Epsilon Archive for Student Projects

Abstract

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 (P<0.05), while cysteine supplementation improved post-thaw motility and DNA integrity (P<0.05). However, antioxidant supplementation had no significant positive effect on post-thaw acrosome integrity (P>0.05). 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.