LEPTIN GENE (LEP) POLYMORPHISM IN PIC HYBRID FATTENERS FREE FROM STRESS SUSCEPTIBILITY GENE (RYR1^T)

Artur Rybarczyk¹, Marek Kmieć², Roman Szaruga¹, Daniel Polasik³, Arkadiusz Terman³, Wanda Natalczyk-Szymkowska^1
1 Department of Evaluation Livestock Products, Westpomeranian University of Technology, Szczecin, Poland
² Department of Genetics and Animal Breeding, Westpomeranian University of Technology, Szczecin, Poland
³ Department of Genetics and Animal Breeding, West Pomeranian University of Technology in Szczecin, Szczecin, Poland

ABSTRACT

The studies were carried out on 126 hybrid porkers from crossing sows Camborough22 and PIC337 boars. The taken up study aimed at determining the T3469C and G2728A polymorphism at the leptin gene (LEP) as well as establishing an association with the quality of carcass and meat in PIC hybrid fatteners that are free from stress susceptibility gene (RYR1^T). Basing on the carried out study, a higher frequency of allele G (0.976) and a lower one of allele A (0.024) at locus G2728A was showed. Genotype distribution was as follows: GG (0.960), GA (0.032), and AA (0.008). On the other hand, occurrence of different alleles at locus T3469C was not found. All tested pigs were characterised by genotype TT. Fatteners with genotypes GG G2728A and TT T3469C were characterised mean percent carcass meatiness amounted almost 57%, out of which 73.5% were classified into class S and E of the EUROP system. The analysis quality of their meat showed since 85.1% carcasses of normal meat, while participation of the PSE type meat amounted to 4.8% only, keeping at the same time optimum intramuscular fat content. Also, a more favourable slaughter value of gilt carcasses in relation to barrows was confirmed, with similar carcass weight, meat quality and its basic chemical composition.

Key words: pigs, LEP and RYR1 gene, carcass and meat quality.

INTRODUCTION

It is well known that animals belonging to different breeds but being characterised by the same genotype at locus RYR1 show significant differences in carcass musculature as well as meat quality traits. Therefore, it should be expected that influence of other genes may turn out to be less significant [13]. The leptin gene (LEP), which has been mapped to chromosome 18 in pigs, can be included among such genes [15]. The LEP gene and its receptor (LEPR) are taken into consideration as candidate genes associated with meat quality and fatness traits in livestock [2]. Leptin is a hormone secreted by adipose  cells (adipocytes) and is being known as a product of the obesity gene. Its most important task is to regulate food intake and maintain equilibrium between reserves and energy expenditures [18]. Within the LEP gene, seven polymorphic sites, i.e. C3469T [19], G3714T, C867T, A1112G [6], A2845T, T3996C and G2728A [8] have been identified in pigs. Studies show an association between the T3469C (locus LEP/HinfI) polymorphism and growth rate in Landrace pigs [8, 12] and carcass slaughter value traits in Large White [6] and Landrace pigs [12] as well as Torhyb and PIC hybrid lines [14]. Moreover, Szydłowski et al. [21] showed a weak association between the T3469C polymorphism and the fat content of meat in Large White pigs and the loin weight in synthetic line 990 pigs. In the study of Kennes et al. [8] referring to the G2728A (locus LEP/HindIII) polymorphism, no significant association was found with production traits of Duroc, Landrace and Yorkshire pigs, whereas Kaczor et al. [7] showed its association with fattening duration and feed conversion in Landrace pigs.

The taken up study aimed at determining the T3469C and G2728A polymorphism at the leptin gene (LEP) as well as establishing an association with the quality of carcass and meat in PIC hybrid fatteners that are free from stress susceptibility gene (RYR1^T).

MATERIAL AND METHODS

Examination was carried out on 126 PIC hybrid fatteners which came from one of pig production farms in the Western Pomeranian Province. The study covered the offspring of PIC337 boars and Camborough22 sows which were housed under the same environmental conditions and fed using a balanced feed mixture ad libitum. All fatteners were transported to a meat processing plant in the evening, using one transport facility, and slaughtered on next day in the morning, after 4 hours of transport at a distance of 200 km.

During animal slaughtering, blood was sampled after CO₂ stunning into test-tubes containing EDTA in order to identify RYR1 and LEP genotypes. Moreover, the sex of fatteners was recorded on the slaughtering line, among which gilts constituted 68 and barrows 58 animals. Thereafter, carcass slaughter value was measured with a Sydel CGM optic-needle apparatus on the left half-carcass [3] as well as hot carcass weight of the examined fatteners was determined. Mean percent carcass meatiness amounted to 56.90 ± 3.75 and hot carcass weight to 78.98 ± 4.33 kg. After 24-hour carcass chilling, the longissimus lumborum (LL) muscle samples were collected from the 1th-4th lumbar vertebra region of the right half-carcass, in which meat pH₂₄ was measured (Elmetron CP-311 pH-meter), as well as the value of drip loss from the muscle tissue determined after 48 hours after slaughter according to Prange [17].

About 48 hours after slaughter, on the ground muscle tissue, pH measurement in water solution was made, meat colour traits, i.e. L* (lightness), a* (redness) and b* (yellowness), were established by means of a HunterLab Mini Scan XE Plus 45/0 with light illuminant D65 and observer 10° [5], and meat water-holding capacity was determined according to the method of Grau and Hamm as modified by Pohja and Niinivaara [16], as well as thermal drip according to Walczak [22], water-soluble protein content by the method of Kotik [10] and basic meat chemical composition, i.e. total protein, intramuscular fat, ash and dry matter [1]. The frequency of normal and defective meat was determined based on pH₂₄, drip loss and meat colour lightness (L*) according to Koćwin-Podsiadła et al. [9]. For normal meat, the limit values for these meat quality traits amounted respectively to: 5.5 6.0; 2 6%; 52 58, whereas for defective meat of the PSE type they were < 5.5; > 6.0%; > 58, for partly PSE meat < 5.5; > 6.0%; 52 58, and for DFD meat > 6.0; < 2.0%; < 52.

Genomic DNA was extracted from blood using MasterPure kit of Epicentre Technologies, according to the instruction (Madison, WI, USA). Genotypes of RYR1 and leptin (LEP)gene were analyses using the PCR-RFLP method. The RYR1/ Hin6I genotypes were identified using sequence of primers according to Brening and Brem [4]. All of the pigs were CC/RYR1 genotype. Genotypes of leptin (LEP) were identified using primers sequences reported by Neuenschwander et al. [15] – C3469T and Kennes et al. [8] – G2728A respectively. The PCR reactions were performed in a total volume of 25 ľl using 100 ng porcine genomic DNA, 15 pmol of each primer, 100 µM of each dNTP, 1.5 mM MgCl₂ and 0.6 units of Taq DNA polymerase (MBI Fermentas) in a standard PCR buffer. Thermal conditions were as follows: 94°C for 2 min followed by 35 cycles of 60 s at 94°C, 60 s at 55°C (C3469T) and 66°C (G2728A), 60 s at 72°C  and a last extension for 5 min at 72°C. Digestion of the PCR product was performed with 5 I.U. of the appropriate restriction endonuclease: HinfI for C3469T and HindIII for G2728A at 37°C overnight. The restriction fragments of DNA were separated by electrophoresis in 3% agarose gel stained with ethidium bromide. The results were visualized using UV rays.

The study results were processed by means of non-orthogonal one-factor analysis of variance, while significance of differences in sexes was determined by the Tukey test. The findings were processed statistically with Statistica 8.0 PL computer software.

RESULTS AND DISCUSSION

Basing on the carried out study, no different alleles were found to occur at locus T3469C in the examined PIC fatteners. All analysed pigs proved to be monomorphic, having genotype TT (Table 1). In other studies on PIC pigs, a high frequency of genotype TT (0.63) and a lower one of genotype TC (0.37) at locus T3469C were found [14]. In the study of Szydłowski et al. [21] on Polish Landrace, Polish Large White and line 990 pigs, a higher frequency of allele T (0.89–0.90) was found, while the frequency of genotype TT was from 0.79 to 0.81. Also Kennes et al. [8] showed a high frequency of allele T (0.85 to 0.94) in Duroc, Landrace and Yorkshire pigs. In all aforesaid studies referring to the T3469C gene polymorphism in pigs, genotype CC was not found or its frequency was very low.

Table 1. Frequency of leptin (LEP) alleles and genotypes in PIC pigs

G2728A			T3469C
GG n = 121	GA n = 4	AA n = 1	TT n = 126	TC n = 0	CC n = 0
0.960	0.032	0.008	1.000	–	–
G = 0.976			T = 1.000
A = 0.024

When analysing the G2728A leptin gene polymorphism, a higher frequency of allele G (0.976) and a lower one of allele A (0.024) were found. Basing on the analysis of genotypes G2728A of the examined fatteners, a high frequency of genotype GG (0.960) and a low one of genotypes GA (0.032) and AA (0.008) were found. Also in the study of Kennes et al. [8] in Duroc, Landrace and Yorkshire pigs, when analysing the G2728A polymorphism, a high frequency of allele G (0.85–1.0) was found. In the study of Kaczor et al. [7] on Polish Landrace fatteners, allele G was observed to dominate in the pigs examined, while genotype distribution was as follows: GG (0.62) and GA (0.38). In the studies of the aforesaid authors [7,8], also the T3996C leptin gene polymorphism was analysed and all animals were found to be monomorphic, being characterised by genotype TT.

Due to the fact that PIC fatteners appeared to be monomorphic (TT) with regard to genotype T3469C and a low frequency of genotypes GA and AA was observed in case of the G2728A gene polymorphism, a characteristic of carcass and meat quality of the fatteners that were characterised by the same genotypes TT and GG for locus LEP was made (Table 2 and 3). Carcasses of the fatteners with genotype TT and GG were characterised mean percent meatiness amounted almost 57%, out of which 73.5% were classified into class S and E of the EUROP system. The quality of their meat can be also found very high since 85.1% of carcasses showed traits of normal meat, while participation of the PSE type meat amounted to 4.8% only. Moreover, it was confirmed that the slaughter value of gilt carcasses is more favourable than that of barrow carcasses, with a non-significant difference in carcass weight. No significant differences were found between sexes with regard to meat quality and basic meat chemical composition.

Table 2. Mean values and their standard deviations (SD) for slaughter value and meat quality traits pigs monomorphic at locus LEP and CC/RYR1

Traits	GG G2728A × TT T3469C at locus LEP
	barrows n=65	gilts n=56	total n=121
Hot carcass weight, kg	79.06 ± 4.40	78.96 ± 3.97	79.01 ± 4.19
Meatiness, %	55.56A ± 3.86	58.32B ± 3.00	56.85 ± 3.74
Thickness muscle LL, mm	55.42 ± 7.53	55.70 ± 7.98	55.55 ± 7.72
Backfat thickness, mm (between 3 and 4 the rib)	14.40A ± 3.75	11.60B ± 2.57	13.10 ± 3.52
Total protein, %	22.24  ± 0.67	22.40 ± 0.53	22.31 ± 0.61
Intramuscular fat, %	2.13 ± 0.61	2.01 ± 0.55	2.07 ± 0.58
Ash, %	1.16 ± 0.05	1.16 ± 0.03	1.16 ± 0.04
Dry matter, %	25.54 ± 0.68	25.62 ± 0.66	25.58 ± 0.66
pH24	5.80 ± 0.13	5.81 ± 0.10	5.81 ± 0.11
pH48	5.71 ± 0.13	5.69 ± 0.10	5.70 ± 0.11
L*	55.94 ± 2.12	55.50 ± 2.04	55.74 ± 2.08
a*	6.81 ± 1.03	6.95 ± 1.17	6.87 ± 1.10
b*	15.64 ± 0.73	15.72 ± 0.80	15.68 ± 0.76
Drip loss, %	5.15 ± 1.72	5.01 ± 2.11	5.09 ± 1.90
WHC, % of free water	17.93 ± 3.43	17.78 ± 3.95	17.86 ± 3.67
WHC, % of bound water	75.94 ± 4.58	76.10 ± 5.33	76.01 ± 4.92
Thermal drip, %	25.50 ± 1.89	25.26 ± 2.23	25.39 ± 2.05
Water-soluble protein, %	10.07 ± 1.22	9.97 ± 1.23	10.02 ± 1.22

A,B Mean values in rows marked by different letters differ significantly at p ≤ 0.01.

Table 3. Frequency of carcasses in  carcass class EUROP system and frequency of normal and defective meat pigs monomorphic at locus LEP and CC/RYR1

EUROP carcass class	LEP genotype GG G2728A × TT T3469C		Meat quality classes	LEP genotype GG G2728A × TT T3469C
	n	%		n	%
S (> 60%)	28	23.1	Normal	103	85.1
E (55–60%)	61	50.4	Partly PSE	10	8.3
U (50–54.9%)	25	20.7	PSE	5	4.1
R (45–49.9%)	7	5.8	DFD	3	2.5

In the study conducted by Jiang and Gibson [6], a relationship was sought between polymorphism in the leptin gene and carcass fatness and slaughter value in four pig breeds (Duroc, Hampshire, Landrace and Large White). The presence of relationship between the leptin gene polymorphism at position T3469C and carcass fatness was showed in Large White pigs. However, the study results referring to differences between genotypes at T3469C are not clear-cut, which can show that the LEP polymorphism may be associated with different productivity of pigs. The observed phenotypic variability may be caused by different genes cooperating with locus LEP [20]. In the study of Kurył et al. [14] on fatteners of different breeds and lines, i.e. Pietrain, Złotnicka Spotted, Polish Landrace and Torhyb, Stamboek and PIC lines, significant differences in the level of some carcass slaughter traits were found depending on genotype T3469C in PIC and Torhyb pigs, but not each relationship occurring in pigs of one line was confirmed in another one. In the study of the aforesaid authors, higher meat content in ham in Torhyb pigs was found for genotype CT in relation to TT, whereas genotype TT at locus T3469C in PIC pigs proved to be more favourable for decreasing the weight and fat content in ham in relation to genotype CT. Also in the study of Křenková et al. [11] was analysed the T3469C gene polymorphism in White Large and Landrace hybrids, in which more favourable growth traits were observed, i.e. daily gains, dead weight and right half-carcass weight, in fatteners with genotype TT in relation to genotype CT. Different results were presented by Kulig et al. [12], who showed in Polish Landrace fatteners that animals with genotype CT had higher mean daily gains and higher carcass lean content than those with genotype TT. On the other hand, Bauer et al. [2] did not observe significant differences in carcass lean content and mean backfat thickness between three genotypes at T3469C in White Improved pigs, with only mean daily gains being significantly more favourable in pigs with genotype CC than TT.

In the study of Kaczor et al. [7], in which the G2728A polymorphism was analysed, genotype GA was found to be associated with smaller feed conversion and shorter fattening duration in relation to genotype GG in Polish Landrace pigs. The authors did not find differences between the aforesaid genotypes in carcass and meat quality traits, except water-holding capacity which was more favourable in pigs with genotype GG. Moreover, mean intramuscular fat content in pigs with genotype GG amounted to 2.06% and was one and the same with that obtained in the present study in PIC pigs with the same genotype.

CONCLUSIONS

Summing up, in fatteners being crossbreeds of Camborough22 sows and PIC337 boars, free from stress susceptibility gene (RYR1^T), a higher frequency of allele G (0.976) and a lower one of allele A (0.024) at locus G2728A was showed. Genotype distribution was as follows: GG (0.960), GA (0.032), and AA (0.008). On the other hand, occurrence of different alleles at locus T3469C was not found. All tested pigs were characterised by genotype TT. Fatteners with genotypes GG G2728A and TT T3469C were characterised mean percent carcass meatiness amounted almost 57%, out of which 73.5% were classified into class S and E of the EUROP system. The analysis quality of their meat showed since 85.1% carcasses of normal meat, while participation of the PSE type meat amounted to 4.8% only, keeping at the same time optimum intramuscular fat content. Also, a more favourable slaughter value of gilt carcasses in relation to barrows was confirmed, with similar carcass weight, meat quality and its basic chemical composition.

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14. Kurył J., Kapelański W., Pierzchała M., Bocian M., Grajewska S., 2003. A relationship between genotypes at the GH and LEP loci and carcass meat and fat deposition in pigs. Anim. Sci. Pap. Rep. 21, 15–26.

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Accepted for print: 18.07.2010

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Artur Rybarczyk
Department of Evaluation Livestock Products,
Westpomeranian University of Technology, Szczecin, Poland
Doktora Judyma 24, 71-466 Szczecin, Poland
phone: 048 091 449 67 08
email: Artur.Rybarczyk@zut.edu.pl

Marek Kmieć
Department of Genetics and Animal Breeding,
Westpomeranian University of Technology, Szczecin, Poland
Doktora Judyma 6, 71-466 Szczecin, Poland
phone: 048 091 449 67 80
email: Marek.Kmiec@zut.edu.pl

Roman Szaruga
Department of Evaluation Livestock Products,
Westpomeranian University of Technology, Szczecin, Poland
Doktora Judyma 24, 71-466 Szczecin, Poland
phone: 048 091 449 67 08

Daniel Polasik
Department of Genetics and Animal Breeding, West Pomeranian University of Technology in Szczecin, Szczecin, Poland
Doktora Judyma 6, 71-466 Szczecin, Poland
Phone: +48 91 449 67 80
email: daniel.polasik@zut.edu.pl

Arkadiusz Terman
Department of Genetics and Animal Breeding, West Pomeranian University of Technology in Szczecin, Szczecin, Poland
Doktora Judyma 6, 71-466 Szczecin, Poland
Phone: +48 91 449 67 83
email: Arkadiusz.Terman@zut.edu.pl

Wanda Natalczyk-Szymkowska
Department of Evaluation Livestock Products,
Westpomeranian University of Technology, Szczecin, Poland
Doktora Judyma 24, 71-466 Szczecin, Poland
phone: 048 091 449 67 08
email: Wanda.Natalczyk-Szymkowska@zut.edu.pl

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