Genetic polymorphisms of calpain1 and calpain3 genes and their effects on growth, carcass, and meat quality traits in Betong chicken (KU line)

2.1 Animal care

Animal care in this research was reviewed and approved by the Kasetsart University's Institutional Animal Care and Use Committee, Bangkok, Thailand (ACKU60-AGR-003).

2.2 Animal and phenotypic data

Initially, the Betong chicken (KU line) population was established from a poultry farm in southern Thailand. Cocks and hens were selected based on the original breed characteristics, namely, feathering rate, golden brown feather color, and growth performance. Additionally, mating based on full-sib and half-sib relationships was avoided in this closed flock (Sopannarath & Bunchasak, 2015).

In the current study, phenotypic measurements (growth, carcass, and meat quality traits) were tested in two progeny flocks. The progeny flocks were produced by artificially inseminating hens from the same cock (using 27 cocks and 96 hens) on a weekly basis at the poultry farm located at Kasetsart University, Bangkok, Thailand. Identification of pedigree information was conducted by daily collection of the eggs and marking them according to dam number. Prior to incubation, the collected eggs were disinfected with formaldehyde and subsequently placed in an incubator for 21 days (18 days in the setter and 3 days in the hatchery). Finally, chicks aged 1 day were individually weighed and wing banded.

The chickens were placed within a brooder with incandescent lighting (23 h light:1 h darkness) on a litter floor. From 4 weeks of age, the chickens were grouped according to sex and then moved to a slat-floored barn with fluorescent lighting (23 h light:1 h darkness) until age 16 weeks. Fresh water and feed were provided ad libitum. The feed formulation, which excluded antimicrobial agents, was based on the method of Putsakul et al. (2010).

At age 16 weeks, approximately three birds from a full-sib family were randomly sampled to determine carcass characteristics and meat quality traits. The slaughtering process for ante-mortem and post-mortem followed good manufacturing practices for a poultry abattoir (Thai Agricultural Standard, 2005); carcass traits were dissected by cutting parts according to the standards for chicken meat (Thai Agricultural Standard, 2006). The performance data for the two progeny flocks were measured using the following parameters at age 16 weeks: BW, live body weight (LW), CW, BMW, LEG), wing weight (WW), cooking loss percentage (CL), drip loss percentage (DL), shear force value (SF), lightness (L*), redness (a*), and yellowness (b*).

The left side of the breast muscle was weighed, placed in a plastic bag, hung on the shelf, and stored at 4°C for 24 h. Subsequently, each sample was then removed from the plastic bag, excess surface moisture was wiped off, and the sample was re-weighed to determine the DL values (Northcutt et al., 1994). The breast meat colors, L*, a*, and b*, were measured using a Hunter Lab colorimeter (Reston, Virginia, USA) at 24 h post-mortem according to the CIE (Commission International de I'Eclairage) method (International Commission on Illumination, 1978).

Cooking loss of the right breast muscle was measured following the method of Papinaho and Fletcher (1996). The sample was weighed and cooked for 15 min at 95°C in steam, cooled to room temperature for 20 min, and then re-weighed. For the SF, the sample was cut to 2.0 × 1.0 × 1.5 cm (Saláková et al., 2009) and analyzed using a Lloyd Texture analyzer with a Warner-Bratzler blade attachment (Bognor Regis, West Sussex, UK).

2.3 Genotypic data

A blood sample was collected from the jugular vein during the slaughtering process. Genomic DNA was isolated using the alkaline lysis method (Sambrook et al., 1989). The PCR primer pairs are shown in Table 1 according to Okumura et al. (2006). The PCR conditions of CAPN1 were initial denaturation at 94°C for 5 min, 35 cycles of denaturing at 94°C for 30 s, annealing at 65°C for 30 s, extension at 72°C for 30 s, and final extension at 72°C for 10 min. The PCR samples of CAPN1 were run on 4.5% polyacrylamide gel at the DNA Technology Laboratory, Kamphaeng Saen, Thailand. Subsequently, the homozygous individuals were sequenced using a 3500xL Genetic Analyzer (Applied Biosystems, USA). Nucleotide sequences were annotated using a nucleotide BLAST program from the National Center for Biotechnology Information (NCBI). Differences in the homologous genotypes were aligned using the Clustal X software (Larkin et al., 2007) and were analyzed using GeneDoc program (Nicholas & Nicholas, 1997).

The PCR conditions for CAPN3 were initial denaturation at 95°C for 5 min, 30 cycles of denaturing at 95°C for 30 s, annealing at 68°C for 30 s, extension at 72°C for 3 min, and final extension at 72°C for 5 min. Restriction fragment length polymorphism (RFLP) was used to identify the CAPN3 polymorphisms. The PCR product of the CAPN3 (5 μL) was digested with 10 units of HhaI enzyme (New England Biolabs Inc., USA) at 37°C for 4 h. After digestion, the DNA fragments were separated on a 1% agarose gel using electrophoresis. The gel was stained using the SERVA DNA Stain G (SERVA, Germany) and visualized under ultraviolet light.

2.4 Statistical analysis

All statistical testing, including the genotypic and allelic frequencies for each locus based on the PROC FREQ procedure, was carried out using the SAS® on Demand for Academics (SAS Institute Inc., Cary, North Carolina, USA). Hardy–Weinberg equilibrium was tested using the chi-square test ($$$ {\chi}^2 $$$) based on the method of Falconer and Mackay (1996) The genotypic and allelic substitution effects of the CAPN1 and CAPN3 were analyzed using the PROC GLM procedure, while least squares means (LSM) and the comparison of the LSM of the genotypic effect of those genes were analyzed using Tukey's test in the SAS® on Demand for Academics (SAS Institute Inc., Cary, North Carolina, USA). The model was y_ijklm = μ + H_i + S_j + G_1k + G_2l + G_1k * G_2l + e_ijklm, where y_ijklm is the individual performance BW, LW, CW, BMW, LEG, WW, CL, DL, SF, L*, a*, and b*; μ is the overall mean; H_i is the fixed effect of hatching batch i (i = 1 and 2); S_j is the fixed effect of sex j (j = male and female); G_1k is the fixed effect of the CAPN1 genotypes k (k = AA, AB, and BB); G_2l is the fixed effect of the CAPN3 genotypes l (l = CC, CT, and TT); G_1k * G_2l is the interaction between the CAPN1 and CAPN3 genotypes; and e_ijklm is the random residual effect ~ NID (0, $$$ {\sigma}_e^2 $$$).

The regression coefficients were estimated to determine the allele substitution effects of the CAPN1 and CAPN3. The fixed genotypic effects were replaced by coding 0, 1, and 2 for the BB, AB, and AA genotypes of the CAPN1 or the TT, CT, and CC genotypes of the CAPN3, respectively. The significance of the allele substitution effect was examined using the PROC GLM procedure.

3.1 Performance of growth, carcass, and meat quality traits

The descriptive statistics are shown in Table 2 for growth (BW and LW), carcass yield (CW, BMW, LEG, and WW), meat quality (CL, DL, and SF), and meat color (L*, a*, and b*) in Betong chicken (KU line). The chickens reached a BW of 1939.15 g, which is close to the preferred market size (2 kg) at 16 weeks of age. Furthermore, the mean BMW in the current study was 275.55 g. The mean DL in the current study was 2.32%. Meat color was described by the values of L*, a*, and b* (62.01, 5.57, and 29.80, respectively). The mean of SF was 29.80 N (Table 2).

3.2 Genotypic polymorphisms of CAPN1 and CAPN3

The blood samples were extracted for DNA analysis. Fragments of the CAPN1 were amplified and denatured using PCR and were visible in a 4.5% polyacrylamide gel. The CAPN1 polymorphisms were classified into three genotype groups: AA, AB, and BB (Figure 1). The product sizes of the AA and BB genotypes were 225 and 219 bp, respectively, whereas the product sizes of the AB genotype covered both 225 and 219 bp. The DNA sequences of all genotypes are shown in Figure 2. The difference between the AA and BB genotypes was caused by a 6-bp single insertion (CAACCC). From DNA sequencing, a 2-bp (TT) insertion was found in a sample, as shown in line 2 in Figure 2.

Polymorphisms of the CAPN3 were found in this population based on PCR-RFLP. Consequently, three genotypes were identified: CC, TT, and CT. The CC genotype presented a single 6-kbp fragment, whereas the TT genotype presented two fragments of 2 and 4 kbp. The heterozygous genotype CT had three fragments of 2, 4, and 6 kbp (Figure 3).

The allelic and genotypic frequencies of the CAPN1 and CAPN3 are shown in Table 3. For the CAPN1, the allelic frequency of the A allele (0.22) was lower than that of the B allele (0.78). Consequently, the genotypic frequency of AA (0.05) was lower than that of BB (0.61) and the frequency of AB was 0.34. On the other hand, for the CAPN3, the allelic frequencies of C (0.54) and T (0.46) were found in nearly half the occurrences. Therefore, the striking genotypic frequency was the CT genotype (0.50), while the genotypic frequencies of CC and TT were 0.29 and 0.21. Moreover, the genotypic frequencies of CAPN1 and CAPN3 were in Hardy–Weinberg equilibrium.

3.3 Genotypic effects of the CAPN1 and CAPN3 on growth, carcass, and meat quality traits

The genotypic effects of CAPN1 and CAPN3 with the interaction between the genes for all traits were analyzed, and the interactions were not significant except for the L* trait (Table 4). The LSM of the growth, carcass, and meat quality traits for each of the CAPN1 and CAPN3 genotypes are shown in Table 5. The genotypic effects of CAPN1 were not significant for all traits, whereas the genotypic effect of the CAPN3 showed association with BMW. The TT genotype was more favorable than the other genotypes for BMW (P < 0.05).

Regardless of the interaction between the genotypic effect of the genes, the allele substitution effect of the CAPN1 had a significant association with L* (P < 0.05) (Table 6). Substitution of the B allele with the A allele led a reduction in the L* value in meat (−0.78 ± 0.30). Furthermore, the allele substitution effect of the CAPN3 was significantly associated with BW, LW, CW, and BMW (P < 0.05). Substitution of the T allele with the C allele reduced BW, LW, CW, and BMW, accounting for −40.58 ± 14.77, −44.53 ± 13.39, −25.14 ± 11.02, and −8.63 ± 2.61 g, respectively (Table 6).