Therefore, this study was conducted to investigate plasma oxidant (malondialdehyde [MDA]) and antioxidant (glutathione [GSH], ß-carotene, vitamin E, glutathione peroxidase [GSH-Px], catalase [CAT]) status in mares with uterine cysts. For this purpose, blood samples were collected from 10 mares with uterine lymphatic cyst and 8 healthy Arabian mares. The age of the mares varied from 15 to 20 years.

There were no significant differences in the concentrations of vitamin E, GSH, MDA and activity of CAT and GSH-Px between groups. However, the concentration of plasma ß-carotene in the mares with uterine cyst was significantly lower (P<0.01) than in the healthy mares.

These results suggested that lower ß-carotene concentration may be one of factors which play role in the pathogenesis of uterine lymphatic cyst.

Cysts are characterized as either glandular or lymphatic. Lymphatic cysts arise from obstructed lymphatic channels and appear more commonly in multiparous mares with uterus that have undergone fibrotic changes associated with mixed endometrial disorders ². The incidence of cysts increases with age and most affected mares are >10 years. The incidence of endometrial cysts in subfertile and older mares has been reported to be 55% ³. Cysts may affect vesicle fixation, conceptus mobility, and placentation. Improper implantation adjoining a cyst can result in inadequate blood flow and nutrient provision for the conceptus, leading to early embryonic loss. Multiple or large cysts may reduce placental exchange leading to inadequate placentation and potentially abortion ⁴. This suggests that the presence of uterine cysts may play an important role in the reduction of fertility of mares. Reactive oxygen species (ROS), which are produced as by products of normal metabolism, are capable of causing cellular damage, leading to cell death, and tissue injury. In organisms, extensive antioxidant defense system includes natural molecules such as urate, cysteine, vitamin E, vitamin C, β protective mechanisms have evolved to prevent ROS damage.-carotene, and enzymes including superoxide dismutase, CAT and GSH-Px ⁵. Oxidative stress has been defined as a disturbance in the prooxidant/ antioxidant balance, resulting in several pathological processes such as inflammation, carcinogenesis, and ischaemia-reperfusion ⁶. ROS also play a number of significant, diverse roles in female reproductive biology including uterine environment, oocyte maturation and ovulation, corpus luteum function and regression ⁷. Accumulated evidence suggests that ROS have been associated with disorders of the reproductive system such as cervicitis, uterine myoma ⁸ and endometritis ^9,10.

Although uterine cysts are a contributing factor to subfertility in the mare, to our knowledge, there are no data concerning the effects of blood oxidant and antioxidant status in mares with lymphatic uterine cysts. Therefore, the present investigation was undertaken to compare plasma antioxidant status in normal mares and mares with uterine lymphatic cyst.

Lipid peroxidation (MDA) level in plasma was estimated according to the method of Placer et al. ¹² modified by Matkovics et al. ¹³ by monitoring thiobarbituric acid-reactive substances (TBARS). The quantification of TBRAS was determined by comparing the absorption to the standard curve of malondialdehyde equvalents generated by 1,1,3,3 tetramethoxypropane at 532 nm with spectrophotometer (Jainway 6100, UK). Vitamin E level was determined according to the method of Desai ¹⁴.

The plasma samples were saponified by the addition of KOH and 1% ascorbic acid, followed by heating at 70ºC for 30 min. After cooling the samples on ice, water and n-hexane were added, and then placed aside for 30 min to allow phase separation. The n-hexane extract was dried under nitrogen and redissolved by using methanol. Methanol extracts were measured at 536 nm after addition bathophenanthroline-ortho-phosphoric acid reagent. The levels of β-carotene in plasma samples were determined according to the method of Suzuki and Katoh ¹⁵. Two milliliters of n-hexane were mixed with 0.25 ml plasma and absorbance of hexane was measured at 453 nm in the spectrophotometer.

The GSH content in plasma was measured by using the method of Sedlak and Lindsay ¹⁶. The samples were precipitated with trichloracetic acid and then centrifuged at 1000 g for 5 min. The reaction mixture including supernatant, tris buffer (pH 8.9) and 5,5’dithiobis- 2-nitrobenzoic acid were kept at room temperature for 5 min, and read at 412 nm on the spectrophotometer.

GSH-Px activity of plasma was measured according to Lawrence and Burk ¹⁷. Briefly, the reaction mixtures including 0.1 ml plasma samples, tris buffer (pH 7.6), GSH and cumene hydroperoxide were incubated for 10 min at room temperature. Tricholoroacetic acid and 5,5- dithiobis-2 nitrobonzoic acid were added to the reaction mixtures and were kept at room temperature for 5 min, and absorbances at 412 nm were recorded. Catalase activity was determined according to the method of Goth ¹⁸. Reaction mixture including hydrogen peroxide and sodium-potassium buffer (pH 7.4) and 0.1 ml plasma samples were incubated at 37ºC for 1 min. Then ammonium molibdat was added to mixture and the yellow complex was measured at 405 nm against a blank by using a spectrophotometer.

Data were analyzed by using student’s t test on SPSS software, 10.1.0 (SPSS Inc. Chicago, IL, USA). The results were expressed as mean ± SD. Differences were considered as significant when P< 0.05.

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Tablo 1: Mean (± SD) plasma oxidant (Malondialdehyde [MDA]) and antioxidant (Glutathione [GSH], ß-carotene, Vitamin E, Glutathione peroxidase [GSH-Px], Catalase [CAT]) status in healthy mares and mares with uterine lymphatic cysts.

It is known that ROS play a number of significant, diverse roles in female reproductive biology including events in the uterine environment, corpus luteum function and regression ⁷. An unbalanced prooxidantantioxidant state has been postulated to be involved in the pathogenesis of a number of uterine disorders such as cervicitis, uterine myoma ^8, and endometriosis ^9,10. However, our results failed to show imbalance between oxidant and antioxidant levels in mares used in this study except for β-carotene.

Plasma concentration of β-carotene in the mares with uterine lymphatic cyst was significantly lower (P<0.01) than in healthy mares. Differences in the levels of β- carotene between healthy mares and mares with uterine cysts could have been due to a number causes. Beta carotene, precursor of vitamin A is known to be necessary for different reproductive functions. It plays important roles in ovarian steroidogenesis and the uterus. β-carotene stimulated progesterone production in bovine luteal cells ^21,22. Lotthammer and Ahlswede ²³ have also reported that β--carotene supplemented cows have a much lower incidence of ovarian cysts than unsupplemented cows. Some reports indicate that the uterus requires vitamin A for normal epithelial differentiation and for the maintenance of fertility ²⁴. Vitamin A deficiency causes alterations in epithelial differentiation, proliferation of squamous cells and shedding of mucous cells. Dietary retinoic acid, vitamin A metabolit permits the replacement of squamous metaplastic cells by mucus-secreting cells and thus, reestablishes normal epithelial morphology and function ²⁵. Oestrogen-dependent keratinisation of the vagina and cervical epithelium occurs during the oestrus phase of the ovarian cycle in rodents. Vitamin A administration was found to reduce the oestrogen-induced proliferation of rat uterine stromal and myometrial cells ²⁴ and inhibits the progression of rat mammary carcinoma ²⁶. These observations indicate that vitamin A can alter the responses of several oestrogen-sensitive tissues.

Lymphatic cysts may be associated with a normal uterus or with chronic inflammation of the uterus ²⁷. β- carotene has been reported to possess immune modulator activities in humans and animals ²⁸. Carotenoids stimulate the phagocytic and bacteria-killing ability of blood neutrophils and peritoneal macrophages. Vitamin A deficiency decreases antigen-specific secretory immunoglobulin A concentrations and mucosal-associated immune cell function ²⁹. Michal et al. ³⁰ have also reported that dairy cows fed β- carotene had lower incidence of metritis compared with non-supplemented cows. The genital tract may be susceptible to infections as a result of a dysfunction of protective epithelia due to β-carotene deficiency ²⁵. Briefly, β-carotene may prevent uterine cyst formation by regulating ovarian steroid production, immune cell function and epithelial differentiation through its biological actions other than its antioxidant properties. On the other words, age-related degenerative changes was shown increasingly in the uteri of mares at the age of 15 onwards ^19, meanwhile lower β-carotene concentration may accelerate the age-related degenerative diseases such as uterine cyst in mares.

In conclusion, it was observed no imbalance between plasma oxidant and antioxidant levels except for β- carotene in mares with uterine lymphatic cyst. Lower β- carotene concentration may be one of factors which expedite age-related uterine degeneration such as lymphatic cyst in mares. Antioxidants such as β-carotene and vitamin A may be advice to prevent possible agerelated degenerative changes in uterine of the mares at the age of 15 onwards.

This study may be considered as a preliminary study because of the small number of animals. Further studies are required to investigate the role of β-carotene in the pathogenesis of uterine cysts.

* A summary of this study was presented in Proceedings of the Ninth International Symposium on Equine Reproduction on 7-11 August 2006 in Kerkrade, The Netherlands.

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