The effect of antioxidants on motility, viability, acrosome integrity and DNA integrity of post-thaw epididymal cat spermatozoa

Living cells stored under aerobic conditions require oxygen (O2) to support their metabolism. Their metabolite products are reactive oxygen species (ROS). In general, the ROS need to be continuously inactivated to keep a minimal amount necessary to maintain normal cell function but an excessive accumulation can cause cell damage. In addition, ROS can be produced and accumulated during the freezing and thawing process of mammalian spermatozoa. However, an adverse effect of ROS can be reduced by antioxidants. The aim of this study was to investigate effects of antioxidant (cysteine or water soluble vitamin E analogue Trolox) supplementation of a tris egg yolk extender on post-thaw epididymal cat spermatozoa. Epididymal spermatozoa were collected from eight male cats. The sperm sample from each cat was divided into three aliquots, resuspended with (1) tris egg yolk extender I (EE-I), (2) tris egg yolk extender I with cysteine (EE-C) or (3) tris egg yolk extender I with vitamin E (EE-Ve) and extended with tris egg yolk extender with Equex STM paste (EE-II) for freezing. Sperm motility, progressive motility, membrane integrity stained with SYBR-14/EthD-1 and acrosome status stained with FITC-PNA/PI were evaluated after collection, prior to freezing at 4 ºC and 0, 2, 4, and 6 h post-thaw. Acridine orange was used to evaluate DNA integrity at 0 and 6 h post-thaw. Vitamin E supplementation had positive effects on post-thaw motility, progressive motility and membrane integrity (P0.05). These results demonstrated that cysteine and vitamin E supplemented to the egg yolk tris extender can improve post-thaw epididymal cat spermatozoa qualities such as motility, progressive motility, membrane integrity and DNA integrity but not acrosome integrity. In conclusion, when the tris egg yolk extender containing Equex STM paste is used, the addition of cysteine or vitamin E is recommended in order to protect post-thaw epididymal cat spermatozoa from ROS-induced sperm damage

Role of oxidative stress and antioxidants in domestic and non-domestic cat spermatozoa

Living cells, including spermatozoa, stored under aerobic conditions require oxygen to support their normal metabolism. Excessive levels of metabolites (reactive oxygen species; ROS) can cause cell damage or lipid peroxidation. The ROS and lipid peroxidation can be detected during preparation and cryopreservation of mammalian spermatozoa. The negative effects of ROS can be partially ameliorated by the action of antioxidants. The aims of this thesis were to 1) study the effects of antioxidants on cryopreserved epididymal spermatozoa from the domestic cats and to apply the results from domestic cats to cryopreserved wild felid ejaculated spermatozoa (flat-headed cats; Prionailurus planiceps) in Thailand; 2) detect lipid peroxidation and its effects on post-thaw epididymal cat spermatozoa and 3) evaluate basic seminal characteristics of captive flat-headed cats. In the first study, the semen extender was supplemented with two types of non-enzymatic antioxidants (cysteine and a water soluble vitamin E analogue Trolox). Vitamin E had positive effects on post-thaw sperm motility, progressive motility and membrane integrity. In the second study, centrifuged post-thaw epididymal cat spermatozoa were resuspended in media with or without egg yolk and with or without a transition metal (Fe2+) (which induces lipid peroxidation). Lipid peroxidation was measured by using a lipophilic dye probe (BODIPY581/591 C11). The results demonstrated that BODIPY581/591 C11 could be used to detect lipid peroxidation in cat spermatozoa. This reaction did not significantly increase during incubation unless induced by Fe2+. In the third study, enzymatic antioxidants (catalase, glutathione peroxidase; GPx and superoxide dismutase) were added to the semen extender. Lipid peroxidation was induced after thawing. Supplementation of GPx resulted in positive effects on post-thaw motility, linear motility, mitochondrial activity, membrane and DNA integrity. Although antioxidants had positive effects on sperm quality, the concentration of antioxidants used in this study did not reduce lipid peroxidation. In the last study, the basic seminal traits of captive flat-headed cats were evaluated, as were the effects of adding the vitamin E and GPx to the semen extender for flat-headed cat spermatozoa. The results demonstrated that the captive flat-headed cats were affected by teratozoospermia. The GPx had positive effects on the post-thaw quality of flat-headed cat spermatozoa in a similar manner to domestic cat sperm quality but it did not significantly affect in vtiro heterologous fertility tests. In conclusion, oxidative stress might be one of the factors causing damage during preservation of domestic and non-domestic cat spermatozoa. Post-thaw sperm quality was enhanced by the action of antioxidants