Animal Feed Science and Technology 174 (2012) 79–85 Contents lists available at SciVerse ScienceDirect 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.