Induced sub-lethal oxidative damage affects osmotic tolerance and cryosurvival of spermatozoa.

If the physiological balance between production and scavenging of reactive oxygen species (ROS) is shifted towards production of ROS this may result in accumulation of cell damage over time. In this study stallion spermatozoa were incubated with xanthine and xanthine oxidase (X-XO) to artificially generate defined levels of superoxide and hydrogen peroxide resulting in sub-lethal oxidative damage. The effects of X-XO treatment on various sperm characteristics were studied. Special emphasis was placed on sperm osmotic tolerance pre-freeze and its correlation with cryosurvival, given that cryopreservation exposes cells to osmotic stress. ROS accumulation occurred predominantly in the sperm midpiece region, where the mitochondria are located. Exposing spermatozoa to increasing X-XO concentrations resulted in a dose-dependent decrease in sperm motility. Percentages of plasma membrane-intact spermatozoa were not affected, whereas stability of membranes towards hypotonic stress decreased with increasing levels of induced oxidative stress. Infrared spectroscopic studies showed that X-XO treatment does not alter sperm membrane phase behaviour. Spermatozoa exposed to higher oxidative stress levels pre-freeze exhibited reduced cryosurvival. Centrifugation processing and addition of catalase were found to have little beneficial effect. Taken together, these results show that treatment of spermatozoa with X-XO resulted in different levels of intracellular ROS, which decreased sperm osmotic tolerance and cryosurvival.

Equine endometrial vascular pattern changes during the estrous cycle examined by Narrow Band Imaging hysteroscopy.

The aim of this study was to evaluate the uterine blood supply and endometrial vessel architecture, during the equine estrous cycle. Narrow Band Imaging (NBI) hysteroscopy was used for evaluating changes in the endometrial vasculature during the estrous cycle [six mares, d 0 (representing the day of ovulation), d 6 and 11 in four locations]. In addition, endometrial biopsy samples were used for immunodetection of markers for angiogenesis (Vascular Endothelial Growth Factor A, its receptor 2, as well as angiopoietin-2 and its receptor-tyrosine-kinase Tie2) during the estrous cycle (three mares, d 0, 5 and 10; one biopsy per mare). Detailed analysis of hysteroscopic images revealed an increase in the vascular density from estrus towards diestrus. In contrast, microscopic specimens prepared from biopsies revealed no evidence for changes in the endometrial vessel number during the estrous cycle. Studies on expression of angiogenesis markers indicated that cyclic changes in the endometrial vascular density observed by NBI-hysteroscopy were not due to formation of new vessels. It is concluded that vessels are involved in blood supply of a smaller area during diestrus, facilitating better distribution of nutrients during this phase.

Identification of vessel degeneration and endometrosis in the equine endometrium, using narrow-band imaging hysteroscopy.

In this study, endometrosis and angiosclerosis in mares were studied. Endometrosis is a severe, progressive, and irreversible fibrotic condition that affects the endometrium, whereas angiosclerosis refers to thickening of vessel walls due to degenerative changes leading to reduced elasticity of the walls and lower perfusion. Histologic evaluations were performed on biopsies and compared with vascular features of the endometrial surface obtained via narrow-band imaging (NBI) hysteroscopy. First, it was determined if hysteroscopic evaluation of the endometrium using NBI resulted in a better visualization of the vascular pattern (i.e., vessel-versus-background contrast was increased) compared with using white light. This was found to be the case for examinations in vivo (n = 10), but not when using abattoir uteri (n = 3). In the second part of this study, it was determined if vascular densities and sizes as derived from NBI images could be used as indicators for the degree of degenerative changes of the equine endometrium and its vessels. Narrow-band imaging hysteroscopic evaluations were performed (n = 10), and endometrial biopsies (n = 32) were collected. Histologic specimens were evaluated for degree of endometrosis and angiosclerosis, and they were classified in Kenney categories. Narrow-band imaging images were analyzed for vascular pattern. Samples classified to Kenney category I, or without signs of vessel degeneration, had significantly higher vascular densities than samples from Kenney category IIa or with angiosclerosis. In conclusion, narrow-band imaging facilitates enhanced visualization of the vasculature of the equine endometrium during hysteroscopies, which has applications in detection of endometrosis and angiosclerosis.