MATERIALS AND METHODS

Ethical approval

The experimental protocol was reviewed and approved by the Animal Care and Use Ethics Committee, Faculty of Veterinary Medicine, Universitas Airlangga, Indonesia (Approval No. 1.KEH.039.03.2024). All procedures involving KUB chickens were conducted in accordance with the Indonesian national guidelines for the ethical care and use of animals in research, the institutional animal welfare requirements of Universitas Airlangga, and the principles outlined in the ARRIVE 2.0 guidelines.

Throughout the 90-day experimental period, the birds were housed under appropriate environmental, hygienic, and husbandry conditions, with unrestricted access to clean drinking water and nutritionally adequate feed in accordance with their assigned dietary treatments. The chickens were observed regularly for general health, behavior, feed and water consumption, mobility, signs of pain or distress, injury, and adverse responses to the experimental diets. All handling, weighing, sampling, and husbandry procedures were performed by trained personnel using methods designed to minimize fear, discomfort, restraint time, and unnecessary stress.

Predefined humane considerations were applied throughout the experiment. Any bird showing persistent, severe distress; inability to access feed or water; marked loss of body condition; serious injury; or clinical deterioration that could not be promptly alleviated was to be evaluated by qualified veterinary personnel and either removed from the experiment or, when necessary, humanely euthanized. At the end of the feeding trial, the chickens were slaughtered using the humane procedure authorized in the approved institutional protocol. Tissue collection was performed only after confirmation of death. Muscle samples were collected aseptically for meat quality assessment and molecular analysis, immediately processed or preserved as required, and stored under appropriate conditions.

The number of birds was limited to the minimum considered necessary to address the study objectives while permitting the planned statistical analyses. All reasonable measures were taken to refine the experimental procedures, reduce bird use, and minimize pain and distress. No procedures were performed solely for purposes unrelated to the approved scientific objectives.

Study period and location

This study was conducted from June 5 to September 6, 2024 at the Faculty of Veterinary Medicine, Airlangga University.

Study design

A total of 25 one-day-old male KUB chicks were randomly assigned to five dietary treatments (n = 5 per treatment). The experiment followed a 5 × 3 factorial arrangements comprising five dietary treatments and three TGFβ2 genotypes (CC, TC, and TT), with genotypes determined post hoc by molecular analysis. The dietary treatments were: F0P0 (control, no enzymes), F4P0 (400 mg/kg phytase), F4P3 (400 mg/kg phytase + 300 mg/kg protease), F4P5 (400 mg/kg phytase + 500 mg/kg protease), and F4P7 (400 mg/kg phytase + 700 mg/kg protease). Enzyme inclusion levels were selected based on commercial recommendations and previous studies in native chickens. Birds were housed individually and fed ad libitum twice daily, with free access to water. All procedures were approved by the institutional ethics committee and conducted in accordance with animal welfare guidelines.

Evaluation of performance

Body weight (BW) was recorded individually using a digital hanging scale with an accuracy of 0.01 g at day 1 and at the end of the experimental period (90 days of age). Birds were weighed weekly to monitor growth performance. Average daily gain (ADG) was calculated using the following formula: ADG (g/day) = (IBW − BW) / days, where BW represents BW (g). Feed intake (FI) was calculated as the difference between feed offered and feed refused for each bird, adjusted for any feed spillage. Daily FI (DFI) was determined by dividing total feed consumption by the number of feeding days. Feed conversion ratio (FCR) was calculated as: FCR = Total FI (g) / Total BW gain (g)

Meat quality analysis

Meat quality parameters were analyzed including water-holding capacity (WHC), pH, texture, and cooking loss (CL), which were measured using 5 g of raw meat samples as the initial weight, with two replicates for each treatment. Meat color was evaluated using a colorimeter (12 mm aperture, U 59730-30, Cole-Parmer International Inc., Pittsford, NY, USA) on first use at the same measurement point for each sample. Meat color characteristics were then expressed in Lightness (L*), Redness (a*), and Yellowness (b*) values [12].

Meat sampling and DNA isolation

Muscle samples weighing approximately 0.5-1.0 g of lean tissue were taken and placed into sterile cryovial tubes. Following instant flash freezing in liquid nitrogen, the samples were stored at −80 °C until DNA extraction and additional molecular analysis. The specific primer sequences used for the chicken TGFβ2 target gene KUB (GenBank accession no. X58071) were as follows: forward primer, 5’-GCC ATA GGT TCA GTG CAA G-3’; reverse primer, 5’-TGA CAG AAG CTC TCA AGC C-3’. The manufacturer's instructions (Promega, Madison, WI, USA) on first use were followed to generate the polymerase chain reaction (PCR) reaction mixture, which included 12.5 μL of master mix, 1 μL of forward primer, 1 μL of reverse primer, 3 μL of DNA template, and 7.5 μL of distilled water. The ideal conditions for amplification were 5 min of initial denaturation at 95°C, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 54°C for 30 seconds, extension at 72°C for 30 seconds, and a final extension at 72°C for 7 min.

PCR-Restriction fragment length polymorphism (RFLP) analysis

PCR-RFLP analysis involved digesting 20 μL of PCR product with 1 μL of the restriction enzyme AluI for 3 h at 37°C. Following digestion, the pieces were separated using 1.5% agarose gel electrophoresis, stained with ethidium bromide, and examined under an ultraviolet lamp. Additionally, 20 μL of the PCR product was sent to Macrogen (Seoul, Korea) for sequencing analysis on first use [13].

Screening and functional analysis of differentially expressed genes (DEGs)

DEGs were subjected to hierarchical clustering analysis using the Gplots package in the R software environment (R Foundation for Statistical Computing, Vienna, Austria). Following gene identification, STRING functional protein association networks were used to build a regulatory enrichment network, and Cytoscape was used to visualize it. The web-based program DAVID was used for Gene Ontology (GO) enrichment analysis, and the significance threshold for relevant GO categories was set at p < 0.05. Additionally, KOBAS 3.0 was used to conduct Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis [14].

Statistical analysis

The Benjamini-Hochberg method was used to calculate adjusted p-values for DEG analysis and KEGG pathway enrichment. For growth performance and meat quality traits, data were analyzed using a two-way analysis of variance to evaluate the main effects of TGFβ2 genotype and enzyme supplementation level, as well as their interaction (genotype × enzyme level). When significant effects were detected (p < 0.05), treatment means were compared using Duncan’s Multiple Range Test. All statistical analyses were performed using SPSS version 20.0. Results are presented as mean ± standard deviation (SD).