving the above treatments to supplement cobalt.
^■Vitamin B12 (Cyanocobalamin) courtesy of Med-Tech, A TechAmerica Company, Elwood, Kansas.
2Cobalt Bullets courtesy of Wellcome Research Laboratories, Kansas City, Kansas. 96
TABLE 4 Composition of diet for preliminary study (100% DM Basis)
Inaredient Beet pulp (%) 10.00 Ground corn (%) 60.00 Soybean meal 44% (%) 20.00 Premix1 (%) 10.00
Analysis Crude protein (%) 18.97 Calcium (%) 0.93 Phosphorus (%) 0.69 Potassium (%) 0.97 NaCl (%) 0.25 Magnesium (%) 0.25 Sulfur (%) 0.19
Zinc (ppm) 139 Iron (ppm) 447 Manganese (ppm) 43 Copper (ppm) 15 Molybdenum (ppm) 2.5
Cobalt (ppm) 0.10 Lasalocid (ppm) 57
1 Premix contained (%) corn, 37.698; soybean meal, 20.5; limestone, 15.0; Biophos, 15.5; Potassium chloride, 3.2; Iodized salt, 2.5; Magnesium oxide, 0.48; Sodium sulphate, 3.2; Zinc oxide, 0.125; Ferrous sulphate, 0.343; Manganese sulphate, 0.043; Copper sulphate, 0.050; Sodium selenite, 0.0006; Potassium iodide, 0.404; Vitamin A (30,000 IU/gm), 0.067; Vitamin D3 (3000 IU/gm), 0.067; Vitamin E (20,000 IU/lb), 0.454; Bovatec-68 ^ lasalocid premix (68 gm/lb), 0.386. Cobalt bullets were manufactured to CSIRO specifications and contained 60% cobalt oxide (w/w). Assignment of treatment combinations is shown in table 5.
TABLE 5 Treatment assignment for Preliminary Study
Lamb I.D. Cobalt bullet Vitamin
576 + + 575 + - 572 - + 570 •
Lambs were weighed after 20 days to determine weight gain. Lambs were kept in a common pen with the pelleted diet available on a free-choice basis. Feed intake was determined during a 5-day period with the lambs kept in individual metabolism crates fed free-choice. Two 24-hour collections of urine were taken, and blood samples were collected over a six-hour period following feeding to determine propionate clearance rates. Feed intake was determined as the average of the feed intake for five days while the lambs were in the metabolism crates. 98
A second preliminary study was conducted in the
summer of 1985 to determine if performance of lambs was
enhanced by injecting 1000 meg cyanocobalamin3 per week
compared with 1 cobalt bullet4 per os at the initiation of
the study. The study was a completely randomized design
experiment with a 2 x 2 factorial assignment of treatments
of the cobalt bullet and vitamin B^2 injections.
Sixty-four weanling lambs were allotted to eight pens and
one of four treatment combinations. Lambs were from
outcome groups of sex, birth type, and weight using
stratified randomization. This provided approximately
equal numbers of wether and ewe lambs, singles, twins and
triplets in each pen and across each treatment
combination. Lambs were fed creep feed Sh-117-81 on a
free-choice basis prior to weaning and for 14 days post-weaning. Formulation of the creep feed is shown in
table 6. After the 14 d adjustment period, they were weighed and allotted to the pens and treatment
combinations for the trial period.
3Vitamin B 12 (cyanocobalamin) courtesy of Med-Tech, A TechAmerica Company, Elwood, Kansas.
4Cobalt bullets courtesy of Wellcome Research Laboratories, Kansas City, Kansas. 99
TABLE 6 Composition of creep feed
Ingredient (%)
Alfalfa meal 30.000 Ground corn 38.135 Soybean meal 44% 27.168 Biophos 0.871 Limestone 0.555 Sodium bentonite 2.000 Trace mineralized salt1 0.500 Ammonium chloride 0.500 Selenium-90 0.100 Z-73 (ZnO), 73% zinc 0.008 Aureomycin 50 0.050 Vitamin A (30,000 IU/gm) 0.007 Vitamin D3 (200,000 IU/gm) 0.018 Vitamin E (2 0,000 IU/lb.) 0.088
1 Trace mineralized salt, Morton Salt Co. contained (%) salt, 93-98; zinc, 0.35; manganese, 0.28; iron, 0.175 copper, 0.035; iodine, 0.007; cobalt, 0.007.
Lambs were fed grower diet SH-10-85 on a free-choice basis during the trial. This diet was formulated to contain 33 ppm lasalocid and was a pelleted corn and soybean meal-based diet. Composition of the grower diet
is shown in table 7. During the 70 day trial, lambs had
free-choice access to water and the pelleted grower diet.
Lambs were weighed and feed intake was determined every 14 days.
Data were analyzed using the general linear models program of the Statistical Analysis Systems Institute,
Inc., Cary, N.C. for main effects of cobalt and vitamin 100
B12 and the interaction between cobalt and vitamin B12.
Mean weight gain per pen, feed intake per pen and feed
efficiency (feed/gain) per pen were the variables compared
in the statistical analysis.
Table 7 Composition of grower diet (100% DM Basis)
Ingredient Corn oil 1.271 Ground corn 72.759 Soybean meal 44% 21.919 Limestone 1.389 Biophos 1.610 Dynamate 0.3 02 Iodized salt 0.250 Magnesium oxide 0.051 Selenium-90 0.100 Zinc oxide 0.013 Potassium chloride (Dyna-K) 0.205 Vitamin A (30,000 IU/gm) 0.007 Vitamin D3 (3000 IU/gm) 0.007 Vitamin E (20,000 IU/lb) 0.045 Trace mineral premix-i 0.050 Lasalocid premix (Bovatec-68) 0.022
Analysis Crude protein (%) 19.46 Calcium (%) 1.15 Phosphorus (%) 0.78 Potassium (%) 1.03 Sodium (%) 0.15 Magnesium (%) 0.28 Sulfur (%) 0.30 Zinc (ppm) 145 Iron (ppm) 318 Manganese (ppm) 50 Copper (ppm) 11 Molybdenum (ppm) 2.9
1 premix contained (%) Ground corn, 33.679; Cupric sulphate (5 H20), 7.852; Ferrous sulphate, 40.0; Manganese sulphate, 18.469. No differences were detected between treatments,
therefor, the following conclusions were drawn: 1)
Synthesis and/or absorption of vitamin B12 was not
limiting performance when abundant cobalt was present in
the rumen since no difference in performance was observed
between lambs given cobalt bullets and lambs given cobalt
bullets plus vitamin B12 by injection and/or; 2) Liver
storage of vitamin B12 may be sufficient for the length of
the study since the lambs were fed a creep feed containing
cobalt prior to the study and fed a diet containing
lasalocid, but no added cobalt during the study and no
differences in performance were observed due to cobalt or vitamin B12 compared to lambs on the control treatment.
Based on data gathered and methodology developed with
the preliminary studies, the following experiments were
conducted to test the hypothesis. 102
B. EXPERIMENT 1
A study was conducted in the Spring and Summer 1987 to determine the effect of cobalt, monensin, and lasalocid on feedlot performance and jugular propionate clearance rates in lambs.
One-hundred and two fall-born and ninety spring-born lambs were randomly assigned to one of six treatment combinations of cobalt (0.0 or 0.4 ppm added) and feed additive (0, 27 ppm monensin or 54 ppm lasalocid) in a randomized complete block design experiment. To comply with federal regulations and enable the lambs to be slaughtered for food, two Investigational New Animal Drug
Exemptions (INADs 4878 and 4881) were obtained from the
Food and Drug Administration (Appendix A) . Lambs were assigned to treatment combinations from outcome groups of sex, birth type, and weight using stratified randomization. Approximately equal numbers of wether and ewe lambs, singles, twins, and triplets were in each pen and assigned to each treatment combination. Three replicates were conducted with fall-born lambs and three replicates were conducted with spring-born lambs with season of birth serving as blocks. Treatment diets were randomly assigned to pens with five or six lambs per pen, giving a total of 6 pens per treatment combination. 103
Lambs were fed pelleted creep feed (SH-26-86) free-choice during the lactation period, and were weaned when they weighed approximately 15.9 kg. The composition of the creep feed is shown in table 8.
TABLE 8 Composition of creep feed diet
Incrredient (%) Ground corn 59.113 Soybean meal 44% 36.075 Limestone 1. 686 Biophos 1. 385 Iodized salt 0. 500 Magnesium oxide 0. 075 Selenium-90 0.150 Zinc oxide 0.014 Copper sulphate 0.002 Propionic acid 1.000
Calculated analysis Crude protein (%) 23. 64 Calcium (%) 1.13 Phosphorus (%) 0.81 Magnesium (%) 0.23 Potassium (%) 1.07 Zinc ppm 104 Copper ppm 8 Selenium ppm 0.32
During the gestation period, ewes were fed a mineral supplement (SH-21-86) which contained lasalocid, but no cobalt in addition to free access to fescue pasture.
Formulation of this supplement is shown in table 9. This theoretically would reduce vitamin B 12 stores of the ewes and therefore reduce the B ^2 stores of the lambs. 104
TABLE 9 Composition of mineral supplement ingr.e.di.e?xt (%) Iodized salt 89.270 Copper sulphate 0.708 Zinc oxide 2.038 Sodium selenite 0.011 Ferrous sulfate 1.021 Bovatec-68 6.959
During lactation, mature fescue grass hay was fed at a rate of approximately 500 gm/hd/day to the ewes. Ewes were also fed 1000-2000 gm of a concentrate mix (SH-25-86) per day depending upon the level of milk production. Ewes with twins or triplets were fed 1500 or 2000 gm/hd/d, respectively, while ewes with singles were fed 1000 gm/hd/d. This diet contained no supplemental cobalt. The composition of the concentrate mix is shown in table 10. 105
TABLE 10 Composition of concentrate mix1
Inaredient (%} Shelled corn 75.418 Ground corn 0.004 Soybean meal 44% 21.270 Limestone 1.130 Biophos 0.300 Iodized salt 1.000 Magnesium oxide 0.120 Sweetone dry molasses 0.600 Selenium-90 0.150 Zinc oxide 0.008 Copper sulphate 0.004
Calculated Analvsis Crude protein (%) 17.75 Calcium (%) 0.70 Phosphorus (%) 0.51 Magnesium (%) 0.23 Potassium (%) 0.82 Zinc ppm 109 Copper ppm 8 Selenium ppm 0.32
1 All ingredients except shelled corn were mixed am pelleted, then mixed back with shelled corn.
Feedlot studv-Lambs were fed the creep diet described
above for 14 d post-weaning, then weighed, vaccinated with
Covexin-8R , de-wormed with TramisolR , and moved into pens with pelleted treatment diets available on a freechoice
basis. Lambs were weighed and feed weighed-back every 14
d to determine individual gain and pen feed intake,
respectively, for the 84 d trial. Average daily gain
(ADG) and feed efficiency (FE) were computed from the mean pen gain and pen feed intake. Water was available on a free-choice basis in each pen, and wood chips were used as bedding. After the feeding period, lambs were placed on the respective control diet (with or without cobalt, but no feed additives) for at least 10 days prior to slaughter at Rocco Further Processing, Inc., Timberville, Virginia.
Urine and serial blood samples were collected from four lambs from each treatment combination after the lambs had completed the 84 day trial, and while still being fed treatment diets. Liver samples were collected at slaughter for vitamin B12 assay. Fifty-two fall-born ewe lambs were kept for replacement breeding stock and were not slaughtered. Dietary treatment combinations of cobalt
(0.0 or 0.4 ppm added) and feed additive (0, 27 ppm monensin or 54 ppm lasalocid) are shown in table 11.
TABLE 11 Dietary Treatment Assignment for feedlot study1
Diet Cobalt2 Monensin3 Lasalocid4
Co- CON _ — Co- MON - + - Co- LAS -- + C0+ CON + - - Co+ MON + + - Co+ LAS + +
1 Diets Co- CON and Co+ CON also used in EXPERIMENT 2 2 0.4 ppm added cobalt as cobalt chloride 3 27 ppm monensin added 4 54 ppm lasalocid added
The analysis and formulation of these diets are shown in table 12. 107
TABLE 12 Composition of treatment diet
Ingredient (%)
Corn oil 1.270 Ground corn 72.776 Soybean meal 44% 21.919 Limestone 1.389 Biophos 1.616 Dynamate 0.302 Iodized salt 0.250 Magnesium oxide-58% 0.051 Selenium-90 0.150 Zinc oxide 0.013 Potassium chloride 0.205 Vitamin A (30,000 IU/gm) 0.007 Vitamin D3 (3000 IU/gm) 0.007 Vitamin E (20,000 IU/lb) 0.045 Trace mineral premix-1- 0.050 Feed additive premix2
Analysis (100% Drv Matter Basis! Crude protein (%) 19.08 Calcium (%) 1.16 Phosphorus (%) 0.78 Potassium (%) 1.04 Sodium (%) 0.13 Magnesium (%) 0.30 Sulfur (%) 0.30 Zinc (ppm) 142 Iron (ppm) 430 Manganese (ppm) 41 Copper (ppm) 11 Molybdenum (ppm) 2.5 Selenium (ppm) 0.33
1 Premix was included according to treatment. Co premix contained (%) : Corn, 61.57; Cobalt chloride (6 H20), 0.20; Cupric sulphate (5 H20) , 7.85; Ferrous sulphate, 24.23; Manganese sulphate, 6.15. No-Co premix contained (%) : Corn, 33.679; Cupric sulphate (5 H20), 7.852; Ferrous sulphate, 40.0; Manganese sulphate, 18.469.
2 Diet contained: a) no feed additive; b) 0.021 % Rumensin-60 premix; or c) 0.037% Bovatec-68 premix replacing corn according to treatment. Feed samples were collected at the OARDC feed mill
from each batch of feed and compiled for analysis. Feed samples were kept frozen at -18° C. until being submitted
for analysis in February 1988. Nutrient composition was determined at Holmes Laboratory, Inc., the Research-
Extension Analytical Laboratory, A & L Great Lakes
Laboratory, Agway Technical Services Laboratory, and
Hazelton Laboratories, Inc. by methods listed under
Section D of this chapter. Lasalocid activity was determined using high-performance liquid chromatography
(HPLC) by Analytics Laboratory-a subsidiary of Roche
Biomedical Laboratories, Inc., Richmond, Virginia, through the courtesy of Hoffmann-La Roche, Inc., Nutley, New
Jersey. Activity of monensin was determined by Lilly
Research Laboratories division of Eli Lilly and Company,
Indianapolis, Indiana using HPLC, through the courtesy of
Elanco Products, Inc. and Lilly Research Laboratories,
Greenfield, Indiana. 109
Statistical analysis of data-Data were analyzed using the general linear models program of the Statistics Analysis
Systems Institute, Inc. Data were analyzed using season as a block (fall-born versus spring-born lambs). The model included diet as the variable with planned contrast statements to determine main effects of cobalt, monensin, lasalocid, and the interactions of cobalt*monensin, and cobalt*lasalocid. Comparison of means for monensin and lasalocid were also made. The ANOVA table for the feedlot data is shown in table 13 below.
Table 13 ANOVA for EXPERIMENT 1
Source Degrees of Freedom fDF)
Model Diet 5 Season 1
Error 29
Corrected total 35
Contrast
No Co vs. Co No Mon vs. Mon No Las vs. Las Co*Mon Co*Las Mon vs. Las 110
C. EXPERIMENT 2-
Vitamin B12 & Monensin Metabolism Study
Sixteen wether lambs of similar weight and age were
moved to OARDC, Wooster, Ohio and randomly assigned to one
of four treatment combinations in a 2 X 2 factorial
arrangement of vitamin B12 and monensin. Lambs were born
from the ewes described in the feedlot study, above, and were fed the same diets and treated identically with those
ewes. Treatment combinations were as follows: treatment
one (positive control) received diet Co- MON (27 ppm monensin) and 1000 meg. cyanocobalamin by IM injection per week; treatment two received diet Co- MON and 1 cc physiological saline solution by IM injection per week; treatment three received diet Co- CON (no feed additive) and the B12 injections; and treatment four (negative control) received diet Co- CON and the saline injections.
Lambs were housed in individual wooden metabolism crates with free-choice access to feed and water. Lambs were weighed at the beginning of the trial and periodically throughout the test period. Liver biopsy samples were taken prior to application of the treatments, and urine was collected twice during the test period for
FIGLU analysis. Liver samples were collected at slaughter
for B 12 assay. I l l
Statistical analysis of data-The analysis of data was conducted as in the feedlot study, .above. The ANOVA for this experiment is shown in table 14, below.
Table 14 ANOVA for EXPERIMENT 2
Source Degrees of Freedom (DF)
M o d e l : Monensin 1 Vitamin 1 Monensin*Vitamin B12 1
Error 12
Corrected total 15 112
D. EXPERIMENTAL METHODS
Methods employed in analysis of feeds-The feed analyses
(a. - i.) were conducted by Holmes Laboratory, Inc.5,
Millersburg, Ohio.
a. Dry Matter = 100% - moisture%. Moisture was determined using a convection oven at 105° C. overnight using method 7.008, p. 135, AOAC, 1975.
b. Crude Protein (CP) determination was done using the Kjeldahl nitrogen determination (Scales and Harrison,
1920). CP = Kjeldahl N x 6.25.
c. Acid detergent fiber (ADF) and Neutral detergent
Fiber (NDF) were determined using the methods of Goerring and Van Soest, 1970.
d. Mineral Analyses (Calcium, Magnesium, Potassium,
Copper, Manganese, Zinc, Iron, Molybdenum, Sodium) was done with a Perkin-Elmer Model 560 double-beam Atomic
Absorption Spectrophotometer using methods no. 7.077 and
2.096 (AOAC, 1975).
5Methodology herein reported was provided courtesy of Mr. Gary Horrisberger, Manager. 113
e. Phosphorus determination was done using the
colorometric method no. 7.103 (AOAC, 1975).
f. Chloride was determined using a solid-state
specific ion electrode with known addition of chloride.
(Cantliffe, et al. 1970.)
g. Sulfur was determined gravimetrically using
method 3.059 on page 43 (AOAC, 1975).
h. Cobalt was analyzed by A & L Laboratories6,
Memphis, Tennessee, using atomic absorption spectro
photometry by wet ashing the sample in nitric and per
chloric acids, diluting to volume, and aspirating directly
into the flame.
Cobalt was determined by the Research-Extension
Analytical Laboratory7, Ohio Agricultural Research and
Development Center, Wooster, Ohio, using Inductively—
coupled plasma spectroscopy.
Cobalt determination by Agway Technical Services8,
Ithaca, New York, was done using a Perkin-Elmer Model 503
Atomic Absorption Spectrophotometer with a Model HGA 2100
Graphite Furnace. Sample size was increased to 5 gm and
6Methodology provided courtesy of Mr. Dan Moffett.
7Methodology provided courtesy of Dr. Maurice Watson.
8Methodology provided courtesy of Mr. Richard Rice. 114
ash was dissolved in 10 ml of 20% HC1. Cobalt was
extracted with ammonium pyrrolidine dithiocarbamate and
methyl isobutyl ketone (Popova, et al., 1974).
Cobalt was analyzed by Hazelton Laboratories, Inc.,
Madison, Wisconsin using a Perkin-Elmer atomic absorption
spectrophotometer. The method used was found in Perkin-
Elmer Analytical Methods for Atomic Absorption
Spectrophotometer. Norwalk, Connecticut (Jan. 1982), and
Methods for Chemical Analysis of Water and Waste (1979),
Metals 1-19, Method 219.1, US EPA, Cincinnati, Ohio.
i. Selenium was analyzed by the Research Extension
Analytical Laboratory9, Ohio Agricultural Research and
Development Center, Wooster, Ohio, using the method of
Olson, et al. (1975. J. Assoc. Official Anal. Chem.
58:117).
Urine collection-
The 24-hour collection of urine was accomplished by
placing the lamb into a galvanized steel (preliminary
study) or wooden (experiments 1 and 2) metabolism crate.
The lamb was given free access to feed and water during
the collection period. Urine was collected into an
acidified 2 1/2 gallon plastic bucket through a large
funnel which had six layers of cheese cloth to prevent
9Methodology was provided courtesy of Dr. Maurice Watson. 115
fecal material, wool, or other foreign material from contaminating the urine.
After 24 hours, the urine was measured using a 1000 ml graduated cylinder and a 50 ml sample was taken to which 1.0 ml concentrated HCL was added. The samples were then frozen until assayed for formimino-L-glutamic acid
(FIGLU). Blood propionate clearance studies-The propionate infu sions and serial blood samples were collected from sheep restrained in metabolism crates. Sheep were fasted for 14 h prior to collecting the first sample and fed following the last sample. Wool was shorn from the throat, and the throat scrubbed with Betadine surgical scrub and flushed with ethanol. A 14 gauge, 5 1/4 inch Deseret Angiocath IV
Catheter placement unit was inserted into the jugular vein, and blood samples were taken through the catheter.
Infusions were made over a 5 minute period with an IV bag with a flow adjustment. Ten ml of sterile physiological saline solution was slowly infused through the catheter immediately prior to taking each of the serial blood samples. Samples were kept in a refrigerator at 5° C after being collected and kept on ice while being transported from EORDC to OSU. The samples were then centrifuged (1325 x g) for 15 minutes and frozen until preparation for VFA analysis.
Blood collection-individual blood samples were taken with a Monoject 10 ml blood collection tube with sodium heparin. The collection needles were 20 gauge, 1 1/2 inch single sample B-D Vacutainer needles with a plastic sleeve holder. Samples were taken prior to feeding (tQ), 15 minutes (t]_), 30 min. (t2 ), one hour (t3), two hours (t4), four hours (t5) , and six hours (t6) post-feeding. The 117 blood samples were centrifuged (1325 x g) for 15 minutes and frozen until preparation for VFA analysis.
Blood VFA Analysis-Blood VFAs were analyzed using gas- liquid chromatography. The procedure was adapted from
Jenkins, (1989) which was a modification of the procedure of Bjorkman and Forslund (1986). Cold ethanol (5 ml at 0°
C +/- 2) was added to a test tube containing 1.0 ml blood plasma sample and 50 ul internal standard (ISTD). The test tube was covered with ParafilmR, mixed, using a
Vortex GenieR and allowed to stand for 10 min. The tube was then centrifuged (1325 x g) for 10 min. The supernatant was then transferred into a test tube containing 500 ul 10 mM sodium hydroxide. The tube was allowed to stand for 30 minutes prior to being placed into a vacuum oven at 50° C overnight to complete the drying process. The residue was dissolved into 500 ul metaphosphoric acid immediately prior to injection into the chromatograph.
All samples (1 ul) were injected into the Hewlet
Packard model 5890 gas-liquid chromatograph equipped with a model 3392A Hewlet Packard integrator. Samples were injected manually using a Hamilton 700 N syringe.
Nitrogen was used as a carrier gas with a flow of 35.0 ml/min. The range was set at 4 with column, FID, and inlet temperatures of 110, 200, and 180° C, respectively. 118
Integrator was set at attenuation of 3, chart speed of
0.5, peak width of 0.04, threshold of 2, and area rejection of 500.
The column used for VFA analysis was packed with 10%
AT-1200 with 1% phosphoric acid on 80/100 mesh Chromosorb
W HP support. Column was an Alltech 6087 (6' x 1/4" x 2 mm) DEACTIGLASS configuration A. Column was conditioned after 6-8 samples for 1-3 hours at 125° C, then re-packed and conditioned after 4-6 additional samples.
Retention time on propionic acid was approximately
3.9 minutes with the ISTD eluting at 5.25 minutes.
Analysis was temperature programmed to maintain a temperature of 120° C for 3 minutes following 8 minutes at the initial temperature. Program included a rate change of 5° per minute before reaching the final temperature.
The ISTD was prepared by bringing 11.6 gm 2-ethyl butyrate to volume in a 100 ml volumetric flask with distilled water. This was labelled "primary ISTD". The working ISTD was prepared by bringing 1 ml of primary ISTD to volume in a 100 ml volumetric flask with distilled water. The working ISTD was prepared daily from the primary ISTD and was used for addition to plasma samples.
FIGLU Assay-The assay for FIGLU in urine was a spectro- photometric assay using Sigma Diagnostics kit no. 365 from
Sigma Chemical Co., St. Louis, Mo. The method is based on 119 that of Tabor and Wyngarden (1958). Urine samples were centrifuged for 10 minutes in an Ependorf microcentrifuge before initiating the assay procedure. Duplicate samples of 0.25 ml urine, 0.20 ml tetrahydrofolate solution, and
0.3 0 ml nanopure water were made. To one labeled "test",
0.25 ml of FIGLU enzyme was added, and to the tube marked
"blank", 0.25ml nanopure water was added. Tubes were incubated in a 25°C water bath for 30 minutes, after which
0.3 ml 10% perchloric acid was added. Solutions were mixed using a vortex genie and placed into a boiling water bath for 1 minute, followed by an ice bath for 1 minute.
The contents of the tube were then transferred to a micro centrifuge tube and centrifuged for 10 minutes. The test sample was read against the blank at 365 nm and the absorbance recorded. The FIGLU concentration was determined by comparing the absorbance of the test to the enzyme blank reported by Sigma for the particular lot of enzyme.
Liver biopsv-Skin was shaved and scrubbed with Deseret
Solo-Prep topical gel and flushed with ethanol. A mixture of Rompun (0.04 mg/lb) and Ketamine (2 mg/lb) was injected into the jugular vein. After the lamb was anesthetized, approximately 6 ml Lidocaine HCL (2.0% plus 10.0 meg. per ml) was injected subcutaneously in several sites. Using a
15.8 cm Tru-Cut biopsy needle (Travenol Laboratories) with 120 a 20 mm sample notch, the liver biopsy was taken from the
10th interspace, approximately 1/3 of the distance down the body wall from the transverse process to the midline.
Skin was flushed with the surgical scrub and a 3 cc injection of Combiotic was given to the lamb, after taking the sample. Biopsy samples were frozen until ready to assay for vitamin B^.
Collection of liver samples at slauahter-Lambs were slaughtered at Rocco Further Processing, Inc., Timber- ville, Virginia. After evisceration, liver was inspected by the USDA inspector, liver abscesses (if any) were noted, and liver sample was taken and placed into a zip-lock freezer bag. Samples were placed in ice in an ice chest and transferred into a freezer upon arriving at
The Ohio State University, approximately ten hours after being taken and kept frozen until ready for assay for vitamin B12. 121
Assay of vitamin B^2 ln sheep liver-This procedure for
assay of vitamin B12 is adapted from several sources.
Among these are: Methods of Vitamin Assay by The
Association of Vitamin Chemists, Inc. (1966), Vitamin £12
by Lichtenstein, et al. (1961), The Society for Analytical
Chemistry (1956), and the Ross Laboratories, Inc. Vitamin
B ^2 assay method from Ross Laboratories division of Abbott
Laboratories10. This method was developed from the Merck
Index (10th ed.), United States Pharmacopeia (20th ed.),
Food Chemicals Codex (3rd ed.), and Skeggs (1963) in
Analytical Microbiology edited by F. Kavenaugh (Vol. 1,
AOAC, 14th ed.).
Vitamin B12 was extracted from the liver homogenate
using sodium cyanide, then assayed using the Lactobacillus
leichmanii microbiological method.
10Methodology for the assay of vitamin B12 was provided courtesy of Dr. William Thomson, Ross Laboratories, and is used by permission. 122
Extraction procedure: A 1 gram (0.8500 - 1.1500) sample of liver was homogenized for approximately 15 seconds in 5 ml of 1% sodium acetate buffered solution (pH=4.8). The test tube containing the liver sample was kept in ice during the homogenation process.
Homogenate was then transferred to a 150 ml erlenmeyer flask using 25 ml of the sodium acetate solution, bringing the volume of sodium acetate solution to 30 ml. While gently shaking the flask, 0.5 ml of 1% sodium cyanide solution was added and the flask allowed to stand for 30 minutes at room temperature. The flask was then placed into a boiling water bath for 30 minutes, then cooled to room temperature and transferred to a 100 ml volumetric flask. After bringing the solution to volume with nanopure water, the flask was stoppered and mixed by inverting and shaking three times for ten seconds each.
Contents were filtered through Whatman no. 1 filter paper. Three further dilutions were made using nanopure water and extraction solution to the following final volumes: 5 ml extraction solution into a volume of 100 ml; 10 ml extraction solution into 50 ml; and 20 ml of extraction solution into 50 ml. Final dilutions were equivalent to the vitamin B^2 from 1 gram of liver diluted to 2000 ml, 500 ml, or 250 ml, respectively. These three dilutions were then used to assay for B^2 at Ross Labs. 123
Microbiological Assay; Assay of vitamin B ^2 was conducted by the author at Ross Laboratories division of Abbott
Laboratories Inc., Columbus, Ohio. Growth of the test organism, Lactobacillus Leichmannii (ATCC # 7830) in the test tubes containing liver extractant sample was compared to growth in test tubes containing known amounts of cyanocobalamin. Initial Vitamin B 12 Standard Solution was prepared by diluting 0.0802 gm of cyanocobalamin (USP lot # M-l) with activity of 7.8 micrograms B12 Per into a 1000ml volumetric flask with 25 % ethanol. This solution contained approximately 625ng B^/ml. The Stock
Standard Solution contained approximately 125 ng Bi2/ ml and was prepared by diluting 50.0ml Initial Vitamin B12
Standard Solution into a 250ml volumetric flask with 25% ethanol. The Intermediate Standard Solution contained approximately 5 ng B^/ml anc* was prepared by diluting
10.0 ml of Vitamin B12 Stock Standard Solution into 250ml volumetric flask with 25% ethanol. Standard curve solutions contained approximately 50, 100, 200, 300, 4 00,
500, 600, 700, 800, 900,and 1000 pg cyanocobalamin per ml and was made by diluting 0.5, 1.0, 2.0, 3.0, 4.0, 5.0,
6.0, 7.0, 8.0, 9.0,or 10.0 ml of Vitamin B^2 Intermediate
Standard Solution, respectively, into 50.0 ml volumetric flasks with Milli-Q water.
All of the microbiological assay procedures were carried out under yellow light. Sample and standard 124 dilutions were accomplished by using an Autoturb I diluter. Sample loops delivered 0.10 and 0.15 ml of sample into 10.0 ml of assay media into the test tubes.
Assay media was prepared by adding 85 gm of Difco
Bacto B12 Assay Medium USP (0457-15-1) to 1000 ml Milli-Q water and bringing it to a boil for 2-3 minutes. An additional 1000 ml of Milli-Q water was added, and the flask placed into a cooling water bath after mixing. The pH was adjusted to 5.8 with 2.0 M acetic acid or 25% sodium hydroxide when cool.
After diluting samples and standards, test tubes were then covered and autoclaved at 122°C and 15 psi for 10 minutes. When cool, the test tubes were inoculated with
0.05 ml of Lactobacillus leichmannii inoculum, prepared by adding one cryule to 400 ml of B12 assay medium. The media used was Difco Bacto B12 Assay Medium (0360-15-7) and was prepared by combining 15.2 gm of the medium with
0.8 gm ascorbic acid and 400 ml Milli-Q water. This was then autoclaved at 122°C and 15 psi for 15 minutes. When cool, the cryule was transferred and mixed. Inoculated test tubes were placed into a circulating water bath at
37°C for 20 hours. Tubes were then mixed with the
Autoturb mixer 10 times and read on the Autoturb I reader for turbidity (% Transmittance) at 650 nm. 125
Calculation of Vitamin B12- Vitamin B12 content was determined by plotting the absorbance response against the known B^2 content (pg/tube). This was done using the SAS regression program. The manual calculation of vitamin B12 content per tube is as follows:
Vit. B12 = STD WT X 1000 ma X A microarams X 1 X (pg/tube) (g) g mg 1000 ml
1000 na X 50 ml X 10 ml X 1000 pq X VI X V2 microgram 250 ml 250 ml ng 50 ml
WHERE: SDT WT is the weight of USP cyanocobalamin reference standard in grams;
1000 mg/g is the conversion factor for grams to milligrams;
A is the activity of the USP cyanocobalamin reference standard in micrograms/milligram;
1/1000 ml is the dilution of the initial vitamin B^2 standard solution;
1000 ng/microgram is the conversion factor for micrograms to ng;
50/250 ml is the dilution factor of the vitamin B i 2 stock standard solution;
10/250 ml is the dilution factor of the vitamin B i 2 intermediate standard solution;
1000 pg/ng is the conversion factor for picograms to nanograms;
VI is the aliquot,in ml, of the working standards, and;
V2 is the amount of working standard, either 0.10 ml or 0.15 ml, delivered to each tube by the Autoturb I dilutor. CHAPTER IV
RESULTS AND DISCUSSION
126 127
This chapter discusses results from the experiments
conducted to determine if a metabolic deficiency of
vitamin B12 is responsible for feed intake reduction when
ionophores are fed in all concentrate diets. The
hypothesis discussed in Chapter II, section C points out
that ionophores increase ruminal propionic acid
production. Increased metabolic production of propionic
acid or dietary addition of propionic acid have both been
shown to increase vitamin B12 requirement in the rat.
Therefore ruminants fed ionophores may respond by
exhibiting an increased vitamin B12 requirement supplied
by increased dietary cobalt or by I.M. injection of the
vitamin.
The first question which needed resolution concerned
the synthesis and absorption of vitamin B12. Could the
need for vitamin B^2 be met by increased dietary cobalt,
or would synthesis and/or absorption of vitamin B12 limit
it's availability for metabolism? If synthesis and/or
absorption is limiting, can injectable B^2 meet the need?
Once these questions were answered, it would be possible to conduct appropriate experiments to test the hypothesis. Would the lamb exhibit metabolic signs of vitamin B12 deficiency when an ionophore was added, even
if feedlot performance were not affected? Would an
interaction between the addition of an ionophore and addition of dietary cobalt occur? 128
Addition of an ionophore results in increased ruminal production of propionic acid with a concomitant reduction in acetic and butyric acids. Propionic acid is an initial substrate in the synthesis of glucose in liver. Vitamin
B 12 is involved in this pathway as a coenzyme in methyl- malonyl-CoA mutase. This enzyme is necessary for the conversion of methylmalonyl-CoA to succinyl-CoA and if vitamin B 12 is limiting, methylmalonic acid is excreted in the urine, and the resulting elevated propionate in the blood causes a reduction in feed intake as long as propionate is elevated. This is observed in uncomplicated cobalt deficiency. Feed intake reduction is often also observed as a response to an ionophore being added to a high-concentrate diet (which has significantly higher propionate production than high roughage diets already). OUTLINE OF RESULTS AND DISCUSSION
A. Preliminary Studies - 1984-85
B. EXPERIMENT 1
C. EXPERIMENT 2
D. General Discussion and Conclusions 130
A. Preliminary Studies - 1984-85
1. Purpose-This study was initiated primarily to learn the methodology needed for this project.
Experimental data provided an estimate of the range of values measured and allowed for some procedural problems to be realized without interfering with a major feeding trial.
2. Results-Data from four lambs are inconclusive due to lack of replication. Weight gain and feed intake are shown in Table 15. Gain by the lambs fed supplemental cobalt is greater than gain by the negative control lamb or the lamb given vitamin B 12 injections. The amount of b 12 given by injection may have been less than the B12 supplied by dietary cobalt in supplemented lambs. It is also possible cobalt elicits an effect in the rumen unrelated to host animal metabolism through improved ruminal fermentation. Feed intake data support the hypothesis, but lacks replication on which to draw conclusions. Feed intake data obtained from animals in metabolism crates are not always representative of feed intakes obtained in pen or group feeding situations. This is especially true of lambs. Further studies must include both field work with feedlot variables measured by pen, and studies with individual animals to collect metabolic 131 data. Average intake was greater for lambs given supplemental cobalt than lambs not given cobalt.
TABLE 15 Results for Preliminary Study
Lamb I .D. 576 575 572 570 Cobalt + + - - Vitamin B^2 + - + - vt‘ib k9 35.6 36.5 34.3 36.5 Wt.f2 , kg 49.5 48.1 43.6 41.3 Gain, kg 13.9 11.6 9.3 4.8 Intake3 , gm 853 860 658 466 Urine, ml/d 1030 1555 700 900 FIGLU4 0.02 1.86 1.79 5.43
1 Wt.i = initial weight 2 Wt.f = final weight 3 Intake = ave. feed intake/d for 5 day test period 4 millimole FIGLU/kg Body Weight/d
The lesser response to injections of Bi2 may indicate the dosage was lower than the Bi2 supplied toy ruminal synthesis at this cobalt intake or a ruminal response to cobalt different from metabolic effects of vitamin B12.
Although FIGLU output was greatest for the lamb not supplemented with cobalt or vitamin B12, and lowest for the lamb which was supplemented with both cobalt and vitamin B12, all FIGLU levels are within the normal range for sheep with adequate vitamin B^2 •
3. Conclusions-Puroose of this study was to learn methodology and realize procedural problems expected in a larger experiment. Data obtained from this study are consistent with the objective of the research, but are 132 without replication. Valid conclusions cannot be drawn.
Data obtained from this experiment were within the ranges reported in the literature. Animal and laboratory methodology were developed so that results were obtained and estimates of time needed and costs of analysis were determined.
4. Puroose-The objective of the second preliminary study was to determine if intramuscular injection of cyanocobalamin would improve feedlot performance of lambs when dietary cobalt was supplemented. Improved feedlot performance in B12 injected lambs fed adequate cobalt would indicate that synthesis and/or absorption of B ^2 may limit the amount available for metabolism.
5. Results-Averaae daily gain, feed intake and feed efficiency were not different (Pc.10) for pens of lambs given any of the treatment combinations. Least squares means for main effects of Co and B12 on weight, weight gain, feed intake, and feed efficiency are shown in table
16. There were no significant interactions of cobalt and vitamin B12 for any of the variables measured. 133
TABLE 16 Effect of Cobalt and Vitamin B 12 on Performance of Lambs no. lambs/treatment 16 16 16 16
Cobalt1 + + Vitamin Bi2 2 - + — + SE-3.
ADG, gm 332 .8 310.0 324.3 321.4 18.4 Feed Intake/d, kg 1.163 1.169 1.179 1.159 0.041 Feed/gain 3.5 3.8 3.6 3.6 0.11
1 Cobalt bullet and grinder, per os 2 Weekly I.M. injection of 1000 meg. cyanocobalamin 3 SE=standard error of least squares means
From the preceding tables, performance was nearly identical for lambs on each treatment. Cobalt and vitamin
B 12 main effect least squares means were not significantly different for any variable. Performance of lambs on all treatment combinations was considered excellent for crossbred lambs.
6 . Conclusions-Since performance was not different in lambs injected with vitamin B12 when compared to lambs fed cobalt bullets, there must be sufficient microbial B12 synthesis and absorption from the G.I. tract to support growth at the observed rate when fed all concentrate diets. It is possible lambs had sufficient vitamin Bi2 liver stores at initiation of the trial to carry through the experiment. 134
Had a significant difference been detected due to Bi2
injection, further studies would have required
supplementation of the vitamin by injection rather than
dietary cobalt addition. Data from this experiment were
contrary to data obtained in the initial study discussed
above. In that study, gain and feed intake were greater
in the lambs which received supplemental cobalt or vitamin
B 12 than the lamb which received neither treatment.
Differences between this study and the preliminary study
are: 1) lambs in preliminary study were Dorset lambs
whose dams were fed corn silage as a main constituent of
the diet, while lambs in this study were crossbred lambs
whose dams were fed fescue grass pasture or hay as a major
portion of the diet; 2) lambs in this study were fed 33
ppm lasalocid, while lambs in the preliminary study were
fed 54 ppm lasalocid; 3) lambs in the preliminary study were given water from the Columbus municipal water supply while lambs in this study were given water from a surface pond on the EORDC farm; 4) lambs in the preliminary study
received 1000 meg vitamin B 12 bi-weekly by I.M. injection,
lambs in this study were given 1000 meg vitamin B^2 weekly by I.M. injection. Any or all of these factors could have
contributed to the different results observed between these two studies. 135
B. EXPERIMENT 1
1. Purpose-This study was conducted to determine if the performance response to cobalt supplementation is effected when ionophores are fed in all concentrate diets.
Dietary treatment combinations of cobalt (0 or 0.4 ppm added), monensin (0 or 27 ppm added), and lasalocid (0 or
54 ppm added) were fed to growing lambs to determine effects on feedlot performance, liver vitamin B ^2 level, urinary formimino-L-glutamic acid excretion, and jugular propionate disappearance rates.
2. Results-This study included six replicates of six pens of lambs-one on each dietary treatment combination.
The first three replicates were fall-born, Targhee-sired lambs and replicates four through six were spring- born lambs sired by Hampshire, Targhee or Dorset rams. There were significant block effects with fall-born or spring-born being blocks. For example, fall-born lambs had lighter birth weights and lighter weaning weights, however, they had superior feedlot performance when compared to spring-born lambs. There were no significant interactions between any feedlot variables and blocks.
Table 17 compares feedlot performance, mortality, liver vitamin B 12 levels, and jugular propionate disappearance rates in lambs fed the treatment diets. TABLE 17 Effect of Cobalt, Monensin and Lasalocid on Performance of Feedlot Lambs
Diet Co- Co- Co- CO+ 1 Co+ CO+ QOH m o n £ las-2. CONWONLAS SE— pens n= 6 6 6 6 6 6 lambs n= 31 32 32 32 32 32
ADG5 ,gm 241 248 255 269 260 284 9. 3ab
DDMI6, gm 1114 1129 1133 1217 1111 1233 3 6.5bcd
F/G7 4.70 4.60 4.51 4.52 4.31 4.36 0.17
Mort 8 6.7 16.1 3.3 3.3 0.0 6.1 3.5efg
LB i 2 9 560 670 460 571 626 625 54.2 fh
C 31 0 , min <30 <30 <30 <30 <30 <30
1 0.4 ppm added cobalt 2 27 ppm Monensin 3 54 ppm Lasalocid 4 SE=standard error of mean 5 Average Daily Gain over 84 d 6 Daily Dry Matter Intake over 84 d 7 Feed/gain over 84 d 8 % Mortality 9 Liver vitamin B 12 at slaughter-ng B^/g® wet liver 10 Jugular propionate disappearance time a Cobalt response (P<.05) in lambs fed CON or LAS b MON and LAS differ (P<.05) in lambs fed cobalt c Cobalt response (Pc.10) in lambs fed CON or LAS d Negative monensin response (P<.10) in lambs fed cobalt e Monensin response (Pc.10) in lambs not fed cobalt f MON and LAS differ (P<.05) in lambs not fed cobalt g Cobalt response (Pc.Ol) in lambs fed MON h Cobalt response (P<.05) in lambs fed LAS 137
Cobalt increased (P<.05) average daily gain (ADG, gm/d)
in lambs fed control diet (241 vs. 269) or lasalocid diet
(255 vs. 284). ADG of lambs fed LAS was higher (P<.05) than
lambs fed MON only when cobalt was added to the diets (284 vs
260, respectively). ADG of lambs fed LAS was not different
(P<.10) from lambs fed MON when lambs were fed diets without added cobalt.
Daily dry matter intake (DDMI, gm/d) was different
(P<.05) for lambs fed MON and lambs fed LAS only when cobalt was fed (1111 vs 1233, respectively). Lambs fed cobalt had greater (P<.10) DDMI than lambs not fed cobalt for the CON and LAS diets, but no difference (P<.10) was detected for lambs fed MON with or without cobalt.
No differences (Pc.10) were detected in efficiency of feed conversion (feed/gain or F/G) with cobalt, monensin, or lasalocid, however, numerical improvements were observed which favored monensin or lasalocid over control, and cobalt supplemented diets over unsupplemented diets. These improvements were apparently additive.
Lambs fed cobalt generally had lower mortality than lambs not fed supplemental cobalt. Lambs fed Co-MON had higher (Pc.10) mortality than lambs fed Co-CON (16.1 vs 6.7%, respectively). Lambs fed diets without supplemental cobalt were affected differently (Pc.05) by MON (16.1%) and LAS
(3.3%) with respect to mortality. Cobalt had a profound effect (P<.01) on mortality when MON was fed. 138
Lambs fed Co- MON had 16.1% mortality, while lambs fed Co+
MON had 0.0% mortality during the 84 d trial.
Liver vitamin B ^2 levels were effected by cobalt and t h e
ionophores. Lambs fed cobalt and LAS (625 ng B12/gm wet
liver) had higher (Pc.05) liver vitamin B 12 levels than lambs
not fed cobalt when LAS was also fed (460 ng B12/gm wet
liver). Lambs not fed cobalt, but fed MON had higher (P<.05)
liver vitamin B^ 2 levels than lambs not fed cobalt, but fed
LAS (670 vs 460, respectively). Since liver samples were
taken at slaughter, lambs which died during the experiment
are not included in liver vitamin B ^2 data. Since a higher
proportion of lambs fed MON died, and these lambs were not
included in mean liver vitamin B 12 content, this may effect
interpretation of results for lambs fed MON.
Jugular disappearance of propionate was not different
(P<.10) for lambs receiving any of the treatment combinations
(Figure 1). Regardless of cobalt status, or presence or
absence of ionophore, clearance of propionate from jugular
blood was less than 30 minutes. In classic vitamin 12
deficiency, elevated propionate is detectable through 3-5
hours after infusion (Marston et al., 1972). All lambs fed
treatment combinations of cobalt, MON, and LAS returned to pre-infusion levels within 3 0 minutes in experiment 1.
Sample means are shown for all dietary treatment combinations
in table 18. 139
mmol/l plasma 16
12 10
0 50 100 150 200 250 minutes after initial sample
— Co- CON Co- MON Co- LAS Co* CON Co* MON Co* LAS
Figure 1. Blood plasma propionate levels vs time 140
Table 18 Plasma propionate levels vs time u O Diet Co- c o l CO+ 1 Co+ Co+ CON m m LAS COJJ MON LAS
T 0 0.232 0.48 0.56 0.43 0.48 0. 52 Tl 15.14 12.72 13.18 13.78 12.30 12.51 t 2 1.05 0.82 0.39 0.56 0.28 0.61 t 3 0.25 0.28 0.33 0.29 0.38 0.31 t 4 0.13 0.24 0.16 0.35 0.59 0.35 T 5 0.19 0.19 0.30 0.42 0.41 0.30 T 6 0.25 0.22 0.22 0.24 0.48 0.40 1 Samples were taken at the following times with respect to infusion: T0, prior to; , immediately after; T2, 30 minutes after; T3, 60 minutes after; T4, 120 minutes after; T 5 180 minutes after; T 6 240 minutes after infusion of 3 mmol C3/kg body weight.
2 All values expressed as mmol C3/litre plasma.
Propionate disappearance rate constant was computed for each sample using the formula:
2.3 log (C1/C 1-C2 ) K= ______T2-T!
Where: is concentration at time 1 (T^)
and C 2 is concentration at time 2 (T2 )
No significant differences were detected between treatment groups for propionate disappearance rate constant. 141
Formimino-L-glutamic acid concentrations in urine were not detectable, apparently due to some interfering substance.
This was not the case in the preliminary studies. The relationship of the blank and test would indicate a negative amount of FIGLU present in most of the samples. This was not due to enzyme, water used, acid used, or urine itself. Cause of these problems was not determined.
3. Conclusions-Data collected in this experiment clearly answers the research question: Is the performance response to cobalt supplementation effected when ionophores are fed in all concentrate diets.? The jugular disappearance of propionate was not different in lambs fed any of the treatment diet combinations, however, growth rate and feed intake were effected. This is a significant finding.
A metabolic deficiency of vitamin B 12 in any of the lambs would have resulted in plasma propionate levels being elevated for several hours. All lambs had propionate levels close to the pre-infusion sample level 30 minutes post infusion. This indicates a metabolic deficiency of Vitamin
B 12 was not responsible for feed intake reduction when ionophores were fed in these all-concentrate diets.
The observed biological interaction of cobalt and monensin is noteworthy, however. Daily dry matter intake
(DDMI) of lambs fed Co+ MON (1111 gm/d) was lower (Pc.10) than DDMI of lambs fed Co+ CON (1217 gm/d). This observed reduction in feed intake did not occur when lambs were fed diets without supplemental cobalt. Lambs fed Co- CON consumed 1114 gm/d, while lambs fed Co- MON consumed 1129 gm/d. A positive response to cobalt was observed in ADG and
DDMI when lambs were fed CON or LAS diets. Performance (ADG and DDMI) of lambs fed MON was not significantly different to performance of lambs fed Co- CON. In essence, MON negated the cobalt response which was observed in lambs fed CON or
LAS diets. 143
C . EXPERIMENT 2
1. Purpose-This study was conducted to examine the
effect of MON and vitamin B 12 on liver vitamin B 12 levels and
the change in these levels over time.
2. Results-Treatments were applied for 74 days. Lambs
were fed control diet (SH-30-87) for 10 days in accordance to
INAD 4847 and slaughtered. Data were analyzed using the SAS
General Linear Models program for main effects of MON and B ^2
and interaction of MON*B^2. Least squares means for MON and
B 12 effects and effects of MON and B 12 treatment combinations
are shown in table 19. There was some imbalance in the
experiment due to one lamb developing urinary calculi and being removed from the study.
Liver B ^2 level in lambs fed MON and injected with B 12 was significantly lower (P=.0372) than lambs on either treatment combination without B^2. This was a chance occurrence and not related to treatments, since biopsies were taken prior to any treatment application.
Although initial levels were different, change in liver b 12 was nak significantly different when M0N+Bi2+ lambs were compared to M0N-Bi2- or M0N-Bi2+ lambs. There was a highly significant difference (P=.0052) in change in B^2 between
M0N+B^2+ lambs and M0N+B^2- lambs. Change in B^2 was not significantly different from zero for lambs given MON+Bi2-. 144
TABLE 19 Effect of Monensin and Vitamin B ^2 on Liver Vitamin B 12
MON - MON - MON + MON + Sample B 12 " B 12 + -Sl 2— - ^ 12— ± SE—
Initial2 473.8 413.5 539.5 287.3 6 6 .7ab
Final 733.5 868.7 457.3 677.0 87.0*cd
Change 259.8 525.0 -82.3 389.8 9 6 .i**ef
1 SE = standard error of LS Mean 2 All values reported as ng B12/gm wet liver. a MON-B^2- and MON+B12+ are different (Pc.10). b MON+Bi2- and MON+Bi2+ are different (Pc.05). c &12 effect in MON+ lambs (Pc.10). d MON+B12- is different (Pc.05) from MON-B12- or MON-B12+. e Bi2 effect in MON- lambs (Pc.io). f MON effect in Bi2- lambs (Pc.05).
* SE of MON-B12+ is 100.4. ** SE of MON-B12+ is 110.9.
3. Conclusions-Treatment differences in liver vitamin
B 12 at slaughter, and change in B 12 indicate MON has an effect on liver vitamin B 12 in lambs. Injections of vitamin
B 12 also had an effect on level of B ^2 in liver and change in
B 12 as one might expect. From Table 19, one can see that the effect of MON on change in B 12 was profound when no supplemental B 12 was given. Lambs given MON-Bi2- had an increase in liver B ^2 of 259.8 ng/gm wet liver, while lambs given MON+B12- had a decrease in liver Bi2 of 82.3 ng/gm wet liver. This -82.3 is not significantly different from zero, 145 but is different from the 259.8 (P<.05). The effect of MON is similar in lambs given Bi2 injections. Liver B 12 of lambs given MON-B^t increased 525.0 ng/gm wet liver, while lambs given M0N+B^2+ hac* an increase in liver B^2 389.8 ng/gm wet liver. These differences were not significant (P<.10).
A more precise experiment may demonstrate these differences to be significant, and biological significance may supercede statistical significance. As one might expect, increase in liver B ^2 due to weekly injections of B^2 was profound, as lambs with lowest initial level had B 12 levels at slaughter similar to levels in lambs not fed MON. It was unfortunate to have differences in initial B 12 level, however, the lowest level was in lambs given positive control which minimized the effect of this difference on the results of the study. Since all lambs were twin wether lambs, born within 7 days, and of similar weaning and on-test weights, any significant differences in initial liver B 12 level was entirely due to chance and was totally unexpected.
One would expect ionophores to increase metabolic need for vitamin B 12 through increased ruminal propionate production, however, the mechanism of MON effect on liver vitamin B±2 level was not investigated, and is not clear.
Data suggest MON has an effect on liver Bi2 level, but it is unclear what mechanism causes this effect. 146
D. General Discussion and Conclusions
At the conclusion of any research project, one must evaluate whether the project uncovered new questions or discovered new answers. This research has done both.
The response in animal performance to cobalt and monensin appear to be related to ruminal fermentation, rather than a metabolic response related to vitamin Bi2 . This was unexpected. Effect of cobalt in the rumen could be related to bacteria and/or protozoa which utilize cobalt or cobalt- containing compounds or bacteria which synthesize other 13- vitamins. Other workers have shown interactions between vitamin B 12 anc* B 6 ' biotin, folic acid, thiamin, and ascorbic acid (Marchetti and Testoni, 1963; Marchetti and Testoni,
1964; MacPherson et al., 1976; Mann et al., 1983; Marston et al., 1961). Low cobalt concentration can affect both fermentation (Gall et al., 1949; Latteur, 1962; MacDonald and
Suttle, 1986; Marston and Lee, 1949) and the animal (Latteur,
1962; Mann et al., 1983; McLoughlin et al., 1984).
Response to cobalt was different with monensin than with lasalocid. Feedlot performance and liver vitamin Bi2 data indicate monensin and lasalocid, though similar in basic modes of action, differ in their effect upon performance and liver vitamin Bi 2 in lambs. It remains to be shown whether feed intake depression, observed in ionophore-fed feedlot beef cattle, is due to a metabolic lack of vitamin B^2. 147
Guy (unpublished data) showed a dramatic reduction in
feed intake, and increased urinary excretion of methylmalonic
acid (MMA) in rats fed 70% of total calories as C 9 fatty
acids (increased propionic acid formed in metabolism)
compared to rats fed 70% of total calories as a mixture of C8
and Cj_q fatty acids in meal-fed animals. The feed intake depression and increased urinary excretion of MMA was
alleviated by a 10-fold increase in vitamin B 12 intake even though the diets were formulated to meet N.R.C. requirements
for B 1 2 • The feed intake depression was only observed when
feed intake was controlled with meal feeding twice per day.
Rats fed the C9 fatty acids ad libitum did not have reduced
feed intake compared to rats fed the mixture of C 8 and C^q
fatty acids. Rats fed ad libitum ate numerous small,
frequent meals, rather than infrequent large meals.
Marston et al. (1972) showed similar changes in eating habits in cobalt deficient sheep. Although lambs in studies reported herein were fed ad libitum, propionate clearance was determined using a large dose of sodium propionate. Baile
(1971) showed propionate infusions reduced feed intake in sheep and goats. He hypothesized propionate receptors in the ruminal area are involved in signaling satiety in sheep.
Orskov and Allen (1966) and Orskov et al. (1966) showed lambs efficiently utilized a mixture of calcium propionate and sodium propionate for body weight gain. More recently,
Istasse and Orskov (1984) showed an insulin response to large 148
doses of propionate, while the same amount of propionate,
given as continuous infusion, showed no insulin response.
This may help explain observed differences between ad libitum
and meal-fed animals.
Previous research reported by Hembry et al. (1979), and
Daugherty et al. (1986) have suggested a relationship between
cobalt/vitamin B12 status and the observed intake response to
addition of an ionophore. These studies were conducted with
cattle and sheep, but have not shown a definite relationship
between vitamin B 12 status in the animal and feed intake
response to ionophores.
Lindemans et al. (1986) have shown monensin inhibited the intracellular processing of transcobalamin II-bound
cobalamin by the isolated rat kidney tubule. This process is temperature- and calcium-dependant and was inhibited by monensin. The normal reabsorption of protein-bound cobalamin was impaired, presumably due to an inhibition of endocytosis. Excessive urinary excretion of cobalamin was expected and found in a patient with overflow proteinuria.
If this process occurs in the ruminant, the host animal could be excreting B 12 via the kidneys at a rate faster than synthesis and/or absorption of B ^2 from the gut. This could cause problems in cobalt-deficient or marginally deficient areas. 149
Feed intake mechanisms are quite complex and would require more space than this work allows. It is sufficient for this work to reiterate the gross lack of information which adequately explains the variability of feed intake response to the addition of an ionophore to ruminant diets.
This author has observed an increased feed consumption of 4-6% with the inclusion of 33 or 54 ppm lasalocid to diets of growing lambs. Dr. Millard Calhoun, Texas Ag. Exp.
Station, reports (1988) a very consistent feed intake depression when lasalocid is added to high-concentrate diets for lambs. The feed reportedly contained 0.08 ppm cobalt on a DM basis. Diets in experiments herein reported contain cobalt at reported values ranging from less than 0.1 ppm to over 1.2 ppm. Data are shown in table 20. No pattern was established between or within laboratories for cobalt values. 150
TABLE 20 Cobalt analytical results for Experiment 1
REAL 1 REAL2 A&L 3 AGWAY DIET oora pom nom DDm
Co- CON < 0.1 0.643 0.32 0.260
Co- MON < 0.1 0.730 0.28 0.244
Co- LAS < 0.1 0.210 0.10 0.150
Co+ CON < 0.1 0.487 1.20 0.390
Co+ MON < 0.1 0.333 0.08 0.250
Co+ LAS < 0.1 0.455 0.13 0.215
TEXAS MIX 0.06 0.080
Prelim diet 0.10 0.220
+ Cobalt premix5 = 684.5 ppm
- Cobalt premix5 = 34.0 ppm
1 Research and Extension Analytical Laboratory, Wooster, Ohio, 02/22/88
2 Research and Extension Analytical Laboratory, Wooster, Ohio, 03/02/88
3 A&L Agricultural Laboratories, Memphis, Tennessee, 05/13/88
4 Agway Technical Services, Inc. Ithaca, N.Y. 05/17/88
5 Hazelton Laboratories, Madison Wisconsin. 11/09/90
Results from laboratory analysis of feed samples from the six diets, sent to three different laboratories, which provided four sets of samples are shown in table 20. Diets were formulated to contain less than 0.10 ppm cobalt for the
no cobalt diets (diets 30, 31, and 32) and an additional 0.4
ppm cobalt in the diets containing supplemental cobalt (diets
33, 34, and 35). Formulation of these diets was based upon
the diet used in the preliminary studies (diet SH-10-85),
reported to contain 0.063 ppm by Agway Technical Services
Laboratory, Ithaca, New York in 1986. Results of the
laboratory analyses show no apparent pattern or similarity
between samples or between laboratories. Since copper, iron
and manganese analysis were similar and near levels
calculated to have been added by the premix, and these
minerals were included in the premix which either contained
or did not contain added cobalt, and since the eighteen pens
of lambs fed supplemental cobalt showed a response in
performance when compared to the eighteen pens of lambs not
fed supplemental cobalt, two conclusions were drawn. First,
laboratory analysis of cobalt was not a reliable estimate of
the cobalt content of the feed, and secondly, since a
response to supplemental cobalt was detected, the true level
of cobalt was not known, nor was it necessary for the true
cobalt level to be known. Production records (and deduction
from laboratory analysis of other minerals) indicate premixes were added, and cobalt was known to have been added to the premix. The response, therefore, was attributed to differences in cobalt level. As discussed previously in this work, Daugherty et al. (1986) reported a cobalt content of 152
0.25 ppm in the pelleted diet fed in their experiments. They
reported the addition of a trace mineralized salt which
contained 0.011% cobalt at the rate of 0.5% of the pelleted
diet. This should have added 0.55 ppm cobalt on a total diet
basis, but the reported cobalt content was 0.25 ppm. This
supports the conclusion that laboratory analysis of cobalt
may not be a reliable estimate of cobalt content of the diet.
Cobalt requirement for sheep has been based upon early
work with cobalt deficiency in sheep in Australia. The
wethers growing in these early studies averaged 125 gm/wk on
pasture. Assuming this level of cobalt is adequate to meet
the needs of modern lambs gaining 500 gm/day is unreasonable without data to support such an assumption. Requirements
established for dairy and beef cattle were originally based
on work with sheep as well.
Observations with the lactating dairy cow of reduced
serum vitamin B 12 during the peak of lactation is evidence of
the huge demand for B 12 to support milk synthesis and loss via the milk. The effect of diet, cobalt, and ionophores and
other feed additives on total water-soluble vitamin synthesis
in the rumen is not known. There have been suggestions in
the literature (Mac Pherson, et al. 1976) of an interaction between vitamin B^2 and thiamin. It is possible rumen microbes which synthesize thiamin may have a need for Bi2 or
other cobalt-containing compounds. 153
Reduced ruminal acetate and methane are commonly
observed when ionophores are added to ruminant diets.
Methane synthetase and acetate synthetase are vitamin
b 12“dependent
enzymes in certain rumen bacteria. It is possible the
ionophore effects B ^2 metabolism in the rumen as well.
Regardless of the mechanisms involved, lambs fed monensin had lower feed intake than lambs fed control diet without monensin when cobalt was added to both diets. Lambs
fed monensin without supplemental cobalt had feed intake
similar to lambs fed control diet without monensin and without supplemental cobalt. This effect is apparently not due to liver propionate clearance, since jugular plasma propionate disappearance was similar for all treatments.
Data suggest monensin and lasalocid have different effects on liver vitamin B ^2 level in lambs, depending on supplemental I.M. vitamin B12 or dietary cobalt content.
This is at variance with work reported by Daugherty, et al.
(1986) in which liver vitamin B 12 levels changed equally when monensin was supplemented with or without vitamin B12.
Lambs in that study were supplemented with cobalt in the basal diet to simulate common feedlot practices in New
Mexico. The trace mineralized salt added to their diets should have added twice the .25 ppm cobalt reportedly present in the diets. This illustrates the lack of reliability of laboratory data concerning cobalt level in feeds. The reader is reminded that to meet FDA requirements for the Investigational New Animal Drug Exemptions, all lambs in experiments 1 and 2 were fed the respective control diet
(with or without cobalt) for at least 10 d prior to slaughter to insure adequate drug withdrawal. Although speculation, it is possible differences in liver vitamin B ^2 level between lasalocid fed lambs and the control diet (diets 32 and 30) and between monensin fed lambs and the control diet (diets 31 and 30) at conclusion of the 84 d trial may not have been representative at slaughter since the drugs had been withdrawn 10 d. It was not feasible to take biopsy samples of liver at the termination of the trial, however, it may be beneficial for future research to determine liver vitamin B ^2 content prior to, during, and at the conclusion of a large feeding study to determine the effects of monensin and lasalocid on vitamin B 12 metabolism of the lamb. Urinary excretion of vitamin B 12 would be of great interest as well.
The true mechanism of ionophore action is not clear, and was not tested in this research. Logically, increased ruminal propionate should increase the need for B12r since
B 12 is needed to convert methylmalonyl-Co A to succinyl-Co
A. One would expect an increased need for biotin as well since it is needed for the conversion of propionyl-Co A to methylmalonyl-Co A. The ionophore may have an effect upon the rumen fermentation which increases the B 12 requirement in the rumen. This could be due to the ionophore suppressing 155 bacteria which synthesize true B 12 relative to bacteria which synthesize pseudo-B12/ or by some suppression of bacteria which convert cobalt into other organic cobalt-containing compounds. The ionophore may affect the host animal in a direct manner such as increasing the excretion of B 12 in the urine by impairing the re-absorption of cobalamin by the kidney. If cobalt were shown to improve feed intake in monensin-fed cattle or lasalocid-fed cattle, it would be of great economic importance. Increased feed intake could increase average daily gain (ADG). Given the choice between improved feed efficiency and increased ADG, most feedlot operators would choose increased ADG since it has a greater bearing on overall profitability. Cobalt supplementation is relatively cheap, however, if biotin is shown to be primarily responsible, supplementation may not be cost-effective since biotin is rather expensive under current economic conditions.
Future research to fully understand the questions raised by this research should include studies to determine the true requirement for cobalt in the diets of sheep, beef cattle, and dairy cattle under different physiological states.
Methodology needs to be developed to quantitatively and reliably determine the cobalt content of feedstuffs. The total synthesis of water soluble vitamins in the rumen and the effect of diet (roughage versus high energy density) and feed additive (ionophores, other antibiotics) on vitamin synthesis needs to be researched. Interaction of cobalt 156 level and water soluble vitamin synthesis in the rumen needs to be addressed as well. During the 1988 Ohio Sheep Day, a sheep producer from Canada related to this author he had a considerable number of lambs die in the past several years, attributed to polio encephalomalacia. The veterinarians advising him instructed him to give vitamin B 12 injections as well as thiamin injections. The flock has been fed monensin in all diets for a number of years. The incidence of polio in his flock has decreased since the B^2 injections have been initiated and was essentially zero prior to monensin feeding.
Response to cobalt and vitamin B 12 needs to be investigated in beef when ionophores are fed. Effect on animal and ruminal metabolism needs further study. Metabolic effects of monensin on vitamin B]^ metabolism in the animal and water-soluble vitamin metabolism in the rumen also needs to be investigated.
Finally, meal feeding and acl libitum feeding differences need to be addressed. Much of the variability of feed intake response to ionophores may be due to differences in feed management at the bunk. Other factors such as ventilation, temperature, space, age and genetics of the animals, and season may influence intake response, but have not been related to use of ionophores. Our current level of knowledge does not fully explain mechanisms involved in response to ionophores and many major factors are as yet unidentified. APPENDIX
157 158
DEPARTMENTOF HEALTH & HUMAN SERVICES Public Health Service
Food and Drug Administration Rockville MD 20S67
INAD-4878 October 27, 1986
Drew A. Uermeire Graduate Research Associate Ohio State University Ohio Cooperative Extension Service Animal Science 2029 Fyffe Road Columbus, OH 43210-2098
Dear Mr. Uermeiret
We acknowledge receipt of your submissiondated October 21, 1986 which pertains to the investigational useof Monensinin lambs.
Your submission has been assigned INAD number 4873 and is being forwarded to the proper reviewer for consideration. Please refer to this number when submitting any future correspondence pertaining to the use of the aforementioned drug inthis species.
This is not an authorization letter.
Sincerely yours
Doriel J. Christensen, Supervisor Document Control Staff Center for Veterinary Medicine 159
DEPARTMENT OF HEALTH & HUMAN SERVICES Public Maalth Service
Food and Drug Administration Rockville MD 20857
INAD-4831 October 27, 1380
Drew A. Vermeire Graduate Research Associate Ohio State University Ohio Cooperative Extension Service Animal Science 2029 Fyffe Road Columbus, OH 43210-2098
Dear Mr. Uermeire:
We acknowledge receipt of your submission dated October 21, 198G which pertains to the investigational use of Lasalocid inlambs.
Your submission has been assigned INAD number 4881 and is being forwarded to the proper reviewer for consideration. Please refer to this number when submitting any future correspondence pertaining to the use of the aforementioned drug inthis species.
This is not an authorization letter.
Sincerely yours,
Doriel J. Christensen, Supervisor Document Control Staff Center for Veterinary Medicine 160
DEPARTMENT OF HEALTH & HUMAN SERVICES Public Health Service
Food and Drug Administration 4 Rockville MD 20857
INAD 4878 JAN - 9 1987 Mr. Drew A. Vermeire Animal Science Department Ohio State University 2029 Fyffe Road Columbus, Ohio 43210-1095
Dear Mr. Vermel re:
We refer to your letter dated October 21, 1968, and to the project proposal/protocol for a study In which lambs fed 1n confinement w ill receive monensln a t 25 grams per ton o f complete feed.
As the proposed study 1s a part o f your Ph.D. dissertation research, and 1s not Intended for submission to FDA in support of any New Animal Drug Application, we w ill have no comment concerning the proposal/protocol. However, you w ill be required to comply with the lab eling , recordkeeping, and drug accountability provisions of 21 CFR 511.1(b).
By separate le tte r, you w ill be advised of a drug withdrawal period and slaughter notification requirements.
I f we can be o f fu rth e r assistance, please c a ll me a t (301) 443-5247.
Sincerely yours,
d^Jack C. Taylor, Chief Ruminant Drugs Branch Division of Biometrics S Production Drugs Center fo r Veterinary Medicine
Enclosure DEPARTMENT OF HEAL! 11 & HOMAN SERVICES Public H®ihh Servic*
Food and Drug Administration Rockville MD 20857
JAN - 9 !987 Mr. Drew A. Vermeire Animal Science Department Ohio State University 2029 Fyffe Road Col unbus, Ohio 43210-1095
Dear Mr. Vermeire:
We refer to your letter dated October 21, 1968, and to the project proposal/protocol for a study In which lambs fed In confinement w ill receive lasalocid at 50 grams per ton of complete feed.
As the proposed study Is a part o f your Ph.D. dissertation research, and Is not Intended fo r submission to FDA In support o f any New Animal Drug Application, we w ill have no comment concerning the proposal/protocol. However, you w ill be required to comply w ith the la b e lin g , recordkeeping, and drug accountability provisions of 21 CFR 511.1(b).
By separate letter, you w ill be advised of a drug withdrawal period and slaughter notification requirements.
If we can be of further assistance, please call me at (301) 443-5247.
Sincerely yours,
Jack C. Taylor, Chief Runlnant Drugs Branch Division of Biometrics A Production Drugs Center fo r Veterinary Medicine
Enclosure DEPARTMENT OF HEALTH & HUMAN SERVICES Public Health Service
Food and Drug Administration Rockville MD 20B57 INAD 4878 INAD 4881 JAN - 9 I98T Hr. Drew A. Vermeire Animal Science Department •Ohio State University 2029 Fyffe Road Columbus, Ohio 43210-1095
Dear Mr. Vermeire:
We acknowledge receipt of your le tte r dated October 21, 1986, requesting authorization to market sheep (lambs and ewes) treated with monensin (INAD 4878) and lasalocid (INAD 4881) with and w ithout supplemental cobalt, to study the dietary vitamin and cobalt requirements In diets containing lonophores.
You Indicated the In itia l tria l Involving 120 weanling lambs w ill have the treatment groups and dosing levels shown below. In a ddition, the ewes from which the lambs are born w ill be held In confinement and w ill receive lasalocid prior to lambing.
Investigational Diets
Diet ID Cobalt* Monensin** Lasalocid***
SH-30-86 - SH-31-86 - + SH-32-86 + SH-33-86 + SH-34-86 + + SH-35-86 + - +
*0.4 ppm cobalt **27 ppm monensin ***54 ppm lasalocid
The lambs w ill receive the Investigational diets for 70 days after which they w ill receive a basal d ie t during a 10-day withdrawal period.
In a telephone conversation on January 6, 1987, you advised th is o ffic e that a total of 800 lambs would be used In tria ls over the next two-year period.
We have reviewed your submission and find your request to be consistent with the public health. As you are aware, monensin 1s not cu rre n tly approved fo r use In sheep, and the use level you have proposed fo r lasalocid 1s higher than the regulated level for that drug. 163
Page 2 INAD 4878 INAD 4881
Accordingly, based on the treatment levels Indicated above, we authorize the slaughter o f a to ta l o f 800 sheep provided you observe a 10-day w ith drawal period p rio r to slaughter. That withdrawal time should ensure that residues of the two drugs are well below th e ir tolerances at the time o f slaughter.
To enable us to complete the f i l e on those INADs, please submit a copy of the Investigational label which contains the appropriate "caution" statement In accordance with 21 CFR 511.1(b)(1).
The New Animal Drug Regulations, Section 511.1(b) require the sponsor to submit specific Information prior to each shipment or other delivery of the drug fo r c lin ic a l Investigation In animals. The agency has devised a form (copy enclosed) which you as the sponsor may reproduce to report c lin ic a l tria ls. Three copies of the completed form should be submitted for each t r i a l .
Clinical Investigations for this INAD cover only the treatment regimen stated above. The combining o f treatment w ith any other drug w ill require a separate authorization. Drugs given control animals must be administered 1n fu ll compliance with the currently approved use. Your Investigators should be made aware of their responsibilities under Section 511.1(b)(7)(H) and Section 511.1(c)(1).
Clinical tests conducted under the provisions of this letter do not exempt Investigational animals and their products from compliance with other applicable Inspection requirements.
The date and place of slaughter must be reported to this office and to Ms. Leotta R. Hensley, D irector, Residue Evaluation and Planning S ta ff, FSIS, USDA; Room 602 Annex B uilding; 12th and C S treets, S.W.; Washington, D.C. 20250, at least 10 days prior to shipment for slaughter. Experimentally treated subjects must be Identified to the Inspector 1n charge of the slaughtering establishment when presented for antemortem Inspection.
Upon request, the reporting of the date and place of slaughter may be waived provided that the Investigational animals are maintained under Investigational conditions and under your supervision for an additional 30-day observation period following the established drug withdrawal period.
In order for us to complete our file s, the disposition of all Investigational animals and unused drugs must be reported to this office as well as any adverse reactions observed. Please re fe r to th is le tte r by date and INAD number when reporting the details of clinical Investigations or disposition of Investigational animals. We w ill not acknowledge re p e titiv e tr ia l submissions unless they are not In accord with the authorization. 164
Page 3 INAD 4878 INAD 4881
We remind you th a t the Investigational new animal drug must be manufactured, processed, packaged, and labeled 1n such a way as to maintain appropriate standards of Identity, strength, quality, and purity as needed for safety and to give significance to Investigations made with the drug.
Sincerely yours,
Marvin A. Norcross, VMD, PhD Associate Director for New Animal Drug Evaluation Center fo r Veterinary Medicine
Enclosure Department of Health & Human Services, PHS DATS Center for Veterinary Medicine (HFV-16) INAD NO. Fooa ana Drug Administration NAME OF DRUG Rockville, Maryland 20857 TRIAL NUMBER Dear Sir:
The sponsor, , submits a notice of claimed investigational exemption fo r the shipment or delivery of a new animal drug under the provisions o f Section 512 of the Federal Food, Drug and Cosmetic Act, and information is submitted in triplicate (original and two copies).
Name of drug: Proposed use of drug: Date of drug shipment (or received): Name of investigator: Address of inve stiga to r:
Location of Trials:
Approximate nunber of animals: Treated Controls Number of animals previously used: Date of authorization letter: Approximate date of t r i a l : Species o f animals: Size and type of animals: Maxdmixn daily dose(s) and duration:
Methods(s) of administration:
Withdrawal period:
If the investigation is discontinued, the Food and Drug Administration should be n o tifie d , giving the reason and disposition of the drug.
A waiver of requirements for notification of date and place of slaughter □ after a 30-day holding and observation period following the required withdrawal period is requested.
The date and place of slaughter w ill be reported to FDA and to Ms. Leotta □ R. Hensley, Residue Evaluation and Planning S ta ff, FSIS, OSDA, Room 602, Annex B uilding, 12th and C. Streets, S.W., Washington, D.C. 20250 at least 10 days p rio r to shipment fo r slaughter. Experimentally treated animals will be identified to the inspector in charge of the slaughtering establishment when presented fo r antemortem inspection.
Si gned ______Sponsor
EXAMPLE OF FORMAT TO BE FOLLOWED TO REPORT CLINICAL TRIALS. WHEN REPORTING ON MORE 'HAN ONE INAD, BE SURE TO INDICATE EACH INAD, DRUG, TRIAL NUMBER AND NUMBER OF TREATED AND CONTROL ANIMALS. 'HIS r0RMAT MAY BE 'NC0RP0RATEn ’NT" 'OUR WORD PROCESS ING SYSTEM ON v0UR .ETTERHEAD. LITERATURE CITED
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