Marek Halenár

Assessment of a potential preventive ability of amygdalin in mycotoxin-induced ovarian toxicity

The possible effects of a natural substance amygdalin and its combination with the mycotoxin deoxynivalenol (DON) on the steroid hormone secretion (progesterone and 17-β-estradiol) by porcine ovarian granulosa cells (GCs) were examined in this in vitro study. Ovarian GCs were incubated without (control group) and with amygdalin (1, 10, 100, 1,000 and 10,000 μg mL1), or its combination with DON (1 μg mL1) for 24 h. The release of steroid hormones was determined by ELISA. The progesterone secretion by porcine ovarian GCs was not affected by amygdalin in comparison to the control. However, the highest amygdalin dose (10,000 μg mL1) caused a significant stimulation of the 17-β-estradiol release. A combination of amygdalin with DON significantly (P < 0.05) increased the progesterone release at all concentrations. Similarly, a stimulatory effect of amygdalin co-administered with DON was detected with respect to the 17-β-estradiol secretion at the highest dose (10,000 μg mL1) of amygdalin and 1 μg mL1 of DON. Noticeable differences between the effects of amygdalin alone and its combination with DON on the progesterone release were detected. In contrast, no differences between the stimulatory effects of amygdalin and its combination with DON on the 17-β-estradiol synthesis by porcine GCs were observed. Findings from this in vitro study did not confirm the expected protective effect of amygdalin on mycotoxin induced reprotoxicity. Our results indicate that the stimulatory effect of amygdalin combined with DON on the progesterone release was clearly caused by the DON addition, not by the presence amygdalin per se. On the other hand, the stimulation of 17-β-estradiol production was solely caused by the presence of amygdalin addition. These findings suggest a possible involvement of both natural substances into the processes of steroidogenesis and appear to be endocrine modulators of porcine ovaries.

The evaluation of endocrine regulators after intramuscular and oral application of cyanogenic glycoside amygdalin in rabbits

Abstract The present in vivo study was designed to reveal whether amygdalin is able to cause changes in the endocrine profile and thus alter the key reproductive and physiological functions, using rabbits as a biological model. 40 adult female rabbits were randomly divided into five groups: the control group without any amygdalin administration, two experimental groups received a daily intramuscular injection of amygdalin at dose 0.6 and 3.0 mg kg-1 b.w. and other two groups were fed by crushed apricot seeds at dose 60 and 300 mg kg-1 b.w., mixed with commercial feed over the period of 28 days. The body weight of each experimental animal was recorded weekly during the whole study. Plasma levels of steroid (progesterone, 17β-estradiol, testosterone), thyroid (triiodothyronine, thyroxine, thyroid-stimulating hormone), as well as anterior pituitary (prolactin, luteinizing hormone) hormones were assessed by ELISA. Intramuscular application of amygdalin did not affect (P ≥ 0.05) the plasma levels of none of the endocrine regulators selected. Oral form of amygdalin did not induce significant changes in the plasma levels of examined hormones either. Similarly, no clinically noticeable changes in the average body weight of experimental animals were observed. Our findings indicate that intramuscular and oral application of amygdalin did not significantly affect the plasma levels of studied endocrine regulators in experimental rabbits. In this in vivo study, no obvious beneficial or negative effects of amygdalin on the physiological functions of female rabbits were demonstrated. Short-term intake of amygdalin at the recommended doses does not represent risk for animal health.

Effect of apricot seeds on renal structure of rabbits

Amygdalin is the major cyanogenic glycoside present in apricot seeds and is degraded to cyanide by chewing or grinding. The animal data available did not provide a suitable basis for acute human health hazard. The apricot seeds are potentially useful in human nutrition and for treatment of several diseases especially cancer. The present study demonstrates the potential effect of short-term oral application of apricot seeds on renal structure of rabbit as a biological model. Meat line P91 Californian rabbits from the experimental farm of the Animal Production Research Centre Nitra (Slovak Republic) were used in the experiments. The animals were randomly divided into the three groups (C-control, P1, P2 - experimental groups) leading to 8 rabbits in each group. The control group received no apricot seeds while the experimental groups P1 and P2 received a daily dose 60 and 300 mg.kg -1 b.w. of crushed apricot seeds mixed with feed during 28 days, respectively. After 28 days all animals were slaughtered and kidney tissue was processed by standard histopathological techniques. Tissue sections were observed under an optical microscope with camera Olympus CX41 (Olympus, Japan) at a magnification of 10 x 0.40. The basic morphometric criteria of the preparations were quantified using image program MeasurIT (Olympus, Japan). From each sample (n = 24) three histological sections with five different fields of view in each section were analysed and followed parameters were analysed: diameter of renal corpuscles (RC), diameter of glomeruli (G), diameter of tubules (T) and the height of epithelial tubules (E). In our study, we observed a slight increase in the most frequent occurrence parenchyma dystrophy experimental animals. These changes were more pronounced in the experimental group (P2) rabbits received a daily dose of 300 mg.kg -1 of body weight of apricot seeds. Most often, we have found enlarged glomeruli filling the entire space of the capsule, and also glomerular basement membrane thickening. The most frequent alterations of tubular organs manifested by thickening and dilatation of proximal tubules and in the lumen of the occurrence fuchsinophilic mass, grains and hyaline cylinders. The occurrence of the vacuole and parenchymal atrophy was mostly balanced groups. Changes in P2 group are also reflected in morphometric evaluation structures. We have found significant decrease ( p <0.001) in the average of all renal structures (diameter of renal corpuscles, diameter of glomeruli, diameter of tubules, and the height of epithelial tubules). Inversely, oral administration a daily dose of 60 mg.kg -1 of body weight of apricot seeds had no significant impact on these parameters. The change displays only the increase of renal tubule diameter. Our data may provide more specific evidence of oral application of apricot seeds on renal structure but further detailed studies are also required.

Blood plasma levels of anterior pituitary hormones of rabbits after apricot seed exposure in vivo

The present study describes possible changes in plasma levels of anterior pituitary hormones induced by bitter apricot (Prunus armeniaca L.) seeds in young female rabbits in vivo. Prunus armeniaca L. is an important medicinal edible plant species commonly known as “apricot”. The apricot is a member of the Rosaceae and subfamily Prunoideae. It is one of the most delicious and commercially traded fruits in the world. Apricot kernel is the inner part of the seed of the apricot fruit. The kernel is used to produce oil and other chemicals used for medicinal purposes. The seeds are potentially useful in human nutrition and for treatment several diseases especially cancer. In the present study apricot seeds were mixed with feed at different doses 0, 60, 300, 420 mg*kg-1 of body weight. ELISA was used to determine the levels of follicle stimulating hormone (FSH), luteinizing hormone (LH) and prolactin (PRL). 58- days application of apricot seeds did not affect the concentration (P≥0.05) of PRL, LH in blood plasma. Significant (P≤0.01) inhibition of FSH levels induced by the seeds was found at the dose of 420 mg*kg-1 but not at 60 and 300 mg*kg-1 of body weight. These results are suggesting that the natural substances present in apricot seeds may be involved in mechanisms of ovarian folliculogenesis.

Assessment of rabbit spermatozoa characteristics after amygdalin and apricot seeds exposure in vivo

Highlights • Rabbit spermatozoa parameters after amygdalin and apricot seeds exposure.• Evaluation of spermatozoa motility by the CASA system.• Decrease of spermatozoa motility after intramuscular AMG application.• Oral consumption of apricot seeds had no effect on the spermatozoa motility.• Our data suggest the potential impact of AMG and apricot seeds on male reproduction.

Green tea extract affects porcine ovarian cell apoptosis

Green tea is a commonly used beverage and green tea extract is a common dietary herbal supplement manufactured into different over-the-counter products. The aim of this in vitro study was to examine the steroid hormone secretion (progesterone and 17-β estradiol), proliferation and apoptosis of porcine ovarian granulosa cells after addition of green tea extract. Granulosa cells were incubated with green tea extract at five doses (0.1, 1, 10, 100 and 200 μg/ml) and the release of hormones by granulosa cells was assessed by EIA after 24 h exposure. The presence of proliferation and apoptotic markers was assessed by immunocytochemistry. Secretion of steroid hormones was not affected by green tea extract at all the doses in comparison to control. Also, markers of proliferation (PCNA and cyclin B1) were not affected by green tea extract. However, the highest dose (200 μg/ml) of green tea extract used in this study increased the accumulation of apoptotic markers caspase-3 and p53 in granulosa cells. In conclusion, our results indicate the impact of green tea extract at the highest dose used in this study on ovarian apoptosis through pathway that includes activation of caspase-3 and p53. Potential stimulation of these intracellular regulators could induce the process of apoptosis in ovarian cells.

IN VITRO EFFECTS OF ACINETOBACTER BAUMANNII AND SELECTED NATURAL BIOMOLECULES ON RABBIT SPERMATOZOA MOTILITY

The aim of this study was to assess the potential efficiency of selected biologically active substances on the motility behaviour of rabbit spermatozoa subjected to in vitro induced A. baumannii contamination. The semen samples used for A. baumannii detection were collected from 10 New Zealand white male rabbits and the presence of the bacterium was confirmed using MALDI-TOF Mass Spectrometry. For the in vitro experiments rabbit spermatozoa were re-suspended in PBS, containing mineral supplements, BSA and glucose in the presence of 3x105 CFU A. baumannii and diverse concentrations of selected biomolecules (resveratrol - RES, quercetin - QUE, curcumin - CUR, epicatechin - EPI, isoquercitrin - ISO). The sperm motility was assessed using the computer-aided sperm analysis at 0h, 2h, 4h and 6h. A. baumannii significantly decreased the sperm motility (P<0.001) at Time 2h and maintained this negative impact throughout the in vitro culture. Meanwhile, the motility at Time 2h was significantly higher in the samples subjected to A. baumannii together with 10 μmol/L RES (P<0.01); 5, 10 and 50 μmol/L QUE (P<0.001); 1 μmol/L CUR (P<0.05); 10, 50 and 100 μmol/L EPI (P<0.01) as well as 50 μmol/L (P<0.05) and 100 μmol/L ISO (P<0.001) in comparison to the control exposed to the bacterium exclusively. After 4h, the motility remained significantly higher in the groups co-treated with the inoculum and 10 μmol/L RES (P<0.05), 50 μmol/L QUE (P<0.05) as well as 50 μmol/L EPI (P<0.05) when compared to the positive control. Nevertheless, none of the biomolecules was effective against the rapid decline of sperm motility caused by A. baumannii during later stages of the experiment (Time 6h). Based on these results, one can conclude that RES, QUE and EPI exhibit antibacterial properties providing a selective advantage to spermatozoa in the presence of A. baumannii, particularly during short-term rabbit semen handling.

Does apricot seeds consumption cause changes in human urine

Natural substances, such as amygdalin, used in alternative medicine gained high popularity. Common people as well as patients with different diseases have almost unlimited access to various natural supplements. To protect human health, it is very important to study effect of these substances. Amygdalin is a cyanogenic glucoside derived from seeds of rosaceous plants, for example seeds of bitter almonds ( Prunus dulcis ), or apricot, cherry, apple, peach, plum, etc. It is a natural product that owns antitumor activity, it has also been used for the treatment of asthma, bronchitis, emphysema, leprosy and diabetes and produces a kind of antitussive and antiasthmatic effects. The present in vivo study was designed to reveal whether amygdalin in apricot seeds has got an effect on human urine composition, pH value and urine associated health status after six weeks of oral administration. The study group finally consisted of 34 healthy adult volunteers (21 females and 13 males). All participants were asked to consume 60 mg.kg -1 body weight of bitter apricot seeds daily (approximately 3.0 mg.kg -1 of amygdalin) during 6 weeks. During the experiment, three urine collections were carried out (first collection - at the beginning of the experiment; second collection - after 21 days; third collection - after 42 days). Quantification of urine calcium (Ca), magnesium (Mg), phosphorus (P), sodium (Na), potassium (K), chlorides (Cl - ), urea and pH value after apricot seeds supplementation was performed. Statistical analysis of variance showed, that consumption of bitter apricot seeds during 42 days had a significant ( p <0.01) effect on amount of calcium excreted in urine, though this decrease shifted its level from elevated mean value in control collection into normal physiological range. Significant changes were observed in urea ( p <0.05) and phosphorus ( p <0.01) levels in urine after apricot seed ingestion, but gender was also considered to be a source of their variation.

Oral and intramuscular application of cyanogenic glycoside amygdalin did not induce changes in haematological profile of male rabbits

Amygdalin is a cyanogenic glycoside initially obtained from the seeds of bitter almonds. It is composed of one molecule of benzaldehyde, two molecules of glucose and one molecule of hydrocyanic acid. Various ways of amygdalin application play a different role in recipient organism. Intravenous infusion of amygdalin produced neither cyanidemia nor signs of toxicity, but oral administration resulted in significant blood cyanide levels. The present in vivo study was designed to reveal whether amygdalin is able to cause changes in the haematological profile and thus alter the physiological functions, using rabbits as a biological model. Adult male rabbits (n = 20) were randomly divided into five groups: the control group without any amygdalin administration, two experimental groups received a daily intramuscular injection of amygdalin at a dose 0.6 and 3.0 mg.kg -1 b.w. and other two groups were fed by crushed apricot seeds at dose 60 and 300 mg. kg -1 b.w., mixed with commercial feed over the period of 14 days. After two weeks, haematological parameters in whole blood were analysed (WBC - total white blood cell count, LYM - lymphocytes count, MID - medium size cell count, GRA - granulocytes count, RBC - red blood cell count, HGB - haemoglobin, HCT - haematocrit, MCV - mean corpuscular volume, MCH - mean corpuscular hemoglobin, MCHC - mean corpuscular hemoglobin concentration, RDWc - red cell distribution width, PLT - platelet count, PCT - platelet percentage, MPV - mean platelet volume, PDWc - platelet distribution width) using haematology analyser Abacus junior VET. Our findings indicate that intramuscular and oral application of amygdalin for two weeks did not significantly affect the haematology parameters in experimental animals. In this study, no obvious beneficial or negative effects of amygdalin administration on the blood of male rabbits were observed.

The influence of deoxynivalenol and zearalenone on steroid hormone production by porcine ovarian granulosa cells in vitro

ABSTRACT Fusarium mycotoxins deoxynivalenol (DON) and zearalenone (ZEA) are frequently occurring in feed of pigs together. The aim of this study was to evaluate the possible in vitro effects of DON and ZEA, alone or their combination on steroid secretion of porcine ovarian granulosa cells (GCs). A species-specific model with porcine ovarian GCs was used to study the potential endocrine disrupting effects of DON and ZEA alone and in co-exposure. Progesterone (P4) and estradiol (E2) were determined by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). The results of this study demonstrate that DON alone at the higher concentrations may act to stimulate P4 (at 1,000, 2,000, 3,000 and 5,000 ng mL−1 but not 10 and 100 ng mL−1) and E2 (at 2,000, 3,000 and 5,000 ng mL−1 but not 10, 100 and 1000 ng mL−1) secretion. The effects of ZEA on P4 and E2 secretion were not confirmed. DON in combination with the other fusariotoxin ZEA may impair steroidogenesis. Results aslo demonstrate different toxicological effects of fusariotoxins on follicle stimulating hormone-induced secretion of P4 and E2. All these results taken together suggest that fusariotoxin and their interactions can impact ovarian steroidogenesis, thereby demonstrating their potential reproductive effects in pigs.