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.
Coyan K
，Başpınar N
，Bucak MN
，Akalın PP
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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.
Başpınar N
，Coyan K
，Bucak MN
，Tuncer PB
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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.
Tuncer PB
，Bucak MN
，Sariözkan S
，Sakin F
，Yeni D
，Ciğerci IH
，Ateşşahin A
，Avdatek F
，Gündoğan M
，Büyükleblebici O
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Cryopreservation of epididymal cat spermatozoa: effects of in vitro antioxidative enzymes supplementation and lipid peroxidation induction.
Reactive oxygen species and lipid peroxidation reaction, causes of sperm damage, can be diminished by action of antioxidative enzymes. This study aimed to investigate effects of (1) the antioxidative enzymes; catalase, glutathione peroxidase and superoxide dismutase, on epipididymal cat sperm quality and (2) the lipid peroxidation reaction induced by a transition metal (ferrous ion (II); Fe(2+)) on sperm quality during the cryopreservation process. Epididymal spermatozoa harvested from 39 male cats were pooled and divided into 13 aliquots (n=13). Each aliquot was resuspended with either a Tris egg yolk extender I (control; EE-I), or the Tris egg yolk extender I supplemented with 200 U/mL catalase (EE-CAT), or 10 U/mL glutathione peroxidase (EE-GPx), or 600 U/mL superoxide dismutase (EE-SOD), and then cryopreserved. After thawing, each sperm sample was subdivided into two groups; with and without lipid peroxidation induction (EE-I plus Fe(2+), EE-CAT plus Fe(2+), EE-GPx plus Fe(2+) and EE-SOD plus Fe(2+)). Subjective sperm motility, membrane, and acrosome integrity were evaluated at the time of collection, after cooling, and at 0, 2, 4, and 6h after thawing. Motility patterns assessed by computer-assisted sperm analysis (CASA), mitochondrial activity, and DNA integrity were evaluated during post-thaw incubation, whereas percentage of lipid peroxidation was detected at 0 and 6h after thawing. The results demonstrate that catalase supplementation reduced linear motility and subjective motility immediately and 2h after thawing (P<0.05). Catalase supplementation, however, improved DNA integrity at 4h (P<0.05). Supplementation with glutathione peroxidase, compared to the control group, had a statistically significant positive effect on subjective motility at 0 and 6h, linear motility at 6h, mitochondrial activity at 6h, membrane integrity at 2 and 6h, and DNA integrity at 4h after thawing. Although superoxide dismutase had a positive effect on sperm membrane integrity at 2h after thawing (P<0.05), it significantly reduced membrane integrity after cooling, linear motility at thawing, and acrosome integrity at 2h after thawing. None of the three selected antioxidative enzymes significantly influenced acrosome integrity and none reduced the level of lipid peroxidation. Furthermore, induction of the lipid peroxidation reaction by Fe(2+) negatively affected most of the sperm quality parameters, i.e., motility and DNA integrity, during post-thaw sperm incubation (P<0.05). After thawing, there were, however, no significant differences between the control plus Fe(2+) and the antioxidative enzymes supplementation plus Fe(2+) groups. We can conclude that (1) glutathione peroxidase exhibits positive effects on post-thaw epididymal cat spermatozoa; but (2) none among the selected antioxidative enzymes could improve all sperm quality parameters; and (3) the lipid peroxidation reaction may be one cause of post-thaw epididymal sperm damage in cats, but the concentrations of antioxidative enzymes used in this study could not protect cat spermatozoa from lipid peroxidation induction.
Thuwanut P
，Chatdarong K
，Johannisson A
，Bergqvist AS
，Söderquist L
，Axnér E
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ResearchGate
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