Effect of RYR 1 T Gene Polymorphism on the Initial Growth and Fattening and Slaughter Values of Polish Synthetic Line 990 Pigs Reared in Standardized Litters

Pietruszka A., A. Sosnowska: Effect of RYR1T Gene Polymorphism on the Initial Growth and Fattening and Slaughter Values of Polish Synthetic Line 990 Pigs Reared in Standardized Litters. Acta Vet. Brno 2008, 77: 217-224. The aim of this study was to determine the effect of the ryanodine receptor gene RYR1T polymorphism on the initial growth and fattening and slaughter values of Polish Synthetic Line 990 pigs reared in standardized litters. The study was carried on 276 offspring of hyperprolific sows. The sows gave birth to at least 12 live-born piglets. On the first day after birth, litters were equalised to 12 piglets in litter. The body weight was examined on the 21st (21BW), 28th (28BW), 63rd (63BW) and 180th (180BW) days of life. During evaluation, the live average daily gain (LADG) from birth to day 180 of life and the average daily gain (ADG) from day 63 to day 180 of life were determined. The percentage meat content (PMC), backfat thickness (BFT) and loin eye thickness (LET) was determined using PIGLOG 105 ultrasound apparatus. Two alleles of the RYR1 gene (RYR1C, RYR1T) and three genotypes (C/C, C/T and T/T) were identified. The 21BW and 28BW of the T/T genotype were significantly lower than that of the C/C and C/T. The highest PMC was characteristic of the T/T genotype, whereas the lowest one of the C/C genotype (p ≤ 0.05). The T/T genotype had a higher LET than the C/C genotype (p ≤ 0.05). No significant differences with respect to LADG, ADG and BFT between RYR1 genotypes were found. It can be stated that early identification of homozygous animals with respect to the RYR1T gene may allow the prediction of the body weight of animals in the initial period of their rearing. RYR1 genotype, body weight, meatiness, gilts, barrows As generally known, piglets that are heavier on the day of birth are also stronger and grow better when compared to lighter piglets from the same litter. Heavier piglets distinguish themselves by a higher survival rate than lighter piglets (Hermesch et al. 2001). Furthermore, heavier piglets are characterised by a higher growth rate than lighter ones (Roehe 1999). The tendency to keeping a higher growth rate is also maintained after piglets have been weaned from a sow. The body weight of piglets on the day of birth and in the subsequent rearing period depends on many factors. One of them is the litter size (Johansson 1981). The results of research by Milligan et al. (2002) and Quiniou et al. (2002) suggest that selection towards increasing the litter size results in the rise of the number of lighter piglets in litter. With the increase of litter size by one piglet, individual piglet body weight decreases by 35 (Quiniou et al. 2002) to 44 g (Roehe 1999). Pig production traits can be shaped, to a certain degree, by genes and genetic markers. First of all, main genes (of large effects) with considerable influence on the level of production traits have taken the interest of many researchers. One of the main genes with proven influence on meat tissue development is the ryanodine receptor gene RYR1T (Leach et al. 1996; Fisher et al. 2000). Due to its negative influence on the ACTA VET. BRNO 2008, 77: 217-224; doi:10.2754/avb200877020217 Address for correspondence: Dr. Arkadiusz Pietruszka University of Agriculture Department of Pig Breeding ul. Dr. Judyma 10, 71-460 Szczecin, POLAND Phone +91 454 1433 Fax +91 454 1642 E-mail: Arkadiusz.Pietruszka@biot.ar.szczecin.pl http://www.vfu.cz/acta-vet/actavet.htm quality of meat (Barton-Gade and Christensen 1998), breeding programmes provide for its elimination from pig populations of different breeds. The results of studies of some authors point out also the effect of the RYR1 genotype on sow reproduction performance results and piglet rearing (Stalder et al. 1997; XunPing et al. 1999). Considering the foregoing, an examination was undertaken that aimed at determining the effect of the RYR1T gene polymorphism on the initial growth of reared gilts and barrows of the Polish Synthetic Line 990. It is assumed that homozygous individuals with respect to the RYR1T gene will distinguish themselves by lower body weight during the rearing period than heterozygous ones and those not loaded with the carrierstate of this gene. Materials and Methods The study was carried out at a farm of the Pig Hybridisation Centre at Pawłowice, Poland. In total, 276 animals were covered in the study, originating from the Polish Synthetic Line 990 sows. The study included piglets from sows that gave birth to at least 12 live-born piglets. On the first day after birth, sow litters were equalised with respect to their size. Each sow fed 12 piglets per litter; in this way the chances of piglet access to mother’s milk were equalised. Piglets remained with their mothers for 28 days. During this time they were weighed twice (on day 21 and 28 after birth). On day 63 of life, all litters under examination were taken out for test evaluation, which lasted until day 180 of life. During this time, animals were also weighed (on day 63 and 180). All examined individuals were kept under uniform environmental conditions. During test evaluation, barrows and gilts were fed individually with the same feed mixture. On day 180 of life, a live evaluation of fattening and slaughter values was made. The percentage meat content was determined using a PIGLOG 105 ultrasound apparatus. According to its operation manual, measurements were made in two points on the back on the right-hand side: P2 backfat thickness (P2) between the 13th and the 14th thoracic vertebrae, 3 cm away from the middle line of the back; P4 backfat thickness at the level of the last thoracic vertebra (P4) and thickness of the longissimus dorsi muscle (P4M), 8 cm away from the middle line of the back. The percentage meat content in the evaluated animals was automatically calculated based on the aforesaid measurements. The second indicator of live evaluation determined was the piglet live growth rate (daily live body weight gain). Furthermore, the growth rate was also determined during test evaluation, i.e. from day 63 to 180 of life (daily body weight gain). In order to identify RYR1 (ryanodine receptor) genotypes, the blood collected from the jugular vein of animals under examination was used. The blood was sampled to vacuum test tubes, containing K3EDTA anticoagulant. The DNA isolation was made using the sampled blood by means of Master Pure Kit of Epicentre Technologies (Madison, WI, USA). For a direct analysis of genotypes, PCR-RFLP method was used. Using the polymerase chain reaction, DNA fragments with 134 base pairs were amplified, applying the following primer sequences (Brenig and Brem 1992): forward primer: 5’-GTGCTGGATGTCCTGTGTTCCCT-3’ reverse primer: 5’-CTGGTGACATAGTTGATGAGGTTTG-3’. The polymerase chain reaction was made using a thermostable Taq polymerase. The reaction mixture of a final volume of 20 μl contained ca 100 ng genomic DNA, 10 pmol of each primer, 2 μl 2 mM dNTP mixture, 1.2 μl 25 mM MgCl2, 0.5 U Taq DNA polymerase (MBI Fermentas Burlington, Ontario, Canada) and 2 μl 10 × PCR buffer. The amplification reaction was carried out in a thermocycler (Whatman Biometra®, Göttingen, Germany) under the following conditions: denaturation at 94 oC for 5 min, and 35 cycles comprising denaturation DNA at 94 oC for 40 s, annealing at 59 oC for 40 s, complementary strand polymerisation at 72 oC for 40 s, and final extension at 72 oC for 5 min. The amplified PCR product was digested with 6 U of the restriction enzyme Hin6I (MBI Fermentas Burlington, Ontario, Canada) at 37 oC for 3 h. The digested PCR product was separated by electrophoresis on 4% agarose gel, stained with ethidine bromide (Sigma-Aldrich Chemie Gmbh P.O., Steinheim, Germany). After electrophoresis, gels were analysed with using UV. Statistical analysis of the collected performance results was done with one-factor analysis of variance. The significance of differences between groups was calculated according to Duncan’s multiple-range test. When making these calculations, a computer statistical software package Statistica 6.0 PL was used. The following equation was used: yijk = μ + ai + sj + eijk where: yijk observed value, μ overall mean, ai effect of i-th genotype (i = CC, CT, TT), sj effect of j-th sex, eijk random residual effect. 218

As generally known, piglets that are heavier on the day of birth are also stronger and grow better when compared to lighter piglets from the same litter.Heavier piglets distinguish themselves by a higher survival rate than lighter piglets (Hermesch et al. 2001).Furthermore, heavier piglets are characterised by a higher growth rate than lighter ones (Roehe 1999).The tendency to keeping a higher growth rate is also maintained after piglets have been weaned from a sow.The body weight of piglets on the day of birth and in the subsequent rearing period depends on many factors.One of them is the litter size (Johansson 1981).The results of research by Milligan et al. (2002) and Quiniou et al. (2002) suggest that selection towards increasing the litter size results in the rise of the number of lighter piglets in litter.With the increase of litter size by one piglet, individual piglet body weight decreases by 35 (Quiniou et al. 2002) to 44 g (Roehe 1999).
Pig production traits can be shaped, to a certain degree, by genes and genetic markers.First of all, main genes (of large effects) with considerable influence on the level of production traits have taken the interest of many researchers.One of the main genes with proven influence on meat tissue development is the ryanodine receptor gene -RYR1 T (Leach et al. 1996; Fisher et al. 2000).Due to its negative influence on the quality of meat (Barton-Gade and Christensen 1998), breeding programmes provide for its elimination from pig populations of different breeds.The results of studies of some authors point out also the effect of the RYR1 genotype on sow reproduction performance results and piglet rearing (Stalder et al. 1997;XunPing et al. 1999).
Considering the foregoing, an examination was undertaken that aimed at determining the effect of the RYR1 T gene polymorphism on the initial growth of reared gilts and barrows of the Polish Synthetic Line 990.It is assumed that homozygous individuals with respect to the RYR1 T gene will distinguish themselves by lower body weight during the rearing period than heterozygous ones and those not loaded with the carrierstate of this gene.

Materials and Methods
The study was carried out at a farm of the Pig Hybridisation Centre at Pawłowice, Poland.In total, 276 animals were covered in the study, originating from the Polish Synthetic Line 990 sows.The study included piglets from sows that gave birth to at least 12 live-born piglets.On the first day after birth, sow litters were equalised with respect to their size.Each sow fed 12 piglets per litter; in this way the chances of piglet access to mother's milk were equalised.Piglets remained with their mothers for 28 days.During this time they were weighed twice (on day 21 and 28 after birth).On day 63 of life, all litters under examination were taken out for test evaluation, which lasted until day 180 of life.During this time, animals were also weighed (on day 63 and 180).All examined individuals were kept under uniform environmental conditions.During test evaluation, barrows and gilts were fed individually with the same feed mixture.On day 180 of life, a live evaluation of fattening and slaughter values was made.
The percentage meat content was determined using a PIGLOG 105 ultrasound apparatus.According to its operation manual, measurements were made in two points on the back on the right-hand side: P2 -backfat thickness (P2) -between the 13 th and the 14 th thoracic vertebrae, 3 cm away from the middle line of the back; P4 -backfat thickness at the level of the last thoracic vertebra (P4) and thickness of the longissimus dorsi muscle (P4M), 8 cm away from the middle line of the back.
The percentage meat content in the evaluated animals was automatically calculated based on the aforesaid measurements.The second indicator of live evaluation determined was the piglet live growth rate (daily live body weight gain).Furthermore, the growth rate was also determined during test evaluation, i.e. from day 63 to 180 of life (daily body weight gain).
In order to identify RYR1 (ryanodine receptor) genotypes, the blood collected from the jugular vein of animals under examination was used.The blood was sampled to vacuum test tubes, containing K3EDTA anticoagulant.The DNA isolation was made using the sampled blood by means of Master Pure Kit of Epicentre Technologies (Madison, WI, USA).For a direct analysis of genotypes, PCR-RFLP method was used.Using the polymerase chain reaction, DNA fragments with 134 base pairs were amplified, applying the following primer sequences (Brenig and Brem 1992): forward primer: 5'-GTGCTGGATGTCCTGTGTTCCCT-3' reverse primer: 5'-CTGGTGACATAGTTGATGAGGTTTG-3'.
The polymerase chain reaction was made using a thermostable Taq polymerase.The reaction mixture of a final volume of 20 µl contained ca 100 ng genomic DNA, 10 pmol of each primer, 2 µl 2 mM dNTP mixture, 1.2 µl 25 mM MgCl 2 , 0.5 U Taq DNA polymerase (MBI Fermentas Burlington, Ontario, Canada) and 2 µl 10 × PCR buffer.The amplification reaction was carried out in a thermocycler (Whatman Biometra ® , Göttingen, Germany) under the following conditions: denaturation at 94 ºC for 5 min, and 35 cycles comprising denaturation DNA at 94 ºC for 40 s, annealing at 59 ºC for 40 s, complementary strand polymerisation at 72 ºC for 40 s, and final extension at 72 ºC for 5 min.The amplified PCR product was digested with 6 U of the restriction enzyme Hin6I (MBI Fermentas Burlington, Ontario, Canada) at 37 ºC for 3 h.The digested PCR product was separated by electrophoresis on 4% agarose gel, stained with ethidine bromide (Sigma-Aldrich Chemie Gmbh P.O., Steinheim, Germany).After electrophoresis, gels were analysed with using UV.
Statistical analysis of the collected performance results was done with one-factor analysis of variance.The significance of differences between groups was calculated according to Duncan's multiple-range test.When making these calculations, a computer statistical software package Statistica 6.0 PL was used.
The following equation was used: y ijk = μ + a i + s j + e ijk where: y ijk -observed value, μ -overall mean, a i -effect of i-th genotype (i = CC, CT, TT), s j -effect of j-th sex, e ijk -random residual effect.
In the examined material, two alleles of the RYR1 gene were identified (dominant -RYR1 C , and recessive -RYR1 T ) that determine the occurrence of three genotypes (C/C; C/T, and T/T) (Table 1).The RYR1 C allele occurred at a frequency of 0.54, whereas the RYR1 T allele at a frequency of 0.46.Participation of the C/T heterozygous genotype was the highest and amounted to 63%.The analysed experimental material was characterised by a relatively high percentage of the recessive homozygotes T/T -14.5%.However, the dominant homozygotes C/C occurred at a frequency of 22.5%.
The results presented in Table 2 suggest that an important source of information on the body weight of animals in the initial period of their rearing can be their RYR1 genotype.In this table, mean values for the body weight of the examined animals in respective rearing stages are presented according to the RYR1 genotype.On day 21 of life, mean body weight of recessive homozygotes (T/T) was considerably lower than that of the dominant homozygotes (C/C) and heterozygotes (C/T).The obtained differences were confirmed statistically (p ≤ 0.05).The differences with respect to body weight were also maintained in the later period of rearing.On day 28 of life, animals with the T/T genotype also distinguished themselves by a lower body weight when compared to piglets with the C/C and C/T genotypes.In this case, the observed differences were also confirmed statistically (at p ≤ 0.01 and p ≤ 0.05, respectively).It was observed that the body weight of heterozygotes on day 28 of life was similar to that of homozygotes C/C.In the later period of rearing, i.e. on day 63 of life, lower body weight was still observed in animals with the 219 T/T genotype.However, this difference was not confirmed statistically.Similar situation was observed on day 180 of life.
The results for the fattening and slaughter traits of the examined material are presented in Table 3.The highest daily live body weight gains and the gains during test evaluation (i.e. from day 63 to day 180 of life) were obtained by dominant homozygotes, whereas the lowest ones by recessive homozygotes.Heterozygotes distinguished themselves by the intermediate values of that trait between homozygotes.Despite obtaining the lowest body weight gains by animals with the T/T genotype, which is also reflected by a lower body weight of these animals in the respective rearing stages, these differences were not confirmed statistically.Most likely, this was caused by body weight equalisation in the examined animals in the later stage of rearing.
As expected, the highest percentage of meat content was found in animals with the T/T genotype, whereas the lowest one in animals with the C/C genotype.The differences found were confirmed statistically (p ≤ 0.05).Animals with the T/T genotype, being an experimental material in the present study, had the thickest loin muscle.Significant differences (p ≤ 0.05) with regard to this trait were demonstrated between the T/T and C/C genotypes.

Discussion
In an earlier study (Pietruszka et al. 2001) with the Polish Synthetic Line 990 pigs, allele frequency distribution was as follows: RYR1 C -0.68, and RYR1 T -0.32.Similar distribution of allele frequency in the population of Polish Synthetic Line 990 pigs was also found by Janik (1999).According to these authors, frequency of the RYR1 C allele amounted to 0.70, whereas that of the RYR1 T allele was 0.30.220 Similar results with respect to daily body weight gains were obtained by Jensen and Barton-Gade (1985).Also Leach et al. (1996) did not find significant differences with respect to the growth rate between RYR1 genotypes either, although they demonstrated that C/T animals had slightly higher gains than animals with the C/C genotype.Nonsignificant differences with respect to the growth rate between RYR1 genotypes were also demonstrated by Pommier et al. (1992).
Regarding the percentage of meat content, similar results were obtained by Fisher et al. (2000).Moreover, these authors demonstrated significant differences between T/T and C/T genotypes.The results of research works carried out in Scandinavia (Nyström and Andersson 1993) also confirmed the superiority of animals with the T/T genotype over those with the C/T genotype with regard to meatiness.The highest carcass meat content in recessive homozygotes with respect to the RYR1 T gene was also found by De Smet et al. (1996).The results of the present study confirm the effect of RYR1 genotypes on the size of the loin eye area.Also McPhee and Trout (1995) and Piedrafita et al. (2001) when analysing animals with three different genotypes demonstrated that the largest area of loin eye muscle were found in animals (T/T) that were susceptible to stress.
In conclusion, the application of the DNA test for the RYR1 T gene identification in pigs of the Polish Synthetic Line 990 showed that the pig population of that line is considerably loaded with this gene.Furthermore, a negative effect of the RYR1 T gene on the body weight in the initial period of pig growth was demonstrated, which confirmed the hypothesis framed earlier.Based on the obtained results, it can be stated that early identification of homozygous animals with respect to the RYR1 T gene may allow for prediction of the body weight of animals in the initial period of their rearing.Should these results be also confirmed in pig populations of other breeds, this would be an argument supporting the necessity of eliminating the allele T from parental pig herds.

Table 1 .
The frequency of RYR1 genotypes and alleles

Table 3 .
Fattening and slaughter values depending on RYR1 genotype