Research note: Reference genes selection for gene expression analyses in reproductive turkey (Meleagris gallopavo) with yellow semen syndrome

Molecular biology techniques, including qPCR, are reliable, powerful, and commonly used tools for diagnostic purposes. qPCR might be useful for determining the etiology of yellow semen syndrome (YSS), an endemic condition in the turkey population, leading to decreased reproductive potential of this species. The current study aimed to evaluate the most accurate qPCR internal controls that might be used as first-choice-reference genes in studies on the reproductive tract, liver, and immune tissues and cells of adult male turkey breeders. RNA was isolated from testis, epididymis, ductus deferens, liver, thymus, bursa of Fabricius, spleen, whole blood, and peripheral blood mononuclear cells (PBMC) from healthy adult individuals producing white semen (WS, n = 6) and with yellow semen syndrome (YSS, n = 6). The expression of seven commonly used housekeeping genes, i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), phosphoglycerate kinase (PGK1), 60S ribosomal protein L13 (RPL13),ribosomal protein L19 (RPL19), transferrin receptor protein (TFRC), and vimentin (VIM) was tested with qPCR followed by computational calculation of these genes' expression stability in selected tissues/cells. For YSS studies we recommend using ACTB/GAPDH gene pair as a reference for male reproductive organs i.e. testis, epididymis, and ductus deferens as well as in the liver, RPL13/RPL19 for central immune organs, i.e. bursa of Fabricius and thymus, and RPL13/VIM, ACTB/RPL19, and PGK1/RPL19 in the spleen, whole blood, and PBMC research, respectively. A careful investigation of the stability of these genes in each following experiment is required to maintain high-accuracy results. Our results may help optimize protocols for better investigation of molecular mechanisms in physiological and pathological conditions in male turkey breeders for further improving commercial flock welfare and livestock production, and are a prerequisite for future studies determining YSS etiology.