Pürhan Barbaros Tuncer

The influence of cysteine and taurine on microscopic-oxidative stress parameters and fertilizing ability of bull semen following cryopreservation.

Oxidative stress significantly damages sperm functions such as motility, functional integrity, endogenous antioxidant enzyme activities and fertility due to lipid peroxidation induced by reactive oxygen species (ROS). The aim of this study was to determine the effects of antioxidants such as taurine and cysteine in Bioxcell extender on standard semen parameters, fertilizing ability, lipid peroxidation (LPO) and antioxidant activities comprising reduced glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD) after the cryopreservation/thawing of bull semen. Nine ejaculates for each bull were included in the study. Three groups, namely taurine (2mM), cysteine (2mM), and control, were designed to analyze the antioxidants in Bioxcell. Insemination doses were processed so that each 0.25-ml straw contained 15 x 10(6) sperm. The addition of cysteine led to higher motility, compared to the other groups (P<0.001). Cysteine showed a greater protective effect on the percentages of acrosome damage and total abnormalities in comparison to the other groups (P<0.001). No significant differences were observed in hypo-osmotic swelling test (HOST), following supplementation with antioxidants during the freeze-thawing process. No significant difference was observed in non-return rates among groups. In biochemical assays, the additives did not show effectiveness on the elimination of malondialdehyde (MDA) formation and maintenance of GSH and GSH-Px activities, when compared to controls. CAT activity (35.1+/-8.1 kU/g) was demonstrated to be significantly higher upon the addition of 2mM taurine (P<0.001), while the level of MDA increased, indicating oxidative stress in this group. SOD activity (21.4+/-2.9 U/g protein) was significantly elevated in the group with cysteine, compared to the other groups (P<0.001).

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.

The effect of cysteine and glutathione on sperm and oxidative stress parameters of post-thawed bull semen.

The aim of this study was to determine the effects of antioxidants such as reduced glutathione (GSH) and cysteine in Laiciphose® extender on semen parameters, fertilizing ability, lipid peroxidation (LPO) level and glutathione peroxidise (GPx) activity of post-thawed bull semen. Totally 54 ejaculates of three bulls were used in the study. Five groups, namely; GSH (0.5 and 2 mM), cysteine (5 and 10 mM) and control group, were conducted to test the antioxidants in Laiciphose®. Insemination doses were processed that each 0.25-mL straw contained 15 x 10⁶ sperm. The addition of antioxidants did not present any significant effect on the percentages of post-thaw sperm morphology (acrosome and total abnormalities), subjective, CASA and progressive motilities, as well as sperm motility characteristics (VAP, VSL, VCL, LIN and ALH), compared to the control groups (P > 0.05). GSH 0.5mM (55.5±7.38%) and cysteine 10 mM (48±5.65%) led to lower rates of DNA damage, compared to control (P < 0.05). As regards to MDA level, cysteine at 10 mM dose gave the highest level (4.99±0.44 nmol/L) (P < 0.001). GPx activity was demonstrated to be higher level upon the addition of 5 mM cysteine when compared to the other groups (P < 0.05). With respect to fertility results based on 60-day non-returns, the supplementation of antioxidants did not present significant differences (P > 0.05). The results of this study may provide an useful information for the future studies in this area. So, further studies could be suggested to achieve better information in terms of the DNA damage and fertilizing capacity of bull sperm frozen with effective antioxidants.

Effects of various cryoprotectants on bull sperm quality, DNA integrity and oxidative stress parameters ☆

The objectives of this study was to compare the effects of type and concentration of cryoprotectants glycerol (G), ethylene glycol (EG) and dimethyl sulfoxide (DMSO) on the plasma membrane and DNA integrity as well as antioxidant activity of cryopreserved Eastern Anatolian red bull sperm. Ejaculates were collected from the three bulls using an artificial vagina twice a week. The ejaculates were pooled to increase the semen volume for replication and to eliminate variability among the evaluated samples. The pooled ejaculates were also split into seven equal experimental groups and diluted with the modified base extender to a final spermatozoa concentration of 15×10(6)/ml. The extended samples were cooled slowly to 4°C and equilibrated for 4h. They were then loaded into 0.25ml French straws and frozen using a digital freezing machine at 3 programmed rates: -3°C/min from +4°C to -10°C, -40°C/min from -10°C to -100°C, and -20°C/min from -100°C to -140°C. Thereafter, the straws were plunged into liquid nitrogen at -196°C. Frozen straws were thawed individually at 37°C for 30s in a water bath to analyse progressive motility and sperm motion characteristics as well as membrane integrity using hypo-osmotic swelling test. Biochemical assays were performed in a spectrophotometer using commercial kits. DNA damage was evaluated by Comet Assay using Image Analysis System. 6% G exhibited the greatest percentages of CASA (43.7±2.92%) and progressive (26.4±2.64%) motilities when compared to the other groups (P<0.001). 6% G and 6% EG showed the greatest values of preserved membrane integrity (P<0.001). 6% DMSO and 3% EG + 3% DMSO resulted in greater chromatin damage than the other groups (P<0.001). The antioxidant activities of GPx, GSH, and CAT as well as the total antioxidant activity were affected by the type of cryoprotectant; notably, 2% G+2% EG+2% DMSO yielded the lowest activities when compared to the other groups (P<0.001). In conclusion, no advantages were found in using EG or DMSO to replace G in bull sperm cryopreservation. Freezing with cryoprotectant 6% G yielded the best post-thaw sperm characteristics for Eastern Anatolian Red bull spermatozoa.

The effect of raffinose and methionine on frozen/thawed Angora buck (Capra hircus ancryrensis) semen quality, lipid peroxidation and antioxidant enzyme activities.

The aim of the present study was to determine the effects of different doses of raffinose and methionine on post-thawed semen quality, lipid peroxidation and antioxidant enzyme activities of Angora buck (Capra hircus ancryrensis) sperm following cryopreservation. Ejaculates collected from three Angora bucks were evaluated and pooled at 37 degrees C. Semen samples, which were diluted with a Tris-based extender containing the additives raffinose (2.5, 5, 10mM) and methionine (2.5, 5, 10mM) and an extender containing no antioxidants (control), were cooled to 5 degrees C and frozen in 0.25 ml French straws. Frozen straws were thawed individually at 37 degrees C for 20s in a water bath for evaluation. The freezing extender supplemented with 2.5 and 5mM methionine led to higher percentages of CASA motility (63.6+/-7.0; 63.4+/-3.1%, respectively), in comparison to the controls (P<0.01) following the freeze-thawing process. The addition of antioxidants did not provide any significant effect on the percentages of post-thaw subjective and CASA progressive motilities as well as sperm motion characteristics (VSL and VCL), compared to the control groups (P>0.05). The freezing extender with raffinose (5 and 10mM) and methionine at three different doses (2.5, 5 and 10mM) led to lower percentages of acrosome abnormalities, in comparison to the controls (P<0.001). In the comet test, raffinose (5 and 10mM) and methionine (10mM) gave scores lower than those of the controls, and thereby reduced DNA damage (P<0.05). Malondialdehyde formation was found to be lower (1.8+/-0.1 nmol/L) in the group of 5mM raffinose, compared to the controls following the freeze-thawing process (P<0.01). The additives did not show any effectiveness on the maintenance of SOD, GSH-PX and GSH activities, when compared to the controls (P>0.05). In conclusion, methionine and raffinose play a cryoprotective role against sperm CASA motility, acrosome abnormality and DNA damage. Raffinose 5mM exhibited antioxidative properties, decreasing MDA levels. Further studies are required to obtain more concrete results on the characterization of microscopic parameters and antioxidant activities in cryopreserved goat sperm with different additives.

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.

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).

Effect of glutamine and sugars after bull spermatozoa cryopreservation.

The objective of this study was to evaluate the effects of the addition of different sugars (raffinose, sucrose, and trehalose) on bull spermatozoa cryopreserved in a commercial extender (Optidyl) supplemented with glutamine on semen parameters, fertilizing ability and superoxide dismutase (SOD) activity. Nine ejaculates for each bull were used in the study. Semen was frozen in five different extenders: raffinose 25 mM plus glutamine 3 mM (RGO), sucrose 25 mM plus glutamine 3 mM (SGO), trehalose 25 mM plus glutamine 3 mM (TGO), glutamine 3 mM (GO) and control (O). Insemination doses were processed so that each 0.25 mL straw contained 15 x 10(6)sperm. Groups of GO and RGO resulted in the higher rates of subjective (54.0 ± 1.7% and 64.0 ± 1.1%; P < 0.01) and CASA motilities (53.0 ± 2.7% and 61.0 ± 4.4%; P < 0.001), respectively compared to the other groups. The supplementation of additives did not provide an effect on the level of post-thaw sperm CASA progressive motilities, the sperm motion characteristics and pregnancy rates. GO and RGO provided the better protective effect for sperm acrosome (4.0 ± 0.5% and 12.0 ± 0.6%) and total abnormalities (5.0 ± 0.3% and 13.0 ± 0.7%; P < 0.001), respectively. At the HOST values, the additives did not give to result the protective effect in comparison to Optydil extender without additives (P > 0.05). For pregnancy rates, there were no significant differences among the groups. The supplementation of additives did not provide any significant difference on the level of SOD activity (P > 0.05). It can be also thought that these sugars might have worked with glutamine in a synergy. Thereby, sugars such as raffinose and sucrose with glutamine in freezing extender may be recommended to facilitate bull semen freezability.

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.

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).