Animal Feed Science and Technology 174 (2012) 79–85
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Animal Feed Science and Technology
journal homepage: www.elsevier.com/locate/anifeedsci
Zearalenone enhances reproductive tract development, but does not
ଝ
alter skeletal muscle signaling in prepubertal gilts
a,∗ a b b c b
W.T. Oliver , J.R. Miles , D.E. Diaz , J.J. Dibner , G.E. Rottinghaus , R.J. Harrell
a
USDA, ARS, U.S. Meat Animal Research Center, Clay Center, NE, United States
b
Novus International, Inc., St. Charles, MO, United States
c
Veterinary Medical Diagnostic Laboratory, University of Missouri, Columbia, MO, United States
a r t i c l e i n f o a b s t r a c t
Article history: Zearalenone is a potent mycotoxin that has estrogenic properties. In vitro results indicate
Received 23 March 2011
that zearalenone metabolites down-regulate proteins associated with protein synthesis
Received in revised form 24 February 2012
(protein kinase B, Akt) and cellular proliferation (extracellular signal-regulated kinase, ERK).
Accepted 27 February 2012
The objectives of this study were to determine the effect of zearalenone on (1) growth
performance and signaling for protein synthesis, and (2) reproductive tract development.
At 28 d of age, gilts were randomly assigned to consume a commercial basal diet (C) or
Keywords:
Mycotoxin C+1.5 mg/kg zearalenone (n = 10) for 4 wk, at which time gilts were euthanized, urine col-
lected, and tissue collected. No differences were observed in average daily gain, average
Skeletal muscle
Swine daily feed intake, or gain:feed (P>0.28). Reproductive tract weight (2.4-fold) and uterine
Uterus endometrial gland development (50%) were increased in zearalenone fed gilts (P<0.01). In
Zearalenone uterus, estrogen receptor (ER)-␣ expression was unchanged (P>0.28), but gilts consuming
zearalenone had 2.0- and 3.5-fold higher abundance of ER- mRNA and protein, respec-
tively (P<0.01). No differences were observed in Akt, mammalian target of rapamycin, or
ERK abundance or phosphorylation in muscle (P>0.36). Zearalenone had no effect on growth
performance or skeletal muscle signaling in prepubertal gilts, but zearalenone increased
reproductive tract size and glandular development, possibly due, in part, to altering the
expression of ER-.
Published by Elsevier B.V.
1. Introduction
The mycotoxin zearalenone is a potent estrogenic secondary metabolite produced by several Fusarium species (Riley and
Petska, 2005). Zearalenone is a resorcylic acid lactone that is most commonly found in many cereal crops and swine are
one of the most sensitive species with tolerance limits in feed as low as 50 g/kg proposed (BML, 2000). Zearalenone and
its metabolites ␣-zearalenol and -zearalenol have been shown, in vitro, to down-regulate signaling for protein synthesis
and cellular proliferation (Wollenhaupt et al., 2004). In addition, zearalenone, as well as ␣-zearalenol and its metabolite
Abbreviations: ADFI, average daily feed intake; ADG, average daily gain; Akt, protein kinase B; BW, body weight; C, commercial basal diet; COX2,
prostaglandin-endoperoxide synthase 2; DON, deoxynivalenol; ER, estrogen receptor; ERK, extracellular signal-regulated kinase; ESI, electrospray ioniza-
tion; G:F, gain to feed ratio; GLM, general linear model; HPLC/MS, high-performance liquid chromatography/mass spectrometry; mTOR, mammalian target
of rapamycin; tcRNA, total cellular ribonucleic acid.
ଝ
Mention of trade names, proprietary products, or specified equipment does not constitute a guarantee or warranty by the USDA and does not imply
approval to the exclusion of other products that may be suitable. USDA is an equal opportunity provider and employer.
∗
Corresponding author at: USDA, ARS, P.O. Box 166, U.S. Meat Animal Research Center, Clay Center, NE, United States. Tel.: +1 402 762 4206;
fax: +1 402 762 4209.
E-mail address: [email protected] (W.T. Oliver).
0377-8401/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.anifeedsci.2012.02.012
80 W.T. Oliver et al. / Animal Feed Science and Technology 174 (2012) 79–85
Table 1
a
Composition and calculated nutrient content of the diets.
Item Diets
b
Phase 2 Phase 3
Control Zearalenone Control Zearalenone
Ingredients, g/kg
Maize 477.5 470.0 622.2 614.7
Soybean meal, 465 g/kg 240.0 240.0 299.9 299.9
Lactose 120.0 120.0 0.0 0.0
Poultry meal 37.0 37.0 0.0 0.0
Fishmeal 35.0 35.0 0.0 0.0
Choice white grease 35.0 35.0 35.0 35.0
Monocalcium phosphate 13.3 13.3 14.1 14.1
Limestone 4.0 4.0 8.9 8.9
c
Mecadox, 5.5 g/kg 10.0 10.0 5.0 5.0
Lysine HCl,788 g/kg 3.0 3.0 3.0 3.0
l-Threonine,985 g/kg 14.0 14.0 1.1 1.1
Methionine hydroxy analog, 880 g/kg 2.2 2.2 1.8 1.8
Salt 4.0 4.0 5.0 5.0
CuSO4 1.0 1.0 0.0 0.0
d
Vitamin premix 2.5 2.5 2.5 2.5
e
Mineral premix 1.5 1.5 1.5 1.5
Toxin 0.0 7.5 0.0 7.5
f
Calculated nutrient content
Metabolizable energy, MJ/kg 14.3 14.2 14.4 14.3
Crude protein, g/kg 208.0 207.0 196.0 195.0
Total ileal digestible lysine, g/kg 12.5 12.0 12.5 12.0
Ca, g/kg 8.6 8.6 7.2 7.2
Available P, g/kg 5.2 5.2 3.7 3.7
a
Expressed on as-fed basis.
b
A common phase 1 diet was fed for one week prior to the initiation of treatments.
c
For control of enteritis and improved growth performance.
d
Provided the following per kilogram of diet: vitamin A, 9000 IU; vitamin D3, 200 IU; vitamin E, 19 IU; vitamin K, 2.2 mg; thiamine, 2.2 mg; riboflavin,
4.4 mg; niacin, 33 mg; pantothenic acid, 22 mg; vitamin B12, 0.028 mg; vitamin B6, 2.2 mg; folic acid, 1.35 mg; and biotin, 0.11 mg.
e
Provided the following per kilogram of diet: Fe, 78 mg; Cu, 7 mg; Co, 0.80 mg; Zn, 168 mg; Mn, 60 mg; I, 0.79 mg; and Se, 0.13 mg.
f
Calculated nutrient content based on standard feed tables (NRC, 1998).
␣-zearalanol, accumulate in muscle tissue, in vivo (Zöllner et al., 2002). Thus, one objective of the current experiment
was to determine the effect of zearalenone contaminated feed on growth performance and protein synthesis signaling in
prepubertal gilts.
The effect of zearalenone on the reproductive performance of pigs is highly dependent on their reproductive status
(Diekman and Green, 1992) and prepubertal gilts are particularly susceptible to zearalenone-contaminated feed (Kordic
et al., 1992). In gilts, high levels of zearalenone (>22.0 mg/kg, Tiemann and Dänicke, 2007) can have deleterious effects on
reproductive performance including a decrease in corpora lutea, decreased ovary size, decreased fertility, and an increase
in abortion rates. More moderate doses (>2.0 mg/kg, Kordic et al., 1992; Döll et al., 2004) will increase vulva and total
reproductive tract size in gilts. The effects of zearalenone and its metabolites are mediated through the estrogen receptors
(ER), and these metabolites have an affinity for both the -␣ and - isoforms of the ER (Mueller et al., 2004; Takemura
et al., 2007). Thus, the second objective of this experiment was to determine the effect of zearalenone contaminated feed
on the reproductive tract development, including the abundance of key regulators of reproductive tract development and
maintenance.
2. Materials and methods
2.1. Animal care and treatments
All animal procedures were reviewed and approved by NOVUS International Animal Care and Use Committee. Gilts
(n = 10 per treatment; PIC cross, landrace × large white females × large white × duroc × pietran males) were weaned from
their sow at 21 d of age, blocked by weight and placed in a pen containing two pigs per pen. A three-phase feeding program
was implemented for 35 d with diets formulated to meet or exceed NRC requirements (NRC, 1998). Dietary ingredients
were screened, and found negative, throughout the purchasing process for levels of most common mycotoxins, including
zearalenone. Gilts were allowed to adjust for 1 wk on a commercial diet, at which time gilts were randomly assigned to
consume a control basal diet or the control diet + 1.5 mg/kg zearalenone for 4 wk (Table 1). Gilts were weighed gravimetrically
at d 0, 7, 14, 21, and 28 of treatment. In addition, vulva height, width, and length were measured as the gilts were weighed.
W.T. Oliver et al. / Animal Feed Science and Technology 174 (2012) 79–85 81
Table 2
a
Primer sequences for real time RT-PCR analysis.
mRNA Primer Sequence Fragment size (bp)
PR F 5 -AAGTCACTGCCAGGTTTTCG 209
R 5 -TGCCACATGGTAAGGCATAA
COX2 F 5 -TCGACCAGAGCAGAGAGATGAGAT 134
R 5 -ACCATAGAGCGCTTCTAACTCTGC
ER␣ F5-AGCACCCTGAAGTCTCTGGA 160 R5-TGTGCCTGAAGTGAGACAGG
ER F 5 -GGCAACGACTTCAAGGTTTC 239
R 5 -CTGCTGCTGGGAGGAGATAC
ERK1 F 5 -CAGTCTCTGCCCTCCAAGAC 218
R 5 -GGGTAGATCATCCAGCTCC
ERK2 F 5 -AGGGGCGGTTTCTGATAGTT 225
R 5 -GAGGAACAGGGTCAGCAGAG
mTOR F 5 -GAAGAGCACGACCTGGAGAG 249
R 5 -GTCCAGCTTCTCCCCTTTCT
18S F 5 -ATGGCCGTTCTTAGTTGGTG 217
R 5 -CGCTGAGCCAGTCAGTGTAG
a ◦ ◦ ◦ ◦
The PCR conditions included denaturation (95 C, 2 min) followed by 40 amplification cycles of 95 C for 15 s, 60 C for 15 s, and 70 C for 45 s. F, forward
primer; R, reverse primer; PR, progesterone receptor; COX2, prostaglandin-endoperoxide synthase 2; ER␣, estrogen receptor alpha; ER, estrogen receptor
beta; ERK, extracellular signal-related kinase; mTOR, mammalian target of rapamycin; 18S, 18S ribosomal protein.
2.2. Zearalenone culture method
To culture the zearalenone, shelled maize (100 g) was added to 1 quart wide mouth canning jars and 40 mL of water is
◦
added. The jars were autoclaved for 20 min at 121 C and 138 kilopascal, then inoculated with a sterile aqueous suspension
of Fusarium graminearum R5454 from the Penn State Fusarium culture collection. The jars were shaken, the lids loosened for
◦
additional respiration, and then stacked lying on their sides. They were incubated in the dark for 4 weeks at 23 C. After 4 wk
◦
the jars were autoclaved and the culture material was dried, ground, blended and stored at −20 C. Multiple subsamples of
the culture material were analyzed for zearalenone, aflatoxins, ochratoxin A, fumonisins, DON and T-2 toxin concentration
by HPLC with fluorescence detection.
2.3. Sample collection and analytical procedures
After 4 wk of treatment, gilts were euthanized and urine was collected from the bladder. At this time, the reproductive
tract was removed intact and weighed. A sample of the uterine body and muscle (longissimus dorsi, approximately at the
◦
last rib) were collected and snap-frozen in liquid nitrogen and stored at −80 C for later analyses. Urine was treated with
beta-glucuronidase/sulfatase (Sigma, St. Louis, MO, USA), affinity column cleanup (R-Biopharm, Darmstadt, Germany) and
analyzed by HPLC/MS using ESI in the negative mode with single ion monitoring for zearalenone and ␣- and -zearalenol
(adapted from Songsermsakul et al., 2006). Uterine samples were collected, fixed in 10% formalin for 24 h, and embedded
in paraffin. Cross-sections of the uterine segments were cut approximately 8 m thick with a microtome and were stained
with hematoxylin and eosin-Y. Morphometric measurements were performed by one person using light microscopy with a
computer assisted morphometric system. The area of endometrial and total uterine tissue was measured.
2.4. RNA isolation and real-time PCR
Total cellular RNA (tcRNA) was isolated from the uterus and longissimus dorsi using RNeasy Mini kits (Quiagen, Valencia,
CA, USA) and samples were DNase I treated on the column using DNase I provided by the manufacturer. Total RNA was
quantified using a ND-1000 spectrophotometer (Nano-Drop Technologies, Wilmington, DE, USA). Total cellular RNA samples
(1 g) were reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Each cDNA sample was
assayed in duplicate (25 ng tcRNA equivalents) for progesterone receptor, prostaglandin-endoperoxide synthase 2 (COX2),
ER-␣, ER-, extracellular signal-related kinases (ERK) 1 and 2, and mammalian target of rapamycin (mTOR) with appropriate
0.25 M forward and reverse primers (Table 2), 12.5 L of Taq, SYBR green, and ROX supermix (Bio-Rad, Hercules, CA, USA)
included in a 25 L reaction. Ribosomal 18S primers (Table 2) were used to measure 18S mRNA concentrations, which were
not different between treatments (data not shown). Therefore, 18S was used as the reference control transcript to calculate
relative quantities of each target transcript using the comparative cycle threshold method (Livak and Schmittgen, 2001).
Amplification was performed using a PTC-200 thermocycler fitted with a chromo 4 fluorescence detector (Bio-Rad, Hercules,
◦ ◦ ◦
CA, USA). The PCR conditions included denaturation (95 C, 2 min) followed by 40 amplification cycles of 95 C for 15 s, 60 C
◦
for 15 s, and 70 C for 45 s. Melting curve analysis and gel electrophoresis was used to confirm the amplification of a single
product of the predicted size.
82 W.T. Oliver et al. / Animal Feed Science and Technology 174 (2012) 79–85
Table 3
a
Urine levels of zearalenone and its metabolites and performance of prepubertal gilts fed control or control + zearalenone (1.5 mg/kg) diets for four weeks.
Variable Treatment P value
Control Zearalenone
Zearalenone, g/kg 4.0 ± 85.3 292.4 ± 76.2 <0.025
␣-zearalenol, g/kg 1.3 ± 21.9 113.3 ± 19.6 <0.002

-zearalenol, g/kg 0.0 ± 3.5 14.6 ± 3.1 <0.007
Live weight, kg
d 0 6.45 ± 0.21 6.97 ± 0.19 0.10
± ±
d 7 9.27 0.50 10.48 0.44 0.11
d 14 12.19 ± 0.46 12.96 ± 0.41 0.25
d 21 17.85 ± 0.91 19.70 ± 0.81 0.17
d 28 21.55 ± 0.94 23.26 ± 0.85 0.21
Average daily gain, g/d
d 0–14 442 ± 28 461 ± 25 0.63
d 14–28 624 ± 42 686 ± 37 0.30
d 0–28 539 ± 28 582 ± 25 0.29
Average daily feed intake, g/d
d 0–14 608 ± 67 601 ± 60 0.93
d 14–28 1068 ± 47 1068 ± 42 0.99
d 0–28 838 ± 48 834 ± 43 0.96
Gain:feed, g/g
d 0–14 0.77 ± 0.07 0.77 ± 0.06 0.96
d 14–28 0.58 ± 0.02 0.64 ± 0.02 0.06
d 0–28 0.65 ± 0.03 0.70 ± 0.03 0.28
a
Values are least squares means ± SEM; for zearalenone, ␣-zearalenol, -zearalenol, live weight, and average daily gain, n = 10; for average daily feed
intake and gain:feed, n = 5.
2.5. Western immunoblotting
Uterine and muscle samples were analyzed for total and amino acid phosphorylation of Akt, ERK 1/2, and mTOR by
Western immunoblot analysis. In addition, uterine samples were analyzed for total ER-␣ and -. Samples were powdered
at liquid nitrogen temperature and a crude soluble protein extract was prepared in buffer that contained 50 mM Tris HCl
(pH 7.5); 3 mM EDTA; 100 mM NaCl; protease inhibitors (Complete Mini (1836153), 1 tab/10 mL, Roche Applied Science,
Indianapolis, IN, USA); phosphatase inhibitors (Phosphatase Inhibitor Cocktail 1 (P2850) and 2 (P5726), each at 1:100 v:v;
Sigma–Aldrich, St. Louis, MO, USA); and 1% IGEPAL A-630 (USB Corp., Cleveland, OH, USA). The protein concentration of the
extracts was determined using the bicinchoninic acid reagent (Smith et al., 1985). Sixty micrograms of soluble protein were
resolved on 10% SDS-PAGE gels and electroblotted onto polyvinylidene fluoride membranes (PALL Co., Pensacola, FL, USA).
Pooled samples of known phosphorylation status were used on each block as a positive control. For Akt, ERK 1/2, and mTOR,
the membranes were probed for phosphorylated protein using specific anti-phosphoprotein antibodies (1:2000, Akt-Ser
202 204 2448
473, 1:2000, ERK-Thr /Tyr , 1:2000, mTOR-Ser ; Cell Signaling Technology, Danvers, MA, USA). After washing in Tris-
buffered saline with 0.1% Tween (TBS-T), the blots were reprobed for total protein with anti-Akt (1:2000, detects isoforms
1–3; Cell Signaling Technology, Danvers, MA, USA), anti-ERK (1:2000, detects isoforms 1 and 2; Cell Signaling Technology,
Danvers, MA, USA), and anti-mTOR (1:2000, Cell Signaling Technology, Danvers, MA, USA). For the ERs, the membranes were
probed for total protein with anti-ER-␣ (1/2000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-ER- (1/1000,
Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies.
2.6. Statistical analysis
Data were subjected to analysis of variance using the GLM procedure of SAS (Version 9.2, 1998; SAS Inst., Cary, NC, USA).
Data were evaluated for the effects of diet, week, and their interaction. For performance measures, initial body weight was as
a covariate. Similarly to previous experiments (Oliver et al., 2005; Oliver and Miles, 2010), pen of pigs was the experimental
unit for evaluation of the effects of diet on feed intake and feed efficiency and individual pig was the experimental unit for
all other statistical procedures. The significance level for all tests was set at P<0.05.
3. Results
3.1. Zearalenone and metabolites
Zearalenone and the zearalenone metabolites ␣- and -zearalenol increased in urine from gilts consuming zearalenone
contaminated feed, compared to gilts consuming the control diet (Table 3; P<0.007). Prepubertal gilts weighed 6.71 ± 0.20 kg
initially (P=0.10) and gained 560 ± 26 g/d (Table 3; P=0.29) during the 4-wk study, which resulted in an ending BW of
W.T. Oliver et al. / Animal Feed Science and Technology 174 (2012) 79–85 83
Table 4
Reproductive tract measurements and estrogen receptor-␣ and - abundance of prepubertal gilts fed control or control + zearalenone (1.5 mg/kg) diets for a
four weeks.
Variable Treatment P value
Control Zearalenone
Estrogen receptor-␣
mRNA, relative units 0.25 ± 0.08 0.24 ± 0.06 0.65
Protein, arbitrary units 83.1 ± 8.06 81.8 ± 5.49 0.32
Estrogen receptor-
± ±
mRNA, relative units 0.39 0.08 0.79 0.09 <0.01
Protein, arbitrary units 15.12 ± 4.42 51.89 ± 5.86 <0.01
Reproductive tract weight, g 20.91 ± 4.30 50.62 ± 3.81 <0.001
Endometrial gland development, glandular:total uterine area 0.18 ± 0.02 0.27 ± 0.02 0.02
Vulva height, mm
d 0 11.09 ± 0.70 10.96 ± 0.63 0.9
d 7 11.48 ± 0.80 14.64 ± 0.72 0.03
d 14 13.65 ± 1.36 22.09 ± 1.21 0.002
± ±
d 21 13.25 1.36 18.77 1.22 0.02
d 28 13.73 ± 1.02 23.89 ± 0.91 <0.001
Vulva width, mm
d 0 9.21 ± 0.55 8.36 ± 0.49 0.28
d 7 10.55 ± 0.66 12.39 ± 0.59 0.08
d 14 13.06 ± 0.80 20.28 ± 0.71 <0.001
d 21 12.53 ± 0.91 17.92 ± 0.82 0.004
d 28 13.43 ± 0.65 23.49 ± 0.58 <0.001
Vulva length, mm
d 0 8.32 ± 0.76 7.71 ± 0.68 0.57
d 7 13.57 ± 0.63 11.00 ± 0.57 0.62
d 14 13.05 ± 0.74 15.23 ± 0.66 0.06
d 21 12.09 ± 0.77 14.61 ± 0.69 <0.05
d 28 14.00 ± 1.07 19.44 ± 0.96 0.007
a
Values are least squares means ± SEM; n = 10.
23.91 ± 0.90 kg (P=0.21), regardless of treatment. Average daily feed intake did not differ between gilts consuming the
control and zearalenone diets (836 ± 45 g/d; P=0.96), which resulted in similar feed efficiency (G:F; 0.68 ± 0.03; P=0.28).
3.2. Reproductive tract measurements
Vulva measurements (height, width, and length) did not differ between groups at the initiation of treatments (Table 4;
P>0.28). By d 7, gilts consuming zearalenone contaminated diets had larger vulva heights (14.64 ± 0.72 vs. 11.48 ± 0.80 mm;
P=0.03) compared to control gilts. Vulva heights were 74% larger at d 28 of study in zearalenone treated compared to control
gilts (23.89 ± 0.91 vs. 13.73 ± 1.02 mm; P<0.001). Vulva width and length increased in zearalenone treated gilts compared to
control gilts by d 14 and 21 of treatment, respectively (P<0.05). By 28 d of age, vulva width and length were increased by 75
and 39%, respectively, in zearalenone treated compared to control gilts (23.49 ± 0.58 vs. 13.43 ± 0.65 mm and 19.44 ± 0.96
vs. 14.00 ± 1.07 mm; P<0.008). After 4 wk of consuming zearalenone contaminated diets, prepubertal gilts had a 2.4-fold
increase in total reproductive tract weight compared to gilts consuming the control diet (Table 4; 50.6 ± 3.8 vs. 20.9 ± 4.3 g;
P<0.001). In addition, endometrial gland development, expressed as a ratio of glandular to total uterine area, was increased
50% in pigs consuming the zearalenone contaminated diets compared to control gilts (Table 4; 0.264 ± 0.027 vs. 0.117 ± 0.016;
P<0.02).
3.3. Cellular signaling
No differences in ERK 1/2 (0.82 ± 0.11 vs. 0.85 ± 0.13 relative units) or mTOR (0.73 ± 0.08 vs. 0.81 ± 0.10 relative units)
mRNA abundance were observed in skeletal muscle of pigs consuming the zearalenone or control diets (P>0.43). Sim-
ilarly, no difference was observed in the total abundance of ERK 1/2 (81 ± 8 vs. 88 ± 9 arbitrary units), Akt (92 ± 8 vs.
86 ± 7 arbitrary units), or mTOR (62 ± 4 vs. 63 ± 4 arbitrary units) protein (P>0.61). In addition, the activity of ERK 1/2
(0.22 ± 0.4 vs. 0.28 ± 0.05), Akt (0.41 ± 0.06 vs. 0.40 ± 0.04), and mTOR (0.39 ± 0.04 vs. 0.41 ± 0.04), as measured by the ratio
of phosphorylated protein to total protein, was not different between zearalenone and control gilts (P>0.22).
In the uterus, no differences in the mRNA abundance of the progesterone receptor, COX2, ERK 1/2, or mTOR were observed
between zearalenone treated and control gilts (data not shown; P>0.44). In addition, ER-␣ mRNA and protein abundance
was similar between zearalenone and control gilts (Table 4; P>0.32). However, the mRNA and protein abundance of ER-
were increased 2.0- and 3.5-fold, respectively, in prepubertal gilts consuming zearalenone compared to gilts consuming the
control diet (Table 4; P<0.01).
84 W.T. Oliver et al. / Animal Feed Science and Technology 174 (2012) 79–85
4. Discussion
In vivo, zearalenone is predominantly converted into ␣- and -zearalenol and excreted as zearalenone, ␣-zearalenol, or

-zearalenol in feces and urine (Zöllner et al., 2002). In the current experiment, zearalenone delivery to prepubertal gilts
was confirmed by analyzing zearalenone and its metabolites, ␣- and -zearalenol, in urine. As expected, control gilts had
very low or undetectable levels while zearalenone gilts excreted high levels of these compounds.
There are few reports on the effect of zearalenone or its metabolites on growth performance in pigs. Döll et al. (2003)
observed decreased feed intake and growth rates in gilts fed diets with zearalenone (1.2 mg/kg of maize), but the decrease in
performance was likely due the presence of the related Fusarium toxin deoxynivalenol (DON, 8.6 mg/kg of maize). It is well
established that DON decreases feed intake and growth (Rotter et al., 1996). However, ␣-zearalanol (also called zeranol),
which is converted from ␣-zearalenol (Zöllner et al., 2002), has long been used successfully as an implant to improve
growth performance in cattle (Simms et al., 1988). Generally, cattle administered zeranol implants do not have altered feed
intake and body composition differences in response to implants are breed specific (Williams et al., 1991). To the authors
knowledge, no genetic differences have been reported in response to the consumption of zearalenone or its metabolites
in swine. Although zearalenone, ␣-zearalenol, and zeranol are present in muscle of pigs consuming zearalenone (Zöllner
et al., 2002), prepubertal gilts consuming zearalenone-contaminated feed in the current experiment gained at a similar rate
and consumed a similar amount of feed compared to gilts consuming the control diet. This is likely because pigs produce
only trace amounts (<1 g/L urine) of zeranol from ␣-zearalenol (Zöllner et al., 2002). In gilts, Jiang et al. (2010) observed
increased ADG and ADFI in pigs consuming zearalenone contaminated feeds. This differs from the current study and is
possibly due to the fact that the gilts in the Jiang et al. (2010) study were older than those in the current study. The age of the
pigs is unknown, but their initial body weight was twice that of gilts in the current experiment. In addition, gilts consuming
zearalenone in the study by Jiang et al. (2010) appear to be heavier at the initiation of treatments, compared to control pigs
(12.12 vs. 11.01 kg; SEM = 0.131), which confounds the performance data.
Contrary to the anabolic effects of zeranol in cattle, ␣- and -zearalenol have been shown, in vitro, to reduce the phospho-
rylation of proteins involved in protein synthesis and cellular proliferation (Wollenhaupt et al., 2004). In addition, Abid-Essefi
et al. (2004) observed that zearalenone inhibited protein and DNA synthesis in Vero (kidney) and Caco-2 (human colon
cancer) cell lines. Cellular proliferation and protein synthesis are driven by a complex interaction of signaling proteins.
Wollenhaupt et al. (2004) observed changes in phosphorylation of ERK 1/2, Akt, and the translation initiation factor 4E-BP1
by -, and to a lesser extent, ␣-zearalenol that would result in decreased cellular proliferation and protein synthesis. Mam-
malian target of rapamycin is considered a master protein kinase that is regulated, in part, by Akt and contributes to the
control of protein synthesis by regulating components of cellular signaling that directly affect protein synthesis (Avruch
et al., 2005). In the current experiment, no differences in skeletal muscle ERK1/2, Akt, or mTOR were observed between gilts
consuming zearalenone and gilts consuming the C diet, which is in agreement with the lack of a performance difference
in the current study. The gilts in the current study were gaining at a high rate (>500 g/d). Thus, it is likely that the normal
anabolic signals (insulin like growth factor-I, insulin) not present in the in vitro studies precluded any potential changes
in signaling for reduced cellular proliferation and protein synthesis that may have occurred due to zearalenone and its
metabolites.
The effect of zearalenone on the reproductive performance of pigs is highly dependent on their reproductive status
(Diekman and Green, 1992) and prepubertal gilts are particularly susceptible to zearalenone-contaminated feed (Kordic
et al., 1992). In the current experiment, prepubertal gilts had large increases in edematous swelling of the vulva throughout
the study and increased total uterus weight and endometrial gland development after 4 wk consuming zearalenone con-
taminated diets. An increase in vulva size is well documented in pigs consuming zearalenone (Kordic et al., 1992). Similarly,
zearalenone has been shown to increase the size of the reproductive tract in prepubertal gilts (Döll et al., 2004). However,
Döll et al. (2004) did not observe histological differences in the uterine glands of these gilts. These data likely differ from the
current experiment due to the lower dose of zearalenone used (0.42 vs. 1.5 mg/kg). In addition, it is likely that pigs in the
current study had considerably less developed uteri before exposure to zearalenone and its metabolites compared to the
older gilts (70 d, 32.6 kg vs. 28 d, 6.6 kg) used in the study by Döll et al. (2004).
The effect of zearalenone and its metabolites on the in vivo transcription and translation of reproductive genes and
mRNA is not well established. In the current experiment, we did not observe any changes in the expression of mRNA for
progesterone receptor, COX2, or ER-␣. In addition, protein abundance for ER-␣ was unchanged. Zearalenone, ␣-zearalenol,
-zearalenol, and zeranol readily bind to ER, with ␣-zearalanol and zeranol having the highest affinities for the receptor
(Le Guevel and Pakdel, 2001; Leffers et al., 2001). Upon ligand binding to the ER, translocation of the ER to the nucleus
occurs where the ER binds to ER-responsive elements to initiate transcription of specific genes in target tissues (Edwards,
2000; Nettles and Greene, 2005). As observed in the current study, estrogen-mediated responses include maintenance and
development of secondary female sex characteristics, as well as uterine hypertrophy. Contrary to ER-␣, we observed 2.0-
and 3.5-fold increases in ER- mRNA and protein, respectively, in the uterus of prepubertal gilts consuming zearalenone.
Estrogen receptor- is widely distributed in tissues, although not highly expressed in the uterus (Shughure et al., 1998; Wang

et al., 1999). In addition, the uteri of ER- knockout mice appear comparable to wildtype mice and respond to estrogen due
to the presence of ER-␣. These mice have decreased fertility compared to wildtype control mice, but are capable of successful
pregnancies (Krege et al., 1998). Thus, ER␣ is the predominant isoform in the uterus, and it is likely that uterine hypertrophy
observed in the current study was largely mediated through this isoform. However, due to the changes in ER- in gilts
W.T. Oliver et al. / Animal Feed Science and Technology 174 (2012) 79–85 85
consuming zearalenone contaminated feed, it is also likely that ER- was, in part, responsible for uterine growth. Thus, a
larger role for the ER- in uterine growth and development cannot be ruled out.
Contrary to some reports, zearalenone did not increase growth performance in the current study. In addition, activation
of key proteins, as measured by protein phosphorylation, involved in protein synthesis was not down-regulated. Thus, in
spite of cell culture-based evidence, zearalenone and its metabolites, at these doses, do not regulate skeletal muscle protein
synthesis in prepubertal gilts, in vivo. This study confirms earlier reports showing the estrogenic effects on prepubertal gilts,
in that reproductive tract size and endometrial gland development increased in gilts consuming zearalenone contaminated
feed. Zearalenone did not affect several mRNA abundances associated with uterine growth and maintenance, including ER-␣.
However, novel to the current experiment, ER- mRNA and protein abundances increased in gilts consuming zearalenone.
While it is likely that the ER-␣ is the main estrogenic mediator in uterine tissue, the changes in ER- observed indicates a
major role in the growth and (or) maintenance of the uterus in response to zearalenone contaminated feed in prepubertal
gilts. Thus, we conclude that zearalenone had no effect on growth performance or skeletal muscle signaling in prepubertal
gilts, but increased reproductive tract size and glandular development which was due, in part, to altering the expression of ER-.
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