Morphological Traits, Productive Performance and Genotyping Fat Deposition PPAR Gene in Gama Ayam Crossbreeds of Female F1 Kamper and Male BC1 Kambro

On the recent classification of native Indonesian chicken there are 31 breeds of chicken. Highly circulated chicken breed in Indonesia is fast-growing broiler which include several strains such as Cobb 500, Hubbar, Hybro, Isa Hyline and Hisex. Fast-growing broiler chickens have a rapid growth, with excessive fat deposition on chicken carcass which implicates a health problem and unfavorable meat quality. Gama Ayam Research Team conducted a selective breeding which produces two new chicken breed Kamper and Kambro. A further selective breeding then crossbred female F1 Kamper and male BC1 (backcross I) Kambro. In this research morphological traits, productive performance and genotyping of PPAR gene related to fat deposition gene and blood lipid content in Gama Ayam were identified. Based on data analysis hybrid chickens morphological traits it concluded that Gama Ayam have a significant variation based on feather color. Productive performance was determined with feed-conversion ratio (FCR) value which was 3.17. Genotyping of PPAR gene resulted in four polymorphisms which formed 14 haplotypes groups. Based on blood lipid content analysis of cholesterol content, triglycerides content, HDL and LDL, Gama Ayam have significantly lower content of cholesterol (107.05 mg/dl), LDL (44.71 mg/dl) and triglycerides (22.41 mg/dl). PPAR gene polymorphisms is not correlated with blood lipid content in Gama Ayam. A significantly strong correlation between PPAR gene polymorphism on the body weight of Gama Ayam at 49-days-old. Triglyceride level, cholesterol level and LDL level in Gama Ayam were lower than broiler chicken. Further research with larger population size and sex classification of hybrid chicken must be conducted to validate the results.


INTRODUCTION
Over the past few decades, the main purpose of poultry production in many countries has been to improve animal growth. Ditjen PKH (2018) showed that Indonesia poultry livestock populations in 2018 consisted of 1.8 billion broiler-type/broiler chickens, 181.752 layer chickens and 310.960 native chickens. However, modern broiler strain often tend to have excessive abdominal fat deposits (Xu et al., 2003;Liang et al., 2015). Fat deposition needs to be controlled hence it has a negative impact on poultry production, as evidenced by increases in feed costs during maintenance, decreased final meat quality, and significant economic losses for the poultry processing industry (Peng et al., 2019;Wu et al., 2006;Lu et al., 2007).
Currently, excessive fat is one of the main problem faced by the poultry industry, because it's not only causes reduced carcass and feed efficiency, but also customers refusal of meat (Colmenero, 2000;wumová & Teimouri, 2010;Wu et al., 2000). Efforts to reduce fat in chicken are one of the focuses in research on Broiler commodities (Ferrini et al., 2010). It is because consumers want healthier food products from chicken. High levels of fat in food products that come from animal are known to be a source of body obesity and coronary heart disease (Sartika, 2008;Gesta et al., 2007;Popkin, 2001). The intense genetic selection for rapid growth in broilers has resulted in an increase in voluntary feed intake and growth rate, accompanied by increased fat deposition in adipose tissue depots throughout the body Vol 7, December 2019 Biogenesis 107 (Wang et al., 2017). On this basis, producing chickens that contain low fat content is one of the goals sought by many poultry nutrition researchers. In the last decade, genetic mechanisms in chicken fat deposition have been widely studied to examine genotypes of genes involved in adipose regulation in chicken (Wang et al., 2008). One gene that expresses fat deposition in chickens is PPAR gene (peroxisome proliferator-activated gene receptor). This gene can mediate regulation of adipose differentiation and fat metabolism (Hattori et al., 2004;Wang et al., 2008).

RESULT AND DISCUSSION
Based on visual observation of phenotypic traits in Table 1 Gama Ayam chicken showed a similarity in comb shape (100%). Significant variations can be visually observed from feather color with 5 phenotypic groups: black barred (37.5 %), white barred (25 %), black (12.5 %), white (12.5 %) and brown (12.5 %). Visual observation of phenotypic traits of Gama Ayam chicken is shown in Figure 1 with representative image of the breeding structure.      Sequencing results indicates several SNP points in PPAR gene. PPAR gene SNPs polymorphism points found in the 172 bp, 183 bp, 297 bp, and 454 bp nucleotide sequence. Several haplotypes were determined based on the points of the PPAR gene polymorphism related to blood lipid contents and body weight of Gama Ayam chicken. The types of mutations and haplotypes that were determined compared to blood lipid contents and body weight were presented in Table 2. In Table 3 the sample tag 16_I to 20_I refer to parentals, 1_I to 8_I refer to Gama Ayam, 9_I to 15_I refer to broiler chicken and 21_I to 23_I refer to Pelung chicken. Based on the table, it can be seen that there are 4 substitution points (G172T, A183G, C297T, and A454T) in exon 2 of PPAR gene. Amongst the four points of mutation, there is one point of polymorphism that was obtained by Meng et al. (2005) and Sun et al., (2013) specified as C297T nucleotide substitution. Sun et al., (2013) stated that there are mutations in native Chinese chickens with uncorrelated changes of amino acids to growth or fat deposition in chickens, these mutations called silent mutation. Silent mutation is a mutation that changes DNA but does not change the composition of the protein or amino acid that is coded (Chamary & Hurst, 2009;Rosaiah et al., 2014;Zhang et al., 2012). Sun et al., (2013)  Han et al. (2011) reported that the analysis of the polymorphism association in the chicken PPAR gene, specified as (g-1784_-1768del16, c. -1241G> A and c.-75G> A) is not only significantly associated with AFW and AFP (P <0.05) but also significantly associated with LFW and LFP (P <0.05). PPAR gene mutations have also been reported to affect cardiovascular damage and death such as obesity, insulin resistance, and hypertension, this is because the PPAR gene regulates adipocyte differentiation and fat metabolism and is a molecular target of insulin sensitivity. Genetic variation in PPAR gene expression is also a potential contributor to the metabolic syndrome (Wu et al., 2006). Zhou (2008) stated that abdominal fat accumulation in Chinese local chickens (yellow-haired broilers) decreases along with decreasing PPAR mRNA expression in abdominal adipose tissue. Royan et al., (2011) confirmed Zhou's (2008) findings by testing unsaturated fatty acids in the commercial strain of Ross 308 Broiler chicken strain, which was able to cause a significant reduction in abdominal fat percentage, due to a decrease in PPAR mRNA expression in abdominal adipose tissue.
Changes in amino acid composition due to polymorphism can be analyzed in relation to differences in chicken weight with Pearson correlation test. Haplotypes that have identical nucleotide bases with GeneBank references are considered to be normal phenotypes whereas haplotypes that have different nucleotide bases with GeneBank references are considered mutant phenotypes. The phenotype was tested for correlation at each point of polymorphism on blood lipid content and 49-days Gama Ayam body weight using Pearson correlation test. Pearson correlation test was only carried out on the polymorphism that occurred among hybrid chickens, specified as A183G substitution and C297T substitution. This analysis was performed to avoid data refraction of Pelung chicken, broiler chicken, BC1 Kambro, and F1 Kamper.
The results showed that PPAR gene polymorphism has no correlation with blood lipid content (cholesterol, LDL, HDL, and triglyceride). This can be caused by the possibility of PPAR gene polymorphism has significantly more effects on fat deposition in adipocyte region. In addition to the correlation test of blood lipid content, Pearson's correlation test was performed on the body weight of Gama Ayam at day 49 th . The correlation test results of PPAR gene polymorphism on the body weight of 49-days-old Gama Ayam can be seen in Table 3. Based on Table 4, PPAR gene polymorphism in Gama Ayam has a significantly strong positive correlation with body weight 49-days-old Gama Ayam. The Adenine substitution mutations to Guanine in nucleotides 183 and 297 PPAR gene correlates with the increase of body weight of Gama Ayam. Takada & Kobayashi (2013) found a similar findings that polymorphism in PPAR gene is correlated with body weight of chickens and the increase of body mass index in chickens.
Mutations that occur in hybrid chickens affect changes in weight and chicken fat, this is consistent with the theory which states that the PPARγ gene is reported as a candidate gene that influences the growth and character of fat in chickens due to its large role in muscle fiber specialization and adipogenesis (Wu et al., 2006). Based on blood lipid content analysis, the average cholesterol level, average LDL level, average HDL level, and the average triglyceride levels in Gama Ayam chicken, broiler chicken and Pelung chicken were presented in Figure 4.  Sulistyoningsih et al. (2014) stated that lower cholesterol levels will reduce abdominal fat levels so that the carcass gets bigger. Low LDL levels indicate that the cholesterol level in the individual is also low. Gama Ayam chicken shows a higher HDL levels compared to Pelung chicken and broiler chicken. Harini & Astirin (2009) stated that high HDL levels prevent the risk of atherosclerosis by transporting cholesterol from the peripheral tissues to the liver and reducing excessive cholesterol. Triglyceride levels in Gama Ayam placed in the lowest position compared to Pelung chicken and broiler chicken, low triglyceride levels will reduce cholesterol levels in the blood. Based on the description above, it can be assumed that hybrid chickens shows a prominent prospects to be used as parental generation in further selective breeding process.

CONCLUSION
Gama Ayam (407.5 grams/49-days-old) has significantly higher body weight compare to Pelung chicken and significantly lower body weight than broiler chicken. Gama Ayam has a FCR value of 3.17. Four points of PPAR gene polymorphism (G172T, A183G, C297T, and A454T) which produced 14 haplotypes of four haplotypes in hens, four haplotypes in Gama Ayam, three haplotypes in broiler chickens and three haplotypes in Pelung chicken. PPAR gene polymorphisms is not correlated with blood lipid content in Gama Ayam. A significantly strong correlation between PPAR gene polymorphism on the body weight of Gama Ayam at 49-days-old. Triglyceride level, cholesterol level and LDL level in Gama Ayam were lower than broiler chicken.