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Selenium tolerance in sheep and selenium supplementation methods for beef cattle

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Selenium tolerance in sheep and selenium supplementation methods for beef cattle
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Davis, Paul Armand
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Blood ( jstor )
Calves ( jstor )
Ewes ( jstor )
Lambs ( jstor )
Liver ( jstor )
Milk ( jstor )
Minerals ( jstor )
Plasmas ( jstor )
Selenium ( jstor )
Sodium ( jstor )
Animal Sciences thesis, Ph. D
Dissertations, Academic -- Animal Sciences -- UF
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Thesis (Ph. D.)--University of Florida, 2004.
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Includes bibliographical references.
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Printout.
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Vita.
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by Paul Armand Davis.

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SELENIUM TOLERANCE IN SHEEP AND SELENIUM SUPPLEMENTATION
METHODS FOR BEEF CATTLE
















By

PAUL ARMAND DAVIS


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2004












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Copyright 2004

by

Paul Armand Davis































To our Heavenly Father and my loving family.














ACKNOWLEDGMENTS


The author wishes to express the utmost gratitude to Dr. Lee R. McDowell,

chairman of the supervisory committee, for his guidance, patience, encouragement and

direction throughout the Ph.D. program and during preparation of this dissertation.

Appreciation is given to Drs. Tim Marshall, Claus Buergelt, and Richard Weldon for

service and dedication on the graduate committee and for advice and understanding.

Special thanks are extended to Dr. McDowell for his unselfish attitude and including me

on several experiments, publications, and educational opportunities. Gratefulness beyond

words is in order to Dr. McDowell for showing me that one will get further by lifting

others up than by putting anyone down. Dr. Todd Thrift deserves thanks for his

willingness to listen and provide both professional and personal advice.

A huge debt of gratitude and appreciation is extended to Mr. Bert Faircloth, Mr.

Charles Stephens, Mr. Steve Chandler, Mr. Jesse Savell, and Mr. Brantley Ivey at the

Santa Fe Beef Unit for their help in conducting the experiments and collecting data. Mr.

Dean Glicco deserves particular thanks for his care of the experimental animals and for

becoming a trusted friend and confidant to the author. The author thanks Ms. Nancy

Wilkinson for sharing her invaluable knowledge of lab procedures and for her patience in

instruction of laboratory techniques. Mrs. Lorraine McDowell deserves thanks for her

work with electron microscopy on animal tissues.








Fellow graduate students Deke Alkire, Carlos Alosilla, Bradley Austin, Nathan

Krueger, Edgar Rodriguez, and Oswaldo Rosendo deserve thanks for their help with

sample collection. Also, Eric Matsuda-Fugisaki provided some needed assistance in the

laboratory and is much appreciated. Likewise, appreciation is extended to all graduate

students in the Animal Sciences Department for support and camaraderie during this

program of study. Pam Gross deserves recognition for her unyielding willingness to help

others and her wonderful attitude.

United States Sugar Corporation and its employees deserve thanks and

recognition for their donations of liquid feeds. A note of appreciation goes to Dr. Jon

Nelson and Southeastern Minerals, Bainbridge, Georgia, for donation of mineral

supplements, to Flint River Mills for transportation of mineral supplements, to Alltech,

Nicholasville, Kentucky, for donation of Sel-Plex, and to Mr. Dane Bernis for

preparation of experimental diets.

The author wishes to acknowledge, though not necessarily in a positive manner,

Hurricanes Charley, Frances, Ivan, and Jeanne. In the worst hurricane season in many

decades, the storms caused chaos across the state of Florida by damaging property and

generally disrupting daily life.

Last, but certainly not least, the author wishes to thank Ms. Rachel Van Alstyne

for her assistance with laboratory analyses and for her unselfishness and dedication as a

friend and colleague. She has truly been the kind of friend that a friend would like to

have.













TABLE OF CONTENTS

ACKNOW LEDGM ENTS ............................................................................................ iv

ABSTRACT ..................................................................................................................... viii

CHAPTER

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

2 REVIEW OF LITERATURE ................................................................................... 3

Benefits of Selenium Supplem entation to Livestock ............................................... 3
M ethods of Selenium Supplementation to Livestock ............................................... 8
Absorption, Transport, Storage, and Excretion of Selenium ................................. 12
Differences in Efficacy of Selenium due to Source ............................................... 14
Selenium Toxicosis ................................................................................................. 17

3 TOLERANCE OF INORGANIC SELENIUM IN RANGE-TYPE EWES DURING
GESTATION AND LACTATION ........................................................................ 22

Introduction ................................................................................................................. 22
M aterials and M ethods ............................................................................................ 23
Results and Discussion ............................................................................................ 26
Implications ................................................................................................................. 39
Summary ..................................................................................................................... 40

4 EFFECTS OF SELENIUM LEVELS IN EWE DIETS ON SELENIUM IN MILK
AND PLASMA AND TISSUE SELENIUM CONCENTRATIONS OF LAMBS ... 48

Introduction ................................................................................................................. 48
M aterials and M ethods ............................................................................................ 49
R esu lts ......................................................................................................................... 5 1
Discussion ................................................................................................................... 56
Implications ................................................................................................................. 61
Summary ..................................................................................................................... 61








5 COMPARATIVE EFFECTS AND TOLERANCE OF VARIOUS DIETARY
LEVELS OF SE AS SODIUM SELENITE OR SE YEAST ON BLOOD, WOOL,
AND TISSUE SE CONCENTRATIONS OF WETHER SHEEP ......................... 67

Introduction ................................................................................................................. 67
M aterials and M ethods ............................................................................................ 68
Results and D iscussion ............................................................................................ 71
Im plications ................................................................................................................. 81
Sum m ary ..................................................................................................................... 82

6 EFFECTS OF FORM OF PARENTERAL OR DIETARY SELENIUM
SUPPLEMENTATION ON BODY WEIGHT AND BLOOD, LIVER, AND MILK
CON CEN TRA TION S IN BEEF COW S ............................................................... 88

Introduction ................................................................................................................. 88
M aterials and M ethods ............................................................................................ 89
Results ......................................................................................................................... 92
Discussion ................................................................................................................... 95
Implications ................................................................................................................. 99
Sum m ary ..................................................................................................................... 99

7 TISSUE AND BLOOD SELENIUM CONCENTRATIONS AND PERFORMANCE
OF BEEF CALVES FROM DAMS RECEIVING DIFFERENT FORMS OF
SELEN IUM SUPPLEM EN TATION ....................................................................... 106

Introduction ............................................................................................................... 106
M aterials and M ethods .............................................................................................. 107
Results ....................................................................................................................... 110
D iscussion ................................................................................................................. 113
Im plications ............................................................................................................... 118
Sum m ary ................................................................................................................... 118

8 SUM M ARY AN D CON CLU SION S ....................................................................... 125

LITERATURE CITED ................................................................................................... 133

BIOGRA PHICAL SKETCH .......................................................................................... 145














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
SELENIUM TOLERANCE IN SHEEP AND SELENIUM SUPPLEMENTATION

METHODS FOR BEEF CATTLE

By

Paul Armand Davis

December 2004

Chair: Lee Russell McDowell
Major Department: Animal Sciences
A series of experiments to evaluate and compare methods, sources, and dietary

levels of selenium was carried out utilizing sheep and cattle. Experiments using sheep

were conducted to gather further data on 1) the tolerance of dietary inorganic Se by ewes

during lamb production, 2) the effects of high levels of dietary Se fed to ewes on their

lambs, and 3) the tolerance of organic or inorganic Se by mature wethers. A cow-calf

herd was used to evaluate and compare effects of using different forms of dietary or

parenteral Se on weight change and blood, milk, and liver Se concentrations of beef cows

and their calves. In ewes fed Se, as sodium selenite, above requirements, Se

concentrations in blood, wool, and soft tissues generally increased (P < 0.05) as dietary

Se increased. Ewes tolerated up to 20 mg/kg dietary Se without suffering from toxicosis.

Lambs born to ewes receiving high levels of dietary Se had increased plasma Se (P <

0.05) as Se in ewe diets increased. No signs of Se toxicosis were observed in lambs

regardless of Se concentration in the ewe diets. Wethers, fed up to 40 mg/kg Se as








sodium selenite or Se yeast for 60 wk, had Se concentrations in serum, whole blood,

wool, and soft tissues which increased as dietary Se increased (P < 0.05). In general, Se

yeast vs selenite was more effective at increasing Se in blood, wool, and soft tissues (P <

0.05). Enzyme activity and histopathological evaluation of soft tissues from ewes and

wethers indicated no evidence of Se toxicosis. From the two sheep experiments,

maximum tolerance for both forms of dietary Se is greater than 40 mg/kg. Cows

receiving Se supplementation as Se yeast maintained adequate concentrations of Se in

plasma, whole blood, and liver and generally had higher (P < 0.05) concentrations than

cows receiving inorganic Se. Calves from cows receiving Se via free-choice minerals

had higher (P < 0.05) weight gains than from cows receiving injectable selenate. Calves

whose dams received Se yeast generally had higher Se (P < 0.05) in blood and liver.













CHAPTER 1
INTRODUCTION

Selenium (Se) has had a long and storied history in animal nutrition. Since its

discovery, at the bottom of a vat of sulfuric acid, by Jbns Jacob Berzelius, a Swedish

chemist, in 1817, Se has played the role of toxic element, essential nutrient, carcinogen,

and contributor in cancer prevention. However, it seems that selenium's greatest legacy

is one of a toxic agent to livestock. As early as 1295, Se was documented as detrimental

as Marco Polo described a poisonous plant which, when eaten by horses, caused their

hooves to drop off (Komroff, 1926). Likewise, a U.S. Army surgeon, stationed at Fort

Randall in 1856, described much the same conditions afflicting horses in the Nebraska

Territory (Madison, 1860). Selenium was identified as the principal toxic agent in

conditions described as "blind staggers" and "alkali disease" throughout Wyoming and

the Dakotas. In 1957, Se was shown to prevent liver necrosis in rats and afterward was

deemed an essential nutrient (Schwarz and Foltz, 1957). Though much of the world is

afflicted with Se deficiency, supplementation of Se using dietary or parenteral forms will

generally resolve the problem. Selenium toxicities require more effort but can be

successfully combated, by not overdosing livestock with supplemental Se, monitoring Se

content of feedstuffs, and by using certain animal management techniques.

With its many implications as a toxic element, the use of Se, as a supplement to

livestock, garners much caution from feed manufacturers, animal scientists, and

nutritionists. The current estimate of the maximum tolerable level for dietary Se in








domestic animals is 2 mg/kg (National Research Council [NRC], 1980). This estimate

does not consider differences inmetabolism of Se by different species and makes no

differentiation in the maximum tolerable level for the different chemical forms of Se,

such as Se yeast or sodium selenite. Previous research has shown that the absorption of

an oral dose of inorganic Se differs between ruminant and monogastric species (Wright

and Bell, 1966). Likewise, studies in cattle and swine have shown a marked difference in

the efficacy of organic vs inorganic Se to increase blood, milk, and tissue Se

concentrations (Pehrson et al., 1999; Kim and Mahan, 2001; Gunter et al., 2003).

Furthermore, some evidence exists to suggest that the maximum tolerable level of Se for

livestock is grossly underestimated and to discredit the notion that the range between

optimal and toxic levels of Se is narrow (Glenn et al., 1964a; Kim and Mahan, 2001;

Cristaldi et al., in press).

To further the body of knowledge in this subject area, a series of experiments were

carried out with sheep and cattle. Experiments using sheep were conducted to gather

further data on 1) the amount of dietary inorganic Se that can be tolerated by ewes during

lamb production, 2) the effects of Se supplementation to ewes on their lambs, and 3) the

amount of organic or inorganic Se that can be tolerated by mature wethers. A cow-calf

herd was utilized to evaluate and compare effects of using different forms of dietary or

injectable Se on body weight change and blood, milk, and liver Se concentrations of beef

cows and their calves.













CHAPTER 2
REVIEW OF LITERATURE

Benefits of Selenium Supplementation to Livestock

Selenium's role in animal nutrition was drastically changed when it was identified

as the third factor involved in preventing liver necrosis in rats (Schwarz and Foltz, 1957).

After this first evidence for the essentiality of Se, benefits for many other species were

discovered. Patterson et al. (1957) demonstrated that Se would prevent exudative

diathesis in chicks and Eggert et al. (1957) showed that hepatosis dietetica (liver necrosis)

could be prevented in swine by feeding Se. In calves and lambs, Se was successful in

preventing white muscle disease (WMD), a condition also known as nutritional

myodegeneration (Hogue, 1958; Muth et al., 1958).

Corah and Ives (1991) reported that insufficient Se could be linked to a variety of

disorders in beef cattle. Among the reproductive disorders observed were retained

placenta, infertility, abortions, births of premature, weak, or dead calves, cystic ovaries,

metritis, delayed conception, erratic estrus periods, and poor fertilization. In addition to

problems in reproduction, a condition known as "ill-thrift" has also been reported in

cattle (Corah and Ives, 1991; Underwood and Suttle, 1999) and also affects sheep. "Ill-

thrift" is defined as a syndrome that includes subclinical growth deficit, clinical

unthriftiness with rapid loss in weight, as well as some mortality. Selenium deficiency

has also been linked to cases of mastitis in dairy cattle that occurred more frequently and

lasted longer than mastitis in cattle with adequate Se intake (Smith et al., 1985).








Perhaps Se is best known for its role as an essential constituent of glutathione

peroxidase (Rotruck et al., 1973) and four Se-dependent glutathione peroxidases have

been identified and designated as glutathione peroxidase 1, 2, 3, and 4 (Lei et al., 1998).

These four enzymes benefit animal health by protecting cellular and subcellular

membranes against oxidative damage. Also, it appears that Se-dependent glutathione

peroxidases provide a second line of defense against peroxidation of vital phospholipids

(McDowell, 2003). Vitamin E provides the first line of defense against the peroxidation

of phospholipids in membranes.

Adequate dietary or supplemental Se is an effective way to combat the

aforementioned problems in growth and reproduction of livestock. Likewise, in the

presence of adequate Se, the glutathione peroxidase system works in synergy to protect

animal cells against lipid peroxidation. In general, livestock species have minimum

requirements of dietary Se which range from 0.05 to 0.30 mg/kg (McDowell, 2003).

There are numerous examples throughout the scientific literature that cite benefits in

growth, reproduction, and prevention of WMD and other anomalies due to adequate

dietary or supplementary Se.

The dietary Se requirement for all classes of sheep ranges from 0.10 to 0.20 mg/kg

(NRC, 1985). However, the minimum dietary Se level necessary to prevent WMD varies

as reported in the literature. Oldfield et al. (1963) reported that 0.06 mg/kg was the

minimum dietary Se level required to prevent WMD in lambs. However, researchers in

New Zealand indicated that lambs had normal growth and remained free of clinical signs

of Se deficiency when grazing pastures containing 0.03 to 0.04 mg/kg (Hartley and

Grant, 1961). Oldfield et al. (1963) further reported that ewes fed a ration containing








only 0.02 mg/kg Se gave birth to lambs with WMD, but by supplementing 0.10 mg/kg Se

in the ewe diet, WMD was prevented consistently. It is suggested that at least some of

the variation in the Se requirements necessary to prevent WMD is due to sparing or

interfering nutrients, such as Vitamin E or sulfur, and that differences also reflect Se

losses due to drying, as well as analytic error (McDowell, 2003). Maas et al. (1984)

suggested that even in cases of Se deficiency, lambs returned to a normal Se status with

one or two i.m. injections containing 1 mg Se and 68 IU of vitamin E. Selenium

supplementation to ewes at a level of 2.25 mg/d reduced both the incidence and severity

of WMD in white-faced lambs (Gardner and Hogue, 1967).

Selenium supplementation has been reported to have some effects on growth and

rate of gain in sheep and cattle. Spears et al. (1986) reported increased summer gains in

calves that received Se and vitamin E supplementation vs those calves receiving no Se

supplementation. Likewise, Perry et al. (1976) reported a 10% increase in ADG of steers

when feedlot diets were supplemented with 0.1 mg/kg Se. Furthermore, an 8% increase

in ADG of finishing beef cattle was again reported when 0.1 mg/kg Se was added to the

diet (Burroughs et al., 1963). Increases in BW gains of 20% were attained when Friesian

heifer calves were supplemented with Se at a rate of 3 mg/d (Wichtel et al., 1996). In

lambs, data from Oldfield et al. (1963) indicated that lambs with the lowest blood Se had

the lowest BW at six wk of age. However, reports of a positive response in growth or

BW gain are inconsistent in sheep and cattle. Ammerman et al. (1980) reported no

differences in weaning weights of calves nursing Se supplemented mothers vs calves

whose dams had received no supplemental Se. Hereford x Angus calves showed no

difference in ADG from birth to weaning due to supplemental Se (Castellan et al., 1999).








Likewise, ADG, feed consumption, and gain:feed were not affected by supplementation

of 0.1 mg/kg dietary Se fed for 13 wk or 0.2 mg/kg dietary Se fed for six wk in separate

studies, two using sheep and one using cattle (Ullrey et al., 1977).

Newborn and suckling calves and lambs can receive Se via their dams from either

maternal transfer or increased Se in milk. Recent studies indicate that blood Se in

newborn calves can be increased through Se supplementation of their dams

(Abdelrahman and Kincaid, 1995; Gunter et al., 2003; Valle et al., 2003). Likewise,

positive correlations between Se concentration in dam's milk and Se concentration of calf

whole blood have been observed in calves up to 70 d of age (Pehrson et al., 1999).

Evidence also exists that milk Se can be increased by level and duration of Se

supplementation in lactating cows (Conrad and Moxon, 1979). Like blood and tissue,

milk Se is affected by dietary Se level (Conrad and Moxon, 1979; Givens et al., 2004)

and Se readily crosses the placenta to the fetus (Van Saun et al., 1989). A strong

relationship of dietary Se to Se in milk of dairy cows was reported with up to 18.08% of

dietary Se being recovered in milk (Maus et al., 1980). Koller et al. (1984) supplemented

first-calf Hereford heifers with dietary Se and concluded that Se readily crosses the

placenta in beef cattle. Furthermore, those authors added that low Se concentrations in

the blood of dams could cause the fetus to gather more Se and result in fetal blood Se that

is higher than that of the mother. In sheep, Cuesta et al. (1995) showed increased

colostrum Se from ewes receiving supplemental Se and that milk Se was higher after one

mo of lactation. Also, Jacobsson et al. (1965) concluded that Se administered to ewes

could be transmitted to lambs through the placenta and the milk after a study using radio-

labeled sodium selenite and selenomethionine. In a study utilizing swine, Wuryastuti et








al. (1993) documented the importance of Se and vitamin E for maintaining immune

function in livestock. Those authors measured immune responses of blood, colostrum

and milk leukocytes of sows and concluded that greater phagocytic and microbicidal

activity could be realized in milk and colostrum through supplementation with Se and

vitamin E.

Reproductive problems in beef cattle such as retained placenta, infertility,

abortions, births of premature, weak, or dead calves, cystic ovaries, metritis, delayed

conception, erratic estrus periods, and poor fertilization may be successfully overcome

with Se supplementation (Corah and Ives, 1991). Awadeh et al. (1998a) concluded that

Se intakes of pregnant cows could be an important factor in weak calf disorders and that

passive immunity and heat production by newborn calves using brown adipose tissue

could both be influenced by maternal Se intakes. A Se deficiency in the diet of dairy

cattle was reported to be a contributor to a high incidence of retained placentas (Trinder

et al., 1973). Data from studies using dairy cows have shown that supplemental Se and

vitamin E to animals receiving Se deficient diets are beneficial in decreasing the

incidence of retained placentas (Julien et al., 1976; Hemken et al., 1978). Smith et al.

(1988) studied the effects of Se on disease resistance in dairy cattle and concluded that

many dairy herds have inadequate dietary intakes of Se and vitamin E. Those authors

added that insufficient intakes of these nutrients could result in increased cases of

mastitis, metritis, and retained placenta, and recommended Se supplementation at a level

to maintain blood Se at a minimum of 200 gg/L. Weiss et al. (1990) studied the

relationships between Se and mammary gland health in commercial dairy herds and








concluded that high serum Se concentrations were associated with reduced rates of

clinical mastitis and low somatic cell counts in the milk tank.

Reproductive problems such as increased services per conception or increased

calving interval which could, at least in part, be attributed to male fertility may also be

improved by Se supplementation. Heimann et al. (1981) showed that the pituitary gland

and reproductive tissues exhibited higher Se concentrations than many other body tissues.

Julien and Murray (1977) reported that percent motility in bovine spermatozoa increased

significantly as concentration of Se in sperm increased. Thus, supplemental Se may have

a positive effect on sperm quality and ultimately on male fertility. However, Segerson

and Johnson (1981) observed no differences in sperm number, viability, or Se content

from Se supplemented bulls compared to sperm from unsupplemented controls.

Methods of Selenium Supplementation to Livestock

The benefits of Se supplementation to livestock are many and Se deficiencies are

easily combated with adequate Se supplementation. Several methods of Se

supplementation exist and successful uses of all methods have been reported. The

method of Se supplementation chosen by livestock producers may be dependent on

factors such as Se content of soils, local grains and forages, species produced, class of

livestock and stage of production, facilities for animal handling, as well as knowledge,

previous experience, and personal preference.

Many areas of the United States have Se deficient soils (McDowell, 2003) and thus

produce grains and forages which are low in Se. Likewise, many regions of the world

have been mapped as Se deficient and may benefit from the administration of Se to

livestock (Oldfield, 2002). In a survey of blood Se status in beef cattle encompassing

more than 250 herds in 18 states in several regions of the U.S., more than 18% of cattle








were classified as marginally deficient (51 to 80 gg/L) or severely deficient (< 50 gg/L)

in blood Se (Dargatz and Ross, 1996). Percentages of cattle classified as deficient varied

with region of the country. Herds in the Central U.S. had the least occurrence of Se

deficiency, while the Southeast, including Florida, had the greatest incidence of Se

deficiency at more than 40%. Stowe and Herdt (1992) also suggest that many cattle in

the U.S. are in a state of Se deficiency.

Selenium supplementation to livestock is accomplished using three or four primary

methods. Addition of Se to livestock feeds and/or minerals, use of injectable Se

preparations (usually in combination with vitamin E), use of sustained release

intrareticular Se supplements, and possibly the use of seleniferous grains or forages

grown on high Se soils (Ammerman and Miller, 1975) are the methods most often used to

supplement Se. One additional option to increase Se intake of livestock is the use of Se

containing fertilizers on forage and pasture (Valle, 2001). The addition of Se to

feedstuffs was not an option until 1974 when the Food and Drug Administration (FDA)

allowed for supplementation of up to 0.1 mg/kg Se as selenite or selenate for swine and

poultry (Schmidt, 1974). An amendment to this FDA order allowed the use of

supplemental Se for sheep in 1978 and a subsequent amendment in 1979 allowed for use

in dairy and beef cattle. Currently, use of 0.3 mg/kg dietary Se is approved for

supplementation in poultry, swine, sheep, and cattle (McDowell, 2003).

Regardless of method chosen for Se supplementation, Se deficiencies are more

easily combated than are toxicities, which generally require more animal and/or pasture

management. In sheep and beef cattle production systems, producers most often choose

to use injectable Se products or supplement Se through free-choice mineral mixtures.








Judson et al. (1991) evaluated long-acting Se treatments for ewes and lambs in a 200 wk

experiment. Those authors reported that a 100 mg injection of barium selenate was more

effective at increasing blood Se of ewes and their lambs than was an intraruminal Se

pellet or no Se supplementation. Data show a near five-fold increase in blood Se from

lambs from injectable selenate treated ewes vs lambs from unsupplemented dams.

Norton and McCarthy (1986) evaluated injectable Se products for prevention of WMD in

lambs and reported increased plasma and milk Se in ewes that received the injectable Se

vs unsupplemented controls. Likewise, those authors showed increases in lamb plasma

Se due to the frequency of use of injectable Se. In a series of University of Florida

studies, the use of injectable Se, as sodium selenite and barium selenate, in a cow-calf

herd was evaluated and compared to inclusion of organic Se in free-choice mineral

mixtures (Valle et al., 2002; 2003). Those authors reported that, in general, injectable Se

as selenate and selenite affected plasma, liver, colostrum, and milk in a similar manner.

Though the injectable products did increase Se levels in blood, milk, and tissue compared

with blood, milk, and tissue Se concentrations from unsupplemented animals, both

injectable forms of Se were generally less effective than the addition of organic Se to

free-choice minerals. The calves born to and suckling cows that received injectable Se

had plasma Se concentrations which were similar to plasma Se concentrations of calves

from unsupplemented dams. Selenium supplementation via free-choice minerals proved

more effective at raising and maintaining Se status of Florida beef cows and their calves.

Gunter et al. (2003) compared effects of Se supplementation as sodium selenite or

Se yeast added to free-choice minerals on performance and Se status of beef cows and

calves in Arkansas. Mineral mixtures were formulated to contain 26 mg/kg Se and were








offered free-choice. No differences in performance between unsupplemented controls or

cattle receiving either form of Se were observed. However, differences in blood Se of

supplemented vs unsupplemented cattle were reported. Likewise, Se yeast treated cows

and their calves had higher blood Se than cows and calves receiving selenite Se. Those

authors concluded that calves are at risk for Se deficiency if their dams are not

supplemented with Se and that even when selenite Se is provided, calves may still be at

risk. Sheep may also be supplemented with Se which is included in mineral mixtures and

salt licks. Norwegian researchers reported no incidences of WMD in lambs and

increased Se in blood and colostrum when Se fortified mineral mixtures and salt licks

were offered to ewes and lambs (Overnes et al., 1985).

In addition to the use of injectable Se or the inclusion of Se in free-choice mineral

mixtures, livestock producers may use an intraruminal or intrareticular bolus or pellet

which provides a sustained release of Se. Judson et al. (1991) reported that the use of an

intraruminal Se pellet and steel grinder increased blood Se of ewes and lambs compared

with controls. However, the Se pellet and grinder system was not as effective as an

injection of barium selenate at increasing blood Se. Campbell et al. (1990) used

crossbred beef cows to evaluate the safety and efficacy of Se boluses and Se pellets.

Both methods of Se supplementation were shown to be both safe and effective; however,

blood Se of cows receiving either method of Se supplementation increased until d 119 of

the study and was decreased by d 220. As in previous studies, both methods produced

blood Se higher than the blood Se from unsupplemented controls. Abdelrahman and

Kincaid (1995) evaluated the effects of administration of an intraruminal Se bolus on

colostrum, plasma, and whole blood Se concentration of dairy cows. Those authors








reported that the Se bolus was an effective method of increasing Se in colostrum, plasma,

and whole blood. Likewise, calves born to Se supplemented cows had higher Se

concentrations in plasma, whole blood, and liver than calves born to cows receiving no

supplemental Se. In this study, the administration of a sustained release Se bolus to cows

proved to be an effective method of Se supplementation to newborn calves.

The need for Se supplementation to livestock is great as evidenced by the many

benefits of supplemental Se on animal health and performance. This need is further

elucidated by surveys such as reported by Dargatz and Ross (1996), which reported a

relatively high percentage of beef cattle in the U.S. that were classified as Se deficient.

Selenium supplementation generally adds only a negligible amount to the cost of

livestock production and producers have several effective means of Se supplementation

to choose from.

Absorption, Transport, Storage, and Excretion of Selenium

Ruminant animals differ from monogastric animals in their ability to absorb and/or

retain Se. Wright and Bell (1966) reported retention of a dose of sodium selenite to be

29% for sheep and 77% for swine. In both sheep and swine, Se absorption occurred in

the small intestine and cecum with some additional absorption in the colon for swine. No

absorption of Se occurred in the rumen of sheep or the stomach of swine (Wright and

Bell, 1966). These authors also reported net absorption of Se to be 36% for sheep and

86% for swine. Less absorption of Se in ruminants seems to be due to the reduction of

inorganic Se to insoluble forms by rumen microorganisms (Butler and Peterson, 1961;

Peterson and Spedding, 1963; Hidiroglou et al., 1968). Inorganic Se is more readily

reduced within the rumen than organic forms of Se such as Se yeast. Diet also affected








Se absorption as sheep on a high concentrate diet had higher plasma Se than sheep

receiving a high forage diet. (Koenig et al., 1997).

In contrast to many other minerals consumed by livestock, which use homeostasis

as a primary status regulator, Se status of animals seems to have little effect on intestinal

absorption. In a study utilizing rats, urinary excretion was shown as the only relevant

means of Se homeostasis (Windisch and Kirchgessner, 2000) as urinary excretion is

directly related to dietary level while fecal Se excretion is quite static (Burk et al., 1972).

When Se absorption was regressed on Se intake of dairy cows, a strongly linear

relationship was observed (Harrison and Conrad, 1984). However, Se intakes reported in

that study were relatively low and ranged from 0.437 to 3.136 mg/d. Most dairy cows

consume closer to 6 mg Se/d, based on supplementation in the diet of 0.3 mg/kg Se.

Absorbed Se is associated with plasma protein and transported in the blood plasma

until it enters tissues (McDowell, 2003). Selenoprotein P is the plasma protein with

which most Se is associated in individuals with adequate or deficient dietary Se, while

most plasma Se is associated with albumin when Se intake is in excess (Xia et al., 2000).

In addition to plasma, Se is also found in muscle and glandular tissues.

Generally, when ranked on a Se concentration basis, tissues follow the general order of

kidney > liver > heart > skeletal muscle, regardless of species, when Se is fed at an

adequate or deficient level (Comb and Combs, 1986). The kidney may be the highest in

Se concentration as it is primary organ of excretion. However, when Se is fed at levels

above requirement, liver surpasses kidney in terms of Se concentration (Cristaldi et al., in

press).








Urine, feces, and exhalation are the primary excretion routes of Se. Amount and

distribution of excreted Se within these routes are affected by chemical form of Se, total

Se intake, and diet composition including antagonists (McDowell, 2003). Urine is the

major excretory pathway and Se excretion via urine increases with Se status of the

animal. Fecal excretion remains nearly constant and exhalation of Se becomes a major

route only when Se concentrations are at a toxic level (McDowell, 2003). The amount of

Se exhaled increases as dietary Se increases (McConnell and Roth, 1966) and one

characteristic of animals which excrete Se via respiration is breath with a garlicky odor.

Selenium excretion in ruminant animals is dependent on method of administration.

When Se is provided orally, ruminants excrete more Se in feces. However, when Se is

given parenterally, more Se is excreted in urine (Wright and Bell, 1966). This is

supported by the concept that rumen microorganisms reduce dietary Se to insoluble forms

(Butler and Peterson, 1961; Peterson and Spedding, 1963; Hidiroglou et al., 1968) and

thus increase fecal excretion of unabsorbed Se.

Differences in Efficacy of Selenium due to Source

The efficacy of Se to increase blood and tissue Se concentrations in animals varies

with source of Se. In general, Se is deposited in tissues and blood Se is more increased

when supplemental Se is of the organic form (McDowell, 2003). The primary sources of

inorganic Se are sodium selenite and sodium or barium selenate, while Se yeast and

seleniferous grains and plants are the primary sources of organic Se. Sodium selenite and

selenate are often added to free-choice mineral mixtures for livestock. Likewise, those

two chemical forms are used in injectable Se products. Selenomethionine is the major Se

compound found in grains used for livestock feeds and in Se yeast. Se-

methylselenocystine is the Se compound found most abundantly in seleniferous plants,








while some inorganic Se is found in grains and plants (Whanger, 2002). In animal tissues,

selenate is the major inorganic form and selenocystine is the predominant organic form.

Selenomethionine is found initially when this amino acid is fed; however seleno-

methionine is converted to selenocystine after some time (Whanger, 2002). With such

differentiation in the sources of Se within plant and animal tissues, it seems reasonable

that differences in efficacy due to form of Se administered would exist and there are

numerous examples in the scientific literature to support this concept.

Goehring et al. (1984a) evaluated the effects of high dietary levels of Se from

selenite or seleniferous grains on blood and tissue concentrations in swine. Those authors

documented that Se from seleniferous grains increased Se in blood and tissue compared

to selenite Se fed at the same level. Awadeh et al. (1998a) reported increased blood Se in

crossbred beef cows consuming free-choice minerals containing 60 mg/kg Se as Se yeast

compared to 60 mg/kg selenite Se. Furthermore, cows receiving free-choice minerals

containing 60 mg/kg Se as Se yeast had a lower percentage protein in albumin compared

to cows receiving minerals with 60 mg/kg selenite Se. Selenium from Se yeast has been

documented by several groups of researchers as more effective than Se selenite or

selenate at increasing blood and liver Se levels in beef cows (Pehrson et al., 1999; Valle

et al., 2002; Gunter et al., 2003) and in dairy cattle (Ortman and Pehrson, 1999).

As with blood and tissue Se, milk Se has been more effectively increased by using

organic Se vs inorganic Se in beef cattle, dairy cattle, and swine. Selenium yeast

produced milk Se more than 100% higher than selenite or selenate Se when 3 mg of Se

from each source were fed to Swedish dairy cows (Ortman and Pehrson, 1999). Hereford

cows supplemented with Se yeast produced milk with markedly higher Se concentrations








than did cows receiving supplemental selenite Se in early and late lactation (Pehrson et

al., 1999). In a two-yr study utilizing Florida beef cows, milk Se was consistently higher

from cows receiving free-choice minerals with Se yeast compared to cows receiving Se

as selenite or selenate injections (Valle et al., 2002). Also, calves suckling the cows

which received the organic Se had higher Se concentrations in plasma and liver (Valle et

al., 2003). Researchers at Ohio State University fed Se as Se yeast or sodium selenite at

dietary levels of 0.15 and 0.30 mg/kg to gestating and lactating sows. Colostrum and

subsequent milk Se concentrations were consistently at least two-fold higher from sows

receiving organic Se than from sows receiving selenite Se (Mahan, 2000). Data from

New Zealand indicated that the transfer of Se into cows' milk was markedly more

efficient, up to three-fold more, with selenized yeast than with sodium selenate (Knowles

et al., 1999).

The effectiveness of different sources of Se for supplementation continues to be

evaluated even though Se has been recognized as nutritionally essential since the late

1950s. Selenium provided by different supplementation methods and from different

sources leads to different physiological responses in the animals that serve mankind.

With evidence of an increasing ability to manipulate the Se content of milk and animal

tissues which are commonly consumed by humans, it seems to be possible to supplement

Se to humans through method and source of Se supplementation to livestock. Givens et

al. (2004) reported a decline in Se intake by the people of Great Britain. After

conducting an experiment which validated previous findings that the milk of dairy cows

could be increased by feeding an organic Se source, those authors further explored the

idea of increasing human consumption of Se by altering the Se content of foods. It seems








that a worthy challenge exists for animal scientists and food scientists to work

collaboratively to identify effective programs for administration of Se to dairy and food

animals so that the milk and meat subsequently produced can be more nutritious for

humankind.

Selenium Toxicosis

Selenium deficiencies are prevalent in many parts of the world (McDowell, 2003)

and the benefits of Se supplementation continue to be elucidated. However, it seems that

Se is still most often implicated as an element which is toxic to livestock. This belief

most likely stems from diary-style documentation, observations, and research findings

beginning as early as 1295 when Marco Polo described an agent in plants which when

eaten by horses caused their hooves to fall off (Komroff, 1926). Six hundred years later

similar afflictions began to be described in the Great Plains region of the United States.

In 1856, a U.S. Army surgeon reported the occurrence of a disease, fatal to U.S. Cavalry

horses, similar to the affliction described by Marco Polo (Madison, 1860). The horses in

the Nebraska Territory near Fort Randall lost hair and had debilitating conditions of the

hoof. By the 1890s, farmers and stockmen who settled in northern Nebraska and South

Dakota observed similar conditions in livestock (Moxon and Rhian, 1943). Selenium

toxicity in livestock and laboratory animals has been reported from the 1930s to the

present day. Some reports were observations of animals receiving seleniferous grains or

grazing seleniferous plants (Franke, 1934; Franke and Potter, 1935; Moxon, 1937). Other

researchers have intentionally induced or attempted to induce Se toxicities, while several

reports of Se toxicity are a result of accidental overdosing with injectable Se.

Rosenfeld and Beath (1964) suggested that Se poisoning in livestock occurs in

three distinct phases: acute toxicity and the two phases of chronic toxicity, alkali disease








and blind staggers. Acute Se toxicity can be caused by ingesting a large amount of

supplemental Se, overdosing with parenteral Se, or by ingesting a large amount of

seleniferous plants. Certain plants, mostly species of Astragalus, may contain up to

10,000 mg/kg Se and cereal crops, grasses, and other forages may contain up to 50 mg/kg

Se (Aitken, 2001). Fatalities of sheep, cattle, and hogs have been reported in regions

known to grow seleniferous plants (National Academy of Sciences [NAS], 1983), with

some deaths occurring within 24 h (Rosenfeld and Beath, 1964). Clinical signs of acute

Se toxicity may include elevated body temperature, labored breathing, diarrhea, and often

death. Alkali disease and blind staggers types of Se toxicosis occur with more time and

involve feedstuffs containing less Se. Clinical signs of chronic Se toxicity include

anorexia, apathy, diarrhea, weight loss, hair loss, and hoof malformations (Glenn et al.,

1964a). Animals in the blind staggers phase of Se toxicosis may wander, stumble, and

lack appetite initially and then become somewhat paralyzed and almost blind in the later

stage. The later stage appears suddenly and death usually occurs within hours (Rosenfeld

and Beath, 1964). Chronic forms of selenosis have been induced by feeding grains

containing 5 to 40 mg/kg Se (Schoening, 1936; Rosenfeld and Beath, 1964).

Glenn et al. (1964a) induced death in ewes after oral dosing of up to 50 mg/d of Se

as sodium selenate for 93 d and concluded that minimum toxic oral dose of Se as selenate

depended on susceptibility, level of Se administered, and duration of administration.

Those authors later reported liver Se concentrations of up to 29 mg/kg in experimentally

poisoned ewes (Glenn et al., 1964c). Evaluation of the tissues of the ewes in the previous

study showed that most tissue damage in Se toxicosis is confined to the heart (Glenn et

al., 1964b). No kidney damage and few instances of liver damage were reported.








Blodgett and Bevill (1987) induced death in sheep by feeding 0.7 to 1.0 mg Se/kg BW as

selenite in as little as 6.75 h. Liver Se concentrations of more than 17 mg/kg and whole

blood Se of 2.7 mg/L were reported. After receiving an oral 5 mg selenite Se/kg BW,

lambs died within 6 h and when the same dosage was given as an injection, lambs lived

up to 60 h (Smyth et al., 1990). After evaluating the major organs, those authors

concluded that the heart is most damaged in a case of Se toxicosis as the heart has great

affinity for Se especially in lethal doses. Caravaggi et al., (1970) injected Merino lambs

with 0.425 to 0.500 mg Se/kg BW, induced death, and determined the LD50 for lambs to

be 0.455 mg Se/kg BW. Twenty lambs received 10 mg of selenite Se orally in an attempt

to prevent WMD. Of those 20, seven died within 16 h, eight developed diarrhea but

recovered, and five lambs were apparently unaffected (Marrow, 1968). Cristaldi et al.

(2004) fed up to 10 mg/kg dietary Se as selenite to growing wether sheep and reported no

signs of Se toxicity. Those authors reported whole blood Se concentrations of up to 1.2

mg/L, wool Se of 2.5 mg/kg, and liver Se concentrations of nearly 15 mg/kg Se on a dry

basis and no evidence of Se toxicity from histopathological evaluation.

Selenium toxicity studies have also been conducted using swine. Goehring et al.

(1984b) fed young pigs up to 20 mg/kg Se as sodium selenite for 5 wk. No pigs on the

study died; however, feed intake and growth rate decreased as dietary Se concentration

increased. Whole blood Se concentrations of up to 3.5 mg/L and hair Se of more than 11

mg/kg were observed. Organic and inorganic Se was included in the diet of growing pigs

at levels of up to 20 mg/kg for 12 wk (Kim and Mahan, 2001). Feed intakes of those pigs

declined as Se level increased and daily gains were decreased when Se was fed at more

than 5 mg/kg, and inorganic Se had a more detrimental effect on performance than did








organic Se. Those authors reported plasma Se concentrations of more than 3.3 mg/L,

liver Se of more than 17 mg/kg, and hoof Se of more than 28 mg/kg and no deaths

regardless of dietary Se level. The authors concluded that the higher retention of organic

Se in tissues and blood cells may effectively reduce the amount of Se available to cause

Se toxicosis.

Tolerance of Se as selenite or selenomethionine was evaluated using yearling steers

in a 4 mo study (O'Toole and Raisbeck, 1995). Those authors observed the highest

incidence of hoof lesions in steers fed organic Se at a rate of 0.80 mg/kg BW. Likewise,

it was shown that the steer with the most severe hoof lesions also had the highest Se

concentrations in hair, liver and kidney. The findings of that study indicated that dietary

exposure of 0.8 mg Se/kg BW, in either form, for 4 mo produces subclinical to clinical

signs of Se toxicosis. The authors concluded that selenomethionine is more likely to

cause alkali disease than sodium selenite. Holstein cows were fed inorganic Se up to 100

mg/d and whole blood and liver Se concentrations of up to 4.9 mg/L and 15 mg/kg DM,

respectively, were reported (Ellis et al., 1997). It was concluded that dairy cattle could

tolerate Se intakes of up to 100 mg/d for several wk without suffering adverse affects.

It seems logical, based on previous findings, that the minimum lethal dosages and

maximum tolerable levels of Se are variable and may be affected by various factors such

as Se source (organic or inorganic), diet composition, method of Se supplementation, and

Se status of the animal. The current maximum tolerable level for dietary Se in domestic

animals is 2 mg/kg (NRC, 1980). This estimate does not consider differences in species,

source of Se, or duration of exposure. Early reports of toxicities are likely reasons for the

conservative estimate of maximum tolerable level and the notion that the range between





21

optimal and toxic level of Se is narrow. Surely, further studies which are long in duration

and use high dietary levels and different sources of Se are necessary to better estimate the

tolerance of Se for dairy and food animals.












CHAPTER 3
TOLERANCE OF INORGANIC SELENIUM IN RANGE-TYPE EWES DURING
GESTATION AND LACTATION

Introduction

Since its discovery by Berzelius in 1817, Se has had a rich and colorful history in

animal agriculture. Though much of the world is troubled with Se deficiencies

(McDowell, 2003), Se toxicities present a greater problem to control. In 1957, Se was

established as an essential nutrient and the benefits of Se supplementation to livestock

continue to be elucidated. Current estimates put the maximum tolerable level of Se at 2

mg/kg for the major livestock species (NRC, 1980) and no differentiation exists for

tolerable levels between ruminants and monogastric animals. However, the work of

Butler and Peterson (1961) and Hidiroglou et al. (1968) suggests that inorganic Se (e.g.,

sodium selenite) may be reduced to insoluble selenide by microorganisms in the rumen,

thus reducing overall absorption of Se by ruminant animals. Wright and Bell (1966)

reported that swine retained 77% of an oral dose of inorganic Se, which is nearly three-

fold the retention by sheep. Selenium toxicities have been often produced by researchers

in ruminants, but they are generally induced by Se injections (Marrow, 1968; Caravaggi

et al., 1970; Shortridge et al., 1971) or by feeding Se above maximum tolerable levels (5

to 196 ppm) to monogastric animals (Franke and Potter, 1935; Miller and Schoening,

1938; Kim and Mahan, 2001). More recently, Cristaldi et al. (2004) demonstrated that

wether sheep did not display signs of Se toxicosis after receiving up to 10 mg/kg dietary

Se for one yr. Based on these and other previous findings, it seems that the current

maximum tolerable level of Se for ruminants is underestimated. Most Se toxicity








research in ruminants has been documented in lambs or wethers. Controlled experiments

using ewes during stresses of production (e.g., gestation and lactation) are lacking in the

scientific literature. The objective of this long-term (72 wk) study were to evaluate and

compare effects of feeding Se as sodium selenite at supranutritional levels on ewe serum,

blood, wool, and tissue Se concentrations during two lambing periods and to determine

maximum tolerable level of Se.

Materials and Methods

All animal procedures were conducted within the guidelines of and approved by

the University of Florida Institutional Animal Care and Use Committee. This experiment

utilizing ewes during two lambings was conducted from December 18, 2001 to May 5,

2003 at the University of Florida Sheep Nutrition Unit located in North Central Florida.

Forty-one, four-yr-old, Rambouillet ewes, that originated from a single range flock in

Texas and had been pasture exposed to rams during October and early November 2001

(average 57 d gestation), were weighed (57.4 5.7 kg) and administered 2-ml ivermectin

dewormer s.c. (Ivomec; Merial Ltd., Iselin, NJ). Ewes were randomly assigned to one of

six dietary treatments for a 72-wk study. Six dietary treatments were 0.2, 4, 8, 12, 16, or

20 mg/kg Se as sodium selenite (as-fed basis) added to a corn-soybean meal basal diet

(Table 3-1). The basal diet was formulated to meet animal requirements for protein,

energy as TDN, vitamins, and minerals for this class of sheep (NRC, 1985). Animal

numbers per treatment were six for 0.2 (control) and seven each for 4, 8, 12, 16, and 20

mg/kg added Se treatments. Ewes were housed by treatment group in covered wooden

pens (53.5 m2) with earth floors and ad libitum water.








Diets were fed at 909 g-ewe'ld' from d 0 until lambing began, increased to 1000

g'ewe-l'dl during lambing, and again increased to 1135 g-ewe-'-d-1 during lactation. Ewes

received 909 g'ewe-l*d of their respective diets after the first lamb crop was weaned. On

August 15, 2002, ewes were pen exposed to rams for 35 d. Diets were offered at the

same increments during the second lambing and lactation as during the first. Diets were

sampled every 28 d, ground (1 mm), and frozen at 0C until analysis.

Ewe BW was recorded on d 0 and for every four wk thereafter, for the remainder

of the study. A 10-mL blood sample for serum analysis was collected using an 18-gauge

needle into a vacutainer tube with no additive (Vacutainer; Becton Dickinson, Franklin

Lakes, NJ) every four wk, via jugular venipuncture, allowed to stand for 20 min,

centrifuged at 700 x g for 25 min, and serum stored frozen at 0C until Se analysis.

Starting at wk 12, an additional 10-mL blood sample was collected into a heparinized

vacutainer tube (Vacutainer; Becton Dickinson, Franklin Lakes, NJ). This additional 10-

mL sample was collected every 12 wk for the remainder of the experiment and stored

frozen at 0C as whole blood until analysis.

The wool around the jugular was shorn initially and regrowth was collected

beginning at wk 12 and every 12 wk thereafter. The collected wool was washed with a

commercial hair shampoo (Alberto VO5; Alberto-Culver Co., Melrose Park, IL), to

remove oil and dirt, rinsed well with deionized water, dried, stored at room temperature,

and later analyzed for Se concentration.

At the termination of the experiment (wk 72), ewes were slaughtered following

approved USDA procedures at the University of Florida Meats Laboratory. An

additional 1 0-mL sample of blood was collected using an 18-gauge needle into a








vacutainer, centrifuged at 700 x g for 25 min, and serum frozen at 0C for analysis of

albumin and the following enzymes: alkaline phosphatase (Alk Phos), alanine

transaminase (ALT), aspartate transaminase (AST), creatinine phosphokinase (CK), and

gamma glutamyl transferase (GGT). Albumin and the enzymes were analyzed in order to

determine possible tissue breakdown as a result of Se toxicosis.

Samples of brain, diaphragm, heart, hoof tip, kidney, liver, and psoas major

muscle were collected, and frozen (00) until analyzed for Se. Sections (1 cm3) of liver,

heart, kidney, diaphragm, and psoas major muscle from all animals were placed in 10%

neutral-buffered formaldehyde for subsequent microscopic evaluation for evidence of Se

toxicosis.

For histopathological evaluation, the tissue samples fixed in buffered formalin

were embedded in paraffin and sectioned at 6 microns. All sections were stained with

hematoxylin and eosin, and examined under a light microscope (lOX, 20X, and 40X).

Serum albumin, Alk Phos, ALT, AST, CK, GGT were evaluated on a Hitachi 911

analyzer with reagents from Sigma (Sigma Chemical Co., St. Louis, Mo.). These

procedures were established by the Veterinary Medical Teaching Hospital at the

University of Florida.

Serum, whole blood, wool, tissue, and feed samples were analyzed for Se

concentration using a fluorometric method described by Whetter and Ullrey (1978). To

help ensure reliability of the analytical method, a certified standard (National Bureau of

Standards Bovine Liver SRM-1577a; U.S. Department of Commerce, National Institute

of Standards and Technology, Gaithersburg, MD) was frequently analyzed.








Brain, diaphragm, heart, hoof tip, kidney, liver, and psoas major muscle Se data

were analyzed for effects of treatment using PROC GLM in SAS (SAS for Windows 8e;

SAS Inst., Inc., Cary, NC) in a completely randomized design. Pre-planned orthogonal

contrast statements were used to compare means as described by Littell et al. (1998;

2000). PROC MIXED of SAS was used to analyze effects of treatment, time, and the

interaction of treatment x time on BW, serum Se, whole blood Se, and wool Se as

repeated measures with a spatial power covariance structure with respect to d and a

subplot of animal nested within treatment. Pre-planned orthogonal contrast statements

were written to determine differences in means at different sampling intervals. Means

were separated at P < 0.05 and regression analysis was used to determine relationships

between dietary Se and Se concentration of various tissues.

Results and Discussion

Performance

Ewe BW was not affected by dietary Se level (P = 0.69) or dietary Se level x time

interaction (P = 0.56). However, time did affect BW (P < 0.001). Initial BW was 57.4

5.7 kg and BW at the termination of the experiment was 61.2 15.1 kg. These findings

agree with previous studies in ruminants. Supplemental selenium fed up to 0.4 mg/kg

which is above requirement but below maximum tolerable level had no effect on rate of

gain in feedlot steers (Perry et al., 1976) and BW gains in wether sheep, fed sodium

selenite up to 10 mg/kg, was unaffected by dietary Se level (Cristaldi et al., in press).

Glenn et al. (1964a) also reported no effect of dietary Se on BW when sodium selenate

was fed to ewes as a single oral dose of up to 50 mg/d. The ewes utilized by those

authors were very similar in breed type and BW to the animals used in the present study.








Effect of time on ewe BW can be explained by changes in BW associated with gestation

and lactation over two lambings during the study.

Ten of 41 ewes died over the course of this 72-wk study. Gross necropsies were

performed on eight ewes following death. Tissues from two ewes were too severely

decomposed to allow for evaluation for pathological changes. Necropsy of eight ewes

cited causes of death as lymphadenitis associated with injury (two ewes), endoparasitism

(two ewes), ketosis (three ewes) and pneumonia (one ewe). Pathological evidence of Se

toxicosis was not found in any ewe that died before the termination of the experiment.

In the first yr, 53 lambs were born over 20 d from March 9, 2002 to March 28,

2002. Fifty-two lambs were born alive and unassisted (Table 3-2). One lamb was very

large (8 kg) and died shortly after a difficult birth. The lambs born in yr one represent a

129% lamb crop when calculated as lambs born alive per ewe exposed. In the second yr,

36 lambs were born over 34 d from January 17, 2003 to February 20, 2003 (Table 3-2).

All lambs were born alive and unassisted. Thirty-six lambs in yr two represent a 109%

lamb crop as only 33 ewes were exposed in the second yr. Number of lambs born per

ewe did not affect serum Se concentration (P > 0.54) of ewes receiving any level of

dietary Se. Glenn et al. (1964a), who fed higher levels of dietary Se than in the present

experiment, did not observe an effect of dietary Se level on reproduction in 2-yr-old

range ewes. Those researchers observed a similar number of pregnancies in each

treatment group and no malformations in lambs. In contrast, Rosenfeld and Beath (1947)

observed lamb deformities in a field study and attributed the anomalies to excess Se in

ewe diets. However, seleniferous plants were the Se source, rather than inorganic sources

used in the present experiment. Furthermore, in a grazing situation, it is possible that








lamb deformities were due to toxic elements other than Se. In both yr of our study, all

lambs were born free of congenital deformities, but the number of pregnancies were

lowest in ewes receiving 16 mg/kg dietary Se, but not 20 mg/kg. However, breeding

soundness evaluations were not performed on ewes or rams used in this study and thus, to

incriminate or exclude dietary Se level as a detriment to ewe reproduction would be

observational.

Blood

Serum Se concentrations from wk 4, 8, and 12 were analyzed together and will be

referred to throughout the results and discussion as late gestation yr 1. Lactation yr 1

includes serum Se concentrations from wk 12, 16, 20, and 24. Week 12 is included in

both late gestation and lactation for yr 1 as some ewes were lactating and some remained

in late gestation when wk 12 sampling occurred. Weeks 28, 32, 36, 40, and 48 compose

the dry, rebreeding period. Late gestation in yr 2 includes serum Se measurements from

wk 52, 56, and 60. Lactation in yr 2 includes wk 60, 64, 68, and 72. Similar to yr 1, one

sampling date (wk 60) was common to both late gestation and lactation and was included

in both periods.

During all stages of lamb production, serum Se increased in a linear fashion (P <

0.001) as dietary Se level increased (Table 3-3). This agrees with previous Se toxicity

research as Se concentrations in serum of wether sheep (Cristaldi et al., in press) also

increased linearly as dietary selenite Se was increased. All ewes had similar (P > 0.82)

serum Se at the initiation of this experiment. Initial serum Se ranged from 90 to 120

[ig/L, which is below the normal range (120 to 180 gg/L) for adult sheep (Aitken, 2001).

A cubic response within treatment (P = 0.02) was observed in serum Se across the stages

of production (time) from wk four to wk 72. Ewe serum Se, in general, was higher








during the dry, rebreeding stage. One plausible explanation for this is the lack of

placenta, fetal tissue, and milk for deposition and excretion of Se. During late gestation

in yr 1, dietary Se level affected serum Se concentration (P < 0.001), ewes receiving 8,

12, 16, and 20 mg/kg Se all had higher (P < 0.05) serum Se than did controls. Likewise,

ewes receiving 16 or 20 mg/kg Se had serum Se higher (P < 0.05) than ewes receiving 4

mg/kg Se. During lactation in yr 1, ewes receiving 16 and 20 mg/kg Se were similar (P =

0.32) and both groups were higher (P < 0.05) than controls and ewes receiving 4 and 8

mg/kg Se in serum Se concentration. During the 20 wk that ewes were not lactating and

were either open or rebreeding, ewes receiving 16 and 20 mg/kg dietary Se had similar (P

= 0.44) serum Se which was higher (P < 0.05) than from all other treatments. Ewes

receiving the intermediate levels of Se (8 and 12 mg/kg) had similar serum Se (P = 0.31)

which was higher (P < 0.05) than from controls and ewes receiving 4 mg/kg Se. Ewes in

late gestation during yr 2 generally produced numerically higher serum Se than in late

gestation the previous yr. Ewes receiving 20 mg/kg Se had serum Se which was similar

(P = 0.69) only to serum Se from ewes receiving 16 mg/kg Se and higher (P < 0.05) than

all other treatments. Ewes receiving 16 mg/kg Se produced serum Se which tended (P =

0.07) to be higher that serum Se from ewes receiving 12 mg/kg Se and was higher (P <

0.05) than serum Se from controls and ewes receiving 4 or 8 mg/kg Se. During lactation

in yr 2, ewes receiving 20 mg/kg Se had higher (P < 0.05) serum Se than serum Se from

all other treatments. Serum Se from ewes receiving 8, 12, or 16 mg/kg Se was similar (P

> 0.20) and only serum Se from ewes receiving 4 mg/kg dietary Se was similar (P = 0.21)

to controls. Throughout the experiment, serum Se concentrations in these ewes remained

below 1500 R~g/L, which is described as a toxic level in horses (Aitken, 2001) and were at








most 37% of a reported toxic level (3700 [tg/L) in swine (Aitken, 2001). Caravaggi et al.

(1970) established an LD50 for sheep at 455 Rg/kg BW. When our data are described on a

gg/kg BW basis using the highest dietary concentration (20 mg/kg), highest daily intake

(1135 g/d), and average ewe BW (60 kg), our ewes were consuming, at maximum, 378

gg/kg BW. This is 17% less than the LD50 for sheep as previously described. The ewes

in the present study were mature and maintained healthy ruminal function throughout the

study. This is contrasted with the unweaned lambs used by Caravaggi et al. (1970).

Those lambs may have received Se via i.m. injection. Administration of Se parenterally

disallows the reduction of selenite Se to insoluble selenide via ruminal microorganisms as

described by (Whanger et al., 1968). This would suggest that the LD50 for sheep could be

considerably higher than previously thought. Glenn et al. (1964a) fed sodium selenate at

high levels to range ewes that were similar in BW to ewes on the present study. Those

researchers did not induce death by Se toxicosis with daily oral doses less than 25 mg

Se/ewe. Of the 17 deaths reported in their experiment, only one was induced with a daily

dose of 25 mg Se/ewe. Eight deaths were induced with a daily dose 37.5 mg Se/ewe and

eight deaths were induced with a daily dose 50 mg Se/ewe. Those deaths were not by

acute Se toxicosis. The ewes received experimental Se doses for at least 80 d before

death by Se toxicosis was induced. In the same experiment, Glenn et al. (1964a) further

suggested an average minimum toxic level of Se for adult sheep to be 0.825 mg/kg BW

when fed for 100 d. Using this estimate, the minimum toxic level of Se for ewes of the

size used in our study would be 50.3 mg/d. Selenium consumption, at the highest dietary

level of 20 mg/kg, never reached even 50% of that previously reported level throughout

our study. Also, Blodgett and Bevill (1987) reported an LD50 for sheep, using sodium








selenite via i.m. injection, at 0.7 mg/kg BW. Other researchers (Rosenfeld and Beath,

1946) reported death in sheep with less Se (30 mg/d); however, the Se maximum intake

level used in our study was approximately 25% less. It is important to note that we used

sodium selenite as our Se source whereas previous research (Rosenfeld and Beath, 1946;

Caravaggi et al., 1970) used sodium selenate as the source of additional Se. Henry et al.

(1988) reported a higher relative bioavailability for selenate than selenite. This suggests

the possibility of a higher tolerance for sodium selenite vs selenate.

Whole blood Se was measured at wk 12, 24, 36, 48, 60, and 72 (Table 3-4).

Dietary Se level, time, and dietary Se level x time affected (P < 0.05) ewe whole blood

Se. Whole blood Se increased linearly (P < 0.001) as dietary Se increased. Response of

whole blood Se from all treatments over time was cubic (P < 0.01) which agrees with the

time response of serum Se. Maas et al. (1992) reported a strong correlation (0.88) for

whole blood Se and serum Se. Our data support this relationship, as serum Se and whole

blood Se responded to dietary Se level in a similar fashion. The cubic response of whole

blood Se over time may be attributed to ewes having no fetal tissue and producing no

milk to use as a route of excretion during the dry, rebreeding period, which encompassed

the midpoint of this study. Each dietary Se level was evaluated individually over time

and control and 8 mg/kg neither increased nor decreased with time (P > 0.20). Whole

blood Se from ewes receiving 4 mg/kg Se responded cubically (P = 0.019) and 16 mg/kg

dietary Se tended (P = 0.07) tended to respond cubically. Whole blood Se concentration

changed more sporadically over time in ewes receiving 12 or 20 mg/kg dietary Se and

each treatment produced a fifth degree polynomial (P < 0.05). At wk 12, ewes in all

treatment groups had higher (P < 0.05) whole blood Se than did controls. Ewes receiving








20 mg/kg Se had higher whole blood Se than controls and ewes receiving 4, 8, or 16

mg/kg Se and tended be higher (P = 0.13) in whole blood Se than ewes receiving 12

mg/kg dietary Se. At wk 24, ewes receiving 20 mg/kg Se had higher whole blood Se

than ewes from all other treatment groups and only ewes receiving 4 mg/kg Se had whole

blood Se similar to controls. At wk 36, ewes receiving 12, 16, and 20 mg/kg Se had

similar (P > 0.05) whole blood Se and again, only ewes receiving 4 mg/kg Se had whole

blood Se similar to controls. At wk 48, whole blood Se concentrations from ewes

receiving 16 and 20 mg/kg Se were similar (P > 0.10) and higher than (P < 0.05) from

ewes on all other treatments. Ewes receiving 8 and 12 mg/kg Se had similar (P = 0.88)

whole blood Se concentrations which were higher (P < 0.05) than those from controls

and ewes receiving 4 mg/kg Se. Only whole blood Se from ewes receiving 4 mg/kg Se

was similar (P = 0.16) to controls at wk 48. Whole blood Se concentrations at wk 60

followed a pattern similar to wk 48, in terms of differences among treatments. At wk 72,

whole blood Se from four of six dietary levels had numerically decreased from wk 60.

Whole blood from ewes receiving 20 mg/kg Se was higher (P < 0.05) in Se concentration

than in ewes from all other treatments. Ewes receiving 4, 8, 12, and 16 mg/kg dietary Se

produced similar (P > 0.10) whole blood Se and only ewes receiving 12 mg/kg Se had

higher (P < 0.05) whole blood Se than did controls. Cristaldi et al. (2004) also reported a

linear increase in whole blood Se as dietary Se was increased. Likewise, those authors

noted differences in treatment means over controls as dietary Se levels were increased up

to 10 mg/kg. Increased whole blood Se concentrations were reported in dairy cows as

their salt-based mineral mixtures were increased from 20 mg/kg to 120 mg/kg selenite Se








(Awadeh et al., 1998a). Whole blood Se increased linearly in young swine as dietary Se

was fed up to 20 mg/kg (Goehring et al., 1984b).

Wool

Selenium concentration in new growth wool was measured at wk 12, 24, 36, 48,

60, and 72 (Table 3-5). Dietary Se level, time, and dietary Se level x time affected (P <

0.001) wool Se. Wool Se increased linearly (P < 0.001) as dietary Se increased.

Response of wool Se over time was quadratic (P < 0.001) and time response for each

dietary Se level was evaluated individually. Wool Se from controls and ewes receiving 8,

12, and 16 mg/kg dietary Se responded quadratically (P < 0.03) from wk 12 to wk 72.

Wool Se from ewes receiving 4 mg/kg Se responded cubically (P < 0.05) and wool Se

from ewes receiving 20 mg/kg Se increased linearly (P < 0.01) over time. Increased Se

in hair has been reported in other livestock species. Kim and Mahan (2001) observed a

linear response in the hair of pigs as Se in their diet was increased. Goehring et al.

(1984b) reported a quadratic response in the hair of swine as dietary Se as sodium

selenite was increased up to 20 mg/kg. Likewise, Perry et al. (1976) reported increased

Se in the hair of feedlot steers as dietary selenite Se was increased. Cristaldi et al. (2004)

reported a linear increase in the wool of growing sheep as dietary Se was increased and

also observed differences in wool Se of wethers receiving 6, 8, or 10 mg/kg Se vs

controls. These authors did not report a significant treatment x time interaction.

However, wool Se in the present study was affected by time and the interaction of

treatment x time as wool Se increased and then seemed to reach a plateau around wk 48.

Kim and Mahan (2001) and Cristaldi et al. (2004) used 10 mg/kg Se as the highest

dietary level and reported linear responses in hair and wool. However, with 20 mg/kg as

the highest dietary level, the quadratic responses observed by Goehring et al. (1984b) and








in our study suggest that Se in wool and hair does not continue to increase linearly as

dietary Se is increased above 10 mg/kg. At wk 12, only ewes receiving 20 mg/kg Se had

wool Se higher (P < 0.05) than controls, however wool Se from ewes receiving 12 and 16

mg/kg Se tended (P < 0.15) to be higher than from controls. At wk 24, wool Se from

ewes receiving 16 or 20 mg/kg Se was higher than from controls and ewes receiving 4

mg/kg Se. Wool Se from ewes receiving 8 or 12 mg/kg Se tended (P < 0.07) to be higher

than wool Se from ewes receiving 4 mg/kg Se. At wk 36, wool Se from ewes on all

treatment groups was higher (P < 0.05) than from controls, and Se concentrations in wool

from ewes receiving 16 mg/kg Se were higher (P < 0.05) than wool Se from ewes

receiving 4 or 12 mg/kg dietary Se. Wool Se concentrations from ewes on all treatment

groups were similar (P > 0.15) and higher (P < 0.05) than wool Se from controls at wk

48. At wk 60, again, wool Se concentrations from ewes on all treatment groups were

higher (P < 0.05) than wool Se from controls. At the termination of the experiment, wool

Se from ewes receiving 20 mg/kg Se was higher than from ewes on all other treatments

and ewes from all treatment groups produced higher (P < 0.05) wool Se than did controls.

Some wool loss was observed in two ewes receiving 20 mg/kg dietary Se during lactation

in yr one. However, after lambs were weaned and lactation had ceased, both ewes regrew

a full fleece.

Tissues

Selenium concentrations in all tissues were affected (P < 0.001) by dietary Se

level. Selenium concentrations in brain ranged from 1.90 to 6.45 mg/kg DM and

increased linearly (P < 0.05) as dietary Se increased (Figure 3-1). Regressing brain Se

(mg/kg DM) on dietary Se concentration (mg/kg) produced the following relationship:

Brain Se = 1.89 + 1.56 Dietary Se (r2 = 0.52; P < 0.05).








Ewes consuming 12 or 20 mg/kg Se had higher (P < 0.05) brain Se than controls and

ewes consuming 20 mg/kg Se had higher (P < 0.05) brain Se than ewes consuming Se at

all levels except 12 mg/kg.

Diaphragm Se ranged from 1.27 to 4.01 mg/kg DM increased (P < 0.05) in a

linear manner as dietary Se was increased. (Figure 3-1). Regressing diaphragm Se

(mg/kg DM) on dietary Se concentration (mg/kg) produced the following relationship:

Diaphragm Se = 1.27 + 1.33 Dietary Se (r2 = 0.63; P < 0.05).

Ewes receiving 20 mg/kg Se had higher (P < 0.05) diaphragm Se than ewes receiving all

other treatments and only ewes receiving 12 or 20 mg/kg Se had higher diaphragm Se

than controls (P < 0.005).

Heart tissue Se (Figure 3-1) ranged from 1.83 to 6.24 mg/kg DM and increased in

a linear fashion (P < 0.001). Regressing heart Se (mg/kg DM) on dietary Se

concentration (mg/kg) produced the following relationship:
2
Heart Se = 1.83 + 1.99 Dietary Se (r = 0.70; P < 0.05).

Ewes receiving 12, 16, and 20 mg/kg Se had higher (P < 0.05) heart Se than controls and

ewes receiving 4 and 8 mg/kg Se tended to have higher (P < 0.12) heart Se than controls.

Heart Se concentrations from ewes receiving 20 mg/kg Se were higher (P < 0.05) than

those from ewes receiving all other dietary Se levels.

Selenium concentration in hoof ranged from 0.93 to 7.68 mg/kg DM and

increased cubically as dietary Se increased (Figure 3-2). Regressing hoof Se (mg/kg

DM) on dietary Se concentration (mg/kg) produced the following relationship:

Hoof Se = 0.93 + 1.95 Dietary Se 0.49 Dietary Se2 + 0.06 Dietary Se3 (r2 = 0.60;

P < 0.05).








Ewes receiving 16 and 20 mg/kg Se had higher hoof Se (P < 0.05) than controls.

Likewise, ewes receiving 20 mg/kg Se had higher hoof Se (P < 0.05) than ewes receiving

4, 8, and 12 mg/kg dietary Se.

Selenium concentrations in psoas major muscle (i.e. tenderloin), a muscle

commonly consumed by humans, ranged from 0.60 to 3.66 mg/kg DM and increased

linearly as dietary Se increased (Figure 3-2). Regressing psoas major muscle Se (mg/kg

DM) on dietary Se concentration (mg/kg) produced the following relationship:

Psoas major muscle Se 0.59 + 1.42 Dietary Se (r2 = 0.62; P < 0.05).

Selenium concentrations in psoas major muscle from controls were lower (P < 0.05) than

from ewes receiving 4, 12, 16, and 20 mg/kg Se and tended to be lower (P = 0.06) than

psoas major muscle Se concentrations from ewes receiving 8 mg/kg Se. Ewes receiving

20 mg/kg Se had higher (P < 0.05) psoas major muscle Se than ewes receiving all other

Se levels.

Kidney Se ranged from 5.18 to 31.61 mg/kg DM and responded to increased

dietary Se in a cubic fashion (Figure 3-2). Regressing kidney Se (mg/kg DM) on dietary

Se concentration (mg/kg) produced the following relationship:

Kidney Se = 5.18 + 6.64 Dietary Se- 2.28 Dietary Se2 + 0.32 Dietary Se3 (r2 =

0.62; P < 0.05).

Ewes receiving 20 mg/kg Se had higher (P < 0.01) kidney Se than ewes from all other

treatment groups. Ewes receiving 12mg/kg Se tended (P = 0.09) to have higher (P <

0.01) kidney Se than controls.








Liver Se concentration ranged from 4.20 to 230.36 mg/kg DM and responded

quadratically as dietary Se level increased (Figure 3-3.) Regressing liver Se (mg/kg DM)

on dietary Se concentration (mg/kg) produced the following relationship:

Liver Se = 4.19 + 26.59 Dietary Se 9.31 Dietary Se2 (r2 = 0.66; P < 0.01).

Ewes receiving 20 mg/kg dietary Se had higher (P < 0.05) liver Se than ewes from all

other treatments. No other differences (P > 0.05) existed among controls and Se

treatment groups.

Linear increases in the Se concentration of loin, liver, kidney and hoof were

reported in swine (Kim and Mahan, 2001) and sheep (Cristaldi et al., in press). Likewise,

Echevarria et al. (1988) reported linear responses of sheep liver, kidney, heart, and

muscle to dietary Se as sodium selenite Se. In our study, loin, diaphragm, heart, and

brain responded linearly, where kidney and hoof responded cubically and liver responded

quadratically. These higher degree polynomials may be due to changes in metabolism of

Se as dietary Se concentration approaches 20 mg/kg. Most previous research used 10

mg/kg Se as the highest dietary concentration.

Enzymes and Histopathology

Serum for evaluation of albumin and enzyme activities was collected at wk 72

along with samples of brain, diaphragm, heart, hoof tip, kidney, psoas major muscle, and

liver for histopathological evaluation. Concentrations of albumin and activities of Alk

phos, ALT, GGT, AST, and CK in serum were in or below the normal range for adult

sheep (Table 3-6). In instances of Se toxicosis, the activities of these enzymes would

have been increased due to tissue necrosis. Our observations agree with those reported by

Cristaldi et al. (2004) as albumin and enzyme activities in wether sheep after receiving up

to 10 mg/kg Se were in the normal ranges.








Most of the tissues collected at slaughter were free from pathological changes.

The findings of lymphocytes in the portal triads were deemed to be a background finding

and insignificant. Likewise, the findings of lymphocytic foci in the heart tissue were

determined to be associated with sarcocystic parasites. Mineral precipitations were

observed in kidney tissue of some ewes and are incidental, background findings.

Contraction bands present in the diaphragm and psoas major muscle were a result of

stunning during humane slaughter. Adipose tissue was present in the heart and psoas

major muscle, which is an indication of adequate nutrition. Hepatic lipidosis was

diagnosed in four ewes. Two cases (one severe, one moderate) were diagnosed in ewes

receiving 16 mg/kg dietary Se. In the moderate case, there was also evidence of bile

retention. Neither of these ewes lambed in either yr. This would indicate that the hepatic

lipidosis could be treatment related rather than due to metabolic changes associated with

gestation, parturition, and lactation. One ewe receiving 12 mg/kg Se and one ewe

receiving 4 mg/kg were diagnosed with mild hepatic lipidosis, however, both ewes

lambed in both yr. Thus, the hepatic lipidosis was likely due to metabolic changes

associated with lamb production. No evidence of significant pathological changes was

observed in ewes receiving 20 mg/kg dietary Se, which was the highest Se level used in

this study. Cristaldi et al. (2004) found no abnormalities after microscopic evaluation of

heart, liver, kidney, diaphragm, and muscle from wethers consuming up 10 mg/kg Se for

one yr. Likewise, only one instance of abnormal pathology was observed in ewes

consuming less than 10 mg/kg Se on our study. Furthermore, our study was

approximately 40% longer in duration, utilized treatments of up to 100% more Se, and

introduced stresses of production, all of which should have helped to induce Se toxicosis








and thus, the finding of abnormal organ pathology. However, abnormal pathological

findings were few and did not follow a pattern with respect to dietary level which would

be indicative of Se toxicosis.

No clinical signs of Se toxicosis such as abnormal hoof growth or loss of wool

were observed in ewes receiving > 16 mg/kg Se. However, some excessive hoof growth

was observed after approximately one yr in ewes receiving 16 and 20 mg/kg Se and wool

loss was observed during lactation in two ewes receiving 20 mg/kg Se. Livestock

suffering from alkali disease were reported to have hair Se concentrations of up to 45

mg/kg and whole blood Se of 4.1 mg/L, while hooves, liver, and kidney of affected

animals contained 10 mg/kg Se or more (NAS, 1983). At no time during our study did

wool Se reach even 10 mg/kg and whole blood Se remained less than 50% of the

aforementioned 4.1 mg/L concentration. Also, hoof Se remained under 8 mg/kg for all

treatments during the course of our study. Liver and kidney Se concentrations from our

study were higher than the 10 mg/kg previously reported. The elevated concentrations of

Se in the liver and kidney of ewes consuming 16 and 20 mg/kg, and the observation of

some clinical signs of Se toxicosis and limited pathological abnormalities in ewes

consuming these Se levels may indicate that some ewes were beginning to suffer from Se

toxicosis. However, definitive evidence was not observed. Therefore, it is necessary that

either dietary Se concentration or duration of experiment be increased in order to induce a

definitive Se toxicosis using inorganic Se.

Implications

The maximum tolerable level of selenium as sodium selenite for ruminants is

higher than 2 mg/kg. Feeding up to 12 ppm selenite selenium to ewes under stresses of

production (i.e., gestation and lactation) for 72 wk did not produce any clinical or








pathologic signs of selenium toxicosis. Ewes fed 16 and 20 mg/kg produced some signs

of selenium toxicosis; however, general metabolic disorders could not be ruled out and no

deaths of ewes consuming these levels of selenium were attributed to selenium toxicosis.

Further studies of this nature should further prove that the current suggested tolerable

level of Se is underestimated.

Summary

The objectives of this 72-wk study were to evaluate and compare the effects of six

dietary levels of inorganic Se on serum, whole blood, wool, and tissue Se concentrations

of mature ewes during lamb production and determine maximum tolerable level of Se

during lamb production. Forty-one range-type ewes were used in a completely

randomized design with six dietary treatments. Sodium selenite was added to a corn-

soybean meal basal diet to provide 0.2 (control), 4, 8, 12, 16, and 20 mg/kg dietary Se to

ewes during lamb production. Serum Se and ewe BW were measured at 4-wk intervals,

whole blood Se, and wool Se were measured every 12 wk, and samples of brain,

diaphragm, heart, hoof, kidney, liver, and psoas major muscle were collected at the

termination of the experiment. Dietary Se did not affect ewe BW during the study (P =

0.69). Serum Se increased linearly as dietary Se level increased (P < 0.001) and

responded cubically (P = 0.02) over time. Selenium in whole blood increased linearly (P

< 0.001) with increased dietary Se and cubically (P < 0.01) over time. Wool Se increased

linearly (P < 0.001) as dietary Se increased and response over time was quadratic (P <

0.001). Brain, diaphragm, heart, and psoas major muscle Se increased linearly as Se in

the diet increased, liver Se responded quadratically, and hoof and kidney Se responded

cubically to treatment (P < 0.05). In general, serum, whole blood, and tissue Se








concentrations from ewes receiving 12, 16, or 20 mg/kg dietary Se were higher (P < 0.05)

than from controls and ewes receiving less dietary Se. Though serum, whole blood, and

wool Se concentrations were elevated in ewes receiving increased dietary Se, at no time

did serum, whole blood, or wool Se concentrations reach levels previously reported as

toxic and a pattern of clinical signs of Se toxicosis was not observed. Microscopic

evaluation of liver, kidney, diaphragm, heart, and psoas major muscle did not reveal

evidence of Se toxicosis in ewes on any dietary Se level. Ewes under our experimental

conditions and during the stresses of production were able to tolerate up to 20 mg/kg

dietary Se as sodium selenite for 72 wk. These findings suggest that the maximum

tolerable level of inorganic Se for sheep to be much higher than 2 mg/kg as was

suggested previously. Experiments which are longer in duration and utilize higher dietary

Se concentrations may be used to clearly define the maximum tolerable level.








Table 3-1. Diet composition (as-fed) for selenite-Se supplemented ewes'
Ingredient % as-fed
Ground yellow corn 53.75
Cottonseed hulls 22.00
Soybean meal (47.5% CP) 16.00
Alfalfa meal (14% CP) 3.00
Soybean oil 3.00
Trace mineral mixb 1.00
Ground limestone 1.25
Vitamins A & D C
aSelenium levels in diet (as analyzed): 0.29, 3.77, 7.54, 11.01, 15.48, and 19.05 ppm
for Se levels 0.2, 4, 8, 12, 16, and 20 ppm, respectively.
bTrace mineral mixture supplied between 96.5% and 98.5% NaCl, and provided per kg
of diet: 1.0 mg Co (as carbonate), 5.0 mg Cu (as oxide), 0.7 mg I (as iodate), 35 mg Fe
(as oxide), 25 mg Mn (as oxide), and 35 mg Zn (as oxide).
cProvided per kg of diet: 5,000 IU of Vitamin A and 500 IU of Vitamin D3.


Table 3-2. Lamb production of ewes receiving different concentrations of dietary Se
Year 1a Year 2b
Dietary Se, ppm Ewes lambed Lambs born Ewes lambed Lambs bornd
0.2 5 9 4 5
4 7 11 7 10
8 5 6 5 7
12 5 8 6 11
16 4 5 0 0
20 7 14 2 3
Total 33 53 24 36
aEwes began receiving experimental diets at 57 d average gestation in yr 1.
bEwes were fed experimental diets continuously during breeding and gestation yr 2.
CLamb crop as lambs born (53) per ewe exposed (41) was 129% in yr 1.
dLamb crop as lambs born (36) per ewe exposed (33) was 109% in yr 2.









Table 3-3. Effect of dietary inorganic Se level on serum Se concentration of mature
ewes at various stages of lamb production a
Dietary Se, mg/kg
0.2 4 8 12 16 20
Stage of Production Serum Se, gg/L
Late Gestation, yr 1 149g 67 242gh 67 354hi 79 414 79 463 79 707 + 81
Lactation, yr 1V 151g+56 2729 54 486h63 623hi 63 718-4 j63 811- i66
Dry, rebreeding d 1629 110 298gh 95 458hi- 100 604 99 120 Y 106 1084 113
Late Gestation, yr2' 1409+ 137 313gh 124 4469b+ 142 596hi 142 986i 158 1072i + 149
Lactation, yr 2f 127 114 3259h+ 103 536hi 114 718i 114 769 136 1355 121
aData represent least squares means SE.
bLate gestation, yr 1, defined as 56 d prepartum and includes serum Se concentrations for wk 4, 8, and 12.
cLactation, yr 1, defined as 84 d postpartum and includes serum Se concentrations for wk 12, 16, 20, and 24.
dDry, rebreeding period, 168 d, includes serum Se concentrations for wk 28, 32, 36, 40, 44, and 48.
eLate gestation, yr 2, defined as 56 d prepartum and includes serum Se concentrations for wk 52, 56, and 60.
rLactation, yr 2, defined as 84 d postpartum and includes serum Se concentrations for wk 60, 64, 68, and 72.
gahijMeans within rows lacking a common superscript differ (P < 0.05).


Table 3-4. Effect of dietary inorganic Se level on whole blood Se concentration of
mature ewesa


Dietary Se, mg/kg
Week of 0.2 4 8 12
experiment Whole blood Se, gg/L
12 386h 168 839i 151 902ij 151 1241jk + 139
24C 420h 140 60lhi- 130 852ij 130 1047jk 130
36 d 438h 152 635h 149 1079i 138 1314 138
48e 378h- 154 661h -130 1117i 139 1145 130
60f 497h 154 802hi 138 1070 -151 1131i 150
729 410h 154 618hi 139 721hi 140 916 -140
aData represent least squares means SE.
n 4, 5, 5, 6, 7, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
cn = 6, 7, 7, 7, 7, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
dn = 5, 5, 6, 6, 6, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
'n = 5, 7, 6, 7, 5, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
fn = 5, 6, 5, 5, 4, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
gn = 5, 6, 6, 6, 3, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
hIjik lMeans within rows lacking a common superscript differ (P < 0.05).


16 20

1053'j 130 1558k 154
1312k _4 130 1822' 154
1668j 139 1373ij 154
1616 151 1951j 154
1892j 167 1796j 154
841hi 194 1855 - 154









Table 3-5. Effect of dietary inorganic Se level on wool Se concentration of mature
ewesa
Dietary Se, mg/kg
Week of 0.2 4 8 12 16 20
experment Wool Se, mg/kg (DM basis)
12 0.50' 0.58 0.71h 0.54 1.36hi 0.54 1.67h 0.54 2.00h + 0.54 2.23 0.64
24c 0.62' 0.62 143hi 0.54 2.76j 0.54 2.79j 0.54 3.58jk 0.54 4.64k 0.64
36' 1.58h 0.63 3.72' 0.54 4.86j 0.54 3.96' 0.54 5.57i 0.57 5.27'j 0.64
48' 1.18h 0.64 4.86' 0.54 4.64i 0.54 4.821 0.54 5.47 0.57 5.53' 0.64
60' 1.25' 0.64 4.06i 0.54 6.09i 0.57 5.50'j 0.57 5.63'j 0.68 5.17'j 0.64
729 0.96h 0.64 3.42i 0.57 3.67'j 0.57 5.12' 0.58 5.1 li 0.80 7.69k 0.64
aData represent least squares means SE.
bn = 6, 7, 7, 7, 7, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
Cn = 5, 7, 7, 7, 7, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
dn = 5, 7, 7, 7, 6, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
en = 5, 7, 6, 7, 6, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
fn = 5, 7, 6, 6, 4, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
gn = 5, 6, 6, 6, 3, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
h'ij'kMeans within rows lacking a common superscript differ (P < 0.05).


Table 3-6. Amount of albumin and tissue enzyme activities present in serum of Se
supplemented ewesa"bc
Dietary Se, mg/kg
Item Normal concentration 0.2 4 8 12 16 20
Albumin 2.4-4.0 g/dL 2.9 2.5 1.940 2.2 2.3 2.6
Alk Phos 68-387 IU/L 91.0 60.2 65.3 95.5 36.3 89.0
AST 60-280 IU/L 53.8 131.7 32.8 52.3 64.0 24.6
ALT 11-40 IU/L 15.4 34.7 2.7 13.3 17.7 24.0
GGT 15-60 IU/L 67.6 63.2 39.8 55.0 63.7 59.6
CK 0-584 IU/L 67.4 108.2 36.3 58.7 47.7 55.0
aSerum sample collected at wk 72.
bAlbumin and tissue enzyme activities presented in same units as normal concentration ranges.
CGGT and CK ranges were established by University of Florida Veterinary Teaching Hospital.








-- Brain
7
Diaphragm
6 -0- Heart
e5
4

g2

0


0 4 8 12 16 20

Dietary Se, mg/kg

Figure 3-1. Effect of dietary inorganic Se level on Se concentrations in brain, diaphragm,
and heart of ewes; SE = 0.6 to 0.9, 0.3 to 0.4, and 0.4 to 0.6 for brain, diaphragm, and
heart, respectively.








35 -
-c- Kidney

30 -Hoof
!- Loin
,. 25

20



0
15





0
5



0 4 8 12 16 20
Dietary Se, mg/kg

Figure 3-2. Effect of dietary inorganic Se level on Se concentrations in kidney, hoof, and
loin (psoas major muscle) of ewes; SE = 3.0 to 3.3, 0.8 to 1.1, and 0.3 to 0.5 for kidney,
hoof, and loin, respectively.








250 -
225 -Liver
a 200
175
S150
125
r 100
75
S50
25
0 I
0 4 8 12 16 20
Dietary Se, mg/kg
Figure 3-3. Effect of dietary inorganic Se level on liver Se concentration in ewes; SE =
27.5, 25.4, 24.5, 24.5, 34.6, and 26.9 for 0.2, 4, 8, 12, 16, and 20 mg/kg dietary Se,
respectively.













CHAPTER 4
EFFECTS OF SELENIUM LEVELS IN EWE DIETS ON SELENIUM IN MILK
AND PLASMA AND TISSUE SELENIUM CONCENTRATIONS OF LAMBS

Introduction

Selenium has long been implicated as a toxic element to livestock (Oldfield,

2002). Animals grazing seleniferous plants in certain regions of the world are subject to

Se toxicosis and conditions such as alkali disease and "blind staggers" (McDowell,

2003). The estimated maximum tolerable level of Se for ruminant livestock is 2 mg/kg

(NRC, 1980). However, recent research (Cristaldi et al., in press) has shown that sheep

may consume up to 10 mg/kg Se as sodium selenite in the total diet for one yr, without

showing signs of selenium toxicosis. Although it was concluded that these wethers were

not suffering from Se toxicity, they did have increased serum, whole blood, and tissue Se

concentrations. Like blood and tissue, milk Se is affected by dietary Se level (Conrad and

Moxon, 1979; Givens et al., 2004) and Se readily crosses the placenta to the fetus (Van

Saun et al., 1989). Furthermore, positive correlations exist between blood Se of cows and

blood Se of their calves (Kincaid and Hodgson, 1989; Enjalbert et al., 1999; Pehrson et

al., 1999). In sheep, Cuesta et al. (1995) showed increased colostrum Se from ewes

receiving supplemental Se, and that milk Se was higher after one mo of supplementation.

Thus, it seems that neonates from dams consuming high dietary levels of Se would have

increased blood Se at birth, and subsequently would be exposed to high Se intake from

increased Se in milk.








Acute Se toxicosis has been evaluated by injecting ewe lambs with sodium

selenite (Blodgett and Bevill, 1987), and Abdennebi et al. (1998) evaluated the toxic

effects of dosing lambs with extracts of milk vetch (Astragalus lusitanicus). In both

studies, weaned lambs were utilized. Newborn and pre-weaned lambs differ from older

sheep in ruminal and digestive function (Church, 1979; Goursand and Nowak, 1999), and

may respond differently than older animals to increased Se intake. We hypothesized that

when Se in gestating ewe diets is increased, colostrun Se, milk Se and plasma Se of their

lambs will increase. The objective of this experiment was to follow ewes through two

lamb crops and evaluate and compare the effects of six levels of dietary Se on ewes' milk

and the Se status of their lambs prior to weaning.

Materials and Methods

All animal procedures were conducted within the guidelines of and approved by

the University of Florida Institutional Animal Care and Use Committee. This 504-d

experiment utilizing ewes and two lamb crops was conducted from December 18, 2001 to

May 5, 2003 at the University of Florida Sheep Nutrition Unit located in southwestern

Alachua County, FL. Thirty-three, four year old, Rambouillet ewes that originated from

a single flock in Texas and were previously confirmed pregnant (average 57 d gestation)

were weighed (57.4 + 5.7 kg) and received 2-ml ivermectin (Ivomec; Merial Ltd. Iselin,

NJ). Ewes were randomly assigned to one of six dietary treatments for a 504 d (two

lambing seasons) study. Six dietary treatments were 0.2, 4, 8, 12, 16, or 20 mg/kg Se as

sodium selenite (as-fed basis) added to a corn-soybean meal basal diet (Table 4-1). The

basal diet was formulated to meet animal requirements for protein, energy as TDN,

vitamins, and minerals for this class of sheep (NRC, 1985). Animal numbers per

treatment were 5, 7, 5, 5, 4, and 7 for 0.2 (control), 4, 8, 12, 16, and 20 mg/kg added Se,








respectively. Ewes were housed by treatment group in covered wooden pens (53.5 in2)

with earth floors and ad libitum water. Diets were fed at 909 g/ewe/d from d 0 until

lambing began (d 81), increased to 1 OOOg/ewe/d during lambing (d 81 to 101), and again

increased to 1135 g/ewe/d during lactation (d 101 to 171). Diets were sampled every 28

d, ground (1 mm), and frozen at 0C until analysis.

In the first year, 52 lambs were born over 20 d from March 9, 2002 to March 28,

2002. Prior to lambing, ewes were fitted with a device to cover the udder and prevent

lambs from nursing until a blood sample could be obtained. The udder cover was crafted

from nylon pantyhose (Eeggs Products, Winston-Salem, NC) and polyester elastic (2.54

cm wide) and held in place with safety pins (Figure 4-1). A blood sample for plasma

analysis was collected from lambs immediately after birth via jugular venipuncture into

10-ml heparinized tubes (Vacutainer; Becton-Dickinson, Franklin Lakes, NJ). The udder

cover was then removed from the ewe and five ml of pre-suckled colostrum was collected

into a 15-ml plastic centrifuge tube (Fisher; Fisher Scientific, Pittsburgh, PA). Additional

blood samples were collected from lambs and milk samples from ewes at 3, 28, and 56 d

postpartum. Blood samples were centrifuged at 700 x g and the plasma then frozen at

0C. Ewe milk samples were also stored frozen at 0C for later analysis. Lambs were

weaned at 70 d of age and ewes then received 909 g/ewe/d of their respective diets until

next lambing. At 70 d, ram lambs were surgically castrated and the testes were frozen at

0C until analysis.

On August 15, 2002, ewes were pen exposed to rams for 35 d. In the second year,

36 lambs were born over 34 d from January 17, 2003 to February 20, 2003. All sampling

intervals, procedures, feeding levels, and materials used were duplicated from the first








year. Plasma, milk, testes, and feed samples from both years were analyzed for Se

concentration using a fluorometric method described by Whetter and Ullrey (1978). To

help ensure reliability of the analytical method, a certified standard (National Bureau of

Standards Bovine Liver SRM-1577a; U.S. Department of Commerce, National Institute

of Standards and Technology, Gaithersburg, MD) was frequently analyzed.

Effects of treatment on lamb testicular and colostrum Se were analyzed using

PROC MIXED in SAS (SAS for Windows 8e; SAS Inst. Inc., Cary, NC) in a completely

randomized design. Contrast statements were used to compare means as described by

Littell et al. (1998; 2000). PROC MIXED of SAS was also used to analyze effects of

treatment, d, and the interaction of treatment x d on milk Se and plasma Se as repeated

measures with a spatial power covariance structure with respect to d and a subplot of

animal nested within treatment. Contrast statements were written to determine

differences in means for different sampling d. PROC CORR was used to determine

correlations of ewe milk Se to lamb plasma Se.

Results

In year one, 11 of 52 lambs were removed from the study before 56 d of age. Five

lambs were born to ewes which produced little or no milk, one ewe had extremely

enlarged or "bottle" teats and her two lambs were unable to suckle, two lambs died of

weakness/dehydration, and two died of "joint ill." There were no apparent signs of

selenium toxicosis in any lambs regardless of dietary Se level of their dams.

In year one, colostrum Se was affected by Se concentration of the ewes' diet (P =

0.008) and increased linearly (P < 0.001) as dietary Se increased (Table 4-2). Ewes

receiving 16 or 20 mg/kg dietary Se produced higher (P < 0.05) colostrum Se than did

controls. Ewes receiving 20 mg/kg dietary Se also produced higher (P < 0.05) colostrum








Se than did those ewes receiving 4 mg/kg dietary Se and tended (P < 0.12) to produce

higher colostrum Se than ewes receiving 8 and 12 mg/kg dietary Se. Likewise, colostrum

Se from ewes receiving 16 mg/kg tended to be higher (P = 0.052) than colostrum Se from

ewes receiving 4 mg/kg dietary Se. Colostrum Se from ewes receiving 8, 12, or 16

mg/kg dietary Se was similar (P > 0.20).

In year two, 12 of 36 lambs were removed from the study before d 56. Seven

lambs were removed due to their dams having either no milk or enlarged teats that were

unable to be suckled, four lambs were lost to predation, and one lamb was removed due

to physical injury. No lambs were lost or removed from the study due to dietary Se in the

diet of their dam. As in year one, colostrum Se was affected by dietary Se (P < 0.05) and

increased linearly (P < 0.01) as dietary Se increased (Table 4-2). Ewes receiving 8, 12,

or 20 mg/kg dietary Se produced colostrum with similar (P > 0.19) Se concentrations,

which were higher (P < 0.05) than colostrum Se from controls. Ewes consuming 8

mg/kg dietary Se produced colostrum Se higher (P < 0.05) than those ewes consuming 4

mg/kg dietary Se. Likewise, ewes consuming 20 mg/kg dietary Se tended to produce

colostrum Se higher (P = 0.10) than ewes consuming 4 mg/kg dietary Se. No ewes

receiving 16 mg/kg dietary Se lambed in year two and are not represented in these

comparisons.

Ewe milk Se collected at 3, 28, and 56 d postpartum increased linearly (P <

0.001) as dietary Se increased in year one (Table 4-3). Day of sampling also had an

effect (P = 0.002), but there was no treatment x d interaction. At d 3 postpartum, ewes

receiving 4 and 8 mg/kg dietary Se produced similar (P = 0.60) milk Se and milk Se from

ewes receiving 8 mg/kg dietary Se tended (P = 0.06) to be higher than from controls.








Ewes consuming 12, 16, and, 20 mg/kg Se produced milk Se higher (P < 0.05) than

controls. Milk Se from ewes consuming 16 mg/kg Se tended to be higher (P = 0.09) than

that from ewes consuming 20 mg/kg Se and was higher (P < 0.05) than milk Se from all

other treatments. At d 28 postpartum, ewes consuming 20 mg/kg dietary Se produced

milk Se concentrations higher (P < 0.05) than did controls or ewes consuming 4 mg/kg

dietary Se. Likewise, milk Se from ewes consuming 20 mg/kg dietary Se tended to be

higher (P < 0.075) than milk Se from ewes receiving 8 or 12 mg/kg Se. Milk Se from all

other treatment groups was similar (P > 0.20). At the final milk collection in year one (d

56), milk Se concentrations from controls and ewes consuming 4, 8, and, 12 mg/kg Se

were similar (P > 0.17). Ewes consuming 16 and 20 mg/kg Se produced similar milk Se

(P = 0.43), which was higher (P < 0.05) than milk Se from all other treatments. A linear

increase (P < 0.01) in milk Se as dietary Se increased was observed at each sampling d as

well as over all sampling d. Milk Se concentrations in year one remained below 1000

gg/L from d 3 to d 56.

In year two, dietary Se concentration had an effect on milk Se (P < 0.05), as did

the interaction of dietary Se concentration x sampling d (P <0.05). Milk Se

concentrations from ewes consuming 8, 12, and 20 mg/kg Se were similar (P > 0.38) to

each other and higher (P < 0.05) than controls at d 3 (Table 4-4). Ewes consuming 12

mg/kg Se had higher (P < 0.05) milk Se than did those consuming 4 mg/kg Se. Ewes

consuming 20 mg/kg Se produced milk Se that tended (P = 0.09) to be higher than milk

Se from ewes consuming 4 mg/kg Se. There were no differences (P > 0.05) in milk Se

among treatment groups at d 28. However, ewes consuming either 12 or 20 mg/kg Se

had milk Se which tended to be higher (P < 0.12) than control. At d 56, milk Se








concentrations from ewes consuming 20 mg/kg Se were higher than from all other

treatments (P < 0.05). Ewes consuming 12 mg/kg Se had milk Se which was higher (P <

0.05) than controls and tended to be higher than from ewes receiving 4 mg/kg Se (P =

0.07). Milk Se concentrations, at d 56, from all other treatment groups were similar (P >

0.18). Milk Se concentrations increased linearly (P < 0.001) as dietary Se increased over

all sampling d.

Lamb plasma Se was affected by dietary Se concentration of their dams (P <

0.001) and increased linearly as dietary Se of dams increased (P < 0.001) in year one

(Table 4-5). Likewise, d of sampling affected lamb plasma Se concentration (P < 0.01).

On d 3 to 56, lamb plasma Se was positively correlated to ewe milk Se (r = 0.29; P <

0.00 1). At birth, lambs suckling ewes consuming 20 mg/kg Se had higher plasma Se than

controls (P < 0.05) and lambs suckling ewes consuming 16 mg/kg Se tended to have

higher plasma Se than controls (P = 0.15). All other lambs had similar plasma Se (P >

0.25). At 3 d of age, lambs from ewes consuming 20 mg/kg Se had higher plasma Se (P

< 0.01) than all other treatment groups. Plasma Se concentrations from control lambs

were lower (P < 0.05) than plasma Se from lambs suckling ewes consuming 8, 12, or 16

mg/kg Se. At 28 d of age, lambs suckling ewes receiving 12, 16, or 20 mg/kg Se had

higher plasma Se than did controls (P < 0.01). Likewise, lambs suckling ewes receiving

4 or 8 mg/kg Se tended to have higher plasma Se than controls (P < 0.14). Plasma Se

from lambs suckling ewes receiving 20 mg/kg Se was higher than from lambs suckling

dams that received 4, 8, or 12 mg/kg Se (P < 0.05) and tended to be higher than from

lambs suckling dams that received 16 mg/kg Se (P 0.09). At 56 d, plasma Se from

lambs suckling ewes receiving 4, 12, 16, or 20 mg/kg Se was higher than controls (P <








0.05) and lambs suckling ewes that received 8 mg/kg Se tended to have higher plasma Se

than controls (P = 0.067). Plasma Se from lambs suckling ewes receiving 16 or 20

mg/kg Se was higher than plasma Se from lambs suckling ewes receiving 4 mg/kg Se (P

< 0.05) and tended to be higher than plasma Se from lambs suckling ewes receiving 8

mg/kg Se (P: <0.08).

Lamb plasma Se, in year two (Table 5-6), was affected by the concentration of Se

in the diet of their dams (P < 0.001) and increased linearly as Se concentration in dams'

diet increased (P < 0.001). Day of sampling and the interaction of dietary Se

concentration x d of sampling also affected lamb plasma Se (P < 0.01). At birth, lambs

from ewes receiving 12 mg/kg Se had higher plasma Se than did controls (P < 0.05).

Likewise, lambs from ewes receiving 20 mg/kg Se had higher (P < 0.05) plasma Se than

did controls and lambs from dams receiving 4 or 8 mg/kg Se. From 3 to 56 d of age,

lamb plasma Se was positively correlated to ewe milk Se (r = 0.44; P < 0.001). At 3 d of

age, lambs from all treatment groups had higher plasma Se than did controls (P < 0.05)

and lambs suckling ewes receiving 8, 12, or 20 mg/kg Se had higher plasma Se than did

those suckling ewes receiving 4 mg/kg Se (P < 0.05). At d 28, lamb plasma Se from all

treatment groups was higher than controls (P < 0.05). Lambs suckling ewes receiving 20

mg/kg Se had plasma Se higher than all other treatment groups (P < 0.05). Also, lambs

suckling ewes receiving 12 mg/kg Se had plasma Se higher than lambs suckling ewes

receiving 4 or 8 mg/kg Se (P < 0.05). At d 56, lambs suckling dams receiving 4, 8, or 12

mg/kg Se had higher plasma Se than did controls (P < 0.05) and lambs suckling dams

receiving 20 mg/kg Se tended to have higher plasma Se than did controls (P = 0.067).








Plasma Se from lambs suckling ewes receiving 12 mg/kg Se was higher than (P < 0.05)

than plasma Se from lambs suckling ewes receiving 4 and 8 mg/kg.

Selenium concentration in testis (dry basis) taken from ram lambs at 70 d of age

(weaning) increased linearly (P < 0.00 1) as dams' dietary Se concentration increased

(Figure 4-2) in year one. Testicular Se from lambs suckling ewes receiving 20 mg/kg Se

was higher than testis Se from controls and lambs suckling ewes receiving 4 or 8 mg/kg

Se (P < 0.05). Lambs suckling ewes receiving 16 mg/kg Se had testicular Se which was

higher (P < 0.05) than testicular Se from controls and lambs suckling ewes receiving 4

mg/kg Se. Lambs suckling ewes receiving 12 mg/kg tended to have higher testicular Se

than did controls or lambs suckling ewes receiving 4 mg/kg Se (P < 0.11). Likewise,

lambs suckling ewes receiving 8 mg/kg tended to have higher testicular Se than did

controls (P = 0.14). There was no effect of treatment on testicular Se in year two (P -

0.70). Testicular Se concentrations were 2.05, 3.16, 2.96, and 3.24 mg/kg for controls

and lambs suckling ewes receiving 4, 8, and 12 mg/kg Se, respectively.

Discussion

Colostrum Se increased with dietary Se level in both years. Cuesta et al. (1995)

reported higher colostrum Se from vitamin E + Se supplemented ewes versus their

unsupplemented counterparts. These findings are further supported by Mahan (2000),

who demonstrated that colostrum Se was increased by increasing Se in prepartum and

postpartum sow diets. Colostrum Se from Se supplemented crossbred ewes was

increased over unsupplemented controls (Norton and McCarthy, 1986), however, those

researchers used injectable vitamin E + Se as the supplemental Se source rather than

dietary Se. Overnes et al. (1985) also reported an effect on colostrum Se from ewes

receiving Se fed via free-choice salt and mineral mixtures. In the present study, ewe








colostrum Se concentrations from controls in year one were lower at 257 Pig/L than

values in cow colostrum from Romania reported by Serdaru et al. (2004). However, in

year two, after our ewes had been receiving their respective diets for approximately 13

mo, colostrum Se from controls had more than doubled to 705 gg/L. The increase in

colostrum Se after a longer duration of Se supplementation is substantiated by Maus et al.

(1980). Those authors reported that Se in cows' milk increased with time when fed at 0.2,

0.3, 0.4, and 0.7 mg/kg in a corn-brewers' grain dairy diet. As dietary Se was increased

by increments of 4 mg/kg from 4 mg/kg up to 20 mg/kg, colostrum Se increased by 45.3,

8.8, 55.2, and 10.1%, respectively in year one. Colostrum Se was numerically higher in

year two when Se was fed at 0.2, 4, 8, and 12 mg/kg. This furthers the idea that

colostrum Se, when Se is supplemented at equivalent concentrations, may be increased as

animals are supplemented for an extended period of time. The use of increased Se in

gestating animals may prove beneficial to their offspring as it provides greater

antioxidative protection through increased colostrum Se and thus provides greater

phagocytic and microbicidal activity (Wuryastuti et al., 1993).

As with colostrum Se, subsequent milk Se also increased as dietary Se increased.

Givens et al. (2004) reported increased milk Se as selenite Se increased from 0.38 to 1.14

mg/kg and that a strong positive correlation exists between milk Se and dietary Se (r =

0.979). Gardner and Hogue (1967) reported up to five-fold increases in milk Se when

sodium selenite was added to ewe diets at 1 mg/kg. In the present study, a five-fold or

greater increase was observed in milk Se as dietary Se was increased from control (0.2

mg/kg) to 8 mg/kg for d 3, from control (0.2 mg/kg) to 20 mg/kg for d 28, and from

control (0.2 mg/kg) to 12 mg/kg for d 56 in year one. In year two, a four-fold or greater








increase in milk Se was observed as dietary Se increased from control to 8 mg/kg on each

sampling d. Few increases of that magnitude were observed between groups receiving Se

at high concentrations. This indicates that the proportion of Se transferred to milk

decreases as dietary Se concentration increases. Waite et al. (1975) suggest that Se is

subject to a bioreducing process as it is transferred from plasma to milk. These authors

report that only 1.5% of dietary selenite Se appeared in milk. Givens et al. (2004)

observed increases of more than four-fold in cows' milk Se when dietary Se was doubled

and tripled using Se yeast. However, in our study, increases of such magnitude were not

observed when dietary Se was doubled and tripled using sodium selenite. These

observations would indicate that, though milk Se can be increased using an inorganic Se

source, a greater proportion of organic Se is transferred to milk. This concept is supported

by several studies using cattle (Knowles et al., 1999; Ortman and Pehrson, 1999; Pehrson

et al., 1999).

One objective of this study was to quantitate the effects on lambs that were

suckling ewes that received dietary Se above the maximum tolerable level of 2 mg/kg

(NRC, 1980). No lambs were born with congenital deformities or abnormalities, nor did

any lamb display signs of selenium toxicosis (e.g., wool loss, hoof malformation,

anorexia) from birth to weaning. Selenium included in ewe diets has previously been

shown to be transmitted to the lamb via the placenta and milk (Jacobsson et al., 1965). In

our study, plasma Se in lambs increased as Se concentration in their dams' diet increased

and was positively correlated to milk Se. Lambs from ewes receiving the control diet had

plasma Se at birth that averaged 81 4g/L in year one and 85[tg/L in year two. These

values are only slightly above the normal range (50-80 gg/L) for neonate lambs (Aitken,








2001) and more than double the plasma Se concentration suggested by Bostedt and

Schramel (1990) for normal growth and health in newborn calves. Lamb plasma,

collected before nursing, increased in Se as Se concentration in the diet fed to ewes

during gestation increased in both years. Ewes receiving 12 mg/kg dietary Se gave birth

to lambs with up three-fold higher plasma Se than did controls. Likewise, ewes receiving

20 mg/kg dietary Se, which is ten fold higher than the established maximum tolerable

level for Se, gave birth to lambs with only approximately four-fold higher plasma Se than

did controls. These results indicate that Se does cross the placenta to the fetus. Koller et

al. (1984) demonstrated maternal transfer of Se in beef cattle and Kim and Mahan (2001)

reported elevated serum and tissue Se in neonate pigs when dietary Se levels of sows

were increased. This does not concur with (Wright and Bell, 1964) who reported no

increase in lamb plasma Se when their dams were fed increased Se and demonstrated a

defined placental barrier for Se.

Plasma Se remained elevated in lambs which were suckling ewes receiving

increased dietary Se and from d 3 to d 56 ranged from 196 to 648 gg/L in year one and

244 to 775 jug/L in year two. These plasma Se concentrations were much higher than the

> 70 gg/L suggested as adequate by Zachara et al. (1993). However, at no time did any

lamb have plasma Se near or above 1400 jug/L which has been suggested as the plasma

level when signs of Se toxicosis appear in sheep (Glenn et al., 1964c) and swine (Kim

and Mahan, 2001). Marrow (1968) reported that death occurred within 16 hours in 35%

of nursing lambs which were dosed with 10 mg of sodium selenite orally in an attempt to

prevent nutritional muscular dystrophy. Smyth et al. (1990) observed death as soon as

six hours after an oral dose of 5 mg Se/kg BW. Contrarily, Lagace et al.(1964) dosed








lambs from two to 14 wk of age with 5 mg of sodium selenite via subcutaneous injection

every two wk and did not induce Se toxicity. Lambs on our study did not receive nearly

the amount of Se that others reported to be deadly, even from nursing dams supplemented

with Se up to 20 mg/kg during gestation and lactation. However, our lambs were

subjected to elevated milk Se concentrations. Based on data from Mellor and Murray

(1986) and Wohlt et al. (1984) milk intake in lambs from birth to 56 d ranges from 866-

1246 g/d. Given those intake estimates and the colostrum and milk Se concentrations

from the present study, lambs consuming the colostrum or milk with the highest Se

concentration at the highest intake would ingest 4.39 mg of Se/d. In newborn lambs (3

kg BW), that amount of Se would translate to 1.46 mg Se/kg BW and to 0.29 mg Se/kg

BW in 8 wk old lambs (15 kg BW). These levels are considerably less than levels

previously reported to cause death in young lambs.

Testes taken from ram lambs at 70 d were evaluated for Se concentration.

Selenium is implicated in sperm quality and reproductive function of livestock

(Hidiroglou, 1982; Marin-Guzman et al., 2000) and concentrations in testes are less than

in liver and generally greater than in heart, spleen, and pancreas. As with plasma Se in

the suckling lambs, testicular Se of lambs increased as Se increased in the ewe diets and

ranged from 1.67 mg/kg in controls to 4.25 mg/kg in lambs whose dams received 20

mg/kg dietary Se. These Se concentrations lie between those concentrations found in the

liver and heart of wethers consuming up to 10 mg/kg Se as sodium selenite for one year

(Cristaldi et al., in press). Those wethers were reported to also have elevated

concentrations of Se in serum, whole blood, wool, and other organs. However, they

displayed no clinical signs of selenium toxicosis








Implications

Feeding Se to gestating and lactating ewes above the current maximum tolerable

level (2 mg/kg) does increase the Se concentration in colostrum and subsequently

produced milk. However, this practice does not increase milk Se concentrations to a

level at which their nursing lambs suffer from Se toxicosis. Likewise, feeding increased

Se to ewes does increase plasma and tissue Se in lambs but not to a concentration above

those previously found in sheep determined not to be suffering from Se toxicity.

Moreover, data from other species even suggests that feeding increased Se to gestating

and lactating animals may produce colostrum of higher quality that may be beneficial to

their offspring. Data from this and other recent research has now established that the

maximum tolerable level of Se, as selenite, for sheep to be considerably higher than the

previously suggested 2 mg/kg.

Summary

The objective of this 504-d experiment was to evaluate and compare the effects of

six levels of dietary selenium (Se) on ewes' milk and the Se status of their lambs prior to

weaning. Sodium selenite was added to a basal diet to provide 0.2 (control), 4, 8, 12, 16,

and 20 mg/kg dietary Se for ewes during gestation and lactation over two lambings.

Colostrum Se ranged from 257 to 3542 gg/L and increased linearly as dietary Se

increased (P < 0.001) in both years. Ewe milk Se ranged from 75 to 2228 ptg/L and also

increased linearly as dietary Se increased (P < 0.01). In general, ewes receiving > 12

mg/kg Se produced higher milk Se than controls. Blood samples were collected from

lambs before nursing and at 3, 28, and 56 d of age to evaluate plasma Se concentrations.

At birth, lamb plasma Se ranged from 74 to 775 gg/L and was affected (P < 0.001) by the

Se concentration of the ewe diets, which indicates placental transfer of Se. Lambs from








ewes receiving dietary Se at 20 mg/kg had higher (P < 0.05) plasma Se than controls at

birth and 3, 28, and 56 d of age in both years. Selenium concentration in lamb testes

collected at 70 d of age was also affected by Se content of ewe diets. In year one, lambs

whose dams received 16 or 20 mg/kg Se had higher (P < 0.05) testicular Se than controls,

and no differences in testicular Se were observed in year two. No signs of Se toxicosis

were observed in lambs regardless of dietary Se concentration of the ewes' diet. These

results suggest that ewes consuming up to 20 mg/kg inorganic Se may give birth to

normal lambs, and that the lambs may not suffer from Se toxicosis before weaning.

Selenium as sodium selenite may be fed to ewes at concentrations greater than the current

maximum tolerable levels (2 mg/kg) without adversely affecting their offspring.








Table 4-1. Diet composition (as-fed) for Se (selenite) supplemented ewes
Ingredient % as-fed
Ground yellow corn 53.75
Cottonseed hulls 22.00
Soybean meal (47.5% CP) 16.00
Alfalfa meal (14% CP) 3.00
Soybean oil 3.00
Trace mineral mixb 1.00
Ground limestone 1.25
Vitamins A & D C
aSelenium levels in diet (as analyzed): 0.29, 3.77, 7.54, 11.01, 15.48, and 19.05 ppm
for treatments 0.2, 4, 8, 12, 16, and 20 ppm, respectively.
bTrace mineral mixture supplied between 96.5% and 98.5% NaCl, and provided per kg
of diet: 1.0 mg Co (as carbonate), 5.0 mg Cu (as oxide), 0.7 mg I (as iodate), 35 mg Fe
(as oxide), 25 mg Mn (as oxide), and 35 mg Zn (as oxide).
CProvided per kg of diet: 5,000 IU of Vitamin A and 500 IU of Vitamin D3.


Table 4-2. Colostrum selenium concentrations ([tg/L) of ewes receiving different levels
of selenium supplementation as sodium selenitea
Year of experiment
Added Se, mg/kg 11 22
0.2 257b 624 705' 517
4 1300bc 543 1452 bd 421
8 1889bcd 767 3256c 480
12 2072bcd 704 2373cd 462
16 3216c 767
20 3542 d 537 2925 d 741
aData represent least squares standard errors.
b, dMeans within columns lacking a common superscript differ (P < 0.05)
in = 4, 5, 3, 3, 3, and 6 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
2n = 4, 6, 5, 5, and 2 for Se levels 0.2, 4, 8, 12, and 20 mg/kg, respectively.








Table 4-3. Milk Se concentrations ( tg/L) from ewes receiving different levels of
dietary Se as sodium selenite in year one'


31
75 b 117
312 117
400bc 117


12 490c 144
16 920d 117
20 628ra 117
aData represent least squares means + SE.
b'c'dMeans within columns lacking a common
1n = 3, 3, 3, 2, 3, and 3 for Se levels 0.2, 4, 8,
2n 5, 5, 3, 3, 2, and 5 for Se levels 0.2, 4, 8,
3n = 5, 6, 3, 3, 3, and 6 for Se levels 0.2, 4, 8,


Table 4-4. Milk Se concentrations ([tg/L)
dietary Se as sodium selenite in year twoa


Days postpartum
28
660 91
121b91
163bc 117
189bc- 117
253bc 144
466c 91


56'
32 b91
165' - 83
160b 117
241' 117
653c 117
538c 83


superscript are different (P < 0.05).
12, 16, and 20 mg/kg, respectively.
12, 16, and 20 mg/kg, respectively.
12, 16, and 20 mg/kg, respectively.


from ewes receiving different levels of


Added Se, mg/kg
0.2
4


31
57b 4- 238
574 b 194
1442d 213


Days postpartum
282
81b 238
646b 180
462b 213


56 3
69b 238
339bc 194
493 b 4- 213


12 895cd 238 689b 238 914c 238
20 933cd 337 923b 475 2228d 476
aData represent least squares means SE.
b,'cdMeans within columns lacking a common superscript are different (P < 0.05).
in = 4, 5, 5, 5, and 2 for Se levels 0.2, 4, 8, 12, and 20 mg/kg, respectively.
2n = 4, 6, 5, 4, and 1 for Se levels 0.2, 4, 8, 12, and 20 mg/kg, respectively.
3n = 4, 5, 5, 4, and 1 for Se levels 0.2, 4, 8, 12, and 20 mg/kg, respectively.


Added Se, mg/kg
0.2
4
8






65


Table 4-5. Plasma Se concentrations ([tg/L) of lambs suckling ewes receiving different
levels of dietary Se as sodium selenite in year onea
Age of lamb, d
Se in ewe diet,mg/kg 01 32 28' 564
0.2 81b 66 111b 66 76b 52 92b 52
4 127be 60 204bc 56 196b 49 246c 49
8 131be 85 330c 73 209bc 73 258bed 73
12 188be 66 333c 85 374 d 66 354cd 66
16 238bc 85 387c 73 297c 73 431 d 66
20 2940 47 648d 60 508d 44 419d 47
aData represent least squares means SE.
b'cdMeans within columns lacking a common superscript are different (P < 0.05).
in 5, 6, 3, 5, 3, and 6 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
2n = 5, 7, 4, 3, 4, and 6 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
3n = 8, 9, 4, 5, 5, and 11 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
4n = 8, 9, 4, 5, 5, and 10 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.


Table 4-6. Plasma Se concentrations (gg/L) of lambs suckling ewes receiving different
levels of dietary Se as sodium selenite in year twoa
Age of lamb, d
Se in ewe diet,mg/kg 01 32 283 564
0.2 85' 62 74b - 55 81b 55 86b 55
4 182be 41 325c 44 244c 47 287c 47
8 186be 47 601d 47 314cd 55 263c 51
12 253 d 41 686d 51 553e 62 430d 55
20 353' 71 737d 71 775f 87 3401cd 124
aData represent least squares means SE.
b' 'd'e' Means within columns lacking a common superscript are different (P < 0.05).
in = 4, 9, 7, 9, and 3 for Se levels 0.2, 4, 8, 12, and 20 mg/kg, respectively.
2n = 5, 8, 7, 6, and 3 for Se levels 0.2, 4, 8, 12, and 20 mg/kg, respectively.
3n = 5, 7, 5, 4, and 2 for Se levels 0.2, 4, 8, 12, and 20 mg/kg, respectively.
4n = 5, 7, 6, 5, and I for Se levels 0.2, 4, 8, 12, and 20 mg/kg, respectively.



























Figure 4-1. Rambouillet ewe in late gestation fitted with a device to cover the udder and
prevent lambs from suckling until a blood sample was obtained. Device was made from
nylon pantyhose and elastic straps, and held in place with safety pins. (Device courtesy of
Dr. Donald J. Davis, Crossville, TN)


5.5

)5
0E 4.5

4

_3.5
'-,

0 3
2.5

S1.2
E
1.5-

1


0.2 4 8 12 16 20


Se content of ewe diet, mg/kg

Figure 4-2. Effects of Se concentration of ewe diet on testicular Se concentration of their
lambs in year one. Testicular selenium concentrations were 1.67, 1.83, 2.88, 3.26, 3.76,
and 4.25 mg/kg (dry matter basis) for ram lambs suckling ewes receiving 0.2, 4, 8, 12,
16, and 20 mg/kg dietary Se, respectively. SE = 0.56.













CHAPTER 5
COMPARATIVE EFFECTS AND TOLERANCE OF VARIOUS DIETARY LEVELS OF
SE AS SODIUM SELENITE OR SE YEAST ON BLOOD, WOOL, AND TISSUE SE
CONCENTRATIONS OF WETHER SHEEP

Introduction

Selenium was first implicated as an essential nutrient for animals by Schwarz and

Foltz (1957). Prior to that, Se was viewed primarily as a detriment to livestock which

was documented by Franke (1934) and Moxon (1937). Selenium deficiency is far more

prevalent worldwide than toxicity. However, Se toxicity is a greater concern to livestock

producers and nutritionists, as toxicities are more difficult than deficiencies to control.

Current estimates put the maximum tolerable level of Se at 2 mg/kg for the major

livestock species (NRC, 1980) and no differentiation exists for tolerable levels between

ruminants and monogastric animals. However, previous research suggests that inorganic

Se (e.g., sodium selenite) may be reduced to insoluble selenide by microorganisms in the

rumen, thus reducing overall absorption of Se by ruminant animals (Butler and Peterson,

1961; Hidiroglou et al., 1968). Wright and Bell (1966) reported that swine retained 77%

and sheep retained 29% of an oral dose of inorganic Se. The NRC makes no distinction

between inorganic and organic (e.g., Se yeast or seleno-methionine) forms of Se for

current maximum tolerable levels, though the chemical form of dietary Se leads to

markedly different physiological responses of livestock (Knowles et al., 1999; Pehrson et

al., 1999; Gunter et al., 2003). Kim and Mahan (2001) reported more accumulation of Se

in the plasma and tissues of swine fed high dietary levels of Se as Se yeast compared to

the same Se levels as sodium selenite, and that Se toxicity occurred sooner and its clinical








signs were more severe when inorganic Se was used as the dietary source. In concluding

that > 5 mg/kg dietary Se, regardless of source, did produce signs of Se toxicity in

growing swine, those authors postulated that the greater tissue retention of organic Se

may reduce the incidence of Se toxicity. Based on these findings and the increasing use

of organic forms of Se for supplementation to livestock, an experiment was conducted to

evaluate and compare effects of feeding Se as sodium selenite or Se yeast at high dietary

levels on serum, whole blood, wool, and tissue Se concentrations of wether sheep.

Materials and Methods

All animal procedures were conducted within the guidelines of and approved by

the University of Florida Institutional Animal Care and Use Committee. This experiment

was conducted from June 4, 2002 to July 29, 2003 at the University of Florida Sheep

Nutrition Unit located in southwestern Alachua County, FL. Twenty-eight, 2-yr-old,

Rambouillet-crossbred wethers were weighed (62.3 8.5 kg) and received 2-ml

ivermectin dewormer s.c. (Ivomec; Merial Ltd. Iselin, NJ). Wethers were randomly

assigned to one of eight dietary treatments for a 60-wk study. Dietary treatments were

arranged as a 2 x 4 factorial with 0.2, 20, 30, and 40 mg/kg Se (as-fed) as four dietary

levels and Se yeast and sodium selenite as two Se sources added to a corn-soybean meal-

cottonseed hull basal diet (Table 5-1). Feed-grade yeast was used as a carrier for the

sodium selenite in order to alleviate differences in the palatability and protein content of

the diets. The basal diet was formulated to meet animal requirements for protein, energy

as TDN, vitamins, and minerals for this class of sheep (NRC, 1985). Animal numbers

per treatment were three for 0.2 (control) and 20 mg/kg Se, and four each for 30 and 40

mg/kg Se treatments for both Se sources. Wethers were housed by treatment group in

covered wooden pens (53.5 m2) with earth floors and ad libitum water.








Diets were fed at 909 g'wether''d- throughout the experiment. Samples of each

diet were taken every 28 d, ground (1 mm), and frozen at 0C until analysis.

Wether BW was recorded on d 0 and for every eight wk thereafter, for the

remainder of the study. A 10-mL blood sample for serum analysis was collected using an

18-gauge needle into a vacutainer tube with no additive (Vacutainer; Becton Dickinson,

Franklin Lakes, NJ) every 12 wk, via jugular venipuncture, allowed to stand for 20 min,

centrifuged at 700 x g for 25 min, and serum stored frozen at 0C until Se analysis. An

additional 10 mL of blood was collected into a heparinized vacutainer tube (Vacutainer;

Becton Dickinson, Franklin Lakes, NJ). This additional 10-mL sample was also

collected every 12 wk for the remainder of the experiment and stored frozen at 0C as

whole blood until analysis.

The wool around the jugular was shorn initially and regrowth was collected

beginning at wk 12 and every 12 wk thereafter. The collected wool was washed with a

commercial hair shampoo (Alberto V05; Alberto-Culver Co., Melrose Park, IL), to

remove oil and dirt, rinsed well with deionized water, dried, stored at room temperature,

and later analyzed for Se.

At the termination of the experiment (wk 60), wethers were slaughtered by

stunning and exsanguination, following USDA procedures at the University of Florida

Meats Laboratory. Immediately prior to slaughter, a 10-mL sample of blood was

collected using an 18-gauge needle into a vacutainer, centrifuged at 700 x g for 25 min,

and serum frozen at 0C for analysis of albumin and the following enzymes: alkaline

phosphatase (Alk Phos), alanine transaminase (ALT), aspartate transaminase (AST),

creatinine phosphokinase (CK), and gamma glutamyl transferase (GGT).








Samples of brain, diaphragm, heart, hoof tip, kidney, liver, and psoas major

muscle were collected, and frozen (00C) until analyzed for Se. Sections (1 cm3) of liver,

heart, kidney, diaphragm, and psoas major muscle from all animals were placed in 10%

neutral-buffered formaldehyde for subsequent microscopic evaluation for evidence of Se

toxicosis.

For histopathological evaluation, the tissue samples fixed in buffered formalin

were embedded in paraffin and sectioned at 6 microns. All sections were stained with

hematoxylin and eosin, and examined under a light microscope (1OX, 20X, and 40X).

Serum albumin, Alk Phos, ALT, AST, CK, GGT were evaluated on a Hitachi 911

analyzer with reagents from Sigma (Sigma Chemical Co., St. Louis, Mo.). These

procedures were established by the Veterinary Medical Teaching Hospital at the

University of Florida.

Samples of kidney, heart, and liver were evaluated for cell structure changes using

transmission electron microscopy. Tissues were transferred to Trump's fixative, pH 7.2,

for 2 h at room temperature (240 C). Samples were then rinsed in O.1M sodium

cacodylate buffer at room temperature for 1 h. After three 15-min rinses with deionized

water, the tissues were placed in a 1% aqueous uranyl acetate solution for 45 min.

Samples were then dehydrated through a graded ethanol-acetone series at room

temperature. Tissue samples were then infiltrated with and embedded in Spurr's resin.

Silver sections (0.06 um) were cut using an RMC MT 6000XL ultramicrotone (RMC

Products; Boeckeler Instruments, Inc.,Tuscon, AZ) and mounted on formvar-coated

copper mesh grids. Sections were stained with 5% acidic uranyl acetate and Reynold's

lead citrate and examined in a Zeiss 100 microscope (Carl Zeiss, Inc, Thornwood, NY).








Serum, whole blood, wool, tissue, and feed samples were analyzed for Se

concentration using a fluorometric method described by Whetter and Ullrey (1978). To

help ensure reliability of the analytical method, a certified standard (National Bureau of

Standards Bovine Liver SRM-1577a; U.S. Department of Commerce, National Institute

of Standards and Technology, Gaithersburg, MD) was frequently analyzed.

Brain, diaphragm, heart, hoof tip, kidney, liver, and psoas major muscle Se data

were analyzed for effects of treatment using PROC GLM in SAS (SAS for Windows 8e;

SAS Inst., Inc., Cary, NC) in a 2 x 4 factorial arrangement. Pre-planned orthogonal

contrast statements were used to compare means as described by Littell et al. (1998;

2000). PROC MIXED of SAS was used to analyze effects of treatment, time, and the

interaction of treatment x time on BW, serum Se, whole blood Se, and wool Se as

repeated measures with a spatial power covariance structure with respect to d and a

subplot of animal nested within treatment. Pre-planned orthogonal contrast statements

were written to determine differences in means at different sampling intervals.

Regression analysis was used to determine relationships between dietary Se and Se

concentration in serum, whole blood, wool, and tissues.

Results and Discussion

Wether BW was affected by dietary Se level (P < 0.05), source of dietary Se (P <

0.05), time (P < 0.05), and average BW decreased linearly (P < 0.10) as dietary Se level

increased (Table 5-2). Body weights of wethers receiving 30 or 40 mg/kg dietary Se as

Se yeast decreased from wk 0 to wk 60, whereas wethers receiving all other dietary Se

treatments gained weight from wk 0 to wk 60. Previous studies have reported no effect

of Se fed above requirements on BW of feedlot cattle (Perry et al., 1975) and no effects

on BW when included up to 10 mg/kg in the diets of wether sheep (Cristaldi et al., in








press). Likewise, Ullrey et al. (1977) reported that lamb BW were unaffected by dietary

Se level in feeds containing differing proportions of organic and inorganic Se. However,

Kim and Mahan (2001) reported a quadratic decrease in final BW of swine as dietary Se

level was increased using sodium selenite or Se yeast. Those authors observed the most

drastic decreases when selenite Se was added above 10 mg/kg and when Se yeast was

added at 20 mg/kg. Our results differ from the findings with swine, as organic Se had a

more dramatic deleterious affect on BW than did selenite Se. This could be explained by

organic Se not being subject to reduction to selenides by rumen microorganisms as

suggested by previous research (Whanger et al., 1968; van Ryssen et al., 1989; Whanger,

2002) and thus, being more available to cause toxic effects in ruminant livestock.

Seven of 28 wethers died during the course of our study and were subjected to

gross necropsy by pathologists at the University of Florida Veterinary Teaching Hospital.

All wethers were described in good physical condition at time of necropsy with adequate

adipose tissue. Causes of death were determined to be spinal cord compression trauma,

endoparasitism, pulmonary edema, and unknown. One wether from the 20 mg/kg

organic Se group had mild hepatic lipidosis and one wether from the 30 mg/kg organic Se

group exhibited signs of mild myocarditis. However, definitive evidence of death due to

Se toxicosis was not found and the gross lesions seemed to be due to metabolic changes

or were merely background findings.

Serum selenium values (105 23 gg/L) were below the normal range (120 to 150

gg/L) for adult sheep (Aitken, 2001) and did not differ among treatment groups (P =

0.36) at the initiation of the experiment. Serum Se concentrations measured at wk 12, 24,

48, and 60 ranged from 110 to 3922 ptg/L and increased linearly (P < 0.05) as dietary Se








level increased, while a quadratic response (P < 0.05) was observed at wk 36 (Table 5-3).

Serum Se concentrations of wethers were affected by dietary Se level, Se source, and the

interaction of dietary Se level x Se source interaction (P < 0.05). Likewise, over the

entire trial serum Se increased quadratically (P < 0.05) as dietary Se level increased.

Wethers receiving organic Se had higher (P < 0.001) serum Se than did selenite treated

animals throughout the study. Kim and Mahan (2001) reported a linear increase in

plasma Se of swine as dietary Se was increased as organic or inorganic Se. Likewise,

those authors reported an effect of source of Se, with pigs supplemented with organic Se

having higher plasma Se than their inorganically supplemented counterparts. Cristaldi et

al. (2004) reported a linear increase in serum Se as dietary Se was increased, however

those authors used a maximum level of 10 mg/kg dietary Se as selenite. The quadratic

response observed in the present study suggests homeostatic regulation of Se in blood as

dietary levels exceed 30 mg/kg. Serum Se in wethers fed up to 10 mg/kg selenite Se for

52 wk reached 870 gg/L (Cristaldi et al., in press). Wethers in the present experiment

exceeded 870 jig/L when receiving either Se source at 20, 30, or 40 mg/kg and at wk 24

wethers receiving 30 mg/kg organic Se had more than four-fold higher serum Se than the

maximum serum Se reported by Cristaldi et al. (2004). Our data show that at most

collections organic Se produced serum Se of more than double the concentration

produced by feeding selenite Se at the same level. Wethers receiving 20, 30 or 40 mg/kg

organic Se had serum Se above 1500 gg/L throughout the experiment. Aitken (2001)

reported serum Se of 1500 gg/L as a level at which signs of toxicity appear in horses.

Likewise, Aitken (2001) reported that serum Se of 3700 gg/L was evident of Se toxicosis





74

in swine. At wk 24, wethers receiving 30 mg/kg organic Se had serum Se of 3922 gg/L.

At no other time during our study did wether serum Se exceed 3700 gg/L.

Whole blood Se was measured in addition to serum Se because of the possibility

of a more accurate Se measurement since use of whole blood eliminates the possibility of

falsely high Se readings in serum due to hemolysis (Maas et al., 1992). Whole blood Se

responded to Se supplementation in much the same fashion as did serum Se (Table 5-4).

This response supports the high correlation between serum Se and whole blood Se

previously described by Maas et al. (1992) and Cristaldi et al. (2004). Whole blood Se

concentrations of wethers were affected by dietary Se level, Se source, and the interaction

of dietary Se level x Se source interaction (P < 0.05) ranged from 392 to 6259 gg/L, and

overall increased quadratically (P < 0.01) as Se concentration of the wether diets was

increased. Whole blood Se concentrations measured at wk 12, 24, 48, and 60 increased

linearly (P < 0.10) as dietary Se level increased. Whole blood Se in swine increased

linearly as dietary Se as sodium selenite was increased from 0 to 20 mg/kg (Goehring et

al., 1984b) and whole blood in sheep responded linearly to increased dietary Se (Cristaldi

et al., in press). Likewise, those authors reported a strong correlation between Se

concentrations of serum and whole blood. In the present study, whole blood Se

responded in neither a linear nor quadratic manner at wk 36 (P > 0.15). It seems that

organic Se was used in place of inorganic Se for the selenite control diet during that

feeding period, which created a whole blood Se concentration of 1004 gg/L for the

selenite control group at wk 36. On average, whole blood Se responded quadratically (P

< 0.05) as dietary Se level was increased, again suggesting the influence of homeostatic

regulation when dietary Se is increased above 30 mg/kg. Wethers receiving organic Se








had higher (P < 0.001) whole blood Se than did wethers receiving inorganic Se

throughout the study. The maximum whole blood Se concentration observed during our

study was 6259 gg/L. This concentration is well above the range of 2000 to 4000 Ptg/L

for whole blood Se, where clinical signs of Se toxicosis should appear (Rosenfeld and

Beath, 1945; 1946) and likewise is greater than a whole blood Se concentration of

4000gg/L, that Maag and Glenn (1967) described as the blood concentration above which

steers became depressed and inactive. However, wethers on the present study, with the

highest whole blood Se concentrations (> 6200 pg/L) did not exhibit signs of Se toxicosis

(e.g. wool loss, anorexia, abnormal hoof growth). Glenn et al. (1964a) fed sodium

selenate at high levels to range ewes that were similar in BW and breed type to the

wethers on the present study. Those researchers did not induce death by Se toxicosis

with daily oral doses less than 25 mg Se/ewe. Of the 17 deaths reported in their

experiment, only one was induced with a daily dose of 25 mg Se/ewe. Eight deaths were

induced with a daily dose 37.5 mg Se/ewe and eight deaths were induced with a daily

dose 50 mg Se/ewe. Those reported deaths were not by acute Se toxicosis; rather the

ewes received experimental Se doses for at least 80 d before death by Se toxicosis was

induced. In the same experiment, Glenn et al. (1964a) further suggested an avg minimum

toxic level of Se for adult sheep to be 0.825 mg/kg BW when fed for 100 d. Using this

estimate, the minimum toxic level of Se for sheep of the size used in our study would be

51.4 mg/d. Selenium consumption of wethers receiving the highest dietary Se level (40

mg/kg) was 78% of the aforementioned minimum toxic level for sheep. Blodgett and

Bevill (1987) reported the LD50 for sheep, using sodium selenite via i.m. injection, to be

0.7 mg Se/kg BW. Wethers of avg BW, on our study, receiving 1 kg of diet containing








40 mg/kg Se received 91.1% of that LD50 for sheep throughout the experiment.

Furthermore that LD50 for sheep (Blodgett and Bevill, 1987) was established using

injectable Se. Administration of Se parenterally disallows the reduction of selenite Se to

insoluble selenide via ruminal microorganisms as described by (Whanger et al., 1968).

This would suggest that the LD50 for Se in sheep could be considerably higher than

previously thought.

Selenium concentration in new growth wool was measured at wk 12, 24, 36, 48,

and 60 (Table 5-5). Dietary Se level, Se source, time, dietary Se level x Se source, and

dietary Se source x time affected (P < 0.05) wool Se. Wool Se ranged from 1.19 to 39.09

mg/kg and increased linearly (P < 0.001) as dietary Se increased. Wool Se from wethers

receiving organic Se was often more than three-fold higher (P < 0.001) than from wethers

receiving selenite Se at the same dietary level. Increased Se in hair has been reported in

other livestock species. Kim and Mahan (2001) observed a linear response in the hair of

pigs as Se in their diet was increased. Goehring et al. (1984b) reported a quadratic

response in the hair of swine as dietary Se as sodium selenite was increased up to 20

mg/kg. Likewise, Perry et al. (1976) reported increased Se in the hair of feedlot steers as

dietary selenite Se was increased. Cristaldi et al. (2004) reported a linear increase in the

wool of wether sheep as dietary Se was increased. Those authors did not report a

significant Se level x time interaction. However, wool Se of sheep, on the present study

was affected by time and the interaction of Se source x time as wool Se continued to

increase from wethers fed selenite Se and wool Se from wethers receiving organic Se

increased then seemed to reach a peak around wk 48. This suggests that wool Se may

reach a plateau when animals are fed high dietary concentrations of organic Se. Wool Se








concentrations in the present study were more than ten-fold higher than concentrations of

2 to 2.5 mg/kg in wool from wethers fed up to 10 mg/kg dietary Se as selenite (Cristaldi

et al., in press), but never exceeded 40 mg/kg which is less than 45 mg/kg which was

described as the Se concentration in hair of animals suffering from alkali disease

(National Academy of Sciences [NAS], 1971).

Selenium concentrations in brain, diaphragm, heart, kidney, and loin muscle were

affected (P < 0.05) by dietary Se level, Se source, and dietary Se x Se source interaction.

Hoof Se concentration was affected by source (P < 0.05) and liver Se was affected (P <

0.05) by dietary Se level and dietary Se x Se source interaction, and tended to be affected

(P = 0.11) by Se source. Selenium concentrations, on a DM basis, were highest in liver

followed by kidney, heart, hoof, brain, loin, and diaphragm (Table 5-6). This pattern is

similar to a ranking of Se concentrations in tissues of farm animals by (Combs and

Combs, 1986) with the exception of liver and kidney being reversed. However, in

animals fed Se at or below requirements, kidney generally has a higher concentration of

Se than does the liver, but when dietary Se is increased, liver Se quickly becomes higher

in Se than kidney. This supported by the work of Ewan et al. (1968) and the findings of

Cristaldi et al. (2004) in sheep, and McDowell et al. (1977) in swine. Those authors

reported higher liver Se than kidney Se when dietary Se was increased. Unlike minerals

such as Zn and Mn, the status of Se is reflected in many tissues (McDowell, 2003). Brain

Se concentrations ranged from 1.28 to 32.3 mg/kg and brain Se concentrations from

wethers receiving organic Se were higher (P < 0.001) than brain Se from wethers

receiving selenite Se. These results suggest that Se does cross the blood-brain barrier and

that brain Se is influenced by dietary Se. Previous research using sheep supports our








findings of increased Se in brain as dietary Se is increased (Yeh et al., 1995;1997;

Ouazzani et al., 1999). Diaphragm Se ranged from 0.82 to 26.34 mg/kg and tended to

increase linearly (P = 0.089) as dietary Se increased. Diaphragm Se was higher (P <

0.001) in wethers receiving organic Se than from wethers receiving selenite Se. Heart Se

ranged from 1.59 to 33.93 mg/kg and, like brain and diaphragm Se was higher (P <

0.001) in wethers receiving organic Se than from wethers receiving selenite Se.

Selenium concentrations in the hoof tip ranged from 3.44 to 29.20 mg/kg and increased

linearly as dietary Se increased (P < 0.05). Selenium concentrations of hoof tip taken

from wethers receiving organic Se tended (P = 0.07) to be higher than from wethers

receiving inorganic Se. Both Se sources produced hoof Se concentrations higher than 10

mg/kg which was previously reported in animals with alkali disease (NAS, 1971).

Kidney Se tended (P = 0.07) to respond linearly to increased dietary Se and ranged from

8.43 to 77.61 mg/kg. Kidney Se concentrations from wethers receiving organic Se were

higher (P < 0.01) than from wethers receiving selenite Se. Kidney Se concentrations

from the present study are much higher than those reported by Maag and Glenn (1967)

where death due to Se toxicosis was produced in 245 kg Hereford steers. However, the

calves used by those authors received approximately 270 mg Se-steerl-d-1 as sodium

selenite and death was induced within 6 wk. Liver Se concentrations ranged from 2.66

to 132.73 mg/kg and increased linearly (P < 0.001) as dietary Se level increased.

Selenium concentrations in liver from wethers receiving organic Se were not different (P

= 0.34) than liver Se concentrations from wethers receiving selenite Se. Selenium

concentrations in the loin muscle (psoas major), which is often consumed by mankind

ranged from 0.71 to 26.87 mg/kg and tended (P = 0.12) to increase linearly as dietary Se








was increased. Organic Se was more effective (P < 0.001) at increasing Se

concentrations in edible tissue than was selenite Se. As daily intake of Se by humans

declines in some parts of the world, increasing the Se content of foods for human

consumption by manipulating source and level of Se supplementation to livestock is now

of interest to food scientists. Givens et al. (2004) suggested that the Se content of cows'

milk could be increased through the use of Se yeast as the supplemental form of Se to

dairy cows. Our findings indicate that Se content of muscle and organ tissue can be

influenced by source and level of Se supplementation to food animals. In general, Se

concentrations of brain, heart, kidney, liver, and muscle were much higher than those

reported in studies with cattle (Maag and Glenn, 1967) and sheep (Glenn et al., 1964c).

Deaths due to Se toxicosis were induced in both species. In contrast, Se death due to Se

toxicosis was never produced during our study. It is important to note that in the two

previous studies that animals were fed Se at higher levels and for a shorter period of time.

Our findings further agree with Smith et al. (1937) who found that the effects of

continued dosing of Se were cumulative and that Se from organic sources was

accumulated in higher quantities in tissues than Se from inorganic sources.

Most of the heart, diaphragm, loin, liver, and kidney tissues subjected to

histopathological evaluation were free from pathological changes. The findings of

lymphocytes in the portal triads were deemed to be a background finding and

insignificant. Three instances of vacuolic degeneration associated with the cytoplasm of

hepatocytes suggesting fatty degeneration were noted. However, no pattern associating

abnormal pathology to either dietary Se level or source could be established. Therefore,

lesions could not be definitively linked to treatment and could have been metabolic in








nature. Cristaldi et al. (2004) found no abnormalities after microscopic evaluation of

heart, liver, kidney, diaphragm, and muscle from wethers consuming up 10 mg/kg Se for

one yr. Examination of kidney, heart, and liver tissues by transmission electron

microscopy did not reveal any apparent changes in cell structure as related to Se toxicosis

and no differences in tissue cells from controls and wethers receiving 20, 30, or 40 mg/kg

Se were observed.

Concentrations of albumin and activities of Alk phos, ALT, GGT, AST, and CK

in serum collected at the termination of the experiment were, in general, in or below the

normal range for adult sheep (Table 5-7). In instances of Se toxicosis, the activities of

these enzymes would have been increased due to tissue necrosis. Our observations agree

with those reported by Cristaldi et al. (2004) as albumin and enzyme activities in wether

sheep after receiving up to 10 mg/kg Se were in the normal ranges. The lack of elevated

enzymes, which are suggestive of tissue necrosis, further indicates that the wethers on our

study were not suffering from Se toxicosis.

Throughout this 60-wk experiment clinical signs of Se toxicosis (e.g., lameness,

wool loss, and abnormal hoof growth) were not observed, though serum and whole blood

Se concentrations were frequently higher than those described in livestock diagnosed

with hyperselenosis. However, wool Se concentrations from wethers on our study never

reached the levels previously reported in the hair of livestock suffering from alkali

disease. Loin muscle and diaphragm showed no gross lesions at slaughter and no

abnormality was observed with microscopic evaluation. Abnormal pathology in the

kidney, heart, and liver was rare and could, in each case, be attributed to a cause other

than Se toxicosis. No pale focal areas were observed in the myocardium, though








previous research (Glenn et al., 1964b; Smyth et al., 1990) has shown the heart to be the

target organ in instances of Se toxicosis. No abnormalities were prevalent enough to

establish a treatment-related pattern and no wethers receiving the maximum level of

dietary Se on our study (40 mg/kg) showed any abnormal tissue lesions. Further

evaluation of kidney, heart, and liver using transmission electron microscopy also

revealed no cellular abnormalities and enzymes, suggestive of tissue necrosis, were in or

below normal ranges at the termination of the experiment. Without the presence of tissue

damage and clinical signs, it seems that Se toxicosis was not induced in wether sheep fed

up to 40 mg/kg dietary Se as Se yeast or sodium selenite. However, Se concentrations in

serum, blood, wool, and tissues from wethers receiving organic Se indicate that Se

toxicity is dependent on Se source and that much inorganic dietary Se is reduced to

insoluble forms. The work of Cousins and Caimey (1961), Whanger et al. (1968), and

Koenig et al. (1997) support our findings.

Implications

The current estimate of the maximum tolerable dietary level of selenium for sheep

(2 mg/kg) seems to be grossly underestimated. Selenium, whether organic or inorganic in

form, can be fed as high as 40 mg/kg for up to 60 wk without inducing Se toxicosis.

Previously the range between optimal and toxic levels of selenium was reported as

narrow; however data from the present study would suggest that the range is relatively

wide. Increasing dietary selenium level, regardless of source, is an effective means of

increasing selenium in blood and tissues. Organic selenium is more greatly accumulated

by organs and tissues. Manipulation of dietary selenium source and level is an effective

way to change the selenium content of animal tissues commonly consumed by mankind.








Summary

The objectives of this 60-wk experiment were to evaluate and compare effects of

feeding Se as sodium selenite or Se yeast at high dietary levels on serum, whole blood,

wool, and tissue Se concentrations in wether sheep and determine maximum tolerable

level of Se. Twenty-eight, 2-yr-old, Rambouillet-crossbred wethers (62.3 8.5 kg initial

BW) were utilized in a 2 x 4 factorial arrangement with 0.2, 20, 30, and 40 mg/kg dietary

Se (as-fed) from sodium selenite or Se yeast added to a corn-soybean meal basal diet.

Wethers were weighed at 8-wk intervals, serum Se, whole blood Se, and wool Se were

measured every 12 wk, and samples of brain, diaphragm, heart, hoof, kidney, liver, and

loin muscle and serum samples for evaluation of albumin and enzyme activities were

collected at the termination of the experiment. Wether BW was affected by dietary Se

level (P < 0.05), source of dietary Se (P < 0.05), and time (P < 0.05). Average BW

decreased linearly (P < 0.10) as dietary Se level increased, though most wethers gained

BW. Serum Se, whole blood Se, and wool Se concentrations were affected (P < 0.05) by

dietary level of Se and source of Se. Serum Se and whole blood Se ranged from 110 to

3922 gg/L and 392 to 6259 gg/L, respectively, and increased in a quadratic fashion as

dietary Se level increased (P < 0.05) and wool Se ranged from 1.19 to 39.09 mg/kg and

responded linearly (P < 0.05) to increased dietary Se. Serum Se, whole blood Se, and

wool Se concentrations from wethers receiving organic Se were higher (P < 0.01) than

those from wethers receiving inorganic Se. Selenium concentrations in brain, diaphragm,

heart, hoof, kidney, liver, and loin muscle were affected (P < 0.05) by dietary Se level,

with higher Se concentrations generally observed in tissues from wethers receiving

organic Se. Though Se concentrations in serum, blood, wool, and major organs at most

times exceeded concentrations previously reported in livestock suffering from Se








toxicosis, a pattern of clinical signs of Se toxicosis was not observed in this experiment.

Microscopic evaluation of liver, kidney, diaphragm, heart, and psoas major muscle did

not reveal definitive evidence of Se toxicosis in wethers on any dietary Se treatment.

Wethers under our experimental conditions tolerated up to 40 mg/kg dietary Se for 60

wk, though differences in Se source were observed. Contrary to previous thought, the

range between optimal and toxic dietary level of Se is not narrow. The maximum

tolerable level of dietary Se, regardless of source, is much higher than the current

estimate of 2 mg/kg.









Table 5-1. Diet composition (as-fed) for Se supplemented wethersa
Ingredient % as-fed
Ground yellow corn 58.00
Cottonseed hulls 30.00
Soybean meal (47.5% CP) 6.50
Soybean oil 3.00
Trace mineral mixb 1.00
Ground limestone 1.00
Ammonium chloride 0.50
Vitamins A & D C
aSelenium levels in diet (as analyzed): 0.48, 20.48, 30.86, and 38.10 mg/kg for Se levels 0.2, 20, 30, and
40 mg/kg from sodium selenite, respectively; 0.54, 20.26, 30.71 and 37.65 mg/kg for Se levels 0.2, 20,
30, and 40 mg/kg Se from Se yeast, respectively
bTrace mineral mixture supplied between 96.5% and 98.5% NaC1, and provided per kg of diet: 1.0 mg
Co (as carbonate), 5.0 mg Cu (as oxide), 0.7 mg I (as iodate), 35 mg Fe (as oxide), 25 mg Mn (as oxide),
and 35 mg Zn (as oxide).
'Provided per kg of diet: 5,000 IU of Vitamin A and 500 IU of Vitamin D3.


Table 5-2. Effects of four dietary levels of Se as sodium selenite or Se yeast on BW of
wethersa
Se source
Sodium selenite- Se yeast
-Dietary Se level, mg/kg
0.2 20 30 40 0.2 20 30 40
Week Wether BW, kg SEM
0 61.2 65.0 65.8 58.9 57.0 55.5 67.9 64.3 4.5
8 59.1 63.6 59.8 49.3 56.8 51.5 54.3 50.6 4.5
16 61.8 68.2 57.9 48.2 57.9 55.1 54.8 50.0 5.70
24 65.1 70.0 59.2 52.4 60.0 57.3 53.3 48.2 7.5e
32 70.9 76.7 63.6 56.1 68.2 59.5 51.1 52.7 9.0e
40 70.5 61.8 61.8 57.3 74.7 51.8 47.3 50.9 8.4
48 77.6 81.8 69.8 62.9 70.5 65.5 38.6 54.5 9.0c
60 83.3 85.6 76.5 67.9 78.2 61.8 50.2 54.5 10.4
Avg 68.7 71.6 64.3 56.6 65.4 57.3 52.2 53.2 7.2bcde
aData represent least squares means and pooled SE.
bDietary Se level response (P < 0.05).
'Selenium source response (P < 0.05).
dTime response (P < 0.05).
e Dietary Se level linear response (P < 0.10).






85


Table 5-3. Serum Se concentrations of wethers fed four dietary levels of Se as sodium
selenite or Se yeast
Se source
-Sodium selenite Se yeast
Dietary Se level, mg/kg
0.2 20 30 40 0.2 20 30 40
Week Serum Se, tg/L SEM
12 157 548 788 1000 412 2583 3210 2458 249bcdc
24 130 1683 1487 1724 354 2639 3922 1585 826be
36 444 851 960 1083 540 3283 2086 1409 250bdf
48 110 822 1219 1496 292 2428 2076 1831 253bce
60 119 610 886 1250 424 1699 2712 2549 331bee
Avg 192 903 1068 1311 404 2526 2801 1966 395bedf
aData represent least squares means and pooled SE.
bDietary Se level response (P < 0.05).
'Selenium source response (P < 0.05).
dDietary Se level x Se source interaction (P < 0.05).
eDietary Se level linear response (P < 0.05).
fDietary Se level quadratic response (P < 0.05).


Table 5-4. Whole blood concentrations of wethers fed four dietary levels of Se as
sodium selenite or Se yeasta
Se source
Sodium selenite-- Se yeast
Dietary Se level, mg/kg-
0.2 20 30 40 0.2 20 30 40
Week Whole blood Se, gtg/L SEM
12 392 1172 1484 1315 1183 4344 4290 5484 475bcde
24 420 1551 2228 2353 1661 4521 6259 5780 415bee
36 1004 1021 1708 2406 1549 5018 4841 1972 635c
48 393 1772 1977 2416 1068 5061 5220 4929 298bcde
60 402 1258 1621 2043 1500 1759 3629 4914 435bee
Avg 522 1355 1804 2107 1392 4028 4803 4408 534bcdf
aData represent least squares means and pooled SE.
bDietary Se level response (P < 0.05).
cSelenium source response (P < 0.05).
dDietary Se level x Se source interaction (P < 0.05).
e Dietary Se level linear response (P < 0.10).
fDietary Se level quadratic response (P < 0.05).






86


Table 5-5. Wool Se concentrations of wethers fed four dietary levels of Se as sodium
selenite or Se yeast
Se source
Sodium selenite Se yeast
Dietary Se level, mg/kg
0.2 20 30 40 0.2 20 30 40
Week Wool Se, mg/kg SEM
12 1.37 3.27 6.69 4.15 3.78 12.67 21.09 24.26 3.80ce
24 1.47 3.57 5.72 11.92 7.04 31.58 35.69 37.30 2.87bd
36 1.68 6.02 9.85 10.85 5.70 18.99 22.79 21.29 4.72ce
48 1.19 3.15 5.64 7.23 6.39 24.81 39.09 29.65 2.22bcd
60 1.29 3.90 5.01 6.23 4.38 23.22 25.65 25.99 2.01bcd
Avg 1.40 3.98 6.58 8.08 5.46 22.25 28.87 27.70 3.38bcdefg
aData represent least squares means and pooled SE.
bDietary Se level response (P < 0.05).
cSelenium source response (P < 0.05).
dDietary Se level x Se source interaction (P < 0.05).
'Dietary Se level linear response (P < 0.10).
fTime response (P < 0.05).
gTime x Se source interaction (P < 0.05).


Table 5-6. Effects of four dietary levels of Se as sodium selenite or Se yeast on tissue
Se of wethersa
Se source
Sodium selenite- Se yeast
Dietary Se level, mg/kg
0.2 20 30 40 0.2 20 30 40
Tissue Se concentration, mg/kg SEM
Brain 1.28 4.22 4.74 6.87 6.12 21.90 32.30 18.71 0.99bcd
Diaphragm 0.82 4.74 3.33 7.81 5.28 10.30 26.34 20.71 2.69bdc
Heart 1.59 3.80 5.13 6.23 6.35 23.77 28.71 33.93 2.43cd
Hoof 3.44 8.79 9.68 13.78 6.26 12.53 29.20 23.66 5.52ce
Kidney 8.43 19.94 27.93 27.89 22.26 33.96 77.61 36.28 6.87bcde
Liver 2.66 31.72 41.42 78.18 15.67 23.42 132.73 41.24 18.17de
Loin 0.71 3.13 4.41 5.13 5.73 14.69 23.51 26.87 1.05bcd
aData represent least squares means and pooled SE.
bDietary Se level response (P < 0.05).
'Selenium source response (P < 0.05).
dDietary Se level x Se source interaction (P < 0.05).
eDietary Se level linear response (P < 0.10).






87


Table 5-7. Amount of albumin and tissue enzyme activities present in serum of Se
supplemented wethers'ab
Se source
Sodium selenite-- Se yeast
Dietary Se level, mg/kg
0.2 20 30 40 0.2 20 30 40 Normal
Enzyme Se concentration, mg/kg .. range
Albumin, g/dL 3.0 3.0 2.9 2.7 2.8 2.0 2.5 2.9 2.4 4.0
AlkPhos, IU/L 127.3 141.7 128.8 153.5 105.7 32.0 50.5 172.0 68-387
ALT, IU/L 12.0 12.0 10.8 9.3 9.7 3.0 2.5 6.0 11 -40
AST, IU/L 81.7 100.7 108.8 81.8 94.7 83.0 65.0 53.0 60-280
GGT, IU/L 49.3 51.7 61.8 60.5 54.3 42.0 46.5 59.0 15 -60
CK, IU/L 123.7 139.7 75.0 103.0 109.0 48.0 50.0 51.0 00-584
aSerum sample collected at wk 60.
bGGT and CK ranges were established by University of Florida Veterinary Teaching Hospital.













CHAPTER 6
EFFECTS OF FORM OF PARENTERAL OR DIETARY SELENIUM
SUPPLEMENTATION ON BODY WEIGHT AND BLOOD, LIVER, AND MILK
CONCENTRATIONS IN BEEF COWS

Introduction

Many areas of the United States have selenium deficient soils (McDowell, 2003)

and may produce forages and grains which are unable to provide adequate Se to

livestock. Selenium deficient brood cows may give birth to calves which are stillborn,

premature, weak, or afflicted with nutritional muscular degeneration (Maas, 1983; Corah

and Ives, 1991). Likewise, even with adequate blood Se at birth, calves suckling Se

deficient dams are susceptible to becoming Se deficient (Pehrson et al., 1999). Without

adequate dietary or parenteral Se supplementation, brood cows may suffer from

infertility, retained placentas, ovarian cysts, metritis, silent estrus periods, and/or poor

weight gains (Dargatz and Ross, 1996).

In cattle, it has been well established that Se crosses the placenta (Koller et al.,

1984; Van Saun et al., 1989), that dietary Se is transferred to milk (Conrad and Moxon,

1979), and that positive correlations exist between blood Se of cows and blood Se of their

calves (Kincaid and Hodgson, 1989; Enjalbert et al., 1999; Pehrson et al., 1999). The

chemical form of Se affects its metabolism and previous research has shown differences

in blood, milk, and liver Se concentrations due to form (organic vs inorganic) of

supplemental Se (Knowles et al., 1999; Gunter et al., 2003; Valle et al., 2002).

Selenium is often supplemented as sodium selenite and included in free-choice

livestock mineral mixtures. However, Se may be supplemented through subcutaneous








injection of barium selenate, sodium selenate or sodium selenite and, in ruminants, with

slow-release, long lasting ruminal Se boluses or pellets. With the recent Food and Drug

Administration approval of Se yeast for use in ruminant diets, livestock producers now

have more choices of form and method of Se supplementation. The objective of this

experiment was to evaluate and compare effects of form and method of Se

supplementation on blood, liver, and milk Se concentrations in beef cows.

Materials and Methods

All animal procedures were conducted within the guidelines of the University of

Florida Institutional Animal Care and Use Committee. Animals were housed at the

University of Florida Boston Farm-Santa Fe Beef Unit located in Northern Alachua

County, Florida. On August 6, 2002, 43 Angus cows, aged 2-3 yr, (mean age = 2.67 yr)

were palpated to diagnose pregnancy and estimate d in gestation. All cows were

determined pregnant and gestation estimates ranged from 115 to 130 d. Each animal

received a chemically altered modified live 4-way viral + vibriosis and leptospirosis

vaccination (Cattlemaster 4+VL-5; Pfizer Animal Health, Exton, PA) and fly control

(Permectrin 10% EC pour-on; Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO)

according to manufacturer directions. Cows were weighed (average initial BW = 417

46 kg), stratified by age and assigned to one of five treatment groups for a 365 d study.

The five treatments were 1) no Se supplementation, control group, 2) one subcutaneous

injection of 9 mL (50 mg Se'mL1) barium selenate (Deposel Multidose; Novartis New

Zealand, Ltd., Auckland, NZ) at the initiation of the experiment, 3) three subcutaneous

injections of 5 mL (5 mg SemL1) of sodium selenite + 68 IU vitamin E as dl-alpha

tocopheryl acetate (Mu-Se; Schering-Plough Animal Health, Union, NJ), one at the

initiation of the experiment and one every four mo thereafter, 4) free-choice access to a








mineral mixture containing 26 mg Se/kg as sodium selenite (Southeastern Minerals, Inc.,

Bainbridge, GA), or 5) free-choice access to a mineral mixture containing 26 mg Se/kg

as Se yeast (Sel-Plex; Alltech, Inc, Nicholasville, KY). All cows grazed bahiagrass

(Paspalum notatum) pastures and were supplemented with bahiagrass (Paspalum

notatum) hay, molasses-based liquid supplement ad libitum, and whole cottonseed and

pelleted citrus pulp at rates of 0.68 kg-cow-ld1 and 1.81 kg-cowl-d1, respectively, from

November, 2002 through March, 2003.

Treatment groups receiving no Se or injectable Se were housed together and had

access to a free-choice mineral mixture containing no Se (Table 6-1). Cows receiving Se

via free-choice mineral mixtures were housed in separate groups and had access to the

same mineral mixture with added Se as sodium selenite or Se yeast for treatments 4 and

5, respectively (Table 6-1). All free-choice mineral mixtures were offered in wooden

mineral feeders and protected from rain.

Blood samples for plasma analyses were collected via jugular venipuncture into

10-mL heparinized tubes (Vacutainer; Becton-Dickinson, Franklin Lakes, NJ) at the

initiation of the study (d 0) and at d 365. Calving occurred over a 24 d span between

December 31, 2002 and January 23, 2003 and whole blood samples were collected in the

same manner from all cows immediately after parturition and at 30, 90, and 205 d

postpartum. A colostrum or milk sample was also collected on those days into a 15-mL

plastic centrifuge tube. Forty-one calves, 24 male, 17 female, were born alive and

unassisted. One cow in the free-choice selenite group had a stillbirth and one control cow

aborted very early in the experiment and both were removed from the study. Liver

biopsies were performed on all cows using the technique described by Chapman et al.




Full Text
Fellow graduate students Deke Alkire, Carlos Alosilla, Bradley Austin, Nathan
Krueger, Edgar Rodriguez, and Oswaldo Rosendo deserve thanks for their help with
sample collection. Also, Eric Matsuda-Fugisaki provided some needed assistance in the
laboratory and is much appreciated. Likewise, appreciation is extended to all graduate
students in the Animal Sciences Department for support and camaraderie during this
program of study. Pam Gross deserves recognition for her unyielding willingness to help
others and her wonderful attitude.
United States Sugar Corporation and its employees deserve thanks and
recognition for their donations of liquid feeds. A note of appreciation goes to Dr. Jon
Nelson and Southeastern Minerals, Bainbridge, Georgia, for donation of mineral
supplements, to Flint River Mills for transportation of mineral supplements, to Alltech,
Nicholasville, Kentucky, for donation of Sel-Plex, and to Mr. Dane Bemis for
preparation of experimental diets.
The author wishes to acknowledge, though not necessarily in a positive manner,
Hurricanes Charley, Frances, Ivan, and Jeanne. In the worst hurricane season in many
decades, the storms caused chaos across the state of Florida by damaging property and
generally disrupting daily life.
Last, but certainly not least, the author wishes to thank Ms. Rachel Van Alstyne
for her assistance with laboratory analyses and for her unselfishness and dedication as a
friend and colleague. She has truly been the kind of friend that a friend would like to
have.
v


68
signs were more severe when inorganic Se was used as the dietary source. In concluding
that > 5 mg/kg dietary Se, regardless of source, did produce signs of Se toxicity in
growing swine, those authors postulated that the greater tissue retention of organic Se
may reduce the incidence of Se toxicity. Based on these findings and the increasing use
of organic forms of Se for supplementation to livestock, an experiment was conducted to
evaluate and compare effects of feeding Se as sodium selenite or Se yeast at high dietary
levels on serum, whole blood, wool, and tissue Se concentrations of wether sheep.
Materials and Methods
All animal procedures were conducted within the guidelines of and approved by
the University of Florida Institutional Animal Care and Use Committee. This experiment
was conducted from June 4, 2002 to July 29, 2003 at the University of Florida Sheep
Nutrition Unit located in southwestern Alachua County, FL. Twenty-eight, 2-yr-old,
Rambouillet-crossbred wethers were weighed (62.3 8.5 kg) and received 2-ml
ivermectin dewormer s.c. (Ivomec; Merial Ltd. Iselin, NJ). Wethers were randomly
assigned to one of eight dietary treatments for a 60-wk study. Dietary treatments were
arranged as a 2 x 4 factorial with 0.2, 20, 30, and 40 mg/kg Se (as-fed) as four dietary
levels and Se yeast and sodium selenite as two Se sources added to a corn-soybean meal-
cottonseed hull basal diet (Table 5-1). Feed-grade yeast was used as a carrier for the
sodium selenite in order to alleviate differences in the palatability and protein content of
the diets. The basal diet was formulated to meet animal requirements for protein, energy
as TDN, vitamins, and minerals for this class of sheep (NRC, 1985). Animal numbers
per treatment were three for 0.2 (control) and 20 mg/kg Se, and four each for 30 and 40
mg/kg Se treatments for both Se sources. Wethers were housed by treatment group in
covered wooden pens (53.5 m ) with earth floors and ad libitum water.


47
Figure 3-3. Effect of dietary inorganic Se level on liver Se concentration in ewes; SE
27.5, 25.4, 24.5, 24.5, 34.6, and 26.9 for 0.2, 4, 8, 12, 16, and 20 mg/kg dietary Se,
respectively.


54
concentrations from ewes consuming 20 mg/kg Se were higher than from all other
treatments (P < 0.05). Ewes consuming 12 mg/kg Se had milk Se which was higher (P <
0.05) than controls and tended to be higher than from ewes receiving 4 mg/kg Se (P =
0.07). Milk Se concentrations, at d 56, from all other treatment groups were similar (P >
0.18). Milk Se concentrations increased linearly (P < 0.001) as dietary Se increased over
all sampling d.
Lamb plasma Se was affected by dietary Se concentration of their dams (P <
0.001) and increased linearly as dietary Se of dams increased (P < 0.001) in year one
(Table 4-5). Likewise, d of sampling affected lamb plasma Se concentration (P < 0.01).
On d 3 to 56, lamb plasma Se was positively correlated to ewe milk Se (r = 0.29; P <
0.001). At birth, lambs suckling ewes consuming 20 mg/kg Se had higher plasma Se than
controls (P < 0.05) and lambs suckling ewes consuming 16 mg/kg Se tended to have
higher plasma Se than controls (P = 0.15). All other lambs had similar plasma Se (P >
0.25). At 3 d of age, lambs from ewes consuming 20 mg/kg Se had higher plasma Se (P
<0.01) than all other treatment groups. Plasma Se concentrations from control lambs
were lower (P < 0.05) than plasma Se from lambs suckling ewes consuming 8, 12, or 16
mg/kg Se. At 28 d of age, lambs suckling ewes receiving 12, 16, or 20 mg/kg Se had
higher plasma Se than did controls (P < 0.01). Likewise, lambs suckling ewes receiving
4 or 8 mg/kg Se tended to have higher plasma Se than controls (P < 0.14). Plasma Se
from lambs suckling ewes receiving 20 mg/kg Se was higher than from lambs suckling
dams that received 4, 8, or 12 mg/kg Se (P < 0.05) and tended to be higher than from
lambs suckling dams that received 16 mg/kg Se (P = 0.09). At 56 d, plasma Se from
lambs suckling ewes receiving 4, 12, 16, or 20 mg/kg Se was higher than controls (P <


33
(Awadeh et al., 1998a). Whole blood Se increased linearly in young swine as dietary Se
was fed up to 20 mg/kg (Goehring et al., 1984b).
Wool
Selenium concentration in new growth wool was measured at wk 12, 24, 36, 48,
60, and 72 (Table 3-5). Dietary Se level, time, and dietary Se level x time affected (P <
0.001) wool Se. Wool Se increased linearly (P < 0.001) as dietary Se increased.
Response of wool Se over time was quadratic (P < 0.001) and time response for each
dietary Se level was evaluated individually. Wool Se from controls and ewes receiving 8,
12, and 16 mg/kg dietary Se responded quadratically (P < 0.03) from wk 12 to wk 72.
Wool Se from ewes receiving 4 mg/kg Se responded cubically (P < 0.05) and wool Se
from ewes receiving 20 mg/kg Se increased linearly (P <0.01) over time. Increased Se
in hair has been reported in other livestock species. Kim and Mahan (2001) observed a
linear response in the hair of pigs as Se in their diet was increased. Goehring et al.
(1984b) reported a quadratic response in the hair of swine as dietary Se as sodium
selenite was increased up to 20 mg/kg. Likewise, Perry et al. (1976) reported increased
Se in the hair of feedlot steers as dietary selenite Se was increased. Cristaldi et al. (2004)
reported a linear increase in the wool of growing sheep as dietary Se was increased and
also observed differences in wool Se of wethers receiving 6, 8, or 10 mg/kg Se vs
controls. These authors did not report a significant treatment x time interaction.
However, wool Se in the present study was affected by time and the interaction of
treatment x time as wool Se increased and then seemed to reach a plateau around wk 48.
Kim and Mahan (2001) and Cristaldi et al. (2004) used 10 mg/kg Se as the highest
dietary level and reported linear responses in hair and wool. However, with 20 mg/kg as
the highest dietary level, the quadratic responses observed by Goehring et al. (1984b) and


69
Diets were fed at 909 g-wether'-d'1 throughout the experiment. Samples of each
diet were taken every 28 d, ground (1 mm), and frozen at 0C until analysis.
Wether BW was recorded on d 0 and for every eight wk thereafter, for the
remainder of the study. A 10-mL blood sample for serum analysis was collected using an
18-gauge needle into a vacutainer tube with no additive (Vacutainer; Becton Dickinson,
Franklin Lakes, NJ) every 12 wk, via jugular venipuncture, allowed to stand for 20 min,
centrifuged at 700 x g for 25 min, and serum stored frozen at 0C until Se analysis. An
additional 10 mL of blood was collected into a heparinized vacutainer tube (Vacutainer;
Becton Dickinson, Franklin Lakes, NJ). This additional 10-mL sample was also
collected every 12 wk for the remainder of the experiment and stored frozen at 0C as
whole blood until analysis.
The wool around the jugular was shorn initially and regrowth was collected
beginning at wk 12 and every 12 wk thereafter. The collected wool was washed with a
commercial hair shampoo (Alberto V05; Alberto-Culver Co., Melrose Park, IL), to
remove oil and dirt, rinsed well with deionized water, dried, stored at room temperature,
and later analyzed for Se.
At the termination of the experiment (wk 60), wethers were slaughtered by
stunning and exsanguination, following USDA procedures at the University of Florida
Meats Laboratory. Immediately prior to slaughter, a 10-mL sample of blood was
collected using an 18-gauge needle into a vacutainer, centrifuged at 700 x g for 25 min,
and serum frozen at 0C for analysis of albumin and the following enzymes: alkaline
phosphatase (Aik Phos), alanine transaminase (ALT), aspartate transaminase (AST),
creatinine phosphokinase (CK), and gamma glutamyl transferase (GGT).


73
level increased, while a quadratic response (P < 0.05) was observed at wk 36 (Table 5-3).
Serum Se concentrations of wethers were affected by dietary Se level, Se source, and the
interaction of dietary Se level x Se source interaction (P < 0.05). Likewise, over the
entire trial serum Se increased quadratically (P < 0.05) as dietary Se level increased.
Wethers receiving organic Se had higher (.P < 0.001) serum Se than did selenite treated
animals throughout the study. Kim and Mahan (2001) reported a linear increase in
plasma Se of swine as dietary Se was increased as organic or inorganic Se. Likewise,
those authors reported an effect of source of Se, with pigs supplemented with organic Se
having higher plasma Se than their inorganically supplemented counterparts. Cristaldi et
al. (2004) reported a linear increase in serum Se as dietary Se was increased, however
those authors used a maximum level of 10 mg/kg dietary Se as selenite. The quadratic
response observed in the present study suggests homeostatic regulation of Se in blood as
dietary levels exceed 30 mg/kg. Serum Se in wethers fed up to 10 mg/kg selenite Se for
52 wk reached 870 pg/L (Cristaldi et al., in press). Wethers in the present experiment
exceeded 870 pg/L when receiving either Se source at 20, 30, or 40 mg/kg and at wk 24
wethers receiving 30 mg/kg organic Se had more than four-fold higher serum Se than the
maximum serum Se reported by Cristaldi et al. (2004). Our data show that at most
collections organic Se produced serum Se of more than double the concentration
produced by feeding selenite Se at the same level. Wethers receiving 20, 30 or 40 mg/kg
organic Se had serum Se above 1500 pg/L throughout the experiment. Aitken (2001)
reported serum Se of 1500 pg/L as a level at which signs of toxicity appear in horses.
Likewise, Aitken (2001) reported that serum Se of 3700 pg/L was evident of Se toxicosis


56
Plasma Se from lambs suckling ewes receiving 12 mg/kg Se was higher than (P < 0.05)
than plasma Se from lambs suckling ewes receiving 4 and 8 mg/kg.
Selenium concentration in testis (dry basis) taken from ram lambs at 70 d of age
(weaning) increased linearly (P < 0.001) as dams dietary Se concentration increased
(Figure 4-2) in year one. Testicular Se from lambs suckling ewes receiving 20 mg/kg Se
was higher than testis Se from controls and lambs suckling ewes receiving 4 or 8 mg/kg
Se (P < 0.05). Lambs suckling ewes receiving 16 mg/kg Se had testicular Se which was
higher (P < 0.05) than testicular Se from controls and lambs suckling ewes receiving 4
mg/kg Se. Lambs suckling ewes receiving 12 mg/kg tended to have higher testicular Se
than did controls or lambs suckling ewes receiving 4 mg/kg Se (P < 0.11). Likewise,
lambs suckling ewes receiving 8 mg/kg tended to have higher testicular Se than did
controls (P = 0.14). There was no effect of treatment on testicular Se in year two (P =
0.70). Testicular Se concentrations were 2.05, 3.16, 2.96, and 3.24 mg/kg for controls
and lambs suckling ewes receiving 4, 8, and 12 mg/kg Se, respectively.
Discussion
Colostrum Se increased with dietary Se level in both years. Cuesta et al. (1995)
reported higher colostrum Se from vitamin E + Se supplemented ewes versus their
unsupplemented counterparts. These findings are further supported by Mahan (2000),
who demonstrated that colostrum Se was increased by increasing Se in prepartum and
postpartum sow diets. Colostrum Se from Se supplemented crossbred ewes was
increased over unsupplemented controls (Norton and McCarthy, 1986), however, those
researchers used injectable vitamin E + Se as the supplemental Se source rather than
dietary Se. Ovemes et al. (1985) also reported an effect on colostrum Se from ewes
receiving Se fed via free-choice salt and mineral mixtures. In the present study, ewe


Change in cow BW, kg
105
Control Deposel Mu-Se Selenite Se Yeast
Source of selenium supplementation
Figure 6-1. Effect of source of selenium supplementation on change in BW in grazing
beef cows; Changes in BW lacking a common superscript differ (P < 0.05); SEM = 6.92.


101
Table 6-1. Composition of mineral mixtures offered free-choice to brood
COWS
Free-choice mineral mixtures
Component
No Sea
Sodium selenite
Se yeastc
%, as-fed
Calcium
18.73
18.73
18.49
Phosphorus
8.00
8.00
7.90
Sodium chloride
26.73
26.73
26.39
Magnesium
2.00
2.00
1.97
ppm, as-fed
Iron
5695
5695
5695
Zinc
4015
4015
4015
Manganese
2225
2225
2225
Copper
500
500
500
Iodine
50
50
50
Cobalt
50
50
50
Selenium

26
26
IU/kg, as-fed
Vitamin A
102272
102272
102272
Vitamin D3
10227
10227
10227
Vitamin E
23
23
23
Manufactured by Southeastern Minerals, Inc., Bainbridge, GA; served as basal mineral mixture.
bManufactured by Southeastern Minerals, Inc., Bainbridge, GA, by addition of 26 mg/kg Se as sodium
selenite to basal mineral mixture.
Created by addition of 1.3% Se yeast (Sel-Plex 2000; Alltech, Inc., Nicholasville, KY) to basal mineral
mixture.
Table 6-2. Frequency, daily amount, and total of amount of supplemental Se
administered to cows
Source of supplemental Se
Selenium
supplementation
interval, d
Avg
supplementation,
mg Se-cow'1-d'1
Total Se
supplementation,
mg
No Se supplementation
6
6
6
Barium selenate injection1
365
1.23
450
Sodium selenite injection2
125
0.21
75
Sodium selenite via free-choice minerals3
l5
1.08
393
Selenized yeast via free-choice minerals4
l5
2.22
811
Cows received a subcutaneous injection of 9 mL Deposel at initiation of study.
2Cows received an injection of 5 mL Mu-Se at initiation of study and re-injection every 4 mo.
3Cows had continuous access to free-choice mineral mix containing 26 mg Se/kg as sodium selenite and
consumed mineral mix at avg of 41.5 g-cow',-d'1 .
4Cows had continuous access to free-choice mineral mix containing 26 mg Se/kg as Se yeast and
consumed mineral mix at an avg of 85.5 g-cow d .
5 Access to free-choice minerals containing Se was continuous throughout the study.
6 Cows receiving no Se supplementation or injectable Se had free-choice access to and consumed the
basal free-choice mineral mix (no Se) at an avg of 62.2 g-cow d'1.


146
Tennessee, and with Moormans Inc. as a sales representative, all while preconditioning
and shipping cattle in partnership with brother Ryan.
Paul began work on a Master of Science in ruminant nutrition and beef cattle
management in August 1999 at the University of Florida. During his studies, he became
involved in Florida Blue Key, Omicron Delta Kappa, Alpha Zeta, Savant UF, Gamma
Sigma Delta, Sigma Xi, American Society of Animal Science, and the Animal Sciences
Graduate Student Association. He held a teaching assistantship and served as course
coordinator for the large animal practicum undergraduate course. In 2001, Paul received a
travel fellowship to the International Livestock Congress in Houston, Texas, and was
recognized as Graduate Student of the Year in the Animal Science Department. In
August 2001, Paul entered a doctoral program in ruminant nutrition concentrating on
minerals at the University of Florida and received his Master of Science in August 2002.
As a doctoral student, Paul was the first graduate student invited as a speaker for
the Florida Ruminant Nutrition Symposium and gave an invited talk at the AFIA Liquid
Feed Symposium in Indianapolis, Indiana. He aided in the teaching of the graduate
Vitamins, and Mineral Nutrition and Metabolism courses by giving lectures in the
absence of his advisor, Dr. McDowell. Paul was elected to the University of Florida Hall
of Fame in April 2003, named the Graduate Student of the Year in the College of
Agricultural and Life Sciences in November 2003, and graduated with his Master of
Agribusiness in December 2003. During his time at the University of Florida, Paul
cultivated a great appreciation for Gator Football, attended 47 games, and never missed a
game at Ben Hill Griffin Stadium. Paul was undecided on plans for the future and his
possibilities included academia, industry, or production agriculture.


89
injection of barium selenate, sodium selenate or sodium selenite and, in ruminants, with
slow-release, long lasting ruminal Se boluses or pellets. With the recent Food and Drug
Administration approval of Se yeast for use in ruminant diets, livestock producers now
have more choices of form and method of Se supplementation. The objective of this
experiment was to evaluate and compare effects of form and method of Se
supplementation on blood, liver, and milk Se concentrations in beef cows.
Materials and Methods
All animal procedures were conducted within the guidelines of the University of
Florida Institutional Animal Care and Use Committee. Animals were housed at the
University of Florida Boston Farm-Santa Fe Beef Unit located in Northern Alachua
County, Florida. On August 6, 2002, 43 Angus cows, aged 2-3 yr, (mean age = 2.67 yr)
were palpated to diagnose pregnancy and estimate d in gestation. All cows were
determined pregnant and gestation estimates ranged from 115 to 130 d. Each animal
received a chemically altered modified live 4-way viral + vibriosis and leptospirosis
vaccination (Cattlemaster 4+VL-5; Pfizer Animal Health, Exton, PA) and fly control
(Permectrin 10% EC pour-on; Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO)
according to manufacturer directions. Cows were weighed (average initial BW = 417
46 kg), stratified by age and assigned to one of five treatment groups for a 365 d study.
The five treatments were 1) no Se supplementation, control group, 2) one subcutaneous
injection of 9 mL (50 mg Se-mL'1) barium selenate (Deposel Multidose; Novartis New
Zealand, Ltd., Auckland, NZ) at the initiation of the experiment, 3) three subcutaneous
injections of 5 mL (5 mg Se-mL'1) of sodium selenite + 68 IU vitamin E as dl-alpha
tocopheryl acetate (Mu-Se; Schering-Plough Animal Health, Union, NJ), one at the
initiation of the experiment and one every four mo thereafter, 4) free-choice access to a


43
Table 3-3. Effect of dietary inorganic Se level on serum Se concentration of mature
ewes at various stages of lamb production3
Dietary Se, mg/kg
02 4 8 12 16 20
Stage of Production Serum Se, pg/L
Late Gestation, yr lb
149s 67
242gh 67
354hi 79
414hi 79
463' 79
707J81
Lactation, yr 1
151s 56
272s 54
486h 63
623hl 63
718'J 63
811J 66
Dry, rebreedingd
162s110
298gh 95
458h' 100
604' 99
1205^ 106
1084J 113
Late Gestation, yr 2e
140s 137
313gh 124
446sh 142
596hl 142
986J 158
1072J 149
Lactation, yr 2f
127s 114
325sh 103
536h' 114
718' 114
769* 136
1355j 121
aData represent least squares means SE.
bLate gestation, yr 1, defined as 56 d prepartum and includes serum Se concentrations for wk 4, 8, and 12.
cLactation, yr 1, defined as 84 d postpartum and includes serum Se concentrations for wk 12, 16, 20, and 24.
dDry, rebreeding period, 168 d, includes serum Se concentrations for wk 28, 32, 36, 40, 44, and 48.
eLate gestation, yr 2, defined as 56 d prepartum and includes serum Se concentrations for wk 52, 56, and 60.
fLactation, yr 2, defined as 84 d postpartum and includes serum Se concentrations for wk 60, 64, 68, and 72.
g.h.ijMeans within rows lacking a common superscript differ (P < 0.05).
Table 3-4. Effect of dietary inorganic Se level on whole blood Se concentration of
mature ewesa
Dietary Se, mg/kg-
Week of 0.2 4 8 12
experiment
12b
386h 168
24c
420h 140
36d
438h 152
48e
378h 154
60f
497h 154
72g
410h 154
Whole blood Se, pg/L
839 151
601hi 130
635h 149
66 lh 130
802hi 138
618hi 139
902,J 151
852ij 130
1079* 138
1117' 139
1070' 151
721hi 140
1241jk 139
1047jk 130
1314ij 138
1145' 130
1131* 150
916* 140
"Data represent least squares means SE.
b = 4, 5, 5, 6, 7, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
cn = 6, 7, 7, 7, 7, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
n = 5, 5, 6, 6, 6, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
c = 5, 7, 6, 7, 5, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
{n = 5, 6, 5, 5, 4, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
gn = 5, 6, 6, 6, 3, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
h1'i,klMeans within rows lacking a common superscript differ (P < 0.05).
16
1053ij 130
1312k 130
1668j 139
1616i 151
1892j 167
841hi 194
20
1558k 154
1822' 154
1373ij 154
195 lj 154
1796s 154
1855J 154


75
had higher (P < 0.001) whole blood Se than did wethers receiving inorganic Se
throughout the study. The maximum whole blood Se concentration observed during our
study was 6259 pg/L. This concentration is well above the range of 2000 to 4000 gg/L
for whole blood Se, where clinical signs of Se toxicosis should appear (Rosenfeld and
Beath, 1945; 1946) and likewise is greater than a whole blood Se concentration of
4000gg/L, that Maag and Glenn (1967) described as the blood concentration above which
steers became depressed and inactive. However, wethers on the present study, with the
highest whole blood Se concentrations (> 6200 gg/L) did not exhibit signs of Se toxicosis
(e.g. wool loss, anorexia, abnormal hoof growth). Glenn et al. (1964a) fed sodium
selenate at high levels to range ewes that were similar in BW and breed type to the
wethers on the present study. Those researchers did not induce death by Se toxicosis
with daily oral doses less than 25 mg Se/ewe. Of the 17 deaths reported in their
experiment, only one was induced with a daily dose of 25 mg Se/ewe. Eight deaths were
induced with a daily dose 37.5 mg Se/ewe and eight deaths were induced with a daily
dose 50 mg Se/ewe. Those reported deaths were not by acute Se toxicosis; rather the
ewes received experimental Se doses for at least 80 d before death by Se toxicosis was
induced. In the same experiment, Glenn et al. (1964a) further suggested an avg minimum
toxic level of Se for adult sheep to be 0.825 mg/kg BW when fed for 100 d. Using this
estimate, the minimum toxic level of Se for sheep of the size used in our study would be
51.4 mg/d. Selenium consumption of wethers receiving the highest dietary Se level (40
mg/kg) was 78% of the aforementioned minimum toxic level for sheep. Blodgett and
Bevill (1987) reported the LD50 for sheep, using sodium selenite via i.m. injection, to be
0.7 mg Se/kg BW. Wethers of avg BW, on our study, receiving 1 kg of diet containing


15
while some inorganic Se is found in grains and plants (Whanger, 2002). In animal tissues,
selenate is the major inorganic form and selenocystine is the predominant organic form.
Selenomethionine is found initially when this amino acid is fed; however seleno
methionine is converted to selenocystine after some time (Whanger, 2002). With such
differentiation in the sources of Se within plant and animal tissues, it seems reasonable
that differences in efficacy due to form of Se administered would exist and there are
numerous examples in the scientific literature to support this concept.
Goehring et al. (1984a) evaluated the effects of high dietary levels of Se from
selenite or seleniferous grains on blood and tissue concentrations in swine. Those authors
documented that Se from seleniferous grains increased Se in blood and tissue compared
to selenite Se fed at the same level. Awadeh et al. (1998a) reported increased blood Se in
crossbred beef cows consuming free-choice minerals containing 60 mg/kg Se as Se yeast
compared to 60 mg/kg selenite Se. Furthermore, cows receiving free-choice minerals
containing 60 mg/kg Se as Se yeast had a lower percentage protein in albumin compared
to cows receiving minerals with 60 mg/kg selenite Se. Selenium from Se yeast has been
documented by several groups of researchers as more effective than Se selenite or
selenate at increasing blood and liver Se levels in beef cows (Pehrson et al., 1999; Valle
et al., 2002; Gunter et al., 2003) and in dairy cattle (Ortman and Pehrson, 1999).
As with blood and tissue Se, milk Se has been more effectively increased by using
organic Se vs inorganic Se in beef cattle, dairy cattle, and swine. Selenium yeast
produced milk Se more than 100% higher than selenite or selenate Se when 3 mg of Se
from each source were fed to Swedish dairy cows (Ortman and Pehrson, 1999). Hereford
cows supplemented with Se yeast produced milk with markedly higher Se concentrations


107
age (Pehrson et al., 1999). Evidence also exists that milk Se can be increased by level
and duration of Se supplementation in lactating cows (Conrad and Moxon, 1979). Given
these findings it seems logical that calf blood Se can be increased from birth to weaning
by supplementing the cow herd with Se. We hypothesized that Se status of calves could
be improved through Se supplementation of their dams and that an organic source of Se
may be more efficient at maintaining adequate blood and tissue Se. Thus, this
experiment was designed to evaluate and compare effects on blood and tissue Se
concentrations of calves bom to and suckling dams that received different sources and
physical forms of supplemental Se. It should be noted that each supplementation method
evaluated in this study is readily available to livestock producers.
Materials and Methods
All animal procedures were conducted within the guidelines of and approved by
the University of Florida Institutional Animal Care and Use Committee. Animals were
housed at and all sampling took place at the University of Florida Boston Farm-Santa Fe
Beef Unit located in Northern Alachua County, Florida. On August 6, 2002, 43 Angus
cows, aged 2-3 yr, (mean age = 2.67 yr) were palpated to diagnose pregnancy and
estimate d in gestation. All cows were determined pregnant and gestation estimates
ranged from 115 to 130 d. Each animal received a chemically altered modified live four
way viral + vibriosis and leptospirosis vaccination (Cattlemaster 4+VL-5; Pfizer Animal
Health, Exton, PA) and fly control (Permectrin 10% EC pour-on; Boehringer Ingelheim
Vetmedica, Inc., St. Joseph, MO) according to manufacturers directions. Cows were
weighed (average BW = 417 46 kg), stratified by age, and assigned to one of five
treatment groups for a 365 d study. The five treatments were 1) no Se supplementation,
(control), 2) one subcutaneous injection of 9 mL (50 mg Se-mL'1) barium selenate


53
Ewes consuming 12, 16, and, 20 mg/kg Se produced milk Se higher (P < 0.05) than
controls. Milk Se from ewes consuming 16 mg/kg Se tended to be higher (P 0.09) than
that from ewes consuming 20 mg/kg Se and was higher (P < 0.05) than milk Se from all
other treatments. At d 28 postpartum, ewes consuming 20 mg/kg dietary Se produced
milk Se concentrations higher {P < 0.05) than did controls or ewes consuming 4 mg/kg
dietary Se. Likewise, milk Se from ewes consuming 20 mg/kg dietary Se tended to be
higher (P < 0.075) than milk Se from ewes receiving 8 or 12 mg/kg Se. Milk Se from all
other treatment groups was similar (P > 0.20). At the final milk collection in year one (d
56), milk Se concentrations from controls and ewes consuming 4, 8, and, 12 mg/kg Se
were similar (P > 0.17). Ewes consuming 16 and 20 mg/kg Se produced similar milk Se
(P = 0.43), which was higher (P < 0.05) than milk Se from all other treatments. A linear
increase (P <0.01) in milk Se as dietary Se increased was observed at each sampling d as
well as over all sampling d. Milk Se concentrations in year one remained below 1000
|ig/L from d 3 to d 56.
In year two, dietary Se concentration had an effect on milk Se (P < 0.05), as did
the interaction of dietary Se concentration x sampling d (P <0.05). Milk Se
concentrations from ewes consuming 8, 12, and 20 mg/kg Se were similar (P> 0.38) to
each other and higher (P < 0.05) than controls at d 3 (Table 4-4). Ewes consuming 12
mg/kg Se had higher (P < 0.05) milk Se than did those consuming 4 mg/kg Se. Ewes
consuming 20 mg/kg Se produced milk Se that tended (P = 0.09) to be higher than milk
Se from ewes consuming 4 mg/kg Se. There were no differences (P > 0.05) in milk Se
among treatment groups at d 28. However, ewes consuming either 12 or 20 mg/kg Se
had milk Se which tended to be higher (P <0.12) than control. At d 56, milk Se


131
In conclusion, ewes under our experimental conditions and during the stresses of
production were able to tolerate up to 20 mg/kg dietary Se as sodium selenite for 72 wk.
These findings suggest the maximum tolerable level of inorganic Se for sheep to be much
higher than 2 mg/kg as was suggested previously. Experiments which are longer in
duration are necessary to clearly define the maximum tolerable level. Furthermore, our
results suggest that ewes consuming up to 20 mg/kg inorganic Se can give birth to normal
lambs and that the lambs do not suffer from Se toxicosis before weaning. Selenium as
sodium selenite can be fed to ewes at concentrations greater than the current maximum
tolerable levels (2 mg/kg) without adversely affecting their offspring. Wether sheep,
under our experimental conditions, tolerated up to 40 mg/kg dietary Se as sodium Se and
Se yeast for 60 wk, though differences in response to Se source were observed. These
results further indicate that the current maximum tolerable level of Se, regardless of
source, is much higher than the current estimate of 2 mg/kg and the range between
optimal and toxic Se levels is not so narrow. On the contrary, using a maximum tolerable
level for Se of 10 mg/kg and 0.1 as the Se requirement, then the range between
requirement and toxic level is 100 units. At that range, Cu, Mn, Co, Fe, and Zn then all
have a more narrow range from requirement to toxic level than does Se.
In a beef cow-calf herd, Se supplementation with organic or inorganic Se via free-
choice minerals or injectable selenate maintained adequate Se concentrations in whole
blood, plasma, and liver for 365 d. However, inorganic Se was limited in its ability to
increase milk Se, whereas Se yeast increased milk Se at parturition and at weaning.
Supplementation of Se as Se yeast to brood cows produced adequate calf blood Se
throughout our experiment, whereas control and the other treatments produced calf blood


SELENIUM TOLERANCE IN SHEEP AND SELENIUM SUPPLEMENTATION
METHODS FOR BEEF CATTLE
By
PAUL ARMAND DAVIS
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2004


42
Table 3-1. Diet composition (as-fed) for selenite-Se supplemented ewesa
Ingredient
% as-fed
Ground yellow com
53.75
Cottonseed hulls
22.00
Soybean meal (47.5% CP)
16.00
Alfalfa meal (14% CP)
3.00
Soybean oil
3.00
Trace mineral mixb
1.00
Ground limestone
1.25
Vitamins A & D
C
aSelenium levels in diet (as analyzed): 0.29, 3.77, 7.54, 11.01, 15.48, and 19.05 ppm
for Se levels 0.2, 4, 8, 12, 16, and 20 ppm, respectively.
bTrace mineral mixture supplied between 96.5% and 98.5% NaCl, and provided per kg
of diet: 1.0 mg Co (as carbonate), 5.0 mg Cu (as oxide), 0.7 mg I (as iodate), 35 mg Fe
(as oxide), 25 mg Mn (as oxide), and 35 mg Zn (as oxide).
cProvided per kg of diet: 5,000 IU of Vitamin A and 500 IU of Vitamin D3.
Table 3-2. Lamb production of ewes receiving different concentrations of dietary Se
Year Ia
Year 2b
Dietary Se, ppm
Ewes lambed
Lambs bomc
Ewes lambed
Lambs bomd
0.2
5
9
4
5
4
7
11
7
10
8
5
6
5
7
12
5
8
6
11
16
4
5
0
0
20
7
14
2
3
Total
33
53
24
36
aEwes began receiving experimental diets at 57 d average gestation in yr 1.
bEwes were fed experimental diets continuously during breeding and gestation yr 2.
cLamb crop as lambs bom (53) per ewe exposed (41) was 129% in yr 1.
dLamb crop as lambs bom (36) per ewe exposed (33) was 109% in yr 2.


124
Cows' milk selenium, ¡ug/L
Figure 7-3. Correlation between the concentration of Se in cows milk and the concentration
of Se in the whole blood of their calves.


115
Both the injectable and the free-choice selenite treatments produced whole blood Se
similar to control. This agrees with Valle et al. (2003) who measured calf whole blood
Se at 60 d. Such decreases are likely due to calves relying solely on a milk diet and
depleting blood Se and liver reserves which are reported to be bolstered by Se
supplementation to pregnant cows (Koller et al., 1984; Abdelrahman and Kincaid, 1995).
Calf blood Se had dropped below marginally adequate levels in the control and
both injectable treatment groups at d 30. Calves from the free-choice selenite group had
blood Se in the marginally adequate range and the Se yeast treatment had blood Se values
nearly two-fold higher than each of the other treatments. Gunter et al. (2003) also
reported whole blood Se from Se yeast treatments to be at least two-fold higher than
sodium selenite or unsupplemented calves at about 110 d of age. Results from Valle et
al. (2003) followed a similar pattern, as plasma Se in calves from Se yeast supplemented
dams was three to four higher than calves whose dams had no Se supplementation or
injectable Se supplementation. At weaning (d 205), calves from control, injectable
selenate, and injectable selenite treatment groups had blood Se of 42, 34, and 36 pg Se/L,
respectively, all of which were considerably below the deficient threshold. Calves from
dams that received free-choice selenite were classified as marginally deficient and the Se
yeast treatment produced blood Se well over the adequate level and more than five-fold
higher than either injectable product at weaning. Blood Se of calves from dams receiving
injectable Se or the free-choice selenite were similar during the majority of this study and
decreased with calf age. Days 90 and 205 of this study were in April and early August.
At these times, in Florida and most of the United States, grasses are growing rapidly and
contain higher concentrations of unsaturated fatty acids. It has been postulated by


12
reported that the Se bolus was an effective method of increasing Se in colostrum, plasma,
and whole blood. Likewise, calves bom to Se supplemented cows had higher Se
concentrations in plasma, whole blood, and liver than calves bom to cows receiving no
supplemental Se. In this study, the administration of a sustained release Se bolus to cows
proved to be an effective method of Se supplementation to newborn calves.
The need for Se supplementation to livestock is great as evidenced by the many
benefits of supplemental Se on animal health and performance. This need is further
elucidated by surveys such as reported by Dargatz and Ross (1996), which reported a
relatively high percentage of beef cattle in the U.S. that were classified as Se deficient.
Selenium supplementation generally adds only a negligible amount to the cost of
livestock production and producers have several effective means of Se supplementation
to choose from.
Absorption, Transport, Storage, and Excretion of Selenium
Ruminant animals differ from monogastric animals in their ability to absorb and/or
retain Se. Wright and Bell (1966) reported retention of a dose of sodium selenite to be
29% for sheep and 77% for swine. In both sheep and swine, Se absorption occurred in
the small intestine and cecum with some additional absorption in the colon for swine. No
absorption of Se occurred in the rumen of sheep or the stomach of swine (Wright and
Bell, 1966). These authors also reported net absorption of Se to be 36% for sheep and
86% for swine. Less absorption of Se in ruminants seems to be due to the reduction of
inorganic Se to insoluble forms by rumen microorganisms (Butler and Peterson, 1961;
Peterson and Spedding, 1963; Hidiroglou et ah, 1968). Inorganic Se is more readily
reduced within the rumen than organic forms of Se such as Se yeast. Diet also affected


26
Brain, diaphragm, heart, hoof tip, kidney, liver, and psoas major muscle Se data
were analyzed for effects of treatment using PROC GLM in SAS (SAS for Windows 8e;
SAS Inst., Inc., Cary, NC) in a completely randomized design. Pre-planned orthogonal
contrast statements were used to compare means as described by Littell et al. (1998;
2000). PROC MIXED of SAS was used to analyze effects of treatment, time, and the
interaction of treatment x time on BW, serum Se, whole blood Se, and wool Se as
repeated measures with a spatial power covariance structure with respect to d and a
subplot of animal nested within treatment. Pre-planned orthogonal contrast statements
were written to determine differences in means at different sampling intervals. Means
were separated at P < 0.05 and regression analysis was used to determine relationships
between dietary Se and Se concentration of various tissues.
Results and Discussion
Performance
Ewe BW was not affected by dietary Se level (P = 0.69) or dietary Se level x time
interaction (P = 0.56). However, time did affect BW (P < 0.001). Initial BW was 57.4
5.7 kg and BW at the termination of the experiment was 61.2 15.1 kg. These findings
agree with previous studies in ruminants. Supplemental selenium fed up to 0.4 mg/kg
which is above requirement but below maximum tolerable level had no effect on rate of
gain in feedlot steers (Perry et al., 1976) and BW gains in wether sheep, fed sodium
selenite up to 10 mg/kg, was unaffected by dietary Se level (Cristaldi et al., in press).
Glenn et al. (1964a) also reported no effect of dietary Se on BW when sodium selenate
was fed to ewes as a single oral dose of up to 50 mg/d. The ewes utilized by those
authors were very similar in breed type and BW to the animals used in the present study.


8
concluded that high serum Se concentrations were associated with reduced rates of
clinical mastitis and low somatic cell counts in the milk tank.
Reproductive problems such as increased services per conception or increased
calving interval which could, at least in part, be attributed to male fertility may also be
improved by Se supplementation. Heimann et al. (1981) showed that the pituitary gland
and reproductive tissues exhibited higher Se concentrations than many other body tissues.
Julien and Murray (1977) reported that percent motility in bovine spermatozoa increased
significantly as concentration of Se in sperm increased. Thus, supplemental Se may have
a positive effect on sperm quality and ultimately on male fertility. However, Segerson
and Johnson (1981) observed no differences in sperm number, viability, or Se content
from Se supplemented bulls compared to sperm from unsupplemented controls.
Methods of Selenium Supplementation to Livestock
The benefits of Se supplementation to livestock are many and Se deficiencies are
easily combated with adequate Se supplementation. Several methods of Se
supplementation exist and successful uses of all methods have been reported. The
method of Se supplementation chosen by livestock producers may be dependent on
factors such as Se content of soils, local grains and forages, species produced, class of
livestock and stage of production, facilities for animal handling, as well as knowledge,
previous experience, and personal preference.
Many areas of the United States have Se deficient soils (McDowell, 2003) and thus
produce grains and forages which are low in Se. Likewise, many regions of the world
have been mapped as Se deficient and may benefit from the administration of Se to
livestock (Oldfield, 2002). In a survey of blood Se status in beef cattle encompassing
more than 250 herds in 18 states in several regions of the U.S., more than 18% of cattle




109
heparinized tubes (Vacutainer; Becton-Dickinson, Franklin Lakes, NJ) immediately after
birth (d 0) and before nursing. If technicians were unable to determine whether or not the
calf had nursed, then no sample was taken. This was done so that calf blood Se
concentration at birth could be attributed to maternal transfer of Se rather than from Se
obtained via colostrum. Additional blood samples were collected in the same manner
from all calves on d 30, d 90, and d 205. On these dates, calves were an average age of
33, 88, and 208 d, respectively. Twenty-two of the male calves were surgically castrated
immediately after birth and the testes were then frozen (0C) until analysis. Liver
biopsies were performed on all calves at d 205, which coincided with weaning, and liver
samples were placed on ice and then frozen (0C) until analysis. Body weight was
recorded for all calves at d 0 and at weaning (d 205). Body weight at weaning and actual
age of calf in d was used to calculate ADG. Additionally, samples of cow blood and milk
were collected immediately following parturition and on the same subsequent dates as the
calves. Samples from cows were collected and stored in the same manner as those taken
from the calves.
Pastures were sampled in October 2002 (before calving season), March 2003
(after calving season), and August 2003 (at weaning). Likewise, hay, molasses-based
liquid supplement, whole cottonseed, and pelleted citrus pulp were sampled during winter
supplementation. Whole blood, liver, testes, and all feedstuffs were analyzed for Se
concentration using a fluorometric method described by Whetter and Ullrey (1978). To
help ensure reliability of the analytical method, a certified standard (National Bureau of
Standards Bovine Liver SRM-1577a; U.S. Department of Commerce, National Institute
of Standards and Technology, Gaithersburg, MD) was frequently analyzed.


9
were classified as marginally deficient (51 to 80 pg/L) or severely deficient (< 50 pg/L)
in blood Se (Dargatz and Ross, 1996). Percentages of cattle classified as deficient varied
with region of the country. Herds in the Central U.S. had the least occurrence of Se
deficiency, while the Southeast, including Florida, had the greatest incidence of Se
deficiency at more than 40%. Stowe and Herdt (1992) also suggest that many cattle in
the U.S. are in a state of Se deficiency.
Selenium supplementation to livestock is accomplished using three or four primary
methods. Addition of Se to livestock feeds and/or minerals, use of injectable Se
preparations (usually in combination with vitamin E), use of sustained release
intrareticular Se supplements, and possibly the use of seleniferous grains or forages
grown on high Se soils (Ammerman and Miller, 1975) are the methods most often used to
supplement Se. One additional option to increase Se intake of livestock is the use of Se
containing fertilizers on forage and pasture (Valle, 2001). The addition of Se to
feedstuffs was not an option until 1974 when the Food and Drug Administration (FDA)
allowed for supplementation of up to 0.1 mg/kg Se as selenite or selenate for swine and
poultry (Schmidt, 1974). An amendment to this FDA order allowed the use of
supplemental Se for sheep in 1978 and a subsequent amendment in 1979 allowed for use
in dairy and beef cattle. Currently, use of 0.3 mg/kg dietary Se is approved for
supplementation in poultry, swine, sheep, and cattle (McDowell, 2003).
Regardless of method chosen for Se supplementation, Se deficiencies are more
easily combated than are toxicities, which generally require more animal and/or pasture
management. In sheep and beef cattle production systems, producers most often choose
to use injectable Se products or supplement Se through free-choice mineral mixtures.


116
Pehrson et al. (1999) that during such times cows would have greater oxidative stresses
due to diet. If so, inorganic Se supplements may not be adequate to maintain Se
concentrations in cow blood and milk. Results from this study, as well as results from
Gunter et al. (2003), Valle et al. (2003), and Pehrson et al. (1999) all demonstrate
decreases in blood Se of calves, whose dams received Se via inorganic sources at times of
lush forage growth. These results support the aforementioned postulation, but
investigation of antioxidants such as blood a-tocopherol levels are likely needed for
definitive support.
In the present study, testes of male calves taken at birth were used as an
alternative to liver in an effort to evaluate maternal transfer of Se to calf tissues.
Selenium concentrations in calf testes taken at birth were not statistically different but did
follow a similar numeric pattern to the liver Se concentrations of beef cows receiving
either Se yeast or injectable Se (Valle, 2001). In our study, testis Se concentration in
calves whose dams received Se yeast or injectable barium selenate was approx 29%
higher than controls. Researchers in India demonstrated greater oxidative stress in the
testes of mice when less Se was included in their diets (Kaur and Bansal, 2004), but data
relating to Se concentrations in calf testes is limited.
Liver samples, on a dry basis, taken at weaning had Se concentrations (pg/kg
DM) that were on average 7.89 times greater than whole blood Se (pg/L) collected on the
same d. Likewise, liver and whole blood Se followed a very similar pattern to whole
blood, as calves from dams receiving Se yeast were higher than all other calves in whole
blood and liver Se. Control and both injectable products were similar for most all
measurements and free-choice selenite produced liver and whole blood Se higher than


21
optimal and toxic level of Se is narrow. Surely, further studies which are long in duration
and use high dietary levels and different sources of Se are necessary to better estimate the
tolerance of Se for dairy and food animals.


57
colostrum Se concentrations from controls in year one were lower at 257 pg/L than
values in cow colostrum from Romania reported by Serdaru et al. (2004). However, in
year two, after our ewes had been receiving their respective diets for approximately 13
mo, colostrum Se from controls had more than doubled to 705 pg/L. The increase in
colostrum Se after a longer duration of Se supplementation is substantiated by Maus et al.
(1980). Those authors reported that Se in cows milk increased with time when fed at 0.2,
0.3, 0.4, and 0.7 mg/kg in a corn-brewers grain dairy diet. As dietary Se was increased
by increments of 4 mg/kg from 4 mg/kg up to 20 mg/kg, colostrum Se increased by 45.3,
8.8, 55.2, and 10.1%, respectively in year one. Colostrum Se was numerically higher in
year two when Se was fed at 0.2, 4, 8, and 12 mg/kg. This furthers the idea that
colostrum Se, when Se is supplemented at equivalent concentrations, may be increased as
animals are supplemented for an extended period of time. The use of increased Se in
gestating animals may prove beneficial to their offspring as it provides greater
antioxidative protection through increased colostrum Se and thus provides greater
phagocytic and microbicidal activity (Wuryastuti et al., 1993).
As with colostrum Se, subsequent milk Se also increased as dietary Se increased.
Givens et al. (2004) reported increased milk Se as selenite Se increased from 0.38 to 1.14
mg/kg and that a strong positive correlation exists between milk Se and dietary Se (r =
0.979). Gardner and Hogue (1967) reported up to five-fold increases in milk Se when
sodium selenite was added to ewe diets at 1 mg/kg. In the present study, a five-fold or
greater increase was observed in milk Se as dietary Se was increased from control (0.2
mg/kg) to 8 mg/kg for d 3, from control (0.2 mg/kg) to 20 mg/kg for d 28, and from
control (0.2 mg/kg) to 12 mg/kg for d 56 in year one. In year two, a four-fold or greater


20
organic Se. Those authors reported plasma Se concentrations of more than 3.3 mg/L,
liver Se of more than 17 mg/kg, and hoof Se of more than 28 mg/kg and no deaths
regardless of dietary Se level. The authors concluded that the higher retention of organic
Se in tissues and blood cells may effectively reduce the amount of Se available to cause
Se toxicosis.
Tolerance of Se as selenite or selenomethionine was evaluated using yearling steers
in a 4 mo study (OToole and Raisbeck, 1995). Those authors observed the highest
incidence of hoof lesions in steers fed organic Se at a rate of 0.80 mg/kg BW. Likewise,
it was shown that the steer with the most severe hoof lesions also had the highest Se
concentrations in hair, liver and kidney. The findings of that study indicated that dietary
exposure of 0.8 mg Se/kg BW, in either form, for 4 mo produces subclinical to clinical
signs of Se toxicosis. The authors concluded that selenomethionine is more likely to
cause alkali disease than sodium selenite. Holstein cows were fed inorganic Se up to 100
mg/d and whole blood and liver Se concentrations of up to 4.9 mg/L and 15 mg/kg DM,
respectively, were reported (Ellis et al., 1997). It was concluded that dairy cattle could
tolerate Se intakes of up to 100 mg/d for several wk without suffering adverse affects.
It seems logical, based on previous findings, that the minimum lethal dosages and
maximum tolerable levels of Se are variable and may be affected by various factors such
as Se source (organic or inorganic), diet composition, method of Se supplementation, and
Se status of the animal. The current maximum tolerable level for dietary Se in domestic
animals is 2 mg/kg (NRC, 1980). This estimate does not consider differences in species,
source of Se, or duration of exposure. Early reports of toxicities are likely reasons for the
conservative estimate of maximum tolerable level and the notion that the range between


CHAPTER 8
SUMMARY AND CONCLUSIONS
Selenium has had a long and storied history in animal nutrition and has played the
role of toxic element, essential nutrient, carcinogen, and contributor in cancer prevention.
However, it seems that seleniums greatest legacy is one of a toxic agent to livestock.
With its many implications as a toxic element, the use of Se as a supplement to livestock,
gamers much caution from feed manufacturers, animal scientists, and nutritionists. The
current estimate of the maximum tolerable level for dietary Se in domestic animals is 2
mg/kg. This estimate does not consider differences in metabolism of Se by different
species and makes no differentiation in the maximum tolerable level for the different
chemical forms of Se, such as Se yeast or sodium selenite. Furthermore, some evidence
exists to suggest that the maximum tolerable dietary concentration of Se for livestock is
grossly underestimated and to discredit the notion that the range between optimal and
toxic levels of Se is narrow. Likewise, studies in cattle and swine have shown a marked
difference in the efficacy of organic vs inorganic Se to increase blood, milk, and tissue Se
concentrations.
At the University of Florida, two types of experiments were completed. With
sheep, studies of tolerance in relation to maximum tolerance levels for ewes and wethers
were undertaken. Experiments using sheep were conducted to gather further data on 1)
the amount of dietary inorganic Se that can be tolerated by ewes during lamb production,
2) the effects of high levels of dietary Se fed to ewes on their lambs, and 3) the amount of
125


85
Table 5-3. Serum Se concentrations of wethers fed four dietary levels of Se as sodium
selenite or Se yeast3
Se source
Sodium selenite Se yeast
Dietary Se level, mg/kg
0.2 20 30 40 0.2 20 30 40
Week Serum Se, pg/L SEM
12
157
548
788
1000
412
2583
3210
2458
249bcde
24
130
1683
1487
1724
354
2639
3922
1585
826bc
36
444
851
960
1083
540
3283
2086
1409
250bcdf
48
110
822
1219
1496
292
2428
2076
1831
253bce
60
119
610
886
1250
424
1699
2712
2549
331bce
Avg
192
903
1068
1311
404
2526
2801
1966
395bdf
Data represent least squares means and pooled SE.
bDietary Se level response (P < 0.05).
Selenium source response (P < 0.05).
dDietary Se level x Se source interaction (P < 0.05).
Dietary Se level linear response (P < 0.05).
fDietary Se level quadratic response (P < 0.05).
Table 5-4. Whole blood concentrations of wethers fed four dietary levels of Se as
sodium selenite or Se yeast3
Se source
Sodium selenite Se yeast
Dietary Se level, mg/kg
0.2 20 30 40 0.2 20 30 40
Week Whole blood Se, pg/L SEM
12
392
1172
1484
1315
1183
4344
4290
5484
475bcd
24
420
1551
2228
2353
1661
4521
6259
5780
415bc
36
1004
1021
1708
2406
1549
5018
4841
1972
635
48
393
1772
1977
2416
1068
5061
5220
4929
298bcde
60
402
1258
1621
2043
1500
1759
3629
4914
435b
Avg
522
1355
1804
2107
1392
4028
4803
4408
534bcdf
Data represent least squares means and pooled SE.
bDietary Se level response (P < 0.05).
Selenium source response (P < 0.05).
dDietary Se level x Se source interaction (P < 0.05).
Dietary Se level linear response (P < 0.10).
fDietary Se level quadratic response (P < 0.05).


81
previous research (Glenn et al., 1964b; Smyth et al., 1990) has shown the heart to be the
target organ in instances of Se toxicosis. No abnormalities were prevalent enough to
establish a treatment-related pattern and no wethers receiving the maximum level of
dietary Se on our study (40 mg/kg) showed any abnormal tissue lesions. Further
evaluation of kidney, heart, and liver using transmission electron microscopy also
revealed no cellular abnormalities and enzymes, suggestive of tissue necrosis, were in or
below normal ranges at the termination of the experiment. Without the presence of tissue
damage and clinical signs, it seems that Se toxicosis was not induced in wether sheep fed
up to 40 mg/kg dietary Se as Se yeast or sodium selenite. However, Se concentrations in
serum, blood, wool, and tissues from wethers receiving organic Se indicate that Se
toxicity is dependent on Se source and that much inorganic dietary Se is reduced to
insoluble forms. The work of Cousins and Caimey (1961), Whanger et al. (1968), and
Koenig et al. (1997) support our findings.
Implications
The current estimate of the maximum tolerable dietary level of selenium for sheep
(2 mg/kg) seems to be grossly underestimated. Selenium, whether organic or inorganic in
form, can be fed as high as 40 mg/kg for up to 60 wk without inducing Se toxicosis.
Previously the range between optimal and toxic levels of selenium was reported as
narrow; however data from the present study would suggest that the range is relatively
wide. Increasing dietary selenium level, regardless of source, is an effective means of
increasing selenium in blood and tissues. Organic selenium is more greatly accumulated
by organs and tissues. Manipulation of dietary selenium source and level is an effective
way to change the selenium content of animal tissues commonly consumed by mankind.


CHAPTER 4
EFFECTS OF SELENIUM LEVELS IN EWE DIETS ON SELENIUM IN MILK
AND PLASMA AND TISSUE SELENIUM CONCENTRATIONS OF LAMBS
Introduction
Selenium has long been implicated as a toxic element to livestock (Oldfield,
2002). Animals grazing seleniferous plants in certain regions of the world are subject to
Se toxicosis and conditions such as alkali disease and blind staggers (McDowell,
2003). The estimated maximum tolerable level of Se for ruminant livestock is 2 mg/kg
(NRC, 1980). However, recent research (Cristaldi et al., in press) has shown that sheep
may consume up to 10 mg/kg Se as sodium selenite in the total diet for one yr, without
showing signs of selenium toxicosis. Although it was concluded that these wethers were
not suffering from Se toxicity, they did have increased serum, whole blood, and tissue Se
concentrations. Like blood and tissue, milk Se is affected by dietary Se level (Conrad and
Moxon, 1979; Givens et al., 2004) and Se readily crosses the placenta to the fetus (Van
Saun et al., 1989). Furthermore, positive correlations exist between blood Se of cows and
blood Se of their calves (Kincaid and Hodgson, 1989; Enjalbert et al., 1999; Pehrson et
al., 1999). In sheep, Cuesta et al. (1995) showed increased colostrum Se from ewes
receiving supplemental Se, and that milk Se was higher after one mo of supplementation.
Thus, it seems that neonates from dams consuming high dietary levels of Se would have
increased blood Se at birth, and subsequently would be exposed to high Se intake from
increased Se in milk.
48


65
Table 4-5. Plasma Se concentrations (gg/L) of lambs suckling ewes receiving different
levels of dietary Se as sodium selenite in year one3
Se in ewe diet,mg/kg
o'
ngc vji lamu, u
32
283
564
0.2
81b 66
11 lb 66
76b 52
92b 52
4
127bc 60
204bc 56
196b 49
246c 49
8
13 lbc 85
330c 73
209bc 73
258bcd 73
12
188bc 66
333 85
374cd 66
354cd 66
16
238bc 85
387 73
297c 73
43 ld 66
20
294 47
648d60
508d 44
419d 47
Data represent least squares means SE.
b,cdMeans within columns lacking a common superscript are different (P < 0.05).
ln = 5, 6, 3, 5, 3, and 6 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
2n = 5, 7, 4, 3, 4, and 6 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
3 = 8, 9,4, 5, 5, and 11 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively,
4 = 8, 9, 4, 5, 5, and 10 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
Table 4-6. Plasma Se concentrations (gg/L) of lambs suckling ewes receiving different
levels of dietary Se as sodium selenite in year two3
Age of lamb, d
Se in ewe diet,mg/kg 01 3^283 564
0.2
85b 62
74b 55
81b 55
86b 55
4
182bc 41
325 44
244c 47
287c 47
8
186bc 47
601d 47
314cd 55
263 51
12
253cd 41
686d 51
553e 62
430d 55
20
353d 71
737d 71
775f 87
340bcd 124
aData represent least squares means SE.
fM.dx.tjyjeans within columns lacking a common superscript are different (P < 0.05).
1n = 4, 9, 7, 9, and 3 for Se levels 0.2, 4, 8, 12, and 20 mg/kg, respectively.
2n = 5, 8, 7, 6, and 3 for Se levels 0.2,4, 8, 12, and 20 mg/kg, respectively.
3n = 5, 7, 5, 4, and 2 for Se levels 0.2, 4, 8, 12, and 20 mg/kg, respectively.
4n = 5, 7, 6, 5, and 1 for Se levels 0.2, 4, 8, 12, and 20 mg/kg, respectively.


59
2001) and more than double the plasma Se concentration suggested by Bostedt and
Schramel (1990) for normal growth and health in newborn calves. Lamb plasma,
collected before nursing, increased in Se as Se concentration in the diet fed to ewes
during gestation increased in both years. Ewes receiving 12 mg/kg dietary Se gave birth
to lambs with up three-fold higher plasma Se than did controls. Likewise, ewes receiving
20 mg/kg dietary Se, which is ten fold higher than the established maximum tolerable
level for Se, gave birth to lambs with only approximately four-fold higher plasma Se than
did controls. These results indicate that Se does cross the placenta to the fetus. Koller et
al. (1984) demonstrated maternal transfer of Se in beef cattle and Kim and Mahan (2001)
reported elevated serum and tissue Se in neonate pigs when dietary Se levels of sows
were increased. This does not concur with (Wright and Bell, 1964) who reported no
increase in lamb plasma Se when their dams were fed increased Se and demonstrated a
defined placental barrier for Se.
Plasma Se remained elevated in lambs which were suckling ewes receiving
increased dietary Se and from d 3 to d 56 ranged from 196 to 648 pg/L in year one and
244 to 775 pg/L in year two. These plasma Se concentrations were much higher than the
>70 pg/L suggested as adequate by Zachara et al. (1993). However, at no time did any
lamb have plasma Se near or above 1400 pg/L which has been suggested as the plasma
level when signs of Se toxicosis appear in sheep (Glenn et al., 1964c) and swine (Kim
and Mahan, 2001). Marrow (1968) reported that death occurred within 16 hours in 35%
of nursing lambs which were dosed with 10 mg of sodium selenite orally in an attempt to
prevent nutritional muscular dystrophy. Smyth et al. (1990) observed death as soon as
six hours after an oral dose of 5 mg Se/kg BW. Contrarily, Lagace et al.(1964) dosed


100
other treatments. Plasma Se and liver Se concentrations were not initially different. At d
365, plasma Se in cows receiving Se yeast was higher (P < 0.05) at 90 pg/L than from all
other treatments. Injectable selenate was intermediate and produced higher plasma Se
than control and both forms of selenite. Liver Se at d 365 was adequate (> 1200 pg/kg)
and higher (P < 0.05) in Se yeast treated cows than all others. Cows receiving injectable
selenate also had adequate liver Se concentrations that were higher (P < 0.05) than the
inadequate levels from control, free-choice selenite and injectable selenite. Whole blood
Se was adequate (> 100 pg/L) for all treatment groups at calving, 30 and 90 d
postpartum. At 205 d postpartum, cows receiving injectable selenate and both free-
choice treatments were adequate in whole blood Se, while controls and cows receiving
injectable selenite had inadequate whole blood Se. Cows receiving Se yeast produced
higher (P < 0.05) colostrum Se than all other treatments. No differences were observed
in milk Se at 30 and 90 d postpartum among treatment groups, however, both free-choice
and the injectable selenate treated cows had milk Se numerically higher than controls and
cows receiving injectable selenite. At weaning (205 d postpartum), cows receiving Se
yeast had at least two-fold higher (P < 0.05) milk Se than cows receiving other
treatments. Selenium supplementation with organic or inorganic Se via free-choice
minerals or injectable selenate maintained adequate Se concentrations in whole blood,
plasma, and liver. Inorganic Se was limited in its ability to increase milk Se, whereas Se
yeast increased milk Se at parturition and at weaning.


51
year. Plasma, milk, testes, and feed samples from both years were analyzed for Se
concentration using a fluorometric method described by Whetter and Ullrey (1978). To
help ensure reliability of the analytical method, a certified standard (National Bureau of
Standards Bovine Liver SRM- 1577a; U.S. Department of Commerce, National Institute
of Standards and Technology, Gaithersburg, MD) was frequently analyzed.
Effects of treatment on lamb testicular and colostrum Se were analyzed using
PROC MIXED in SAS (SAS for Windows 8e; SAS Inst. Inc., Cary, NC) in a completely
randomized design. Contrast statements were used to compare means as described by
Littell et al. (1998; 2000). PROC MIXED of SAS was also used to analyze effects of
treatment, d, and the interaction of treatment x d on milk Se and plasma Se as repeated
measures with a spatial power covariance structure with respect to d and a subplot of
animal nested within treatment. Contrast statements were written to determine
differences in means for different sampling d. PROC CORR was used to determine
correlations of ewe milk Se to lamb plasma Se.
Results
In year one, 11 of 52 lambs were removed from the study before 56 d of age. Five
lambs were bom to ewes which produced little or no milk, one ewe had extremely
enlarged or bottle teats and her two lambs were unable to suckle, two lambs died of
weakness/dehydration, and two died of joint ill. There were no apparent signs of
selenium toxicosis in any lambs regardless of dietary Se level of their dams.
In year one, colostrum Se was affected by Se concentration of the ewes diet (P =
0.008) and increased linearly (P < 0.001) as dietary Se increased (Table 4-2). Ewes
receiving 16 or 20 mg/kg dietary Se produced higher (P < 0.05) colostrum Se than did
controls. Ewes receiving 20 mg/kg dietary Se also produced higher (P < 0.05) colostrum


108
(Deposel Multidose; Novartis New Zealand, Ltd., Auckland, NZ) at the initiation of the
experiment, 3) three subcutaneous injections of 5 mL (5 mg Se-mL'1 + 68 IU vitamin E as
dl-a-tocopheryl-mL'1) of sodium selenite (Mu-Se; Schering-Plough Animal Health,
Union, NJ) at the initiation of the experiment and every four mo thereafter, 4) free-
choice access to a mineral mixture containing 26 mg Se/kg as sodium selenite
(Southeastern Minerals, Inc., Bainbridge, GA), or 5) free-choice access to a mineral
mixture containing 26 mg Se/kg as Se yeast (Sel-Plex; Alltech, Inc, Nicholasville, KY).
All cows grazed bahiagrass {Paspalum notatum) pastures at a stocking rate of 1.7
cows/ha and were supplemented with bahiagrass {Paspalum notatum) hay, molasses-
based liquid supplement ad libitum, and whole cottonseed and pelleted citrus pulp at
rates of 0.68 kg-cow_1-d 1 and 1.81 kg-cow^-d'1, respectively, from November 2002
through March 2003.
Treatment groups receiving no Se or injectable Se were housed together and had
access to a free-choice mineral mixture containing no Se (Table 7-1). Cows receiving Se
via free-choice mineral mixtures were housed in separate groups and had access to the
same mineral mixture with added Se as sodium selenite or Se yeast for treatments 4 and
5, respectively (Table 7-1). All free-choice mineral mixtures were offered in wooden
mineral feeders and protected from rain and at a height to prevent calves from consuming
the mineral mixtures.
Forty-two calves (25 male, 17 female) were bom over a 24 d span between
December 31, 2002 and January 23, 2003. Average age of calf did not differ among
treatment groups {P = 0.40) and calves had an average birth date of January 10, 2003.
Blood samples from calves were collected via jugular venipuncture into 10-mL


7
al. (1993) documented the importance of Se and vitamin E for maintaining immune
function in livestock. Those authors measured immune responses of blood, colostrum
and milk leukocytes of sows and concluded that greater phagocytic and microbicidal
activity could be realized in milk and colostrum through supplementation with Se and
vitamin E.
Reproductive problems in beef cattle such as retained placenta, infertility,
abortions, births of premature, weak, or dead calves, cystic ovaries, metritis, delayed
conception, erratic estrus periods, and poor fertilization may be successfully overcome
with Se supplementation (Corah and Ives, 1991). Awadeh et al. (1998a) concluded that
Se intakes of pregnant cows could be an important factor in weak calf disorders and that
passive immunity and heat production by newborn calves using brown adipose tissue
could both be influenced by maternal Se intakes. A Se deficiency in the diet of dairy
cattle was reported to be a contributor to a high incidence of retained placentas (Trinder
et al., 1973). Data from studies using dairy cows have shown that supplemental Se and
vitamin E to animals receiving Se deficient diets are beneficial in decreasing the
incidence of retained placentas (Julien et al., 1976; Hemken et al., 1978). Smith et al.
(1988) studied the effects of Se on disease resistance in dairy cattle and concluded that
many dairy herds have inadequate dietary intakes of Se and vitamin E. Those authors
added that insufficient intakes of these nutrients could result in increased cases of
mastitis, metritis, and retained placenta, and recommended Se supplementation at a level
to maintain blood Se at a minimum of 200 pg/L. Weiss et al. (1990) studied the
relationships between Se and mammary gland health in commercial dairy herds and


122
Table 7-5. Testis and liver Se concentrations (jug/kg, dry basis) of beef calves suckling
dams that received different sources of Se supplementation3
Source of Se Supplementation
Testes Se, gg/kg
SE
Liver Se, ug/kg
SE
Control (No Se)
162b
21
372b
82
Barium Selenate1 (Deposel)
210b
17
297b
65
Sodium Selenite2 (Mu-Se)
177b
19
308b
61
Free Choice Mineral3 (selenite)
169b
14
600c
69
Free Choice Mineral4 (Sel-Plex)
207b
21
132 ld
69
Testes taken at birth; Liver biopsies at 205 d of age; data represent least squares means, n = 22 and 36,
respectively.
b c,dMeans within columns lacking a common superscript differ (P < 0.05).
'Dams of these calves received a subcutaneous injection of 9 mL Deposel at an avg of 125 d gestation.
2Dams of these calves received an injection of 5 mL Mu-Se every 4 mo beginning at an avg of 125 d
gestation.
3Dams of these calves consumed free-choice mineral mix containing 26 mg/kg Se as sodium selenite at
an avg of 41.5 g cow''-d'' beginning at an avg of 125 d gestation.
4Dams of these calves consumed free-choice mineral mix containing 26 mg/kg Se as Se yeast at an avg
of 85.5 g-cow '-d'1 beginning at an avg of 125 d gestation.


40
pathologic signs of selenium toxicosis. Ewes fed 16 and 20 mg/kg produced some signs
of selenium toxicosis; however, general metabolic disorders could not be ruled out and no
deaths of ewes consuming these levels of selenium were attributed to selenium toxicosis.
Further studies of this nature should further prove that the current suggested tolerable
level of Se is underestimated.
Summary
The objectives of this 72-wk study were to evaluate and compare the effects of six
dietary levels of inorganic Se on serum, whole blood, wool, and tissue Se concentrations
of mature ewes during lamb production and determine maximum tolerable level of Se
during lamb production. Forty-one range-type ewes were used in a completely
randomized design with six dietary treatments. Sodium selenite was added to a corn-
soybean meal basal diet to provide 0.2 (control), 4, 8, 12, 16, and 20 mg/kg dietary Se to
ewes during lamb production. Serum Se and ewe BW were measured at 4-wk intervals,
whole blood Se, and wool Se were measured every 12 wk, and samples of brain,
diaphragm, heart, hoof, kidney, liver, and psoas major muscle were collected at the
termination of the experiment. Dietary Se did not affect ewe BW during the study (P =
0.69). Serum Se increased linearly as dietary Se level increased (P < 0.001) and
responded cubically (P = 0.02) over time. Selenium in whole blood increased linearly (P
< 0.001) with increased dietary Se and cubically (P <0.01) over time. Wool Se increased
linearly (P < 0.001) as dietary Se increased and response over time was quadratic (P <
0.001). Brain, diaphragm, heart, and psoas major muscle Se increased linearly as Se in
the diet increased, liver Se responded quadratically, and hoof and kidney Se responded
cubically to treatment (P < 0.05). In general, serum, whole blood, and tissue Se


I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.
A- ^./H
Lee R. McDowell, Chairman
Professor of Animal Sciences
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.-
&
X-
Timothy T. Marshall
Professor of Animal Sciences
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.
Claus D. Buergelt
Professor of Veterinary Medicine
I certify that I have read this study and that in my opinion it conforms to acceptable
standards of scholarly presentation and is fully adequate, in scope and quality, as a
dissertation for the degree of Doctor of Philosophy.
Richard N. Weldon
Associate Professor of Food and Resource
Economics
This dissertation was submitted to the Graduate Faculty of the College of
Agricultural and Life Sciences and to the Graduate School and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy. A
December, 2004
r,,, ~ -S. ^ >.
Dean, College^of Agriculturatand Life"
Sciences
Dean, Graduate School


27
Effect of time on ewe BW can be explained by changes in BW associated with gestation
and lactation over two lambings during the study.
Ten of 41 ewes died over the course of this 72-wk study. Gross necropsies were
performed on eight ewes following death. Tissues from two ewes were too severely
decomposed to allow for evaluation for pathological changes. Necropsy of eight ewes
cited causes of death as lymphadenitis associated with injury (two ewes), endoparasitism
(two ewes), ketosis (three ewes) and pneumonia (one ewe). Pathological evidence of Se
toxicosis was not found in any ewe that died before the termination of the experiment.
In the first yr, 53 lambs were bom over 20 d from March 9, 2002 to March 28,
2002. Fifty-two lambs were bom alive and unassisted (Table 3-2). One lamb was very
large (8 kg) and died shortly after a difficult birth. The lambs bom in yr one represent a
129% lamb crop when calculated as lambs bom alive per ewe exposed. In the second yr,
36 lambs were bom over 34 d from January 17, 2003 to February 20, 2003 (Table 3-2).
All lambs were bom alive and unassisted. Thirty-six lambs in yr two represent a 109%
lamb crop as only 33 ewes were exposed in the second yr. Number of lambs bom per
ewe did not affect serum Se concentration (P > 0.54) of ewes receiving any level of
dietary Se. Glenn et al. (1964a), who fed higher levels of dietary Se than in the present
experiment, did not observe an effect of dietary Se level on reproduction in 2-yr-old
range ewes. Those researchers observed a similar number of pregnancies in each
treatment group and no malformations in lambs. In contrast, Rosenfeld and Beath (1947)
observed lamb deformities in a field study and attributed the anomalies to excess Se in
ewe diets. However, seleniferous plants were the Se source, rather than inorganic sources
used in the present experiment. Furthermore, in a grazing situation, it is possible that


30
most 37% of a reported toxic level (3700 pg/L) in swine (Aitken, 2001). Caravaggi et al.
(1970) established an LD5o for sheep at 455 pg/kg BW. When our data are described on a
pg/kg BW basis using the highest dietary concentration (20 mg/kg), highest daily intake
(1135 g/d), and average ewe BW (60 kg), our ewes were consuming, at maximum, 378
pg/kg BW. This is 17% less than the LDsofor sheep as previously described. The ewes
in the present study were mature and maintained healthy ruminal function throughout the
study. This is contrasted with the unweaned lambs used by Caravaggi et al. (1970).
Those lambs may have received Se via i.m. injection. Administration of Se parenterally
disallows the reduction of selenite Se to insoluble selenide via ruminal microorganisms as
described by (Whanger et al., 1968). This would suggest that the LD50 for sheep could be
considerably higher than previously thought. Glenn et al. (1964a) fed sodium selenate at
high levels to range ewes that were similar in BW to ewes on the present study. Those
researchers did not induce death by Se toxicosis with daily oral doses less than 25 mg
Se/ewe. Of the 17 deaths reported in their experiment, only one was induced with a daily
dose of 25 mg Se/ewe. Eight deaths were induced with a daily dose 37.5 mg Se/ewe and
eight deaths were induced with a daily dose 50 mg Se/ewe. Those deaths were not by
acute Se toxicosis. The ewes received experimental Se doses for at least 80 d before
death by Se toxicosis was induced. In the same experiment, Glenn et al. (1964a) further
suggested an average minimum toxic level of Se for adult sheep to be 0.825 mg/kg BW
when fed for 100 d. Using this estimate, the minimum toxic level of Se for ewes of the
size used in our study would be 50.3 mg/d. Selenium consumption, at the highest dietary
level of 20 mg/kg, never reached even 50% of that previously reported level throughout
our study. Also, Blodgett and Bevill (1987) reported an LD50 for sheep, using sodium


CHAPTER 5
COMPARATIVE EFFECTS AND TOLERANCE OF VARIOUS DIETARY LEVELS OF
SE AS SODIUM SELENITE OR SE YEAST ON BLOOD, WOOL, AND TISSUE SE
CONCENTRATIONS OF WETHER SHEEP
Introduction
Selenium was first implicated as an essential nutrient for animals by Schwarz and
Foltz (1957). Prior to that, Se was viewed primarily as a detriment to livestock which
was documented by Franke (1934) and Moxon (1937). Selenium deficiency is far more
prevalent worldwide than toxicity. However, Se toxicity is a greater concern to livestock
producers and nutritionists, as toxicities are more difficult than deficiencies to control.
Current estimates put the maximum tolerable level of Se at 2 mg/kg for the major
livestock species (NRC, 1980) and no differentiation exists for tolerable levels between
ruminants and monogastric animals. However, previous research suggests that inorganic
Se (e.g., sodium selenite) may be reduced to insoluble selenide by microorganisms in the
rumen, thus reducing overall absorption of Se by ruminant animals (Butler and Peterson,
1961; Hidiroglou et al., 1968). Wright and Bell (1966) reported that swine retained 77%
and sheep retained 29% of an oral dose of inorganic Se. The NRC makes no distinction
between inorganic and organic (e.g., Se yeast or seleno-methionine) forms of Se for
current maximum tolerable levels, though the chemical form of dietary Se leads to
markedly different physiological responses of livestock (Knowles et al., 1999; Pehrson et
al., 1999; Gunter et al., 2003). Kim and Mahan (2001) reported more accumulation of Se
in the plasma and tissues of swine fed high dietary levels of Se as Se yeast compared to
the same Se levels as sodium selenite, and that Se toxicity occurred sooner and its clinical
67


ACKNOWLEDGMENTS
The author wishes to express the utmost gratitude to Dr. Lee R. McDowell,
chairman of the supervisory committee, for his guidance, patience, encouragement and
direction throughout the Ph.D. program and during preparation of this dissertation.
Appreciation is given to Drs. Tim Marshall, Claus Buergelt, and Richard Weldon for
service and dedication on the graduate committee and for advice and understanding.
Special thanks are extended to Dr. McDowell for his unselfish attitude and including me
on several experiments, publications, and educational opportunities. Gratefulness beyond
words is in order to Dr. McDowell for showing me that one will get further by lifting
others up than by putting anyone down. Dr. Todd Thrift deserves thanks for his
willingness to listen and provide both professional and personal advice.
A huge debt of gratitude and appreciation is extended to Mr. Bert Faircloth, Mr.
Charles Stephens, Mr. Steve Chandler, Mr. Jesse Saveli, and Mr. Brantley Ivey at the
Santa Fe Beef Unit for their help in conducting the experiments and collecting data. Mr.
Dean Glicco deserves particular thanks for his care of the experimental animals and for
becoming a trusted friend and confidant to the author. The author thanks Ms. Nancy
Wilkinson for sharing her invaluable knowledge of lab procedures and for her patience in
instruction of laboratory techniques. Mrs. Lorraine McDowell deserves thanks for her
work with electron microscopy on animal tissues.
IV


66
Figure 4-1. Rambouillet ewe in late gestation fitted with a device to cover the udder and
prevent lambs from suckling until a blood sample was obtained. Device was made from
nylon pantyhose and elastic straps, and held in place with safety pins. (Device courtesy of
Dr. Donald J. Davis, Crossville, TN)
Figure 4-2. Effects of Se concentration of ewe diet on testicular Se concentration of their
lambs in year one. Testicular selenium concentrations were 1.67, 1.83, 2.88, 3.26, 3.76,
and 4.25 mg/kg (dry matter basis) for ram lambs suckling ewes receiving 0.2, 4, 8, 12,
16, and 20 mg/kg dietary Se, respectively. SE = 0.56.


4
Perhaps Se is best known for its role as an essential constituent of glutathione
peroxidase (Rotruck et ah, 1973) and four Se-dependent glutathione peroxidases have
been identified and designated as glutathione peroxidase 1, 2, 3, and 4 (Lei et ah, 1998).
These four enzymes benefit animal health by protecting cellular and subcellular
membranes against oxidative damage. Also, it appears that Se-dependent glutathione
peroxidases provide a second line of defense against peroxidation of vital phospholipids
(McDowell, 2003). Vitamin E provides the first line of defense against the peroxidation
of phospholipids in membranes.
Adequate dietary or supplemental Se is an effective way to combat the
aforementioned problems in growth and reproduction of livestock. Likewise, in the
presence of adequate Se, the glutathione peroxidase system works in synergy to protect
animal cells against lipid peroxidation. In general, livestock species have minimum
requirements of dietary Se which range from 0.05 to 0.30 mg/kg (McDowell, 2003).
There are numerous examples throughout the scientific literature that cite benefits in
growth, reproduction, and prevention of WMD and other anomalies due to adequate
dietary or supplementary Se.
The dietary Se requirement for all classes of sheep ranges from 0.10 to 0.20 mg/kg
(NRC, 1985). However, the minimum dietary Se level necessary to prevent WMD varies
as reported in the literature. Oldfield et al. (1963) reported that 0.06 mg/kg was the
minimum dietary Se level required to prevent WMD in lambs. However, researchers in
New Zealand indicated that lambs had normal growth and remained free of clinical signs
of Se deficiency when grazing pastures containing 0.03 to 0.04 mg/kg (Hartley and
Grant, 1961). Oldfield et al. (1963) further reported that ewes fed a ration containing


93
treated cows had similar changes in BW and cows receiving Mu-Se tended (P = 0.07) to
gain more weight than cows receiving Deposel.
Cow whole blood Se concentrations at intervals postpartum are summarized in
Table 6-4. Significant effects of treatment (P < 0.001), d (P < 0.001), and treatment x d
(P = 0.013) were observed. At parturition, whole blood Se concentrations from cows
receiving Deposel or Sel-Plex were higher (P < 0.05) than whole blood Se from controls
and cows receiving Mu-Se or free-choice selenite. Also, Mu-Se tended (P = 0.13) to
produce blood Se higher than control. At 30 d postpartum, no differences (P > 0.05) in
whole blood Se were observed among Deposel, Mu-Se, and free-choice selenite
treatment groups. Cows receiving Deposel had higher (P < 0.05) blood Se than controls
and tended (P = 0.08) to have higher blood Se than cows receiving Mu-Se. Cows
receiving Sel-Plex tended to have higher (P = 0.053) blood Se concentrations than did
controls and cows receiving any other form of Se supplementation at 30 d postpartum.
At 90 d postpartum, Sel-Plex produced blood Se concentrations higher (P < 0.05)
than did free-choice selenite, Mu-Se, or no Se supplementation. Deposel treated cows had
blood Se higher (P < 0.05) than Mu-Se treated cows and the controls and tended to be
higher (P = 0.14) in blood Se than cows receiving free-choice selenite. Blood Se from
the free-choice selenite group was similar (P = 0.46) to that from Mu-Se and higher (P <
0.001) than from controls. Again, at 205 d postpartum, which coincided with weaning of
calves, Sel-Plex treated cows had blood Se higher (P < 0.05) than cows from all other
treatment groups. Free-choice selenite and Deposel treated cows had similar (P = 0.92)
blood Se, which was higher (P < 0.05) than blood Se from controls and the Mu-Se group.
A third order polynomial response (P = 0.001) was observed, as overall, whole blood Se


120
Table 7-1. Composition of mineral mixtures offered free-choice to brood cows
Free-choice mineral mixtures
Component
No Sea
Sodium seleniteb
Selenized yeastc
%, as-fed
Calcium
18.73
18.73
18.49
Phosphorus
8.00
8.00
7.90
Sodium chloride
26.73
26.73
26.39
Magnesium
2.00
2.00
ppm, as-fed
1.97
Iron
5695
5695
5695
Zinc
4015
4015
4015
Manganese
2225
2225
2225
Copper
500
500
500
Iodine
50
50
50
Cobalt
50
50
50
Selenium

26
IU/kg, as-fed
26
Vitamin A
102272
102272
102272
Vitamin D3
10227
10227
10227
Vitamin E
23
23
23
aManufactured by Southeastern Minerals, Inc., Bainbridge, GA; served as basal mineral mixture.
bManufactured by Southeastern Minerals, Inc., Bainbridge, GA, by addition of 26 mg/kg Se as sodium
selenite to basal mineral mixture.
cCreated by addition of 1.3% Se yeast (Sel-Plex 2000; Alltech, Inc., Nicholasville, KY) to basal mineral
mixture.
Table 7-2. Frequency, daily amount, and total of amount of supplemental Se
administered to cows
Source of supplemental Se
Selenium
supplementation
interval, d
Avg
supplementation,
mg Se cow '-d-1
Total Se
supplementation,
mg
No Se supplementation
6
6
6
Barium selenate injection1
365
1.23
450
Sodium selenite injection2
125
0.21
75
Sodium selenite via free-choice minerals3
l5
1.08
393
Selenized yeast via free-choice minerals4
l5
2.22
811
'Cows received a subcutaneous injection of 9 mL Deposel at initiation of study.
2Cows received an injection of 5 mL Mu-Se at initiation of study and re-injection every 4 mo.
3Cows had continuous access to free-choice mineral mix containing 26 mg Se/kg as sodium selenite and
consumed mineral mix at avg of 41.5 g-cow''-d 1 .
4Cows had continuous access to free-choice mineral mix containing 26 mg Se/kg as Se yeast and
consumed mineral mix at an avg of 85.5 g-cow1d1.
5Access to free-choice minerals containing Se was continuous throughout the study.
6 Cows receiving no Se supplementation or injectable Se had free-choice access to and consumed the
basal free-choice mineral mix (no Se) at an avg of 62.2 g-cow1-d'1.


49
Acute Se toxicosis has been evaluated by injecting ewe lambs with sodium
selenite (Blodgett and Bevill, 1987), and Abdennebi et al. (1998) evaluated the toxic
effects of dosing lambs with extracts of milk vetch (Astragalus lusitanicus). In both
studies, weaned lambs were utilized. Newborn and pre-weaned lambs differ from older
sheep in ruminal and digestive function (Church, 1979; Goursand and Nowak, 1999), and
may respond differently than older animals to increased Se intake. We hypothesized that
when Se in gestating ewe diets is increased, colostrum Se, milk Se and plasma Se of their
lambs will increase. The objective of this experiment was to follow ewes through two
lamb crops and evaluate and compare the effects of six levels of dietary Se on ewes milk
and the Se status of their lambs prior to weaning.
Materials and Methods
All animal procedures were conducted within the guidelines of and approved by
the University of Florida Institutional Animal Care and Use Committee. This 504-d
experiment utilizing ewes and two lamb crops was conducted from December 18, 2001 to
May 5, 2003 at the University of Florida Sheep Nutrition Unit located in southwestern
Alachua County, FL. Thirty-three, four year old, Rambouillet ewes that originated from
a single flock in Texas and were previously confirmed pregnant (average 57 d gestation)
were weighed (57.4 5.7 kg) and received 2-ml ivermectin (Ivomec; Merial Ltd. Iselin,
NJ). Ewes were randomly assigned to one of six dietary treatments for a 504 d (two
lambing seasons) study. Six dietary treatments were 0.2, 4, 8, 12, 16, or 20 mg/kg Se as
sodium selenite (as-fed basis) added to a corn-soybean meal basal diet (Table 4-1). The
basal diet was formulated to meet animal requirements for protein, energy as TDN,
vitamins, and minerals for this class of sheep (NRC, 1985). Animal numbers per
treatment were 5, 7, 5, 5, 4, and 7 for 0.2 (control), 4, 8, 12, 16, and 20 mg/kg added Se,


BIOGRAPHICAL SKETCH
Paul Armand Davis was bom in Winchester, Tennessee on July 11, 1973. The
fifth-generation stockman is the elder son of Dr. Donald J. and Carol Davis and has one
younger brother, Ryan. He lived in Belvidere, Tennessee, until the age of 3, and then
moved to Crossville, Tennessee, where he graduated from Cumberland County High
School in 1991. Throughout elementary and high school, Paul was heavily involved in
numerous 4-H and FFA livestock projects, was recognized as Cumberland County FFAs
Star Greenhand and traveled to Japan as a 4-H exchange student. In January 1992, he
began work on a Bachelor of Science in Agriculture at Tennessee Technological
University and graduated in May 1996.
As an undergraduate in the animal science concentration, Paul became active in
the Block and Bridle Club and held the office of club president. He was honored as
Pledge of the Year by his Kappa Sigma Fraternity brothers in 1993, was inducted into the
Delta Tau Alpha Agricultural Honor Society, and served on the Animal Science
Academic Quadrathlon team in 1994. Paul received the first Tennessee Cattlemens
Association Future Cattle Industry Leader Scholarship, the Joe Scott Memorial
Scholarship, and the W. Clyde Hyder Animal Science Award for outstanding senior in
animal science.
Following graduation, Paul worked in the livestock industry at Peoples
Stockyards in Cookeville, Tennessee, East Tennessee Livestock Center in Sweetwater,
145


CHAPTER 7
TISSUE AND BLOOD SELENIUM CONCENTRATIONS AND PERFORMANCE OF
BEEF CALVES FROM DAMS RECEIVING DIFFERENT FORMS OF SELENIUM
SUPPLEMENTATION
Introduction
Selenium is required as a coenzyme for glutathione peroxidase, which acts as part
of the cellular antioxidant defense system (NRC, 1996), and is associated with the
pathogenesis of white muscle disease. White muscle disease, a degeneration of striated,
skeletal muscles is the major outward sign of Se deficiency in newborn calves and may
develop in tero or shortly after birth (McDowell, 2003). Selenium is also important for
adequate immune function. Underdeveloped, weak, dead, or generally unthrifty calves
may be bom to Se deficient brood cows (Corah and Ives, 1991). Unfortunately, affliction
with white muscle disease and poor animal performance due to insufficient Se is not
limited to newborn livestock. Calves are susceptible to a delayed WMD, developing
after birth, usually from one to four mo of age. Likewise, in Florida and other parts of
the southeastern U.S., conditions known as buckling and shoulder lameness are observed
in Se deficient feeder calves (McDowell et al., 2002). The costs associated with these
conditions and deficiencies may seriously reduce the profit margin of Stocker or feedlot
operations (Pirelli et al., 2000).
Recent studies indicate that blood Se in newborn calves can be increased through
Se supplementation of their dams (Abdelrahman and Kincaid, 1995; Gunter et al., 2003;
Valle et al., 2003). Likewise, positive correlations between Se concentration in dams
milk and Se concentration of calf whole blood have been observed in calves up to 70 d of
106


41
concentrations from ewes receiving 12, 16, or 20 mg/kg dietary Se were higher (P < 0.05)
than from controls and ewes receiving less dietary Se. Though serum, whole blood, and
wool Se concentrations were elevated in ewes receiving increased dietary Se, at no time
did serum, whole blood, or wool Se concentrations reach levels previously reported as
toxic and a pattern of clinical signs of Se toxicosis was not observed. Microscopic
evaluation of liver, kidney, diaphragm, heart, and psoas major muscle did not reveal
evidence of Se toxicosis in ewes on any dietary Se level. Ewes under our experimental
conditions and during the stresses of production were able to tolerate up to 20 mg/kg
dietary Se as sodium selenite for 72 wk. These findings suggest that the maximum
tolerable level of inorganic Se for sheep to be much higher than 2 mg/kg as was
suggested previously. Experiments which are longer in duration and utilize higher dietary
Se concentrations may be used to clearly define the maximum tolerable level.


28
lamb deformities were due to toxic elements other than Se. In both yr of our study, all
lambs were bom free of congenital deformities, but the number of pregnancies were
lowest in ewes receiving 16 mg/kg dietary Se, but not 20 mg/kg. However, breeding
soundness evaluations were not performed on ewes or rams used in this study and thus, to
incriminate or exclude dietary Se level as a detriment to ewe reproduction would be
observational.
Blood
Serum Se concentrations from wk 4, 8, and 12 were analyzed together and will be
referred to throughout the results and discussion as late gestation yr 1. Lactation yr 1
includes serum Se concentrations from wk 12, 16, 20, and 24. Week 12 is included in
both late gestation and lactation for yr 1 as some ewes were lactating and some remained
in late gestation when wk 12 sampling occurred. Weeks 28, 32, 36, 40, and 48 compose
the dry, rebreeding period. Late gestation in yr 2 includes serum Se measurements from
wk 52, 56, and 60. Lactation in yr 2 includes wk 60, 64, 68, and 72. Similar to yr 1, one
sampling date (wk 60) was common to both late gestation and lactation and was included
in both periods.
During all stages of lamb production, serum Se increased in a linear fashion (P <
0.001) as dietary Se level increased (Table 3-3). This agrees with previous Se toxicity
research as Se concentrations in serum of wether sheep (Cristaldi et al., in press) also
increased linearly as dietary selenite Se was increased. All ewes had similar (P > 0.82)
serum Se at the initiation of this experiment. Initial serum Se ranged from 90 to 120
pg/L, which is below the normal range (120 to 180 pg/L) for adult sheep (Aitken, 2001).
A cubic response within treatment (P = 0.02) was observed in serum Se across the stages
of production (time) from wk four to wk 72. Ewe serum Se, in general, was higher


130
injectable selenite. At weaning (205 d postpartum), cows receiving Se yeast had at least
two-fold higher (P < 0.05) milk Se than cows receiving other treatments.
Cows utilized in this study calved in early January 2003 and effects of Se
supplementation on performance and blood and tissue Se concentrations of their calves
were also compared and evaluated. No differences in calf weight at birth or weaning
were observed among treatment groups. Average daily gain was higher (P < 0.05) for
calves from dams that received Se via free-choice minerals. Calf whole blood Se was
determined at birth, 30, 90 and 205 d of age. Testicles were collected at birth from males
and a liver biopsy was performed on all calves at d 205 for liver Se determination. At
birth, calf blood Se was in the adequate range (>100 pg Se/L) for all treatments. At d 30
and d 90, control, both injectable products, and free-choice sodium selenite treatments
produced calf blood Se in or near the adequate range and Se yeast treated calves
remained well above adequate range at 166 and 182 pg Se/L, respectively. At 205 d,
whole blood Se concentrations for control and both injectables were in the deficient range
(< 50 pg Se/L), calves from the free-choice sodium selenite treatment were marginally
deficient (< 75 pg Se/L) and the Se yeast group had blood Se well above adequate at 188
pg Se/L. Selenium in calf testes ranged from 162 to 210 pg Se/kg and did not differ
among treatments. Liver Se taken at weaning ranged from 297 to 1321 pg Se/kg and
calves from the two free-choice treatments had Se concentrations higher (P < 0.05) than
both injectable treatments and the controls. Positive correlations (r = 0.57 and 0.64; P <
0.01) between calf liver and whole blood Se and cow whole blood Se were observed.
Correlation between concentration of Se in calf whole blood and in cow milk was also
positive (r = 0.49; P < 0.002).


72
press). Likewise, Ullrey et al. (1977) reported that lamb BW were unaffected by dietary
Se level in feeds containing differing proportions of organic and inorganic Se. However,
Kim and Mahan (2001) reported a quadratic decrease in final BW of swine as dietary Se
level was increased using sodium selenite or Se yeast. Those authors observed the most
drastic decreases when selenite Se was added above 10 mg/kg and when Se yeast was
added at 20 mg/kg. Our results differ from the findings with swine, as organic Se had a
more dramatic deleterious affect on BW than did selenite Se. This could be explained by
organic Se not being subject to reduction to selenides by rumen microorganisms as
suggested by previous research (Whanger et al., 1968; van Ryssen et ah, 1989; Whanger,
2002) and thus, being more available to cause toxic effects in ruminant livestock.
Seven of 28 wethers died during the course of our study and were subjected to
gross necropsy by pathologists at the University of Florida Veterinary Teaching Hospital.
All wethers were described in good physical condition at time of necropsy with adequate
adipose tissue. Causes of death were determined to be spinal cord compression trauma,
endoparasitism, pulmonary edema, and unknown. One wether from the 20 mg/kg
organic Se group had mild hepatic lipidosis and one wether from the 30 mg/kg organic Se
group exhibited signs of mild myocarditis. However, definitive evidence of death due to
Se toxicosis was not found and the gross lesions seemed to be due to metabolic changes
or were merely background findings.
Serum selenium values (105 23 pg/L) were below the normal range (120 to 150
pg/L) for adult sheep (Aitken, 2001) and did not differ among treatment groups (P =
0.36) at the initiation of the experiment. Serum Se concentrations measured at wk 12, 24,
48, and 60 ranged from 110 to 3922 pg/L and increased linearly (P < 0.05) as dietary Se


16
than did cows receiving supplemental selenite Se in early and late lactation (Pehrson et
al., 1999). In a two-yr study utilizing Florida beef cows, milk Se was consistently higher
from cows receiving free-choice minerals with Se yeast compared to cows receiving Se
as selenite or selenate injections (Valle et al., 2002). Also, calves suckling the cows
which received the organic Se had higher Se concentrations in plasma and liver (Valle et
al., 2003). Researchers at Ohio State University fed Se as Se yeast or sodium selenite at
dietary levels of 0.15 and 0.30 mg/kg to gestating and lactating sows. Colostrum and
subsequent milk Se concentrations were consistently at least two-fold higher from sows
receiving organic Se than from sows receiving selenite Se (Mahan, 2000). Data from
New Zealand indicated that the transfer of Se into cows milk was markedly more
efficient, up to three-fold more, with selenized yeast than with sodium selenate (Knowles
et al., 1999).
The effectiveness of different sources of Se for supplementation continues to be
evaluated even though Se has been recognized as nutritionally essential since the late
1950s. Selenium provided by different supplementation methods and from different
sources leads to different physiological responses in the animals that serve mankind.
With evidence of an increasing ability to manipulate the Se content of milk and animal
tissues which are commonly consumed by humans, it seems to be possible to supplement
Se to humans through method and source of Se supplementation to livestock. Givens et
al. (2004) reported a decline in Se intake by the people of Great Britain. After
conducting an experiment which validated previous findings that the milk of dairy cows
could be increased by feeding an organic Se source, those authors further explored the
idea of increasing human consumption of Se by altering the Se content of foods. It seems


139
Marrow, D. A. 1968. Acute selenite toxicosis in lambs. J. Am. Vet. Med. Assoc 152:
1625-1629.
Maus, R. W., F. A. Martz, R. L. Belyea, and M. F. Weiss. 1980. Relationship of dietary
selenium to selenium in plasma and milk from dairy cows. J. Dairy Sci. 63: 532-
537.
McConnell, K. P., and D. M. Roth. 1966. Respiratory excretion of selenium. Proc. Soc.
Exper. Biol. Med. 123:919-921.
McDowell, L. R. 2003. Minerals in animal and human nutrition. 2nd ed. Elsevier
Science, Amsterdam.
McDowell, L. R., J. A. Froseth, R. C. Piper, I. A. Dyer, and G. H. Kroening. 1977.
Tissue selenium and serum tocopherol concentrations in selenium-vitamin E
deficient pigs fed peas (Pisum sativum). J. Anim. Sci. 45: 1326-1333.
McDowell, L. R., G. Valle, L. Cristaldi, P. A. Davis, O. Rosendo, and N. S. Wilkinson.
2002. Selenium availability and methods of selenium supplementation for
grazing ruminants. Pages 86-101 in Proc. 13th Annual Ruminant Nut. Symp.,
Univ of Florida, Gainesville, FL.
Miller, W. T., and H. W. Schoening. 1938. Toxicity of selenium fed to swine in the
form of sodium selenite. J. Agrie. Res. 56: 831-835.
Mellor, D. J., and L. Murray. 1986. Making the most of colostrum at lambing. Vet. Rec.
118:351-353.
Moxon, A. L. 1937. Alkali disease or selenium poisoning. South Dakota Agrie. Exp. Stn.
Bull. 311. South Dakota State Univ., Brookings, SD.
Moxon, A. L., and M. A. Rhian. 1943. Selenium poisoning. Physiol. Rev. 23: 305-337.
Muth, O. H., J. E. Oldfield, L. F. Remmert, and J. R. Schubert. 1958. Effects of
selenium and vitamin E on white muscle disease. Science. 128: 1090-1091.
National Academy of Sciences (NAS). 1971. Selenium in Nutrition. Natl. Acad. Sci.
USA, Washington, DC.
National Academy of Sciences (NAS). 1983. Selenium in Nutrition. Revised ed. Natl.
Acad. Press., Washington, DC.
Norton, S. A., and F. D. McCarthy. 1986. Use of injectable vitamin E and selenium-
vitamin E emulsion in ewes and suckling lambs to prevent nutritional muscular
dystrophy. J. Anim. Sci. 62: 497-508.



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PAGE 158

)WIO@O_OM A /n


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
SELENIUM TOLERANCE IN SHEEP AND SELENIUM SUPPLEMENTATION
METHODS FOR BEEF CATTLE
By
Paul Armand Davis
December 2004
Chair: Lee Russell McDowell
Major Department: Animal Sciences
A series of experiments to evaluate and compare methods, sources, and dietary
levels of selenium was carried out utilizing sheep and cattle. Experiments using sheep
were conducted to gather further data on 1) the tolerance of dietary inorganic Se by ewes
during lamb production, 2) the effects of high levels of dietary Se fed to ewes on their
lambs, and 3) the tolerance of organic or inorganic Se by mature wethers. A cow-calf
herd was used to evaluate and compare effects of using different forms of dietary or
parenteral Se on weight change and blood, milk, and liver Se concentrations of beef cows
and their calves. In ewes fed Se, as sodium selenite, above requirements, Se
concentrations in blood, wool, and soft tissues generally increased (P < 0.05) as dietary
Se increased. Ewes tolerated up to 20 mg/kg dietary Se without suffering from toxicosis.
Lambs bom to ewes receiving high levels of dietary Se had increased plasma Se (P <
0.05) as Se in ewe diets increased. No signs of Se toxicosis were observed in lambs
regardless of Se concentration in the ewe diets. Wethers, fed up to 40 mg/kg Se as


61
Implications
Feeding Se to gestating and lactating ewes above the current maximum tolerable
level (2 mg/kg) does increase the Se concentration in colostrum and subsequently
produced milk. However, this practice does not increase milk Se concentrations to a
level at which their nursing lambs suffer from Se toxicosis. Likewise, feeding increased
Se to ewes does increase plasma and tissue Se in lambs but not to a concentration above
those previously found in sheep determined not to be suffering from Se toxicity.
Moreover, data from other species even suggests that feeding increased Se to gestating
and lactating animals may produce colostrum of higher quality that may be beneficial to
their offspring. Data from this and other recent research has now established that the
maximum tolerable level of Se, as selenite, for sheep to be considerably higher than the
previously suggested 2 mg/kg.
Summary
The objective of this 504-d experiment was to evaluate and compare the effects of
six levels of dietary selenium (Se) on ewes milk and the Se status of their lambs prior to
weaning. Sodium selenite was added to a basal diet to provide 0.2 (control), 4, 8, 12, 16,
and 20 mg/kg dietary Se for ewes during gestation and lactation over two lambings.
Colostrum Se ranged from 257 to 3542 pg/L and increased linearly as dietary Se
increased (P < 0.001) in both years. Ewe milk Se ranged from 75 to 2228 pg/L and also
increased linearly as dietary Se increased (P < 0.01). In general, ewes receiving > 12
mg/kg Se produced higher milk Se than controls. Blood samples were collected from
lambs before nursing and at 3, 28, and 56 d of age to evaluate plasma Se concentrations.
At birth, lamb plasma Se ranged from 74 to 775 pg/L and was affected (P < 0.001) by the
Se concentration of the ewe diets, which indicates placental transfer of Se. Lambs from


64
Table 4-3. Milk Se concentrations (jixg/L) from ewes receiving different levels of
dietary Se as sodium selenite in year onea
Added Se, mg/kg
31
Days postpartum
282
563
0.2
75b 117
66b 91
32b 91
4
312bc 117
12lb91
165b 83
8
r-
-H
O
O
O
163bc 117
160b 117
12
490c 144
189bc 117
N)
4^
cr
n-
--U
16
920d 117
253bc 144
653c 117
20
628cd 117
466c 91
538c 83
aData represent least squares means SE.
b,cdMeans within columns lacking a common superscript are different (P < 0.05).
]n = 3, 3, 3, 2, 3, and 3 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
2n = 5, 5, 3, 3, 2, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
3 = 5, 6, 3, 3, 3, and 6 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
Table 4-4. Milk Se concentrations (pg/L) from ewes receiving different levels of
dietary Se as sodium selenite in year twoa
Added Se, mg/kg
31
Days postpartum
282
563
0.2
57b 238
81b 238
69b 238
4
574bc 194
646b 180
339bc 194
8
1442d 213
462b 213
493bc 213
12
895cd 238
689b 238
914c 238
20
933cd 337
923b 475
2228d 476
aData represent least squares means SE.
bc,dMeans within columns lacking a common superscript are different (P < 0.05).
ln = 4, 5, 5, 5, and 2 for Se levels 0.2, 4, 8, 12, and 20 mg/kg, respectively.
2n = 4, 6, 5, 4, and 1 for Se levels 0.2, 4, 8, 12, and 20 mg/kg, respectively.
3n = 4, 5, 5, 4, and 1 for Se levels 0.2, 4, 8, 12, and 20 mg/kg, respectively.


113
between Se in cows whole blood and liver Se of their calves at d 205. The positive (P <
0.003) correlation (r = 0.049) between Se in cows milk and Se in whole blood of their
calves is illustrated in Figure 7-3.
Discussion
Birth weights of the calves (average 35 kg) were similar to those reported in other
studies of this type (Ammerman et al., 1980; Gunter et al., 2003; Valle et al., 2003).
Likewise, these authors reported no significant differences in calf birth weight among
treatment groups. Unadjusted weaning weight of calves on our study did not differ
among treatment groups, though calves whose dams received Se yeast were numerically
heavier than those from all other treatments. Gunter et al. (2003) compared effects of Se
supplementation via sodium selenite or Se yeast to beef cows on calf performance and
found no differences in total weight gain and ADG in calves. Flowever, Spears et al.
(1986) found significant differences in summer weight gain and adjusted weaning weight
in crossbred beef calves supplemented with injectable Se and vitamin E. Hidiroglou and
Jenkins (1975) also reported weight gain in calves administered Se with vitamin E.
Additionally, Castellan et al. (1999) observed increased ADG in Angus x Hereford calves
that were supplemented with injectable sodium selenite at intervals before weaning.
Differences observed in calf ADG in this study somewhat follow the pattern of
differences in calf blood Se at weaning. At weaning, the control and both injectable
groups had similar blood Se and similar ADG. In our experiment, calves from both free-
choice treatments had higher blood Se than calves whose dams received injectable Se and
ADG was higher in the free-choice groups than in the injectable selenate group. Wichtel
et al. (1996) reported an increase of 20% in ADG of Se supplemented Friesian heifers vs
unsupplemented controls. Those authors also indicated the possibility of altered growth


118
from our study, where a correlation value of r = 0.49 was observed between the
concentration of Se in cows milk and the Se in the whole blood of their calves. This
value may have been more positive if Se concentrations from Se rich colostrum were not
included in the correlation.
Implications
Supplementation of selenium to the cow herd with injectable selenium or with
sodium selenite in free-choice minerals was not shown to be an effective method to attain
adequate blood selenium in pre-weaned calves. Therefore, calves whose dams are
receiving either no selenium or selenium supplementation via these methods may be
susceptible to disease and suppressed growth due to selenium deficiency. Calves
suckling dams that were supplemented with organic selenium as selenium yeast,
however, have been found to have adequate blood selenium from birth to weaning.
These calves should be at little or no risk of disease due solely to selenium deficiency.
Summary
The objective of this experiment was to evaluate and compare effects of different
sources and physical forms of supplemental Se fed to cows on blood and tissue Se
concentrations of their calves. Forty-three Angus cows (115-130 d gestation) were
randomly assigned to five groups and received either no Se supplementation (control),
one 9 mL barium selenate s.c injection, 5 mL sodium selenite via s.c. injection, every
four mo, or sodium selenite or Se yeast at 26 mg Se/kg in free-choice mineral mixtures.
Cows grazed bahiagrass (Paspalum notatum) pastures, received routine dietary
supplementation and calved mostly in early January 2003. No differences in calf weight
at birth or weaning were observed between treatment groups. Average daily gain was
higher (P < 0.05) for calves from dams that received Se via free-choice minerals. Calf


17
that a worthy challenge exists for animal scientists and food scientists to work
collaboratively to identify effective programs for administration of Se to dairy and food
animals so that the milk and meat subsequently produced can be more nutritious for
humankind.
Selenium Toxicosis
Selenium deficiencies are prevalent in many parts of the world (McDowell, 2003)
and the benefits of Se supplementation continue to be elucidated. However, it seems that
Se is still most often implicated as an element which is toxic to livestock. This belief
most likely stems from diary-style documentation, observations, and research findings
beginning as early as 1295 when Marco Polo described an agent in plants which when
eaten by horses caused their hooves to fall off (Komroff, 1926). Six hundred years later
similar afflictions began to be described in the Great Plains region of the United States.
In 1856, a U.S. Army surgeon reported the occurrence of a disease, fatal to U.S. Cavalry
horses, similar to the affliction described by Marco Polo (Madison, 1860). The horses in
the Nebraska Territory near Fort Randall lost hair and had debilitating conditions of the
hoof. By the 1890s, farmers and stockmen who settled in northern Nebraska and South
Dakota observed similar conditions in livestock (Moxon and Rhian, 1943). Selenium
toxicity in livestock and laboratory animals has been reported from the 1930s to the
present day. Some reports were observations of animals receiving seleniferous grains or
grazing seleniferous plants (Franke, 1934; Franke and Potter, 1935; Moxon, 1937). Other
researchers have intentionally induced or attempted to induce Se toxicities, while several
reports of Se toxicity are a result of accidental overdosing with injectable Se.
Rosenfeld and Beath (1964) suggested that Se poisoning in livestock occurs in
three distinct phases: acute toxicity and the two phases of chronic toxicity, alkali disease


37
Liver Se concentration ranged from 4.20 to 230.36 mg/kg DM and responded
quadratically as dietary Se level increased (Figure 3-3.) Regressing liver Se (mg/kg DM)
on dietary Se concentration (mg/kg) produced the following relationship:
Liver Se = 4.19 + 26.59 Dietary Se 9.31 Dietary Se2 (r2 = 0.66; P < 0.01).
Ewes receiving 20 mg/kg dietary Se had higher (P < 0.05) liver Se than ewes from all
other treatments. No other differences (P > 0.05) existed among controls and Se
treatment groups.
Linear increases in the Se concentration of loin, liver, kidney and hoof were
reported in swine (Kim and Mahan, 2001) and sheep (Cristaldi et al., in press). Likewise,
Echevarria et al. (1988) reported linear responses of sheep liver, kidney, heart, and
muscle to dietary Se as sodium selenite Se. In our study, loin, diaphragm, heart, and
brain responded linearly, where kidney and hoof responded cubically and liver responded
quadratically. These higher degree polynomials may be due to changes in metabolism of
Se as dietary Se concentration approaches 20 mg/kg. Most previous research used 10
mg/kg Se as the highest dietary concentration.
Enzymes and Histopathology
Serum for evaluation of albumin and enzyme activities was collected at wk 72
along with samples of brain, diaphragm, heart, hoof tip, kidney, psoas major muscle, and
liver for histopathological evaluation. Concentrations of albumin and activities of Aik
phos, ALT, GGT, AST, and CK in serum were in or below the normal range for adult
sheep (Table 3-6). In instances of Se toxicosis, the activities of these enzymes would
have been increased due to tissue necrosis. Our observations agree with those reported by
Cristaldi et al. (2004) as albumin and enzyme activities in wether sheep after receiving up
to 10 mg/kg Se were in the normal ranges.


80
nature. Cristaldi et al. (2004) found no abnormalities after microscopic evaluation of
heart, liver, kidney, diaphragm, and muscle from wethers consuming up 10 mg/kg Se for
one yr. Examination of kidney, heart, and liver tissues by transmission electron
microscopy did not reveal any apparent changes in cell structure as related to Se toxicosis
and no differences in tissue cells from controls and wethers receiving 20, 30, or 40 mg/kg
Se were observed.
Concentrations of albumin and activities of Aik phos, ALT, GGT, AST, and CK
in serum collected at the termination of the experiment were, in general, in or below the
normal range for adult sheep (Table 5-7). In instances of Se toxicosis, the activities of
these enzymes would have been increased due to tissue necrosis. Our observations agree
with those reported by Cristaldi et al. (2004) as albumin and enzyme activities in wether
sheep after receiving up to 10 mg/kg Se were in the normal ranges. The lack of elevated
enzymes, which are suggestive of tissue necrosis, further indicates that the wethers on our
study were not suffering from Se toxicosis.
Throughout this 60-wk experiment clinical signs of Se toxicosis (e.g., lameness,
wool loss, and abnormal hoof growth) were not observed, though serum and whole blood
Se concentrations were frequently higher than those described in livestock diagnosed
with hyperselenosis. However, wool Se concentrations from wethers on our study never
reached the levels previously reported in the hair of livestock suffering from alkali
disease. Loin muscle and diaphragm showed no gross lesions at slaughter and no
abnormality was observed with microscopic evaluation. Abnormal pathology in the
kidney, heart, and liver was rare and could, in each case, be attributed to a cause other
than Se toxicosis. No pale focal areas were observed in the myocardium, though


23
research in ruminants has been documented in lambs or wethers. Controlled experiments
using ewes during stresses of production (e.g., gestation and lactation) are lacking in the
scientific literature. The objective of this long-term (72 wk) study were to evaluate and
compare effects of feeding Se as sodium selenite at supranutritional levels on ewe serum,
blood, wool, and tissue Se concentrations during two lambing periods and to determine
maximum tolerable level of Se.
Materials and Methods
All animal procedures were conducted within the guidelines of and approved by
the University of Florida Institutional Animal Care and Use Committee. This experiment
utilizing ewes during two lambings was conducted from December 18, 2001 to May 5,
2003 at the University of Florida Sheep Nutrition Unit located in North Central Florida.
Forty-one, four-yr-old, Rambouillet ewes, that originated from a single range flock in
Texas and had been pasture exposed to rams during October and early November 2001
(average 57 d gestation), were weighed (57.4 5.7 kg) and administered 2-ml ivermectin
dewormer s.c. (Ivomec; Merial Ltd., Iselin, NJ). Ewes were randomly assigned to one of
six dietary treatments for a 72-wk study. Six dietary treatments were 0.2, 4, 8, 12, 16, or
20 mg/kg Se as sodium selenite (as-fed basis) added to a corn-soybean meal basal diet
(Table 3-1). The basal diet was formulated to meet animal requirements for protein,
energy as TDN, vitamins, and minerals for this class of sheep (NRC, 1985). Animal
numbers per treatment were six for 0.2 (control) and seven each for 4, 8, 12, 16, and 20
mg/kg added Se treatments. Ewes were housed by treatment group in covered wooden
pens (53.5 m2) with earth floors and ad libitum water.


143
Waite, R., H. R. Conrad, and A. L. Moxon. 1975. Metabolism of selenium and
selenium-75 in dairy cows. J. Dairy Sci. 58: 749. (Abstr.)
Weiss, W. P., J. S. Hogan, K. L. Smith and, K. H. Hoblet. 1990. Relationships among
selenium, vitamin E, and mammary gland health in commercial dairy herds. J.
Dairy Sci. 73:381-390.
Whanger, P. D. 2002. Selenocompounds in plants and animals and their biological
significance. J. Am. Coll. Nutr. 21:223-232.
Whanger, P. D., P. H. Weswig, and O. H. Muth. 1968. Metabolism of 75Se-selenite and
75Se-selenomethionine by rumen microorganisms. Fed. Proc. 27: 418 (Abstr.).
Whetter, P. A., and D. E. Ullrey. 1978. Improved fluorometric method for determining
Se. J. Assoc. Off. Anal. Chem. 61:927-930.
Wichtel, J. J., A. L. Craigie, D. A. Freeman, H. Varela-Alvarez, and N. B. Williamson.
1996. Effect of selenium and iodine supplementation on growth rate and on
thyroid somatotropic function in dairy calves at pasture. J. Dairy Sci. 79: 1895-
1902.
Windisch, W., and M. Kirchgessner. 2000. True absorption, excretion and tissue
retention of selenium at widely varying selenium supply to rats. In: A.M. Roussel,
R.A. Anderson and A.E. Favier (eds). Trace Elements in Man and Animals 10.
Plenum Publishers, New York.
Wohlt, J. E., W. L. Foy, Jr., D. M. Kniffen, and J. R. Trout, J. R. 1984. Milk yield of
Dorset ewes as affected by sibling status, sex and age of lamb, and measurement.
J. Dairy Sci. 67: 802-807.
Wright, P. L., and M. C. Bell. 1964. Selenium-75 metabolism in the gestating ewe and
fetal lamb: Effects of dietary a tocopherol and selenium. J. Nutr. 84: 49-57.
Wright, P. L., and M. C. Bell. 1966. Comparative metabolism of Se and tellurium in
sheep and swine. Am. J. Physiol. 211: 6-10.
Wuryastuti, H., H. D. Stowe, R. W. Bull, and E. R. Miller. 1993. Effects of vitamin E
and selenium on immune responses of peripheral blood, colostrum, and milk
leukocytes of sows. J. Anim. Sci. 71: 2464-2472.
Xia, Y., J. Butler, M. Janghorbani, P. Ha, P. Whanger, J. Olesik, and L. Daniels. 2000.
Effects of selenium status on selenium incorporation into plasma fractions and
excretion in urine in men infused with 74Se selenite. In: A.M. Roussel, R.A.
Anderson and A.E. Favier (eds). Trace Elements in Man and Animals 10. Plenum
Publishers, New York.


CHAPTER 3
TOLERANCE OF INORGANIC SELENIUM IN RANGE-TYPE EWES DURING
GESTATION AND LACTATION
Introduction
Since its discovery by Berzelius in 1817, Se has had a rich and colorful history in
animal agriculture. Though much of the world is troubled with Se deficiencies
(McDowell, 2003), Se toxicities present a greater problem to control. In 1957, Se was
established as an essential nutrient and the benefits of Se supplementation to livestock
continue to be elucidated. Current estimates put the maximum tolerable level of Se at 2
mg/kg for the major livestock species (NRC, 1980) and no differentiation exists for
tolerable levels between ruminants and monogastric animals. However, the work of
Butler and Peterson (1961) and Hidiroglou et al. (1968) suggests that inorganic Se (e.g.,
sodium selenite) may be reduced to insoluble selenide by microorganisms in the rumen,
thus reducing overall absorption of Se by ruminant animals. Wright and Bell (1966)
reported that swine retained 77% of an oral dose of inorganic Se, which is nearly three
fold the retention by sheep. Selenium toxicities have been often produced by researchers
in ruminants, but they are generally induced by Se injections (Marrow, 1968; Caravaggi
et al., 1970; Shortridge et al., 1971) or by feeding Se above maximum tolerable levels (5
to 196 ppm) to monogastric animals (Franke and Potter, 1935; Miller and Schoening,
1938; Kim and Mahan, 2001). More recently, Cristaldi et al. (2004) demonstrated that
wether sheep did not display signs of Se toxicosis after receiving up to 10 mg/kg dietary
Se for one yr. Based on these and other previous findings, it seems that the current
maximum tolerable level of Se for ruminants is underestimated. Most Se toxicity
22


86
Table 5-5. Wool Se concentrations of wethers fed four dietary levels of Se as sodium
selenite or Se yeast3
Se source
Sodium selenite Se yeast
Dietary Se level, mg/kg
0.2 20 30 40 0.2 20 30 40
Week -Wool Se, mg/kg SEM
12
1.37
3.27
6.69
4.15
3.78
12.67
21.09
24.26
3.80b"
24
1.47
3.57
5.72
11.92
7.04
31.58
35.69
37.30
2.87bcd
36
1.68
6.02
9.85
10.85
5.70
18.99
22.79
21.29
4.72
48
1.19
3.15
5.64
7.23
6.39
24.81
39.09
29.65
2.22bcd
60
1.29
3.90
5.01
6.23
4.38
23.22
25.65
25.99
2.01bcd
Avg
1.40
3.98
6.58
8.08
5.46
22.25
28.87
27.70
3 3gkcdefg
aData represent least squares means and pooled SE.
bDietary Se level response (P < 0.05).
'Selenium source response (P < 0.05).
dDietary Se level x Se source interaction {P < 0.05).
'Dietary Se level linear response (P < 0.10).
fTime response (P < 0.05).
gTime x Se source interaction (P < 0.05).
Table 5-6. Effects of four dietary levels of Se as sodium selenite or Se yeast on tissue
Se of wethers8
Se source
Sodium selenite Se yeast
Dietary Se level, mg/kg
0.2
20
30
40
0.2
20
30
40
SEM
Tissue
CP
concentration,
DC
mg/Kg
Brain
1.28
4.22
4.74
6.87
6.12
21.90
32.30
18.71
0.99bcd
Diaphragm
0.82
4.74
3.33
7.81
5.28
10.30
26.34
20.71
2.69bcdc
Heart
1.59
3.80
5.13
6.23
6.35
23.77
28.71
33.93
2.43bcd
Hoof
3.44
8.79
9.68
13.78
6.26
12.53
29.20
23.66
5.52
Kidney
8.43
19.94
27.93
27.89
22.26
33.96
77.61
36.28
6.87bcde
Liver
2.66
31.72
41.42
78.18
15.67
23.42
132.73
41.24
18.17bde
Loin
0.71
3.13
4.41
5.13
5.73
14.69
23.51
26.87
1.05bcd
aData represent least squares means and pooled SE.
bDietary Se level response (P < 0.05).
'Selenium source response (P < 0.05).
dDietary Se level x Se source interaction (P < 0.05).
'Dietary Se level linear response (P < 0.10).


103
Table 6-5. Milk Se concentrations of cows receiving different sources and forms of Se
supplementation at various d postpartum3
Source of Se supplementation
Days postpartum
0
30
90
205
Milk Se, ug/L
Control (No Se)
39b 7
14b 7
6b 7
15b 7
Barium Selenate1 (Deposel)
34b 6
15b6
15b 6
21b 6
Sodium Selenite2 (Mu-Se)
35b 6
13b 6
6b 6
16b 6
Free Choice Mineral3 (Selenite)
39b 7
26b 6
16b 6
15b 7
Free Choice Mineral4 (Sel-Plex)
71c 6
27b 6
15b 6
42c 6
aData represent least squares means and standard errors; n=41 for each sample d.
bcMeans within columns lacking a common superscript differ (P < 0.05).
!Cows received a subcutaneous injection of 9 mL Deposel at d 0.
2Cows received an injection of 5 mL Mu-Se every 4 mo beginning at d 0.
3Cows consumed free-choice mineral mix containing 26 mg/kg Se as sodium selenite
at an avg of 41.5 g-cow^-d"1 beginning at d 0.
4Dams of these calves consumed free-choice mineral mix containing 26 mg/kg Se as Se
yeast at an avg of 85.5 g-cow'1-d'1 beginning at d 0.
Table 6-6. Plasma Se concentration at d 0 and d 365 of beef cows that received
different sources and forms of Se supplementation8
do d 365
Source of Se Supplementation
Plasma Se, gg/L
SE
Plasma Se, ug/L
SE
Control (No Se)
62b
5
35b
5
Barium Selenate' (Deposel)
61b
4
71 =
4
Sodium Selenite2 (Mu-Se)
56b
4
28b
4
Free Choice Mineral3 (selenite)
52b
4
54d
5
Free Choice Mineral4 (Sel-Plex)
56b
4
90e
4
aData represent least squares means and standard errors; n = 42 and 41 for d 0 and d 365, respectively;
Adequate Se level in plasma is > 70 pg/L.
b c d eMeans within columns lacking a common superscript differ (P < 0.05).
'Cows received a subcutaneous injection of 9 mL Deposel at d 0.
2Cows received an injection of 5 mL Mu-Se every 4 mo beginning at d 0.
3Cows consumed free-choice mineral mix containing 26 mg Se/kg as sodium selenite at an avg of 41.5
g-cow '-d'1 beginning at d 0.
4Cows consumed free-choice mineral mix containing 26 mg Se/kg as Se yeast at an avg of 85.5 g-cow"
'd~' beginning at d 0.


97
ruminants than selenite. From d 90 to d 205 postpartum, whole blood Se decreased in
controls and cows receiving Mu-Se, and both were below the adequate whole blood Se
level (> 100pg/L) as defined by Gerloff (1992) and Dargatz and Ross (1996). Cows
receiving Se from Deposel or either free-choice mineral mix maintained whole blood Se
above the adequate level from parturition to 205 d postpartum. At d 205 postpartum, all
controls and 89% of cows receiving Mu-Se had whole blood Se below the adequate level.
Pehrson et al. (1999) reported whole blood Se concentrations above adequacy in
Hereford cows while nursing calves, and observed no differences between organic and
inorganic forms of Se supplementation.
Differences in milk Se concentrations from this study, agree with findings of
Valle et al. (2002) as, in both experiments, cows supplemented with Se yeast consistently
produced milk Se higher than or similar to cows receiving selenite or selenate. Likewise,
colostrum Se from cows receiving organic Se was higher than in controls and inorganic
Se treated cows. Ortman and Pehrson (1999) reported milk Se values more than 100%
higher from Swedish dairy cows receiving Se yeast than from cows receiving inorganic
or no Se supplementation. Also, beef cows supplemented with Se yeast were consistently
higher in milk Se at calving and at approximately 35 d postpartum than cows receiving
sodium selenite (Pehrson et al., 1999). Results have been similar in other species; Mahan
(2000) reported higher milk Se at seven and 14 d postpartum from sows receiving organic
vs inorganic Se fed at the same levels. In our study, though differences were observed in
blood Se from selenate vs selenite, no differences in milk or colostrum Se were observed
between the two inorganic sources. Our findings agree with those of Ortman and
Pehrson (1999) and support their statement that there is no difference in effect of selenate


11
offered free-choice. No differences in performance between unsupplemented controls or
cattle receiving either form of Se were observed. However, differences in blood Se of
supplemented vs unsupplemented cattle were reported. Likewise, Se yeast treated cows
and their calves had higher blood Se than cows and calves receiving selenite Se. Those
authors concluded that calves are at risk for Se deficiency if their dams are not
supplemented with Se and that even when selenite Se is provided, calves may still be at
risk. Sheep may also be supplemented with Se which is included in mineral mixtures and
salt licks. Norwegian researchers reported no incidences of WMD in lambs and
increased Se in blood and colostrum when Se fortified mineral mixtures and salt licks
were offered to ewes and lambs (Ovemes et al., 1985).
In addition to the use of injectable Se or the inclusion of Se in free-choice mineral
mixtures, livestock producers may use an intraruminal or intrareticular bolus or pellet
which provides a sustained release of Se. Judson et al. (1991) reported that the use of an
intraruminal Se pellet and steel grinder increased blood Se of ewes and lambs compared
with controls. However, the Se pellet and grinder system was not as effective as an
injection of barium selenate at increasing blood Se. Campbell et al. (1990) used
crossbred beef cows to evaluate the safety and efficacy of Se boluses and Se pellets.
Both methods of Se supplementation were shown to be both safe and effective; however,
blood Se of cows receiving either method of Se supplementation increased until d 119 of
the study and was decreased by d 220. As in previous studies, both methods produced
blood Se higher than the blood Se from unsupplemented controls. Abdelrahman and
Kincaid (1995) evaluated the effects of administration of an intraruminal Se bolus on
colostrum, plasma, and whole blood Se concentration of dairy cows. Those authors


Ill
affected by Se supplementation of dams (P = 0.04) and there were differences (P < 0.05)
in ADG among treatment groups. Both free-choice treatments produced higher (P <
0.05) calf ADG than did the injectable barium selenate treatment and calf ADG from
both free-choice treatments tended to be higher (P = 0.10) and {P = 0.07), respectively,
than ADG in calves whose dams received Se supplementation as injectable sodium
selenite (Table 7-3).
Treatment and treatment x d affected (P < 0.001) Se concentrations in calf whole
blood (Table 7-4). Differences (P < 0.05) existed among treatment groups on all
sampling d. At d 0, calves from Se yeast treated cows had higher (P < 0.05) whole blood
Se than did those on control, injectable selenate, or free-choice selenite treatments and
tended (P < 0.10) to have higher blood Se than calves from injectable selenite treated
cows.
Whole blood Se concentrations were higher (P < 0.05) in calves from the Se yeast
treatment than those from all other treatment groups and no other differences or
tendencies (P = 0.30) were observed at d 30. Again at d 90, the Se yeast treatment
produced blood Se concentrations higher (P < 0.05) than all other treatments. The two
injectable treatments were similar to control (P 0.64) and the free-choice selenite
treatment was higher (P < 0.05) than injectable selenite. Furthermore, the free-choice
selenite treatment tended (P = 0.15) to be higher than control and injectable selenate
treatments.
At d 205 (weaning), the two injectable treatments produced calf whole blood Se
similar (P = 0.56) to controls. The free-choice selenite treatment produced calf whole
blood Se higher (P < 0.05) than either injectable product and tended (P = 0.08) to be


32
20 mg/kg Se had higher whole blood Se than controls and ewes receiving 4, 8, or 16
mg/kg Se and tended be higher (P = 0.13) in whole blood Se than ewes receiving 12
mg/kg dietary Se. At wk 24, ewes receiving 20 mg/kg Se had higher whole blood Se
than ewes from all other treatment groups and only ewes receiving 4 mg/kg Se had whole
blood Se similar to controls. At wk 36, ewes receiving 12, 16, and 20 mg/kg Se had
similar (P > 0.05) whole blood Se and again, only ewes receiving 4 mg/kg Se had whole
blood Se similar to controls. At wk 48, whole blood Se concentrations from ewes
receiving 16 and 20 mg/kg Se were similar (P >0.10) and higher than (P < 0.05) from
ewes on all other treatments. Ewes receiving 8 and 12 mg/kg Se had similar (P = 0.88)
whole blood Se concentrations which were higher (P < 0.05) than those from controls
and ewes receiving 4 mg/kg Se. Only whole blood Se from ewes receiving 4 mg/kg Se
was similar (P = 0.16) to controls at wk 48. Whole blood Se concentrations at wk 60
followed a pattern similar to wk 48, in terms of differences among treatments. At wk 72,
whole blood Se from four of six dietary levels had numerically decreased from wk 60.
Whole blood from ewes receiving 20 mg/kg Se was higher (P < 0.05) in Se concentration
than in ewes from all other treatments. Ewes receiving 4, 8, 12, and 16 mg/kg dietary Se
produced similar (P > 0.10) whole blood Se and only ewes receiving 12 mg/kg Se had
higher (P < 0.05) whole blood Se than did controls. Cristaldi et al. (2004) also reported a
linear increase in whole blood Se as dietary Se was increased. Likewise, those authors
noted differences in treatment means over controls as dietary Se levels were increased up
to 10 mg/kg. Increased whole blood Se concentrations were reported in dairy cows as
their salt-based mineral mixtures were increased from 20 mg/kg to 120 mg/kg selenite Se


62
ewes receiving dietary Se at 20 mg/kg had higher (P < 0.05) plasma Se than controls at
birth and 3, 28, and 56 d of age in both years. Selenium concentration in lamb testes
collected at 70 d of age was also affected by Se content of ewe diets. In year one, lambs
whose dams received 16 or 20 mg/kg Se had higher (P < 0.05) testicular Se than controls,
and no differences in testicular Se were observed in year two. No signs of Se toxicosis
were observed in lambs regardless of dietary Se concentration of the ewes diet. These
results suggest that ewes consuming up to 20 mg/kg inorganic Se may give birth to
normal lambs, and that the lambs may not suffer from Se toxicosis before weaning.
Selenium as sodium selenite may be fed to ewes at concentrations greater than the current
maximum tolerable levels (2 mg/kg) without adversely affecting their offspring.


Copyright 2004
by
Paul Armand Davis


39
and thus, the finding of abnormal organ pathology. However, abnormal pathological
findings were few and did not follow a pattern with respect to dietary level which would
be indicative of Se toxicosis.
No clinical signs of Se toxicosis such as abnormal hoof growth or loss of wool
were observed in ewes receiving > 16 mg/kg Se. However, some excessive hoof growth
was observed after approximately one yr in ewes receiving 16 and 20 mg/kg Se and wool
loss was observed during lactation in two ewes receiving 20 mg/kg Se. Livestock
suffering from alkali disease were reported to have hair Se concentrations of up to 45
mg/kg and whole blood Se of 4.1 mg/L, while hooves, liver, and kidney of affected
animals contained 10 mg/kg Se or more (NAS, 1983). At no time during our study did
wool Se reach even 10 mg/kg and whole blood Se remained less than 50% of the
aforementioned 4.1 mg/L concentration. Also, hoof Se remained under 8 mg/kg for all
treatments during the course of our study. Liver and kidney Se concentrations from our
study were higher than the 10 mg/kg previously reported. The elevated concentrations of
Se in the liver and kidney of ewes consuming 16 and 20 mg/kg, and the observation of
some clinical signs of Se toxicosis and limited pathological abnormalities in ewes
consuming these Se levels may indicate that some ewes were beginning to suffer from Se
toxicosis. However, definitive evidence was not observed. Therefore, it is necessary that
either dietary Se concentration or duration of experiment be increased in order to induce a
definitive Se toxicosis using inorganic Se.
Implications
The maximum tolerable level of selenium as sodium selenite for ruminants is
higher than 2 mg/kg. Feeding up to 12 ppm selenite selenium to ewes under stresses of
production (i.e., gestation and lactation) for 72 wk did not produce any clinical or


55
0.05) and lambs suckling ewes that received 8 mg/kg Se tended to have higher plasma Se
than controls (P = 0.067). Plasma Se from lambs suckling ewes receiving 16 or 20
mg/kg Se was higher than plasma Se from lambs suckling ewes receiving 4 mg/kg Se (P
< 0.05) and tended to be higher than plasma Se from lambs suckling ewes receiving 8
mg/kg Se (P < 0.08).
Lamb plasma Se, in year two (Table 5-6), was affected by the concentration of Se
in the diet of their dams (P < 0.001) and increased linearly as Se concentration in dams
diet increased (P < 0.001). Day of sampling and the interaction of dietary Se
concentration x d of sampling also affected lamb plasma Se (P < 0.01). At birth, lambs
from ewes receiving 12 mg/kg Se had higher plasma Se than did controls (P < 0.05).
Likewise, lambs from ewes receiving 20 mg/kg Se had higher (P < 0.05) plasma Se than
did controls and lambs from dams receiving 4 or 8 mg/kg Se. From 3 to 56 d of age,
lamb plasma Se was positively correlated to ewe milk Se (r = 0.44; P < 0.001). At 3 d of
age, lambs from all treatment groups had higher plasma Se than did controls (P < 0.05)
and lambs suckling ewes receiving 8, 12, or 20 mg/kg Se had higher plasma Se than did
those suckling ewes receiving 4 mg/kg Se (P < 0.05). At d 28, lamb plasma Se from all
treatment groups was higher than controls (P < 0.05). Lambs suckling ewes receiving 20
mg/kg Se had plasma Se higher than all other treatment groups (P < 0.05). Also, lambs
suckling ewes receiving 12 mg/kg Se had plasma Se higher than lambs suckling ewes
receiving 4 or 8 mg/kg Se (P < 0.05). At d 56, lambs suckling dams receiving 4, 8, or 12
mg/kg Se had higher plasma Se than did controls (P < 0.05) and lambs suckling dams
receiving 20 mg/kg Se tended to have higher plasma Se than did controls (P = 0.067).


25
vacutainer, centrifuged at 700 x g for 25 min, and serum frozen at 0C for analysis of
albumin and the following enzymes: alkaline phosphatase (Aik Phos), alanine
transaminase (ALT), aspartate transaminase (AST), creatinine phosphokinase (CK), and
gamma glutamyl transferase (GGT). Albumin and the enzymes were analyzed in order to
determine possible tissue breakdown as a result of Se toxicosis.
Samples of brain, diaphragm, heart, hoof tip, kidney, liver, and psoas major
muscle were collected, and frozen (0) until analyzed for Se. Sections (1 cm3) of liver,
heart, kidney, diaphragm, and psoas major muscle from all animals were placed in 10%
neutral-buffered formaldehyde for subsequent microscopic evaluation for evidence of Se
toxicosis.
For histopathological evaluation, the tissue samples fixed in buffered formalin
were embedded in paraffin and sectioned at 6 microns. All sections were stained with
hematoxylin and eosin, and examined under a light microscope (10X, 20X, and 40X).
Serum albumin, Aik Phos, ALT, AST, CK, GGT were evaluated on a Hitachi 911
analyzer with reagents from Sigma (Sigma Chemical Co., St. Louis, Mo.). These
procedures were established by the Veterinary Medical Teaching Hospital at the
University of Florida.
Serum, whole blood, wool, tissue, and feed samples were analyzed for Se
concentration using a fluorometric method described by Whetter and Ullrey (1978). To
help ensure reliability of the analytical method, a certified standard (National Bureau of
Standards Bovine Liver SRM-1577a; U.S. Department of Commerce, National Institute
of Standards and Technology, Gaithersburg, MD) was frequently analyzed.


132
Se concentrations that were mostly in the marginal or deficient range. Supplementation
of Se, as Se yeast, to the cow herd is an effective means of maintaining adequate blood
Se in pre-weaned calves and possibly increasing calf ADG.


695Ft3$fl]l|lj
03/21/05 347 60^ L'


63
Table 4-1. Diet composition (as-fed) for Se (selenite) supplemented ewesa
Ingredient
% as-fed
Ground yellow com
53.75
Cottonseed hulls
22.00
Soybean meal (47.5% CP)
16.00
Alfalfa meal (14% CP)
3.00
Soybean oil
3.00
Trace mineral mixb
1.00
Ground limestone
1.25
Vitamins A & D
C
aSelenium levels in diet (as analyzed): 0.29, 3.77, 7.54, 11.01, 15.48, and 19.05 ppm
for treatments 0.2, 4, 8, 12, 16, and 20 ppm, respectively.
bTrace mineral mixture supplied between 96.5% and 98.5% NaCl, and provided per kg
of diet: 1.0 mg Co (as carbonate), 5.0 mg Cu (as oxide), 0.7 mg I (as iodate), 35 mg Fe
(as oxide), 25 mg Mn (as oxide), and 35 mg Zn (as oxide).
Provided per kg of diet: 5,000 IU of Vitamin A and 500 IU of Vitamin D3.
Table 4-2. Colostrum selenium concentrations (pg/L) of ewes receiving different levels
of selenium supplementation as sodium selenite3
Added Se, mg/kg
0.2
4
8
12
16
20
Year of experiment
r f
257b 624
1300bc 543
1889bcd 767
2072bcd 704
3216cd 767
3542d 537
705b 517
1452bd 421
3256c 480
2373cd 462
2925cd 741
aData represent least squares standard errors.
b,c,dMeans within columns lacking a common superscript differ (P < 0.05)
' = 4, 5, 3, 3, 3, and 6 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
2n = 4, 6, 5, 5, and 2 for Se levels 0.2, 4, 8, 12, and 20 mg/kg, respectively.


90
mineral mixture containing 26 mg Se/kg as sodium selenite (Southeastern Minerals, Inc.,
Bainbridge, GA), or 5) free-choice access to a mineral mixture containing 26 mg Se/kg
as Se yeast (Sel-Plex; Alltech, Inc, Nicholasville, KY). All cows grazed bahiagrass
(Paspalum notation) pastures and were supplemented with bahiagrass (Paspalum
notatum) hay, molasses-based liquid supplement ad libitum, and whole cottonseed and
pelleted citrus pulp at rates of 0.68 kg-cow'1-d"1 and 1.81 kg-cow'1-d'1, respectively, from
November, 2002 through March, 2003.
Treatment groups receiving no Se or injectable Se were housed together and had
access to a free-choice mineral mixture containing no Se (Table 6-1). Cows receiving Se
via free-choice mineral mixtures were housed in separate groups and had access to the
same mineral mixture with added Se as sodium selenite or Se yeast for treatments 4 and
5, respectively (Table 6-1). All free-choice mineral mixtures were offered in wooden
mineral feeders and protected from rain.
Blood samples for plasma analyses were collected via jugular venipuncture into
10-mL heparinized tubes (Vacutainer; Becton-Dickinson, Franklin Lakes, NJ) at the
initiation of the study (d 0) and at d 365. Calving occurred over a 24 d span between
December 31, 2002 and January 23, 2003 and whole blood samples were collected in the
same manner from all cows immediately after parturition and at 30, 90, and 205 d
postpartum. A colostrum or milk sample was also collected on those days into a 15-mL
plastic centrifuge tube. Forty-one calves, 24 male, 17 female, were bom alive and
unassisted. One cow in the free-choice selenite group had a stillbirth and one control cow
aborted very early in the experiment and both were removed from the study. Liver
biopsies were performed on all cows using the technique described by Chapman et al.


98
and selenite on milk Se concentrations. The overall depression in whole blood and milk
Se concentrations at 90 d postpartum supports the idea set forth by Pehrson et al. (1999)
and supported by the work of Valle et al. (2002), that the increase in consumption of lush,
growing forage increases oxidative stress on the animals. This stress is likely due to
increased unsaturated fatty acids, such as linolenic acid, and thus, Se content of milk and
whole blood was decreased due to the cows need for antioxidants. Ninety d postpartum
on our study was April 8th. In Florida and much of the southeastern U.S., cattle will
consume more fresh grass than hay around that date.
Sel-Plex supplemented cows had higher liver Se at the end of our study than did
cows receiving any other treatment. Sel-Plex produced liver Se concentrations up to
three-fold higher than Mu-Se. Such dramatic increases and differences in liver Se among
treatments after one yr reported by Valle et al. (2002) and are similar to ours, as Se yeast
treated cows had liver Se higher than cows from control or inorganic Se treatments.
Likewise, those authors reported Deposel treated cows as having intermediate in liver Se
concentrations. Deposel performed similarly in the present study, and liver Se
concentrations from controls and Mu-Se decreased in both experiments. All cows
receiving Mu-Se and 83% of controls had liver Se concentrations below the adequate
level (> 1200 pg/kg). Liver and plasma Se concentrations were highly correlated and
followed a similar pattern with respect to increases and decreases over the course of this
study. Plasma Se concentrations and differences among treatments were, again, similar to
those reported by Valle et al. (2002). Control cows, cows receiving Mu-Se, and cows
receiving free-choice selenite had lower plasma Se after one yr, whereas Deposel and Se-
Plex treated cows were able to maintain or increase plasma Se, respectively. At the


123
Cow whole blood selenium, ig/L
Figure 7-1. Correlation between the concentration of Se in cows whole blood and the
concentration of Se in the whole blood of their calves.
Cow whole blood selenium, fig/L
Figure 7-2. Correlation between the concentration of Se in cows whole blood and the
concentration of Se in the liver of their calves.


99
termination of the experiment, 100% of cows supplemented with selenite, free-choice or
injectable, and cows receiving no supplemental Se had plasma Se concentrations below
the critical level of > 70 pg/L.
Implications
Organic forms of selenium supplemented in free-choice mineral mixtures and the
selenate form of selenium as an injection were able to increase or maintain selenium
levels in plasma and liver of beef cows. Either of these forms of selenium
supplementation may be effective for open or gestating beef cows. However, grazing
beef cows receiving no selenium supplementation or selenium as injectable sodium
selenite had inadequate plasma and liver selenium. During times of stress such as calving
and lactation, organic selenium was superior to other forms of supplementation in
maintaining adequate blood selenium and milk selenium adequate for nursing calves.
Summary
In a 365 d study, the effects of chemical form and method of Se supplementation
on blood, milk, and tissue Se in grazing beef cows were evaluated and compared. Forty-
three Angus cows (115-130 d gestation) were randomly assigned to one of five groups
and received either no Se supplementation (control), one 5-mL sodium selenite injection
s.c. every four mo, one 9-mL barium selenate injection s.c. at the initiation of the study,
or free-choice mineral mixtures containing 26 mg/kg Se as sodium selenite or Se yeast.
Cows grazed bahiagrass (Paspalum notatum) pastures, received routine dietary
supplementation, and calved mostly in early January 2003. Body weight, plasma Se, and
liver Se were measured at d 0 and d 365. Whole blood and milk samples were taken at
calving and 30, 90, and 205 d postpartum. Cows receiving Se in free-choice minerals
were heavier and had a greater increase in BW at d 365 (P < 0.05) than cows receiving all


CHAPTER 6
EFFECTS OF FORM OF PARENTERAL OR DIETARY SELENIUM
SUPPLEMENTATION ON BODY WEIGHT AND BLOOD, LIVER, AND MILK
CONCENTRATIONS IN BEEF COWS
Introduction
Many areas of the United States have selenium deficient soils (McDowell, 2003)
and may produce forages and grains which are unable to provide adequate Se to
livestock. Selenium deficient brood cows may give birth to calves which are stillborn,
premature, weak, or afflicted with nutritional muscular degeneration (Maas, 1983; Corah
and Ives, 1991). Likewise, even with adequate blood Se at birth, calves suckling Se
deficient dams are susceptible to becoming Se deficient (Pehrson et al., 1999). Without
adequate dietary or parenteral Se supplementation, brood cows may suffer from
infertility, retained placentas, ovarian cysts, metritis, silent estrus periods, and/or poor
weight gains (Dargatz and Ross, 1996).
In cattle, it has been well established that Se crosses the placenta (Roller et al.,
1984; Van Saun et al., 1989), that dietary Se is transferred to milk (Conrad and Moxon,
1979), and that positive correlations exist between blood Se of cows and blood Se of their
calves (Kincaid and Hodgson, 1989; Enjalbert et al., 1999; Pehrson et al., 1999). The
chemical form of Se affects its metabolism and previous research has shown differences
in blood, milk, and liver Se concentrations due to form (organic vs inorganic) of
supplemental Se (Knowles et al., 1999; Gunter et al., 2003; Valle et al., 2002).
Selenium is often supplemented as sodium selenite and included in free-choice
livestock mineral mixtures. However, Se may be supplemented through subcutaneous
88


144
Yeh, J. Y., M. A. Beilstein, J. S. Andrews, and P. D. Whanger. 1995. Tissue distribution
and influence of selenium status on levels of selenoprotein W. FASEB J. 9: 392-
396.
Yeh, J. Y. Q. P. Gu, M. A. Beilstein, N. E. Forsberg, and P. D. Whanger. 1997.
Selenium influences tissue levels of selenoprotein W in sheep. J. Nutr. 127: 394-
402.
Zachara, B. A., U. Trafikowska, H. Labedzka, and J. Mikolajczak. 1993. Effect of
dietary Se intake on blood Se levels and glutathione peroxidase activities in
lambs. Small Rumin. Res. 9: 331-340.


38
Most of the tissues collected at slaughter were free from pathological changes.
The findings of lymphocytes in the portal triads were deemed to be a background finding
and insignificant. Likewise, the findings of lymphocytic foci in the heart tissue were
determined to be associated with sarcocystic parasites. Mineral precipitations were
observed in kidney tissue of some ewes and are incidental, background findings.
Contraction bands present in the diaphragm and psoas major muscle were a result of
stunning during humane slaughter. Adipose tissue was present in the heart and psoas
major muscle, which is an indication of adequate nutrition. Hepatic lipidosis was
diagnosed in four ewes. Two cases (one severe, one moderate) were diagnosed in ewes
receiving 16 mg/kg dietary Se. In the moderate case, there was also evidence of bile
retention. Neither of these ewes lambed in either yr. This would indicate that the hepatic
lipidosis could be treatment related rather than due to metabolic changes associated with
gestation, parturition, and lactation. One ewe receiving 12 mg/kg Se and one ewe
receiving 4 mg/kg were diagnosed with mild hepatic lipidosis, however, both ewes
lambed in both yr. Thus, the hepatic lipidosis was likely due to metabolic changes
associated with lamb production. No evidence of significant pathological changes was
observed in ewes receiving 20 mg/kg dietary Se, which was the highest Se level used in
this study. Cristaldi et al. (2004) found no abnormalities after microscopic evaluation of
heart, liver, kidney, diaphragm, and muscle from wethers consuming up 10 mg/kg Se for
one yr. Likewise, only one instance of abnormal pathology was observed in ewes
consuming less than 10 mg/kg Se on our study. Furthermore, our study was
approximately 40% longer in duration, utilized treatments of up to 100% more Se, and
introduced stresses of production, all of which should have helped to induce Se toxicosis


79
was increased. Organic Se was more effective (P < 0.001) at increasing Se
concentrations in edible tissue than was selenite Se. As daily intake of Se by humans
declines in some parts of the world, increasing the Se content of foods for human
consumption by manipulating source and level of Se supplementation to livestock is now
of interest to food scientists. Givens et al. (2004) suggested that the Se content of cows
milk could be increased through the use of Se yeast as the supplemental form of Se to
dairy cows. Our findings indicate that Se content of muscle and organ tissue can be
influenced by source and level of Se supplementation to food animals. In general, Se
concentrations of brain, heart, kidney, liver, and muscle were much higher than those
reported in studies with cattle (Maag and Glenn, 1967) and sheep (Glenn et al., 1964c).
Deaths due to Se toxicosis were induced in both species. In contrast, Se death due to Se
toxicosis was never produced during our study. It is important to note that in the two
previous studies that animals were fed Se at higher levels and for a shorter period of time.
Our findings further agree with Smith et al. (1937) who found that the effects of
continued dosing of Se were cumulative and that Se from organic sources was
accumulated in higher quantities in tissues than Se from inorganic sources.
Most of the heart, diaphragm, loin, liver, and kidney tissues subjected to
histopathological evaluation were free from pathological changes. The findings of
lymphocytes in the portal triads were deemed to be a background finding and
insignificant. Three instances of vacuolic degeneration associated with the cytoplasm of
hepatocytes suggesting fatty degeneration were noted. However, no pattern associating
abnormal pathology to either dietary Se level or source could be established. Therefore,
lesions could not be definitively linked to treatment and could have been metabolic in


70
Samples of brain, diaphragm, heart, hoof tip, kidney, liver, and psoas major
muscle were collected, and frozen (0C) until analyzed for Se. Sections (1 cm3) of liver,
heart, kidney, diaphragm, and psoas major muscle from all animals were placed in 10%
neutral-buffered formaldehyde for subsequent microscopic evaluation for evidence of Se
toxicosis.
For histopathological evaluation, the tissue samples fixed in buffered formalin
were embedded in paraffin and sectioned at 6 microns. All sections were stained with
hematoxylin and eosin, and examined under a light microscope (10X, 20X, and 40X).
Serum albumin, Aik Phos, ALT, AST, CK, GGT were evaluated on a Hitachi 911
analyzer with reagents from Sigma (Sigma Chemical Co., St. Louis, Mo.). These
procedures were established by the Veterinary Medical Teaching Hospital at the
University of Florida.
Samples of kidney, heart, and liver were evaluated for cell structure changes using
transmission electron microscopy. Tissues were transferred to Trumps fixative, pH 7.2,
for 2 h at room temperature (24 C). Samples were then rinsed in 0.1M sodium
cacodylate buffer at room temperature for 1 h. After three 15-min rinses with deionized
water, the tissues were placed in a 1% aqueous uranyl acetate solution for 45 min.
Samples were then dehydrated through a graded ethanol-acetone series at room
temperature. Tissue samples were then infiltrated with and embedded in Spurrs resin.
Silver sections (0.06 um) were cut using an RMC MT 6000XL ultramicrotone (RMC
Products; Boeckeler Instruments, Inc.,Tuscon, AZ) and mounted on formvar-coated
copper mesh grids. Sections were stained with 5% acidic uranyl acetate and Reynolds
lead citrate and examined in a Zeiss 100 microscope (Carl Zeiss, Inc, Thomwood, NY).


119
whole blood Se was determined at birth, 30, 90 and 205 d of age. Testicles were
collected at birth from males and a liver biopsy was performed on all calves at d 205 for
liver Se determination. At birth, calf blood Se was in the adequate range (>100 pg Se/L)
for all treatments. At d 30 and d 90, control, both injectable products, and free-choice
sodium selenite treatments produced calf blood Se in or near the adequate range, and
calves from Se yeast treated cows maintained blood Se well above the adequate range at
166 and 182 pg Se/L at d 30 and 90, respectively. At 205 d, whole blood Se
concentrations for control and both injectables were in the deficient range (< 50 pg Se/L),
calves from the free-choice sodium selenite treatment were marginally deficient (< 75 pg
Se/L) and the Se yeast group had blood Se well above adequate at 188 pg Se/L.
Selenium in calf testes ranged from 162 to 210 pg Se/kg DM and did not differ among
treatments. Liver Se taken at weaning ranged from 297 to 1321 pg Se/kg DM and calves
from the two free-choice treatments had Se concentrations higher (P < 0.05) than calves
from both injectable treatments and the controls. Positive correlations (r = 0.57 and 0.64;
P < 0.01) between calf liver Se and calf whole blood Se, to cow whole blood Se were
observed. Correlation between concentration of Se in calf whole blood and in cow milk
was also positive (r = 0.49; P < 0.002). Supplementation of Sel-Plex to cows produced
adequate calf blood Se throughout this experiment, whereas control and the other
treatments produced calf blood Se concentrations that were mostly in the marginal or
deficient range. Supplementation of Se as Se yeast to the cow herd is an effective means
of maintaining adequate blood Se in pre-weaned calves.


128
Wethers were weighed at 8-wk intervals, serum Se, whole blood Se, and wool Se were
measured every 12 wk, and samples of brain, diaphragm, heart, hoof, kidney, liver, loin
muscle, and serum samples for evaluation of albumin and enzyme activities were
collected at the termination of the experiment. Wether BW was affected by dietary Se
level (P < 0.05), source of dietary Se (P < 0.05), and time (P < 0.05), and avg BW
decreased linearly (P <0.10) as dietary Se level increased though most wethers gained
BW. Serum Se, whole blood Se, and wool Se concentrations were affected (P < 0.05) by
dietary level of Se and source of Se. Serum Se and whole blood Se ranged from 110 to
3922 |ig/L and 392 to 6259 pg/L, respectively, and increased in a quadratic fashion as
dietary Se level increased (P < 0.05) and wool Se ranged from 1.19 to 39.09 mg/kg and
responded linearly (P < 0.05) to increased dietary Se. Serum Se, whole blood Se, and
wool Se concentrations from wethers receiving organic Se were higher (P <0.01) than
those from wethers receiving inorganic Se. Selenium concentrations in brain, diaphragm,
heart, hoof, kidney, liver, and loin muscle were affected (P < 0.05) by dietary Se level,
with higher Se concentrations generally observed in tissues from wethers receiving
organic Se. Though Se concentrations in serum, blood, wool, and major organs at most
times exceeded concentrations previously reported in livestock suffering from Se
toxicosis, a pattern of clinical signs of Se toxicosis was not observed in this experiment.
Histopathological, microscopic evaluation of liver, kidney, diaphragm, heart, and loin
muscle did not reveal definitive evidence of Se toxicosis in wethers on any dietary Se
treatment, and albumin and enzyme activity levels were in their normal respective ranges.
An Angus cow-calf herd was utilized to evaluate and compare effects of using
different forms of dietary or parenteral Se on BW change and blood, milk, and liver Se


82
Summary
The objectives of this 60-wk experiment were to evaluate and compare effects of
feeding Se as sodium selenite or Se yeast at high dietary levels on serum, whole blood,
wool, and tissue Se concentrations in wether sheep and determine maximum tolerable
level of Se. Twenty-eight, 2-yr-old, Rambouillet-crossbred wethers (62.3 8.5 kg initial
BW) were utilized in a 2 4 factorial arrangement with 0.2, 20, 30, and 40 mg/kg dietary
Se (as-fed) from sodium selenite or Se yeast added to a corn-soybean meal basal diet.
Wethers were weighed at 8-wk intervals, serum Se, whole blood Se, and wool Se were
measured every 12 wk, and samples of brain, diaphragm, heart, hoof, kidney, liver, and
loin muscle and serum samples for evaluation of albumin and enzyme activities were
collected at the termination of the experiment. Wether BW was affected by dietary Se
level (P < 0.05), source of dietary Se {P < 0.05), and time (P < 0.05). Average BW
decreased linearly (P <0.10) as dietary Se level increased, though most wethers gained
BW. Serum Se, whole blood Se, and wool Se concentrations were affected (P < 0.05) by
dietary level of Se and source of Se. Serum Se and whole blood Se ranged from 110 to
3922 pg/L and 392 to 6259 pg/L, respectively, and increased in a quadratic fashion as
dietary Se level increased (P < 0.05) and wool Se ranged from 1.19 to 39.09 mg/kg and
responded linearly (P < 0.05) to increased dietary Se. Serum Se, whole blood Se, and
wool Se concentrations from wethers receiving organic Se were higher (P <0.01) than
those from wethers receiving inorganic Se. Selenium concentrations in brain, diaphragm,
heart, hoof, kidney, liver, and loin muscle were affected (P < 0.05) by dietary Se level,
with higher Se concentrations generally observed in tissues from wethers receiving
organic Se. Though Se concentrations in serum, blood, wool, and major organs at most
times exceeded concentrations previously reported in livestock suffering from Se


CHAPTER 1
INTRODUCTION
Selenium (Se) has had a long and storied history in animal nutrition. Since its
discovery, at the bottom of a vat of sulfuric acid, by Jons Jacob Berzelius, a Swedish
chemist, in 1817, Se has played the role of toxic element, essential nutrient, carcinogen,
and contributor in cancer prevention. However, it seems that seleniums greatest legacy
is one of a toxic agent to livestock. As early as 1295, Se was documented as detrimental
as Marco Polo described a poisonous plant which, when eaten by horses, caused their
hooves to drop off (Komroff, 1926). Likewise, a U.S. Army surgeon, stationed at Fort
Randall in 1856, described much the same conditions afflicting horses in the Nebraska
Territory (Madison, 1860). Selenium was identified as the principal toxic agent in
conditions described as blind staggers and alkali disease throughout Wyoming and
the Dakotas. In 1957, Se was shown to prevent liver necrosis in rats and afterward was
deemed an essential nutrient (Schwarz and Foltz, 1957). Though much of the world is
afflicted with Se deficiency, supplementation of Se using dietary or parenteral forms will
generally resolve the problem. Selenium toxicities require more effort but can be
successfully combated, by not overdosing livestock with supplemental Se, monitoring Se
content of feedstuffs, and by using certain animal management techniques.
With its many implications as a toxic element, the use of Se, as a supplement to
livestock, gamers much caution from feed manufacturers, animal scientists, and
nutritionists. The current estimate of the maximum tolerable level for dietary Se in
1


129
concentrations of beef cows. In a 365 d study, 43 gestating Angus cows were randomly
assigned to five groups and received either no Se supplementation (control), one 5-mL
sodium selenite injection s.c. every four mo, one 9-mL barium selenate injection s.c. at
the initiation of the study, or free-choice mineral mixtures containing 26 mg/kg Se as
sodium selenite or Se yeast. Body weight, plasma Se, and liver Se were measured at d 0
and d 365. Whole blood and milk samples were taken at calving and 30, 90, and 205 d
postpartum. Cows receiving Se in free-choice minerals were heavier and had a greater
increase in BW at d 365 (P < 0.05) than cows receiving all other treatments. Plasma Se
and liver Se concentrations were not initially different. At d 365, plasma Se in cows
receiving Se yeast was higher (P < 0.05) at 90 pg/L than from all other treatments.
Injectable selenite was intermediate and produced higher plasma Se than control and both
forms of selenite. Liver Se at d 365 was adequate (> 1200 pg/kg) and higher (P < 0.05)
in Se yeast treated cows than all others. Cows receiving injectable selenate also had
adequate liver Se concentrations that were higher (P < 0.05) than the inadequate levels
from control, free-choice selenite and injectable selenite. Whole blood Se was adequate
(> 100 pg/L) for all treatment groups at calving, 30 and 90 d postpartum. At 205 d
postpartum, cows receiving injectable selenate and both free-choice treatments were
adequate in whole blood Se, while controls and cows receiving injectable selenite had
inadequate whole blood Se. Cows receiving Se yeast had higher {P < 0.05) colostrum Se
than other treatments. No differences were observed in milk Se at 30 and 90 d
postpartum among treatment groups, however, both free-choice and the injectable
selenate treated cows had milk Se numerically higher than controls and cows receiving


110
Effects of treatment on birth weight, weaning weight, ADG, liver Se, and testes
Se were analyzed using PROC GLM (SAS for Windows 8e; SAS Inst., Inc., Cary, NC) in
a completely randomized design. Pre-planned contrast statements were used to compare
means as described by Littell et al. (1998; 2000). PROC MIXED (SAS for Windows 8e;
SAS Inst., Inc., Cary, NC) was used to analyze effects of treatment, d, and the interaction
of treatment x d on calf whole blood as repeated measures with a spatial power
covariance structure with respect to d and subplot of animal nested within treatment. Pre
planned contrast statements were written to determine differences in means for different
sampling d. PROC CORR was used to determine correlations of calf whole blood Se and
calf liver Se to cow whole blood and milk Se.
Results
Selenium concentration of pasture and hay for all groups averaged 0.071 and
0.045 mg/kg DM basis, respectively. Crude protein was 12.57% for hay and 12.79%
1.76 for standing forage. Forage CP did not differ (P = 0.92) during the study. Whole
cottonseed, pelleted citrus pulp, and liquid supplement analyzed 0.002, 0.002, and 0.744
mg Se/kg, (as-fed) respectively. Free-choice mineral consumption was monitored and
recorded. Mineral consumptions and amount of Se via injection are summarized in Table
7-2. All free-choice mineral mixtures had been formulated for an expected intake of 85-
113 g-cow'l-d'1. However, only the cows on the Se yeast treatment had mineral
consumption in this range (Table 7-2). Treatment groups receiving free-choice minerals
with no Se consumed 62.19 g-cow''-d'' and cows receiving free-choice minerals with
sodium selenite consumed 41.5 g-cowI-dl.
There was no effect of form Se supplementation to cows on calf birth weight (P =
0.83) or weaning weight (P =0.75) at 205 d (Table 7-3). Conversely, calf ADG was


141
Pirelli, G. J., S. Weedman-Gunkel, and D. W. Weber. 2000. Beef production for small
farms. Tech. Bull. No. EC 1514. Oregon State Univ., Corvallis, OR.
Podoll, K. L., J. B. Bernard, D. E. Ullrey, S. R. DeBar, P. K. Ku, and W. T. Magee.
1992. Dietary selenate versus selenite for cattle, sheep and horses. J. Anim. Sci.
70: 1965-970.
Rosenfeld, I., and O. A. Beath. 1945. The elimination and distribution of selenium in the
tissues in experimental selenium poisoning. J. Nutr. 30: 445-449.
Rosenfeld, I., and O. A. Beath. 1946. Pathology of selenium poisoning. Wyoming
Agricultural Experiment Station Bulletin no. 275. University of Wyoming,
Laramie, WY.
Rosenfeld, I., and O. A. Beath. 1947. Congenital malformations of eyes of sheep. J.
Agrie. Res. 75: 93-103.
Rosenfeld, L, and O.A. Beath. 1964. Selenium: Geobotany, Biochemistry, Toxicity and
Nutrition. Academic Press, New York.
Rotruck, J. T., A. L. Pope, H. E. Ganther, A. B. Swanson, D. G. Hafeman and W. G.
Hoekstra. 1973. Selenium: biochemical role as a component of glutathione
peroxidase. Science. 179: 588-590.
Schmidt, A. M. 1974. Selenium in animal feed. Fed. Regis. 39:1355.
Schoening, H. W. 1936. Production of so called alkali disease in hogs by feeding com
grown in affected areas. North Am. Veterinarian. 17: 22-28.
Schwarz, K., and C. M. Foltz. 1957. Selenium as an integral part of factor 3 against
dietary necrotic liver degeneration. J. Am. Chem. Soc. 79: 3292-3293.
Segerson, E. C., and B. H. Johnson. 1981. Selenium and reproductive function in
yearling Angus bulls. J. Anim. Sci. 51: 395-401.
Serdaru, M., L. Vladescu, and I. Tolea. 2004. Fluorimetric study of the selenium course
in the dam-calf relationship. Biol. Trace Elem. Res. 99: 113-122.
Shortridge, E. H., P. J. O'Hara, and P. M. Marshall. 1971. Acute selenium poisoning in
cattle. N. Z. Vet. J. 19: 47-50.
Smith, K. L., J. H. Harrison, D. D. Hancock, D. A. Todhunter, and H. R. Conrad. 1985.
Effect of vitamin E and selenium supplementation on incidence of clinical
mastitis and duration of clinical symptoms. J. Dairy Sci. 67: 1293-1300.


117
both injectable products. These data agree with Abdelrahman and Kincaid (1995) where
liver Se at d 0 and d 42 was more than two-fold higher for Se supplemented than for
unsupplemented Holstein calves. These authors continue by stating that their data help
emphasize the need for Se supplementation to newborn calves in Se deficient areas. Our
findings help support such a need. Liver Se of 2200 pg/kg in calves is required for
normal growth and health (Van Saun et al., 1989). Though liver samples were not taken
at birth, Se concentrations in liver at weaning failed to even approach this threshold and
in control animals liver Se concentrations were less than 20% of this recommended
concentration. Also, only calves from dams that received Se yeast had liver Se
concentrations at weaning that would be considered to be in the normal range (Stowe and
Herdt, 1992). Valle (2001) reported liver Se that averaged 730 pg/kg in yearling cattle
that had been supplemented with sodium selenite and 930 pg/kg when Se was
supplemented via Se yeast. Our current findings concur with these concentrations as well
as concentrations in dairy cows reported by Knowles et al. (1999). Regardless of liver or
whole blood Se status, no signs of WMD or buckling were observed in the calves on this
study, even during times of stress (e.g., liver biopsy or weaning).
The correlation between cow whole blood and calf whole blood in our study was
positive (r = 0.64) and suggests that Se supplementation of the cow herd is an effective
means of supplementing Se to nursing calves. The correlation between calf liver Se at
weaning and cow whole blood Se concentration (r = 0.57) further supports this
suggestion. Pehrson et al. (1999) reported positive correlations of r = 0 .64, 0.68, and
0.59 between calf blood Se, calf plasma Se, and glutathione peroxidase activity in calf
blood to cow milk Se concentration. These correlation values are somewhat higher than


95
Se than did control or Mu-Se, and Deposel produced higher (P < 0.001) plasma Se than
did control, free-choice selenite, or Mu-Se. After one yr, only cows receiving Sel-Plex
had increased (P < 0.001) plasma Se. Deposel tended to increase (P = 0.13) plasma Se,
while plasma Se decreased (P < 0.001) in control and Mu-Se treated cattle, and plasma
Se concentrations in cows receiving Se as free-choice selenite were unchanged (P =
0.69).
Liver from biopsies at d 0 and d 365 was evaluated for Se concentration (Table 6-
7). As with plasma Se, treatment and treatment x d had significant effects (P < 0.001).
Liver Se (946 to 1136 jig/kg) did not differ among treatment groups at d 0 (P > 0.31).
However, at d 365, liver Se from Sel-Plex treated animals was higher (P < 0.02) than
from animals on all other treatments. Liver Se concentrations from cows receiving Se
from Deposel or free-choice selenite were similar, (P = 0.21) and both were higher (P <
0.05) than those of controls and cows receiving Mu-Se. At the end of this study, liver Se
had increased (P < 0.001) in cows receiving Sel-Plex. Cows receiving Mu-Se had
decreased (P <0.01) liver Se and liver Se tended to decrease (P = 0.07) in controls. Liver
Se remained unchanged (P = 0.48; 0.73) in cows receiving Deposel and free-choice
mineral with sodium selenite, respectively. Liver and plasma Se concentrations were
highly correlated (r = 0.71; P < 0.001).
Discussion
No differences in cow BW were observed among the control, Mu-Se, and Deposel
treatment groups. However, BW of cows receiving Se supplementation from Se yeast or
sodium selenite in free-choice minerals was higher than controls and cows receiving
injectable Se. Gunter et al. (2003) compared effects of Se yeast and sodium selenite on
cow performance and found no differences in BW between cows that received Se yeast or


2
domestic animals is 2 mg/kg (National Research Council [NRC], 1980). This estimate
does not consider differences inmetabolism of Se by different species and makes no
differentiation in the maximum tolerable level for the different chemical forms of Se,
such as Se yeast or sodium selenite. Previous research has shown that the absorption of
an oral dose of inorganic Se differs between ruminant and monogastric species (Wright
and Bell, 1966). Likewise, studies in cattle and swine have shown a marked difference in
the efficacy of organic vs inorganic Se to increase blood, milk, and tissue Se
concentrations (Pehrson et al., 1999; Kim and Mahan, 2001; Gunter et ah, 2003).
Furthermore, some evidence exists to suggest that the maximum tolerable level of Se for
livestock is grossly underestimated and to discredit the notion that the range between
optimal and toxic levels of Se is narrow (Glenn et al., 1964a; Kim and Mahan, 2001;
Cristaldi et al., in press).
To further the body of knowledge in this subject area, a series of experiments were
carried out with sheep and cattle. Experiments using sheep were conducted to gather
further data on 1) the amount of dietary inorganic Se that can be tolerated by ewes during
lamb production, 2) the effects of Se supplementation to ewes on their lambs, and 3) the
amount of organic or inorganic Se that can be tolerated by mature wethers. A cow-calf
herd was utilized to evaluate and compare effects of using different forms of dietary or
injectable Se on body weight change and blood, milk, and liver Se concentrations of beef
cows and their calves.


71
Serum, whole blood, wool, tissue, and feed samples were analyzed for Se
concentration using a fluorometric method described by Whetter and Ullrey (1978). To
help ensure reliability of the analytical method, a certified standard (National Bureau of
Standards Bovine Liver SRM-1577a; U.S. Department of Commerce, National Institute
of Standards and Technology, Gaithersburg, MD) was frequently analyzed.
Brain, diaphragm, heart, hoof tip, kidney, liver, and psoas major muscle Se data
were analyzed for effects of treatment using PROC GLM in SAS (SAS for Windows 8e;
SAS Inst., Inc., Cary, NC) in a 2 x 4 factorial arrangement. Pre-planned orthogonal
contrast statements were used to compare means as described by Littell et al. (1998;
2000). PROC MIXED of SAS was used to analyze effects of treatment, time, and the
interaction of treatment x time on BW, serum Se, whole blood Se, and wool Se as
repeated measures with a spatial power covariance structure with respect to d and a
subplot of animal nested within treatment. Pre-planned orthogonal contrast statements
were written to determine differences in means at different sampling intervals.
Regression analysis was used to determine relationships between dietary Se and Se
concentration in serum, whole blood, wool, and tissues.
Results and Discussion
Wether BW was affected by dietary Se level (P < 0.05), source of dietary Se (P <
0.05), time (P < 0.05), and average BW decreased linearly (P < 0.10) as dietary Se level
increased (Table 5-2). Body weights of wethers receiving 30 or 40 mg/kg dietary Se as
Se yeast decreased from wk 0 to wk 60, whereas wethers receiving all other dietary Se
treatments gained weight from wk 0 to wk 60. Previous studies have reported no effect
of Se fed above requirements on BW of feedlot cattle (Perry et al., 1975) and no effects
on BW when included up to 10 mg/kg in the diets of wether sheep (Cristaldi et al., in


5 COMPARATIVE EFFECTS AND TOLERANCE OF VARIOUS DIETARY
LEVELS OF SE AS SODIUM SELENITE OR SE YEAST ON BLOOD, WOOL,
AND TISSUE SE CONCENTRATIONS OF WETHER SHEEP 67
Introduction 67
Materials and Methods 68
Results and Discussion 71
Implications 81
Summary 82
6 EFFECTS OF FORM OF PARENTERAL OR DIETARY SELENIUM
SUPPLEMENTATION ON BODY WEIGHT AND BLOOD, LIVER, AND MILK
CONCENTRATIONS IN BEEF COWS 88
Introduction 88
Materials and Methods 89
Results 92
Discussion 95
Implications 99
Summary 99
7 TISSUE AND BLOOD SELENIUM CONCENTRATIONS AND PERFORMANCE
OF BEEF CALVES FROM DAMS RECEIVING DIFFERENT FORMS OF
SELENIUM SUPPLEMENTATION 106
Introduction 106
Materials and Methods 107
Results 110
Discussion 113
Implications 118
Summary 118
8 SUMMARY AND CONCLUSIONS 125
LITERATURE CITED 133
BIOGRAPHICAL SKETCH 145
vii


45
Figure 3-1. Effect of dietary inorganic Se level on Se concentrations in brain, diaphragm,
and heart of ewes; SE = 0.6 to 0.9, 0.3 to 0.4, and 0.4 to 0.6 for brain, diaphragm, and
heart, respectively.


58
increase in milk Se was observed as dietary Se increased from control to 8 mg/kg on each
sampling d. Few increases of that magnitude were observed between groups receiving Se
at high concentrations. This indicates that the proportion of Se transferred to milk
decreases as dietary Se concentration increases. Waite et al. (1975) suggest that Se is
subject to a bioreducing process as it is transferred from plasma to milk. These authors
report that only 1.5% of dietary selenite Se appeared in milk. Givens et al. (2004)
observed increases of more than four-fold in cows milk Se when dietary Se was doubled
and tripled using Se yeast. However, in our study, increases of such magnitude were not
observed when dietary Se was doubled and tripled using sodium selenite. These
observations would indicate that, though milk Se can be increased using an inorganic Se
source, a greater proportion of organic Se is transferred to milk. This concept is supported
by several studies using cattle (Knowles et al., 1999; Ortman and Pehrson, 1999; Pehrson
et al., 1999).
One objective of this study was to quantitate the effects on lambs that were
suckling ewes that received dietary Se above the maximum tolerable level of 2 mg/kg
(NRC, 1980). No lambs were bom with congenital deformities or abnormalities, nor did
any lamb display signs of selenium toxicosis (e.g., wool loss, hoof malformation,
anorexia) from birth to weaning. Selenium included in ewe diets has previously been
shown to be transmitted to the lamb via the placenta and milk (Jacobsson et al., 1965). In
our study, plasma Se in lambs increased as Se concentration in their dams diet increased
and was positively correlated to milk Se. Lambs from ewes receiving the control diet had
plasma Se at birth that averaged 81 pg/L in year one and 85 pg/L in year two. These
values are only slightly above the nomial range (50-80 pg/L) for neonate lambs (Aitken,


137
Hidiroglou, M. D., P. Heanley, and K. J. Jenkins. 1968. Metabolism of inorganic
selenium in rumen bacteria. Can. J. Physiol. Pharm. 46: 229-232.
Hidiroglou, M., and K. J. Jenkins. 1975. Effects of selenium and vitamin E, and copper
administrations on weights gains of beef cattle raised in a selenium-deficient area.
Can. J. Anim. Sci. 55: 307-313.
Hogue, D. E. 1958. Vitamin E, selenium and other factors related to nutritional
muscular dystrophy in lambs, pp 32-39. In Proc. Cornell Nutr. Conf. Feed
Manuf., Ithaca, NY.
Jacobsson, S. O., H. E. Oksanen, and E. Hansson. 1965. Excretion of selenium in the
milk of sheep. Acta Vet. Scand. 6: 299-312.
Judson, G. H., N. J. Ellis, B. R. Kempe, and M. Shallow. 1991. Long acting selenium
treatments for sheep. Aust. Vet. J. 68: 263-265.
Julien, W. E., H. R. Conrad, and A. L. Moxon. 1976. Selenium and vitamin E and
incidence of retained placenta in parturient dairy cows II. Prevention in
commercial herds with prepartum treatment. J. Dairy Sci. 59:1960-1962.
Julien, W. E., and F. A. Murray. 1977. Effect of selenium and selenium with vitamin E
in vitro motility of bovine spermatozoa. J. Dairy Sci. 60: 174 (Abstr.).
Kaur P., and M. P. Bansal. 2004. Effect of experimental oxidative stress on
steroidogenesis and DNA damage in mouse testis. J Biomed Sci. 11: 391-397.
Kim, Y.Y., and D.C. Mahan. 2001. Comparative effects of high dietary levels of organic
and inorganic Se on Se toxicity of growing finishing pigs. J. Anim. Sci. 79: 942-
948.
Kincaid, R. L., and A. S. Hodgson. 1989. Relationship of selenium concentrations in
blood of calves to blood selenium of the dam and supplemental selenium. J. Dairy
Sci. 72: 259-266.
Knowles, S. O., N. D. Grace, K. Wurms, and J. Lee. 1999. Significance of amount and
form of dietary selenium in blood, milk and casein concentrations in grazing
cows. J. Dairy Sci. 82: 429-437.
Koenig, K. M., L. M. Rode, R. D. H. Cohen, and W. T. Buckley. 1997. Effects of diet
and chemical form of selenium on selenium metabolism in sheep. J. Anim. Sci.
75: 817-827.
Koller, L. D., G. W. Whitbeck, and P. J. South. 1984. Transplacental transfer and
colostral concentrations of selenium in beef cattle. Am. J. Vet. Res. 45: 2507-
2510.


31
selenite via i.m. injection, at 0.7 mg/kg BW. Other researchers (Rosenfeld and Beath,
1946) reported death in sheep with less Se (30 mg/d); however, the Se maximum intake
level used in our study was approximately 25% less. It is important to note that we used
sodium selenite as our Se source whereas previous research (Rosenfeld and Beath, 1946;
Caravaggi et al., 1970) used sodium selenate as the source of additional Se. Henry et al.
(1988) reported a higher relative bioavailability for selenate than selenite. This suggests
the possibility of a higher tolerance for sodium selenite vs selenate.
Whole blood Se was measured at wk 12, 24, 36, 48, 60, and 72 (Table 3-4).
Dietary Se level, time, and dietary Se level x time affected (P < 0.05) ewe whole blood
Se. Whole blood Se increased linearly (P < 0.001) as dietary Se increased. Response of
whole blood Se from all treatments over time was cubic (P <0.01) which agrees with the
time response of serum Se. Maas et al. (1992) reported a strong correlation (0.88) for
whole blood Se and serum Se. Our data support this relationship, as serum Se and whole
blood Se responded to dietary Se level in a similar fashion. The cubic response of whole
blood Se over time may be attributed to ewes having no fetal tissue and producing no
milk to use as a route of excretion during the dry, rebreeding period, which encompassed
the midpoint of this study. Each dietary Se level was evaluated individually over time
and control and 8 mg/kg neither increased nor decreased with time (P > 0.20). Whole
blood Se from ewes receiving 4 mg/kg Se responded cubically (P = 0.019) and 16 mg/kg
dietary Se tended (P = 0.07) tended to respond cubically. Whole blood Se concentration
changed more sporadically over time in ewes receiving 12 or 20 mg/kg dietary Se and
each treatment produced a fifth degree polynomial (P < 0.05). At wk 12, ewes in all
treatment groups had higher (P < 0.05) whole blood Se than did controls. Ewes receiving


46
Figure 3-2. Effect of dietary inorganic Se level on Se concentrations in kidney, hoof, and
loin (psoas major muscle) of ewes; SE = 3.0 to 3.3, 0.8 to 1.1, and 0.3 to 0.5 for kidney,
hoof, and loin, respectively.


77
concentrations in the present study were more than ten-fold higher than concentrations of
2 to 2.5 mg/kg in wool from wethers fed up to 10 mg/kg dietary Se as selenite (Cristaldi
et al., in press), but never exceeded 40 mg/kg which is less than 45 mg/kg which was
described as the Se concentration in hair of animals suffering from alkali disease
(National Academy of Sciences [NAS], 1971).
Selenium concentrations in brain, diaphragm, heart, kidney, and loin muscle were
affected (P < 0.05) by dietary Se level, Se source, and dietary Se x Se source interaction.
Hoof Se concentration was affected by source (P < 0.05) and liver Se was affected (P <
0.05) by dietary Se level and dietary Se x Se source interaction, and tended to be affected
(P = 0.11) by Se source. Selenium concentrations, on a DM basis, were highest in liver
followed by kidney, heart, hoof, brain, loin, and diaphragm (Table 5-6). This pattern is
similar to a ranking of Se concentrations in tissues of farm animals by (Combs and
Combs, 1986) with the exception of liver and kidney being reversed. However, in
animals fed Se at or below requirements, kidney generally has a higher concentration of
Se than does the liver, but when dietary Se is increased, liver Se quickly becomes higher
in Se than kidney. This supported by the work of Ewan et al. (1968) and the findings of
Cristaldi et al. (2004) in sheep, and McDowell et al. (1977) in swine. Those authors
reported higher liver Se than kidney Se when dietary Se was increased. Unlike minerals
such as Zn and Mn, the status of Se is reflected in many tissues (McDowell, 2003). Brain
Se concentrations ranged from 1.28 to 32.3 mg/kg and brain Se concentrations from
wethers receiving organic Se were higher (P < 0.001) than brain Se from wethers
receiving selenite Se. These results suggest that Se does cross the blood-brain barrier and
that brain Se is influenced by dietary Se. Previous research using sheep supports our


5
only 0.02 mg/kg Se gave birth to lambs with WMD, but by supplementing 0.10 mg/kg Se
in the ewe diet, WMD was prevented consistently. It is suggested that at least some of
the variation in the Se requirements necessary to prevent WMD is due to sparing or
interfering nutrients, such as Vitamin E or sulfur, and that differences also reflect Se
losses due to drying, as well as analytic error (McDowell, 2003). Maas et al. (1984)
suggested that even in cases of Se deficiency, lambs returned to a normal Se status with
one or two i.m. injections containing 1 mg Se and 68 IU of vitamin E. Selenium
supplementation to ewes at a level of 2.25 mg/d reduced both the incidence and severity
of WMD in white-faced lambs (Gardner and Hogue, 1967).
Selenium supplementation has been reported to have some effects on growth and
rate of gain in sheep and cattle. Spears et al. (1986) reported increased summer gains in
calves that received Se and vitamin E supplementation vs those calves receiving no Se
supplementation. Likewise, Perry et al. (1976) reported a 10% increase in ADG of steers
when feedlot diets were supplemented with 0.1 mg/kg Se. Furthermore, an 8% increase
in ADG of finishing beef cattle was again reported when 0.1 mg/kg Se was added to the
diet (Burroughs et al., 1963). Increases in BW gains of 20% were attained when Friesian
heifer calves were supplemented with Se at a rate of 3 mg/d (Wichtel et al., 1996). In
lambs, data from Oldfield et al. (1963) indicated that lambs with the lowest blood Se had
the lowest BW at six wk of age. However, reports of a positive response in growth or
BW gain are inconsistent in sheep and cattle. Ammerman et al. (1980) reported no
differences in weaning weights of calves nursing Se supplemented mothers vs calves
whose dams had received no supplemental Se. Hereford x Angus calves showed no
difference in ADG from birth to weaning due to supplemental Se (Castellan et al., 1999).


136
Glenn, M. W., R. Jensen, and L. A. Griner. 1964b. Sodium selenate toxicosis: Pathology
and pathogenesis of sodium selenate toxicosis in sheep. Am. J. Vet. Res. 25:
1486-1494.
Glenn, M. W., J. L. Martin, and L.M. Cummins. 1964c. Sodium selenate toxicosis: The
distribution of selenium within the body after prolonged feeding of toxic
quantities of sodium selenate to sheep. Am. J. Vet. Res. 25: 1495-1499.
Goehring, T. B., I. S. Palmer, O. E. Olson, G. W. Libal, and R. C. Wahlstrom. 1984a.
Effect of seleniferous grains and inorganic Se on tissue and blood composition
and growth performance of rats and swine. J. Anim. Sci. 59: 725-732.
Goehring, T. B., I. S. Palmer, O. E. Olson, G. W. Libal, and R. C. Wahlstrom. 1984b.
Toxic effects of selenium on growing swine fed corn-soybean meal diets. J.
Anim. Sci. 59: 733-737.
Goursand, A., and R. Nowak. 1999. Colostrum mediates the development of mother
preference by newborn lambs. Physiol. Behav. 67: 49-56.
Gunter, S. A., P. A. Beck, and J. M. Phillips. 2003. Effects of supplementary selenium
source on the performance and blood measurements in beef cows and their calves.
J. Anim. Sci. 81: 856-864.
Hansen, D., R. Hathaway, and J. E. Oldfield. 1991. White muscle and other selenium-
responsive disease of livestock. Tech. Bull. No. PNW 157. Oregon State Univ.,
Corvallis, OR.
Harrison, J. H., and H. R. Conrad. 1984. Effect of selenium intake on selenium
utilization by the nonlactating dairy cow. J. Dairy Sci. 67: 219-223.
Hartley, W. J., and A. B. Grant. 1961. A review of selenium responsive diseases of New
Zealand livestock. Fed. Proc. 20: 679-688.
Heimenn, E. D., T. J. Gall, M. F. Smith, R. E. Morrow, and R. G. Elmore. 1981.
Selenium levels in tissues, semen, and blood plasma in yearling bulls. J. Anim.
Sci. 52: 328 (Abstr.).
Hemken, R. W., D. Olds, R. L. Botts, and L. S. Bull. 1978. Selenium injection prior to
calving on prevention of retained placenta. J. Dairy Sci. 61: 209-210 (Abstr.).
Henry, P. R., M. G. Echevarria, C. B. Ammerman, and P. V. Rao. 1988. Estimation of
the relative biological availability of inorganic selenium sources for ruminants
using tissue uptake of selenium. J. Anim. Sci. 66: 2306-2312.
Hidiroglou, M. 1982. Selenium in the ruminant genital system and mammary glands: A
review. Ann. Rech. Vet. 13:133-141.


13
Se absorption as sheep on a high concentrate diet had higher plasma Se than sheep
receiving a high forage diet. (Koenig et ah, 1997).
In contrast to many other minerals consumed by livestock, which use homeostasis
as a primary status regulator, Se status of animals seems to have little effect on intestinal
absorption. In a study utilizing rats, urinary excretion was shown as the only relevant
means of Se homeostasis (Windisch and Kirchgessner, 2000) as urinary excretion is
directly related to dietary level while fecal Se excretion is quite static (Burk et ah, 1972).
When Se absorption was regressed on Se intake of dairy cows, a strongly linear
relationship was observed (Harrison and Conrad, 1984). However, Se intakes reported in
that study were relatively low and ranged from 0.437 to 3.136 mg/d. Most dairy cows
consume closer to 6 mg Se/d, based on supplementation in the diet of 0.3 mg/kg Se.
Absorbed Se is associated with plasma protein and transported in the blood plasma
until it enters tissues (McDowell, 2003). Selenoprotein P is the plasma protein with
which most Se is associated in individuals with adequate or deficient dietary Se, while
most plasma Se is associated with albumin when Se intake is in excess (Xia et ah, 2000).
In addition to plasma, Se is also found in muscle and glandular tissues.
Generally, when ranked on a Se concentration basis, tissues follow the general order of
kidney > liver > heart > skeletal muscle, regardless of species, when Se is fed at an
adequate or deficient level (Comb and Combs, 1986). The kidney may be the highest in
Se concentration as it is primary organ of excretion. However, when Se is fed at levels
above requirement, liver surpasses kidney in terms of Se concentration (Cristaldi et ah, in
press).


LITERATURE CITED
Abdelrahman, M. M., and R. L. Kincaid. 1995. Effect of selenium supplementation of
cows on maternal transfer of selenium to fetal and newborn calves. J. Dairy Sci.
78: 625-630.
Abdennebi, E. H., N. el Ouazzani, and D. Lamnaouer. 1998. Clinical and analytical
studies of sheep dosed with various preparations of Astragalus lusitanicus. Vet.
Hum. Toxicol. 40: 327-331.
Aitken, P. 2001. Se toxicity. In Practice. 23:286-289.
Ammerman, C. B., H. L. Chapman, G. W. Bowman, J. P. Fontenot, C. P. Bagley, and L.
X. Moxon. 1980. Effect of supplemental selenium for beef cows on the
performance and tissue selenium concentrations of cows and suckling calves. J.
Anim. Sci. 51: 1381-1386.
Ammerman C. B., and S. M. Miller. 1975. Selenium in ruminant nutrition: A review. J.
Dairy Sci. 58: 1561-1577.
Awadeh, F .T., M. M. Abdelrahman, R. L. Kincaid, and J. W. Finley. 1998a. Effect of
selenium supplements on the distribution of Se among serum proteins in cattle. J.
Dairy. Sci. 81: 1089-1094.
Awadeh, F. T., R. L. Kincaid., and K. A. Johnson. 1998b. Effect of level and source of
dietary selenium on concentration of thyroid hormones and immunoglobulins in
beef cows and calves. J. Anim. Sci. 76: 1204-1215.
Blodgett, D. J., and R. F. Bevill. 1987. Acute selenium toxicosis in sheep. Vet. Hum.
Toxicol. 29:233-236.
Bostedt, H., and P. Schramel. 1990. The importance of selenium in the prenatal and
postnatal development of calves and lambs. Biol. Trace Elem. Res. 24: 163-171.
Bruce, L. B. 1997. Effects of selenium on cold adapted beef cattle. Asian-Aust. J.
Anim. Sci. 11:265-267.
Burk, R. F., D. G. Brown, R. J. Seely, and C. C. Scaief III. 1972. Influence of dietary and
injected selenium on whole body retention, route of excretion, and tissue retention
of75 Se03 in the rat. J. Nutr. 102: 1049-1055.
133


78
findings of increased Se in brain as dietary Se is increased (Yeh et al., 1995; 1997;
Ouazzani et al., 1999). Diaphragm Se ranged from 0.82 to 26.34 mg/kg and tended to
increase linearly (P = 0.089) as dietary Se increased. Diaphragm Se was higher (P <
0.001) in wethers receiving organic Se than from wethers receiving selenite Se. Heart Se
ranged from 1.59 to 33.93 mg/kg and, like brain and diaphragm Se was higher (P <
0.001) in wethers receiving organic Se than from wethers receiving selenite Se.
Selenium concentrations in the hoof tip ranged from 3.44 to 29.20 mg/kg and increased
linearly as dietary Se increased (P < 0.05). Selenium concentrations of hoof tip taken
from wethers receiving organic Se tended (P = 0.07) to be higher than from wethers
receiving inorganic Se. Both Se sources produced hoof Se concentrations higher than 10
mg/kg which was previously reported in animals with alkali disease (NAS, 1971).
Kidney Se tended (P = 0.07) to respond linearly to increased dietary Se and ranged from
8.43 to 77.61 mg/kg. Kidney Se concentrations from wethers receiving organic Se were
higher (P <0.01) than from wethers receiving selenite Se. Kidney Se concentrations
from the present study are much higher than those reported by Maag and Glenn (1967)
where death due to Se toxicosis was produced in 245 kg Hereford steers. However, the
calves used by those authors received approximately 270 mg Se steer''-d'1 as sodium
selenite and death was induced within 6 wk. Liver Se concentrations ranged from 2.66
to 132.73 mg/kg and increased linearly (P < 0.001) as dietary Se level increased.
Selenium concentrations in liver from wethers receiving organic Se were not different (P
= 0.34) than liver Se concentrations from wethers receiving selenite Se. Selenium
concentrations in the loin muscle (psoas major), which is often consumed by mankind
ranged from 0.71 to 26.87 mg/kg and tended (P = 0.12) to increase linearly as dietary Se


36
Ewes receiving 16 and 20 mg/kg Se had higher hoof Se (P < 0.05) than controls.
Likewise, ewes receiving 20 mg/kg Se had higher hoof Se (P < 0.05) than ewes receiving
4, 8, and 12 mg/kg dietary Se.
Selenium concentrations in psoas major muscle (i.e. tenderloin), a muscle
commonly consumed by humans, ranged from 0.60 to 3.66 mg/kg DM and increased
linearly as dietary Se increased (Figure 3-2). Regressing psoas major muscle Se (mg/kg
DM) on dietary Se concentration (mg/kg) produced the following relationship:
Psoas major muscle Se = 0.59 + 1.42 Dietary Se (r2 = 0.62; P < 0.05).
Selenium concentrations in psoas major muscle from controls were lower (P < 0.05) than
from ewes receiving 4, 12, 16, and 20 mg/kg Se and tended to be lower (P = 0.06) than
psoas major muscle Se concentrations from ewes receiving 8 mg/kg Se. Ewes receiving
20 mg/kg Se had higher (P < 0.05) psoas major muscle Se than ewes receiving all other
Se levels.
Kidney Se ranged from 5.18 to 31.61 mg/kg DM and responded to increased
dietary Se in a cubic fashion (Figure 3-2). Regressing kidney Se (mg/kg DM) on dietary
Se concentration (mg/kg) produced the following relationship:
Kidney Se = 5.18 + 6.64 Dietary Se 2.28 Dietary Se2 + 0.32 Dietary Se3 (r2 =
0.62; P < 0.05).
Ewes receiving 20 mg/kg Se had higher (P <0.01) kidney Se than ewes from all other
treatment groups. Ewes receiving 12mg/kg Se tended (P = 0.09) to have higher (P <
0.01) kidney Se than controls.


134
Burroughs, W., R. Kohlmeier, R. Barringer, R. Kawashima, and A. Trenkle. 1963.
Selenium and vitamin E and K additions to a no hay finishing cattle ration. J.
Anim. Sci. 22: 929-933.
Butler, G. W., and P. J. Peterson. 1961. Aspects of the fecal excretion of selenium by
sheep. New Zealand J. Agr. Res. 4: 484-491.
Campbell, D. T., J. Maas, D. W. Weber, O. R. Hedstrom and B. B. Norman. 1990.
Safety and efficacy of two sustained release intra reticular selenium supplements
and the associated placental and colostrum transfer of selenium in beef cattle.
Am. J. Vet. Res. 51: 813-817.
Caravaggi, C., F. L. Clark, and A. R. B. Jackson. 1970. Acute selenium toxicity in
lambs following intramuscular injection of sodium selenite. Res. Vet. Sci. 2: 146-
149.
Castellan, D. M., J. P. Maas, I. A. Gradner, J. W. Oltjen, and M. L. Sween. 1999.
Growth of suckling beef calves in response to parenteral administration of
selenium and the effect of dietary protein provided to their dams. J. Am. Vet.
Med. Assoc. 214: 816-821.
Chapman, H. L., D. H. Cox, C. H. Haines, and G. K. Davis. 1963. Evaluation of the
liver biopsy technique for mineral nutrition studies with beef cattle. J. Anim. Sci.
22: 733-741.
Church, D. C. 1979. Digestive physiology and nutrition of ruminants. 2nd ed. Oxford
Press, Inc., Portland. OR.
Combs, G. F., Jr., and S. B. Combs. 1986. The role of Se in Nutrition. Academic Press,
New York.
Conrad, H. R., and A. L. Moxon. 1979. Transfer of dietary selenium to milk. J. Dairy
Sci. 62: 404-411.
Corah, L. R., and S. Ives. 1991. The effects of essential trace minerals on reproduction
in beef cattle. Vet. Clin. North Am. Food Anim. Pract. 7: 41-53.
Cousins, F. B., and I. M. Caimey. 1961. Some aspects of selenium metabolism in sheep.
Aust. J. Agr. Res. 12: 927-943.
Cristaldi, L. A., L. R. McDowell, C. D. Buergelt, P. A. Davis, N. S. Wilkinson, and F. G.
Martin. In press. Tolerance of inorganic selenium in wether sheep. Small
Rumin. Res.


91
(1963) on d 0 and d 365. After each collection, liver, whole blood, and/or milk samples
were placed on ice, transported to the University of Florida Animal Nutrition Laboratory,
and then frozen (0C) until analyses. Blood collected for plasma analyses was
centrifuged at 700 x g for 25 min; following centrifugation, plasma was frozen for later
analyses. Cows were weighed again at the cessation of the experiment and change in BW
was calculated.
Pastures were sampled in October 2002 pre-calving, March 2003 post-calving,
and August 2003 at weaning. Likewise, hay, molasses-based liquid supplement, whole
cottonseed, and pelleted citrus pulp were sampled during winter supplementation. Whole
blood, liver, plasma, milk, and all feedstuffs were analyzed for Se concentration using a
fluorometric method described by Whetter and Ullrey (1978). To help ensure reliability
of the analytical method, a certified standard (National Bureau of Standards Bovine Liver
SRM-1577a; U.S. Department of Commerce, National Institute of Standards and
Technology, Gaithersburg, MD) was frequently analyzed.
Effects of treatment on change in body weight were analyzed using PROC
MIXED in SAS (SAS for Windows 8e; SAS Inst., Inc., Cary, NC) in a completely
randomized design with a diagonal covariance structure. Contrast statements were used to
compare means as described by Littell et al. (1998; 2000). PROC MIXED was also used
to analyze effects of treatment, d, and the interaction of treatment x d on body weight,
whole blood Se, milk Se, plasma Se, and liver Se as repeated measures with a spatial
power covariance structure with respect to d and subplot of animal nested within
treatment. Contrast statements were written to determine differences in means for
different sampling d. PROC CORR was used to determine correlations of milk Se to


29
during the dry, rebreeding stage. One plausible explanation for this is the lack of
placenta, fetal tissue, and milk for deposition and excretion of Se. During late gestation
in yr 1, dietary Se level affected serum Se concentration (P < 0.001), ewes receiving 8,
12, 16, and 20 mg/kg Se all had higher (P < 0.05) serum Se than did controls. Likewise,
ewes receiving 16 or 20 mg/kg Se had serum Se higher (P < 0.05) than ewes receiving 4
mg/kg Se. During lactation in yr 1, ewes receiving 16 and 20 mg/kg Se were similar (P =
0.32) and both groups were higher (.P < 0.05) than controls and ewes receiving 4 and 8
mg/kg Se in serum Se concentration. During the 20 wk that ewes were not lactating and
were either open or rebreeding, ewes receiving 16 and 20 mg/kg dietary Se had similar (P
= 0.44) serum Se which was higher (P < 0.05) than from all other treatments. Ewes
receiving the intermediate levels of Se (8 and 12 mg/kg) had similar serum Se (P = 0.31)
which was higher (P < 0.05) than from controls and ewes receiving 4 mg/kg Se. Ewes in
late gestation during yr 2 generally produced numerically higher serum Se than in late
gestation the previous yr. Ewes receiving 20 mg/kg Se had serum Se which was similar
(P = 0.69) only to serum Se from ewes receiving 16 mg/kg Se and higher (P < 0.05) than
all other treatments. Ewes receiving 16 mg/kg Se produced serum Se which tended (P =
0.07) to be higher that serum Se from ewes receiving 12 mg/kg Se and was higher (P <
0.05) than serum Se from controls and ewes receiving 4 or 8 mg/kg Se. During lactation
in yr 2, ewes receiving 20 mg/kg Se had higher (P < 0.05) serum Se than serum Se from
all other treatments. Serum Se from ewes receiving 8, 12, or 16 mg/kg Se was similar (P
> 0.20) and only serum Se from ewes receiving 4 mg/kg dietary Se was similar (P = 0.21)
to controls. Throughout the experiment, serum Se concentrations in these ewes remained
below 1500 pg/L, which is described as a toxic level in horses (Aitken, 2001) and were at


14
Urine, feces, and exhalation are the primary excretion routes of Se. Amount and
distribution of excreted Se within these routes are affected by chemical form of Se, total
Se intake, and diet composition including antagonists (McDowell, 2003). Urine is the
major excretory pathway and Se excretion via urine increases with Se status of the
animal. Fecal excretion remains nearly constant and exhalation of Se becomes a major
route only when Se concentrations are at a toxic level (McDowell, 2003). The amount of
Se exhaled increases as dietary Se increases (McConnell and Roth, 1966) and one
characteristic of animals which excrete Se via respiration is breath with a garlicky odor.
Selenium excretion in ruminant animals is dependent on method of administration.
When Se is provided orally, ruminants excrete more Se in feces. However, when Se is
given parenterally, more Se is excreted in urine (Wright and Bell, 1966). This is
supported by the concept that rumen microorganisms reduce dietary Se to insoluble forms
(Butler and Peterson, 1961; Peterson and Spedding, 1963; Hidiroglou et ah, 1968) and
thus increase fecal excretion of unabsorbed Se.
Differences in Efficacy of Selenium due to Source
The efficacy of Se to increase blood and tissue Se concentrations in animals varies
with source of Se. In general, Se is deposited in tissues and blood Se is more increased
when supplemental Se is of the organic form (McDowell, 2003). The primary sources of
inorganic Se are sodium selenite and sodium or barium selenate, while Se yeast and
seleniferous grains and plants are the primary sources of organic Se. Sodium selenite and
selenate are often added to free-choice mineral mixtures for livestock. Likewise, those
two chemical forms are used in injectable Se products. Selenomethionine is the major Se
compound found in grains used for livestock feeds and in Se yeast. Se-
methylselenocystine is the Se compound found most abundantly in seleniferous plants,


92
whole blood Se from parturition to 205 d postpartum and to determine the correlation of
plasma Se to liver Se.
Results
Selenium concentration of pasture and hay for all groups averaged 0.071 0.014
and 0.045 mg/kg DM basis, respectively. Crude protein was 12.57% for hay and 12.79%
1.76 for standing forage. Forage CP did not differ (P = 0.92) among pastures during the
study. Whole cottonseed, pelleted citrus pulp, and liquid supplement analyzed 0.002,
0.002, and 0.744 mg Se/kg, respectively. Free-choice mineral consumption was
monitored and recorded. Mineral consumptions and amount of Se via injection are
summarized in Table 6-2. All free-choice mineral mixtures were formulated for an
expected intake of 85-113 g-cow -d'1. However, only the cows on the Se yeast treatment
had mineral consumption (85.5 g-cow''-d ') in this range (Table 6-2). Treatment groups
receiving free-choice minerals with no Se consumed 62.19 g-cow'1-d1 and cows receiving
free-choice minerals with sodium selenite consumed 41.5 g-cowl-d'1.
There was an effect of d and treatment x d (P < 0.001) on cow BW. Initial BW of
cows did not differ (P > 0.40) among treatment groups (Table 6-3) and all cows gained
weight {P < 0.03). At the end of study (d 365), cows that received Sel-Plex were heavier
(P <0.05) than controls, and cows receiving either injectable Se source. Cows from the
free-choice selenite treatment were heavier (P < 0.05) than those receiving Deposel and
tended (P = 0.085) to be heavier than controls and Mu-Se treated cows. Form of Se
supplementation had an effect (P < 0.001) on change in cows BW (Figure 6-1). Both
free-choice treatment groups were similar and had a greater increase in BW (P < 0.05)
than did control and the injectable Se treated groups. Control, Mu-Se, and Deposel


To our Heavenly Father and my loving family.


114
rate due to a Se deficiency and its effects on thyroid hormones. In the present study,
increases in ADG of 8, 11, and 19% were observed in calves from cows supplemented
with Se via free-choice minerals over those calves from control, injectable selenite, and
injectable barium selenate treated cows, respectively. These increases in ADG could be
somewhat due to calves enhanced immune function and decreased affliction with
subclinical illnesses.
The critical whole blood Se concentration for cattle is controversial; however >
100 pg Se/L is defined as adequate by several authors (Hansen et ah, 1991; Gerloff,
1992; Dargatz and Ross, 1996). Those authors also define a blood Se of < 75 pg Se/L as
marginal or subclinical. Blood Se < 50 pg Se/L is accepted as deficient by Pehrson et al.
(1999) and Gerloff (1992). Furthermore, Dargatz and Ross designate that concentration
as severely deficient. We will use the aforementioned concentrations and designations to
discuss our findings as they relate to calf whole blood Se.
At birth, all calves on this study had adequate blood Se concentrations ranging
from 104 pg Se/L (control) to 200 pg Se/L (Se yeast). Both free-choice and the injectable
barium selenate treated cows had calves with higher blood Se than did controls.
Injectable selenite also produced numerically higher concentrations than control. These
results concur with the findings of Gunter et al. (2003) where higher blood Se was found
in calves whose dams were supplemented with Se yeast (203 pg Se/L) vs sodium selenite
(134 pg Se/L) in their free-choice minerals. In the same study, both forms of Se
supplementation produced higher blood Se than did no Se supplementation.
By d 30, whole blood Se was still lowest at 86 pg Se/L for the control animals
and whole blood Se remained highest for the Se yeast treated animals at 166 pg Se/L.


50
respectively. Ewes were housed by treatment group in covered wooden pens (53.5 m2)
with earth floors and ad libitum water. Diets were fed at 909 g/ewe/d from d 0 until
lambing began (d 81), increased to 1000g/ewe/d during lambing (d 81 to 101), and again
increased to 1135 g/ewe/dduring lactation (d 101 to 171). Diets were sampled every 28
d, ground (1 mm), and frozen at 0C until analysis.
In the first year, 52 lambs were bom over 20 d from March 9, 2002 to March 28,
2002. Prior to lambing, ewes were fitted with a device to cover the udder and prevent
lambs from nursing until a blood sample could be obtained. The udder cover was crafted
from nylon pantyhose (Eeggs Products, Winston-Salem, NC) and polyester elastic (2.54
cm wide) and held in place with safety pins (Figure 4-1). A blood sample for plasma
analysis was collected from lambs immediately after birth via jugular venipuncture into
10-ml heparinized tubes (Vacutainer; Becton-Dickinson, Franklin Lakes, NJ). The udder
cover was then removed from the ewe and five ml of pre-suckled colostrum was collected
into a 15-ml plastic centrifuge tube (Fisher; Fisher Scientific, Pittsburgh, PA). Additional
blood samples were collected from lambs and milk samples from ewes at 3, 28, and 56 d
postpartum. Blood samples were centrifuged at 700 x g and the plasma then frozen at
0C. Ewe milk samples were also stored frozen at 0C for later analysis. Lambs were
weaned at 70 d of age and ewes then received 909 g/ewe/d of their respective diets until
next lambing. At 70 d, ram lambs were surgically castrated and the testes were frozen at
0C until analysis.
On August 15, 2002, ewes were pen exposed to rams for 35 d. In the second year,
36 lambs were bom over 34 d from January 17, 2003 to February 20, 2003. All sampling
intervals, procedures, feeding levels, and materials used were duplicated from the first


52
Se than did those ewes receiving 4 mg/kg dietary Se and tended (P <0.12) to produce
higher colostrum Se than ewes receiving 8 and 12 mg/kg dietary Se. Likewise, colostrum
Se from ewes receiving 16 mg/kg tended to be higher (P = 0.052) than colostrum Se from
ewes receiving 4 mg/kg dietary Se. Colostrum Se from ewes receiving 8, 12, or 16
mg/kg dietary Se was similar (P > 0.20).
In year two, 12 of 36 lambs were removed from the study before d 56. Seven
lambs were removed due to their dams having either no milk or enlarged teats that were
unable to be suckled, four lambs were lost to predation, and one lamb was removed due
to physical injury. No lambs were lost or removed from the study due to dietary Se in the
diet of their dam. As in year one, colostrum Se was affected by dietary Se (P < 0.05) and
increased linearly (P < 0.01) as dietary Se increased (Table 4-2). Ewes receiving 8, 12,
or 20 mg/kg dietary Se produced colostrum with similar (P >0.19) Se concentrations,
which were higher (P < 0.05) than colostrum Se from controls. Ewes consuming 8
mg/kg dietary Se produced colostrum Se higher (P < 0.05) than those ewes consuming 4
mg/kg dietary Se. Likewise, ewes consuming 20 mg/kg dietary Se tended to produce
colostrum Se higher (P = 0.10) than ewes consuming 4 mg/kg dietary Se. No ewes
receiving 16 mg/kg dietary Se lambed in year two and are not represented in these
comparisons.
Ewe milk Se collected at 3, 28, and 56 d postpartum increased linearly (P <
0.001) as dietary Se increased in year one (Table 4-3). Day of sampling also had an
effect (P = 0.002), but there was no treatment x d interaction. At d 3 postpartum, ewes
receiving 4 and 8 mg/kg dietary Se produced similar (P = 0.60) milk Se and milk Se from
ewes receiving 8 mg/kg dietary Se tended (P = 0.06) to be higher than from controls.


10
Judson et al. (1991) evaluated long-acting Se treatments for ewes and lambs in a 200 wk
experiment. Those authors reported that a 100 mg injection of barium selenate was more
effective at increasing blood Se of ewes and their lambs than was an intraruminal Se
pellet or no Se supplementation. Data show a near five-fold increase in blood Se from
lambs from injectable selenate treated ewes vs lambs from unsupplemented dams.
Norton and McCarthy (1986) evaluated injectable Se products for prevention of WMD in
lambs and reported increased plasma and milk Se in ewes that received the injectable Se
vs unsupplemented controls. Likewise, those authors showed increases in lamb plasma
Se due to the frequency of use of injectable Se. In a series of University of Florida
studies, the use of injectable Se, as sodium selenite and barium selenate, in a cow-calf
herd was evaluated and compared to inclusion of organic Se in free-choice mineral
mixtures (Valle et al., 2002; 2003). Those authors reported that, in general, injectable Se
as selenate and selenite affected plasma, liver, colostrum, and milk in a similar manner.
Though the injectable products did increase Se levels in blood, milk, and tissue compared
with blood, milk, and tissue Se concentrations from unsupplemented animals, both
injectable forms of Se were generally less effective than the addition of organic Se to
free-choice minerals. The calves bom to and suckling cows that received injectable Se
had plasma Se concentrations which were similar to plasma Se concentrations of calves
from unsupplemented dams. Selenium supplementation via free-choice minerals proved
more effective at raising and maintaining Se status of Florida beef cows and their calves.
Gunter et al. (2003) compared effects of Se supplementation as sodium selenite or
Se yeast added to free-choice minerals on performance and Se status of beef cows and
calves in Arkansas. Mineral mixtures were formulated to contain 26 mg/kg Se and were


CHAPTER 2
REVIEW OF LITERATURE
Benefits of Selenium Supplementation to Livestock
Seleniums role in animal nutrition was drastically changed when it was identified
as the third factor involved in preventing liver necrosis in rats (Schwarz and Foltz, 1957).
After this first evidence for the essentiality of Se, benefits for many other species were
discovered. Patterson et al. (1957) demonstrated that Se would prevent exudative
diathesis in chicks and Eggert et al. (1957) showed that hepatosis diettica (liver necrosis)
could be prevented in swine by feeding Se. In calves and lambs, Se was successful in
preventing white muscle disease (WMD), a condition also known as nutritional
myodegeneration (Hogue, 1958; Muth et al., 1958).
Corah and Ives (1991) reported that insufficient Se could be linked to a variety of
disorders in beef cattle. Among the reproductive disorders observed were retained
placenta, infertility, abortions, births of premature, weak, or dead calves, cystic ovaries,
metritis, delayed conception, erratic estrus periods, and poor fertilization. In addition to
problems in reproduction, a condition known as ill-thrift has also been reported in
cattle (Corah and Ives, 1991; Underwood and Suttle, 1999) and also affects sheep. Ill-
thrift is defined as a syndrome that includes subclinical growth deficit, clinical
unthriftiness with rapid loss in weight, as well as some mortality. Selenium deficiency
has also been linked to cases of mastitis in dairy cattle that occurred more frequently and
lasted longer than mastitis in cattle with adequate Se intake (Smith et al., 1985).
3


127
were in their respective normal ranges at the end of the study. This further indicates no
tissue damage occurred due to Se toxicosis.
Most of the ewes receiving different levels of dietary Se lambed twice during the
study. The effects of the six levels of dietary selenium on ewes milk and the Se status of
their lambs prior to weaning were also compared and evaluated. Colostrum Se ranged
from 257 to 3542 pg/L and increased linearly as dietary Se increased (P < 0.001) in both
years. Ewe milk Se ranged from 75 to 2228 pg/L and also increased linearly as dietary
Se increased (P < 0.01). In general, ewes receiving > 12 mg/kg Se produced higher milk
Se than controls. Blood samples were collected from lambs before nursing and at 3, 28,
and 56 d of age to evaluate plasma Se concentrations. At birth, lamb plasma Se ranged
from 74 to 775 pg/L and was affected (P < 0.001) by the Se concentration of the ewe
diets, which indicates placental transfer of Se. Lambs from ewes receiving dietary Se at
20 mg/kg had higher (P < 0.05) plasma Se than controls at birth and 3, 28, and 56 d of
age in both yr. Selenium concentration in testes collected at 70 d of age was also affected
by Se content of ewe diets. In yr one, lambs whose dams received 16 or 20 mg/kg Se had
higher (P < 0.05) testicular Se than controls, but no differences in testicular Se were
observed in yr two. No signs of Se toxicosis were observed in lambs regardless of
dietary Se concentration of the ewes diet.
A 60-wk experiment was conducted to determine maximum tolerable of Se by
feeding Se as sodium selenite or Se yeast at high dietary levels to wether sheep. Criteria
use to determine maximum tolerable levels were the same as those evaluated in the ewe
experiment. Twenty-eight crossbred wethers received 0.2, 20, 30, or 40 mg/kg dietary Se
(as-fed) from sodium selenite or Se yeast added to a corn-soybean meal basal diet.


44
Table 3-5. Effect of dietary inorganic Se level on wool Se concentration of mature
ewesa
-Dietary Se, mg/kg
Week of 0,2 4 8 12 16 20
experiment
12
0.50h 0.58
0.71h 0.54
- Wool Se, mg/kg (DM basis)
1.36hi 0.54 1.67h 0.54
2.00h 0.54
2.23* 0.64
24c
0.62h 0.62
1.43hl 0.54
2.76'J 0.54
2.79J 0.54
3.58jk 0.54
4.64k 0.64
36d
1.58h 0.63
3.72 0.54
4.86,J 0.54
3.96'0.54
5.57J 0.57
5.27IJ 0.64
48c
1.18h 0.64
4.86' 0.54
4.64' 0.54
4.82'0.54
5.47'0.57
5.53' 0.64
60f
1.25h0.64
4.06' 0.54
6.09* 0.57
5.50,J 0.57
5.63,J 0.68
5.17,J 0.64
72g
0.96h 0.64
3.421 0.57
3.67IJ 0.57
5.12J 0.58
5.11,J 0.80
7.69k 0.64
aData represent least squares means SE.
bn = 6, 7, 7, 7, 7, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
crc = 5, 7, 7, 7, 7, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
d = 5, 7, 7, 7, 6, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
cn = 5, 7, 6, 7, 6, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
rn = 5, 7, 6, 6, 4, and 5 for Se levels 0.2,4, 8,12, 16, and 20 mg/kg, respectively.
gn = 5, 6, 6, 6, 3, and 5 for Se levels 0.2, 4, 8, 12, 16, and 20 mg/kg, respectively.
ll'l'i'kMeans within rows lacking a common superscript differ (P < 0.05).
Table 3-6. Amount of albumin and tissue enzyme activities present in serum of Se
supplemented ewesa,b,c
Dietary Se, mg/kg
Item
Normal concentration
0.2
4
8
12
16
20
Albumin
2.4-4.0 g/dL
2.9
2.5
1.940
2.2
2.3
2.6
Aik Phos
68-387 IU/L
91.0
60.2
65.3
95.5
36.3
89.0
AST
60-280 IU/L
53.8
131.7
32.8
52.3
64.0
24.6
ALT
11-40 IU/L
15.4
34.7
2.7
13.3
17.7
24.0
GGT
15-60 IU/L
67.6
63.2
39.8
55.0
63.7
59.6
CK
0-584 IU/L
67.4
108.2
36.3
58.7
47.7
55.0
Serum sample collected at wk 72.
bAlbumin and tissue enzyme activities presented in same units as normal concentration ranges.
CGGT and CK ranges were established by University of Florida Veterinary Teaching Hospital.


102
Table 6-3. Initial and ending BW of beef cows receiving different sources and forms of
Se supplementation3
Source of Se supplementation
Initial BW, kg
Ending BW, kg
Control1 (No Se)
419b 21
45 Lc 21
Barium Selenate2 (Deposel)
402b 19
418b 19
Sodium Selenite3 (Mu-Se)
419b 18
453bc 18
Free Choice Mineral4 (Selenite)
422b 19
502cd 19
Free Choice Mineral5 (Sel-Plex)
421b 19
509d 19
aData represent least squares means and SE, n = 39 for initial and ending BW.
b,c,dMeans within columns lacking a common superscript differ (P < 0.05).
Cows consumed free-choice mineral mix with no Se at an avg of 62.2 g-cow'1-d"1 beginning at d 0.
2Cows received a subcutaneous injection of 9 mL Deposel at d 0.
3Cows received an injection of 5 mL Mu-Se every 4 mo beginning at d 0.
4Cows consumed free-choice mineral mix containing 26 mg Se/kg as sodium selenite at
g-cow''-d' beginning at d 0.
5Cows consumed free-choice mineral mix containing 26 mg Se/kg as Se yeast at an avg
d"1 beginning at d 0.
an avg of 41.5
of 85.5 g-cow'
Table 6-4. Whole blood Se concentrations of cows receiving different sources and
forms of Se supplementation at various d postpartum3
Days postpartum
0 30 90 205
Source of Se supplementation Whole blood Se, pg/L
Control (No Se)
143b

15
162b

15
121b
15
74b

15
Barium Selenate1 (Deposel)
235c

12
207cd

12
166de
12
156c
:
12
Sodium Selenite2 (Mu-Se)
173b

13
178bc

12
127bc
12
89b

12
Free Choice Mineral3 (Selenite)
159b

12
x>
00

13
140cd
13
155c
:
13
Free Choice Mineral4 (Sel-Plex)
216

12
24 ld

12
185e
12
198c
'
12
Data represent least squares means and standard errors; n = 41/d; Adequate Se level in whole blood is >
100 pg/L.
b c d =Means within columns lacking a common superscript differ (P < 0.05).
Cows received a subcutaneous injection of 9 mL Deposel at d 0.
2Cows received an injection of 5 mL Mu-Se every 4 mo beginning at d 0.
3Cows consumed free-choice mineral mix containing 26 mg/kg Se as sodium selenite at an avg of 41.5
g-cow'-d'1 beginning at d 0.
4Dams of these calves consumed free-choice mineral mix containing 26 mg/kg Se as Se yeast at an avg
of 85.5 g-cow'1-d'1 beginning at d 0.


94
values increased from d 0 to d 30 postpartum and decreased from d 30 to d 205
postpartum.
Effects of treatment and d (P < 0.001) were observed in Se concentration of milk
collected at the same postpartum intervals as whole blood (Table 6-5). Cows receiving
Sel-Plex had higher (P < 0.05) Se concentrations in post-suckled colostrum than did cows
receiving all other treatments. Colostrum Se was similar (P > 0.54) from control,
Deposel, Mu-Se and free-choice selenite treated cows. No significant differences in milk
Se were observed at 30 d postpartum, however, Sel-Plex treated cows tended (P = 0.13)
to produce milk with higher Se concentration than did cows that received Mu-Se. At 90
d postpartum, no differences or tendencies were observed in milk Se among treatment
groups (P > 0.28). Selenium in milk collected at 205 d postpartum, was similar (P >
0.50) among control, Mu-Se, Deposel, and free-choice selenite treatments. Cows
receiving Sel-Plex produced higher (P <0.01) milk Se than cows receiving any other
form of Se supplementation. Milk Se from all treatment groups decreased quadratically
(P<0 .001) from parturition to 205 d postpartum. Selenium concentrations in whole
blood and milk collected at parturition and 30, 90, and 205 were positively correlated (.P
< 0.01; r = 0.25).
Plasma Se concentrations were evaluated at d 0 and at d 365 (Table 6-6).
Treatment and treatment x d had significant effects (P < 0.001) on plasma Se. At d 0, all
cows had similar plasma Se (52 to 62 pg/L). At the end of the study, only cows from the
control and Mu-Se treatments had similar (P = 0.32) plasma Se. Plasma Se
concentrations in Sel-Plex treated cows were higher (P < 0.005) than from cows
receiving any other treatment. Free-choice selenite produced higher (P < 0.012) plasma


Table 5-1. Diet composition (as-fed) for Se supplemented wethers3
Ingredient
% as-fed
Ground yellow com
58.00
Cottonseed hulls
30.00
Soybean meal (47.5% CP)
6.50
Soybean oil
3.00
Trace mineral mixb
1.00
Ground limestone
1.00
Ammonium chloride
0.50
Vitamins A & D
C
aSelenium levels in diet (as analyzed): 0.48, 20.48, 30.86, and 38.10 mg/kg for Se levels 0.2, 20, 30, and
40 mg/kg from sodium selenite, respectively; 0.54, 20.26, 30.71 and 37.65 mg/kg for Se levels 0.2, 20,
30, and 40 mg/kg Se from Se yeast, respectively
bTrace mineral mixture supplied between 96.5% and 98.5% NaCl, and provided per kg of diet: 1.0 mg
Co (as carbonate), 5.0 mg Cu (as oxide), 0.7 mg I (as iodate), 35 mg Fe (as oxide), 25 mg Mn (as oxide),
and 35 mg Zn (as oxide).
Provided per kg of diet: 5,000 IU of Vitamin A and 500 IU of Vitamin D3.
Table 5-2. Effects of four dietary levels of Se as sodium selenite or Se yeast on BW of
wethersa
Se source
Sodium selenite Se yeast-
Dietary Se level, mg/kg
Week
0.2
20
30
40
0.2
20
30
40
SEM
0
61.2
65.0
65.8
58.9
57.0
55.5
67.9
64.3
4.5
8
59.1
63.6
59.8
49.3
56.8
51.5
54.3
50.6
4.5
16
61.8
68.2
57.9
48.2
57.9
55.1
54.8
50.0
5.7e
24
65.1
70.0
59.2
52.4
60.0
57.3
53.3
48.2
7.5e
32
70.9
76.7
63.6
56.1
68.2
59.5
51.1
52.7
9.0e
40
70.5
61.8
61.8
57.3
74.7
51.8
47.3
50.9
8.4
48
77.6
81.8
69.8
62.9
70.5
65.5
38.6
54.5
9.0C
60
83.3
85.6
76.5
67.9
78.2
61.8
50.2
54.5
10.4
.Ayg
68.7
71.6
64.3
56.6
65.4
57.3
52.2
53.2
Data represent least squares means and pooled SE.
bDietary Se level response (P < 0.05).
cSelenium source response (P < 0.05).
dTime response (P < 0.05).
'Dietary Se level linear response (P < 0.10).


76
40 mg/kg Se received 91.1% of that LD5o for sheep throughout the experiment.
Furthermore that LD5o for sheep (Blodgett and Bevill, 1987) was established using
injectable Se. Administration of Se parenterally disallows the reduction of selenite Se to
insoluble selenide via ruminal microorganisms as described by (Whanger et al., 1968).
This would suggest that the LD50 for Se in sheep could be considerably higher than
previously thought.
Selenium concentration in new growth wool was measured at wk 12, 24, 36, 48,
and 60 (Table 5-5). Dietary Se level, Se source, time, dietary Se level x Se source, and
dietary Se source x time affected (P < 0.05) wool Se. Wool Se ranged from 1.19 to 39.09
mg/kg and increased linearly (P < 0.001) as dietary Se increased. Wool Se from wethers
receiving organic Se was often more than three-fold higher (P < 0.001) than from wethers
receiving selenite Se at the same dietary level. Increased Se in hair has been reported in
other livestock species. Kim and Mahan (2001) observed a linear response in the hair of
pigs as Se in their diet was increased. Goehring et al. (1984b) reported a quadratic
response in the hair of swine as dietary Se as sodium selenite was increased up to 20
mg/kg. Likewise, Perry et al. (1976) reported increased Se in the hair of feedlot steers as
dietary selenite Se was increased. Cristaldi et al. (2004) reported a linear increase in the
wool of wether sheep as dietary Se was increased. Those authors did not report a
significant Se level x time interaction. However, wool Se of sheep, on the present study
was affected by time and the interaction of Se source x time as wool Se continued to
increase from wethers fed selenite Se and wool Se from wethers receiving organic Se
increased then seemed to reach a peak around wk 48. This suggests that wool Se may
reach a plateau when animals are fed high dietary concentrations of organic Se. Wool Se


126
organic or inorganic Se that can be tolerated by mature wethers. With a cow-calf herd,
methods and sources of Se supplementation were evaluated.
A 72-wk study was conducted to determine the maximum tolerable level of
selenite Se for ewes during lamb production. The criteria for determining Se tolerance
included clinical signs, Se in blood, wool, and tissues, histopathological evaluation, and
the activity levels of enzymes related to Se toxicosis. Forty-one range-type ewes were
fed Se as sodium selenite which was added to a corn-soybean meal basal diet at levels of
0.2 (control), 4, 8, 12, 16, and 20 mg/kg. Serum Se and ewe body weight (BW) were
measured at 4-wk intervals, whole blood Se and wool Se were measured every 12 wk,
and samples of brain, diaphragm, heart, hoof, kidney, liver, and loin muscle were
collected at the termination of the experiment. Ewe BW was unaffected by dietary Se
level (P = 0.69). Ewe serum Se, whole blood Se, and wool Se increased linearly as
dietary Se increased (P < 0.05). Brain, diaphragm, heart, and psoas major muscle Se
increased linearly as Se in the diet increased, liver Se responded quadratically, and hoof
and kidney Se responded cubically to treatment (P < 0.05). In general, serum, whole
blood, and tissue Se concentrations from ewes receiving 12, 16, or 20 mg/kg dietary Se
were higher (P < 0.05) than from controls and ewes receiving less dietary Se. Though
serum, whole blood, and wool Se concentrations were elevated in ewes receiving
increased dietary Se, at no time did serum, whole blood, or wool Se concentrations reach
levels previously reported as toxic and a pattern of clinical signs of Se toxicosis was not
observed. Ehstopathological microscopic evaluation of liver, kidney, diaphragm, heart,
and psoas major muscle did not reveal evidence of Se toxicosis in ewes on any dietary Se
treatment. Levels of albumin and the activity of Aik phos, ALT, AST, CK, and GGT


sodium selenite or Se yeast for 60 wk, had Se concentrations in serum, whole blood,
wool, and soft tissues which increased as dietary Se increased (P < 0.05). In general, Se
yeast vs selenite was more effective at increasing Se in blood, wool, and soft tissues (P <
0.05). Enzyme activity and histopathological evaluation of soft tissues from ewes and
wethers indicated no evidence of Se toxicosis. From the two sheep experiments,
maximum tolerance for both forms of dietary Se is greater than 40 mg/kg. Cows
receiving Se supplementation as Se yeast maintained adequate concentrations of Se in
plasma, whole blood, and liver and generally had higher (P < 0.05) concentrations than
cows receiving inorganic Se. Calves from cows receiving Se via free-choice minerals
had higher (P < 0.05) weight gains than from cows receiving injectable selenate. Calves
whose dams received Se yeast generally had higher Se (P < 0.05) in blood and liver.
IX


142
Smith, K. L., J. S. Hogan, and H. R. Conrad. 1988. Selenium in dairy cattle: Its role in
disease resistance. Vet. Med. 83: 72-78.
Smith, M. I., E. F. Stohlman, and R. D. Lillie. 1937. The toxicity and pathology of
selenium. J. Pharmacol. Exp. Therap. 61:89-102.
Smyth, J. B. A., J. H. Wang, R. M. Barlow, D. J. Humphreys, M. Robins, and J. B. J.
Stodulski. 1990. Experimental acute Se intoxication in lambs. J. Comp. Path.
102: 197-208.
Spears, J. W., R. W. Harvey, and E. C. Segerson. 1986. Effects of marginal selenium
deficiency and winter protein supplementation on growth, reproduction, and
selenium status of beef cattle. J. Anim. Sci. 63: 586-594.
Stowe, H. D. and T. H. Herdt. 1992. Clinical assessment of selenium status of livestock.
J. Anim. Sci. 70: 3928-3933.
Trinder, N., R. J. Hall, and C. P. Renton. 1973. The relationship between the intake of
selenium and vitamin E on the incidence of retained placenta in dairy cows. Vet.
Rec. 93:641-642.
Ullrey, D. E., P. S. Brady, P. A. Whetter, P. K. Ku, and W. T. Magee. 1977. Selenium
supplementation of diets for sheep and beef cattle. J. Anim. Sci. 46: 559-565.
Underwood, E. J. and N. F. Suttle. 1999. The Mineral Nutrition of Livestock. 3rd ed.
CABI Publishing, Oxon, UK.
Valle, G. 2001. Effects of different, methods, sources, and levels of selenium
supplementation and fertilization on beef cattle and forage tissue levels. Ph.D.
Diss., Univ. of Florida, Gainesville, FL.
Valle, G., L. R. McDowell, D. L. Pritchard, P. J. Chenoweth, D. L. Wright, F. G. Martin,
W. E. Kunkle, andN. S. Wilkinson. 2002. Selenium status of beef calves from
dams receiving selenium supplementation. J. Anim. Vet. Adv. 2: 338-342.
Valle, G., L. R. McDowell, D. L. Pritchard, P. J. Chenoweth, D. L. Wright, F. G. Martin,
W. E. Kunkle, and N. S. Wilkinson. 2003. Effects of supplementing selenium to
a beef cattle cow-calf herd on tissue selenium concentration. J. Anim. Vet. Adv.
2: 126-132.
van Ryssen, J. B. J., J. T. Deagen, M. A. Beilstein, and P. D. Whanger. 1989.
Comparative metabolism of organic and inorganic selenium by sheep. J. Agrie.
FoodChem. 37: 1358-1363.
Van Saun, R. J., T. H. Herdt, and H. D. Stowe. 1989. Maternal and fetal selenium
concentrations and their interrelationships in dairy cattle. J. Nutr. 119: 1128-1137.


138
Komroff, M. 1926. The travels of Marco Polo. Revised from Mardens translation, pp.
81. Liverright, NY.
Lagace A., A. H. Hamdy, A. L. Trapp, D. S. Bell, and W. D. Pounden. 1964. Serum
transaminase in selenium-treated and hysterectomy-derived lambs. Am. J. Vet.
Res. 25: 483-486.
Lei, X. G., H. M. Dann, D. A. Ross, W. S. Cheng, G. F. Combs, and K. R. Roneker.
1998. Dietary selenium supplementation is required to support full expression of
three selenium-dependent glutathione peroxidases in various tissues of weanling
pigs. J. Nutr. 128: 130-135.
Littell, R. C., P. R. Henry, and C. B. Ammerman. 1998. Statistical analysis of repeated
measures data using SAS procedures. J. Anim. Sci. 76: 1216-1231.
Littell, R. C., J. Pendergast, and R. Natarajan. 2000. Modelling covariance structure in
the analysis of repeated measures data. Statist. Med. 19: 1793-1819.
Maag, D. D., and M. W. Glenn. 1967. Toxicity of selenium: Fram Animals, pp. 127-
140. In O. H. Muth (ed.) Symposium: Selenium in Biomedicine. AVI Publishing
Co., Westport, CT.
Maas, J. 1983. Diagnosis and management of selenium responsive diseases in cattle.
Compend. Contin. Educ. Pract. Vet. 7: 393-399.
Maas, J., M. S. Bulgin, B. C. Anderson, and T. M. Frye. 1984. Nutritional
myodegeneration associated with vitamin E deficiency and normal selenium
status in lambs. J. Am. Vet. Med. Assoc. 184: 201-204.
Maas, J., D. Galey, J. R. Peauroi, J. T. Case, E. S. Littlefield, C. C. Gay, L. D. Roller, R.
O. Crisman, D. W. Weber, D. W.Warner, and M. L. Tracy. 1992. The
correlation between serum Se and blood Se in cattle. J. Vet. Diagn. Invest. 4: 48-
52.
Madison, T. C. 1860. Sanitary report-Fort Randall. In R. H. Coolidge (ed.). Statistical
Report on the Sickness and Mortality in the Army in the United States. Senate
Exch. Doc. 52: 37.
Mahan, D. C. 2000. Effect of organic and inorganic selenium sources and levels on sow
colostrum and milk selenium content. J. Anim. Sci. 78: 100-105.
Marin-Guzman, J., D. C. Mahan, and J. L. Pate. 2000. Effect of dietary selenium and
vitamin E on spermatogenic development in boars. J. Anim. Sci. 78: 1537-1543.


87
Table 5-7. Amount of albumin and tissue enzyme activities present in serum of Se
supplemented wethersa,b
Se source
Sodium selenite Se yeast
Dietary Se level, mg/kg
Enzyme
0.2
20
30
40
0.2
20
30
40
Normal
range
Se
concentration,
I Ufa/
Albumin, g/dL
3.0
3.0
2.9
2.7
2.8
2.0
2.5
2.9
2.4 4.0
Aik Phos, IU/L
127.3
141.7
128.8
153.5
105.7
32.0
50.5
172.0
68 387
ALT, IU/L
12.0
12.0
10.8
9.3
9.7
3.0
2.5
6.0
11-40
AST, IU/L
81.7
100.7
108.8
81.8
94.7
83.0
65.0
53.0
60 280
GGT, IU/L
49.3
51.7
61.8
60.5
54.3
42.0
46.5
59.0
15-60
CK, IU/L
123.7
139.7
75.0
103.0
109.0
48.0
50.0
51.0
00 584
aSerum sample collected at wk 60.
bGGT and CK ranges were established by University of Florida Veterinary Teaching Hospital.


60
lambs from two to 14 wk of age with 5 mg of sodium selenite via subcutaneous injection
every two wk and did not induce Se toxicity. Lambs on our study did not receive nearly
the amount of Se that others reported to be deadly, even from nursing dams supplemented
with Se up to 20 mg/kg during gestation and lactation. However, our lambs were
subjected to elevated milk Se concentrations. Based on data from Mellor and Murray
(1986) and Wohlt et al. (1984) milk intake in lambs from birth to 56 d ranges from 866-
1246 g/d. Given those intake estimates and the colostrum and milk Se concentrations
from the present study, lambs consuming the colostrum or milk with the highest Se
concentration at the highest intake would ingest 4.39 mg of Se/d. In newborn lambs (3
kg BW), that amount of Se would translate to 1.46 mg Se/kg BW and to 0.29 mg Se/kg
BW in 8 wk old lambs (15 kg BW). These levels are considerably less than levels
previously reported to cause death in young lambs.
Testes taken from ram lambs at 70 d were evaluated for Se concentration.
Selenium is implicated in sperm quality and reproductive function of livestock
(Hidiroglou, 1982; Marin-Guzman et al., 2000) and concentrations in testes are less than
in liver and generally greater than in heart, spleen, and pancreas. As with plasma Se in
the suckling lambs, testicular Se of lambs increased as Se increased in the ewe diets and
ranged from 1.67 mg/kg in controls to 4.25 mg/kg in lambs whose dams received 20
mg/kg dietary Se. These Se concentrations lie between those concentrations found in the
liver and heart of wethers consuming up to 10 mg/kg Se as sodium selenite for one year
(Cristaldi et al., in press). Those wethers were reported to also have elevated
concentrations of Se in serum, whole blood, wool, and other organs. However, they
displayed no clinical signs of selenium toxicosis


121
Table 7-3. Birth weights, weaning weights, and ADG of calves from dams receiving
different sources and forms of Se supplementation8
Source of Se supplementation
Birth wt, kg
Weaning wt, kg
ADG, kg
Control (No Se)
35.3b 3.1
213.5b 17.8
0.85bc .04
Barium Selenate1 (Deposel)
34.0b 2.5
194.9b 15.5
0.78b .04
Sodium Selenite2 (Mu-Se)
34.3b 2.5
209.2b 14.6
0.83bo .03
Free Choice Mineral3 (selenite)
37.5b 2.5
195.5b 15.5
0.92c .04
Free Choice Mineral4 (Sel-Plex)
33.6b2.5
219.3b 15.5
0.93 .04
aData represent least squares means, n = 42, 39, 39 for birth wt, 205 d weaning wt, and ADG,
respectively.
b,c,dMeans within columns lacking a common superscript differ (P < 0.05).
Dams received a subcutaneous injection of 9 mL Deposel at an avg of 125 d gestation.
2Dams received an injection of 5 mL Mu-Se every 4 mo beginning at an avg of 125 d gestation.
3Dams of these calves consumed free-choice mineral mix containing 26 mg/kg Se as sodium selenite at
an avg of 41.5 g-cow'-d'1 beginning at an avg of 125 d gestation.
4Dams of these calves consumed free-choice mineral mix containing 26 mg/kg Se as Se yeast at an avg
of 85.5 g-cow''-d 1 beginning at an avg of 125 d gestation.
Table 7-4. Calf whole blood Se at various ages from dams receiving different sources
and forms of Se supplementation3
Age of calf, d
0
30
90
205
Source of Se supplementation
Whole blood Se, gg/L
Control (No Se)
104b 24
86b 10
65bc 10
42bc 10
Barium Selenate1 (Deposel)
160 12
100b 8
71bc 8
34b 8
Sodium Selenite2 (Mu-Se)
155bcd 24
98b 8
59b 8
36b 8
Free Choice Mineral3 (selenite)
156c 11
99b 9
88c 9
67 10
Free Choice Mineral4 (Sel-Plex)
200d 14
166 9
182d 9
188d9
Data represent least squares means, n = 14, 39, 39, and 39 for d 0, 30, 90, and 205, respectively. At d 0
samples were collected only from calves which had not yet nursed.
b,c,dMeans within columns lacking a common superscript differ (P < 0.05).
Dams of these calves received a subcutaneous injection of 9 mL Deposel at an avg of 125 d gestation.
2Dams of these calves received an injection of 5 mL Mu-Se every 4 mo beginning at an avg of 125 d
gestation.
3Dams of these calves consumed free-choice mineral mix containing 26 mg/kg Se as sodium selenite at
an avg of 41.5 g-cow '-d'1 beginning at an avg of 125 d gestation.
4Dams of these calves consumed free-choice mineral mix containing 26 mg/kg Se as Se yeast at an avg
of 85.5 g-cow^-d'1 beginning at an avg of 125 d gestation.


112
higher than control. Again at d 205, as was observed at d 30 and d 90, calves on the Se
yeast treatment had higher (P < 0.05) blood Se than calves on all other treatments. The
two injectable products produced blood Se that was similar (P = 0.33) on all sampling d.
A linear decrease (P < 0.001) in calf whole blood Se was observed across all sampling d.
Selenium concentrations in testes taken from calves at birth did not differ among
treatment groups (Table 7-5). Although, testicular Se from the injectable selenate group
tended to be higher (P = 0.07) than testicular Se from the free-choice selenite treatment.
Liver Se (DM basis) taken from calves at 205 d was affected (P < 0.001) by form of Se
supplementation administered to their dams (Table 7-5). Calves whose dams received Se
as Se yeast had higher (P < 0.05) liver Se than calves from all other treatment groups.
Likewise, calves whose dams received sodium selenite via free-choice minerals had
higher (P < 0.05) liver Se than calves on control and both injectable treatments. Calf
liver Se from control did not differ (P = 0.48) from either injectable treatment, and calf
liver Se from the two injectable Se sources were similar (P = 0.90).
Selenium concentrations from whole blood collected from cows and their calves
on d 30, 90, and 210 were used to correlate calf blood Se to cow blood Se. Correlations
between Se concentration in cow whole blood and calf liver were calculated from
samples collected at d 205. Figure 7-1 exhibits the positive correlation (r = 0.64; P <
0.001) between Se concentration in cows whole blood and Se concentration in the whole
blood of their calves across all sampling d. When correlations between cows and calves
whole blood Se were calculated on individual sampling d, positive (P < 0.001)
correlations were also observed (r = 0.65, 0.68, and 0.52) in samples taken at d 30, 90,
and 205, respectively. Figure 7-2 shows the positive (P < 0.001) correlation (r = 0.57)


135
Cuesta, P. A., L. R. McDowell, W. E. Kunkle, N. S. Wilkinson, and F. G. Martin. 1995.
Effects of high-dose prepartum injections of Se and vitamin E on milk and serum
concentrations in ewes. Small Rumin. Res. 18: 99-103.
Dargatz, D. A., and P. F. Ross. 1996. Blood selenium concentrations in cows and heifers
on 253 cow-calf operations in 18 states. J. Anim. Sci. 74: 2891-2895.
Echevarria M. G., P. R. Henry, C. B. Ammerman, and P. V. Rao. 1988. Effects of time
and dietary selenium concentration as sodium selenite on tissue selenium uptake
by sheep. J. Anim. Sci. 66: 2299-2305.
Eggert, R. G., E. Patterson, W. T. Akers, and E. L. R. Stockstad. 1957. The role of
vitamin E and selenium in the pig. J. Anim. Sci. 16: 1037.
Ellis, R. G., T. H. Herdt and H. D. Stowe. 1997. Physical, hematologic, biochemical and
immunologic effect of supranutritional supplementation with dietary selenium in
dairy cows. Am. J. Vet. Res. 58: 760-764.
Enjalbert, F., P. Lebreton, O. Salat, and F. Schelcher. 1999. Effects of pre- or
postpartum selenium supplementation on selenium status in beef cows and their
calves. J. Anim. Sci. 77: 223-229.
Ewan, R. C., C. A. Baumann, and A. L. Pope. 1968. Retention of Se by growing lambs.
J. Agrie. Food Chem. 16:216-219.
Franke, K. W. 1934. A new toxicant occurring naturally in certain samples of plant
foodstuffs. I. Results obtained in preliminary feeding trials. J. Nutr. 8: 597-608.
Franke, K. W., and V. R. Potter. 1935. A new toxicant occurring naturally in certain
samples of plant foodstuffs. IX. Toxic effects of orally ingested selenium. J. Nutr.
8: 597-608.
Gardner, R. W. and D. E. Hogue. 1967. Milk levels of selenium and vitamin E related to
nutritional muscular dystrophy in the suckling lamb. J. Nutr. 93: 418-424.
Gerloff, B. J. 1992. Effect of selenium supplementation on dairy cattle. J. Anim. Sci.
70: 3934-3940.
Givens, D. I., R. Allison, B. Cottrill, and J. S. Blake. 2004. Enhancing the selenium
content of bovine milk through alteration of the form and concentration of
selenium in the diet of the dairy cow. J. Sci. Food Agrie. 84: 811-817.
Glenn, M. W., R. Jensen, and L. A. Griner. 1964a. Sodium selenate toxicosis: The
effects of extended oral administration of sodium selenate on mortality, clinical
signs, fertility, and early embryonic development in sheep. Am. J. Vet. Res. 25:
1479-1485.


19
Blodgett and Bevill (1987) induced death in sheep by feeding 0.7 to 1.0 mg Se/kg BW as
selenite in as little as 6.75 h. Liver Se concentrations of more than 17 mg/kg and whole
blood Se of 2.7 mg/L were reported. After receiving an oral 5 mg selenite Se/kg BW,
lambs died within 6 h and when the same dosage was given as an injection, lambs lived
up to 60 h (Smyth et al., 1990). After evaluating the major organs, those authors
concluded that the heart is most damaged in a case of Se toxicosis as the heart has great
affinity for Se especially in lethal doses. Caravaggi et al., (1970) injected Merino lambs
with 0.425 to 0.500 mg Se/kg BW, induced death, and determined the LD50 for lambs to
be 0.455 mg Se/kg BW. Twenty lambs received 10 mg of selenite Se orally in an attempt
to prevent WMD. Of those 20, seven died within 16 h, eight developed diarrhea but
recovered, and five lambs were apparently unaffected (Marrow, 1968). Cristaldi et al.
(2004) fed up to 10 mg/kg dietary Se as selenite to growing wether sheep and reported no
signs of Se toxicity. Those authors reported whole blood Se concentrations of up to 1.2
mg/L, wool Se of 2.5 mg/kg, and liver Se concentrations of nearly 15 mg/kg Se on a dry
basis and no evidence of Se toxicity from histopathological evaluation.
Selenium toxicity studies have also been conducted using swine. Goehring et al.
(1984b) fed young pigs up to 20 mg/kg Se as sodium selenite for 5 wk. No pigs on the
study died; however, feed intake and growth rate decreased as dietary Se concentration
increased. Whole blood Se concentrations of up to 3.5 mg/L and hair Se of more than 11
mg/kg were observed. Organic and inorganic Se was included in the diet of growing pigs
at levels of up to 20 mg/kg for 12 wk (Kim and Mahan, 2001). Feed intakes of those pigs
declined as Se level increased and daily gains were decreased when Se was fed at more
than 5 mg/kg, and inorganic Se had a more detrimental effect on performance than did


6
Likewise, ADG, feed consumption, and gaimfeed were not affected by supplementation
of 0.1 mg/kg dietary Se fed for 13 wk or 0.2 mg/kg dietary Se fed for six wk in separate
studies, two using sheep and one using cattle (Ullrey et al., 1977).
Newborn and suckling calves and lambs can receive Se via their dams from either
maternal transfer or increased Se in milk. Recent studies indicate that blood Se in
newborn calves can be increased through Se supplementation of their dams
(Abdelrahman and Kincaid, 1995; Gunter et ah, 2003; Valle et ah, 2003). Likewise,
positive correlations between Se concentration in dams milk and Se concentration of calf
whole blood have been observed in calves up to 70 d of age (Pehrson et ah, 1999).
Evidence also exists that milk Se can be increased by level and duration of Se
supplementation in lactating cows (Conrad and Moxon, 1979). Like blood and tissue,
milk Se is affected by dietary Se level (Conrad and Moxon, 1979; Givens et ah, 2004)
and Se readily crosses the placenta to the fetus (Van Saun et ah, 1989). A strong
relationship of dietary Se to Se in milk of dairy cows was reported with up to 18.08% of
dietary Se being recovered in milk (Maus et ah, 1980). Koller et ah (1984) supplemented
first-calf Hereford heifers with dietary Se and concluded that Se readily crosses the
placenta in beef cattle. Furthermore, those authors added that low Se concentrations in
the blood of dams could cause the fetus to gather more Se and result in fetal blood Se that
is higher than that of the mother. In sheep, Cuesta et ah (1995) showed increased
colostrum Se from ewes receiving supplemental Se and that milk Se was higher after one
mo of lactation. Also, Jacobsson et ah (1965) concluded that Se administered to ewes
could be transmitted to lambs through the placenta and the milk after a study using radio-
labeled sodium selenite and selenomethionine. In a study utilizing swine, Wuryastuti et


96
sodium selenite. Likewise, those authors did not report a difference in BW of Se
supplemented cows vs unsupplemented controls. Likewise, Awadeh et al. (1998b)
reported no differences in the BW of Angus and crossbred cows supplemented with Se
yeast and various levels of sodium selenite. Bruce (1997) also reported no differences in
BW of cows in Alaska regardless of Se supplementation. Cow age, when reported,
indicated that cows used in previous studies were mature and may have had less potential
for growth than the younger cows utilized in our study.
At calving, cows receiving Se via Deposel or Sel-Plex had higher whole blood Se
than did cows receiving no Se, Mu-Se, or selenite in free-choice minerals. Whole blood
Se measured at 30 and 90 d postpartum followed a pattern similar with respect to
treatment to whole blood Se at calving. Deposel and Sel-Plex produced similar and
consistently higher whole blood Se than sodium selenite or no Se supplementation. In an
Arkansas study, Gunter et al. (2003) found higher whole blood Se at calving in cows
receiving Se yeast than in cows receiving sodium selenite. Knowles et al. (1999)
reported whole blood Se in cows supplemented with Se yeast to be similar to, or higher
than from cows receiving sodium selenate in a 19-wk study during mid-lactation.
However, Awadeh et al. (1998b) did not report a difference in whole blood Se when
comparing dietary Se at equal concentrations as Se yeast or sodium selenite. Podoll et al.
(1992) did report a slight increase in serum Se for selenate vs selenite fed to lactating
Holstein cows and Awadeh et al. (1998a) reported an increase in whole blood Se at
calving for Se yeast vs selenite when added to free-choice salt at 60 mg/kg. Increases in
whole blood Se from Deposel over either selenite form of Se are further supported by the
work of Henry et al. (1988) as those authors reported that selenate was more available to


18
and blind staggers. Acute Se toxicity can be caused by ingesting a large amount of
supplemental Se, overdosing with parenteral Se, or by ingesting a large amount of
seleniferous plants. Certain plants, mostly species of Astragalus, may contain up to
10,000 mg/kg Se and cereal crops, grasses, and other forages may contain up to 50 mg/kg
Se (Aitken, 2001). Fatalities of sheep, cattle, and hogs have been reported in regions
known to grow seleniferous plants (National Academy of Sciences [NAS], 1983), with
some deaths occurring within 24 h (Rosenfeld and Beath, 1964). Clinical signs of acute
Se toxicity may include elevated body temperature, labored breathing, diarrhea, and often
death. Alkali disease and blind staggers types of Se toxicosis occur with more time and
involve feedstuffs containing less Se. Clinical signs of chronic Se toxicity include
anorexia, apathy, diarrhea, weight loss, hair loss, and hoof malformations (Glenn et al.,
1964a). Animals in the blind staggers phase of Se toxicosis may wander, stumble, and
lack appetite initially and then become somewhat paralyzed and almost blind in the later
stage. The later stage appears suddenly and death usually occurs within hours (Rosenfeld
and Beath, 1964). Chronic forms of selenosis have been induced by feeding grains
containing 5 to 40 mg/kg Se (Schoening, 1936; Rosenfeld and Beath, 1964).
Glenn et al. (1964a) induced death in ewes after oral dosing of up to 50 mg/d of Se
as sodium selenate for 93 d and concluded that minimum toxic oral dose of Se as selenate
depended on susceptibility, level of Se administered, and duration of administration.
Those authors later reported liver Se concentrations of up to 29 mg/kg in experimentally
poisoned ewes (Glenn et al., 1964c). Evaluation of the tissues of the ewes in the previous
study showed that most tissue damage in Se toxicosis is confined to the heart (Glenn et
al., 1964b). No kidney damage and few instances of liver damage were reported.


24
Diets were fed at 909 g-ewe '-d'1 from d 0 until lambing began, increased to 1000
g-ewe'1-d'1 during lambing, and again increased to 1135 g-ewe -d'1 during lactation. Ewes
received 909 g-ewe'^d"1 of their respective diets after the first lamb crop was weaned. On
August 15, 2002, ewes were pen exposed to rams for 35 d. Diets were offered at the
same increments during the second lambing and lactation as during the first. Diets were
sampled every 28 d, ground (1 mm), and frozen at 0C until analysis.
Ewe BW was recorded on d 0 and for every four wk thereafter, for the remainder
of the study. A 10-mL blood sample for serum analysis was collected using an 18-gauge
needle into a vacutainer tube with no additive (Vacutainer; Becton Dickinson, Franklin
Lakes, NJ) every four wk, via jugular venipuncture, allowed to stand for 20 min,
centrifuged at 700 x g for 25 min, and serum stored frozen at 0C until Se analysis.
Starting at wk 12, an additional 10-mL blood sample was collected into a heparinized
vacutainer tube (Vacutainer; Becton Dickinson, Franklin Lakes, NJ). This additional 10-
mL sample was collected every 12 wk for the remainder of the experiment and stored
frozen at 0C as whole blood until analysis.
The wool around the jugular was shorn initially and regrowth was collected
beginning at wk 12 and every 12 wk thereafter. The collected wool was washed with a
commercial hair shampoo (Alberto V05; Alberto-Culver Co., Melrose Park, IL), to
remove oil and dirt, rinsed well with deionized water, dried, stored at room temperature,
and later analyzed for Se concentration.
At the termination of the experiment (wk 72), ewes were slaughtered following
approved USDA procedures at the University of Florida Meats Laboratory. An
additional 10-mL sample of blood was collected using an 18-gauge needle into a


74
in swine. At wk 24, wethers receiving 30 mg/kg organic Se had serum Se of 3922 gg/L.
At no other time during our study did wether serum Se exceed 3700 gg/L.
Whole blood Se was measured in addition to serum Se because of the possibility
of a more accurate Se measurement since use of whole blood eliminates the possibility of
falsely high Se readings in serum due to hemolysis (Maas et ah, 1992). Whole blood Se
responded to Se supplementation in much the same fashion as did serum Se (Table 5-4).
This response supports the high correlation between serum Se and whole blood Se
previously described by Maas et al. (1992) and Cristaldi et al. (2004). Whole blood Se
concentrations of wethers were affected by dietary Se level, Se source, and the interaction
of dietary Se level x Se source interaction (P < 0.05) ranged from 392 to 6259 gg/L, and
overall increased quadratically (P < 0.01) as Se concentration of the wether diets was
increased. Whole blood Se concentrations measured at wk 12, 24, 48, and 60 increased
linearly (P <0.10) as dietary Se level increased. Whole blood Se in swine increased
linearly as dietary Se as sodium selenite was increased from 0 to 20 mg/kg (Goehring et
al., 1984b) and whole blood in sheep responded linearly to increased dietary Se (Cristaldi
et al., in press). Likewise, those authors reported a strong correlation between Se
concentrations of serum and whole blood. In the present study, whole blood Se
responded in neither a linear nor quadratic manner at wk 36 (P > 0.15). It seems that
organic Se was used in place of inorganic Se for the selenite control diet during that
feeding period, which created a whole blood Se concentration of 1004 gg/L for the
selenite control group at wk 36. On average, whole blood Se responded quadratically (P
< 0.05) as dietary Se level was increased, again suggesting the influence of homeostatic
regulation when dietary Se is increased above 30 mg/kg. Wethers receiving organic Se


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS
ABSTRACT
CHAPTER
1 INTRODUCTION 1
2 REVIEW OF LITERATURE 3
Benefits of Selenium Supplementation to Livestock 3
Methods of Selenium Supplementation to Livestock 8
Absorption, Transport, Storage, and Excretion of Selenium 12
Differences in Efficacy of Selenium due to Source 14
Selenium Toxicosis 17
3 TOLERANCE OF INORGANIC SELENIUM IN RANGE-TYPE EWES DURING
GESTATION AND LACTATION 22
Introduction 22
Materials and Methods 23
Results and Discussion 26
Implications 39
Summary 40
4EFFECTS OF SELENIUM LEVELS IN EWE DIETS ON SELENIUM IN MILK
AND PLASMA AND TISSUE SELENIUM CONCENTRATIONS OF LAMBS... 48
Introduction 48
Materials and Methods 49
Results 51
Discussion 56
Implications 61
Summary 61
vi


140
National Research Council (NRC). 1980. Mineral Tolerance of Domestic Animals. Natl.
Acad. Press, Washington,
DC.
National Research Council (NRC). 1985. Nutrient Requirements of Sheep. Natl. Acad.
Press, Washington, DC.
National Research Council (NRC). 1996. Nutrient Requirements of Beef Cattle. 7th Ed.
Natl. Acad Press, Washington, DC.
Oldfield, J. E. 2002. A brief history of selenium research: From alkali disease to prostate
cancer (from poison to prevention) Available: http://www.asas.org/bios/
oldfieldhist.pdf. Accessed Sept. 10, 2004.
Oldfield, J.E., J. R. Schubert, and O. H. Muth. 1963. Implications of selenium in large
animal nutrition. J. Agr. Food Chem. 11: 388-390.
Ortman K., and B. Pehrson. 1999. Effect of selenate as a feed supplement to dairy cows
in comparison to selenite and selenium yeast. J. Anim. Sci. 77: 3365-3370.
O'Toole, D., and M. F. Raisbeck. 1995. Pathology of experimentally induced chronic
selenosis (alkali disease) in yearling cattle. J. Vet. Diagn. Invest. 7: 364-373.
Ouazzani, N., D. Lamnaouer, and E. H. Abdennebi. 1999. Toxicology of Astragalus
lusitanicus Lam. Therapie. 54: 707-710.
Ovemes, G., K. Moksnes, A. Froslie, J. G. Gunnar, and J. Flaat. 1985. The effect of
different levels of selenium in mineral mixtures and salt licks on selenium status
in sheep. Acta Vet. Scand. 26:405-416.
Patterson, E. L., R. Milstrey, and E. L. R. Stokstad. 1957. Effect of selenium in
preventing exudative diathesis in chicks. Proc. Soc. Exp. Biol. Med. 95: 617-620.
Pehrson B., K. Ortman, N. Madjid, and U. Trafikowska. 1999. The influence of dietary
selenium as selenium yeast or sodium selenite on the concentration of selenium in
the milk of suckler cows and on the selenium status of their calves. J Anim. Sci.
77: 3371-3376.
Perry, T. W., W. M. Beeson, W. H. Smith, and M. T. Mohler. 1976. Effect of
supplemental Se on performance and deposit of Se in blood and hair of finishing
beef cattle. J. Anim. Sci. 42: 192-195.
Peterson P. J., and D. J. Spedding. 1963. The excretion by sheep of 75-selenium
incorporated into red clover (Trifolium pratense L.): the chemical nature of the
excreted selenium and its uptake by three plant species. New Zealand J. Agrie.
Res. 6: 13-23.


104
Table 6-7. Liver Se concentration (DM basis) at d 0 and d 365 of beef cows that
received different sources and forms of Se supplementation3
d0 d 365
Source of Se Supplementation
Liver Se, pg/kg
SE
Liver Se, ug/kg
SE
Control (No Se)
973b
129
642b
129
Barium Selenate1 (Deposel)
1136b
105
1240c
105
Sodium Selenite2 (Mu-Se)
946b
105
537b
105
Free Choice Mineral3 (selenite)
1089b
105
1046c
111
Free Choice Mineral4 (Sel-Plex)
101 lb
105
1604d
105
aData represent least squares means and standard errors; n = 42 and 41 for d 0 and d 365, respectively;
Adequate Se concentration in liver is > 1200 pg/kg.
b c'dMeans within columns lacking a common superscript differ (P < 0.05).
'Cows received a subcutaneous injection of 9 mL Deposel at d 0.
2Cows received an injection of 5 mL Mu-Se every 4 mo beginning at d 0.
3Cows consumed free-choice mineral mix containing 26 mg Se/kg as sodium selenite at an avg of 41.5
g-cowl-d'1 beginning at d 0.
4Cows consumed free-choice mineral mix containing 26 mg Se/kg as Se yeast at an avg of 85.5 g-cow'
'd'1 beginning at d 0.


35
Ewes consuming 12 or 20 mg/kg Se had higher (P < 0.05) brain Se than controls and
ewes consuming 20 mg/kg Se had higher (P < 0.05) brain Se than ewes consuming Se at
all levels except 12 mg/kg.
Diaphragm Se ranged from 1.27 to 4.01 mg/kg DM increased (P < 0.05) in a
linear manner as dietary Se was increased. (Figure 3-1). Regressing diaphragm Se
(mg/kg DM) on dietary Se concentration (mg/kg) produced the following relationship:
Diaphragm Se = 1.27 + 1.33 Dietary Se (r2 = 0.63; P < 0.05).
Ewes receiving 20 mg/kg Se had higher (P < 0.05) diaphragm Se than ewes receiving all
other treatments and only ewes receiving 12 or 20 mg/kg Se had higher diaphragm Se
than controls (P < 0.005).
Heart tissue Se (Figure 3-1) ranged from 1.83 to 6.24 mg/kg DM and increased in
a linear fashion (P < 0.001). Regressing heart Se (mg/kg DM) on dietary Se
concentration (mg/kg) produced the following relationship:
Heart Se = 1.83 + 1.99 Dietary Se (r2 = 0.70; P < 0.05).
Ewes receiving 12, 16, and 20 mg/kg Se had higher (P < 0.05) heart Se than controls and
ewes receiving 4 and 8 mg/kg Se tended to have higher (P <0.12) heart Se than controls.
Heart Se concentrations from ewes receiving 20 mg/kg Se were higher (P < 0.05) than
those from ewes receiving all other dietary Se levels.
Selenium concentration in hoof ranged from 0.93 to 7.68 mg/kg DM and
increased cubically as dietary Se increased (Figure 3-2). Regressing hoof Se (mg/kg
DM) on dietary Se concentration (mg/kg) produced the following relationship:
Hoof Se = 0.93 + 1.95 Dietary Se 0.49 Dietary Se2 + 0.06 Dietary Se3 (r2 = 0.60;
P < 0.05).


34
in our study suggest that Se in wool and hair does not continue to increase linearly as
dietary Se is increased above 10 mg/kg. At wk 12, only ewes receiving 20 mg/kg Se had
wool Se higher (P < 0.05) than controls, however wool Se from ewes receiving 12 and 16
mg/kg Se tended (P <0.15) to be higher than from controls. At wk 24, wool Se from
ewes receiving 16 or 20 mg/kg Se was higher than from controls and ewes receiving 4
mg/kg Se. Wool Se from ewes receiving 8 or 12 mg/kg Se tended (P < 0.07) to be higher
than wool Se from ewes receiving 4 mg/kg Se. At wk 36, wool Se from ewes on all
treatment groups was higher (P < 0.05) than from controls, and Se concentrations in wool
from ewes receiving 16 mg/kg Se were higher (P < 0.05) than wool Se from ewes
receiving 4 or 12 mg/kg dietary Se. Wool Se concentrations from ewes on all treatment
groups were similar (P >0.15) and higher (P < 0.05) than wool Se from controls at wk
48. At wk 60, again, wool Se concentrations from ewes on all treatment groups were
higher (P < 0.05) than wool Se from controls. At the termination of the experiment, wool
Se from ewes receiving 20 mg/kg Se was higher than from ewes on all other treatments
and ewes from all treatment groups produced higher (P < 0.05) wool Se than did controls.
Some wool loss was observed in two ewes receiving 20 mg/kg dietary Se during lactation
in yr one. However, after lambs were weaned and lactation had ceased, both ewes regrew
a full fleece.
Tissues
Selenium concentrations in all tissues were affected (P < 0.001) by dietary Se
level. Selenium concentrations in brain ranged from 1.90 to 6.45 mg/kg DM and
increased linearly (P < 0.05) as dietary Se increased (Figure 3-1). Regressing brain Se
(mg/kg DM) on dietary Se concentration (mg/kg) produced the following relationship:
Brain Se = 1.89 + 1.56 Dietary Se (r2 = 0.52; P < 0.05).


83
toxicosis, a pattern of clinical signs of Se toxicosis was not observed in this experiment.
Microscopic evaluation of liver, kidney, diaphragm, heart, and psoas major muscle did
not reveal definitive evidence of Se toxicosis in wethers on any dietary Se treatment.
Wethers under our experimental conditions tolerated up to 40 mg/kg dietary Se for 60
wk, though differences in Se source were observed. Contrary to previous thought, the
range between optimal and toxic dietary level of Se is not narrow. The maximum
tolerable level of dietary Se, regardless of source, is much higher than the current
estimate of 2 mg/kg.