Study of the molecular relationship between the jejunum angiogenesis pathway and feed efficiency in Iranian native turkeys

Introduction: Real-time PCR has become a powerful tool for quantifying nucleic acids and assessing gene expression. Its accuracy and sensitivity have made it indispensable in biological research, allowing for detailed analysis of gene expression changes in response to various stimuli. In the livestock and poultry industry, improving feed efficiency is a key goal in breeding programs due to its significant economic benefits. Defined as the ratio between feed intake and weight gain or production, feed efficiency is influenced by genetic, physiological, nutritional, and environmental factors. Understanding its molecular mechanisms can lead to targeted breeding strategies and the selection of superior genotypes. The intestine is critical for feed utilization efficiency, serving as the main site of nutrient digestion and absorption; its epithelial structure, including villi and microvilli, is essential for effective absorption. Furthermore, a rich vascular network, formed through angiogenesis, is crucial for efficient feed metabolism and nutrient absorption within the intestinal tissue. This study investigated the expression of RHOA, ACTA2, and ENO1 genes in the jejunum of high feed efficiency (HFE) and low feed efficiency (LFE) turkeys. RHOA is involved in angiogenesis. Meanwhile, ACTA2 is essential for maintaining vascular structure and integrity. Additionally, ENO1 provides the adenosine triphosphate (ATP) required for endothelial cell proliferation and migration, processes involved in angiogenesis. The expected higher expression of these genes in HFE turkeys suggests improved intestinal function, energy metabolism, and angiogenesis, contributing to better feed efficiency. This research aimed to lay the groundwork for identifying genetic markers to enhance feed efficiency in turkey, contributing to more sustainable animal protein production.
Materials and methods: Samples from native Iranian turkeys were used in this study. A total of 500 male turkey poults were raised under standard production protocols until 20 weeks of age. Subsequently, 75 turkeys were selected and transferred to individual cages to allow for precise monitoring of feed intake and weight gain. Body weight was measured at 20 weeks of age (the initiation of the cage period) and at 24 weeks of age (the termination of the experimental period). The turkeys were then ranked based on their feed conversion ratio (FCR), with FCR ranging from 3.59 to 16.346. Jejunum tissue samples were collected post-slaughter from three turkeys exhibiting HFE and three turkeys exhibiting LFE. The collected tissue samples were immediately stored at -80°C to preserve RNA integrity. Total RNA was extracted from 250 mg of jejunum tissue samples using the RNeasy Midi Kit (Qiagen). The quality and concentration of the extracted RNA were assessed using a spectrophotometer and agarose gel electrophoresis. Primers for the target genes (RHOA, ACTA2, and ENO1) and the reference gene (RPS7) were designed using specialized software. Real-time PCR was performed to quantify the expression levels of the target genes, and gene expression levels were calculated using the Pfaffl method, which normalizes the data to the reference gene. Statistical analyses, including Shapiro-Wilk and Spearman's rank correlation, were performed using GraphPad Prism 8, with significance set at P<0.05.
Results and discussion: High-quality RNA was confirmed by spectrophotometry and agarose gel electrophoresis. RPS7 was selected as the stable reference gene, its stability having been confirmed in previous studies. Melting curve analysis confirmed primer specificity and lack of non-specific products. Real-time PCR analysis showed that the expression of all three genes was significantly higher in the HFE group (P<0.05). Specifically, RHOA expression was approximately 7.52 times higher, ACTA2 about 46.17 times higher, and ENO1 around 33.08 times higher in the HFE group compared to the LFE group. Spearman's rank correlation analysis revealed significant positive correlations for ACTA2 and ENO1 with feed efficiency (r_s=1, P=0.0028), while RHOA showed a strong positive correlation (rs=0.8) approaching statistical significance (P=0.058). The high correlation between ACTA2 and ENO1 also suggests shared regulatory mechanisms, reinforcing their combined role in feed efficiency.
Conclusions: The elevated expression of RHOA, ENO1, and ACTA2 in HFE turkeys enhanced intestinal function, vascular network development, and energy supply, underscoring their importance in nutrient absorption and overall feed efficiency. These findings align with existing evidence in other species and provide a foundation for identifying potential genetic markers and developing nutritional and genetic strategies to improve poultry production efficiency. Further research is necessary to fully elucidate the complex interactions among these genes and other factors contributing to turkey.