The effect of superoxide dismutase mimetic and catalase on the quality of postthawed goat semen.
Manganese(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin chloride (MnTE) is a cell-permeable superoxide dismutase mimetic agent which can convert superoxide to hydrogen peroxide (H2O2). Supplementation of MnTE to a commercial semen extender can protect sperm from superoxide but not H2O2. Therefore, we proposed that addition of catalase (0.0, 200, or 400 IU/mL) in combination with MnTE (0.1 μM) may further improve the cryopreservation efficiency of goat semen in commercially optimized freezing media such as Andromed. Therefore, ejaculates were obtained from three adult bucks twice a week during the breeding season and diluted with Andromed supplemented with or without MnTE and catalase and were frozen in liquid nitrogen. Sperm parameters and reactive oxygen species contents were evaluated 2 hours after dilution (before freezing) and after freezing/thawing. The results revealed that all the treatments significantly (P ≤ 0.05) improved sperm motility, viability, and membrane integrity after freezing and reduced reactive oxygen species content compared with the control group, but maximum improvement was obtained in MnTE + 400 IU/mL catalase. In addition, supplementation with these antioxidants significantly (P ≤ 0.05) increases the cleavage rate after IVF. In conclusion, the results of present study suggest that addition of antioxidant MnTE or catalase to commercial optimized media, such as Andromed, improves total motility, membrane integrity, and viability of goat semen samples after thawing. But the degree of improvement for these parameters significantly (P ≤ 0.05) higher when MnTE and catalase were simultaneously added to the cryopreservation media.
Shafiei M
，Forouzanfar M
，Hosseini SM
，Esfahani MH
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Can permeable super oxide dismutase mimetic agents improve the quality of frozen-thawed ram semen?
This study was carried out to assess the effects of MnTBAP, a cell permeable antioxidant, on motility, membrane integrity, capacitation status and in vitro fertilization ability of frozen-thawed ram semen. Fresh semen ejaculates were collected with artificial vagina from five rams, mixed and divided into five equal fractions, and diluted (1:20 v/v) with commercial extender, Bioxell®, containing 0 (control), 50, 100, 150 and 200 μM of MnTBAP. All diluted sperm suspensions were cooled to 5°C for 2h followed by transfer into 0.5 ml French straws before being stored in liquid nitrogen. The results showed that MnTBAP supplementation of extender improved ram semen quality in a dose-dependent manner. Accordingly, the extender supplemented with 150μM MnTBAP resulted in higher sperm motility and improved acrosomal membrane integrity compared to control. However, further supplementation (200μM) with MnTBAP not only did not improve the results but inversely affected motility and membrane integrity. The results of in vitro fertilization (IVF) indicated that the presence of MnTBAP in semen extender has a marginal beneficial effect on developmental competence of inseminated oocytes, though this improvement was not significant. In conclusion, this study demonstrated that semen extender supplemented with MnTBAP can reduce the oxidative stress provoked by freeze/thaw processes. Moreover, beneficial effect of 100 μM of MnTBAP on preservation of spermatozoa in a non-capacitated state post freezing, an important criterion for in vitro or in vivo fertilization, was observed. However, at 150 μM of MnTBAP, the harmful effects of cryopreservation on membrane integrity were decreased. Regarding to importance of non-capacitated spermatozoa during IVF or artificial insemination, the optimum MnTBAP concentration appears to be 100 μM for commercial ram semen extender tested here.
Forouzanfar M
，Fekri Ershad S
，Hosseini SM
，Hajian M
，Ostad-Hosseini S
，Abid A
，Tavalaee M
，Shahverdi A
，Vosough Dizaji A
，Nasr Esfahani MH
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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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Dietary flax seed oil and/or Vitamin E improve sperm parameters of cloned goats following freezing-thawing.
Semen cryopreservation is affected by individual differences and use of clones animal from the same source is the main tool to eliminate genetic variation. Among many nutrients that are necessary for fertility, essential fatty acids and antioxidants are vital for production of healthy sperm by improving sperm membrane integrity and protecting sperm from oxidative stress. The goal of the current study was to investigate whether a flax seed oil or/and Vitamin E dietary supplementation could improve semen quality of cloned bucks following semen cryopreservation. Accordingly, eight adult cloned Bakhtiari bucks were divided randomly into four groups. Bucks were offered a base diet of hay and concentrate. The concentrate was enriched with flax seed oil, 30 gr/kg body weight/day (OIL), Vitamin E (VIT), 3 gr/kg body weight/day, or combined flax seed oil and the vitamin E (OIL-VIT). The concentrate with no supplements was considered as control group (CONT). Both flax seed oil and Vitamin E supplements were added to the total diet. The bucks were fed with their corresponding diets for a total of 9 weeks while sperm collection was carried out within 10-14 weeks. Ejaculates were diluted with Andromed® and were frozen in liquid nitrogen. Sperm parameters and reactive oxygen species (ROS) contents were evaluated following freezing/thawing. According to the results of our study, dietary supplementation with flax seed oil, or/and Vitamin E can improve sperm motility, vitality and number of sperm with intact plasma membrane following freezing-thawing. But the degree of improvement in these parameters was significantly higher when Flax seed oil and vitamin E were co-supplemented.
Kargar R
，Forouzanfar M
，Ghalamkari G
，Nasr Esfahani MH
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