Effect of astaxanthin in extenders on sperm quality and functional variables of frozen-thawed boar semen.

The aim of the study was to determine whether the presence of astaxanthin (ASX) protects boar spermatozoa against damage related to cryopreservation. Pooled ejaculates extended in Beltsville Thawing Solution (BTS) were used. Three experiments were conducted: 1) sperm samples were pre-incubated overnight (17 °C) with ASX (0, 0.5, 5, 15 µM) prior to freezing and then frozen using cooling and thawing extenders supplemented with ASX (0, 0.5, 5, 15 µM); 2) sperm samples were treated with ASX (0, 0.5, 5, 15 µM) only during overnight pre-incubation (17 °C) prior to cryopreservation; and 3) a thawing extender was supplemented with ASX (0, 0.5, 5, 15 µM). The groups were as follows: control (C; no treatment), ASX 1 (0.5 µM), ASX 2 (5 µM) and ASX 3 (15 µM). Total (TM) and progressive (PM) motility was analyzed using CASA, while sperm viability, reactive oxygen species generation, lipid peroxidation and apoptoticlike changes were analyzed using flow cytometry. Sperm variables were evaluated prior to freezing as well as 30 and 150 min after thawing. In Experiment 1, the values of TM and sperm viability post-thaw were less in the ASX 3 than C group. In Experiment 2, there was no effect of ASX on any of the sperm variables evaluated, while in Experiment 3, apoptotic-like changes were less in the ASX 1 than C group. In conclusion, there was a subtle beneficial effect on cryopreserved boar spermatozoa after addition of ASX to thawing media.

Dietary omega-3 polyunsaturated fatty acids induce plasminogen activator activity and DNA damage in rabbit spermatozoa.

The aim of this study was to determine the effect(s) of dietary omega-3 polyunsaturated fatty acids (ω-3 PUFA) on rabbit semen. Adult rabbit bucks were assigned to two groups that were given two diets, a standard diet (control) and a diet supplemented with ω-3 PUFA. Sperm samples were collected from all bucks with the use of an artificial vagina in 20-day intervals, for a total period of 120 days. The enrichment of membranes in ω-3 PUFA was manifested by the elevation of the 22:5 ω-3 (docosapentaenoic acid [DPA]) levels within 40 days. This increase in DPA content did not affect semen characteristics (i.e., concentration, motility and viability). However, it was associated with the induction of lipid peroxidation in spermatozoa, as determined on the basis of the malondialdehyde content. Lipid peroxidation was associated with DNA fragmentation in ω-3 PUFA-enriched spermatozoa and a concomitant increase in plasminogen activator (PA) activity. The effects of ω-3 PUFA on sperm cells were evident within 40 days of ω-3 PUFA dietary intake and exhibited peack values on day 120. Our findings suggest that an ω-3 PUFA-rich diet may not affect semen characteristics; however, it may have a negative impact on the oxidative status and DNA integrity of the spermatozoa, which was associated with an induction of PAs activity.

Effect of N-acetyl-L-cysteine supplementation in semen extenders on semen quality and reactive oxygen species of chilled canine spermatozoa.

The objective of this study was to evaluate the quality of chilled dog semen processed with extenders containing various concentrations of N-acetyl-L-cysteine (NAC). Ejaculates from five dogs were collected, pooled and evaluated for concentration, motility, rapid steady forward movement (RSF-movement), viability, acrosomal integrity and by the hypo-osmotic swelling test (HOST). In addition, superoxide anion (O(2)(-*)) production, hydroxyl radicals (OH(*)) and total reactive oxygen species (tROS) were determined. The pool was divided into five aliquots, which were diluted to a final concentration of 66.66 x 10(6) spermatozoa/ml with Tris-glucose-egg yolk extender containing one of the following concentrations of NAC (0, 0.5, 1, 2.5 or 5 mm). The semen aliquots were chilled and preserved at 4 degrees C. Semen quality was evaluated after rewarming at 72 h. Sperm motility was significantly higher with the 0.5 mm concentration compared with the control group (p = 0.001). Rapid steady forward movement was higher with the 0.5 and 1 mm concentrations compared with the control and 5 mm group (p < 0.001). Viability and HOST percentages were not significantly altered. Compared with the control, the 5 mm concentration showed significantly reduced percentages of spermatozoa with normal acrosomes (p = 0.049). None of the ROS values at 72 h were significantly affected by the presence of NAC in semen extenders, although all NAC concentrations showed lower O(2)(-*) and OH(*) values compared with the control. Only the concentrations of 1 and 5 mm inhibited the significant increase of tROS values after 72 h, compared with the fresh semen value. In conclusion, NAC supplementation of semen extenders is beneficial to semen motility of canine spermatozoa during chilling with the 0.5 mm concentration being the most effective, although no significant ROS inhibition was observed at 72 h.

Effect of antioxidant supplementation in semen extenders on semen quality and reactive oxygen species of chilled canine spermatozoa.

The objective of this study was to evaluate quality of chilled dog semen processed with extenders containing various antioxidants. Single ejaculates from five dogs were always pooled and evaluated for concentration, sperm motility, progressive motility (RSF-movement), viability, acrosomal integrity and by the hypo-osmotic swelling (HOS)-test. Also, superoxide (O(2)(-)) production, hydroxyl radicals (OH) and total reactive oxygen species (tROS) were determined. Pooled semen was divided in seven aliquots (for control and test conditions), which were diluted to a final concentration of 67x10(6)spermatozoa/ml with TRIS-glucose-egg yolk extender with or without the following supplements: control (without antioxidants), vitamin C (0.5mM), N-acetyl-l-cysteine (NAC; 0.5mM), taurine (0.2mM), catalase (100u/ml), vitamin E (0.1mM) and 5-(4-dimethylamino-phenyl)-2-phenyl-penta-2,4-dienoic acid (B16; 0.1mM). The semen aliquots were chilled and preserved at 4 degrees C. Portions of chilled semen were removed at 24 and 72h, and semen quality was evaluated after rewarming. At 24h the mean (+/-S.E.M.) sperm motility was higher (p<0.001) when vitamin E, taurine and B16 were added in the extender, whereas more spermatozoa with RSF-movement were observed (p<0.001) in the vitamin E, catalase, B16 and taurine groups. Sperm viability was higher (p=0.040) in B16 and vitamin E groups and the percentage of swollen spermatozoa was higher (p=0.002) only in the B16 group. Acrosomal integrity and OH were not significantly influenced by any of the antioxidants tested. Superoxide production was significantly lower when vitamin C, B16 and vitamin E were added in semen extenders compared with the control (p=0.017). All antioxidant groups, except vitamin C and NAC, contained less tROS compared to the control group, but only the B16 group value differed significantly (p=0.05). At 72h sperm motility was higher (p<0.001) when vitamin E, catalase, B16, taurine and NAC were added in the extender. More spermatozoa with RSF-movement were observed (p<0.001) in the vitamin E, catalase, B16, taurine and NAC treatment groups. Sperm viability was higher (p=0.001) when vitamin E, B16, taurine and vitamin C were added in semen extenders. HOS-test percentages were higher (p=0.016) in the B16, vitamin E, catalase and NAC groups. Acrosomal integrity was not influenced in any case. Production of O(2)(-) was significantly higher using catalase compared to all the other groups (p=0.006), while OH was not significantly influenced by any of the antioxidants tested. The addition of vitamin E, catalase and B16 in semen extenders resulted in significantly lower tROS values compared with the controls (p<0.0005). The results suggest that vitamin E and B16 had the most pronounced effect in preserving semen quality of chilled dog spermatozoa.

Quality and reactive oxygen species of extended canine semen after vitamin C supplementation.

The objective of this study was to evaluate the quality of extended dog semen processed with diluents containing various concentrations of vitamin C. Ejaculates from five dogs were collected, pooled and evaluated for concentration, sperm motility, rapid steady forward movement (RSF-movement), viability, acrosomal integrity and by the hypo-osmotic swelling test. Also, superoxide (O(2)(-)*) production, hydroxyl radicals (OH*) and total reactive oxygen species (tROS) were determined. The pool was divided in five aliquots, which were diluted to a final concentration of 66 x 10(6) spermatozoa/ml with a Tris-glucose-egg yolk extender containing one of the following concentrations of vitamin C (0, 0.1, 0.5, 1 or 2.5 mM). The semen aliquots were chilled and preserved at 4 degrees C. Portions of chilled semen were removed at 24 and 72 h, and semen quality was evaluated after rewarming. This process was repeated 10 times in pooled semen of the same origin and data were analysed by one-way analysis of variance. At both times, none of the semen quality parameters were positively influenced (p>0.05) by vitamin C supplementation. At 24 h, none of the reactive oxygen species (O(2)(-)*, OH*, tROS) were significantly altered. At 72 h, significant reductions of O(2)(-)* production were observed by the concentrations of 0.1, 0.5 and 2.5 mM compared with the 0 mM concentration (p=0.049). Also, at 72 h, the 2.5 mM concentration showed significantly lower OH* values in comparison with the control group (p=0.048). In conclusion, addition of vitamin C to semen extenders does not benefit the quality of canine extended spermatozoa.