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

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.

Environment contamination by mycotoxins and their occurrence in food and feed: Physiological aspects and economical approach.

The contamination of food and feed by mycotoxins as toxic metabolites of fungi is a risk not only for consumers resulting in various embarrassment regarding health status and well-being, but also for producers, companies and export market on the ground of economic losses and ruined stability of economic trade. As it is given in historical evidence, the contamination of food by mycotoxins is a topic as old as a history of mankind, finding some evidence even in the ancient books and records. Nowadays, the mycotoxins are used in modern biotechnological laboratories and are considered an agent for targeting the specific cells (e.g., defected cells to eliminate them). However, this promising procedure is only the beginning. More concern is focused on mycotoxins as abiotic hazard agents. The dealing with them, systematic monitoring, and development of techniques for their elimination from agricultural commodities are worldwide issues concerning all countries. They can be found alone or in co-occurrence with other mycotoxins. Thus, this review aims to provide widened information regarding mycotoxins contamination in environment with the consequences on health of animals and humans. The inevitability for more data that correctly determine the risk points linked to mycotoxins occurrence and their specific reactions in the environment is demonstrated. This review includes various symptoms in animals and humans that result from mycotoxin exposure. For better understanding of mycotoxin's impact on animals, the sensitivities of various animal species to various mycotoxins are listed. Strategies for elimination and preventing the risks of mycotoxins contamination as well as economical approach are discussed. To complete the topic, some data from past as historical evidences are presented.

Assessment of T-2 toxin effect and its metabolite HT-2 toxin combined with insulin-like growth factor I, leptin and ghrelin on progesterone secretion by rabbit ovarian fragments.

Assessment of A-trichothecene mycotoxins (T-2 and HT-2 toxins) effect combined with growth factor IGF-I, and the metabolic hormones leptin and ghrelin on progesterone secretion by rabbit ovarian fragments was studied. Rabbit ovarian fragments were incubated without (control group) or with T-2/HT-2 toxin, or their combinations with insulin-like growth factor I (IGF-I), leptin or ghrelin at various concentrations for 24 h. Secretion of progesterone was determined by ELISA. First, T-2 toxin and HT-2 toxins at all doses used (0.01, 0.1, 1, 10, and 100 ng mL(-1)) were not shown to be potential regulators of progesterone secretion in rabbit ovarian fragments. Second, T-2 toxin but not HT-2 toxin combined with IGF-I was shown to be potential regulator of progesterone secretion in rabbit ovarian fragments. T-2 toxin at all doses used (0.01; 0.1; 1; 10; and 100 ng mL(-1)) combined with IGF-I (at dose 100 ng mL(-1)) significantly (P < 0.05) decreased progesterone secretion by rabbit ovarian fragments. Third, T-2 toxin and HT-2 toxin at all doses used in the study (0.01, 0.1, 1, 10, and 100 ng mL(-1)) combined with leptin (at dose 1000 ng mL(-1)) were not shown to be potential regulators of progesterone secretion in rabbit ovarian fragments. Furthermore, T-2 toxin and HT-2 toxin at all doses used in the study (0.01, 0.1, 1, 10, and 100 ng mL(-1)) combined with ghrelin (500 ng mL(-1)) were not shown to be potential regulators of progesterone secretion in rabbit ovarian fragments. Results in this study showed that trichothecene as T-2 toxin combined with IGF-I but not HT-2 toxin was able to decrease progesterone secretion in rabbit ovarian fragments in vitro. Experimental results of T-2 and HT-2 toxins combined with leptin and ghrelin did not confirm ability to modulate progesterone secretion by ovarian fragments in rabbits.

In vitro changes in porcine ovarian granulosa cells induced by copper.

Objective of this in vitro study was to examine the secretion activity (progesterone and insulin-like growth factor I) of porcine ovarian granulosa cells after copper (Cu) addition and to outline a potential intracellular mediator (cyclin B1) of its effects. It also aimed at investigating the apoptotic potential of Cu on porcine ovarian granulosa cells after addition in vitro. Ovarian granulosa cells were incubated with copper sulphate (CuSO4·5H2O) at the doses 0.33, 0.40, 0.50, 1.0 and 2.0 µL mL(-1) for 18 h and compared with control group without Cu addition. Release of progesterone (P4) and insulin-like growth factor I (IGF-I) by granulosa cells was assessed by RIA, expression of cyclin B1 by immunocytochemistry and apoptosis by TUNEL assay. Observations show that P4 release by granulosa cells was inhibited while the release of IGF-I and cyclin B1 was stimulated significantly (P < 0.05) by CuSO4·5H2O addition at the dose 2.0 µL mL(-1). Also, addition of CuSO4.5H2O at the lowest dose used in the study (0.33 µL mL(-1)) significantly (P < 0.05) decreased apoptosis in granulosa cells. In conclusion, results indicate dose dependent effect of Cu on (1) secretion of steroid hormone progesterone and growth factor IGF-I, (2) expression of cyclin B1 as marker of proliferation of porcine ovarian granulosa cells, (3) apoptosis of porcine ovarian granulosa cells and, (4) that the effect of Cu on ovarian cell proliferation could be mediated by IGF-I and cyclin B1. Obtained data suggest interference of Cu in the pathways of proliferation of porcine ovarian granulosa cells through hormonal and intracellular peptide cyclin B1.

T-2 toxin and its metabolite HT-2 toxin combined with insulin-like growth factor-I modify progesterone secretion by porcine ovarian granulosa cells.

The aim of this study was to examine the effect of A-trichothecenes T-2 and HT-2 toxins combined with insulin-like growth factor I (IGF-I) on the release of steroid hormone progesterone (P4) by porcine ovarian granulosa cells (GCs). The cells were incubated without (control) or with treatments of A-trichothecenes T-2 (100 and 1000 ng/mL)/ HT-2 (100 and 1000 ng/mL) combined with IGF-I (1, 10 and 100 ng/mL) for 24 h. Progesterone secretion was determined by RIA. The release of P4 by GCs after addition of T-2 toxin (at 100 ng/mL) combined with IGF-I (at 10 but not at 1 and 100 ng/mL) and HT-2 toxin (at 100 ng/mL) combined with IGF-I (at all doses) was significantly (P < 0.05) inhibited. On the other hand the release of P4 after addition of T-2/ HT-2 toxin (at 1000 ng/mL) combined with IGF-I (at all doses) was significantly (P < 0.05) stimulated. Alone IGF-I addition (at 10, 100 but not at 1 ng/mL) significantly (P < 0.05) stimulated P4 release by GCs. The results of our in vitro study indicate the T-2 and HT-2 toxins combined with IGF-I could modify progesterone secretion by porcine ovarian granulosa cells and potentially regulate process of steroidogenesis in the ovaries. Currently, occurrence of mycotoxins in food and feed is a worldwide problem and therefore study of these toxins as well as their interaction with different substances such as growth factors could be beneficial for better understanding of mechanism of their toxic effects in organism.

The effect of deoxynivalenol on the secretion activity, proliferation and apoptosis of porcine ovarian granulosa cells in vitro.

The aim of this in vitro study was to examine the secretion activity, markers of proliferation and apoptosis in porcine ovarian granulosa cells (GCs) after deoxynivalenol (DON) addition. Ovarian granulosa cells were incubated with DON for 24h: 10, 100 and 1000 ng/mL, while the control group received no DON. The secretion of insulin-like growth factor I (IGF-I) and progesterone was determined by radioimmunoassay (RIA) and expression of cyclin B1, PCNA and caspase-3 by immunocytochemistry. IGF-I release by GCs was inhibited by DON, while progesterone release and the expression of cyclin B1 was stimulated by DON (at 1000 ng/mL but not at 10 and 100 ng/mL). PCNA expression was stimulated by DON (at 100 and 1000 ng/mL but not at 10 ng/mL). Caspase-3 expression was not influenced by DON treatment (at all doses). In conclusion, our results indicate, (1) a direct effect of DON on secretion of growth factor IGF-I and steroid hormone progesterone, (2) expression of markers of proliferation (cyclin B1 and PCNA) but not on the (3) expression of marker of apoptosis (caspase-3) in porcine ovarian granulosa cells. This in vitro study suggests the dose-dependent association of DON on porcine ovarian functions.

Occurrence of chromosomal aneuploidy in rabbit oocytes and embryos at different developmental stages.

The aim of our study was to analyse chromosomal aneuploidy occurrence in rabbit oocytes and embryos. Chromosomal analysis was done in rabbit oocytes and rabbit preimplantation embryos at 2-, 4- and 8-cell stages derived from in vivo fertilization. For mitotic cycle synchronization at the metaphase stage, 2-, 4- and 8-cell embryos were incubated in k-DMEM medium, supplemented with colcemid (1 microg/ml), for 7, 12 or 13h respectively. Success of metaphase synchronization was at values of 100, 86.1 and 92.2% for 2-, 4- and 8-cell embryos respectively. Recovery rate of analysable metaphase plates was reached at 58.8%, 83.9% and 59.8% for 2-, 4- and 8-cell embryos and 100% for oocytes. Significant difference (p < 0.01) in aneuploidy rate between oocytes (40.7%) and 2-cell embryos (62.5%) was found. These results demonstrate higher efficiency of synchronization of embryo cells at the metaphase stage, what may contribute to elevating the proportion of analysable nuclei.

Cadmium toxicity at low concentration on rabbit spermatozoa motility, morphology and membrane integrity in vitro.

In this study the effect of cadmium on various parameters of spermatozoa motility, morphology as well as on the spermatozoa membrane integrity in rabbits was analyzed in vitro, experimental concentrations ranging from 0.62 to 0.98 micro g CdCl(2)/mL. Pooled rabbit (n = 5) semen was cultured in vitro with cadmium and subsequently diluted to various experimental concentrations apart from control which received no cadmium exposure. Using computer assisted semen analysis method (CASA) we detected decrease of total motility with in the higher concentration range at Time 0. However, with increasing time (after 1 and 2 h of culture), cadmium exerted deleterious effect leading to significant motility reduction in comparison to control. A similar trend was exhibited in case of progressive motility, too. Most of the spermatozoa distance and velocity parameters detected no significant change in comparison to control at the beginning of culture (Time 0), although the toxic effect became significant (P < 0.05) with the passage of culture time (Times 1 and 2 h) in all concentrations. Analysis of spermatozoa morphology detected significant (P < 0.05) alterations at higher concentrations. At higher concentrations acrosomal changes, head without flagellum/separated flagellum, broken flagellum and other abnormalities were significantly higher (P < 0.05), while knob-twisted flagellum and small heads differed significantly (P < 0.05) in comparison to control at all concentrations. In regards to flagellum torso, flagellum ball and retention of cytoplasmic drop statistically higher values (P < 0.05) were noted at the maxium experimental concentration only. Annexin analysis for detection of spermatozoa with disordered membranes revealed higher occurrence of positive spermatozoa in cadmium exposed groups. Annexin-positive reactions suggested alterations in anterior part of head (acrosome) and in flagellum (mitochondrial segment) of spermatozoa. This paper underlines that cadmium is highly toxic for rabbit spermatozoa, as visualized by the toxic effects on parameters of spermatozoa motility, morphology and membrane integrity. The toxic effect is more drastic at higher concentrations. This study also indicates that cadmium requires a minimum one hour incubation time to exert its deletorious effects on various parameters of spermatozoa, particularly at low concentrations.

In vitro copper toxicity on rabbit spermatozoa motility, morphology and cell membrane integrity.

In this in vitro study the effects of copper sulphate on the motility, morphology and structural integrity of rabbit spermatozoa were investigated. The spermatozoa motility was evaluated by CASA method and Annexin analysis was used for detection of structural changes. For analysis of morphology samples of rabbit semen were fixed with Hancock's solution and stained with Giemsa, and for each sample at least 500 spermatozoa were evaluated. The concentration of copper in the medium varied from 3.57 to 4.85 microg CuSO4/mL. At Time 0 the highest motility was detected in the control group (57.78 +/- 3.90%). Motility in groups with copper administration was lower in comparison to control. Significant differences were detected in groups with 3.70-4.85 microg CuSO4/mL (P<0.05) at Time 0. After 1 h of incubation with copper sulphate the motility significantly decreased almost in all experimental groups. However, at Time 2 h significant increase of total motility was observed in groups with lower concentrations of copper (3.57 and 3.63 microg CuSO4/mL). After 24 and 48 h of incubation almost all the spermatozoa were dead recording no motility at all concentrations. The concentration- dependent decrease of spermatozoa motility up to 50% of control was detected for the group receiving highest copper administration (4.85 microg CuSO4/mL) at Times 1 and 2 h. Progressive motility had an identical trend to that of motility in all experimental groups, at all culture times and for all concentrations. Evaluation of distance and velocity parameters indicated that a sort of stress tolerance developed in lower concentrations (3.57 and 3.63 microg CuSO4/mL). At lower concentrations, an increase was noted for distance parameter DCL and velocity parameter VCL, indirectly confirming the significant motility and progressive motility increase. Other motility parameters (straightness index, linearity index, wobble and amplitude of lateral head displacement) revealed decrease in the group with the highest copper concentration (4.85 microg CuSO4/mL) in comparison to the control group after 2 h of incubation, only. No significant alteration was noted for these parameters in comparison to control at Times 0 and 1 h. The total percentage of morphologically abnormal spermatozoa was significantly higher (P<0.05) in the group with the highest copper concentration (46.20+/-5.54%) in comparison to control (30.60+/-2.91). Predominant morphological abnormalities were acrosomal changes, knob-twisted flagellum and small heads. Detection of spermatozoa with disordered membrane was carried out for groups with higher copper concentrations and control, using Annexin analysis. Analysis showed higher occurrence of positive spermatozoa in the copper-exposed groups. Some Annexin positive reactions from all spermatozoa were detected in the control group. In copper-exposed groups positive reaction proved alteration in anterior part of head (acrosome) and in connection segment (mid-piece) of spermatozoa. Detected data evidently confirm adverse effects of high copper sulphate concentrations in rabbit semen on parameters of spermatozoa motility, morphology and membrane integrity. This paper also indicates the lowest possible toxic concentration of copper (3.70 microg CuSO4/mL) to rabbit spermatozoa in relation to motility.

In vitro gossypol induced spermatozoa motility alterations in rabbits.

The objectives of the present study were to: (i) examine the in vitro dose response of rabbit spermatozoa motility to the antifertility agent gossypol (GOS) and (ii) determine whether filtered (FIL) and unfiltered (UNFIL) GOS differ in their magnitude of effect. Rabbit semen belonging to adult males (n = 5; 12-14 months) were cultured with UNFIL GOS and FIL GOS (5% solution) and subsequently diluted (1:1-7) for analysis using a Computer Assisted Semen Analyzer (CASA) system in 5 time periods (0, 60, 120, 180 and 360 minutes). At Time 0, no significant change in rabbit spermatozoa motility (MOT) and progressive motility (PROG) with GOS FIL was noted, while increases were observed with GOS UNFIL. At Time 60, weak changes were noted for MOT and PROG. After 120 minutes of culture with both GOS FIL and GOS UNFIL, MOT and PROG decreased significantly in some experimental groups. However, no differences were recorded for both the parameters at Times 180 and 360, with the exception of PROG in the GOS UNFIL category (groups A, B, E, F and G), where a significant decrease was noticed. Detailed evaluation of the distance and velocity parameters revealed reduction in all these studied markers after 60 and 120 minutes of in vitro culture with both GOS FIL and GOS UNFIL, indirectly confirming the PROG decrease. Straightness (STR), linearity (LIN), wobble (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) mostly remained unaltered at all time periods for GOS FIL, where as some minor alterations were noticed in GOS UNFIL category for STR, LIN, WOB, ALH and BCF parameters at Time 0, 60 and 120. The present study confirms the dose and time dependent alterations of rabbit spermatozoa motility parameters by GOS. The GOS dynamics in our experiment shows that rabbit spermatozoa as a biological material can indicate a GOS inhibition of motility. Obtained data for the first time indicates a higher immobilizing potential of unfiltered GOS in comparison to filtered GOS in its inhibitory action of spermatozoa motility parameters in rabbits.

The effect of hFVIII transgene on the chromosomal aneuploidy rate in rabbits.

The aim of this study was to compare the chromosomal aneuploidy rate between transgenic and non-transgenic rabbits derived from the F4 generation. Chromosomal analysis was carried out on bone marrow samples of New Zealand White transgenic (carrying human factor VIII gene) and non-transgenic rabbits (F4 generation) each having a different genetic background (female no. 1-3-5 line I and female no. 1-9-7 line II). C-metaphase plates were obtained from the bone marrow lymphocytes synchronized by the addition of 0.25 microg/ml colcemide. No significant difference in chromosomal aneuploidy between transgenic (61%) and non-transgenic (51.27%) rabbits of line I was observed. A higher but non-significant aneuploidy rate between transgenic and non-transgenic rabbits was found in line II, on the other hand a significant difference (P<0.05) was observed in diploidy rate. In conclusion, chromosomal aneuploidy rates in this experiment were higher than published previously in other reports.

Simultaneous detection of malignant hyperthermia and genetic predisposition for improved litter size in pigs by multiplex PCR-RFLP.

The ryanodine receptor gene (RYR1) and the estrogen receptor gene (ESR) are the best commercially used markers for predisposition of stress susceptibility (malignant hyperthermia--MH) and increased litter size, respectively. A simplified method of simultaneous detection of MH and ESR genotypes has been developed. The method is based on simultaneous amplification of fragments of two genes by multiplex PCR and subsequent digestion of the products with two restriction enzymes. The PCR and the digestion could be performed in a single tube and all genotypes could be detected by electrophoretic separation on the same agarose gel. Thus, the development of the method can decrease the cost of the sample analysis and increase the speed and efficiency of the analysis. In our study, frequencies of mutated T allele of the RYR1 gene in Large White (LW), White Meaty (WM) and Landrace (L) were 0.11, 0.13, and 0.15, respectively. Frequencies of the preferred B allele of the ESR gene in the same breeds were 0.35, 0.26, and 0.06, respectively.

Influence of a 50 hz extra low frequency electromagnetic field on spermatozoa motility and fertilization rates in rabbits.

Effects of a 50 Hz extra-low frequency electromagnetic field (ELF EMF) on in vitro rabbit spermatozoa motility were analyzed, as well as the effect on fertilization rates after insemination. Pooled semen samples and a control were exposed to 50 Hz ELF EMF. The difference of the samples of the test groups G1 and G2 with the control group CG (75.56%) for spermatozoa motility were found to be significant (P < 0.01). Differences were significant (P < 0.01) for curvilinear velocity (VCL) between the test group G3 (122.38 microm/s) and the control group CG (112.02 microm/s). Hormonally stimulated adult (9-12 months) females (n = 140) were inseminated with semen samples from G1, G2, G3 and CG (0.88 x 109 spermatozoa/0.5 mL average insemination portion) immediately after ELF EMF exposure and fertilization (kindling) rates were calculated. For the G2 it was 54.28% data indicate 50 Hz ELF EMF induced alterations of spermatozoa motility and kindling rate in rabbits, therefore influencing fertility.