292 CHANGES IN MEMBRANE SULFHYDRYL STATUS OF BOAR SPERMATOZOA BY FREEZING

Abstract

The processes of cooling and freezing/thawing produce physical and chemical stresses on the sperm membrane that reduce the viability and fertilizing capacities. The cold shock and freezing of spermatozoa are associated with an oxidative stress, with reactive oxygen species (ROS) generation (Chatterjee et al., 2001, Mol. Reprod. Dev. 60, 498–506) and with a significant reduction of the GSH content (Gadea et al., in press). In the processes of capacitation, fertilization and freezing, qualitative and quantitative changes in protein membrane composition occurs, including changes in distribution of sulphydryl groups on the sperm membrane. The aim of this work was to evaluate the changes in the sulfhydryl groups of proteins from the sperm surface after cooling and freezing procedures as a marker of membrane changes. Ejaculate-rich fractions from three mature Pietrain boars were diluted in Beltsville Thaw Solution (BTS) extender and cooled to 15°C over 2 h (control). Thereafter sperm were centrifuged and diluted in lactose/egg-yolk extender cooled to 5°C over 2 h and later frozen with glycerol and equex by classic methodology (Westendorf et al., 1975, Dtsch. Tierärztl. Wschr. 82, 261–267). Sperm parameters were measured in extended semen (control) at 0, 1 and 2 h after cold shock at 5°C and after freezing-thawing. The structure of the sperm membrane was evaluated with carboxyfluorescein diacetate/propidium iodide (DCF) (Harrison and Vickers, 1990, J. Reprod. Fert. 88, 343–352), and the sulfhydryl status of proteins from spermatozoa surface are evaluated with fluorescent-staining 5-iodoacetamidofluoresceine (5-IAF) and by acrosome integrity (normal apical ridge, NAR). Some seminal parameters to evaluate functionality such as motility (MOT), forward progressive motility (FPM, 0–5), and mitochondria activity with Rhodamine 123(MIT) were also evaluated. Data from 11 freezing batches were analyzed by one-way ANOVA. When ANOVA revealed a significant effect, values were compared by the Tukey test. The freezing process significantly affected all the sperm parameters studied. Motility was negatively affected from the onset of cooling to 5°C. However, DCF, NAR and 5-IAF were only affected after freezing process. Mitochondria activity decreased in the last period of the cooling procedure (2 h) and it was lower after freezing. An inverse significant relation was found between 5-IAF and motility, viability, NAR and mitochondria functionality (P < 0.01). These results show that freezing damage produces an alteration in the structure of the sperm membranes (DCF, NAR, 5-IAF) and sperm functionality (motility and mitochondrial). However, only motility (MOT and FPM) was affected by cold shock when lactose/egg-yolk extender was used. Previous studies of cold shock with no cryo-protective medium (BTS) showed a marked effect on sulphydryl membrane characteristics (Marco and Gadea, 2003). These preliminary results in the use of 5-IAF in boar semen showed that freezing produces an alteration in the structure of the sperm membranes, which could be detected by simple fluorescent staining. This research was supported by grant AGL 2000-0485-C02-01.