Acta agriculturae Slovenica, suplement 2 (september 2008), 93–98. 
 
16th Int. Symp. “Animal Science Days”, Strunjan, Slovenia, Sept. 17–19, 2008. 
  
Agris category codes: L10, Q04 COBISS Code 1.08 
INVESTIGATING PRKAG3 POLYMORPHISMS FOR TRAITS OF INTEREST IN DRY 
HAM PRODUCTION 
Martin ŠKRLEP a), Marjeta ČANDEK-POTOKAR a), Tatjana KAVAR a), Blaž ŠEGULA a), 
Veronique SANTÉ-LHOUTELLIER b) and Pere GOU c) 
a) Agricultural Institute of Slovenia, Hacquetova 17, SI-1000 Ljubljana, Slovenia  
b) INRA, Qualité des produits animaux, FR-63122 Saint Genés Champanelle, France 
c) IRTA, Food Processing and Engineering, Finca Camps i Armet, E-17121 Monells, Spain 
ABSTRACT 
Genetic markers affecting proteolysis could prove useful for selection of pigs suitable for dry 
ham production with lower salt content and/or prolonged maturation. One of the candidate genes 
to affect muscle metabolism, and consequently meat and dry-cured ham quality, is the I199V 
polymorphism of PRKAG3 gene which is currently studied within the 6th FP project 
TRUEFOOD (FOOD-CT-2006-016264) in three countries (France, Spain and Slovenia). 
Presented results involve 569 Slovenian Duroc×Hampshire sired commercial pigs slaughtered in 
10 batches from which 723 hams were harvested and processed into dry hams “kraški pršut”. 
Pigs were genotyped (PCR-RFLP) and traits of interest measured (backfat thickness, meat%, 
carcass weight, ham weight, ham subcutaneous fat thickness, pH in semimembranosus muscle, 
colour, processing losses). Genotype frequencies on codon 199 of PRKAG3 gene were 12.7%, 
55.5% and 31.8% for I/I, I/V and V/V, respectively. Results indicate an effect of PRKAG3 gene 
polymorphism on carcass traits (carcass weight, ham weight, ham subcutaneous fat thickness), 
meat quality (m.gluteus medius colour score and Minolta L*) and processing losses (weight 
losses at the end of salting, resting and drying phase).  
Key words: pigs / meat quality / processing losses / dry ham / PRKAG3 / genotype frequencies / carcass traits 
RAZISKOVANJE POLIMORFIZMOV GENA PRKAG3 ZA LASTNOSTI POMEMBNE 
PRI PROIZVODNJI PRŠUTA 
IZVLEČEK 
Genski markerji, povezani s proteolizo v mišičnini, bi lahko precej pripomogli k selekciji 
prašičev, namenjenih za predelavo v pršut, z manjšo vsebnostjo soli in/ali podaljšanim zorenjem. 
V projekt 6. OP TRUEFOOD (FOOD-CT-2006-016264) je vključena tudi raziskava vpliva 
kandidatnih genov. Eden od preučevanih polimorfizmov je I199V na genu PRKAG3. V 
raziskavi sodelujejo tri države (Francija, Španija in Slovenija), predstavljamo pa slovenske 
rezultate na materialu, ki vključuje 569 križancev, potomcev očetovske linije durok×hempšir, 
zaklanih v 10 serijah. Prašiči so bili genotipizirani s pomočjo PCR-RFLP metode. Odbranih je 
bilo 723 stegen, ki so bila poslana v predelavo v “kraški pršut”. Izmerili smo zanimive lastnosti 
(debelina hrbtne slanine, mesnatost, masa trupa, masa stegna, debelina slanine na stegnu, pH v 
m. semimembranosus, barva in izgube mase med predelavo). Frekvence genotipov na kodonu 
199 gena PRKAG3 so bile 12,7 % za I/I, 55,5 % za I/V in 31,8 % za V/V genotip. Analize 
rezultatov kažejo na možen vpliv polimorfizmov PRKAG3 gena na lastnosti klavnega trupa 
(masa toplih trupov, masa stegna in debelina stegenske slanine), kakovosti mesa (ocena barve in 
vrednost L* v m.gluteus medius) ter izgube med predelavo (izgube po soljenju, podaljšanem 
soljenju in sušenju).  
Ključne besede: prašiči / meso / kakovost / predelava mesa / izgube / pršut / PRKAG3 / frekvence genotipov / 
klavne lastnosti 
http://aas.bf.uni-lj.si 
Acta agriculturae Slovenica, suplement 2 (september 2008). 
 
94 
INTRODUCTION 
One of the main health threats in particular for cardiovascular diseases in the contemporary 
human diet is the increased salt ingestion due to its excessive use in food processing. High 
content of salt is still present in many traditional dry hams (e.g. Spanish “Jamon Serrano”, 
French “Jambon de Bayonne” and Slovenian “kraški pršut”). One of the reasons is that salt plays 
an important role as an enzymatic inhibitor (especially for proteolytic enzymes) and as such 
prevents the occurrence of quality defects related to excessive proteolysis (Toldra et al., 1992). 
Reducing salt content may cause defects in texture (excessive softness, pastiness), aroma, taste 
and colour (Buscailhon and Monin, 1994). Proteolysis is strongly affected by the properties of 
raw material (fat, water content and meat pH, Morales et al., 2007). Therefore it has been 
suggested that the selection of pigs (based on genetic polymorphisms) would be of interest so as 
to obtain appropriate raw material for salt reduction and/or prolonged maturation. Among the 
investigated genes known to affect meat quality in pigs the PRKAG3 is a good candidate. The 
gene encodes a specific isoform of γ subunit of the adenosine monophosphate dependant protein 
kinase (AMPK), an enzyme with the key role in cell energy metabolism regulation. Five 
nonsynonymous substitutions (T30N, G52S, L53P, I199V and R200Q) have been detected in the 
PRKAG3 gene (Milan et al., 2000). In addition to R200Q substitution also known as RN- 
mutation, well known for causing a defect named “acid meat” (Sellier and Monin, 1994), the 
I199V substitution (Ile →Val on the codon 199) turned out to be the most interesting. It was 
shown to affect carcass leanness, muscle glycogen content, ultimate muscle pH and water 
holding capacity (Ciobanu et al., 2001; Enfält et al., 2006; Lindahl et al., 2004a). The allele that 
seems to be favourable for meat quality is the 199I. Low incidence of I/I genotype (< 10%) have 
been reported for the majority of modern breeds and crosses, with the exception of Berkshire 
breed (Ciobanu et al., 2001; Josell et al., 2003; Huang et al., 2004; Lindahl et al., 2004 a, b; 
Stalder et al., 2005; Otto et al., 2007). The aim of the present study was firstly to screen the 
PRKAG3 genotype frequencies in one Slovenian commercial crossing and secondly to evaluate 
the effect of I199V polymorphisms on the aptitude of raw material (green hams) for dry ham 
processing.  
MATERIAL AND METHODS 
The material consists of 569 approximately 6.5 months (200 days) old pigs, commercial 
crosses originating from one herd and line i.e. landrace (LN) × large white (LW) as maternal and 
DU (duroc) × hampshire (HA) as paternal line. Pigs from this herd were previously demonstrated 
to be free of RYR1 and RN- mutation (Škrlep et al., 2008 in press). All animals were slaughtered 
in the same commercial abattoir according to the routine procedure (i.e. CO2 stunning, vertical 
exsanguination, vapour scalding, dehairing and evisceration, followed by veterinary inspection 
and carcass classification). The carcasses were cooled (by storage at 0–2 °C) until the internal 
temperature dropped below 5 °C. 
For genotype determination, small pieces of ear laps were taken from each animal at the end 
of the slaughter line and frozen until the analysis. All animals were genotyped for PRKAG3 
I199V polymorphisms according to Milan et al. (2000). Backfat thickness (Fat-DM5), muscle 
thickness (Muscle-DM5), warm carcass weight and meat% were measured according to the 
approved method for carcass classification (OJ EU L324/87 10.12.2005). The following day, the 
hams were selected against visible lesions (skin lacerations, haemorrhages, hematomes and 
extensive veining) on the surface of the hams (Šegula et al., 2007), and sent to two processors of 
dry ham “kraški pršut”, where they were cut into the prescribed shape, and further selected 
according to weight (≥ 9.5 kg), subcutaneous fat thickness (≥ 10 mm) and meat quality 
consistent with the rules for “kraški pršut”. Subcutaneous fat under caput femoris, pH in m. 
Škrlep, M. et al. Investigating PRKAG3 polymorphisms for traits of interest in dry ham production. 
 
95
semimembranosus (MP120 Mettler Toledo pH Meter) and Minolta L* value (Minolta Chroma 
Meter CR-300) on m. gluteus medius were measured. Colour on m. gluteus medius was also 
assessed using Japanese 1–6 colour scale (Nakai et al., 1976). The hams were processed 
according to the specifications for “kraški pršut”. i.e. two salting phases, resting phase, drying 
phase and maturation phase (in total 60 weeks). Weight losses after each processing phase were 
recorded.  
Analysis of variance was performed using a statistical package SAS (SAS Inst., Inc., Cary, 
NC) and procedure GLM. The model included the fixed effects of the processor, PRKAG3 gene 
and slaughter batch within processor. In case of significant genotype effect (P < 0.05) least 
square means were compared using PDIFF option, adjust = Tukey. 
RESULTS AND DISCUSSION 
In the present study, the observed frequencies of PRKAG3 codon 199 genotypes (Table 1) 
were 12.7% (I/I), 55.5% (I/V) and 31.8% (V/V). Low incidence of I/I genotype is coherent with 
the available literature reports for the majority of modern breeds or crosses (Ciobanu et al., 2001; 
Josell et al., 2003; Huang et al., 2004; Lindahl et al., 2004 a, b; Stalder et al., 2005; Otto et al., 
2007). Also, the observed frequencies for the I199V genotypes are similar to the ones obtained in 
our previous study on Slovenian pigs (9.8% I/I, 51.1% I/V and 39.1% V/V, Škrlep et al., 2008 in 
press) The only breed for which the literature reports higher frequency of I/I genotype (74%) is 
the Berkshire breed (Ciobanu et al., 2001), also known for its good meat quality, especially 
appropriate for cured products (Breeds of livestock, 2008)). The results presented in Table 1 
show, that I199V genotypes at PRKAG3 gene significantly affected green ham weight and fat 
thickness over m. gluteus medius at carcass split line and below caput femoris bone. There was 
also notable effect (P < 0.1) for leanness, carcass weight, meat colour and Minolta L* value. Pigs 
of I/I genotype had lower carcass and green ham weights than I/V or V/V pigs. In agreement 
with our previous study (Škrlep et al., 2008 in press) the heterozygous pigs (I/V) exhibited the 
thickest subcutaneous fat at two anatomical locations. Overall, there is not much available 
literature about the effect of I199V PRKAG3 polymorphism on carcass composition (especially 
for I/I genotype). The related studies of Josell et al. (2003), Lindahl et al. (2004a, b) and Enfält 
et al. (2006) pooled the results of I/I and I/V genotype into one group, due to very low 
frequencies of I/I. Even so, according to Enfält et al. (2006) the presence of allele 199I is related 
to lower lean meat content. Our study showed no significant effect on the ultimate pH, which is 
against our expectations, since the available literature indicates an effect of this gene on muscle 
pH, i.e. higher pH for the I/I in comparison to V/V pigs (Ciobanu et al., 2001; Otto et al., 2007). 
On the other hand, notable effect (P < 0.1) of I199V polymorphism was observed on colour; both 
Minolta L* and subjective colour note. Pigs with I/I genotype had darker LD muscle colour than 
V/V, which corroborates with other literature data (Ciobanu et al., 2001; Lindahl et al., 2004b, 
Otto et al., 2007). 
The results presented in Table 2 show, that I199V polymorphism in PRKAG3 gene affected 
significantly ham processing weight losses during first salting and resting phase and as a 
consequence total weight losses. Again it was the heterozygote genotype that exhibited the 
lowest processing losses (significantly different from V/V, but not from I/I), in agreement with 
the thickest fat, which represents the obstacle for salt diffusion and water removal. In the only 
comparable study available in the literature (on American country-style dry-cured hams), Stalder 
et al. (2005), did not report any significant differences for processing yields although the values 
they obtained show similar trend as in our case i.e. the I/V genotype exhibited the highest 
processing yield or the lowest processing weight loss compared to other two genotypes.  
 
Acta agriculturae Slovenica, suplement 2 (september 2008). 
 
96 
Table 1. Least squares means (standard errors) of I199V genotypes for carcass and ham 
properties  
 
 I/I I/V V/V P 
Number of pigs 72 316 181  
Muscle-DM5, mm 74.2 (0.6) 74.7 (0.3) 75.0 (0.4) 0.42 ns  
Fat-DM5, mm 15.4 (0.4) ab  15.9 (0.2) a  15.0 (0.3) b  0.03* 
Meat, % 57.9 (0.4) 57.6 (0.2) 58.2 (0.2) 0.09 † 
Carcass weight, kg 96.4 (0.4) 98.3 (0.4) 98.3 (0.5) 0.06 † 
Number of hams 94 394 235  
Green ham, kg 10.8 (0.08) a 11.0 (0.04) b 11.1 (0.05) b  0.005** 
Ham fat, mm 12.9 (0.3) a  13.8 (0.1) b 13.3 (0.2) a  0.008** 
pHu (m. semimembranosus) 5.70 (0.02) 5.69 (0.01) 5.70 (0.01) 0.83 ns 
Colour (1–6) 3.7 (0.06) 3.6 (0.03) 3.5 (0.04) 0.07 † 
Minolta L* 47.6 (0.3) 48.3 (0.2) 48.6 (0.2) 0.08 † 
a,b,c Least squares means followed by a different letter are significantly (P < 0.05) different; P = level of significance, 
P > 0.1 = statistically insignificant (ns); P < 0.1 = tendency (†); statistically significant: P < 0,05 (*), P < 0.01(**), 
P < 0.001(***); Muscle-DM5 = muscle thickness according to DM5 method; Fat-DM5 = subcutaneous fat thickness 
according to DM5 method; Meat = % of meat in the carcass according to DM5 method; Carcass weight = hot 
carcass weight; Green ham = weight of the ham prior to processing; Ham fat = subcutaneous fat thickness below 
caput ossis femoris; pHu = ultimate pH value of m. semimembranosus; colour (1–6) = colour of m. gluteus medius 
according to Japanese scale (Nakai et al., 1976); Minolta L* = CIE L parameter measured with Minolta 
chromameter on m.gluteus medius. 
 
Table 2. Least squares means (standard errors) of I199V genotypes for ham weight processing 
losses 
 
 I/I I/V V/V P 
Number of hams 94 394 235  
Salting 7d, % 2.30 (0.03) ab 2.26 (0.02) a 2.37 (0.02) b 0.0005***
Salting 7–14d, % 1.77 (0.03) 1.75 (0.01) 1.75 (0.02) 0.79 ns 
End of salting 14d, % 4.02 (0.05) ab 3.97 (0.02) a 4.08 (0.03) b 0.02* 
Resting, % 17.64 (0.11) ab 17.43 (0.06) a 17.78 (0.07) b 0.0006***
End of resting, % 20.95 (0.13) ab 20.70 (0.07) a 21.13 (0.09) b 0.0005***
Drying, % 7.18 (0.07) 7.14 (0.04) 7.17 (0.05) 0.79 ns 
End of drying, % 26.61 (0.16) ab 26.36 (0.08) a 26.80 (0.10) b 0.003** 
Maturation, % 13.11 (0.15) 12.48 (0.07) 12.98 (0.10) 0.23 ns 
End of maturation, % 36.25 (0.23) ab 35.81 (0.11) a 36.27 (0.15) b 0.02* 
a,b Least squares means followed by a different letter are significantly (P < 0.05) different; P = level of significance, 
P > 0.1 = statistically nonsignificant (ns), P < 0.1 = tendency (†), statistically significant: P < 0,05 (*), P < 0.01(**), 
P < 0.001(***); Salting 7d = weight loss after 7 days of salting; Salting 7–14d = weight loss between 7 and 14 days 
of salting; End of salting = weight loss at the end of salting period; Resting = weight loss in the resting period; End 
of resting = weight loss from the beginning of the processing to the end of resting; Drying = weight loss in the 
drying period; End of drying = weight loss from the beginning of the processing to the end of drying period; 
Maturation = weight loss in the maturation period; End of maturatiom = weight loss from the beginning of the 
processing to the end of maturation period. 
Škrlep, M. et al. Investigating PRKAG3 polymorphisms for traits of interest in dry ham production. 
 
97
CONCLUSIONS 
Our study indicates an effect of I199V polymorphism in PRKAG3 gene on green ham 
properties and dynamics of ham weight losses, all susceptible to influence salt intake and 
diffusion. As the endogenous proteolytic activity is directly (pH, Aw, salt concentration) or 
indirectly (ham fatness, marbling and weight) affected by green ham properties, we expect to be 
able to observe the effect of the PRKAG3 polymorphism also on the properties of processed dry 
hams. 
ACKNOWLEDGEMENT 
This study was made within the research project TRUEFOOD (“Traditional United Europe 
Food”), an Integrated Project financed by the European Comission under the 6th Framework 
Programme for RTD, contract n. FOOD-CT-2006-016264. The information in this document 
reflects only the author’s views and the Community is not liable for any use that may be made of 
the information contained therein.  
The authors would like to thank companies Farme Ihan d.d., KRAS d.d. and MIP d.d. for 
cooperation (providing material and technical assistance) within the presented research. 
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