S. Sancho1, Isabel Casas2, H. Ekwall3, F. Saravia3, Heriberto Rodríguez‐Martinez3, Joan E. Rodríguez‐Gil4, E. Flores4, Elisabeth Pinart2, M. Briz2, N. García-Gil2, Judit Bassols2, A Pruneda2, E Bussalleu2, Marc Yeste2, Sergi Bonet2
1Biotechnology of Porcine Reproduction, University of Girona, Campus de Montilivi, s/n, 17071 Girona, Spain. [email protected] 2Biotechnology of Porcine Reproduction, University of Girona, Campus de Montilivi, s/n, 17071 Girona, Spain
3Division of Comparative Reproduction, Obstetrics and Udder Health, Faculty of Veterinary Medicine and Animal Sciences, Swedish University of Agricultural Sciences (SLU), SE-750 07, Uppsala, Sweden and
4Unit of Animal Reproduction, Department of Animal Medicine and Surgery, School of Veterinary Medicine, Autonomous University of Barcelona, Barcelona E-08193, Spain
Tóm tắt
This study evaluated the effects of cooling, freezing and thawing on the plasma membrane integrity, kinetics and expression of two sugar transporters glucose transporter-3 and -5 (GLUT-3 and GLUT-5) in spermatozoa from Iberian boars. Semen samples were collected twice weekly from eight young, fertile Iberian boars of the ‘Entrepelado’ and ‘Lampiño’ breeds. The samples were suspended in a commercial extender and refrigerated to 17 °C for transport to the laboratory (step A), where they were further extended with a lactose–egg yolk-based extender and chilled to 5 °C (step B) prior to freezing in the presence of glycerol (3%). Spermatozoa were assessed for plasma membrane integrity and sperm motility at each of the steps, including post-thaw (step C). Aliquots were also prepared for immunocytochemical localisation of the sugar transporters (fixed and thin smears for transmission and scanning electron microscopy levels respectively) and for SDS–PAGE electrophoresis and subsequent western blotting, using the same antibodies (rabbit anti-GLUT-3 and anti-GLUT-5 polyclonal antibodies). The results showed lower percentages of progressively motile spermatozoa at step C in both breeds, while the percentage of live spermatozoa was significantly lower only in the ‘Entrepelado’ breed. The results obtained from electron microscopy clearly showed that Iberian boar spermatozoa expressed the hexose transporters, GLUT-3 and GLUT-5. The pattern of expression, in terms of location and concentration, was characteristic in each case but, in the case of isoform GLUT-5, it remained constant during the different steps of freezing–thawing protocol. These results indicate that cryopreservation affects the status of sperm cells of Iberian boars by altering the distribution of some membrane receptors and decreasing the percentage values of parameters linked to sperm quality.
