The objective of this work was analyze the PSS genotype by PCR-RFLP in a F2 segregant swine population, produced by outbred crossing using commercial sows and Brazilian [r]
Trang 1PSS GENE GENOTYPING AND ASSOCIATIONS TO MEAT QUALITY
TRAITS IN A SWINE F2 POPULATION 1
G.O Band, 1*S.E.F Guimarães, 1P.S Lopes, 3A.V Pires, 1D.A Faria, 1K M
Silva, 2A A Benevenuto Júnior, 2L.A M Gomide
1Animal Science Department and 2Food Technology Department, UFV, Viçosa, MG, Brasil, CEP:
36571-000,
3FAESA -Campus II - Faculdade de Saúde,Rodovia Serafim Derenzi, 3115,São Pedro, Vitória, ES,
CEP: 29030-001
*Corresponding author Financial Support: FAPEMIG, CNPq, CAPES
ABSTRACT PSS gene is a major gene with known effects in commercial herds, affecting important traits in swine production The objective of this work was to evaluate the effects of PSS gene in meat quality traits in a F2 segregant population, produced by outbred crossing using commercial sows (18) and Brazilian native boars (2) Genotypes of the PSS gene were obtained for 596 F2 pigs by PCR-RFLP technique Meat quality traits were evaluated after slaughter Among the 596 animals, 493 animals (82.72%) were characterized as NN and 103 animals (17.28%) as Nn Both genotypes didn’t differ for
pH 24 hours after slaughter (5.71 vs 5.70, for NN and Nn, respectively), intra muscular fat (1.55% vs 1.65% to NN and Nn animals), objective tenderness (5551.60g/1.2cm vs 5506.60g/1.2cm to NN and Nn), lightness (44.96 vs 45.01 to NN and Nn), hue angle (84,28 vs 83,41 to NN and Nn) and chroma (6.68 vs 6.73 to NN and Nn) However, Nn animals presented lower results (P<0.05) for pH 45 minutes after slaughter (6.51 vs 6.41), higher drip (3.06% vs 3.92%), cooking (32.50% vs 33.29%) and total (34.01% vs 35,67%) losses These results confirm that PSS gene carriers present inferior meat quality and that even in divergent crosses gene effects are observed
KEY WORDS:
PSS syndrome, PCR-RFLP, meat quality, pigs
INTRODUCTION Due to genetic selection, pig has became fast growing, lean meat producer with a high food convertion Some negative effects of the leanness, such as increased stress susceptibility, leading to a poor meat quality condition have been found very frequently It is known that the negative genetic correlation between meat quality and lean pork is greatly due to the PSS (Porcine Stress Syndrome) gene PSS affected pigs present hyperthermia, dyspnea, red to purple skin and a marked stress response, with cardiac arrest After slaughter, the findings include pale, soft, exudative (PSE) meat, and rapid development of rigor mortis One of the main consequences of PSE meat is the inferior quality of the products, for example, cured products made from PSE meat have a lower net gain, due to reduced water holding capacity, when compared to normal meat The defect in PSS susceptible animals is a hypersensitive gating of the Ca2+ -release channel (ryanodine receptor – coded by ryr1 gene) of the skeletal
Trang 2muscle sarcoplasmatic reticulum, in this situation, channel opening is facilitated and closing is inhibited
The first method developed to detect PSS susceptible pigs was the halothane challenge test After the challenge, pigs that developed muscle rigidity were assigned as PSS susceptible However, halothane test was not able to detect heterozygous pigs, so a DNA test was developed by FUJII et al., (1991) based in a sequence comparison of full length ryadodine receptor cDNA from a PSS susceptible pig and a PSS normal pig The authors found a polymorphism (replacement of a C at nucleotide 1843 from the PSS normal animal to a T in the cDNA from the PSS susceptible one) that could be implicated in the development of the syndrome The consequence of this replacement
is an alteration in amino acid sequence from an arginine at protein position 615 in the PSS normal, to a cysteine in the PSS susceptible pig
The objective of this work was analyze the PSS genotype by PCR-RFLP in a F2 segregant swine population, produced by outbred crossing using commercial sows and Brazilian native boars and statistically associate genotypes to several meat quality traits measured after F2 animals slaughter
MATERIALS AND METHODS DNA extraction: In this investigation, blood was collected from 596 F2 animals during slaughter These animals were produced by outbred crossing using commercial sows (18) and Brazilian native boars (2) The F2 animals were raised and slaughtered at the Pig Breeding Farm, located at the Animal Science Department, Federal University of Viçosa, Viçosa, Minas Gerais State, Brazil DNA was extracted by the sodium chloride protocol After extraction, DNA was quantified in spectrophotometer (260 and 280 nm) and diluted in 10 mM TRIS (pH 8.0) - 1mM EDTA (pH 8.0) solution to a working concentration of 25 ng/µL and preserved at 4oC The stock DNA was also diluted in TRIS-EDTA solution and maintained at -20oC
PSS gene amplification and mutation detection: DNA samples from the F2 animals were amplified in a MJ Research PTC 100-96 thermocycler (MJ Research) The amplification program consisted of the following steps: denaturation at 94oC, anneling temperature at 64 oC, and extention at 72oC This cycle was repeated 35 times The PCR reaction consisted of 1U of Taq DNA polymerase, 0.2 uM of each dNTP, 20 mM TRIS-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, primers forward and reverse 0.2 µM each (forward - 5’-TCCAGTTTGCCACAGGTCCTACCA-3’ and reverse – 5’-TTCACCGGAGTGGAGTCTCTGAGT-3’, O’BRIEN et al., 1993), and 25 ng of genomic DNA
in a volume of 20µL The success of the PCR reactions was observed in 5% PAGE, stained with silver nitrate when a 659 bp (base pairs) fragment was visualized The causative mutation of the PSS syndrome (C Þ T replacement at nucleotide 1843) was detected by digestion of the 659 bp fragment with the restriction enzyme BsiHKA I (New England Biolabs) After enzymatic digestion, genotypes were assigned as follow: homozygous pigs for the mutation (nn) were characterized by fragments at 358 bp,
166 bp and 135 bp For normal animals (NN), it was observed bands at 524 bp and
135 bp Heterozygote carriers (Nn) for the mutation presented bands at 524 bp, 358bp, 166 bp and 135 bp
Meat quality trait analysis: In this experiment 596 F2 animals slaughtered at 65 Kg of live weight were analyzed for the following meat traits: pH 45 minutes (pH45) and pH
24 hours after slaughter (pHu), intra muscular fat (IMF), drip loss (DL), cooking loss (CL), total loss (TL), objective tenderness (OT), lightness (L), redness (A), yellowness (B), hue angle (h) and chroma (c) These analyses were conducted at the Meat Science Laboratory, in the Food Science Department at the Federal University of Viçosa Details can be found at BENEVENUTO Jr (2001)
Trang 3Statistical analysis: Statistical analysis for association of genotypes with meat quality traits were made by SAS PROC GLM System, according to the model:
Yijkl = µ+ Gi + Sj + Lk + eijkl
Where:
Yijkl = observation at the animal;
µ= population mean
Gi = genotype effect i (NN or Nn)
Sj = sex effect j (1 = castrated male e 2 = female)
Lk = batch effect k (k = 1, 2, 3, 4 and 5)
eijkl = random error
The differences between the genotypes were tested by F test at 5%
RESULTS AND DISCUSSION F2 animals genotyping: The genotypic frequency for the 596 F2 analyzed animals was
493 (82.72%) normal homozygotes (NN) and 103 (17.28%) heterozygotes carriers (Nn) No recessive homozygotes (nn) were found
Meat quality traits results: The analyzed traits pH45, DL, CL and TL showed statistical difference between both genotypes (table 1) at 5% of probability The values for genotypes NN and Nn were respectively: 6.51±0.26 and 6.41±0.26 for pH45, 3.06±1.68 and 3.92±1.68 for DL, 32.50±2.54 and 33.29±2.54 for CL and 35.67±2.67 and 34.01±2.67 for TL As genotype Nn presented the highest values for DL, CL and
TL, it could be confirmed that allelic variant n has a negative effect on meat quality, since Nn animals had a worst water holding capacity, that is associated to a lower meat pH and a high degree of protein denaturation, and in this condition, an increase
in water loss is observed Although no homozygous animals were found, the effect of the PSS gene in pork quality was detected, even in this experimental segregant population, confirming that this gene is one of the most important in swine production
An important point to consider in the development of a strategy for the use of PSS mutation in commercial herds is its deleterious effects in meat quality, confirmed by the present investigation, that is negatively associated to food conversion and leanness The increased profit generated by lean and fast growing animals can be lost
if a poor quality meat is produced, so the decision of how the PSS mutation will be used in a selection program is related to the objectives of these program and to the management conditions offered to the pigs not only in the farms but specially during transportation and slaughter
REFERENCES BENEVENUTO JÚNIOR, A A., 2001 Avaliação de rendimento de carcaça e de qualidade
da carne de suínos comerciais, de raça nativa e cruzados Dissertação de mestrado, 93 págs., Dep de Tecnologia de Alimentos, UFV
FUJII, J., OTSU, K., ZORZATO, F., et al., 1991 Identification of a mutation in the porcine ryanodine receptor associated with malignant hyperthermia Science 253:
448-451
O’BRIEN, P T., SHEN, H., CORY, C R., et al., 1993 Use of a DNA-based test for the mutation associated with porcine stress syndrome (malignant hyperthermia) in 10.000 breeding swine JAVMA, Vol 203, No 6