Improving the effect of incubation and oxidative stress on thawed spermatozoa from red deer by using different antioxidant treatments

P. 856-870Antioxidants could improve sperm media, extending the viability of spermatozoa and protecting their DNA. The protective ability of lipoic acid, melatonin, Trolox and crocin was tested on red deer spermatozoa incubated at 37°C. Cryopreserved spermatozoa were thawed and incubated with 1 mM or 0.1 mM of each antioxidant, with or without oxidative stress (100 μM Fe2+). Motility (CASA), viability, mitochondrial membrane potential and acrosomal status were assessed. Lipoperoxidation (malondialdehyde production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were checked at 4 h. Incubation alone increased ROS and decreased motility. Oxidative stress intensified these effects, increasing lipoperoxidation and DNA damage. Lipoic acid had little protective effect, whereas 1 mM melatonin showed limited protection. Trolox lowered ROS and lipoperoxidation both in oxidised and non-oxidised samples. In oxidised samples, Trolox prevented DNA and acrosomal damage, and ameliorated motility. Crocin at 1 mM showed similar results to Trolox, but noticeably stimulated motility and had no effect on lipoperoxidation. In a second experiment, a broader range of crocin and melatonin concentrations were tested, confirming the effects of crocin (positive effects noticeable at 0.5–0.75 mM), but showing an increase in lipoperoxidation at 2 mM. Melatonin was increasingly effective at 2.5 and 5 mM (ROS, lipoperoxidation and DNA status). Crocin seems a promising new antioxidant, but its particular effects on sperm physiology must be further studied, especially the consequences of motility stimulation and confirming its effect on lipoperoxidation. Melatonin might be useful at relatively high concentrations, compared to Trolox.S

Fertility of cryopreserved ovine semen is determined by sperm velocity

P. 102-109The present study aims to examine the predictive value of some sperm parameters on male fertility. Semen samples from six Manchega rams were collected and cryopreserved. Sperm quality was assessed after thawing and after 2 h of incubation, either in the freezing extender (37 °C) or after dilution in Synthetic Oviductal Fluid (SOF) (38 °C, 5% CO2), attempting to mimic the physiological conditions of the female reproductive tract. The following sperm parameters were evaluated: motility and kinetic parameters by computer-assisted semen analyzer (CASA), and sperm viability (propidium iodide), mitochondrial membrane potential (JC-1), apoptotic-like membrane changes (YO-PRO-1), acrosomal status (PNA-FITC), and intracellular calcium (fluo-3) by flow cytometry. Results showed no significant differences between incubation media neither after thawing nor after incubation. There were no significant correlations between fertility and sperm parameters assessed by flow cytometry. However, after incubation in the freezing extender, sperm samples from males with poor fertility yielded less linearity and velocity (P < 0.05) as indicated by motility parameters analyzed by CASA. These results indicate that kinematic sperm motility parameters evaluation by CASA might be useful to identify samples with poor fertility.S

Response of thawed epidi dymal red deer spermatozoa to increasing concentrations of hydrogen peroxide, and importance of individual male variability

Oxidative stress represents a challenge during sperm manipulation. We have tested the effect of increasing hydrogen peroxide (H2O2) levels on red deer spermatozoa after cryopreservation, and the role of male-to-male variation in that response. In a first experiment, eight thawed samples were submitted to 0, 25, 50, 100 and 200 μM H2O2 for 2 h at 37 °C. Intracellular ROS (H2DCFDA-CM) increased with H2O2 concentration, but we only detected a decrease in sperm function (motility by CASA and chromatin damage by SCSA) with 200 μM. Lipoperoxidation (TBARS) increased slightly with 50 μM H2O2 and above. In a second experiment, samples from 7 males were submitted to 0 and 200 μM H2O2 for 2 h, triplicating the experiment within each male. Males differed at thawing and regarding their response to incubation and H2O2 presence. We found that the kinematic parameters reflected male-to-male variability, whereas the response of the different males was similar for lipid peroxidation and viability. A multiparametric analysis showed that males grouped differently if samples were assessed after thawing, after incubation without H2O2, or after incubation with H2O2. Red deer spermatozoa are relatively resilient to H2O2 after thawing, but it seems to be a great male-to-male variability regarding the response to oxidative stress. The acknowledgement of this individual variability might improve the development of optimized sperm work protocols