Download The effect of dietary soy daidzein on pig growth and viral replication

The effect of dietary soy daidzein on pig growth and viral replication
during a viral challenge1,2
L. L. Greiner*, T. S. Stahly*3, and T. J. Stabel†
*Department of Animal Science, Iowa State University, Ames 50011 and
†National Animal Disease Center, Ames, IA 50010
ABSTRACT: Twelve replications of four littermate
pigs from a porcine reproductive and respiratory syndrome (PRRS) naive herd were weaned (11 ± 2 d of age)
and penned individually in isolation rooms. Pigs were
randomly allotted within litter to one of four dietary
soy daidzein concentrations (0, 200, 400, or 800 ppm)
to quantify the effect of daidzein on growth and immune
response during a PRRS challenge. Daidzein was provided as the soy aglycone. At 27 ± 2 d of age (4.9 ±
1.4 kg BW), pigs were oronasally inoculated with 104.3
PRRS virus/mL from strain JA142 in a 2-mL dose.
Blood was collected every 4 d from d 0 to 24 after inoculation and analyzed for serum PRRS virus, interferon,
and alpha-1-acylglycoprotein (AGP) concentrations. Serum virus and interferon peaked at 105.3 virus/mL and
79% protection, respectively, at 4 d after inoculation
and then declined steadily. Serum AGP concentration
peaked at 12 d after inoculation. Each log increase in
serum virus was associated with an increase in serum
interferon, which resulted in a decrease of pig ADG and
daily feed intake of 0.019 kg and 0.023 kg, respectively,
in 5.8-kg pigs and a feed intake reduction of 0.024 kg
in 12.5-kg pigs. Dietary daidzein additions did not (P
> 0.3) alter the serum concentration after inoculation
of PRRS virus (101.79, 101.94, 101.86, 101.93 virus/mL of
serum) or AGP. Serum concentrations of interferon responded cubically (30.3, 28.9, 29.4, and 31.1% protection) as dietary daidzein concentrations increased; however, the magnitude of the response decreased over
time. Dietary daidzein additions resulted in improvements in daily pig gain, daily feed intake, and gain/
feed during periods of peak viremia (d 4 to 16 after
inoculation), but not in periods when systemic virus
concentrations were minimized (d 16 to 24 after inoculation), resulting in a daidzein × days after inoculation
interaction. Based on these data, the magnitude of the
growth responses that occur in pigs infected with a
virus is quantitatively related to the animal’s serum
concentration of the virus and interferon, and dietary
soy daidzein at 200 or 400 ppm is a weak enhancer of
body growth in virally challenged pigs.
Key Words: Growth, Pigs, Viral Replication
2001 American Society of Animal Science. All rights reserved.
Introduction
Isoflavones are a subgroup of flavonoids that are
found in soybeans and clover (Reinli and Block, 1996)
in concentrations ranging from 100 to 5,000 ppm
1
Journal paper no. J-19101 of the Iowa Agric. and Home Econ.
Exp. Sta., Ames, Iowa, project no. 3142 and supported by Hatch Act,
State of Iowa funds, and the Illinois Soybean Program Operating
Board.
2
The authors wish to express their appreciation to William Mengeling and Ann Vorwald (USDA/ARS/National Animal Disease Center,
Ames, IA) for providing the PRRS virus used in the study and for
their guidance in the analysis of serum virus titer and to Ruth Wilson
(USDA/ARS/National Animal Disease Center, Ames, IA) for the analysis of serum interferon.
3
Correspondence: 201 Kildee Hall (phone: 515/294-5009; fax: 515/
294-1399; E-mail: [email protected]).
Received January 9, 2001.
Accepted July 31, 2001.
J. Anim. Sci. 2001. 79:3113–3119
(Wang and Murphy, 1994a). Two of the primary
isoflavones found in soybeans and soybean feed products are genistein and daidzein. Previous work performed at our station demonstrated that soy genistein,
an orally active immune modulator, reduces virus replication and increases body growth in virally challenged pigs (Greiner et al., 2001).
Soy daidzein also has been reported in vitro to elicit
potential immune modulation properties, including
enhanced phagocytosis rate of macrophages and
greater antibody production and cytotoxic T cell activity (Zhang et al., 1997).
The objective of this study was to quantify the effects
of dietary soy daidzein on pig growth and viral replication during a viral challenge.
Materials and Methods
Animals
Twelve sets of four littermate pigs from a high-leanstrain herd naive (uninfected, unvaccinated) for por-
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Greiner et al.
cine reproductive and respiratory syndrome (PRRS)
virus were used. Pigs were weaned at 11 ± 2 d of age
and penned individually on slatted floors in 61- × 122cm pens. Pigs were reared in the disease isolation
rooms at the National Animal Disease Center
(NADC), Ames, IA, to minimize the animals’ exposure
to other antigens. During the first 3 d after weaning,
each pig was administered intramuscularly 4.4 mg of
ceftiofur sodium (Naxcel, Pharmacia & Upjohn Animal
Health, Kalamazoo, MI) per kilogram per day. Subsequently, no therapeutic or subtherapeutic antimicrobial agents were provided. A thermal climate of 24° to
29°C was maintained.
At weaning, the pigs were randomly allotted within
litter to the basal diet supplemented with one of four
dietary concentrations of daidzein (0, 200, 400, or 800
ppm). The diets contained nutrient concentrations
that met or exceeded the nutrient requirement of highlean pigs fed from 5 to 18 kg (NRC, 1998). Pigs were
allowed to consume feed and water ad libitum.
Seventeen days following weaning, the pigs were
oronasally inoculated with 104.3 PRRS virus/mL of
strain JA142 in a 2-mL dose (courtesy of William
Mengeling, USDA/ARS/National Animal Disease Center, Ames, IA). From d 0 to 24 after inoculation, body
temperatures were measured daily in the control pig
from each replication using a digital rectal thermometer. Feed intake and BW gain also were measured
every 4 d before and after inoculation. Blood samples
were collected via venipuncture every 4 d before and
after inoculation for determination of serum concentrations of virus, interferon, and alpha-1-acylglycoprotein (AGP). Blood was also collected at weaning,
inoculation, and at the removal from test (15 kg BW)
for determination of serological titers for five major
pathogens: Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae, PRRS virus, swine influenza
virus, and transmissible gastroenteritis.
At BW of 15 ± 2 kg, pigs were killed and the spleens
and thymuses were rapidly harvested and weighed.
One pig died after inoculation due to a twisted gut.
The Committee on Animal Care at NADC approved
the animal care procedures employed.
Experimental Diets
The basal diet consisted of a corn, soy protein concentrate, and whey mixture fortified with minerals and
vitamins (Table 1). The experimental diets consisted
of the basal diet supplemented with 0, 200, 400, or
800 ppm of soy daidzein. Soy daidzein was provided
primarily in the aglycone form as a 93.7% pure extract
(CSI Metabolics, Newport Beach, CA). The daidzein
concentrations of the basal diet and daidzein extracts
were analyzed via high-performance liquid chromatography method (Wang and Murphy, 1994b) and are reported in Table 2. The analyzed concentrations of
daidzein on an aglycone-equivalent basis in the experimental diets were 33, 232, 432, and 805 ppm.
Table 1. Basal diet composition (%)
Item
Yellow corn
Soy protein concentrate
Dried whey
Dried skim milk
Choice white grease
L-Lysine HCl
L-Threonine
D,L-Methionine
Tryptosine
Dicalcium phosphate
Limestone
Salt
Choline chloridea
Trace mineral and vitamin premixb
Daidzein carrierc
% of Diet
36.76
28.85
20.00
5.00
4.00
0.20
0.15
0.30
0.15
2.74
0.33
0.40
0.20
0.52
0.40
a
Choline chloride was provided as a 60% choline chloride mixture.
Provided the following per kg of diet: biotin 0.15 mg, folacin 1.8 mg,
niacin 90 mg, pantothenic acid 60 mg, riboflavin 21 mg, pyridoxine 4.5
mg, thiamin 3 mg, vitamin A 13,200 IU, vitamin D3 1,320 IU, vitamin
E 96 IU, vitamin K 3 mg, vitamin B12 105 µg, vitamin C 100 mg, Zn
212 mg, Cu 17.5 mg, Fe 175 mg, Mn 60 mg, I 0.20 mg, and Se 0.30
mg.
c
Daidzein source added at the expense of starch carrier.
b
Serological Titers
The presence of serum titers for PRRS virus, transmissible gastroenteritis virus, swine influenza virus,
Mycoplasma hyopneumoniae, and Actinobacillus pleuropneumoniae were determined by the Iowa State University Veterinary Diagnostic Laboratory, Ames, via
methods outlined by Greiner et al. (2000). These samples were taken to verify that the animals did not have
passive or active titers for PRRS before inoculation
and to determine the exposure status of the pigs to
other prevalent pig antigens.
Serum Virus and Immune Parameters
Serum PRRS virus concentrations were analyzed by
the procedure provided by William Mengeling, USDA/
ARS/NADC, as described by Greiner et al. (2000). Serum interferon concentrations were assayed using a
modified cell bioassay (Rubinstein, 1981) as described
by Greiner et al. (2000). A 1:64 dilution was used in
the assay. The interferon-gamma concentration consisted of 76.1% of the total interferon present in the
serum. Serum AGP concentrations were analyzed using a radial-immunodiffusion assay (Cardiotech Services, Louisville, KY) as described by Greiner et al.
(2000).
Data Analysis
Pigs were placed into blocks by litters and then randomly allotted within litter to one of four dietary treatments. Data were analyzed as a randomized complete
block design by analysis of variance technique using
the GLM procedure of SAS (SAS Inst. Inc, Cary, NC).
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Daidzein, pig growth, and immune response
Table 2. Isoflavone concentration of basal diet and daidzein sourcea
As-is basis, ppm
Isoflavone
Daidzein
Malonyl daidzin
Acetyl daidzin
Daidzin
Daidzein
Total
Genistein
Malonyl genistin
Acetyl genistin
Genistin
Genistein
Total
Glycitin
Malonyl glycitin
Acetyl glycitin
Glycitin
Glycitein
Total
Aglycone equivalent
Basal diet
Daidzein source
Basal diet
Daidzein source
13
23
21
0
57
0
0
0
937,000
937,000
10
11
12
0
30
0
0
0
572,000
572,000
19
32
30
0
81
0
0
0
0
0
10
20
20
0
50
0
0
0
0
0
0
14
0
0
14
0
0
0
0
0
0
10
0
0
10
0
0
0
0
0
a
Determined by Patricia Murphy, Iowa State University, Ames, by high-performance liquid chromatography.
Pig weight at inoculation was used as a covariate when
analyzing pig growth, serum virus concentration, and
immune parameters after inoculation. Responses of
body weight gain, feed intake, gain/feed, serum concentrations of virus, interferon, and AGP, as well as
plasma concentration of endotoxin, over the 4-d periods were analyzed as repeated measures. The error
terms used to test the effects of daidzein, period, and
daidzein × period were, respectively, replicate × daidzein, replicate × period, and replicate × daidzein ×
period.
The relationship of serum virus concentrations, pig
performance traits, and immune response was analyzed by multiple regression techniques using the
backward stepwise regression model procedures of
SAS. Variables that were not significant (P > 0.10)
were deleted from the regression model. The independent variables included in the analysis were the linear
and quadratic effects of serum concentrations of PRRS
virus, interferon, and AGP and BW for d −4 to 24 after
inoculation. The dependent variables were pig BW
gain and feed intake for each 4-d period immediately
prior to the measurement of the independent variable
from −4 to 24 d after inoculation. The pig was considered the experimental unit. Data were reported as
least squares means.
also naive for Mycoplasma hyopneumoniae at weaning, before inoculation, and at the termination of the
study. Passively acquired titers for Actinobacillus
pleuropneumoniae, transmissible gastroenteritis, and
swine influenza virus were present in 8, 73, and 75%
of the pigs at weaning, which subsequently reached
25, 67, and 25% at inoculation and 8, 58, and 0% of
the animals, respectively, at termination (15 kg BW)
of the study.
Effect of Virus Inoculation
Pigs were not infected until after 21 d of age to
ensure that the immune system was developed and
Results and Discussion
Serological Titer Status
Serological results confirmed that pigs were PRRSnaive at weaning and immediately prior to inoculation.
Based on the level of serum antibody titers, pigs were
Figure 1. Mean daily rectal temperatures from d 0 to 24
d after inoculation. Data are represented as least squares
means of 12 pigs (pig fed 0 ppm daidzein in each replication). SEM = standard error of the mean.
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Greiner et al.
Figure 2. Mean daily pig weight gain (g) during 4-d
periods from −8 to 24 d after inoculation. Data are pooled
across dietary soy daidzein concentrations. SEM = standard error of the mean.
functional in the experimental animals (Varley, 1995).
Pigs were fed their experimental diets before and after
inoculation to allow potential effects of daidzein on the
animal’s initial susceptibility to the PRRS virus and
the animal’s subsequent ability to eliminate the virus
after inoculation to be expressed.
Within 4 d after inoculation, pigs experienced elevated temperatures (≅ 40.6°C), coughing, and anorexia
(Figure 1). In agreement with our previous report
(Greiner et al., 2000), pig serum viral concentration
peaked at 4 d after inoculation (105.3/mL of blood) and
then declined linearly. Serum PRRS virus existed in
100 and 50% of the pigs, respectively, on d 4 and 24
after inoculation.
Serum concentrations of interferon, a signaling compound for stimulation of macrophages (Kuby, 1997),
peaked at 4 d after inoculation in correspondence with
virus concentration, which was similar to the pattern
of response reported by Greiner et al. (2000). However,
serum AGP did not peak until 8 to 12 d after inoculation (685 µg/mL). This response was expected, because
AGP is produced by liver hepatocytes in response to
proinflammatory cytokine release by macrophages and
requires 8 to 12 d to be synthesized and released in
circulation (Kuby, 1997).
Figure 3. Mean daily feed intake (g) during 4-d periods
from −8 to 24 d after inoculation. Data are pooled across
dietary soy daidzein concentration. SEM = standard error
of the mean.
Following inoculation, daily pig gain decreased from
260 g for the 4-d period before inoculation to 100 g
for d 4 to 8 after inoculation (Figure 2). Feed intake
decreased from 279 g/d for the 4-d preinoculation period to 175 g/d for 4 to 8 d after inoculation (Figure
3). Based on these data, the viral exposure used in this
study created a prolonged and substantial immune
response and resulted in growth inhibition.
Dietary Daidzein and Daidzein × Day Effects
From weaning to inoculation, pig ADG decreased
linearly (P < 0.04) as soy daidzein concentrations increased (Table 3). Daily feed intake tended to respond
cubically (P < 0.10) to increased concentrations of dietary soy daidzein (Table 3). However, pig weight before inoculation was not altered between dietary
groups (P = 0.34).
After inoculation, dietary daidzein additions did not
(P > 0.10) alter serum concentrations of virus (Figure
4) or AGP (data not shown). Dietary additions of 200
and 800 ppm, but not 400 ppm, soy daidzein resulted
in greater serum interferon during the initial period
of high viremia (d 4), but not in subsequent periods
Table 3. Effect of dietary daidzein on pig performance from weaning to inoculationa
Dietary daidzein concentration, ppm
Criterion
Number of pigs
Pig weight, kg
Weaning
Inoculation
Growth and feed utilization
Pig gain, g/d
Feed intake, g/d
Gain/feed
a
0
12
200
12
400
12
11
SEM
P-valueb
—
—
3.41
6.06
3.40
6.06
3.41
5.60
3.38
5.64
0.10
0.13
0.99
0.34
192
196
0.903
189
214
0.839
155
184
0.746
162
182
0.710
6
6
0.02
0.04L
0.10C
0.01L
Least squares means reported.
Linear (L) and cubic (C) effect of dietary daidzein concentration.
b
800
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Daidzein, pig growth, and immune response
Figure 4. Effect of dietary soy daidzein (Daid) concentration on serum concentration of virus (10x/mL) from d
0 to 24 after inoculation. Data are represented as least
squares means with pig weight at inoculation (d 0) used
as a covariate. SEM = standard error of the mean.
(— 0, — — 200, — ⴢ — 400, ⴢ ⴢ ⴢ 800 ppm Daid).
Figure 5. Effect of dietary soy daidzein (Daid) concentration on serum concentration of interferon-gamma (%
protection) from d 0 to 24 after inoculation. Data are
represented as least squares means with pig weight at
inoculation (d 0) used as a covariate. SEM = standard
error of the mean. (— 0, — — 200, — ⴢ — 400, ⴢ ⴢ ⴢ 800
ppm Daid).
after inoculation, which resulted in a cubic daidzein ×
day after inoculation interaction (P < 0.01, Figure 5).
The effects of dietary daidzein addition on pig
growth and feed utilization after inoculation were dependent on dietary daidzein concentrations and days
after inoculation. Dietary soy daidzein additions resulted in improvements in pig ADG (Figure 6), daily
feed intake (Figure 7), and gain/feed (Figure 8) in periods of high viremia (d 4 to 16 after inoculation), but not
in periods when systemic virus concentrations were
minimized (d 16 to 24 after inoculation), resulting in
a daidzein × days after inoculation interaction (P <
0.07). Specifically, ADG, and to a lesser degree daily
feed intake and gain/feed, were improved by addition
of 200 ppm and(or) 400 ppm daidzein but were depressed by the addition of 800 ppm daidzein. Based
on these data, the magnitude of the improvement was
greatest during d 4 to 16 after inoculation when viremia was high and then was lost during d 20 to 24 after
inoculation, when systemic virus concentration had
been minimized. Over the duration of the study, di-
Table 4. Effect of dietary daidzein on pig performance and immune measurements from d 0 to 24
after inoculation (PI)a
Dietary daidzein concentration, ppm
Criterion
0
200
400
800
SEM
P-valueb
—
Number of pigs
12
12
12
11
—
Postinoculation pig weight, kg
D 0 PI
D 24 PI
6.06
13.40
6.06
13.96
5.60
12.58
5.64
12.52
0.13
0.15
0.34
0.60
294
388
0.776
307
388
0.775
309
393
0.781
291
371
0.748
8
10
0.01
0.30
0.40
0.50
1.79
30.3
376.7
1.94
28.9
374.0
1.87
29.4
391.4
1.93
31.1
385.6
0.06
0.79
7.04
0.30
0.35
0.47
2
1
0.03L
0.30
Growth and feed utilization (pooled across d 0 to 24 PI)cd
Pig gain, g/d
Feed intake, g/d
Gain/feed
Serum immune measurements (pooled across d 0 to 24 PI)de
Virus, 10x/mL
IFN, % protection
AGP, µg/mL
Immune organ weights, g (at 15 kg BW)d
Spleen
Thymus
a
77
50
88
46
71
50
87
54
Least squares means reported.
Linear (L) effect of dietary daidzein concentration.
c
Means of 4-d body weight gain, feed intake, or gain/feed ratio pooled across d 0 to 24 after inoculation.
d
Pig body weight at d 0 after inoculation used as covariate.
e
Means of 4-d serum concentrations of PRRS virus, interferon (IFN), or alpha-1-acylglycoprotein (AGP) pooled across d 0 to 24 after
inoculation.
b
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Greiner et al.
Figure 6. Effect of dietary soy daidzein (Daid) concentration on mean daily pig weight gain (g) during 4-d
periods from d (D) 0 to 24 after inoculation. Data are
represented as least squares means with pig weight at
inoculation (d 0) used as a covariate. SEM = standard
error of the mean, Quad = Quadratic (— 0, — — 200,
— ⴢ — 400, ⴢ ⴢ ⴢ 800 ppm Daid).
etary soy daidzein additions resulted in a linear increase (P < 0.03) in spleen weight, which signifies an
increase in B cell production, but did not alter (P =
0.30) thymus weight (Table 4).
The supplemental levels of 200, 400, and 800 ppm
daidzein used in the study are estimated to represent
moderate, high, and very high concentrations relative
to those present in commercial soybean meal-based
diets for high-lean nursery pigs. These estimations
are based on the following. Soy-based feedstuffs are
frequently included in nursery diets of high-lean pigs
at concentrations ranging from 30 to 50%. The daidzein content of soybeans used for human consumption
has been reported to range from 240 to 600 µg/g (Wang
and Murphy, 1994b), with 235 to 588 µg/g being present in the glycone form. The processing of soybeans
Figure 7. Effect of dietary soy daidzein (Daid) concentration on mean daily feed intake (g) during 4-d periods
from d (D) 0 to 24 after inoculation. Data are represented
as least squares means with pig weight at inoculation (d
0) used as a covariate. SEM = standard error of the mean,
Lin = Linear (— 0, — — 200, — ⴢ — 400, ⴢ ⴢ ⴢ 800 ppm Daid).
Figure 8. Effect of dietary soy daidzein (Daid) concentration on mean gain:feed during 4-d periods from d (D)
0 to 24 after inoculation. Data are represented as least
squares means with pig weight at inoculation (d 0) used
as a covariate. SEM = standard error of the mean, Quad
= Quadratic (— 0, — — 200, — ⴢ — 400, ⴢ ⴢ ⴢ 800 ppm Daid).
into soybean meal via hexane extraction likely has
minimal effect on isoflavone concentration because of
the low solubility of isoflavones in organic solvents and
lipids. The aglycone form of daidzein represents 2 to
3% by weight of the glycone forms present in most soy
products, although the aglycone form must be achieved
for maximum absorption (Izmi et al., 2000). Therefore,
the supplemental levels of 200 and 400 ppm daidzein
represent moderate and high levels of daidzein possibly present in nursery diets for high-lean pigs. The
800-ppm daidzein diet was used to determine whether
daidzein could elicit negative effects when fed at extremely high doses and whether the palatability of
isoflavones could negatively influence feed intake. In
this study, high levels of daidzein over the duration
of the study did not alter feed intake or reduce pig
body weight gain.
Daidzein has been shown in vivo to increase B and
T lymphocyte activity and phagocytosis rate of rat
macrophage cells (Zhang et al., 1997). However, in the
current study, dietary daidzein did not alter the rate
of serum virus elimination, even though it did enhance
pig growth and feed utilization during periods of high
viremia. In a previous study performed by our group,
the addition of soy genistein did decrease serum PRRS
virus concentration and enhance pig growth after inoculation (Greiner, 2001). However, in both studies, the
soy isoflavones daidzein and genistein exhibited their
greatest efficacy during periods of high viremia.
Specifically, factors that minimized serum PRRS virus concentration by one log within the first 4 d after
inoculation resulted in an increase in daily pig weight
gain in pigs of similar weight by 0.034 kg. The improvement in weight gain by each additional log reduction
in virus would result in an exponential increase in
pig weight gain (Greiner et al., 2000). In our previous
work, serum concentrations of PRRS virus in pigs was
shown to be quantitatively related to the animals body
3119
Daidzein, pig growth, and immune response
Table 5. Quantitative relationship of serum virus concentration and immune
measurements on individual 4-d pig gain in 48 pigs from d −4 to 24 after inoculation.
(day-0 to -4 pig gain and feed intake values were regressed to d- −4 virus
concentration, interferon, and alpha-1 acylglycoprotein)
Pig gain, kg/4 da
Factor
Intercept
Pig weight, kg
Virus, 10x/mL
Virus × virus
Alpha-acylglycoprotein (AGP), 1 µg/mL
Interferon (IFN), %
AGP × IFN
IFN × IFN
bb
SE
P-value
0.54351
0.13426
−0.14172
−0.01929
−0.00005
−0.01514
−0.00001
0.00015
0.183
0.010
0.036
0.010
0.001
0.005
0.001
0.001
0.01
0.01
0.01
0.02
0.85
0.01
0.06
0.01
R2 for pig gain = 0.78.
b = Coefficient for each factor.
a
b
growth rate after inoculation (Greiner et al., 2000). In
the current study, each log reduction in serum virus
was associated with a reduction in serum interferon,
and an increase in daily pig gain and daily feed intake
of 0.019 kg and 0.023 kg, respectively, in 5.8-kg pigs
and an increase in feed intake of 0.024 kg in 12.5-kg
pigs (Table 5).
Implications
Orally active compounds, such as the soy isoflavone
daidzein, influence the growth of virally challenged
animals. The biological response of animals to pathogens and(or) vaccines may be altered by the type and
level of soy isoflavones they ingested.
Literature Cited
Greiner, L. L., T. S. Stahly, and T. J. Stabel. 2000. Quantitative
relationship of systemic virus concentration on growth and immune response in pigs. J. Anim. Sci. 78:2690−2695.
Greiner, L. L., T. S. Stahly, and T. J. Stabel. 2001. The effect of
dietary soy genistein on pig growth and viral replication during
a viral challenge. J. Anim. Sci. 79:1272–1279.
Izumi, T., M. K. Piskula, S. Osawa, A. Obata, K. Tobe, M. Saito,
S. Kataoka, Y. Kubota, and M. Kikuchi. 2000. Soy isoflavone
aglycones are absorbed faster and in higher amounts than their
glucosides in humans. J. Nutr. 130:1695–1699.
Kuby, J. 1997. Immunology. 3rd ed. W. H. Freeman and Company,
New York.
NRC. 1988. Nutrient Requirements of Swine. 9th ed. National Academy Press, Washington, DC.
Reinli, K., and G. Block. 1996. Phytoestrogen content of foods—a
compendium of literature values. Nutr. Cancer 26:123−148.
Rubinstein, S., P. C. Familetti, and S. Pestka. 1981. Convenient assay
for interferons. J. Virol. 37: 755−758.
Varley, M. A. 1995. The Neonatal Pig: Development and Survival.
CAB International, Oxon, U.K.
Wang, H.-J, and P. A. Murphy. 1994a. Isoflavone composition of
American and Japanese soybeans in Iowa: Effects of variety,
crop year, and location. J. Agric. Food Chem. 42:1674−1677.
Wang, H., and P.A. Murphy. 1994b. Isoflavone content in commercial
soybean foods. J. Agric. Food Chem. 42:1666–1673.
Zhang, R., L. Yaping, and W. Weiqun. 1997. Enhancement of immune
function in mice fed high doses of soy daidzein. Nutr. Cancer
29:24−28.