Kenan Çoyan

Effects of antioxidants on post-thawed bovine sperm and oxidative stress parameters: antioxidants protect DNA integrity against cryodamage.

This study was conducted to determine the effects of methionine, inositol and carnitine on sperm (motility, abnormality, DNA integrity and in vivo fertility) and oxidative stress parameters (lipid peroxidation, total glutathione and antioxidant potential levels) of bovine semen after the freeze-thawing process. Nine ejaculates, collected with the aid of an artificial vagina twice a week from each Simmental bovine, were included in the study. Each ejaculate, splitted into seven equal groups and diluted in Tris-based extender containing methionine (2.5 and 7.5 mM), carnitine (2.5 and 7.5 mM), inositol (2.5 and 7.5 mM) and no additive (control), was cooled to 5 °C and then frozen in 0.25 ml straws. Frozen straws were then thawed individually at 37 °C for 20s in a water bath for the evaluation. The extender supplemented with 7.5 mM doses of carnitine and inositol led to higher subjective motility percentages (61.9±1.3% and 51.3±1.6%) compared to the other groups. The addition of methionine and carnitine at doses of 2.5 and 7.5 mM and inositol at doses of 7.5mM provided a greater protective effect in the percentages of total abnormality in comparison to the control and inositol 2.5 mM (P < 0.001). As regards CASA motility, 7.5 mM carnitine (41.6±2.9% and 54.2±4.9%) and inositol (34.9±2.0% and 47.3±2.2%) caused insignificant increases in CASA and total motility in comparison to the other groups. All of the antioxidants at 2.5 and 7.5 mM resulted in lower sperm with damaged DNA than that of control, thus reducing the DNA damage (P < 0.05). No significant differences were observed in CASA progressive motility and sperm motion characteristics among the groups. In fertility results based on 59-day non-returns, no significant differences were observed in non-return rates among groups. As regards biochemical parameters, supplementation with antioxidants did not significantly affect LPO and total GSH levels in comparison to the control group (P > 0.05). The maintenance of AOP level in methionine 2.5 mM was demonstrated to be higher (5.06±0.38 mM) than that of control (0.96±0.29 mM) following the freeze-thawing (P < 0.001). Supplementation with these antioxidants prior to the cryopreservation process protected the DNA integrity against the cryodamage. Furthermore, future research should focus on the molecular mechanisms of the antioxidative effects of the antioxidants methionine, carnitine and inositol during cryopreservation.

Effects of cysteine and ergothioneine on post-thawed Merino ram sperm and biochemical parameters

The aim of the current study was to evaluate the effects of cysteine and ergothioneine on the post-thawed sperm parameters, lipid peroxidation and antioxidant activities. Semen samples from 5 mature Merino rams were used in the study. Semen samples, which were diluted with a Tris-based extender containing l-Cysteine and l-(+)-Ergothioneine and no antioxidant (control), were cooled to 5°C and frozen in 0.25 ml French straws. Frozen straws were then thawed individually at 37°C for 20s in a water bath for evaluation. Ergothioneine at doses of 2 and 4mM increased percentages of subjective motility, VSL and VCL, compared to controls following the freeze-thawing (P<0.001). Ergothioneine at three different doses led to higher rates of progressive motility and VAP, compared to control groups (P<0.001). Cysteine and ergothioneine at three doses provided the higher rates of ALH, in comparison to no antioxidant group (P<0.001). As regards CASA motility, supplementation with antioxidants did not provide any significant difference on the percentage of post-thaw sperm CASA motilities, in comparison to the control. In regards of sperm membrane integrity, only cysteine 1mM provided a greater protective effect, compared to control (P<0.001). Percentages of sperm with high mitochondrial activity were dramatically increased with cysteine at doses of 1 and 2mM, compared to control (P<0.05). No significant differences were observed in sperm acrosome integrities among groups. CAT activity was increased significantly only in cysteine1mM compared to control group (P<0.001). Cysteine at doses of 2 and 4mM showed a tendency of increased activities of CAT when compared to control. But these increases were not statistically significant. Supplementation with antioxidants did not significantly affect activities of SOD and GPx. Findings of this study showed that ergothioneine supplementation in semen extenders, was of greater benefit to motility and motion characteristics of frozen-thawed ram sperm.

Effects of hypotaurine, cysteamine and aminoacids solution on post-thaw microscopic and oxidative stress parameters of Angora goat semen.

This study was conducted to determine the effects of cysteamine, hypotaurine and aminoacids solution (BME) on standard semen parameters, lipid peroxidation and antioxidant activities of Angora goat semen after the freeze-thawing process. Ejaculates collected from four Angora goats were evaluated and pooled at 37 degrees C. Semen samples, which were diluted with a Tris-based extender containing the antioxidants hypotaurine (5 mM) and cysteamine (5 mM), and an aminoacid solution (13%), and an extender containing no antioxidants (control), were cooled to 5 degrees C and frozen in 0.25-ml French straws in liquid nitrogen. Frozen straws were thawed individually at 37 degrees C for 20s in a water bath for evaluation. Supplementation with cysteamine, hypotaurine and BME caused significant (P<0.05) increases in sperm motility, and significant (P<0.05) decreases in total abnormality rates in comparison to the control group. While all in vitro treatments did not affect the acrosomal abnormality rates, hypotaurine and BME but not cysteamine significantly (P<0.05) increased the HOST results as compared to the control group. Supplementation with antioxidants and BME did not significantly affect MDA levels and CAT activity in comparison to the control group (P>0.05). The antioxidants hypotaurine and cysteamine decreased SOD activity when compared to the BME group and controls (P<0.001).

Influence of methionine and dithioerythritol on sperm motility, lipid peroxidation and antioxidant capacities during liquid storage of ram semen.

The aim of this study was to investigate the effects of methionine and dithioerythritol, added to the Tris extender, on ram sperm motility and LPO (lipid peroxidation) and antioxidant capacities during liquid storage up to 72 h at 5°C. Ejaculates collected from five Merino rams, were evaluated and pooled at 37°C. This study included two experiments. In experiment 1, each pooled ejaculate was divided into four equal aliquots and diluted (37°C) with the base extender, containing 0 (control), 1, 2 and 4 mM methionine, at a final concentration of approximately 4×10(8)sperms/ml (single step dilution), in a 15-ml plastic centrifuge tube. In experiment 2, dithioerythritol, at concentrations of 0 (control), 0.5, 1 and 2 mM, was used as an additive in the extender, and the procedure explained above was applied for the division of aliquots and the dilution of semen. Diluted semen samples were kept in glass tubes and cooled from 37 to 5°C in a cold cabinet, and maintained at 5°C. Sperm motility and LPO and total glutathione (GSH) and glutathione peroxidase (GPx) capacities were determined at 5°C for periods of 0, 24, 48 and 72 h of liquid storage. The extender supplemented with 1 mM methionine led to higher motility percentages (77.0±1.2%), in comparison to the control group (66.0±4.9%), during 72 h of liquid storage (P<0.05). As regards dithioerythritol, it did not statistically improve the motility rates for any of the storage times at 5°C. In biochemical assays, differences in LPO levels between the groups with antioxidants and the control groups were not statistically significant. Compared to the control group, no significant difference was observed in GSH and GPx activities following the addition of methionine, during 72 h of storage. Total GSH and GPx activities did not increase significantly upon supplementation with 0.5 and 1 mM of dithioerythritol, compared to the control group, at any of the time points (P>0.05). Dithioerythritol at 2 mM led (P<0.01) to elevating GSH activity, compared to the control group, during 72 h of liquid storage. GPx activity was approximately 10 times higher for 2 mM of dithioerythritol (P<0.001), compared to that of the control group at all time points. The question regarding the sustainability of sperm survival, LPO and antioxidant capacities following liquid storage of semen remains unanswered. Further studies are required for a better understanding of the biochemical changes and to obtain more information on the determination of lipid peroxidation and antioxidant capacities during cooled storage of ram semen.

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 mm dose 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 dithioerythritol on ram semen after the freeze-thawing process.

The aim of this study was to evaluate the effects of dithioerythritol added to cryopreservation extender on the post-thawed sperm parameters, lipid peroxidation and antioxidant activities of Merino ram sperm. Semen samples from 5 mature Merino rams (1 and 2 years of age) were used in the study. Semen samples, which were diluted with a Tris-based extender containing 0.5, 1, and 2mM dithiothreitol and no antioxidant (control), were cooled to 5°C and frozen in 0.25 ml French straws. Frozen straws were then thawed individually at 37°C for 20s in a water bath for evaluation. The addition of dithioerythritol at 0.5 and 2mM doses led to higher percentages of subjective motility (62.9±4.2% and 63.6±1.8%) compared to control (52.0±4.9%, P<0.05). As regards CASA motility, dithioerythritol 0.25 and 2 mM (60.2±4.5% and 59.6±1.2%) groups were higher from that of control (44.2±8.7%, P<0.05). For the CASA progressive motility, 0.25, 0.5 and 2 mM doses of dithioerythritol (22.0±2.1%, 21.7±2.5% and 24.0±1.2%) had increasing effect in comparison to control (15.0±2.5%). Dithioerythritol at 1 and 2 mM doses for ALH provided higher values compared to the control (P<0.001) following the freeze-thawing process. Supplementation with dithiothreitol did not significantly affect the integrities of sperm membrane and acrosome, and mitochondrial activities. No significant differences were observed in biochemical parameters among the groups (P>0.05). Findings of this study showed that dithioerythritol supplementation in semen extenders, was of greater benefit to sperm motility of frozen-thawed ram sperm.