Biol Trace Elem Res (2014) 161:69–77 DOI 10.1007/s12011-014-0078-5

Supplementation Dietary Zinc Levels on Growth Performance, Carcass Traits, and Intramuscular Fat Deposition in Weaned Piglets H. B. Zhang & M. S. Wang & Z. S. Wang & A. M. Zhou & X. M. Zhang & X. W. Dong & Q. H. Peng

Received: 14 May 2014 / Accepted: 14 July 2014 / Published online: 23 July 2014 # Springer Science+Business Media New York 2014

Abstract This study was conducted to estimate dietary zinc (Zn) levels on growth performance, carcass traits, and intramuscular fat (IMF) deposition in weaned piglets. Sixty piglets were randomly divided into five groups, as follows: control (basal diet), Zn250, Zn380, Zn570, and Zn760 with supplementation of 250, 380, 570, and 760 mg Zn/kg of the basal diet, respectively. The final weight, average daily gain (ADG), gain/feed (G/F), lean meat percentage, fat meat percentage, lean eye area, backfat thickness, and IMF content were dosedependently increased in all groups of Zn treatment. The serum total triglycerides (TG) and free fatty acid (FFA) were significantly higher in all Zn treatments than in the control. The enzyme activities of lipoprotein lipase (LPL), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) were markedly higher, while enzyme activities of hormonesensitive lipase (HSL) and carnitine palmitoyltransferase-1 (CPT-1) were significantly lower in all Zn treatments than in the control. The messenger RNA (mRNA) levels of sterol regulatory element-binding protein 1 (SREBP-1), stearoylCoA desaturase (SCD), FAS, ACC, peroxisome proliferatoractivated receptor γ (PPARγ), LPL, and adipocyte fatty acidbinding protein (A-FABP) were significantly higher, while the mRNA levels of CPT-1 and HSL were significantly lower in all Zn treatments compared with the control. These results indicated that high levels of Zn increased IMF accumulation by up-regulating intramuscular lipogenic and fatty acid M. S. Wang and A. M. Zhou contributed equally to this work. H. B. Zhang : M. S. Wang : Z. S. Wang (*) : A. M. Zhou : X. M. Zhang : X. W. Dong : Q. H. Peng Animal Nutrition Institute, Sichuan Agricultural University, Ya’an 625014, China e-mail: [email protected] M. S. Wang Life Sciences Department, Zao Zhuang University, Zao Zhuang 277160, China

transport gene expression and enzyme activities while downregulating lipolytic gene expression and enzyme activities. Keywords Dietary Zn levels . Intramuscular fat . Longissimus muscle . Weaned piglets

Introduction Zinc (Zn) has been recognized as an essential element for the activity of lots of enzymes and also plays an important role in numerous essential processes including cell proliferation and differentiation and protein and nucleic acid synthesis [1–3]. Odutuga et al. reported that the symptoms of Zn deficiency were similar to those of essential fatty acid deficiency [4]. Some researchers also reported that Zn plays an important role in cell lipid metabolism as a structural and functional group for some lipid metabolic enzymes [5, 6]. These studies suggested that Zn plays an important role in fatty acid metabolism. Intramuscular fat (IMF) content is one of the most important traits of meat quality [7]. Increasing IMF content could be a useful way to improve the pig meat quality by improving the sensory meat quality, such as favor, juiciness, and tenderness [8]. Zn supplementation provided as either ZnO or Zn methionine increased fat thickness and yield grade compared with control steers [9]. It also reported that ZnO and Zn proteinate increased quality grade, marbling score, and backfat thickness [10]. Spears and Kegley also reported that the marbling of steers increased with Zn supplementation [11]. At the metabolic level, IMF content results from the balance between uptake, synthesis, and degradation of triacylglycerols, involved in metabolic pathways of adipocytes and myofibers, which are regulated by metabolic enzymes and controlled by functional genes in the muscle tissue. Several key factors are involved in three key lipid pathways including fatty acid

70

synthesis genes (sterol regulatory element-binding protein 1 (SREBP-1), acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and stearoyl-CoA desaturase (SCD)) [12–15], fatty acid uptake genes (hormone-sensitive lipase (HSL) and carnitine palmitoyltransferase-1 (CPT-1)) [16, 17], and fatty acid transport genes (lipoprotein lipase (LPL), PPAR (PPARγ), and adipocyte fatty acid-binding protein (AFABP)) [18–21] in adipose tissues. However, there is little information on how dietary Zn levels regulate the balance between synthesis, uptake, and transport of fatty acids in longissimus muscle. Previous studies were focused on the effects of high Zn levels on the hepatic lipid metabolism in vitro [4, 22]. Few studies investigated the positive effects of dietary Zn levels on the IMF accumulation by gene level in weaned piglets. Therefore, the objectives of this work were to determine different levels of Zn on the IMF deposition relating gene expression (fatty acid synthesis genes, fatty acid uptake genes, and fatty acid transport genes) in weaned piglets, thus to clarify the underlying mechanism of IMF accumulation regulated by Zn level in gene level, and to continue to provide some valuable information to better understand the biological function of Zn on the lipid metabolism in longissimus muscle.

Materials and Methods

Zhang et al. Table 1 Composition and nutritional contents of experimental diets

a

Provided per kilogram of diet: VA, 32,000 IU; VD, 38,000 IU; VE, 200 IU; VK, 340 mg; VB1, 16 mg; VB2, 128 mg; VB6, 4.8 mg; VB12, 0.24 mg; D-calcium pantothenate, 200 mg; nicotinic acid, 280 mg; folic acid, 4 mg b

Minerals provided the following per kilogram of diet: Fe, 100 mg/kg; Cu, 10 m g/kg; Mn , 10 mg/kg; Se, 0.3 mg/kg; I, 0.3 mg/kg c

The experimental protocols were approved by the Animal Care Advisory Committee of the Sichuan Agricultural University. Veterinary supervision was provided to the animals throughout the experiment. Animals, Experimental Design, Diets, and Housing A total of 60 pigs (Duroc×Landrace×Yorkshire), with 7.73± 0.035 kg initial body weight (BW) and 28±1 days of age, were randomly divided into five dietary treatment groups. There were four replicates for each treatment and three pigs for each replicate. Five treatments consisted of the control group (basal diet) and four experimental groups with supplementation of ZnO, provided by GuangZhou Wisdom BioTechnology Co., Ltd., in which 250 mg Zn/kg (Zn250), 380 mg Zn/kg (Zn380), 570 mg Zn/kg (Zn570), and 760 mg Zn/kg (Zn760), respectively, were added to the basal diet. Taking into consideration the bioavailability of ZnO (50 to 80 %) in piglets [23], the Zn250 treatment was in the recommended requirement in several European countries [24] and the Zn380–Zn760 treatments were significantly higher than the recommended amount. All of the treatments were supplemented without other Zn sources. The basal diet was formulated on the basis of nutrient requirements for weaned piglets [25]. The formulations of the basal diet and chemical profiles are listed in Table 1.

Zn was a measured value and others were calculated values

Ingredient composition

%

Corn Rapeseed oil Soybean Fish meal Acid whey powder

51.45 2.00 22.50 3.80 6.00

Sucrose Glucose Wheat bran Plasma protein powder CaHPO4 CaCO3 NaHCO3 NaCl L-Lys·HCl DL-Met Choline chloride Vitamina Mineral premixb Nutrition level DE (MJ/kg) CP (%) Lys (%) Met (%)

2.00 3.00 2.00 4.00 1.00 0.65 0.05 0.20 0.31 0.02 0.10 0.08 0.40 14.29 20.19 1.36 0.35

Ca (%) TP (%) Znc (mg/kg)

0.80 0.65 29.39

Three pigs with the same replication were placed together in an individual pen equipped with woven wire floors, stainless steel nipple drinkers, and feeders. The room temperature was maintained at 28 °C. During the experiment, the piglets were allowed ad libitum access to the diets and water; the diets were added to the feeder four times daily at 8:00, 12:00, 16:00, and 20:00 hours, respectively.

Growth Performance The trial was carried out in the Research Base of the Institute of Animal Nutrition in Sichuan Agricultural University. The adaptation period was 5 days. The experiment began at the age of 28 days. The experiment was 40 days long. The amounts of diet offered per animal were recorded and adjusted according to feed refusals to determine average daily feed intake (ADFI). Any feed refusals were collected and weighed; the data were used for the determination of feed consumption. The pigs were individually weighed at the end of the trial to calculate average daily gain (ADG). The ADG was calculated with the formula (final weight − initial weight)/40. The feed/gain ratio

Dietary Zinc on Intramuscular Fat Deposition

was calculated with the formula (total feed intake/total weight gain)×100. Carcass Evaluation and Muscle Sample Collection At the end of the trial, the pigs were transported to the Sichuan Agricultural University Center Meats Laboratory and were slaughtered by exsanguination after electrical stunning. Slaughter weight was determined by weighing the animals after feed deprivation ≥24 h. The whole carcass was dissected into muscle, fat (subcutaneous fat, testicles, kidney, and pelvic fat), and bone, then lean meat percentage was calculated with the formula (muscle weight/slaughter weight)×100 and fat meat percentage was calculated with the formula (fat weight/ slaughter weight)×100. The loin eye area was measured at the position of the last rib. Backfat thickness was measured at the position of the 11th and last thoracic vertebrae, and the mean of these two measurements was used as the backfat thickness value. The muscle sample was taken from this slice, carefully avoiding intermuscular fat depots surrounding the muscle, and immediately frizzed at −20 and −80 °C for further analysis. Blood Collection and Analysis Ten milliliters of blood, collected from the jugular vein using heparin sodium-coated tubes before the slaughter trial, was centrifuged at 3,000×g for 20 min, and then the serum was then frozen at −20 °C. The blood sample was analyzed for total triglycerides (TG) and glucose using an automatic biochemical analyzer (Hitachi 902; Hitachi, Tokyo, Japan). Serum insulin and free fatty acid (FFA) were determined with commercial kits (Nanjing Jiancheng Biochemical Reagent Co., Nanjing, China). Enzyme Activity Analysis The enzyme activities of FAS, HSL, LPL, ACC, and CPT-1 were determined in longissimus tissue homogenates. Approximately 1 g longissimus muscle tissue was homogenized in 3 mL 10 mM HEPES buffer containing 0.25 M sucrose, 1 mM EDTA, and 1 mM dithiothreitol, and then centrifuged at 100,000×g for 30 min at 4 °C. Supernatants were collected and used for the enzyme assays. FAS, LPL, and HSL enzyme activities were determined using a commercial kit (Nanjing Jiancheng Biochemical Reagent Co., Nanjing, China) according to the manufacturer’s instructions. The enzyme activity of ACC was assayed by the H14CO3-fixation method [26]. Whole mitochondrial isolations were ultrasonicated after being resuspended in 0.3 M sucrose containing 10 mM Tris-HCl and 1 mM EGTA at 0 °C with pH 7.4 and then centrifuged at 20,000 × g for 40 min at 4 °C to obtain a mitochondrial supernatant for determining the CPT-1 activity [27].

71

Chemical Analysis Longissimus muscles were sampled for IMF content evaluation at 24 h after slaughtering by following the Soxhlet petroleumether extraction method. IMF content was quantified as the weight percentage of dry muscle tissue. Basal diet samples were prepared for Zn analysis using nitric-percholoric acid wet digestion. The determination of Zn concentration was carried out with a nov-AA400 flame atomic absorption spectrometer (Analytik Jena AG, Jena, Germany). Real-Time Quantitative PCR According to the published sequences of β-actin, PPARγ, CPT-1, SREBP-1, A-FABP, ACC, FAS, HSL, LPL, and SCD messenger RNA (mRNA) at GenBank, the oligonucleotide primer sets for the three genes were designed using Primer Premier 5.0 software, and the details are described in Table 2. Total RNA was extracted using the TRIZOL reagent (TaKaRa, Dalian, China) according to the manufacturer’s specifications. The extracted RNA was dissolved in DEPCtreated water, and the concentration, purity, and integrity were assessed by using a spectrophotometer at 260/280 nm (OD260/OD280 = 1.8 ~ 2.0) and by electrophoresis with ethidium bromide staining. Two micrograms of total RNA was extracted for reverse transcription using the AMV First Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). The 10 μL reverse transcription (RT) system contains 2 μL 5× Prime Script® Buffer, 0.5 μL Prime Script® RT Enzyme Mix I, 0.5 μL Oligo dT Primer (50 μM) and 0.5 μL Random 6 mers (100 μM), 4 μL Total RNA, and RNase Free dH2O up to 10 μL. Real-time PCR (RT-PCR) was performed with a Mastercycler (Eppendorf, Hamburg, Germany) in two consecutive separate steps: 37 °C for 15 min and 85 °C for 5 min. RT-PCR was performed in triplicate using SYBR Green PCR Mix (TaKaRa, Dalian, China) and a Chromo4 Gradient thermocycler (Bio-Rad, Hercules, CA, USA). Briefly, 10 μL 2× SYBR® Premix Ex TaqTM II (TaKaRa Ex Taq HS, dNTP Mixture, Mg2+, SYBR® Green I), 0.8 μL each of primers, and 2.0 μL cDNA were included in a 20 μL PCR. RT-PCR conditions were as follows: 95 °C for 2.5 min, 40 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s, a melt curve of increasing temperature of 2 °C every 10 s starting at 55 °C followed by a hold at 4 °C. The reactions were repeated thrice for every sample. All target genes were normalized to the endogenous reference gene β-actin by employing an optimized comparative Ct (2−ΔΔCt) value method. Statistical Analyses All data were analyzed using SPSS 13.0 by one-way ANOVA and multiple comparisons of Duncan analysis to determine statistical differences between groups. The results were

72

Zhang et al.

Table 2 Specific primers used for real-time quantitative PCR Gene

Primer

Product size (bp)

Accession

β-Actin

F: 5′-ACTGCCGCATCCTCTTCCTC-3′ R: 5′-CTCCTGCTTGCTGATCCACATC-3′ F: 5′-TGCACTCGCACTTACGAGAA-3′ R: 5′-AACCGGAACTGAACGCAGG-3′ F: 5′-ATGTTTCGGCAGTCCCTGAT-3′ R: 5′-TGTGGACCAGCTGACCTTGA-3′ F: 5′-AGCCTAACTCCTCGCTGCAAT-3′ R: 5′-TCCTTGGAACCGTCTGTGTTC-3′ F: 5′-GCG ACG GTGCCTCTG GTA GT-3′ R: 5′-CGC AAG ACGGCGGAT TTA-3′ F: 5′-AAC TTG TGGCTGCCC TAT-3′ R: 5′-GAC CCT CTGGTGAAT GTG-3′ F: 5′-GCT CCC ATCGTCAAG AAT C-3′ R: 5′-TAA AGC GAA TGCGGT CC-3′ F: 5′-GAT TTC TCCAGCATT TCC A-3′ R: 5′-GCT CTT CGT GAGGTT TGT T-3′ F: 5′-ATG GTG GGCGACTAA CT-3′ R: 5′-TGC CTG CTGTCTGTG AG-3′

115

BC063166

180

AJ416019

133

EF618729

196

AY183428

218

AF102873

367

X62984

262

AJ000482

184

DQ437884

321

AY181062

A-FABP ACC FAS SREBP-1 LPL HSL PPARγ CPT-1

ACC acetyl-CoA carboxylase, CPT-1 carnitine palmitoyltransferase 1, FAS fatty acid synthase, A-FABP adipocyte fatty acid-binding protein, HSL hormone-sensitive lipase, LPL lipoprotein lipase, PPARγ peroxisome proliferator-activated receptors, SCD stearoyl-CoA desaturase, SREBF-1 sterol regulatory element-binding transcription factor 1

presented as the mean ± SD. Significant differences were set at P0.05), but final BW, ADG, and G/F had significant differences (P