Gábor Tornyos

Subsequent effect of subacute T-2 toxicosis on spermatozoa, seminal plasma and testosterone production in rabbits

Pannon White (n=12) male rabbits (weight: 4050 to 4500 g, age: 9 months) received 2 ml of a suspension containing purified T-2 toxin by gavage for 3 days. The daily toxin intake was 4 mg/animal (0.78 to 0.99 mg/kg body weight (BW)). Control animals (n=12) received toxin-free suspension for 3 days. Since a feed-refusal effect was observed on the second day after T-2 administration, a group of bucks (n=10) were kept as controls (no toxin treatment) but on a restricted feeding schedule, that is, the same amount of feed was provided to them as was consumed by the exposed animals. On day 51 of the experiment (i.e. 48 days after the 3-day toxin treatment), semen was collected, and pH, concentration, motility and morphology of the spermatozoa, as well as concentration of citric acid, zinc and fructose in the seminal plasma, were measured. After gonadotropin-releasing hormone (GnRH) analogue treatment, the testosterone level was examined. One day of T-2 toxin treatment dramatically decreased voluntary feed intake (by 27% compared to control, P<0.05) and remained lower (P<0.05) during the first 2 weeks after the withdrawal of the toxin. BW of the contaminated rabbits decreased by 88% on days 17 and 29 compared to controls (P<0.05). No effect of toxin treatment was detected on pH and quantity of the semen or concentration of spermatozoa. The ratio of spermatozoa showing progressive forward motility decreased from 65% to 53% in the semen samples of toxin-treated animals compared to controls (P>0.05). The ratio of spermatozoa with abnormal morphology increased (P<0.05) in the ejaculates collected from the toxin-treated animals. T-2 toxin applied in high doses decreased the concentration of citric acid in seminal plasma (P<0.05). No effect of T-2 toxin on the concentrations of the other seminal plasma parameters (fructose and zinc) was observed. T-2 toxin decreased the basic testosterone level by 45% compared to control (P<0.01) and resulted in lower (P<0.05) GnRH-induced testosterone concentration. Feed restriction, that is, less nutrient intake, resulted in more morphologically abnormal spermatozoa in the semen, but it did not cause significant loss in BW, motility of the spermatozoa, composition of the seminal plasma or testosterone concentration--its effect needs further examination.

Effect of chronic T-2 toxin exposure in rabbit bucks, determination of the No Observed Adverse Effect Level (NOAEL)

T-2 toxin (T-2) was administered to adult Pannon White (n = 10/group) male rabbits for 65 days, first in a suspension by gavage (0.05, 0.1 or 0.2 mg/animal/day), and secondly mixed into the feed (0.33 and 0.66 mg/kg feed). In the first experiment 0.1 mg T-2 exposure resulted in temporary decrease in feed intake, slower increase in the gonadotropin-releasing hormone (GnRH) induced testosterone synthesis, slight centrolobular infiltration in the liver and a slight hyperplasia of the Leydig cells. In addition to the temporary feed refusal effect, 0.2 mg T-2 caused a temporary decrease in plasma albumin and urea concentrations, lesser glutathione peroxidase (GPx) activity in the seminal plasma, a greater (by 320%) ratio of spermatozoa with cytoplasmic droplets, slower increase in the GnRH-induced testosterone synthesis, centrolobular infiltration in the liver, slightly hyperaemic testes and increased proliferative activity of the Leydig cells. The two smaller doses applied in feed (0.33 and 0.66 mg/kg) did not cause any significant adverse effect, and no feed refusal was observed. According to these results the No Observed Adverse Effect Level (NOAEL) of T-2 for adult rabbit males was found to be <0.1 mg/animal/day (<0.02 mg/kg b.w./day).

Individual and Combined Effects of Subchronic Exposure of Three Fusarium Toxins (Fumonisin B, Deoxynivalenol and Zearalenone) in Rabbit Bucks

Objective of the study was to determine reproductive toxicity of Fusarium toxins orally at three subchronic doses on adult Pannon White male rabbits. The four treatments were: control (C, toxin-free diet), F (5 mg/kg FB1), DZ (1 mg/kg DON+0.25 mg/kg ZEA), FDZ (5 mg/kg FB1+1 mg/kg DON+0.25 mg/kg ZEA) for 65 days (n=15/treatment). The doses were pre-determined according the EU limits in finished feed for young pig (in the absence of limits for rabbits’ feed; based on the European Commission Recommendation 2006/576/EC and the European Commission Directive 2003/100/EC). The most pronounced effects of the toxins were exerted on the reproductive processes. The ratio of spermatozoa showing progressive forward motility decreased (P<0.05) from 80% to 67% in the FDZ group by day 60. Differences were found between the groups DZ (66.3% ± 23.7) and C (80.2% ± 11.2) in spermatozoa morphology. GnRH treated animals produced less testosterone in FDZ animals, compared to the other three groups (P<0.05). In the comet assay the individual fumonisin treatment resulted in significantly less 0 comets (intact cells), compared to all others. Based on the prevalence of score, lower (P<0.0001) damage was observed in FDZ group, as compared to F and DZ. Among the mycotoxins studied, additive or less than additive effect was found in case of spermatogenesis and sperm cell morphology, synergism in testosterone production, while FB1 acted antagonistically against DON+ZEA in comet assay. All mycotoxins provoked moderate lipid-peroxidation, based on the changes of glutathione concentration, glutathione peroxidase activity and formation of malondialdehyde and conjugated dienes and trienes, and exerted slight genotoxicity based on comet assay, FB1 being antagonistic towards DON+ZEA. In F, DZ and FDZ animals the intensity of spermatogenesis decreased by 43, 31 and 64%, respectively, which was reflected by lack of differentiated spermatozoa, thinning of the germinal epithelium, the appearance of multinuclear giant cells, indicative of the disturbance of meiosis and mitosis of the germinal epithelial cells and in some cases the lack of spermatogonia.

Effect of different weaning ages (21, 28 or 35 days) on production, growth and certain parameters of the digestive tract in rabbits

The effect of different weaning ages, that is, 21 (G21), 28 (G28) or 35 (G35) days, on growth and certain parameters of the digestive tract was examined in rabbits to assess the risk of early weaning attributable to the less-developed digestive system. On days 35 and 42, G35 rabbits had 10% to 14% and 10% higher BW, respectively (P < 0.05), than those weaned at days 21 and 28. In the 4th week of life, early weaned animals had 75% higher feed intake than G28 and G35 rabbits (P < 0.05). The relative weight of the liver increased by 62% between 21 and 28 days of age, and thereafter it decreased by 76% between 35 and 42 days of age (P < 0.05), with G21 rabbits having 29% higher weight compared with G35 animals on day 35 (P < 0.05). The relative weight of the whole gastrointestinal (GI) tract increased by 49% and 22% after weaning in G21 and G28 rabbits, respectively (P < 0.05). On day 28, the relative weight of the GI tract was 19% higher in G21 than in G28 rabbits, whereas on day 35 G21 and G28 animals had a 12% heavier GI tract compared with G35 rabbits (P < 0.05). Age influenced the ratio of stomach, small intestine and caecum within the GI tract; however, no effect of different weaning age was demonstrated. The pH value of the stomach and caecum decreased from 5.7 to 1.6 and from 7.1 to 6.3, respectively, whereas that of the small intestine increased from 6.8 to 8.4 (P < 0.05); the differences between groups were not statistically significant. Strictly anaerobic culturable bacteria were present in the caecum in high amounts (108), already at 14 days of age; no significant difference attributable to weaning age was demonstrable. The concentration of total volatile fatty acids (tVFA) was higher in G21 than in G28 and G35 throughout the experimental period (P < 0.05). The proportion of acetic and butyric acid within tVFA increased, whereas that of propionic acid decreased, resulting in a C3 : C4 ratio decreasing with age. Early weaning (G21) resulted in higher butyric acid and lower propionic acid proportions on day 28 (P < 0.05). No interaction between age and treatment was found, except in relative weight of the GI tract and caecal content. In conclusion, early weaning did not cause considerable changes in the digestive physiological parameters measured, but it resulted in 10% lower growth in rabbits.

Quantitative PCR with 16S rRNA-gene-targeted specific primers for analysis of caecal microbial community in growing rabbits after dietary supplementation of thyme (Thymus vulgaris) and spirulina (Arthrospira platensis)

Abstract Objective of this study was to evaluate the effect of supplementation of growing rabbits’ diet with 5% spirulina (Arthrospira platensis) and/or 3% (Thymus vulgaris L.), on composition and amount of rabbits caecal microbiota. After weaning, rabbits were randomly sorted to 4 groups (n = 42/group). The control group received a pellet without any supplementation, in treated groups the dietary supplementation was provided until 77 days of age. On 49, 63 and 77 days of age, six healthy animals/group were randomly selected and slaughtered. From the caecal content the amount of total bacteria, Bacteroides, Clostridium leptum and Clostridium coccoides were determined by quantitative polymerase chain reaction (qPCR), with the aid of bacterial ribosome coding DNA. The copy number of total bacteria, C. leptum, C. coccoides and Bacteroides varied between 2.75 × 1012 to 2.24 × 1013, 5.25 × 1011 to 1.82 × 1012, 2.5 × 1010 to 6.91 × 1011 and 5.89 × 1010 to 1.10 × 1012, respectively. The amount of investigated bacteria represented 0.6%-13.4% of the total bacteria. The use of spirulina and/or thyme supplements influenced the amount of the copy number of each bacteria examined between 49 and 77 days of age. Their effect on the total bacteria number was temporary. Spirulina resulted in more Bacteroides on day 63 but significantly lower amount of Clostridia at 63 and at 77 days of age. The antimicrobial effect of thyme on the absolute Clostridia number was temporary, prevailing on day 63. Thyme resulted in significantly decreasing percentage ratio (within total bacteria) of C. leptum and C. coccoides by 77 days of age.

Individual and combined effects of feed artificially contaminated with with fumonisin B1 and T-2 toxin in weaned rabbits

Co-contamination of feed and feed raw materials with two or more mycotoxins is frequently reported, however, only a few studies have investigated the combined effects of low doses of multiple mycotoxins. In the present study the individual and combined effects of 10 mg/kg fumonisin B1 and 2 mg/kg T-2 toxin (n=12/group) were investigated in weaned rabbits. Mycotoxin contaminated feed was produced by adding fungal cultures of Fusarium verticillioides and Fusarium sporotrichioides, and fed to 40 days old rabbits during 28 days. Feed intake and body weight were measured weekly, serum biochemistry and antioxidant parameters on day 0, 14 and 28, while histopathological examination and comet assay were performed at the end of the experiment. T-2 exposure both alone and in combination resulted in 15-18% less final body weight compared to the control and FB1 treatment. There was a significant increase in the concentration of plasma total protein, albumin, fructosamine and creatinine in the group treated with FB1 c...

Effect of dietary supplementation of Spirulina (Arthrospira platensis) and thyme (Thymus vulgaris) on serum biochemistry, immune response and antioxidant status of rabbits

Abstract Growing rabbits’ (42 rabbits/group, 3 rabbits/cage, 14 cages/treatment) diet was supplemented with 5% Spirulina (Arthrospira platensis) and 3% thyme (Thymus vulgaris L.) powder single (S or T) and in combination (ST) between 35 and 77 days of age. On day 0 (weaning at 35 days of age) 14 rabbits were vaccinated with 100 μg/animal ovalbumin to provoke immune response. Blood samples were taken on days 0, 14, 28 and 42 of the experimental period. Sampling dates significantly influenced total protein, albumin, glucose, cholesterol, urea, creatinine concentration and enzyme (AST, ALT, GGT) activities, with a significant age × diet interaction in the case of TP and CREA. There was a significant increase in ALT (+45 and 74%) and GGT (+87 and 102%) activity after immunisation. While Spirulina and thyme significantly ameliorated the rise in AST activity, their effect was inefficient in the case of GGT. Spirulina, both single and in combination showed a tendency in higher IgG level as compared to control (P<0.05). No significant effect of sampling date or treatment on phagocytic activity or secretory IgA was demonstrable (P>0.05). Higher MDA concentration was measured in the red blood cells of S, T and ST animals, while no other significant diet effect on the antioxidant parameters was detected, however, significant sampling date × diet interaction was found in the case of GPx activity. Plasma GGT (increase by 19-66%) was inversely associated with GSH (decrease by 66-113%) between days 0 to 42 of the experimental period (r=-0.57, P<0.05). It can be concluded that Spirulina supplementation alone resulted in higher IgG production, but none of the phytobiotics, at the dose used, affected significantly the antioxidant status of blood.