Association between a non-synonymous HSD17B4 single nucleotide polymorphism and meat-quality traits in Berkshire pigs

Carcass trait analyses

The Berkshire pigs were slaughtered by electrocution with electrical tongs (300 V for 3 s) after 12 h of feed restriction. The carcasses were placed in a dehairer at 62°C for 5 min and any remaining hair was removed using a knife and flame following exsanguination. Carcasses were eviscerated and split before being placed in a chiller set at 2°-4°C for 12 h. Various meat-quality traits including carcass weight, backfat thickness, meat color (lightness, redness, and yellowness), drip loss, cooking loss, chemical composition (collagen, fat, moisture, and protein), water holding capacity, shear force, and post-mortem pH 24 h (measurement of pH after 24 h of slaughter) were measured (Cho et al., 2015b).

Backfat thickness was measured on the 10th rib region positioned at three-quarters distance along the longissimus thoracis (LT) toward the belly, within 30 min of slaughter. The concentrations of moisture, crude protein, and crude fat from the LT muscles were determined according to the methods of the Association of Official Analytical Chemists (AOAC, 2006) in muscle samples taken 24 h after slaughter. Collagen content was measured as previously described (Cho et al., 2015b). The LT (6th to 13th rib) was excised and stored at 2°-4°C before it was transported to the laboratory to determine the chemical composition (Cho et al., 2015b).

Meat and fat color were recorded after 30 min blooming at 1°C by using a Minolta Chromameter (CR400; Minolta, Japan) with 8 mm in aperture size, illuminant D65, a 2° closely matches CIE1931 standard observer, and measurement/illumination of ɸ8/ɸ11 mm. The instrument was standardized using white plate (L* = +97.83, a* = -0.43, b* = +1.98) (Cho et al., 2015b).

The drip loss was measured as the percentage weight loss of a standardized (3 x 3 x 3 cm) LT muscle sample placed on a sealed Petri dish at 4°C for 2 days. A 3-cm thick slice (weight 100 ± 5 g) separated from the LT muscle was used to measure cooking loss. LT muscle was placed into a polypropylene bag, cooked for 40 min at 70°C in a water bath, and then cooled to room temperature. Cooking loss was calculated by the weight difference before and after heating (Cho et al., 2015b).

The water holding capacity was determined according to a previously described method (Kristensen and Purslow, 2001). Warner-Bratzler shear force was measured using LT muscle, which was allowed to thaw to room temperature for about 2 h before measurement using 5 cores (1.23 cm in diameter) following the direction of the fibers and sheared at a crosshead speed of 240 mm/min (Cho et al., 2015b).

The post-mortem pH 24 h was measured using 10 g LT muscle that was stored at 4°C for 24 h. The LT muscle was cut into small pieces to which 90 mL distilled water was added and an slurry was made using a homogenizer (T25B, IKA Sdn, Bhd., Malaysia). The post-mortem pH 24 h of the slurry was then measured using a digital pH meter (8603, Metrohm, Swiss).

Identification of the non-synonymous SNP (nsSNP) by RNA-Seq and genotyping

Total RNA from liver tissue was isolated using an RNA-Seq sample preparation kit as previously described (Illumina, Inc., San Diego, CA, USA) (Gispert et al., 2010). The SNP was identified as previously described (Jung et al., 2012). The nsSNP within the HSD17B4 gene was identified as rs81216713 using the dbSNP database. Genomic DNAs were isolated using the Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA) from individual LT muscles of Berkshire pigs. To analyze the association between the nsSNP in the HSD17B4 gene and pork quality traits, genomic DNAs of 377 Berkshire LT muscles were employed using a Veracode GoldenGate assay kit (Illumina). Oligonucleotides utilized for genotype analyses are indicated in Table 1.

RT-qPCR amplification

To investigate the mRNA expression of HSD17B4 in various tissues of Berkshire, Duroc, Landrace, and Yorkshire pigs, total RNA was isolated from liver, stomach, lung, kidney, large intestine, small intestine, and spleen of three female pigs using TRI Reagent^® according to the manufacturer’s protocol (Molecular Research Center, Cincinnati, OH, USA). To elucidate the mRNA expression of marker genes for myogenesis, total RNA was prepared from C2C12 (mouse myoblast) cells. The quality of isolated RNA was measured as A₂₆₀/A₂₈₀ >1.8. The mRNA (500 ng) was reverse-transcribed into cDNA using Superscript II (Invitrogen, Carlsbad, CA, USA) in a total reaction volume of 20 μL.

Gene-specific exon spanning primers against pig and mouse HSD17B4, genes that were used for normalization (PPIA, TBP, TOP2B, RPL4, and RPS18 for pig and GAPDH for mouse), and genes associated with myogenesis (MyoD, MyoG, and MyHC-2B) were designed using the Primer3 program (http://frodo.wi.mit.edu/primer3/; Table S1) (Rozen and Skaletsky, 2000). BLASTs were performed to confirm gene specificity of the primer sequences, and the results showed the absence of multi-locus matching at individual primer sites. Standard curves were determined by increasing the quantity of template DNA, a t-vector clone created with RT-PCR products from each gene. Amplification efficiencies and correlation coefficients (R² values) were measured using the slopes of the standard curves obtained from the serial dilutions. Standard curves with a 10-fold dilution series were used to calculate amplification efficiency according to the following formula: efficiency (%) = (10^(-1/slope) - 1) x 100. The range of efficiency of the real-time RT-PCR amplifications was 88-99%.

RT-qPCR was performed using SYBR Green Chemistry with a Rotor Gene Q thermocycler (Qiagen). For each reaction, 1 µL cDNA (500 ng RNA was used for reverse transcription in a total volume of 20 µL) was used as a template and added to a 19-µL reaction mixture containing 7 µL H₂O, 10 µL Rotor Gene SYBR Green PCR mastermix (Qiagen), and 1 µL each of forward and reverse primer (10 pmol). Real-time RT-PCR amplification was performed as described previously (Park et al., 2015). In brief, PCR conditions were as follows: 40 cycles at 94°C for 5 s and 60°C for 10 s. The melting curve, performed at 70° to 95°C for 5 s, was used to confirm the specificity of each reaction. Three independent reactions were performed to determine statistical significance. Target genes were normalized for relative quantification by the normalization factor derived from geometric means delta-Cq of PPIA, TBP, and HSPCB in Berkshire pigs; PPIA, TBP, RPL4, and RPS18 in Landrace pigs; PPIA and TBP in Duroc pigs; and PPIA, TOP2B, RPL4, and RPS18 in Yorkshire pigs. The fold induction of HSD17B4 was analyzed by 2^-∆∆Cq after determining ∆∆Cq based on the relative difference between the experimental target and a control, which was extemporaneously selected based on the tissue with the lowest level of HSD17B4 expression. All experiments were performed according to guidelines of Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) (Bustin et al., 2009). The significance between results was analyzed by the Student t-test with P < 0.05 being considered significant.

Cell culture and myoblast differentiation

Mouse C2C12 myoblast cells were purchased from the ATCC (Manassas, VA, USA). C2C12 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Daegu, Korea) supplemented with 10% of fetal bovine serum (Gibco, Grand Island, NY, USA) in the presence of 100 U/mL penicillin and 100 µg/mL Streptomycin (Welgene) at 37°C in humidified atmosphere of air and 5% CO₂. For myogenesis, C2C12 cells were switched to a differentiation medium containing DMEM supplemented with 2% heat-inactivated horse serum (Gibco) at 80% confluence for 8 days.