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EFFECT OF SELENIUM ON SELENOPROTEIN ACTIVITY
AND THYROID HORMONE METABOLISM IN CATTLE

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EFFECT OF SELENIUM ON SELENOPROTEIN ACTIVITY AND THYROID
HORMONE METABOLISM IN CATTLE

By

Jason Edward Rowntree

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY

Department of Animal Science

2003

ABSTRACT

EFFECT OF SELENIUM ON SELENOPROTEIN ACTIVITY AND THYROID
HORMONE METABOLISM IN CATTLE

By
Jason Edward Rowntree
Selenium (Se) is essential for anti-oxidation and thyroid hormone function in
cattle. However, factors that influence its requirement are not well understood. Three
experiments were conducted to determine the influence of cattle breed and age on
selenoprotein activity and the effect of maternal Se supplementation on cow and calf
selenoprotein activity and neonatal thyroid hormone production. In Exp. 1, four
cowherds of differing ages representing three breeds were bled to determine the influence
of breed and age on erythrocyte glutathione peroxidase activity (RBC GPX-l). All
females were non—lactating, bred, and consumed total mixed rations (Holstein) or grazed I
pasture (Angus and Hereford). In beef breeds, yearlings had higher (P < 0.05) RBC
GPX-l activity than mature cows; however, age did not influence RBC GPX-l activity in
Holstein females (23.00 vs. 19.14 vs. 20.79 EU/g hemoglobin, for yearlings, two year-old
and mature cows, respectively). In Exp. 2, neonatal Holstein heifers (n = 8) were bled
daily from O to 6 d of age to determine their thyroid hormone profile. Thyroxine (T4) and
triiodothyronine (T3) concentrations were highest on d O and decreased (P < 0.05)
continuously until (1 5 postpartum (156.13 to 65.88 and 6.69 tol.95 nmol/L, d 0 to 5 for
T4 and T3, respectively). Reverse triiodothyronine concentrations were 3.1 nmol/L on d O
and decreased (P < 0.05) to 0.52 nmol/L by d 5. In Exp. 3, multiparous Hereford cows
were drenched weekly with: 1) a placebo containing 10 mL double deionized H20 (n =

14), or 2) 20 mg Se as sodium selenite (n = 13). After two months of treatment, Se

drenched cows had higher (P < 0.01) plasma Se concentrations than control cows (84.92
vs. 67.08 ng/mL) and at parturition had plasma Se concentrations two-fold higher than (P
< 0.05) control cows (95.51 vs. 47.14 ng/mL). After four months, cows receiving Se had
higher (P < 0.05) RBC GPX-l activity than controls and this trend continued until
parturition. Colostrum Se concentration was two-fold higher (P < 0.05) in Se drenched
cows than control cows (169.97 vs. 87.00 ng/mL). Calves born to cows drenched with Se
had higher (P < 0.05) plasma Se concentration, RBC GPX-l and plasma glutathione
peroxidase activity (P GPX-3) on d 0 compared with calves born to control cows. By d
7, no differences in P GPX-3 activity in calves were observed. Maternal Se
supplementation did not influence calf thyroid hormone concentrations.

Key Words: Selenium, Glutathione Peroxidase, Cows, Thyroid Hormones

Acknowledgements

I would first like to recognize Dr. Gretchen Hill and Dr. David Hawkins for not
only agreeing to mentor my degree work, but also directing my character development as
well. With this, I am equally appreciative of Mrs. Jane Link and Mr. Mike Rincker, who
without their help I would forever be a graduate student. I will forever be impressed with
Jane’s dedication to career and family and Mike’s inability to say no! Dr. Harlan Ritchie
has also been integral in my program at MSU and I am honored to work with him. I
would also like to thank Drs. Steve Bursian and Kent Ames for agreeing to serve on my
dissertation committee.

The MSU farms have been very supportive of my research. Mr. Geof Bednar and
Mr. Brett Barber, both of the Purebred Beef Unit and Mr. Bob Kreft of the Dairy
Teaching and Research Center along with the capable staff of both facilities were
tremendous. I would also like to acknowledge, the AHDL Endocrinology section, Dr.
Raymond Nachreiner and Susan Lombardini for laboratory assistance.

I would like to thank my two families: church and home. Holt Baptist Church has
been instrumental in my spiritual growth and I also thank you for prayer support. My
parents, Dr. Richard and Kaye Rowntree, and sister, Dr. Kara Polk, have supported me

incredibly. Lastly, Cara and Asa, my two best friends, thank you.

iv

Table of Contents

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

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

LITERATURE REVIEW ................................................................................................ 1

Importance of Selenium ..................................................................................... 1

Selenoproteins .................................................................................................... l

Glutathione Peroxidase-1 ......................................................................... 2

Glutathione Peroxidase as an Antioxidant ............................................... 6

Glutathione Peroxidase-3 ......................................................................... 7

Deiodinase ................................................................................................ 8

Selenium in Livestock ......................................................................................... l 1

Glutathione Peroxidases ............................................................................ 11

Status Indicators ....................................................................................... 15

Selenium Supplementation of Cattle ......................................................... 17

Organic Se vs Sodium Selenite ................................................................ 21

Selenium, Thyroid and Irnmunoglobulin Interaction ............................... 22

Selenium Toxicity ............................................................................................. 24

Literature Cited ................................................................................................. 26

ABSTRACT ................................................................................................................... 35

INTRODUCTION ........................................................................................................... 37

MATERIALS AND METHODS .................................................................................... 39
Experiments

Experiment 1 ............................................................................................ 39

Experiment 2 ............................................................................................ 4O

Experiment 3 ............................................................................................ 40

Laboratory Assays ............................................................................................. 42

Statistical Analysis ............................................................................................ 43

RESULTS AND DISCUSSION .................................................................................... 45

Experiment 1 ..................................................................................................... 45

Experiment 2 ..................................................................................................... 47

Experiment 3 ..................................................................................................... 49

IMPLICATIONS ............................................................................................................ 56

LITERATURE CITED .................................................................................................. 7O

List of Tables

Table 1. Erythrocyte glutathione peroxidase activity in cooperator
Angus (CH) and Michigan State University herds:
Angus (MA), Hereford (MHE) and
Holstein (MH) in Experiment 1 ....................................................................... 57

Table 2. Neonatal calf plasma Se and thyroid hormone concentrations
during the first week of life in Experiment 2 ................................................... 59

Table 3. Effect of Se drench on monthly prepartum cow plasma Se
concentrations and erythrocyte glutathione peroxidase
activity in Experiment 3 ................................................................................... 61

Table 4. Mineral composition of feedstuffs offered to
cows in Experiment 3 ...................................................................................... 63

Table 5. Effect of Se drench on cow Se status indicators at parturition
in Experiment 3 ................................................................................................ 64

Table 6. Neonatal calf thyroid hormone concentrations
during the first week of life in Experiment 3 ................................................... 66

vi

Figure 1.

List of Figures

Effect of maternal weekly drench (Se or placebo) and time on:

Panel A: calf plasma Se during wk 1 after birth; treatment x time P < 0.05.
Panel B: calf erythrocyte glutathione peroxidase activity (RBC GPX-l).
One enzyme unit (EU) is the activity needed to oxidize lumol of NADPH
per minute. Panel C: plasma glutathione peroxidase activity (P GPX-3);
treatment x time P < 0.05. Data for plasma Se concentration (panel A) and
RBC GPX-l activity (panel B) are LS means :1: SEM. Statistical analysis
of P GPX—3 activity was performed on loge-transformed LS means,
therefore, data for P GPX-3 activity (panel C) are back-transformed means
with error bars for corresponding 95 % confidence

intervals in Experiment 3 . ............................................................................... 68

vii

Literature Review

Importance of Selenium

Selenium (Se) is an important nutrient in livestock production and has many roles
in mammalian life. The identification of alkali disease and blind staggers, both originally
thought to be associated with Se toxicity, wad made in the Great Plains (Moxon, 1937).
These developments triggered an interest in Se and ultimately led to increased research
on Se concentration and associated enzyme activity in soils, plants and animal tissues.
More research led to Se’s role in preventing liver necrosis in rats (Schwarz and Foltz,
1957) and nutritional muscular degenerative disease (NMD) in ruminants (Muth et al.,
1958). Also known as white muscle disease in sheep and cattle, NMD was initially
diagnosed in Oregon and New Zealand (Schubert et al., 1961), but it is also found in
Michigan, a recognized Se deficient state (Ullrey, 1974). Rotruck et a1. (1973) also
reported Se was a component of cytosolic glutathione peroxidase (GPX-l). In this
enzyme reaction, glutathione functions as a reducing agent of hydrogen peroxide (H202)
in the body in the presence of GPX-l. This discovery spurned more research in the area
of Se biochemistry and the role of Se in livestock production (Underwood and Suttle,
1999).
Selenoproteins

There are two primary groups of selenoproteins: glutathione peroxidases and
deiodinases. The four primary glutathione peroxidases are: classical glutathione
peroxidase (GPX-l; EC 1.11.1.9), gastrointestinal glutathione peroxidase (GPX-2;
1.11.1.9), plasma glutathione peroxidase (GPX-3; 1.11.1.9), and phospholipid

hydroperoxide glutathione peroxidase (GPX-4; EC 1.11.1.12). The GPX-2 enzyme has

66 % amino acid sequence homology with GPX-1 (Chu et al., 1993), and GPX-2 mRN A
is highly expressed in the mucosa] epithelium (Esworthy et al., 1998). There are no
reports on dietary Se’s influence on GPX-2 activity and expression. The GPX-4 enzyme
is 40% homologous to GPX-l (Sunde et al., 1993), and Lei et al. (1995) reported that
dietary Se does not influence GPX-4 mRNA in rats. Research is in agreement that
dietary Se influences both GPX-1 and GPX-3 (Holben and Smith, 1999).

There are three primary deiodinases: type 1 iodothyronine deiodinase (ID—1; EC
3.8.1.4), type 2 iodothyronine deiodinase (ID-2; 3.8.1.4) and type 3 iodothyronine
deiodinase (ID-3; 3.8.1.4). Although all deiodinases require Se, the 03-1 and 113-2
enzymes are responsive to only extreme Se deficiency while [13-3 is unresponsive to Se
status (Bianco et al., 2002).

Gluthathione peroxidase-1. Much attention has been given to the influence of Se
on the GPX protein, its activity and synthesis. Rotruck et al. (1973) demonstrated Se
incorporation into GPX-l by injecting 75 Se, as sodium selenite, into Se-adequate rats.
Erythrocytes (RBC) were later collected and GPX-1 was purified from the sample. The
75 Se was found to be primarily in the RBC GPX-1 protein as compared to other body
tissues. Later, this laboratory reported that rat liver perfused with either 75Se as sodium
selenite, or 75Se as selenocysteine preferentially incorporated sodium selenite into
GPX-1(Sunde and Hoekstra, 1980). Utilizing Se normal rats, labeled selenocysteine and
unlabeled sodium selenite were injected simultaneously; very little radioactive Se was
incorporated into liver GPX-1. In another Se normal treatment group, labeled Se as
sodium selenite and unlabeled selenocysteine were injected. The labeled Se from sodium

selenite was incorporated into GPX-1 indicating that Se from sodium selenite was most

likely the precursor for GPX-1 protein in the rat (Sunde and Hoekstra, 1980). In a similar
experiment conducted by Whanger and Butler (1988), rats were fed a Se deficient basal
diet or the basal diet supplemented with 0.2, 1.0, 2.0, 4.0 ppm Se as either sodium
selenite or selenomethionine. The GPX-1 activity did not differ between Se sources,
however the percentage of Se incorporated into GPX-1 from sodium selenite was higher
in liver and kidney compared to selenomethionine. For the 2 ppm treatment, liver and
kidney had 26% and 25% higher Se incorporation respectively, for GPX-1 synthesis in
the sodium selenite vs selenomethionine groups.

Much attention has been given to the biochemistry of Se incorporation into
protein. Utilizing primer extension sequencing experiments, Chambers et al. (1986)
elucidated that Se coding in glutathione peroxidase mRNA was encoded by the stop
codon UGA. The UGA codon in RNA requires a selenocysteine insertion sequence
(SECIS). The SECIS is a sequence of nucleotides located upstream, in the 3’
untranslated region (3’UTR) from the UGA codon that encodes selenocysteine amino
acid insertion into protein. Without the SECIS, termination of translation would precede
(Copeland and Driscoll, 2001). Building from prior Se moiety research involving GPX-
1, Sunde and Evenson (1987) injected labeled cysteine or serinc into rats and 4 hr later
isolated GPX-1 in liver perfusant. Serine was identified as the carbon skeleton precursor
for selenocysteine production. Knight and Sundc (1988) studied the effects of
progressive Se deficiency and repletion on GPX-1 concentration and activity. Rats were
fed a Se deficient diet (< 0.02 ppm) or a Se supplemented diet (0.2 ppm Se as sodium
selenite). The rats were sacrificed serially at the onset of the trial and 3, 7, 14, 21, or 28 (1

later. Liver GPX-1 activity increased 66% and GPX-1 protein increased 50% over the 28

(1 period with Se supplementation compared with rats fed the Sc deficient diet. The liver
GPX-l activity and protein concentration decreased exponentially throughout the trial
when < 0.02 ppm Se was fed. When Se deficient rats were fed 0.5 ppm Se as sodium
selenite, liver GPX-1 protein and activity increased after 1 d of Se inclusion in the diet,
and the GPX-1 parameters continued to improve and plateaued at d 14 (Knight and
Sunde,1988)

The effect of Se on regulation of GPX-1 gene expression has been investigated by
several groups. Selenium deficiency reduces the amount of GPX-1 mRN A without
altering gene transcription (Weiss et al., 1996). Thus, because Se deficiency does not
influence transcription of selenoprotein mRNA, the Sc deficient induced degradation of
GPX—1 mRNA is the key step in gene expression (Christensen and Burgener, 1992; Lei et
al., 1995; Berrnano et al., 1996). To further elucidate the cytosolic degradation
mechanism, Weiss and Sunde (1997), utilizing gene transfection, demonstrated that when
the 3’ untranslated region (UTR) was deleted from cloned liver GPX mRNA, Se had no
regulatory effect on GPX mRNA. Thus, under low Se conditions, a site within the UTR
could be responsible for GPX degradation. The site hypothesized by Weiss and Sunde
(1997) was later discerned to be the original codon, UGA, which programs the decay of
cytoplasmic mRNA. Under Se deprivation, the UGA codon reduces the abundance of
cytoplasmic Se-GPX—l mRNA. The reduction is attributed to a nonsense-mediated decay
(N-MD) of cytoplasmic mRNA (Moriarty et al., 1998). Although still unclear as to the
actual link between Se deprivation and N-MD, later research has acknowledged that the
intron position in the 3’ UTR identifies the site of decay in the mRNA strand. Sun et al.

(2000) demonstrated that N-MD only occurs when the Sec codon was located more than

59 base pairs upstream of the intron. Further, N-MD did not occur when the
selenocysteine codon was located less than 43 base pairs from the intron. A model for
the intron decay proposes that after introns are spliced, proteins near the newly formed
exon-exon junction act as constituents of messenger ribonucleo—protein that mediate the
decay or recruit other proteins to complete the process (Sun and Maquat, 2002).

The SECIS binding proteins (SBP-l , and SBP-2) have also been identified as a
potential mediator of N-MD (Copeland and Driscoll, 2001). The SBP-2 protein is widely
expressed and is in high concentrations in the testis. Presumably, the SBP-2 is needed for
phospholipid glutathione peroxidase (GPX-4), a selenoprotein necessary for normal
sperm function (Copeland et al., 2000). Specifically, SBP-2 binds to the SECIS element
and this mechanism is required for selenocysteine insertion in eukaryotes. It is also
known that SBP-2 is the limiting factor in selenocysteine insertion in reticulocytes and
that SBP-2 affinity could determine the efficiency of synthesis and mRNA stability
(Copeland and Driscoll, 2002). Thus, SBP-2 could be the mediator for N-MD. Recently,
Zavacki et al. (2003) proposed that SECIS elements recode UGA codons from
termination to “sense”. Their model suggests that the SECIS element recruits SBP-2,
which in turn, is anchored to a selenocysteine-specific elongation factor- selenocysteinyl
tRNA complex. The trimeric complex promotes a conformational change of the C-
terminal domain. The change in conformation provides an opportunity for the complex
to deliver the selenocysteine tRNA to bind the UGA codon on the ribosome. Following
insertion of selenocysteine into the protein, the ribosome initiates release of the
elongation factor, disfavoring SBP-2 binding, and ultimately allows the elongation factor

to bind a new selenocysteine tRNA. In a recent review, Copeland (2003) maintains that

SBP-2 stabilizes the selenocysteine-specific elongation factor- selenocysteinyl tRNA
complex and without its presence, N-MD occurs. The author also discusses the
possibility of an unknown factor downstream that influences SBP-2 affinity in vivo.
Hence, more research is necessary to elucidate the regulation of selenoprotein synthesis
and termination.

Glutathione Peroxidase as an Antioxidant. Wu et al. (2003) also investigated the
effects of long-term Se deficiency on glutathione peroxidase activities and expressions in
rat aorta. Rats were fed either an adequate Se diet (0.32 ppm Se), or a Se deficient diet
(0.017 ppm Se) for 12 mo. Animals in some treatments were supplemented with an
additional 1 ppm as selenite in drinking water. Water intake and blood Se values were
not presented. Aortic GPX-1 and Cu, Zn superoxide dismutase (SOD; EC 1.15.1.1)
activities were reduced (P < 0.05) in the Sc deficient group. The SOD and GPX enzymes
work synchronistically. The SOD converts superoxide radical (02’) into H202
(Fridovich, 1975) and GPX reduces H202 into H20 and 02. Because the GPX activity
was interrupted, 02' concentration increased and ultimately overwhelmed the capacity of
the SOD enzyme to function. Hence, they hypothesized that due to interruption of GPX
activity, the SOD reaction was impaired as well.

Other researchers have compared GPX and other antioxidant activity in tissue of
young and old rodents. Muradian et al. (2002) investigated the activities of GPX, SOD
and catalase (EC 1.11.1.6), an iron requiring peroxidase, in the livers of young (n = 15, 3
to 5 mo) and old mice (n = 15, 23 to 26 mo). Only SOD activity was lower (P < 0.01)
with age. No Se values were presented in the trial. Lawler and Demaree (2001)

compared GPX-1 activity in skeletal muscle (soleus, red and white gastrocnemius

muscle) in young (4 mo), middle aged (18 mo) and old (24 mo) rats. Rats in the old
group had the highest GPX-1 activity (P < 0.05) in all muscle tissues, suggesting age may
affect antioxidative responses.

Glutathione Peroxidase-3. Plasma or extracellular glutathione peroxidase (GPX-
3) was discovered by Cohen et al. (1985). It is synthesized in the kidney and found in
milk, placenta and plasma. Because GPX-3 is not found in red blood cells, this enzyme
responds to acute changes in Se status by sensing circulating plasma Se concentrations
distal to the kidney (Cohen et al., 1985; Cohen and Avissar, 1994). Hill et al. (1987)
reported that GPX-3 activity fell to less than 5% of basal activity in Se deficient rats and
the enzyme’s activity was the first to decrease with inadequate Se status compared with
GPX-l. However, the GPX-3 protein is found in very low quantities in plasma, leading
researchers to question the importance of this enzyme in metabolism (Broderick et al.,
1987). The role of GPX-3 is largely unknown. It seems logical that GPX-3 serves as an
antioxidant in plasma, but reduced glutathione, a component of GPX-3, is found well
below saturating conditions in plasma. There is a possibility that GPX-3 reduces
peroxides in circulation and subsequent enzyme reduction occurs later (Sunde, 1997).
Cohen and Avissar (1994) suggested that the primary action of GPX-3 could be in renal
extracellular space. Whitin et al. (2002) later established that GPX—3 is secreted
basolaterally from human proximal tubule cells. Furthermore, mice induced with colitis
have increased GPX-3 in the plasma and kidney. The increase of GPX-3 in the plasma is
also associated with increases in renal GPX-3 mRNA, indicating that renal GPX-3
production is sensitive to challenges away from the kidney (Tham et al., 2002). There is

no known mechanism for GPX-3 upregulation during injury or challenge. The GPX-3

enzyme has been cloned in bovine plasma (Martin-Alonso et al., 1993), and the
corresponding amino acid sequence showed an overall identity of 46.4% with GPX-1.

Mannan and Picciano (1987) investigated the influence of maternal Se status on
human milk Se concentration and milk GPX-3 activity. Ten lactating women (mean age
30 i 5.6 yr) provided milk and blood samples. During gestation, women took vitamin
and mineral supplements, without Se. Blood samples from eight non-lactating women
(no age given) were used as a control. Foremilk and hindmilk Se concentrations and
GPX-3 activities were 15.6 and 18.1 ng/ml and 68.1 and 90.4 EU/L, respectively. Milk
Se concentration and GPX-3 activity were positively correlated (r = 0.81, P < 0.001).

Amid discussion of whether milk GPX-3 was a unique extracelluar glutathione
peroxidase or the same as plasma GPX-3, Avissar et al. (1991) sequenced the enzymes in
human milk and plasma. They determined that 90 % of human milk GPX-3 could be
precipitated by anti-p-plasma GPX-3-immunoglobulin G. Allen and Miller (1981)
observed when 7SSe was administered intravenously to lactating goats, 6.2 % of the 75 Se
was found in mammary tissue. Eighty percent of the labeled Se was associated with
casein fraction of milk. Debski et a1. (1987) hypothesized that a substantial quantity of
Se in casein was contributed by glutathione peroxidase. Since cow’s milk and colostrum
can contain 50 and 200 ng/ml Se, respectively (Maus et al., 1980), the potential for
mammary GPX-3 protein synthesis seems logical.

Deiodinase. Thyroxine (T4), the major hormone of the thyroid gland, is
deiodinated (5’) in the outer ring to the more metabolically active thyroid hormone,
triiodothyronine, 3, 5, 3’ by ID-l (Braverman et al., 1970) and 113-2 (Visser et al., 1982)

iodothyronine deiodinases that require Se for function (Arthur et al., 1988). The T4

hormone is also converted to the inactive hormone, reverse triiodothyronine (rT3) by 5
deiodination in the inner ring by 03-3 iodothyronine deiodinase, which is not regulated
by Se (St. Germain et al., 1994). However, the [13-1 enzyme also has limited ability to
catalyze inner ring deiodinations as well. On the other hand, ID-2 and 113-3 strictly
deiodinate outer and inner rings, respectively. Further, the 113-] and [13-2 enzymes are
the most responsive to Sc supplementation (Bianco et al., 2002).

Thyroid stimulating hormone (T SH) regulates production of T4. Typically, in
hyperthyroid conditions, TSH is undetectable. Circulating T4 concentration is controlled
by feedback inhibition of T4 on synthesis and secretion of TSH by the pituitary gland.
The control requires intrapituitary conversion of T4 to T3, by 03-2, and the T3 binds
specific nuclear receptors which elicits regulatory response (Beckett et al., 1989).

Arthur et al. (1988) were the first to associate Se deficiency with interrupted
thyroid hormone metabolism while studying Se metabolism in cattle and rats. Twelve
Friesian steers were fed either a Se deficient torulla yeast diet (< 0.015 ppm Se) or this
same diet supplemented with 0.1 ppm Se as sodium selenite. Blood samples were
collected after 23 wk to measure plasma T4, T3, whole blood Se and whole blood GPX
activity. Cattle fed the Sc deficient diet had whole blood Se concentrations of 0.008
mg/L compared to 0.081 mg/L in the Sc supplemented cattle. The corresponding whole
blood GPX activity was 0.19 and 17.09 EU/L for the Sc deficient and supplemented
groups, respectively. Plasma T4 concentrations increased by 62% in the Sc deficient
group and T3 concentrations decreased by 35% when compared to the Sc supplemented
cattle. The lowered T3 and increased T4 concentrations in Se deficient cattle, indicate

impaired conversion of T4 to T3. This was also demonstrated in Se deficient rats (Beckett

et al., 1987). To assess Se influence on 113-1 and ID-2, rats were fed a Se deficient diet
for 6 wk and then killed. Blood, brain, liver and kidney samples were collected.
Thyroxine was added to the organ homogenates, and T3 concentrations were measured.
Selenium deficiency caused an inhibition of T3 formation in plasma, kidney and liver
indicating suppressed activity of 113-1. There were also suppressed T3 concentrations in
the brain homogenate indicating ID-2 suppression.

Arthur et al. (1990) validated the hypothesis that 113-] was a selenoprotein by
using 125 I coupled with a 75Se labeling procedure. The unstable ID-l enzyme is about
0.01% of total microsomal protein. However, utilizing 125I, the researchers isolated the
enzyme and high levels of 75Se were found in the 113-] fraction. Arthur’s laboratory also
confirmed that the loss of hepatic ID-l activity in Se deficient mice was due to decreases
in 03-1 synthesis and not inhibition of activity (Beckett et al., 1992). In severely Se
deficient rats, 113-] activity was maintained in the thyroid gland and 113-1 mRNA levels
were increased indicating a greater conversion of T4 to T3 to account for diminished
conversion in the periphery. The increase in thyroidal T3 production does not
compensate for impaired conversion during Se deficiency and the hypersecretion of T4
eventually impairs biochemical function. Similar to RBC GPX-1, there is no decrease in
113-] gene transcription during Se deficiencies (Bermano et al., 1995).

The metabolic control of increased T4 to T3 conversion in the thyroid gland during
Se deficiency may be attributed to Sc status interaction with TSH. In cultured thyroid
cells (FRTL-S), high levels of ID-l and RBC GPX-l mRNA are expressed when cultured
in TSH enriched, Se deficient media. Alternatively, very little increases in mRNA are

detected in TSH enriched Se normal media. In Se deficient animals, the thyroid gland

lO

has higher concentrations of TSH, partitioning available Se to the 3’ UTR during
translation of ID—l mRNA (Villette et al., 1998). These data provide a likely mechanism
for impact of Se deficiency on the thyroid in vivo.

Typically, in Se deficient rats, liver and kidney deiodinase activities decrease to
very low levels. Pituitary Se remains largely unchanged, which could support the TSH
partitioning theory (Beckett et al., 1989). An alternative hypothesis for deiodinase
activity mentioned in the literature is that deiodinase activity is related to organ Se
concentration during deficiency. Under Se deficient conditions, certain organs are able to
maintain adequate Se concentrations because they need the mineral more so than others
for function. For instance, the ovary and testes maintain Se concentrations and
deiodinase activity during severe Se deficiency. However, this phenomenon is not seen
in brown adipose tissue and other tissues implying that more regulatory factors are
involved in partitioning Se (Bates et al., 2000).

Selenium in Livestock

Glutathione Peroxidases. The discovery of Se deficiencies leading to NMD in
lambs (Muth et al., 1958) greatly increased the amount of research in livestock species.
The report that Se was incorporated into GPX-1 (Rotruck et al., 1973) led to research on
the correlation of the enzymes activity with Se status in livestock. The GPX—1 assay is
simpler and more time efficient than the fluorimetric procedures used to assay Se
(W hetter and Ullrey, 1978).

Thompson et al. (1976) compared the correlations between whole blood GPX-1
and Se in sheep, cattle and pigs. Sheep whole blood GPX was highly correlated (r = 0.92,

P < 0.001) followed by cattle (r = 0.59, P < 0.001) and then pigs (r = 0.27, P < 0.1). Age

11

within species, diet and blood storage time and temperature were not accounted for in the
trial. Later, Anderson et al. (1978) reported high correlations (P < 0.001) between whole
blood GPX and Se in Friesan influenced calves fed a diet containing 0.01 ppm Se for 10
wk. A subset of calves was injected with 2.5 mg Se as sodium selenite and 750 mg
vitamin E, and an increase in RBC GPX-1 activity was documented as early as wk 2.
Typically, RBC turnover in the bovine is 120 (1, so little change due to RBC GPX-1
activity would be expected. However, the authors acknowledge that RBC were not
washed as they felt plasma GPX (not documented as GPX-3 until 1985) was negligible
(Anderson et al., 1978). Thus, differences could be due the plasma GPX-3 (P GPX-3)
itself and not Se incorporation into GPX-1. In both trials (Thompson et al., 1976;
Anderson et al., 1978), samples were frozen at -20° C and whole blood GPX activity was
run within a wk. Decreases in plasma GPX-3 activity, over time, have been reported in
swine. The P GPX-3 activity for the samples frozen at -15° C decreased by 8.9 % from d
0 to d 1 and this continued through d 56 (Zhang et al., 1986).

Whanger et al. (1978) evaluated the effects of various methods of Se
administration on NMD and GPX activity in sheep. Ewes were administered sodium
selenite by subcutaneous injection, a heavy ruminal pellet, a feed supplement (whole
oats), aqueous drench, or in salt offered ad libitum. Three injections of Se prepartum (5
mg Se per sodium selenite injection), the pellet (no concentration given) or Se in the salt
(50 ppm) fed free choice protected lambs born to these ewes from NMD. There were,
however, instances of NMD in lambs whose dams were fed 0.1 or 0.2 ppm Se or
supplemented a 5 mg Se as sodium selenite drench given twice, prepartum. There were

no NMD cases in lambs whose dams received 10 mg Se as sodium selenite in a drench,

12

given twice prepartum. The RBC GPX-1 activities and plasma Se concentrations were
highest (P < 0.05) in ewes and their lambs fed the Sc supplemented salt (0.024 p g/ml and
121 EU/g Hb, respectively). The authors concluded the lambs of ewes receiving 0.1 ppm
Se/d were not protected from NMD. They recommended supplementing Se in a drench
(10 mg/ hd Se as sodium selenite, administered four times annually) as an excellent way
to supply Se and prevent Se deficiency related diseases.

In a trial comparing amounts of Se in supplements relative to Sc concentration
and GPX activity in tissues, fifteen newborn Holstein influenced calves were fed milk
replacer containing 0.03, 0.23 or 0.53 pg Se/g solids (Scholz et al., 1981). Calves were
fed the diet for 12 wk and bled weekly. Selenium status parameters were monitored, and
calves were slaughtered at the end of the trial for tissue collection. Calves consuming the
high Se milk replacer had higher whole blood Se and GPX activity compared to the two
lower Se treatments beginning at d 14 of supplementation (P < 0.05). However, by wk
12, there were no differences in calves’ blood Se concentration between Se supplemented
groups (0.147 vs 0.153 pg/ml Se for the 0.23, or 0.53 pg Se/g treatment groups,
respectively). Calves consuming 0.03 pg Se/ g had lower whole blood Se concentrations
(0.041 pg/ml) compared with calves in the higher supplemented groups. By wk 12, the
whole blood GPX activity was highest (P < 0.05) in the group receiving 0.53 pg Se/g
milk solids and decreased as Se supplementation decreased (70.7 vs 53.1 vs 14.0 EU/g
Hb). There were no differences across treatment groups for plasma GPX activity (not
documented as GPX-3 until 1985). Hence, GPX-1 (the difference between whole blood
GPX and plasma GPX) appeared to be more sensitive to differing long-term Se intakes.

The highest Se solid tissue concentrations were found in the renal cortex followed by the

13

liver of calves in all treatments (P < 0.05). Liver Se concentration was highest in the
group receiving 0.53 Se/g milk solids and decreased as Se supplementation decreased
(0.397 vs 0.300 vs 0.141 p g/g Se). Treatments also influenced renal cortex Se
concentration in a comparable manner (0.733 and 0.870 vs 0.985 p g/g Se for the 0.03,
0.23, or 0.53 pg Se/g solids treatments, respectivley).

Stevens et a1. (1985) further investigated the correlation of serum Se and RBC
GPX-1 activity by sampling 15 herds of Holstein cattle (n = 210). Five herds were from
Se deficient areas in central Wisconsin, five from variable Se areas in eastern Minnesota
and five from Se toxic regions in South Dakota. Age and differences in cow production
status (dry vs low vs high producing) existed in all herds. Production status did not
influence serum Se concentrations and RBC GPX-1 activity, using the Paglia and
Valentine (1967) method. However, geographic region did (P < 0.05). Cattle from the
Sc deficient area averaged 0.042 pg/ml serum Se, compared with 0.057 and 0.317 pg/ml
for cattle in the variable and toxic areas, respectively. The RBC GPX-1 activities were
37.8, 46.8, 111.5 EU/mg of Hb for cattle residing in the deficient, variable, and toxic
areas, respectively. The r-value for the correlation was 0.9493. Within the toxic
areas,RBC GPX-1 activities were correlated to serum Se concentrations as high as 0.789
pg/ml. Backall and Scholz (1979) sampled Angus and Friesian cows across
Pennsylvania and reported RBC GPX-1 activity correlated to serum Se concentrations of
0.10 pg/ml. Counotte and Hartmans ( 1989) sampled Holstein cows on soils varying in Se
concentrations and reported high correlation for RBC GPX-1 activity and Se
concentration in whole blood (r = 0.933, P < 0.001). They reported a significant age

affect on enzyme activity with calves and mature cows having higher activities than

14

yearlings (P < 0.01). No significant differences due to time of year (spring vs autumn)
for RBC GPX-1 or whole blood Se concentration were found.

Plasma glutathione peroxidase (GPX-3) activity has also been documented in
bovine milk. Hojo (1982) was the first to detect GPX-3 activity in raw bovine milk.

Milk Se, assayed fluorometrically, and GPX-3 activity correlated (r = 0.778). Twelve
percent of the milk Se was bound to GPX but only 0.003% of the protein found in milk is
GPX. Hence, although milk GPX activity is low, there is a relationship between Se status
and milk GPX-3 activity. Debski et al. (1987) compared GPX-3 activity in human, cow,
and goat milk. No diet, age or stage of lactation information was presented. Regardless
of species, lipid fraction contained less than 3% of GPX-3 activity. They found goat milk
had higher (P < 0.05) GPX-3 activity than human or cow milk (57.3 vs 36.0 and 25.9
EU/ml, respectively). Since all species contained GPX-3, the authors concluded that
presence of GPX—3 has a metabolic role in the neonate and/or mammary gland during
lactation.

Status Indicators. Much of the Se research conducted in the last thirty years is
associated with Se supplementation and quantifying responding Se status indicators.
Breed, production status, geographical location, genetics, age and management confound
many responses to Sc supplementation. Large cattle trials have been conducted to
determine adequate concentrations of circulating Se. Selenium concentrations in plasma
and serum correlate well with oral (10 mg Se drench) and parenteral (5 mg Se injection as
sodium selenite) supplementation in ewes up to 90 (I post supplementation (Whanger et
al., 1978). Whole blood Se also correlates well with Se consumption, but responds

slower than plasma and serum Se (Perry et al., 1976). The primary source of whole blood

15

Se is RBC GPX-l. Since adult RBC turnover ranges from 90 to 120 d, acute Se status
changes are not detected in whole blood Se assays (Stowe and Herdt, 1992).

Diagnostic laboratories assume normal serum Se concentrations in adult
Holstein cattle to range from 70 to 100 ng/ml (Stowe and Herdt, 1992). The normal
range in the neonatal Holstein calf (0 to 30 d of age) is 50 to 75 ng/ml (Stowe and Herdt,
1992). Marginally Se deficient adult cattle have serum Se concentrations in the range of
40-70 ng/ml while cattle deficient in Se have serum concentrations below 40 ng/ml
(Gerloff, 1992). Dargatz and Ross (1996) sampled 253 beef cow-calf operations in 18
states to evaluate Se status and concluded that cattle with less than 50 ng/ml whole blood
Se or between 51 and 80 ng/ml whole blood Se were severely or moderately Se deficient,
respectively. Further, they reported that 19.0, 54.7 and 61.4 % of western, central and
southeastern producers regularly supplement Se in a trace salt to their cattle, respectively.
However, 14.3 % of Se supplemented southeast cowherds were severely deficient and
22.9% were marginally deficient. Over 16% of supplemented southeastern cattle were
severely deficient. Deficiencies in supplemented cattle were also documented in other
areas of the country. Within the western United States, 4.4 % of Se supplemented
cowherds were severely deficient and 8 % were marginally deficient for Se. Although no
severe deficiencies were reported, 3.6 % of supplemented central United States cattle
were marginally deficient. Supplementation concentrations or daily Se intake were not
provided in this study and no Midwest operations were utilized. It would, however,
appear that Se supplementation in trace mineral salt does not guarantee adequate Se

status in mature cattle.

l6

Selenium Supplementation of Cattle. Many Se studies in cattle have attempted to
quantify what Se supplementation amounts are necessary to maintain adequate Se status
in cattle. Utilizing feedlot cattle originating from Oklahoma, Perry et al. (1976) fed cattle
in a control group with 0.08 ppm Se from feed or an additional 0.1, 0.2, 0.4 ppm Se as
sodium selenite in feed for 113 d. Cattle receiving the additional 0.4 ppm Se had serum
Se concentrations of 73 and 83 ng/ml at d 29 and 113, respectively. Cattle receiving
additional 0.2 ppm Se had serum Se concentrations of 57 ng/ml at both sampling times.
The control group (0.08 ppm Se) had serum Se concentrations of 24 and 44 ng/ml at d 29
and 113, respectively. In Holstein cows, Schingoethe et al. (1982) administered an
injection of 68 IU vitamin E and 5 mg Se/45.4 kg body weight as sodium selenite
approximately 21 d prepartum or gave no injection, and fed a diet adequate (0.1 ppm to
2.0 ppm) for Se. Prepartum cows receiving the injection had 75 ng/ml serum Se
concentrations prior to injection and 150 ng/ml by d 2 post injection. However, Se
concentrations decreased to original concentrations by d 3. There were no differences in
colostrum Se concentrations or incidence of retained placenta at parturition between the
two treatments. The Se in serum was not determined in the calves.

Other reports have compared Se supplementation and subsequent Se status in
Holstein cows and calves (deToledo and Perry, 1985). Pregnant cows (average age 2.9 -_i-_
0.9 yr, 11 = 30) were divided into five groups: (1) control, (2) orally supplemented with 1
mg Se from sodium selenite/daily for the last 60 d of gestation, (3) 2 mg Se from sodium
selenite/daily for the last 60 d of gestation, (4) two injections of 50 mg Se as sodium
selenite 40 and 20 d prepartum or (5) three injections of 50 mg Se as sodium selenite 60,

40 and 20 d prepartum, respectively. For oral treatments, sodium selenite was mixed into

17

corn and placed on top of alfalfa silage. Cow serum Se concentration was assessed every
10 d, from 60 d prepartum to 10 d postpartum and colostrum was collected within 8 hr of
parturition for Se analysis. Calf serum Se concentration was also assessed every 10 d,
from birth to 20 d of age. All prepartum treatments increased serum Se concentration
except the daily 1 mg dose in feed. No cumulative effect was gained from administering
three injections vs two injections throughout the trial. Calves from dams receiving the
injections had higher Se serum concentrations at birth compared to calves from cows in
the other treatment groups. However, calves from all treatment groups had similar serum
Se concentrations by 20 d of age. There were no differences in colostrum Se
concentrations (deToledo and Perry, 1985).

The results of deToledo and Perry (1985) differ from Stowe et al. ( 1988) who fed
0 or 2 mg Se/d as sodium selenite in feed for two lactation-gestation cycles and reported
higher concentrations of Se in colostrum (53.7 ng/ml colostrum vs 62.4 ng/ml,
respectively) from supplementation. Koller et al. (1984) also reported increased Se in
colostrum when Se supplementation was utilized in Hereford bred heifers (n = 30). Cows
consuming trace mineral salt containing 90 ppm Se as sodium selenite had 124 ng/ml
colostrum Se compared to 70 ng/ml colostrum Se for cows consuming 0.313 mg/kg Se in
feed.

Weiss et al. (1984) analyzed Se concentrations in serum of calves whose dams
were supplemented daily with 0, 1 or 5 mg Se from sodium selenite beginning
approximately 60 d prepartum. At birth, calves were assigned to one of three treatment
groups: (1) no injection, (2) one injection at birth or (3) two injections at birth and 14 d

of age of 0.078 mg Se and 5.4 IU vitamin 13/ kg body weight (BO-SE, Schering- Plough,

18

Kenilworth, NJ .). Average serum Se concentrations of cows were 14, 32, and 58 ng/ml at
parturition for the 0, 1 and 5 mg Se supplementation treatment groups, respectively. At
birth, calves reflected the Sc status of their dam. Calves from cows receiving 1 mg
supplementation had 41 % higher serum Se (pre-injection) than calves from cows
receiving no Se supplementation. Calves (preinjection) from dam’s receiving 5 mg Se
injections had higher serum Se concentrations at birth than calves (preinjection) from
cows given 1 mg Se (48 vs 32 ng/ml). However, the Sc concentrations in serum were not
different 14 d after birth. Calves from cows in the 5 mg Se group receiving injections
had higher serum Se concentrations than injected calves from cows in the 1 mg and
control groups. Calves from cows in the control group (no supplementation) with one
and two Se injections had higher serum Se concentrations than calves from the control
cows receiving no injection. Calves from both injection groups had higher serum Se
concentration at d 14 of age than calves from the control group given no injection. By (I
28 of age, calves receiving two injections, regardless of dam’s treatment, had higher
serum Se concentrations than calves given 1 mg injection and control group calves.
However, by d 56 of age, serum Se concentrations of calves receiving injections did not
differ from controls (no injection). The results from this study indicate postnatal Se
injections can increase serum Se concentration in calves born to dams receiving low or
modest Se supplementation, but circulating Se does not remain elevated (Weiss et al.,
1984).

A study on the effect of supplementing Se to deficient (no Se status indicators
given) multiparous Salers cows and calves (n = 72), before or after calving was

conducted in two different experiments (Enjalbert et al., 1999). In the initial experiment,

19

deficient cows were fed 13, 32.5 or 45.5 mg of Se/d from sodium selenite mixed into
barley for 15 (1 prior to calving. The RBC GPX-1 and plasma GPX-3 were measured on
d 15 and between d 17 and 88, postpartum. At (1 0, the RBC GPX-1 activity for each
treatment group was 2.33, 1.67, 5.30 EU/g Hb for 13, 32.5, 45.5 mg Se/d, respectively.
The RBC GPX-1 activity differed (P < 0.05) at d 15 postpartum (122.4 vs 161.7 vs 197.8
EU/g Hb for the 13.0, 32.5, 45.5 mg/ Se (1 treatment groups, respectively). However, by
88 d postpartum, GPX-1 activity did not differ (P < 0.05). There were no differences in
plasma GPX-3 activity for the cows at any bleeding time. Calves born from cows
receiving 45.5 mg Se/d had higher (P < 0.05) RBC GPX-1 activity at d 15 of age than
calves from cows fed 13.0 or 32.5 mg/Se d (378.8 vs 186.2 and 236.1 EU/g Hb,
respectively). Plasma GPX-3 activity of calves from cows fed 32.5 and 42.5 mg Se/d
were higher than calves born to control cows (346.1 and 346.2 vs 263.8 EU/L). In the
second experiment, Se deficient Salers cows were supplemented 0, 13 or 32.5 mg Se/d as
sodium selenite for 15 d postpartum. Calves were administered two 1.38 mg injections of
Se as sodium selenite at 2 d and 49 d of age. At 30 d of age, RBC GPX—1 activities of
calves from the control cows were significantly lower (P < 0.05) than activity of calves
from cows supplemented with 32.5 mg Se/d (46.0 vs 119.7 EU/g Hb). The RBC GPX-1
activity of calves from cows receiving 13.0 mg Se/d did not differ from either group.
Calf RBC GPX-1 activity did not differ at the final sampling period, which was 77 to 115
d after the last injection at 49 d of age. No differences in cow or calf GPX-3 activity
were observed. Abdelrahman and Kincaid (1995) also reported that a selenite
intraruminal bolus (designed to release 3 mg Se/d) administered to cows in late

pregnancy increased colostrum Se and hepatic Se of their calves.

20

Organic Se vs Sodium Selenite. While much of the research has been done with
sodium selenite, Se containing yeasts are now approved for supplementation to cattle.
Gunter et al. (2003) reported calves born from cows receiving 26 mg Se as Se yeast in
free choice salt had higher (P < 0.05) whole blood Se and whole blood GPX activity
compared with calves from cows receiving the same amount of Se provided as sodium
selenite (203 vs 134 ng/ml and 115 vs 62 EU/g Hb). Koenig et a1. (1997) investigated the
influence of diet and chemical form of Se on intestinal entry, absorption and retention on
ruminal and duodenal cannulated sheep. Utilizing radioisotopes ([77Se] yeast, [szse]
selenite), sheep were fed forage (alfalfa hay) or concentrate (barley) based diets
containing 0.37 ppm and 0.27 ppm Se, respectively. Selenium isotopes of the two
sources were ruminally infused four times daily for 7 d. Feces and urine were collected
daily. Duodenally, there was a higher proportion of Se associated with bacteria in the
particulate fraction than fluid portion. Approximately 63.7 and 48.4% of Se was
available for absorption from the concentrate and forage diets, respectively (P < 0.053).
The 82Se selenite was more available for absorption (P < 0.05) than 77Se yeast in the
forage (41.8 vs 36.7 %) and concentrate (53.1 vs 50.9%) diets, respectively. Thus, the
availability of Se from inorganic and organic sources in Suffolk wethers was influenced
by type of diet. However, Peter et al. (1982) reported that absorption and retention of
ruminally infused selenomethionine was greater ( P < 0.05) than infused selenite in
wethers consuming 0.01 ppm Se in an 80% oat hay, 20% whole oat diet. In yeast and
feedstuffs only 50% of Se is found as selenomethionine (Korhola et al., 1986). The
remainder is Se bound to several small peptides. Recent research indicates that Se yeasts

maybe more effective in transferring Se into bovine milk. (Pehrson et al., 1999; Knowles

21

et al., 1999). Pehrson et al. (1999) reported higher (P < 0.05) milk Se concentrations in
Hereford cows after 90 d postpartum supplementation of 30 ppm Se as Se yeast versus
cows supplemented the same concentration as sodium selenite. Knowles et a1. (1999)
reported Se yeast was 50 % more effective (P < 0.05) than sodium selenite in improving
Holstein milk Se concentrations when supplemented for 90 d in a 2 or 4 mg Se drench
given three times weekly.

Selenium, Thyroid and Immunoglobulin Interaction. Dietary sources of Se and
their effect on thyroid hormones and immunoglobulins have been investigated in beef
cows and calves (Awadeh et al., 1998). Mature cows were assigned to treatments of 20,
60, 120 ppm supplemented Se as sodium selenite or 60 ppm Se as selenomethionine from
yeast in free choice salt for 90 d prepartum until 7 mo after the second parity. Selenium
status, thyroid status and immunoglobulin G (IgG) production of cows and calves were
monitored. However, Se concentration in the feedstuffs was 0.82 ppm on a DM basis,
which is considerably higher than the requirement of 0.1 to 0.3 ppm on a dry matter basis
(NRC, 1996). Cows receiving 120 ppm Se had higher whole blood Se concentrations
than cows consuming all other diets between calving periods. There were no differences
in whole blood Se concentration of cows supplied 60 ppm Se as yeast or selenite in their
free choice salt. Additionally, there were no differences between cows fed 120 ppm Se
as selenite and 60 ppm Se as yeast for the 7 mo of supplementation after second
parturition. Whole blood Se in cows fed 60 ppm Se as selenite in salt was lower
(P < 0.05) 7 mo after second parturition compared with that of cows provided 60 ppm Se
as yeast or 120 ppm Se as selenite in free choice salt (100 vs 125 and 124 ng Se/ml,

respectively). Interestingly, cows fed 20 ppm Se as selenite in salt had the highest whole

22

blood GPX activity compared to cows on the other treatments; however, there were no
differences due to treatment at second calving. Two month-old calves from dams
receiving the Sc yeast in salt for one year had higher whole blood Se concentrations at
birth than calves born to the dams in the other treatment groups. However, this effect
was not seen in calves from the next parity where cows were provided a longer period of
free choice salt supplementation.

In this same study, there were no differences in colostrum Se following the first
parturition. However, colostrum Se was higher (P < 0.05) in cows supplemented with the
Sc yeast relative to all other treatments after the second parturition. Colostrum from the
Sc yeast group was 60 ng Se/ml higher than the prior year while there were no increases
in colostrum Se from year to year in the other treatments. The IgG concentrations of
calves from cows in the different treatment groups did not differ. In the second year,
calves from dams receiving 120 ppm Se in free choice salt had higher T3:T4 ratios at birth
indicating greater deiodinase activity. However, there were no differences in thyroid
parameters at 2 mo of age for calves from cows given any of Se treatments in free choice
salt. These very limited data indicate that maternal Se intake may influence calf
deiodinase activity.

Lacetera et al. (1996) did not see an influence of Se supplementation on passive
immunity in the calf, but Swecker et al. (1995) reported improved (P < 0.05) IgG
concentration in calves from Se supplemented cows compared to marginally Se deficient
cows receiving no Se supplementation. Eighty beef cows considered to be marginally Se
deficient (50 ng/L Se, whole blood) were assigned to four treatments: (1) no

supplemental Se, (2) injection of 0.1 mg Se and 1 mg Vitamin E/ kg body weight, (3)

23

available free choice salt with 120 ppm Se as sodium selenite, or (4) both injection and
free choice salt. On (I 28 and 56 of the study, gestating cows were inoculated with 200
pg lysozyme in 0.5 m1 saline and 0.5 ml Freunds incomplete adjuvant. Blood was
collected at d 84, 70, 56, 42, 28 prepartum and at parturition and from calves immediately
after birth. Concentrations of IgG and IgM were measured in colostrum and postsuckle
serum. There were no differences in the specific lysozome antibody titers in cows or
calves, regardless of cow’s treatment. Cows supplemented with 120 ppm sodium selenite
or 120 ppm sodium selenite plus SeNitamin E injection had higher (P < 0.002) colostral
IgG concentrations (Lacetera et a1, 1996). These results indicate Se supplementation can
influence colostral IgG production; however, no reports of calf health were given.
Selenium Toxicity

Selenium toxicity was originally documented in the United States in 1937
(Moxon, 1937), and it may occur because of consumption of Se accumulating plants
and/or over supplementation of Se. Kubota et al. (1967) reported incidence of Se
accumulating plants throughout the Rocky Mountain region, however no Se accumulators
have been documented east of the Mississippi River. The primary plants known for high
Se concentrations are in the Astragalas family, commonly known as locoweed. Byers
(1936) reported 6500 ppm Se in the shoots of Astragalus bisulcatus. Other reports of Se
concentrations in the A. bisulcatus range up to 3000 ppm Se (NRC, 1996). Trelease et al.
(1960) reported the primary form of Se in A. bisulcatus is Se-methyl selenocysteine.

Overfeeding Se can be detrimental to livestock. Young Holstein calves
consuming 10 mg Se/d in a milk replacer for 42 (1 had suppressed weight gain and feed

efficiency (Jenkins and Hidiroglou, 1986). Pregnant cows consuming 12.0 ppm Se as

24

sodium selenite for 80—120 (1 prepartum had suppressed immune response to pokeweed
mitogen and gave birth to weak calves (Yaeger et al., 1998). Selenium induced liver
lesions have been reported in sheep and swine fed 20 ppm Se as sodium selenite
(Raisbeck, 2000; Goehring etal., 1984). MacDonald et a1. (1981) reported pulmonary
edema in freshly weaned dairy calves injected with 2 mg Se/kg BW as sodium selenite.
Ahmed et al.(1990) reported liver lesions in goats dosed with 5, 20, 40, 80, and 160 ppm
Se as sodium selenite for 21 d. Eighteen of 28 goats across all treatment groups died
during the trial. Hemorrhages were seen in the rumen, reticulum, omasum, and
abomasum along with liver necrosis.

Selenium substitution for sulfur (S) in protein has been reported to be detrimental
to metabolism as well. Daniels (1996) reported that excess Se analogs of typically S
containing enzymes play a role in embryonic deformities. Further, a primary sign of
selenomethionine toxicity in mammals is the loss of hair and sloughing of hooves.
O’Toole and Raisbeck (1995) reported alopecia of the tail and cracking of hooves in
selenotic cattle, which may be due to improper keratinocyte maturation. Witte et a1.
(1993) also reported alopecia of the mane and cracking of hooves in chronic selenotic
horses. Hypothetically, selenotic conditions may promote substitution of Se for S which

weakens disulfide bridges in keratin.

25

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34

ABSTRACT

Selenium (Se) is essential for anti-oxidation and thyroid hormone function in
cattle. However, factors that influence its requirement are not well understood. Three
experiments were conducted to determine the influence of cattle breed and age on
selenoprotein activity and the effect of maternal Se supplementation on cow and calf
selenoprotein activity and neonatal thyroid hormone production. In Exp. 1, four
cowherds of differing ages representing three breeds were bled to determine the influence
of breed and age on erythrocyte glutathione peroxidase activity (RBC GPX-1). All
females were non-lactating, bred, and consumed total mixed rations (Holstein) or grazed
pasture (Angus and Hereford). In beef breeds, yearlings had higher (P < 0.05) RBC
GPX-1 activity than mature cows; however age did not influence RBC GPX-1 activity in
Holstein females (23.00 vs 19.14 vs 20.79 EU/g Hb, for yearlings, two year-old and
mature cows, respectively). In Exp. 2, neonatal Holstein heifers (n = 8) were bled daily
from 0 to 6 d of age to determine their thyroid hormone profile. Thyroxine (T4) and
triiodothyronine (T3) concentrations were highest on d 0 and decreased (P < 0.05)
continuously until d 5 postpartum ( 156.13 to 65.88 and 6.69 tol.95 nmol/L, d 0 to 5 for
T4 and T3, respectively). Reverse triiodothyronine concentrations were 3.1 nmol/L on d 0
and decreased (P < 0.05) to 0.52 nmol/L by d 5. In Exp. 3, multiparous Hereford cows
were drenched weekly with: 1) a placebo containing 10 ml double deionized H2O (n =
14), or 2) 20 mg Se as sodium selenite (n = 13). By mo 2 of treatment, Se drenched cows
had higher (P < 0.01) plasma Se concentrations than control cows (84.92 vs 67.08 ng
Se/ml) and at parturition had plasma Se concentrations two-fold that of (P < 0.05) control

cows (95.51 vs 47.14 ng Se/ml). By mo 4, cows receiving Se had higher (P < 0.05) RBC

35

GPX-1 activity than controls and this trend continued until parturition. Colostrum Se
concentration was two-fold higher (P < 0.05) in Se drenched cows than control cows
(169.97 vs 87.00 ng Se/ml). Calves born to cows drenched with Se had higher (P < 0.05)
plasma Se concentration, RBC GPX-1 and plasma glutathione peroxidase activity (P
GPX-3) on d 0 compared with calves born to control cows. By d 7, no differences in P
GPX-3 activity in calves were observed. Maternal Se supplementation did not influence
calf thyroid hormone concentrations.

Key Words: Selenium, Glutathione Peroxidase, Cows, Thyroid hormones

36

Introduction

Selenium is an essential trace mineral for anti-oxidative and thyroid hormone
function. Erythrocyte glutathione peroxidase (RBC GPX-1; EC 1.11.1.9) and plasma
glutathione peroxidase (P GPX-3; EC 1.11.1.9), both require Se (Rotruck et al., 1973;
Martin-Alonso et al., 1993) and reflect long- and short-term Se status, respectively
(Cohen et al., 1985). Thyroxine (T4) is deiodinated to the more metabolically active
triiodothyronine (T3) by the Sc requiring enzyme type 1 deiodinase (DI-l; EC 3.8.1.4;
Beckett et al., 1987).

Michigan forages are marginally Se deficient containing 0.1 to 0.2 ppm Se
(Kubota et al., 1967; Mortimer et al., 1999). Cow Se status can be further compromised
as Se is transferred across the placenta during late pregnancy and during early lactation in
colostrum. Consequently, calves born from Se deficient or marginally deficient dams
may have compromised Se status.

Schrama et al. (1993) reported neonatal calves are susceptible to cold stress in
temperatures below 14.6°C. Approximately 70 % of Michigan calves are born between
January and April when typical temperatures range from -6.7° to 78°C (Ritchie, 1991).
Therefore, calves must generate therrnogenic responses to cold. A major form of
facultative thennogenesis is through metabolism of brown adipose tissue which is
regulated by T3.

Arthur et al. (1988) reported impaired T4 to T3 conversion in Se deficient Friesian
steers. Further, Awadeh et al. (1998b) reported that Se status of the dam could influence
neonatal calf thyroid production. Hence, because calves born in Michigan could be prone

to a combination of cold stress and Se deficiency, three experiments were conducted to:

37

(1) investigate the influence of breed and age of cows on Se status, (2) determine the
normal thyroid profile of neonatal calves, and (3) determine the effect of Se
supplementation on maternal and calf selenoprotein activity and Se transfer on neonatal

thyroid hormone status.

38

Materials and Methods

Animal Use and Care

The All University Committee on Animal Use and Care approved all procedures
in these experiments (AUF number: 12/01-183-00).
Animal Experiments

Experiment I. The initial experiment was conducted in fall of 2001 to assess the
influence of breed and age on RBC GPX-1 activity. Four non-lactating cow herds
consisting of yearling heifers (< 730 d of age), two-year old (730 to 1095 d of age) and
mature cows (> 1095 d of age) were bled via coccygeal venipuncture into evacuated
blood tubes (BD Vacutainer, Franklin Lakes, NJ). These herds were: 1) cooperator
Angus (CH), n = 49; 2) Michigan State Universtiy (MSU) Angus (MA), n = 38; 3) MSU
Hereford (MHE), n = 43; and 4) MSU Holstein (MHO), n = 32. The CH was composed
of 13 yearlings, 12 two-year olds and 24 mature cows. The MA herd contained 8
yearlings, 8 two-year olds and 22 mature cows. The MHE herd included 19 yearlings, 5
two-year olds and 19 mature cows. The MHO herd consisted of 10 yearlings, 8 two-year
olds and 14 mature cows. The CH was located 15 km from MSU and had been
assembled between 1997 and 2000 from herds in Montana, Virginia and Georgia, where
forage Se concentration is variable to normal. The yearling heifers in this herd were born
in Michigan. The MSU herds are located near campus. All two-year old and mature
cows had calved the previous year. The CH, MA and MHE herds grazed a mixture of
alfalfa, blue grass and orchard grass ad libitum and were supplied a free choice trace
mineral salt containing 60 ppm Se as sodium selenite. The MHO yearlings were housed

in a solid floor, partially sheltered, pole-ham and fed a haylage based total mixed ration

39

(TMR), balanced to contain 0.3 ppm Se. The MHO two-year old and mature cows were
housed in a slatted, enclosed tie-stall barn and consumed a predominately silage and
haylage TMR with 0.3 ppm Se.

Experiment 2. The second experiment was conducted to determine the normal
thyroid hormone status profile of newborn calves. Holstein heifer calves (n = 8) born to
cows with an adequate Se status were bled at < 12 hr of age and daily thereafter for 7 d.
Following the initial bleeding, calves were injected with 1 mg of Se as sodium selenite
and 68 IU vitamin E as d-alpha tocopheryl acetate (BO-SE, Schering-Plough,
Kenilworth, NJ). All blood samples were obtained via jugular venipuncture in
heparinized evacuated tubes for plasma Se and RBC GPX-1 enzyme activity. An
additional 5 mL of blood was collected into clot activated evacuated tubes (Vacutainer
plus SST®, Franklin Lakes, NJ) for determination of T4, T3, free T4 (ff 4), free
triiodothyronine (fI‘3), and reverse triiodothyronine (rT3). On d 0, before initial bleeding,
calves consumed 3 L of colostrum and 12 hr later were offered an additional 3 L of
colostrum. Thereafter, calves were offered 2 L of a commercial milk replacer (Calvita
Supreme, Milk Specialties, Dundee, IL) twice daily containing 0.4 ppm Se on a DM
basis. On (1 2, calves were placed in individual hutches located in an open front pole-barn
for the remainder of the trial.

Experiment 3. The third experiment was conducted to investigate the influence of
maternal Se supplementation on cow and calf plasma Se concentration, P GPX-3 and
RBC GPX-1 activity, and to determine thyroid hormone status through the first week of
life. Twenty-seven multiparous Hereford cows were assigned to one of two treatment

groups: 1) weekly placebo drench of 10 mL double deionized H2O (n = 14) or 2) weekly

4O

drench with 20 mg Se from sodium selenite (n = 13). The Se drench was prepared by
dissolving 4.38 g sodium selenite in 1 L of double deionized water. Cattle were allotted
to treatments by initial RBC GPX-1 activity, parity and date of last calving. Cows were
provided a trace mineral salt, free choice, that contained no added Se. In April, prior to
initiation of the trial, cows were artificially inseminated and thereafter cows were
pastured with a bull until mid-August. Initial drenching began in mid-July with cows
ranging from 0 to 3 mo of pregnancy and continued to within 1 wk of parturition.
Monthly, cows were bled using heparinized evacuated tubes via coccygeal venipuncture.
Cattle grazed a mixture of alfalfa, blue grass and orchard grass ad libitum until the end of
October, and were then offered locally harvested comstalk round bales ad libitum and
corn silage (3.4 kg/d, DM) for the remainder of the trial. Beginning in January, locally
harvested grass hay was supplied every third day until calving. Feedstuffs were sampled
periodically and analyzed for mineral content. Approximately one month prior to
parturition, cows were removed from pasture and relocated to a pole-bam enclosure until
calving.

Within 12 hr postpartum, environmental temperature was recorded and cows were
separated from calves for initial sample collection. Calf rectal temperature and weight
were recorded. Colostrum was taken from the left rear teat for analysis of colostrum Se
and glutathione peroxidase activity (C GPX-3) and 5 mL of blood was collected from
cows and calves for analysis of plasma Se concentration, RBC GPX-1 and P GPX-3
activities. An additional 5 mL of blood was collected from calves for determination of
serum thyroid hormone. Calves were tattooed, ear-tagged, vaccinated and returned to

their dam. Vaccinations included: 1) a corona and E. coli — K99 bolus (First Defense,

41

ImmuCell, Portland, ME) and 2) infectious bovine rhinotracheitis (IBR) and para
influenza-3 (P13) intranasal (TSV-2, Pfizer, Exton, PA). In addition, a vitamin A and D
injection (500,000 and 75,000 IU, respectively; Veterinary Laboratories, Lenexa, KS)
was administered in the platysma muscle. All sample and data collection from calves,
except weight, were repeated on d 3 and d 7.
Laboratory Analysis

Blood was centrifuged (2000 x g, 15 min, 4°C) to separate plasma or serum from
RBC. After initial centrifugation, plasma was frozen (-80° C) in aliquots for
determination of P GPX-3 activity and Se concentration. Red blood cells were washed
and hemoglobin was determined (Hill etal., 1999). Plasma protein concentrations were
analyzed by the method of Lowry et al. (1951). Colostrum was mixed thoroughly,
separated into aliquots, and frozen (- 80°C) for later determination of C GPX-3 activity
(Debski et al., 1987) and Se concentration. All glutathione peroxidase activity in RBC,
plasma and colostrum was determined by the method of Paglia and Valentine (1967).
One enzyme unit (EU) of activity represents 1pmol NADPH oxidized per minute using a
molar extinction coefficient of 6.22 x 103 for NADPH and the stoichiometry of reaction
of 2 moles GPX formed per mole NADPH oxidized.

Feed samples were digested (Shaw et al., 2002) and minerals, excluding P and Se,
were analyzed by atomic absorption spectroscopy. Phosphorus was analyzed by the
method of Gomori (1942). Instrument accuracy for all mineral analysis was established
using bovine liver standard (1557b; National Institute of Standards and Technology,
Gaithersburg, MD). Plasma, colostrum and feed Se were analyzed by the fluorometric

method of Whetter and Ullrey (1978) with a modification to the digestion procedure as

42

described by Reamer and Veillon (1983). Fluorescence was analyzed with a Cytofluor H
Fluorescence Multi-Well Plate Reader, Series 4000 (Perseptive Biosystems;
Framingham, MA) at an excitation and emission of 360 and 530 nm, respectively. For
accuracy in Se determination, a bovine plasma pooled sample (acceptable range 60 to 70
ng Se/mL) was utilized.

Serum T4, ff 4 and fT 3 were determined with commercial radioimmunoassay kits
(Clinical Assay, GammaCoat, DiaSorin, Stillwater, MN). A commerical
radioimmunoassay kit was used to determine rT3 (code 10834U, Adaltis, Rome, Italy).
Serum T3 was determined by the method of Refsal et a1. (1984) and measured on an
Apex 10/600 gamma counter (Titertek Instruments, Huntsville, AL). A commercial
radioimmunoassay kit was utilized to analyze TSH (Coat-A-Count procedure, Diagnostic
Products, Los Angeles, CA).

Statistical Analysis

Data in Exp. 1 were analyzed by least squares AN OVA using the PROC MIXED
procedure of SAS (SAS Inst. Inc, Cary NC). The model contained animal as a random
effect and breed, age and breed x age interaction as fixed effects. Significant differences
in all experiments were separated using the LS Means procedure of SAS. In Exp. 2 and
3, calf Se indicators and thyroid hormones were analyzed by least squares ANOVA in a
repeated measures design using the PROC MIXED procedure of SAS. The model
contained calf and time as random and fixed effects, respectively. In Exp. 3, time of
supplementation and air temperature were used as covariates for Se status indicators and
thyroid hormone analysis. The model contained cow as a random effect and treatment,

time and time x treatment interaction as fixed effects. Monthly cow plasma Se

43

concentration and RBC GPX-1 activity were analyzed using repeated measures over time
using the PROC MD(ED procedure of SAS. A Sattherwaithe’s degree of freedom
adjustment was used to account for decreasing sample size over time. Correlations
between dependent variables were analyzed by Pearson’s correlation procedure in SAS.
When necessary, to meet AN OVA heterogeneous variability requirements, loge-
transforrnation on response variables (cow P GPX-3 and C GPX-3 activity, calf P GPX-3
activity, fl‘ 4, fI‘ 3 and rT3) were performed. For consistency, all loge-transformed least
squares means are reported as back transformed LS means with confidence intervals as

indices of variability.

Results and Discussion

Experiment 1

In all beef herds (CH, MA, MHE) mature cows had lower (P < 0.05) RBC GPX-l
activity than yearlings (Table 1). Within the Angus herds, RBC GPX-l activity was
decreased in yearlings compared to two-year olds, while the decrease in activity for
Herefords was not significant (P > 0.71). To meet the Sc needs of the neonatal calf, Se is
primarily transferred across the placenta during late pregnancy (VanSaun et al., 1989),
and colostrum and milk Se concentrations are reflective of cow’s Se status (Stowe et al.,
1988; Knowles et al., 1999). Even when cows have reduced RBC GPX-1 activity,
reports have indicated that RBC GPX-1 activity of the calf is often adequate (Koller et
al., 1984). Hence, the dam will reduce her stores to provide Se for the calf. A free choice
trace mineral salt containing 60 ppm Se as sodium selenite was supplied to all beef cows.
Dargatz and Ross (1996) reported reduced Se status can occur in beef herds even when
trace mineralized salt containing Se was provided. Backall and Scholz (1981) reported
that Holstein and Angus cows with adequate whole blood Se status (90-105 ng/mL) had
whole blood GPX-1 activity between 27-35 EU/g hemoglobin while cows with marginal
status had RBC GPX-1 activity of 11.5 EU/g hemoglobin. Likewise, Maas et al. (1993)
reported Hereford heifers with whole blood Se concentrations of 100 ng/mL correlated
with whole blood GPX-1 of 30.00 EU/g hemoglobin. Based on these values, it appears
that two-year old and mature cows in the beef herds studied were losing Se with
advancing parity and may not have had adequate Se status.

Age, however, did not influence RBC GPX-1 activity in the MHO herd (Table 1).

Supplementation of sodium selenite in TMR diets provided 0.3 ppm Se, the NRC

45

requirement for dairy cattle (NRC, 2001). The dairy diets contained a higher proportion
of grain compared with the beef cow diets. Rumen pH is responsive to type of diet and
drops rapidly as NDF level decreases (Allen, 1997). In vitro studies suggest rumen
bacteria can influence Se absorption. Hudman and Glenn (1984, 1985) reported greater
Se absorption by Selenomonas ruminantium and Butyvibrio fibrisolvens, bacterium more
prevalent in acidic rumen environments, compared with Prevotella ruminicola, bacteria
associated with more basic rumen pH. Additionally, Koenig et al. (1997) reported that Se
absorption from labeled selenite was greater for wethers consuming concentrate vs.
forage based diets. Counotte and Hartmans (1989) reported that yearling Holstein heifers
fed high roughage diets adequate in Se had lower (P < 0.01) RBC GPX-1 activity
compared with calves and mature cows, supporting the hypothesis that type of diet may
influence Se status. In addition to diet differences between beef and dairy herds, MHO
females received semi-annual Se injections (50 mg Se as selenite; MU-SE, Schering-
Plough, Kenilworth, NJ) while beef cows did not, which could further influence the RBC
GPX-1 activity. Consequently, because of diet, injections and perhaps other facets of
management, age did not influence MHO RBC GPX-1 activity.

The CH yearling females had higher (P < 0.05) RBC GPX-l activity than the
MA, MHE and MHO herd yearling females. The activity of CH, MHE and MHO two-
year olds were higher (P < 0.05) than MA two-year olds. However, it is of interest that
the MHE two-year olds grazed the same pastures and consumed the same preserved
feedstuffs as the MA two-year olds. The CH and MHO mature cows also had higher

(P < 0.05) RBC GPX-1 activity than the MA and MHE mature cows.

46

Between the two Angus herds, all CH age group females had higher RBC GPX-1
activity than those in the MA herd. The cattle grazed comparable forages, were managed
similarly and were offered a similar trace salt containing 60 ppm Se as sodium selenite.
Possible reasons for the within breed differences could be genetics or area of origin. The
CH two-year old and mature cows were assembled from areas (Georgia, Montana, and
Virginia) known to be variable to normal for forage Se concentrations (Kubota et al.,
1967). The CH yearling females received in utero Se from cows brought into Michigan,
theoretically their stores were greater than the Michigan born cows that were dams to MA
yearlings. Stevens et a1. (1985) reported Holstein cattle residing on Se variable soils had
higher RBC GPX-1 activity compared with cattle residing on Se deficient soil. Further,
the amount of nutrients transferred from dam to offspring is based on nutritional status of
the dam (VanSaun et al., 1989). Consequently, the higher RBC GPX-l activity in the CH
vs. MA herd could be because cattle originating from higher Se areas had higher Se status
in late gestation and transferred more Se to the fetus. Thus, RBC GPX-1 activities were
influenced by breed and age as well as nutrition and management.

Experiment 2

On (1 0, the mean neonatal calf plasma Se concentrations was 72.93 ng/mL (Table
2). This agrees with Stowe and Herdt (1992) who reported that newborn Holstein calves
had serum Se concentrations ranging from 50 to 70 ng/mL. Following Se injection
(post-bleeding on d 0), Se plasma concentrations rose 39 % (P < 0.05) on d 1. Plasma Se
concentrations decreased (P < 0.05) from d 1 to d 6 when they were not different from d
0. Intramuscular Se injections are commonly used to administer Se to calves and cows in

Se deficient areas. Maas et a1. (1993) reported selenite injections (0.05 mg Se/kg body

47

weight) improved ( P < 0.05) serum Se concentrations in Se deficient Hereford heifers
for 56 (1. However, Weiss et al. (1984) reported selenite injections (0.078 mg Se/kg body
weight) did not improve serum Se concentration of young calves born to dams fed 5 mg
Se daily, but improved serum Se concentrations up to 56 d post injection of calves born
from dams fed 1 mg or no Se daily. Thus, calf Se status prior to injection may influence
how long serum Se remains elevated. In our study, calf serum Se was only elevated for 5
d, post injection. Close-up diets of the calves’ dams met NRC requirements for Se

(0.3 ppm Se; NRC, 2001) and farm standard operating procedure included administration
of selenite injections (50 mg Se) twice annually to cows. Consequently, newborn calves
had adequate Se status and a majority of the injected Se was likely cleared through the
urine or stored in the liver and kidney.

Serum T4 concentrations decreased (P < 0.05) from d 1 until d 5 (Table 2).
Although T3 concentration was not different between (1 0 and 1, decreases (P < 0.05) were
observed from d 2 to d 5. The f1“ 4 and fl‘ 3 concentrations follow the same trend as T4 and
T3, respectively. In young calves, T4 and T3 concentrations decrease with low energy
intake (Kinsbergen et al., 1994). Colostrum is high in lipids and protein and more
energy-dense than milk (Rauprich et al., 2000), thus concentrations of T4 and T3 are high
at birth and decrease until d 6, which is comparable to other reports (Hadom et al., 1997;
Nussbaum et al., 2002). However, Hadom et al. (1997) and Nussbaum et al. (2002)
reported considerably higher (1 0 T4 and T3 concentrations when blood of Holstein calves
was taken within 6 hr of birth; but by d 6, calf T4 and T3 concentrations were comparable
to our observations. In our trial, blood was collected within 12 hr of birth which could

explain the difference in initial thyroid hormone concentration.

48

Serum rT3 concentrations were highest (P < 0.05) on d 0 and decreased by d 5
(Table 2). Type III deiodinase is induced by high levels of T3, and it inactivates T4 to the
inactive metabolite, rT3 (Kohrle, 2000). Because T3 concentrations are high on d 0,
corresponding rT3 concentrations were highest on d 0 and both continued to decrease
until (1 5. The T32T4 ratios were highest on d 0, 1 and 2 but decreased (P < 0.05) by d 5.
The conversion of T4 to T3 is catalyzed by the selenoprotein type 1 deiodinase (DI-1;
Beckett et al., 1989) and its activity is reflected by the T3:T4 ratio. The observed T3 : T4
ratios on d 0 are consistent with other reports (Awadeh et al., 1998b; Hammon et al.,
2002) and appear to stabilize on d 5 and 6, consistent with ratios reported by Arthur et al.
(1988) in Holstein calves 3 mo of age.
Experiment 3

After one month of Se supplementation, plasma Se concentrations did not differ
between treatment groups (Table 3). However, after two months, the plasma Se
concentrations were higher (P < 0.01) for cows in the Sc supplemented group compared
to control cows, and this difference continued until conclusion of the trial. Diagnostic
laboratories assume normal serum Se concentrations in adult cattle to range from 70-100
ng/mL (Stowe and Herdt, 1992). Marginally Se deficient adult cattle have serum Se
concentrations between 40-70 ng/mL while cattle deficient in Se have serum
concentrations below 40 ng/mL (Gerloff, 1992). According to these values, by month
eight, control cow plasma Se concentrations were reduced to 41.08 ng/mL, approaching
deficient concentrations. Removal of the sodium selenite from the trace salt and Se
transfer from dam to fetus in the last trimester of pregnancy (VanSaun et al., 1989) both

likely contributed to the declining plasma Se concentrations observed over time. Further,

49

adequate Se in Michigan grown feedstuffs (Table 4) was not sufficient to maintain
adequate status as measured by the plasma Se concentrations in control cows. Twenty
mg Se/wk supplemented as a sodium selenite drench, adequately maintained cow plasma
Se status for the entire trial (Table 3). The RBC GPX-1 activity of cows in both
treatment groups did not differ during the first three months of the study (Table 3).
However, after four months, Se drenched cows had higher RBC GPX-1 activity and
maintained this difference for the remainder of the trial. The delay in RBC GPX-1
activity’s response to Se is likely due to the 90 to 120 (1 RBC life expectancy, resulting in
only limited monthly incorporation of GPX-1 enzyme into RBC during erythropoesis
(Stowe and Herdt, 1992). However, Enjalbert et al. (1999) reported 35-fold increases in
RBC GPX-1 activity after 15 d of supplementing large amounts of Se (45 mg) as sodium
selenite in Se deficient Salers cows. Thus, concentration of Se could also influence
response time of RBC GPX-l activity to Sc supplementation. Peak RBC GPX—1
activities in our trial agree with Stevens et al. (1985) who reported whole blood RBC
GPX-1 activities between 27-35 EU/g hemoglobin for Se adequate Holstein and Angus
cows. However, more recently Gunter et al. (2003) reported British cross and Simmental
cows in Arkansas consuming trace salt with 26 ppm Se as sodium selenite or Se yeast for
four months had RBC GPX-1 activities of 101 or 106 EU/g hemoglobin, respectively.
These activities are considerably higher than seen in our trial, but differences could also
be attributed to laboratory variation in assessing enzyme activity.

At parturition, Se supplemented cows had two-fold greater plasma Se
concentrations than control cows, and this increase was also seen in RBC GPX-1 activity

(Table 5). The correlation between the two variables was 0.75 (P < 0.001). The plasma

50

Se concentrations in our cows were higher than Weiss et al. (1984) who reported
parturition serum Se concentrations of 32 and 58 ng/mL in cows supplemented with l or
5 mg/d Se as sodium selenite beginning 60 d prepartum. Counotte and Hartmans (1989)
reported high correlation for RBC GPX-1 activity and Se concentration in whole blood
(r = 0.933, P < 0.001) of dairy cattle. More recently, Knowles et a1. (1999) reported
whole blood GPX-1 and Se correlations of 0.85 (P < 0.001) in Holstein cows receiving 2
or 4 mg Se as sodium selenite or selenium yeast drenches administered three times
weekly for 19 wk. However, Awadeh et al. (1998a; 1998b) reported no differences in
whole blood GPX activity in beef cows offered differing amounts of supplemental Se as
sodium selenite or yeast in free choice trace salt.

Plasma GPX-3 (Table 5) activity tended to be higher at parturition for cows
receiving Se supplementation. However, since this is a short-terrn indice, and the time
from drench until blood sampling was highly variable, differences may be obscured.
Cows in this trial received Se supplementation weekly, thus, a 6 (1 difference between
drench and blood collection at parturition could exist. In nonruminants, serum GPX-3 is
used as a short term Se status indicator (Mahan and Parrett, 1996; Mahan et al., 1999),
however in the ruminant, few trials have examined P GPX-3 activity and Se status.
Podoll et al. (1992) reported that supplemental Se increased Holstein serum Se, but serum
GPX-3 was not affected. Likewise, Enjalbert et a1. (1999) reported no influence of beef
cow Se intake on P GPX-3 activity. Only a small portion of Se found in blood is
associated with P GPX-3 in the rat (Deagen et al., 1993) and bovine (Awadeh et al.,

1998a). Importantly, 98% of GPX activity is associated with erythrocytes (Scholz and

51

Hutchinson, 1979) and P GPX-3 represents only a very small portion of Se and total GPX
activity.

Colostrum Se concentration at parturition was almost 50% greater in cows
drenched with Se than in control cows (Table 5). Schingoethe et al. (1982) reported that
dietary Se concentrations (0.1 or 2 ppm Se) or selenite injection (5 mg Se/45.4 kg BW)
did not influence dairy cow colostrum Se concentrations in Se adequate cows.
Additionally, deToledo and Perry (1985) reported that colostrum Se concentrations were
not different between Se adequate and Se deficient Holstein cows receiving 1 or 2 mg Se
in feed as sodium selenite for 60 d prepartum. Alternatively, Koller et al. (1984) reported
Hereford cows consuming high Se soybean meal (0.3 ppm Se) and Se supplemented trace
salt (90 ppm Se as sodium selenite) had higher Se concentrations in colostrum than cows
consuming only low Se hay. Intraruminal boluses (3mg Se/d as selenite) given 120 d
prepartum has also increased colostrum Se concentrations in Holstein cows
(Abdelrahman and Kincaid, 1995).

Although our supplemented cows had 39 % higher C GPX-3 activity compared
with control cows, the difference was not significant (P < 0.17). To our knowledge,
GPX-3 activity has never been assayed in bovine colostrum. Hojo (1982) was the first to
detect GPX-3 activity in raw bovine milk. However, only twelve percent of the milk Se
was bound to GPX-3 and only 0.003% of the protein found in milk was GPX. Thus, the
small percentage of Se associated with milk GPX-3 could explain why differences were
not detected.

Treatment did not influence calf birth weights (41.60 :i: .79 kg). Awadeh et al.

(1998b) and most recently, Gunter et al. (2003) reported no influence of maternal Se

52

supplementation on calf birth weights. Conversely, Castellan et al. (1999) reported Se
injections (0.05 mg Se/ kg BW as selenite) improved gain in Hereford x Angus calves
from birth to 70 d.

In our study, calves born to dams receiving drenches containing Se had higher
(P < 0.05) plasma Se concentrations at d 0, 3 and 7 compared with calves born to control
cows (Figure 1, panel A). However, since plasma Se concentrations decreased 10 ng/mL
between d 3 and 7 in calves born to Sc supplemented cows, there was a significant time x
treatment interaction. No decrease was observed in calves born to control cows. Stowe
and Herdt ( 1992) reported that serum Se concentrations in newborn Holstein calves range
from 50 to 70 ng/mL. Based on these values, calves born from cows receiving weekly 20
mg Se drenches had adequate plasma Se, while calves born to control cows had plasma
Se concentrations approaching less than adequate Se status. However, calves born to
cows receiving the placebo were numerically higher for plasma Se concentrations than
their dam. This observation agrees with Koller et al. (1984) who reported calves born to
Se deficient Hereford cows had higher whole blood Se concentrations than their dam.

Calves born to Sc drenched cows had a two-fold greater (P < 0.05) RBC GPX-

1 activity compared to calves born to control cows (Figure 1, panel B) at birth, (1 3 and 7.
Scholz et al. (1981) reported Angus x Holstein calves with low plasma Se concentrations
(24 ng/mL) had whole blood GPX-1 activity of 14.0 EU/g hemoglobin. Awadeh et al.
(1998b) observed that neonatal calves born to cows offered trace mineralized salt
containing 60 or 120 ppm Se as selenite or 60 ppm Se as yeast had higher whole blood
GPX activity compared with calves born from cows receiving 20 ppm Se as selenite,

when supplied for 90 d prepartum. However when supplementation continued through

53

the next parity, amount or source of Se supplemented to cows did not influence newborn
calf whole blood GPX activity. Gunter et al. (2003) reported no differences in neonatal
calf whole blood GPX activity when their dams were offered either no Se, or trace salt
containing 26 ppm Se as selenite or yeast.

Although cow P GPX-3 activity did not differ between treatments, a treatment x
time interaction for calf P GPX—3 activity was observed (Figure 1, panel C). Calves born
from Se supplemented cows had higher P GPX-3 activity compared with control calves at
d 0 and 3. However, by d 7, maternal Se supplementation had no influence on P GPX-3
activity. Koller et al. (1984) reported colostrum Se concentrations decrease rapidly from
d 0 to 7, postpartum. Because P GPX-3 responds to immediate Se intakes (Cohen et al.,
1985), the higher colostrum Se intake of calves born to Sc supplemented dams could
have influenced d 0 and 3 calf P GPX-3 activity.

Mean environmental temperature at initial bleeding was — 2 : 1.1°C, well below
temperatures reported to induce cold stress in neonatal calves (Schrama et al., 1993). The
combined mean calf rectal temperature on d 0, 3 and 7 was 39 i 0.1 C° and was not
influenced by maternal Se supplementation. Calves from both treatments appeared
thrifty at birth and no illness was noted during the study duration.

Maternal Se supplementation did not influence neonatal calf thyroid hormone
concentrations (data not shown). This differs from Awadeh et al. (1998b) who reported
improved T3: T4 ratios at birth from calves whose dams received 120 ppm Se as sodium
selenite in a trace salt for 15 mo. They found no differences, however, in T3: T4 ratios of
calves born from cows receiving 20 or 60 ppm Se as selenite or 60 ppm Se as Se yeast in

trace salt. Arthur et al. (1988) reported Se deficient Holstein steers had increased T4 and

54

decreased T3 concentrations when fed torulla yeast diets. These deficient steers had
whole blood Se concentrations of 8 ng/mL, considerably lower than the plasma Se
concentrations observed in our control cows and calves.

Calf thyroid hormone concentrations were influenced by post-partum time of
measure (Table 6). The T4 concentrations were highest on d 0 and decreased (P < 0.05)
on d 3 and 7, similar to Holstein calves in Exp. 2. Thyrotropin (TSH) concentration
decreased between (1 0 and 7, post-partum. The T3 concentrations were not different on d
0 and d 3 but decreased (P < 0.05) by d 7. This observation differs from our Holstein
calves in Exp. 2 (Table 2) and other reports (Hadom et a1, 1997; Nussbaum et al., 2002)
where large decreases in calf T3 concentrations were observed by d 3 of life. However,
fl‘ 4 and f1‘3 concentrations both decreased from d 0 to d 7 and followed a trend similar to
the Holstein calves in Exp. 2. Further, rT3 concentrations decreased (P < 0.05)
dramatically to low concentrations by d 3, similar to the observed rT3 concentrations in
the Holsteins of Exp. 2.

The T4 and T3 concentrations are also numerically higher in the Hereford calves in
Exp. 3 compared with the Holsteins in Exp. 2. The Hereford calves nursed their dams
and consumed colostrum and transition milk to mature milk through wk 1 compared with
Holstein calves in Exp. 2 who were fed a milk replacer, after receiving 6 L of colostrum
on d 0. This observation was also apparent for fT4 and ff 3 concentrations on d 3 and 7.
Recently, Pezzi et al. (2003) reported high concentrations of T4 and T3 in Holstein cow
colostrum which decreased (P < 0.01) by d 5 of milk. Thus, our Hereford calves could
have absorbed more colostral thyroid hormones before gut closure; however, we did not

measure colostrum thyroid hormone concentrations.

55

Implications
Cows consuming exclusively Michigan grown feedstuffs may be at risk for
selenium deficiency, even when selenium is offered in a trace salt. The results that older
cows have reduced selenium status compared with yearling females suggest that
producers should be cognizant of replenishing nutrients that are transferred to the calf.
Furthermore, more research is needed to fully understand the influence of breed and

management on selenium status and thyroid hormone concentrations.

56

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Figure 1. Experiment 3- Effect of maternal weekly drench (20 mg Se as sodium
selenite or placebo) and time on: Panel A: calf plasma Se concentration post partum
during wk 1; treatment x time P < 0.05. Panel B: calf erythrocyte glutathione peroxidase
activity (RBC GPX-1), treatment P < 0.05. One enzyme unit (EU) is the activity needed
to oxidize 1pmol of NADPH per minute. Panel C: plasma glutathione peroxidase
activity (P GPX-3); treatment x time P < 0.05. Data for plasma Se concentration (panel
A) and RBC GPX-1 activity (panel B) are LS means :1: SEM. Statistical analysis of P
GPX-3 activity was performed on loge-transformed LS means, therefore, data for P GPX-
3 activity (panel C) are back-transformed means with error bars for corresponding 95 %

confidence intervals.

69

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