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Growth characteristics of Indonesian thin tail Sheep (TTS) based on calpastatin (CAST) gene genotype variation.

ABSTRACT

Background: Calpastatin involved in various physiological processes in the body such as the protein turnover, growth, fusion and myoblast migration. Thus, allegedly Calpastatin gene diversity (CAST) have an association with growth and potential use as candidate genes for growth trait. Objective: This study aims to identify the association between the genetic diversity of CAST gene with some growth properties such as body dimension (morphometric), body weight and daily weight gain in sheep. Methods: A total of 157 heads of Thin Tail Sheep (TTS) reared intensively for fattening purposes in the uniform environmental conditions. Overall sheep used were male, and maintained for 3 months. The parameters of growth properties were measured among others: body weight gain (ADG) (g/head / day), body weight (kg), body length (cm), chest circumference (cm), height (cm). All the sheep were genotyped by using PCR-SSCP (single strand conformational polymorphism) methods. CAST gene in locus fragment intron 5 - exon 6 were amplified with a predicted length of about 254 bp PCR products. Then the sheep were stratified based on their CAST genotypes. Results: The result of this research showed that no association were found between the CAST gene variations with morphometric body weight, but there was a significant association with daily body weight gain (ADG) in sheep observed. CAST-23 and CAST-33 genotypes has higher average daily gain than other genotypes. Conclusion: It is conclude that CAST-23 and CAST-33 genotypes that carrying the CAST-2 and CAST-3 alleles potential to be used in the selection of the nature of the growth trait of the TTS sheep.

KEYWORDS: Body weight, Calpastatin, Genotype, Growth trait, Thin tail sheep

INTRODUCTION

Calpastatin involved in various physiological processes in the body, such as the regulation of protein turnover, and growth [1], fusion [2] and myoblast migration [3]. Thus, allegedly genetic diversity of calpastatin (CAST) has a relationship with growth and potential used as candidate genes for growth trait in sheep.

Based on previous research that reported by [4; 5], it is known that calpastatin (CAST) gene on the Indonesian local sheep have fairly high diversity. There are six genotypes of CAST genes were identified in the local sheep population ie. CAST-11, CAST-12, CAST-22, CAST-13, CAST-23 and CAST-33. Groups of local sheep that has high diversity is Thin Tail sheep (TTS), which is derived from the Sukabumi, Jonggol and Kissar sub population with the discovery of three kinds of alleles in a population (CAST-1, CAST-2 and CAST-3), while the population in Fat Tailed sheep (FTS) and Priangan found only two kinds of alleles (CAST-1 and CAST-2). Based on this, so that more information is needed regarding the relationship of this gene diversity with the growth characteristics of the local sheep.

Livestock growth, particularly the growth of muscle is related to the process of synthesis and degradation of proteins, and this process may affect the quality of the meat and the carcass. The aims of this study to identify the association between the genetic diversity of CAST genes with growth characteristics, such as body size (morphometric), body weight, and average daily gain on TTS sheep reared intensively.

MATERIALS AND METHODS

Livestock management:

The research was conducted in Tawakkal Fattening Sheep Farm, Cimande Village, Bogor regency, West Java. The research used in total 157 heads of Thin Tail sheep (TTS) ram reared intensively for fattening purposes in uniform environmental and management conditions. Ram used in the range of 1 year of age (over 12 months). Parameters measured growth characteristics, among others: body weight gain (ADG) (g/head/day), body weight (kg), body length (cm), chest circumference (cm), body height (cm). Sheep kept for 3 months and body weight were measured every month.

CAST gene genotyping:

Sheep CAST gene fragment identified by using PCR-SSCP (single strand conformational polymorphism) methods. CAST gene locus in intron 5 - exon 6 regions amplified by the primer sequences F:5'-GTTATGAATTGCTTTCTACTC-3' and R: 5'-ATACGATTGAGAGACTTCAC-3' based on [6], with a predicted amplicon length 254 bp. Amplification was done by PCR conditioned at 25 mL reaction volume consisting of 50-100 ng DNA template, 0.25 [micro]M primer, 150 [micro]M dNTPs (Fermentas), 2.5 mM Mg2+, 0.5 U Taq DNA polymerase (Fermentas) and 1x buffer.

Amplification starting with an initial denaturation at 94 [degrees]C for 5 minutes, followed by 35 subsequent cycles, each 94 [degrees]C for 30 seconds, 56 [degrees]C for 45 seconds, 72 [degrees]C for 45 seconds, ending with one cycle of final extension at 72 [degrees]C 5 minutes by using PCR machine (Mastercycler, Eppendorf, Germany). The PCR products were then electrophoresed on 1.5% agarose gel, with 0.5x TBE buffer containing 200 ng/ml Ethidium Bromide. Then visualized on a UV transiluminator.

To identify the sheep genotype, a PCR-SSCP procedure was used to identify variation in the amplicon of CAST locus. A 5 [micro]l aliquot of each PCR product was mixed with 5 [micro]l of loading dye (98% formamide, 10 mM EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol). After denaturation at 95 [degrees]C for 5 minutes, samples were rapidly cooled on wet ice and then loaded on 12% acrylamide : bisacrylamide (29 : 1) gels. Electrophoresis was performed using Protean II xi cells (Bio-Rad), at 300 V for 18 h at refrigerator condition (5 [degrees]C) in 0.5 x TBE buffer. Then, gels were silver stained based on the method of [7] with modification in staining solution (0.15% AgN[O.sub.3], 0.04% NaOH 10 N and 0.4% N[H.sub.3]). The sheep, whose genotype has been identified then their growth characteristics were measured.

Statistic analysis:

Association of CAST gene variation with the growth traits were analyzed by t-test with statistical equation model as follows:

[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]

[sigma] = [square root of ([summation.sup.n.sub.i=1][([bar.X.sub.i] - [bar.X.sub.1]).sup.2] + [summation.sup.n.sub.i=1] [([bar.X.sub.i] - [bar.X.sub.2]).sup.2])/[[n.sub.1] + [n.sub.2] - 2]]

Note:

[bar.X.sub.1] and [bar.X.sub.2] = average traits of genotype 1 and genotype 2 n1 and n2 = individual number of genotype 1 and 2

[sigma] = combined variance

The growth traits data were corrected in advance using statistical equation as follows:

[X.sub.i] correction = [[bar.X] standard/[bar.X] observed] x [X.sub.i] observed

Note:

Xi correction = trait values after correction for age

[bar.X.sub.standard] = average trait of a standard population

[bar.X.sub.observed] = average trait of observed population

Xi observed = trait values before correction for age

RESULTS AND DISCUSSION

CAST gene genotype distribution in Thin Tail sheep (TTS):

The CAST gene region were succesfully amplified by PCR with predicted amplicons length 254 bp. PCR-SSCP analysis showed polymorphism in the CAST gene region with three different SSCP banding patterns (Figure 1). The three band corresponding to three different CAST alleles, CAST-1, CAST-2 and CAST-3. Either one or two different band were found in each individual sheep that consistent with either homozygous or heterozygous with six different genotypes (CAST-11, CAST-12, CAST-13, CAST-22, CAST-23 and CAST-33).

Calpastatin (CAST) gene genotype distribution obtained in the TTS sheep population used, are presented in Table 1. Common genotypes obtained in this study were the CAST-11, CAST-12 and CAST-22, while genotypes CAST-13, CAST-23 and CAST-33 tend to be fewer in number (rare) in the TTS population used. However, the distribution of genotypes showed that the CAST gene diversity within TTS sheep populations used was quite high.

Association of CAST variation with growth trait:

Growth appearance, such as body weight, daily weight gain (ADG), as well as the animal's body size is a parameter of growth that is often used to measure the growth performance in livestock [8; 9]. The average value of body weight and body weight gain obtained was within the same range as obtained by [9]. The association of CAST variation with growth trait in TTS Sheep are presented in Table 2. T-test results showed no significant differences (P> 0.05) between the different genotypes on the parameters body weight and morphometric size (chest circumference, body length and height).

Similar results were reported by [10] in TTS sheep in UP3J Jonggol, but in contrast to those reported in [11] identified the six local sheep populations in Indonesia. Similarly to that reported in [12], reported an association of CAST genotype variations with body weight in Kurdi sheep, but had no effect on growth properties when weaning until the age of six months, the growth of six to nine months and six months until one year.

Average parameters of daily body weight gain (ADG) between genotypes showed significant differences (P<0.05), ADG of CAST-23 and CAST-33 genotypes, significantly higher than CAST-11, CAST-12 and CAST-13 but not significantly different with CAST-22 genotype. The results differ from [13] reported no correlation between CAST genotype variation with growth until weaning in sheep, but related to the birth weight traits. The body weight and growth traits is controlled by many genes (polygenic) and is greatly influenced by environmental factors [14].

The rate and degree of skeletal muscle growth is dependent on several factors including the rate of muscle protein synthesis and degradation, as well as the size and number of muscle cells. Calpain activities required in the myoblast fusion process and the proliferation of cell growth [15]. Calpain system can also affect the number of fibers of the muscle cells in animals by affecting the rate of proliferation and modulate fusion myoblast. The system is also important in normal growth of the skeletal muscles.

The increase in the rate of growth of skeletal muscle could be caused by a decrease in the rate of muscle protein degradation, and it is associated with a decrease in the activity of calpain system. This occurs due to a large increase in calpastatin activity [15]. One example of that support is Callipyge phenotype in sheep. Callipyge sheep have extreme muscular, especially in the hindquarter, as well as a less fatty [16; 17]. The hypertrophy characteristics caused a decrease in levels of muscle protein degradation [18]. In callipyge sheep, there was increased calpastatin activity in the muscles [19; 20], and cause the meat was very tough [19].

Conclusion:

The diversity of calpastatin gene (CAST) has no association to body dimensions in TTS sheep studied, but such diversity affects the daily body weight gain (average daily gain/ADG). CAST-23 and CAST-33 relatively has higher ADG than other genotypes. CAST-23 and CAST-33 that carrying the allele-CAST-2 and CAST-3, suspected of having an association with the growth performance and potential to be used in the selection of the nature of the growth.

ACKNOWLEDGEMENTS

Authors thank H. Bunyamin (Tawakkal Farm, Cimande Village, Bogor, West Java) for sheep samples and management and technical assistance for this research and Erik Andreas for blood samples technical assistance.

REFERENCES

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[5] Dagong, M.I.A., 2012. Polymorphisms of Calpastatin (CAST) Gene and its Association with the Growth Trait and Carcass Quality of Local Sheep. Disertation. Postgraduate Program. Bogor Agricultural University. Bogor, West Java. Indonesia.

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[20] Duckett, S.K., G.D. Snowder and N.E. Cockett, 2000. Effect of the callipyge gene on muscle growth, calpastatin activity, and the tenderness of three muscle across the growth curve. Journal of Animal Science, 78: 2836-2841.

(1) Muhammad Ihsan Andi Dagong, (2) Cece Sumantri, (2) Ronny Rachman Noor, (2) Rachmat Herman, (2)Mohamad Yamin

(1) Animal Production Department, Faculty of Animal Science, Hasanuddin University, 90245. Makassar, South Sulawesi Indonesia

(2) Animal Production and Technology Department, Animal Science Faculty, Bogor Agricultural University, Jalan Agatis, Kampus IPB Darmaga, Bogor 16680

Address For Correspondence:

Muhammad Ihsan Andi Dagong, Hasanuddin University, Animal Production Department, Faculty of Animal Science, Perintis Kemerdekaan Street, KM. 10, Tamalanrea, 90245, Makassar, South Sulawesi, Indonesia.

This work is licensed under the Creative Commons Attribution International License (CC BY).

http://creativecommons.org/licenses/by/4.0/

Received 23 August 2016; Accepted 1 November 2016; Published 20 November 2016

Table 1: CAST genotype and allele distribution in TTS sheep population

CAST Genotype  N   Genotype frequency  Allele Frequency
                                       CAST-1  CAST-2  CAST-3

CAST-11        54  0.344               0.525   0.389   0.086
CAST-12        48  0.306
CAST-13         9  0.057
CAST-22        31  0.197
CAST-23        12  0.076
CAST-33        3   0.019

Note: N = number of samples

Table 2: Average body weight, ADG and body dimension of Thin Tail sheep
based on CAST genotype variation

Characteristics    Genotype (n)
                   CAST-11 (54)         CAST-12 (48)

Intial Body        21.93[+ or -]2.91    23.09[+ or -]3.28
Weight (kg)
Final Body Weight
(kg)               26.41[+ or -]3.01    27.41[+ or -]3.81
ADG (g/head/day)   75.05[+ or -]33.29a  72.13[+ or -]34.28a
Morphometric
CC (cm)            69.01[+ or -]4.08    70.05[+ or -]4.66
BL (cm)            60.96[+ or -]3.50    61.93[+ or -]3.31
BH (cm)            58.67[+ or -]4.05    59.79[+ or -]3.56

Characteristics
                   CAST-13 (9)          CAST-22 (31)

Intial Body        23.43[+ or -]4.96     21.79[+ or -]3.39
Weight (kg)
Final Body Weight
(kg)               26.87[+ or -]5.54    26.64[+ or -]3.72
ADG (g/head/day)   57.24[+ or -]30.48a  81.35[+ or -]36.58ab
Morphometric
CC (cm)            69.88[+ or -]5.42    68.58[+ or -]4.56
BL (cm)            61.33[+ or -]4.74    62.04[+ or -]4.20
BH (cm)            58.67[+ or -]3.67    60.05[+ or -]3.57

Characteristics
                   CAST-23 (12)         CAST-33 (3)

Intial Body        22.00[+ or -]3.28    20.73[+ or -]4.09
Weight (kg)
Final Body Weight
(kg)               27.76[+ or -]3.17    27.12[+ or -]4.91
ADG (g/head/day)   97.44[+ or -]25.55b  108.66[+ or -]38.41b
Morphometric
CC (cm)            68.83[+ or -]3.42    68.46[+ or -]3.36
BL (cm)            63.00[+ or -]5.64    61.50[+ or -]4.50
BH (cm)            60.51[+ or -]3.08    61.06[+ or -]2.50

Superscript followed by different letters in the same row indicate
significant differences at the level of 5% (P<0.05). ADG = average
daily gain, CC = chest circumference, BL = body lenght, BH = body
height
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Author:Dagong, Muhammad Ihsan Andi; Sumantri, Cece; Noor, Ronny Rachman; Herman, Rachmat; Yamin, Mohamad
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:9INDO
Date:Nov 1, 2016
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