Physical Meat Characteristics of Local Thin Tail Sheep based on
Calpastatin (CAST) Genotype variation
M.I.A. Dagong1, *, C. Sumantri2, R.R. Noor2, R. Herman2, & M. Yamin2
1Animal Production Department, Faculty of Animal Science, Hasanuddin
University, Makassar, 90245, Indonesia. *e-mail: iccangdagong@yahoo.com
2 Department of Animal Production and Technology, Faculty of Animal Science,
Bogor Agricultural University, Bogor, 16680, Indonesia
Abstract
Calpastatin (CAST) is an indigenous inhibitor of calpain that involved in regulation of protein turn over and growth. The objective of this research was to identify genetic polymorphisms in the part of intron 5 - entire exon 6 of CAST gene in local sheep and their association with meat quality and muscle composition. A PCR-SSCP method was carried out to identify genetic variation of CAST gene. In total 401 heads of sheep from 8 subpopulations were investigated, three groups of samples were thin tail sheep from Sukabumi, Jonggol and Kissar. The rest samples were Priangan sheep from Margawati and Wanaraja and fat tail sheep from Donggala, Sumbawa and Rote islands. Twenty one heads from Jonggol were used for meat and muscle identification. SSCP analysis revealed that three different SSCP patterns corresponded to three different alleles in the CAST locus (CAST-1, 2 and 3 allele) with six different genotypes. Genetic variation between local sheep populations were calculated based on genotypic and allelic frequencies. Most populations studied were polymorphic, with genotype frequencies of CAST-11, CAST-12, CAST-22, CAST-13, CAST-23, and CAST-33 were 29.7%, 38.2%, 24.2%, 2.5%, 4.5% and 0.7% respectively. CAST-1 and 2 alleles were most commonly found in all populations with total frequency 95.8%, while rare allele was CAST-3 (4.2%) and only found in thin tail population. Based on sequence analysis identified a non-synonymous amino acid variation in exon 6 induced Gln/Leu substitusion. There was no association between CAST alleles and genotypes with meat quality.
Key words: calpastatin, local sheep, meat quality, PCR-SSCP
Introducton
envronments, dsease resstance, shorter producton cycles and relatvely requres small captal. In addton, n some densely populated areas lke Java, sheep are able to substtute some beef that have to be mported each year. Based on 2008 data, specal needs of sheep n West Java alone reach the range of 3.343.365 heads (Dtjennak 2009). The amount of the request ndcates that the prospects for sheep farmng s stll wde open.
In relaton to sheep development efforts, some weaknesses of the local sheep s that ther slaughter weghts and daly body weght gan are low vared between 54 - 174 g/head/day (Yamn et al. 2009), n addton, the carcass qualty and meat s also hghly varable and do not meet nternatonal market standards. In order to solve the problem the genetc qualty mprovement efforts are needed to ncrease productvty, carcass and meat qualty so the mpact on lamb producton wll n-crease the contrbuton of lamb to total meat producton n the country that currently only around 5% (Dtjennak 2009).
Molecular botechnology advances allow the selecton can be done at the DNA level through the use of marker genes that have an assocaton wth the hghly eco-momc trats. One of marker genes assocated wth body weght n local sheep was calpastatn (Sumantr et al. 2008). Numerous other studes have also shown a re-latonshp of calpastatn gene wth carcass qualty (Schenkel et al. 2006), and meat qualty, especally tenderness (Casas et al. 2006; Cur et al. 2009).
Calpastatn (CAST) s a member of the calpan-calpastatn system nvolvng
three molecules of the enzyme μ-calpain, m-calpain and calpastatin that serves as
both calpan nhbtor. Ths system plays a mportant role n dverse physologcal processes such as regulaton of proten turn over and growth (Goll et al. 1992), and myoblast mgraton (Dedeu et al. 2003), therefore CAST s beleved as a good canddate gene for growth, carcass and meat qualty.
Informaton on meat qualty based on varous calpastatn gene genotype n the local sheep currently s not yet avalable, so the nformaton s needed n order to descrbe the effect of ths gene on meat qualty.
Materals and Methods
Sample and Genotyping
Ths study used 21 heads of Thn Tal Sheep (TTS) from UP3J Jonggol that reared ntensvely. Sheep then grouped based on ther genotype varaton based on Dagong et. al. (2011) methods. A par of PCR prmer, forward: 5’-GTTATGAATT-GCTTTCTACTC-3’ and reverse: 5’-ATACGATTGAGAGACTTCAC-3’ was de-sgned to amplfy part of ntron 5 and whole exon 6 of CAST gene, as descrbed by
the reacton buffer. The condton of thermal cyclng conssted of pradenaturaton at 95 oC for 5 mn, followed by 35 cycles of denaturaton 95 oC for 30 s, annealng
56 oC for 45 s, and extenson 72 oC for 45 s. The fnal extenson step was at 72 oC
for 5 mn. Amplfcaton was carred out n a thermal cycler (Mastercycler Personal 22331, Eppendorf, Germany). The PCR amplcon were checked on 1.5% agarose gels n 0.5 x TBE buffer contanng 10% of ethdum bromde at 100 volt for 45 mn and vsualzed by UV translumnator. A SSCP procedure was used to dentfy vara-ton n the amplcon of CAST locus. The sheep that have been known to represent the CAST genotype then slaughter each genotype to dentfy ther physcal meat charactrerstcs.
Physical Meat Characteristics
Meat qualty was measured based on the physcal parameters whch nclude: pH measurement by pH meter and measured after agng for 24 hours. Meat tender-ness was shown by the enormous strength (kg/cm2) requred to cut the meat cores
ndcated by the needle cutter Warner Bratzler Shear Force (WBSF) that moves over the scale wth a measurement senstvty of 0.1 kg/cm2. Water Holdng
Capac-ty was measured wth a planmeter by fndng out the amount of water (mg H2O). Cookng loss was measured by substractng the ntal weght wth the weght after sample cooked at 80 °C for 1 hour.
Assocaton of CAST gene polymorphms wth physcal meat qualty was analyzed by t-test wth the followng statstcal equaton:
Where :
= Mean value n genotype 1 and genotype 2
n1 and n2 = Number of sample n genotype 1 and genotype 2
= Total varans
Physcal meat qualty data corrected n advance usng the followng statstcal equaton:
Where :
standard = mean of physcal meat characterstcs of standard populaton
observed = mean of physcal meat characterstcs of observed populaton X observed = value of physcal meat characterstcs before beng corrected by
sex and age
Results and Dscusson
Differences of physical meat characteristic
Means value of physcal meat qualty from dfferent CAST genotypes on the local sheep are shown n Table 1. There was no sgnfcant dfference (P>0.05) ether n tenderness, water holdng capacty, cookng loss and pH from three dfferent CAST genotyes (CAST-11, CAST-12 and CAST-22) n local sheep. Smlar results wth a prevous study by Zhou et al. (2008), who reported that all allelc varaton (CAST-1, 2 dan 3) or varatons of genotypes were dentfed n the CAST locus dd not sgnfcantly affect the lamb tenderness.
Tenderness value of research results n the range 2 - 3 n a tender category, but no dfferences among the three genotypes. In contrast to some prevous studes that dentfed a sgnfcant assocaton between CAST varaton wth beef tenderness. In cattle, CAST gene varatons have been used commercally as genetc markers. Two markers are currently avalable commercally were GeneSTAR Tenderness and
Igenity TenderGENE. GeneSTAR usng SNP G/A n 3’UTR regon (Barendse 2002),
Tabel 1. Mean value of physcal meat characterstcs from varous CAST genotypes of local Thn Tal Sheep
Cookng loss (%) 49.54±3.24 6.54 43.76±3.61 8.25 46.32±6.53 14.10
Water Holdng
pHult (24 h) 5.57±0.07 1.26 5.75±0.31 5.39 5.80±0.32 5.54
whle Igenity TenderGENE usng SNP G/C n ntron 5 regon (Van Eenennaam et al. 2007).
Dfferences n CAST gene effect sheep meat tenderness and beef probably caused by the fact that sheep meat s more tender due to the rate of myofbrls pro-teolyss of sheep meat s faster than beef (Koohmarae et al. 1991), therefore, the dfferences n sheep meat tenderness has smaller effect on meat tenderness.
Concluson
There was no dfference between physcal meat qualty wth CAST genotype varaton n local sheep.
Acknowledgements
Ths research was funded by DP2M Dkt through Hibah Kompetensi project wth contract No: 224/SP2H/PP/DP2M/III/2010 and DIPA IPB through Hibah Disertasi Doktor wth contract No : 10/I.3.24.4/SPK/PDD/2011.
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