title: Determining ACTB, ATP5B and RPL32 as optimal reference genes for quantitative RT-PCR studies of cryopreserved stallion semen.
abstract: Equine germplasm bank management involves not only the conservation and use of semen doses, in addition it can also be a resource to study stallion semen quality and after thawing semen properties for reproductive purposes. A possible criterion to measure quality may be based on differential gene expression of loci involved during spermatogenesis and sperm quality maturation. The rapid degradation of sperm after thawing affects the integrity and availability of RNA. In this study we have analyzed genes expressed in equine cryopreserved sperm, which provided an adequate amplification, specificity, and stability to be used as future reference genes in expression studies. Live spermatozoa were selected from cryopreserved semen straws derived from 20 stallions, through a discontinuous concentration gradient. RNA purification followed a combination of the organic and column extraction methods together with a deoxyribonuclease treatment. The selective amplification of nine candidate genes was undertaken using reverse transcription and real-time polymerase chain reaction (qPCR) carried out in a one-step mode (qRT-PCR). Specificities were tested by melting curves, agarose gel electrophoresis and sequencing. In addition, gene stabilities were also calculated. Results indicated that five out of the nine candidate genes amplified properly (β-Actin, ATP synthase subunit beta, Protamine 1, L32 ribosomal protein and Ubiquitin B), of which β-Actin and the L32 Ribosomal protein showed the highest stability thus being the most suitable to be considered as reference genes for equine cryopreserved sperm studies, followed by the ATP synthase subunit beta and Ubiquitin B.
authors.affiliation: Laboratorio de Investigación Aplicada, Cría Caballar de las Fuerzas Armadas (Ministry of Defense), Apdo. de Correos 2087, 14080 Córdoba, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Pérez-Rico
authors.affiliation: Centro Militar de Cría Caballar de Ávila, Cría Caballar de las Fuerzas Armadas (Ministry of Defense), Acuertelamiento "El Pradillo", Calle Arsenio Gutierrez Palacios s/n, 05005 Ávila, Spain.
authors.forename: F
authors.initials: F
authors.lastname: Crespo
authors.affiliation: Laboratorio de Investigación Aplicada, Cría Caballar de las Fuerzas Armadas (Ministry of Defense), Apdo. de Correos 2087, 14080 Córdoba, Spain.
authors.forename: M L
authors.initials: ML
authors.lastname: Sanmartín
authors.affiliation: Centro Militar de Cría Caballar de Écija, Cría Caballar de las Fuerzas Armadas (Ministry of Defense), Calle Nueva 2, 41400 Écija, Sevilla, Spain.
authors.forename: A
authors.initials: A
authors.lastname: De Santiago
authors.affiliation: Laboratorio de Investigación Aplicada, Cría Caballar de las Fuerzas Armadas (Ministry of Defense), Apdo. de Correos 2087, 14080 Córdoba, Spain. Electronic address: jvegpla@gmail.com.
authors.forename: J L
authors.initials: JL
authors.lastname: Vega-Pla
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000662:veterinary*; D006736:Horses / Q000502:physiology*; D008297:Male; D025261:Mitochondrial Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D012661:Semen / Q000502:physiology*; D012662:Semen Preservation / Q000662:veterinary*
chemical_list: D012269:Ribosomal Proteins; D025261:Mitochondrial Proton-Translocating ATPases
keywords: Cryopreserved sperm; Houskeeping gene; Quantitative polymerase chain reaction; Stallion