Animal Biotechnology, 21: 77–87, 2010 Copyright # Taylor & Francis Group, LLC ISSN: 1049-5398 print=1532-2378 online DOI: 10.1080/10495390903500607 EXAMINATION OF TESTICULAR GENE EXPRESSION PATTERNS IN YORKSHIRE PIGS WITH HIGH AND LOW LEVELS OF BOAR TAINT Maxwell C. K. Leung, Kiera-Lynne Bowley, and E. James Squires Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada Boar taint refers to the objectionable odor and flavor in meat of some uncastrated male pigs, which is primarily due to high levels of androstenone, a steroid produced in the testis, and 3-methylindole (skatole) which is produced by bacterial degradation of tryptophan in the intestinal tract. We determined testicular gene expression patterns of Yorkshire pigs with high and low levels of boar taint using swine DNA microarrays with two-color hybridization. The microarrays contained 19486 annotated probes; the expressions of 8719 genes were detected. Fifty-three genes were significantly up-regulated in the high boar taint group and four were significantly down-regulated (p < 0.05; fold change > Æ1.55). Gene ontology (GO) analysis short-listed 11 significant GO terms (p < 0.05), most of which are associated with steroid metabolism and mitochondrial components. Comparing the results of this study with published work on Duroc and Norwegian Landrace boars,1 eleven genes (HSB17B4, FDX1, CYP11A1, DHRS4, PRDX1, CYB5, CYP17A1, FTL, IDI1, SULT2A1, and RDH12) were over-expressed in all three breeds with a high androstenone level. The current findings confirmed a number of candidate genes identified in previous functional studies and suggest several new genes differentially expressed with different levels of boar taint. Keywords: Boar taint; Candidate genes; Testis Boar taint refers to the objectionable odor and flavor in meat of some intact male pigs (boars).2 Since a significant percentage of intact male pigs can possess this undesirable meat quality trait,3 most pigs in North America are castrated shortly after birth to remove the potential for taint.4 Castration, however, results in a lower feed efficiency, a lower lean yield, as well as animal welfare concerns.3,5 Selective breeding of intact pigs would be a more preferable method to prevent boar taint, so a better understanding of the genes involved in boar taint is warranted in order to develop genetic markers for boar taint. Boar taint is primarily caused by high levels of androstenone and=or skatole in carcasses.6 Androstenone is a steroid produced by the testis near sexual maturity,7,8 The authors thank Yanping Lou for technical assistance. This work was supported by a NSERC Discovery grant and funding from the Ontario Ministry of Agriculture and Food. Address correspondence to E. James Squires, Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada, N1G 2W1. E-mail: [email protected] 77 78 M. C. K. LEUNG ET AL. whereas skatole is produced by bacteria in the hindgut of the pig and then absorbed into the blood stream.9 The production of androstenone is mainly affected by genetic factors rather than diet and environmental factors.4 Several functional studies, furthermore, have identified a number of genetic markers associated with increased production of androstenone.10–15 A recent study was published on differences in gene expression profiles in Duroc and Norwegian Landrace boars with high and low fat androstenone,1 and a total of 563 and 160 genes were differentially expressed between the two groups, respectively. However, only 53 genes were differentially expressed in both breeds, suggesting that there were both breed specific changes in gene expression as well as general changes affecting boar taint. Moreover, the degree of sexual maturity and steroidogenic potential of the boars was not reported by Moe et al.1 Previous studies by Stewart et al.16 comparing gene expression profiles between boars with high and low levels of plasma estrone sul- fate using a smaller (1,700 gene) human microarray identified a number of the same genes as the study by Moe et al.1 comparing low and high androstenone boars. Thus, in order to identify those genes that are specific to boar taint, it is important to differ- entiate between boars with high and low levels of steroidogenesis and boar with high and low levels of androstenone. This allows the separation of desirable genes for ‘‘maleness,’’ from genes that are important for boar taint, which we want to eliminate. The objective of the current experiment was to examine the differential gene expressions associated with high and low levels of boar taint in Yorkshire boars using a swine protein-annotated oligonucleotide microarray.17 The results were further analyzed using gene ontology and KEGG pathway analyses. The current results are also compared with the previous findings from Duroc and Norwegian Landrace boars. This will provide useful information for future studies identifying breed specific and general genetic markers associated with boar taint. MATERIALS AND METHODS Tissue Samples and Biochemical Analysis Ten Yorkshire boars (144 Æ 40 kg) were selected from a larger pool obtained from local breeders in Ontario. Testis tissue samples were taken at time of slaughter, frozen in liquid nitrogen, and stored at À70C until use. Plasma and back fat samples were collected and stored at À20C. Sexual maturity of the boars was confirmed 18 by measuring the length of bulbourethral glands, as well as the plasma concentration of estrone sulfate (E1S) by radioimmunoassay.19 Fat and plasma androstenone levels were assayed using an ELISA method modified from Claus et al.20 as described by Squires and Lundstro¨m.21 Pigs having a fat androstenone level of less than 0.5 mg=g and greater than 1.0 mg=g were defined as low and high boar taint samples, respect- ively. Concentrations of skatole in plasma and fat were determined by a HPLC method modified after Claus et al.22 and Denhard et al.23 as described by Lanthier et al.24 Design of Microarray Experiment Five testis samples from boars with high levels of boar taint were paired with five testis samples from low taint boars for a competitive two-color microarray TESTICULAR GENE EXPRESSION AND BOAR TAINT 79 analysis. Relative gene expression of each pair of samples was assessed by a microarray with a replicate of dye swap for each pair for a total of ten microarrays used. The porcine 70-mer oligonucleotide-microarrays used in the current study were provided by the laboratory of David W. Galbraith at the University of Arizona. The oligonucleotides printed on the microarrays were supplied in part by the contri- bution of the US Pig Genome Coordinator, Max Rothschild, Iowa State University, and the contributions of the swine genome array coordination committee, Scott Fahrenkrug, University of Minnesota, Chair. Each slide contains 19486 annotated probes (18244 protein-annotated probes, 198 unrepresented porcine RefSeq genes, and 1044 TIGR porcine consensus sequences), 914 controls (mismatch and scramble sequences), and blanks. A full description of the porcine array and a complete list of EST annotation can be found at http://www.pigoligoarray.org/. Preparation of RNA Samples and Dye-Labeled cDNA Total RNA was extracted from 200 mg of frozen testis sample using RNeasy Midi kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. The quality and quantity of RNA was assessed using an Agilent 2100 Bioanalyzer (Santa Clara, CA). cDNA was prepared using 20 mg of total RNA, 8 ml of 5x first-strand buffer, 100 pmol of oligo(dT) primer, 20 mM dNTP-dTTP (6.67 mM each of dATP, dGTP, and dCTP), 2 mM of 2-aminoallyl-dUTP (AA-dUTP, Sigma-Aldrich Inc., St. Louis, MO), 2 mM dTTP, and 20 mM DTT in a reaction volume of 40 ml. The mixture was incubated at 65C for 5 minutes and then 42C for 5 minutes. Then, 4 ml of Super- script II Reverse Transcriptase (Invitrogen Corp., Carlsbad, CA) was added and the reactions were incubated for 4 hours at 42C. The reaction was then heated to 95C for 5 minutes to inactivate the enzyme. RNA was hydrolyzed by adding 8 ml of 1 M NaOH followed by a 15 minute incubation at 65C. Then 8 mlof1MHCl and 4 ml of 1 M Tris-Cl (pH 7.5) were added to neutralize the reaction. The PureLink PCR purification kit (Invitrogen, Carlsbad, CA) was used according to the manufac- turer’s instructions to purify the cDNA in a final volume of 6 ml. Cyanine 3 (Cy3) and cyanine 5 (Cy5) monofunctional reactive dyes (Amersham Pharmacia Ltd., Piscataway, NJ) were each dissolved in 72 ml of dimethylsulphoxide and each dye was then aliquoted into 4.5 ml of dye per tube and stored at À70C until use. The dye aliquot and 3 ml of 0.3 M sodium bicarbonate was added to each sample and incubated for 2 hours in the dark at room temperature to allow for coupling of the dyes. The Qiaquick nucleotide removal kit (Qiagen) was used according to the manufacturer’s instructions to remove unincorporated dye. Following purification, the samples labeled with two different dyes were combined and 3.0 ml glycogen, 30 ml of 3 M sodium acetate, and 333 ml of isopropanol were added. The samples were incubated for 2 hours at À70C to precipitate the labeled cDNA. Following precipi- tation, the samples were reconstituted in 15 ml of water. Hybridization of Microarray and Data Acquisition The labeled cDNA was added to 2.5 ml of yeast tRNA (Invitrogen; 10 mg=ml) and 2.5 ml of calf thymus DNA (Sigma; 10 mg=ml) and 50 ml DIG Easy Hyb solution (Roche, Basel, Switzerland). The mixture was incubated at 65C for 2 minutes, 80 M.