Frozen semen can be preserved for a potentially indefinite time, however sperm cells fertilizing ability is lower, in part due to the increased levels of Reactive Oxygen Species which cause a damage to spermatozoal membranes, proteins and DNA, and reduce motility (1). In dogs, several anti-oxidants were tested (e.g. 2). Trolox®, a water-soluble synthetic tocopherol analogue, has been evaluated for canine semen preservation only at 200μM (3). The aim of this study was to evaluate if Trolox® at different concentrations in the freezing extender would improve post-thaw motility and plasma membrane integrity. Semen from 8 dogs was frozen in two steps in a Tris-citrate-fructose extender with a final concentration of 5% glycerol and 0.5% Equex STM paste (CONTR) to which 100, 200 or 400 μM Trolox® were added (T100, T200 and T400, respectively). Post thaw, motility was evaluated at hours 0, 1, 2 and 3 of incubation at 37°C by a Computer Assisted Sperm Analyser; while plasma membrane integrity (HOS-test) was evaluated at hours 0 and 2. Motility data were evaluated by Friedman test within each evaluation time. HOS-test data were analyzed by one way ANOVA. There was no difference between treatments at hour 0 and 1 (mean total motility h0: CONTR: 50.9%, T100: 46.9%, T200: 49.2%, T400: 47.2%), while T400 had a statistically significant lower total, progressive and rapid motility than CONTR at hours 2 (e.g. mean total motility CONTR: 17.0%, T100: 12.3%, T200: 13.4%, T400: 10.1%) and a significantly lower mean total motility at h3 (CONTR: 11.2%, T100: 9.1%, T200: 8.1%, T400: 4.7%). The proportion of intact plasma membranes was never significantly different between groups (h0: CONTR: 51.0%, T100: 43.4%, T200: 47.9%, T400: 42.5%; h2: CONTR: 42.1%, T100: 37.8%, T200: 39.3%, T400: 38.7%). Trolox® was able to improve post-thaw semen quality in swine (200 μM, 4), ram (60-120 μM, 5), and human (40 μM, 6) species. The present results disagree with those data and confirm what observed at 200 μM (3). It cannot be excluded that lower Trolox® concentrations may have positive effects, however the inclusion of 100 to 400μM in the freezing extender did not improve post-thaw canine semen characteristics, on the contrary Trolox® 400 μM worsened motility parameters.
EFFECT OF DIFFERENT CONCENTRATIONS OF TROLOX IN A CANINE SEMEN FREEZING EXTENDER ON POST THAW SPERM CHARACTERISTICS
ROTA, ALESSANDRA;PANZANI, DUCCIO;CAMILLO, FRANCESCO;VANNOZZI, IACOPO
2015-01-01
Abstract
Frozen semen can be preserved for a potentially indefinite time, however sperm cells fertilizing ability is lower, in part due to the increased levels of Reactive Oxygen Species which cause a damage to spermatozoal membranes, proteins and DNA, and reduce motility (1). In dogs, several anti-oxidants were tested (e.g. 2). Trolox®, a water-soluble synthetic tocopherol analogue, has been evaluated for canine semen preservation only at 200μM (3). The aim of this study was to evaluate if Trolox® at different concentrations in the freezing extender would improve post-thaw motility and plasma membrane integrity. Semen from 8 dogs was frozen in two steps in a Tris-citrate-fructose extender with a final concentration of 5% glycerol and 0.5% Equex STM paste (CONTR) to which 100, 200 or 400 μM Trolox® were added (T100, T200 and T400, respectively). Post thaw, motility was evaluated at hours 0, 1, 2 and 3 of incubation at 37°C by a Computer Assisted Sperm Analyser; while plasma membrane integrity (HOS-test) was evaluated at hours 0 and 2. Motility data were evaluated by Friedman test within each evaluation time. HOS-test data were analyzed by one way ANOVA. There was no difference between treatments at hour 0 and 1 (mean total motility h0: CONTR: 50.9%, T100: 46.9%, T200: 49.2%, T400: 47.2%), while T400 had a statistically significant lower total, progressive and rapid motility than CONTR at hours 2 (e.g. mean total motility CONTR: 17.0%, T100: 12.3%, T200: 13.4%, T400: 10.1%) and a significantly lower mean total motility at h3 (CONTR: 11.2%, T100: 9.1%, T200: 8.1%, T400: 4.7%). The proportion of intact plasma membranes was never significantly different between groups (h0: CONTR: 51.0%, T100: 43.4%, T200: 47.9%, T400: 42.5%; h2: CONTR: 42.1%, T100: 37.8%, T200: 39.3%, T400: 38.7%). Trolox® was able to improve post-thaw semen quality in swine (200 μM, 4), ram (60-120 μM, 5), and human (40 μM, 6) species. The present results disagree with those data and confirm what observed at 200 μM (3). It cannot be excluded that lower Trolox® concentrations may have positive effects, however the inclusion of 100 to 400μM in the freezing extender did not improve post-thaw canine semen characteristics, on the contrary Trolox® 400 μM worsened motility parameters.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
