Published December 3, 2014

Fatty acid composition and lipogenic enzyme protein expression in subcutaneous adipose tissue of male pigs vaccinated against boar taint, barrows, and entire boars J. Mackay,* M. C. Pearce,† S. Thevasagayam,† and O. Doran*1 *Centre for Research in Biosciences, Faculty of Health and Life Sciences, University of the West of England, Coldharbour Lane, Bristol, BS16 1QY, UK; and †Pfizer Animal Health, 10 Hoge Wei, 1930 Zaventem, Belgium

pigs. Total MUFA content was increased (P < 0.001) in barrows compared with entire and vaccinated pigs. This was not accompanied (P > 0.05) by an increase in expression of stearoyl-CoA desaturase protein, the enzyme catalyzing MUFA biosyntheses. Total n-6 PUFA content did not differ (P < 0.001) between entire and vaccinated pigs but was lower in barrows. Expression of ∆6-desaturase protein, one of the key enzymes of PUFA biosynthesis, was greater (P < 0.05) in vaccinated pigs than in barrows but did not differ significantly between vaccinated and entire animals. We conclude that fatty acid profile of animals vaccinated against boar taint is similar to that of entire male pigs and that the effect of physical castration and vaccination on fatty acid composition involves changes in lipogenic enzyme protein expression.

ABSTRACT: Objectives of this study were to compare fatty acid composition of subcutaneous adipose tissue of entire boars, barrows, and male pigs vaccinated against boar taint with a vaccine containing a GnRH analogue-protein conjugate (Improvac, Pfizer Animal Health) and to investigate the association between fatty acid composition and protein expression of key lipogenic enzymes in entire boars, barrows, and vaccinated pigs. Differences between groups were observed in the content of total SFA (P ≤ 0.001), MUFA (P = 0.035), and n-6 PUFA (P ≤ 0.001) but not n-3 PUFA (P = 0.373). Total SFA were greater (P < 0.001) in barrows and vaccinated pigs compared with entire animals. This was accompanied by an increase (P < 0.05) in the protein expression of the lipogenic enzyme fatty acid synthase in barrows and vaccinated

Key words: castration, fatty acid composition, Improvac, pig, protein expression © 2013 American Society of Animal Science. All rights reserved.

INTRODUCTION Boar taint is an offensive odor of pork from some entire boars due to an excessive accumulation of androstenone and skatole in adipose tissue (Walstra et al., 1999), and it can be prevented by physical castration of piglets (Claus et al., 1994). Percentage of pigs affected by boar taint varies from 1 to 30% (Xue and Dial, 1997). Physical castration has, however, been discontinued in Europe (Prunier et al., 2006), and the European Commission is committed to a plan to voluntarily end surgical castration of pigs in Europe by 2018 (European Commission, 2011).

1Corresponding

author: [email protected] Received September 10, 2011. Accepted September 7, 2012.

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J. Anim. Sci. 2013.91:395–404 doi:10.2527/jas2011-4685

Therefore, there is an increasing need for alternatives to surgical castration for prevention of boar taint. One alternative is vaccination against GnRH with Improvac (Pfizer Animal Health, New York, NY), which has been accepted and implemented in a number of countries. Although the effect of Improvac on animal performance and behavior has been extensively studied (Bonneau et al., 1994; Jaros et al., 2005), little is known about the effect of Improvac on fatty acid composition, which is a major determinant of meat quality characteristics (Wood et al., 1999). The level of dietary SFA, MUFA, and PUFA has a strong impact on human health (Rose, 1997; Li et al., 2008). It is known that fatty acid composition of pig tissues is associated with expression of lipogenic enzymes (Doran et al., 2006; Missotten et al., 2009) and that physical castration affects fatty acid composition (Gunn et al., 2004). However, the effect

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of vaccination with Improvac on fatty acid composition has only been reported in one paper (Pauly et al., 2009). The aims of this study were 1) to investigate effects of vaccination against GnRH with Improvac and physical castration on the fatty acid composition of subcutaneous adipose tissue and 2) to determine whether the effect of vaccination and physical castration is mediated via regulation of protein expression of the key lipogenic enzymes. MATERIALS AND METHODS No approval was required from the University of West of England for these experiments because only samples of subcutaneous adipose tissue from carcasses were used in the studies performed at the University. The study was conducted in accordance with the regulations for the humane care and use of animals. Animals and Sample Collection Samples of subcutaneous adipose tissue were taken at slaughter from the same area of the belly of 30 randomly selected male pigs farrowed by Topigs Dalland and sows crossed with Piétrain boars enrolled on a large field trial. The animals were raised under the same management conditions. Growing pens (plastic slats) had 0.31 to 0.36 m2 floor space per animal and 30 pigs per pen. Finishing pens (concrete slats) had minimum 0.65 m2 floor space per animal and no more than 15 pigs per pen. The samples originated from 10 males physically castrated (“barrows” group) at age 2 to 7 d, 10 males vaccinated with 2 mL Improvac when aged 9 to 10 wk and 20 to 21 wk (“vaccinated” group), and 10 untreated entire boars (“entire” group). The pigs were slaughtered at age 169 to 184 d. All the pigs were fed the same commercial diet (Promor, Hartswater, South Africa) to exclude potential effect of the diet on outcomes of the study. In weaning pens, pigs were fed ad libitum by feed hoppers. In finishing pens, pigs were fed ad libitum, feed delivered to troughs serving 2 pens each. The finishing diet contained 3,160 Kcal/kg ME, CP (17%), lysine (1%), crude fat (3%), cellulose (4%), ash (4.6%), Ca (0.7%), and P (0.47%). The mean values for the BW at the slaughter were 105.3, 107.6, and 102.2 kg for the barrows, vaccinated, and entire pigs, respectively. Samples of subcutaneous adipose tissues (10 to 15g) were collected within 30 to 60 min after slaughter, frozen, and kept at –20°C until analyzed. Fatty Acid Analysis Fatty acid composition of subcutaneous adipose tissue was analyzed by high resolution gas chromatography using appropriate fatty acid standards as described previously

(Doran et al., 2006). In brief, lipids from adipose tissue were extracted into chloroform containing 2,6-di-tert-butylp-cresol as an antioxidant and anhydrous sodium sulphate was added to remove water. After filtration, duplicate subsamples were hydrolyzed for 1 h at 60°C with 2 M KOH in 50% aqueous methanol, containing hydroquinone as an antioxidant, and a known amount of C21:0 as an internal standard. Distilled water was added to the cooled samples, and nonsaponifiable lipids were extracted into light petroleum ether at 40 to 60°C and then discarded. The hydrolysates were acidified with 5 M H2SO4 and fatty acids were extracted into petroleum ether. Methyl esters were prepared using diazomethane in diethyl ether and analyzed by gas chromatography using a CP Sil88 WCOT capillary column (Chrompack Varian, Inc., Walnut Creek, CA), a split-splitless injector set with a split of 50:1, and a flame ionization detector. Helium gas was used as a carrier and fatty acids were identified by comparison with standards from Sigma (Poole, Dorset, UK). Quantification was achieved by use of the internal standard added before hydrolysis. The linearity of the detector response was tested using a reference monoenoic fatty acid methyl ester (FAME) mix (FAME5; Thames Rstek UK Ltd., Bucks, UK). Subcellular Organelle Isolation Stearoyl-CoA desaturase (SCD) and delta-6 desaturease (Δ6-d) are membrane-bound enzymes and fatty acid synthase (FAS) is a cytosolic enzyme. Therefore, both the cytosol and microsomal fractions were isolated from subcutaneous adipose tissue samples by differential centrifugation with Ca2+ as described previously (Doran et al., 2004). Approximately 5 to 10 g of tissue was thawed and homogenized in a Tris-sucrose buffer (10 mM Tris and 250 mM sucrose, pH 7.4). The postmitochondrial fraction containing cytosol and microsomes was obtained by centrifugation at 10,000 × g for 10 min at 4°C. The supernatant was removed and CaCl2 was added to a final concentration of 8 mM. The mixture was centrifuged at 43,000 × g for 50 min at 4°C to separate microsomes from cytosol. Approximately 1 mL of the cytosolic fraction was collected from each sample and frozen at –20°C for FAS expression analysis. The remaining supernatant was discarded. The microsomal pellet was resuspended in Tris-KCl buffer (pH 7.4) and frozen at –20°C for SCD and Δ6-d protein expression analysis. This buffer contained the protease inhibitors antipain hydrochloride, pepstatin A, and leupeptin hydrochloride at concentrations of 1.5, 1.5, and 2 μM, respectively, to prevent possible protein degradation during storage. Total protein concentration in microsomes and cytosol was determined by using the method described by Bradford (1976).

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Enzyme Expression Analyses To determine the amount of FAS, Δ6-d, and SCD proteins present in microsomal fractions (in case of SCD and Δ6-d) or incytosolic fractions (in case of FAS), protein expression was analyzed by Western blotting. To compare expression of the lipogenic enzyme proteins between samples run on different blots, a reference sample was used. The reference sample was a microsomal preparation in the case of SCD and Δ6-d or a cytosolic preparation in the case of FAS. These preparations were made from subcutaneous adipose tissue of 2 randomly chosen animals used in this study. The microsomal or cytosolic preparations from the 2 samples were combined together and called reference samples. Combination of preparations from 2 samples was necessary to ensure sufficient amount of the reference sample for all experiments. The reference sample was present on all blots and the intensity of signal of the reference sample was always taken as 100 arbitrary units. The signal intensity of other samples present on the same blot was expressed as a fraction of the signal intensity of the reference sample. The reference sample was run on all blots in duplicate to eliminate experimental error. Fatty acid synthase protein expression was analyzed using 5 μg of cytosolic proteins, which were separated by SDS-PAGE and transferred onto a nitrocellulose membrane at 100 V for 1 h. The membrane was then blocked with a 10% milk solution in PBS Tween (PBST) for 1 h and probed overnight at 4°C with primary antibody (rabbit polyclonal anti-FAS IgG), which recognizes the porcine FAS immunoreactive protein (Abcam, Cambridge, UK). The membrane was washed with PBST and incubated with secondary antibody [goat anti-rabbit conjugated to horseradish peroxidise (Santa Cruz Biotechnology, Santa Cruz, CA)] for 1 h at room temperature. Protein bands were visualized by developing the membranes with commonly used Enhanced Chemiluminescence Reagent (GE Healthcare, Buckinghamshire, UK). The intensities of the protein bands were quantified using ImageQuant software (Molecular Dynamics; GE Healthcare, Buckinghamshire UK). The optimum amount of cytosolic protein used for the analyses (5 μg) was determined by calibration. All blots were performed in duplicate. If duplicates differed by more than 10%, a triplicate was run. All repeats were averaged for statistical analysis. Delta-6-desaturase and SCD protein expression was analyzed in the microsomal fraction. The amount of microsomal protein used in the analysis was determined by calibration and was either 6 μg (for ∆6-d) or 8 μg (for SCD). Microsomal proteins were separated by SCD-PAGE and electroblotted onto a nitrocellulose membrane as described above for FAS. Incubation

conditions were similar to those described for FAS with the exception of the primary and secondary antibodies used. In the case of ∆6-d, we used rabbit polyclonal anti-∆6-desaturase antibodies produced against the synthetic peptide containing the AA sequence from regions that are conserved in the rat, pig, and human near the C-terminus of corresponding proteins (SigmaGenosys Ltd., Cambridge, UK). Steroyl-CoA desaturase antibodies were produced by Abcam (Cambridge, UK) in rabbits and demonstrated to cross-react with corresponding porcine proteins. For both SCD and ∆6-d, we used goat anti-rabbit secondary antibody conjugated to horseradish peroxidise (Santa Cruz Biotechnology). The SCD and ∆6-d signals were detected and quantified as described for FAS. Statistical Analysis Fatty acid composition and the relative expression of FAS, ∆6-d, and SCD proteins were analyzed using 1-way ANOVA. The experimental group in the statistical analyses was a group of animals receiving the same treatment (i.e., vaccinated, barrows, or entire male pigs). Post hoc comparison of the means was done using the Tukey method. A P-value < 0.05 was considered statistically significant. The relationship between SFA and FAS, C9c11tCLA and SCD, C18:1n-9 and SCD, n-3 and ∆6-desaturase, and n-6 PUFA and ∆6-d were analyzed using Pearson’s correlation analysis and P < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS software version 17.0 (SPSS, Portsmouth, UK). RESULTS Saturated Fatty Acids There was a difference (P < 0.001) in mean concentration of total SFA between groups. Mean total SFA concentration was 17% greater (P = 0.036) in vaccinated males and 31% greater (P < 0.0001) in barrows compared with entire males (Table 1). Differences in the mean concentration of C14:0, C15:0, C16:0, C18:0, and C20:0 SFA were observed between groups. In vaccinated pigs, the mean concentration of C15:0 was 21% lower (P = 0.007) and the mean concentration of C20:0 32% greater (P = 0.034) compared with entire boars. The mean concentration of C16:0 was 11% less (P < 0.001) in vaccinates than in barrows. Compared with entire males, the mean concentrations of SFA C14:0, C16:0, C18:0, and C20:0 were 17 (P = 0.009), 28 (P < 0.001), 41 (P = 0.002), and 54% (P < 0.001) greater in barrows, respectively, but the mean concentration of C15:0 was 43% less (P < 0.001) in barrows.

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Table 1. Saturated fatty acid, MUFA, and PUFA content of subcutaneous adipose tissue in barrows, males vaccinated with Improvac (Pfizer Animal Health, New York, NY), and entire boars1 Item

Individual fatty acid

SFA

C12:0 C14:0 C15:0 C16:0 C17:0 C18:0 C20:0 ΣSFA2

MUFA

C16:1 C18:1n-7 Ct18:1n-7 C18:1n-9 C20:1 ΣMUFA3

Barrows 0.76 (0.02) 11.51a (0.29) 0.4a (0.02) 177.1a (4.02) 2.67 (0.15) 86.24a (2.95) 1.79a (0.09) 280.56a (6.62) 21.56a (1.01) 22.45a (0.96) 2.12a (0.05) 308.8a (5.31) 6.49a (0.22) 361.50a (6.71) 0.75a (0.02) 5.86 (0.626) 0.12 (0.01) 0.44 (0.02) 0.11 (0.02) 88.89a (1.82) 0.63a (0.02) 1.44a (0.04) 0.57a (0.02) 6.53 (0.26)

Mean fatty acid, mg/g adipose tissue and SE Vaccinated 0.74 (0.03) 10.48ab (0.4) 0.55a (0.03) 157.34b (7.1) 2.82 (0.18) 77.41a (5.59) 1.53a (0.11)

Entire 0.75 (0.03) 9.84b (0.38) 0.7b (0.04) 138.20b (4.95) 3.15 ( 0.19) 61.10b (3.02) 1.16b (0.1)

P-value 0.824 0.011