Assessment of the genotoxicity of imidacloprid and metalaxyl in cultured human lymphocytes and rat bone-marrow

Imidacloprid and metalaxyl are two pesticides that are widely used in agriculture, either separately, or in combination. These agents were studied for their possible genotoxic effects with respect to the following cytogenetic end-points: (1) in vitro micronucleus (MN) formation and sister-chromatid exchange (SCE) induction in human lymphocytes and (2) in vivo micronucleus induction in polychromatic erythrocytes (PCEs) of the rat bone-marrow. The results of the MN analysis indicate that MN frequencies after treatment with both pesticides, separately or as a mixture, do not significantly differ from those in the controls except after treatment with metalaxyl alone at 50 μg/ml (p < 0.05). The results of the SCE analysis show that SCE frequencies after treatment with imidacloprid do not differ significantly from those in the controls. A statistically significant increase (p < 0.05) in SCE frequency resulted from treatments with metalaxyl at 5, 10 and 100 μg/ml and with the combination of imidacloprid and metalaxyl at 100 and 200 μg/ml. Finally, the in vivo micronucleus assay with rat bone-marrow polychromatic erythrocytes showed a statistically significant effect upon separate treatments with imidacloprid and metalaxyl at doses of 300 mg/kg body weight (b.w.) (p < 0.01) or upon combined treatment with 200 mg/Kg b.w. (p < 0.001) and 400 mg/kg b.w. (p < 0.05).     

Vicia root-mirconucleus and sister chromatid exchange assays on the genotoxicity of selenium compounds

Selenium (Se) is an important metalloid with industrial, environmental, biological and toxicological significance. Excessive selenium in soil and water may contribute to environmental selenium pollution, and affect plant growth and human health. By using Vicia faba micronucleus (MN) and sister chromatid exchange (SCE) tests, possible genotoxicity of sodium selenite and sodium biselenite was evaluated in this study. The results showed that sodium selenite, at concentrations from 0.01 to 10.0 mg/L, induced a 1.9–3.9-fold increase in MN frequency and a 1.5–1.6-fold increase in SCE frequency, with a statistically significantly difference from the control (P < 0.05 and 0.01, respectively). Sodium selenite also caused mitotic delay and a 15–80% decrease in mitotic indices (MI), but at the lowest concentration (0.005 mg/L), it slightly stimulated mitotic activity. Similarly, the frequencies of MN and SCE also increased significantly in sodium biselenite treated samples, with MI decline only at relatively higher effective concentrations. Results of the present study suggest that selenite is genotoxic to V. faba root cells and may be a genotoxic risk to human health.     

Fenvalerate-induced chromosome aberrations and sister chromatid exchanges in the bone marrow cells of mice in vivo

Fenvalerate, a synthetic pyrethroid insecticide, is commonly used in agriculture and other domestic applications due to its high insecticidal activity and low mammalian-, avian- and phyto-toxicities. However, the genotoxic effect of fenvalerate is highly equivocal. In the present study the genotoxic effects of fenvalerate was evaluated using structural chromosome aberration (CA) and sister chromatid exchange (SCE) assays in mice. Out of the three doses (5, 10 and 20 mg/kg) tested, statistically significant increase in CA was found following intra peritoneal (i.p.) treatment of 20 mg/kg of fenvalerate for 24 h (P<0.01) and 48 h (P<0.05) only. Neither the acute doses of 5 and 10 mg/kg, nor the sub-acute dose (5×4 mg/kg) of fenvalerate could induce any significant effect. All the three acute doses induced significant increase in the frequency of SCEs (P<0.01) in the bone marrow cells, which showed a significant dose-response correlation (r=0.9541, P<0.05). With certain reservations to possible impurities, from the present findings technical grade fenvalerate may be considered as a weak clastogen and a potent inducer of SCEs in mice.     

Genotoxic effects of the pesticides Rubigan, Omite and Rovral in root-meristem cells of Crepis capillaris L.

Three pesticides have been studied for their genotoxicity by the use of assays in the plant Crepis capillaris, aimed at measuring chromosomal aberrations, micronuclei and sister chromosome exchange (SCE). The fungicides Rubigan 12 EC (fenarimol) and Rovral 25 Flo (iprodione) and the insecticide Omite 57 E (propargite) are all widely used nowadays. The aim of our study was to evaluate the genotoxic effects of these pesticides at concentrations corresponding to those applied in agricultural practice. In preliminary experiments we found that these concentrations do not influence cell proliferation and do not inhibit the growth of root meristems. In all experiments formulated commercial products were used. From the results we conclude that the three pesticides did not induce chromosomal aberrations as estimated by metaphase and anaphase analyses. They were also not capable to induce SCE. Rubigan did not induce micronucleus formation even at the highest concentration tested, but Omite and Rovral markedly increased micronucleus formation. The MN response depended on the sampling time and the concentration used, which showed a significant dose–response correlation (r = 0.978, P < 0.01 and r = 0.941, P < 0.01, respectively). A greater increase in micronucleus frequency was observed after Rovral treatment, where the highest concentration gave a response 8–10-fold above the negative control. Both pesticides induced high frequencies of lagging chromosomes, even after exposure to the lower test concentrations. The presence of lagging chromosomes is an indication of anti-microtubule activity of the pesticides tested. This effect was more strongly expressed after exposure to the two higher concentrations of Omite and Rovral. In this case a complete destruction of the mitotic spindle was observed, resulting in C-mitoses as well as in numerical aberrations—polyploidy and aneuploidy. The present findings suggest that Omite and Rovral at concentrations comparable to those used in practice can be regarded as potential aneugens.     

Radiation protection of human lymphocyte chromosomes in vitro by orientin and vicenin

Orientin (Ot) and Vicenin (Vc), two water-soluble flavonoids isolated from the leaves of Indian holy basil Ocimum sanctum have shown significant protection against radiation lethality and chromosomal aberrations in vivo. In the present study the protective effect of Ot and Vc against radiation induced chromosome damage in cultured human peripheral lymphocytes was determined by micronucleus test. In order to select the most effective drug concentration, fresh whole blood was exposed to 4 Gy of cobalt-60 γ-radiation with or without a 30 min pre-treatment with 6.25, 12.5, 15.0, 17.5 or 20 μM of Ot/Vc. Micronucleus (MN) assay was done by cytochalasin induced cytokinesis block method. Radiation significantly increased the MN frequency (16 times normal). Pre-treatment with either Ot or Vc at all concentrations significantly (P<0.05–0.001) reduced the MN count in a concentration dependent manner, with the optimum effect at 17.5 μM. Therefore, fresh blood samples were incubated with/without 17.5 μM Ot/Vc for 30 min and then exposed to 0.5–4 Gy of γ-radiation. Radiation increased the MN frequency linearly (r²=0.99) with dose. Pre-treatment with Ot or Vc significantly (P<0.01–0.001) reduced the MN counts to 51–67% of RT alone values, giving DMFs of 2.62 (Ot) and 2.48 (Vc). Both the compounds showed significant antioxidant activity in vitro at the above concentrations, which was significantly higher than that of DMSO at equimolar concentrations. Thus, the results demonstrate that both the flavonoids give significant protection to the human lymphocytes against the clastogenic effect of radiation at low, non-toxic concentrations. The radioprotection seems to be associated with their antioxidant activity. The clinical potential of these protectors in cancer therapy needs to be investigated.     

The chlorophenoxy herbicide dicamba and its commercial formulation banvel induce genotoxicity and cytotoxicity in Chinese hamster ovary (CHO) cells

The sister chromatid exchange (SCE) frequency, the cell-cycle progression analysis, and the single cell gel electrophoresis technique (SCGE, comet assay) were employed as genetic end-points to investigate the geno- and citotoxicity exerted by dicamba and one of its commercial formulation banvel^® (dicamba 57.71%) on Chinese hamster ovary (CHO) cells. Log-phase cells were treated with 1.0–500.0 μg/ml of the herbicides and harvested 24 h later for SCE and cell-cycle progression analyses. All concentrations assessed of both test compounds induced higher SCE frequencies over control values. SCEs increased in a non-dose-dependent manner neither for the pure compound (r = 0.48; P > 0.05) nor for the commercial formulation (r = 0.58, P > 0.05). For the 200.0 μg/ml and 500.0 μg/ml dicamba doses and the 500.0 μg/ml banvel^® dose, a significant delay in the cell-cycle progression was found. A regression test showed that the proliferation rate index decreased as a function of either the concentration of dicamba (r = −0.98, P < 0.05) or banvel^® (r = −0.88, P < 0.01) titrated into cultures in the 1.0–500.0 μg/ml dose-range. SCGE performed on CHO cells after a 90 min pulse-treatment of dicamba and banvel^® within a 50.0–500.0 μg/ml dose-range revealed a clear increase in dicamba-induced DNA damage as an enhancement of the proportion of slightly damaged and damaged cells for all concentrations used (P < 0.01); concomitantly, a decrease of undamaged cells was found over control values (P < 0.01). In banvel^®-treated cells, a similar overall result was registered. Dicamba induced a significant increase both in comet length and width over control values (P < 0.01) regardless of its concentration whereas banvel^® induced the same effect only within 100.0–500.0 μg/ml dose range (P < 0.01). As detected by three highly sensitive bioassays, the present results clearly showed the capability of dicamba and banvel^® to induce DNA and cellular damage on CHO cells.     

The effect of donor sex and age on the number of sister chromatid exchanges in human lymphocytes growing in vitro

Summary After analysing the distribution of the numbers of sister chromatid exchanges (SCE) in 200 cells from one individual, we found no evidence to reject the hypothesis of a normal distribution (P>0.50). We then compared the mean numbers of SCE per cell in 18 individuals, three males and three females of each of three age groups (0–10, 30–40, and 60–70 years of age) by means of a one-way analysis of variance, and found that there was no significant difference among them at the level of 5%. When these data were analysed by means of a two-way analysis of variance to test separately the effect of sex and age, we found that the number of SCE per cell does not differ significantly between sexes, but differs with age (P>0.05). People in the age bracket 30–40 years have a higher number of SCE per cell. Age seems to affect both sexes equally.Supported by The Medical Research Council of Ireland     

Effect of maternal fatness on fetal steroids and semi-quantitative real-time PCR expression of receptor genes in sheep

Sexual differentiation of the brain occurs between d 30 and 70 in the fetal lamb. The objective of this experiment was to determine if maternal fatness affects fetal steroid production and expression of their receptors which may ultimately alter endocrine systems postnatally. Fetuses were collected from ewes fed at either 100% (Control; n = 5) or 150% (Fat; n = 6) of NRC recommendations from 60 d prior to breeding until collection at 75 d of gestation. Hypothalamic and amygdala neural tissues were collected from twin male/female fetuses. Serum concentrations of testosterone were greater (P < 0.001) in male fetuses compared to female fetuses. Further, male fetuses from Fat ewes had greater (P < 0.05) serum concentrations of testosterone than male fetuses from Control ewes, but differences in testicular steroidogenic enzyme mRNA were not detected (P = 0.18). Quantity of hypothalamic mRNA for estrogen receptor (ER) β tended (P = 0.1) to be influenced by a sex by treatment interaction. Messenger RNA for ER-β was greater in female fetuses than male fetuses from Control ewes (P = 0.05). Although amount of ER-β mRNA did not differ among male fetuses (P = 0.7), amounts tended to be less (P = 0.07) in female fetuses from Fat ewes compared to those from Control ewes, and did not differ (P ≥ 0.8) from male fetuses. Hypothalamic ER-α mRNA tended (P = 0.1) to be less in fetuses from Fat ewes compared to Control fetuses but was not influenced (P = 0.3) by fetal sex or their interaction. Amount of mRNA for hypothalamic progesterone receptor tended (P = 0.06) to be greater in male fetuses than female fetuses and tended to be less (P = 0.06) in fetuses from Fat ewes than in Control fetuses, but did not differ by any sex by treatment interaction (P = 0.6). Hypothalamic RNA for the androgen receptor did not differ by sex, dam nutritional treatment, or the interaction. Likewise, amygdala RNA for the estrogen or androgen receptor did not differ (P ≥ 0.3) by sex, treatment, or their interaction. Dam fatness appears to decrease the expression of progesterone receptor, ER-α, and decrease amount of ER-β in the female fetuses while increasing circulating concentrations of testosterone in male fetuses. Altered expression of hypothalamic receptor genes by the uterine environment may affect adult responses to stress, sexual behavior and/or the pattern of gonadotropin release in response to gonadal steroids.     

Protective effects of vanadium against DMH-induced genotoxicity and carcinogenesis in rat colon: Removal of O-methylguanine DNA adducts, p53 expression, inducible nitric oxide synthase downregulation and apoptotic induction

Previous studies have shown that dietary micronutrient vanadium can protect neoplastic development induced by chemical carcinogens. Current investigation is an attempt to evaluate the role of vanadium (4.27 μmol/l) in inhibiting 1,2 dimethyhydrazine (DMH) (20 mg/kg body weight) induced rat colon carcinogenesis. We investigated the effect of vanadium against the formation of DMH-induced O⁶-methylguanine (O⁶-Meg) DNA adduct, a potent cytotoxic and mutagenic agent for colon cancer. Supplementation of vanadium significantly reduced the hepatic (P < 0.05), and colonic (at three sequential time points; ANOVA, F = 4.96, P < 0.05) O⁶-Meg DNA adduct levels in rats, indicating vanadium's potency in limiting the initiation event of colon carcinogenesis. Removal of initiated and damaged precancerous cells by apoptosis can prevent tumorigenesis and further malignancy. DNA fragmentation study revealed the vanadium-mediated apoptotic induction in colon tumors. The increased value of apoptotic index (AI) (62.27%; P < 0.01) in subsequent TUNEL assay further confirmed the apoptosis induction by vanadium. This paralleled the nuclear immunoexpression of p53. A significant positive correlation between p53 immunoexpression and AI (P = 0.0026, r = 0.83, r² = 0.69) links its association with vanadium-mediated apoptotic induction. Vanadium treatment also abated the mRNA expression of iNOS (54.03%), reflecting its protective effect against nitric oxide-mediated genotoxicity and colon tumorigenesis. These studies cumulatively provide strong evidence for the inhibitory actions of vanadium against DMH-induced genotoxicity and carcinogenesis in rat colon.     

Intake, rumen fermentation and nutrient flow to the omasum in beef cattle fed grass silage fortified with sucrose and/or supplemented with concentrate

The objective was to determine the relative effects of a specific increase in grass silage sucrose concentration, or a specific supplement of a starch-based concentrate, on rumen fermentation and nutrient supply to the omasum in beef cattle. Four ruminally cannulated Holstein–Friesian steers were fed grass silage only (G), G plus 3 kg concentrates/day (GC), G plus 90 g sucrose/kg dry matter (DM) (GS) and G plus 90 g sucrose/kg DM plus 3 kg concentrates/day (GCS) in a 4 × 4 Latin Square design experiment. Omasal flow was estimated using Co-EDTA, Yb-acetate and indigestible neutral detergent fibre (INDF) as digesta flow markers and purine bases as microbial markers. Concentrate supplementation reduced (P < 0.01) silage and increased (P < 0.001) total DM intake whereas sucrose had no effect. There was a sucrose × concentrate interaction (P < 0.05) for rumen pH whereby addition of sucrose to grass silage alone decreased pH and to grass silage plus concentrate had no effect. Rumen ammonia N (P < 0.01), total volatile fatty acid (VFA) concentration (P < 0.05) and the molar proportions of valerate (P < 0.05) and butyrate (P < 0.001) increased with concentrate supplementation whereas, sucrose supplementation had no effect on rumen fermentation parameters. Organic matter (OM) intake, omasal OM flow, the quantities of OM apparently (OMAD) and truly digested (OMTD) in the rumen (P < 0.001) and total tract OM digestibility (P < 0.01) increased, and apparent and true ruminal OM digestibility decreased (P < 0.05) with concentrate supplementation. Supplementation with concentrate decreased (P < 0.05) ruminal neutral detergent fibre (aNDFom) digestibility and increased (P < 0.05) aNDFom omasal flow. There was a tendency for addition of sucrose to increase (P < 0.1) ruminal OMAD and OMTD, while there was no effect of sucrose addition on intake or digestion of aNDFom. Concentrate supplementation increased (P < 0.001) N intake, flows of N, non-ammonia N (NAN), microbial N (MN) (P < 0.05) and non-ammonia non-microbial N (NANMN) (P < 0.01) and apparent total tract digestibility of N (P < 0.01), whereas sucrose reduced (P < 0.05) N intake and apparent ruminal N digestibility. There was no effect of concentrate or sucrose on N use efficiency or efficiency of microbial protein synthesis. Concentrate supplementation increased (P < 0.001) plasma β-hydroxybutyrate levels. In comparison to supplementing unwilted, well preserved grass silage of moderate digestibility with 3 kg starch-based concentrate per day, the limited response to the rate of sucrose supplementation employed suggests that increasing the water-soluble carbohydrate (WSC) concentration of grass silage through agronomic and/or ensiling practices will have relatively little effect on intake, rumen digestion or efficiency of microbial N synthesis.     

High-Density Genomewide Linkage Analysis of Exceptional Human Longevity Identifies Multiple Novel Loci

Background

Human lifespan is approximately 25% heritable, and genetic factors may be particularly important for achieving exceptional longevity. Accordingly, siblings of centenarians have a dramatically higher probability of reaching extreme old age than the general population.

Methodology/Principal Findings

To map the loci conferring a survival advantage, we performed the second genomewide linkage scan on human longevity and the first using a high-density marker panel of single nucleotide polymorphisms. By systematically testing a range of minimum age cutoffs in 279 families with multiple long-lived siblings, we identified a locus on chromosome 3p24-22 with a genomewide significant allele-sharing LOD score of 4.02 (empirical P = 0.037) and a locus on chromosome 9q31-34 with a highly suggestive LOD score of 3.89 (empirical P = 0.054). The empirical P value for the combined result was 0.002. A third novel locus with a LOD score of 4.05 on chromosome 12q24 was detected in a subset of the data, and we also obtained modest evidence for a previously reported interval on chromosome 4q22-25.

Conclusions/Significance

Our linkage data should facilitate the discovery of both common and rare variants that determine genetic variability in lifespan.     

A note on alarm cues in juvenile African catfish, Clarias gariepinus Burchell: Indications for opposing behavioural strategies

Following exposure of African catfish to alarm cues, a relationship (r_p = 0.60, N = 10, P = 0.07) between feed efficiency (measured as residual feed intake) and the change in percentage of time spent swimming in response to damage-released alarm cues was observed. Feed-efficient animals responded with a decrease in percentage of time spent swimming (P < 0.05) whereas feed-inefficient animals responded with an increase in percentage of time spent swimming (P < 0.05). This study shows opposing behavioural strategies in African catfish in response to damage-released alarm cues being related to feeding efficiency.     

The Importance of Social Condition in the Hormonal and Behavioral Responses to an Acute Social Stressor in the Male Siberian Dwarf Hamster (Phodopus sungorus)

Social condition is an important factor in determining the behavioral and hormonal responses to a social stressor in the Siberian dwarf hamster (Phodopus sungorus). We predict that males housed with a female or a family (female and pups) will show an increase in the magnitude of the behavioral and hormonal responses to a male intruder compared to those of individually housed males. Three treatment groups were studied: individually housed males that had been previously group-housed in same-sex colonies (males,n= 10), males housed with a female (male + female,n= 9), and males housed with their female and pups (male + family,n= 12). Males were monitored for aggressive behavior toward an intruder male for 10 min. Blood samples were taken at baseline and after the encounter. Male + female and male + family groups spent more time in aggressive behavior (P< 0.05), such as attacking (P< 0.05) and fighting (P<0.05), than did individually housed males. These same groups showed significant increases in plasma cortisol after the encounter (P< 0.01) whereas there were no significant increases in plasma cortisol in solitary males. All groups showed significantly lower levels of plasma testosterone (male,P< 0.001, male + female,P< 0.05; male + family,P< 0.01) whereas a significant increase in prolactin occurred only in the male + family group (P< 0.05). There was a significant positive correlation between postencounter cortisol levels and total number of minutes spent in aggressive behavior (P< 0.05). These results demonstrate that the introduction of a novel intruder male results in an activation of the hypothalamic–pituitary–adrenocortical axis and a suppression of the reproductive axis. Furthermore, pairing of a male with a female alters the behavioral and hormonal responses to an intruder male.     

Assessment of cytogenetic damage in lymphocytes and in exfoliated nasal cells of dental laboratory technicians exposed to chromium, cobalt, and nickel

Dental laboratory technicians may be exposed to metal alloys that are used in the production of crowns, bridges and removable partial dentures. These alloys consist of 35–65% cobalt, 20–30% chromium, 0–30% nickel, and small amounts of molybdenum, silica, beryllium, boron and carbon. The aim of this study was to assess whether dental technicians are occupationally exposed to chromium, cobalt and nickel, by analyzing urinary excretion levels of these metals and to investigate the genotoxic effects of occupational exposure associated with dental prostheses production operations by analyzing cytokinesis-blocked micronucleus (CB-MN) frequencies in peripheral lymphocytes and micronucleus (MN) frequencies in exfoliated nasal cells from 27 dental laboratory technicians and 15 control subjects. The differences in the urinary excretion of metals between technicians and controls were statistically significant. The mean (±S.D.) CB-MN frequencies (‰) in peripheral lymphocytes were 4.00 (±2.98) among the dental technicians and 1.40 (±1.30) among the controls, a statistically significant difference (P<0.005). The mean (±S.D.) MN frequencies (‰) in nasal cells were 3.50 (±1.80) among the dental technicians and 1.19 (±0.53) among the controls, which was also a statistically significant difference (P<0.005). There was a significant correlation between duration of exposure and MN frequencies in lymphocytes (r=0.642, P<0.01), but not in nasal cells of technicians. Our data reveal that in vivo exposure to chromium, nickel and cobalt metals is evident and that this occupational exposure may contribute to the observed genotoxic damage in two types of cells, e.g. lymphocytes and exfoliated nasal cells. However, it cannot be determined which compound(s) are responsible for the genotoxic damage observed in this study.     

Increased lymphocyte DNA strand breaks in rubber workers

Objective: To study the effect of occupational exposure to rubber processing, smoking, and alcohol drinking on lymphocyte DNA damage. Subjects and Methods: Of 371 employees (197 men and 174 women) from a rubber factory in Guangzhou, 281 were rubber processing workers from five production sections and 90 were managerial workers. Information on occupational exposure, smoking, and drinking was collected by interviews. Blood samples were taken in the morning by venipuncture. DNA damages were measured by the Comet assay. Possible DNA-protein crosslinks were broken down by proteinase K. Tail moment, measured by Komet 4.0 image analysis software, was the measure of DNA damage. Results: The rubber processing workers had larger tail moment than the managerial workers (Geometric mean, 95%CI) [1.77 μm (1.64–1.90) versus 1.52 μm (1.36–1.71), P=0.04]. Both smoking [1.93 μm (1.74–2.13) versus 1.59 μm (1.47–1.71), P=0.003] and alcohol drinking [2.21 μm (1.87–2.62) versus 1.63 μm (1.53–1.74), P<0.001] increased tail moment. Tail moment differed significantly among job categories (F=3.21, P=0.008), the largest was observed in mixers. In the non-smoking and non-drinking workers, rubber processing workers had larger tail moment than managerial workers after adjusting for age (P=0.033). General linear model analysis showed that after adjusting for each other, occupational exposure (P=0.027), smoking (P=0.012), and alcohol drinking (P=0.013) was associated with larger tail moment, whereas age and gender had no effect. Conclusions: Occupational exposure to rubber processing, smoking, and alcohol drinking can cause DNA damage.     

The relationship between micronuclei in human lymphocytes and selected micronutrients in vegetarians and non-vegetarians

A vegetarian diet results in higher intake of vitamins and micronutrients, which – although providing antioxidant defence – may lead to deficiency in other micronutrients involved in DNA metabolism and stability (such as vitamins belonging to the B group). The principal difference among various vegetarian diets is the extent to which animal products are avoided. We have performed a pilot study to determine the relationship between the micronucleus frequency in lymphocytes and diet, and we compared the levels of Vitamins C and E, β-carotene, B₁₂, folic acid, homocysteine and total antioxidant capacity in healthy vegetarians and non-vegetarians. The vegetarian group, consisting of 24 volunteers (13 women and 11 men), were matched for age and sex with 24 volunteers (12 women and 12 men) with a traditional dietary habit. Among the vegetarians were 13 lacto-ovo-vegetarians with average duration of vegetarian diet 10.8 years (ranging from 5 to 26 years) and 11 lacto-vegetarians with average duration of vegetarian diet 8.2 years (ranging from 3 to 15 years). Homocysteine, Vitamins C and E and β-carotene levels in plasma were assayed by HPLC, and serum folate and Vitamin B₁₂ were determined with Elecsys Immunoassay tests. The total antioxidant capacity of plasma was estimated by measuring the ferric-reducing activity in a spectrophotometric assay. Micronuclei were measured in cytokinesis-blocked lymphocytes. Vegetarians had significantly higher levels of Vitamin C and β-carotene (but not Vitamin E) in plasma compared with non-vegetarians (P < 0.001). There were no significant differences in serum levels of folic acid and Vitamin B₁₂ between the monitored groups. Levels of folic acid in vegetarians correlated with length of vegetarianism (r = 0.62, P = 0.001, N = 24). Vegetarians had elevated levels of homocysteine compared with non-vegetarians (P = 0.007), as did vegetarian women compared with non-vegetarian women (P = 0.031). We did not find any differences in total antioxidant capacity or in micronucleus frequency between the groups. Micronuclei correlated with age (r = 0.62, P < 0.001, N = 48), women having higher frequencies than men. Multifactorial regression analysis showed significant effects of age, sex and total antioxidant capacity on micronucleus frequency (N = 48, P < 0.001).     

Interrelationships among morphology, echotexture, and function of the bovine corpus luteum during the estrous cycle

It has been suggested that ultrasound image attributes are a potential indicator of physiological and functional status of the corpus luteum (CL) in several species, including cattle. The aims of this study were to evaluate CL morphological, functional and echotextural characteristics, and also to investigate the hypothesis that those attributes are correlated and change similarly throughout an estrous cycle. Ovaries of crossbred (Bos taurus taurus × Bos taurus indicus) heifers were evaluated using ultrasonography daily throughout an interestrus interval using a B-mode, real-time ultrasound machine equipped with a 5 MHz linear-array rectal transducer, during a natural estrous cycle (Experiment 1; n = 12) or during a shortened cycle, with luteolysis induction 10 d after estrus (Experiment 2; n = 6). Blood samples were collected for assay of plasma progesterone concentrations. Corpora lutea areas were measured and daily images of each CL were videotaped and digitized for computer-assisted analysis using custom-developed software. In Experiment 1, area of luteal tissue increased until a maximum value 10 d after estrus (P < 0.001), followed by a plateau phase, and then a decline beginning 14 d after estrus. Luteal tissue area was highly correlated to plasma progesterone concentrations (r = 0.86; P < 0.001). When luteolysis was induced in Experiment 2, loss of CL function (decrease in plasma progesterone concentrations to metestrous values) preceded tissue regression by 48 h (24 h compared with 72 h; P < 0.001). The mean pixel value of ultrasound images did not change in Experiment 1 (P > 0.70), but a day effect on this attribute was observed in Experiment 2 (P = 0.052). In contrast, mean pixel value was correlated to plasma progesterone concentrations in Experiment 1 (r = −0.63; P < 0.05), but not in Experiment 2 (r = −0.28; P > 0.10). In regard to CL heterogeneity, defined as the standard deviation of the mean pixel value of the luteal tissue, a time effect was observed following both natural (Experiment 1; P < 0.009) and luteolysis-induced (Experiment 2; P < 0.05) estrous cycles (P < 0.05). Moreover, this variable was correlated with plasma progesterone concentrations (r = −0.71 and −0.58 in Experiments 1 and 2, respectively; P < 0.01), indicating that CL images were more heterogeneous during metestrus and after luteolysis (functional regression). In summary, morphological and echotextural attributes were correlated with CL function and underwent similar changes during the estrous cycle. Luteal tissue heterogeneity, assessed by ultrasonography, is considered a potential indicator of CL functional status, because it is correlated to circulating progesterone concentrations.     

The influence of handling by humans on the behavior, growth, and corticosteroids in the juvenile female pig

The influence of two handling treatments by humans on the behavioral response to the presence of humans, growth performance, and total and free corticosteroid concentrations in the presence and absence of humans was studied in the female pig. The handling treatments, considered as pleasant and unpleasant, were imposed three times per week for 2 min in duration from 11 to 22 weeks of age. Gilts in the unpleasant handling treatment spent less (P < 0.01) time within 0.5 m of the experimenter and displayed fewer (P < 0.01) interactions with the experimenter in a 3-min test conducted at 25 weeks of age. In addition, gilts in this treatment had a slower (P < 0.05) growth rate from 11 to 22 weeks of age and had higher (P < 0.001 and P < 0.01) corticosteroid concentrations at rest and in response to the presence of the experimenter, respectively, at 24 weeks of age. It was concluded that the unpleasant handling treatment resulted in both chronic and acute stress responses.     

Influence of exogenous enzymes on nutrient digestibility, extent of ruminal fermentation as well as milk production and composition in dairy cows

This experiment studied effects of a mixture of exogenous enzymes (ZADO^®) from anaerobic bacteria on ruminal fermentation, feed intake, digestibility, as well as milk production and composition in cows fed total mixed rations (TMRs; 0.7 corn silage and 0.3 of a concentrate mixture). Twenty lactating multiparous Brown Swiss cows (500 ± 12.4 kg live weight) were randomly assigned into two experimental groups of 10 immediately after calving and fed a TMR with or without (CTRL) addition of 40 g/cow/d of enzymes for 12 weeks. Addition of enzymes increased (P<0.05) rumen microbial N synthesis. Intake of dry matter (DM) and organic matter (OM) was positively influenced (P<0.05) by supplementation, and digestibility of all nutrients was higher (P<0.05) in the total tract of supplemented cows, although the magnitude of the improvement varied among nutrients, with the highest improvement in aNDFom and ADFom (418–584 and 401–532 g/kg respectively; P<0.05) than the other nutrients. Supplementation of enzymes also increased (P<0.05) rumen ammonia N and total short chain fatty acid (SCFA) concentrations, and individual SCFA proportions were also altered with an increase in acetate (61.0–64.8 mol/100 mol; P=0.05) before feeding, and acetate and propionate increased 3 h post-feeding (60.0–64.0 and 18.3–20.8 mol/100 mol respectively; P<0.05). Milk and milk protein production was higher (12.8–15.7 and 0.45–0.57 kg/d respectively; P<0.05) for cows fed the ZADO^® supplemented diet. This exogenous enzyme product, supplemented daily to the TMR of cows in early lactation, increased milk production due to positive effects on nutrient intake and digestibility, extent of ruminal fermentation and microbial protein synthesis.     

Effects of inclusion of neutral detergent soluble fibre sources in diets varying in forage particle size on feed intake, digestive processes, and performance of mid-lactation Holstein cows

The aim of this study was to evaluate effects of partial replacement of neutral detergent soluble fibre (NDSF) for starch in diets varying in particle size (PS) of alfalfa hay on chewing activities, ruminal fermentation, nutrient digestibility and performance in mid-lactation dairy cows. Eight multiparous Holstein cows (146 ± 6.0 d in milk; 36.7 ± 2.57 kg milk/d) were used in a replicated 4 × 4 Latin square design with four 21 d periods with the last 7 d for data collection. The experiment was a 2 × 2 factorial arrangement with 2 levels of NDSF (low = 85 g/kg or high = 130 g/kg diet dry matter) each combined with 2 PS (short = 20 mm or long = 40 mm) of alfalfa hay. Results show that forage PS alone, or in combination with NDSF inclusion, had no effect on dry matter (DM) intake. Although total chewing, eating and ruminating times were not affected by treatments, eating time per kg of neutral detergent fibre (NDF) ingested was higher in long versus short alfalfa hay-based diets (P<0.05). Feeding long forage PS increased sorting of the diet against particles >19 mm, and in favor of those <8 mm (P<0.05). Feeding diets high in NDSF lowered DM intake (P<0.05), but increased apparent digestibility of all nutrients including NDF (P<0.05) independent of forage PS. Ruminal pH and concentrations of total volatile fatty acids were unaffected by dietary treatments, however the proportion of butyrate was higher in ruminal fluid of cows fed high NDSF diets (P<0.05). Changes in milk composition included lower milk crude protein content in high NDSF diets and higher lactose content for short hay-based diets (P<0.05). That milk yield and milk energy output were similar in low versus high NDSF diets suggests that high NDSF-fed cows had higher energy efficiency due to lower DM intake. Results suggest that, independent of forage PS, NDSF sources can be successfully included to partly replace starchy grains in diets exceeding minimum fibre recommendations.