The cryoprotective potential of propolis supplemented in frozen-thawed bull semen; biochemical and physiological findings.

In this study, the cryoprotective effect of different doses of propolis (P) on bull semen, which has solid pharmacological properties thanks to its rich phenolic components, was investigated biochemically and physiologically. Semen samples were collected from Simmental breed bulls via the artificial vagina and pooled. After dividing into five groups, control (C: no additive) and four different P (200, 100, 50, and 25 µg/mL) groups, the final concentration was diluted to 16×106 per straw. Semen samples were equilibrated at 4°C for approximately 4 hours, then placed in French straws and frozen. After thawing, sperm motility and kinetic parameters, DNA integrity by single-cell gel electrophoresis, sperm abnormalities by liquid fixation, and lipid peroxidation levels by the colorimetric method was analyzed by Computer-Assisted Semen Analyzer. P added to the diluent showed no effect on motility and kinetic parameters at P25 and P50 (p>0.05), while P100 and P200 had a negative effect (p⟨0.001). The addition of P (25 and 50) showed a treatment effect on tail abnormality compared to C (p⟨0.05). Especially P50 had a positive effect on tail length, tail DNA, and tail movement, while P100 and P200 caused DNA damage (p⟨0.001). MDA levels increased in all P dose groups compared to C (p⟨0.001). This study has clearly demonstrated that P25 and P50 supplements could be used therapeutically to treat sperm tail abnormalities and prevent DNA damage in post-thawed bull sperm.

Supplementation of Rosmarinic Acid Has Reduced Oxidative Stress on Bull Spermatozoa Following the Freeze Thawing Process.

BACKGROUND: Cryopreservation has a side effect on the motility, chromatin integrity and viability of sperm cells. OBJECTIVE: The present study investigated the effects of supplementation with rosmarinic acid (RA) Tris extender on sperm quality parameters, plasma and acrosome membrane damage, antioxidant enzyme activity and chromatin integrity following the freeze thawing process on bull spermatozoa. MATERIALS AND METHODS: Ejaculates were split into five aliquots and diluted to a final concentration of 15x106 spermatozoa per ml with the Tris extender containing RA (25, 50, 100 and 200 microgram per ml) and (control) and then frozen at a controlled rate. RESULT: Treatments did not give better results on the percentages of sperm progressive, total motility and sperm motion characters (P >0.05); however, RA25 and RA50 exhibited favourable chromatin integrity. In conclusion, RA25 and RA50 increased total antioxidant activity. As a consequence, the amount of MDA and chromatin damage were reduced in sperm cells.

Antioxidative effects of cysteamine, hyaluronan and fetuin on post-thaw semen quality, DNA integrity and oxidative stress parameters in the Brown Swiss bull.

The aim of this study was to compare the effectiveness of antioxidants including cysteamine (2.5, 7.5 mm), hyaluronan (0.25, 1 mg ml(-1) ) and fetuin (5, 10 mg ml(-1) ) in the freezing of Brown Swiss bull semen. The best percentages of CASA motilities were achieved with 10 mg ml(-1) of fetuin and 2.5 mm of cysteamine. For sperm morphology, 10 mg ml(-1) of fetuin and 2.5 mm of cysteamine had better protective effects (P < 0.001). The results of hypo-osmotic swelling test showed that the percentage values of membrane integrity in all the groups, excluding that supplemented with 5 mg ml(-1) of fetuin, were higher than those of the control group (P < 0.001). Results obtained for the DNA damage of sperm cells demonstrated that 0.25 mg ml(-1) of hyaluronan, and 2.5 and 7.5 mm of cysteamine led to lower rates of spermatozoa with damaged DNA, compared with the control group (P < 0.001). The maintenance of superoxide dismutase and glutathione peroxidase antioxidant activities following freeze-thawing with 2.5 and 7.5 mm of cysteamine and 10 mg ml(-1) of fetuin was demonstrated to be at a higher level in comparison with the control group (P < 0.001). Malondialdehyde formation was found to be lower in the groups supplemented with 0.25 mg ml(-1) of hyaluronan and 7.5 mm of cysteamine after the freeze-thawing process (P < 0.001).

Comparison of cryoprotective effects of lycopene and cysteamine in different cryoprotectants on bull semen and fertility results.

The objectives of this study were to compare glycerol and ethylene glycol at different concentrations as cryoprotectants and lycopene or cysteamine (with/without) as antioxidants in Tris extender for bull semen. Twenty-four ejaculates were obtained from three bulls. Each ejaculate was split into four equal aliquots and diluted using both of the Tris extenders with glycerol (5% or 7%) or ethylene glycol (3% or 5%). After that, each extenders were split into three equal aliquots and added using both of the cysteamine 5 mm or lycopene 500 µg/ml, and control (without additives). The addition of 7% glycerol with cysteamine, 5% ethylene glycol with cysteamine and 3% ethylene glycol with cysteamine groups gave the lowest CASA motility than the other groups. However, 7% glycerol and 7% glycerol with lycopene resulted in a better rate of CASA progressive motility compared with that of other groups. Generally, all the lycopene groups signed better protective effects on acrosome and total morphology than the other groups. Glycerol 7% and 3% ethylene glycol with lycopene groups yielded to slight higher percentages of membrane integrity assessed by HOST than that of the other groups, but 7% glycerol with cysteamine and 3% ethylene glycol with cysteamine showed the worst percentages of membrane integrity. Glycerol 7% and 5% glycerol with lycopene gave rise to a higher value of VAP, VSL and VCL compared with that of the other groups. On the contrary, adding to 5% glycerol with cysteamine showed negative effect for VAP, VSL, VCL and ALH values. All cryoprotectant groups with lycopene decreased chromatin damage than the other groups. Ethylene glycol 3% led to lower non-return rates of inseminated cows. However, this result was not considered to be statistically important.

Effects of curcumin and dithioerythritol on frozen-thawed bovine semen.

The aim of this study was to determine the effects of curcumin and dithioerythritol added into bull semen extender on sperm parameters, lipid peroxidation, total glutathione and antioxidant potential levels of bull spermatozoa following the freeze/thawing process. Twenty-seven ejaculates obtained from three bulls were included in the study. Each ejaculate that was splitted into five equal groups and diluted in a Tris-based extender containing curcumin (0.5 and 2 mM), dithioerythritol (0.5 and 2 mM) and no additive (control) was cooled to 5 °C and frozen in 0.25-ml French straws. The extender supplemented with 0.5 mMdose of curcumin led to lower percentage of total abnormality (20.40 ± 2.36%) when compared to the control (30.60 ± 1.47%, P < 0.05). Curcumin and dithioerythritol at 0.5 mM provided a greater protective effect in the membrane functional integrity (54.40 ± 2.09% and 50.00 ± 2.68%), in comparison with control (37.20 ± 1.77%, P < 0.001). Supplementation with antioxidants did not significantly affect the lipid peroxidation and antioxidant potential levels, while the maintenance of total glutathione levels in curcumin 0.5 mM was demonstrated to be higher than that of control, following the freeze/thawing (P < 0.05). Supplementation with these antioxidants prior to the cryopreservation process may be recommended to facilitate the enhancement of sperm cryopreservation techniques.

Effects of different extenders and centrifugation/washing on postthaw microscopic-oxidative stress parameters and fertilizing ability of Angora buck sperm.

The main objective of the current study was to evaluate the effects of extender type and centrifugation/washing prior to cryopreservation on the postthaw sperm parameters, lipid peroxidation, and superoxide dismutase activity of Angora buck (Capra hircus ancryrensis) sperm. Ejaculates collected from three Angora bucks were used in this study. Two consecutive ejaculates from each buck were pooled and split into equal parts in four Falcon tubes. Two tubes were diluted at 37 degrees C and then centrifuged to remove semen plasma. After centrifugation, two sediment parts were diluted with a Tris-based extender and commercial Bioxcell extender, respectively. The remaining two parts, which were not centrifuged/washed, were diluted with the above-mentioned extenders, respectively. Diluted samples were cooled to 5 degrees C and frozen in 0.25-mL French straws to be stored in liquid nitrogen. Frozen straws were thawed individually at 37 degrees C for 20 sec in a water bath for evaluation. The semen part with centrifugation/washing in the Bioxcell extender (BC) demonstrated a higher rate of subjective motility (58.1+/-3.0%) compared with that of groups with (TC) or without (T) centrifugation/washing in the Tris-based extender (P<0.01). Angora buck sperm frozen with (BC) or without (B) centrifugation/washing in the Bioxcell extender demonstrated higher percentages of motility (60.6+/-2.7% and 54.3+/-4.8%, respectively) compared with that of groups T and TC. The postthaw progressive motility rate (22.3+/-2.7%) was significantly greater for semen parts diluted in B compared with that of other groups. BC gave rise to a lower value of average path velocity (90.0+/-5.2 microm/sec) compared with that of other groups (P<0.01). For straight linear velocity and linearity index, the highest values (103.2+/-4.7 microm/sec, 47.5+/-1.6% and 94.8+/-3.0 microm/sec, 44.8+/-1.1%, respectively) were obtained from B and TC (P<0.001). For sperm acrosome and total abnormalities, TC gave the highest values (11.2+/-0.6% and 26.6+/-1.5%, respectively, P<0.01). In the group frozen in BC, the percentage of membrane integrity assessed by hypo-osmotic swelling test was higher (61.2+/-2.2%) than that of the other groups (P<0.001). With respect to fertility results based on 35-d pregnancy rates, BC gave a higher rate (76.5%) than that of TC (27.8%, P<0.05). Malondialdehyde formation was found to be lower (1.64+/-0.26 nmol/L) in BC than in the other groups after the freeze-thawing process (P<0.001). In the semen part frozen in BC, superoxide dismutase activity was higher (0.18+/-0.02 U/mg protein) compared with that of the other groups (P<0.05). Further studies are required to obtain more precise results for the characterization of oxidative stress parameters and fertilizing ability in cryopreserved buck spermatozoa.