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—._

This is to certify that the

thesis entitled

EFFECT OF EXCESS DIETARY SELENIUM
SUPPLEMENTATION 0N HOLSTEIN COWS

presented by
Roger George Ellis

has been accepted towards fulfillment
of the requirements for

Master of Science degree in Large Animal Clinical
Sciences

flee/W

Major professor

 

 

Date November 30, 1992

 

0—7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

 

 

 

 

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.

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MSU Is An Affirmative Action/Equal Opportunity Institution
, emana-pd

 

 

EFFECT OF EXCESS DIETARY SELENIUM SUPPLEMENTATION ON
HOLSTEIN COWS

By

Roger George Ellis

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Large Animal Clinical Sciences

1992

ABSTRACT

EFFECT OF EXCESS DIETARY SELENIUM SUPPLEMENTATION ON
HOLSTEIN COWS

By

Roger George Ellis

Twenty-four non-lactating cows were fed 0, 3, 20, 50 or 100 mg supplemental
Se/head/ day. Over the treatment period, mean Se concentrations (serum, whole-blood,
liver, urine, feces) did not differ between the unsupplemented control group and the 3-mg
Se/head/day group. However, within two days of initial supplementation, serum Se in
both the 20-and SO-mg groups exceeded controls (P<0.01). Whole—blood Se exceeded
controls (P < 0.01) at one week post supplementation in the SO-mg group and at seven
weeks in the 20-mg group. Liver Se concentrations of the 20- and SO-mg groups were
higher than controls at 90 days (P <0.01). No significant differences between groups
were detected at any time for complete blood counts; serum activities of AST, CPK,
SDH and GGT; immunological variables and general health. Sodium selenite
supplementation at as much as SO—mg Se/head/day for 100 days and loo-mg Se/head/day

for 28 days had no detectable harmful effects in non-lactating cattle.

DEDICATION

To my family, Claudia, Lisa and Timothy for their
never failing support. THANK YOU!

ACKNOWLEDGMENTS

The author would like to acknowledge the help and support of many individuals
who made this project possible.

Sincere thanks to my graduate committee, Drs. Tom Herdt (chairman), Howard
Stowe, Paul Bartlett, and Robert Bull for their assistance and support. A special thanks
to Dr. N. Kent Ames who helped with surgical techniques for sample collection. I wish
to recognize the help and encouragement of my Residency Advisor Dr. Paul Coe who
worked hard to make time available for me to complete this project.

Funding was generously provided in part from a BRSG grant, and Schering—
Plough Corporation. My thanks also to the Department of Large Animal Clinical
Sciences for funding and the opportunity to do this project.

Thanks and appreciation are also due to Anne House, Tonic Thiel and Kathy
O’Hare for their patience and help with sample analyses. A special thanks to both
Pamela Bunce and Sheila Wing-Proctor who were so helpful as technicians during saniple
collection. The assistance of MaryEllen Shea with slides and document preparation was
excellent. Finally thanks, to Dr. Allaire Smith who donated her valuable student time
to help with sample collection.

Thanks also to Dennis Frie, Dale Pollock, Lloyd Sheffield, Howard Spicer and

Mike Stevens for their help in preparing stalls and daily care of the animals.

iii

TABLE OF CONTENTS

Ease
LIST OF TABLES ....................................... vi
LIST OF FIGURES ...................................... ix
LITERATURE REVIEW
Toxicity ............ ‘ ............................. 1
Essentiality and Biochemical Function ....................... 2
Sources and Factors Influencing Requirements . . . . . . .~ ........... 3
Absorption ........................................ 4
Biological Availability ................................. 7
Excretion ......................................... 8
Deficiency ........................................ 9
Retained Placenta .................................... 10
Cystic Ovarian Disease ................................ l4
Fertilization ....................................... l4
Sperm Viability ..................................... 15
Abortion ......................................... 16
Selenium Status ..................................... l7
Supplementation .................................... 19
Bibliography ....................................... 20
INTRODUCTION ....................................... 26
MATERIALS AND METHODS
General .......................................... 28
Trial 1 .......................................... 31
Trial 2 .......................................... 34
RESULTS
Trial 1 .......................................... 36
Trial 2 .......................................... 43

iv

TABLE OF CONTENTS (continued)

DISCUSSION .......................................... 50
APPENDICES
Appendix A ....................................... 59
Appendix B ....................................... 79
BIBLIOGRAPHY ....................................... 89

Table Bass:
TEXT
1 Assessment of selenium status in cattle ....................... 18
2 Composition of concentrate fed to cows during the trials ............ 29
3 Sampling schedule for trial 1 ............................. 33
4 Sampling schedule for trial 2 ............................. 35
APPENDIX 1
1 Serum selenium concentrations ............................ 59
2 Whole blood selenium concentrations ........................ 60'
3 Selenium concentrations of liver biopsies obtained on days 0 and 90 ..... 61
4 Urinary and fecal selenium concentrations ..................... 62
5 White blood cell concentrations ........................... 63
6 Erythrocyte concentrations .............................. 64
7 Hemoglobin concentrations .............................. 65
8 Packed cell volumes .................................. 66
9 Serum aspartate amino transferase activities .................... 67
10 Serum gamma-glutamyl transferase activities ................... 68

LIST OF TABLES

LIST OF TABLES (continued)

my. Bags
11 Serum sorbitol dehydrogenase activities ...................... 69
12 Serum creatine phosphokinase activities . V ..................... 70
13 Body weights ...................................... 71
14 Body condition scores ................................. I 72
15 Average daily hoof growth .............................. 73
16 Rabies titers ....................................... 74
17 [’Ifl-thymidine uptake of unstimulated lymphocytes ............... 75
18 [3H]-thymidine uptake of phytohemagglutinin-stimulated lymphocytes . . . . 76
19 [3H]-thymidine uptake of concanavalin-stimulated lymphocytes ........ 77
20 [’Hl-thymidine uptake of pokeweed-stimulated lymphocytes .......... 78
APPENDIX 2
1 Serum selenium concentrations ............................ 79
2 Whole blood selenium concentrations ........................ 79
3 Selenium concentrations of liver biopsies ..................... 8O
4 Urinary selenium concentrations ........................... 80
5 Fecal selenium concentrations ............................ 81
6 White blood cell concentrations ........................... 81
7 Erythrocyte concentrations .............................. 82
8 Hemoglobin concentrations .............................. 82

LIST OF TABLES (continued)

Ialzle Rage

9 Packed cell volumes .................................. 83
10 Serum aspartate amino transferase activities .................... . 83
11 Serum gamma-glutamyl transferase activities ................... 84
12 Serum sorbitol dehydrogenase activities ...................... 84
13 Serum creatine phosphokinase activities ...................... 85
14 Body weights ......... ' ............................. 85
15 Body condition scores ................................. 86
16 [3H]-thymidine uptake of unstimulated lymphocytes ............... 86

17 [’H]-thymidine uptake of phytohemagglutinin-stimulated lymphocytes . . . . 87
18 [’Ifl-thymidine uptake of concanavalin-stimulated lymphocytes ........ 87

19 [3H]-thymidine uptake of pokeweed-stimulated .................. 88

viii

10

11

12

13

LIST OF FIGURES

Page
Selenium concentrations in forages and grains from different regions
of the United States and Canada ........................... 5
Mean serum selenium concentrations for cows in trial 1 ............ 37
Mean whole blood selenium concentrations for cows in trial 1 ........ 38
Mean selenium concentrations of liver biopsies obtained on days 0 and
90 in trial 1 ....................................... 39
Mean urine selenium concentrations of cows in trial 1 on day 90 ....... 41
Mean fecal selenium concentrations of cows in trial 1 on day 90 . . . . . . 42
Mean serum selenium concentrations of cows in trial 2 ............. 4-4
Mean whole blood selenium concentrations of cows in trial 2 ......... 45
Mean selenium concentrations of liver biopsies of cows in trial 2
on days 100, 128 and 184). ............................. 46
Mean urine selenium concentrations of cows in trial 2 ............. 48
Mean fecal selenium concentrations of cows in trial 2 ............. 49
Mean ratio of whole blood selenium to serum selenium concentrations . . . 52
Regression of estimated daily fecal and urinary losses ............. 55

LITERATURE REVIEW

Selenium (Se) is an essential trace mineral in animal nutrition. The discovery of
Se is attributed to the Swedish scientist Berzelius in 1818.1 The biological importance
of high concentrations of Se became evident when it was associated with "alkali disease"
and ”blind staggers” in the 1930’s. These syndromes were related to feeds grown on
seleniferous soils (soils having high Se concentrations) of the northern Great Plains of
the United States.2 Descriptions of toxic feed producing similar syndromes have been
reported dating back to the writings of Marco Polo. The perceived negative effects of
Se were supported by reports in the 1940’s that Se had carcinogenic properties.
Consequently, for the next two decades Se was viewed as a toxic element with no

beneficial nutritional effect.

Toxicity:

Toxicity in dairy cattle is usually of the chronic form (alkali disease or blind
staggers); however, there have been reports of acute toxicity or ”high-concentration"
toxicity. Signs of acute toxicity include anorexia and decreased milk production with
single doses of 7-10 mg Se/kg of body weight.’ Single doses above 11 mg Se/kg
produce signs of excessive salivation, respiratory distress, garlic-smelling breath, and

death within 48 hours. These eases usually only occur when cattle have no other feed

2 .
except seleniferous plants (plants which accumulate high concentrations of Se) or when
large amounts of Se are accidentally or experimentally administered. Chronic selenosis,
in the form of alkali disease, is characterized by signs of alopecia, hoof malformations
and loss, emaciation and reproductive failure accompanied by increases in serum
transaminases and alkaline phosphatase.‘ Chronic ingestion of seleniferous plants also
produces the syndrome of blind staggers. Animals affected wander aimlessly, stumble,
appear to have impaired vision, and have signs of respiratory distress. This characterizes
the blind staggers type of toxicity and can be reproduced with Se-free water extracts from
the plants. Based on these findings it has been proposed that the cause of these signs is .
the water soluble alkaloids found in seleniferous plants and that Se is not the cause of

”blind staggers”.‘

Essentiality and Biochemical Functiom:

The dietary essentiality of Se became evident in the late 1950’s. Researchers
working independently discovered that Se played an important role in preventing liver
necrosis in rats’ and exudative diathesis in chicks.“ During the next three deeades, Se
deficiency has been associated with many diseases. Selenium supplementation of animal
feeds today, is a relatively common practice. A common concern is the perceived ease
of producing toxicity with small deviations from recommended concentrations.

The biochemieal importance of Se is related to Sc being a component of the
enzyme glutathione peroxidase (GSH-Px)." This enzyme is located in the cytosol where

it is important in converting toxic free radicals to water. Research continues to identify

other biochemical functions of Se which may help to explain further the causes of Se

3
deficiency diseases. The functions of Se are closely related to vitamin E. Se alone may

alleviate or decrease the severity of some vitamin E-responsive diseases.“

Sources and Factors Influencing Requirements:

Selenium is normally obtained by the body through the diet. The Se content of
a feed is related to the content and availability of Se in the soil on which the feed is
grown, the plant species and any supplemental source of Se added to the feed. The Se
availability to plants depends upon soil type, pH and climatic conditions. Soils are
divided into three groups based on Se availability: toxic seleniferous, nontoxic
seleniferous and low Se soils.9 Plant species utilize and accumulate Se to various
degrees producing different concentrations of Se. Plants have been divided into three
groups: primary, secondary, and non-Se accumulators. ‘0 Primary accumulators such
as the genera Mus and Stanley; have the ability to accumulate Se in concentrations
of from 1000 to greater than 7000 ppm.“ Secondary accumulators such as the genera
mm and Mentzelia accumulate Se up to 100 to 200 ppm. Non-Se accumulators
include plants routinely used as animal feeds such as cereal grains, grasses, and legumes.
These plants may accumulate Se up to 20-50 ppm depending on soil condition and Se
content. Grasses tend to have higher Se concentrations than legumes.

The many factors which influence the Se concentration in plants make it possible
for animal feed Se concentrations to vary from less than 0.01 ppm to greater than 10,000
ppm. Forages produced on neighboring farms may be very different in Se
concentration.12 Maps defining areas with forages containing low, variable, adequate,

and toxic Se concentrations are constantly being expanded and updated as new

4

information becomes available (Figure 1). In North America, areas containing forages
with toxic concentrations of Se are limited to the Great Plains of the United States.
Areas in North Ameriea having forages low in Se content include the Eastern and
Western coastal regionsandtheareassurroundingtheGreatIakesinCanadaandthe
Northeast, Atlantic Seaboard, areas around the Great Lakes, and the Pacific Northwest

in the United States.

Absorption:

The absorption of Se is significantly lower in ruminants than monogastrics. The
retention of oral Se as sodium selenite was 77% in swine while only 29% in sheep.‘3
Absorption from the stomach area is essentially absent in both monogastrics and
ruminants. Most absorption in the ruminant occurs in the small intestine and the cecum.
In monogastric species, absorption occurs in the last part of the small intestine, cecum,
and colon. In the rat, the availability of Se has been shown to be related to the form of
Se. Organic Se compounds were found to be 13896-18096 more available when
compared to inorganic forms.“ A significant amount of the availability at low
concentrations of Se supplementation is related to the amount of Se absorbed. Everted
intestinal sacs of hamsters have demonstrated that selenomethionine is transported against
a concentration gradient where selenite and selenocystine are not. Transport of
selenomethionine is inhibited by methionine where selenite and selenocystine are not
inhibited. ‘5 This indicates selenite is passively absorbed along concentration gradients

while selenomethionine is actively transported during absorption.

 

 

 

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m Low-arraoxmaruv sort or ALL FORAGE mo enam comam (0.10 PPM snemum
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C3 ADEQUATE-80% OF ALL FORAGES AND GRAIN CONTAIN )OJO PPM SELENIUM
(INCLUDES HAWAIII

Figure l. Selenium concentrations in forages and grains from different regions of
the United States and Canada.‘

 

' National Research Council. 1983. Selenium in Nutrition, revised ed. National
Academy Press, Washington, D.C. pp 24.

6
Chemically, the differences in availability of Se from various seleno—compounds

hasbeenrelatedtoanumberofcriteriaintherat. Thetwocarbonchainsattachedto
the Sc atom affect Se availability. Even numbered chains have low‘availability while un-
even numbered chains have relatively high availability. The location of the Sc atom near
the center of the molecule, combined with odd numbered earbon chains, gives very high
availability. Short alkyl chains with 3 or 4 earbons significantly depress Se availability
by allowing the formation of 5-6 member rings. Finally acid amides of seleno-mrboxylic
acids are more potent than the free acids unless the compound contains more than one
Se atom. “ Further, availability was greatly influenced by the structure of the alkanoic
moiety of the molecule, and the presence of a quaternary carbon atom in the chain almost
totally eliminated availability. Biopotency was further decreased by the introduction of
methyl or nitro groups in the fourth position of the ring in the benzylseleno-earboxylic
compounds." Unfortunately, no data could be found comparable to these data for
ruminants. Rumen microbes do play a major role in changing the form of ingested Se.

The absorption of Se is also related to whether Se is bound or unbound in protein
with inorganic Se being absorbed more readily.“ Biosynthesis of seleno-compounds
from inorganic Se or unbound Se occurs in the rumen of sheep. '9 Specifically Se"s
selenomethionine has been found to be formed after incubation of Se” selenite with
rumen microbes.20 Rumen microbes have been shown to not only convert Se to
insoluble forms by reduction but also to incorporate Se, in the form of selenomethionine,
into bacterial protein. Studies have also shown that inorganic Se may be substituted for
inorganic sulfur during rumen microbial amino acid synthesis and that the resulting

seleno-amino acids are incorporated into microbial protein.21 The preceding would

7

indicate that rumen microorganisms are responsible for the lower absorption of Se in the .
ruminant compared to the non-ruminant. The ruminant microbes impair absorption by
reducing ingested Se to insoluble forms (bound or unbound Se) which occurs to a greater
extend with inorganic Se compared to organic forms. Further data indicate that a
ruminant consuming a high carbohydrate diet provides a better environment for the
conversion of Se to insoluble forms than when consuming a high roughage diet.” The
solubility of inorganic Se is related to the oxidation states of Se which include -2
(selenide), 0 (elemental Se), +4 (selenite), and +6 (selenate). The selenate form of Se

is the most soluble with elemental Se being insoluble and selenite in between.

Biological Availability:

All Se absorbed is not utilized physiologically. Biologieal availability is a
measure of how much Se is available to the tissues from a specific Se compound after
the compound has been exposed to several physiologieal and metabolic processes. These
include digestion, absorption, and metabolism which may be affected by the Se status of
the animal. Selenium bioavailability is only an estimate of Se utilization derived from
experimental values, and must be considered in the light of biologieal response(s).’°
Selenium compounds are classified into three different groups based on biological
availability. The first group consists of the more reduced and insoluble forms which
have very low bioavailability. Secondly, Se in most animal products other than protein
has low to moderate bioavailability. Thirdly, the common selenoamino acids like

selenomethionine or selenocystine which have relatively good bioavailability.

Excretion:

Selenium is excreted from the body by three major routes. These include feeal,
urinary, and respiratory excretion. Respiratory excretion is minimal at dietary Se
concentrations less than 1 ppm. At higher concentrations, respiratory excretion becomes
more significant.” Excretion of Se depends on the form of Se ingested and the amount
of Se fed. In sheep fed sodium selenite and selenomethionine at the same low
concentrations, the amount of Se excreted in the feces was about equal for each source.
The major route of excretion is through the feces. However, the Se in the feces of the
sheep fed sodium selenite was much more insoluble than the feces of sheep fed
selenomethionine. This supports the theory that rumen microbes convert ingested Se into
insoluble forms, especially if the Se is in the inorganic form.

Urinary excretion of Se was higher in the sheep fed sodium selenite compared to
sheep fed selenomethionine. This suggests that selenomethionine is better utilized by
tissues (higher biological availability) than sodium selenite leaving more sodium selenite
available for excretion in the urine.” Selenomethionine may also be incorporated into
non Se requiring proteins making very little Se available for body needs. It is however,
important to note that selenite is more easily excreted by the kidney when compared to
seleno amino acids. Toxic concentrations of Se eause Se excretion in the urine to
approach and even exceed Se excretion in the feces.” The major excretory product in
rat urine fed excessive amounts of Se is trimethylselenonium.26 In animals receiving
adequate concentrations of Se, little or no trimethylselenonium is found in the urine;
however, at excess concentrations of Se, the concentration of trimethylselenonium

increases.” This has been suggested as a possible method for evaluating the adequacy

9

of the concentration of Se supplementation. However, the necessity of a twenty four
hour urine collection for accuracy limits it’s practical applieation. The observations of
this excretory product have been limited to the urine of monogastrics. Presence of this
compound in ruminant urine must, therefore, be confirmed. It has been observed in
ruminants that Se excretion in the urine does increase with excess concentrations of

supplementation.”

Deficiency:

Numerous livestock problems have been related both independently and in
conjunction with other nutrients, to Sc deficiency. Selenium most frequently is
associated with vitamin E but also has been associated with the elements calcium, copper,
sulfur, arsenic and cadmium. In ruminants, Se deficiency has been associated with a
multiplicity of disease syndromes including nutritional muscular dystrophy (White Muscle
Disease) in sheep, cattle, pigs, goats and horses,” infertility in sheep, pigs, and
cattle,” retained placenta in cattle-”'3‘ cystic ovarian disease in eattle,32 abortion in
sheep, cattle, pigs, and horses,33 and untliftiness in both sheep and cattle.34 Recently,
Se deficiency has also been related to impaired function of both the humoral and cell-
mediated immune systems in ruminants.35""37 In eattle the influence of Se on the
immune system has been specifically manifest in the duration of clinical signs of
mastitis."39

Reproductive problems in cattle, including retained placenta, cystic ovarian
disease, fertilization, sperm viability and abortion, have been related to Se deficiency.

The literature in these areas is both supportive and non-supportive of these relationships.

’10

It is impossible to disassociate Se from vitamin E in most eases. The following review

will focus on the effects of Se.

Retained Placenta:

Retention of the placenta during the early postpartum period is a common problem
in dairy eattle and has received much attention over the years. The overall incidence of
retained placenta is reported by numerous investigators to be ten percent in North
America.”""" Sporadic incidences exceeding fifty percent have been reported.“

Retained placenta in cattle is characterized as failure of the fetal placenta to separate
from the maternal crypts, which normally occurs within 2-8 hours postpartum. Retention
is defined as the placenta remaining intact for greater than 12 hours.‘4 Retained
placenta is associated with decreased fertility in the postpartum dairy cow as measured
by increases in calving interval and days to conception due to various factors. A major
factor is an increased rate of uterine infection, up to 54% from a normal of 10%.“
Historically, nutrition has been associated with the various causes of increased incidence
of retained placenta.“

During the late 1960’s and early 1970’s, an association between white muscle
disease and retained placenta was first reported by Trinder in the British Isles.” This
was the first reported association of Se and vitamin E deficiency with retained placenta.
Since that time many investigations of this relationship have been conducted in different
locations in the world with some investigators, interestingly, concluding Se and/or
vitamin E to be the cure for retained placenta and some investigators finding no evidence

to support a relationship.

1 1
Effective experimental prevention of retained placentas with Se supplementation

has been reported in Ohio herds consuming a ration formulated with feeds containing 20-
40 ng Se/g DW.“ This was accomplished either by injecting 50 mg of Se 1-3 weeks
prepartum or supplementing the ration with 1 mg sodium selenite daily (a very low,
concentration; of supplementation). The same group of investigators reported at about the
same time that a single dose of 50 mg of sodium selenite and 680 IU of vitamin E as
alpha-tocopherol, administered 21 days prepartum, reduced the incidence of retained
placenta from 51% to 9% in 113 cows.‘3 At the same time, no improvement in retained
placenta incidence was observed with sodium selenite supplementation in South Dakota
where Se is adequate in feeds.“ Collectively, of these observations seemed to support
Se deficiency being related to the incidence of retained placenta and making Se.
supplementation a logical preventative.

Subsequent observations indicated that the concentration of Se supplementation
to prevent retained placenta varied relative to the concentration of protein and type of
diet during the prepartum period. The same concentration of Se supplementation was
shown to be more effective in preventing retained placean when cows were fed pasture
compared with higher protein alfalfa silage as the main component of their prepartum
diet.”

Calcium concentlations in the ration are related to the amount of Se absorbed;
however, this was not directly related to the incidence of retained placenta. Maximal Se
absorption was reported when Ca represented 0.8% of dry matter intake. Higher or
lower concentrations of calcium in the diet resulted in decreased Se absorption.‘0 The

combination of these two observations may be related due to the higher predicted ealcium

12
content of alfalfa silage when compared to pasture. The high protein concentration of

the silage may not be related to the lower Se tissue concentrations and higher incidence
of retained placenta.

Selenium has been closely linked with vitamin E in a controlled experiment. It
showed no effect of vitamin E or Se supplementation individually when administered
orally, or by injection, however, a reduction of the incidence of retained placenta from
17.5% to 0% was observed in animals supplemented with both vitamin E and Se.‘1
Adding even more confusion, a study in Israel of cattle consuming Se-low diets showed
a decreased incidence of retained placenta (approx. 10%) in relatively low doses of
supplemental Se (2.3 mglday, 21 days prepartum) and that Se alone was just as effective
as vitamin E and Se together.‘2

A specific mechanism has not been demonstrated relating Se deficiency to retained
placenta. The theory of decreased uterine muscle function in the early postpartum period
has been considered. In a limited number of cows with retained placenta, the Se
concentrations in the cotyledons and caruncles were 27 .5 % and 33 % lower, respectively,
than for cows with normal placenta expulsion.” A similar observation has been made
in both the placenta and caruncle of the ewe suggesting that these tissues might be
particularly prone to Se deficiency.“

Numerous investigators, however, have found no reduction of retained placenta
incidence when Se and/or vitamin B were supplemented.” Some of these observations
were made in geographical locations (Nebraska) with adequate concentrations of Se in
foodstuffs and with confirmed adequate serum Se concentrations of .082 ug Selml.“

Another large study recently examined 627 parturitions at a large university research

13
farm in the province of Ontario in Canada.” These investigators concluded that

retained placenta was not a Se-responsive disease. This conclusion was based on the lack
of signifieant differences in the retained placenta incidence between controls, Se and/or
vitamin E supplemented cows. It is important to note, however, that the parturition
plasma Se concentrations of control animals were in the low adequate range (.07 ug/ ml)
and the concentrations of the treated animals were in the middle of the expected adequate
range (.083-.089 pg/ml). This fact would support a different conclusion; e.g. that
retained placenta is not Se-responsive in cattle which have an adequate level of Se. The
study did not demonstrate what the incidence of retained placenta was in deficient
animals.

The most logical approach to the relationship of Se and vitamin E to the incidence
of retained placenta in cattle is demonstrated in a study conducted in four herds in North
Carolina during the early 1980’s." A single injection of 50 mg Se and 680 IU alpha-
tocopherol was used at 21 days prior to expected parturition. The Se status of the cattle,
based on serum Se concentration prior to treatment, was correlated with the incidence
of retained placenta. There was no effect of Se and vitamin E supplementation in cattle
with a pretreatment Se status of adequate (2 0.08 ppm) or extremely deficient
(s 0.05 ppm). A significant difference was demonstrated in eattle with borderline Se
status (0.05—0.08 ppm). This supports the hypothesis that one of the factors affecting the
incidence of retained placenta is the deficiency of Se and/or vitamin E. Since the
concentration of vitamin E provided by the injection is low, it is most likely Se is the
important component. Further, it demonstrates that the incidence of retained placenta

will not be changed if Se status is adequate and additional Se is provided. Likewise,

14

insufficient Se provided to severely deficient eattle to correct their Se status will not
decrease the incidence of retained placenta. Indiscriminant use of standard dosages of
Se and vitamin E injections in prepartum dairy cattle are not indieated. Pretreatment
evaluation of Se status of the population should be accomplished prior to making the

clinical decision to supplement the population.

Cystic Ovarian Disease:

A negative correlation has been demonstrated between cystic ovarian disease and
plasma Se concentrations (r = 0.83) and GSH-Px activity (r = 0.69). Correlation,
however does not indicate a causal relationship. Subsequently, it was demonstrated that
injected Se reduced the incidence of cystic ovaries by 28% compared to controls.33 It
is important to note that the incidence of cystic ovaries has been shown to be related to
other post-partum problems such as retained placenta and milk fever. The investigations
do not allow conclusions regarding whether Se has a direct eausal effect on cystic ovarian
disease incidence or whether the reduced incidence is simply related to a decrease in the

incidence of retained placenta which has been related to Sc.

Fertilization:

Decreased fertility in a group of cattle used for embryo transfer in Ohio has been
related to low Se concentrations.” This group of cattle was also consuming a diet low
in protein, energy and vitamin A. A subsequent trial showed 100% fertilization of ova
when eattle were provided a adequate diet supplemented with Se and vitamin E and only

a 40% rate of fertilization in unsupplemented group. Other investigators have reported

15
no change in number of fertilized ova in Se- and vitamin E-treated Charolais cattle but

did report an increased number of sperm associated with ova in supplemented eattle. On
this basis these investigators suggested that ova fertilization has no association with Se
status but that Se supplementation may increase sperm transport.‘o Supporting data
relating Se to fertility are reported for sheep where in Se supplementation was related to
increased ova fertilization, embryonic survival and stronger contractions of uterine
muscle, possibly improving sperm transportmm However, one paper reports
normal ealving percentages in cattle grazing the same pasture with sheep exhibiting very
low lambing percentages. Iambing percentages were improved from as low as 25 % to
80-120% with Se supplementation immediately prior to breeding.

The evidence for a relationship behveen Se and fertilization in cattle is unclear.
The only evidence suggestive of a relationship is an increased number of sperm
associated with fertilized ova. This was in a relatively small group of cattle with no

increase in numbers of fertilized ova in Se supplemented animals.

Sperm Viability:

The addition of Se at the rate of 1 ppm to diluted semen was reported to increase
sperm motility and sperm oxygen consumption in 13 of 15 ejaculates.“ However, other
investigators, working with twenty-four yearling Angus bulls, were able to show
increased Se concentration in serum and various semen components as a result of Se
supplementation but no difference in percent viability thawed semen.“ They concluded

that Se was not associated with in vitro sperm viability; however, no in vivo fertility

 

l6
observations were made. Conflicting evidence about the association of Se to semen

viability is the result.

Abortion:

In the past decade, reports have surfaced associating the frustrating problem of
undiagnosed abortion in the bovine with Se deficiency. In western Canada liver Se
concentrations in 69 of 243 aborted fetus were in the severely Se-deficient category.
Thirty-five of the Sc- deficient fetuses had no other detectable cause of abortion while
most of the other thirty-four had bacterial and viral isolates usually not associated with
bovine abortion.“ In Michigan, of seventy-four bovine fetuses with an undetermined
cause of abortion after complete necropsy, histology, bacteriology and virology,
nutritional analysis demonstrated that 32 % of the liver Se values were in the deficient
category and that 28 % had deficient concentrations of Vitamin E. 16 More recently
another study in Michigan demonstrated a 31% incidence of low liver Se in 301 fetuses
reviewed. One hundred and forty one fetuses in this group had no other demonstrable
cause of abortion; of these, twenty-eight had low liver Se and thirty-eight had both low
liver Se and vitamin E concentrations.” A review of aborted fetuses from 1976-86
submitted to the Veterinary Diagnostic Laboratory at Oregon State University showed an
increase in Se—deficient fetuses compared to liver Se between 1982-86.“

A hypothesis is proposed by Taylor to explain the relationship of Se to abortion.
A common thread through the many abortion syndromes suggests vascular damage which
leads to degeneration and necrosis of cells in the particular target organ. Taylor

proposed that a common denominator is the protection of biological membranes by Se

17
and vitamin E. In the deficient state, more severe damage may be prostaglandin-

mediated. Arachidonic acid is released from the damaged membranes via phospholipase
A2 which is activated by many stimuli, including membrane damage comparable to that
which may occur with Se deficiency. Two of the resulting prostaglandins (F2a and
TXA2) have. physiological effects which include producing thrombosis and vascular
damage. This is the common histological lesion in nutritional muscular dystrophy
(WMD) as well as in aborted fetuses and placenta. Likewise PGF2a is produced which
has a luteolytic effect and initiates strong uterine contractions.“ A strong case is

developing for a relationship between Se deficiency and abortion in the bovine.

Selenium Status:

Selenium status is assessed directly by measuring the concentration of Se in
serum, plasma, whole blood and/or tissues flurometrically.“9 An indirect measure of
Se status is plasma, erythrocyte or tissue GSH-Px activity using a coupled colormetric
procedure.7o Plasma and serum Se concentrations indieate the present Se status."
Whole blood Se concentration and erythrocyte GSH-Px activity, tend to reflect prior Se
status."2 This is thought to be due to the incorporation of GSH-Px and Se into
erythrocytes during erythropoiesis. The rate of erythrocyte turnover is reflected in the
whole blood Se concentration and erythrocyte GSH-Px activity .7’ Table 1 provides a

summary of values used in the assessment of Se status in cattle."

18

 

 

Table 1. Assessment of Selenium status in cattle.‘
Won
Reference Tissue Units Adequate Marginal Deficient
Julien et al. 1976b; Serum jig/ml > .08 .05-.08 < .05
Sergerson et al. 1981
Puls 1981 Serum jig/ml .07-.3 .02-.04 .002-.008
Liver jig/g W .25-.50 .12-.25 .02-07
Koller et al. 1983 Whole rig/ml > .10 .051-.10 < .05
Blood
GSH-PX3 mU/ mg Hb < 15 15-30 > 30a
Maas 1983; Maas and Whole rig/m1 .07-> .10 .05-.06 .01-.04
Koller 1985 Blood
GSH—Px mU/mg Hb 0-15 15-25 25-500
Liver ug/g DW < .2-.3
Miller and Thompson Whole umole/L .8-2.5 .4-.8 < .4
1983 Blood
' Liver umole/kg < 3.0
DW
Van Vleet 1980 Liver jig/g WW <10
Whole ug/ml < .05
Blood .
Carlstrom et al. 1979 GSH-Px ukat/L > 500 200-500 <200
Thompson et al. 1980 Whole pmole/kg > .191 .127—.19l < 127
Blood
Liver umole/kg > .635 .254-.635 <254
WW

 

1from Van Saun 1988
2Liver wet weight (WW) or dry weight (DW)

3Erythrocyte glutathione peroxidase activity

l9
Supplementation:

Supplemental Se has been provided to livestock per 03 and by parenteral
injection.” The parenteral (injection) Se supplementation method has several
limitations. These include labor requirements to administer repeated individual
injections, relative short period of effect (1-2 weeks), drug and tissue reactions to the
parenteral product, and the additional cost required to provide a sterile product suitable
for injection. In fact, Se supplementation on a unit per unit basis of Se costs greater than
twenty-five times more in the parenteral form compared to oral Se supplementation.
Selenium supplementation has only been allowed in some livestock feeds since
1974.767” This was due to the concern of possible toxicosis resulting from over
supplementation. The allowable concentrations have also been very conservative
reflecting data collected mainly in monogastric animals."9 The current allowable
concentration of supplementation is 0.3 ppm of the total ration for ruminants.”

Researchers have speculated that ruminants probably have a higher tolerance for
supplemental Se.12 Concentrations approaching 10 ppm have been fed to lactating dairy
cattle for a short period of time (8 days) with no apparent problems.’o Livestock
producers and veterinarians report an apparent biological effect in the absence of toxicity
at concentrations of Se supplementation above allowable concentrations.81 The exposure
of supplemental Se to the microbial flora probably is a signifieant factor affecting the

amount of absorption of Se.“2

BIBLIOGRAPHY

1. Schamberger RJ, Biochemistry of Selenium. Plenum Press, New York (1983).

2. Franke KW, A new toxicant occurring naturally in certain samples of plant
foodstuffs. J Nutr l934;8:597.

3. Miller WT, Williams KT. Minimum lethal doses of selenium, as sodium
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2O

21

13. Wright PL, Bell MC. Comparative metabolism of selenium and tellurium in
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14. Schwarz K, Fredga A. Biologieal potency of organic selenium compounds.
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16. Schwarz KA, Fredga A. Biological potency of organic selenium compounds.
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17. Schwarz K, Fredga A. Biological potency of organic selenium compounds,
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18. Thomson CD, Robinson BA, Stewart RDH, Robinson MF. Metabolic studies
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19. Rosenfeld I. Biosynthesis of seleno-compound from inorganic selenium; Proc
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20. Hidiroglou M, Heaney DP, Jenkins KJ. Metabolism of inorganic selenium in
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21. Paulson GD, Baumann CA, Pope AL. Metabolism of 75-Se-selenite, 75-Se-
selenate, 75-Se-se1enomethionine, and 35-S-sulfate by rumen microorganisms in vitro.
J Ani Sci 1968b;27:497.

22. Whanger PD, Wewig PH, Muth OH. Metabolism of 75-Se-selenite and 75-Se-
. selenomethionine by rumen microorganisms. Fed Proc 1968;27:418.

23. Burk RF, Brown DG, Seeley RJ, Scaief CC. Influence of dietary and injected
selenium on whole body retention, route of excretion, and tissue retention of 75-Se(-2)
in the rat. J Nutr 1972;102:1049.

24. Ehlig CF, Hogue DE, Allaway WH, Hamm DJ. Fate of selenium from
selenite or selenomethionine with or without vitamin E, in Lambs. J Nutr 1967;92: 121.

25. Rosenfeld 1. Metabolic effects and metabolism of selenium in animals.
University of Wyoming Agricultural Exp Sta Bull 1964:414.

26. Palmer 18, Fischer DD, Halverson AW, Olson OE. Identification of a major
selenium excretory product in rat urine. Biochimica et Biophysica Acta 1969;177:336.

27. Nahapetian AT, Janghorbani M, Young, VR. Urinary Trimethylselenonium
excretion by the rat: Effect of level and source of selenium-75. J Nutr 1983;113:401.

22

28. Muth OH, Oldfield JB, Schubert JR, Remmert LF, White muscle disease
(myopathy) in lambs and calves. VI. Effects of Selenium and Vitamin E on lambs. Am
J Vet Res 1959;20:231.

29. Hartley WJ, Selenium and ewe fertility. Proceedings of the N Z Soc Ani Prod
1963;23:20.

30. Trinder N, Woodhouse CD, Rentan CP. The effect of vitamin E and selenium
on the incidence of retained placentae in dairy cows. Vet Rec 1969;85:550.

31. Julien WE, Conrad HR, Jones JE, Moxon AL, Selenium and Vitamin E and
the incidence of retained placenta in parturient dairy cows. J Dairy Sci 1976;59: 1954.

32. Harrison JH, Hancock DD, Conrad HR. Selenium deficiency and ovarian
function in dairy cattle. Fed Pro 1982;41:786.

33. Yarnini B, Mullaney TP, Vitamin E and selenium deficiency as a possible
cause of abortion in food animals. Proc 28th Annual Meeting Am Assoc Vet Lab
Diagnosticians 1985;131-144.

34. Andrews ED, Hartley, WJ, Grant AB. Selenium-responsive diseases of
animals in New Zealand. N 2 Vet J 1968;16:3.

35. Norman BB, Johnson W. Selenium responsive disease. Ani Nutr Health
1976;31:6.

36. Gyang E0, Stevens JB, Olson WG, Tsitsamis SD, Usen KEA. Effects of
selenium-vitamin E injections on bovine polymorpho-nucleated leukocytes phagocytosis
to killing of Staphylococcus aureus. Am J Vet Re 1984;45:175.

37. Swecker WS, Eversole DE, Thatcher CD, Blodgett DJ, Schurig GG,
Meldrum JB. Influence of supplemental selenium on humoral immune responses in
weaned beef ealves. Am J Vet Res 1989;50: 1760.

38. Smith KL, Harrison JH, Hancock DD, Todhunter DA, Conrad H,R. Effect
of vitamin E and selenium supplementation on the incidence of clinical mastitis and
duration of clinical symptoms. J Dairy Sci 1984;67:1293-1300.

39. Erskine RJ, Eberhart RJ, Grasso MS, Scholz RW. Induction of Escherichia
coli mastitis in cows fed selenium-deficient or selenium-supplemented diets. Am J Vet
Res 1989;50:2093.

40. Erb RE, Hinze PM, Gildow EM, Morrison RH. Retained Fetal Membranes -
The effect of prolifieacy of Dairy Cattle. J Am Vet Med Assoc 1958;133:489.

41. Muller LD, Owens MJ. Factors associated with the incidence of retained
placentas. J (Dairy Sci 1974;57:725.

23

42. Wetherhill GD, Retained placenta in the bovine. A brief review. Can Vet J
1965;6z290.

43. Julien WE, Conrad, HR, Moxon AL, Selenium and Vitamin E and incidence
of retained placenta in parturient dairy cows. 11. Prevention in commercial herds with
prepartum treatment. J Dairy Sci 1976b;59: 1960.

44. Black WG, Ulberg LC, Kidder HE, Sinvi J, McNutt SH, Casida LE.
Inflammatory response of the bovine endometrium. Am J Vet Res 1953;14:179.

45. Callahan CJ, Post parturient infection of dairy cattle. J Am Vet Med Assoc
1969;155:1963.

46. Guieero RTC. Retained fetal membranes in eattle. J Am Vet Med Assoc
1959;135:475.

47. Trinder N, Rentan CP, The relationship between the intake of selenium and
vitamin E on the incidence of retained placenta in dairy cows. Vet Rec 1973;93:641.

48. Williams WF, Yuer DR, Diefeuderfer DL, Douglas L.W., and Vandersall,
Influence of prepartum selenium-vitamin E on retained placenta in dairy cattle, Proc
Forage Research Farm Field Days, Maryland Agri Exp Sta, Univ Maryland 1977 :24.

49. Reinhardt TA, Conrad HR, J ulien WE, Moxon AL. Alfalfa silage, selenium
injections and retained placentas. J Dairy Sci l978;61(supp. l):185.

50. Harrison JH, Conrad HR. Effect of dietary calcium on selenium absorption
by the non-lactating dairy cow. J Dairy Sci 1984;67:1860.

51. Harrison JH, Hancock DD, Conrad HR. Vitamin E and selenium for
reproduction of the dairy cow. J Dairy Sci 1984;67:123.

52. Eger S, Drori D, Kadoori 1, Miller N, Schindler H. Effects of selenium and
vitamin E on incidence of retained placenta. J Dairy Sci 1985;68:2119.

53. 80th H, Schramel P. Investigation to the Influence of Selenium in
Veterinary Medicine: Nutritional Muscle Dystrophy and Retained Placenta. Trace
Element Analytical Chemistry in Medicine and Biology, Walter de Gruyter and Co. ,
New York, N.Y., pg 83 (1980).

54. Hidiroglou M, Hoffman I, Jenkins KJ. Selenium distribution and
radiotocopherol metabolism in the pregnant ewe and fetal lamb. Can J Physi Pharm
1969;47:953.

55. Gwazdauskas FC, Bibb ML, McGillard ML, Lineweaver JA. Effect of
prepartum selenium-vitamin E injection on time for the placenta to pass and on
reproductive functions. J Dairy Sci 1979;62:978.

24

56. Ishak MA, Larson LL, Owen FG, Lowry SR, Erickson ED. Effects of
selenium, vitamins and ration fiber on placental retention and performance in dairy cattle,
J Dairy Sci 1983;66:99.

57. Hidiroglou M, McAllister A], Williams CJ. Prepartum supplementation of
selenium and vitamin E to dairy cows: Assessment of selenium status and reproductive
performance, J Dairy Sci 1987;70: 1281.

58. Segerson EC, Rivierc GJ, Dalton HL, Whitacre MD. Retained placenta of
Holstein cows treated with selenium and vitamin E. J Dairy Sci 1981;64:1833.

59. Segerson EC, Murray FA, Moxon AL, Redman DR, Conrad HR. Selenium
and Vitamin E: Role in fertilization of the bovine ova. J Dairy Sci 1977;60: 1001.

60. Segerson EC, Libby DW. Ova fertilization and sperm number per fertilized
ovum for selenium and vitamin E-treated Charolais cattle. Theriogenology 1982; 17:333.

61. Hartley WJ, Grant AB. A review of selenium responsive diseases of New
Zealand livestock. Fed Proc 1961;20:679.

62. Segerson EC, Ganapathy SN. Fertility of ova in ewes receiving selenium and
vitamin E supplementation. J Ani Sci 1979;49 (supp. 1):335.

63. Segerson EC, Ganapathy SN. Fertilization of ova in selenium/vitamin E
treated ewes maintained on two planes of nutrition. J Ani Sci 1983;51:386.

64. Julien WE. Murray FA. Effect of selenium and selenium and vitamin E on
in vitro motility of bovine spermatozoa. Proceedings of the Ameriean Society of Animal
Science, 69th annual meeting, Madison, U. of Wisconsin, pg. 174 .

65. Segerson EC, Johnson BH. Selenium/Vitamin E and reproductive function in
yearling Angus Bulls. J Ani Sci 1980;51:395.

66. Taylor RF, Puls R, MacDonald (KR. Bovine abortions associated with
selenium deficiency in Western Canada. Proceedings of the 22nd Annual Meeting of the
Am Assoc Vet Lab Diagnos 1979:77.

67. Yarnini B, Trapp AL, Stowe HD. Congenital myopathy, cardiomyopathy,
Purkinjie cardiocyte degeneration and abortion associated with Vitamin E and/or selenium
deficiency in the bovine Am J Vet Res 1989? (accepted for publication).

68. Hedstrom OR, Maas JP, Hultgren DD, Lassen ED, Wallner-Pendelton EA,
Synder SP. Selenium deficiency in bovine, equine, and ovine with emphasis on its
association with chronic disease. Proceedings of the 29th Annual Meeting of the Am
Assoc Vet Lab Diagno 1986:101.

25

69. Olson OE. Flourometric analysis of selenium in plants. J Assoc Off Anal
Chemists 1969;52:627-634.

70. Agergarrd N, Jensen PT. Procedure for blood glutathione peroxidase
determination in eattle and swine. Acta Vet Seand 1982;23:515-527.

71. Thompson KG, Fraser AJ, Harrop BM, Kirk JA. Glutathione peroxidase
activity in bovine serum and erythrocytes in relation to selenium concentrations of blood
serum and liver. Res Vet Sci 1980;28:321-324.

72. Thompson KG, Fraser AJ, Harrop BM, Kirk JA, Bullians J, Cordes DO.
Glutathione peroxidase activity and selenium concentration in bovine blood and liver as
indicators of dietary selenium intake. N Z Vet J 1981;29:3—6.

‘ 73. Oh SH, Sunde RA, Pope AL, Hoekstra WG, Glutathione peroxidase response
to selenium intake in lambs fed a tonrla yeast-based, artificial milk. J An Sci
1976a;42:977—983.

74. VanSaun RJ. Selenium and Vitamin E: Relationships between the pregnant
dairy cow and fetus. MS Thesis 1988:36.

75. Muth OH, Schubert JR, Oldfield JE, White muscle disease (myopathy) in
lambs and calves. VII. Etiology and prophylaxis Am J Vet Res 1961;22:466.

76. Food and Drug Administration. Food Additives: selenium in animal feed. Fed
Reg 1974;39:1355.

77. Food and Drug Administration. Food additives permitted in feed and drinking
water of animals: selenium. Fed Reg 1979;44:5392.

78. Food and Drug Administration. Food Additives: selenium in animal feed. Fed
Reg 1987;52: 10,668.

79. Combs, GF, Combs SB. W Aeademic Press,
New York 1986:465.

80. Fisher LJ, Hoogendom C, Montemurro J. The effect of added dietary
selenium on the selenium content of milk, urine and feces. Can J An Sci 1980;60:79

81. Ellis RG. (personal communications).

82. National Research Council Subcommittee on Selenium; W
1983:72.

INTRODUCTION

Selenium (Se) is an essential trace mineral in the nutrition of eattle. However,
cattle feed that contains high Se concentrations is reported to produce acute and chronic
toxicity. "2'3 Signs of acute toxicity include anorexia, decreased milk production, excess
salivation, respiratory distress, breath with a garlic-like odor, and occasionally death.‘
Clinical signs of chronic toxicity in cattle are frequently that of a chronic wasting
disease. These signs include anorexia, emaciation, dullness, listlessness, rough hair coat,
alopecia, hoof sloughing, joint erosions, liver cirrhosis and death.‘

Research during the last three decades has related numerous livestock problems
to Sc deficiency; both independently and in conjunction with other nutrients. Selenium
deficiency is frequently associated with vitamin E deficiency but also has been
demonstrated to interact with and be affected by, the elements calcium, copper, sulfur,
arsenic and cadmium. In eattle, Se deficiency has been associated with numerous disease
syndromes. These include nutritional muscular dystrophy (White Muscle Disease),s
infertility,‘5 retained placenta,“ cystic ovarian disease,’ abortion,10 untriftiness,"
impaired function of both the humoral and cell-mediated immune systems?” and
mastitis.“ Selenium’s major biological function is related to it’s structural role in the

enzyme glutathione peroxidase (GSH-Px).“ Selenium is also associated with the

26

27
function of hepatic enzymes involved with metabolism and detoxification of drugs and

other foreign substances.“

The maximum legal concentration of Se in feed for dairy cows has recently been
raised from 0.1 to 0.3 ppm of total dry matter." Consequently the Sc concentration
in supplements and premixes has been raised. These changes, while most likely being
beneficial to animal health and production, also, increase the possibility of dietary Se
intake inadvertently exceeding legal limits. This could result from multiple Se sources
or errors in feed formulation. The authors are aware of some herds consuming 19 mg '
of supplemental Se/head/day and have seen bags of Se-supplement premix containing 50
mg Se/ounce (approximately 10X higher than intended) mistakenly delivered to dairy
farms on Se supplementation programs.

The need to document any signs of excess dietary Se intake in Holstein cows is
evident. Likewise it is necessary to identify tests to determine the Sc status of cattle
suspected to be receiving excess dietary Se whether from natural or supplemental
sources.

The objectives of this experiment were twofold. First, to determine the
response(s) of Holstein cows to high concentrations of dietary Se as sodium selenite.
Secondly, to compare the values of Various measures of Se status in Holstein cows to
three different dietary Se concentrations. The experiment consisted of two concurrent

trials to be referred to as trial one and two.

MATERIALS AND METHODS

General:

The research cows were housed in individual tie stalls in a common stable .
Feeders were partitioned to assure that consumption of the Sc supplement was limited to
the designated cow. All cows were fed mixed hay (ad libitum). A corn meal concentrate
(Table 2) containing the supplemental Se was fed once daily. Blood samples were
collected from the coccygeal vessels. into tubes containing heparin, EDTA or no
anticoagulant. Samples of whole blood and serum for Se analysis and rabies titers were
frozen at -20°C for future analysis. Blood samples for hematology studies, enzyme
activity determinations, and lymphocyte response testing were analyzed immediately.

Antemortem liver samples were obtained using a percutaneous liver biopsy
technique." This procedure uses a illuminating endoscopic device‘ to visualize the
liver. The endoscope has a plastic stylet which, after making a small skin incision, is
bluntly introduced into the abdomen via the eleventh or twelfth right intercostal space.
After removal of the stylet, a custom-made biopsy instrument was introduced through the
endoscope to collect a 3—6 gram sample of liver tissue. Postmortem liver samples were
obtained when the cows were slaughtered at the end of the trials. Liver samples were

frozen at - 0°C until analyzed.

 

' Welch Allyn, Skaneateles Falls, New York
28

29

 

 

Table 2. Composition of concentrate fed to cows during the trials."2
WW

Ingredients units 0 3 20 50 100
Ground com 96 87 87 87 87 87
Trace mineral salt % 6.7 6.7 6.7 6.7 6.7

‘ Molasses 95 3.7 3.7 3.7 3.7 3.7
Limestone x 102 96 2.5 2.4 2.0 1.2 0.0
Sodium Selenite X 10'2 % 0.0 .074 .5 1.3 2.5
Vitamin A IU3/kg 55 55 55 55 55
Vitamin D IU’lkg 5 5 5 5 5
Vitamin E u/kg 166 166 166 166 166

 

‘.9 kg of concentrate fed daily

2in addition to ad libitum mixed legume hay - estimated Se content .05 ppm

30
Variables tested included Se concentrations in serum, whole blood and liver.

Urinary and fecal Se concentrations were also measured. Hematologic measurements
included packed cell volume and the concentrations of white blood cells, red blood cells
and hemoglobin. Serum enzyme activities of aspartate arninotransferase (AST), gamma
glutamyl transferase (GGT), sorbitol dehydrogenase (SDI-I) and creatine phosphokinase
(CPK) were also determined. General health variables included daily measurements of
body temperature, heart rate and respiratory rate. At the beginning of the trial, a groove
was cut 5mm below the coronary band on the dorsal and lateral surfaces of diagonal feet.
Monthly, the distance between the groove and the coronary band was measured to
quantify hoof growth, body weight was estimated by heart girth measurement, body-
condition scores were recorded and the animals were videotaped. Lymphocyte response
tests to the non-specific mitogens coneanavalin A, phytohemagglutinin and pokeweed
mitogen were measured approximately every 45 days during the trial. All samples were
collected in the morning prior to the feeding of the concentrate containing the Sc
supplement.

Analysis for serum, whole blood, liver, urine and fecal Se concentrations used
the improved flurometric method. ‘9 Hematologieal analysis of fresh, EDTA-treated
blood used the Technicon H-l automated hematology analyzer.” Analysis of serum

enzymes was done on fresh serum using a Flexi-Gem centrifugal analyzer.c

 

”Technicon Instruments Corp., Tarrytown, N .Y.
cPharrrlacia E.N.I. Diagnostics Inc., Fairfield, N.J.

31
Trial One:

Twenty-four adult, non-lactating Holstein cows were used as research animals.
After a two-week period of adjustment on the base diet, they were divided into four
groups balanced for serum Se concentrations. The groups were supplemented with 0,
3, 20 and 50-mg Se/head/day (Control, 3, 20, and 50-mg groups, respectively) for 90
days. A summary of the sampling dates for each variable measured is presented in
(Table 3).

The cows were vaccinated on days 45 (primary) and 73 (secondary) with a
commercial rabies vaccine‘I licensed for use in cattle. Rabies vaccine was used because
it is safe and effective and cows were not likely to have baseline titers. Rabies titers
were measured on days 0, 7 and 14 after primary and secondary vaccinations to
measure primary and secondary immune response.

On the last day of the trial, voided urine samples and random samples of fresh
feces were collected from each cow. ‘

Variables measured were tested by a split-plot, repeated measures analysis of

variance with main effects of dietary Se and time over treatment.20 The linear model

is as follows:
Yiill = +S,- + A0),- + T, + ST, + E“,
Where
Y = the individual dependent variables measured
S = dietary Se; 0, 3, 20, 50 mg/head/day
A = animal, A0,- = animal within treatment group = error term for testing
treatment effects.
T = time
ST = time by Se interaction
E = random error

 

‘Rabguard TC, Norden Laboratories Inc. , Lincoln, Neb.

32
An example of the AN OVA table used follows:

TYPE III

Won 11! as MS halite 81:
Among Subjects

Treatrnemts 3 74.469 24.823 17.68 .0001
Subjects (error) 20 28.077 1.4038

With Subjects
Time 1 57.028 57.028 56.74 .0001
Time * Treatments 3 63.644 21.214 21.11 .0001
Error, 20 20.101 1.005

Minimum Significant Difference (MSD) equations used are:
l) MSD = V [q,] (t,), where

2) Vs = (0)2/ {[(MSDIA + (3‘1) (MSafi/abzan}. 811d
3) tD,.05,3,20) 33d

4) V (Qt) = (Caz) (Oz/1‘). 311d

5) o2 = [MSW + (b-1)MSB] lb

Specific contrasts between controls (0mg Se) and treatments were assessed with
Dunnet’s t distribution.
In this analytical design, time trends, as well as overall mean differences in

dependent variables were assessed.21

33

 

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34
Trial two:

Trial two was conducted beeause no detectable signs of toxicity had developed in
any of the groups after 90 days of daily feeding in trial one.

At the end of trial one, the 6 cows in the 50-mg group continued to receive the
concentrate feed containing 50 mg of supplemental Se for 10 additional days. Starting
on day 100 they were fed the same concentrate reformulated to provide lOO-mg
Se/head/day (lOO-mg group) for an additional 28 days. After day 128 no concentrate
was fed so that changes occurring in response to the withdrawal of Se could be
monitored. A summary of the sampling dates to assess each variable can be found in
(Table 4). Analysis of variables measured at the beginning and end of the 100 mg

supplementation was done using paired t tests.

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RESULTS

Trial One:

Mean serum Se concentrations increased with treatment in all groups (Figure 2 and
Table l of Appendix A). Differences between the mean serum Se concentrations of the
control and 3-mg groups were never significant (P > .05) and remained below reference
concentrations for the entire trial.”23 Serum Se concentrations increased rapidly in
the 20 and 50-mg groups becoming and remaining significantly different (P < .05) from
the control group within 2 days after the beginning of Se supplementation. The mean
serum Se concentration of the 20-mg group remained within, while the 50-mg group
exceeded the reported reference range at the end of the trial.

Whole-blood Se concentrations increased gradually with treatment in all groups
with values becoming significantly different from the control group (P < .05) at days
5 and 70 of the trial in the 50- and 20-mg groups, respectively (Figure 3 and Table 2 of
Appendix A). The mean values of whole blood Se concentrations in the 3-mg group
were never significantly different (P 2 .05) from the control group.

Mean liver Se concentrations in the control and 3-mg groups did not change
significantly between day 0 and day 90 of the trial (Figure 4 and Table 3 of Appendix
A). In the 20- and 50-mg groups Se concentrations were significantly different

(P < .05) from the controls at day 90.

36

37

 

 

Serum Selenium

 

 

 

o l l l l l l l l l
0 10 20 30 40 50 60 70 80 90
Days of Trial
Figure 2. Mean serum selenium concentrations for cows in trial 1 supplemented with
0 (control)¢—O, 3 Anna, 20 I--I and 50 O' '0 mg
Sc/head/day. IEEEEEEEEEI reference range (70 - 100 ng/ml),

— significantly different from control group (P S .01).

E
5
OH
H
2
a)
U)
'6
Q
G
_
m
o
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G
E 50
0
Figure 3.

 

38

 

I I l I l l l l l

10 20 30 4o 50 60 70 80 90
Days of Trial

Mean whole blood selenium concentrations for cows in trial 1
supplemented with 0 (control) 0—0 , 3 A - . - oa, 20 I—-I and 50
O- -0 mg Se/head/day. IEEEEEEEEE | reference range (150 - 220 ng/ml),
— signifieantly different from control group (P s .01).

39

 

 

 

 

pg/g dry weight
8 ..
t
50
I ’ ’
I
6- ,”
S ”
H I ’ ’
. I t
5 , ’
_ I ’ J
0 4 — a r ’ , - -
8 ””’,
.2 , - , ’
A ’ I I ’ a
t'{é}.-.{GEE}?!{iiii-Ital-2+2?Inf-lift}?Z-Z-I-Ii-Ii-I-I-I'li-I'2'Ii-Ii-Iii-EI-I'I-I'I-Ii-I-I'2'2-2'}lii'I-Z-I'li-I-I-Iii-I'l'li'I'I-I-I
- - - - 213,? 3.3.4].35.3.13.3}.35.3.32:333:35:313:?:3:1:3:3:':':':':':'3'1':°:':'11:3:3:313:2}:3.133335‘j'3'3'3'3'?5'3“3:333'i'3;3:3:3:1:3:l:';3:3:l:32121:3331
................................... H
l L l L l _l
40 so 60 7o 80 90
Days of Trial

Figure 4. Mean selenium concentrations of liver biopsies obtained on days 0 and 90
in trial 1 for cows supplemented with 0 (control) 0—0 , 3 A - . - - A, 20 '
I--I and 50 O' '0 mg Se/head/day. IEEEEEEEEE I reference range (1.2
(P10 erg/g dry wt.), * significantly different from control group
S. .

40
The mean concentrations of urinary and fecal selenium were significantly greater

(P < .05) in the 20- and 50-mg groups when compared to the control at day 90 of the
trial. There was no difference between the mean urine and fecal Se concentrations in the
control and 3-mg groups at day 90 of the trial (Figures 5 and 6 and Table 4 and 5 of
Appendix A).

Means for the hematologic variables measured, including packed cell volumes and
concentrations of white blood cells, red blood cells and hemoglobin did not deviate from
reference ranges or from controls (P > .05) (Tables 5-8 of Appendix A). Likewise,
there was no deviation from reference values in the mean activities of serum enzymes
which included AST, GGT, SDH and CPK (Fables 9-12 of Appendix A). No significant
differences (P _<_ .05), in the above variables were detected in any groups compared to
controls.

The cows gained weight in all groups and maintained their body condition score
throughout the trial (Tables 13-14 of Appendix A). No significant difference (P 2_ .05)
in hoof growth was observed between treatment groups, compared to the control (Table
15 of Appendix A).

Following vaccination there were no differences in rabies titers among groups. All
rabies titers were negative initially in all groups and rose after the first and second
vaccinations, showing comparable primary and secondary immune responses (Table 16
of Appendix A). Baseline lymphocyte blastogenesis, as well as the response to three
non-specific mitogens, revealed no significant differences (P > .05) between

supplemented groups and the controls (Tables 17-20 of Appendix A).

 

 

 

 

 

 

 

 

 

 

 

§

' mg Selenium/head/day

Mean urine selenium concentrations of cows

intriallonday90,

* signifieantly different from control group (P S .01).

 

 

 

 

 

 

\\\\\\\\.

 

 

 

 

 

 

 

 

'um/hea

43
The other health variables monitored including attitude, body temperature, heart

rate, respiratory rate showed no apparent deviation from reference ranges or differences

between groups.

Trial Two:

Mean serum Se concentration increased very rapidly during the first ten days after
Se intake was increased from 50 to 100 mg/head/day (100-mg group) and continued to
increase for the next 18 days to a concentration about two and one half times reference
concentrations (Figure 7. and Table 1 of Appendix B). When the Sc supplementation
was terminated at day 128, mean serum Se concentration fell very rapidly to
approximately one half of the peak values by day 132 and continued to fall gradually,
reaching the reference range by day 156 of the trial. The mean concentration declined
below the reported reference concentrations by day 184.

The mean whole-blood Se concentration rose gradually throughout the 28 days of
100-mg Se/head/day supplementation reaching a peak mean concentration of 485 ng/ml
(Figure 8 and Table 2 of Appendix B). , After supplementation was terminated, mean
whole-blood Se concentration also fell very rapidly to about four fifths peak concentration
by day 132 and then very slowly decreased to about two times the reported reference
concentrations and slightly above the predicted reference concentrations at day 184.

Mean liver Se concentration from liver biopsies at day 128 of 100-mg group was
seven times higher than reference concentrations and two and one half times beginning
* concentrations (Figure 9 and Table 3 of Appendix B). After 56 days with no additional

Se supplementation, mean liver Se concentration had returned to normal.

 

 

11 ml
300 -g/
250 — , , ‘°.
8 ’ ’ I '-
3' 200 — Ir 2
fl 1 .
m 150 - ,' °.
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100 . :3. .................. 3G .......................................
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'3}}?Eff-I'.CC'ZC'ZCCCCf' ......................................................................................................... 3.23:; '0
50 —
1 1 1 1 1 1 1 1 1
100 110 120 130 140 150 160 170 180 190
Days of Trial
Figure 7.

Mean serum selenium concentrations for cows previously fed 50 mg
Se/head/day for 100 days in trial 1 and (in trial 2) supplemented with 100
mg Se/head/day for 28 days O ' ' O , followed by no supplemental

selenium for 56 days .00.. IEEEEEEEEEI reference range (70 - 100
ng/ml).

45

 

 

11 ml
600 — g/
500 —
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03 ’l’ 2
5 400 — ,z . o
a I .‘ e
m I. 9 . . . e. e . . ‘
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é 5:323:35?5:323:22?:3:?:3:?:3:?:3:355:35:23???13:3:3z3i3t3t3:3:3:3:1:3:311:3:313:35:52???3:1:3:31?23:3:3:3:3:3:3:i:3:3:3:3:3:3:3:3:?:33:313:33:
100 -
0 l l l l l 1 l l A
100 110 120 130 140 150 160 170 180 190
Days of 'Il'ial

Figure 8. Mean whole blood selenium concentrations for cows previously fed 50 mg
Se/head/day for 100 days in trial 1 and (in trial 2) supplemented with 100
mg Se/head/day for 28 days 0 ' - O , followed by no supplemental
selenium for 56 days .00.. liiiiiiiiil reference range (150 - 220
ng/ml).

46

jig/g dry weight

 

 

20
D
15 — ’I ' .
E ’ ° .
.E ’ I e .
a x o.
'3 10 —- 1’ ° .
U) 1’ e
3 ” O 0
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5 L e e
1223;352:2223:1:323:3:?:3:?:i:3:3:3:35:3:331:323:?:3:3:3:3:3:3:?:?:3:3:1:3:3:15:32?13:3:32221:3123?1323123323132323:23:23:3:313:23:3132353352-52'6
o l l l l l l ' J I l l
90 100 110 120 130 140. 150 160 170 180 190
Days of Trial

Figure 9. Mean selenium concentrations of liver biopsies from cows previously fed
50 mg Se/head/day for 100 days in trial 1 and (in trial 2) supplemented
with 100 mg Se/head/day for 28 days 0" '0, followed by no
supplemental selenium for 56 days .00.. liiiiiiiiil reference range
(1.2 - 2.0 ug/g dry wt).

47

The mean concentrations of urinary and fecal selenium was markedly increased at
the end of supplementation. By the end of the trial, concentrations had returned to
values comparable to the original control group on day 90 of trial one (Figures 10 and
11 and Tables 4 and 5 of Appendix B). 7

Means for the hematologic variables measured, including packed cell volumes and
concentrations of white blood cells, red blood cells and hemoglobin, did not deviate from
reference ranges (Tables 6—9 of Appendix B). Likewise the mean activities of serum
enzymes, which included aspartate aminotransferase, gamma-glutamyl transferase,
sorbitol dehydrogenase, and creatine phosphokinase, did not deviate from normal (Tables ,
10-13 of Appendix B). The cows gained weight and maintained their body condition
throughout the trial (Table 14 and 15 of Appendix B).

Tests of lymphocyte responses to the three non-specific mitogens showed no
differences at any time during the trial. (Tables 16-19 of Appendix B).

The other health variables monitored, including attitude, body temperature, heart
rate, respiratory rate and mucus membrane color, showed no apparent deviation from

normal ranges during the trial.

48

 

 

11 ml
1600 — g/ a;
I o
I e
I e
I ¢.
1200 '— I ’ e .
a x .,
.a ,I .
.5. . X n
a 800 ..
0 o
0:: ‘.
t: .
:9 ° ,
400 — . .
0 l l l l l l l l .. J
100 110 , 120 130 140 150 160 170 180 190
Days of Trial

Figure 10. Mean urine selenium concentrations of cows previously fed 50 mg
Se/head/day for 100 days in trial 1 and (in trial 2) supplemented with 100
mg Se/head/day for 28 days O " ' O , followed by no supplemental
selenium for 56 days O00 O. * significant different from 50 mg group
(P < .01).

12

on
I

Fecal Selenium
a
I

 

100

Figure 11.

49

jig/g dry weight ,

1 1 1 1 1 1 . 1 1’
110 120 130 140 150 160 170 180

Days of 'h‘ial

Mean fecal selenium concentrations of cows previously fed 50 mg
Se/head/day for 100 days in trial 1 and (in trial 2 supplemented with 100
mg Selhead/day for 28 days O ' ' O , followed by no supplemental
selenium for 56 days O 00 O. * significant different from 50 mg group
(P < .01).

DISCUSSION

The major purpose of this experiment was to study clinical responses of Holstein
cows to dietary Se concentrations which ranged from allowable (.3ppm) to excessive as
might result accidentally in the preparation of diets. The responses were evaluated on
the basis of changes over time in serum, whole—blood, liver, urine and fecal Se
concentrations. From these observations, an effort was made to identify the most
sensitive measure of Se status.

Serum Se concentrations changed much more rapidly in response to treatment than
did whole-blood Se (Figure 2 and 3 and Table l and 2 Appendix A). These different
rates of response have occurred due to differences in Se dynamics in serum, compared
to red cells. Se in red cells is present almost entirely as glutathione peroxidase, an
enzyme protein synthesized during development of the cell, prior to release into the
circulation. Serum Se, on the other hand, consists of several protein and nonprotein
bound forms of Se. Therefore, that portion of whole-blood Se that is contributed by the
red cells, probably represents Se availability during development of the current red cell
population. Because the red cell life span is 100-120 days, red cell Se may better reflect
long-term changes in Se availability while serum Se reflects short term changes. This
interpretation is consistent with the data of this experiment. It is interesting to note that

whole-blood Se concentrations first became significantly different from controls after

50

51
about 10 days of Se supplementation in the 50-mg Se/head/day group versus 70 days in

the 20-mg group after the beginning of supplementation (Figure 3 and table 2 of
Appendix A). The more rapid increase in whole-blood Se concentrations in the 50-mg
group compared to the 20-mg group probably reflects the proportionally higher Se
concentration in the serum fraction of the whole-blood in the 50-mg group.

When the ratio of whole blood Se to serum Se for a given sample day is calculated,
the overall mean ratio is approximately 2.2 (Figure 12). Reference concentrations for
serum Se are reported to be 70 - 100 ng/ml.24 If whole-blood Se is on average 2.2
times serum Se reference, concentrations the predicted whole blood Se reference
concentrations should be 150 - 220 ng/ ml. These reference concentrations are supported
by various other investigations,”"“"7 but are inconsistent with 80 - 120 ng/ ml as
proposed by others.28 This observation is important when evaluating the Sc status of
cows when different methods of Se testing have been used.” In this experiment, mean
whole-blood Se concentrations in all groups exceeded the lower (80-120 ng/ ml) reported
reference concentrations at all times.’”‘ However, mean whole-blood Se
concentrations exceeded the higher (150-220 ng/ ml) proposed reference concentrations
only in the 50-mg group at the end of the trial.

As evident in this experiment, this 50-mg group of cows would be diagnosed as
having a low Se status based on serum Se concentrations and an adequate Se status based
on whole blood Se concentrations if the lower whole—blood Se reference concentrations
were used(Figures 2 .& 3 and table 1 & 2 of Appendix A). However, using the higher
proposed whole blood Se reference, concentrations the cows would be diagnosed as

having low Se status based upon both serum and whole-blood Se tests.

P :9
. N a

to Serum Selenium

Whole Blood Selenium

LA

Figure 12.

 

52

 

1 1 1 1 1 1 1 1 1
10 20 30 40 50 60 70 80 90

Days of Trial

Mean ratio of whole blood selenium concentrations to serum selenium
concentrations in cows fed 0 0—0, 3 A- . . u, 20 I--I and 50
O' 'O mg Sc/head/day and the overall mean ratio x .. .. x.

53
The whole-blood Se concentration in cows, after being fed lOO—mg'Se for 28 days,

was approximately five times the lower reported reference concentrations and only two
times the higher proposed reference concentrations. This difference in reference
concentrations could lead to an erroneous diagnosis of toxicity.

Because of the need for surgical intervention to obtain liver biopsy samples to
measure liver Se concentration, liver biopsies were only collected at the beginning and
end of each trial. Liver Se concentrations were at normal reference values32 at the
beginning of the trial in all four groups. There was no difference between the control
and 3-mg group during the 90 days of feeding (Figure 4 and Table 3 of Appendix A).
Liver Se concentrations in the 20- and 50-mg groups were significantly higher than the
control group at the end of the first trial (P < 0.01). Liver Se concentrations in the 100-
mg group, which initially were about 3 times the reference values, increased to 8 times
reference values in the 28 day feeding period. Liver Se values declined to the reference
range within 60 days after discontinuing the 100 mg Se/head/day supplementation (Figure
9 and Table 3 of Appendix B).

The concentrations of Se in the urine and feces were highest in cows fed the
highest amounts of Se (Figures 5,6,10 & 11 and tablei4 Appendix A and table 4 & 5
Appendix B). It is important to note that the urine and fecal Se concentrations were only
from one-time catch samples and that 24-hour urine and fecal collections would be
necessary to quantify, more accurately, the actual amount of Se excreted. Measurement
of the ratio of urinary total Se and trimethylselenonium Se concentration has also been
used in the rat and man to help establish guidelines for adequate supplementation.33"‘

This is an area that should receive attention in cows.

54
Assuming that a Holstein cow produces 4 kg of fecal dry matter and 20 liters of

urine daily, the Sc retained by the cows fed the four concentrations of supplemental Se
was estimated. A regression line, (Figure 13) based on the estimated retention of Se on
the four diets predicts the amount of supplemental Se necessary to maintain Se balance
in cows receiving low Se forages. Based on the above assumptions, the requirement
appearstobeabout6-8mgofSedaily. Inareas whereforagesandgrainsarelowin
Se (< 0.05 mg Se lkg), a lactating Holstein cow consuming 20 Kg of dry matter
supplemented at .3 ppm would obtain about 1 mg of Se naturally from forages and grains
and would receive about 6 mg of Se from the supplementation. This would appear to be
adequate to maintain Se balance in the lactating cow and is in agreement with other
investigations.” However, a dry cow consuming 10 Kg of dry matter supplemented
at 0.3 ppm would be obtaining only about 0.5 mg of Se from forages and grains and only
about 3 mg from supplemental Se. This would not be adequate to maintain Se balance.
This apparent Se imbalance could be particularly harmful during the period of rapid fetal
growth during late gestation."5 More extensive balance studies, including 24-hour
collection of urine and feces, are needed to confirm these interpretations.

Selenium toxicity, while expected, was not observed at the amounts of Se
supplementation in this experiment. This is probably due to at least two factors.
Namely, the differences between the ruminant and monogastric digestive tracts, and the
Sc excretion rates via urine and feces. Proposed toxic concentrations for ruminants
appear to have been extrapolated from data collected in monogastric animals. Studies
have shown that monogastrics absorb as much as 2.5 times more Se than ruminants.”

The lower absorption of Se by the ruminant is likely due to the reduction of dietary Se

55

25 mglday

15—

lo-

 

Estimated Selenium Retained

 

Figure 13.

mg Selenium/head/day

Regression of estimated daily fecal and urinary selenium losses by adult
cows orally supplemented with selenium at 0, 3, 20 and 50 mg

Se/head/day. R2 =.958. Daily fecal dry matter and urine volumes were
estimated at 4 kg and 20 L, respectively.

56
to less available forms, such as elemental Se, by rumen microbes prior to the opportunity

for absorption in the small intestine. Biosynthesis of seleno-compounds from inorganic
or unbound Se has been demonstrated in the rumen of sheep and in in vitro incubations
of Se” with rumen microbes?”39 Selenium absorption is related to whether Se is in
bound or unbound forms. Unbound, inorganic Se. forms are absorbed more completely
than bound forrns.‘o Inorganic Se in the soluble forms of selenate and selenite is
reduced in the rumen to insoluble elemental Se.“

It is clear from the data presented that absorption and excretion increased with
dietary intake. This is supported by the increases in all three measures of Se status and
both urine and fecal Se concentrations in Cows supplemented at higher concentrations.
Reasons for the lack of toxicity therefore are likely related to both decreased absorption
and increased excretion of Se.

The lack of any significant affect on the hematologic, serum enzyme and health
variables measured, as well as the fact that animals maintained or gained weight,
maintained body condition and remained in good health during the experiment, indicate
that no apparent negative affects occurred at these excessive amounts of Se
supplementation in Holstein cows. These observations indicate that there is a large
difference between allowable and toxic supplemental Se concentrations in animal feeds.
However, it is important to note that no apparent benefits to health or immunological
variables were observed to be associated with high Se supplementation concentrations.

The addition of supplemental Se to cattle diets is a routine nutritional
management practice in the United States. This is due to the convenience and modest

cost of oral supplementation when compared to other methods of supplementation. A

 

57

common method of incorporating Se into grain mixes is to mix either 3 pounds of Se
supplement (200 ppm) or 1 pound of Se supplement (600 ppm) per ton of grain, to create
a final dietary Se concentration of .3ppm. A cow consuming 10 kilograms of the
concentrate daily would then receive 3 mg of supplemental Se. A sixteen fold mistake
in formulation for 90 days would be necessary to equal the Sc intake in the 50-mg group
in this experiment. A thirty three fold mistake for 28 days would be necessary to equal
the Sc intake rate of the 100-mg supplemental Se group.

A second method of supplementation is to top dress 200 ppm or 600 ppm Se
premixes daily on the feed for each cow or alternatively incorporate supplements into a
total-mixed ration. Commonly, 1 ounce of the 200 ppm Se premix is fed daily per head.
This provides 5.5 mg of supplemental Se per head, an amount approximating the
maximum allowed by FDA. Depending on the type of animal housing and the
completeness of feed mixing, cows might get two to three times the expected amount of
Se by consuming feed intended for other animals. A feeding or mixing error of about
9 times the target amount daily for 90 days would be required to equal the amount of Se
provided to the experimental 50-mg group. An error of 19 times the daily targeted
amount for 28 days would be necessary to equal the amount of Se provided to the 100-
mg experimental group. Based on the results of this experiment, the above scenarios
would not cause short term problems with Holstein cows. NRC guidelines suggest Se
concentrations in excess of 2 ppm Se fed are toxic to cattle.‘2 This guideline could
lead to the incorrect, presumptive diagnosis of Se toxicity in cows consuming diets with

Se concentrations of 2 ppm or greater.

58
Legallytheconcentrationof.3ppmSeoftotaldietarydrymatterisnottobe

exceeded.‘3 In this experiment cows fed the 3 mg supplemental Se/head/day would
have consumed .3 ppm dietary Se, assuming a dry matter intake of 10 kg/day. Serum
and whole blood Se concentrations in the 3 mg group were never significantly different
(P g .05) from cows in the group receiving no supplemental Se (controls). The Se
status of both the control and 3-mg group remained below the reference ranges for all
three measures of- Se status. This observation, combined with the absence of detectable
signs of toxicity at the higher rates of supplementation, suggests that the current legal
rate (0.3 ppm) of oral Se supplementation as sodium selenite is certainly in a very safe
range and indeed may be lower than optimal. It is important to note that no benefits of
feeding the excessive concentrations of Se used in this experiment were detected. This
research indicates that in ruminants, the margin of safety between legal (.3 ppm) and
toxic Se intake is greater than was previously thought. Therefore, Se supplementation
within reasonable boundaries should not be avoided because of the concern for a low

margin of safety and the production of accidental toxicity.“

APPENDIX A‘

 

Includes data (individual, mean, SD) for the respective variables from cows fed
0, 3, 20, and 50 mg supplemental Se/hd/d for 90 days. Unless otherwise
indicated reference ranges are from the Animal Health Diagnostic and Clinical
Pathology Laboratories at the College of Veterinary Medicine, Michigan State
University.

59

 

 

 

 

APPENDIX A
Table 1. Serum selenium concentrations.
Cow Wide!
Se ID 0 2 6 13 18 23 28 42 56 70 84 90
mg/d ng/ml"
0 1 26 45 46 49 54 60 63 59 48 50 56 61
2 33 44 45 38 34 41 44 35 40 38 38 43
3 35 51 53 44 57 56 60 57 72 58 52 64
4 35 40 43 39 40 41 45 48 52 58 58 68
5 73 72 70 58 52 59 55 46 47 50 46 57
6 41 52 52 44 48 48 45 47 46 53 51 63
Mean 40 51 52 45 48 51 52 49 51 51 50 59
SD :1; 15 10 9 7 8 8 8 8 10 7 7 8
3 7 24 37 48 59 59 60 73 60 75 60 47 69
8 38 55 64 60 59 62 62 60 60 61 53 63
9 41 48 56 47 53 56 61 49 55 56 54 64
10 37 54 63 56 52 78 61 58 62 55 54 64
1 l 40 55 57 50 84 53 64 56 56 52 55 62
12 26 43 45 49 53 56 65 54 55 54 49 62
Mean 34 49 56 54 60 61 64 56 60 56 52 64
SD :1; 7 7 7 5 1 l 8 4 4 7 3 3 2
20 13 28 53 70 79 7 1 72 80 67 64 65 70 85
14 33 54 77 95 76 78 83 67 74 76 88 88
15 40 63 60 87 89 . 77 85 71 76 78 81 83
16 36 60 70 102 68 75 80 79 75 72 78 93
17 36 79 77 66 70 77 78 76 76 76 78 85
18 48 73 77 73 84 77 75 78 83 80 81 90
Mean 37 64 72 84 76 76 80 73 75 74 79 87
SD 3; 6 10 6 12 8 2 3 5 6 5 5 3
50 19 32 74 92 107 130 114 117 92 89 96 112 102
20 35 66 88 84 85 106 106 87 93 96 103 l 15
21 44 86 102 82 101 110 110 88 108 93 107 129
22 28 7 1 86 87 94 106 106 91 106 94 106 l 12
23 48 66 97 86 102 117 117 95 106 99 112 104
24 43 82 108 100 102 147 147 100 122 101 117 125
Mean 38 74 96 91 102 99 117 92 104 96 110 114
SD :1; 7 8 8 9 14 9 14 4 11 3 5 10

 

* Reference range 70-100

 

 

 

 

APPENDIXA
Table 2. Whole blood selenium concentrations.
Cow Quantum!
Se ID 0 2 6 13 18 23 28 42 56 70 84 90
mg/d nglml'll
-0 1 63 59 62 74' 82 81 84 94 85 81 87 114
2 139 117 117 129 131 131 138 137 129 118 132 154
3 145 123 122 127 150 133 140 137 156 138 133 144
4 157 146 135 137 154 124 146 148 143 143 143 184
5 153 174 156 171 190 160 170 152 169 142 135 175
6 88 112 86 111 112 96 107 99 103 99 92 125
Mean 124 122 113 129 136 121 131 128 131 120 120 149
so :1: 36 35 31 29 34 26 28 23 29 24 22 25
3 7 148 56 65 90 100 92 91 102 112 105 102 139
8 149 137 129 131 155 154 171 163 162 143 155 154
9 158 148 140 145 152 150 147 139 144 125 124 144
10 130 125 132 135 140 122 142 144 152 136 135 184
11 154 140 144 147 158 132 157 158 167 145 143 175
12 100 98 97 109 126 130 139 126 134 119 147 125
Man 140 117 118 126 138 130 141 139 145 129 134 154
so :1: 20 32 28 20 20 20 25 20 18 14 17 20
20 13 106 99 101 128 143 150 137 132 150 142 151 199
14 139 134 175 145 170 160 165 164 177 164 185 220
15 109 107 119 115 124 120 132 132 141 136 169 206
16 137 144 143 162 159 162 160 174 165 148 192 234
17 115 106 113 129 131 136 142 147 164 170 160 194
18 140 130 148 163 151 147 152 143 162 158 149 185
Mean 124 120 133 140 146 146 148 149 160 153 168 206
so a 15 17 25 18 16 14 12 16 12 12 16 16
50 19 115 106 142 161 165 169 173 167 174 170 179 223
20 146 146 162 177 186 186 200 202 222 214 248 240
21 155 150 167 190 201 180 216 199 215 203 241 235
22 106 117 127 151 162 158 195 183 217 205 250 237
23 139 124 148 164 202 175 220 184 233 194 244 193
24 153 156 166 191 235 206 228 216 267 212 301 261
Mean 136 133 152 172 192 179 205 192 221 200 244 232
so a: 19 18 14 15 25 15 18 16 28 15 35. 21

 

* Reference mge 150-220 (derived from Figure 12)

61

 

 

 

 

APPENDIX A
Table 3. Selenium concentrations of liver biopsies obtarned' on days 0 and 90.
Cow
Se ID 0 23 28 90
Ins/d usigdry wt"
0 1 0.69 0%
2 1.08 0.$
3 1.54 1.39
4 1.85 1.43
5 2.04 0.‘fl
6 1.87 0.83
Mean 1.51 1.01
SD :1: 0.48 0.1)
3 7 0.50 1.38
8 0.98 1.(B
9 1.36 1.56
10 0.64 1.39
1 1 0.64 1.34
12 0.89 1.32
Mean 0.84 1.35
SD :1: 0.29 0.14
20 13 0.41 6.22
14 1.34 6.67
15 0.70 4,52
16 2.19 4.0
17 0.40 1.63
18 0.37 2.37
Mean 0.90 4.33
SD 1: 0.67 1.84
50 19 1.33 4.56
20 0.89 6.47
21 1.97 4.47
22 1.48 7.32
23 2.31 9.39
24 1.28 8.76
Mean 1.54 6.8
SD :1: 0.47 1.89

 

"' Reference range 1.2-2.0

62

APPENDIXA

Table 4. Urinary and fecal selenium concentrations.l

 

 

Se Cow Urine Selenium Fecal Selenium
mg/d ID ng/ml leg/g dry wt
0 1 36 0.28
2 35 0.26
3 58 0.33
4 144 0.71
5 52 0.26
6 17 0.24
Mean 57 0.35
SD :1; 41 0.16
3 7 107 0.84
8 88 0.54
9 105 0.44
10 69 0 52
11 68 0.45
12 92 0.57
Mean 88 0.56
SD :1; 15 0.13
20 13 100 2.78
14 377 2 58
15 390 1 81
16 711 3 56
17 402 1 37
18 504 2 03
Mean 414 2.36
SD i 181 0.71
50 19 672 2.32
20 744 3.14
21 954 4.02
22 966 3.95
23 577 3.20
24 1002 3.58
Mean 819 3.37
SD :1: 163 0.58

 

lSamples obtained on day 90

70

 

28 42

63

cells/moi x 10"!

18

13

 

White blood cell concentrations.

 

 

APPENDIX A
mg/d

Table 5.

838830 21

786997

1.8

Mean

SD21:

OOOOOO

7.237.447.
606.190
1 l l

48

7.1

59
9.1

29
91.

as
91..

.I

33
83

i

6.
0.

7.6
1.1

Sth

 

* Reference range 4.7-11.5

APPENDIX A

Table 6. Erythrocyte concentrations.

 

 

 

 

Cow
Se ID 0 2 6 13 18 56 90
mg/d cells/mm’ x 10""
0 1 6.46 5.92 6.39 6.23
2 9.07 7.42 8.02 80)
3 10.40 6.75 7.10 6.5
4 11.30 7.44 7.20 8.4)
5 6.36 6.23 7.03 6.4)
6 6.62 5.22 5.87 6.15
Mean 8.37 6.50 6.94 7.01
SD :1: 2. 0.80 0.67 0.88
3 7 5.92 5.09 5.71 5.3)
8 8.61 7.63 8.25 7.”
9 6.78 5.61 6.09 6.“)
10 6.63 6.75 7.50 7.!)
11 7.64 6.74 7.47 6.”
12 7.37 6.27 6.95 65)
Mean 7.16 6.35 7.00 6.73
SD :t 0.85 0.83 0.87 0.73
20 13 7.17 6.04 6.70 6.3
14 8.34 7.40 7.78 7.74
15 6.12 4.68 5.93 525
16 7.21 6.69 6.99 6.“)
17 6.24 5.27 6.20 5.4)
18' 6.90 5.77 6.21 5.95
Mean 7.00 5.98 6.64 6.”
SD :1: 0.73 0.89 0.62 0.3
50 19 8.14 8.16 8.28 7.3
20 7.55 6.17 5.94 5.&
21 7.49 6.39 7.02 6.5
22 8.28 7.36 7.99 7.07
23 6.72 5.89 6.68 6.”
24 7.11 6.30 6.46 6.19
Mean 7.55 6.71 7.06 6.$
SD :1: 0.54 0.79 0.83 0.71

 

* Reference range 5.29-9.19

APPENDIXA

Table 7. Hemoglobin concentrations.

 

 

 

 

Cow 1231mm
Se ID 0 2 6 13 1s 23 28 42 56 70 84 90
mg/d gldl"
0 1 118 . . . 10.9 . . . 11.1 . . 11.1
2 14.3 . . . 118 . . . 12.3 . . 12.3
3 12.1 . . . 11.1 . . . 11.4 . . 11.5
4 14.0 . . . 128 . . . 12.2 . . 14.6
5 11.5 . . . 11.2 . . . 12.5 . . 108
6 13.0 . . . 10.4 . . . 11.5 . . 12.0
Mean 12.3 11.4 . . . 118, . . 12.1
SD :1: 11 03 . . . 0.5 . . 1.2
3 7 12.4 . . . 10.7 . . . 11.5 . . 11.5
s 11.1 . . . 10.2 . . . 11.1 . . 11.0
9 12.5 . . . 10.5 . . . 11.2 . . 10.9
10 9.6 . . . 98 . . . 10.7 . . 10.6
11 12.0 . . . 10.6 . . . 118 . . 10.9
12 12.9 . . . 10.9 . . . 11.6 . . 11.0
Mean 11.8 . . . 10.5 . . . 11.3 . . 11.0
SD :1: 11 . . . 0.4 . . . 0.4 . . 0.3
20 13 128 108 . . . 11.7 . . 10.7
14 11.5 . . . 10.2 . . . 10.6 . . 10.6
15 13.0 . . . 9.6 . . . 12.0 . . 10.9
16 131 12.0 ' . . . 12.0 . . 10.2
17 11.3 . . . 98 . . . 11.3 . . 10.2
18 12.1 . . . 10.4 . . . 10.9 . . 10.5
Mean 12.3 . . ., 10.5 . . . 11.4 . . 10.7
SD 1 0.7 . . . 08 . . . 0.5 . . 0.3
50 19 12.0 . . . 118 . . . 11.6 . . 11.2
20 14.7 . . . 12.0 . . . 11.3 . . 11.0
21 12.6 . . . 10.9 . . . 11.5 . . 10.5
22 13.0 . . . 11.5 . . . 12.5 . . 11.3
23 108 . . . 9.5 . . . 10.5 . . 10.9
24 11.3 . . . 10.2 . . . 10.6 . . 10.5
Mean 12.4 . . . 11.0 . . . 11.3 . . 10.9
SD 1 1.3 . . . 0.9 . . . 0.7 . . 0.3

 

" Reference range 8.8-15.6

APPENDIX A

Table 8. Packed cell volumes.

 

 

 

 

Cow W
Se ID 0 2 6 13 18 23 28 42 56 70 84 90
mg/d %*
0 1 31.4 . . . 28.3 . . . 29.7 28.5
2 41.0 . . . 31.9 . . . 34.5 33.0
3 32.7 . . . 28.8 . . . 31.4 29.1
4 38.1 . . . 34.0 . . . 33.1 37.8
5 31.1 . . . 30.1 . . . 33.8 28.5
6 34.3 . . . ‘ 27.1 . . . 30.8 30.9
Mean 34.8 30.0 32.2 31.3
SD :1; 3.6 2.3 1.7 3.3
3 7 33.3 27.9 307 29.8
8 34.4 31.1 34 31.0
9 32.9 27.3 29.5 27.8
10 27.3 28 31.5 29.4
11 33.4 29.1 32 5 28.3
12 34.9 28.9 31 9 28.8
Mean 32.7 28.7 31.7 29.2
SD i 2.5 1.2 1.4 1.0
20 13 35.1 28.6 30.8 27.2
14 33.3 29.6 32.0 30.3
15 34.5 25.4 31.7 27.8
16 35.1 31.7 32.3 29.5
17 29.2 24.9 29.8 26.0
18 32.8 26.6 29.3 27.4
Mean 33.3 27.8 31.0 28.0
SD :1; 2.0 2 4 1.1 1.4
50 19 34.3 407 34.2 30.5
20 38.1 307 29.6 27.9
21 34.5 282 31.2 28.4
22 36.1 321 35.0 30.0
23 29.1 25.3 28.8 29.2
24 31.3 28.2 29.6 27.2
Mom 33.9 . . . 30.9 31.4 28.9
SD :1: 3.0 . . . 4.9 2.4 1.2

 

* Reference range 23.7-41.4

67

 

 

 

 

APPENDIX A
Table 9. Serum aspartate ammo' transferase activities.
Cow W
Se ID 0 2 l3 18 23 28 42 56 70 90
mg/d IUIL"
0 l 58 60 52 57
2 46 48 49 70
3 64 48 45 48
4 38 36 35 45
5 54 49 49 48
6 48 40 47 49
Mean 51.3 46.8 46.2 52.8
SD :1: 8 5 7.6 5.4 8.5
3 7 39 46 41 39
8 50 43 48 42
9 48 45 53 49
10 61 56 50 54
11 41 38 49 37
12 81 50 62 57
Mean 53 3 46.3 50.5 46.3
SD 1 14 3 5.6 6.3 7.5
20 13 53 44 51 49
14 54 46 45 55
15 41 39 45 39
16 58 56 77 62
17 45 40 42 48
18 53 50 42 49
Mean 50 7 45.8 50.3 50.3
SD :1: 5 8 5.8 12.3 7.0
50 19 63 64 48 48
20 45 47 40 40
21 45 49 58 50
22 60 70 51 44
23 44 46 42 58
24 51 44 55 50
Mean 51.3 53.3 49.0 48.3
SD :1: 7.6 9.9 6.5 5.6

 

"' Reference range 48-109

68

 

 

 

 

APPENDIX A
Table 10. Serum gamIm-glutamyl transferase activities.
Cow W
Se ID 0 2 13 18 23 28 42 56 70 90
mgld .'.U/L‘-I
0 1 33 25 40 36
2 38 36 36 47
3 50 38 39 38
4 42 25 31 38
5 28 32 29 24
6 37 32 32 43
Mean 38.0 31.3 34.5 37.7
SD :1; 6 9 5.0 4.1 7.1
3 7 34 20 31 26
8 49 35 33 31
9 39 28 33 35
10 25 23 29 33
11 30 31 27 31
12 28 23 26 29
Mean 34.2 26.7 29.8 30.8
SD :1: 8 0 5.2 2.7 2.9
20 13 36 30 29 32
14 41 24 29 27
15 31 34 35 30
16 41 40 38 46
17 40 38 37 41
18 46 35 38 36
Mean 39.2 33.5 34.3 35.3
SD :1: 4.7 5.3 3.9 6.5
50 19 24 6 26 24
20 24 13 22 20
21 73 46 38 22
22 27 17 27 19
23 25 22 25 23
24 40 34 31 23
Mean 35.5 23.0 28.2 21.8
SD :1; 17.7 13.4 5.1 1.8

 

I"ReferwencerangeO-40

 

69

 

 

APPENDIX A
Table 11. Serum sorbitol debydrogenase activities.
Cow Days of the Trial
Se ID 0 13 18 23 28 42 56 70 90
mg/d IU/L*
0 1 27 22 14 32
2 23 18 14 26
3 29 15 14 21
4 21 13 8 14
5 19 27 19 11
6 29. 19 17 26
Mean 24.7 19.0 14.3 21.7
SD :1; 3.9 4.6 3.4 7.3
3 7 22 17 28 15
8 24 16 16 18
9 19 12 10 14
10 38 32 36 51
11 17 14 44 13
12 79 25 35 37
Mean 33.2 19.3 28.2 24.7
SD :1: 21.6 7.0 11.8 14.3
20 13 33 22 22 25
14 30 21 22 51
15 24 28 31 17
16 29 17 24 23 -
17 24 17 17 17
18 13 26 14 19
Mean 25.5 21.8 21.7 25.3
SD 1; 6.4 4.1 5.4 11.9
50 19 33 26 25 33
20 31 13 14 11
21 43 22 23 16
22 63 15 20 17
23 27 18 20 25
24 28 18 25 18
Mean 37.5 18.7 21.2 20.0
SD :1: 12.5. 4.3 3.8 7.1

 

 

 

* Reference range 24-42

 

70

 

 

 

 

APPENDIX A
Table 12. Serum creatin' e phosphokmase' activities.
Cow W
Se ID 0 2 13 18 23 28 42 56 70 90
mg/d IUIL"
0 1 62 46 105 102
2 32 44 97 105
3 232 42 94 86
4 28 25 71 96
5 36 37 115 158
6 42 39 169 466
Mean 72.0 38.8 108.5 168.8
SD :1; 72.4 6.9 30.2 134.9
3 7 23 30 78 75
8 30 35 98 76
9 15 19 48 54
10 78 63 143 138
ll 26 34 93 65
12 39 39 145 167
Mean 35.2 36.7 100.8 95.8
SD :1: 20.5 13.3 34.4 41.6
20 13 303 32 120 76
14 43 34 108 110
15 27 14 78 61
16 39 40 124 121
17 37 37 105 93
18 35 24 90 74
Mean 80 7 30.2 104.2 89.2
SD 3; 99 5 8.8 16.0 21.1
50 19 33 41 191 92
20 28 194 97 69
21 21 43 96 114
22 51 37 143 115
23 45 102 63 63
24 36 40 116 78
Men 35.7 76.2 117.7 88.5
SD :1: 10.0 57.3 40.6 20.4

 

* Reference range 23-118

71

 

 

 

APPENDIX A
Table 13. Body weights.
Cow M
So ID 0 2 6 13 18 23 28 42 56 70 90
Ins/d 3:8
0 1 591 569 599 614
2 742 673 694 694
3 577 569 644 629
4 562 584 562 569
5 603 606 610 659
6 518 547 569 606
Mean 599 592 613 629
SD :1; 69 41 45 40
3 7 591 591 621 614
8 462 456 497 489
9 701 701 680 666
10 429 429 448 476
11 614 599 621 629
12 435 448 469 497
Mean 539 538 556 562
SD :1: 103 100 88 76
20 13 448 448 476 497
14 396 409 448 448
15 701 715 715 709
16 483 504 555 547
17 629 621 651 673
18 533 555 577 606
Mean 532 542 570 580
SD :1: 105 104 93 92
50 19 422 455 462 483
20 635 701 660 673
21 584 599 ' 614 614
22 422 469 483 511
23 544 544 569 569
24 533 544 533 599
Mean 523 552 553 575
SD :t 79 83 70 64

 

28 42 56 70

72

18

Body condition ricores.l
13

APPENDIX A
mg/d

Table 14.

 

 

3.6
0.4

3.7

3.8

SD :1: 0.4

Mean

000000

3.5
0.4

005500

0:4

1Range 1(thin) to 5(fat) (from Mulvany 1977).

 

APPENDIX A

 

 

 

 

Table 15. Average daily hoof growth.
Cow W
Se ID 0 2 6 13 18 28
rug/d mm/day
0 1 0.0 .38 .17 22
2 0.0 .38 .16 m
3 0.0 .38 .18 .21
4 0.0 .38 .20 24
5 0.0 .50 .20 25
6 0.0 .42 .17 21
Mean 0.0 .41 .18 .22
SD :1: 0.0 .04 .02 .01
3 7 0.0 .38 .18 24
8 0.0 .42 .16 m
9 0.0 .33 .18 m
10 0.0 .38 .20 24
11 0.0 .38 .20 21
12 0.0 .36 .20 22
Mean 0.0 .38 .19 .22
SD :1; 0.0 .03 .01 .01
20 13 0.0 .38 .21 . m
14 0.0 .38 .18 24
15 0.0 .38 .21 21
16 0.0 .38 .20 m
17 0.0 .38 .18 a
18 0.0 .44 .20 24
Men 0.0 .40 .20 22
SD :1; 0.0 .02 .01 .01
50 19 0.0 .38 .20 .24
20 0.0 .38 .16 m
21 0.0 .38 .18 22
22 0.0 .38 .20 24
23 0.0 .38 .18 .21
24 0.0 .38 .21 22
Mean 0.0 .38 .19 .2
SD 4 0.0 0.0 .02 .01

 

74

APPENDIX A

Rabies titers.

Table 16.

 

70

42

18

13

 

 

IU/ml

 

000000

17.48
9 5

1.46 15.3
16 13.1

1.73
1 38

.067
.094

000000

000000

1.83 17.72 19.87

1.34 8.05 5.55

2.35
1 3

000000

......

1.98 6.5

1.88 .93 6.68 16$
.11

.03
.075 1.21

000000

000000

7
5.

1.45 0.90 14.7 1.27
16

0.97 0.22 8.1

 

APPENDIX A

Table 17. [’H]-thymidine uptake of unstimulated lymphocytes.

75

 

 

 

 

Cow '
Se ID 0 2 6 13 18 23 28 42 90
Inc/‘1 cpmflosnd'
0 1 3.41 3.49. 3.9
2 3.81 4.13 4.37
3 2.98 3.02 2.77
4 3.41 3.70 3.64
5 3.17 4.53 4.$
6 3.72 3.23 4.12
Mean 3.42 3.71 3.94
SD :1: 0 29 0.52 0.57
3 7 2.87 3.71 3.94
8 3.46 4.05 4.58
9 3.69 3.48 3.5
10 4.67 3.51 4.57
11 4.10 4.42 4.56
12 3.62 3.70 33
Mean 3.73 3.81 4.18
SD :1; 0 56 0.33 0.4)
20 13 2.85 3.12 1%
14 3.72 4.11 3.8
15 3.44 3.94 3.64
16 3.60 3.61 4.6
17 4.52 4.24 4.fi
18 3.10 3.43 3.72
Mean 3.54 3.74 4.02
SD :1: 0.53 0.39 0.33
50 19 3.41 3.18 3.91
20 2.91 3.62 3.71
21 3.21 2.64 3.56
22 4.49 3.08 3.79
23 3.26 3.42 3.0
24 3.60 3.31 413
Mean 3.48 3.21 335
SD :1: 0 50 0.31 0.2)

 

76

 

 

 

 

APPENDIX A
Table 18. [’H]-thymidine uptake of phytohemagglutinin-stinndated lymphocytes.
Cow
Se ID 0 13 18 23 28 42 70 90
Ina/d 9119100810'
0 1 4.99 3.94 5.0
2 5.39 5.24 5.3
3 5.17 3.92 5.3
4 4.89 3.92 3.45
5 5.00 4.83 5.43
6 5.54 4.87 5.47
Mean 5 16 4.67 515
SD :1; 0 23 0.55 0.72
3 7 4.61 4.04 5.41
8 4.92 4.90 5.37
9 4.92 5.20 5.38
10 4.67 3.77 5.3
11 4.83 5.31 5.5
12 4.44 4.83 5.10
Mean 4.73 4.68 5.32
SD :1: 0.17 0.57 0.11
20 13 5.26 4.37 5.48
14 4.17 3.57 5.41
15 4.72 4.37 5.10
16 4.86 3.66 5.31
17 5.10 4.36 5.15
18 4.37 4.27 5.19
Mean 4.75 4.10 5.27
SD :1: 0.38 0.34 0.14
50 19 3.91 4.07 5.15
20 5.11 4.89 5.6
21 5.41 4.16 5.37
22 5.05 3.79 5.57
23 4.88 5.00 5.12
24 5.27 5.22 5.38
Mean 4.94 4.52 5.32
SD :t 0.49 0.53 0.18

 

'Counts per minute

AH’ENDIXA

Table 19. [’Ifl-thymidine uptake of concanavalin A-stimulated lymphocytes.

77

 

 

 

Cow '
S6 ID 0 2 6 13 18 23 28 42 90
Ina/d 99910081.?
0 1 4.98 4.61 5.56
2 5.39 4.93 5.46
3 4.93 4.69 5.47
4 4.59 5.31 527
5 5.10 5.15 5.48
6 5.36 5.12 534
Mean 5.06 4.97 5.43
SD :1 0.27 0.25 0.10
3 7 4.27 3.14 5.45
8 4.33 4.19 4.32
9 4.74 5.40 522
10 4.77 3.97 4.96
11 4.71 5.30 5.36
12 4.47 5.29 5.43
Mean 4 55 4.55 5.12
SD 4 0 20 0.85 0.39
20 13 4.95 4.83 5.43
14 4.54 4.75 5.41
15 4.85 5.07 558
16 5.43 4.83 5.14
17 5.25 4.93 524
18 4.52 4.52 525
Mean 4.92 4.82 5.34
SD 4: 0.34 0.17 0.15
50 19 3.98 4.49 523
20 4.50 5.00 5.04
21 5.03 4.83 5.38
22 4.77 4.46 554
23 4.63 5.14 4.44
24 5.34 5.25 5.42
Mean 4 71 4.86 5.18
SD 1 0 43 0.30 0.36

 

 

78

APPENDIXA

Table 20. [’H]-thymidine uptake of pokeweedustimulated lymphocytes.

 

 

 

 

Cow W
Se ID 0 2 6 13 18 23 28 42 56 70 90
Ina/d 90940081.?
0 1 4.82 ' 4.35 5.32
2 5.22 5.38 5.17
3 5.05 4.55 529
4 5.16 5.41 4.77
5 4.97 5.14 5.43
6 5.36 5.19 5.29
Mean 5.10 5.01 521
SD 1 0.17 0.41 021
3 7 4.62 . . . . . . 4.43 5.46
8 4.67 . . . . . . 4.94 5.16
9 4.59 . . . . . . 5.43 5.32
10 5.24 . . . . . . 4.19 5.31
11 5.08 . . . . . . 5.41 523
12 4.42 . . . . . . 5.32 529
Mean 4.77 . . . . . . 4.95 529
SD :1: 0.29 . . . . . . 0.49 0.09
20 13 5.02 . . . . . . 4.93 5.58
14 4.50 4 79 558
15 4.74 5 28 522
16 4.83 4 67 5.37 -
17 5.13 5.37 5.10
18 4.40 4.30 524
Mean 4.77 4.89 5.35
SD 1 0.26 0 36 0.18
50 19 4.03 4 52 5.08
20 4.68 4 95 553
21 5.29 5 25 5.41
22 4.62 4 02 5.38
23 4.69 4 96 526
24 4.70 5 29 5.62
Mean 4.67 4 83 5.38
SD 1 0.36 0 44 0.17

 

‘APPENDIXB"

 

b

Includes data (individual, mean, SD) for the respective variables for cows which
had comprised the 50 mg group in trial 1. These cows were subsequently fed
(trial 2) 100 mg supplemental Se/hd/d for 28 days, followed by no supplemtal Sc
for 56 days. Unless otherwise indicated reference ranges are from the Animal
Health Diagnostic and Clinical Pathology Laboratories at the College of
Veterinary Medicine, Michigan State University.

79

APPENDIXB

Table 1. Serum selenium concentrations.

 

 

 

 

 

 

 

 

 

Cow 12432213112124!
ID 100 102 108 120 128 132 136 149 156 163 176 184
ng/ml'!'
19 91 99 131 300 175 132 100 94 77 83 56 55
20 98 113 196 333 289 154 128 94 87 M 64 71
21 138 108 177 232 186 116 117 76 70 86 65 66
22 140 144 255 435 311 176 152 116 93 105 75 73
23 97 136 191 342 250 151 127 107 90 105 78 73
24 130 154 302 425 338 202 168 120 106 111 82 75
Mean 116 126 209 344 258 155 132 101 87 82 70 69
SD :1: 21 20 55 70 61 28 22 15 12 38 9 7
"' Reference range 70-100
Table 2. Whole blood selenium concentrations.
COW mm
ID 100 102 108 120 128 132 136 149 156 163 176 184
ng/ml“
19 234 237 232 . 329 293 242 220 223 279 216 176
20 246 326 369 . 542 304 357 313 374 357 372 345
21 325 298 346 . 542 304 357 313 374 357 372 345
22 275 276 393 . 535 491 567 368 338 455 411 310
23 258 238 300 . 417 422 403 273 270 356 261 232
24 304 306 470 . 633 383 389 325 420 340 371 334
Mean 274 280 352 . 485 366 382.3 295 322 342 .321 271
SD :1: 32 34 74 . 98 74 97.5 47 65 62 69 62

 

* Reference range 150-220 (derived from Figure 12)

 

 

 

 

 

 

 

 

 

APPENDIX B
Table 3. Selenium concentrations of liver biopsies.
Cow mm
ID 100 102 108 120 128 132 136 149 156 163 176 184
148/8417 “It"
19 4.56 . . . 14.91 .. . . . . . 13
20 6.47 . . . 22.94 . . . . . . 0.3
21 4.47 . . . 8.49 . . . . . . 2.07
22 7.32 . . . 16.86 . . . . . . 1.50
23 4.65 . . . M . . . . . . 1.61
24 8.76 . . . 12.74 . . . . . . 1.5)
Mean 6.0 . . . 15.2 . . . . . . 1.60
SD :1; 1.6 . . . 4.8 . . . . . . 0.1)
* Reference range 1.2-2.0
Table 4. Urine selenium concentrations.
Cow W
ID 100 102 108 120 128 132 136 149 156 163 176 184
ng/ml
19 672 . . . 1400 . . . . . . 40
20 744 . . . 1410 . . . . . . 28
21 954 . . . 1280 . . . . . . 47
22 966 . . . 1670 . . . . . . 19
23 577 . . . 1560 . . . . . . 64
24 1002 . . . . . . . . . . 57
Mean 819 . . . 1464 . . . . . . 42.5
SD :1; 163 . . . 136 . . . . . . 15.6

 

81

APPENDIX B

Table 5. Fecal selenium concentrations.

 

 

 

 

 

 

 

 

 

Cow was!
ID 100 102 108 120 128 132 136 149 156 163 176 184
u8/8dry M
19 2.32 . . . 7.75 . . . . . . 0.15
20 3.14 . . . 11.04 . . . . . . 0.16
21 4.02 . . . 9.02 . . . . . . 0.15
22 3.95 . . . 9.82 . . . . . . 0.16
23 3.20 . . . 10.99 . . . . . . 0.21
24 3.58 . . . 9.99 . . . . . . 0.19
Mean 3.37 . . . 9.77 . . . . . . 0.17
SD 3: 0.58 . . . 1.14 . . . . . . 0.02
Table 6. White blood cell concentrations.
Cow 19212188021113!
ID 100 102 108 120 128 132 136 149 156 163 176 184
cells/mm’ x 103*
19 7.5 . . . 6.8 . . . . 5.63 . 7.7
20 5.8 . . . 6.06 . . . . 6.0 . 7
21 . 6.3 . . . 8.20 . . . . 6.08 . 10.9
22 7.5 . . . 6.41 . . . . 6.84 . 8.7
23 7.7 . . . 7.25 . . . . 6.4 . 8.2
24 6.4 . . . 5.91 . . . . 5.24 . 6.6
Mean 6.9 . . . 6.8 . . . . 6.0 . 8.2
SD :1: 0.7 . . . 0.8 . . . . 0.5 . 1.4

 

"' Reference range 4.7-11.5

82

APENDIX B

Table 7. Erythrocyte concentrations.

 

 

 

 

 

 

 

 

 

Cow W
ID 100 102 108 120 128 132 136 149 156 163 176 184
cells/mm’ x 10"!
19 7.9 . . . 6.9 . . . . 8.2 8.(B
20 5.7 . . . 6.2 . . . . 6.4 6.5
21 6.4 . . . 7.0 . . . . 6.0 6.28
22 7.1 . . . 6.0 . . . . 7.0 6.97
23 7.0 . . . 8.3 . . . . 6.8 6.25
24 6.2 . . . 6.4 . . . . 5.7 5.63
Mean A 6.7 . . . 6.8 . . . . 6.7 6.60
SD :1: 0.7 . . . 0.8 . . . . 0.8 0.1)
* Reference range 5.29-9.19 x 10‘
Table 8. Hemoglobin concentrations.
Cow 23332111211131
ID 100 102 108 120 128 132 136 149 156 163 176 184
g/dl"
19 11.2 . . . 11.2 . . . . 11.4 11.7
20 11.0 . . . 10.5 . . . . 12.3 12.2
21 10.5 . . . 11.4 . . . . 10.9 11.3
22 11.3 . . . 10.2 . . . . 12.2 12.1
23 10.9 . . . 11.5 . . . . 11.4 10.6
24 10.5 . . . 12.3 . . . . 9.8 9.9
Mean 10.9 . . . 11.2 . . . . 11.3 11.3
0.8

SD :1: 0.3 . . . 0.7 . . . . 0.8

 

* Reference range 8.8-15.6

83

 

 

 

APPENDIX B
Table 9. Packed cell volumes.
Cmv W31
ID 100 102 108 120 128 132 136 149 156 163 176 184
71
19 30.5 . . . 29.4 . . . . 31.6 . 31.6
20 27.9 . . . 26.7 ' . . . . 31.5 . 31.4
21 28.4 . . . 30.2 . . . . 28.1 . 29.7
22 30.0 . . . 26.8 . . . . 31.5 . 31.8
23 29.2 . . . 30.8 . . . . 30.0 . 28.1
24 27.2 . . . 31.6 . . . . 25.5 . 25.3
Mean 28.9 . . . 29.3 . . . . 29.7 . 29.7
SD :t 1.2 . . . 1.9 . . . . 2.3 . 2.3

 

"' Reference range 23.7-41.4

Table 10. Serum aspartate aminotransferase activities.

 

 

 

 

Cow W
1]) 100 102 108 120 128 132 136 149 156 163 176 184
IU/U'
19 48 51 . . . . 54 . 75
20 40 57 . . . . 48 . 49
21 50 63 . . . . 62 . 66
22 44 50 . . . . 47 . 59
23 58 70 . . . . 81 . 58
24 50 49 . . . . 52 . 55
Mean 48 57 57 . 60
SD :1; 6 8 12 8

 

* Reference range 48-109

APENDIX B

Table 11. Serum gamm-glutamyl transferase activities.

 

 

 

 

 

 

 

 

 

Cow W
ID 100 102 108 120 128 132 136 149 156 163 176 184
IUIL"
19 24 34 . . . . 26 . 20
20 20 18 . . . . 16 . 11
21 22 28 . . . . 24 . 25
22 19 26 . . . . 21 . 19
23 23 29 . . . . 28 . 21
24 23 29 . . . . 24 . 21
Mean 22 . . . 27 . . . . 23 . 20
SD :1: 2 . . . 5 . . . . 4 . 4
"' Reference range 0-40
Table 12. Serum sorbitol dehydrogenase activities.
Cow mm
ID 100 102 108 120 128 132 136 149 156 163 176 184
IU/L"
19 33 . . . 25 . . . . 24 64
20 l 1 . . . 14 . . . . 12 13
21 16 . . . 19 . . . . 30 25
22 17 . . . 20 . . . . 16 26
23 25 . . . 42 . . . . 54 20
24 18 . . . 14 . . . . 16 14
Men 20 . . . 22 . . . . 25 . 27
SD :1; 7 . . . 10 . . . . 14 . 17

 

* Reference range 24-42

APENDIXB

85

Table 13. Serum creatine phosphokinase activities.

 

 

 

 

 

 

 

 

Cow Willi

ID 100 102 108 120 128 132 136 149 156 163 176 184
IU/U'
19 92 109 103 146
20 69 129 85 87
21 1 14 145 97 228
22 1 15 108 138 145
23 63 80 68 86
24 78 73 96 105
Mean 88 107 98 133
SD :1; 20 25 21 49
* Reference range 23-118
Table 14. Body weights.

Cow Wrist

ID 100 102 108 120 128 132 136 149 156 163 176 184
1:8

19 483 483 483 497
20 673 687 621* 621
21 614 629 636 636
22 51 1 525 540 533
23 569 577 591 591
24 599 584 569 622
Mean 575 581 573 583
SD 1 64 66 52 51

 

* Cow calved since previous body weight

APENDIXBA

Table 15. Body condition score.‘

86

 

 

 

 

 

 

 

Cow M1081
ID 100 102 108 120 128 132 136 149 156 163 176 184
19 3 3 3 3
20 4 4 4 4
21 3.5 3 3 3
22 3 5 3.5 3.5 3.5
23 3 3 3.5 3
24 3 3.5 3.5 3.5
Mean 3.3 3.3 3.4 3.3
SD 1; 0.4 0.4 0.3 0.4
'Range 1(thin) to 5(fat) (from Mulvany, 1977)
Table 16. [’Ifl-thymidine uptake of unstimulated lymphocytes.
Cow W041
II) 100 102 108 120 128 132 ' 136 149 156 163 176 184
Wmaogm)‘
19 3.91 3.62
20 3.71 3.66
21 3.56 3.75
22 3.79 3.82
23 3.49 3.77
24 4.08 3.74
Mean 3.76 3.73
SD :1: 0.2 0.07

 

lConntsperminute

87

APPENDIX B

Table 17. [’Hthmidine uptakes of phytohemgglutinin—stinmlated lymphocytes.

 

 

 

 

 

Cow mm

ID 100 102 108 120 128 132 136 149 156 163 176 184
chOOSm)‘

19 5.05 . . . 4.97
20 5.45 . . . 5.19
21 5.37 . . . 5.45
22 5.57 . . . 5.40
23 5.12 . . . 5.49
24 5.38 . . . 5.59

Mean 5.32 . . . 5.35

SD :1: 0.18 . . . 0.21

lCounts per minute

Table 18. [’Ifl-thymidine uptakes of concanavalin A-stimulated lymphocytes.

 

 

 

 

Cow 12412912021113!

ID 100 102 108 120 128 132 136 149 156 163 176 184
9139400816?

19 5.23 . . . 5.26

20 5.04 . . . 5.42

21 5.38 . . . 5.30

22 5.54 . . . 5.34

23 4.44 . . . 5.42

24 5.42 . . . 5.49

Mean 5.18 . . . 5.37

SD i 0.36 . . . 0.08

 

‘Counts per minute

88

APPENDIXB

Table 19. [’Hl-thymidine of pokeweed-stimulated lymphocytes.

 

Cow mm
ID 100 102 108 120 128 132 136 149 156 163 176

 

 

 

4‘1"“ (10810)’

19

21

23

Mean
SD :t

5.08
5.53
5.41
5.38
5.26
5.62

5.38
0.18

4.96
5.43
5.54
5.60
5.61
5.59

5.45
0.23

3.0
4.0
3.0
3.5
3.0
3.5

3.3
0.4

 

‘Counts per minute

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