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thesis entitled

EFFECT OF VITAMIN E AND PORCINE STRESS SYNDROME (PSS)
GENOTYPE 0N ANTIOXIDANT
STATUS AND CARCASS CHARACTERISTICS OF PIGS

presented by

Gerry Ann McCully

has been accepted towards fulfillment
of the requirements for

Master of Science degree in Animal Science

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EFFECT OF VITAMIN E AND PORCINE STRESS SYNDROME (PSS)
GENOTYPE ON ANTIOXIDANT
STATUS AND CARCASS CHARACTERISTICS OF PIGS
By

Gerry Ann McCully

A THESIS
Submitted to
Michigan State University

in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Animal Science

2002

ABSTRACT
EFFECT OF VITAMIN E AND PORCINE STRESS SYNDROME (PSS)
GENOTYPE ON ANTIOXIDANT STATUS AND CARCASS CHARACTERISTICS
OF PIGS
By

Gerry Ann McCully

The effects of vitamin E (injected or fed) and porcine stress syndrome
genotype on antioxidant status in young pigs and carcass characteristics in
market pigs were evaluated. Vitamin E injected pigs had greater serum E than
saline injected pigs for 7 d post injection. Homozygous dominant (NN) pigs had
lower (P < 0.01) glutathione peroxidase and higher (P < 0.05) CuZn superoxide
dismutase activities than Mn 1 d post injection. Vitamin E injection at weaning
had no affect on serum E, or enzyme activities measured 28 d later. Serum
ceruloplasmin activity in non-injected pigs was lower (P < 0.05) in nn pigs than in
NM while the opposite was true in injected pigs. Carcasses of nn pigs were
shorter, had less BF, larger LEA, greater kg lean, lower 45 min muscle pH, and
were more pale than NN or Nn carcasses (P < 0.05), and nn had greater drip
loss (P < 0.05) than NN carcasses regardless of vitamin E injection. When
vitamin E was fed, muscle vitamin E was greater (P < 0.01 ). Carcasses of nn
pigs were Shorter, paler, had less BF, and greater drip loss (P < 0.05) than NN or
Nn. Also, nn had lower 45 min muscle pH, larger LEA and greater kg lean (P <
0.05) than NN carcasses. Nn and nn pigs had altered antioxidant activity and
carcass characteristics compared with NN pigs and may have different dietary

requirements for nutrients associated with antioxidant capability.

ACKNOWLEGEMENTS

I want to express great appreciation to all the faculty and staff at Michigan
State University, especially to Dr. Gretchen M. Hill for her support, expertise,
patience, never-ending encouragement and friendship. To Jane Link for always
going above and beyond in every situation including organization, friendship and
an amazing ability to remember every detail of the hundreds of experiments
conducted with her guidance. To Dr. Dale Rozeboom for his knowledge,
assistance and his positive attitude. To C. Gerald Haarer, Dr. David Hawkins,
and Dr. Maynard Hogberg for their leadership and confidence in my ability and
for presenting my family and me with outstanding opportunities at Michigan State
University that created memories and friendships that will last a lifetime. Vera
Thompson, my mother, for helping with my children while I finished my thesis.
Mark McCully, my husband, for always supporting and assisting me in every

venture and for being my best friend in life.

iii

.v'.

I»).

TABLE OF CONTENTS

LIST OF TABLES ......................................................................................... v
LITERATURE REVIEW ............................................................................... 1
wmmME ........................................................................................... 1
Forms ..................................................................................... 1

Functions ................................................................................ 1

Biological activity and biopotency .......................................... 2

Tissue concentrations ............................................................ 2

Effect on growth ..................................................................... 4

Effect on meat quality ............................................................. 4

Antioxidant System ........................................................................... 5
Malignant Hyperthennia Gene .......................................................... 6
Relationship with vitamin E .................................................... 7

Genotype Characteristics .................................................................. 7
Performance ........................................................................... 7

Meat quality and quantity ....................................................... 8
INTRODUCTION ......................................................................................... 12
MATERIALS AND METHODS ..................................................................... 14
Experiments ...................................................................................... 14
Experiment 1 .......................................................................... 14

Experiment 2 .......................................................................... 15

Experiment 3 .......................................................................... 1 5

Experiment 4 .......................................................................... 16

Laboratory Assays ............................................................................ 16
Experiment 1 and 2 ................................................................ 16

Slaughter Procedures and Carcass Characteristic Evaluation ......... 18
Experiment 3 and 4 ................................................................ 18

Statistical Analysis ............................................................................ 19
RESULTS AND DISCUSSION ..................................................................... 21
Experiment 1 ..................................................................................... 21
Experiment 2 ..................................................................................... 23
Experiment 3 ..................................................................................... 24
Experiment 4 ..................................................................................... 26
IMPLICATIONS ............................................................................................ 30
APPENDIX ................................................................................................... 41
REFERENCES ............................................................................................ 75

iv

LIST OF TABLES

Table 1. Experimental Diets (as-fed basis) ................................................. 31

Table 2. Effect of treatment, genotype and time post injection on
serum vitamin E and ceruloplasmin and red cell
glutathione peroxidase 1, catalase and CulZn superoxide
dismutase — experiment 1 ............................................................. 32

Table 3. Effect of treatment, genotype and time post injection on
serum vitamin E and ceruloplasmin and red cell
glutathione peroxidase 1, catalase and CuZn superoxide
dismutase - experiment 2 ............................................................. 34

Table 4. Effect of injected vitamin E and genotype on carcass
characteristics - experiment 3 ...................................................... 35

Table 5. Effect of injected vitamin E and genotype on pork quality -
experiment 3 ................................................................................. 36

Table 6. Effect of vitamin E (injected or fed) and genotype on 10th
rib chop vitamin E concentration (umoI/g tissue) in
experiments 3 and 4 ..................................................................... 37

Table 7. Effect of feeding vitamin E and genotype on pork quality -
experiment 4 ................................................................................. 38

Table 8. Effect of feeding vitamin E and genotype on carcass
characteristics — experiment 4 ...................................................... 39

Table 9. Effect of feeding vitamin E on serum vitamin E
concentration (umol L") - experiment 4 ....................................... 40

LITERATURE REVIEW

Vitamin E

Forms. The term vitamin E is used to describe all derivatives of
tocopherol and tocotrienol that exhibit the biological activity of a—tocopherol.
Eight naturally occurring substances have been found to have vitamin E activity:
d-or-tocopherol, d-B-tocopherol, day-tocopherol, d-S-tocopherol, d-a-tocotrienol, d-
B-tocotrienol, d-y-tocotrienol and d-S-tocotrienol. Natural forms of vitamin E are
all d-stereoisomers while synthetic forms of vitamin E, typically found in
commercial diets, are mixtures of stereoisomers prepared by coupling
trimethlythydroquinone (TMHQ) with isophytl (Kayden and Traber, 1993).

Functions. Numerous functions have been reported for vitamin E in
biological systems. It is probably best known as a non-specific anti—oxidant in
lipid membranes where its activity is believed to protect membrane integrity.
\fitamin E has also been reported to be involved in the synthesis of co-enzyme
Q, the metabolism of vitamin C and sulfur amino acids, maintenance of structure
and health of the nervous, gastro-intestinal, immune and muscular systems
(Coelho, 1991 ). However, its role as a natural anti-oxidant is thought to be the
underlying factor in most of these functions.

Vitamin E is the major lipid-soluble antioxidant in biological systems and
serves as a free radical scavenger (Halliwell, 1987; Duthie et al., 1989; Dutta-
Roy et al., 1994). Free radicals are molecules or molecular fragments with an
unpaired electron. The presence of an unpaired electron can convey

1

considerable reactivity to the free radical which by hydrogen abstraction can
damage a wide range of biological materials, such as DNA, nucleotide co-
enzymes, proteins and lipids (Slater, 1984; DiMaSCio et al., 1991). Vitamin E
disrupts the sequence of lipid peroxidation and prevents the formation of
damaging lipid hydroperoxides.

Biological activity and biopotency. The biological activity of tocopherol has
been assessed by the ability of a form of this vitamin to prevent a specific
deficiency-related symptom. For example, RRR-a-tocopherol is the most
effective form when activity is assessed by the rat resorption-gestation assay, but
units of activity are expressed relative to that of all-rac-a-tocopheryl acetate
(Chow, 2001 ). Therefore, the biological activity of RRR-a-tocopheryl acetate is
1.49 lUlmg compared to all-raC-tocopheryl acetate which is 1.00 lUlmg. How
well a tocopherol form functions or its biopotency is related to its kinetics in a
tissue. Since the activity of RRR-a-tocopherol is highest, it is believed that the
phytyl tail is important for optimal biopotency. In serially sampled sows,
Lauridson et al. (2002) recently reported that RRR-a-tocopherol was twice as
effective in maintaining plasma or-tocopherol concentrations than synthetic all-
rac-a-tocopherol.

Tissue concentrations. Tocopherols , like dietary fat, are absorbed from
the small intestine in triglyceride containing Chylomicrons into the lymph system
to be transported to the liver. Released from the liver in lipoproteins, many
tissues including the liver, muscle and adipose tissue have the ability to take-up

car—tocopherol. Jensen et al. (1988) reported that skeletal muscle had higher (P <

2

0.001) vitamin E tissue concentrations after 3 wk of feeding as little as 15 mg
vitamin Elkg of diet compared with pigs fed unsupplemented diets. When
Cannon et al. (1996) fed diets containing 0 or 100 mg of vitamin E for 84 d, a-
tocopherol concentration was increased 10-fold in the longissimus muscle. In a
dietary vitamin E depletion and repletion study by Jensen et al. (1990), high
levels of vitamin E (405 mg/kg diet) were fed for a 7-wk period. Following this
dietary regime, a depleted vitamin E diet was fed. On d 2 of depleted diet
feeding, liver vitamin E concentrations dropped by 80%. Fat and muscle vitamin
E concentrations remained elevated after 7 d of feeding the depleted diet.
Jensen et al. (1990) concluded that serum vitamin E and liver concentrations are
short-term indicators of vitamin E status; however, adipose and skeletal muscle
tissues are long-term indicators. Moreira and Mahan (2002) fed different levels
of vitamin E (0, 20, 40, 60, 80, 100, 150, or 200 IU dl-a-tocopherol acetate/kg of
diet) to newly weaned pigs (n = 248; age = 15 d :t: 1 d; weight = 4.8 kg) for 42
days and found that serum (ii-tocopherol concentrations increased quadratically
in the 7 d period following weaning but linearly thereafter. Serum car-tocopherol
was highest for all the pigs at weaning, declining in all treatments by d 7. In a
second experiment (n = 248) feeding 0, 20, 40, or 60 lUIkg diet, similar findings
were reported in car-tocopherol concentrations. Additionally, tissue (liver, heart
and adipose) (TI-tocopherol concentrations increased as dietary vitamin E
increased. When 5% fat was added to the diets, liver and adipose car-tocopherol

concentration increased; however, heart muscle tissue concentration was

unchanged.

Effect on growth. The effect of vitamin E on growth performance was
recently reported by Moreira and Mahan (2002). In two experiments (n = 496)
utilizing newly weaned pigs fed various levels (up to 200 lU/kg of diet) of vitamin
E (all-rac—tocopherol) they found no advantages in pigs gains, feed intake, or
gain2feed for any period during the forty-two days following weaning. Chung et
al. (1992) and Cannon et al. (1996) also reported that increased dietary vitamin E
did not improve growth performance. Most researchers agree that supplemental
vitamin E above typical NRC guidelines does not improve the overall growth rate
of the pig at any stage.

Effect on meat quality. Numerous research trials have been conducted
utilizing various levels of vitamin E supplementation as it pertains to meat quality,
although not all agree on the effect regarding pork meat quality. Some research
has shown that increased levels of dietary vitamin E (200 mglkg) in the form of
acetate improves color (Asghar et al., 1991; Monahan et al., 1992), decreases
drip loss and enhances shelf life stability (Asghar et al., 1991) of pork Chops.
Cheah et al. (1995) reported that dietary supplementation of 500 mg vitamin Elkg
of diet potentially reduced drip loss in longissimus thoracis (LT) muscle by 45% in
porcine stress syndrome (PSS) carriers (Nn) and 54% in stress-free pigs (NN).
They also concluded that supplementation of 1,000 mg vitamin Elkg diet for 46 d
could actually prevent the formation of pale, soft, and exudative (PSE) carcasses
when analyzing pre-slaughter biopsy and post-slaughter evaluation of the LT
muscle. Contradicting these findings, Jensen et al. (1997) fed diets

supplemented with car-tocopherol (100, 200 or 700 mglkg feed) to pigs from

weaning to slaughter and concluded that drip loss and color stability were not
affected by vitamin E treatment. Color measurement, pH, drip loss and growth
were also reported by Cannon et al. (1996) to be unaltered by vitamin E
supplementation in pigs fed 100 mglkg diet for 84 d prior to slaughter compared
with those fed a control diet containing no supplemental vitamin E. Corino et al.
(1999) fed varying levels of vitamin E (25, 50, 100, 200 and 300 mglkg
tocopherol acetate of diet) for the last 60 d prior to slaughter and found vitamin E
treatment did not alter drip loss. Past research does not fully agree on the
overall effect of dietary vitamin E supplemented at varying levels and how it
affects meat quality in swine.
Antioxidant System

Vitamin E is not the only antioxidant in biological systems, but it is an
integral part of a network of antioxidants (Lauridsen et al., 1999a), including:
ceruloplasmin (Cp; EC 1.12.3.1), catalase (CAT; EC 1.11.1.6), copper/zinc
superoxide dismutase (Cu/Zn SOD; EC 1.15.1.1), and glutathione peroxidase
(GPX1; EC 1.11.1.9). DiMascio et al. (1991) reported oxidative damage is
controlled by a system of enzymatic and nonenzymatic antioxidants that
eliminate pro-oxidants and scavenge damaging free radicals. Tocopherols are
the most abundant and efficient of the antioxidants.

The protein Cp is made in the liver, binds 90 to 95% of the circulating Cu
in the body, and has non-specific antioxidant activity and acts as an acute phase
reactant protein. The enzyme CAT helps to rid the body of damaging peroxides.

Hill et al. (1999) reported that young pigs, 3 to 14 d of age, did not have a change

in Cp activity when injected with Fe (200 mg/mL Fe) and vitamin E (300 lU) at 6 d
of age, but Cp activity increased with age from 3 to 7 d of age. Milne and
Matrone (1970) also reported an increase in Cp activity of the young pig through
two weeks of age.

Copper/Zinc superoxide dismutase is a copper/zinc dependent enzyme of
the oxidoreductase class that catalyzes the reduction of superoxide anions to
hydrogen peroxide, protecting cells against dangerous levels of superoxide.
Glutathione peroxidase (GPX1) is a selenium dependent enzyme of the ligase
class that catalyzes the ATP dependent-formation of glutathione from glycine and
y-glutamylcysteine. In a study by Lauridsen et al. (1999b) pigs (8W = 25 kg)
were fed three levels of vitamin E: 1) basal diet (9 mglkg vitamin E); 2) basal diet
+ 100 mg vitamin E; 3) basal diet + 200 mg vitamin E. They found that hepatic
SOD and GPX1 activities were not affected by dietary vitamin E regime. In a
second study, feeding the same diets, Lauridsen et al. (19993) found no effect of
diet on CulZn SOD and GPX1 in the M. Longissimus dorsi (LD) and M. Psoas
major (PM) muscles. Duthie and Arthur (1989) reported that pigs consuming a
diet containing 10 IU vitamin Elkg had increased GPX1 activity in the longissimus
dorsi muscle of pigs homozygous for the porcine stress syndrome (PSS) gene
(nn) compared with the normal pig of similar genetics.

Malignant H yperthermia Gene

Pigs that carry porcine stress syndrome (PSS), also known as the

halothane gene, are commonly referred to as being “stress-susceptible”. When

exposed to relatively minor stresses, such as halothane, transportation, weaning

and co-mingling, mating and parturition, PSS homozygous recessive (nn) pigs
may develop malignant hyperthermia; a rapid increase in body temperature,
muscle rigidity, hyperventilation, rapid heart rate, and potentially death (Williams,
1985; Geers et al., 1992).

Relationship with vitamin E. According to research at the Rowett Institute
(Duthie and Arthur, 1989), pigs that are deficient in vitamin E display similar
antioxidant abnormalities to pigs with the genetic mutation known as PSS. As
with vitamin E deficient pigs, PSS pigs have an increased susceptibility to free
radical mediated peroxidation compared with pigs free of the stress gene (Duthie
and Arthur, 1989), or “normal” (NN) pigs, and increased vitamin E
supplementation may reduce the negative effects of PSS at the biochemical
level.

Current HAL-1843 DNA technology (Fujii et al., 1991) makes it possible to
determine the presence or absence of the PSS gene; hence, the ability to identify
the heterozygote (Nn). Prior stress testing utilizing halothane was limited to only
identifying the homozygous recessive (nn) individual. Therefore, past research
of the heterozygote (Nn) is somewhat limited.

Genotype Characteristics

Performance. Christian and Rothchild (1981) found no differences in ADG
due to presence of the PSS gene. Leach et al. (1996) studied PSS carriers (Nn)
and negative (NN) pigs produced within the same litter and concluded that daily
gains were similar. They also reported that Nn pigs had higher feedzgain ratio

than NN pigs. Byrem et al. (1999) found improved gain in nn pigs in the finishing

stage.

Meat quality and quantity. The inheritance of PSS is thought to be
through a recessive gene although not all researchers agree. Some studies
indicate complete recessiveness of the PSS gene (Jensen et al., 1981; Webb,
1980) while other research has shown some dominance effects of the gene on
meat quality (Sather et al., 1991b; Simpson and Webb, 1989). Cannon et al.
(1995) concluded the mode of inheritance seems to be an autosomal recessive
trait that demonstrates incomplete penetrance. Homozygous recessive (nn) pigs
for the trait demonstrate the characteristic limb rigidity when exposed to
halothane; however, the heterozygotes (Nn) do not demonstrate the limb rigidity,
but produce inferior meat quality. Murray et al. (1989) reported that the
heterozygote (Nn) is intermediate between both homozygotes. In terms of meat
quality, Lundstrom et al. (1989) concluded that the heterozygote (Nn) is
intermediate between the homozygote (rm and NN) pigs. It has also been
suggested that the dominance of the PSS gene as it affects the quality of pork
may rise with increased slaughter weight (Sather et al., 1991a). The interaction
between the pre-slaughter handling and the genotype could be the cause of
contradicting results (Oliver et al., 1993).

Carriers of PSS, both homozygous recessive (rm) and heterozygous (Nn),
are leaner, heavier muscled and produce carcasses with a higher percentage of
lean muscle (Oliver et al., 1993; McCully et al., 1996; Weavers et al., 1996),
unfortunately their meat is more likely to be pale, soft and exudative (PSE;

Casteels et al., 1995). Van Laack et al. (1993) noted that homozygous recessive

(nn) pigs produced carcasses with larger LEA and less BF, but meat quality was
often sacrificed since their carcasses were more pale and exudative. On the
other hand, pigs that were heterozygous (Nn) for the stress gene produced
carcasses with larger LEA and a greater amount of lean product than pigs that
were free of the stress gene (NN) although muscle color and drip loss did not
differ (van Laack et al., 1993). Stalder et al. (1998) reported heterozygotes were
intermediate for carcass traits. Leach et al. (1996) studied Nn and NN within the
same litter and concluded Nn pigs had an advantage in feed efficiency and
carcass yield but had a higher incidence of PSE.

According to Klont et al. (1993) PSE meat is associated with rapid decline
in pH in the muscle during the first hour following slaughter. Sather et al. (1991a)
reported lower pH at 45 min post mortem for nn pigs than for NN or Nn and Nn
pigs were lower than NN. Greater incidence of pale color by the L* score in pigs
of nn genotype was also reported by Pommier and Houde (1993) and Sather et
al. (1991a) with NN loins being darker in color. Van Laack et al. (1993) stated
that nn pigs (n = 4) demonstrated lower pH at 45 min postmortem and had
higher L* values at 48 h than NN (n = 4) or Nn (n = 4) pigs. Klont et al. (1993),
utilizing pre-slaughter anesthesia, found that differences in color and drip loss are
not explained by the stress state of pigs or differences in pH and 45 min carcass
temperature. Sather et al. (1991a) reported with heavier carcasses, drip loss
from nn or Nn was not different. However, both differed from NN heavy
carcasses.

Many factors contribute to the onset of PSE, including genetics, pre-

slaughter stress, early post-mortem pH drop, carcass temperature, chilling
procedures, feed restriction during transport and additional Slaughter techniques
(Murray et al., 1989; Klont et al., 1993; Buckley et al., 1995). Murray et al. (1989)
reported that the mixing of unfamiliar animals, physical abuse and environmental
Changes may increase PSE while restriction of feed during transport prior to
slaughter decreases PSE. Stalder et al. (1998) wanted to determine if
preslaughter handling, mixing or resting, influence the quality of muscle in pigs of
differing genotypes (29 Nn; 14 NN; 9 nn). They found that 16 h of resting prior to
slaughter improved the color and water holding capacity of heterozygotes (Nn).
A 1995 report by the National Genetic Evaluation Board (Anonymous, 1996)
suggested that 35% of carrier pigs raised under good handling and management
conditions, had inferior pork quality, while only 20% of pigs free of the stress
gene had inferior pork quality. Thus, the majority of PSE pork in today‘s
marketplace is thought to be caused by the management of certain genotypes
especially those carrying PSS. Because much of the reduction in pork quality is
oxidative in nature, antioxidants appear to have the greatest impact in
maintaining pork quality. Although many factors contribute to PSE in today‘s
marketplace, researchers agree that PSS genetics play a major role.

Pale, soft, and exudative pork is detrimental to all aspects of the swine
industry, including producers, packers and ultimately the consumer (Anonymous,
1995). Millions of dollars are estimated to be lost annually due to PSE (Kauffman
and Weller, 1992; Marbery, 1992). Lost revenue from purge and undesireable

meat case display is difficult to quantify; however, the U. S. Livestock

10

Conservation Institute estimates the United States loses $ 32 million annually
due to PSE-related losses.

It is agreed upon by past research that homozygous recessive (nn) pigs
for the stress gene have increased lean, along with increased incidence of PSE;
however, the overall meat quantity and quality of the heterozygote (Nn) is still in
question. Also, the benefit of vitamin E on the reduction of PSE and
improvement of overall meat quality in PSS pigs (rm and Nn) is not clear.
Perhaps the utilization of today’s molecular laboratory techniques can clarify the

quantity of vitamin E needed for acceptable pork quality from the various

genotypes.

11

INTRODUCTION

Pigs that carry an inherited genetic mutation known as porcine stress
syndrome (PSS) are referred to as being stress-susceptible. Carriers of PSS,
both homozygous recessive (rm) and heterozygotes (Nn), are leaner, heavier
muscled and produce carcasses with a higher percent lean muscle (Oliver et al.,
1993; McCully et al., 1996; Weavers et al., 1996), but their meat is more likely to
be pale, soft and exudative (PSE). Leanness advantages are potentially lost due
to lower yields from reduced water holding capacity in the muscle (Pommier and
Houde, 1993). According to a survey of packing plants, Kauffman et al. (1992)
reported 26% of pig carcasses in the United States were considered undesirable.

Pale, soft and exudative pork composed up to 16% of these undesirable
carcasses.

Stress-susceptible pigs have altered antioxidant activity under sustained
oxidative stress from situations such as weaning, transport, mating and
parturition (Duthie et al., 1989). Signs of free radical mediated damage to cell
membranes, similar to normal (NN) pigs that are deficient in vitamin E, are found
in stress-susceptible pigs (Duthie and Arthur, 1989). Vitamin E, the major lipid
soluble antioxidant in biological systems, serves as a free radical scavenger.

Vitamin E does not function as the only antioxidant, but it is an integral
part of a network of antioxidants (Lauridsen et al., 1999b), including: CulZn
superoxide dismutase (Cu/Zn SOD; EC 1.15.1.1), glutathione peroxidase (GPX1;

EC 1.11.1.9), catalase (CAT; EC 1.11.1.6) and ceruloplasmin (Cp; EC 1.12.3.1).
12

h... .t

V
\\.
t

Therefore, our objective in these studies was to determine the impact of vitamin
E, either fed or injected, antioxidant status, growth rate, and carcass
characteristics of pigs representing all stress genotypes (NN, Nn, nn) at various

stages of growth.

13

MATERIALS AND METHODS

The All-University Committee on Animal Care and Use at Michigan State
University approved (AUF number: 02194-037-03) animal usage in all
experiments.

Experiments

Experiment 1. In Exp. 1, our goal was to determine if carriers (Nn) of the
PSS gene, given an exogenous antioxidant, vitamin E, demonstrate a metabolic
shift in antioxidant parameters when compared to non-carriers (NN). Pigs (n =
39) were selected from the offspring of crossbred boars (Hampshire X Pietrain)
and crossbred sows (Duroc X Pietrain). Genotype was established by DNA
determination using the HAL 1843 method (University of Toronto, Innovations
Foundation, Toronto, Ontario) at the Universal Gene Laboratories, Eldora, lovva.
Pigs (mean BW = 38.5 kg) were grouped by weight and genotype in 13 pens of
three (10 pans of Nn; 3 pens of NN). They were housed in 1.5 m X 5 m pens in
an open front building for 21 d prior to the initiation of the study for an adjustment
period. A 16% crude protein, corn-soybean meal grower diet (Table 1) that met
or exceeded National Research Council (NRC, 1988) nutrient recommendations
and water were provided ad libitum.

Pigs within a pen were randomly injected i.m. (20 ga, 2.5 cm needle) in
the cervical portion of the trapezius muscle, approximately 15 cm posterior to the
ear with one of the following: (1) placebo (2 mL saline/22.7 kg BW); (2) 600 IU d-

a—tocopherol (Vital E300; Schering-Plough Animal Health, Kenilworth, NJ)I22.7

14

kg BW; or (3) 1200 IU d-or -tocopheroll22.7 kg BW. Blood samples were
collected, via the anterior vena cava, immediately prior to injection and at 1, 4
and 7 d post injection.

Experiment 2. To determine the effect of vitamin E and genotype on
antioxidant activity and growth during and following the stress of weaning, 154
pigs (BW 7.84 kg, age 24 d) from crossbred boars (Hampshire X Pietrain) and
crossbred sows (Duroc X Pietrain) and of known PSS (HAL 1843) genotype (37
NN, 80 Nn, and 37 nn) were randomly assigned by weight, litter and sex to
receive either an i.m. injection of vitamin E (900 IU d-a-tocopherol) or no injection
at weaning. Pigs were provided water and a complex phase I starter diet (Table
1), ad libitum, for 7 d post weaning followed by a phase II diet (Table 1) for d 8 to
28 post weaning. Both diets met or exceeded all dietary recommendations of
NRC (1988). Blood was collected prior to injection and at 28 d post injection.
Weights were recorded weekly.

Experiment 3. For determination of the effect of vitamin E and PSS
genotype on carcass characteristics, 60 pigs (BW = 104.3 kg) of known genotype
(16 NN, 32 Nn, and 16 nn) were randomly injected i.m. 24 h prior to slaughter
with one of two treatments: (1) placebo, 2 mL saline/22.7 kg BW or (2) 600 IU d-
or-tocopheroll22.7 kg BW. Prior to study, pigs were fed a com-soybean meal
finishing diet (Table 1) that met or exceeded NRC (1988) requirements and
contained 19.3 mg rat-tocopherol per kg diet by analysis. Pigs were transported
for approximately 2 h to a commercial packing plant and rested for a minimum of

2 h prior to slaughter.

15

Experiment 4. For evaluation of dietary vitamin E and PSS genotype on
carcass characteristics during the finishing phase, 56 pigs (BW = 110.7 kg) of
known genotype (14 NN, 28 Nn, and 14 nn) were grouped by weight and
genotype into 1.5 m X 5 m pans in an open front building. Each pen of four pigs
contained 1 NN, 2 Nn, and 1 nn pig. Forty-seven days prior to slaughter, pens
were allotted to one of two corn-soybean commercial diets (Table 1) meeting or
exceeding NRC (1988) requirements with either: (1) 33 mglkg vitamin E or (2)
500 mglkg vitamin E. Growth rates were recorded weekly. Pigs were
transported to a commercial packing plant as in Exp. 3.

Laboratory Assays

Experiment 1 and 2. Blood samples were collected by venipuncture in 10
mL heparinized vacutainers (Vacutainer, Becton Dickinson and Co., Frankling
Lakes, NJ) and kept on ice. For serum analyses, blood was collected in

vacutainers with no anticoagulant and again kept on ice. Serum was separated
by centrifugation (2000 x g, 10 min, 4°C) and frozen for Cp and vitamin E
analysis. The heparinized tubes were centrifuged (2000 x g, 10 min, 4°C), and

the harvested red blood cells (RBC) were washed (ice-cold 0.9% saline) three
times. Between washes, saline was removed by aspiration. Washed RBC were

lysed in an equal volume of ice-cold deionized distilled (DD) water and frozen (-

800 C) until analysis for determination of CulZn SOD and CAT activities. Packed

RBC’s were frozen (-80°C) for later GPX1 activity measurement.

Copper/Zinc superoxide dismutase was determined by the method of Hill

16

et al., (1999). Units of CulZn SOD activity were expressed per gram of
hemolysate protein (Lowry et al., 1951). Red blood cell GPX1 activity was
determined utilizing the method of Sunde and Hoekstra (1980). Red blood cell
CAT activity was measured by the method by Aebi (1984), and serum Cp was
determined by the method of Lehmann et al. (1974). Chemicals for antioxidant
analysis were purchased from Sigma Chemical (St. Louis, MO).
Alpha-tocopherol concentration of plasma was determined by HPLC as
previously described (Hill et al., 1999). Briefly, 100 pL of 6-tocopherol (25
umoles/L) in absolute ethanol (Quantum Chemical Corporation, Tuscola, IL) was
introduced into each plasma sample (100 pL) as an internal standard.
Tocopherols were extracted with heptane (200 uL). Extracts were evaporated to
dryness under a stream of N2 gas, then resuspended in methanol (150 uL).
Muscle a-tocopherol determination was carried out following a procedure
adapted from Taylor et al. (1976). Briefly, frozen tissue samples (0.5 g) were
weighed then homogenized in 10 volumes of 10 mM EDTA that contained 100 pL
of 0.054 M BHT in absolute ethanol. One mL of homogenate was then added to
2 mL of 0.057 M ascorbic acid to which 100 pL of internal standard, S-tocopherol
(25 liL) had previously been added. Tocopherols were extracted from the
homogenates with hexane. Extracts were evaporated to dryness under a stream

of N2, then resuspended in 0.15 mL of methanol.

A 100 uL aliquot of each sample extract was injected into a high

performance liquid Chromatograph (Gilson HPLC) equipped with a C-18 reverse-

l7

phase column (15 cm x 4.6 mm; 3 mm; Supelco lnc., Bellefonte, PA). The
mobile phase used consisted of methanol and water (98:2, by volume) with a
flow rate of 1.5 mUmin. The effluent was monitored at a UV wavelength of 292
nm (Gilson UV detector 117). Sample or-toc0pherol concentration was calculated
from peak area responses using a standard curve that was established from
known amounts of pure (yr-tocopherol and 8—tocopherol. Values were corrected
for losses during processing by following the recovery of the internal standards,
which generally exceed 90%.
Slaughter Procedures and Carcass Characteristic Evaluation

Experiment 3 and 4. At the commercial slaughter plant, pigs were
electrically stunned and exsanguinated. Carcasses were then scalded,
mechanically de-haired, eviscerated, and washed. Hot carcass weights were
recorded and muscle pH was measured (Solomat MPM2000, Norwalk, CT;
Radiometer PHM82, Copenhagen, Denmark) on the surface of the longissimus
muscle (LM) at the 10th rib, 45 min postmortem. After chilling overnight,
carcasses were transported in a refrigerated truck to a commercial cutting plant.

At 24 h postmortem, loin eye area (LEA), backfat thickness (BF), carcass
length and muscle pH were measured and two 10th rib chops (2.5 cm thickness)
were removed for drip loss and color evaluation. Initial (24 h post mortem) chop
weight and light reflectance values (L*, Minolta CR-30 Chroma Meter, Minolta

Camera Corp., Osaka, Japan) were recorded. Boneless Chops were stored in a
—3°C cooler under florescent light. At 48, 72 and 96 h postmortem light

reflectance values (L*) were again recorded. For drip loss calculation, chops

18

were placed on an absorbent meat-packaging pad and stored in unsealed plastic
vacuum packaging bags at —3°C. At 48, 72 and 96 h postmortem chops were

removed, patted dry and weighed. Percent drip loss was calculated from the
weight loss of chops over the 96 h post mortem. Carcass lean (kg) was
calculated (7.231 + 0.437 * adjusted warm carcass weight — 18.746 * 10th rib fat
depth + 3.877 * 10th rib loin eye area; (NPPC, 1991)).
Statistical Analysis

Experiment 1 was analyzed as a completely randomized design with a 3 x
2 factorial arrangement of treatments and repeated measures over time. The
GLM procedure of SAS (SAS Institute, Cary, NC) was used to test for treatment
and genotype effects. The model included the main effects of injection and
genotype and injection x genotype interaction. Mean differences between
injections were ascertained by least significant difference. Effect of time was
ascertained using the repeated statement.

Experiment 2 was analyzed as a completely randomized design with a 3 x
2 factorial arrangement of treatments using the GLM procedure of SAS. The
model included the main effects of treatment and genotype and the interaction.
Mean differences between genotypes were ascertained by least significant
difference. Pen served as the experimental unit for ADG, while individual pig
was the experimental unit for enzyme activity.

Experiments 3 and 4 were analyzed as completely randomized designs
with a 3 x 2 factorial arrangement of treatments using the GLM procedure of

SAS. In Exp. 3, pig served as the experimental unit and the model contained the

19

main effects of genotype and injection and the interaction. In Exp. 4, pig served
as the experimental unit for carcass characteristics and pork quality. The model
contained the main effects of genotype and diet and the interaction. Differences

between genotypes were ascertained by least significant difference.

20

RESULTS AND DISCUSSION

Experiment 1. Serum vitamin E concentrations of the pigs injected with
vitamin E were elevated (> 20-fold, P < 0.001) 24 h post injection, compared with
pigs of both genotypes that were injected with saline only (Table 2). There were
no differences in serum vitamin E concentration between genotypes at any
sampling time. However, those injected with vitamin E had higher circulating
1 concentrations of this antioxidant (~ two-fold). On d 7 post injection, the vitamin
E concentrations of those pigs injected with vitamin E remained higher (< two-
fold, P < 0.05) than those injected with saline which were similar to initial, pre-
injection samplings. In a dietary vitamin E depletion and repletion study by
Jensen et al. (1990), high levels of vitamin E (405 mglkg diet) were fed for a 7-wk
period. Following this dietary regime, a depleted vitamin E diet was fed. On d 2
of depleted diet feeding, liver vitamin E concentrations dropped by 80%. Fat and
muscle vitamin E concentrations remained elevated after 7 d of feeding the
depleted diet. Jensen et al. (1990) concluded that serum vitamin E and liver
concentrations are short-term indicators of vitamin E status; however, adipose
and skeletal muscle tissues are long-tenn indicators.

Duthie et al. (1989) reported that carriers of the stress gene have altered
antioxidant activity when compared to pigs that are free of the stress gene. In
our study, pigs that carried the stress gene (Nn) had higher (P < 0.002) GPX1
activity 24-h after receiving the vitamin E or placebo injection than non-carriers

regardless of treatment (Table 2). However, at 4 and 7 d post injection, neither

21

treatment nor genotype influenced the activity of this enzyme. Copper/zinc SOD
activity in the red blood cell was lower (P < 0.05) in Nn than in NN pigs 1 d after
Injection with vitamin E. This change in concentration was enhanced in the 1200
IU treatment. At d 4 and 7 neither treatment nor genotype influenced CulZn SOD
activity. While the activity of GPX1 and CulZn SOD were not affected by vitamin
E injection the genotypes responded with differing activities of these antioxidant
enzymes when an increase in vitamin E was administered. In a study by
Lauridsen et al. (1999a) pigs (BW = 25 kg) were fed three levels of vitamin E: 1)
basal diet (9 mglkg vitamin E); 2) basal diet + 100 mg vitamin E; 3) basal diet +
200 mg vitamin E. They found that hepatic SOD and GPX1 activities were not
affected by dietary vitamin E regime. In a second study, feeding the same diets,
Lauridsen et al. ( 1999b) found no effect of diet on CulZn SOD and GPX1 in the
M. Longissimus dorsi (LD) and M. Psoas major (PM) muscles. Our study
measured these changes in a more transient tissue (blood) and was the result of
an i.m. injection as compared with a dietary intervention.

The protein Cp is made in the liver, binds 90 to 95% of the circulating Cu
in the body, and has non-specific antioxidant activity. The enzyme CAT helps to
rid the body of damaging peroxides (Hill et al., 1999). Ceruloplasmin and CAT
activities from pigs in this study were not affected by treatment or genotype;
however, Cp activity tended to increase during the 7 d post injection period while
CAT activity remained unchanged (Table 2). Hill et al. (1999) reported that young
pigs, 3 to 14 d of age, did not have a change in Cp activity when injected with Fe

(200 mglMl Fe) and vitamin E (300 lU) at 6 d of age, but Cp activity increased

22

increased with age from 3 to 7 d of age. Similarly an increase in Cp activity was
noted in this experiment using older pigs. This increase may not be an age
effect, but perhaps a response to the stress of handling since Cp is also an acute
phase reactant protein.

Data from Exp. 1 indicates that PSS genotype influences antioxidant
activity and might affect the requirements for nutrients associated with
antioxidant defense in the growing stress susceptible pig.

Experiment 2. Antioxidant enzyme activities (Table 3) 28 d post weaning
were not affected by vitamin E injection. Ceruloplasmin and GPX1 activities
increased during the 28 (I post injection period (Table 3). This is consistent with
Chung et al. (1992) who reported that varying levels of vitamin E in the diet (16,
48, 96 lUlkg) did not influence GPX1 activity or growth performance in the
weanling pig. There was a treatment x genotype interaction (P < 0.05) for Cp
activity in pigs 28 d after injection with vitamin E. Homozygous recessive pigs
had higher Cp activity than homozygous dominant animals when vitamin E was
not injected. The reverse was true when vitamin E was given. This may indicate
that the need for added antioxidant enzyme activity protection necessary for PSS
pigs is reduced by the addition of vitamin E. Catalase and CulZn SOD activities
remained unchanged during the experiment and were not influenced by
treatment or genotype. However, nn pigs had higher initial catalase activity than
NN or Nn pigs.

Homozygous recessive pigs had lower (P < 0.05) ADG over the 28 d when

compared with Nn and NN (0.37 vs 0.41, 0.41 kgld, respectively). Previously,

23

Christian and Rothchild (1981) had reported no differences in ADG, and Byrem
et al. (1999) found improved gain in nn pigs. From this experiment it is apparent
that injectable vitamin E (900 IU d-a-tocopherol), given only at the time of
weaning, is not beneficial to the performance and antioxidant activity of the
weanling pig when the activities were measured 4 wk after injection. However,
increasing vitamin E may have a beneficial influence on the overall antioxidant
network if measures of antioxidant status were evaluated at the time of stress
(weaning).

Experiment 3. Injection of vitamin E (600 IU d-a-tocopheroI122.7 kg BW)
24 h prior to slaughter did not improve any of the measured carcass parameters
in this study (Table 4). Presence of the PSS gene, however, influenced the
quality and quantity of the carcass (Table 4 and 5). Homozygous recessive (nn)
pigs had shorter carcasses (P < 0.05) with larger LEA (P < 0.01) and less BF (P
< 0.01) compared with NN and Mn carcasses (Table 4). Since all carcasses
were of adequate length the biological importance of this difference is probably
not relevant. Huff-Lonergan et al. (2002) reported a highly significant correlation
coefficient (-0.57) between LEA and BF thickness. As expected, nn pigs
produced more lean pork as calculated from the National Pork Producers Council
(NPPC, 1991) formula that utilizes 10"1 rib fat depth and LEA. Chops from
homozygous recessive (nn) pigs had the highest (P < 0.05) L* values and lowest
(P < 0.01) LM pH at 45 min post mortem (Table 5). A Significant interaction (P <
0.05) between treatment and genotype was observed in pH change between 45

min postmortem and 24 h postmortem. The pH Change was lowest in nn pigs

24

not treated with vitamin E, perhaps because the 45 min pH was already lower
(5.88) in this group. If the Clearance of injected vitamin E from plasma was
similar to that found by Lauridsen (2002) with dietary vitamin E, perhaps this
Circulating antioxidant may have been important in reducing the rapid pH decline
observed in nn pigs injected with vitamin E (Table 5). No pH differences were
found at 24 h postmortem. Similarly, Kocwin-Podsiadla et al. (1995) found that
the 30 min postmortem pH was lowest in nn pigs compared with MN and Nn,
(units of pH Change were similar to ours between 45 min and 24 hr post-mortem)
and LM samples from nn pigs were more pale (Momcolor—D3098) compared with
NN. Klont et al. (1993) found that carcasses from Nn and nn pigs had higher
Hunter L* values than carcasses from NN pigs. However, they did not report a
drop in pH at 45 min or 18 h post mortem. Relative to our results, the units of pH
change were similar, but our initial pH at 45 min was almost one unit lower than
that reported by Klont et al. (1993). In our study drip loss was higher (P < 0.05)
in chops from nn pigs during all time periods (Table 5). Similar results were
reported by Klont et al. (1993). Contrary to the results obtained when dietary
vitamin E is fed to pigs (Jensen et al., 1997; Cannon et al., 1996), injection of
vitamin E 24 h prior to slaughter did not increase vitamin E content of 10th rib
chops (Table 6). While not significant with the small sample size (n = 2 per
treatment and genotype) the higher cit-tocopherol concentration in the injected
pigs of nn genotype may be of importance if this genotype has a higher
antioxidant need as suggested by Duthie (1989). These results indicate that

carrier (Nn) pigs produced carcasses with a greater amount of lean product than

25

NN but without sacrificing color quality and drip loss as found in nn.

Experiment 4. The vitamin E content of 10th rib chops was increased (P <
0.01) when 500 mg vitamin Elkg diet was fed for 47 d (Table 6). While vitamin E
is known to be rapidly cleared from the plasma to the liver, Jensen et al. (1988)
reported that skeletal muscle had higher (P < 0.001) vitamin E tissue
concentrations after 3 wk of feeding as little as 15 mg vitamin Elkg of diet
compared with pigs fed unsupplemented diets. When Cannon et al. (1996) fed
diets containing 0 or 100 mg of vitamin E for 84 d, car-tocopherol concentration
was increased 10-fold in the longissimus muscle. Hence it is not surprising that
our results reflect an increase. However, with this small sample size (n = 2 per
genotype and treatment), it is not possible to discern if genotype influences
tissue concentration. In our study, dietary vitamin E did not influence carcass or
pork quality measurements or characteristics (Table 7 and 8). Similarly, Jensen
et al. (1997) fed diets supplemented with rat-tocopherol (100, 200 or 700 mglkg
feed) to pigs from weaning to Slaughter and concluded that drip loss and color
stability were not affected by vitamin E treatment. Color measurement, pH, drip
loss and growth were also reported by Cannon et al. (1996) to be unaltered by
vitamin E supplementation in pigs fed 100 mglkg diet for 84 d prior to slaughter
compared with those fed a control diet containing no supplemental vitamin E.
Corino et al. (1999) fed varying levels of vitamin E (25, 50, 100, 200 and 300
mglkg tocopherol acetate of diet) for the last 60 d prior to slaughter and found
vitamin E treatment did not alter drip loss. However, not all researchers agree on

the effect of vitamin E supplementation on drip loss. Cheah et al. (1995)

26

 

reported that dietary supplementation of 500 mg vitamin Elkg of diet potentially
reduced drip loss in longissimus thoracis (LT) muscle by 45% in carriers (Nn) and
54% in stress-free pigs (NN). They also concluded that supplementation of
1,000 mg vitamin Elkg diet for 46 days could actually prevent PSE carcasses.
Asghar et al. (1991) reported that pork Chops (following three months frozen
storage) from pigs supplemented with 200 mglkg vitamin E had decreased drip
loss (P < 0.05).

Loin pH, associated with oxidative capacity, at 45 min postmortem was
highest for NN pigs (P < 0.05), but there were no genotype differences in LM pH
at 24 h (Table 7). This is similar to the results we observed when vitamin E was
injected in study 3, and typical of pigs carrying the PSE gene. Sather et al.
(1991a) reported lower pH at 45 min post mortem for nn pigs than for NN or Mn
and Nn pigs were lower than NN. Pommier and Houde (1993) noted that the
ultimate pH of loins was significantly higher from NN pigs than from Nn pigs, but
nn did not differ from the other two genotypes. Minolta light reflectance (L*) of
10th rib chops at 24, 48, 72 and 96 h was the highest for nn pigs when compared
with either Nn or NN genotypes (Table 7). The greater incidence of pale color by
the L* score in pigs of nn genotype was also reported by Pommier and Houde
(1993) and Sather et al. (1991a) with NN loins being darker in color. Van Laack
et al. (1993) stated that nn pigs (n = 4) demonstrated lower pH at 45 min post
mortem and had higher L* values at 48 h than NN (n = 4) or Nn (n = 4) pigs. Drip
loss was higher (Table 7) in nn pigs when compared with NN. Klont et al. (1993),

utilizing pre-slaughter anesthesia, found that differences in color and drip loss are

27

not explained by stress state of pigs or differences in pH and 45 min carcass
temperature. Sather et al. (1991a) reported in heavier carcasses (85 to 105 kg),
muscle drip loss from nn or Nn genotypes was not different. However, drip loss
from both (rm and Nn) were greater than from NN heavy carcasses. Results of
both study 3 and 4 (83 to 86 kg carcasses) support these findings.

In the present study, genotype influenced both carcass measurements
and quality. Homozygous recessive pigs had shorter length (P < 0.05) and less
BF (P < 0.01) than NN pigs. Both Nn and nn pigs produced carcasses with
larger (P < 0.05) LEA and a greater amount (P < 0.05) of lean pork when
compared to NN genotypes (Table 8). Similar results were reported by van
Laack et al. (1993). These researchers noted that homozygous recessive (nn)
pigs produced carcasses with larger LEA and less BF, but meat quality was often
sacrificed since their carcasses were more pale and exudative. On the other
hand, pigs that were heterozygous (Nn) for the stress gene produced carcasses
with larger LEA and a greater amount of lean product than pigs that were free of
the stress gene (NN) although muscle color and drip loss did not differ (van
Laack et al., 1993). These findings are similar to ours in both Exp. 3 and 4.

While initial bleeding vitamin E concentrations in serum (Table 9)
appeared to vary, there were no genotype differences. Serum vitamin E
increased (approximately 1.5-fold) in the three genotypes fed the 33 mglkg
vitamin E diets as pigs approached market weight. In humans and rodents, the
strong relationship between serum tocopherol and serum lipid has been

recognized for many years (Horwitt et al., 1972). While it is reasonable to

28

assume that pigs would be similar to humans and rodents in this regard, we are
not aware of any data in the literature that directly support this conclusion. Pigs
fed the diet containing 500 mglkg vitamin E had 3- to 5-fold greater (P < 0.01)
serum vitamin E concentrations than those fed the 33 mglkg vitamin E diet. In
addition, as they approached market weight, pigs fed the 500 mglkg vitamin E
diet had increased (5- to 6-fold) serum vitamin E concentrations with no
differences between the genotypes. Duthie and Arthur (1989) previously
reported that when 10 or 235 IU vitamin Elkg were fed, plasma vitamin E
concentrations did not differ for stress-susceptible vs stress-resistant pigs.
However, Moreira and Mahan (2002) reported a linear increase in serum vitamin
E when nursery pigs were fed 0 to 200 IU vitamin E [kg of diet. Lauridsen et al.
(2002) recently reported in sows, plasma vitamin E, when supplied as deuterated
all-raC-or-tocopheryl acetate, reached maximum concentrations 12 h post-
prandially and returned to pre-ingestion concentrations by 36 h. Our data does
not indicate that dietary vitamin E is managed differently due to differing stress

genotype in market weight pigs.

29

IMPLICATIONS

These results indicate that feeding 500 mglkg of vitamin E or injecting 600
IUI22.7 kg BW does not consistently influence carcass characteristics. Further
research is needed to determine if higher amounts of (eg. 1,000 mglkg) vitamin
E could be fed to prevent PSE. Form and duration of feeding must be evaluated
with these genotypes that appear to have altered antioxidant capability. Hence
not only vitamin E needs should be evaluated, but also other nutrients involved in

antioxidant responses such as Se, Cu, and Zn.

3O

Table 1. Experimental dietsa (as-fed basis)

 

 

. . 1, 3 Ex . 2 Ex . 2

“mm“ 9" °f d'“ 5:: 43b (PhaZe I)c (Phage II)cl
Dent yellow corn 68.88 36.40 37.96
Dried whey - 20.00 22.50
Dehulled soybean meal, 44% 22.87 14.46 32.54
Dried skim milk - 10.00 -
Spray-dried animal plasmae - 7.50 -
Oat groats - 5.00 -
Tallow 4.03 - -
Soybean oil - 3.00 3.00
Monocalcium phosphate, 21% P 1.71 1.04 1.48
Limestone 1 .06 0.90 0.87
Vrtamin and trace mineral premixf 0.50 0.50 0.50
Selenium and vitamin E premixg 0.50 0.50 0.50
NaCI 0.30 0.30 0.30
Antibacterial premixh - 025 025
L-Lysine'HCL 0.1 5 - -
Copper sulfate - 0.10 0.10
DL-methionine - 0.05 -

 

aFormulated to contain 16 % CP, 0.95 Lysine, 0.8 % Ca and 0.7 % total P.

bCorn was removed for additional vitamin E (500 mglkg) diet. By analysis, diet contained 568 mg
Int-tocopherol per kg of diet.

°Pelleted diet formulated to contain 23 % OF, 1.5 % Lysine, 0.89 96 Ca, 0.79 % total P and 0.56 %
available P.

dFormulated to contain 22 % CP, 1.3 % Lysine, 0.89 % Ga, 0.79 % total P and 0.50 % available P.
°AP-920, American Protein Corporation, Ames, IA.

fProvided per kg of complete diet: 3300 IU vitamin A, 660 IU vitamin D, 2.2 mg menadione, 3.3 mg
riboflavin, 17.6 mg niacin, 13.2 mg pantothenic acid, 111.7 mg choline, 19.8 mg vitamin 812, 37.4
mg Mn, 59.4 mg Fe, 9.9 mg Cu, 0.5 mg l, and 74.8 mg Zn.

9Provlded per kg of complete diet: 16.5 IU vitamin E and 0.3 mg Se. By analysis, the diet contained
19.3 mg (ii-tocopherol per kg diet.

"Mecadox—I 0, 2.2 % carbadox antibacterial agent, Pfizer Animal Health Company, Exton, PA.

31

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APPENDIX

41

Effect of Vitamin E and Porcine

 

Stress Syndrome (PSS) on
Antioxidant Status and
Carcass Characteristics of Pigs

Presented by:
Gerry Ann McCully

Vitamin E

 

- Vitamin E is the major lipid-soluble
antioxidant in biological systems. It
is an integral part of a network of
antioxidants, including Cu/Zn SOD,
GPX1, CAT and Cp (Lauridsen et al.,
1999).

 

 

42

 

[Antioxidant Reactions

 

 

S do
02 ”+0; +2H+fiuup7—::se—' H202+02

 

Hao2 + 2GSH GPX1 2H10 + GSSH

 

2H¢o2 ““1“” 2HzO+‘/202

02.- Fe(III) .OH 02H——\’ Peroxidation
D C D (1'11 Vitamin]:
02

F601)

 

 

Porcine Stress Syndrome
(PSS)

 

- Halothane or Stress gene
a DNA testing (Fuji et al., 1991)
- Ability to identify heterozygote (Nn)
. Limited research on heterozygote (Nn)
a Increased muscle and lean
- Increased incidence PSE

- Increased mortality when exposed
to relatively minor stresses

 

43

 

Stress Positive (nn)

 

 

 

 

 

‘ fi'g’fi‘iw» 4&3?

~ 1 17.; u

 

 

 

 

Introduction

 

- PSS shows certain etiologic
similarities to inadequacies in
cellular antioxidant defense
mechanisms, such as may be
incurred in vitamin E deficiency
(Duthie et al., 1987)

 

 

 

Objective

 

- Four studies were conducted to
determine the effects of Vitamin E
and the halothane gene on
antioxidant status and carcass
characteristics

 

 

45

Objective — Study 1

 

- To determine if carriers (Nn) of the
halothane gene given an exogenous
antioxidant (d-a-tocopherol)
demonstrate a metabolic shift in
antioxidant parameters

 

 

Methods — Study 1

 

:- Crossbred pigs (n = 39)
- Pietrain x Duroc Sows and Pietrain x
Hampshire Boars
- Average weight = 38.5 kg
- Genotype established by DNA
determination (HAL 1843*)
- 30 “Carriers” (Nn)
- 9 “Normal” (NN)

 

 

   

Methods cont. - Study 1

 

:- Housed in an open front building
I 13 pens (1.5 m x 5 m)

u 10 pens “Carriers” (Nn)

. 3 pens “Normal” (NN)

 

Housing Facility — Study 1

 

 

 

 

47

 

 

 

Methods cont. — Study 1

 

a Corn-soybean diet (16% crude
protein)

3 Vitamin E (on-tocopherol)
- Formulated: 16.5 IU per kg diet
. Actual: 19.3 IU per kg diet

 

Methods cont. — Study 1

 

n Treatments were injected i.m.
- Control = 2 ml sterilized
saline/22.7 kg of BW
- 1X Vit E = 600 IU d-a-tocopherol/22.7
kg of BW
- 2X Vit E = 1200 IU d—a—tocopherol/22.7
kg of BW

 

48

 

 

Methods cont. — Study 1

I Blood samples were collected prior
to injection and 1, 4 and 7 day:
. RBC Superoxide Dismutase (SOD)
. RBC Glutathione Peroxidase (GPX1)
. RBC Catalase (CAT)
. Serum Ceruloplasmin (CP)
. Serum Vitamin E (Vit E)

 

 

Statistical Analysis - Study 1

- Completely randomized design with
a 3 x 2 factorial arrangement of
treatment and repeated measures
over time

- GLM procedure of SAS was used to
test for treatment and genotype
effects

 

49

 

 

Statistical Analysis — Study 1

 

- Model included main effects of
injection and genotype and injection
x genotype interaction

 

 

Effect of Treatment, Genotype and Time
on Serum Vitamin E (umol/L‘l) - Study 1

 

120

100

 

DNN-NOE
Elfin-NOE
INN-IXE
INn-lXE
DNN-ZXE
INn-ZXE

40

 

 

 

20

 

1XE ZXE

24 h Post Injection T”: P < 0-01

 

50

 

Effect of Treatment, Genotype and Time
on Serum Vitamin E (pmol/L‘l) — Study 1

 

 

 

 

 

 

 

7 d Post Injection

 

 

 

Effect of Treatment, Genotype and Time on
RBC GPX1 (EU/g hemoglobin) — Study 1

 

 

 

EINN-NoE
Dun-NOE
INN-1XE
INn-IXE
CINN-2XE
INI‘I-ZXE

 

 

 

 

 

 

 

24 In Post Injection
Geno P < 0.01

 

 

51

Effect of Treatment, Genotype and Time
on CU/ZN SOD (U/mg protein) — Study 1

 

 

120-

100-

IO<

60‘

40<

 

20-

 

 

 

 

0‘

24 h Poet Injection
Geno P < 0.05

 

 

 

Implications — Study 1

 

. These data indicate that the
halothane gene influences
antioxidant activity and might affect
the nutritional requirements
associated with antioxidant defense

 

 

52

Objective — Study 2

 

To determine the impact of
injectable vitamin E on performance
and antioxidant activity of the newly
weaned pig

 

Methods cont. — Study 2

 

- Crossbred pigs (n = 154)
. Pietrain x Duroc sows and Pietrain x
Hampshire boars
- Average weight = 7.84 kg
- Average age = 24 days

 

53

 

 

Methods cont. — Study 2

 

a Genotype established by DNA
determination (HAL 1843*)
u 37 “Positive” (nn)
- 80 “Carriers” (Nn)
- 37 “Normal" (NN)

 

 

Methods cont. — Study 2

 

a At the time of weaning
- Control = no injection
- Vit E = i.m. injection of 900 IU d-a-

tocopherol

a Randomly allotted to treatment by
weight, litter and sex

a All genotypes and treatments were
represented within each pen

 

 

54

 

Methods cont. — Study 2

 

a Weights were recorded weekly
! Blood samples collected at weaning
and 28 day post—weaning:
- RBC Superoxide Dismutase (SOD)
- RBC Glutathione Peroxidase (GPX1)
- RBC Catalase (CAT)
- Serum Ceruloplasmin (CP)

 

 

Methods cont. — Study 2

 

- Diets (met or exceeded NRC)

- 0 - 7 d: complex pelleted starter diet
that contained spray—dried plasma and
dried skim milk

- 8 - 28 (1: phase II diet containing
primarily corn/soybean

 

55

 

A Statistical Analysis — Study 2

 

- Completely randomized design with
a 3 x 2 factorial arrangement of
treatments using the GLM procedure
of SAS

- Model included the main effects of
treatment and genotype and
treatment x genotype interaction

 

 

Statistical Analysis — Study 2

 

a Mean differences between
genotypes ascertained by least
significant difference

a Pen served as the experimental unit
for growth data, while individual pig
was the experimental unit for
enzyme activity

56

 

xv .
\
a...
~
L: s
‘
.. ..
D
r.

ADG — Study 2

 

   
  
  
   
 
   

0.42
0.41

0.4
0.39

0.37

Control Vlt I

Geno P < 0.01

 

Results — Study 2

 

. Vitamin E injection at weaning did

not alter antioxidant activity
a CP activity increased with time
n GPX1 increased with time

- CAT and Cu/Zn SOD remained
unchanged

 

57

 

 

Summary - Study 2

 

- Injectable Vitamin E at the time of
weaning did not alter gain or
antioxidant activity

- nn pigs had reduced ADG compared
with NN and Mn

 

Objective — Study 3 and 4

 

a To determine the impact of
genotype and vitamin E on carcass
characteristics of pigs (NN, Nn, nn)

 

58

 

 

PSE Carcass

 

 

 

Objective — Study 3

 

- To determine the impact of the
halothane gene and injectable
vitamin E 24 hours prior to slaughter
on carcass characteristics of pigs of
known genotype (nn, Nn, NN)

 

59

 

 

Methods — Study 3

 

. Crossbred pigs (n = 60)
- Pietrain x Duroc Sows and Pietrain x
Hampshire Boars

. Average weight = 69.25 kg

. Genotype established by DNA
determination (HAL 1843*)
u 14 “Positive” (nn)
- 32 “Carriers” (Nn)
. 14 “Normal" (NN)

 

Methods cont. — Study 3

 

- 24 hours prior to slaughter pigs
were injected i.m.

. Control = 2 ml sterilized saline/22.7 kg
of BW

. Vit E = 600 IU d-a-tocopherol/22.7 kg of
BW

60

 

 

Methods cont. — Study 3

 

a Pigs were transported for
approximately 2 h to a commercial
harvesting facility

a Pigs rested for a minimum of 2 h

a Carcasses were chilled overnight
and transported to a commercial
cutting plant

 

Methods cont. — Study 3

 

- pH at 45 min and 24 h on the
surface of the LM at the 10th rib

a At 24 h, two boneless chops (2.5 cm)
- Minolta light reflectance (L*)
I Drip loss (DL)

a 10th rib loin eye area (cmZ)

a 10th backfat (BF)

a Lean kg of pork produced

 

 

 

61

Effect of Vitamin E on Loin pH in
nn pigs- Study 3

 

 

 

Inn-VIII

 

 

 

 

Geno P < 0.05

 

 

Effect of Genotype on 10th Rib
Loin Eye Area, cm2 — Study 3

 

Geno P < 0.01

 

 

 

62

Effect of Genotype on 10th
Rib Backfat, cm — Study 3

2.5
2
1.5
1
0.5

 

Geno P < 0.01

 

 

Effect of Genotype on Lean
Pork (kg) — Study 3

 

50
40

30

Geno P < 0.05

20

10

 

Leen Pork

 

 

63

Effect of Genotype of Minolta
L* Values — Study 3

 

 

70-
‘01
50-
40-
30-
20-
10.

 

 

 

 

04
24h 48h 72h

Geno P < 0.01

 

Effect of Genotype on Drip
Loss (%) — Study 3

 

 

4.
3.5<
3.
2.5-
2.
1.5-
1.

 

 

 

 

 

 

0.5.
o.
“-4“. 40-72:. 72-95:-
GenoP < 0.05

 

 

 

 

Summary - Study 3

- Homozygous recessive (nn) pigs
were leaner with larger LEA

- Pigs carrying the stress gene (nn,
Nn) produced a greater amount of
lean pork

- Chops from homozygous recessive
(nn) pigs were more pale and had
the greatest amount of drip loss

 

 

Summary cont. - Study 3

. Injectable vitamin E did not affect
drip loss or color

- Injectable vitamin E did improve pH
at 45 min in the homozygous
recessive (nn) pig

 

 

 

 

65

Objective — Study 4

 

- To determine the impact of.
halothane genotype and dietagy
vitamin E on carcass characteristics
of pigs of know genotype (nn, Nn,
NN)

 

Methods — Study 4

 

- Crossbred pigs (n = 56)
- Pietrain x Duroc Sows and Pietrain x
Hampshire Boars
e Genotype established by DNA
determination (HAL 1843*)
a 14 “Positive” (nn)
- 28 “Carriers” (Nn)
- 14 “Normal” (NN)

 

 

 

Methods cont. — Study 4

 

a Two dietary treatments fed 42 days
prior to slaughter
- Control = finisher diet containing 33 IU
Vitamin E/kg diet
- High E = Formulated: control diet plus
500 IU Vitamin E/kg diet
a Blood samples were collected 72 h
prior to slaughter for serum Vitamin
E analysis

Methods cont. — Study 4

 

n Pigs were transported for
approximately 2 h to a commercial
harvesting facility

- Pigs rested for a minimum of 2 h

u Carcasses were chilled overnight
and transported to a commercial
cutting plant

67

 

 

Methods cont. — Study 4

 

- pH at 45 min and 24 h on the
surface of the LM at the 10th rib

. At 24 h, two boneless chops (2.5 cm)

- Minolta light reflectance (L*)
. Drip loss (DL)
:- 10th rib loin eye area (cmz)
- 10th backfat (BF)
- Lean kg of pork produced

 

Effect of Genotype, Treatment and
Time on Serum Vitamin E — Study 4

 

OHNUOGOV

Initial Flnel Final

 

Tl't P < 0.01

 

68

 

 

Effect of Dietary Vitamin E on Vitamin
E Content (umol/g tissue) in Chops -
Study 4

 

12

10

°°""°' m" E

 

 

 

Effect of Genotype on 10th Rib
Loin Eye Area, cm2 — Study 4

 

49
48
47
46
45
44
43
42
41
40

 

Geno P < 0.05

 

 

69

Effect of Genotype on 10th
Rib Backfat, cm — Study 4

2
1.3
1.6
1.4
1.2

1
0.8
0.6
0.4
0.2

0

 

Geno P < 0.01

 

 

Effect of Genotype on Lean
Pork (kg) — Study 4

 

40
43
47

40

Geno P < 0.05

45

44

43

 

 

70

 

Effect of Genotype of
Minolta L* Values — Study 4

 

 

24h 43h 72h

Geno P < 0.01

 

Effect of Genotype on Drip
Loss (%) — Study 4

 

 

OHNUQUION.

24-43II 43-72h 72-96h

Geno P < 0.05

 

71

 

 

Effect of Genotype on pH in
chops — Study 4

 

6.4

6.2
6

5.3

5.4

5.2

 

45 min 24 h

 

 

Summary — Study 4

 

g Dietary vitamin E (500 mg/kg diet)
fed for 42 (1 prior to slaughter
increased vitamin E content in chops

a Homozygous recessive (nn) pigs
were leaner than pigs free of the
stress gene (NN)

a Nn and nn pigs had larger LEA and
produced more lean pork than NN

 

72

 

Summary cont. — Study 4

 

- Dietary vitamin E did not influence
any of the measured carcass
characteristics

a Carriers (Nn) produced increased kg
of lean product without sacrificing
color or drip loss

 

 

Implications

 

- 500 mg/kg vitamin E or injecting
600 IU/22.7 kg BW does not
consistently influence carcass
characteristics

in Further research is needed to
determine if higher amounts of
vitamin E could prevent PSE

 

73

 

Implications cont.

 

n Stress positive (rm) and carriers
(Nn) appear to have altered
antioxidant capability and may have
differing dietary requirement than
pigs that are free of the stress gene
(NN)

 

Implications cont.

 

- Not only should vitamin E be
evaluated, but also other nutrients
involved in antioxidant responses
such as Se, Cu, and Zn

 

74

 

 

REFERENCES

Aebi, H. 1984. Catalase in vitro. Methods Enzymol. 1052121-126.

Anonymous. 1995. The porcine stress syndrome: a review of genetics,
environmental factors, and animal well-being. Agri-Practice 16(8):24—30.

Anonymous. 1996. Keeping Current - evaluating pork quality and nutritional
influences on pale, soft and exudative (PSE) syndrome. BASF Corp,
Kansas City.

Asghar, A, J. I. Gray, A. M. Booren, E. A Gomaa, M. M. Abouzied, E. R. Miller,
and D. J. Buckley. 1991. Effects of supranutritional dietary vitamin E levels
on subcellular deposition of alpha-tocopherol in the muscle and on pork
quality. J. Sci. Food Agric. 57:31.

Buckley, D. J., P. A. Morrissey, and J. 1. Gray. 1995. Influence of dietary vitamin
E on the oxidative stability and quality of pig meat. J. Anim. Sci. 73:3122-
31 30.

Byrem, T. M., A. M. Booren, G. M. Hill, F. S. Chu, and G. M. Strausburg. 1999.
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exudative pork from pigs of defined malignant hypertherrnia genotype. J.
Anim. Sci. 77:166-172.

Cannon, J. E., J. B. Morgan, J. Heavner, F. K McKeith, G. C. Smith, and D. L.
Meeker. 1995. Pork quality audit: a review of the factors influencing pork
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