First transcriptomic insight into the working muscles of racing pigeons during a competition flight

Molecular Biology Reports (2024) 51:625 https://doi.org/10.1007/s11033-024-09566-7 ORIGINAL ARTICLE First transcriptomic insight into the working muscles of racing pigeons during a competition flight Monika Stefaniuk‑Szmukier1,2 · Tomasz Szmatoła1,3 · Agnieszka Pustelnik1 · Katarzyna Ropka‑Molik1 Received: 25 January 2024 / Accepted: 18 April 2024 © The Author(s) 2024 Abstract Background The currently known homing pigeon is a result of a sharp one-sided selection for flight characteristics focused on speed, endurance, and spatial orientation. This has led to extremely well-adapted athletic phenotypes in racing birds. Methods Here, we identify genes and pathways contributing to exercise adaptation in sport pigeons by applying nextgeneration transcriptome sequencing of m.pectoralis muscle samples, collected before and after a 300 km competition flight. Results The analysis of differentially expressed genes pictured the central role of pathways involved in fuel selection and muscle maintenance during flight, with a set of genes, in which variations may therefore be exploited for genetic improvement of the racing pigeon population towards specific categories of competition flights. Conclusions The presented results are a background to understanding the genetic processes in the muscles of birds during flight and also are the starting point of further selection of genetic markers associated with racing performance in carrier pigeons. Keywords Racing pigeons · Sport pigeons · Transcriptome profiling · RNA-seq · Pectoralis muscle Introduction The currently known homing pigeon is a result of crossing many lines of pigeons and a sharp one-sided selection for flight characteristics focused on speed, endurance, and spatial orientation. The purpose of selection focuses on breeding an extraordinarily motivated to get a home bird, that in the shortest possible time in various weather conditions at * Monika Stefaniuk‑Szmukier Tomasz Szmatoła Katarzyna Ropka‑Molik 1 Department of Animal Molecular Biology, National Research Institute of Animal Production, Krakowska 1, Balice 32‑083, Poland 2 Department of Animal Reproduction, Anatomy and Genomics, The University of Agriculture in Kraków, Al. Mickiewicza 24/28, Kraków 30‑059, Poland 3 Center for Experimental and Innovative Medicine, The University of Agriculture in Krakow, Rędzina 1C, Kraków 30‑248, Poland an average speed greater than 70 km/h will cover a certain distance to the loft [1]. For over 200 years of sports competition, breeders developed a highly specialised breed of pigeon called Racing Homer, paying great attention to improving their ability to approximate the direction to home from foreign locations defeating hundreds of kilometers, avoiding hazards and coping with unexpected weather conditions [2]. The competition aims to compare bird individuals’ flight performance by the speed of returning to the loft. Contests are held at various distances from, short ranging from 95 km to long exhaustive marathons with more than 700 km to cope [3]. When displaced to an unfamiliar location, homing pigeons apply a spatial navigation system and outstanding physiological adaptations to returning to the loft [4] including cardiorespiratory properties [5], energy expenditure supported by anaerobic and aerobic metabolic pathways with efficient circulatory system and oxygen transport [6, 7]. Avian flight is powered primarily by large pectoralis muscles (m. pectoralis pars thoracicus which accounts for up to 11% of total body mass and generates up to 95% of the power used for flight. The large m.pectoralis extends from the sternum, clavicle, and ribs to the humerus, and consists of two anatomical parts, the sternobrachialis, and Vol.:(0123456789) 625 Page 2 of 9 the thoracobrachialis, separated by an aponeurotic central tendon [8]. The fibre type composition contains mainly IIb type, referred to as fast oxidative (~ 85% in pigeons) adapted to anaerobic glycolytic metabolism [11]. Fibers possessed representative sarcomere structures, however, with shorter resting sarcomere lengths compared to mammalians [8]. The avian flying muscles introduce the most energetically expensive muscle work with the highest mass-specific metabolic rates in vertebrates, in comparison to exercising mammals, flapping is energetically more costly than running [6, 9]. To cope with these demands, several mechanisms have been described. Fuel selection of avian muscles during locomotion supports lipid oxidation, with minimum changes in blood glucose concentrations. Pigeons flying at their maximum rate of O2 uptake (V̇O2,max) and a respiratory quotient (RQ) at 0.73 indicating dependence on fat oxidation [10] whilst mammals exercising at similar V̇O2,max uptake reach RQ at 0.9 reflect dependence on carbohydrates and finally induce fatigue, with low muscle glycogen and blood glucose [11]. However, birds maintain a very high plasma glucose concentration (1–2 times) compared to mammals of equal body mass but with no harmful physiological effect. It is believed that endogenous antioxidant mechanisms such as free radical scavenging, DNA protection and uric acidmediated inhibition of lipid peroxidation help with homeostasis maintenance [12]. The dominant role and large size of the m.pectoralis, enable an assessment of adaptation and muscle function tailored to meet the mechanical and energetical requirements of exercising flight, compared to exercising mammals of different body masses and aerobic capabilities [13]. Several studies have been undertaken to find molecular pathways modified in skeletal muscles during exercise for example in humans and horses. It has been established that repeated sets of exercises lead to new basal levels of gene expression [14–16]. The molecular mechanism underlying the genetic adaptation during pigeon flight and training remains poorly understood. Since the sport and breeding of racing pigeons is a profitable business covering areas such as nutrition, supplementation, and genetic markers, the aim of the present study was transcriptome profiling of pigeons m.pectoralis muscle, collected from untrained birds and trained birds after competing 300 km competition flight with the use of high throughput RNA-sequencing. Material and methods Animals and study design The present study was performed on 13 muscle samples of m. pectoralis collected from 13 racing pigeons (Columba livia). Samples were collected from adult birds never trained Molecular Biology Reports (2024) 51:625 for racing (k; n = 5) and birds after competing in a 300 km race (f; n = 8), who had undergone earlier flight training. All birds were bred, and raised on a private loft owner, at the same location, with the same environmental and feeding conditions. The E group was basketed the day before and transported to the place of release (300 km away from the loft). The racing group was released at 6 a.m. Upon their return, both groups were sedated and euthanized using the (...truncated)