PL-ISSN 0015-5497 (print), ISSN1734-9168 (online) Folia biologica (Kraków), vol. 59 (2011),No 1-2@ Institute of Systematics andEvolution ofAnimals, PAS,Kraków, 2011 doi:10.3409/fb59_1-2.77-82

Effect of g.2728G>A and g.3996T>C Polymorphisms at the Leptin Gene

Locus on Microstructure and Physicochemical Properties

of longissimus lumborum Muscle of Polish Landrace Pigs*

Dorota WOJTYSIAK and Urszula KACZOR

Accepted October 05, 2010

WOJTYSIAK D., KACZOR U. 2011. Effect of g.2728G>A and g.3996T>C polymorphisms atthe leptin gene locus on microstructure and physicochemical properties of longissimuslumborum muscle of Polish Landrace pigs. Folia biologica (Kraków) 59: 77-82.The influence of HindIII (g.2728G>A) and BglII (g.3996 T>C) polymorphisms at the leptingene locus on muscle fibre characteristics and meat quality of longissimus lumborum musclewas studied in 146 barrows of the Polish Landrace breed. Leptin gene polymorphism wasidentified by PCR-RFLP. Fibre type percentage, fibre diameter and the followingtechnological parameters of meat were also determined: pH45, pH24, L*a*b* colour, drip loss,water holding capacity, shear force and intramuscular fat content. Polymorphism was notdetected in the locus studied in the Landrace pig herd analysed with the BglII restrictionenzyme (g.3996 T>C). For the HindIII enzyme (g.2728G>A), there was a high frequency ofGG homozygotes (0.78) and G allele (0.89), but the AA genotype was not present. Moreover,the genotypes of leptin gene RFLP-HindIII polymorphism had no effect on intramuscular fatcontent and muscle fibre type percentage, but had a significant effect on muscle fibre size.Heterozygous GA fatteners had a significantly larger (PT, 1112A>G, 3469C>T,3714G>T, A2845T, 3996T>C and 2728G>A(STRATIL et al. 1997; JIANG & GIBSON 1999;KENNES et al. 2001). The 3469T>C polymor-phism was observed to have a significant effect onthe fat depth and daily weight gain in Large Whiteand Landrace pigs (JIANG & GIBSON 1999; KEN-NES et al. 2001), and on the lean meat content inLandrace pigs (KULIG et al., 2001). SZYD£OWSKIet al. (2004) observed a weak association betweenthe 3469T>C polymorphism and intramuscular fatcontent in Polish Large White pigs and loin weightin the synthetic line 990. The 2845A>T polymor-

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*Supported by grant no. N N311 086034.

phism may be associated with feed intake in Lan-drace pigs (KENNES et al. 2001).

The relationship between polymorphism of theleptin gene and histological characteristics (mus-cle microstructure) is still not clear. It is wellknown that many characteristics of muscle tissuesuch as size, proportion of muscle fibres, collagenand intramuscular fat content directly affect meatquality (RYU & KIM 2005; LEPETIT 2008;OSHIMA et al. 2009; YANG et al. 2010). Therefore,the purpose of this paper is to evaluate HindIII(g.2728G>A) and BglII (g.3996T>C) polymor-phisms at the leptin gene locus of Polish Landracepigs and their relationship with histological char-acteristics of muscle and several other traits thataffect meat quality.

Material and Methods

A total of 146 Polish Landrace barrows (only CCgenotypes in analysis of mutation g.1843C>T inthe ryanodine receptor 1 gene) were kept in indi-vidual pens equipped with automatic drinkers andfed ad libitum. All animals were reared under thesame environmental and production regime. Ani-mals were fattened from 50 to 105 kg body weightusing a complete diet containing 13.3 MJ metabo-lizable energy and 161 g crude protein. This levelwas calculated based on diet composition, usingtabular data for individual components (NORMY¯YWIENIA ŒWIÑ 1993). When the pigs had at-tained the appropriate slaughter weight they wereelectrically stunned and exsanguinated. Pigs wereslaughtered at a commercial slaughterhouse. Feedwas withdrawn 12 h before slaughter but waterwas freely available in lairage.

DNA was isolated from whole blood drawn intosterile tubes containing EDTA. Isolation was car-ried out using a MasterPure™ Genomic DNA Pu-rification Kit (Epicentre Technologies, USA),according to manufacturer’s instructions. Poly-morphisms at the leptin (LEP) locus g.2728G>Aand g.3996T>C were identified using the PCR-RLFPmethod described by KENNES et al. (2001). Prim-ers were synthesized by Genomed (Poland) andthe PCR (Eppendorf Thermal Cycler, Germany)reactions were performed for each polymorphismin 25 FL reaction mixtures containing 150 ng ofgenomic DNA, standard PCR buffer, 2.5 mMMgCl2, 0.2 mM each dNTP, 0.15 FM of eachprimer, and 2.0 units of Taq DNA polymerase(MBI Fermentas, Lithuania). The following cy-cling protocol was used: 94oC for 3 min, followedby 38 cycles of 94oC for 60 s, 65oC for 60 s and72oC for 60 s, and a final extension step at 72oC for6 min. The PCR products were digested overnightwith 3 units of restriction enzyme HindIII

(g.2728G>A) and 1.5 units of the enzyme BglII(g.3996T>C) (MBI Fermentas, Lithuania) at 37oC.The result was read from 1.5% agarose gelsstained with ethidium bromide and visualized us-ing the UVI-KS4001/Image PC system (SyngenBiotech, Poland).

Shortly after slaughter (15-30 min) samples forimmunohistochemical analysis were taken fromthe right carcass side from the m. longissimus lum-borum at the level of the 5th lumbar vertebra anddeep within the muscle. Muscle samples were im-mediately cut into 1 cm3 pieces (parallel to themuscle fibres) and frozen in isopentane that wascooled using liquid nitrogen and the samples werestored at -80oC until histochemical analyses wereperformed. Samples were mounted on a cryostatchuck with a few drops of tissue-freezing medium(Tissue-Tek; Sakura Finetek Europe, Zoeter-woude, The Netherlands). Transverse sections(10-Fm thick) were cut at -20oC in a cryostat (SleeMEV, Germany). To determine the muscle histo-chemical composition we used a modified com-bined method of NADH-tetrazolium reductaseactivity and immunohistochemical determinationof slow myosin heavy chain on the same section.First, sections were air-dried for 1 h and incubatedfor 1 h at 37oC with medium for the determinationof NADH-TH (DUBOVITZ et al. 1973). Next, onthe same section, immunohistochemical stainingwith monoclonal antibodies against skeletal slowmyosin heavy chain was performed for 1 h at RT(clone WB-MHCs Leica, Germany, dilution 1:80).The reaction was visualized by the NovoLinkTMPolymer Detection System (Leica, Germany), ac-cording to the manufacturer’s instruction. Finally,all sections were dehydrated in a graded series ofethyl alcohol, cleared in xylene and mounted inDPX mounting medium (Fluka, Buchs, Switzer-land). On the same section three different fibretypes were recognized, namely, type I (red, slowtwitch oxidative) which were blue-brown granulated;blue granulated type IIA (intermediate, fast-twitchoxidative glycolytic); and unstained type IIB(white, fast-twitch glycolytic) fibres. A minimumof 300 fibres were counted in each section using aNIKON E600 light microscope.

Moreover, frozen sections were also stainedwith Oil red O (Sigma, Germany) to evaluate lipidcontent. Intramuscular fat content and distributionwas evaluated based on 10 randomly chosen areas(1000 m2) in each preparation.

The percentage and diameter of muscle fibretypes and percentage of intramuscular fat werequantified with an image analysis system using theMulti Scan v. 14.02 computer program.

The samples for meat quality assessment weretaken from the right carcass side from the longis-

D. WOJTYSIAK, U. KACZOR78

simus lumborum muscle at the level of the 2nd 4thlumbar vertebrae. The meat colour was assessedby the L* (lightness), a* (redness) and b* (yellow-ness) system (CIE 1976) using a Minolta color-imeter (Chroma Meter CR-310, Minolta CameraC, Osaka, Japan). The sample values were meansfrom three measurements. The pH was measuredusing a Matthäus (Germany) pH meter with a glasselectrode standardized for pH 4.0 and 7.0 accord-ing to Polish Standard PN-77/a-82058, with auto-matic correction for muscle temperature. Intramus-cular fat content (IMF) was determined in dupli-cate according to the methods of BUDS£AWSKI &DRABENT (1972). Water holding capacity (WHC)was determined as the amount of free water ac-cording to the filter paper press method of GRAU &HAMM (1953). For determination of drip loss twochops from each muscle were weighed (e=0.001g)45 min postmortem (initial weight). Next, thechops were stored in a sealed plastic bag at 4°C.After 24 h of storage, the chops were blotted, andreweighed (final weight). Drip loss percentagewas calculated by the following equation: [(initialweight – final weight) / initial weight] × 100. Formeasurements of tenderness, samples of meat (80 g)were packed separately in plastic bags and cookedin a water bath at 95oC until the core temperaturereached 80oC. Then the samples were cooled,weighed for thermal loss determination, and pre-pared for the shear force measurements. One core(1.27 cm in diameter) was excised from each sam-ple parallel to the muscle fibre orientation, throughthe thickest portion of the cooked muscle. Shearforce was determined as maximum force (kg/cm2)perpendicular to the fibres, using an INSTRON5542 equipped with a Warner-Bratzler blade.

Associations of the pig genotypes with parame-ters of meat quality were calculated by analysingvariance of quantitative traits, which included thepercentage (%) and diameter (Fm) of muscle fibretypes and physico-chemical properties of longis-simus lumborum muscle using GLM of Statisticav.8.0PL (StatSoft, 2009).

Results and Discussion

In the present study, we identified g.2728G>Aand g.3996T>C polymorphisms at the leptin genelocus of the Polish Landrace population of pigs us-ing HindIII and BglII restriction enzymes. Analy-sis of the genetic structure of Landrace pigs usingBglII restriction enzyme (g.3996T>C) revealed nopolymorphism at the locus studied. All the pigswere TT homozygotes.

The identification of g.2728G>A polymorphismusing HindIII enzyme showed a high frequency ofGG homozygotes (0.78) and the G allele (0.89),

but no presence of the AA genotype. An earlierstudy by KENNES et al. (2001), which analysedthis mutation in Duroc, Landrace and Yorkshirepopulations showed equally high G allele frequen-cies of 0.91, 0.85 and 1.0, respectively.

Identification of the RFLP-HindIII polymor-phism made it possible to investigate its possibleinfluence on the composition of muscle fibres andquality parameters of the meat from the analysedfatteners.

The effect of genotypes of the leptin gene onmuscle fibre traits of m. longissimus lumborum ofPolish Landrace pigs is shown in Table 1 and Fig.1A-B.

The data revealed that the genotypes of the leptingene had no effect on muscle fibre type percent-age, but it affected the size of muscle fibres. Bothtype I and type IIB fibres from pigs with the GAgenotype were characterized by considerablygreater diameter compared to GG fatteners. Theseresults were not confirmed by the literature data.However, the differences found in the presentstudy in the diameter of muscle fibres of both typesindicate a higher gain in muscle tissue of m. longis-simus lumborum in GA genotype pigs compared toGG pigs. This was also indicated by KENNES et al.(2001), who showed that LEP/HindIII polymor-phism is associated with the growth rate of fat-tened animals. In addition, the greater musclefibres in GA pigs found in the present study wouldindicate that these animals have a greater loin eye.It must be remembered, however, that loin eye sizeis determined not only by the size but also by theamount of muscle fibres. The increase in the di-ameter of muscle fibres, especially white fibres(IIB) with glycolytic metabolism, is connectedwith the increase in carcass meat content and lowerfat content. On the other hand, it contributes topoorer meat quality, as reflected in a decreasednumber of capillaries and the associated muscleoxygen deficiency and increased production oflactic acid during glycolysis (RUUSUNEN &PUOLANNE 2004). This may ultimately be indica-tive of poorer meat quality in heterozygous (GA)animals.

The intramuscular fat content of meat is an im-portant factor to be considered when evaluatingthe consumption quality of meat. DASZKIEWICZ etal. (2005) reported that a certain amount of intra-muscular fat, which results in the marbling of meatand relaxation of connective tissue, is essential forthe sensory traits of meat to be favourable. The re-sults of the present analysis did not show a signifi-cant effect of the leptin gene g.2728G>Apolymorphism on the intramuscular fat content(IMF) of m. longissimus lumborum from PolishLandrace pigs (Table 1). Meanwhile, KENNES et

Polymorphism of Leptin Gene and Meat Quality 79

al. (2001) suggest that lower frequency of the A al-lele (0.09) in Duroc pigs is related to the highestfatness of animal carcasses (0.66) compared toLandrace pigs, in which the frequency of this allelewas 0.15 and carcass fatness level was 0.20. Dif-ferent results were obtained by JIANG & GIBSON(1999), who studied C®T substitution at position3469 (exon III) of the leptin gene, identified bySTRATIL et al. (1997). Experiments with four dif-ferent populations of pigs (Duroc, Hampshire,Landrace, Large White) showed a relationship be-tween higher frequency of the C allele and lowerfatness of Large White pigs. Meanwhile, KENNESet al. (2001), who studied the same polymorphismat the leptin gene locus obtained results suggestingthat in the group of analysed animals (Duroc, Lan-drace, Yorkshire) the higher frequency of the C al-lele was responsible for greater carcass fatness.This indicates that the inconsistency of the men-tioned results may be due to differences in the genepools of the analysed populations (breeds), as evi-denced by different genotype effects. It shouldalso be recalled that these mutations are localizedin the non-coding regions of the leptin gene.3’UTR is an untranslatable region, but it is conjec-tured that these mutations may determine mRNAstability or translation efficiency, although this hy-pothesis needs to be confirmed. They may occur as

molecular markers conjugated with specific re-gions controlling growth and feed intake (KENNESet al. 2001). These mutations may also determinethe level of gene expression, as reported by TE PASet al. (1999), who showed that point mutation inthe 3’ region of the myogenin gene has a signifi-cant effect on carcass meat content. It is also im-portant that the quantity and quality of fat rawmaterial results from the interaction between ge-netic type and many other factors such as the ani-mal’s age and sex, management and feedingconditions, and location of adipose tissue. The lat-est research (VILLALBA et al. 2009) on g.3469C>Tpolymorphism confirms the association betweenthe leptin gene polymorphism and the level of car-cass fatness according to the age of pigs and loca-tion of adipose tissue. Based on the measurementsof Duroc pigs at different developmental stages, itwas found that the genotype effect changes duringindividual growth and thus animal’s age may in-fluence the effect being estimated. In the investi-gated population, it was found that in the case ofg.3469C>T leptin gene polymorphism, 160-day-oldTT pigs had a 0.096 mg/kg higher intramuscularfat content compared to TC pigs, whereas at 220 daysTT pigs had a 1.64 mg/kg lower intramuscular fatcontent compared to TC animals. In addition,GARDAN et al. (2006) discovered that the leptin

Table 1

Percentage (%) and diameter (µm) of muscle fibre types and physico-chemical properties oflongissimus lumborum muscle as related to leptin gene polymorphism

ItemGG

N=114

GA

N=32SEM P-value

Fibre type percentage (%)IIBIIAI

71.0915.2713.64

71.5314.3814.09

0.920.590.63

0.370.250.08

Fibre diameter (Fm)IIBIIAI

56.6842.4947.45

65.2644.3255.93

0.791.120.63

0.020.140.03

pH45 6.68 6.71 0.02 0.31pH24 5.63 5.65 0.01 0.47Colour

L*a*b*

53.0714.35

3.57

54.2614.184.08

0.681.190.84

0.040.690.12

Drip loss (%) 2.63 2.48 0.31 0.38

WHC (%) 37.62 39.78 0.34 0.02

Shear force (kg/cm2) 6.64 6.35 0.63 0.09IMF % 1.72 1.86 0.27 0.42

IMF-H %1 1.23 1.39 0.41 0.25

1intramuscular fat content evaluated histochemically

D. WOJTYSIAK, U. KACZOR80

mRNA level between 160 and 210 days of agechanges for intramuscular fat adipocytes but nosuch differences were found for subcutaneous fatadipocytes. It is believed that this results from dif-ferences in metabolism and from dissimilar hor-monal expression profiles of different types ofadipose tissue.

From the viewpoint of technological usefulnessand fitness for human consumption, quality ofpork is significantly affected not only by intramus-cular fat content but also its distribution (Table 1,Fig. 1C-F). The lack of significant differences inintramuscular fat percentage, histochemically de-termined in the present study between the analysedGG and GA genotypes is due to large differencesin intramuscular fat distribution and content be-tween individual animals. Similar to what has beenshown in the present study, intramuscular fat oc-curs not only between but also inside muscle bun-dles, forming clusters of lipid droplets locatedbetween single muscle fibres (LESEIGNEUR--MEYNIER & GANDEMER 1991) but also sur-rounding mainly type I (red) muscle fibres(ESSÉN-GUSTAVSSON et al. 1994). Intramuscularfat is also composed of small lipid droplets locatedinside red fibres (type I) in the form of intracellularfat. This is consistent with ESSÉN-GUSTAVSSON etal. (1994) who showed, in an analysis of the micro-structure of pig muscles, that lipids are also depos-

ited in type I and IIA fibres, i.e. in fibres with higheroxidative capacity, and to a lesser extent in white fi-bres (type IIB) with glycolytic metabolism.

The comparison of meat quality traits (Table 1)did not reveal differences in pH45min and pH24h be-tween the analysed genotypes. Our results aresimilar to those reported by BORGES & GOULART(2002), who identified g.3469T>C polymorphismin the populations of Landrace, Large White andPietrain pigs but also did not find the effect of ani-mal genotype on pH of meat. In addition, in thepresent study, no significant differences in shearforce values were identified between GA and GGgenotypes. In the literature there are only a fewstudies on the influence of polymorphism at theleptin gene locus on meat quality of pigs. An ear-lier study with two sheep populations (Dorset, Suf-folk) revealed the association between 103A>Gpolymorphism at the leptin gene locus and greatertenderness of longissimus muscle in the Suffolkbreed (BOUCHER et al. 2006). On the other hand,marked differences were noted in the meat colouras affected by genotypes. L* values were higher inGA than in GG genotypes, which suggests poorerquality of meat raw material from GA pigs. Thelighter meat colour in GA pigs is probably relatedto the greater diameter of type IIB fibres in theseanimals. This is confirmed by LARZUL et al.(1997) and RYU & KIM (2005), who showed posi-tive correlations between parameters of L* colourand parameters of type IIB muscle fibres. Mean-while, BORGES & GOULART (2002), who identi-fied 3469T>C polymorphism in the leptin gene inLandrace, Large White and Pietrain pigs, showedno relationship between animal genotype (TT, TC)and meat colour. This may be due to the fact thatmeat colour, like other meat traits, is affected byseveral factors such as IMF content, pH and waterbinding in muscle, the interaction of which deter-mines the variation of this parameter.

Some of the major quality traits of pork, impor-tant for technological processing, are water hold-ing capacity and drip loss. In our study we did notfind the effect of polymorphism, identified usingHindIII, on drip loss. Significantly higher waterholding capacity was found, however, in m. long-issimus lumborum of pigs with the GA genotypecompared to GG animals. SAFFLE & BRATZLER(1959) hold that muscles with a higher content ofintramuscular fat have higher water holding ca-pacity, which is probably due to the relaxation ofmuscle fibre microstructure by intramuscular fat,which allows for higher water activity in tissue(HAMM 1960). This finding is reflected in the re-sults of the present study, in which GA pigsshowed higher water holding capacity in m. long-issimus lumborum and a tendency towards greaterdepositionof intramuscular fat compared toGGpigs.

Fig. 1. Exemplary cross-sections of m. longissimus lumborumof Polish Landrace fatteners: NADH-TH and immuno-histochemical MHCslow staining: I – red fibres; IIA –intermediate fibres; IIB – white fibres; A – genotype GA;B – genotype GG; C, D – intramuscular fat between musclebundles (*); E – inside muscle bundles (); F – intracellularfat (p). Scale bar 50 Fm.

Polymorphism of Leptin Gene and Meat Quality 81

Analysis of the microstructure and technologicalparameters of ML in the pigs studied revealed theeffect of g.2728G>A polymorphism at the leptingene locus on muscle fibre diameter and meat col-our and water holding capacity. The results ob-tained do not provide a basis for definiteconclusions because each of the analysed traits ismost often modulated by the interaction of a poolof different genes and environmental factors. It isalso possible that the leptin gene may influenceother muscle parameters for which no genotype ef-fect was found.

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