RESULTS

Functional and pathway enrichment analysis of target genes

Over 80% of the reads were successfully mapped to the reference genome of the chicken (Gallus gallus). After the mapping process, the expression level of each gene was calculated as the average, and genes showing significant DEGs in KUB chickens were identified (Figure 1A). The three biological replicates of KUB chickens showed very similar expression patterns, according to hierarchical clustering analysis, suggesting that the sequencing data were highly biologically reproducible. A volcano plot visualized the distribution of DEGs identified using DESeq2, with the X-axis representing changes in gene expression (log2 Fold Change) and the Y-axis indicating statistical significance (-log10 Adjusted p-value). Genes with significantly higher expression are indicated by red dots, whereas genes with significantly lower expression are indicated by blue dots (Figure 1B). Overall, these results indicate that KUB chickens have several candidate genes that warrant further exploration to elucidate the regulatory mechanisms of gene expression related to muscle tissue growth and development (Figure 1C).

Effect of the TGFβ2 gene on FI, FCR, and ADG in KUB chickens

The results in Table 1 show no significant difference between the three TGFβ2 genotypes in improving the performance of KUB chickens. However, there was a significant difference (p < 0.05) in BW in the F4P7 treatment with the TC genotype, but no significant difference was found between the C and T genotypes. The dominant BW of KUB chickens with the CT genotype in the F4P7 treatment was 1586 g/bird, 1584 g/bird, and 1582 g/bird, respectively. The TC genotype in the F4P5 treatment was 1537 g/bird, followed by the CC genotype at 1535 g/bird and the TT genotype at 1532 g/bird. The increase in BW is suspected to be related to the KUB chicken genotype.

Table 1 also shows significant differences between the three TGFβ2 genotypes in carcass weight (CW). Meanwhile, there was a significant difference in CW in the control treatment genotypes, namely TT 848 g/head, 852 g/head, and 850 g/head. The F4P7 treatment with the dominant TC genotype was higher at 1198 g/head, followed by the CC genotype at 1194 g/head, and then the TT genotype at 1190 g/head. The data obtained from this study are presented in Table 1. The TGFβ2 genotype did not differ significantly in FI. However, in the F4P5 treatment, the CT genotype had a higher FI compared to the other treatments, namely 3881 g/head/90 days, followed by the CC genotype at 3880 g/head/90 days. In general, Table 1 shows a significant difference between the three TGFβ2 genotypes in the FCR in the F4P3 treatment of TC genotypes 2.70, CC 2.72, and TT 2.76. While there was no significant difference with the F4P7 treatment, a high FCR was detected in the CC genotype control treatment, namely 4.21.

Figure 1: Functional characterization and interaction analysis of DEGs in KUB chickens. (A) Pathway enrichment analysis (http://www.bioinformatics.com.cn/). (B) The gene interaction of DEGs (https://cn.string-db.org/). (C) The Venn Plot of DEGs (http://www.bioinformatics.com.cn/).

Treatment | Feed Intake (g/ekor/90 day) CC (n = 5) | FCR | BW (g/ekor/90 day) CC (n = 5) | CW (g/ekor/90 day)
TC (n = 14) | TT (n = 6) | CC (n = 5) | TC (n = 14) | TT (n = 6) | TC (n = 14) | TT (n = 6) | CC (n = 5) | TC (n = 14) | TT (n = 6)
F0P0 | 3872 ± 1.27 | 3871 ± 1.17 | 3870 ± 1.21 | 4.18d ± 0.67 | 4.15d ± 0.47 | 4.21d ± 0.37 | 1142d ± 1.96 | 1141d ± 1.06 | 1143d ± 1.11 | 850d ± 2.61 | 852d ± 1.91 | 848d ± 2.01
F4P0 | 3873 ± 1.22 | 3873 ± 1.11 | 3874 ± 1.09 | 3.16c ± 0.24 | 3.13c ± 0.27 | 3.19c ± 0.14 | 1438c ± 1.85 | 1440c ± 1.02 | 1436c ± 1.15 | 1014c ± 1.19 | 1018c ± 1.24 | 1010c ± 2.19
F4P3 | 3878 ± 1.77 | 3877 ± 1.10 | 3879 ± 1.12 | 2.73a ± 0.35 | 2.70a ± 0.36 | 2.76a ± 0.17 | 1499b ± 1.67 | 1502b ± 1.06 | 1494b ± 1.19 | 1118c ± 1.53 | 1121bc ± 1.59 | 1115c ± 2.13
F4P5 | 3880 ± 1.21 | 3881 ± 1.12 | 3879 ± 1.11 | 2.84a ± 0.16 | 2.85a ± 0.22 | 2.86a ± 0.19 | 1535ab ± 1.20 | 1537ab ± 1.03 | 1532ab ± 1.13 | 1152b ± 2.36 | 1154b ± 1.76 | 1150b ± 2.06
F4P7 | 3877 ± 1.13 | 3877 ± 1.15 | 3878 ± 1.15 | 2.94b ± 0.15 | 2.92b ± 0.29 | 2.96b ± 0.12 | 1584a ± 1.06 | 1586a ± 1.07 | 1582a ± 1.12 | 1194a ± 2.20 | 1198a ± 1.24 | 1190a ± 2.09

Meat quality of KUB chicken

Dietary multi-enzyme affected various meat quality traits, such as pH, texture, CL, and WHC (Table 2), as well as color parameters (Table 3). Meat quality analysis showed that pH and texture effects were not significantly different (p > 0.05) between the multienzyme addition treatment and the treatment without multienzyme in the feed, but were significantly different (p < 0.05) in CL and WHC, as seen in Table 2. Color parameters were significantly higher (p < 0.05) in the multienzyme feed group compared to the control group, and there was a significant difference (p < 0.05) between the lower (F4P0) and higher (F4P3, F4P5 and F4P7) multienzyme concentrations; consequently, the results for other different analyses were based on the Lightness of KUB chicken meat. Redness of KUB chicken was significantly increased (p < 0.05) between the control group (F0P0) and the multienzyme treatments (F4P3, F4P5, and F4P7). The results of the analysis of the yellowness of KUB chicken meat showed a significant decrease (p < 0.05) in the F4P5 and F4P7 multienzyme groups when compared with the F4P0 and F4P3 groups; then, the group with the highest dose of multienzyme (F4P7) decreased significantly (p < 0.05) when compared with the control group (F0P0) without multienzymes in the feed.

Treatment | pH | Texture (N) | CL (%) | WHC (%)
CC (n = 5) | TC (n = 14) | TT (n = 6) | CC (n = 5) | TC (n = 14) | TT (n = 6) | CC (n = 5) | TC (n = 14) | TT (n = 6) | CC (n = 5) | TC (n = 14) | TT (n = 6)
F0P0 | 5.87 ± 0.47 | 5.82 ± 0.72 | 5.77 ± 0.52 | 19.37d ± 1.97 | 18.99d ± 1.07 | 19.37d ± 1.17 | 35.58d ± 0.69 | 34.18d ± 0.55 | 34.39d ± 0.92 | 21.31d ± 1.82 | 22.22d ± 1.42 | 21.31d ± 1.62
F4P0 | 5.61 ± 0.39 | 5.55 ± 0.59 | 5.91 ± 0.69 | 22.71a ± 1.22 | 21.22c ± 1.12 | 21.76b ± 1.41 | 30.65c ± 1.41 | 29.22b ± 1.41 | 30.73c ± 1.43 | 28.63c ± 1.31 | 27.19 ± 1.11 | 28.33c ± 1.17
F4P3 | 5.68 ± 0.26 | 5.72 ± 0.46 | 5.56 ± 0.36 | 22.40a ± 1.48 | 22.63a ± 1.09 | 21.35b ± 1.11 | 29.66b ± 1.72 | 29.54b ± 1.72 | 29.72b ± 1.63 | 28.78c ± 1.19 | 29.29b ± 1.09 | 28.48c ± 1.21
F4P5 | 5.49 ± 0.31 | 5.81 ± 0.61 | 5.64 ± 0.51 | 21.39b ± 1.15 | 21.18c ± 1.02 | 21.77b ± 1.10 | 28.82a ± 1.54 | 28.78a ± 1.54 | 29.52b ± 1.34 | 29.98a ± 1.12 | 29.47b ± 1.16 | 29.55b ± 1.19
F4P7 | 5.79 ± 0.22 | 5.49 ± 0.32 | 5.75 ± 0.42 | 21.52b ± 1.25 | 21.68b ± 1.14 | 21.49b ± 1.21 | 28.74a ± 1.76 | 28.55a ± 1.76 | 28.99b ± 1.66 | 29.98a ± 1.14 | 29.63a ± 1.13 | 29.77a ± 1.16

Treatment | L* | a* | b*
CC (n = 5) | TC (n = 14) | TT (n = 6) | CC (n = 5) | TC (n = 14) | TT (n = 6) | CC (n = 5) | TC (n = 14) | TT (n = 6)
F0P0 | 43.73d ± 2.09 | 42.66d ± 2.09 | 40.62d ± 2.19 | 4.13d ± 1.72 | 4.73d ± 1.22 | 4.99d ± 1.12 | 14.15d ± 1.41 | 14.62d ± 1.11 | 14.55d ± 1.01
F4P0 | 48.99b ± 2.11 | 48.12c ± 2.11 | 48.42c ± 2.17 | 8.75b ± 1.11 | 7.75c ± 1.21 | 8.15c ± 1.17 | 9.28b ± 1.18 | 9.87c ± 1.23 | 9.28b ± 1.03
F4P3 | 49.25b ± 2.12 | 49.57a ± 2.12 | 48.29b ± 2.32 | 8.19b ± 1.71 | 9.25a ± 1.32 | 8.19c ± 1.11 | 9.05b ± 1.21 | 9.55c ± 1.24 | 8.73a ± 1.11
F4P5 | 49.41a ± 2.54 | 49.34a ± 2.54 | 48.73b ± 2.44 | 9.45a ± 1.59 | 9.65a ± 1.35 | 8.45b ± 1.19 | 8.59a ± 1.22 | 9.39b ± 1.27 | 8.32a ± 1.04
F4P7 | 49.57a ± 2.54 | 49.14b ± 2.54 | 49.77a ± 2.14 | 9.27a ± 1.43 | 9.99a ± 1.32 | 8.87b ± 1.13 | 8.09a ± 1.27 | 9.01b ± 1.09 | 8.17a ± 1.06

Distribution of genotype and allele frequency

Table 4 presents the genotypic distribution of the TGFβ2 gene coding region, as determined by distinct restriction patterns obtained from enzymatic digestion. The frequencies of the CC, TC, and TT genotypes were 20%, 56%, and 24%, respectively. Moreover, these genotype proportions showed highly significant differences (p > 0.05). According to the Hardy–Weinberg equilibrium, the allele frequencies for C and T were 0.48 and 0.52, respectively. Chi-square analysis revealed a strong association between the chicken strain and the AluI restriction enzyme. Such variations could be attributed to factors including gene flow across populations, interbreeding among groups, and differences in sample size.

Genetic variation in the TGFβ2 genes

Amplification was performed to identify SNPs by DNA sequencing (Figure 2). SNPs were detected using the F4P3 primer pair, whereas no polymorphisms were observed with the TGFβ2 primer pair (Figure 3). Two SNPs were identified within the exon 1 region (c.103C/T and c.99G/A). The g.1079C/T substitution altered the codon from GGC to GGT, representing a synonymous mutation that still encodes glycine (Figure 4). DNA sequencing successfully genotyped the c.103C/T and c.99G/A SNPs, with the g.103C/T locus exhibiting CC and CT genotypes, while the g.99G/A locus showed GG and GA genotypes.

Genotype | Number | Percentage (%)
CC (Wild) | 5 | 20
TC (Heterozygous) | 14 | 56
TT (Mutant) | 6 | 24
Total | 25 | 100

In this study, two SNPs were identified for the TGFβ2 gene target (Figure 3). SNP identification was performed by comparing the KUB chicken sequence and two local chicken sequences published in NCBI (Acc. X58071). Consequently, two SNPs were identified in the TGFβ2 gene target. One SNP was detected in the F4P5 treatment, and another in the F4P7 treatment (Figure 4). However, these SNPs were not confirmed among the F0P0, F4P0, and F4P3 samples. In fact, two other SNPs were found in the same gene target (Figure 3).

Figure 2: Electrophoresis of the PCR amplified TGFβ2 gene showing bands of approximately 284 bp on 1.5% agarose gel. M = 100 bp DNA ladder.

Figure 3: Alul restriction fragment patterns of TGFβ2 by PCR-RFLP on 1.5% agarose gel; marker 284 pb, 155 bp, 129 bp; CC/TT/CT = genotype.

Figure 4: Partial nucleotide sequence of the TGFβ2 gene in KUB chickens obtained by DNA sequencing.