Vitamin E status in swine as affected by form or level of dietary vitamin E and/or by supplementation of vitamin A

MISSING IMAGE

Material Information

Title:
Vitamin E status in swine as affected by form or level of dietary vitamin E and/or by supplementation of vitamin A
Physical Description:
ix, 71 leaves : ill. ; 29 cm.
Language:
English
Creator:
Anderson, Lee E., 1944-
Publication Date:

Subjects

Subjects / Keywords:
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1993.
Bibliography:
Includes bibliographical references (leaves 66-70).
Statement of Responsibility:
by Lee E. Anderson.
General Note:
Typescript.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 001941170
oclc - 30991190
notis - AKB7343
System ID:
AA00004732:00001

Full Text











VITAMIN E STATUS IN SWINE AS AFFECTED BY FORM OR LEVEL OF
DIETARY VITAMIN E AND/OR BY SUPPLEMENTATION OF VITAMIN A















By

LEE E. ANDERSON, SR.


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

1993


UNIVERSITY OF FLORIDA LIBRARIES

































This dissertation is dedicated to my wife, Erma, and
children, Valerie, Lee Jr., Jenaya, Calvin, and Kelvin, and
to the memory of my parents, Florence Anderson, Myrtis
Stepherson and Leon Anderson, for their love, support and
encouragement.














ACKNOWLEDGMENTS


The author sincerely appreciates the efforts of all

members of his supervisory committee. Dr. Robert 0. Myer is

greatly appreciated for his understanding, guidance, advice

and companionship over the past several years. Sincere

thanks go to Dr. Joel Brendemuhl for his help and guidance

and his expertise and assistance during data collection.

The author is very appreciative of Dr. Lee McDowell's help

and advice, especially his assistance with acquiring the

vitamins. The advice, assistance and encouragement of Dr.

Jimmy Cheek and Dr. Joseph Conrad are deeply appreciated.

Thanks go to Dr. George Combs for his assistance. Without

the support of the above-mentioned individuals, this

dissertation would not have been possible.

Special gratitude is kindly extended to Dr. Jack Fry,

Dr. Roderick McDavis and Dr. Charles Kidd for their

encouragement and assistance during the course of this

program. Thanks are extended to Mr. Stephen Linda for the

valuable assistance he has given in the statistical analysis

of the data collected.

To his fellow graduate students, Maria Soler, Kim and

Kelly Sheppard, Dave Davis and Ken Mooney, the author

extends his thanks for their contributions and sacrifices in

iii








assisting him in this research. Special appreciation is

extended to Maria Soler and Kim Sheppard for their time and

involvement in trial 3.

Deep appreciation is extended to Alvin Boning for his

companionship and enthusiastic assistance in the laboratory.

Thanks go to Nancy Wilkinson for her assistance in the

laboratory. The author would like to take this opportunity

to thank Dane Bernis, Tom Crawford, Dennis Perry, Jack

Stokes, Dean Glicco and Rome Williams for their involvement

in the preparation of diets, care and management of the

experimental animals. Special appreciation is extended to

Leroy Washington, Larry Eubanks and Art Rogers for their

assistance in collecting tissue samples.

The author thanks Dr. Scot Williams and Hoffmann-La

Roche for providing the vitamins used in trials 2 and 3.

A special thanks is also extended to Mary Chambliss for

typing the manuscripts for review and publication.
















TABLE OF CONTENTS


Page

ACKNOWLEDGMENTS.... ............................ ... iii

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

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

CHAPTERS

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

2 POTENCY OF VARIOUS VITAMIN E COMPOUNDS FOR
FINISHING SWINE ................................ 9

Introduction................................ .... 9
Experimental Procedures....................... 10
Results and Discussion......................... 14
Summary........................................ 25

3 THE EFFECT OF EXCESSIVE DIETARY VITAMIN A ON
PERFORMANCE AND VITAMIN E STATUS IN SWINE FED
DIETS VARYING IN DIETARY VITAMIN E............. 27

Introduction .................................. 27
Experimental Procedures......................... 28
Results and Discussion.......................... 32
Summary........................................ 42

4 EFFECT OF INJECTED VITAMIN A AND DIETARY
SUPPLEMENTATION OF VITAMIN E ON REPRODUCTIVE
PERFORMANCE AND TOCOPHEROL STATUS IN GESTATING
GILTS ........................................... 44

Introduction............................... .... 44
Experimental Procedures........................ 45
Results and Discussion......................... 48
Summary............................................. 59

5 GENERAL CONCLUSIONS........................... 63

REFERENCES.......................................... 66

BIOGRAPHICAL SKETCH......................... ........ 71














LIST OF TABLES


Table Page
2-1 Composition of finisher diet...................... 11

2-2 Performance of finishing pigs fed diets containing
various vitamin E compounds...................... 15

2-3 Vitamin E (a-tocopherol) concentrations in feed... 17

2-4 Adjusted serum vitamin E (tocopherol) concentrations
in finishing swine fed diets supplemented with
different vitamin E compounds..................... 19

2-5 Relative biopotency of vitamin E compounds (%).... 22

2-6 Adjusted tissue a-tocopherol concentrations in
finishing swine fed diets supplemented with
different vitamin E compounds..................... 23

3-1 Composition of diets (%) ......................... 29

3-2 Performance of growing-finishing swine fed diets
with different dietary levels of vitamin E
and vitamin A..................................... 33

3-3 Mean a-tocopherol concentrations in blood serum
due to dietary additions of vitamins E and A...... 34

3-4 Main means of serum a-tocopherol due to dietary
additions of vitamins E and A.................... 35

3-5 Main means of serum retinol due to dietary additions
of vitamins E and A............................... 38

3-6 Main means of tissue a-tocopherol concentrations due
to dietary additions of vitamins E and A.......... 40

3-7 Main means of tissue retinol concentrations due to
dietary additions of vitamins E and A............. 41

4-1 Composition of diet fed to gestating gilts........ 46








4-2 Mean reproductive response criteria of gestating
gilts given dietary additions of vitamin E and
injected with vitamin A.......................... 49

4-3 Mean serum a-tocopherol concentrations in gestating
gilts given dietary additions of vitamin E and
injected with vitamin A............................ 52

4-4 Main mean serum a-tocopherol concentrations in
gestating gilts given dietary additions of vitamin
E and injected with vitamin A..................... 53

4-5 Mean serum retinol concentrations in gestating gilts
given dietary additions of vitamin E and injected
with vitamin A.................................... 54

4-6 Main mean serum retinol concentrations in gestating
gilts given dietary additions of vitamin E and
injected with vitamin A........................... 55

4-7 Mean tissue a-tocopherol concentrations of gestating
gilts given dietary additions of vitamin E and
injected with vitamin A.......................... 57

4-8 Main mean tissue a-tocopherol concentrations in
gestating gilts given dietary additions of vitamin E
and injected with vitamin A....................... 60


vii














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

VITAMIN E STATUS IN SWINE AS AFFECTED BY FORM OR LEVEL OF
DIETARY VITAMIN E AND/OR BY SUPPLEMENTATION OF VITAMIN A

By

Lee E. Anderson, Sr.

August, 1993

Chairman: R. O. Myer
Major Department: Animal Science

Experiment one used 40 finishing pigs (80 kg) to

determine the potency of vitamin E compounds. Pigs were

divided among five nutritionally adequate diets supplemented

with DL-a-tocopherol, DL-a-tocopheryl acetate, D-a-

tocopherol, D-a-tocopheryl acetate or no vitamin E. Blood

and tissue samples were collected. Vitamin E forms

increased (P < .05) serum a-tocopherol concentrations by d 2

of the feeding period. Serum tocopherol in pigs fed acetate

forms remained elevated through out the study; serum

concentrations declined (P < .01) in pigs fed alcohol forms.

D-a-tocopheryl acetate resulted in highest serum and tissue

tocopherol. The potency of D-acetate form was greater for

swine than that predicted from bioassays with the rat.

Experiment two evaluated excessive dietary vitamin A on

vitamin E status and performance of growing-finishing pigs.


viii








Eighty-four pigs were fed corn-soybean meal based diets

supplemented with DL-a-tocopheryl acetate to provide 0, 15

or 150 IU of vitamin E/kg and with retinyl acetate to

provide 2,000 or 20,000 IU of vitamin A/kg of diet. Serum

and tissue tocopherol increased (P < .01) as dietary levels

of vitamin E increased. The data indicated that 20,000 IU

of vitamin A/kg of feed did not affect performance or serum

and tissue tocopherol.

In experiment three, 32 gilts were used to determine

the effects of vitamins A and E on reproductive performance

and on serum and tissue concentrations of vitamin E during

early gestation. Treatments consisted of corn-soybean meal

based diets supplemented with DL-a-tocopheryl acetate to

provide either 25 or 500 IU of vitamin E/kg of diet,

beginning d -7 prebreeding through d 25 of gestation. Half

of the gilts were injected with 350,000 IU of vitamin A

(retinyl palmitate) at d -7, again at breeding (d 0), and at

d 7 postbreeding. Reproductive performance was not affected

by treatment. Serum tocopherol increased (P < .01) with 500

IU of vitamin E. High (500 IU/kg) dietary vitamin E

increased tocopherol level (P < .01) in all tissues except

adipose. High vitamin A (350,000 IU) via injections had no

consistent effect on reproductive performance or on serum or

tissue concentrations of a-tocopherol or retinol.














CHAPTER 1
INTRODUCTION


Vitamin E was discovered in 1922 as a missing, needed

dietary factor (Brandner, 1971; Ullrey, 1981; Raacke, 1983;

McDowell, 1989). Vitamin E was isolated as alpha-

tocopherol. The name tocopherol means to bring forth

offspring (McDowell, 1989). George M. Calhoun, a professor

of Greek at the University of California, Berkeley, named

the new vitamin tocopherol in 1936 (tocos for childbirth,

phero to confer, and ol for alcohol) (Evans, 1962; Ullrey,

1981; Raacke, 1983).

It was recognized in 1920 that reproductive failure

occurred in rats consuming diets thought to be nutritionally

adequate. An unknown dietary factor, then called X and

later determined to be vitamin E, was deficient, which

resulted in fetal death and embryo resorption in the

laboratory rat (Evans, 1962; Mason, 1980; Diplock, 1985;

McDowell, 1989). Estrus and mating were normal, but fetuses

died and were resorbed unless the diet was supplemented with

small amounts of wheat germ, dried alfalfa leaves, or fresh

lettuce, which contained the deficient vitamin E (Evans,

1962; Mason, 1980; McDowell, 1989). Degeneration of the

germinal epithelium in male rats was prevented by








2

supplements of fresh lettuce (Mason, 1980). Other animal

species (cattle, sheep, mink, and chickens) were able to

reproduce without dietary vitamin E, but in each case their

offspring died prematurely (Brandner, 1971).

Vitamin E became known as the fertility vitamin.

Many studies were done to determine if vitamin E affected

reproduction in humans. In most cases vitamin E had little

or no effect (McDowell, 1989).

Vitamin E deficiency in swine results in reduced

reproductive efficiency, locomotor incoordination, muscular

and hepatic necrosis, fibrinoid degeneration of blood vessel

walls and muscular dystrophy (McDowell, 1977).

Vitamin E is a hydrophobic, peroxyl radical-trapping,

chain-breaking antioxidant found in the lipid fraction of

living organisms. Its principal function is to protect the

lipid material of an organism from oxidation (Machlin, 1980;

Burton et al., 1983; Diplock, 1985; McDowell, 1989; Coelho,

1991). Lipid peroxidation of membranes of cells and

cellular constituents can be very damaging. Damage may be

as simple as breaking a membrane and allowing leakage of

contents, or as complex as breaking a membrane containing

destructive enzyme systems. Hemolysis of red blood cells is

an example of relatively simple membrane breakage. Membrane

damage to lysosomes can be particularly devastating.

Lysosomes are sometimes called the "suicide bags" of the

cell, and when their membranes are broken they release








3

enzymes that hydrolyze tissue constituents and magnify

tissue damage (Tappel, 1962). Damage to the membrane of

such other cellular components as mitochondria and

microsomes, which contain 25 and 40 % unsaturated lipid,

respectively, have profound effects. In both microsomes and

mitochondria, vitamin E is the only known lipid antioxidant

(Tappel, 1962).

Selenium (Se) is a trace mineral that is known to spare

some of the requirement for vitamin E. Selenium is a

component of the enzyme glutathione peroxidase, which is a

selenoprotein containing four atoms of selenium per molecule

of protein (Scott, 1969; Draper, 1980). Glutathione is a

hydrogen donor. Vitamin E functions as a fat soluble

antioxidant, and selenium functions as a water soluble

antioxidant (Cunha, 1977). Vitamin E is the first line of

defense against peroxidation of fats in cells. If peroxides

are formed, selenium through the enzyme glutathione

peroxidase destroys the peroxides before tissue damage can

occur. Thus, selenium is considered the second line of

defense (Diplock, 1985; McDowell, 1989) and as a result,

both selenium and vitamin E are capable of preventing some

of the same nutritional diseases (McDowell, 1989). Vitamin

E can also reduce the selenium requirement by inhibiting

production of peroxides.

Pigs exhibiting clinical vitamin E and Se deficiency

signs have a pale, white discoloration of the skeletal









4

muscle, an enlarged, friable heart, associated with

hydropericardium, and sometimes intestinal edema and

hepatosis dietetica (Mahan and Moxon, 1980).

There are many factors that affect the bioavailability

of vitamin E. These include the form of vitamin E compound,

potency of compound, stability, absorption, other fat

soluble vitamins (e.g., retinol), mineral interactions, and

unsaturated fat. Bioavailability is defined as the

percentage of a drug or nutrient (in this case, vitamin E)

that enters the systemic circulation after administration

and the rate of entry into the general circulation for

distribution throughout the body as well as tissue

accumulation (Koch-Weser, 1974; "The American Heritage

Dictionary", 1982).

Eight forms of vitamin E are known to occur in nature,

four of which are referred to as tocopherols and four as

tocotrienols. They have been given Greek letter names to

distinguish them from one another (Diplock, 1985; NRC,

1988). The compounds differ in the placement of methyl

groups on the ring and the degree of saturation in the side

chain (McDowell, 1989).

Alpha-tocopherol is a yellow oil, soluble in certain

organic solvents. It is common practice to assay only this

isomer rather than all eight compounds because a-tocopherol

is the most biologically active, naturally occurring vitamin

E source (Ullrey, 1981).








5

DL-a-tocopherol has a potency of 1.1 IU/mg and its

acetate (DL-a-tocopheryl acetate) has a potency of 1 IU/mg

as determined by bioassays with rats. Activity of naturally

occurring a-tocopherol, D-a-tocopherol (also called RRR-

tocopherol) is 1.49 IU/mg and of its acetate, 1.36 IU/mg.

D-a-tocopherol is the most biologically active form (IU per

unit of weight; NRC, 1988).

Loss of vitamin E potency occurs in mixed feed from a

number of factors. The naturally occurring tocopherols have

relatively poor stability during processing, grinding,

pelleting, and storing at high temperatures or under moist

conditions. Vitamin E will also readily interact with other

ingredients in feed formulations (Adams, 1978; NRC, 1988).

More pigs are being raised in confinement without

access to pasture, which is an excellent source of vitamin

E. Heating and pelleting feed grains lower their vitamin E

values. The use of high moisture grain increases the need

for vitamin E supplementation due to the destruction of the

vitamin. Feeds formulated with fats containing high

quantities of unsaturated fatty acids are susceptible to

rancidity, which destroys vitamin E (Cunha, 1977). Malm et

al. (1976) reported that diets high in polyunsaturated fatty

acids increased vitamin E requirement and that pigs fed a

polyunsaturated fatty acid, low vitamin E diet throughout

the postweaning period resulted in some degree of red blood

cell destruction.








6

The alcohol form, a-tocopherol, is easily destroyed by

oxidation. Oxidative destruction of a-tocopherol is

accelerated by heat, light, moisture, unsaturated fats,

sulfates, nitrates and molds, and in diets containing

increased levels of copper, iron, zinc and manganese

(Ullrey, 1981; McDowell, 1989; Dove and Ewan, 1991; Mahan,

1991; Thompson, 1993). A more stable source of vitamin E is

a-tocopheryl acetate. Alpha-tocopheryl acetate is

chemically synthesized by esterification of a-tocopherol

with acetic acid. DL-a-tocopheryl acetate is the

international standard for vitamin E activity.

Vitamin E is fat soluble and as such its absorption is

associated with that of lipids. Vitamin E is absorbed in

the alcohol form. Vitamin E acetate is hydrolyzed to the

alcohol form in the small intestine prior to absorption.

Droplets of triglycerides are degraded by lipase and bile

into monoglycerides and free fatty acids, which form into

micelles. Micelles contain the lipid components including

the fat soluble vitamins. Vitamins are absorbed with the

fatty acids and monoglycerides. Triglycerides are re-formed

in the intestinal cell and packaged into chylomicrons.

Chylomicrons are absorbed into the lacteal ducts and carried

into the lymphatic system until they enter the general

circulation and are distributed to various tissues.

Factors interfering with digestion and absorption of lipid

affect the bioavailability of vitamin E.








7

Competition for absorption sites in the small

intestines among the fat soluble vitamins may affect

bioavailability of vitamin E. Vitamin A (retinol) may

interfere with both absorption and blood concentrations of

vitamin E. This has been demonstrated in chicks (Sklan and

Donoghue, 1982; Abawi and Sullivan, 1989) and rats (Blakely

et al., 1991). This effect appears to be due to increased

oxidation of vitamin E prior to the digesta reaching the

duodenum. This would result in vitamin E concentration

being lower at the major absorption sites in the upper small

intestine (Sklan and Donoghue, 1982). In this case, vitamin

E is oxidized at the expense of vitamin A. Erdman et al.

(1988) reported that vitamin E may protect vitamin A from

oxidation in the gastrointestinal tract and within cell

membranes. Reports that vitamin A toxicity in chicks has

been completely reversed with high dietary vitamin E

supplementation (Arnrich and Arthur, 1980) also indicate an

additional loss of vitamin E resulting in an increased need.

Young children, who were vitamin A deficient, absorbed more

vitamin A when given high supplemental levels of vitamin E

(Kusin et al., 1974) indicating that vitamin A may also

affect the availability of vitamin E. However, there is

very little or no research regarding the influence of

vitamin A on the vitamin E status of swine.

Recently there has been an interest in increasing

supplemental vitamin A levels via i.m. injections in








8

gestating gilts and sows. Extra vitamin A given just

before, during, and shortly after breeding has been reported

to improve reproductive performance in breeding swine (Brief

and Chew, 1985; Coffey and Britt, 1993). The elevation of

maternal plasma vitamin A is believed to improve embryonic

survival (NRC, 1988). The elevated vitamin A may also

affect bioavailability of vitamin E and/or its requirement.

Selection for increased growth rate and reproductive

performance increases dietary vitamin E requirements. In

addition, confinement rearing and feeding cereal-soybean

meal diets that vary considerably in vitamin E content, make

it important to insure that adequate levels of nutrients are

included in the diet. Fortification of diets adequately

supplemented with vitamins is extremely important in

optimizing performance under current production conditions.

More research is necessary to ascertain the significant

aspects of vitamin E and its enhancement or impediment on

performance under conventional swine production systems.

Therefore the focus of this manuscript is on the

bioavailability of vitamin E as affected by the type of

vitamin E compound fed, and the influence of vitamin A

supplementation on vitamin E status of growing-finishing

pigs and during early gestation of gilts.














CHAPTER 2
POTENCY OF VARIOUS VITAMIN E COMPOUNDS FOR FINISHING SWINE


Introduction


Vitamin E is an essential nutrient for normal growth,

health and reproduction in swine. Vitamin E requirement for

swine ranges between 10 and 22 IU/kg of diet (NRC, 1988).

Swine diets consisting mainly of corn and soybean meal

usually do not contain adequate amounts of vitamin E needed

to meet the pig's requirement (NRC, 1988). In addition, the

stability of all naturally occurring vitamin E forms are

very poor in mixed feed (Ullrey, 1981; Dove and Ewan, 1991;

Hidiroglou et al., 1992). Therefore, supplementation of

swine diets with a readily available form of vitamin E

assures that swine will receive the correct amount for

optimum performance.

Eight forms of vitamin E occur in nature (4

tocopherols, 4 tocotrienols). D-a-tocopherol has the

greatest biological activity (highest IU per unit weight;

NRC, 1988) but acetate and succinate forms are more stable

(Erdman et al., 1988). This experiment evaluated the

relative biopotencies of four forms of vitamin E (DL-a-

tocopheryl acetate, D-a-tocopheryl acetate, DL-a-tocopherol,

and D-a-tocopherol) when supplemented in the diet of

9








10

finishing swine. The concentration of a-tocopherol in blood

serum and tissue was used as an indicator of potency.


Experimental Procedures


Forty crossbred finishing pigs, 20 barrows and 20

gilts, with an average initial weight of 80 kg were randomly

assigned by sex to individual pens. Treatments were

randomly assigned to the pens such that each treatment

consisted of 8 pigs (4 barrows, 4 gilts). Treatments

consisted of the following supplemented vitamin E forms: DL-

a-tocopherol, DL-a-tocopheryl acetate, D-a-tocopherol, and

D-a-tocopheryl acetate. A negative control, which received

no supplemental vitamin E, was also included to give a fifth

treatment. Vitamin E forms used were pure forms supplied in

an unprotected oil solution (Sigma Chemical Co., St. Louis,

MO). Supplemental vitamin E was added to the diets such

that pigs consuming 3.2 kg of feed would received 200 IU per

day. Pigs were fed a corn-soybean meal finishing diet

formulated with a modified vitamin premix (exclusive of

vitamin E) and .1 ppm of added selenium. Diets were

otherwise formulated following NRC (1988) guidelines.

Composition of the corn-soybean meal basal diet is given in

Table 2-1. The pigs were fed the finisher diet for 28 days.

Prior to the start of this trial all pigs were fed a diet

that contained 22 IU of vitamin E/kg of diet. Feed and

water were available ad libitum throughout the experiment.











Table 2-1. Composition of finisher diet


Ingredient %, as fed



Ground corn 82.05
Soybean meal (48%) 15.00
Dynafos 1.70
Ground limestone 0.80
Salt 0.25
Trace mineral 0.10
Vitamin mixb 0.05
Se premix' 0.05



aProvided 200 ppm zinc, 100 ppm iron, 55 ppm manganese,
11 ppm copper, and 1.5 ppm iodine.

bProvided 2.2 mg riboflavin, 11 mg niacin, 9 mg
pantothenic acid, 150 mg choline chloride, 11 ug vitamin
B,2, 1.5 mg vitamin K, 2750 IU vitamin A, and 440 IU
vitamin D3 per kg of diet.


cProvided 0.1 ppm selenium.








12

Pigs were housed in an open-sided building with solid

concrete floors. Individual pig weights and feed

consumption were recorded biweekly. The trial was carried

out in the spring (April-May) of the year. Pigs were

managed according to acceptable management practices

throughout the experiment. Protocol for animal care had

been approved by the University Animal Use Committee.

Blood samples were collected by jugular vein puncture

from each pig on d 0, 1, 2, 7, 14, 21, and 28 of the feeding

period. Blood samples were centrifuged after collection,

and serum was frozen and stored at -200C until analyzed.

Feed samples were taken from the feeders on d 0, 5, 14 and

21, frozen and stored at -200C until analyzed for a-

tocopherol concentration. On d 29, the 20 barrows were

slaughtered, using accepted slaughter procedures, at the

University of Florida meats laboratory and tissue samples

collected. Tissue samples included liver, muscle

(rhomboideus and semimembranosus), back fat (10th rib area)

and leaf fat. Tissue samples were frozen following

collection and stored at -200C until analyzed.

Procedures used for the extraction and determination of

a-tocopherol in blood serum were as previously described

(Njeru et al., 1992). Procedures were similar to those used

by McMurray and Blanchflower (1979a,b) except in our study,

propanol was used in the serum extraction instead of

ethanol. Most of the vitamin E activity in serum and tissue








13

was assumed to be a-tocopherol (Ullrey, 1981). Extraction

of vitamin E from tissues and feed was done using a

procedure outlined by Chung et al. (1992). This procedure

was a modification of that of McMurray and Blanchflower

(1979b) and Hatam and Kayden (1979).

Alpha-tocopherol concentration was determined using 50

ul of the reconstituted sample (serum, tissue or feed)

injected onto a LiChrosorb SI 60 column (Hibar Fertigsaule

RT pre-packed column RT 250-4 E, Merck, Darmstadt, Germany)

250 mm x 4 mm I.D. and using a Perkin Elmer 550 terminal

(Perkin-Elmer Corp. Analytical Instruments, Norwalk, CT), a

Perkin Elmer ISS-100 auto sampler, and a Perkin Elmer Series

4 Liquid chromatograph pump. The mobile phase consisted of

900:99:1 HPLC grade iso-octane, tetrahydrofuran and acetic

acid. The detector was a Perkin Elmer LS-4 Fluorescence

Spectrometer with an excitation wavelength of 290 nm and an

emission wavelength of 320 nm. Data were collected by a

Perkin Elmer LCI-100 Laboratory Computing Integrator. Flow

rate was 1 ml/min. The retention time of a-tocopherol was

5.2 minutes. Alpha-tocopherol (Eastman Kodak Company,

Rochester, NY) was used as a standard, and sample peaks and

retention times were compared to those of the standards.

Standard concentration was calculated to give a peak of 250

or 500 ng. Alpha-tocopherol concentration of samples was

calculated by the external standard method. Spiked samples

were found to have a mean recovery rate of 97%.








14

Potencies of the various vitamin E compounds were

determined by comparing areas under the time curve (AUC)

within the serum, feed and tissue samples. Serum and tissue

concentrations were adjusted based on actual feed intake and

a-tocopherol levels in the feed. Alpha-tocopherol

concentrations reported were adjusted to a constant feed a-

tocopherol concentration (d 0 feed level). Serum and tissue

means were analyzed using the general linear model procedure

(SAS, 1988). Analysis of variance compared treatment

differences in serum and tissue tocopherol concentrations.

Analysis of covariance was also applied to the serum data

using baseline (d 0 serum tocopherol) data as a covariate.

Treatment means were compared using the least significant

difference multiple comparison procedure.


Results and Discussion


Growth rate of all pigs was good over the duration of

the 28 d study. Daily feed intake and feed-to-gain ratio

were not affected (P > .1) by supplementation of the vitamin

E sources (Table 2-2); however, a slight improvement in

growth rate (P < .09) was obtained in pigs fed the D-a-

tocopherol form compared to the negative control. Asghar et

al. (1991) reported improved growth rates in growing-

finishing pigs fed dietary levels of DL-a-tocopheryl acetate

at 100 IU/kg of diet compared to pigs fed 10 IU/kg of diet.

In contrast, Chung et al. (1992) found no difference in











Table 2-2. Performance of finishing pigs fed diets containing
various vitamin E compounds


Vitamin E sourcea
Item DL-a-TAC D-a-TAC DL-a-TOH D-a-TOH Neg.
control
No. of pigs 8 8 8 8 8
ADG, kg 1.05b 1.0b 1.09bc 1.18c 1.02b
ADF, kg 3.87 3.5 3.66 3.79 3.4

F/G 3.78 3.61 3.37 3.28 3.55

"DL-a-TAC = DL-a-tocopheryl acetate; D-a-TAC = D-a-
tocopheryl acetate; DL-a-TOH = DL-a-tocopherol; D-a-TOH =
D-a-tocopherol.

"Means within the same row with a different superscript
differ significantly (P < .09).








16

growth performance due to vitamin E source (encapsulated D-

a-tocopherol or DL-a-tocopheryl acetate) or level (16, 48

and 96 IU/kg) in trials with young, starting swine.

Alpha-tocopherol analysis of the diets containing the

vitamin E forms are reported in Table 2-3. The vitamin E

forms were included in the diet so that pigs would consume

62 IU of added vitamin E/kg of feed (72 IU/kg total).

However, there was considerable variation in analyzed levels

among the dietary treatments. Also, there was some

variation in feed consumption among treatments. Therefore,

data reported were adjusted based on analysis of diets and

mean treatment group feed consumption (Table 2-3).

The indicator used to determine biopotency of the

vitamin E compounds was the concentration of a-tocopherol in

serum and selected tissues. Bratzler et al. (1950) found

that plasma tocopherol concentration reflected level of

tocopherol ingested in trials with young pigs fed different

concentrations of tocopherol. They also observed increases

of tocopherol in various tissues. Other researchers

indicated that an animal's vitamin E status can be

determined by measuring a-tocopherol concentration in serum

and various tissue after oral administration: Baker et al.

(1986) with humans, Hidiroglou and McDowell (1987) with

sheep, and Jensen et al. (1990) and Asghar et al. (1991)

with pigs. Numerous studies have shown that dietary vitamin

E compounds are effective in elevating blood tocopherol










Table 2-3. Vitamin E (a-tocopherol) concentrations in feed


Treatment Sampling daya
(vitamin E source) 0 5 14 21

--------------IU/kg-------------
DL-a-tocopheryl acetate 94(72) 70(54) 64(49) 56(43)
D-a-tocopheryl acetate 113(72) 104(66) 74(47) 72(46)
DL-a-tocopherol 94(72) 24(17) 21(16) 13(10)
D-a-tocopherol 83(72) 22(19) 19(16) 15(13)
Neg. control 10 5 5 5


'Day samples were taken after start of trial. Samples
were taken directly from feeder then frozen until analyzed.
Numbers in parenthesis represent adjusted levels adjusted
to a constant IU/kg extrapolated from d 0 levels.








18

concentration, and also that blood tocopherol concentration

increased with increasing dietary level of vitamin E

(Hidiroglou et al., 1988; Jensen et al., 1988; Behrens and

Madere, 1991; Asghar et al., 1991; Chung et al., 1992).

As expected, mean serum concentrations of tocopherol at

d 0 (baseline) were similar across all treatments (Table 2-

4). All vitamin E compounds fed in this experiment

increased (P < .01) serum tocopherol concentration. The

increase in serum tocopherol concentration was rapid. The

increase started on d 1, grew further by d 2 (P < .01), and

plateaued by d 7. Horwitt et al. (1984) noted in a study

with humans that serum a-tocopherol concentrations were

increased at 8 to 24 hr after ingestion of various vitamin E

forms. Howard et al. (1990) with pigs weaned at 28 d,

depleted of vitamin E for the next 38 d, and then fed 30 IU

of supplemental vitamin E in the form of D-a-tocopheryl

acetate or DL-a-tocopheryl acetate per kg of diet noted a

rapid increase in blood a-tocopherol. Jensen et al. (1990),

in a study with pigs, 49 d old, also observed a rapid

increase in serum tocopherol concentrations after feeding

supplemental DL-a-tocopheryl acetate. In both of the above

swine studies, the first blood samples were taken 7 d after

the start of the feeding trial.

Serum tocopherol concentrations of pigs fed both

acetate forms were maintained beyond d 7; however, levels

dropped steadily in pigs fed the alcohol forms and were











Table 2-4. Adjusted serum vitamin E (tocopherol)
concentrations in finishing swine fed diets
supplemented with different vitamin E compounds


Vitamin E source"
Day DL-a-TAC D-a-TAC DL-a-TOH D-a-TOH Neg.
control
----------------- g/ml------------------

0 .8 .8 .8 .8 .9
Slb9 1.2b 1.3b .8c
2 1.3c 1.8b 1.4c 1.6bc .8d
7 1.5c 2.2b 1.4c 1.4c .6d
14 1.4C 1.8b i. i 1.3' .4d
21 1.4c 1.8b .6d .9d .3e
28 1.4c 1.7b .5d .5d .4d

Note: Each mean is based on eight observations. Adjusted to
constant intake of 72 IU/kg diet based on d 0 feed
analyses (Table 2-3). Day 0 serum values were not
adjusted.

"DL-a-TAC = DL-a-tocopheryl acetate; D-a-TAC = D-a-
tocopheryl acetate; DL-a-TOH = DL-a-tocopherol; D-a-TOH =
D-a-tocopherol.

c"Means within the same row with a different superscript
differ (P < .01).








20

lower (P < .01) on d 21 and 28 than the acetate forms. This

drop was probably due to poor stability of the alcohol forms

in the feed (Table 2-3). Degradation of vitamin E occurs

through oxidation, and is accelerated by light, alkali, heat

and trace minerals (Ullrey, 1981; Erdman et al., 1988; Dove

and Ewan, 1991; Hidiroglou et al., 1992). In the absence of

oxygen, tocopherols are relatively heat, light, and alkali

stable (Ullrey, 1981). Stability of a-tocopherol is

increased by acylation of the compound (Ullrey, 1981).

Acetate forms of vitamin E were found to be quite stable in

feed in our study. Acetate forms of vitamin E have also

been noted to be stable compounds by other researchers

(Harris and Ludwig, 1949a,b; Ullrey, 1981; Dove and Ewan,

1991; Chung et al., 1992). In general, serum tocopherol

concentrations observed in the present study were similar to

those of other studies in which pigs were fed diets

containing similar levels of added vitamin E (Jensen et al.,

1988; Asghar et al., 1991).

While all vitamin E forms evaluated rapidly increased

serum tocopherol concentrations, there was some evidence of

a slight difference in the rate of this increase. Average

serum concentration in pigs fed the DL acetate form was not

increased (P > .05) until d 2, whereas serum concentrations

of pigs fed the other compounds were increased (P < .05) on

d 1. Horwitt et al. (1984) found that D-tocopherol raised

blood a-tocopherol concentrations faster than the D- or DL-








21

a-tocopherol acetate forms in research done with humans.

D-a-tocopheryl acetate resulted in higher serum

tocopherol levels than the DL- acetate form. Relative to

DL-a-tocopheryl acetate, the D- form of the same compound

had an average biopotency of 146% (IU basis; Table 2-5) or

199% (weight basis; 146 X 1.36). Howard et al. (1990)

determined a relative biopotency of 218% (weight basis) for

D-a-tocopheryl acetate relative to the DL- form of the same

compound in trials with growing pigs. Thus it would appear

that the D- acetate form has a higher biopotency for swine

than that determined from the traditional rat fetal-

resorption bioassays. However, Ames (1979) presented

evidence that the commonly accepted conversion value of 1.36

may be too low in trials evaluating the relative biopotency

of several vitamin E compounds using the rat fetal-

resorption assay. Both alcohol forms in our study exhibited

similar biopotencies, and there was evidence during the

early portion of the study that these forms were slightly

more biopotent than DL-a-tocopherol acetate (Table 2-5).

Alpha-tocopherol in tissues in general followed a

similar pattern to that observed with serum (Table 2-6).

Overall, pigs fed any of the compounds had tissue tocopherol

concentrations higher than the negative control. Other

researchers have also found that adding vitamin E to the

diet increased tissue concentrations (Bratzler et al., 1950;

Jensen et al., 1990; Asghar et al., 1991).









22

Table 2-5. Relative biopotency of vitamin E compounds (%)


day day day day day day
Compound 1 2 7 14 21 28 Avg.

D-a-TAC 154 146 151 151 141 132 146
DL-a-TOH 122 110 123 124 32 83 99
D-a-TOH 124 114 118 140 46 73 103

Note: Based on serum values. DL-a-tocopheryl acetate = 100;
IU basis.






























C 4
a)

0 4-4
-H 4-4

CU
41 *H
(0 l






00a
4-
C 41


0 -0
5
4-1
r-1 C

0 0)

4 r-4
a
o o
u 0

0 -
o 4

0
10


'0 H0 U

I :
t3 Q) 0

4J -H 0





9 C
C -r4 C
* 4 >





(0


. .


a)
0-4



0 v

Sto

j >4
-a






* m
CU



1 : 0
UO (

>0
0 *



D
H0 Hn

Ot a)


OC 3

r. 0
4-40




S3
a ) (0



0 0)
0 U0
o rS
S-H '0



0 -3 1

a) a)





Q C0
-H -l
a)0


Q)
*P

4-)
Q)







0
0


I
0
4-i) 4

1 Q)






I
0
a






o


u
4 -
QU





C11







0
4a)





00
04

i 4-


II




U0
S-a
l




a
O


Q)
4-4
4-4
-l



4-
-1
a






*4-i
Q)
a)



04
4-1





0)
4-1
-H

(U

4-)
-H
:s

0



0 *
V
aQ)


H O

C CU


-rl

c-H
0 C
Q) 0
s *m
S U








24

Generally, acetate vitamin E forms resulted in greater

tissue tocopherol concentrations than alcohol forms, and the

difference was significant (P < .02) in the liver and

rhomboideus. The greater tissue levels reflected the

greater stability of the acetate forms in the feed over the

duration of the study. Liver tocopherol averaged 4.6 Ag/g

for the acetate forms and 2.6 Ag/g for the alcohol forms.

Between the two acetate forms, D-a-tocopheryl acetate

resulted in the same tissue tocopherol concentrations as the

DL- form, with exception of the semimembranosus in which the

D- form resulted in higher concentrations (P < .02). Tissue

tocopherol concentrations were similar for the two alcohol

forms.

Liver had the highest tocopherol concentration of all

tissues evaluated. The liver could be an indicator of

dietary vitamin E status or reflect immediate status.

Jensen et al. (1990) indicated that serum and liver a-

tocopherol concentrations reflected the short term vitamin E

status of the pig and that muscle and fat tissue

concentrations reflected the pig's long-term vitamin E

status.

The concentration of tocopherol in all tissues other

than the liver was similar (P > .10) in pigs fed the two

acetate forms, with the exception of semimembranosus as

noted above. Jensen et al. (1988), in trials with growing

pigs, also noted that the liver had the highest









25

concentration of tocopherol, followed by adipose tissue and

skeletal muscle. Also, Asghar et al. (1991), feeding

growing pigs DL-a-tocopheryl acetate at 100 IU/kg of diet,

observed higher tocopherol concentrations in the liver,

followed by the heart, lung and kidney.

In conclusion, all vitamin E compounds evaluated almost

immediately (by d 1) began to increase serum a-tocopherol

concentrations upon ingestion. Tissue a-tocopherol

concentrations were reflective of serum concentrations.

Vitamin E acetate forms fed to finishing pigs resulted in

higher serum and tissue tocopherol concentrations than the

alcohol forms due to their greater stability in mixed feed.

Among the two acetate forms evaluated, the D- form had a

greater biopotency for swine than that determined by

traditional assays.


Summary


Relative biopotencies of four chemical forms of vitamin

E supplemented in diets of finishing swine for 28 d were

evaluated. Forty crossbred pigs (80 kg), individually

penned, were divided equally among five treatments.

Treatments consisted of corn-soybean meal based diets

supplemented with DL-a-tocopherol, DL-a-tocopheryl acetate,

D-a-tocopherol or D-a-tocopheryl acetate. A treatment

without vitamin E supplementation (negative control) served

as the fifth treatment. Each compound was supplemented at








26

62 IU/kg of diet. Blood samples were collected on d 0, 1,

2, 7, 14, 21, and 28. On d 29, half of the pigs were

slaughtered to obtain tissue samples. Feed samples, taken

from feeders, were collected on d 0, 5, 14, and 21. All

vitamin E forms fed increased (P < .05) serum a-tocopherol

concentration by d 2 and the concentration peaked by d 7.

Serum tocopherol concentrations in pigs fed either acetate

form remained elevated beyond d 7; serum concentrations

steadily declined and were lower (P < .01) on d 21 and 28 in

pigs fed either alcohol form in comparison to acetate-fed

pigs. The decrease was probably a reflection of reduced

stability of the alcohol forms in the feed; the acetate

forms were found to be stable in the feed over the 28 d

study. Dietary supplementation of D-a-tocopheryl acetate

resulted in the highest serum tocopherol concentrations

throughout the study, compared to concentrations obtained

for pigs fed the other compounds. A similar trend was

observed in tissue (liver, back fat, leaf fat,

semimembranosus, rhomboideus) tocopherol concentrations as

with serum concentrations, with the liver having the highest

concentration. The biopotency of D-a-tocopheryl acetate for

swine appears to be greater than predicted from traditional

bioassays with rats.














CHAPTER 3
THE EFFECT OF EXCESSIVE DIETARY VITAMIN A ON PERFORMANCE
AND VITAMIN E STATUS IN SWINE FED DIETS
VARYING IN DIETARY VITAMIN E


Introduction


Both vitamins A and E are fat soluble vitamins. There

is evidence that high dietary vitamin A may interfere with

both vitamin E absorption and blood a-tocopherol

concentration. High dietary vitamin A reduced absorption of

a-tocopherol in trials with chicks (Sklan and Donoghue,

1982). Abawi and Sullivan (1989) noted decreased plasma

vitamin E concentration when chicks received high (100,000

IU/kg) levels of dietary vitamin A. Blakely et al. (1991)

also reported that high dietary vitamin A (100 times

requirement) plus high levels of beta carotene (480 mg/kg of

diet) reduced plasma vitamin E concentration by 77% in

rats.

Limited research is available on the effect of dietary

vitamin A on vitamin E status in pigs. Our study was done

to evaluate the effect of excessive dietary vitamin A on

performance and on serum and tissue concentrations of a-

tocopherol of growing-finishing pigs fed diets supplemented

with varying levels of vitamin E.











Experimental Procedures


Eighty-four crossbred pigs with an average initial

weight of 26 kg were divided by sex, weight and litter

origin into pens of two pigs each (1 barrow, 1 gilt). Each

pen was assigned to one of six dietary treatments within

each of seven replications. The treatments for the 2 x 3

trial consisted of a basal corn-soybean meal diet

supplemented with DL-a-tocopheryl acetate (Hoffmann-La Roche

Inc., Nutley, NJ) at levels of 0, 15, or 150 IU/kg, and

retinyl acetate (Hoffmann-La Roche Inc., Nutley, NJ) at

levels of 2,000 or 20,000 IU/kg of diet. Pigs were fed a

grower diet (Table 3-1), formulated to meet NRC (1988)

requirements (except for vitamins A and E), until they

reached an average body weight of 57 kg and continued on a

finisher diet (Table 3-1) with the same treatment until they

reached an averaged body weight of 107 kg. The vitamin

premix used did not contain vitamins A and E. Pigs were

given feed and water ad libitum. Pigs were housed in an

open-sided building in pens with solid concrete floors.

Pigs were weighed at the start of the feeding phase and

biweekly thereafter. Average daily weight gains, feed to

gain ratios, and average daily feed intakes were determined.

Prior to the start of the study the pigs were fed a nursery

diet that contained 2750 IU of added vitamin A and 22 IU

added vitamin E/kg of feed. The trial was carried out










Table 3-1. Composition of diets


Ingredient Grower Finisher



Ground corn 75.00 82.05
Soybean meal (48%) 22.00 15.00
Dynafos 1.70 1.70
Ground limestone .80 .80
Salt .25 .25
Trace mineral premix" .10 .10
Vitamin premix .10b .05c
Se premixd .05 .05



aProvided 200 ppm zinc, 100 ppm iron, 55 ppm manganese,
11 ppm copper, and 1.5 ppm iodine.

bProvided 4.4 mg riboflavin, 22 mg niacin, 18 mg
pantothenic acid, 300 mg choline chloride, 22 ug
vitamin B,2 3 mg vitamin K, and 880 IU vitamin D3 per
kg of diet.

cProvided 2.2 mg riboflavin, 11 mg niacin, 9 mg
pantothenic acid, 150 mg choline chloride, 11 ug
vitamin B,, 1.5 mg vitamin K, and 440 IU vitamin D3 per
kg of diet.


dProvided .1 ppm selenium.








30

during late spring and early summer (March through June).

Pigs were managed following acceptable care and management

practices. Protocol for animal care had been approved by

the University Animal Use Committee.

Blood samples were collected by jugular venipuncture

from each pig at the start (d 0) and on d 3, 7, 21, 35, 63,

and 77 of the feeding period thereafter. Blood samples were

shielded from direct sunlight. Blood samples were

centrifuged after collection, serum harvested, frozen and

stored at -200C until analyzed. During storage, blood

samples were covered with foil to avoid exposure to light.

Upon termination of the feeding phase, one pig (barrow)

per pen was slaughtered, following accepted slaughter

procedures, at the University of Florida meats laboratory

and tissue samples collected. Tissue samples included

liver, leg (semimembranosus) and neck (rhomboideus) muscle,

back fat (10th rib area) and leaf fat. Tissue samples were

frozen following collection and stored at -200C until

analyzed.

Vitamin E (a-tocopherol) was extracted from serum

samples using the procedure described by McMurray and

Blanchflower (1979a) with modifications described by Njeru

et al. (1992). Extraction of vitamin E from tissue and feed

samples was done using the procedure of Chung et al. (1992).

This procedure was similar to that of McMurray and

Blanchflower (1979b) and Hatam and Kayden (1979) with








31

modifications (Njeru et al., 1992). Alpha-tocopherol was

determined by injecting 20 ul of the reconstituted sample

(serum, or tissue) into an HPLC (Anderson et al., In Press).

Alpha-tocopherol concentration of samples was calculated

from the known concentration of standards. Spiked samples

of a-tocopherol were found to have a mean recovery rate of

97 3%.

Vitamin A was extracted from serum and tissues as

described by Chew et al. (1991) and Mooney (1992).

Extraction procedures were performed under dark conditions

with either yellow filtered or subdued light. Vitamin A was

assayed and determined by the method of Mooney (1992). The

only modifications were that HPLC prepared samples were

eluted using an 80:20 (vol/vol) mixture of iso-octane:

tetrahydrofuran with 1% acetic acid and retention time was

approximately 10.5 min. All trans retinol (Sigma Chemical

Co., St. Louis, MO) standards were prepared and used to

determine concentration of samples. Several liver samples

were spiked to determine the recovery rate and validate the

extraction procedure. Recovery rate of retinol was found to

be 100 4.7%.

Data collection included serum and tissue

concentrations of vitamin A and E and performance data (body

weight gain, feed-to-gain and feed intake). Tissue data

were log transformed prior to analysis to improve

homogeneity of variance. A univariate repeated measures








32

ANOVA was performed on serum data. Data were analyzed using

the GLM procedure of SAS (1988). Orthogonal polynomial

contrasts were performed to compare treatment means.


Results and Discussion


Growth performance data of the pigs in this study are

summarized in Table 3-2. Pigs grew well on all dietary

treatments (NRC, 1988). An increase in average daily gain

of pigs approached significance (P = .15) in linear fashion

as dietary vitamin E increased; feed to gain was not

affected (P > .1). Dietary vitamin A levels had no effect

on pig performance (P > .1). Hoppe et al. (1992) fed pigs

54 IU of supplemental vitamin E in combination with 5,000,

10,000, 20,000 or 40,000 IU of retinol/kg of diet and also

observed no differences in pig performance. Other

researchers have noted similar results in the chick (Sklan

and Donoghue, 1982; Abawi and Sullivan, 1989) and rat

(Blakely et al., 1991).

Serum a-tocopherol concentrations are reported in

Tables 3-3 and 3-4. Serum tocopherol concentrations were

affected by dietary a-tocopherol on all sampling days except

d 0 (Table 3-4). When no supplemental vitamin E was added

to the diet, there was a steady decline in serum tocopherol

concentration from d 0 to d 77 (Table 3-4). When the diet

was supplemented with 15 IU of a-tocopheryl acetate per kg

of diet, serum tocopherol concentrations declined (P < .05)











Table 3-2. Performance of growing-finishing swine fed diets
with different dietary levels of vitamin E and
Vitamin A


Added
Vit. E, Added vitamin A, IU/kg
IU/kg 2,000 20,000 Mean SE
-- ----------Avg. daily gain, kg-----------------

0 .91 .91 .91
15 .92 .96 .94
150 .96 .92 .95

Mean .93 .94 .01
SE .02
-------------------Avg. daily feed, kg---------------

0 3.02 3.23 3.13
15 3.10 3.11 3.11

150 3.17 3.08 3.12
Mean 3.09 3.14 .05
SE .09
--------------------Avg. feed/gain --- -----------

0 3.33 3.56 3.44
15 3.39 3.25 3.32

150 3.29 3.32 3.30

Mean 3.34 3.38 .07
SE .08

Note: Seven pens per treatment with 2 pigs per pen.

'P>F:E = .19, A = .75, E*A = .22, E linear = .15

bP>F:E = .97, A = .52, E*A = .26, E linear = .94

cP>F:E = .41, A = .67, E*A = .26, E linear = .36











Table 3-3. Mean a-tocopherol concentrations in blood serum
due to dietary additions of vitamins E and A


Vit. E and A, IU/kcg
Days 0/2b 0/20b 15/2 15/20 150/2 150/20 SE
----------------------g/ml-----------------

0 1.14 1.17 1.18 1.24 1.03 1.25 .11
3 .60 .66 .75 .75 2.75 2.04 .09
7 .55 .59 .92 1.02 3.18 2.92 .11
21 .37 .53 .75 .84 3.15 2.99 .10
35 .39 .45 .87 .78 3.18 2.99 .09
63 .31 .32 .99 .78 3.29 3.24 .08
77 .33 .31 .98 .70 2.84 3.02 .11

Note: Each mean is based on 14 observations.

aVitamin E linear effect P < .01 all days except d 0.
E x A P < .01 d 3 and 21 only.


b2 = 2,000; 20 = 20,000 IU of retinyl acetate.











Table 3-4. Main means of serum a-tocopherol due to dietary
additions of vitamins E and A


Vit. Ea, IU/kg Vit. Ab, IU/kg
Days 0 15 150 SE 12,000 20,000 SE
------------------ig/ml----------------------

0 1.15 1.21 1.14 .07 1.11 1.22 .06
3 .63 .75 2.40 .06 1.37 1.15 .05
7 .57 .97 3.05 .08 1.55 1.51 .06
21 .45 .79 2.92 .07 1.42 1.35 .06
35 .41 .83 3.08 .06 1.48 1.40 .05
63 .31 .88 3.26 .05 1.53 1.45 .04
77 .31 .84 2.93 .08 1.38 1.34 .06

Note: Each mean is based on 28 or 42 observations.

"Vitamin E linear effect P < .01 all days except d 0; E
quadratic P < .02, d 63 and 77 only.

bVitamin A effect P < .01, d 3 only.








36

from their initial concentration by d 3 and then stabilized

throughout the remainder of the trial (Table 3-4). Serum

tocopherol concentration increased (P < .01) with the

highest vitamin E supplementation level by d 3 and continued

to increase (P < .01) to d 7 after which serum concentration

was maintained throughout the study (Table 3-4). Serum

tocopherol was highest (P < .01) at the highest level of

vitamin E supplementation on all days except d 0. Overall,

as dietary levels of supplemental vitamin E increased, serum

concentration of tocopherol also increased (linear; P <

.01). Other studies have shown dietary vitamin E compounds

are effective in increasing blood tocopherol and that blood

concentrations increased with increasing dietary vitamin E

(Jensen et al., 1988; Asghar et al., 1991 and Anderson et

al., In Press).

Serum tocopherol concentrations due to supplementing

the diet with vitamin A at 2,000 or 20,000 IU/kg of diet are

summarized in Table 3-4. Pigs fed the low level of vitamin

A tended to have higher concentrations of serum tocopherol

than pigs fed the high level of vitamin A on all blood

sampling days, but was significant only on d 3 (P < .01).

Among the three dietary levels of vitamin E, the highest

dietary vitamin E tended to be decreased by the high dietary

vitamin A to the greatest extent, but was significantly

lower (P < .01) on d 3 and d 21 only (Table 3-3). Although

significant differences were noted above, the magnitude of










these changes was very small. Therefore vitamin A level of

20,000 IU/kg had only a minimal effect on blood tocopherol

in swine. Weaver et al. (1989), in a trial with young

starting pigs, observed that high dietary levels of vitamin

A (9,900 and 16,500 IU/kg) fed with 33 IU of added vitamin

E/kg tended to lower plasma tocopherol concentrations

slightly. In trials with chicks and rats, however, a

decrease in blood tocopherol was noted upon feeding diets

with excessive vitamin A. In these trials, very high

vitamin A concentrations (> 100,000 IU/kg of diet) were fed

(Sklan and Donoghue, 1982; Blakely et al., 1991). This

negative effect is thought to be attributed to competition

for absorption sites in the small intestine or enhanced

oxidation of tocopherol prior to tocopherol reaching the

small intestine (Sklan and Donoghue, 1982).

Although some differences were observed on individual

sampling days, supplemental vitamin A had no effect (P >

.10) on serum retinol (Table 3-5). Differences that were

noted were very small and within normal values for serum

retinol usually found in the pig (Kaneko, 1980). Blood

retinol was also not affected by dietary supplementation of

various levels of vitamin A in studies done by other

researchers (Abawi and Sullivan, 1989; Blakely et al., 1991;

Hoppe et al., 1992).

With two exceptions, there was no effect (P > .10) on

serum retinol due to supplementation of vitamin E at any of










Table 3-5. Main means of serum retinol due to dietary
additions of vitamins E and A


Vit. Ea, IU/kg Vit. Ab, IU/kg
Days 0 15 150 SE l 2,000 20,000 SE
--------------------g/ml---------------

0 .34 .34 .39 .02 .31 .41 .02
3 .31 .33 .35 .01 .30 .35 .01
7 .34 .32 .35 .01 .33 .34 .01
21 .34 .36 .35 .01 .35 .35 .01
35 .37 .34 .32 .01 .35 .34 .01
63 .39 .35 .37 .01 .38 .36 .01
77 .47 .49 .46 .02 .52 .44 .02

Note: Each mean is based on 28 or 42 observations.

'Vitamin E linear effect P < .05 on d 3 and 35 only.

bVitamin A effect P < .01 on d 3 and 77 only.








39

the dietary levels evaluated in this study. Increasing

dietary vitamin E (Table 3-5) resulted in higher (P < .02)

retinol serum concentration on d 3 in pigs fed the highest

supplemental vitamin E, but also resulted in the lowest (P <

.02) serum retinol concentration on d 35. Although both

were significant, the differences were small. Weaver et al.

(1989) in trials with growing pigs also noted that plasma

vitamin A was not affected by dietary level of vitamin E.

Tissue a-tocopherol increased (linear; P < .001) as

dietary vitamin E increased (Table 3-6). As dietary vitamin

E increased from 15 to 150 IU per kg of diet, tissue

tocopherol increased by at least a factor of 2 or more in

all tissues evaluated. This finding is in agreement with

others who have observed similar responses in pigs (Jensen

et al., 1988; Asghar et al., 1991). Among the tissues

sampled, the highest concentration of a-tocopherol due to

treatment was found in the adipose tissue followed by liver

and muscle tissue, respectively (Table 3-6).

High supplementation of vitamin A had no effect (P >

.1) on tocopherol concentration in any of the tissues

studied (Table 3-6). The retinol concentration in the liver

was greatly enhanced (P < .001) when pigs were fed the high

level of vitamin A (Table 3-7). Retinol concentration in

other tissues (back fat, leaf fat, rhomboideus and

semimembranosus) was not detected and are not reported.

Dietary vitamin E had no effect (P > .10) on liver retinol











40



>4
H W(N I N N ml
Q) .



0 o






0 0


C I
O ** IO
H0 I V (N 'c ?





it > 0

--T 0 0 ( 0 O O
0 I



O > 0 L n







U r i n M O
0 W a)






0 0
r0 (




o) I 0



L0 4-4 H O *

o H H H 4 o
0 0
41U) 4 0









10 1 4 -J c O U
M 4-1 a)
SZ0 '0 0-












0 4
* r M U) 'O I





Ul C 0
I -4 r














fl)O Q ) Q) 0 '4-4 4-4 5
Sg 4 4-4 Q)
0 r 0o u o > u

U E)-,Qi 0 V
E- 0)
'0-H Qr 'a4) 4








41

Table 3-7. Main means of tissue retinol concentrations
due to dietary additions of vitamins E and A

Vit. E, IU/kq Vit. Aa, IU/kg
Tissue 0 15 150 SE 12,000 20,000 SE
---------------------------- /--------------------

Liver 503 470 496 44 103 876 36

Note: Each mean is based on 14 or 21 observations.

aVitamin A effect P < .001.








42

concentration (Table 3-7). Hoppe et al. (1992) found that

liver retinol was linearly related to dietary retinol in a

trial involving young, growing pigs as was also observed in

the present study. They also found that 10,000 IU of

dietary retinol did not affect tissue a-tocopherol other

than the heart in which a-tocopherol was slightly depressed.

However, Pudelkiewicz et al. (1964) feeding vitamin A

depleted chicks 0, 1,453, 14,535, 1,453,488 and 14,534,883

IU of vitamin A acetate/kg of diet observed a marked decline

in tocopherol concentration in liver tissue at the highest

dietary vitamin A levels.

In conclusion, there was no consistent evidence that

excessive dietary vitamin A (20,000 IU/kg of diet) affected

growth performance or serum or tissue a-tocopherol in

growing-finishing pigs fed diets supplemented with varying

levels of vitamin E. Likewise serum retinol was not

affected by dietary vitamin E.


Summary


A 2 X 3 factorial experiment was conducted to evaluate

excessive dietary vitamin A on vitamin E status and

performance of growing-finishing pigs fed diets supplemented

with varying levels of vitamin E. Treatments consisted of

corn-soybean meal based diets supplemented with DL-a-

tocopheryl acetate to provide 0, 15 or 150 IU added vitamin

E/kg and with retinyl acetate to provide 2,000 or 20,000 IU








43

vitamin A/kg of diet. The trial involved 84 crossbred pigs

(26 kg) divided by sex, weight, and genetic background into

pens of two pigs each. Treatment was assigned at random to

pens within each of seven replications. Pigs were fed

grower diets (.75% lysine) until they reached 57 kg average

weight and were then switched to finisher diets (.60%) until

107 kg. Serum was collected on day 0, 3, 7, 21, 35, 63, and

77 of the feeding period. Tissue samples (liver, muscle,

back fat and leaf fat) were collected from one pig (barrow)

in each pen at the end of the feeding phase. Overall

average daily gain and feed-to-gain were .93 kg and 3.35

respectively, without treatment differences (P > .1).

Excessive dietary vitamin A had no effect (P > .1) on serum

retinol concentrations except d 3 in which there was a small

(P < .01) increase. Serum tocopherol was increased (P <

.01; linear) by d 3 with dietary vitamin E supplementation

and was maintained (P < .01) throughout the feeding period.

High dietary vitamin A resulted in a small but significant

(P < .01) decrease in serum tocopherol on d 3; serum

tocopherol concentrations were not affected on other days.

Tissue tocopherol was increased (P < .001; linear) as

dietary vitamin E increased from 15 to 150 IU/kg. Liver

retinol increased (P < .001) by a factor of eight. No

consistent evidence was found that high dietary vitamin A

interfered with performance or with serum or tissue

tocopherol in growing-finishing swine.















CHAPTER 4
EFFECT OF INJECTED VITAMIN A AND DIETARY SUPPLEMENTATION OF
VITAMIN E ON REPRODUCTIVE PERFORMANCE AND TOCOPHEROL STATUS
IN GESTATING GILTS


Introduction


Supplemental vitamin A and/or B-carotene given via

injection just before and/or shortly after breeding appears

to enhance reproductive performance of gilts and sows (Brief

and Chew, 1985; Coffey and Britt, 1993). There is evidence

that high dietary vitamin A may interfere with both vitamin

E absorption and blood a-tocopherol concentrations. High

dietary vitamin A reduced absorption of a-tocopherol in

trials with chicks (Sklan and Donoghue, 1982). Abawi and

Sullivan (1989) noted a decrease in plasma vitamin E

concentrations when depleted chicks were administered high

(100,000 IU/kg) levels of dietary vitamin A. Blakely et al.

(1991) also reported that high levels of vitamin A (100

times requirement) plus high levels of B-carotene (480 mg/kg

of diet) reduced vitamin E plasma concentration by 77% in

rats.

This study was conducted to evaluate the effect of

injecting vitamin A just before, during and shortly after

breeding, and dietary supplementation of vitamin E on

reproductive performance and on blood and tissue

44








45

concentrations of a-tocopherol during early gestation of

gilts.


Experimental Procedures


The trial was a 2 x 2 factorial design and involved 32

(7 to 8 month old) crossbred gilts. The gilts were divided

into pens of 8 gilts each. Gilts used in this study were

from a previous trial that involved the feeding of diets

supplemented with either 2,000 (L) or 20,000 (H) IU vitamin

A/kg of diet. This was taken into consideration in the

allotment of gilts to treatment (4L and 4H per pen). Each

pen was randomly assigned to one of four treatments.

Treatments consisted of a basal corn soybean meal diet

(Table 4-1) supplemented with DL-a-tocopheryl acetate

(Hoffmann-La Roche Inc. Nutley, NJ) at levels of either 25

or 500 IU/kg of diet. Gilts were fed experimental diets

beginning 7 d prior to breeding through d 25 of gestation.

Half of the gilts were given three injections (i.m. in the

neck) of 350,000 IU each of vitamin A (vitamin A palmitate

Hoffmann-La Roche Inc. Nutley, NJ); the other half were

injected with vehicle only. The gilts were injected at 7 d

prebreeding (d -7), at breeding (d 0) and 7 d postbreeding

(d 7). Gilts were fed 1.9 kg of feed/hd once daily and

given free access to water. Gilts were housed in an open-

sided building with solid concrete floors. Gilts were

checked twice daily for estrus during the trial and doubled











Table 4-1. Composition of diet fed to gestating gilts


Ingredient


Ground corn 84.66
Soybean meal (48%) 12.40
Dynafos 1.54
Ground limestone .65
Salt .50
Trace mineral .10
Vitamin premixb .10
Se premixc .05



'Provided 200 ppm zinc, 100 ppm iron, 55 ppm manganese,
11 ppm copper, 1.5 ppm iodine, and 1 ppm cobalt.

bProvided 4.4 mg riboflavin, 22 mg niacin, 18 mg
pantothenic acid, 300 mg choline chloride, 22 ug vitamin
B,, 3 mg vitamin K, 880 IU vitamin D3, and 4000 IU
vitamin A per kg of diet.


'Provided .1 ppm selenium.








47

mated on their second or third observed estrus to duroc x

hampshire x yorkshire boars.

Blood samples were collected by jugular vein puncture

from each gilt on d -7, 0, 7 and 24 of gestation to monitor

the serum a-tocopherol (vitamin E) and retinol (vitamin A)

concentrations. Blood samples were covered with foil to

prevent exposure to direct sunlight, taken to the

laboratory, centrifuged and the serum harvested. Serum was

stored at -200C until analyzed for a-tocopherol and retinol

concentration. During storage, serum samples were covered

with foil to prevent exposure to light.

Gilts were slaughtered, following accepted slaughter

procedures, on d 25 of gestation at the University of

Florida meats laboratory. Reproductive tracts were

immediately removed and refrigerated for later counting of

corpora lutea (CL) and recovery of embryos. Tissue samples

were also collected which consisted of endometrium, embryo,

ovary, uterus, liver, leaf fat, back fat and muscle

(semimembranosus and rhomboideus). Tissue samples were

stored at -200C until analyzed for a-tocopherol and retinol

concentrations.

The trial was carried out during the summer and early

fall (July through October). Pigs were managed following

acceptable care and management practices throughout the

study. Protocol for animal care had been approved by the

University Animal Use Committee. Three pigs from different









48

treatment groups were eliminated from the study due to

death, lameness and failure to conceive.

Alpha-tocopherol was extracted from serum and tissues

using procedures as described earlier (Anderson et al., In

Press). Alpha-tocopherol concentration was determined by

injecting 10 ul of the extracted sample (serum and tissue)

into the HPLC system. Retinol was extracted, assayed and

concentration determined from the serum and tissues by the

method as described previously (Mooney, 1992).

Experimental data included serum and tissue

concentrations of a-tocopherol and retinol, and reproductive

performance. Tissue data was log transformed prior to

analysis to improve homogeneity of variance. A univariate

repeated measures ANOVA was performed on serum data (SAS,

1988). Data were analyzed as a 2 X 2 factorial design with

the factors being dietary vitamin E level and whether or not

gilts were injected with vitamin A.


Results and Discussion


Levels of vitamin E used were chosen to reflect NRC

(1988) requirement and to give a very high level. The

injected level for vitamin A was chosen because it was

thought to be the upper limit that would elicit a response

and not be toxic to the gilts (also some evidence of this

dosage was being used).

Reproductive performance data are summarized in Table









49

Table 4-2. Mean reproductive response criteria of gestating
gilts given dietary additions of vitamin E and injected
with vitamin A


Item

No. of CL
No. of Embryob
Embryo wt., g
Ovary wt., g
Uterus wt., kg


Vit. E, IU/kg and Vit. A inji.
25/No 25/Yes 500/No 500/Yes

14 13 15 15
12 13 12 15
9 9 7 8
13 13 14 15
3 2 2 2


SE

.76
1.13
.98
.90
.22


Note: Each mean is based on 7 or 8 observations.

"Three injections of 350,000 IU each.

bEffect of vitamin E (P = .14), effect of
vitamin A (P = .13).








50

4-2. Although none of the differences noted with

reproductive data were significant (P > .1), due to the

small number of gilts per treatment and the inherent nature

of swine reproductive data, nevertheless, some positive

trends were observed due to treatment. Gilts receiving the

high vitamin E and high vitamin A treatment had larger (P =

.16) litters than gilts given other treatments. Embryonic

survival was 86% in gilts given the low dietary vitamin E

with no injected vitamin A and 80% in gilts given the high

dietary vitamin E with no injected A. Embryonic survival

was 100% in gilts injected with vitamin A and fed either the

low or high vitamin E diets. Brief and Chew (1985) reported

larger litter size and higher embryonic survival in gilts

receiving weekly injections of vitamin A (12,300 IU) and B-

carotene (33 mg) compared to gilts fed vitamin A and B-

carotene at the same levels. However, these gilts in the

study of Brief and Chew (1985) were depleted of vitamin A

and B-carotene for 5 weeks before the start of the study.

Coffey and Britt (1993) observed on average .5 pig increase

in the number of pigs born alive and higher embryonic

survival in sows given i.m. injections of vitamin A

palmitate (50,000 IU) compared to sows given vehicle only

(corn oil) on day of weaning, mating and 7 d postbreeding.

These sows were also supplemented with 11,000 IU of vitamin

A acetate per kg of diet.

Serum concentrations of a-tocopherol in gilts fed diets








51

supplemented with two levels of vitamin E with and without

injected vitamin A are reported in Tables 4-3 and 4-4.

Initial a-tocopherol serum concentrations in gilts were

similar (d -7). When dietary levels were increased from 25

to 500 IU of DL-a-tocopheryl acetate per kg of diet, serum

a-tocopherol concentrations increased (P < .01) by d 0 and

were maintained throughout the duration of the study (Table

4-4). This finding is in agreement with other studies that

have shown that dietary vitamin E compounds are effective in

increasing serum tocopherol, and that serum concentration

increased with increasing dietary vitamin E (Jensen et al.,

1988; Asghar et al., 1991; Mahan, 1991 and Anderson et al.,

In Press).

Serum a-tocopherol was not affected (P > .1) by

injecting gilts with vitamin A (retinyl palmitate) except on

d 7 (Table 4-4). On d 7, the gilts fed the low vitamin E

diet had similar (P > .1) serum tocopherol concentrations

regardless of vitamin A treatment, whereas gilts fed the

high vitamin E diet had higher (P < .08) serum tocopherol

concentrations when injected with vitamin A than gilts not

injected with vitamin A (Table 4-3).

Serum concentrations of retinol due to treatment are

shown in Tables 4-5 and 4-6. In general, there was no

consistent effect on serum retinol due to dietary vitamin E

supplementation level or injection of vitamin A. However, a

difference in serum retinol (P < .08) was observed on d 0 in











Table 4-3. Mean serum a-tocopherol concentrations in
gestating gilts given dietary additions of vitamin
E and injected with vitamin A


Vit. E, IU/kg and vit. A inj.a"'
Sampling 25/No 25/Yes 500/No 500/Yes SE
day
----------------- g/ml---------------------

-7 .8 .9 1.1 .6 .17
0 1.2 1.2 3.6 3.6 .18
7 1.1 1.1 3.4 4.0 .15
24 1.3 1.3 3.8 3.6 .15

Note: Each mean is based on 7 or 8 observations.

aThree injections of 350,000 IU each.

bEffect of vitamin E (P < .01), d 0, 7, 24.


CE x A effect (P < .08), d 7.









53

Table 4-4. Main mean serum a-tocopherol concentrations
in gestating gilts given dietary additions of
vitamin E and injected with vitamin A


Vit. E. IU/kgq Vit. A inj.b.c
Sampling 25 500 No Yes SE
day
-------------------g/ml---------------------

-7 .8 .9 .9 .8 .12
0 1.2 3.6 2.4 2.4 .13
7 1.1 3.7 2.3 2.6 .11
24 1.3 3.7 2.5 2.5 .11


Note: Each mean is based on 14, 15 or 16

aEffect of vitamin E (P < .01), d 0, 7 an

bThree injections of 350,000 IU each.

cEffect of vitamin A (P < .06), d 7 only.


observations.

d 24.









54

Table 4-5. Mean serum retinol concentrations in gestating
gilts given dietary additions of vitamin E and
injected with vitamin A


Vit. E. IU/kq" and vit. A inij.b.
Sampling 25/No 25/Yes 500/No 500/Yes SE
day
-------------------g/ml-----------------
-7 .51 .55 .52 .51 .03
0 .44 .49 .51 .59 .03
7 .40 .43 .47 .42 .03
24 .46 .47 .48 .45 .03

Note: Each mean is based on 7 or 8 observations.

aEffect of vitamin E (P < .03), d 0 only.

bThree injections of 350,000 IU each.

cEffect of vitamin A (P < .08), d 0 only.









55

Table 4-6. Main mean serum retinol concentrations in
gestating gilts given dietary additions of vitamin
E and injected with vitamin A


Vit. E, IU/kqg Vit. A inj.b.c
Sampling 25 500 No Yes SE
day
------------------p g/ml------------------

-7 .53 .52 .52 .53 .02
0 .46 .55 .47 .54 .02
7 .41 .44 .43 .42 .02
24 .46 .47 .47 .46 .01

Note: Each mean is based on 14, 15 or 16 observations.

"Vitamin E effect (P < .03) d 0 only.

bThree injections of 350,000 IU each.

cVitamin A effect (P < .08) d 0 only.








56

that serum retinol concentration was highest in gilts fed

the high vitamin E and injected with vitamin A, and lowest

in gilts fed low vitamin E without injected A. The

difference noted was very small and within normal values for

serum retinol concentrations usually found in the pig. Our

findings agree with Weaver et al. (1989) in that plasma

vitamin A concentration was not affected by dietary level of

vitamin E. Serum retinol concentrations in the chick and

rat have also been reported not to be affected by dietary

supplementation of vitamin E in studies done by Abawi and

Sullivan, (1989) and Blakely et al. (1991), respectively.

Mooney (1992) injected gilts with vitamin A palmitate

(ranging from 53,200 to 106,400 IU given once weekly), or B-

carotene (106.4 to 425.6 mg) and observed no difference in

plasma concentrations of either retinol or B-carotene. In

contrast, Brief and Chew (1985) noted increased plasma

vitamin A concentration upon injecting vitamin A, however,

gilts used in their research were depleted of vitamin A

prior to the study and they also received injected B-

carotene. Serum retinol may have been elevated with

injection of vitamin A early in our study but may have been

missed since blood samples were taken 7 d after injection.

Tissue a-tocopherol concentration in gestating gilts

increased (P < .01) as dietary supplementation of vitamin E

increased (Table 4-7) in all tissues except adipose. This

finding is in agreement with others who have observed











Table 4-7. Mean tissue a-tocopherol concentrations of
gestating gilts given dietary additions of vitamin E
and injected with vitamin A


Vit. E, IU/kg and vit. A inj.b
Tissuea 25/No 25/Yes 500/No 500/Yes SE
--g/---- -g--..-----------------g/g_

Liver 4 4 24 23 1.6
Back fat 6 8 9 9 1.5
Leaf fat 9 10 12 12 2.2
Semimembranosus 2 3 4 4 .3
Rhomboideus 3 3 7 7 .6
Endometriumd 2 1 4 5 .3
Embryo .4 .4 .8 .7 .05
Oviduct 1 1 4 3 .3
Uterus 1 1 4 4 .2
Ovary 20 19 104 90 5.6

Note: Each mean is based on 7 or 8 observations.

aEffect of vitamin E (P < .01) for all tissues except
back fat and leaf fat.

bThree injections of 350,000 IU each.

CWet tissue basis.

dEffect of vitamin A (P < .08); E x A (P < .04).








58

similar responses in the pig (Jensen et al., 1988; Asghar et

al., 1991; Mahan, 1991). Average tocopherol concentration

increased by a factor of 2 in embryos upon high dietary

supplementation indicating that tocopherol is transferred

from the dam to the developing embryo. Vitamin A injections

had no effect (P > .1) on tissue a-tocopherol concentrations

except in the endometrium where there was a vitamin E x

vitamin A interaction (P < .04). In the endometrium gilts

fed low vitamin E and injected with vitamin A had slightly

lowered tocopherol concentration, while gilts fed the high

vitamin E and injected with vitamin A had increased

tocopherol concentration over that of the non injected gilts

(Table 4-7). Vitamin A injections appear to have no effect

on the transfer of tocopherol into the developing embryos as

tocopherol concentration in the embryos was not influenced

by vitamin A injection (P > .1).

Injecting vitamin A had no effect (P > .1) on retinol

concentration in any of the tissues studied including the

liver. Retinol concentrations in tissues other than liver,

however, were very small or nonexistent. Average

concentration in the liver was 386 gg/g. Mooney (1992)

found no differences in concentration of retinol in uterine

flushings in gilts that were injected with varying levels of

vitamin A; liver retinol was not determined.

Among the tissues sampled, the highest average

concentration of a-tocopherol upon supplementation of high








59

level of vitamin E was found in the ovary, followed by

liver, adipose, rhomboideus and endometrium, respectively

(Table 4-8). Three other tissues followed (semimembranosus,

oviduct, uterus) that had similar average concentrations and

embryo tissue had the lowest a-tocopherol concentration

(units per wet tissue basis).

In conclusion, there was no consistent evidence found

in this study that injecting a relatively large amount of

vitamin A (3 injections of 350,000 IU) just before, during

and shortly after breeding, significantly improved

reproductive performance, or interfered with serum or tissue

concentrations of a-tocopherol in gestating gilts fed diets

supplemented with 25 or 500 IU of vitamin E/kg of diet.

However, tocopherol concentration was increased further in

the endometrial tissue when vitamin A was given along with

high dietary vitamin E. No evidence was found that

injections of vitamin A interfered with the transfer of a-

tocopherol to the developing embryo. Likewise, serum

retinol concentrations were not affected by treatment.


Summary


A 2 x 2 factorial experiment was conducted to determine

the effects of injecting vitamin A and feeding vitamin E on

reproductive performance and on blood serum and tissue

concentrations of tocopherol during early gestation of

gilts. Thirty-two crossbred gilts were fed corn soybean-









60

Table 4-8. Main mean tissue a-tocopherol concentrations in
gestating gilts given dietary additions of vitamin E
and injected with vitamin A


Vit. E, IU/kg Vit. A inj.b
Tissues' 25 500 No Yes SE
---------------- g/g-----------------

Liver 4 24 14 14 1.1
Back fat 7 9 7 9 1.0
Leaf fat 9 12 10 11 1.5
Semimembranosus 3 4 3 3 .2
Rhomboideus 3 7 5 5 .4
Endometriumd 1.4 4.6 2.8 3.3 .18
Embryo .4 .8 .6 .6 .03
Oviduct 1 4 3 2 .2
Uterus 1 4 3 3 .1
Ovary 19 97 61 55 3.8

Note: Each mean is based on 14, 15 or 16 observations.

aEffect of vitamin E (P < .01) for all tissues except
back fat and leaf fat.

bThree injections of 350,000 IU each.

Wet tissue basis.

dEffect of vitamin A (P < .08).








61

meal based diets supplemented with DL-a-tocopheryl acetate

to provide either 25 or 500 IU of added vitamin E/kg of

diet. Gilts were fed daily 1.9 kg/h beginning d -7

prebreeding through d 25 of gestation. Half of the gilts

were injected (i.m.) with 350,000 IU of retinol palmitate at

d -7 prebreeding, breeding (d 0) and d 7 postbreeding; the

other half were injected with vehicle only. All gilts were

double mated during their second or third estrus. Blood

samples were collected on d -7, 0, 7 and 24 of gestation.

Gilts were slaughtered on d 25 of gestation following

accepted slaughter procedures. Twenty-nine gilts conceived.

The number of corpora lutea and embryos was not affected (P

> .1) by treatment. Serum tocopherol concentrations

increased with 500 IU of vitamin E by d 0 and were stable

through d 24 of gestation (P < .01). Vitamin A injections

had no effect (P > .1) on serum tocopherol concentrations

except on d 7 when a small increase (P < .06) was noted.

High dietary vitamin E increased tocopherol concentration (P

< .01) in all tissues examined except fat. A vitamin E x

vitamin A interaction (P < .04) was noted in endometrium

tissue. Low dietary vitamin E and injections of vitamin A

slightly lowered tocopherol concentration, while high

vitamin E and vitamin A injections increased tocopherol

concentration in the endometrium. Increasing dietary

vitamin E increased serum and tissue tocopherol

concentrations. Vitamin A injections had little or no









62

effect on these concentrations during early gestation of

gilts.














CHAPTER 5
GENERAL CONCLUSIONS


Three experiments were conducted, one to determine the

bioavailability of four forms of vitamin E compounds, and

two to assess the effect of high levels of vitamin A on the

vitamin E status of growing finishing pigs or gestating

gilts.

In experiment 1 the biopotency of four forms of vitamin

E were determined. Generally, the acetate forms resulted in

greater serum and tissue concentrations of vitamin E (a-

tocopherol) than the alcohol forms, due to the greater

stability of the acetate forms that was noted in mixed feed.

Serum tocopherol increased rather rapidly when the four

compounds were fed. Dietary supplementation of D-a-

tocopheryl acetate resulted in the highest serum tocopherol

throughout the study, compared to concentrations obtained

for pigs fed the other compounds indicating a greater

biopotency (IU/mg) for swine than determined by the

traditional rat bioassay. A similar trend was observed with

tissue (liver, back fat, leaf fat, and muscle) tocopherol

concentrations as with serum concentrations, with the liver

having the highest concentration. In general, all forms

would probably be suitable dietary supplemental sources if








64

the stability of the alcohols were improved. Encapsulating

the alcohol forms to protect them from destruction would

increase their suitability for use in mixed feed.

Experiment 2 was conducted to evaluate the effect of

feeding excessive vitamin A on growth performance, and on

blood and tissue a-tocopherol (vitamin E) levels of growing-

finishing pigs. High dietary vitamin A (20,000 IU/kg of

diet) was found not to affect or have little affect on pig

performance, or on blood or tissue concentrations of a-

tocopherol. A threefold increase (P < .01) in serum

tocopherol occurred on all sampling days when dietary

supplementation increased from 15 to 150 IU/kg. Tissue

tocopherol also increased (P < .001) as dietary vitamin E

increased from 15 to 150 IU/kg. Tissue tocopherol

concentration increased (P < .001) by a factor of at least

two in all tissues evaluated. Liver retinol increased (P <

.001) eightfold with a tenfold increase in dietary vitamin

A. Even in the liver when vitamin A (retinol) concentration

was high, a-tocopherol concentration was not affected.

Thus, the form of vitamin A within the liver is not in a

form which can lead to oxidative destruction of a-tocopherol

or the concentrations encountered may not have been high

enough to affect a-tocopherol.

Experiment 3 evaluated the effect of giving a high

level of vitamin A via intramuscular injections on

reproductive performance, and on serum and tissue a-








65

tocopherol concentrations during early gestation of gilts.

High levels of vitamin A (350,000 IU per week) did not

affect reproductive performance, or serum or tissue

concentrations of a-tocopherol or retinol in this study. As

observed in the previous studies, increasing dietary levels

of vitamin E increased blood serum and tissue tocopherol

concentrations including reproductive tissues. Alpha-

tocopherol concentration also increased in the embryo when

dietary vitamin E was increased. The increased

concentration of a-tocopherol indicates a transfer of

tocopherol from the dam to the developing embryo.

Fluctuations of retinol concentrations in serum may have

been missed due to weekly sampling of blood. Blood sampling

on d 2, 3, or on a more frequent routine after vitamin A

injections would provide more answers.

Further research would be desirable to determine

vitamin E bioavailability other than by oral administration.

Fecal sample tocopherol analysis may provide more

information from which better conclusions might be drawn

concerning digestion and absorption. Studies using larger

numbers of gilts and/or sows is recommended to determine if

vitamin A injection alone or in combination with high

vitamin E, either orally or by injection, would enhance

reproductive performance.















REFERENCES


Abawi, F. G., and T. W. Sullivan. 1989. Interaction of
vitamins A, D3, E, and K in the diet of broiler chicks.
Poult. Sci. 68:1490.

Abawi, F. G., T. W. Sullivan, and S. E. Scheideler. 1985.
Interaction of dietary fat with levels of vitamin A and
E in broiler chicks. Poult. Sci. 64:1192.

Adams, C. R. 1978. Vitamin product forms for animal
feeds. In: Vitamin Nutrition Update, Seminar Series
2. Roche Publ. Nutley, NJ.

Ames, S. R. 1979. Biopotencies in rats of several forms
of alpha-tocopherol. J. Nutr. 109:2198.

Anderson, L. E.,Sr., R. O. Myer, J. H. Brendemuhl, and L.
R. McDowell. Bioavailability of various vitamin E
compounds for finishing swine. J. Anim. Sci. In press.

Arnrich, L., and V. A. Arthur. 1980. Interaction of fat-
soluble vitamins in hypervitaminoses. Ann. of N. Y.
Acad. Sci. 355:109.

Asghar, A., J. I. Gray, E. R. Miller, P. K. Ku, A. M.
Booren, and D. J. Buckley. 1991. Influence of
supranutritional vitamin E supplementation in the feed
on swine growth performance and deposition in different
tissues. J. Sci. Food Agric. 57:19.

Baker, H., G. J. Handelman, S. Short, L. J. Machlin, H.
N. Bhagavan, E. A. Dratz, and 0. Frank. 1986.
Comparison of plasma a and gamma tocopherol levels
following chronic oral administration of either all-
rac-a-tocopheryl acetate or RRR-a-tocopheryl acetate in
normal adult male subjects. Am. J. Clin. Nutr. 43:382.

Behrens, W. A., and R. Madere. 1991. Tissue discrimination
between dietary RRR-a- and all-rac-a-tocopherols in
rats. J. Nutr. 121:454.

Blakely, S. R., G. V. Mitchell, M. Y. Jenkins, E.
Grundel, and P. Whittaker. 1991. Canthaxanthin and











excess vitamin A alter a-tocopherol, carotenoid and
iron status in adult rats. J. Nutr. 121:1649.

Brandner, G. 1971. "Vitamin E." Bell Publishing Company,
New York.

Bratzler, J. W., J. K. Loosli, V. N. Krukousky, and L. A.
Maynard. 1950. Effect of the dietary level of
tocopherols on their metabolism in swine. J. Nutr.
42:59.

Brief, S., and B. P. Chew. 1985. Effects of vitamin E and B-
carotene on reproductive performance in gilts. J.
Anim. Sci. 60:998.

Burton, G. W., K. H. Cheeseman, T. Doba, K. U. Ingold, and
T. F. Slater. 1983. Biology of Vitamin E. Ciba
Foundation Symposium 101. Pitman, London.

Chew, B. P., T. S. Wong, J. J. Michael, F. E. Standaert, and
L. R. Heirman. 1991. Kinetic characteristics of B-
carotene uptake after an injection of B-carotene in
pigs. J. Anim. Sci. 69:4883.

Chung, Y. K., D. C. Mahan, and A. J. Lepine. 1992. Efficacy
of dietary d-a-tocopherol and dl-a-tocopheryl acetate
for weanling pigs. J. Anim. Sci. 70:2485.

Coelho, M. B. 1991. "Vitamin E in Animal Nutrition and
Management." BASF Corp. Parsippany, NJ.

Coffey, M. T., and J. H. Britt. 1993. Enhancement of sow
reproductive performance by B-carotene or vitamin A.
J. Anim. Sci. 71:1198.

Cunha, T. J. 1977. "Swine Feeding and Nutrition."
Academic Press, New York.

Diplock, A. T. 1985. "Fat-Soluble Vitamins." Technomic
Publishing Co. Inc., Lancaster, Pennsylvania.

Dove, C. R., and R. C. Ewan. 1991. Effect of trace
minerals on the stability of vitamin E in swine grower
diets. J. Anim. Sci. 69:1994.

Draper, H. H. 1980. Nutrient relationships. In: Machlin,
L. J. "Vitamin E: A Comprehensive Treatise." Marcel
Dekker, New York.

Erdman, J. W., Jr., C. L. Poor, and J. M. Dietz. 1988.
Factors affecting the bioavailability of vitamin A,
carotenoids, and vitamin E. Food Technol. 42:214.










Evans, H. M. 1962. The pioneer history of vitamin E.
Vitam. and Horm. 20:379.

Harris, P. L., and M. I. Ludwig. 1949a. Vitamin E
potency of a-tocopherol esters. J. Biol. Chem.
180:611.

Harris, P. L., and M. I. Ludwig. 1949b. Relative
vitamin E potency of natural and of synthetic a-
tocopherol. J. Biol. Chem. 170:1111.

Hatam, L. J., and H. J. Kayden. 1979. A high-performance
liquid chromatographic method for the determination of
tocopherol in plasma and cellular elements of the
blood. J. Lipid Res. 20:639.

Hidiroglou, N., N. Cave, A. S. Atwal, E. R. Farnworth, and
L. R. McDowell. 1992. Comparative vitamin E
requirements and metabolism in livestock. Ann. Rech.
Vet. 23:337.

Hidiroglou, N., and L. R. McDowell. 1987. Plasma and
tissue levels of vitamin E in sheep following
intramuscular administration in an oil carrier.
Internat. J. Vit. Nutr. Res. 57:261.

Hidiroglou, N., L. R. McDowell, and R. Pastrana. 1988.
Bioavailability of various vitamin E compounds in
sheep. Internat. J. Vit. Res. 58:189.

Hoppe, P. P., F. J. Schoner, and M. Frigg. 1992. Effects of
dietary retinol on hepatic retinol storage and on
plasma and tissue a-tocopherol in pigs. Internat. J.
Vit. Nutr. Res. 62:121.

Horwitt, M. K., W. H. Elliott, P. Kanjananggulpan, and C. D.
Fitch. 1984. Serum concentrations of a-tocopherol after
ingestion of various vitamin E preparations. Am. J.
Clin. Nutr. 40:240.

Howard, K. A., S. V. Radecki, E. R. Miller, A. J. Thulin,
and D. E. Ullrey. 1990. Relative bioavailability of
vitamin E of natural or synthetic origin in growing
pigs. Res. Rep. 502. Michigan State Univ. Agric.
Experiment Station, East Lansing, MI.

Jensen, M., J. Hakkarainen, A. Lindholm, and L. Jonsson.
1988. Vitamin E requirement of growing swine. J. Anim.
Sci. 66:3101.

Jensen, M., A. Lindholm, and J. Hakkarainen. 1990. The
vitamin E distribution in serum, liver, adipose and











muscle tissues in the pig during depletion and
repletion. Acta Vet. Scand. 31:129.

Kaneko, J. J. 1980. "Clinical Biochemistry of Domestic
Animals." (3rd Ed.). Academic Press, New York.

Koch-Weser, J. 1974. Medical intelligence. The New England
J. of Med. 29(5):233.

Kusin, J. A., V. Reddy and B. Sivakumar. 1974. Vitamin E
supplements and the absorption of a massive dose of
vitamin A. Am. J. Clin. Nutr. 27:774.

Machlin, L. J. 1980. "Vitamin E: A Comprehensive
Treatise." Marcel Dekker, New York.

Mahan, D. C. 1991. Assessment of the influence of dietary
vitamin E on sows and offspring in three parities:
reproductive performance, tissue tocopherol, and
effects on progeny. J. Anim. Sci 69:2904.

Mahan, D. C., and A. L. Moxon. 1980. Effect of dietary
selenium and injectable vitamin E-selenium for weanling
swine. Nutr. Rep. Int. 21:829.

Malm, A., E. F. Walker Jr., M. Homan, D. Kirtland, A. Aydin,
and W. G. Pond. 1976. Glutathione peroxidase and other
enzymes in serum of sow and their progeny fed vitamin
E adequate or deficient diets with added Se. Nutr. Rep.
Int. 14:185.

Mason, K. E. 1980. The First Two Decades of Vitamin E
History. In: Machlin, L. J., "Vitamin E: A
Comprehensive Treatise." Marcel Dekker, Inc., New
York.

McDowell, L. R. 1989. "Vitamins in Animal Nutrition."
Academic Press, Inc., San Diego, CA.

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 pea (Pisam sativum). J. Anim. Sci.
45:1326.

McMurray, C. H., and W. J. Blanchflower. 1979a. Application
of a high-performance liquid chromatographic
fluorescence method for the rapid determination of a-
tocopherol in the plasma of cattle and pigs and its
comparison with direct fluorescence and high-
performance liquid chromatography-ultraviolet detection
methods. J. Chromatogr. 178:525.











McMurray, C. H., and W. J. Blanchflower. 1979b.
Determination of a-tocopherol in animal feedstuffs
using high-performance liquid chromatography with
spectrofluorescence detection. J. Chromatgr. 176:488.

Mooney, K. 1992. The effects of supplemental vitamin A
or B-carotene on reproduction and the localization
of B-carotene during gestation in gilts. M.S. Thesis.
Univ. of Florida, Gainesville.

Njeru, C.A., L. R. McDowell, N. S. Wilkinson, S. B. Linda,
S. N. Williams, and E. L. Lentz. 1992. Serum a-
tocopherol concentration in sheep after intramuscular
injection of DL-a-tocopherol. J. Anim. Sci. 70:2562.

NRC. 1988. "Nutrients Requirements of Swine." (9th Ed.).
National Academy Press, Washington, DC.

Pudelkiewicz, W. J., L. Webster, and L. D. Matterson. 1964.
Effects of high levels of dietary vitamin A acetate on
tissue tocopherol and some related observations. J.
Nutr. 84:113.

Raacke, I. D. 1983. Herbert McLean Evans: a biographical
sketch. J. Nutr. 113(5):929.

SAS. 1988. SAS User's Guide: Statistics." SAS Inst. Inc.,
Cary, NC.

Scott, M. L. 1969. Studies of vitamin E and related
factors in nutrition and metabolism. In: DeLuca,
H. F., and J. W. Suttie. "The Fat Soluble Vitamins."
The University of Wisconsin Press, Madison.

Sklan, D., and S. Donoghue. 1982. Vitamin E response to
high dietary vitamin A in the chick. J. Nutr. 112:759.

Tappel, A. L. 1962. Vitamin E as the biological lipid
antioxidant. Vitamins and Hormones. 20:493.

"The American Heritage Dictionary." 1982. (2nd Ed.).
Houghton Mifflin, Boston.

Thompson, J. 1993. Vitamin E injections improve pig
survival. Feedstuffs 65(21):13.

Ullrey, D. E. 1981. Vitamin E for swine. J. Anim. Sci.
53:1039.

Weaver, E. M., G. W. Libal, C. R. Hamilton, and I. S.
Parker. 1989. Relationship between dietary vitamin A
and E on performance and vitamin E status of the weaned
pig. J. Anim. Sci. 67(Suppl. 2):113. (Abstr.).














BIOGRAPHICAL SKETCH


Lee E. Anderson, Sr., was born December 1944 in

Pineland, South Carolina. He is married to Erma L. Anderson

and the father of five children. He graduated from

Middleton Senior High School in 1962. He graduated with an

Associate of Arts degree in 1964 from Gibbs Junior College.

He received a Bachelor of Science in Agriculture in 1969

from Florida A and M University. He received the Master of

Science in reproductive physiology from the University of

Florida in 1972. Since receiving the M.S. he has been

employed at Alcorn State University, Fort Valley State

University and Florida A and M University, in extension,

research and instructional programs. He is presently a

candidate for the Ph.D.








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.



Robert 0. Myer, Chairman
Associate Professor of Animal
Science

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.



Joel H. Brendemuhl'
Associate Professor of Animal
Science

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.



Ji'mmy G. cyeek
Professor of Agricultural
'Education and Communication

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 dre of Doctor of Philosophy.



J ph HU C nr
ofessor of Animal Science

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.



Lee R. McDowell
Professor of Animal Science








This dissertation was submitted to the Graduate Faculty
of the College of Agriculture and to the Graduate School and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.


August, 1993


Dean, C Yege of Agric ture


Dean, Graduate School










































UNIVERSITY OF FLORIDA


3 1262 08556 9464