Dragan Žikić

Genistein affects ovarian folliculogenesis: A stereological study

The effects of short‐term genistein exposure on ovarian folliculogenesis in immature rats were examined stereologically. To determine whether genistein acts as an estrogen agonist or antagonist, the results were compared with the effects of 17α‐ethynylestradiol. Immature female rats received 50 mg/kg/bw of genistein in dimethyl sulfoxide subcutaneously daily for three consecutive days from 18 to 20 days. The second group was injected with 1 μg/kg/bw of 17α‐ethynylestradiol in olive oil in the same schedule. Each group had a corresponding control. Genistein increased ovary and ovarian stroma volumes by 18.50% (P < 0.05) and 53.40% (P < 0.05), respectively, and changed the parenchyma to stroma ratio in favor of stroma. Genistein induced decreases in the number of primordial (by 17.23%; P < 0.05), primary (16.62%; P < 0.05), and secondary follicles (12.29%: P < 0.05), whereas the number of atretic secondary follicles increased (5.10‐fold; P < 0.05). The number of healthy large follicles was raised by 27.3% (P < 0.05), accompanied by 35.64% more atretic large follicles (P < 0.05). Similarly to genistein, estradiol changed the parenchyma to stroma ratio in favor of stroma, and reduced the number of primordial follicles, but the number of primary follicles was elevated. There were more healthy and atretic small and large follicles. In conclusion, genistein acted as an estrogen antagonist and had an inhibitory effect on the initial phase of folliculogenesis. In the other phases, genistein acted as an estrogen agonist, stimulating transition from the preantral to antral stage of folliculogenesis, and altering the ratio of follicular parenchyma and ovarian stroma in favor of stroma. Microsc. Res. Tech. 2012.

Effects of genistein on gonadotropic cells in immature female rats

The effects of genistein on pituitary gonadotropic cells of immature female rats were examined and compared to actions of the synthetic estrogen, 17α-ethynylestradiol. Immature female rats received 50mg/kg/bw of genistein in dimethylsulfoxide (DMSO) subcutaneously (s.c.) daily for 3 days at 18, 19 and 20 days of age. A second group was injected with 1μg/kg of 17α-ethynylestradiol in olive oil in the same schedule. The genistein control group received DMSO only, while 17α-ethynylestradiol controls were given sterile olive oil only. Changes in cell number per mm(2), cell volume and volume density of follicle-stimulating (FSH) and luteinizing (LH) immunolabeled cells were evaluated by morphometry and stereology. Genistein induced significant increases in the number of FSH cells (by 21%) and LH cells (by 20%) per mm(2) compared to corresponding controls. Volumes of FSH and LH cells were significantly increased by 19.7% and 20% and their volume densities by 20% and 20.2%, respectively. Estradiol markedly affected gonadotropes in the same manner, but to a greater extent. It can be concluded that genistein acted as an estrogenic agonist in the pituitaries of immature female rats, and as such, stimulated gonadotropic cells.

The influence of EPO and hypothermia on the kidneys of rats after perinatal asphyxia

The aim of this study was to determine the effects of erythropoietin (EPO), moderate hypothermia, and a combination thereof on the kidneys of newborn rats damaged during perinatal asphyxia. An animal model of perinatal asphyxia (Wistar rats) was used in which after birth, newborn rats were divided into four groups of 15 animals each: G1, rats exposed only to asphyxia; G2, rats exposed to asphyxia and hypothermia (rectal temperature 32°C) and which received EPO (darbepoetin alpha) intraperitoneally; G3, rats exposed to asphyxia and hypothermia; G4, rats exposed to asphyxia and which received EPO. The rats were sacrificed on the 7th day of life and histopathological evaluation of kidneys was performed. Damage to the proximal tubules was significantly higher in group G1 rats than in groups G2, G3, and G4 rats (p < 0.01). Damage to the distal tubules was found only in group G1 rats. Histological changes in the proximal tubules were more prominent than in the distal tubules (p < 0.01). The immature glomeruli zone was less expressed in group G4 rats than in groups G1, G2, and G3 rats (p < 0.01). Based on these results, we conclude that EPO and hypothermia, as well as the combination thereof, have a protective effect on rats’ kidneys damaged during perinatal asphyxia.

Effects of Fullerenol C60(OH)24 Nanoparticles on a Single-dose Doxorubicin-induced Cardiotoxicity in Pigs: An Ultrastructural Study

Abstract Cardioprotective effects of fullerenol C60(OH)24 nanoparticles (FNP) were investigated in pigs after a single treatment with doxorubicin (DOX). Semithin and ultrathin sections of myocardial tissue routinely prepared for transmission electron microscopy were analyzed. Extensive intracellular damage was confirmed in cardiomyocytes of DOX-treated animals. By means of ultrastructural analysis, a certain degree of parenchymal degeneration was confirmed even in animals treated with FNP alone, including both the oral and the intraperitoneal application of the substance. The cardioprotective effects of FNP in animals previously treated with DOX were recognized to a certain extent, but were not fully confirmed at the ultrastructural level. Nevertheless, the myocardial morphology of DOX-treated animals improved after the admission of FNP. Irregular orientation of myofibrils, myofibrillar disruption, intracellular edema, and vacuolization were reduced, but not completely eliminated. Reduction of these cellular alterations was achieved if FNP was applied orally 6 h prior to DOX treatment in a dose of 18 mg/kg. However, numerous defects, including the inner mitochondrial membrane and the plasma membrane disruption of certain cells persisted. In FNP/DOX-treated animals, the presence of multinuclear cells with mitosis-like figures resembling metaphase or anaphase were observed, indicating that DOX and FNP could have a complex influence on the cell cycle of cardiomyocytes. Based on this experiment, further careful increase in dosage may be advised to enhance FNP-induced cardioprotection. These investigations should, however, always be combined with ultrastructural analysis. The FNP/DOX interaction is an excellent model for the investigation of cardiomyocyte cell death and cell cycle mechanisms.

Effects of supplementation with α-tocopherol, ascorbic acid, selenium, or their combination in linseed oil-enriched diets on the oxidative status in broilers

&NA; A trial was conducted to investigate whether, and if so to what extent, the combined supplementation of vitamin E, vitamin C, and selenium was superior to their sole supplementation concerning the oxidative stress induced by a high n‐3 dietary polyunsaturated fatty acids (PUFA) intake in broilers. Four hundred 21‐day‐old Ross 308 male broilers were allocated to 5 experimental groups fed the following linseed oil (5%)‐enriched finisher diets: Cont (no supplement), +E (200 IU vitamin E/kg feed), +C (250 mg vitamin C/kg feed), +Se (0.2 mg selenium/kg feed), or +ECSe (concentrations as in the sole supplementation, combined). Analyses of malondialdehyde (MDA), vitamin C, and &agr;‐ and &ggr;‐tocopherols in plasma, antioxidant capacity of water‐ (ACW) and lipid‐ (ACL) soluble compounds in serum, and glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities in whole blood were performed. In breast muscle, analyses of MDA, &agr;‐ and &ggr;‐tocopherols, ACW, selenium, and fatty acid (FA) composition were carried out. Birds fed the combination of antioxidants showed reduced average daily gain (ADG) and average daily feed intake (ADFI) and, as +E, lower MDA and &ggr;‐tocopherol, together with raised &agr;‐ tocopherol levels in plasma and lower MDA and raised &agr;‐ tocopherol levels in breast muscle compared to the control. The combination of antioxidants in the +ECSe group raised GPx activity in whole blood compared to the control. In conclusion, results indicated that vitamin E is the most effective antioxidant to alleviate oxidative stress caused by high dietary PUFA and that the supplementation with additional vitamin C and selenium did not have clear synergistic effect.

Circulation Index as a Quantitative Indicator of Angiogenesis in Chorioallantoic Membrane of Chicken Broilers

Summary Chorioallantoic membrane (CAM) is an extraembryonic membrane very frequently used for in vivo studies in various researches. Since researches require a fast method for quantifying the CAM angiogenic response, there is a need to develop a new precise and unbiased method of quantification of angiogenesis in CAM, which would be easy to perform and suitable for analysis of a large number of samples. The objective of this paper is to apply a new method of quantification of angiogenesis in investigation of the development of blood vessels in the CAM, in particular days of embryonic life considered essential for CAM development. The present research included 75 fertilized eggs of heavy hybrid Ross 308. CAM sampling for stereological analyses was in key phases of embryonic development, namely on the 12th, 15th and 19th day. The results of the present investigation show that the increase in embryonic age results in increase in circulation index, which is also an indicator of angiogenic processes developing in CAM. The lowest value of circulation index (0.1952) was recorded on the first sampling day (E12), while the highest value (0.2666) was recorded on the last sampling day (E19). This method may be applied in researching different factors which affect angiogenesis in CAM.

The Effect of Thermal Conditioning on Thyroid Hormones, Hatchability and Embrionic Mortality of Broilers during the Incubation Period

Summary The aim of this study was to examine the effect of thermal conditioning on the embryonic mortality, on the time of embryonic mortality, as well as to examine the effect of this temperature regime on hatching and on thyroid gland hormone levels. 400 fertile eggs, randomly divided into two groups with two repetitions, were used in this research. The control group was exposed to the standard temperature (37.8°C) through the whole embryonic development. The second group was incubated under the standard conditions as the control group, up to 15th day of embryonic development. During 16th, 17th and 18th day of embryonic development, the incubation temperature was increased to 39°C for 3 hours. From 19th day up to the end of the incubation period the conditions of incubation were identical to those of the control group. Results of this study show that the applied treatment leads to an increase of concentration of free T3 hormone on the day 19 of embryonic development, as opposed to a decrease of free T4 hormone in the embryo’s blood compared to the control group. The percentage of hatchability was 82.2% in the control group and 83.3% in the second group. The result shows that this way of thermal manipulation did not affect the embryonic mortality, which was 9.5% in the control group and 8.9% in the experimental group. It can be concluded that in this manner thermal conditioning may manifest the positive effect on the thyroid gland hormone levels without increasing embryonic mortality or disturbing the hatching percentage.