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 ... -  《-》

The effect of antioxidants on motility, viability, acrosome integrity and DNA integrity of frozen-thawed epididymal cat spermatozoa.

Thuwanut P ，Chatdarong K ，Techakumphu M ，Axnér E ... -  《THERIOGENOLOGY》

The effect of Equex STM paste and sperm morphology on post-thaw survival of cat epididymal spermatozoa.

Axnér E ，Hermansson U ，Linde-Forsberg C 《ANIMAL REPRODUCTION SCIENCE》

The cryoprotective effects of ascorbic acid supplementation on bovine semen quality.

To investigate the effects of ascorbic acid supplementation on standard semen quality parameters and antioxidant activities after thawing of bovine frozen semen, antioxidant ascorbic acid was added at concentrations of 2.5, 4.5, 6.5 and 8.5 mg/ml to bovine semen cryoprotective medium. The results showed that the sperm motility and motion characteristics were improved in the presence of ascorbic acid in extender, as compared to the control. The motility and straight linear velocity (VSL), linearity index (LIN), average path velocity (VAP), wobble coefficient (WOB), lateral head displacement (ALH) values and the percentage of "grade A" sperm in the extender supplemented with 4.5 mg/ml ascorbic acid were significantly higher than that of other treatment groups (P<0.05). The acrosome integrity and membrane integrity were significantly improved (P<0.05) by supplementing with 4.5 mg/ml ascorbic acid in the extender compared with a control. The extender supplemented with ascorbic acid did not lead to any improvement in superoxide dismutase (SOD) levels. The catalase (CAT) activity was higher in the extender supplemented with ascorbic acid at 4.5 mg/ml, when compared with other groups (P<0.05) and the extender supplemented with ascorbic acid significantly decreased glutathione peroxidase (GSH-Px) activity, whereas reduced glutathione (GSH) activities were significantly enhanced, compared with the control (P<0.05). Increasing the doses level of ascorbic acid decreased GSH-Px and GSH activity, the supplementation of 8.5 mg/ml ascorbic acid produced the lowest level of GSH-Px and GSH activity among groups (P<0.05). The extender supplemented with ascorbic acid could reduce the oxidative stress provoked by freezing-thawing and improve bovine semen quality. The particular properties of ascorbic acid are poorly related to its effectiveness in membrane cryopreservation. Further studies are required to determine lipid peroxidation and antioxidant capacities of ascorbic acid in cryopreserved bovine semen.

Hu JH ，Tian WQ ，Zhao XL ，Zan LS ，Wang H ，Li QW ，Xin YP ... -  《-》

The effect of antioxidants on the quality of cryopreserved semen in two salmonid fish, the brook trout (Salvelinus fontinalis) and the rainbow trout (Oncorhynchus mykiss).

Until now the supplementation of cryopreservation extenders with antioxidants has not been examined in teleost fish. Therefore, the present study investigated whether addition of antioxidants (catalase, superoxide dismutase, peroxidase, reduced glutathione, reduced methione, mixtures of reduced and oxidized glutathione or methionine) to the cryopreservation extenders could increase the quality of frozen-thawed semen of brook trout, Salvelinus fontinalis, and rainbow trout, Oncorhynchus mykiss. In brook trout and rainbow trout semen post-thaw fertility and motility were evaluated and in brook trout additionally the membrane integrity, DNA integrity, and sperm lipid peroxidation were evaluated. The tested antioxidants affected the motility parameters, DNA integrity, and fertility of cryopreserved semen, but not the membrane integrity. Most of the observed effects were negative and only minor positive effects were found. In brook trout 1.5 mmol/l reduced methionine and a mixture of 1.5 mmol/l oxidized and reduced glutathione increased the swimming velocity of frozen-thawed semen. One hundred U/l catalase, 1.5 mmol/l reduced glutathione, and 1.5 mmol/l reduced methionine slightly, but not statistically significantly increased the semen post-thaw fertility. However, these effects were not detectable in rainbow trout. Antioxidative stress or damage seems to play no role during cryopreservation, as also in the lipid peroxidation test no differences were obtained between fresh and cryopreserved semen. Therefore, for routine cryopreservation extender supplementation with antioxidants is not recommended in brook trout and rainbow trout.

Lahnsteiner F ，Mansour N ，Kunz FA 《-》