PSS GENE GENOTYPING AND ASSOCIATIONS TO MEAT QUALITY

TRAITS IN A SWINE F2 POPULATION

1
G.O. Band,
1
*S.E.F. Guimares,
1
P.S. Lopes,
3
A.V. Pires,
1
D.A. Faria,
1
K. M.
Silva,
2
A. A. Benevenuto Jnior,
2
L.A. M. Gomide.

1
Animal Science Department and
2
Food Technology Department, UFV, Viosa, MG, Brasil, CEP:
36571-000,
3
FAESA -Campus II - Faculdade de Sade,Rodovia Serafim Derenzi, 3115,So Pedro, Vitria, 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 didnt 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
muscle 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
Viosa, Viosa, 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/L 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 M each
(forward - 5-TCCAGTTTGCCACAGGTCCTACCA-3 and reverse 5-
TTCACCGGAGTGGAGTCTCTGAGT-3, OBRIEN et al., 1993), and 25 ng of genomic DNA
in a volume of 20L. 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 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 Viosa. Details
can be found at BENEVENUTO Jr. (2001).
Statistical analysis: Statistical analysis for association of genotypes with meat quality
traits were made by SAS PROC GLM System, according to the model:

Yijkl = + Gi + Sj + Lk + eijkl
Where:
Yijkl = observation at the animal;
= 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: 6.510.26 and 6.410.26 for pH45,
3.061.68 and 3.921.68 for DL, 32.502.54 and 33.292.54 for CL and 35.672.67
and 34.012.67 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 JNIOR, A. A., 2001. Avaliao de rendimento de carcaa e de qualidade
da carne de sunos comerciais, de raa nativa e cruzados. Dissertao de mestrado, 93
pgs., 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.

OBRIEN, 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.